journal of applied botany and food quality 93, 121 (2020), doi:10.5073/jabfq.2020.093.015 fresh fruit and vegetables are the major source of essential vitamins and minerals, which are needed for human health and well-being. they are, however, perishable living products that request continuous measures for quality keeping by growers, storage operators, processors, and retailers. sampling of fresh produce for assessing appearance, texture, flavour, and nutritional value have been established quality criteria, whereas non-invasive measurements on each individual product preand postharvest with traceability along the supply chain are becoming important for all role players. the frutic-2019 provided a platform for researchers and practitioners to engage in technical discussions about innovations and new technologies, and explore further areas of research needed in the industry to promote quality and safety of fruit and vegetables. the first symposium of the frutic series took place in israel 1983, followed by usa, spain, japan, france, germany, italy and chile. in 2017 and 2018 frutic was organized in cooperation with fruit logistica in berlin, germany, in september 2019 frutic was held in hong kong in cooperation with asia fruit logistica. scientists presented their topics at the site of the asia fruit logistica trade fair at the asiaworld-expo of hong kong, in industry-oriented sections. some of the selected talks are now published as full articles in this special issue. the frutic-2019 event provided a concerted action that brought together academic scientists and the role players from fresh produce industry, to interact with each other for the purpose of information dissemination, sharing practical experience and developing road maps for the most effective way to reach the common goals. we would like to thank all participants for their contributions to the symposium program and for their contributions to this special issue. we also express our sincere thanks to the journal of applied botany and food quality team for publishing this special issue on time. pramod mahajan, manuela zude-sasse editorial preface to the frutic 2019 symposium frutic conference in hongkong. von links: wilfried wollbold (global brand manager, fruit logistica), dr. manuela zude-sasse (atb), john hey (keynote speaker, fruitnet media international), dr. pramod mahajan (photo: von haselberg/atb) journal of applied botany and food quality 95, 174 (2022) book review cannabis – ein handbuch für wissenschaft und praxis andreas s. ziegler although cannabis was already widely used in various traditional medical systems in ancient times, it is only in recent years that the medicinal drug has advanced to become one of the most exciting medical-pharmaceutical topics of the present day. in hardly any other field of pharmacotherapy has the state of knowledge developed with comparable dynamism in recent times. in the book, renowned experts from various scientific disciplines describe the current scientific evidence for various therapeutic ap plications of cannabis or cannabinoids and promote quality-assured care of patients assisted by cannabis medicines. in the individual chapters of the handbook, reference is made to all relevant disciplines that are important for a sound understanding of the use of cannabis as a medicinal product: history, botany, biosynthesis, breeding and cultivation, import, supply chain, quality assurance and quality control, pharmacology, biopharmacy and pharmacokinetics, indications and evidence of pharmacotherapy, risks and side effects, driving safety, legal status of cannabinoid-containing medicinal products and non-medicinal products, dosage forms and modes of administration, forms of consumption in recreational use, identity verification of starting materials, production of prescription and defecture medicines, supply of cannabis prescriptions, reimbursement and taxation, narcotic documentation, legal framework of supply, and patient counseling. the individual scientists, who have provided their specific knowledge from the various fields in a representative manner, have understood very well how to combine their respective expertise into a holistic overall picture. in addition, the book offers a scientifically sound source of information as an alternative to the sometimes targeted disinformation on dubious websites or half-truths circulating in social media. thanks to this concentrated expertise, a unique overall picture emerged that summarizes the current legal and scientific framework of medical cannabis application in a larger context for the first time. the appendix contains an extensive subject index that allows the reader to quickly research the points covered in each chapter on specific topics. this book can be recommended without any reservation as an exciting and instructive information to a broad range of readers. not only does it provide valuable support to professional groups such as plant breeders, farmers, plant physiologists, botanists, pharmacists, medical doctors as well as students of the mentioned faculties, but the book also offers an excellent opportunity for interested laypeople to obtain comprehensive information on the current state of knowledge of this important pharmaceutical drug. a number of scientific articles are listed in each annex of the 17 chapters, allowing readers to obtain more detailed information on the individual topics. dr. h. schulz e-mail: hs.consulting.map@t-online.de bibliography: andreas s. ziegler, cannabis – ein handbuch für wissenschaft und praxis, wissenschaftliche verlagsgesellschaft mbh, stuttgart, with contributions from renowned experts from various scientific fields, 1st edition 2022, 527 pages, 275 mostly color illustrations, 83 tables, price: 98,€, isbn: 978-3-8047-4152-2. angewandte botanik. 112 kleine mitteilungen. kleine mitteilungen. zur geschichte der quassia amara. – das surinam-bitterholz oder echte quassia-holz wurde durch eine linnésche dissertation „lignum quassiae, quod praeside d. d. car. v. linné, pro gradu doc toris proposuit carolus m. blom, smolandus, upsala 1763, maji 28“ (auch in linné amoenitates academicae vi, 1764, s. 416–430 mit 1 tafel) in den europäischen arzneischatz eingeführt. ein göttliches heilmittel, das den apothekern nicht genug empfohlen werden könne, heißt es am schlusse jener dissertation, die unter linnés autorität zur folge hatte, daß man sich lebhaft für die pflanze interessierte und sich material davon zu verschaffen suchte. 1788 fand das surinam quassiaholz aufnahme in die londoner pharmakopoe. weniger bekannt ist, daß nicht nur das holz, sondern auch wurzel, rinde, blätter, blüten und frucht ebenfalls stark bitter schmecken. barbosa rodriguez beobachtete (nach peckolt in ber. deutsch. pharmazeut. ges. viii, 1898, s. 428) in manaos (amazonas) zur blütezeit unter dem baume stets eine anzahl der verschiedensten toten insekten. nach fermins angaben sollen in surinam schon 1714 die hochroten blumen des baumes als gutes magenmittel geschätzt worden sein. auf diese verwendung wirft ein dokument licht, das ich zufällig auffand und hier mitteilen will, da die geschichtlichen an gaben über quassia amara sehr abweichend sind (vergl. zörnig, arznei drogen i, 1909, s. 316). das schriftstück, das an den apotheker witting, direktor des apotheker-vereins, in hoexter gerichtet ist, lautet: „cassel, 30. juli 1823. hochgeehrtester herr und freund! als ein zu den ihnen vor einiger zeit überschickten flores quassiae gehöriger nachtrag, habe ich die ehre, ihnen noch zu bemerken, daß selbige 1791 von einem herrn beyrodt aus surinam mitgebracht worden sind, wo man sich ihrer als ein magen stärkendes mittel bedient, herr schwabe hält sie von quassia amara abstammend und glaubt ihre natürliche farbe sei rot, weil die noch nicht geöffneten blumen durch anwendung von säuren gerötet werden. mit der bitte diese ergänzung gütig aufzunehmen und gehörigen orts davon anwendung zu machen, habe ich die ehre hochachtungsvoll zu verbleiben ew. hochwohlgeb. ergebenster fiedler.“ (karl wilhelm fiedler, geb. 4. xii. 1758 zu malchin, war anfangs apotheker in cassel, ward 1797 lehrer am forstinstitut zu waldau bei cassel, 1800 prof. und 1840 lehrer der chemie und bergbaukunst bei der kurfürstl. lehranstalt für die bergwerksalumnen.). j. schuster. die krankheit des „fadenziehenden brotes“ und seine ver hütung. häufiger als vor dem kriege tritt jetzt an warmen tagen eine krankheit des brotes auf, die sich darin äußert, daß beim durch brechen oder durchschneiden des brotes die krume desselben klebrig erscheint und in langen fäden auseinanderziehbar ist. dem brote ent strömt ein anfangs etwa erdbeerartiger, aromatischer, späterhin aber loheartiger, widerlicher, scharfer geruch. die krume is t braun verfärbt, das brot sieht unappetitlich aus und schmeckt ekelerregend. für einen normalen geschmack wenigstens ist solches brot ungenießbar. kleine mitteilungen. 113 der erreger der krankheit ist ein spaltpilz, bacillus mesentericus, der auf stärkehaltigen pflanzenteilen überall vorkommt, mit dem, korn in die mühle und mit dem mehl in die bäckerei gelangt. er bildet so hitzebeständige keime (sporen), daß selbst der backprozeß den schäd ling nicht zu töten vermag. bei günstiger feuchtigkeit und tempera turen von über 20° c vermag sich der spaltpilz zu entwickeln und die oben beschriebenen erscheinungen hervorzubringen. zur verhütung der krankheit empfiehlt sich folgendes: während der heißen zeit sollte nach möglichkeit kein ungesäuertes brot (großgebäck) hergestellt werden. dasselbe bietet infolge seiner mehrere tage anhaltenden hohen feuchtigkeit den spaltpilzen günstige lebensbedingungen. großgebäck ist entweder mit sauer zu führen oder auf andere weise genügend zu säuern. das kann z. b. durch saure molken (ersatz des zur teigbereitung erforderlichen wassers ganz oder, bei stark sauren molken, teilweise durch molken) oder durch zusatz von mindestens 0,5% milchsäure oder 0,1% essigsäure (5 g milchsäure pro kg mehl = 8 g pro 1 wasser oder 2 g essigsäure pro kg mehl = 3 g pro 1 wasser). in genügend sauren broten vermögen sich die spaltpilze nicht zu entwickeln. soll ungesäuertes gebäck hergestellt werden, so muß auf kleingebäck zurückgegriffen werden. dasselbe trocknet schnell aus und wird schnell verbraucht. durch genügend langes und scharfes ausbacken, wo durch die feuchtigkeit herabgesetzt wird, schnelles ab kühlen, saubere, luftige, kühle lagerung und raschen ver brauch der backware kann das auftreten der krankheit in der regel ebenfalls verhütet werden. fadenziehende backware ist vom verkehr auszuschließen und ist scharf getrocknet als futtermittel, z. b. für hühner, bei geringerer entwicclung der krankheit eventuell auch noch als nahrungsmittel, z. b. wie es in der schweiz geschieht, zu suppenwürfeln u. dgl. zu verwerten. dr. w. herter. vorstand der botanisch-bakteriologischen abteilung der versuchsanstalt fürgetreide verarbeitung. literatur. einecke, a., farbenänderungen der kartoffelblüte im januar 1918 und saatenanerkennung. deutsche landwirtschaftliche presse xlvi (1919), s. 356. alle ausnahmefälle in der blütenfarbe (variationen, knospen oder sproßmutationen) sind bei der saatenbesichtigung abzuerkennen. rabanus. gerum, i. , über den stärkegehalt der haferflocken. zeitschrift für äuns der nahrungsund genußmittel xxxvii, s . "1s angewandte botanik i. 8 nahrungs mittel. uc1.b3299303_page_130_cut uc1.b3299303_page_131_cut angewandte botanik. vereinigung für angewandte botanik. bericht über die 15 . hauptversammlung der vereinigung für angewandte botanik in hann.-münden vom 4.–5. august 1919. in der herkömmlichen weise fand auch in diesem jahre zusammen mit der deutschen botanischen gesellschaft und der vereinigung für systematik und pflanzengeographie die haupt versammlung in hann.-münden statt. es hatten sich hierzu nachstehende 2 7 mitglieder eingefunden: benary-erfurt naumann-dresden bredemann-berlin neger-tharandt brick-hamburg plaut-bernburg brunner-hamburg raschberlin buchwald-berlin schulz-halle duysen-berlin seeliger-berlin engelmann-elberfeld simon-dresden falck-hann.-münden thiele-witzenhausen fischer-essen a. r. voigt-hamburg gilg-berlin wächter-berlin gropengießer-leverkusen . wehnert-kiel lindner -berlin westerdijk -amsterdam ludwigs-berlin wollen weber-berlin müller-augustenberg als gäste nahmen noch 18 personen teil. um 9° uhr eröffnete der vorsitzende prof. voigt-hamburg die sitzung. den geschäftsbericht erstattete der 1. schriftführer dr. müller-augustenberg. während des geschäftsjahres hat di e vereinigung durch den tod verloren: apotheker kruer in ahrens burg, dr. raatz in kl.-wandsleben bei hamburg, ökonomierat wanner in straßburg und saatzüchter strube in schlanstedt. die jahresberichte der vereinigung gingen nach beschluß der hamburger versammlung ein und a n deren stelle trat di e neue zeitschrift angewandte botanik, die von nun a n das organ bericht über die 15. hauptversammlung der vereinigung für ang. bot. 187 der vereinigung für angewandte botanik darstellen wird. die mitglieder erhalten sie kostenlos geliefert. den kassenbericht erstattete für den erkrankten rechner der vorsitzende. das jahr schließt mit einer mehreinnahme von 248,54 / ab. dem rechner wird vorbehaltlich der nachprüfung durch die rechnungsprüfer entlastung erteilt. bei der vorstandswahl wurden die bisherigen vorstands mitglieder einstimmig wiedergewählt. eine zeitweilige erhöhung des mitgliedsbeitrages wurde ab gelehnt, dagegen dem vorstand die ermächtigung erteilt, in schwierigen zeiten eine erhöhung eintreten lassen zu können. die satzungen der vereinigung sollen einer durchsicht unter zogen und, wenn nötig, abgeändert werden. der vorstand wird ermächtigt, die satzungen neu abzufassen und im nächsten jahr der versammlung vorzulegen. als nächstjähriger versammlungsort wird breslau gewählt. von 9"–10” sprach dann dr. bredemann über die bisherigen erfahrungen und die aufgaben weiterer forschung über den feldmäßigen anbau der nessel zur fasergewinnung. die bisherigen erfahrungen lassen die kultur der nessel in unseren gegenden aussichtsreich erscheinen. auch im felde an gebaut, liefert die nessel hohe bestände. eine überfrucht würde die bodenfeuchtigkeit in den oberen schichten besser festhalten, solange die nesselpflanzen noch klein sind. als windschutz emp fiehlt der vortragende 1–2 drillreihen hanf quer durch die felder. auf eine ernte ist in allen fällen im ersten jahr noch nicht zu rechnen. als bester boden für nesselkulturen hat sich niede rungsmoor erwiesen. hier sind zwei ernten wohl möglich. un geklärt ist noch die sortenfrage, da nicht alle rassen gleiche erträge und gleiche faser-qualitäten liefern. von 10°–11 sprach dr. h. fischer über den gegen wärtigen stand der kohlensäurefrage für pflanzen kulturen. den kohlensäuredüngungsfragen sind praxis und wissen schaft bisher recht gleichgültig gegenüber gestanden. eine größere anlage zum studium der kohlensäuredüngung is t seit kurzem in horst a . d . r . vorhanden. dort werden die abgase eines hoch 0fens für pflanzenkulturen verwendet. die kohlenoxydgase werden zunächst z u kohlendioxyd verbrannt, dann gereinigt und in zement 188 bericht über die 15 . hauptversammlung der vereinigung für ang. bot. röhren nach glashäusern geleitet. im ganzen stehen 9 glashäuser und etwa 4 h a land zu versuchszwecken zur verfügung. ver gleiche zwischen begasten und unbegasten kulturen zeigen ganz erhebliche (doppelte bis dreifache) mehrerträge in den begasten häusern. der vortragende liefert dafür zahlreiche exakte an gaben. viele fragen, die mit der kohlesäuredüngung zusammen hängen, sind aber noch ungeklärt und erfordern dringend eine arbeitsstätte zur wissenschaftlichen behandlung dieser praktisch so bedeutsamen fragen. (vergl. s . 138.) von 11” uhr bis 12 uhr hielt prof. falck einen vor trag über holzkonservierung und über eine methode zur laboratoriumsmäßigen beurteilung von pflanzenschutz mitteln. nach einleitenden bemerkungen über frühere kulturen von holzzerstörenden pilzen, die ein gemenge von pilzen darstellten, ging redner auf die neuen arbeiten seines laboratoriums über, sowie auf die mittel, die heutzutage zur holzkonservierung in betracht kommen. versuche mit einem neuen mittel „resinol“ ergaben seine unbrauchbarkeit für d ie holzkonservierung, dagegen seine verwendungsmöglichkeit für den pflanzenschutz. in welcher weise das im laboratorium festzustellen ist, wurde ausführlich erläutert und schließlich noch die herstellung einer resinolkalk und resinolmagnesiabrühe besprochen. (vergl. s. 157.) um 1 2 uhr sprach prof. neger über ein untrügliches erkennungsmerkmal für rauchschäden bei laubhölzern. er weist daraufhin, wie schwierig oft rauchgasbeschädigungen festzustellen sind, d a manches, was bisher als charakteristisches merkmal bezeichnet wurde, nicht immer zutrifft. als ein untrüg liches merkmal nach seinen bisherigen beobachtungen sind jedoch einbuchtungen um die lentizellen, wie sie durch keine andere ursache hervorgerufen werden. (vergl. s . 129.) hierauf sprach von 12”–12” uhr als letzter redner prof. simon über die beurteilung des anbauwertes französischer rotkleesamen. der anbauwert des saatgutes hängt von seiner herkunft ab. da der in deutschland erzeugte rotkleesamen für unseren futter bau nicht genügt, so sind wir gezwungen, ausländischen rotklee einzuführen und als solcher kam vor allem französische saat in betracht, die jedoch je nach der ursprungsgegend recht ver schiedenen anbauwert besitzt. ungeeignet ist der südfranzösische bericht über die 15. hauptversammlung der vereinigung für ang. bot. 189 jº klee. über den mittel-, westund nordfranzösischen klee sind d ie ansichten der forscher, die sich mit diesen fragen befaßt haben, verschieden, weil die klimatischen verhältnisse deutsch lands eben auch verschieden sind. referent schlägt eine neuein teilung der ursprungsbezeichnungen französischer rotkleesaaten w r, der beim neuaufbau unserer handelsbeziehungen mit frank reich geltung verschafft werden sollte. (vergl. s . 146.) die vorträge werden, mit ausnahme des ersten vortrages, d e r bereits in einer anderen zeitschrift erschienen ist, ausführlich in dieser zeitschrift zum abdruck gelangen, zum größten teile befinden sie sich bereits in der vorliegenden nummer. im anschluß a n die versammlung fand a n einem nach mittage eine forstliche exkursion in den wald bei münden statt, w o verschiedene versuchsparzellen besichtigt und die ver slche vom direktor der forstakademie, oberforstmeister prof. schilling erläutert wurden. an einem andern nachmittage erfolgte e in ausflug nach witzenhausen zur besichtigung der dortigen kolonialschule (direktor fabarius). sie stellt eine privat anstalt dar, die sich zur aufgabe gestellt hat, den schülern mög ichst vielerlei wissen auf landwirtschaftlichem und kolonialem gebiete beizubringen. dazu finden außer theoretischem unter richt und seminaristischen übungen auch praktische unterweisungen in dem 800 morgen gelände umfassenden landwirtschaftlichen betriebe statt. k. müller. uc1.b3299303_page_204_cut uc1.b3299303_page_205_cut uc1.b3299303_page_206_cut uc1.b3299303_page_207_cut art10_fähnrich.indd journal of applied botany and food quality 85, 73 74 (2012) institute for applied botany and pharmacognosy, university of veterinary medicine, vienna, austria effects of gibberellic acid as a gametocide on different genotypes of german chamomile (matricaria recutita [l.] rauschert) b. faehnrich, ch. franz (received february 1, 2012) summary the objective of the present research was to determine statistically before made observations on reactions of chamomile after treatment with gibberellic acid undertaken to fi nd a feasible way of chemical castration to create maternal lines as a base for hybrid progeny. chamomile plants of three cultivars were set up in a split plot design, with treated and untreated parts. four traits: ‘percentage affected capitula’, ‘seeds per capitulum’, ‘percentage of germination’ and ‘percentage infertile pollen’ were valuated, twice per plant. analyses showed a signifi cant effect of treatment on pollen viability and a strong tendency on the visible affection of capitula, but no infl uence on number of seeds or germination rate. neither did cultivars show any infl uence, nor did an interaction between cultivar and treatment appear. according to the aim to fi nd a suitable method to generate male sterile maternal lines, the reactions, affecting male, but not female fertility, seem to be highly appreciated, but the repeated spray application in a necessarily sensible stage of fl ower development and a reduction of pollen viability of only about 10% constrain the practicability. introduction looking for a suitable gametocide for german chamomile in order to produce male sterile lines comprehensive research work was undertaken. in publications treating other asteraceae in this context mostly gibberellic acid (ga3, c19h22o6) was mentioned as a useful agent (schuster and liu, 1983; baydar and gökmen, 2003; miller and fick, 1978; spirova, 1975). additionally there is a patent web entry on the use of sulfonyl urea derivative as a gametocide on sun fl owers (patent-de, 2008). basing on the recommended use of gibberellic acid in a threefold spray application on carthamus tinctorius in a concentration of 100ppm and a result of reduced pollen viability from 81.6% to 6.7% (baydar and gökmen, 2003) similar trials with german chamomile were undertaken. material and methods in a split plot design with two factors (cultivars ‘bona’, ‘manzana’, ‘lutea’ and treatment with ga3, treated or not treated, respectively) 18 chamomile plants were set up under green house conditions with six replications per combination of factors. in the treated plot eight spray applications of gibberellic acid in a concentration of 100ppm started in a very early fl owering stage and were continued with always three days interval, while the second plot stayed untreated. after that the four traits ‘percentage affected fl ower heads’, ‘seeds per fl ower head’, ‘percentage of germination’ and ‘percentage infertile pollen’ were evaluated, each twice per plant. the valuation of the primarily mentioned trait started a few days after the last application and was repeated two weeks later. ‘percentage of germination’ was tested in petri dishes with wet fi lter paper and controlled after a two weeks period – in accordance to recommended germination tests in heeger (1989). each petri dish was fi lled with the seeds of one capitulum. pollen viability was estimated by analyzing fresh, mature pollen after acetocarmine staining (2% acetocarmine solution), according to lambrou et al. (2001) and gerlach (1984). the percentage of affected fl ower heads concerns visually cognizable damages of the disc fl owers and/or the ray fl owers. results ga3-treatment showed a signifi cant negative infl uence on pollen viability (p = 0.023) and a strong tendency on affection of fl ower heads (p = 0.054), at a level of signifi cance of α = 0.05. the mean for the ga3-treated plot was 9.8 % of infertile pollen vs. 1.4 % for the untreated plot and 9.3 % of affected fl ower heads vs. 0 % for the untreated plot, respectively (fig. 1 and 2). fig. 1: means of percentage of infertile pollen in treated and untreated plots. the traits ‘seeds per fl ower head’ and ‘percentage of germination’ showed no signifi cance or tendency for the infl uence of ga3treatment. neither did the factor ‘cultivar’ cause any signifi cant infl uence, nor did interactions between ‘ga-treatment’ and ‘cultivar’ occur. discussion considering the intended aim to fi nd a suitable agent for the production of maternal lines with male sterility the application of gibberellic acid initially seems to be highly suitable due to the negative effect on male fertility (‘percentage infertile pollen’, ‘percentage affected capitula’) and the non-effect on female fertility 74 b. faehnrich, ch. franz (‘seeds per capitulum’, ‘percentage of germination’). additionally the result shows no infl uence of cultivars and no interactions between the factors. but reduction of pollen fertility is less than ten percent (9.8 % vs. 1.4 %) and even if having in mind that this refers only to despite affection of fl ower heads yet developed pollen this extent of reduction is too little to be used in practice. also due to a necessarily reiterate application in a sensible fl owering stage, the danger of damaging the whole plant in case of a too-much of the agent and the dependency of weather conditions the use of gibberellic acid as a gametocide for chamomile cannot be recommended. acknowledgements with many thanks to prof. dr. johannes novak who gave very helpful suggestions and comments to experimental design, statistical analyses and writing efforts. references baydar, h., gökmen, o., 2003: hybrid seed production in safflower (carthamus tinctorius) following the induction of male sterility by gibberellic acid. plant breed. 122, 459-461. gerlach, d., 1984: botanische mikrotechnik, eine einführung. georg thieme verlag stuttgart, new york. heeger, e., 1989: handbuch des arzneiund gewürzpfl anzenanbaues, drogengewinnung. veb deutscher landwirtschaftsverlag, berlin. lambrou, m., bein-lobmaier, b., franz, c., 2001: cytological analysis of diand tetraploid plants of matricaria recutita (asteraceae (asteraceae ( ) and their hybrid progeny, poster presentation. miller, j., fick, g., 1978: adaptation of reciprocal full sib selection in sunfl ower breeding using gibberellic acid induced male sterility. crop sci. 18, 161-162. patent-de, 2008: verwendung von sulfonylharnstoffen als gametozide. http://www.patent-de.com/19890302/de3727629a1.html. schuster, w., liu, s., 1983: über die gametozide wirkung von gibberellinsäure auf unterschiedliche genotypen der sonnenblume. j. appl. bot. food qual. – angew. bot. 57, 85-98. spirova, m., 1975: new data on the male sterility in sunfl owers induced by gibberellic acid. plant sci. 12, 10-17. address of the authors: dipl.-ing. dr. bettina faehnrich* and o. univ. prof. dr. chlodwig franz, institute of applied botany and pharmacognosy, university of veterinary medicine, veterinärplatz 1, a-1210 vienna, austria (*corresponding author, e-mail: bettina.faehnrich@vetmeduni.ac.at) fig. 2: means of percentage of affected fl ower heads in treated and untreated plots. angewandte botanik. 74 th. sabalitschka, verbreitung falscher ansichten über denwert pflanzlicher nahrungsmittel im wolke. von dr. th. sabalitschka. wie nötig es ist, daß der heutige standpunkt der nahrungs mittelchemie und der angewandten botanik, soweit er für das er nährungsproblem u. dergl. von bedeutung ist, endlich auch einmal weiteren kreisen bekannt wird, dürfte folgendes klar beweisen. in der festbeilage des „berliner lokalanzeiger“ ostern 1919 findet sich eine abhandlung von prof. dr. med. h. rosin: „formen der abmagerung und ihre beseitigung“. es sei gestattet, auf einige ausführungen des autors, welche die angewandte botanik be treffen, hier kurz einzugehen. der verfasser schreibt über die pflanzenkost: „am nahr haftesten sind die hülsenfrüchte, erbsen, bohnen, linsen, die neben mehl auch reichlich eiweiß enthalten. aber auch sonst sind viele – mehlhaltigen nahrungsmittel für die ernährung sehr günstig, vor allem getreidemehle, in denen, wenn die außenhülle der getreide körnerunter der schale, die sogenannte kleberschicht, mit ver mahlen wird, sogar etwas eiweiß steckt. zum getreidemehl ge sellt sich als vorzügliches nahrungsmittel das reismehl, das mais mehl und die kartoffel, letztere ist wegen des wasserreichtums etwas weniger wertvoll als die vorgenannten, aber wie bekannt ein enorm wichtiger nahrungsstoff. von größter bedeutung sind so dann die fette, besonders speck, sowie die pflanzlichen und tierischen öle. sehr nahrhaft ist ferner der zucker. die bisher genannten nahrungsmittelgruppen können als nahrhaft bezeichnet werden. schon kleinere mengen von ihnen, namentlich in mischung, erhalten die kraft des körpers.“ was stellt sich der autor unter dem mehl, das außer eiweiß in den hülsenfrüchten enthalten ist, wohl vor? meint er damit stärke? daß getreidemehl mehlhaltig ist, wird wohl niemand be zweifeln, enthält das wasser ja auch wasser! bekanntlich ver steht der laie und der eingeweihte wissenschaftler unter mehl die in der mühlenindustrie verarbeiteten von der äußeren gewebe verbreitung falscher ansichten über denwert pflanzlicher nahrungsmittel. 75 schicht möglichst befreiten und zu einem feinen pulver zerriebenen getreidefrüchte u. dergl. was will der autor damit sagen, daß das getreidemehl auch eiweiß enthält, wenn die kleberschicht m it vermahlen wird? einmal sitzt ja auch in den endosperm zellen des getreidekorns neben der stärke auch eiweiß (kleber). außerdem ist beim mahlen eine vollkommene scheidung der kleber schicht von dem inneren teil des getreidekernes nicht möglich. durch künstliche entfernung des klebers aus dem getreidemehl wird ja bekanntlich die stärke dargestellt. auch die feinsten mehle haben ja immer eiweiß enthalten, also ohne daß das mehl 8 0 hoch ausgemahlen war, wie e s im kriege gesetz wurde. nach könig”) beträgt der gehalt des feinsten weizenmehles a n stick stoffsubstanz 10,68°/o, der des roggenmehles 9,62%. ferner er scheint e s sehr gewagt, einer ernährung mit „kleineren“ mengen von getreidemehl, kartoffel usw. eine erhaltung der körperkraft nachzusagen. oder hat der verfasser das am eigenen leibe ex perimentell festgestellt? nach rubner würden brot oder mais auch in größeren mengen allein genommen, eine wahre hungerkost sein”). weiter lesen wir: „im gegensatz zu diesen nahrungs reichen gruppen stehen nun andere nahrungsarme pflanzensub stanzen, die nur in enormen mengen genossen, einigermaßen ersatz fü r die anderen bieten können. hierher gehören viele gemüse, die salate und das obst. sie sind wenig nahrhaft, weil sie überaus wasserreich sind, weil ihr hauptinhalt, nämlich die pflanzenzell wand, vom menschlichen verdauungsapparat – im gegensatz zu dem der pflanzenfresser – nicht aufgenommen und unausgenützt ausgeschieden wird. unter den gemüsen sind diejenigen, die mehl enthalten, noch am nahrhaftesten, so besonders die rüben, die karotten, die erdschocken und kohlrüben. ganz nahrungsarm sind z . b . spinat, spargel und die kohlarten. im obste is t der zucker alleinige nahrung.“ was stellt sich der autor vor unter gemüsen, die mehl ent halten? daß die gemüse nicht hochwertige nahrungsmittel sind, is t wohl richtig. nicht verständlich ist, weshalb der spinat mit einem gehalt von 3,7% stickstoffsubstanz, 0,5% fett und 3,5% stickstofffreien extraktstoffen schlechter sein soll als die 1,39% stickstoffsubstanz, 0,18% fett und 7,31% stickstofffreie extrakt *) könig, chemie der menschlichen nahrungsund genußmittel, i. *) wandlungen der volksernährung, 1913. 76 th. sabalitschka, stoffe enthaltende kohlrübe!). keineswegs stimmt die angabe, daß die pflanzenzellwand vom menschlichen verdauungsapparat nicht aufgenommen und unausgenützt ausgeschieden wird, mit dem er gebnis der rubnerschen untersuchungen, die zeigten, daß z. b. die zellmembran des wirsingkohles ausgezeichnet resorbiert wird”). rubner hat festgestellt, daß die zellmembranen des obstes und gemüses bis zu 90°/o verdaut werden. so dürfte daher das obst nicht nur durch seinen zuckergehalt zur ernährung beitragen. von den so geringen eiweißstoffen der kohlrübe bezeichnet rubner”) nur die hälfte als verdaulich. er sagt: „für die deckung der eiweißbedürfnisse haben selbst so große nahrungsaufnahmen wie 1500–2500 g kohlrüben für den tag gar keine bedeutung.“ ich persönlich war im berliner kohl rüben-winter 1916/1917 zu großzügigen ernährungsversuchen am eigenen körper mit kohlrüben gezwungen, wobei sich die kohlrübe gerade nicht besonders geeignet für den menschlichen verdauungs apparat erwies. ich möchte kohlrüben doch in zukunft lieber unseren haustieren überlassen. rubner”) ist weiter der ansicht, daß die gemüse einen besonderen, zweckmäßigen reiz auf den darm ausüben, der wahrscheinlich durch bestimmte reizstoffe be dingt wird. als solche können auch die vitamine gelten. die not wendigkeit der vitamine oder ergänzungsstoffe für die menschliche ernährung is t erst in der jüngsten zeit richtig erkannt worden und e s is t heute auch der vitamingehalt der naturprodukte bei der beurteilung ihres wertes für die menschliche ernährung zu be rücksichtigen. die gemüse gelten im allgemeinen besonders reich a n vitaminen. jürgensen“) schreibt in seinem ausgezeichneten buche: „allgemeine diätetische praxis“ wie folgt: „frisches, grünes gemüse wird im ganzen wie alle in stärkerem wachstum sich be findenden pflanzenteile als vitaminkräftiges nahrungsmittel auf gefaßt, bei skorbut, barlowscher krankheit, pellagra, sprue. in der richtung werden salat, kohl, zwiebel besonders benannt. auch löwenzahn und karotten sind als antiskorbutika genannt.“ daß frisches gemüse bedenkliche krankheitserscheinungen, die bei seinem längeren mangel in der zusammensetzung der kost des *) sabalitschka, berichte d. deutsch. pharmaz. ges., 23, s. 7 (1918). – könig, chemie der menschlichen nahrungsund genußmittel, i, s. 809. *) archiv für anat. und physiol., physiol. abteil, 1916, 221. *) archiv für anat. und physiol., physiol. abteil, 1916, 227. *) jürgensen, allgemeine diätetische praxis, s. 116. verbreitung falscher ansichten über den wert pflanzlicher nahrungsmittel. 77 menschen auftreten, sofort heilen kann, habe ich”) an anderer stelle berichtet. die gemüse enthalten weiter noch pentosen, die nach den feststellungen von könig und reinhard*) vom menschen wohl verdaut werden und die rubner neuerdings auch bei der wert beurteilung der nahrungsmittel berücksichtigt. dann sind die ge müse wegen ihres im allgemeinen reichen gehalts an mineralstoffen am aufbau organischer körpersubstanz, sowie am stoffwechsel be teiligt. auch dem eisengehalt der gemüse als blutbildender sub stanz wird eine wichtige aufgabe bei der ernährung zugeschrieben. gerade der vom verfasser so verachtete spinat wird wegen seines relativ beträchtlichen eisengehaltes als speise für blutarme, kinder und rekonvaleszenten öfters ärztlicherseits verordnet. nach den untersuchungen von moneyrat*) enthält spinat in 100 g trocken substanz 35–45 mg eisen. haensel*) fand einen noch höheren eisengehalt bei winterkohl, kopfsalat und kohlrabiblättern. diese von irrtümern strotzenden angaben in einem von der großen masse viel gelesenen berliner blatt fordern dringend ab hilfe von ähnlichen entgleisungen. es erscheint schon im interesse des bildungsniveaus der bevölkerung wenig erwünscht, daß solche falschen ansichten im volke verbreitet werden. wenn es sich aber, wie hier, um für das ernährungsproblem und den gesund heitszustand des deutschen volkes so wichtige dinge handelt, kann man wohl verlangen, daß die der bevölkerung erteilten ratschläge auch mit dem augenblicklichen stand unseres wissens überein stimmen. es wird eine vornehme aufgabe der angewandten bo tanik sein nicht nur eifrigst weiter zu forschen, sondern auch die ergebnisse ihrer forschungen möglichst bald gemeingut werden zu lassen, wenn "es sich um für die bevölkerung wissenswerte wichtige tatsachen handelt. so wird das volk am besten vor solchen irreführungen geschützt, vor schaden an der gesundheit bewahrt und sein wohl gefördert. *) sabalitschka, über das konservieren und blanchieren der pilzund gemüsekonserven. pharm. zeit., 63, s. 234 (1918). *) buchka, das lebensmittelgewerbe, bd. ii, s. 255. *) compt. rend., 1907, 144, 1067. *) biochemische zeitschrift, 1909, 16, 9. uc1.b3299303_page_092_cut uc1.b3299303_page_093_cut uc1.b3299303_page_094_cut uc1.b3299303_page_095_cut journal of applied botany and food quality 93, 276 279 (2020), doi:10.5073/jabfq.2020.093.034 institut für pflanzenwissenschaften und mikrobiologie, universität hamburg reinhard lieberei − die gabe, vernetzt zu denken petra schwarz (submitted: october 26, 2020; accepted: december 7, 2020) summary ausstellungen universitärer museen verkörpern eine wissenschaftlich-kuratorische transferleistung, die mehr ist als ein „übersetzen“ oder „vereinfachen“ akademischen wissens. reinhard lieberei hat das loki schmidt haus, das museum für nutzpflanzen der universität hamburg von der neukonzeption an begleitet und systemisches denken in den museumsalltag eingeführt. er erkannte in den mög lichen lesarten von ausstellungen ansatzpunkte für wissenstransfer. sein vertrauen darauf, studierende früh in den wissenschaftlichen dialog einzubinden und an die eigenständige entwicklung von kommunikationsformaten heranzuführen, hat die basis und den impuls für neue herangehensweisen in der lehre gegeben. universität und museum universitäten sind bezogen auf ihre kernaufgaben orte, an denen wissen geschaffen wird, wissenschaftler_innen sich beruflich qualifizieren und wissenschaftlicher nachwuchs ausgebildet wird. wissenschaftliche sammlungen spiegeln als sacharchive diese prozesse in vergangenheit und gegenwart wider. sie veranschaulichen das wissen und die vielfältigen ansätze, fragestellungen, herangehensweisen und philosophien ihrer zeit in forschung und lehre. sie können als speicher des universitären gedächtnisses betrachtet werden (habsburg-lothringen, 2015). wenn in deutschland von wissenschaftlichen sammlungen die rede ist, liegt der fokus meist auf historisch gewachsenen, objektbasierten sammlungen, bezogen auf die begriffsbestimmung des wissenschaftsrats (2011). die universität hamburg beherbergt demnach rund 40 wissenschaftliche sammlungen. eine momentaufnahme am biozentrum klein flottbek der universität hamburg im wintersemester 2014/2015 (hammerla, 2015) ergab, dass neben den klassischen vier botanischen sammlungen 23 weitere, bis zu 61 jahre alte und bis zu mehrere tausend objekte umfassende forschungsspezifische sammlungen vorhanden waren. diese nahmen die nutzer_innen in einem spektrum von dnaund proteinsequenzen, viren und bakterien bis hin zu blütenpflanzen eher als objekte und werkzeuge aktueller forschung, denn als wissenschaftliche (teil-)sammlungen wahr. während die arbeit an und mit universitätssammlungen in diesem erweiterten verständnis – das beschaffen, bewahren und beforschen – als akademische tätigkeiten gelten, werden das bekanntmachen und ausstellen nicht zu den genuinen hochschul-aufgaben gezählt bzw. als gleichwertig anerkannt. das verhältnis von universitäten und ihren museen ist vielerorts ambivalent. der hochschulbetrieb belohnt in der regel die forschung an einzelproblemen und die veröffentlichung wissenschaftlicher aufsätze. die öffentlichkeitswirksame darstellung des eigenen faches nach außen hin erfährt im vergleich dazu (noch) geringere wertschätzung. 20 jahre nach publikation des push memorandums (push-memorandum, 1999) deutscher forschungseinrichtungen – public understanding of science and humanities – bevorzugen universitäten die kommunikation über professionalisierte, spezielle abteilungen. ein anreizund reputationssystem für die beteiligung der forschenden in der kommunikation (lugger, 2020) blieb weitgehend unrealisiert. wofür fördermöglichkeiten zur entwicklung von wissenschaftskommunikation und wissenstransfer genutzt werden, hängt stark von lokalen sichtweisen und interpretationen dieser begrifflichkeiten ab. museen im wissenschaftlich-universitären umfeld werden durch ihre verortung vom publikum als fachöffentliche institution verstanden und das kommunikationsangebot universitäre ausstellung als eine „spezifische form der publikation“ wahrgenommen. diese verräumlichte vorstellung wissenschaftlicher inhalte zeigt analogien zum wissenschaftlichen artikel. die ergebnisse systematischer, wissenschaftlicher recherchen werden im kontext von leitthese und botschaft der ausstellung nach transparenten kriterien analysiert, in neue zusammenhänge gestellt und dokumentiert. der prozess bis zur fertigen ausstellung trägt experimentalcharakter und beinhaltet rückkopplungsschleifen mit fachwissenschaftler_ innen. zunehmend wird dieser prozess in der ausstellung sichtbar reflektiert. die dritte dimension erfordert darüber hinaus besondere fähigkeiten, bietet gleichwohl besondere möglichkeiten, inhalte räumlich zu denken, zielgruppenbezogen zu argumentieren und raum für individuelle lesarten, assoziationen und interpretationen der besucher_innen zu lassen bzw. dazu anzuregen (schnalke, 2008). das eröffnet universitätsmuseen die chance, als instrument der kommunikation dazu beizutragen, „... den kulturwert „wissen“ und die kulturtechnik „wissenschaft“ im dialog zwischen wissenschaft und gesellschaft öffentlich neu zu verhandeln“ (balsinger, 2010). dieses „verhandeln“ impliziert, dass „übersetzen“ oder „verein fachen“ akademischen wissens nicht ausreicht. kuratorische praxis erfordert die aktive kritische auseinandersetzung mit evidenzen des faches bzw. der fächer und evidenzen der besucher_innen – evidenzen im sinne von tyradellis (2014) als gewissheiten, über zeugungen, argumentationsfiguren und kausalketten. die „hohe kunst“ der wissenschaftlich-kuratorischen transferleistung liegt in der synthese von wissenschaftlichen inhalten, ausstellungexponaten und jenen evidenzen. metaphorisch gesehen ist jede ausstellung mehr als ein fenster zum hinausund hineinschauen oder eine brücke an der schnittstelle zwischen wissenschaft und öffentlichkeit. sie gleicht im idealfall eher einem balancieren abseits befestigter wege mit der bereitschaft, klüfte im mutigen sprung zu überwinden. im medium wissenschaftliche ausstellung liegen innovationsreserven. die realen objekte universitärer sammlungen können mit ihrer unmittelbarkeit des originalen mit einer neuen, zur beobachtung und zum diskursiven austausch anregenden konstellation auch eine neue bildende und kommunikationsfördernde wirkung entfalten (parmentier, 2001). das format ausstellung bietet im handling mit bedeutungsebenen von objekten vielfältige möglichkeiten, forschung über objekte und anhand von objekten zu thematisieren (vgl. dinge zum sprechen bringen; was bleibt). vom handelsmuseum zum loki schmidt haus hamburg, april 2005: das botanische museum der universität hatte umzugsbedingt fünf jahre zuvor seine ca. 60.000 objekte umfas sende sammlung verpacken und den ausstellungsbetrieb schließen müssen. im gespräch war der bau eines ausstellungsgebäudes auf dem gelände des botanischen gartens hamburg. der neubau soll reinhard lieberei − die gabe, vernetzt zu denken 277 te den namen der forscherin und botschafterin für die natur, loki schmidt, erhalten. reinhard lieberei hatte am biozentrum klein flottbek die professur für nutzpflanzenbiologie inne. gemeinsam mit der neu ernannten museumsleiterin und interessierten kollegen entstanden aus einem intensiven gedankenaustausch erste schwer punkte eines neuen inhaltlichen, didaktischen und gestalterischen konzeptes für das museum. das loki schmidt haus sollte an eine tradition anknüpfen, die auf das engste mit der stadt hamburg verbunden war. seine wurzeln gehen auf die schenkung einer carpologischen sammlung des stadt physikus bueck aus früchten und samen von mehr als 10.000 arten an die stadt hamburg von 1879 zurück. sie wurde in der folgezeit ergänzt und weitere sammlungen wurden eingegliedert mit besonderem augenmerk auf merkantil und technisch wichtige objekte. 1883 entstand daraus das botanische museum zu hamburg mit der inten tion, die wirtschaftlich wichtigsten pflanzen der welt der allgemein heit in einer zweckmäßigen darstellung nahezubringen (voigt, 1897). bereits 1885 wurde die schausammlung eröffnet. pflanzen als primärprodukte brachten einen beachtlichen teil des hamburger wohlstandes. durch die zusammenstellung von handelsobjekten aus dem pflanzenreich besaß das museum von anfang an auch die werte eines typischen hamburger handelsmuseums. in den sammlungen wurde „auf die erzeugnisse der deutschen kolonien eine besondere rücksicht genommen“ (sadebeck, 1899). das museum wurde in den folgejahren durch laboratorien für warenkunde und saatgutprüfung erweitert. als ende des 19. jahrhunderts der umfang der importierten handelsprodukte im hamburger hafen und damit der bedarf an qualitätsuntersuchungen stark zunahm, kamen eine abteilung für pflanzenschutz und ein chemisches laboratorium hinzu. die ständige vertretung der labore an der hamburger börse war ausdruck der engen verbindung mit dem handel. fragen aus handelskreisen konnten so direkt vor ort beantwortet werden. 1901 wurden botanisches museum und botanischer garten zu den botanischen staatsinstituten vereint. daraus gingen 1912 die staatsinstitute für angewandte botanik mit botanischem museum und allgemeine botanik mit botanischem garten hervor. 1919 erfolgte deren eingliederung in die neu gegründete universität hamburg. die trennung der institute blieb bis 2003 bestehen. vernetztes denken mit dem neubau des loki schmidt hauses 2006 sollte das museum der stadt und der bevölkerung wieder zugänglich gemacht werden. reinhard lieberei brachte nicht nur die expertise für nutzpflanzen mit. er war es, der die kunst vernetzten denkens (vester, 2000) in den museumsalltag einführte. das planungsteam machte sich auf den weg, das museum für einen neuen ort, mit neuem namen und einem neuen konzeptionellen ansatz zu den vom menschen genutzten pflanzen weit über den charakter der früheren schausammlung hinausgehend zu entwickeln. aus der präsentation nur zum betrachten sollte in einer verknüpfung von tradition und moderne ein begreifbares, erlebbares museum entstehen. ziel war es von anfang an, das verständnis und die handlungskompetenz für eine nachhaltige nutzung natürlicher ressourcen aus dem pflanzenreich in einer breiten öffentlichkeit zu fördern. was zeichnet seine systemische herangehensweise aus? reinhard lieberei besaß nicht nur ein außerordentlich breites pflanzenbiologisches wissen in pflanzenphysiologie, phytopathologie, biochemie und ökologie. seine forschungen an tropischen nutzpflanzen und nachhaltigen agrikultursystemen waren durch multiperspektivische, fachübergreifende ansätze charakterisiert. er sah den menschen als betrachter und gleichzeitig als beteiligten, eingebunden und selbst in ständiger wechselwirkung und veränderung mit anderen teilen größerer systeme. dieses denken und handeln bestimmte auch seine vorstellungen von museumsarbeit. er war ein meister im erkennen und analysieren von grundmustern und beziehungsgeflechten. er schaute über den tellerrand hinaus. er konnte perspektiven wechseln, sich hineindenken und hineinfühlen. für ihn war es selbstverständlich, das eigene wie menschliches handeln an sich zu reflektieren. die konzeptionelle und praktische arbeit im museum profitierte davon. dinge zum sprechen bringen vernetztes denken bedeutet ganzheitliche betrachtung musealer prozesse einschließlich der rückkopplungen und nebenwirkungen. wenn ein ding in die museumssammlung kommt, erfährt es eine transformation. es verliert seinen ursprünglichen gebrauchswert, erhält einen zeugniswert und wird nach pomian (1988) zum semiophor, zum zeichenträger. zunächst dekontextualisiert wird es später als objekt in sammlung und ausstellung wieder rekontex tualisiert. wissenschaftler_innen und kurator_innen kategorisieren, selektieren, präsentieren, arrangieren, inszenieren, interpretieren. jedes objekt trägt zunächst eine fülle von bedeutungen aus objektbiografie und überlieferungskontext in sich. weitere können ihm zugeschrieben werden. abhängig von deutungsabsicht und funktionalisierung in der ausstellung werden bedeutungsdimensi onen betont oder vernachlässigt. das objekt kann zum beispiel als relikt, beleg, sachzeugnis, symbol, symptom für etwas stehen. es kann einen schatz, eine trophäe, einen katalysator für erinnerungen darstellen (muttenthaler, 2016). es kann als imageobjekt im mittelpunkt stehen oder teil eines ensembles sein. bedeutungen werden gelenkt und verstehbar durch die (an-)ordnung der ob jekte im raum, durch ihre inszenierung und kontextualisierung (thiemeyer, 2014). die ausstellenden müssen sich bewusst machen, welcher intention und welchem duktus der anordnung und ge staltung die ausstellung grundsätzlich folgen soll. den absichten der ausstellenden stehen immer auch bewusste oder unbewusste wahrnehmungen, (be-)deutungsvermutungen und decodierungen des publikums gegenüber. das ganze ist mehr als die summe seiner teile (thiemeyer, 2015). reinhard lieberei war sich der verantwortung im verlauf konstru ierter und nicht intendierter wandlungen sehr wohl bewusst. er wusste nicht nur mit deutungen und bedeutungen umzugehen. er erkannte in den möglichen lesarten ansatzpunkte für wissenstrans fer. er hatte die vision, im loki schmidt haus mit sammlungsobjek ten und ausstellungsexponaten mehr als nur botanik zu vermitteln oder wie menschen pflanzen nutzen. er wollte mit der information zur pflanze die verbindung zur pflanze herstellen und verdeutlichen, in welcher dimension etwas passiert, bis in den botanischen garten oder mikroskopische bereiche hinein. daraus entstand die idee eines „zooms“ in den drei ausstellungsetagen des loki schmidt hauses – vom großen ins kleine, vom weitwinkel der einführung in die dauerausstellung zur vertiefenden nahaufnahme in sonderausstellungen bzw. den botanischen garten hinein. sein bild vom universitären museum war das vom ort eines lebendigen austausches. die systemischen und kuratorischen betrachtungen fanden ihre praktische umsetzung in der ersten großen sonderausstellung 2008 „kakao – der schatz der tropen“. in zehn kapiteln vom kakaobaum bis zu den geheimnissen der kakaoverarbeitung, von der kulturellen reise des kakao bis zum welthandelsgut wurden biologische, ökologische, ökonomische und soziale, historische, aktuelle und voraus denkende „brillen“ thematisiert und untereinander in beziehung gesetzt. exponate aus wissenschaft und praxis, aus kunst und kultur spannen weitere fäden im netz. die integrierte kinderebene ermöglichte generationenübergreifende erlebnisse und erfahrungen. ein vielseitiges begleitprogramm mit vorträgen und verkostungen, führungen, gesprächen und werkstätten bereicherte die aus stellung. reinhard lieberei war in verschiedenen rollen zu hause: 278 petra schwarz wissenschaftlicher ratgeber, unterstützender netzwerker, kreativer impulsgeber, beeindruckender erklärer und brillanter erzähler. diese ausstellung wurde zum „gesellenstück“ des loki schmidt hauses und setzte maßstäbe für alle späteren aktivitäten. die planung und umsetzung der dauerausstellung ein jahr später profitierte von diesen gemeinsamen erfahrungen. ein weiterer ausdruck systemischer herangehensweise: dynamik ist inbegriffen. es gehört dazu kontinuierlich herauszufinden, was verstetigt werden kann bzw. einer veränderung bedarf. reinhard lieberei bot sich als brücke zwischen wissenschaft und kommunikation, zwischen forschungsabteilungen und museum an, einschließlich der refle xionsrunden mit ihm – ein glücksfall für das junge loki schmidt haus. spätere sonderausstellungen setzten diese entwicklung fort. für die ausstellung „bilderwelten der botanik – unsichtbares sichtbar machen“ 2012 wurde der radius der zusammenarbeit auf alle forschungsabteilungen des botanischen institutes erweitert und bei „sammeln – von leidenschaft, die wissen schafft“ 2015 auch öffentlichkeit beteiligt (schwarz, 2015). was bleibt die zusammenarbeit am museumskonzept und der sonderausstellung zum kakao hat zwischen ihm und dem museumsteam eine ver bundenheit wachsen lassen, die ungebrochen bis zu seinem letzten atemzug währte. er hat viel bewegt. ungeachtet seiner terminfülle ging er mit offenen augen und armen durch die welt. er konnte überzeugend argumentieren und dinge auf den punkt bringen. er blieb er selbst, authentisch und ohne schnörkel. er hatte ein gespür für die töne zwischen den zeilen. er inspirierte, motivierte und moderierte. zwei entwicklungen seinen genannt, die auf dem urvertrauen fußen, das er ausstrahlte. erstens: studierende bereits in der lehre mit wissenschaftlichen kommunikationsprozessen in berührung zu bringen, erschöpft sich nicht darin, ihnen eine beobachteroder rezipientenrolle zuzuschreiben. über lehrformate können studierende in den wissenschaftlichen dialog eingebunden und an die eigenständige entwicklung von kommunikationsformaten herangeführt werden. seit 2016 schlüpfen studierende im 5. semester des studienganges „bachelor of science biologie“ jedes wintersemester in die rolle von ausstellungsmachenden auf zeit und bringen pflanzenforschung ins museum. arbeitsgruppen des institutes für pflanzenwissenschaften und mikrobiologie ermöglichen ihnen einblick in aktuelle pro jekte. vor den studierenden steht die aufgabe, sich in den forschungskontext hineinzudenken und einen selbst gewählten themenaspekt öffentlichkeitsorientiert aufzuarbeiten. ziel des dreiwöchigen kurses mit der autorin (museumsleiterin) ist ein fertiges ausstellungskonzept für das „fenster in die wissenschaft“ im loki schmidt haus. die studierenden definieren zielgruppe, leitthese und kernbotschaft und entscheiden eigenverantwortlich über die art der gestaltung. die ergebnisse werden vom museumsteam umgesetzt. „was pflanzen im inneren bewegt“ nahm die welt der pflanzlichen motorproteine in ihrer funktion als innerzelluläre transporthelfer in analogie zu verkehrsmitteln in der stadt unter die lupe. „wissenschaft im all-tee-glichen“ beleuchtete prozessdetails pflanzenökologischer forschung zum einfluss des klimawandels auf die salzmarschen der nordsee. im fokus standen teebeutel als standardisierte „litter bags“, mit denen die dynamik des in den marschböden gespeicherten kohlenstoffes untersucht wird. „vom ursprung zur zukunft – wie mikroalgen das land erobern“ thematisierte vertreter der organismen, die vor ca. 500 mil lionen jahren als pioniere das land besiedelten und deren heutige einsatzbereiche im alltag. das „fenster in die wissenschaft“ symbolisiert jährlich neu eine momentaufnahme universitärer gegenwart. die jungen ausstellungsmachenden stellen ihr format der wissenschaftskommunikation anlässlich eines aktionstages für familien gemeinsam mit ihren forscherpaten vor (schwarz, 2020). zweitens: vernetztes denken bildet eine unabdingbare voraus setzung für eine erfolgreiche bildung für nachhaltige entwicklung und entsprechende vermittlungsformate. studierende im 2. semes ter des studienganges „master of education“ setzen sich mit möglichkeiten und grenzen musealer lernorte und der bedeutung von sammlungsobjekten für wissenschaft und bildung ausein ander. sie erproben, objekte zum sprechen zu bringen und über deren rolle von requisiten hinaus im inhaltlichen kontext wis sensbildungsprozesse zu fördern. sie experimentieren, mit mög lichkeiten offener herangehensweise raum für kommunikation, selbständige tätigkeit und lernen mit allen sinnen zu geben und über die erweiterung des eigenen denk-, erlebnis-, erfahrungsund kreativitätshorizontes gestaltungskompetenz für eine nachhaltige entwicklung zu fördern. sie reflektieren ihren lernprozess in einem portfolio (schwarz, 2019). reinhard lieberei strahlte eine gesunde mischung aus idealismus und realismus aus. für ihn war selbstverständlich, im universitätsmuseum nicht nur forschungsergebnisse, sondern auch den prozess der forschung an sich zu thematisieren. das schloss eine kritische sicht auf wissenschaftliche arbeit selbst und deren ergebnisse ein. solch ein museum sah er jenseits einer auf motive strategischen eigeninteresses reduzierten wissenskommunikation als beitrag zum dialog, auch um die vorläufigkeit und fragilität erzeugten wissens. den traum vom eigenen nutzpflanzenmuseum in seiner wahlheimat wendland konnte er sich nach der pensionierung nur noch für kurze zeit erfüllen. dem evolutioneum, dem neuen naturkundemuseum für hamburg, würde er wahrscheinlich mit auf den weg geben, eine transdisziplinäre ausrichtung zu verfolgen mit schnittstellen zu verschiedenen bereichen des gesellschaftlichen lebens und einen ausstellungsbereich zu verwirklichen, der als „third place“ (steelcase, 12.10.2020) ein gemeinschaftlich genutzter öffentlicher ort, ein raum der begegnung und des austausches, des denkens, der auseinandersetzung und der reflexion werden kann. nutzpflanzen würden gut hineinpassen. conflict of interest statement the author declares no conflict of interest. references balsinger, p., 2010: das museum in der universität – überlegungen zu einer form künftiger wissenschaftskommunikation. in: weber, c., mauersberger, k. (eds.), universitätsmuseen und sammlungen im hoch schulalltag, 105-117. hermann von helmholtz-zentrum für kultur technik, humboldt-universität zu berlin, berlin. lugger, b., 2020: verständlichkeit ist nur der anfang. in: schnurr, j., mäder, a. (eds.), wissenschaft und gesellschaft. ein vertrauensvoller dialog, 139-150. springer-verlag gmbh, berlin. doi: 10.1007/978-3-662-59466-7 muttenthaler, r., 2016: beredsam und wirkungsvoll. dimensionen der dinge aus museologischer perspektive. in: griesser, m., haupt-stummer, c., höllwart, r., jaschke, b., sommer, m., sternfeld, n., ziaja, l. (eds.), gegen den stand der dinge. objekte in museen und ausstellungen, 3547. edition angewandte, walter de gruyter gmbh, berlin, boston. habsburg-lothringen, b., 2015: andere ausgangslage, vergleichbare fragen. universitäre sammlungen aus museologischer perspektive. in: neues museum. die österreichische museumszeitschrift, 15(1-2), 8-11. hammerla, s., 2015: die forschungsspezifischen sammlungen am bio zentrum klein flottbek. in: schwarz, p. (ed.), sammeln. von leidenschaft, die wissen schafft. ausstellungskatalog, 44-62. universität hamburg, hamburg. parmentier, m., 2001: der bildungswert der dinge oder: die chancen des museums. zeitschrift für erziehungswissenschaft 4(1), 39-50. reinhard lieberei − die gabe, vernetzt zu denken 279 pomian, k., 1988: der ursprung des museums. vom sammeln. verlag klaus wagenbach, berlin, 73-90. push-memorandum, 1999: dialog wissenschaft und gesellschaft. re trieved 12th october 2020, from https://www.wissenschaft-im-dialog. de/fileadmin/user_upload/ueber_uns/wid_dokumente/push_memo randum_1999.pdf sadebeck, r., 1899: die kulturgewächse der deutschen kolonien und ihre erzeugnisse. iii, verlag von gustav fischer, jena. schnalke, t., 2008: auf leben und tod. ausstellen im berliner medizin historischen museum. in: hermannstädter, a., sonnabend, m., weber, c. (eds.), wissenschaft kommunizieren. die rolle der universitäten, 58-61. edition, stifterverband, essen. schwarz, p., 2015: sammeln. von leidenschaft, die wissen schafft. aus stellungskatalog, universität hamburg, hamburg. schwarz, p., 2019: wissen im quadrat – das universitätsmuseum als fenster in die wissenschaft. natur im museum 9, 69-72. schwarz, p., 2020: fenster in die wissenschaft, vom ursprung zur zukunft – wie mikroalgen das land erobern. biospektrum 26(1), 122. steelcase: interview mit ray oldenburg. retrieved 12th october 2020, from https://www.steelcase.com/eu-de/forschung/artikel/interview-mitray-oldenburg/ thiemeyer, t., 2014: die sprache der dinge. in: stieglitz von, l., brune, t. (ed.), hin und her – dialoge in museen zur alltagskultur, 41-54. edition museum, transcript verlag, bielefeld. thiemeyer, t., 2015: inszenierung. in: museen verstehen. begriffe der theorie und praxis, 45-62. wallstein verlag, göttingen. tyradellis, d., 2014: müde museen, oder: wie ausstellungen unser denken verändern können, 149-159. edition körber stiftung, hamburg. vester, f., 2002: die kunst vernetzt zu denken, 16-30. deutscher taschenbuch verlag, münchen. voigt, a., 1897: die botanischen institute der freien und hansestadt hamburg. verlag von leopold voss, hamburg, leipzig, 64. wissenschaftsrat, 2011: empfehlungen zu wissenschaftlichen samm lungen als forschungsinfrastrukturen 16, drs. 10464-11. retrieved 04th december 2020, from https://wissenschaftliche-sammlungen.de/ files/9213/7474/4488/10464-11-1.pdf address of the author: petra schwarz, institut für pflanzenwissenschaften und mikrobiologie, universität hamburg, ohnhorststraße 18, 22609 hamburg, germany e-mail: petra.schwarz@uni-hamburg.de © the author(s) 2020. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). journal of applied botany and food quality 92, 88 93 (2019), doi:10.5073/jabfq.2019.092.012 1department of field crops, faculty of agriculture, eskişehir osmangazi university, eskişehir, turkey 2department of field crops, faculty of agriculture, namık kemal university, tekirdağ, turkey autotetraploid plant production in endemic onobrychis elata with colchicine treatments süleyman avci1*, metin tuna2, mehmet demir kaya1 (submitted: december 10, 2018; accepted: february 22, 2019) * corresponding author summary this study aimed to induce autotetraploidy in endemic onobrychis elata plants by colchicine treatment of seeds or seedlings. colchicine was applied to o. elata directly on germinated seeds, pre-germinated seeds (root length of 3-8 mm), and apical regions (using cotton) under in vivo conditions. out of a total of 1,210 colchicine-treated seeds that were evaluated, only 203 survived. there was an inverse re lationship between the number of surviving plants and colchicine concentration and exposure time. the highest percentage of tetraploidy in surviving plants (50%) was obtained by applying 0.2% colchicine for 6 hours to pre-germinated seeds. no significant tetra ploidy was achieved by colchicine application to seedlings. flow cytometry observations indicated that dna content varied between 0.99 and 1.06 pg in diploid plants (controls), while dna content varied between 2.22 and 2.48 pg in tetraploid plants. it was con cluded that tetraploid plants were induced successfully only in seedlings obtained from pre-germinated seeds, with their ploidy level confirmed via flow cytometry analysis. key words: onobrychis, colchicine, flow cytometry, polyploidy introduction sainfoin (onobrychis viciifolia scop.) is an important forage legume for hay and pasture in calcareous and arid lands because of its well-developed root system and adaptability to water limitations (jefferson et al., 1994; borreani et al., 2003). the nutritive values of hay are similar to alfalfa, and because hay contains appreciable amounts of condensed tannins, animals grazed in pure pasture are not at risk of bloat (wang et al., 2015; bhattarai et al., 2016). there are approximately 170 species of the genus onobrychis around the world, located mainly in southwestern asia, the mediterra nean, and temperate regions of europe and asia (cronquist, 1981; zohary, 1987; aktoklu, 2001). wild onobrychis species constitute important genetic resources for breeding sainfoin and improving grassland (avci et al., 2013; özaslan parlak and parlak, 2008). in turkey, there are 55 onobrychis species with two subgenera (onobrychis and sisyrosema) and five sections (dendobrychis, laphobrychis, onobrychis, hymenobrychis, and heliobrychis); 28 of them are endemic (aktoklu, 2001; avci et al., 2013). onobrychis elata boiss. et bal. is a valuable native species that is widely scattered due to its morphological similarity to cultivated o. viciifolia (avci et al., 2013). o. elata is diploid (2n = 14) and has a basic chromosome number of x = 7 (cartier, 1976). it has been considered an important potential wild genetic source for hybridization with o. viciifolia to help further forage production under biotic and abiotic stresses; therefore, it would be advantageous to double its chromosome number. autopolyploids originate from the combination of unreduced ga metes in nature and can also sometimes be artificially stimulated (chen, 2010). chromosome doubling is used to more efficiently breed superior varieties with high yields of forage crops (acquaah, 2007; meru, 2012). moreover, polyploidy plays an important role in the fertility of interspecific hybridization between cultivated species and their wild relatives (falcinelli, 1999; aversano et al., 2012). colchicine treatment is the most effective and widely used method of producing polyploidy in forage legumes. anderson et al. (1991) obtained polyploidy in trifolium plants and their hybrids by treating seeds, seedlings, and growing shoot apices with colchicine. similar results were achieved by dibyendu (2010) in lathyrus sativus l., zeinab et al. (2012) in trifolium alexandrinum l., and wu et al. (2015) in stylosanthes guianensis (aublet) sw. however, research on polyploidy in onobrychis species has not been found in the literature. this study aimed to produce autotetraploid plants from diploid o. elata by colchicine treatment under in vivo conditions. materials and methods o. elata is a perennial with erect stems measuring approximately 60-120 cm (fig. 1a). leaves include 5-7 pairs of narrow elliptical leaflets (fig. 1b). the raceme is many flowered with a loose structure (fig. 1c), and the corolla of the flower is rose with deeper striations (fig. 1d). the fruit is suborbicular and includes long and short spiny teeth on the crest and disk, respectively (fig. 1e). the seed is roughly kidney-shaped (reniform), and seed color varies from light brown to dark brown (fig. 1f). seeds of o. elata were collected from mount erciyes (1,443 m), kayseri, turkey, and stored at 4 °c until used. the seed coats were very hard and impermeable; consequently, they were abraded with sandpaper as described by avci and kaya (2013). the seeds were then surface sterilized in 96% alcohol for 2 minutes and rinsed twice with distilled water. germination was performed in petri dishes (100 mm × 20 mm) on top of two filter papers moistened with distilled water. each petri dish was then sealed with laboratory film, and the seeds were allowed to germinate in the dark at 20 ± 1 °c. colchicine (sigma catalog no.: c9754) was dissolved in water at room temperature, and the aqueous solution was stored at 4 °c. the colchicine was directly applied to germinated seeds at two concentrations (0.05% and 0.1%) and three exposure times (24, 48, and 72 hours). pre-germinated seeds with 3-8 mm root lengths were treated with four concentrations (0.1%, 0.2%, 0.3%, and 0.4%) and three exposure times (6, 12, and 24 hours), followed by rinsing. in addition, three concentrations of colchicine (0.05%, 0.1%, and 0.2%) at two exposure times (48 and 72 hours) were applied with cotton to the apical regions of 10-day-old seedlings. the surviving seedlings were transferred into pots containing a mixture of peat and perlite (3:1 v/v) and incubated for a 16-hour photoperiod day of 8,00010,000 lux, at 20 ± 1 °c and 60% humidity. ploidy analysis was performed using flow cytometry, which is the newest, fastest, most accurate, and most economical method for this purpose. first, rice, tomato, common vetch, barley, and safflower plants were analyzed separately with diploid o. elata, and common vetch (vicia sativa l.) and safflower (carthamus tinctorius l.) were selected as the most suitable internal standards. partec kits were used to isolate nuclear dna from fresh leaf samples derived from both in vivo induction of tetraploid plants in onobrychis elata 89 fig. 1: morphological characteristics of o. elata. (a) general view with erect stem; (b) leaf; (c) raceme; (d) corolla with calyx; (e) fruits; (f) seeds. 90 s. avci, m. tuna, m.d. kaya diploid and colchicine-treated two-month-old plantlets, and dna content was calculated for each sample using the standard plants. the resulting flow histogram was analyzed with the flomax packet program, and fluorescence intensity, mean, and cv values were determined for each sample. the nuclear dna content of each o. elata sample was calculated according to the following formula: dna content = (fluorescence intensity of sample) / (fluorescence intensity of standard) × dna content of standard. results and discussion out of a total of 480 germinated seeds treated with colchicine, 113 seedlings (23.5%) survived, and chromosome doubling was not observed (data not shown). in similar studies of legume species, direct treatment of germinated seeds with colchicine did not successfully induce polyploidy. patil (1992) reported that germinated seeds of crotalaria linifolia linn. treated with colchicine at concentrations of 0.10%, 0.15%, 0.20%, 0.25%, and 0.30% for 6, 12, and 24 hours did not produce polyploid plants. arya et al. (1988) found that colchicine treatment at a concentration of 0.15% for 10 hours was a lethal dose in germinated seeds of fenugreek, and gandhi and patil (1997) stated that direct treatment of germinated seeds of clitoria ternatea l. was not a practically efficient means of inducing tetraploid tissue. colchicine treatment of pre-germinated seeds resulted in 75 survi ving plantlets (12.5%) out of a total of 596 seedlings. of those 75, 15 plants (20%) were determined to be tetraploid by flow cytometry (tab. 1). as the concentration and exposure time increased, the number of surviving plants decreased, and plants did not survive when treated at concentrations of 0.2%, 0.3%, and 0.4% for 24 hours. tetraploid plants were observed for all exposure times at 0.1% concentration, as well as for 6 hours of exposure time at 0.2% concentration, which was determined to produce the highest chromosome doubling percentage (50%) in surviving plantlets. similar results were obtained from astragalus membranaceus, with the mortality rate higher than 70% at a concentration of 0.3% colchicine over a 24hour exposure time; the highest chromosome doubling (50%) among surviving plants was obtained from treatment with 0.2% concentration for 6 hours (chen and gao, 2007). tulay and unal (2010) and joshi and verma (2004) reported that when seeds of vicia villosa roth and vicia faba l. were first soaked in water for 20 hours, tetra ploid plants were attained with colchicine treatment at lower doses (0.005% concentration for 8 hours). pathak et al. (2015) supported the finding that soaking seeds in water prior to colchicine treatment was quite efficient at promoting polyploidy. when colchicine was applied to the apical regions of 134 seedlings, only 15 (11%) of them survived, and no polyploidic plants were observed (data not shown). increasing concentration and exposure time negatively affected seedling survival. treatment with a concentration of 0.2% for 48 and 72 hours led to death of the plantlets. dabkeviciene et al. (2016) determined that colchicine treatment to trifolium pratense l. embryos resulted in 3.3 times more polyploid plants than overhead application to cotyledon leaves of sixto eightday-old seedlings. wu et al. (2015) obtained 10% tetraploid induction by applying colchicine to the apical region of s. guianensis at the highest concentration (0.2%) for 48 hours. bewal et al. (2009) stated that the highest polyploidy rate obtained by colchicine treatment to the apical region of cyamopsis tetragonoloba l. was observed at 0.2% concentration and 10 hours of exposure time for two days. in contrast to the current findings, previous studies have demon strated that polyploid plants were obtained from applications to the apical regions of different legume species, and a reduced survival rate was observed at colchicine concentrations over 0.2%. in the future, new approaches to exposure frequency and times and application type on the apical region should be evaluated. colchicine was applied to germinated seeds, pre-germinated seeds, and the apical region of seedlings, and out of a total of 1,210 seedlings, 203 survived. of the surviving plantlets, 15 of them, inclu ding those originating from pre-germinated seeds, were determined to be tetraploid by using flow cytometry (tab. 2). when safflower (c. tinctorius) was used as the internal standard, nuclear dna peaks of tetraploid (2n = 28) o. elata overlapped (fig. 2), but no overlap was observed in nuclear dna peaks of diploid (2n = 14) plants (fig. 3). after analysis of all samples with safflower as the internal standard, those with overlapping nuclear dna peaks and thus considered to be tetraploid were again analyzed with the nuclear dna of common vetch (v. sativa). as a result of these analyses, tetraploid plants were successfully identified as those without overlap (fig. 4). the content of nuclear dna in diploid and tetraploid plants ranged from 0.99 to 1.06 pg and 2.22 to 2.48 pg, respectively (tab. 2). in conclusion, tetraploid o. elata plants were successfully obtained at a high percentage by treating pre-germinated seeds of diploid plants with colchicine. increased concentrations of colchicine adversely affected the survival rate of the plants. for unsuccessful treatments of the apical region and germinated seeds, modifications can be made to increase the success of chromosome doubling. thirteen tetraploid tab. 1: results of colchicine application to germinated seeds (3-8 mm root length) colchicine application time number of planted number of plantlet survival rate number of tetraploid plant rate concentration(%) (hours) seedlings surviving plantlets (%) tetraploid plants (%) in surviving plantlets 0.1 6 30 14 47 3 21 12 50 4 8 1 25 24 60 10 17 2 20 0.2 6 50 18 36 9 50 12 50 1 2 0 0 24 41 0 0 0 0 0.3 6 45 12 27 0 0 12 50 1 2 0 0 24 70 0 0 0 0 0.4 6 30 14 47 0 0 12 50 1 2 0 0 24 70 0 0 0 0 total — 596 75 — 15 — in vivo induction of tetraploid plants in onobrychis elata 91 tab. 2: fluorescence intensity, average dna content, and cv values in samples with and without colchicine treatment plant sample fluorescence standard fluorescence sample standard cv1 cv2 ploidy number intensity intensity dna content (pg) dna content (pg) (o. elata) (standard) level 14 (control) 119.24 238.72 1.00 2.00* 4.75 3.00 diploid 24 (control) 115.42 233.24 0.99 2.00 6.15 3.68 diploid 32 (control) 128.94 244.13 1.06 2.00 3.95 4.99 diploid 69 (control) 133.78 266.69 1.00 2.00 5.24 5.99 diploid 57 129.28 197.37 2.39 3.65** 5.32 3.43 tetraploid 59 134.12 199.27 2.46 3.65 5.52 2.75 tetraploid 64 119.63 186.20 2.35 3.65 3.97 2.54 tetraploid 71 93.97 150.29 2.28 3.65 6.33 4.13 tetraploid 77 125.74 187.67 2.45 3.65 5.98 3.02 tetraploid 79 149.51 234.60 2.33 3.65 3.28 3.88 tetraploid 82 123.29 196.15 2.29 3.65 6.39 4.09 tetraploid 85 122.39 201.41 2.22 3.65 7.81 4.25 tetraploid 89 139.64 211.13 2.41 3.65 3.63 2.47 tetraploid 90 139.56 205.61 2.48 3.65 3.78 2.56 tetraploid 91 118.97 182.09 2.38 3.65 3.82 2.89 tetraploid 92 129.82 202.40 2.34 3.65 3.63 2.82 tetraploid 94 141.21 218.06 2.36 3.65 3.27 2.74 tetraploid 96 129.02 200.04 2.35 3.65 4.48 4.36 tetraploid 97 143.96 218.05 2.41 3.65 3.92 3.16 tetraploid * safflower (c. tinctorius) was used as the standard dna content. ** common vetch (v. sativa) was used as the standard dna content. fig. 2: overlap of g1 peaks in tetraploid o. elata and internal standard (safflower) plants were transferred to field conditions for further morpholo gical, cytological, and palynological observation, and to hybridization facilities in the sainfoin breeding program. acknowledgments the author thanks eskişehir osmangazi university for its financial support (project number: 2016-1030). references acquaah, g., 2007: principles of plant genetics and breeding. uk, oxford: blackwell publishing. aktoklu, e., 2001: two new varieties and a new record in onobrychis from turkey. turk. j. bot. 25, 359-363. anderson, j.a., mousset-déclas, c., williams, e.g., taylor, n.l., 1991: an in vitro chromosome doubling method for clovers (trifolium spp.). genome, 34(1), 1-5. doi: 10.1139/g91-001 fl1 uv led co un ts 92 s. avci, m. tuna, m.d. kaya fig. 4: positions of g1 peaks belonging to tetraploid o. elata and standard plants (common vetch). fig. 3: positions of g1 peaks belonging to diploid o. elata and standard plants (safflower) arya, 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(ed.), flora palaestina, 158164, vol. 2. jerusalem: academic science human. orcid süleyman avci https://orcid.org/0000-0002-4653-5567 metin tuna https://orcid.org/0000-0003-4841-8871 mehmet demir kaya https://orcid.org/0000-0002-4681-2464 address of corresponding author: süleyman avcı: department of field crops, faculty of agriculture, uni versity of eskişehir osmangazi, odunpazarı, eskişehir, turkey, 26160. e-mail: savci@ogu.edu.tr © the author(s) 2019. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). angewandte botanik. sachregister aaskäfer-imagines 126 abbau pflanzlicher zellmembranen 78 achillea millefolium 50 ackerbau in argentinien 224 ackererbse 195 ackersenf 268 ackerunkräuter (verbreitung und be kämpfung) 213 agriculturchemiker 13 agrotis segetum 221 agrumenfrüchte (öl) 264 akarinose des weinstocks 125 algen, meeres(pferdefutter) 53 alkoholerzeugung aus holz 49 alkoholgärung und chloride 115 analyse, chemische (zwetschen) 24 – – (johannisbeersäfte) 22 – – (himbeersäfte) 17, 20 –– (rangoonbohne) 29 – (typha) 37 – – (nährstoffgehalt der typha grundachsen) 100 – der zellwandbestandteile 79 – pharmakognostische eines verfälsch ten und mit brechweinstein ver mischten enzianpulvers 56 – von pflaumenkernen 262 anaptychia ciliaris var. glaberrima 196 anatomie (typha angustifolia) 40 anbau medizinischer pflanzen in schweden 115 anfärbbarkeit (fasern) 264 antherenbrand 217 äpfel (äppleträdens skorosjuka) 268 apfelbaumkrebs 219 apparate zur anwendung von pflanzen schutzmitteln 8 appel, o. 271 arachis hypogaea 202 areka-alkaloide 118 argentinien (auslandswegweiser) 223 arion-arten 88 arnica 50 arsenbrühe(ersatz für nikotinbrühe) 122 arthrolobium scorpioides 153, 155 artischocken 260 arundo phragmites 271 arzneipflanzenkultur 117– auf almböden 57 – rationelle 58 arzneipflanzen, deutsche 58 arzneiwarenerzeugung in deutsch österreich 117 aspergillus 81, 86 astacus fluviatilis 88 ausbildung des landwirtes 223 ausmahlungsgrad der mehle 114 azaleen-schädling (gracilaria zachrysa) 219 azofarbstoffe 265 bacillus asterosporus 87 – comesii 87 – meseutericus 113 bacterium solanacearm 270 baldrian 266 balata 264 bambus 267 banane 208 bastfasern des flachsstengels 118 bastard von typha angustifolia un d typha latifolia 3 5 baumarten javas (mikrographie de s holzes) 205 baumwolle in deutschland 120 baumwollen-kultur 204 baumwolle, versorgung mit 204 behrens, j. 271 beizapparate, transportable 8 beizbehandlung des saatguts 220 beizung deswinterweizens gegen stein brand 215 beizverfahren gegen brandkrankheit 27 0 sachregister. 273 belladonna 116 beobachtung und bekämpfung der pflanzenkrankheiten 5, 6 bergahorn 132 bergbau in argentinien 224 besenginster (als faserpflanze) 61 bespritzungsversuche an kartoffeln 220 bienenzucht 206 bier 259 bilsenkrautsamen im mohn 115 bingelkraut und hauhechel sind ge nießbar 195 biosbegriff 221 blattlaus 206 blattrollkrankheit der kartoffel 63, 125, 215, 216, 217 blausäuregas 267 blistercanker of apple-trees 219 blumenbinse als faserpflanze 121 blumenzucht 266 bodenkolloide 64 bodenverbesserungsmittel 267 bohne 28, 215 adam, paigya, portal, kidney, fève de kratok, haricot de siève, pois d'achery, amer, kratokbohne, java »bohne, limabohne, duffinbohne, burmabohne, du cap 27 – busch-, anbau 266 borassus flabelliformis 85 bordeauxbrühe 127 botrytis 86, 93, 206 botrytis cinerea auf raps 268 brennessel 66, 204, 260 brennesselfaser (zellonieren, lüstrieren) 203 brennesselschädlinge 269 brennfleckenkrankheit der bohnen 215 brombeerkrankheit 103 brombeerkrebs 105 brotbacken mit zusatz von flechten in ägypten 196 brot, fadenziehendes 112 brot (i n krieg und frieden) 5 2 buche 132 buchenschwamm 6 0 bucheckeröl 118 buchweizen 195 buckeye-rot o f tomatofruit 269 bulbus scillae (chem.untersuchung) 115 butomus umbellatus 121 calandra granaria (bekämpfung) 6 3 capita papaveris 5 6 capsella bursa pastoris (altes und neues) 200 carpocapsa pomonella 125 carex brizoides 120 carthamus tinctorius 59 cassia auriculata 56 catechu, über substitution von 117 catha edulis 55 centaurea solstitialis 154, 155 cephalaria transsilvanica 155 ceratonia 85 champignonkultur 5 4 chenopodium quinoa 4 9 chloride und alkoholgärung 115 cidaris granularia 202 cladium mariscus 59 cladosporium herbarum 8 7 cnicus benedictus l . 199 cocoa production 199 coniferenharz 263 coniothyrium fuckelii 104 – diplodiella 109 – tumaefaciens 105, 109 – concentricum 109 convallaria 60 cronartium viticola 269 crownrust 217 cryptomyces pteridis 207 cucurbitaria piceae 6 3 darwins zuchtwahl 63 dauerwaren 15 dauerwarenprüfung 25, 2 6 deli-versuchsstation 271 denitrifikation bei gegenwart schwer zersetzlicher organischer substanzen 128 digitalis 57, 5 9 distel 268 – bekämpfung 121 drogen und wohlgerüche in kairo, bazar der 201 drogen (verfälschungen und verschlech terungen) 5 9 274 sachregister. düngemittel 17, 20 – künstliche 221 dünger 270 düngewirkung des guanols 221 düngungsbedürfnis der ackerböden und wiesen 208 düngungsfragen, praktische 212 düngungsversuche mit lein 209 – in der buschobstpflanzung der guts verwaltung deiner-ittendorf 121– mit gaswasser 207 dunstfrüchte 16 durchfrieren 214 edelpilzzucht 54 efeu 264 eiche 132 einsammeln und anbau medizinischer pflanzen in schweden 115 einsäuerungsmethoden, neuzeitliche 114 empusa 126 entbitterung der lupine 193, 197– der reismeldesamen 193 entomophtoreae 126 entwicklungsrythmus treides 51 enzianpulver 56 erdbeeren 198 erdbeersorten 267 erdflöhe 121 erdraupe 221 erkennungsmerkmale für rauchschäden 129 ertragssteigerung durch so, 142 erythrina indica 57 esche 132 eschenmanna, stammpflanze der 116 evernia furfuracea 196 fadenziehendes brot 112 farbstoffe 265 farbstoff der beeren des efeus 265 farbstoffindustrie (englische) 265 faser (wirkung der kupfersalze) 265 fasergehalt von gespinstpflanzen 65 faserforschung 204 faserpflanzen, heimische (anatomischer bau und verwertbarkeit) 120 faserstoff aus torfmasse 204 des winterge faserstoffe (deckung des bedarfs in deutschland) 60 feldbohne 195 feldkresse als ackerunkraut 208 feldversuche in der landwirtschaft 209 feld und wald (kampf zwischen) 64 feinde der kulturpflanzen 124 feinde der obstbäume und sträucher 126 fettchemie 262 fettgehalt 25, 26 fetthefefabrikation 262 fettindustrie 262 fettsäuren 262 fettbildung in hefen auf festen nähr böden 221 feuchtigkeitsgehalt beim mahlen 19 5 fichte 61 285 fichtennadelmark-wickler 206 fichtenscharrharz 264 firnis 261 flachs 66, 67, 118 flachsbau in bayern 211 flachsverkauf 60 flachsstengelbastfasern 118 flechten als watteersatz 50 flechten (kohlehydratgehalt) 115 flußkrebs 88 s folia sennae 55 fomes fomentarius 60 forst und weide, trennung von 49 forstwissenschaft 121 fraxinus excelsior 82 frostschäden an reben 124 frostspannerbekämpfung 268 fruchtfolgen 209 fruchtsäfte 15 fruchtwechsel 8 fungus laricis 5 9 futtergewinnung aus der heimische" pflanzenwelt 51, 54 futtermittelkontrolle, mikroskopische 114, 194 futtermittelhandel 260 fusarium-arten 90 fusarium blight o f potatoes 217 fusicladium pirinum 206 galega 5 6 galeopsis 5 0 sachregister. 275 gallobelicus nicotianae 260 garnalen -en zeesternenmeel 114 gartenbau 206 gartenbibliothek, illustrierte 271 gartenbohne 27 gartenbuch 266 gartenfreunde (taschenbuch für) 271 gartengehölze (krankheiten) 218 gärtnerberuf 64 gärtnerische betriebslehre 223 gasvergiftungsversuche (so,) 132 geheimmittel 50 geigenharz 263 gemüsebau 266 gemüsesamenbau 54, 214 gemüseverwertung 206 gentiana 57 gentiana-wurzeln 116 genußund nahrungsmittel 114, 197 gerberinden 264 gerbstoffhaltige rinden 60 gerbstoffverbindungen 264 gerste 270 gerstenkreuzungen 124 gesundheitszustand der felder 8 getreideblasenfuß 206 getreidegarbentrocknung 260 getreidemehl 74 gewürze und gewürz-ersatz im kriege 199 gewürzund heilpflanzeneinfuhr nach deutschland 117 gewü1z-, heilund teepflanzen 199 gewürzpflanzen als honigspender 54 ginster 66 gips im brot 190 glossostemon brugieri 202 glyceria aquatica 99 gracilaria zachrysa 219 gräserarten (mikroskopische scheidung) 196 gräser, echte (bestimmungen) 194 gründüngung im gartenbau 207 grünfutter im winter 194 guayule 264 gummifluß 268 gummikulturpflanzen 263 guttapercha 264 unter guvacin 118 haardtwald, abholzung des 50 habitusbild (diagnostik von pflanzen krankheiten) 219 hafer 54, 270 – (einfluß des lichtes) 122 haferflocken (stärkegehalt) 113 haferpflanzen (gehalt an stickstoff, phosphorsäure und kali) 223 hagelbeschädigte reben 125 hainbuche 132 hanf 66, 67, 119, 265 hanfanbau 122, 203 haricot à acide cyanhydrique 27 harzgewinnung 263, 264 haselnußernte des jahres 1917 51 hauhechel und bingelkraut, sind – genießbar? 195 hauszwetsche 24 hedera helix 264 hederich 268 heidemoor 20 heil-, gewürzund teepflanzen 199 heilpflanzen als honigspender 54 heilund gewürzpflanzeneinfuhr nach deutschland 117 heilwerte heimischer pflanzen 55 helix pomatia 88 helminthia echioides 153, 154, 155 hemoeserna nebulella (sonnenblumen schädling) 127 heßdörfer, m. † 271 heuund sauerwurm (bekämpfung durch nikotin-schmierseifenbrühe 219 hexenbesen 127 hexenmehl (verfälschung)30 himbeersäfte 16, 17 himbeersorten 16, 17, 18, 20, 21 holz (alkoholerzeugung) 49 holzaufschließung zu futterzwecken 54 hölzer, in der tischlerwerkstatt ver nachlässigte 61 holz der baumarten auf java (mikro graphie) 205 – in volksund kriegswirtschaft 62 – als sparsamer baustoff 62 holzindustrie schwedens 62 276 sachregister. holzindustrie u. -handel ostpreußens 62 holzkonservierung 188 holzschutzmittel 177 holzterpentinöl 261 holzwirtschaft in deutsch-österreich 61 honigspender (heilu. gewürzpflanzen) 54 hopfenfaser 120, 203 hoering, paul + 36 homarus vulgaris (hummer) 88 hyoscyamus muticus 202 immunesande 215 impatiens (penicillium) 86 indigo 208 indigogelbreihe 264 indigo of nigeria 203 industrie in argentinien 224 – und landwirtschaft 64 inkarnatklee 195 inkubations-kalender spora) 64 inocybe 51 insektenvertilger (pflanzen) 269 jod in pflanzen 118 johannisbeerkernöl 262 johannisbeersäfte 21, 24 johannisbeersorten 16, 21, 24 isoguvacin 118 juniperus oxycedrus 121 – sabina 165, 166 juteersatz (convallaria) 60 kaffee-ersatzstoffe 54 kakao 115, 208 kakaoerzeugnisse mitschalengehalt 115 kakaoöl 262 kalidüngesalze 270 kali-endlaugen (wirkung auf boden und pflanze) 64 kalkempfindlichkeit des leins 213 kalken des sommerweizens 216 kapokbaum 125, 204 kardi 260 kardobenediktenkrautöl 199 karotten 76 kartoffeln 8, 74, 142 kartoffelbau 267 kartoffel (blattrollkrankheit) 125, 251, 216, 217 (reben-perono kartoffelblüte (farbenänderung) 113 kartoffelernteschätzungen 266 kartoffelfeind, sonderbarer (lecanium corni beté) 215 kartoffel in der deutschen volkswirt schaft 51 -knollenkrankheiten 221 -krankheiten 63, 215, 217 -krebs 6, 220 -land (umwandlung von wald in 49 -prüfung (ermittlung des spezifi schen gewichts) 209 -stecklinge 207 – -trocknung 260 (vermehrung) 197, 212 – -züchtung 213 kaseinspaltendes vermögen von milch säurebakterien 270 kauriharz 263 kautschuk 208, 264 kautschuksorten (madagaskar) 264 keimfähigkeit von sämereien (nach uspulunbeize) 208 keimkraftdauer 213 keimschimmel 270 keimungsenergie des kiefernsamens 63 keimungsverhältnisse bei nesselsamen 21 1 kiefernsamen (keimungsenergie) 63 kiefernsämling 207 kienöl 261 kindelbildung 270 kirschbäume 268 kirschgummi 263 kirschkerne 25, 26 kirschsorten 21, 22, 24, 25 kleegrasbau 210 klima-provinzen frankreichs 152 knospenvariationen an kartoffeln 267 kobert, rudolf 117 kohl 76 kohlenaschen als düngemittel 205 kohlensäurefrage für pflanzenkulturen 138, 178, 211 kohlenstoffernährung 266 kohlerdflöhe 214 kohlhernie 126 kohlrabi 77 sachregister. kohlraupe 88 kohlrübe 76 kohlweißlinge (bekämpfung) 217 kohn-abrest 28 kokosfett 261 kokosöl 262 koksaschen als düngemittel 205 kolbenschilf als faserpflanze 60 kolophonium 263 koniferen 266 korkersatz 121 kornkäfer (bekämpfung) 63 kotonisierung 61 krabbe 88 krankheit des bambus 267 krankheiten unserer waldbäume und gartengehölze 218 – von kulturgewächsen 124, 268 kräuselkrankheit des weinstocks 125 krebs 216 – auf obstbäumen 103 kresse 268 kriegsfuttermittel 53 kronenlichtnelke 212 kulturpflanzen (widerstandsfähigkeit gegen parasiten) 207 kulturversuche mit lein 209 kümmel 210 kunstdünger 221 kupferkalkbrühe setzung) 220 kupfervitriol 268 kürbis 267 lachenalia tricolor 212 lack 261 lactuca scariola oleifera 2 lamia 126 landwirtschaft der eingeborenen afri kas 208 landwirtschaftl. unterrichtswesen 223 landwirtschaft nach dem kriege 64 und industrie 64 lattichöl 262 lebensmittelgewerbe 251 lecanium corni 215 lein (kalkempfindlichkeit) 213 leinölersatz 261 leiusaaten 266 (chem. zusammen leukoverbindungen 264 lichteinfluß auf hafer 122 lichtnelke, kronen212 lignum quassiae 112 limax-arten 88 linde 122 lindenblätter 50 linsenmehl 202 literaturnachweise (stammbuch des apothekers mergenthaler) 115 loffa-kräuter 50 löslichkeitsverhältnisse mitteln 183 löwenzahn 76, 268 lupine (s . lupinus) 142, 195, 212, 267 – (entbitterung) 193, 197 – als faserpflanze 119, 120 lupinenverwertung 115, 259 lupinus albus 8 5 – angustifolius 197 – luteus 197 – termis 197 lüstrieren der brennesselfaser 203 mahagoniholz in guatemala 205 maja squinado 8 8 maifaser 98 mais 263 maisblütenstände 207 maismehl 74 maisöl 262 bei schutz mandragore, la 5 7 mandrake 57 manihotsamen (techn. ausnutzung) 201 mäusefraß 266 mechanisches gewebe (typha) 3 8 medicinali piante, coltivazione della 116 medizinische pflanzen (einsammeln u . anbau in schweden) 115 melilotusklee 120 mengsaat 209 mentha piperita 5 9 menta da essenza 50 menthol 264 milbenbefall von futtermitteln 1 14 mich 259 milchsäurebakterien 270 mineraldüngung 15, 16 mineralölversorgung 262 278 sachregister. mineralstoffe 16, 20, 23, 24 mohn 199 – mit bilsenkrautsamen 115 molinia coerulea 82 mondbohne 27 moorbödenerschließung 207 moorböden-nitrifikation 64 moorkultivierung 210 moosknopfkäfer 206 monilia-gefahr 219 moniliakrankheit der kirschbäume 268 mucor 81 – stolonifer 87 mutterkorn 270 mykozide wertzahlen 181 myzaphis abietina 206 nachtkerze, fette öl der 118 nacktschneckenplage in nordfrankreich 219 nadelhölzer 266 nahrungsmittel aus getreide 195 –, pflanzliche 74 – und genußmittel 114, 197 nessel 212 – -anbau 203, 204 – -faser 60 – -samen (keimungsverhältnisse) 211 nikotinbrühe ersetzt durch arsenbrühe 127 nitrifikation des stallmiststickstoffs 270 – in moorböden 64 obstarten 15 obstbau 206, 266 – (sortenelend) 123 obstbäume und sträucher (feinde und krankheiten) 126 obstbaumschädlinge 269 obstbaumzwischenpflanzung 24 obstdauerwaren 15, 16 obstkernsammlung 261 obstkernöl 203, 261 obstund gemüsegut der neuzeit 54 obstwickler 125 oidium 126 öl (physikal. u. chem. konstitution) 59 öle, ätherische 263, 264 –, flüchtige 264 ölgewächse (schädlinge) 219 ölpalme, schildkröten-202 ölsaaten 262 ölige produkte, reindarstellung 26 5 oliven der kinder israel 202 oenothera biennis 118 opium(verfälschung durch capita papa veris) 56 otiorrhynchus rotundatus 125, 126 papyrus-brikettes 32 parasiten 207 patella vulgata 8 8 pecan catcins 268 penicillium 81, 8 6 periodische erscheinungen a n wurzeln 62 peronospora 64, 206, 217 – viticola 5 petroleum 262 pfefferminzenrost 126 pfefferminzkultur 267 pfefferminzöl 264 pfeilkresse als ackerunkraut 208 pflanzenbau 205 –-düngung mit harn u . sulfitlauge 6 4 – -gallen in japan 220 – -krankheiten 219, 270 – -pathologie 170 – -physiologie als theorie der gärt nerei 62 – -schutzdienst 219 – -schutz in baden 121 – -schutz in deutschland 3, 4, 7, 10 pflanzenschutzmittel 156, 177 – chemische 6, 8 – (schlick) 62 pflanzenschutzstelle 7 , 9 , 1 5 pflaumen 259 pflaumenkerne 262 phalaris arundinacea 99 phaseolus lunatus 27 phoma destructiva 125 phoenix canariensis 85 – dactylifera 82 phosphorsäurekalkdüngung 270 phylloxorafrage 127 physoderma disease o f corn 220 phytelephas 8 9 phytopathologie 7 , 8 , 10, 11, 1 2 , 1 3 , 1 4 sachregister. 279 piassavafett 261 pieris brassicae 88 pilzangriffe, schutz gegen 181 pilze, wildfrüchte (verwertung) 52 pilzfreunde, vademecum für 196 pilzinfektionen (temperatur, feuchtig keit) 217 pilzkochbuch 51 pilzkunde 190 pilzvergiftungen (inocybe und tricho loma) 5l plantagenbau im mexikanischen tief lande 208 plantagerubber 264 plantago 50 plectridium pectinovorum 87 plocaederus obesus 125 polyporus betulinus, imbricatus, offi cinalis, resinosus, sulfurens 59 – fomentarius 60 podophyllum peltatum 57 produktionssteigerung, landwirtschaftl. 209 prüfung der pflanzenschutzmittel 7 pseudodematophora 90, 92 puccinia coronata 217 – graminis 217 – menthae 126 – pringsheimiana 219 – subnitens 267 pulververstäuber 8 quaet-faslem, g. † 224 quassia amara 112 quecke 268 ramalina graeca 196 rangoonbohne 27, 28, 29 raps 268 –(düngungsversuche) 60 rapserdflöhe 214 rapsglanzkäfer 128, 214, 215 rapsverborgenrüßler 128 rasenröste des flachses 118 rauchgase 129 rauchschäden 129, 207 – (erkennungsmerkmale) 188 raute 268 rebenperonospora 64, 217 reben, gelbsüchtige 219 reben, hagelbeschädigte 125 reblaus 64 reblausbekämpfung 267 reblauswiderstandsfähigkeit nischer reben 220 rebschädlinge (bekämpfung) 63 rebsorten, widerstandsfähige 123 reifegrad 26 reinhefezucht 270 reinsaat 209 reis 192, 208 reismelde 49 reismeldesamen (entbittern) 193 reizund rauschmittel 199 „resinol m“ 184 revalenta arabica 202 rhabarberarten in europa 202 rhabarber, chinesischer (in rußland kultivierter) 58 rhapontik 58, 118 rheum 57, 58, 118 – anglicum 58 – austriacum 58 – chinense 58 – gallicum 58 – palmatum (var. tanguticum) 58 rhizoctonia 268 rhizopus nigricans 270 ricinus 59 riebel + 271 rigolen 267 rinden, gerbstoffhaltige 60 rippel, a. 128 römer, th. 224 roggenstengelbrand 268 rohrernte 265 rohrzucker 262 – aus pflanzlichen objekten 115 rohrzuckersorten 266 rohsaft 16, 17, 20, 21 rohstoffversorgung deutschlands 60 römer, stephan (gedenkschrift) 117 rosenöl 263 rost (white pine blister-rust) 269 rostpilze 215 rotbrenner 269 rothea 28 rotklee 146 ff. amerika 28() sachregister. rotkleesaaten, französische 146, 147, 188 rübenbauer 52 rübenmehl 260 rübenschäden 270 rübstiel 142 rundbohne 27 saatgutbeizmittel 268, 269 saatwicke 195 saatzucht 205 safran 57 saftgehalt 25 salat 76 samenanerkennungen 124 samenbau im kleingarten 63 sandwicke 195 sarothamnus scoparius (l.) koch 61 sauerwurm 268 schädling auf picea pungens (cucur bitaria piceae) 63 schädlinge unserer ölgewächse, pflanz liche 219 schädlingsbekämpfung im winter 268 schalengehalt in kakaoerzeugnissen 115 scheba 196 schibutter (-baum und -industrie) 223 schildkröten-ölpalme 202 schilfkultur 60 schilfmehl 271 schilfrohr 33 schimmelpilze 21, 90 – des brotes 51, 114 schlick als pflanzenschutzmittel 62 schmierlaus 206 schmieröle 262 schwarzbeinigkeit 269 schwarzfleckigkeit der tomaten 125 schutzmittelprüfung 178 ff . scincus officinalis 201 scirpus holoschoenus 42 – lacustris l 18 sclerotinia 86 scopolia carniolica (litauen) 5 0 scopoliawurzel 200 seegras als textilfaser 120 seeigel 202 seetang (verwendung als faser 5 0 von kohlpflanzen seidenkultur 204 seifenfabrikation 202, 263 selen (im pflanzlichen und tierischen organismus) 115 senf, weißer 195 sennesblätter 55, 56, 202 serologische untersuchungen pflanzen bau und -zucht) 122 serradella 195 1 . serradellabau 211 sesamöl 263 sessous, g . 224 silphiden 126 sisalagave 208 soja (anbau und akklimatisation) 19 8 sojabohne 262 sojabohnenöl 263 solenostemma argel 202 sommergetreide (umzüchtung v.winter getreide in) 213 sommerweizen (kälken) 216 sortenanbauversuche 211 sortenelend im obstbau 123 sortenempfänglichkeit von getreide pflanzen gegen rostpilze 215 sparassis radicata 270 speisefette 262 speiseöle 262 speisepilze 53 spelzengehalt 195 spinat 142, 269 spinnfasern 265 spinnfaserversorgung deutschlands 6 0 spinnpflanzen 265 – (sind die einheimischen jetzt über flüssig?) 1 18 spiraea 104 spitzahorn 132 spörgel 195 sprengstoffe im obstbau 194 spritzen 8 sproßpilze in mineralischen nähr lösungen 222 stachelbeermehltau 267 stachelbeerrost (puccinia pringsheim ana) 219 standweite von kulturpflanzen 122,20 staphylea pinnata 5 9 sachregister. 281 stärke aus roßkastanien 115 steine (einfluß auf daswachstum) 270 steinbrand 5, 160, 164, 215, 269 steinbrandbekämpfung 127 steinobst 268 stickstoffdüngemittel, künstliche 212 stickstoffha shalt der böden 270 stopfmaterial für kissen 30 stoppelfruchtbau 195 streptococcus lacteus 270 strophanthus semina 116 strohaufschließung für futterzwecke 195, 196 strohfaser in der textilindustrie 205 strohfütterung 94 strohstoff und seine verdaulichkeit 197 stroh (verfahren zur behandlung) 61 sudanlettuce seed as a source of oil 203 surinam-bitterholz 112 süßholz 50 süßkirschen 1 6 , 2 4 süßpreßfutter 194 süßpreßfutterverfahren (i n silos) 197 süßstoffe 259 tabak 5 4 , 55, 208, 260 tabakbau 261 tabakfermentation 261 tabakkunde 115 tafeltraubenkultur 261 tanne 6 1 tannensterben im frankenwald 128 tarichium 126 t e e 260, 261 tee-,heilund gewürzpflanzen 199 teerfarbstoffe in der mikroskopischen technik 118 teeröl 262 teichbinse 118 terpentinöl 263 theezaden 260 thephrosia anthylloides, apollinea, villosa 56 timothyfrüchte 266 tobler, fr. 271 tomaten (schwarzfleckigkeit) 125 tomatenkernöl 262 torffaser (technologie) 204 torfmasse, faserstoffe aus 204 angewandte botanik i. torfmehl 267 torilis nodosa 153, 155 tortrix pygmaea 206 trapezeule 206 traubenkernöl 262 traubensaftkonserven 260 treiben der zierpflanzen 6 2 tricholoma 51 trichothecium 81, 86 trifolium supinum 155 trockendesinfektion 220 trockendestillation des holzes von juniperus oxyocedrus und einiger koniferen 121 trockenpilze (behandlung und unter suchung) 114 trocknungskosten 2 tussilago farfara 5 0 typha 30ff, 66, 98ff. – angustifolia 31, 34ff. – glauca 35 – latifolia 32ff. – minima 30 – hoeringii, shuttleworthii 36 – zinziae 36 – als faserstoff 30, 98, 119 typhafaser (nachweis in geweben) 203 ungeziefervertilgung 267 unkrautbekämpfung 62, 268 untersuchungen, chemische 1 6 uredineen 215 urocystis occulta 268 urteer 262 usnea plicata 196 ustilago violacea 217 waleriana officinalis 115 vanille 208 vegetationsversuch 6 2 verbaumwollung 61, 120 veredelungsunterlagen 267 verfälschungen von drogen 5 9 verfälschung von opium durch capita papaveris 5 6 vermehrung der kartoffel 197, 212 verwelkungskrankheiten 268 verwertung von pilzen und wild früchten 52 wezelstoffen 61 19 282 sachregister. violae odoratae radix 116 violae tricoloris radix 116 viola tricolor 50 vogelfraß 266 volldüngung 16, 19, 25, 26, 27 wachstumskurve 266 waldbäume (krankheiten) 218 waldes, erträge des deutschen 62 wald (umwandlung in kartoffelland) 49 wald und feld (kampf zwischen) 64 waldwolle als spinnfaser 120 walnußbaummotte 206 walnußblätter 50 watteersatz (flechten) 50 wegwart 268 wegweiser für pilzfreunde 51 weichholzhandel 61 weide, trennung von forst und 49 weidenröschen 66 weinbau 121, 206 weinbergschnecke 88 weinrebenfaser 119 weinstock (kräuselkrankheit) 125 weinstockkrebs 104 weinstock-schädlinge und -krankheiten 124 weintrauben 259 weißährigkeit der wiesengräser 220 weizen 270 weltvorräte 121 weymouthkiefer 61 widerstandsfähigkeit der französischen rotkleesaaten 149 – unserer kulturpflanzen gegen para siten 207 wiesengräser (weißährigkeit) 220 wildfrüchte 193 –, pilze (verwertung) 52 wildgemüse 193 wildpflanzen-lexikon 196 wildtee 193 winterfutter 193 wintergemüse 260, 266 wintergerste 198 wintergetreide (entwicklungsrhythmus 51 – (umzüchtung in sommergetreide)213 winterhafer 266 winterweizen (beizung gegen stein brand) 215 wirsingkohl 76 withamia somnifera 202 witterungseinflüsse und -verhältnisse 5 wohlgerüche und drogen in kairo, ba zar der 201 wühlmausbekämpfung 267 wundkorkbildung an kartoffeln 26 9 wurzelbrand a n spinat 269 wurzelentwicklung der gemüsepflanzen 53 wurzeln heimischer gräser als faser stoffe 61 würzpflanzen 210 yucca-blätter 109 zeesternenen garnalenmeel 114 zellmembranen, pflanzliche 78 zellonieren der brennesselfaser 203 zellstoff 61 zellwandverdauung (mikroskopische u r tersuchung) 193 zitrone (sour rot) 269 s zostera 120 zottelwicke 195 zuchtwahl darwins 63 zuckergewinnung aus ahornbäumen” zucker, holländisch-indiens 199 zuckerindustrie 261 zuckerrohr 208 zuckerrübe 259 – (feinde) 128 zuckerrübenbau 266 – krankheiten 270 zwergmaus 269 zwergobstbau 210 zwetschen 16, 2 4 zwiebel 76 uc1.b3299303_page_290_cut uc1.b3299303_page_291_cut uc1.b3299303_page_292_cut uc1.b3299303_page_293_cut uc1.b3299303_page_294_cut uc1.b3299303_page_295_cut section 10 uc1.b3299303_page_296_cut uc1.b3299303_page_297_cut uc1.b3299303_page_298_cut uc1.b3299303_page_299_cut uc1.b3299303_page_300_cut journal of applied botany and food quality 93, 54 58 (2020), doi:10.5073/jabfq.2020.093.007 1food research institute, national agriculture and food research organization (naro), tsukuba, ibaraki, japan 2headquarters, national agriculture and food research organization (naro), tsukuba, ibaraki, japan 3school of agricultural engineering and food science, shandong university of technology, zibo, shandong, china effect of moisture-proof corrugated boxes on water loss from cabbage during storage daniel z. k. wambrauw1, yuko sato1, naoki sugino1,2, saki matsumoto1, ling li3, hiroaki kitazawa1* (submitted: july 17, 2019; accepted: december 23, 2019) * corresponding author summary reducing water loss from cabbages during storage is essential to extend the shelf life of this widely consumed horticultural crop. in this study, we evaluated the effect of moisture-proof corrugated boxes (mpbs) on water loss from cabbages during storage. we first evaluated the water vapour barrier property of mpb material and found it to be superior to that of conventional corrugated box (ccb) material. cabbages were then stored in mpbs and ccbs for 9 days, during which their water loss was measured. cabbages stored in mpbs showed significantly less water loss than those in ccbs. moreover, storage in the mpbs did not negatively affect the fundamental qualities of the cabbages, such as the green colouration, the soluble solid content, and the ascorbic acid content. the use of mpbs was demonstrated to be an effective and viable way to reduce water loss from cabbages during storage. keywords: corrugated box, moisture-proof, storage, water loss, water vapour barrier property introduction cabbage is one of the most widely grown, traded, and consumed horticultural crops worldwide. in japan, approximately half of all produced cabbages are used for processed foods (takada, 2012). therefore, the maintenance of cabbage qualities such as colour, nutrition, and water content is important. however, leafy vegetables, especially cabbages, are known to have generally low storage po tentials in terms of their quality traits and a high perishability as a result of high respiration and water loss during the post-harvest period (kays and paull, 2004; wang, 2003). water loss is one of the major causes of post-harvest deterioration because it directly results in quantitative losses (loss of saleable weight) and qualitative losses such as wilting and decreased textural quality (i.e. loss of crispness, softening, and flaccidity) (nunes and emond, 2007). therefore, the prevention of water loss from cabbages is a major concern for the maintenance and extension of the shelf life of fresh produce. past studies have demonstrated the effectiveness of certain edible coatings in reducing water loss from fresh produce (ali et al., 2015; de león-zapata et al., 2015). however, considering the size and structure of cabbage heads, and depending on the regulation on agrichemicals and food additives, it is difficult to coat cabbages with such agents to reduce their transpiration. nevertheless, ad vances in packaging technology, including modified atmospheres, have provided possibilities for quality improvement and shelf life extension of horticultural crops, including leafy vegetables (li et al., 2014; scully and horsham, 2007; srinivasa et al., 2006; zagory and kader, 1988). the use of packaging materials with moisture-proof abilities are expected to reduce water loss from cabbages during the storage period. however, previous studies on modified atmosphere packaging using plastic films, which usually have a high water vapour barrier property (wvbp), focused on the control of oxygen and carbon dioxide inside the packaging. recently, the use of corrugated boxes has been proposed for the modified atmosphere packaging of fresh produce. it is important that such box materials maintain their wvbp as well as their oxygen and carbon dioxide permeability because the water vapour permeability of paper-based materials is usually higher than that of plastic-based materials. however, little is known about the relationship between wvbp of such paper-based packaging materials and water loss from cabbages during storage. therefore, in this study, we investigated the storage potential of moistureproof corrugated boxes (mpbs) with regards to their effectiveness in preventing water loss from cabbages. we also investigated the changes in fundamental qualities of cabbages, such as the green colour, soluble solids content, and ascorbic acid, throughout the ex perimental storage duration. materials and methods corrugated boxes we used two different types of single-layered corrugated boxes: a conventional corrugated box (control, ccb; da012, danball one, japan) without any coating on the surface of the linerboard of the box and an mpb (damp-proof 2, rengo, japan) with dried latex films and a filler on both surfaces of the linerboard of the box. the thickness of both the corrugated boards was ~5 mm, and the kind of corrugating medium was ‘a flute’. the external dimensions of both the boxes were approximately 400 × 600 × 180 mm (width × length × height). evaluation of wvbp of corrugated boards (experiment 1) we used a gas-tight acrylic chamber and a beaker containing water to evaluate the wvbp of the boards of both corrugated boxes (fig. 1). the corrugated boards of ccb and mpb were cut into discs with a diameter of 120 mm. the discs were then placed in a room at 10 °c for 48 h to equilibrate the materials to this room temperature. the relative humidity of the room was 85-90%. a 100-ml glass beaker containing 50 g (ml) water was placed into the gas-tight chamber with 1 l capacity. the disc of the corrugated boards was placed between the chamber and a ring made of acrylic resin, which had the same internal and external diameters as the gastight chamber. this assembly comprising the opening of the chamber, the ring, and the disc of the corrugated boards was sealed with a piece of stretch paraffin film (parafilm® m, bemis, wi, u.s.a.) to avoid the leakage of water vapour. in addition, the stretch film was secured using plastic polyvinyl chloride tape (19 mm; yamato, tokyo, japan). the gas-tight chamber, which contained 50 g (ml) of water and the discs of the corrugated boards and the acrylic ring, was kept in the same room to equilibrate the discs of the corrugated boards to the temperature of the storage room. the experiments commenced on moisture-proof corrugated boxes reduce water loss from cabbage 55 15 april 2019. the weight change of each beaker was measured on days 2, 4, 7, and 10 of the experimental periods. the chamber was opened each day for a maximum of 3 min. analysing the effects of mpb on the quality of cabbages during storage (experiment 2) cabbages: cabbages (brassica oleracea var. capitata) with compact heads were harvested from a commercial farm in aichi prefecture, japan. the cultivar was unknown. the initial weights of the cabbages were ~1.4 kg ± 0.14 of standard deviation. the cabbages were randomly divided, packed in ccbs, and transported to a laboratory at the food research institute, tsukuba, japan. all cabbages were stored overnight at 5 °c with ~90% of relative humidity before commencing with the storage experiments. storage conditions: the experiment commenced on 16 april 2019. the cabbages were stored at 10 °c with a relative humidity of 8590% under dark conditions. eight cabbages were placed into each box type. in total, eight boxes were prepared: four ccbs and four mpbs. all boxes were stacked in four columns with five tiers each (fig. 2). the column space between the boxes was set at ~40 mm (for air circulation). both the samples (n = 4 boxes for ccb and n = 4 boxes for mpb) were randomly placed (dark grey boxes in fig. 2) in front of the cooling fan (23 m3 h-1, 70 w). the other boxes (light grey boxes in fig. 2) were used as balancers or dummy boxes. the boxes were shuffled every day throughout the experimental storage duration. weight loss and colour change: weight loss and colour change are related to the apparent quality of cabbage, and the change in weight is assumed to be the result of water loss. the change in cabbage weight during storage was measured on days 0 (16 april 2019, initial weight before storage), 2, 3, 6, and 9 of the experimental storage period. the change in green colour of the leaves was also measured on the same sampling days, using a non-destructive chlorophyll meter (spad502plus, konica minolta optics, tokyo, japan). the fourth to sixth leaves from the outer part of five cabbages were used. three parts on each leaf were selected randomly and their soil plant analysis development (spad) values were measured using the chlorophyll meter, and the three measured values were averaged. the spad value is used for indirect measurement of the chlorophyll content of plant leaves (kasim and kasim, 2017; león et al., 2007). soluble solids content and ascorbic acid: to measure the soluble solids content (brix %) and ascorbic acid (l-ascorbic acid), stored cabbages were cut diagonally into eight parts and the core part was removed. after cutting the sample into small pieces and thoroughly mixing the cut pieces, ~5 g was sampled for use in the analysis of the soluble solids content (brix %), and ~10 g was sampled for use in the analysis of the ascorbic acid. the exudates from the 5-g cabbage samples, obtained with the use of an extractor, were dropped onto the prism of a saccharimeter (pr-201α, atago, tokyo, japan), which was used to measure the soluble solids content. to analyse the ascorbic acid of the 10-g samples, the cabbage pieces were immediately placed into 90 ml of 5% metaphosphoric acid solution, homogenised using a homogeniser (hg30, hitachi koki, tokyo, japan), and filtrated using filter paper (no. 6, 110 mm, advantech toyo, tokyo, japan). the content of the ascorbic acid in the filtered liquid was measured using a reflectometer (rqflex 20, merck, darmstadt, germany). both the soluble solids content and ascorbic acid analyses were conducted with five replications and on the same sampling days as the weight loss and colour changes were measured. statistical analysis all results are shown as means ± standard deviations. we used welch’s t-test for pairwise comparisons of data within the same measurement period using a spreadsheet program (excel 2016, microsoft japan, tokyo japan). p-values < 0.05 were considered statistically significant. results wvbps of the corrugated boards (experiment 1) the weights of the water beaker on days 2, 4, 7, and 10 of the storage period were 49.8, 49.6, 49.2, and 48.9 g, respectively, in the experiment with discs from ccb (i.e. cc; fig. 3) and 49.7, 49.6, 49.4, and 49.1, respectively, in the experiment with discs from mpb (i.e. mp). significant differences were found between cc and mp on days 7 and 10 of the experimental storage period. fig. 1: method for evaluation of the water vapour barrier property of corrugated boards. a) gas-tight chamber containing a beaker with 50 ml of water. b) top view of the assembly. c) disc of corrugated board. fig. 2: position of corrugated boxes inside the storage room. dark grey boxes indicate the positions of the sample boxes. light grey boxes in the top and bottom rows and one third of dark grey boxes indicated empty boxes for balance. 56 d.z.k. wambrauw, y. sato, n. sugino, s. matsumoto, l. li, h. kitazawa effects of mpb on the quality of cabbages during storage (experiment 2) weight loss: for the control, the weight loss (g) of cabbages on days 2, 3, 6, and 9 after starting the experiment was 3.2, 5.1, 11.0, and 15.1, respectively (fig. 4a). on the other hand, for the mpb, the values on days 2, 3, 6, and 9 were 3.2, 4.3, 7.5, and 10.8, respectively. significant differences were found in the cabbage weight loss values for both boxes at 6 and 9 days after starting the experiment; the weight loss for mpb was 30% lower than that of the control during storage (fig. 4b). colour, soluble solids content, and ascorbic acid: the change of green colour (spad value), soluble solids content, and ascorbic acid of cabbages during storage for 9 days are shown in tab. 1. the spad values for both boxes decreased in the days after starting the experiment. the values of soluble solids content and ascorbic acid were unchanged during storage. thus, for all measurement items and periods, there were no significant differences between control and mpb. discussion in the present study, we first evaluated the wvbp of cc and mp. compared to cc, mp showed lower water vapour permeability (fig. 3). for cc, a major decrease in the beaker water level was observed on days 7 and 10 of the experiment. in contrast, the decrease in the beaker water level of mp was less significant than that of cc on the same sampling days, indicating that less water was lost. in accordance with the above-mentioned results, we then conducted storage experiments with cabbages for 9 days. weight loss analysis revealed significant differences between cabbages stored in the ccbs and mpbs at 6 and 9 days (fig. 4a). the most important factor of weight loss in fresh produce is the decrease in water content via transpiration (kays and paull, 2004; kitazawa et al., 2011). although, several factors, including moisture (bovi et al., 2016; kader, 2002), affect the degree of transpiration of fresh produce, the maintenance of a high relative humidity inside packaging contri butes to the reduction of water loss from fresh produce (mampholo * * 48.0 48.4 48.8 49.2 49.6 50.0 0 2 4 7 10 c ha ng e of w at er w ei gh t ( g) days after starting cc mp a b fig. 3: the change in weight of water inside beakers placed in the gastight chambers sealed with conventional corrugated board (cc) and moisture-proof corrugated board (mp) to determine the water vapour permeability of the storage materials. error bars indicate standard deviations (n = 5). asterisks indicate significant differences by welch’s t-test (p < 0.05). fig. 4: effect of storage in different corrugated boxes on weight loss from cabbages. (a) weight loss from cabbages (g) and (b) weight loss rate (%) for cabbages stored in conventional corrugated boxes (ccbs) and moisture-proof corrugated boxes (mpbs). error bars indicate standard deviations (n = 5). asterisks indicate significant differences by welch’s t-test (p < 0.05). tab. 1: effects of two types of corrugated boxes on the colour, soluble solids content, and ascorbic acid content of cabbage during storage. storage duration green colour soluble solids content ascorbic acidz (days) (spad value) (brix %) (mg kg-1 fw) ccb mpb ccb mpb ccb mpb 0 6.2 ± 2.4y 7.2 ± 1.1 530 ± 52 2 5.8 ± 1.3 5.4 ± 2.6 7.0 ± 0.7 8.0 ± 0.6 554 ± 46 542 ± 54 3 7.1 ± 2.0 5.0 ± 1.7 8.0 ± 0.7 7.0 ± 0.6 542 ± 57 504 ± 27 6 3.4 ± 1.0 3.2 ± 1.0 6.7 ± 1.8 7.5 ± 1.1 520 ± 56 538 ± 23 9 4.0 ± 1.5 2.8 ± 0.7 7.7 ± 1.5 7.6 ± 0.2 522 ± 56 522 ± 31 ccb, conventional corrugated box (control); mpb, moisture-proof corrugated box zl-ascorbic acid ymean ± standard deviation (n = 5) a b moisture-proof corrugated boxes reduce water loss from cabbage 57 et al., 2013; tzoumaki et al., 2009). in experiment 1, we demonstrated that mp had a high wvbp, and experiment 2 showed that the water loss from cabbages stored in mpbs was lower than that from cabbages stored in ccbs. for leafy vegetables, the loss of green colour usually indicates the end of the shelf life of the produce. we therefore used the spad value, which represents the chlorophyll content, to evaluate the decrease in green colour of cabbages leaves. no significant differences were observed in the spad value between the ccb and mpb for each measurement period (tab. 1); however, the values for each box tended to decrease with the increase in storage duration. the loss of green colour of fresh produce is reportedly caused by chlorophyll degradation (king and morris, 1994; mampholo et al., 2013; yamauchi, 2013; zhuang et al., 1997) and accelerated by the absence of light or dark conditions (büchert et al., 2011). thus, the decrease in green colour of cabbages packaged in both types of boxes in the present study was presumably induced by the dark-light conditions during storage. however, our results demonstrate that the use of mpbs, specifically, did not induce any negative changes in the green colour of the cabbages. there were no significant differences between the soluble solids content and ascorbic acid content of the cabbages stored in the ccbs and mpbs for each measurement period (tab. 1). the soluble solids content remained stable throughout the storage period in the present study, and this result is consistent with the findings of similar studies on cabbages and asparagus (brueckner et al., 2010; nilsson, 1993; shou et al., 2007). the ascorbic acid content also remained stable throughout the storage period in the present study. several studies have reported that post-harvest water loss from leafy vegetables results in the rapid loss of ascorbic acid (lee and kader, 2000). therefore, considering the observed water loss from cabbages stored in ccbs, the ascorbic acid content of such cabbages may decrease to a greater extent than that of cabbages stored in mpbs over an extended storage period; however, further studies are needed to confirm this expectation. nevertheless, the different packaging conditions in the present study did not affect the soluble solids content and ascorbic acid content of cabbages over the storage durations used, and mpb did not induce any negative changes to the assessed fundamental qualities during the storage period. conclusion our results demonstrate that storage in mpbs effectively reduced water loss from cabbages and did not induce any negative effects on other fundamental qualities; 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(in japanese). tzoumaki, m.v., biliaderis, c.g., vasilakakis, m., 2009: impact of edible coating and packaging on quality of white asparagus (asparagus officinalis l.) during cold storage. food chem. 117, 55-63. doi: 10.1016/j.foodchem.2009.03.076 wang, c.y., 2003: leafy, floral and succulent vegetables. in: bartz, j.a., brecht, j.k. (eds.), postharvest physiology and pathology of vegetables, 599-623. new york, usa, marcel dekker. yamauchi, n., 2013: quality maintenance of postharvest horticultural crops by stress treatments and approach for the elucidation of its mechanism. j. jpn. soc. hort. sci. 82, 1-10. doi: 10.2503/jjshs1.82.1 zagory, d., kader, a.a., 1988: modified atmosphere packaging of fresh produce. food technol. 42, 70-77. zhuang, h., hildebrand, d.f., barth, m.m., 1997: temperature influenced lipid peroxidation and deterioration in broccoli buds during postharvest storage. postharvest biol. technol. 10, 49-58. doi: 10.1016/s0925-5214(96)00054-3 orcid hiroaki kitazawa http://orcid.org/0000-0003-1470-3658 li ling http://orcid.org/0000-0003-4918-0893 address of the corresponding author: hiroaki kitazawa, food research institute, national agriculture and food research organization (naro), 2-1-12, kannondai, tsukuba, ibaraki, 3058642, japan e-mail: ktz@affrc.go.jp © the author(s) 2020. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1016/j.scienta.2006.12.048 http://dx.doi.org/10.1002/jsfa.2472 http://dx.doi.org/10.1016/j.foodchem.2009.03.076 http://dx.doi.org/10.2503/jjshs1.82.1 http://dx.doi.org/10.1016/s0925-5214(96)00054-3 journal of applied botany and food quality 93, 130 135 (2020), doi:10.5073/jabfq.2020.093.017 1szent istván university, department of physics and control, budapest, hungary 2industrial university of ho chi minh city, institute of biotechnology and food technology, ho chi minh, vietnam 3szent istván university, department of refrigeration and livestock product technology, budapest, hungary 4szent istván university, department of postharvest science and sensory evaluation, budapest, hungary evaluation of precooling temperature and 1-mcp treatment on quality of ‘golden delicious’ apple lászló baranyai1*, lien le phuong nguyen2,3, mai sao dam2, tamás zsom4, géza hitka4 (submitted: september 22, 2019; accepted: january 17, 2020) * corresponding author summary the presented study simulates commercial practice and focuses on the effect of time gap between harvest and the beginning of cold storage. apple fruit ʻgolden delicious’ was harvested in hungary and randomly separated into 3 groups for precooling at 1, 4 and 10 °c. after 7 d cold storage, groups were randomly split half to control and the other half was subjected to gaseous 1-methylcyclopropene (1-mcp) treatment for 24 h on their cold storage temperature (1, 4 and 10 °c). all samples were stored for 6 months at 1 °c followed by 7 d shelf-life at ambient temperature. ethylene production, firmness, total soluble solid content, surface color and disorder in cidence were determined. significant correlation was found between color parameters hue and normalized green with firmness, ssc and ethylene production. precooling temperature and 1-mcp treatment significantly affected apple quality (p < 0.01). initial storage at 10 °c and application of 1-mcp on this temperature had no clear effect on maintaining fruit quality compared to control after 6 months storage. on the other hand, 1 °c and 4 °c precooling and applied 1-mcp treatment could slow the softening of samples during 6 months storage and in the following shelf-life. apple quality was observed to change faster for group of 10 °c precooling and slower for groups of 1 °c and 4 °c. according to the results, precooling of apple fruit at 1 °c can be recommended in case the 1-mcp treatment is delayed. introduction apple (malus × domestica) is important fruit with annual production of 83 million tonnes (fao, 2019). it is the second most consumed fruit after banana. the majority of apple production is used for fresh fruit consumption, therefore postharvest technology applied to maintain its best quality is important. harvested apple is typically stored for months in cold rooms of 0 5 °c using refrigerated air or controlled atmosphere (delong et al., 2004; matthes and schmitz-eiberger, 2009). postharvest technology subject apples to treatment before long term storage to keep both quality and commercial value. controlled atmosphere of %co2:%o2 of 1.5:1.5 and 5:3 were found to maintain apple firmness after long term storage and shelf-life the best among other combinations (jeziorek et al., 2010). the controlled atmosphere of 1.5% o2 and 0.6-0.9% co2 at 3 °c and 5 °c was applied on ‘honyecrisp’ apple during 6 months storage (delong et al., 2004). desired low o2 concentration was obtained by flushing n2 gas into storage chamber. controlled atmosphere cold storage of 1.5 °c, 1.2% o2 and 2.5% co2 was also reported to successfully maintain polyphenol content in apple (matthes and schmitz-eiberger, 2009). ripening of climacteric fruit can be controlled by 1-methylcyclopropene (1-mcp) successfully (blankenship and dole, 2003). the advantage of application of 1-mcp is its effect on respiration and ethylene production. as a result, 1-mcp treatment also affects ethy lene induced postharvest disorders. gago et al. (2015) evaluated effect of harvest date and gaseous 1-mcp treatment on apple qua lity during and after 6 months storage. it was observed that 1-mcp was able to maintain firmness, reduce electrolyte leakage and slow surface color change. during the experiments, apples from certain orchards developed bitter pit at higher rate as a result of 1-mcp treatment. those fruit were found to have low ca and high mg content and therefore bitter pit is more affected by cultivation technique. authors also concluded that optimal harvest date is essential, affecting both postharvest disorders and following shelf-life. milinkovic et al. (2018) investigated 5 apple cultivars and did not find significant difference in their k:ca ratio, but observed fluctuation of mineral content in consecutive years. ericsson and tahir (1996) evaluated possible factors on bruising of 3 cultivars. pre cooling with cold air of 3 4 °c was found to make fruit more resistant to bruising, but treatment was less effective for late harvested fruit. harvest date affected bruising sensitivity significantly. delay of 10 d could increase damage made by bruising with 20 40% in ‘aroma’ and 12 25% in ‘ingrid marie’. the effect of harvest date was found to depend on cultivar. timing is also important in application of postharvest treatments. delong et al. (2004) delayed cooling treatment with a warming period of 20 °c on ‘honeycrisp’ apple in order to decrease soft scald and low temperature breakdown. it was found that delayed cooling of 7 d at 20 °c was beneficial and resulted in less disorder incidence during 4 6 months storage in case of earlyand late harvested apple. positive effect of warming period and delay was confirmed by watkins et al. (2004). they found that soft scald incidence can be decreased with higher storage temperature for sensitive cultivars. it was also highlighted that low production quantity of such sensitive fruit may not be enough to separate them and fill a storage facility to ensure different storage temperature. delay before cooling was found to be effective, 1 d at 20 °c was able to prevent soft scald and soggy breakdown disorders during storage at 0 and 0.5 °c. common parameters measured to monitor apple quality are fruit firmness, soluble solid content and titratable acidity (lu and lu, 2016; watkins et al., 2004). nondestructive methods are offered by computer vision in wide range from color imaging of surface defects to hyperspectral imaging of quality based on spatial spectral information. advanced techniques such as x-ray, mri (magnetic resonance imaging) and ct (computed tomography) can detect internal structural changes, while diffuse reflectance showed its potential in firmness assessment (lu and lu, 2016). surface color measurement with digital image processing is preferred in postharvest handling due to the high speed and ability to deploy in-line. more sophisti cated and time consuming methods are utilized primarily in research. the main goal of the presented research was to simulate the commercial application of 1-mcp. during gradual loading of the storage facility, temperature fluctuates and fruit also have different temperature according to the length of their prior storage. this may affect the efficiency of 1-mcp treatment. therefore, this study evaluated the apple response to precooling and 1-mcp treatment 131 effect of precooling temperature of 1, 4 and 10 °c as well as 1-mcp treatment on apple quality during 6 months storage. another objective of the experiment was to analyze relationship between quality indices and color parameters measured by digital image processing. materials and methods apple fruit and 1-mcp treatment apple fruit (malus × domestica) of ‘golden delicious’ cultivar have been collected from orchard in hungary (at city of ráckeve in pest county). samples were collected during middle of harvest season in september 2018. the experimental design was made to simulate whole harvesting procedure including the time gap between picking and long-term storage. the total amount of 900 fruit was selected for experiment and randomly split into 3 groups. groups were subjected to 7 d cooling at 1 °c, 4 °c and 10 °c. after this initial precooling, groups were divided into two subgroups. half was kept as control and another half was subjected to gaseous 1-mcp treatment at the precooling temperature. after 1-mcp treatment, apple fruit were stored for 6 months at 1 °c. forty fruit of each group was left at ambient temperature for 7 d to test their behavior during shelf-life right after the 1-mcp treatment. after long term storage of 6 months, all groups were withdrawn to ambient temperature for 7 d shelf-life. experimental plan is introduced on flowchart (fig. 1). ethylene production ethylene production was determined by an ica-56 handheld ethylene analyzer (international controlled atmosphere ltd., uk). apple was placed in a hermetically closed plastic container of 4 l for 1 h before measurement was performed. measurement was repeated in triplicates. results were expressed in microliter gas produced per kilogram of fruit in one hour (μl kg-1 h-1) calculated on fresh weight basis. soluble solid content soluble solid content (ssc, %) was determined by a hand-held temperature-compensated atago pal-1 digital refractometer (atago co. ltd., tokyo, japan). firmness measurement fruit firmness was recorded with a handheld fruit firmness tester (ft 327, italy). the instrument was mounted on a stand to make measurement more stable. cylindrical probe of 10 mm diameter was penetrated into the tissue of peeled apple until 10 mm depth. the maximum force was measured at two opposite points on the external circumference. the average was recorded for each fruit. the instrument provided values in kg cm-2, what was transformed to the unit of n. disorder incidence fruit were visually tested for rot and bitter pit on the skin during storage period. the disorder was determined when a sign of those symptoms occurred. the incidence was calculated as percentage of the total number of fruit. surface color digital images of hd size (high definition, 1920 × 1080 pixel) and 24 bit/pixel color depth were recorded with a camera (samsung wb350f). the optical zoom of 4× was applied. apples were placed on white paper and this background was used as white color reference during processing. the roi (region of interest) was selected based on saturation with the threshold of 0.2. recorded color information was transformed into hsl (hue, saturation, luminosity) space. green color dominance was calculated as well, with linear normalization (eq. 1). gn = g / (r + g + b) (1) where gn is the normalized green component, r, g and b are red, green, blue intensity values, respectively. statistical analysis all data were subjected to statistical analysis with r version 3.6.0 (r foundation for statistical computing, austria) using analysis of variance (anova). the effect of the factors of precooling temperature and storage time was evaluated. the anova f value was used to compare effects to the natural variability of collected data. tukey’s method was used as post-hoc test to compare selected groups with p<0.05. the results are reported on charts with mean and standard deviation (sd). figures were created using microsoft excel (microsoft co., usa). results and discussion effect of precooling on quality indices the comparison of initial readings with measurements after 7 d precooling (tab. 1) showed that apple subjected to 10 °c precooling changed more than that of 1 °c and 4 °c. during the 7 d precool fig. 1: experimental plan from harvest (h) with 7 d precooling, 6 months storage and 7 d shelf-life. steps are simulating: short-term storage in orchard and transportation (7 d, 1-10 °c); long-term storage (6 months, 1 °c); storage in shop and household (7 d, 20 °c). the gaseous 1-mcp treatment was performed with commercial tablets (0.14% 1-mcp) as an application of the smartfresh® system, provided by rohm and haas polska sp.z.o.o. (warsaw, poland). measurements measurements were performed at initial stage 0 d, at 7 d precooling, after 7 d shelf-life following 1-mcp treatment, at the end of 6 months storage and 7 d shelf-life following the 6 months storage. starch in dex was measured at 0 d and after 7 d precooling. the ethylene production, firmness, soluble solid content (ssc), disorder incidence, and surface color were measured at 20 °c during the experiment. starch index starch index was determined using lugol’s solution (aqueous iodine) on half cut fruit and surface pattern was compared to starch index reference guide. 132 l. baranyai, l.l.p. nguyen, m.s. dam, t. zsom, g. hitka ing after harvest, starch index and ssc increased while firmness decreased. on the other hand, no significant difference was observed among fruit according to ssc and firmness. regarding color attributes, hue angle value decreased indicating that surface color started to change toward yellow. the green dominance was still similar after precooling, which means that fruit color changed within the green range. ethylene production ethylene measurements were conducted to evaluate the effect of precooling and 1-mcp application on postharvest life of apple. precooling temperatures affected the ethylene production. apples kept at 1 °c and 4 °c showed lower ethylene production during shelf-life both after 1-mcp treatment and after storage than those at 10 °c (fig 2). tab. 1: quality parameters before and after precooling treatment (mean value ± sd) parameter 0 d 7 d, 1 °c 7 d, 4 °c 7 d, 10 °c starch index 6.51 ± 0.22a 7.15 ± 0.24ab 7.82 ± 0.21bc 8.75 ± 0.27c soluble solid content, % 12.87 ± 0.22a 13.30 ± 0.16a 13.20 ± 0.16a 13.40 ± 0.16a firmness, n 72.81 ± 2.52a 71.15 ± 2.52a 69.32 ± 1.89a 66.25 ± 1.89a hue angle 108.24 ± 0.94a 105.12 ± 1.28ab 103.45 ± 1.19bc 100.25 ± 0.91bc green dominance, % 44.79 ± 0.73a 43.59 ± 0.22a 43.71 ± 0.29a 44.12 ± 0.48a different superscript letters in a row show significant difference at p<0.05. fig. 2: ethylene production during experiment (sl: shelf-life). presented values are mean ± sd. the 1-mcp inhibited strongly the ethylene production. after 6 months of storage and during the following shelf-life, treated samples showed lower values than control but at different rate. fruit of 1 °c and 4 °c precooling treated with 1-mcp had lower level in ethylene production than those of 10 °c. at the same time, all control fruit showed high ethylene production, with increasing value from 1 °c precooling temperature to 10 °c. samples of 1 °c or 4 °c precooling were not significantly different in ethylene production. firmness after 7 d of precooling, firmness decreased slightly for fruit kept at 1 °c and 4 °c, whereas apple from 10 °c had significantly lower values compared to the initial time (tab. 1). firmness declined dramatically for all groups throughout shelf-life and storage. the results also showed that 1-mcp reduced the softening of apple for all temperature groups compared to control during the experiment but at different rates (fig. 3). fig. 3: firmness during experiment (sl: shelf-life). presented values are mean ± sd. fig. 4: soluble solid content during experiment (sl: shelf-life). presented values are means ± sd. precooling temperature influenced the efficiency of 1-mcp application. 1-mcp strongly affected only the fruit of 1 °c and 4 °c groups. there was no significant difference in firmness between 1 °c and 4 °c precooling either at storage or shelf-life, whereas 1-mcp treated fruit from 10 °c group were much softer. 1-mcp treatment did not show expected effect on apple of 10 °c since no significant difference was detected between control and 1-mcp treated fruit using this precooling temperature. soluble solid content apple obtained similar ssc values after 7 d precooling in all groups (fig. 4). however, it increased slightly during shelf-life before storage and after storage. fruit subjected to 10 °c precooling had higher values in ssc after 6 months of storage, then declined in the following shelf-life. nonetheless, there were significant changes and significant differences among groups throughout the experiment. apple response to precooling and 1-mcp treatment 133 bitter pit and rot the disorder of bitter pit was detected more frequently in 1-mcp treated samples than in control after storage (tab. 2). after precooling and shelf-life before long term storage, no bitter pit symptom was observed. there were no significant differences in bitter pit occurrence among groups at the end of storage and in the following shelflife. the rot infection was detected less frequently for 1-mcp treated fruit than for control in all temperature. the symptom of fungal development only occurred after 6 months of storage. samples of 1 °c and 4 °c precooling suffered less decay than those of 10 °c (tab. 2). fruit subjected to precooling at 1 °c showed the lowest level of bitter pit and rot, while at 10 °c it became very high. no significant difference was observed in bitter pit and rot occurrence between 1-mcp treated and control apples for group of 10 °c. surface color the color development of apple surface was expressed by hue angle values. the surface color of apple continuously changed during the experiment according to the mean value of groups, but no significant difference was found between initial values and that of after 7 d precooling. the hue angle decreased and standard deviation increased after storage for all groups (fig. 5). the standard deviation was similar for all groups after shelf-life following both precooling and long term storage. correlation between parameters pearson’s correlation coefficient was calculated between para meter pairs (tab. 3). the highest correlation was observed between hue angle and firmness (r = 0.9597). similarly high correlation was found between firmness and starch index, but starch index is not included in the overall comparison because it was measured until the beginning of long term cold storage. firmness was highly correlated (r = -0.7715) with ethylene production according to the expectations. among quality parameters, firmness gained the highest correlation with color, r = 0.9597 for hue angle and r = 0.7721 for green dominance. ethylene production and ssc obtained lower but still significant correlation values with color. 1 °c 4 °c 10 °c 0 40 80 120 shelf-life-control shelf-life-1-mcp storage-control storage-1-mcp storage+sl – control storage+sl – 1-mcp precooling temperature h u e , d e g tab. 2: bitter pit and rot occurrence after 6 months storage and after shelf-life after storage after storage + 7 d shelf-life precooling treatment bitter pit, % rot, % bitter pit, % rot, % 1 °c control 8.45 9.86 13.49 11.32 1-mcp 10.69 7.33 16.74 9.12 4 °c control 8.96 10.45 14.22 12.29 1-mcp 11.85 7.84 17.48 9.43 10 °c control 12.89 12.39 20.83 15.79 1-mcp 14.97 11.92 21.75 14.61 tab. 3: correlation between measured parameters ethylene ssc hue gn firmness -0.7715** -0.3425 0.9597** 0.7721** ethylene 1 -0.0256 -0.6733* -0.4829+ ssc 1 -0.5199* -0.3294 hue 1 0.8198** ** significant at p < 0.001; * significant at p < 0.01; + significant at p < 0.05 fig. 5: hue angle value during experiment (sl: shelf-life). presented values are means ± sd. similar tendency was observed for green dominance. less difference was found in green dominance before 6 months storage. green color became less dominant after shelf-life following both precooling and long term storage. together with decreasing hue angle, they indicate that color changed into the direction of yellow. discussion maturity stage is the most important factor influencing the efficiency of 1-mcp treatment. the efficacy of 1-mcp is low at advanced maturity stage (jia et al., 2014; sozzi and beaudry, 2007). our results also indicated that the effectiveness of 1-mcp treatment depends on fruit maturity, in agreement with the previous report (jia et al., 2014). application of 1-mcp on apple subjected to 10 °c precooling had a little effect in reducing ethylene production after 6 months storage, while 1-mcp treatment on fruit of 1 °c and 4 °c precooling was effective in suppressing the ethylene production. the reason for this behavior might be that samples of 10 °c precooling for 7 d reached the advanced maturity, therefore 1-mcp did not show the efficacy as strong as other groups. according to this observation, if delay in 1-mcp treatment cannot be avoided due to transportation or optimization of bulk treatment, short-term cold storage at 1 °c is recommended. the decline in firmness during storage results in soft and mealy fruit decreasing the quality value of fruit (kader, 1999). in our work, the softening of fruit increased during the precooling period from 1 °c to 10 °c corresponding to the increase of starch index due to advanced ripening (tab. 1). 1-mcp treatment on fruit of 1 °c and 4 °c precooling delayed the softening of apple during storage and shelflife, whereas the softening of control samples rose rapidly. similar behavior was observed for pear using gaseous 1-mcp of 1.0 ml l-1 (gao et al., 2015). less efficacy of 1-mcp at 10 °c precooling was found perhaps due to incomplete blocking of the ethylene receptors, 134 l. baranyai, l.l.p. nguyen, m.s. dam, t. zsom, g. hitka thus ethylene could exert its action partly in ripening (blankenship and dole, 2003; watkins, 2008). regarding to delayed 1-mcp treatment, previous reports also indicated that the applied treatments at earlier maturity stage increased the possible storage periods (kubo et al., 2003; watkins and nock, 2005). depending on the cultivar and circumstances of treatment, the 1-mcp treated fruit can have higher, lower, or same values of soluble solid content compared to control samples (gago et al., 2015). our results showed that 1-mcp had small effect on ssc and ssc of fruit of 10 °c precooling was lower than those at 1 °c and 4 °c at the end of experiment, but not significantly different. surface color was found to change gradually from green toward yellow. hue angle decreased in this direction but green dominance measured by normalized green value did not change significantly. apple color in control group changed more than groups of 1-mcp treatment. similar results were found for ‘red chief delicious’ apples (mir et al., 2001). ethylene plays an important role in the ripening process. 1-mcp inhibited the ripening by occupying ethylene receptors, so that ethylene is unable to elicit its action (blankenship and dole, 2003). other fruit, including papaya and melon also showed similar results (bron et al., 2004; ergun et al., 2005). color para meters were found to correlate strongly with firmness, followed by ethylene production and ssc. the percentage of bitter pit was higher in 1-mcp treated fruit than control. in earlier reports, similar results were found for ‘golden delicious’ apple (gago et al., 2015), ‘granny smith’ apple (calvo and candan, 2010). the symptom of bitter pit related to the ca, k, mg concentration and cultural technique (gago et al., 2015). rot is also one of the main problems during postharvest transport and storage. as shown in tab. 2, fruit was more susceptible to decay at the end of experiment due to aging and senescence. in addition, ambient temperature during shelf-life also favor the microbial growth (miccolis and saltveit, 1995; yang et al., 2003). conclusions precooling treatment of 7 d was applied on apple fruit ‘golden delicious’ before gaseous 1-mcp treatment, simulating commercial practice. results show that 1-mcp treatment successfully delayed fruit ripening, but differences were observed depending on the shortterm storage temperature between harvest and treatment. precooling temperature of 1 °c and 4 °c were more effective, while samples subjected to 10 °c did not respond sensitively to 1-mcp by means of ethylene production, firmness and surface color. the 1-mcp treatment should be applied as soon as possible to prevent progressed aging. in case transportation or optimization of bulk treatment make several days delay, short-term cold storage at 1 °c should be applied to maintain quality and effectiveness of 1-mcp treatment. color parameters correlated significantly with fruit firmness, ssc and ethylene production. this observation suggests that surface color can follow changes during ripening. the normalized green color component shown similar behavior to hue angle offering another parameter to use. acknowledgements the project is supported by the european union and co-financed by the european social fund (grant agreement no. efop3.6.3-vekop-16-2017-00005). the project is supported by the european structural and investment funds (grant agreement no. vekop-2.3.3-15-2017-00022). this research was supported by the ministry for innovation and technology within the framework of the higher education institutional excellence program (nkfih-1159-6/2019) in the scope of plant breeding and plant protection researches of szent istván university. conflict of interest no potential conflict of interest was reported by the authors. references blankenship, s.m., dole, j.m., 2003: 1-methylcyclopropene: a review. postharvest biol. tec. 28, 1-25. doi: 10.1016/s0925-5214(02)00246-6 bron, i.u., ribeiro, r.v., azzolini, m., jacomino, a.p., machado, e.c., 2004: chlorophyll fluorescence as a tool to evaluate the ripening of ‘golden’ papaya fruit. postharvest biol. tec. 33(2), 163-173. doi: 10.1016/j.postharvbio.2004.02.004 calvo, g., candan, a., 2010: 1-methylcyclopropene (1-mcp) affects physio logical disorders in ‘granny smith’ apples depending on maturity stage. acta hortic 857, 63-70. doi: 10.17660/actahortic.2010.857.5 delong, j.m., prange, r.k., harrison, p.a., 2004: the influence of prestorage delayed cooling on quality and disorder incidence in ‘honeycrisp’ apple fruit. postharvest biol. tec. 34, 353-358. doi: 10.1016/j.postharvbio.2004.02.009 ericsson, n.a., tahir, i.i., 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273-304, isbn 9780128022320. doi: 10.1016/b978-0-12-802232-0.00011-6 matthes, a., schmitz-eiberger, m., 2009: polyphenol content and antioxidant capacity of apple fruit: effect of cultivar and storage conditions. j. appl. bot. food qual. 82, 152-157. miccolis, v., saltveit, m.e., 1995: influence of storage period and temperature on the postharvest characteristics of six melon (cucumis melo l., inodorus group) cultivars. postharvest biol. tec. 5(3), 211-219. doi: 10.1016/0925-5214(94)00024-m milinkovic, m. lalevic, b., raicević, v., paunovic, s.m., 2018: application of 1-methylcyclopropene in fruit of five apple cultivars grown in serbia. j. appl. bot. food qual. 91, 296-303. doi: 10.5073/jabfq.2018.091.038 mir, n.a., curell, e., khan, n., whitaker, m., beaudry, r.m., 2001: harvest maturity, storage temperature, and 1-mcp application frequenhttp://dx.doi.org/10.1016/s0925-5214(02)00246-6 http://dx.doi.org/10.1016/j.postharvbio.2004.02.004 http://dx.doi.org/10.17660/actahortic.2010.857.5 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of air-stored and controlledatmosphere-stored apples. hortscience 40(7), 2096-2101. watkins, c.b., nock, j.f., weis, s.a., jayanty, s., beaudry, r.m., 2004: storage temperature, diphenylamine, and pre-storage delay effects on soft scald, soggy breakdown and bitter pit of ‘honeycrisp’ apples. postharvest biol. tec. 32, 213-221. doi: 10.1016/j.postharvbio.2003.11.003 yang, b., shiping, t., hongxia, l., jie, z., jiankang, c., yongcai, l., weiyi, z., 2003: effect of temperature on chilling injury, decay and quality of hami melon during storage. postharvest biol. tec. 29(2), 229232. doi: 10.1016/s0925-5214(03)00104-2 orcid lászló baranyai https://orcid.org/0000-0001-6177-7364 lien phuong le nguyen https://orcid.org/0000-0002-5975-9906 mai sao dam https://orcid.org/0000-0002-3170-0785 géza hitka https://orcid.org/0000-0003-0942-8102 adress of the corresponding author: e-mail: baranyai.laszlo@etk.szie.hu © the author(s) 2020. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.21273/jashs.126.5.618 http://dx.doi.org/10.1016/j.postharvbio.2003.11.003 http://dx.doi.org/10.1016/s0925-5214(03)00104-2 journal of applied botany and food quality 92, 199 203 (2019), doi:10.5073/jabfq.2019.092.028 1 institute for plant biology, tu braunschweig, braunschweig, germany 2 repha gmbh – biologische arzneimittel, langenhagen, germany modern applied botany: changes in the perception of applied botanists to themselves and others during the last century. three recent examples of the scientific potential of this field.# dirk selmar 1*, maik kleinwächter 2 (submitted: may 2, 2019; accepted: may 16, 2019) # this treatise is dedicated to böle biehl and reinhard lieberei who passed away in january and march 2019. both were outstanding scientists who had a strong positive impact on the entire field of applied botany. we greatly appreciate their innovative and successful research on post-harvest treatment of cocoa and the cultivation of other tropical crops and food plants as well as their outstanding roles as academic teachers and mentors. * corresponding author summary subsequent to a short chronicle of the history of applied research in plant biology in germany, the relevance of modern applied botany is illustrated by three relevant post-harvest processes. the metabolic reactions that play a key role in the determination of quality of the related plant-derived commodities from each are presented. increased understanding of the processes involved in these treatments has facilitated improvement of product quality in the resulting products. in each instance, it has been necessary to regard plant metabolism comprehensively and not to focus on a single physiological process. moreover, the various interactions with the environment have to be considered. these illustrations demonstrate that transfer and application of basic plant knowledge into product-related research can provide significant information that is valuable for improvement of plant-derived products. in some instances, these correlations can even account for traditional and well-established processes, as illustrated for the malting process. however, interdisciplinary work and intensive cooperation with growers and producers must be an integral part of developing feasible and economically acceptable solutions that can be transferred into practice. ultimately, the major challenge in applied botany today is the implementation of new concepts and ideas into product-related research. in consequence, modern applied botany acts as a mediator between basic plant science and industrial, product-related research. keywords: applied botany; cocoa; coffee; malting; coffea arabica; theobroma cacao; hordeum vulgare; plant-derived commodities; post-harvest treatments; product quality. historical notes within the first decades of the past century, major improvements in experimental capabilities occurred. one major result of these advancements was improved scientific knowledge of the major metabolic processes of life and metabolism. in plant science, photosynthesis was a major center of focus, but our understanding of other metabolic processes also was greatly improved. as result of these major advances, many scientists involved in basic or fundamental research began to feel that only these studies represent respectable research. as a consequence, research of colleagues involved in more applied topics, were often not considered to be of significance. this was despite the fact that their studies provided many new important insights for forestry, agriculture, and food production. in the wake of this contempt, many botanists involved in applied research felt that the german botanical society − deutsche botanische gesellschaft, which was founded in 1882 and was supposed to deal with the scientific interests of all plant biologists, no longer played that role. because of their dissatisfaction, in 1902, scientists working on applied aspects of botanical research established the society for applied botany – vereinigung für angewandte botanik. this new society gathered scientists working in forestry, horticulture, agri culture, plant protection, post-harvest management and food qua lity. to enhance their scientific communication, in 1919, an official journal for the new society, the zeitschrift für angewandte botanik, was initiated. eventually, to also make the journal accessible for non-german speakers, it was refocused as the international research organ journal of applied botany. due to partial overlap in fields of research, in 2004, the journal was extended by including the german society for quality research in plant foods − deutsche gesellschaft für qualitätsforschung. today the journal of applied botany and food quality is well established as a successful online-only and open access journal. at present, research combining fundamental and applied science is not stigmatized − quite the contrary. due to the fascinating possibilities offered by modern molecular tools, the alliance between fundamental or basic and applied research is omnipresent and the lines within the research of many are blurred. increasing inclusion of university education on the one hand, and elevated public perception of research on the other, even requires such a combination. this paradigm change is also nicely reflected by changes in university studies: the traditional university education in the sense of humboldt is substituted by bachelor and master studies in which the acade mic education is complemented − or even exchanged − by vocational training. apart from scientific considerations, focus on job training aspects requires integration of applied research in modern basic science. thus, within the last few decades, the perception of applied botanists to themselves in regard to the conception of scientists involved in fundamental research has undergone major changes. the lines between applied and fundamental research are no longer important. as a result, in 2015, the vereinigung für angewandte botanik was abandoned as independent scientific organization and integrated as the sektion für angewandte botanik in the deutsche botanische gesellschaft. applied botany − a broad array of actual research topics at the time of its inception, research positioned within the ver einigung für angewandte botanik was related to and focused on agriculture, forestry, horticulture, plant protection, post-harvest management and food quality. although much of the research was done by well-known techniques, even at this early time, quite modern approaches in respect to plant biotechnology were studied by a number of members of the society. an intriguing example for such contemporary research is given by an article from hugo fischer (1919), who outlined the usage of elevated co2 concentrations to enhance journal of applied botany 200 d. selmar, m. kleinwächter plant growth and production. fischer’s innovative work represents the basis for the modern approaches of co2 fertilization, a technique presently used in modern greenhouse technology. other than a few modern day topics, e.g., the consequences of global warming or contamination of plant derived commodities, the core areas of interest for applied botany been little altered over the last hundred years. however, the strategy − and especially the modi operandi − changed significantly. two main alterations should be noted: a shift in experimental and field work, and consideration of appropriate metabolic relationships. descriptive and empirical studies in applied botany have largely been substituted by carefully outlined experimental approaches and sound field trials. however, the approaches used are often very broad. accordingly, the required spectrum of methods is far more diverse than in most other biological disciplines. these range from highly sophisticated analytical and phytochemical methods such as lc-ms, nmr, or biochemical studies, to techniques of modern molecular biology. one major distinguishing feature of modern applied botany is the effective and successful implementation of research into praxis. in consequence, many research projects that are funded by various federal scientific organizations require recognition and approval of the practical relevance of the corresponding projects. presently, such institutional funding exhibits two major advantages: first, smalland medium-sized companies that cannot afford the costs for requisite research, get access to novel insights relevant for their production. secondly, the required fund raising does not depend on a direct sponsorship by the industry, and thus, conflicts of interest are reduced. nonetheless, to transfer research results into praxis requires combining the expertise of plant biologists and industrial researchers. this frequently is attained by cross-functional committees associated with the project that consist of the plant biologists conducting the research and industrial representatives. in this manner, the objectives have to be defined, results have to be evaluated, and corresponding solutions must be elaborated. such strategy, however, creates major drawbacks in the evaluation of concurrent research projects, e.g., dealing with the underlying scientific bases. unfortunately, a research proposal that is directed toward applied content frequently still is assessed as a “knock-out criterion” for projects submitted to research foundations that mainly support fundamental or basic research. nonetheless, apart from problems related to fund raising, research in the broad field of applied botany exhibits many advantages. in this context, the application of basic plant biology to find relevant answers and to develop problem-oriented solutions opens fascinating fields of research. to illustrate these intriguing possibilities, three interesting examples are now described. consideration of metabolic processes frequently is lacking in industrial research physiological processes influence the composition of plants and, accordingly, plant-derived commodities. as a result, these metabolic events determine product quality. as plant metabolism is significantly influenced by various exogenous factors, such as light, temperature, and water availability, metabolic processes can be – at least in part – deliberately modulated by altering these factors. unfortunately, despite the relevance of these factors for product quality of plant-derived commodities, until the present, the related physiological processes and their regulation frequently are not adequately considered. this is obvious when appropriate cultivation and post-harvest processing techniques must be optimized or when alternative approaches need to be developed. basic metabolic relationships often remain unseen when technologists and mechanical engineers not trained in botany dominate and predefine industrial research. plant scientists with required plant biological expertise as well as a broad range of methods are quite rare in industrial research and development divisions. awareness of the importance of physiological processes on product quality is frequently lacking. apparently, the flux of knowledge from basic plant sciences to industrial applied research and vice versa is quite limited and must be enhanced in the future, e.g. by increasing cross-linkages and communication platforms. in this context, modern applied plant biology, considering and focusing on fundamental metabolic processes, exhibits the potential to serve as an important mediator for transfer of basic know ledge and analytical knowhow into industrial research approaches. metabolic changes in tropical seeds during post-harvest treatments all major tropical plant-derived commodities such as coffee, cocoa, or tea, are generated by various post-harvest treatments including specific drying procedures. in this context, we have to consider that the seeds of tropical plants − in contrast to the orthodox seeds of crops grown in europe − are not dormant; but recalcitrant1. accordingly, as soon as they are exposed to favorable conditions, the seeds germinate and − as a result − metabolism is released. this normally requires removal of the fruit flesh, which is involved in suppression of germination in the fruits. the timing and manner of induction of metabolic processes, which influence the composition of the seeds in major ways, strongly impacts the quality of the plant-derived pro ducts. these relationships are seen in the post-harvest processing of the two most important tropical commodities, cocoa and coffee. cocoa fermentation traditionally, post-harvest processing of cocoa beans entails fermentation in small heaps: in the course of this procedure, the sticky, sugary pulp that surrounds the cocoa or cacao seeds, is degraded anaerobically by yeasts (for review see: amoa-awua, 2015; voigt and lieberei, 2015). after some days, the major share of the pulp is gone, and the formerly compact and solid heap is interspersed by numerous cavities and caverns. as a result, the heap is aerated, in ducing massive development of aerobic bacteria that oxidize the ethanol produced by the yeasts to yield acetic acid (biehl et al., 1985; schwan et al., 2015). consequently, the ph of the heap decreases strongly, and, due to influx of acetic acid, the so far still vital (living) cocoa seeds are killed. in the course of the nascent de compartmentation, proteases obtain access to the storage proteins, which are hydrolysed to yield large amounts of amino acids and peptides (voigt et al., 1994; voigt and lieberei, 2015). these essential aroma precursors react with reducing sugars during the roasting of the cocoa beans generating maillard products that are characteristic for typical cocoa aroma (afoakwa et al., 2008). due to tremendous increases in demand for raw cocoa, processing of that commodity has been more and more mechanized. heap fermentation has largely been replaced by fermentation in shallow boxes, which hold batches of up to one ton of material. unfortunately, the corresponding raw cocoa, especially that produced in malaysia, had a very high content of acetic acid and was weak in flavour (biehl, 1985). when considering the biological background, the solution for improving the quality was obvious: the amount of ethanol produced in the anaerobic phase of fermentation must be reduced. to achieve this goal, two main strategies were developed: bean spreading and pod storage. in the case of pod storage, the intact cocoa fruits after harvest were stored on the plantation for certain time period, usually for one week (meyer et al., 1989). as a result, due to active metabolism within the fruits, the amount of sugars, which are fermented in the anaerobic phase of fermentation, is strongly reduced (biehl et al., 1989). moreover, this preconditioning of the pulp reduced the time span required to liquefy the pulp. consequently, the liquid drains faster and aerobic conditions are attained earlier (meyer 1 according to roberts (1973), recalcitrant seeds do not undergo a maturation drying and are not capable of withstanding water loss. applied botany: three recent examples of the scientific potential of this field 201 et al., 1989). the same relationships apply for the “bean spreading” process (biehl et al., 1990). however, in this case, the fruits are opened directly after harvesting, and the seeds are extracted. instead of transferring them instantly into the fermentation boxes, the cocoa seeds are spread on drying patios for a certain time, before they are subjected to the fermentation process. both of these methods of pulp preconditioning, which were deve loped by considering the basic metabolic processes of post-harvest physiology, are now standard procedures. they significantly increase the quality of cocoas fermented in shallow boxes (amoa-awua, 2015). green coffee processing dried seeds of the coffee tree (coffea arabica l.), denoted as green coffee, represent one of the top ten commodities in global trade. traditionally, in the coffee industry, green coffee had always been considered as an inanimate plant material. consequently, all attempts to explain the characteristic differences of wetand dry-processed green coffees were premised solely on the basic chemical and physical parameters of green coffee (selmar et al., 2015). the same accounts for the approaches of the coffee producers to modify the sensory properties and to increase product quality. however, inspired by the major advances in optimization of cocoa fermentation by consideration of the metabolic events occurring in that process, ana logous approaches were initiated for green coffee processing. as is true for cocoa beans, green coffee also represents germinable seeds that lack dormancy. however, in contrast to the recalcitrant seeds of cocoa, coffee seeds are classified as intermediate seeds, because they can tolerate water loss without losing their germinability (ellis et al., 1990; radwan et al, 2014). because of these similarities, induction of germination during post-harvest processing is of particular interest (selmar et al., 2006; bytof et al., 2007). in addition, in coffee seeds, as in other living cells, any decrease in water content during drying will also induce typical drought stress responses (kramer et al., 2010). both metabolic syndromes, i.e., germination and stress, will change the composition of the affected cells, and thus of the green coffee seeds. inexplicably, these relationships had not been taken into consideration before plant biologists insistently undertook research on green coffee processing. by combining classical as well as novel and innovative approaches of modern applied botany, the causes of the well-known quality differences of dryand wet-processed green coffees could be determined: the characteristic sensory differences of wetand dry-processed coffees could be attributed to differences in metabolic activities in coffee beans during the course of the various post-harvest treatments. as predicted, in the first phases of green coffee processing, the coffee seeds do indeed germinate (kramer et al., 2010). however, water availability of the seeds which are differentially processed differs greatly. loss of water is much faster in the depulped and wet-processed seeds than in those that are still surrounded by the fruit flesh during dry processing. as a consequence, the extent and velocity of germination is greater during wet processing and the composition of the differentially-processed green coffee seeds differs. in addition, the drought stress responses also are induced differently in the wetand dry-processed coffees. these differences in stress-induced metabolism also generate significant differences in the composition of the dried coffee beans (kramer et al., 2010; kleinwächter and selmar, 2010), in particular in the content of γ-aminobutyric acid (bytof et al., 2005). as a consequence of these plant physiological insights, a paradigm change in the coffee industry was initiated. today, it is common knowledge that green coffee represents living organisms that exhibit active metabolism. that observation provides major potential for quality improvement during processing (selmar and bytof, 2006; kleinwächter and selmar, 2010). research on green coffee processing is one of the most intriguing examples in applied botany for establishing that the knowledge and conside ration of underlying basic plant physiological relationships opened new doors: deliberate alterations of the corresponding processing conditions could induce desired changes in the quality of plant-derived commodities (kleinwächter et al., 2014). as a result, new approaches have been introduced and become established in praxis − the most intriguing one is undoubtedly the successful implementation of an additional stationary storage phase in the so-called becolsub-process. becolsub represents a type of green coffee processing that is in-between classical dryand wet-processing. in analogy to wet-processing, the coffee cherries are depulped. how ever, the fermentation step, which is inherently responsible for the degradation of residues of the fruit flesh, is substituted by mechanical removal of the sticky pulp. because of its economic advantages, this procedure was favored by the coffee producers. unfortunately, the quality of the corresponding coffees was far lower than that of the wet-fermented ones. based on biochemical insight, it was postulated fig. 1: introduction of an intermediate storage phase in the becolsub green coffee processing. the removal of the pulp of coffee fruits induces seed germination. during the course of drying, the seed moisture content (m.c.) decreases from initially 50% down to 12%. when the water content falls below about 25%, the metabolic activities are shut down. whereas the related time span of metabolic activity is about two to four days in the case of classical wet processing, due to the omission of a fermentation step, it is much shorter in the case of becolsub, where mucilage is removed mechanically (for details see kleinwächter et al., 2014). an introduction of an interim storage phase enhances this time span and thereby leading to an improvement of green coffee quality 202 d. selmar, m. kleinwächter that extension of the phase exhibiting an active metabolism will solve this problem (selmar et al., 2005). accordingly, an additional sto rage step of the mechanically cleaned beans was introduced (fig. 1). indeed, as predicted, when the beans were stored for one day after pulp removal, the quality of the coffees generated by becolsub processing increased significantly. this interim storage phase has now been implemented worldwide by coffee producers. malting − an ancient biotechnological approach still lacks phy siological insight for plant biologists, it is obvious that many metabolic processes occur in living cells. unfortunately, this basic observation is frequently disregarded with respect to its significance for post-harvest treatments or food processing. indeed, in dormant seeds, e.g., cereals, metabolic processes are almost completely shut down. after imbibition, ger mination is induced and metabolic activity is regained. these co herences have been exploited for centuries in numerous malting processes. it is matter of common knowledge that germination of barley and wheat has to be induced as a precondition for starch degradation. this is necessary to produce maltose, the required product for the alcoholic fermentations by yeasts. nonetheless, there are many additional metabolic reactions that occur in the course of germination, and, thus, during the malting process. nonetheless, so far, these apparently simple relationships had not been considered adequately and comprehensively. in this context, an intriguing issue concerns the role and impact of carbon dioxide (co2). maltsters always appraised co2 as toxic, although any scientific data were lacking. it is a general maltsters̀ s rule that “this poisonous substance” has to be removed efficiently from the germinating seedlings. yet, plant phy siologists consider co2 as an inert gas for plants. accordingly, they explain the negative effect of this “toxic compound” by the lack of oxygen, which results from respiration of the seedlings in the large malting vessels. in order to clear up this apparently contrary situation and to determine the real impact of co2 during malting, the relevant physiological processes occurring in the barley seeds in the steep tanks was analyzed (kleinwächter et al., 2012). as predicted, co2 does not reveal any toxic effects for barley seeds; but, high concentrations of this gas greatly inhibit germination, and, thus, biosynthesis of the relevant enzymes. even more surprisingly, lack of oxygen was not responsible for this observed inhibition of germination. indeed, the exact mode of action is still unknown, but there may be competitive interactions between oxygen and carbon dioxide (kleinwächter et al., 2012). based on this insight, one of the major problems in industrial-scale malting, the blocking of the filters during lautering (separation of the wort from the spent grains), can be explained. this problem is especially relevant when steeping is performed in high layers today tanks with a height of 3-4 m are frequently used. inhomogeneous aeration results in massive differences in the distribution of co2 and o2. these corresponding spatial differences in the partial pressures of oxygen and carbon dioxide are responsible for large variation in the progression of germination, and, thus, in an asynchronous degradation of carbohydrates. in this context, the hydrolysis of galactomannans located in the cell wall is of special interest. insufficient degradation entails the generation of gels, which in turn, cause severe problems by blocking the filters during lautering. as predicted, comprehensive analyses of the physiological processes occurring in the barley seeds during malting verified the presence of heterogeneities, and clarified the biochemical background of this phenomenon. simple modifications in process control, e.g., changes in the extent of aeration or reduction in the height of the malt bed enable the production of malts exhibiting enhanced homogeneity (kleinwächter et al., 2014; müller et al., 2013). this example nicely demonstrates that even well-established processes can be optimized by transferring plant basic knowledge into industrial research. conclusions the three examples outlined above confirm that metabolic pro cesses during post-harvest treatments play a key role in the quality manifestation of plant-derived commodities. consequently, increased understanding of the relevant processes enables us to take steps to improve product quality. however, these striking examples also underline that it is not sufficient to focus only on one single physiological process, but that it is necessary to consider comprehensively the plant metabolism and its complexity. this is especially important in studies of various interactions with the environment. moreover, these examples demonstrate that the transfer of plant basic knowledge into product-related research should initiate strong impulses for product-related research. as noted above, this even applies to traditional and well-established processes, as is illustrated for the malting process. continuous interdisciplinary work and collaboration in project accompanying committees assists development of feasible and economically acceptable solutions that can be transferred directly into practice. apart from consideration of the basic scientific approaches, implementation of new concepts and ideas into product-related research is a great challenge. in this context, a major future goal is to optimize the related transfer of knowledge. for this reason, modern applied botany has to act as a mediator between basic plant science and industrial, product-related research. acknowledgements we wish to thank prof. dr. david seigler (university of illinois, urbana-champaign) for fruitful discussions and critical reading the manuscript. references afoakwa, e.o., paterson, a., fowler, m., ryan, a., 2008: flavor formation and character in cocoa and chocolate: a critical review. crit. rev. food sci. 48, 840-857. doi: 10.1080/10408390701719272 amoa-awua, w.k., 2015: methods of cocoa fermentation and drying. in: schwan, r.f., fleet, g.h. (eds.), cocoa and coffee fermentations, 431476. crc press. biehl, b., brunner, e., passern d., quesnel, v.c. adomako, d., 1985: acidification, proteolysis and flavour potential in fermenting cocoa beans. j. sci. food agric. 36, 583-598. doi: 10.1002/jsfa.2740360710 biehl, b., meyer, b., bin said, m., samarakoddy, r.j., 1989: chemical and physical changes in the pulp during ripening and post-harvest storage of cocoa pods. j. sci. food agric. 48, 189-208. doi: 10.1002/jsfa.2740480207 biehl, b., meyer, b., crone, g., pollmann, l., bin said, m., 1990: bean spreading: a method for pulp preconditioning to impair strong nib acidification during cocoa fermentation in malaysia. j. sci. food agric. 51, 35-45. doi: 10.1002/jsfa.2740510105 bytof, g., knopp, s.-e., kramer, d., breitenstein, b., bergervoet, j.h.w., groot, s.p.c., 2007: transient occurrence of seed germination processes during coffee post-harvest treatment. ann bot. 100(1), 61-66. doi: 10.1093/aob/mcm068 bytof, g., knopp, s.-e., schieberle, p., teutsch, i., selmar, d. 2005: influence of processing on the content of γ-aminobutyric acid in green coffee beans. eur. food res. technol. 220, 245-250. doi: 10.1007/s00217-004-1033-z ellis, r.h. hong, t.d., roberts e.h., 1990: an intermediate category of seed storage behaviour?. j. exp. bot. 41, 1167-1174. doi: 10.1093/jxb/42.5.653 fischer, h., 1919: der gegenwärtige stand der kohlensäurefrage für pflan zenkulturen. j. appl. bot. food qual. 1, 138-146. kleinwächter, m., meyer, a.-k., selmar, d., 2012: malting revisited: germination of barley (hordeum vulgare l.) is inhibited by both oxygen http://dx.doi.org/10.1080/10408390701719272 http://dx.doi.org/10.1002/jsfa.2740360710 http://dx.doi.org/10.1002/jsfa.2740480207 http://dx.doi.org/10.1002/jsfa.2740510105 http://dx.doi.org/10.1093/aob/mcm068 http://dx.doi.org/10.1007/s00217-004-1033-z http://dx.doi.org/10.1093/jxb/42.5.653 applied botany: three recent examples of the scientific potential of this field 203 deficiency and high carbon dioxide concentrations. food chem. 132, 476-481. doi: 10.1016/j.foodchem.2011.11.027 kleinwächter, m., müller, c., methner, f.-j., selmar, d., 2014: biochemical heterogeneity of malt is caused by both biological variation and differences in processing: i. individual grain analyses of biochemical parameters in differently steeped barley (hordeum vulgare l.) malts. food chem. 147, 25-33. doi: 10.1016/j.foodchem.2013.09.090 kleinwächter, m., selmar, d., 2010: influence of drying on the content of sugars in wet processed green arabica coffees. food chem. 119(2), 500-504. doi: 10.1016/j.foodchem.2009.06.048 kleinwächter, m., bytof, g. selmar, d., 2014: coffee beans and processing. in: preedy, v.r. (ed.), coffee in health and disease prevention, 73-81, elsevier. kramer, d., breitenstein, b., kleinwächter, m., selmar, d., 2010: stress metabolism in green coffee beans (coffea arabica l.): expression of dehydrins and accumulation of gaba during drying. plant cell physiol. 51(4), 546-553. doi: 10.1093/pcp/pcq019 meyer, b., biehl, b., bin said, m., samarakoddy, r.j., 1989: post-harvest pod storage: a method for pulp preconditioning to impair strong nib acidification during cocoa fermentation in malaysia. j. sci. food agric. 48, 285-304. doi: 10.1002/jsfa.2740480305 müller, c., kleinwächter, m., selmar, d., methner, f.j., 2013: the influence of elevated steeping temperatures on the resulting malt homogeneity and malt quality. brewing science. 66, 114-122. radwan, a., hara, m., kleinwächter, m., selmar, d., 2014: dehydrin expression in seeds and maturation drying − a paradigm change. plant biol. 16, 853-855. doi: 10.1111/plb.12228 roberts, e.h., 1973: predicting the storage life of seeds. seed sci. technol. 1, 499-514. schwan, r., de mello pereira, g.v., fleet, g.h., 2015: microbial activities during cocoa fermentation. in: schwan, r.f., fleet, g.h (eds.), cocoa and coffee fermentations, 129-192. crc press. selmar, d., bytof, g., 2006: green coffee is alive! a review on the metabolic processes taking place in coffee beans during processing and their implication for modern coffee research. proceedings of the asic. 21, 423-436. selmar, d., bytof, g., kleinwächter, m., 2015: metabolic responses of coffee beans during processing and their impact on coffee flavour. in: schwan, r.f., fleet, g.h. (eds.), cocoa and coffee fermentations, 431476. crc press. selmar, d., bytof, g., knopp, s.-e., bradbury, a.g.w., wilkens, j., becker, r., 2005: biochemical insights into coffee processing: quality and nature of green coffees are interconnected with an active seed metabolism. in: 20ème colloque scientifique international sur le café, 111-119. asic, paris. selmar, d., bytof, g., knopp, s.-e., breitenstein, b., 2006: germination of coffee seeds and its significance for coffee quality. plant biol. 8, 260264. doi: 10.1055/s-2006-923845 voigt, j., biehl, b., heinrichs, h., kamaruddin, s., gaim marsoner, g., hugi, a, 1994: in-vitro formation of cocoa-specific aroma precursors: aroma-related peptides generated from cocoa-seed protein by cooperation of an aspartic endoprotease and a carboxypeptidase. food chem. 49, 173-180. doi: 10.1016/0308-8146(94)90155-4 voigt, j., lieberei, r., 2015: biochemistry of cocoa fermentation. in: schwan, r.f., fleet, g.h. (eds.), cocoa and coffee fermentations, 193225. crc press. address of the corresponding author: dirk selmar, institute for plant biology, tu braunschweig, mendelssohn straße 4, 38106 braunschweig, germany e-mail: d.selmar@tu-bs.de © the author(s) 2019. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1016/j.foodchem.2011.11.027 http://dx.doi.org/10.1016/j.foodchem.2013.09.090 http://dx.doi.org/10.1016/j.foodchem.2009.06.048 http://dx.doi.org/10.1093/pcp/pcq019 http://dx.doi.org/10.1002/jsfa.2740480305 http://dx.doi.org/10.1111/plb.12228 http://dx.doi.org/10.1055/s-2006-923845 http://dx.doi.org/10.1016/0308-8146(94)90155-4 angewandte botanik. die zukunft des pflanzenschutzes in deutschland. vortrag, gehalten bei der hauptversammlung der ver einigung für angewandte botanik am 24. september 1918 in hamburg!). von prof. dr. 0. appel, geh. reg.-rat. wenn wir von der zukunft des pflanzenschutzes in deutsch land sprechen wollen, so müssen wir uns zunächst darüber klar sein, ob der pflanzenschutz für deutschland notwendig is t oder nicht. seine notwendigkeit ist schon häufig dargetan worden, d a aber immer dieselben beispiele als beweis angeführt worden sind, hat sich bei manchen die auffassung herausgebildet, daß wir einen allgemeinen pflanzenschutz gar nicht brauchen, sondern daß e s genüge, einzelne pflanzenkrankheiten z u bekämpfen. andererseits liegt eine gewisse anerkennung der notwendigkeit eines allgemeinen pflanzenschutzes darin, daß eine organisation für den pflanzen schutz, die sich über ganz deutschland ausbreitet, schon vor einer reihe von jahren geschaffen worden ist. aber diese organisation hat sich bis jetzt nicht so entwickelt, daß es möglich wäre, von einem einheitlichen und ausreichenden pflanzenschutz in deutsch land z u sprechen. immerhin ist aber durch sie das verständnis für die bedeutung der pflanzenkrankheiten und ihre bekämpfung gefördert worden und e s hat sich in weiten kreisen die über zeugung bahn gebrochen, daß wir tatsächlich eines kräftigen *) dieser vortrag is t z u einer zeit gehalten, als man den zusammenbruch deutschlands noch nicht ahnen konnte. die verhältnisse, die inzwischen ein getreten sind, zwingen uns, der landwirtschaft die allergrößte sorgfalt zu widmen, d a si e eine der hauptgrundlagen für den aufbau des neu entstehenden deutsch lands sein wird. dadurch erhält auch der pflanzenschutz eine größere bedeutung, und e s dürfen keine mittel gescheut werden, so schwer si e auch aufzubringen sind, um durch ihn unsere produktion zu sichern und zu erhöhen. 1* 4 * . o. appel, pflanzenschutzes bedürfen. außerdem muß aber auch darauf hin gewiesen werden, daß in andern ländern der pflanzenschutz große fortschritte gemacht hat und daß man anerkennen muß, ohne dabei in eine verhimmelung ausländischen wesens zu verfallen, daß er in einzelnen ländern auf dem wege ist, den unsrigen zu überflügeln. gegner eines kräftig durchgeführten pflanzenschutzes weisen darauf hin, daß die vorstellungen, die man sich über den schaden, der durch pflanzenkrankheiten verursacht wird, macht, übertrieben seien. aber diejenigen, die solche schäden am eignen hab und gut erleiden, sind darin anderer meinung und stehen auf dem standpunkt, daß schon die beseitigung oder überwindung einzelner krankheiten eine großzügige durchführung des pflanzen schutzes bezahlt macht und damit rechtfertigt. ich glaube, daß d ie notwendigkeit des pflanzenschutzes a ls voraussetzung für uns schon länger gegeben war, ich glaube aber weiter, daß wir in den kommenden zeiten, in denen wir auf alle fälle unsere landwirt schaft auf die höchste produktionsfähigkeit steigern und in der wir auch auf landwirtschaftlichem gebiet mehr wie je qualitäts arbeit leisten müssen, des pflanzenschutzes nicht entraten können. auch die weltwirtschaft hat sich schon den pflanzenschutz dienstbar gemacht, wie das die gesetze verschiedener länder für die einfuhr von pflanzen und samen zeigen, und e s is t sehr wahrscheinlich, daß man in dieser richtung weiter fort schreiten wird. eine aufgabe. des pflanzenschutzes, die schon vielfach an geregt und deren lösung versucht worden ist, ist die sogenannte statistik. gegen das wort statistik wird in dieser verbindung verschiedentlich einspruch erhoben, und ich will deshalb kurz aus führen, wie ich e s meine. ich halte es für notwendig, daß wir uns endlich einmal klar werden über die verbreitung der häufigsten pflanzenkrankheiten, die höhe der von ihnen verursachten schäden und damit über die einflüsse, die auf die stärke ihres auftretens einwirken. besonders die praktischen landwirte werden erst dann das richtige bild von der bedeutung des pflanzenschutzes bekommen, wenn ihnen a n der hand von zahlen die durch pflanzenkrankheiten verursachten schäden nachgewiesen werden. seinerzeit hat die deutsche land wirtschaftsgesellschaft berichte herausgegeben, die diesem ziele dienen sollten; aber d a weder ein geordneter pflanzenschutzdienst, noch eine leitende stelle d a war, so konnten diese berichte das die zukunft des pflanzenschutzes in deutschland. 5 gesteckte ziel nicht erreichen. so sind sie mehr zu einer sammlung gelegentlicher beobachtungen geworden als zu der grundlage, auf der man einen einigermaßen genauen überblick über diese fragen bekommen kann. auch die hauptsammelstellen für pflanzen schutz sollten in ähnlicher weise tätig sein, und die zusammen fassung ihrer in der biologischen anstalt bearbeiteten berichte über die beobachtung und bekämpfung der pflanzenkrankheiten sollten ein bild von dem auftreten der hauptsächlichsten krank heiten in den verschiedenen jahren geben. auch jetzt ist das ziel nicht erreicht worden. ich brauche nur darauf hinzuweisen, daß wir heute noch nicht genauer darüber unterrichtet sind, wo und in welcher ausdehnung z. b. der steinbrand in deutschland vor kommt. wenn man auf diesem wege eine sichere grundlage schaffen will, so wird man nicht umhin können, zunächst einmal einzelne krankheiten nach gleichmäßigen gesichtspunkten hin vor zunehmen und mehrere jahre lang nach denselben gesichtspunkten das material zu sammeln. krankheiten, die im wesentlichen von witterungseinflüssen abhängig sind und sich über bestimmte ge biete allgemein verbreiten, sind durch kartenmäßige darstellung leicht zu veranschaulichen. bei anderen, wie z. b. solchen, die durch saatgut verbreitet werden, wird man zu anderen hilfsmitteln greifen müssen. die versuche, solche erhebungen durch aussendung von fragebogen zu machen, haben keinen erfolg gehabt, weil durch die belastung der landwirte mit schriftwerk die fragebogen meist nur in ganz geringem prozentsatz und dann meist noch ungenau beantwortet werden. dagegen is t e s sehr wohl möglich, daß die organisation des pflanzenschutzes so ausgebaut und gegliedert wird, daß ihre organe die bewegungen der wichtigsten pflanzen krankheiten in ihrem gebiet dauernd verfolgen können. der nutzen solcher erhebungen und darstellungen is t ein mehrfacher. auf diese weise würde festgestellt werden können, o b einzelne krankheiten a n bestimmte gegenden gebunden sind und wie weit e s nötig ist, eine allgemeine bekämpfung durchzu führen oder die bekämpfung auf einzelne gebiete z u beschränken. es würde sich feststellen lassen, wie weit das auftreten von kli matischen faktoren abhängig ist, so daß sich die bekämpfung nach den witterungsverhältnissen richten könnte, wie das z. b . für die peronospora viticola jetzt in baden durchzuführen versucht wird. bei krankheiten, bei denen saatgut, baumschulen oder ähnliche 6 o. appel, verbreitungsorte in frage kommen, wird man in die möglichkeit versetzt, das übel an der wurzel zu fassen. ferner würden sich bei solchen erhebungen diewiderstandsfähigkeit bezw. anfälligkeit der verschiedenen sorten feststellen lassen u. a. m. ganz allgemein aber würde man endlich einmal material in die hand bekommen, um einwandfrei die höhe der schäden festzustellen und den nach weis zu führen, welche summen durch einen ungenügend durch geführten pflanzenschutz verloren gehen. das würde wieder seine rückwirkung haben auf die bereitwilligkeit, den pflanzenschutz, der bis jetzt bei uns sehr stiefväterlich behandelt worden ist, in der nötigen weise zu unterstützen.neben der dauernden beobachtung und feststellung über die verbreitung der wichtigsten krankheiten is t eine ständige über wachung notwendig, um neu auftretende krankheiten möglichst rechtzeitig aufzufinden und womöglich zu unterdrücken, solange sie noch kein größeres verbreitungsgebiet haben. daß dies bis jetzt nicht in ausreichendem maße geschehen ist, zeigt das beispiel des kartoffelkrebses, der zweifellos schon viel länger vorhanden war, als e r nachgewiesen worden ist. diese beobachtungen lassen sich mit den erstgenannten vereinigen. weiter muß aber unsere stellung zu den chemischen pflanzen schutzmitteln noch eine andere werden. bis vor dem kriege waren e s verhältnismäßig wenig pflanzenschutzmittel, die allgemeiner an gewandt wurden. in neuerer zeit mehren sich die mittel, aber wenn sie auch zum teil da und dort geprüft werden und ihre wirksamkeit in das rechte licht gerückt wird, so geschieht es doch häufig genug, daß unwirksame mittel, mindestens zeitweise, auf den markt kommen. dadurch wird aber nicht nur die an wendung wirksamer. mittel verhindert, sondern das ansehen des pflanzenschutzes im allgemeinen geschädigt. häufig genug kann man die erfahrung machen, daß landwirte, die durch solche mittel schaden erlitten haben, sich in zukunft ganz abweisend gegen den pflanzenschutz verhalten und nur schwer dazu bewegt werden können, andere mittel, selbst solche, deren wirksamkeit man ver bürgen kann, anzuwenden. es muß daher gefordert werden, daß mittel erst in den handel kommen dürfen, wenn ihre wirksamkeit einwandfrei festgestellt ist. damit würde hand in hand gehen müssen, daß die im handel befindlichen mittel ständig kontrolliert werden, denn die erste prüfung würde nur dann einen dauernden wert haben, wenn auch die zusammensetzung der mittel und vor die zukunft des pflanzenschutzes in deutschland. 7 allen dingen ihr gehalt an wirksamen bestandteilen dauernd gleich bleibt. dazu ist es nicht nötig, daß eine zentralstelle geschaffen wird, die allein die berechtigung hat, diese prüfungen vorzunehmen, sondern dies könnte sehr wohl der pflanzenschutzorganisation über tragen werden. es wäre dann allerdings nötig, daß diese organi sation enger und einheitlicher zusammenarbeitet. gewissenhafte hersteller von pflanzenschutzmitteln bringen auch jetzt ihre er zeugnisse erst in den handel, wenn sie dieselben geprüft haben. sollte eine zwangsmäßige prüfung eingeführt werden, so würde ein zeitverlust nicht eintreten, da die offizielle prüfung gleichzeitig mit den prüfungen der hersteller in angriff genommen werden könnte. schon vielfach ist mir von inhabern derartiger firmen zum ausdruck gebracht worden, daß sie die einrichtung solcher prüfungen auch ihrerseits als durchaus wünschenswert anerkennen, und daß sie darin ein wirksames mittel erblicken, unlauteren wett bewerb auf diesem gebiet auszuschalten. eine derartige maßnahme würde noch den weiteren vorteil haben, daß eine engere fühlungnahme zwischen den herstellern der pflanzenschutzmittel und den ausübenden pflanzenschutzorganen zustande kommt und daß dadurch die herausarbeitung neuer mittel und verbesserung vorhandener erleichtert wird. meistens haben die pflanzenschutzstellen nicht den überblick über die chemischen rohstoffe, daß sie ihrerseits neue mittel auffinden oder zusammen setzen könnten. andererseits sind die fabriken nicht in der lage, sich besondere pflanzenpathologen zu halten, die auf diesem gebiet ausschließlich für sie tätig sind. jedenfalls würde die zusammen arbeit einen unparteiischeren charakter annehmen, wenn die fa briken mit der pflanzenschutzorganisation gemeinsam solche fragen bearbeiteten, a ls wenn e s so bleibt, wie e s jetzt vielfach ist, näm lich daß jede gruppe bei der andern nicht das gemeinsame, sondern das trennende sieht. wie weit sich die pflanzenschutzstellen selbst mit dem ver triebe der mittel befassen sollen, ist eine frage, die sorgfältiger erwägung bedarf. in den letzten jahren hat es sich für einige mittel mehr und mehr eingebürgert, daß die pflanzenschutzstellen den vertrieb selbst übernommen haben. dies gilt besonders für den formaldehyd. zweifellos hat eine derartige maßnahme viele vorteile. für den landwirt ist es am einfachsten, wenn er sich den 8 o. appel, rat erholt, die mittel gleichzeitig da zu beziehen. außerdem liegt natürlich in einem solchen vertrieb die größte sicherheit für die zuverlässigkeit der mittel. die andere frage ist, ob nicht dem pflanzenschutz mehr gedient ist, wenn der landwirt überall, wo er sonst einkauft, auch die pflanzenschutzmittel erhält. ich er innere aber nur daran, daß früher vielfach den landwirten statt kupfervitriol eisenvitriol oder statt 40proz. formaldehydlösung 30proz. gegeben wurde. es wäre daher anzustreben, daß alle mittel in bestimmten, leicht kontrollierbaren packungen von den fabriken ausgegeben und nur so weiterverkauft werden dürfen. im engsten zusammenhang mit den chemischen mitteln stehen die apparate, die zu ihrer anwendung nötig sind. daher müssen die pflanzenschutzstellen auch mehr wie bisher für die einführung solcher apparate sorge tragen. am leichtesten wird dies möglich sein, wenn sie selbst die geeigneten apparate zur hand haben und diese zunächst, soweit es sich nicht um ortsfeste apparate handelt, verleihen. ich denke dabei an spritzen und pulver verstäuber, aber auch an transportable beizapparate u. a. m. bei dem verleihen würde die nötige bedienung mitgeschickt und da mit die gewähr für richtige anwendung gegeben werden können. die landwirte werden auf diese weise rascher an die anwendung solcher apparate gewöhnt und werden sich entweder allein oder zu genossenschaften vereinigt solche apparate anschaffen. orts feste apparate, wie z. b. größere beizanlagen, müßten, soweit es irgend tunlich ist, für die benutzung der umwohnenden landwirte zur verfügung gestellt werden. auch eine prüfungsstelle für apparate besteht noch nicht! eine solche wäre aber sehr nützlich, da sie auch an der weiteren ausgestaltung der apparate mitarbeiten könnte. aber auch den änderen bekämpfungsmitteln müßte sich der pflanzenschutz mehr wie bisher zuwenden. zu diesen mitteln rechne ich den ersatz anfälliger sorten durch weniger anfällige, den einfluß des fruchtwechsels, die bedeutung bestimmter kultur maßnahmen und die saatenanerkennung. bei der letzteren wirken heute noch die pathologen nicht genügend mit. dies gilt be sonders für die kartoffel, bei der die anerkennung mit der richtigen erkenntnis des gesundheitszustandes der felder steht und fällt. alle diese maßnahmen werden an sich die pflanzenschutz stellen mehr mit der praxis in berührung bringen, aber es is t auch die zukunft des pflanzenschutzes in deutschland. 9 heute noch notwendig, daß alle anderen mittel, die dazu dienen können, den pflanzenschutzstellen das vertrauen der landwirte zu erwerben, in bewegung gesetzt werden. auch jetzt schon is t die presse hierzu vielfach herangezogen worden. e s kann dies aber in zukunft noch mehr geschehen und zwar am wirksamsten wohl in der form, daß auf alle wichtigeren vorkommnisse durch kurze notizen in den tageszeitungen hingewiesen wird. auch das vor tragswesen kann noch mehr ausgebaut werden und gerade dieses wird sich wirksam gestalten, wenn die einzelnen pflanzenschutz stationen in möglichst enge fühlung mit den landwirtschaftlichen schulen treten und dabei das interesse der landwirtschaftslehrer fü r den pflanzenschutz anregen. leider wird ja der pflanzenschutz b e i der ausbildung der landwirtschaftslehrer sehr stiefmütterlich behandelt und die unsicherheit, die dadurch gerade in diesen kreisen vielfach herrscht, ist der ausbreitung der kenntnisse im pflanzen schutz hinderlich. erst wenn der unterricht an den hochschulen in dieser beziehung verbessert wird und zwischen den pflanzen schutzstellen und den landwirtschaftsschulen ein innigeres ver hältnis besteht, wird man auf die allgemeine mitwirkung der landwirtschaftslehrer. rechnen können. durch diese aber geht der weg zur jugend und wenn bei ihr das richtige verständnis ge weckt wird, so wird auch der einfluß auf die ältere generation damit erweitert. noch eine frage bleibt kurz zu erörtern, es ist die: wie weit sollen die inhaber von pflanzenschutzstellen sich wissenschaft lich betätigen? sie von der forschungsarbeit ausschließen z u wollen, wäre meiner ansicht nach verkehrt, denn ihre nahen be ziehungen zur praxis, die ihnen meist gebotene möglichkeit zur anstellung von versuchen, die notwendigkeit, daß sie sich stets mit der neueren literatur befassen, ihre vorbildung und der bei den meisten wohl vorhandene drang nach wissenschaftlicher be tätigung macht sie durchaus geeignet für forschungsarbeit. dem gegenüber steht allerdings vielfach der mangel a n zeit, teilweise auch a n den notwendigen einrichtungen. es wird sich daher die forschungstätigkeit der sammelstellen nicht einheitlich gestalten, vielmehr wird der eine mehr, der andere weniger sich auf diesem gebiet betätigen. wo aber eine geeignete person ist, die den willen hät, in dieser richtung zu arbeiten, sollte man ihr jede nur mögliche unterstützung angedeihen lassen, denn die noch z u 10 o. appel, lösenden fragen im pflanzenschutz sind so außerordentlich zahl reich, daß jegliche mitarbeit daran willkommen ist. um diese aufgaben zu bewältigen, ist es aber unbedingt nötig, daß wir unsere einrichtungen verbessern und ergänzen. es muß angestrebt werden, daß die organisation des pflanzen schutzes, die heute noch sehr ungleichmäßig ist, gleichmäßig durch geführt wird. es ist dabei weniger an eine vermehrung der stellen gedacht, als an eine richtige besetzung und einen festeren zu sammenschluß. ein solcher zusammenschluß is t in den beteiligten kreisen schon lange als ein dringendes bedürfnis empfunden worden und e s is t anzunehmen, daß e s gelingt, ihn durchzuführen, sobald wieder einigermaßen normale verhältnisse eingetreten sind. die besetzung der stellen ist aber abhängig von den dazu vorhandenen persönlichkeiten und dem heranwachsenden nachwuchs. letzterer bedarf unserer besonderen beachtung, d a auf ihm ein großer teil des fortschrittes beruhen wird. vergegenwärtigen wir uns, wie die lage jetzt ist. für die phytopathologen haben. wir zurzeit in deutschland weder ordent liche professuren noch irgend welches examen. wer dieses studium ergreifen will, hat zwar a n manchen orten gelegenheit, ein kleines kolleg darüber zu hören, aber eine gründliche ausbildung an universitäten und hochschulen haben wir nicht. das zeigt sich schon darin, daß kein akademischer lehrer dieser richtung schule gemacht hat. im allgemeinen wird diese richtung mehr oder weniger aus liebhaberei ergriffen und diejenigen, die sich ihr widmen, sind zum großen teil autodidakten. ihrer vorbildung nach kommen si e aus solchen berufen, z u deren grundlage die naturwissenschaften gehören, und zwar sind es entweder solche, die das studium der oberlehrer ursprünglich ergriffen hatten, landwirte, die sich nach dieser richtung hin spezialisiert haben, oder apotheker, die zur phytopathologie übergegangen sind. sie alle dringen mehr oder weniger einseitig in die phytopathologie ein, d a sie in ihrer tätigkeit als assistenten einer versuchsanstalt usw. bestimmte fragen zur bearbeitung bekommen und wenige zeit haben, sich nebenbei einen vollständigen überblick über das gesamt gebiet z u verschaffen. je nachdem ihre vorbildung eine einseitige oder vielseitige war und je nachdem sie mehr praktisch oder theoretisch veranlagt sind, werden sie auch auf ihrem neuen gebiet arbeiten. die assistenten bleiben meist in den stellungen, in die sie anfänglich eingetreten sind, und suchen in der versuchsstation, die zukunft des pflanzenschutzes in deutschland. 11 in der si e einmal sind, vorwärts z u kommen. ein häufigerer wechsel tritt im allgemeinen nicht ein. dadurch lernen sie vorwiegend nur mit den methoden arbeiten, die am ort ihrer tätigkeit üblich sind. auch das material, das si e durch d ie hand bekommen, ist mehr oder weniger einseitig, und da sie meist sehr viel technische arbeit zu leisten haben, haben sie wenig gelegenheit, ihre ausbildung auf dem gebiet der pathologie zu vertiefen und zu verallgemeinern. daß ein häufiger wechsel nicht eintritt, liegt an zweierlei, erstens daran, daß die aussichten für das vor wärtskommen im ganzen fach sehr gering sind, zum andern aber7 auch daran, daß auch den leitern der institute nicht viel a n einem häufigen wechsel gelegen ist, weil ja hilfskräfte mit all gemeiner phytopathologischer vorbildung, die ohne weiteres die arbeiten aufnehmen könnten, nicht vorhanden sind und infolge dessen der leiter die neu eintretenden immer von neuem ein arbeiten muß. aber selbst unter diesen umständen des verbleibens im gleichen institut sind die aussichten für junge phytopathologen außerordentlich schlechte. das liegt daran, daß für phytopatho logen nur wenige lebensstellungen vorhanden sind und im all gemeinen immer noch die tendenz vorherrscht, die arbeit des pflanzenschutzes nebenamtlich oder durch assistenten ausführen zu lassen. gegen die nebenamtliche ausübung ist a n sich nichts ein zuwenden, sofern die aufgaben, die erfüllt werden sollen, die dem hetreffenden zur verfügung stehende zeit nicht überschreiten. das wird aber nur dort der fall sein, wo es sich um einen kleinen aufgabenkreis handelt. auch besteht dabei immer die gefahr, daß die arbeit nicht nur nebenamtlich sondern auch nebensächlich geführt wird und daher eine den bedürfnissen entsprechende ent wicklung ausschließt. für ganz kleine bezirke wie lübeck und reuß ä . l . haben oberlehrer die funktion des pflanzenschutz dienstes mit übernommen. vielfach sind aber für größere bezirke diese arbeiten eine nebenarbeit der landwirtschaftsdirektoren und landwirtschaftslehrer, und es gehört für diese herren eine große liebe zur sache dazu, wenn sie bei der großen arbeitslast und vielseitigen inanspruchnahme diesem teil ihrer berufsgeschäfte die nötige zeit und sorgfalt angedeihen lassen. für größere bezirke is t e s ausgeschlossen, daß der pflanzenschutz in der erforderlichen weise nebenamtlich ausgeübt wird. man hat daher auch im 12 o. appel, allgemeinen in der organisation des pflanzenschutzes die landwirt schaftlichen versuchsstationen herangezogen. in einigen derselben sind auch selbständige stellen geschaffen worden, in einer größeren anzahl aber läßt man die arbeiten durch assistenten ausführen. es is t nun nicht zu leugnen, daß ein tüchtiger assistent, der längere zeit beim fach ist, eine segensreiche tätigkeit entfalten kann. im allgemeinen is t aber dieserweg nicht der richtige, weil der betreffende als fachmann nicht die nötige selbständigkeit hat und sich nicht in einer stellung befindet, die ihm als lebens stellung genügen kann, und weil e r auch gegenüber der praxis schwer die nötige autorität erlangt. durch diese verhältnisse springt ein teil derer, die sich den aufgaben zugewandt haben, wieder ab. andere erlahmen all mählich in dem wenig aussichtsreichen kampf um eine gesicherte zukunft. manchen, der zweifellos die fähigkeit hatte, tüchtiges z u leisten, habe ich in diesem kampf erlahmen sehen und habe e s immer wieder bedauert, daß dadurch so viel nützliche kräfte der sache verloren gehen. es erhebt sich daher die frage: wie sind diese verhältnisse z u ändern? zunächst muß darauf hingewirkt werden, daß die phytopathologie nicht im praktischen betriebe der versuchsstationen erlernt wird, sondern daß die, die sich ihr zuwenden, zunächst auf der universität oder hochschule eine entsprechende ausbildung erfahren. in andern ländern wie in den vereinigten staaten von nordamerika, in schweden, dänemark, holland sind schon seit längerer zeit professuren für phytopathologie vorhanden. in amerika gibt e s zahlreiche hochschulen, a n denen dozenten im hauptfach phytopathologie lehren und die vielen phytopathologen, die dort aus gebildet werden, sind über das ganze land verbreitet. bei dem praktischen sinn der amerikaner würde das nicht der fall sein, wenn man nicht d ie überzeugung hätte, daß diese einrichtungen nützlich sind. und zweifellos hat sich auch diese einrichtung bewährt, denn der phytopathologe hat in amerika einen großen anteil a n dem emporblühen der landwirtschaft. aber auch in europa liegen ähnliche verhältnisse vor. s o bestehen in holland professuren an der universität utrecht und der landwirtschaftlichen hochschule in wageningen und zurzeit geht man mit dem ge danken um, auch in amsterdam eine professur für phytopathologie zu errichten. die zukunft des pflanzenschutzes in deutschland. 13 in deutschland hatten wir schon einmal eine professur für phytopathologie, wenn sie auch nicht so hieß, an der landwirt schaftlichen hochschule zu berlin, solange frank daselbst war. heute aber wird dieser wissenszweig allgemein nur nebenamtlich gelehrt. meiner ansicht nach müssen wir, wenn wir in der phyto pathologie auf die richtige höhe kommen wollen, unbedingt sorge tragen, daß die phytopathologie wenigstens an einigen universi täten und landwirtschaftlichen hochschulen die richtige stellung erhält, denn nur so ist es möglich, daß wir das geeignete material bekommen, um unsern pflanzenschutz gleichmäßig ausbauen zu können. diese professuren für pflanzenschutz würden es außerdem mit sich bringen, daß mehr forschungsstellen wie bisher tätig sind und die zahlreichen und großen probleme dieses interessanten und wichtigen gebietes rascher wie bisher gefördert würden. außerdem würde dadurch auch im examen der landwirt schaftslehrer der pflanzenschutz eine andere stellung einnehmen wie bisher, und sind erst die landwirtschaftslehrer von derwichtig keit des pflanzenschutzes durchdrungen, so werden auch sie mehr in der praxis dafür wirken. außerdem könnte man auch in erwägung ziehen, ein examen zu schaffen, durch das die befähigung für das selbständige arbeiten im pflanzenschutz nachgewiesen wird. es wäre das etwas ähn liches, wie es lemmermann für die agrikulturchemiker verlangt”), und das von solchen abzulegen wäre, die später den pflanzenschutz als hauptberuf ergreifen wollen. analoge examina haben wir bereits in dem examen für tierzüchter, pflanzenzüchter usw. will man aber von jemandem verlangen, daß er ein solches studium ergreift und ein solches examen macht, so muß man ihm anderes bieten können wie bisher, denn um vielleicht einmal eine befriedigende stellung nach jahrelanger weiterarbeit zu finden, wird sich niemand dazu herbeilassen, besondere mühe und kosten aufzuwenden. zunächst muß die assistentenfrage einheitlich geregelt werden. früher war ein assistent ein wissenschaftler, der kurz vor dem examen stand oder es eben gemacht hatte und nun zu seiner weiterausbildung in einem institut arbeitete, und dessen arbeits *) lemmermann: zur frage der ausbildung von agrikulturchemikern und der organisation agrik.-chemischer anstalten. die landwirtschaftlichen versuchsstationen, verlag p. parey, berlin 1913. 14 o. appel, kraft der professor teilweise in anspruch nahm. dafür erhielt er eine kleine bezahlung, die ihm diese art der fortbildung erleichterte. allmählich sind aber assistentenstellen geschaffen worden, die die volle arbeitskraft eines solchen mannes in an spruch nehmen, ihm aber keine höheren bezüge gewähren. solche stellen haben nur dann eine berechtigung, wenn sie in anderer weise dem inhaber vorteile bieten. ein solcher vorteil wird viel fach darin gesehen, daß der inhaber einer solchen stelle hofft, in eine höhere stelle hineinzuwachsen. diese hoffnung erfüllt sich wohl manchmal, meist aber nicht oder nach sehr langer zeit. derartige zustände müssen aber den ganzen beruf notwendiger weise schädigen, denn es kommt doch mehr oder weniger dabei auf eine unberechtigte ausnutzung junger arbeitskräfte hinaus. daher haben sich auch allmählich die verhältnisse etwas gebessert, so daß es heute schon mehr assistentenstellen gibt, die wenigstens nicht mehr zu armselig bezahlt werden, aber gelöst ist die frage noch keineswegs. » für eine gesunde lösung der frage scheint mir folgendet weg der richtige zu sein: diejenigen studierenden der phytopathologie, die ihr studium mit dem examen abgeschlossen haben, treten in ein institut ein, in dem sie zunächst bei geringer bezahlung (sagen wir 200 m. monatlich) ein probejahr ablegen. haben sie dieses hinter sich und bleiben sie in dem institut tätig, so steigt ihr gehalt jährlich um 200 m., bis es 4000 m.!) erreicht hat. sie würden dann in einem probejahr und 8 jahren das mindestgehalt eines gymnasiallehrers einschl. des wohnungsgeldes der klasse ia erreichen und damit ein existenzminimum erhalten. vor allen dingen fiele aber das außerordentlich drückende gefühl für sie weg, daß ihre arbeit zum großen teil nicht bezahlt wird. weiter müßte aber dafür gesorgt werden, daß die assistenten stellen, die allmählich zu selbständigen stellungen geworden sind, entsprechend ausgestaltet werden, und dann muß der betreffende, mag man ihn nun abteilungsvorsteher oder mitarbeiter oder sonst wie nennen, in die stufenfolge der andern akademischen berufe eingefügt werden. es müßte dafür gesorgt werden, daß die zahl der assistentenstellen ins richtige verhältnis zu den lebens *) diese zahlen wären sinngemäß den sich neu entwickelnden verhätnissen anzupassen. die zukunft des pflanzenschutzes in deutschland. 15 stellungen gebracht wird. das läßt sich dadurch erreichen, daß die arbeiten, die eine akademische vorbildung nicht erfordern, von nicht akademischen hilfskräften (kriegsbeschädigten, damen) aus geführt werden. in der samenkontrolle ist dies ja schon zum größten teil durchgeführt. aber auch im pflanzenschutz, sowohl in den wissenschaftlichen laboratorien wie in den pflanzenschutz stellen wird es noch möglich sein, an manchem platz durch ein stellung einer hilfskraft den assistenten mehr für wissenschaftliche arbeit frei zu bekommen und dadurch seine stellung mit zu heben. besonders wo mehrere assistenten sind, wird oft die möglichkeit bestehen, durch eine derartige arbeitsteilung die mechanischen arbeiten durch billigere arbeitskräfte ausführen zu lassen und -dadurch die zahl der assistenten einzuschränken, das einkommen der vorhandenen aber zu steigern. wenn ich hier auf die wirtschaftlichen verhältnisse unseres standes etwas ausführlicher eingegangen bin, so geschah dies aus der überzeugung, daß wir eine erhöhte leistung auf dem gebiet des pflanzenschutzes nur durchführen können, wenn wir aus denen, die sich diesen wissenszweig erwählen, einen berufsstand machen, der mit andern akademischen berufen wenigstens einigermaßen den vergleich aushält. untersuchungen über den einfluß verschiedenartiger mineral düngung auf die zusammensetzung von 0bstdauerwaren. mitgeteilt von dr. j. kochs, versuchsstation für obstund gemüseverwertung an der gärtnerlehranstalt dahlem. seit verschiedenen jahren wurden dem laboratorium der obigen versuchsstation auf veranlassung des kalisyndikates g. m. b. h., agrikulturabteilung, durch die versuchsansteller proben ver schiedener obstarten von düngungsversuchen übersandt, um aus diesen fruchtsäfte oder sonstige dauerwaren herzustellen. neben den teilweise vorgenommenen qualitätsprüfungen dieser dauerwaren, angewandte botanik. 190 kleine mitteilungen. kleine mitteilungen. gips im brot. zu den beliebtesten verfälschungen des mehles gehörte in früheren zeiten der gips; man sollte meinen, daß heutzu tage eine derartig plumpe verfälschung kaum noch vorkommt, da gips zusatz doch leicht mikroskopisch wie chemisch nachweisbar ist. in unserem mit immer neuen streckungsmitteln beschwerten brot liegen die dinge allerdings nicht so einfach. brotuntersuchüngen sind an sich schwieriger als mehluntersuchungen und haben daher zu beginn des krieges, als sie vielerorts notwendig wurde, den untersuchungs ämtern häufig schwierigkeiten bereitet. seit 1914 habe ich reichlich gelegenheit gehabt, gebäcke aller art aus den verschiedensten gegenden nach eigener methode zu untersuchen, worüber ieh mehrfach (u . a . zeitschr. f. d. ges. getreidewesen, jg. 6 , nr. 10/11; jg. 7 , nr. 2 ; jg. 8, nr. 10/11 und 12; jg. 9 , nr. 2 und 78; jg. 10, nr. 1/2: jg. 11, nr. 12) berichten konnte. während der ganzen zeit is t mir aber nicht ein einziges mal gipszusatz im brot vorgekommen. kürzlich fand ich nun doch in einem aus einer berliner vorortbäckerei stammenden brot eine verfälschung mit gips, auf die ich bei der seltenheit des falles hier besonders hinweisen möchte. e s handelt sich um ein vormals angeschobenes roggenbrot, das beim durchschneiden zunächst keinerlei besonderheiten erkennen ließ. erst auf der angetrockneten schnittfläche zeigten sich einige winzige, selten bis 1 mm große körnchen, die man bei oberflächlichem hin sehen für mehlteilchen halten konnte. die mikroskopische untersuchung der körnchen ergab indessen feine kristalle von aussehen der gips kristalle; dieselben waren in verdünnter salzsäure bei zimmertemperatur schwerer, bei höherer temperatur leicht löslich; bariumchlorid ergab einen in säuren unlöslichen niederschlag. durch die quantitative ana lyse des brotes wurden sodann 4% gips in der trockensubstanz ermittelt. dr. w. herter. neue billige pilzbücher. während des krieges, als es galt, alle für die menschliche und tierische ernährung irgendwie geeigneten stoffe auszunutzen, hat auch die pilzkunde weitgehende förderung erfahren. pilzausstellungen, pilzwanderungen, pilzvorträge sind überall im reiche veranstaltet worden und haben dazu beigetragen, die kenntnis des großen schatzes a n nahrung, den wir in unseren wäldern fast völlig ungenutzt umkommen lassen, z u erweitern. s o wertvoll diese ver anstaltungen auch sein mögen, sie ersetzen nicht einen guten führer in buchform, der stets zur hand is t und den wir zu rate ziehen können, wo und wann e s uns beliebt. zur einführung recht geeignet sind kleine ganz populäre schrift chen, wie z. b . das im jahre 1917 in zweiter auflage erschienene heftchen „unsere pilze“ von k . butz (verlag bernhard kraus, schwäb. gmünd), in welchem über eine pilzwanderung und die auf derselben anzutreffenden pilze berichtet wird. in dem nur 20 seiten umfassenden schriftchen findet sich manch nützlicher ratschlag; u. a. auch im gegensatz z u der bekannten weit verbreiteten, aber durch s kleine mitteilungen. 191 nichts gerechtfertigten vorschrift über das abschneiden der pilze der rat, si e abzudrehen, nicht abzuschneiden. weniger glücklich is t die beigabe der recht mäßigen, von der reichsstelle für gemüse und obst herausgegebenen farbtafel. originell is t ein „untrüglicher ratgeber für pilzsucher“ von walther th. prym (verlag otto nemnich, münchen und leipzig, 4 7 seiten, 5 tafeln), der auf die frage: „wie erkennen wir die gift pilze“ mit einfachen regeln antwortet, die es jedem laien ermöglichen sollen, die eßbaren pilze von den giftigen zu unterscheiden. das büchlein, das zum preise von 1,85 m . angeboten wird, enthält zunächst allgemeine erläuterungen über die giftpilze, sodann eine genaue be schreibung der beiden giftigsten arten: gelblicher und grüner knollen blätterpilz und im anschluß daran die regeln: 1. iß nur, was ver lockend aussieht! koste mit der zunge! verwirf die bitter und wider lich schmeckenden pilze! 2 . mißtraue den weißblätterigen pilzen! 3 . mißtraue den röhrlingen mit rotem futter oder rot am stiele! 4 . mißtraue den stiellosen kartoffelähnlichen pilzen! ein weiteres kriegspilzbüchlein, das 1917 in erster und schon 1918 in zweiter auflage (51. bis 60. tausend) erschienen ist und nur l,50 m . kostet, ist das „taschenbuch für pilzsammler“ von ernst walther (verlag hesse u. becker, leipzig). e s bringt auf 96 seiten mit zahlreichen textfiguren und 24 farbigen tafeln eine kurze be schreibung und gute abbildung der wichtigsten speiseund giftpilze unserer deutschen wälder. einige angaben über bau und leben der pilze, gestalt der pilze, über die bedeutung der pilze im haushalte der natur und des menschen, über das sammeln, die zubereitung, die verwendung der pilze in der küche als pilzgemüse, pilzsuppe, pilz klößchen, pilzpfanne, pilzgebäck, pilzsalat, die herstellung von dauer ware, die weitere wirtschaftliche ausnutzung der pilze, über pilz vergiftungen, über pilzzucht usw. werden jedem leser erwünscht sein. das wertvollste der mir bekannten populären pilzbücher ist und bleibt der „führer für pilzfreunde“ von edmund michael, der im jahre 1896 zum ersten male erschienen ist und im jahre 1917 eine gründliche, nach dem neuesten stande der wissenschaft bearbeitete auflage erlebt hat (verlag förster u. borries, zwickau sa.). es er scheint in mehreren ausgaben. in der dreibändigen ausgabe (b) sind 345 gruppen von pilzen farbengetreu in natürlicher größe abgebildet und beschrieben. außer dieser großen ausgabe (von der jeder band 8 m . kostet) und den tafelausgaben (a und d ) is t eine billige volks ausgabe (c) erschienen (preis 2,50 m.), die anfängern sehr zu empfehlen is t. beide ausgaben sind augenblicklich – die eine im 21.–28. tausend, die andere im 101.–110. tausend– wieder im handel erhältlich. nach michael gibt es nur ein mittel gegen pilzvergiftung: genaue kenntnis d e r pilze, die nur durch vorzügliche abbildungen mit zutreffenden er läuterungen erreicht werden kann. minderwertige, schlechte abbildungen sind die größte gefahr für den pilzverbraucher, vor ihnen kann nicht dringend genug gewarnt werden. – in der großen ausgabe sind auch beachtenswerte kapitel über den nährwert der pilze – worüber neuer dings sabalitschka (ber. d. deutschen pharm. ges. 1918) ausführliche untersuchungen veröffentlicht hat – und die zubereitung, die pilz vergiftungen und die pilzzüchtung enthalten. bei seinen züchtungs versuchen des champignons lernte michael „einen bisher in keinem pilzbuch als eföbar verzeichneten pilz“, den blauen lacktrichterling, 192 kleine mitteilungen. nahrungs mittel. clitocybe laccata, als einen vorzüglichen speisepilz kennen. hierzu se i bemerkt, daß der pilz bereits in meiner bearbeitung der pilze in der kryptogamenflora der mark brandenburg (bd. 6 , heft 1 , 1910) unter dem namen russuliopsis laccata z u den „beliebten speisepilzen“ gestellt worden ist. schließlich sei noch auf die im jahre 1918 gegründete zeitschrift „der pilzund kräuterfreund“ (verlag a . henning jr., nürnberg) hin gewiesen, die monatlich erscheint und zahlreiche lesenswerte aufsätze über speiseund giftpilze enthält. besonders interessant sind in den letzten nummern dieser zeitschrift die artikel namhafter pilzkenner wie ricken, dittrich und herrfurth über die pilzvergiftungen der letzten jahre. namentlich über den pantherpilz und die morchel sind die akten noch nicht geschlossen. s o wird z. b . von michael und walther der pantherpilz, amanita pantherina, ebenso wie der perlpilz, a . rubescens, als „eßbar ohne oberhaut“, von butz und walther die morchel, morchella esculenta, als „eßbar“ (ohne weiteren zusatz) b e zeichnet, während durch diese pilze nachweislich vergiftungsfälle – für den pantherpilz (ohne oberhaut!) durch kolkwitz (verhandl. d. bot. vereins d . prov. brandenb. 59, s . 151, 1918), für die morchel (nicht die lorcheln, helvella-arten!) durch dittrich (ber. d. deutschen bot. ges. 35, s . 27, 1917) verbürgt – vorgekommen sind. hierauf müßte in neuauflagen hingewiesen werden. die neuerdings vertretene ansicht, die sich z. b . auch bei michael findet, daß die meisten pilz vergiftungen entstehen, wenn zu alte oder zu wässerige pilze ge nommen oder die pilze zu lange aufbewahrt werden, ehe sie zur ver wendung kommen, kann ich nicht teilen. die mehrzahl der vergiftungen ist wohl sicher auf frische, unverdorbene, giftige arten zurückzuführen. hat man früher, so wie man den nährwert der pilze unterschätzt hat, die giftigkeit vieler arten überschätzt, so scheint man jetzt in da s andere extrem verfallen zu wollen. in volksbüchern kann m. e . nicht genug vorsicht anempfohlen werden. herter. literatur. der reis, sein anbau, seine gewinnung, seine verwendung und seine wirtschaftliche bedeutung. da we wa-bücher nr. 1. allgemeine verlagsgesellschaft, münchen. 41 seiten mit 2 abbildungen, einer verbreitungskarte und mehreren statistischen tabellen und figuren. der verfasser des büchleins ist nicht genannt. dem titel ent spricht der inhalt nur zum teil. das werk is t mehr für den kaufmann als für den landmann geschrieben. nach sehr gedrängter schilderung der geschichte, des anbaues wie der hauptsächlich kultivierten arten werden eingehender die handelssorten, die chemische zusammensetzung des reises wie die der reisprodukte behandelt. den schluß bildet eine aufzählung der produktionswie der verbrauchsländer mit angaben ihrer anbauflächen, ihrer ausfuhr und ihrer einfuhr. meyer-hamburg (my). uc1.b3299303_page_208 uc1.b3299303_page_209_cut uc1.b3299303_page_210_cut journal of applied botany and food quality 93, 105 111 (2020), doi:10.5073/jabfq.2020.093.013 1college of life sciences, longyan university, longyan, china 2fujian provincial key laboratory of agroecological processing and safety monitoring, fujian agriculture and forestry university, fuzhou, china 3college of tea and food, wuyi university, wuyishan, china effects of tea garden soil on aroma components and related gene expression in tea leaves haibin wang1,2#, xiaoting chen1,2#, yuhua wang1, jianghua ye3, xiaoli jia3, qi zhang3, haibin he2* (submitted: august 13, 2019; accepted: april 7, 2020) * corresponding author # these authors contributed equally to this work summary in order to explore the effect of soil on the synthesis of aroma components in tea leaves, tea seedlings replanted in tea rhizosphere soil of different ages were used as research materials. tea seedlings were replanted in soils aged 0, 4, 9, and 30 years, and after one year of growth, 34, 37, 29, and 26 substances were detected in the tea leaves, respectively, using gas chromatography-mass spectrometry (gc-ms). the relative contents of terpenoids and alcohols in the tea leaves dropped from 66.40% to 44.52% and 5.21% to 2.61%, respectively, as the age of the rhizosphere soil increased. aldehydes, esters, and nitrogen compounds increased from 3.80% to 22.36%, 1.33% to 12.02%, and 3.13% to 19.96%, respectively, as the age of the rhizosphere soil increased. gene differential expression measured by fluorescence quantitative pcr (qrt-pcr) showed that the number of nerolidol synthetase and linalool synthase genes in tea leaves increased significantly, and the terpineol synthetase, phellandrene synthase, myrcene synthetase, ocimene synthase, limonene synthetase, germacrene synthase, and farnesene synthase genes declined significantly with the increase in soil age. in summary, as the number of years tea had been planted in the soil increased, the soil significantly affected the expression of terpene synthase genes in tea leaves, and then the composition and content of aroma substances in tea leaves changed. the results provide a theoretical basis for the improvement of tea quality. key word: tea tree; rhizosphere soil; aroma components; terpenoid synthases; gene expression introduction tea trees are acidophilic crops with an adequate soil ph range of 4.0-6.5 and an optimal ph value of 5.0-5.5. when the soil ph is lower than 4.0, the growth of tea trees is restricted, and the yield and quality of tea leaves decline (mehra and baker, 2007; mohammad et al., 2014). wang et al. (2018a, 2018b) investigated the acidification of tea garden soil in anxi county, china, and they found that 37.67% of the tea soil had become acidic (ph < 4.5), and 10.03% of the soils were not suitable for tea cultivation. further, their results showed that the number of continuous planting years and rhizosphere soil ph value exhibited a significant negative correlation; whereas, the yield and quality of tea and the rhizosphere soil ph showed a significant positive correlation. ye et al. (2016a, 2016b) found that the ph of tea soil decreased as planting years increased, the potential of soil autotoxicity increased, and the yield and quality of tea declined. they suggested that this phenomenon was related to the accumulation of acidic substances in the rhizosphere soil. all the results indicated that as the number of continuous planting years of tea increase, the soil becomes increasingly acidified, which could lead to a reduction in tea yield and quality. professional tea evaluators generally use sensory assessment to evaluate the quality of tea. for example, the maximum total score for oolong tea is 100 points, including 15 points for appearance, 10 points for soup color, 35 points for aroma, 30 points for taste and 10 points for leaf base (gb/t 30357.2-2013). in the evaluation index system of tea, taste and aroma account for 65% of the score, constituting the most important factor in the index. therefore, many researchers evaluate the quality of tea primarily on aroma and taste. for example, in terms of taste, the content of polyphenols, theanine, caffeine, and amino acids in tea leaves are commonly assessed to evaluate the quality of tea (jia et al., 2017, 2018). in terms of aroma, tea quality is evaluated by quantitative or qualitative analysis of aroma substances extracted by different methods. the aroma of tea mainly comes from volatile aroma substances. as a key factor of sensory quality of tea, aroma accounts for 30-35% of the score in the evaluation system of all kinds of tea (gb/t 30357.2-2013). therefore, many researchers study differences in the content of aroma substances by type of tea, processing method, and harvesting time, so as to evaluate the quality of tea leaves from an objective perspective (lv et al., 2015; kowalsicka et al., 2014; yi et al., 2015). however, there are few reports on whether planting soil affects the content of aroma substances in tea tree leaves after long-term planting of tea trees. in this study, rhizosphere soil was collected from tieguanyin tea trees planted in different years and used to replant new tea seedlings. the aroma substances in tea leaves were measured after tea trees had been replanted in order to compare aroma substances from plants grown in tea soils of different ages. in addition, fluorescent quantitative pcr was used to analyze the differential expression of the aroma sub stances synthetase gene. this research aims to lay a theoretical foundation for the improvement and promotion of tea quality. materials and methods sampling sites and processing in this study, rhizosphere soil of the tieguanyin tea cultivar was collected from the huaxiangyuan tea garden, located in zhuta village, longjuan town, anxi county, fujian province of china (n24°57'53.89'', e117°40'8.74''). tieguanyin tea trees that had been planted for 4, 9 and 30 years in this tea garden were selected, and 100 tea trees of each age were selected as a sample site, three replicates per sample. in each sample site, 15 tea trees were randomly selected to collect 15 kg of rhizosphere soil as a sample. three independent soil samples were collected from tea trees of each age. the tea rhizosphere soil was collected using the method of wang et al. (2018b) and then used to replant the tea seedlings. briefly, for each sample, leaf litter was removed from the soil surface, tea trees were dug out, and rhizosphere soil was collected from the tea tree. the control sample was nearby soil in which no tea trees (0 year) had 106 h. wang, x. chen, y. wang, j. ye, x. jia, q. zhang, h. he grown. the control soil was collected from a depth of 15-25 cm was collected and the collection was repeated three times. basic physical and chemical indexes of rhizosphere soil of tea trees with different years are shown in tab. 1. planting tea seedlings the soil sample collected as described above was dried, ground, and sifted through 40 meshes. then 8 kg of ground soil was put into a pot, and three pots were prepared for each sample. five one-year-old tea seedlings were planted in each pot. the experiment was conducted for one year from april 2017 to april 2018. during the planting process, fertilizer was applied according to the routine management of tea trees. after one year, the tea leaves with one bud and two leaves were picked for the following assessments. determination of aroma substances in tea leaves the aroma substances contained in the tea leaves obtained as described above were determined using a gas chromatography-mass spectrometer (gc-ms) with three replicates per sample. the fresh tea leaves were cut into 0.2 cm pieces; 2 g of tea leaves were placed into a sample bottle with 20 ml of headspace, and 10 ml of boiling ultrapure water was added to each bottle. then the bottles were placed in a water bath at 90 °c for 60 min and inserted immediately thereafter into a manual sampler fitted with a 50/30 μm dvd/car/ pdms extraction head. the extraction head was then immediately inserted into the gc-ms (7890a-5975c). injection port temperature was 250 °c and thermal desorption was 5 min. the chromatographic column was an elastic quartz capillary column of hp-5ms (30 m × 0.25 mm × 0.25 μm). the temperature procedure was as follows: initial column temperature was 40 °c, maintained for 5 min, and then increased to 240 °c at 5 °c/min for 60 min. the carrier gas was high purity helium, the flow rate was 1 ml/min, the ion source was ei source, the electron energy was 70 ev, the ion source temperature was 230 °c, the four-stage bar temperature was 150 ℃, and the interface temperature was 280 °c. the mass scanning range was 30-500 amu, and the solvent delay was 1 min. the substance was identified using the nist.11 library, and the relative content of substances was quantified by the peak area normalization method. the substance whose matching rate was greater than 80% was used for analysis. differential expression of terpene synthase genes in tea leaves the total rna of the tea leaves obtained as previously described was extracted using an rnaiso plus (takara) kit, and the residual dna was removed by dnase; 1% agarose gel electrophoresis was used to detect rna integrity. next, the rna was reversed into cdna by using a primescript rt reagent kit (takara). the fluorescence quantitative pcr (qrt-pcr) was performed using a transstart top green qpcr mix kit (transgen). the reaction system measured 25 μl, including 1.0 μl of template dna, 12.5 μl of sybr premix ex taq, and 1 μl each of positive and negative primers, and the remaining was ddh2o. the pcr reaction procedure was as follows: predenaturation at 94 °c for 1 min, 94 °c for 10s, 55 °c for 30s, 72 °c for 20s, repeated for 35 cycles, and fluorescence signals were collected in each cycle. after that the pcr reaction system extended at 72 °c for 5 min. five technical replicates per sample were performed. the average of the five technical replicates of each sample was used as a single measurement. the standard deviation was cal culated from the three independent samples of tea leaves. the primer of terpenoid synthase genes are shown in tab. 2 (li et al., 2014; liu et al., 2014; ma et al., 2014). the glyceraldehyde-3-phosphate dehydrogenase gene (csgapdh) was used as an internal gene. the segments of product size was 206 bp, the expression quantity of all terpenoid synthase genes were calculated by the method of 2-ääct. data analysis excel was used for data classification and percentage calculation analysis. statistical analyses for comparing the average results of the different samples were performed using a one-way analysis of varitab. 1: physico-chemical properties of tea rhizosphere soil of different ages planting total n total p total k available n available p available k years (g/kg) (g/kg) (g/kg) (mg/kg) (mg/kg) (mg/kg) 0 2.63±0.08 1.37±0.11 1.72±0.04 27.2±1.23 79.3±2.57 305.2±4.85 4 2.58±0.12 1.29±0.07 1.64±0.07 29.3±1.54 87.4±3.62 312.1±3.97 9 2.47±0.06 1.21±0.05 1.71±0.05 28.1±1.29 88.5±2.84 320.2±3.26 30 2.49±0.09 1.24±0.13 1.69±0.08 28.7±1.38 89.2±2.16 324.5±2.53 tab. 2: the qrt-pcr primers of terpenoid synthase genes in tea tree leaves gene name primer sequence product size (bp) 3-hydroxy-3-methylglutaryl coenzyme a reductase 5-ctcttcctcctcctcctcct-3 5-cctttgtgcccttggatagt-3 200 farnesene synthase 5-taatgctctattctttaggctcc-3 5-tgaattggtgatattcttcagaat-3 226 germacrene synthase 5-agtgagaaagatgttagtggcag-3 5-tccttgttgtctaagtaaccgaa-3 217 limonene synthase 5-aacactacctttggtgctctg-3 5-aggcatgtccttatattgtgtg-3 211 ocimene synthase 5-cttctgttcagttggaatggtc-3 5-gaagcatagtttcaggcagtct-3 205 myrcene synthase 5-acgatgggaatttcaaacc-3 5-agacctagtcatttgccattgt-3 233 phellandrene synthase 5-ctaaaaatctcaaagaaaacctca-3 5-ttcagatcttcttggtaagttgtt-3 220 terpineol synthase 5-atctgcgaactaccaacctcc-3 5-ccttgaagtgatagaacactcca-3 209 linalool synthase 5-cagcacaaacgaaatttcct-3 5-cattccatgacccaagagaa-3 226 nerolidol synthase 5-attcttaaaatggacgggct-3 5-tgaggacatcttcgaacaag-3 226 csgapdh (internal gene)* 5-ttggcatcgttgagggtct-3 5-cagtgggaacacggaaagc-3 206 * csgapdh – glyceraldehyde-3-phosphate dehydrogenase gene as internal gene. soil affect on the synthesis of tea tree aroma substances 107 ance (anova) followed by scheffe’s test for multiple comparisons. significance and correlation analysis were performed using spss software package 11.0, and principal component analysis was performed using dps software package 7.05 results and discussion analysis of aroma components in tea leaves the aroma substance analysis detected 34, 37, 29, and 26 substances in tea leaves from tea seedlings replanted in soils aged 0, 4, 9, and 30 years, respectively (tab. 3). the substances detected can be divided into the following six types: terpenoids, alcohols, aldehydes, ketones, esters, and nitrogen compounds. the number of terpenoids and ketones exceeded the number of other types of aroma substances. in leaves of tea seedlings replanted in 0-, 4-, 9-, and 30-year-old soils, the proportions of terpenoids were 52.94%, 56.76%, 58.62%, and 53.85%, respectively, and the proportions of ketones were 17.65%, 13.51%, 10.34%, and 15.38%, respectively (fig. 1). aroma substances are one of the key indicators in tea quality evaluation, and they come from secondary metabolites of tea trees, which have attracted the attention of many researchers in recent years. wan and xia (2015) found that the main aroma substances of tea leaves were terpenes, alcohols, phenols, ketones, acids, esters and heterocyclic. ma et al. (2014) used hs-spme (headspace solid-phase microextraction) combined with gc-ms to analyze and compare the aroma components between conventional treatment and freezing treatment of dangui varieties of wuyi rock tea. they established the hs-spme–gc-fid detection method with nerolidol as an evaluation index and then analyzed the changes of nerolidol content in three grades of oolong tea during processing. kowalsicka et al. (2014) studied the aroma substances of 201 spring tea samples and 196 postseason tea samples, finding that 59 aroma substances were unique substances, which were mainly composed of oxygenated monoterpene. our results indicate that the relative contents of terpenoids and alcohols in tea leaves decreases with increasing age of tea rhizosphere soil (tab. 3). when the soil age was 0 years, the relative contents of terpenoids and alcohols were the greatest, measuring 66.40% and 13.09%, respectively. in contrast, the relative contents of aldehydes, esters and nitrogen compounds increased with increasing age of the soil. when the soil age was 30 years, these compounds had the largest presence, measuring 22.36%, 12.02% and 16.96%, respectively. the results indicate that tea soil age could significantly affect the composition and content of aroma substances in tea leaves, especially terpenoids. principal component analysis of aroma substances in tea leaves the results of the principal component analysis (pca) showed that the aroma substances in tea leaves could be divided into three major components (pc1, pc2, and pc3), and the contribution rates of the three principal components were 80.44%, 12.39%, and 7.17%, respectively (fig. 2). the tea leaves sampled from the 0-, 4-, 9-, and 30-year-old soils could be effectively divided into different areas. the correlation analysis results showed that 29 substances were significantly correlated with pc1 (with 80.44% contribution rate; tab. 4), 16 of the 29 substances were significantly positively corre lated, and 13 of the 16 substances were terpenoids. the results showed that the proportion of positively correlated terpenoids accounting for 81.25%. furthermore, 13 of the 29 substances were significantly negatively correlated, and 6 of the 13 substances were terpenoids, accounting for 46.15%. the results suggest that aroma substances could effectively be distinguished from the four different samples, and terpenoids play a major role in the process of differentiation. fig. 1: proportion of each type number to total number of detected sub stances in hot-water extract of tea leaves. -3 -2 -1 0 1 2 3 -10 -5 0 5 10 -3 -2 -1 0 1 2 3 -10 -5 0 5 10 principal component 1 (80.44%) pr in ci pa l c om po ne nt 2 (1 2. 39 % ) pr in ci pa l c om po ne nt 3 (7 .1 7% ) principal component 1 (80.44%) fig. 2: principal component analysis of aroma substances in tea leaves. ▲: 0 a, : 4 a, ■: 9 a, : 30 a. fig. 1: proportion of each type number to total number of detected substances in hot-water extract of tea leaves. 0 25 50 75 100 0 a 4 a 9 a 30 a pr op or tio n (% ) t erpenoid ket one alcohol aldehyde est er nit rogen compound 108 h. wang, x. chen, y. wang, j. ye, x. jia, q. zhang, h. he tab. 3: analysis of aromatic substances in tea leaves by gc-ms classification retention time compound molecular formula relative content of aromatic substances in tea leaves (p/%) (min) 0 a 4 a 9 a 30 a 11.395 2,6-dimethyl-2-trans-6-octadiene c10h18 2.443±0.105 0.661±0.033 nd nd 14.456 α-terpinene c10h16 1.386±0.028 0.996±0.042 nd 0.214±0.035 14.463 β-myrcene c10h16 nd 1.125±0.038 nd 0.394±0.037 15.162 d-limonene c10h16 2.963±0.125 1.633±0.026 1.486±0.034 0.398±0.026 18.809 o-cymene c10h14 2.747±0.131 1.242±0.084 0.708±0.048 nd 19.121 α-ocimene c10h16 1.950±0.087 1.106±0.045 0.616±0.028 nd 23.928 2,4,6-octatriene, 2,6-dimethyl-, (e,z)c10h16 2.298±0.142 0.451±0.082 nd nd 27.144 1,3-cyclohexadiene, 1,5,5,6-tetramethylc10h16 0.383±0.035 0.332±0.042 nd nd 29.239 isodurene c20h20 0.749±0.021 0.682±0.054 0.504±0.047 0.434±0.039 30.844 4-carene c10h16 0.011±0.009 0.694±0.025 0.782±0.028 1.582±0.023 46.132 β-ocimene c10h16 0.598±0.054 1.384±0.047 1.635±0.083 2.550±0.052 19.136 terpinen-4-ol c10h18o 1.143±0.058 1.102±0.032 1.007±0.041 nd 32.314 linalool c10h18o nd 0.827±0.024 0.969±0.032 1.773±0.042 35.048 β-cyclocitral c10h16o 0.986±0.037 1.193±0.081 1.326±0.067 1.528±0.057 50.336 carvenone c10h16o 1.849±0.036 0.717±0.027 0.673±0.032 nd 52.483 l-menthol c10h20o 2.037±0.128 1.296±0.018 0.408±0.032 0.317±0.042 31.141 α-ionone c13h20o 0.559±0.038 0.606±0.042 1.107±0.038 2.909±0.037 37.864 2-undecanone, 6,10-dimethylc13h26o 1.897±0.107 1.069±0.076 0.866±0.046 0.610±0.017 43.925 5,9-undecadien-2-one, 6,10-dimethylc13h22o 3.592±0.083 3.072±0.054 1.498±0.057 0.898±0.038 48.063 nerolidol c15h26o nd nd 0.108±0.035 0.431±0.051 49.415 phytone c18h36o nd 0.420±0.032 0.636±0.054 1.941±0.064 51.406 isophytol c20h40o 0.631±0.048 0.359±0.025 0.131±0.018 nd 21.357 cyclohexanone, 2,2,6-trimethyl c9h16o 0.439±0.042 0.327±0.016 0.538±0.052 nd 22.850 5-hepten-2-one, 6-methylc8h14o 0.643±0.025 0.534±0.038 0.220±0.052 0.114±0.018 34.394 6-methyl-3,5-heptadiene-2-one c8h12o nd 0.272±0.029 nd 1.572±0.028 36.400 acetophenone c8h8o 0.427±0.043 0.184±0.036 nd 0.054±0.004 38.123 2,6,6-trimethyl-2-cyclohexene-1,4-dione c9h12o2 1.610±0.058 nd nd nd 41.192 ethanone, 1-(2-methylphenyl)c9h10o 1.683±0.104 0.890±0.053 0.806±0.041 0.794±0.049 42.967 1-(4-tert-butylphenyl)propan-2-one c13h18o 0.761±0.024 nd nd nd 44.698 benzyl alcohol c7h8o nd 0.177±0.018 0.238±0.029 0.938±0.058 45.612 phenylethyl alcohol c8h10o 0.436±0.035 0.401±0.023 nd nd 49.913 1,3-benzenediol, 5-pentylc11h16o2 1.778±0.132 0.604±0.041 0.393±0.027 nd 30.012 2,4-heptadienal, (e,e)c7h10o 0.231±0.027 0.399±0.031 0.776±0.047 4.376±0.053 33.451 2-furancarboxaldehyde, 5-methylc6h6o2 1.386±0.082 2.529±0.026 3.201±0.043 2.282±0.046 44.178 2-propenal, 3-(2-furanyl)c7h6o2 nd nd 0.308±0.038 0.401±0.042 45.225 benzaldehyde, 2,4,5-trimethylc10h12o nd nd 0.379±0.045 0.968±0.058 41.080 methyl salicylate c8h8o3 1.127±0.035 1.938±0.047 2.194±0.045 4.313±0.053 50.567 hexadecanoic acid, methyl ester c17h34o2 0.202±0.023 0.209±0.028 nd nd 15.422 1-ethylpyrrole c6h9n 2.537±0.039 0.937±0.039 nd nd 35.947 benzenamine, 4-methoxy-2-methylc8h11no 0.659±0.054 1.577±0.039 2.238±0.061 2.806±0.032 53.330 indole c8h7n 0.225±0.041 0.108±0.028 nd nd 53.858 1h-indole, 3-methylc9h9n 0.138±0.021 0.186±0.032 0.274±0.048 1.296±0.053 note: nd: not detected. means standard error (± se) from three replications for each sample is shown. analysis of the differential expression of terpenoid synthase genes in tea leaves the synthesis of terpenoid substances was closely related to the gene expression of key enzymes in the terpenoid metabolic pathway. according to previous reports, the isoprene pyrophosphate and its isomer come from the terpenoid precursor synthesis pathway and could be catalyzed by isoamyl alkenyl transferase and produced geranyl pyrophosphate and faranyl pyrophosphate. these products could be catalyzed by monoterpene and sesquiterpene synthases to produce volatile monoterpenes and sesquiterpenes (degenhardt et al., 2009; aharoni et al., 2003; xiang et al., 2013). the results of this study (fig. 3) show that the expression of nerolidol synthase and linalool synthase increased with increasing soil age. with comparison to the internal reference gene, the two genes of tea leaves corresponding to 0and 30-year-old soils showed that nerolidol synthase gene was upregulated 3.86 and 6.21 times, and linalool synthase was up-regulated 2.02 and 5.18 times, respectively. the expression of terpineol synthase, phellandrene synthase, myrcene synthase, ocimene synthase, limonene synthase, germacrene synthase and farnesene synthase decreased with the increase in soil planting years. corresponding to terpenoids ketones alcohols aldehydes esters nitrogen compound soil affect on the synthesis of tea tree aroma substances 109 tab. 4: correlation analysis of different compounds and principal component 1 compound r compound r 2,6-dimethyl-2-trans-6-octadiene 0.91* 2,4,6-octatriene, 2,6-dimethyl-, (e,z) 0.89* 1,3-cyclohexadiene, 1,5,5,6-tetramethyl 0.90* 5-hepten-2-one, 6-methyl 0.96** 2-undecanone, 6,10-dimethyl 0.96** 5,9-undecadien-2-one, 6,10-dimethyl 0.97** acetophenone 0.94* phenylethyl alcohol 0.89* d-limonene 0.98** α-ocimene 1.00** pinolene 0.88* isodurene 0.97** carvenone 0.96** l-menthol 0.96** 1,3-benzenediol, 5-pentyl 0.96* isophytol 0.99** phytone -0.93* 2-propenal, 3-(2-furanyl)-0.91* benzaldehyde, 2,4,5-trimethyl-0.91* linalool -0.98** benzenamine, 4-methoxy-2-methyl-1.00** β-ocimene -0.99** 4-carene -0.97** 1-ethylpyrrole 0.93* o-cymene 0.98** methyl salicylate -0.93* indole 0.95* benzyl alcohol -0.89* β-cyclocitral -1.00** *: significant correlation at the 0.05 level; **: significant correlation at the 0.01 level. fig. 3: analysis of differential expression of terpene synthase genes in tea leaves. the bars represent standard errors of the mean (n = 3). different letters indicate significant differences at p < 0.05 among soils of different ages. fig. 3: analysis of differential expression of terpene synthase genes in tea leaves. the bars represent standard errors of the mean (n = 3). different letters indicate significant differences at p < 0.05 among soils of different ages. conclusion volatile terpenoids play an important role in the formation of tea aroma quality and are often used to evaluate the quality of tea (owuor, 1992; zhu et al., 2017). ravichandran (2002) used terpenoid content to evaluate the aroma quality of tea and found that the higher the terpenoid content, the better the aroma quality of tea. in this study, we found that as soil age increased, the expression of terpene synthase genes in tea leaves was down-regulated, and the content of terpene aroma substances in tea relative expression level nerolidol synthase linalool synthase terpineol synthase phellandrene synthase myrcene synthase ocimene synthase limonene synthase germacrene synthase farnesene synthase 3-hydroxy-3-methylglutaryl coenzyme a reductase a a a a a a a a d d a b b b b b b c c b b b c c c c c c c a a a a d d d d d d d 0 5 10 15 30 a 9 a 4 a 0 a 110 h. wang, x. chen, y. wang, j. ye, x. jia, q. zhang, h. he 0and 30-year-old soils, they were showed that terpineol synthase gene was up-regulated 2.39 and 0.58 times, phellandrene synthase gene was up-regulated 4.27 and 1.05 times, myrcene synthase gene was up-regulated 11.05 and 6.23 times, ocimene synthase gene was up-regulated 13.49 and 5.23 times, limonene synthase gene was up regulated 10.26 and 3.18 times, germacrene synthase gene was up regulated 3.25 and 0.83 times, and farnesene synthase gene was upregulated 5.27 and 3.04 times, respectively, compared to the internal reference gene. no significant difference in expression of 3-hydroxy3-methylglutaryl coenzyme a reductase (fig. 3) was observed. the expression of terpene metabolic genes in tea leaves showed significant differences among tea seedlings transplanted into soil of dif ferent ages. among 10 terpene synthase genes, 2 genes showed significant upward trends as soil age increased, 7 genes showed significant downward trends, and only 1 gene showed no significant difference in expression. the amount of terpenoids produced was related to the expression intensity of terpenoid metabolic pathway genes. the higher the expression of terpenoid metabolic genes, the more terpenoids were synthesized, and the opposite was also observed (keeling and bohlmann, 2006). yahyaa et al. (2015) found that increased expression levels of terpene synthase, sesquiterpene synthase, and monoterpene synthase genes in carrot tissues could promote increased terpenoid content in carrot tissue. in our study, it was observed that with increased tea tree planting years, soil could significantly affect expression of terpene synthase genes in tea leaves. the expression of most terpene synthase genes decreased significantly as the age of the soil increased, leading to the reduction of terpene content in tea leaves. conclusion volatile terpenoids play an important role in the formation of tea aroma quality and are often used to evaluate the quality of tea (owuor, 1992; zhu et al., 2017). ravichandran (2002) used terpenoid content to evaluate the aroma quality of tea and found that the higher the terpenoid content, the better the aroma quality of tea. in this study, we found that as soil age increased, the expression of terpene synthase genes in tea leaves was down-regulated, and the content of terpene aroma substances in tea leaves decreased, which led to a decline in tea quality. in addition, some non-terpene aroma substances have been identified, and the effects of these substances on tea quality and their contributions needs to be further studied. these results provide a basis for further improvement of tea quality. acknowledgments this work was supported by china postdoctoral science foundation (2016m600493), national 948 project (2014-z36), natural science foundation of fujian province (2017j05057), science & technology project of longyan city (2017ly71), the project of scientific re search of young and middle-aged teachers, fujian province (jat170573), fujian outstanding research talent cultivation pro ject, national program for innovation and entrepreneurship training for college students (201911312001), longyan college youth top talent training program (2019jz19). conflict of interest no potential conflict of interest was reported by the authors. references aharoni, a., giri, a.p., deuerlein, s., griepink, f., 2003: terpenoid meta bolism in wild-type and transgenic arabidopsis plants. plant cell, 5(12), 2866-2884. doi: 10.1105/tpc.016253 degenhardt, j., köllner, t.g., gershenzon, j., 2009: monoterpene and sesquiterpene synthases and the origin of terpene skeletal diversity in plants. phytochemistry, 70, 1621-1637. doi: 10.1016/j.phytochem.2009.07.030 gb/t 30357.2-2013, 2013: onlong tea – part 2: tieguanyin. the national standards of the people’s republic of china. jia, x.l., ye, j.h., wang, h.b., li, l., wang, f.q., zhang, q., chen, j.b., zheng, x.y., he, h.b., 2018: characteristic amino acids in tea leaves as quality indicator for the evaluation of wuyi rock tea in different culturing regions. j. appl. bot. food qual. 91, 187-193. doi: 10.5073/10.5073/jabfq.2018.091.025 jia, x.l., ye, j.h., zhang, q., li, l., hu, y.l., zheng, m.z., hong, y.c., wang, f.q., wu, c.z., 2017: soil toxicity and microbial community structure of wuyi rock tea plantation. allelopathy j. 41(1), 113-126. doi: 10.26651/2017-41-1-1088 keeling, c.i., bohlmann, j., 2006: genes, enzymes and chemicals of terpenoid diversity in the constitutive and induced defence of conifers against insects and pathogens. new phytol. 170, 657-675. doi: 10.1111/j.1469-8137.2006.01716.x kowalsicka, a., kfoury, n., jr, r.a., ahmed, s., orians, c., griffin, t., cash, s.b., stepp, j.r., 2014: metabolite profiling of camellia sinensis by automated sequential, multidimensional gas chromatography/ mass spectrometry reveals strong monsoon effects on tea constituents. j. chromatogr a, 130(130), 230-239. doi: 10.1016/j.chroma.2014.10.058 li, y.h., lu, j.l., fan, f.y., shi, y.t., 2014: gene cloning and expression analysis of hmgr in tea plant roots. j. tea sci. 34(6), 583-590. 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hierarchical cluster analysis. lwt food sci. © the author(s) 2020. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). technol. 62, 194-201. doi: 10.1016/j.lwt.2015.01.003 zhu, y., shao, c.y., lv, h.p., zhang, y., dai, w.d., guo, l., tan, j.f., peng, q.h., lin, z., 2017: enantiomeric and quantitative analysis of volatile terpenoids in different teas (camellia sinensis). j. chromatogr. a, 1490, 177-190. doi: 10.1016/j.chroma.2017.02.013 address of the corresponding author: haibin he, fujian provincial key laboratory of agroecological processing and safety monitoring, fujian agriculture and forestry university, fuzhou, 350002 china e-mail: alexhhb@163.com http://dx.doi.org/10.1021/acs.jafc.5b00546 http://dx.doi.org/10.1007/s11738-016-2216-5 http://dx.doi.org/10.1016/j.lwt.2015.01.003 http://dx.doi.org/10.1016/j.chroma.2017.02.013 art11_shi.indd journal of applied botany and food quality 85, 75 78 (2012) faculty of forestry resources, southwest forestry university, kunming, yunnan, p.r. china infl uence of different fruit loads on the starch accumulation in the pistils of chinese chestnut cv. ‘zaodali’ z. shi, r. he (received january 3, 2011) summary the experimental samples were collected from chinese chestnut trees (castanea mollissima blume) of two different fruit loads, i.e., “high fruit load” and “low fruit load”. no starch accumulation in the needle-shaped stigmas of all female fl owers was observed while rapid starch accumulation took place in the transmitting tissues of the style just before the formation of complete ovules. in the cells of the ovary wall and the ovule, starch accumulation was obvious and reached the peak as the embryo sac was fully mature. starch grains of ovule were mainly stored in the inner and outer integuments. fruit loads of chestnut trees greatly affected level of starch accumulation in each part of the pistil. the starch accumulation in the ovary wall and the ovule of trees of “high fruit load” apparently surpassed that of trees of “low fruit load”. the starch content in the outer integument could be of 17.50% for trees of “high fruit load” compared to a low percentage of 2.92% for trees of “low fruit load” in same time. introduction chinese chestnut (castanea mollissima blume) is an important crop in yunnan province, southwestern china, but low yields and alternate-bearing affect the production development. the main reasons for low yield are the high empty cupule rate and fewer than 3 nuts per cupula at harvest. the rate of empty cupules in many chestnut orchards is usually 10 to 20%, but sometimes it can be as high as 90% (bai, 1988; xia et al., 1989; shi, 2003; shi and stösser, 2005). starch is the most important carbohydrate reserve in woody plants species (chapin et al., 1990). according to previous studies, the accelerated growth of pollen tube when it entered into the transmitting tissues through the stigma was related to the change from autotrophy to heterotrophy, which means that the growth of pollen tube relied on the carbohydrate reserve in pistil for vegetative nutrition (herrero and dickinso, 1980; mulcahy and mulcahy, 1983). it was proved that starch accumulation existed in both the stigma and the transmitting tissues in apple pistil (braun et al., 1986). as for peach trees, starch accumulation occurred in embryo sacs, integuments and the top of nucellus from anthesis to fertilization until the maturity of ovule (arbeloa and herrero, 1991). during fertilization, many large starch grains were observed in the integuments (mogensen and suthar, 1979). accumulation and consumption of starch in the reproductive organs of fruit trees was of great signifi cance for the pollen tube growth and the fertilization process as well as the stability of fruiting (stösser, 2002). the above cited data (mogensen and suthar, 1979; mogensen and suthar, 1979; stösser, 2002) indicate that starch accumulation in the fl ower is critical for the fruiting process of fruit trees. the purpose of this paper is to determine if the levels of starch accumulation in pistils of the chinese chestnut during the time of anthesis to fertilization could be correlated with the fruit set ability and thereby it would substantiate the delusive female fl ower quality concept. materials and methods material trees of the cultivars ‘zaodali’ were selected for the study between 2005 and 2006 at the yiliang county experimental station in yunnan province, southwest china. three “high fruit load” (hfl) and three “low fruit load” (lfl) trees planted in 1998 were selected. the “hfl” trees in 2004 and 2005 yielded less than 5% empty cupules, and the “lfl” trees yield 30% to 40% empty cupules during the same period. from each tree, three female catkins (cupules) per tree were sampled when the stigmas appeared in late april. thereafter, sampling was done every 7 or 8 days until the end of may. the samples were immediately fi xed in a solution fpa70 (containing 40% formaldehyde : propionic acid : 70% ethanol = 5: 5: 90 [v/v/ v]). methods the sampled tissues were dehydrated with an ethanol series (70%, 80%, 90%, 96%), at least 4 hours in each step, and the dehydrated samples embedded in glycolmethacrylate-methyl methacrylate (rudell, 1967). after dehydration, the tissues were transferred to 50:50 (v/v) mixture of methacrylic-acid-hydroxyethyl ester and methyl methacrylate and stored for 24 hours at 4 °c. they were then transferred to a mixture containing 60ml methacrylic acidhydroxyethyl ester, 20ml methyl methacrylate, 16ml ethylene glycol monobutyl ether, 2ml polyethylene glycol 400 (serva), and 270mg benzoyl peroxide (solution a), and stored for 24 hours at 4 °c. polymerization was achieved by transferring the tissue to the same amount of solution a and by adding 100µl n,n-dimethylaniline (solution b). all reagents were obtained from merck, darmstadt, germany. sections (5µm thick) were obtained using a reichert-jung 2050 microtome (leica, bensheim/germany), and sections were stained with the lugol’s solution and tested for starch. starch content of the ovary and ovule cells was determined by percentage starch (grains) coverage over the total cell area, and three measurements were done in each typical area. the image analyzing system consisted of a microscope (axioskop, fa. zeiss) and a digital camera (axioskop, fa. zeiss) linked to a computer. the analyzing software of the firma sis (soft imaging system) was used (lai-dihn and stösser, 2004). the paraffi n embedding method (shi, 2003) was used to observe the development of female gametophyte. statistical signifi cance of starch content (%) for the pistil between the fruit loads was analyzed by using the software program sas, version 6.12 (cary, usa), with p<0.05. results starch accumulation in the stigma and style the female fl owers of chestnut have a needle-shaped stigma and an ovary with 6 to 9 locules (shi, 2003). no starch accumulated in the stigmas from the emergence of the cupula to the formation of the embryo (fig. 1a). however, once the integument primordium appeared, starch grains appeared mainly in the transmitting tissues 76 z. shi, r. he starch accumulation in the pistils of chinese chestnut fig. 3: starch accumulation in the ovule (vertical section): a) ovule of tree of lfl (on may 1st, 2006), an arrow (→) is indicating a ovule, b) ovule of tree of lfl ((on may 8th, 2006), c) ovule of tree of hfl (sampled on may 16th, 2006), an arrow (→) is indicating a starch grain, d) ovule of tree of hfl (on may 24th, 2006). fig. 1: starch accumulation in the ovary: a) the needle-shaped stigma of hfl trees (may 8th, 2006), b) cells in the style of lfl trees (may 1st, 2006), c) cells at the ovary bottom of lfl trees (may 1st, 2006). an arrow (→) is indicating a starch grain. fig. 2: starch accumulation in the tissues of the ovary wall etc. (vertical section): a) ovary wall cells of trees of lfl (on may 1st, 2006), b) ovary wall cells of trees of lfl (on may 8th, 2006), c) ovary wall cells of trees of hfl (on may 16 th, 2006), d) ovary wall cells of trees of hfl (on 24 may 24th, 2006). starch accumulation in the pistils of chinese chestnut 77 of the style (fig. 1b). these disappeared after the completion of ovule formation or during the formation of megaspore mother cell, with obvious starch grains only apparent in the ovary tissue cells connecting the cupula (fig. 1c). starch accumulation in the ovary wall starch grains began to accumulate in a few cells of the ovary walls when the ovule was club-shaped in late april (fig. 2a). these then grew when the integument primordium appeared. they were accumulated remained in the ovary wall (especially in the outer wall) as the ovule and the female gametophyte developed (figs. 2b, c, d and tab. 1). for “hfl” trees, starch content increased to a maximum value of 2.90% on may 16th when the embryo sac was fully matured, and then decreased to 2.30% on may 24th. in contrast, the corresponding starch contents for “lfl” were 0.60% on may 24th (tab. 1). starch accumulation in the ovule when the integument primordium emerged, a few starch grains were observed only at the chalaza (fig. 1a). in the “lfl” trees this occurred on may 1st. when the ovule was fully developed, starch accumulated in both the inner and outer integuments (fig. 3b and tab. 2). when the embryo sac was mature, the starch content was a maximum value of 17.5% in the outer integument and 4.9% in the inner integument of the “hfl” trees (fig. 3c and tab. 2). no starch accumulated in the nucellus cells (figs. 3b and 3c). the starch level in the ovule decreased after fertilization (fig. 3d and tab. 2). infl uence of different fruit loads on starch accumulation before the integument primordium developed, there had been few differences in the accumulation of starch in pistil of trees with different fruit loads. however, later stages of growth were more responsive. the levels of starch in the ovary wall and the ovule of the “hfl” trees were apparently higher than those of the “lfl” trees (tab. 1 and 2). the female gametophytes of the “lfl” trees developed later than those of the “hfl” trees. for instance, the proembryo of 3 to 4 cells was developed in the “hfl” trees on may 24th, while the “lfl” trees only had 4to 8-nucleate embryo sacs (fig. 3d and tab. 2). fruit loads of the chestnut ‘zaodali’ affected starch accumulation in the style. on may 24th, the levels of starch in the outer integument of the “hfl” trees was a maximum value of 14.08% followed by that in the “lfl” trees only 2.94%. at the same stage of female fl ower development (mature embryo sacs), the level of starch of the “hfl” trees was 17.50% in the outer integument and 2.92% in the “lfl” trees (tab. 2). discussion starch accumulated in the transmitting tissues of the style before the formation of megaspore mother cell. the starch grains then disappeared. this is similar to which hu (1982) described for wheat when the pollen tube pierced the style and also for peach trees (herrero and arbeloa, 1989). in the present study, no starch accumulated in the needle-shaped stigmas of chinese chestnut. the nutrients required by the pollen tube to penetrate stigma cells were mainly supplied from the secretions of the top of the stigma. there was no starch grain in the tissues of the nucellus. instead tab. 1: starch content (%) in the ovary wall of chinese chestnut in 2006. date low fruit load high fruit load starch content1 stage of development2 starch content stage of development april 24th beginning club-shaped ovule 0.17 club-shaped ovule may 1st 0.28a inte. 3primordium appeared 0.73b inte. parallels the nucellus may 8th 0.39a inte. completely enclosed nucellus 0.89b tetrad stage may 16th 0.51a ovule fully developed 2.90b mature embryo sac may 24th 0.60a embryo sac of 4 to 8 nucleates 2.30b 2 to 4 cells proembryo 1 starch content: percentage of area coverage by starch grains over the total area of ovary wall. 2 develop.: development of female fl ower. 3 inte.: integument. *statistical difference is indicated by different letters in same date. tab. 2: starch content (%) in the ovule of chinese chestnut in 2006. date low fruit load high fruit load starch content1 stage of development2 starch content stage of development outer inner inte.3 outer inner inte. may 1st none none inte. primordium appeared none none inte. parallels the nucellus may 8th beginning beginning inte. completely enclosed nucellus 1.44 0.52 tetrad stage may 16th 0.03a 0.05a ovule fully developed 17.50c 4.87b mature embryo sac may 24th 2.92b 1.35a embryo sac of 4 to 8 nucleates 14.08c 2.46b 2 to 4 cells proembryo 1 starch content: percentage of area coverage by starch grains over the total area of ovary wall. 2 develop.: development of female fl ower 3 inte.: integument. *statistical difference is indicated by different letters in same date. 78 z. shi, r. he starch grains accumulated in the inner and mostly outer integument. starch accumulation and depletion occurred the pistil and the female gametophyte development. starch accumulation in the ovary and the ovule was a maximum when the embryo sac was mature. this was presumably to support the growth of the pollen tube in the ovary and fertilization. levels of starch were low in the pistils of the “lfl” trees under supply of poor nutrition the development of embryos arrested and embryos aborted in chinese chestnut (shi, 2003). therefore, this experiment should be repeated in the future to determine that there is a relation between the starch accumulation in the pistils and actual fruit load of the chestnut cv. ‘zaodali’. acknowledgements we thank prof. wünsche of university hohenheim, germany for his generous assistance during the experiment in his laboratory. this work was partly supported by a fund for scientifi c research from the p. r. china, and by a scholarship from the daad project of 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befruchtungsbiologie der zwetschensorte “valjevka”. erwerbsobstbau 44, 71-75. xia, r., ma, m., 1989: a study on the causes of empty cupule of chinese chestnuts (castanea mollissima b1.) i. infl uence of pollination and fertilization the empty copule of chinese chestnuts. journal of huazhong agricultural university 8, 242-247. yuan, y., du, g., xie, z., 1997: change of main nutritional substances of empty bur occurrence in chestnut. journal of wuhan botanical research 15, 243-247. address of the authors: prof. dr. zhuogong shi (corresponding author) and mr. runxi he, faculty of forestry, southwest forestry university, 650224 kunming, yunnan, p. r. china journal of applied botany and food quality 92, 1 6 (2019), doi:10.5073/jabfq.2019.092.001 1 laboratory for plant breeding, bioplant center, university of ciego de avila, ciego de ávila, cuba 2 laboratorio de carbocat, departamento de ingeniería química, facultad de ingeniería, universidad de concepción, región del bio bio, chile 3 escuela superior politécnica agropecuaria de manabí manuel félix lópez (espammfl), campus politécnico el limón, carrera de ingeniería agrícola, calceta, manabí, ecuador 4 school of life sciences, university of kwazulu-natal, durban, south africa mutagenic effects of sodium azide on pineapple micropropagant growth and biochemical profile within temporary immersion bioreactors daviel gómez1, 2, lázaro hernández1, julia martínez1, janet quiñones1, byron e. zevallos3, sershen4, lourdes yabor1, josé carlos lorenzo1 (submitted: october 4, 2018; accepted: november 22, 2018) summary sodium azide (nan3) is widely used to induce mutagenesis within in vitro plant systems. however, since this mutagenesis is un directed, its unintended effects demand characterization. this study investigated the mutagenic effects of sodium azide (0 0.45 mm) on selected growth (shoot multiplication rate and shoot cluster fresh weight) and biochemical (aldehydes, chlorophylls, carotenoids and phenolics) parameters in pineapple micropropagants within temporary immersion bioreactors (tibs). the content of soluble phenolics in the culture medium was also evaluated. irrespective of the concentration nan3 decreased shoot multiplication rate (by 87% relative to the control at 0.45 mm) and fresh weight (by 66% relative to the control at 0.45 mm). levels of chlorophyll a and b, and soluble phenolics in the culture medium were also negatively correlated with nan3 concentration. interestingly, nan3 application increased shoot carotenoid and soluble phenolic levels but had no significant effect on a range of established plant stress biomarkers: cell wall-linked phenolic levels, malondialdehyde and other aldehydes. given that 0.19 mm nan3 decreased shoot multiplication rate by 50% and resulted in propagants that displayed no morphologically abnormalities, increased levels of photoprotective pigments (relative to the control) and no significant increase in lipid peroxidation products, the mutagen can be used at this concentration to induce pineapple mutagenesis in tib based studies aimed at producing agriculturallyuseful mutants. keywords: in vitro stress; in vitro mutagenesis; plant metabolites; ananas comosus (l.) merr.; plant breeding abbreviations: sodium azide (nan3); temporary immersion bio reactor (tib). introduction plant breeders have generated new varieties using chemical mutagens for many decades (dubey et al., 2017). sodium azide (nan3) is widely regarded as a relatively safe to handle and very efficient chemical mutagen that is both inexpensive and non-carcinogenic (salvi et al., 2014). this common laboratory chemical is used widely in organic synthesis, agricultural and medical research (most often as bactericide, pesticide and nitrogen gas generator) but it is most famous for its mutagenic effects in plants and animals (mendiondo et al., 2016). this mutagenic capacity is based on nan3’s production of an organic metabolite, β-azidoalanine, which is valued for its ability to induce chromosomal aberrations at a rate much lower than other mutagens (dubey et al., 2017). the concentrations of nan3 that are used for the treatment of plant tissues vary widely across species: e.g. 3 10 mm (castillo et al., 2001) or 0.5 4.0 mm in hordeum vulgare (szarejko et al., 2017); 1.0 3.0 mm in pisum sativum (divanli-türkan et al., 2006); 0.04 1.1 mm in phaseolus vulgaris (chen et al., 2011); 15.5 77.6 mm in trigonella foenum-graecum (siddiquia et al., 2007); and 31.1124.3 mm in brassica napus (hussain et al., 2017). however, we have not found any report on the use of nan3 during in vitro production of axillary buds. despite the wide range of concentrations at which nan3 is used, the mutagen generally acts by crea ting point mutations in the genome, disturbing metabolic activity, growth and development, and inhibiting protein and dna replica tion (ragunathan and panneerselvam, 2007). these effects, the severity of which are concentration dependent (vainstein, 2002), change the balance between growth promoters and their antagonists (gruszka et al., 2012). nevertheless, a number of traits have been improved in a variety of species using nan3, including days to germination, flowering and silique maturation in brassica napus (hussain et al., 2017); related to quality, yield and disease resistance in wheat (dubey et al., 2017); germination, seedling survival, root length and height, height at maturity, number of leaves, and fruit yield in tomato (adamu and aliyu, 2007); and groundnut yield (mensah and obadoni, 2007). the mutagen has also been applied to a variety of explant types: e.g. cultures of pisum sativum seeds (divanli türkan et al., 2006), tomato seeds (el kaaby et al., 2015), brassica napus cotyledons (he et al., 2011), and hordeum vulgare anthers and microspores (castillo et al., 2001). although the tib technique is known to increase multiplication rates in many crop species (jiménez et al., 1999), the use of this culture technique for the nan3 application has not been reported to date. the increased multiplication rates in tibs might be caused by the combined advantages of using a solid and liquid culture medium. micropropagation on solid culture medium allows plant air exchange but nutrient uptake is limited to the explant basal surface (escalona et al., 1999). on the other hand, whilst micropropagation in liquid culture medium increases nutrient uptake, it often leads to hyperhydricity (ziv, 1995). hyperhydricity is characterized by various degrees of morphological and physiological disorders which can include a glassy, waterlogged-tissue appearance and disordered shoot growth (more specifically in the leaves). unlike the classic liquid culture procedure (alvard et al., 1993), in a tib explants are co vered with the culture medium only for a few minutes and immersion allows nutrient uptake through the entire explant surface. the plant air exchange is restored after removal of the culture medium. irrespective of the in vitro culture method, explant type or species, the mutations underlying the changes induced by chemical mutagens like nan3 are undirected and as alluded to above can have negative effects on plant growth and performance. this necessitates the characterization of the unintended effects of nan3 exposure and identification of the optimum concentration for application within specific species. 2 d. gómez, l. hernández, j. martínez, j. quiñones, b.e. zevallos, sershen, l. yabor, j.c. lorenzo pineapple is the main commercial species of the bromeliaceae glo bally (martín et al., 2015). fruit production in pineapple reached 25.4 million tons in 2013 (faostat, 2016) but to maintain and/ or improve the quality of pineapple production in many parts of the world, researchers are now developing new varieties (yabor et al., 2016). given that pineapple breeding through conventional techniques is extremely costly (loison-cabot and lacoeuilhe, 1989), biotechnological approaches (e.g. induced mutagenesis) provide great potential for improving selected clones more affordably. in vitro-induced mutagenesis has been employed to produce improved genetic variants in numerous crop species, including pineapple (ibrahim et al., 2009). research on the efficacy of chemical mutagens, specifically nan3, in inducing mutagenesis in pineapple is limited and hence, forms the focus of the present research. the study investigated the mutagenic effects of nan3, applied at a range of concentrations (0 0.45 mm), on selected growth (shoot multiplication rate and shoot cluster fresh weight) and biochemical (aldehydes, chlorophylls, carotenoids and phenolics) parameters in pineapple micropropagants tibs. soluble phenolic content in the culture medium was also assessed. the selection of tibs for the study is based on the high multiplication rate of pineapple (1:66) using this tech nique, compared with conventional micropropagation containers (1:8) (escalona et al., 1999). materials and methods plant material, in vitro methods and growth measurements feld-grown pineapple plants (cv. md2) served as the source of explants for initiating in vitro cultured buds (daquinta and benegas, 1997). these axillary buds, excised after removal of the crown leaves, were decontaminated (with 1% (w:v) ca(clo)2 for 10 min) followed by rinsing with tap water. the buds were excised with a portion of basal tissue and established in 300 ml glass containers with 5 ml of liquid culture medium per explant. ms salts (murashige and skoog, 1962), 100 mg l-1 myo-inositol, 0.1 mg l-1 thiamine-hcl, 30 g l-1 sucrose, 4.4 μm 6-benzyladenine (ba), and 5.3 μm naphthaleneacetic acid (naa) were included in the medium. at 45 d of culture, shoots were transferred to the multiplication medium (original medium supplemented with 9.3 μm ba and 1.6 μm naa). the shoots were subcultured and multiplied for 6 months, at 45-d intervals, and then placed in tibs with 3.0 μm paclobutrazol (after escalona et al. (1999)). immersions (2 min in duration) occurred every 3 h for 30 d. free shoots were located in the bottom of glass vessels (300 ml volume) filled with 200 ml of liquid medium with 5 explants within each of three containers per treatment (40 ml medium / explant). at the beginning of the 30-d-long subculture, different levels of nan3 (0, 0.15, 0.30 and 0.45 mm) were supplemented in the culture medium. culture conditions were as follows: 25±1 oc and 80 μmol m-2 s-1 cool fluorescent light for an 8 h photoperiod. shoot multiplication rate, shoot cluster fresh weight and all biochemical parameters described below were measured after 30 d of culture. biochemical parameters plant tissues (100 mg per replicate) were sampled from each of three bioreactors. phenolics were quantified according to gurr et al. (1992), chlorophylls following porra (2002) and malondialdehyde and other aldehydes after heath and packer (1968). for chlorophyll pigments a and b, the tissue was extracted in 5.0 ml acetone (80%, v:v), centrifuged (14086.8 g, 4 °c, 15 min) and the subsequent supernatants read for absorbance at 646.6 and 663.6 nm using a spectrophotometer (rayleigh, vis-723g). similarly, carotenoids levels were measured by reading the absorbance of acetone (80%, v:v) extracts at 470 nm (lichtenthaler, 1987). phenolic compounds were extracted and quantified (mg gallic acid equivalents per g fresh weight) using a colorimetric assay which involves the reaction of phenols with folin ciocalteu reagent (gurr et al., 1992). the reaction of malondialdehyde and other aldehydes with thiobarbituric acid formed the basis of the colorimetric method used to quantify the products of lipid peroxidation (molar extinction coefficient: 1.57 . 105m-1 cm-1) (albro et al., 1986; heath and packer, 1968). phenolic exudation was quantified using a modification of the hoagland (1990) procedure. this involved mixing the culture medium (0.5 ml; one sample per tib) with 4.5 ml distilled water, after which 0.5 ml folin ciocalteau reagent (50% v/v) was added. this mixture was shaken and then left to stand for 5 min before saturated sodium carbonate (1 ml) was added. the mixture was then shaken again, left to stand for 60 min, and then read for absorbance at 725 nm. phenolic concentration was calculated based on a gallic acid standard curve. statistical analysis all statistical analyses were performed using spss (version 8.0 for windows, spss inc., new york, ny). where data was parametric, tested for normality using a kolmogorov-smirnov test, means were compared using a one-way analysis of variance (anova) in combination with a tukey post-hoc test (p≤0.05). additionally, the overall coefficients of variation (ocv) were calculated as follows: (standard deviation/average) * 100. in this formula, the average values of the four nan3 levels were compared (treatments) to calculate the standard deviation and average. the higher the difference between the four treatments compared, the higher the ocv (lorenzo et al., 2015). ocvs from 2.94 to 30.00% were classified as low, from 30.00 to 57.06% as medium and from 57.06 to 84.12% as high. results exposure to nan3 decreased pineapple shoot multiplication rate and fresh weight in a concentration-dependent manner (fig. 1a, 1b, 1c). at the maximum concentration (0.45 mm) shoot multiplication rate was 12.65% of that obtained in the control, while fresh weight was reduced by 66.42% relative to the control. despite this reduction in growth and multiplication rate, shoot production was observed across all nan3 treatment concentrations and the shoots displayed no morphological abnormalities when compared to the control (fig. 1a). it should also be noted that the inhibitory effects of increasing nan3 concentration were more severe on shoot multiplication rate than cluster fresh weight. in terms of plant pigment contents, nan3 decreased chlorophyll a (fig. 2a) levels at the maximum concentration and chlorophyll b (fig. 2b) at the two highest concentrations, relative to the control. in contrast, nan3 increased carotenoid levels, compared with the control, at all concentrations tested (fig. 2c). ocv values were, how ever, low (13.65-24.13%) across all three plant pigments and the shoots produced on nan3 supplemented media showed no signs of chlorosis (fig. 1a) throughout the growth period. while tissue soluble phenolics (fig. 2d) increased relative to the control, soluble phenolics levels in the culture medium (fig. 2f) decreased within increasing nan3 concentrations, but ocvs were again low (18.15 and 28.22%, respectively). interestingly, nan3 had no significant effects on the levels of cell wall-linked phenolics (fig. 2e), malondialdehyde (fig. 2g) or other aldehydes (fig. 2h). discussion in light of a predicted reduction in agricultural productivity in many parts of the world due to climate change, induced mutation techno logy for crop improvement has become an increasing important area of research over the last few decades (dubey et al., 2017). in this effects of sodium azide on pineapple 3 regard, chemical mutagens are now useful tools in crop improvement and have been used to produce abiotic stress tolerance and disease resistance in various susceptible crops, improving their yield and quality traits (olawuyi and okoli, 2017). there are several mutagens available for crop improvement and like nan3, the mutagen investigated here, each has its own positive or negative effect on plants (heslot, 1977). mutagenesis has been employed to introduce many useful traits, including plant size, fruit ripening and resistance to pathogens in a wide range of fruit crops (reviewed by lamo et al. (2017)). however, studies on the effects of chemical mutagens on pineapple are scarce (paull et al., 2017), while no published reports on the effects of nan3 on the in vitro growth and biochemistry of pineapple explants were available at the time of this study. in the present study nan3 decreased shoot multiplication rate and cluster fresh weight (fig. 1b, 1c). nan3 has been reported to have similar inhibitory effects on in vitro explant growth in other species (dubey et al., 2017; hussain et al., 2017; olawuyi and okoli, 2017; szarejko et al., 2017). however, at 0.19 mm nan3, the multiplication rate (1:19) in the tibs used was lower than the control but still higher to that achieved for pineapple grown in the absence of nan3 using conventional culture methods (1:8) (escalona et al., 1999). furthermore the propagants generated displayed no morphological abnormalities, when compared to the control (fig. 1a). sodium azide did, however, induce significant changes in a number of biochemical parameters relative to the control. most plants grown in vitro have a different metabolism than in vivo. in the former, they are provided with a carbon source, growth regulators, high humidity, and less carbon dioxide and light, which promote proliferation but also change the autotrophic metabolism to heterotrophic or mixotrophic (chandra et al., 2010). once transferred to the ex vitro environment, micropropagated plantlets must then adapt their metabolism to the new conditions, which can represent a significant stress (teixeira et al., 2017). the biochemical profile of micropropagated plantlets can therefore influence, either negatively or positively, their survival and performance during acclimatization and subsequent ex vitro growth (hazarika et al., 2006). in the present study while in vitro exposure to nan3 decreased chlorophyll levels (a and b) in pineapple shoots, it increased carotenoid levels. carotenoids are important components of the antioxidant system in photosynthetic organisms and their absence can increase the extent of photoinhibition (cabrera, 2002). both the decreased chlorophyll and increased carotenoid levels suggest that nan3 exposure may reflect a nan3-stress-related photosynthetic down-regulation fig. 1: effects of sodium azide exposure on pineapple micropropagation in temporary immersion bioreactors. values represent means ± se (n=3) and are significantly different when labelled with different letters (anova, tukey, p≤0.05). overall coefficient of variation (ocv) = (standard deviation/ average)*100. to calculate this coefficient, the four average values were considered. the higher the difference among results, the higher is the overall coefficient of variation: low = 2.94 to 30.00%, medium = 30.00 to 57.06% and high = 57.06 to 84.12%. effects of sodium azide exposure on pineapple micropropagation in temporary immersion bioreactors. values represent means ± se (n=3) and are significantly different when labelled with different letters (anova, tukey, p≤0.05). overall coefficient of variation (ocv) = (standard deviation/average)*100. to calculate this coefficient, the four average values were considered. the higher the difference among results, the higher is the overall coefficient of variation: low = 2.94 to 30.00%, medium = 30.00 to 57.06% and high = 57.06 to 84.12%. maximum multiplication rate = 32.93 (0 mm nan3) minimum multiplication rate = 4.17 (0.45 mm nan3) 50%-reduction in multiplication rate = min. + ((max. – min.)/2) = 4.17 + ((32.93 – 4.17)/2) = 18.55 (~0.19 mm) fig. 1: 4 d. gómez, l. hernández, j. martínez, j. quiñones, b.e. zevallos, sershen, l. yabor, j.c. lorenzo capacity and enhanced photoprotection. increased carotenoid-based photoprotection is a commonly reported stress response (pompelli et al., 2010). this may have in turn decreased the potential for photooxidation, which can arise as a consequence of stress induced damage to the photosynthetic machinery. this suggestion is supported by the fact that nan3 exposure did not result an increase in malon dialdehyde and other aldehyde levels (fig. 2g, 2h) which are common products of cellular damage caused by (photo) oxidative stress (dumet and benson, 2000). under normal growth conditions, basal levels of aldehydes remain low in plant tissues but with exposure to abiotic stress they can accumulate to much higher levels (sershen et al., 2016). stress-induced aldehydes can act as generic signal mo lecules for plants under adverse environmental conditions, inhibi ting developmental and metabolic processes (mostofa et al., 2015). yadav et al. (2005) for example, showed that the accumulation of aldehydes can inhibit shoot growth. phenolic compounds are some of the most common products of secondary metabolism in plants and some are essential for plant survival, given their involvement in defense mechanisms under stress situations (sharma et al., 2012). the regulation of the biosynthesis of phenolics is generally brought about by biotic and abiotic stimuli (bettaieb et al., 2011) and generally accumulate in plant tissues during a stress (joseph et al., 2015). in the present study, though nan3 exposure induced a reduction in growth, an unambiguous indication of stress, it did not increase phenolic levels in shoot tissues (fig. 2d, 2e) and or the culture medium (fig. 2f). this is encouraging, since cell wall-linked or insoluble phenolics, in particular, can make cell walls more rigid and less porous inhibiting nutrient uptake and growth (kabera et al., 2014). their accumulation in in vitro cultures is therefore not beneficial for growth (machakova et al., 2008). the results suggest that though nan3 decreased pineapple shoot multiplication rate within tibs, propagants displayed no morphologically abnormalities, and when produced using 0.19 mm nan3 exhibited enhanced levels photoprotective pigments and no obvious signs of enhanced lipid peroxidation. the mutagen can therefore be used at this concentration to induce pineapple mutagenesis in tib based studies aimed at producing agriculturally-useful mutants. fig. 2: effects of sodium azide exposure on pineapple micropropagation in temporary immersion bioreactors. values represent means ± se (n=3) and are significantly different when labelled with different letters (anova, tukey, p≤0.05). overall coefficient of variation (ocv) = (standard deviation/ average)*100. to calculate this coefficient, the four average values were considered. the higher the difference among results, the higher is the overall coefficient of variation: low = 2.94 to 30.00%, medium = 30.00 to 57.06% and high = 57.06 to 84.12%. effects of sodium azide exposure on pineapple micropropagation in temporary immersion bioreactors. values represent means ± se (n=3) and are significantly different when labelled with different letters (anova, tukey, p≤0.05). overall coefficient of variation (ocv) = (standard deviation/average)*100. to calculate this coefficient, the four average values were considered. the higher the difference among results, the higher is the overall coefficient of variation: low = 2.94 to 30.00%, medium = 30.00 to 57.06% and high = 57.06 to 84.12%. fig. 2: effects of sodium azide exposure on pineapple micropropagation in temporary immersion bioreactors. values represent means ± se (n=3) and are significantly different when labelled with different letters (anova, tukey, p≤0.05). overall coefficient of variation (ocv) = (standard deviation/average)*100. to calculate this coefficient, the four average values were considered. the higher the difference among results, the higher is the overall coefficient of variation: low = 2.94 to 30.00%, medium = 30.00 to 57.06% and high = 57.06 to 84.12%. fig. 2: effects of sodium azide exposure on pineapple micropropagation in temporary immersion bioreactors. values represent means ± se (n=3) and are significantly different when labelled with different letters (anova, tukey, p≤0.05). overall coefficient of variation (ocv) = (standard deviation/average)*100. to calculate this coefficient, the four average values were considered. the higher the difference among results, the higher is the overall coefficient of variation: low = 2.94 to 30.00%, medium = 30.00 to 57.06% and high = 57.06 to 84.12%. fig. 2: effects of sodium azide on pineapple 5 author contribution dg, lh, jm, jq, bez, s, ly and jcl designed the research; 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(ed.), biotechnologies for plant mutation breeding. international atomic energy agency. teixeira, j., hossain, m.m., sharma, m., dobránszki, j., cardoso, j.c., songjun, z., 2017: acclimatization of in vitro-derived dendrobium. hort. plant j. 3, 110-124. doi: 10.1016/j.hpj.2017.07.009 vainstein, a., 2002: breeding for ornamentals: classical and molecular approaches. springer science & business media. yabor, l., valle, b., rodríguez, r.c., aragón, c., papenbrock, j., tebbe, c.c., lorenzo, j.c., 2016: the third vegetative generation of a field-grown transgenic pineapple clone shows minor side effects of transformation on plant physiological parameters. plant cell tiss. org. cult. doi: 10.1007/s11240-016-0950-4 yadav, s.k., singla-pareek, s.l., ray, m., reddy, m., sopory, s., 2005: methylglyoxal levels in plants under salinity stress are dependent on glyoxalase i and glutathione. biochem. biophys. res. comm. 337, 61-67. doi: 10.1016/j.bbrc.2005.08.263 ziv, m., 1995: in vitro acclimatization. automation and environmental control in plant tissue culture. springer. address of the authors: daviel gómez, lázaro hernández, julia martínez, janet quiñones, lourdes yabor, josé carlos lorenzo, laboratory for plant breeding, bioplant center, university of ciego de avila, ciego de ávila, 69450, cuba e-mail: jclorenzo@bioplantas.cu daviel gómez, laboratorio de carbocat, departamento de ingeniería química, facultad de ingeniería, universidad de concepción, región del bio bio, chile byron e. zevallos, escuela superior politécnica agropecuaria de manabí manuel félix lópez (espammfl), campus politécnico el limón, carrera de ingeniería agrícola, calceta, manabí, ecuador sershen, school of life sciences, university of kwazulu-natal, durban, 4001, south africa © the author(s) 2019. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). journal of applied botany and food quality 95, 175 181 (2022), doi:10.5073/jabfq.2022.095.022 1fruit research institute of jeollanamdo agricultural research and extension services, haenam, jeonnam, korea 2department of food science & technology, chonnam national university, korea 3institute of agricultural science and technology, chonnam national university, korea nutritional and neuroprotective characterization of ‘tadanishiki’ yuzu according to harvesting period or extraction condition bo-bae lee1, young-min kim2, seung-hee nam3* (submitted: september 29, 2022; accepted: november 9, 2022) * corresponding author summary the present study investigated the phenolic profile, antioxidant activity, and neuroprotective properties of ‘tadanishiki’ yuzu (citrus junos, a seedless variety of yuzu) according to harvesting period and extraction condition. high-performance liquid chromatography (hplc) was used to identify the functional components. to evaluate the neuroprotective properties, scopolamine was used to induce cholinergic dysfunction in human neuroblastoma sh-sy5y cells pretreated with yuzu extracts. among the harvesting periods, september provided the optimum fruit weight of yuzu and relatively high amounts of total phenolics (3.67 mg/g dw), flavonoids (10.13 mg/g dw), and 2,2-diphenyl-1-picrylhydrazyl (dpph) scavenging activity (29.10 μg vit. c eq.). of the functional compounds, hesperidin (13.57 mg/100 g dw) and naringin (5.84 mg/100 g dw) were the highest in 5% (w/v) yuzu extracted with 80% ethanol and this extract showed the highest dpph (289.2 μg vit. c eq.) scavenging activity. this same extract showed the highest cell viability and lowest cortisol or acetylcholinesterase content in scopolaminetreated sh-sy5y cells. these results indicate that ‘tadanishiki’ yuzu harvested in september should be extracted at 5% (w/v) yuzu with 80% etoh, and this extract might be useful for application as a natural functional additive. keywords: ‘tadanishiki’, harvest, extraction, neuroprotection introduction yuzu (citrus junos) is a type of citrus grown in japan and china. in korea, it is mainly grown in southern coastal regions, including jeju island, goheung, wando, and geoje (shin et al., 2008), and is commonly used as a raw material for beverages and herbal medicines due to its unique flavor and effectiveness against colds. unlike other citrus fruits, the pulp, peel, and juice of yuzu are used in japanese and korean cuisine, so it is easy to consume active ingredients in the peel (fukutome, 2020). studies suggest that the yuzu fruit peel contains more nutritionally beneficial and biologically active com ponents than the fruit pulp (yoo and moon, 2016). as one of the fruits in the citrus genus, yuzu has attractive aroma and notable contents of vitamin c, carotenoids, flavonoids, and citric acid, among various nutritive and non-nutritive compounds (ji et al., 2008). it is well known that the polyphenols and flavonoids found in yuzu fruit have potent antioxidant properties and the capacity to scavenge free radicals (vinson et al., 2001). hesperidin, naringin, and neohesperidin, the main flavonoids in yuzu, have antioxidant properties and can penetrate the blood-brain barrier, indicating that they prevent neurodegeneration and improve mental performance (hirata et al., 2005). by reducing free radicals and inflammation, oral hesperidin treatment lessens the severity of rat brain damage after stroke (raza et al., 2011). yuzu has higher concentrations of vitamin c and phenolics than other citrus fruits, and the peel and pulp of immature yuzu contain more vitamin c as well as more overall antioxidant activity than the mature fruit because of the decline in the quantities of hesperidin, naringin, and total phenolics with fruit ripening (yoo et al., 2004). the composition and biological activity of fruit extracts are affected by a number of variables, including the extraction solvent, the extraction time, and the extraction temperature (lee et al., 2015). re search has demonstrated that the total polyphenol, tannin content, and radical scavenging capacity of the 70% etoh extract of c. junos are higher than those of the water extract; however, the scavenging capability of both types of extracts was dependent on their concentration (lee and lee, 2017). another study reported that the antioxidant and immunological activities of 80% etoh extracts from c. junos peel increased with increasing phenolics concentration (park et al., 2008). in both cell culture and mouse models, the 70% etoh extract of yuzu peel exerted antidiabetic effects via ampactivated protein kinase (ampk) and peroxisome proliferator-activated receptor-gamma (pparg) signaling (kim et al., 2013). most studies on yuzu have focused on its physiochemical or nutritional characteristics, little was known about the influences of harvesting period or extraction way on the nutritional characteristics of yuzu. moreover, our group previously assessed the physiochemical and functional characteristics of three major yuzu cultivars in korea (nam et al., 2021). this study analyzed and compared the functional compounds by hplc and neuroprotective function of ‘tadanishiki’ yuzu using human neuroblastoma cells according to harvesting period or extraction condition. materials and methods reagents and materials the ‘tadanishiki’ yuzu fruits in this study were grown at the fruit research facility of jeonnam agricultural research & extension services (wando, jeonnam province, korea). the yuzu fruits were harvested on the 10th of each month from july to november 2020. the yuzu fruits were cut into slices, freeze-dried (fd8512, ilshin, korea), and pulverized (fm681c, hanil electric, korea). yuzu samples were prepared at different yuzu powder contents (1~10%, w/v; s 1%, s 3%, s 5%, s 7.5%, and s 10%) or ethanol (etoh) content (v/v; e 10%, e 30%, e 50%, e 80%, and e 100%) and extracted by soxhlet extraction (60 °c for 3 h) and evaporated to dryness under vacuum. ascorbic acid, gallic acid, quercetin, 2,2-diphenyl-1-picrylhydrazyl (dpph), and the folin-denis reagent were purchased from sigmaaldrich (st. louis, mo, usa). naringin, narirutin, hesperidin, and neohesperidin were purchased from chromadex (irvine, ca, usa). all analytical reagents used for testing had a purity level of at least 90%. 176 b.-b. lee, y.-m. kim, s.-h. nam functional phenolics by high-performance liquid chromato graphy-diode array detection (hplc-dad) the method reported by wang et al. (2008) was modified to determine the phenolic contents in yuzu using an agilent 1216 infinite lc series system (agilent technologies, palo alto, ca, usa). the zorbax eclipse plus c18 column (4.6 × 250 mm, 5 mm; agilent technologies) was used for the analysis, with mobile phases of 0.1% formic acid (solvent a) in distilled water and methanol-acetonitrile (solvent b). the absorbance was measured at 280 nm. the gradient elution program of the two solvents was as follows: a:80, b:20 at 5-10 min; a:60, b:40 at 10.1-15 min; a:50, b:50 at 15.1-20 min; a:30, b:70 at 20.1-25 min; and a:0, b:100 at 25.1-30 min. the flow rate was 0.5 ml/min, and the column temperature was 35 °c. total phenolic and flavonoids contents the folin and denis (1912) method was used to determine the total phenolic content. sample aliquots of 30 μl were diluted by adding 32.5 μl of distilled water, followed by the addition of 12.5 μl of the folin-denis reagent and 6 min of reaction time in total darkness. afterward, 12.5 μl of 7% sodium carbonate and 250 μl of distilled water were added and mixed. after 1 h, the absorbance at 760 nm was measured using a microplate spectrophotometer (synergy htx, biotek epoch, agilent, santa clara, ca, usa). the total phenolic contents were calculated from a calibration curve of gallic acid as the standard. diethylene glycol (200 μl), 2n sodium hydroxide (20 μl), and 20 μl of sample were added and reacted at 37 °c for 30 min, followed by measurement of the absorbance at 420 nm using a microplate reader. flavonoid content was measured using a standard curve of rutin. dpph radical scavenging activity the modified blois (1958) method was used to determine the dpph radical scavenging capacity. after sample aliquots of 50 μl were continuously diluted, 250 μl of 1 mm dpph was added. the mixture was incubated at room temperature for 10 min, followed by measurement of the absorbance at 517 nm using a microplate spectrophotometer (biotek epoch) with ascorbic acid as the reference compound. the following equation was used to compute the dpph radical scavenging activity: dpph radical scavenging activity (%) = 1 − (sample absorbance / control absorbance) × 100. cell culture and sample treatment sh-sy5y human neuroblastoma cells were purchased from the korean cell line bank (seoul, korea). in rpmi 1640 medium (gibco, waltham, ma, usa), 10% fetal bovine serum and 1% antimycotics/antibiotics (gibco) were added to refine sh-sy5y cells. cells were incubated at 37 °c in a fully humidified atmosphere of 5% co2 until 70-80% confluency. cells were seeded in a 96-well plate for 24 h, and yuzu samples (10, 100, and 500 μg/ml) from 1~10% (w/v) yuzu extract (s 1%, s 3%, s 5%, s 7.5%, and s 10%) or 10~100% (v/v) etoh content (e 10%, e 30%, e 50%, e 80%, and e 100%) were pretreated for 24 h. theanine (10 μg/ml) was used as the positive control. scopolamine (5 mm) was added to the sh-sy5y cells as the stimulant. the survival rate was compared and examined, with the percentage representing the difference between the absorbance of the sample and the control. for acetylcholinesterase (ache) quantification, 100 μg/ml of yuzu sample was used in the assay. cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt assay) and acetylcholinesterase (ache) content by enzyme-linked immunosorbent assay (elisa) cell viability was measured using the 3-(4,5-dimethyl-2-thiazolyl) 2,5-diphenyl-2h-tetrazolium bromide kit (sigma). briefly, cells were seeded onto a 96-well plate at a density of 5 × 104 cells/well. different concentrations of yuzu extract were treated for 24 h. mtt solution was added to reach a final 0.45 mg/ml and incubated at 37 °c for 4 h. mtt solution was removed, and dimethyl sulfoxide (dmso) was added. the absorbance was measured at 570 nm using an elisa reader. the cell viability (%) was calculated using the following equation: cell viability (%) = (mean absorbance of the sample / mean absorbance of the control) ×100 ache was quantified using an ache elisa kit (cusabio, wuhan, china). sample-treated cells were prepared at a 500-fold dilution, and then 100 μl of diluted sample and standard were dispensed into each well and incubated at 37 °c for 2 h. then, the liquid was removed from the well and incubated with 100 μl of biotin-antibody at 37 °c for 1 h. conversion to ache concentration according to absorbance was converted to a formula according to standard value. statistical analysis all values were represented as the mean ± standard deviation (sd). significant differences were determined by one-way analysis of va riance (anova) and duncan’s multiple range test. significant differences were identified at p < 0.05. the spss 23.0 for windows program (spss, inc., chicago, il, usa) was used to conduct statistical analyses. results and discussion the appearance of yuzu fruits cultivated from july to november is shown in fig. 1. the weight or size of ‘tadanishiki’ yuzu fruit increased depending on the harvest time. it is commonly assumed in korea that the general harvesting time is november when the yuzu fruit is fully mature, the external color of the fruit is fully yellow, and the fruit attains the characteristic flavor (phi and sawamura, 2008). yuzu peel gradually changes from green to yellow as the fruit matures (fig. 1) because of the degradation of chlorophyll and the increase in carotenoids. fig. 1(b) shows that the extracts of the ‘tadanishiki’ yuzu fruit harvested every month from july to november contain four major flavonoids, including naringin, narirutin, hesperidin, and neohesperidin. the primary phenolic compounds in citrus fruits are flavonoids, which have been linked to numerous health benefits, such as anti oxidant, anti-inflammatory, anti-carcinogenic, and antibacterial properties (yoo and moon, 2016). the concentrations of the indivi dual flavonoids in ‘tadanishiki’ yuzu are shown in tab. 1. narirutin was present at the highest concentration, followed by hesperidin, neohesperidin, and naringin. all four flavonoids were detected at the highest concentrations in yuzu harvested in july, the immature period, and tended to decrease as the harvest time was delayed. this trend was consistent with the study by nam et al. (2021), in which the contents of hesperidin and naringin were high in immature yuzu harvested in july and then decreased steadily. among citrus flavonoids, naringin, hesperidin, and naringenin are the most commonly studied (zou et al., 2016). results of total phenolics, total flavonoids, and antioxidant activity according to harvest time of ‘tadanishiki’ yuzu are shown in tab. 1. consistent with the total flavonoid content, the total phenolic content was highest in yuzu harvested in july, presenting 4.88 mg/g dw. the subsequent decrease in the total phenolic content may reflect the ini characterization of ‘tadanishiki’ yuzu 177 (a) fig. 1: (a) photographs and (b) hplc chromatograms of ‘tadanishiki’ yuzu by harvesting period. (b) tab. 1: functional components of yuzu extracts by harvesting period. harvesting total phenolics total flavonoids dpph radical functional compounds by hplc period (mg/g dw) (mg/g dw) scavenging activity (mg/100 g dw) (μg vit. c eq.) naringin narirutin hesperidin neohesperidin july 4.88 ± 0.00a 26.33 ± 0.32a 69.09 ± 0.47a 298.60 ± 6.80a 992.90 ± 23.00a 690.20 ± 18.80a 305.00 ± 11.30a august 4.02 ± 0.05b 16.29 ± 0.04b 53.83 ± 0.34b 194.40 ± 23.00b 674.30 ± 10.30b 430.10 ± 26.10b 191.80 ± 1.80b september 3.67 ± 0.04c 10.13 ± 0.07c 29.10 ± 0.85c 111.00 ± 4.40c 354.40 ± 19.80c 273.00 ± 21.20c 111.40 ± 21.8c october 3.22 ± 0.03d 9.71 ± 0.14c 26.40 ± 0.44d 88.20 ± 16.40c 341.80 ± 4.40c 261.00 ± 1.70c 110.60 ± 6.60c november 3.05 ± 0.04e 9.11 ± 0.51d 24.90 ± 0.16e 96.90 ± 3.00c 352.00 ± 6.50c 250.50 ± 14.00c 108.70 ± 1.00c 1)means with the same letter in each column are not significantly different by duncan’s multiple range test (p < 0.05). values are represented as mean ± sd (n = 3). tiation of lignification of parenchyma cells around the secretory cavities of the peel (frei, 2013). color, flavor, and astringency are some other properties affected by the presence of phenolic compounds. the flavonoid content of ‘tadanishiki’ yuzu was investigated for each sample from immature in july to mature in november. the total flavonoid content in citrus fruits generally differs according to the varie ty, maturity period, and fruit area of the sample (díaz-mulab et al., 2009). the yuzu harvested in july showed a total flavonoid content about three times higher than the yuzu harvested in november. a rapid decline in the flavonoid content started in september. as a result, flavonoid content was found to be the highest in the immature fruit and tended to decrease as maturity progressed. similarly, rhyu et al. (2002) reported that the flavonoid content of tangerines by harvest period generally decreased as the harvest period was delayed. yuzu had the highest content of narirutin from 352.00-992.90 mg/ 100 g (tab. 1). this result was consistent with that of tangerines in that it showed the highest content of hesperidin and narirutin as the representative flavonoid components (yang et al., 2019). gattuso et al. (2007) also reported that oranges, mandarins and lemons contained the most hesperidin but grapefruit showed the highest content of naringin among functional phenolics. antioxidant activity was measured using the dpph assay. the results showed a comparable ranking of radical scavenging activity for the yuzu collected according to the harvest period from july to november (tab. 1). the dpph free radical scavenging activity was three-fold higher in ‘tadanishiki’ yuzu harvested in july (69.09 μg vit. c eq.) than in november (24.90 μg vit. c eq.), rapidly decreasing from september to the final harvest (29.10 μg vit. c eq.). among the several antioxidant methods reported to date, the dpph radical scavenging activity method is popular and straightforward (mathew and abraham, 2006). among harvesting periods, yuzu fruit on september is optimal for practical application with fruit yield and sugar content(data not shown) with relatively high amounts of total phenolics, flavonoids, and antioxidant activity as shown in tab. 1. an extraction procedure is required to obtain the antioxidant components from citrus fruits (altemimi et al., 2017). extraction is a process used to obtain plant secondary metabolites, such as alkaloids, phenolics, flavonoids, and glycosides, using selective solvents. solvent selection is an important step in establishing the extraction procedure (azwanida, 2015). etoh is safe for human consumption when used as a solvent for natural compounds in food and natural medicine. phenolic derivative antioxidant compounds have been effectively extracted from natural substances using absolute etoh and aqueous etoh (sultana et al., 2009). in this study, etoh was used as the extraction solvent, and for the fruit harvested in september only, yuzu sample contents of 1-10% w/v (s 1%, s 3%, s 5%, s 7.5%, and s 10%) were analyzed for the functional components of each extract and antioxidant activity by the same methods used above. additionally, cellular assays for cell viability and neuroprotective properties were also performed. yuzu fruit harvested in september demonstrated higher fruit weight and size than those harvested in july and august (nam et al., 2021). from the results of the functional content of each extract and antioxidant activity (tab. 2), naringin and neohesperidin did not show any significant difference depending on the sample content but tended to decrease as the sample content reached 10%. hesperidin and narirutin also showed the same tendency. the flavonoid contents were highest for s 5% and s 7.5%. the total phenolic content decreased as the sample content increased in the order of 7.10 mg/g (s 3%) > 6.82 mg/g (s 5%) > 5.18 mg/g (s 7.5%) > 4.72 mg/g (s 10%). the dpph radical scavenging activity was contrary to the tendency of total phenols and flavonoids. following etoh extraction, the functional components, total phenolics, and total flavonoids showed no significant difference between s 3% and s 5%; however, s 5% had a higher hesperidin concentration compared to s 1%, so s 5% was chosen as the sample content for the subsequent analyses. ‘tadanishiki’ yuzu (s 5%) was extracted according to the etoh content of 10-100% (e 10%, e 30%, e 50%, 178 b.-b. lee, y.-m. kim, s.-h. nam e 80%, and e 100%) to investigate the functional components and antioxidant effect (tab. 3). the naringin content was 3.88-5.84 mg/g, narirutin was 9.83-18.34 mg/g, hesperidin was 9.38-13.57 mg/g, and neohesperidin was 4.56-6.57 mg/g. naringin and narirutin contents were highest for e 50% and e 80%, without a significant difference. hesperidin content was highest for e 30%, e 50%, and e 80%, presenting 12.5, 13.2 and 13.5 mg/g, respectively. neohesperidin was highest for e 80%, showing 6.57 mg/g. phenolic compounds are mainly found in plants and are known to exhibit antioxidant power. the total phenolic content of the ‘tadanishiki’ yuzu extract according to the extraction solvent concentration was 4.75-6.62 mg/g. the total flavonoid concentration ranged from 50.0 to 65.6 mg/g, with e 80% providing the highest contents of total flavonoids and total phenolics (p < 0.05). flavonoids display different degrees of dissolution in water and etoh depending on their chemical structure. therefore, it is thought that the flavonoid content dissolved in the extraction solvent varies depending on the types of flavonoid compounds present in the sample. it was confirmed that the dpph radical scavenging activity of the ‘tadanishiki’ yuzu extract was significantly affected by the concen tration of etoh as the extraction solvent, increasing as the etoh content increased. according to al-dabbagh et al. (2018), the phenol content and the radical scavenging activity are proportional. therefore, it is believed that the most efficient extraction method in this study would use 5% (w/v) immature yuzu harvested in september and 80% etoh, taking into account the content of functional components and the antioxidant activity of yuzu. these results can be used as basic data for broadening the applications of yuzu in food formulations. as a result of measuring cell viability to confirm the limit showing no toxicity in sh-sy5y cells, toxicity was shown from the concentration of 500 μg/ml in all samples treated according to the sample concentration in the extraction (fig. 2 and 3). however, in the s 1% and s 7.5% groups, the cell viability decreased significantly even at a concentration of 100 μg/ml, but it was 89.2% and 94.5% higher compared to the control, so concentrations up to 100 μg/ml were considered to be safe for the cell line tested. in addition, as a result of sample treatment according to the concentration of the extraction solvent, toxicity also appeared from the concentration of 500 μg/ml, and cell viability was significantly reduced even at the concentration of 100 μg/ml in e 50% and e 10%, presenting 89.75% and 92.07% compared to the control, so the experiment was conducted at a concentration of 100 μg/ml or less. scopolamine, a non-selective muscarinic receptor, impairs short-term memory function and learning in both humans and animals (marisco et al., 2013). furthermore, scopolamine significantly increases the levels of ache and malon dialdehyde in the cortex and hippocampus (tota et al., 2012a, b). treatment of sh-sy5y cells with scopolamine caused cell damage and death (baral et al., 2015). the present study confirmed that cell viability was significantly reduced, and 5 mm scopolamine caused damage to nerve cells. yuzu extract reversed scopolamine-induced neurotoxicity in sh-sy5y cells in a concentration-dependent manner. cell viability was most effectively increased in the group treated with s 5% and e 80% at a concentration of 100 μg/ml. acetylcholine is a neurotransmitter supplied to the brain by cholinergic neurons. acetylcholine is decomposed into choline and acetate by ache, thereby terminating neurotransmission (shaikh et al., 2014). ache is present in high concentrations in the synaptic cleft and generally helps to rapidly remove acetylcholine for the normal functioning of skeletal muscle (kalamida et al., 2007). however, it has been reported that when ache acts excessively, it suppresses the action of acetylcholine, causing nerve damage and inducing diseases, such as alzheimer’s (kihara and shimohama, 2004). therefore, in this experiment, the effect of sample treatment on the content of ache was confirmed to check the protective effect of yuzu extract in scopolamine-treated nerve cells. according to the sample and extractab. 2: functional components of yuzu extracts by yuzu powder content. yuzu powder total phenolics total flavonoids dpph radical functional compounds by hplc content (mg/g dw) (mg/g dw) scavenging activity (mg/100 g dw) (w/v) (vit. c eq. μg) naringin narirutin hesperidin neohesperidin 1.0% 8.62 ± 0.00a 68.43 ± 2.21a 35.61 ± 7.60e 4.58 ± 0.27bc 14.72 ± 0.02b 10.81 ± 0.68b 5.15 ± 0.09d 2.5% 7.10 ± 0.13b 68.49 ± 2.21a 141.77 ± 5.07d 5.40 ± 1.11ab 16.68 ± 0.30b 12.54 ± 0.32a 6.02 ± 0.06c 5.0% 6.82 ± 0.02c 66.47 ± 0.35ab 287.97 ± 4.53c 5.64 ± 0.17a 18.19 ± 0.08a 13.27 ± 0.35a 6.36 ± 0.15b 7.5% 5.18 ± 0.00d 64.06 ± 2.77b 368.40 ± 20.10b 5.84 ± 0.19a 18.34 ± 0.39a 13.57 ± 0.50a 6.57 ± 0.08a 10.0% 4.72 ± 0.02e 68.91 ± 1.81a 443.76 ± 24.45a 3.88 ± 0.08c 9.83 ± 0.15c 9.38 ± 0.76c 4.56 ± 0.03e a-e mean with the same letter in each column are not significantly different by duncan’s multiple range test (p < 0.05). values are represented as mean ± sd (n = 3). tab. 3: functional components of yuzu extracts by extraction condition. extraction total phenolics total flavonoids dpph radical functional compounds by hplc solvent (mg/g dw) (mg/g dw) scavenging activity (mg/100 g dw) vit. c eq. μg naringin narirutin hesperidin neohesperidin etoh 10% 4.75 ± 0.05e 50.02 ± 1.23d 166.23 ± 9.24d 4.58 ± 0.27bc 14.72 ± 0.02b 10.81 ± 0.68b 5.15 ± 0.09d etoh 30% 5.40 ± 0.05d 58.69 ± 0.00c 219.67 ± 0.36c 5.40 ± 1.11ab 16.68 ± 0.30b 12.54 ± 0.32a 6.02 ± 0.06c etoh 50% 6.31 ± 0.01b 63.29 ± 1.59b 260.25 ± 10.14b 5.64 ± 0.17a 18.19 ± 0.08a 13.27 ± 0.35a 6.36 ± 0.15b etoh 80% 6.62 ± 0.04a 65.67 ± 0.26a 289.23 ± 5.43a 5.84 ± 0.19a 18.34 ± 0.39a 13.57 ± 0.50a 6.57 ± 0.08a etoh 100% 6.12 ± 0.06c 57.54 ± 0.97c 262.79 ± 4.71b 3.88 ± 0.08c 9.83 ± 0.15c 9.38 ± 0.76c 4.56 ± 0.03e a-e mean with the same letter in each column are not significantly different by duncan’s multiple range test (p < 0.05). values are represented as mean ± sd (n = 3). etoh: ethanol concentration (% v/v). characterization of ‘tadanishiki’ yuzu 179 tion solvent concentrations, the ache content was most effectively reduced by yuzu extracted at s 5% with e 80%. the aforementioned information also revealed that this extract successfully protected the sh-sy5y nerve cells. conclusions this research revealed the phenolic compounds and functional activity (dpph antioxidant activity and neuroprotective properties) of the extract of ‘tadanishiki’ yuzu fruit according to harvest time or extraction conditions, including sample content (1-10%, w/v) and extraction solvent (etoh 10%, 30%, 50%, 80%, and 100%, v/v). although the yuzu harvested in july has higher total phenolics and total flavonoids, ‘tadanishiki’ harvested in september was finally chosen in this study due to its reasonable fruit weight while still containing relatively high contents of functional compounds. in addition, it was confirmed that the sh-sy5y nerve cell protection effect was most effective when the sample was extracted with 80% etoh at 5% yuzu content. therefore, it is suggested that this work will inform about the harvesting period of ‘tadanishiki’ yuzu and its extraction condition for the highest neuroprotective effect as a functional material. acknowledgments this study was financially supported by the agriculture science and technology development program (pj016161) of the rural development administration, funded by the korean government. conflict of interest no potential conflict of interest was reported by the authors. fig. 2: neuroprotective effects of yuzu extracts by yuzu powder contents on human neuroblastoma cells (sh-sy5y). (a) cell toxicity; (b) cell viability after scopolamine treatment; (c) acetylcholinesterase content (ache) by elisa. data are shown as mean ± sd. significance levels (*p < 0.05, **p < 0.01, ***p < 0.001) compared to respective parameter value of control group. different letters above the bar represent a significant difference between groups by duncan’s multiple range test (p < 0.05). “s” denotes yuzu powder content (%) in extraction conditions with 80% etoh solvent. fig. 3: neuroprotective effects of yuzu extracts by extraction condition on human neuroblastoma cells (sh-sy5y). 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http://dx.doi.org/10.2174/18715273113126660166 http://dx.doi.org/10.5352/jls.2008.18.12.1745 http://dx.doi.org/10.3390/molecules14062167 http://dx.doi.org/10.1016/j.bbr.2012.03.015 http://dx.doi.org/10.1007/s00213-012-2639-7 http://dx.doi.org/10.1021/jf0009293 http://dx.doi.org/10.1016/j.foodchem.2007.05.086 http://dx.doi.org/10.7235/hort.20190065 http://dx.doi.org/10.1021/jf0498158 http://dx.doi.org/10.1016/j.foodchem.2015.09.079 http://dx.doi.org/10.1016/j. foodchem.2015.09.072 characterization of ‘tadanishiki’ yuzu 181 orcid: bo-bae lee http://orcid.org/0000-0001-8436-8686 young-min kim http://orcid.org/0000-0002-2559-9182 seung-hee nam http://orcid.org/0000-0001-5271-1275 address of the corresponding author: seung-hee nam, institute of agricultural science and technology, college of agriculture and life sciences, chonnam national university, gwangju 500-757, republic of korea e-mail: namsh1000@ hanmail.net © the author(s) 2022. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://orcid.org/0000-0001-8436-8686 http://orcid.org/0000-0002-2559-9182 http://orcid.org/0000-0001-5271-1275 angewandte botanik. ein neues, untrügliches merkmal für rauchschäden bei laubhölzern. won f. w. neger, tharandt. (vortrag, gehalten am dienstag, den 5. august 1919 in der vereinigung für angewandte botanik zu hann.-münden.) die botanische diagnostik der rauchschäden bei unseren laubund nadelbäumen liegt – das wird jeder, der sich mit dieser frage eingehend befaßt hat, zugeben – noch sehr im argen. es gibt z. z. noch kein absolut sicheres erkennungs merkmal für die beschädigung durch rauchgase. alle, die im laufe der zeit namhaft gemacht worden sind, erwiesen sich a ls mehr oder weniger unzuverlässig. wenn z. b . von „sach verständigen“ bald die form und farbe der flecken a n laub blättern, bald die nuance der verfärbung von koniferennadeln als sicheres diagnostisches merkmal gerühmt wird, so ist dem entgegen zuhalten, daß die fleckenbildung a n laubblättern in vollkommen gleicher äußerer erscheinung ebensowohl durch trockenheit bezw. frost hervorgerufen werden kann!), und daß die rotfärbung der koniferennadeln nur ein postmortaler vorgang ist, der hervor gerufen wird durch das licht auf die abgestorbenen gewebe und daher in vollkommen gleicher nuancierung ebensowohl durch andere vegetationsfeindliche faktoren (frost, hitze usw.) bewirkt werden kann”), wie durch giftige in der atmosphäre enthaltene gase, also keinerlei diagnostischen wert besitzt. auch das von r . hartig beschriebene merkmal der schließ zellenrötung bei koniferennadeln erwies sich – wie wieler, sorauer und der verf. dieser zeilen ausführten – als durchaus unzuverlässig, indem diese erscheinung einerseits bei einwirkung *) neger, die bedeutung des habitusbildes für die diagnostik von pflanzenkrankheiten. zentralb. bakt. par. 1918, ii. abt., s. 171. *) neger, rauchwirkung; spätfrost und frosttrocknis und ihre diagnostik. thar. forstl. jahrb. 1915. angewandte botanik i. 9 130 f. w. neger, hochkonzentrierter saurer gase vollkommen ausbleiben, anderer seits auch durch andere lebensfeindliche einflüsse (frost, heißluft, pilze usw.) hervorgerufen werden kann. es besteht also zweifellos das bedürfnis nach einem untrüg lichen merkmal für das vorhandensein eines rauchschadens bei pflanzen, insbesondere bei bäumen. ich glaube ein solches – wenigstens für akute rauchschäden – in dem verhalten der lenti zellen gefunden zu haben und bringe hier zunächst nur eine kurze vorläufige mitteilung darüber, indem ich mir die weitere aus arbeitung dieses diagnostischen merkmals vorbehalte. eine ausführliche darstellung der versuchsresultate werde ich – zusammen mit herrn dr. kupka, der mich bei dieser untersuchung getreulich unterstützte und dem ich auch für die herstellung der dieser mitteilung beigegebenen figuren zu danken habe – an anderer stelle bringen. nachdem ich schon vor mehreren jahren – zusammen mit meinem damaligen assistenten dr. lakon – den beweis geliefert hatte!), daß die giftigen gase in koniferennadeln ausschließ lich durch die spaltöffungen eintreten (topfpflanzen von fichte, tanne u. a. wurden, nachdem zuerst eine anzahl zweige geknickt worden waren, einer mäßig konzentrierten atmosphäre von s02 ausgesetzt, wobei sich zeigte, daß die nadeln nur unterhalb der knickungsstelle erkrankten und abstarben, während sie oberhalb jener stelle grün blieben, offenbar weil sich hier infolge von wassernot die spaltöffnungen geschlossen und kein giftiges gas hatten eintreten lassen), griff f. weber”) diese methode auf, um die wegsamkeit der lentizellen für gase zu veranschaulichen. er fand, daß schon nach kurzer einwirkung von nh3-gas das unter den lentizellen befindliche rindengewebe in mehr oder weniger weitem umkreis abstirbt und in der folge zusammen sinkt, so daß schließlich die lentizelle von einem kreisförmigen hof umgeben erscheint. *) neger und lakon, studien über den einfluß von abgasen auf die lebensfunktionen der bäume. mitt. k. s. forstl. versuchsanstalt tharandt, bd. i, 1914. *) weber, f ., über eine neue methode die wegsamkeit der lentizellen z u demonstrieren (gasdiffusionsmethode). ber. deutsche bot. ges. xxxiv, 1916, s . 73. ein neues, untrügliches merkmal für rauchschäden bei laubhölzern. 131 diese „gasdiffusionsmethode“ leistet gute dienste, um die wegsamkeit jedes durchlüftungsorganes der pflanzen (auch der spaltöffnungen) zu veranschaulichen, und verdient vielleicht wegen der einfachheit der versuchsanstellung sogar den vorzug vor den vonmolisch, stahl und dem verf. dieser zeilen vorgeschlagenen infiltrationsmethoden. es lag nun nahe, diese lentizellenbeschädigung durch giftige gase auch zum nachweis des vorhandenseins von rauchschäden zu verwenden. # fig. 1. querschnitt durch eine lentizelle von frarinus. 4 wochen nach der einwirkung des giftigen gases. an der grenze des gesunden und getöteten rindengewebes ein bogenförmig verlaufender wundkorkstreifen. (nach der natur gezeichnet von dr. kupka.) voraussetzung dafür ist allerdings, daß nicht nur hoch konzentrierte gase – wie sie f. weber verwandte – sondern auch sehr verdünnte gase – wie sie in der natur in der nähe von rauchquellen vorkommen – in die lentizellen eindringen und schädigungen des rindengewebes bewirken. es mußte also: 1. experimentell ermittelt werden, ob derartige verdünnte gase die von weber beschriebenen wirkungen aus üben; 2. beobachtet werden, ob auch in der freien natur in der nähe von rauchquellen solche beschädigungen auftreten. 9* 132 f. w. neger, a. laboratoriumsversuche mit sehr verdünnten gasen. die versuche wurden in der weise angestellt, daß nur das in der praxis am meisten in betracht kommende gas, die schweflige säure, verwendet wurde: die versuchsobjekte – teils topfpflanzen, teils abgeschnittene in wasser stehende zweige von esche, linde, ahorn, buche u. a.–wurden unter einem nahezu luftdicht schließen den glaskasten einer bestimmten konzentration von so2 aus gesetzt. durch einen elektrisch betriebenen ventilator wurde dafür gesorgt, daß das gasgemisch gleichmäßig durchwirbelt wurde, und schwadenbildung unterblieb. im folgenden führe ich nur einige der wichtigsten versuche an; ausführlicher soll – wie gesagt – an anderer stelle berichtet werden: versuch 1. (16–18 mai.) nach zweimaliger räucherung mit */1000 so2) am 18. mai deutliche reaktion bei spitzahorn und eiche, nicht bei esche, hainbuche, bergahorn, linde. nach weiteren zwei behandlungen mit der gleichen kon zentration deutliche reaktion auch bei bergahorn, nicht bei linde, esche. versuch 2. (26. mai) versuchspflanze esche (abgeschnittene zweige) und zwar a) noch im winterzustand, b) schon ausgeschlagen. so2 konzentration */1000, einmalige behandlung, bei a) nach 24 stunden fast keine reaktion „ b) „ 24 »an 3 zweigen 67 leutizellen beschädigt. versuch 3. (29. mai.) versuchspflanzen: buche und eiche, einmalige räucherung mit /1000 so2. buche: keine reaktion. eiche: deutliche einsenkung der umgebung zahlreicher leuti zellen; wo äußerlich nichts zu sehen war, war bräunung des gewebes mikroskopisch nachzuweisen. *) d. h. 1 ccm so, auf 1000 ccm luft. ein neues, untrügliches merkmal für rauchschäden bei laubhölzern. 133 in den versuchen 1–3 wurde diewirkung des sauren gases nur auf die lentizellen vorjähriger triebe beobachtet. lentizellen diesjähriger triebe sind in der regel weit empfindlicher und reagieren schneller als lentizellen vorjähriger triebe. meist ist d iewirkung am deutlichsten bei jenen lentizellen, die den knoten a m nächsten liegen, w o also die atmung – mit rücksicht auf d ie hier befindlichen knospen – am lebhaftesten ist. swirkunglentiellen . esche fig. 2 . in der mitte: querschnitt durch lindenzweig. lentizelle selbst nicht sichtbar. korkgewebe erst in bildung begriffen; unter dem getöteten rinden gewebe – ein streifen wundkork; links und rechts davon von höfen umgebene lentizellen a n linde und esche, schwächer und stärker vergrößert. (nach der natur gezeichnet von dr. kupka.) versuch 4 . a ) am 6 . juni kaltes, trübes wetter. b ) am 9 . juni heißes, sonniges wetter. konzentration /2ooo so. versuchszweige: esche, linde, spitzahorn, eiche (diesjährig und vorjährig). erfolg: a ) esche, diesjährig – keine wirkung, vorjährig – 77 77 134 f. w. neger, linde, diesjährig – deutlicher hof an zahlr. lentizellen, vorjährig – undeutliche wirkung, ahorn, diesjährig – keine wirkung, vorjährig – „ 77 eiche, diesjährig – „ ** vorjährig – „ »b) esche, diesjährig – deutliche wirkung, vorjährig – 77 7» linde, diesjährig – » 17 vorjährig – ** ** ahorn, diesjährig und vorjährig – deutliche wirkung, eiche, diesjährig – deutliche wirkung, vorjährig – keine wirkung. der versuch zeigt, daß– wie oben ausgeführt – diesjährige triebe im allgemeinen besser reagieren als vorjährige, sowie daß die reaktion bei warmem wetter kräftiger ist als bei kaltem. konzentrationen von */2000 so2 können als sehr hoch be zeichnet werden. solche kommen in der freien natur nur selten und nur in unmittelbarer nähe starker rauchquellen vor. es wurden daher noch versuche angestellt mit konzentrationen von /sooo, /1oooo und 20000 so2. versuch 5. (25. juni bis 3. juli.) konzentration /6000 so.. versuchszweige: linde, esche, spitzahorn. täglich wirkte etwa 1 stunde lang das gas in der angegebenen konzentration ein. an den ersten 6 tagen war keine reaktion zu erkennen, nur bei spitzahorn zeigte sich am 6. tag deutliche reaktion (a n einjährigen trieben), am 7 . und 8 . tag wurde die einwirkung auch bei linde und esche deutlich, bei letzterer a n vorjährigen trieben auffallender als a n diesjährigen. versuch 6. 5–8. juli, mit */1oooo so2. linde – keine reaktion, spitzahorn – vorjährige triebe 0, diesjährige triebe: deutliche höfe an zahl reichen lentizellen (eingesunkenes gewebe): auch mikroskopisch nachweisbar. esche – vorjährige triebe: hofbildung an zahlreichen lentizellen (mikroskopisch: bräunung des rindengewebes unter der lentizelle), diesjährige triebe – keine reaktion. ein neues, untrügliches merkmal für rauchschäden bei laubhölzern. 135 versuch 7. mitte juli bi s ende august, soooo so. topfpflanzen von linde, esche, spitzahorn und abgeschnittenen zweigen der gleichen baumarten. abgesehen von einigen pausen von 3–4 tagen wurden die versuchspflanzen alltäglich einige stunden der atmosphäre von 2oooo so2 ausgesetzt. schon nach 8 tagen wurden die ersten wirkungen sichtbar und zwar a n zweijährigen trieben von esche. merkwürdigerweise war nach wochenlang wiederholter einwirkung nicht mehr z u sehen als nach jener verhältnismäßig kurzen zeit. e s scheint daher, daß langandauernde einwirkung äußerst ver dünnter rauchgase doch kurze behandlung mit höher konzentrierten gasen nicht z u ersetzen vermag. gleichzeitig dürfte aus diesem versuch hervorgehen, daß die grenzkonzentration, bei welcher die lentizellenreaktion eintritt, (für so2) zwischen */1oooo und */2oooo liegt, konzentrationen, die in der nähe gefährlicher rauchquellen nicht selten vorkommen. zusammenfassung. die versuche 1–7 zeigen: als besonders empfindlich erwiesen sich esche, linde, spitz ahorn, weniger eiche, während buche, apfel, edelkastanie, eber esche, birke u. a. als wenig empfindlich gelten können. esche, linde, spitzahorn wären daher gewissermaßen als „fangpflanzen“ z u bezeichnen. besonders deutlich reagieren die lentizellen (bei esche) a n sehr kräftigen stark atmenden und transpirierenden trieben, weniger a n dünnen, spärlich mit lentizellen besetzten trieben. dies erklärt, warum topfpflanzen die reaktion weniger deutlich zeigten als abgeschnittene kräftige triebe älterer bäume. b . beobachtungen im freien in der nähe von rauchquellen. um zu ermitteln, o b die bei künstlichen versuchen auftreten den lentizellenbeschädigungen unter geeigneten umständen auch im freien beobachtet werden können, untersuchte ich die vege tation in der nähe anerkannter, schwere rauchschäden ver ursachender rauchquellen. leider is t hierzu die gelegenheit gegenwärtig wenig günstig, weil viele industrielle anlagen infolge d e s mangels a n betriebsmitteln ihre tätigkeit sehr eingeschränkt haben. immerhin gelang e s mir in drei fällen die genannten lenti zellenbeschädigungen im freien nachzuweisen und damit is t der 136 f. w. neger, beweis geliefert, daß das merkmal in der tat diagnostischen wert hat. a) schönheidehammer bei eibenstock. das werk is t seit langer zeit als rauchquelle schlimmster art bekannt. an einem (ca. 800 m entfernten) abhang östlich des werkes, auf welchen bei westwind die abgase getrieben und niedergeschlagen werden, wurden in einem pflanzgarten a n jungen buchen deutliche, die lentizellen umgebende höfe von eingesenk tem rindengewebe in großer anzahl beobachtet. es ist dies insofern besonders beachtenswert, als wie die künstlichen räucherungen zeigten, die buche z u den weniger empfindlichen holzarten gehört. b ) blaufarbenwerk bockau i. erzgeb. hier wurden etwa 100 m östlich der rauchquelle gleichfalls a n jungen buchen unverkennbare lentizellenbeschädigungen de r angegebenen art in großer anzahl nachgewiesen. besonders auf fallend war hier, daß die lentizellenhöfe stets nur an der der rauchquelle zugewendeten seite (luvseite) der zweige auftreten, nicht aber an der abgewendeten (leeseite). das mikroskopische bild is t hier – ebenso wie in schönheidehammer – genau das gleiche wie bei den künstlich erzeugten lentizellenbeschädigungen. c ) ölsnitz bei zwickau. durch freundliche vermittlung von herrn forstrat gerlach, tharandt, wurden mir aus ölsnitz im erzgebirge laubholzzweige (linde, esche, eiche) aus der nähe einer gefürchteten rauchquelle gesandt. hier zeigte sich, daß a n den lentizellen der linde offenbar durch andauernde einwirkung saurer gase tiefe löcher im rinden gewebe, ausgehend von den lentizellen, entstanden waren. wenn auch somit kein zweifel darüber bestehen kann, daß das merkmal geeignet ist die anwesenheit schwerer (akuter) rauchschäden einwandfrei nachzuweisen, so wäre doch wünschens wert weitere beobachtungen a n geeigneter stelle in der freien natur anzustellen und ich wäre daher für jede derartige zusen dung von herzen dankbar. ich habe das merkmal als „untrüglich“ bezeichnet und glaube dies aus folgenden gründen tun z u dürfen: es ist ausgeschlossen, daß durch andere vegetationsfeindliche faktoren derartige krankheitsbilder erzeugt werden, wie wir si e ein neues, untrügliches merkmal für rauchschäden bei laubhölzern. 137 unter dem einflusse saurer gase an lentizellen haben entstehen sehen: tötung und bräunung des gewebes unter der lentizelle in fast genau kugeliger ausdehnung – entsprechend der nach allen richtungen des raumes gleichmäßig erfolgenden ausbreitung eines gases. frost, trockenheit, hitze können wohl ganze sprosse zum absterben bringen, niemals aber derartige kugelige gewebekom plexe (mit der lentizelle als mittelpunkt) in lokaler begrenzung abtöten. während also, wie wir eingehends gesehen haben, durch hitze, frost, trockenheit usw. an blättern krankheitsbilder erzeugt werden können, die sich von jenen der rauchgaswirkung nicht unter scheiden, kann das oben beschriebene krankheitsbild an lenti zellen nur durch giftige gase hervorgebracht werden. der wert des merkmals liegt ferner darin, daß laubbäume (bes. esche, linde, ahorn) sich fast überall auch in unmittelbarer nähe von rauchquellen befinden, die möglichkeit des nachweises daher fast überall gegeben ist. wo äußerlich höfe um die lentizellen nicht erkennbar sind, wird man die mikroskopische untersuchung zu hilfe nehmen– bräunung des rindengewebes unter den lenti zellen und abgrenzung des abgestorbenen gewebes gegen das gesunde durch wundkorkbildung (s . fig.) ein faktor, der die reaktion der lentizellen auf saure gase beeinträchtigt, is t folgender: im winter sind die lentizellen bei den meisten laubhölzern geschlossen!) und lassen daher kein saures gas eintreten; im sommer schützt die belaubung, solange sie durch die sauren gase nicht getötet und zum abfall gebracht ist, die lentizellen teil weise vor einwirkung der rauchgase. diese tatsachen sind bei der anwendung des merkmals im freien in betracht zu ziehen. zum schluß möchte ich noch auf eine erscheinung aufmerk sam machen, über die ich aber noch kein abschließendes urteil abgeben kann: angenommen, a n einem zweig sind alle lentizellen beschädigt und das darunter befindliche abgestorbene rindengewebe gegen d a s gesunde durch eine wundkorkschicht abgeschlossen: werden unter den alten, ausgeschalteten lentizellen neue entstehen? wenn nicht, wie vermag der betreffende zweig weiter zu atmen? viel leicht ist das allmähliche absterben von laubholzzweigen in der *) vergl. klebahn, die rindenporen. jenaische z . f. naturw. 1884. 138 f. w. neger, ein neues, untrügliches merkmal für rauchschäden. nähe von rauchquellen auf eine art von erstickungstod infolge der ausschaltung der lentizellen zurückzuführen. diese und mehrere andere sich anschließende fragen werden den gegenstand weiterer untersuchungen bilden. nachtrag: einen weiteren fall von lentizellenbeschädigung habe ic h inzwischen bei dohna (bez. dresden) beobachtet, und zwar an apfelfrüchten (wahrscheinlich durch einwirkung von flußsäure). äußerlich war die beschädigung a n höfen, welche die lentizellen umgaben, erkennbar. merkwürdig ist, daß das fruchtfleisch des apfels nicht die fähigkeit besitzt, das abgestorbene gewebe durch wundkork abzugrenzen. infolgedessen greift hier die schädigung sehr schnell um sich. der gegenwärtige stand der kohlensäurefrage für pflanzenkulturen. von dr. hugo fischer. als kolumbus seine erste entdeckerfahrt plante, sprach sich ein kollegium hochgelehrter männer mit allen gegen eine stimme dahin aus, daß der gedanke einer erdumsegelung barer unsinn sei. als die eisenbahn erfunden war, gab ein rat sachverständiger männer das urteil ab, es sei das gewiß eine recht interessante sache, aber davon könne keine rede sein, daß das neue verkehrs mittel jemals zur überwindung großer entfernungen oder zur beförderung größerer lasten dienen könne. dieses urteil erfolgte einstimmig. die mendelschen spaltungsregeln, das einmaleins der ver erbungsforschung, haben rund 3 5 jahre in verborgenheit ge schlummert, ehe sie die verdiente anerkennung fanden. e s is t nicht richtig zu meinen: „die zeit sei nicht reif gewesen“; man würde den führenden männern von damals doch gar zu wenig zu trauen, wollte man sagen, jene regeln seien ihnen „zu hoch“ gewesen – im gegenteil, zu einfach waren sie. uc1.b3299303_page_147_cut uc1.b3299303_page_148_cut uc1.b3299303_page_149_cut uc1.b3299303_page_150_cut uc1.b3299303_page_151_cut uc1.b3299303_page_152_cut uc1.b3299303_page_153_cut uc1.b3299303_page_154_cut uc1.b3299303_page_155_cut uc1.b3299303_page_156_cut art16_bratek.indd journal of applied botany and food quality 85, 100 104 (2012) 1department of plant physiology and molecular plant biology, eötvös loránd university, budapest, hungary 2 department of botany, faculty of veterinary science, szent istván university, budapest, hungary 3vác, hungary mineral composition of hypogeous fungi in hungary á.k. orczán1, j. vetter2, z. merényi1, é. bonifert3, z. bratek1* (received october 20, 2011) * corresponding author introduction in the course of the work, 93 samples from 17 hypogeous fungus species belonging to 6 genera were taken from various habitats in hungary and were analysed for the concentrations of 22 elements using the inductively coupled plasma spectroscopy icp method. all the measurements were made in three independent replications. the data were compared with the element contents of 625 epigeous fungi, previously determined using the same method. for all the genera, the elements present in the highest concentrations on a dry matter basis were potassium (6990-29590 ppm) and phosphorus (3400-9140 ppm). these were followed by the macroelements calcium (330-2190 ppm), magnesium (810-1000 ppm) and sodium (110-2990), and the microelements aluminium (30-450 ppm), zinc (60-340 ppm), iron (30-120 ppm) and copper (25-75 ppm), in different orders for each genus. until now the element contents of fungi have mostly been analysed to determine the nutritional value of edible fungi, and the data on other elements for instance total minerals are insuffi cient for further comparisons (mattila et al., 2001). very little work has been published on the mineral contents of hypogeous large fungi, despite the fact that these include commercially important species such as tuber aestivum and t. melanosporum (ian et al., 2003). most of the previous papers exhibited the following characteristics: (1) some species (e.g. terfezia species, tuber melanosporum) were investigated more frequently, and others rarely, if at all; (2) the analyses concentrated chiefl y on toxicological and/or environmental aspects; (3) measurements were only made on a few elements (important from the nutritional point of view); (4) only cultivated fungi were included in the studies. the aim of the present work was to determine the element contents of various species of hypogeous fungi in order to answer the following questions: (1) which characteristic differences can be observed between the element contents of hypogeous and epigeous fungi? (2) which differences characterise the element contents of various genera of hypogeous fungi? (3) is there any signifi cant difference between the element contents of hypogeous ascomycota and basidiomycota genera? (4) can any signifi cant difference be observed between the element contents of edible and non-edible hypogeous fungi? materials and methods a total of 93 hypogeous fungus samples were collected from habitats covering the whole area of hungary, and were shown by macroand microscopic analysis to represent 17 species belonging to 6 genera. the species names and number of samples are listed in tab. 1. after cleaning and drying, the fruit bodies were ground. the samples (200 mg of fungi powder) were digested in closed tefl on bombs in triplicate (2 cm3 hno3 + 2 cm3 h2o2 /30%/) at 1.56×105 pa pressure for 20 min. the digested materials were fi ltered and diluted to 10 cm3, after which the mineral element contents were determined by inductively coupled plasma spectroscopy. the data were evaluated using the graphpad instat program (version 3.00, 32 bit for win 95/nt, graphpad software, san diego, california, usa, www.graphpad.com) and the kolmogorov-smirnov test was used to determine the normality of the data. for data with normal distribution, one-way anova was carried out using the bonferroni post hoc test and the d (welch) test, while in the case of non-normal distribution the kruskall-wallis test was applied, using dunnett’s post test (dunn, 1964) to identify signifi cant differences. results and discussion the results obtained for the icp analysis of 22 elements are presented in tab. 2 for the six hypogeous fungus genera (elaphomyces, gautieria, mattirolomyces, melanogaster, rhizopogon, tuber) in comparison with a database containing data on 625 samples of epigeous fungi (vetter, 2003). by comparing the mean quantities of each element, three groups of elements could be distinguished, as illustrated in tab. 3. as can be seen in the tab. 3, the fruit bodies of the two fungal groups had very similar mean contents of aluminium, boron, copper, strontium and titanium, while hypogeous fungi generally contained larger quantities of barium, calcium, molybdenum, sodium and zinc. on the other hand, the fruit bodies of hypogeous species had lower mean contents of arsenic, cadmium, cobalt, chromium, iron, potassium, magnesium, manganese, nickel, phosphorus, selenium and vanadium. three of the macroelements (potassium, magnesium and phosphorus) were thus found in this group. the higher mean contents of arsenic, cadmium, selenium and vanadium in epigeous tab. 1: species names and number of the samples species name: number of samples: elaphomyces aculeatus vittad. 6 elaphomyces granulatus fries 2 elaphomyces muricatus fries 33 elaphomyces persoonii vittad. 1 elaphomyces reticulatus vittad. 2 elaphomyces virgatosporus hollós 6 gautieria borealis states, fogel & hosford 3 mattirolomyces terfezioides (mattir.), e. fisch 2 melanogaster ambiguus (vittad.) tulasne & c. tulasne 2 rhizopogon roseolus (corda) th. m. fries 3 rhizopogon vulgaris var. intermedius svrcek 10 tuber aestivum vittad. 11 tuber brumale vittad. 1 tuber excavatum vittad. 4 tuber ferrugineum vittad. 1 tuber mesentericum vittad. 3 tuber rapaeodorum tul. 3 mineral composition of hypogeous fungi in hungary 101 fungi can be explained by the fact that many fungal taxa, such as the amanita genus, are known to accumulate these elements (vetter, 2005). giaccio et al., 1992 detected that copper and cadmium might be accumulated in the 7 studied truffl e species, zinc appeared to be accumulated only in a certain range, while other examined elements did not seem to be accumulated. granetti et al., 2005 also detected copper, cadmium and zinc accumulation in t. melanosporum. maser et al., 2008 found high phosphorus and potassium, while low sodium and calcium content in fruit bodies, especially in comparison with eggs, milk and vegetables. potassium, sodium and calcium as well as other micronutrients, however, could vary markedly among and within species, but in general, fruit bodies were particularly rich in manganese, copper and zinc. based on the complete database of the present studies in case of hypogeous species the difference between the minimum and maximum concentration of each element was smallest for phosphorus (8x) and magnesium (9x) and greatest for molybdenum (1780x), arsenic (835x) and boron (940x). in case of epigeous fungi the ranges were wider. the smallest difference was found for magnesium (13x) and the greatest for boron (3300x). it could also be seen that the most important elements (potassium, phosphorus, calcium, magnesium) were among those with the smallest coeffi cient of variation in the fruit bodies of both fungal groups. when the deviation values were compared between genera it was observed that sodium was generally among the elements exhibiting the greatest deviation, with the exception of the elaphomyces genus, where it had the smallest deviation in both relative and absolute terms. of the four major elements, the deviation for calcium was the greatest in all the genera except rhizopogon, where it was almost the smallest. averaged data groups are presented in tab. 4 for the six hypogeous fungus genera to which most of the samples belonged (elaphomyces, tuber, rhizopogon, gautieria, mattirolomyces, melanogaster), together with the databases of the epigeous fungi used for comparative purposes. the potassium contents of all the hypogeous fungal genera proved to be lower than that in epigeous fungi that tend to concentrate potassium 20-40-fold in fruit bodies (seeger, 1978), but were the highest amounts in all hypogeous genera. granetti et al., 2005 also showed potassium to be the highest content element in t. melanosporum. the lowest value of potassium (6994 ppm) was recorded for the elaphomyces genus, which was very low even compared with the other hypogeous genera. the opposite tendency was observed for sodium, an element of great importance from the biological point of view, as the sodium contents of the hypogeous fungi were higher than those measured in epigeous fungi, particularly in the case of elaphomyces. the only exception was the tuber genus, where the sodium content was approximately tuber genus, where the sodium content was approximately tuber the same as in epigeous fungi, and mattirolomyces, which exhibited a value of only 111 ppm, far lower than the mean na content of epigeous fungi and even lower than the aluminium content of this genus. in the case of calcium the average content was higher in hypogeous fungi, but this was principally due to the high values for tuber and tuber and tuber melanogaster, as the calcium contents of the other genera were similar to the average for epigeous fungi. in fact, that of gautieria was extremely low (330 ppm). the mean phosphorus content of the hypogeous fungi (5625 ppm) was well below the level recorded for epigeous fungi (7800 ppm). the phosphorus content of the tuber genus hardly surpassed the epigeous mean (7810 ppm), while that of mattirolomyces was far higher (9140 ppm). in the case of magnesium, which content depends on species and genus (seeger and manfred, 1979), none of the hypogeous genera had contents as high as the mean for the epigeous fungi (1440 ppm), with an average level of 870 ppm. it is interesting to note that the calcium: phosphorus and potassium:sodium ratios were also very different for the various genera. among the biologically important microelements, no signifi cant differences could be detected in the contents of copper and boron. when the data series for the hypogeous fungi were analysed at the genus level (tab. 4), it could be seen that, despite the similarity of the means, samples from the gautieria, mattirolomyces and melanogaster genera had very low, hardly detectable contents melanogaster genera had very low, hardly detectable contents melanogaster of boron. the average quantity of manganese (17 ppm) in the hypogeous fungi was considerably lower than the mean value for the comparative database (40 ppm), yet the two data available for the melanogaster genus averaged 300 ppm! a comparison of the mean contents of zinc indicated that the hypogeous fungi contained considerably more of this element (260 ppm) than their epigeous counterparts (115 ppm). when the genera were evaluated separately it could be clearly seen that the elaphomyces genus, which was tab. 2: average element concentration of hypogeous and epigeous fungi with standard deviation average of average ofaverage ofa elements 97 hypogeous materials 625 epigeous materials (ppm) (ppm) al 150 ± 140 150 ± 217 as 4 ± 12 9 ± 15 b 13 ± 18 16 ± 45 ba 8 ± 8 5 ± 5 ca 1770 ± 1900 1460 ± 1270 cd 2 ± 4 3 ± 7 co 1 ± 1 1 ± 1 cr 2 ± 1 3, ± 9 cu 60 ± 40 60 ± 50 fe 100 ± 80 240 ± 300 k 14800± 10500 34900 ± 14800 mg 870 ± 300 1440 ± 640 mn 17 ± 57 43 ± 45 mo 2 ± 12 0,7 ± 0,6 na 1880 ± 1600 325 ± 380 ni 2 ± 2 4 ± 6 p 5625 ± 2625 7800 ± 4300 se 0,3 ± 0,8 5 ± 4 sr 5 ± 3 5 ± 5 ti 2 ± 5 3 ± 3 v 0,4 ± 0,4 2 ± 14 zn 260 ± 160 115 ± 110 total 25600 46558,00 tab. 3: grouping of elements based on their mean concentrations in hypogeous and epigeous fungi mean element concentration elements nearly identical al, b, cu, sr, ti hypogeous species > epigeous species ba, ca, mo, na, zn hypogeous species < epigeous species as, cd, co, cr, fe, k, mg, mn, ni, p, se, v 102 á.k. orczán, j. vetter, z. merényi, é. bonifert, z. bratek represented by a large number of samples, had the highest zinc concentration (340 ppm), while that of gautieria and melanogaster was much lower (around 60 ppm). among the elements of interest from the toxicological point of view, the arsenic content was below the mean value recorded for the epigeous fungi, with the exception of the gautieria genus, though it must not be forgotten that the relatively high average value (9 ppm) for epigeous fruit bodies was infl uenced by the presence of several accumulator genera (particularly species belonging to the agaricus and macrolepiota genera). this was also true of the concentrations of cadmium and chromium. seeger (1978a) found highly scattered values of cadmium content in 402 epigeous fungi, and demonstrated that it was clearly species-dependent, and to a lesser extent genusdependent. the quantity of nickel was also lower in the hypogeous fungus group (1,9-3,6 ppm), though samples from the gautieria genus had a surprisingly high content (14,7 ppm). the concentrations of selenium and vanadium must be viewed with similar reservations. in both cases the hypogeous fungi had very low values (in many cases below the detection limit) compared with most of the epigeous fungi, but here again the very high values recorded for a number of accumulating taxa (boletus species in the case of selenium and amanita muscaria for vanadium) were responsible for the higher average values for this fungal group. in terms of the total mineral elements (tab. 2 and 5), considerable differences were observed, as the total element content of the hypogeous fungi (25 ppm) was substantially less than that of fungi with epigeous fruit bodies (45 ppm). the analysis of individual genera revealed that the mineral contents of tuber and tuber and tuber mattirolomyces were closest to those of the group used for comparison. in tab. 5 the hypogeous fungi were divided according to their taxonomic groupings into ascomycota (76 samples) and basidiomycota species (21 samples). when the data for these groups were compared the following differences were found: a. both taxonomic groups of hypogeous fungi contained approximately equal quantities of the elements b, ba, ca, fe, cr, cu, mg, p and sr; b. the element content of the ascomycota group was greater than that of the basidiomycota group in the case of al, na, ti, v and zn; c. the basidiomycota had higher concentrations of as, cd, co, k, mn, mo, ni and se. a comparison of the total element quantities showed that hypogeous fungi belonging to the basidiomycota had considerably higher element contents than the ascomycota, mainly due to the great difference in the potassium concentrations. it should be noted, however, that the ascomycota contained more zinc. the hypogeous fungi were also grouped on the basis of edibility (tab. 6), with 14 samples in the edible group and the majority (83) being inedible. the two groups exhibited a number of substantial differences, with higher quantities of boron, calcium, copper and magnesium, much higher contents of potassium and phosphorus, and lower quantities of zinc, manganese and sodium in the edible species. all in all, these fungi contained an average of 38580 ppm mineral elements, as compared with 23370 ppm in the inedible group. tab. 4: average element concentration of six hypogeous fungi taxa and epigeous fungi with standard deviation elements average of average of average of average of average of average of average of 50 elaphomyces 3 gautieria 2 mattirolomyces 2 melanogaster 13 rhizopogon 23 tuber 625 epigeous materials materials materials materials materials materials fungi materials (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) al 175 ± 120 30 ± 3 450 ± 480 250 ± 210 70 ± 40 130 ± 160 150 ± 210 as 3 ± 10 20 ± 5 0,1 ± 0,0 4 ± 5 7 ± 25 0,1 ± 0,0 9 ± 15 b 12 ± 15 0,1 ± 0,0 0,1 ± 0,0 6 ± 8 13 ± 25 15 ± 20 15 ± 45 ba 9 ± 75 2 ± 0,0 3 ± 0,5 15 ± 1 9 ± 10 5 ± 3 4 ± 5 ca 1455 ± 2030 330 ± 130 1260 ± 330 2190 ± 1300 1570 ± 730 2170 ± 1250 1460 ± 1270 cd 1 ± 45 1 ± 0,2 0,6 ± 0,0 0,1 ± 0,0 3 ± 6 3 ± 2 3 ± 7 co 0,5 ± 0,65 4 ± 2 0,1 ± 0,0 0,3 ± 0,2 1 ± 1 0,2 ± 0,4 1 ± 1 cr 1,3 ± 1 4 ± 0,3 2 ± 0 0,6 ± 0,6 1 ± 1 1 ± 0,5 3 ± 9 cu 50 ± 20 25 ± 3 80 ± 60 35 ± 15 50 ± 70 65 ± 35 60 ± 50 fe 100 ± 65 32,20 ± 3 55 ± 6 75 ± 5 120 ± 95 110 ± 110 240 ± 305 k 6990 ± 4590 18400 ± 1400 29585 ± 210 17295 ± 8000 23395 ± 13750 24110 ± 4245 34865 ± 14760 mg 820 ± 335 890 ± 80 1000 ± 50 810 ± 320 830 ± 390 955 ± 210 1440 ± 640 mn 8 ± 7 20 ± 5 10 ± 0 305 ± 365 13 ± 15 10 ± 7 45 ± 45 mo 0,2 ± 0,1 0,1 ± 0,0 0,2 ± 0,1 0,5 ± 0,5 10 ± 35 0,2 ± 0,2 0,7 ± 0,6 na 2990 ± 995 420 ± 170 110 ± 2,76 570 ± 505 870 ± 440 330 ± 280 325 ± 385 ni 1 ± 1 15 ± 1 1 ± 0,4 1 ± 0,2 2 ± 2 1 ± 0,5 3 ± 6 p 4515 ± 2090 4970 ± 455 9140 ± 460 3405 ± 150 6030 ± 3410 7810 ± 2015 7800 ± 4320 se 0,2 ± 0,6 0,1 ± 0,0 0,1 ± 0,0 0,1 ± 0,0 1 ± 1 0,1 ± 0,0 5 ± 4 sr 3 ± 2 2, ± 1 5 ± 3,30 7 ± 4 4 ± 2 7 ± 4 5 ± 5 ti 2 ± 1 0,2 ± 0,2 0,4 ± 0,1 1 ± 0,2 0,8 ± 0,5 4 ± 10 3 ± 3 v 1 ± 1 0,1 ± 0,0 0,2 ± 0,1 0,5 ± 0,5 0,2 ± 0,1 0,3 ± 0,2 2 ± 14 zn 340 ± 130 60 ± 10 90 ± 1 55 ± 15 135 ± 105 190 ± 65 115 ± 110 total 17480 25225 41800 25015 33145 35915 46560 mineral composition of hypogeous fungi in hungary 103 tab. 5: average element concentration of hypogeous ascomycetes and basidiomycetes and epigeous fungi with standard deviation elements average of average of average of 76 ascomycetes materials 21 basidiomycetes materials 625 epigeous fungi materials (ppm) (ppm) (ppm) al 170 ± 150 85 ± 80 150 ± 220 as 2 ± 10 7± 20 9 ± 15 b 13 ± 18 12 ± 20 16 ± 45 ba 8 ± 6 10 ± 10 5 ± 5 ca 1770 ± 2000 1760 ± 1490 1470 ± 1270 cd 2 ± 3 3 ± 5 3 ± 7 co 0,4 ± 0,5 1 ± 1 1 ± 2 cr 1 ± 0,8 2 ± 2 3 ± 9 fe 100 ± 80 100 ± 85 200 ± 300 cu 60 ± 30 50 ± 60 60 ± 50 k 13000 ± 9400 21600 ± 12000 34900 ± 14800 mg 900 ± 300 845 ± 320 1400 ± 600 mn 9 ± 7 50 ± 120 40 ± 45 mo 0,2 ± 0,2 6 ± 30 0,7 ± 0,6 na 2100 ± 1500 1100 ± 1750 325 ± 380 ni 1,0 ± 0,7 4 ± 4 4 ± 6 p 5680 ± 2600 5400 ± 2800 7800 ± 4320 se 0,1 ± 0,5 0,7 ± 1 5 ± 5 sr 5 ± 3 4, ± 2 5 ± 5 ti 2 ± 6 0,8 ± 0,5 3 ± 4 v 0,4 ± 0,4 0,2 ± 0,2 2 ± 14 zn 300 ± 150 120 ± 90 115 ± 110 total 24000 31200 45700 tab. 6: average element concentration of edible (tuber aestivum, tuber brumale, mattirolomyces terfezioides) and non-edible hypogeous and epigeous fungi with standard deviation elements average of average of average of 14 edible hypogeous fungi materials 83 non-edible hypogeous fungi materials 625 epigeous fungi materials (ppm) (ppm) (ppm) al 225 ± 250 140 ± 110 150 ± 220 as 0,1 ± 0,0 4 ± 13 9 ± 15 b 15 ± 18 10 ± 18 15 ± 45 ba 6 ± 3 9 ± 8 5 ± 5 ca 2535 ± 1200 1600 ± 1970 1500 ± 1270 cd 3 ± 2 2 ± 4 3 ± 7 co 0,1 ± 0,0 1 ± 1 1 ± 1 cr 1 ± 0,4 2 ± 1 3 ± 9 cu 75 ± 40 50 ± 40 60 ± 50 fe 125 ± 120 100 ± 70 240 ± 300 k 25800 ± 2700 13000 ± 10200 34865 ± 14760 mg 1000 ± 200 840 ± 310 1440 ± 640 mn 10 ± 7 20 ± 60 40 ± 45 mo 0,2 ± 0,2 2 ± 10 0,7 ± 0,6 na 240 ± 200 2200 ± 1600 325 ± 380 ni 1 ± 0,5 2 ± 2 4 ± 6 p 8300 ± 1700 5200 ± 2480 7800 ± 4320 se 0,1 ± 0,0 0,3 ± 1 5 ± 5 sr 8 ± 3 4 ± 3 5 ± 5 ti 2 ± 3 2 ± 6 3 ± 3 v 0,1 ± 0,1 0,5 ± 0,4 2 ± 14 zn 170 ± 60 270 ± 165 115 ± 110 total 38582,27 23460 46590 104 á.k. orczán, j. vetter, z. merényi, é. bonifert, z. bratek conclusion in our work we got a comprehensive view of the element content of several hypogeous fungi genera. compared to the epigeous fungi we found higher element content in the hypogeous fungi. the main general difference was in the potassium level, however that was the highest of all the hypogeous genera and the epigeous fungi of the investigated elements. the most important elements (potassium, phosphorus, calcium, magnesium) in both groups belong to elements with the smallest coeffi cient of variation. we found signifi cant difference in the studied questions, although the content of elements is strongly dependent on genera and species. acknowledgements the authors would like to express their grateful thanks to józsef garay, of the department of plant taxonomy and ecology, eötvös loránd university (elte), budapest for his help with the statistical analyses, and to szabolcs rudnóy and gabriella tamaskó, of the department of plant physiology and molecular plant biology, elte, budapest and to hanna dóra orczán for their constant support. this research was funded by a grants from the hungarian ministry of education (no. om-00055/2006 and om-00-312/2008). references giaccio, m., di giacomo, f., marchegiani, r., 1992: chromium, manganese, nickel, cooper, zinc, cadmium and lead content in some species off truffl es. micologia e vegetazione mediterranea, vii(2): 287-294. granetti, b., angelis, a., materozzi, g., 2005: umbria terra di tartufi, gruppo micologico ternano, terni, composizione chimica e valore nutritivo del tartufo, 76-78. hall, i.r., wang, y., amicucci, a., 2003: cultivation of edible ectomycorrhizal mushrooms. trends in biotechnology 21, 433-438. mattila, p.h., könkö, k., eurola, m., pihlava, j.m., astola, j., vahteristo, l., hietaniemi, v., kumpulainen, j., valtonen, m., piironen, v., 2001: contents of vitamins, mineral elements, and some phenolic compounds in cultivated mushrooms. j. agric. food chem. 49, 2343-2348. maser, c., claridge, a.w., trappe, j.m., 2008: tress, truffles, and beasts how forests function. rutgers university press, new brunswick, new jersey, london, 94-95. dunn, o.j., 1964: multiple contrasts using rank sums. technometrics 5, 241-252. seeger, r., 1978a: cadmium in pilzen. z. lebensmittel untersuchung und forschung 166, 23-43. seeger, r., 1978: kaliumgehalt höherer pilze. z. lebensmittel untersuchung und forschung 167, 23-31. seeger, r., beckert, m., 1979: magnesium in höheren pilzen. z. lebensmittel untersuchung und forschung 168, 264-281. vetter, j., 2003: monograph of the mineral composition of basidiomes of higher fungi (in hungarian). a fi nal report of the research program otka (hungarian scientifi c research foundation) no. 31702. manuscript, budapest, 1-99. vetter, j., 2005: mineral composition of basidiomes of amanita species. mycological research 109, 746-750. address of the author: z. bratek (corresponding author), e-mail: bratek@caesar.elte.hu journal of applied botany and food quality 95, 167 173 (2022), doi:10.5073/jabfq.2022.095.021 1institute of botany, university of the punjab, lahore, pakistan domestication and element analysis of the giant edible macrocybe gigantea from pakistan aneeqa ghafoor1, abdul rehman niazi1*, najam-ul-sehar afshan1 (submitted: january 11, 2022; accepted: june 22, 2022) * corresponding author summary during a survey of mushrooms of pakistan, macrocybe gigantea was collected from university of the punjab, lahore, pakistan under the morus species. for the domestication of this wild species, its culturability and cultivation potential was assessed by using different synthetic culture media and substrates. among these different media used, maximum cultural growth was observed on potato dextrose agar (pda) medium at 30 ℃ followed by malt extract agar (mea), compost extract agar (cea), glucose peptone agar (gpa), and saboraud dextrose agar (sda). strains on pda medium were used for production of spawning material on wheat, sorghum and barley grains. sorghum grains at 30 ℃ were the best combination for spawn production. a mixed substrate of wheat straw and tea waste at 30 ℃ produced the highest yield. mineral analysis of the wild and cultivated strain revealed that both forms enrich potassium and calcium. these findings show that this giant edible mushroom species could be domesticated on the number of media and substrates. its domestication can provide nutritional, economical, medicinal and tasty food to the growing population that would otherwise be restricted to natural production at a specific time of the year. keywords: biological efficiency, culturability, cultivation potential, macrocybe gigantea, spawn. introduction the genus macrocybe pegler & lodge emerged out in 1998 as separate entity from the genus tricholoma on the basis of morphological and molecular evidences (pegler et al., 1998). species of tricholoma are obligatory ectomycorrhizal with clampless hyphae while the species of macrocybe are large, saprophytic with clamped hyphae (pegler et al., 1998; razzaq et al., 2017). recent comparative genomic approaches also support that macrocybe is a stand-alone genus from tricholoma (kui et al., 2021). it has nine well-recognized species, distributed in the tropical regions of the world, which include m. gigantea (massee) pegler & lodge, m. pachymeres (berk. & broome) pegler & lodge, m. praegrandis (berk.) m. spectabilis (peerally & sutra) pegler & lodge, m. crassa (sacc.) pegler & lodge, m. praegrandis (berk.) pegler & lodge, m. titans (h.e. bigelow & kimbr.) pegler, lodge & nakasone, m. lobayensis (r. heim) pegler & lodge and macrocybe sardoa vizzini, consiglio & m. marchetti and most of these species are edible (pegler et al., 1998; duong et al., 2017; vizzini et al., 2020). initially, the macrocybe species were only studied for pharmaceutical purposes (chatterjee et al., 2011), while their farming is relatively new as compared to other commonly cultivable mushrooms. m. gigantea (massee) pegler & lodge belongs to family tricholomataceae under the order agaricales and was first reported as tricholoma giganteum (massee, 1912). this giant mushroom is mainly confined to the pantropical regions (asia and africa) and commonly found in the west bengal region. however, it is also reported from the japan, usa, and korea (khatua and acharya, 2016; ghosh and acharya, 2022). it grows gregariously in shady, grassy areas or with angiospermic trees in high temperature and humidity and occurs in hylaea and subtropical rain forests in africa and asia (huang, 2001). it is mostly grown in cluster form of about 20 to 30kg (kui et al., 2021). it has a smooth cap, whitish to greyish white in color, the gills are straw yellow in color and are crowded. the stipe is 15-18 cm in length and 6 cm in diameter and its color is same as that of the cap. the spore print is white (pegler et al., 1998; kui et al., 2021). it contains water-soluble polysaccharides and certain bioactive compounds that have a pharmaceutical value (kui et al., 2021) as anti tumor, anticancer, antioxidant and antimicrobial agent (chatterjee et al., 2011; gaur and rao, 2016). macrocybin, a natural triglyce ride present in macrocybe species reduces tumor cells both in vitro and in vivo by interacting with the actin cytoskeleton (vilarino et al., 2020). ethanolic extracts of m. gigantea revealed the presence of antioxidants like vitamin e, colchicines, and 5-methyldioadenosine. (acharya et al., 2012). it is also rich in mineral elements such as ca (38-470 mg/kg), mg (84-550 mg/kg) and zn (16-160 mg/kg) (liu et al., 2012). m. gigantea is also valuable because it sustains high temperature range 30~38 ℃ in addition to nutrient and taste (amin et al., 2010). due to these nutritional and therapeutic attributes, (chatterjee et al., 2011; niazi and ghafoor, 2021) it could be advantageous to grow this fungus at industrial scale for maximum benefits. m. gigantea can meet the demand of food of a growing population due to both nutritional and therapeutic peculiarities. however, in wild form, there is a chance of radioactive contamination, which can be overcome by the cultivation under controlled conditions (falandysz et al., 2015). cultivation practices of m. gigantea, m. crassa, and m. lobayense are described in literature but none of the species farming status reached the industrial scale in any country. initially, m. gigantea was commonly cultivated as tricholoma gigentium (yoshikazu and takashi 1997; dadwal, 1984; lu, 1992; kim et al., 1998; kinjo and miyagi, 2006; chen et al., 2012; inyod et al., 2016) as one of the largest edibles tricholomatoid agarics of southeast asia. it is being cultivated on a small scale in india and china but requires more research and refinement. generally, it requires a temperature between 25-35 °c, 70-80% relative humidity and light of 8-10 h for its growth (razaq et al., 2016; inyod et al., 2016; verma et al., 2017; akhtar et al., 2019; devi and sumbali, 2021). from pakistan, only one species of m. gigantea is reported (razaq et al., 2016). pakistan’s climatic conditions are favourable for its natural growth but its domestication was never tried before. the aim of this study was to optimize the standard cultivation requirements of the m. gigantea and to compare the element composition of the wild and artificially cultivated fruiting bodies to get maximum benefit from this edible mushroom. its domestication can compete with the nutritional peculiarities of the widely growing edible fungal species like button and oyster strains. 168 a. ghafoor, a.r. niazi, n.-u.-s. afshan a step towards sustainable development goal 2 “zero hunger” the findings will help open new possibilities for food production, namely large scale production of macrocybe gigantea, and contribute to sustainability by optimizing the use of waste materials as substrates for food production. material and methods sampling and experimental design basidiomata of the m. gigantea were collected in gregarious form under the morus species from university of the punjab, lahore pakistan. the collected specimen were photographed using an android camera and identified by macro-microscopically and phylogenetically according to the literature available (razzaq et al., 2016). the experiments were carried out in fungal biology and systematics research lab, institute of botany, university of the punjab, lahore. specimen m. gigantea (lah03821), was deposited in the herbarium, institute of botany, university of the punjab, lahore, pakistan (lah) for ready reference. evaluation of culturability culturability of m. gigantea was assessed according to the method described by siddiq et al., 2018. small tissues from inner unexposed part of the fruiting body were placed onto five different nutrient agar media i.e, malt extract agar (2% mea: agar 20 g, malt extract 20 g dissolved into 1000 ml dh2o), potato dextrose agar (2% pda: thin potato slices 200 g, glucose 20 g, agar 20 g per liter of dh2o), glucose peptone agar medium (2% gpa: 20 g peptone, 20 g dextrose, 5 g nacl, 15 g agar dissolved into 1000 ml dh2o), saboraud dextrose agar ( 2% sda: 15 g agar, 40 g dextrose, 10 g peptone dissolved into 1000 ml dh2o) and compost extract agar (2% cea: 20 g agar,10 g glucose dissolved into 1000 ml wheat straw water based filtrate). inoculated petri plates were sealed with paraffin film and then incubated at different temperatures i.e., 15 ℃, 20 ℃, 25 ℃, 30 ℃, and 35 ℃. mycelial growth characteristics were observed on regular basis. each effect was determined in triplicates. these mushroom cultures were also deposited in the herbarium culture collection of university of the punjab, lahore (as lah#072021c(abcde). spawn production spawn was prepared by following pal and thupa (1979) described methodology. three types of cereal grains viz., sorghum, wheat, and barley grains were used as the substrate to determine the spawn production efficiency. for spawn preparation, grains were washed and soaked for overnight, boiled for half an hour and excess water from grains was removed by spreading them on blotting paper. three quarters of the each 1 l filter jars was filled with boiled grains supplemented with gypsum (2 g) and lime (1 g) and then autoclaved. spawns were prepared by inoculating mycelial discs from pure pda culture on sterilized grains in a laminar air flow cabinet. inoculated grains were incubated at 30 ℃. effect of grains on production of spawning material was determined in triplicates. substrate production wheat straw, sawdust and tea waste were used as the raw material for substrate production. dried wheat straw collected from the field area, university of the punjab, lahore, sawdust collected from the furniture shop, while tea-waste collected from the hostel canteens, university of the punjab, lahore. tea waste is just the left-over residues of tea (water containing tea) after usage. it is enriched with cellulose, hemicellulose, proteins, lipids, polyphenols as well as many minerals (zhu et al., 2013). six types of substrates were prepared. three of pure types i.e., wheat straw, sawdust and tea-waste while three were of mixed type i.e., sawdust and wheat straw, tea waste and saw dust and tea waste and wheat straw. for substrate production (pure and mixed types), raw materials were sprinkled with water and piled up, 65% moisture maintained during the substrate production process of ten days. piles were turned every second day. chicken manure and urea (one fourth of the substrates (25 g/1 kg) were added as supplements for nitrogen (n) and carbon (c) source on the second and last turning while gypsum (15 g/kg) was added and thoroughly mixed before the pasteurization process. when substrates were prepared, 700 g/bag were filled in polypropylene bags (20 × 15cm) and autoclaved for 3 to 4 hours for sterilization purpose. experiment was performed in triplicates. spawning sterilized substrate bags were kept until their temperature reached 28 ℃ (for spawning), then they were inoculated with the spawn prepared on sorghum grains (25 g/700 g). the bag mouths were loosely tied with the rubber bands and incubated at different temperatures. spawn running spawn running period was observed on different substrates at different temperatures. during the spawn running, relative humidity (70%) was maintained by humidifier and ventilation fan at different incubation temperatures. when the spawn running was almost completed, casing with autoclaved tea-waste (tea containing water) was done to maintain the moisture of the substrates. after pinhead emergence, bags were transferred to the cropping room with relative humidity of 85%, maintained by continuous ventilation. biological efficiency biological efficiency (dry weight basis) of different types of substrates up to three flushes was observed as per 700 g of the substrate bags. weight of fresh mushroom biological efficiency = × 100 % dry weight of substrate element analysis element analysis of the wild and cultivated fruiting bodies on the mixed substrate of wheat straw and tea waste was pursued by using standard procedures described by horwitz et al. (1970). powder samples were subjected to digestion by wet digestion method to check the mineral contents present in the samples. minerals i.e., potassium (k), zinc (zn), calcium (ca), nickle (ni), copper (cu) and cobalt (co) were analyzed through wet digestion method. standard solutions of 5, 10, 15 20 and 25 ppm were prepared from the stock solution of the five required metals except potassium. for potassium, standard solution of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 ppm were prepared from the stock solution (1000 ml) of potassium and were analyzed in atomic absorption (aa) spectrophotometer (perkinelmer aanalyst100) and in flame photometer (janway pfp 7). statistical analysis of the data completely randomized design was used to determine the different parameters i.e., culturability, spawn production and cultivation potential. all the treatments were evaluated in triplicates and twoway analysis of variance was applied to determine the significant differences between different treatments. spss software package was used for the statistical analysis. data is also expressed as mean value ± s.e. domestication and element analysis of the macrocybe gigantea 169 results and discussion screening the most effective culture medium and optimization of temperature mycelium extension rate and density always rely on the suitable culture medium utilized for culturing (reyes et al., 2009). mycelial features of m. gigantea were evaluated on various nutritive solid media at different temperatures. white colored mycelium appeared in cultures without any exudates, with regular pattern and moderate to abundant density on different solid media. (fig. 1). cottony mycelium with fibrillar growth was observed on all the media used for the evaluation of mycelial growth potential. findings of this study were similar with dulay et al., 2021; in which they observed the same mycelial characteristics for l. tigrinus on the majority of carbon sources used for its culturing. amongst the different media utilized for the culturing of m. gigantea, pda proved the most efficient in terms of mycelium extension rate (13.96±0.033 mm/day) and density (abundant). sda medium (6.43±0.035 mm/day) at 15 ℃ was found to be the least appropriate for the mycelial growth of this species (fig. 1). the current results coincide with sardar et al., 2015 as they found that pda is the best medium for the growth of mycelium of various pleurotus species. our findings were also in concurrent with the roy and krishnappa, 2018; they evaluated the medium growth specificity of the m. gigan tea on different nutritive solid media such as pda, mea, sda and czapek dextrose agar (cda). they determined the maximum mycelial growth on the pda medium followed by mea. jo et al., 2002 evaluated the cultural potential of ganoderma lucidum and screened the pda as the appropriate medium for the mycelial growth. however, our findings were contrasted with the shim et al., 2005 as they found the minimum mycelial density of macrolepiota procera on the pda medium. vegetative growth of mushrooms needs a specific range of temperature for proliferation due to its effect on metabolic reactions. different basidiomycetes species grow in a wide range of temperatures, while the best temperature is between 20-30 ℃ (vahidi et al., 2004; lai et al., 2014). for the vegetative growth of m. gigantea, temperature suitability was evaluated and 30 °c was determined as the optimum temperature on all the solid nutritive media used for culturability testing. at 35 ℃, mycelium growth slowed down. our findings were also in line with the temperature response of various tropical mushrooms like lentinus squarrosulus (leon et al., 2017), collybia re inakeana (reyes et al., 1997) and volvariella volvacea (reyes et al., 1998). all media used for the evaluation of culturability of m. gigantea were proven to be supportive for its growth, with pda being the best option at 30 ℃. the mycelium extension rate (mm/ day) on different media at different temperatures differed significantly at p<0.001 as shown in tab. 1. spawn production spawn is the medium for the transformation of the mushroom my celium to the growing substrate (wozniak, 2009). it promotes quick colonization of the mushroom substrate and initiates successful fruiting (chang, 2009). the colonization rate of the active mycelium (cultured on the pda medium) was checked on cereal grains (sorghum, wheat and barley) at 30 ℃. mycelium colonized more quickly on sorghum (sorghum bicolor) followed by wheat (triticum aestivum) and barley (hordeum vulgare) (fig. 2). full spawning material (whole grains covered with the mycelium) was ready on sorghum grains after 13 days of inoculation. the ample growth of mycelium on sorghum grains was due to its moisture and nutrient composition (leder, 2004). this is in agreement with devi and sumbali (2021) for the spawn production of fig. 1 (a-f): a: basidiomata of m. gigantea; b-f: cultures on different nutrient agar media at 30 oc after 12 days of inoculation; b: on pda; c: on mea; d: on cea; e: on gpa; f: on sda. scale bar: a: 2 cm, b-f: 1 cm table 1: mycelial growth rate (mm/day) of m. gigantea on different media at different temperatures. cea, compost extract agar; pda, potato dextrose agar ; mea, malt extract agar ; sda, saboraud dextrose agar ; gpa glucose peptone agar values given are mean ± standard error. media type and temperature have significant impact over mycelium growth rate (p<0.001). moreover, the joint effect of media and temperature has also a significant impact over mycelium extension rate (p<0.001). in addition, lsd test was applied and found the significant differences between all possible combinations of media types (p<0.001) and between all possible combinations of temperatures (p<0.001). ty p e s o f media mycelium extension rate (mm/day) temperatures 15 °c 20 °c 25 °c 30 °c 35 °c p-value pda 7.33±0.033 10.27±0.057 12.33±0.035 13.96±0.033 12.85±0.033 <0.001 mea 7.25±0.036 9.83±0.035 11.28±0.033 12.87±0.033 11.95±0.033 <0.001 cea 6.95±0.043 9.3±0.033 10.4±0.057 12.26±0.631 11.36±0.036 <0.001 gpa 6.76±0.033 8.96±0.033 9.9±0.057 11.24±0.033 10.96±0.036 <0.001 sda 6.43±0.035 6.96±0.033 7.86±0.033 8.56±0.036 7.93±0.035 <0.001 p-value <0.001 <0.001 <0.001 <0.001 <0.001 25 tab. 1: mycelial growth rate (mm/day) of m. gigantea on different media at different temperatures. cea, compost extract agar; pda, potato dextrose agar; mea, malt extract agar; sda, saboraud dextrose agar; gpa glucose peptone agar types of media mycelium extension rate (mm/day) temperatures 15 °c 20 °c 25 °c 30 °c 35 °c p-value pda <0.001 mea <0.001 cea <0.001 gpa <0.001 sda <0.001 p-value <0.001 <0.001 <0.001 <0.001 <0.001 values given are mean ± standard error. media type and temperature have significant impact over mycelium growth rate (p<0.001). moreover, the joint effect of media and temperature has also a significant impact over mycelium extension rate (p<0.001). in addition, lsd test was applied and found the significant differences between all possible combinations of media types (p<0.001) and between all possible combinations of temperatures (p<0.001). (green-yellow-red) scheme was applied on the tables for quick readability of the most and least effective treatment on all the parameters. green color indicated the best treatment while red color showed the least effective. 170 a. ghafoor, a.r. niazi, n.-u.-s. afshan m. gigantea. leon et al. (2017) observed the shortest incubation period for spawn production of wild strain of l. squarrosulus on sorghum grains from the philippines. these grains were also found to be the good spawning material for agaricus blazei and agrocybe aegerita (galamgam, 2009; marcelo, 2011). bharti (2019) found wheat grains while pamitha (2014) and duong et al. (2017) found paddy grains as the suitable substrate for spawn preparation of m. gigantea. tab. 2 shows that the days required to complete spawn production of m. gigantea on sorghum, wheat and barley grains at 30 ℃ significantly differed at (p<0.001). determination of efficient lignocellulosic substrate for fruiting and yield of m. gigantea the success of a newly cultivated strain depends on both economical and biological factors (thawthong et al., 2014). temperature is one of the key biological factors for successful fruiting of any mushroom or the conversion of the dikaryotic mycelium into the fruiting body. mata et al. (2005) reported that different factors like, temperature, light and humidity of the incubation room influence the spawn runn ing of mushrooms. spawn prepared on sorghum grains was used to determine the spawn running time of m. gigantea on six different substrates (three were of pure type and three mixed substrate) at various temperatures, 20 ℃, 25 ℃, 30 ℃, 35 ℃, and 40 ℃. at 30 ℃, minimum spawn running period was observed on the tea waste + wheat straw (19.86±0.033d) followed by pure wheat straw (21.34±0.035d) with 70% humidity level of the incubation rooms (tab. 3). this is similar to the work of devi and sumbali (2021), who found the lowest spawn running period (16 days) on wheat straw substrate. furthermore, cultivation potential, in the form of fruiting bodies and yield, was evaluated on six types of substrates, three were of pure substrates, (wheat straw, tea waste and saw dust) and other three were the combination of two substrates (wheat straw+ tea waste, tea fig. 2 (a, b, c): spawn production of m. gigantea on a: sorghum grains; b: wheat grains; c: barley grains at 30 oc after 17 days of inoculation. scale bar: a-c: 1 cm. tab. 2: efficiency of wheat, sorghum and barley grains for spawn production of m. gigantea types of grains days required to complete spawn production at 30 °c sorghum 13 d 15 h ±1.15h wheat 16 d 22.3 h ± 1.20 h barley 20 d 22 h ± 1.15 h the results reported were run in triplicates and stated as mean± standard error. table 3: days required to complete spawn running period on different substrates at variable temperatures values given are mean ± standard error. substrate types and temperature have significant impact over spawn running time (p<0.001). moreover, the joint effect of substrates and temperature has also a significant impact over spawn running time (p<0.001). in addition, lsd test was applied types of days required to complete spawn running period substrates temperatures 15 °c 20 °c 25 °c 30 °c 35 °c p value tea waste & wheat straw 29.96±0.033 25.93±0.035 22.96±0.033 19.86±0.033 21.86±0.006 <0.001 pure wheat straw 27.93±0.035 26.93±0.035 24.96±0.036 21.34±0.035 24.93±0.035 <0.001 saw dust & wheat straw 28.92±0.039 27.96±0.033 26.85±0.033 24.96±0.033 26.96±0.033 <0.001 tea waste & saw dust 31.93±0.035 28.96±0.033 28.33±0.035 27.85±0.036 29.95±0.033 <0.001 p u r e s a w dust not initiated 30.93±0.035 29.96±0.033 27.96±0.033 30.93±0.035 <0.001 p u r e t e a waste not initiated 32.91±0.044 30.56±0.036 29.93±0.035 31.3±0.033 <0.001 p-value <0.001 <0.001 <0.001 <0.001 <0.001 27 tab. 3: days required to complete spawn running period on different substrates at variable temperatures types of substrates days required to complete spawn running period temperatures 15 °c 20 °c 25 °c 30 °c 35 °c p-value tea waste & wheat straw pure wheat straw saw dust & wheat straw tea waste & saw dust pure saw dust pure tea waste p-value <0.001 <0.001 <0.001 <0.001 <0.001 values given are mean ± standard error. substrate types and temperature have significant impact over spawn running time (p<0.001). moreover, the joint effect of substrates and temperature has also a significant impact over spawn running time (p<0.001). in addition, lsd test was applied and found the significant differences between all possible combinations of substrate types (p<0.001) except between tea waste & wheat straw and pure tea waste; and between all possible combinations of temperatures (p<0.005) except 15 oc & 20 oc (p=0.086) and 20 oc and 25 oc (p=0.737). (green-yellow-red) scheme was applied on the tables for quick readability of the most and least effective treatment on all the parameters. green color indicated the best treatment while red color showed the least effective. <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 domestication and element analysis of the macrocybe gigantea 171 waste+ saw dust and saw dust+ wheat straw) at 30 ℃ with humidity level 85%. the mixture of tea waste and wheat straw was found to be the best substrate for the cultivation of m. gigantea at 30 ℃ in the term of fruiting and yield (86.77±0.035g) followed by the wheat straw (79.85±0.035). tea-waste was used for the first time as the growing medium for the cultivation of m. gigantea that was proved effective. yang et al. (2016) revealed the tea-waste as the economic and suitable substrate for the fruiting and yield of p. ostreatus. lignin cellulosic waste supplemented with tea-waste could be an efficient source of growing substrate for the cultivation of various saprophy tic mushrooms like ganoderma lucidum (peksen and yakupoglu, 2009). as far as the efficiency of substrates were concerned, inyod et al. (2016) obtained the maximum yield of m. crassa on saw-dust. atri and lata (2013) obtained the maximum yield of l. cladopus from the substrate of the mixture of wheat straw and paddy straw. our research was also related to the findings of dulay et al., 2021. they observed the combination of rice straw and saw dust at different temperatures as the suitable substrate for the cultivation of lentinus species. tab. 4 revealing the yield obtained from varieties of substrates and fig. 3 representing the different stages of fruiting body production of m. gigantea. element analysis the element analysis was conducted to determine macro and trace elements in the nutritionally and medicinally significant macrocybe gigantea. this species was enriched with macronutrients like potassium and calcium while trace elements (nickel and cobalt) were not detected in both wild and cultivated basidiomata (tab. 5). enrichment of essential elements and absence of toxic metals showed their suitability to enrich a diet. these results were in agreement with the findings of liu et al., 2012 and mallikarjuna et al., 2013. conclusion it can be concluded that m. gigantea could easily be grown on different media but pda medium at 30 °c was proved the best combination for the growth of this mushroom. tea waste medium was used for the first time as the growth medium for m. gigantea and found very effective. however, different combinations of the substrates and media like paddy straw with tea waste, cotton waste with tea waste, tea waste with banana peels etc at 30 ℃ should be investigated in more detail to enhance the yield and biological efficiency of this nutritious mushroom. conflict of interest no potential conflict of interest was reported by the authors. authors contribution aneeqa ghafoor: collection, analysis, methodology and writing abdul rehman niazi: identification, supervision, analysis and methodology najam-ul-sehar afshan: formal analysis, visualization tab. 4: yield (g/700 g) obtained from different types of substrates at 30 ℃ types of substrates biological efficiency (yield g/700 g) 1st flush yield 2nd flush yield 3rd flush yield total yield tea waste & wheat straw 39.96±0.033 25.96±0.033 20.85±0.033 86.77±0.035 pure wheat straw 37.96±0.036 22.96±0.033 18.93±0.033 79.85±0.035 sawdust & wheat straw 30.96±0.036 21.93±0.035 17.95±0.033 70.84±0.033 tea waste & saw dust 26.96±0.033 19.94±0.047 not appeared 46.9±0.035 pure saw dust 21.40±0.057 18.93±0.065 not appeared 40.33±0.057 pure tea waste 20.93±0.033 17.93±0.033 not appeared 38.86±0.044 p-value <0.001 <0.001 <0.001 <0.001 values given are mean ± standard error. substrate types have significant impact over the total yield (p<0.001). fig. 3 (a, b, c): a: spawned compost; b: pinheads and c: harvested fruiting body of m. gigantea. scale bar (a-c): 2 cm tab. 5: minerals concentration present in the macrocybe gigantea mushrooms essential and trace minerals (mg/g) ca co cu k zn ni m. gigantea wild 1.54 <lod 0.524 69 0.20 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oyster mushroom (pleurotus spp.) on different growing media. j. agric. res. 56(3), 187-91. thawthong, a., karunarathna, s.c., thongklang, n., chukeatirote, e., kakumyan, p., chamyuang, s., hyde, k.d., 2014: discovering and domesticating wild tropical cultivatable mushrooms. chiang mai j. sci. 41(4), 731-764. vahidi, h., kobarfard, f., namjoyan, f., 2004: effect of cultivation conditions on growth and antifungal activity of mycena leptocephala. afr. j. biotechnol. 3(11), 606-609. vilariño, m., garcía-sanmartín, j., ochoa-callejero, l., lópezrodríguez, a., blanco-urgoiti, j., martínez, a., 2020: macrocybin, a mushroom natural triglyceride, reduces tumor growth in vitro and in vivo through caveolin-mediated interference with the actin cytoskele ton. molecules. 25(24), 6010. doi: 10.3390/molecules25246010 vizzini, a., consiglio, g., marchetti, m., alvarado, p., 2020: insights into the tricholomatineae (agaricales, agaricomycetes): a new arrangement of biannulariaceae and callistosporium, callistosporiaceae fam. nov., xerophorus stat. nov., and pleurocollybia incorporated into callistosporium. fungal divers. 101(1), 211-259. doi: 10.1007/s13225-020-00441-x wozniak, w., 2009: production and quality appraisal of mycelium of parasol mushroom macrolepiota procera (scop. ex fr.) sing, herba. pol. 55(3), 285-291. yang, d., liang, j., wang, y., sun, f., tao, h., xu, q., wan, x., 2016: tea waste: an effective and economic substrate for oyster mushroom cultivation. j. sci. food agric. 96(2), 680-684. doi: 10.1002/jsfa.7140 zhu, l.g., cheng, x.x., zhang, w.j., 2013: research progress on the reuse of solid wastes in tea industry and its application in environmental improvement. fujian j. agric. sci. 28, 1310-1315. address of the corresponding author: abdul rehman niazi, institute of botany, university of the punjab, lahore, 54590, pakistan e-mail: drarniazi.botany@pu.edu.pk © the author(s) 2022. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.3390/molecules25246010 http://dx.doi.org/10.1007/s13225-020-00441-x http://dx.doi.org/10.1002/jsfa.7140 journal of applied botany and food quality 93, 215 (2020) book review the papaya – botany, production and uses sisir mitra papaya (carica papaya l.) is well known as the third most widely produced tropical fruit worldwide after mango and pineapple. the global papaya production has grown significantly over the last few years, mainly as a result of increased production in india. global sharing of information on papaya was advanced by the 1st internatio nal papaya symposium held at genting highlands, malaysia, in 2005. this congress has been followed by regular symposia every 4 years in different papaya-producing countries. since more than 100 years, numerous articles related to production and use of papaya fruits have been published in various scientific and popular journals. however, a comprehensive review that summarised these long-standing research results and findings in a suitable form has been lacking until now. so, it is a very important step that this first comprehensive book authorized by an international team of experts at the forefront of research covering all important aspects of botany, biotechnology, production, postharvest physiology and processing, is now available. the book starts by providing a detailed overview about the origin, history, valuable ingredients and processing of papaya fruits. sub sequent sections deal mainly with the taxonomy, propagation and breeding of papayas. further on, the fruit formation, the ripening process, various plant diseases, greenhouse cultivation, optimal storage conditions and quality characteristics are discussed in detail on a high scientific level. as mentioned by the authors, today analysis of the papaya genome promises new, faster breeding techniques to obtain improved cultivars. these and other advances are helping to tackle diseases like papaya ring spot viruses and major pests which still cause significant losses. this book can be recommended without any reservation as an ex citing and instructive information to a broad range of readers. not only does it provide valuable support to professional groups such as plant breeders, farmers, plant physiologists, food technologists and chemists, but it also provides an excellent opportunity for interested laypeople to obtain comprehensive information on the current state of knowledge of this important tropical fruit. a number of scientific articles are listed in each annex of the 16 sections, allowing readers to obtain more detailed information on the individual topics. dr. h. schulz e-mail: hs.consulting.map@t-online.de bibliography: sisir mitra, the papaya – botany, production and uses, cab international 2020, 269 pages includes bibliographical references and index, price: 113,27 €, isbn-13: 9781789241907 (hardback), isbn 9781789241914 (epdf), isbn 9781789241921 (epub) art18_engert.indd journal of applied botany and food quality 84, 111 118 (2011) institute of agronomy and plant breeding, justus-liebig-university giessen, germany effect of sprouting on the concentration of phenolic acids and antioxidative capacity in wheat cultivars (triticum aestivum ssp. aestivum l.) in dependency of nitrogen fertilization n. engert, a. john, w. henning, b. honermeier (received october 26, 2010) summary the study was conducted to analyze the effects of sprouting on content and composition of phenolic acids (pa) and antioxidative capacity by the folin-ciocalteu (total phenolic content (tpc)) and orac (oxygen radical absorbance capacity) assay depending on nitrogen application and cultivar. three wheat cultivars (cv. tommi, cv. privileg and cv. estevan) were treated with two different nitrogen (n) levels (n1 without n and n2 with 150 kg n/ha). three fractions of the grinded wheat caryopses (free, free-insoluble and bound phenolic acids) were extracted. mean values for total phenolic acids (tpa) ranged between 498.1 and 1726.2 µg gae/g with signifi cant differences for the cultivars in not sprouted samples. nitrogen fertilization showed signifi cant differences with lower tpa contents for not fertilized grain. even the interaction cultivar x nitrogen was signifi cant for not sprouted grain. the statistical analysis of sprouting time did not show effects on tpa values after 24 h and 48 h of sprouting. in the present study no signifi cant infl uence of cultivar or nitrogen application was identifi ed for tpc and orac values. in contrary to that, the effects of sprouting time on the antioxidative capacity by folin-ciocalteu and orac were signifi cant. after 48 h of sprouting the values of tpc (2575.6 µg gae/g) and orac (32.6 µmol te/g) were signifi cantly higher than for not sprouted wheat samples (tpc: 2054.8 µg gae/g, orac: 28.2 µmol te/g). the longer the sprouting time was, the higher the antioxidative capacity of free phenolic acids, indicating that more free phenolic acids were released and other phytochemicals might have been present after sprouting. since there are only little information about the effect of sprouting time on phenolic acids and antioxidative capacity in dependence on nitrogen application further studies need to be conducted. introduction cereals, especially wheat (triticum aestivum l. ssp. aestivum), provide an important contribution to the human diet with their basic nutrients carbohydrates, protein, dietary fi ber, vitamins and minerals. besides, they contain secondary plant metabolites such as carotinoids, fl avonoids and phenolic acids that complement a balanced diet by means of their antioxidative potential. as a result of enhanced health awareness during the last years, natural antioxidants gained considerable interest due to their health benefi cial functions. e. g. phenolic acids show health affecting potentials because of their antioxidativity (serpen, 2008). potential effects of phenolic compounds are to reduce the risk of cardiovascular diseases and cancer (knekt et al., 2002; peterson et al., 2003; sesso et al., 2003; arts and hollman, 2005; graf et al., 2005). wheat phenolic acids (pa), such as ferulic (fa), p-cumaric (pca), vanillic (va), sinapic (sia), syringic (sya) and caffeic acid (ca) are valuable antioxidativ ingredients (mpofu, 2006). it is reported that phenolic acids inhibit lipid oxidation by scavenging free radicals such as hydroxyl radicals (cuppett et al., 1997). in addition, sprouts are considered to be health benefi cial, even more than the cereal grain itself (kahlil and mansour, 1995; prodanov et al., 1997; yang et al., 2001, arora et al., 2010, tian et al., 2010). however, for some processes, like the production of bread, it is extremely necessary that wheat caryopses are of a very high quality with no degradation of their compounds. sprouting is a result of humid conditions during and after grain development or storage. by reason of sprouting the starchy endosperm can be reduced because of enzymatic degradation processes, e.g. by α-amylase. little information is available about the effect of nitrogen fertilization on the content of phenolic acids. germination of the caryopses induces a higher amylase activity in caryopses resulting in lower starch contents. during this process other components like phenolic acids might be changed, too. thus, the aim of this study was to investigate phenolic acids, their composition and their variation in three different wheat cultivars in dependency on sprouting time and nitrogen fertilization. materials and methods in 2008 winter wheat cultivars were grown in fi eld plots of the experimental station “rauischholzhausen” of the institute of agronomy and plant breeding (degree of longitude: 8° 52’ 05” e, degree of latitude: 50° 46’ 41” n, altitude: 222 m). three cultivars (cv. tommi, cv. privileg, cv. estevan) of t. aestivum ssp. aestivum l. with four randomized replications were obtained from the fi eld experiment. two nitrogen levels were analyzed: n1 without n fertilization and n2 with 150 kg n/ha (40, 40, 70 kg n/ha). to avoid lodging of wheat plants all plots were treated with the plant growth regulator chlorcholin chloride (ccc, 720 g/l) at two times. to protect the wheat against diseases fungicides were applied at two times (fi rst: 0.8 l/ha proline + 1.5 l/ha pugil and second: 0.7 l/ha diamant + 0.7 l/ha champion). after harvesting the samples were stored in darkness at 20 °c until they were used for the sprouting experiment. to induce sprouting 30 g wheat were steeped with 15 ml aqua dest. in petri dishes which were covered with ashless circular fi lters (mn 640 m, 90 mm, macherey-nagel). three different sprouting times were tested at room temperature, without direct solar radiation: 0 h (no sprouting), 24 h (presprouted wheat samples) and 48 h (fully sprouted wheat samples) (fig. 1). to stop the sprouting process, all samples were dried for 24 h at 36 °c. in total 72 wheat samples (3 cultivars x 3 sprouting levels x 2 n fertilization doses x 4 replications) were included and ground with a 500 µm sieve on a cyclotec 1073 sample mill (foss, germany) to get a whole wheat fl our. the samples were mixed thoroughly to ensure homogeneity. extraction followed immediately. extraction: for the analysis of phenolic acids, samples were extracted using a modifi ed method by krygier et al. (1982) and adom and liu (2002) as follows. whole wheat flour (1 g) was extracted with 10 ml methanol/acetone/water (7:7:6, v/v/v) and divided into three fractions: (1) soluble free, (2) soluble conjugated, and (3) bound phenolic acids. the supernatant provided fraction 1 and 2, the residue fraction 3. before all fractions were extracted with ethyl acetate, bound and soluble conjugated phenolic acids had to be hydrolyzed with 5 m naoh for three hours. the reaction was stopped with 5 m hcl and the extracts were shaken out with ethyl acetate. the organic extracts were transferred into fl asks and ethyl acetate was evaporated. referring to that, extracts were resumed in 10 ml of 10 % methanol and stored at -18 °c for all further analysis (hplc, folin, orac). quality parameters: generally, parameters concerning grain quality were measured to characterize the used wheat cultivars before starting the sprouting experiment. according to icc and ista standard methods thousand grain weight (tgw), crude protein (cp), falling number (fn), gluten index and sedimentation test by zeleny (sedi) were analyzed. the parameter falling number was additionally used to determine the state of sprouting. that means, grain with no sprouting showed highest mean falling numbers (419 s), grain which was presprouted showed lower (104 s) and fully sprouted grain had lowest falling numbers (62 s). hplc: the hplc method according to weidner et al. (2000) and zielinski et al. (2001) was used with some modifi cations for determination of phenolic acids (pa) and total phenolic acids (tpa) as the sum of all analyzed phenolic acids. the analysis was performed on a hplc-dad system by knauer on a c18 column (ec 250 x 4 nucleodur sphinx rp, 5 µm (mn)) at a temperature of 25 °c, a fl ow rate of 1 ml min-1 and an injection volume of 100 µl. the detection was carried out at 250 nm (hydroxybenzoic acids) and 290 nm (hydroxycinnamic acids) following the program: 0-3 min 20 % a, 3.5-7.5 min 0 % a, 8-14 min 8 % a, 14.5-32 min 30 % a, 33-39 min 95 % and 40-45 min 20 %. the gradient system consisted of (a) 95 % methanol with acetic acid (about 200 µl) adjusted to ph 4.5 and (b) water adjusted to ph 4.5 with about 5 µl acetic acid. folin-ciocalteu assay: the folin-ciocalteu assay is an electron transfer based reaction assay, and measures the reducing capacity of the sample (huang et al., 2005). in the present analysis it is called total phenolic content (tpc) and the folin-ciocalteau micro method by waterhouse (2001) was used. folin-ciocalteu reagent consisted of phosphotungstic (h3pw12o40) and phosphomolybdic (h3pmo12o40) acids, additionally gallic acid was used as standard. to measure the absorbance spectrophotometically at 765 nm with a specord 205 – 222a430 (analytik jena ag, germany), the folinciocalteu reagent had to be reduced to blue oxides of tungsten and molybdenum. the total phenolic content was expressed as gallic acid equivalent (gae). orac assay: orac (oxygen radical absorbance capacity) is a hydrogen atom transfer based reaction assay to measure the antioxidative capacity towards peroxyl radicals (huang et al., 2005). a previously described method by huang et al. (2002) was used and the analysis was operated on a 96-well plate fl uorescence reader fluoroskan (fisher scientifi c gmbh, germany). very briefl y, fl uorescein (6-hydroxy-9-(2-carboxyphenyl)-(3h)-xanthen-3-on) and the sample were mixed and incubated at 37 °c. to initiate the reaction the ros-generator aaph (2,2’-azobis(2-methylpropion amidine)dihydrochloride) was added to measure fl uorescence at excitation wavelength 485 nm and emission wavelength 538 nm every 60 s for 90 min. as a standard trolox® (6-hydroxy-2,5,7,8tetramethylchromane-2-carboxylic acid), a vitamin e analogue, was used and the antioxidative capacity was expressed as trolox equivalent (te). statistical analysis: one-way analysis of variance (anova) was performed with pasw statistics 18 (spss inc., chicago, il) for all wheat cultivars (cv. tommi, cv. privileg, cv. estevan) and nitrogen levels (n1 and n2) within all sprouting times (no sprouting, presprouted, fully sprouted) to determine the effects on the measured parameters. when signifi cant differences occurred (p≤0.05) multiple mean comparisons were performed using the tukey t-test. to determine the infl uence of sprouting time on the analyzed parameters a paired samples test (t-test) was performed. spearman’s correlation (rs) factors (bivariate) were calculated between total phenolic acids (tpa), total phenolic content (tpc) and antioxidative capacity (orac). results are reported as mean values and the signifi cance level was at a 95 % confi dence interval (α= 0.05). results quality parameters: thousand grain weight (tgw) ranged from 40.1 to 45.0 g with signifi cant differences (≤ 0.001) between all three cultivars (tab. 1). cv. privileg could be characterized as the cultivar with largest grain (45.0 g) followed by cv. tommi (42.8 g) and cv. estevan (40.1 g). analyzed falling numbers (fn) ranged from 323 to 388 s signifi cantly affected by the cultivars (p ≤0.001) and n fertilization (p ≤0.001). cv. tommi with a mean value of fig. 1: different sprouting levels of wheat: apresprouted grain after 24 h; bfully sprouted grain after 48 h of sprouting a b 112 n. engert, a. john, w. henning, b. honermeier 388 s showed the highest falling number which was signifi cantly higher than cv. estevan with 323 s. cv. tommi and cv. privileg (377 s) were at the same level, which indicates a very low amylase activity in the caryopses. nitrogen fertilized samples had higher fn (381 s) than not fertilized (345 s) ones. sedimentation volumes (fig. 2) differed between 33 ml and 58 ml with signifi cantly (p=0.003) interaction effects for cultivar and nitrogen treatment. nitrogen application to cv. estevan (58 ml) and cv. tommi (55 ml) induced signifi cantly higher sedimentation volumes than nitrogen application to cv. privileg (44 ml). gluten indices (gi) ranged between 81 and 92 indicating a high gluten quality. n fertilization (p≤ 0.001) and cultivar (p ≤0.001) were signifi cant with regard to gi. fertilized wheat samples had signifi cantly lower gi than not fertilized wheat samples. the cultivar estevan (92) reached signifi cantly higher gi than cv. tommi (81) and cv. privileg (84) which were at the same level. crude protein (cp) ranged between 12.5 and 12.9 %. as to expect, nitrogen treatment (13.6 %) led to higher contents of crude protein (p≤ 0.001) in wheat caryopses than in unfertilized (11.6 %) ones, whereas between cultivars no signifi cant cp differences were observed. phenolic acids (pa): the analyzed total phenolic acids (tpa) consisted of vanillic acid (va), syringic acid (sya), caffeic acid (ca), p-coumaric acid (pca) and ferulic acid (fa), measured by the hplc method. results were expressed as mean values in sum of the measured phenolic acids, ranging between 498.1 and 1726.2 µg gae/g. cultivars which were not sprouted showed signifi cantly differences (p ≤0.001) in their tpa contents. however, cv. privileg (1066.1 µg gae/g) and cv. estevan (1154.3 µg gae/g) had signifi cantly lower values than cv. tommi (1726.2 µg gae/g). tpa values of non-fertilized wheat (n1=1085.7 µg gae/g) were signifi cantly lower than of fertilized ones (n2=1545.3 µg gae/g). moreover, signifi cantly (p≤0.001) interaction effects between cultivar and nitrogen occurred for not sprouted wheat samples mainly resulting from cv. tommi x n2 which was about twice as high than the other cultivar x nitrogen interactions. after a sprouting time of 24 h an interaction effect between cultivar and nitrogen fertilization (p=0.044) was observed. the combination of cv. tommi x n2 showed once more the highest tpa value (1669.0 µg gae/g) which signifi cantly differed only from cv. tommi x n1 (760.5 µg gae/ g). after a sprouting time of 48 h a signifi cant cultivar effect on tpa was noticeable (tab. 2). the cultivars cv. tommi (1642.2 µg gae/g) and cv. estevan (1554.0 µg gae/g) had the same level of tpa contents which was signifi cantly higher than the tpa of cv. privileg (498.1 µg gae/g). nitrogen application had no signifi cant effects on tpa contents of the analyzed wheat samples but a tendency of higher tpa contents of n2 fertilized samples in comparison with the n1 fertilization level was observed. looking at the means of tpa values of the three sprouting treatments, which varied from 1315.5 µg (0 h) to 1099.2 µg (24 h) and 1231.4 µg gae/g (48 h), no signifi cant differences were detectable. total phenolic acids (tpa) were most concentrated in fraction 3 (tpa3) for all three sprouting times (fig. 3). unsprouted wheat samples contained 86 % (1128.4 µg gae/g) in fraction 3 followed by fraction 1 (tpa1, 77.9 µg gae/g) and fraction 2 (tpa2, 109.3 µg gae/g) with less than 10 %. while sprouting for 24 h the relative and absolute values did not change much. after 48 h tpa1 increased to 28 % (346.3 µg gae/g) and tpa3 provided 59 % (722.3 µg gae/g). tpa2 rose up to 13 % (162.8 µg gae/g). looking at the composition of the fi ve analyzed phenolic acids, the predominant phenolic acid was ferulic acid. during sprouting the proportion of each analyzed phenolic acid shifted. nevertheless, ferulic acid (fa) was the main phenolic acid with 67 % (881.8 µg gae/g) for not sprouted, 74 % (809.3 µg gae/g) for presprouted and 54 % (669.9 µg gae/g) for fully sprouted grain (fig. 4). fig. 2: interaction effect of n fertilization and cultivar on sedimentation by zeleny in wheat samples (mean ± sd, α=0.05) tab. 1: effect of n fertilization and cultivar on thousand grain weight (tgw, [g]), falling number (fn, [s]), gluten index (gi) and crude protein (cp, [% dm]) in wheat samples treatment tgw fn gi cp (cultivar/nitrogen) g s % dm main effect cv. estevan 40.1 c 323 b 92 a 12.9 n.s. privileg 45.0 a 377 a 84 b 12.5 n.s. tommi 42.8 b 388 a 81 b 12.5 n.s. main effect n n1 42.7 n.s. 345 a 93 a 11.6 a n2 42.6 n.s. 381 b 78 b 13.6 b p cultivar (cv.) ≤ 0.001 ≤ 0.001 ≤ 0.001 n.s. p nitrogen (n) n.s. ≤ 0.001 ≤ 0.001 ≤ 0.001 p cv. x n n.s. n.s. n.s. n.s. sprouting and nitrogen effects on phenolic acids and antioxidative capacity 113 total phenolic content (tpc): the total phenolic content (tpc) analyzed by the folin-ciocalteu assay ranged between 1523.8 and 2836.7 µg gae/g (tab. 3). at the beginning of the experiment not sprouted wheat samples were characterized by a small variability of tpc values. neither main effects (cultivars, nitrogen) nor interaction effects (cv. x n) could be observed. contrary to that after 24 h of sprouting there was a signifi cant nitrogen effect of tpc expressed by lower tpc for n1 (1464.6 µg gae/g) in comparison to n2 (2038.6 µg gae/g). after 48 h of sprouting the treatments cultivar (p=0.008) and fertilization (p=0.011) showed signifi cantly effects, however, the interaction between both parameters is not signifi cant. cv. estevan (2836.7 µg gae/g) and cv. tommi (2684.9 µg gae/g) had signifi cantly higher tpc values than cv. privileg (2205.2 µg gae/g). tpc values for fertilized wheat (n2=2790.1 µg gae/g) were signifi cantly higher than for nonfertilized samples (n1=2361.0 µg gae/g). a sprouting time of 48 h signifi cantly (p ≤0.001) infl uenced the tpc. after 48 h (2575.6 µg gae/g) of sprouting the tpc were higher than after 24 h (1751.6 µg gae/g) and for unsprouted wheat samples (2054.8 µg gae/g) (tab. 3). the highest tpc provided fraction 1 (tpc1) for all three sprouting times. unsprouted wheat samples showed a total phenolic antioxidative capacity of 46 % (940.2 µg gae/g for tpc1, followed by fraction 3 (tpc3) with 28 % (574.7 µg gae/g) and fraction 2 (tpc2) 26 % (539.7 µg gae/g) (fig. 5). during 24 h of sprouting, tpc1 (38 %, 678.1 µg gae/ g) lost some of its antioxidative capacity while tpc3 (35 %, 574.2 µg gae/g) gained. after 48 h tpc1 (46 %, 1194.3 µg gae/g) and tpc2 (31 %, 799.8 µg gae/g) values increased whereas tpc3 (23 %, 581.5 µg gae/g) decreased (fig. 5). tab. 2: effect of sprouting time on total phenolic acids (tpa) [µg gae/g] in wheat samples depending on n fertilization and cultivar treatment sprouting time [h] (cultivar/nitrogen) 0 24 48 main effect cv. estevan 1154.3 a 1148.6 n.s. 1554.0 a privileg 1066.1 a 934.4 n.s. 498.1 b tommi 1726.2 b 1214.8 n.s. 1642.2 a main effect n n1 1085.7 a 1048.5 n.s 1151.7 n.s. n2 1545.3 b 1149.9 n.s 1311.2 n.s. interaction effect estevan x n1 1136.9 a 1500.5 ab 1542.7 n.s. estevan x n2 1171.8 a 796.7 ab 1565.3 n.s. privileg x n1 1057.3 a 884.5 ab 359.1 n.s. privileg x n2 1074.9 a 984.2 ab 637.1 n.s. tommi x n1 1063.0 a 760.5 a 1553.3 n.s. tommi x n2 2389.3 b 1669.0 b 1731.1 n.s. mean 1315.5 n.s. 1099.2 n.s. 1231.4 n.s. p cultivar (cv.) ≤ 0.001 n.s. 0.002 p nitrogen (n) ≤ 0.001 n.s. n.s. p cv. x n ≤ 0.001 0.044 n.s. treatment fig. 3: effect of sprouting time on the composition of the extracted fractions (tpa1, tpa2, tpa3) of total phenolic acids (tpa) in wheat samples fig. 4: effect of sprouting time on the composition of phenolic acids (tpa) in wheat grain 114 n. engert, a. john, w. henning, b. honermeier orac: the antioxidative capacity by the orac assay ranged from 24.4 to 35.1 µmol te/g whole wheat fl our (tab. 4). in unsprouted wheat samples no signifi cant effects could be observed, neither for main effects nor for the interaction of both treatments (cv x n). after 24 h of sprouting the factor cultivar (p ≤0.001) and the interaction cv. x n had signifi cant infl uence (p=0.006) on the antioxidative capacity. cv. privileg x n1 (28.3 µmol te/g), cv. privileg x n2 (22.6 µmol te/g) and cv. tommi x n2 (36.5 µmol te/g) showed signifi cant differences whereas cv. estevan x n1 (24.1 µmol te/g), cv. estevan x n2 (24.6 µmol te/g) and cv. tommi x n1 (28.2 µmol te/g) were at the same level. after 48 h of sprouting no signifi cant differences neither for main effects nor for the interaction effect could be observed. the results showed a tendency that wheat samples fertilized with 150 kg n/ha (n2) had higher orac values than n1 ones. furthermore, tab. 4 reveals a signifi cant infl uence of sprouting time on the antioxidative capacity. the values were signifi cantly higher after 48 h (32.6 µmol te/g) of sprouting than after 24 h (27.4 µmol te/g) or with no sprouting (28.2 µmol te/g). the highest antioxidative capacity contributed fraction 3 (orac3) for not sprouted wheat samples (39 %, 10.9 µmol te/g) and presprouted cultivars (40 %, 10.8 µmol te/g). after a sprouting time of 48 h the values of orac1 increased to 48 % (15.7 µmol te/g) whereas orac3 (26 %, 8.6 µmol te/g) decreased (fig. 6). tab. 3: effect of sprouting time on the total phenolic content (tpc) [µg gae/g] in wheat samples depending on n fertilization and cultivar treatment sprouting time [h] (cultivar/nitrogen) 0 24 48 main effect cv. estevan 2012.0 n.s. 1523.8 n.s. 2836.7 a privileg 2170.5 n.s. 1646.7 n.s. 2205.2 b tommi 1981.9 n.s. 2084.4 n.s. 2684.9 a main effect n n1 1970.6 n.s. 1464.6 a 2361.0 a n2 2139.0 n.s. 2038.6 b 2790.1 b interaction effect estevan x n1 2090.9 n.s. 1031.6 n.s. 2634.9 n.s. estevan x n2 1933.1 n.s. 2015.9 n.s. 3038.5 n.s. privileg x n1 2005.9 n.s. 1656.9 n.s. 2151.2 n.s. privileg x n2 2335.2 n.s. 1636.6 n.s. 2259.1 n.s. tommi x n1 1815.1 n.s. 1705.4 n.s. 2297.0 n.s. tommi x n2 2148.7 n.s. 2463.5 n.s. 3072.7 n.s. mean 2054.8 a 1751.6 a 2575.6 b p cultivar (cv.) n.s. n.s. 0.008 p nitrogen (n) n.s. 0.029 0.011 p cv. x n n.s. n.s. n.s. treatment treatment fig. 5: effect of sprouting time on the composition of the extracted fractions (tpc1, tpc2, tpc3) of total phenolic contents (tpc) in wheat samples tab. 4: effect of sprouting time on the antioxidative capacity by the orac assay [µmol te/g] in wheat samples depending on n fertilization and cultivar treatment sprouting time [h] (cultivar/nitrogen) 0 24 48 main effect cv. estevan 27.9 n.s. 24.4 a 35.1 n.s. privileg 26.4 n.s. 25.4 a 29.5 n.s. tommi 30.2 n.s. 32.3 b 33.3 n.s. main effect n n1 28.0 n.s. 26.9 n.s. 30.8 n.s. n2 28.3 n.s. 27.9 n.s. 34.5 n.s. interaction effect estevan x n1 28.4 n.s. 24.1 ab 35.1 n.s. estevan x n2 27.3 n.s. 24.6 ab 35.1 n.s. privileg x n1 27.6 n.s. 28.3 a 29.3 n.s. privileg x n2 25.2 n.s. 22.6 b 29.8 n.s. tommi x n1 28.1 n.s. 28.2 ab 27.9 n.s. tommi x n2 32.4 n.s. 36.5 c 38.7 n.s. mean 28.2 a 27.4 a 32.6 b p cultivar (cv.) n.s. ≤ 0.001 n.s. p nitrogen (n) n.s. n.s. n.s. p cv. x n n.s. 0.006 n.s. discussion the present study focused on the effects of sprouting time on phenolic acids and antioxidative capacity of whole wheat samples sprouting and nitrogen effects on phenolic acids and antioxidative capacity 115 depending on cultivar and nitrogen fertilization. sprouting is the fi rst important stage during biogenesis of wheat. enzymatic processes, like starch degradation by α-amylase, take place to provide the embryo with nutrients. normally sprouting occurs after a certain time of dormancy when the caryopsis has come to full ripeness (haumann and dietzsch, 2000). if it starts too early, because of humid conditions during ripening, it is a negative parameter for grain quality, e.g. for fl our processing. nevertheless it is necessary to know, if deliberately induced sprouting has an infl uence on phenolic acids and their antioxidative capacity with regard to their health benefi cial effects (kahlil and mansour, 1995; prodanov et al., 1997; yang et al., 2001; tian et al., 2004; arora et al., 2010; tian et al., 2010). in the conducted study analysis of phenolic acids (tpa) and antioxidative capacity by the folin-ciocalteu and orac assay was carried out in whole wheat fl our because of highest concentrations of phenolic acids in the bran and aleurone layer of caryopses (pussayanawin et al., 1988; zhou et al., 2004; adom et al., 2005; anson et al., 2008). the analyzed phenolic acids (tpa) didn’t show a signifi cantly infl uence for the different sprouting times, indicating that enzymatic processes during sprouting did not affect phenolic acids while differences for the analyzed cultivars were visible. after 48 h of sprouting in the current study cultivar estevan (1554.0 µg gae/g) showed higher tpa values than after 0 h of sprouting (1154.3 µg gae/g), whereas cv. privileg and cv. tommi showed lower values (tab. 2). the cultivar seems to have an infl uence whether the tpa concentration is higher in sprouted wheat samples than in not sprouted ones or not. until now it is known that genotype and environment infl uence the content of phenolic acids (abdelaal et al., 2001; mpofu et al., 2006; moore, 2006). the current study presents higher results than in literature which could be due to different extraction methods. stracke et al. (2009) reported concentrations between 282 µg/g and 1262 µg/g in soft wheat samples whereas zhou et al. (2007) only reported concentrations between 219.7 µg/g and 389.1 µg/g in eleven soft red winter wheat grain cultivars. moore et al. (2005) analyzed eight soft red winter wheat genotypes with tpa contents of 486.6 µg/g 656.0 µg/g and mpofu et al. (2006) conducted a study with six western canadian wheat genotypes where tpa values ranged between 580.2 µg/g and 724.7 µg/g. different cultivars were used in these studies which displayed a variation in tpa values between the used cultivars. even though only three cultivars were analyzed in the current study and a genetic determination was found for not sprouted cultivars, as well. n fertilization was signifi cantly infl uencing tpa contents for not sprouted grain resulting in higher values after application. margna (1977) traced this effect back to the suffi ciently available amino acid phenylalanine which is primarily used for protein biosynthesis and which can be released by protein molecules. since there is only little information about the correlation between n application and phenolic acid content, especially for wheat caryopses, further studies need to be conducted, to get a greater knowledge about the infl uence of nitrogen on phenolic acids in wheat caryopses. fraction 3 (tpa 3) represented cell wall bound phenolic acids and provided the biggest proportion of all three fractions, being well in line with further studies (stracke et al., 2009; abdel-aal et al., 2001). according to other authors free phenolic acids (fraction 1) contributed the smallest part to total phenolic acids ranging between 1 % and 2 %. this is in agreement with the detected results for not sprouted grain but not for fully sprouted samples (li et al., 2008; abdel-aal et al., 2001). generally, phenolic acids are bound to hydrolysable tannins, lignins, cellulose and proteins which are mainly structural components of bran, building a protective layer to inner parts of the caryopsis. furthermore, important functions of phenolics, including phenolic acids, are defending mechanisms against pathogens, parasites and predators in plants (graf, 1992; liu, 2004; parr and bolwell, 2000). the highest ratio in all sprouting times was observed for tpa 3 (fig. 3). nevertheless, it is noticeable that after 48 h of sprouting tpa 3 decreases from 86 % (not sprouted) to 59 % for fully sprouted grain and tpa 1 increases from 6 % (not sprouted) to 28 %. it can be assumed that conjugated phenolic acids are released from the breakdown of cell walls, maybe to protect the inner parts of the caryopsis which is still needed to support the developing germ. a study by cheng et al. (2006) suggests, that the conjugation of polyphenolics, e.g. tannins, are broken and simple phenolics are released. it is reported that the main phenolic acid in wheat is ferulic acid (fa) which is proposed to enhance wall extensibility during cell elongation (graf, 1992). fig. 4 shows that the composition of the phenolic acids shifted from 67 % fa and 7 % caffeic acid (ca) for not sprouted wheat samples to 54 % fa and 21 % ca for fully sprouted samples. since ca is a precursor of fa it suggests itself that breakdown processes of cellular constituents are in progress during sprouting, e.g. when the coleoptile breaks through. another assumption is that fa and ca, as intermediates in the biosynthesis of lignin, are released to provide substrates for the lignifi cation. for human nutrition the use of fully sprouted grain could provide positive effects because of the higher amounts of free phenolic acids. in most fi ndings the bioavailability and bioaccessibility of free phenolic acids is higher than of bound forms, which leads to a better resorption, e.g. of ferulic acid, in the small intestine. bound phenolic acids fi rstly have to be released by bacterial hydrolytic enzymes during fermentation in the large intestine (kroon et al., 1997; zhao et al. 2003; anson et al., 2009). in contrary to tpa values the antioxidative capacity by the folinfig. 6: effect of sprouting time on the composition of the extracted fractions (orac1, orac2, orac3) of the antioxidative capacity by the orac assay in wheat samples 116 n. engert, a. john, w. henning, b. honermeier ciocalteu (tpc) and by the orac assay showed signifi cant differences for sprouting times. after 48 h of sprouting the tpc was signifi cantly higher (2575.6 µg gae/g) than for not sprouted wheat samples. the same was visible for orac values, which were higher for fully sprouted samples (32.6 µmol te/g) than for not sprouted samples (28.2 µmol te/g). in the present study signifi cant infl uences of the cultivar on tpc or on orac in not sprouted samples were not observed. according to other authors, there should be a signifi cant variation in orac and tpc values of different cultivars due to genetic variability (moore et al., 2005; moore et al., 2006; mpofu et al., 2006; zhou et al., 2007). one reason for the differing results might be the small number of treatments (three cultivars) analyzed in the present study. the effect of n fertilization on the antioxidative capacity by folinciocalteu and orac assay was not signifi cant for not sprouted grain. however, there was the tendency of higher tpc and orac values after nitrogen application which needs to be analyzed further to get more information about the infl uence of nitrogen application on the antioxidative capacity. these two tests of the antioxidative capacity are working differently with the folin-ciocalteu assay being an electron transfer based reaction assay and orac a hydrogen atom transfer based reaction assay (huang et al., 2005). nevertheless, they both are testing the antioxidative activity which is the reason for the same tendency. moore et al. (2006) and okarter et al. (2010) reported a correlation between tpc and orac values. in the current study tpc were correlating strongly with orac values after 48 h of sprouting (rs=0.714, p ≤0.001). for not sprouted samples it is at least a positively tendency (rs=0.327). highest tpc were present for free phenolic acids with 46 % in not sprouted grain and fully sprouted grain, even though fraction 3 supplied the highest amount of phenolic acids. the same was visible for the orac values where orac 1 and orac 3 were about at the same level in unsprouted grain. after 48 h of sprouting orac 1 was the predominant fraction in terms of antioxidative capacity by the orac assay. these results indicate that sprouting alters the phenolic acids and other free phytochemicals might be present, leading to an improved antioxidative capacity in fully sprouted grain. the presence of tocopherols in wheat grain including the vitamers α-tocopherol, γ-tocopeherol and δ-tocopherol and carotenoids including β-carotene, zeaxanthin and lutein was reported by several studies (zhou et al., 2004; moore et al., 2005; okarter et al., 2010). during the biosynthetic pathway the different tocopherol isomers can be transformed into α-tocopherol which has the highest vitamin e activity (bramley et al., 2000). the antioxidative capacity of the tocopherol isomers seems to differ, too. it is possible that tocopherols and carotenoids are released to a greater extent from the aleurone fraction and improved the antioxidative capacity after 48 h of sprouting. zhou et al. (2004) reported a correlation between δ-tocopherol and tpc, with the strongest antioxidative capacity among the tocopherols. those phytochemicals, including phenolic acids could be the reason for a health benefi t of whole wheat products or sprouted products, which is in line with the assumption of okarter et al. (2010). in conclusion, the current study showed that the tpa composition was comparable with the literature while tpa values were higher than reported. phenolic acids contribute together with other phytochemicals in wheat, especially in sprouted wheat, a high potential health benefi t by scavenging free radicals in caryopses as well as in the human organism. the composition of free, freeinsoluble and bound phenolic acids changed during the process of sprouting for the benefi t of free phenolic acids after 48 h. furthermore, the antioxidative capacity by the folin-ciocalteu (tpc) and orac assay was increased after the caryopses were fully sprouted. the composition of the three extracted fractions changed for the benefi t of the antioxidative capacity of free phenolic acids. this indicated that the more free phenolic acids and other free phytochemicals were present, the higher was the antioxidative capacity. sprouted grain is a negative quality parameter with regard to standard backing processes. nevertheless, it should be taken in consideration for human diets, e.g. in bread with whole cereals or in special breads named “essener bread” with up to 100 % spouted grain fl our (beck et al., 2010). it is assumed that bioavailability along with bioaccessibility is upgraded because of free phytochemicals. further studies need to be conducted with more than three cultivars to show whether the interaction between cultivar and fertilization has an effect on phenolic acids and antioxidative capacity of sprouted grain. references abdel-aal, e.-s. m., hucl, p., sosulski, f.w., graf, r., gillott, c., pietrzak, l., 2001: screening spring wheat for midge resistance in relation to ferulic acid content. j. agric. food chem. 49, 3559-3566. adom, k., liu, r., 2002: antioxidant activity of grains. j. agric. food chem. 50, 6182-6187. adom, k., sorrells, m.e., liu, r.h., 2005: phytochemicals and antioxidant activity of milled fractions of different wheat varieties. j. agric. food chem. 53, 2297-2306. anson, n.m., berg, van den, r., havenaar, r., bast, a., haenen, g.r.m.m., 2008: ferulic acid from aleurone determines the antioxidant potency of wheat grain (triticum aestivum l.). j. agric. food chem. 56, 5589-5594. anson, n.m., berg, van den, r., havenaar, r., bast, a., haenen, g.r.m.m., 2009: bioavailability of ferulic acid is determined by its bioaccessibility. j. cereal sci. 49, 296-300. arora, s., jood, s., khetarpaul, n., 2010: effect of germination and probiotic fermentation on nutrient composition of barley based food mixtures. food chem. 119, 779-784. arts, i.c., hollman, p.c., 2005: polyphenols and disease risk in epidemiologic studies. am. j. clin. nutr. 81, 317-325. arts, i.c., hollman, p.c., bas bueno de mesquita, h., feskens, e.j., kromhout, d., 2001: dietary catechin and epithelial cancer incidence: the zutphen elderly study. 92, 298-302. bramley, p.m., elmadfa, i., kafatos, a., kelly, f.j., manios, y., roxborough, h.e., schuch, w., sheehy, p.j.a., wagner, k.-h., 2000: review vitamin e. j. sci. food agric. 80, 913-938. beck, a., busch, k.g., erik, d., deinert, c., dylla, r., gruhn, a., 2010: essener brot – herstellung und verwendung von keimlingen in der bäckerei. 1-24, fibl deutschland e.v., hrsg., frankfurt / main. cheng, z., su, l., moore, j., zhou, k., luther, m., yin, j.-j., 2006: effects of post harvest treatment and heat stress on availability of wheat antioxidants. j. agric. food chem. 54, 5623-5629. cuppett, s., schnepf, m., hall, c., 1997: natural antioxidants – are they a reality?. in: shahidi, f., 1997: natural antioxidants: chemistry, health effects, and applications. aocs press, champaign, usa. graf, e., 1992: antioxidant potential of ferulic acid. free radical bio. med. 13, 435-448. graf, b.a., milbury, p.e., blumberg, j.b., 2005: flavonols, flavones, fl avanones, and human health. epidemiological evidence. 8, 281-290. haumann, g., dietzsch, h., 2000: winterund sommerweizen. in: lütke entrup, n., oehmichen, j. 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wheat grain. int. j. food sci. nutr. 52, 319-330. zielinski, h., kozlowska, h., lewczuk, b., 2001: bioactive compounds in the cereal grains before and after hydrothermal processing. innov. food sci. emerging technol. 2, 159-169. zhao, z., egashira, y., sanada, h., 2003: digestion and absorption of ferulic acid sugar esters in rat gastrointestinal tract. j. agric. food chem. 51, 5534-5539. zhou, k., su, l., yu, l.l., 2004: phytochemicals and antioxidant properties in wheat bran. j. agric. food chem. 52, 6108-6114. zhou, k., hao, j., griffey, c., o’keefe, s.f., chen, j., hogan, s., 2007: antioxidant properties of fusarium head blight-resistant and – susceptible soft red winter wheat grains grown in virginia. j. agric. food chem. 55, 3729-3736. address of the authors: institute of agronomy and plant breeding, ludwigstrasse 23, 35390 giessen, germany corresponding author: bernd.honermeier@agrar.uni-giessen.de 118 n. engert, a. john, w. henning, b. honermeier art11_shabaz.indd journal of applied botany and food quality 84, 192 199 (2011) 1department of botany, university of agriculture, faisalabad pakistan is foliar-applied glycinebetaine effective in mitigating the adverse effects of drought stress on wheat (triticum aestivum l.)? m. shahbaz1*, y. masood1, s. perveen1, m. ashraf1, m. ashraf1, m. ashraf (received april 11, 2011) * corresponding author summary drought stress is a serious threat to crop growth and development. exogenous application of various compatible solutes is an effective means to lessen the adverse drought effects on plants. to explore the effectiveness of exogenously applied gb as foliar spray in mitigating the harmful effect of drought on wheat crop, an experiment was conducted using fi ve wheat cultivars (sarc-i, inqlab-91, mh-97, bhakkar and s-24) and three levels (0, 50 and 100 mm100 mm100 m ) of gb applied as foliar spray under well-watered and m) of gb applied as foliar spray under well-watered and m water-stressed (60% fi eld capacity) conditions. growth and yield attributes, gas exchange characteristics, and root and shoot n, p, and k+ were determined in the wheat cultivars. drought stress signifi cantly reduced shoot and root fresh and dry biomass, shoot length, leaf area, grain yield, photosynthetic (alength, leaf area, grain yield, photosynthetic (alength, leaf area, grain yield, photosynthetic ( ) and transpiration rates (e), stomatal conductance (gs), and shoot and root p and k+ contents. however, foliar-applied gb mitigated the adverse effects of drought stress by enhancing plant biomass, shoot length, transpiration rate, root p, and n contents and in shoot only k+ in both cultivars under stress conditions, while its effect was not prominent on leaf area per plant, water use effi ciency (wue), and shoot p and n, and root k+. the cultivar response to varying gb levels was variable. overall, foliar-applied gb @ 50 mmlevels was variable. overall, foliar-applied gb @ 50 mmlevels was variable. overall, foliar-applied gb @ 50 m showed m showed m better performance in reducing the adverse effects of drought stress on wheat crop under water defi cit conditions. cultivars sarc-i and inqlab-91 were better as compared to the others in their response to foliar-applied gb under water defi cit conditions. introduction environmental factors play important roles in plant growth and development. any adverse change in these factors negatively affects physiological and biochemical processes of plants (garg, 2010). for example, scarcity of water (drought) causes adverse effects on crop yield and quality (blum, 2005; neumann, 2008; abd el-rhman, 2010). under drought stress conditions, alteration in various morphological, physiological and biochemical attributes occur, that help plants to protect themselves against the stress (demiral and türkan, 2006; nawaz and ashraf, 2010; ashraf, 2010). of various, adaptations, accumulation of compatible solutes is an effective phenomenon which plays a signifi cant role in improving stress tolerance (chen et al., 2000; munns, 2005). of these solutes, glycinebetaine (gb) is an effective osmoprotectant which accumulates in a number of plants under drought stress (zhang et al., 2009) thereby playing a vital role in drought tolerance of plants (yang et al., 2007; mahmood et al., 2009; khan et al., 2009). gb stimulates a multitude of physiological phenomena including net co2 assimilation rate (wang et al., 2010). it protects the quaternary structure of proteins, maintains enzyme activity, stabilizes membrane structure of psii complex, prevents oxidative damage to membranes and enhances antioxidative defense system under osmotic stress (ma et al., 2006; raza et al., 2007; yang et al., 2007; chen and murata, 2008; hassine et al., 2008; hossain and fujita, 2010). the accumulation of glycinebetaine depends not only on type of plant species (moghaieb et al., 2004), plant varieties (cha-um et al., 2007; hassine et al., 2008), plant organelles (zhu et al., 2003), but also on environmental factors, such as salinity (longstreth et al., 2004; saneoka et al., 2001; girija et al., 2002), drought (zhang et al., 2008), alkaline stress (cui et al., 2008), and extreme temperatures (zhang et al., 2008), etc. exogenous application of a variety of compatible organic solutes has been advocated as one of the prospective means of improving plants stress tolerance (raza et al., 2007; ashraf et al., 2008; mahmood et al., 2009). it has been widely reported that exogenous application of gb regulates some key physiological attributes in plants subjected to stress conditions (mahmood et al., 2009). for example, gb protects photosynthetic machinery (zhao et al., 2001; allakhverdiev et al., 2003) by preventing photoinhibition (ma et al., 2006), partially preserves the net photosystem-ii effi ciency (demiral and turkan, 2006), and enhances the tolerance of photosynthetic apparatus of wheat plants subjected to various stress conditions (cherian et al., 2006; wang et al., 2010a; 2010b). however, mechanism involved in gb protection of this machinery is still unclear and thus needs to be investigated. glycinebetain biosynthesis has been reported to increase in most crop species under water defi cit conditions (martinez et al., 2005; hessine et al., 2009), while in others the natural synthesis of gb is considerably lower than the required level to protect the plants from the stress conditions (subbarao et al., 2001). however, exogenous application of glycinebetaine is known to have benefi cial effects on growth and fi nal yield of a number of crops, particularly those which do not normally accumulate signifi cant amount of gb under these stresses including drought stress (raza et al., 2006; 2007; athar et al., 2009; wang et al., 2010a; 2010b; cha-um and kirdmanee, 2010). for example, foliar spray of glycinebtaine mitigated the unpleasant drought stress effects on sunfl ower achene weight (iqbal et al., 2005), improved gas exchange characteristics and biomass production in sunfl ower (iqbal et al., 2009), and increased dry matter, grain yield and osmolytes in maize (zhang et al., 2009). in view of naidu et al. (1998) even under mild fi eld stress conditions, crop yield could be improved from 10 to 50% with foliar application of gb. effectiveness of exogenous application of gb depends on the type of species, developmental stage of plant, application level and the number of application, etc. (ashraf and foolad, 2005; 2007; ashraf et al., 2008). keeping in the view the role of glycinebtaine in plants under water defi cit conditions, we hypothesize whether or not exogenously applied gb as foliar spray alter morpho-physiological and yield responses of various wheat cultivars under water defi cit conditions. the chief objective of the current study was to assess whether foliarly-applied gb could alleviate the adverse effects of drought on growth, gas exchange attributes and mineral nutrition of wheat plants. role of glycinebetaine in wheat under drought stress 193 materials and methods a pot experiment was conducted to assess the infl uence of gb as foliar spray on the growth, physiological and biochemical attributes of wheat (triticum aestivum l.) under non-stress (control) and stress (drought) conditions. there were fi ve wheat cultivars (sarci, inqlab91, mh-97, bhakkar and s-24) obtained from ayub agriculture research institute, faisalabad (inqlab-91, mh-97, bhakkar), department of plant breeding and genetics (sarc-i) and from the department of botany, university of agriculture, faisalabad (s-24). the study was carried out in the net-house under natural conditions. during to course of experiment day and night average temperatures were 31.2 ± 5 °c and 26.5 ± 4 °c, respectively, relative humidity was from 32.6 to 64.7%, and day-length from 11 to 12 h. the experiment was laid out in a completely randomized design with three replications of each experimental unit. equal weight plastic pots (diameter 20 cm and 24 cm depth) were fi lled with 9 kg dry sandy loam soil. soil in each pot was completely saturated with normal irrigation water. when the moisture contents were at fi eld capacity, 12 healthy seeds (surface sterilized with 1% sodium hypochloride for 5 minutes) of each cultivar were hand sown. thinning of wheat plants was done to maintain 6 plants per pot after 15 days of germination. two levels of water i.e., (well-watered (control) and irrigation at 60% fi eld capacity (drought) were started after 6 weeks of seed sowing. the moisture contents of drought pots were regularly monitored and maintained by keeping weight of every pot equal to that calculated for 60% fi eld capacity through watering with normal irrigation water if required on daily basis till the maturation of the crop. three levels of glycinebetaine (gb) (m. wt. = 117.15 of sigma aldrich) i.e., 0, 50 and 100 mmof sigma aldrich) i.e., 0, 50 and 100 mmof sigma aldrich) i.e., 0, 50 and 100 m were applied after four m were applied after four m weeks of drought treatment (10-weeks of seed sowing) @ 32 ml per pot as foliar spray (tween-20 (0.1%) was used as a surfactant). data for various growth attributes, gas exchange characteristics and mineral nutrients were collected after 3-weeks of glycinebetaine application. two uniform plants from each pot were uprooted carefully, washed with distilled water and recorded their mean shoot and root fresh weights, shoot length, and leaf area per plant. the samples were oven dried at 65 °c for 72 hours and then their dry biomass was computed. gas exchange characteristics: an irga (infrared gas analyzer, model lca-4; analytical development company, hoddesdon, england) was used to measure gas exchange attributes including photosynthetic (aphotosynthetic (aphotosynthetic ( ) and transpiration (e) rates, stomatal conductance (gs), etc. all attributes were measured from 10.00 to 13.00 hours with the following leaf chamber specifi cations: air fl ow per unit leaf area 410.3 mmol m-2 s-1, atmospheric pressure 99.5 kpa, chamber water vapor pressure from 7.0 to 9.1 mbar, maximum par at leaf surface was up to 1620 µmol m-2 s-1, leaf temperature from 26.3 to 30.5 °c, 21.4 to 23.8 °c ambient temperature, and ambient co2 concentration was 350 µmol mol-1. determination of mineral elements: the (0.1g) dried ground material of leaves or roots tissues was digested according to the method described by allen et al. (1986). potassium ion was determined by a fl ame photometer (jenway pfp 7), while nitrogen was measured according to micro-kjeldhal’s method (bremner, 1965) and phosphorus by spectrophotometer according to jackson (1962). yield attributes: yield parameters i.e., grain yield per plant, and 100-grain weight were determined at maturity. statistical analysis: computer program mstat-c (mstat development team 1989) was used for the analysis of variance of the data. the least signifi cance difference test (lsd) was used to compare mean values of each attribute according to snedecor and cochran (1980). results drought stress (60% of fi eld capacity) markedly reduced shoot fresh and dry weights of all fi ve wheat cultivars (tab. 1; fig. 1). however, foliar-applied glycinebetaine (gb) enhanced shoot fresh and dry biomass of some cultivars under drought stress conditions. for example, positive effect of gb was observed in inqlab-91, bhakkar, and slightly in sarc-1, while in mh-97 and s-24 the infl uence of gb was non-signifi cant. overall, 50 mmof gb was non-signifi cant. overall, 50 mmof gb was non-signifi cant. overall, 50 m gb level was more m gb level was more m effective as compared to 100 mmeffective as compared to 100 mmeffective as compared to 100 m in causing growth improvement. m in causing growth improvement. m sarc-1 and inqlab-91 also showed better response in shoot dry weight to exogenous-applied 50 mmweight to exogenous-applied 50 mmweight to exogenous-applied 50 m gb under normal watering m gb under normal watering m conditions. root fresh and dry weights of all wheat cultivars reduced signifi cantly under different water stress (tab. 1; fig. 1). foliar-applied 50 mmunder different water stress (tab. 1; fig. 1). foliar-applied 50 mmunder different water stress (tab. 1; fig. 1). foliar-applied 50 m gb signifi cantly improved root fresh and dry weights in sarc-1, inqlab-91 and mh-97 under well-watered conditions and, in inqlab91 under water defi cit conditions. overall, sarc-i and inqlab-91 showed better performance under well-watered and water defi cit conditions in terms of root biomass. shoot length of all wheat cultivars decreased signifi cantly under drought regimes (tab. 1; fig. 1). however, foliar spray of gb slightly increased the shoot length of all cultivars under both drought regimes. total leaf area per plant of all wheat cultivars decreased markedly due to water defi cit conditions (tab. 1; fig. 1). foliar-applied gb did not mitigate the unpleasant effects of drought stress in terms of leaf area per plant. drought stress signifi cantly reduced grain yield per plant, however, 100-grain weight was not affected markedly (tab. 1; fig. 1). the exogenous application of gb slightly improved the grain yield of all cultivars under non-stress conditions, while under water defi cit conditions, the interaction between gb and drought stress was nonsignifi cant. drought stress signifi cantly reduced the photosynthetic rate (adrought stress signifi cantly reduced the photosynthetic rate (adrought stress signifi cantly reduced the photosynthetic rate ( ) of all fi ve wheat cultivars (tab. 1; fig. 2). response of all cultivars to foliar applied gb was variable under well-watered and drought stress conditions. in mh-97 gb enhanced net photosynthetic rate under well-watered conditions while in s-24 under drought conditions. in all remaining cultivars, gb did not alter net co2 assimilation rate effectively. transpiration rate of all wheat cultivars decreased due to imposition of drought stress conditions. while, gb application showed a contradictory role in this attribute. transpiration rate in cvs. mh-97 and bhakkar increased, while that in s-24 decreased with the application of gb under different water stress regimes (tab. 1; fig. 2). stomatal conductance in all wheat cultivars was markedly higher under normal watering as compared to that under drought stress conditions (tab. 1; fig. 2). foliar-applied gb did not alter this attribute under well-watered or water defi cit conditions. there was a slight difference among the cultivars in stomatal conductance under normal watering with high value being in sarc-1 as compared to the others. water use effi ciency was higher in all cvs under drought regimes as compared to that under non-stress. of different cultivars, s-24 showed better performance in wue due to gb application under drought stress conditions (tab. 1; fig. 2). sub-stomatal internal co2 concentration and ci/ca ratio showed a non-signifi cant behavior under drought stress conditions (tab. 1; fig. 2). of different cultivars, sarc-1 showed high value for substomatal co2 concentration at 100 mm concentration at 100 mm concentration at 100 m gb under drought stress m gb under drought stress m 194 m. shahbaz, y. masood, s. perveen, m. ashraf tab. 1: mean square values from analyses of variance of data for growth, yield, gas exchange and mineral nutrients of different cultivars of wheat (triticum aestivum l.) when 71-d old well-watered and water-stressed plants were subjected for 21-d to various levels of foliar-applied glycinebetaine. source of variation d.f. shoot f. wt. shoot d. wt. root f. wt. root d. wt. shoot length glycinebetaine (gb) 2 109. 3ns 0. 241* 1. 278*** 0. 036* 348. 1*** drought (d) 1 978. 3*** 10. 48*** 10. 16*** 0. 555*** 1291. 8*** cultivars (cvs) 4 13. 34ns 0. 053ns 0. 682*** 0. 106*** 69. 50*** gb x d 2 206. 9* 1. 517*** 0. 208ns 0. 043** 25. 90*** gb x cvs 8 63. 42ns 0. 928*** 0. 707*** 0. 07*** 58. 66*** d x cvs 4 48. 63ns 0. 588*** 0. 804*** 0. 044*** 14. 87*** d x gb x cvs 8 74. 25ns 0. 758*** 0. 545*** 0. 018* 48. 08*** error 60 64. 83 0. 055 0. 076 0. 008 2. 05 source of variation d.f. leaf area grain yield 100-seed a e per plant per plant weight glycinebetaine (gb) 2 10869. 2* 0. 397ns 0. 397ns 0. 608ns 7. 27*** drought (d) 1 867025. 7*** 85. 79*** 0. 121ns 5269. 0*** 76. 40*** cultivars (cvs) 4 125620. 2*** 2. 71*** 0. 546ns 53. 84*** 2. 14*** gb x d 2 3402. 5ns 0. 023ns 0. 061ns 51. 03*** 5. 33*** gb x cvs 8 21917. 3*** 0. 84* 0. 493ns 25. 01*** 1. 35*** d x cvs 4 69483. 0*** 2. 31*** 0. 367ns 50. 74*** 1. 48*** d x gb x cvs 8 14754. 8*** 0. 92** 0. 422ns 11. 94*** 0. 795*** error 60 2202. 4 0. 317 0. 315 0. 77 0. 165 source of variation d.f. gsgsg a/e cicic cicic/ci/ci a/ca/c shoot p glycinebetaine (gb) 2 752. 7ns 64. 12*** 26. 26ns 0. 007ns 31. 1*** drought (d) 1 20091. 3*** 25. 70** 13603. 8** 0. 233*** 87. 52*** cultivars (cvs) 4 1798. 14* 32. 95*** 3764. 9* 0. 04*** 0. 197ns gb x d 2 215. 24ns 5. 13ns 170. 4ns 0. 011ns 0. 736ns gb x cvs 8 1650. 29ns 28. 55*** 2436. 3ns 0. 025*** 0. 491ns d x cvs 4 3599. 63** 31. 91*** 5622. 4** 0. 018* 2. 512*** d x gb x cvs 8 1419. 80ns 7. 52* 1614. 9ns 0. 014* 1. 023* error 60 813. 95 2. 76 1429. 7 0. 055 0. 448 source of variation d.f. shoot n shoot k+ root p root n root k+ glycinebetaine (gb) 2 111. 61** 166382 *** 0. 626* 11. 32ns 263. 40*** drought (d) 1 35. 84ns 4885380 *** 1. 048** 164. 02*** 302. 5** cultivars (cvs) 4 70. 34** 45795.1 *** 0. 126ns 15. 35* 35. 13ns gb x d 2 5. 22ns 217785 *** 1. 361*** 20. 32* 601. 45*** gb x cvs 8 12. 28ns 18691. 1*** 0. 452** 6. 295ns 88. 83* d x cvs 4 53. 45* 20106. 6*** 0. 496* 3. 43ns 113. 61* d x gb x cvs 8 5. 13ns 27563. 2*** 0. 035ns 9. 662* 169. 60*** error 60 14. 71 5689. 3 0. 143 4. 52 33.611 *, **, *** = signifi cant at 0.05, 0.01, and 0.001 levels, respectively. ns = non-signifi cant conditions. the response of all the remaining four cultivar was almost not uniform in this attribute. water defi cit conditions signifi cantly reduced shoot and root p contents, however, under drought regimes, foliar-applied gb improved root p but not those of shoot p contents (tab 1; fig. 3). drought had a signifi cant reducing effect on root n contents while shoot n contents did not change signifi cantly under water defi cit condition (tab. 1; fig. 3). foliar application of gb did not improve shoot n contents but slightly increased root n contents. the interaction among cultivars for foliar application of gb was nonrole of glycinebetaine in wheat under drought stress 195 � � �� �� �� � � � � �� �� �� �� �� � �� �� � �� � � ������� �������� ��������� � � � � � � � � � �� � �� � �� �� � �� �� � �� � � ������� �������� ��������� � ��� � ��� � ��� � � � � �� �� �� �� �� � �� �� � �� � � � ��� ��� ��� ��� � � � �� � �� � �� �� � �� �� � �� � � � �� �� �� �� � � � � �� �� � � �� �� � � � ��� ��� ��� ��� ���� � � � �� � �� � �� �� � �� �� � � � � � � � � � � � � � � � � � � � � ����� � ������� � � � �� � � ������� �� � � � � � � �� �� �� �� �� � �� �� � � � � � � � � � � � � � � � � � ����� � ������� � � � �� � � ������� �� � � � � � �� � � � �� � �� � �� �� � ������������������������������������������������������������������������������������������������� ���� ��������������� � ������� ����� ���������� ���� ��� ����� ��� �������� ������� ��� �������� �������� ������������������������������������������ fig. 1: growth and yield attributes of wheat (triticum aestivum l.) when 71-day old well-watered and water-stressed plants were subjected for 21 days to various levels of foliarapplied glycinebetaine. (c = control; d = drought) signifi cant for both shoot and root n contents. water defi cit conditions signifi cantly reduced shoot and root k+ contents. foliar-applied gb slightly improved shoot k+ under water defi cit conditions in all wheat cultivars except sarc-1, while the reverse was true for root k+. of various gb levels, 100 mm. of various gb levels, 100 mm. of various gb levels, 100 m gb was m gb was m more effective in increasing shoot k+ under water defi cit conditions (tab. 1; fig. 3). discussion drought stress triggers a series of biochemical and physiological processes in plants, which impose adverse effects on plant growth and biomass production (gomes and prado, 2010). in the present study, water stress reduced the growth of all wheat cultivars which is in agreement with some earlier investigations in which growth of different crops was markedly decreased due to water stress, e.g., in sugar beet (bloch et al., 2006), wheat (passioura, 2006), and maize (ashraf et al., 2007). however, foliar-applied gb proved to be effective in enhancing shoot fresh and dry biomass of all cultivars under drought regimes. the benefi cial role of gb in promoting shoot and root fresh biomass in wheat under drought stress has already been reported (mahmood et al., 2009). genetic variation for drought tolerance is reported in various crops like grass species (ashraf et al., 1986), and maize cultivars (ashraf et al., 2007; zhang et al., 2009). similarly, in our study of the wheat cultivars examined, sarc-1, inqlab-91 and mh-97 were relatively better under drought stress. generally, water stress is known to reduce the growth of plants by reducing the photosynthetically active leaf area, one of the most important factors affecting crop productivity (dubey, 1997). similarly, in our experiment leaf area was decreased under water defi cit conditions, while foliar-applied gb did not reduce the bad effect of drought stress in terms of leaf area which did not agree to the fi ndings of mahmood et al. (2009) ) 196 m. shahbaz, y. masood, s. perveen, m. ashraf who reported an increase in leaf area by exogenous application of gb. enhancement in dry weight and grain yield by exogenous gb application has been earlier reported in maize under drought stress, but not under well-watered conditions (zhang et al., 2009). cultivar s911 (drought sensitive) of maize was more responsive to exogenous applied gb compared to drought tolerant cultivar s9 (zhang et al., 2009). in our study, a marked reduction was observed in grain yield per plant under drought stress. however, foliarapplied gb slightly increased the grain yield of all cultivars under normal watering, but not under drought stress. the net photosynthetic and transpiration rates, stomatal conductance (gs), and intercellular co2 concentration (ci) in most plants decrease under drought stress (perez-perez et al., 2009; wang et al., 2010). however, over-accumulation of gb increased rate of photosynthesis in wheat under drought and other stress conditions (wang et al., 2010). in our study, drought stress caused a signifi cant reduction in photosynthetic rate of all wheat cultivars. however, the cultivars differed in their response to foliarapplied gb under well watered and drought stress conditions. for example, under water defi cit conditions, photosynthetic rate (aexample, under water defi cit conditions, photosynthetic rate (aexample, under water defi cit conditions, photosynthetic rate ( ) is slightly increased in all the wheat cultivars while, under wellwatered conditions, it markedly increased in mh-97, and slightly in inqlab-91 and s-24 as compared to sarc-1 and bhakkar. it has been reported that exogenously-applied gb enhances net co2 assimilation rate under drought stress conditions particularly in plants which naturally accumulate low levels of gb (makela et al., 1999; lopez et al., 2002) as compared to those with higher levels of endogenous gb (gorham et al., 2000; meek et al., 2003). iqbal et al. (2009) reported that sunfl ower line with high endogenous gb level showed higher stomatal conductance. in our experiment, stomatal conductance was higher in the well-watered plants of all cultivars as compared to that under water defi cit conditions. foliar application of gb showed a variable role in transpiration rate and water use effi ciency. foliar-applied gb increased transpiration rate under drought stress, while wue under well watered conditions. mahmood et al. (2009) reported that exogenous application of gb proved ineffective in altering the gas exchange characteristics in wheat. however, in contrast in our study, of all cultivars, sarc-1 was better than the other cultivars particularly at 100 mmwas better than the other cultivars particularly at 100 mmwas better than the other cultivars particularly at 100 m gb. these m gb. these m results reinforce the statement that effectiveness of gb application varies on the basis of species type and level of gb used (ashraf and foolad, 2005; 2007; ashraf et al., 2008). under long-term exposure to water defi cit conditions a signifi cant decrease in inorganic p concentration in younger and older leaves occur in sugar beet (choluj et al., 2008). in our experiment, water defi cit conditions reduced both shoot and root p contents in all wheat cultivars. however, under drought stress foliar-applied gb improved root p than that of shoot. drought stress in our study decreased root n but not shoot n in all wheat cultivars. these � � � � � � �� µ � � �� � � � �� �� �� �� � ������� �������� ��������� � � � � � �� � � �� � � � �� �� �� �� � ������� �������� ��������� � ��� ��� ��� ��� � � �� � � �� � �� �� �� � � � � � � � � � ��µ � � �� � � � �� � � �� � � � �� ��� ��� ��� ��� ��� ��� ��� � � � � � � � � � � ����� � ������� � � � ��� � ������� ��� � � � �� µ � � �� � � �� � � � ��� ��� ��� ��� � ��� � � � � � � � � � � ������ ������� � � � ��� � ������� ��� � � ��� � �� �� � ����� ��� ���� ��������� ��������������������� ����������� ���� �������� ���������� ��� ������ ���������� ��������� ���� ����� � ������� ���� ������������� ���� ��������������� ������� ����� �������������������������������������������������������������������������������������������� �������� fig. 2: gas exchange characteristicsyield attributes and mineral nutrients of wheat (triticum aestivum l.) when 71-day old well-watered and water-stressed plants were subjected for 21 days to various levels of foliar-applied glycinebetaine. (c = control; d = drought) role of glycinebetaine in wheat under drought stress 197 results can be explained with the help of an earlier report in which leaf drought was reported to decrease plant n uptake and availability of leaf n (zhen and zhou, 2006). earlier sarwar et al. (1991) examined the effect of drought regimes on different wheat varieties and found a marked increase in n concentration under drought stress conditions. however, in our study, foliar application of gb did not improve shoot n, but slightly increased root n. likewise, mahmood et al. (2009) found that exogenously-applied gb did not alter the leaf mineral nutrients in wheat. overall, drought stress decreased the growth, yield components, gas exchange characteristics and mineral nutrients of all fi ve genetically diverse wheat cultivars. of all cultivars, sarc-1 and inqlab-91 showed relatively better performance in terms of plant biomass, net photosynthetic rate and stomatal conductance under well watered and drought stress conditions. foliar-applied gb was found to be effective in enhancing various growth attributes and grain yield as well as the levels of some key metabolites of all wheat cultivars. of various gb levels, 50 mmwheat cultivars. of various gb levels, 50 mmwheat cultivars. of various gb levels, 50 m was found to be more m was found to be more m effi cient than the other gb levels for promoting wheat growth under water defi cit conditions. references abd el-rhman, i.e., 2010: a study on some treatments which mitigate drought effects on barrani grapevines cv. j. appl. sci. res. 6, 704-711. allakhverdiev, s.i., hayashi, h., nishiyama, y., ivanov, a.g., aliev, j.a., klimov, v.v., murata, n., carpentier, r., 2003: glycinebetaine protects the d1/d2/cytb559 complex of photosystem ii against photoinduced and heat-induced inactivation. j. plant physiol. 160, 41-49. allen, s.e., grimshaw, h.m., rowland, a.p., 1986: chemical analysis. in: moore p.d., chapman, s.b. 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peroxidation of a perennial grass leymus chinensis. planta 224, 10801090. zhu, m.y., ahn, s.j., matsumoto, h., 2003: inhibition of growth and development of root border cells by al. physiol. plant. 117, 359-367. address of the author: e-mail: shahbazmuaf@yahoo.com journal of applied botany and food quality 94, 75 81 (2021), doi:10.5073/jabfq.2021.094.009 1facultad de ciencias, departamento de biología, laboratorio de fisiología y bioquímica vegetal. universidad nacional de colombia, bogotá, colombia 2facultad de ciencias agrarias, departamento de agronomía. universidad nacional de colombia, bogotá, colombia photosynthesis, biochemical activity, and leaf anatomy of tree tomato (solanum betaceum cav.) plants under potassium deficiency claudia helena ramírez-soler1,2,*, stanislav magnitskiy2, sandra esperanza melo martínez2, fagua álvarez-flórez1, luz marina melgarejo1,* (submitted: february 16, 2021; accepted: march 28, 2021) * corresponding author summary the effects of potassium (k) deficiency on the physiological, biochemical, and anatomical parameters of leaves in the tree tomato plants (solanum betaceum cav.) were evaluated during vegetative growth. the experiment was carried out for 135 days after treatment applications under greenhouse conditions, employing the nutrient solutions with the following treatments: control plants (without k deficiency) and the plants with k deficiency. the light response curve, photosynthesis at light saturation (amax), light compensation point (ic), transpiration rate (e), stomatal resistance (sr), and pigment contents in leaves were evaluated. additionally, the maximum photochemical efficiency of psii (fv/fm), contents of malondialdehyde (mda), total soluble sugars, proline, and leaf anatomy parameters were assessed. in the k-deficient plants, the reduction in amax (66%), ic (63.7%), e (66%), fv/fm (17.3%), contents of total chlorophyll (77.4%) and chlorophyll a (52%), thickness of leaf blade l (28.5%), palisade parenchyma pp (6.5%), and spongy parenchyma sp (9.5%) were observed, compared to the control plants. in contrast, the variables that increased significantly were sr (65%), mda (52%), upper epidermis thickness (ue) (27.1%), and lower epidermis thickness (le) (22.3%). the potassium deficiency caused alterations in the plant development due to the influence on physiological, biochemical, and anatomical parameters, which suggests the importance of mineral nutrition with k for this plant. introduction the tree tomato (solanum betaceum cav.), also known as tamarillo, belongs to the solanaceae family, originating from the andean fo rests of southern bolivia and northern argentina (acosta-quezada et al., 2015). it is a perennial plant that reaches around 2 to 3 m height in its natural habitat and has a semi-woody stem that ramifies and forms the crown. the tree tomato is one of the most important fruit crops in the colombian andean region, with a national production of 196,558 t (asohofrucol, 2020), due to its potential for food processing, pigment, cosmetics, and pharmacy industries, fresh consumption, and antioxidant content (acosta-quezada et al., 2015; mohd nor et al., 2018). chemical and biological factors can affect crop development, including nutrient management (garza-alonso et al., 2019). mineral nutrition plays a key role in the growth and development of plants and, consequently, in crop production. potassium (k) is one of the crucial nutrient elements for meristem functioning, defense, signaling, and transport processes (armengaud et al., 2009; demidchik, 2014). in addition, this element participates in stomatal opening, movement of solutes via phloem, cellulose synthesis, osmoregulation, induced resistance to pathogen attack (demidchik, 2014), and water absorption (hawkesford et al., 2012). it participates in about 60 enzymatic reactions, involving photosynthesis, respiration, protein synthesis, and carbohydrate metabolism (dong et al., 2010). it is also a key element for the establishment of transmembrane ph gradient required for atp synthesis (hawkesford et al., 2012; zörb et al., 2014). various experiments in hydroponics systems and in pots with a substrate lacking k show a negative effect of the k deficiency on the physiological and growth parameters of plants (wang et al., 2015; srinivasarao et al., 2016; du et al., 2019). the absence of k altered the distribution of assimilates and this translated into the changes in metabolite contents in vegetative organs (hawkesford et al., 2012). however, in the k-deficient leaves, a concentration of internal co2 increased, indicating that the reduction in photosynthesis was more affected by the resistance of leaf mesophyll than by the stomatal resistance (römheld and kirkby, 2010; zörb et al., 2014). in addition, along with a decrease in the k+ concentration in leaves, not only the photosynthesis rate and rubp carboxylase activity decreased but also the photorespiration rate (hawkesford et al., 2012). in this way, the diagnostics of the nutrient status of k in plants is important for the optimal production and quality of the crops (mattiello et al., 2015; lu et al., 2016; sanadi et al., 2018). given its importance as an exotic fruit crop on the international markets (acosta-quezada et al., 2015), the tree tomato has positioned itself as one of the main fruit and vegetable crops produced in colombia (asohofrucol, 2020) due to its high potential for bioprospecting. to our knowledge, there were no published reports on the effects of k deficiencies in the tree tomato at the physiological, biochemical, and anatomical levels. due to the above, it would be necessary to characterize the effects of k deficiency on photosynthesis, biochemical composition (malondialdehyde (mda), proline, total sugars), and leaf anatomy in the tree tomato plants. therefore, the objective of this study was to evaluate the effect of k deficiency on the physiological, biochemical, and anatomical parameters in the tree tomato plants during vegetative growth. material and methods plant material and growth conditions the experiment was carried out under the 7-gauge polyethylene plasticized greenhouse, with daily average air relative humidity of 70%, average temperature of 20.3 °c, and photosynthetically active radiation (par) of 200 μmol photons m2 s-1. three-month-old common red ecotype tree tomato (solanum betaceum cav.) seedlings were used, which were subjected to root wash with distilled water to remove soil particles from the substrate. subsequently, these were transplanted into the black plastic bags with 8 kg capacity (0.40 × 0.60 m) containing quartzite sand of two par76 c.h. ramírez-soler, s. magnitskiy, s. esperanza melo martínez, f. álvarez-flórez, l.m. melgarejo ticle sizes (0.7 and 1.5 mm) in a 1:1 v/v ratio, electrical conductivity (ec) of 0.012 ds m-1 s-1, and ph of 6.7. each bag was positioned 1 m apart to avoid the effects between the plants. once transplanted, these were subjected to pretreatment (acclimatization) for one month, by supplying the complete modified nutrient solution of hoagland et al. (1938), where the control treatment received the complete nutrient solution without mineral deficiencies (tab. 1). gas exchange and chlorophyll a fluorescence in two fully expanded leaves of the middle-third stratum of four plants per treatment, the maximum photochemical efficiency of psii was measured (fv/fm) at 0, 15, 30, 45, 60, 75, 90, 105, 120 and 135 dat with a handypea unmodulated fluorometer (hansatech instruments, norfolk, uk) with a saturating par of 3000 μmol photons m2 s-1. in the same plants and leaves, the transpiration rate (e) and stomatal resistance (sr) were determined between 7:00 and 10:00 am, using a porometer li-cor li-1600 steady (lincoln, nebraska, usa). biochemical parameters these were analyzed at the end of the experiment (135 dat). photosynthetic pigment contents in three individual plants per treatment, the leaves were collected from the middle-third stratum for extraction of the total chlorophyll content (total chl) according to lichtenthaler and wellburn (1983). the 0.05 g leaf tissue without ribs was used and the extraction was carried out with 80% acetone previously kept at -4 °c. absorbance at 470, 646 and 633 nm was measured using a bio-rad smart spectm 3000 spectrophotometer (bio-rad, philadelphia, usa) expressing the results obtained in fresh weight mg g-1 (fw), for the concentration of chlorophyll a (chl a), chlorophyll b (chl b), total chlorophyll (total chl), and carotenoids. malondialdehyde content the content of malondialdehyde (mda) was determined with thiobarbituric acid based on that described by wang et al. (2012). the 0.05 g of fresh leaf tissue was obtained from the middle-third stratum of three individual plants per treatment, then used for maceration and extraction, homogenized with 2 ml of 10% trichloroacetic acid (tca) solution. to 1 ml of the supernatant, 4 ml of 0.5% tba solution (prepared in 10% tca) was added and stirred. subsequent ly, it was heated at 95 °c for 30 min and then cooled on ice. it was centrifuged at 5000 rpm for 15 minutes. absorbance was read with a spectrophotometer (bio-rad smart spectm 3000, philadelphia, usa) at 450, 532 and 600 nm. the blank was 10% tca. the mda contents was expressed in μmol ml-1 fw. total sugars the soluble sugars were extracted according to dubois et al. (1956) modified by melgarejo et al. (2010). the 0.05 g of fresh plant material was weighted from leaves obtained from the middle-third stratum of three plants per treatment. subsequently, it was macerated with liquid nitrogen, then 5 ml of distilled water were added, and it was stirred at room temperature for 60 min. it was centrifuged at 6,000 rpm for 30 min at 12 °c. the 30 μl of the supernatant were taken per sample, 180 μl of distilled water were added, homo genized, and then 200 μl of 80% phenol were added. subsequently, 1.0 ml of concentrated sulfuric acid was added, stirring in vortex for 1 min. finally, absorbance was read at 490 nm with a spectro photometer (bio-rad smart spectm 3000, philadelphia, usa). the content of total sugars was expressed in μg mg-1 fw. proline content the proline content was determined according to bates (1973), with adjustments (melgarejo et al., 2010). the 0.05 g of fresh plant ma terial was obtained from the leaves taken from the middle-third stratum of three individual plants per treatment. subsequently, 5.0 ml of 3% (w/v) sulfosalicylic acid extracting solution was introduced, stirred for 60 minutes at 10 °c, and centrifuged at 6,000 rpm for 30 minutes at 10 °c in darkness. next, in dark glass tubes, 1.0 ml of the supernatant was placed and 1.0 ml of freshly prepared ninhytab. 1: concentration of mineral elements (ml of the stock solutions 1 m to be applied to 7.5 l h2o) modified based on the stock solutions (hoagland et al., 1938) and the nutrient requirements reported for the species (fischer and miranda, 2012). macronutrients (ml) treatments source without k control nh4no3 69.72 55.78 kh2po4 0.00 13.02 ca(h2po4)2.h2o 32.16 32.16 kcl 0.00 43.94 kno3 0.00 37.86 cacl2 2.70 2.70 ca(no3)2.4h2o 23.00 23.00 caso4.2h2o 20.96 12.58 mg(no3)2.6h2o 41.19 51.48 mgso4 4.83 0.00 k2so4 0.00 31.83 micronutrients h3bo3 0.28 0.28 mncl2.4h2o 0.70 0.70 zns04 0.24 0.24 cuso4.5h2o 0.19 0.19 feso4.7h2o 0.73 0.73 treatments the hoagland solution (hoagland et al., 1938) was modified and adjusted based on the needs of the crop (fischer and miranda, 2012) (tab. 1); this was prepared in distilled water with an ec < 3 μs m-1 to generate the stock solutions; later it was diluted in 7.5 l of water to carry out the applications. the following two treatments were used: without k deficiency (control) and with k deficiency (without k), which were applied to the plants two times per week. a volume of 100 ml of the nutrient solution was supplied per plant (according to the substrate moisture retention curve) during vegetative growth. physiological parameters photosynthesis at the end of the experiment (135 days after the treatment application (dat)), three plants were taken per treatment and light response curves (a/pfd) were obtained with an irga lcipro + kit (bioscientific ltd. hoddesdon, uk). the measured radiation points were 1,200, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 50, 40, 30, 20, 10, and 0 μmol photons m2s-1 between 7:00 10:00 h (a range of time previously determined, where a highest rate of photosynthesis was observed). the curves were made using the 4th leaf fully expanded of the middle-third stratum of the plants. the data obtained were adjusted using the mitscherlich hyperbolic model (aleric and kirkman, 2005; melgarejo et al., 2010; barrera et al., 2012). the parameters derived from the a/pfd curves were determined as amax = photosynthesis at saturation by light or maximum photosynthesis (μmol co2 m-2 s-1), ic = light compensation point (μmol photons m-2 s-1). analysis of tree tomato plants under potassium deficiency 77 drin and 1.0 ml of warm glacial acetic acid were added, which were brought to the boil for 60 min. then these were left at room temperature and 3ml of toluene was added. finally, the upper phase was collected and the absorbance was read at 520 nm in a spectrophotometer (bio-rad smart spectm 3000, philadelphia, usa), expressing the content of proline in μg g-1 fw. the concentration of proline was determined using the standard curve of l-proline from sigma®. leaf anatomy at the end of the experiment (135 dat), leaf sections (approximately 30 mm2) were collected from the middle region of the same leaf used for the gas exchange and fv/fm measurements. the sections were fixed in a mixture (formalin: 10, ethanol: 5, glacial acetic acid: 85), later they were subjected to different solutions of ethanol and histochoice® according to megías et al. (2018). the paraffin blocks were cut with a model 820 spencer rotary microtome (american optical, delhi, usa), making transverse cuts in the leaves and covering the midrib with a thickness of 13 μm. the cross sections were arranged and fixed on slides, later they were stained with the double stain of astra-blue, basic fuchsin (kraus et al., 1998). digital images with a resolution of 100x were obtained from the assemblies in olympus® cx31 microscope (new york microscope company, ny), with camera adaptation. using the image-proplus® program (media cybernetics, rockville, md), the measurements were made of the leaf thickness (l), spongy parenchyma thickness (ps), palisade parenchyma thickness (pp), upper epidermis (ue), and lower epidermis thickness (le). experimental design and statistical analysis a completely randomized design and two repetitions of the same trial were used in time; in the present study, the data of the second repetition in time are presented. each trial had four replicates and twenty individual plants per treatment. the data were statistically analyzed using anova to identify the presence of significant differences between the treatment means due to their effects on the evaluated physiological, biochemical, and anatomical variables. the sas version 9.2 package was used. once the significant differences between treatment means were found, the tukey ś multiple comparison test was used (p≤0.05). results physiological parameters light response curve (a/pfd) the response curves to light a/pfd indicate the response that plants present under different light intensities (pérez and melgarejo, 2014). the tree tomato plants during vegetative growth under potassium deficiency (without k) had a decrease in amax under the different radiation points (pfd), presenting a final value of 2.3 μmol co2 m-2 s-1 and differing from the control plants with 6.8 μmol co2 m-2 s-1 at 135 dat (fig. 1 and tab. 2). the light compensation point (ic) decreased in the plants deficient in k with a value of 12.17 μmol photons m-2 s-1 as compared with the control, which obtained a value of 35.1 μmol photon m-2 s-1 (fig. 1, tab. 2). transpiration (e), stomatal resistance (sr) and fluorescence of chlorophyll a (fv/fm) the tree tomato plants without k registered a significant reduction (p≤0.05) in the transpiration rate (e) at 90, 120, and 135 dat by 45, 58 and 66%, respectively, compared to the control plants (fig. 2a). the stomatal resistance (sr) presented an inverse behavior to the transpiration, that is, the lower was the transpiration, the higher was the stomatal resistance, as a physiological adaptation of plants to avoid water loss. the sr significantly increased in the plants without k at 120 and 135 dat by 55 and 65%, respectively, compared to the control plants (fig. 2b). the plants without k registered a significant decrease in fv/fm at 105, 120 and 135 dat (0.74, 0.72 and 0.67, respectively) unlike the control plants, which remained with an average fv/fm of 0.81 (fig. 2c). photosynthetic pigment contents the tree tomato plants grown without k at 135 dat showed a significant reduction in the content of chl a and total chl (by 51.7 and 77.4%, respectively) as compared to the control (tab. 3). however, the plants without k had the tendency to decrease the chl b content by 33% and a slight increase in the carotenoid content by 14%, unlike the control plants (tab. 3). the lipid peroxidation, measured as mda contents, indicates a possible damage at the cellular membrane level. the plants without k presented significant increases of 0.85 ± 0.07 μmol ml-1 mda, compared to the control, which registered 0.52 ± 0.03 μmol ml-1 mda (tab. 3). the contents of total soluble sugars in leaves did not present significant differences between the treatments, however, the tendency to decrease was evidenced in the tree tomato plants cultivated without k (24.87 ± 0.85 μg mg-1 leaf fw), unlike of the control plants (29.35 ± 0.98 μg mg-1 leaf fw) (tab. 3). the content of free proline did not present statistical differences between the treatments; however, an increasing trend was registered for the treatment without k (0.20 ± 0.05 μg g-1), unlike the control (0.16 ± 0.01 μg g-1) (tab. 3). leaf anatomy the differences were observed in the plants with k deficiency, compared to the control at 135 dat. in contrast to the control, the leaves of the plants grown without k exhibited less thickness of leaf lamina (l), which was 28.5% thinner (tab. 4). the leaves with k deficiency zns04 0.24 0.24 cuso4.5h2o 0.19 0.19 feso4.7h2o 0.73 0.73 350 351 352 fig. 1: light saturation curves of photosynthesis in the tree tomato plants during vegetative 353 growth (a vs. ppfd). control (without k deficiency) (r2 = 0.94); plants grown without 354 potassium k (potassium deficiency) (r2 = 0.99). at the bottom of each figure the equation is 355 derived from the fit to a mitscherlich model. n = 3 plants per treatment. 356 357 tab. 2: parameters obtained through the light response curve in the tree tomato plants during 358 vegetative growth at 135 dat. control (without k deficiency) and plants grown without k 359 (potassium deficiency). these were calculated by fitting to a mitscherlich model. a max = 360 photosynthesis at saturation by light or maximum photosynthesis, ic = light compensation 361 point. n = 3 plants per treatment. 362 treatment r2 amax (μmol co2 m-2s-1) ic (μmol photons m-2s-1) control 0.94 6.8±0.044 ±35.1 without k 0.99 2.3±0.047 ±12.17 363 -8,0 -6,0 -4,0 -2,0 0,0 2,0 4,0 6,0 8,0 10,0 12,0 0 100 200 300 400 500 600 700 800 a (µ m ol c o 2/ m -2 s1 ) pfd (µmol m2 s of photons) control without k 7 6 5 4 2 1 0 -1 -2 -3 control: p= 6.787*(1-exp(-4.25e-03*(pfd-35.133))) without k: p= 2.2286*(1-exp(-0.0127*(pfd-12.170))) fig. 1: light saturation curves of photosynthesis in the tree tomato plants during vegetative growth (a vs. ppfd). control (without k deficiency) (r2 = 0.94); plants grown without potassium k (potassium deficiency) (r2 = 0.99). at the bottom of each figure the equation is derived from the fit to a mitscherlich model. n = 3 plants per treatment. tab. 2: parameters obtained through the light response curve in the tree tomato plants during vegetative growth at 135 dat. control (without k deficiency) and plants grown without k (potassium deficiency). these were calculated by fitting to a mitscherlich model. a max = photosynthesis at saturation by light or maximum photosynthesis, ic = light compensation point. n = 3 plants per treatment. treatment r2 amax (μmol co2 m-2s-1) ic (μmol photons m-2s-1) control 0.94 6.8±0.044 ±35.1 without k 0.99 2.3±0.047 ±12.17 78 c.h. ramírez-soler, s. magnitskiy, s. esperanza melo martínez, f. álvarez-flórez, l.m. melgarejo showed a reduction by 6.5% in the thickness of the palisade parenchyma (pp) (tab. 4) and the cells were irregular (fig. 4d), unlike in the control (fig. 4b). the thickness of the spongy parenchyma (sp) was reduced by 9.5%, compared to the control (tab. 4), and also exhibited an irregular shape of the cells (fig. 4d). on the contrary, the k deficiency increased the thickness of the upper epidermis (ue) and lower epidermis (le), surpassing the control plants by 27.1 and 22.3%, respectively (tab. 4). the leaves of the k-deprived plants presented variations in the size of the middle vein (midrib) (data not shown) and had irregular distribution of the vascular bundles (v) and irregular parenchyma cells (p), unlike the control leaves (fig. 4c, a, respectively). discussion our results show that the tree tomato plants during vegetative growth under k deficiency severely reduced the photosynthetic rate (fig. 1 and tab. 2), presenting a behavior similar to that reported for arabi dopsis plants (armengaud et al., 2009), soybean (dong et al., 2010) and corn (du et al., 2019) under deficiency of this element. lu et al. (2016) pointed out that the k deficit affects the photosynthetic rate due to the lower activity of rubisco and the activation of the pyruvate kinase, which catalyzes the phosphoenolpyruvate to produce atp and pyruvate in glycolysis (wang and wu, 2010). in the same way, the activity of rubp carboxylase could be negatively affected due to the fact that k maintains a high ph in the stroma and its deficiency alters the photorespiration (hawkesford et al., 2012). in this study, as the k deficiency progressed, the transpiration rate (e) decreased and stomatal resistance (sr) increased, which indicates that starting from 90 dat the plants experienced a stress, which was reflected with the reduction of fv/fm (fig. 2) and was further translated into a reduction in photosynthesis (tab. 2). similar results were reported by tang et al. (2015) in tea, wang et al. (2015) in soybean, and du et al. (2019) in corn, where k deficiency significantly reduced the variable e, unlike in the control plants. this response is due to the important role of k in maintaining turgor pressure, a fundamental variable for stomatal opening (hawkesford et al., 2012). it was, possibly, due to the fact that the reductions in e (fig. 2a) and amax (tab. 2) are caused by the stomatal limitations (römheld and kirkby, 2010; jin et al., 2011). andrés et al. (2014) pointed out that the k deficiencies affect the opening of stomata, decreasing the photosynthetic rate due to the limitation in the assimilation of co2 from the atmosphere to the internal spaces of leaves, causing a negative effect on the carboxylation sites within the chloroplasts (flexas 364 365 366 0 2 4 6 8 10 12 0 15 30 45 60 75 90 105 120 135 e (m ol h 2o m -2 s1 ) dat (days after transplanting) control without k * a) * * 0 1 2 3 4 5 6 7 8 0 15 30 45 60 75 90 105 120 135 st om at al r es is ta nc e (s .c m ) dat (days after transplanting) control without k * b) 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 0 15 30 45 60 75 90 105 120 135 fv /f m dat (days after transplanting) control without k * * * * * c) fig. 2: physiological parameters in the tree tomato plants during vegetative growth. treatments: control (without deficiency) and plants grown without k (potassium deficiency). a) transpiration rate (e), b) sto matal resistance (sr); and c) maximum photochemical efficiency of photosystem ii (fv/fm). bars represent the standard error (n = 4 plants per treatment). significant differences at the 0.05 level of significance are indicated by * (the tukey’s test). tab. 3: contents of photosynthetic pigments (mg g-1 leaf fw), malondialdehyde (mda) (μmol ml-1), total sugars (μg mg-1 leaf fw), and proline (μg g-1) in the leaves of tree tomato plants during vegetative growth. means ± standard error (n = 3 per treatment) are presented. control (without deficiencies) and plants grown without k (potassium deficiency). different letters indicate significant statistical differences with a p value <0.05 (the tukey’s test). biochemical parameter chl a chl b total chl carotenoids mda total sugars proline control 0.58 ±0.029 a 0.31 ±0.004 a 0.89 ±0.025 a 0.56±0.060 a 0.52±0.03 a 29.35±0.98 a 0.16±0.01 a without k 0.30±0.064 b 0.24±0.042 a 0.54±0.100 b 0.64±0.195 a 0.85±0.07 b 24.87±0.85 a 0.20±0.05 a tab. 4: anatomical parameters registered in the cross sections of the tree tomato leaves during vegetative growth. control (without deficiencies) and plants grown without k (potassium deficiency). means ± standard error (n = 3 per treatment) are presented. different letters indicate significant statistical differences with a p value <0.05 (the tukey’s test). treatment leaf blade palisade parenchyma spongy parenchyma upper epidermis lower epidermis thickness (l) thickness (pp) (μm) thickness (sp) (μm) thickness (ue) (μm) thickness (le) (μm) control 247.6±13.8 b 51.9±2.5 a 87.6±7.8 a 9.6±0.7 b 10.3±0.4 b without k 177.1±10.7 a 48.5±2.83 a 79.3±6.1 a 12.2± 0.5 a 12.6±1.21 a analysis of tree tomato plants under potassium deficiency 79 et al., 2008). similarly, as k is important for the opening of stomata, the lack of this element would reduce its accumulation in the vacuole, affecting the stomatal opening (jordan-meille and pellerin, 2008) and altering the stomatal and non-stomatal regulation of co2 (tang et al., 2015). the response of e was opposite to sr (fig. 2a, b) as a strategy of the plants to avoid the loss of water inside the leaf towards the atmosphere (quezada et al., 2002). the k deficiency generated a reduction in fv/fm (fig. 2c), suggesting a possible photoinhibition starting from day 105 (maxwell and johnson, 2000). this photoinhibition was, probably, related to the chlorophyll content (tab. 3), which agrees with that reported by hu et al. (2016). kitajima and butler (1975) proposed that fv/fm alterations generate changes in the photochemical conversion of energy on the reaction centers of psii and induce a possible photoinhibition. according to our results, it can be inferred that the reduction in the content of pigments, such as chla, chlb and total chl (tab. 3), due to the treatment without k were related to the low photosynthetic rate (amax) (tab. 2) and possible stomatal limitations. results similar to those found in this study have been reported by jin et al. (2011) and cavalcante et al. (2015), indicating that the k deficiencies could be associated with the degradation of chlorophylls, biochemical alteration of chloroplasts and, therefore, less absorption of light. jin et al. (2011) and wang et al. (2012) suggested that the k deficiency gene rates production of reactive oxygen species (ros) due to the null or low consumption of atp and nadph in the calvin cycle due to the absence of the electron acceptor nadp+, causing the degradation of the chloroplast pigments (cavalcante et al., 2015). additionally, the reduction in the chlorophyll contents was, possibly, due to the fact that the k deficiency is associated with the nitrogen deficiencies, which is key element in the synthesis of proteins destined for growth and development. potassium is involved in the protein synthesis in roots as well as in the root uptake and assimilation of nitrogen (coskun et al., 2017). in particular, k participates in the regulation of nrt2 nitrate transporters in roots and the activity of nitrate reductase (coskun et al., 2017; hu et al., 2016; armengaud et al., 2009). tab. 3 shows the increase in the mda content in leaves with the k deficiency. these results agree with that reported by hu et al. (2016) in cotton and du et al. (2019) in corn, which, under the k deficiency, presented the high mda contents and were associated with high h2o2 production (a variable not measured in the present study). similarly, hernandez et al. (2012) found significant differences in tomato with low k deficiency, presenting high accumulation of ros, simultaneously increasing the amount of mda, which suggests that k deficiency caused the oxidative damage to lipids and, finally, caused a chlorosis in the plants. our results show that the k deficiency decreased the leaf thickness including the thickness of the spongy and palisade parenchyma (tab. 4) resulting in a decrease in amax (tab. 2), which shows a close relationship between the leaf anatomy and the photosynthetic process. the diffusion of co2 during photosynthesis can be regulated by carbon allocations, which is determined by the leaf thickness and the size of the mesophyll cells (hu et al., 2020). this is in accordance with data reported in corn by du et al. (2019) who observed that the k deficiency negatively affected the anatomical structure of leaves, significantly reducing the size of the cells. in the same way, lin and yeh (2008) indicated that the thickness and size of leaf cells are reduced in plants because the water storage tissues in cells decreases along with the increment of k deficiency. the damage to the leaf anatomy, apparently, influences the photosynthetic process, the transport of nutrients and water (mattiello et al., 2015). faced with the stress due to the k deficiency, the leaves tend to be smaller, and the growth and cell expansion decrease (armengaud et al., 2009; elise et al., 2020). it has been reported that the mesophyll cells reduce the rate of cell division due to the k deficiency (toshio et al., 2009; hu et al., 2020) and k regulates cell expansion and proliferation of the mesophyll, which affects the leaf area (battie-laclau et al., 2014; lu et al., 2020). for this reason, the less thickness was observed in the spongy and palisade parenchyma of the tomato tree plants grown without k (tab. 4). in general, it was observed that the k deficiency negatively affected the physiology, biochemical components, and anatomical parameters in the tree tomato plants. fig. 4: cross-sections of leaves in the tree tomato plants at 135 dat. control plants (without deficiencies) (a, b) and plants grown without k (k deficiency) (c, d). midrib (a, c), same magnification, bar = 100 μm. leaf blade (b and d), same magnification, bar = 50 μm. p: parenchyma; v: vascular bundles; co: colenquima; midrib: rib; pp: palisade parenchyma; sp: spongy parenchyma, ue: upper epidermis, le: lower epidermis, t: trichomes, and cu: cuticle. 381 fig. 4: cross-sections of leaves in the tree tomato plants at 135 dat. control plants (without 382 deficiencies) (a, b) and plants grown without k (k deficiency) (c, d). midrib (a, c), same 383 magnification, bar = 100 𝜇𝜇m. leaf blade (b and d), same magnification, bar = 50 𝜇𝜇m. p: 384 parenchyma; v: vascular bundles; co: colenquima; midrib: rib; pp: palisade parenchyma; 385 sp: spongy parenchyma, ue: upper epidermis, le: lower epidermis, t: trichomes, and cu: 386 cuticle. 387 388 389 390 391 392 393 80 c.h. ramírez-soler, s. magnitskiy, s. esperanza melo martínez, f. álvarez-flórez, l.m. melgarejo conclusions the tree tomato plants grown with the k deficiency reduced the physiological parameters amax, ic, e, fv/fm, content of chlorophylls a, b and total. on the other hand, these plants increased the sr, and the mda contents in leaves. compared with the control plants, the deficiency of k altered the leaf anatomy, reducing the thickness of the leaf along with the thickness of spongy and palisade parenchyma. in addition, an increase in the thickness of the adaxial and abaxial epidermis was evidenced. this research presents the physiological, biochemical and anatomical indicators of the tree tomato plants subjected to potassium deficiency during vegetative growth, 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facultad de ciencias, departamento de biología, laboratorio de fisiología y bioquímica vegetal. universidad nacional de colombia, sede bogotá. ciudad universitaria, carrera 30 #45-03, bogotá, colombia e-mail: chramirezs@unal.edu.co luz marina melgarejo, facultad de ciencias, departamento de biología, laboratorio de fisiología y bioquímica vegetal. universidad nacional de colombia, sede bogotá. ciudad universitaria, carrera 30 #45-03, bogotá, colombia e-mail: lmmelgarejom@unal.edu.co © the author(s) 2021. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1016/0005-2728(75)90209-1 http://dx.doi.org/10.3109/10520299809141117 http://dx.doi.org/10.1042/bst0110591 http://dx.doi.org/10.21273/hortsci.43.1.146 http://dx.doi.org/10.1093/jxb/eraa356 http://dx.doi.org/10.1038/srep21725 http://dx.doi.org/10.1016/j.jplph.2015.05.014 http://dx.doi.org/10.1093/jexbot/51.345.659 http://dx.doi.org/10.30880/jst.2018.10.03.005 http://dx.doi.org/10.15446/abc.v20n1.42196 http://dx.doi.org/10.1007/s11104-010-0520-1 http://dx.doi.org/10.1016/j.jphotobiol.2016.03.052 http://dx.doi.org/10.1007/s11738-015-1901-0 http://dx.doi.org/10.1111/j.1365-313x.2008.03672.x http://dx.doi.org/10.1016/j.jphotobiol.2012.02.002 http://dx.doi.org/10.1016/s2095-3119(14)60848-0 http://dx.doi.org/10.1093/mp/ssq006 http://dx.doi.org/10.1016/j.jplph.2013.08.008 journal of applied botany and food quality 92, 274 280 (2019), doi:10.5073/jabfq.2019.092.038 institute of plant nutrition and soil science, kiel university, kiel, germany foliar magnesium supply increases the abundance of rubisco of mg-deficient maize plants julian wolf, stefanie thor straten, britta pitann, karl-hermann mühling* (submitted: march 4, 2019; accepted: september 5, 2019) * corresponding author summary magnesium is a vital macronutrient for plants and is involved in a series of essential physiological processes, e.g., carbohydrate partitioning and photosynthesis. the latter is strictly mg-dependent, as mg is the central atom of chlorophyll, and is also required for the de novo synthesis of sugars; this pathway revolves around the activity of the enzyme rubisco. when plants are subject to mglimiting conditions, development as well as yield is reduced. the foliar resupply of mg, contrary to traditional resupply to the roots, has the advantage of delivering the element directly to the site of highest physiological demand. thus, the aim of this research is to compare the effects of both resupply methods on the physiology and nutritional status of mg-deficient plants to see whether foliar application compared to root fertilization can properly improve plant growth after mg deficiency conditions. maize plants were cultivated in hydroponics in order to set up a mgdeficient root environment. mgso4 was then resupplied alternatively to the leaves or to the root. under mg-limiting conditions, rubisco abundance, as well as the total content of mn, zn, fe, and cu were severely reduced. this state was significantly ameliorated by the foliar resupply of mgso4, although the highest increase in biomass production was observed in response to root resupply. the foliar resupply of mgso4 upregulated the process of photosynthesis in mg-deprived plants. in this context, the foliar mgso4 application was able to return rubisco abundance to control levels in mg-deficient plants. key words: magnesium deficiency, maize, foliar application, photosynthesis, rubisco, micronutrients introduction magnesium (mg) is an essential macronutrient for the optimal growth and development of plants. although mg is mostly known for its role as the central atom of chlorophyll, this element is utilized for numerous other physiological and biochemical processes. the loading of photosynthates into the phloem and their redistribution throughout the plant, for example, are mg-dependent processes (cakmak and kirby, 2008; cakmak and yazici, 2010). the driving force of these mechanisms is the activity of the plasma membrane h+-atpases, which require mg-atp as a substrate (hanstein et al., 2011). moreover, mg is known to be involved in the activity of over 300 enzymes, including photosynthetic enzymes, such as ribulose-1,5-biphosphate carboxylase/oxygenase (rubisco) and phosphoenolpyruvate carboxylase (pepc) (chen et al., 2018). mg is crucial for the process of protein synthesis, as it is required for the association of ribosomal subunits and the maintenance of their activity (chen et al., 2018). due to the multiple roles of mg in the plant system, mg deficiency (mgd) is detrimental for crop growth and yield (farhat et al., 2016). this nutritional stress is very common in acidic soils that have a high content of aluminum (al) and manganese (mn), and in saline soils with a high content of sodium (na). these elements are present in the soil as cations and can replace and cause mg to be leached, since mg is only weakly bound to negatively charged soil particles. sandy soils are also particularly prone to leaching as mg adheres mainly to clay minerals. moreover, since mg reaches the root surface through diffusion and mass flow, mg nutrition is additionally hampered if the soil’s water content is low (jezek et al., 2015a). a way to overcome these issues is by delivering mg directly to the site of highest nutritional demand through foliar application. the efficacy of this resupply method has been previously studied in faba bean and soybean (vrataric et al., 2006; neuhaus et al., 2013, 2014). here it was shown that a greater share of mg is taken up by the plant following foliar application when compared to root application (neuhaus et al., 2013, 2014; jezek et al., 2015a, b). this is due to the fact, that mg can enter the plant more directly via the stomata and cuticula, while application to the soil causes a greater portion of the fertilizer to not reach the root due to secondary soil interactions and leaching (chen et al., 2018). moreover, soil fertilization is best performed either directly at the moment of sowing or at the seedling stage, as the field is more easily accessible (kannan, 2010). delivering the fertilizer to the plant any later via soil application during the growing season can be more labor intensive and cause mechanical damage to the shoot. conversely, mg can be delivered to the leaves of more numerous plants at once via spraying the plants or by taking advantage of the water irrigation sprinklers. the shoot is where the main processes that affect biomass production take place, namely the light-dependent and -independent reactions of photosynthesis. both pathways rely on mg and take place in the chloroplast, where most of the plant’s mg is found (15-35%) (wanli et al., 2016). the former converts the sun’s radiation energy into plant-available chemical energy, namely adenosine triphosphate (atp). chlorophyll plays a crucial role as it captures photons and channels their energy along the electron transport chain (etc). the relationship between mg-availability and chlorophyll content is substantial, as multiple authors observed a decrease in the content of the pigment under mgd (cakmak and yazici, 2004; mengutay et al., 2013; broadley et al., 2012). this is most likely caused by the inhibition de novo synthesis of chlorophyll, as well as the de gradation of existing chlorophyll in response to mgd (chen et al., 2018). through the latter, in particular, the plant gains access to the organically-complexed mg pool, which can then be reallocated from mature tissues to the young shoot (senbayram et al., 2015; wanli et al., 2016). the light-independent reactions of photosynthesis revolve around the fixation of carbon dioxide (co2), using the newly-generated atp. the first step of this pathway is catalyzed by the enzyme rubisco. this complex globular protein has both a carboxylase activity, which is prevalent under physiological con ditions and accepts co2 as a substrate, as well as an oxygenase activity, requiring molecular oxygen (o2). a prerequisite to enable the carboxylation reaction to occur is that a mg atom anneals to the active site of the enzyme; only then can a co2 molecule bind to the enzyme. moreover, mg is required also indirectly, during the day, to foliar magnesium supply increases the abundance of rubisco of mg-deficient maize plants 275 balance the charge difference across the thylakoid membrane and ensure the stroma reaches the correct ph for optimal catalytic activity (spreitzer and salvucci, 2002). however, little is known regarding the response of rubisco to mgd. a study by yuguan et al. (2009a) observed how mgd in spinach caused the expression level of the genes coding for rubisco’s big and small subunit to decrease, as well as an overall reduction of its catalytic activity. these results are in accordance with the decreased net co2 assimilation rate observed by jezek et al. (2015a) in response to mgd in maize. this study was performed using maize, as it is one of the most extensively cultivated cereal crops in the world (pechanova et al., 2013). the grain of this plant, as well as the whole portion growing above-ground, are used as silage material. in addition to this, its high starch content, on average 72%, justify its use as raw material for the production of biofuel and biogas (ksiezak et al., 2008; ksiezak et al., 2012; ranum et al., 2014). the aim of this research was firstly to verify that severe mgd had a negative effect on the photosynthetic machinery, specifically on rubisco abundance. secondly, in this research the effects of foliar and root resupply of mg on the physiology and nutritional status of mg-deficient plants were investigated. additionally, the question should be answered whether the foliar resupply of magnesium sulphate (mgso4) is comparably or even better suited than the re supply to the root to amend the detrimental effects of mgd. material and methods experimental setup maize (zea mays l.) plants from the cultivar susann (nordsaat saatzucht, langstein, germany) were cultivated hydroponically in a greenhouse from november to december 2017. plants were grown under an artificial light system (lamps: professional lighting, sonk 400, philips deutschland gmbh, hamburg, germany; bulbs: philips son-t agro 400 watt, philips deutschland gmbh; light intensity: 350 μmol m-2 s-1) with a 16/8 h day/ night regime, at 20-25 °c and 65% relative humidity. seeds were soaked in an aerated 2 mm caso4 solution overnight and germinated between two sheets of filter paper moistened with 2 mm caso4 solution. six-day-old seedlings were transferred to 10 l containers (four plants per container) containing 25% strength nutrient solution (ns); this day is hereafter considered as day 0. within the next 3 d, the ns was increased step-wise up to 100%. the full-strength ns was exchanged weekly and had the following composition: 1.3 mm ca(no3)2, 0.7 mm nh4no3, 2.0 mm cacl2, 1.0 mm k2so4, 0.2 mm kh2po4, 200 μm fe-edta, 5 μm h3bo3, 2 μm mnso4, 0.5 μm znso4, 0.3 μm cuso4, and 0.01 μm (nh4)2mo7o24. plants were divided into four discrete experimental groups with four independent biological replicates per treatment [“positive control” (contr+), “negative control” (contr–), “root application” (root) and “foliar application” (leaf)]. containers were set up in a completely randomized design. contr+ plants were given sufficient mg, namely the full ns with the addition of 0.5 mm mgso4, throughout the experiment. the other three variants were supplied with 0.02 mm mgso4 for the first week, in order to ensure proper seedling development, and only 0.01 mm mgso4 from thereafter. this concentration was chosen in order to induce clear mgd symptoms. two weeks after the transfer to 10-l containers (day 14), the first mgd symptoms became clearly visible on the leaves of plants growth in mg-deficient ns, e.g., intervenial chlorosis. the same day, and until the end of the experiment, the mg concentration of root was raised to the level of contr+. concurrently and over the course of two weeks, leaf was sprayed with a 200 mm mgso4 solution, splitted into four single applications; each one of these had a total volume of 80 ml. by this, the total mg amount applied to the leaves was stepwise increased to avoid salt effects (e.g., burning) possibly caused by a single higher dose. silwet (spiess-urania chemicals gmbh) in the dose of 0.1% was added as a wetting agent to the foliar application solution. plants were harvested on day 29 and shoots and roots were sampled separately. moreover, the shoots were additionally subdivided into two discrete samples: the two youngest leaves (without the stalk) were sampled as “young”; the following three leaves were sampled as “old”. in order to calculate the total shoot fresh weight of one plant, the weight of the discrete shoot fractions (“young” and “old”) were summed up. thus, for each treatment, 12 samples were taken (4 “old”, 4 “young”, and 4 “root”), for a total of 48 samples. samples were homogenized with a mortar and pestle in the presence of liquid n2 before being stored at -80 °c for further analysis. determination of growth parameters during harvest, the average height and the weight of both shoots and roots were measured for each container. the height was defined as the distance from the top of the hydroponic culture container to the tip of the highest leaf. the same portion was subsequently weighted to obtain the weight of the shoot. the leaves of the leaf variant were thoroughly rinsed with deionised water before storage to remove adhering spraying solution. protein analysis total protein extraction total protein was extracted using the tca/acetone protocol adapted from zörb et al. (2004). for this, aliquots of 300 mg ground material were extracted with 1.6 ml of ice-cold 10% (w/v) trichloroacetic acid in acetone with the addition of 50 mm dithiothreitol (dtt). after incubating at -20 °c overnight, the protein fraction was separated from the liquid phase by centrifugation (19,125 g, 4 °c, 15 min). pellets were suspended in 1 ml of ice-cold acetone plus 50 mm dtt and incubated again overnight at -20 °c. vacuum-dried protein pellets were suspended in 1 ml of lysis buffer (urea 8 m, thiourea 2 m, dtt 30 mm, tris base 20 mm, chaps 4%) to which 0.625 μl of protease inhibitor (sigma) was added. after incubating for 2 h at 3 °c, the protein fraction was separated by centrifugation (19,125 g, 4 °c, 30 min) and stored at -80 °c. the protein concentration was determined using 2d quant kit according to the manufacturer’s manual (ge healthcare life sci ences). the calibration curve was created using bovine serum albumin (bsa) as a standard. western blot a western blot (wb) analysis was performed on the samples associated with old and young leaves; for this, the method suggested by zörb et al. (2005) was used. briefly, the rubisco large subunit (55 kda) was employed as the primary antibody (rubisco, anti bodies-online.com). the four polyacrylamide gels used for this ana lysis were a combination of a 6.3% stacking gel and a 15% separation gel. the equivalent of 8 μg of protein was loaded into each well. page ruler pre-stained marker (thermo fisher scientific) was used as a protein ladder. the gels were run at 20 ma per gel in an electrophoresis chamber. once the gels had been run (20 ma per gel for 1 h), they were incubated in towbin buffer (for 1 l: 3.03 g tris base, 14.4 g glycine and 100 ml of methanol, ph 8.3) for 15 min. simultaneously, four gel-sized pieces of polyvinylidene difluoride (pvdf) membrane were soaked for 3 s in methanol and subsequently incubated in towbin buffer for 10 min. the “blot-sandwich” was run at 110 ma for 1 h using the hoefer semiphor chamber (pharmacia biotech). after the transfer, the pvdf membranes were incubated in a 5% bsa/tbs solution (bovine serum albumin, merck; for 1 l: 1.21 g tris-hcl 276 j. wolf, s. thor straten, b., k.-h. mühling and 8.77 g nacl) for 2 h. after 3 washing steps with tbs-t (tbs and 0.1% polysorbate 20 (tween 20, sigma)), the solution containing the antibody for rubisco, diluted 1:5.000 with pbs (for 1 l: 8 g nacl, 0.2 g kcl, 0.76 g na2hpo4, 0.24 g kh2po4, ph 7.4.), was added to the pvdf membranes. the membranes were incubated 1 h at rt and at 4 °c overnight. the following morning, the membranes were washed twice for 10 min with tbs-t and once for 10 min with a 1.5% milk powder in tbs-t solution. next, the membranes were incubated for 2 h in a solution of the second antibody (anti-rabbit igg, alkaline phosphatase conjugate, sigma), diluted 1:30.000 in pbs. to remove the second antibody, the membranes were washed for 10 min a total of 5 times, 4 of which with tbs-t and one with tbs. after the last washing step, the membranes were incubated for 5 min in the ap-buffer (for 1 l: 12.11 g tris base, 5.84 g nacl and 1.02 g mgcl2, ph, 9.5). finally, the ap-buffer was removed and the membranes were covered with the developing solution (10 ml ap-buffer, 32 μl bcip solution (100 mg bcip in 1.9 ml h2o), 66 μl nbt solution (100 mg of nbt in 1.9 ml 70% dmf/h2o solution)). membranes were washed multiple times with purified h2o and in cubated overnight in the dark. the developed pvdf membranes were scanned with an epson perfection v700 photo scanner and the silverfast epson se program. subsequently, the pictures were analyzed using the gelanalyzer software (lazer software, version 2010a). determination of nutritional status between 280 and 330 mg of frozen sample were dissolved in 10 ml of 69% hno3 solution. digestion of the sample occurred using a microwave (cem, one touch technology, mars 6) with the following regime: 2 min at 100 °c, 1 min at 120 °c, 20 min at 180 °c, and allowed to cool for 20 min. the digested samples were diluted with purified water to a total volume of 50 ml. the mg, ca, and k contents of each sample were measured using the atomic absorption spectrometer (aas, thermo electron corporation, s series). wavelength was set to 285.5, 422.9, and 766.5 nm, respectively. mn, zn, cu, fe and mo content, on the other hand, was measured using inductively coupled plasma-mass spectrometry (icp-ms, agilent technologies 7700 series, böblingen, germany). two different analytical methods were used to measure the macro and micro-nutrient contents of the samples because of the different abundance at which these elements are present in plants; micronutrients make up only 0.02% of the total dry weight and are, thus, 1-4 orders of magnitude less abundant than macronutrients (sabeeha, 2010). accordingly, the aas and icp-ms were chosen to account for these differences while preserving the necessary precision of the measurements. statistical treatment significant differences (p-value ≤ 0.05) between means were identified through a one-way analysis of variance (anova) using spss (version 17.0). post-hoc analysis for multiple comparisons (tukey’s range test) was additionally carried out to discriminate among the significant differences of the variant’s means. shown values are given as means of at least three replicates with standard deviations (sd). results effect of mgd and resupply on overall plant growth plant height was significantly reduced after prolonged exposure to mg-limiting conditions; on average, maize plants grew up to 75.5 cm which accounted for 63.3% of contr+ plants (fig. 1a). of the two resupply variants, only the root variant did not differ significantly from the positive control; the plants that were treated with foliar mgso4 grew less than the root variant and were statistically indistinguishable from the negative control. similar results were observed for the shoot’s fresh weight (fw) (fig. 1b): mgd led to a drastic reduction in plant’s fw, namely 90.1% of the positive control. the root and leaf variant, on the other hand, reached only 45% and 29%, respectively, of the fw of contr+. with this, the resupply variants exhibited an intermediate value between the two controls. in the end, only root was significantly different from the controls. finally, when measuring the root biomass formation, both resupply variants showed a higher mass than the negative control, although not statistically different (fig. 1b). fig. 1: heights and weights (fws) of shoot and root. (a) height (cm) of shoots and (b) fws of shoot and root at harvest (35 days after germination). contr+, 0.5 mm mgso4 in ns; contr–, 0.01 mm mgso4 in ns; root, 0.01 mm mgso4 in ns for 2 weeks and 0.5 mm mgso4 in ns for 2 weeks; leaf, 0.01 mm mgso4 in ns and foliar application of 200 mm mgso4 over 2 weeks; mean (n = 4 biological replicates) ± sd; small letters indicate significant difference between treatments (anova, with tukey adjustment, p ≤ 0.05). effect of mgd on macroand micronutrient composition the concentrations of mg, ca, and k were measured for aliquots of each sample. in order to eliminate dilution effects, the total content of each element was displayed. positive control plants were supplied with 0.5 mm of mgso4 throughout the experiment and always displayed the highest mg contents. these values were the highest in the old leaves with 67.8 mg plant-1, and decreased in the roots (40.0 mg plant-1) and young leaves (11.1 mg plant-1) (fig. 2a). conversely, the growth of maize plants deficient in mg severely limited total mg content across all organs. in the old leaves, root was the only variant to show a significantly increased mg content (13.1 mg plant-1 against 1.7 mg plant-1 of the negative control); in contrast, in the young leaves it was the leaf variant, with more than double the content of the negative control, which diverged most significantly from contr–. the fluctuation of the total k content: mgd led to a very pronounced decrease in total nutrient accumulation (10.8%, 4.4% and 5.4% of the contr+, respectively), while the resupply variants did not differ significantly from the negative control (fig. 2b). the only exception was the total content of k accumulated in the old leaves after resupply of mgso4 to the ns; this value was significantly higher than the negative control, yet lower than the positive control. the ca content in the old leaves, as well as in the young leaves and the roots, did not exhibit any particular differences (fig. 2c). overall, the positive control represented the highest value (176.9, 159.1 and 140.7 mg plant-1, respectively), while the contents of all other variants foliar magnesium supply increases the abundance of rubisco of mg-deficient maize plants 277 were drastically lower (on average, 5%, 2%! and 3% of the contr+, respectively) and did not differ significantly from each other. the total amount of mn accumulated in the plants showed significant differences only in the old leaves: here, the leaf variant yielded the highest mn content (1.49 mg plant-1), yet no significant difference to the positive control could be found (1.08 mg plant-1) (fig. 3a). additionally, the value of the root variant (0.46 mg plant-1 ) was increased compared to the negative control (0.13 mg plant-1) but not enough so to be considered different. the total content of zn was measured as well (fig. 3b). the old leaves were the only organ to show a particular response, specifically in the leaf variant: the zn content was markedly increased (2.26 mg plant-1) compared to the positive control (1.41 mg plant-1). moreover, the root variant also accumulated a significantly higher amount of zn compared to the negative control (0.74 and 0.15 mg plant-1, respectively), yet less than the positive control. the pattern of fe accumulation differs among the three organs (fig. 3c). in the old leaves, the value of leaf (2.35 mg plant-1) was increased to the point of showing no significant difference to the positive control (3.2 mg plant-1). also in the young leaves, the leaf variant showed an increased value (0.69 mg plant-1), not nearly as large as the positive control (1.77 mg plant-1), yet sufficiently high to be considered different from both controls. lastly, in the radical apparatus, the root variant (1.56 mg plant-1) reached the iron content of the positive control (1.7 mg plant-1). no statistical difference could be found between these two measurements. the cu content of the old leaves is the highest in the leaf variant (1.02 mg plant-1 against 0.23 mg plant-1 of contr+) (fig. 3d); the other variants do not differ significantly from one another. in the young leaves, the highest cu content is one of the positive controls (0.58 mg plant-1). in this organ, the leaf variant represents an intermediate (0.34 mg plant-1) between the positive control and the negative control (0.001 mg plant-1) and root (0.04 mg plant-1). finally, the roots exhibit a similar patter as the old leaves with the leaf variant showing the highest cu content (0.48 mg plant-1). the positive control (0.25 mg plant-1) is statistically independent from the fig. 3: micronutrient content in various leaves and roots. (a) total content of mn, (b) zn, (c) fe, (d) cu and (e) mo per plant at harvest (35 days after germination). contr +, 0.5 mm mgso4 in ns; contr –, 0.01 mm mgso4 in ns; root, 0.01 mm mgso4 in ns for 2 weeks and 0.5 mm mgso4 in ns for 2 weeks; leaf, 0.01 mm mgso4 in ns and foliar application of 200 mm mgso4 over 2 weeks; mean (n = 4 biological replicates) ± sd; small letters indicate significant difference between treatments (anova, with tukey adjustment, p ≤ 0.05). fig. 2: macronutrient content in various leaves and roots. (a) total content of mg, (b) k and (c) ca per plant at harvest (35 days after germination). contr+, 0.5 mm mgso4 in ns; contr–, 0.01 mm mgso4 in ns; root, 0.01 mm mgso4 in ns for 2 weeks and 0.5 mm mgso4 in ns for 2 weeks; leaf, 0.01 mm mgso4 in ns and foliar application of 200 mm mgso4 over 2 weeks; mean (n = 4 biological replicates) ± sd; small letters indicate significant difference between treatments (anova, with tukey adjustment, p ≤ 0.05). 278 j. wolf, s. thor straten, b., k.-h. mühling leaf, as well as from the contr– (0.01 mg plant-1) and root (0.09 mg plant-1). finally, mo content showed a similar trend across all organs; the amount stored by the leaf variant was always higher than the negative control but lower than the positive control (fig. 3e). addi tionally, in the roots, also the root variant showed an increased value compared to the negative control. rubisco abundance as influenced by mg deficiency and resupply due to the tight link between mg nutrition and the process of photosynthesis, the abundance of rubisco was measured through a wb analysis. since no rubisco is present in the roots, the analysis was performed only on old and young leaves. in the old leaves, mgd caused rubisco abundance to decrease by a 0.7 fold change. the resupply variants displayed discrete results: the foliar mgso4 application was able to return rubisco abundance to control levels; this was not the case for the root variant, which scored an intermediate value between the two controls (fig. 4). a slightly different situation was visible in the young leaves. here the two control variants were not significantly different from each other. the behavior of the resupply variants was similar to the old leaves: the root application did not manage to return the enzyme’s abundance to control levels, while the leaf application did much better, scoring the best overall. ing conditions; in multiple instances, mgd was associated with a decline of the chlorophyll content (yuguan et al., 2009a; jezek et al., 2015a; wanli et al., 2015). the effect of the absence and of the resupply of mg on the rubisco abundance was studied because this enzyme plays a pivotal role in the process of photosynthesis and was previously shown to be downregulated under mgd on a transcriptional level (yuguan et al., 2009a). effect of mgd and mg resupply on plant growth the reduction in plant growth is a common consequence of a mgd and has been studied in a wide variety of plants, like arabidopsis thaliana, vicia faba, beta vulgaris, spinacia oleracea, brassica napus and zea mays (hermans et al., 2010; neuhaus et al., 2014; hermans et al., 2004; yuguan et al., 2009b; billard et al., 2016; jezek et al., 2015a, b). in accordance to other studies, this research found additional evidence for the deleterious effects of mgd on maize growth, speci fically on plant height, as well as on shoot and root weight (fig. 1). the reduced height and weight of mg-deficient plants is an indicator of the inability of the plants to redistribute sugars from source organs to the sites of active tissue development, such as young leaves and roots. in this context, it is concluded that the inhibition of the plasma membrane h+-atpase primarily limited maize growth under mg deficiency (jung et al., 2017) due to the lack of the enzyme’s co-substrate mg. afterwards, the reduced apoplastic acidification caused a reduced cell-extension growth. literature is scarce regarding the effects of resupplying mg after prolonged mgd. in this experiment, the reapplication of mg to the root had an overall better effect on plant growth than the foliar application: it returned plant height to the levels of the control and increased shoot and root fresh weight more than the leaf application. this was surprising since the latter delivered mg straight to the site of the most severe mgd damage, the shoot; the former, on the other hand, was presumed to be less efficient due to the additional time required for nutrient uptake from the ns and subsequent transport of mg to the shoot through the xylem. the observed results point towards the ability of root resupply fertilizers to remedy the detrimental effects of mgd more promptly than the foliar resupply. future research might focus on studying the long-term effect of the two discrete resupply methods by extending the plant’s growth period. these results show that the resupply of mg after a period of deficiency has a beneficial effect on overall plant development. additionally, the data indicate that the mg applied through the leaves was successfully taken up by the leaves and translocated to the site of meristematic tissue growth. effect of mgd and mg resupply on maize plant’s total mg content in vivo, newly taken-up mg ions are translocated through the xylem to the shoot (kobayashi and tanoi, 2015). this process is driven by the transpiration of water through the leaves and increases in strength relative to the leaf area through which water transpires and the metabolic rate of the tissues (kramer and boyer, 1995). with this in mind, it was assumed that newly taken up mg would reach both the old and young leaves, with a slight preference for the latter due to their greater metabolic activity. the resupply of mg to the roots did significantly raise the mg content in the old leaves (fig. 2a). the response to root resupply was visible also in the young leaves; here, the mg content was no significantly different from the negative control due to the inherently higher mg content in young organs in response to mgd. this physiological fig. 4: rubisco abundance in old and young leaves. (a) rubisco abundance, measured in raw pixel volume, in the old and (b) young leaves at harvest (35 days after germination). contr+, 0.5 mm mgso4 in ns; contr–, 0.01 mm mgso4 in ns; root, 0.01 mm mgso4 in ns for 2 weeks and 0.5 mm mgso4 in ns for 2 weeks; leaf, 0.01 mm mgso4 in ns and foliar application of 200 mm mgso4 over 2 weeks; mean (n = 4 biological replicates) ± sd; small letters indicate significant difference between treatments (anova, with tukey adjustment, p ≤ 0.05). discussion magnesium is involved in a plethora of physiological processes due to its divalent positive charge and ionic radius. not only does this element serve as a cofactor for over 300 enzymes, it also stabilizes the conformational structure of many different kinds of macromole cules, like proteins, ribosomes, nucleic acids, and the cell membrane and wall (wanli et al., 2015). due to its widespread biological function, mgd negatively affects overall plant growth as well as crop yield (senbayram et al., 2015). the aim of this research was twofold: on the one hand, it aimed at elucidating how the absence and the resupply of mg affect the micronutrient content of maize plants. these were expected to fluc tuate because of their role as cofactors and as structural elements in the process of photosynthesis. the process of chlorophyll synthesis, in particular, was anticipated to be severely impaired under mg-limit foliar magnesium supply increases the abundance of rubisco of mg-deficient maize plants 279 response to mgd is fairly well documented (senbayram et al., 2015; wanli et al., 2016); this mechanism implies the reallocation of mg resources from old tissues, where mg is being released as a result of catabolic degradation, and their reallocation through the phloem to young developing organs. keeping this in mind, the fact that the mg accumulated in the young leaves, as a consequence of root resupply, was not significantly different from the negative control, points to the conclusion that mg reallocation from the old to the young leaves was not induced. if this process was not initiated, it means that the shoot did not perceive the decrease in mg content since the xylem transport from the root delivered mg at a sufficiently high rate. another aspect that emerges from the results is that the foliar resupply of mg leads to an accumulation of mg solely in the young tissues (fig. 2a). previous studies by jezek et al. (2015a, b) already demonstrated the beneficial effect of reapplying mg through the leaf on the plant’s total mg content. contrary to this research, they found that the foliar resupply of mg enhanced the mg content only of the leaves that were directly treated with mgso4 solution. no translocation to the young leaves appeared to occur. in the present study, mg was applied solely to the old leaves, which were the third, fourth and fifth leaf from the top of the plant; based on this and the available literature, it was expected for the old leaves to show a significant increase in mg content. however, it was the young leaves that exhibited the highest increase of two and half fold. the measured doubling in mg content might be seen as proof that the reallocation of mg to the young leaves is taking place. moreover, even if the increase in mg content was not enough to be considered statistically significant, it was sufficient to markedly alter both micronutrient content as well as rubisco abundance (fig. 3 and 4). mgd and mg resupply alter the micronutrient content of maize plants micronutrients play an important role in the process of photosynthesis. they aid enzymatic activity as prosthetic groups of metalloproteins (römheld and marschner, 1991). during this study, the change in leaf content of mn, zn, cu, and fe in response to varying mg conditions were investigated. mgd caused a marked decrease in micronutrient content across all organs; conversely, the foliar resupply of mgso4 caused these contents to significantly increase (fig. 3). manganese, together with fe, cu, and zn, is involved in the biosynthetic pathway that leads to the formation of chlorophyll (shenker et al., 2004; marsch et al., 1963; maksymiec, 1997; hu and sparks, 1991). this process is also heavily influenced by the availability of mg, as multiple authors have linked mgd to a decrease in chlorophyll content (cakmak and yazici, 2010; mengutay et al., 2013; broadley et al., 2012). cu and fe additionally play an important role in ensuring a continuous flow of electrons along the electron transport chain (etc), the former as part of plastocyanin, the latter as a constituent of cytochromes (raven et al., 1999; broadley et al., 2012). finally, mn is also involved in the process of oxygen evolution, as part of the oxygen evolving complex (oec) of the photosystem ii (psii). fashui’s group published multiple articles (yuguan et al., 2009a; yin et al., 2009; zhao et al., 2012) in which they linked mgd to a decrease in the rate of oxygen evolution. the observed decrease in micronutrient content might therefore reflect the reduced requirement for cofactors of the synthesis reactions and the limited flow of electrons along the etc. in other words, it is suggested that mgd inhibits not only the biosynthetic pathway of chlorophyll but causes the whole process of photosynthesis to be down-regulated. the foliar resupply of mg, on the other hand, caused micronutrient content to increase (fig. 3). this effect was predominantly visible in the old leaves and, to a lesser extent, in the young ones. there is already proof for the positive effect of mg resupply on the plant’s chlorophyll content (jezek et al., 2015a; yuguan et al., 2009a). keeping this in mind, it is reasonable to assume that the observed increase in mn, cu, zn, and fe content in the old leaves in response to the foliar mg application might reflect an up-regulation of the de novo synthesis of chlorophyll, and possibly of the whole process of photosynthesis. the effect of mgd and mg resupply on rubisco abundance the enzyme rubisco is referred to as the most abundant globular protein in the world (ellis, 1979). the importance of this complex oligomer in plant physiology is due to its fundamental role in the process of co2 fixation from the atmosphere. mg2+ ions play both a direct and an indirect role in the activity of this enzyme: first of all, for catalytic activity to initiate, mg has to physically bind to the active site of a rubisco. secondly, mg is required during the day to balance the charge difference across the thylakoid membrane (spreitzer and salvucci, 2002). during this experiment, mgd led to a reduction of rubisco abundance in the old leaves (fig. 4). these results were partially expected since previous studies already showed the negative effects of mgd on rubisco’s expression levels and catalytic activity (yuguan et al., 2009a); the novelty of this study lies in proving that mgd affects rubisco also on a translational level. potentially promising results were obtained for the resupply variants. it was shown in this study for the first time how the resupply of mg through the root or leaf supply has very discrete effects on the rubisco abundance of mg-deficient plants (fig. 4). the former caused a slight response only in the old leaves, while the latter was notably able to return enzyme abundance to control levels. this effect was already evident in the old leaves but became even more pronounced in the young leaves. the reason for the different efficacy of the two resupply methods might be ascribed to the distance that mg had to travel before interacting with the chloroplasts. the foliar application delivered mg directly to the site where mgd had the most detrimental effect, which coincidentally is also the location of rubisco transcription and translation. conclusions mg deficiency leads to a large reduction in rubisco abundance, as well as in the total content of mn, zn, fe and cu. the foliar resupply of mgso4 was shown to cause a marked increase of these physiological traits, proving that mg application to the leaves up-regulates the process of photosynthesis. while the resupply of mg to the root was shown to have a better effect on the overall biomass formation of mg-deficient plants, compared to the foliar application, both methods appear to be suited as a rescue measure against the detrimental effects of mgd. references billard, v., maillard, a., coquet, l., jouenne, t., cruz, f., garciamina, j.m., yvin, j.c., ourry, a., etienne, p., 2016: mg deficiency affects leaf mg remobilization and the proteome in brassica napus. plant physiol. bioch. 107, 337-343. doi: 10.1016/j.plaphy.2016.06.025 broadley, m., brown, p., cakmak, i., rengel, z., zhao, f., 2012: function of nutrients: micronutrients. in: marschner, h. 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liu, c., qu, c., si, w., hong, f., 2012: magnesium deficiency results in damage of nitrogen and carbon cross-talk of maize and improvement by cerium addition. biol. trace elem. res. 148, 102-109. doi: 10.1007/s12011-012-9340-x zörb, c., stracke, b., tramnitz, b., denter, d., sümer, a., mühling, k.h., yan, f., schubert, s., 2005: does h+ pumping by plasmalemma atpase limit leaf growth of maize (zea mays) during the first phase of salt stress? j. plant nutr. soil sci. 168, 550-557. doi: 10.1002/jpln.200520503 orcid: britta pitann https://orcid.org/0000-0002-5476-3244 karl h. mühling https://orcid.org/0000-0002-9922-6581 address of the corresponding author: prof. dr. karl h. mühling, institute of plant nutrition and soil science, kiel university, hermann-rodewald-straße 2, 24118 kiel, germany e-mail: khmuehling@plantnutrition.uni-kiel.de © the author(s) 2019. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1071/cpv66n12_fo http://dx.doi.org/10.1016/j.semcdb.2017.08.005 http://dx.doi.org/10.1016/0968-0004(79)90212-3 http://dx.doi.org/10.1007/s11738-016-2165-z http://dx.doi.org/10.1042/bj20101414 http://dx.doi.org/10.1007/s00425-004-1340-4 http://dx.doi.org/10.1111/j.1469-8137.2010.03257.x http://dx.doi.org/10.3389/fpls.2014.00781 http://dx.doi.org/10.1007/s00425-015-2371-8 http://dx.doi.org/10.1016/j.plaphy.2017.02.009 http://dx.doi.org/10.1007/978-90-481-8741-6 http://dx.doi.org/10.1016/b978-012425060-4/50003-6 http://dx.doi.org/10.3390/ijms160923076 http://dx.doi.org/10.1007/s11104-013-1761-6 http://dx.doi.org/10.1002/jpln.201300127 http://dx.doi.org/10.1007/s11104-013-1711-3 http://dx.doi.org/10.1002/pmic.201200275 http://dx.doi.org/10.1111/nyas.12396 http://dx.doi.org/10.2136/sssabookser4.2ed.c9 http://dx.doi.org/10.1104/pp.110.161810 http://dx.doi.org/10.1071/cp15104 http://dx.doi.org/10.1078/0176-1617-00931 http://dx.doi.org/10.1146/annurev.arplant.53.100301.135233 http://dx.doi.org/10.1556/crc.34.2006.1.177 http://dx.doi.org/10.1016/j.cj.2015.11.003 http://dx.doi.org/10.1007/s12011-009-8392-z http://dx.doi.org/10.1007/s12011-009-8354-5 http://dx.doi.org/10.1007/s10534-009-9246-z http://dx.doi.org/10.1007/s12011-012-9340-x http://dx.doi.org/10.1002/jpln.200520503 https://orcid.org/0000-0002-5476-3244 https://orcid.org/0000-0002-9922-6581 journal of applied botany and food quality 91, 332 340 (2018), doi:10.5073/jabfq.2018.091.042 1plant nutrition department, national research centre, giza, egypt 2 vegetable research department, national research centre, giza, egypt 3 institute of plant sciences and resource conservation, division of horticultural sciences, university of bonn, bonn, germany tomato yield, physiological response, water and nitrogen use efficiency under deficit and partial root zone drying irrigation in an arid region m.a. badr1*, w.a. el-tohamy2, s.d. abou hussein2, n. gruda3 (submitted: july 17, 2018; accepted: october 19, 2018) * corresponding author summary water scarcity in arid regions is a serious problem, which calls for innovative irrigation water management. partial root zone drying (prd) technique can considerably reduce irrigation amount for crops. to investigate this further, tomato plants were imposed to either surface drip (sur) with full irrigation (fi) at 100% of evaporative demands and regulate deficit irrigation (rdi) at 50% water of fi or subsurface drip irrigation (sdi) with fixed prd at 75 (prd75) and 50% (prd50) of the fi. surface evaporation under sur with fi constitutes a large fraction of water losses from cropped fields while sdi with prd75 preserved more water for plant uptake. plants grown under water saving treatments showed lower stomatal conductance and transpiration rates compared to fi plants. tomato yield under sdi with prd75 was comparable to yield under sur with fi for both tested seasons along with 25% water saving and 30% increase in water use efficiency (wue). otherwise, prd50 reduced yield by 18-20%, but a substantial amount of irrigation water was saved along a 60 and 65% higher wue compared to fi treatment. fruit dry weight and harvest index (hi) were significantly higher with prd75 compared to the other treatments. seasonal n uptake and in turn n recovery was higher in prd75 than any other treatment associated with improving n use efficiency. keywords: deficit irrigation, prd irrigation, n uptake and recovery, soil moisture, wue, solanum lycopersicum. introduction limited resources of fresh water and severity of droughts in arid and semi-arid regions are continuously threats for food productions, so that it is difficult to grow more crops or even to meet full biological plant demands. moreover, due to climate change, the climatic water balance between precipitation and evapotranspiration is projected to become increasingly negative in the future, where water is required in large quantities (bisbis et al., 2018). therefore, more efficient use of water is the major target to cope with the growing water shortage. for the different ways of water application, drip irrigation is one of the most efficient methods of applying water and nutrients to the crops. this method of irrigation provides various unique agronomic, water and energy conservation benefits that address many of the challenges facing irrigated lands. consequently, the use of drip irrigation is rapidly increasing in arid and semi-arid regions with the aim to improve water use efficiency (wue) of plants. in the design of a drip irrigation system for improving water use and optimizing crop production, factors to be considered include plant spacing and plant canopy cover as well as soil texture and topography, potential evaporation, water quality (cetin and uygan, 2008). especially subsurface drip irrigation has the ability to minimizes soil evaporation compared to surface drip (hanson et al., 2006; badr et al., 2010). deficit irrigation (di) has been developed to meet the minimum crop water requirement without significant reduction in crop yield (davies et al., 2002). moreover, a novel deficit irrigation technique named partial root zone drying (prd) has been raised and attracted considerable interest (davies et al., 2000; kang and zhang, 2004). in prd one half of the root zone is irrigated while the other half is allowed to dry out. the treatment is then periodically reversed, allowing the previously watered side of the root system to dry out while irrigating the previously dry side (stoll et al., 2000; topcu et al., 2007). the wetting side of the plant has plentiful supply of water and therefore the plant never became stressed as deficit as that of conventional irrigation method. in most cases, prd has the potential to increase wue, decrease plant growth and maintain yield and quality when compared with classical irrigation methods (davies et al., 2000; tahi et al., 2007). fixed prd is one form of this irrigation technique where water is applied only from one side of the root system while the other side is exposed to continuous dry conditions. fixed prd was used as water saving irrigation technique compared to alternate prd and conventional irrigation. moreover, fixed prd can be employed in row crops as a cost effective and less energy consuming method since it requires lower irrigation equipment and does not involve complexities compared to typical prd (lekakis et al., 2011). the practical use of prd was developed based on the knowledge of physiological regulations of plants grown under dry soil conditions. however, stomatal conductance of leaves might decline with in creasing the measurable abscisic acid (aba) that flow from roots to leaves through the transpiration stream to reduce stomatal aperture and leaf growth. the increased concentration of aba in the xylem flow from roots to leaves triggers closure of stomata was proved in tomato (campos et al., 2009) and other crops (kang and zhang, 2004). as consequences of plant physiological response, the aperture of stomata can be regulated so that a partial closure of stomata at a certain level of soil water deficit may lead to limit transpiration rate and increase wue (liu et al., 2005a). prd causes spatially and temporally heterogeneous distribution of soil moisture and hereby causing uneven availability of nutrients in the soil and uneven absorptions by the roots in different root zones (hu et al., 2006; li et al., 2007). more recently, hu et al. (2009) used alternate and fixed prd technique to investigate the dynamic change of plant n absorption and accumulation from root zones. the authors reported increased root n absorption in the irrigated zone significantly when compared to that of conventional irrigation and the re-irrigated half resumed high n inflow rate, suggesting that alternate prd had compensatory effect on n uptake. otherwise, under fixed prd, the n accumulation in plant was mainly from the irrigated root zone and the recovery rate, loss percentage of n applied to the irrigated zone was higher, and the residual percentage of n in soil was lower if compared to those of the non-irrigated zone. physiological characterization of tomato under deficit and partial root zone drying irrigation in an arid region 333 both fixed and alternate prd increased n and water use efficiencies but only consumed about 70% of the irrigated water when compared to conventional irrigation. however, the extended wetting and drying processes under prd give local soil water content close to field capacity and thus may enhance the radial flow rate of nitrate to the root surfaces. tomato plants have the highest cultivated area of any vegetable crop in the world and can tolerate drought to some degree (hanson and may, 2004; abdelmageed and gruda, 2009). under arid and semi-arid conditions, adoption of irrigation management strategy that utilizes deficit irrigation may be a viable option to improve irrigation wue. therefore, greater emphasis is being placed on water management for dry conditions with the aim of crop yield maintenance, which is highly dependent on improving wue. the projected shift in precipitation pattern, as well as increasing weather variability and extreme weather events such as heat waves, or drought, make furthermore water management a key factor in combating the adverse impacts of climate change (bisbis et al., 2018). the present study was carried out to describe the soil moisture variations in the root zone from different drip line positions. furthermore, yield performance, physiological response, n uptake and recovery and wue were investigated under different drip irrigation treatments in an arid region where irrigation is the only way for crop production. materials and methods site and soil description field experiments were conducted in a vegetable farm located at serapium area, ismailia province east of nile delta, egypt during the late summer growing season (august-december) 2015 and 2016 using drip irrigation system. this area is a desert region included in the agricultural expansion program and recently became productive lands for many crops. the site is located in the arid climate at latitude 30°58 n and longitude 32°23 e with an elevation of 13 m above mean sea level. the area has hot and dry summer months with some ineffective rains in winter and usually bright, sunny days with mild and cold nights. the mean monthly evapotranspiration ranged from 6.8 to 2.5 mm in the respective cropping season. the climate parameters recorded during the growing season of tomato are summarized in (tab. 1). the soil of the experimental site was deep, well drained sandy profile which was classified as an entisoltypic torripsamments comprising of 84.2% sand, 11.5% silt, 4.3% clay and 0.46% organic matter in the topsoil (0-80 cm depth) with an alkaline ph 8.2, ec 0.78 ds m−1, caco3 1.4% and bulk density 1.46 g cm−3. the average soil water content at field capacity from surface soil layer down to 60 cm depth at 20 cm intervals was 0.21 (v/v) and the permanent wilting point for the corresponding depth was 0.11 (v/v), respectively. average available n, p and k from surface soil layer down to 60 cm depth at 20 cm intervals was 15, 7 and 78 mg kg−1 soil, respectively prior to experiment initiation. experimental design and treatments the experiment was laid in a complete randomized block design with three replications. four drip irrigation treatments were in vestigated: surface drip (sur) with full irrigation (fi) at 100% of et crop and regulate deficit irrigation (rdi) at 50% of fi where the water amount was applied uniformly to the entire plant root zone or subsurface drip (sdi) with two fixed partial root zone drying (prd75) and (prd50) at 75 and 50% of fi, respectively in which half of the root zone is irrigated while the other half is exposed continuously to dry conditions. the experimental design also included unfertilized treatment which was used for calculation of n recovery. the plot area was 120 m2 (6 m × 20 m), with one meter between two neighboring plots to protect water from lateral movement. drip irrigation (twinwall, 15 mm inner diameter, in-line drippers at 40 cm distance delivering 2.5 liter h–1 at operating pressure 100 kpa) was either laid out directly on soil surface with fi and di or buried at 15 cm beneath soil surface with both prd treatments. twenty-five-day old seedlings of tomato (solanum lycopersicum l.) cultivar ‘ty 70/70’f1 hybrid was directly transplanted to the main field at 25 cm intervals along drip lines (32 000 plants ha–1) on the middle of august. plants were arranged in north south oriented soil beds pre-furrowed to receive 40 t ha–1 of organic manure and arranged either in single rows at 100 cm apart for fi and di or in paired rows at 40/160 cm alternately for both prd treatments, so that the total number of plant rows was the same for all the treatments. before tomato transplanting, one drip line was placed along each row in fi and di or in the center of the paired rows in both prd treatments as the treatments specified in (fig. 1). all treatments received 150 kg p ha–1 as single super phos phate and 250 kg k ha–1 as potassium sulfate before transplanting which incorporated into the soil beds. nitrogen fertilizer was applied at the rate of 320 kg n ha–1 as ammonium nitrate in water soluble form at 7 days interval through the drip irrigation system using venturi type injector. fertigation events of n were started two weeks after planting in 12 equal doses and stopped 30 days prior to the end of the growing season. tab. 1: average monthly maximum (tmax) and minimum (tmin) temperature, relative humidity, rainfall, evapotranspiration (et0) and wind speed during the growing season. month tmax tmin relative humidity rain fall et0 wind speed (°c) (°c) (%) (mm) (mm d−1) (km h-1) 2015 august 36.6 22.1 56 0.0 7.2 8.9 september 35.5 20.2 56 0.0 6.3 8.9 october 31.5 17.4 58 0.0 4.7 7.2 november 27.4 13.5 59 6.7 3.2 6.9 december 25.7 13.1 59 8.3 2.6 8.3 2016 august 35.6 20.2 53 0.0 6.8 8.9 september 32.8 18.4 50 0.0 5.8 9.3 october 30.2 16.2 56 2.3 4.5 8.5 november 25.7 12.8 59 7.5 2.9 7.8 december 21.2 8.8 60 4.4 2.5 7.8 334 m.a. badr, w.a. el-tohamy, s.d. abou hussein, n. gruda soil water and n measurements the volumetric water content on the wetted soil volume of the bed was monitored every 12 hours during the development stage (30-75 dat) by time domain reflectometry (tdr) probes (pico-bt).the tdr probes were installed vertically under the dripper to measure the soil moisture in the profile at 10 (range 0-20) and 30 (range 2040) cm intervals for each treatment. the time interval of measuring of soil water content was daily during sampling and no less than 2 h after an irrigation event which was considered enough time for irrigation water to infiltrate within sand particles and provide the appropriate balance between the soil and the access tube. to determine ammonium and nitrate in the root zone at the last harvest, soil samples were collected from below the drippers at depths of 10 cm down to 40 cm using tube auger from the wetted area and composite samples were placed on ice and refrigerated until further analysis. the samples from each depth increment were air-dried and ground to pass through 2-mm sieve. analysis of 2 m kcl extractable ammonium and nitrate were performed by steam distillation and total n was determined by the micro-kjeldahl method modified to recover no3–-n (bremner and mulvaney, 1982). physiological measurements and sampling leaf area index (lai) was measured monthly on cut plant samples taken on fully expanded youngest leaves in each replicate with a leaf area meter (3050 li-cor inc., lincoln en usa). monitoring diurnal change of stomatal conductance, photosynthetic rate and transpiration rate were conducted with a portable photosynthesis system (adc bio-scientific, uk) and leaf water potential (lwp) using a pressure chamber (pms, corvallis, usa). measurements were taken on the central section of a mature leaflet of the last youngest fully expanded leaf, between 07:00 and 19:00 h at flowering stage on two plants per replicate. estimation of crop water requirement collected meteorological data were calculated from weather station of the central laboratory of agricultural climate for ismailia province located near the experimental field. reference crop evapotranspiration (et0) was calculated on a daily basis using penmanmonteith’s empirical formula (allen et al., 1998). the actual irrigation water was calculated following the crop evapotranspiration (etc) method according to soil water balance equation: etc = et0 × kc where etc is the maximum daily evapotranspiration (mm); et0 is the reference evapotranspiration (mm); kc is the crop coefficient for different months based on crop growth stages, 0.45 initial; 0.75 de velopmental; 1.15 middle; 0.85 maturity for growth stages 30/40/ 40/25 days (allen et al., 1998). cumulative etc was calculated to be 390 mm in fi treatment for a growing period of 135 days. the entire water requirements were supplied daily during the initial stage of growth to encourage plant establishment, but thereafter irrigation frequency was running at 3 days intervals to deliver the calculated amount of water for each treatment. measurements of crop parameters biomass accumulation throughout the entire growth period was determined by harvesting three representative plants per treatment replicate at 50, 80, 105 and 135 dat intervals. total nitrogen uptake of whole plant parts was determined at maximum biomass accumulation in shoot and fruit tissues. the different plant samples were separated and dried at 70 oc in a forced air oven for subsequent dry weight determination. tissue samples were ground to pass through a 0.5 mm screen and stored for dry weight analysis, with a thoroughly mixed 5 g portion of each sample stored. tissue material was digested using h2so4 in the presence of h2o2 and analyzed for total kjeldahl n at the analytical research lab (national research center, are) using the method described by bremner and mulvaney (1982). seasonal n uptake was derived from the whole plant sample (shoots + fruits) data and as the product of the crop biomass (dry weight) and the n concentrations in plant materials from which the uptake per hectare was derived based on plant population. harvesting of the tomatoes was made on last week of november until end of december. total fruit yield was recorded during the harvest on at least 25 plants in a row in each treatment in all the replications and data were presented as ton per hectare. water use efficiency was calculated from the total fruit yield (kg ha−1) divided by seasonal crop water applied for each irrigation treatment during the growing season and expressed as kg yield−1 mm−1. nitrogen use efficiency (nue) was calculated using the following equation: yt −y0 nue = n where yt equals total yield under treatment, y0 equals total yield under control and n equals applied nitrogen. all equation variables are in units of kilogram per hectare. postharvest n recovery was calculated using the following equation: nt−n0 n recovery = × 100 n where nt equals total crop n uptake (shoots + fruits) under treatment, n0 equals total n uptake under unfertilized treatment and n equals applied nitrogen. the average crop n uptake from the unfertilized sur-fi 1 m 2 m 6.0 m 6.0 m 6.0 m 1 m 6.0 m drip line crop rows 2 m drip line crop rows sur-rdi sdi-prd75 sdi-prd50 fig. 1: layout of the experimental plots showing drip line positions and arrangement of tomato plants under different drip irrigation treatments. fi = full irrigation; prd = partial root zone drying; rdi = regulate deficit irrigation; sdi = subsurface drip irrigation; sur = surface drip irrigation. physiological characterization of tomato under deficit and partial root zone drying irrigation in an arid region 335 field plots (n0) and total yield from the same plots (y0) were 12 kg n ha−1 and 0.815 t ha−1, respectively for the whole growing season. statistical analysis all data were subjected to the analysis of variance (anova) appropriate to the experimental design to evaluate the effects of treatments on yield and yield components of tomato (total biomass, shoot dry weight and lai). costat (version 6.311, cohort, usa, 1998-2005) was used to conduct the analysis of variance. comparison of treatment means was carried out using the least significant difference (lsd) at significant level of p≤0.05. results soil moisture content analysis of soil moisture content was conducted directly at the position of drip lines during the period of maximum growth rate for sur with fi and sdi with water saving treatment (prd75). regardless of the amount of water applied, soil moisture content under sur fluctuated in wide range (0.32 and 0.11 cm3 cm–3) for soil layer (0-20 cm) due to the effect of high temperatures (average tmax = 31.5 °c and tmin = 17.3 °c) for the period from 15th sep to 30th of oct (fig. 2). for sdi treatment a relatively lower soil moisture fluctuation (0.27 and 0.15 cm3 cm–3) was observed at the corresponding soil depth as the movement of water by capillary forces was not enough to reach top soil. on the other hand, moisture content at soil layer (2040 cm) recorded lower values (0.28 and 0.13 cm3 cm–3) with sur than with sdi (0.30 and 0.17 cm3 cm3) indicating that most of the applied water was remained in the root zone although sdi received lower amount of irrigation water. moreover, soil moisture content under sdi remains in higher levels during the growing season in relation to sur because of intensive plant canopy shading for soil surface (two plant rows per one drip line) as was observed in the field. physiological responses the evaluation of the plant physiological state showed remarkable differences between fi in sur and the three water saving treatments following water restriction. after the beginning of the light period, stomatal conductance in plants grown under water saving treatments began to diverge with fi plants particularly when the temperature rises at midday. the highest lwp was observed under fi irrigation throughout the day, but the difference became most obviously in the afternoon (fig. 3a). mean values of lwp in control plants was significantly greater than in water saving treatments, however, the measured values in the prd75 had experienced little bit lower lwp than in the other treatments at midday. plants under rdi showed the lowest stomatal conductance suggesting that this method of irri gation imposes the greatest restriction on stomatal aperture (fig. 3b). further, the photosynthetic rate measured under prd75 was nearly the same as the fi treatment; whereas, the rdi exhibited the lowest rate (fig. 3c). similarly, plant transpiration rate showed appreciable decrease following partial stomatal closure in water saving treatments, particularly in rdi plants, which recorded approximately 50% lower values than those of fi plants. yield and water use efficiency although prd75 plants in sdi received 25% lower water compared to fi plants, shoot biomass and fruit yield were not significantly affected but almost the same yield was obtained (tab. 2). when applying prd50 under sdi, remarkable water savings was obtained while the yield reduced to only 20 and 18% during first and second season, respectively. otherwise, tomato fruit yield depressed under rdi in sur by 34 and 32% during first and second season, respectively mainly due to the decline in fruit weight and high fruit losses. this result indicated that the prd technique had higher yield benefit along with substantial amount of water saving (50%) as compared to conventional rdi. moreover, both prd treatments facilitates 7-10 days early fruit harvest compared with fi plants which give an advantage for fresh market tomato over other treatments. water saving treatments had higher percentage of fruit dry weight at which maximum value was obtained under both prd treatments. fruit harvest index (hi = fruit dry weight/total above ground dry weight at harvest) were higher under prd75 compared to other treatments which related to greater fruit dry weight. however, harvest index tended to decrease with increasing water stress, so that rdi in sur caused the lowest value. the highest dry biomass accumulation throughout the growth period was obtained under prd75 in sdi although leaf area index (lai) reduced the most under all water saving treatments compared to the corresponding value of fi in surface drip (fig. 4a, b). water use efficiency was positively affected by both prd treatments, suggesting that the crop yield was benefit from the water applied to the crop for the whole season (tab. 2). as the amount of water applied was lower by 25% in prd75, the wue increased by 30% over than fi treatment (from 177 to 230 kg ha−1 mm−1). moreover, when water amount was reduced by 50% in prd50 the wue increased by 65% (from 177 to 291 kg ha−1 mm−1). higher wue in both prd treatments might be owing to more water available to each plant due to retardation of water evaporation from surface soil area because of intensive plant cover (paired rows per one drip line). 0.0 0.1 0.2 0.3 0.4 0.5 0.6 30 35 40 45 50 55 60 65 70 75 s oi l m oi st u re c on te n t ( cm 3 cm –3 ) days after transplanting range (0-20 cm) sur-fi sdi-prd75 0.0 0.1 0.2 0.3 0.4 0.5 0.6 30 35 40 45 50 55 60 65 70 75 s oi l m oi st ur e co nt en t ( cm 3 cm –3 ) days after transplanting range (20-40 cm) sur-fi sdi-prd75 fig. 2: temporal changes of moisture content in two different soil layers during the developmental stage for sur with fi and sdi with prd75 (pooled data of the two years). fi = full irrigation; prd = partial root zone drying; sdi = subsurface drip irrigation; sur = surface drip irrigation. 336 m.a. badr, w.a. el-tohamy, s.d. abou hussein, n. gruda nitrogen uptake, recovery and nitrogen use efficiency tomato fruits took up the largest portion of n compared to other plant parts, irrespective of drip irrigation treatment (tab. 3). although n translocation to the shoots was slightly higher with fi plants in sur, the translocation was selectively towards fruits with prd75 in sdi which accumulated 17 and 15% higher n in the fruits during 2015 and 2016, respectively. total dry biomass (shoot + fruit) with prd75 showed relatively higher total n uptake and recovery than with fi due to relative higher fruit dry weight and better utilization of n from the root zone. on the other hand, the significantly lower yield with di and prd50 may be partially due to lower n uptake and recovery because of soil drying coincided with limited nutrient diffusion and mass flow to the root surface. the highest nue was noted under the fi treatment whereas; it was the least under di treatment although the same amount of n was applied. this result shows how deficit irrigation reduces capacity of the crop for uptake and efficient use of nitrogen particularly when n deficiency may cause drastic yield reductions. soil nitrogen content the content of residual soil nh4+-n and no3–-n were determined for sur with fi and sdi with prd75 in the different soil layers at the last harvest of tomato (fig. 5). there was a relatively higher level -1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 4 6 8 10 12 14 16 18 20 le av e w at er p ot in tia l ( m p a) time day (hour)a sur-fi sur-rdi sdi-prd75 sdi-prd50 0.00 0.10 0.20 0.30 0.40 0.50 0.60 6 8 10 12 14 16 18 20 s to m at al c on du ct an ce (m ol m −2 s− 1 ) time day (hour) b sur-fi sur-rdisdi-prd75 sdi-prd50 0 5 10 15 20 25 30 35 6 8 10 12 14 16 18 20 c o 2 as si m ila tio n (u m ol m −2 s− 1 ) time day (hour) c sur-fi sur-rdi sdi-prd75 sdi-prd50 0 4 8 12 16 6 8 10 12 14 16 18 20 tr an sp ira tio n ra te (m m ol .m −2 s −1 ) time day (hour) d sur-fi sur-rdi sdi-prd75 sdi-prd50 fig. 3: diurnal changes of leaf water potential (a), stomatal conductance (b), co2 assimilation rate (c) and transpiration rate (d) of tomato grown under different drip irrigation treatments at flowering stage. data points are two years pooled data of three replications. least significant differences (lsds) at p ≤ 0.05 are presented as vertical line bars. fi = full irrigation; prd = partial root zone drying; rdi = regulate deficit irrigation; sdi = subsurface drip irrigation; sur = surface drip irrigation. tab. 2: tomato fruit yield, shoot dry weight (dw), total dry weight, harvest index (hi) and water use efficiency (wue) as affected by different drip irrigation treatments. treatments yield (t ha−1) hi fruit dw i wue fruit shoot dw total dry weight (%) (mm) (kg ha−1 mm−1) year 2015 sur-fi 65.32a 2.31a 5.65ab 0.59 5.12b 420 156c rdi 43.29c 1.91b 4.48c 0.57 5.95a 210 206b sdi-prd75 63.87a 2.19a 5.95a 0.63 5.90a 315 203b prd50 52.14b 2.11ab 5.21b 0.60 5.95a 210 248a year 2016 sur-fi 68.87a 2.36a 5.89ab 0.60 5.12b 390 177c rdi 46.74c 1.97c 4.70c 0.58 5.85a 195 240b sdi-prd75 67.29a 2.27ab 6.25a 0.64 5.92a 293 230b prd50 56.72b 2.08bc 5.44b 0.62 5.92a 195 291a values within the column followed by different letters are significantly different based on least significant difference (lsd) at p ≤ 0.05. fi = full irrigation; prd = partial root zone drying; rdi = regulate deficit irrigation; sdi = subsurface drip irrigation; sur = surface drip irrigation. physiological characterization of tomato under deficit and partial root zone drying irrigation in an arid region 337 of nh4+-n content at the proximity of the water source in both drip irrigation systems because of adsorption on soil particles. except for the surface soil layer (0-10 cm), residual nh4+-n content in prd75 was relatively lower compared to fi treatment. on the other hand, no3–-n content decreased from the topsoil to deeper soil layers for the both drip irrigation systems. the residual no3–-n content in the whole root zone was significantly 17% lower with prd75in sdi than with fi in sur. the total residual n mineral content in the root zone was significantly 27% less in prd75 than fi in accordance with higher n uptake (221 kg ha−1) than any other treatments. discussion water scarcity has become a significant limitation in agricultural/ horticultural production worldwide. hence the need to accurately estimate the crop water needs, under different conditions in order to 0 50 100 150 200 250 0 20 40 60 80 100 120 140 d ry b io m as s (g p la n t − 1 ) days after tranplanting asur-fi sur-rdi sdi-prd75 sdi-prd50 0.0 1.0 2.0 3.0 4.0 0 20 40 60 80 100 120 140 le af a re a in de x (l a i) days after tranplanting bsur-fi sur-rdi sdi-prd75 sdi-prd50 0 10 20 30 40 0–10 10–20 20–30 30–40 n h 4+ -n (m g kg s oi l−1 ) soil depth (cm) asur-fi sdi-prd75 0 10 20 30 40 0–10 10–20 20–30 30–40 n o 3− -n (m g kg s oi l−1 ) soil depth (cm) bsur-fi sdi-prd75 fig. 4: seasonal changes of dry biomass (a) and lai (b) during the progress of growth season of tomato under different drip irrigation treatments. data points are two years pooled data of three replications. least significant difference (lsds) at p ≤ 0.05 is presented as vertical line bars. fi = full irrigation; prd = partial root zone drying; rdi = regulate deficit irrigation; sdi = subsurface drip irrigation; sur = surface drip irrigation. tab. 3: nitrogen uptake, n recovery and n use efficiency (nue) of tomato as affected by different drip irrigation treatments. treatments nitrogen uptake (kg ha−1) n recovery nue fruit shoot total % (kg yield kg−1 n) 2015 sur-fi 124b 73a 197b 58 202a rdi 101c 47c 148d 42 133c sdi-prd75 144a 71a 215a 63 197a prd50 116b 54b 170c 50 160b 2016 sur-fi 130b 75a 205b 60 213a rdi 105c 50c 155d 45 144c sdi-prd75 149a 72a 221a 65 208a prd50 126b 56b 182c 53 175b values within the column followed by different letters are significantly different based on least significant difference (lsd) at p ≤ 0.05. fi = full irrigation; prd = partial root zone drying; rdi = regulate deficit irrigation; sdi = subsurface drip irrigation; sur = surface drip irrigation. fig. 5: soil residual nh4+-n (a) and no3–-n (b) contents at different soil depths for sur with fi and sdi with (prd75) at the last harvest of tomato in 2016. error bars indicate standard error of the means (n = 3) ± s.e. fi = full irrigation; prd = partial root zone drying; sdi = subsurface drip irrigation; sur = surface drip irrigation. 338 m.a. badr, w.a. el-tohamy, s.d. abou hussein, n. gruda optimize irrigation and increase the water saving. hence, customizing irrigation is a multi-faceted activity (gruda and tanny, 2014). soil moisture status through the evaluation of both drip irrigation systems, the water delivered on top soil in sur with fi exposed directly to evaporation components, which resulted in higher soil moisture fluctuation at surface soil layer (0-20 cm) compared to sdi treatment (fig. 2). on the other hand, soil surface was usually dry in sdi treatment where the water was provided at a certain depth (15 cm from top soil) which ensures that a substantial fraction of applied water becomes available to the plant root system. however, sdi resulted in a relatively small increase in soil moisture content at soil surface where the upward capillary movement of water was not sufficient to reach top soil, which resulted in lower soil evaporation loss there by repressing the upward capillary movement of water (patel and rajpat, 2008; meshkat et al., 2000). although prd75 plants in sdi received lower water the buried drip line in sdi treatment produced relatively higher soil moisture content over time in the root zone (2040 cm), where a reasonable amount of water below soil surface was maintained and helped in store and increase wetted soil volume more than sur treatment (patel and rajpat, 2008). similar indication of water distribution in the soil was noted by zotarelli et al., (2009) who found that water applied through sdi remained at the root zone for utilization of plants and was not lost due to deep percolation. this finding holds, both in terms of amount of available water and distribution uniformity by placing the drip line sufficiently below the soil surface, which ensures that the applied water becomes available to the active part of crop root zone and cuts of evaporation losses due to restricted upward capillary flow (patel and rajpat, 2008). therefore, sdi seems to be more suited to effectively address the limitation of low water storage capacity of sandy soils. leaf water potential and stomatal control fixed prd technique, involves frequent irrigation of only one-half of the root zone in each irrigation event while the other half was always kept in a drying state. this made it possible that half root system absorb water easily, whereas the other half was subjected to partial water deficit. the roots in the dry side can sense soil drying and produce signals (mainly aba) that move through the transpiration stream to the shoots. these signals can play a vital role in maintaining a highly water status in the shoots through the reduction of stomatal conductance and leaf expansion (holbrook et al., 2002; bacon, 2003; liu et al., 2006). as expected, the limited water supply under deficit irrigation, induced water competition among the growing plants, evaporative demand as fruits of tomato are highly water demanding (mingo et al., 2003). however, less water availability in water saving treatments modified lwp of tomato with the minimal effect observed with both prd plants (fig. 3). with inadequate water supply, or extreme heat and drought, the stomata close much earlier with negative consequences for gas ex changes and co2 assimilation. because of the reduction in co2 assimilation in leaves, the metabolic processes are impacted re sulting in many of the integrated physiological and biochemical processes that cause yield and quality reduced (gruda and tanny, 2014; gruda and tanny, 2015). although it is difficult to determine whether stomatal closure limited photosynthetic rate, but this seems likely when the strong relationship of transpiration rate and yield is taken into account. this would explain the significant reduction in tomato yield under deficit irrigation (rdi) whereas loose-leaf turgidity adversely affects photosynthesis between the irrigations. diurnal stomatal conductance was most significantly reduced under rdi suggesting that this method of irrigation cannot control stomatal aperture, which imposes the greatest water stress on plant growth. this would explain the reason behind the greatest yield reduction under deficit irrigation (rdi). however, coordinates of stomatal conductance under prd75 fall closely to that of fi treatment whereas; photosynthetic rate almost follows the same rate or affected least between the irrigations. the outcome of this process is reasonably good yield with considerable water savings and higher wue, which is of paramount importance in areas of limited water resources. earlier studies indicated that at similar soil water deficit, prd could intensify aba signaling relative to the rdi treatment resulting in better control of plant water loss and avoiding water luxury causing further improvement of wue (dodd, 2007; wang et al., 2010). fruit production and water use efficiency coincide with changes in leaves physiology and growth, prd75 did not significantly decrease yield of tomato, expressed as fresh fruit weight but 25% water saving was achieved during the completely growing season (tab. 2). similar results have been obtained in other studies with tomato (zegbe et al., 2003; kirda et al., 2004). although prd50 exert negative effect on vegetative growth, fruit yield reduced by only 18% corresponded with 50% lower of water applied as compared to fi, suggested that prd technique induces a photosynthetic assimilates towards fruit growth to maintain yield production (topcu et al., 2007). this may explain the minimal effect on fruit yield in comparison with the pronounced yield reduction (32%) occurred on the plants grown under regulate deficit irrigation (rdi). consistent with previous study by topcu et al. (2007), our results suggest that the prd technique facilitates 7-10 days early harvest with high cash profit compared to fi plants. fruit dry weight was greater in water saving treatments, particularly in both prd treatments compared with fi. this represents a production of firmer fruits, a very important quality parameter for tomato in post-harvest process. according to gruda (2005) and gruda et al. (2018), product quality of vegetables is a complex issue and apart from visual characteristics and properties such as texture, the content of minerals and vitamins, flavor and other organoleptic characteristics should be considered. in our study, these characteristics were not investigated. according to bogale et al. (2016) prd can enhance health-promoting qualities of tomato by increasing contents of vitamin c, lycopene, and -carotene in fruits as well as total phenolic content (tpc) and antioxidant activity. however, the impact on vitamin c, lycopene, external color and tpc appear to be cultivar dependent. therefore, the authors emphasized that the choice of appropriate cultivars under different irrigation techniques is crucial for maintaining or modulating the quality and nutritional contents in tomato, while allowing for water savings. overall fruit harvest index declined significantly in response to water deficit in rdi but it was higher under both prd treatments, which related to greater water use and fruit dry biomass. tomato plants under prd75 accumulated higher dry biomass than fi plants because of higher fruit dry matter translocation from shoots to reproductive organs (fig. 4a). leaf area index was remarkably lower in the all water saving treatments compared with fi (fig. 4b). however, leaf expansion is highly sensitive to soil drying as observed on tomato (topcu et al., 2007) and other crops (kang and zhang, 2004). water use efficiency was markedly improved when less amount of water was applied in prd75 and this effect was more expressed in prd50, which relatively maintained tomato yield with lower amount of water but this effect was not apparent when the yield decreased markedly in rdi treatment. accumulated evidence has shown that, prd technique was found able to reduce irrigation water relative to fi and in turn wue was appreciably increased (zegbe et al., 2004; campos et al., 2009).the observed higher wue obtained at the prd50 treatment was a result of a higher ratio of water reduction to yield reduction. tomato is highly consumed of water in order to physiological characterization of tomato under deficit and partial root zone drying irrigation in an arid region 339 increase yields however; the important comparison with respect to wue is the compromise between conventional fi and prd technique. thus, taking together wue and yield, prd50 seems to be the best water management option when plants utilized 50% less amount of water and resulted in substantial 65% increase in wue in the drier environment. plant nitrogen indices growth conditions, n availability and water consumption by plants greatly affect the total n uptake in the plants (tab. 3). although prd75 plants consumed less water (75% of fi) but also accumulated relative higher n amount and higher n recovery than fi plants. these results indicated that prd treatment could improve translocation of n from shoot to fruits and increases dry biomass allocation, which in turn increases harvest index (topcu et al., 2007). in addition, the higher uptake of soil nitrogen, under sdi coupled with prd75, should be attributed to delivering n fertilizer amount directly to plant root zone (camp et al., 1997). these results therefore, confirm the earlier studies that the water deficit like in prd technique could improve crop n nutrition and optimize n distribution in the canopy thereby recovering higher portion of n costs compared to fi or conventional rdi treatments (stoll et al., 2000; zegbe et al., 2003). less n uptake in rdi and prd50 might be caused by lower n uptake from the relatively dry soil zones where soil moisture content determines the soil n availability and its transport to the roots (bahrun et al., 2002). however, soil nutrient availability is a function of soil che mistry and regulated by the dynamic changes of soil moisture. for the nutrient transport from the soil to the root surface, mass flow and diffusion are two different mechanisms. kang and zhang (2004) show further that soil drying reduced plant vegetative growth and also possibly reduced the total nutrient absorption as a consequence. this is because of reduced n absorption from the dry zones, which may result in n stress in this zone, and thus induce plant adaptation to the nutrient stress (hu et al., 2009). the nue under prd75 was slightly lower compared to fi where such technique could maintain fruit yield of tomato. however, it has been shown that prd can greatly induce the initiation and growth of secondary roots, which improve the ability of the plant to absorb both water (liang et al., 1996) and nutrients from the soil matrix, which may increase the nutrient use efficiency (han and kang, 2002). status of soil nitrogen for both drip irrigation systems, soil nh4+-n with (fi and prd75) remained higher at the proximity of the water source (fig. 5a) due to adsorption on soil particles (li et al., 2004; hanson et al., 2006). the content of nh4+-n was relatively lower in prd75 compared to fi although only in the top soil layer the reverse was true, which can be ascribed because of nh4+-n losses via nitrification and/ or volatilization. however, there was no evidence on nh4+-n accumulation occurred in the soil profile at last harvest of tomato. by contrast, the residual soil no3–-n content decreased from the topsoil to deeper soil layers for both drip irrigation systems (fig. 5b) as nitrate tended to accumulate at the periphery of the wetted soil volume (li et al., 2004; gardenas et al., 2005). however, in all soil layers the residual total n mineral content (nh4+-n + no3–-n) for the water saving treatment (prd75) tended to be lower than that of fi in the root zone of tomato. the lowered residual n in the soil under prd75 treatment can be explained by enhancing n uptake, deeper roots and increasing root density. these effects of prd technique have been reported by poni et al. (1992) and fort et al. (1997) who observed that root systems extend to deeper layers along with prd induces the initiation and growth of the secondary roots which improves the ability of the plant to absorb water and nutrients from the active part of the root zone (liang et al., 1996). conclusion design of drip irrigation system included sdi coupled with water saving treatment such as prd represents unique practical benefits for tomato production and water saving. partial root zone drying with moderate water saving (75% of et crop) could enhance the balance of yield and wue, which may provide a useful approach to apply this technique in arid regions. on the other hand, prd with more water saving (50% of et crop) can be realized by losses of only 18% fruit yield but with substantially water saving and a remarkable improvement in wue. the sacrifice of some yield losses, but with a sustainably saving in irrigation water along with improving wue could be acceptable under these conditions. special attention should be paid to the importance of sdi with prd technique for the reproductive of crops in areas where water shortage dominates or expensive in the view of water saving and yield maintenance. references abdelmageed, a.h.a, gruda, n., 2009: influence of high temperatures on gas exchange rate and growth of eight tomato cultivars under controlled heat stress conditions. europ. j. hort. sci. 74(4), 152-159. allen, r.g., pereira, l.s., raes, d., smith, m., 1998: crop evapo transpiration. guidelines for computing crop water requirements. fao irrigation and drainage. paper no. 56, fao, rome, italy. bacon, m.a., 2003: partial root drying: a sustainable irrigation system for efficient water use without reducing fruit yield. the lancaster environmental center, lancaster university, lancaster, uk. badr, m.a., abou hussein, s.d., el-tohamy, w.a., gruda, n., 2010: efficiency of subsurface drip irrigation for potato production under different dry stress conditions. healthy plants 62, 63-70. doi: 10.1007/s10343-010-0222-x bahrun, a., jensen, c.r., asch, f., mogensen, v.o., 2002: droughtinduced changes in xylem ph, ionic composition, and aba concentration act as early signals in field-grown maize (zea mays l.). j. exp. bot. 53, 251-263. doi: 10.1093/jexbot/53.367.251 bisbis, m.b., gruda, n., blanke, m., 2018: potential impacts of climate change on vegetable production and product quality – a review. j. cleaner prod. 170, 1602-1620. doi: 10.1016/j.jclepro.2017.09.224 bogale, a., spreer, w., gebeyehu, s., aguila, m., müller, j., 2016: alternate furrow irrigation of four fresh-market tomato cultivars under semi-arid condition of ethiopia-part i: effect on fruit yield and quality. j. agric. rural. dev. trop. subtrop. 117, 255-268. urn:nbn:de:he bis:34-2016100451011. bremner, j.m., mulvaney, c.s., 1982: nitrogen-total. in: page a.l. et al. 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‘petopride’ processing tomato (lycopersicon esculentum, mill.). sci. hort. 1916, 1-6. doi: 10.1016/s0304-4238(03)00036-0 zotarelli, l., dukes, m.d., scholberg, j.m.s., munoz-carpena, r., icerman, j., 2009: tomato nitrogen accumulation and fertilizer use efficiency on a sandy soil, as affected by nitrogen rate and irrigation scheduling. agric. water manage. 96, 1247-1258. doi: 10.1016/j.agwat.2009.03.019 address of the corresponding author: e-mail: badrmab@hotmail.com © the author(s) 2018. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 93, 225 233 (2020), doi:10.5073/jabfq.2020.093.027 1 research and development centre, bharathiar university, coimbatore, tamil nadu, india 2department of botany, bharathiar university, coimbatore, tamil nadu, india 3department of biotechnology, k.s.rangasamy college of technology, tiruchengode, tamil nadu, india 4centre for advanced studies in botany, university of madras, chennai, tamil nadu, india 5department of biotechnology, sri krishna arts and science college, coimbatore, tamil nadu, india evaluation of different native streptomyces spp. for effective management of rhizome rot of turmeric muthusamy nithya1, ponnusamy ponmurugan2*, balasubramanian mythili gnanamangai3, jayachandran philip robinson3, narayanasamy mathivanan4, jeyaraj senthilkumar5 (submitted: february 19, 2020; accepted: october 15, 2020) * corresponding author summary the efficacy of talc based bioformulations containing various biocontrol agents against rhizome rot disease caused by pythium aphanidermatum in turmeric plants was evaluated under field condition. indigenous biocontrol agents such as streptomyces lydicus, streptomyces griseus and streptomyces sannanensis belonging to actinomycetes group, pseudomonas fluorescens (bacterial) and trichoderma atroviride (fungal) were selected for the biological control of rhizome rot of turmeric. the results indicated a significantly stronger reduction in disease severity in trial plots treated with bacillus subtilis based commercial fungicide ‘companion’ when compared to plants treated with indigenous biocontrol agents. however, it was reverse in trial plots in terms of turmeric rhizome yield potential, yield attributes, physiological components, biochemical constituents and quality characteristics of rhizomes. among 17 treatments, a dual mixture of s. griseus and t. atroviride achieved the best disease control as well as plant growth improvement when compared to single and triple combinations of biocontrol agents. the present study confirms that exploration of microbial formulations containing streptomyces spp. as soil inoculant to turmeric plants exhibited some benefits to turmeric plant growth as well as controlling rhizome rot disease, which ultimately enhance the overall quality characteristics of rhizomes. further, our results suggest that a dual combination of biocontrol agents represent a promising method for effective manage ment of rhizome rot of turmeric. keywords: turmeric, rhizome rot, companion, biocontrol agents, streptomyces, pythium aphanidermatum, curcumin. introduction turmeric (curcuma longa l.) is an herbaceous annual plant belonging to the family zingiberaceae that is a potential cash crop in india. it has a large oval rhizome with sessile cylindrical tubers containing curcumin and oleoresin contents and commonly used as a flavouring, colouring agent and preservatives including biomedical applications (chattopadhyay et al., 2004). turmeric is a tropical rhizomatous crop cultivated in a wide variety of soil (parthasarthy et al., 2007) and climatic conditions in india. it prefers to grow in the range of soil ph 6.5 to 8.5, electrical conductivity 0.50 to 1.85 ds/m, total organic carbon 2.5 to 8.5%, clay/silt content 145.5 to 275.5 g kg-1, water holding capacity 64.3 to 75.5%, temperature 25 to 37 °c, relative humidity 60 to 75%, annual rainfall 1500 to 2000 mm and sunshine duration of 5.3 to 7.0 hrs/day (akpan, 2018). the important turmeric varieties grown in india are alleppey finger, salem turmeric, raja pore, erode local, bsr-1 and 2, pts-10, roma, suguna, sudarsana, sangli turmeric and nizamabad bulb (sumathi et al., 2008). indian turmeric is considered the best in the world in terms of quality, especially in high curcumin content. india produced 6,62,200 tons of turmeric from an area of 1,86,000 ha during 2017-18 (choudhary and rahi, 2018). the major turmeric exporting countries are india, china, myanmar and bangladesh. india has occupied around 60% of the world trade in turmeric production (ponmurugan et al., 2017). the rhizome rot disease caused by the fungal pathogen pythium graminicolum f.sp. aphanidermatum is more prevalent in turmeric plantations of india (ramarethinam and rajagopal, 1999). it is a serious problem in certain pockets of southern india and there was a significant reduction in turmeric export since 2010 due to this disease severity. in advanced stages of the disease, the rhizomes get decomposed; decayed and rotten rhizome emits foul smell. symptoms are stunted growth of the plant, pseudo-stem at the base and yellowing of leaves and necrotic spots. in advanced stages, the bright orange colour of the tissues of rhizomes changes to different shades of brown. in severe cases, rotting of rhizomes with white fungal filaments on the rhizome surface, complete dryness and death of the whole plant are the symptoms (ponmurugan et al., 2016). in india, all turmeric cultivars available today are highly susceptible to rhizome rot and no resistance or tolerant source has been identified yet. the quality of turmeric rhizomes is being affected by a number of root and leaf diseases in which rhizome rot is considered to be very important in terms of capital loss and poor quality of rhizomes. biocontrol agents, especially actinomycetes and streptomyces spp. in particular, have been proven as efficient biocontrol agents in controlling various plant diseases as well as enhancing the plant growth significantly by producing a wide spectrum of growth pro moting and antibiotics like substances (etebarian, 2006). strepto myces spp. are having high g+c content (80%) in the genome and they are potential secondary metabolite producer with substantial antagonistic properties (sanglier et al., 1993). streptomyces spp. are able to suppress the growth of pathogens by parasitizing the mycelium and degrading the spores (moreno et al., 2003). with the reported exploration of various pgprs against rhizome rot in turmeric (chenniappan et al., 2019) in mind, efforts were made to evaluate the bioefficacy of streptomyces spp. consortia along with various other biocontrol agents for their efficacy against rhizome rot and for improvement of plant growth and metabolism. material and methods field experiments fields with history of rhizome rot disease in turmeric plantations were selected for the present study. field experiments were conducted for four consecutive years with three cycle of the crop cultivation at thondamuthur turmeric fields located in coimbatore district of tamil nadu, india during 2015-2018 (latitude 10.9753 and longitude 76.8586). erode turmeric variety was selected for planting which carried out during may-june with the receipt of pre-monsoon 226 m. nithya, p. ponmurugan, b.m. gnanamangai, j.p. robinson, n. mathivanan, j. senthilkumar showers. the field experiments were devised as completely randomized block design with different treatments implemented and the plots were also analyzed randomly using quadrat method to study the consequence. each experimental block contained 100 plants, re plicated three times. the sampling timeline is given in fig. 1. the field layout is provided as supplememental material. effectual indigenous streptomyces strains such as s. lydicus (bu 013), s. griseus (ksr 348) and s. sannanensis (bu 254) and other biocontrol agents such as pseudomonas fluorescens (pf 017) and trichoderma atroviride (uta 9041) were identified by screening the pgpr traits tests like, hcn, indole, phosphate solublization, siderophore production and confirmed species level by sequencing were applied (ponmurugan et al., 2017). the treatment details are represented in tab. 1. selection of fungicides the systemic fungicide companion, which contains the spore forming bacteria bacillus subtilis gb03 with 5.5x106 cfu/ml. was preliminarily screened under in vitro condition (data not shown). 0.05% of companion was applied at 100 ml/plant dosage to the trial plots two times per month as per the recommendations of indian institute of spices research, kozhikode, kerala, india. preparation of bioformulations actinomycetes (s. lydicus, s. griseus and s. sannanensis), bacterial (p. fluorescens) and fungal (t. atroviride) antagonists were grown on casein nitrate agar (kuster and williams, 1964), nutrient agar and trichoderma selective media (elad and chet, 1983), respectively. all the biocontrol agent strains were formulated using talc powder as carrier for delivery as per the protocol made by elango et al. (2015). for field application, each antagonist was mixed with steri lized talcum powder at a concentration of 400 ml broth (8×108 cfu/ ml) per kg of talc and incubated for 3 days under shade condition. later, this talc formulation was further mixed with dried farm yard manure with 60-70% moisture content before application to trial plots. it was applied to the soil around the plant (at a depth of 10 cm) at 100 gm/plant at two months intervals (initiated from the vegetative stage of turmeric where the establishment of bulb occurred at the 4-5 months from planting) for a period of six months. the pretreatment data was taken between the planting until the initiation of treatments in the trial plots. periodically soil samples were collected from the experimental plots for the enumeration of biocontrol agents with specific medium following the dilution plate technique to estimate the survival rate of each antagonist in the soil samples. disease assessment the disease incidence was recorded in all the trial plots periodically by partial removal, 25 plants from each block were observed carefully without disturbing root establishments by digging the soil around the rhizosphere and are completely removed if infection arises severely. this has ensured number of plant reduction in that blocks. percentage of disease incidence (pdi) was calculated as proposed by elango et al. (2015) for disease assessment. soil analysis the soil samples were collected periodically in two-week intervals from the experimental fields and air dried at room temperature. various soil edaphic parameters like soil ph, electrical conductivity (ec), total organic matter (om) as carbon content (walkley and black, 1934), available nitrogen (aoac, 1990), available phosphorous (jackson, 1973), exchangeable potassium (murphy and riley, 1962), calcium, magnesium and sodium (bhargava and raghu fig. 1 timeline chart for the field trial conducted in the turmeric fields marker timeline p1, p2, p3 planting: jun 15, jun 16, jun 17 t1, t2, t3 treatment: sep 15, oct 16, oct 17 h1,h2,h3 harvest: feb-mar 16, feb-mar 17, feb-mar 18 pre-treatment (jun-sep) of every year two sampling intervals@every 14 days indicated by 1 mm line fig. 2 population densities of individual biocontrol agents after imposing various treatments in turmeric soils* *the mean values of each biocontrol agents for a period of four consecutive years with three harvest cycles 0 100 200 300 400 500 600 jun aug oct dec feb apr jun aug oct dec feb apr jun aug oct dec feb apr jun aug oct dec feb apr jun aug oct dec feb apr s. lydicus s. griseus s. sannanensis p. fluorescens t. atroviride companion# s. lydicus s. griseus s. sannanensis p. fluorescens t. atroviride s. lydicus + p. fluorescens s. lydicus + t. atroviride s. griseus + p. fluorescens s. griseus + t. atroviride s. sannanensis + p. fluorescens s. sannanensis + t. atroviride p. fluorescens + t. atroviride s. griseus + p. fluorescens + t. atroviride s. lydicus + p. fluorescens + t. atroviride s. sannanensis + p. fluorescens + t. atroviride untreated control 2015 2016 2017 2018 p1 h1 p2 h2 p3 h3 t1 t2 t3 fig. 1 timeline chart for the field trial conducted in the turmeric fields marker timeline p1, p2, p3 planting: jun 15, jun 16, jun 17 t1, t2, t3 treatment: sep 15, oct 16, oct 17 h1,h2,h3 harvest: feb-mar 16, feb-mar 17, feb-mar 18 pre-treatment (jun-sep) of every year two sampling intervals@every 14 days indicated by 1 mm line fig. 2 population densities of individual biocontrol agents after imposing various treatments in turmeric soils* *the mean values of each biocontrol agents for a period of four consecutive years with three harvest cycles 0 100 200 300 400 500 600 jun aug oct dec feb apr jun aug oct dec feb apr jun aug oct dec feb apr jun aug oct dec feb apr jun aug oct dec feb apr s. lydicus s. griseus s. sannanensis p. fluorescens t. atroviride companion# s. lydicus s. griseus s. sannanensis p. fluorescens t. atroviride s. lydicus + p. fluorescens s. lydicus + t. atroviride s. griseus + p. fluorescens s. griseus + t. atroviride s. sannanensis + p. fluorescens s. sannanensis + t. atroviride p. fluorescens + t. atroviride s. griseus + p. fluorescens + t. atroviride s. lydicus + p. fluorescens + t. atroviride s. sannanensis + p. fluorescens + t. atroviride untreated control 2015 2016 2017 2018 p1 h1 p2 h2 p3 h3 t1 t2 t3 tab. 1: treatment details and rates of application of biocontrol agents treattreatment dosage mixture ment details ratio t1 companion 100 ml/plant 0.05% t2 s. lydicus 100 gm/plant 100% t3 s. griseus 100 gm/plant 100% t4 s. sannanensis 100 gm/plant 100% t5 p. fluorescens 100 gm/plant 100% t6 t. atroviride 100 gm/plant 100% t7 s. lydicus + p. fluorescens 100 gm/plant 50+50% t8 s. lydicus + t. atroviride 100 gm/plant 50+50% t9 s. griseus + p. fluorescens 100 gm/plant 50+50% t10 s. griseus + t. atroviride 100 gm/plant 50+50% t11 s. sannanensis + p. fluorescens 100 gm/plant 50+50% t12 s. sannanensis + t. atroviride 100 gm/plant 50+50% t13 p. fluorescens + t. atroviride 100 gm/plant 50+50% t14 s. griseus + p. fluorescens + 100 gm/plant 50+25+25% t. atroviride t15 s. lydicus + p. fluorescens + 100 gm/plant 50+25+25% t. atroviride t16 s. sannanensis + p. fluorescens + 100 gm/plant 50+25+25% t. atroviride t17 infected control 100 gm/plant fig. 1: timeline chart for the field trial conducted in the turmeric fields native streptomyces spp. for rhizome rot management in turmeric 227 pathi, 2001) were estimated at the maturity stage of turmeric. the biocontrol agents s. lydicus, s. griseus, s. sannanensis, p. fluorescens and t. atroviride were enumerated in soil samples periodically as mentioned above and were reported at two-month intervals for enumeration of microflora using suitable basal medium following the dilution plate technique to know their survival rate in soil (kuster and williams, 1964; elad and chet, 1983). determination of physiological and biochemical constituents net photosynthetic rate (pn), transpiration rate (tr) and stomatal conductance (sc) were measured using an infrared gas analyzer (adc lca3, uk) and an open type parkinson leaf chamber (adc plc-3). water use efficiency (wue) was calculated from the ratio between net pn rate and rate of tr (premkumar et al., 2008). the biochemical composition of leaf samples such as chlorophyll, carotenoid (welburn, 1994), total sugar (dubois et al., 1956) nitrogen (aoac, 1990), amino acids (moore and stein, 1948) and protein (lowry et al., 1951) contents were analyzed. the study was carried out throughout the stages of turmeric plant and were reported during the maturation stage of the plants that exhibited the trends of treatment schedules. analysis of quality parameters of turmeric rhizomes the crop was harvested after maturity during the experimental period; rhizomes were separated carefully and cleaned; and the fresh weight was recorded. mean yield and yield attributes in terms of productivity index (pi) were calculated from the yield data recorded with individual trial plots. according to sharma and sathyanarayana, (1990) pi = yield of the treated plot/mean yield of the field. the number of primary, secondary and tertiary finger rhizomes ori ginated from the mother rhizomes and its dry weight was enumerated carefully. the extraction and quantification of curcumin and oleo resin contents from the turmeric rhizomes of different treatments were carried out according to the methods of bagchi (2012) and kumar et al. (2014). statistical analysis all the data collected for three cropping cycles with 25 plants per block (total 100 plants) in three replicates for all parameters in each block were subjected to analysis of variance (anova) and newman keuls test (at p = 0.05) using spss 17.0 statistical package (spss, inc. chicago, il). if the data did not fulfill the assumptions of normal distribution and homogeneity of variances, non-parametric test were carried out using kruskal-wallis test and when the significant differences were obtained mann-whitney u test (at p = 0.05) was used for pair’s comparison (gomez and gomez, 1984). results rhizome rot disease protection in turmeric in response to soil application of companion (commercial fungicide) and indigenous biocontrol agents, there was a significant reduction in rhizome rot disease incidence in turmeric plants (tab. 2). the results revealed that the former was significant in disease protection when compared to later treatments. among the 17 treatments, the actinomycete was found to be better than fungal and bacterial antagonists in protecting the plants against the rhizome rot. systemic fungicide, companion showed a maximum disease protection of 60.3%. among the streptomyces spp. tested against p. aphanidermatum, s. griseus (42.7%) was found better. other biocontrol agents, t. atroviride showed nearly 42.4% disease reduction when compared to p. fluorescens (40.1%). dual combination of s. griseus and t. atroviride strains achieved the best disease control (50.8%) against p. aphanidermatum. the results clearly indicated that all the triple combination of biocontrol agents were found to be inferior to dual combinations in terms of disease reduction that ranged between 33.9 and 34.3%. the untreated control plants exhibited lowest disease protection (-13.85%), with all finger and mother rhizomes rotten and decayed. rhizome yield and their attributes a significant difference in rhizome yield and their attributes as productivity index was recorded after imposing various treatments tab. 2: effect of various biocontrol agents and fungicide on rhizome rot disease incidence, yield and yield attributes in turmeric plants. treatment details disease incidence (%) disease rhizome yield productivity pre treatment post treatment protection (t ha-1) index (%) companion# 57.8 ± 0.7a 22.3 ± 0.3a 60.3 ± 7.0g 22.3 ± 1.5b 0.48 ± 0.08b s. lydicus* 57.9 ± 0.3a 35.0 ± 0.3f 41.0 ± 0.8c 44.2 ± 1.7f 1.09 ± 0.02cd s. griseus* 58.7 ± 0.5a 32.2 ± 0.6e 42.7 ± 2.4cd 45.8 ± 1.4f 1.61 ± 0.31e s. sannanensis* 58.7 ± 0.4a 35.2 ± 0.2f 41.8 ± 2.1c 45.0 ± 2.5f 1.51 ± 0.37e p. fluorescens** 57.4 ± 0.5a 33.5 ± 0.2e 40.1 ± 3.0c 40.8 ± 2.0e 0.93 ± 0.21c t. atroviride*** 57.2 ± 0.0a 32.7 ± 0.6e 42.4 ± 2.7c 42.8 ± 2.9e 1.95 ± 0.28e s. lydicus + p. fluorescens 57.8 ± 0.5a 29.6 ± 0.2cd 48.3 ± 2.1de 52.7 ± 0.8gh 2.12 ± 0.02f s. lydicus + t. atroviride 59.3 ± 0.3a 27.1 ± 0.3c 47.1 ± 1.9d 51.0 ± 1.4g 2.43 ± 0.36f s. griseus + p. fluorescens 58.9 ± 0.2a 26.0 ± 0.4b 48.7 ± 0.8de 52.7 ± 0.7gh 2.21 ± 0.34f s. griseus + t. atroviride 58.0 ± 0.3a 25.0 ± 0.6b 50.8 ± 0.7f 55.7 ± 0.6i 2.93 ± 0.06fg s. sannanensis + p. fluorescens 58.5 ± 0.2a 29.4 ± 0.3cd 48.0 ± 1.4de 50.2 ± 0.7g 2.50 ± 0.01fg s. sannanensis + t. atroviride 58.0 ± 1.0a 27.8 ± 0.4c 46.6 ± 2.3d 50.6 ± 1.4g 2.78 ± 0.15fg p. fluorescens + t. atroviride 57.1 ± 1.2a 29.8 ± 0.5cd 45.3 ± 1.5d 51.4 ± 0.7g 2.12 ± 0.02f s. griseus +p. fluorescens + t. atroviride 58.0 ± 0.5a 35.2 ± 0.4f 34.3 ± 2.3b 33.7 ± 1.5c 0.81 ± 0.39c s. lydicus + p. fluorescens + t. atroviride 59.2 ± 1.0a 34.5 ± 0.3f 35.0 ± 2.2b 35.9 ± 0.8cd 0.87 ± 0.37c s. sannanensis + p. fluorescens + t. atroviride 59.2 ± 0.4a 33.5 ± 0.2e 33.9 ± 3.8b 32.9 ± 2.2c 0.98 ± 0.23c untreated control 58.5 ± 0.5a 66.6 ± 0.7g -13.5 ± 1.1a 11.4 ± 3.1a 0.04 ± 0.31a # systemic fungicide, *actinomycete biocontrol agents, ** bacterial biocontrol agent, *** fungal biocontrol agent identical superscript letters denote that values are not significantly different at p < 0.05. a higher letter denotes improvements acquired due to treatment and lower alphabet denotes decline acquired due to treatment. 228 m. nithya, p. ponmurugan, b.m. gnanamangai, j.p. robinson, n. mathivanan, j. senthilkumar in the trail plots (tab. 2). among 17 different treatments, the highest rhizome yield of 55.7 t/ha was recorded in t10, s. griseus and t. atroviride with productivity index of 2.93%. this was followed by the treatment containing s. lydicus with p. fluorescens, s. griseus with p. fluorescens, s. lydicus with t. atroviride s. sannanensis with t. atroviride and s. sannanensis with p. fluorescens combinations in which the rhizome yield was in the range of 50.2 52.7 t/ha and productivity index was in the range of 2.12 2.78%. there was a striking difference in the rhizome yield potential between single and triple combination mixture of biocontrol agents treated plants which ranged from 40.8 45.8 t/ha to 32.9 35.9 t/ha; respectively. the companion treatment registered least rhizome yield of 22.3 t/ ha with the productivity index of 0.48%. wherein the rhizome yield of the companion treatment was 22.3 and the mean yield of all the plot in that field was 46.5. hence for t1 (companion treatment) 22.3/46.5 = 0.48%. untreated control plants registered the yield of 11.4 t/ha with the productivity index of 0.04%. among the individual treatments, s. griseus treated plots produced a higher rhizome yield of 45.8 t/ha with the productivity index of 1.6%. similarly, among triple combination mixtures tested, the highest rhizome yield of 35.9 t/ha was recorded in a treatment containing s. lydicus + p. fluorescens + t. atroviride with productivity index of 0.87% (tab. 2). rhizome quality characteristics of treated plants to fungicide and biocontrol agents it is evident from the tab. 3 that turmeric rhizome quality charac teristics like number of primary, secondary and tertiary finger rhizomes were influenced by various bioinoculant treatments. the rhizome quality characteristics like curcumin and oleoresin contents were also shown in the tab. 3. improvement with increased yield and their attributes was reflected in rhizome quality characteristic para meters of treated plants. in the case of biocontrol agents treated plants, the maximum number of primary, secondary and tertiary finger rhizomes was noticed in s. griseus and t. atroviride mixtures with 5.3, 4.9 and 5.8 followed by other treatments combinations. and no significant increase was observed between single and triple combination treatments as per the mann-whitney u test (at p = 0.05) method of statistical analysis. in case of biocontrol treated plants, there was significant increase in curcumin and oleoresin contents of 8.7% and 9.5%; respectively in plants treated with s. griseus and t. atroviride mixtures with no appreciable changes in single and triple combination of biocontrol agents. both curcumin and oleoresin contents were least in untreated control plants (1.7 and 1.5%; respectively) but these were superior to companion treatment (4.0 and 4.4%; respectively). physiological and biochemical response of treated plants to fungicide and biocontrol agents both physiological and biochemical traits were appreciably enhanced in turmeric plants treated with biocontrol agents, especially in combination treatments (tab. 4 and 5). among 17 treatments imposed against rhizome rot of turmeric, combination of two bio control agents (t7 t13) performed better than single (t2 t6) and triple combinations (t14 t16) in terms of enhancement in physiolo gical and biochemical response. the physiological parameters such as photosynthetic rate, transpiration rate, water use efficiency and stomatal conductance were improved maximum in s. griseus and t. atroviride treated plants with 13.1 μm min-2m-2, 4.6 m s-2m-2, 3.5 wue and 2.7 mol s-2m-2; respectively. the minimum pn and tr rates was recorded in the companion treatment which accounted nearly about 7.7 μm min-2m-2 and 1.8 m s-2m-2, respectively. similarly, water use efficiency and stomatal conductance recorded were 1.6 wue and 0.9 mol s-2m-2 in companion treated plants (tab. 4). further the results indicated that there was no significant difference (p<0.05) in physiological traits recorded between single and triple combination treatments. biochemical constituents were also increased to a greater extent in biocontrol agents treated plants when compared to companion schedule (tab. 5). the highest amount of chlorophyll and carotenoid contents was registered as 6.1 and 3.1 mg g-1 fresh weight of leaves in t10 treatment. similarly, the total sugar (7.3%) and nitrogen (5.8%) contents was found to be highest in the same t10 treatment. the tab. 3: effect of various biocontrol agents and fungicide on quality characteristics of turmeric rhizomes treatment details no. of no. of no. of curcumin oleoresin primary fingers secondary fingers tertiary fingers (%) (%) companion# 2.3 ± 0.3b 2.2 ± 0.9b 3.5 ± 1.3b 4.0 ± 0.4b 4.5 ± 1.1b s. lydicus* 3.0 ± 0.1cd 2.8 ± 0.3c 4.1 ± 0.4c 4.9 ± 0.3c 6.6 ± 0.4e s. griseus* 3.3 ± 0.2d 3.4 ± 0.6d 4.7 ± 0.6de 5.4 ± 0.3cd 6.9 ± 0.1ef s. sannanensis* 3.4 ± 0.2d 3.7 ± 0.6e 4.5 ± 0.7d 6.2 ± 0.3e 7.4 ± 0.6efg p. fluorescens** 3.0 ± 0.3cd 3.0 ± 0.3cd 4.0 ± 0.3cd 5.7 ± 0.3ef 7.1 ± 0.5f t. atroviride*** 3.0 ± 0.4cd 3.1 ± 1.7cd 4.4 ± 0.8d 6.6 ± 0.4f 8.6 ± 1.1gh s. lydicus + p. fluorescens 3.6 ± 0.3e 3.6 ± 0.6d 4.8 ± 0.3cde 6.1 ± 0.1e 8.5 ± 0.1h s. lydicus + t. atroviride 3.8 ± 0.9ef 3.8 ± 0.6e 5.2 ± 0.6e 7.1 ± 0.1g 9.0 ± 0.9i s. griseus + p. fluorescens 4.5 ± 0.2f 4.0 ± 0.4e 5.4 ± 0.8e 7.0 ± 0.5g 9.0 ± 0.5i s. griseus + t. atroviride 5.3 ± 0.1g 4.9 ± 0.1f 5.8 ± 0.6f 8.7 ± 0.6h 9.5 ± 0.4j s. sannanensis + p. fluorescens 3.6 ± 0.1e 3.6 ± 0.5d 5.0 ± 0.4e 6.3 ± 0.2ef 8.1 ± 0.5g s. sannanensis + t. atroviride 3.6 ± 0.4e 3.6 ± 1.4d 4.8 ± 1.3cde 7.4 ± 0.3fgh 7.4 ± 1.8efg p. fluorescens + t. atroviride 3.6 ± 0.1e 3.4 ± 0.6d 4.8 ± 0.2cde 6.3 ± 0.3ef 7.1 ± 0.4f s. griseus + p. fluorescens + t. atroviride 2.8 ± 0.1c 2.6 ± 0.3c 3.6 ± 0.6b 5.0 ± 0.0c 5.8 ± 0.9d s. lydicus + p. fluorescens + t. atroviride 2.7 ± 0.2c 3.2 ± 0.2cd 3.4 ± 1.3b 6.3 ± 0.4ef 5.3 ± 0.8c s. sannanensis + p. fluorescens + t. atroviride 2.6 ± 0.1c 2.8 ± 0.8c 3.6 ± 1.0b 5.5 ± 1.1d 5.9 ± 1.0cd untreated control 1.5 ± 0.4a 1.9 ± 1.8a 2.3 ± 1.8a 1.7 ± 0.5a 1.5 ± 1.6a # systemic fungicide, *actinomycete biocontrol agents, ** bacterial biocontrol agent, *** fungal biocontrol agent identical superscript letters denote that values are not significantly different at p < 0.05. a higher letter denotes improvements acquired due to treatment and lower alphabet denotes decline acquired due to treatment. native streptomyces spp. for rhizome rot management in turmeric 229 tab. 4: effect of various biocontrol agents and fungicide on physiological variations in rhizome rot disease infected turmeric plant leaves treatment details pn rate tr rate wue sc companion# 07.7 ± 1.4b 1.8 ± 0.2b 1.6 ± 0.2b 0.9 ± 0.3b s. lydicus* 10.7 ± 0.8e 2.5 ± 0.3bc 1.8 ± 0.1b 1.4 ± 0.0c s. griseus* 10.3 ± 1.8cd 2.7 ± 0.3bc 1.9 ± 0.2c 2.0 ± 0.4e s. sannanensis* 12.3 ± 0.3fgh 2.9 ± 0.5d 1.8 ± 0.1bc 2.1 ± 0.3e p. fluorescens** 11.1 ± 1.0ef 2.0 ± 0.3bc 1.9 ± 0.3c 1.5 ± 0.3cd t. atroviride*** 11.1 ± 2.4ef 2.4 ± 0.5c 1.8 ± 0.3bc 1.6 ± 0.3cd s. lydicus + p. fluorescens 11.3 ± 1.0f 4.1 ± 0.3gh 1.5 ± 0.1b 2.2 ± 0.1f s. lydicus + t. atroviride 12.0 ± 2.4gh 3.5 ± 0.3ef 2.4 ± 0.4d 2.1 ± 0.2e s. griseus + p. fluorescens 12.3 ± 0.6fgh 4.0 ± 0.8f 3.0 ± 0.3f 2.0 ± 0.3e s. griseus + t. atroviride 13.1 ± 1.2i 4.6 ± 0.4h 3.5 ± 0.6g 2.7 ± 0.2g s. sannanensis + p. fluorescens 11.2 ± 0.3ef 3.6 ± 0.2ef 2.9 ± 0.1e 2.1 ± 0.1e s. sannanensis + t. atroviride 10.2 ± 1.7d 3.4 ± 0.3e 2.7 ± 0.3de 1.6 ± 0.3cd p. fluorescens + t. atroviride 11.8 ± 0.6g 4.0 ± 0.2g 2.7 ± 0.1de 1.9 ± 0.1d s. griseus + p. fluorescens + t. atroviride 09.0 ± 0.9c 3.3 ± 0.1e 2.7 ± 0.7de 1.5 ± 0.1cd s. lydicus + p. fluorescens + t. atroviride 10.2 ± 0.5d 2.9 ± 0.4d 3.0 ± 0.3f 2.0 ± 0.6e s. sannanensis + p. fluorescens + t. atroviride 09.5 ± 0.6c 2.7 ± 0.5bc 2.6 ± 0.7de 1.4 ± 0.1c untreated control 03.6 ± 2.4a 1.1 ± 0.5a 1.2 ± 0.5a 0.4 ± 0.5a # systemic fungicide, *actinomycete biocontrol agents, ** bacterial biocontrol agent, *** fungal biocontrol agent pn rate: photosynthetic rate (per mol min-2 m-2); tr rate: transpiration rate (mol s-2 m-2); wue: water use efficiency (ratio of pn/tr rates); sc: stomatal conductance (mol s-2 m-2). identical superscript letters denote that values are not significantly different at p < 0.05. a higher letter denotes improvements acquired due to treatment and lower alphabet denotes decline acquired due to treatment. lipids and protein content was increased substantially up to 3.5 and 4.6% in the plants treated with s. griseus and t. atroviride (t10). all the biochemical parameters were found to be elevated more in dual combinational treatments than in single and triple combinations. the untreated control plants exhibited least chlorophyll and carotenoid contents of 2.2 and 1.0 mg g-1respectively, with the least other biochemical constituents like total sugar, nitrogen, protein and lipids but superior to companion treated plants (tab. 5). determination of nutrient status and analysis of soil edaphic parameters in turmeric soils turmeric plants prefers between acidic to alkaline ph condition to grow and thrive abundantly. the result on the ph values of turmeric soils showed that it was found to be in the range of 7.5-8.3. similarly, the results on the estimation of soil electrical conductivity (ec) indicated that it was in the range between 0.7 and 1.8. the highest amount of organic matter (om) about 8.1% was recorded in soil tab. 5: effect of various biocontrol agents and fungicide on biochemical constituents in rhizome rot disease infected turmeric plant leaves treatment details chlorophyll carotenoid sugar nitrogen amino acids protein (mg g-1) (mg g-1) (%) (%) (%) (%) companion# 3.2 ± 0.4b 1.3 ± 0.2b 3.2 ± 0.6b 2.1 ± 0.4b 1.6 ± 0.3b 2.1 ± 0.5b s. lydicus* 3.4 ± 0.5b 1.3 ± 0.2b 4.7 ± 0.3cd 3.3 ± 0.2c 2.0 ± 0.2b 2.9 ± 0.5c s. griseus* 5.6 ± 0.3de 2.5 ± 0.3d 4.7 ± 0.6cd 4.0 ± 0.5d 2.3 ± 0.2bc 4.3 ± 0.3f s. sannanensis* 6.0 ± 0.5e 2.9 ± 0.5e 5.1 ± 0.3e 5.0 ± 0.4g 2.3 ± 0.2bc 4.2 ± 0.4f p. fluorescens** 3.7 ± 0.3b 1.7 ± 0.3c 4.2 ± 0.3c 3.8 ± 0.3cd 2.1 ± 0.1b 3.0 ± 0.3d t. atroviride*** 3.5 ± 0.8b 1.7 ± 0.3c 5.7 ± 0.5f 3.4 ± 0.5c 2.5 ± 0.3c 3.8 ± 0.4de s. lydicus + p. fluorescens 3.5 ± 0.1b 1.6 ± 0.2b 5.9 ± 0.3f 4.4 ± 0.1ef 2.6 ± 0.5bc 3.8 ± 0.6de s. lydicus + t. atroviride 4.1 ± 0.8c 1.9 ± 0.3c 6.1 ± 0.7g 5.0 ± 0.2g 2.4 ± 0.7bc 3.2 ± 0.6d s. griseus + p. fluorescens 5.1 ± 0.5c 2.4 ± 0.5e 6.0 ± 0.6g 5.0 ± 0.3g 3.0 ± 0.3d 4.0 ± 0.4f s. griseus + t. atroviride 6.1 ± 0.5e 3.1 ± 0.2f 7.3 ± 0.6h 5.8 ± 0.3h 3.5 ± 0.3e 4.6 ± 0.2g s. sannanensis + p. fluorescens 3.6 ± 0.2bc 1.4 ± 0.4bc 5.4 ± 0.5ef 4.6 ± 0.3ef 2.4 ± 0.5bc 3.7 ± 0.2de s. sannanensis + t. atroviride 3.4 ± 0.8b 1.7 ± 0.3c 5.5 ± 0.7ef 4.5 ± 0.6ef 2.7 ± 0.2bc 3.9 ± 0.5de p. fluorescens + t. atroviride 3.4 ± 0.3b 1.3 ± 0.2b 5.5 ± 0.3ef 4.2 ± 0.1e 2.7 ± 0.4bc 4.1 ± 0.4f s. griseus + p. fluorescens + t. atroviride 4.6 ± 0.5cd 2.0 ± 0.3e 4.6 ± 0.7d 4.0 ± 0.5d 2.4 ± 0.7bc 3.6 ± 0.4e s. lydicus + p. fluorescens + t. atroviride 5.2 ± 0.4c 3.1 ± 0.5f 4.3 ± 0.3c 5.0 ± 0.4g 2.8 ± 0.3d 2.5 ± 0.3c s. sannanensis + p. fluorescens + t. atroviride 4.2 ± 0.7c 2.0 ± 0.3e 5.7 ± 0.6e 4.4 ± 0.4ef 2.5 ± 0.3c 2.8 ± 0.4c untreated control 2.2 ± 0.9a 1.0 ± 0.4a 2.4 ± 1.1a 1.6 ±0.6a 1.2 ± 0.2a 1.2 ± 0.5a # systemic fungicide, *actinomycete biocontrol agents, ** bacterial biocontrol agent, *** fungal biocontrol agent identical superscript letters denote that values are not significantly different at p < 0.05. a higher letter denotes improvements acquired due to treatment and lower alphabet denotes decline acquired due to treatment. 230 m. nithya, p. ponmurugan, b.m. gnanamangai, j.p. robinson, n. mathivanan, j. senthilkumar samples collected from the plots amended with the combination of s. griseus and t. atroviride formulation. similarly, the estimation of available n, p, exchangeable k, ca, mg and na contents were found to be higher in the same treatment than in other treatments. these results were reflected similar to that of biochemical and physiological traits. the available n and p contents recorded were 3.1% and 381 ppm; respectively in the trail plots treated with s. griseus and t. atroviride combination. the amount of exchangeable k, ca, mg and na contents recorded was 270.3, 586.7, 97.7 and 76.1 ppm in the same treatment. it was superior to fungicide, single and triple combinational treatments of biocontrol agents. total om, available n and p contents were found to be 4.5%, 0.8% and 194.3 ppm; respectively to soils of untreated control plants. in the same way, the level of exchangeable k, ca, mg and na contents were found to be 145, 192, 45.3 and 24.9 ppm in the same treatment (tab. 6). survival of biocontrol agents in turmeric soils the population density of all biocontrol agents increased their population level from june to december and thereafter there was a reduction in the population density with the age of the sample. the enumeration graph strictly showed that there was a general decrease in population density of biocontrol agents after soil delivery in the experimental plots (fig. 2). at the time of application, the total population density of all the biocontrol agents were 8×108 colony forming unit per ml. finally, towards the end of experimentation (april) the population density of biocontrol agents was declined in varying levels. the mean population level of s. griseus was 120×105 cfu g-1 soil dry weight followed by s. lydicus (95.5) and s. sannanensis (95.0) in the month of april. it was about 50.5 and 60.5×105 cfu g-1 soil dry weight of p. fluorescens and t. atroviride; respectively in the same period (fig. 2). discussion in our studies, there was a significant (p < 0.05) reduction in rhizome rot incidence in trial plots due to fungicide and biocontrol agent treatments (tab. 2). the companion fungicide protected the maximum disease control (61.55%) followed by the dual combination of s. griseus and t. atroviride (56.97%) and minimum with the triple combination of biocontrol agents (ranged between 39.31 and 43.41%). the treatments containing the combination of three biocontrol agents (t14 t16) were inefficient in disease reduction when compared to single (t2 t6) and dual application (t7 t13) of biocontrol agents. it is due to competition between biocontrol agents in nutrient absorption from the rhizosphere and their growth. a large number of soil-borne plant diseases in plantation crops were successfully controlled through bacterial, fungal and actinomycete antagonists (gnanamangai and ponmurugan, 2011). when compared to biocontrol agents in terms of disease control, companion fungicide gave a better protection than that of biocontrol agents (tab. 2). rhizome rot of ginger can be controlled effectively by the application of various contact and systemic fungicides like bavistin 50wp, ridomil gold mz-72, captan, dithane m-45, copper oxychloride and bordeaux mixture. many researchers worked on the chemical control of the disease and reported to be very promising effect (kapratwar et al., 2016). due to the treatment of various biocontrol agents, rhizome yield and their attributes were significantly improved in which the highest rhizome yield of 26.77 t/ha was registered in the treatment of s. griseus and t. atroviride mixture. the same had been reflected in produc tivity index also (tab. 2). there was a remarkable difference in the rhizome yield between single and triple combination mixture of biocontrol agent treated plants. the companion fungicide treated trial plots registered with the least yield (17.35 t/ha) which is inferior to tab. 6: effect of various biocontrol agents application on nutrient status of turmeric soils treatment ph electric organic n p k ca mg na conductivity matter (ds/m) (%) (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) companion# 7.8±0.3bc 1.1±0.2ab 5.7±0.3b 1.3±0.3b 269.0±18.4b 178.3±34.3a 287.3±20.7b 60.7±2.3b 36.3±0.1b s. lydicus* 7.5±0.7a 1.0±0.3b 6.5±0.6d 2.3±1.4cd 292.7±15.1bc 199.3±32.7b 357.7±20.3c 68.3±3.8b 61.2±3.6d s. griseus* 7.6±0.4a 1.2±0.4b 7.1±0.4e 1.8±0.2c 293.3±15.8bc 201.3±23.3b 373.3±24.3c 76.7±7.0c 51.6±4.8c s. sannanensis* 8.0±0.6cd 1.2±0.2b 5.9±0.2b 2.6±0.3d 307.3±11.6c 203.3±33.3b 362.7±17.5c 77.7±9.8c 63.9±6.2d p. fluorescens** 8.3±0.4de 1.1±0.4ab 6.3±0.4c 1.6±0.4bc 338.3±14.3cd 232.7±26.9bc 432.0±29.1d 77.3±5.3c 63.2±5.4d t. atroviride*** 7.9±0.4c 1.3±0.3cd 6.6±0.8de 1.6±0.3bc 340.7±15.2cd 234.7±33.2bc 385.0±28.8c 80.0±3.1d 58.8±5.3cd s. lydicus + p. fluorescens 7.5±0.3a 1.1±0.5ab 7.1±0.3e 2.4±0.2cd 321.0±24.8cd 253.7±25.8c 586.3±20.0g 91.3±1.5e 68.0±2.1cd s. lydicus + t. atroviride 8.2±0.3d 1.5±0.5cd 7.4±0.5efg 2.6±0.2d 359.3±11.1d 266.3±32.3d 564.7±21.8ef 94.3±1.7ef 71.6±2.9e s. griseus + p. fluorescens 8.3±0.3de 1.5±0.4cd 7.1±0.4e 2.6±0.7d 333.3±17.7cd 255.7±25.3c 533.3±17.3e 90.3±2.7e 72.2±1.9e s. griseus + t. atroviride 8.2±0.4d 1.8±0.6de 8.1±0.2gh 3.1±0.5e 381.0±21.0f 270.3±20.9d 586.7±18.8gh 97.7±2.0ef 76.1±1.5f s. sannanensis + 8.2±0.1d 1.7±0.4d 7.1±0.3e 2.7±0.3de 365.0±12.5e 266.3±32.3d 580.0±27.2g 90.7±4.2e 70.4±1.9e + p. fluorescens s. sannanensis + t. atroviride 8.2±0.2d 1.5±0.3cd 7.2±0.5ef 2.6±0.2d 351.3±13.1d 240.3±30.7bc 584.0±27.2g 82.0±8.1d 71.4±2.0e p. fluorescens + t. atroviride 8.0±0.3cd 1.8±0.4de 7.8±1.4f 2.7±0.4de 361.3±19.2e 236.7±32.8bc 523.7±25.9e 82.3±8.6d 70.3±1.0e s. griseus + p. fluorescens + 7.7±0.6b 1.8±0.3de 7.5±0.4f 2.4±0.5cd 356.0±15.3e 232.7±26.9bc 434.3±25.8d 80.0±9.2d 68.8±7.7cd t. atroviride s. lydicus + p. fluorescens + 7.6±0.4a 1.4±0.5c 6.8±1.3de 2.6±0.3d 373.3±14.7e 234.7±33.2bc 471.7±25.5d 83.7±7.8d 70.3±6.3e t. atroviride s. sannanensis + 7.7±0.3b 1.2±0.03b 6.2±0.8c 2.4±0.5cd 371.0±12.1e 236.7±33.8bc 465.3±15.1d 82.7±6.4d 73.5±5.1e p. fluorescens + t. atroviride untreated control 7.5±0.1a 0.7±0.03a 4.5±0.4a 0.8±0.5a 194.3±13.3a 145.0±21.2a 192.0±12.3a 45.3±2.8a 24.9±2.5a # systemic fungicide, *actinomycete biocontrol agents, ** bacterial biocontrol agent, *** fungal biocontrol agent identical superscript letters denote that values are not significantly different at p < 0.05. a higher letter denotes improvements acquired due to treatment and lower alphabet denotes decline acquired due to treatment. native streptomyces spp. for rhizome rot management in turmeric 231 triple combination mixture of biocontrol agents (tab. 2). according to gnanamangai and ponmurugan (2011) biocontrol agents are potential microorganisms in soil system to the recovery of plants from infection. it has been reported that biocontrol agents enhance plant growth by producing growth stimulating hormones such as auxins, cytokinins and gibberellins (ajay et al., 2004). a moderate yield was recorded in the plots treated with companion fungicide which is not only due to the disease control but also due to their phytotonic effect (baćmaga et al., 2016). actinomycetes particularly streptomyces spp. inhabiting the rhizosphere, which have the ability to colonize plant root systems can sti mulate plant growth by secreting antibiotics like substances, enzymes and phytohormones (manjukarunambika et al., 2013; elango et al., 2015). utilization of such streptomyces spp. in turmeric fields is a promising approach for the biological control of rhizome rot as well as for the improvement of plant growth and rhizome yield poten tial. sabaratnam and traquair (2002) and gopalakrishnan et al. (2013) reported the plant growth promoting and biocontrol activities of streptomyces spp. in rice, sorghum and tomato. turmeric rhizome quality characteristics like number of primary, secondary and tertiary finger rhizomes and amount of curcumin and oleoresin contents were significantly increased due to bioinoculants treatment. a significant improvement was noted in the combination of s. griseus and t. atroviride treated plants (tab. 3). there are enormous research reports coincide with significant improvement in yield and productivity in various crops by exploring soil application of biocontrol agents (gupta et al., 2000; yan et al., 2002). curcumin followed by oleoresin contents are the bioactive compounds of turmeric, which are responsible for the wide spectrum of medicinal properties lead to commercial exploitation. the present study revealed the increased values in biochemical and physiological constituents, advance in progress of rhizome quality in response to bioformulation. both physiological and biochemical traits were enhanced in biocontrol agent treatments, especially in combinational treatments (tab. 4 and 5). the substantial increase of all biochemical and physiological constituents in biocontrol agent treated plants may be due to their utilization by the pathogen during host-pathogen interaction (ponmurugan and baby, 2007). increase in protein, nitrogen and amino acid due to biocontrol agent treatments assail to increase in translation process (protein synthesis) and hydrolysis of protein for cellular metabolism of the host plant (reddy et al., 2003). in our present study a minor variation of ph and ec was observed between treatments with the application of biocontrol agents. the values of ph positively coincided with exchangeable calcium level content because high calcium content is required for maintaining turmeric soil at alkaline ph and ec, which is suitable for turmeric plant growth. in soils with ph above 6.0, accumulation of calcium and the h+ and al3+ ions are likely to interfere with uptake of potassium which reflected in our results. due to biocontrol applications, soil nutrients were improved significantly in turmeric soils which could be competent enough with soil-borne pathogens like p. aphanidermatum (srinivasan et al., 2016). periodical monitoring of soil nutrients status revealed the uptake and fertility status of soil due to single and combined biocontrol treatments. curcumin synthesis in turmeric requires soil nitrogen, since it forms the structural unit of many proteins by undergoing phenyl propanoid pathway (sandeep et al., 2015). the population density of biocontrol agents in turmeric soils was positively coincided with the result of soil nutrient could survive well in the turmeric soils for a prolonged duration (fig. 1 and tab. 6). from our field experiments, bioformulations containing streptomyces spp. were found to be superior in terms of rhizome rot suppression and growth promotion in turmeric when compared to pseudomonas and trichoderma spp. effective biological control of phytopathogens results could be expected from mixtures of antagonists rather than from high populations of a single antagonist due to mimic the natural soil dynamics, broaden the spectrum of biocontrol activity and the efficacy with reliability of disease control (duffy and weller, 1995). among streptomyces spp. tested for the biological control of fig. 1 timeline chart for the field trial conducted in the turmeric fields marker timeline p1, p2, p3 planting: jun 15, jun 16, jun 17 t1, t2, t3 treatment: sep 15, oct 16, oct 17 h1,h2,h3 harvest: feb-mar 16, feb-mar 17, feb-mar 18 pre-treatment (jun-sep) of every year two sampling intervals@every 14 days indicated by 1 mm line fig. 2 population densities of individual biocontrol agents after imposing various treatments in turmeric soils* *the mean values of each biocontrol agents for a period of four consecutive years with three harvest cycles 0 100 200 300 400 500 600 jun aug oct dec feb apr jun aug oct dec feb apr jun aug oct dec feb apr jun aug oct dec feb apr jun aug oct dec feb apr s. lydicus s. griseus s. sannanensis p. fluorescens t. atroviride companion# s. lydicus s. griseus s. sannanensis p. fluorescens t. atroviride s. lydicus + p. fluorescens s. lydicus + t. atroviride s. griseus + p. fluorescens s. griseus + t. atroviride s. sannanensis + p. fluorescens s. sannanensis + t. atroviride p. fluorescens + t. atroviride s. griseus + p. fluorescens + t. atroviride s. lydicus + p. fluorescens + t. atroviride s. sannanensis + p. fluorescens + t. atroviride untreated control 2015 2016 2017 2018 p1 h1 p2 h2 p3 h3 t1 t2 t3 fig. 2: population densities of individual biocontrol agents after imposing various treatments in turmeric soils* *the mean values of each biocontrol agents for a period of four consecutive years with three harvest cycles 232 m. nithya, p. ponmurugan, b.m. gnanamangai, j.p. robinson, n. mathivanan, j. senthilkumar p. aphanidermatum, s. griseus is better than s. lydicus and s. sannanensis. the present investigation provides information for better management of turmeric plants using biocontrol agents against rhizome rot infection. hence, biological control might be an alternative, ecofriendly and sustainable technique for turmeric farmers towards the management of rhizome rot. acknowledgements the authors are thankful to university grant commission, new delhi, government of india (ref. no. mrp-major-biot-2013-12463) for financial support through major research project. moreover, the authors are grateful to mr.t.rajan, thontamuthur, coimbatore district, tamil nadu, india for providing turmeric fields to conduct field experiments. we also acknowledge dst-fist (ref. no. sr/fst/ ls-ii/2017/106) and ugc-sap (ref. no. f.5-16/2016/drs-1/sapii) grant, government of india given to the department of botany, bharathiar university for their infrastructural support. we also acknowledge dbt star scheme, new delhi, 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http://dx.doi.org/10.1006/bcon.2001.1014 http://dx.doi.org/10.1016/0923-2508(93)90066-b http://dx.doi.org/10.1016/j.indcrop.2016.03.027 http://dx.doi.org/10.1097/00010694-193401000-00003 http://dx.doi.org/10.1016/s0176-1617(11)81192-2 http://dx.doi.org/10.1094/phyto.2002.92.12.1329 i supplementary material layout of the conducted field trial in a turmeric cultivation fields 1-17 treatment trial plots fa-fd series fields with treatment trial plots ca-cd series fields with control plot * for every cropping cycles the alternative fields and the opposite plots were selected 1 3 2 4 3 5 3 6 3 7 8 3 10 11 12 13 14 15 16 17 9 3 f1 f2 f3 f4 f5 f6 f7 f8 f9 ca1 ca2 ca3 ca4 ca5 ca6 ca7 ca8 ca9 cb1 cb2 cb3 cb4 cb5 cb6 cb7 cb8 cb9 cc1 cc2 cc3 cc4 cc5 cc6 cc7 cc8 cc9 cd1 cd2 cd3 cd4 cd5 cd6 cd7 cd8 cd9 fa1 fa2 fa3 fa4 fa5 fa6 fa7 fa8 fa9 fb1 fb2 fb3 fb4 fb5 fba 6 fb7 fb8 fb9 fc1 fc2 fc3 fc4 fc5 fc6 fc7 fc8 fc9 fd1 fd2 fd3 fd4 fd5 fd6 fd7 fd8 fd9 1 3 2 4 5 6 7 8 10 11 12 13 14 15 16 17 9 ca1 ca2 ca3 ca4 ca5 ca6 ca7 ca8 ca9 cb1 cb2 cb3 cb4 cb5 cb6 cb7 cb8 cb9 cc1 cc2 cc3 cc4 cc5 cc6 cc7 cc8 cc9 cd1 cd2 cd3 cd4 cd5 cd6 cd7 cd8 cd9 fa1 fa2 fa3 fa4 fa5 fa6 fa7 fa8 fa9 fb1 fb2 fb3 fb4 fb5 fb6 fb7 fb8 fb9 fc1 fc2 fc3 fc4 fc5 fc6 fc7 fc8 fc9 fd1 fd2 fd3 fd4 fd5 fd6 fd7 fd8 fd9 layout of the conducted field trial in a turmeric cultivation fields 1-17 treatment trial plots fa-fd series fields with treatment trial plots ca-cd series fields with control plot * for every cropping cycles the alternative fields and the opposite plots were selected 1 3 2 4 3 5 3 6 3 7 8 3 10 11 12 13 14 15 16 17 9 3 f1 f2 f3 f4 f5 f6 f7 f8 f9 ca1 ca2 ca3 ca4 ca5 ca6 ca7 ca8 ca9 cb1 cb2 cb3 cb4 cb5 cb6 cb7 cb8 cb9 cc1 cc2 cc3 cc4 cc5 cc6 cc7 cc8 cc9 cd1 cd2 cd3 cd4 cd5 cd6 cd7 cd8 cd9 fa1 fa2 fa3 fa4 fa5 fa6 fa7 fa8 fa9 fb1 fb2 fb3 fb4 fb5 fba 6 fb7 fb8 fb9 fc1 fc2 fc3 fc4 fc5 fc6 fc7 fc8 fc9 fd1 fd2 fd3 fd4 fd5 fd6 fd7 fd8 fd9 journal of applied botany and food quality 93, 280 288 (2020), doi:10.5073/jabfq.2020.093.035 1julius kühn institute (jki) – federal research centre for cultivated plants, germany, institute for plant protection in horticulture and forests, braunschweig, germany 2inoq gmbh, schnega, germany directed inoculum production of arbuscular mycorrhizal fungi – the state of the art falko feldmann1*, carolin schneider2 (submitted: september 7, 2020; accepted: december 7, 2020) * corresponding author summary since the mutualistic nature of arbuscular mycorrhiza (am) had been discovered, an enormous amount of experiments elucidated the huge potential of mycorrhizal technology. nevertheless, the predictability of mycorrhizal effectiveness (i.e. the quantitative measure of a pronounced effect) in agro-ecosystems and plant production systems remained low. on this background, directed inoculum production (dip) had been developed based on quantitative genetics. during the inoculum production process the ecological niche of an arbuscular mycorrhizal fungus (amf) is considered, the effective ecological niche widened and population engineering included in large-scale inoculum production. this is done in order to place effective mycorrhiza products on the market. this article reviews briefly the historical development and the idea of dip and its performance in the last 25 years. recent and current scientific explanations for the variabili ty of mycorrhizal effectiveness and future demands of research are outlined. introduction in september 1985, 100 years after the description of the symbiotic nature of mycorrhizal fungi, an international symposium on “mycorrhiza and stress in plants” was organised by the society for applied botany in hannover, germany (dehne, 1986). the impact of this symposium was huge. several research groups in germany and europe became aware of the status quo of mycorrhizal research and decided to develop it on the background of their specific fields of interest. the applied botanist, late prof. dr. reinhard lieberei (*04.07.1948, 06.03.2019) initially compared physiological changes in roots colonized by arbuscular mycorrhizal fungi (amf) using the cyanogenic tropical rubber tree hevea brasiliensis (lieberei and feldmann, 1990). basing on the findings he induced a research group starting its work in amazonia, brazil (lieberei et al., 1989) in order to evaluate the agricultural potential of mycorrhiza in tropical plant production (feldmann et al., 1990). they found mycorrhizal effects in agro-ecosystems of perennial plants like reduction of plant loss during plantation, better nutrition of host plants and even enhanced resistance of leaves against biotrophic fungi due to mycorrhizal co lonization. all these findings resulted in the chance to re-cultivate abandoned degraded areas in the amazon region (feldmann et al., 1995). the observation of negative mycorrhizal population dynamics induced by management practices (feldmann et al., 2002) could be overcome by appropriate inoculum production techniques previously adapted to tropical demands (feldmann and idzcak, 1991). after return to germany, we had to learn that under european agri cultural and climatic conditions cultivated plants expressed a very different responsiveness to mycorrhizal inoculation. we observed all principal effects of the mycorrhizal symbiosis (nicely reviewed by koide and mosse, 2004), but the predictability of mycorrhizal effectiveness as the quantitative measure of an effect remained low – a major constraint for mycorrhizal products to be placed on the market as biostimulants in europe. the lack of predictability was in the 90ies of the last century one of the major reasons why companies had to quit after short time on the market (feldmann, 2003). what is behind the variability of mycorrhizal effectiveness? what could an inoculum producer do to develop a product with stable characteristics? to answer these questions, we brought together the knowledge on environment-fungal genotype/plant genotype interactions published over decades, focused on a few fungal species only (e.g. claroideoglomus etunicatum, formerly glomus etunicatum, rhizoglomus irregularis, formerly glomus irregularis, rhizoglomus intraradices, formerly glomus intraradices; wijayawardene, 2020) and analysed the amf/host interrelationships in a special test system (feldmann et al., 1998c) based on quantitative genetics (feldmann, 1998b). out of these studies an inoculum production procedure was developed, described in detail as “directed inoculum production process” (dip) (feldmann and grotkass, 2002). the differentiation between the qualitative mycorrhizal “effect” and its quantitative measure “effectiveness” is of major importance at this point. dip is targeted to increase the predictability of effectiveness towards an economically satisfying process with ensured quality (alten et al., 2002). consequently, we published the description of a best practice procedure for inoculum production (feldmann et al., 2009). this paper reviews the historical development of dip and the main reasons for variable mycorrhizal effectiveness, and furthermore, the quantitative genetic situation in our mycorrhizal inoculum as starting point for population engineering in plant cultivation systems. all empirical findings, which led to dip, are discussed on the background of newest outcomes of mycorrhiza workgroups from all over the world. finally, definitions like “strain”, “isolate”, “genotypes” or “genetic population” of amf fungi are discussed explaining “adaptation” processes within the inoculum. in honour of late prof. dr. lieberei we focus on the findings of our group initiated by him. variability of mycorrhizal effectiveness is the variability of mycorrhizal phenotypes selling amf inoculum on the market means to provide mycorrhizal propagules (spores, mycelia, colonized roots, and associated microbiomes of the production system) to users growing different plant species under several, often changing environments. from these propagules, the amf has to colonize the roots of the hosts. the symbiosis should modify the plant’s physiology in a way, which results in a practical advantage for mycorrhizal plants in comparison to nonmycorrhizal plants. the effects desired by plant growers are very different and reach from higher fresh produce, better rooting, more intense flower directed amf inoculum production 281 ing, prolongation of flowering, higher seed production, survival in drought periods or under suboptimal fertilization situations and much more (feldmann, 1998a). the amf economic threshold of effectiveness as the quantitative outcome of an effect desired by an inoculum user can vary depending on the effect observed: root colonization only already supports the development of cucumber plants in presence of meloidogyne hapla in greenhouses with soil infestation (feldmann et al., 2008). only 5% more stem thickening is sufficient for significantly earlier budding of trees (feldmann et al., 1990), 10% less plant losses can economize in vitro production of baptisia tinctoria (schneider et al., 2008), 20% more tubers are desired in production systems of potato in china (cheng et al., 2008). in bell pepper, cotton and marigold biotic stress reduction of only 10% already showed significant economic net benefit for the producer (long et al., 2008). the variability of effectiveness becomes clear during the selection of fungal isolates belonging to different amf species. as an example, baltruschat (1993) tested 21 amf isolates on two strawberry cultivars in order to identify the “best isolate”. he repeated the experiment in a short time after another under changed light conditions in the greenhouse and found very different rankings of the isolates’ effectiveness (fig. 1). exactly this variability of effectiveness is a marketing problem for inoculum producers. they simply cannot guarantee a certain quantitative outcome of the symbiosis after inoculation with their product. the results reasoned to accept the impossibility to find “the best mycorrhizal isolate” for inoculum production in those days. consequently, it was necessary to identify reasons for variability of effectiveness and options to reduce it. the example shows that host plant genotype/amf genotype/environment interrelationships are relevant for the quantitative outcome of the symbiosis. from a quantitative genetical point of view the quantitative outcome of the symbiosis, the effectiveness, reflects the phenotype of the mycorrhizal symbiosis. the results of baltruschat (1993) indicated that there was a phenotypic plasticity in an inoculum with a huge potential to react to changes in the environment. but at this point we did not yet understand which part of the reaction was induced by the plant and which by the fungal partner. environment-induced changes of mycorrhizal effectiveness reflect the host dependency of plants on am symbiosis in natural ecosystems mycorrhizal species have relationships with heterogeneous plant communities of different species frequencies and changing abundances, seasonal variations, long-term changes, and successions over time are observed (allen, 1992). host species can be colonized by various amf species at the same time or subsequently in a seasonal succession (gange et al., 1993). hosts are interlinked with each other forming a widespread fungal internet, in which nutrients are exchanged between host individuals and host species (newman, 1988). it is hard to estimate mycorrhizal effectiveness in situ under natural growth conditions. however, recently, there were interesting results indicating within-species (“strain”) differences of mycorrhizal phenotypes in plant communities: savary et al. (2018) found that within-species differences in rhizoglomus irregularis did not strongly influence the performance of individual plants or the structure of the overall plant community. however, the evenness of the plant community was affected by the phylogeny of the fungal isolates, where more closely-related amf isolates were more likely to affect plant community evenness in a similar way compared to more genetically distant isolates. this study underlined the effect of within amf species variability on plant community structure. while differential effects of the amf isolates were not strong, a single amf species had enough functional variability to change the equilibrium of a plant community in a way that is associated with the evolutionary history of the fungus. this observation implies the differential effectiveness of the fungal partner at a given site. furthermore, the mycorrhizal effectiveness can be different under changing environmental conditions. this phenomenon was explained by variable “dependency” on symbioses of hosts and was already observed by stahl (1900). the dependency of hosts under certain environmental conditions is quantified by their responsiveness to amf colonization. the idea behind is the action of the “law of the minimum”, a principle already developed in agricultural science in 1828 by carl sprengel (gröger, 2010). mycorrhizal dependency of a host is genetically fixed (azcon and ' e ls an ta 1 . e ls an ta 2 . con con 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10 11 1112 12 13 13 14 14 15 15 16 16 17 1718 18 19 19 20 2021 21 amf strain e lv ir a 1. e lv ir a 2. con con 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10 11 11 12 12 13 13 14 14 15 15 16 16 17 17 18 18 19 19 20 20 21 21 amf strain mei feb mei may mei maymei feb strawberry cultivar elsanta strawberry cultivar elvira ranked mycorrhizal effectiveness index (mei) first experiment in february (feb), repetition in may (may) amf: arbuscular mycorrhizal fungi; con: without mycorrhiza fig. 1: variability of mycorrhizal effectiveness on fresh weight after inoculation of two strawberry cultivars in two subsequent repetitions (ranked from „best“ isolate (top) to „worst“). the mycorrhizal effectiveness index (mei) is the percentage difference between mycorrhiza inoculated and not inoculated plants (con). numbers are the isolate numbers used for inoculation. ranking below con means fresh weight reduction, above con fresh weight increase of mycorrhiza inoculated plants. the figure was drawn based on data derived from baltruschat (1993). 282 f. feldmann, c. schneider ocampo, 1981). the degree of mycorrhizal dependency is expressed on individual level as a gradient within the host’s fundamental ecological niche and relevant environmental conditions. if a plant cannot explore a resource in its actual ecological niche without mycorrhiza, romell (1939) speaks of “obligate mycotrophy”. he assumes that effectiveness is then always positive. to his opinion, “facultative mycotrophy” leads to a shift of actual niche characteristics, too, if mycorrhiza is developed: in a facultative symbiosis mycorrhiza would either increase the acquisition of a limiting resource (e.g. p) or decrease it (e.g. carbon gain under low light). allen (1992) supports this hypothesis. from our point of view, the cited hypothesis is applicable to the practice of plant production with some considerations, which have to be kept in mind. first, advanced plant production normally bases on long selection processes of plant cultivars grown without consideration of mycorrhizal fungi. the plants are, therefore, selected to be independent on mycorrhiza. furthermore, long term experiences normally led to optimized procedures and growing conditions during plant cultivation. the actual ecological niche of a cultivated, intensively screened and selected plant cultivar, therefore, should make nearly optimal use of resources without any mycorrhiza. this circumstance leads to the impression that it is stress, which creates some mycorrhizal dependency of useful plants in production systems. deviations from the optimal growing system, suboptimal periods, and temporary depletion of resources, upcoming disease and unknown limiting factors open the window for use of mycorrhiza in advanced plant production. if primary selections are used, wild collections tested or even plant varieties with well-documented host characteristics are produced, at least facultative mycotrophy can be assumed. higher degree of plants’ mycotrophy, low extent of their genetic selection and suboptimal growing conditions in tropical and subtropical agriculture lead to much better situations for mycorrhizal use than under conditions of the northern hemisphere. a practical complication to classify a host as facultatively mycotrophic or obligate symbiont is the multifactorial nature of an ecological niche. important effects of mycorrhiza might be masked because of not influenced limiting factors existing in a cultivation system. in fig. 2 we show the ecofactor strength on a scale from zero to one hundred. a plant can explore the resources necessary for optimal growth without restrictions if the ecofactor strength is at maximum. reduced ecofactor strength opens the opportunity for mycorrhizal dependency. the plant can be responsive to mycorrhizal colonization if the symbioses can increase the ecofactor strength. because mycorrhizal fungi might influence only specific ecofactors, the law of the minimum leads to apparent or hidden mycorrhizal effects if the limiting factor is not influenced by the mycorrhizal symbiosis. for instance, a host might be obligatory dependent on mycorrhiza with respect to the survival under heavy metal stress and mycorrhiza overcomes this stress. but because of light deficiency it may not grow better than non-mycorrhizal plants even if the heavy metal stress has been overcome by mycorrhization in mycorrhizal plants. the result would be a hidden effect of heavy metal stress reduction if the light deficiency would not be reduced. concluding, our ability to predict mycorrhizal dependency of a host under specific conditions depends on the knowledge of stress tolerance characteristics and growth limiting factors of that host. additionally, to host dependency, the amf are interacting with the environmental factors, too. adaptations of amf to environmental conditions occur (read, 1999) and are well documented. even environment mediated selection on amf species takes place. if environmental conditions change drastically, a loss of amf species can be the consequence. for instance, in plant production systems, the amf communities are negatively influenced by management practices (könig et al., 2014). such selection pressure by cultivation methods leads to stress tolerant amf communities, which might be of low effectiveness (feldmann et al., 2002). in horticultural practice, in nearly all potting substrates used in plant production, amf are normally completely missing if not added (feldmann, 1998a). in these systems, especially the soil and sometimes many more parameters (e.g. in greenhouse production) are optimised for the plant to be produced. this leads to the observation that the same host is differently colonized by amf under different environments. nevertheless, the prediction of effectiveness in production systems is much easier than in nature. here, the widely controlled practical conditions and non-mycorrhizal treatments as comparisons in tests facilitate the application of the mycorrhizal technology for users and inoculum producers. the more practical experience in plant cultivation a grower has, the better he can predict success of mycorrhizal symbiosis in his growing system because the difference between performance of actual niche and fundamental host niche defines the maximum expression of mycorrhizal effectiveness in the symbiosis. selection phenomenons between the symbionts at this point, it is necessary to introduce some population genetical definitions regarding amf inoculum. as “isolate” we define descendants of mycorrhizal spores isolated from certain sites (proveniences) whether taxonomically determined or not. isolates often are morphologically identical spores but not descendants of a single spore. all spores produced from one single spore we call “strain”. in our studies, we developed descendants of single spores of strains and called them “sub-strains”. the expression “population” is used to describe all taxonomically defined descendants of one amf species in a given inoculum. population refers, here, to pure strains in the inoculum production system or to mixtures of strains of the same species. if amf species are mixed in an inoculum or the relations in situ to other, e.g. naturally occurring amf are studied the expression “amf community” is used. as shown in the example of fig. 1, amf isolate characteristics change with changing environment. but in a given stable environment further specificity phenomenons occur. for instance, a preference of partners forming the symbiosis can be observed, which leads to the support of one specific host being planted in dual-culture (feldmann, 1998a; croll et al., 2008). such an observation could be obtained in a maize field as well, where symbiotic weeds could favour the cultivated plant. in contrast, non-mycorrhizal weeds re duced maize fresh weight (feldmann and boyle, 1999). conse quently, we could find a competition of mycorrhizal isolates for the host plant resulting in the exclusion of certain amf species in mycorrhizal communities from colonizing the root system of a test plant. such specificity phenomena with enormous consequences for sym biotic effectiveness were postulated even on the level of amf popu' fig. 2: ecofactor strength and apparent, hidden and no effect of mycorrhizal symbioses. mycorrhizal effects might be hidden by not influencing the limiting factor (explanation see text). directed amf inoculum production 283 lations of one single strain (feldmann, 1997): we produced inoculum of a claroideoglomus etunicatum strain over four years on two maize cultivars, “badischer landmais” and “felix”. after two years, the effectiveness on the host plant decreased on “felix” while this was not observed on the other cultivar. the shift of effectiveness continued over four years in case of zea mays var. “felix” could be reproduced in a further experiment and could be demonstrated to depend on the plant genotype. after the fourth year, we changed the inoculum produced on the maize cultivars. the low effectiveness of inoculum produced on “felix” in the fourth year was improved over three subsequent years by var. “badischer landmais“, while the highly effective inoculum produced on “badischer landmais” lost its effectiveness on “felix” (fig. 3). because “badischer landmais“ is an old maize cultivar and “felix“ a modern hybrid the maintenance of higher effectiveness correlates with higher genetic heterogeneity of the host. whether this was the cause could not be proved here. however, if – in case of high host dependency – genetic hetero geneity of host would guarantee stable effectiveness of amf, the influence of biodiversity of host and fungal communities could have an important impact on symbiotic relevance in natural ecosystems and production systems. these observations were a clear indication for us that host genotype/ amf genotype selection processes take place with relevance for the effectiveness of the symbiosis. therefore, we developed a functional genotyping test system to study such processes. is, therefore, the “principal environment” for the biotrophic microsymbiont, while all the other abiotic and biotic factors can be seen as “secondary environment”. if we want to argue to observe the function of amf genes, we have to make sure that the principal and the secondary environment is as stable as possible. consequently, we designed the quantitative genetic analysis as „common garden experiment“, i.e. we standardised both environments as much as possible, including the plant material (feldmann et al., 1998c). from the host’s point of view, the micro-symbiont is as well the principal environment, and all other environmental parameters belong to the secondary environment. in our experiment, the secondary environment was standardized. changes of the host phenotypic performance of polygenic characters should, therefore, be modified by the heterogeneity of the principal environmental factors, the amf. if we, then, inoculate with one spore only, we should be able to observe the action of a certain amf spore phenotype resulting from a standardized principal and secondary environment and its related genotype and epigenetics. to ease the communication, we, therefore, speak of “functional amf genotypes” from here on. we exemplarily worked with the metric trait „host plant fresh weight“ as a result of the expression of a set of non-allelic, quanti tative polygenes under the influence of a standardized secondary environment and the tested functional amf genotype. the variation observed should be due to amf induced variation at several or many loci, each with a rather slight contribution to the variation in host phenotype. because of the standardization of the environment for the host, the total phenotypic variation should result of host gene – amf gene interactions. against this background, an inoculum of identical functional amf genotypes, including epigenetics, should have a nearly identical reaction norm under a given environment, including the host. we started with the experiments with the development of strains of an initial c. etunicatum spore population multiplied over subsequent multiplication cycles. single spores of sub-strains of each multiplication cycle were tested for their effectiveness on petroselinum crispum as outlined in feldmann (1998c) and feldmann (1998b). it could be shown that the inoculation with single spores increased the variance of p. crispum fresh weight drastically, while the standard reaction of non-inoculated plants did not change significantly (feldmann, 1998b). the spread of effectiveness values reached from negatively effective over neutral effectiveness to positive response. in case of positive effectiveness, the plant response was positively correlated with the degree of root colonization. effectiveness of spores with neutral effectiveness and negative effectiveness did not show this correlation. high root colonization was not a guarantee for positive effectiveness. even when descendants of a single spore (“strain”) were compared, within this strain positively, neutrally and negatively effective substrains could be derived (fig. 4, data derived from feldmann, 1998b). testing multispore inocula of these defined sub-strains for their effectiveness in subsequent multiplication cycles, originally negative strains appeared neutrally effective, neutrally effective strains remained neutrally effective and positively effective strains lost their positive effectiveness after two multiplication cycles (feldmann, 1998b). by these data, we could demonstrate the existence of different functional amf genotypes within an inoculum of a defined amf strain for the first time. in a next step, we estimated the “heritability” of the trait from one multiplication cycle to the next. a statistical measure of variation is the variance of measures. the proportion of the phenotypic variance that is genetic variance is the heritability of a trait. here, we calculated the genetic component of variation by the correlations of effectiveness between spore descendants in the subsequent multipli“functional amf genotype” in inoculum of arbuscular mycorrhizal fungi effectiveness of am symbioses, measured e.g. as fresh weight, number of flowers, survival under stress and so on normally is a continuous or metric character of a host plant. while genes individually associated with pronounced phenotypic effects (oligogenes) control the production of discontinuous, qualitative characters, polygenes individually have a small effect but are the basis of metric traits. oligogenes segregate clearly and are easily subject to mendelian analysis, polygenes are not. each character is always determined by a series of genes, and depends on allelic and non-allelic gene interactions (futuyma, 1998). for instance, physiologically induced effects are an important drivers of mycorrhizal response and colonization, in particular hormonal balance and respiration patterns, which concentrate response from both genomic and bio/abiostimuli effects (bedini et al., 2018; mercy et al., 2017). up to date, all attempts to link quantitative trait loci (qtl) with a given response pattern failed, because there are likely multifactorial components that regulate the symbiotic partners. here, we have clearly to keep in mind that we are studying the phenotype of the host to describe the action of fungal genotypes. the host ' zea mays cultivar multiplication cycle badischer landmais felix i 35 41 ii 30 38 iii 31 16 iv 36 9 exchange of host/inoculum v 20 25 vi 44 17 vii 36 3 fig. 3: mycorrhizal effectiveness [%] of claroideoglomus etunicatum on zea mays (varieties badischer landmais and felix) over seven years of subsequent inoculum production. (the inoculum was exchanged after the fourth year; derived from feldmann, 1997). 284 f. feldmann, c. schneider cation cycles c1/c2 and c2/c3. there was no correlation between spore effectiveness in c1 and c2 calculated from the slope (r=0.41), but a significant “heritability” of spore characteristics between c2 and c3 (r=0.80). these calculations were supported by the observation of distinct strain characteristics described above. finding no heritability for the trait between the first multiplication cycles might be due to the particular spore population studied having not yet sufficient genetic variation to show because of the small sample size (25 observations in c1 and 50 in c2). the next question to be answered was the clarification whether quantitative or qualitative interaction exist between host and functional amf genotypes. therefore, we repeated the experiment with a second host, the clonally multiplied host anagallis arvensis (data derived from feldmann and grotkass, 2002). in anagallis arvensis clones, the inoculation with single amf spores showed a variability of effectiveness from slightly effective to highly effective (c1). the multiplication of single spores from sub-strains with distinct effectiveness conserved the characteristics in the next propagation cycle (c2) indicating a purely quantitative interaction of functional amf genotypes with the “new” host. the variability of effectiveness increased after a further propagation cycle (c3) but followed the pattern of quantitative interactions (see more details in feldmann and grotkass, 2002). genetic homogeneity of plant material seems to “conserve“ strain characteristics better than selected seedlings. “heritability” of strain characteristics on anagallis clones were calculated 0.94 between c1/ c2, 0.86 between c2/c3 and 0.88 between c1/c3. going back to the p. crispum test system, we started to develop an inoculum with known characteristics. we produced populations derived from sub-strains with distinct characteristics in c3 and inoculated them as populations – not as single spores. would the host/amf interaction further allow increase of variation? alternatively, could a selective host maintain a certain level of effectiveness? surprisingly, plants, which were inoculated with a population of c3 containing negative and neutral effective origins exclusively expressed neutral or positive effective symbioses in multiplication cycle c4. in the following two cycles c5 and c6 this characteristic was maintained with the majority of measurements mainly in the standard reaction range (neutral effectiveness). a certain sub-strain with a broad range of effectiveness (from negative to positive, but mainly neutral) did also not express negative growth responses but positive and mainly neutral effective symbioses. another nearly completely positive effective sub-strain showed a trend to lower effectiveness in the sub sequent multiplication cycles c4-c6. in summary, it was obvious that the actually expressed effectiveness of the p. crispum/c. etunicatum symbiosis rapidly tended to result in neutral effective combinations, if the host was inoculated with spore populations of mixed sub-strains (feldmann, 1998b). environment/amf functional genotype interactions changing the host led to quantitative host/functional amf genotype interactions. changing the secondary environment for both partners modified the physiological reaction norm of the principal environment for the fungus. additionally, changed secondary environment might influence the strain characteristics directly. in order to understand more about the amf population dynamics, we made a simple test. we let anagallis arvensis grow together with a positively effective strain under different environmental conditions (ph of the substrate). the root colonization appeared low under low ph and the effectiveness in this treatment remained low. if we then multiplied spores produced under such conditions and inoculated again, we could achieve higher effectiveness under unfavourable conditions (more details see feldmann and grotkass, 2002). the experiment showed that selection of amf genotypes occurred under sub-optimal conditions possibly allowing only parts of the population to colonize the plant (qualitative functional amf geno type/environment interactions). furthermore, if this part was enhanced technologically, the inoculum could be optimized for use under sub-optimal conditions. this was the breakthrough for the design of the directed inoculum production (dip). the directed inoculum production as the consequence of the observations described above, we designed a very simple protocol for inoculum production (feldmann et al., 2009) integrating the population dynamical aspects with and without dominating environmental factors. the protocol recognizes that the relevant taxonomic units for specificity phenomenons are plant variety and fungal strain. we select only plants and plant cultivars for inoculum production showing high root colonization. as fungal partners, we use amf from in situ conserved natural sites with certain environmental characteristics (e.g. drought or salt influenced). they should be highly sporulating and competitive. this fungal material undergoes the following processing steps: · we develop strains from single spores, resulting in sub-strains, which might cause different effects with different effectiveness on the plants involved in the production process · we stress the plants during the first step of the production process (or longer) and select spores, which can reproduce and colonize under this pressure. doing so, we adapt the inoculum to certain environments. · we then mix inocula (sub-strains) with certain characteristics to populations of certain species or even communities of different species because the later target plants of customers are not pre-visible before inoculum production, we try to achieve a high functional amf genetic heterogeneity in the inoculum. this can be reached by mixing different inocula from different strains and/or by producing them on diverse plants separately. the idea is that the target plant should have the possibility to choose the partner according to their dependency. this dip allowed us a continuous inoculum production over 25 years. the ideas were followed scientifically: bedini et al. (2018) presented first results of the adaptation of amf as a tool to overcome phosphate inhibition in practical condition. successful adaptation to high inorganic phosphate pi would allow the use of am fungi in conventional agriculture field, providing to crops the beneficial effects induced by the symbiosis and reducing the need of chemicals. ' fig. 4: increase of variance of fresh weight of petroselinum crispum by mycorrhizal strains after three subsequent multiplication cycles (data derived from feldmann, 1998b). directed amf inoculum production 285 r. irregulare was propagated in parallel, for five generations, in pre sence or not of high pi concentrations (up to 3.25 mm) in root organ cultures. inoculation of potato with in vitro spores of r. irregulare exposed to high pi for one generation did not highlight any effect on plant growth and root colonization. 5th generation grown under high pi, instead, was associated with improved plant biomass and root colonization. suggesting that in this case, at least five generations are necessary for the adaptation. a very important example for strain adaptation was worked out by van bui and franken (2018). they found that amf in root organ cultures are able to gain heavy metal tolerance when they were grown in high heavy metal conditions. moreover, the zn-adapted strains could confer its higher heavy metal tolerance to plants. plants inoculated with zn-adapted strains showed higher biomasses than plants inoculated with a non-adapted strain. to our knowledge, this is the first research group outside of our own lab who reproduced the adaptation process with success following the dip protocol. we hope that more such attempts will result in elucidating more details about the underlying mechanisms of the adaptation process already in use for so much years without knowing much detail about the mode of action. discussion one important question needs to be discussed: what constitutes a “functional amf genotype”? we define it as “all genetic information of a single spore transmitt able over spore multiplication cycles” this genetic information is expressed in tied interaction with the principal environment (the host) under the influence of (secondary) environmental factors. this definition is the result of a long research process. three decades ago, inoculum producers ignored the fact that effectiveness of symbioses resulting from inoculum was very variable because of genetical differences between propagules. they assumed not to have the right strain for the right environment or the right plant species if the inoculum failed to achieve the desired effect. amf inoculum was thought to be genetically homogenous in a wide range because of the potentially exclusive asexual and clonal reproduction of spores (stukenbrock and rosendahl, 2005). the assumed genetic homogeneity of amf inoculum was the basis for all screening projects on amf strains (baltruschat, 1993). but the genetic homogeneity of an amf strain does not exist (summarised in clapp et al., 2001). twenty-five years ago we analysed the interrelationship between amf and host plants with quantitative genetical methods. a description of functional amf genotypes as described above does of course not describe the actual genetic differences on the dna level between strains but is focussed on active functional genes for specific interactions, including epigenetics, and transmitted characteristics of gene expressions. nevertheless, the chosen definition reflects genotypes as targets for eco-factor actions and, therefore, gives a strict orientation for the design of the mycorrhizal technology in practice without knowing the actual physiological and molecular mechanisms. over more than one decade biochemical and molecular methods have been developed to distinguish between amf species (e.g. simon et al., 1993), determine phylogenetic relationships (e.g. (redecker et al., 2000) and genetic heterogeneity (e.g. pringle et al., 2000) of amf. heterogeneity of genes within single spores of amf was described (e.g. sanders et al., 1995) by restriction fragment length polymorphism analysis (pcr-rflp) and sequencing studies and had also been reported for the 18s rrna genes (e.g. schüssler, 1999). other studies (pringle et al., 2000) indicated that the level of genetic diversity in amf rrna genes was very high. this was supported by studies of the whole genome using other molecular techniques including randomly amplified polymorphic dna (rapd, e.g. wyss and bonfante, 1993), m13-primed minisatellites (zeze et al., 1997), pcr-generated microsatellite loci (longato and bonfante, 1997) and amplified fragment length polymorphism (aflp, rosendahl and taylor, 1997). intraand intersporal diversity of its rdna sequences was assessed by cloning and sequencing, and by small subunit (ssu) rdna analysis (vandenkoornhuyse and leyval, 1998). different hypotheses existed about the organization of am fungal genomes (ropars and corradi, 2015). spores of amf can contain more than a thousand of nuclei, which are proposed to represent the mycorrhizal „individual“ (pringle et al., 2000). these nuclei can be genetically different from each other. nuclear exchange exists in some amf fungi through anastomosis (giovannetti et al., 1999). more recently, daubois et al. (2016) could show independent mito chondrial and nuclear exchanges through anastomoses of strains from different proveniences. furthermore, indications of recombination were found in glomus populations (vandenkoornhuyse et al., 2001), but were not confirmed by stukenbrock and rosendahl (2005). additionally, they pointed out that populations with mainly asexual reproduction are characterized by multilocus associations and identical genotypes composed of unique allels and proved this. testing this in natural amf populations they confirmed that all spore genotypes were unique and subdivision of genotypes existed at the experimental site within plots. the question of inter-nucleus recombinations was supported by chen et al. (2018), but auxier and bazzicalupo (2019) could not confirm their results. overall, it seems to be clear that genetically very different propagules in an inoculum meet the plant symbiont. but still in 2006, koch et al. had doubts that genetic differences would have an effect on the outcome of a symbiosis. in own experiments our group investigated with partners genetical differences between single spores on basis of the alternative oxidase (aox), which is an enzyme of the alternative respiratory chain already described in different taxa, including various fungi, which decreases the damage caused by oxidative stress. the analysis of riaox polymorphisms in single spores of three different isolates showed a reduced variability in one spore relatively to a group of spores. a high number of polymorphisms occurred in introns; nevertheless, some putative amino acid changes resulting from non-synonymous variants were found, offering a basis for selective pressure to occur within the populations. given the aox relatedness with stress responses, differences in gene variants amongst r. irregularis isolates are likely to be related with its origin and environmental constraints. (campos et al., 2015). we think, that “functional amf genotypes” are constituted by more or less genetically heterogenous nuclei assemblages in all progagules involved in the formation of the symbiosis, including the coenocy tium of arbuscular mycorrhizal fungi in and outside of host roots. it probably depends on the quantity of certain genetically defined nuc lei and their activity pattern how the development of the symbiosis starts and continues. a selection of nuclei or activation of nuclei by plant and environment may be the basis for adaptation processes to plant properties (e.g. dependency) or environmental factors utilized in the directed inoculum production process. van bui and franken (2018) state that differential gene expression could be based on the selective accumulation of particular nuclei during the adaptation (what they call “acclimatization process”). the consequence would be the expression of different alleles from one locus in the adapted and not adapted strains (ehinger et al., 2012). further sequencing of transcripts and genomes of individual nuclei must clarify, if differential expression of the same allele or the occurrence of different genomes accompanies variations in adapted strains. unfortunately, attempts to determine whether sequence diversity found in single spores originates in polymorphic genes within single nuclei or within different nuclei had long time not been successful (e.g. hijri et al., 1999). in any case it became clear that genetic exchange and altered mycorrhiza-specific gene transcription occurs (colard et al., 2011). 286 f. feldmann, c. schneider currently, indications rise that genetic variation of the symbionts regulate the physiological interaction of microand macrosymbiont (savary et al., 2020). the group found a clear link between mycorrhizal fungal genetic variation and plant molecular reprogramming that reflect the evolutionary history of closely related amf involving the production and transport of one essential currency that is fundamental to the symbiosis. while previous molecular studies have qua litatively demonstrated the switching-on of this important pathway in symbiosis, the study shows that in order to understand the regulation of the currency involved in this important mutualism, incorporate fungal genetic variation rather than using more simple experimental designs is necessary. furthermore, dual rna-seq revealed largescale non-conserved genotype × genotype-specific genetic reprograming and molecular crosstalk in the symbiosis (mateus et al., 2020). outlook for a producer of amf inoculum, dip was a decisive step forward in enhancing the predictability of the inoculum effectiveness. in fig. 5 the most important factors influencing mycorrhizal effectiveness are summarized. the viewpoint is strictly host oriented, because it is the effectiveness on the host we want to achieve. the genotypes of host and fungus form the mycorrhizal host phenotype under the influence of concurrent environmental conditions. the mycorrhizal host phenotype in relation to non-mycorrhizal host plants reflects the mycorrhizal effectiveness (“dependency”) with regard to the evaluated effect. roughly summarized, variability of environmental factors causes variability of effectiveness via qualitative and quantitative changes of inoculum characteristics, root cha racteristics or root colonization rates. mycorrhizal dependency results from the host’s genetically and epigenetically fixed characteristics under given environmental conditions, which define the physiological status. this includes e.g. cultivar specific pattern of growth and development, the tolerance and resistance metabolism, developmental state and root morphology. knowledge of plant’s growth, therefore, allows exact application time for inoculations with amf. at the same time, the growth of amf and the production of propagules is well known even in commercial scale (vosatka et al., 2012). overall, there is no real problem to bring both partners together to colonize the host’s roots and to form efficient symbioses. for the amf, the micro-symbiont, there are two quantitative aspects of major importance: the inoculum potential, i.e. the number of pro pagules of amf inoculum, and the amf community and population composition. these two parameters are influenced during technical inoculum production. the other parameters cited in fig. 5 are processing factors, which guarantee the desired coincidence of the right developmental stage of roots/plants and infective micro-symbionts. against this background, high quality inoculum has to provide sufficient fungal material with characteristics realising desired effects with commercially reasonable effectiveness. because the plant cultivar is defined by the target production system, the question returns, which fungus should be used. at this point, dip is a process to adapt the fungal material to later, anticipated environmental conditions. the next step is definitely much more complicated: to develop an inoculum with desired characteristics for a number of plants and target environments. on the one hand, the key lies in the inoculum production process. we need to find plants, which are not selecting amf genotypes but multiply all. we need ideas how to adapt inocula to various environments at the same time. the reason are simply the costs of the inoculum. it is mandatory to reduce the developmental costs of inoculum to be able to inoculate more propagules to a given target scenario. this would favour adapted amf in competition situations. still, another question is unanswered: should we breed crops for mycorrhizal symbiosis (feldmann et al., 2013)? the future might be a completely different inoculum: ecologically optimised inoculum for large-scale use in cash crops, which are genetically adapted for use of this important symbiosis. on the other hand, we have to learn much more about “amf population engineering”. this is mainly directed to understand a quantitative aspect of inoculum application. changing the quote of different isolates, strains or functional genotypes within a commercial inoculum should result in guaranteed establishment of the elaborated strains under practical conditions. furthermore, it has a qualitative aspect by influencing the functional potential of strains by environmental adaptation processes. such qualitative population engineering will result in the option that the fundamental ecological niche of both, host and amf, is utilized to create a widened actual ecological niche of the symbiosis resulting in advantages for the host to be explored. ' fig. 5: mycorrhizal effectiveness as phenotype of host/amf interactions and population biological measures (after feldmann, 1998a). directed amf inoculum production 287 we worked now 25 years on engineering functional amf genotypes in arbuscular mycorrhizal inoculum in order to get a good product for use in agriculture and horticulture. our long way started with the simple question of late prof. dr. reinhard lieberei who asked: “why shouldn’t you be able to find out how to use them?” we are thankful for this encouraging initiating push. conflict of interest no potential conflict of interest was reported by the authors. references allen, m., 1992: mycorrhizal functioning. an integrative plant-fungal process, new york, ny, chapman and hall. alten, h. von, blal, b., dodd, j.c., feldmann, f., vosatka, m., 2002: quality control of arbuscular mycorrhizal fungi inoculum in europe. in: gianinazzi, h., schuepp, h., barea, j.m., haselwandter, k. 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m13 minisatellite-primed pcr. appl. environ. microb. 63, 676-678. doi: 10.1128/aem.63.2.676-678.1997 orcid carolin schneider https://orcid.org/0000-0001-8936-2793 adresses of the authors: falko feldmann, julius kühn-institut, messeweg 11-12, 38104 braunschweig, germany e-mail: falko.feldmann@julius-kuehn.de carolin schneider, inoq gmbh, solkau 2, 29465 schnega, germany e-mail: schneider@inoq.de © the author(s) 2020. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1002/ciuz.201000533 http://dx.doi.org/10.1006/fgbi.1998.1112 http://dx.doi.org/10.1111/j.1461-0248.2005.00853.x http://dx.doi.org/10.1007/s00572-004-0307-4 http://dx.doi.org/10.1016/0167-8809(90)90283-j http://dx.doi.org/10.1017/s0953756296002766 http://dx.doi.org/10.1038/s41396-020-0694-3 http://dx.doi.org/10.3389/fpls.2017.00417 http://dx.doi.org/10.1016/s0065-2504(08)60182-8 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proanthocyanidins with antinutritional and antimicrobial properties from the forage legume acaciella angustissima alexandra h. smith1, fayez e. kandil2, david s. seigler2, roderick i. mackie1 (received may 13, 2011) summary the proanthocyanidins of acaciella angustissima (mill.) britton & rose foliage have antinutritional and antimicrobial effects as proanthocyanidin-containing extracts reduced intake and weight gain of weanling rats when added to conventional diets and caused changes in fecal bacterial diversity and metabolic activity. purifi ed fractions of a. angustissima were shown to be more inhibitory to rat fecal bacteria than schinopsis spp. (quebracho) proanthocyanidin extracts. in this study, it was determined that a. angustissima proanthocyanidins consist largely of 5-deoxyfl avan3-ols and proanthocyanidin dimers through hexamers, based on guibourtinidol, fi setinidol, and robinetinidol, with a predominance of fi setinidol units. these are accompanied by smaller amounts of higher oligomers (heptamers-decamers) and deoxyfl avan-3-ols, dimers and trimers containing a p-hydroxybenzoate ester moiety. introduction many parts of the world have large tracts of arid and semi-arid lands that cannot be cultivated, but which can be used to raise livestock, mainly cattle, sheep and goats. it has often been suggested that leguminous, multi-purpose trees could be useful as livestock fodder in these regions (osuji et al., 1995); however, secondary metabolites found in these potentially useful plants can be toxic to animals or cause a reduction in their productivity by reducing feed intake (barry and blaney, 1987). in many cases, the antinutritional compounds have not been identifi ed and little is known about their specifi c effects on ruminant metabolism. one such leguminous shrub/tree acaciella angustissima (mill.) britton & rose [syn. acacia angustissima (mill.) kuntze] has great agronomic and nutritional potential, but is limited by the presence of antinutritional compounds (smith et al., 2001, 2003). proanthocyanidins from several species of the genus acacia sensu lato and the segregate genus acaciella have previously been examined. best known are those from acacia mearnsii, wattle or mimosa, widely cultivated in south africa and brazil for industrial tannin production (roux, 1972; roux et al., 1980; seigler, 2003). in that instance, the condensed tannins are largely based on 5-deoxyfl avan3-ol units. the proanthocyanidins of another commercial tannin source, quebracho (schinopsis spp., anacardiaceae) are similar, but typically possess opposite confi guration at positions 2 and 3 of the chain extending units to those from wattle. in some instances, 5-deoxyfl avan-3-ols occur in combination with phloroglucinoltype a-rings (for example, catechin and epicatechin as in acacia baileyana) (foo, 1984). in a similar manner, the “upper” units of (4→8)and (4→6)-linked prorobinetinidins from the stem bark of stryphnodendron adstringens (fabaceae: mimosoideae) all are based on (-)-robinetinidol, but the lower or terminating units are based on (+)-gallocatechin and (-)-epigallocatechin (de mello et al., 1996). proanthocyanidins based entirely on guibourtinidol/ epiguibourtinidol (258 da), fi setinidol/epifi setinidol (274 da), and robinetinidol/ epirobinetinidol (290 da) (fig. 1) also have previously been reported from legumes. fisetinidinol and robinetidinol occur both as initiating or extender units and as terminal units (de mello et al., 1996; malan et al., 1988; mathisen et al., 2002). (-)-fisetinidol(4β→6)-(-)-robinetinidol has been reported from the heartwood of the wild seringa, burkea africana (fabaceae: caesalpinioideae) (malan et al., 1988). both (2r,3s)and (2s,3r)-absolute confi gurations for profi setinidins are known and, although almost all in legumes are (2r,3s)-, several instances with opposing confi guration have now been documented (ferreira et al., 2000). nonetheless, profi setinidins based on units such as (+)-(2s,3r)-fi setinidol, (+)-(2s,3r)-robinetinidol and (2s,3r)-guibourtinidol] from legumes are rare (ferreira et al., 2000; nel et al., 1999; steynberg et al., 1997). 2,3-cis-5-deoxy compounds are even more uncommon. (2r,3r)-2,3-cis-epifi setinidol chain extender units are known only from the bark of guamúchil, pithecellobium dulce (fabaceae: mimosoideae), where they make up the major type of 5-deoxyfl avan-3-ol present (steynberg et al., 1997). acylated proanthocyanidins also have been reported from a number of legumes. most of these involve gallic acid, typically attached as an ester at c-3 of one or both of the ring systems of the parent compound. for example, (-)-fi setinidol (4α→8)-(+)-catechin 3o-galloyl ester has been reported from the heartwood of burkea africana (fabaceae: caesalpinioideae) (malan et al., 1988). in addition, several of the prorobinetinidins of stryphnodendron adstringens (fabaceae: mimosoideae) occur as galloyl esters (de mello et al., 1996). in synthetic studies with (+)-mollisacacidin and (-)-fi setinidol, the principal dimeric products were (4α→6)and (4β→6)-linked bisprofi setinidins (steenkamp et al., 1983). the conspicuous absence of (4→8)-coupled analogs among this class of oligofl avanoids contrasts with the involvement of both c-6 and c-8 in interfl avanyl bonding that occurs when the a-rings of the terminal or “lower” groups are of the phloroglucinol type (foo, 1984). however, it should be noted that a series of 4α→6, 4β→6 and 4α→8 (-)-fi setinidol and (+)-entepifi setinidols were isolated in a later study from the heartwood of mopane, colophospermum mopane (fabaceae: caesalpinioideae) (steenkamp et al., 1988). the small amounts of (4→8)-linkedprofi setinidins and proguibourtinidins in that instance were based on (-)-fi setinidol, (+)-ent-epifi setinidol, and (-)-guibourtinidol (malan et al., 1990a, b). most proanthocyanidins, p-hydroxybenzoic acid, and p-hydroxybenzoic acid esters possess antioxidant activity. however, because most proanthocyanidins have the ability to bind to protein, nucleic acids and carbohydrates to various degrees, they are at least weakly toxic in animal systems. they are known to inhibit some enzymes and not others (zucker, 1983). in other instances, they exhibit more specifi c and well-defi ned effects. this may be related to stereospecifi c binding processes and in the case of larger (4→8)-linked oligomers, may involve helical forms of the proanthocyanidins (haslam, 1979). there is compelling evidence that proanthocyanidins have quite selective interactions with proteins and, in particular, with known receptors (zucker, 1983; zhu et al., 1997). certain proanthocyanidins arrest the cell cycle proanthocyanidins of acaciella angustissima 143 in the g0/g1 phase (kozikowski et al., 2003). proanthocyanidins also have been shown to possess antimicrobial properties, possibly because of binding to proteins, interactions with membranes, and by deprivation of metal ions (scalbert, 1991; smith et al., 2005). p-hydroxybenzoic acid is widespread in nature. esters of this compound (parabens) are commonly used as food additives and as common ingredients in cosmetics such as underarm deodorants to inhibit bacterial growth. this series of compounds is clearly antibiotic, but also has been shown to have estrogenic activity in mice and to appear in human breast cancer cells (darbre et al., 2003; lemini et al., 1997). p-hydroxybenzoic acid also has been demonstrated to affect plant-water balance and has allelopathic activity (barkosky and einhellig, 2003). plants of acaciella angustissima (miller) britton and rose, can produce biomass yields as high as 5 tons per hectare per year. further, the nitrogen content of the leaves of this plant is about 3%, i.e., almost 19% crude protein (ahn et al., 1989). results of a study comparing supplementation of sheep maize stover diets with various legume forages indicated that a. angustissima may be a suitable alternative to leucaena leucocephala, an established high-quality tree forage, that is not appropriate, however, for all agroecological conditions (masama et al., 1997). despite these outstanding features, air-dried a. angustissima plant material has been shown to be highly toxic to ethiopian highland sheep at levels higher than 50 grams per day when fed without adaptation. symptoms in the sheep suggested that the antinutritional factor(s) is a neurotoxin and that the cardiovascular system also is targeted (odenyo et al., 1997). further evidence of toxicity involves suppression of fermentation by mixed rumen organisms in vitro (osuji and odenyo, 1997) and suppression of growth of pure bacterial cultures by aqueous extracts of a. angustissima (osuji and odenyo, 1997; el hassan et al., 1995). samples of a. angustissima plant material contain 6.5% proanthocyanidin according to the vanillin-hcl method, but no tannins were detectable using the butanol-hcl method (ahn et al., 1989). these workers speculated that catechin gallates, which react positively in the vanillin-hcl method, but not the butanolhcl method, are present in a. angustissima. however, because the proanthocyanidins of woody legumes often are based on 5deoxyfl avan-3-ols and others are acylated with other than galloyl units, it seems probable that these tannins are insensitive to the butanol-hcl reaction for other reasons. gravimetric determination of soluble phenolics using trivalent ytterbium allowed other investigators to estimate the tannin content of a. angustissima (ilri accession no. 15132, the same accession as used in this study), to be between 21 and 24% (odenyo et al., 1997). in our laboratories, the tannin content of four a. angustissima accessions from texas and mexico ranged from 1.9 to 14.7% as determined by casein precipitation; proanthocyanidins (vanillin-hcl method) varied from 2-11.0% and hydrolysable tannins from 1.4-25.4% (iodate method) (readel et al., 2001). the plant material used in this study was found to contain 17.8% total phenolics (folin-denis method) and 17.4% proanthocyanidins (vanillin-hcl method). gallic acid, used as an indication of hydrolysable tannin after acid hydrolysis, was below detectable limits using the rhodanine assay (smith et al., 2001). hammer and cole (1965) found the proanthocyanidins of a. angustissima to be based primarily on fi setinidinol as evidenced by conversion to fi setinidin. they also reported the presence of the corresponding leucoanthocyanidin, 7,3’,4’-trihydroxyfl avan-3,4diol. although the most obvious potentially toxic secondary metabolites identifi ed from a. angustissima are non-protein amino acids (seigler, 2003), based on the results of previous studies summarized in tab. 1 (smith et al., 2001, 2003; smith and mackie, 2004), the deleterious substances appear to be proanthocyanidins. the ethyl acetate soluble portion from partition of lyophilized 70% acetone extract from milled air-dried foliage of a. angustissima between ethyl acetate and water (aa5) was previously used in a rat bioassay developed to evaluate palatability and toxicity and to determine the identity of the antinutritional factor(s) contained in the extract (smith et al., 2001). the major components involved in the antinutritional effects of this extract in rat diets were phenolics (smith et al., 2003), in particular, proanthocyanidins. hydrolysable tannins were not detected. inhibition of intake and weight gain with diets containing these phenolic substances was reversed by polyethylene glycol, a substance that complexes with proanthocyanidins (smith et al., 2003; makkar et al., 1995). tannin resistant bacteria have been isolated from gastrointestinal tab. 1: summary of biological effects of acaciella angustissima proanthocyanidin-containing extracts. a. angustissima extract major biological effects reference 70% acetone 2.3% proanthocyanidins in rat diets resulted in intake and average daily gain (smith et al., 2001) (similar to aa1) reduction (37% and 94% respectively). increases in fecal nitrogen excretion and fecal proline, glycine and glutamic acid were most likely due to production of tannin-binding salivary gland proteins. ethyl acetate extract 0.7% proanthocyanidins in rat diets resulted in intake and average daily gain (smith et al., 2001) (similar to aa5) reduction (20% and 42% respectively). increases in fecal nitrogen excretion and fecal proline, glycine and glutamic acid were detected. ethyl acetate extract the negative effects of 4.2% proanthocyanidins in rat diets were neutralized (smith et al., 2003) (similar to aa5) by polyphenolic-binding polyethylene glycol. ethyl acetate extract the proportion of rat fecal tannin-resistant bacteria increased signifi cantly (smith and mackie, 2004) (aa5) from 0.3% to 25.3% and 47.2% with 0.7% and 2% proanthocyanidin-containing diets respectively. predominant bacteria shifted towards tannin-resistant enterobacteriaceae and bacterioides species with a corresponding decrease in the clostridium leptum group. sephadex lh-20 extract the a. angustissima extract was more inhibitory to the growth of bacteria than (smith et al., 2005) (aa1.2) a quebracho extract with a similar concentration of phenolics. 144 a.h. smith, f.e. kandil, d.s. seigler, r.i. mackie tract ecosystems where they are thought to prevent detrimental effects of dietary tannins on the animal. the mechanism of action of the protective effect of tannin-resistant bacteria in animals exposed to proanthocyanidins is unknown, but is likely to involve shifts in bacterial populations and modifi cations to their metabolic activity. after feeding a. angustissima extracts for three weeks, there was a signifi cant shift in the proportion of tannin-resistant bacteria in the feces of rats (smith and mackie, 2004). 16s rdna sequence analyses indicated that tannins selected for gram-negative enterobacteriaceae and bacteriodes species, whereas there was a corresponding decrease in low gc gram-positive groups. the present work was done to determine the structures and the antimicrobial effects of the proanthocyanidins present in foliage of the legume a. angustissima. materials and methods general all solvents were from fisher scientifi c (pittsburgh, pa.) and were reagent grade or better. tlc analyses were carried out on silica gel tlc plates (sigma chemical co., st. louis, mo, silica gel 60 f 254 on aluminum, 0.20 mm) and developed with ethyl acetate:methanol: water (79:11:10) or on cellulose plates (fisher scientifi c, fairlawn, nj, avicel, 0.20 mm) with 2% acetic acid unless otherwise stated. proanthocyanidins were visualized on tlc plates with vanillin-hcl reagent followed by brief heating at 110 c (pink spots). measurement of total phenolics in plant extracts was performed using the folin-denis method (readel et al., 2001; seigler et al., 1986). quantifi cation was based on a standard curve generated with tannic acid (fisher scientifi c co., a-310, fairlawn, nj). in this instance, the vanillin reaction (price et al., 1978) was used to determine the proanthocyanidin concentration of plant extracts, but purifi ed a. angustissima tannin, obtained by absorption of tannins to sephadex lh-20 and elution with 50% acetone (strumeyer and malin, 1975), was used to prepare the standard curve. appropriate amounts of extract were dissolved in methanol. sample and reagent volumes were reduced so reactions could be performed in eppendorf tubes. mass spectrometry mass spectra were determined by fast atom bombardment (fab) on a micromass zab-se spectrometer and by matrix-assisted laser desorption/ionization (maldi) on a micromass tofspec spectrometer in the mass spectrometry laboratory of the school of chemical sciences, university of illinois, urbana, il. fab measurements were obtained in negative ion or positive ion modes. es-ms analyses were made on an lcq deca xp mass spectrometer, thermo finnigan corp., san jose, ca.) nmr spectrometry nmr spectra were recorded on a varian 400 spectrometer operating at 400 mhz for 1h-spectra and 100 mhz for 13c-spectra in the nuclear magnetic resonance laboratory of the school of chemical sciences, university of illinois, urbana, ill. extraction of proanthocyanidin-containing fractions from acaciella angustissima milled air-dried foliage from a. angustissima (ilri accession #15132, 1 mm screen), grown in the ethiopian highlands was obtained from the international livestock research institute (ilri), debre zeit, ethiopia. the extraction procedures can be visualized in fig. 1-2. isolation of purifi ed proanthocyanidin extracts aa1 and aa1.2 (fig. 1) the plant material (100 g) was immersed six times in 70% acetone at room temperature for 1-2 hours and subsequently fi ltered (whatman 4 paper). the extracts were combined and acetone removed under vacuum. material that was insoluble in the remaining aqueous portion of the extract was removed by fi ltration (whatman 4 paper) and the fi ltrate lyophilized to yield a blackish-brown amorphous substance (26.5 g, 26.5% yield). the tlc on silica gel gave a series of poorly resolved spots ranging between rf 0.8-0.1 when visualized f 0.8-0.1 when visualized f with vanillin-hcl reagent. in order to prepare a purifi ed proanthocyanidin fraction, lyophilized 70% acetone extract (1 g) dissolved in ethanol was added to a column of sephadex lh-20 (25 g) packed from a slurry in 80% ethanol (100 ml) (asquith and butler, 1985). the column was washed with 95% ethanol until no further material was eluted as judged by tlc, followed by washing with 50% aqueous acetone until elution of material ceased. the combined acetone washes were evaporated and extracted with an equal volume of etoac to obtain a solid sample (aa1.2) that consisted of 97% phenolics, as determined by the folin-denis method. tlc analysis of this fraction indicated the presence of three spots (rf 0.76, 0.81 and 0.83). two compounds that were visible, but that did not react with vanillin-hcl also were present (rf 0.47, and 0.54). six spots were visible on cellulose tlc f 0.47, and 0.54). six spots were visible on cellulose tlc f plates (rf 0.02, 0.07, 0.15, 0.47, 0.60 and 0.73). the three spots with f 0.02, 0.07, 0.15, 0.47, 0.60 and 0.73). the three spots with f lowest rf values reacted with this reagent.f values reacted with this reagent.f nmr spectra. 1h-nmr δ (acetone-d6d6d ) 7.4-8.4 (broad multiplet), 6.2-6.8 (broad multiplet), 6.0-6.15 (multiplet), 5.1-5.6 (multiplet), 4.4-5.0 (multiplet), 4.1 (broad singlet), 4.0 (broad singlet), 3.8 (broad singlet), 3.7 (broad singlet), 3.5 (singlet), 3.38 (broad singlet, h2o), 3.1 (singlet), 3.05 (singlet, h2o), 2.4-2.8 (broad multiplet), 1.9-2.4 (multiplets), 2.0-2.1 (acetone methyl group), 1.3 (singlet), 1.25 (singlet). 13c-nmr δ (acetone-d6d6d ) 206.870, 206.673 (carbonyl groups), 156.880 (1), 152.316 (1) 145.729 (4), 133.352 (1), 132.329 (1), 128.761 (1), 118.192 (<1), 115.298 (1), 108.844 (1), 105.742 (3), 105.649 (3), 104.004 (1), 103.584 (1), 79.982 (1), 78.622 (1), 78.363 (1), 78.105 (1), 76.124 (1), 75.515 (<1), 73.185 (<1), 49.354 fig. 1: flow chart of solvent extraction of acaciella angustissima foliage resulting in proanthocyanidin-enriched fractions aa1 and aa1.2. details of solvent extractions are in the text under materials and methods. proanthocyanidins of acaciella angustissima 145 (1), 35.910 (1), 30.384 29.223 (methyl groups of acetone), 19.491 (1), 14.326 (1). approximate carbon integrals estimated from the spectrum. mass spectrum. fab-ms (positive ion spectrum) (m/z) 329 (50), 361.1 (66), 393.1 (55) [c22h16o7+h]+, 411.1 (72%) [c22h18o8+h]+, 447.1 (20), 547.1 (29) [c30h26o10+h]+, 563.2 (46) [c30h26o11+h]+, 667.2 (34) [c37h30o12+h]+, 683.1 (100) [c37h30o13+h]+, 699.2 (28), [c37h30o14+h]+, 818.0 (20) [c45h38o15+h]+, 834.3 (46) [c45h38o16+h]+, 857.2 (14) [c45h38o16+na]+, and 874.3 (12) [c45h38o17+na]+, 939 (12) [c52h42o17+h]+, 955.3 (20) [c52h42o18+h]+, 971.3 (8) [c52h42o19+h]+, 1107.4 (5) [c60h50o21+h]+. maldi-ms (m/z) 563.22 (80% of base peak) [c30h26o11+h]+, 583.22 (90) [c30h26o11+na]+, 683.31 (97) [c37h30o13+h]+, 699.32 (78) [c37h30o14+h]+, 857.37 (100) [c45h38o16+na]+, 873.35 (96) [c45h38o17+na]+, 955.3 (83) [c52h42o18+h]+, 978.26 (53) [c52h42o18+na]+, 1114.43 (82) [c60h50o20+na]+, 1130.35 (94) [c60h50o21+na]+, 1146.38 (85) [c60h50o22+na]+, 1266.80 (40) [c67h54o24+na]+, 1386.66 (50) [c75h62o25+na]+, 1402.74 (74) [c75h62o26+na]+, 1419.19 (63) [c75h62o27+na]+, 1674.13 (43) [c90h74o31+na]+, 1691.27 (46) [c90h74o32+na]+, and smaller series of peaks corresponding to heptamers, octamers, nonamers, and decamers. ethyl acetate soluble materials (aa5) from partition of lyophilized 70% acetone axtract with water (fig. 2) an acetone soluble fraction was derived by extraction of dried foliage (5 kg) from a. angustissima (ilri accession #15132, 1 mm screen) in 1 kg batches with 70% acetone (smith et al., 2003). acetone was removed and the resulting material lyophilized. analysis by folin-denis reaction indicated that the lyophilized extract consisted of 60% proanthocyanidins. this solid residue was dissolved in the minimum amount of ethyl acetate needed to effect solubility and partitioned with an equal volume of water. ethyl acetate was removed from the organic phase and the remaining aqueous material lyophilized to yield an amorphous solid (720 g, 14.4% yield). tlc analysis of this fraction (aa5) on silica gel revealed the presence of two proanthocyanidin-rich spots (rf 0.84 and 0.81). three additional spots (rf 0.58, 0.54, and 0.51) were visible, but did not react with f 0.58, 0.54, and 0.51) were visible, but did not react with f the vanillin-hcl reagent. analysis on cellulose plates indicated the presence of at least six compounds (rf values: 0.8, 0.46, 0.13, 0.4, f values: 0.8, 0.46, 0.13, 0.4, f 0.06, and 0.03). mass spectrum. fab-ms (negative ion spectrum) (m/z) 529.1 (12) [c30h26o9-h]-, 545.1 (29) [c30h26o10-h]-, 561.2 (82) [c30h26o11h]-, 575.1 (100%) [c31h28o11-h]-, 591.2 (17) [c31h28o12-h]-, 607.2 (42) [c30h24o14-h]-, 785.2 (4) [c45h38o13-h]-, 801.2 (10) [c45h38o14-h]-, 817.2 (17) [c45h38o15-h]-, 833.2 (50) [c45h38o16h]-, and 849.2 (2) [c30h26o17-h]-, 1089 (2) [c60h50o20-h]and 1105.4 (4) [c60h50o21-h]-. vacuum column chromatography (vlc). a portion of the ethyl acetate soluble material from partition with water (aa5) (1 g) was dissolved in ethyl acetate and mixed with silica gel (5 g, merck, rahway, nj, gf-254 type 60). the ethyl acetate was permitted to evaporate and the silica gel mixture placed onto a vacuum column containing silica gel (60 g) that had been fl ushed with petroleum ether (50 ml). thirty fractions were collected: 1 (petroleum ether), 2 (20 % etoac in petroleum ether), 3 (40 %), 4 (60 %), 5 (80 %), 6 (etoac), 7 (1% of 1:1 meoh:h2o in etoac), 8 (2 %), 9 (3 %), 10 (4 %), 11 (5 %), 12 (6 %), 13 (7 %), 14 (8 %), 15 (9 %), 16 (10 %), 17 (12 %), 18 (14 %), 19 (16 %), 20 (18 %), 21 (20 %), 22 (25 %), 23 (30 %), 24 (35 %), 25 (40 %), 26 (45%), 27 (50 %), 28, 29, and 30 (100 % meoh-h2o, 1:1). fractions 1-6 were 50 ml, all others 100 ml. after removal of solvents under vacuum, the fractions were analyzed by tlc (silica gel, vanillin-hcl reagent). fractions 1-6 contained only traces. tlc of fractions 7 and 8 revealed the presence of two proanthocyanidin-containing spots (rf 0.65 and f 0.65 and f 0.75) in each instance, fraction 9 also had two spots (rf 0.53 and f 0.53 and f 0.65); fractions 7-9 were kept separate. fractions 10-13 had one proanthocyanidin (rf 0.75), as well as other spots that did not react f 0.75), as well as other spots that did not react f with vanillin-hcl reagent (rf 0.35, 0.42 and 047), fractions 16-17 f 0.35, 0.42 and 047), fractions 16-17 f had one vanillin-hcl positive spot (rf 0.24), as did fractions 23-25 f 0.24), as did fractions 23-25 f (rf 0.05). based on tlc, fractions 10-11, 12-13, 14-15, 16-17, 1822, 23-25, and 26-30 were combined. mass spectrum of fraction 7 from vlc (aa5.1.7). fab-ms (positive ion spectrum) (m/z) 309.0 (magic bullet), 329.0 (70), 345.0 (36), 371.0 (54), 387.0 (52), 395.1 (48) [c22h18o7+h]+, 411.1 (100) [c22h18o8+h]+, 427.0 (30) m-152, reverse diels-alder fragment, 443 (36), 530.1 (18) [c30h26o9+h]+, 547.1 (41) [c30h26o10+h]+, 563.1 (76) [c30h26o11+h]+, 651.1 (12) [c37h30o11+h]+, 667.2 (22) [c37h30o12+h]+, 683.1 (32) [c37h30o13+h]+, 699.1 (12) [c37h30o14+h]+, 803.1 (7) [c45h38o14+h]+, 819.2 (12) [c45h38o15+h]+, and 835.1 (7) [c45h38o16+h]+. nmr spectrum of fraction 7 from vlc (aa5.1.7). 1h-nmr δ (acetone-d6d6d ) 7.4-8.4 (broad multiplet); 6.2-7 (multiplet), 5.9-6.1 (multiplet), 5.2-5.4 (multiplet), 4.4-5.1 (multiplet), 3.0-3.5 (broad singlet, h2o), 2.4-2.9 (multiplet), 1.8-2.4 (multiplet, includes acetone peak), 1.3-1.4 (broad singlet), 1.2 (singlet). 13c-nmr δ (acetone-d6d6d ) 206.552 (acetone carbonyl), 157.505 (1), other smaller peaks 153-159, 146.352 (6), 134.108 (2), 132.962 (2), 129.472 (2), 115.771 (3), 109.277 (2), 108.556 (2), 105.999 (4), 105.787 (4?), 105.476 (4?), 104.019 (2), 103.579 (2), 79.545 (2), 77.406 (2), 76.055 (2), 73.409 (1), 30.377-29.216 (acetone methyl groups), 20. the number of carbon atoms is estimated from the spectrum. fig. 2:fig. 2: flow chart of solvent extraction of flow chart of solvent extraction of acaciella angustissimaacaciella angustissima foliage foliage resulting in ethyl acetate extracted sample aa5 and further proanthocyanidin-enriched samples. details of solvent extractions are in the text under materials and methods. 146 a.h. smith, f.e. kandil, d.s. seigler, r.i. mackie fractionation of the ethyl acetate soluble material from partition with water (aa5) on sephadex lh-20 (aa5.2.1-12) (fig. 3). a purifi ed fraction of a. angustissima proanthocyanidins was obtained by chromatography of the ethyl acetate soluble material from partition with water (aa5) on sephadex lh-20 (hagerman, accessed 2011). a sample of this substance (1 g) was dissolved in 95% ethanol and applied to a sephadex lh-20 column (50 g) (asquith and butler, 1985; hagerman, accessed 2011). the column was washed with 95% ethanol (fraction 1) until material was no longer eluted as indicated by tlc. fractions 2-7 were then eluted with 80% ethanol, fraction 8 with 60% ethanol, and fractions 9-12 with a mixture of acetone, ethanol and water (2:1:1). the column was fi nally washed with acetone (fraction 13) until no additional material was eluted. fraction 6 from this separation contained primarily one proanthocyanidin-rich spot (rf 0.67). based on tlc results, fractions f 0.67). based on tlc results, fractions f 6-8 were combined and used for further analyses (320 mg). this sample (aa5.2.6-8) consisted of 72% condensed tannins by the vanillin-hcl assay. nmr spectra. 1h-nmr δ (dmso-d6d6d ) 7.4-9.6 (broad multiplet), 5.8-7.0 (multiplets), 5.2-5.4 (multiplet), 4.2-5.0 (multiplet), 3.66 (broad singlet, h2o), 3.459 (singlet), 3.445 (singlet), 3.431 (multiplet), 2.5-3.0 (broad multiplet), 2.471 (singlet), 2.096 (singlet), 1.6-2.05 (broad multiplet), 1.131 (singlet). 13c-nmr δ (acetone-d6d6d ) 206.954 (carbonyl groups of acetone), 160.645 (<1), 158.172 (2), 157.435 (1), 153.522, 146.245 (4), 134.168 (1), 132.856 (1), 129.305 (1), 123.107 (<2), 115.786 (1), 109.262 (1), 108.519, 106.068 (3), 105.764 (3), 103.974 (1), 79.538 (1), 76.101, 57.673, 36.446 (1), 30.377-29.223 (methyl groups of acetone). number of protons estimated by peak height from spectrum. mass spectrum. fab-ms (positive ion spectrum) (m/z) 563.1 (20% of base peak) [c30h26o11+h]+, 573.1 (12), 665.1 (25) [c37h28o12+h]+, 683.2 (100) [c37h30o13+h]+, 699.2 (28) [c37h30o15+h]+, 817.3 (14) [c45h38o15+h]+, 835.3 (50) [c45h38o16+h]+, 857.2 (40) [c45h38o16+na]+, 874 (22) [c45h38o17+na]+, 937.2 (10) [c52h42o17+h]+, 955.2 (18) [c52h42o18+h]+, 971.3 (4) [c52h42o20+h]+, 1129 (4) [c60h50o21+na]+. results and discussion based on previous studies, it was known that sizable quantities (6-25%) of proanthocyanidins are found in forage material of a. angustissima (smith et al., 2001, 2003; ahn et al., 1989; odenyo et al., 1997; readel et al., 2001). it has been established that these compounds are toxic to livestock and have antibacterial activity (smith and mackie, 2004) (tab. 1). however, the composition of these proanthocyanidins has not been described previously. extracts of a. angustissima and their purifi cation in order to evaluate the relative effectiveness of various chromatographic methods, lyophilized 70% acetone extracts of a. angustissima were fractionated in several ways. none of these extraction and chromatographic methods was completely effective in resolving the mixtures of proanthocyanidins from a. angustissima. however, prior silica gel or sephadex lh-20 fractionation was important for carrying out subsequent mass spectrometric analyses. many mass spectrometric peaks cannot be seen in crude mixtures and are only detectable in purifi ed fractions (prior et al., 2001; taylor et al., 2003). crude deoxyfl avan-3-ols and proanthocyanidins lyophilization of a 70% acetone extract of air-dried foliage from a. angustissima yielded an amorphous solid that consisted of 60% condensed tannins (readel et al., 2001; seigler et al., 1986) (26.5% yield). tlc of this material visualized with vanillin-hcl reagent indicated the presence of a complex series of proanthocyanidins. a purifi ed proanthocyanidin sample (aa1.2) was prepared by absorption of the lyophilized material above on sephadex lh-20, washing the column with 95% ethanol, and fi nally elution with 50% aqueous acetone. this fraction (aa1.2) consisted of 97% phenolics, as determined by the folin-denis method. based on analysis by tlc (silica gel) and comparison to the original 70% fig. 3: 5-deoxyfl avan-3-ols fig. 4: a representative acylated proanthocyanidin. proanthocyanidins of acaciella angustissima 147 acetone extract, this product appears to contain a full complement of the proanthocyanidins found in a. angustissima. the series of fractions terminated with those consisting mostly of acylated proanthocyanidins. positive ion faband maldi-ms, indicated that this material contains a complement of proanthocyanidins consisting mostly of dimers through hexamers (m/z 547.1, 563.2, 818.0, 834.3; sodium adducts m/z 857.2, 874.3, 1114, 1130.4, 1146.4, 1386.7, 1402.7, 1419.2, 1674.1, 1691.3) based on monomeric units of m/z 258, 274, and 290. in addition, peaks greater by 120 da were present at 411.1, 683.1, 699.2, 939, 955.3, and 971.3; a na adduct at 1266.8 or 120 da greater than the acylated tetramer also was observed. these signals were accompanied by smaller peaks corresponding to proanthocyanidin heptamers through decamers. few signals other than those associated with proanthocyanidins were present. because of atropisomerism (shoji et al., 2003), the 1h-nmr spectrum of this fraction (aa1.2) contains a series of poorly defi ned multiplets between δ 6-7 ppm (2’-, 5’and 6’-protons of 3’,4’and 3’,4’,5’-substituted b-rings, and 5-, 6-, and 8-protons of 5-deoxyfl avan-3-ol a-rings (steynkamp et al., 1988; malan et al., 1990b; qa’dan et al., 2003). peaks in the 1h-nmr spectrum corresponding to the presence of both robinetinidol (a singlet at 6.58 ppm) and fi setinidol (multiplet, 6-7 ppm) are present. signals for 2-, 3-, and 4-c-ring protons of fl avan-3-ols are found between 5.0 and 3 ppm. multiplets in the region between 2 and 3 ppm corresponding to unsubstituted 4-protons (methylene groups) are largely obscured by solvent and impurity peaks in these samples. because of the complex nature of the compounds and mixtures involved, the resonances of most protons in the 1h-nmr spectrum cannot be assigned exactly. the 13c-nmr spectrum is more informative: the peak at 156.880 ppm coincides with that expected for c-7 (mathisen et al., 2002; czochanska et al., 1980). signals centered at 145.729 ppm correspond to carbons at the 3’and 4’-positions of 3’,4’-disubstituted b-rings and 3’, 5’-carbons of 3’,4’,5’trisubstituted b-rings. the intensity of this peak (approximately 4 times greater than the peak for c-7) supports this assignment. resonances at 133.352 and 132.329 ppm correspond to those expected for 1’-positions of 3’,4’-disubstituted b-rings and 4’ positions of 3’,4’,5’-trisubstituted b-rings (czochanska et al., 1980; behrens et al., 2003; morais et al., 1999). signals at 118.192 ppm are compatible with those produced by 6’-carbons of 3’,4’-disubstituted b-rings. peaks at 115.298 ppm can be assigned to 2’and 5’-carbons of 3’,4’-disubstituted b-rings (behrens et al., 2003; porter et al., 1982). among the peaks at higher fi eld are those for 6or 8-carbons involved in linkage of proanthocyanidin ring systems. signals at 104.004 and 103.584 may correspond to either c-8 in (4→6)-linked systems or to c-6 in (4→8)-linked systems based on 5-deoxyfl avan3-ols (foo, 1984). resonances assignable to either 6or 8-carbons in (4→6)or (4→8)-linked oligomers involving the linkage usually are found between 106-108.5 ppm (porter et al., 1982). the lack of c-6 and c-8 resonances at 96-97 ppm and of a peak for c-5 (~ 152155 ppm) for phloroglucinol-type proanthocyanidins in these spectra confi rm that catechin/epicatechin, gallocatechin/epigallocatechin, or afzelechin/epiafzelechin are not present as either the “upper” initiating or “lower” terminal unit (czochanska et al., 1980; porter et al., 1982; sun et al., 1988). peaks at 79.982, 78.622, 78.363, 78.105, 76.124, and 75.515 ppm in the condensed tannin sample (aa1.2) correspond to carbons at the 2 and 3-positions of c-rings and similar rings in oligomers. the peaks immediately below 80 ppm may be attributed to c-2 absorptions of 2,3-cis-units (foo, 1984; czochanska et al., 1980), whereas those at higher fi eld are of c-3 carbons. in contrast, resonances above 80 ppm, corresponding to c-2 of 2,3-trans-units, are absent. peaks for 1’ (~ 121 ppm), 4a (~ 102 ppm), and 8a (~ 157 ppm) are often weak or not observed as in published spectra (qa’dan et al., 2003; sun et al., 1988). because the materials examined consist mainly of oligomers and acylated species in which the peak for c-3 is shifted both by acylation and by substitution at position 4, the peak for c-3 at 67-68 ppm (czochanska et al., 1980) should be low in intensity and is not seen in most spectra. the c-4 peak of terminal or “lower” units of acylated monomers (28-29 ppm) in the 13c-nmr of the purifi ed condensed tannin sample (aa1.2) is reduced in intensity for the same reasons, but in addition often is obscured by solvent absorptions. proanthocyanidins from partitioning between water and ethyl acetate another purifi ed proanthocyanidin extract was prepared by partitioning lyophilized 70% acetone extract between water and ethyl acetate (aa5 14.4% yield). the ethyl soluble portion (aa5) consisted largely of low molecular weight proanthocyanidin oligomers. although tlc (silica gel) of this fraction revealed only the presence of two spots that reacted strongly with vanillin-hcl reagent (rf 0.84 and 0.81), tlc analysis (cellulose) indicated the presence of at least six compounds. a negative ion fab-ms of the ethyl acetate soluble materials from partition (aa5) has peaks at m/z 529, 545.1, 561.2, 785.2, 801.2, 817.2, 833.2, 849.2, 1089 and 1105.4, corresponding to dimers, trimers, and tetramers based on monomeric units of m/z 258, 274, and 290. peaks at 575.1, 591.2, and 607.2 are unidentifi ed, but may correspond to fl avonoid glycosides. proanthocyanidins from vlc fractionation of the material obtained by partitioning between water and ethyl acetate by vacuum chromatography (vlc, silica gel) yielded a series of proanthocyanidin-rich fractions as indicated by positive vanillin-hcl reactions on tlc. based on the relative intensity of peaks in an fab-ms, the major component of aa5.1.7 from this fractionation had m/z 411.1 (290 + 120), but this substance was accompanied by a series of dimeric and trimeric proanthocyanidins. a positive ion fab-ms (fraction aa5.1.7) had peaks at m/z 411.1 (290 monomer + 120), 547.1, 563.1 (dimers based on monomers of 274 and 290). these peaks correspond to masses at 545.1 and 561.2 seen in the negative ion fab-ms of sample aa5. peaks of lower intensity corresponding to the proanthocyanidin trimers of aa5 occur at m/z 803.1, 819.2 and 835.1. peaks at 667.2 and 683.1 (dimers + 120) in this sample correspond to acylated proanthocyanidin dimers. ms data suggest that at least 14 compounds were present. however, based on peak intensities, this mixture contained at least 50% acylated monomers and dimers. proanthocyanidins from partition between water and ethyl acetate purifi ed on sephadex lh-20 an even more-highly purifi ed fraction of proanthocyanidins was obtained by chromatography of the ethyl acetate soluble material from partition with water (aa5) on sephadex lh-20 (asquith and butler, 1985; hagerman, accessed 2011). as indicated by tlc (silica gel), the material eluted with 80% ethanol (aa5.2.6-8) contained primarily one proanthocyanidin-rich spot (rf 0.7), but as f 0.7), but as f indicated by ms, this fraction also consisted of a mixture of several proanthocyanidins. in a positive ion fab-ms spectrum, peaks corresponding to dimers (low intensity, 563.1) and trimers (higher intensity, 817.3, 835.3; na adduct peaks m/z 857.2, 874, 1129) based on the same series of monomeric units were observed. signals with mass 120 da greater were found at m/z 665.3, 683.2, 699.2, 937.2, and 955.2. 148 a.h. smith, f.e. kandil, d.s. seigler, r.i. mackie structure of proanthocyanidins of a. angustissima the condensed tannins of a. angustissima consist of a series of 5-deoxyfl avan-3-ols and dimers to hexamers based on monomeric units of 258, 274, and 290 da (guibourtinidol, fi setinidol, and robinetinidol) and a series of compounds greater in mass by 120 da based on the same series of monomeric units, probably corresponding to acylation. the presence of smaller amounts of oligomers as large as decamers is indicated by fab-ms and maldi-ms spectra. several common and widely distributed fl avan-3-ols in legumes (plants of the fabaceae) have identical mass values in mass spectra, but peaks in the 1h-nmr spectra corresponding to h-6 and h-8 of catechin/epicatechin, gallocatechin/epigallocatechin, or afzelechin/ epiafzelechin-based proanthocyanidins (~5.9, ~6.1 ppm) (de mello et al., 1996; shoji et al., 2003; tanaka et al., 2000), or the aa’bb’ patterns of the b-ring of afzelechin/epiafzelechin (7.5 and 7.0 ppm, j = 8 hz) (j = 8 hz) (j tanaka et al., 2000) do not occur in the spectra of proanthocyanidins from a. angustissima, precluding the presence of phloroglucinol a-ring-based proanthocyanidins. further, peaks at ~157 ppm characteristic for c-5 and 96-97 ppm characteristic for c-6 and c-8 of phloroglucinol-type a rings are absent from 13cnmr spectra. proanthocyanidins based on oritin or epioritin (5deoxy-7,8-dihydroxy substitutions) may be ruled out (bennie et al., 2002). in previous studies, it has been found that (4β→6)-linkages predominate in condensations of 5-deoxyfl avan-3-ols and other appropriate precursors (steenkamp et al., 1988; malan et al., 1990a, b), although small amounts of (4β→8)-, (4α→6)-, and (4α→8)-linkages are sometimes encountered (steenkamp et al., 1988). these observations suggest that the proanthocyanidins of a. angustissima contain largely (4β→6)-linkages, although this remains to be established. the presence of a number of signals 120 da greater than the monomers, dimers and trimers in mass spectra, strongly suggests the presence of acyl derivatives. this is supported by corresponding shifts in the positions of c-2, c-3, and c-4. the presence of an acyl group (gallate) at position c-3 of a phloroglucinol-type system with 2,3-cis-confi guration effects an upfi eld shift in the position of c-2 of approximately 2.5 ppm in the “upper” ring system (77.1 to 75.8 ppm) (sun et al., 1988). this corresponds to peaks at 79.050 and 76.124 that are present in the 13c-nmr spectra of purifi ed proanthocyanidins from a. angustissima (aa1.2). the presence of an acyl group (gallate) at c-3 of a phloroglucinoltype system with 2,3-cis-confi guration causes a downfi eld shift of approximately 1.5 ppm in the “upper” ring system (73.2 to 74.7 ppm) (sun et al., 1988). the shift for a c-3 of the “lower” ring system with gallate attached is 69.2 ppm and is relatively independent of whether a gallate is attached to c-3 of the “upper” ring system or not (mathisen et al., 2002; sun et al., 1988). peaks at 73.185 and 75.515 ppm in the a. angustissima spectra correspond to the peaks predicted for acylated and non-acylated forms of the proanthocyanidins. a peak at 69.254 appears in some spectra, corresponding to the “lower” unit of oligomers, but this peak is weak in most instances. the observed m/z of the most prominent peak of one of the peaks differing by 120 m/z from high resolution mass spectra (683.176300 observed, calculated for c37h31o14: 683.176467) confi rms the assigned empirical formula and strongly supports identifi cation of this compound as the p-hydroxybenzoate derivative of a proanthocyanidin dimer. proanthocyanidins acylated by p-hydroxybenzoic acid have previously been reported in a type-a proanthocyanidin from prunus armeniaca (prasad et al., 1998) and type-b proanthocyanidins from camellia sinensis (hashimoto et al., 1987). the 13c-nmr of phydroxybenzoate moieties includes peaks at 166.4 (ester carbonyl), 121.7 (1’-), 132.2 (2’and 6’-), 116 (3’and 5’-), and 162.8 (4’-) ppm (hashimoto et al., 1987). although peaks corresponding to 2’-, 3’-, 5’-, and 6’were observed, peaks corresponding to the carbonyl 1’ (~121.7 ppm), and 4’-carbons (~162.8 ppm) of proanthocyanidins of a. angustissima were not observed in the spectra. signals for the aa’bb’-pattern of the p-hydroxybenzoate moiety (7.76-7.50 and 6.84-6.73 ppm, depending on the solvent, temperature, and composition of the mixture examined) of proton spectra were weak and diffi cult to observe in all fractions. the presence of gallate esters can be ruled out by the absence of a singlet at 7.03-7.05 ppm (hashimoto et al., 1987; dauer et al., 1998, 2003) in the 1h-nmr spectrum, a characteristic peak at 139 ppm in the 13c-nmr spectrum (mathisen et al., 2002), and peaks with an increase of m/z 152 in the mass spectra of proanthocyanidins. in summary, the proanthocyanidins of a. angustissima leaf material consist largely of 5-deoxyfl avan-3-ols, and dimers through hexamers accompanied by smaller amounts of higher molecular weight oligomers (heptamers-decamers). these are accompanied by 5-deoxyfl avan-3-ols, dimers and trimers that contain a phydroxybenzoate ester moiety. acknowledgements a. h. smith would like to acknowledge the agricultural research council, south africa, for partial support during her studies at the university of illinois and ilri for providing the plant material for the study. this research was supported in part by funds provided by the international arid land consortium (ialc), by the usda forest service and by the usda-csrees. fayez e. kandil wishes to acknowledge support by value-added 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polyphenolics from myrica esculenta bark. phytochemistry 27, 579583. tanaka, t.k., kondou, k., kouno, i., 2000: oxidation and epimerization of epigallocatechin in banana fruits. phytochemistry 53, 311-316. taylor, a.w., barofsky, e., kennedy, j. a., deinzer, m. l., 2003: hop (humulus lupulus l.) proanthocyanidins characterized by mass spectrometry, acid catalysis, and gel permeation chromatography. j. agric. food chem. 51, 4101-4110. zhu, m.j.d., phillipson, p.m., greengrass, p.m., bowery, n.e., cai, y., 1997: plant polyphenols: biologically active compounds or nonselective binders to protein? phytochemistry 44, 441-447. zucker, w.v., 1983: tannins: does structure determine function? an zucker, w.v., 1983: tannins: does structure determine function? an zucker, w.v ecological perspective. am. nat. 121, 335-365. addresses of the authors: alexandra h. smith, roderick i. mackie, department of animal sciences, university of illinois, urbana, illinois 61801, usa. e-mail: r-mackie@ illinois.edu alexandra h. smith’s present address: danisco cultures division, w227 n752 westmound dr., waukesha, wisconsin 53186, usa fayez e. kandil, david s. seigler, department of plant biology, university of illinois, urbana, illinois 61801, usa. e-mail: seigler@life.illinois.edu journal of applied botany and food quality 94, 82 91 (2021), doi:10.5073/jabfq.2021.094.010 1division urban plant ecophysiology, faculty of life sciences, humboldt-universität zu berlin, germany 2department of horticulture, college of agriculture and natural resources, kwame nkrumah university of science and technology, kumasi, ghana 3department of biotechnology (food processing technology unit), faculty of agriculture, university for development studies, nyankpala campus, tamale, ghana 4department of food science and technology, college of science, kwame nkrumah university of science and technology, kumasi, ghana 5department of horticulture and crop production, school of agriculture and technology, university of energy and natural resources, sunyani, ghana effect of terroir on the glucosinolate content of moringa oleifera grown in three agro-ecological zones of ghana olivia naa ayorkor tetteh1*, susanne huyskens-keil1, newton kwaku amaglo2, francis kweku amagloh3, ibok nsa oduro4, charles adarkwah5, daniel obeng-ofori5, christian ulrichs1, nadja förster1 (submitted: december 22, 2020; accepted: april 25, 2021) * corresponding author summary environmental factors and cultural practices significantly influence the secondary metabolites in plants, e.g., glucosinolates, depending on the cultivar of each species. the present study analyzed the in fluence of specific environmental factors (e.g., temperature, rain fall, and relative humidity), elevation, and fertilization (i.e., nitrogen and sulfur) on the glucosinolate content in leaves of wild-grown moringa oleifera from three agro-ecological zones in ghana and selected m. oleifera accessions cultivated under semi-controlled field conditions. the results showed that climate did not significantly influence total glucosinolate content in leaves of both wild-grown and cultivated accessions of m. oleifera, while elevation significantly influenced the total glucosinolate content of wild-grown plants. fertilization had no significant impact on the total glucosinolate content of the cultivated accessions. furthermore, wild-collected m. oleifera leaves from the three agro-ecological zones did not reveal a significant difference in their total glucosinolate content. for the cultivated accessions of m. oleifera, the agro-ecological zone, harvest time, and accession and the interactions among these factors significantly influenced the total glucosinolate content. the results suggest that selecting suitable accessions, choosing suitable locations, and applying appropriate cultivation practices could contribute to optimizing the production of health-promoting moringa plants with special emphasis on glucosinolate content. keywords: agro-ecological zone, climate, fertilization, glucosinolates, moringa oleifera accession introduction moringa oleifera lam. has been described as a multipurpose pe rennial plant with high nutritional and medicinal value. the high nutritional value is attributed to high contents of protein, vitamin a, and iron in the plants’ leaves compared to other nutritionally valuable legumes such as soybeans (makkar and becker, 1997; rweyemamu, 2006). additionally, it has high contents of vitamin c, vitamin b complex, as well as micro-, and macronutrients such as potassium (k), calcium (ca), magnesium (mg), selenium (se), and zinc (zn) (fuglie, 2001). due to these benefits, m. oleifera is a highly valuable food source in tropical and sub-tropical areas and has been included in many nutritional programs (fuglie, 2001; thurber and fahey, 2009). besides its high nutritional value, the different morphological plant parts of m. oleifera, especially the leaves, comprise health-promoting compounds with medicinal benefits (bharali and azad, 2003; oluduro et al., 2010; waterman et al., 2014). these medicinal benefits, including anti-inflammation (waterman et al., 2014), anti-bacterial (oluduro et al., 2010), and anti-carcinogenic effects (bharali and azad, 2003), are often traced back to the secondary plant metabolites, especially glucosinolates (gs) and their breakdown products (bharali and azad, 2003; oluduro et al., 2010). gs provide plants with unique survival or adaptive strategies (variyar et al., 2014) and are classified as aliphatic, aromatic, and indolic compounds, depending on the respective amino acid precursor, i.e., methionine, tryptophan, and phenylalanine (fahey et al., 2001). high contents of aromatic gs have been identified in the leaves of m. oleifera, namely, 4-α-rhamnopyranosyloxy-benzyl gs (gs1) and three isomers of acetyl-4-α-rhamnopyranosyloxy-benzyl gs (gs2, gs3, and gs4) (bennett et al., 2003). the profile, i.e., the content and composition, of secondary meta bolites such as gs in plants is influenced by the environmental and cultural conditions of the plant (zandalinas et al., 2012). for instance, gs content in brassicaceae is known to be influenced by temperature, relative humidity, and radiation (bohinc and trdan, 2012). the variations in gs profile are also associated with soil type and water supply (fenwick and heaney, 1983). moreover, for the effect of fertilization on gs levels, authors of a previous study have suggested that sulfur (s) supply to broccoli has a strong influence on the nitrogen (n) availability, and hence on gs concentration in the plant (schonhof et al., 2007a). in contrast, it was found that s supply, regardless of the n content, has no significant influence on gs content in different ecotypes of m. oleifera (förster et al., 2015). the effects of environmental factors and growing conditions on gs content in plants are species-specific and even cultivar-specific (charron et al., 2005; bohinc and trdan, 2012). the latter has been confirmed by studies on multiple accessions of m. oleifera cultivated under field conditions (doerr et al., 2009) and for ecotypes cultivated under greenhouse conditions (förster et al., 2015). so far, studies on moringa have not been replicated across different geographical environments (e.g., in tropical areas) that are known to support the optimal growth of the plant. meanwhile, these different geographical environments have different abiotic and biotic factors, which could modulate the gs content in the plant. thus, there is limited information on the impact of eco-physiological parameters on the gs dynamics in m. oleifera, i.e., the impact of environmental conditions at different agro-ecological zones in ghana on the gs effect of terroir on the glucosinolate content of moringa 83 profile of m. oleifera plants. moreover, no study has been conducted so far on the gs profile of wild-grown m. oleifera in ghana. therefore, the objectives of the present study were to determine the influence of specific environmental factors (e.g., temperature, rainfall, and relative humidity), elevation, and soil minerals (i.e., n and s) on the gs content in leaves of wild-grown m. oleifera from three selected agro-ecological zones in ghana, and in the leaves of selected m. oleifera accessions cultivated under semi-controlled field conditions. moreover, the study was designed to determine the impact of the agro-ecological zone, the selection of accessions, fertilization, and harvest time on the total gs content in leaves of the selected m. oleifera accessions. materials and methods description of the agro-ecological zones three agro-ecological zones in ghana were selected for the study: guinea savannah, transitional forest, and deciduous forest. whereas the guinea savannah zone is located within the northern regions of ghana, the transitional and deciduous forest zones span from the east to the west along the middle portions of the country. the transitional zone spans from the middle portions towards the northern part of the country, while the deciduous forest zone stretches from the middle portions towards the southern part of the country. the guinea savannah zone records a unimodal rainfall pattern, which starts in april and ends in september/october with a mean annual rainfall of 1100 mm and a minimum temperature of 15 °c in january and a maximum of 42 °c in march (food and agriculture organization of the united nations (fao, 2005). the rainfall pattern of the deciduous forest zone is bi-modal, with the major rainy season starting in early march, reaching its peak in june, and tapering off gradually through july. the minor season begins in late august and reaches its peak in september/november. the mean annual rainfall is 1500 mm, and the mean monthly temperature is between 24 °c in august and 30 °c in march (fao, 2005). the transitional zone is characterised by a bi-modal rainfall pattern with a mean annual rainfall of 1300 mm (fao, 2005). temperatures in the zone range between 17 and 33 °c, with the lowest recorded in august and the highest in december, january, and february (nketia et al., 2018). collection of wild-grown moringa oleifera leaves three collection points within each agro-ecological zone were selected for the study (tab. 1). m. oleifera leaflets were collected from five mature plants in each of the three collection points for each of the agro-ecological zones in june 2017. the five plants’ leaflets were thoroughly mixed to form a composite for each collection point. leaflets from the pinnae were harvested as described by tetteh et al. (2019) from the lower, mid, and upper parts of the tree canopy. harvested leaf material was transported over a period of 2 h, 4 h, and 8 h from the harvest locations of deciduous forest, transitional and guinea savannah zones, respectively, to the laboratory in the department of food science and technology, kwame nkrumah university of science and technology, kumasi, ghana. upon ar rival, the leaves were oven-dried at 40 °c for 48 h. all samples were packaged in airtight and waterproof packages and stored in a -20 °c freezer prior to gs analysis. moringa oleifera cultivation under semi-controlled conditions the effects of elevation, temperature, relative humidity, as well as soil minerals and fertilizer application on the gs profile of m. olei fera accessions cultivated in the three agro-ecological zones in ghana were investigated. the point values, representing the experi mental fields for the m. oleifera cultivation within the agro-ecological zones of ghana, were recorded (tab. 1) using a gps device (garmin gps 60, model 010-00322-01, usa). for this experiment, three accessions of m. oleifera, including 1) 16016fl-161a pkm 1 (origin: india), 2) 04024fl-041a (origin: kenya), and 3) 08025fl-081d (origin: uganda) were used. all accessions were obtained from the educational concerns for haiti organization (echo), florida, usa. a total plot size of 15 × 12 m2 was demarcated, and soil was prepared to a fluffy state in all three experimental fields. each field was divided into two blocks as fertilized and unfertilized blocks. further, each block was divided into nine subplots of 1 m2, each with 0.6 m wide walkways in between the individual plots and borders. a 0.8 m wide border was created around the 18 subplots. plants were sown in a randomised complete block design factorial field layout, and each accession was replicated three times. the seeds were sown directly without any pre-treatment at a 0.2 m × 0.2 m spacing on each of the 1 m2 subplots at the three experimental fields in february 2017. they were sown at 0.02 m depth with one seed per drill. in total 36 plants per subplot were planted (250,000 plants/ha) and watered soon after sowing in all three locations. m. oleifera landrace (local accessions) were sown on the 0.8 m wide borders. the borders were created around the treatment plots (accessions) at each experimental field as a transition zone to prevent predators, pests, and diseases that may directly have a negative impact on the densely populated experimental plants. germination generally occurred within a maximum of 10 days after sowing at all the experimental fields, and the needed refills on the plots were done with extra seedlings, which were grown specifically for refilling. the seedlings were then left to grow without any treatment for another 14 days before the poultry manure compost was applied. watering was done every other day on each of the three experimental fields, except on days with rainfall. deep litter poultry manure was collected from aban farms in dormaa ahenkro, ghana, and analysed for n using the kjeldahl method (din-iso-13878, 1998), while the other minerals and organic matter composition were determined as described by motsara and roy (2008). the mean contents (±standard deviations) were: 1.08 ± 0.01% n, 2.89 ± 0.02% p, 2.04 ± 0.08 cmol/kg k, 1.70 ± 0.01 cmol/kg na, 4.26 ± 0.03 cmol/kg ca, 0.42 ± 0.01 cmol/kg mg, 1.30 ± 0.14 cmol/ kg h+, 0.14 ± 0.01 cmol/kg al, 0.25 ± 0.01% s, 34.67 ± 0.37% organic carbon, 6.17 ± 0.11 ph, and 59.77 ± 0.78% organic matter. the poultry manure was then composted for 21 days while turning (aeration) every three days. after 14 days of seed germination, 5 kg tab. 1: gps coordinates and elevations for the collection points agro-ecological zone gps coordinates elevation [m] for wild-grown moringa oleifera transitional zone n7 16.175 w2 50.950 281 n7 26.866 w2 34.729 322 n7 18.955 w2 18.356 300 deciduous forest zone n6 43.963 w1 28.436 277 n6 41.458 w1 37.639 261 n6 30.460 w1 32.396 252 guinea savannah zone n9 29.902 w0 53.971 169 n9 26.961 w0 51.885 188 n9 25.333 w0 51.140 191 for the cultivated accessions of moringa oleifera transitional zone n7 16.421 w2 48.039 315 deciduous forest zone n6 40.650 w1 33.980 264 guinea savannah zone n9 35.432 w1 01.667 109 84 o.n.a. tetteh, s. huyskens-keil, n.k. amaglo, f.k. amagloh, i.n. oduro, c. adarkwah, d. obeng-ofori, c. ulrichs, n. förster of the decomposed manure were applied per 1 m2 subplot (50 tons/ ha) on the block marked for fertilization. the poultry manure compost was worked into the soil around the seedlings on the subplot and watered afterward. harvest and postharvest preparation at the first harvest of m. oleifera, 70 days after sowing, leaf material was manually cut at 0.4 m above ground level (fig. 1). the leaflets from the plants on each subplot were harvested separately. a composite of leaflets from each subplot was collected into perforated a4-sized envelopes and put on ice packs in ice chests to keep the leaf material at a cold temperature during transportation to the laboratory for drying. upon arrival in the laboratory, the leaflets were oven-dried at 40 °c for 48 h. the oven-dried samples were kept in a freezer (-18 °c) prior to the gs analysis. samples were sent to germany within one month for gs analysis. after the first harvest, another round of 5 kg per 1 m2 poultry manure compost was applied to the fertilized plots in all locations. plants were left to regrow uninterrupted for a period of 35 days un til the second harvest. leaf samples were prepared in the same procedure as described for the first harvest. climate data with respect to the wild-grown m. oleifera plants, climatological data for the three agro-ecological zones was obtained from the ghana meteorological agency. daily climatic data for five months (february to june 2017) of rainfall (mm/year), relative humidity (%), as well as minimum and maximum temperature (°c) recorded before the harvest of m. oleifera leaf material, were used. the relative humidity data for the transitional zone were not available in the climatological dataset from the ghana meteorological agency. the data obtained for the cultivated m. oleifera accessions at the same agro-ecological zone were hence also used for the wild-collected variants. means (± standard deviation) of each climate variables’ daily records were calculated (tab. 2). for the cultivated accessions of m. oleifera at the experimental fields, relative humidity (%) and temperature (°c) were recorded during the experimental period, from february to june 2017 with hobo data logger (u23 series pro v2 loggers, onset company, bourne, ma) within 30 min intervals. the devices were set up at the experimental fields in the transitional and the deciduous forest zones, whereas daily records for relative humidity and temperature for the experimental field in the guinea savannah zone were provided by the potsdam institute for climate impact research, germany. the mean fig. 1: moringa oleifera morphological sections used for the experiments (source: tetteh et al., 2019 with modifications) tab. 2: temperature (maximum and minimum), rainfall, and relative humidity recorded daily from the three agro-ecological zones in ghana. agro-ecological zone mean daily mean daily mean daily mean daily maximum temperature minimum temperature relative humidity rainfall [ºc] [ºc] [%] [mm] a transitional zone 34.57 ± 2.60 a 23.18 ± 1.28 c 74.27 ± 9.60 a 4.32 ± 8.99 a deciduous forest zone 33.21 ± 1.98 b 23.78 ± 1.25 b 73.14 ± 14.95 a 5.40 ± 10.56 a guinea savannah zone 35.16 ± 2.88 a 25.44 ± 2.10 a 63.87 ± 14.87 b 3.52 ± 8.34 a b transitional zone 31.96 ± 3.00 b 25.71 ± 1.52 a 74.27 ± 9.60 a deciduous forest zone 31.25 ± 2.12 b 26.33 ± 1.38 a 72.60 ± 11.54 a guinea savannah zone 33.87 ± 9.49 a 24.88 ± 6.85 b 58.10 ± 15.84 b the results presented in the table are mean ± standard deviation of daily climate data recorded from february to june 2017. the mean values were compared using one-way anova followed by tukey’s test, p ≤ 0.05; same lowercase letters in the same column indicate no significant differences in means of climate data among the agro-ecological zones for wild-grown m. oleifera (a) and cultivated accessions of m. oleifera under semi-controlled field conditions (b). effect of terroir on the glucosinolate content of moringa 85 (± standard deviation) of daily climate variables in the three experimental fields within the three agro-ecological zones for m. oleifera cultivation were calculated (tab. 2). soil collection for wild-grown m. oleifera plants, topsoil was collected with a hand auger from 0 1 m depth around the mature plants. soils collected around the plants from the three collection points within each agro-ecological zone were mixed as composite soil. the individual composite soils from the three agro-ecological zones were packed in airtight and waterproof containers and transported to the soil science laboratory in the department of agriculture, kwame nkrumah university of science and technology (knust), kumasi, for soil mineral analysis. prior to the cultivation of m. oleifera accessions at the three experi mental fields, soil samples were taken with a hand auger from a depth of 0 1 m from all the three experimental fields for routine analysis (tab. 3). soil samples were also taken at the first and second harvest times of the experiment at five spots on each of the subplots for all the experimental fields in the three agro-ecological zones. the soils from the same accessions from either fertilized or non-fertilized blocks were mixed as composite soil and packaged and transported as described for the soil samples from the wild-collected m. oleifera plants. (buck scientific model 280 g). the determination was done after s extraction with cacl2 and hno3 (motsara and roy, 2008). the method employed in the analysis of phosphorous (p) was based on the production of a blue complex of molybdate and thiophoshate in an acid solution where p was determined spectrophotometrically (buck scientific, model 280 g, usa) at 630 nm (motsara and roy, 2008). exchangeable bases determination (potassium (k) and sodium (na)) was determined using the volumetric sodium tetra phenyl boron method after dry ashing digestion of the soil, and the composite samples were analysed with a flame photometer (jenway, model pfp7, uk) (motsara and roy, 2008). all soil analyses were conducted in duplicate. for soil samples collected prior to the cultivation of the m. oleifera accessions, the means were calculated for the various soil properties analysed (tab. 3). due to the strong influence of n and s on the biosynthesis of gs, n and s contents were determined in the soil samples of the wild-grown (tab. 4) and the cultivated accessions of m. oleifera plants (tab. 5). tab. 3: soil composition of the three experimental fields prior to the cultivation of moringa oleifera accessions (mean ± standard deviation). mineral/ agro-eclological zone transitional deciduous guinea savannah zone forest zone zone n [%] 0.13 ± 0.007 0.15 ± 0.014 0.04 ± 0.007 p [%] 0.49 ± 0.014 2.31 ± 0.014 0.15 ± 0.003 s [%] 0.42 ± 0.008 0.25 ± 0.008 0.07 ± 0.004 k [cmol/kg] 0.35 ± 0.007 0.46 ± 0.021 0.08 ± 0.007 na [cmol/kg] 0.46 ± 0.014 0.53 ± 0.007 0.49 ± 0.014 ca [cmol/kg] 7.18 ± 0.035 5.86 ± 0.085 3.06 ± 0.085 mg [cmol/kg] 4.15 ± 0.071 0.85 ± 0.071 1.18 ± 0.035 h+ [cmol/kg] 0.66 ± 0.014 0.66 ± 0.021 2.35 ± 0.007 al [cmol/kg] 0.58 ± 0.035 0.52 ± 0.021 0.83 ± 0.021 cation exchange 13.37 ± 0.134 8.86 ± 0.141 7.97 ± 0.028 capacity [cmol/kg] organic matter [%] 6.51 ± 0.146 3.34 ± 0.079 1.56 ± 0.147 organic carbon [%] 3.78 ± 0.085 1.94 ± 0.046 0.90 ± 0.086 ph 6.59 ± 0.042 6.10 ± 0.057 5.12 ± 0.014 sand [%] 50.88 72.80 70.96 clay [%] 24.00 16.12 7.92 silt [%] 25.12 11.08 21.12 textural class sandy clay loam sandy loam sandy loam physicochemical soil analysis the particle size analysis of the soil was carried out using the hydrometer method (bouyoucos, 1962), and the textural class was determined (motsara and roy, 2008). organic matter content was determined by loss of weight on ignition with the aid of muffle furnace (naberthem, model l9/11/skm, germany), and the soil ph level was determined with an electrode (wtw ph meter, model ph3110, germany) in 5:1 water to soil extract (motsara and roy, 2008). nitrogen (n) was determined by the kjeldahl method (din-iso-13878, 1998). to determine sulfur (s) in the soil, the turbidometric method using a spectrophotometer was applied tab. 4: mineral composition of soils collected from the three agro-ecological zones. agro-ecological zone n [%] s [%] transitional zone 0.13 ± 0.06 a 0.40 ± 0.11 a deciduous forest zone 0.14 ± 0.03 a 0.36 ± 0.09 a guinea savannah zone 0.06 ± 0.03 a 0.41 ± 0.26 a results presented in the table are mean ± standard deviation of soil minerals. the mean values were compared using one-way anova, p ≤ 0.05; same lowercase letters in the same column indicate no significant difference in the means of the soil minerals from the three agro-ecological zones. tab. 5: mineral composition of soil collected for moringa oleifera accessions cultivated. fertilization n [%] s [%] fertilized 0.14 ± 0.05 a 0.2 ± 0.10 a unfertilized 0.11 ± 0.05 b 0.21 ± 0.11 a results presented in the table are mean ± standard deviation of soil minerals of fertilized and unfertilized soils at first and second harvest. the mean values were compared using the t-test, p ≤ 0.05; same lowercase letters in the same column indicate no significant difference in the means of the soil minerals of fertilized and unfertilized soils from the experimental fields. extraction and analysis of intact glucosinolates intact gs in the leaves were extracted according to the method described by förster et al. (2015). for the extraction, 20 mg of dried and ground leaf material was used. leaf material was extracted in 750 μl of 70% methanol (75 °c) and 100 μl of internal standard (1 mm sinigrin, 2-propenyl gs, isolated from brassica juncea seeds), followed by two additional re-extraction steps with 500 μl of the 70% methanol. the collected supernatants were concentrated in a vacuum concentrator (thermo scientific savent spd111v concentrator, vacuum pump: vacuumbrand pc 3001 series, cvc 3000) to a final volume of 150 μl. a volume of 200 μl of 0.4 m barium acetate was added to each tube and made up to 1 ml with ultrapure water (milliq quality). samples were incubated for 30 min at room temperature and centrifuged at 16,000 g (thermo scientific, heraeus megafuge 11r centrifuge) for 10 min. supernatants were decanted into new eppendorf tubes and filled to 2 ml with ultra86 o.n.a. tetteh, s. huyskens-keil, n.k. amaglo, f.k. amagloh, i.n. oduro, c. adarkwah, d. obeng-ofori, c. ulrichs, n. förster pure water. of each sample, 1 ml was filtered using costar® spinx tubes and transferred to hplc vials. the remaining amount of each sample (about 1 ml) was incubated with 0.05 u myrosinase (thioglucosidase, sigma aldrich) for 8 h at 37 °c to hydrolyze gs to their corresponding breakdown products. hydrolyzed samples of the leaves were filtered through a costar® spinx tube and transferred to hplc sample vials. extracts of intact gs were qualitatively and quantitatively analyzed by hplc (thermo scientific) equipped with an autosampler, pump, dad detector, and column oven. volumes of 10 μl of the samples were injected on a sb-c18 column (zorbax 5 μm, 4.6 × 250 mm, agilent). the following gradient program was used: 0 – 2 min: 0 – 1% b, 2 – 20 min: 1 – 50% b, 20 – 24 min: 50 – 100% b, 24 – 26 min: 100% b, 26 – 27 min: 100 – 1% b, and 27 – 35 min: 1 – 0% b at a flow rate of 1.5 ml/min. buffered eluents with solvent a: 100% 0.1 m ammonium acetate, b: 40% acetonitrile/0.1 m ammonium acetate were used for the extracts. detection was at 229 nm, and components were identified by retention time and quantified against internal standard. the peak area remaining after myrosinase treatment of the intact gs extract was subtracted from the peak area of the gs of the intact gs extract without myrosinase treatment (förster et al., 2015). gs content was determined on a dry weight (dw) basis. statistical analysis the software, statistical package for social sciences (spss) (ibm spss statistics for windows, version 25.0), was used for the statistical analysis. the mean values were compared using analysis of variance (anova) followed by tukey’s honest significant difference (hsd) test for pairwise comparison. spearman’s correlation analysis was used to study the relationship between local conditions (climate, soil minerals, elevation in the specific locations) and gs content in the leaves for both experiments. results glucosinolate profile of wild-grown moringa oleifera leaf material from the three agro-ecological zones four different gs, 4-α-rhamnopyranosyloxy-benzyl gs (gs1), and three isomers of acetyl-4-α-rhamnopyranosyloxy-benzyl gs (gs2, gs3, and gs4) were identified in the leaves of m. oleifera trees harvested from the three agro-ecological zones. the total gs content analysed in the leaves from all the agro-ecological zones was not significantly different (fig. 2). their total gs content varied on average between 41.18 μmol/g dw and 53.42 μmol/g dw. in addition, the contents of single gs were not significantly different comparing the different agro-ecological zones (fig. 2). correlation analysis of total glucosinolate content of wild-grown moringa oleifera leaves with soil minerals, elevation, and climate data for the three agro-ecological zones results showed no statistically significant correlation between total gs in leaves of m. oleifera, and the climate data (tab. 6). however, a significant negative correlation between total gs and n (r = -0.85, p < 0.05), as well as elevation (r = -0.68, p < 0.05) was found (tab. 6). elevation correlated negatively with minimum temperature (r = -0.95, p < 0.05) and positively with relative humidity (r = 0.95, p < 0.05) (tab. 6). maximum temperature had a strong negative correlation with rainfall (r = -1.00 p < 0.05) (tab. 6). cultivation under semi-controlled conditions leaf glucosinolate profiles of three moringa oleifera accessions cultivated with/without fertilizer at two harvest times the results revealed that the independent factors, i.e., harvest time, accession, and ago-ecological zone, had a significant influence on total gs content in m. oleifera, while fertilization had no significant influence on the total gs content (tab. 7a). interaction effects 20 573 fig. 2: glucosinolates (gs) profile of leaves from wild moringa oleifera plants in three agro-574 ecological zones. one-way anova followed by tukey's test, p ≤ 0.05. 4-α-575 rhamnopyranosyloxy-benzyl gs (gs1), three isomers of acetyl-4-α-rhamnopyranosyloxy-576 benzyl gs (gs2, gs3, gs4). same lowercase letters indicate no significant differences for 577 means of single gs among agro-ecological zones; same uppercase letters indicate no 578 significant differences for means of total gs content (comprising of gs1-gs4). error bars 579 indicate standard deviations of mean total gs content. 580 581 582 583 584 585 586 587 588 0 10 20 30 40 50 60 70 decidious forest zone guinea savannah zone transitional zone g lu co si no la te s co nt en t [ µm ol /g d w ] gs1 gs2 gs3 gs4 a a a a a a a a a a a a a a a fig. 2: glucosinolates (gs) profile of leaves from wild-grown moringa oleifera plants in three agro-ecological zones. one-way anova followed by tukey’s test, p ≤ 0.05. 4-α-rhamnopyranosyloxy-benzyl gs (gs1), three isomers of acetyl-4-α-rhamnopyranosyloxy-benzyl gs (gs2, gs3, gs4). same lowercase letters indicate no significant differences for means of single gs among agro-ecological zones; same uppercase letters indicate no significant differences for means of total gs content (comprising of gs1-gs4). error bars indicate standard deviations of mean total gs content. tab. 6: spearman’s correlation analysis between the total glucosinolate (gs) content in the leaves of moringa oleifera and the local conditions of the three agro-ecological zones. variables (1) (2) 3) (4) (5) (6) (7) (8) (1) total gs [μmol/g dw] 1 (2) n [%] -0.85** 1 (3) s [%] -0.23 0.30 1 (4) elevation [m] -0.68* 0.67* 0.1 1 (5) maximum temperature [°c] 0.47 -0.74* 0.05 -0.47 1 (6) minimum temperature [°c] 0.63 -0.61 -0.21 -0.95** 0.50 1 (7) rainfall [mm] -0.47 0.74* -0.05 0.47 -1.00** -0.50 1 (8) relative humidity [%] -0.63 0.61 0.21 0.95** -0.50 -1.00** 0.50 1 ** correlation is significant at the 0.01 level (2-tailed). * correlation is significant at the 0.05 level (2-tailed). effect of terroir on the glucosinolate content of moringa 87 were identified for agro-ecological zone×fertilization, agro-ecological zone×accession, agro-ecological zone×harvest time, and agro-ecological zone×fertilization×harvest time on total gs content (tab. 7a). additionally, the comparison of group means revealed that transitional and guinea savannah zones showed significantly higher total gs content than those of the deciduous forest zone (tab. 7b). furthermore, group mean comparison showed significantly higher total gs content in the accession 04024fl-041a than that of the accession, 08025fl-081d, which was also significantly higher than that of 16016fl-161a pkm 1 (tab. 7c). also, based on group mean comparison, it was found that total gs content at the first harvest was significantly lower than that of the second harvest (tab. 7d). results on group mean comparison of the factor fertilization showed that the total gs content in the fertilized variants was not significantly dif ferent from the unfertilized variants (tab. 7e). correlation analysis of total gs content of cultivated moringa oleifera accessions with the soil minerals, elevation, and climate data from three agro-ecological zones the results showed no significant correlation between the total gs content of m. oleifera leaves and the soil minerals and climate data (tab. 8). however, a significant correlation was observed between elevation and soil minerals and between elevation and climate data measured. discussion impact of temperature on glucosinolate profile of moringa oleifera the lack of significant correlation between temperature and total gs content in the leaves in both studies (tab. 6 and tab. 8), even though significant differences were observed in mean maximum and minitab. 7: glucosinolates (gs) profile of moringa oleifera leaves of three accessions cultivated with and without fertilizer and harvested at two harvest times from the three agro-ecological zones. a treatment effects total gs (p-value) harvest (h) p = 0.00 agro-ecological zone (ag) p = 0.00 accession (ac) p = 0.00 fertilization (f) p = 0.18 h×ag p = 0.00 h×ac p = 0.14 h×f p = 0.42 ag×ac p = 0.00 ag×f p = 0.00 ac×f p = 0.91 h×ag×ac p = 0.17 h×ag×f p = 0.01 h×ac×f p = 0.92 ag×ac×f p = 0.65 h×ag×ac×f p = 0.12 treatment gs content [μmol/g dw] gs1 gs2 gs3 gs4 total gs b agro-ecological zone transitional 19.33 ± 10.27 a 6.12 ± 3.01 a 2.97 ± 1.83 a 13.89 ± 6.37 a 42.31 ± 15.42 a deciduous forest 19.02 ± 6.56 a 3.32 ± 2.73 b 1.44 ± 1.42 b 10.20 ± 5.64 b 33.97 ± 14.98 b guinea savannah 23.07 ± 9.17 a 4.06 ± 2.29 b .85± 1.22 b 10.32 ± 4.45 b 39.30 ± 13.58 a c accession 16016fl-161a pkm 1 17.09 ± 6.81 b 4.25 ± 3.38 ab 2.02 ± 1.96 a 8.95 ± 4.37 b 32.31 ± 13.12 c 04024fl-041a 22.74 ± 9.86 a 5.57 ± 3.05 a 2.52 ± 1.72 a 14.93 ± 6.53 a 45.77 ± 15.68 a 08025fl-081d 21.58 ± 9.04 a 3.76 ± 1.96 b 1.75 ± 1.06 a 10.78 ± 4.69 b 37.86 ± 13.32 b d fertilization fertilized 22.73 ± 9.50 a 4.27 ± 3.00 a 1.95 ± 1.67 a 11.14 ± 6.41 a 40.09 ± 16.12 a unfertilized 18.03 ± 7.61 b 4.76 ± 2.86 a 2.23 ± 1.62 a 11.86 ± 5.06 a 36.87 ± 13.65 a e harvest 1 18.13 ± 8.27 b 3.39 ± 2.37 b 1.41 ± 1.26 b 10.63 ± 5.76 a 33.57 ± 12.52 b 2 22.72 ± 9.00 a 5.63 ± 3.03 a 2.76 ± 1.72 a 12.35 ± 5.71 a 43.45 ± 15.69 a a: h, ag, ac, f, h×ag, h×ac, h×f, ag×ac, ag×f, ac×f, h×ag×ac, h×ag×f, h×ac×f, ag×ac×f, and h×ag×ac×f effects on total gs content were compared using anova, p ≤ 0.10. b and c: the results presented in the tables are the means ± standard deviations of single gs (gs1: 4-α-rhamnopyranosyloxy-benzyl gs; gs2, gs3, gs4: three isomers of acetyl-4-α-rhamnopyranosyloxy-benzyl gs) and total gs (comprising of gs1 – gs4) content in m. oleifera leaves. the mean values were compared using one-way anova followed by tukey’s test, p ≤ 0.10. same lowercase letters in the same column indicate no significant difference in means of single gs, and total gs content among the agro-ecological zones (b) and accessions (c) d and e: the results presented in the tables are the means ± standard deviations of single gs (gs1: 4-α-rhamnopyranosyloxy-benzyl gs; gs2, gs3, gs4: three isomers of acetyl-4-α-rhamnopyranosyloxy-benzyl gs) and total gs (comprising of gs1 – gs4) content in m. oleifera leaves. the mean values were compared using the t-test, p ≤ 0.10. same lowercase letters in the same column indicate no significant difference in means of single gs, and total gs content between fertilized and unfertilized variants of moringa oleifera (d) and first harvest and second harvest (e). 88 o.n.a. tetteh, s. huyskens-keil, n.k. amaglo, f.k. amagloh, i.n. oduro, c. adarkwah, d. obeng-ofori, c. ulrichs, n. förster mum temperatures (tab. 2), was not consistent with findings in the literature, where higher temperatures were reported to significantly increase gs content in different brassica crops (ciska et al., 2000). however, the transferability of the present results to other studies is difficult, especially when plants are of different species and are grown in an uncontrolled environment combined with high variabili ty of abiotic factors (ahuja et al., 2010). this assertion holds, parti cularly for wild-grown m. oleifera from the three agro-ecological zones. here, various parameters such as previous climate events during early plant growth, age, and stage of development of the wildgrown m. oleifera plants, which might have had an impact on gs were unknown. therefore, it is difficult to trace the results back to one particular parameter. in the wild-grown m. oleifera, the lack of significant difference in total gs content for all three agro-ecological zones (fig. 2) could be due to the perennial nature of m. oleifera plants (duke, 1983). with trends of increasing temperatures in the last several decades (leary et al., 2013), physiological adaptations might have developed in the plant to deal with the repeated temperature stress and consequently increased the plants’ secondary metabolite accumulation (yang et al., 2018). therefore, over time, the plants probably retain nearly similar total gs content in the leaves at the three agro-ecolo gical zones irrespective of the climate conditions. on the other hand, m. oleifera is a tropical plant (roloff et al., 2009), and the wildgrown plants could be well adapted to the climate at the three agroecological zones. in this context, the plants seem to grow well under nearly optimal temperature and other abiotic conditions such as light, carbon dioxide, water, and nutrients supply. thus, the plants did not encounter extreme stress at any specific location during the experimental period to cause them to significantly increase their secondary plant metabolites. impact of rainfall and relative humidity on glucosinolate profile of moringa oleifera with respect to wild-grown m. oleifera, no significant correlation was found between rainfall and total gs content in the leaves (tab. 6), which is not in accordance with previous studies (stamp, 2004; ramakrishna and ravishankar, 2011; förster et al., 2015). these authors reported that water availability affects gs accumulation in such a manner that plants under water stress conditions accumulate higher gs content than well-watered plants. thus, due to the lack of stress on well-watered plants, gs accumulation is not enhanced (selmar and kleinwächter, 2013). because of the long periods of drought resulting from reduced or no rainfall from september/ october to march (padi, 2017), we expected that the wild-grown plants in their respective environments would respond with a stressmediated increase in secondary plant metabolites, e.g., gs. in our study, it had been assumed that other edaphic and climate conditions in the three agro-ecological zones might have also impacted the gs content of the wild-collected m. oleifera leaves. however, in the present results, rainfall was not significantly different during the experimental period (tab. 2a). indeed, at the time of harvest (june 2017), the rainy season in all the three agro-ecological zones was generally at its peak (one-way anova, p ≤ 0.05, detailed results not shown). therefore, it is assumed that the increased rainfall might have resulted in a sufficient water supply for the vegetative growth of the plants in all three agro-ecological zones, and consequently, plants did not experience water stress conditions. furthermore, the increased rainfall at the time of harvest was accompanied by a significant decrease in maximum temperature (r = -1.00, tab. 6). thus, the absence of high-temperature stress on the plants, in turn, might have led to an inhibition of accelerated gs synthesis. although not confirmed by the presented results, rainfall might be an important environmental factor for the gs accumulation in m. oleifera leaves, as reported. therefore, a more thorough understanding of the changes in within-season variability of rainfall and its close relation to temperature and their effects on gs content in m. oleifera is warranted in future studies. further, no significant correlation between relative humidity and total gs content was observed for both the wild-grown and cultivated accessions (tab. 6 and tab. 8), even though relative humidity data among the three agro-ecological zones were significantly different (tab. 2). this contradicts reports suggesting that relative humidity influences gs accumulation in cabbage (bohinc and trdan, 2012). indeed, water requirement in plants is significantly influenced by re lative humidity, and consequently, relative humidity influences plant growth, although the effects differ depending on plant species (ford and thorne, 1974). low relative humidity increases transpiration and thus results in water deficiency, which is being caused by stomata closure and consequently reduction of carbon dioxide intake by plants (talbott et al., 2003). based on such water stress conditions, there is a significant decrease in nadph + hydrogen ion (h+) consumption for carbon dioxide fixation and reduction in the calvin cycle (selmar and kleinwächter, 2013). therefore, the concentration of the oxidized form of nadph, nadp+, decreases considerably, which significantly decreases the levels of adequate electron acceptors for the electron transport chain of photosynthesis. as a result, no more electrons from the electron transport chain are transferred to the reduction equivalents, leading to the production of reactive oxygen species. this respective metabolic change could damage the photosynthetic apparatus, referred to as oxidative photodestruction. however, a protective mechanism is induced, which shifts all reactions involving nadph + h+ consumption towards an enhanced synthesis of secondary plant metabolites, such as gs (selmar and kleinwächter, 2013). therefore, we expected a correlation between relative humidity and total gs content in our experiments which could, however, not be confirmed in our studies. this is attri butable to the absence of water stress of the wild-grown m. oleifera plants, as previously explained in the preceding paragraph. similarly, tab. 8: spearman’s correlation analysis between the total glucosinolate (gs) content of the leaves of the cultivated moringa oleifera accessions, and the local conditions of the three experimental fields in the three agro-ecological zones. variables (1) (2) (3) 4) (5) (6) (7) (1) total gs [μmol/g dw] 1 (2) n [%] 0.16 1 (3) s [%] 0.19 0.78** 1 (4) elevation [m] 0.07 0.87** 0.78** 1 (5) maximum temperature [°c] 0.18 -0.40** -0.16 -0.47** 1 (6) minimum temperature [°c] -0.18 0.40** 0.16 0.47** -1.00** 1 (7) relative humidity [%] 0.07 0.87** 0.78** 1.00** -0.47** 0.47** 1 * correlation is significant at the 0.05 level (2-tailed); ** correlation is significant at the 0.01 level (2-tailed). effect of terroir on the glucosinolate content of moringa 89 the cultivated accessions were well-watered during the experimental period except on days with rainfall. this also indicates that the plants had sufficient water supply for vegetative growth; hence gs synthesis was not enhanced. impact of elevation on glucosinolate profile of moringa oleifera for the wild-grown m. oleifera, total gs content in the leaves significantly increased as elevations decreased (tab. 6). presumably, high elevations have been associated with stresses, e.g., lower air temperatures, leading to poorer plant growth (boyer, 1982). however, in the present study, the stresses exist at the zone in the lowest elevation, i.e., the guinea savannah zone, rather than at the zones in the higher elevations (tab. 1). this has been reported in studies characterizing the guinea savannah zone as the zone with very harsh environmental conditions accompanied by lower annual rainfall and higher temperatures than the transitional and the deciduous forest zones (leary et al., 2013; padi, 2017). therefore, due to the longevity of perennial plants such as m. oleifera trees, the repeated seasonal stress in this zone probably increased gs accumulation (yang et al., 2018). in conclusion, this might have tendentiously increased total gs content in the guinea savannah zone in comparison to the two other zones, although the differences were non-significant (fig. 2). contrarily, for m. oleifera accessions cultivated under semi-controlled field conditions, no significant correlation was found between elevation and the total gs content (tab. 8). we expected that because temperature decreased significantly with increasing elevation and relative humidity increased significantly with increasing elevation (tab. 8), total gs content would decrease at the higher elevation. presumably, reduced impact of stress factors on plants prevents gs accumulation (stamp, 2004; yang et al., 2018). however, the pre sent study under semi-controlled field conditions contradicted this hypothesis. it is assumed that the differences in temperature and relative humidity values along the elevation gradient at the time of the experiment were not strong enough to trigger a stress condition. therefore, a trade-off between primary metabolism and secondary metabolism, as depicted by hartmann (2007), did not occur to enhance gs accumulation. although not considered in the pre sent study, other environmental factors, including radiation, could also interact with, e.g., temperature to influence gs accumulation (schonhof et al., 2007b). these authors suggested that temperature and radiation significantly contributed to influence gs content in broccoli in a manner that increasing temperature in combination with increasing radiation significantly increased gs content. putatively, secondary plant metabolites can be altered in response to changing environmental conditions along the elevational gradient, and this phenomenon can vary across species. it, therefore, becomes more complex to elucidate the specific impacts observed in the pre sent study. therefore, for m. oleifera, further studies, including other environmental factors, are of particular relevance in the area of the present study. impact of different accessions on glucosinolate profile of moringa oleifera wild-collected leaf material for each agro-ecological zone was a composite of leaf material from different wild-grown m. oleifera with diverse genetics as well as origins. as sampling was not carried out based on accession/origin of the m. oleifera plants, the impact of accession on total gs content will not be discussed. however, for m. oleifera cultivated under semi-controlled conditions, in general, accession significantly differed in their total gs content (tab. 7a), with higher total gs content found in the accession 04024fl-041a than in the other two accessions (tab. 7c). this result is in accordance with a study, which found significant varying trends in the gs content of 30 different accessions of m. oleifera (doerr et al., 2009), and another study, where six ecotypes of m. oleifera showed high variability in their gs profile (förster et al., 2015). the authors attributed these differences to the plant genotypes. plants of different genotypes have different ways to adapt to their environments, altering the biosynthesis and accumulation of secondary metabolites (yang et al., 2018). the presented results showed significantly higher total gs content in the accession 04024fl-041a than in the accession, 08025fl-081d (tab. 7c), although the latter revealed a significantly higher number of leaves than the accession 04024fl-041a (tetteh, unpublished data). this could be attributed to the fact that gs synthesized in leaves of the accession 08025fl-081d were transported into more leaves than in the accession 04024fl-041a with fewer leaves. thus, some form of dilution effect occurred to produce a lower yield of gs in the accession 08025fl-081d than in the accession 04024fl-041a. this assumption is confirmed by a study on white cabbage in which leaf expansion was found to be accompanied by dilution of gs (pocock et al., 1987). impact of fertilization and cultivation practices on glucosinolate profile of moringa oleifera results of the wild-collected m. oleifera leaf material revealing no influence of s on total gs content (tab. 6) conforms with a study identifying no influence of s supply on gs content in leaves of m. olei fera (förster et al., 2015). further, our results showed that increasing n content decreased total gs (tab. 6), which corroborates, in part, with a study on broccoli heads indicating a decline in gs content with increasing n content, however only in combination with insufficient s supply (schonhof et al., 2007a). the present study suggests that regardless of s supply, increasing n content decreased total gs content significantly in wild-grown m. oleifera (tab. 6). in contrast, n and s contents had no significant influence on total gs content in the cultivated accessions of m. oleifera (tab. 8). this observation indicates that plants were presumably growing under optimal cultivation conditions, including suitable temperature (roloff et al., 2009), adequate water supply, and rainfall. plants had also been fertilized with compost manure, a rich source of nutrients, including n and s (amanullah and muthukrishnan, 2010), which had a significant positive correlation between n and s (tab. 8). consequently, increasing n and s contents increased the leaf numbers of the cultivated accessions significantly (tetteh, unpublished data). this suggests that plants growing in a resource-rich environment invested their resources into leaf production instead of the enhanced production of secondary plant metabolites (stamp, 2004). for m. oleifera accessions cultivated under semi-controlled conditions, no influence of fertilizer application on the total gs content in leaves was found (tab. 7a). the results were supported by the hypothesis of growth differentiation balance. this hypothesis describes the physiological trade-off between growth and differentiation processes regarding nutrient availability, influencing the accumulation of secondary metabolites (herms and mattson, 1992). therefore, in plants grown in nutrient-rich soil, biomass production is being promoted to a greater extent than the synthesis of secondary metabolites such as gs (stamp, 2004; hartmann, 2007). accordingly, fertilization did not significantly influence total gs content (tab. 7d). however, fertilization significantly influenced the number of leaves of m. oleifera, i.e., the fertilized variants had significantly higher leaf numbers than the non-fertilized plants (tetteh, unpublished data). for further studies, different fertilizer rates and a higher frequency of poultry manure compost application for a longer period than six months might be recommended to better understand the impact of fertilization on gs synthesis in m. oleifera growing under semi-controlled field conditions. 90 o.n.a. tetteh, s. huyskens-keil, n.k. amaglo, f.k. amagloh, i.n. oduro, c. adarkwah, d. obeng-ofori, c. ulrichs, n. förster an additional trend observed in the cultivated accessions under semicontrolled field conditions was that total gs content in the second harvest was significantly higher than in the first harvest (tab. 7e), which is consistent with findings of studies on m. oleifera (doerr et al., 2009). the significant interaction effect of agro-ecological zone×fertilization×harvest time for total gs content (tab. 7a) contributed to this trend observed. assumingly, seasonal variation at the two harvest times contributed to the differences in total gs content, and thus, m. oleifera accessions adapted differently under prevailing environmental conditions at the two harvest times resulting in their different total gs content identified (charron et al., 2005). with these findings, it has been shown that under semi-controlled field conditions, the various independent factors, i.e., accession, agro-ecological zone, fertilization, and harvest time, interacted significantly in terms of changes in the total gs content in m. oleifera. however, for further studies, the impact of single climate parameters and other abiotic factors on gs content needs to be more specific. to summarize, choosing the appropriate accession and cultivating under appropriate cultural practices at a location with suitable environmental conditions is essential to produce plants with high healthpromoting properties in m. oleifera in terms of gs content. conclusion this study provides clear evidence that temperature, relative humi dity, and rainfall had no significant influence on gs content in the leaves of both the wild-grown and cultivated accessions of m. olei fera. rainfall in the three agro-ecological zones was at its peak at the time of harvest, and their amounts were not significantly different. therefore, wild-grown plants, assumingly, did not experience water stress conditions, and thus, no significant changes in gs content occurred. this implies that wild-grown m. oleifera leaves from the three agro-ecological zones would have nearly similar gs contents when harvested during the rainy season, which is a beneficial outcome for moringa producers and processors in ghana. this outcome could also be applicable in other tropical areas that have a similar climate as ghana. regardless, further studies considering different seasons (i.e., the dry and rainy seasons) in the study area may help to better understand the specific impacts of each of the different climate parameters on the dynamics of gs synthesis in m. oleifera leaves. in determining the influence of elevation on the total gs content for both the wild-grown and cultivated accessions of m. oleifera, contradictive results have been identified. future work is certainly required to further elucidate how the changes of eco-physiological factors along the elevational gradient influence gs accumulation in both wild-grown and cultivated accessions of m. oleifera. by determining the influence of soil minerals on the gs profile, we identified that for wild-grown m. oleifera, increasing n content significantly increases gs accumulation irrespective of s supply. however, for cultivated accessions, increasing composted poultry manure to increase n and s content during the rainy season did not affect gs accumulation. finally, the appropriate selection of m. oleifera accessions, which differed in their total gs content, and cultivation under favorable environmental conditions, should be envisaged to produce healthpromoting m. oleifera plants. considering that genetics and environmental conditions interact and influence the performance of accessions, genome analyses of accessions and their impact on gs could be of further interest. acknowledgment the authors are grateful to the daad in partnership with the govern ment of ghana scholarship secretariat, humboldt-universität zu berlin, germany, and the university of energy and natural resources for co-funding this study. also, we would like to thank echo, florida, usa, for providing the seeds for this study. heartfelt gratitude to dr. iddrisu wahab abdul of the university of energy and natural resources, sunyani, 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y.x., wei, f., wang, q., 2018: response of plant secondary metabolites to environmental factors. molecules 23, 1-26. doi: 10.3390/molecules23040762 zandalinas, s.i., vives-peris, v., gómez-cadenas, a., arbona, v., 2012: a fast and precise method to identify indolic glucosinolates and camalexin in plants by combining mass spectrometric and biological information. j. agric. food chem. 60, 8648-8658. doi: 10.1021/jf302482y orcid christian ulrichs https://orcid.org/0000-0002-6733-3366 susanne huyskens-keil https://orcid.org/0000-0002-7174-4176 nadja förster https://orcid.org/0000-0002-9732-8939 olivia tetteh https://orcid.org/0000-0002-0227-107x ibok nsa oduro https://orcid.org/0000-0003-3731-2684 newton kwaku amaglo https://orcid.org/0000-0002-8447-3256 francis kweku amagloh https://orcid.org/0000-0001-7243-0972 charles adarkwah https://orcid.org/0000-0003-4619-2434 address of the corresponding author: olivia naa ayorkor tetteh, division urban plant ecophysiology, faculty of life sciences, humboldt-universität zu berlin, germany e-mail: olivia.tetteh@uenr.edu.gh © the author(s) 2021. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1016/j.phytochem.2007.09.017 http://dx.doi.org/10.1017/cbo9781107415324.004 http://dx.doi.org/10.4324/9781315067179 http://dx.doi.org/1017/s0021859697004292 http://dx.doi.org/10.1007/s12223-010-0071-0 http://dx.doi.org/10.4172/2332-2594.1000211 http://dx.doi.org/10.4161/psb.6.11.17613 http://dx.doi.org/10.1002/jpln.200620639 http://dx.doi.org/10.1016/j.agee.2006.06.018 http://dx.doi.org/10.1093/pcp/pct054 http://dx.doi.org/10.1111/j.0030-1299.2004.12039.x http://dx.doi.org/10.1093/jxb/erg215 http://dx.doi.org/10.1016/j.scienta.2018.11.089 http://dx.doi.org/10.1080/03670240902794598 http://dx.doi.org/10.1016/b978-0-12-800876-8.00012-6 http://dx.doi.org/10.1016/j.phytochem.2014.03.028 http://dx.doi.org/10.3390/molecules23040762 http://dx.doi.org/10.1021/jf302482y journal of applied botany and food quality 92, 187 191 (2019), doi:10.5073/jabfq.2019.092.025 research and development, repha gmbh – biologische arzneimittel, germany four examples demonstrating the impact of applied botany on plant-based industrial processes m. kleinwächter* (submitted: december 21, 2018; accepted: july 18, 2019) * corresponding author summary currently, many producers of plant-derived commodities indicate a scarcity of associates whose skills cover the entire field of plant biology and who bolster industrial research by linking it to basic plant biology. this scarcity is of particular concern to small and medium sized companies. to illustrate the benefit of appropriate mediation between basic science and product-oriented research, four innovative examples of collaborative research are presented here. the examples cover a broad range of economically relevant issues, including green coffee processing, malting, production of spice and medicinal plants, and prevention of contamination with toxic natural products, such as nicotine or pyrrolizidine alkaloids. these examples illustrate that applied botany has the potential to improve even well-established production processes. this article argues that innovative product-oriented research must focus on the relevant physiological processes occurring in the plants. in particular, the impact of cultivation and post-harvest processes on related metabolic processes should be considered, rather than placing continued focus on physical parameters or on economic aspects. in order to achieve practical and feasible solutions that also meet economic demands, interdisciplinary and cross-functional approaches between partners are essential. keywords: applied botany; physiological processes; product quality; interdisciplinary work, feasible solutions applied botany: modern plant biology in the context of industrial research physiological processes determine the material composition and the variability of living organisms. the material composition is of particular interest because it determines the product quality of crops and plant-derived commodities. this applies to physiological processes occurring during plant growth and development as well as during postharvest treatment and further processing. thus, physiological processes are directly linked to product quality. in principal, numerous publications exist dealing with the influence of physiological processes on the quality of plant-derived products. a well-known example of the deliberate utilization of physiological processes to enhance plant growth and development is the use of carbon dioxide (co2) to fertilize plants in greenhouse cultivation. it is widely understood that raising atmospheric co2 concentration increases the photosynthetic rate (e.g., schopfer and brennicke, 2010), which results in higher plant productivity and greater yields. related experimental trials have been performed with various crops, such as tomatoes (solanum lycopersicum; e.g., wittwer and robb, 1964; zhang et al., 2014), cucumbers (cucumis sativus; e.g., ito, 1973) and sweet peppers (capsicum annuum; e.g. nederhoff, 1994). for a review of this topic, see prior et al., 2012. due to the highly beneficial effects of enhanced atmospheric co2 concentrations, it is commonplace in industrial greenhouse production to apply elevated co2 concentrations. while the impact of various growth and cultivation conditions on the quality of crops has been relatively well analyzed, knowledge regarding the effects of postharvest procedures and processing techniques on plant material is scarce. this is especially true for delibe rate manipulations of physiological processes through alteration of complex processing techniques. however, a prominent exception can be found in the postharvest physiology of several economically relevant fruits. a common method to influence fruit ripening is the deliberate application of the plant signal transducer ethylene or, depending on the desired purpose, the prevention of its biosynthesis. ethylene is known to induce fruit ripening and senescence processes, so it is frequently administered after fruit storage in order to induce and synchronize fruit ripening (e.g., cornelius and barry, 2007; bapat et al., 2010). on the other hand, if the retardation of ripening processes is desired during fruit storage, the synthesis of ethylene is deliberately prevented by inhibiting the activity of the key enzyme in its biosynthesis, namely the 1-aminocyclopropane-1-carboxylic acid (acc) synthase. since acc synthase is inhibited by high co2 concentrations (e.g., de wild et al., 2005), increasing co2 concentration in the storage compartment or during transport can prolong the shelf life of various fruits, such as apples, pears, bananas and tomatoes, up to several months (for a review, see paul and pandey, 2014). however, in many other areas the underlying plant physiological processes have not been adequately considered, despite their tremendous relevance to product quality. related activities stand isolated and no systematic approaches are available to exploit the potential of physiological processes. this deficit is particularly clear in the development of alternative cultivation and postharvest processing techniques. one reason for overlooking basic plant physiological aspects may be that engineers involved in industrial research are primarily trained in technical subjects. scientists exhibiting comprehensive experience in plant physiology as well as ample knowledge of metabolic processes are quite rare. moreover, economic aspects often play a superior role. producers are driven to optimize processing methods mainly due to economic considerations, while lower importance is placed on considerations of plant physiology. a further issue restraining industrial research is the limited availability and readiness of sophisticated plant biological laboratories to analyze fundamental and basic physio logical processes. this is particularly restrictive for small and medium sized companies, which frequently are not capable of performing the necessary sophisticated and expensive molecular-biological, biochemical and phytochemical techniques. moreover, in many cases even awareness of the relevance of physiological processes for pro duct quality is lacking. in addition, it is worth noting that the flow of knowledge between plant basic science and industrial applied research is very limited. the poor exchange between the sectors may be due to a lack of connection and communication platforms. in exaggerated terms, many plant scientists perch in a kind of ivory tower of basic science, while companies emphasize the protection of intellectual property rights and the thwarting of competitors. in this regard, a modern applied botany that is focused on plant physiology has the potential to act 188 m. kleinwächter as a mediator for the transfer of botanical knowledge and analytical methods from basic plant science to industrial research, and vice versa. as mentioned previously, further research is needed that focuses on the metabolic impacts of cultivation and postharvest production processes on the quality of plant-derived commodities. improved understanding of plant physiological processes and their regulation is an important tool to improve product quality. accordingly, the relevant processes should be analyzed thoroughly using modern plant biological methods such as genomic, proteomic and metabolomic approaches. this, however, requires combining the expertise of plant physiologists and industrial researchers. interdisciplinary and cross-functional project committees, consisting of plant biologists and industrial representatives, should be formed to define objectives, evaluate results and elaborate corresponding solution strategies. continuous interdisciplinary exchange and collaboration promotes the development of solutions to ongoing problems and ensures rapid transfer into practice. in the next section, four innovative approaches in modern applied botany highlight how the application of basic plant physio logy can improve traditional industrial production processes and the quality of the plant-derived commodities produced. exemplification of the ample capabilities of applied botany as outlined above, the quality of plant-derived commodities can be modulated by altering either the conditions of plant growth (preharvest) or those of postharvest processing. however, in many cases no clear differentiation can be made. for example, malting of barley and processing of green coffee are frequently characterized as postharvest seed treatments; however, with respect to germination and seedling development, they in principle represent pre-harvest processes. in addition to these instances, whose denomination as preor post-harvest process seems to be problematic, two further examples are described that represent unambiguously pre-harvest processes. these are the impact of drought stress to increase the quality of medicinal and spice plants and the horizontal transfer of natural plant products as a source of contamination. drought stress: optimizing the cultivation of medicinal and spice plants it is well known that spice and medicinal plants grown in the mediterranean region produce a more intense flavor than the same plants cultivated in central europe. it may be tempting to explain this phenomenon by the much higher light intensity in the mediterranean area, but from the perspective of a plant physiologist, this deduction can be ruled out because even in central europe plants are gene rally exposed to an excess of light (wilhelm and selmar, 2011). however, another factor that differs markedly between central and southern europe corresponds to the water supply. a comprehensive literature survey revealed that in many plant species drought stress causes enhanced concentrations of secondary metabolites, and that in many cases also their overall contents are increased (selmar and kleinwächter, 2013a). until recently, a sound scientific rationale for this common phenomenon was lacking. today the basic relation is understood: water shortage induces stomata closure, which strongly reduces co2-influx into the leaves. as result, far fewer reduction equivalents (nadph+h+) are consumed and re-oxidized via the calvin cycle. despite the fact that the various energy dissipating mechanisms are up-regulated, the reduction status of the chloroplasts increases significantly. in consequence, the ratio of nadph+h+ to nadp+ is greatly increased, and, according to the law of mass action, all processes consuming nadph+h+ (e.g., the biosynthesis of highly reduced secondary plant products) will then be favoured even without any change in enzyme activity (selmar and kleinwächter, 2013a). these fundamental plant physiological conside rations have been verified by ingenious drought stress experiments employing sage (salvia officinalis) as a model plant (nowak et al., 2010). the exogenous co2 concentration was varied to compensate for the stress-related decrease of endogenous co2 concentration. in consequence, the over-reduction related to drought stress was decreased, and accordingly, the stress-related increase in biosynthesis of secondary plant products (monoterpenes) was diminished. based on these findings, general cultivation practices for the production of medicinal and spice plants exhibiting enhanced contents of natural products have been elaborated (selmar and kleinwächter, 2013b; paulsen et al., 2014; bloem et al., 2014, kleinwächter et al., 2015). by deliberately inducing drought stress, either by reducing irrigation or by enhancing drainage, the quality of spice and medicinal plants can be improved. another approach, which is directly derived from this nexus and is very promising with respect to the “non-arid” conditions prevailing in central europe, concerns the application of methyl jasmonate (me ja). analogous to drought, the application of this signal transducer should induce stress-related metabolic responses, including the increased content of seconda ry plant products. similar approaches have already been successfully introduced for the cultivation of grapevines (vitis vinifera l.). vezulli et al. (2007) demonstrated that me ja treatment significantly increased the resveratrol content in cultivated grapes. these results have been verified by corresponding experiments employing nasturtium (tropaeolum majus l.) in which the me ja application resulted in an increase in glucosinolate content of more than 70% (bloem et al., 2014). meanwhile, this approach was also successfully scaledup to field conditions. consequently, the next step would be the approval of a corresponding preparation for commercial use. horizontal natural product transfer: contaminations of tea, spice and medicinal plants with nicotine and pyrrolizidine alkaloids the contamination of plant-derived commodities, such as herbal teas, spices and phytopharmaceuticals, with toxic natural products, like nicotine and pyrrolizidine alkaloids (pas), is a great challenge for producers and the processing industry (efsa, 2011; bfr, 2013). in the case of pas, one putative path of contamination has been identified, the mistaken co-harvesting of pa-containing weeds, but the source of nicotine contamination was fully unknown until recently. similar to the well-known uptake of organic xenobiotics (e.g., trapp and legind, 2011), it was assumed that the alkaloidal contaminations could result from the uptake of these natural products from the soil. by applying cigarette tobacco to the soil of various experimental plants, this hypothesis was proven; all plants took up high amounts of the alkaloid leached out from the decaying tobacco (selmar et al., 2015). in order to outline the relevance for practical application, this issue was also investigated under field conditions. it was illustrated that even one discarded cigarette butt per square meter is sufficient to generate nicotine concentrations in the acceptor plants ten times higher than the allowed maximum residue level (selmar et al., 2018). in consequence, any discarding of cigarette butts in the fields has to be prevented, especially when extensive manual work is required in regions where smoking is common. accordingly, this practice needs to be included in the code of good agricultural and collection practice (gacp). similar to the uptake of nicotine, plants also import pas in significant amounts from the soil. corresponding studies have revealed that the application of only 1 g of dried pa-containing plant material to the soil surface results in contaminations of up to 500 μg/kg dry weight in all plants grown in the vicinity. in consequence, these plants cannot be utilized as phytopharmaceuticals (nowak et al., 2016). these findings have important implications for weed ma the impact of applied botany on plant-based industrial processes 189 nagement. when pa-containing weeds (e.g., senecio vulgaris l.) are killed with herbicides or hoed up, the plant material must be removed from the fields in order to prevent any pa transfer to the crop plants and thus entrance of pas into the food chain. based on these insights, the immediate removal of toxic weeds has already become common practice for producers of spice and medicinal plants. for instance, large firms such as the martin bauer group and waldland international instantly adopted this approach and now insist on the removal of pa-containing weeds from the fields. recent studies have revealed that the translocation of natural pro ducts from decaying plant materials into other plant species is a common occurrence that depends primarily on the physicochemical properties of the compounds, specifically membrane permeability and solubility (selmar, radwan and nowak, 2015). in analogy to horizontal gene transfer, the interspecific transfer of natural pro ducts has been denoted as “horizontal natural product transfer.” it is important to be aware that apart from nicotine and pas many other natural products leached out from decomposing plant materials re present potential sources of further contaminations. in this regard, it is noteworthy that the european food safety authority (efsa) recently published a survey on the occurrence of tropane alkaloids in herbal teas (efsa, 2018). it is a well-known feature in chemical ecology that plants release allelochemicals in the soil, which can affect the germination and growth of competing plants in the vicinity (e.g., bertin, yang and west, 2003; kalinova, vrchotova and triska, 2007). accor dingly, a direct transfer of allelochemicals between living plants has to be assumed. despite this well-established knowledge, a corresponding transfer of natural products that do not have an allopathic effect, was not taken into consideration by industry as a potential contamination source. meanwhile, it was demonstrated that not only rotting plant materials but also living plants can act as pa donors (selmar et al., 2018). accordingly, the concept of horizontal natural product transfer was extended. indeed, up to now, the exact mechanism of transfer is still unknown. however, co-culture experiments with ragwort (senecio jacobaea) and parsley (petroselinum crispum) clearly demonstrated that the alkaloids are transferred via the soil, either due to an active exudation or as a result of minor injuries to the roots. with respect to the prevention of contamination, these results indicate that current weed management needs to be improved: instead of hoeing pa-containing weeds, they should be extracted entirely, including the roots, and removed from the site. the corresponding transfer into practical application is currently on the way. in addition to the relevance for contamination of plant-derived commodities, the phenomenon of horizontal transfer of natural products may have significance for basic plant biology and agricultural production in general. with respect to agricultural practices, it may improve understanding of various hitherto unexplained effects in agricultural practice, such as the benefits of crop rotation and co-cultivation. germination and stress: metabolic changes in coffee seeds during green coffee processing just like oil, wheat or soybeans, coffee represents one of the top ten commodities in global trade. in the past, green coffee, the tradable seeds of the coffee tree (coffea arabica l.), had always been regar ded as inanimate plant material. in the coffee processing industry, only the basic chemical and physical parameters had been consi dered in the approaches developed to increase product quality. this perspective applied to all aspects of green coffee processing, inclu ding postharvest treatments like depulping and drying. in contrast, the perspective of plant biologists is quite different, and the various metabolic reactions of green coffee are in the center of focus. with respect to germination physiology, one has to be aware that the seeds of tropical plants are not dormant (e.g. pammenter and berjak, 2000; chin, 1978). accordingly, as soon as they are exposed to favorable conditions, they will germinate. moreover, any decrease in the water content of vital cells (e.g., during drying) induces various meta bolic stress responses (radwan et al., 2014). substantial research on this topic was triggered by the elucidation of well-known quality differences between wet and dry processed green coffees. since the conditions affecting metabolic responses are quite different in each type of processing, the underlying metabolic bases were intensively studied. it was revealed that in the first phases of raw coffee processing, especially in the course of wet processing, the seeds do indeed germinate (selmar and bytof, 2006). subsequently, depending on the speed of the drying, drought stress induced metabolic responses are dominant and substantially change the composition of the coffee beans (knopp et al., 2006; kleinwächter and selmar, 2010; kramer et al., 2010). as consequence of these plant physiological insights, a paradigm change in the coffee producing industry was initiated (selmar, bytof and kleinwächter, 2014). nowadays, it is common knowledge that raw coffee seeds are living organisms with an active metabolism, offering great potential for quality improvement (selmar and bytof, 2006; kleinwächter and selmar, 2010). it is intriguing to realize that by understanding and conside ring the plant’s physiology, it became possible to deliberately change composition of green coffee by altering the processing conditions. thus, plant physiology was the key for improvement of green coffee quality by altering conditions impacting the most relevant aroma precursors (kleinwächter, bytof and selmar, 2014). meanwhile, such approaches led to physiologically based optimizations of various steps in coffee processing in order to improve the aroma potential of raw coffees (kleinwächter, bytof and selmar, 2014). a relevant example for the successful implementation of plant physio logical knowledge into green coffee processing practice concerns the modification of the so-called becolsub process. this type of green coffee processing falls in between the classic dry and wet processing. similar to wet processing, the coffee cherries are depulped; however, the fermentation step, which is responsible for the degradation of residues of the fruit flesh, is substituted by mechanical removal of the sticky pulp. because of its economic advantages, coffee producers immediately favored this procedure. unfortunately, the quality of the corresponding coffees was far lower than that of the wet fermented ones. based on this research, it was possible to predict that additional storage of the mechanically cleaned beans could solve this problem. today, such interim storage is implemented worldwide by coffee producers. germination and stress: the impact of malting on the physiology of barley seedlings archeological findings indicate that malting and beer brewing was practiced in ancient egypt. thus, the malting process surely is one of the oldest techniques invented for food processing. one of the basic ingredients of beer apart from yeast and hope is malt. malt is produced by germination of cereal seeds, like barley (hordeum vulgare l.). the malting process can be divided into three steps; the steeping, the germination and the kilning. in the first step, the seeds are imbibed for several hours in water in order to induce seed germination. then, the germination process induces the mobilization of starch and the breakdown of cellular structures. in the final malting step, the germination process is terminated by drying the seedlings in kiln ovens. the high temperatures in kilning are responsible for the gene ration of the typical color and flavor compounds of malts (for detailed information on malting, refer to kunze, 2010; narziss, 1999). as previously noted, the malting process is among the earliest food processing techniques established by man. despite its long history, many unanswered questions remain if one starts to analyze the 190 m. kleinwächter processing through the eyes of a plant physiologist. with respect to malting physiology, the most intriguing issue concerns the role and impact of co2. plant physiologists consider co2 an inert gas that is the limiting factor of the photosynthesis. in contrast, in the malting business co2 is considered a toxic compound that has to be efficiently removed from the seedling by excessive aeration. in order to explore this contradiction and to elucidate the actual impact of co2 during malting, the relevant physiological processes occurring in the barley seeds in the steep tanks were analyzed. as expected, it was demonstrated that co2 does not exhibit any toxic effects. however, high concentrations of this gas severely inhibit the biosynthesis of germination-related enzymes, although the seeds are still viable even after being incubated under extremely high co2 concentrations (kleinwächter, meyer and selmar, 2012). indeed, up to now, the exact mode of action is still unknown, but there are some indications of possible competitive interactions between oxygen and co2 (kleinwächter, meyer and selmar, 2012). another important problem in industrial-scale malting comes from biochemical heterogeneities in the malt. these variations result in asynchronous degradation of carbohydrates. in consequence, insufficient hydrolysis of galactomannans located in the cell wall causes severe problems in the brewing process by blocking filters during lautering (palmer, 2000; de sá and palmer, 2004). based on comprehensive analyses of the physiological processes occurring in the barley seeds during malting, the biochemical background of these heterogeneities was unveiled. the combination of high layers of malt beds (3-4 m) and inhomogeneous aeration in the steeping tanks causes significant spatial differences in the partial pressures of oxygen and carbon dioxide (kleinwächter et al., 2014). as consequence, the progression of germination differs, creating large biochemical heterogeneities. in conclusion, the primary cause of insufficient hydrolysis of galactomannans, and thus the blockage of filters during lautering, is related to asynchronous germination depending on position in the steep tank. accordingly, simple modifications in the process control (changes in the extent of aeration and enhancement of the steeping temperatures) enable the production of malts exhibiting enhanced homogeneity (kleinwächter et al., 2014; müller et al., 2013). this example clearly demonstrates that even well-established processes can be optimized by transferring basic plant physiology knowledge into industrial research. in this sense, it may be worth revisiting other well-established production processes through the eyes of a plant physiologist. conclusions these four examples highlight that plant physiological processes play a key role in the quality of plant-derived products. consequently, an increased understanding of the relevant processes enables delibe rate improvement of product quality. yet, it is not sufficient to focus solely on physiological process. rather, the greatest improvements can be made by comprehensively considering plant metabolism and its complexity in a systemic manner, especially in interaction with the environment. in addition, these examples demonstrate that the transfer of basic plant science knowledge into industrial research could initiate strong impulses for product-related research. this even accounts for traditional and well-established processes, as has been clearly illustrated for the malting process. continuous interdisciplinary work and collaboration in project committees can enable the development of feasible and economically acceptable solutions that are directly transferable into practice. apart from the basic scientific approaches, a great challenge is the implementation of new concepts and ideas into product-related research. one future goal is to optimize knowledge transfer. thus, modern applied botany should seek to act as a mediator between basic plant science and industrial product-related research. in order to support sophisticated applied botanical approaches in germany, reconsideration of research funding practices and of the evaluation of scientific performance is required. indeed, the deutsche forschungsgemeinschaft (dfg) offers a variety of research programs that unfortunately are not available for applied research. moreover, when scientific performance is evaluated on the basis of funding raised, dfg projects are frequently rated two times better than projects funded by other sources. in this regard, a new approach is required to achieve the same support for basic and applied research. acknowledgements i thank dirk selmar (institute for plant biology, technische universität braunschweig) for critical counter-checking of the manuscript. in addition, i am deeply grateful to holger preibisch (german coffee association, hamburg), maximilian wittig (german tea association, hamburg), erika hinzmann (wifö der deutschen brauwirtschaft, berlin), barbara steinhoff (german medicines manufacturers’ association, bonn) and birgit grohs (forschungsvereinigung der arzneimittel-hersteller e.v., bonn) for their support and close cooperation. the same accounts for the “fachagentur für nachwachsende rohstoffe” (fnr), „arbeitsgemeinschaft industrieller forschungsgemeinschaften” (aif) and the „german egyptian research fund” (gerf), which delivered financial support. references bapat, v.a., trivedi, p.k., gosh, a., sane, v.a., ganapathi, t.r., nath, p., 2010: ripening of fleshy fruit: molecular insight and the role of ethy lene. biotechnol. adv. 28(1). doi: 10.1016/j.biotechadv.2009.10.002 barry, c.s., giovannoni j.j., 2007: ethylene and fruit ripening. j. plant growth regul. 26, 143-159. doi: 10.1007/s00344-007-9002-y bertin, c., yang, x., west, l.a., 2003: the role of root exudates and allelochemicals in the rhizosphere. plant soil 256, 67-83. doi: 10.1023/a:102629050 bfr, 2013: pyrrolizidinalkaloide in kräutertees und tees. stellungnahme 018/2013. retrieved 5th 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(ed.), dealing with contaminated sites, 369-408. springer, netherlands. vezzulli, s., civardi, s., ferrari, f., bavaresco, l., 2007: methyl jasmonate treatment as a trigger of resveratrol synthesis in cultivated grapevine. am. j. enol. vitic. 58, 530-533. wilhelm, c., selmar, d., 2011: energy dissipation is an essential mechanism to sustain the viability of plants: the physiological limits of improved photosynthesis. j. plant physiol. 168(2), 79-87. doi: 10.1016/j.jplph.2010.07.012 wittwer, s.h., robb, w.m., 1964: carbon dioxide enrichment of greenhouse atmospheres for food crop production. econ bot. 18(1), 34-56. zhang, z., zhang, m., zhang, y., wang, q., 2014: effect of carbon dioxide enrichment on health-promoting compounds and organoleptic properties of tomato fruits grown in greenhouse. food chem. 153, 157-163. doi: 10.1016/j.foodchem.2013.12.052 address of the corresponding author: maik kleinwächter, research and development, repha gmbh – biologische arzneimittel, alt godshorn 87, 30855 langenhagen, germany e-mail: maik.kleinwaechter@repha.de © the author(s) 2019. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1016/j.foodchem.2011.11.027 http://dx.doi.org/10.1016/j.foodchem.2013.09.090 http://dx.doi.org/10.1016/j.indcrop.2014.10.062 http://dx.doi.org/10.1016/j.foodchem.2009.06.048 http://dx.doi.org/1007/s00217-005-0172-1 http://dx.doi.org/10.1093/pcp/pcq019 http://dx.doi.org/10.1016/j.foodchem.2016.06.069 http://dx.doi.org/10.1002/j.2050-0416.2000.tb00056.x http://dx.doi.org/10.1017/s096025850000034 http://dx.doi.org/10.1007/s13197-011-0583-x http://dx.doi.org/10.21273/hortsci.46.2.158 http://dx.doi.org/10.1111/plb.12228 http://dx.doi.org/10.1007/978-3-319-76887-8_10-1 http://dx.doi.org/10.1007/s13593-015-0298-x http://dx.doi.org/10.1093/pcp/pct054 http://dx.doi.org/10.1016/j.indcrop.2012.06.020 http://dx.doi.org/10.1016/j.envpol.2018.01.113 http://dx.doi.org/10.4172/2161-0525.1000287 http://dx.doi.org/10.1016/j.jplph.2010.07.012 http://dx.doi.org/10.1016/j.foodchem.2013.12.052 mailto:maik.kleinwaechter@repha.de journal of applied botany and food quality 92, 151 159 (2019), doi:10.5073/jabfq.2019.092.021 1university of goettingen, department of crop sciences, division of quality of plant products, goettingen, germany 2university of eldoret, department of seed, crop and horticultural sciences, eldoret, kenya 3world agroforestry centre (icraf), nairobi, kenya (currently rhine-waal university of applied sciences, faculty of life sciences, kleve, germany) variation in fruit chemical and mineral composition of kenyan guava (psidium guajava l.): inferences from climatic conditions, and fruit morphological traits josiah chiveu1,2, marcel naumann1, katja kehlenbeck3, elke pawelzik1* (submitted: november 1, 2018; accepted: may 23, 2019) * corresponding author summary there is limited knowledge about the impact of climatic conditions and fruit morphological traits on the nutritional composition of the guava fruit. fruits were gathered from 128 guava trees across four geographically diverse regions of kenya. the fruits were morphologically characterized and analysed for their chemical and mineral composition. the ascorbic acid content correlated positively only with total annual precipitation, while total soluble solids (tss) correlated positively with mean annual temperature. tss correlated negatively with pulp weight and was higher in white-fleshed fruits than in the red-fleshed types. the mineral content of the fruits correlated negatively with most of the fruit weightand size-based morphological traits, as well as with the total annual precipitation, but positively with fruit seed proportion. this information could act as a guide in the selection of specific regions for upscaling guava production and aid in the selection of accessions for improvement programmes that enhance guava fruit nutritional composition. key words: ascorbic acid, fruit minerals, guava, psidium guajava l., pulp colour, tss introduction tropical fruits have considerable importance for developing countries from both nutritional and economic perspectives, with about 90% of these fruits being consumed in their countries of origin, while 10% are traded internationally as fresh fruits and processed products (available: http://www.fao.org/docrep/meeting/028/ma937e.pdf – accessed 22.02.2019). despite some efforts seen in the production of tropical fruits such as mangoes and avocados, the opportunities to grow, consume, and export more fruits from tropical regions remain under-exploited compared to those in temperate regions (griesbach, 2007). for instance, the supply of fruits and vegetables in lower income countries fall on average 58% short based on nutritional recommendations (siegel et al., 2014). consequently, low-quality nutritionally unbalanced diets are common in these regions, leading to high risks of nutrient deficiencies (arimond et al., 2010). research to improve fruit production, therefore, offers tremendous opportunities for raising the incomes of small-scale farming families in these regions while also improving their nutritional status, as observed by keding et al. (2017). guava (psidium guajava l.) is an important tropical fruit tree grown mainly for its edible fruits which are eaten raw or made into purée (pulp), jam, jelly, paste, juice, syrup, chutney, among other products (leite et al., 2006). the guava tree is cultivated in orchards and in home gardens in many tropical countries (cabi, 2013). in kenya, for example, the guava tree exists in all regions of the country (hcd, 2014) and mainly grows unattended. despite the lack of attention to guava tree husbandry, guava fruit production in kenya has recently seen an increase (hcd, 2014). however, most of these guava fruits are collected from the wild, and not much effort is put to improve tree husbandry and the production potential (mbuvi and boon, 2009). recent studies have reported an appreciable amount of antioxidant phytochemicals including ascorbic acid, carotenoids, flavonoid compounds and polyphenols in the guava fruit (araújo et al., 2015, flores et al., 2015; gull et al., 2012), which are essential dietary components (flores et al., 2013). besides, guava has been repor ted to contain substantial amounts of minerals such as k, p, and ca (ogoloma et al., 2013; natale et al., 2007), which could significantly contribute to meeting a person’s daily dietary requirements. detailed nutritional evaluation of guava for other mineral nutrients such as fe and zn is still needed. the nutritional composition of a fruit reflects the geographic region where the fruit tree grows and the mineral composition of the soil there (wall, 2006; forster et al., 2002). the traits also vary with climate (rodriguez-amaya et al., 2008), fruit maturity (gull et al., 2012), and cultivar (burlingame et al., 2009; toledo and burlingame, 2006a). soil quality determines the sustainability and productivity of any agro-ecosystem (forster et al., 2002). hence, the growth and development of a plant is a function of the soil-plant interaction and the prevalent weather conditions (haque et al., 2009). the nutritional composition of fruits may thus vary from continent to continent, from country to country, as well as from region to region within the same country due to changes in climatic conditions (haque et al., 2009) and soil quality parameters. however, there is limited data on the nutrient content of the guava fruit in relation to these variables (natale et al., 2007). the objectives of this study were as follows: (1) to characterize and correlate the variation in the fruit chemical and mineral composition of guava with climatic variables (temperature and precipitation), and (2) to determine if the fruit morphological traits (flesh colour, and sizeand weight-based traits) influence the chemical and mineral composition of the guava fruit and if they could be correlated. it is assumed that variations in fruit chemical and mineral composition are correlated to climatic and fruit morphological traits, which lead to their differences in guava fruits. the information would help establish the species’ actual and potential contributions to nutritional security, especially in relation to these factors. materials and methods sampling the regions for sampling in kenya were chosen based on their high guava fruit production trends (hcd, 2014). fruit sampling was carried out between september and november 2015. this was when the fruits were available and ready for harvesting in the specific regions. with the help of key informants and field guides, the main guavaproducing locations within the regions were identified. households http://www.fao.org/docrep/meeting/028/ma937e.pdf 152 j. chiveu, m. naumann, k. kehlenbeck, e. pawelzik and institutions were randomly selected within these locations, and trees with ripe fruits were targeted for fruit collection. the geo graphical locations of the trees were recorded with a handheld global positioning system (gps) (tab. s1). the latitudes and longitudes also enabled the retrieval of the mean annual temperature and annual precipitation data from worldclim—global climate data: http://www. worldclim.org/bioclim (fick and hijmans, 2017) for individual accessions (tab. s1). fig. s1 shows the monthly meteorological data (temperature, relative humidity [rh], and precipitation) based on the nearest meteorological station within the regions. healthy and clean fruits from 128 trees were collected from the coast (36 trees), eastern (12 trees), rift valley (19 trees), and western (61 trees) regions (fig. 1). fruit morphological characterization a descriptor list for mango (ipgri, 2006) was modified to accommodate guava fruit traits for characterization. the modification also considered the results of other guava characterization studies (e.g. singh et al., 2015; mehmood et al., 2014; nasution and hadiati, 2014; sharma et al., 2010) and the authors’ own observations. twenty fruits per tree were randomly collected for measurements in the laboratory at the world agroforestry centre (icraf), nairobi. during morphological fruit characterization, fruits that were found to be infested by maggots and could only be discovered after longitudinal dissection were not characterized. this reduced the number of accessions for morphological characterization. as the minimum number of fruits for sizeand weight-based fruit morphological cha racterization was set to be at least 20, the characterization was eventually carried out for fruits from 105 trees (coast = 23, eastern = 12, rift valley = 17, and western = 53), except for the characterization of fruit flesh colour, for which at least one fruit per tree was used. therefore, all the 128 trees were used for the determination of flesh colour. fig. 2 depicts the fruit and the various fruit parts which were measured. determination of fruit chemical and mineral composition since the chemical and mineral characterization of the fruits considered between five to 20 healthy and undamaged ripe fruits, fruits from all the 128 trees were characterized for their chemical and mine ral content. the ripeness of the fruits was determined as the yellow colour of the skin based on the colour chart of the royal horticultural society besides the softness of the fruits to touch (araújo et al., 2015; gull et al., 2012). the fruits were cleaned and separated into skin, pulp, and seeds, while the edible portion (pulp plus skin) was divided into two sub-samples. one fresh sample was used for the analysis of ascorbic acid content, tss, and titratable acidity (ta) immediately after processing. the other sub-sample was weighed and then freeze-dried. the freeze-dried sample was weighed again to determine the percentage of water loss. the sample was later used to analyse the protein, sugar (glucose and fructose), phenolic compounds, and mineral contents. all the results were expressed in fruit fresh weight (fw). fruit chemical analyses based on fresh weight the content of ascorbic acid was determined in fresh samples by reduction with 2,6-dichloroindophenol solution to a colorless dye using the titration method according to the procedure developed by puwastien et al. (2011). for better precision, the samples were measured titrimetrically. tss was measured by placing a few drops of guava juice squeezed from fresh fruits on a handheld refractometer. the values were read directly as % brix. ta was determined on the extracted guava juice from fresh fruits by titrating to a ph of 8.1 by adding 0.1n naoh according to the method by lmbg (1983). the result was expressed as mg of citric acid per 100 g of sample. fruit chemical and mineral analyses based on freeze-dried weight the phenolic compounds were extracted from 0.25 g of freeze-dried sample by adding 5 ml of 80% ethanol in a falcon tube. the tube was thoroughly vortexed and then centrifuged at 5,000 g for 10 minutes. the supernatant was transferred to a 10-ml flask. the extraction was repeated and the supernatants combined. the flask was then filled up to the 10-ml mark with 80% ethanol. the amount of the phenolic compounds was estimated in triplicate photometrically at 735.8 nm, immediately after extraction with the folin-ciocalteu reagent and expressed as mg per gallic acid equivalent (mg/gae), following the protocol developed by singleton and rossi (1965). sugars (glucose and fructose) were extracted from 200 mg of freezedried and milled guava fruit samples by adding 8 ml of pure water, vortexing, and then shaking the samples for one hour. thereafter, 0.5 ml of 0.25 m carrez i (containing potassium hexacyanoferrate (ii) trihydrate, k4[fe(cn)6] · 3h2o) and 0.5 ml of 0.09 m carrez ii (containing zinc sulphate heptahydrate, znso4 · 7h2o) were added to each sample and mixed by vortexing. the tubes were then centrifuged at 5,000 rpm for 20 minutes and the supernatant transferred into 25 ml volumetric flasks. the extraction was repeated by adding 7 ml of pure water. the volumetric flasks were then filled up to the 25-ml level and the extract filtered into scintillation vessels. soluble carbohydrates were separated according to the procedure used by keutgen and pawelzik (2008), and the sugar content (glucose and fig. 1: sample collection locations for guava accessions (crosses) from four regions of kenya with coast = 36, eastern = 12, rift valley = 19, and western = 61. http://www.worldclim.org/bioclim http://www.worldclim.org/bioclim variation in fruit chemical and mineral composition of kenyan guava 153 fructose) detected by high-performance liquid chromatography (hplc) (jasco, 26600 mary’s court easton, md 21601). proteins were extracted using the phenol protocol developed by faurobert et al. (2007) on 200 mg of milled freeze-dried samples. the proteins were measured photometrically, each in three replications, according to bradford (1976). fruit mineral contents − namely calcium (ca), magnesium (mg), potassium (k), sodium (na), phosphorus (p), sulphur (s), iron (fe), boron (b), zinc (zn), and copper (cu) − were extracted from 100 mg of each milled freeze-dried sample according to the procedure developed by wheal et al. (2011), and determined using inductively coupled plasma atomic emission spectroscopy (icp-aes) (vista-rl icp-oes, varian inc., usa). data analysis analysis of variance, mean separation, and correlation analyses for fruit traits were conducted using spss (version 20.0) (ibm, 2011). the test for normality and the resulting histograms showed that the data for morphological traits and fruit chemical composition was slightly skewed to the right. therefore, these datasets were log transformed to make the distribution normal. the data was thus analysed by analysis of variance (anova), followed by either a post hoc tukey test or independent sample t-test for mean separation. regarding the fruit chemical and mineral composition, analysis was first done per region to check for regional variations. next, mean annual temperature and annual precipitation data obtained from worldclim–global climate data: http://www.worldclim.org/bioclim (fick and hijmans, 2017) for individual accessions was correlated to the fruit chemical and mineral composition data. furthermore, the fruit chemical and mineral composition data was again tested for variation based on the colour of the fruit pulp and also correlated with the fruit morpholo gical traits. results fruit chemical and mineral composition and morphological traits based on region and climate the chemical and mineral composition and important morphological fruit traits of guava from the four mentioned regions of kenya are provided in tab. 1. the ascorbic acid content ranged between 10 mg/100 g fw and 360 mg/100 g fw, and was on average, highest in the eastern region and lowest in the rift valley region. the eastern region had 58% of the trees recording the ascorbic acid content of more than 100 mg/100 g fw, followed by western (36%), coast (19%), and rift valley (11%). on the other hand, tss ranged between 6-20% brix with the highest mean value recorded at the coast. consequently, higher fructose values were also recorded in samples from the coastal region, with the other regions recording similar lower values. however, the glucose content was higher and similar for the rift valley, western, and coastal regions. more than 79% of trees in each region had tss values above 9% brix. the least ta values were recorded in fruits from the rift valley region, while the highest was in the eastern region, although high variations were observed within regions. with regard to protein, mineral, and water content of the fruits, a significant variation among regions was observed, but also high variations were noted within regions. fruit weight was fairly uniform and did not differ among regions, but other fruit components like proportion of pulp and seed varied. the pulp proportion ranged between 11-48% of the total fruit fresh weight with the highest value being recorded in the eastern region. fruits from the coastal and western regions were seedier while those from the rift valley region were less seedy. tab. 2 shows a correlation of fruit chemical and mineral composition of individual accessions, with mean annual temperature and annual precipitation at their growth location. the ascorbic acid content correlated positively with annual precipitation (fig. s2) but negatively with mean annual temperature. similarly, the fruit water content cor fig. 2: guava fruit and fruit parts used in morphological characterization: (a) entire guava fruit, (b) fruit longitudinally cut into two parts, (c) pulp and seed removed with a spoon, (d) seed and pulp, hence their combined weight measured (e) pericarp, hence pericarp thickness and weight measured, (f) mesocarp removed from pericarp with a spoon and weight measured, (g) fruit exocarp/skin after removing the mesocarp, thickness and weight measured, (h) seed and pulp separated by a fruit mill and pulp weight measured, and (i) guava seeds washed and dried for weighing. http://www.worldclim.org/bioclim 154 j. chiveu, m. naumann, k. kehlenbeck, e. pawelzik related positively with annual precipitation, but it had a negative correlation with the mean annual temperature. however, tss (see also fig. s2), fructose, protein, and most of the fruit minerals (e.g. k, mg, na, p, s, and b) correlated positively with the mean annual temperature but negatively with annual precipitation. fruit chemical and mineral composition, and morphological traits based on pulp colour the chemical and mineral composition of the fruits based on fruit pulp colour is depicted in tab. 3. there was no variation in the as corbic acid content of the fruits. however, the content of phenolic compounds of the red-fleshed fruits (151.0 mg/100 g fw) was significantly higher than that of the white-fleshed fruits (137.6 mg/100 g fw). the ta did not vary with the fruit pulp colour, but the tss of the rather few fruits with white pulp was significantly higher than that of fruits from the red-fleshed group (12.6% vs 10.7%). similar to tss, the fructose content was also higher in the white-fleshed fruits than in the red-fleshed fruits. there was no variation in the glucose content with regard to fruit pulp colour. there was no variation in the fruit mineral contents of ca, fe, zn, and cu with respect to the fruit flesh colour. however, interestingly, the white-fleshed fruits were superior to the red-fleshed ones with regard to their protein content and the content of k, mg, na, s, p, and b. in contrast, the water content was higher in the pulpier red-fleshed group (84.4%) than in the less pulpy white-fleshed group (80.7%). correlation of fruit morphological traits and fruit chemical and mineral composition results of a correlation analysis of fruit chemical and mineral composition traits vs fruit morphological traits are presented in tab. 4. fig. s3 specifically depicts the correlation between pulp proportion and tss, as well as seed proportion and fruit water content. the contents of ascorbic acid, ta, fructose, and glucose, as well as the fruit mineral concentrations of zn and cu, showed no correlation with the fruit morphological characteristics; hence, they are not depicted in tab. 4. fruit weight generally correlated negatively with most fruit mine rals and protein, but positively with fruit water content. the pericarp proportion correlated positively with tss, but negatively with k. the proportion of the exocarp (fruit skin) correlated positively with tss, fructose, ca, and b, but negatively with the fruit water content. however, the percent mesocarp negatively correlated with the content of phenolic compounds, k, and b. the proportion of pulp negatively correlated with tss, protein, mg, na, and b, but was found to have a positive correlation with the fruit water content. seed proportion of the entire fruit positively correlated with tss, fructose, protein and most minerals such as ca, k, mg, s, b, and cu. seed proportion however negatively correlated with the fruit water content. discussion the effect of temperature and precipitation on fruit chemical and mineral composition the mean ascorbic acid content of fruits from the 128 guava trees tab. 1: fruit chemical and mineral composition, and morphological traits of 128 guava accessions sampled from four regions of kenya region of collection fruit chemical and mineral composition rift valley (n = 19) western (n = 61) coast (n = 36) eastern (n = 12) mean (n = 128) p-value ascorbic acid (mg/100 g fw) 43.7c (19%) 79.3ab (13%) 48.5bc (21%) 128.1a (11%) 66.1 (18%) < 0.001 total phenolic compounds(mg/100 g fw) 141.1a (2%) 148.6a (4%) 148.2a (4%) 157.9a (3%) 148.2 (4%) 0.425 tss (% brix) 8.98c (9%) 11.0b (6%) 13.0a (7%) 9.47c (5%) 11.0 (9%) < 0.001 ta (mg/100 g fw) 0.77c (60%) 0.93bc (55%) 0.98ab (53%) 1.14a (75%) 0.94 (64%) < 0.001 fructose (g/100 g fw) 2.42b (20%) 2.62b (22%) 3.64a (15%) 2.25b (17%) 2.81 (21%) < 0.001 glucose (g/100 g fw) 1.05ab (28%) 0.99ab (43%) 1.43a (36%) 0.87b (20%) 1.11 (39%) < 0.004 protein (mg/100 g fw) 1.42b (18%) 1.48b (27%) 1.68a (19%) 1.38b (16%) 1.52 (28%) < 0.001 ca (mg/100 g fw) 14.4a (13%) 12.5a (15%) 15.0a (13%) 9.27b (16%) 13.1 (15%) 0.001 k (mg/100 g fw) 259.1b (6%) 267.7b (7%) 393.3a (4%) 239.3b (4%) 293.7 (7%) < 0.001 mg (mg/100 g fw) 7.64b (18%) 8.96b (17%) 12.7a (9%) 8.36b (11%) 9.60 (17%) < 0.001 na (mg/100 g fw) 3.58a (48%) 1.57b (141%) 5.44a (28%) 1.15b (172%) 2.44 (91%) < 0.001 p (mg/100 g fw) 17.5a (11%) 11.3b (17%) 18.1a (10%) 10.7b (12%) 13.7 (16%) < 0.001 s (mg/100 g fw) 10.2b (15%) 10.1b (17%) 16.5a (10%) 9.93b (8%) 11.6 (17%) < 0.001 fe (mg/100 g fw) 0.45a (11%) 0.27b (6%) 0.37ab (7%) 0.36ab (9%) 0.33 (8%) < 0.001 b (mg/100 g fw) 0.15c (25%) 0.21b (33%) 0.27a (26%) 0.18bc (18%) 0.21 (33%) < 0.001 zn (mg/100 g fw) 0.09ab (0.8%) 0.04b (0.9%) 0.11ab (1.9%) 0.13a (0.8%) 0.08 (1.3%) 0.006 cu (mg/100 g fw) 0.13a (12%) 0.11ab (9%) 0.10ab (4%) 0.07b (4%) 0.11 (8%) 0.062 water content (%) 86.3a (0.7%) 85.1a (1.2%) 78.4b (1.5%) 88.4a (0.5%) 83.6 (1.5%) < 0.001 fruit morphological trait rift valley (n = 17) western (n = 53) coast (n = 23) eastern (n = 12) mean (n = 105) p-value fruit weight (g) 46.4a (8%) 48.9a (10%) 41.9a (10%) 51.9a (9%) 47.2 (9%) 0.283 % pulp 31.4ab (10%) 27.5bc (6%) 24.0c (7%) 34.4a (6%) 28.0 (8%) 0.001 % seed 7.43b (22%) 10.4a (10%) 10.1a (15%) 8.21ab (20%) 9.52 (15%) < 0.001 values within the same row show the geometric mean (antilog of the transformed means), followed by the percent standard deviation of the log transformed means in parenthesis. the mean values in the same row followed by the same letter are not significantly different at p < 0.05 according to anova followed by tukey hsd pairwise comparisons. ascorbic acid, tss, and ta were determined on extracted juice from edible portion of the fruit, total phenolic compounds were determined from 0.25 g of freeze dried sample, fructose, glucose, and proteins were determined from 200 mg of freeze-dried sample, while minerals were determined from 100 mg of freeze-dried sample. water content was determined as the difference between freeze-dried and fresh sample weights. pulp proportion (% pulp), and seed proportion (% seed) are based on the weight of the entire fresh fruit (g). variation in fruit chemical and mineral composition of kenyan guava 155 from four kenyan regions was 66.1 mg/100 g fw. according to food composition tables, the ascorbic acid content of guava is estimated to be 228.3 mg/100 g edible portion (lukmanji et al., 2008), which is higher than the observed value in our sample. the observed differences could be due to variation in the determination methods and the state of the sample at the time of analysis. the samples used in this study were partly transported over long distances to the laboratory and could only be analysed for ascorbic acid content the following day. however, variations ranging from 11% to 21% of the mean values were observed within the regions. based on mean annual temperature and total annual precipitation data from worldclimglobal climate data: http://www.worldclim.org/bioclim (fick and hijmans, 2017) for individual accessions, annual precipitation correlated positively with the ascorbic acid content of the fruits in this study, while the mean annual temperature correlated negatively with the ascorbic acid content. the effect of precipitation on the ascorbic acid content of guava has so far not been reported. however, gull et al. (2012) determined the ascorbic acid content in the pulp and peel of fully ripe guava fruits from three diverse regions of pakistan as ranging between 129.5 mg/100 g and 247.9 mg/100 g. the ascorbic acid composition of the fully ripe fruits was found to vary regionally, with higher values recorded in the higher-temperature regions (mean max./min. air temperature: 35/24 °c) than in moderate and colder areas (mean max./min. air temperature: 33/21 °c and 33/18 °c). the variation was attributed to climatic and soil factors. contrarily, thaipong and boonprakob (2005) found higher contents of ascorbic acid in the slightly colder winter season (mean max./min. air temperature: 31.8/20.8 °c) rather than in the hot summer season (mean max./min. air temperature: 33.6/24.5 °c) in guava fruits grown in thailand. the authors concluded that the effect of lower temperatures in winter during fruit development could not only retard the excessive loss of respiratory substrates but also increase the translocation of photosynthates to other parts of the plant, including the fruits. it should be noted that the mean annual temperature for individual accessions in the present study (21.2 °c), the mean minimum (16 °c), and the mean maximum (23.7 °c) are far below that reported by gull et al. (2012) and thaipong and boonprakob (2005), making a comparison of the results difficult. however, it can be assumed that the cooling effect and slightly lower temperatures associated with precipitation are a possible reason for the positive correlation between the ascorbic acid content and precipitation. thus in this study, an increase in precipitation was found to be important as it resulted in an increase in the ascorbic acid content of guava fruits. the observed tss value in the present study (mean = 11.0% brix) is similar to that reported by el-sisy (2013) in eight-year-old guava genotypes growing under uniform conditions for two seasons (ranging from 9.4% to 14.07%). el-sisy (2013) recorded higher values in the first season than in the second season, although both were under a uniform irrigation scheme, which highlights the variation in temperature as one of the factors influencing tss. the tss in the present study positively correlated with mean annual temperature but negatively with annual precipitation. these results partly agree with those of thaipong and boonprakob (2005), where lower tss values during the summer season were attributed to the higher moisture content. however, the findings of thaipong and boonprakob (2005) contrast with the positive correlation observed between temperature and tss in the present study. a possible explanation for the negative correlation of tss with preci pitation could be the dilution effect, which is a result of a higher soil moisture content leading to more water uptake and, hence, to water accumulation in the fruits, as also shown by the positive correlation between fruit water content and precipitation (tab. 2). in line with the observations of the present study, and using a 14c tracer to compare the effects of elevated temperature on sugar and acid accumulation in mandarin fruit grown under tunnel house experiments, marsh et al. (1999) reported a positive correlation between temperature and tss. fruit labelling with 14c showed that rising canopy temperatures reduced the amount of incoming photosynthates partitioned to citrate and increased the amount allocated to sugars. a likely scenario could also have occurred in our study with guava. the mean protein content of the guava fruit samples of the present study was 1.52 g/100 g fw, which is slightly lower than that reported in the food composition tables (2.6 g/100 g edible portion; lukmanji et al., 2008). similar to tss, the protein content also positively correlated with the mean annual temperature but negatively with the total annual precipitation. this trend was also observed with regard to most of the fruit minerals, which were also lower than that given in the food composition tables (except for na and fe) for ca (18 mg/ 100 g), k (417 mg/100 g), mg (22 mg/100 g), na (2.0 mg100 g-1), p (40 mg/100 g), fe (0.3 mg/100 g), zn (0.2 mg/100 g), and cu (0.2 mg/ 100 g) (lukmanji et al., 2008). the state of the sample at the time of determination, the method of determination and possibly genetic differences among the accessions are the likely reasons for the observed variations. there is presently no report on the relationship between climatic conditions and guava fruit protein and mineral composition for comparison. however, the dilution effect as a result of higher moisture content is also the likely reason for the negative correlation between precipitation and these fruit components. this can also be confirmed by the positive correlation between the fruit water content and annual precipitation. the observed positive correlation of protein and some fruit minerals with temperature requires further investigation. fruit chemical and mineral composition tab. 2: pearson correlation coefficients between fruit chemical and mineral composition traits with annual mean temperature and annual precipitation based on the individual accession climatic data (tab. s1) of 128 guava accessions climatic data mean annual annual temperature precipitation ascorbic acid -0.22* 0.37*** total phenolic compounds ns ns tss 0.36*** -0.31*** ta ns ns fructose 0.18* -0.36*** glucose ns -0.30*** protein 0.35*** -0.47*** ca ns -0.25** k 0.30*** -0.45*** mg 0.27** -0.40*** na 0.44*** -0.71*** p 0.21* -0.54*** s 0.32*** -0.51*** fe ns -0.32*** b 0.28** -0.28** zn ns -0.24** cu ns ns water content -0.38*** 0.52*** ***correlation is significant at p ≤ 0.001 level. **correlation is significant at p ≤ 0.01 level. *correlation is significant at p ≤ 0.05 level. ns = correlation was not significant at p ≤ 0.05 level. ascorbic acid, tss, and ta were determined on extracted juice from edible portion of the fruit, total phenolic compounds were determined from 0.25 g of freeze dried sample, fructose, glucose, and proteins were determined from 200 mg of freeze-dried sample, while minerals were determined from 100 mg of freeze-dried sample. water content was determined as the difference between freeze-dried and fresh sample weights. http://www.worldclim.org/bioclim 156 j. chiveu, m. naumann, k. kehlenbeck, e. pawelzik tab. 4: pearson correlation coefficients between fruit morphological characteristics and fruit chemical and mineral composition from 105 guava trees summarized for all the regions fruit chemical and mineral composition total tss fructose protein ca k mg na p s b fe cu water phenolic compounds fruit weight ns ns ns -0.23* -0.49*** -0.28** -0.23* -0.23* -0.19* -0.26** -0.20* -0.33*** ns 0.26** % pericarp ns 0.22* ns ns ns -0.21* ns ns ns ns ns ns ns ns % exocarp ns 0.32*** 0.19* ns 0.20* ns ns ns ns ns 0.30** ns ns -0.19* % mesocarp -0.22* ns ns ns ns -0.25* ns ns ns ns -0.20* ns ns ns % pulp ns -0.45*** ns -0.31** ns ns -0.27** -0.22* ns ns -0.25** ns ns 0.28** % seed ns 0.42*** 0.28** 0.49*** 0.37*** 0.61*** 0.48*** ns ns 0.45*** 0.59*** ns 0.25** -0.48*** ***correlation is significant at p ≤ 0.001 level. **correlation is significant at p ≤ 0.01 level. *correlation is significant at p ≤ 0.05 level. ns = correlation was not significant at p ≤ 0.05 level. total phenolic compounds were determined from 0.25 g of freeze dried sample, tss was determined on extracted juice from edible portion of the fruit, fructose and proteins were determined from 200 mg of freeze-dried sample, while minerals were determined from 100 mg of freeze-dried sample. water content was determined as the difference between freeze-dried and fresh sample weights. pericarp proportion (% pericarp), exocarp proportion (% exocarp), mesocarp proportion (% mesocarp), pulp proportion (% pulp), and seed proportion (% seed) are based on the weight of the entire fresh fruit (g). fruit morphological characteristics tab. 3: chemical and mineral composition, and morphological traits of fruits from 128 guava accessions based on the fruit pulp colour irrespective of the region of collection fruit pulp colour fruit chemical and mineral composition white (n = 26) red (n = 102) mean (n = 128) p-value ascorbic acid (mg/100 g fw) 67.6a (17%) 65.7a (18%) 66.1 (18%) 0.860 total phenolic compounds (mg/100 g fw) 137.6b (3%) 151.0a (4%) 148.2 (4%) 0.020 tss (% brix) 12.6a (8%) 10.7b (8%) 11.0 (9%) <0.001 ta (mg/100 g fw) 0.92a (58%) 0.94a (65%) 0.94 (64%) 0.681 fructose (g/100 g fw) 3.29a (20%) 2.70b (21%) 2.81 (21%) 0.018 glucose (g/100 g fw) 1.28a (36%) 1.06a (40%) 1.11 (39%) 0.120 protein (g/100 g fw) 1.60a (26%) 1.50b (28%) 1.52 (28%) 0.007 ca (mg/100 g fw) 12.9a (17%) 13.1a (14%) 13.1 (15%) 0.852 k (mg/100 g fw) 346.2a (7%) 281.7b (6%) 293.7 (7%) 0.011 mg (mg/100 g fw) 11.8a (13%) 9.11b (17%) 9.60 (17%) 0.002 na (mg/100 g fw) 3.44a (74%) 2.24b (95%) 2.44 (91%) 0.016 p (mg/100 g fw) 16.0a (16%) 13.2b (16%) 13.7 (16%) 0.036 s (mg/100 g fw) 14.1a (16%) 11.0b (16%) 11.6 (17%) 0.006 fe (mg/100 g fw) 0.31a (6%) 0.34a (9%) 0.33 (8%) 0.368 b (mg/100 g fw) 0.24a (29%) 0.21b (34%) 0.21 (33%) 0.030 zn (mg/100 g fw) 0.07a (1.1%) 0.08a (1.3%) 0.08 (1.3%) 0.780 cu (mg/100 g fw) 0.09a (4%) 0.11a (9%) 0.11 (8%) 0.248 water content (%) 80.7b (1.8%) 84.4a (1.4%) 83.6 (1.5%) 0.002 fruit morphological trait white (n = 18 ) red (n = 87 ) mean (n = 105) p-value fruit weight (g) 49.5a (12%) 46.7a (9%) 47.2 (9%) 0.545 % pulp 23.6b (8%) 29.0a (7%) 28.0 (8%) 0.002 % seed 9.29a (17%) 9.57a (15%) 9.52 (15%) 0.745 values within the same row show the geometric mean (antilog of the transformed means), followed by the percent standard deviation of the log transformed means in parenthesis. the mean values in the same row followed by the same letter are not significantly different at p < 0.05 according to independent sample t-test. ascorbic acid, tss, and ta were determined on extracted juice from edible portion of the fruit, total phenolic compounds were determined from 0.25 g of freeze dried sample, fructose, glucose, and proteins were determined from 200 mg of freeze-dried sample, while minerals were determined from 100 mg of freeze-dried sample. water content was determined as the difference between freeze-dried and fresh sample weights. pulp proportion (% pulp), and seed proportion (% seed) are based on the weight of the entire fresh fruit (g). pulp colour influences chemical and mineral composition of guava fruits the findings related to the fruit flesh colour depicted red-fleshed fruits as having a higher phenolic content than the white-fleshed types. the results of this study were in agreement with those of santos and corrêa (2012), in which the pinkand red-fleshed guava accessions recorded greater values for phenolic compound concentrations. in contrast, hassimotto et al. (2005) found a higher phenolic content in white guava pulp than in red guava pulp (160 vs. 124 mg/100 g fw, respectively). phenolic compounds have been reported to be variation in fruit chemical and mineral composition of kenyan guava 157 affected by many other factors such as variety, cultivation, species, area, and climatic conditions (iqbal and bhanger, 2006; wang and lin, 2000). the total phenolic compounds have been reported to also vary with the cultivars (flores et al., 2015; wang and lin, 2000) and thus could indicate genotypic variation among accessions of guava. accordingly, we found that the white-fleshed guavas accumulated more tss and fructose than the red-fleshed types, which is in agreement with the findings of choudhary et al. (2012), who studied the chemical composition of four guava cultivars under similar cultural conditions and found variations in tss and non-reducing sugars, which were attributed partly to the variety of the fruit. moreover, we also observed that the white-fleshed guava fruits accumulated more protein and some minerals (k, mg, na, s, and b) than the red-fleshed ones. the variation in the accumulation of mine rals in the fruit, such as k, mg, s, and b, support the observation by natale et al. (2002, 2007) that different guava cultivars vary in their nutrient uptake. larger and heavier fruits negatively correlate with their chemical and mineral composition fruit weight correlated negatively with protein and with most fruit mineral contents. fruits with higher pulp weight negatively correlated with tss, protein, and a few minerals as mg, na, and b. in this regard, the results of the present study were similar to those of thaipong and boonprakob (2005) − larger fruits resulted in lower tss and total sugar content in guava. mehmood et al. (2014) and singh et al. (2015) also reported poor accumulation of chemical compounds in large guavas, including tss. similarly, negative observations of fruit size and weight with tss were observed during guava selection and breeding (dinesh and yadav, 1998), where the genotypic correlation was lower than the phenotypic correlation for tss, indicating a greater effect of fruit sizeand weight-based traits, along with external factors such as soil and environment, on tss. accordingly, pulp weight positively correlated with the fruit water content − an indication that much of the juicy pulp core in large fruits mainly consisted of water. this could be confirmed by the positive correlation between the pericarp and exocarp proportions with tss, implying a higher dilution effect of tss in the pulp core but no or less effect in the peel portion. in addition, the pulp proportion of the fruits negatively correlated with the fruit protein content and some of the minerals (mg, na, and b). similarly, negative correlations were also observed between the mesocarp proportion and some fruit minerals, mainly k and b. these negative correlations could still be attributed to the dilution effect of increasing fruit size on fruit mineral accumulation, as was also observed by singh et al. (2015), mehmood et al. (2014), and dinesh and yadav (1998) in guava. however, the peel part of the fruit (exocarp) is not likely to be affected by the dilution effect as evidenced by the positive correlation between the exocarp proportion with tss, fructose, ca and b. it was notable that seed proportion correlated positively with most of the fruit mineral and chemical consti tuents, but negatively with fruit water content. one may assume that increased seed proportion is likely to take up the position of water in the fruit pulp, thus reducing water accumulation in the pulp core of the guava fruit, as evidenced by the negative correlation observed between seed proportion and fruit water content. reduced water in the pulp core implies a reduced dilution effect for the fruit minerals and chemicals in the pulp. the fruit qualities of guava define their uses and vary by genotype, developmental stage, and growing environment (moon et al., 2018). qualities preferred for industrial/commercial production are out lined in pommer and murakami, (2009). for instance, preferred pulp colour for industrial production is dark rose, rosy or red. in our study, the red-fleshed fruits were more frequent, comprising of 80% of the total sample collected and were found in all the sampled regions. despite significant variations in important traits such as tss, the preferred threshold for industrial processing of at least 9% brix was met by most trees in all the regions, such as western (100%), coastal (97%), eastern (83%), and rift valley (79%). similarly, the preferred ta range of 1.25-1.5 mg/100 g fw was observed in fruits from 64% of trees in western region, 55% from coastal region, 50% from eastern region, and 32% from the rift valley region. substantial amount of ascorbic acid of at least 100 mg/ 100 g fw was found in fruits from 58% of the trees in eastern region, 36% in the western region, 19% at the coast, and 11% in the rift valley region. this implies that despite some regions having low mean values for certain traits, individual trees superior in these traits could be targeted for breeding and commercial production. for the kenyan guava, the similar low values observed with regard to fruit size of 47.2 g against the preferred size of 200-300 g, and pulp yield of 48% against the preferred >70% for industrial processing could be improved through agronomic practices. quantification of these traits in each production area to identify the best genotypes for specific markets and selection of superior parents for breeding is vital. conclusions the ascorbic acid content positively correlated with annual precipitation. tss positively correlated with temperature, and was found to be higher in white-fleshed fruits and fruits having lower pulp weight and more seeds. red-fleshed fruits had a higher content of phenolic compounds than the white-fleshed types, while the white-fleshed fruits had more tss, protein, and some minerals. larger fruits were generally observed to have a dilution effect on the fruit mineral content. generally, most of the correlations were not strong, implying that more than just the studied factors influence the nutritional and chemical content of guava. the relationship between climatic data on fruit traits such as ascorbic acid and tss could aid in the choice of guava production regions that are rich in these chemical components. the flesh colour of fruits provides the information necessary for the selection of fruits for various purposes − for example, sweeter white-fleshed fruits with a higher mineral content could be preferred for fresh consumption; 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https://orcid.org/0000-0001-9329-4375 address of the corresponding author: university of goettingen, department of crop sciences, division of quality of plant products carl-sprengel-weg 1, 37075, goettingen, germany e-mail: epawelz@gwdg.de © the author(s) 2019. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). mailto:epawelz@gwdg.de i supplementary material 27 692 supplementary information 693 tab. s1: geographical coordinates, altitude, mean annual temperature, and annual 694 precipitation of the 128 guava accessions collected from four regions of kenya 695 accession number accession code region latitude [n°/s°] longitude [e°] altitude (m) mean annual temperature (°c) annual precipitation (mm) 1 kil001 coast 03.69568 °s 039.72340 °e 208 23.7 425 2 kil002* coast 03.69580 °s 039.72343 °e 199 23.7 425 3 kil003* coast 03.69679 °s 039.72604 °e 202 23.7 425 4 kil004* coast 03.69518 °s 039.72219 °e 200 23.7 425 5 kil009* coast 03.92239 °s 039.74352 °e 23 23.7 410 6 kil010* coast 03.92240 °s 039.74314 °e 25 23.7 410 7 kil011* coast 03.92226 °s 039.74282 °e 22 23.7 410 8 kil012* coast 03.92228 °s 039.74283 °e 22 23.7 410 9 kil013* coast 03.91339 °s 039.74015 °e 18 23.7 410 10 kil014 coast 03.91348 °s 039.74015 °e 17 23.7 410 11 kil015* coast 03.91338 °s 039.73997 °e 18 23.7 410 12 kil016* coast 03.91332 °s 039.73999 °e 21 23.7 410 13 kil017* coast 03.91347 °s 039.73988 °e 20 23.7 410 14 kwa001 coast 04.16923 °s 039.59783 °e 23 22.7 458 15 kwa002 coast 04.16853 °s 039.59749 °e 19 22.7 458 16 kwa003 coast 04.16856 °s 039.59748 °e 19 22.7 458 17 kwa005* coast 04.16494 °s 039.57737 °e 104 22.5 475 18 kwa006* coast 04.16495 °s 039.57743 °e 97 22.5 475 19 kwa007* coast 04.16496 °s 039.57764 °e 119 22.5 475 20 kwa008* coast 04.16782 °s 039.56780 °e 108 22.7 458 21 kwa009 coast 04.16837 °s 039.56796 °e 92 22.7 458 22 kwa010* coast 04.16860 °s 039.56822 °e 94 22.7 458 23 kwa011* coast 04.34928 °s 039.53458 °e 22 22.3 469 24 kwa014 coast 04.34318 °s 039.51459 °e 35 22.3 469 25 kwa015* coast 04.33752 °s 039.44971 °e 117 22.1 491 26 kwa016* coast 04.33753 °s 039.44975 °e 118 22.1 491 27 kwa017* coast 04.49746 °s 039.25124 °e 39 21.6 518 28 kwa018 coast 04.49765 °s 039.25125 °e 45 21.6 518 29 kwa019* coast 04.49763 °s 039.25131 °e 41 21.6 518 30 kwa020* coast 04.49715 °s 039.25139 °e 45 21.6 518 31 kwa021 coast 04.60348 °s 039.18504 °e 25 21.7 505 32 kwa023 coast 04.60352 °s 039.18509 °e 20 21.7 505 33 kwa024 coast 04.60323 °s 039.18452 °e 21 21.7 505 34 mom006 coast 03.96482 °s 039.73122 °e 15 23.7 410 35 mom007* coast 03.96493 °s 039.73089 °e 14 23.7 410 36 mom008 coast 03.96229 °s 039.73233 °e 16 23.7 410 37 mer001* eastern 00.17234 °s 037.64283 °e 1564 20.5 1149 supplementary information tab. s1: geographical coordinates, altitude, mean annual temperature, and annual precipitation of the 128 guava accessions collected from four regions of kenya*accessions used for fruit morphological characterization. supplementary material ii 28 38 mer002* eastern 00.17239 °s 037.64275 °e 1545 20.5 1149 39 mer005* eastern 00.17249 °s 037.65120 °e 1479 20.5 1149 40 mer009* eastern 00.08721 °s 037.66675 °e 1455 20.5 1382 41 mer010* eastern 00.08726 °s 037.66695 °e 1452 20.5 1382 42 mer012* eastern 00.08564 °s 037.66451 °e 1478 16.8 1582 43 mer013* eastern 00.08536 °s 037.66438 °e 1481 16.8 1582 44 mer014* eastern 00.11461 °s 037.69637 °e 1384 20.5 1382 45 mer016* eastern 00.18701 °s 037.69572 °e 1290 20.8 1335 46 mer017* eastern 00.18693 °s 037.69600 °e 1288 20.8 1335 47 mer018* eastern 00.12048 °s 037.72087 °e 1393 20.5 1382 48 mer019* eastern 00.12024 °s 037.72074 °e 1385 20.5 1382 49 elg001* rift valley 00.64776 °n 035.51977 °e 2089 21.2 950 50 elg002* rift valley 00.64203 °n 035.52221 °e 2064 21.2 950 51 elg003* rift valley 00.64265 °n 035.52145 °e 2077 21.2 950 52 elg004* rift valley 00.64264 °n 035.52150 °e 2071 21.2 950 53 elg005* rift valley 00.67029 °n 035.51809 °e 2214 20.3 954 54 elg007* rift valley 00.64350 °n 035.51839 °e 2104 21.2 950 55 elg008* rift valley 00.64349 °n 035.51843 °e 2104 21.2 950 56 elg009* rift valley 00.64338 °n 035.51852 °e 2102 21.2 950 57 elg010* rift valley 00.64505 °n 035.51627 °e 2132 21.2 950 58 elg011* rift valley 00.63185 °n 035.52095 °e 2024 21.2 950 59 elg012 rift valley 00.63469 °n 035.52243 °e 2031 21.2 950 60 elg013* rift valley 00.63766 °n 035.51977 °e 2079 21.2 950 61 elg014* rift valley 00.57152 °n 035.30377 °e 2142 17.1 1055 62 elg015* rift valley 00.57151 °n 035.30377 °e 2150 17.1 1055 63 elg016* rift valley 00.58574 °n 035.46054 °e 2317 16 1104 64 elg019* rift valley 00.58788 °n 035.46055 °e 2322 16 1104 65 elg020 rift valley 00.66651 °n 035.53149 °e 1972 21.2 950 66 elg021* rift valley 00.66682 °n 035.53004 °e 1985 20.3 954 67 uag018* rift valley 00.64256 °n 035.52145 °e 2067 21.2 950 68 hom001* western 00.59582 °n 034.57717 °e 1308 20.8 1659 69 hom003* western 00.59585 °n 034.57596 °e 1307 20.8 1659 70 hom004* western 00.59594 °n 034.57690 °e 1306 20.8 1659 71 hom006* western 00.59596 °n 034.57690 °e 1306 20.8 1659 72 hom007* western 00.59593 °n 034.57692 °e 1307 20.8 1659 73 hom009* western 00.59600 °n 034.57698 °e 1305 20.8 1659 74 hom010* western 00.59596 °n 034.57703 °e 1307 20.8 1659 75 hom011* western 00.59603 °n 034.57717 °e 1302 20.8 1659 76 hom012* western 00.60963 °n 034.58897 °e 1329 20.8 1659 77 hom013* western 00.60974 °n 034.58366 °e 1335 20.8 1659 78 hom014* western 00.60961 °n 034.58369 °e 1339 20.8 1659 79 hom016* western 00.60961 °n 034.58374 °e 1337 20.8 1659 80 hom017* western 00.60984 °n 034.58377 °e 1336 20.8 1659 81 hom018* western 00.60610 °n 034.63214 °e 1463 20.8 1659 82 hom019* western 00.60611 °n 034.63223 °e 1456 20.8 1659 83 hom020* western 00.61762 °n 034.64497 °e 1498 20.8 1659 29 84 hom021* western 00.61760 °n 034.64495 °e 1502 20.8 1659 85 hom022* western 00.53904 °n 034.50943 °e 1242 20.8 1659 86 hom023* western 00.53907 °n 034.50946 °e 1238 20.8 1659 87 hom024* western 00.53907 °n 034.50945 °e 1240 20.8 1659 88 hom025* western 00.53907 °n 034.50941 °e 1237 20.8 1659 89 hom026* western 00.53908 °n 034.50942 °e 1238 20.8 1659 90 hom027 western 00.53906 °n 034.50946 °e 1242 20.8 1659 91 hom028* western 00.53905 °n 034.50951 °e 1239 20.8 1659 92 hom029* western 00.53893 °n 034.50956 °e 1240 20.8 1659 93 hom030* western 00.53880 °n 034.50989 °e 1239 20.8 1659 94 hom032* western 00.53987 °n 034.50855 °e 1246 20.8 1659 95 hom035* western 00.72481 °n 034.45610 °e 1289 21.1 1526 96 hom036* western 00.72479 °n 034.45597 °e 1290 21.1 1526 97 hom039* western 00.72471 °n 034.45581 °e 1292 21.1 1526 98 hom042* western 00.72455 °n 034.45533 °e 1283 21.1 1526 99 hom043* western 00.72442 °n 034.45531 °e 1283 21.1 1526 100 hom045* western 00.72436 °n 034.45530 °e 1285 21.1 1526 101 hom046* western 00.72439 °n 034.45518 °e 1283 21.1 1526 102 hom047* western 00.72412 °n 034.45534 °e 1265 21.1 1526 103 hom048* western 00.72412 °n 034.45539 °e 1275 21.1 1526 104 kak001* western 00.27951 °n 034.67358 °e 1419 20.6 1917 105 kak002* western 00.27863 °n 034.67363 °e 1409 20.6 1917 106 kak003 western 00.27861 °n 034.67367 °e 1420 20.6 1917 107 kak004* western 00.27791 °n 034.69564 °e 1447 20.6 1917 108 kak005* western 00.27700 °n 034.69589 °e 1441 20.6 1917 109 kak006* western 00.27777 °n 034.69579 °e 1443 20.6 1917 110 kak007* western 00.24446 °n 034.82470 °e 1571 20.6 1917 111 kak008* western 00.24442 °n 034.82479 °e 1572 20.6 1917 112 sia001 western 00.19481 °n 034.34081 °e 1297 21.8 1774 113 sia002* western 00.19376 °n 034.33390 °e 1286 21.8 1774 114 sia003* western 00.19423 °n 034.33385 °e 1280 21.8 1774 115 sia004* western 00.13007 °n 034.42597 °e 1358 21.6 1740 116 sia005 western 00.13003 °n 034.42687 °e 1357 21.6 1740 117 sia006* western 00.12687 °n 034.42089 °e 1340 21.6 1740 118 sia007 western 00.12680 °n 034.42102 °e 1342 21.6 1740 119 sia008* western 00.12804 °n 034.42337 °e 1347 21.6 1740 120 sia009* western 00.12810 °n 034.42309 °e 1347 21.6 1740 121 sia010* western 00.13046 °n 034.42354 °e 1348 21.6 1740 122 sia011* western 00.13008 °n 034.42255 °e 1349 21.6 1740 123 unk001 western 00.84360 °n 034.79930 °e 1684 16.8 1455 124 unk002 western 00.08413 °n 034.79875 °e 1688 20.3 1864 125 vih001* western 00.08540 °n 034.79936 °e 1680 20.3 1864 126 vih002 western 00.08539 °n 034.79936 °e 1679 20.3 1864 127 vih003* western 00.08532 °n 034.79938 °e 1682 20.3 1864 128 vih004* western 00.84470 °n 034.79931 °e 1683 16.8 1455 *accessions used for fruit morphological characterization. 696 697 iii supplementary material 30 697 698 fig. s1: mean monthly temperature, precipitation, and relative humidity (rh) of the four regions (a) the rift 699 valley, (b) coast, (c) western, and (d) eastern of guava fruit collection based on data from the nearest 700 meteorological station for the year 2015. the period when sampling was carried out is indicated. 701 702 fig. s2: pearson correlation between (a) annual precipitation and ascorbic acid content in 128 guava fruit 703 samples from four regions of kenya, and between (b) mean annual temperature and tss in 128 guava fruit 704 samples from four regions of kenya. ascorbic acid and tss were determined on extracted juice from edible 705 portion of the fruit. 706 707 708 fig. s1: mean monthly temperature, precipitation, and relative humidity (rh) of the four regions (a) the rift valley, (b) coast, (c) western, and (d) eastern of guava fruit collection based on data from the nearest meteorological station for the year 2015. the period when sampling was carried out is indicated. fig. s2: pearson correlation between (a) annual precipitation and ascorbic acid content in 128 guava fruit samples from four regions of kenya, and between (b) mean annual temperature and tss in 128 guava fruit samples from four regions of kenya. ascorbic acid and tss were determined on extracted juice from edible portion of the fruit. supplementary material iv 31 708 709 fig. s3: pearson correlation between (a) pulp proportion and tss of 105 guava fruit samples from four 710 regions of kenya, and between (b) seed proportion and fruit water content of 105 guava fruit samples from 711 four regions of kenya. tss was determined on extracted juice from edible portion of the fruit, and water 712 content was determined as the difference between freeze-dried and fresh sample weights. pulp proportion (% 713 pulp), and seed proportion (% seed) are based on weight of the entire fresh fruit (g). 714 715 716 717 fig. s3: pearson correlation between (a) pulp proportion and tss of 105 guava fruit samples from four regions of kenya, and between (b) seed proportion and fruit water content of 105 guava fruit samples from four regions of kenya. tss was determined on extracted juice from edible portion of the fruit, and water content was determined as the difference between freeze-dried and fresh sample weights. pulp proportion (% pulp), and seed proportion (% seed) are based on weight of the entire fresh fruit (g). v supplementary material journal of applied botany and food quality 93, 216 (2020) book review strawberries 2nd edition james f. hancock this new and updated edition provides a broad, balanced review of the scientific knowledge of strawberries and their cultivation. the worldwide strawberry industry has grown substantially since the first edition was published 20 years ago. furthermore, methods of cultivation have undergone extensive modifications. important changes have been made to the taxonomy of strawberries and there is a much better understanding today of how its ancestors evolved. new disease and pest control methods have been developed and a large amount of genomic information has been generated. this information has greatly broadened our knowledge of how flowering and fruiting is regulated and will revolutionize the breeding of strawberries. this book covers various important aspects from taxonomy, ecology, morphology and genetics to environmental physiology, disease and pest control, fruit ripening, storage and processing. drawing on extensive research and practical experience, the author provides in 8 chapters a thorough review of the evolution of strawberries (chapter 1) and the history of strawberry cultivation (chapter 3). chapter 3 and 4 summarize the major cultivation systems employed across the world and describe the physiology behind these practices. chapter 5 addresses strawberry anatomy and developmental physiology including temperature and photoperiod control of flowering. chapter 6 highlights research work on the fruiting and postharvest physiology. chapter 7 deals with the diseases and pests of strawberry and describes the individual consequences that came up with the abandonment of methyl bromide. the last chapter reviews strawberry breeding and genetic research, an area where tremendous progress continues to be made. particularly in mediterranean and other sub-tropical environments breeding activities increased dramatically over the last two decades. it is reported that strawberry breeders have begun to widely use molecular approaches in their programs including also marker-assisted and genomic selection. this book provides an excellent, concise overview of strawberries and their cultivation and can be therefore recommended without any reservation as an exciting and instructive information to a broad range of readers. not only does it provide valuable support to students of horticulture and various professional groups such as plant breeders, strawberry farmers, plant physiologists, food technologists and chemists, but it also provides an excellent opportunity for interested laypeople to obtain comprehensive information on the current state of knowledge of strawberry science and cultivation. a number of scientific articles are listed in each annex of the 8 chapters, allowing readers to obtain more detailed information on the individual topics. dr. h. schulz e-mail: hs.consulting.map@t-online.de bibliography: james f. hancock, strawberries 2nd edition, cab international 2020, 275 pages includes bibliographical references and index, price: 53,29 €, isbn-13: 9781789242270 (hardback), isbn 9781789242287 (epdf), isbn 9781789242294 (epub) journal of applied botany and food quality 95, 114 120 (2022), doi:10.5073/jabfq.2022.095.015 1college of life science, sichuan agricultural university, ya’an, china 2college of agricultural sciences, xichang university, xichang, china morphology and oil quality of introduced olive cultivars (olea europaea l.) in southwest china li liu1,a, jie liu1,a, zhou xu2, siyuan luo1, zhi huang1, qingbo kong1, shiling feng1, tao chen1, lijun zhou1, chunbang ding1,* (submitted: october 15, 2021; accepted: may 30, 2022) * corresponding author a these authors contributed equally to this manuscript and worked as co-first authors summary there are enormous benefits to olive cultivation in china. however, little research has been conducted into the characteristics of the morphology and oil quality of introduced olive cultivars in southwest china. this study investigates the morphological and biochemical characteristics of seven introduced olive cultivars and one indigenous cultivar grown in the county of jintang in sichuan province. the results reveal that all cultivars have adapted to their new environment and exhibit unique characteristics. the coratina, koroneiki and grossanne varieties have all proved to be excellent oil cultivars, with a fresh oil content of 20.42%, 18.58% and 16.46%, respectively. the free acidity and peroxide values of these olive oils are within the range of the extra virgin olive oil category, while the extracted oils are rich in unsaturated fatty acids, α-tocopherol, squalene, stigmasterol, β-sitosterol and phenolic compounds. moreover, the olive cultivated in southwest china possesses a higher content of moisture, oleic acid and unsaturated fatty acids compared with the mediterranean region. therefore, the region around jintang, a new environment, is shown to have tremendous potential for the cultivation and development of olives in the future. in addition, these results can provide theoretical guidance for olive planting and cultivar selection in southwest china. keywords: olea europaea l., introduced cultivar, morphological characteristics, oil quality, chemical composition introduction the olive tree (olea europeae l.) is a medium-sized evergreen tree and an important oil crop species, and planted originally in the me diterranean region. (ghanbari et al., 2012). due to the nutritional benefits of olive oil and its great commercial value, the olive has been widely introduced elsewhere, such as the united states, australia, south africa and china, among others (laaribi et al., 2017). since 1956, when they were introduced to china, olive trees have been regarded as an important woody oil plant. the first large-scale plantation of olive trees began in 1964. at present, the olive trees are mostly distributed in yunnan, gansu, sichuan, chongqing, hubei, jiangsu and shaanxi provinces (rao et al., 2019). after several decades of cultivation and domestication, plenty of introduced olives have adapted to the local environment, and then some new cultivars have been bred. in china, the planting regions of olive trees were increased to 800 km2 in 2018, and the total areas are expected to be 1,670 km2 by the end of 2020 (deng, 2018). morphological characteristics, including aspects of the leaf, inflorescence, fruit and endocarp have been extensively used to describe and identify olive cultivars. noting these is the first step in the classification of olive germplasm resources (rotondi et al., 2003). nevertheless, the morphological characteristics often vary greatly within an olive variety depending on environmental conditions, phenological stage, agricultural practices, and others (zaher et al., 2011). the production of olive oil is the principal purpose of planting olive trees. olive oil, a key component of the traditional “mediterranean diet” is extracted by mechanically pressing the olive fruit without any chemical treatment (guasch-ferrĕ et al., 2014). the chemical composition of olive oil primarily consists of triacylglycerols (tag, about 99%), followed by free fatty acids (ffa), diacylglycerols, and other bioactive components. some of these compounds help contri bute to the unique character of olive oil (blekas et al., 2006). because of the high content of monounsaturated fatty acid (mufa) and various functional bioactive components in olive oil, long-term intake can reduce morbidity and mortality of cardiovascular disease (such as hypertension, coronary artery disease, etc.), and prevent skin cancer, breast cancer, neurodegeneration and others (foscolou et al., 2018). however, the quality of olive oil is easily affected by geographic location, including climate (mailer et al., 2010). previous works have reported on different olive cultivars grown in china. for example, the cold and drought resistance of young trees has been evaluated and the growth, fruit production, and oil quality of eight olive cultivars in wudu in the southeast gansu province have also been assessed (wang et al., 2018). xiang et al. comprehensively valued the quality, composition, and antioxidant activity of virgin olive oil from four introduced varieties at liangshan (xiang et al., 2017). authorities in southwest china, and notably jintang county of sichuan province, aim to develop olive cultivation as a leading industry, integrating the construction of a complete olive industry into the huge tourism market of sichuan, as well as encouraging a healthy pastoral lifestyle in the region (chen et al., 2019). there have already been some studies on the cultivation techniques and photosynthetic characteristics of olive trees cultivated in jintang. however, research into the morphological and oil qualitative characteristics of the introduced olive cultivars in southwest china is rare. therefore, this study aims to characterize these regional olive cultivars and select the most superior ones. this work is expected to provide valuable information on olive cultivation and research to both olive growers as well as researchers. materials and methods plant materials seven introduced olive cultivars, including arbequina (from spain), koroneiki (from greece), coratina (from albania), picholine (from france), grossanne (from france), leccino (from italy), manzanilla (from israel), as well as the indigenous olive cultivar ezhi-8 (from china) have been planted in southwest china (jintang county of sichuan province, 30°45’30.03’’ n; 04°32’52.07’’ e, at an altitude of 800-1100 m. the average annual precipitation is 1000 mm, the mean annual temperature is 16 °c, and during harvest time, the temperature fluctuates between 9 °c (minimum) and 25 °c (maximum). in morphology and oil quality of olive cultivars (olea europaea l.) 115 the orchards identified for this study, the olive trees are six years old, and are planted with spacing of 5 × 5 m, with consistent cultivation conditions. the analysis was conducted as described in the following sections. morphological characteristics and chemical composition of fruits morphological characteristics were assessed according to the primary descriptor methodology of olive trees (hosseini-mazinani and samaee, 2004). the leaf characters (leaf length, leaf width and leaf area), inflorescence characters (number of flowers/inflorescence, perfect flowers percentage/inflorescence and blooming periods), fruit and endocarp characters (fruit weight, stone weight and pulp/ stone ratio), yield per tree, moisture and oil contents of olive fruit were measured and recorded. three olive trees of each cultivar were randomly selected for determination with uniform cultivation conditions and tree shape. for each tree, 100 mature leaves, 10 branches and 30 olive fruits were hand-picked from the inner and outer part of the four subdivided quadrants of the canopy with uniform cha racteristics in total from the orchard (fayek et al., 2014). five olive trees from each cultivar were randomly selected for measurement of the yield. the contents of oil and moisture were measured according to national standard methods of china (gb 5009.6-2016) and previous reports, respectively (nsprc, 2016; cheng et al., 2017). experiments were performed in triplicate for each cultivar. olive oil quality characteristics oil extraction some 5 kg of olive fruits were collected during harvest time and extracted according to the previous method (xiang et al., 2017). the fruits were crushed, and the crushed slurry was fused in a hot water bath for 30 min at 30 °c, then centrifuged at 3,000 r/min for 5 min to obtain an oil-water mixture. finally, the olive oil was separated from the mixture after the mixture was placed at 4 °c overnight. the collected oil was then stored in dark glass bottles at 4 °c for further analysis, described below. free acidity and peroxide value the free acidity and peroxide value of the olive oil samples were determined according to international standards (iso 660-2009 and iso 3960-2007, respectively) (iso, 2007; 2009). fatty acid composition the fatty acid composition was evaluated by gas chromatography mass spectrometry (gc-ms) (issaoui et al., 2011). firstly, the oil was simply methyl esterified (fames). then, the gc-ms analysis was conducted using an agilent 7890a gas chromatograph and 5977c mass spectrometry (agilent technologies, usa), equipped with a capillary column hp-5ms (30 m×0.25 mm; 0.25 μm, agilent technologies, usa). detection conditions were listed as follows: capillary column temperature program was set from 80 °c, holding for 5 min, and increased at 10 °c/min to 180 °c. holding for 2 min, then increased to 260 °c at a rate of 5 °c/min, maintained for 3 min. the carrier gas was helium at a flow rate of 0.6 ml/min with a split ratio of 1:10. the injector temperature was 245 °c, and the temperature of the detector was 260 °c. ei ion source and mass scan ranged from 45 to 550 m/z. the fames profiles were identified by comparing them with the database of the national institute of standard technology library, nist. in addition, the individual fatty acid content was expressed as a percentage of the total fatty acid. contents of α-tocopherol, squalene, stigmasterol and β-sitosterol the results of the α-tocopherol, squalene, stigmasterol and β sitosterol analysis were determined using an agilent 1260 hplc (agilent technologies, usa) equipped with a zorbax sb-c18 column (150×4.6 mm, 5.0 μm). the content of α-tocopherol was evaluated according to the pre vious method (cao et al., 2015). a 1 g oil sample was dissolved in n-hexane, the volume fixed to 10 ml, then mixed and filtered for hplc analysis. detection conditions included a fluorescence detector; the excitation wavelength was 295 nm, and the emission wavelength was 325 nm; the mobile phase was methanol at a flow rate of 0.8 ml/min, and the column temperature was 35 °c. the content of squalene was evaluated as described in previous studies (liu, 2017). firstly, the oil was saponified using a potassium hydroxide-ethanol solution, then the sample was analysed by hplc. the detection conditions were set as follows: an ultraviolet detector, a wavelength of 325 nm, and a column temperature of 30 °c; the mobile phase was methanol: acetonitrile (60:40, v:v) at a flow rate of 1.0 ml/min. the contents of stigmasterol and β-sitosterol were evaluated according to the previous method with some modifications (sivakumar et al., 2006). firstly, the oil sample was briefly saponified. then, detection conditions were set as follows: an ultraviolet detector, with a wavelength of 210 nm and column temperature of 35 °c; the mobile phase was methanol at a flow rate of 1.0 ml/min. total polyphenol and phenolic compounds the total polyphenol fraction was extracted according to the previous method described by bouarroudj (bouarroudj et al., 2016). the mixture was vortexed and centrifuged at 4000 rpm for 15 min. the extraction was repeated three times and concentrated by rotary evaporator at 40 °c, then adjusted to 10 ml with methanol. the content of total polyphenol was determined by the folin-ciocalteu method described by baiano with some modifications (baiano et al., 2009). a 0.1 ml polyphenol extract was mixed with 0.02 ml of folin-ciocalteu and a 0.08 ml 10% sodium carbonate solution for 5 min. the absorbance of the mixture was read at 765 nm by spectra max m2 microplate reader (molecular devices corp., ca) after incubation in dark for 1 h. the polyphenol quantity was given as milligram gallic acid equivalents per kilogram. the phenolic compounds analysis was carried out in accordance with previous studies (xiang et al., 2017). the analytical equipment used is mentioned in section 2.3.4. the conditions for analysis were set as follows: wavelength at 280 nm; column temperature of 35 °c; mobile phase: (a) water: acetic acid (99.5:0.5, v:v), (b) methanol, and (c) isopropyl alcohol, with a flow rate of 1 ml/min. gradient elution: 0 to 14 min 92% (a), 4% (b), 4% (c); 14 to 45 min 82% (a), 9% (b), 9% (c) and 45 to 60 min 70% (a), 15% (b),15% (c). statistical analysis the average values between different cultivars of oil samples were compared using duncan’s multiple tests, and significant differences were established at p<0.05. the ibm spss statistics version 20.0 (spss inc., chicago, il, usa) was used to perform analysis of variances (anova). results and discussion morphological characteristics and chemical analyses of fruits leaf and flower characteristics leaves are the main sites of photosynthesis in plants, playing an important role in the growth and development of fruits and trees (liu et al., 2011). the adult leaf length of the eight olive cultivars 116 l. liu, j. liu, z. xu, s. luo, z. huang, q. kong, s. feng, t. chen, l. zhou, c. ding ranged from 45.80 to 64.24 mm, and the leaf width varied from 10.53 to 14.73 mm (tab. 1). the leaf area of coratina was the biggest (674.80 mm2) while arbequina was the smallest (395.06 mm2). in addition, as shown in tab. 1, the number of flowers per inflorescence differed among the eight cultivars, ranging from 3 to 36. more importantly, the numbers of perfect flowers are closely related to the fruit set percentage. the percentage of perfect flowers per inflores cence of eight olive cultivars varied from 58.76 to 82.12%. accordingly, the fruit set percentage of arbequina was the highest and manzanilla was the lowest. olive production depends mainly on this fruit set percentage (mezghani et al., 2012). most olive cultivars are self-incompatible and have low self-pollination rates (saumitou-laprade et al., 2017). however, an overlap of blooming periods exists in the eight cultivars. for example, the blooming periods of koroneiki are closer to those of leccino. planting cultivars with similar blooming periods together can therefore improve the pollination rate when growing olives. fruit and stone characteistics at harvest, as shown in tab. 2, the fruit weight, stone weight and ratio of pulp on stone differed significantly between the eight olive cultivars. the fruit weight ranged from 1.37 g to 4.06 g. the stone weight ranged from 0.22 g (koroneiki) to 0.84 g (ezhi-8). the coratina was recorded as the highest value (6.59) in terms of pulp/stone ratio, followed in order by manzanilla, koroneiki, arbequina, grossanne, picholine, leccino and ezhi-8 (5.55, 5.23, 4.97, 4.61, 4.52, 4.12 and 2.65, respectively). the fruit weight of coratina, koroneiki and arbequina was higher than that of previous research results (cheng et al., 2017). however, the fruit weight of ezhi-8 and manzanilla was lower than previous studies (wang et al., 2018). simultaneously, tab. 2 shows that the fruit yield per tree of grossanne was the highest (11.79 kg per tree), followed by picholine (8.37 kg per tree), while the lowest was manzanilla (4.19 kg per tree). the fruit moisture content of eight cultivars ranged from 56.02 to 65.77% (tab. 2). in addition, the fresh fruit oil content was highest in coratina (20.42%), followed in descending order by koroneiki (18.58%), grossanne (16.46%), picholine (15.89%), arbequina (15.54%), leccino (15.07%), ezhi-8 (14.27%) and lastly manzanilla (13.05%). in this study, the moisture content of olive fruit was much higher and the fresh fruit oil content was low compared with other studies (lee et al., 2018; dabbou et al., 2009). proietti et al. have indicated that sufficient irrigation in the fruiting stage would raise the fruit water content (proietti and antognozzi, 1996). in addition, irrigation has also been reported to affect oil content (rinaldi et al., 2010). hence, the difference of moisture and oil content between this study and previous research may be caused by climate, especially precipitation. on the basis of this current study, the manzanilla cultivar is con sidered to be promising for its use for table or eating olives because of its low oil content and high pulp/stone ratio. the result is in agreement with previous research reported by del río’ (del rĺo and caballero, 2008). similarly, the high pulp/stone ratio and the moderate oil content of picholine and arbequina demonstrate that both of them could be applied as dual-purpose cultivars (for both oil and table olives), while koroneiki, coratina and grossanne remain excellent oil cultivars. oil qualitative characteristics free acidity and peroxide value the free acidity and peroxide values are an important index of qua lity in olive oil (gomez-caravaca et al., 2016). above all, free acidity has been used exclusively as the traditional classification criterion for olive oil. the free acidity and peroxide values of eight cultivars were shown in tab. 3. the different olive oils show significant differences in terms of free acidity, ranging from 0.24 to 0.47%. their peroxide value ranged from 9.08 to 14.66 meq/kg. the tab. 1: leaf and inflorescence characteristics of eight cultivars leaf length/mm leaf width/mm leaf area/mm2 number of flowers/ perfect flowers percentage/ blooming periods inflorescence inflorescence (%) arbequina 45.80±0.79a 10.98±0.54ab 395.06±25.25a 9~27 82.12±2.33e april 19 to may 04 koroneiki 49.24±1.11b 11.36±0.60ab 439.45±33.16a 6~30 70.27±0.61c april 22 to may 08 coratina 62.29±1.56d 13.81±1.24c 674.80±55.81e 6~36 73.70±2.34c april 15 to 29 picholine 57.30±0.97c 11.55±0.52ab 519.76±32.33bc 4~24 67.28±1.47b april 17 to 30 grossanne 64.24±1.60d 12.38±0.90b 624.82±59.82de 8~29 75.92±0.98d april 21 to may 06 leccino 51.43±1.39b 14.73±1.11c 595.48±59.41cd 3~19 73.61±1.39c april 23 to may 09 manzanilla 54.82±1.81c 10.53±0.58a 452.72±20.24ab 5~25 58.76±1.67a april 16 to may 01 ezhi-8 48.78±3.68ab 14.45±0.32c 553.08±39.26cd 3~31 77.99±2.14d april 16 to may 03 data are expressed as the mean±standard deviations, n=3. different letters in the same column indicate significantly different values, p<0.05. tab. 2: fruit characteristics and yield per tree of eight cultivars fruit weight (g) stone weight (g) pulp/stone ratio fruit yield (kg/tree) moisture content (%) fresh fruit oil content (%) arbequina 1.91±0.09b 0.32±0.00b 4.97±0.04d 6.53±0.66bc 60.91±2.09c 15.54±0.35cd koroneiki 1.37±0.03a 0.22±0.01a 5.23±0.11e 6.24±1.52abc 59.24±1.79bc 18.58±0.42e coratina 4.63±0.26f 0.61±0.02d 6.59±0.07g 8.36±0.76c 57.19±0.99ab 20.42±0.54f picholine 3.59±0.05e 0.65±0.01e 4.52±0.04c 8.37±1.23c 64.32±1.57d 15.89±0.35cd grossanne 2.02±0.04c 0.36±0.01c 4.61±0.13c 11.79±1.87d 59.06±1.08bc 16.46±0.11d leccino 3.02±0.07d 0.59±0.01d 4.12±0.05b 5.18±0.60ab 56.02±1.55a 15.07±0.23bc manzanilla 4.06±0.37e 0.62±0.01d 5.55±0.18f 4.19±0.50a 65.77±0.72d 13.05±0.48a ezhi-8 3.07±0.10d 0.84±0.03f 2.65±0.08a 7.89±1.17c 64.05±1.54d 14.27±0.60b data are expressed as the mean±standard deviations, n=3. different letters in the same column indicate significantly different values, p<0.05. morphology and oil quality of olive cultivars (olea europaea l.) 117 free acidity and peroxide values of all olive oils tested were within the range of the extra virgin olive oil category (where free acidity ≤ 0.80% and peroxide value ≤ 20.0 meq/kg) (ioc, 2019). therefore, the olive oil of eight cultivars is of excellent quality and can be ranked as extra virgin olive oil. fatty acid composition the content of unsaturated fatty acids and saturated fatty acids in the olive oil are shown in tab. 4 and 5, respectively. the main unsaturated fatty acid in olive oil is palmitoleic acid (c16:1, 0.40~3.44%), followed by heptadecenoic acid (c17:1, 0.09~0.26%), oleic acid (c18:1, 61.64~79.30%), linoleic acid (c18:2, 4.04~8.17%), linolenic acid (c18:3, 0.39~0.95%) and eicosenoic acid (c20:1, 0.14~0.40%). in addition, the main saturated fatty acid in olive oil was palmitic acid (c16:0, 10.76~19.48%), followed by heptadecanoic acid (c17:0, 0.11~0.31%), stearic acid (c18:0, 1.16~4.64%), arachidic acid (c20:0, 0.31~0.58%) and behenic acid (c22:0, 0.05~0.16%). in this study, oleic acid was the mainly constituent in all oils tested, and a high dietary intake of oleic acid has been proved to reduce the risk of coronary artery disease (cad), hypertension and other diseases (bermudez et al., 2011). the oil of the eight cultivars were also rich in the essential fatty acids of linoleic acid and linolenic acid. arbequina did not contain heptadecenoic acid. arbequina, picholine and ezhi-8 were also short of heptadecanoic acid. there was also an absence of behenic acid in coratina and ezhi-8. taken together, the fatty acid composition of the oil from the eight culti vars complies with the established limits of extra virgin olive oil (ioc, 2019). the results are also in agreement with the finding that the cultivar is closely related to the composition of fatty acids in olive oil (chapagain et al., 2009). the content of fatty acids in the olive oil samples from southwest china is different from other studies. for example, the regional content of oleic acid is higher than the results of other researches (zarrouk et al., 2009). previous studies have shown that geographic location seriously affects the fatty acid composition and content of olive oil (mailer et al., 2010). it has also been discovered that olive fruits living at a high altitude and in cooler areas are apt to exhibit a high content of unsaturated fatty acids (ouni et al., 2011). previous studies report that the oil extracted from olives grown in high temperature sites contains lower levels of oleic acid than olives grown in a milder environment (nissim et al., 2020). tab. 3: free acidity and peroxide value of olive oil free acidity (%) peroxide value (meq/kg) arbequina 0.38±0.01d 14.66±0.21d koroneiki 0.30±0.00b 14.00±0.15c coratina 0.30±0.01b 9.08±0.09a picholine 0.41±0.00e 12.68±0.18b grossanne 0.24±0.01a 12.94±0.11b leccino 0.46±0.02f 12.66±0.18b manzanilla 0.47±0.01f 12.94±0.12b ezhi-8 0.32±0.01c 12.73±0.18b data are expressed as the mean±standard deviations, n=3. different letters in the same column indicate significantly different values, p<0.05. tab. 4: unsaturated fatty acid composition of olive oil (%) c16:1 c17:1 c18:1 c18:2 c18:3 c20:1 evoo 0.30-3.50 ≤0.60 55.00-83.00 2.50-21.00 ≤1.00 ≤0.40 arbequina 0.58±0.02b 77.20±0.80d 5.94±0.65cd 0.87±0.03d 0.39±0.01d koroneiki 2.36±0.08g 0.09±0.01a 73.01±1.14b 6.12±0.54cd 0.95±0.05e 0.39±0.02d coratina 1.64±0.05e 0.21±0.01d 73.66±1.23b 5.24±0.66bc 0.95±0.01e 0.35±0.03c picholine 2.17±0.05f 0.15±0.01b 75.63±0.94c 4.04±0.55a 0.94±0.07e 0.27±0.01b grossanne 1.35±0.06d 0.22±0.01d 79.21±0.36e 4.62±0.50ab 0.60±0.01c 0.14±0.00a leccino 0.40±0.01a 0.16±0.01b 74.74±0.62b 6.85±0.52d 0.47±0.01b 0.25±0.01b manzanilla 3.44±0.07h 0.26±0.01e 61.64±0.37a 8.17±0.54e 0.93±0.01e 0.33±0.01c ezhi-8 0.92±0.02c 0.17±0.00c 79.30±0.24e 4.65±0.54ab 0.39±0.01a 0.40±0.02d unsaturated fatty acid: c16:1, palmitoleic acid; c17:1, heptadecenoic acid; c18:1, oleic acid; c18:2, linoleic acid; c18:3, linolenic acid; c20:1, eicosenoic acid. the “-” represents not detected in the identification process. data are expressed as the mean±standard deviations, n=3. different letters in the same column indicate significantly different values, p<0.05. tab. 5: saturated fatty acid composition of olive oil (%) c16:0 c17:0 c18:0 c20:0 c22:0 evoo 7.5-20.00 ≤0.40 0.50-5.00 ≤0.60 ≤0.20 arbequina 13.47±0.67b 1.16±0.02a 0.31±0.01a 0.08±0.00c koroneiki 14.90±0.48c 0.11±0.00a 1.40±0.07a 0.56±0.01e 0.11±0.01d coratina 14.81±0.58c 0.31±0.01d 2.33±0.09b 0.49±0.01c picholine 14.83±0.38c 1.24±0.03a 0.58±0.02f 0.16±0.00e grossanne 10.76±0.66a 0.27±0.01c 2.44±0.05b 0.33±0.01a 0.05±0.00a leccino 14.64±0.39d 0.16±0.00b 1.75±0.05b 0.41±0.01b 0.16±0.01e manzanilla 19.48±0.12e 0.15±0.01b 4.64±0.05c 0.54±0.01d 0.07±0.00b ezhi-8 11.37±0.48a 2.23±0.08b 0.58±0.03g saturated fatty acid: c16:0, palmitic acid; c17:0, heptadecanoic acid; c18:0, stearic acid; c20:0, arachidic acid and c22:0, behenic acid. the “-” represents not detected in the identification process. data are expressed as the mean±standard deviations, n=3. different letters in the same column indicate significantly different values, p<0.05. 118 l. liu, j. liu, z. xu, s. luo, z. huang, q. kong, s. feng, t. chen, l. zhou, c. ding analysis of the contents of α-tocopherol, squalene, stigmasterol and β-sitosterol tocopherols are important components in olive oil and contribute to its nutritional values. they also play a key role in preserving oil from rancidity during storage. the antioxidant a-tocopherol, the main tocopherol homolog found in olive oil, can reduce the risk of cardiovascular diseases and cancers (malheriro et al., 2009). in this study, the content of a-tocopherol ranged from 97.54 mg/kg in arbequina oil to 164.47 mg/kg in oil from the leccino cultivar (tab. 6). these values are consistent with previously reported results (casal et al., 2010; xiang et al., 2017). the content of α-tocopherol is associated with plenty of factors, such as cultivar, climatic condition, agronomic practice and extraction processes, etc. (beltrăn et al., 2010). the content of α-tocopherol varies greatly among different cultivars (psomisdou et al., 2000). sterols are the major components of the unsaponifiable fraction in olive oil. and stigmasterol and β-sitosterol are dominant sterols in olive oil, especially β-sitosterol, which accounts for more than 80% of total sterols (lukic et al., 2013). as shown in tab. 6, the stigmasterol in the olive oils tested ranged from 31.76 mg/kg (picholine) to 90.67 mg/kg (arbequina). it was interesting that β-sitosterol content exhibited significant differences between the eight cultivars. the highest was coratina, up to 1135.28 mg/kg, while the lowest was leccino, with 144.45 mg/kg. these results are consistent with other studies that show the existence of differences according to cultivar (sivakumar et al., 2006). the sterol component and content contri bute significantly to evaluating the nutritional value and quality control of olive oil, as they are typically used as the reference measures to detect adulteration (ilyasoglu et al., 2010). total phenol and phenolic composition the phenolic compounds present in olive oil, consisting of all kinds of natural antioxidants, have been proved to possess anti-oxidation, anti-inflammatory and anti-cancer activities (martín-peláez et al., 2013). in olive oil, phenolic compounds contribute to olive oil’s oxidative stability and can extend its shelf life, as well as to its sensory characteristics, such as its bitter, astringent and pungent tastes (alarcon flores et al., 2012). the main classes of phenols present in olive oil are phenolic acids, phenolic alcohols, flavonoids, secoiridoids and lignans (servili and montedoro, 2002). in this study, the total phenols and common phenolic compositions, together with their contents, are shown in tab. 7. the total phenolic contents of the eight oil samples exhibit significant differences. the highest proportion was detected in arbequina, with 102.21 mg/kg, followed by ezhi-8, with 93.88 mg/kg, while the lowest was in picholine with 28.83 mg/kg. these results are lower than previous data (cioffi et al., 2010; loubiri et al., 2016). simultaneously, the common phenolic compounds were evaluated by hplc including hydroxyltyrosol, p-hydroxybenzoic acid, caffeic acid, p-coumaric acid, ferulic acid, rutin and oleuropein (tab. 7). there were significant differences in phenolic compounds among the eight cultivars. while p-hydroxybenzoic acid and caffeic acid were present in all oils, their content ranged from 0.70 mg/kg (picholine) to 4.51 mg/kg (arbequina) and 3.28 mg/kg (ezhi-8) to 5.56 mg/kg (grossanne), respectively. it was notable that rutin was detected in arbequina and other five cultivars, ranging from 0.88 to 11.93 mg/kg, while it has scarcely been reported in previous research (cioffi et al., 2010; gutierrez-rosales et al., 2003). in addition, hydroxytyrosol, p-coumaric acid, ferulic acid and oleuropein were also detected in different quantities in some cultivars, ranging from 0.26 to 2.75 mg/ kg, 0.08 to 0.18 mg/kg, 3.11 to 9.19 mg/kg and 0.70 to 3.05 mg/kg, respectively. vinha et al. have reported that the presence of phenolic compounds is strongly influenced by geographical origins and cultivars (vinha et al., 2005). furthermore, the phenolic compounds in tab. 7: total phenol and phenolic composition in olive oil (mg/kg) total phenol hydroxytyrosol p-hydroxybenzoic acid caffeic acid p-coumaric acid ferulic acid rutin oleuropein arbequina 102.21±3.10e 4.51±0.11f 4.19±0.10bc 3.30±0.12a 11.93±1.27c koroneiki 37.90±1.17b 3.76±0.08e 4.77±0.12d 0.18±0.01c 2.64±0.25b 0.97±0.06b coratina 60.58±3.26c 2.75±0.02d 1.43±0.04c 4.43±0.09bcd 0.08±0.00a 5.30±0.10b picholine 28.83±1.51a 0.26±0.01a 0.70±0.02a 4.56±0.08cd 0.70±0.02a grossanne 36.29±1.10b 1.03±0.03b 5.56±0.14e 0.08±0.00a 3.11±0.06a 0.88±0.04a leccino 30.59±0.68a 1.46±0.03c 4.15±0.53bc 1.17±0.14a 1.28±0.04c manzanilla 93.07±4.40d 0.74±0.02b 2.43±0.06d 4.00±0.10b 0.13±0.00b 5.86±0.36c 2.98±0.15b 1.84±0.04d ezhi-8 93.88±1.94d 2.28±0.05c 1.10±0.03b 3.28±0.51a 9.19±0.14d 3.05±0.07e data are expressed as the mean±standard deviations, n=3. different letters in the same column indicate significantly different values, p<0.05. tab. 6: α-tocopherol, squalene, stigmasterol and β-sitosterol contents of olive oil (mg/kg) α-tocopherol squalene stigmasterol β-sitosterol arbequina 97.54±2.37a 1794.27±34.39b 90.67±2.83h 231.95±11.80b koroneiki 130.65±3.77d 2296.10±74.61c 62.33±0.46e 286.81±8.98c coratina 120.26±3.39c 1924.71±124.64b 58.64±2.00d 1135.28±45.88g picholine 121.71±2.67c 2292.19±88.73c 31.76±0.65a 502.49±20.07d grossanne 101.86±2.20a 1617.07±23.20a 47.13±0.80c 685.82±23.47f leccino 164.47±2.41e 2398.33±151.36c 40.83±1.09b 144.45±11.56a manzanilla 113.79±1.18b 1802.97±48.21b 66.74±1.36f 574.91±22.86e ezhi-8 161.71±1.98e 3152.04±14.12d 86.74±2.43g 667.60±25.02f data are expressed as the mean±standard deviations, n=3. different letters in the same column indicate significantly different values, p<0.05. olive oil is one of the richest sources of squalene. the squalene content in virgin olive oil typically ranges from 200 to 7500 mg/kg (cayuela and garcĺa, 2018). in this study, the content of squalene in olive oil from eight cultivars is summarised in tab. 6. the highest content of squalene was found in ezhi-8 (3152.04 mg/kg oil), while the lowest results are for grossanne (1617.07 mg/kg oil). these results are consistent with owen et al. but higher than those reported by sagratini et al. (owen et al., 2000; sagratini et al., 2012). squalene is a triterpene hydrocarbon and natural antioxidant, which possesses antitumor activity against different cancer types and is used as a component of parenteral emulsions for drugs and vaccines (fox, 2009). in addition, it may be useful for the treatment of cardiovascular diseases (spanova and daum, 2011). therefore, the oil from cultivars such as ezhi-8 that contain a high proportion of squalene could be applied in the manufacture of food supplements and the pharmacological industry. morphology and oil quality of olive cultivars (olea europaea l.) 119 olive oil are also closely related to climate, fruit ripening, processing and storage methods (ghanbari shendi et al., 2018). conclusions in this study, eight olive cultivars cultivated in southwest china showed excellent morphological and oil qualitative characteristics. they exhibit unique characteristics consistent with the climate of the region in which they are grown. the olive fruit from southwest china shows higher moisture content and lower fresh fruit oil content. in addition, the olive oil from all eight cultivars is ranked as extra virgin olive oil. all oils exhibited a high oleic content and were rich in various functional bioactive components. conclusively, southwest china (jintang) has a clear potential for the future development of olives. however, this study only records and measures the complete data for one year, which cannot fully reflect the morphological variability and stability of olive trees in a changing environment. in the following years, we will continue to record and measure the characteristics of olives cultivated in southwestern china. conflict of interests no potential conflict of interest was reported by the authors acknowledgments this study was supported by sichuan science and technology program (2020yfh0207). references alarcon flores, m.i., romero-gonzalez, r., gaeeido frenich, a., martinea vidal, j.l., 2012: analysis of 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aceites 60, 500-508. doi: 10.3989/gya.021109 orcid chunbang ding https://orcid.org/0000-0002-4829-2554 address of the corresponding author: chunbang ding, college of life science, sichuan agricultural university, ya’an 625014, china. e-mail: dcb@sicau.edu.cn © the author(s) 2022. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1016/j.crvi.2017.03.003 http://dx.doi.org/10.1021/acsomega.8b02491 http://dx.doi.org/10.16736/j.cnki.cn41-1434/ts.2017.11.030 http://dx.doi.org/10.7150/ijbs.7.1161 http://dx.doi.org/10.1007/s00217-016-2760-7 http://dx.doi.org/10.1016/j.foodchem.2012.08.005 http://dx.doi.org/10.1016/j.fct.2008.10.014 http://dx.doi.org/10.1002/mnfr.201200421 http://dx.doi.org/10.1007/s11746-010-1608-8 http://dx.doi.org/10.1016/j.scienta.2012.07.030 http://dx.doi.org/10.1371/journal.pone.0231956 http://dx.doi.org/10.1016/j.foodchem.2011.01.068 http://dx.doi.org/10.1097/01.cej.0000130221.19480.7e http://dx.doi.org/10.1080/01140671.1996.9513950 http://dx.doi.org/10.1021/jf990993o http://dx.doi.org/10.1016/j.foodchem.2019.125246 http://dx.doi.org/10.17660/actahortic.2011.924.57 http://dx.doi.org/10.1016/j.scienta.2011.08.005 http://dx.doi.org/10.1007/s12161-012-9422-6 http://dx.doi.org/10.1111/eva.12457 http://dx.doi.org/10.1002/1438-9312(200210)104:9/10<602::aid-ejlt602>3.0.co;2-x http://dx.doi.org/10.1016/j.foodchem.2005.04.003 http://dx.doi.org/10.1002/ejlt.201100203 http://dx.doi.org/10.1016/j.foodchem.2004.03.012 http://dx.doi.org/10.1016/j.lwt.2016.12.029 http://dx.doi.org/10.3989/gya.021109 http://dx.doi.org/https://orcid.org/0000-0002-4829-2554 journal of applied botany and food quality 93, 186 196 (2020), doi:10.5073/jabfq.2020.093.023 1department of horticultural science, faculty of agricultural science and engineering, college of agriculture and natural resources, university of tehran, karaj, iran 2medicinal plants and drugs research institute, shahid beheshti university, tehran, iran 3julius kühn-institut, institute for ecological chemistry, plant analysis and stored product protection, berlin, germany morphological and phytochemical screening of some thymus ecotypes (thymus spp.) native to iran in order to select elite genotypes siavash mohammadi1, leila tabrizi1*, majid shokrpour1, javad hadian2, hartwig schulz3, david riewe3 (submitted: may 14, 2020; accepted: september 11, 2020) * corresponding author summary thymus spp. is one of the most important medicinal plants widely used in food, pharmaceutical, and cosmetic industries. in this research, different ecotypes of three thymus species including t. daenensis, t. kotschyanus and t. lancifolius native to iran were compared to two commercial cultivars of t. vulgaris (i.e. 'varico 3' and 'deutscher winter') under identical conditions. based on the results, there was a remarkable diversity among different ecotypes of thymus species. the highest plant dry weight was found in t. daenensis (malayer 2), t. kotschyanus (azerbaijan gharbi), and t. lancifolius (fars). the highest thymol percentage (> 75%) was obtained by t. daenensis. the ecotype of ilam belonging to t. daenensis gained highest essential oil percentage (7.83%). in all ecotypes of t. daenensis, thymol was the major constituent in their essential oil. five chemotypes of citral, carvacrol-thymol, thymol-carvacrol, p-cymene-carvacrol, and geranyl acetate-citral were found in t. kotschyanus ecotypes, while four chemotypes of thymol, α-terpineol-linalool, carvacrol-thymol and thymolgeraniol were identified for t. lancifolius. in addition, in terms of growth, yield, and phytochemical traits, the elite genotypes within ecotypes were selected. elite ecotypes and genotypes detected during this research could be used in thymus breeding programs. keywords: chemotype, essential oil, medicinal plants breeding, thymol introduction thymus species, belonging to the family lamiaceae, includes more than 215 species in the world, in which 14 species are native to iran, while 4 species are endemic to iran including t. persicus, t. carmanicus, t. daenensis, and t. trautvetteri (jamzad, 2009; safaeighomi et al., 2009; amiri, 2012). essential oil of thymus species has many applications in different industries such as pharmaceutical, food, sanitary, cosmetic and others, due to its antibacterial, antifungal and antioxidant properties, thereby occupying the top position in world trade of medicinal plants (alizadeh et al., 2013; mojab et al., 2008; rasooli and mirmostafa, 2002). in thymus species, the major constituents of its essential oil include thymol, carvacrol, and p-cymene (tohidi et al., 2019). in addition to its essential oil, its aerial parts containing tannin, flavonoids, saponin and bitter materials have been received great attention. thyme is used as an expectorant for curing cough, sore throat, bronchi, and asthma (petrović et al., 2017; edeoga et al., 2005). regarding the application of thymus in different industries, the breeding and creating thyme genotypes with high qualitative and quantitative yield is imperative. the main breeding objective of thymus species is to produce plants with higher yield of dry matter, ratio of leaf/shoot, essential oil, thymol content, crop uniformity, freezing tolerance in winter, upright form of growth, and finally possibility of mechanizing harvesting (carlen et al., 2010). in this regard, the simplest way for thyme breeding is evaluation of their populations and then selecting the elite genotypes based on their growth and chemotype traits (hadian et al., 2016; heydari et al., 2019). approximately, 60 to 75% of current medicinal crops have been produced through selecting elite genotypes among wild or landraces populations (bernat, 2000; nemet, 2000). in terms of dry matter yield, essential oil content, and chemical composition, thyme with higher qualitative traits and uniformity, are highly respected. progress in plant breeding requires an extended genetic pool (rahim malek et al., 2009). awareness of genetic diversity in crops and their wild relatives is an underlying requirement to improve crop yield (govindaraj et al., 2015). there are basic necessities for achieving high amount of bioactive compounds and their homogeneity in pharmaceutically important plants, such as sufficient genetic diversity in populations for creating favorable traits, appropriate genotypes for different cultivation systems, high harvest index, and low sensitivity to diseases and climate perturbations (deng et al., 2019; islam et al., 2019; deng et al., 2019). therefore, progress in breeding being in line with the mentioned requirements depends on genetic diversity created by plant breeders (rahim malek et al., 2009). thyme usually possesses high genetic diversity in terms of morphological and phytochemical traits (imbrea et al., 2010). according to loapez-pujol et al. (2004), higher genetic diversity was found in different ecotypes of t. loscosil; this was attributed to its polyploidy, resulted naturally from its heterozygosity as well as its biochemical and genetic diversities. also, the study of morphological diversity of 5 t. glabrescens ecotypes, showed a significant difference in leaf length and width as well as number of leaf secreting trichomes; which are useful traits in thyme breeding programs (dajic-stevanovic, 2008). hadian et al. (2016) evaluated morphological and essential oil of 6 populations of t. daenensis on their natural habitats, and found a remarkable morphological diversity between the populations; the content of essential oil in these ecotypes varied from 1.53 to 4.28%, with the highest essential oil content in ilam ecotype. however, major components of essential oil in different ecotypes of t. kotschyanus ranged from 1 to 1.41%, and the amount of main components in their essential oil was as follows: thymol (31.2%), carvacrol (19.5%), linalool (2.38-20.06%), alpha-terpineol (0.16-11.64%), and geraniol (0.36-11.37%) (tohidi et al., 2018). mostly, breeding thyme (especially those native to iran) has been focused on selecting naturally ecotypes and elite genotypes on the basis of morphological, phytochemical, and genetic traits. compared to breeding of agricultural crops, there is limited progress in breeding of medicinal plants. therefore, the present study aimed to evaluate different ecotypes of three thymus species (i.e. t. daenensis, screening of thymus spp. ecotypes for selection of elite genotypes 187 t. kotschyanus, and t. lancifolius) under field condition and also their comparison with two commercial cultivars of t. vulgaris (i.e. 'varico 3' and 'deutscher winter') under identical conditions in order to select elite genotypes and ecotypes qualified with high qualitative and quantitative yield for thyme breeding and cultivation goals. materials and methods plant materials the seeds of different ecotypes of thymus spp., provided by different places including research institute of forests and rangelands (rifr), medicinal plants and drugs research institute of shahid beheshti university (mdri-sub) and department of horticultural science and landscape engineering, university of tehran (hsdut) (tab. 1), were planted in february 2013 in plant growing trays, containing peat moss, grown in the research greenhouse of hsdut and irrigated regularly. in may 2013, the seedlings of studied ecotypes of different thymus spp. such as t. daenensis (12 ecotypes), t. kotschyanus (5 ecotypes) and t. lancifolius (5 ecotypes) along with two commercial cultivars of t. vulgaris ('varico 3' and 'deutscher winter') (tab. 1) were transferred into an experimental field located in research station of hsd-ut (located at 35° 46´ n; 50° 55´ e, at an altitude of 1320 m.a.s.l.). the experiment was laid out in randomized complete block design (rcbd) with three replications. the interand intra-planting spaces were 60 and 30 cm, respectively. each ecotype entailed three blocks containing 16 plants (3×16=48 plants). after transferring transplants into the field, they were irrigated using drip system and weed control was carried out in spring and summer seasons. the average day and night temperatures during growing season were 26.14 °c and 19.92 °c, respectively and precipitation average was 5.47 mm. physico-chemical characteristics of the soil of field used in this study were: texture: clay loam, ph: 8.7, electrical conductivity (ec): 1.3 ds/ m, organic matter: 0.64%, nitrogen (n): 0.07%, phosphorus (p): 1.03 mg/kg and potassium (k): 350 mg/kg. assessment of growth and yield traits as thyme is a perennial herbal plant, its growth and yield performance is not considerable in first year of cultivation. therefore, they were solely maintained during first year and then in the second year, their growth and yield traits were evaluated at full flowering stage (in spring and summer of 2014). in our research, morphological traits consisted of plant height, number of lateral shoot per plant, internode length, internode number, main fluorescence’s length, leaf length, leaf width, plant form (from 1, as recumbent to 5, as upright plants), shoot dry weight, leaf dry weight, ratio of leaf/shoot, and percentage of male sterile plants. after measuring growth traits, the plants were harvested at full flowering stage and then dried in shade at room temperature in order to perform phytochemical analysis of essential oil including its content, yield, and constituents. essential oil extraction and analysis after drying, at room temperature, essential oil was extracted using hydrodistillation method for 3 h with a clevenger type apparatus based on british pharmacopoeia (1988) and then, was kept in a vial at 4 °c until to be analysed. analysis of essential oil was carried out using gas chromatography/mass spectrometry (gc/ms) and gas chromatography/flame ionization detector (gc/fid) in phytochemistry department of julius kühn institut (jki), institute for ecological chemistry, plant analysis and stored product protection, berlin, germany. afterwards, essential oil of different ecotypes was injected to gc/ms (agilent, msd 5972/hp) and gc/fid, in which, thegc/ms was equipped with a column of ms-5 hp (30 m × 0.25 mm i.d. and film thickness 0.5 μm). the column temperature was set to start with 60-120 °c at 3 °c/min and then it was held with 220 °c at 8 °c/min and it held for 10 min at this temperature. the temperature of injector was 250 °c. helium was used as a carrier gas (1.0 ml min-1). mass spectrometry operates in electronic ioni zation mode at 70ev and calculation was carried out using chem station software. the detection of spectrometry was carried out using retention index and resulted data were compared with the indexes found in reference books (adams, 2017) and information found in computer library. the system of gc/fid (agilent, n hp6890) was equipped with a column ms-5 hp (30 m × 0.25 mm i.d. and film thickness 0.5 μm). the program of column temperature was set from 60-220 °c at 8 °c/min and then held for 10 min at this temperature. also, the temperature of injector was 250 °c and helium was used as a carrier gas (1.0 ml min-1). data analysis the anova was made based on rcbd and the means were compared by duncan’s multiple range test (dmrt) at 5% probability level. the analyses were done using spss (ver. 20) and the plotting of graphs was carried out using excel 2013. tab. 1: plant materials and seed collection origins of thyme samples. species ecotype name origin in iran species ecotype name origin in iran (province) (province) 1. khan-e-miran markazi 1. tehran tehran 2. lorestan lorestan 2. zanjan zanjan 3. isfahan 1 isfahan t. kotschyanus 3. ghazvin ghazvin 4. ilam ilam 4. az. gharbi west azarbaijan 5. malayer 1 hamedan 5. kordestan kordestan t. daenensis 6. arak markazi 1. lorestan lorestan 7. zagheh lorestan 2. kordestan kordestan 8. malayer 2 hamedan t. lancifolius 3. isfahan isfahan 9. fars fars 4. fars fars 10. isfahan 2 isfahan 5. markazi markazi 11. markazi 1 markazi 1. 'deutscher winter' germany 12. markazi 2 markazi t. vulgaris 2. varico 3 switzerland 3. isfahan isfahan 188 s. mohammadi, l. tabrizi, m. shokrpour, j. hadian, h. schulz, d. riewe results and discussion yield and morphological traits the analysis of variance (anova) results of morphological traits in thyme species and ecotypes are shown in tab. 2. as shown in tab. 3, the highest plant height was found in t. vulgaris (39.4 cm on average) that was significantly higher than those in other species (i.e. t. daenensis, t. kotschyanus, and t. lancifolius). in t. vulgaris, 'varico 3' gained the highest plant height (43.6 cm) compared to 'deutscher winter' and isfahan ecotype. carlen et al. (2010) substantiated the superiority of 'varico 3' over 'deutscher winter' in terms of plant height. in other studies, it has been confirmed that the height of t. vulgaris ecotypes were taller than t. daenensis ecotypes (kaveh et al., 2013; mondak et al., 2014; askari et al., 2018; mohammadi et al., 2019). in t. daenensis, the highest plant height (33 cm) was in markazi 1 which did not show a significant difference with markazi 2 and malayer 1. likewise, the highest plant height in t. kotschyanus and t. lancifolius was observed in azerbaijan gharbi (18.6 cm) and markazi (22.5 cm), respectively (tab. 4). our findings are in agreement with those obtained by kaveh (2013) who concluded that azerbaijan gharbi is the tallest plant among t. kotschyanus ecotypes. the height of plants is important because of its role in mechanizing and facilitating harvest activities. also, the highest biomass yield was obtained by 'varico 3' and azerbaijan gharbi ecotype, possessing highest plant height. according to results of previous studies carried out on t. pubescens (yavari et al., 2010) and different thymus species native to iran (mondak et al., 2014), there is a positive relationship between plant height and plant dry weight, and this is in agreement with our findings. in thyme, number of shoots per plant is attributed effectively in yield performance. among thymus species evaluated in this research, the highest shoot number (163.2) was observed in t. vulgaris that significantly higher than t. daenensis (8.5), t. kotschyanus (91.2), and t. lancifolius (85.6) (tab. 3). also, between ecotypes of the three last-mentioned thymus species, no significant difference was found between them in terms of shoot number (tab 3). the number of shoot in the mentioned species was as follows: t. daenensis (ilam ecotype, 97), t. kotschyanus (azerbaijan gharbi, 120), and t. lancifolius (kurdistan, 95) (tab. 4). the highest internode length (2.4 cm) belonged to t. daenensis which was significantly higher than that in the other three species. also, there was no significant difference in terms of internode length between t. vulgaris, t. kotschyanus, and t. lancifolius (tab. 3). in t. daenensis, the highest (2.93 cm) and lowest (1.27 cm) internode length were observed in markazi 1 and ilam, respectively (tab. 4). while in t. vulgaris, the highest internode length (2.3 cm) belonged to 'varico 3' which significantly was higher than 'deutscher winter' and isfahan (with no significant difference between the last two ecotypes); (tab. 4). also, the highest internode length in t. kotschyanus (2 cm) and t. lancifolius (1.83 cm) were belonged to tehran and isfahan, respectively (tab. 4). t. vulgaris showed the highest internode number (15.9) that significantly differs from that in the other three species, although the difference between these three thyme species was not significant (tab. 3). the highest internode number (10.7) in t. daenensis was allocated to ilam, being significantly higher than other ecotypes belonged to this species. likewise, the highest internode number (16.5) was found in 'deutscher winter' that significantly longer than 'varico 3' (14.7), but did not significant difference with isfahan (tab. 4). azerbaijan gharbi (9.6) and kurdistan (7.6) gained the highest internode number in t. kotschyanus and t. lancifolius ecotypes, respectively (tab. 4). of all thymus species investigated in this research, the highest florescence length (3.6 cm) was found in t. daenensis which higher than t. kotschyanus and t. lancifolius, whereas it did not have a significant difference with t. vulgaris (tab. 3). in t. daenensis, the longest florescence (5.5 cm) was in markazi 1 which did not have significant difference with markazi 2 (tab. 4). tab. 2: analysis of variance (anova) of morphological traits in thyme species and ecotypes. sources df anova of variance mean of the squares (ms) (s.o.v) traits plant lateral internode internode infloresleaf leaf plant shoot leaf to male height shoot length number cence length width form dry weight shoot sterile number length ratio plants block 2 12.058* 102.01ns 0.033ns 0.333ns 0.029ns 0.030* 0.0002ns 0.599* 648.6* 8.093ns 46.45ns species 3 1331.6** 583.8** 0.411** 179.5** 14.45** 4.61** 0.061** 7.28** 43912.6** 1153.3** 6990.4** ecotypes 21 36.3** 15535.5** 3.33** 3.74** 0.86** 0.065** 0.0067** 0.585** 1724.6** 52.82** 606.1** within species error 50 172.2 312.4 0.026 0.316 0.114 0.007 0.0009 0.088 42.49 2.87 22.67 coefficient of 7.6 18.4 8.1 6.5 11.7 6.3 6.7 8.4 8.2 2.9 13.3 variation (cv) (%) * and ** indicate statistical significance at 95% and 99%; ns show statistical insignificance. tab. 3: morphological traits in different thymus species species plant lateral internode internode infloresleaf leaf plant shoot leaf to male height shoot length number cence length width form dry weight shoot ratio sterile (cm) number (cm) length (cm) (cm) (cm) (g) (%) plants (%) t. daenensis 26.9 b 85.5 b 2.4 a 8.0 b 3.9 a 1.8 a 0.44 a 3.4 b 68.4 b 62.1 a 25.7 b t. vulgaris 39.4 a 163.2 a 1.9 b 15.9 a 3.8 a 0.6 c 0.32 b 5.0 a 192.4 a 46.2 b 61.2 a t. kotschyanus 15.1 c 91.2 b 1.6 b 7.6 b 2.2 b 1.0 b 0.49 a 3.3 b 65.9 b 49.0 b 27.3 b t. lancifolius 18.9 c 85.6 b 1.6 b 7.1 b 2.4 b 1.3 b 0.46 a 3.3 b 53.9 b 63.0 a 59.2 a any two means within a column followed by same letters are not significantly different at p ≤ 0.05, n = 3. screening of thymus spp. ecotypes for selection of elite genotypes 189 according to tab. 3, the highest and lowest leaf length were found in t. daenensis (1.8 cm) and t. vulgaris (0.6 cm), respectively. in t. daenensis, the highest leaf length was seen in khane-e-miran and lorestan (1.9 cm) that, except for isfahan 2, markazi 1 and 2, did not have a significant difference with other ecotypes belonging to this species. in terms of leaf length, the ecotypes of t. vulgaris (with leaf length of 0.6 cm on average), did not show a significant difference with each other. also, the highest leaf length in t. kotschyanus and t. lancifolius was allocated to tehran and markazi, respectively (tab. 4). among the four investigated thymus species, the highest leaf width (0.49 cm) was seen in t. kotschyanus which did not have a significant difference with t. daenensis and t. kotschyanus species. the lowest leaf width was observed in t. vulgaris (0.32 cm) (tab. 3). the ecotypes of zagheh (0.53 cm) (t. daenensis), azerbaijan gharbi (0.60 cm) (t. kotschyanus), and fars (0.51 cm) (t. lancifolius) were superior in leaf width among ecotypes of the three mentioned species. moreover, the leaf width in ecotypes of t. vulgaris was insignificant (tab. 4). in terms of mechanizing harvest, plants’ form plays a significant role in breeding program of thymus spp. (carlen et al., 2010). as stated before, the plants’ form is a qualitative trait and ratified by digits in this research as follows: 5 signifies completely upright plant, 3: semiupright plants and 1: completely recumbent plant. in this regard, t. vulgaris significantly ranked 5 and other thymus species occupied other positions, although difference between plants’ form of other species (i.e. t. daenensis. t. kotschyanus and t. lancifolius) was not statistically significant and they were qualified with semi-upright plants (tab. 3). in this regard, ilam (4.8 on average) remarkably demonstrated more upright form in comparison to other ecotypes of t. daenensis. in t. kotschyanus and t. lancifolius, the most upright plants belonged to azerbaijan gharbi and fars, respectively (tab. 4). the results of our research revealed that shoot dry weight of t. vulgaris (192.4 g) was significantly higher than other thymus species. also, there was no significant difference between shoot dry weight of t. daenensis (68.5 g), t. lancifolius (65.9 g), and t. kotschyanus (53.9 g) (tab. 3). our findings are in agreement with those found by kaveh et al., 2013; mondak et al., 2014; askari et al., 2018; mohammadi et al., 2019 who stated that shoot dry weight of t. vulgaris is higher than in the other three mentioned thymus species. the highest shoot dry weight in t. vulgaris belonged to 'varico 3' (213 g) that was significantly higher than in 'deutscher winter' and isfahan (tab. 4). it is necessary to mention that 'varico 3' is a hybrid produced from t. vulgaris in the agroscope research institute, switzerland. the results of carlen et al. (2010) also proved the superiority of 'varico 3' over 'deutscher winter' in terms of shoot dry weight. according to tab. 4, the highest shoot dry weight was in malayer 2 (85 g) (t. daenensis), azerbaijan gharbi (152 g) (t. lancifolius) and fars (61 g) (t. kotschyanus). the highest leaf/shoot ratio (63%) was found in t. lancifolius which did not have a significant difference with t. daenensis (62.2%), and the lowest (42.2%) was obtained by t. vulgaris (tab. 3). in t. daenensis, the highest ratio of leaf/shoot was observed in zagheh (69%) which did not show a significant difference with ilam (66%), although it had a significant difference with other ecotypes of t. daet. d ae ne ns is t. la nc ifo liu s t. k ot sc hy an us t. v ul ga ri s tab. 4: morphological traits in different thymus ecotypes species ecotype plant lateral internode internode inflorescence leaf leaf plant shoot leaf to male height shoot length number length length width form dry weight shoot ratio sterile (cm) number (cm) (cm) (cm) (cm) (g) (%) plants (%) khane miran 28.4 bc 96 a 2.63 ab 8.5 b 3.5 de 1.90 a 0.45 bc 3.5 bcd 83.8 a 63.0 bc 27.7 cd lorestan 26.0 cd 94 a 2.17 c 8.7 b 3.4 de 1.90 a 0.45 bc 2.9 d 62.2 de 61.0 c 0.0 h esfahan1 26.3 cd 95 a 2.27 bc 8.3 b 4.0 bcd 1.87 a 0.36 d 3.1 d 68.3 cd 60.7 c 31.0 bc ilam 23.5 d 97 a 1.27 d 10.7 a 4.4 bc 1.83 a 0.46 bc 4.6 a 48.5 f 66.3 ab 7.7 gh malayer 1 29.4 abc 86 ab 2.63 ab 7.8 bc 3.6 cde 1.83 a 0.42 bcd 4.0 ab 77.1 abc 60.3 c 21.7 def arak 26.8 cd 67 bc 2.67 ab 7.0 cd 3.8 cd 1.80 ab 0.45 bc 3.1 d 59.1 e 62.3 c 38.7 b zagheh 18.2 e 55 c 2.00 c 6.2 d 2.8 e 1.80 ab 0.53 a 3.3 bcd 47.1 f 69.0 a 28.0 cd malayer 2 26.9 cd 69 bc 2.63 ab 6.1 d 3.7 cde 1.77 ab 0.48 ab 3.9 bc 84.6 a 60.0 c 55.3 a fars 28.6 bc 94 a 2.70 a 7.9 bc 3.9 bcd 1.73 ab 0.39 cd 3.2 d 62.6 de 62.7 bc 26.0 cde esfahan 2 23.7 d 84 ab 2.17 c 7.6 bc 3.4 de 1.60 bc 0.43 bcd 2.9 d 70.4 bcd 60.7 c 38.0 gh markazi 1 33.0 a 94 a 2.93 a 8.5 b 5.5 a 1.60 bc 0.42 bcd 3.1 d 78.7 ab 60.7 c 16.0 fg markazi 2 32.2 ab 96 a 2.80 a 8.4 b 4.8 ab 1.43 c 0.46 abc 3.3 cd 79.3 ab 59.3 c 18.3 ef 'deutscher winter' 37.2 b 155 b 1.8 b 16.5 a 3.73 a 0.57 a 0.31 a 5.0 a 183.1 b 52.3 a 58.7 b esfahan 37.3 b 156 b 1.7 b 16.3 a 3.67 a 0.53 a 0.31 a 5.0 a 182.6 b 52.0 a 57.3 b 'varico 3' 43.6 a 179 a 2.3 a 14.7 b 3.87 a 0.57 a 0.33 a 5.0 a 212.6 a 34.0 b 67.7 a tehran 14.4 b 88 b 2.0 a 6.4 c 2.4 a 1.1 a 0.49 b 3.2 bc 49.3 bc 48.3 a 18.0 b zanjan 15.4 b 82 b 1.7 ab 8.1 ab 2.3 a 1.0 ab 0.49 b 3.5 ab 33.2 d 48.3 a 28.7 ab ghazvin 13.6 b 89 b 1.7 ab 6.6 bc 2.2 a 1.0 a 0.42 b 2.9 c 54.5 b 50.0 a 41.7 a az. gharbi 18.6 a 120 a 1.4 b 9.6 a 2.1 a 0.8 c 0.60 a 3.9 a 152.1 a 48.0 a 28.7 ab kordestan 13.7 b 77 b 1.3 b 7.2 bc 2.1 a 0.9 bc 0.46 b 2.9 c 40.4 cd 50.3 a 19.7 b lorestan 14.6 c 89 a 1.5 a 6.8 a 2.2 a 1.0 c 0.48 a 2.8 b 49.1 bc 56.7 d 33.3 c kordestan 17.6 bc 95 a 1.5 a 7.6 a 2.5 a 1.1 c 0.47 ab 3.1 ab 46.2 c 63.7 bc 48.3 bc esfahan 20.4 ab 89 a 1.8 a 6.7 a 2.7 a 1.3 b 0.39 b 3.4 ab 54.7 abc 67.3 a 46.0 bc fars 19.2 ab 86 a 1.6 a 7.4 a 2.7 a 1.4 b 0.51 a 3.7 a 61.3 a 65.0 b 52.7 c markazi 22.5 a 69 a 1.8 a 6.8 a 2.1 a 1.6 a 0.43 ab 3.5 a 58.1 ab 62.3 c 84.3 a any two means within a column followed by same letters are not significantly different at p ≤ 0.05, n = 3. 190 s. mohammadi, l. tabrizi, m. shokrpour, j. hadian, h. schulz, d. riewe nensis (tab. 4). in t. vulgaris, 'varico 3' significantly gained the lowest ratio of leaf/shoot (34%) which was lower than 'deutscher winter' and isfahan (tab. 4). also, there was no significant difference between ecotypes of t. kotschyanus in terms of ratio of leaf/ shoot (tab. 4). in t. lancifolius, the highest ratio of leaf/shoot (67%) was shown by isfahan that significantly was higher than that in other ecotypes belonging to this species (tab. 4). the ratio of leaf/shoot (called as a harvest index) is of important traits suitable for breeding of thymus species. regarding higher essential oil content in leaf and flower compared to shoot and also high density of trichrome in leaf and flower secreting essential oil (hadian et al. 2016), the yield of essential oil in thymus species with high ratio of leaf/shoot is higher than the other species with low ratio of leaf/shoot. in our research, the ratio of leaf/shoot in t. vulgaris was lower than the other species investigated here. although 'varico 3' showed highest leaf dry weight (34%), its leaf/shoot ratio was lower than 'deutscher winter' (52%). because of depressing leaf/shoot ratio of 'varico 3' compared to 'deutscher winter', high harvest index (or ratio of leaf/shoot) is one of the most important advantages in thymus species native to iran such as t. daenensis. ratio of leaf/shoot is directly related to leaf number and essential oil yield; accordingly, it plays a crucial role in cultivation and breeding of thymus species. compared to t. vulgaris, the three-mentioned species also possess higher shoot dry weight and ratio of leaf/shoot, thereby having higher leaf number and essential oil yield. percent of male sterile plant the percent of male sterile plant among different ecotypes of t. daenensis is one of most important traits useful in thyme breeding via hybridization. among the four investigated thymus species, the highest percentage of male sterile plant (61.2%) was seen in t. vulgaris that did not show a significant difference with t. lancifolius (59.2%) (tab. 3). the highest percentage of male sterile plant in t. vulgaris was found in 'varico 3' (68%) that showed a significant difference with 'deutscher winter' and isfahan (tab. 4). in t. daenensis, the highest percentage of male sterile plant was obtained by malayer 2 (55%) that was significantly higher than that in other ecotypes of this species. also, in lorestan ecotype, even one male sterile plant was not found (tab. 4). the highest percentage of male sterile plant (42%) was found in qazvin ecotype belonged to t. kotschyanus which did not show a significant difference with azerbaijan gharbi and zanjan ecotypes of this species (tab. 4). among ecotypes belonging to t. lancifolius, the highest percentage of male sterile plant (84%) was obtained by markazi which significantly differed to other t. lancifolius ecotypes (tab. 4). as described earlier, male sterility plays an important role in thyme breeding. in general, emasculation of thyme flowers is difficult, due to flower smallness and high density of flower on a fluorescence as well as non-simultaneity in time of opening flowers (rey, 1993; mewes and pank, 2006). accordingly, identifying male sterile plant in thyme populations is important for hybridization activities. the variety 'varico 3' is produced from t. vulgaris via hybridization using male sterile plant (carlen et al., 2010). also, the percentage of male sterile plant in 'varico 3' was higher than that in 'deutscher winter', and this may be due to exploiting male sterile plant in hybridization as maternal parents (carlen et al., 2010). moreover, the type of male sterility in thyme is cytoplasmic; accordingly, male sterility modulation is a complex process and controlled by at least 5 genes (belhassen et al., 1991). cluster analysis of thymus ecotypes in our research, growth and yield traits were subjected to cluster analysis using ward method. the results showed that all ecotypes were categorized into four groups (fig. 1). the first group consisted of 11 ecotypes classified into two subclusters. the first sub-cluster consisted of 9 ecotypes of t. daenensis (khan-e-miran, malayer 1 and 2, markazi 1 and 2, lorstan, isfahan 1, arak and isfahan 2). in the second sub-cluster, only ilam belonging to t. daenensis was placed (fig. 1). in is noteworthy that 11 ecotypes of t. daenensis in this group were qualified with high phytochemical attributes (such as thymol) and ordinary growth and yield. in the second group, 10 ecotypes were found in forms of two sub-clusters; at first sub-cluster, 5 ecotypes of t. lancifolius including lorstan, kurdistan, isfahan, and fars and 1 ecotype of t. daenensis including zagheh were observed, and in second sub-cluster, just 4 ecotypes of t. kotschynanus including tehran, zanjan, kurdistan, and qazvin were placed (fig. 1). this group was qualified with high phytochemical (especially essential oil content and thymol) and low growth and yield. zagheh, belonging to t. daenensis, solely occupied this place, because of its unique characteristics such as low essential oil percentage and low chemical compounds such as thymol, growth, and yield, fig. 1: clustering different ecotypes and species of thymus based on morphological traits using ward method screening of thymus spp. ecotypes for selection of elite genotypes 191 in comparison to the other t. daenensis’s ecotypes. in the third group, only azerbaijan gharbi, belonging t. kotschyanus and qualified with relatively high growth and yield, but low phytochemicals (especially essential oil and thymol) was placed. in the fourth group, 3 ecotypes of t. vulgaris were placed in forms of two sub-clusters: isfahan and 'deutscher winter' in the first sub-cluster and 'varico 3' in the second sub-cluster (fig. 1). this group is characterized by high growth and yield and a moderate rate of essential oil and thymol. in general, it can be concluded that the cluster analysis categorized different ecotypes of thymus species into four groups, in such a way that 11 groups belonged to the first group, 10 ecotypes to the second, one ecotype to the third, and finally three ecotypes to the fourth group (fig. 1). in a research carried out by mondak et al. (2014), 22 ecotypes of thymus native to iran were placed into two main groups in terms of morphologic and essential oil traits. the first group included t. daenensis ecotypes and the second group belonged to t. lancifolius and t. kotschynanus ecotypes. phytochemical traits essential oil content among four thymus species investigated in this research, t. daenensis gained the highest essential oil content (5.76%) and its content in other three species was as follows: t. vulgaris (3.59%), t. kotschyanus (2.43%), and t. lancifolius (3.96%) (fig. 2). in t. daenensis, the highest essential oil content was observed in ilam being significantly higher when compared to other ecotypes of this species (fig. 2). in t. vulgaris, 'varico 3' showed the highest essential oil content (4.42%) which was significantly higher than in 'deutscher winter' (3.21%) and isfahan (3.16%) (fig. 2). azerbaijan gharbi, belonging to t. kotschyanus, gained the highest essential oil content (2.79%), although it did not have a significant difference with that in zanjan and qazvin belonging to this species (fig. 2). among ecotypes belonging to t. lancifolius, fars significantly obtained the highest essential oil (5.38%) in comparison to the other ecotypes belonging to this species (fig. 2). as presented in fig. 2, there was a relatively high diversity among ecotypes and species of thymus in this regard. the highest essential oil content (7.83%) was found in t. daenensis (ilam). also, essential oil in t. daenensis ecotypes varied from 2.5 to 5%. moreover, khorshidi et al. (2018) reported a remarkable diversity among t. daenensis ecotypes (2.5-5%). based on rustaiee et al. (2010), the content of essential oil among t. daenensis ecotypes, naturally growing in iran, was 1.8 to 3.83%, and the highest essential oil and thymol content were found in arak, while the lowest was observed in daenensis ecotypes. in another study, the content of essential oil in t. daenensis ecotypes, gathered from hamadan regions (nicaver et al., 2005), was about 2.4%, and its content in ecotypes gathered from 5 regions of isfahan province, iran, was about 3.3%. the previous studies showed a higher essential oil content in t. daenensis ecotypes, as compared to t. vulgaris (mondak et al., 2014; askari et al., 2018; mohammadi et al., 2019). according to our findings, the essential oil content in 'varico 3' was significantly higher than in 'deutscher winter' (fig. 2), and this is in agreement with that obtained by carlen et al. (2010), who recorded a superiority of 'varico 3' over 'deutscher winter' in terms of essential oil content. in our research, the lowest essential oil content was observed in t. kotschyanus (fig. 2). also, kaveh et al. (2013) reported various levels of essential oil in t. kotschyanus, from 0.42 to 2.17%. in other research, the amount of essential oil in this species was 1.1 to 2.5% (khoshsokhan et al., 2014). also, in this research, essential oil content in t. lancifolius, depending on the individual ecotype, varied from 2.93 to 5.38%. furthermore, the essential oil content in t. lancifolius under greenhouse condition was reported to have 0.93 to 2.32% (mondak et al., 2014). essential oil yield as shown in fig. 3, the highest essential oil yield was found in t. vulgaris (3.08 ml per plant), although it did not have a significant difference with that in t. daenensis (2.47 ml per plant) and conversely had a significant difference with t. kotschyanus (0.8 ml per plant) and t. lancifolius (1.38 ml per plant) (fig. 3). the highest yield of essential oil (3.19 ml per plant) was obtained by 'varico 3', although it did not show a significant difference with 'deutscher winter' (3.07 ml per plant) (fig. 3). among ecotypes of t. kotschyanus, azerbaijan gharbi gained the highest yield of essential oil (2.04 ml per plant) that was significantly higher than other ecotypes belonging to this species (fig. 3). in t. lancifolius, the highest yield of essential oil was in fars (2.14 ml per plant) that was significantly higher than other ecotypes of t. lancifolius species (fig. 3). in thymus species, essential oil yield is of the most important traits gained through multiplying essential oil and leaf dry weight. as shown in fig. 3, the highest yield of essential oil was obtained by t. vulgaris which did not show a significant difference with t. daenensis. although dry weight of t. vulgaris was higher than t. daenensis and, conversely, ratio of leaf/shoot in t. daenensis was higher than t. vulgaris, but the essential oil yield in t. vulgaris and t. daenensis was the same. also, in our research, the yield of essential oil in 'varico 3' and 'deutscher winter' did not show a significant difference (fig. 3). although the rate of essential oil and shoot dry weight in 'varico 3' was higher than b d c a bc b f b e de b c b b a a a b a b c c b a b 0 1 2 3 4 5 6 7 8 k ha ne m ir an lo re st an e sf ah an 1 ila m m al ay er 1 a ra k za gh eh m al ay er 2 fa rs e sf ah an 2 m ar ka zi 1 m ar ka zi 2 d . w in te r e sf ah an v ar ic o 3 te hr an za nj an g ha zv in a z. g ha rb i k or de st an lo re st an k or de st an e sf ah an fa rs m ar ka zi t. daenensis t. vulgaris t. kotschianus t. lancifolius es se n ti al o il co n te n t (% ) 5.76 a 3.59 bc 2.34 c 3.96 b fig. 2: mean essential oil content in different thymus ecotypes and species 192 s. mohammadi, l. tabrizi, m. shokrpour, j. hadian, h. schulz, d. riewe 'deutscher winter', the ratio of leaf/shoot in 'deutscher winter' (52%) remarkably was higher than 'varico 3' (34%); so, leaf dry weight in 'deutscher winter' was higher than 'varico 3', and this leads to insignificant difference in both cultivars in terms of essential oil yield (fig. 3). major essential oil constitutes of t. daenensis’s ecotypes according to tab. 5, the main components found in different ecotypes of t. daenensis include thymol (47.1-82%), carvacrol (0.7724.39%), p-cymene (2.76-5.37%) and γ-terpinen (1.65-4.07%). thy mol was the major constituent widely found in all ecotypes of t. daenensis; all ecotypes, except for ecotype zagheh (47.08%), contained more than 75% thymol. in contrast, zagheh showed highest rate of carvacrol (24.39%) in comparison to the other t. daenensis ecotypes which contained lower than 5% of carvacrol. in different studies, thymol has been found as a main constituent of t. daenensis ecotypes (nickavar et al., 2005; mondak et al., 2014; khorshidi et al., 2018, haydari et al., 2019). also, the rate of carvacrol in zagheh was 24.4%, while carvacrol in other ecotypes of t. daenensis was lower than 5% (tab. 5). in a study carried out by khorshidi et al. (2018), zagheh was shown to have higher carvacrol rather than the other ecotypes of t. daenensis. also, among 12 ecotypes of t. daenensis, 11 ecotypes were introduced as thymol chemotypes and one of them (i.e. zagheh) as carvacrol-thymol chemotype. major essential oil constitutes of t. vulgaris in t. vulgaris, the main components of essential oil included thymol (38.33-42.23%), p-cymene (22.06-24.48%), γ-terpinene (9.9714.37%), and carvacrol (2.83-2.94%) (tab. 6). in this respect, 'varico 3' gained the highest thymol (42.23%). in terms of carvacrol, there was not found a remarkable diversity between ecotypes of t. vulgaris, and its rate in this species was reported 2.9% (tab. 6). carlen et al. (2010) recorded high essential oil content in 'varico 3' compared to 'deutscher winter'. in this regard, mondak et al. (2014) a gh def cde bc efg i a h fgh ab cd a a a b c b a c c c b a b 0 1 2 3 4 k ha ne m ir an lo re st an e sf ah an 1 ila m m al ay er 1 a ra k za gh eh m al ay er 2 fa rs e sf ah an 2 m ar ka zi 1 m ar ka zi 2 d . w in te r e sf ah an v ar ic o 3 te hr an za nj an g ha zv in a z. g ha rb i k or de st an lo re st an k or de st an e sf ah an fa rs m ar ka zi t. daenensis t. vulgaris t. kotschianus t. lancifolius es se n ti al o il yi el d ( m l p er p la n t) 2.47 a 3.08 a 0.79 b 1.38 b fig. 3: mean essential oil yield in different thymus ecotypes and species tab. 5: major constituents in essential oil of different ecotypes of t. daenensis ecotype p-cymene (%) γ-terpinene (%) thymol (%) carvacrol (%) chemotype khane miran 2.83 b 3.61 a 78.93 a 1.38 b thymol lorestan 5.37 a 4.07 a 75.98 a 3.52 b thymol isfahan 1 3.32 b 2.94 a 79.48 a 3.38 b thymol ilam 3.9 b 3.69 a 78.47 a 3.83 b thymol malayer 1 3.11 b 3.13 a 80.25 a 2.37 b thymol arak 4.73 ab 1.065 b 78.15 a 2.67 b thymol zagheh 5.08 a 3.32 a 47.08 b 24.39 a thymol/carvacrol malayer 2 2.76 b 2.33 a 82.01 a 0.77 b thymol fars 3.57 b 2.36 a 76.26 a 4.01 b thymol isfahan 2 3.94 b 2.12 ab 77.27 a 3.15 b thymol markazi 1 3.08 b 2.58 a 78.19 a 3.17 b thymol markazi 2 3.05 b 2.32 a 76.69 a 2.63 b thymol any two means within a column followed by same letters are not significantly different at p ≤ 0.05, n = 3. tab. 6: major constituents in essential oil of t. vulgaris cultivar p-cymene (%) γ-terpinene (%) thymol (%) carvacrol (%) β-caryophyllene (%) chemotype 'deutscher winter' 24.68 a 14.37 a 38.33 b 2.94 a 0.85 a thymol isfahan 24.16 a 14.3 a 39.1 b 2.9 a 0.79 a thymol 'varico 3' 22.06 b 9.97 b 47.23 a 2.83 a 1.0 a thymol any two means within a column followed by same letters are not significantly different at p ≤ 0.05, n = 3. screening of thymus spp. ecotypes for selection of elite genotypes 193 also reported that thymol was the major constituent of essential oil in t. vulgaris. also, it was introduced thymol (51.2%) as a major constitute of monoterpene index in essential oil of t. vulgaris; however, with low rate of carvacrol (4%) in this species. in general, thymol was introduced as a major constituent in essential oil of t. vulgaris (carlen et al., 2010). major essential oil constitutes of t. kotschyanus’s ecotypes in t. kotschyanus, the main components of its essential oil were different, due to not only type of ecotype but also type and content of main components of essential oil (tab. 7). the main essential oil components of t. kotschyanus were as follows: citral (0.48-42.09%), thymol (7.32-34.07), carvacrol (2.34-19.37%), geranial astat (0.9524.81%), p-cymene (0.43-18.98%) and gamma terpinene (0.2411.23%) (tab. 7). according to type of ecotype, the main essential oil components of t. kotschyanus’s ecotypes were consisted of tehran (citral, carvacrol, thymol, and geranial astat), zanjan (thymol, carvacrol, geranyol, p-cymene), qazvin (carvacrol, thymol, p-cymene, and beta-terpineol), azerbaijan gharbi (p-cymene, carvacrol, gamma terpinen and thymol), and kurdistan (geranial astat, citral, thymol, and linalool) (tab. 7). however, for nickavar et al. (2006), the main components of t. kotschyanus were thymol (38.6%) and carvacrol (33.9%). in another study, the main components found in this species were as follows: polygon (18.7%), isomenton (17.8%), thymol (14.3%), eucalyptol (9%), piperiton (6.3%) and carvacrol (5.5%) (semnani et al., 2006). in this regard, it has also been reported that the major constituents found in essential oil of t. kotschyanus ecotypes were different and included thymol (31.2%), carvacrol (19.5%), p-cymene (11.2%), and γ-terpinene (8.4%) (kilic and özdemir, 2017). according to tohidi et al. (2018), major constituents of essential oil in different ecotypes of t. kotschyanus varied from 1 to 1.41% and main components in its essential oil were consisted of thymol (31.2%), carvacrol (19.5%), linalool (2.38-20.06%), α-terpineol (0.16-11.64%), and geraniol (0.36-11.37%). also, among 43 chemicals found in essential oil of t. kotschyanus, sajadi and khatamsaz (2003) reported that thymol, carvacrol, and p-cymene were major constituents. major essential oil constituents of t. lancifolius’s ecotypes as shown in tab. 8, the main components of essential oil in t. lancifolius were largely different, depending on type of ecotype. the main components of essential oil in ecotypes of t. lancifolius were as follows: thymol (13.81-68.67), carvacrol (3.64-48.74), geraniol (0.57-25.57), α-terpineol (4.19-14.81), linalool (3.56-12.15), citral (7.88-10.08), geranil astat (1.06-7.33), α-terpinel astat (3.72-6.75), pcymene (2.10-5.12), and borneol (1.47-4.08) (tab. 8). also, the main components of essential oil in all ecotypes of t. lancifolius were as follows: lorstan (carvacrol, thymol and p-cymene), kurdistan (alpha terpineol, thymol, linalool, carvacrol and citral), isfahan (thymol, linalool, and alpha terpineol), fars (thymol, geraniol, citral, and geranil astat), and markazi (thymol and alpha terpineol astat) (tab. 8). furthermore, mondak et al. (2014) found thymol, carvacrol, and geraniol as major constituents of essential oil in t. lancifolius. elite genotypes by evaluating different ecotypes of thymus species in terms of growth, yield, and phytochemical traits, some genotypes were selected within ecotypes qualified with interesting traits. the preliminary selection of elite genotypes was carried out on the basis of some visual traits such as plant height, growth form, shoot dry weight, and ratio of leaf/shoot; subsequently the qualified genotypes were evaluated in terms of phytochemical traits such as content and yield of essential oil as well as major constituents of essential oil such as thymol, and eventually three genotypes (numbered as 1, 2, and 3), as elite genotypes, were identified. the results showed that all three selected genotypes belong to t. daenensis and their characteristics a presented in tab. 9. these genotypes were qualified with some important traits such as plant height, plant form, number of lateral shoots, dry and fresh weight of shoot, ratio of leaf/shoot, the percent and yield of essential oil, and rate of thymol. the form of plant is important because of its role in mechanizing crop harvest. in our research, the elite genotypes (1, 2, and 3) were in upright form and suitable for harvesting mechanically (tab. 9). shoot dry weight in elite genotypes remarkably was higher than that in t. daenensis and even in t. vulgaris. the ratio of leaf/shoot in elite genotypes was similar to tab. 7: major constituents in essential oil of different ecotypes of t. kotschyanus ecotype p-cymene γ-terpinene beta-terpineol linalool citral geraniol thymol carvacrol geranyl chemotype (%) (%) (%) (%) (%) (%) (%) (%) acetate (%) tehran 3.37 c 0.81 c 0.17 b 3.5 b 42.09 a 0.46 bc 7.32 c 19.37 a 5.1 b citral zanjan 8.74 b 2.68 b 0.09 b 1.3 b 0.11 e 9.08 a 34.07 a 18.74 a 0.95 c thymol/carvacrol ghazvin 7.00 b 4.44 b 6.79 a 0.25 c 5.49 c 0.12 c 18.62 b 19.35 a 0.16 d thymol/carvacrol az. gharbi 18.98 a 11.23 a 0.21 b 2.27 b 0.48 d 0.18 c 11.09 c 17.31 a 0.12 d p-cymene /carvacrol kordestan 0.43 d 0.24 c 0.08 b 8.25 a 22.41 b 2.13 b 14.79 bc 2.34 b 24.81 a geranyl acetate/citral any two means within a column followed by same letters are not significantly different at p ≤ 0.05, n = 3. tab. 8: major constitutes in essential oil of different ecotype of t. lancifolius ecotype linalool borneol α-terpineol citral geraniol thymol carvacrol α-terpineol geranyl chemotype (%) (%) (%) (%) (%) (%) (%) acetate (%) acetate (%) lorestan 3.56 c 1.84 b 0.09 c 0.22 c 1.12 b 19.46 d 48.74 a 0.20 c 1.06 c carvacrol kordestan 12.15 a 4.08 a 14.81 a 7.88 b 0.57 b 13.81 e 8.4 b 0.16 c 2.38 b α-terpineol isfahan 6.63 b 0.11 c 4.19 b 0.15 c 0.10 c 60.14 b 4 c 3.72 b 0.12 d thymol fars 0.21 d 1.47 b 0.13 c 10.08 a 25.57 a 28.69 c 4.5 c 0.11 c 7.33 a thymol/geraniol markazi 0.14 d 0.18 c 0.11 c 0.18 c 0.18 c 68.67 a 3.64 c 6.75 a 0.14 d thymol any two means within a column followed by same letters are not significantly different at p ≤ 0.05, n = 3. 194 s. mohammadi, l. tabrizi, m. shokrpour, j. hadian, h. schulz, d. riewe that in t. daenensis, but larger than t. vulgaris. essential oil content in elite genotypes of 1, 2, and 3 (8.22%, 7.80%, and 9.07%, respectively) was remarkably higher than those in t. vulgaris (3.56%) and t. daenensis (5.76%). the essential oil yield, as an important traits in thymus species, in elite genotypes of 1, 2, and 3 (11.89, 10.27, and 17.55 ml per plant, respectively), was remarkably higher than essential oil yield in t. daenensis (2.47 ml per plant) and t. vulgaris (3.18 ml per plant). in terms of thymol, elite genotypes contained 73.61, 73.91 and 75.19%, being similar to those in t. daenensis (75.73%), and conversely larger than that in t. vulgaris (41.55%) (tab. 9). the results of our research revealed a remarkable diversity between and within thymus species in terms of morphological and phytochemical traits. among four thymus species investigated in this research, the highest shoot dry weight was found in t. vulgaris that was significantly higher than that in the other three thymus species. an average yield of dry matter in the mentioned three species, compared to t. vulgaris, was relatively low. depending on the individual ecotype, the rate of dry matter among thymus ecotypes was different and khan-e-miran and malayer 2 belonging to t. daenensis and azerbaijan gharbi belonging to t. kotschyanus and fars of t. lancifolius had higher dry matter in comparison to the other ecotypes. moreover, among the four thymus species, the highest essential oil content was found in t. daenensis which was remarkably higher than in the others. in t. daenensis, ilam (7.83%) gained highest essential oil content and its essential oil yield did not show a significant difference to t. vulgaris; also, the ecotypes of khan-e-miran, malayer 2 and markazi 1 had higher yield in comparison to the other thymus species, especially t. vulgaris. according to the results, the essential oil content and major constituents of essential oil were clearly dependent on the individual species and ecotype. in this regard, our findings exhibited high diversity in the thymus species and their ecotypes. in a nutshell, thymol was found to be a major component in the essential oils of all t. daenensis ecotypes, and all the ecotypes contained approximately 75% thymol, except for zagheh ecotype. also, thymol was a major constituent in essential oil of t. vulgaris (43% on average). in addition to thymol, other main components including p-cymene and γ-terpinene were found in t. kotschyanus and t. lancifolius. it is necessary to mention that the type and content of essential oil in the thymus species was different, dependent on type of ecotype. in our research, five chemotypes (citral, carvacrol-thymol, thymol-carvacrol, p-cymene-carvacrol and geranial astat) and four chemotypes (thymol, alpha terpineol-linalool, carvacrol-thymol, thymol-geraniol) were identified in different ecotypes of t. kotschyanus and t. lancifolius, respectively. therefore, such high diversity in type and content of thyme essential oil is suitable for employing in different industries. finally, the three elite genotypes were recognized based on their morphological and phytochemical traits. so, after propagating elite genotypes and examining their yield, they can be used as candidate parents for thyme breeding and cultivation. acknowledgments hereby we want to appreciate department of horticultural science of university of tehran and julius kühn-institut (jki), institute for ecological chemistry, plant analysis and stored product protection, for supporting this research. we also thank dr. torsten meiners for his detailed comments and suggestion which highly improved this project. conflict of interest no potential conflict of interest was reported by the authors. references adams, r.p., 2017: identification of essential oil components by gas chromatography/mass 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leaf plant shoot plant shoot shoot leaf essential essential thymol length node cence length width height number growth fresh dry to shoot oil content oil yield (%) (cm) number length (cm) (cm) (cm) (cm) form weight (g) weight (g) ratio (%) (ml per plant) t. daenensis 2.4 a 8.0 b 3.9 a 1.8 a 0.44 a 26.9 b 85.5 b 3.4 b 134.3 b 68.5 b 62.1 a 5.76 a 2.47 a 75.73 a t. vulgaris 1.9 b 15.9 a 3.8 a 0.6 c 0.32 b 39.4 a 163.2 a 5.0 a 405.7 a 192.4 a 46.2 b 3.59 bc 3.18 a 41.55 b t. kotschyanus 1.6 b 7.6 b 2.2 b 1.0 b 0.49 a 15.1 c 91.2 b 3.3 b 153.4 b 65.9 b 49 b 2.34 c 0.80 b 17.18 c t. lancifolius 1.6 b 7.1 b 2.4 b 1.3 b 0.46 a 18.9 c 85.6 b 3.3 b 150.8 b 53.9 b 63 a 3.96 b 1.38 b 38.15 b genotype 1 1.5 11 10 2 0.5 38 250 5 576.5 226.5 63 8.33 11.89 73.61 genotype 2 2.2 8 5 2.1 0.5 32 190 4 536.5 219.5 60 7.8 10.27 73.91 genotype 3 1.7 11 6 2 0.5 32 300 4 765.5 322.5 60 9.07 17.55 75.19 any two means within a column followed by same letters are not significantly different at p ≤ 0.05, n = 3. 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phytochemical properties of thymus species in iran. ind. crops prod. 134, 89-99. doi: 10.1016/j.indcrop.2019.02.038 yavari, a.r., nazeri, v., sefidkon f., hassani. m.e., 2010: study on some ecological factors, morphological traits, ploidy levels and essential oil composition of thymus pubescens boiss. & kotschy ex celak in two natural regions of east azerbaijan province, iranian journal of medicinal and aromatic plants 26, 500-512. in persian. orcid siavash mohammadi https://orcid.org/0000-0001-7718-2648 leila tabrizi https://orcid.org/0000-0002-8878-7412 majid shokrpour https://orcid.org/0000-0001-9377-554x javad hadian https://orcid.org/0000-0002-5033-8857 hartwig schulz https://orcid.org/0000-0002-7551-3906 david riewe https://orcid.org/0000-0002-9095-5518 addresses of the authors: siavash mohammadi, department of horticultural science, faculty of agricultural science and engineering, college of agriculture and natural resources, university of tehran, karaj, 31587, iran leila tabrizi, department of horticultural science, faculty of agricultural science and engineering, college of agriculture and natural resources, university of tehran, karaj, 31587, iran e-mail: l.tabrizi@ut.ac.ir majid shokrpour, department of horticultural science, faculty of agri cultural science and engineering, college of agriculture and natural http://dx.doi.org/10.12162/jbb.v4i4.012 http://dx.doi.org/10.5897/ajb2005.000-3127 http://dx.doi.org/10.1155/2015/431487 http://dx.doi.org/10.1016/j.indcrop.2019.01.033 http://dx.doi.org/10.1016/j.aaf.2019.02.007 http://dx.doi.org/10.2478/cerce-2014-0016 http://dx.doi.org/10.1093/aob/mch039 http://dx.doi.org/10.17660/actahortic.2006.723.6 http://dx.doi.org/10.1016/j.indcrop.2018.12.046 http://dx.doi.org/10.31421/ijhs/6/3/124 http://dx.doi.org/10.1016/j.foodchem.2004.04.020 http://dx.doi.org/10.1016/j.jff.2016.11.007 http://dx.doi.org/10.1007/s10528-009-9281-z http://dx.doi.org/10.1016/j.bse.2009.06.002 http://dx.doi.org/10.1016/s0367-326x(02)00064-3 http://dx.doi.org/10.17660/actahortic.1993.344.46 http://dx.doi.org/10.1080/0972060x.2010.10643862 http://dx.doi.org/10.1016/j.foodchem.2009.01.051 http://dx.doi.org/10.1080/10412905.2003.9712257 http://dx.doi.org/10.1080/10412905.2006.9699085 http://dx.doi.org/10.1080/0972060x.2018.1533435 http://dx.doi.org/10.1016/j.indcrop.2019.02.038 196 s. mohammadi, l. tabrizi, m. shokrpour, j. hadian, h. schulz, d. riewe resources, university of tehran, karaj, 31587, iran javad hadian, medicinal plants and drugs research institute, shahid beheshti university, g. c., evin, 1983969411 tehran, iran hartwig schulz, julius kühn-institut, institute for ecological chemistry, plant analysis and stored product protection, königin-luise-strasse 19, 14195 berlin, germany david riewe, julius kühn-institut, institute for ecological chemistry, plant © the author(s) 2020. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). analysis and stored product protection, königin-luise-strasse 19, 14195 berlin, germany art19_buchbesprechung.indd journal of applied botany and food quality 84, 119 (2011) buchbesprechung flower and fruit morphology, ontogeny. phylogeny, function and ecology peter leins und claudia erbar bei dem buch handelt es sich um eine englische übersetzung der im jahre 2008 erschienenen 2. überarbeiteten aufl age von „blüte und frucht“. der inhaltliche aufbau gliedert sich in die drei abschnitte, die auf die morphologie der blütenpfl anzen, auf deren evolutionäre entwicklung und schließlich auf die früchte sowie die fruchtentwicklung bezug nehmen. zu beginn gehen die autoren auf den aufbau und die entwicklung der blüten näher ein und illustrieren diesen teil mit zahlreichen rasterelektronenmikroskopischen aufnahmen. in diesem zusammenhang werden die einzelnen baupläne, die evolutionäre entwicklung, die stammesgeschichte, die funktion sowie die ökologie der blütenpfl anzen und der sich daraus entwickelnden früchte sehr anschaulich auf hohem wissenschaftlichen niveau dargestellt (z.b. verschiedene blütenund fruchttypen, wachstum und abgrenzung einzelner organe, anpassung an die befruchtung und ausbreitung der samen durch wind, tiere und wasser). die anschaulichkeit wird dabei vor allem auch durch zahlreiche schwarz-weiß fotos, mikrofotos und zeichnungen unterstützt. der hauptanteil des buches ist dann vor allem der funktion und ökologie gewidmet. so wird in zwei umfassenden kapiteln sehr detailliert auf die pollenverteilung der blütenpfl anzen sowie die verbreitung der resultierenden früchte eingegangen. dabei werden die phylogenetischen, funktionellen und ökologischen fragestellungen vor allem auf grundlage der morphologie und entwicklungsgeschichte behandelt. so wird z.b. die entwicklung von spiraligen zu zyklischen blüten beschrieben, die evolutionären prozesse zur verhinderung bzw. einschränkung von inzucht näher beschrieben und schließlich auf aspekte der pollen und samenportionierung sowie unterschiedliche strategien der bestäubung und samenausbreitung bezug genommen. dieser teil liefert auch zahlreiche beispiele zur koevolution von blütenpfl anzen und bestäuber-insekten mit faszinierenden details. auch wenn die autoren einige aspekte wie z.b. die genetik der blütenpfl anzen in diesem zusammenhang nur anreißen, erhält der leser doch zumindest einen guten einstieg in die einzelnen themenstellungen und hat darüber hinaus die möglichkeit, sich anhand der umfangreichen literaturhinweise am ende jedes kapitels, noch detaillierter mit den einzelnen sachthemen auseinanderzusetzen. hierbei hilft auch das am ende des bandes aufgeführte literaturverzeichnis, das mehr als 12 seiten umfasst. den abschluss des buches bildet ein sorgfältig erstelltes sachregister. im buchanhang befi ndet sich außerdem noch eine ausführliche klassifi zierung der im textteil behandelten blütenpfl anzen, sodass auch taxonomisch weniger geschulte leser einen guten überblick hinsichtlich der blütensystematik erhalten. das vorgestellte system versucht darüber hinaus, die klassische morphologisch orientierte systematik mit den aus molekularen daten stammenden systemvorschlägen zu verknüpfen. der text wird durch zahlreiche zeichnungen und fotos (allerdings überwiegend in schwarz-weiß) begleitet, wodurch die vermittelten inhalte sehr gut veranschaulicht werden. die lektüre ist spannend zu lesen, didaktisch gut aufbereitet und liefert daher auch dem botanisch interessierten laien faszinierende einblicke in die abläufe der pfl anzlichen reproduktion. das buch wendet sich vorrangig an studenten sowie gymnasialund hochschullehrer. diesem leserkreis kann es als lehrbuch und nachschlagewerk wärmstens empfohlen werden. dr. h. schulz e-mail: hartwig.schulz@jki.bund.de bibliographie e. schweizerbart’sche verlagsbuchhandlung (nägele u. obermiller), stuttgart, 2010. 440 seiten, engl. übersetzung der zweiten aufl age von „blüte und frucht, 258 abbildungen, 3 tabellen, hardcover, preis 69 €, isbn 978-3-510-65261-7. journal of applied botany and food quality 94, 99 (2021) book review / buchbesprechung heilpflanzenlexikon – ein leitfaden auf wissenschaftlicher grundlage dietrich frohne die neuauflage des bereits zum klassiker avancierten heilpflanzen lexikons wurde vollständig durchgesehen, behutsam aktualisiert und mit einem modernen layout versehen. dabei wurde jedoch bewusst darauf verzichtet, das über jahrzehnte von den begründern der lexi kalischen sammlung zusammengetragene wissen zu den einzelnen arzneidrogen durch neue texte zu ersetzen. so sind die bereits in den früheren auflagen angeführten anekdoten zu einzelnen medizinaldrogen sowie ausgewählte historische inhalte unverändert auch wieder in der neuen auflage zu finden. der autor informiert wissenschaftlich fundiert über knapp 500 altbekannte und hochaktuelle arzneipflanzen. die in kompakter weise präsentierten monographien geben dabei u.a. auskunft über ihre jeweilige regionale herkunft, wirksame inhaltsstoffe, empfohlene dosierungen bei deren unterschiedlichen applikation sowie den in der literatur beschriebenen therapeutischen nutzeffekten; auch auf mögliche risiken bei der anwendung einiger pflanzlicher präparate wird kurz hingewiesen. bereits in der letzten auflage wurde eine reihe zusätzlicher pflanzenarten wie z.b. aronia melanocarpa, ballota nigra, isatis tinctoria, prunus africana, rhodiola rosea, macadamia integrifolia, leontopodium alpinum, limonia acidissima, lippia dulcis, papaver rhoeas, prilla frutescens und andere arten zusätzlich in den monographie-katalog aufgenommen. in entsprechender weise wurden die literaturhinweise ergänzt und auch der aktuelle kenntnisstand der bestehenden monographien entsprechend berücksichtigt. ergänzt wurden in dem einleitenden kapitel auch die hinweise auf alternative und komplementäre naturheilverfahren, auf die aromatherapie, ayurveda-medizin, hildegard-medizin sowie die traditionelle chinesische medizin. das nachschlagewerk richtet sich insbesondere an ärzte, apotheker und heilpraktiker, kann aber durchaus auch biologen, lebensmittelchemikern sowie studenten der genannten fachrichtungen empfohlen werden. auch für interessierte laien ist das buch als nachschlagewerk geeignet, da es aufgrund der zahlreichen informationen einen ersten einblick in die vielfalt der heute in der westlichen welt gebräuchlichen medizinalpflanzen liefert. dr. h. schulz e-mail: hs.consulting.map@t-online.de bibliography: dietrich frohne, heilpflanzenlexikon – ein leitfaden auf wissenschaftlicher basis, deutscher apotheker verlag, stuttgart, 9. auf lage, 2021, 686 seiten, preis: 54,€, isbn: 978-3-8047-3700-6 (printausgabe), isbn: 978-3-8047-4200-0 (e-book, pdf). art_15344_02.indd journal of applied botany and food quality 94, 53 60 (2021), doi:10.5073/jabfq.2021.094.007 1plant toxicology and molecular biology of microorganisms, faculty of sciences of bizerte, zarzouna, tunisia 2biology and environmental department. insitute of applied biology of medenine (isbam), university of gabes, tunisia 3dry land and oases cropping laboratory, arid land institute of medenine (ira), medenine, tunisia screening of the effects of zinc oxide based nanofertilizers on the germination of lathyrus sativa l. seeds hiba arfaoui1,§, inès karmous1,2,§,*, yethreb mahjoubi1, oussama kharbech1, samir tlahig2,3, mohammed loumerem3,abdelilah chaoui1 (submitted: july 5, 2020; accepted: november 27, 2020) * corresponding author § equal contributions of authors summary zinc based nanofertilizers may be useful tools in improving crop culture, especially in zinc deficient soil. the present study aims to investigate the role of nanosized zinc oxide particles (zno nps, diameter<100 nm) in modulating seed germination, embryo nutrition and growth of grass pea (lathyrus sativus l.). our data revealed ameliorating or inhibiting effects depending of the concentration of zno nps administrated. at metabolic level, the growing embryonic axes seem to cope with induced oxidative stress, by enhancing hydrogen peroxide scavenging capacity. we revealed interesting regulatory mechanisms evolved within the embryonic cells to limit the oxidative damages induced by zno nps and zinc sulfate when applied at low concentrations (0.01 mg ml-1, 0.1 mg ml-1). nonetheless, at high concentrations (1 mg ml-1, 10 mg ml-1), zno nps led to drastic perturbations in the metabolism, which resulted in the inhibition of root and seedling growth. our work may bring novel insight into the mechanistic understanding of the physiological role of the nanosized zno in enhancing the efficacy of fertilization. we also assess the critical role of applied concentration of nano fertilisers to avoid toxicity to plants. such phytotoxicity is not only affecting crops yield, but also may alter the biological properties and the nutritive quality of plant-derived food products, which may endanger or risk human health. keywords: germination, lathyrus sativus l., nutrition, response, zinc oxide nanoparticles. introduction zinc oxide nanoparticules (zno nps) are nanoscale (1-100 nm) materials, with novel applications in cosmetics, bioimaging, drug delivery, health care, bioremediation, environmental and bioprocess control (naveed et al., 2017; keskinbora and jameel, 2018). in agriculture, nanotechnology provides novel “tools” to improve plant nutrient, productivity and resistance against the environmental stresses (mahakham et al., 2017). zno nps were used as nanopesticides and nanofertilizers (kah, 2015). nanofertilizers have the capacity to synchronize the release of nutrients with their plant uptake, which can decrease nutrient loss and limit the risk of groundwater contamination. they can offer efficient delivery systems to plants via encapsulating nutrients, and their selective discharge to be directly inter nalized by the plant (prasad et al., 2010). this also limits nitrogen and nutrients loss due to leaching, emissions, and long-term assimilation by soil microorganisms (tarafdar et al., 2014). on the other hand, zinc (zn) is one of the essential micronutrients required for proper growth and yield of crops in plants. it is required for germination, and it is involved in maintaining the structural and functional integrity of cells and proteins (das et al., 2016). zn also serves as a cofactor for many structural and catalytic proteins (romeo et al., 2014). hence, zn deficiencies can lead to negative effects on all actors of the whole chain, notably humans. this results in the impetus on the importance to improve the uptake of zn by crops and subsequently humans. therefore, zno nps were shown to be efficient in improving plants growth (rizwan et al., 2018), as well as in promoting plants defense against environmental abiotic and biotic stresses (kah, 2015). nonetheless, the use of agrochemicals and fertilizers may cause the accumulation of zn ions in the agricultural soil (kah, 2015). besides, the mechanisms by which nps exert their effects in plants are not well clear, and heavy metals released from metaland metal oxide nps can be a major point of concern (owen and handy, 2007). in literature, differential effects of nps on the plant biological pathways were shown to be dependent on their physical and chemical properties (surface coating, type, size), their concentration, bioavailability, methods of exposure, as well as the plant species and life cycle stage (raliya et al., 2015; dimkpa et al., 2020). for instance, the phytotoxicity of zno nps to plants has been reported in many studies (lee et al., 2010; dimkpa et al., 2020). indeed, among the negative effects of metal oxide nps are the inhibition of crops yield and biomass (ebbs et al., 2016; bradfield et al., 2017). they also induce cellular and genetic toxicity, via the generation of ros. the phytotoxicity of zno nps has been shown on many plant species, such triticum aestivum l. (yahyaoui et al., 2017) and pisum sativum l. (mukherjee et al., 2014). when zno nps are dissolved in water, they release zn2+ ions, which are one of the principle sources of toxicity. this toxicity can be added to a supplementary toxicity due to nanosized particles themselves. excess of zn ions also resulted in toxicity, via the alteration of proteins function and activity, and the disruption of cellular metabolism, expression of genes involved in metal homeostasis and binding, thus leading to cell death (mustafa and komatsu, 2016). the current study aims to investigate the effects of zno nps on the germination of grass pea (lathyrus sativa l.) seeds, particularly the growing embryonic axes. lathyrus sativus l. is a legume species of fabaceae, cultured as human food and animal feed, for its important nutritional and economic properties, mostly in the rgions of india, asia, south america, mediterranean, south europe and north of africa (hanbury et al., 2000). grass pea seeds are charac terized by 48% of starch, 1% of lipids and 28% of proteins (xu et al., 2017), including low contents in cystein and methionin, and high content in lysine and threonine (yan et al., 2006). nonetheless, lathyrus contains high level of neurotoxinem known as β-n-oxalyll-α, β-diaminopropionic acid (β-odap), which is responsible of lathyrism (grela et al., 2001). jiao et al. (2006) also showed that zn deficiency caused the increase of β-odap. lathyrus sativus l. is, however, able to thrive in arid and semi-arid areas, in several tropical 54 h. arfaoui, i. karmous, y. mahjoubi, o. kharbech, s. tlahig, m. loumerem, a. chaoui and subtropical countries, therefore it is well adapted to unfavorable agricultural conditions, such as floods, drought, salinity, low soil fertility, infected soils and pathogens (hanbury et al., 2000). in the present study, the response of lathyrus sativa l. germinating seeds to increasing concentrations of zno nps, commonly used as nanofertilizers, was evaluated. the eventual mechanisms by which zno nps may interfere at the physiological and metabolic levels were discussed by the comparison between the assays of nanosized zno nps and soluble zn ions (treatment with zn sulfate, znso4). materials and methods treatments, germination and embryo growth seeds of lathyrus sativa l. were surface sterilized by sodium hypochlorite 20% for 2 min, followed by ethanol 70% for 2 min, and thouroughtly washed with distilled water. seeds were then germinated on filter paper in the presence of distilled water (control), or solutions of zinc oxide nanoparticles (zno nps) and zinc sulfate (znso4), at concentrations of 0.01 mg ml-1, 0.1 mg ml-1, 1 mg ml-1 and 10 mg ml-1. for each treatment, we carried 6 petri-dishes, with 10 seeds per petri-dish (six biological replicates/ treatment). germination was performed in culture room in dark at 28 °c. the solutions of zno nps were dispersed by vortexing and sonication, to avoid nps aggregation or precipitation. same volumes of 5-10 ml of distilled h2o, and solutions of zno nps and znso4 were added daily, in order to maintain seeds imbibition. the whole experiment was repeated as three technical replications. after 4 days, the length of roots and embryonic axes were measured. fresh biomass (fm) of embryos was measured. the embryonic axes were stored at -20 °c, to use for the biochemical assays. other samples of embryonic axes were dried at 60 °c in oven (thermoline scientific) to constant weights, then dry biomass (dm) was measured, and used for the determination of mineral content. all the chemicals, reagents (znso4, zno nps diameter <100 nm) were purchased from sigma-aldrich. measurement of levels of hydrogen peroxide (h2o2) the embryonic axes were homogenized in the presence of 0.1% (w/v) trichloroacetic acid (tca) (1:20, w/v), then the homogenate was centrifuged at 12 000 × g for 20 min at 4 °c. an aliquote of supernanant was added to 10 mm potassium phosphate buffer (ph 7.0) and 1 m potassium iodide (ki). levels of h2o2 were determined by measuring the absorbance at λ = 390 nm (sergiev et al., 1997). assays of antioxidant enzymes the embryonic axes were homogenized in the buffer containg 25 mm potassium phosphate (kh2po4/k2hpo4), ph 7.0 and 5 mm sodium ascorbate. homogenate was centrifuged at 20 000 × g for 30 min, using the sorvall x1r general purpose refrigerated centrifuge (analytical bionanotechnology equipment core (antec), according to manufacture instructions. the resulting supernatant was considered as the enzymatic extract. catalase (cat; ec 1.11.1.6) activity was measured by monitoring the decrease of absorbance at 240 nm (ε240 = 36 × 10-6 m-1. cm-1) (aebi, 1984). the reaction consisted in 25 mm kh2po4/k2hpo4 (ph 7) and 10 mm h2o2. ascorbate peroxidase (apx; ec 1.11.1.11) activity was measured in the reactional mixture containing 50 mm kh2po4/k2hpo4 (ph 7), 0.5 mm sodium ascorbate, 5 mm h2o2 and 0.1 mm edta. the decrease of absorbance at 290 nm was measured using the extinction coefficient ε290 = 2.8 × 10-3 m-1. cm-1 (nakano and asada, 1981). guaiacol peroxidase (gpox; ec 1.11.1.7) activity was measured by monitoring the increase of absorbance at at 470 nm (ε470 = 26.6 m-1. cm-1) (fielding and hall, 1987). the reactional mixture contains 10 mm h2o2 in 25 mm kh2po4/k2hpo4 (ph 7.0) and 9 mm guaiacol. glutathione reductase gr (ec 1.6.4.2) activity was measured using 50 mm kh2po4/k2hpo4 (ph 7.0) buffer, 0.2 mm nadph and 0.5 mm gssg. the decrease of absorbance was measured at 340 nm (ε340 = 6.22 × 103 m-1. cm-1, foyer and halliwel, 1976). activities were expressed in units of enzyme activity: μmol min-1 mg-1 fm. all measurements were carried out using jenway 6405uv/vis spectrophotometer. determination of mineral elements by atomic absorption spec trometry (aas) dry samples of embryonic axes (0.1-0.2 g) were digested in a mixture of 4 m nitric acid (hno3) and 1 m perchloric acid (hclo4), by heating process 500 °c. the resultant ashes were resolubilised in 0.7% hno3 to a final adjusted volume of 15 ml, and then filtered using cender free filter paper of 70 mm diameter. the obtained solutions were analyzed for the content of some mineral microelements (zn, cu, mn, fe) by aas, according to manufacture instructions (thermo scientific, type 138 ice3500aa system, nc942350023500, ser no c103500104). the operation conditions used to operate aas instrument were as recommended by the manufacturer. data were rounded off properly based on the value of standard deviation from measurement conducted in triplicate. final results are means of values obtained from three biological replicates. statistical analysis statistical analyses were carried out using spss 20.0 and xlstat version 9.0 software 2014. data were subjected to analysis of variance (two ways) anova at α=0.05%, and duncan post hoc multi-range test at 5%, to compare the effect of the treatments (zno nps and znso4), the effect of concentrations, and the interaction of effects (treatment×concentration). results effects of zno nps on the physiology of seed germination and embryo growth in the present study, the effects of zno nps on seeds of lathyrus sativa l. were investigated. our results showed variations of seed germination rate with the increasing concentrations of zno nps (fig. 1a) and znso4 (fig. 1b). the variations between days were estimated significant (p<0.05) for each treatment separately (fig. 1). in comparison with control, concentrations of 0.01 mg ml-1, 0.1 mg ml-1 and 1 mg ml-1 enhanced germination at day 2, but concentration of 10 mg ml-1 decreased germination capacity. however, these differences between applied concentrations (0.01 mg ml-1, 0.1 mg ml-1, 1 mg ml-1 and 10 mg ml-1) were estimated not significant. also, differences between treatments (zno nps and znso4) were found not significant. similarly, at days 3 and 4, the delays in germination of 1 mg ml-1 and 10 mg ml-1 treated seeds were found not significant in comparison with controls. however, fig. 2 showed that the length of embryonic axis (fig. 2a) and the length of root (fig. 2b) were enhanced significantly (p<0.001) at low concentrations (0.01 mg ml-1, 0.1 mg ml-1), wheareas they were inhibited at higher ones (1 mg ml-1, 10 mg ml-1). the effect of treatments (zno nps and znso4) was found not significant, but the effect of concentration was highly significant (p<0.001) (fig. 2). furthermore, the negative effect of znso4 was found more pronounced than zno nps, particularly when applied at high doses. besides, the concentrations of 0.01 mg ml-1 and 0.1 mg ml-1 of both treatments resulted in embryonic fm increase. at higher concentra zno nps use as nanofertilizers 55 fig. 1: germination rate of lathyrus sativa l. seeds during time (days) in the presence of distilled water (control) or different concentrations of zno nps and znso4; 0.01 mg ml-1, 0.1 mg ml-1, 1 mg ml-1 and 10 mg ml-1. values are means (±sd) which are average of two independant germinations. differences between treatments and within concentrations estimated according to duncan test (α=0.05) were non significant (p<0.05). fig. 2: length of the embryonic axes (a) and the roots (b) of lathyrus sativa l. seeds germinated for 4 days in the presence of distilled water (control) or solutions of zno nps and znso4 at concentrations 0.01 mg ml-1, 0.1 mg ml-1, 1 mg ml-1 and 10 mg ml-1. values are means (±sd) of 5 independent measurements. letters a,b and a-c denote statistic classes, respectively, for zno np and zn sulfate, using duncan test (α=0.05). differences are significative, at *: 0.01≤p<0.05, **: 0.001≤p<0.01, ***: p<0.001, and non significant ns at p≥0.05. tions of 1 mg ml-1 and 10 mg ml-1, a highly significant (p<0.001) reduction of fm (fig. 3a) was recorded. the effects of zno nps on the dm of embryonic axes were not clearly noticeable, except a decrease at high concentrations (1 and 10 mg ml-1). variations of the cotyledonary fm were, however, detected in zno nps treated seeds (fig. 4a). these variations were estimated significant (p<0.01) with treatments and highly significant (p<0.001) with concentrations. no significant changes of the dm of cotyledons were registered with zno nps or znso4 (fig. 4b), which could be attributed to the short duration of assay; 4 days of germination might not be enough to detect or monitor the cotyledonary biomass changes, in the contrary of the embryonic axes. effects of zno nps on the antioxidant status and mineral nutrition of the growing embryos the effects of 0.1 and 10 mg ml-1 of zno nps and znso4 were assayed on the antioxidative response of the growing embryonic axes. fig. 5 revealed that the concentration 0.1 mg ml-1 of both treatments did not affect significantly the levels of h2o2 (hereby playing the role of assignaling molecules) in comparison with control. in the contrary, the higher concentration (10 mg ml-1) caused a highly significant (p<0.001) increase in the level of h2o2 (herein becoming toxic molecules). besides, fig. 6 showed several changes within the activities of antioxidant enzymes; a reduction of cat activity was estimated significant at p<0.01 as the effect of treatments zno nps and znso4, and highly significant (p<0.001) as the effect of concentration (fig. 6a). combined effect of treatment and concentration was found significant at p<0.01. apx and gpox activities (fig. 6 b, c) showed highly significant variations (p<0.001) with the concentrations applied. apx activity (fig. 6b) decreased in the presence of both treatments, particularly znso4. gpox activity was however stimulated in the presence of 0.1 mg ml-1 zno nps, and in the pre sence of znso4 (0.1 mg ml-1 and 10 mg ml-1) (fig. 6c). these variations were estimated highly significant (p<0.001) with treatments, concentrations and their combined effect. gr activity increased significantly with 10 mg ml-1 zno nps and 0.1 mg ml-1 zn so4 (fig. 6d). moreover, the evaluation of the levels of total soluble proteins revealed an increase in the presence of 0.1 mg ml-1 of zno nps, but remained not significant, while at higher concentration of 1 mg ml-1 zno nps a significant decrease was recorded (fig. 7). 56 h. arfaoui, i. karmous, y. mahjoubi, o. kharbech, s. tlahig, m. loumerem, a. chaoui fig. 3: fresh matter (a) and dry matter (b) of the embryonic axes of lathyrus sativa l. seeds germinated for 4 days in the presence of distilled water (control) or solutions of zno nps and znso4 at concentrations 0.01 mg ml-1, 0.1 mg ml-1, 1 mg ml-1 and 10 mg ml-1. values are means (±sd) of 5 independent measurements. letters a-c and a-c denote statistic classes, respectively, for znonp and zn sulfate, using duncan test (α=0.05). differences are significative, at *: 0.01≤p<0.05, **: 0.001≤p<0.01, ***: p<0.001, and non significant ns at p≥0.05. fig. 5: level of proxide hydrogen in the embryonic axes of lathyrus sativa l. seeds germinated for 4 days in the presence of distilled water (control) or solutions of zno nps and znso4 of 0.1 mg ml-1 and 10 mg ml-1. values are means (±sd) of 5 independent measurements. letters a-b and a-b denote statistic classes, respectively, for zno nps and zn sulfate, using duncan test (α=0.05). differences are significative, at *: 0.01≤p<0.05, **: 0.001≤p<0.01, ***: p<0.001, and non significant ns at p≥0.05. fig. 4 fresh matter (a) and dry matter (b) of the cotyledons of lathyrus sativa l. seeds germinated for 4 days in the presence of distilled water (control) or solutions of zno nps and znso4 at concentrations 0.01 mg ml-1, 0.1 mg ml-1, 1 mg ml-1 and 10 mg ml-1. values are means (±sd) of 5 independent measurements. letters a-c and a-c denote statistic classes, respectively, for zno nps and zn sulfate, using duncan test (α=0.05). differences are significative, at *: 0.01≤p<0.05, **: 0.001≤p<0.01, ***: p<0.001, and non significant ns at p≥0.05. in zn so4 treated embryonic axes, differently to zno nps, no significant variations were found with both concentrations of zn so4 (fig. 7). additionally, the determination of the content of zn and other micronutrients (mn, fe and cu) by aas in the embryonic axes showed a significant increase in the accumulation of zn ions particularly with the highest concentration of 10 mg ml-1 of zno nps and znso4. besides, a significant decrease in the content of fe was found after exposure to zno nps (tab. 1). similar decrease was also registered with the highest dose of znso4. variation of mn content was estimated not significant, except an increase with 0.1 mg ml-1 znso4. similarly, cu content decreased with 0.1 mg ml-1 and 10 mg ml-1 in both treatments, especially with 10 mg ml-1 znso4. discussion the use of nanosized zno in fertilization represents a novel technique to improve the nutrition and growth of plants especially when cultured in the presence of critical soil and water factors. nevertheless, the risk of a potential hazard of zno nps on plant system is not none. in order to shed more light on this point, the effects of zno nps on seeds of lathyrus sativa l. were investigated. in order to ascertain wheither the effects of zno nps can be attributed to zn ions, seed zno nps use as nanofertilizers 57 anova results (p<0,05) cat apx gpox gr effet traitement (t) ** ns *** ns effet concentration (c) *** *** *** *** effet combiné t×c ** *** *** *** fig. 6: activities of the antioxidant enzymes cat (a), apx (b), gpox (c) and gr (d) in the embryonic axes of lathyrus sativa l. seeds germinated for 4 days in the presence of distilled water (control) or solutions of zno nps and znso4 at concentrations of 0.1 mg ml-1 and 10 mg ml-1. values are means (±sd) of 5 independent measurements. letters a-c and a-c denote statistic classes, respectively, for zno np and zn sulfate, using duncan test (α=0.05). differences are significative, at *: 0.01≤p<0.05, **: 0.001≤p<0.01, ***: p<0.001, and non significant ns at p≥0.05. fig. 7: levels of total soluble proteins in the embryonic axes of lathyrus sativa l. seeds germinated for 4 days in the presence of distilled water (control) or solutions of zno nps and znso4 at concentrations of 0.1 mg ml-1 and 10 mg ml-1. values are means (±sd) of 5 independent measurements. letters a-c and a-c denote statistic classes, respectively, for zno np and zn sulfate, using duncan test (α=0.05). differences are significative, at *: 0.01≤p<0.05, **: 0.001≤p<0.01, ***: p<0.001, and non significant ns at p≥0.05. germinations in the presence of zno nps and zn sulfate were compared to scheme their mechanism of response. besides, we aim to determine the concentrations of zn nanofertilizers that are possibly improving growth or in contrary causing toxicity, which may bring insight at the choice of concentration to apply. our results revealed the significant interference of zno nps and znso4 on the germinative process and the early growth of embryo, dependent on the applied concentration; indeed, low concentrations (0.01 mg ml-1, 0.1 mg ml-1) seem to promote seeds germination, as well as the elongation and biomass of the embryonic axis. higher doses had, however, toxic and inhibitory effects (1 mg ml-1, 10 mg ml-1). overall data showed that zno nps affected negatively fm and dm of the embryonic axes, while zn sulfate affected fm more than dm, which could probably be attributed to the process of water absorption following seed imbibition. we may hypothesize that the low doses of zno nps and zn sulfate induced the intense absorption of water, without significant modification of dry biomass, which can explain the increase of fm versus no change of dm, as compared with respective controls. hence, this increasing absorption of water as compared with control could be an adaptative response in seeds to improve tolerance and defense mechanism against the metallic stress and the osmotic stress imposed by zno nps or znso4. this leads to the hypothesis that seeds try to cope with stressfull condition, via the enhancement of a vital phenomenon associated with waper uptake, which may explain in part the finding that no significant variation was recorded for the length of embryonic axes and roots. this 58 h. arfaoui, i. karmous, y. mahjoubi, o. kharbech, s. tlahig, m. loumerem, a. chaoui adaptative response seems to be possibly disrupted or altered with the highest concentration (10 mg ml-1) that triggers a significant decrease of the embryonic growth (figs. 2, 3). besides, the decrease of fm and dm at 10 mg ml-1 suggests that zno nps may act via the inhibition of water absorption (similar effect with zn sulfate), which brings evidence of the involvement of zn2+ ions possibly released from zno nps and zn sulfate. additionnally, embryo dm seems to be negatively affected more by zno nps than znso4, which suggests that the reduction of embryo biomass could be the proper effect of nps themselves. consequently, nps might interfere with the mobilization of biomass and allocation of nutrients in cotyledons. in this study, overall data discussed above may suggest that lathyrus sativa l. evidenced tolerance capacity towards zno nps up to 10 mg ml-1. therefore, we may ascertain that lower concentrations of zno nps are probably required as fertilizers. our findings agree with other reports that the effects of zn ions and zno nps depend upon their concentration applied, and the biological properties of plant species, such as the permeability of seed coat to nps and their internalization in root tissues (dimkpa et al., 2020). indeed, upadhyaya et al. (2017) demonstrated that rice exposure to lower concentrations of zn nps (5, 10, 15, 20 and 50 mg l-1) showed better potential of seed germination, as well as radicle and plumule growth. de la rosa et al. (2013) also reported that zno nps improved germination capacity of cucumber seeds. in addition to zno nps use as fertilizers in improving plant growth, yield and zn biofortification, they can also be promising tool to alleviate environmental stresses, such as sali nity and drought ((dimkpa et al., 2020; sun et al., 2020). therefore, in our work, the exposure to low concentration of zno nps and znso4 might play a cellular modulating role. in the contrary, a higher concentration (10 mg ml-1) caused severe physiological and metabolic disturbances. in another study, zno nps at the levels of 400 and 1600 mg l-1 increased the germination of cucumber seeds, but became toxic at higher concentrations (de la rosa et al., 2013). similar toxicity studies reported that exposure to metal and metal oxide nps, including zno nps, inhibited seed germination, root elongation and plantlet growth (lee et al., 2010). in arabidopsis thalinana, the inhibition of seed germination by 400 mg l-1 zno nps was shown by lee et al. (2010). first hypothesis behind the dual effect of zno nps consists into the controversal effects of zn ions depending on the concentration. for instance, appropriate amount of zn as essential micronutrients is needed in several physiological processes in plants (samreen et al., 2017). zn ions were also shown to modulate the abundance of proteins related to the antioxidant system, carbohydrate/energy, and amino acid metabolism (romeo et al., 2014). in our study, indeed, the obtained increase in embryo fm with increasing concentrations of zno nps can be attributed, in part, to the role displayed by zn in growth modulation, as well as the mitigation of dehydration induced damages, and the improvement of post stress rehydration responses in plant (upadhyaya et al., 2017). taking into account the similar responses of the embryonic axes towards both treatments zno nps and znso4, the mechanism of action of nanosized zno nps seems to exhibit via released zn2+ ions, thereby leading to either positive effects or phytotoxicity, depending on zn amount (lee et al., 2010). zn excess indeed was shown to cause a drastic delay in the growth of seedling, root and shoot in many plant species (de la rosa et al., 2013). alternative possible mechanism of action of zno nps consists into the interference of nps themselves. our data suggests that the effects of zno nps can also be attributed to the nanosized particles. this hypothesis was further investigated in order to provide a significant clue about the magnitude of oxidative stress under the exposure to 0.1 mg ml-1 (lower concentration) and 10 mg ml-1 (higher concentration) of zno nps. the analysis of the antioxidant enzymatic activi ties revealed that the inhibition of cat activity by 0.1 mg ml-1 zno nps or znso4 suggests the possible role of zn2+ to restrain the oxidative balance at lower concentration. this effect was displayed more significantly with the metallic soluble form of zn, in comparison with nanosized zn. in contrary, at higher concentration (10 mg ml-1), cat activity was enhanced, probably resulting from the activation of the defense response of the stressed embryonic axes in attempt to detoxify the cells and protect the cellular components and molecules. the negative effects of 10 mg ml-1 of both treatments on the embryo growth may be associated in major part with the increased induction of oxidative stress. in addition, the changes (stimulation/inhibition) of the antioxidant activities of apx, gpox and gr in the presence of zno nps in comparison with those occurring in the presence of znso4 revealed, in some part, either the positive/negative interference of zn+2 ions within the enzymatic protein, or else the interference of both zn ions and nanosized particles. in literature, the toxic effects of nanosized particles were evidenced to be due to their size, surface area ratio, morphology, nature, composition, reactivity, and others (zaka et al., 2016; dimkpa et al., 2020). at cellular and molecular levels, metal/metal oxide nps can easily enter the cells, and interact with the metabolic processes (dimkpa et al., 2020). they can induce cellular generation of oxidative stress (koce et al., 2014) and interfere with the functional groups of biological macromolecules, leading to dna denaturation, lipid peroxidation, enzymes deactivation, and protein alteration (koce et al., 2014). in addition, it has been hypothesized that nps and released ions impede cell metabolism by altering redox status and antioxidative responses (gene expression, enzyme activity) (koce et al., 2014). other studies reported the upregulation in the expression of different genes, mainly those involved in defense signaling pathways (zafar et al., 2016). higher concentrations of nps can also promote tissues damage, cytotoxicity, genotoxicity, as well as the inhibition of cell division and ultimately cell death (zafar et al., 2016). furthermore, we inquired into the possible influence of bioavailability, thus the impact of solubility, dissolution and adsorption cha racteristics of zno nps. interestingly, saa data revealed the higher solubility of znso4 compared to zno nps. hence, the accessibility of zn ions released from znso4 was higher than zno nps (tab. 1). this finding suggests that zno nps toxicity might be considered due to the internalization of nps, which may operate by means of their tab. 1: content (mg l-1) of mineral elements (mn, zn, cu, fe) in the embryonic axes of 4 day-germinated seeds, measured by atomic absorption spectrometry (aas). letters a-c and a-c denote statistic classes, respectively, for zno nps and zn sulfate, using duncan test (α = 0.05). treatment concentration mn zn cu fe control 1.263±0.079 aa 2.195±0.124 bb 1.3065±0.310 aa 3.536±0.025 aa znonp 0.1 mg ml-1 1.176±0.005 a 1.4735±0.044 c 1.1365±0.170a 1.1335±0.161b 10 mg ml-1 1.275±0.098 a 11.355±0.134 a 1.0555±0.054 a 1.731±0.463 b zn sulfate 0.1 mg ml-1 1.397±0.031ab 1.642±0.043 c 0.9775±0.050 a 2.3355±0.159 b 10 mg ml-1 1.5065±0.021a 12.83±0.056 a 0.827±0.005 a 1.4915±0.086 c anova: all differences are significant at p<0.05. zno nps use as nanofertilizers 59 physical and chemical properties. other studies pointed out to a more hamful risk caused by np on plant species than their bulk counterparts or their soluble metallic ions. additionally, the determination of other micronutrients (mn, zn, fe and cu) in the embryonic axes suggests that bioavailability of zno nps is associated with many factors related to the nps themselves or to zn ions. besides, the intracellular zn homeostasis could be affected through zn influx, efflux, translocation, accumulation and intracellular compartmentalization. moreover, in this study, we evidenced antagonistic relationships between zn and some nutrient elements of two-capacity cations, mainly fe. indeed, fe showed competitive behavior with zn in the presence of higher concentrations of zno nps and znso4, however mn and cu contents were not significantly changed with zn increase. this may lead to changes within the accessibility of the essential microelements to the embryonic cells, which can result in changes of the metabolic and physio logical balance by local competition in different places. in other studies, it was indeed reported that zn has a high chemical similari ty to fe, therefore it can substitute for this metal ion in the active sites of enzymes, and consequently interfere with cellular functions (zargar et al., 2015). for example, fe is involved in the activation of many metabolic, physiological and biochemical pathways in plants, and it serves as a prosthetic group constituent of many enzymes (rout and sahoo, 2015). besides, zno nps were able to interrupt the apoplastic and symplastic pathways (yahyaoui et al., 2017), thus inhibiting the process of absorption of water and microand oligo elements such as fe and mn (dimkpa et al., 2014). conclusion our study may bring novel insight at the potential application of zinc based nanofertilizers to efficiently correct zinc deficiency and to enhance plant growth. in addition, we could reveal more understanding of the physiological mechanisms of zno nps to improve early growth of lathyrus seedlings. we suggest either the dissolution of zinc ions from zno nps and their interference with the metabolic pathways in plant system. however, for lathyrus, it appears that zno nps were benefecial when applied at low levels, but became toxic at higher concentrations. hence, in attempts to avoid nps toxicity, further analyses are needed to elucidate their response depending factors, such as applied concentration, plant species, stage of deve lopment and culture condition. conflicts of interest no potential conflict of interest was reported by the authors. acknowledgments this work was supported by the tunisian ministry of high education and research. we also acknowledge the help of mr abbes wacharine from fsb and dr lamjed bouslama from cbbc borj cedria for 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https://orcid.org/0000-0002-8857-4839 samir tlahig https://orcid.org/0000-0002-2742-2428 mohammed loumerem https://orcid.org/0000-0003-1003-6915 abdelilah chaoui https://orcid.org/0000-0001-8168-6582 address of the corresponding author: inès karmous, plant toxicology and molecular biology of microorganisms, faculty of sciences of bizerte, 7021 zarzouna, tunesia biology and environmental department, higher insitute of applied biology of medenine (isbam), university of gabes, 4119 medenine, tunisia e-mail: ineskarmouss@gmail.com © the author(s) 2021. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1039/c5mt00168d http://dx.doi.org/10.1016/j.chemosphere.2018.09.120 http://dx.doi.org/10.1016/j.jplph.2014.03.016 http://dx.doi.org/10.7831/ras.3.1 http://dx.doi.org/10.1016/j.arabjc.2013.07.005 http://dx.doi.org/10.1080/03650340.2020.1723003 http://dx.doi.org/10.1007/s40003-014-0113-y http://dx.doi.org/10.29328/journal.jpsp.1001008 http://dx.doi.org/10.3389/fpls.2016.00535 http://dx.doi.org/10.1049/iet-nbt.2015.0039 http://dx.doi.org/10.1002/pmic.201400467 art02_hirsch.indd journal of applied botany and food quality 85, 6 -16 (2012) institute of food science and biotechnology, chair of plant foodstuff technology, hohenheim university, stuttgart, germany validation and applicability of a standardized procedure for evaluating freshness of citrus juices based on pectin methylesterase activity quantitation angelika r. hirsch, reinhold carle, sybille neidhart (received january 30, 2012) summary a method developed for freshness authentication of freshly squeezed citrus juices (fscj) was evaluated for routine application. it involved titrimetric assessment of pectin methylesterase (pe) activity after enzyme extraction from pulp-standardized juice samples. standard test conditions enabled reliable discrimination between fscj and chilled citrus juices that had comparative advantages due to extended shelf life. unlike the latter, fscj always displayed pe activities in the linear range between the limit of identifi cation (loi, 0.42 units g-1 of juice) and the maximum activity found for fscj (1.94 units g-1), equivalent to 0.00350.016 units during titration. however, for model samples having activities < loi due to production by respective dilution of fscj, the responses abruptly fell to unspecifi c levels below the limit of detection (lod, 0.21 units g-1). accuracy was substantiated by 100 -106 % recovery for model juices with pe activities of 0.87 -1.22 units g-1 resulting from fscj dilution or pe standard addition, but it was lower (76 80 %) near loi. the average of the mean activities, which were detected by 3 analysts with intraassay precision ≤ 8.4 %, varied with relative standard deviations of 8.2 % for fscj and 3.9 % for a sample of the same juice diluted to 60 % (w/w), thus proving reproducibility. fscj batches were unambiguously distinguished from four commercial chilled juices, because the activities detected for the latter were by far ≤ lod and thus confi rmed labeled mild preservation. 1. introduction orange fruit accounts for the most popular fl avor of fruit juices and nectars in europe (~35 %) with a portion of even 55-62 % in some eu countries (aijn, 2010). the total of ~7.5 billion l of fruit juice, which was consumed in europe in year 2009 regardless of the fl avor, mostly comprised ambient products, either from concentrate (fc) or not from concentrate (nfc), with a shelf life of up to 18 months. chilled commodities though added up to notable 18 % (aijn, 2010). the latter are uniformly perceived by consumers to be premium (crupi and rispoldi, 2002), but their role in the juice market greatly varied among countries (e.g., 610 % of fruit juice in germany and france, 55 69 % in the uk and sweden; aijn, 2010). their distribution involves a cold chain, which is either needed for the retention of food quality and safety or chiefl y associated with premium market positioning. since freshly squeezed orange juices (fsoj), i.e. freshly squeezed citrus juices (fscj) in general, are offered without previous pasteurization, their shelf life is limited to a few days despite chilled supply chain (crupi and rispoldi, 2002), as expressed by a labeled best-before date within two weeks after juice extraction (fsa, 2008). however, the diverse group of chilled citrus juices also includes nfc with a shelf life of a few weeks owing to mild pasteurization (collet et al., 2005; sentandreu et al., 2005). while they most resemble fscj with respect to fresh taste (ruiz perez-cacho and rouseff, 2008), microbial load and pectin methylesterase (pe; ec 3.1.1.11) activity causing decay (kopelman and rauchwerger, 1984) and rapid cloud loss (hirsch et al., 2008), respectively, have been reduced by minimal preservation (crupi and rispoldi, 2002). the same shelf life-extending effects may be attained by various non-thermal preservation techniques (crupi and rispoldi, 2002; guiavarc’h et al., 2005), as recently discussed by hirsch et al. (2011) as to pe deactivation. by contrast, due to complete pe deactivation during their production, chilled fc constituting the third subgroup would only require cold supply, if the pasteurization of the juice that was reconstituted from concentrate was merely mild (crupi and rispoldi, 2002) or if fc was blended with fscj or gently pasteurized nfc. fc even prevailed among the chilled juices in individual eu countries (aijn, 2010). hence, freshness is an outstanding feature of the fi rst subgroup, demanding strictest sanitation standards during processing and distribution (crupi and rispoldi, 2002; hirsch et al., 2008). analytical distinction of fscj from other chilled citrus products via authentication of juice freshness by food inspection boards is thus crucial when labeled specifi cation of product types comes under scrutiny. in view of the great impact of processing on citrus juice aroma, analysis of aroma compounds has been used for discrimination between different juice types of this highly diverse product group (tønder et al., 1998). by classifying juices via multivariate analysis of aroma profi les, fsoj were found among various kinds of ambient orange juices (fc and nfc) (shaw et al., 1993). being basically characteristic of fscj, activities of endogenous enzymes, e.g., acid phosphatase (gel moretó, 2000), have generally been regarded as the criterion of choice to distinguish this juice type from ambient products, provided that the test enzyme is stable throughout juice distribution. enzyme stability of peroxidase during cold juice storage was unacceptable for a freshness indicator of fscj, unlike that of pe (hirsch et al., 2008). the latter is also the technologically most relevant enzyme for the design of citrus juice preservation (duvetter et al., 2009). suitability of pe as a freshness indicator (hirsch et al., 2008) was substantiated by its universal applicability across citrus cultivars and species, as deduced from the activity range and the overall uniform thermal resistance observed for pe of numerous fscj of known production history (hirsch et al., 2011). however, discrimination between different types of chilled juices, especially fscj and chilled nfc, has appeared by far less trivial, because partial pe deactivation by mild preservation may lead to activities in a broad range (hirsch et al., 2011). the analytical method suggested for freshness evaluation (hirsch et al., 2008, 2011) was though deemed suitable for the distinction of fscj from those chilled nfc that have comparative advantages in terms of signifi cantly enhanced shelf life, when mild pasteurization almost completely inactivated their thermo-labile pe fraction. freshness authentication implied a standardized procedure aiming at routine assessment of pe activity in unknown samples. unlike other citrus juice types, specifi cally fscj may contain notable amounts of entire juice sac particles, following the organoleptic characteristics of the fresh fruit (crupi and rispoldi, 2002). consequently, the analytical method involved initial juice standardization by manual fi nishing via fi ltration to exclude interference by varying contents verifi cation of citrus juice freshness 7 of pulp with adherent pe (hirsch et al., 2008). solubilization of tightly bound thermo-stable pe fractions was reported to be always incomplete (wicker et al., 1988). enzyme extraction was thus adjusted to the release of the cell wall-bound enzyme under standard conditions (hirsch et al., 2011) that were chiefl y selective for the heat-sensitive pe isoenzymes (wicker et al., 1988), because the latter were considered crucial for discrimination between fscj and chilled nfc (hirsch et al., 2011). for the analysis of the crude extract based on recorded titration of liberated carboxyl groups (schols and voragen, 2003), assay temperature and ph were chosen with respect to reasonable rates of enzymatic pectin deesterifi cation (wicker et al., 1987), but minimal concurrent alkaline de-esterifi cation and β-eliminative degradation of the substrate (hirsch et al., 2008). unlike the well-established titrimetric pe assay for monitoring pasteurization via relative deactivation percentages (collet et al., 2005), routine evaluation of citrus juice freshness relies on precise and accurate pe activities that are to be quantitated in extracts of standardized juices. this study aimed at validation of the complete analytical procedure of hirsch et al. (2008, 2011) for freshness evaluation of citrus juices as regards precision, accuracy, linearity, and sensitivity prior to its application to the analysis of commercial chilled products. in this way, the possibilities to distinguish between various types of chilled citrus juices were to be further substantiated. concurrently, the limits for freshness authentication were to be identifi ed in order to refi ne the procedure for routine application. 2. materials and methods 2.1 chemicals and reagents a commercial preparation of pure pe from aspergillus niger (rapidase® fp 15000) was kindly provided by dsm food specialties (seclin, france). apple pectin with a degree of esterifi cation (de) of 77 % (pektin classic au-l) was supplied by herbstreith & fox (neuenbürg, germany). polyvinylpolypyrrolidone (pvpp) and saponin were obtained from fluka (buchs, switzerland). all other chemicals were of analytical grade and supplied by vwr international (darmstadt, germany), including titrisol® ampoules for volumetric analyses. ultra-pure water (milli-q system, millipore, bedford, usa) was used for analytical purposes. to dissolve the pectin for the preparation of the substrate solution for the enzyme assay (cf. 2.4.3), the pectin (0.5 % w/v) and nacl (0.15 m) were slowly strewed into water (55 ± 3 °c) under stirring in a beaker until complete dispersion of the polysaccharide prior to adjustment of the volume in a volumetric fl ask (20 °c). the fi nal substrate solution was used after a swelling time of ~16 h in the refrigerator. 2.2 plant material and juice samples fresh orange fruit (citrus sinensis (l.) osbeck cvs. ‘navelina’ and ‘salustiana’) of class i from spain (harvests 2004 and 2005, respectively) was obtained from retailers in stuttgart, germany, as raw material for the production of freshly squeezed orange juices on the laboratory scale (cf. 2.3) directly after purchase. the size codes were 6 -7 for ‘navelina’ fruit and 6 for ‘salustiana’. commercial citrus juices, all of which were distributed in a cold chain, were purchased from various retailers in france and germany in year 2004 as sample material for an application study (cf. 3.4) in order to apply the analytical method for freshness evaluation (cf. 2.4) to chilled citrus juices of unknown production history. sample collection was at random, based on the availability of respective chilled products in retail markets. as specifi ed on the packages by the producers, three juices had been produced from oranges and one from mandarin fruit and they had been subjected either to (gentle) pasteurization or, in the case of one orange juice, to high-pressure treatment. storage in the refrigerator or at maximally 8 °c was indicated by the producers in each case. after purchase (approx. 2 4 weeks prior to the specifi ed best-before date), the samples were immediately deep-frozen until analysis of the defrosted product according to 2.4. 2.3 production of freshly squeezed orange juices and model juices for the manufacture of freshly squeezed orange juices (fsoj), fruit (cf. 2.2) was de-juiced in the semi-automatic orange x-press extractor (brimato maschinenbau, hilter, germany), which had previously been used for citrus juice production on the pilot plant scale (hirsch et al., 2008, 2011). on the small production scale of this study, ‘navelina’ fsoj was obtained by extracting 4.5 kg of fruit of the respective cultivar, ‘salustiana’ fsoj by the use of 21 kg. since the fi nisher used in the past (hirsch et al., 2008, 2011) was inapplicable on this scale, the juices were fi nished by passing them through a stainless steel strainer of approx. 1 mm mesh size for removal of coarse pulp particles and manual disruption of remaining juice sacs. besides an aliquot used for the analysis of fsoj according to 2.4, further aliquots of the ‘navelina’ fsoj were immediately processed after fi nishing into a series of freshly squeezed model orange juices (fsmoj) by dilution for the analysis of products, which clearly differed in their pe activities owing to different contents of original fsoj. after fi nishing, ‘salustiana’ fsoj was completely fi lled into 1 l plastic bottles and deep-frozen at -20 °c until analysis of the fsoj or preparation of fsmoj from defrosted juice for different types of validation experiments (cf. 2.5). for the production of these model juices, an aliquot of the respective fsoj was subjected to the same standardization procedure based on juice fi ltration (cf. 2.4.1), which was otherwise applied as sample preparation to those fsoj aliquots that were directly used for freshness evaluation (cf. 2.4). proportional differences in pulp contents and pe activity could thus be assumed among the individual samples of each dilution series. for each fsmoj, the exactly weighed amount of pulp-standardized fsoj was made up to a total mass of 100 g of model juice with an aqueous solution, which consisted of glucose (28 g/l), fructose (30 g/l), sucrose (33 g/l), citric acid (9.4 g/l), and malic acid (1.7 g/l) and had been adjusted to the ph value of the fsoj with 5 n potassium hydroxide (hirsch et al., 2008). fsmoj from ‘navelina’ fsoj comprised a series of nine model juices with fsoj contents of 0.1-75 % (w/w). the ‘salustiana’ fsmoj series covered model juices containing the respective original fsoj at ten levels from 1 to 75 % (w/w). the model juices were directly subjected to the enzyme extraction step (cf. 2.4.2) of the freshness evaluation method. 2.4 analytical method for evaluating citrus juice freshness 2.4.1 sample preparation for standardization of the juice sample to uniform pulp content prior to enzyme extraction, it is shaken up and an aliquot of ~110 g is strained through a screen din 4188 (retsch, haan, germany) of 0.5 mm mesh size and 10 mm diameter (hirsch et al., 2008, 2011). in this study, this procedure was accordingly applied to the fsoj (cf. 2.3) and the commercial products (cf. 2.2), respectively. for fsmoj, this step was omitted, because it had already been applied to the respective fsoj prior to its conversion into fsmoj by dilution (cf. 2.3). 8 a.r. hirsch, r. carle, s. neidhart 2.4.2 enzyme extraction the enzyme extract is prepared following the standard procedure described by hirsch et al. (2008, 2011). accordingly, the exactly weighed mass of ~100 g of pulp-standardized juice sample (cf. 2.4.1) or fsmoj (cf. 2.3) was added to 3 g of pvpp, 877 mg of nacl, and 100 mg of saponin. after immediate adjustment to ph 6.0 with 10 n and 1 n naoh, the mixture was stirred at 4 °c for 120 min and subsequently centrifuged at 25,000 × g for 30 min at 4 °c in a suprafuge 22 with a rotor of the type 14290 (heraeus sepatech, osterode, germany). the centrifugate was decanted through a folded fi lter (schleicher & schuell no. 597 1⁄2, dassel, germany) at 4 °c into a volumetric fl ask (200 ml). the residue was washed twice with ultra-pure water (4 °c) and was subsequently discarded. the combined volume of centrifugate and washing water was made up to 200 ml with ultra-pure water (4 °c). this extract was divided into portions of 20 ml, fi lled into plastic vials, and stored at -80 °c until analysis of pe activity (cf. 2.4.3). as concluded from a preliminary comparison of this one-time extraction method according to hirsch et al. (2008, 2011) with a procedure involving repeated extraction, the former was clearly acceptable for the analytical method of freshness evaluation. in the other procedure, extraction was repeated by resuspension of the aforementioned residue in 20 ml of ultra-pure water (4 °c), centrifugation (25,000 × g, 30 min, 4 °c), resuspension of the second residue in 20 ml of ultra-pure water (4 °c), further centrifugation (25,000 × g, 30 min, 4 °c), and pooling of all three centrifugates and the washing water in the volumetric fl ask (200 ml). pe activities after one-time extraction and this repeated extraction procedure, respectively, were insignifi cantly different (data not shown). 2.4.3 pectin methylesterase assay pe activity (ape), expressed in units g-1 of pulp-standardized juice sample (cf. 2.4.1) according to eq. 1 as the liberation rate of carboxyl groups during enzymatic pectin de-esterifi cation, is quantitated as detailed by hirsch et al. (2008, 2011). hence, titrimetric analysis was carried out in a thermostated cell at 30 °c and ph 7.0 for 30 min with 0.01 n naoh using an automatic titrator titrino 718 stat (metrohm, herisau, switzerland). the 0.5 % (w/v) solution of apple pectin (de 77 %), containing 0.15 m nacl, served as the substrate. after adjusting 59 ml of substrate solution to ph 7.0, 1 ml of enzyme extract (cf. 2.4.2) was added. the ph value was set to 7.0 again, and the titration was performed at constant ph within 30 min. another aliquot of the enzyme extract was boiled for 3 min and analyzed by analogous titration with 0.01 n hcl (blank). as calculated from the naoh equivalents required for ph retention (eq. 1), ape (units g-1 of sample, i.e., juice or fsmoj) resulted from the volumes of naoh and hcl used to titrate the extracts of sample (vnaoh, ml) and blank (vhcl,b, ml), the corresponding naoh and hcl concentrations (cnaoh, chcl = 0.01 mol l-1), the observation time at ph 7.0 (δt, s), the extract volumes of sample (vextract, ml) and blank (vblank, ml), and the factor of enzyme extract volume per sample mass (nominal: f = 200 ml/100 g of sample). one unit of pe activity denoted a liberation rate of carboxyl groups of 1 µmol min-1 (i.e., 60 µkat). each extract was analyzed in triplicate, if not otherwise stated. 000,60 blank hclbhcl, extract naohnaoh pe ⋅⋅      ⋅∆ ⋅ + ⋅∆ ⋅ = f vt cv vt cv a (1) unless otherwise declared, the standard assay with an extract volume of 1 ml was used. however, when no naoh consumption was induced because of too low pe activity, titration of sample and blank was additionally performed with 5 ml of active and boiled extract, respectively, while the substrate volume was reduced to 55 ml for a constant total volume of 60 ml. 2.5 methods used for validating the procedure of freshness evaluation 2.5.1 evaluation of repeatability intra-assay precision and time-dependent intermediate precision (green, 1996) of the analytical method described in 2.4 were explored by analyzing defrosted ‘salustiana’ fsoj 2-3 times per day by the same person and with the same equipment on 3 different days within a period of 4 months. each of the 2-3 enzyme extracts (cf. 2.4.2) obtained per day was titrated 3-4 times according to 2.4.3 for evaluating the precision of the titration step in comparison with that of the whole method. intra-assay precision of the analytical method detailed in 2.4 (i.e., precision under repeatability conditions; thompson et al., 2002) was concluded from the standard deviations (sd) and the relative standard deviations (rsd) observed on each day i (sdr,i, rsdr,i, n = 2-3). time-dependent intermediate precision (i.e., precision under day-to-day conditions) was evaluated based on the sd and rsd of the mean calculated from the average results of 3 different days (sdday, rsdday, n = 3). total precision was expressed by sdrun and rsdrun of all individual runs that were performed on these three days (n = 7) (ich, 1996; thompson et al., 2002). 2.5.2 evaluation of reproducibility for standardization of an analytical method that is designated for offi cial application, reproducibility is usually deduced from the application of this method to aliquots of homogeneous samples by different analysts in multiple laboratories (ich, 1996). this concurrently provides information on the ruggedness of the method (thompson et al., 2002). since an inter-laboratory trial was beyond the scope of this study, reproducibility was estimated as the analystdependent intermediate precision. for this purpose, defrosted ‘salustiana’ fsoj and a fsmoj prepared by dilution of the former to a juice content of 60 % (w/w) (cf. 2.3) (in this experiment referred to as fsmoj 1 and fsmoj 2, respectively) were analyzed by three analysts (pa, pb, pc) on different days in the same laboratory. each analyst investigated both samples in triplicate, following the analysis specifi cation as detailed in 2.4. however, since sample preparation (juice standardization; cf. 2.4.1) had to be applied to the fsoj before its conversion into fsmoj (cf. 2.3), each analyst began the analysis of fsmoj 2 by standardizing the respective fsoj aliquot prior to dilution of the pulp-standardized juice to 60 % (w/w) and subsequent enzyme extraction. the activity resulting from each individual fsmoj 2 run was thus corrected to a juice content of exactly 60 % (w/w) by multiplying the reading by the quotient of nominal to real fsoj content before any averaging calculation for fsmoj 2. in addition to the intra-assay precision of each analyst (sdr,p, rsdr,p, n = 3 per analyst and sample), reproducibility was expressed by sdr and rsdr of the average of the three analyst-specifi c mean pe activities. moreover, the grand mean characterized by sdr,run and rsdr,run was computed from the total of 9 complete runs for each sample, regardless of the analyst. additionally, the relative deviation of the means determined by analyst pb and pc, respectively, from the mean obtained by analyst pa was calculated for each sample (green, 1996). 2.5.3 evaluation of recovery accuracy of the analytical method specifi ed in 2.4 was assessed by two different approaches. the fi rst one was based on the comparison of the measured value with the true value, the other one involved standard addition at different levels to the sample material (green, 1996). verifi cation of citrus juice freshness 9 for the fi rst purpose, the data set produced for evaluating reproducibility (cf. 2.5.2) was explored accordingly. the grand mean obtained for the pe activity of the fsmoj with a juice content of 60 % (w/w) (fsmoj 2), representing the measured value, was compared with the value predicted as the ape mean of the corresponding fsoj (fsmoj 1), multiplied by the juice dilution factor of fsmoj 2. recovery was calculated as the percentage of found ape relative to the predicted value. the enzyme preparation rapidase® fp 15000 (microbial pe) served as the standard for the second approach. as specifi ed by the producer based on titration at ph 4.5 and 30 °c with citrus pectin (0.5 %) as the substrate, its pe activity was ≥ 5000 units g-1. its activity at ph 7.0 was assessed according to 2.4.3 by examination of six enzyme solutions obtained by diluting rapidase® fp 15000 with ultra-pure water in the range of 1:20 -1:60 (2-4 titrimetric runs per enzyme solution). respective mean pe activity of rapidase® fp 15000 was 55.6 ± 2.7 units g-1 of commercial enzyme preparation with a 95 % confi dence interval of ± 5.7 units g-1. the latter activity was used for the calculation of the spiking doses in terms of added ape. fsmoj prepared from defrosted ‘salustiana’ juice by dilution to three different juice contents (30, 60, and 75 % w/w; cf. 2.3) served as different types of authentic test material and were analyzed as described in 2.4, both before and after addition of known doses of rapidase® fp 15000. each fsmoj was used for standard addition at 1-2 different concentrations to assess the recovery as the percentage of found ape relative to the predicted pe activity of the spiked fsmoj for fi ve different samples. the exact mass of ~100 g of fsmoj was spiked by adding a defi ned volume of the enzyme standard (either without or after aqueous dilution of rapidase® fp 15000) by weight. the predicted activity of the spiked fsmoj was calculated as the sum of the activity, which was added via the standard, and the activity, which was found for the unspiked fsmoj after correcting the latter by the ratio of the juice contents of spiked and unspiked sample. this correction factor was necessary, because standard addition slightly lowered the juice content of the spiked fsmoj. to fsmoj 3 (juice content 60 % w/w), the exact mass of 1 ml of pre-diluted enzyme solution containing 2.5 and 1.67 % (v/v) of rapidase® fp 15000, respectively, was added. for fsmoj 4 (juice content 75 % w/w), the mass of 0.25 ml of rapidase® fp 15000 was used without previous dilution. fsmoj 5 (juice content 30 % w/w) was spiked by weight with 0.5 and 1 ml of undiluted rapidase® fp 15000, respectively. from each fsmoj, one enzyme extract was prepared (cf. 2.4.2) before and after spiking, respectively. the titrimetric assay (cf. 2.4.3) was carried out 3-4 times per enzyme extract. 2.5.4 evaluation of linearity and the limits of detection, identifi cation, and quantifi cation to verify the linearity of the analytical method specifi ed in 2.4, authentic sample material differing in known pe activity over a wide range was provided by the fsmoj series produced from ‘navelina’ and ‘salustiana’ fsoj by dilution to juice contents in the range of 0.1-75 % (w/w) (cf. 2.3). at the top, the range was thus naturally limited by the maximum pe activity observed for fsoj. linearity was verifi ed by plotting the predicted value (x) versus the measured one (y) for each fsmoj, irrespective of the cultivar. predicted ape was computed as the product of the mean pe activity of the fsoj, which had been diluted to produce this fsmoj, and the respective juice dilution factor. from each fsmoj, one enzyme extract was obtained (cf. 2.4.1-2.4.2) and subjected to the titrimetric enzyme assay (cf. 2.4.3) 3 times, sporadically up to 6 times (m = 1 complete analysis according to 2.4 per fsmoj). concurrently, information regarding the limits of detection (lod), identifi cation (loi), and quantifi cation (loq) was deduced according to din 32645 (1994) from the cultivar-independent calibration line (ich, 1996) that was generated from the combined data sets of the ‘navelina’ and ‘salustiana’ fsmoj series for the linearity check. the critical minimum ape level that was unequivocally measurable (yc) under the conditions specifi ed for the method in 2.4 was calculated as the sum of the ordinate intercept (a) of the calibration line and the width of its one-sided prediction interval (δa), with the latter originating from the residual standard deviation of the measured values used for calibration (sdy,x), the quantile of the student’s t-distribution (p = 0.05) for f = n-2 degrees of freedom (t0.05,n-2), the mean ( ) and the deviance (qx) of all specifi ed values (x) of the calibration line, the number of calibration measurements (n), and the number of analyses per sample (m = 1) (din 32645, 1994). the lod, being the lowest analyte concentration producing a detectable response above the noise level in analytical chemistry (green, 1996), consequently resulted as the ape level expected (xlod) for yc from the linear calibration function (y = a + bx) with the slope b. in this context, the lod represented the decision limit for the presence of pe activity (p ≤ 0.05) (din 32645, 1994). the loi, defi ned as the minimum analyte concentration that can be detected with a specifi ed probability, was calculated by multiplication of the lod by the factor 2 for a probability of 95 %, assuming the same rates of type i and ii errors (α = β = 0.05) (din 32645, 1994). the loq, being the lowest analyte level that can be measured accurately and precisely (green, 1996), was estimated on the basis of p = 0.05 and the standard deviation of the procedure (sdxo = sdy,x/b) for a relative uncertainty of 33.3 % (k = 3) by solving eq. 2 (din 32645, 1994) iteratively. the loq was thus the specifi ed pe activity (xloq), when the relative uncertainty, defi ned as the quotient of the half two-sided prediction interval and xloq, equaled k-1 = 1/3. iteration of eq. 2 was started by using (k · lod x)2 as approximation of (loq x)2 (din 32645, 1994). ( ) xn qxnmtk /-loqsdloq 2112,05.0xo ++⋅⋅= −−− (2) 2.6 statistical analyses besides sd, rsd, and the standard error (se), the half twosided confi dence intervals (ci) of the quantitated ape means were calculated based on the student’s t-distribution (p = 0.05) with estimation of the quantiles t0.05; n-1 via the tinv function of microsoft offi ce excel 2003. if indicated by deviations between arithmetic and trimmed means, the arithmetic means were checked for outliers by means of dean-dixon tests and grubbs tests. signifi cant differences (p = 0.05) between variants were identifi ed by multiple pairwise comparisons of means (duncan tests) with the glm procedure of sas 9.1 (sas institute, cary, nc, usa). 3. results and discussion 3.1 precision of the analytical procedure under study the same analyst determined the pe activity of the pulp-standardized ‘salustiana’ fsoj after previous enzyme extraction on 3 individual days (tab. 1). the relative standard deviations of the daily means of 2-3 runs only amounted to 3.1-4.3 % (rsdr, i) on two days, but greater dispersion (13.0 %) occurred as well. intra-assay precision of the whole three-step method specifi ed in 2.4 was thus ≤ 13 %. by contrast, the rsd of the titrimetric enzyme assays (2.4.3) performed for each enzyme extract only varied between 0.9 and 7.5 % around a median of 2.9 % among all 7 enzyme extracts of this juice sample (tab. 1). greater dispersion of the whole analytical procedure of 2.4 on analysis day 2 thus indicated that the performance of sample 10 a.r. hirsch, r. carle, s. neidhart tab. 1: repeatability of the method for pectin methylesterase activity quantifi cation after enzyme extraction from pulp-standardized juice: pe activity (ape) of freshly squeezed ‘salustiana’ orange juice, which was immediately frozen after juice production and defrosted for analysis on different days. enzyme extraction no. ape [units g-1 of juice] mean sd rsd [%] se ntitr [ ] ntotal [ ] ci (p=0.05) analysis day 1 after 6 days of frozen juice storage run 1 1.28 c 0.01 0.9 0.007 3 0.028 run 2 1.23 c 0.01 1.2 0.008 3 0.036 subsample meanr,i (i = day 1) 1.25 0.04 3.1 0.027 2 0.345 analysis day 2 after 35 days of frozen juice storage run 3 1.23 c 0.03 2.2 0.016 3 0.068 run 4 1.02 d 0.08 7.5 0.038 4 0.122 subsample meanr,i (i = day 2) 1.13 0.15 13.0 0.104 2 1.317 analysis day 3 after 128 days of frozen juice storage run 5 1.41 b 0.09 6.1 0.050 3 0.215 run 6 1.48 ab 0.05 3.2 0.027 3 0.117 run 7 1.54 a 0.04 2.6 0.023 3 0.098 subsample meanr,i (i = day 3) 1.48 0.06 4.3 0.036 3 0.157 analyses of days 1-3 grand meanrun 1.31 0.18 13.5 0.067 7 0.164 mean values with different letters (a-d) are signifi cantly different (p ≤ 0.05). sd, standard deviation; rsd, relative standard deviation; se standard error; ntitr, number of individual titrations performed per enzyme extract; ntotal, number of complete runs consisting of enzyme extraction from the juice and subsequent titration (i.e., number of extractions); ci, confi dence interval ( nt n sd1;05.0 ⋅− ). standardization regarding the pulp content (cf. 2.4.1) and enzyme extraction (cf. 2.4.2) could sporadically be suboptimal. analysis of the ‘salustiana’ fsoj after frozen storage for 128 days resulted in signifi cantly increased pe activity (1.48 units g-1; tab. 1). it was 1.2 -1.3 times higher than the daily mean activities found by the same analyst after shorter periods up to ~1 month (1.25 and 1.13 units g-1). the average of the mean activities detected on 3 individual days was 1.29 ± 0.10 units g-1 (rsdday = 13.8 %). its 95 % confi dence interval (1.3 ± 0.44 units g-1) refl ected the greater variation on analysis day 2 and the signifi cantly higher pe activity at the end. the time-dependent intermediate precision of 13.8 % (rsdday) though came quite close to the intra-assay precision (maximum rsdr,i). the grand mean of all 7 runs performed by the same analyst regardless of the day of analysis implied a total precision of 13.5 % (rsdrun) for the method and a pe activity of 1.31 ± 0.07 units g-1 for the ‘salustiana’ fsoj (tab. 1). minimum and maximum activities suggested by runs 4 and 7, respectively, were beyond its 95 % confi dence interval (1.3 ± 0.16 units g-1). analysis of the same sample in triplicate by each of 3 people on different days (fsmoj 1 in tab. 2) overall confi rmed the pe activity of ‘salustiana’ fsoj presented in tab. 1. identical results were obtained by the analysts pa and pc with individual intra-assay precision of 4.3-4.9 % (rsdr,p). the pe activity found by the analyst pb (1.27 ± 0.004 units g-1) with an rsdr,p of 0.6 % was signifi cantly lower (tab. 2), but consistent with the values reported in tab. 1 for short frozen storage. the results of the analysts pc and pb deviated from that achieved by analyst pa by 1.5 % (insignifi cant) and 14.1 %, respectively. to mimic an inter-laboratory trial for best possible estimation of reproducibility, reagents were prepared by each analyst individually. the average of the mean activities that were found by the 3 analysts was 1.40 ± 0.07 units g-1 with a 95 % confi dence interval of ± 0.29 units g-1, and the reproducibility of the method was hence 8.2 % (rsdr). the grand mean of all 9 runs irrespective of the analyst ranged at 1.40 ± 0.04 units g-1 with a 95 % ci of ± 0.085 units g-1 (tab. 2), thus suggesting a total precision of the method of 7.9 % (rsdr, run) for the involvement of different analysts. pe activity quantifi cation after enzyme extraction from pulp-standardized fsoj was thus reproducible and overall robust. investigation of the defrosted sample by various analysts (tab. 2) or at different times (tab. 1) did not further increase the dispersion that had to be expected from intra-assay precision in the worst case. examination of a sample, which ranged at a lower pe activity level because of adjustment to 60 % juice content during fsmoj production, even provided more uniform results among the 3 analysts (fsmoj 2 in tab. 2). however, one run of analyst pb was probably an outlier (tab. 2). the variance of the titrimetric assays performed for the enzyme extract of this run was extreme (rsd = 21.4 %). without consideration of this outlier, analyst-specifi c intra-assay precision (rsdr,p = 5.3-8.4 %) was slightly inferior to that usually observed at the higher activity levels of fsoj, but rsdr,p though remained clearly below the maximum rsdr,i listed in tab. 1 for the intra-assay precision. the average calculated from the respective mean results of three analysts was 0.87 ± 0.02 units g-1 with a 95 % ci of ± 0.085 units g-1. its rsdr (3.9 %) even indicated enhanced reproducibility at the lower activity level. accordingly, the average results of the analysts pb and pc deviated from that of pa only by 4.7 and 7.4 %, respectively. these deviations were comparable verifi cation of citrus juice freshness 11 tab. 2: reproducibility of the method for pectin methylesterase activity quantifi cation after enzyme extraction from pulp-standardized juice: pe activity (ape) of freshly squeezed model orange juices (fsmoj)a as determined by three different analysts (pa, pb, pc). analyst ape [units g-1 of model juice] mean sd rsd [%] se ntitr [ ] ntotal [ ] ci (p=0.05) fsmoj 1 analyst pa run pa 1 1.41 bc 0.09 6.1 0.050 3 0.215 run pa 2 1.48 ab 0.05 3.2 0.027 3 0.117 run pa 3 1.54 a 0.04 2.6 0.023 3 0.098 subsample meanr,p pa 1.48 0.06 4.3 0.036 3 0.157 analyst pb run pb 1 1.26 d 0.02 1.9 0.014 3 0.060 run pb 2 1.26 d 0.04 3.4 0.021 4 0.067 run pb 3 1.28 d 0.08 6.0 0.044 3 0.191 subsample meanr,p pb 1.27 0.007 0.6 0.004 3 0.018 analyst pc run pc 1 1.49 ab 0.06 3.8 0.033 3 0.142 run pc 2 1.50 ab 0.007 0.5 0.005 2 0.061 run pc 3 1.37 c 0.01 1.0 0.010 2 0.128 subsample meanr,p pc 1.46 0.07 4.9 0.041 3 0.177 grand meanr,run fsmoj 1 1.40 0.11 7.9 0.037 9 0.085 fsmoj 2 analyst pa run pa 1 0.97 a 0.02 1.6 0.009 3 0.038 run pa 2 0.88 abc 0.04 4.4 0.022 3 0.096 run pa 3 0.88 abc 0.05 6.0 0.026 4 0.084 subsample meanr,p pa 0.91 0.05 5.3 0.028 3 0.119 analyst pb run pb 1 0.82 bc 0.02 2.1 0.010 3 0.042 run pb 2 0.57b d 0.12 21.4 0.055 5 0.152 run pb 3 0.92 ab 0.01 1.6 0.008 3 0.036 subsample meanr,p pb 0.77c (0.87)d 0.18c (0.07)d 23.4c (8.4)d 0.104c (0.05)d 3c (2)d 0.446c (0.66)d analyst pc run pc 1 0.91 ab 0.02 2.5 0.016 2 0.200 run pc 2 0.84 bc 0.04 4.8 0.023 3 0.100 run pc 3 0.78 c 0.0004 0.05 0.0003 2 0.004 subsample meanr,p pc 0.84 0.06 7.6 0.037 3 0.159 grand meanr,run fsmoj 2 0.84c (0.87)d 0.12c (0.06)d 13.8c (6.9)d 0.039c (0.02)d 9c (8)d 0.089c (0.05)d mean values with different letters (a-d and a-d, respectively) are signifi cantly different (p ≤ 0.05). sd, standard deviation; rsd, relative standard deviation; se standard error; ntitr, number of individual titrations performed per enzyme extract; ntotal, number of complete runs consisting of enzyme extraction from the juice and subsequent titration (i.e., number of extractions); ci, confi dence interval ( nt n sd1;05.0 ⋅− ). a ‘salustiana’ fsoj was immediately frozen after juice production and defrosted for analysis by each analyst on the individual day, either without (fsmoj 1) or after (fsmoj 2) previous dilution of defrosted fsoj to a juice content of 60 % (w/w). b outlier according to the dean-dixon test (p ≥ 0.02) and grubbs test (p ≥ 0.05). c calculated irrespective of potential outliers. d calculated after removal of the probable outlier. enzyme extraction no. to the dispersion indicated by the range of the analyst-specifi c rsdr, p. consequently, the grand mean of all 8 runs, irrespective of the analysts, was justifi ed and amounted to 0.87 ± 0.04 units g-1 (rsdr, run = 6.9 %), with the 95 % ci being ± 0.05 units g-1 (tab. 2). however, even if the debatable run of analyst pb was no outlier (n = 9), rsdr, run would be 13.8 % (tab. 2), thus meeting the worst intra-assay precision found for fsoj (tab. 1). 3.2 accuracy of the analytical procedure under study since fsmoj 2 in tab. 2 was prepared from ‘salustiana’ fsoj (fsmoj 1 in tab. 2) by dilution to a juice content of 60 % (w/w), the pe activity of the former could be predicted by multiplication of the mean pe activity of the latter (1.40 ± 0.04 units g-1; tab. 2) by the juice dilution factor (0.6). the pe activity expected for fsmoj 2 (0.84 units g-1) was compared with the measured value of this sample (0.87 ± 0.02 units g-1; n = 8; tab. 2). a recovery percentage of 104.1 % was derived from the ratio of measured to predicted activity. its standard error of ± 3.7 % resulted from error propagation of the standard errors, which were reported in tab. 2 for the ape grand means of fsmoj 1 (‘salustiana’ fsoj) and fsmoj 2 and consequently specifi ed the errors of the predicted and the measured value (± 0.04 and 0.02 units g-1, respectively). high accuracy of the analytical method was thus indicated. fsmoj 3-5, which were used for the standard addition experiment (tab. 3), were also produced from ‘salustiana’ fsoj. analysis of a single extract per sample by means of 3-4 titrimetric assays led to the measured pe activities reported in tab. 3 for these fsmoj before spiking with the commercial enzyme preparation. when those activities were likewise related to the ape grand mean of ‘salustiana’ fsoj (1.40 ± 0.04 units g-1; fsmoj 1 in tab. 2), high accuracy was confi rmed by the recovery of 100.8 ± 3.9 % that was estimated for unspiked fsmoj 4 (75 % juice content). after standard addition to fsmoj 4, comparison of measured and predicted pe activity of the spiked sample equally showed 12 a.r. hirsch, r. carle, s. neidhart good recovery (106 ± 4.4 %; tab. 3). accuracy of the analytical method was thus also proven by adding microbial pe to model juice as the second approach (green, 1996) applied, although the additional analytical error of the pe activity detected at ph 7.0 for the commercial enzyme preparation (cf. 2.5.3) further raised the variance of the recovery percentage. since the doses of microbial pe added to fsmoj 3, which was the fi rst model juice batch prepared for the standard addition experiment, turned out to be in the range of the standard errors of the pe activities detected before and after pe admixture (tab. 3), spiking of fsmoj 3 was not expected to cause demonstrable changes. the pe activities of the spiked fsmoj 3 batches though signifi cantly exceeded that of the unspiked model juice. apparently excessive recovery percentages of 111 ± 3.5 % and 120 ± 4.6 % after standard addition (tab. 3) were predominantly ascribed to a too low result for the pe activity of the unspiked fsmoj 3 batch (0.61 units g-1). the latter was 30.6 % below the ape level that was uniformly confi rmed by different analysts for fsmoj 2 (0.87 ± 0.02 units g-1; tab. 2), although both fsmoj batches contained 60 % juice. a possible outlier ranging in the same order as ape of unspiked fsmoj 3 was among the results of another analyst for fsmoj with this juice content (tab. 2). hence, the fi ndings for fsmoj 3 in tab. 3 pointed out to a weakness of the analytical method that rather concerned its robustness than limited accuracy. two notable doses of microbial pe added to fsmoj 5 with a juice content of only 30 % (w/w) were interrelated by a ratio of 1 : 2.1. despite signifi cant increments in ape relative to the unspiked sample, the pe activities of both spiked juices were only related to each other by a ratio of 1 : 1.4. consistently, merely 92 ± 2.6 and 82 ± 2.7 % of the ape predicted after spiking, respectively, were found (tab. 3). for fsmoj 5 without admixture of microbial pe, detection of the ape level, which was expected from the content and the pe activity of the parent fsoj of fsmoj 5, was limited to 80 ± 2.7 %. hence, the risk of underestimation seemed to be enhanced, when only ~30 % of the pe activity of fsoj could be expected (fsmoj 5). accuracy was though overall acceptable (recovery ≥ 80 %) and, at least for the activity range of fsoj (hirsch et al., 2011), even good (~100 % recovery). samples analyzed with high accuracy among those listed in tab. 2 and 3 (fsmoj 2, 4, and 1) displayed pe activities of 0.87, 1.06, and 1.40 units g-1 of model juice. this equated to activity levels of ~0.0073, 0.0088, and 0.0117 units, respectively, in the test system of the titrimetric assay. activity analyses for standard solutions of commercial orange peel pe by means of a spectrophotometric assay, which was adapted to a kinetic microplate reader for quantitating the liberation rate of free carboxyl groups, confi rmed high accuracy (100 % recovery) for the same range (0.009-0.015 units; cameron et al., 1992). however, for a sample with 0.006 units, these authors only reported ~50 % recovery. a respective fsmoj analyzed with the method of 2.4 would have a pe activity of 0.72 units g-1, corresponding to ~51 % of the activity of fsmoj 1 (‘salustiana’ fsoj; tab. 2). even for fsmoj 5 with a juice content of only 30 %, recovery was ≥ 80 % (tab. 3). hence, accuracy of the titrimetric assay according to 2.4.3 was superior in the lower activity range. moreover, high accuracy in the upper range was obviously not affected by the procedures of juice standardization and enzyme extraction, being part of the quantitation method detailed in 2.4. 3.3 range, linearity, and limits of the analytical procedure under study the range of the analytical method was estimated by comparing the measured pe activities of fsmoj (y) with their expected levels (x) for a series of 10 ‘navelina’ and 12 ‘salustiana’ samples that contained 0.51-100 and 1.02-100 % of the respective fsoj (fig. 1). after different times of frozen storage, two ‘salustiana’ fsoj batches were defrosted for the determination of their pe activity and the use as parent juices for fsmoj production for this experiment. since their mean pe activities signifi cantly differed from each other (tab. 1), the batch-specifi c mean (1.25 ± 0.03 and 1.48 ± 0.04 units g-1, respectively) served as the reference value for predicting the ape levels of model juice batches that were prepared from the respective parent fsoj batch. however, pe activity of the ‘navelina’ fsoj batch (1.94 ± 0.05 units g-1) notably exceeded the ape records obtained for the ‘salustiana’ fsoj at different times (tab. 1) and by different analysts (tab. 2) and thus represented the experimental upper limit of the range (fig. 1). the pe activity of a fsmoj with 29.86 % (w/w) of juice (expected ape: 0.44 ± 0.01 units g-1) marked the bottom of the linear range (white rhombs in fig. 1). a slope closely approximating 1.0 suggested that the measured values confi rmed the predicted ones rather accurately, tab. 3: recovery of pectin methylesterase activity (ape) after addition of a respective enzyme preparation (diluted or undiluted rapidase fp 15000) to freshly squeezed model orange juices (fsmoj)a prior to enzyme extraction. model juice sample ape [units g-1 of model juice] recoveryb [%] added predicted measuredc rsd [%] ci (p=0.05) fsmoj 3 0 --0.61 ± 0.013 e 4.4 0.04 -- 0.010 0.613 0.68 ± 0.015 d 3.9 0.07 111.2 ± 3.5 0.015 0.619 0.75 ± 0.023 cd 5.4 0.10 120.4 ± 4.6 fsmoj 4 0 --1.06 ± 0.030 b 4.8 0.13 -- 0.150 1.22 1.30 ± 0.043 a 6.6 0.14 106.0 ± 4.4 fsmoj 5 0 --0.34 ± 0.007 f 3.6 0.03 -- 0.307 0.64 0.59 ± 0.007 e 2.1 0.03 92.1 ± 2.6 0.650 0.99 0.82 ± 0.006 c 1.3 0.03 82.4 ± 2.7 mean values with different letters (a-f) are signifi cantly different (p ≤ 0.05). rsd, relative standard deviation of the measured ape value; ci, confi dence interval of the measured ape value ( nt n sd1;05.0 ⋅− ). a ‘salustiana’ fsoj was immediately frozen after juice production and defrosted for analysis after previous dilution of defrosted fsoj to a juice content of 60 % (w/w) (fsmoj 3), 75 % (w/w) (fsmoj 4), or 30 % (w/w) (fsmoj 5) and addition of differently diluted rapidase fp 15000. b recovery calculated as the ratio of measured to predicted ape of the spiked fsmoj (mean ± standard error based on error propagation). c mean ± standard error of 3-4 titrimetric assays per extract. verifi cation of citrus juice freshness 13 although the coeffi cient of determination (r2 = 0.962) indicated deviations of individual records. accordingly, the residual standard deviation of the measured values (sdy,x) was 0.0949 units g-1. as expected from the ape result of the unspiked fsmoj 5 (tab. 3; cf. 3.2), the negative ordinate intercept (a = -0.1247 units g-1) confi rmed slight underestimation of pe activity near the bottom of the range also for other model juices. irrespective of juice content and cultivar, fsmoj with expected pe activities ≤ 0.39 ± 0.01 units g-1 due to juice contents ≤ 20 % (w/w) could not be distinguished analytically (gray rhombs in fig. 1). for those samples, the enzyme assay was even performed with an extract volume of 5 ml, whereas the standard assay with 1 ml was applicable to all samples in the linear range (cf. 2.4.3). hence, the selectivity of the method was abruptly lost, when the expected pe activity (x) declined from 0.44 to 0.39 units g-1 of model juice. this corresponded to a drop from 0.0037 to 0.0032 units in the test system of the standard titrimetric assay. the expected pe activity at the experimental upper limit of the linear range caused 0.0162 units in this test system, i.e. the 4.4-fold value of the expected level at the bottom of the linear range. however, if even the 5-fold extract volume was insuffi cient for proper analysis of both fsmoj samples with 20 % (w/w) of ‘navelina’ and ‘salustiana’ fsoj, respectively, the crucial factor limiting sensitivity was not the enzyme assay (cf. 2.4.3), but obviously the enzyme extraction (cf. 2.4.2) from the diluted sample. the regression line of fig. 1 was analyzed according to the calibration line method of din 32645 (1994) in order to specify the sensitivity of the analytical procedure of 2.4. presence of pe activity was indicated by a record of at least 0.095 units g-1 of model juice (yc), being the critical minimum ape value that could unambiguously be measured under the conditions applied (p = 0.05). the minimum activity (xlod) producing this lowest detectable response (yc) was an expected ape level of 0.207 units g-1 of model juice, which was thus defi ned as the limit of detection (lod). it equated to 0.00173 units in the titrimetric standard test system with 1 ml of enzyme extract (cf. 2.4.3). as deduced from the lod, the expected pe activity that represented the minimum level for ape detection with 95 % probability (xloi) accounted for 0.415 units g-1 of model juice (limit of identifi cation, loi). such a sample would yield 0.00346 units in the test system during titration. the loi ranged between the predicted ape levels of the two fsmoj batches, which marked the zone of abrupt selectivity loss in fig. 1. the value recorded for a sample with an activity equal to the loi would be 0.314 units g-1 of model juice (yloi) according to the regression equation. the recovery at the loi would thus be 76 %, similar to the recovery found for the unspiked fsmoj 5 of tab. 3 with an expected pe activity coming close to the loi (cf. 3.2). as iteratively estimated from eq. 2 based on a standard deviation of the procedure (sdxo) of 0.0897 units g-1 of fsmoj, precise and accurate ape detection with a relative uncertainty ≤ 33.3 % was assumed for model juices with expected pe activities (xloq) ≥ 0.537 units g-1 of fsmoj (≥ 0.00448 units in the titrimetric test system). this was considered as the limit of quantifi cation (loq). for a sample with a pe activity ranging at the loq, the regression equation of fig. 1 suggested a measured value of 0.444 units g-1 (yloq) and thus a recovery of 82.5 %. if all titrimetric records within the linear range were treated as individual runs, a regression line based on 52 values could be computed, with the slope (1.0546) and the ordinate intercept (-0.1222 units g-1) deviating somewhat less from 1.0 and 0, respectively. whereas the regression based on mean activities (fig. 1) refl ected linearity and sensitivity of the whole analytical method, this approach rather addressed the quality of the titrimetric assay itself. the dispersion of the titrimetric records reduced r2 to 0.942 and enhanced sdy,x to 0.1100 units g-1 and sdxo to 0.1043 units g-1. estimation of yc, lod, and loi from that regression line suggested slightly lower values of 0.08, 0.19, and 0.38 units g-1, respectively. however, that loi would already be located in the noise range found for fsmoj (fig. 1). on the whole, good linearity could be assumed for the method of 2.4 above an ape bottom limit represented by the loi (xloi = 0.415 units g-1 of model juice; cf. fig. 1). activities > ~0.0035 units should fi nally be ensured in the test system for the enzyme assay. consequently, the method was more sensitive than the microplate reader method of cameron et al. (1992), requiring ≥ 0.009 units (cf. 3.2). sampedro et al. (2008) reported a detection limit of 0.057 units for a titrimetric assay that was likewise performed at ph 7.0, but at 22 °c under other concentration ratios and for shorter reaction times. although estimates of lod, loi, and loq greatly depend on the approach used (ich, 1996), fig. 1 and the comparison of our loi with that detection limit suggested that the facilitated reaction at the higher temperature of 30 °c (cf. 2.4.3) probably contributed most to the increase in sensitivity by 16 times relative to the assay of sampedro et al. (2008). for their macro and semi-micro enzyme assays performed at 30 °c, but only for 3 min at ph 7.5, bartolome and hoff (1972) estimated a sensitivity of 3 ppm of methanol in the test system prior to conversion of methanol to methyl nitrite for gas chromatographic quantitation. according to the description of those test systems, this indicated a minimum release of methanol of ~0.2-0.3 µmol min-1. the minimum pe activities required were thus ~85 times greater than our loi (~0.0035 units), but were though acceptable for the analysis of pe activity in plant tissue. sensitivity of the analytical method described in 2.4 overall appeared superior. however, because of the reduced accuracy near loi and loq with anticipated recoveries of 76 and 82.5 %, respectively, analysis should be repeated with the 3-fold enzyme extract volume in case of records ranging in the order of the loi or loq. as suggested by the results obtained for y = 1.0575x 0.1247 r 2 = 0.9619 0 0.5 1 1.5 2 2.5 0 0.5 1 1.5 2 2.5 a pe expected (x ) [units g -1 of model juice] a p e m e a su re d (y ) [u n its g -1 o f m o d e lj u ic e ] fig. 1: range and linearity of the procedure validated for pectin methylesterase activity quantifi cation after enzyme extraction from standardized model juices: pe activity (ape; rhombs) of 22 freshly squeezed model orange juices (fsmoj), which comprised ‘navelina’ and ‘salustiana’ fsoj and various dilutions thereof with juice contents of 0.5-75.1 % w/w. the expected ape levels of the model juices (x) were calculated as the mean ape of the respective fsoj, multiplied by the juice fraction of the model juice prepared thereof by dilution. open rhombs represent values included in the linear regression model (n = 12 model juices with juice contents of 30 -100 % w/w). 14 a.r. hirsch, r. carle, s. neidhart fsmoj 4 (tab. 3) and ‘navelina’ fsoj (fig. 1), best performance is expected, when the enzyme assay is run in the presence of 0.0088-0.0162 units. 3.4 applicability to citrus juices of unknown production history for freshness evaluation the pe activities of the fsoj batches, which were prepared on the small scale for this study from ‘navelina’ (1.94 ± 0.05 units g-1; fig. 1) and ‘salustiana’ fruit (tab. 1-2), represented the ~1.3fold values relative to those of the fsoj, which had previously been produced from the respective cultivars on the pilot plant scale (hirsch et al., 2011). hence, this earlier report was overall confi rmed. slightly higher activities in the present case could be caused by the less effi cient manual fi nishing process, but the differences were within the wide range documented by snir et al. (1996) for the natural pe activity variation among fruit based on an assay at 30 °c and ph 7.5. according to the authors, the ratio of maximum to minimum activity varied from 1.4 to 3.5 among cultivar-specifi c fscj batches and overall amounted to 4.5 for the total of 17 fscj batches from 8 citrus cultivars. when different titrimetric assays involving the same temperature (30 °c) were used for quantitation at ph 7.0, three ‘valencia’ fsoj batches produced throughout the season and six ones of ‘valencia late’, which were consecutively manufactured from the same fruit lot, displayed pe activities of 2.22 ± 0.86 units ml-1 (wicker et al., 1987) and 1.02 ± 0.05 units g-1 (hirsch et al., 2008), respectively. since the pe activities of the cultivar-specifi c citrus juices produced on the pilot plant scale by hirsch et al. (2008, 2011) originated from exactly the same analytical method, they were directly compared with the results of this study in fig. 2. the former comprised both freshly squeezed and thermally treated juices from oranges [c. sinensis cvs. ‘valencia late’, ‘salustiana’, ‘navelina’, and ‘navelate’], lemons [c. limon (l.) burm. f. cvs. ‘verna’ and ‘primofi ori’], clementine mandarins (c. reticulata blanco cvs. ‘clemenules’ and ‘marisol’), and the mandarin hybrid cultivars ‘clemenvilla’ and ‘ortanique’. the records for all fscj batches accumulated between 0.6 and 1.4 units g-1 within the full range of 0.44 -1.94 units g-1 (fig. 2), clearly exceeding the lod of the method. ‘navelina’ samples ranged on the top, followed by those of ‘salustiana’. the pe activity was within the linear range of the method (fig. 1) for all fscj samples, with the maximum equating to the 4.4-fold level of the minimum activity (fig. 2) by analogy with the range observed by snir et al. (1996). however, the minimum (hirsch et al., 2011) was hardly above the loi (0.42 units g-1; fig. 2). if the recovery was assumed to gradually decline to 76-80 % near the loi, as suggested by fig. 1 and the unspiked fsoj 5 of tab. 3, the difference in the true activities between the fsoj, which displayed the minimum ape, and the majority of the other samples might have been lower than that indicated by fig. 2. whereas chilled fc products accounted for 57 and 44 % of the chilled citrus juices on the german market in years 2005 and 2009, respectively, their portion on the french market was almost negligible (3.0-2.5 %) (aijn, 2010). as far as quantitative results were attainable at all, the pe activities found for the four commercial chilled citrus juices (tab. 4) were far below the lod of 0.21 units g-1 (fig. 2), despite the use of the 5-fold extract volumes in the enzyme assays of all four samples (cf. 2.4.3). in view of the lod defi nition (cf. 3.3), the labeled treatments (tab. 4) thus apparently inactivated the pe isoenzymes (fig. 2). however, confi rmation of complete deactivation would require a more sensitive approach, such as the semi-quantitative pasteurization test of ifu method no. 46 (ifu, 1972, 2005). the ape records obtained for the four tab. 4: pectin methylesterase activities (ape) of commercial citrus juices distributed in cold chain. product code ja jb jc jd package size 1 l 1 l 1 l 1 l citrus species processed c. sinensis c. sinensis c. sinensis c. reticulata source: retail markets in france france germany france preservation as labeled on the package pressurized pasteurized gently pasteurized pasteurized ape [units g-1 of juice] 0.030 ± 0.016 nd tr 0.014 ± 0.014 nd, not detected; tr, traces. fig. 2: discrimination between different types of citrus juices by means of the evaluated analytical method: pe activities (ape; rhombs) detected in the freshly squeezed ‘navelina’ and ‘salustiana’ orange juices produced on the laboratory scale for this study (fsoj: ls), the 4 chilled commercial citrus juices of tab. 4 (cj: comm cc), as well as the pilot plant scale products of hirsch et al. (2008, 2011), comprising 15 freshly squeezed citrus juices (fscj: pps) and citrus juices that were continuously heat-treated at 42-92 °c, respectively, for 12 s (cj: pps, 12s / 42…92 °c). the juice samples of hirsch et al. (2008, 2011) are marked by asterisks. dotted and dashed lines indicate the limits of detection (lod) and identifi cation (loi), respectively, as deduced from the linear range (fig. 1) by means of the calibration method according to din 32645. 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 f s o j, 2 x: ls f s c j, 1 5 x: p p s* c j, 1 0 x: p p s , 1 2 s / 4 2 ˚c * c j, 1 0 x: p p s , 1 2 s / 5 2 ˚c * c j, 1 0 x: p p s , 1 2 s / 6 2 ˚c * c j, 1 0 x: p p s , 1 2 s / 7 2 ˚c * c j, 1 0 x: p p s , 1 2 s / 8 2 ˚c * c j, 1 0 x: p p s , 1 2 s / 9 2 ˚c * c j, 4 x: co m m cc a p e [u n its g -1 o f ju ic e ] orange juices mandarin juices lemon juices lod loi verifi cation of citrus juice freshness 15 commercial products of tab. 4 were similar to those reported by hirsch al. (2008, 2011) for juices after continuous thermal processing of fscj at 72-92 °c (partly also at 62 °c) with dwell times of 12 s in a tube-type actijoule® heating system, which was integrated in a tubular heat exchanger (fig. 2). if soft treatments like the non-thermal and thermal ones indicated by tab. 4 for the commercial juices are applied to nfc products, incomplete deactivation of thermostable pe fractions can be assumed (nienhaber and shellhammer, 2001; sentandreu et al., 2005; carbonell et al., 2006), but the residual total activities can though be expected to be low enough under chilled conditions to extend the shelf life relative to fscj due to signifi cant retardation of juice clarifi cation until consumption (hirsch et al., 2008). the ape differences between fscj and minimally processed juices were discussed in detail by hirsch et al. (2011) for different thermal and non-thermal mild preservation techniques. the abrupt selectivity loss below the loi (fig. 1) enabled clear and reliable distinction of fsoj from chilled juices like those of tab. 4 (fig. 2). 4. conclusions freshness evaluation requires reliable appraisal of the pe activity for unknown citrus juice samples. ape quantitation via the tested method was considered reproducible and accurate within the linear range, but complete recovery obviously required the presence of at least ~0.009 units in the titration jar. although analysis was mostly very repeatable, extreme results that were sporadically observed overall limited the intra-assay precision to ~13 %. this suboptimal repeatability, which resulted from unidentifi ed factors constricting robustness, constituted the greatest weakness of the method. hence, strict consideration of standardized operation conditions is necessary. however, the validated method overall proved suitable for verifi cation of citrus juice freshness via the activity of pe in the product because of two reasons: (1) the wide cultivar-independent ape range of fscj (hirsch et al., 2011) conformed to the linear range of the method, which was limited by the loi (0.42 units g-1 of juice). (2) the fact that the standardized method involving an assay with an enzyme extract volume of 1 ml per 60 ml abruptly became insensitive below the loi ensured reliable distinction of fscj from other chilled juices having comparative advantages due to enhanced convenience. thermal or non-thermal treatments of the latter group had inactivated at least the thermo-labile pe fractions almost completely and thus extended the shelf life signifi cantly. hence, limited sensitivity of the analytical method, which prevented applications as to specifi cation of pasteurization results, conversely enabled its use as regards authentication of freshness for citrus juices. samples that could not be distinguished from fscj solely by ape comprised juices with residual activities > loi, which may be caused by minor partial reduction of initial ape via preservation techniques or by blending of pasteurized juice with fscj. but for such products, the risk of clarifi cation (hirsch et al., 2008) and limitation of shelf life would be the same as for fscj (hirsch et al., 2011). if a highly active fscj like the one of this study from ‘navelina’ orange fruit was used for blending with a juice reconstituted from concentrate, addition of ~22 % of fsoj would cause a residual activity equal to loi. in conclusion, 3 complete runs of the standard procedure according to 2.4 were recommended for authentication of juice freshness. when the accurate ape values are needed for samples with activities near loi for unambiguity or to evaluate the risk of clarifi cation more precisely, 3 further runs with extract volumes ensuring ≥ 0.009 units in the titration jar additionally become necessary. acknowledgments the authors thank dr. s. schilling and mr. k. mix (hohenheim university) for their valuable assistance in estimation of analystdependent intermediate precision. abbreviations used a, ordinate intercept of the regression line (cf. 2.5.4, fig. 1); ape, pectin methylesterase activity; b, slope of the regression line (cf. 2.5.4, fig. 1); ci, half two-sided confi dence interval; de, degree of esterifi cation; fscj, freshly squeezed citrus juice; fsmoj, freshly squeezed model orange juice (derived from fsoj by dilution); fsoj, freshly squeezed orange juice; fc, product from concentrate; lod, limit of detection (cf. 2.5.4); loi, limit of identifi cation (cf. 2.5.4); loq, limit of quantifi cation (cf. 2.5.4); nfc, product not from concentrate; pe, pectin methylesterase; pvpp, polyvinylpolypyrrolidone; rsd, relative standard deviation; rsdday, time-dependent intermediate precision based on analyses under day-to-day conditions (cf. 2.5.1); rsdr, reproducibility as the analyst-dependent intermediate precision (cf. 2.5.2); rsdr,i, intra-assay precision based on the analyses under repeatability conditions on day i (cf. 2.5.1); rsdr, p, intra-assay precision based on the analyses of a given analyst under repeatability conditions (cf. 2.5.2); rsdr,run, total precision as deduced from all individual runs performed by all analysts (cf. 2.5.2); rsdrun, total precision as deduced from all individual runs performed by the same analyst irrespective of the time of analysis (cf. 2.5.1); sd, standard deviation; sdy,x, residual standard deviation of the measured values used for regression (cf. 2.5.4); sdxo (= sdy,x/b), standard deviation of the procedure (cf. 2.5.4); se, standard error; x, expected values (predicted pe activities) of the regression line (cf. 2.5.4, fig. 1); xlod, expected pe activity of a sample ranging at the limit of detection; y, responses (measured pe activities) of the regression line (cf. 2.5.4, fig. 1); yc (= a + bxlod), critical minimum pe activity that is unequivocally measurable for the sample ranging at the limit of detection (cf. 2.5.4). references aijn, 2010: market report 2010 – liquid fruit. aijn european fruit juice association, brussels, belgium. http://www.aijn.org/pages/main/factsfi gures.html (retrieved october 19, 2011). bartolome, l.g., hoff, j.e., 1972: gas chromatographic methods for the assay of pectin methylesterase, free methanol, and methoxy groups in plant tissues. j. agric. food chem. 20, 262-266. cameron, r.g., buslig, b.s., shaw, p.e., 1992: adaptation of a spectrophotometric assay for pectinmethylesterase to a kinetic microplate reader. j. food sci. 57, 1006 -1008. carbonell, j.v., contreras, p., carbonell, l., navarro, j.l., 2006: pectin methylesterase activity in juices from mandarins, oranges and hybrids. eur. food res. technol. 222, 83-87. collet, l.s.f.c.a., shigeoka, d.s., badolato, g.g., tadini, c.c., 2005: a kinetic study on pectinesterase inactivation during continuous pasteurization of orange juice. j. food eng. 69, 125-129. crupi, f., rispoldi, g., 2002: citrus juices technology. in: dugo, g., di giacomo, a. (eds.), citrus – the genus citrus, 77-113. taylor & francis, london, uk. duvetter, t., sila, d.n., van buggenhout, s., jolie, r., van loey, a., hendrickx, m., 2009: pectins in processed fruit and vegetables: part i – stability and catalytic activity of pectinases. compr. rev. food sci. food saf. 8, 75-85. din deutsches institut für normung e.v., 1994: chemische analytik. nachweis-, erfassungsund bestimmungsgrenze. din 32645. beuth verlag, berlin, germany. fsa, 2008: criteria for the use of the terms fresh, pure, natural etc. 16 a.r. hirsch, r. carle, s. neidhart in food labelling. food standards agency, london, uk. http: //www.food.gov.uk/multimedia/pdfs/markcritguidance.pdf (retrieved october 19, 2011). gel moretó, n., 2000: analytische untersuchungen zur unterscheidung von frisch-gepresstem, direktund konzentratorangensaft. flüss. obst 67, 137-140. green, j.m., 1996: a practical guide to analytical method validation. anal. chem. news & features 68, 305a-309a. guiavarc’h, y., segovia, o., hendrickx, m., van loey, a., 2005: purifi cation, characterization, thermal and high-pressure inactivation of a pectin methylesterase from white grapefruit (citrus paradisi). innovative food sci. emerging technol. 6, 363 -371. hirsch, a.r., förch (née resch), k., neidhart, s., wolf, g., carle, r., 2008: effects of thermal treatments and storage on pectin methylesterase and peroxidase activity in freshly squeezed orange juice. j. agric. food chem. 56, 5691-5699. hirsch, a.r., knauss (née resch), a., carle, r., neidhart, s., 2011: impact of minimal heat-processing on pectin methylesterase and peroxidase activity in freshly squeezed citrus juices. eur. food res. technol. 232, 71-81. ich, 1996: validation of analytical procedures: methodology. ich guideline q 2 b. international conference on harmonization of technical requirements for the registration of pharmaceuticals for human use, geneva, switzerland. http://www.nihs.go.jp/drug/validation/ q2bwww.html (retrieved november 18, 2004), http://www.ich.org/ fi leadmin/public_web_site/ich_products/guidelines/quality/q2_r1/ step4/q2_r1_guideline.pdf (retrieved october 20, 2011). ifu, 1972, 2005: determination of pectin esterase (pe) activity in citrus juices and their concentrates. ifu methods of analysis no. 46 (1972, revised 2005). international federation of fruit juice producers, paris, france. kopelman, i.j., rauchwerger, m., 1984: shelf-life and microbial growth kinetics of fresh citrus juices: shamouti orange. j. food process. preserv. 8, 241-250. nienhaber, u., shellhammer, t.h., 2001: high-pressure processing of orange juice: kinetics of pectinmethylesterase inactivation. j. food sci. 66, 328-331. ruiz perez-cacho, p., rouseff, r., 2008: processing and storage effects on orange juice aroma: a review. j. agric. food chem. 56, 9785-9796. sampedro, f., rodrigo, d., hendrickx, m., 2008: inactivation kinetics of pectin methyl esterase under combined thermal-high pressure treatment in an orange juice-milk beverage. j. food eng. 86, 133-139. schols, h.a., voragen, a.g.j., 2003: pectic polysaccharides. in: whitaker, j.r., voragen, a.g.j., wong, d.w.s. (eds.), handbook of food enzymology, 829-843. marcel dekker, new york, ny, usa. sentandreu, e., carbonell, l., carbonell, j.v., izquierdo, l., 2005: effects of heat treatment conditions on fresh taste and on pectinmethylesterase activity of chilled mandarin and orange juices. food sci. technol. int. 11, 217-222. shaw, p.e., buslig, b.s., moshonas, m.g., 1993: classification of commercial orange juice types by pattern recognition involving volatile constituents quantifi ed by gas chromatography. j. agric. food chem. 41, 809-813. snir, r., koehler, p.e., sims, k.a., wicker, l., 1996: total and thermostable pectinesterases in citrus juices. j. food sci. 61, 379-382. thompson, m., ellison, s.l.r., wood, r., 2002: harmonized guidelines for single-laboratory validation of methods of analysis. pure appl. chem. 74, 835-855. tønder, d., petersen, m.a., poll, l., olsen, c.e., 1998: discrimination between freshly made and stored reconstituted orange juice using gc odour profi ling and aroma values. food chem. 61, 223-229. wicker, l., braddock, r.j., vassallo, m., 1987: effect of assay temperature on activity of citrus pectinesterase in fresh orange juice. j. food sci. 52, 378-380. wicker, l., vassallo, m.r., echeverria, e.j., 1988: solubilization of cell wall bound, thermostable pectinesterase from valencia orange. j. food sci. 53, 1171-1174, 1180. address of the authors: dipl.-lm-ing. angelika hirsch, institute of food science and biotechnology, chair of plant foodstuff technology, hohenheim university, garbenstrasse 25, 70599 stuttgart, germany. present address: institute of biological process engineering, mannheim university of applied sciences, paul-wittsack-straße 10, 68163 mannheim, germany. prof. dr. habil. dr. h.c. reinhold carle, institute of food science and biotechnology, chair of plant foodstuff technology, hohenheim university, garbenstraße 25, 70599 stuttgart, germany. dr. sybille neidhart (corresponding author), institute of food science and biotechnology, chair of plant foodstuff technology, hohenheim university, garbenstraße 25, 70599 stuttgart, germany. phone: +49(0)711 459 22317. fax: +49(0)711 459 24110. e-mail: sybille.neidhart@uni-hohenheim.de. angewandte botanik. 250 georg lakon, über d ie bezahnung der kiele der vorspelze be i lolium perenne l . und l . multiflorum lmk. von priv.-dozent dr. georg lakon (hohenheim-stuttgart). (mit 4 textfiguren.) die scheinfrüchte des englischen raigrases (lolium perenne l.) lassen sich bekanntlich von denen des italienischen raigrases (lolium multiflorum lmk.; lolium italicum a . br.) im allgemeinen durch das fehlen der granne unterscheiden. dieses merkmal ist indessen nur in den fällen, bei welchen es sich um die identität einer bekanntermaßen unvermischten probe handelt, durchaus zuverlässig. auch kann über die zugehörigkeit begrannter früchte zu l . multiflorum kein zweifel bestehen. anders bei grannenlosen früchten, die man in schwankender anzahl unter typischen, begrannten früchten des italienischen raigrases findet! solche scheinfrüchte können sowohl dem englischen, wie auch der grannenlosen form des italienischen raigrases angehören. die feststellung der identität solcher raigrasfrüchte is t schwierig und läßt sich nur durch die bezahnung der kiele der vorspelze durchführen. auf dieses merkmal hat meineswissens zum ersten male alexander braun hingewiesen. in einer abhandlung über das italienische raigras) schreibt er bezüglich der hauptcharaktere dieser art im vergleich zu den anderen verwandten arten: die vorspelze der blüte „zeigt a n ihren beiden kielen stärkere, unter sich getrenntere wimpern als die anderen lolche; bei l . perenne sind sie viel gedrängter und feiner; bei l . temulentum kann man sie an den beiden scharfen leisten kaum unterscheiden, si e sind wie verschmolzen“. eingehender hat sich später wittmack vom standpunkt der samenkontrolle mit der frage der unter scheidung der beiden raigräser befaßt. in einem vortrage führte e r aus*): „besonders charakteristisch ist aber die borstige be *) flora. xvii, 1. (1834), (s. 241–253, 257–269). s. 266. *) verhandlungen d . botan. vereins d . provinz brandenburg. jahrg. xviii, (1876), s . li–lii. (vortrag gehalten am 28. oktober 1876 in der 25. haupt versammlung des vereins). bezahnung d. kiele d. vorspelze bei lolium perenne l. u. l.multiflorum lmk. 251 zahnung der oberen spelze. diese spelze is t bei l . perenne an textur derber, aber kürzer und meist unterbrochener bezahnt a ls bei l . italicum, weshalb schon unter einer guten lupe l . italicum stärker gezähnt erscheint. noch deutlicher wird dies unterm mikroskop. die borstenzähnchen der oberen spelze a m reifen samen messen bei l . perenne an der längsten (äußeren) seite 74–171 u meist ca. 85–100 u ; bei l . italieum dagegen 114–208 u , meist ca. 114–150 u , a n der inneren seite. bei ersterem meist 43–57, bei letzterem meist 67–71. da die dicke a n der basis bei beiden gleich ist, so erscheinen daher die zähn chen bei l . italicum schlanker, und gewöhnlich stehen sie auch dichter. – bei lolium temulentum sind sie noch kürzer als bei l . perenne (57–85 u an der äußeren seite) und stark verdickt, hier ist auch das lumen meist mit einer bräunlichen masse an gefüllt; bei l . remotum schrk. (l. arvense schrd.) sind die zähn chen am kürzesten (28–57 u) und gleichfalls stark verdickt.“ in den handbüchern über landwirtschaftliche samenkunde drücken sich die autoren nur kurz und keinesfalls eindeutig aus. in harz samenkunde!) heißt e s bei l . perenne: obere spelze kurz mikroskopisch-borstig; bei l . italicum: obere spelze lang borstig gewimpert. nach settegast”) sind die ränder der bauchspelze b e i l . italicum „durch stärkere borstenhaare bewimpert“ als bei l . perenne. eine bildliche darstellung des unterschieds finden wir bei rostrup”) und bei stebler*). die rostrupsche zeich nung der bezahnung des italienischen und englischen raigrases gebe ich auf fig. 1 in der original-größe wieder. von der steblerschen zeichnung is t ein teil in zweifacher vergrößerung auf fig. 2 reproduziert. stebler sagt an dieser stelle unter hinweis auf seine figur: „schon a . braun führt aus, daß d ie stachelhaare auf den kielen der vorspelze beim italienischen raigrase dichter stehen, als beim englischen.“ bei einer tabellari schen gegenüberstellung der unterschiede zwischen englischem und italienischem raigrase steht aber in demselben werke") bezüg *) landwirtschaftliche samenkunde. 2 . bd. berlin 1885, s . 1341–1342. *) die landwirtsch. sämereien usw. leipzig 1892, s . 231. * aarsberetning fra dansk frokontrol for 1901–1902 a f o . rostrup. kopenhagen 1903, s . 41. *) stebler und volkart. die besten futterpflanzen. 1. bd, 4. aufl., s . 5 9 , abb. 46. *) s . 47. 17* 252 georg lakon, lich der bezahnung der kiele der vorspelze für das englische raigras: „auf den nerven fein und dicht gewimpert“; für das italienische raigras: „auf den nerven gröber und entfernter gewimpert“. vergleichen wir die vorstehenden angaben der verschiedenen autoren untereinander, so können wir feststellen, daß sie sich a 2 -e-htfz -z-z fig. 1. die bezahnung der vorspelze bei l. perenne (oben) und l. multforum (unten) nach rostrup. (in der größe des rostrupschen originals). zum teil widersprechen. die steblerschen angaben stehen sogar gegenseitig im widerspruch. zunächst is t die oben zitierte behauptung, daß nach a . braun die zähne bei italienischem rai grase dichter stehen sollen als bei englischem, falsch; a . brauns– fig. 2. die bezahnung der vorspelze bei l. perenne (unten) und l . multiflorun (oben) nach stebler. (etwa aufs zweifache des steblerschen originals vergrößert). schildert, wie wir bereits oben gesehen haben, den sachverhalt gerade umgekehrt. die steblersche schilderung stimmt anderer seits mit der von diesem autor gegebenen figur überein (vgl. fig. 2), steht aber (ebenso wie die figur) in direktem widerspruch zu den in der oben zitierten tabellarischen zusammen stellung enthaltenen angaben. die ausführungen von a . braun, bezahnung d. kiele d. vorspelze bei lolium perenne l. u. l. multiflorum lmk. 253 wittmack, harz, settegast") und die abbildung von rostrup stimmen, was die länge der zähne anbelangt, miteinander über ein, nämlich darin, daß die zähnchen bei l. multiflorum länger sind als bei l. perenne, während nach der figur von stebler d ie bezahnung des italienischen raigrases nur feiner aber nicht länger als die des englischen erscheint. was dagegen die ent fernung der zähne voneinander betrifft, so paßt die angabe wittmacks, daß die zähne von l . perenne meist unterbrochener stehen als die von l . multiflorum, wohl auf das steblersche, aber nicht auf das rostrupsche bild, nach welchem letzteren die zähne des englischen raigrases gedrängter, die des italienischen mehr unterbrochen stehen. mit der zeichnung rostrups steht andererseits die beschreibung von a . braun im einklang. die in der literatur vorhandenen widersprüche veranlaßten mich, die frage einer eingehenden untersuchung zu unterwerfen. hierbei stellte e s sich bald heraus, daß die abbildung im stebler schen buch den sachverhalt keinesfalls richtig illustriert, daß sie eher geeignet ist, irrezuführen. da sich dieses ausgezeichnete werk mit recht der größten verbreitung und beliebtheit erfreut, s 0 scheint mir eine richtigstellung im allgemeinen interesse zu liegen. andererseits macht die mannigfaltigkeit der formen eine genauere darlegung der verhältnisse notwendig. zu meinen untersuchungen standen mir außer zahlreichen samenproben verschiedenen ursprungs auch mannigfaches frisches und herbar-material zur verfügung. in der überwiegenden anzahl der fälle war die bezahnung der kiele der vorspelze für jede art sehr charakteristisch und je einem bestimmten typus ent sprechend, doch konnten auch ausnahmen festgestellt werden, die eine geringere oder beträchtlichere abweichung vom typus dar stellen. die interessantesten formen habe ich auf fig. 3 und 4 vereinigt. die form fig. 3 f stellt die typische bezahnung von l . perenne dar: die zähnchen gehen bei auffallender gleichmäßigkeit der form unmittelbar ineinander über ohne zwischen räume, sie stehen also gedrängt, sind sehr kurz und im ver hältnis hierzu a n der basis sehr breit (die spitze daher stumpf), und zeigen eine gleichmäßige starke einseitige ') die von a . braun und von settegast für die borstenhaare des italienischen raigrases gebrauchte bezeichnung „stärker“ is t nicht völlig ein deutig: e s müßte eher „länger“ heißen. 254 georg lakon, neigung (nach der spitze der spelze hin), wobei die eine (die längere) flanke leicht konvex, die andere (die kürzere konkav ist (zahn daher schnabelförmig, von plastischem aussehen). die form stimmt mit der abbildung von stebler vollkommen über ein. auch die abbildung rostrups entspricht dieser form, nur daß hier an zwei stellen zwei größere zwischenräume vorhanden sind. solche zwischenräume kommen tatsächlich nicht selten bei l. perenne in wechselnder anzahl und größe vor. einen solchen fall stellt z. b. fig. 3a dar; die zähne stehen hier einzeln, durch größere zwischenräume voneinander getrennt, ihre form is t nicht die typische: sie sind schlanker, nämlich länger und a n der basis fig. 3. die bezahnung der kiele der vorspelze bei l. perenne nach eigenen untersuchungen (mit dem abbeschen zeichenapparat gezeichnet; auf das 75fache der nat. größe reduziert). f typische form. a-e mehr oder weniger vom typus abweichende formen. schmäler (daher am gipfel mehr zugespitzt) als die der typischen form und weniger schnabelförmig gebogen (von mehr starrem aussehen). die figuren 3b-e stellen übergänge von der typischen (fig. 3f) zu der vom typus extrem abweichenden form (fig. 3a) dar. abb. 4a stellt die typische bezahnung von l . multiflorum dar. die zähnchen gehen zwar auch hier ohne zwischenräume ineinander über, sind aber ungleichmäßig gebaut und haben eine von der des englischen raygrases bedeutend abweichende form: die länge der einzelnen zähnchen ist im vergleich zu der breite der basis außerordentlich groß (daher die zähne bezahnung d. kiele d. vorspelze bei lolium perenne l. u. l.multiflorum lmk. 255 am gipfel scharf zugespitzt); die beiden flanken der zähne sind geradlinig oder kaum merklich bogenförmig und was ihre länge anbelangt nur geringfügig voneinander ver schieden, so daß die zähnchen eine ungleichmäßige, kaum ausgesprochene neigung nach der spitze der spelze hin zeigen. die zähnchen bieten also hier in ihrergesamtheit ein ungleich mäßiges, ausgesprochen starres aussehen. der dichtere stand der zähne von fig. 4a im vergleich zu fig. 4f is t nicht auf einen mehr lückenlosen zusammenhang, sondern auf die geringere breite -z->z-z-<<s-<szz. f -z-c-z-zez-e-zz-zz fig. 4 . die bezahnung der kiele der vorspeize bei l . multiflorum nach eigenen untersuchungen (mit dem abbeschen zeichenapparat gezeichnet; auf das 75fache der natürl. größe reduziert. a-d typische formen. e-f seltene, vom typus abweichende formen. d e r einzelnen zähne (an der basis) zurückzuführen. die figur steblers wird den tatsächlichen verhältnissen nicht gerecht, d a si e den unterschied in länge und form der zähne nicht zum ausdruck bringt. dies tut die rostrupsche figur, welche aber einen mehr isolierten stand der einzelnen zähne zeigt. solche zwischenräume kommen in der tat bei l . multiflorum noch öfter v0r als bei l . perenne. einen solchen fall illustriert insbesondere fig. 4d. fig. 4b-c stellen zwischenformen dar. diese drei, fast 8 0 0 ſt wie der typus vorkommenden formen weisen einen mehr unterbrochenen und weniger starren stand der zähne auf. in 256 georg lakon, seltenen fällen kann die abweichung in form und größe der zähne vom typus sogar so weit gehen, daß die bezahnung von der des englischen raigrases kaum zu unterscheiden ist. solche äußerst seltenen fälle stellen fig. 4e und f dar. fig. 4f nähert sich außerordentlich der typischen form des englischen raigrases (fig. 3f) oder zum mindesten der ihr nächsten übergangsformen (fig. 3e). fig. 4e ähnelt anderen selteneren formen des eng lischen raigrases (etwa fig. 3b, c, d) . auch formen, wie die bereits erwähnte fig. 4d kommen bei l . perenne selten vor (vgl. fig. 3a). vergleichen wir die oben zitierten literaturangaben mit der typischen form der bezahnung bei l . perenne und l . multiflorum, so können wir folgendes feststellen: die darstellung a . brauns ist im wesentlichen richtig, aber nicht klar genug. die eingehenden darlegungen wittmacks entsprechen bis auf zwei punkte den tatsachen: erstens kommt die bezahnung nicht bei l . perenne sondern eher bei l . multiflorum meist unterbrochener vor, und zweitens ist der unterschied in der länge der beiden flanken der zähne wohl bei l . perenne, nicht aber bei l . multiflorun s0 groß, wie aus den von diesem autor angegebenen zahlen zu schließen wäre. bei dieser gelegenheit möchte ich nicht unerwähnt lassen, daß die angabe brauns, daß die zähne bei l. temulentum wie verschmolzen und kaum zu unterscheiden wären, den tatsachen nicht entspricht. nach meinen beobachtungen sind die zähnchen bei l . temulentum wohl ausgebildet, erinnern in der form an die des italienischen raigrases und stehen, was die größe betrifft, zwischen diesen und denjenigen des englischen raigrases (sind also nicht – wie wittmack angibt – noch kürzer als die der zuletzt genannten art); die wand ist – wie wittmack richtig hervorhebt – stark verdickt. im übrigen scheint wittmack die angaben a . brauns nicht gekannt zu haben, desgleichen stebler, sowie rostrup die oben zitierte arbeit wittmacks. die feststellung von formen, welche (wie bei fig. 3a-b; 4e-f) vom typus derart abweichen, daß die artzugehörigkeit der betreffenden scheinfrucht daran schwierig oder kaum erkannt werden kann, führt z u der frage nach dem werte dieses unter scheidungsmerkmals. daß dieses merkmal nicht in allen fällen absolut zuverlässig ist, muß nach den obigen ausführungen al s feststehend angesehen werden. diese ausnahmefälle verlieren bezahnung d. kiele d. vorspelze bei lolium perenne l. u. l. multiflorum lmk. 257 indessen an bedeutung, sobald ihre häufigkeit in betracht gezogen wird. die von mir beobachteten wenigen ausnahmefälle konnten in der tat nur nach mühsamem suchen an einem sehr umfang reichen material gefunden werden. kein einziger posten zeigte eine besondere häufigkeit solcher ausnahmen. besonders wichtig is t d ie tatsache, daß bei l . multiflorum ein zusammenhang der typischen bezahnung mit dem vorhandensein der granne keines falls existiert. mir gelang es sogar in keinem einzigen falle, an grannenlosen scheinfrüchten des italienischen raigrases zweifel hafte bezahnung festzustellen; sie war hier stets typisch aus gebildet. ausnahmefälle dürften demnach bei den grannenlosen früchten zum mindesten nicht häufiger vorkommen als bei den begrannten. die ausnahmefälle sind also infolge der großen seltenheit ihres auftretens keinesfalls geeignet, der be zahnung als unterscheidungsmerkmal abbruch zu tun. am schluß meiner ausführungen möchte ich nochmals die charakteristik der bezahnung der kiele der vorspelze beim englischen und italienischen raigrase kurz zusammenfassen: lolium perenne: zähnchen im vergleich zu der breiten basis sehr kurz, die eine flanke sehr lang und leicht konvex, die andere sehr kurz und leicht konkav (raubvogel schnabelförmig), gleichmäßig stark einseitig geneigt, a n der spitze stumpf; die ganze reihe der zähne meist von auffallender gleich imäßigkeit der form, von plastischem aussehen, ohne größere zwischenräume (dichter stand der zähne, die unmittelbar in einander übergehen) (vgl. fig. 3f). lolium multiflorum: zähnchen im vergleich zu der (ebenso breiten oder noch schmäleren als bei l . perenne) basis sehr lang, mit fast gleichlangen, geradlinigen oder kaum merklich bogen förmigen flanken, nur leicht und ungleichmäßig einseitig geneigt, a m gipfel scharf zugespitzt; die ganze reihe der zähne unregel mäßig gebaut, von starrem aussehen, oft von größeren zwischen räumen unterbrochen (vgl. fig. 4a-d). uc1.b3299303_page_268_cut uc1.b3299303_page_269_cut uc1.b3299303_page_270_cut uc1.b3299303_page_271_cut uc1.b3299303_page_272_cut uc1.b3299303_page_273_cut uc1.b3299303_page_274_cut uc1.b3299303_page_275_cut journal of applied botany and food quality 92, 216 225 (2019), doi:10.5073/jabfq.2019.092.030 lehrstuhl für pharmazeutische biologie, erlangen exploiting plant cell culture for natural product formation wolfgang kreis* (submitted: may 27, 2019; accepted: july 9, 2019) * corresponding author summary the initiation of callus cultures and in vitro cultivation of plant cells, even in large bioreactors, has become a routine task. despite the fact, that permanent cell and organ cultures can produce a whole range of small natural compounds (snaps) used in medicine, only a few could be produced at commercial scale. however, plant cell cultures provide very useful systems to study the biosynthetic pathways leading to snaps at the enzyme and gene level. they turned out to be ‘a pot of gold’ for those chasing the enzymes and genes involved in natural product formation. the use of genetically modified yeast and bacteria for the production of snaps is an emerging technique that will take research into the coming decades. this review contemplates and revisits developments in the field of using plant cell and tissue culture as tools to elucidate snap formation and means to produce bioactive plant natural products over the past 40 years alongside my own work. keywords: secondary metabolism, specialized metabolism, cell suspension culture, bioreactor, elicitation, biochemical pathways, natural products introduction a multitude of small natural products (snaps), such as alkaloids, small peptides, isoprenoids, phenolics, and polyketides generated by plants are used as pigments, food additives or pharmaceuticals. some of them are bioactive in an ecological or co-evolutionary sense. in an average plant about 90% of the individual products formed can be characterized as snaps (firn, 2010) although some are only produced in trace amounts. as early as the 1970s my interest in the biochemistry, physiology, chemistry, and analysis of snaps was sparked and still i regard each discipline as equally important when dealing with snap formation. at that time snaps were sometimes regarded as ’flotsam and jetsam on the metabolic beach‘ (haslam, 1986). the model of chemical co-evolution eventually brought them into focus (hartmann, 1996) and the vast structural diversity kindled much enthusiasm and attracted many researchers. jones and firn (1991) introduced the screening hypothesis in order to explain the existence of hundreds of thousands of individual snaps. their hypothesis is based on the assumption that organisms making snaps face the same challenges as pharmaceutical companies trying to find new drugs. only recently, noda-garcia et al. (2018) have offered an integrative metaboliteenzyme co-evolution hypothesis proposing that metabolite pools change alongside the appearance of newly evolved enzymes. if an emerging snap provides a selective advantage for its producer, enzymes will be recruited or shaped resulting in improved synthesis. any of these hypotheses try to explain the occurrence of the multiplicity of snaps. these hypotheses rather complement than contradict each other and may even be merged. in the 1970s reports on the biosynthesis of snaps were still limited. how did we study snap biogenesis in these days? beadle and tatum (1941) used neurospora crassa as a model organism to elucidate biochemical pathways. they established that individual gene mutations were responsible for single metabolic steps (‘one geneone enzyme’ hypothesis) by constructing mutant strains requiring specific amino acids or vitamins for their survival. this approach, however, could not be successfully employed in elucidating the pathways leading to snaps. they are not necessary for the survival of the producing cell, lethal mutants could not be generated and their deficiencies then repaired by adding appropriate precursors. the only change in phenotype that could easily be observed was pig ment formation (see selection and characterization of biochemical mutants). many of the pathways shown in the textbooks were based on chemical plausibility or studies using radiolabeled compounds assumed to be precursors in a particular pathway. experiments were usually carried out with whole plants or excised plant organs. one particular booklet (bu’lock, 1965), translated into german and also slightly edited by wolfgang barz and hans grisebach (bu’lock, 1972), was sufficient to convince me that the field ‘biosynthesis of natural products’ is large enough to nurture generations of scientists. traditionally snaps are isolated from intact plants since chemical synthesis proved to be complicated and expensive. production of snaps by plant cell cultures could provide an alternative source of snaps. articles reviewing this idea generally begin with the statement that hundreds of plants have been used to establish cell cultures. among these plants are many endangered species and a lot of plants containing compounds with, e.g., anti-cancer, neuroprotec tive or anti-malarial activities. most of the reviews repeat the following statements like a mantra: as an alternative way to produce snaps, cell and tissue culture have the advantages that (1) fields can be freed up for growing crops instead, that (2) plant cell culture systems are not affected by weather and season changes and that (3) their metabolism can be manipulated to maximize production. one may challenge all of these statements and find arguments contradicting them. for example, in germany medicinal and aromatic plants are cultivated on only 13,000 ha (iva, 2019) whereas cereals occupy about 6,000,000 ha (agravis, 2019). these figures warrant criticism against the first argument. the overwhelming amount of literature on plant cell cultures and their use in snap formation made it impossible to write a non-biased review covering the developments over the past 40 years. a literature search conducted on the google scholar website using the search terms “natural products” and “plant cell suspension” and covering the years 2015 2019 yielded 386 hits, among them 52 reviews or book chapters (e.g., dias et al., 2016; ochoa-villarreal et al., 2016; yue et al., 2016). a recent meta-analysis study came to the conclusion that plant cell technology is the best strategy for production of natural-based drugs (davoodi et al., 2019). contemplating on this astonishing statement and the plentitude of quite recent reviews i here tried to put my own experiences in the field in a methodological and historical context and give my very own assessment of the state-of-the-art (fig. 1). i took the book entitled ‘plant tissue culture and its biotechnological application’ (barz et al., 1977) as my personal starting point. the proceedings, edited by wolfgang barz (münster), ernst reinhard (tübingen), and meinhart zenk (münchen), documented the journal of applied botany natural products in plant cell cultures 217 state-of-the-art in the field at the time. well-reputed scientists from all over the world shared their experiences and the titles of their contributions could still be used to structure a review article today. in these proceedings alfermann et al. (1977) put forward the results of ernst reinhards’s group in tübingen in a contribution entitled ’biotransformation of cardiac glycosides by plant cell cultures’ and i was lucky enough to get a postgraduate position in this particular group in 1983 where i got the chance to combine in my research analytical, plant physiological, biochemical and biotechnological aspects of plant cell culture (see growth of plant cells in bioreactors). plant cell cultures are nowadays also considered to produce recombinant proteins, such as antibodies. however, this issue will not be touched here and the reader is referred to other reviews dealing with various aspects of protein production by suspension-cultured plant cells (fisher et al., 1999; huang and mcdonald, 2009; tekoah et al., 2015). initiation of plant cell cultures in 1934, white and gautheret independently demonstrated that plant cells and tissues can be grown and propagated for long periods of time in complex media. by including indole-3-acetic acid and vitamins in his media, gautheret (1934) extended the culture period of callus to 18 months by including indole-3-acetic acid and vitamins in his media and he was able to subculture the tissue aseptically. routien and nickell (1956) described the submerged cultivation of tissues of several plant species and were granted the first patent for the cultivation of plant cells in vitro. today, the initiation of plant cell and tissue cultures and cultivation of plant cells and tissues in vitro has become routine. cell behavior can be triggered with phytohormones and even regeneration of intact plants became possible. a number of chemical factors like media components, phytohormones and ph as well as physical factors like light, aeration and temperature, which can affect plant cell growth in vitro have been investigated. several textbooks, proceedings and lab manuals have been published over the past four decades to cover the topic more comprehensively than possible here (reinert and yeoman, 1982; allen, 1991; gamborg and philipps, 1995; coleman et al., 2003; loyola-vargas and ochoa-alejo, 2018). under certain conditions plant cells can grow as dedifferentiated callus masses. culture media usually in clude salts of inorganic macro-nutrients (n, p, s, k, na, ca, mg) and micro-nutrients (i, ni, fe, cu, b, mn, co) as well as vitamins and an appropriate carbohydrate source. the most common media for plant cell culture are those devised by murashige and skoog (1962), gamborg et al. (1968), schenk and hildebrandt (1972), and white (1934). under certain conditions plant cells can grow as de-differentiated callus masses. after several medium changes these cell masses can be used to study various aspects of snap formation and eventually plant cell culture turned out to be an excellent system to understand and manipulate snap formation. application of precursors or inhibitors is simple and analysis is facilitated by the fact that bulk compounds, such as phenolics or chlorophyll are not as abundant as, for example, in plant leaves. long-distance transport does not happen in plant cell culture, an obvious advantage for studying snap metabolism. in intact plants precursors can be transported, sequestered and/or modified in various ways prior to reaching the site of snap biosynthesis. precursors may even be degraded to very small molecules, such as acetate, which in turn can be used ‘second hand’ for snap formation. for example, the discovery of the nonmevalonic acid terpenoid pathway (lichtenthaler et al., 1997) supported the view that studies with radio-labeled precursors can lead to ambiguous conclusions. this and other examples demonstrate that even ‘trodden’ pathways like those depicted in generations of textbooks, do need confirmation by using modern techniques (see chasing enzymes of snap formation in plant cell cultures). plant cell cultures are an appropriate tool to do so provided they express the pathway to be examined. snap formation in callus cultures extensive lists of plant cell cultures accumulating bioactive snaps of most of the biosynthetic classes known were compiled (mulabagal and tsay, 2004; karuppusamy, 2009). many of the reports cited used callus cultures, which provide a reliable and easily accessible material for conducting biosynthetic studies. callus cultures of portulaca grandiflora hook. were the first cell cultures to catch my interest. they produce betalains and depending on their biosynthetic capacity they accumulate red and yellow pigments possessing a betalamic acid moiety. betalamic acid is derived from l-tyrosin needing a 4,5-dioxygenase, which has been isolated from p. grandiflora (christinet et al., 2004). l-tyrosine is also a precursor of cyclo-dopa and the conjugation of betalamic acid with cyclo-dopa yields betanidine. we found that betanidine biosynthesis is favored in the dark and that several catecholamines, including adrenaline, also accumulate in darkness. methyldopa, a drug developed for the treatment of high blood pressure, inhibited target producer explant callus induction fungi culture conditions selection protocols elicitation immobilization scale-up strategies market analysis fermenter design biotransformation operating mode target product natural product protein other product bacteriaplant cell culture root culture target plant microalgae co-cultivation suspension culture product harvest pathway enzymes genes manipulation metabolism flux storage degradation commercial process fig. 1: strategies for the alternative production of natural products (snaps). special emphasis is only put on plant cell suspension cultures. the latest results gained from the cultivation of genetically transformed roots are not taken into account. the use of microorganisms, including microalgae, in the production of plant-borne snaps is not addressed in a comprehensive way here. neither are methods for product isolation and release. other products, like proteins, are not addressed in this review. the areas not reviewed in detail are shown in grey. issues related to large-scale culture and commercialization are shown in blue. methods using plant cell suspension cultures for pathway elucidation are shown in red. the black arrow indicates the path from a target product to an industrial process using plant cell suspension cultures. the arrow also defines the structure of this review. 218 w. kreis catecholamine biosynthesis in p. grandiflora callus (endress et al., 1984). some questions began to puzzle me: (1) do plants somehow respond to the mammalian stress hormone adrenaline? (2) could it be a ‘secondary’ metabolite in plants but a ‘primary’ one in humans? (3) is the metabolism associated with its formation ‘secondary’? much later, firn and jones (2009) stressed that the same biochemical rules apply for all pathways, albeit to a different extent because they operate under different evolutionary constraints. there is no theoretical basis for separating metabolisms into ‘primary’ and ‘secondary’ and i decided to avoid these terms where ever possible. actually, only a few basic metabolic principles provide access to the majority of snaps: (1) starting from biogenic amines mannich-type reactions lead to a variety of alkaloids (‘alkaloid pathways’), (2) the isopentenyl-diphosphate/dimethyl allyl diphosphate system provides building units for a large number of terpenoid skeletons (‘terpenoid pathways’), (3) phenolic acids derived from phenylalanine and tyrosine provide building blocks for a large number of phenolics (‘phenylpropanoid pathway’), (4) malonyl-coa derived from acetylcoa provides a universal and sequential c2-group donor used in the formation of a plentitude of polyketides (‘polyketide pathway’). (5) small peptides can be produced via the processing of ribosomally produced precursors or with the help of nonribosomal peptide syn thetases (barber et al., 2013). the rich diversity of snaps found arises from modifications by only a limited number of chemical substitution types, such as hydroxylation, glycosylation, acylation, prenylation, and methylation. the formation of members of all the biogenetic groups mentioned have been investigated in plant cell cultures (see chasing enzymes of snap formation in plant cell cultures). selection and characterization of biochemical mutants callus can be grown for months and even years. it is, however, a quite heterogeneous population of cells. small clumps or aggregates can be picked from these cell masses to develop virtually homogeneous clones of fast growing viable cell cultures. several screening and selection protocols have been developed (widholm, 1977; berlin and sasse, 1985) and are still valid today. elite cell lines can be obtained by selection following various strategies including macroscopical, microscopical and chemical inspection. cell-aggregates or proto plasts can be used as the starting material for selection (dix, 1990; kreis et al., 1992). variation can be enhanced employing induced physical or chemical mutagenesis (ahloowalia et al., 2001; donini and sonnino, 1998). selection can be achieved easily if the product of interest is a pigment. for example, anthocyanin-rich euphorbia milii des moul. cells and cell clusters were selected and a cell strain was established producing 7 times as many anthocyanins as the initial cell culture (yamamoto et al., 1982). screening and selecting high-producing cell lines was not always successful and ‘recalcitrant’ cell cultures were the rule rather than the exception (kreis, 2003). cell cultures of papaver somniferum l., for instance do not produce quantities of morphinanes worth considering. low or no apparent productivity of alkaloids in plant cell cultures was explained by insufficient cell differentiation (farrow et al., 2012). this is supported by the fact, that non-producing plant cells may recover their biosynthetic capacity during morphogenesis. for example, in a duboisia leichhardtii (f. muell.) f. muell. callus scopolamine production and accumulation was restored after root induction, but not after shoot induction (yamada and endo, 1984). root cultures, by the way, have also turned out to be suitable systems for producing snaps and for studying snap formation (oksman-caldentey and arroo, 2000; srivastava and srivastava, 2007; yue et al., 2016) but this topic will not be reviewed here. plant tissues, when cultured in vitro, show genetic instability even without mutagenic treatment (stafford, 1991). cells can become habituated and ultimately exhibit hormone-autotroph callus growth which may explain the loss of regeneration potential after a pro longed callus phase. for example, in callus of digitalis mariana ssp. heywoodi (p. silva & m. silva) hinz morphogenesis could still be induced after 24 months but not after 32 months. cardenolide production was impaired in plants regenerated after several months of cultivation as callus (kreis et al., 2014). the variations occurring in plants regenerated from tissue culture or in permanent plant cell culture were termed ‘somaclonal variation‘ (larkin and scowcroft, 1981), which will also occur in permanent plant cell culture. the ‘apparent’ stability (meta-stability) of a plant cell culture ‘strain’ may be the effect of a strict subculture regime (ulbrich et al., 1985; fowler, 1988) rather than the result of the clonal origin of the cells. the loss in productivity reported for many plant cell suspension cultures might be caused by rapidly dividing cells overgrowing the snap accumulating cells (stafford, 1991). this phenomenon may constitute a problem for the establishment of a commercially viable plant cell culture process (see commercialization of plant cell culture processes). snap production using plant cell suspension cultures callus culture is not well suited as a method to commercially produce snaps. it cannot be used for the large-scale production of secondary metabolites because of slow growth rates and the lack of suitable bioreactor systems for their cultivation. however, calluses might disintegrate into small cell aggregates when submerged in liquid media yielding cell suspension cultures. actually, most of the physiological and biochemical experiments designed for elucidating metabolic pathways have been carried out with suspension-cultured cells. the cells are kept in flasks on appropriate shakers to allow for aeration under various light-dark regimes. reports on the production of snaps by plant cell cultures generally used small tissue samples grown in standard shake flasks allowing for easy sampling and additions to the suspensions. growth curves are usually determined during process optimization. for this purpose, we introduced a simple, sample-free method to allow for rapid estimation of cell growth in erlenmeyer flasks (blom et al., 1992). the method proved to be very suitable to determine the increase of cell mass during each phase of the growth cycle and it is still used in our laboratory today. because metabolic activity is a function of the surface of an organism, growth and production rates of plant cell aggregates are much lower than those of microbial cells. in microbes secondary metabolite production is not necessarily associated with biomass production. products are generally released into the bathing medium, production rates are high and production phases are short. the growth of plant cell cultures is quite slow, metabolite production is low and the products are usually stored in the cells (scragg, 1995). for example, rosmarinic acid accumulates to 36% dw in cultured cells of salvia officinalis l. (hippolyte et al., 1992), and berberine (kobayashi et al., 1988) or its derivative jatrorrhizine (breuling et al., 1985) can reach about 10% of the cells’ dry mass. although the idea of using plant cell cultures for the production of snaps is reasonable and even hyped in the 1980s and 1990s, tables of successes are still quite short and should be regarded with caution as far as productivity claims are concerned (see commercialization of plant cell culture processes). the list of plant cell cultures not producing the compounds of interest in amounts large enough to consider commercialization is much longer (kreis, 2007). elicitation of snap formation in plant cell cultures though all approaches to produce morphinanes in plant cell cultures so far have failed (see selection and characterization of biochemical natural products in plant cell cultures 219 mutants), compounds sharing the same precursors and intermediates as the morphinanes, such as benzophenanthridines, can accumulate in rather large amounts. it was demonstrated that the production of sanguinarine was even enhanced by adding preparations from fungal mycelia (eilert and constabel, 1986) and many studies have shown, that snap production can be provoked by exposing cultured plant cells to fungal cell wall fragments (barz, 1988; park et al., 1992; gundlach et al., 1992). preparations like these belong to the so-called ‘elictors’, exogenous molecules inducing physiological abnormalities that are often associated with plant pests. a broader definition of them includes exogenous and endogenous elicitors, as well as compounds released from plants in direct response to the pathogen. the label ’chemical elicitors‘ is now even used for substances like methyl jasmonate, methyl salicylate or benzoic acid, which are plant hormones and not elicitors in the original sense (patel and krishnamurthy, 2013). for example, methyl jasmonate ’elicitation‘ induced paclitaxel and related taxane production in taxus cells (tabata, 2004; see commercialization of plant cell cul ture processes). the elicitor approach was not successful in all cases: morphinane biosynthesis, for example, could not be elicited as yet. anyway, the application of elicitors (in the broader sense) has been considered as one of the most effective methods to improve the synthesis of snaps in plant cell cultures and elicitors can also be used to increase yield in field-grown plants (baenas et al., 2014) demonstrating the impact of basic research conducted with plant cell cultures. biotransformation of snaps by plant cell cultures suspension-cultured cells can be used in so-called biotransformation processes. biotransformation studies have been carried out with a view to (1) produce new chemicals, (2) produce known chemicals more economically, (3) investigate the metabolic fate of xenobiotics, and (4) elucidate metabolic pathways (reinhard and alfermann, 1980; suga and hirata, 1990; stepan-sarkissian, 1991; giri et al., 2001). for example, none of the cell cultures investigated so far was able to produce scopolamine, a drug used in urinary incontinence and motion sickness, but the ultimate biosynthetic step, the conversion of hyoscyamine into scopolamine was realized in transgenic tobacco cell cultures (moyano et al., 2007). in one of our own more recent biotransformation studies cell suspension cultures of digitalis lanata erh. were fed with 21-o-acetyl-deoxycorticosterone and three new pregnane compounds were formed and their structures elucidated (type (1) approach; pádua et al., 2012). in a type (2) approach, the commercially unavailable glucoevatromonoside, a natural carde nolide inducing cancer type-specific cell death (schneider et al., 2018), was produced in d. lanata cell cultures (munkert et al., 2017). already in the 1970s d. lanata cells cultivated in vitro were shown to convert digitoxin derivatives into digoxin derivatives (reinhard and alfermann, 1980). from this line of research a cell culture process has emerged where viable amounts of β-methyldigoxin are generated from β-methyldigitoxin, a semi-synthetic cardenolide with almost no side reactions. the β-methyldigoxin process was suc cessfully scaled up to a volume of 200 l (alfermann et al., 1983; reinhard et al., 1989). at that stage the reason why digitoxin could not be used to estab lish a putative digoxin process was unclear. storage, transport and sequestration had not be considered to understand the cellular organization of the processes involved in cardenolide biotrans formation. the vacuole was assumed to be a storage site for cardenolides but it could not be explained why some of the bio transformation products were released into the bathing medium while others remained in the cells. eventually, procedures to isolate intact vacuoles from plant cells cultivated in vitro became available and were used to investigate snap formation and storage (deusneumann and zenk, 1984; kreis and reinhard, 1985; mende and wink, 1987; hopp and seitz, 1987). after feeding digitoxin to suspension-cultured d. lanata cell we were able to demonstrate that only cardenolides possessing a terminal glucose moiety were stored in the vacuole. moreover, we found that cardenolides without a terminal glucose enter the cells by simple diffusion, whereas those containing the glucose tag (termed ‘primary glycosides‘) are taken up actively (kreis and reinhard, 1987). once accumulated, the primary glycosides were retained by the cells even if cyanide was added to the cell suspensions indicating that no energy was required to keep the primary glycosides stored. of course, the enzymes involved in cardenolide biosynthesis had to be identified and characterized (see chasing enzymes of snap formation in plant cell cultures). finally, a model featuring the mechanisms involved in cardenolide biotransformation and storage was proposed to help with the development of new biotransformation processes on a large scale (kreis et al., 1992; see growth of cell cultures in biorectors). chasing enzymes of snap formation in plant cell cultures to characterize enzymes and genes of a metabolic pathway the target enzyme needs to be purified, the protein sequenced and the appropriate nucleotide primers synthesized. this classical approach benefitted from some of the characteristics of plant cell cultures which can be cultivated under standardized conditions and manipulated in a multitude of ways. protein extraction and purification of snap forming enzymes, which may occur in low concentrations only, turned out to be very straightforward and efficient (zenk, 1991). for example, ajmalicine was the first alkaloid whose biosynthesis was elucidated at the enzyme level using catharanthus roseus (l.) g. don cell suspension cultures (zenk, 1980). much of the biosynthetic pathway leading to morphinane alkaloids was investigated using plant cell cultures (hagel and facchini, 2013). others and we isolated the enzymes involved in cardenolide biotransformation (kreis et al., 1998; see biotransformation of snaps by plant cell cultures) and also investigated early steps of the cardenolide pathway. for instance, we isolated the δ5-3β-hydroxysteroid de hydrogenase (dl3βhsd) from d. lanata tissues (seidel et al., 1990). this enzyme catalyzes the formation of pregnenolone from progesterone. a more recent discovery is that dl3βhsd, unlike other known mammalian enzymes, only has oxido-reductase activity but not δ5-δ4-ketosteroid-isomerase activity (meitinger et al., 2016). progesterone is further metabolized to 5β-pregnane-3,20-dione by the progesterone 5β-reductase (gärtner et al., 1994) which was proposed to be the first regulatory ’key‘ enzyme in the 5β-cardenolide pathway. however, later it was shown that the enzyme was active and the respective gene expressed in suspension-cultured cells (not capable of producing cardenolides) and cardenolide-free tissues of d. lanata (ernst et al., 2010). enzymes at the start of a biosynthetic pathway are sometimes described as ‘key’ enzymes, but they are not necessarily ratelimiting for production. the bottleneck(s) often occur further down the pathway. rate-limiting or pathway-pivotal enzymes do not necessarily have to catalyze spectacular reactions but may instead only adorn scaffolds with small structural elements. this was nicely demonstrated in berberine-producing thalictrum cell cultures. zenk’s group and later kutchan and co-workers (frenzel and zenk, 1990; frick and kutchan, 1999) tried to ascertain why some cell culture strains were producing berberine-type alkaloids while others failed. they found that most of the enzymes necessary for the formation of protoberberines were present in non-producing cells but the full set of all four methyltransferases involved in protoberberine biosynthesis was only expressed in producing cells. methyl groups attached to a scaffold by s-adenosyl-l-methionine220 w. kreis dependent methyltransferases can decide the destiny of a precursor. the precursor might not be funneled into the target pathway if the substitution pattern facilitates inappropriate condensation reactions due to the lack of protecting groups. meinhart zenk once stated that “the development of plant cell cultures for the study of the biosynthesis of secondary metabolites in the 1970s revolutionized the field. it became possible to identify, characterize and ultimately, in specific cases, to purify the biocatalysts involved in individual transformations. the precise knowledge of the biosynthetic pathways provided by the identification of these enzymes, of the stereoand substrate-specificity of the reactions they catalyze and of the sequence of these reactions in situ opened new fields for study in plant sciences” (zenk, 1991) and indeed, for more than 30 years plant cell cultures have become a central tool to study snap formation. it turned out that snap formation appears to utilize multidimensio nal grids rather than linear pathways and that various factors can control the flux through a biosynthetic grid. these factors are: (1) enzyme amount/activity, (2) metabolite inhibition/stimulation, (3) pathway competition, (4) co-factor availability, and (5) compartmentalization (verpoorte et al., 2000). potential flux limitations can be investigated by feeding different precursors to cultured plant tissues or cells. the question arose of how specialized snap metabolism actually is because the multitude of metabolites found in living organisms does not correspond with the small number of metabolic genes identified in the various genome studies (schwab, 2003). convergent, parallel, and divergent evolution can be seen in metabolic processes (pichersky and gang, 2000; delgoda and murray, 2017). gene duplication (ohno, 1970) and divergence to more specialized enzymes may be important for snap formation (ober, 2005). however, enzymes with a relaxed substrate specificity increase the possibility of producing a greater number of new chemicals (weng and noel, 2012; kreis and munkert, 2019). enzymes may compete for common intermediates. a particular enzyme may catalyze a series of parallel reactions involving structurally related substrates. there are still metabolic uncertainties in every step of a given pathway or in a metabolic grid. the flux to a certain compound is predominantly decided by grid architecture. at enzymatic level only a few pathways/grids have been studied extensively enough to allow for serious pathway engineering (see outlook). in most cases snap pathways are still far from being elucidated. as an example the biosynthesis of tropic acid and the ester alkaloid formation in scopolamine biosynthesis has been the subject of continued discussion and remains an unresolved issue (qiu et al., 2018). eventually snap formation research slipped into the genomic and post-genomic area. screening of cdna or genomic libraries and the use of pcr approaches made it possible to identify, isolate and clone genes involved in snap formation. it became feasible to express them heterologously in microorganisms with the aim to obtain enough protein for characterization and structure elucidation. we also contributed to this field by elucidating the crystal structures of two progesterone 5β-reductases (egerer-sieber et al., 2006; schmidt et al., 2018). during this phase plant cell cultures became nearly obsolete. this is underlined by the fact that about 10 years ago the dsmz (german collection of microorganisms and cell cultures) maintained more than 700 different plant cell lines from more than 80 different plant families (kreis, 2007). in april 2019, 41 plant cell lines were still provided as growing or cryopreserved cultures (dszm, 2019a) but now (dsmz, 2019b) the catalogue of strains does not offer plant cell cultures any longer. either way plant cell cultures with their limited potential and complexity turned out to be a good system for metabolome studies. the metabolome is defined as the final downstream product of the genome and consists of all metabolites in a cell, tissue or organism. a snap metabolome may be defined as a part of the metabolome related to a specific group of snaps. the ‘taxane metabolome’ of a taxus cell culture will hence comprise all taxanes, including taxa4,11-diene, formed by the initial action of a diterpene cyclase from geranyl-geranyl diphosphate and the highly complex paclitaxel that may be regarded as the end product of the taxane pathway. over 400 taxanes described so far are represented in this constrained metabolome (croteau and ketchum, 2006) but most of them are not part of the actual paclitaxel biosynthetic pathway(s). taxol bio synthesis requires 19 enzymatic steps, but so far only 15 enzymes have been characterized (mcelroy and jennewein, 2018). it involves eight oxidation steps, two coa esterifications, n-benzoylation, five acetyl/aroyl transferase steps, a c4β,c20-epoxidation reaction, and a phenylalanine aminomutase step. understanding the relationship among all intermediates in the metabolic grid may provide clues for metabolic engineering of plant cell cultures for increased taxol production. it has been stated that to increase taxol yields in taxus cell cultures these „numerous and apparently diversionary taxoid bio synthetic side-routes and dead-ends” have to be taken into account (croteau et al., 2006; see commercialization of plant cell culture processes). plant cell cultures have also been used to decipher multiple levels of cellular control of lignin deposition, composition, structure, and quantity. in this case, plant cell cultures have provided insights into the lignification processes that could not have been easily obtained by using whole plants (pesquet et al., 2019). growth of plant cell cultures in bioreactors already in the 1960s john d. bu’lock (see introduction) saw that biotechnology (at that time usually referred to as ‘fermentation technology’) would be the next big development to happen for the production of snaps. large scale cultivation of plant cells in bioreactors was the obvious next step (kreis and reinhard, 1989; scragg, 1995; sharma and shazad, 2013; eibl and eibl, 2008). in the 1970s pneumatically agitated air-lift bioreactors were advertised and routinely used for the cultivation of plant cell aggregates that were assumed to be highly shear-sensitive (verpoorte et al., 2002). today many different designs and configurations of bioreactors for plant in vitro systems are available. the most common type of reactor for growing plant cells is the stirred-tank bioreactor, a mechanically agitated vessel. this type of reactor uses impellers for gas-liquid transfer and mixing. though bioreactors are scalable, reproducing the physical environment (shear, mixing, gas transfer) is often difficult (e.g., scragg, 1991). big challenges in bioreactor design are the efficient illumination of phototrophic cell cultures (huang et al., 2017) and the use of disposable bioreactors (eibl and eibl, 2008). some engineering aspects will have to be addressed before citing examples of processes developed (see commercialization of plant cell culture processes). the ‘operating mode’ refers to how the nutrient and product streams are supplied to or removed from a culture. when all nutrients are supplied from the beginning the operating mode is termed (1) batch culture. in a (2) fed-batch operation one or more nutrients are added to the bioreactor during cultivation, either in batches or continuously. fed-batch mode is more suitable than standard batch cultivation if a nutrient or precursor is detrimental to the cells when supplied all at once. in (3) repeated fed-batch operation a substantial portion of the spent medium or the cell suspension is withdrawn and replaced with fresh culture medium. (4) two-stage batch mode involving a change of culture media is an option to allow for rapid growth in the first stage and product synthesis in a second stage. for example, a two-stage process for the production of deacetyllanatoside c was designed and it was demonstrated, that this process can be run in a semi-continuous mode (kreis and reinhard, 1990). (5) chemostat operation refers to a fermentation mode in which fresh medium is continuously fed to the bioreactor and a stream of cell suspension is continuously withdrawn for pro natural products in plant cell cultures 221 duct harvest or metabolite analysis. in (6) perfusion operation fresh medium is added to the vessel continuously and only spent medium is withdrawn. for example, berberine production using coptis japonica (thunb.) makino cells was improved by a factor of 6.5 when a continuous turbidostat set-up was used instead of the batch mode (matsubara et al., 1989). the perfusion reactor is also appropriate for immobilized producers. immobilization is a technique which traps catalytically active cells or enzymes in a matrix that prevents them from entering the liquid phase. plant cells immobilized in alginate or on porous glass beads have been used for the de novo biosynthesis of valuable snaps as well as for biotransformations (yeoman, 1987; ramachandra rao and ravishankar, 2002; brodelius, 2018). this issue will not be elaborated further here and the reader is referred to the respective reviews. commercialization of plant cell culture processes during the dynamic 1980s i had the opportunity to visit japan and some of the companies using plant cell cultures for the production of snaps already then. mitsui petrochemical industries were about to establish processes for vinca alkaloids, rubia dyes, berberine, and shikonin, while shiseido focused on an arbutin process using catharanthus roseus (l.) g. don cell cultures. nitto denko produced ginsenoside-rich biomass using ginseng root cultures and nippon paint and sanei chemical ran their own plant cell culture projects. optimization of berberine production from coptis japonica (thunb.) makino cells resulted in a yield of 3.5 g/l after a scale up to 4 m3 (matsubara et al., 1989). however, as recently pointed out by lange (2018) the introduction of plant tissue culture-derived fine chemicals to the market has been confined to few examples, including the 1988 ‘one-hit wonder‘ shikonin (fujita, 1988) and the ‘showpiece‘ paclitaxel. phyton catalytic (now phyton biotech) began the scale-up of cell suspension cultures of taxus chinensis (miq.) de laub. in the 1990s. after extensive studies, a large-scale culture process was developed where the addition of the ‘elicitor’ methyl jasmonate increased the productivity to 23.4 mg l-1 d-1 (ketchum et al., 1999). a production volume of 75 m3 was established and in 1995 the plant cell culture process was licensed to bristol-myers squibb. eventually several new tissue culture processes for cytostatic taxanes emerged (choi et al., 2002; mcelroy and jennewein, 2018). it seems appropriate to comment on the profitability of plant cell culture at this point. first of all there has to be a sufficiently large market for the product. the most important parameter for possible commercialization of a cell culture process is its productivity. there are many different definitions of productivity. we defined it as the amount of product formed per volume and time. this figure is dependent on growth and product formation rates. it takes into account unproductive phases and the time required for preparation and follow-ups of a production run. it was estimated that the wholesale kilo price must be between 250 u.s. $ and 8,000 u.s. $ for any plant cell culture process with a production rate between 0.025 to 1.0 g of product l-1 d-1 (kreis, 1993). lange (2018) addressed various con siderations for commercial targets like market value/volume, structural complexity/chemical synthesis, regulatory burden and consu mer acceptance. he also provided a list of possible new targets, yet avoiding compounds that have ’fallen out of favor among clinicians‘, and focused on structurally complex and expensive snaps not easily accessible from the source plants. commercialization is hindered when there is a complex system of players. for example the anti-malaria drug artemisinin was regarded a good target compound and lots of efforts have been made to establish plant cell cultures to study artimisinin biosynthesis and to use them as a source for this valuable compound. so far commercialization of alternative processes, including a very promising yeast process generating semi-synthetic artemisinin, has not been successful (peplow, 2016). this illustrates the complexities of economic forces that affect the market for certain drugs. they also had an adverse impact on the metildigoxin process established in ernst reinhard’s group in tübingen. the process was scaled-up to 5 m3 by boehringer mannheim (now roche) and was regarded economically feasible. but the market for cardiac glycosides deteriorated and the high-yield plants obtained by selection breeding yielded enough digoxin to sa tisfy the needs in the end. but what happened to the shikonin process? the demand for shikonin in 1988 was only around 150 kg per year with a market value of 600,000 u.s. $. it turned out that production was not significantly cheaper than the extraction from lithospermum erythrorhizon roots (lange, 2018) and hence the process abandoned. only recently, a bilberry cell culture process for the production of a polyphenol extract as a dietary supplement was announced by diana plant sciences. they had already launched a cocoa product derived from theobroma cacao l. suspension-cultured cells in 2013 (lange, 2018). eibl et al. (2018) gave an overview of plant cell culture extracts containing snaps such as polyphenols, vitamins, fatty acids, peptides for applications in the cosmetics and food industries. teoside 10, an extract prepared from suspension-cultured ajuga reptans cells was the first plant cell culture extract approved as a food supplement ingredient in europe. only time will tell whether this gives notice of a renaissance of plant cell culture or of a sudden hype prone to extinction. outlook although plant cell culture technologies have been around for decades, it still seems to be difficult to commercialize plant cell culture processes, mainly due to the competition with already existing ways of manufacturing. but without any doubt, plant cell suspension cultures left their mark on various aspects of applied botany rela ted to the elucidation of biochemical pathways and the production of snaps. in the genomic and post-genomic era plant cell cultures became less important tools to study biochemical pathways. new technologies emerged and homology search in genome and protein databases allowed for new strategies in pathway elucidation. even if not all genes and enzymes of a given snap pathway are known the snap itself may be produced after merging genes from different organisms in an appropriate host. however, each step in a given pathway is subject to uncertainties and there are many cases where the genetic engineering approach did not give the expected results. for example, the tryptophan decarboxylase gene introduced and constitutively expressed in catharanthus roseus (l.) g. don cells resulted in an increase of tryptamine, but not in an increase of indole alkaloid production (berlin and fecker, 2000). in recent years, microorga nisms were genetically engineered with a view to synthesize natural products usually derived from plants. among these engineered pathways a lot of different target structures can be found, such as phenols, isoprenoids, alkaloids, and polyketides (siddiqui et al., 2012). for example, a saccharomyces cerevisiae strain has been developed, which is capable of synthesizing hydrocortisone from a simple carbon source (dumas et al., 2006) and more recently, morphinanes have been produced in e. coli (nakagawa et al., 2016). pathway engineering seems to be a promising approach for the production of a multitude snaps. only recently, we have constructed a yeast expression plasmid containing a cardenolide biosynthetic module. it enables a yeast strain to produce 5β-pregnane-3β,21-diol-20-one, a central intermediate in 5β-cardenolide biosynthesis, starting from pregnenolone which we added to the culture medium. with this approach, five consecutive steps in cardenolide biosynthesis were realized in baker’s yeast (rieck et al., 2019). two of the enzymes 222 w. kreis used, namely δ5-3β-hydroxysteroid dehydrogenase and progesterone 5β-reductase were isolated for the first time in the 1980s from digitalis cell and tissue cultures demonstrating again the impact of plant cell culture for basic research and applied botany. references agravis, 2019: retrieved 25th june, 2019, from www.agravis.de/de/ueberagravis/newsroom/publikationen/infografiken/ ahloowalia, b.s., ahloowalia, b., maluszynski, m., 2001: induced mutations – a new paradigm in plant breeding. euphytica 118, 167. doi: 10.1023/a:1004162323428 alfermann, a.w., boy, h.m., döller, p.c., hagedorn, w., heins, m., wahl, j., reinhard, e., 1977: biotransformation of cardiac glycosides by plant cell cultures, 125-141. in: barz, w., reinhard, e., zenk, m.h. 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(eds.), cell culture and somatic cell genetics of plants, volume 4. san diego, usa: academic press. yue, w., ming, q., lin, b., rahman, k., zheng, c.-j., han, t., qin, l., 2016: medicinal plant cell suspension cultures: pharmaceutical applications and high-yielding strategies for the desired secondary metabolites. crit. rev. biotechnol. 36, 215-232. doi: 10.3109/07388551.2014.923986 zenk, m.h., 1980: enzymatic synthesis of ajmalicine and related indole alkaloids. j. nat. prod. 43, 438-451. zenk, m.h., 1991: chasing the enzymes of secondary metabolism: plant cell cultures as a pot of gold. phytochemistry 30, 3861-3863. doi: 10.1016/0031-9422(91)83424-j address of the author: wolfgang kreis, lehrstuhl für pharmazeutische biologie, staudtstraße 5, 91058 erlangen, germany e-mail: wolfgang.kreis@fau.de © the author(s) 2019. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1111/j.1567-1364.2011.00774.x http://dx.doi.org/10.1080/07388550601173918 http://dx.doi.org/10.1016/0031-9422(90)85155-9 http://dx.doi.org/10.1007/b13538 http://dx.doi.org/10.1007/bf00269273 http://dx.doi.org/10.1111/pbi.12428 http://dx.doi.org/10.1007/978-3-642-70717-9_28 http://dx.doi.org/10.1023/a:1015871916833 http://dx.doi.org/10.1101/sqb.2012.77.014787 http://dx.doi.org/10.1007/bf00270196 http://dx.doi.org/10.1007/bf00273877 http://dx.doi.org/10.3109/07388551.2014.923986 http://dx.doi.org/10.1016/0031-9422(91)83424-j mailto:wolfgang.kreis@fau.de journal of applied botany and food quality 93, 20 25 (2020), doi:10.5073/jabfq.2020.093.003 1institute of genetics and physiology, hebei academy of agriculture and forestry sciences, pr china 2plant genetic engineering center of hebei province, pr china 3college of chemistry and chemical engineering, xingtai university, pr china the involvement of phenolic metabolism in superficial scald development in ‘wujiuxiang’ pear shuo zhou1,2a, yunxiao feng1,2a, zhe zhao1,3, yudou cheng1,2, junfeng guan1,2 (submitted: august 16, 2019; accepted: december 6, 2019) * corresponding author a these authors contributed equally to the article. summary superficial scald often occurs after a long term of cold storage in apples and pears. in this study, the superficial scald index, the contents of major phenolic compounds, polyphenol oxidase (ppo) activity and its related genes expression in peel was investigated during cold storage period and at shelf life in ‘wujiuxiang’ pear (pyrus communis l. cv. wujiuxiang) with or without 1-mcp treatment. it showed that arbutin, chlorogenic acid, catechin and epi catechin were the main phenolic compounds in the peel, and 1-mcp treatment significantly inhibited scald development while altering the composition of phenolic compounds, inhibited ppo activity and the expression of phenylalanine ammonia ligase (pal1, pal2), cinnamate 4-hydroxylase (c4h1, c4h2) and ppo (ppo1, ppo5) and up-regulated the expression of hydroxycinnamoyl-coa shikimate/ quinate hydroycinnamoyltransferase (hct1), p-coumarate-3-hydroxylase (c3h) and ppo (ppo4 and ppo6) in the peel. these results suggested that the phenolic metabolism iss closely related to the scald development, and several genes related to phenolic metabolism were involved in scald development. keywords: superficial scald; 1-methylcyclopropene; polyphenol; polyphenol oxidase; gene expression introduction superficial scald, as a physiological disorder causing brown or black patches on fruit peel, often occurs after a long term of cold storage in apples and pears (lurie and watkins, 2012). the development of the scald was associated with α-farnesene metabolism (yazdani et al., 2011; zhou et al., 2017), and antioxidant system (gao et al., 2015; larrigaudière et al., 2016, 2019; li et al., 2016; sabban-amin et al., 2011; zhou et al., 2016), which could be dependent on ethylene action (feng et al., 2018; xie et al., 2016; zhi et al., 2018). however, a full understanding about the mechanism of scald development remains elusive. it has been showed that phenolic metabolism may be associated with the scald development (abbasi et al., 2008; busatto et al., 2018; busatto et al., 2014; lu et al., 2014; piretti et al., 1996). it is not only because most phenolic compounds have antioxidant activity which could resist against scald development (andrés-lacueva et al., 2010; lee et al., 2003), but also phenolic oxidation products catalyzed by polyphenol oxidase (ppo) most likely results in browning reaction during the development of scald symptom (mayer and harel, 1991; nishimura et al., 2003). in general, after a long term of cold storage, loss of membrane integrity allows the contacting of phenolic substrates with ppo, in which phenolic substrates could be catalyzed into oxidized forms by ppo, such as quinones, and then, quinones could react with thiol and amines groups, finally leading to the formation of brown or black pigments in peel (valentines et al., 2005). however, in pears few evidence confirmed the involvement of phenolic metabolism in scald development, and multiple ppo gene members existed which usually exhibited various expression patterns in the process of growth and development or in response to stress (thipyapong et al., 2007), which specific ppo members might play important role in scald development is unknown. it shows that chlorogenic acid may be main substrate for ppo-cata lyzed reaction during scald development (busatto et al., 2014). previous researches indicated that chlorogenic acid is synthesized via phenylpropanoid pathway, which is initiated from the deamination of phenylalanine to cinnamic acid by phenylalanine ammonia ligase (pal), followed by hydroxylation of cinnamic acid by cinnamate 4-hydroxylase (c4h) and methylation by 4-hydroxycinnamoylcoa ligase (4cl), respectively, to produce p-coumaric acid, and then enters hydroxycinnamoyl-coa shikimate/quinate hydroycinnamoyltransferase (hct/hqt) pathway, finally leading to chlorogenic acid synthesis, which was hydroxylated by p-coumarate-3-hydroxylase (c3h), (mahesh et al., 2007; niggeweg et al., 2004; zhao et al., 2013). however, the involvement and regulation of chlorogenic acid synthesis in scald development was not fully understood in pears. therefore, the objective of this study is to reveal the involvement and regulation of phenolic metabolism in scald development, and which genes may function in scald development in ‘wujiuxiang’ pear (pyrus communis l. cv. wujiuxiang). materials and methods materials and treatments ‘wujiuxiang’ pears were harvested in a commercial orchard in jinzhou county (hebei, china) at the commercial maturity stage (september 2, 2012). fruit were transported to the laboratory in 2 h. uniform fruit (average weight each fruit: 289.0 g) without any visual defects were selected and randomly divided into two lots of 150 fruit each. one lot was exposed to 1.0 μl/l 1-mcp (rohm and haas china inc., beijing) for 24 h at 25 ± 2 °c, and the other lot was exposed to air as control. after treatment, all fruit were stored at 0 °c. after 90 and 120 days of cold storage, fruit were removed for biochemical measurements and gene expression analysis. after 120 days of cold storage, all fruit left were transferred to 20 ± 2 °c for 7 days of shelf life. each treatment contained three replicates of 10 fruit each at each sampling time. scald index measurement the scald index was measured based on the percentage of the fruit surface area affected (zanella, 2003), where no scald = 0, <25% = 1, 25-50% = 2, and >50% = 3. the scald index = ∑ (score level × number of fruit at the level) / [3× (total number of fruit)]. phenolic compounds extraction and content analysis phenolic compound was determined as described by awad et al. (2000) with some modifications. a 2.0 g ground frozen peel tissue phenolic metabolism involved in superficial scald 21 were used to extract phenolic compound with 10 ml of 80% (v/v) methanol for 20 min under ultrasonic condition, and then centrifuged at 10,000 g for 10 min at 4 °c. a 1.0 ml aliquot of supernatant was first filtered through a c18-spe (bonna-agela technologies inc., tianjin, china) and then filtered via a 0.45 μm microporous membrane before being injected into the high performance liquid chromatograph (hplc, hitachi l-2000 system, hitachi, tokyo, japan), which consisted of a hitachi pump l-2130, a hitachi automatic sample injector l-2200, and a hitachi uv detector l-2400 equipped with a c18 column. the flow rate was maintained at 1 ml min-1 and the injection volume was 10 μl. the phenolic content was determined at wavelength of 280 nm. the integrated peaks were calculated by comparison with standard solutions of different known phenolic compounds. data are expressed in mg g-1 fw. extraction and assay of ppo activity the extraction and assay of ppo activity were performed as described by serradell et al. (2000). a 1.0 g ground frozen peel was homogenized in 3 ml of phosphate buffer (0.1 mol/l, ph 7.8) with 1% polyvinylpyrrolidone (pvp), and then centrifuged at 20,000 g for 15 min at 4 °c. the supernatant was collected as a crude ppo extract. the reaction mixture contained 0.1 mol/l catechol containing in 0.05 mol/l phosphate buffer (ph 6.0). changes in the absorbance at 410 nm were measured. one unit of ppo activity was defined as a change of 0.01 at 410 nm in the absorbance per min. rna isolation and quantitative rt-pcr analysis total rna was isolated by ctab (cetyltrimethylammonium bromide) (sangon biotech, shanghai, china) method (gasic et al., 2004). after isolation, 1.0 μg of total rna was reverse-transcribed into first-strand cdna using primescripttm rt reagent kit with gdna eraser (takara biomedicals). quantitative rt-pcr was performed using the tb green® premix ex taqtm (tli rnaseh plus) kit (takara biomedicals) with the 7500 real-time pcr system (applied biosystems, usa). the pcr primers were designed using primer premier 6.0. the gene name, genbank accession number, forward and reverse primers were shown in tab. 1. the qrt-pcr reaction was performed in a final volume of 20 μl, containing 10 μl of tb green® premix ex taqtm mix, 0.4 μl of rox ii dye, 0.4 μl of forward and reverse primer each, and cdna equivalent to 10 ng of rna. the reaction conditions were carried out as follows: 10 s at 95 °c, 40 cycles of 95 °c for 5 s and 60 °c for 34 s. the melting temperature of the amplification products was analyzed using a dissociation curve to confirm the specificity of amplification. all quantitative rt-pcr reactions were normalized using a ct value corresponding to the pcactin gene. the amplification efficiency of primers was between 95 and 105%, which was calculated by serial dilutions of cdna samples. the relative expression levels of target genes were calculated with the formula 2-δδct (livak and schmittgen, 2001), with samples harvested at day 0 used as a calibrator (assigned an arbitrary value of 1.0). statistical analysis all values are expressed as the means ± standard errors (se) of three replicates. the significance of the differences between means was calculated by student t test using spss software (version 19.0, spss inc., chicago, il, usa). differences were considered to be significant at p < 0.05. results scald development during cold storage and shelf life in the control fruit, scald symptom was appeared after 120 days of cold storage, and after 7 days of shelf life, scald developed more severely, while no scald was found in 1-mcp-treated fruit during all of cold storage and shelf life (fig. 1). tab. 1: primers for quantitative rt-pcr analysis. gene genbank no. forward (5’-3’) reverse (5’-3’) pcpal1 gu906268 gcaaagaggactttaacaactgg tactccctatcgacaactttaagc pcpal2 gu906269 ccgggaaataaccaaacc atggctccctttcattgc pcc4h1 xm_009376113 aacttcgagcttctgcctcc ccccaagcatcaatctacgc pcc4h2 xm_009356593 caagcacacgggctacaac gatcgaccacaacgtggttt pchct1 jq280303 ccccctccagtctgacca ccaatgaaaacacaaacacgtc pcc3h xm_009357051 ggtgcaacaaaaggctcaag ttagtggtgttggagggtgc pcppo1 hq729709 tccctactcacaaagcccaag gacctccaagaccaagaagca pcppo4 gu906265 aaggtgacaatgataacccagac tgccgcaccgtagagacc pcppo5 gu906266 accaaaataaaaacccttccac cagccactccaccatacagg pcppo6 gu906267 agaaggcggaacgagagga ggtctggctgggctgactt pcactin ab190176.1 gctgagagattccggtgccc ttgacccaccactgagcacg fig. 1: scald index in ‘wujiuxiang’ pear during cold storage and shelf life. the error bars represent se of the means. the asterisk “*” indicates a significant difference (p<0.05). the content of phenolic compounds during scald development the phenolic compounds, including arbutin, chlorogenic acid, gallic acid, catechin, epicatechin, coumaric acid, and caffeic acid, were detected in peel after 90, 120 days of cold storage and 7 days of shelf life. it showed that arbutin, chlorogenic acid, catechin, and epicatechin were the main phenolic compounds in the peel of ‘wujiux22 s. zhou, y. feng, z. zhao, y. cheng, j. guan iang’ pear. in general, the content of arbutin, chlorogenic acid, gallic acid, epicatechin, and caffeic acid were increased during cold storage and shelf life, while catechin and coumaric acid were decreased. for arbutin, chlorogenic acid, gallic acid, epicatechin, and caffeic acid, 1-mcp treatment could significantly inhibit the accumulation of these phenolic compounds, especially after 7 days of shelf life (fig. 2). ppo activity during scald development the ppo activity in peel showed no significant changes after 90 and 120 days of cold storage in both control and 1-mcp-treated fruit. however, it increased in control at shelf life, while it was still stable and significantly lower than control in 1-mcp-treated fruit (fig. 3). fig. 2: the content of phenolic compounds during cold storage and shelf life with and without 1-mcp treatment in ‘wujiuxiang’ pear: arbutin (a), chlorogenic acid (b), gallic acid (c), catechin (d), epicatechin (e), coumaric acid (f), and caffeic acid (g). the error bars represent se of the means. the asterisk “*” indicates a significant difference (p<0.05). the expression of genes associated with phenolic compounds synthesis during scald development in this study, the expression levels of phenolic compounds synthesis related genes, including pcpal1, pcpal2, pcc4h1, pcc4h2, pchct1, and pcc3h were detected during cold storage and shelf life with and without 1-mcp treatment in ‘wujiuxiang’ pear. the expression levels of pcpal1 and pcpal2 increased constantly during cold storage and shelf life in both control and 1-mcp-treated fruit, while 1-mcp treatment significantly inhibited their expression (fig. 4a, b). the expression of pcc4h1 was slightly increased after 90 days of cold storage, and then declined, while 1-mcp-treatment inhibited the expression except for day 120 (fig. 4c). however, no significant change was observed in the expression of pcc4h2 (fig. 4d). phenolic metabolism involved in superficial scald 23 fig. 3: ppo activities in peel of ‘wujiuxiang’ pear during cold storage and shelf life. the error bars represent se of the means. the asterisk “*” indicates a significant difference (p<0.05). fig. 4: expression levels of chlorogenic acid synthesis-related genes during cold storage and shelf life with and without 1-mcp treatment in ‘wujiuxiang’ pear: pcpal1 (a), pcpal2 (b), pcc4h1 (c), pcc4h2 (d), pchct1 (e), and pcc3h (f). the error bars represent se of the means. the asterisk “*” indicates a significant difference (p<0.05). the expression of pchct1 and pcc3h were slightly inhibited during cold storage in both control and 1-mcp-treated fruit, while it was increased to a higher level after 7 days of shelf life in 1-mcp-treated fruit (fig. 4e, f). the expression of ppo gene members during scald development the expression of pcppo1 and pcppo5 increased dramatically during cold storage and afterwards declined at shelf life in control, whereas they were significantly suppressed by 1-mcp treatment (fig. 5a, c). the expression levels of pcppo4 and pcppo6 were decreased during cold storage and shelf life in control, while 1-mcptreatment could up-regulate their expression. (fig. 5b, d). discussion superficial scald could result in severe fruit quality loss in apples and pears, and many postharvest methods have been established to inhibit scald development (lurie and watkins, 2012). in this study, 1-mcp treatment significantly inhibited scald development in ‘wujiuxiang’ pear (fig. 1), in good agreement with our previous reports (gao et al., 2015; zhou et al., 2016, 2017), confirming the crucial role of ethylene action during scald development in ‘wujiuxiang’ pear. phenolic compounds are particularly important in fruit, not only because they contribute to color and flavor, but also they could be antioxidant function in fruit, which may involve in multiple stress and senescence-related processes (andrés-lacueva et al., 2010). in this study, it was found that arbutin, chlorogenic acid, catechin, and epicatechin were main phenolic compounds in peel of ‘wujiuxiang’ pear (fig. 2). rich and varied phenolic compounds make ‘wujiu 24 s. zhou, y. feng, z. zhao, y. cheng, j. guan conclusion that after a long term cold storage, phenolic compounds released from the vacuole can get in contact with ppo enzyme and activate it, being oxidized which results in tissue browning (abbasi et al., 2008). the expression levels of pcppo1 and pcppo5 showed a significant up-regulation in control during cold storage, and were effectively inhibited by 1-mcp treatment (fig. 5a, c). it suggests that pcppo1 and pcppo5 may involve in browning reactions during scald development. in contrast, the expression levels of pcppo4 and pcppo6 were down-regulated in control, while they were up-regulated slightly in 1-mcp-treated fruit (fig. 5b, d), implying pcppo4 and pcppo6 may play positive role in cold acclimation and senescence process in fruits. our results are analogous to other reports, which showed mdppo was linked with scald development in apple (busatto et al., 2018; busatto et al., 2014; sabban-amin et al., 2011). however, only one ppo gene member was analyzed in their reports. our results indicate that different member of ppo genes may have different functions during scald development in pears. in summary, these results showed the involvement of phenolic metabolism in the scald development in pears, in which arbutin, chlorogenic acid, and epicatechin might be the main phenolic compounds, and pcpal1, pcpal2, pcppo1 and pcppo5 were the key genes related with scald development. meanwhile, 1-mcp inhibited the scald development by regulating expression of above phenolic metabolismrelated genes. acknowledgments this study was funded by the earmarked fund for modern pear industry technology research system of china (cars-29-20), the key r & d project of hebei province (19227111d), and the haafs agriculture science and technology innovation project (2019-2-01). conflict of interest no potential conflict of interest was reported by the authors. references abbasi, n., kushad, m., hafiz, i., maqbool, m., 2008: relationship of superficial scald related fruit maturity with polyphenoloxidase and sufig. 5: expression levels of ppo genes during cold storage and at shelf life with and without 1-mcp treatment in ‘wujiuxiang’ pear: pcppo1 (a), pcppo4 (b), pcppo5 (c), and pcppo6 (d). the error bars represent se of the means. the asterisk “*” indicates a significant difference (p<0.05). xiang’ pear more nutrient, however, they may also contribute to high scald incidence (busatto et al., 2014; piretti et al., 1996). some studies have suggested that the phenolic metabolism could be involved in scald development and could be altered by 1-mcp treatment in apples (abbasi et al., 2008; busatto et al., 2014; pesis et al., 2007). in this study, the content of arbutin, chlorogenic acid, and epicatechin increased constantly in control fruit during cold storage and shelf life (fig. 2a, b, e), while the scald symptom appeared and developed (fig. 1). it suggested the involvement of phenolic compounds in cell senescence after a long cold storage and providing 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http://dx.doi.org/10.1016/j.scienta.2016.12.025 http://dx.doi.org/10.5073/jabfq.2016.089.040 journal of applied botany and food quality 94, 182 191 (2021), doi:10.5073/jabfq.2021.094.022 1department of crop sciences, division quality of plant products, university of goettingen, goettingen, germany 2 institute for ecological chemistry, plant analysis and stored product protection, julius kühn-institute (jki), federal research centre for cultivated plants, quedlinburg, germany assessment of sensory profile and instrumental analyzed attributes influenced by different potassium fertilization levels in three tomato cultivars bashar daoud1*, marcel naumann1, detlef ulrich2, elke pawelzik1, inga smit1 (submitted: august 1, 2021; accepted: october 17, 2021) * corresponding author summary sensory properties are an essential quality aspect when the consumption of fresh tomato is under consideration. the flavor of tomato is defined as a combination of taste sensations (sweetness, sourness), aroma (volatile compounds), and texture (firmness, mealiness), some of which are proven to be affected by insufficient nutrient supply − especially potassium (k). this study intends to undertake a holistic assessment of the k fertilization effect on the flavor of tomato by connecting the use of sensorial and instrumental methods. an optimal k supply significantly increased the sensory descriptors sweetness, sourness, and aroma as well as the instrumental estimated color, firmness, total soluble solids (tss), titratable acids (ta), and dry matter (dm) in a cultivar-specific manner. the volatile organic compounds (vocs) were not significantly affected by k fertilization. the evaluation by the panelists confirmed the results of the instrumental analyses, by which an increment in the fruit quality with rising k supply could be detected. an optimal k supply of 3.66 g/plant could be suggested to increase tomato flavor in the cocktail cultivars studied: primavera and yellow submarine. cultivar effects should, therefore, be considered for defining the optimal k fertilizer dose that favors high tomato fruit quality and, hence, better flavor. keywords: solanum lycopersicum l., potassium, sensory evaluation, instrumental analyses, volatile organic compounds introduction the tomato is one of the most important vegetables in the world. in 2019, around 181 million tons of tomatoes were produced (faostat, 2021). the increasing annual demand for tomato can be attributed to its versatility and suitability for several dishes (adegbola et al., 2019), as well as its fruitfulness in nutrients like minerals and antioxidants (afzal et al., 2015). in the european union, 40% of tomatoes are consumed fresh and 60% are processed for different products (european, 2020). fresh fruits can be described by their extrinsic (e.g. color, shape, and firmness) as well as the intrinsic cha racteristics (e.g. taste and aroma) (oltman et al., 2014). the flavor is a complex attribute and derived from the interaction between the volatile compounds, such as hexanal and 2-isobutylthiazole, and nonvolatile components like sugar, acids, and minerals (beckles, 2012). the flavor of tomato is frequently described as a sweet-sour taste accompanying special aromatic aspects like ‘fruity’ and ‘floral’ (baldwin et al., 2008). however, consumers have often complained about the poor flavor of fresh tomato (tieman et al., 2012). therefore, the flavor of the tomato needs to be comprehensively considered, and not only for the consumers, but also for the producers (piombino et al., 2013). moreover, the extrinsic and intrinsic characteristics of the flavor are remarkably influenced by many factors like weather conditions and the nutrient status of the plant and soil (mattheis and fellman, 1999). this study focused on the effect of potassium (k) nutrition on the flavor of tomato. being an essential macronutrient, k is involved in many physiological and biochemical processes in plants (hawkesford et al., 2012). cellular k plays a role in catalyzing many enzymes, in addition to having major functions in osmotic pressure adjustment (zörb et al., 2014). furthermore, sufficient k nutrition reinforces the resistance of the plants against biotic stresses like diseases and insects (bidari and hebsur, 2011). k is also involved in the relocation of photosynthetic assimilates to sink organs, resulting in an increment in the sugar content in the cytosol (lester et al., 2006). consequently, increasing the k fertilizer dose has demonstrated a positive enhancement on total soluble solids (tss), titratable acids (ta) (sonntag et al., 2019), dry matter (dm) (javaria et al., 2012), and firmness (lester et al., 2010). serio et al. (2007) and taber et al. (2008) could state a significant influence of k supply on the lycopene content and, hence, on skin color. the positive effect of k fertilization on increasing yields and fruit quality has been pointed out by many studies (afzal et al., 2015; amjad et al., 2014). volatile organic compounds (vocs) have been considered as sensory indications for flavor preferences (goff and klee, 2006). though around 400 volatile compounds have been detected in tomatoes, only 15-20 compounds, such as hexanal, 2-isobutylthiazole, and 6-methyl5-hepten-2-one, have been found to characterize the flavor of the tomato (kanski et al., 2020). most volatile compounds are derived from essential nutrient precursors like amino acids, carotenoids, and fatty acids (rambla et al., 2014). flavor can be measured by instrumental analyses, e.g. tss, ta, and color, and by sensory evaluation. several studies investigated the interaction between sensory evaluation and instrumental analyses in tomatoes (kanski et al., 2020; tandon et al., 2003). to the best of our knowledge, very few studies (afzal et al., 2015; wang et al., 2009) have been measuring the effect of k nutrition on the instrumental and sensory characteristics and their interactions in tomato. our work attempts to investigate the effect of k fertilization on sensory and physicochemical traits. it also aims to verify whether the results obtained by instrumental methods can be confirmed by the human senses. we hypothesize that: (i) increasing the k supply modifies the values of instrumental analyzed traits and the intensity of sensory quality; (ii) the effect of k fertilization on the sensory quality can be recognized by human senses; (iii) instrumental analyzed traits will distinctly correlate with sensory quality; and (iv) the effect of k fertilization will be cultivar-dependent. materials and methods experimental set-up in summer 2016, an outdoor experiment was conducted with three tomato cultivars. two cocktail tomato cultivars − primavera and yellow submarine (kiepenkerl, everswinkel, germany) − and one potassium influences sensory and instrumental analyzed traits in tomato fruits 183 salad tomato cultivar − lyterno f1 (rijk zwaan, de lier, netherlands) − were chosen. the cocktail cultivars were used in previous experiments and showed a good response to k fertilization (sonntag et al., 2019). the salad cultivar was chosen based on the breeders’ description highlighting this cultivar as being high in lycopene. therefore, it was expected that lyterno f1 would respond well to varying k supply as regards its color, which has been shown for high lycopene cultivars by taber et al. (2008) and serio et al. (2007). all cultivars were sown on march 30 in planting trays with capa cities of 0.1 l. after three weeks, the seedlings were transplanted into 11 cm pots with capacities of 1 l in a greenhouse. greenhouse conditions comprised 16 hours of daylight, with a mean temperature of 22 °c during the day and 18 °c at night. the soil in the trays and pots was a mixed peat (‘a 400’, stender, schermbeck, germany). the final transplantation to the outdoor location took place after seven weeks of sowing on may 25. the seedlings were planted into 20 cm mitscherlich vessels with capacities of 6.2 l filled with peat substrate (gartentorf, naturana, vechta, germany). three different concen trations of k − k1 low with 0.5 g k/plant; k2 medium with 2.19 g k/ plant; and k3 optimal with 3.66 g k/plant − in the form of liquid k2so4 were applied weekly during the growing season. nitrogen (n) was applied on a weekly basis along with k − as a mixture of nh4no3 and ca(no3)2 · 3h2o − for k3 treatment and every two weeks for k1 and k2 treatments. another n solution − (nh4)2so4 − was applied for k1 and k2 treatments, alternating with the previous mixture every two weeks to balance the sulfate supply. other plant macroand micronutrients were applied at the final transplantation and two more times during the season (tab. a1). the plants were irrigated with distilled water when required and were pruned to one shoot weekly. they were arranged in a randomized design, with four blocks representing four replicates per cultivar and k level. during harvest, the fruits of each sample were split into three subsamples. one sample set was used for the sensory evaluation by the panelists; the second subsample was used for extraction of vocs; and the third for instrumental analyses. the number of fruits used for each type of quality analysis is given in the table a2. instrumental analyses the k concentration, color, firmness, ta, tss, dm, and volatile compounds were estimated at fruit maturity. based on the method of koch et al. (2019), the k concentration was determined by digesting 100 mg fine powder of lyophilized tomato fruits in 4 ml of 65% nitric acid and 2 ml of 30% hydrogen peroxide for 75 min at 200 °c and 40 bar in a microwave (ethos terminal 660, milestone, sorisole, italy). subsequently, the samples were analyzed using inductively coupled plasma-optical emission spectrometry (icp-oes; vista-rl icp-oes, varian, palo alto, usa). fruit color was determined by minolta chroma meter cr-400 (konica minolta optics, japan) at the two equatorial sides of each fruit in the lab modus, where the a value represents the red color intensity of lyterno and primavera fruits, while the b value represents the yellow color intensity of yellow submarine fruits. afterwards, the firmness was estimated by a penetration test (5 mm staple micro cylinder, speed: 6 mm/s, distance: 6 mm) on the equatorial side of these fruits with a texture analyzer (ta.xt2, stable micro system, surrey, uk). tss, ta, and dm were estimated for the same fruits. the fruits were mixed for two minutes with a kitchen blender (mq 5000 soup, braun, neu-isenburg, germany) to achieve a homogenized puree. an amount of 10 g of this puree was dried for estimating dm, and the rest of the puree was centrifuged for 20 minutes at room temperature and at 5000 g (centrifuge 5804 r, eppendorf, hamburg, germany) to estimate tss and ta based on sonntag et al. (2019). immediately after harvest, vocs were extracted from fresh fruits, as described by ulrich and olbricht (2013). the fruits were rinsed with distilled water, cut into quarters, and homogenized in a solution with 20% (m/v) nacl by a kitchen blender (mq 5000 soup, braun, neu-isenburg, germany). the homogenate was centrifuged for 30 minutes at 4 °c and 3000 g (centrifuge 5804 r, eppendorf, hamburg, germany). to 8 ml of the supernatant and 4 g of nacl, 16 μl of the internal standard (5 μl octanol + 10 ml ethanol) were added. the samples were vortexed and stored at -20 °c until analysis by gas chromatography − fid, as previously described by ulrich and olbricht (2013). sensory evaluation a group of 12 panelists had been trained weekly over two months, resulting in eight training sessions in accordance with the iso 13299 (2016) sensory analysis guidance (iso 8586, 2014), by focusing especially on the quantitative descriptive analysis of the type of tomato fruits used in this study. the panel performance was checked after each training session and the result was used for improving the training. the sensory descriptors color and odor intensity, juiciness, sweetness, sourness, bitterness, skin strength, aroma, and aftertaste were elaborated with the sensory panel (table a3). the scale from 0% (minimum intensity) to 100% (maximum intensity) was used to determine the intensity of all descriptors that were studied. the final sensory evaluation was performed during the second week of august on fully ripe fruits for three consecutive days, with a single cultivar being evaluated each day with respect to sensory fatigue of the panelists. the experimental set-up of the outdoor trial was retained during the sensory evaluation. the replicate samples deriving from the four blocks were evaluated separately by the sensory panel. overall, each panelist evaluated 12 samples that derived from four field plots multiplied by three fertilization levels. the samples were provided to the panel in a randomized design generated by the eyequestion software. the evaluation was accomplished in a sensory laboratory that provided separated booths, in accordance with iso 8589 (2007). the fruits of cocktail cultivars were cut into halves, while those of the salad cultivar were cut into quarters immediately before being served in transparent plates that were coded with threedigit numbers. the panelists performed the evaluation and the data were acquired digitally using the eyequestion software. between the served samples, the panelists were directed to consume a piece of bread and tap water to naturalize the basic tastes. statistical analyses statistical analyses were performed mainly by using the spss software, version 22 (ibm corporation, new york, united states). data were proven to be normally distributed with the shapiro-wilk test (p<0.05), and the variance homogeneity was verified with welch’s test. general fertilizer effects were tested at the significance level of p<0.05 with one-way anova before separating the means of each fertilization treatment within the cultivars by using tukey’s post-hoc test. in order to connect sensory and physicochemical traits, pearson’s correlation analysis was calculated with spss and a principal component analysis (pca) was calculated with the statistica software, version 13.3 (tibco statistica, tulsa, united states). the panel performance was calculated by a 2-way anova with assessor and sample as main effects with the software panelcheck v1.4.0. results fruits’ k concentration the fruits’ k concentration was significantly influenced by the fertilization level (tab. 1). as anticipated, the level k1 significantly displayed the lowest values. the supply of the fertilizer level k3 could 184 b. daoud, m. naumann, d. ulrich, e. pawelzik, i. smit only significantly raise the k concentrations in the two cocktail tomato cultivars compared to level k2. instrumental and sensory determined color instrumental analyzed color values increased significantly with rising k fertilization only in the cocktail cultivars, where the color-b value (yellow) of yellow submarine fruits and the color-a value (red) of primavera fruits were more intense in k3 (tab. 1). based on the panelists’ evaluation, color intensity was increased significantly only in primavera (tab. 3). consequently, the color values affected by k fertilizations depended remarkably on the cultivar. the principal component analysis (pca) in three cultivars confirmed the anova results. in the pca, color intensity and instrumental determined color were located closely to each other in primavera (fig. 2) but distanced from each other in lyterno f1 (fig. 1) and yellow submarine (fig. 3). additionally, the correlations of color intensity with the instrumental determined color were low and nonsignificant: color-a (r = 0.23) and color-b (r = 0.45) (tab. a5). volatile organic compounds and odor intensity around 16 known volatile organic compounds (vocs) were distinguished in this study and they comprised around 80% of all the detected vocs (tab. 2). most of them were not influenced significantly by k fertilization, while the main variations were related to a tab. 1: mean values and standard deviation of taste-related attributes calculated for each k level (n=4) within the three cultivars lyterno f1, primavera, and yellow submarine. tss: total soluble solids, ta: titratable acids. color-a: estimated for lyterno f1 and primavera. color-b: determined for yellow submarine. n.d. not determined. letters indicate significant differences at p<0.05 between the k treatments. k1 low 0.5; k2 medium 2.19; and k3 optimal 3.66 g/plant. instrumental lyterno f1 primavera yellow submarine analyzed attributes k1 k2 k3 k1 k2 k3 k1 k2 k3 k-content (%) 1.42b ± 0.11 2.48a ± 0.12 2.44a ± 0.17 1.21c ± 0.07 2.34b ± 0.07 2.66a ± 0.15 1.60c ± 0.11 2.29b ± 0.07 2.54a ± 0.15 color-a 21.09a ± 0.92 20.32a ± 0.7 20.77a ± 0.71 12.16b ± 0.45 16.81a ± 1.72 17.67a ± 1.25 n.d. n.d. n.d. color-b n.d. n.d. n.d. n.d. n.d. n.d. 44.82b ± 2.71 49.48a ± 1.33 50.24a ± 1.94 firmness 1.21b ± 0.34 1.59ab ± 0.08 1.79a ± 0.32 0.69a ± 0.03 0.82a ± 0.09 0.70a ± 0.11 0.75b ± 0.01 0.95a ± 0.10 1.03a ± 0.14 (kg/cm) tss (%) 5.80b ± 0.43 6.45ab ± 0.44 7.30a ± 0.62 6.75b ± 0.3 8.45a ± 0.44 8.52a ± 0.19 8.25b ± 0.01 9.05b ± 0.01 10.45a ± 0.00 ta (%) 0.26c ± 0.03 0.48b ± 0.03 0.53a ± 0.01 0.25b ± 0.02 0.48a ± 0.04 0.51a ± 0.02 0.34c ± 0.02 0.51b ± 0.08 0.65a ± 0.08 dm (%) 7.76a ± 1.18 7.94a ± 0.73 8.99a ± 0.64 8.35b ± 0.42 9.92a ± 0.3 9.95a ± 0.61 10.18b ± 0.66 10.93b ± 0.76 12.44a ± 0.26 tab. 2: mean values and standard deviation of identified and unknown vocs calculated for each k level (n=4) within the three cultivars lyterno f1, primavera, and yellow submarine. values below the limit of detection (lod) were indicated. letters indicate significant differences at p<0.05 between the k treatments. vocs lyterno f1 primavera yellow submarine identified (%) k1 k2 k3 k1 k2 k3 k1 k2 k3 hexanal 32.82a ± 4.93 19.86b ± 6.19 19.46b ± 2.47 40.29a ± 7.47 40.47a ± 1.55 39.24a ± 1.72 27.27a ± 2.64 26.72a ± 2.45 27.38a ± 1.28 (e)-2-hexenal 4.22a ± 0.81 3.65ab ± 0.32 3.01b ± 0.39 8.08a ± 3.73 6.67a ± 1.85 7.01a ± 1.47 9.12a ± 2.73 10.76a ± 3.01 9.89a ± 3.08 octanal 5.26a ± 0.82 4.04ab ± 0.68 3.96b ± 0.35 4.48a ± 1.11 3.64a ± 0.31 4.14a ± 1.89 1.35a ± 0.96 1.73a ± 0.21 1.59a ± 0.12 β-ionone 1.06a ± 0.23 0.36b ± 0.42 0.16b ± 0.33 1.98a ± 0.68 1.73a ± 0.23 1.83a ± 0.73 <lod <lod <lod β-cyclocitral 0.67a ± 0.45 0.15a ± 0.3 0.20a ± 0.4 1.91a ± 0.59 1.52a ± 0.27 1.05a ± 0.7 <lod <lod <lod (z)-3-hexen0.42a ± 0.51 0.18a ± 0.36 0.61a ± 0.43 3.04a ± 0.28 2.41a ± 0.39 2.63a ± 0.57 0.38a ± 0.47 0.57a ± 0.66 0.81a ± 0.71 1-ol linalool 0.67a ± 0.45 0.77a ± 0.55 0.57a ± 0.75 0.41a ± 0.47 0.27a ± 0.32 0.25a ± 0.29 1.23a ± 0.42 1.67a ± 0.84 1.46a ± 0.6 2-isobutyl17.11a ± 3.49 27.28a ± 7.23 27.77a ± 7.61 11.57a ± 1.59 11.49a ± 1.53 11.30a ± 2.25 24.43a ± 3.21 22.64a ± 4.97 23.58a ± 4.01 thiazole eugenol 0.48a ± 0.39 0.47a ± 0.4 0.77a ± 0.47 1.18a ± 0.94 0.77a ± 0.82 0.88a ± 1.07 0.22a ± 0.25 0.38a ± 0.25 0.36a ± 0.04 1-hexanol <lod <lod <lod 1.7a ± 0.28 1.63a ± 0.39 1.34a ± 0.97 0.19a ± 0.39 0.43a ± 0.51 0.51a ± 0.44 β-damascenone 0.31a ± 0.36 0.86a ± 0.77 0.66a ± 1.11 0.86a ± 1.05 0.64a ± 0.81 0.68a ± 0.78 0.75a ± 0.67 1.50a ± 1.09 1.71a ± 1.17 (e)-geranyl10.19a ± 1.99 8.05a ± 2.2 6.98a ± 1.29 4.58a ± 1.83 6.32a ± 1.69 5.94a ± 0.75 <lod <lod <lod acetone 6-methyl-519.93a ± 4.22 28.05a ± 4.62 28.71a ± 5.83 14.39a ± 5.3 17.67a ± 1.58 16.72a ± 5.78 27.59a ± 2.93 23.84a ± 3.69 23.25a ± 2.84 hepten-2-one benzaldehyde 2.84a ± 0.79 1.82a ± 0.59 2.51a ± 1.01 3.07a ± 0.85 1.99a ± 0.64 4.29a ± 5.21 6.62a ± 6.52 7.37a ± 5.54 7.00a ± 2.18 citral 3.81a ± 1.39 3.20a ± 1.17 3.09a ± 1.29 2.25a ± 1.14 2.83a ± 0.61 2.29a ± 0.93 <lod <lod <lod methylsalicylate <lod <lod <lod <lod <lod <lod 0.54a ± 0.36 0.80a ± 0.62 1.07a ± 0.34 unknown (%) 18.98a ± 1.88 17.45a ± 3.05 15.55a ± 3.22 22.91a ± 3.73 20.38a ± 4.71 22.26a ± 12.27 23.29a ± 7.55 22.44a ± 7.46 17.85a ± 1.66 potassium influences sensory and instrumental analyzed traits in tomato fruits 185 tab. 3: mean values and standard deviation of the sensory evaluation calculated for each k level (n=4) within the three cultivars lyterno f1, primavera, and yellow submarine. 0 % refers to minimum intensity and 100 % to maximum intensity. letters indicate significant differences at p<0.05 between the k treatments. k1 low 0.5; k2 medium 2.19; and k3 optimal 3.66 g/plant. sensory lyterno f1 primavera yellow submarine descriptors (%) k1 k2 k3 k1 k2 k3 k1 k2 k3 color intensity 63.5a ± 12.1 64.2a ± 12.2 64.8a ± 12.7 55.9b ± 12.1 73.2a ± 12.9 73.3a ± 15.3 58.9a ± 11.7 60.5a ± 11.4 58.4a ± 8.4 odor intensity 39.0a ± 15.5 43.1a ± 17.5 42.6a ± 18.3 49.2a ± 14.6 53.3a ± 13.5 52.5a ± 10.9 52.0a ± 18.9 53.7a ± 16.4 53.8a ± 14.8 juiciness 67.5a ± 13.9 66.1a ± 15.2 64.7a ± 16.11 78.8a ± 14.1 78.2a ± 14.1 77.5a ± 12.9 72.1a ± 14.5 75.0a ± 14.4 74.7a ± 12.9 skin strength 56.8a ± 15.7 59.3a ± 14.7 62.4a ± 18.5 58.9a ± 12.5 56.9ab ± 12.5 51.6b ± 10.4 56.7b ± 13.0 55.5b ± 13.1 65.1a ± 12.6 sweetness 15.1a ± 12.9 13.6a ± 11.7 12.5a ± 8.1 33.9b ± 15.5 40.0ab ± 15.6 43.7a ± 16.9 50.7b ± 17.1 57.2ab ± 15.7 59.7a ± 13.4 sourness 17.6a ± 12.5 20.4a ± 14.4 21.9a ± 13.1 16.3b ± 12.3 25.2a ± 11.8 28.4a ± 15.3 20.3b ± 13.0 29.3a ± 14.1 35.3a ± 13.8 bitterness 8.2a ± 9.7 7.1a ± 8.1 8.8a ± 11.3 10.1a ± 12.8 7.0a ± 8.7 6.3a ± 6.1 9.87a ± 11.5 9.06a ± 9.7 12.35a ± 14.7 aroma 32.7a ± 18.3 32.0a ± 17.5 37.6a ± 17.3 36.6b ± 14.4 52.9a ± 14.6 56.2a ± 14.2 46.01b ± 17.8 53.7ab ± 18.8 57.4a ± 17.2 aftertaste 31.7a ± 14.0 35.7a ± 15.1 36.4a ± 15.5 39.0a ± 16.8 42.8a ± 14.0 44.3a ± 13.5 43.2a ± 14.4 46.7a ± 13.1 49.3a ± 14.4 fig. 1: principal component analysis (pca) of the sensory evaluation (green), metric data (red), and vocs (blue) for mature fruits of cv. lyterno f1 with k supply as an independent variable. k1: 0.5, k2: 2.19, and k3: 3.66 g/plant weekly k dose, ta: titratable acidity, tss: total soluble solids. color intensity: estimated by the panelists. color-a: measured by minolta chroma-meter. cultivar effect. for instance, in lyterno f1, hexanal, (e)-2-hexanal, octanal, and ß-ionone decreased significantly with rising k supply, while these compounds exhibited no alterations as regards k levels in primavera and yellow submarine. in addition, some of the detected vocs were only found in red-colored cultivars, e.g. ß-ionone, ß-cyclocitral, and (e)-geranylacetone, or only in yellow-colored cultivars like methylsalicylate (tab. 2). odor intensity was not affected by k application along the cultivars that were studied. these observations could be visualized with the previously mentioned pca plots (fig. 1, 2, and 3), in which the odor intensity and most of the vocs were less related to k3. interestingly, odor intensity correlated in a significantly positive manner with only a few compounds − in particular, ß-ionone (r = 0.57**), (e)-2-hexenal (r = 0.64**), and benz aldehyde (r = 0.40**) (tab. a5). texture parameters similar to the vocs, the cultivar background influenced the textural parameters analyzed by instruments or evaluated by the panelists. the firmness determined by a texture analyzer increased significantly only in lyterno f1 and yellow submarine with rising k levels (tab. 1). in terms of the sensory descriptors, skin strength increased significantly in yellow submarine, while a significant reduction in primavera was found (tab. 3). on the other hand, juiciness did not exhibit any significant alterations with k fertilization (tab. 3). in fig. 1 and 3 of the pca plot, the firmness and skin strength exhibited a significant increase with k application and were associated closely with k3. overall, the firmness correlated in a positively significant manner with skin strength (r = 0.42**), while juiciness did not significantly correlate either with firmness or with skin strength (tab. a5). relationship between instrumental analyses and taste attributes tss and ta positively increased with rising k fertilization in the three cultivars (tab. 1). dm was significantly rising in the cocktail cultivars, while in lyterno f1 only a positive trend was observed (tab. 1). from the panelists’ perspective, sweetness and sourness ß-damascenone dm color intensity -1,2 -1,0 -0,8 -0,6 -0,4 -0,2 0,0 0,2 0,4 0,6 0,8 1,0 1,2 factor 1: 43.91 % -1,2 -1,0 -0,8 -0,6 -0,4 -0,2 0,0 0,2 0,4 0,6 0,8 1,0 1,2 f a ct ro 2 : 1 5 .5 2 % hexanal (e)-2-hexenal octanal 6-me-5-hepten-2-one (z)-3-hexen1-ol 2-isobutylthiazole benzaldehyde linalool ß-cyclocitral citral (e)-geranylacetone ß-ionone eugenol odor intensity juiciness sweetnesssourness bitterness aroma skin strength aftertaste firmness t ss t a k-content k1 k2 k3f ac to r 186 b. daoud, m. naumann, d. ulrich, e. pawelzik, i. smit increased significantly with higher k levels, though only in the cocktail cultivars (tab. 3). apparently, the cultivar effect was evident in the instrumental and sensory determined taste attributes. for instance, in lyterno f1, tss, ta, sourness, and dm grouped with optimal k3, while sweetness was decreased with rising k dose; consequently, it dissociated from k3 and approached the low k1 (fig. 1). sweetness correlated significantly positively with tss (r = 0.81**); likewise, sourness with ta (r = 0.76**) and dm correlated in a significantly positive manner as well with tss (r = 0.95**), ta (r = 0.63**), sweetness (r = 0.79**), and sourness (r = 0.63**) (tab. a5). retronasal attributes (aroma) the aroma of the fruits was finally estimated by the panelists at the end of the sensory evaluation represented by aftertaste and aroma for red-colored cultivars − primavera and lyterno f1 − and for yellow hexanal citral (e)-geranylacetone firmness -1,2 -1,0 -0,8 -0,6 -0,4 -0,2 0,0 0,2 0,4 0,6 0,8 1,0 1,2 factor 1: 28.93 % -1,2 -1,0 -0,8 -0,6 -0,4 -0,2 0,0 0,2 0,4 0,6 0,8 1,0 1,2 f a ct o r 2 : 2 3 .4 5 % (e)-2-hexenal octanal 6-methyl-5-hepten-2-one 1-hexanol (z)-3-hexen1-ol 2-isobutylthiazole benzaldehyde linalool ß-cyclocitral ß-damascenone ß-ionone eugenol color intensity odor intensity juiciness sweetness sourness bitterness aroma skin strength aftertaste color a t sst a dm k-content k1 k2 k3 -1,2 -1,0 -0,8 -0,6 -0,4 -0,2 0,0 0,2 0,4 0,6 0,8 1,0 1,2 factor 1: 31.13 % -1,0 -0,8 -0,6 -0,4 -0,2 0,0 0,2 0,4 0,6 0,8 1,0 f a ct o r 2 : 2 2 .3 1 % hexanal (e)-2-hexenal octanal 6-methyl-5-hepten-2-one (z)-3-hexen1-ol 2-isobutylthiazole benzaldehyde linalool methylsalicylate ß-damascenone eugenol color intensity odor intensity juiciness sweetness sourness bitterness aroma skin strength aftertaste color b firmness t ss t a dm k-content k1 k2 k3 fig. 3: principal component analysis (pca) of the sensory evaluation (green), metric data (red), and the vocs (blue) for mature fruits of cv. yellow submarine with k fertilization as an independent variable. k1: 0.5, k2: 2.19, and k3: 3.66 g plant-1 weekly potassium fertilization dose, ta: titratable acidity, tss: total soluble solids. color intensity: estimated by the panelists. color – b: measured by minolta chroma-meter. fig. 2: principal component analysis (pca) of the sensory evaluation (green), metric data (red), and vocs (blue) for mature fruits of cv. primavera with k fertilization as an independent variable. k1: 0.5, k2: 2.19, and k3: 3.66 g plant-1 weekly potassium fertilization dose, ta: titratable acidity, tss: total soluble solids. color intensity: estimated by the panelists. color – a: measured by minolta chroma-meter. potassium influences sensory and instrumental analyzed traits in tomato fruits 187 submarine as well. application of k increased aftertaste in the stu died cultivars though not significantly (tab. 3). aroma increased with rising k supply; significantly in primavera and yellow sub marine and not significantly in lyterno f1. these observations were visually acknowledged by pca plots (fig. 1, 2, and 3). aftertaste was associated with k3 in all cultivars. in the same way, aroma (fig. 1, 2, and 3) were linked to k3, confirming the anova results. correlations among aroma, and the instrumental attributes as well as the voc’s were identified. aroma associated in a significantly positive manner with tss (r = 0.83**), ta (r = 0.51**), dm (r = 0.74**), sweetness (r = 0.82**), sourness (r = 0.67**), odor intensity (r = 0.76**), hexanal (r = 0.44*), and (e)-2-hexenal (r = 0.48**). discussion in the present study, the effects of different k applications on the instrumental as well as the sensory descriptors on three different cultivars were investigated. in all cultivars, increasing k fertilization to the optimal level significantly ameliorated the k concentrations in the fruits (tab. 1). this confirmed our outcomes in the previous research (daoud et al., 2020), in which the fruit’s content of k and the yield of the cocktail tomatoes used in this study were significantly increased by k fertilization. as a major macronutrient, the plants manage to maintain k concentrations in a specific range even under deficient k conditions (zörb et al., 2014). a constant limitation of k nutrition, however, leads to a decrease in k concentrations; in contrast, sufficient k application increases k concentrations (sonntag et al., 2019; zörb et al., 2014), which was confirmed by our results as well (tab. 1). effect of k fertilization on instrumental and sensory determined color the color is the most important external property for the evaluation of tomato fruits (lópez camelo and gómez, 2004). in our study, a positive significant effect of k fertilization was exhibited (tab. 1 and 3) and compatible results were found between the red color intensity and the instrumental analyzed red color measurement in primavera. in lyterno f1 as well, the panelists confirmed the instrumental analyzed color measurement, in which no significant effect of k supply was revealed. fertilization of k has a positive effect on the color intensity, as has been demonstrated by several researches (e.g. afzal et al., 2015; sonntag et al., 2019). arias et al. (2000) demonstrated high significant correlations between carotenoids content in tomato and color-a and color-b, such that in this context, a positive increment of k fertilization on lycopene and phytoene in tomato was confirmed (taber et al., 2008). in yellow submarine, the sensory analysis of color intensity showed no significant effect of k, which contradicted the instrumental analyzed color evaluation. yellow tomatoes are not as widely common as the red ones, and one can presume that the panelists in this matter were not able to differentiate between the yellow color intensity among k fertilization levels, because of their slight experience of yellow tomato consumption. in line with this, the assessor effect on results of sensory descriptor color was proven to be significant for all cultivars (tab. a4), indicating a higher variation between the panelists’ evaluation compared to the samples derived of different k fertilizer levels. all in all, the instrumental and the sensory color attribute affected by k fertilization was cultivar-dependent, as the k fertilization sig nificantly increased the instrumental analyzed color in the cocktail cultivars, while in the salad cultivar, no significant effect was detected. accordingly, several studies pointed out the remarkable cultivar effect on color values under k application (javaria et al., 2012; sonntag et al., 2019). the instrumental analyzed color did not correlate with the color intensity of sensory results and also csambalik et al. (2014) did not find that as well in their study on cherry tomatoes. effect of k fertilization on volatile organic compounds and sensory determined aroma the vocs were analyzed by gc-fid to gain deeper insights into the possible changes in the aroma of tomato fruits by differing k supply. in combination with the instrumental analysis of vocs, the odor intensity as a sensory descriptor was estimated by panelists. of all the vocs determined, only four were influenced significantly − although negatively − by k application in the salad cultivar ‘lyterno f1’. the volatile compounds in tomatoes are derived from secondary metabolites such as fatty acids, phenolics, amino acids, and carote noids (rambla et al., 2014). hexanal and (e)-2-hexanal, which are being formed from the degradation of fatty acids, showed a signi ficant decrease with increasing k fertilization in lyterno f1. that could have been caused by the changes in the peroxidation of the fatty acids under stress conditions (k deficiency), as was observed by wang et al. (2013). in addition, these compounds are classified as green leafy volatiles as they have the fresh aroma of cut grass (klee, 2010). presumably, under sufficient k supply, the fruits developed to the full ripe stage better than under k-deficiency and lessened the green grass odor; as was stated, k provokes early maturity of fruits (varis and george, 1985). similarly, ß-ionone − derived from the apocarotenoids (rambla et al., 2014) − decreased significantly by k supply but only in lyterno f1. apocarotenoids are derived from carotenoids by oxidative clea vage. the cleavage of carotenoid induces rapidly under stress conditions when a non-enzymatic process catalyzed by reactive oxygen species (ramel et al., 2012). taking this into account, the studied plants were exposed to stress conditions represented by k deficiency (k1), thus, the production of ß-ionone increased in lyterno f1 at deficient k supply. the cultivar effect was evident for the vocs, which was confirmed by wang et al. (2018) and kanski et al. (2020), as they found differences in aroma profile among different tomato cultivars. odor intensity correlated in a significantly positive manner with ß-ionone, (e)-2-hexenal, and benzaldehyde. vogel et al. (2010) pointed out that ß-ionone has fruity and floral perceptions, which can be positively associated with the acceptability of tomato flavor. the significant correlation of (e)-2-hexenal with odor intensity is in agreement with baldwin et al. (1998), they found a high positive significant correlation (r = 0.62**) between (e)-2-hexenal and the overall aroma intensity in seven tomato salad cultivars. benzaldehyde is described as having a peach-like/fruitiness perception (baldwin et al., 2015; selli et al., 2014), and it belongs to the group of phenolic volatiles in tomato fruits (rambla et al., 2014). in our study, benz aldehyde correlated in a significantly positive manner with odor intensity. baldwin et al. (2015) also found a positive significant correlation of benzaldehyde with tomato flavor along a seven-year study with 38 tomato cultivars. effect of k fertilization on instrumental and sensory determined texture with the sense of touch, either when the product is picked up by hand or is bitten off in the mouth and is chewed, the textural parameters of vegetables and fruits can be perceived. physiologically, the texture of fruits and vegetables is derived from their turgor pressure, and the combination of individual plant cell walls and the middle lamella, which holds the cells together valente et al. (2011). in this context, it has been stated that k supply can result in an enhancement of the 188 b. daoud, m. naumann, d. ulrich, e. pawelzik, i. smit tissue firmness (sonntag et al., 2019; tong et al., 1999) by increasing the osmotic potential as a result of the increment of cytosolic k and the accumulation of photosynthetic assimilates (lester et al., 2006). instrumental determined firmness increased with optimal k dose (k3) in lyterno f1 and yellow submarine. accordingly, the sensory descriptor skin strength rose significantly in yellow submarine and showed a similar − although not significant − trend in lyterno f1. however, we observed the opposite effect in primavera (tab. 3). consequently, the results of javaria et al. (2012) and tavallali et al. (2018) could be confirmed by our findings that the positive effect of k on the tissues firmness and skin strength was partly revealed. the instrumental determined firmness and the sensory parameter skin strength are highly positive correlated (0.42**). hence, the effect of k fertilization on the texture in this study has been confirmed by instruments as well as by human senses. thus, the second hypothesis − the effect of k fertilization on the sensory properties can be recognized by the human senses − can be demonstrated. juiciness is one of the most important sensory characteristics and a favorable attribute in most food products (meat, fruits, and vegetables). it is highly correlated to the texture of the plant tissues, in which the juiciness is associated with the cell turgor (valente et al., 2011). k has been pointed out to be essential to cell turgor and the accumulation of photosynthetic products into the plant cell (kanai et al., 2011). nonetheless, our results exhibited no significant effect of k on juiciness. chaïb et al. (2007) stated that the firmest tomato fruits with a strengthened skin were less juicy. accordingly, juiciness correlated in a significantly negative manner with firmness and skin strength in the salad tomato (lyterno f1). it has to be considered that the descriptor juiciness was elaborated by the sensory panel to distinguish juicy fruits from other fruits with less juiciness and more granular dry tissues. it can be assumed − based on the evaluation by the panel − that fruits of salad cultivars appeared to have a generally more granular tissue than those of cocktail cultivars. the cultivar effect was also noticeable for the instrumental determined firmness (tab. 1 and 3). our results confirm that the texture is a complex attribute, which can be affected highly by the genetic background of the cultivars (chaïb et al., 2007). effect of k fertilization on instrumental and sensory determined taste the taste of tomatoes is mainly derived from reducing sugars, organic acids, and bitter compounds. however, as it is abundant in relatively high concentrations, the higher impact is related to sugar (2.6 g 100 g/fm) (kader, 2008; verheul et al., 2015). many stu dies have demonstrated that rising k supply increases the contens of sugar and organic acids (asri and sönmez, 2010; sonntag et al., 2019). in line with this, k dose exhibited a positive significant impact on tss and ta contents in the three cultivars (tab. 1). likewise, the sensorial sweetness and sourness increased significantly with high k fertilization, though only in primavera and yellow submarine, but not in lyterno f1 (tab. 3). in this context, kanski et al. (2020) found in their study on three tomato cultivars and two breeding lines that tss and ta as well sweetness and sourness were highly influenced by the genetic background of the cultivars. taking into account the correlations between the instrumental and sensorial attributes, tss and ta correlated highly positive with sweetness (0.81**) and sourness (0.76**) respectively. therefore, the outcomes of kanski et al. (2020) could be confirmed by our findings, as they proved a high positive correlation between tss and sweetness as well as between ta and sourness. apart from tss and ta, dm is considered to exert a strong influence on tomato taste as it is correlated positively with sugars like fructose and glucose (chapagain and wiesman, 2004; kanski et al., 2020). the positive effect of k was stated to increase dm in tomato fruits (constán-aguilar et al., 2014; javaria et al., 2012), which is because of the function of k in translocating and accumulating the assimilated ‘sugar’ in the cytosol (hawkesford et al., 2012). we were able to prove these previous findings, as we showed that increasing the supply of k increased the dm values in the cocktail cultivars but not in the salad one. here, the water content in the cells and the type of the cultivar have a prominent effect on dm content (beckles, 2012). interestingly, dm correlated in a significantly positive manner not just with tss (r = 0.95**) and ta (r = 0.63**) but also with sweetness (r = 0.79**) and sourness (r = 0.63**), which could lead to a new approach to enhance cocktail tomato taste under sufficient k fertilization. the bitter taste is desirable in some products like coffee and beer (goff and klee, 2006). however, the bitter taste in tomato is not much of a favorite as far as consumers are concerned (you and van kan, 2021). interestingly, the sensory evaluation in the present study exhibited no significant increment of k supply on the bitterness. the sweet compounds have been reported to restrain the bitter taste (beckles, 2012), which is compatible with our findings in primavera, in which k can increase sweet taste and reduce bitter taste. moreover, bitterness had neither positive nor negative significant correlations with any instrumental analyzed or sensorial attributes. effect of k fertilization on retronasal attributes (aroma) tomato flavor is defined by several studies as a complex impression caused by sugar content, organic acids, bitter compounds, and volatile compounds percepted retronasally (auerswald et al., 1999; baldwin et al., 2015; kanski et al., 2020). in our study, the descriptors aroma and aftertaste correlated in a significantly positive manner with tss (r = 0.76**), ta (r = 0.46**), sweetness (r = 0.71**), and sourness (r = 0.68**), which are in line with the previous studies of baldwin et al. (2015) and kanski et al. (2020). the positive correlations between tss, sweetness and aroma intensity found in the present study are also in accordance with the findings made in the case of strawberries (schwieterman et al., 2014; ulrich and olbricht, 2016). however, aroma correlated positively with odor intensity but negatively with hexanal and (e)-2-hexenal. this was surprising; hexanal and (e)-2-hexenal together comprised a high percentage (in lyterno f1: 22–37%, in primavera: 36-48%) of the known detected vocs, and were expected to be associated with aroma. rambla et al. (2014) reported that the vocs − hexanal and (e)-2-hexenal − have the most abundant volatile compounds produced in tomato fruits. nevertheless, the influence of these compounds on tomato flavor has been a matter of discussion. some researchers observed a diminution in the effect of these compounds on tomato flavor and no effect on consumer liking (chen et al., 2004; tieman et al., 2012). in our findings, these two compounds were decreased with rising k fertilization and seem not to contribute to the aroma (tab. 5a). instead, the sugar and acid content seemed to be more relevant for this descriptor. it was a consensus of the panel that the typical tomato aroma could be attributed to red-colored cultivars; lyterno f1 and primavera, while the flavor of yellow submarine was different. aroma of redcolored cultivars did not match the flavor of the yellow cultivar. in which, the panel described the aroma of yellow submarine as having a spicy flavored fruit. increasing k supply resulted in a significant increase in the descriptor aroma in yellow submarine. some vocs mainly characterize the spicy aroma in tomato puree (viljanen et al., 2011) − for instance, 4-methyl-1,5-heptadiene and 6-methyl-3,5-heptadien-2-one. in our study, however, these substances were not detected. among the determined vocs, eugenol was stated to be associated with the smoky aroma in fresh tomato fruits (tikunov et al., 2013). it was supposed potassium influences sensory and instrumental analyzed traits in tomato fruits 189 that the attribute aroma described by the panelists in this research is closely related to the attribute smoky. nonetheless, eugenol did not significantly correlate with the aroma as it was found by tikunov et al. (2013). the reason for this finding might be the effect of k on eugenol, because k fertilization was reported to increase eugenol concentrations (sensch et al., 2000), which was consistent with our results. remarkably, the panelists were able to detect the positive increment of optimal k fertilization on aroma; this can enhance the possibilities of a new approach in increasing tomato flavor with rising k application. conclusion in this study, the effect of k on instrumental determined and sensory traits could be demonstrated. in this context, the following conclusions are drawn: (i) optimal k3 application − 3.66 g/plant − increased the instrumental analyzed attributes and some of the sensory de scriptors, such as sweetness, sourness, and aroma. nevertheless, it did not significantly increase the identified vocs. (ii) the panelists were able to distinguish between the three k fertilization levels, as confirmed by the instrumental analyses. (iii) sugars (sweetness and tss), acids (sourness and ta), and odor attributes (odor intensity, hexanal, and (e)-2-hexenal) were positively associated with aroma and aftertaste. (iv) the cultivar background had a fundamental influence on both instrumental analyzed and sensory attributes and, finally, on tomato flavor. in this study, cocktail cultivars − primavera and yellow submarine − exhibited higher aftertaste and aroma compared to salad cultivar lyterno f1. consequently, optimal k supply − 3.66 g/plant − could be suggested to increase tomato flavor in the studied cocktail cultivars. the flavor of the tomato is a complex perception and is affected by many factors from seed-sowing to the harvest, which needs further investigations to elucidate it comprehensively. acknowledgments we would like to thank the assur project of erasmus mundus scholarship for financial support. many thanks to the technical staff: bettina egger, gunda jansen, and evelyn krüger of the division quality of plant products and the students: nadine mesecke, alexandra glawe, and dorothea meine for their great help during the sensory evaluation. we greatly appreciate the efforts of the 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zörb, c., senbayram, m., peiter, e., 2014: potassium in agriculture – status and perspectives. j. plant physiol. 171, 656-669. doi: 10.1016/j.jplph.2013.08.008 orcid bashar daoud https://orcid.org/0000-0003-4047-2507 marcel naumann https://orcid.org/0000-0003-1577-0772 elke pawelzik https://orcid.org/0000-0001-9329-4375 address of the corresponding author: bashar daoud, university of goettingen, department of crop sciences, quality of plant products, carl-sprengel-weg 1, 37075 göttingen, germany e-mail: bdaoud@gwdg.de © the author(s) 2021. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.3390/ijms14047370 http://dx.doi.org/10.1080/01904160903092663 http://dx.doi.org/10.1016/j.jplph.2013.08.008 https://orcid.org/0000-0003-4047-2507 https://orcid.org/0000-0003-1577-0772 https://orcid.org/0000-0001-9329-4375 i supplementary material 1 table a1. application of fertilization during the growing season (may–september 2017) for the cultivars 1 studied. 2 fertilization chemical g plant-1 application k1 k2so4 0.5 weekly k2 k2so4 2.19 weekly k3 k2so4 3.66 weekly n ca(no3)2 + nh4no3 9.22 + 1.56 weekly for level k3, second week for levels k1 and k2 (nh4)2so4 2.32 second week for levels k1 and k2 (to balance the sulfate) mg mgso4•7h2o 19 three times per growing season: -final transplanting 24.05.2016 -second time 16.07.2016 -third time 26.08.2016 fe fe-edta 0.71 mixture of micronutrients mncl2•4h2o + znso4•7h2o + cuso4•5h2o + na2moo4•2h2o + h3bo3 0.26 + 0.05 + 0.02 + 0.0005 + 0.21 p ca(h2po4)2 •xh2o 17.70 one time at final transplanting s k2so4 and (nh4)2so4 sufficient supply by k and n fertilization 3 table a2. number of fruits used from each cultivar for the conducted analyses; tss: total soluble solids; ta: 4 titratable acids; and dm: dry matter. 5 instrumental analyses lyterno f1 primavera yellow submarine aroma extraction 3 to 5 8 to 10 8 to 10 sensory evaluation 4 to 6 12 to 24 12 to 24 tss, ta, and dm 2 to 3 4 to 6 4 to 6 firmness and minerals extraction 6 to 10 15 to 20 15 to 20 color 15 to 20 35 to 40 35 to 40 6 7 8 9 10 11 12 13 table a1. application of fertilization during the growing season (may–september 2017) for the cultivars studied. fertilization chemical g plant-1 application k1 k2so4 0.5 weekly k2 k2so4 2.19 weekly k3 k2so4 3.66 weekly n ca(no3)2 + nh4no3 9.22 + 1.56 weekly for level k3, second week for levels k1 and k2 (nh4)2so4 2.32 second week for levels k1 and k2 mg mgso4•7h2o 19 three times per growing season: -final transplanting 24.05.2016 -second time 16.07.2016 -third time 26.08.2016 fe fe-edta 0.71 mixture of micronutrients mncl2•4h2o + znso4•7h2o + cuso4•5h2o + na2moo4•2h2o + h3bo3 0.26 + 0.05 + 0.02 + 0.0005 + 0.21 p ca(h2po4)2 •xh2o 17.70 one time at final transplanting s k2so4 and (nh4)2so4 sufficient supply by k and n fertilization (to balance the sulfate) supplementary material supplementary material ii 2 table a3. classification of descriptors according to the kind of sensory impression. the evaluation order was 14 established by the panel and is not equal to order of the classification. detailed information is given on the 15 evaluation instructions for the panel. a scale from 0-100% intensity with a setting of anchors inside (e.g. for 16 50% color intensity) was used. 17 18 sensory impression order descriptor evaluation instructions appearance 1 color intensity a self-made reference template with a color standard representing 50% color intensity either for red or for yellow fruits was used by the panelists. the evaluation was carried out on the fruit skin and not on the cross-sectional view of the fruit. smell (orthonasal olfactory impression) 2 odor intensity odor intensity is defined as the smell of the freshly sliced fruit. tactile or haptic impression 3 juiciness the juiciness of the fruit is represented mainly by the mesocarp, placenta, and myxotesta (pulp and jelly) after biting in. these fruit parts had to be mixed by slight chewing. ’weak‘ is defined as a granular dry tissue. ’strong‘ is defined either crispy and fresh but watery tissue or a very soft and watery / liquid tissue of a ripe to overripe tomato. 8 skin strength ’weak‘ means that the peel is easily broken during chewing. ’medium‘ (50 %) means that a peel residue is clearly recognizable. ’strong‘ also means that a peel residue is clearly recognizable and moreover the peel appears to be very thick. taste (gustatory impression) 4 sweetness for evaluating the taste, it was not differentiated between jelly and pulp. the fruit parts had to be mixed by slight chewing. to compare with samples, the reference fruits were provided with a defined sweetness and sourness. sweetness was calculated based on the total soluble solids that were measured with a refractometer in advance, while sourness was calculated based on the titratable acidity. 5 sourness without instruction 6 bitterness without instruction retronasal smell (aroma) 7 aroma taking into account the sensory differences between yellow and red-fruited cultivars, the panel was trained with both, a yellow-fruited cultivar (cultivar yellow nugget) and a redfruited cocktail tomato cultivar (biologically produced date tomato, cultivar unknown) from a local supermarket set as a standard for the aroma. aftertaste 9 aftertaste the intensity of aftertaste was evaluated half a minute after swallowing. 19 20 21 22 table a4. 2-way anova of sensory data showing the main effects of assessor (n=12) and sample (n= 4) deriving of k fertilization level (n= 3) and the interaction of factors assessor*sample. f-values are displayed, and the significance value is indicated by asterisks (*, **, *** significant at p ≤ 0.05, 0.01, 0.001; n.d. not determined). lyterno f1 primavera yellow submarine sensory descriptor assessor assessor* sample assessor sample sample assessor sample assessor* sample color intensity 12.81*** 0.24 1.36 5.41*** 26.41*** 1.76* 7.49*** 1.33 0.39 odor intensity 18.06*** 1.8 0.97 8.55*** 2.38 0.84 22.88*** 0.5 0.99 juiciness 10.71*** 0.7 1.12 28.37*** 0.37 1.26 84.73*** 5.79** 0.32 skin strength 5.73*** 2.02 0.85 5.86*** 6.26** 1.18 3.56** 7.23** 1.61 sweetness 6.87*** 0.62 2.82*** 8.37*** 6.33** 1.32 4.63** 3.77* 2.13** sourness 12.15*** 2.22 1.17 4.44** 8.49** 2.6*** 7.36*** 17.97*** 1.56 bitterness 8.85*** 0.68 0.87 7.97*** 1.94 5.43*** 9.82*** 1.4 1.41 aroma 30.6*** 4.79* 0.97 8.29*** 28.37*** 2.53*** 21.26*** 12.31*** 1.22 aftertaste 39.59*** 6.19** 0.62 11.31*** 2.15 2.51*** 28.24*** 8.68** 0.56 assessor* sample iii supplementary material 4 table a5. pearson correlations between the studied attributes. 46 color intensity odor intensity juiciness skin strength sweetness sourness bitterness aroma aftertaste odor intensity 0.04 1.000 juiciness 0.16 0.58** 1.000 skin strength -0.28* -0.08 -0.327* 1.000 sweetness -0.15 0.79** 0.496** -0.060 1.000 sourness 0.17 0.52** 0.197 0.140 0.564** 1.000 bitterness -0.26 0.14 0.147 0.392** 0.234 0.198 1.000 aroma 0.44* 0.76** 0.540** -0.109 0.824** 0.686** -0.045 1.000 aftertaste 0,23 0.69** 0.453** -0.001 0.712** 0.682** 0.257 0.529** 1.000 k_content 0.42** 0.23 -0.070 0.145 0.114 0.686** -0.047 0.501** 0.331* color_a 0.23 -0.62** -0.691** 0.052 -0.640** 0.042 -0.250 -0.249 -0.436* color_b 0.45 0.027 0.115 0.287 0.652* 0.811** 0.171 n.d. 0.736** firmness 0.06 -0.522** -0.579** 0.422** -0.636** -0.040 0.015 -0.386* -0.335* tss 0.09 0.672** 0.268 0.133 0.808** 0.743** 0.102 0.828** 0.758** ta 0.23 0.360* -0.044 0.302* 0.282 0.759** 0.038 0.512** 0.457** dm 0.01 0.644** 0.230 0.172 0.785** 0.631** 0.117 0.740** 0.734** hexanal 0.21 0.293* 0.620** -0.480** 0,259 -0.056 -0.223 0.436* 0.224 (e)-2-hexenal -0.34 0.642** 0.436** -0,183 0.745** 0.374* 0.036 0.477** 0.433** octanal 0.28 -0.481** -0.069 -0,082 -0.669** -0.468** -0.050 -0.431* -0.534** 6-methyl-5-hepten2-one -0,23 -0.181 -0.618** 0.425** -0.132 0.025 0.211 -0,280 -0,057 (z)-3-hexen-1-ol 0.28 0.297* 0.516** -0.180 0.173 -0.044 -0.067 0.522** 0,238 isobutylthiazole_2 -0.19 -0.285* -0.545** 0.435** -0.172 0.116 0.089 -0.361* 0,004 benzaldehyde -0.05 0.397** 0.382* -0.081 0.495** 0.312* 0.373 -0.116 0.422** linalool -0.44* 0.188 -0,131 0.058 0.349* 0.190 -0.007 -0.290 0,138 citral -0.17 -0.316 -0.431* 0.044 -0,337 -0.255 0.228 -0.329 -0,311 ß-damascenone -0.26 0.194 0.077 0.030 0.276 0.263 -0.189 0.093 0,134 (e)-geranylacetone 0.04 -0.575** -0.556** -0.219 -0.527** -0.324 -0.197 -0.49** -0.489** ß-ionone 0.11 0.568** 0.589** -0.536** 0.701** -0.019 0.126 0.31 0.356* eugenol 0.21 0.179 0.238 0.040 -0.123 -0.044 0.197 0.13 0.088 ß-cyclocitral -0.67 -0,192 -0.156 0.760** 0.072 -0.491 0.241 -0.08 -0.698** 1-hexanol 0.27 -0.393* 0.285 0.003 -0.531** -0.287 -0.239 0.075 -0.297 methylsalicylate 0.03 -0.41 -0.577* 0.064 -0.313 0.361 -0.253 n.d. 0.523* 47 48 supplementary material iv 5 ta bl e a 5. c on tin ue k_ co nt e nt co lo r_ a co lo r_ b fi rm ne ss ts s ta d m he xa na l (e )2he xe na l oc ta na l 6m et hy l-5 he pt en -2 -o ne (z )3he xe n1ol co lo r_ a 0. 40 7* 1. 00 0 co lo r_ b 0. 66 6* n. d. 1. 00 0 fi rm ne ss 0. 28 1 0. 70 1* * 0. 61 6* 1. 00 0 ts s 0. 48 9* * -0 .2 38 0. 68 1* -0 .3 04 * 1. 00 0 ta 0. 88 6* * 0. 37 2* 0. 67 9* 0. 28 5* 0. 67 6* * 1. 00 0 d m 0. 39 2* * -0 .2 26 0. 62 5* -0 .2 67 0. 95 5* * 0. 62 7* * 1. 00 0 he xa na l -0 .2 85 * -0 .6 20 ** 0. 16 3 -0 .6 70 ** 0. 02 3 -0 .3 34 * -0 .0 47 1. 00 0 (e )2he xe na l -0 .0 28 -0 .6 35 ** -0 .0 77 -0 .5 44 ** 0. 56 8* * 0. 15 0 0. 56 6* * 0. 27 8 1. 00 0 oc ta na l -0 .2 04 0. 00 4 0. 01 1 0. 13 9 -0 .6 37 ** -0 .3 64 * -0 .6 22 ** 0. 13 1 -0 .5 78 ** 1. 00 0 6m et hy l-5 -h ep te n2on e 0. 26 4 0. 57 1* * -0 .4 77 0. 47 8* * -0 .0 14 0. 24 3 0. 02 4 -0 .8 21 ** -0 .2 93 * -0 .3 26 * 1. 00 0 (z )3he xe n1ol -0 .1 02 -0 .8 55 ** 0. 15 4 -0 .5 33 ** 0. 09 9 -0 .1 83 0. 04 5 0. 72 9* * 0. 08 6 0. 29 6* -0 .6 54 ** 1. 00 0 2is ob ut yl th ia zo le _2 0. 19 6 0. 60 1* * 0. 08 1 0. 63 2* * 0. 07 9 0. 30 2* 0. 14 2 -0 .8 37 ** -0 .1 87 -0 .3 49 * 0. 61 5* * -0 .6 76 ** be nz al de hy de -0 .0 22 -0 .1 75 0. 06 0 -0 .2 16 0. 41 6* * 0. 16 4 0. 44 6* * -0 .0 53 0. 34 7* -0 .1 65 -0 .1 49 -0 .1 51 lin al oo l 0. 08 6 0. 23 5 -0 .0 04 0. 04 5 0. 27 0 0. 24 1 0. 35 9* -0 .2 73 0. 60 2* * -0 .6 33 ** 0. 25 2 -0 .4 76 ** ci tr al -0 .0 21 0. 34 2 n. d. 0. 15 5 -0 .3 77 * -0 .1 00 -0 .3 33 -0 .3 99 * -0 .6 15 ** 0. 30 5 0. 66 1* * -0 .4 67 * ß_ da m as ce no ne 0. 22 3 -0 .0 81 0. 11 6 -0 .0 37 0. 27 2 0. 33 6* 0. 29 6* -0 .0 02 0. 62 2* * -0 .4 71 ** -0 .0 60 -0 .1 22 (e )ge ra ny la ce to ne -0 .0 66 0. 66 4* * n. d. 0. 29 6 -0 .4 56 * -0 .1 62 -0 .4 10 * -0 .4 19 * -0 .6 08 ** 0. 46 2* 0. 39 7* -0 .6 45 ** ßio no ne -0 .2 81 -0 .6 99 ** n. d. -0 .8 58 ** 0. 32 7 -0 .3 34 0. 30 9 0. 69 7* * 0. 60 1* * 0. 34 6* -0 .6 34 ** 0. 80 0* * eu ge no l -0 .1 31 -0 .3 69 * 0. 37 9 -0 .0 37 -0 .1 03 -0 .0 97 -0 .0 95 0. 25 7 -0 .0 25 0. 49 2* * -0 .4 82 ** 0. 44 2* * ßcy cl oc itr al -0 .4 54 -0 .4 72 n. d. 0. 08 6 -0 .4 43 -0 .4 94 -0 .4 21 -0 .2 42 0. 19 0 -0 .1 92 0. 35 9 0. 15 3 1he xa no l -0 .0 44 -0 .0 82 0. 31 1 -0 .2 67 -0 .4 11 * -0 .2 84 -0 .5 19 ** 0. 69 1* * -0 .5 48 ** 0. 43 1* -0 .3 84 * 0. 77 2* * m et hy ls al ic yl at e 0. 43 8 n. d. 0. 18 3 0. 48 0 0. 28 1 0. 19 8 0. 30 0 -0 .1 50 -0 .1 86 -0 .2 86 0. 19 8 0. 78 6* * 6 ta bl e a 5. c on tin ue 2is ob ut yl th ia zo le be nz al de hy de lin al oo l ci tr al ß_ da m as ce no ne (e )ge ra ny la ce to ne ß_ io no ne eu ge no l ß_ cy cl oc itr al 1he xa no l be nz al de hy de -0 .0 12 1. 00 0 lin al oo l 0. 31 6* 0. 19 3 1. 00 0 ci tr al 0. 01 8 -0 .2 25 -0 .2 20 1. 00 0 ß_ da m as ce no ne 0. 07 6 -0 .0 04 0. 81 2* * -0 .5 46 ** 1. 00 0 (e )ge ra ny la ce to ne 0. 21 2 0. 01 1 -0 .1 30 0. 65 8* * -0 .4 47 * 1. 00 0 ßio no ne -0 .8 30 ** 0. 39 2* -0 .3 56 * -0 .0 85 -0 .1 60 -0 .2 60 1. 00 0 eu ge no l -0 .2 63 0. 11 7 -0 .4 31 ** -0 .3 17 -0 .3 53 * -0 .2 55 0. 36 6* 1. 00 0 ßcy cl oc itr al 0. 27 1 -0 .7 15 ** 0. 23 8 0. 22 6 0. 12 4 -0 .2 72 -0 .0 08 -0 .4 54 1. 00 0 1he xa no l -0 .6 20 ** -0 .6 31 ** -0 .6 48 ** 0. 20 8 -0 .2 34 -0 .1 37 -0 .6 30 * -0 .0 44 0. 61 9* 1. 00 0 m et hy ls al ic yl at e 0. 54 3* -0 .6 67 * -0 .0 87 n. d. 0. 07 1 n. d. n. d. 0. 43 8 n. d. 0. 75 2* * v supplementary material 6 ta bl e a 5. c on tin ue 2is ob ut yl th ia zo le be nz al de hy de lin al oo l ci tr al ß_ da m as ce no ne (e )ge ra ny la ce to ne ß_ io no ne eu ge no l ß_ cy cl oc itr al 1he xa no l be nz al de hy de -0 .0 12 1. 00 0 lin al oo l 0. 31 6* 0. 19 3 1. 00 0 ci tr al 0. 01 8 -0 .2 25 -0 .2 20 1. 00 0 ß_ da m as ce no ne 0. 07 6 -0 .0 04 0. 81 2* * -0 .5 46 ** 1. 00 0 (e )ge ra ny la ce to ne 0. 21 2 0. 01 1 -0 .1 30 0. 65 8* * -0 .4 47 * 1. 00 0 ßio no ne -0 .8 30 ** 0. 39 2* -0 .3 56 * -0 .0 85 -0 .1 60 -0 .2 60 1. 00 0 eu ge no l -0 .2 63 0. 11 7 -0 .4 31 ** -0 .3 17 -0 .3 53 * -0 .2 55 0. 36 6* 1. 00 0 ßcy cl oc itr al 0. 27 1 -0 .7 15 ** 0. 23 8 0. 22 6 0. 12 4 -0 .2 72 -0 .0 08 -0 .4 54 1. 00 0 1he xa no l -0 .6 20 ** -0 .6 31 ** -0 .6 48 ** 0. 20 8 -0 .2 34 -0 .1 37 -0 .6 30 * -0 .0 44 0. 61 9* 1. 00 0 m et hy ls al ic yl at e 0. 54 3* -0 .6 67 * -0 .0 87 n. d. 0. 07 1 n. d. n. d. 0. 43 8 n. d. 0. 75 2* * supplementary material vi angewandte botanik. 138 f. w. neger, ein neues, untrügliches merkmal für rauchschäden. nähe von rauchquellen auf eine art von erstickungstod infolge der ausschaltung der lentizellen zurückzuführen. diese und mehrere andere sich anschließende fragen werden den gegenstand weiterer untersuchungen bilden. nachtrag: einen weiteren fall von lentizellenbeschädigung habe ic h inzwischen bei dohna (bez. dresden) beobachtet, und zwar an apfelfrüchten (wahrscheinlich durch einwirkung von flußsäure). äußerlich war die beschädigung a n höfen, welche die lentizellen umgaben, erkennbar. merkwürdig ist, daß das fruchtfleisch des apfels nicht die fähigkeit besitzt, das abgestorbene gewebe durch wundkork abzugrenzen. infolgedessen greift hier die schädigung sehr schnell um sich. der gegenwärtige stand der kohlensäurefrage für pflanzenkulturen. von dr. hugo fischer. als kolumbus seine erste entdeckerfahrt plante, sprach sich ein kollegium hochgelehrter männer mit allen gegen eine stimme dahin aus, daß der gedanke einer erdumsegelung barer unsinn sei. als die eisenbahn erfunden war, gab ein rat sachverständiger männer das urteil ab, es sei das gewiß eine recht interessante sache, aber davon könne keine rede sein, daß das neue verkehrs mittel jemals zur überwindung großer entfernungen oder zur beförderung größerer lasten dienen könne. dieses urteil erfolgte einstimmig. die mendelschen spaltungsregeln, das einmaleins der ver erbungsforschung, haben rund 3 5 jahre in verborgenheit ge schlummert, ehe sie die verdiente anerkennung fanden. e s is t nicht richtig zu meinen: „die zeit sei nicht reif gewesen“; man würde den führenden männern von damals doch gar zu wenig zu trauen, wollte man sagen, jene regeln seien ihnen „zu hoch“ gewesen – im gegenteil, zu einfach waren sie. hugo fischer, der gegenwärtige stand der kohlensäurefrage usw. 139 zu einfach ist wohl auch der gedanke, den pflanzen mehr kohlensäure zuzuführen, als die natur ihnen für den assimilations vorgang bietet. jeder bauer weiß heutzutage, daß man durch düngung mit stickstoff, kali, phosphor usw. höhere ernten herausholen kann; die pflanzen auch einmal mit mehr kohlen säure zu versorgen, daran hat kaum jemals jemand gedacht, und wenn es einer aussprach, dann blieb er ein prediger in derwüste. die ablehnung dieses gedankens seitens der landwirtschafts wissenschaft ist um so seltsamer, als schon 1837 der vater der rationellen landwirtschaft, albrecht thaer, betont hat, der hauptwert organischer düngung liege in der entwicklung von „kohlensaurem gas“, das an die blätter der pflanzen herantrete und von ihnen aufgenommen werde. wie sehr diese höchst wichtige erkenntnis in vergessenheit geraten war, habe ich in einer kleinen studie im centralbl. f. bakt., 2. abt., xlviii (1918), s. 515 mit einigen beispielen belegt; ic h gehe hier kurz darüber hinweg. betonen möchte ich hier nur: angesichts der erfolge der mistbeetkultur war es doch geradezu widersinnig, z u glauben, ein höherer co2-gehalt der luft als die üblichen 0,02 bis 0,03 v . h.) sei den pflanzen schädlich! – jahrelange bemühungen bei behörden, bei landwirtschaft ichen und gärtnerischen organisationen, d ie frage der kohlen säureversorgung der pflanzen in fluß zu bringen, waren und blieben vergeblich. auch die theoretische wissenschaft brachte der sache kein merkliches interesse entgegen – war es wegen ihres praktischen hintergrundes? die frage is t doch auch rein physiologisch, besonders durch ihre beziehungen zur theorie der blühreife”) von nicht geringer bedeutung, wie überhaupt die zahl reichen noch offenen fragen des zusammenhanges zwischen stoff wechsel und entwicklungsgang der pflanzen. s o war es der großindustrie vorbehalten, als erste dieser ih r ja ursprünglich fernliegenden frage tätiges interesse entgegen *) gerade wenn die pflanzen si e am besten brauchen könnten, scheint die kohlensäure noch knapper zu werden: zwischen grünen pflanzen, b e i günstigem wetter, d . i. bei windstille und sonnenschein, fanden klein und reinau chemiker-ztg. xxxviii, 1914, s. 125) überhaupt keine nachweisbaren spuren von co, *) vergl. flora xciv, (1905), s. 124 u. s. 478; – ebenda xcv, (1905), s. 324. g. klebs, physiologie der fortpflanzung. handwörterbuch der naturwissen schaften, iv, jena 1914, s. 288 ff. 140 hugo fischer, zubringen: auf betreiben von herrn dr. ing. f. riedel ist, er richtet von der deutsch-luxemburgischen bergwerksu. hütten-a.-g., in horst a. d. ruhr (unweit essen) eine anlage entstanden, die dafür bestimmt ist, nach für herrn dr. r. paten tiertem verfahren die abgase eines hochofens für pflanzenkulturen nutzbar zu machen. was ein kleiner hochofen täglich an kohlen verbraucht, entspricht umgerechnet dem stärkegehalt von gut 30000 zentner kartoffeln (nach der formel c.hoos und be i 18"/o stärke); ein großer hochofen leistet das 3–4-fache. selbst verständlich is t die ausnützung dieser mengen von kohlenstoff nur zu einem ganz geringen teil möglich, und ebenso versteht sich im glasbaus eine höhere ausnützung als im freiland, w o diese von witterungseinflüssen sehr beeinflußt zu werden scheint, die hochofenabgase sind verhältnismäßig rein, nur staub und schädliche kohlenwasserstoffe müssen daraus entfernt werden, schweflige säure, die für pflanzen sehr gefährlich wäre, kommt hier kaum in frage, weil im hochofenbetrieb nur koks verwendet wird. den kohlenstoff enthält das abgas infolge unvollkommener verbrennung größtenteils als kohlenoxyd, co, das erst zu kohlen dioxyd, co2, weiter verbrannt werden muß; das geschieht zum größten teil schon im betriebe, wo die brennbaren abgase dazu dienen, in den „vorwärmern“ die in den hochofen eintretende luft zu erhitzen. das kohlensäurereiche gas wird, mit luft ver mischt, durch einen großen, elektrisch betriebenen ventilator in ein röhrensystem gepreßt und so über glashäuser und freiland verteilt. – nach dem riedelschen verfahren lassen sich natürlich heizgase verschiedenster art, u . a . auch die abgase von kalköfen, in gleicher weise verwerten; reinigung von schädlichen bei mengungen ist dabei voraussetzung. es waren i. j. 1917 drei parallele häuser, je 6× 25 m , nebst einem 1 8 m langen verbindungshaus errichtet, i. j. 1918 wurden a n dessen anderer langseite drei andere, je 6× 4 0 m angebaut, so daß jetzt (ohne verbindungshaus) 1170 qm unter glas sind. an freiland stehen etwa 4 hektar (= 16 morgen) zur verfügung. die gegebene gasmenge könnte ein viele male größeres feld ver sorgen. von der vorhandenen fläche is t nur ein kleiner bruchteil für kontrollversuche ohne begasung freigelassen. leider sind di e bodenverhältnisse sehr ungleichartig; etwa */ 4 der fläche sind seit jahren in kultur, aber ohne einheitlichkeit, parzellenweise an gehörigen des werkes überlassen gewesen, und sind nach ihrer der gegenwärtige stand der kohlensäurefrage für pflanzenkulturen. 141 lage zu dem unbegasten stück für wirkliche vergleiche ungeeignet. das näher der kontrollfläche gelegene, mit röhren belegte feld stück is t alte schlackenhalde, erst künstlich durch auffahren von boden in kulturland umgewandelt, so daß die grundlage für exakten vergleich: gleichheit der bodenverhältnisse, erst in einigen jahren erreicht werden kann. jedenfalls ist aber das unbegaste stück das bessere, so daß jeder auf dem begasten feld erzielte erfolg um so höher z u bewerten ist. in den häusern wurde schon i. j. 1917 eine tomatenernte von unbegast 100: begast 275 erzielt; bei höchst ungünstiger witterung und krankheitsbefall hatten wir i. j. 1918 nur 100: 200; d ie begonnene ernte 1919 dürfte aber über 100 : 300 noch hinaus gehen. von am 23. jan. d. j. in töpfe gesäten buschbohnen konnten im begasten haus vom 25. märz a n bereits die schnittreifen hülsen geerntet werden, als die pflanzen im unbegasten haus sich eben zum blühen anschickten. in denselben beiden häusern gezogener blumenkohl entwickelte sich im begasten haus viel kräftiger, die pflanzen um etwa die hälfte höher, die „blumen“ entsprechend größer; gewichtsmäßige feststellung der ernten mußte in diesen beiden fällen unterbleiben. die mittelhäuser beiderseits sind mit gurken bepflanzt, mit denen ein vergleich nicht angesetzt wurde. nur in dem durch eine querwand mit schiebetür geteilten haus 6x40 m war die hintere hälfte anfangs unbegast gelassen; hier zeigten nun die ersten 2–3 pflanzen jeder reihe, die von herein diffundierender kohlensäure mitbekommen hatten, eine ganz auf fallende wachstumsförderung!), wie überhaupt die gurkenpflanzen, bevor sie zur blüte schritten, sich ungemein üppig entwickelten, mit blättern von ganz ungewöhnlicher größe. die ernte a n früchten ist sehr reich, nur fehlt es hier a n vergleichszahlen, weil keines der gurkenhäuser unbegast geblieben war. im sommer 1917 wurden in zwei parallelhäusern gurken geerntet 100: 170. – die beiden äußeren häuser 6× 40 m sind je zur hälfte für wein und für pfirsiche bestimmt. sehr auffallend, namentlich a n den tomatenpflanzen, ist die dunkle, bläulichgrüne farbe des laubes als wirkung der kohlen säurebehandlung; ähnliches zeigte sich im freiland besonders deut lich a n mohnpflanzen. *) diese fällt um so mehr ins gewicht, als die gurkenhäuser, fast stets geschlossen und mit stark gedüngtem boden, a n sich schon eine a n kohlensäure sehr reiche luft, etwa 0,2–0,3 v. h., enthalten. 142 hugo fischer, im freiland wurden auf kleinen flächen schon im spät sommer 1917 versuche angestellt, die folgende ergebnisse hatten: spinat 100: 250; rübstiel 100: 150; kartoffeln 100: 280; lupinen 100: 290; gerste 100: 200 (nicht mehr reif geworden; die zahlen beziehen sich auf das pflanzengewicht); die behandelte gerste stand im anfang november nahe vor der blüte, die unbehandelte war noch kaum am schossen. die freilandversuche d. j. 1918 litten unter den höchst ungünstigen witterungsverhältnissen, teils wurden sie durch die geschilderte unregelmäßigkeit und ungunst des bodens vereitelt; vergleichszahlen konnten kaum festgestellt werden, außer von einigen kartoffelbeeten, wo das höchstmaß der (in einem fall erzielten) ertragssteigerung 100: 421 betrug; di e größte knolle unbegast wog 180 g , die größte begast 320 g , = 100: 177: auch in diesem jahr hat uns die naßkalte witterung) manche enttäuschung bereitet: von erntezahlen liegt erst ei n vergleich mit mangold vor: geerntete blätter mitte juni 100: 170, ende juli 100: 146, summe beider erträge 100: 152,5. nun ist eines zu bemerken: wir stecken mit diesen versuchen ja noch ganz in den anfängen, wir wissen: es geht! – wir wissen aber noch so gut wie gar nichts davon, wie es am besten geht. wüßten wir das, so würden wir wohl noch ganz andere ertragszahlen bekommen. mit stickstoff-, kaliusw. düngung hat ja auch erst viel probiert werden müssen, wie man höchsternten herausholt. s o sind denn bezüglich der co2-behandlung noch zahlreiche bedeutungsvolle fragen zu lösen, deren für die praktische ausnützung wichtigste ich hier zusammenstelle: 1 . welche arten und welche sorten von nutzpflanzen eignen sich besser, welche weniger gut für kultur unter kohlen *) man darf wohl mit gutem grund annehmen, daß windstilles, sonnig warmes wetter für den assimilationsvorgang das günstigste ist. aber auch übertriebene trockenheit, wie im mai d . js, is t der vollausnützung der co, ent gegen; is t das wasser im minimum, so kann alles andere wenig helfen. eine kräftige wasserdurchströmung dürfte für ausgiebige c-assimilation notwendig sein; sie kann aber nur stattfinden bei warm-trockenem wetter, ohne daß es dem boden a n feuchtigkeit gebricht. bei wassermangel schließen sich ja bekannt: lich die spaltöffnungen, damit is t dann der gasaustausch zwischen blattgewebe und außenluft beeinträchtigt. niedere sommertemperatur schädigt aber di e assimilation in doppelter weise, erstens direkt, durch verlangsamung des vor ganges selbst, zweitens indirekt, durch hemmung des ableitenden stromes, der eine stauung der assimilate herbeiführt. der gegenwärtige stand der kohlensäurefrage für pflanzenkulturen. 143 säurezufuhr? gibt es unter ihnen solche, für welche der all gemein sicher ungültige satz, der normale co2-gehalt der luft stelle das bestmaß für die pflanze dar, vielleicht doch geltung hat? 2. gibt es pflanzen, welche für eine größere, oder für eine geringere zunahme der kohlensäure besonders dankbar sind? 3. welche arten von kohlensäure quellen kommen, außer den abgasen der industrie, für praktische verwendung, von fall zu fall, in frage? vgl. hierzu frage 19. 4. wie erklären sich die vereinzelten mißerfolge an pflanzen, d ie ein anderes mal gut reagiert haben? und wie sind mißerfolge z u vermeiden? 5 . für welche pflanzen empfiehlt es sich täglich von morgen bis abend, für welche nur halbtägig – voroder nach mittags, – oder stundenweise, oder nur tag um tag kohlen säure z u geben? 6 . lassen sich mit erfolg im deutschen klima solche pflanzen heranziehen, die sich wegen z u langer vegetationsdauer und verspäteter samenreife bisher nicht einbürgern konnten? 7 . sind die in frage kommenden arten oder sorten zu allen zeiten ihres lebens (ruhezeiten natürlich ausgeschlossen) für kohlensäuregaben gleich dankbar, oder wollen sie in verschiedenen entwicklungszuständen verschieden behandelt sein? 8 . welche wirkung hat vorübergehende behandlung auf pflanzen, welche nachher wieder der gewöhnlichen luft ausgesetzt werden? 9 . wie groß ist die verhältnismäßige ausnützung der gebotenen kohlensäure bei verschiedenen pflanzen? in verschiedenen altern? bei verschiedenen gaben? unter verschiedenen ernährungs zuständen? 10. wie wirkt das wetter auf diese ausnützung? wind oder stille, sonnenschein oder zerstreutes himmelslicht, wärme oder kühle, feuchtigkeit oder trockenheit? 11. wie stellt sich der ausnützungsfaktor der kohlensäure b e i – quantitativ und qualitativ – verschiedener mineral düngung, und umgekehrt die ausnützung der einzelnen minera lischen nährstoffe bei kohlensäurezufuhr, ev. bei düngegaben, die bisher als übernormal galten? 12. verändert sich derwasserhaushalt der pflanze? und wie? 13. kann man, da ja im freien der lichtfaktor in überfluß, d ie kohlensäure im minimum is t (sofern nicht etwa die wasser 144 hugo fischer, versorgung auf diesem punkte steht!) mit aussicht auf erfolg die pflanzweite enger wählen als sonst normal und üblich? 14. hat die kohlensäurezufuhr einen einfluß auf diewurzel bildung? und welchen? 15. wird der gehalt der pflanzen an wichtigen nähr stoffen, an kohlenhydraten, fetten, eiweiß, oder an nutzbaren fasern durch die co2-behandlung gesteigert, und bis zu welchem grade? 16. enthalten arzneiund drogenpflanzen nach kohlen säuredüngung mehr an wirksamen stoffen? 17. macht sich ein einfluß auf die nachkommenschaft geltend? etwa im sinne allgemein besseren saatgutes, oder in häufigerem erscheinen nutzbarer erblicher abänderungen (mu tationen)? 18. läßt sich der samenansatz bei wenig fruchtbaren bastardpflanzen durch kohlensäuregaben steigern? 19. inwieweit gelten alle diese gesichtspunkte nicht nur für künstliche, sondern auch bei zweckmäßiger ausnützung der natür lichen kohlensäurequellen, von stallmist, gründüngung, kompost, moorerde usw. 20. läßt sich bei kohlensäuredüngung in der lichtarmen winterzeit künstliches licht nutzbringend anwenden? 21. bestätigen sich auch weiterhin die beobachtungen, wo nach mit co2 behandelte pflanzen widerstandsfähiger gegen schädlinge sind? – dies die vorläufig wichtigsten der auf die praxis bezüglichen fragen!), auf welche wir nur erst sehr teilweise zu antworten wissen! auf dem bisherigen wege würde es noch vieler langer jahre bedürfen, bis man einigermaßen in diesen dingen klar sehen könnte. plan volle versuchsanstellung – das weiß jeder gebildete – kann diesen zeitraum ganz wesentlich ab kürzen, kann in wenigen jahren antwort auf fragen finden, mit denen die bloße praxis in eben sovielen jahr *) von wissenschaftlichem interesse wäre es, die einwirkung der c0. (zum vergleich auch die wirkung abnorm geringer gaben) auf anatomische merk male und auf die mikroskopie der zelle und ihrer teile, ferner auf physiologische erscheinungen zu studieren; auch hier sind wir über anläufe hinaus! vgl. z. b. farmer u. chandler, proceed. roy. soc, lxx (1902), s. 413. kisselew beih. botan. centralbl. i, xxxii (1914), s. 86. vgl. dazu auch meinen aufsatz „spezifische assimilationsenergie“ im juliheft 1919 der ber. d. d. botan. ges. der gegenwärtige stand der kohlensäurefrage für pflanzenkulturen. 145 zehnten nicht ins reine kommt! darum ist ein dringen des bedürfnis der allernächsten zeit: schaffung einer arbeitsstätte, nur dazu bestimmt, die kohlensäurefrage in ständiger rücksicht auf die praxis, aber unter wissen schaftlicher leitung, auf wissenschaftlicher grundlage und mit den methoden derwissenschaft, nach allen seiten hin durchzuarbeiten. denn diese frage hat ja nicht nur für diejenigen stellen bedeutung, denen die aus heizanlagen, hochöfen, kalköfen usw. abfallende kohlensäure zur verfügung steht (beiläufig sei hier auch der gärungs-kohlensäure gedacht), oder für kulturen unter glas, in denen man auch mit künstlich erzeugter co2 mit erfolg arbeiten würde, wie ich schon vor jahren bewiesen habe. für die landwirtschaft von größter wichtigkeit ist der aus organisch gedüngtem boden aufsteigende kohlensäure strom, den schon thaer (vgl. oben) für die wichtigste unter den wirkungen des stalldüngers erklärt hat. wir brauchen seinen wert a ls nährstoff-, insbesondere stickstoffquelle, und seine leistungen für physikalische bodenverbesserung darum nicht gering z u achten: diese dinge sind ja eingehend untersucht und ziemlich gut bekannt, weil man stalldung, gründüngung usw. jahrzehnte lang ausschließlich danach bewertet hat; seine bedeutung als kohlensäurequelle ist aber mindestens des gleichen inter esses würdig, nur ist es gerade das, was vorerst noch von wenigen anerkannt wird. in dieser sache könnten wir schon vor 12 jahren weiter gewesen sein als wir heute sind. es mag sich jeder selbst fragen, und jeder sich selbst sagen, was e s bedeutet hätte, wenn wir in den letzten fünf jahren auf grund von kenntnissen, die uns leider heut noch größtenteils fehlen, unsere ernten selbst nur um einige hundertstel hätten steigern können!!! warum waren wir rück ständig? weil, dank einer ganz einseitigen schulbildung, unter den maßgebenden selten ein mann z u finden ist, der in einer naturwissenschaftlichen frage ein eigenes, unbefangenes urteil hätte. soll man nun urteilen, dann muß man sachverständige befragen; und was das bedeuten will, dafür weise ich auf die ein leitenden sätze dieses vortrages zurück: auf kolumbus, auf die erste eisenbahn, auf gregor mendel. alles, was in der naturwissenschaft neu (oder wieder neu) ist, kommt so in eine üble lage. denken wir uns, das schießpulver wäre noch unbekannt, angewandte botanik i. 1 () 146 hugo fischer, der gegenwärtige stand der kohlensäurefrage usw. und es käme jemand (ich setze als bekannt voraus, daß solche erfindung nur von der naturwissenschaft ausgehen kann!) zum kriegsminister: „ich bin einer erfindung auf der spur, so und so , welche die ganze kriegführung umgestalten und derjenigen seite, welche in ihrem besitz ist, ungeheure vorteile gewährleisten wird; ich brauche aber zur durchführung der sache ein kleines labora torium mit geeigneter einrichtung, usw.“ und der minister, nach dem e r noch einige fragen gestellt, spräche z u ihm: „schön, was sie brauchen, sollen sie haben! gewinnen sie nur erst mit ihrem „pulver“ ein paar schlachten und erobern sie einige festungen, dann soll die errichtung ihres laboratoriums sofort . . . . . . . in wohlwollende erwägung gezogen werden.“ – so liegt die sache, und e s ist ein schwerer hemmschuh für unsere ganze kulturentwicklung, daß die möglichkeit, einen fruchtbaren gedanken in die tat umzusetzen und ihn der allgemeinheit nutzbar zu machen, von außenbedingungen abhängig ist, die mit seinem wert oder unwert rein gar nichts zu tun haben. die beurteilung des anbauwertes französischer rotkleesaaten. von prof. dr. j. simon-dresden. mit 2 karten. die erste und wichtigste grundlage einer erfolgreichen pflanzenkultur is t die verwendung guten, einwandsfreien saat gutes. die eigenart des heimischen pflanzenbaues, einmal die kulturellen und klimatischen verhältnisse, dann die notwendigkeit des gesteigerten anbaues von getreide und hackfrüchten sowie von futterpflanzen machen es unmöglich, den gesamten bedarf an saatgut für alle pflanzenarten im inlande selbst zu erzeugen: in weitgehendem maße sind und bleiben wir auch für die folge auf den bezug vom auslande angewiesen. ganz besonders behält dies geltung für unsere wichtigste futterpflanze, den rotklee“). *) importiert wurden vor dem kriege jährlich 378000 d z kleesaat. (alves, arbeit. der d . l . g., 1913, bd. 245.) uc1.b3299303_page_156_cut uc1.b3299303_page_157_cut uc1.b3299303_page_158_cut uc1.b3299303_page_159_cut uc1.b3299303_page_160_cut uc1.b3299303_page_161_cut uc1.b3299303_page_162_cut uc1.b3299303_page_163_cut uc1.b3299303_page_164_cut art15_netsere.indd journal of applied botany and food quality 84, 219 222 (2011) 1jimma agricultural research center, jimma, ethiopia 2department of horticulture and plant sciences, jimma university college of agriculture and veterinary medicine, jimma, ethiopia allelopathic effects of parthenium hysterophorus l. aqueous extracts on soybean (glycine max l.) and haricot bean (phaseolus vulgaris l.) seed germination, shoot and root growth and dry matter production anteneh netsere1, esayas mendesil2 (received may 20, 2011) summary aqueous extracts of shoot (stem + branch), leaf, fl ower and root of parthenium hysterophorus l. at 0, 5, 10 and 15% (w/v) concentrations were applied on seeds of soybean (glycine max l.) and haricot bean (phaseolus vulgaris l.) to investigate their effect on percent germination, germination rate, and seedling growth (shoot and root length) and dry matter production under laboratory condition. the treatments were laid out in a completely randomized design with factorial arrangement in four replications. the trial was conducted twice, november 13 26 and december 1 14, 2008. results depicted that signifi cant (p < 0.01) difference between plant parts, concentration levels and their interaction for the aforementioned parameters. aqueous extracts of fl ower at all concentrations, and leaf at 10 and 15% and shoot at 15% concentration levels 100% inhibited germination of both crops. in contrast, aqueous extracts from shoot and root at 5% had lower effect, while at higher concentrations greatly reduced germination percentage and growth of the crop. roots of the crops were more sensitive to allelopathic effect than shoots. it is recommended that integrated weed management strategy should be designed and employed to combat the weed from soybean and haricot bean fi eld to avoid poor germination and seedling growth and ensure sustainable production of the crops introduction parthenium (parthenium hysterophorus l.) is an invasive annual weed which is native to central america and now it is widely distributed in kenya, india, china, australia and africa (khosla and sobti, 1981; aneja et al., 1991). it was introduced to ethiopia in the mid 1970s after noticeable occurrence in eastern part of the country, where it has now emerged as one of the most trouble weed in arable and grazing land of the country as well as abandoned fi elds of the country (mekbib et al., 1996; tamado and milberg, 2000; tadesse et al., 2005; rezene et al., 2005). parthenium can severely compete with annual crops and can cause tremendous yield loss. accordingly, nath (1988), respectively, reported a yield decrease of 40% in agricultural crops and up to 90% reduction in forage production in grass lands due to this weed. similarly, tamado et al. (2002) demonstrated that a 40 to 90% sorghum yield reduction if parthenium weed is left uncontrolled through the cropping season. it also inhibited growth and nodulation of legumes because of the inhibitory effect of allelochemicals on nitrogen fi xing and nitrifying bacteria (kanchan and jayachandra, 1980; deyama, 1986). besides, it adversely affects animal health, production and quality of their produces (tadesse et al., 2005), human health and activities, ecology and biodiversity (rezene et al., 2005). soybean (glycine max l.) and haricot bean (phaseolus vulgaris l.) is one of the most important traditional pulse crop in the midand low-land areas of ethiopia. its grain used for food and cash source for subsistence farmers and it by product, such as stalk and leaves, is used for fi rewood and feed. it provides an important source of protein and minerals, especially fe and zn, in the diet. as a leguminous crop, soybean and haricot bean is important due to its role for crop rotation in area where cereals sole cropping is a common practice in southwestern ethiopia, where maize is the major staple food crop and grown in mono-crop conditions. moreover, as the crops are short maturing and have moderate drought tolerance, it is used as the main or the only food in short growing seasons and poor annual harvest area. thus, it plays a vital role in farmers’ risk aversion strategies. in spite of the importance of the crops, their productivity is seriously threatened by parthenium weed invasion. this calls for further scientifi c investigation on the allelopathic potential of this weed on germination and seedling growth of the crops. the objective of this study was, therefore, to investigate the allelopathic effects of aqueous extracts of shoot, leave, fl ower and root of parthenium on germination, seedling growth and dry matter production of soybean and haricot bean under laboratory condition. materials and methods preparation of aqueous extract of p. hysterophorus parthenium hysterophorus plants growing naturally along the roadside of jimma town, southwestern ethiopia, were randomly uprooted and collected at their fl owering stage in november 1, 2008. the collected plants were brought into the entomology laboratory of jimma agricultural research center, ethiopia, and were immediately partitioned into shoot (stem + branch), leaf, fl ower and root parts. shoot and root part of the fresh plant was cut into 1-2 cm pieces. then after, each plant part put in paper bags separately and dried in oven at 70 °c for 24 hours and pounded using electrical stainless steal wiley mill. five, 10 and 15 g powder of each plant part was weight using sensitive electronic balance and soaked in 100 ml of distilled water using 300 ml fl asks and mixed thoroughly. after 24 hours of soaking at room temperature (21 22 °c), the mixture containing 5, 10 and 15 g parthenium extracts were collected by sieving through cheesecloth and designated as 5, 10 and 15% aqueous extracts, respectively. bioassays and experimental design one kilogram of soybean and haricot bean seeds, variety roba 1 and clerk 63k respectively, were obtained from field crop agronomy research division of jimma agricultural research center was used for the study. the seeds of each variety were treated with sodium hypochlorite in a 500 ml fl ask for 3 minute and washed thoroughly with distilled water. the bioassay was conducted following the procedures of tefera (2002) and wakjira et al. (2005). one hundred uniform size seeds of both crops were separately placed in a petri dish (9 cm diameter) lined with 9 cm whatman fi lter paper®. then after, the seeds were treated with 10 ml of the 5, 10 and 15% of aqueous extracts of shoot, leaf, fl ower and root parts and with 10 ml of distilled water as a control. the treatments were laid out in a completely randomized design with factorial arrangement in 220 a. netsere, e. mendesil four replications and kept at room temperature (21 22 °c) on a laboratory bench. the experiment was conducted twice, november 13 26 and december 1 14. 2008. data collected and statistical analysis number of germinated seeds (number of seedlings with visible shoot and root growth) was collected 15th day after treatment application. germination rate (gr) was calculated following the methodology of wardle et al. (1991) as follows: gr = (n1 * 1) + 1/2(n2 – n1) + 1/3(n3 – n2) +----+ 1/n(nn – nn1), where n1, n2, n3-----nn-1, nn is the proportion of germinated seeds obtained in the fi rst (1), second (2), third (3),------(n – 1), (n) days. shoot and root length (cm) of the respective crop were measured on the same day using a ruler by taking ten seedlings per treatment at random. however, if the germination percentage was less than 10, all the seedlings were used as the sample. seedlings sampled from a treatment for measurement of shoot and root lengths were partitioned in to the respective plant parts and oven dried at 70 °c for 24 hours to a constant weight and dry matter of each part was weighted separately using sensitive electronics balance. finally, the average data obtained from the two experiments were subjected to analysis of variance using sas software (sas institute, 2001) and means separation was made using duncan’s multiple range test at 0.01 probability level (mandefero, 2005). results the result depicted that signifi cant (p < 0.01) difference between parthenium plant parts, concentration levels and their interaction for all the parameters considered (tab. 1 and 2). aqueous extract of fl ower at all concentration levels, leaf at 10 and 15% and shoot at 15% concentration levels 100% inhibited seed germination and subsequent growth of haricot bean and soybean. likewise, very low germination, shoot and root growth and dry matter of the seedlings recorded at 5% leaf extract. in contrast, the 5% shoot and root aqueous extract had lower effect on germination and growth of the crops, while at high concentration greatly reduced germination, growth and dry matter production of the crops. the result also depicted that soybean and haricot bean root was more sensitive to allelopathic effect than shoot (tab. 1 and 2). discussion aqueous extracts of shoot, leaf, fl ower and root of parthenium weed exhibited allelopathic effect on soybean and haricot bean seed germination, germination rate, and shoot and root growth and dry matter production of seedlings. the allelopathic effect varied among the concentration levels of the extracts and the part of the weed from which they were extracted. aqueous extracts of fl ower at 5, 10 and 15%, shoot at 15%, and leaf at 10 and 15% concentration completely inhibited seed germination and subsequent shoot and root growth of the crops (tab. 1 and 2). although the allelopathic effect varied among the plant parts, all plant parts exhibited allelopathic effects (tab. 1 and 2). this is attributed due to the release of different kinds of phytotoxic compounds, viz. phenolics, sesquiterpenes and lactones, from root and vegetative part of living plants as well as from the achen by exudation (guzman, 1988) and leaching (mersie and singh, 1987; stephan and sowerby, 1996; evans, 1997; belz et al., 2007), respectively. the present study also showed that, aqueous extracts of fl ower at all concentration levels and leaf at intermediate and higher concentration levels induced the highest allelopathic effects as indicated by complete failure of germination and seedling growth. this could be due to the presence of high level inhibitory compounds tab. 1: effect of different concentration of aqueous extracts of parthenium plant parts on soybean bean seed germination, shoot and root growth and dry matter production plant part concentration germination germination shoot length root length shoot dry root dry level (%) (%) rate (ngspd)§ (cm) (cm) matter (g) matter (g) control 0 96a 23.45a 4.00a 8.41a 10.64a 31.75a shoot 5 48bc 11.75bc 1.66d 1.84d 1.51d 2.09e 10 32cd 8.52d 1.34e 1.57e 1.05e 1.10ef 15 0f 0f 1.01ef 1.11f 0.6f 0.86f leaf 5 0.4ef 0.55f 1.07ef 1.58e 1.05e 2.10e 10 0f 0f 0f 0g 0f 0f 15 0f 0f 0f 0g 0f 0f flower 5 0f 0f 0f 0g 0f 0f 10 0f 0f 0f 0g 0f 0f 15 0f 0f 0f 0g 0f 0f root 5 65b 14.87b 2.20b 4.30b 6.83b 11.01b 10 40cd 9.90c 2.107c 2.57c 3.90c 9.01c 15 25e 5.42e 1.04d 1.17f 1.89cd 3.79d f-test ** ** ** ** ** ** se (+) 1.83 1.77 2.08 1.75 3.10 1.58 cv (%) 8.10 15.12 8.70 9.06 10.42 7.25 ** = signifi cant at 0.01 probability level. means within a column followed by same superscript letter (s) are not signifi cantly different at 0.01probability level. §ngspd = number of germinated seeds per day. parthenium hysterophorus 221 in these plant parts as compared to shoot and roots (kanchan and jaycharad, 1980). similarly, belz et al. (2007) report parthenin released to the soil during decomposition of parthenium leaves is the leading toxic compound causing phytotoxicity. soybean and haricot bean roots were appeared more sensitive to allelopathic effect than shoot, which is in agreement with the results of tefera (2002) and wakjira et al. (2005). this may be due to direct contact of the root with the extracts and subsequently with inhibitory compounds (qasem, 1995; tefera, 2002). the reduction in seedling roots length may be attributed to the reduced rate of cell division due to the presence of allelochemicals, which might inhibit gibberellin and indoleacetic acid function (tomaszewski and thimann, 1966). the fi ndings of this study demonstrated that parthenium weed has allelopathic effect on soybean and haricot bean as indicated by its inhibitory effect on germination, germination rate, growth and dry matter production of the seedlings. similar results have been reported for cabbage (kohli et al., 1985), pumpkin (cucurbita moschata) (guzman, 1988), tomato (mersie and singh, 1988), multi purpose trees and arable crops (swaminathan et al., 1990; evans, 1997), tef (eragrostis tef) (eragrostis tef) (eragrostis tef tefera, 2002) and maize and sorghum (tamado et al., 2002). from the present preliminary investigation under laboratory condition, it can be concluded that parthenium has the potential of inhibiting seed germination and seedling growth of soybean and haricot bean. this is a clear indication parthenium is going to be a menace in the production of the crop. hence integrated management strategy of parthenium should be sought and this calls for a concerted effort of all institutions affi liated in agricultural production. acknowledgments the authors thank dr. tesfatesfat bogale, the staff of field crop agronomy tab. 2: effect of different concentration of aqueous extracts of parthenium plant parts on haricot bean seed germination, shoot and root growth and dry matter production plant part concentration germination germination shoot length root length shoot dry root dry level (%) (%) rate (ngspd)§ (cm) (cm) matter (g) matter (g) control 0 97a 22.03a 4.62a 11.49a 18.92a 40.21a shoot shoot shoot 5 60b 16.002b 1.15e 1.98e 1.94e 3.89e 10 32cd 11.68c 1.07e 1.52f 1.77f 1.54ef 15 0f 0f 0f 0g 0g 0f leaf leaf leaf 5 8ef 0.86f 0.02f 0.02g 0.33g 0.01f 10 0f 0f 0f 0g 0g 0f 15 0f 0f 0f 0g 0g 0f flower flower flower 5 0f 0f 0f 0g 0g 0f 10 0f 0f 0f 0g 0g 0f 15 0f 0f 0f 0g 0g 0f root root root 5 63b 15.62b 2.60b 5.30b 8.66b 14.74b 10 39c 10.51d 2.07c 3.97c 5.16c 10.24c 15 21de 3.76e 1.40d 1.78d 2.37d 5.79d f-test f-test f-test ** ** ** ** ** ** se (+) 2.12 0.61 0.19 0.05 0.15 0.35 cv (%) 9.35 8.15 7.78 4.43 7.91 6.99 ** = signifi cant at 0.01 probability level. means within a column followed by same superscript letter (s) are not signifi cantly different at 0.01probability level. §ngspd = number of germinated seeds per day. research division of jimma agricultural research center, ethiopia, for providing soybean and haricot bean seeds for the study. references aneja, k.r., dhawan, s.r., sharma, a.b., 1991: deadly weed. parthenium hysterophorus l.. indian journal of weed sciences 23, 4-19. belz, r.g., reinhardt, c.f., foxcroft, l.c., karl, h., 2007: residual allelopathy in parthenium hysterophorus l.. does parthenin play a leading role? crop protection 26, 237-245. deyama, d.p., 1986: allelophatic effect of deyama, d.p., 1986: allelophatic effect of deyama, d.p parthenium hysterophorus l. on growth, nodulation and nitrogen content of leucaena lucocephale. leucaena research report 7, 36-37. evans, h.c., 1997: parthenium hysterophorus: a review of its weed status and possibilities for biological control. biocontrol news and information 18, 89-98. guzman, c.d., 1988: allelopathic effects of seven weed species in pumpkin (cucurbita moschata) under green house conditions. journal of agriculture 72, 491-493. kanchan, s.d., jayachandra, 1980: allelopathic effects of parthenium hysterophorus l. ii. leaching of inhibitors from aerial vegetative part. plant and soil 55, 61-66. khosla, s.n., sobti, s.n., 1981: effective control of parthenium hysterophorus l.. pesticide 15, 18-19. kohli, r.k., anita kumari, r.p., saxena, d.b., 1985: autoand teletoxicity of parthenium hysterophorus l.. acta universitatis agriculturae brno, a facultas agronomica 33, 253-263. mandefero, n., 2005: statistical procedures for designed experiments. ethiopian agricultural research organization, addis ababa, ethiopia. mekbib, f., kebede, s., dejene, m., 1996: prevalence and distribution of parthenium hysterophorus l. in eastern ethiopia. arem 1, 19-26. mersie, w., singh, m., 1987: allelopathic effect of parthenium hysterophorus extract and residue on some agronomic crop and weeds. j. chem. ecol. 13, 1739-1747. 222 a. netsere, e. mendesil mersie, w., singh, m., 1988: effect of phenolic acids and ragweed parthnium hysterophorus l. extracts on tomato (lycopersicum esculentum) growth and nutrient and chlorophyll content. indian journal of weed sciences 36, 278-281. nath, r., 1988: parthenium hysterophorus l., a review. agricultural reviews 9, 171-179. qasem, j.r., 1995: the allelopathic effect of three amaranthus spp. (pigweeds) on wheat (triticum durum). weed research 35, 41-49. rezene, f., mekasha, c., mengistu, h., 2005: spread and ecological consequence of parthenium hysterophorus in ethiopia. arem 6, 11-21. sas, 2001: sas institute, carry, north carolina, usa. stephen, w.a., sowerby, m.s., 1996. allelopathic potential of the weed parthenium hysterophorus l. in australia. plant protection q. 11, 2023. swaminathan, c., vinaya rai, r.s., sureshi, k.k., 1990: allelopathic effects of parthenium hysterophoruseffects of parthenium hysterophoruseffects of l. on germination and seedling growth of a few multipurpose trees and arable crops. journal of int. tree crops 6, 143-150. tamado, t., milberg, p., 2000: weed flora in arable fields of eastern tamado, t., milberg, p., 2000: weed flora in arable fields of eastern tamado, t., milberg, p ethiopia with emphasis on the occurrence of parthenium hysterophorusethiopia with emphasis on the occurrence of parthenium hysterophorusethiopia with emphasis on the occurrence of l. weed research 40, 507-521. tamado, t., ohlander, l., milberg, p., 2002: interference by the weed tamado, t., ohlander, l., milberg, p., 2002: interference by the weed tamado, t., ohlander, l., milberg, p parthenium hysterophorus l. with grain sorghum: infl uence of weed density and duration of competition. international journal of pest management, 48, 183-188. tadesse, b., das, t.k., mohadevappa, m., taye, t., tamado, t., 2005: parthenium distribution, biology, hazards and control measure in ethiopia. pest mgt. j. eth. 9, 1-15. tefera, t., 2002: allelopathic effects of parthenium hysterophorus extracts on seed germination and seedling growth of eragrostis tef. journal of eragrostis tef. journal of eragrostis tef agronomy and crop sciences 188, 306-310. tomaszewski, m., thimann, k.v., 1966: interactions of phenolic acids, tomaszewski, m., thimann, k.v., 1966: interactions of phenolic acids, tomaszewski, m., thimann, k.v metallic ions and chelating agents on auxin induced growth. plant physiology 41, 1443-1454. wardle, d.a., ahmed, m., nicholson k.s., 1991: allelopathic influence of nodding thistle (carduus mutans l.) seeds on germination and radicle growth of pasture plants. j. agric. res. 34, 185-191. wakjira, m., berecha, g., bulti, b., 2005: allelopathic effects of parthenium hysterophorus extracts on seed germination and seedling growth of lettuce. trop sci 45, 159-162. addresses of the authors: mr. anteneh netsere, jimma agricultural research center, p.o. box 192, jimma, ethiopia, e-mail: anetsere@yahoo.com mr. esayas mendesil (corresponding author), department of horticulture & plant sciences, jimma university college of agriculture and veterinary medicine, p.o. box 307, jimma, ethiopia, e-mail: emendesil@yahoo.com journal of applied botany and food quality 93, 59 65 (2020), doi:10.5073/jabfq.2020.093.008 1department of botany, school of mathematical and natural sciences, university of venda, thohoyandou, south africa 2department of plant production, school of agriculture, university of venda, thohoyandou, south africa propagation potential for the conservation of brackenridgea zanguebarica oliv., a critically endangered plant species endemic to vhembe district in limpopo province (south africa) tiawoun makuété andré patrick1*, tshisikhawe milingoni peter1, gwata eastonce tendayi2 (submitted: january 21, 2019; accepted: november 1, 2019) * corresponding author summary brackenridgea zanguebarica oliv. is an important multipurpose tree valued for its medicinal uses in vhembe district. the unsustainable harvesting coupled with poor seed germination in the wild is threatening its regeneration; which poses a challenge in efforts to its conservation. this study was conducted to identify suitable methods for propagating b. zanguebarica species using seeds and stem cuttings. seeds propagation was carried out to evaluate the effect of various pre-treatments. vegetative propagation was tested to assess if b. zanguebarica could be successfully propagated via stem cuttings with appropriate treatments. the results showed that b. zanguebarica seeds did not germinate at all under any of the conditions tested. stem cuttings presented a possibility of propagating this species despite the poor results obtained, where 51% of cuttings across all treatment produced buds and 17% only developed leaves without any root development. the growth media had insignificant (p > 0.05) effect on some vegetative growth parameters, while growth hormones showed significant (p < 0.05) effect in all the vegetative growth parameters of stem cuttings where iaa performed better than iba and nna. however, their interaction were significant (p < 0.05) on all the growth parameters of brackenridgea zanguebarica stem cuttings except on the percentage of cuttings that produced buds (p = 0.107). the findings showed that b. zanguebarica is difficult to propagate sexually and asexually, hence, further studies are needed to identify suitable methods for both seed and vegetative propagation of this plant. key words: multipurpose tree; seed propagation; stem cuttings; auxins; growth media. introduction the on-going disappearance of biological diversity due to various anthropogenic activities has increased drastically during the last few decades, to the point where many species are now threatened with extinction (hayes and hayes, 2013). many plant species are threatened due to indiscriminate overexploitation coupled with insufficient efforts made on the renewal of natural resources, (chen et al., 2016). the most affected species are those with small populations and narrow geographical ranges (wilson et al., 2004). this trend is alarming and immediate conservation measures are required to safeguard many of these species (sharanappa and rai, 2011). many plant species have the potential to be propagated with both sexual and asexual means. these techniques play an important role in the conservation of numerous threatened plant species with poor natural regeneration and offer a promising way for saving plants from extinction. in south africa, brackenridgea zanguebarica oliv. is a species comprised of a small population of plant found only in thengwe village within the vhembe district of limpopo province. it has been identified as an endemic plant species of that region. it is locally known as “mutavhatsindi” and commonly known as “yellow peeling plane”. the plant belongs to the family ochnaceae and can grow into a tree of 10 m high (tiawoun et al., 2018). its roots and barks are widely harvested and traditionally used in the treatment of amenorrhoea, swollen ankles, wounds, diarrhea and various others (arnold and gulumian, 1984; möller et al., 2006; bruschi et al., 2011). due to its multiple uses, the indiscriminate extraction and overexploitation of this important plant is depleting its population at an alarming rate. it has now been categorized according to the international union for conservation of nature (iucn) red list of south africa as a critically endangered plant species (williams et al., 2013). detailed information concerning the propagation is required to increase the availability of this plant species and to expand its distribution to new areas in the region. cultivation is crucial for in situ and ex situ conservation for this threatened plant species. there is therefore a need to take an initiative for mass propagation and reestablishment of the species in areas it used to occur in nature. the natural regeneration of b. zanguebarica from seeds in the wild is unsatisfactory in generating adequate plant material (mutshinyalo, 2011) and requires a long time to germinate. this is probably due to extremely complex seed dormancies that are not well understood and the vegetative regeneration process that has not yet been fully investigated. in order to minimize dependence of propagation by seeds, the use of growth media and hormonal supplements in vegetative propagation can be an effective alternative or supplementary method to seed propagation. many studies showed that growth media and hormonal concentration for vegetative propagation has successfully improved root initiation on stem cuttings (kassahun and mekonnen, 2012; kiuru et al., 2015). to ensure the conservation and the perpetuation of b. zanguebarica, the present study was aimed at enhancing its propagation by de veloping an understanding of sexual and asexual propagation of this species. the specific objectives were to develop a protocol for seed germination by testing several pre-treatments requirements and assess b. zanguebarica stem cuttings’ response to various auxins in different growth media in order to develop an efficient method for vegetative propagation. materials and methods study area and plant collection the collection of fruits and stem cuttings from b. zanguebarica trees was conducted in the brackenridgea nature reserve (bnr) and transported to the university of venda. this reserve is found in the vhembe district of limpopo province, south africa. it is located between the latitudes of 22° 24’ and 23° 36’s, and longitudes of 29° 12’ and 31° 12’e. the climate of the study area is semi-arid (nel and nel, 2009) with an average annual rainfall of about 350 mm (mzezewa et al., 2010); and the mean annual maximum and mini60 m.a.p. tiawoun, m.p. tshisikhawe, e.t. gwata mum temperatures are 26.5 °c and 16 °c respectively. the soil type is mainly stony with shallow sandy loam (mutshinyalo, 2011). to assess the propagation capacity of b. zanguebarica seeds and stem cuttings, the experiments were carried out in the shade house at the school of agriculture, university of venda (south africa). seed propagation the fresh and mature black fruits used in the experiments were picked from five different b. zanguebarica trees (fig. 1a) on the 30th of january 2017. a total of 360 seeds used for all the pre-treatments including control, were manually de-pulped from the fruits (fig. 1b), which were washed with distilled water and left to dry for 24 h at room conditions. preparation of plant hormones (auxins) and growth media each plant hormone (hormone powder) was prepared by dissolving a calculated amount of iaa, iba and naa in distilled water to obtain concentrations of 50 mg/ml, 100mg/ml, and 150 mg/ml respectively. the control solution was distilled water. the basal ends of about 2 cm of the cuttings were dipped for 30 sec in the prepared auxin treatment. each treatment was replicated four times and each repli cation consisted of 5 cuttings. a total of 200 stem cuttings were used for this experiment and the treated stem cuttings were individually planted into a perforated black plastic bags filled with growing media (top site soil and hygromix). each growth medium had 100 cuttings that were maintained in the shade house and watered when necessary every two to three days, until the termination of the experiment (90 d). fig. 1: brackenridgea zanguebarica seed. (a) fresh fruits (b) seed depulped from the fruit. after this procedure, the seeds were subjected to nine pre-treatments: t1: seeds soaked in concentrated sulfuric acid (95-97%) for 30 min, t2: seeds soaked in concentrated sulfuric acid (95-97%) for 60 min, t3: seeds soaked in hot water (100 oc) for 5 mins, t4: seeds soaked in hot water (100 oc) for 10 min, t5: seeds soaked in cold water for 24 hs, t6: seeds soaked in cold water for 48 h, t7: seeds cold stratified at 4 °c for 60 d, t8: seeds cold stratified at 4 °c for 90 d, t9: control (untreated seeds). seeds treated with concentrated sulfuric acid (95-97%) were washed thoroughly under tap water to remove all the acid residuals. forty seeds were used for each pre-treatment and each pre-treatment had four replicates of 10 seeds. each seed was sown to about the same depth as the size of the seed in a perforated black plastic bag containing the hygromix growth medium, and placed on a metal bench in the shade house at the school of agriculture on the 1st of february 2017. watering was done when necessary every two to three days to maintain the moisture content for better seed germination conditions throughout the duration of the experiments of twelve months. source of plant material and preparation of stem cuttings healthy semi hardwoods stem were collected in february 2018 from fresh branches of b. zanguebarica trees growing in the bnr. a total of 200 stem cuttings were uniformly trimmed into 10 cm long pieces, with a diameter of about 5 mm and each cutting having four to five nodes; these were used for all the treatments. after taking all the leaves off, the cuttings were treated with three types of auxins, namely, indole-3-acetic acid (iaa), indole-3-butyric acid (iba) and 1-naphthaleneacetic acid (naa) at different concentrations and the controls were treated with distilled water. all the cuttings were then planted in two types of growing media – top site soils (collected in the reserve) and hygromix. tab. 1: total number of cuttings in different types and concentrations of auxins and different growth media. auxins and concentrations of auxins (mg/ml) total number growth media of cuttings 0 50 100 150 iaa site soil 10 10 10 30 hygromix 10 10 10 30 iba site soil 10 10 10 30 hygromix 10 10 10 30 naa site soil 10 10 10 30 hygromix 10 10 10 30 control site soil 10 10 hygromix 10 10 data recording the following parameters were recorded: (1) days to first buds appearance: it was recorded from the day of planting to the minimum number of days taken to first buds appearance from one cutting of each treatment and then averaged to get the mean value. (2) percentage of cuttings that produced buds = number of cuttings with buds ×100 total number of cuttings planted (3) average number of buds per cutting: the total number of buds per cutting was counted in each treatment after 45 d and then averaged to get the mean value. (4) days to first leaves development: it was recorded from the day of planting to the minimum number of days taken to first leaves development from one cutting of each treatment and then averaged to get the mean value. (5) percentage of cuttings that produced leaves = number of cuttings with leaves ×100 total number of cuttings planted (6) average number of leaves per cutting: the total number of leaves per cutting was counted in each treatment after 90 d and then averaged to get the mean value. (7) percentage of cuttings that produced roots = number of cuttings with roots ×100 total number of cuttings planted improving conservation of brackenridgea zanguebarica through propagation 61 data analysis two-way anovas were performed to evaluate the effects of auxins concentration and growth media on the mean of each growth parameter mentioned above. the means of the significantly different growth parameters were separated using tukey test honestly significant differences (hsd), with p < 0.05 being considered as statistically significant. all statistical analyses were conducted using microsoft excel 2013. results and discussion seed germination in this study, brackenridgea zanguebarica seeds were subjected to numerous pre-treatments known to enhance seed germination in other species. these were pre-treatments with sulfuric acid, hot water, cold water, and stratification at low temperature. however, none of the 360 seeds germinate within 12 months of the experiment. none of the treatments was able to overcome the seed dormancy of b. zanguebarica. the inability of all pre-treated seeds to germinate might be due to the hard seed coat, which prevented water imbibition and gas exchange. brackenridgea zanguebarica seeds seem to exhibit deep physical dormancy leading to poor seed germination. there are reports that revealed that seeds of many species showed delayed germination because of hard seed coats (el-juhany et al., 2009; babashpour-asl et al., 2011; zhang et al., 2015). this physical dormancy is clearly one of the problems that make this species difficult to propagate from seeds and this could explain partially the scarcity of this species in the wild. further experimentations should include the combination of treatments, as well as other pre-treatment methods should be tested. for example, physical dormancy can be broken by fluctuating temperatures, fire and passage through the digestive tracts of animals (baskin and baskin, 1998). baskin (2003) revealed that the fluctuating temperatures promoted dormancy break in impermeable seeds of stylosanthes humilis and s. hamata (fabaceae). additionally, the application of gibberellic acid (ga3) is also necessary in case this seed may present physiological dormancy. lack of information about seed propagation of brackenridgea zanguebarica remains an immense challenge to developing a successful technique for promoting germination and conserving this plant species. thus, at the current state, preference should be given to vegetative propagation rather than propagation from seeds for largescale plant production. effect of various auxins concentration and growth media on the vegetative growth parameters of brackenridgea zanguebarica stem cuttings. stem cuttings of b. zanguebarica with four to five nodes each were treated with three kinds of auxins at three concentrations each, to evaluate the hormonal effect on vegetative growth. the cuttings was planted in soil from the collection site or hygromix, monitored for 90 d and several growth parameters were recorded. for all the treated cuttings including the controls, the first bud emergence (fig. 2a) was in the range of 9.5 to 13 d (tab. 3). the effects of auxins concentrations were highly significant (p < 0.001) for days to first buds emergence while the effects of growth media on days to first buds development were not significant (p > 0.05). however, significant interaction effects (p < 0.05) were observed between auxins concentrations and growth media for days to first buds appearance (tab. 2). percentage of cuttings that produced buds was significantly influenced by growth media and auxins concentration but not significantly affected by their interaction (tab. 2). the highest percentage of cuttings that produced buds was recorded under site soil than those observed under hygromix (tab. 3). cuttings from all treatment groups developed buds and the average number of buds per cutting varied from 0.4 to 3.7. average number of buds per cuttings after 45 d was significantly affected by both hormone concentration and growth media. their interaction also had significant effect on the average number of buds per cuttings (tab. 2). most of the buds wilted and started dying during their development and by day 45 no further bud growth was observed in any of the treatments and control cuttings. only a few buds were able to survive and produce leaves (fig. 2b). there was no significant (p > 0.05) effect of growth media on the average day to first leaves development. but, day to first leaves de velopment reveals highly significant (p < 0.001) effect of auxins concentrations. the interaction between auxins concentrations and growth media showed significant difference (p < 0.05) on time taken for first leaves development (tab. 2). percentage of cuttings that produced leaves was significantly (p < 0.05) affected by growth hormone and the interaction between growth hormone and growth media. but insignificant (p > 0.05) effect of growth media (tab. 2). the highest percentage of cuttings that produce leaves was observed in iaa100 (40%) when propagated in both site soil and hygromix; whereas, no cuttings produced leaves in iba150 and naa100 (tab. 3). at the end of the experiments (90 d) some leaves were well expanded (fig. 2c). the number of leaves per stem cutting treated with differ ent auxin, including control, and planted in different growth media varied from 0.1 2. there was significant (p < 0.05) effect of hormones application on the average number of leaves per stem cutting with no significant (p > 0.05) effect of growth media. the average number of leaves per stem cutting was affected by the interaction of various auxins and growth media at p < 0.05 (tab. 2). overall, two-way anovas (tab. 2) showed that various auxins concentrations were highly significant (p < 0.001) in the mean number of all the vegetative growth parameters tested while only the percentage of cuttings that produced buds and the average number of buds per cuttings after 45 d were significantly affected by the growth media. however, the interaction of auxins concentrations and growth media were significant (p < 0.05) on the growth parameters of brackenridgea zanguebarica stem cuttings except on the percentage of cuttings that produced buds (p = 0.107). remarkably, none of the cuttings had produced roots by the end of the experiment after 90 d (tab. 3). with auxin type and concentration, and their interaction with growth media, it was possible to generate from b. zanguebarica stem cuttings, some of the vegetative growth parameters. however, based on the performance of the untreated cuttings in all the parameters studied, it was clear that the application of all these treatments did not considerably confer any advantage for the vegetative growth para meters. all treated and untreated cuttings were ineffective to induce root growth on stem cuttings. even though, the cuttings were more responsive to auxins treatment than control, the difference was not statistically significant indicating that b. zanguebarica stem cuttings can develop buds and leaves without the requirement of hormones. similar results were also found in lippia javanica and vitex leucoxylon stem cuttings which showed no significant differences between cuttings with growth hormone application and untreated cuttings on some vegetative growth parameters studied (tiwari et al., 2015). different auxins have been successfully used for the rooting of many plant species. according to stuepp et al. (2017), plant growth hormones control all phases of growth and development from embryogenesis to senescence. the same authors reported that, auxins are critical in the regulation of many aspects of plant growth and development and their endogenous levels are considered important in the process of root induction. root growth, however, was not recorded in 62 m.a.p. tiawoun, m.p. tshisikhawe, e.t. gwata fig. 2: example of different stages of vegetative growth parameters used to score the response of brackenridgea zanguebarica stem cuttings to different treatments in both growth media. (a) buds emergence; (b) leaves appearance; (c) leaves expansion. tab. 2: two-way anova of the parameters of brackenridgea zanguebarica stem cuttings showing the effects of various auxins concentrations and growth media and their interaction. source of variation ss df ms f p-value f crit days to first buds appearance growth media 1.8 1 1.8 3.375 0.071 4.001 auxins concentration 51.2 9 5.689 10.667 0.000 2.040 growth media × auxins concentration 10.2 9 1.133 2.125 0.041 2.040 within 32 60 0.533 percentage of cuttings that produced buds growth media 1445 1 1445 10.081 0.002 4.001 auxins concentration 16725 9 1858.333 12.965 0.000 2.040 growth media × auxins concentration 2205 9 245 1.709 0.107 2.040 within 8600 60 143.333 average number of buds per cuttings after 45 d growth media 2.926 1 2.926 9.377 0.003 4.001 auxins concentration 61.145 9 6.794 21.772 0.000 2.040 growth media × auxins concentration 6.190 9 0.688 2.204 0.034 2.040 within 18.723 60 0.312 day to first leaves development growth media 0.0125 1 0.013 0.020 0.889 4.001 auxins concentration 1584.563 9 176.063 276.176 0.000 2.040 growth media × auxins concentration 1669.363 9 185.485 290.956 0.000 2.040 within 38.25 60 0.6375 percentage of cuttings that produced leaves growth media 180 1 180 3.6 0.063 4.001 auxins concentration 10580 9 1175.556 23.511 0.000 2.040 growth media × auxins concentration 1520 9 168.889 3.378 0.002 2.040 within 3000 60 50 average number of leaves per cutting after 90 days growth media 0.144 1 0.145 1.040 0.312 4.001 auxins concentration 29.470 9 3.275 23.558 0.000 2.040 growth media × auxins concentration 2.823 9 0.314 2.257 0.030 2.040 within 8.34 60 0.139 improving conservation of brackenridgea zanguebarica through propagation 63 this study after the application of auxins to the stem cuttings. puri and swamy (1999) and guo et al. (2009) state that, the effects of auxins application on stem cuttings vary between species because of their genetic differences. a growth medium with an adequate supply of nutrients is important as it promotes the growth and development processes of plants. the mean values for the different parameters showed that the cuttings of b. zanguebarica responded positively to both the growth media used in this study (table 3). from these results, it is clear that the growth and development of b. zanguebarica stem cuttings may not only depend of soil nutrients. according to takoutsing et al. (2014), a good growth medium must have low water holding capacity and have a considerable level of porosity. stem cutting is the most frequent technique used for vegetative propagation of many plant species (hassanein, 2013) and growth regulators and growth media were found to be effective factors for rooting many plant species (akinyele, 2010). the performance of untreated cuttings in all the parameters demonstrated that auxin application and growth media may not necessarily be the major factor influencing buds and leaves growth on stem cuttings of this species. wahab et al. (2001); hartmann and kester (1990) stated that the development of buds and leaves from stem cuttings can be attributed to high levels of plant carbohydrate reserve, however, leakey and coutts (1989), reported that carbohydrate content can quickly become depleted in cuttings without leaves or with very small leaf area, but increases if leaf area is appropriate; at least until roots start to develop. stem cuttings without leaves were used in this study and the rapid buds and leaves development was probably due to the use of high carbohydrate reserve and slow reconstitution by stem cuttings, leading to the absence of root development. similar findings have been obtained by takoutsing et al. (2014) in leafless cuttings of garcinia lucida, therefore, the type and concentration of auxins applied on b. zanguebarica stem cuttings may not have any direct effect on bud and leaf development. these results are similar to the findings of ullah et al. (2005) who reported that different growth regulators did not have significant effect on the shoot development of psidium guajava. even though growth regulators and growing media are found to be effective factors for root development of many plant species, several researchers reported that root development from treated cuttings may also depend on a number of factors, such as type of cuttings, growing media, growth regulators, size of cuttings, season of planting (environmental conditions), cutting source (young seedlings or juvenile) and leaf area, (tchinda et al., 2013; naidu and jones, 2009; takoutsing et al., 2014). the inability of root development in b. zanguebarica from stem cuttings may be due to some of these factors and also to the application method of the growing media and growth regulators that did not produce the expected effects. for instance, ahmad (2006) reported that dipterocarp species failed to root when the stem cuttings dropped their leaves one or two days after planting. similarly, several studies have reported that stem cuttings without leaves presented a high mortality rate in vegetative propagation (ofori et al., 1996; akinyele, 2010). however, many other studies revealed that the presence of leaves on cuttings is necessary to stimulate root initiation (hartmann et al., 1990; zem et al., 2016; belniaki et al., 2018). therefore, buds and leaves development on stem cutting of this species was an important stage to stimulate root initiation; this has shown the possibility to propagate this species by stem cuttings if appropriate treatments are provided. for this tab. 3: effect of auxins concentration and growth media on vegetative growth parameters of brackenridgea zanguebarica stem cuttings. data are shown as mean values of four replicates ± se. growth parameters buds leaves roots growth auxins days to firs % of cuttings average number day to firs % of cuttings average number % of cuttings media concentration buds that produced of buds leaves that produced of leaves that produced appearance buds per cutting development leaves per cutting roots after 45 d after 90 d iaa 50 10.5 ± 0.6abc 70 ± 16.3efg 3.1 ± 0.8hij 21 ± 1cd 25 ± 4de 0.5 ± 0.3ab 0 100 09.5 ± 0.6a 60 ± 0.0cdef 3.7 ± 0.6j 18.5 ± 1.3b 40 ± 8.1f 2 ± 0.7d 0 150 11.5 ± 0.6cde 30 ± 11.5a 0.4 ± 0.3a 21 ± 0.8cd 15 ± 4bcd 0.4 ± 0.2ab 0 site soil iba 50 11.5 ± 1.3cde 60 ± 0.0cdef 2.9 ± 0.6ghij 20.5 ± 1c 15 ± 4bcd 0.25 ± 0.1a 0 100 11 ± 0.8bcd 65 ± 25.8defg 3.5 ± 0.7ij 22.5 ± 0.6de 20 ± 11.5cd 1.5 ± 0.6cd 0 150 13 ± 0.0f 40 ± 16.3abc 1 ± 0.5abc 0 ± 0.0a 0 ± 0.0a 0 ± 0.0a 0 naa 50 11.5 ± 6cde 60 ± 11.5cdef 1.6 ± 0.5abcde 21.5 ± 1cde 10 ± 8.1abc 0.7 ± 0.6ab 0 100 10 ± 0.0ab 30 ± 11.5a 0.9 ± 0.4ab 20 ± 0.8bc 10 ± 4abc 0.6 ± 0.4ab 0 150 12 ± 0.8cdef 55 ± 11.5cdef 2.6 ± 0.4efghi 23 ± 0.8e 35 ± 8.1ef 1 ± 0.5bc 0 control 00 12 ± 0.8cdef 85 ± 12.2g 2.8 ± 0.7ghij 20 ± 1bc 15 ± 10.8bcd 0.5 ± 0.2ab 0 iaa 50 10.5 ± 0.6abc 50 ± 4abcde 2.7 ± 0.8fghij 20.5 ± 0.6c 35 ± 0.0ef 0.7 ± 0.2ab 0 100 10 ± 0.8ab 60 ± 0.0cdef 2.7 ± 0.5fghij 18.5 ± 1.3b 40 ± 8.1f 1.6 ± 0.6cd 0 150 10.5 ± 1abc 30 ± 11.5a 1 ± 0.4abc 21.5 ± 0.5cde 5 ± 0.0ab 0.1 ± 0.1a 0 hygromix iba 50 11.5 ± 1.3cde 60 ± 0.0cdef 2.1 ± 0.5defgh 21 ± 0.8cd 20 ± 11.5cd 0.15 ± 0.1a 0 100 11 ± 0.8bcd 35 ± 7ab 2.7 ± 0.7fghij 22.5 ± 0.6de 5 ± 5.8ab 1.5 ± 0.6cd 0 150 12.5 ± 0.6def 40 ± 16.3abc 1 ± 0.5abc 20.5 ± 0.6c 10 ± 8.1abc 0.2 ± 0.1a 0 naa 50 11.5 ± 0.6cde 40 ± 11.5abc 1.2 ± 0.5abcd 21.5 ± 1cde 10 ± 8.1abc 0.1 ± 0.2a 0 100 10.5 ± 0.6abc 30 ± 11.5a 1.5 ± 0.4abcd 0 ± 0.0a 0 ± 0.0a 0 ± 0.0a 0 150 11.5 ± 0.6cde 45 ± 11.5abcd 2 ± 0.4cdefg 20 ± 0.0bc 25 ± 8.1de 1.8 ± 0.5d 0 control 00 10 ± 0.0ab 75 ± 10.8fg 1.8 ± 0.7bcdef 22.5 ± 0.6de 5 ± 7ab 0.1 ± 0.1a 0 mean 11.1 51 2.06 18.8 17 0.69 0 means followed by the same letter within each column are not significantly di ferent at p < 0.05 according to tukey hsd test. 64 m.a.p. tiawoun, m.p. tshisikhawe, e.t. gwata reason, more studies with different interactions of growth media and hormones need to be conducted as these factors may promote root growth and development of b. zanguebarica stem cuttings. conclusion this is the first study to have reported on the propagation by seeds and stem cuttings of b. zanguebarica, with the aim of improving and promoting its conservation. the results of this study demonstrated that seeds subjected to different pre-treatments exhibited difficulties in germination under all the tested conditions and this may partially explain the scarcity of this plants species in the wild. based on the trends observed, the protocol for vegetative propagation has shown, for the first time, promising results despite the very low growth parameters recorded. despite the unsatisfactory results from both techniques, this study, nevertheless has improved the scientific understanding of seeds and vegetative propagation of b. zanguebarica. the nature of seed dormancy of this species is not well understood and further research need to define the type of dormancy before b. zanguebarica can be propagated. for vegetative propagation, mixture of auxins, reliable growth media, size of cuttings, environmental conditions and cutting source are required. tissue culture propagation should be tried as another means for mass production. acknowledgments the authors would like to express their sincere gratitude to the university of venda for financial support. we also thank the management of brackenridgea nature reserve, for allowing us to conduct the study in the reserve and the assistance of mr maluta with field work is acknowledged. conflict of interest no potential conflict of interest was reported by the authors references ahmad, d.h., 2006: vegetative propagation of dipterocarp species by stem cuttings using a very simple technique. in: suzuki, k., ishii, k., sakurai, s.s.s. 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international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.9734/arrb/2018/44847 http://dx.doi.org/10.13057/nusbiosci/n070204 http://dx.doi.org/10.4102/koedoe.v55i1.1072 http://dx.doi.org/10.3923/ajps.2005.238.243 http://dx.doi.org/10.3923/jbs.2001.184.187 http://dx.doi.org/10.1016/j.sajb.2013.01.006 http://dx.doi.org/10.1038/nature03031 http://dx.doi.org/10.1590/0103-8478cr20141486 art13_ercisli.indd journal of applied botany and food quality 85, 86 90 (2012) 1*department of horticulture, faculty of agriculture, ataturk university, erzurum, turkey 2department of food science, faculty of agriculture, ataturk university, erzurum, turkey 3department of food science, faculty of engineering, gumushane university, gumushane, turkey 4faculty of agriculture, zagreb university, zagreb, croatia phytochemical and antioxidant characteristics of medlar fruits (mespilus germanica l.) s. ercisli1*, m. sengul2, h. yildiz3, d. sener2, b. duralija4, s. voca4, d. dujmovic purgar4 (received november 19, 2011) * corresponding author summary eleven medlar (mespilus germanica l.) genotypes sampled from turkey were analyzed for their fruit weight, fruit dimensions, fruit fi rmness, ostiole diameter, shape index, skin color, moisture (%), ash (%), reducing sugar (%), crude protein (%), ph, soluble solid content (%), vitamin c (mg/100 g), minerals (p, k, ca, mg, fe, zn, mn), total phenolic content and total antioxidant capacity. a wide variation among genotypes on most of the searched parameters was evident. fruit weight varied from 11.21 g to 33.24 g indicating high variability among genotypes. determination of antioxidant activities by β-carotene – linoleic acid and 2-diphenyl-1-picrylhydrazyl (dpph) free radical scavenging assays resulted in average 80.8%, and 46.6 µg/ml fw dpph, respectively. the total phenolic contents of eleven medlar genotypes varied from 114 to 293 mg gallic acid equivalent in 100 g fresh weight basis. the medlar fruits were found to be rich in terms of potassium, calcium, phosphorus, magnesium and iron. introduction the association between a diet rich in horticultural crops and a decreased risk of cardiovascular disease and certain forms of cancer is supported by considerable epidemiological evidences (ziegler, 1991; law and morris, 1998). guidelines of healthy nutritions have also directed the general public to eat more fresh horticultural crops, fruit and vegetables, throughout the world for prevention such kind of diseases. it is well known that horticultural crops in particular berries are the main sources of natural antioxidants (heinonen et al., 1998; hegedus et al., 2008; tulipani et al., 2008). more recently underutilized fruits, small fruits, such as cornelian cherry, mountain ash, sea buckthorn, rose hip, service tree, elderberry, bilberry, mulberry, jujube are also being increasingly consumed mainly due to their pleasant fl avor and their perceived health benefi ts related to their vitamins, antioxidants and minerals (ercisli and orhan, 2007; serteser et al., 2008; tomosaka et al., 2008). the human healthy benefi ts such as antioxidants of common consumed fruits have been reported (heinonen et al., 1998; mohamed et al., 2007; netzel et al., 2007; voca et al., 2008). however, chemical composition of under utilized fruits including medlar is scarce. the assessment of such properties remains can be an interesting and useful task, particularly for fi nding new sources for natural antioxidants, functional foods and nutraceuticals. in addition more recently under utilized fruit market, once restricted to local areas, has increasingly expanded to the metropolitan centers in most of the countries. thus, information on the human healthy values of these kinds of fruits could be a great importance (arabshahidelouee and urooj, 2007). the antioxidant activity of fruits varies considerably. these differences may be due to multiple reasons including genetic factors or cultivar differences, different environmental conditions, stage of maturity, growth stage, soil fertilization and the part of the plant used, amongst other factors that propitiate quantitative variation in these phytochemicals (netzel et al., 2007; ercisli and orhan, 2008). medlar, mespilus germanica l. belongs to rosaceae family and it grows mainly in frost-free areas, and on rocks and poor soils. in turkey, they are abundant particularly in north and west-anatolia and marmara regions (browicz, 1972). it is one of the latest maturating fruits and the ripening occurs in late october before frosts in turkey. the fruits are used as a nutrition component by the local population and are prepared by the local people as marmalade or pickle. the fruit is consumed as a medicinal remedy for example treatment of constipation, diuretic, and to rid the kidney and bladder of stones in turkey (baytop, 1999). the increasing demand for natural antioxidants, together with the introduction of new technologies to meet the new quality standards, justifi es the search for new sources of natural antioxidants. the present study is aimed at assessing the phytochemical content of medlar fruits from turkey, paying special attention in order to identify new sources of natural antioxidants. materials and methods collection and preparation of medlar fruits approximately 3 kg fruit from each of eleven medlar genotypes were sampled from coruh valley in turkey. the genotypes were pre-selected according to their rising yield capacity, attractive fruit properties and absence of pest and disease indicators. fruits were harvested at commercial maturation stage (skin brownish, pulp white, fruit hard) by hand and transferred to the laboratory for physical and phytochemical analysis. samples were frozen immediately and then stored in about 100 g batches at -30 °c prior to analysis. determination of fruit weight, dimensions, fi rmness and skin colors in medlar fruits fifty fruits from each genotype were used immediately after harvest for fruit weight, dimensions, fi rmness and color determination. fruit weight was measured by using a digital balance with a sensitivity of 0.001 g (scaltec spb31). linear dimensions of fruits as length (l) and width (w) was measured by using a digital calliper gauge with a sensitivity of 0.01 mm. fruit fi rmness was measured at 22 °c using a non-destructive fi rmness device (aweta, nl). skin color of medlar fruits was measured by using a cr-400 chromometer (konica minolta, japan) and the color of the fruit surface was determined for the l (lightness), a (green chromaticity) and b (yellow chromaticity) values. chroma and hue were then calculated as described by mcguire (1992): chroma = (a2+b2)1/2 hue angle = tan-1 (b/a) color values for every fruit were computed as means of triplicate measures on equidistant points of each fruit. phytochemical and antioxidant characteristics of medlar fruits 87 determination of moisture, ash, soluble solid content (ssc), vitamin c, ph, reducing sugar and crude protein in medlar fruits for each genotype, total 50 fruits were thawed at room temperature and homogenized in a standard food blender. homogenates were assayed for ph, reducing sugar, soluble solid content (ssc) and vitamin c. total soluble solid contents (tss) were determined by a digital refractometer (model ra-250he, kyoto electronics manufacturing co. ltd., japan) at 22 °c. moisture and ash were determined by aoac (1984). the kjeldahl method (bremner, 1996) and a vapodest 10 rapid kjeldahl distillation unit (gerhardt, königswinter, germany) were used to determine total n. ascorbic acid (vitamin c) and reducing sugar of samples was quantifi ed with the refl ectometer set of merck co (merck rqfl ex). determination of total phenolics and antioxidant activity in medlar fruits for extraction, fruit homogenates obtained with a blender were extracted with a buffer containing acetone, water, and acetic acid (70:29.5:0.5, v/v/v) for 1 h in darkness (singleton and rossi, 1965). this extract was fi ltered and used for phytochemical analysis. total phenolics in the methanol extracts were determined colorimetrically using folin-ciocalteu reagent as described by slinkard and singleton (1977). gallic acid was used as the standard and results were expressed as mg gallic acid equivalents per 100 g fresh weight basis. total antioxidant capacity of samples was determined by β-carotene bleaching and 2,2-diphenyl-1-picrylhydrazyl radical (dpph•) assays. in the β-carotene bleaching assay, antioxidant capacity is determined by measuring the inhibition of the volatile organic compounds and the conjugated diene hydroperoxides arising from linoleic acid oxidation (kaur and kapoor, 2002). antioxidant capacities of the samples were compared with those of the synthetic antioxidant butylated hydroxyanisole (bha) and the blank. in dpph assay, 50 µl of various concentrations of the extracts in methanol were added to 5 ml of a 0.004% methanol solution of dpph.. after a 30 min incubation period at room temperature, the absorbance was read against a blank at 517 nm. inhibition of free radical dpph in percent (i%) was calculated in following way: i% = (a= (a= ( blank-asample/a/a/ blank) x100; where ablank is the absorbance of the control reaction (containing all reagents except the test compound), and asample is the absorbance of the test compound. extract concentration providing 50% inhibition (ic50) was calculated from the graph plotting inhibition percentage against extract concentration. tests were carried out in triplicate. results were expressed as µg/ml fw (burits and bucar, 2000). determination of mineral elements fruit samples were oven-dried at 68 °c for 48 h and ground to pass 1 mm. phosphorus content was determined after wet digestion using a hno3-hclo4 acid mixture (4:1 v/v). phosphorus in the extraction solution was measured spectrophotometrically using the indophenol-blue and ascorbic acid method with a uv/vis aquamat spectrophotometer (thermo electron spectroscopy ltd, cambridge, uk). k, ca, mg, fe, mn and zn were determined after wet digestion using a hno3-hclo4 acid mixture (4:1 v/v) with a perkin-elmer 360 atomic absorption spectrophotometer (perkinelmer, waltham, massachusetts, usa). results were expressed in mg/100g fresh mass for p, k, ca, mg, fe, mn and zn. statistical analysis the experiment was a completely randomized design with 5 replications. data were subjected to analysis of variance (anova) and means were separated by duncan’s multiple range test at p<0.05 signifi cant level. results and discussion fruit weight, dimensions, fi rmness and colors of medlar fruits the fruit weight, dimensions, fi rmness, shape index, ostium diameters and colors of fruits in eleven medlar genotypes are shown in tab. 1. statistically signifi cant differences were recovered between the means for all the traits tested (tab. 1). the highest fruit weight was observed in genotype m7 as 33.24 g, and followed by m3 (22.71 g) and m6 (16.42 g), respectively. fruit dimensions are also found very variable among genotypes from 27.45 to 38.88 mm for length and 28.44 to 42.51 mm for diameter (tab. 1). on the other hand shape index was found between 0.81 and 1.09 indicating some genotypes have pear-shaped (m2 and m8) and the others are apple-shaped form. previously a wide variation on fruit weight and dimensions has been observed in medlar genotypes from 9.46 to 40.80 g for fruit weight, 23.67 to 42.51 mm for fruit length and 26.53 to 48.73 mm for fruit diameter (ozkan et al., 1997; bostan, 2002; bostan and islam, 2007). our results are within the range of the values reported tab. 1: fruit weight, dimensions and color characteristics of samples genotypes fruit weight fruit length fruit diameter fruit fi rmness shape index ostiole diameter hue chroma (g) (mm) (mm) (kg/cm2) (mm) (deg) (%) m1 14.32bc 32.23ab 30.75c 0.35ab 0.95ab 18.81bc 67.79bc 40.87ab m2 15.83bc 37.03ab 29.82cd 0.38ab 0.81c 15.51bc 72.85ab 33.45b m3 22.71b 34.76ab 36.62b 0.48ab 1.05ab 20.44b 61.92c 42.45ab m4 11.21c 28.73ab 28.44d 0.31b 0.99ab 16.14bc 80.54a 43.30a m5 12.94c 30.13ab 28.84cd 0.35ab 0.96ab 16.93bc 70.44b 42.21ab m6 16.42bc 32.62ab 31.68bc 0.41ab 0.97ab 18.68bc 69.63bc 39.98ab m7 33.24a 38.88a 42.51a 0.61a 1.09a 26.48a 68.85bc 33.21b m8 14.19bc 31.76ab 29.50cd 0.34ab 0.93b 13.92c 66.96bc 40.07ab m9 15.79bc 31.81ab 31.21bc 0.38ab 0.98ab 17.14bc 69.96bc 38.37ab m10 14.77bc 31.54ab 30.19c 0.35ab 0.96ab 17.30bc 69.03bc 42.45ab m11 13.74bc 27.45b 29.90cd 0.38ab 1.09a 16.52bc 68.28bc 40.67ab *different letters indicate the statistical difference within same column among genotypes at 5% level. 88 s. ercisli, m. sengul, h. yildiz, d. sener, b. duralija, s. voca, d. dujmovic purgar in literature. fruit fi rmness and colors (as chroma and hue) were found between 0.31 and 0.61 kg/cm2 and 33.21-43.30% for chroma and 61.92-80.54 (deg) for hue (tab. 1). moisture, ash, soluble solid content (ssc), vitamin c, ph, reducing sugar and crude protein in medlar fruits there were statistically signifi cant differences among genotypes in terms of above parameters except ash and reducing sugar (tab. 2). ssc content of medlar genotypes were between 16.4-21.4% (tab. 2). notable the genotype m7 had relatively higher soluble solid content. soluble solid contents of medlar genotypes previously reported between 12.5-26.0% (ozkan et al., 1997; bostan, 2002; bostan and islam, 2007). among genotypes vitamin c and ph ranged from 11.5 to 15.0 mg/100 g and 3.3 to 4.2 (tab. 2). the mean of the vitamin c contents of medlar genotypes was 12.7 mg/100 g. the genotype dependent moisture and crude protein of medlar fruits were observed between 67.4-75.6% and 3.3-4.3%, respectively (tab. 2). as in most vegetarian diets, protein quality and quantity are major concerns. lack of adequate proteins, either in quality or quantity contributes to low body mass, growth retardation in children, and developmental defi ciency during pregnancy. the average adult requires approximately 0.8 g of protein per kg of lean body mass per day to maintain normal functions, and so a person weighing 70 kg needs approximately 56 g of protein daily. to a certain extent the use of medlar genotypes in a diet may contribute to fi lling the protein gap. vitamin c, ph, moisture and crude protein of medlar fruits was previously reported between 15.70-24.80 mg/100 g (ozkan et al., 1997; wazbinska, 2007), 2.89-6.15 (ozkan et al., 1997; bostan, 2002; bostan and islam, 2007); 72.2% (haciseferogullari et al., 2005) and 3.7% (haciseferogullari et al., 2005), respectively. the variation of ssc, vitamin c, moisture and crude proteins in medlar fruits could be due to different genotypes used, environmental conditions and the nutritional status of the plantations, as well. total phenolics and antioxidant activity in medlar fruits the total phenolic contents of the fruits of medlar genotypes varied from 114 mg gae/100 g fw in m11 genotype to 293 mg gae/100 g in m5 genotype (tab. 3). the average total phenolic content of genotypes was 194 mg gae/100 g fw. it can be said that medlar germplasm from coruh valley is rich in total phenolics. this phenomenon could be due to an induction of synthesis of antioxidant enzymes and an increase in polyphenolic concentration due to the greater exposure of the unsheltered medlar plants to extremes of temperature, and infecting/damaging organisms in the valley. phenolic compound biosynthesis is a typical stress-defense reaction. total antioxidant capacity of medlar genotypes is shown in tab. 3. the genotype seemed to infl uence the extent of antioxidant activity in medlar fruits. determination of antioxidant activities by β-carotene – linoleic acid and 2-diphenyl-1-picryhydrazyl (dpph) free radical scavenging assays resulted in average 80.8 % and 46.6 µg/ml fw dpph, respectively. in β-carotene linoleic acid assay, antioxidant capacity was in order of 92.85% (m9) > 89.01% (m11) > 87.05% (m5) > 85.42% (m7) > 84.75% (m10) > 83.25% (m8) > 82.07% (m4) > 81.60% (m6) > 69.86% (m1) > 68.90% (m2) > 64.63% (m3) (tab. 3). in dpph assay, the antioxidant activity was between 22.3-57.7 µg/ml fw dpph. the genotype m10 had the highest antioxidant capacity with 22.3 µg/ml fw dpph, whereas the genotype m3 had the lowest one (57.7 µg/ml fw dpph). tab. 2: moisture, ash, reducing sugar, soluble solid content, vitamin c and crude protein of samples genotypes moisture ash reducing sugar ssc vitamin c crude protein (%) (%) (%) (%) (mg/100 g fw) (%) m1 69.3ab 1.9ns 3.3ns 19.4ab 12.7ab 4.1ab m2 71.4ab 2.1 2.9 18.0bc 11.3b 3.6ab m3 68.7ab 1.8 3.2 20.2ab 13.8ab 4.0ab m4 73.4ab 2.3 2.6 17.4bc 11.9ab 3.4ab m5 70.6ab 2.0 2.7 18.6b 14.4ab 3.7ab m6 72.3ab 2.2 2.6 17.6bc 13.3ab 3.5ab m7 67.4b 1.8 3.3 21.4a 15.0a 4.3a m8 74.9ab 2.3 2.4 16.8c 11.9ab 3.5ab m9 75.6a 2.4 2.4 16.4c 12.0ab 3.3b m10 73.1ab 2.4 2.7 17.3bc 12.2ab 3.3b m11 70.4ab 2.0 2.9 18.5b 11.5b 3.6ab *different letters indicate the statistical difference within same column among genotypes at 5% level. tab. 3: total phenolic content (tpc), antioxidant activity (β-carotene) and free radical scavenging capacity (dpph) of samples genotypes tpc dpph β-carotene (mg gae/100 g fw) (µg/ml fw) bleaching assay (%) m1 152d 54.0ab 69.7c m2 199c 43.3bc 68.9cd m3 119e 57.7a 64.6d m4 238bc 44.0bc 82.1b m5 293a 32.3c 87.1ab m6 232bc 53.7ab 81.6bc m7 244b 53.3ab 85.4ab m8 176cd 56.0ab 83.3ab m9 218bc 45.6b 92.9a m10 147d 22.3d 84.8ab m11 114e 50.0ab 89.0ab average 194 46.6 80.8 bha 21.24 94.33 *different letters indicate the statistical difference within same column among genotypes at 5% level. phytochemical and antioxidant characteristics of medlar fruits 89 these results indicate that medlar fruits can function as important natural antioxidant sources. these results agree with those previously reported for medlars in which a good antioxidant capacity had been described (campanella et al., 2003; serteser et al., 2008). it was previously reported that the genotype effects antioxidant capacity in different fruit species such as strawberries (tulipani et al., 2008), mulberries (ercisli and orhan, 2007) and currants (hegedus et al., 2008). many under utilized fruits possess high concentrations of phenolic acids, some fl avonols, and other phenolic classes, which have antioxidant activity in vitro (tomosaka et al., 2008; ikram et al., 2009). the results of our study show large variations on physico-chemical properties of medlar genotypes. a wide diversity among genotypes in turkey, presumably the one of the centre of origin and diversity of mespilus germanica, offers scope for selecting the better ones. the results also imply that dietary polyphenolic phytochemicals from medlar may supply substantial antioxidants, which, in turn, may provide health-promoting effects to consumers. mineral element contents of medlar fruits the mineral contents of medlar genotypes are shown in tab. 4. the statistical differences between the genotypes were observed based on p, k, ca, mg and fe contents (tab. 4). the average p, k, ca, mg and fe values of medlar genotypes were 39, 792, 73, 55 and 7.2 mg/ 100 g (tab. 4), respectively. data obtained from medlar genotypes show that they have very high nutritional potential, particularly ca, fe, p, k, mg and their nutritional value is greater than that of some cultivated fruits presented in tab. 5 (anon., 2007). glew et al. (2003) reported that medlar fruits are richer in ca than in p and mg. macro and trace elements play an important role in many metabolic processes and functions throughout the life cycle. studies on humans as well as on animals revealed that optimal intakes of elements such as potassium, magnesium, calcium, sodium, manganese, copper and zinc could reduce individual risk factors, including those related to cardiovascular disease (mertz, 1982). with respect to their ca and fe content, the medlar genotypes considered by this study may offer a better nutritional potential. due to the high content of k, p and mg, the medlar genotypes have the potential to meet the daily k, p and mg requirements of an adult. references anonymous, 2007: www.healthalternatives2000.com/minerals-nutritionchart.html. aoac., 1984: offi cials methods of analysis (14th ed.). va, usa: association of offi cial analytical chemist, arlington. tab. 4: mineral content of medlar genotypes genotypes mg/100g k ca mg p fe mn zn m1 828ab 73ab 51bc 42bc 7.6ab 0.6ns 0.7ns m2 788ab 72ab 60ab 38cd 7.0ab 0.3 0.5 m3 830ab 76ab 57ab 44b 8.1a 0.5 0.7 m4 754ab 68ab 54b 34d 6.8ab 0.4 0.6 m5 793ab 73ab 61ab 35cd 7.4ab 0.3 0.3 m6 774ab 70ab 56ab 39c 6.2b 0.5 0.3 m7 841a 80a 62a 48a 7.5ab 0.4 0.6 m8 762ab 69ab 50bc 36cd 6.2b 0.5 0.5 m9 740b 67b 49c 30de 7.0ab 0.4 0.6 m10 768ab 72ab 51bc 32de 7.4ab 0.6 0.3 m11 834ab 77ab 50bc 45ab 6.2b 0.5 0.5 average 792 73 55 39 7.2 0.5 0.5 *different letters indicate the statistical difference within same column among genotypes at 5% level. tab. 5: mineral content of some selected fruits compared to medlar fruits k ca mg p fe mn zn fruits mg/100g medlar 792 73 55 39 7.2 0.5 0.5 apple 158 9.5 7 9.5 -* avacado 1204 22 78.4 82.4 2 banana 467 7 43 27 0.4 blackberries 282 46 28 30 0.8 1.9 0.4 grapes 176 13 4.6 9 0.4 kiwi 588 46 53 71 0.7 0.3 mango 323 20.7 18.6 22.8 0.3 orange 237 52 13 18 peach 193 5.0 69 12 * : trace amount 90 s. ercisli, m. sengul, h. yildiz, d. sener, b. duralija, s. voca, d. dujmovic purgar arabshahi-delouee, s., urooj, a., 2007:antioxidant properties of various solvent extracts of mulberry (morus indica l.) leaves. food chem. 102,1233-1240. baytop, t., 1999: therapy with medicinal plants in turkey (past and present). capa-istanbul, nobel tıp press (2nd ed.). bostan, s.z., 2002: interrelationships among pomological traits and selection of medlar (mespilus germanica l.) types in turkey. j. amer. pomolog soc. 56, 215-218. bostan, s.z., islam, a., 2007: a research on breeding by selection of medlar (mespilus germanica l.) types in eastern black sea region of turkey. proceedings of 5th national horticultural congress, 330-337 (in turkish). bremner, j.m., 1996: nitrogen-total. in: sparks, d.l. (ed.), methods of soil analysis. part 3. sssa book ser. no. 5. sssa, 1085-1122. madison, wi. (1996). browicz, k., 1972: mespilus. in: davis, p.h. (ed.), flora of turkey and the east aegean islands, vol. 4, 128-129. edinburgh university press. burits, m., bucar, f., 2000: antioxidant activity of nigella sativa essential oil. phytother. res. 14, 323-328. campanella, l., bonanni, a., favero, g., tomassetti, m., 2003: determination of antioxidant properties of aromatic herbs, olives and fresh fruit using an enzymatic sensor. anal. bioanal. chem. 375, 10111016. ercisli, s., orhan, e., 2007: chemical composition of white (morus alba), red (morus rubra) and black (morus nigra) mulberry fruits. food chem. 103, 1380-1384. glew, r.h., ayaz, f.a., vanderjagt, d.j., millson, m., dris, r., niskanen, r., 2003: a research note mineral composition of medlar (mespilus germanica) fruit at different stages of maturity. j. food qual. 26, 441-447. haciseferogullari, h., ozcan, m., sonmete, m.h., ozbek, o., 2005: some physical and chemical parameters of wild medlar (mespilus germanica l.) fruit grown in turkey. j. food eng. 69, 1-7. hegedus, a., balogh, e., engel, r., sipos, b.z., papp, j., blazovics, a., stefanovist-banyai, e., 2008: comparative nutrient element and antioxidant characterization of berry fruit species and cultivars grown in hungary. hortscience 43, 1711-1715. heinonen, i.m., meyer, a.s., frankel, e.n., 1998: antioxidant activity of berry phenolics on human low-density lipoprotein and liposome oxidation. j. agr. food chem. 46, 4107-4112. ikram, e.h.k., eng, k.h., jalil, a.m.m., ismail, a., idris, s., azlan, a., nazri, h.s.m., diton, n.a.m., mokhtar, r.a.m., 2009: antioxidant capacity and total phenolic content of malaysian underutilized fruits. j. food compos. anal. 22, sp.iss.s1, 388-393. kaur, c., kapoor, h.c., 2002: anti-oxidant activity and total phenolic content of some asian vegetables. int. j. food sci. techn. 37,153-161. law, m.r., morris, j.k., 1998: by how much does fruit and vegetable consumption reduce the risk of ischaemic heart disease? eur. j. clin. nutr. 52, 549-556. mcguire, r.g., 1992: reporting of objective color measurements. hortscience 27, 1254-1255. mertz, w., 1982: trace minerals and atherosclerosis, fed. proc. 41, 28072812. mohamed, r., pineda, m., aguilar, m., 2007: antioxidant capacity of extracts from wild and crop plants of the mediterranean region. j. food sci., 72, 59-64. netzel, m., netzel, g., tian, q., schwartz, s., konczak, i., 2007: native australian fruits-a novel source of antioxidants for food. innov. food sci. emerg. 8, 339-346. ozkan, y., gercekcioglu, r., polat, m., 1997: a study on the determination of fruit properties of medlar types (mespilus germanica l.) from tokat region. proceedings of national pome fruit symposium. 123-129 (in turkish). serteser, a., kargioglu, m., gok, v., gok, v., gok, v baci, y., baci, y., baci, y ozcan, m.m., arslan, d., 2008: determination of antioxidant effects of some plant species wild growing in turkey. int. j. food sci. nutr. 59, 643-651. singleton, v.l., rossi, j.l., 1965: colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. amer. j. enol. viticult. 16, 144-158. slinkard, k., singleton, v.l., 1977: total phenol analyses: automation and comparison with manual methods. amer. j. enol.viticult. 28, 49-55. tomosaka, h., chin, y.w., salim, a.a., keller, w.j., chai h., kinghorn, a.d., 2008: antioxidant and cytoprotective compounds from berberis vulgaris (barberry). phytother. res. 22, 979-981.vulgaris (barberry). phytother. res. 22, 979-981.vulgar tulipani, s., mezzetti, b., capocasa, f., bompadre, s., beekwilder, j., de vos, c.h., capanoglu, e., bovy, a., battino, m., 2008: antioxidants, phenolic compounds, and nutritional quality of different strawberry genotypes. j. agr. food chem. 56, 694-704. voca, s., dobricevic, n., dragovic-uzelac, v., duralija, b., druzic, j., cmelik, z., skendrovic babojelic, m., 2008: fruit quality of new early ripening strawberry cultivars in croatia, food technol.biotech. 46, 292-298. wazbinska, j., 2007: the yield and content of some chemical components in fruits of common medlar (mespilus germanica l.). part ii content of some chemical components in fruits of common medlar. sodininkyste ir darzininkyste 26, 69-73. ziegler, r.g., 1991: vegetables, fruits, and carotenoids and the risk of cancer. am. j. clin. nutr. 53, 251-259. address of the corresponding author: s. ercisli, tel.: +90 442 2312599, e-mail: sercisli@hotmail.com journal of applied botany and food quality 92, 240 245 (2019), doi:10.5073/jabfq.2019.092.033 1department of safety and quality of cereals, max rubner-institut, detmold, germany 2 institute of crop science, quality of plant products (340e), university of hohenheim, stuttgart, germany modern aspects of wheat grain proteins georg langenkämper1*, christian zörb2 (submitted: may 08, 2019; accepted: august 14, 2019) * corresponding author summary the unique baking properties of wheat have contributed to the large variety of food products made of wheat. wheat products are immensely popular, which is reflected in their ubiquitous consumption. concerning wheat quality, a main challenge for intense growing strategies is to adapt wheat plants of unaltered yield and baking qua lity to decreased nitrogen input, which will limit unwanted nitrogen leaching into drinking water and safe resources. a probably more important challenge for wheat adaptation will be caused by global climate change. for a relative small percentage of the human population wheat grain proteins can cause a number of serious diseases including coeliac disease. susceptible persons often have to completely avoid wheat, as well as rye and barley products. methods for detection of gluten protein are well advanced, increasing safety for patients. wheat breeding using traditional breeding and mo dern genome editing approaches are seen to be necessary to develop new wheat cultivars for adaptation to new environmental conditions caused by climate change, reduced nitrogen input and increased production efficiency, as well as reduction of disease potential. wheat grain protein analytical methods are important, e.g. for determination of quality parameters and for decision making in breeding programmes. aspects of protein extraction, proteomic analysis and database coverage of wheat protein sequences are discussed. keywords: baking quality; celiac disease; protein analysis; storage proteins; triticum aestivum; wheat; wheat allergy, wheat sensitivity, gluten introduction wheat grain proteins are classically categorized and separated according to solubility in albumin, globulin, gliadin and glutenin (osborne, 1907), with the latter two fractions together correspon ding to gluten proteins. in more recent times, gliadin and glutenin, making up between 60-80% of total grain protein, have also been classified as prolamins either being poor or rich in sulphur or of high molecular weight (shewry et al., 2009; uthayakumaran and wrigley, 2017). based on comparison of amino acid sequences proteins like puroindolines, α-amylase/trypsin inhibitors and lipid transfer proteins are also regarded to be members of the group of small sulphur rich prolamins (shewry and tatham, 1999; shewry et al., 2009). during dough formation the visco-elastic properties of gluten proteins are chiefly responsible for excellent processing and baking quality of wheat. considering the negative, some proteins of wheat and some closely related proteins of barley and rye can cause a range of health problems in humans (shewry et al., 2009). the aim of this review is to highlight modern and future aspects of wheat grain proteins concerning baking quality, health problems and corresponding analytical strategies. 1. modern aspects of proteins contributing to baking quality baking quality of bread wheat is determined by the wheat cultivar, as well as the interplay of the natural grain constituents’ proteins, carbohydrates and lipids, each of which are dependent on external conditions of the environment (e.g. water, soil, weather, climate) as well as type, amount and timing of fertilisation (békés et al., 2004; xue et al., 2016; rekowski et al., 2019; xue et al., 2019) (fig. 1). the considerable impact of milling and baking technology on baking quality (fig. 1) is not subject of the discussion here; for recent reviews of these topics see bock et al. (2016); miskelly and suter (2017). this chapter will focus on the role of diverse storage proteins in bread making quality. by far the largest part of total protein, approximately 60-80%, is deposited in form of gliadin and glutenin in the starchy endosperm tissue of the wheat grain (shewry et al., 2009; uthayakumaran and wrigley, 2017). research on the role of protein in baking quality of wheat has been focused on various aspects such as total protein concentration, ratio of gliadin and glutenin concentration, occurrence of disulphide bonds mainly in glutenin, extractable mass of sds-insoluble glutenin, the so-called glutenin macropolymer, and occurrence of different forms of glutenin and gliadin proteins in a range of cultivars. as members of the prolamin superfamily, puroindolines are largely determining hardness of the wheat grain, having an important effect on processing properties (shewry et al., 2009; uthayakumaran and wrigley, 2017). many properties of proteins contributing to baking quality have been established using protein-biochemical methods. the physical perspective of baking quality is investigated employing rheological methods, which are mostly performed in dough formulations. fig. 1: baking quality of wheat is determined by wheat cultivar, the interplay of natural grain constituents’ proteins, carbohydrates and lipids, growing conditions of wheat as well as milling and baking technology. journal of applied botany modern aspects of wheat grain proteins 241 rheological methods can broadly be distinguished in empirical and fundamental assay formats, the latter of which can determine structure-function properties (dobraszczyk, 2016; tietze et al., 2016). further elucidation of wheat gluten polymer structures and their functionality in response to cultivar, environment and processing is seen as important to improve of gluten-based products, such as bread (johansson et al., 2013). incentives and challenges for improving baking quality of wheat currently, price finding for wheat is strongly influenced by high crude protein content with the debateable rationale that more protein leads to higher baking quality, i.e. higher loaf volume. wheat with high crude protein content (generally ≥ 13%), is achieved in intense cultivation systems, using high levels of nitrogen-based fertiliser. excess nitrogen-fertilisation has often the consequence of leaching of various chemical forms of nitrogen, which in turn creates multiple problems, e.g. no3is contributing to eutrophication and pollution of drinking water resources, whereas nitrous oxide released into the atmosphere is a very potent greenhouse gas by far exceeding the effect of co2 on a molecule basis (butterbach-bahl et al., 2013). in the face of evident climate change, dwindling energy resources and growing demand for food it is imperative to make wheat production more efficient in terms of fertiliser input (zörb et al., 2018). one option to reach this goal is wheat breeding. analysis of winter wheat cultivars grown between 1983 and 2014 in germany has shown some success in this respect: 31,6% increased grain yield was accompanied by 1,5% rise in protein levels and in these 31 years the baking quality parameters sedimentation value (+45,4%) and loaf volume (+8,3%) increased (laidig et al., 2017). while normally grain yield and protein content are negatively correlated, the authors are pointing out that results above were possible due to newly bred wheat cultivars in combination with agricultural practices. advancing this positive development, in the future “ideal” wheat needs less fertiliser to maintain grain yield at a lower crude protein content and at the same time to feature proteins (and possibly other grain constituents) conferring improved baking quality. ways to achieve these aims are seen in wheat breeding for improved grain yield stability and nitrogen use efficiency, and possibly in targeted deletion of genes coding for storage proteins not needed for baking quality, which in summary are predicted to reduce nitrogen input in wheat cultivation (zörb et al., 2018). wheat breeding might also succeed to improve end-use quality by introducing specific genes into commercial cultivars. as one example, using transcriptomic analysis, the gene wheat bread making (wbm) was shown to occur in some wheat genotypes, where a high expression of wbm was correlated with improved baking quality (furtado et al., 2015; guzmán et al., 2016). the authors are recommending introduction of wbm into wheat breeding programmes, which presently has got only a frequency of 14% in cimmyt wheat germplasm. probably having more complex consequences than decreasing fertilization input, climate change will alter numerous environmental conditions (e.g. heat and drought stress, rise of atmospheric co2 concentration) inevitably leading to changed levels of natural grain constituents (halford et al., 2015). specifically, the same authors note that temperature > 35 °c during grain filling and drought stress causes a decrease in the proportion of glutenin to gliadin, which impacts negatively on baking quality. to meet the challenges of climate change concerning baking quality, a combination of measures will be required, including agricultural practices, choice of existing species and cultivars suitable for altered climate conditions. besides traditional breeding, modern tools like genomic selection and gene editing together with comprehensive transcriptomic and genomic data of wheat are holding promise that new cultivars better adapted to changing growing conditions can be developed in a future world (henry et al., 2016; international wheat genome sequencing consortium, 2018). 2. wheat related disorders consumption of wheat products is ubiquitous. while for a large proportion of humans wheat products are nutritious and healthy, there is a rising number of individuals, which experience health problems when being exposed to products from all triticum species, as well as to products from barley and rye. these wheat-related disorders are connected to the human immune system, where autoimmune, allergic and possibly innate immunity mediated responses are distinguished (fig. 2). this review is giving a brief summary of wheat-related disorders. for an in depth overview of this topic including disease definitions, clinical symptoms, diagnosis and treatment the reader is referred to scherf et al. (2016). coeliac disease (cd), affecting approximately 1% of the worldwide population, is an immune mediated inflammatory disease of the upper small intestine caused by gluten ingestion. patients classically suffer from abdominal pain, diarrhoea and vomiting and often from malabsorption of minerals and vitamins, leading to various forms of malnutrition (scherf et al., 2016). both, dermatitis herpetiformis and gluten ataxia are related to cd, but affecting skin and neurological functions and having a much lower prevalence than cd, respectively (fig. 2). wheat allergy (wa) is caused by immunologic reactions to wheat proteins involving ige antibodies (sapone et al., 2012). expressions of wa are food allergy, wheat-dependent exercise-induced anaphylaxis (wdeia), respiratory allergy and contact urticaria (fig. 2). afflicted fig. 2: categories of wheat related disorders (adapted from sapone et al., 2012). *1wheat-dependent exercise-induced anaphylaxis, *2non-celiac gluten sensitivity 242 g. langenkämper, c. zörb are skin (swelling, itching), gastrointestinal tract (abdominal pain, diarrhoea) or respiratory tract (baker’s asthma), where a number of wheat proteins have been identified as triggers, e.g. α-amylase inhibitors, lipid transfer proteins, as well as various gliadins and glutenins. a severe reaction is anaphylaxis (wdeia) in response to exposure to ω5-gliadins or high molecular weight glutenins in combination with physical exercise (scherf et al., 2016). non-coeliac gluten sensitivity (ncgs) is a recently defined disorder with symptoms comparable to cd, but leading neither to histological changes of the small intestine nor to production of anti-tg2 antibodies. α-amylase trypsin inhibitors possibly in combination with certain carbohydrates are suspected to play a role in causing ncgs (zevallos et al., 2017; dieterich et al., 2018). detection and quantification of gluten peptides at present, strict avoidance of wheat, rye and barley inclusively products thereof is mandatory for persons susceptible to most of the described disorders. in international food standards, it is regulated that food labelled gluten free must not contain more than 20 mg/kg gluten in total (codex standard 118-1979, 2015). determination of gluten in food is routinely performed with elisas based on antibodies that target different epitopes in various gluten proteins (lexhaller et al., 2017). when using these elsia assays it is important to consider variation in commercial test kits as well as to consider influences of wheat cultivars and species (scherf, 2017; schopf and scherf, 2018). recent work has identified novel cd-active target sequences in gliadin and glutenin proteins, which are suitable for generation of new antibodies (röckendorf et al., 2017). the authors are envisaging that the combination of existing and new antibodies will improve detection of gluten due to better coverage of harmful gluten epitopes. in turn, this will increase safety for patients suffering from coeliac disease. advancements in lc-ms/ms techniques together with increased database coverage of wheat protein sequences have spurred efforts to use proteomics based methods for detection and quantification of peptides causing cd (van den broeck et al., 2015; huschek et al., 2016; bromilow et al., 2017; schalk et al., 2018b; a). due to high selectivity, sensitivity, versatility and applicability also for processed gluten, lc-ms/ms methods are seen as the most promising approach, besides immunological assays, for detection and quantification of gluten (scherf and poms, 2016). both, lc-ms/ms and immunological methods will profit substantially using well characterised reference proteins from several cereal species that became available recently (schalk et al., 2017). other methods to detect gluten or gluten genes comprise pcr, next generation sequencing, aptamers, magnetic beads, microarrays and multianalyte profiling. these methods have conceptual disadvantages or are technically not as mature and thus are up to date not widely applied (scherf and poms, 2016). approaches to reduce or eliminate gluten protein approaches to eliminate gluten protein from cereals and cereal pro ducts are followed with the aim to make these cereals available mainly for cd patients, increasing the choice of food available to them. in this direction, one option is to use enzymatic modification and, more effectively, digestion of problematic gluten proteins. these treatments can lead to sourdough based wheat bread, pasta, wheat starch and bran, beer and rye products below the threshold of 20 mg/kg gluten tolerable for cd patients (scherf et al., 2018). a further option is to eliminate genes from the wheat genome, coding for health related proteins. an example for such an approach is the strong reduction of α-gliadins in wheat obtained either after application of an rnai technique (becker et al., 2012) or a genome editing technique using crispr/cas9 (sánchez-león et al., 2018). the recent sequencing of the wheat genome has facilitated a very detailed mapping of an allergy/immune-stimulatory set of genes as potential targets for breeding programmes to further reduce health related proteins (juhász et al., 2018). while molecular breeding and mutagenic approaches appear very promising for reducing individual genes, two main challenges in this area are seen, i.e.: the complex multigenic control of gluten proteins and to retain processing and product functionality in the modified wheat (shewry and tatham, 2016). 3. recent challenges of analysis of wheat storage proteins the analysis of storage proteins in seeds is an important issue since “pre-modern” laboratories started to evolve knowledge about aspects of nutritive value, seed ingredients, technological function of proteins, effects of genotypes or fertilization or other environmental factors on grain proteins, or pure basic science of storage protein knowledge. these analyses were initiated early 20th century (shewry et al., 2009), especially in countries with important wheat production such as uk, germany, denmark, france and the usa. starting point for analysis of storage proteins was a fractionation based on diffe rent solubility where fractions were called albumin, globulin, gliadin and glutenin (osborne, 1907). the terminology in the first half of the 20th century unfortunately is non-regular and up to date, there is some confusion with these terms, (see introduction for a diffe rent classification system of wheat proteins). the so-called 'osborne fractionation' was a great progress in these former times and it still applied today, e.g. for extraction of reference material (see chapter 2) followed by hplc analysis (wieser et al., 1998; schalk et al., 2017) and to investigate the wheat grain proteome using oneor twodimensional gel-electrophoresis (skylas et al., 2000; hurkman and tanaka, 2004). however, the osborne fractionation has got several disadvantages: a considerable overlap between protein fractions can easily occur, many variations of the procedure lead to widely varying results in the literature, a part of the total protein remains as an extractable residue, see shewry et al. (2009) for an in depth discussion. to overcome the problems of fractionation, especially for proteomic analyses, there have been attempts to develop methods that can extract as many grain proteins as possible in one solution only. one example is the extraction using trichloracetic acid in cold acetone, where the protein extracts are dissolved in urea containing solution and subsequently analysed in 2d-ge (damerval et al., 1986; mechin et al., 2007; zörb et al., 2009). using an extraction method with an sds based solution, also followed by 2d-ge and mass spectrometry, dupont et al. (2011) were able to identify the majority of wheat storage proteins and to estimate their relative le vels. currently, proteomics approaches aim to advance lc-ms/ms methods. bromilow et al. (2016) applied a bottom-up lc-ms/ms technique on wheat grain protein and obtained a protein identification rate comparable to the work of dupont et al. (2011). depending on individual questions researchers are pursuing, they have to decide which method is most appropriate. while analysis of high abundant proteins like gliadins and glutenins can be done with one of the me thods discussed above, it will be necessary to use more specific protein extraction techniques when membrane and low abundant proteins, such as regulating proteins, are focussed. these might be interesting to analyse in terms of finding evidence for differences in wheat genotypes and their behaviour in different environments for instance in climate change or different fertilization strategies (juhász et al., 2016; dier et al., 2018). challenges for proteomic approaches besides establishing and improving methods for protein extraction there are at least two further interconnected challenges for proteomic approaches, i.e. to improve identification rates through better database coverage of expressed genes (cdnas) and quantification of modern aspects of wheat grain proteins 243 peptides/proteins when using lc-ms/ms techniques. protein mass based identification rates specifically of gluten proteins are hampered by very high levels of similarity combined with large numbers of these similar protein types. for instance, based on in depth sequence analysis, there are 47 genes for α-gliadins with 26 encoding intact full-length proteins in hexaploid wheat, cv. chinese spring (huo et al., 2018), and these proteins feature very few amino acids diffe rence. for this reason, both works on proteomics mentioned above (dupont et al., 2011; bromilow et al., 2016) stress the importance of carefully curated and comprehensive cdna databases for wheat storage proteins. since it has been found that protein sequences are cultivar specific, dupont et al. (2011) recommend that research on storage proteins of a particular wheat cultivar should comprise a thorough cdna sequencing project. this suggestion appears feasible because cdna sequencing has become straight forward and inexpensive. based on available gluten protein sequences bromilow et al. (2017), constructed a manually curated database (glupro) containing 630 discrete protein sequences, which will greatly assist in identification of gluten proteins. the recent completion of the wheat genome sequencing project in combination with rna sequencing (international wheat genome sequencing consortium, 2018) will be a further asset for protein identification in wheat proteomics projects. in bottom-up lc-ms/ms based proteomics methods it is generally required to select unique marker peptides if quantification is required. these marker peptides can then be used for quantification in high resolution mass spectrometers after lc separation and often electrospray ionisation (juhász et al., 2016). obviously, the pool of available unique peptides becomes larger with increasing know ledge about protein sequences from diverse wheat cultivars, which refers back to the discussion above and underlines the significance of databases. lc-ms/ms based quantification of a smaller number of targeted proteins or peptides might be technically easier to achieve, when standard proteins or labelled peptides for calibration are available (scherf and poms, 2016; schalk et al., 2017). in summary, a fast progress in availability of wheat protein sequences will promote proteomic approaches, which in turn will help to enhance knowledge about wheat grain proteins. conclusions it has been argued that wheat growing needs to use less nitrogen based fertilizers in order to reduce nitrogen pollution of drinking water and to safe resources. a further challenge to wheat growing and quality will arise because of climate change. regarding human health, it is important to consider that a rising number of individuals are suffering from wheat related disorders connected to various wheat proteins. long established grain protein analytical procedures together with modern proteomics methods are important for identification and quantification of proteins connected to both, human di sease and changed wheat baking quality parameters. detailed know ledge about grain proteins will facilitate wheat breeding directed at developing wheat cultivars better adapted to changed environmental conditions caused by climate change, reduced nitrogen input and increased production efficiency, as well as reduction of disease potential. acknowledgments we thank m. dier for his helpful comments improving the manuscript. author contributions both authors conceptualised and wrote this review. the final version of the manuscript was approved by both authors. references becker, d., wieser, h., koehler, p., folck, a., mühling, k.h., zörb, c., 2012: protein composition and techno-functional properties of transgenic wheat with reduced alpha-gliadin content obtained by rna interference. j. appl. bot. food qual. 85, 23-33. békés, f., gianibelli, m.c., wrigley, c., 2004: grain proteins and flour quality. in: wrigley, c., walker, c., corke, h. 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accepted: april 6, 2019) * corresponding author summary this study reports the main phenolic compounds, as well as phenolic profiles and antioxidant activity in nine sun-dried fig cultivars with different skin color, originating from south-eastern and middleeastern tunisia. for all evaluated parameters, a considerable variability with high significant differences was observed among the cultivars studied. dark fruits exhibited a higher total polyphenol contents (201.77 mg gae/100g dm in cultivar saoudi douiret) compared to green fruits (73.74 mg gae/100g dm in cultivar bayoudhi douiret). fatty acid methyl esters, identified by gc-ms, distinguished the presence of (c16: 0), (c18: 1), ((c18: 2) 9, 12), ((c18: 3) 9, 12, 15) and (c20: 0). strong correlations between the amounts of total phenolics, phenolic acids, flavonoids, fatty acids and antioxidant capacity were found. a principal component analysis showed three groups of cultivars regarding their similarity level. key words: bioactive compounds; fatty acids; ficus carica l.; gcms; lc-esi-ms. introduction ficus carica l. is considered one of the world’s oldest fruit trees, belonging to the moraceae botanical family (solomon et al., 2006). this species is characterized by various chemical components and biological activities, which allow it to be a typical element of the mediterranean diet that has been favorable to health for millennia; comparing its phenolic composition with red wine and tea, its level seems to be higher (vallejo et al., 2012). all over the world, figs are consumed fresh or dried because of their richness in minerals, carbohydrates, essential amino acids, vitamins and polyphenols (solomon et al., 2006; veberic et al., 2008; viuda-martos et al., 2015). furthermore, they are exploited in traditional medicine and have been studied and proven their potential therapeutic value (khairuddin et al., 2017; moniruzzaman et al., 2017). this plant is cultivated in all mediterranean regions and countries with similar climatic conditions. about 1,184,884 tons of figs are produced worldwide. turkey is the leading producer of figs providing approximately 23.5% of the total production in the world; about 51.6% of this crop is marketed in dry form (faostat, 2015). in tunisia, total fig production is 26,000 tons/year representing 3% of world production (faostat, 2015), mainly produced in the southeastern regions (34% of the national production) (mars et al., 2008). fig cultivars (cvs) and the wild form of the fig can show a wide variety of fruits with respect to color, shape, weight, acidity, sugar and mineral contents, etc, while the region where the figs are mainly cultivated is at the origin of their name (meziant et al., 2015). thus, a morphological and chemical study must be performed in order to show the variation among cultivars. despite the prominence of this crop in arid, semi-arid and sub-humid regions, research on natural substances and molecules of this species has been a major topic for several years. these molecules, which constitute the active ingredient of medicinal plants, belong predominantly to secondary metabolites such as polyphenols, essential oils and alkaloids. several research programs on figs are being developed in order to analyze their content in polyphenols (soloman et al., 2006; caliskan and polat, 2011; harzallah et al., 2016; konak et al., 2017). since dried figs are a more concentrated form of fruit, and due to its economic benefits which are appreciated by both industrial users and individual consumers, the current work investigated the quantity and quality of antioxidants contained in these fruits to determine the richness in these elements. few studies investigated the phytochemical contents in dried figs. the current study evaluates the contents of phytochemical compounds and the antioxidant activity by using abts and dpph tests and characterizing the phenolic and fatty acid profiles of light and dark skin dried fig cultivars cropped in south-east and mid-east of tunisia. the aim is to determine which cultivars can be recommen ded as natural healthy food products basing on their biochemical and nutritional facts, thus allowing their labeling as a superior product. materials and methods plant material the nine local fig cultivars were harvested by hand in the summer of 2015 and 2016 in several commercial farms located in southern and central tunisia during the ripening stage (tab. 1, fig. 1). figs were then traditionally sun-dried for seven days using the direct solar dryer, consisting of a single piece that is both a drying chamber and a solar collector, which produces an uncontaminated final product of good quality on the hygienic and nutritious plan (ouaouich et al., 2005). after drying, figs were stored in closed jars in cold rooms. tab. 1: origin and description of studied dry fig cultivars. accession name label geographical origin description (color) saoudi douiret sd douiret tataouine black bayoudhi douiret bd (south-east) green – yellow wedleni bir amir wba bir amir tataouine purple hamouri bir amir hba (south-east) light purple zidi bni khdech zbk bni khdech medenine black bayoudhi bni khdech bbk (south-east) light green hamouri bni khdech hbk purple kahli karkenah khk karkenah sfax purple mahdoui karkenah mk (mid-east) light green 144 m. khadhraoui, m. bagues, f. artés, a. ferchichi total polyphenols content determination the polyphenols were extracted with 80% methanol (1 g of each sample is ground with 6 ml of methanol), then the samples were placed in a box with ice and kept in the dark during the extraction which was performed on an orbital shaker (stuart, staffordshire, uk) for one hour at 200 × g. after that, fig extracts were centrifuged at 15,000 rpm (4 °c) for 10 min and the supernatant was recovered and stored for total phenolic compounds (tpc) and total antioxidant capacity (tac) quantification. total phenolic content in fig extracts was evaluated, using the folin-ciocalteu method, in 96-well plates, with a spectrophotometer (tecan infinite m200 mannedorf, switzerland) at the absorbance of 750 nm, estimated by the method of singleton and rossi (1965) with some modifications proposed by martinez-hernandez et al. (2011). in each sample, the total polyphenol contents were expressed in mg of gallic acid equivalent (gae) per 100g of dry matter (mg gae/100g dm) through a calibration curve based on gallic acid at different concentrations (mg/ml). total flavonoids content determination total flavonoids content in the methanolic extracts of dried figs was monitored by the colorimetric method of aluminum chloride (alcl3) (brighente et al., 2007) with slight modifications. then, 1 ml of each fig extract was added to 1 ml of a methanolic solution of alcl3 (2%). after 10 min incubation at room temperature and using a spectrophotometer at 430 nm, the absorbance of the mixture was determined. the flavonoid concentrations were deduced from the calibration curve established with quercetin. the results were expressed as mg quercetin equivalents per 100 g of dry matter (mg qe/100g dm). antioxidant capacity dpph assay the dpph assay (2, 2-diphenyl-1-picrylhydrazil) was determined by a spectrophotometric method at 515 nm, adapted from brandwilliams et al. (1995) with slight modifications made by martinezhernandez et al. (2011). the test is based on the capacity of sca venging free radicals. the dpph radical solution was prepared by dissolving 12 mg of dpph in 50 ml of methanol and then stored at -5 °c in the dark. the absorbance was adjusted to 1.1 ± 0.02. an aliquot of 21 μl of extract was placed in a 96-well plate and 194 μl of dpph solution were added. after incubation for 35 min under dark conditions, the absorbance of the aqueous solution was measured against a blank using a spectrophotometer. the results were expressed as mg trolox equivalent antioxidant capacity (teac) per 100 g dm through a trolox calibration curve at different concentrations (mg/ml). all measurements were performed in triplicate. abts assay the abts assay (2, 2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) was determined by a spectrophotometric method at 734 nm, which was adapted from arnao et al. (2001) with slight modifications. the assay is based on the ability to reduce the radical cation 2, 2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (abts) by antioxidant. the mixture solution contained 14 mm abts and 4.9 mm k2s2o8 solutions diluted in methanol and adjusted to 1.1 ± 0.02 (absorbance), and then incubated for 16 h in the dark. an aliquot of 10 μl of extract was placed in a 96-well plate and added with 190 μl of abts solution. after 35 min of incubation, the absorbance of the aqueous solution was measured against a blank using a spectrophotometer. results were expressed in terms of mg trolox equivalent antioxidant capacity (teac) per 100 g dm through a trolox calibration curve at different concentrations (mg/ml). identification and quantification of phenolic compounds after pressurized liquid extraction and solid-phase extraction, the polyphenolic patterns of figs were identified and quantified using a liquid chromatography-electrospay ionization-tandem mass spectrometry (lc-esi-ms). the extract was filtered through a 0.45 μm membrane filter and then injected into the high performance liquid chromatography system (hplc). quantification was achieved by injection of standard chemical solutions of known concentrations at the purity > 98%. the mobile phase was composed of a mixture of two solutions: the first contains 0.1% formic acid in h2o and the second contains 0.1% formic acid in methanol. the column temperature was set at 40 °c and the flow rate of the mobile phase was 0.4 ml/min. the auxiliary gas used is the n2 which was very pure. the results were expressed as mg/100 g dm. all extracts were analyzed in triplicate. fig. 1: nine fig cultivars with different skin colour at ripening stage. zidi bni khdech kahli karkenah wedleni bir amir saoudi douiret bayoudhi bni khdech (zbk) (khk) (wba) (sd) (bbk) bayoudhi douiret hamouri bni khdech mahdoui karkenah hamouri bir amir (bd) (hbk) (mk) (hba) bioactive substances and fatty acids of dry figs 145 fatty acids composition the total lipid content was determined by the soxhlet method which allows extracting the dry extract as much as possible. the fatty acid methyl esters (fames) were extracted using a hexane solvent and were determined by gas chromatography coupled with gc-ms mass spectrometry (supelcowax tm10 fused silica capillary column), 30 m × 0.25 mm × 0.25 μm film thickness (supelco, milan, italy), according to mondello et al. (2004) with slight modifications. gc-ms analysis was performed using a gcms-qp2010 ultra mass spectrometer (shimadzu, kyoto, japan), with a split/split less injector and a shimadzu aoc-20i auto-injector and a shimadzu aoc-20s auto sampler. the data was obtained by the gcsolution / gcmssolution software. the identification of fatty acid methyl esters (fames) was achieved by comparing their retention time with pure standards analyzed under the same conditions. statistical and multivariate analysis statistical analyzes were performed using an anova with spss version 22.0 software. duncan test was used to compare significant differences with the level of significance at p < 0.05 and pearson’s correlation coefficient (r) involved to determine correlations between studied parameters. each assay was performed three times from the same extract and the value was expressed as mean ± standard deviation (sd). the structure of genetic variability among the figs cultivars based on mean values of total polyphenol contents, phenolic compounds such as phenolic acids and flavonoids, fatty acids and antioxidant activity was analyzed using principal component analysis (pca). these multivariate analyzes were carried out using the xlstat 9.0 software to evaluate the influence of phenolic compounds in the differentiation of tunisian dried fig samples. results and discussion total polyphenol contents (tpc) the tpc of dried fig cultivars are shown in fig. 2. the fig cultivars were classified into seven different homogenous subsets according to the duncan test (p < 0.05). cultivars belonging to different subsets presented significant differences in terms of phytochemical cha racters. the tpc ranged from 73.74 mg gae/100g dm in (bd) cv to 201.76 mg gae/100g dm in (sd) cv. our findings are inferior to those of pourghayoumi et al. (2017), who mentioned that dried iran fig contained 1120 to 2681.8 mg gae/100g dm, but are in accordance with the results reported by capanoglu (2014) who found 169.4 mg gae/100g dm in turkish dried fig. these differences in tpc can be attributed to differences in crop region and cultivar type, as well as to cultivation practices, geographical origin, experimental conditions, and postharvest storage conditions (soufi et al., 2014; hoxha et al., 2015). furthermore, the drying processes used (air or sun drying) may be responsible for these differences (arvaniti et al., 2019). on the other hand, there was no significant difference between cultivars harvested in south-east and mid-east of tunisia in terms of tpc, since these two regions have almost the same climatic conditions (arid climate and semi-arid climate, respectively). mk cv (from mid-east) contained 90.96 mg gae/100g dm while bbk cv (from south-east) contained 81.21 mg gae/100g dm). as expected, the polyphenol contents in dark-skinned figs were significantly higher than those in light ones. the highest values were found on dark colored figs (sd-cv, zbk-cv, hba-cv and khk-cv) which shows that the skin color was the major contributor of this diffe rence. these results were confirmed by debib et al. (2014) who found similar findings and reported that dark-colored dried fig varieties contained a higher level of phenolics than the green and yellow ones (harzallah et al., 2016). comparing the levels found in dried figs with those of fresh figs, several studies have shown that the tpc in dried figs was higher than that of fresh ones (konak et al., 2017). however, other studies have shown that drying methods have a negative impact on the phytochemical contents (bachir bey et al., 2016). nevertheless, these levels remain higher than those of certain fruits (dates for example) (al-turki et al., 2010). it is interesting to note that a large number of biochemical properties are identified in these compounds and are conceivably useful for inhibiting the development of diseases such as cancer, cardiovascular diseases and diabetes (chang et al., 2016). total flavonoid contents (tfc) as shown in fig. 2, tfc of dried fig extracts ranged from 57.96 mg qe/100g dm to 112.28 mg qe/100g dm. statistically significant differences in tfc were found between dark and light cultivars (p < 0.05) (fig. 2); sd cv (south-east) appears to have a significantly higher tf content, while a low bd cv (south-east) content was observed. there is no data available on tfc in black and light dried figs; only one study reported the total number of carotenoids in algerian figs (ouchemoukh et al., 2012; arvaniti et al., 2019). these results are consistent with those obtained by bey and louaileche (2015) who indicated that flavonoids content in dark cultivars (126.55 mg/100 g) was higher than that of light ones (87.24 mg/100 g) which confirm that dark-colored figs had more tfc compared to light-colored ones. regarding the factors that affected this phytochemical composition, our study demonstrated that color and fig cv were responsible for tfc variation, but other studies have shown that other factors were accountable such as drying me thods used and the level of maturity of this fruit (pereira et al., 2017; sedaghat and rahemi, 2018). as antioxidants, flavonoids exert therapeutic effects such as antihepatotoxic, antitumor and prevent diabetes and infections (chang et al., 2016). in addition, a set of flavonoids provides protection against cardiovascular mortality and heart disease (wang et al., 2018). antioxidant capacity (ac) the antioxidant capacity (ac) of dried fig cultivars was assessed by two assays: the dpph and the abts tests. results shown in fig. 3 proved that all fig extracts were characterized by antioxidant fig. 2: contents of total phenolics (mg gae/100 g dm) and flavonoids (mg qe/100 g dw) in nine cultivars of dry figs. data are displayed as mean ± standard deviation of three replications for each cultivar. letters above columns represent the groups given by duncan test (α = 0.05) in arising order. different letters indicate significant difference at p < 0.05. 146 m. khadhraoui, m. bagues, f. artés, a. ferchichi activities with significant differences (p < 0.05). the antioxidant activity (aao) against dpph ranged from 131.55 mg teac/100 g dm to 418.51 mg teac/100 g dm, respectively for bd cv and sd cv. similarly, sd cv exhibited the highest level of teac using the abts test up to 207.43 mg teac/100 g dm, while the bd cv presented the lowest level up to 124.53 mg teac/100 g dm. besides, a significant positive and high correlation was assigned between these two assay methods used to evaluate the antioxidant capacity on the one hand (r = 0.953), and between the ac and the amount of phytochemical compounds on the other hand at the p < 0.05 (tab. 2), which infers that the value of antioxidant capacity is often due to the existence of polyphenols and flavonoids. similarly, ksouda et al. (2018) estimated a high and positive correlation between the values of antioxidant capacities obtained by these two methods for the majority of the plant methanolic extracts of 25 tunisian plant species. furthermore, ouchemoukh et al. (2012) and ammar et al. (2015) showed strong correlations between the amount of phytochemical compounds and the antioxidant capacity of figs. according to brewer (2011), the antioxidant capacity is gene rally due to the presence of phenolic acids such as caffeic, gallic and chlorogenic acids, as well as to flavonoids namely rutin, catechin, kaempferol and quercetin. moreover, the anova study revealed that the difference between the green-yellow bd cv and the light-green bbk cv was not significant, neither between hbk and wba purple cultivars nor between hba and khk purple cultivars. significant differences were found only between cultivars owning different skin color (fig. 3). thus, figs originated from dark-colored cultivars exhibited a higher antioxidant capacity compared to those from light-colored cultivars with a range of 3 to 4 fold differences between cultivars. these findings were confirmed by konak et al. (2017) and pereira et al. (2017). therefore, a strong correlation between skin color and tpc and ac should be expected. these results make it possible to deduce that the two darkcolored figs, sd and zbk, harvested in south-eastern tunisia, are recommended for their phytochemical contents and their antioxidant potential in order to improve their health-promoting properties. regarding the impact of drying on the antioxidant capacity, chang et al. (2016) reported that sun-drying process had a positive effect on antioxidant activity, which increases the antioxidant activity of dried figs compared to that of fresh figs, although bachir bey et al. (2016) found that it reduced the antioxidant capacity of figs. despite the great interest in the ac of dry figs, it is difficult to know which of the phenolic compounds has the most important antioxidant activity. that’s why we use to isolate and identify the antioxidant components in order to evaluate the mechanisms of activity. lc-esi-ms analysis individual antioxidants found in our extracts are shown in tab. 3 and seventeen isolated antioxidants were detected. these compounds included different classes of phenolic acids and flavonoids (flavan3-of, flavanones, flavonols, and flavones). among the phytochemicals, in dried figs, each extracted fraction revealed high contents of rutin (38.05 mg/100 g dm) for sd cv, followed by protocatechuic acid (10.6 mg/100 g dm) for wba cv, cirsiliol (2.72 mg/100 g dm) for sd cv and 4-o-caffeoylquinic acid (2.42 mg/100 g dm) for khk cv. moreover, other phenolic compounds were detected, such as quercetin (1.49 mg/100 g dm) for sd cv and kaempferol (1.13 mg/100 g dm) for zbk cv. a number of phenolic acids were also detected in dried figs, such as gallic acid, p-coumaric, trans cinnamic and trans-ferulic, but at very low concentrations (< 1 g/100 g dm). in addition, salviolinic acid was detected only in wba and sd cultivars with respective amounts of 0.89 and 0.939 mg/100 g dm at a retention time of 20.90 min. the apigegin was presented in low amounts except in zbk cv with 4.85 mg/100 g dm. according to arvaniti et al. (2019), gallic acid, and chlorogenic acid were the most predominant phenolic acids in dried figs, while rutin, quercetin-3-o-rutinoside, and epicatechin exhibited the highest concentration levels among flavonoids. comparing these compounds to those of fresh figs, the same target compounds were found, but with high levels (arvaniti et al., 2019). many factors were responsible for these variations; the ripening phase and drying process used seem to significantly affect the content of phytochemical compounds (pereira et al., 2017; arvaniti et al., 2019), as well as the nature of the solvent used for extraction, the fruit color, the harvest season, the weather conditions, and the fruit variety (sedaghat and rahemi, 2018). moreover, the dark-colored varieties (sd and zbk cultivars) had higher phenolic compounds compared to those from light-colored ones (bd and bbk cultivars). furthermore, it should be emphasized that dark-colored dried figs have the highest content of phytochemicals and exhibited the highest antioxidant capacity due to these compounds. according to pourghayoumi et al. (2017) and based on our current results, dried figs own one of the highest concentrations of poly phenols among the commonly consumed fruits. in addition, the dark colored dried varieties, especially the sd and zbk cultivars, harvested in south-eastern tunisia, represent a rich source of two phytochemical classes such as phenolic acids and flavonoids and can offer valuable value for food and pharmaceutical industries, and should be recommended as a natural and healthy food product. fatty acids determination (fames) fames composition is shown in tab. 4. the total lipid content (tlc) analyzed for the cultivars studied, expressed in gram per 100 g of dry matter (g/100 g dm), was in the order of 1.5 g/100 g. this tab. 2: correlations between antioxidant capacities by abts and dpph assays, tpc and tfc of fig cultivars studied. bold pearson correlation coefficients are significant at 5% of confidence level. variables dpph (teac) abts (teac) tpc tfc dpph (teac) 1 abts (teac) 0.953*** 1 tpc 0.961*** 0.906*** 1 tfc 0.973*** 0.963*** 0.916*** 1 fig. 3: the total antioxidant capacity of nine dry fig cultivars using dpph and abts radicals. data are displayed with mean ± standard deviation of three replications. letters above columns represent the groups given by duncan test (α = 0.05). different letters indicate significant difference at p < 0.05. bioactive substances and fatty acids of dry figs 147 tab. 3: phenolic acid and flavonoid profiles in the nine tunisian dry fig cultivars. zbk khk wba sd bbk phenolic contents m/z cc (mg/100g) rt (min) cc (mg/100g) rt (min) cc (mg/100g) rt (min) cc (mg/100g) rt (min) cc (mg/100g) rt (min) gallic acid 169 0.47 ± 0.02d 4.14 0.12 ± 0.07b 4.14 0.54 ± 0.14e 4.14 0.09 ± 0.01ab 4.12 0.082 ± 0.00a 4.13 protocatechuic acid 153 4.47 ± 0.23e 7.33 1.85 ± 0.05bc 7.31 10.59 ± 0.13f 7.32 1.96 ± 0.10c 7.32 1.91 ± 0.02c 7.32 4-o-caffeoylquinic acid 353 0.92 ± 0.03c 12.70 2.42 ± 0.13d 12.71 0.55 ± 0.02b 12.70 0.88 ± 0.05c 12.71 0.27 ± 0.02a 12.75 p-coumaric acid 163 0.36 ± 0.01b 22.20 0.39 ± 0.01bc 22.18 0.46 ± 0.01cd 22.19 0.89 ± 0.04f 22.20 0.68 ± 0.06e 22.22 trans cinnamic 147 0.49 ± 0.08b 33.56 0.61 ± 0.03bcd 33.56 0.58 ± 0.02bc 33.60 0.85 ± 0.16e 33.61 0.41 ± 0.03ab 33.55 trans ferulic acid 193 0.73 ± 0.05de 24.45 0.53 ± 0.05c 24.46 0.46 ± 0.00c 24.46 0.79 ± 0.03e 24.43 0.25 ± 0.01b 24.50 salviolinic acid 515 n.d n.d 0.89 ± 0.00b 29.63 0.94 ± 0.00c 29.63 n.d rutin 609 34.68 ± 0.74g 25.23 22.34 ± 0.62f 25.35 10.28 ± 0.10cd 25.36 38.05 ± 1.75h 25.36 9.29 ± 0.24c 25.37 luteolin-7-o-glucoside 447 0.43 ± 0.03f 26.07 0.18 ± 0.01d 26.06 0.07 ± 0.01bc 26.06 0.08 ± 0.01c 26.06 0.02 ± 0.00a 26.04 naringin 579 0.02 ± 0.01b 27.60 0.05 ± 0.00d 27.58 0.03 ± 0.00c 27.59 0.03 ± 0.00c 17.59 0.06 ± 0.01e 27.59 apigenin-7-o-glucoside 431 0.91 ± 0.06 d 28.53 0.07 ± 0.00ab 28.52 0.02 ± 0.00 a 28.52 n.d n.d quercetin 301 0.45 ± 0.01b 33.56 1.05 ± 0.03d 33.55 0.24 ± 0.02a 33.56 1.49 ± 0.09e 33.57 0.68 ± 0.05c 33.57 kaempferol 285 1.13 ± 0.04g 33.56 0.24 ± 0.01e 33.56 0.11 ± 0.01c 33.57 0.10 ± 0.01c 33.60 0.08 ± 0.03bc 33.58 naringenin 271 0.43 ± 0.02d 35.62 0.24 ± 0.03c 35.61 0.17 ± 0.01b 35.62 0.06 ± 0.01a 35.62 0.20 ± 0.02bc 35.63 apigenin 269 4.85 ± 0.21d 36.22 0.15 ± 0.01ab 36.21 0.23 ± 0.03ab 36.22 0.05 ± 0.01ab 36.29 0.04 ± 0.00ab 36.26 luteolin 285 0.07 ± 0.01c 36.67 0.08 ± 0.02c 36.66 n.d 0.15 ± 0.01d 36.66 n.d cirsiliol 329 1.79 ± 0.05bc 37.29 1.23 ± 0.06a 37.30 1.46 ± 0.17abc 37.29 2.72 ± 0.07d 37.30 1.89 ± 0.40c 37.30 bd hbk mk hba phenolic contents m/z cc (mg/100g) rt (min) cc (mg/100g) rt (min) cc (mg/100g) rt (min) cc (mg/100g) rt (min) gallic acid 169 0.07 ± 0.01a 4.10 0.08 ± 0.01a 4.16 0.12 ± 0.01b 4.12 0.38 ± 0.01c 4.15 protocatechuic acid 153 0.95 ± 0.02a 7.32 2.60 ± 0.13d 7.32 1.50 ± 0.06 b 7.31 4.28 ± 0.15e 7.34 4-o-caffeoylquinic acid 353 0.10 ± 0.01a 12.73 2.33 ± 0.10d 12.72 0.54 ± 0.00 b 12.71 0.25 ± 0.02a 12.72 p-coumaric acid 163 0.49 ± 0.01d 22.20 0.36 ± 0.01b 22.23 0.32 ± 0.01 b 22.23 0.23 ± 0.01a 22.22 trans cinnamic 147 0.28 ± 0.01a 33.57 0.71 ± 0.01cde 33.51 0.42 ± 0.03 ab 33.58 0.79 ± 0.06de 33.57 trans ferulic acid 193 0.42 ± 0.07c 24.45 n.d 0.68 ± 0.01d 24.45 n.d salviolinic acid 515 n.d n.d n.d n.d rutin 609 2.17 ± 0.16a 25.36 17.99 ± 0.14e 25.35 12.19 ± 0.23d 25.35 6.87 ± 0.33b 25.37 luteolin-7-o-glucoside 447 0.04 ± 0.00ab 26.03 0.28 ± 0.01e 26.06 0.04 ± 0.00a 26.02 0.24 ± 0.01e 26.08 naringin 579 n.d n.d 0.02 ± 0.01b 27.59 0.01 ± 0.01a 27.62 apigenin-7-o-glucoside 431 n.d 0.08 ± 0.01bc 28.52 n.d 0.14 ± 0.01c 28.55 quercetin 301 0.25 ± 0.00a 33.55 0.45 ± 0.01b 33.54 0.95 ± 0.03 d 33.58 0.28 ± 0.01a 33.57 kaempferol 285 0.02 ± 0.00a 33.58 0.17 ± 0.00d 33.56 0.041 ± 0.00ab 33.58 0.43 ± 0.01f 33.60 naringenin 271 0.01 ± 0.01a 35.63 0.17 ± 0.03b 35.61 0.17 ± 0.01b 35.63 0.22 ± 0.02 bc 35.65 apigenin 269 n.d 0.25 ± 0.04b 36.22 n.d 0.53 ± 0.04c 36.25 luteolin 285 n.d 0.04 ± 0.01b 36.68 0.15 ± 0.01d 36.67 n.d cirsiliol 329 0.95 ± 0.04a 37.31 1.49 ± 0.18abc 37.30 1.27 ± 0.09ab 37.31 1.38 ± 0.10abc 37.33 note: mean values ± se, followed by different letters in the same column indicate significant difference at p < 0.05 according to duncan’s multiple range test. rt: retention time; cc: concentration (mg/100 g); n.d: not detected p he no lic a ci ds f la vo no id s p he no lic a ci ds f la vo no id s content varied between 1 g/100 g and 2.25 g/100 g. these levels are slightly higher than those indicated by vidaud et al. (1997) and bostan et al. (1998) who estimated that the content was 1 g/100 g. the analysis of fatty acids presented in tab. 3 led to the following results; palmitic acid (c16: 0) has an average content of 0.27 g/100 g dm, the maximum content of which is detected in hba cv and the lowest in hbk cv of a value equal to 0.91 g/100 g dm and 0.15 g/ 100 g dm respectively for these two cultivars. the average content of oleic acid (c18: 1) is equal to 0.31 g/100 g dm, the highest of which is detected in hba cv (0.51 g/100 g dm), linoleic acid (c18: 2) has an average of 0.35 g/100 g dm. the high content of linolenic acid (c18: 3) is observed in wba cv (0.79 g/100 g), ara chidic acid is present in all cultivars studied but in trace form. its average content is low and equal to 0.08 g/100 g dm. linolenic and linoleic acid were the dominant saturated fatty acids in all samples. these levels are higher than those estimated in the table of nutritional composition of ciqual foods (2013) (palmitic acid 0.118 g/ 100 g dm, linoleic acid 0.39 g/100 g dm, oleic acid 0.18 g/100 g dm, arachidic acid absent). fatty acids play a key role in metabolism as a metabolic fuel, a necessary component of all membranes and as a regulator of genes. in addition, fatty acids are used for many industrial purposes. the level of vitamin e generally depends on the part of the plant. indeed, this content is higher in vegetable leaves and especially those that are green when ripe (chun et al., 2006). 148 m. khadhraoui, m. bagues, f. artés, a. ferchichi chemometric analysis: multivariate classification of dried fig cultivars studied the weight and the impact of the different tested parameters in the dry fig cultivars classification were established using the pca technique. the pca relies on the extraction of the most important quantitative variables in a data table representing the observations called ‘components’, in order to construct the pattern of similarity between the variables and the observations as points on a map (saporta and niang, 2009). in the pca, a multivariate technique was used to distinguish between different cultivars in terms of phytochemical contents (tpc, tfc), and their antioxidant capacity (dpph and abts assays) on the one hand, and the phenolic composition, such as phenolic acids and flavonoids, and fatty acids, on the other hand, to establish the possible grouping of the dried fig cultivars studied. the cluster analysis was performed on the basis of the first two main pcs (pc1 and pc2) (fig. 4). a total of 55.42% of the whole varia bility was explained by the relationship between pc1 vs pc2. pc1 was responsible for 32.61%, and represented the most important component which was positively related to the following variables: phenolic acids, flavonoids, tpc, tfc, tlc, antioxidant capacity using both abts and dpph assays, c18:2, c18:3, and c18:1, that were positively correlated with sd and zbk cultivars and were therefore primarily responsible for the discrimination of dark dried figs. however, light dried figs were negatively associated with pc1 and positively with pc2 which was responsible for 22.81% of the difference. in addition, both pc1 and pc2 were negatively related with c16:0 and c20:0. the cumulative variance of 55.42% shows that the fig cultivars were well discriminated by their biologically active compounds levels, fatty acids and their tac. in fact, pc1 allowed differentiation between dark and green skinned cultivars (fig. 4). the dispersion of the cultivars on the biplot defined by the first two components revealed 6 fig.2: contents of total phenolics (mg gae/100g dm) and flavonoids (mg qe/100g dw) in nine cultivars of dry figs. data are displayed as mean ± standard deviation of three replications for each cultivar. letters above columns represent the groups given by duncan test (α = 0.05) in arising order. different letters indicate significant difference at p < 0.05. fig. 3: the total antioxidant capacity of nine dry fig cultivars using dpph and abts radicals. data are displayed with mean ± standard deviation of three replications. letters above columns represent the groups given by duncan test (α = 0.05). different letters indicate significant difference at p < 0.05. zbk khk wba sd bbk bd hbk mk hba dpph abts tpc tfc ap1 ap2 ap3 ap4 ap5 ap6 ap7 f1 f2 f3 f4 f5 f6 f7 f8 f9 f10 c16 :0 c18 :1 c18:2 c18:3 c20:0 tlc -6 -4 -2 0 2 4 6 8 -10 -8 -6 -4 -2 0 2 4 6 8 10 p c 2 (2 2, 81 % ) pc1 (32,61 %) biplot (pc1 vs pc2: 55,42 %) light cultivars dark cultivars purple and dark cultivars fig. 4: principal component analyses of nine dry fig tunisian cultivars. flavonoids: rutin (f1), luteolin-7-o-glucoside (f2), naringin (f3), apigenin-7-oglucoside (f4), quercetin (f5), kaempferol (f6), naringenin (f7), apigenin (f8), luteolin (f9), cirsiliol (f10), and phenolic acids: gallic acid (ap1), protocatechuic acid (ap2), 4-o-caffeoylquinic acid (ap3), p-coumaric acid (ap4), trans cinnamic acid (ap5), trans ferulic acid (ap6), salviolinic acid (ap7). tab. 4: fatty acid contents and composition (g/100g dm) of studied dry fig cultivars. sfa mufa pufa cultivar lipids c16:0 c20:0 c18:1 ((c18:2) 9, 12) ((c18:3) 9, 12, 15) hbk 1.41 ± 0.04b 0.152 ± 0.01a 0.02 ± 0.00ab 0.32 ± 0.01de 0.41 ± 0.01d 0.43 ± 0.01d zbk 1.60 ± 0.02c 0.163 ± 0.00ab 0.04 ± 0.02ab 0.27 ± 0.01cd 0.47 ± 0.03f 0.60 ± 0.02e bbk 1.73 ± 0.09c 0.190 ± 0.01abc 0.04 ± 0.01ab 0.38 ± 0.01e 0.46 ± 0.01ef 0.56 ± 0.01e wba 2.06 ± 0.07d 0.180 ± 0.01abc 0.04 ± 0.00a 0.38 ± 0.02e 0.57 ± 0.01g 0.79 ± 0.03f sd 1.39 ± 0.02b 0.153 ± 0.00a 0.01 ± 0.00a 0.33 ± 0.00de 0.42 ± 0.00de 0.40 ± 0.01d mk 1.01 ± 0.03a 0.203 ± 0.00bc 0.01 ± 0.02d 0.24 ± 0.03bc 0.19 ± 0.00ab 0.07 ± 0.03a hba 2.25 ± 0.05e 0.913 ± 0.02e 0.28 ± 0.09e 0.51 ± 0.01f 0.23 ± 0.01c 0.23 ± 0.01b bd 1.00 ± 0.03a 0.256 ± 0.00d 0.21 ± 0.01c 0.17 ± 0.01a 0.21 ± 0.00bc 0.30 ± 0.01c khk 1.12 ± 0.04a 0.223 ± 0.01cd 0.13 ± 0.02d 0.19 ± 0.02ab 0.15 ± 0.00a 0.12 ± 0.01a total 1,5 ± 0.05 0.27 ± 0.04 0.08 ± 0.13 0.31 ± 0,02 0.35 ± 0.02 0.39 ± 0.04 anova 68.92** 262.15*** 404.21*** 27.26** 104.59*** 124.31*** sfa: saturated fatty acids; mufa: monounsaturated fatty acids; pufa: polyunsaturated fatty acids one-way anova (α = 0.05) is expressed in f-values followed by the significance level (***) at p < 0.05. mean values ± se, followed by different letters in the same column indicate significant difference at p < 0.05 according to duncan’s multiple range test. bioactive substances and fatty acids of dry figs 149 a strong heterogeneity among cultivars. indeed, the cultivars are significantly individualized. the graphical distribution of the dried fig cultivars presented in fig. 4, depending on their factor scores, indicates that the dark cultivars were distinguished from light ones; the first group on the right side of the graph includes sd and zbk cultivars, while the second on the left includes bd, bbk and mk cultivars, and the third one contains hba, zbk and wba cultivars in the center of the graph. the highest values of tp, tf and ac were achieved on dark colored figs (sd and zbk cultivars) that showed different flavonoids and phenolic acids in the polyphenol profile which are antioxidant compounds. our results are consistent with those of bey and louaileche (2015) who reported that pca plot shows a clear sepa ration between the dark and light dried fig cultivars. the highest values of tlc and fatty acids were detected in the hba, zbk and wba cultivars, which contained also fairly high levels of tp, tf and tac. the third group which included bd, bbk and mk cultivars was poor in all compounds. in our research, using 24 parameters relative to bioactive compounds, fatty acids and antioxidant activity, pca applied to the considered fig cultivars proved that three phenotype groups of tunisian dried figs (black, green, and purple) can be distinguished with significant differences. obtained results showed that dark fig (zbk) cv, harves ted from the south-east, is a relevant food, which provides interesting antioxidant compounds in sufficient amounts to the human diet; especially samples rich in rutin (f1), cirsiliol (f10), apigenin (f8) and kaempferol (f6) (the black cultivars), characterized by a large amount of polyphenols, mainly flavonoids, and fatty acids, particularly polyunsaturated ones. conclusions the nine fig cultivars studied herein have a huge potential and constitute an important heritage that deserves to be valued. they are characterized by an attractive range of fruit skin colors, ranging from dark black to green-yellow. dark cultivars contained the highest levels of flavonoids and phenolics and exhibited high antioxidant capacity, while light skinned cultivars contained the lowest levels. the determined total phenolic content indicates the richness of southern tunisian figs, in particular sd and zbk cultivars. in these, main compounds were rutin, protocatechuic acid, cirsiliol, 4-o-caffeoylquinic acid, kaempferol and quercetin. these dried fruits also 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doi: 10.1016/j.ajps.2017.08.004 orcid marwa khadhraoui https://orcid.org/0000-0002-2530-3214 mohamed bagues https://orcid.org/0000-0002-5703-4442 francisco artés https://orcid.org/0000-0002-0689-7301 address of the corresponding author: khadhraoui marwa, university of tunis el manar (fst), 2092, el manar, tunis and oases aridoculture and culture laboratory of the arid region institute (ira), 4119, medenine, tunis, tunisia e-mail: khadhraoui.marwa88@gmail.com © the author(s) 2019. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). https://doi.org/10.1016/j.indcrop.2018.07.008 https://doi.org/10.17660/actahortic.2008.798.2 https://doi.org/10.1016/j.postharvbio.2011.06.015 http://dx.doi.org/10.1016/j.chroma.2004.02.058 http://dx.doi.org/10.17660/actahortic.2017.1173.40 http://dx.doi.org/10.1016/j.lwt.2012.07.022 http://dx.doi.org/10.1016/j.jfca.2017.09.006 http://dx.doi.org/10.1016/j.scienta.2018.04.003 http://dx.doi.org/10.1021/jf060497h https://doi.org/10.1016/j.foodchem.2014.02.075 http://dx.doi.org/10.1016/j.foodchem.2011.07.032 https://doi.org/10.1016/j.foodchem.2007.05.061 https://doi.org/10.1016/j.indcrop.2015.03.005 https://doi.org/10.1016/j.ajps.2017.08.004 https://doi.org/10.1016/j.ajps.2017.08.004 https://orcid.org/0000-0002-2530-3214 https://orcid.org/0000-0002-5703-4442 https://orcid.org/0000-0002-0689-7301 mailto:khadhraoui.marwa88@gmail.com angewandte botanik. die zukunft des pflanzenschutzes in deutschland. 15 stellungen gebracht wird. das läßt sich dadurch erreichen, daß die arbeiten, die eine akademische vorbildung nicht erfordern, von nicht akademischen hilfskräften (kriegsbeschädigten, damen) aus geführt werden. in der samenkontrolle ist dies ja schon zum größten teil durchgeführt. aber auch im pflanzenschutz, sowohl in den wissenschaftlichen laboratorien wie in den pflanzenschutz stellen wird es noch möglich sein, an manchem platz durch ein stellung einer hilfskraft den assistenten mehr für wissenschaftliche arbeit frei zu bekommen und dadurch seine stellung mit zu heben. besonders wo mehrere assistenten sind, wird oft die möglichkeit bestehen, durch eine derartige arbeitsteilung die mechanischen arbeiten durch billigere arbeitskräfte ausführen zu lassen und -dadurch die zahl der assistenten einzuschränken, das einkommen der vorhandenen aber zu steigern. wenn ich hier auf die wirtschaftlichen verhältnisse unseres standes etwas ausführlicher eingegangen bin, so geschah dies aus der überzeugung, daß wir eine erhöhte leistung auf dem gebiet des pflanzenschutzes nur durchführen können, wenn wir aus denen, die sich diesen wissenszweig erwählen, einen berufsstand machen, der mit andern akademischen berufen wenigstens einigermaßen den vergleich aushält. untersuchungen über den einfluß verschiedenartiger mineral düngung auf die zusammensetzung von 0bstdauerwaren. mitgeteilt von dr. j. kochs, versuchsstation für obstund gemüseverwertung an der gärtnerlehranstalt dahlem. seit verschiedenen jahren wurden dem laboratorium der obigen versuchsstation auf veranlassung des kalisyndikates g. m. b. h., agrikulturabteilung, durch die versuchsansteller proben ver schiedener obstarten von düngungsversuchen übersandt, um aus diesen fruchtsäfte oder sonstige dauerwaren herzustellen. neben den teilweise vorgenommenen qualitätsprüfungen dieser dauerwaren, 16 j. kochs, über deren ausfall an anderer stelle ebenfalls berichtet werden soll, wurden chemische untersuchungen durchgeführt, um festzustellen, ob sich ein einfluß auf die zusammensetzung der obstdauerwaren durch die mineraldüngung geltend macht. außer himbeeren kamen johannisbeeren in form des rohsaftes zur untersuchung, ferner zwetschen und süßkirschen als dunstfrüchte. die proben, welche den einzelnen parzellen entstammten, wurden getrennt versandt, so daß eine verwechslung ausgeschlossen war. neben zucker, säure, extrakt und mineralstoffen wurde auch der alkoholgehalt festgestellt, da die früchte (besonders himbeeren) schon auf dem transport teilweise in gärung übergingen. aus dem zucker und alkohol wurde sodann der „zucker vor der ver gärung“, also der ursprünglich vorhandene zucker, berechnet und aus dem extrakt nach abzug des zuckers der „zuckerfreie extrakt rest“ festgelegt. untersuchung von himbeersäften. himbeeren 1910. tabelle 1. himbeersäfte 1910.----e= = = ## äts = "fäe ## s = = = = = = nr. bezeichnung ## e # # ### # ## ## ###t ====== * s*## 1 (kpn), volldüngung doppelt . 10063307331,30202380,4583631036366 2 kp, kali-phosphors. . 1,00923,563,1381,3160,230 073 3,42,9087,338 3 kn, kalistickstoff 1,015279286011620207046940 7774pn, phosphors.-stick stoff. 100872372584102202860448– 5,011 5 | kpn, volldüngung . 1,00532,65 – 0,420 0,5450,4733,4 – 5,818 6 | o, ungedüngt . . . 1,00903,353,166 1,2880,2460,507 3,8 6,933 die säfte waren bis auf einen geringen zuckergehalt vet goren. es ergab sich, daß bei „doppelter“ volldüngung der gehalt an extrakt und zuckerfreiem extrakt am höchsten war. bei düngung ohne kali war von diesen beiden stoffen am wenigsten vorhanden, auch war hier der niedrigste zuckergehalt „vor der ver gärung“ nachzuweisen, das gleiche gilt für die aschenbestandteile. einfluß verschiedenartiger mineraldüngung. 17 himbeeren 1913. versuchsansteller kärsten in altenweddigen. die verteilung der parzellen und düngemittel ergibt sich aus tabelle 2. tabelle 2. düngungsversuch an himbeersorte fastolf. verteilung der düngemittel zur ernte 1913. düngermenge für 1 a in kg nummer und bezeichnung schwefel der parzellen . ... 40proz. superchilisbulres kainit kali phosphat salpeter am moniak 1 . ungedüngt . . . . . 2 . volldüngung . . . . . – 4,0 3,0 – 2,25 3 . phosphorsäure-stickstoff . – 3,0 | – 2,25 4 . kai stickstoff – 40 | – – 2,25 5 . volldüngung . . . . . 4,0 | 3,0 – 4,00 6 . kali-phosphorsäure . . . – 4,0 3,0 – 7 . volldüngung . . . . . | – 4,0 30 30 8 . volldüngung kainit . . | 16,0 – 3,0 2,25 tabelle 3 . zusammensetzung der himbeersäfte. parzelle n r. . . . | 1 2 3 4 5 6 7 8–--–+– spezit. gewicht b e i 15103581082810368107610377 1021108161025 alkohol . . . . . 1,05 1,29 0,88 256 0,80 1,06 1,47 1,59 extrakt |963 885 9 9 5,75 10,4 112 890725 zucker, berechn. als invertzucker . . . | 4,70 4,99 5,68 1,95 5,11 6,17 4,07 2,08 gesamtzucker vor der vergärung berechn. 663 742 721 714 651 805 690 526 säure (zitronens). |1,46 1,44 1,57 143 1,51 1,48 1,55 1,52 mineralstoffe „0302 0507038 0 º. 02 050 0,451 hosphorsäure (p,0.00ä9005800672 0.51705710.737 000 000 extrakt, zuckerfrei.4,93 386 4,26 380 503 535 4,83 5,17 auch hier waren sämtliche säfte angegoren, trotzdem die himbeeren auf beschleunigtem wege übersandt worden waren. die säfte wurden sofort abgepreßt, pasteurisiert und erst nach völliger klärung untersucht. ein bemerkenswerter einfluß der düngungsmittel auf diezu sammensetzung der rohsäfte ließ sich hier nicht feststellen. angewandte botanik i . 2 d ü n g e sa lz 8 m ., 1 0 0 k g k a in it = 2 ,6 0 m ., 1 0 0 k g h im b e e re n 5 0 m . h im b e e re n 1 9 1 4 . 3. v e rs u c h sa n st e ll e r: g a rt e n in sp e k to r s to ff e rt , o b st a n la g e d e r s im o n sc h e n s ti ft u n g zu p e in e . t a b e ll e 4. (b e w ä ss e rt ). s o rt e m a rl b o ro u g h , g e p fl a n z t 1 9 1 2 . e rn te 1 9 1 4 . 1d 4 d jä h rl ic h e d ü n g u n g a u f 1 a ä s d ü n t a g d e r – t e il – –– e rt ra g – “ – g u n g * |je. ä | o | kpn pn k p n |s tü c k " .de w e rt . ä ä p fl ü c k e z, k a in it nr a rt u n d m e n g e k o st e n ar j1 kg = fü r 6a |6a 6 a 6 a g w ic h t 0 ,5 0 m 3 ja h re kg kg kg kg " 3 0 . 6. 8 5 0 1 0 5 0 9 0 0 9 5 0 1d | ung e d ü n g t ... ... ... .. – | 26 ,7 5 | '– s 2. 7. | 24,0 0 4 4 ,5 0 3 3 ,5 0 3 9 ,0 0 |2d | volld ü n g u n g : g 4. 7. | 3 3 ,5 0 | 6 8 ,5 0 5 9 ,0 0 | 7 4 ,7 5 4 k g 4 0 p ro z . k a li sa lz ... s 1y 7 7 y 3 „ s u p e rp h o sp h a t 1 ,1 7 | 55 ,2 5 | 28 ,5 0 1 4 ,2 5 | 3 ,5 1 | 10, 7 4 * 8. 7. | 29 ,0 0 | 64 ,5 0 5 0 ,5 0 6 9 ,0 0 " ä ä 2 m sc h w e fe ls . a m m o n ia k 1 3 . 7. | 1 5 ,5 0 4 8 ,5 0 3 4 ,0 0 4 9 ,5 0 18. 7. 2 8 ,0 0 4 2 ,5 0 2 9 ,0 0 4 4 ,7 5 |3d | dün g u n g o h n e k a li : 2 3 . 7 1 3 ,5 0 3 1 ,5 0 2 3 ,7 5 3 3 ,5 0 3 k g s u p e rp h o sp h a t . 0 ,8 5 | 42 ,3 7 | 1 5 ,6 2 7 ,8 1 2 ,5 5 5 ,2 6 28. 7. | 8502 ,0 0 1 5 5 0 2 8 5 0 2 n sc h w e fe ls . a m m o n ia k 4d | vol ld ü n g u n g : . z u s. 1 6 0 ,5 0 3 3 1 ,5 0 2 5 4 ,2 5 3 4 3 5 0 1 0 k g k a in it .. .. .. .. . e rt ra g 3 „ s u p e rp h o sp h a t ... . 1 ,1 1 | 57 ,2 5 | 30 ,5 0 1 5 ,2 5 | 3 ,3 3 | 11 ,9 2 v o n 1a | 26 ,7 5 5 5 ,2 5 4 2 ,3 7 5 7 ,2 5 2 „ sc h w e fe ls : a m m o n ia k » d e r b e re c h n u n g z u g ru n d e g e le g t: 1 0 0 kg s u p e rp h o sp h a t = 7 m ., 1 0 0 kg sc h w e fe ls . a m m o n ia k = 32 m ., 1 0 0 kg 4 0 p ro z e n ti g e s k a li "? t a b e ll e 5. (u n b e w ä ss e rt ). u n b e w ä ss e rt ed c|2c 3c 4c t a g d e r – t e il p fl ü c k e k p n st ü c k 1 9 1 4 |9 *” ” k a in it nr. 3 a 6 a 6 6 a * k g k g k g k g – t -3 0 . 6. 4 5 0 8 5 0 1 0 5 0 5 5 0 |1c 2 . 7 8 5 0 3 4 ,0 0 2 6 ,0 0 3 2 ,0 0 |2 c 4. 7. | 10.5 0 5 8 0 0 5 1 ,0 0 6 8 ,5 0 8. 7. oo so ºo 4 ss o ss o 13. 7. | 19 ,0 0 5 1 ,0 0 3 0 ,5 0 5 3 ,5 0 || 18. 7. 2 7 5 0 5 3 5 0 3 7 5 3 5 ,0 0 | 3c » 23. 7. | 11 ,0 0 3 2 ,5 0 2 3 ,5 0 2 6 ,5 0 2 8 . 7. | 50 ,5 0 1 9 ,0 0 1 0 ,5 0 2 3 ,0 0 4 c z u s. | 93 ,5 0 a ls o 2 4 4 ,2 5 3 0 7 ,5 0 e rt ra g v o n 1a | 3 1 ,1 7 4 9 ,3 3 4 0 ,7 1 5 1 ,2 5 s o rt e m a rl b o ro u g h , g e p fl a n z t 1 9 1 2 . e rn te 1 9 1 4 . h jä h rl ic h e d ü n g u n g a u f 1 a m e h re rt ra g v o m a r --ert ra g –a v o ii l w e rt w w 6 k a r g e k a rt u n d m e n g e k o st e n w ic h t g = 0 ,5 0 m m. k g k g m . u n g e d ü n g t ... ... ... .. 3 1 ,1 7 v o ll d ü n g u n g : 4 kg 4 0 p ro z . k a li sa lz ... 3 „ s u p e rp h o sp h a t .. . 1 ,1 7 | 49 ,3 3 | 1 8 ,1 6 9 ,0 8 2 m sc h w e fe ls . a m m o n ia k d ü n g u n g o h n e k a li : 3 kg s u p e rp h o sp h a t ... .) 0 ,8 5 4 0 ,7 1 9 ,5 4 4 ,7 7 2 „ sc h w e fe ls . a m m o n ia k v o ll d ü n g u n g : 10 kg k a in it ... ... ... | 3 „ s u p e rp h o sp h a t ... . 1 ,1 1 | 51 ,2 5 | 20 ,0 8 | 10 ,0 4 2 „ sc h w e fe ls . a m m o n ia k d ü n g u n g s k o st e n fü r 3 ja h re 2 ,5 5 3 ,3 3 g e w in n 2 ,2 2 s 20 j. kochs, tabelle 6. zusammensetzung der aus den himbeeren (s . tab. 4 und 5 ) bereiteten rohsäfte. bezeichnung . . . dit d2 | d 3 | d | c 1 c 2 c3 | c4---– kpn » kpn düngung . . . . . . o kpn pn ät 0 kpn pn kainit bewässert unbewässert== -t-t-–-t spezifisches gewicht 1,01621,0157101641,01351,02691,0099 1,0108 1,0171 alkohol . . . . . . . 0,64 | 0,58 | 0,69 2,10 0,53 |1,82 1,39 | 1,39 extrakt . . . . . . . 4,49 4,31 4,56 4,50 |7,19 3,46 3,98 5,09 zucker (zusatz) . . | 1,23 | 0,99 1,12 | 0,68 3,96 0,25 0,46 1,65 mineralstoffe . . . . . 0,448 | 0,497 0,499 alkalität / n pro 1 g asche in ccm |18,9 10,7 100 22,2 3,58 1,50 phosphorsäure(p„o.) % der asche . . |11,82 12,77 11,85 13,95 | – – zuckerfreier extrakt | 3,26 3,32 3,44 3,82 3,23 3,21 zucker vor der ver gärung (als rohrz.) 2,49 | 2,10 2,44 485 4,82 3,88 3,22 4,35 0,450 0,450 0,480 0,408 0,486 1 6 , 5 5 ? 1 3 3,52 3,44 während auch bei diesen rohsäften ein bemerkenswerter ein fluß der düngemittel nicht hervortrat, war der zuckergehalt vor der vergärung, also in den himbeeren, bei den bewässerten par zellen niedriger wie bei den unbewässerten, aber auch sonst war der zuckergehalt niedriger wie in tabelle 1 und 3 . tabelle 7 . himbeersäfte aus eysselhof. bezeichnung . . . . . . . . . 1 2 3 • , düngung . . . . . . . . . . | o | cakpn | pn kn spezifisches gewicht . . . . . . | 1,0404 1,0368 | 1,0352 | 1,0290 alkohol . . . . . . . . . . 0,00 0,00 0,00 0,11 extrakt . . . . . . . . . . . . | 10,19 5,47 8,79 | 7,55 zucker . . . . . . . . . . . 5,74 5,30 4,57 3,18 mineralstoffe . . . . . . . . . . 0,500 0,554 0,512 0,477 alkalität / n-pro 1 kg asche . . | 9,0 ccm 8, 1 t 8,8 ccm 9,9 ccm phosphorsäure (p,o.) . . . . . | 10,01 | 14,78 10,16 | 6,98 zuckerfreier extrakt . . . . . . 4,45 4,17 4,22 4,37 die himbeeren dieses versuches waren auf heidemoor in der gegend bei gifhorn auf einer plantage des herrn j. röber in braunschweig gewachsen. bemerkenswert a n den säften war das hohe spez. gewicht, sowie der erhöhte gehalt an mineralstoffen sowie zuckerfreiem extrakt. durch die düngung waren bemerkens einfluß verschiedenartiger mineraldüngung. 21 werte unterschiede -nicht hervorgetreten. die himbeeren waren zerdrückt worden, mit einem konservierungsmittel versetzt und in glasgefäßen eingesandt worden. untersuchung von johannisbeersäften. versuchsansteller rittergut poschwitz bei altenburg s.-a. die mit a bezeichneten parzellen waren nur mit der sorte „rote holländische“ bepflanzt worden, die b-parzellen enthielten „rote kirsch“ und „rote holländische“ gemischt. die verteilung der parzellen und die düngung war folgendermaßen: tabelle 8. johannisbeeren, gepflanzt 1907 und 1908.mmmmm lfd. « düngermenge für 1 ar in kg nr. d. düngung " | 40proz. thomaschilikalkparz. kalisalz mehl salpeter 3. 1a ungedüngt 2a cakpn 4 9 2 15 3a capn 9 2 15 4a cakn . 4 2 15 5a ungedüngt – 6a kpn 4 9 2 7a cakp 4 9 5 8a cakpn, 4 9 4 15 9a ungedüngt – 1b ungedüngt 2b cakpn 4 9 2 15 3b capn 9 2 15 4b cakn . 4 2 . 15 5b ungedüngt 6b | kpn 4 9 2 – 7b cakp . 4 9 8b | cakpn, . . . 4 9 4 9b | ungedüngt – | – – die johannisbeeren wurden nach parzellen gesondert in körben als eilgut versandt und kamen in gutem zustande ohne schimmelbeschlag, allerdings in den unteren teilen etwas gedrückt, hier an. in den sofort bereiteten rohsäften konnte daher auch eine teilweise schon begonnene alkoholbildung durch gärung fest gestellt werden. die aus ihnen durch verkochen mit zucker be reiteten sirupe waren, wie die ergebnisse der kochproben ergaben, von erstklassiger beschaffenheit. t a b e ll e 9. jo h a n n is b e e rs ä ft e . p o sc h w it z 1 9 1 2 . # s p e z if is c h . c c m */ 1 a ls v o r d e r “ | gew ic h t a lk o h o l e x tr a k t in v e rt fr e ie r n o rm a lz it ro n e n | as c h e n a tr o n la u g e c e m n a tr o n w e r 1 a 1 ,0 3 6 8 0 ,2 0 8 ,5 2 1 5 ,1 5 6 3 ,3 6 5 2 7 ,4 1 ,9 1 8 0 ,4 5 9 5 ,3 0 2a | 1 ,0 3 2 1 | 0 ,0 7 7 ,6 8 1 | 3 ,9 1 2 | 3 ,7 6 9 2 8 ,6 2 ,0 0 2 0 ,3 4 9 3 ,2 9 ,1 6 3 ,8 5 3a | 1 ,0 3 6 2 | 0 ,2 7 8 ,8 6 8 4 ,2 9 6 4 ,5 7 2 3 0 ,8 2 ,1 5 6 0 ,3 8 6 3 ,4 8 ,8 0 4 ,6 3 s 4 a | 1 ,0 3 7 4 0 ,0 0 8 ,8 7 1 4 ,6 9 2 | 4 ,1 7 9 | 3 0 ,0 2 ,1 0 0 0 ,4 1 8 3 ,8 9 ,0 8 4 ,4 6 > 5 a 1 ,0 3 4 3 0 ,8 7 8 ,3 3 8 4 ,8 1 6 3 ,5 2 2 3 1 ,4 2 ,1 9 8 | 0, 4 8 4 3 ,4 7 ,0 3 6 ,3 2 # 6 a | 1 ,0 3 4 7 | 0 ,5 3 8 ,5 2 9 | 4 ,9 9 6 | 3 ,5 3 3 3 1 ,0 2 ,1 7 0 0 ,4 0 0 3 ,0 7 ,5 0 5 ,8 1 " 7 a | 1 ,0 3 4 5 1 ,2 0 8 ,3 0 8 | 4 ,9 9 6 | 3 ,3 1 2 3 2 ,6 2 ,2 8 2 0 ,4 5 7 3 ,8 8 ,3 1 7 ,1 5 8 a | 1 ,0 3 8 7 0 ,0 0 9 ,3 2 8 | 2 ,6 6 7 6 ,6 6 1 3 0 ,6 2 ,1 4 2 | -0 ,4 2 2 3 ,6 8 ,5 3 2 ,5 4 s ä u re b e z u c k e r a lk a li tä t a lk a li tä ts z e ic h (a ls z u c k e r1 c c m n o rm a lz a h l z u c k e r d e r z u c k e r e x tr a k t n a tr o n sä u re a u f 1 0 0 c c m la u g e a u f ä ru n a rt e n b e re c h n e t) la u g e b e re c h n e t s a ft 1 g a sc h e g a ru n g = 9 a 1 ,0 3 8 0 0 ,7 3 9 ,3 1 7 | 5 ,3 1 6 4 ,0 0 1 3 0 ,4 2 ,1 2 8 0 ,3 6 4 3 ,0 8 ,2 3 6 ,5 1 1b | 1,03 8 7 0 2 0 8 ,7 2 6 | 6 ,1 8 2 | 2 ,5 4 4 2 8 ,6 2 ,0 0 2 0 ,3 3 5 4 ,2 1 2 ,5 3 6 ,2 7 2 b | 1 ,0 3 9 0 0 ,2 7 9 ,0 9 1 | 6 ,0 4 8 | 3 ,0 4 3 2 8 ,6 2 ,0 0 2 0 ,4 0 3 3 ,8 9 ,4 3 6 ,2 9 3 b | 1 ,0 3 4 9 0 ,0 0 , 8068 | 5, 0 7 6 | 29 9 2 2 8 ,4 1 ,9 8 8 0 ,3 9 3 3 ,5 8 ,9 1 4 ,8 3 4b | 10 3 5 1 | 0 ,6 7 . 7 8 3 0 4 ,6 9 9 3 ,1 3 1 3 1 ,6 2 ,2 1 2 0 ,4 3 9 | 4 ,6 1 0 ,4 8 5 ,8 1 5 b | 1 ,0 3 2 0 | 0 ,1 3 7 ,1 8 2 | 4 ,1 1 5 | 3 ,0 6 7 2 6 ,2 1 ,8 3 4 0 ,4 1 9 4 ,0 9 ,5 4 4 ,1 7 6 b | 1 ,0 2 8 2 0 ,1 3 6 ,8 1 3 | 3 ,1 5 6 3 ,6 5 7 3 1 ,8 2 ,2 2 6 0 ,5 3 0 4 ,6 8 ,6 8 3 ,2 6 7 b 1 ,0 3 3 8 0 ,4 7 | 7 ,6 5 2 4 ,3 6 8 3 ,2 8 4 3 1 ,4 2 ,1 9 8 0 ,4 9 8 4 ,6 9 ,2 4 5 ,0 9 8b | 1 ,0 2 4 4 | 1 ,6 8 5 ,6 9 0 | 2 ,1 6 3 3 ,5 2 7 3 4 ,2 2 ,3 9 4 0 ,4 6 1 5 ,4 1 1 ,7 2 | 5 ,4 1 9 b 1 ,0 3 4 5 0 0 0 7 ,3 5 3 4 ,4 4 8 2 ,9 0 5 3 1 ,0 2 7 . 0 ,4 7 8 3 ,8 7 ,9 5 4 ,2 3 einfluß verschiedenartiger mineraldüngung. 23 8 3 'g 'g e r9 9 % , g g '8 || 8 0 ‘9 sv º8 0 'g ' g o ºg , g 1 º9 || 8 6 'g |g lº ,6 0 '# | 6 1 % 9 8 '9 9 f' l ii 'ſ l ºº g , †g ‘9 || 3 ū n u ſ; 3 1 0 a || ſ -| || |1 ø p io a jº x { o n z || | .* , �,, � |º n i{ 4 4 !4 4 � 4 *● { 4 … ſº º� ·s a l ... /9 ^ \ & 6 1 0 '0 , 8 1 0 '0 , 1 3 0 '0 | g 1 0 '0 8 0 0 '0 , 6 1 0 '0 8 1 0 '0 | 9 1 0 '0 , 2 1 0 ‘0 || 8 3 0 '0 ' 3 8 0 '0 |6 1 1 0 '0 | 9 1 0 '0 , ºg 0 '0 , 1 8 0 '0 |� 8 3 0 '0 8 3 0 '0 | 9 3 0 '0 (º o ºā )|| ·'s io q d so q ā . } || |· 0 8 '# | z g '# | z i‘ f| 8 0 '# | 9 6 % | fg 'ſ ºſ ºg † l ºg| 0 9 “g | fo ºg l ºg g 0 % ; †8 ‘g ºg '� 9 9 'g | 9 8 ‘g | fg 'g | 8 9 'g |· ·· n */, || + |* |º g ļſ ſe ſſ y g g f' 0 | 0 iť '0 ] & & f' 0 || # 1 ţ' 0 || 8 1 8 '0 || 6 8 ț¢ ’0 | 0 8 ř' 0 | + 6 8 °0 || o g g '0 || 8 6 8 '0 | 9 0 ſ' 0 ' 0 9 ţ' 0 || 9 8 ř' 0 | 0 f8 '0 | 9 8 8 '0 f9 ę 0 g 0 ț¢ °0 | 6 ig ‘0 || a ų o g s[ e u a u ! w †0 ‘g | 8 3 '3 | g [º a ] [0 '3 || # 0 ‘g | 0 0 ‘g | # 0 ‘g | 6 6 ‘i l 0 6 “i l g g 'g ' 9 3 'g | 0 8 ‘g | o g 'g | 9 3 'g | ig 'g | 8 3 'g | 6 3 'g | ii ‘g |( “s u a u o iq țz ) 9 .i n g sq u e sº ſ) 6 8 'g | 9 iº ț| 9 6 “† | 0 8 ‘f | 8 8 ‘g | g g 'ı ] g ț" # | 0 0 '# | 8 0 ‘f | ig ºț | io ‘f | 9 8 ‘f | 9 g ºț | g [º ] 6 6 'f | 1 9 ‘g , g g '# | g ºl'' q x q u iq x g i ---j ø ļa u ju ºx { o n z 8 3 'i | 9 8 ‘g | ig ‘g | 8 i' g | i6 'g | 9 6 ‘i | 9 8 '# | 8 i“ g | l º' 9 | 0 6 '0 || 8 iº i| 8 8 ‘i | 0 i' i| 6 9 '9 . g g ‘l || # 9 'g | 9 3 '9 | l i‘ 9 ]···· (z je s -n z ) 1 9 x ſo n z l i‘ g | i0 'l i̇ 1 3 '8 || 8 6 '9 || 6 1 '6 || ig ‘6 || ig '6 | 8 i' 6 | 0 ř' o ii ii ºg þ i‘ g | fl ºg | 9 9 'g | # 1 '0 1 | 1 8 ‘i i| i0 'l i̇ ig 'o i| 6 1 °0 1 || ''' q x ſu u q x g i 8 9 '0 | 1 0 .j | 0 8 0 | + 1 '0 ' 1 3 '0 | 1 1 '0 ' 1 3 '0 , g 0 '0 | 9 1 '0 | 9 0 'ſ g ºº i| g ºº i| | 1 8 '1 || 8 0 '0 | 9 1 '0 ' l º' 0 | 9 3 0 fº o |…·· 1 o q o x iv 0 6 io 'i lg g ő 0 '[ |8 i8 0 ‘i |8 g .g 0 ‘i |9 9 9 0 'i lf ºg º0 ‘i |9 9 9 0 ‘i lº g 8 0 ‘i l6 6 8 0 ‘i |8 .l i0 ‘i u w ia e †6 io 'i |g iť 0 ‘i lg g ť 0 ‘i lz ºg o ºſ || g 0 ř0 ‘i 1 8 6 8 0 ‘i |4 ų o ſa o đ ºz ºd s | |� ț |– 4 6 | 4 8 || q || || q 9| q g || q*| q g || q g || qi || 8 6 |w º | w|||| wg || wg || vſ || wg| uz| vi |:·· a lſ ºz iv ă *1 6 1 z a ſa q o so a ºn jº si ºø q w ſu u v ų o r 01 » īņ q v l 24 j. kochs, ein wesentlicher einfluß der düngungsmodifikationen auf die zusammensetzung dieser johannisbeersäfte war nicht festzustellen. doch aber ergab sich bei den 1914er säften die eigentümliche tatsache, daß sowohl zucker wie extrakt bei beiden düngungs reihen mit zunehmender parzellenzahl im gehalt wesentlich her untergingen. ich führe dies auf eine größere beschattung der johannisbeersträucher durch die obstbaumzwischenpflanzung in den genannten parzellen zurück. untersuchung von zwetschen in dunst. zwetschen 1914. versuch an hauszwetsche, teltow b. berlin. die zwetschen wurden entsteint und ohne jeden zusatz in gläsern pasteurisiert. tabelle 11. bezeichnung . . | 1 | 11 | ii i iv. v | w | vit |viii cakpncakpn düngung . . . . | o cakpn pn | kn k p * b . kpn */ , n | kainit= trockensubstanz in prozent . . |22,89 22,60 22,62 22,63 229 24,18 22,37 23,65 säure(zitronens.) in prozent . . | 0,85 0,94 0,84 089 0,85 0,88 | 0,91 | 0,97 mineralstoff in prozent . . . . | 0272 0,255 0,222 0232 0,234 0,237 0.239 | 0228 phosphors.(p„o.) in prozent der asche . . . . . 22,19 22,65 23,08 22,86 22,90 2322 2332 23,62 zucker.ber. in vertzucker . . | 6,80 6,55 6,29 | 6,46 6,72 6,63 6,46 6,37 auch hier ließen sich wesentliche unterschiede noch nicht nachweisen. dahingegen waren die ergebnisse des folgenden ver suches von bedeutung. untersuchung an süßkirschen 194. versuchsansteller hofbesitzer j. eckhoff, neuenkirchen im alten lande. sorte: eckhoffs schwarze knorpelkirsche. einfluß verschiedenartiger mineraldüngung. 25 ergebnisse des düngungsversuches an kirschen. tabelle 12. kirschen. parzellen bezeichnung und -nummer gewicht: gute kirschen. gewicht: gedrückte kirschen gewicht von 1000 ent stielten kirschen mit stein gewicht der steine . gewicht des frucht fleisches . punktierung bei der dauerwarenprüfung 3. xii. 15. iiiii iv volldüng ohne kali, ää ca, unged. ca, ka, sup. c a , sup, ää ammon. ammon. phorsäure –“–“––– – a ) 4470 a ) 4980 a ) 4000 a ) 4150 b ) 4400 b ) 4310 b ) 4050 b ) 4150 a ) 880 a ) 1410 a ) 1750 a)2030 b ) 1230 b ) 1230 b ) 1500 b ) 1830 3530 4160 3660 3970 370 411 380 396 3160 3749 3280 3574 13 17 1 2 15 tabelle 13. kirschkerne, berechnet auf wasserfreie trockensubstanz. parzellen-nummer feuchtigkeit . asche phosphorsäure (p,o.) in 100 teilen asche sind fettgehalt protein stickstofffreie extraktst. rohfaser i | ii iii | iv . . . . . . .– ––– – – – – – 585 58s 57s sº 3,02 4,33 | 6,55 2,07 1,02 | 2,63 3,65 0,97 p,o, . (33,87) (60,68) (55,68) (46,87) 8,65 | 9,02 | 8,83 8,62 7,37 | 7,84 7,14 7,02 2289 | 26,89 2074 1555 58,07 | 52,52 56,74 66,74 die kirschen wurden zunächst beim eintreffen geprobt. die proben von den parzellen „ohne düngung“ und „ohne kali“ waren weniger süß, „mit kali ohne phosphorsäure“ süßer und mit „voll düngung“ am süßesten. wie sich aus nachstehenden tabellen ergibt, waren die entstielten kirschen bei „volldüngung“ am schwersten, von parzelle „ungedüngt“ am leichtesten. dazwischen standen parzelle iii, ohne kali, und parzelle iv, ohne phosphorsäure. der größe und dem saftgehalt entsprechend hatten die guten kirschen 26 e. rost, aus „volldüngung“ den transport weniger gut ertragen, wie die von parzelle „ungedüngt“, welche am wenigsten gedrückte kirschen aufwiesen. die bei der dauerwarenprüfung in erfurt vollzogene kostprobe ergab für volldüngung die höchste punktezahl. doch nicht nur in der kirsche allein machte sich ein in die augen fallender unterschied bei den einzelnen parzellen bemerkbar, sondern auch in dem gewicht der kerne und in dem des frucht fleisches, wie sich aus tabelle 12 ergibt. es wurde fernerhin versucht, auf analytischem wege festzu stellen, ob sich der düngungseinfluß auch auf die ausbildung der kerne geltend macht. die kerne (steinschale samt samen) wurden gemahlen und getrocknet, um dann untersucht zuwerden. falls sich ein einfluß bemerkbar machen würde bei der ausbildung des samens einerseits oder der steinschale andererseits, mußte der gehalt an protein, fett und stickstofffreien extraktstoffen ansteigen, soweit es sich um den samen handelt, bezw. es mußte der rohfasergehalt zurücktreten, soweit die steinschale in betracht kommt. wie sich aus tabelle 13 ergibt, trafen diese voraussetzungen fast genau ein. in tabelle 13 ist der rohfasergehalt am geringsten in parzelle ii , während dort protein, fett und stickstofffreie extraktstoffe einzeln wie auch in gesamtheit am höchsten stehen. hinsichtlich der mineralstoffe se i noch bemerkt, daß derge halt bei den einzelnen parzellen außerordentlich wechselnd ist, daß aber der prozentuale gehalt der aschen an phosphorsäure bei par zelle „volldüngung“ am höchsten war. von demselben anbauer jakob eckhoff, neuenkirchen, trafen am 27. juli 1918 drei körbe knupperkirschen ein... die erste parzelle hatte volldüngung, also kalisalz-superphosphat-ammoniak-kalk. die zweite war mit kalisalz-superphosphat-ammoniak gedüngt und die dritte schließlich mit superphosphat-ammoniak-kalk. die kirschen kamen in gutem zustand hier an. nr. i war im geschmack die süßeste und in der form die größte kirsche. allerdings waren viele von vögeln angefressen. nr. ii und iii waren gleichgroß. im geschmack hatten beide etwas herbes aber angenehm bitterliches. sie erschienen nicht denselben reifegrad zu haben wie nr. i. von nr. i wogen 100 stck.469 g . von „ ii „ 100 „ 420 g. von „ iii „ 100 „ 418 g. einfluß verschiedenartiger mineraldüngung. 27 durch diese ergebnisse werden die befunde des vorhergehenden versuches hinsichtlich zunahme der größenverhältnisse und ge schmacksverbesserung bei „volldüngung“ bestätigt. die analytischen untersuchungen dieses versuches sowie von anderen, welche teilweise als ergänzung einiger oben beschriebener zu gelten haben und daher als wiederholungen aus späteren jahren anzusehen sind, waren noch nicht zum abschluß gelangt und sollen in einer weiteren veröffentlichung niedergelegt werden. die indische rundoder rangoonbohne. von e. rost (berlin). seitdem 1884 von davidson und stevenson vergiftungen durch samen von phaseolus lunatus (pois d'achery) beschrieben wurden, sind durch den genuß dieser bohne (mondbohne) zahl reiche vergiftungsfälle") beobachtet worden. 1906 untersuchte diese haricot à acide cyanhydrique eingehend guignard, nachdem 1904 dunstan und henry das blausäureabspaltende glykosid, das phaseolunatin, näher untersucht hatten. in deutschland gaben die von dammann und behrens 1906 beobachteten massenvergif tungen von pferden, rindern und schweinen anlaß zu chemischen untersuchungen dieser bohnenart. neuerdings brachten tages zeitungen die für den fachmann beunruhigende nachricht, es sollten die 50000 t bohnen der ersten lebensmittellieferung der entente an deutschland aus der rangoonbohne bestehen. die rangoonbohne is t die mondbohne, die auch als kra tok-, java-, lima-, duffin-, burma-, paigya-, kidney bohne, fève de kratok, haricot de siève, pois d'achery, amer, adam, portal oder du cap bezeichnet wird. sie ist im tropischen amerika heimisch, wird auf java, in ostindien, im östlichen binnenafrika, auf madagaskar und mauritius angebaut, ist unserer gartenbohne nahe verwandt und kommt mit verschieden *) e . rost, blausäurepflanzen. encyclopäd. jahrb. d. ges. heilkunde, xvi (1909), s. 83. uc1.b3299303_page_033_cut uc1.b3299303_page_034_cut uc1.b3299303_page_035_cut uc1.b3299303_page_036_cut uc1.b3299303_page_037_cut uc1.b3299303_page_038_cut uc1.b3299303_page_039_cut uc1.b3299303_page_040_cut uc1.b3299303_page_041_cut uc1.b3299303_page_042_cut journal of applied botany and food quality 92, 281 287 (2019), doi:10.5073/jabfq.2019.092.039 1salt-tolerant rice research group, department of biology, faculty of science, khon kaen university, thailand 2 faculty of veterinary science, khon kaen university, thailand 3 program of biology, faculty of science, ubon ratchathani rajabhat university, thailand 4 faculty of science and technology, surindra rajabhat university, thailand nutritional composition and genetic diversity of thai aromatic rice landraces worasitikulya taratima1*, pitakpong maneerattanarungroj2, khwanduean rattana3, wattanachai pathomsirivong4, pradub reanprayoon4 (submitted: october 18, 2018; accepted: september 19, 2019) * corresponding author summary fifty thai aromatic rice landraces and one commercial cultivar (kdml 105) were subjected to proximate nutritional analysis to determine protein, fat, fiber, carbohydrate, ash, moisture, amylose and 2ap contents. genetic diversity was characterized using random amplified polymorphic dna (rapd) and inter simple sequence repeat (issr) markers. fifty-one cultivars were clustered based on rapd and issr markers. various nutrient compositions of the fifty rice landrace cultivars and one commercial cultivar were checked. among 100 rapd primers tested, 15 showed high polymorphism (100%) with an average of 21.2 bands per primer. fifteen issr pri mers out of 100 produced high polymorphism (99.74%) with an average of 26 bands per primer. the upgma dendrogram based on genetic similarity grouped the cultivars into two clusters and several sub-clusters. clustering of aromatic rice using issr markers gave increased clarity and was more effective than using rapd markers for both nutritional composition and polymorphism level. findings will provide practical guidelines for nutritionists and plant breeders in selecting suitable cultivars and genetically diverse parents for plant breeding programs. keywords: aromatic rice, nutritional composition, genetic diversity, rapd, issr introduction rice (oryza sativa l.) represents one of the most important cultivated crops since it supplies food for half of the world’s population (mahathanaseth, 2014; tarang and gashti, 2016). thailand is a global center for rice diversity and important rice-producing countries, devoting 66% of its agricultural area for rice production. more than 17,093 rice cultivars have been collected around thailand since 1937 (department of agriculture thailand, 2000). rice landraces developed from wild progenitors and still retain high genetic diversity (ray et al., 2013). they can be identified by their morphological characteristics and many have been named by local farmers. the thai word “hom” means aroma. the genetic structure of rice landraces is heterogeneous; landraces can show variable phenology and are able to grow in both biotic and abiotic stress environments as a valuable trait for crop production improvement (dwivedi et al., 2016). the diversity of rice landraces serves as a valuable genetic resource for future crop improvement to meet the ever-increasing demand for food production while alleviating poverty and promoting economic growth (choudhury et al., 2013). moreover, some landraces or traditional varieties show higher nutritional and medicinal values than general rice distribution in thailand. therefore, thai rice landraces are a necessary and valuable resource for rice breeding programs (rerkasem and rerkasem, 2002). genetic diversity can be evaluated using morphological traits, seed proteins, isozymes and dna markers (liu et al., 2016). a number of molecular markers have been used to study genetic diversity in rice (devi et al., 2015; emon et al., 2015; edwards et al., 2016). the random amplified polymorphic dna (rapd) technique is simple with low cost and less performing time; prior knowledge on the genotype is not required (rekha et al., 2011). inter simple sequence repeat (issr) is a microsatellite-based multilocus marker technique, which is simple and useful for estimating genetic diversity in several crop plants. the issr technique has advantages over rapd with a higher level of polymorphism, reproducibility and cost-effectiveness (kshirsagar et al., 2014). rapd and issr analyses have been used in genetic diversity studies in several crop plants. in rice, rapd has been used for identification and classification of aromatic rice varieties (choudhury et al., 2001) and recognition of duplicate ac cessions within a rice germplasm collection. meanwhile, issr has been utilized to examine genetic diversity and population structure among landraces to improve rice varieties (kumbhar et al., 2015). usefulness of rapd and issr markers in assessing the diversity of rice landraces from northeast thailand has not been previously examined. thus, this study was conducted to determine nutritional compositions and elucidate genetic diversity information for 50 aro matic rice landraces from northeast thailand and a commercial rice cultivar using these two marker systems. results will provide a foundation for further research to select appropriate parental geno types for plant breeding and crop improvement programs. materials and methods plant materials seeds of 50 aromatic rice landraces and one commercial rice cultivar (hom mali 105 or kdml 105) were provided by the surin rice research center, bureau of rice research and development, surin province, thailand. codes c1-c50 were used for the cultivars with c51 (kdml 105) as the out group for dendrogram analysis (tab. 1). proximate analysis total nitrogen and crude protein were determined by the kjeldahl gerhardt method (n x 5.95) (nitrogen distillation system): velp scientifica (aoac, 2000) while amylose content was measured using a uv-1700 shimadzu spectrophotometer at 620 nm with potato amylose as the standard (aoac, 1996). crude lipids were extracted with hexane using a soxhlet extractor (buchi e-816, switzerland), soxhlet method (aoac, 2000). total crude carbohydrate (cho) was analyzed by the phenol-sulfuric acid method (dubois et al., 1956). ash contents (gravimetric) were determined based on methods outlined in aoac (2000), while moisture content was examined based on induhara et al. (1971). 282 w. taratima, p. maneerattanarungroj, k. rattana, w. pathomsirivong, p. reanprayoon quantification of 2-acetyl-1-pyrroline (2ap) using headspace-solid phase microextraction (hs-spme) followed by gas chromatography coupled with flame ionization detection (gc-fid) in scented rice was investigated based on mathure et al. (2011). 2ap concentrations were calculated using the following formula. 2ap area × tmp conc. (ng/μl) × injection vol. (μl) 2apconcentrations (ng/g) = tmp area × sample weight (g) each treatment consisted of three replicates with 100 g of rice per replicate, except for 2ap when 0.50 g of finely ground rice grains was used. data were expressed as means ± standard deviation and assigned by analysis of mean variance (one-way anova) to investigate nutritional variation. differences of means between clusters based on rapd and issr markers were separated by lsd test (p < 0.05) using spss version 18 software. correlation studies the nutritional composition character associations represented by correlation coefficient between different pairs of characters at the phenotypic levels were calculated based on searle (1961) and singh et al. (2018): where, cov.xy (p) denotes phenotypic covariance between characters x and y, respectively. var.x (p), var.y (p) denotes variance for characters x and y at phenotypic levels, respectively. extraction of genomic dna fresh leaves of 4-5-day-old rice seedlings were collected randomly from each rice landrace and used as the source material. genomic dna was extracted following li and midmore (1999). dna concentrations were determined by measuring the absorbance of diluted dna solution at 260 nm using a spectrophotometer. dna samples were checked by 1.5% agarose gel with 0.5x tbe buffer; those with high band intensity and less smear were selected for polymerase chain reaction (pcr). rapd/issr markers and pcr amplifications a set of 100 decamer primers was used for rapd and a set of 100 primers was screened for issr. out of 100 rapd/issr pri mers screened, 15 were selected for further analysis based on clari ty, scorability, and reproducibility of the banding patterns. rapd and issr amplifications were carried out using a touchdown pcr method in 20 μl reaction mixtures containing 10-20 ng of genomic dna, 2.0 μl of 10x reaction buffer, 1.5 mmol/l mgcl2, 1.13% form amide, 200 μmol/l dntps and l u taq dna polymerase with 1.0 μmol/l rapd or issr primer. a touchdown thermal program was set as follows: 94 ºc, 5 min; 35 × (52 ºc to 48 ºc decreased in 1 ºc step, 1 min); and 72 ºc, 5 min. pcr reactions were carried out on a thermal cycler. amplified products from each sample were separated electrophoretically on 1.5% agarose gel containing sybr green dye in 0.5x tbe buffer at 100 v for 1½ hours. data analysis rapd and issr bands were scored using photocapt program (vilber lourmat, france). each amplified band was considered as a unit character regardless of its intensity and scored in terms of a binary code based on presence (1) and absence (0) of bands. rapd and issr data matrix was constructed based on presence/absence of bands. a combined data matrix was also produced and used to calculate genetic similarity based on the nei and li’s similarity coefficient (nei and li, 1979) using biogene version 98 (bioprofil, vilber lourmat, france). the similarity matrix was used to construct a dendrogram using the unweighted pair-group method with arithmetic averages (upgma). results and discussion nutrient composition and 2ap analysis proximate protein, fat, fiber, carbohydrate, ash, moisture and amylose contents of 50 cultivars and one commercial rice cultivar were investigated. average, minimum, maximum, mean and standard deviations are shown in tab. 1. nutritional composition showed no significant relationship with low standard deviation except for carbohydrate and amylose content. high variances were shown in carbohydrate and amylose contents with standard deviations also high at 5.23 and 6.38, respectively. seven cultivars of aromatic rice and kdml 105 showed 2ap content, with highest value in c36 (hom mali, gs no. 19380) (tab. 1). the 2ap contents of c35, c36, c41, c44 and c50 were all higher than kdml 105. this result concurred with hien et al. (2006) and pitija et al. (2017) with 2ap content of c36 cultivar higher than kdml 105 by a factor of three. many rice cultivars showed lower protein content compared to general rice at around 7-8% protein (chaudhari et al., 2018) and other rice cultivars such as indian aromatic rice (nadiger and kasturiba, 2015), indian non-aromatic rice (verma and srivastav, 2017), landrace rice cv njavara (deepa et al., 2008), basmati, siam, and fragrant rice (mohd fairulnizal et al., 2015) and thai jasmine rice, kdml 105 (laokuldilok et al., 2013; moonghgarm et al., 2012). this finding presents an opportunity to develop or improve some aromatic rice cultivars as low protein rice, a food with special medical properties to support the renal function of patients with chronic kidney disease. these patients need to reduce the amount of protein ingested from rice (takei et al., 2017). correlation studies the estimates of phenotypic correlation coefficients calculated between seven nutritional composition characters were presented in tab. 3. amylose exhibited significant and positive correlation with protein, fat, fiber, carbohydrate and ash. most of nutritional composition characters showed positive correlation except fat and protein (-0.1205), fat and moisture (-0.0320), fiber and moisture (-0.0319), and carbohydrate and moisture (-0.0274). this significant negative correlation indicated that rice variety high in moisture may probable to be low in fat, fiber and carbohydrate. the correlation between moisture and carbohydrate in this result concurred with verma and srivastav (2017) who reported on aromatic and non-aromatic indian rice but not similar in correlation between carbohydrate and protein. however, nutritional composition content may not be consistent as milling time (laokuldilok et al., 2013), rice processing (abbas et al., 2011), different measurement methods (grimm et al., 2011), and diverse varieties, environments and crop management methods all affect nutritional composition content and their correlations. although many reports have been established about nutritional composition in rice varieties, nutrient correlation between some compositions are poorly perceived, including in thai rice. however, correlation between some nutritional compositions in this report may useful as selection criteria for thai aromatic rice breeding program in the future. genetic similarities and clustering among the 100 rapd markers used, 15 were selected to characterize and assess the genetic variability among the 50 rice landraces be cov.xy (p) phenotypic correlation coefficients (rp) = √ var.xp.var.yp thai rice landraces diversity 283 tab. 1: nutrient composition and 2ap contents of the rice landraces. gs no. code-local name percentage (mean) protein fat fiber cho ash moisture amylose 2ap (μg/g) 2720 c1-kee tom hom 6.24 0.40 2.67 71.51 0.13 11.90 2.06 0.00 4488 c2-khao hom 5.80 0.73 2.67 72.03 0.33 12.29 1.56 0.00 4489 c3-niaw hom mali 4.34 0.60 1.67 65.98 0.47 10.96 1.72 0.00 4869 c4-khao hom 6.00 0.73 2.00 74.18 0.53 10.86 2.33 0.00 4870 c5-khao hom 4.59 0.73 1.67 80.20 0.33 11.40 1.61 0.00 4878 c6-khao hom 7.57 0.40 1.67 71.05 0.47 11.23 2.22 0.00 5595 c7-niaw hom 4.97 0.47 3.00 72.00 0.47 10.94 2.61 0.00 5624 c8-hom tung 6.54 0.73 2.33 72.25 0.60 11.89 4.33 0.00 5670 c9-hom tung 5.42 0.40 1.33 63.80 0.27 12.13 3.61 0.00 6724 c10-hom pa ma 4.34 0.80 2.00 72.11 0.27 13.08 2.61 0.00 7608 c11-hom udom 4.55 0.67 2.33 73.91 0.33 11.04 2.72 0.00 12148 c12-mak hom 4.14 0.60 1.67 79.69 0.33 12.31 1.94 0.00 12512 c13-hom udom 4.73 0.40 1.67 64.11 0.13 11.28 2.67 0.00 13911 c14-hom 3.90 1.07 1.67 79.12 0.47 11.65 1.56 0.00 13929 c15-hom 4.05 0.73 2.33 66.21 0.27 11.75 2.39 0.00 13932 c16-hom maled lek 6.76 0.60 2.33 80.99 0.67 11.70 13.89 0.00 13975 c17-doh hom 5.82 0.80 2.67 80.00 0.67 10.59 15.50 0.00 14512 c18-hom nual 5.02 0.87 3.33 80.73 0.67 12.78 15.67 0.00 14518 c19-hom nang nual 4.64 1.07 3.33 79.40 0.67 11.80 13.00 0.00 14539 c20-hom dong 4.83 0.67 3.67 80.20 0.27 10.97 16.17 0.00 16066 c21-hom pa ma 5.84 0.80 2.33 78.02 0.27 11.29 14.89 0.00 16068 c22-pa ma hom 5.72 0.73 2.67 80.53 0.47 14.02 14.33 0.00 18415 c23-hom mali 3.85 0.60 3.33 67.62 0.53 12.20 3.28 0.00 18421 c24-hom mali 4.73 0.73 2.00 79.03 0.40 11.60 15.78 0.00 18424 c25-hom mali 5.12 0.33 2.33 81.21 0.47 10.83 17.78 0.00 19342 c26-hom mali 5.83 0.93 2.33 79.98 0.67 12.31 15.06 9.34 19343 c27-hom mali 5.82 0.73 2.33 81.64 0.33 13.16 18.22 0.00 19344 c28-hom mali 5.82 0.73 2.33 81.04 0.47 11.53 15.22 0.00 19345 c29-hom mali 5.74 0.67 2.00 82.48 0.27 13.30 16.61 0.00 19347 c30-hom mali 5.39 0.67 3.33 78.20 0.40 13.58 14.28 0.00 19348 c31-hom mali 5.16 0.87 2.00 79.64 0.33 11.70 16.61 0.00 19349 c32-hom mali 5.55 0.67 2.67 83.54 0.47 12.13 13.61 0.00 19350 c33-hom mali 5.12 0.73 2.33 77.10 0.33 12.22 14.61 0.00 19378 c34-hom mali 5.83 0.53 3.33 81.55 0.47 12.31 13.39 0.00 19379 c35-hom mali 5.22 0.80 2.67 78.29 0.47 11.97 16.78 30.14 19380 c36-hom mali 5.68 0.60 3.00 81.36 0.40 10.72 14.67 65.88 19381 c37-hom mali 6.16 0.47 3.00 81.31 0.33 12.54 16.61 0.00 19382 c38-hom mali 5.24 0.93 2.33 80.07 0.33 13.57 15.56 0.00 19383 c39-hom mali 4.21 0.47 1.00 67.41 0.33 13.31 2.72 0.00 19384 c40-hom mali 5.19 0.80 3.67 82.88 0.47 9.86 15.44 0.00 19385 c41-hom mali 6.67 0.40 1.33 78.25 0.33 12.83 2.17 29.87 19386 c42-hom mali 4.27 0.60 1.67 82.50 0.27 10.72 4.83 0.00 19387 c43-hom mali 5.94 0.27 2.67 80.27 0.40 11.18 1.56 0.00 21688 c44-hom pa ma 4.80 0.53 1.67 79.44 0.27 10.88 3.50 34.18 21940 c45-hom dok doo 7.11 0.40 3.33 81.07 0.27 12.02 14.89 0.00 22776 c46-hom nual 4.72 0.20 2.33 73.19 0.40 13.45 5.94 0.00 22781 c47-khao hom 6.74 0.47 2.33 78.30 0.27 14.35 14.50 5.52 21383 c48-hom yai 6.55 0.60 1.67 75.99 0.27 12.97 2.39 0.00 23209 c49-khao hom 5.51 0.60 3.00 78.04 0.47 12.29 4.89 0.00 23253 c50-hom udom 4.77 0.93 1.33 77.99 0.33 10.23 3.89 42.75 none c51-kdml 105 5.03 0.73 2.33 80.81 0.47 10.59 14.89 20.43 or hom mali 105* min 3.85 0.20 1.00 63.80 0.13 9.86 1.56 0.00 max 7.57 1.07 3.67 83.81 0.67 14.35 18.22 65.88 mean 5.36 0.65 2.36 77.02 0.40 11.93 9.20 4.86 sd 0.85 0.19 0.65 5.23 0.13 1.01 6.38 13.25 * c51-kdml 105 or hom mali 105 is a commercial cultivar used as the out group. (gs no.-genetic stock number; cho-carbohydrate) 284 w. taratima, p. maneerattanarungroj, k. rattana, w. pathomsirivong, p. reanprayoon tab. 2: efficiency comparison between rapd and ssr markers for identifying polymorphism among the 51 rice cultivars. fifty-one selected rice cultivars rapd issr total amplified bands 347 390 maximum band 5,335 3,580 minimum band 140 170 polymorphic bands 347 389 polymorphic band percentage 100 99.74 number of primers used 15 15 mean of polymorphic bands/primers 21.20 26 nei and li’s similarity coefficient percentage 91 74 tab. 3: estimates of phenotypic correlation coefficients between seven nutrient compositions in aromatic rice. composition protein fat fiber cho ash moisture amylose 0.2001** 0.1722* 0.3638** 0.6606** 0.1843* 0.1263 protein -0.1205 0.0948 0.2050* 0.1145 0.1236 fat 0.0178 0.1437 0.1294 -0.0320 fiber 0.2463 0.2256 -0.0319 cho 0.2054 -0.0274 ash 0.1843 * and **, correlation is significant at p < 0.05 and p < 0.01, respectively. fig. 1: rapd and issr amplification profiles of 50 rice cultivars and a commercial rice cultivar generated using rapd primer a01 in panel a and issr primer 03 in panel b, where lane m is 100–10,000 bp dna ladder. cause of their distinct amplifications and highly reproducible bands. a total of 347 bands were amplified by the selected rapd primers with an average of 21.2 bands per primer and sizes ranging from 140 bp to 5,335 bp. they were all polymorphic as shown in fig. 1 and tab. 2. out of 100 issr markers screened, 15 were selected to assess genetic diversity among the 50 rice landraces. selected issr primers yielded a total of 390 bands with an average of 26 bands per primer, with sizes ranging from 170 bp to 3,580 bp, and 389 as polymorphic (99.74%) as presented tab. 2. the rapd and/or issr amplification patterns were used to assess genetic variation among the 50 rice landraces by cluster analysis. a dendrogram was plotted for each of the amplification patterns using the similarity coefficient derived from the rapd profile data as depicted in fig. 2-a. the dendrogram constructed for the rapd amplification pattern was resolved into two clusters at 91% similarity coefficient. minimum number of rice landraces (6) was represented in cluster i, whereas cluster ii contained 45 rice landraces. cluster i was again divided into two sub-clusters and cluster ii into five subclusters. each sub-cluster of cluster i contained 3 rice landraces, with 12, 14, 13, 5 and 1 rice landrace included in sub-clusters iia, iib, iic, iid and iie, respectively. the dendrogram constructed for the issr amplification pattern was divided into two clusters at 74% similarity coefficient (fig. 2-b). the minimum number of rice landraces (2) was represented in cluster i, whereas cluster ii contained 49 rice landraces. cluster ii was again divided into six sub-clusters. sub-clusters iia, iib, iic, iid, iie and iif contained 2, 14, 13, 9, 10 and 1 rice landrace, respectively. data quality monitoring is important for various utilized applications, especially in the food industry and nutrition-related medicine. in general, a rice grain consists of 90% flour, 8% protein, 0.4-0.6% fat, 0.3-0.6% fiber and 0.4-0.9% ash (piyachomkwan et al., 2001). examples of rice grain applications are as high purity rice flour or low protein flour production which are both useful in the food, pharmaceutical and cosmetics industries. nutritional composition in this aromatic rice group showed no distinguishing differences except for total carbohydrate and amylose contents. some rice cultivars recorded low carbohydrate and amylose content although these had the same local names. for example, cultivar codes c23-c43 had the same local name (home mali) but different genetic stock number (gs no.), nutritional composition and polymorphism level. their nutritional compositions showed various measurement values. hom mali rice varieties (c23-c43) were found in various clusters using the rapd and issr systems (fig. 2). this may involve amylose biosynthesis gene expression (gbss alleles) (fasahat et al., 2014) or other genes in the rice genome. clustering of aromatic rice using the issr system showed more clarity and effectiveness than the rapd system, especially regarding the nutritional composition issue. average nutritional composition value from issr clustering related closely with the measurement data. our results indicated that the 50 aromatic rice landraces showed both genetic and nutrient diversity; however, genetic diversity remains the cornerstone of crop improvement, providing breeders with options to develop new and improved cultivars with desirable characteristics (govindaraj et al., 2015). morphological (a) rapd protein fat cho amylose 0 5 0 0.5 0 20 40 60 80 0 5 10 15 0 5 0 0.5 1.0 0 20 40 60 80 0 5 10 15 0 5 0 0.5 1.0 0 20 40 60 80 0 5 10 15 0 5 0 0.5 0 20 40 60 80 0 5 10 15 0 5 0 0.5 0 20 40 60 80 0 5 10 15 0 5 0 0.5 0 20 40 60 80 0 5 10 15 i (a) rapd (b) issr (b) issr 0 5 0 0.5 0 20 40 60 0 5 10 15 20 0 5 0 0.5 1 0 20 40 60 80 0 5 10 15 0 5 0 0.5 1 0 20 40 60 80 0 5 10 15 0 5 0 0.5 0 20 40 60 80 0 5 10 15 0 5 0 0.5 0 20 40 60 80 0 5 10 15 0 5 0 0.5 0 20 40 60 80 0 5 10 15 0 5 0 0.5 0 20 40 60 80 0 5 10 15 (b) issr iif i protein fat cho amylose thai rice landraces diversity 285 trait analyses, as well as chemical characterization, are useful tools to identify differences in plant cultivars affected by phenotypic expression (roy and sharma, 2014; roy et al., 2016). our results may not reflect real genetic variation due to genotype-environment interaction or unknown genetic control of phylogenic morphological and agronomic traits (kumbhar et al., 2015). instead, classification of individual genotypes into different groups based on polymorphisms at the dna level with molecular markers is considered a powerful tool for estimation of genetic divergence (al-turki and basahi, 2015; roy et al., 2015). here, rapd and issr markers were utilized to assess genetic diversity in 50 aromatic rice landraces from northeast thailand and a commercial rice cultivar (kdml 105). selected rapd and issr markers exhibited distinct amplification and highly reproducible bands, thereby suggesting their suitability for genetic diversity study. grouping of rice landraces based on polymorphic rapd and issr markers indicated low to moderate genetic diversities among the studied genotypes. moderate genetic diversity among the landraces suggested distinct differences in their genetic architecture. among the 100 rapd primers used, 15 generated several specific fig. 2: upmga-based dendrograms depicting genetic relationships among 50 rice landraces and a commercial rice cultivar based on rapd (a) and issr (b) profiles in comparison with some nutritional composition (protein, fat, cho-carbohydrate and amylose). (a) rapd protein fat cho amylose 0 5 0 0.5 0 20 40 60 80 0 5 10 15 0 5 0 0.5 1.0 0 20 40 60 80 0 5 10 15 0 5 0 0.5 1.0 0 20 40 60 80 0 5 10 15 0 5 0 0.5 0 20 40 60 80 0 5 10 15 0 5 0 0.5 0 20 40 60 80 0 5 10 15 0 5 0 0.5 0 20 40 60 80 0 5 10 15 i (a) rapd (b) issr (b) issr 0 5 0 0.5 0 20 40 60 0 5 10 15 20 0 5 0 0.5 1 0 20 40 60 80 0 5 10 15 0 5 0 0.5 1 0 20 40 60 80 0 5 10 15 0 5 0 0.5 0 20 40 60 80 0 5 10 15 0 5 0 0.5 0 20 40 60 80 0 5 10 15 0 5 0 0.5 0 20 40 60 80 0 5 10 15 0 5 0 0.5 0 20 40 60 80 0 5 10 15 (b) issr iif i protein fat cho amylose 286 w. taratima, p. maneerattanarungroj, k. rattana, w. pathomsirivong, p. reanprayoon bands in 23 rice landraces. in particular, primer c07 produced only two specific bands in c51, which can be effectively used for further development of sequence characterized amplified region (scar) markers for precise identification of this rice landrace in the given set of rice genotypes. similarly, out of 100 issr markers, 15 primers produced several specific bands in 32 rice landraces, with primers issr5 and issr15 generating 5 specific bands in c41. in this investigation, 51 rice genotypes were grouped into 2 major clusters through upgma-based clustering using nei and li’s similarity coefficient which indicated low to moderate variation between genotypes. the studied rice landraces belong to different areas of northeast thailand and evolved through different biotic and abiotic factors, thereby displaying differences in their genetic compositions. comparison between rapd and issr markers indicated that clustering using issr markers gave a higher level of polymorphism than rapd markers, similar to kshirsagar et al. (2014). our findings showed that genetic diversity assessed using issr markers was more reliable than evaluated by rapd markers due to the larger size of issr markers and higher temperature in the annealing step, allowing issr markers to generate more specific bands. this is the first report on the nutrient composition of a set of 50 aromatic rice landraces from the northeastern region of thailand. conclusions findings will be useful for plant breeders to selected suitable cultivars to achieve high nutrient composition and improved yield through breeding techniques as the first step in rice improvement. moreover, this study highlighted the utilization of touchdown pcr for rapd and issr amplifications in the identification of 50 aromatic rice landraces and a commercial rice cultivar kdml 105. both marker systems produced specific bands in the studied genotypes, thereby suggesting their suitability for genetic diversity studies. findings also showed low to moderate genetic diversities among the studied rice genotypes. marker-based identification and differentiation of rice genotypes may be applied to maintain the integrity of these rice landraces which will benefit farmers and research workers. this investigation determined five cultivars (c35, c36, c41, c44 and c50) that showed interesting characteristics of low protein and high 2ap content. moreover, their genetic characteristics were similar when considered on rapd (cluster i, iia and iid) and issr (cluster iid and iie) systems. further improvements in breeding programs and cultivation of these five cultivars are highlighted as a future research area. acknowledgements this research was supported by the department of biology, faculty of science, khon kaen university, thailand. the authors are also indebted to the surin rice research center, thailand for providing rice seed materials. references abbas, a., murtaza, s., aslam, f., khawar, a., rafique, s., naheed, s., 2011: effect of processing on nutritional value 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the corresponding author: worasitikulya taratima, department of biology, faculty of science, khon kaen university, khon kaen 40002, thailand e-mail: worasitikulya@gmail.com © the author(s) 2019. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1007/s12161-010-9171-3 http://dx.doi.org/10.1016/j.apcbee.2012.06.014 http://dx.doi.org/10.4061/2011/784719 http://dx.doi.org/10.1371/journal.pone.0141405 http://dx.doi.org/10.1186/s12863-016-0414-1 http://dx.doi.org/10.1007/s12298-014-0221-y http://dx.doi.org/10.3923/ajcs.2018.100.106 http://dx.doi.org/10.17140/aftnsoj-3-142 http://dx.doi.org/10.1016/s2095-3119(15)61221-7 http://dx.doi.org/10.3923/ajps.2005.562.573 http://dx.doi.org/10.1016/j.rsci.2016.05.005 mailto:worasitikulya@gmail.com journal of applied botany and food quality 92, 371 377 (2019), doi:10.5073/jabfq.2019.092.049 1department of botany, university of zululand, kwadlangezwa, south africa 2department of chemistry, university of zululand, kwadlangezwa, south africa agro-morphological changes caused by the accumulation of lead in corchorus olitorius, a leafy vegetable with phytoremediation properties sibongokuhle ndlovu1, rajasekhar v.s.r. pullabhotla2, nontuthuko r. ntuli1* (received: october 3, 2019; accepted: october 21, 2019) * corresponding author summary lead (pb) can enter the food chain through the consumption of contaminated plants and can cause serious health issues. however, research on how pb accumulation affects morphology of leafy vegetables in south africa is minimal. this study tested the effect of lead accumulation on vegetative and reproductive traits of corchorus olitorius. plants were grown under varying pb concentrations, and studied for their variation in vegetative and reproductive traits as well as pb accumulation in leaves, stems and roots. plants grown within allowable soil concentrations of 150 mg kg–1 pb accumulated toxic (≥ 10 mg kg–1) pb in all plant parts without causing any morphological defect, except for a decrease in chlorophyll content. minor reductions in growth and yield were evident only at 900-1000 mg kg–1 concentration. pb accumulation increased as its concentration increased in the soil, with a higher accumulation in roots in comparison to aerial parts. in conclusion, c. olitorius can grow and reproduce under toxic pb levels (≥ 300 mg kg–1) and accumulate toxic amounts of pb (≥ 10 mg kg–1) without visible morphological defects. therefore, it is suitable for phytoremediation but unsafe for consumption when it is collected from sites prone to pb contamination. keywords: corchorus olitorius, lead heavy metal, plant accumulation, vegetative and yield traits introduction contamination of agricultural soils with lead (pb) is a major problem for agricultural production and human health (fattahi et al., 2019). lead, a heavy metal that is not essential for plant growth, can be accumulated in excess by plants and become toxic to them (sheoran et al., 2016). lead soil contamination results from sources such as smelting, combustion of leaded gasoline and application of leadcontaminated sewage sludge as fertilizer (pourrut et al., 2011). the maximum allowable pb concentration in agricultural soils is 300 mg kg–1 (xiao et al., 2018). the lowest detected value is 0.3 mg kg–1 (ahmad et al., 2019) and the maximum permissible limit is 10 mg kg–1 (who, 1998 cited in jezler et al., 2015) pb within edible plant parts. phytoremediation is a process of growing plants in contaminated soils to either remove heavy metals (phytoextraction and phytovolatilization) or stabilize them into a harmless status (phytostabilization or phytoimmobilization) (khalid et al., 2017; liu et al., 2018). hyperaccumulators are plants that can accumulate metals and metalloids at concentrations 100 times greater than that of normal plants growing in the same environment (sheoran et al., 2016). certain plants within the asteraceae and brassicaceae families can accumulate > 1 000 mg kg–1 of pb (sheoran et al., 2016). some wild leafy vegetables are good accumulators of pb, too. amaranthus viridis is recommended for pb phytoextraction, whereas solanum nigrum is recommended for phytostabilization of pb-contaminated soils (malik et al., 2010). corchorus olitorius l., a leafy vegetable commonly known as jute mallow, is an annual erect herb that grows in fields, home gardens (tovihoudji et al., 2015) and on roadsides (sanyaolu et al., 2011). its leaves are in abundance of iron, folate, protein, fibre, calcium, riboflavin, carotene, vitamin c and phenols, and have high zinc bioavailability and appreciable amounts of other proximate components and minerals (isuosuo et al., 2019). cooked leaves and tender shoots that are eaten along with food staples are recommended for pregnant and nursing mothers because of their high iron content (sanyaolu et al., 2011). leaves of c. olitorius are used in folk medicines for different ailments and they possess antioxidant, hepatoprotective and antidiabetic properties (saliu et al., 2019). this species has the ability to accumulate 0.31 mg kg–1 of pb in its leaves when it grows at ≤ 10 m away from the major roads, which is slightly above the lowest detected value (0.3 mg kg–1) for consumption purposes (sanyaolu et al., 2011). consumption of lead contaminated food including vegetables in particular, cause health problems such as brain and kidney diseases (fattahi et al., 2019). lead uptake is primarily through the roots, but some plants such as brassica napus also acquire it through the leaves (rubio et al., 2019). roots can accumulate large amount of pb but restrict its movement towards the shoots (pourrut et al., 2011). the uptake of pb from the soil by plants increases with an increase of its concentration in the soil − availability being the highest in loam and sandy soils − at higher soil moisture levels − and low ph (sheoran et al., 2016). vegetable and medicinal plants such as ocimum basilicum show similar plant height, internode length, leaf length and width as the control when exposed to 400 mg kg–1 pb (fattahi et al., 2019); which is a major concern because this concentration is above the maximum allowable soil concentration (300 mg kg–1) (xiao et al., 2018). lead toxicity can induce different morphological, physiological and biochemical effects on plants at different stages of plant growth and contamination (pourrut et al., 2011). increase in pb levels in the soil retards seed germination, seedling growth and fresh weight of roots and shoots (yahaghi et al., 2019). it also impairs mitosis, root and shoot growth, transpiration, chlorophyll production, lamellar organization in the chloroplast and results in leaf chlorosis (pourrut et al., 2011). increased pb concentration in the soil also reduces the moisture content of roots, stems and leaves (yilmaz et al., 2009). improved and/or similar morphological features of some edible plants such as ocimum basilicum grown under toxic pb concentrations (≥ 300 mg kg–1) and had accumulated toxic concentrations (> 0.3 mg kg–1), when compared with untreated plants (fattahi et al., 2019), have high potential of intoxicating consumers. when people from rural areas accidentally harvest such pb contaminated vegetables for food and medicinal purposes, they can be intoxicated by high concentrations or gradual pb accumulation in their system. as c. olitorius is one of the leafy vegetables that grow in areas prone to pb contamination, it is essential to study its pb accumulation potential. depending on the type of species, plants that have accumulated toxic pb amounts can either have better, retarded or similar 372 s. ndlovu, r.v.s.r. pullabhotla, n.r. ntuli growth and yield than plants without pb. therefore, the objective of this study was to determine variation in vegetative and reproductive traits of c. olitorius accumulating different toxic pb concentrations. materials and methods seed sourcing, study area and experimental design seeds of corchorus olitorius l. were sourced from the agricultural research council in roodeplaat, pretoria. subsequent experiments were conducted at the university of zululand (28.85416° s, 31.84565° e), department of botany, in a rain-free environment. a black, humus-rich soil was collected from the university’s farm and had its properties analysed using a method described by manson and roberts (2000) (tab. 1). twenty-litre plastic pots were filled with soil mixed with lead in the form of lead acetate [pb(no3)2], applied at 0, 150, 300, 600, 900 and 1000 mg kg–1 soil as modified from khodaverdiloo et al. (2011). these values were chosen to include the maximum allowable (≤ 300 mg kg–1) and toxic (> 300 mg kg–1) pb levels in agricultural soils (xiao et al., 2018). the experiment was laid out in a randomized complete block design with five replications. ten seeds of c. olitorius were germinated in each pot and later thinned into one plant per pot, followed by application of 2:3:2 (27) npk fertilizer at a rate of 1 g kg–1 of soil. plants were regularly irrigated with deionised water (50 ml per pot), which was then collected from the base of the pots and reused to irrigate, in order to avoid nutrient or heavy metal loss. measurement of agronomic traits germination percentage was recorded at seven days after sowing (das), prior to thinning. all vegetative traits were measured at 44 das, whereas the number of branches and all reproductive traits were measured at 85 das, in quintuplicate. plant height (cm) was measured from the soil level to the tip of the stem using a ruler. the numbers of leaves and branches were counted manually. vernier callipers were used to measure stem width (mm) at 10 cm from the soil level. leaf area (length × width) (cm2) was recorded on the fourth leaf from the apex using a ruler. the leaf chlorophyll content of the fifth oldest leaf (from the apex of the main stem) was captured with a chlorophyll content meter (ccm-200). five different spots were randomly measured in the leaf and provided the average leaf chlorophyll content. plants were uprooted, washed with distilled water and blot-dried with a paper towel, and had their root length (cm) measured from the root tip to the base of the stem using a ruler. the fresh mass (g) of roots, stems and leaves of uprooted plants were measured separately. separated plant parts were oven dried at 60 °c until constant dry mass. the dry mass (g) and moisture content of roots, stems and leaves were also determined separately. the number of pods per plant was counted manually, and pod length (cm) and width (cm) determined with vernier callipers. seed traits were determined from five pods per plant per treatment. the number of seeds per pod was counted manually; total seed mass (g) per pod and 100-seed mass (g) in each pod were also recorded. lead content of plant parts plants were analysed for lead accumulation in roots, stems and leaves at both seedling (44 das) and termination or maturity (85 das) stages. three pots were selected for harvesting and each pot was used as a replicate for each plant part. plants were carefully uprooted, thoroughly washed with distilled water and blot-dried with a paper towel. plants were then separated into leaves, stems and roots. each plant part (in three separate replicates) was cut into pieces and further dried in an oven at 80 °c until they reached a constant weight. dried samples were ground into powder and packaged in air-tight plastic containers and stored in a fridge (–4 °c) for further analysis. one gram of each milled sample was dissolved in 5 ml of 60% hydrochloric acid and 10 ml of 70% nitric acid, and then digested at a moderate temperature of 50 °c until white fumes evolved, and the solution changed to a brownish colour. the heat was further intensified for few minutes to expel most of the hcl. 50ml distilled water were added, heated for few minutes and allowed to cool. the solution was filtered through a whatman’s no. 1 paper into a transparent plastic container, and was allowed to settle for a few minutes for the aspiration of the lead accordingly. the digested sample was analysed for lead concentration using an atomic absorption spectrophotometer. statistical analysis data were subject to analysis of variance (one-way anova) in genstat 12.1 version. means were separated using tukey’s multiple range test in genstat at a 5% level of significance. correlation matrix analysis also determined the relationship between vegetative and reproductive traits. results vegetative traits some vegetative traits of plants treated with lead differed significantly (p < 0.05) from each other and/or the untreated plants except for root length; number of branches, and leaf fresh mass (tab. 2). lead application at 300 mg kg–1 was associated with the maximum germination rate (100%) of c. olitorius seeds, whereas the minimum germination rate (60%) corresponded with 1000 mg kg–1. a significant reduction in seed germination was recorded for plants exposed to 900-1000 mg kg–1 pb compared with the control. plants exposed to 150-600 mg kg–1 pb were taller than untreated plants, whereas the shortest plants were exposed to 1000 mg kg–1. the plants treated with 1000 mg kg–1 pb had the thinnest stems compared with the control, but their stem girth was similar to all other treated plants. exposure of plants to 150 mg kg–1 pb promoted the formation of numerous leaves, whereas 1000 mg kg–1 pb drastically reduced the number of leaves per plant compared with the control. leaves gradually became smaller as the concentration of pb increased above 300 mg kg–1. plants treated with pb had a significantly lower chlorophyll content than untreated plants. only stems and leaves of plants exposed to 150 mg kg–1 pb had increased fresh and dry mass, respectively. this treatment also caused an increase in stem moisture content, but a reduction in leaf moisture content. however, a reduction in leaf moisture content was only significant when compared with untreated plants and 1000 mg kg–1 pb. tab. 1: properties of the soil used in the research soil property value (mg kg–1 for elements) p 10 k 170 mg 1432 na 525 zn 22 cu 3 mn 6 fe 395 ph 4.5 clay content 23.0% organic matter 4.3% effect of lead on corchorus olitorius 373 reproductive traits reproductive traits of pb-treated plants differed significantly among each other and from the untreated plants, except for the number of pods per plant (tab. 3). a reduction in pod length was recorded in plants treated with 900-1000 mg kg–1 pb, when compared with the control. only pods from plants treated with 1000 mg kg–1 were lighter than those of untreated plants. although the number of seeds per pod of the untreated plants were insignificantly different from all treated plants, 150 mg kg–1 produced plants with more numerous seeds per pod than plants treated with 900–1000 mg kg–1. total seed mass of plants grown under 150 mg kg–1 was higher than seeds of untreated plants and those treated with ≥ 600 mg kg–1. also, treatment with 150 mg kg–1 resulted in plants with a heavier 100-seed weight than untreated plants and those exposed to 600 and 100 mg kg–1. lead accumulation and correlation matrix lead accumulation differed significantly within each plant part and among different plant parts at seedling (44 days after sowing) and termination or maturity (85 days after sowing) stages (tab. 4). among the treated plants, mature roots of plants exposed to 900 mg kg–1 had accumulated the most pb (634.0 mg kg–1); whereas mature leaves treated with 1000 mg kg–1 had the least (22.8 mg kg–1). accumulation of a range from 22.8-78.8 mg kg–1 in immature and mature stems and leaves of plants exposed to different pb concentration in the soil did not differ significantly with the untreated plants without pb. at seedling stage, application of 1000 mg kg–1 resulted in plants with the highest pb accumulation (448.8 mg kg–1) in roots; but the lowest (67.0 mg kg–1) in stems, when compared with all other treatments. at both stages of growth, an increase in pb soil content resulted in its high accumulation in the roots. however, in immature stems and leaves, the increase in accumulation was evident up to the maximum of 900 mg kg–1 pb and then declined drastically at 1000 mg kg–1. in mature stems, the increase was only evident in plants exposed to 1000 mg kg–1; whereas leaves were not significantly different from each other. also, in each pb concentration in the soil, the accumulation was the highest in roots and then decreased relatively similarly in stems and leaves, at both stages of growth. almost all traits had a significant positive correlation with at least 50% of the traits investigated, except for root dry mass and leaf moisture content (tab. 5). most vegetative traits correlated significantly with one another except for leaf chlorophyll content, root dry mass, and moisture content of leaves, stems and roots. further, pod and seed traits were significantly correlated with one another and to most of the vegetative traits, except root fresh mass; leaf and root dry mass, as well as leaf, stem and root moisture content. tab. 2: effect of lead on the vegetative traits of c. olitorius. conc. gp rl ph sg nb nl la lcc lfm sfm rfm ldm sdm rdm lmc smc rmc 0.00 90.0ab 24.7a 38.00c 5.09a 8.0a 25.8bc 54.13a 54.07a 7.27ab 5.55a 1.34abc 1.08b 0.70b 0.51a 84.90b 61.70b 87.20b 150 86.7ab 26.3a 43.60b 4.86ab 8.8a 34.4a 49.90ab 44.54b 8.59a 8.77a 1.93a 2.64a 1.29a 0.16b 69.33d 91.70a 89.30ab 300 100a 26.0a 44.20b 4.68ab 8.0a 30.4ab 47.11b 44.04b 4.79c 9.16a 1.44ab 0.89b 0.83b 0.46a 81.50bc 65.20b 91.07ab 600 70.0ab 23.7a 49.80a 4.79ab 7.8a 24.0c 46.73b 37.77c 6.04bc 6.41a 1.27abc 1.28b 0.83b 0.44a 78.83c 65.90b 87.13b 900 73.3ab 26.3a 36.00c 4.44ab 7.4a 20.8c 36.22c 43.53b 6.02bc 5.15a 0.99bc 1.14b 0.65b 0.32ab 81.10bc 67.90b 87.07b 1000 60.0b 16.0a 29.80d 4.05b 7.2a 14.2d 34.76c 34.52c 6.30bc 6.11a 0.57c 0.72b 0.41c 0.21b 91.33a 71.20b 93.27a gm 80.0 23.8 40.23 4.65 7.87 24.93 44.84 43.08 6.50 6.86 1.26 1.29 0.79 0.35 81.17 70.60 89.17 cv% 16.3 20.3 6.3 9.2 14.7 12.1 6.6 4.5 8.9 38.1 23.9 18.5 9.9 18.8 2.2 8.8 2.0 lsd 23.72 8.81 3.35 0.57 1.53 3.98 3.91 2.56 1.06 4.75 0.55 0.44 0.14 0.12 3.23 11.33 3.19 p-value 0.033 0.159 <.001 0.02 0.36 <.001 <.001 <.001 <.001 0.35 0.005 <.001 <.001 <.001 <.001 0.002 0.006 means followed by different superscript(s) within a column vary significantly (p < 0.05). conc. (concentration (mg kg-1)); gp (germination percentage (%)); rl (root length (cm)); ph (plant height (cm)); sg (stem girth (mm)); nb (number of branches); la (leaf area (cm2)); nl (number of leaves); lcc (leaf chlorophyll content (mg cm–2)); lfm (leaf fresh mass (g)); sfm (stem fresh mass (g)); rfm (root fresh mass (g)); ldm (leaf dry mass (g)); sdm (stem dry mass (g)); rdm (root dry mass (g)); lmc (leaf moisture content (%)); smc (stem moisture content (%)); rmc (root moisture content (%)). tab. 3: effect of lead on the reproduction traits of c. olitorius. conc. np pl pm spp tsm 100-sm 0 7.00a 6.35ab 5.50a 114.4ab 0.12b 0.11bc 150 5.50a 4.97c 5.40a 139.6a 0.25a 0.15a 300 6.75a 6.48ab 5.38a 121.4ab 0.17ab 0.17a 600 6.25a 6.62a 5.37a 113.0ab 0.13b 0.14ab 900 2.25a 4.77c 4.58ab 98.4b 0.15b 0.15a 1000 6.50a 5.42bc 4.11b 86.6b 0.10b 0.09c gm 5.71 5.77 5.06 112.2 0.15 0.13 cv% 40.5 8.4 9.1 16.0 31.3 14.8 lsd 3.48 0.73 0.69 23.74 0.06 0.03 p-value 0.092 <.001 0.003 0.003 0.001 <.001 means followed by different superscript(s) within a column vary significantly (p < 0.05). np (number of pods); pl (pod length (cm)); pm (pod mass (g)); spp (number of seeds per pod); tsm (total seeds mass (g)); 100-sm (100 seed mass (g)). tab. 4: accumulation of pb in roots, stems and leaves of c. olitorius. harvest stage pb in soil pb in plant parts (mg kg–1) (days after (mg kg–1) sowing) 44 0 0.0l 0.0l 0.0l 150 185.5ef 78.8g–l 110.5f–k 300 276.8de 78.2g–l 136.2f–i 600 340.8cd 159.2fgh 179.5efg 900 409.5bc 202.5ef 212.0ef 1000 448.8b 67.0h–l 68.6h–l 85 0 0.0l 0.0l 0.0l 150 129.2f–j 33.2i–l 34.5i–l 300 212.5ef 37.5i–l 39.2i–l 600 483.8b 33.2i–l 42.0i–l 900 634.0a 41.0i–l 26.2jkl 1000 596.8a 116.0f–k 22.8kl means followed by different superscript(s) within a column and a row vary significantly (p < 0.05) roots stem leaves 374 s. ndlovu, r.v.s.r. pullabhotla, n.r. ntuli discussion phytoremediation and toxic consumption potential of c. olitorius exposed to lead contaminated soils corchorus olitorius species accumulated toxic lead content (> 10 mg kg–1) even when grown at concentrations below the maximum allowable soil levels (300 mg kg–1), but had very low morphological defects even at the exposure to high pb concentrations in the soil. however, its herein observed maximum pb accumulation of 634 mg kg–1 does not qualify it as hyperaccumulator (sheoran et al., 2016). this plant is good for phytoextraction purposes, because it can grow well in pb contaminated soil, remove it and accumulate it within its plant parts (liu et al., 2018). it can also be recommended for phytostabilization purpose because it accumulates high levels of pb in its roots and transport limited amount to the aerial parts (khalid et al., 2017). the low phytotoxicity was probably caused by the retention of more pb in roots and less translocation to the stems and leaves (shoots) (shi et al., 2019). although this retention was evident in the study, but the concentration that ranges from 22.8-212.0 mg kg–1 accumulated in its stems and leaves, is very high for the maximum recommended 10 mg kg–1 pb within the consumed plant part (jezler et al., 2015). statistical analysis showed insignificant differences of accumulated concentrations from 0-100 mg kg–1 pb among the c. olitorius plant parts; but this can be disputed and consider the maximum allowable concentration of 10 mg kg–1 pb for consumption purposes. a 150 mg kg–1 pb concentration in the soil, which is half the maximum limit of 300 mg kg–1 (xiao et al., 2018), either promoted growth and yield in c. olitorius or induced traits that were similar to the untreated plants. these included plants with numerous leaves that were relatively large, with higher fresh and dry masses — increased shoot dry mass and moisture content — as well as heavier total and 100-seeds weight. these features can make the plant to be selected for vegetable harvest by rural communities when they had accumulated toxic amounts of 78.8 and 33.2 mg kg–1 in stems as well as 110.5 and 34.5 mg kg–1 in leaves at immature and mature stages, respectively. c. olitorius accumulates pb at low soil concentrations, but pb becomes intensified within the plant leading to toxic amounts for consumption purposes. there was also a significant decline in pb concentration of stems (202.5–41.0 mg kg–1) and leaves (212.0–26.2 mg kg–1) from immature to mature stages of c. olitorius plants exposed to 900 mg kg–1 pb soil concentration, respectively. this might result from an increased level of phytostabilization in this plant (khalid et al., 2017; liu et al., 2018), and enhanced passive mechanisms as even small amount of pb penetrated root cell membranes, interacted with cellular components and increased thickness of the cell walls (pourrut et al., 2011), as plants were forming nodule-like swellings in their roots in concentrations from 900–1000 mg kg–1 at maturity. effects of consumption of plants such as vegetables that are contaminated with pb differs among individuals and their different stages of growth (dinis and fiúza, 2011). as c. olitorius is recommended for pregnant and nursing mothers (sanyaolu et al., 2011), its consumption with trace amounts of pb might lead to severe health issues of both the mother and the foetus. pregnant mothers, foetus and breastfeeding individuals are more susceptible to renal failure, cardiovascular diseases as well as neurological and mental disorders as a result of pb toxicity (dinis and fiúza, 2011; sarwar et al., 2017). therefore, precautions are necessary for the collection sites of these vegetables by pregnant women. high accumulation of pb in c. olitorius roots and relatively less translocation to the aerial parts is similar to the records in edible plants such as amaranthus viridis, malvastrum coromandelianum and chenopodium album (malik et al., 2010), as well as mentha arvensis (jezler et al., 2015). however, in ocimim bacilicum pb concentration was higher in leaves than roots when plants were exposed to 100 and 200 mg kg–1 pb (fattahi et al., 2019). c. olitorius is a potential species for phytoremediation in pb contaminated areas with advantages such as high germination rate, growth and yield rates on pb intoxicated soils. it also quickly accumulates lead at very juvenile stages without any morphological defects (phytotoxicity), which is a requirement for a good species for phytoremediation purposes (sarwar et al., 2017, liu et al., 2018). tab. 5: correlation matrix among vegetative and reproductive traits. traits rl ph sg nb nl la lcc lfm sfm rfm ldm sdm rdm lmc smc rmc np pl pw spp tsm ph 0.53 sg 0.84 0.86 nb 0.83 0.82 0.95 nl 0.52 0.90 0.81 0.76 la 0.56 0.94 0.91 0.87 0.90 lcc 0.40 0.85 0.78 0.73 0.83 0.90 lfm 0.89 0.57 0.85 0.86 0.56 0.63 0.57 sfm 0.90 0.71 0.75 0.79 0.86 0.71 0.59 0.72 rfm 0.90 0.56 0.80 0.77 0.68 0.60 0.45 0.88 0.80 ldm 0.77 0.54 0.71 0.73 0.68 0.56 0.43 0.81 0.90 0.88 sdm 0.86 0.68 0.83 0.86 0.75 0.68 0.49 0.82 0.89 0.90 0.92 rdm 0.64 0.81 0.54 0.56 0.29 0.37 0.39 0.55 0.23 0.47 0.24 0.43 lmc 0.79 0.39 0.71 0.66 0.30 0.44 0.33 0.78 0.42 0.65 0.44 0.53 0.54 smc 0.73 0.28 0.60 0.55 0.35 0.34 0.15 0.75 0.61 0.80 0.68 0.63 0.31 0.84 rmc 0.83 0.48 0.77 0.71 0.43 0.51 0.37 0.83 0.58 0.76 0.58 0.65 0.51 0.98 0.91 np 0.67 0.88 0.88 0.90 0.79 0.90 0.77 0.61 0.64 0.59 0.54 0.74 0.48 0.38 0.23 0.44 pl 0.62 0.87 0.86 0.91 0.75 0.91 0.78 0.60 0.61 0.52 0.45 0.67 0.50 0.43 0.23 0.47 1.00 pw 0.62 0.89 0.88 0.93 0.79 0.93 0.80 0.64 0.69 0.56 0.55 0.72 0.45 0.42 0.27 0.48 0.97 0.99 spp 0.61 0.88 0.87 0.90 0.77 0.93 0.83 0.59 0.59 0.51 0.44 0.64 0.52 0.43 0.21 0.47 1.00 0.99 0.98 tsm 0.61 0.87 0.8 0.86 0.79 0.84 0.69 0.51 0.67 0.53 0.46 0.72 0.45 0.37 0.23 0.44 0.93 0.95 0.93 0.93 1000.66 0.81 0.81 0.90 0.74 0.81 0.65 0.58 0.69 0.58 0.55 0.76 0.45 0.37 0.27 0.45 1.00 0.96 0.95 0.93 0.96 sm values ≥ 0.6 in bold are significant. traits are described in tab. 2 and 3. effect of lead on corchorus olitorius 375 agro-morphological traits insignificant changes in the germination rate of c. olitorius seeds sown in pb treated soils compared with the control indicate that it can germinate successfully even in pb concentrations far above the maximum allowable limit of 300 mg kg–1 in agricultural soils (xiao et al., 2018). however, seeds of ocimum basilicum had a significant decline in their germination percentage with an increase in pb concentration from 0–80 mg l–1 (fattahi et al., 2019). this also reveals that pb toxicity varies between plant species, as plants with phytoremediation properties tolerate more pb than sensitive ones (pourrut et al., 2011). when pb is in excess in the soil for a particular plant, it interferes with hydrolytic enzymes, including proteases and amylases that breaks down the cotyledons to initiate the germination process (pourrut et al., 2011). the insignificant differences between control and pb exposed c. olitorius plants in terms of root length, number of branches, stem and root fresh mass and number of pods indicate that this species possesses some phytoremediation properties. the same holds for insignificances with the control for stem girth, number of leaves, dry mass of leaves, stems and roots, root moisture content, pod length and mass, number of seeds per pod and total seed mass when plants were exposed to toxic soil pb levels (≥ 300 mg kg–1) (xiao et al., 2018). although pb is a non-nutrient heavy metal (sheoran et al., 2016), exposure of c. olitorius to mild soil concentration of 150 mg kg–1 resulted in taller plants, numerous leaves, heavier dried leaves and stems, higher stem moisture content, numerous seeds per pod as well as higher total seed and 100-seed mass than the control. a similar trend was recorded in ocimum basilicum where pb treatments (100– 400 mg kg–1) resulted in taller flowering stems as well as leaf collars and stem that are wider, than the control (fattahi et al., 2019). this might result from prolific cell division and intensive reproduction as plants were exposed to heavy metal stress, which is the similar case as in eclipta prostrata (chandrasekhar and ray, 2019). high dry mass and low moisture content in leaves of plants exposed to 150 mg kg–1, could have resulted from the loading of photosynthetic products in them, as they are the site of photosynthesis. however, the retain of both high dry weight and moisture content of stems was related to the taller plants at this pb concentration. therefore, more secondary tissues contributed to dry mass and moisture content relates to the stem as the passage for the transpiration and assimilation streams which both works with adequate moisture content. roots at this concentration had a drastic reduction in their dry mass because they function primarily for water absorption, although their moisture content was basically the same as that of the control. however, the increase in root moisture content in plants exposed to 1000 mg kg–1 is evident of high pb accumulation (448.8 mg kg–1) at this concentration, which will facilitate more water uptake from the soil (zhang et al., 2019). insignificant effect of pb application on the length of c. olitorius roots was contrary to a decline in root length of solanum melongena (yilmaz et al., 2009) and spinacia oleracea (alia et al., 2015) caused by 150 and 300 mg kg–1 pb levels, respectively. resistance of c. olitorius is an indication of its phytoremediation potential and therefore reacts differently to pb toxicity in the soil. the pb inhibition on root growth depends on the lead and ionic composition and the ph of the medium (pourrut et al., 2011). interaction of pb with the chemicals found in the soil results in reduced cell growth and formation, which results in root and shoot growth inhibition (alia et al., 2015). increase in height of plants exposed to 150–600 mg kg–1, but a decrease only at 1000 mg kg–1 pb concentration probably means that c. olitorius can resist and undergo proper stem cell division and elongation at toxic pb levels (> 300 mg kg–1). on contrary, high pb levels results in a decrease in plant height in spinacia oleracea (lamhamdi et al., 2013). stem girth and number of branches of c. olitorius were not affected by pb application, which is indicative of species resistance towards toxic pb concentrations. similarly, an insignificant decline in stem diameter at increasing pb levels in the soil was also recorded in ligustrum lucidum seedling (zhou et al., 2018). however, diameter of spinacia oleracea shoots decreased as a result of an increase in pb in the growth medium (lamhamdi et al., 2013). also, increasing pb resulted in a decrease in number surviving shoots in salix species (wang et al., 2014). the lowest pb concentration (150 mg kg–1) increased the number of c. olitorius leaves, with a decline only at the highest (1000 mg kg–1) concentration in the soil. increase in pb soil concentration (100– 1 600 mg kg–1) did not affect the number of eclipta prostrata and scoparia dulcis leaves but reduced that of phyllanthus niruri leaves at the maximum dose of 1 600 mg kg–1 (chandrasekhar and ray, 2019). differences in response towards pb contamination in the current and previous studies shows that pb effect is species dependant. the decline in leaf number at highest pb concentrations results from leaf senescence, chlorosis, and later abscission, because of the disturbed plant metabolic activities (chandrasekhar and ray, 2019; shi et al., 2019). a gradual reduction in c. olitorius leaf area in concentrations ≥ 300 mg kg–1 indicated lead toxicity in the soil, which is known to reduce the leaf area in plants, such as taraxacum officinale (bini et al., 2012). the decline in leaf chlorophyll content in the current study is similar to the reduction recorded in triticum aestivum and spinacia oleracea (lamhamdi et al., 2013) when pb concentration was increase in the soil. this was probably a result of toxic levels of pb that altered relative proportion of chlorophyll a and chlorophyll b and thus reduced total chlorophyll production and the rate of photosynthesis (khan et al., 2013; hou et al., 2018). insignificant difference in the fresh and dry weight of c. olitorius leaves, stems and roots treated with pb than the control, with few exceptions, might imply the potential of these plants to grow relatively well under pb contaminated soils. different responses towards pb contamination were recorded in different plants, where fresh and dry weights of shoots and roots of eclipta prostrata were increased by an increase in pb concentrations (100–1 600 mg kg–1); were not affected in scoparia dulcis; but they were drastically decreased in both phyllanthus niruri (chandrasekhar and ray, 2019) and helianthus annus (with pb increase from 300–900 mg kg–1) (saleem et al., 2018). the increase in c. olitorius leaf and stem dry mass at 150 mg kg–1 might result from enhanced growth under pb application as in eclipta prostrata (chandrasekhar and ray, 2019) but reduction in stem and root dry mass at 1000 mg kg–1 was caused by growth retardation at high pb concentrations as in spinacia oleracea and triticum aestivum (lamhamdi et al., 2013), ceratophyllum demersum (afaj et al., 2017), helianthus annus (saleem et al., 2018) and phyllanthus niruri (chandrasekhar and ray, 2019). moisture content of c. olitorius leaves, stems and roots was hardly affected by pb application, except for its decrease in leaves (150 and 600 mg kg–1 pb) and increase in stems (150 mg kg–1) as well as leaves and roots (1 000 mg kg–1). similarly, the relative water content of eichhornia crassipes seedlings increased at low pb concentrations and started to decrease at higher concentrations, when compared with the control (malar et el., 2014). however, increase of pb in the soil resulted in a decline in moisture content in spinacia oleracea (alia et al., 2015). increasing concentrations of pb in the soil interfere with cell formation in the roots which results in reduced plant growth (alia et al., 2015). lead adversely affects plant biomass by reducing the accumulation and translocation of other essential nutrients and by blocking their entry or binding to the nutrient carriers making them unavailable for other elements (pinho and ladeiro, 2012). an insignificant effect of pb in most c. olitorius reproductive traits, with few exceptions, probably means that plants that reach the re376 s. ndlovu, r.v.s.r. pullabhotla, n.r. ntuli productive stages (maturity) are less affected by the amount of pb in the soil. increase in total and 100–seed mass at lower pb concentrations (150 and 300 mg kg–1) might be a response toward stress, where plants maximizes their resources for reproduction than vegetative growth (cho et al., 2017). however, in zea mays the presence of pb in the soil results in a decrease in seeds mass (ghani, 2010). correlation matrix vegetative and reproductive traits correlated with each other in terms of how they are affected by pb content in the soil. moisture content of leaves, stems and roots had a strong positive correlation with one another and they were all less affected by pb content. although leaf chlorophyll content was drastically reduced by the increase in pb concentration, it correlated positively with less-affected reproductive traits as well as stem and leaf traits. significant positive correlations among vegetative and reproductive traits are essential to indicate similar effect of pb on all traits. insignificant correlation of reproductive traits with some vegetative traits such as root fresh and dry mass; leaf dry mass as well as moisture content of roots, stems and leaves probably means that these plants do not entirely depend on these traits for reproduction. the growth of ligustrum lucidum seedlings under pb stress had a significant negative correlation with dry mass of roots, stems and leaves (zhou et al., 2018). several studies mainly focus on correlation matrix among different heavy metals (bini et al., 2012) and their accumulation within plants (hashim et al., 2017; huang et al., 2017; fattahi et al., 2019; shi et al., 2019), but less among plant traits that are affected by heavy metals. conclusions corchorus olitorius accumulates toxic amounts of pb when growing in pb-contaminated soils, but continues to grow successfully. this shows its potential for phytoremediation. however, its consumption can be fatal to humans when it is collected from pb-contaminated areas. pb is less translocated from roots to aerial plant parts, but continuous shoot consumption could lead to gradual pb accumulation in humans over time resulting in its toxicity. therefore, collection and consumption of this vegetable species from areas that are prone to pb contamination is not recommended. future research will focus on variation on growth and yield of pb-treated c. olitorius plants, grown in different soil types under different climatic conditions. this will also include the measurement of ph and ion conductivity of the 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author(s) 2019. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1186/s40529-014-0054-6 http://dx.doi.org/10.1155/2012/369572 http://dx.doi.org/10.1007/978-1-4419-9860-6_4 http://dx.doi.org/10.1016/j.radphyschem.2019.04.041 http://dx.doi.org/10.1016/j.chemosphere.2017.12.117 http://dx.doi.org/10.1111/jfbc.12949 http://dx.doi.org/10.1016/j.chemosphere.2016.12.116 http://dx.doi.org/10.1016/s1002-0160(15)60032-7 http://dx.doi.org/10.1016/j.jes.2018.07.003 http://dx.doi.org/10.4314/jab.v92i1.5 http://dx.doi.org/10.1371/journal.pone.0108568 http://dx.doi.org/10.1016/j.ecoenv.2018.06.095 http://dx.doi.org/10.1016/j.sajb.2019.01.006 http://dx.doi.org/10.1080/01140670909510264 http://dx.doi.org/10.1016/j.scitotenv.2018.10.443 https://doi.org/10.1371/journal.pone.0191139 journal of applied botany and food quality 92, 192 (2019), doi:10.5073/jabfq.2019.092.026 it started as journal for members of the “vereinigung für angewandte botanik (german society for applied botany)” only. on a monthly basis, it delivered the newest research results in the field of applied botany as well as short communications, literature reviews and reports from scientific meetings to the german speaking research community. after a short publication hiatus in the 1940ies, socie ty members continued to report their findings, quickly exhausting the available print pages. throughout the years, english titles and summaries where added, making the journal accessible for a wider, non-german speaking audience. the final step into the international research community was the opening of the journal for authors, who were not society members in the 1980ies. in 2004, the “deutsche gesellschaft für qualitätsforschung − pflanzliche lebensmittel (german society for quality research in plant foods)” joined the journal due to partial overlap in their research focus, thus, making it the journal of applied botany and food quality as we know it today. the latest change happened in 2013, with the switch from a subscription based print journal to an online-only gold open access journal. this once again broadened the readership and gained the attention of even more international researchers, which nowadays make up the majority of our journal authors. the first article in “angewandte botanik” published in 1919 was submitted by otto appel, a phytopathologist well known for his discoveries on bacterial and fungal diseases of crops such as potato and cereals. his work ranged from fundamental research to applied research and reflected in his long-term membership in the board of the association for applied botany. in the first contribution of this special jubilee section hartwig schulz and heike riegler report about “otto appel and his contributions to food quality and safety at the beginning of the 20th century” and give insights into the major challenges in phytopathology at the early years of the 20th century and discoveries by otto appel in this field. maik kleinwächter and dirk selmar prepared a contribution about “modern applied botany − changes in the perception of applied botanists to them selves and others during the last century. three recent examples of the scientific potential of this field”. they give a short outline on the history of applied research in plant biology in germany and illustrate the relevance of modern applied botany in three relevant post-harvest processes. they state that interdisciplinary work and intensive cooperation with growers and producers is an integral part of developing feasible and economically acceptable solutions that can be successfully transferred into practice. the major challenge in applied botany today is the implementation of new concepts and ideas into product-related research. the contribution of muna ali abdalla and karl h. mühling leads us to “plant-derived sulfur containing natural products produced as a response to biotic and abiotic stresses. a review of their structural diversity and medicinal importance” and reveals the diversity in structure and function of secondary compounds in plants. in the article written by wolfgang kreis “exploiting plant cell culture for natural product formation” we get insights into the high expectations researchers and industry had on the exploitation of plant cell cultures from a historical and personal point of view. imke hutter and carolin schneider report on the current state of “commercial micropropagation in germany” from the applied site. in an interdisciplinary project, jan philipp schuchardt, andreas hahn, theresa greupner, paulina wasserfurth, maría rosales-lópez, johann hornbacher and jutta papenbrock work on different aspects concerning “watercress – cultivation methods and health effects”. georg langenkämper and christian zörb submit an article about “modern aspects of wheat grain storage proteins”. finally, on the interaction of plants with microorganisms, in this case fungi, is shed light on by oluwatosin abdulsalam, erika kothe and katrin krause in “the parasitic-neutral-mutual continuum of plantfungal interactions”. journal of applied botany editorial this year we want to celebrate the 100th birthday of our journal, which was first published as angewandte botanik (= applied botany) in 1919. angewandte botanik. 258 kleine mitteilungen. kleine mitteilungen. die lösung der phylloxerafrage durch reformierung der rebenkultur. nach beobachtungen desbulgarischenuniversitätsprofessors popoff, über welche er kurz hintereinander nun schon zum drittenmal berichtet (zuletzt zeitschrift für angewandte entomologie, bd. v, 1919), trotzen in mazedonien reben, die baumartig in oberflächlich nicht gelockertem boden wachsen, den reblausangriffen. aus dieser tat sache leitet nun der verfasser schlüsse von solch allgemeiner trag weite ab, daß wir uns hier kurz mit seinen ausführungen befassen müssen, vor allem in bezug auf unseren deutschen weinbau. schon der verstorbene okonomierat oberlin vertrat den stand punkt, unsere europäischen reben fielen nur deshalb der reblaus zum opfer, weil wir ihnen nicht die nötige freiheit in der entwicklung ließen. er behauptete, reben, die ähnlich den wilden reben lang ge zogen würden (mindestens 5 m lange kordons), fielen der reblaus in folge ihres stärker entwickelten wurzelwerkes nicht zum opfer. daß diese anschauung unrichtig ist, hat sich inzwischen mehrfach gezeigt und popoff bringt selbst beispiele dafür. er führt die anfälligkeit auf die alljährliche bodenlockerung zurück, denn die baumartigen reben, die in mazedonien in reblausverseuchten gegenden gesund bleiben, wachsen an stellen, an welchen der boden niemals gelockert wird, ja sogar teilweise gepflastert ist. die wurzeln solcher pflanzen kann die reblaus dann nicht erreichen. nach popoff muß nämlich „die phylloxera für ihre normale entwicklung und fortpflanzung eine an dauernde wanderung von den wurzeln zur oberfläche und umgekehrt ausführen“. er scheint also anzunehmen, daß, sobald die phylloxera nicht aus dem boden herauskommen, also nur den unterirdischen ent wicklungskreislauf vollenden kann, der durch sie angerichtete schaden auch nicht so groß sei. diese annahme wäre aber ein gewaltiger irrtum, denn in deutschland werden die reblausschäden mit umgehung des oberirdischen entwicklungskreislaufes (geschlechtstiere, gallenlaus) angerichtet! mir scheint der kräftige wuchs der mazedonischen reben selbst in reblausverseuchten gebieten ganz einfach dadurch erklärlich zu sein, daß diese reben gar keine rebläuse an den wurzeln haben. an keiner stelle spricht sich der verfasser darüber klar aus, und doch wäre das für die ganze frage von größter bedeutung, denn nur wenn die reben bei reblausbefall gesund bleiben, kann man von „phylloxera festen“ reben sprechen in dem sinne, wie man dieses wort z. b. bei bestimmten amerikanerreben gebraucht. wenn aber keine rebläuse an den wurzeln sind, dann ist alles nur phantasie. dann könnten wir mit dem gleichen recht unsere in verseuchten rebgegenden noch gut gedeihenden reben als phylloxerafest bezeichnen und doch sind sie es nicht, sobald sich rebläuse auch an ihren wurzeln festsetzen. sollten jedoch rebläuse an den wurzeln der baumartigen reben vorkommen, diesen aber nicht schaden, dann wäre das auch noch keine für die reblausbekämpfung verwertbare tatsache, denn die baumartigen reben mazedoniens sind schon sehr alt, sie hatten also offenbar auch kleine mitteilungen. 259 schon sehr tiefgehende, mit starker borke versehene wurzeläste, denen d ie rebläuse keinen großen schaden mehr zufügen konnten, als diese nach mazedonien eingeschleppt wurden. wie sich der verfasser die erziehung baumartiger reben in reblausverseuchten böden vorstellt, ist mir auch nicht klar. frisch ge pflanzte reben gehen doch – auch wenn wir die bodenoberfläche fest stampfen würden – alsbald zugrunde, weil die für die nahrungsauf nahme in betracht kommenden wurzelenden von den rebläusen an gestochen werden und später verfaulen. man is t also gar nicht in der lage, eine baumartige rebe z u erhalten. daß die popoffsche „lösung der phylloxerafrage“ für unsere deutschen verhältnisse vollkommen aussichtslos ist, wird jedem wein bauer sofort klar sein. auch der verfasser sieht ein, daß sich eine baumartige rebenerziehung in deutschland weder mit dem klima noch mit der weinqualität verträgt. e r glaubt aber, man könne auch für deutschland eine erziehungsform finden, die den rebstock üppige ent wicklung gestattet, ohne daß der boden bearbeitet zu werden braucht. e r wird z u diesem vorschlag in deutschen weingebieten wenig zu trauen finden. die winzer werden ihn also nicht aus theoretischen gründen und bedenken ablehnen, sondern weil e r wissenschaftlich noch gar nicht genügend gestützt und praktisch unmöglich ist. k . müller, augustenberg. literatur. d e s raummangels wegen wird in dieser nummer nur eine liste gegeben. die besprechungen wichtigerer arbeiten folgen im nächsten bande. beckenstedt, h., bessere aussichten für die lupinenverwertung. deutsche landw. presse xlvi (1919), nr. 18, s. 587–588. buchka, k . von, das lebensmittelgewerbe. ein handbuch für nahrungsmittelchemiker, vertreter von gewerbe und handel, apo theker, arzte, tierärzte, verwaltungsbeamte und richter. band iv. akademische verlagsgesellschaft m . b . h., leipzig, 1919. 412 seiten mit 2 1 abbildungen. der vorliegende 4 . band des handbuches behandelt in drei ab schnitten die milch und milcherzeugnisse, die süßstoffe und das bier. meyer. das aufbewahren der weintrauben und pflaumen. reichs-gemüse und obstmarkt iv (1919), nr. 108, s. 1. verschiedene konservierungsverfahren von pflaumen und wein trauben für die kleinwirtschaft. my. ehrenberg, p ., hahn-haslinger, e., zyl, j. p. van, vergleich der trocknungskosten für zuckerrüben auf einem trommeltrockner und einer darranlage. landw. jahrb. liii (1919), heft 4 , s . 525–560. nach den angestellten untersuchungen und berechnungen arbeitet der trommeltrockner rationeller und vorteilhafter und ist der darre entschieden vorzuziehen. my. nahrungs mittel. uc1.b3299303_page_276_cut uc1.b3299303_page_277_cut journal of applied botany and food quality 93, 245 247 (2020), doi:10.5073/jabfq.2020.093.030 institute of plant biology, tu braunschweig, germany opinion: the accustomed inconsistency in biochemical ecology – enhanced knowledge of the evolution and function of natural products frequently implies teleological misinterpretations dirk selmar (submitted: april 04, 2020; accepted: july 15, 2020) summary secondary plant products are the basis for complex interactions between plants and their environment. by protecting plants against pathogens and herbivores or by attracting potential pollinators, they accomplish various and distinct ecological functions. the enor mous diversity of these natural compounds is the result of evolutionary processes that have been driven by the selection of corresponding advantageous properties. unfortunately, when discussing this context, we frequently formulate statements such as “plants have acquired the ability to synthesize secondary plant products in order to…” without realizing that such assertions contradict the darwinian principles of evolution and thus represent the lamarckian view of a teleological evolution. the primary reason for these unconscious misapprehensions seems to be the ambiguous usage of the term “biological function”, whose denotation frequently includes an intention or a special purpose. in this treatise, the related associations and conclusions are outlined and depicted. key words: biological function; ecological biochemistry; evolution; natural products; secondary plant products. preamble professor reinhard lieberei was one of the leading scientists in the field of applied botany. apart from his great achievements in this area, he was also deeply committed to ecological biochemistry. based on a holistic way of thinking combined with comprehensive knowledge of plant physiological coherences, he developed a distinguished understanding of ecological biochemistry. his related statements were sophisticated and descriptive, and he omitted misleading simplifications. he opened new doors, developing the basis for quite novel comprehensive approaches, particularly in the field of applied botany. the broad spectrum of lieberei’s important achievements in this area and his scientific impact become obvious when reading the various articles by his scientific scholars and companions, compiled and displayed in this special section. for lieberei and his scientific self-conception, and in particular for his way of understanding biological processes, darwinian principles had always been essential and inevitable – they came first and foremost also in the field of ecological biochemistry. indeed, we all are convinced to have internalized these general principles. however – especially when addressing biochemical ecology – many scientists seem to forget or even ignore them and frequently drift into lamarckian argumentation and teleological conceptions. i still have strong memories of many divisive and vivid discussions with numerous renowned colleagues in which lieberei tried to pinpoint the respective inconsistencies. together with professor böle biehl, the academic mentor of lieberei, who also passed away in 2019, we discussed these misappre hensions extensively and pervasively many times. inspired by these experiences, in this treatise i outline how our increased understanding of biochemical ecology and the metabolism of secondary plant products frequently generates inaccurate statements concerning their evolution and function. secondary metabolism, its evolution and teleological misinter pretations in recent decades tremendous progress in the understanding of secondary metabolism has been achieved. moreover, many novel insights into ecological biochemistry have been gene rated, verifying that secondary plant products reveal important functions within the complex interactions between plants and their environment (current comprehensive reviews have been compiled, such as by mérillon and ramawat, 2020). today, there is no doubt that plants are pro tected by accumulating toxic, bitter or pungent tasting substances such as alkaloids or phenolics, which effectively repel potential herbivores (for review see: hartmann, 2007; zenk and juenger, 2007). similarly, pathogens are fended off by the synthesis of various phytoalexins or by toxic phytoanticipins. furthermore, we know that pollinators are attracted by pigments or scents of flowers. based on the tremendous progression of molecular and cell biology, the complex biosynthesis of many natural products has been comprehensively elucidated, and the involved genes have been identified and characterized (related reviews are compiled by wink, 2010). finally, in many cases, the underlying regulation processes have been explored and unveiled (e.g. zhang and memelink, 2009; del carpio et al., 2014), and we are aware that the enormous diversity of natural products is the result of evolutionary processes driven by the selection of advantageous properties (e.g. hartmann, 2007; firn and jones, 2009). in addition, extensive studies have indicated that genes involved in secondary metabolism originated by duplication of those responsible for primary metabolism with successive diversification (ober, 2010; moghe and last, 2015). all in all, our general understanding of secondary metabolism is profound, and the major relationships are quite clear. unfortunately, this comprehensive knowledge in combination with deep insights into metabolism often generates an oversimplified perception of the entire issue. many discussions of the topic include not only inaccurate but even erroneous statements, in particular when accounting for the relationships between function and evolution of natural products. in this context, one of the common assertions is: “plants have acquired the ability to produce and accumulate numerous secondary plant products in order to protect themselves against herbivores”. indeed, when considering the numerous excellent examples of plant-herbivore-interactions in the literature and the tremendous approaches in modern chemical ecology, superficially, this statement seems to be correct. however, when carefully scrutinising the meaning, it becomes obvious that the phrase “in order to protect” is inappropriate: this statement implies that the evolutionary processes which have created and established secondary metabolism are directed to a specific goal and to fulfil a certain purpose. this, in turn, would imply that evolution is target-oriented. well, in general, we all are aware that evolution does not follow lamarck’s theory, which depicts a teleological, goal-oriented process. in this regard, we know that giraffes did not develop longer necks in order to feed on treetops. unfortunately, when referring to secondary plant products, this basic nexus is ignored, and even honourable and knowledgeable experts are trapped by this flaw when allegorising or exemplifying this top246 d. selmar ic. accordingly, in many releases – especially in those dealing with biochemical, molecular biological or molecular genetic topics – the corresponding objections seem to be forgotten. obviously, we are so impressed by the tremendous progress we have made and the details we have elucidated that the basic scientific fundamentals are neglected. teleological considerations, and the related deductions, are not reliable and are consequently unscientific. in principle, the relationship between function and evolution of natural products seems to be very simple. caused by mistakes in gene replication or odd recombinations in crossover, mutations occur frequently, which might be accompanied by duplication of genetic material. as the genuine function of the duplicated gene, or the related enzyme, is maintained by the original gene, there is no massive selection power to rapidly knock out the duplicate. accordingly, due to further mutations, successive diversification might take place. indeed, when these processes result in properties that create a disadvantage for the organism, they will be selected out of the gene pool. by contrast, when the mutations create repercussions that enhance fitness (e.g., by increased pollination efficiency, or better protection against herbivores), this organism will be favoured. in this manner, evolution of the large diversity of secondary plant products can be explained. in addition, as a further significant issue with respect to the evolution of secondary metabolism, the presence of promiscuous enzymes has to be considered. indeed, originally, it was postulated that the enzymes involved in secondary metabolism were highly specific (e.g., hartmann, 1996; wink, 1997). however, we have learned that substrate specificity of enzymes is far lower than initially assumed (e.g., atkins, 2015), and various enzymes, denoted as moonlighting proteins, are known to be responsible for the catalysis of different metabolic reactions (jeffery, 1999). in consequence, promiscuous enzymes are also thought to serve as evolutionary starting points with respect to secondary metabolism (khersons ky and tawfik, 2010). nonetheless, with respect to the contribution of promiscuous enzymes in the evolution of secondary metabolism, we have to be aware that evolution is not target oriented but represents the outcome of a selective process (kreis and munkert, 2019). with respect to the evolution of secondary metabolism, another aspect which is frequently is misjudged is the tall story of “cost-benefit considerations”. in this context many related publications have stated: “with respect to secondary metabolism the cost versus benefits have to be balanced”. indeed, due to our recurring experience in daily life, we all have internalised that energy conservation represents one of the most important issues in our subsistence. accordingly, it seems reasonable at first glance to transfer such importance also into plant biology. however, in this context we have to consider that plants – in contrast to heterotrophic organisms – face less of a challenge in covering their energy requirements. indeed, plants generally absorb much more energy than required for photosynthetic co2 fixation (wilhelm and selmar, 2011). as a result, plants have to dissipate a tremendous oversupply of energy in order to avoid massive damage by oxygen radicals, resulting from overreduction of the electron transport chain, which would destroy the leaves (reddy et al., 2004; szabó et al., 2005). apart from the various classical mechanisms for effective energy dissipation (i.e., non-photochemical quenching, photorespiration, or xanthophyll cycle), especially under stress conditions, the synthesis of highly reduced compounds also contributes to effective energy dissipation (selmar and klein wächter, 2013a). in this context the massive emission of isoprene represents one of the most intriguing examples (e.g. fall 1999; sharkey and yeh, 2001). thus, the synthesis of highly reduced secondary plant products does not correspond to a “cost of energy” but contributes to the unequi vocally required energy dissipation (selmar and kleinwächter, 2013b; kleinwächter and selmar, 2015; yahyazadeh et al., 2018). this vividly displays that – even without a distinct ecological function – the synthesis of natural products involves an evolutionarily relevant advantage, facilitating the evolutionary generation and establishment of ecologically relevant natural products. biological function – a term which frequently generates mis apprehensions the primary reason for the unintended teleological statements regarding the ecological significance of secondary metabolism discussed above is certainly the ambiguity of the term “function” and the related discrepancy between its basic, inherent meaning and its extended usage (selmar, 2009). in natural sciences, a function in stricto senso corresponds either to a mathematic relationship, for example y = f(x), or in an extended sense, it describes a link of a cau sal sequence in which one parameter, based on a set of stipulations, determines another parameter. accordingly, the term “function” describes a response to a certain event or the related con sequence without entailing any purpose or intention. in contrast, in our daily life the term “function” is applied teleologically, and routinely it includes a significance or relevance, implying that a function per se accomplishes a certain purpose (selmar, 2009). in this sense, we are accustomed to tools having a distinct function and implementing a specific purpose. as result, the ambiguous meaning of “function” has been adopted and internalized in the biological sciences, generating the equivocalities mentioned. based on approved experimental techniques, plant metabolism has been extensively analysed, and the processes involved have been comprehensively investigated. although in many cases only a limited section of a certain area has been elucidated, based on reliable results and appropriate causal chains, we can use understanding of single parts of the system to comprehend the entire organism. as we have internalised conceptualisation of the operation of mechanical devises by identifying the various functional elements and tools (e.g. clutch, conveyor, energy supplier), we have tried to explain the various metabolic processes and even the entire metabolism analogously. thus, we attribute and assign a “particular function” to a certain metabolic process. although an extended use of the term “function” in the sense of “purpose” virtually contradicts its valid scientific definition by neglecting the sound principle of causality, related statements and deductions are suitable – if they are based on an extended definition including the intent of a function. however, the inaccuracy and the unscientific actions begin when these teleological reflections are expanded with respect to evolutionary considerations in statements like: “…in the course of evolution a certain metabolic process was created in order to fulfil certain functions”. this, unfortunately, is particularly true for secondary metabolism, since it is frequently stated that “…natural products were generated in the course of evolution in order to protect the plants”. in modern biology such ambiguity should be avoided by relying on the darwinian principles of mutation and selection. we always are on a solid scientific basis when – based on reliable experimental methods and by employing sound causal chains of established evidence – we deduce that in phylogenetic history, selection of certain modified reaction patterns has occurred and organisms adapted to special environmental conditions and – in the sense of the meaning outlined above – a certain function is thereby established. but we should be aware that such a statement is not equivalent to the teleological assertion that mutation and selection automatically generate a certain purposeful mechanism, fulfilling a predetermined goal. indeed, such knotty explanation can be quite problematic when explaining the issue to a broad community. nonetheless, we should avoid teleological statements such as “…evolved in order to protect…” and empha size scientifically sound deductions based on darwinian principles. in this context, i remember quite well the outcome of a long and fruitful discussion with reinhard lieberei, who stated appropriately: “in the the accustomed inconsistency in biochemical ecology 247 course of evolution, plants have acquired the ability to synthesize and accumulate a tremendous diversity of natural products. the presence of these compounds entails selective advantages, e.g. by protecting the plants against herbivores or pathogens or by attracting pollinators. accordingly, these natural products reveal an essential role in various plant-environmental-interactions by accomplishing vital ecological functions.” commentary this discourse is a progression of a disquisition published about ten years ago (selmar, 2009). the consistent development of the entire topic was massively expedited by vivid and profound discussions with reinhard lieberei. references atkins, w.m., 2015: biological messiness vs. biological genius: mechanistic aspects and roles of protein promiscuity. j. steroid. biochem. mol. biol. 151, 3-11. doi: 10.1016/j.jsbmb.2014.09.010 del carpio, d.p., basnet, r.k., arends, d., lin, k., de vos, r.c.h., muth, d., kodde, j., boutilier, k., bucher, j., wang, x., jansen, r., bonnema, g., 2014: regulatory network of secondary metabolism in brassica rapa: insight into the glucosinolate pathway. plos one 9, e107123. doi: 10.1371/journal.pone.0107123 fall, r., 1999: biogenic emissions of volatile organic compounds from higher 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(eds.), plant-derived natural products, 181-194. springer, new york, ny. doi: 10.1007/978-0-387-85498-4_8 address of the author: dirk selmar, institute of plant biology, tu braunschweig, mendelssohn straße 4, 38106 braunschweig, germany e-mail: d.selmar@tu-bs.de © the author(s) 2020. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1016/j.jsbmb.2014.09.010 http://dx.doi.org/10.1371/journal.pone.0107123 http://dx.doi.org/10.1016/b978-012346240-4/50003-5 http://dx.doi.org/10.1093/jxb/erp002 http://dx.doi.org/10.1111/j.1570-7458.1996.tb00914.x http://dx.doi.org/10.1016/j.phytochem.2007.09.017 http://dx.doi.org/10.1146/annurev biochem-030409-143718 http://dx.doi.org/10.1007/s13593-014-0260-3 http://dx.doi.org/10.1093/jxb/erz025 http://dx.doi.org/10.1104/pp.15.00994 http://dx.doi.org/10.1111/j.1438-8677.2009.00317.x http://dx.doi.org/10.1016/j.jplph.2004.01.013 http://dx.doi.org/10.1111/j.1438-8677.2009.00190.x http://dx.doi.org/10.1093/pcp/pct054 http://dx.doi.org/10.1146/annurev.arplant.52.1.407 http://dx.doi.org/10.1038/sj.embor.7400460 http://dx.doi.org/10.1016/j.jplph.2010.07.012 http://dx.doi.org/10.1016/j.phytochem.2018.05.007 http://dx.doi.org/10.1016/j.phytochem.2007.07.009 http://dx.doi.org/10.1007/978-0-387-85498-4_8 art04_eichholz.indd journal of applied botany and food quality 84, 26 32 (2011) 1 humboldt-universität zu berlin, division urban plant ecophysiology, section quality dynamics/postharvest physiology, berlin, germany 2 technische universität berlin, institute of food technology and food chemistry, department of food chemistry and analysis, berlin, germany 3 universität hamburg, institute of food chemistry, hamburg, germany phenolic compounds, pectin and antioxidant activity in blueberries (vaccinium corymbosum l.) infl uenced by boron and mulch cover i. eichholz1, s. huyskens-keil1, l.w. kroh2, s. rohn3 (received june 21, 2010) summary highbush blueberry cultivars ‘bluecrop’ and ‘reka’ were growing in two variants of mulching and fertilizing systems on formerly used farmland. particular attention of this work was to study the effect of using pine bark as a mulch layer and foliar application with the plant stimulant wuxal® ascofol (3% boron) on selected bioactive compounds (polyphenols and pectins) of blueberry fruits. the results represented a stress-preventive effect of mulch application. furthermore, these plants exhibited lower calcium content in fruits due to a reduced calcium uptake from the soil. with regard to the bioactive compounds, mulched plants showed a higher content of pectin which was in contrast to the phenolic compounds. they revealed reduced concentrations in fruits accompanied by a lower antioxidant activity. the foliar supply of boron was able to inactivate polyphenols presumably by complex formation and favoured the formation of pectins. introduction highbush blueberries (vaccinium corymbosum l.) gain high popularity in europe due to their proposed health-benefi cial properties. optimum locations for cultivation of blueberries are found on heathland, peat or forest soils. nevertheless, such locations are not always available. however, it is tried to cultivate blueberries on formerly used farmland, which is primarily insuffi cient for blueberry cultivation. unfavorable conditions are the high ph value; the low humus content and the reduced water holding capacity of the soil (haynes, 1986). the improvement of blueberry production on formerly used farmland has been carried out in several investigations, all with the aim of increasing crop yield. unfortunately, changes of the bioactive compound composition have not been investigated up to now. blueberries offer a high content of pectin (ternes et al., 2005) and phenolic compounds (häkkinen, 2000). for both classes the health benefi cial aspects are well known. due to their antioxidant properties especially polyphenols are attributed with several positive physiological properties. they are able to quench free radicals, released by oxidative stress, and are implicated in prevention of cancer and cardiovascular diseases in the human organism (bazzano et al., 2002). in plants, secondary metabolites such as the phenolic compounds are accumulated as a protective defence mechanism against stress conditions (dixon and pavia, 1995). also pectins, as compounds of the primary plant metabolism, are able to react on changed ecophysiological conditions (lesniewska et al., 2004). besides stress-induced effects on the primary and secondary plant metabolism, availability of nutrients can be an infl uencing factor for the biosynthesis of compound classes (stewart et al. 2001). thus, mineral availability may have an effect on both classes, phenolic compounds and pectins by e.g. nitrogen, phosphate (steward et al., 2001; juszczuk et al., 2004) or calcium availability uszczuk et al., 2004) or calcium availability uszczuk (naphun et al., 1997). as a result, there are different possibilities to infl uence the amount and composition of bioactive compounds by specifi c cultivation techniques. the aim of this study was to investigate whether mulch layer and foliar applications using pine bark and a plant stimulant based on 3% boron (wuxal® ascofol) might infl uence mineral availability of blueberries under open land conditions. besides the two minerals boron and calcium, which were mostly affected due to treatments, phenolic compounds and pectins have been analyzed as they are closely related to mineral metabolism. additionally, antioxidant activity was determined. materials and methods plant material plant material was obtained from the highbush blueberry cultivars ‘bluecrop’ and ‘reka’, which have been produced on open land conditions at the research site of the humboldt-universität zu berlin. bushes were planted in peat fi lled holes on formerly used farmland and cultivated in two ground cover (with [m] and without [om] pine bark mulch) and fertilization variants. fertilization was conducted from may to mid july with a nutrient supply of 10 g n 15 g p2o5 24 g k2o 2 g mgo 66 mg b per plant in fi rst fertigation variant (f1). plants of the second nutrient supply system (f2) received an increased nitrogen fertilization (14 g per plant) and additionally boron foliar applications (400 mg per plant). boron foliar applications were given at the start of vegetation cycle (april), at the blossom (may) and at beginning of fruit development (june) in a concentration of 3 l ha-1 (= 340 mg boron per plant per year). samples of ripe berries of each variant and cultivar were frozen immediately after picking and stored at -20 °c. approx. 100 g of fruits of each sample were lyophylized for 48 h (alpha 1-4, fa. christ, osterode am harz, germany), ground and stored in a vaccuum desiccator for further analysis. the experiment was conducted with 6 replicates per variant. chemical analysis minerals for the estimation of the calcium content freeze-dried fruit samples (0.5 g) were ashed 4 h at a temperature of 490 °c. afterwards the ash was transferred in hydrochloric solution (5 ml of 10% hcl) and vaporized on a sand bath (type hc 42, gerhardt, bonn, germany). the residue was heated up once more to 490 °c for 1 h. after addition of 5 ml of hcl (25%) the ash solution was fi ltered (macherey-nagel mn 615 1⁄4, düren, germany). filtrate was fi lled up to a volume of 50 ml. the measurement of calcium was done using atomic absorption spectrometry (aas, type 905 aa, gbc, sydney, australia). results were expressed as mg ca g-1 dw. to analyze boron, freeze-dried fruit samples (1 g) were ashed for 6 h at a temperature of 550 °c. afterwards, the ash was incubated with 0.36 n h2so4 for 1 h. the solution was fi ltered (machereynagel mn 260, düren, germany) and the fi ltrate was fi lled up to a volume of 5 ml. the measurement of boron was conducted spectrophotometrically (sp8-300, pye unicam, ok, usa) at a wavelength of 420 nm according to the azomethine-h method (bingham, 1982). hence, 1 ml of extracted sample was added to a mixture of 1 ml buffer (ph 5.1) and 2 ml colour reagent (0.45 g of azomethine-h and 1 g of ascorbic acid in 100 ml of destilled water). boric acid (merck 10165) was used as a standard. results were expressed as µg b g-1 dw. antioxidant activity and total phenolic content for the determination of the antioxidant activity and total phenolic content 0.5 g freeze-dried fruit samples were extracted in 10 ml of the cooled solvent 0.1% hcl/methanol (v/v; 15/85) according to the method of connor et al. (2002). the samples were diluted in 3 ml solvent, and centrifuged for 10 min at 3000 rpm (heraeus christian labofuge gl, hanau, germany). supernatants were decanted and fi ltered (macherey-nagel, mn 260, düren, germany). this process was repeated twice. extracts were fi lled up to a 10 ml volume and stored at -20 °c until further analysis. antioxidant activity was estimated using electron spin resonance (esr) spectroscopy and the trolox equivalent antioxidant capacity (teac) assay. esr was performed as described by rösch et al. (2003), using a miniscope ms 100 spectrometer (magnettech, berlin, germany). the signal intensity of a stable synthetic radical (fremy’s salt; sigmaaldrich, steinheim, germany) was obtained after 5 min. results were expressed as mmol fremy’s salt g-1 dw. teac assay was carried out spectrometrically as described by rohn et al. (2004). absorbance was measured on a specord 40 spectrophotometer (analytik jena, jena, germany) at a wavelength of 732 nm. abts (2,2’-azinobis-3-ethylbenzothiazonline-6-sulfonic acid, sigma-aldrich, steinheim, germany) was used as a free radical and trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, sigma-aldrich, steinheim, germany) as a standard. results were expressed as mmol trolox g-1 dw. total phenolic content was analyzed spectrophotometrically (sp8300, pye unicam, ok, usa) according to the folin-ciocalteu procedure. absorbance was measured at a wavelength of 765 nm. gallic acid (serva, heidelberg, germany) served as a standard. results were expressed as mg gallic acid (gae) g-1 dw. pectic substances for determination of pectic fractions (water-soluble pectin, alkaliedta soluble pectin, insoluble pectin) and the total pectin content, cell wall extraction (ais, alcohol insoluble substance) was conducted according to the method by blumenkrantz and asboe-hansen (1973) und huyskens (1991). an aliquot of 2 g of freeze-dried samples was diluted in 120 ml of an acetone / 70% ethanol mixture (70/30) and boiled for 30 min. thereafter, the cell wall material was washed under vacuum using glass frits (g3, schott duran, mainz, germany) with acetone and ethanol (70%). glas frits were dried for 24 h at 70 °c (wtc binder, tuttlingen, germany). for the extraction of water-soluble pectins (wsp) 100 mg of ais were mixed with 20 ml of distilled water and dissolved by stirring for 1 h. thereafter, samples were adjusted to a ph value of 4.5 (microprocessor ph meters 526, fa. wtw, weilheim, germany) and 0.1 ml pectinase (= 20 µg) (pectinex ultra sp l, novo nordisk ferment, switzerland) was added. subsequently, samples were stirred for 1 h prior to fi ltration (miracloth fi lter, calbiochem, frankfurt, germany) and centrifugation for 10 min at 4°c and 11000 rpm (biofuge 22r, heraeus sepatech, osterode am harz, germany). the remaining pellets were frozen at -20 °c for extracting further pectin fractions. the fi ltrates were fi lled up with 0.5% edta buffer (ph 4.5) to a 50 ml volume and stored until the spectrophotometric measurement. the extraction of the alkali-soluble pectins (esp) was conducted by adding 20 ml of 0.5% edta solution (ph 6.0) to the pellets of wsp fraction, the insoluble pectin fraction (isp) by adding 20 ml of 0.5% edta solution (ph 11.5) to the pellets of esp fraction similary to the extraction of the water-soluble pectins. the measurement of each pectin fraction was performed spectrophotometrically at a wavelength of 520 nm using mhdp (mhydroxydiphenyl) as a color reagent (mccomp and mccready (1952). as a standard, d-galacturonic acid (5 to 80 µg ml-1, sigma d 4288) was used. results were expressed as mg galacturonic acid (gal) g-1 dw. statistics the statistic evaluation was performed using spss 13.0 (spss inc., chicago, usa 2001). signifi cance of differences was conducted with a tukey-b test (p< 0.05). results and discussion mulch covering (with pine bark) in blueberry cultivation infl uences soil physical properties primarily, especially soil temperature and evaporation (krewer et al., 1997). furthermore, it enriches the soil organic matter, and therefore, helps conserving soil moisture (plizka et al., 1997). on the other hand, soil water availability affects the mineral nutrition of plants. all these factors might have an effect on the biosynthesis of bioactive compounds in fruits. moreover, as far as minerals concerned mulch covering revealed the greatest impact on calcium which will be discussed in this work in detail. boron and calcium content of blueberry fruits as infl uenced by different cultivation practices in blueberry fruits boron content ranged from 4.5 10.7 µg g-1 dw. calcium contents varied between 0.85 1.43 mg g-1 dw (fig. 12). boron application and mulch cover had a signifi cant infl uence on the boron content of blueberry fruits (fig. 1). boron treated plants revealed clearly higher boron concentrations than plants without additional boron supply. furthermore, differences could be observed between the mulched and the unmulched boron variant, i.e. the unmulched boron variant showed the highest boron content in fruits. the calcium content of the fruits was predominantely infl uenced by the mulch treatment (fig. 2). results revealed signifi cantly higher calcium content in fruits of the unmulched variants than in fruits of the mulched variants. however, boron application did not affect the calcium content of blueberry fruits. sd ‘bluecrop’/‘reka’= standard devision of both cultivars fig. 1: boron content of fruits of different blueberry cultivars [µg b/g dw], (n ± sd, tukey-b test, p < 0.05); f1m-mulched without b; f1omunmulched without b, f2m-mulched with b, f2om-unmulched with b. the increased boron content of boron treated fruits demonstrated the uptake of foliar-applied boron by leaves (data not shown) followed ��� ��� ������ ��� ������� ���� ������� ������ ��� ��� ��� ��� ���� ���� ��� ��� ��� cultivation management µ g b / g d w ��� ���� ��� ���� ��� ���� ��� ������� ���� ��� ���� ���������� ������ �� ����������������� ���� � � � � � � �� polyphenols, pectin, and antioxidant activity of blueberries 27 by its translocation to the fruit which was also reported by hahnfeld et al. (2004). moreover, bushes of the unmulched boron variant showed a stronger accumulation of boron in the fruit tissue in comparison to plants of the mulched boron variant. it is assumed that these bushes could have undergone drought stress due to the unprotected ground. hetherington and woodward (2003) reported that during persistent drought conditions leaves developed a higher number of small stomata on the leaf lower surface in order to regulate the gas exchange. an increased number of small stomata could explain the increased boron uptake of the leaves (data not shown), and therefore, the higher content of boron in fruits of the unmulched variant. sd ‘bluecrop’/’reka’= standard devision of both cultivars fig. 2: calcium content of fruits of different blueberry cultivars [mg ca/g dw], (n ± sd, tukey-b test, p < 0.05); f1m-mulched without b; f1om-unmulched without b, f2m-mulched with b, f2omunmulched with b. fruit calcium contents of the unmulched variants were signifi cantly higher than in mulched variants. however, li et al. (2006) observed a higher calcium content in leaves after ground covering with organic matter due to a higher calcium content being released during the rotting process. in the present work the soil (below the ground cover) exhibited increased calcium contents in the mulched variants (data not shown). however, calcium was transported to the leaves (data not shown) as well as to the fruits of the mulched variants to a much lower extent. this might be explained by the fact that calcium was fi xed in the rhizosphere, and thus, uptake of calcium was inhibited. hence, the plants of the mulched variants rooted predominantly in the ground cover (own observations) where they probably absorbed nutrients preferably from this layer. on the basis of a few random samples (roots with adherent mulch and/or soil particles) a clear ph decline was found in the mulched variants ([om]: ph = 5.6; [m]: ph = 4.5). this acidifi cation could have occurred by exudation of organic acids in the rhizosphere resulting from a symbiosis with mycorrhizal fungi or a direct root secretion. though, a mycorrhizal infection could not be found in this work (based on few random samples), it can not be excluded completely. despite high calcium availability, the plants of the mulched variants were able to inhibit an excessive calcium uptake. in contrast, the plants of the unmulched variants completely rooted in the peat bed because mineral soil was generally avoided for rooting (own observation). due to the fact that peat was almost decomposed, these conditions apparently did not inactivate calcium uptake in plants. infl uence of boron and calcium on total phenolic compounds and antioxidant activity (teac, esr) of blueberry fruits higher values of the total phenolic content were determined in the present study (35.1 40.1 mg of gae g-1 dw or 702 802 mg gae 100 g-1 fm), exceeding the contents reported by moyer et al. (2002) with 444 mg gae 100 g-1 fw (fig. 3). this might be due to different local growing conditions. an own comparative study on fruits cultivated on forest soils confi rmed this assumption (eichholz et al., 2007). in particular, the suboptimal growing conditions on farmerly used farmland presumably might have caused stress mediated responses in plants with a subsequently increase of the phenol synthesis and therefore higher values of antioxidant activity. similary, for the teac assay higher values were determined (0.25 0.27 mmol trolox g-1 dw) in comparison to the literature where an average value of 0.19 mmol trolox g-1 dw was reported by garcia-alonso et al. (2004) (fig. 4). the antioxidant activity of the fruits, being measured by esranalysis, ranged between 0.38 0.51 mmol fremy’s salt g-1 dw (fig. 5). in the literature, no data are available on the esr analysis in order to compare the results of the present study. however, the high correlation of this analysis to teac assay (r = 0.76; p < 0.01) suggested that the esr method is suitable for the determination of the antioxidant activity in blueberries. the high amount of anthocyanins in blueberries often leads to diffi culties in spectrophotometrical tests. thus, the esr method indicated to be a good alternative for samples with a high coloration. sd ‘bluecrop’/’reka’= standard devision of both cultivars fig. 3: total phenolic content of different blueberry cultivars as measured by the folin-ciocalteu method [mg gae/g dm], (n ± sd, tukey-b test, p < 0.05); f1m-mulched without b; f1om-unmulched without b, f2m-mulched with b, f2om-unmulched with b. sd ‘bluecrop’/’reka’= standard devision of both cultivars fig. 4: antioxidant activity of different blueberry cultivars as measured by teac assay [mm trolox/g dm], (n ± sd, tukey-b test, p < 0.05); f1m-mulched without b; f1om-unmulched without b, f2mmulched with b, f2om-unmulched with b. ���� ���� �������� ���� �������� ���� �������� �������� ���� ���� ���� ���� ���� ���� cultivation management m g c a / g d w ��� ���� ��� ���� ��� ���� ��� ������� ���� ��� ���� ���������� ������ �� ����������������� � � � � � � � � � � � � ���� �������� ���� �������� ���� �������� ���� �������� ��� ��� ���� ���� ���� ���� ���� ��� ��� ��� cultivation management m g g a e / g d w ��� ���� ��� ���� ��� ���� ��� ������� ���� ��� ���� ���������� ������ �� ����������������� � ���� �� � � � ���� � � ���� ���� �������� ���� ������������ �������� �������� ���� ���� ���� ���� ���� ���� ���� ���� ��� ��� ��� variant m m t r o l o x /g d w ��� ���� ��� ���� ��� ���� ��� ������� ���� ��� ���� ���������� ������ �� ����������������� �� �� �� �� � �� � � 28 i. eichholz, s. huyskens-keil, l.w. kroh, s. rohn sd ‘bluecrop’/’reka’= standard devision of both cultivars fig. 5: antioxidant activity of different blueberry cultivars as measured by esr method [mm fremy’s salt/g dm], (n ± sd, tukey-b test, p < 0.05); f1m-mulched without b; f1om-unmulched without b, f2mmulched with b, f2om-unmulched with b statistical evaluation revealed an infl uence of boron application on total phenol content as well as on antioxidant activity (esr; teac) of fruits (fig. 3 5). in the boron treated variants lower values were observed in comparision to the untreated plants. furthermore, ground covering tended to a decline in total phenolic content and in the antioxidant activity. although single statistical calculation of each cultivar did not always exhibit any differences (no signifi cances in teac assay for both cultivars and in total phenol assay for ‘bluecrop’), results showed a similarity in all analysis due to the treatments (boron, mulching). if plants undergo stress like drought, high light intensity or nutrient defi ciency, they are affected by oxidative damage resulting from reactive oxygen species (ros). ros are formed in plants primarily during the photosynthetic electron transport and the activation of membrane-engaged nadph-oxidases (møller, 2001). to protect themselves from a cellular damage, plants mobilise compounds which inactive ros – the so-called antioxidant compounds. in particular in blueberries, phenolic compounds are responsible for their high antioxidant properties (häkkinen, 2000). this was also confi rmed by the high correlations between the total phenolic content and both analytic assays used for the antioxidant activity (esr: r = 0.86: teac: r = 0.82; p < 0.01) in the present study. hence, due to these correlations the following conclusions, which were stated for the total phenolic content, can also be considered for teac-and esr analysis. the statistical calculation represented a signifi cant decrease of total phenolic content in the boron treated variants. a close relationship between the boron content and the polyphenol metabolism in plants is already known. the accumulation of polyphenols is a typical symptom of boron defi cient plants. boron defi ciency infl uences the polyphenol metabolism which is proved by its impact of the enzymes phenylalanine-ammonialyase (pal) and the polyphenoloxidase (ppo) (camacho-cristóbal et al., 2002). in addition, due to the high complex binding properties boron easily binds polyphenols (brown et al. 2002). during boron defi ciency the content of free boron molecules in plant tissue is low. hence, a complexation and an inactivation of the polyphenols do not occur. furthermore, an increase in phenolic compounds could be associated with the accumulation of carbohydrates induced by boron defi ciency. here, the polyphenol synthesis might be stimulated by a reinforced supply via pentose phosphate cycle (ppp). according to gomez-rodriguez et al. (1987) boron defi ciency resulted in an activation of the glucose-6-phosphates dehydrogenase. this enzyme is known as the starting point of the ppp and oxidizes glucose-6-phosphate to 6-phosphogluconolacton. in addition, it was also reported that boron defi ciency increases activities of the enzyme 6-phosphogluconat-dehydrogenase which is responsible for the 2nd reaction step of the ppp (dugger, 1983). with an adequate boron supply a high portion of free boron ions would be available, also being able to bind polyphenols. this would lead to a depression of pal and also to a degraded phenol oxidation by ppo (brown et al., 2002), possibly explaining the decline of total phenol content of the boron variants in the present study. numerous investigations have been carried out to provoke an activation of the polyphenol metabolism due to moderate stress induction by abiotic and biotic orgins (schreiner and huyskenskeil, 2006). in the present work stress mediated responses of plants might have been caused, primarily, by the lack of ground cover resulting in a low ground water capacity, a low portion of organic matter and an increased calcium uptake of the roots. thus, a trend to higher contents of antioxidant activity and of total phenolic content became evident in the unmulched variants. concerning the minerals a pronounced infl uence should be expected from calcium as a stressor, mainly because blueberries are calcifuge plants. however, apart from the effect of the ground cover and boron availability, several other factors might have had an impact on the calcium uptake. from the present results it is concluded that the stimulation of the polyphenol synthesis might have been a result of stress caused by a high content of calcium in blueberries as it was measured in the unmulched variants. an excess of calcium is known to cause phosphate defi ciency as well as decreased iron availability and uptake in fruit tissue. this could have been stimulated polyphenol synthesis, as it was found in beans (phaseolus vulgaris l.) (juszczuk et al., 2004) and grapes (vitis vinifera l.) (piagnani et al., 2003) revealing phosphate and iron defi ciency, respectively. infl uence of boron and calcium on pectic substances of blueberry fruits contents of total pectin of berries were found to range between 29.4 39.1 µg gal g-1 dw. the single pectin fractions exhibited average contents of 20.1 30.3 µg gal g-1 dw for water-soluble pectins (wsp), 5.5 7.0 µg gal g-1 dw for alkali-soluble pectins (esp) and 1.7 3.6 µg gal g-1 dw for insoluble pectins (isp) (tab. 1). no signifi cant differences of wsp were determined in ground cover and boron application treatments, although both cultivars showed a signifi cant increase of wsp in the boron treated variants compared to the untreated variants. furthermore, variants without boron application (f1) of ‘reka’ revealed signifi cantly higher contents of wsp in the mulched variants in contrast to the unmulched bushes. in the esp fraction was a trend to higher contents in the boron treated variants, especially, in the mulched variant compared to the unmulched variant were established. moreover, for the isp fraction only the cultivar ‘bluecrop’ resulted in signifi cant differences in the mulched boron variant when compared to the mulched untreated variant. thus, the statistical calculation in respect to total pectin content showed clear differences in the variants treated with boron, primarily to unmulched untreated variants. in the literature a total pectin content of 0.51 g gal 100 g-1 fm in blueberries was reported by silva et al. (2005) which underlines the data upraised in the present work (29.4 39.1 µg gal g-1 dw = 0.58 0.78 g gal 100 g-1 fw). furthermore, wsp fraction exhibited the highest portion of pectic substances, followed by esp and isp fraction. the majority of cellular boron is associated with pectin in the cell wall (brown et al., 2002), where the cell wall bound boron forms a tetravalent borate-diol-ester. here, two molecules rhamnogalacturonan-ii (rg ii) are cross-linked with each other to form a stable dimer in the primary cell wall (ishii and matsunaga, 1996). matsunaga and ishii (2006) reported during boron defi ciency ���� ���� �������� �������� ���� �������� ���� �������� ���� ���� ���� ���� ���� ��� ��� ��� cultivation management m m f re m y 's s a lt /g d w ��� ���� ��� ���� ��� ���� ��� ������� ���� ��� ���� ���������� ������ �� ����������������� � �� �� � �� � � � �� � � polyphenols, pectin, and antioxidant activity of blueberries 29 cell wall thickening of leaves is closely related with the formation of a dimeric rg-ii-b complex. adding boric acid to boron defi cient leaves of pumpkin (cucurbita maxima duch.) led to the formation of dimeric rg-ii-b from monomeric rg-ii as well as to the formation of a thinner cell wall. in contrast, boron defi cient plants form a huge, thick cell wall. boron applications also infl uenced the content of pectins in blueberry fruits of the present study. nevertheless, the infl uence of boron due to additionally formed rg-ii-b complexes seems to be of minor importance, because according to brown et al. (2002) such complexes stabilize primarily the protopectin. hence, it is assumed that the presence of boron allowed an improved transport of carbohydrates, the source of pectin synthesis, and/or exerted a direct infl uence on the carbohydrate synthesis. the synthesis of pectin “backbones”, which are mainly galacturonic acid molecules, depends on the availability of carbohydrates (mohnen, 1999). however, an improved transport of sugars by boron is reported to be controversial. the chemical structure of the respective sugar infl uences the stability and the transport ability of those complexes. carbohydrates which could easily react with boric acid are riboses, apiose, sorbitol and other polyols (brown et al., 2002). however, in most plants sucrose is the main transport sugar (williams et al., 2000) and thus, the chemical structure does not allow a stable complex because sucrose shows a trans-diol position of the hydroxyl group (brown et al., 2002). furthermore, han et al. (2008) determined a lower content of sucrose in boron defi cient leaves of citrus seedlings (citrus sinensis l.), while the content of glucose and fructose strongly increased. these authors associated a direct impact of boron on the sucrose synthesis. nevertheless, currently it is unclear whether boron is involved in the synthesis processes of this sugar or if it reduces its degradation. dugger and humphrey (1960) had already suggested a support of sucrose synthesis from glucose-1-phosphate and fructose in the presence of boron. they proved a support of the udpgpyrophosphorylase with simultaneous inhibition of the udpgtransglycosylase in leaves of peas and sugar cane seedlings (pisum sativum l.; saccharum offi cinarium l.). also teare (1974) reported an accumulation of utpand a decrease of udp-glucose in bean roots (phaseolus vulgaris l.) during boron defi ciency. however, han et al. (2008) referred to reinforced sucrose degradation by an increased invertase activity in the absence of boron. the higher content of boron in berries studied here might have led to a reinforced construction of pectins, based on a presumably higher availability of transport sugars. in the presence of boron this would become predominantly for the construction of pectins as cell wall material where boron plays a primary role in the synthesis (brown et al., 2002). with an increase in protopectin the single pectin fractions also increased in the boron treated variants. as a result of ongoing fruit development and maturation the content of protopectin was decomposed increasingly to soluble pectin fractions, as it was also reported by vicente et al. (2007). moreover, an increase of pectins by enhanced calcium content was also proved by other research studies, for example, in strawberry fruits (fragaria x ananassa dutch.) (naphun et al., 1997). in general, the element calcium is able to link galacturonic acid molecules in the hg complex with each other. a decline of calcium results in a dissolution of the gel, leading to a break-down of calcium bridges and to a loss of the cell wall integrity (willats et al., 2001). despite higher calcium contents in leaves and fruits the plants of the unmulched variants were not able to link calcium in the pectin fractions of the fruits in absence of boron (tab. 1). the reason might be associated with the characteristics of the plant as a calcifuge species. calcium preferentially bounds other elements like p, fe or mn and causes a defi ciency of these elements in the tissue (zohlen and tyler, 2004). in the presence of boron, calcium would be also required for the linkage of pectin, in particular in the low esterifi ed fraction. calcium is also required for stabilizing rg-ii-b-complexes. ‘bluecrop’ ‘reka’ sd ‘bluecrop’/’reka’ wsp f1m 29.21 ± 0.73 ab 21.88 ± 1.05 b 25.54 ± 3.93 a f1om 28.27 ± 0.29 a 20.09 ± 1.57 a 24.18 ± 4.59 a f2m 30.10 ± 1.16 b 23.92 ± 0.58 c 27.01 ± 3.34 a f2om 30.30 ± 0.82 b 24.83 ± 0.97 c 27.57 ± 2.98 a esp f1m 6.44 ± 0.57 ab 5.55 ± 0.78 a 6.00 ± 0.80 a f1om 5.55 ± 0.31 a 6.10 ± 0.48 ab 5.83 ± 0.47 a f2m 6.96 ± 0.57 c 6.87 ± 0.43 b 6.91 ± 0.48 b f2om 6.66 ± 0.66 c 6.01 ± 0.78 ab 6.36 ± 0.76 ab isp f1m 1.48 ± 0.20 a 2.84 ± 0.21 a 2.10 ± 0.74 a f1om 1.71 ± 0.07 ab 3.19 ± 0.49 a 2.45 ± 0.86 a f2m 2.05 ± 0.29 b 3.63 ± 0.46 a 2.84 ± 0.90 a f2om 1.81 ± 0.17 ab 3.29 ± 0.66 a 2.55 ± 0.90 a total f1m 37.13 ± 1.26 ab 29.96 ± 1.42 a 33.55 ± 3.96 a pectin f1om 35.53 ± 0.47 a 29.38 ± 0.64 a 32.46 ± 3.40 a f2m 39.10 ± 1.41 b 34.58 ± 0.63 b 36.84 ± 2.58 b f2om 38.78 ± 1.06 b 33.13 ± 2.87 b 35.95 ± 3.60 b sd ‘bluecrop’/’reka’= standard devision of both cultivars tab. 1: pectin content (wsp, esp, isp, total pectin) of different blueberry cultivars [µg gal/ g dw], (n ± sd, tukey-b test, p < 0.05); f1m-mulched without b; f1om-unmulched without b, f2m-mulched with b, f2om-unmulched with b. 30 i. eichholz, s. huyskens-keil, l.w. kroh, s. rohn brown et al. (2002) reported that the rg-ii-b complex consists of two calcium molecules, apart from two boron molecules and two monomere rg-ii chains. however, the pectin content increased in the mulched variants where calcium content of fruits was lower than in unmulched variants. this leads to the conclusion that there are other infl uencing factors. for example, higher potassium contents were found in leaves and fruits of the mulched variants (data not shown). potassium is closely integrated into the regulation of stomata, and therefore enhances its turgor pressure, causing an opening of stomata (humble and raschke, 1971). however, this regulatory mechanism assumes optimum leaf water potential. in the present study, a higher soil water capacity and accordingly higher leaf water potential is suggested in the mulched variants in contrast to the unmulched variants. the shortage of stomata opening under drought stress could decrease the uptake of co2, and could led to a limited photosynthesis activity and a lower production of ribulose biphosphat as reported from flexas and medrano (2002). ribulose biphosphat is responsible for the rebuilding of co2 to glucose in the calvin cycle. in conclusion, it could be shown that plants absorbed foliar applied boron, leading to higher boron contents of fruits. application of boron induced an increase in the pectin content of all fractions (water-soluble, alkali-soluble, and insoluble). however, the content of phenolic compounds decreased and respectively the antioxidant acticvity. in contrast, the increased calcium content in the unmulched variants did not infl uence the biosynthesis of pectin substances, probably as blueberries avoid high calcium content. therefore, they cannot benefi t high calcium content for further pectin synthesis. plants preferred to root in mulch layer benefi ting from a lower ph and reduced calcium uptake in the rhizosphere. mulching appeared to improve growth conditions due to the high amount of organic matter and higher soil water availability. this circumstance tended to higher contents of pectin. however, polyphenol accumulation in fruits was inhibited by mulching. finally, this work indicates that moderate stress conditions during cultivation might improve fruit quality in terms of bioactive compounds, however only to a limited extent. references bazzano, l.a., he, j., ogden, l.g., loria, c.m., vupputuri, s., myers, l., whelton, p.k., 2002: fruit and vegetable intake and risk of cardiovascular disease in us adults: the fi rst national health and nutrition examination survey epidemiologic follow-up study. am. j. clin. nutr. 76, 93-99. bingham, f.t., 1982: boron. in: page, a.l. 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51, 4233-4329. schreiner, m., huyskens-keil, s., 2006: phytochemicals in fruit and vegetables: health promotion and postharvest elicitors. crit. rev. plant sci. 25, 267-278. silva, j.l., marroquin, e., matta, f.b., garner jr., j.o., stonjanovic, j., 2005: physicochemical, carbohydrate and sensory characteristics of highbush and rabbiteye blueberry cultivars. j. sci. food agric. 85, 18151821. stewart, a.j., chapman, w., jenkins, g.i., graham, i., martin, t., crozier, a., 2001: the effect of nitrogen and phosphorus defi ciency on fl avonol accumulation in plant tissues. plant cell environ. 24, 11891197. teare, i.d., 1974: boron nutrition and acid-soluble phosphorus compounds in 32 i. eichholz, s. huyskens-keil, l.w. kroh, s. rohn bean roots. hort. sci. 9, 236-238. ternes, w., täufel, a., tunger, l., zobel, m., 2005: lebensmittellexikon. behr’s verlag gmbh & co. kg. hamburg, germany. vicente, a.r., ortugno, c., rosli, h., powell, a.l.t., greve, l.c., labavitch, j.m., 2007: temporal sequence of cell wall disassembly events in developing fruits. 2. analysis of blueberry (vaccinium species). j. agric. food chem. 55, 4125-4130. willats, w.g.t., orfila, c., limberg, g., buchholt, h.c., van alebeek, g.j., voragen, a.g.j., marcus, s.e., christensen, t.m.i.e., mikkelsen, j.d., murray, b.s., knox, j.p., 2001: modulation of the degree and pattern of methyl-esterifi cation of pectic homogalacturonan in plant cell walls: implications for pectin methyl esterase action, matrix properties, and cell adhesion. j. biol. chem. 276, 19404-19413. williams, l.e., lemoine, r., sauer, n., 2000: sugar transporters in higher plants-a diversity of roles and complex regulation. trends plant sci. 5, 283-290. zohlen, a., tyler, g., 2004: soluble inorganic tissue phosphorus and calcicole-calcifuge behaviour of plants. ann. bot. 94, 427-432. authors address: ines eichholz, humboldt-universität zu berlin, divison urban plant ecophysiology, section quality dynamics/postharvest physiology, lentzeallee 75, d-14195 berlin, germany. phone: + 49-30-314 71447, fax: + 49-30-314 71262, e-mail: ieichholz@gmx.de journal of applied botany and food quality 92, 138 142 (2019), doi:10.5073/jabfq.2019.092.019 1institute of genetics and physiology, hebei academy of agriculture and forestry sciences, shijiazhuang, hebei, pr china 2hebei university of environmental engineering, qinhuangdao, hebei, pr china 3plant genetic engineering center of hebei province, shijiazhuang, pr china 4hebei normal university of science & technology, qinhuangdao, hebei, pr china different response to 1-methylcyclopropene in two cultivars of chinese pear fruit with contrasting softening characteristics jianmei wei1, 2 a, yudou cheng 1, 3 a, yunxiao feng1, 3, xiudong qi 4, jingang he1, 3, junfeng guan1, 3 * (submitted: january 9, 2019; accepted: february 14, 2019) * corresponding author a these authors contributed equally to the article summary in this study, the change in softening and its related genes expres sion under influence of 500 nl l-1 1-methylcyclopropene (1-mcp) was assessed in the two chinese pear fruit, ‘jingbaili’ (pyrus ussuriensis maxim) and ‘yali’ (pyrus bretschneideri rehd), which exhibit different softening characteristics. ‘jingbaili’ pear fruit softened rapidly after harvest, and was strongly inhibited by 1-mcp. in contrast, there was no obvious change of firmness compared to the control after 1-mcp treatment in ‘yali’ pear fruit. the respiration and ethylene production rates were reduced by 1-mcp at early storage in both two cultivars. ‘jingbaili’ pear fruit exhibited dramatically increased expression levels of the softening-related genes, i.e., polygalacturonase1 (pg1), polygalacturonase2 (pg2), β-galactosidase4 (gal4), α-arabinofuranosidase1 (arf1) and α-arabinofuranosidase2 (arf2), and these genes’ expression levels were significantly decreased by 1-mcp treatment. in contrast, ‘yali’ pear fruit showed lower expression levels of the above-mentioned genes, as well as a relatively smaller inhibition effect by 1-mcp treatment before day 27. these results suggest that ‘jingbaili’ pear fruit are more sensitive to 1-mcp/ ethylene than ‘yali’ pear fruit during ripening. keywords: pear; 1-methylcyclopropene; softening; cell wall enzyme; gene expression introduction ‘jingbaili’ (pyrus ussuriensis maxim) and ‘yali’ (pyrus bretschneideri rehd) pear fruit are famous cultivars and belong to typical chinese pear with different softening and ripening behaviors. ‘jingbaili’ pear fruit exhibits an extremely rapid decrease in flesh firmness during ripening, while ‘yali’ pear fruit do not exhibit fruit softening and maintain a crispy texture even during the late stage of ripening (hiwasa et al., 2004; wei et al., 2009, 2015). 1-methylcyclopropene (1-mcp), an effective inhibitor of ethylene action, has been found to delay softening of fruits, such as apple, banana, kiwifruit, mango, tomato and durian by blocking the ethy lene signal transduction pathway, and has been widely used to investigate fruit tissue responses to ethylene during ripening and senescence of climacteric fruit (golding et al., 1998; jiang and joyce, 2002; gamrasni et al., 2010; vilas-boas et al., 2007; piriyavinit et al., 2011; lu et al., 2012; amornputti et al., 2014). cell wall disassembly is believed to contribute to fruit softening (wakabayashi, 2000; sañudo-barajas et al., 2009). the depolymerization and solubilization of the pectin polymer is the major factor involved in fruit softening, and the pectin modification that cause cell dispersion and hydrolysis of the cell wall polymers is associated with texture change (brummell, 2006; goulao et al., 2008; gwanpua et al., 2014). many studies have shown that pectin modification is caused by the action of a series of cell wall-modifying enzymes and proteins, such as polygalacturonase (pg), β-galactosidase (β-gal) and α-arabinofuranosidase (α-arf) (brummell, 2006; gwanpua et al., 2014; wei et al., 2015). these enzymes have the capacity to reduce intercellular connections and the molecular size of pectin polymers by cleaving the backbone or the side chain residues. the modified polymers play different roles in fruit softening (sañudobarajas et al., 2009; wei et al., 2015). in this study, we investigated the effects of 1-mcp on fruit softening of ‘jingbaili’ and ‘yali’ pears. furthermore, we measured changes in the expression patterns of the softening-related genes (pg1, pg2, β-gal4, arf1 and arf2) to analyze the differences in the respon ses to 1-mcp between two cultivars of pear fruits. materials and methods biological materials and treatments ‘jingbaili’ (pyrus ussuriensis maxin.) and ‘yali’ (pyrus bretschneideri rehd.) pear fruit were harvested at commercial maturity (for ‘jingbaili’ pear fruit: average weight: 101.7 ± 5.8 g, tss: 12.2 ± 0.9%; for ‘yali’ pear fruit: average weight: 179.2 ± 10.7 g, tss: 10.9 ± 1.0%) (sep. 15, 2014) from an orchard in changli county, hebei province, china. the fruit without mechanical injury, insects and diseases were placed directly in a sealed container with 500 nl l-1 1-mcp (0.18%, ansip®, taiwan, china), after fumigating for 24 h at 20 ± 1 °c, the fruit were then ventilated and stored at 20 ± 1 °c. control fruit were subjected to the same protocol with the exception of a lack of 1-mcp treatment in the sealed container. fruit were sampled at appropriate intervals based on their softening rates, the flesh tissues were frozen in liquid nitrogen and stored at -70 °c for subsequent analysis. determination of fruit firmness, respiration rate, ethylene production rate flesh firmness was determined using a digital fruit penetrometer (tuopu instruments, model gy-4, zhejiang, china) with ten fruit per sampling time, and three replications per treatment. the respiration and ethylene production rates were measured via the air-stream method with three replications per treatment. ethylene production rate was determined by using a gas chromatograph (lunan ruihong chemical instruments, model gc-sp-6890, shandong, china). 2.0 kg of fruit were placed in a 9.5 l airtight jar for 1 h at 20 ± 1 °c. the 60 μl of gas samples were withdrawn through a septum on top of the jar using a gas-tight syringe, and three replications per treatment. rna extraction and qrt-pcr analysis total rna was extracted from the pear fruit samples using the modified ctab method (gasic et al., 2004). based on our previous research (wei et al., 2015), we choose pg1, pg2, gal4, arf1 two chinese pear response to 1-mcp 139 and arf2 to be measured by quantitative reverse transcriptionpolymerase chain reaction (qrt-pcr). first-strand cdnas were synthesized from dnase-digested rna (0.5 μg) using the takara rna pcr kit (amv) version 3.0 (takara biomedicals, japan). qrt-pcr was performed using the sybr premix ex taqtm (perfect real time) kit (takara biomedicals, japan) on a 7500 real-time pcr system (applied biosystems, usa). qrt-pcr primers for pgs, gal4 and arfs have been reported by wei et al. (2015). the qrtpcr reaction was performed in a final volume of 25 μl containing 12.5 μl sybr green pcr premix ex taqtm, 1 μmol l-1 forward and reverse primers, and 10 ng cdna with cycles as follows: 10 s at 95 °c, 40 cycles of 95 °c for 5 s, and 60 °c for 34 s. the tm of the amplification products was analyzed using a dissociation curve to confirm the quality of the pcr product and primer specificity. actin2 (act2) was used as a reference gene. all qrt-pcr reactions were normalized using ct value corresponding to the actin2 gene. the relative expression levels of the detected genes were calculated with the formula 2 -δδct and the experiment was performed with four replicates per treatment. statistical analysis the results were subjected to anova statistically analyzed using spss 18 software (spss inc., chicago, il, usa). all of the values are expressed as the means ± sd of three replicates. least significant differences (lsd) at a significance level of 0.05 were generated by anova. results respiration and ethylene production rates and flesh firmness in ‘jingbaili’ pear fruit, the respiration and ethylene production rate reached their peaks at the day 9 of storage concomitant with a rapid decrease in flesh firmness during ripening. after exposure to 1-mcp, the respiration and ethylene production rates dramatically decreased at first. and moreover, their climatic peaks were all delayed, and flesh firmness was noticeably kept at an initial value (fig. 1 a, c and e). in contrast, although 1-mcp resulted in a decrease trend in the respiration during storage, as well as the ethylene production rates before day 27 of storage in ‘yali’ pear, it did not exhibit the marked loss of flesh firmness (fig. 1 b, d and f). pg gene expression both the pg1 and pg2 transcripts were expressed at very high levels and were rapidly enhanced before day 12 of storage in ‘jingbaili’ pear fruit, and both of the pg1 and pg2 mrna accumulated slowly and were dramatically reduced in 1-mcp treatment than in control, except at day 15 for pg2 (fig. 2a and c). relatively, the mrna amounts of pg1 and pg2 were much lower in ‘yali’ pear than that in ‘jingbaili’ pear, and the peaks were found at day 36 for pg1and day 18 for pg2. in addition, pg1 and pg2 were significantly decreased by 1-mcp at day 18 and day 27, respectively (fig. 2 b and d). β-gal gene expression the gal4 is the major softening-related gene involved in fruit ripe ning, consistent with the changes in β-gal activity in pear fruit (wei et al., 2015). therefore, the expression level of gal4 was stu died here. the mrna of gal4 accumulated rapidly and was dramatically inhibited by 1-mcp in ‘jingbaili’ pear before day 12 of storage (fig. 3a). the expression of gal4 in ‘yali’ pear rose to a peak at day 18 in control, meanwhile, it increased slowly before day 27 of storage and afterwards reached the peak at day 36 in 1-mcp treatment (fig. 3b). fig. 1: effects of 1-mcp on the rates of respiration (a and b) and ethylene production (c and d), and firmness (e and f) in ‘jingbaili’ (a, c and e) and ‘yali’ (b, d and f) pear fruit. values are the means ± sd. fig.2: effect of 1-mcp on the expression of pgs in ‘jingbaili’ (a and c) and ‘yali’ (b and d) pear fruit. values are the means ± sd. 140 j. wei, y. cheng, y. feng, x. qi, j. he, j. guan α-arf gene expression the expression level of arf2 in ‘jingbaili’ pear was higher and increased more rapid than that of arf1, while the expression levels of the arf1 and arf2 genes increased slowly and showed much lower levels in 1-mcp treatment than that in control (fig. 4a and c). however, both arf1 and arf2 expression were lower and there was a lower level of arf1 expression at day 9 and 18, and arf2 at day 9, 18 and 27, even though there was a higher expression level of arf1 in 1-mcp treatment during the later stage in ‘yali’ pear fruit (fig. 4b and d). et al., 2009; minas et al., 2013), mangosteen (piriyavinit et al., 2011), apple (yang et al., 2013; ireland et al., 2014), pear (xie et al., 2014; escribano et al., 2017), tomato (dong et al., 2013) and muskmelon (supapvanich et al., 2013). in this study, application of 1-mcp after harvest dramatically delayed softening in ‘jingbaili’ pear fruit but had little effect in ‘yali’ pear fruit (fig. 1e and f), suggesting that ‘jingbaili’ pear is more sensitive to ethylene for regulating fruit softening than ‘yali’ pear. an increase in pg activity and mrna level has been reported in several fruit species concomitant with the degradation of pectin polysaccharides and softening (hiwasa et al., 2003, 2004; wei et al., 2015, song et al., 2016; gwanpua et al., 2016). 1-mcp treatment restricted softening and reduced pg activity and the expression level of pgs genes in pear and papaya fruits after the onset of ripening (hiwasa et al., 2003, 2004; zerpa-catanho et al., 2017). a similar result was observed in the ‘jingbaili’ pear fruit, which both pg1 and pg2 expression presented at much higher levels in control and were sharply suppressed by 1-mcp during the onset of softening in the ‘jingbaili’ pear fruit (fig. 2a and c). by contrast, pgs expression in ‘yali’ pear fruit was relatively less affected by 1-mcp treatment (fig. 2b and d). ranwala et al. (1992) found that purified β-gal had the capa city to catalyze an apparent decrease in the molecular size of pectins in vitro. in strawberry, down-regulated of faβ-gal4 increases cell wall galactose levels and reduces fruit softening (paniagua et al., 2016). 1-mcp treatment inhibited mrna accumulation of cmgal in melon, suggesting that the expression of cmgal is likely to be dependent on ethylene and may therefore function as a potential co-operative action partner with pg1 (nishiyama et al., 2007). in this study, we found that the expression of gal4 was dramatically inhibited by 1-mcp treatment in ‘jingbaili’ pear fruit (fig. 3a). in agreement with report that the expression of gal4 was negatively correlated with waterand na2co3-soluble polymers (rich in neutral sugars) in the ‘jingbaili’ pear fruit (wei et al., 2009). in contrast, the expression level of gal4 was maintained at a low level under 1-mcp treatment in ‘yali’ pear fruit with a slower increase at first and then dropped (fig. 3 b). it was in agreement with the results reported by mwaniki et al. (2005) and tateishi et al. (2005). the possible involvement of a glycoside hydrolase such as α-arf in cell wall modification during fruit ripening has been well documented. arf expression could result in the release of in many fruits (sozzi et al., 2002; brummell et al., 2004; nishiyama et al., 2007; wei et al., 2010). in ‘jingbaili’ pear fruit, arfs gene expression rapidly increased and were significantly inhibited by 1-mcp treatment (fig. 4 a and c), while in ‘yali’ pear fruit, they were maintained at a lower level and exhibited only a slight inhibition by 1-mcp (fig. 4 b and d). these results suggested that arfs in ‘jingbaili’ pear fruit were more sensitive to ethylene than those in ‘yali’ pear fruit. furthermore, α-arf may play different roles in pear fruit softening due to the different textural attributes and response to ethylene as shown in tomato (sozzi et al., 2002) and apple (goulao et al., 2007). conclusion in summary, this study indicated that the fruit softening was significantly inhibited by 1-mcp treatment in ‘jingbaili’ pear, while no obvious change was observed in ‘yali’ pear. this was caused by the different change of expression of the softening-related genes, i.e., pg1, pg2, gal4, arf1 and arf2. this suggests that the ‘jingbali’ pear is more sensitive to 1-mcp/ethylene than the ‘yali’ pear during ripening. acknowledgements this work was supported by the emarked fund for the china agriculture research system for national technology system for fig. 3: effect of 1-mcp on the expression of gal4 in ‘jingbaili’ (a) and ‘yali’ (b) pear fruit. values are the means ± sd. fig. 4: effect of 1-mcp on the expression of arfs in ‘jingbaili’ (a and c) and ‘yali’ (b and d) pear fruit. values are the means ± sd. discussion 1-mcp can effectively inhibit fruit tissue responses to ethylene, thereby depress respiration, ethylene production and delaying ripe ning of climacteric fruit (serek et al., 1995). therefore, the application of 1-mcp provides an alternative approach to assess the ripe ning-related mechanism in many fruits. the maintenance of better flesh texture and effective delay in fruit softening by 1-mcp treatment has been widely reported in many fruits such as plum (luo two chinese pear response to 1-mcp 141 pear industry (cars-28-05b), the innovation project of modern agricultural sciences and technology of hebei province, china (494-0402-ysn-c8ra) and finance special foundation of hebei province (f18r060018-9). references amornputti, s., ketsa, s., van doorn, w.g., 2014: effect of 1-methylcyclopropene (1-mcp) on storage life of durian fruit. postharvest biol. tec. 97, 111-114. doi: 10.1016/j.postharvbio.2014.06.011 brummell, d.a., dal cin, v., crisosto, c.h., labavitch, j.m., 2004: cell wall metabolism during maturation, ripening and senescence of peach fruit. j. exp. bot. 55, 2029-2039. doi: 10.1093/jxb/erh227 brummell, d.a., 2006: cell wall disassembly in ripening fruit. funct. plant biol. 33, 103-119. doi: 10.1071/fp05234 dong, x., huber, d.j., rao, j., 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(carica papaya l. ‘pococí’ hybrid). postharvest biol. tec. 115, 42-51. doi: 10.1016/j.postharvbio.2016.11.002 address of the corresponding author: junfeng guan, institute of genetics and physiology, hebei academy of agriculture and forestry sciences, shijiazhuang, hebei 050051, pr china plant genetic engineering center of hebei province, shijiazhuang 050051, pr china © the author(s) 2019. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1016/j.postharvbio.2006.09.010 http://dx.doi.org/10.1007/pl00013932 http://dx.doi.org/10.1016/j.postharvbio.2009.12.003 http://dx.doi.org/10.1016/j.scienta.2015.10.002 http://dx.doi.org/10.1016/j.postharvbio.2014.06.002 http://dx.doi.org/10.1016/j.postharvbio.2012.11.012 https://www.sciencedirect.com/science/article/abs/pii/s0925521416305336#! https://www.sciencedirect.com/science/article/abs/pii/s0925521416305336#! https://www.sciencedirect.com/science/article/abs/pii/s0925521416305336#! https://www.sciencedirect.com/science/article/abs/pii/s0925521416305336#! https://www.sciencedirect.com/science/article/abs/pii/s0925521416305336#! https://www.sciencedirect.com/science/article/abs/pii/s0925521416305336#! http://dx.doi.org/10.1016/j.postharvbio.2016.11.002 art01_ivic.indd journal of applied botany and food quality 84, 121 124 (2011) 1institute for plant protection, croatian centre for agriculture, food and rural affairs, zagreb, croatia 2university of goce delcev, faculty of agriculture, stip, republic of macedonia 3university of mostar, faculty of agriculture and food technology, mostar, bosnia and herzegovina occurrence of potentially toxigenic fusarium verticillioides and low fumonisin b1 content on barley grain in bosnia and herzegovina d. ivic1, biljana kovacevik2, visnja vasilj3, nikolina idzakovic3 (received november 1, 2010) summary a study was conducted to determine the mycobiota, the presence of potential fumonisin producers, and eventual fumonisin b1 contamination on four barley grain samples from bosnia and herzegovina. alternaria spp. were the dominant fungi, detected on 82 %, 77 %, 78 % and 82 % of kernels in different samples. fusarium spp. was found in all samples, on 26 %, 40 %, 49 % and 60 % of kernels. among fusarium spp., f. verticillioides was the most frequent in three samples (40 %, 49 % and 60 %), while f. graminearum was the most frequent in one sample (12 %). f. avenaceum, f. sporotrichioides, f. solani, f. semitectum, f. tricinctum, f. equiseti and f. oxysporum were found in lower percentages. other fungal species found in all samples were cladosporium spp., epicoccum spp., gonatobotrys spp. and drechslera spp. contamination of grain with aspergillus spp. and penicillium spp. was very low. pcr amplifi cation with fum5/6 primer pairs was performed on 29 f. verticillioides isolates from the grain, and all isolates yielded 419 bp amplifi cation products. fumonisin b1 content in grain determined by elisa was very low (5.35, 1.68, 1.48, and 1.01 ng/g), with an average of 2.40 ng/g for all four samples. introduction cereal grain is usually infected or colonised by different pathogenic or saprophytic fungi during the vegetation period. numerous fungal species are reported to occur on barley grain (rohácik and hudec, 2007; medina et al., 2006; andersen et al., 1996), some of which are economically important as seed-borne pathogens (maude, 1996), or producers of mycotoxins, fungal metabolites toxic to humans or animals (bottalico and perrone, 2002). different fusarium species, causal agents of barley head blight (mcmullen et al., 1997) and producers of numerous mycotoxins (de nijs et al., 1996), are among those fungi which regularly colonize barley grain (leslie and summerell, 2006; bottalico and perrone, 2002). barley grain from different european countries was found to be contaminated with one or more mycotoxins produced by fusarium species, mostly with zearalenone (zen) or trichothecenes like deoxinivalenol (don) (malachova et al., 2010; tabuc et al., 2009; bottalico and perrone, 2002). these mycotoxins are produced by fusarium graminearum, f. culmorum or f. crookwellense (leslie and summerell, 2006), pathogenic species which cause head blight of barley and other small-grain cereals. however, numerous other fusarium species can colonize barley grain as secondary invaders or saprophytes. several recent studies showed that f. verticillioides and f. proliferatum are common on barley grain (maenetje and dutton, 2007; medina et al., 2006). as those species are the main producers of fumonisins, cancerogenic mycotoxins (leslie and summerell, 2006), it would be important to fi nd out whether these toxins are produced on barley grain. fumonisins are mycotoxins mainly found in maize (munkvold and desjardins, 1997), but there are reports on their occurrence in other plants (seefelder et al., 2002; logrieco et al., 1998). the presence of fumonisin b1 was detected in wheat (ivic et al., 2008; desjardins et al., 2007), and small quantities of these mycotoxins were found in beer (torres et al., 1998). these studies indicate that fumonisins could be found on barley grain as well. studies on mycotoxin production on molecular and genetic level have lead to the development of pcr-based detection of fungal genes needed for different mycotoxin production (niessen, 2007; nicholson et al., 2004; niessen et al., 2004). pcr primers based on sequence data of polyketide synthase gene (fum 1) were recently developed for the detection of fumonisin-producing f. verticillioides (baird et al., 2008). although not without certain constraints (paterson, 2006), pcr amplifi cation of fum genes could become rapid and reliable method for detecting potential fumonisinproducing fungi in plants or plant parts. it could also be used for screening the potential ability of f. verticillioides isolates to produce fumonisins. in a routine microbiological analysis of stored barley grain from bosnia and herzegovina during the last few years, a relatively high level of contamination with fusarium spp. of the liseola section was observed, resembling mostly f. verticillioides or f. proliferatum. the objectives of this study were to determine the presence of potential fumonisin b1 producers and eventual fumonisin b1 contamination on barley grain in bosnia and herzegovina. the other objectives were to determine eventual differences in mycobiota on several grain samples from the same region, especially regarding the presence of fusarium species. materials and methods grain origin, sampling, and mycological analysis spring barley (cv. novosadski 294) was grown in 2008 on different locations around the area of posusje, bosnia and herzegovina. data on temperatures and rainfall in the area of posusje in 2008 (april, may and june) was obtained from federal hydrometeorological institute in sarajevo. grain was harvested in june, and approximately one kg of randomly selected harvested grain was collected for mycobiota and mycotoxin analysis. for mycological analysis, 100 kernels in four replicates was placed on moist blotter and incubated under 12/12 h photoperiod at 22 °c for 14 days. after incubation, each kernel was checked for the presence of fungi under the stereomicroscope and microscope. fungi other than fusarium spp. on kernels were identifi ed only to the genus level based on the conidial morphology according to descriptions of barnett and hunter (1998) and samson et al. (1984). if a colony of fusarium was detected, kernel was placed on potato-dextrose agar (pda) and incubated at the same conditions as described above for additional seven days. fusarium species were determined according to leslie and summerell (2006) from colonies developed on pda, while non-sporulating colonies or colonies resembling f. graminearum or f. avenaceum were subcultured using a single-spore technique described by leslie and summerell (2006), transferred to carnation leaf agar (cla), and identifi ed. mycobiota on kernels was expressed as a percentage of kernels on which certain fungal genera or species were found. 122 d. ivic, b. kovacevik, v. vasilj, n. idzakovic f. verticillioides isolation, identifi cation, and pcr amplifi cation of fum fragments from pda cultures, 29 isolates identifi ed as f. verticillioides were collected using a single-spore technique (leslie and summerell, 2006). five isolates were collected from the posusje i sample, while 8 isolates were collected fom each of the other three grain samples. a single-spore isolate of f. semitectum collected from the posusje iii sample was used in pcr reaction as a negative control. for dna extraction, isolates were grown on cellophane-layer pda, from where mycelia was harvested and ground to a powder with a liquid nitrogen. fungal dna was extracted using dneasy® plant mini kit (quiagen inc., usa) according to manufacturer’s instructions and checked on agarose gel. two to fi ve µl of dilluted dna extract was used in 50 µl pcr reactions containing 5 µl of 10x pcr buffer, 4 µl of 2.5 mm dntps, 2.5 µl of mgcl2, 1 u of taq polymerase and 25 pmoles of fum5f and fum6r primers (baird et al., 2008). pcr conditions were the following: 95 °c for 3 min, 33 cycles of 95 °c for 1 min, 60 °c for 1 min, and 72 °c for 3 min, with a fi nal extension on 72 °c for 5 min. pcr products were visualised and photographed after electrophoresis through 1.5 % tbe agarose gels, stained with ethidium-bromide. all isolates used in this research were stored on water agar (wa), and they are available at the institute for plant protection collection in zagreb, croatia. fumonisin b1 analysis elisa test (fumonisin b1 eia kit, euro-diagnostica b.v., arnhem, the netherlands) was used for fumonisin b1 quantifi cation on grain of each sample. approximately 50 g of grain was pulverised, extracted with 80 % methanol and fi ltrated through whatman no.1 fi lter paper. an aliquot of 50 µl of extract was used for analysis in two replicates. elisa was performed according to the manufacturer’s instructions. results colonies of different fungi were detected on almost all analysed barley kernels in all periods of analysis (tab. 1). species from two or more genera were often found to be present on the same kernel. mycobiota was very similar in all four samples. alternaria spp. were the dominant fungi on grain, found on average 80 % of kernels. fusarium spp. were the second in prevalence on grain, detected on average 44 % of kernels. f. verticillioides was found to be the dominant fusarium species in three samples, while f. graminearum was found in the highest percentage in one sample. some fusarium species sporulated well on kernels after the incubation (e. g. f. avenaceum, f. sporotrichioides, f. verticillioides or f. semitectum) while most of the colonies later determined to be f. graminearum developed as sterile mycelium on kernels and had to be transferred and identifi ed on pda and cla. average temperatures in 2008 in period from “boot” stage of experimental barley to the harvest (april, may and june) were similar to the 10-year average (tab. 2). however, average rainfall in april and june was higher than average, while in may it was almost 50 mm lower than average. tab. 1: contamination of barley grain with fungi (%) and fumonisin b1 (ng/g). grain samples fungal genera/species posusje i posusje ii posusje iii posusje iv % of kernels contaminated alternaria spp. 82 77 78 82 79.8 cladosporium spp. 24 18 11 3 14 epicoccum spp. 20 10 7 11 12 drechslera spp. 17 9 10 14 12.5 gonatobotrys spp. 14 27 17 21 19.8 khuskia oryzae 0.3 1 3 1 1.4 aspergillus spp. 0.5 0.8 2 0.3 0.9 penicillium spp. 0.5 2 0.8 3 1.6 gliocladium spp. 0.3 0.1 fusarium graminearum 12 10 8 10 10 fusarium avenaceum 2 1 5 0.8 2.2 fusarium verticillioides 6 26 32 42 26.5 fusarium solani 2 0.5 0.6 fusarium sporotrichioides 2 1 2 2 1.8 fusarium semitectum 0.8 0.5 1 5 1.8 fusarium tricinctum 0.5 1 0.3 0.5 fusarium equiseti 0.3 0.1 fusarium oxysporum 0.5 0.5 0.3 0.3 0.4 fumonisin b1 content (ng/g) 5.350 1.680 1.482 1.099 2.40 average tab. 2: average temperatures (°c) and rainfall (mm) in posusje for april, may and june 2008, compared to 10-year average. month 2008 10-year average temperature rainfall temperature rainfall (°c) (mm) (°c) (mm) april 14.0 162.0 16.5 119.2 may 20.0 30.3 21.9 87.8 june 23.5 121.9 24.6 82.7 fungi and fumonisin b1 on barley grain 123 pcr products of 419 bp amplifi ed with fum5f/fum6r primer pairs were recorded for all 29 f. verticillioides isolates. (fig. 1). no products were visible for f. semitectum negative control isolate and for water blanks. amplifi cation of polyketide synthase gene with fum5/6 primer pairs showed that all 29 f. verticillioides isolates tested are potential fumonisin producers. considering that these isolates were randomly collected from the kernels, it can be assumed that the high percentage of f. verticillioides on analyzed barley grain is capable of producing fumonisins. however, fumonisin b1 content in all four samples was very low. it is interesting to notice that the relatively highest amount of fumonisin b1 was found in the sample posusje i, on which low f. verticillioides contamination was detected, while the relatively lowest content of fumonisin b1 was found on sample posusje iv, heavily contaminated with f. verticillioides. to a certain extent, such results can show that in some cases mycotoxin content can not be predicted on the basis of the extent of fungal contamination. beside various environmental factors (marín et al., 1999a; marín et al., 1999b), it is also known that the quantity of fumonisin b1 produced in different conditions is dependent on strain of f. verticillioides (medina et al., 2006; reyonso et al., 2004). average fumonisin b1 content of 2.40 ng/g detected in this study is far below those reported to occur on maize (logrieco et al., 2002). one of the reasons could be in the difference between saprophytic nature of f. verticillioides on barley and parasitic nature of the same fungal species on maize. f. verticillioides is considered to be a saprophyte on barley grain (leslie and summerell, 2006). on the other hand, this fungus is the causal agent of fusarium ear rot of maize, and high fumonisin b1 content in maize grain is often correlated with ear rot (munkvold and desjardins, 1997). when the fungus is a parasite, it has an ability to grow through plant tissue without competing with other micro-organisms, and consequently to develop much more mycelial mass. the other reason for low fumonisin b1 contamination of analyzed grain may be in environmental factors not conductive for fumonisin production. fumonisin production is dependent on many factors, out of which temperature and water availability are among the most important ones (marín et al., 1999a). comparing the kernel development period in barley and maize in europe, it is clear that the maize grain is developing and maturing in warmer periods of the season. it is not known when f. verticillioides colonize barley kernels in the fi eld. if such colonization occurs late in the season, when kernels are approaching maturity, the fungus may simply not have enough time to produce higher quantities of mycotoxins. the moisture content of maturing barley kernels could descend to the level which does not support further f. verticillioides growth or fumonisin production. in cases of fusarium ear rot of maize, the moisture content of grain and humidity under the husks usually allows f. verticillioides or f. proliferatum intensive growth. finally, it could be assumed that barley grain is simply not a good substrate for fumonisin production. marín et al. (1999b) compared fumonisin b1 production by f. verticillioides and f. proliferatum isolates on maize, barley and wheat grain in controlled conditions. fumonisin b1 production by both fusarium species was much higher on maize grain than on wheat or barley grain. considering all of the theories which could explain the results of this study, it can be assumed that barley grain as an unsuitable substrate, late colonisation, and saphrophytic growth of f. verticillioides could all, in certain amount, contribute to the low level of fumonisin production on grain. the results of the present study showed that relatively high contamination levels of fumonisin-producing fusarium species on grain sometimes may not lead to high levels of fumonisin contamination. references andersen, b., thrane, u., svendsen, a., rasmussen, i.a., 1996: associated fi eld mycobiota on malt barley. can. j. bot. 74, 854-858. baird, r., abbas, h.k., windham, g., williams, p., baird, s., ma, p., kelley, r., hawkins, l., scruggs, m., 2008: identifi cation of select fig. 1: pcr products of f. verticillioides isolates amplifi ed with fum5f/ fum6r primer pairs. lanes 1-11: fv26; fv27; fv28; fv29; fv30; fv32; fv33; fv34; fv37; fv39; fv40 (all f. verticillioides); lane 12: fs36 (f. semitectum); lane 13: water blank; lane 14: 1 kb dna ladder. discussion alternaria, gonatobotrys and fusarium species were found in the highest percentage on barley grain during the storage. similar percentages of alternaria spp. contamination was reported from other studies (rohácik and hudec, 2007; medina et al., 2006; andersen et al., 1996). the presence of epicoccum, cladosporium and drechslera species in all samples is not unexpected, as those species often colonize seed and grain of various plants (maude, 1996; samson et al., 1984). population structure of fusarium spp. on grain was also similar in all four samples. the only noticeable difference was between relatively low contamination level of f. verticillioides on posusje i sample and the other three samples, where f. verticillioides was detected in high percentages. considering the presence of f. graminearum, f. avenaceum and other species found in this study, all of them are reported as the usual colonizers of barley grain (leslie and summerell, 2006; krstanovic et al., 2005; bottalico and perrone, 2002). it could be assumed that the high rainfall in june, during the ripening stages of barley, was conductive to the colonization of saprophytic fungi, like alternaria, epicoccum or f. verticillioides, on developing grain. on the other hand, low rainfall conditions in may, during the fl owering stage, might has not be favourable to the development of fusarium head blight and more extensive colonization of grain with pathogenic f. graminearum. relatively high contamination level of f. verticillioides detected (average 27 %) can indicate the possibility of fumonisin contamination. f. verticillioides, f. proliferatum and fumonisin b1 were detected on barley grain in south africa (maenetje and dutton, 2007). f. verticillioides was the most prevalent fungal species found on barley rootlets used as feedstuff raw material in brazil, and all samples were contaminated with fumonisin b1 (cavaglieri et al., 2009). based on their results, cavaglieri et al. (2009) pointed out that the presence of f. verticillioides on barley rootlets could be correlated with fumonisin b1 contamination. in the study of medina et al. (2006), f. verticillioides and f. proliferatum were also among the most frequent fusarium spp. found on barley grain. medina et al. (2006) studied the production of fumonisin b1 by 31 f. verticillioides and 18 f. proliferatum isolates from barley grain, and most of them were capable of fumonisin production on a rice grain medium. 124 d. ivic, b. kovacevik, v. vasilj, n. idzakovic fumonisin forming fusarium species using pcr applications of the polyketide synthase gene and its relationship to fumonisin production in vitro. int. j. mol. sci. 9, 554-570. barnett, h.l., hunter, b.b., 1998: illustrated genera of imperfect fungi. aps press, st. paul, minnesota. bottalico, a., perrone, g., 2002: toxigenic fusarium species and mycotoxins associated with head blight in small-grain cereals in europe. eur. j. plant pathol 108, 611-624. cavaglieri, l.r., keller, k.m., pereyra, c.m., gonzález pereyra, m.l., alonso, v.a., rojo, f.g., dalcero, a.m., rosa, c.a.r., 2009: fungi and natural incidence of selected mycotoxins in barley rootlets. j. stored prod. res. 45, 147-150. de nijs, m., rombouts, f., notermans, s., 1996: fusarium 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occurrence of fumonisin b1 and b2 in fusarium proliferatum infected asparagus plants. j. agr. food chem. 46, 5201-5204. maenetje, p.w., dutton, m.f., 2007: the incidence of fungi and mycotoxins in south african barley and barley products. j. environ. sci. heal. b 42, 229-236. malachova, a., cerkal, r., ehrenbergerova, j., dzuman, z., vaculova, k., hajslova, j., 2010: fusarium mycotoxins in various barley cultivars and their transfer into malt. j. sci. food agric. 90, 24952505. marín, s., magan, n., bellí, n., ramos, a.j., canela, r., sanchis, v., 1999a: two-dimensional profi les of fumonisin b1 production by fusarium moniliforme and fusarium proliferatum in relation to environmental factors and potential for modelling toxin formation in maize grain. int. j. food microbiol. 51, 159-167. marín, s., magan, n., serra, j., ramos, a.j., canela, r., sanchis, v., 1999b: fumonisin b1 production and growth of fusarium moniliforme and fusarium proliferatum on maize, wheat, and barley grain. j. food sci. 64, 921-924. maude, r.b., 1996: seedborne diseases and their control. cab international, wallingford, uk. mcmullen, m., jones, r., gallenberg, d., 1997: scab of wheat and barley: a re-emerging disease of devastating impact. plant dis. 81, 13401348. medina, á., valle-algarra, f.m., mateo, r., gimeno-adelantado, j.v., mateo, f., jiménez, m., 2006: survey of the mycobiota of spanish malting barley and evaluation of the mycotoxin producing potential of species of alternaria, aspergillus and fusarium. int. j. food microbiol. 108, 196-203. munkvold, g.p., desjardins, a.e., 1997: fumonisins in maize – can we reduce their occurrence? plant dis. 81, 556-565. nicholson, p., simpson, d.r., wilson, a.h., chandler, e., thomsett, m., 2004: detection and differetiation of trichothecene and enniatinproducing fusarium species on small-grain cereals. eur. j. plant pathol. 110, 503-514. niessen, l., 2007: pcr-based diagnosis and quantifi cation of mycotoxin producing fungi. int. j. food microbiol. 119, 38-46. niessen, l., schmidt, h., vogel, r.f., 2004: the use of tri5 gene sequences for pcr detection and taxonomy of trichothecene-producing species in the fusarium section sporotrichiella. int. j. food microbiol. 95, 305-319. paterson, r.r.m., 2006: identifi cation and quantifi cation of mycotoxigenic fungi by pcr. process biochem. 41, 1467-1474. reyonso, m.m., torres, a.m., chulze, s.n., 2004: fusaproliferin, beauvericin and fumonisin production by different mating populations among the gibberella fujikuroi species complex isolated from maize. mycol. res. 108, 154-160. rohàcik, t., hudec, k., 2007: fungal infection of malt barley kernels in slovak republic. plant protect. sci. 43, 86-93. samson, r.a., hoekstra, e.s., van oorschot, c.a.n., 1984: introduction to food-borne fungi. centraalbureau voor schimmelcultures, baarn, the netherlands. seefelder, w., gossmann, m., humpf, h.u., 2002: analysis of fumonisin b1 in fusarium proliferatum-infected asparagus spears and garlic bulbs from germany by liquid chromatography-electrospray ionization mass spectrometry. j. agr. food chem. 50, 2778-2781. tabuc, c., marin, d., guerre, p., sesan, t., bailly, j.d., 2009: molds and mycotoxin content of cereals in southeastern romania. j. food protect. 72, 662-665. torres, m.r., sanchis, v., ramos, a.j., 1998: occurrence of fumonisins in spanish beers analyzed by enzyme-linked immunosorbent assay method. int. j. food microbiol. 39, 139-143. address of the authors: dr. dario ivic, institute for plant protection, croatian centre for agriculture, food and rural affairs, svetosimunska cesta 25, 10 000 zagreb, croatia. dr. biljana kovacevik, university of goce delcev, faculty of agriculture, krste misirkov bb, 2000 stip, republic of macedonia. dr. visnja vasilj and nikolina idzakovic, university of mostar, faculty of agriculture and food technology, biskupa cule bb, 88 000 mostar, bosnia and herzegovina. journal of applied botany and food quality 95, 136 142 (2022), doi:10.5073/jabfq.2022.095.018 1seoul metropolitan government research institute of public health and environment, gwacheon, republic of korea 2department of food and nutrition, and research institute of human ecology, seoul national university, republic of korea phenolic compounds and in vitro biological activities of nonanthocyanin fractions from mulberry fruits harvested at different maturity stages yongcheol lee1, keum taek hwang2* (submitted: february 6, 2022; accepted: september 7, 2022) * corresponding author summary mulberry fruits contain substances involved in physiological activities beneficial to human health. to explore potential utilization of mulberry fruits as functional foods, functional compounds and in vitro biological activities of their extracts were determined at different maturity stages. as maturity progressed, nonanthocyanin fraction (naf) decreased, while anthocyanin fraction (af) increased. main phenolic acid contained in the naf was chlorogenic acid, which decreased during ripening. main flavonols present in the naf were rutin, isoquercetin, and morin, which also decreased during ripening. the naf and af exhibited anti-inflammatory properties by lowering nitric oxide production in raw264.7 cells. the naf of immature mulberry fruits promoted hexokinase activity in hepg2 cells and inhibited α-glucosidase, indicating its possible hypoglycemic effect. it is suggested that immature mulberry fruits that are rich in nonanthocyanin phenolics might be a potential functional food source. keywords: mulberry, maturity, phenolic compound, nonanthocyanin, hypoglycemic effect introduction mulberry (morus alba l.) is widely cultivated from the tropics to the subarctic (machii, koyama, and yamanouchi, 2000) and high in phytochemicals having health promoting effects. several studies revealed that mulberry fruits possess a wide range of biological activities attributed to the high content of phenolic compounds. most of the studies on mulberry fruits have concentrated on anthocyanins and bioactive compounds in mature fruits. mulberry extracts and anthocyanins in them have been reported to be associated with anti inflammatory (chen et al., 2016; qian et al., 2015), anti-cancer (chen et al., 2006) and anti-diabetes effects (wang et al., 2013). mulberry fruits are commonly consumed fresh, but they are prone to bruising during storage and distribution because of their soft tissue. mature mulberries are more prone to mold growth than immature ones, making it easier to deteriorate in quality (park et al., 2013). mature mulberries are often processed and consumed as juice, jam, or liquor. processing, however, can degrade the bioactive compounds leading to a decrease in their bio-accessibility (cavalcanti, santos, and meireles, 2011). in terms of the potential availability of mulberry fruits, it is necessary to retain their bioactive compounds asso ciated with biological activity. immature and mature mulberry fruits are used in traditional medicine as tonics, antidiabetics, and immunostimulants (kfda, 2012; kim et al., 2013; tang and eisenbrand, 2011). our previous study revealed that 1-deoxynojirimycin, γ-aminobutyric acid, and amino acids, as well as nonanthocyanin phenolics including phenolic acids and rutin, were higher in immature mulberry fruits than in mature ones. additionally, immature fruits had an advantage retaining their physicochemical characteristics during storage (lee and hwang, 2017). with this result, immature mulberries are likely to be a pro mising resource of functional food; however, only a few studies on biological activity of immature mulberries have been reported (lin et al., 2013; oki et al., 2006). anthocyanins and nonanthocyanin phenolics, including phenolic acids and flavonoids, demonstrated strong biological effects such as antioxidant (zhang et al., 2008), antidiabetic (wang et al., 2013), and α-glucosidase inhibitory effects. chlorogenic acids, rutin, and isoquercetin are known to be the main nonanthocyanins in mulberry fruits (yang et al., 2016; zhang et al., 2008) and are more abundant in immature mulberry fruits than in mature ones (lee and hwang, 2017). nonanthocyanin phenolics in mulberry fruits presented a higher antioxidant activity than trolox (zhang et al., 2008). phenolic compounds (polyphenol-rich extracts or isolated phenolic compounds) affect inhibitory activity on digestive enzymes including α-amylase and α-glucosidase, which contribute to preventing type 2 diabetes (posedek et al., 2014). ethyl acetate extract (nonanthocyanin fraction) of mulberry fruits had a lowering effect on fasting blood glucose level in hyperglycemic mice and higher α-glucosidase inhibitory effect than ethanol, hexane, dichloromethane, butanol, and water extracts (wang et al., 2013). furthermore, rutin and quercetin in mulberry fruits contributed to promoting viability of pancreatic β-cells (li et al., 2017). in this study, therefore, we investigated phenolic compounds and in vitro biological activities in nonanthocyanin and anthocyanin fractions from mulberry fruits at different maturity stages (ms) and evaluated in vitro biological activities of nonanthocyanin phenolics in mulberry fruits in order to enhance the potential availability of mulberry as a functional food source. materials and methods materials and chemicals freeze-dried mulberry powders obtained from seven different ms (ms1-7) as described in the materials and methods section of the previous study (lee and hwang, 2017) were used. mulberry fruits (morus alba l.) were hand-harvested at seven different ms(1-7). ms were set by the color and sizes. the fruits turned green into red, purple, and black as ripening progressed: ms-1 and 2 were reddish green, ms-3 was red, ms-4 was dark red, ms-5 was dark purple, and ms-6, 7 were black. methanol and acetonitrile were from merck chemicals (darmstadt, germany). ethanol, n-hexane, and ethyl acetate were from fisher scientific korea ltd. (seoul, korea). acetic acid (lc-ms grade) was from fisher chemical co. (leicestershire, uk). dulbecco’s modified eagle medium (dmem), dmem/f-12, 10% fetal bovine serum (fbs), 2% 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (hepes), 1% penicillin/streptomycin, and trypsin were from gibco (grand island, ny). phosphate buffered saline (pbs) was from biorad laboratories (hercules, ca). phosphoric acid, hydrochloric acid, sulfuric acid, sodium hydroxide, and dimethyl sulfoxide (dmso) were from samchun pure chemical co. (seoul, korea). perchloric biological activities of mulberry extracts 137 acid and naphtylethylenediamine dihydrochloride were from wako pure chemical industries (osaka, japan). hexokinase assay kit was from abcam (cambridge, uk). protocatechuic acid (reference standard grade) was from hwi analytik gmbh (ruelzheim, germany). all the other reagents and standard materials were purchased from sigma-aldrich co. (st. louis, mo). analysis of total cyanides in mulberry fruits total cyanides were determined by the method of bradbury, egan, and lynch (1991) with some modification. mulberry powder (0.3 g) was vigorously mixed with 10 ml 0.1 m phosphoric acid, followed by centrifugation (1,900 × g) at 4 °c. the residue was re-extracted. the supernatants were filled into a 20 ml volumetric flask with 0.1 m phosphoric acid. the extract (1.0 ml) was hydrolyzed with 1 ml 4 m sulfuric acid at 100 °c for 50 min and neutralized with 2.5 ml 3.6 m sodium hydroxide after cooling. 100 μl of the solution was reacted with 700 μl 2 m phosphate buffer (ph 6.0) and 40 μl 0.5% (w/v) chloramine-t for 5 min. in 60 min after adding 160 μl barbituric acid/pyridine solution absorbance was measured at 600 nm with a microplate reader (spectramax 190, sunnyvale, ca). cyanide standard was used to quantify total cyanides. analysis of proanthocyanidins in mulberry fruits proanthocyanidin content was determined by the method of prior et al. (2010). 80% (v/v) ethanol extracts of mulberry powder at dif ferent ms and blank (70 μl) were reacted with 0.1% (w/v) 4-di methylaminocinnamaldehyde reagent solution (210 μl ) at room temperature for 10 min. absorbance was measured using the microplate reader at 640 nm. catechin was used as standard, and proanthocyanidin content was expressed as mg catechin equivalent (eq)/100 g on dry weight basis (dw). preparation of nonanthocyanin fraction (naf) and anthocyanin fraction (af) from mulberry fruits freeze-dried mulberry powder (approximately 1.5-2.0 g) was de fatted with n-hexane (3 × 20 ml) and extracted with acidified 80% ethanol (ph 2.25) (3 × 20 ml) in a shaking water bath for 1 h at 40 °c. according to the method described by kammerer et al. (2004), the extract was suspended in 10 ml distilled water (ph 3.0) after evaporation of the solvent. 5 ml aliquot of the extract solution was made up to 20 ml with distilled water and ph was adjusted to 1.5, followed by partitioning with 50 ml ethyl acetate. the ethyl acetate layer was entirely evaporated to obtain naf, which was dissolved in 10 ml distilled water for analysis of nonanthocyanin phenolics or 2 ml pbs for biological assay. the water layer was further fractionated by a c18 spe cartridge (waters co., milford, ma) to collect af according to the method described by kammerer et al. (2004). after the cartridge was activated with 3 ml methanol and cleaned with 10 ml distilled water, the af was eluted from the water layer with 10 ml methanol with 0.01% hydrochloric acid (v/v). the af was concentrated in vacuo and then dissolved in 2 ml pbs. hplc-ms/ms analysis of nonanthocyanin phenolics nonanthocyanin phenolics were eluted from the naf using the spe cartridge according to the method described by kammerer et al. (2004). after the cartridge was activated with 3 ml methanol and cleaned with 10 ml distilled water, phenolic acids were eluted from the naf with 10 ml distilled water and 10 ml 0.01% (v/v) hydrochloric acid (ph 2.65). flavonols were subsequently eluted with 20 ml ethyl acetate. the eluates were concentrated in vacuo. the concentrated eluates of phenolic acids and flavonols were dissolved in 2 ml 2% acetic acid and 2 ml methanol, respectively. identification and quantification of nonanthocyanin phenolics in the naf were carried out by hplc (agilent 1200 series, agilent technologies) coupled with an api 4000 triple quadrupole mass spectrometer (applied biosystem, foster city, ca) with an electrospray interface. separation was operated on an atlantis t3 column (3 μm, 2.1 mm × 100 mm; waters co.) with a guard column (xdb c18, 5 μm, 4.6 mm × 12.5 mm; agilent technologies) at 30 °c. mobile phases consisted of deionized water with 2% (v/v) acetic acid (solvent a) and acetonitrile (solvent b). flow rate was 0.5 ml/min with a gradient elution program as follows: 0-3 min, 5% b; 3-5 min linear gradient from 5 to 10% b; 5-7 min 10% b; 7-10 min linear gradient from 10 to 15% b; 10-13 min 15% b; 13-16 min linear gradient from 15 to 20% b; 16-20 min 20% b; 20-25 min linear gradient from 20 to 25% b; 25-30 min 25% b; 30-35 min linear gradient from 25 to 30% b; 35-40 min 30% b; 40-45 min linear gradient from 30 to 95% b; 45-50 min 95% b; 50-51 min linear gradient from 95 to 5% b; and 4 min reconditioning. injection volume was 5 μl. mass spectroscopy was performed in negative mode using multiple reaction monitoring with the optimal conditions obtained by direct infusion of the standard solution. the optimum conditions were -4500 v of ion spray voltage and 50 psi for ion source gas 1 and gas 2. standard compounds used for identification and quantification were purchased from sigma-aldrich co. (chlorogenic acid, protocatechuic acid, caffeic acid, 4-hydroxybenzoic acid, ferulic acid, rutin, isoquercetin, taxifolin, and scopoletin) and wako pure chemicals co. (morin). cell culture mouse macrophage raw264.7 and human hepatoma hepg2 cells obtained from korea cell bank (seoul, korea) were cultured in dmem supplemented with 10% fbs, 2% hepes, and 1% penicillin/streptomycin. all the cells were maintained at 37 °c in a humidified atmosphere of 5% co2. the cells were harvested with trypsin and subcultured when they reached 80 % confluence. determination of cell viability and nitric oxide (no) assay 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt) assay (mosmann, 1983) was conducted to measure cytotoxicity of the af and naf on raw264.7 and hepg2 cells. raw264.7 or hepg2 cells were plated in a 96-well plate at a density of 2.0 × 104 cells/well and incubated at 37 °c for 24 h. after removing the medium, the cells were incubated with serum-free medium including the af and naf at a concentration range of 25-200 μg/ml for 24 h and then added to a serum-free medium with mtt solution (5 mg/ml in pbs). after incubating for 2 h, 100 μl dmso was added to dissolve formazan. absorbance was measured at 540 nm with the microplate reader. the level of no production was determined by measuring accumulation of nitrile (izumi, ohno, and yadomae, 1997). raw264.7 cells were plated in a 96-well plate at a density of 1.0 × 105 cells/well and incubated at 37 °c for 24 h. after the medium was removed, the cells were incubated with serum-free medium including the af and naf at a concentration range of 25-200 μg/ml for 4 h, and then added with 100 μg/l lipopolysaccharides (lps). after incubation for 20 h, the supernatant of the wells was reacted with griess reagent (0.5% (w/v) sulphanilamide with 2.5% phosphoric acid and 0.05% (w/v) naphthylethylenediamine dihydrochloride) at room temperature for 5 min. absorbance was measured at 540 nm with the microplate reader and no production (%) was expressed as the ratio of absorbance of the control and the sample. hexokinase activity of hepg2 cells hepg2 cells were plated in a 96-well plate at a density of 1.0 × 105 cells/well and incubated at 37 °c for 24 h. after removing the me138 y. lee, k.t. hwang dium, the cells were treated with the naf, af (50 μg/ml), or metformin (positive control) and then incubated with high glucose dmem (5 μg/ml of insulin) for an additional 24 h. after removing the medium again and washing with pbs, 200 μl assay buffer was added to the wells. supernatant was obtained by centrifuging (smart r17, micro refrigerated centrifuge, hanil science industrial co., incheon, korea) the lysate at 13,475 × g. hexokinase activity was determined using hexokinase assay kit and measured at 450 nm with the microplate reader. hexokinase activity (%) was calculated as the ratio of absorbance of the control and the sample for which the protein content was corrected. the protein concentration was determined using the bradford assay (bradford, 1976). α-glucosidase inhibition assay a modified bioassay method described by sigma-aldrich (kapustka, annala, and swanson, 1981) was used to determine α-glucosidase inhibitory effect. twelve g maltose was dissolved in 300 ml 50 mm sodium acetate buffer to prepare a 4% maltose solution. α-glucosidase enzyme (ec 3.2.1.20) solution was prepared at 1 unit/ml using icecold distilled water. o-dianisidine (dian) solution and peroxidase/ glucose (pgo) system-color reagent solution which are both colorimetric reagents were prepared by dissolving one dian tablet and one pgo capsule in 25 ml and 100 ml of ice-cold distilled water, respectively. acarbose was obtained from sigma-aldrich co., and acarbose solution (10 μg/ml) was used as positive control. in the first step, 20 μl of sample, distilled water (control) or acarbose was mixed with 20 μl 4% maltose solution, left in an incubator at 37 °c for 5 min. reaction started after adding 50 μl of the α-glucosidase solution, and the mixture was left in an incubator at 37 °c for 30 min. after that, 10 μl 4.2% (v/v) perchloric acid was added to terminate the reaction. in the second step, 50 μl of supernatant obtained by centrifuging (13,475 × g) the reaction tube was reacted with dian and pgo solutions, left in an incubator at 37 °c for 30 min. absorbance of the reactant solution was measured at 500 nm with the microplate reader. α-glucosidase inhibition was expressed as ic50 value (μg/ml), re presenting the concentration of a compound that causes 50% enzyme loss. statistical analysis statistical analysis was performed using spss program (spss version 20.0, chicago, il). data were presented as means ± standard deviations (sd) of three independent experiments. to compare differences among the experiment groups, the data were analyzed by analysis of variance (anova) and t-test (p<0.05). regression analysis was also calculated between nonanthocyanin phenolic compounds and α-glucosidase inhibitory effect. results and discussion total cyanides and proanthocyanidins in mulberry fruits, and yield of mulberry extract since seeds of some immature fruits may have higher amount of cyanides, the total cyanides in mulberry fruits were analyzed for the different ms. total cyanides in the fruits of the ms1-5 ranged from 4.1 to 7.7 mg/100 g dw, and those of the ms6 and 7 were not detectable (tab. 1). cyanides released from a large number of plants and fruit seeds such as apricots and peaches are lethal at a dose of 0.5-3.5 mg/kg body weight (bradbury, egan, and lynch, 1991). it is, therefore, suggested that intake of immature or mature mulberry fruits may not cause a risk of cyanide toxicity. as shown in tab. 1, proanthocyanidins in immature mulberry fruits (ms2-4, 20.3-35.3 catechin eq mg/100 g dw) were significantly (p<0.05) higher than those of mature fruits (ms6 and 7, 4.1 and 8.9 mg/100 g, respectively). proanthocyanidins function as a plant defense against predation by accumulating in different tissues and organs. proanthocyanidins, as strong antioxidants, also have potential to protect against cancers, cardiovascular disease, and alzheimer’s disease (sharma et al., 2011). the yield of the total extract rapidly increased after the ms5, changing from 26.1% (w/w, dw) at the ms1 to 76.2% at the ms7. af also increased from 3.1% at the ms4 to 4.9% at the ms7 whereas naf decreased from 6.2% at the ms3 to 2.8% at the ms7. it is assumed that as fruits matured, sugars and anthocyanins increased, affecting the yield of the extracts. nonanthocyanin phenolics in the naf nonanthocyanin phenolics in the naf analyzed by lc-ms/ms are shown in tab. 2. according to total ion chromatogram (not shown) of the naf, three high peaks observed at 6.6, 10.0, and 12.8 minutes were identified as chlorogenic acid (cga) and their isomers (353 m/z, precursor ion; 191, 85 m/z, product ion). cga is 3-caffeoylquinic acid, of which neochlorogenic acid (neo-cga, 5-caffeoylquinic acid) and cryptochlorogenic acid (crypto-cga, 4-caffeoylquinic acid) are isomers. several studies demonstrated that cga and crypto-cga (zhang et al., 2008), neo-cga and crypto-cga (oki et al., 2006), and all the 3 cga isomers (izabelle et al., 2008; lee et al., 2016) are present in mulberry fruits. phenolic acid contained in the naf was mostly cga (tab. 2); cga content dropped drastically (12,8132,251 mg/100 g dw) as the fruits matured. this observation cor responded to previous studies reporting that cga in mulberry fruits decreased during ripening (lee et al., 2016; oki et al., 2006; yang et al., 2016). neo-cga was the most abundant among the cga isomers in agreement with the results in a previous study (yang et al., 2016). tab. 1: changes in total cyanides and proanthocyanidins in mulberry fruits and yield of extracts from mulberry fruits total cyanides proanthocyanidins yield of mulberry extract (%, dw) total extract naf af ms-1 5.6±2.8 11.3±5.1 cd 26.1±1.9 e 5.2±0.5 a ms-2 5.6±1.8 21.2±8.9 b 27.9±4.3 de 5.9±0.6 a ms-3 7.4±3.0 35.3±3.5 a 32.4±0.8 d 6.2±1.6 a ms-4 7.7±2.5 20.0±5.3 bc 40.9±3.5 c 5.4±0.8 a 3.1±1.2 a ms-5 4.1±0.7 11.3±2.7 cd 51.7±1.8 b 4.7±0.7 a 3.5±1.2 a ms-6 nd 4.1±1.0 d 74.2±2.3 a 2.8±0.5 b 4.2±0.5 a ms-7 nd 8.9±2.9 d 76.2±2.2 a 2.9±0.6 b 4.9±0.6 a ms, maturity stage; eq, equivalent; dw, dry weight basis; naf, nonanthocyanin fraction; af, anthocyanin fraction; values are means ± standard deviations (n=3); values with different lower case letters within the same column are significantly different by duncan’s test at p<0.05; nd, not detected. (mg/100 g, dw) (catechin eq mg/100 g, dw) biological activities of mulberry extracts 139 two major naf flavonols, rutin and isoquercetin, decreased with ripening from 693.1 to 401.1 mg/100 g dw and from 622.8 to 160.6 mg/100 g, respectively (tab. 2). morin ranged from 171.5 to 320.4 mg/100 g. previous studies reported that morin content was 15.7 mg/100 g dw in dried mulberry fruits (yang, yang, and zheng, 2010) and in a range from 0.6 to 1.6 mg/100 g dw in six mulberry cultivars (lee et al., 2004). scopoletin, one of coumarin derivatives, decreased from 13.1 to 3.8 mg/100 g dw, whereas taxifolin increased from 14.2 to 36.4 mg/100 g during ripening. previous studies reported similar trends that main flavonols present in mulberry fruits were rutin and isoquercetin (lin and lay, 2013; yang, yang, and zheng, 2010; zhang et al., 2008), and rutin was present in higher concentrations than isoquercetin (pawloska, oleszek, and braca, 2008; yang et al., 2016). taxifolin level was 0.7 and 3.1 mg/100 g fresh weight in two cultivars of mulberry fruits (zhang et al., 2008), slightly increasing (0.01-0.04 mg/100 g) with ripening (yang et al., 2016). lin and lay (2013) also reported that scopoletin decreased from 22.1 to 5.3 mg/100 g dw with progressing maturity. it is well known that the content of phenolic compounds varies by genetic diversity and environmental conditions although the previous reports demonstrated some similar results to this study. bassoli et al. (2008) reported that chlorogenic acid with 0.5-1.0 mm inhibited glucose-6-phosphatase activity in liver cells of rats by 40% and glucose transport capability of brush-border membrane vesicles separated from small intestine of rats by 80%. in addition, chlorogenic acid and rutin were dominant nonanthocyanin phenolics separated from mulberry fruits using ethyl acetate (zhang et al., 2008). ethyl acetate fraction of mulberry fruits demonstrated higher α-glucosidase inhibitory and radical scavenging activities than ethanol, hexane, chloroform, butanol, and water-soluble fractions (wang et al., 2013). the ethyl acetate fraction could also significantly decrease fasting blood glucose and glycosylated serum protein levels and increase antioxidant enzymatic activities in streptozotocin-induced diabetic mice (wang et al., 2013). it has been reported that formation and coagulation of islet amyloid polypetide worsened symptoms of type 2 diabetes due to apoptosis of pancreatic β-cells (clark et al., 1987), and morin hydrate with 32 µm (10.2 mg/ml) was effective in breaking down islet amyloid polypetide fiber decomposition (noor, cao, and raleigh, 2011). no production in lps-stimulated raw264.7 cells conventional mtt assay was conducted to determine cytotoxicity of anthocyanin (af4-7) and nonanthocyanin (naf1-7) fractions on raw264.7 and the hepg2 cells. the viabilities of all the cells treated with different concentrations (50-400 μg) of all the fractions were higher than 80% (data not shown). fig. 1 shows the amounts of no generated when the extracts were treated in raw264.7 macrophage cells stimulated by lps. the cells treated with the naf from the fruits of all the ms except ms2 and tab. 2: phenolic compounds in nonanthocyanin fractions (naf) from mulberry fruits, measured by lc-ms/ms precursor ion product ion tr (min) phenolic compounds (mg/100 g, dw) in naf ms-1 ms-2 ms-3 ms-4 ms-5 ms-6 ms-7 total cga 12812.9 11141.6 5796.5 6400.0 5059.8 3008.7 2251.5 cga 353 191, 85 6.6 2917.1 2456.0 1284.0 1210.5 859.2 251.0 254.2 neo-cga 353 191, 85 10.0 7669.2 6672.6 3780.3 4179.5 3542.7 1650.5 1785.3 crypto-cga 353 191, 85 12.8 2226.6 2013.0 732.2 1010.0 657.9 264.4 212.0 pca 153 129, 91 5.4 14.0 14.0 11.1 17.7 21.9 78.5 57.4 caffeic acid 179 134, 89 11.6 22.3 17.3 8.0 9.5 5.8 7.8 8.2 4-hba 137 95, 65 9.0 2.4 2.2 1.4 1.7 1.4 5.0 3.4 ferulic acid 193 137, 178 17.9 1.8 1.7 1.0 1.0 1.1 2.2 3.7 rutin 609 300, 271 19.4 693.1 594.9 342.3 491.2 483.9 455.7 401.1 isoquercetin 463 300, 271 20.0 622.8 533.3 275.8 390.8 326.4 235.2 162.6 morin 301 151, 149 28.8 320.4 273.7 171.5 253.0 237.9 263.6 181.1 taxifolin 303 285, 125 19.0 14.2 13.1 13.8 27.9 36.5 44.0 36.4 scopoletin 191 176, 105 17.4 13.1 10.4 7.8 4.3 5.9 4.8 3.8 ms, maturity stage; dw, dry weight basis; cga, chlorogenic acid; pca, protocatechuic acid; 4-hba, 4-hydroxybenzoic acid; tr, retention time of each compound in chromatogram; values are means (n=2). fig. 1: nitric oxide production of raw264.7 cells treated with nonanthocyanin fraction (naf, maturity stage 1-7) and anthocyanin fraction (af, maturity stage 4-7). untreated control cells were set at 100. bars represent means ± standard deviations (n=3). statistical significance is based on the difference when compared with the cells without treating extracts (*p<0.05, **p<0.01, and ***p<0.001). 140 y. lee, k.t. hwang 6 had less no than those with the control at concentrations above 80 μg/ml and tended to inhibit no generation dependent on concentration. all the concentrations of the af from the fruits of the ms4 and 5 retarded production of no compared to the control, whereas the af of the ms6 only showed inhibition effect at concentrations of 50 and 100 μg/ml. qian et al. (2015) observed that nitrite generation was inhibited in raw264.7 macrophage cells treated with mulberry extract at 25-200 μg/ml. cuevas-rodriquez et al. (2010) reported that antioxidant activity and no production inhibition were significantly correlated with polyphenol contents in blackberry extracts. therefore, the abundant phenolic compounds in the mulberry extract may contribute to the inhibition of inflammation in raw264.7 macrophage cells stimulated by lps. hexokinase activity and α-glucosidase inhibition of mulberry extracts hexokinase, an enzyme involved in glucose metabolism in numerous organisms, produces hexose phosphate by phosphorylating hexoses. there are four isoforms of hexokinase, and one of them is glucokinase, which facilitates glycolysis by phosphorylating glucose and producing glucose-6-phosphate in liver cells of animals and humans. it is known that glucokinase is less active in a large number of type 2 diabetes patients (caro et al., 1995). the effect of the mulberry extract fractions (naf and af) at dif ferent maturity stages (ms1-7) on hexokinase activity is shown in fig. 2. hexokinase activity is expressed as a percentage of the untreated group (control). in the assay containing 25 mm glucose, insulin, and naf5, hexokinase activity was significantly higher than in the control (naf5 114.8% compared to control) and in the same range as the assay containing metformin (120.3% compared to control). significantly higher hexokinase activities could also be observed in several naf from immature fruits (ms2-5) when compared to af from mature fruits (ms6-7). primary side effects of currently used α-glucosidase inhibiting drugs such as acarbose include flatulence, abdominal distention, and diarrhea (bischoff, 1994). α-glucosidase is a digestive enzyme located in the brush border of the small intestine and degrades carbohydrates such as disaccharides and oligosaccharides into monosaccharides (balfour and mctavish, 1993). reduction of postprandial hyperglycemia by inhibition of α-glucosidase is essential for prevention and control of type 2 diabetes. in this study, α-glucosidase inhibitory ability of the mulberry fractions was determined and presented in fig. 3. as maturity progressed, ic50 of the naf (ms1-7) increased from 178.0 to 428.3 μg/ml, whereas that of the af (ms4-7) decreased from more than 500.0 to 276.9 μg/ml, indicating α-glucosidase inhibitory activity decreased in the naf but increased in the af. all the fractions except the naf of mature fruits (ms6 and 7) and the af of immature fruits (ms4 and 5) had a significantly (p<0.05) higher activity than acarbose (410.9 μg/ml). also, the naf (ms1-4) showed higher inhibitory effect than the af (ms4-7), suggesting that nonanthocyanin phenolics in immature mulberry fruits might have stronger hypoglycemic effect than anthocyanins in mature mulberry fruits. wang et al. (2013) reported that ethyl acetate extract of mulberry fruits showed a decreasing effect on fasting blood glucose in hyperglycemic mice and higher α-glucosidase inhibitory activity than ethanol, hexane, dichloromethane, butanol, and water extracts. regression analysis was performed with total amount of nonantho cyanin phenolic compounds (naf1-7, 27329.0-5360.7 mg/100 g dw) and ic50 value expressing α-glucosidase inhibitory effect (naf1-7, 178.0-401.3 μg/ml). log-transformed total amount of nonantho cyanin phenolic compounds significantly correlated with ic50 value (p=0.016). a negative linear relationship (r2=0.719) was shown between nonanthocyanin phenolic compounds and ic50 value, indicating that phenolic compounds including chlorogenic acid, rutin, isoquercetin, and morin, which were abundantly found in the naf, were likely to contribute to the biological activity. conclusion the present study investigated phenolic compounds and in vitro biological activities of mulberry extracts at seven different ms in order to explore if mulberry fruits could be a potential source of functional fig. 2: effect of mulberry extracts on hexokinase activity of hepg2 cells. c, control; met, metformin; naf, nonanthocyanin fraction (maturity stage 1-7); and af, anthocyanin fraction (maturity stage 4-7). bars represent means ± standard deviations (n=3). different letters (a-g) above the bars indicate significant differences by duncan’s test at p<0.05. fig. 3: effect of mulberry extracts on α-glucosidase activity. ic50 values (µg/ml) of compounds represent the concentrations causing 50% enzyme activity loss. acar, acarbose; naf, nonanthocyanin fraction (maturity stage1-7); and af, anthocyanin fraction (maturity stage4-7). ic50 of af4 and 5 are more than 500 µg/ml. bars represent means ± standard deviations (n=3). different letters (a-f) above the bars indicate significant differences by duncan’s test at p<0.05. biological activities of mulberry extracts 141 foods. as maturity progressed, naf decreased but af increased. main phenolic acid contained in the naf was cga, which decreased during ripening. furthermore, main flavonols present in the naf were rutin, isoquercetin, and morin, which also decreased during ripening. the naf and af had anti-inflammatory effects by lowering no production in raw264.7 cells. the naf of immature mulberry fruits promoted hexokinase activity in hepg2 cells and inhibited α-glucosidase, suggesting that the naf might have a hypoglycemic effect. cga, rutin, isoquercetin, and morin present in the naf might contribute to biological activities of mulberry fruits. it is suggested that immature mulberry fruits that are rich in nonanthocyanin phenolics might be a potential functional food source. conflict of interest no potential conflict of interest was reported by the authors. references balfour, j., mctavish, d., 1993: acarbose. an update of its pharmacology and therapeutic use in diabetes mellitus. drugs. 46, 1025-1054. doi: 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accepted: january 10, 2022) * corresponding author summary mesembryanthemum crystallinum l. or iceplant is an annual facul tative halophyte adapted to extreme environmental conditions, as salinity. this characteristic has special importance in the mediterranean region, where the drought conditions entail the most important problems of soil and water salinity. furthermore, there is currently a great gastronomic interest for this plant due to its soft texture, fresh and salty taste, high content of water and beneficial compounds for health. therefore, the objective of this work was to establish the optimal salt growth conditions for pot cultivation of iceplant in greenhouse. thus, the effect of different salinity levels (0, 100, 200 and 300 mm nacl) under controlled conditions was evaluated. severe salinity treatments reduced crop production. however, results showed that the 100 mm nacl treatment benefited plant growth. this treatment showed greater leaf fresh weight and area, pigment content and maintained its root and shoot biomass similar to control values. in addition, compared to control plants, salinity increased the spe cific leaf area and leaf relative water content, reduced leaf starch and k concentrations. therefore, results confirm that iceplant pot culture can be strategically perform under low salinity conditions with an enhancement or maintenance of crop yield and quality. keywords: mesembryanthemum crystallinum, salinity, growth, production. introduction it is known that soil salinity can reduce crop, horticulture and forage production in arid and semiarid regions. salt may arise naturally in the subsoil or be introduced by brackish irrigation waters. in any case, salinity is becoming more extensive as a result of land clearing and unsustainable irrigation practices and through pressures for bringing marginal land into production (munns and gilliham, 2015). hence, intensified use of halotolerant crop plants will be necessary, even in europe. in this context, commercial use of halophytes as fresh food is nowadays very limited. several facultative halophytic members of aizoa ceae are used as special crop plants since many years (herppich et al., 2008). per example, an old, but rare salad green is the common iceplant or glacier lettuce, mesembryanthemum crystallinum (vogel, 1996). this species is an annual facultative halophyte with a crassulacean acid metabolism (cam), which is induced by saline stress, among others. it is an ephemeral or pseudo-biennial prostrate white flowering herbaceous weedy plant with simple, ovate, succulent leaves and stems which are densely covered with bladder cells. these cells give the plant a glistening appearance (adams et al., 1998). it is native from south and east africa (winter, 1978) and is introduced in western australia and in the mediterranean amongs others (winter and smith, 1996). with its succulent, mellow, slightly salty tasting leaves and young shoots, this species is a delicious cool flavored salad green. in europe it is also known as a quickly cooked tender vegetable (herppich et al., 2008). on the other hand, m. crystallinum was classified as a highly functional food according to the work of agarie et al. (2009), due to high concentration of polyols that is increased under conditions of saline and water stress. the high salt tolerance and content in bioactive compounds and its excellent capacity for phytoremedia tion, makes the ice plant an important candidate for use as human food and medical care, and in the decontamination of polluted sites (loconsole et al., 2019). the effects of environmental stress factors have frequently been stu died in m. crystallinum, as salt stress (agarie et al., 2009; otsuka et al., 2021). but more studies are needed to determine the optimum conditions in pot cultivation of m. crystallinum to obtain maximum yield and market introduction, to boost the use of this wild edible plant. therefore, the objective of this work was to establish the non detrimental saline growth conditions for the cultivation of glacier lettuce in greenhouse. to this end, the effect of different irrigation salinity levels on edible leaf production and quality were evaluated. materials and methods plant material and growth conditions seeds of mesembryanthemum crystallinum l. were collected from wild plants of alicante (se spain), disinfected with 0.5% naclo during 2 h and pre-hydrated with aerated, deionised water for 22 h. then, they were germinated in vermiculite hydrated with deionised water and mantained in a growth chamber with an air temperature (t) of 24 °c day/night (d/n) and relative humidity (rh) of 70% d/n (thomas et al., 1998). light conditions of the chamber were 16 h light-8 h dark cycle with a photosynthetically active radiation of 400 μmol m-2s-1 supplied by a combination of fluorescent tubes (philips tld 36w/83 germany and silvana f36w/gro, usa). after 20 days, seedlings were transferred to a greenhouse under semi-controlled conditions of t d/n: 25/18 °c; rh d/n: 60/80% and received natural daylight (mean photosynthetic photon flux rate of 400 μmol m-2 s-1) (agarie et al., 2007). then, seedlings were transplanted to 1 l plastic pots with vermiculite watered weekly with 500 ml of hoagland solution. after 55 days of transplantation, plants were divided into 4 homogeneous groups of 6. four saline treatments were compared: control (0 mm), 100 mm, 200 mm and 300 mm of nacl. to avoid an osmotic shock, the concentration of nacl was increased gradually during the first week to reach the desired nacl concentration and maintained for one additional week. after 15 days of salinity conditions, plants were harvested and the different determinations were performed. at the end of the experiment, the elec trical conductivity (ec) of the substrate from non-saline container and the pots cultivated under 100, 200 and 300 mm of nacl was 1.80, 13.67, 19.08, 25.01 ds m-1, respectively. growth parameters plant dry weight (dw) was determined after drying fresh matter at 80 °c in an oven until constant weight. in addition, shoot and root length were measured. leaf area was measured by the app “easy 18 m.c. rodríguez-hernández, i. garmendia leaf area free” (easlon and bloom, 2014) and specific leaf area (sla) was calculated as the ratio of the leaf area and the dry weight of leaves. water status the leaf relative water content (rwc) was calculated according to weatherley’s method (1950), using the following formula rwc (%): (fw-dw) / (tw-dw) × 100, being fw: fresh weight, tw: turgid weight, and dw: dry weight of the tissue, respectively. foliar succulence was measured according to atzori et al. (2017) by the ratio of the fw of the leaves and the foliar area. midday water potential (ψ) of the root was measured by a scholander pressure chamber (skpm 1450/40, skye instruments ltd., united kingdom). all the determinations were performed in expanded young leaves collected at noon. biochemical analysis these analyses were performed on the youngest full-mature leaves harvested at midday, frozen in liquid nitrogen and stored at -20 °c for later quantifications. the concentration of foliar photosynthetic pigments was determined according to sesták et al. (1971). the samples (20 mg fw) were included in 5 ml of 96% ethanol at 80 °c for 10 minutes to extract the pigments. the absorbance of the extracts was spectrophotometrically measured and the equations reported by lichtenthaler (1987) were used to calculate the concentration of chlorophylls and carotenoids. starch, total soluble sugars (tss) and proline in leaves were quantified in potassium phosphate buffer (kpb) (50 mm, ph = 7.5) extracts of fresh tissue (0.1 g). these extracts were filtered through four layers of cheesecloth and centrifuged at 28710 g for 15 min at 4 °c. the pellet was used for starch determinations (jarvis and walker 1993). the supernatant was collected and stored at 4 °c for tss and proline determinations. total soluble sugars were analysed spectrophotometrically with the anthrone reagent (yemm and willis, 1954). free proline was estimated by spectrophotometric analysis at 515 nm of the ninhydrine reaction (irigoyen et al., 1992). mineral analysis leaf samples (0.5 g dw) were dry-ashed and dissolved in hcl according to duque (1971). magnesium, potassium, phosphorus, calcium, sodium, manganese, zinc and iron concentrations were de termined using a perkin elmer optima 4300 inductively coupled plasma optical emission spectroscopy (icp-oes) (perkin elmer, usa). the operating parameters of the icp-oes were: radio frequency power 1300 w, nebulizer flow 0.85 l min-1, nebulizer pressure 30 psi, auxiliary gas flow 0.2 l min-1, sample introduction 1 ml min-1 and three replicates per sample. total carbon and nitrogen were quantified after combustion (950 °c) of leaf dw with pure oxygen y an elemental analyzer provided with a thermal conductivity detector (truspec cn, leco, usa). statistics the results were analyzed with one-way analysis of variance (anova) by the statistical program spss v.26 (ibm corp., usa). the means ± standard deviation (sd) were calculated and, when the f ratio was significant (p<0.05), least significant differences were evaluated by the duncan test. significance levels were set at 5%. results growth parameters showed significant differences regarding to the salt treatment (tab. 1). plants grown with 100 mm of nacl maintained high shoot and root biomass, if we compare with the rest of saline treatments. in addition, iceplant subjected to 100 mm of nacl enhanced significantly leaf area (tab. 1) and leaf fresh weight (fig. 1). values of sla also increased in plants cultivated with salinity conditions comparing with control ones (tab. 1). the results of shoot height and root length showed that there were no differences between salinity levels. with reference to leaf water status, plants grown under salinity over 100 mm nacl had greater rwc than control plants (tab. 2). however, the highest leaf succulence was observed in control plants. in relation to root water potential, this parameter did not show a clear trend, with higher values in 100 and 300 mm nacl treatments in comparison with controls. biochemical analysis (tab. 3) performed on the leaves of iceplant displayed differences between treatments. in relation to the photosynthetic pigments, plants cultivated with 100 mm nacl increased levels of both chlorophylls and carotenoids concentration. more over, although amounts of starch declined in leaves of iceplant due to salt stress, tss concentration only reduced in treatments of 100 and 300 mm of nacl. levels of proline increased in plants subjected to saline conditions over 100 mm. tab. 1: growth parameters in iceplant subjected to different salt conditions. treatment shoot dw leaf dw root dw leaf area sla shoot height root length [nacl] mm (g plant-1) (g plant-1) (g plant-1) (cm2) (cm2 g-1) (cm) (cm) 0-control 1.95 ± 0.40 ab 1.38 ± 0.29 a 0.91 ± 0.20 ab 139.12 ± 20.00 c 96.47 ± 12.95 b 15.87 ± 2.84 a 38.20 ± 6.14 a 100 2.12 ± 0.35 a 1.45 ± 0.41 a 1.08 ± 0.32 a 243.34 ± 16.47 a 178.97 ± 53.94 a 15.48 ± 3.79 a 35.00 ± 2.24 a 200 1.60 ± 0.19 bc 1.22 ± 0.16 a 0.64 ± 0.11 b 172.63 ± 18.38 b 146.43 ± 15.72 a 16.00 ± 3.24 a 34.58 ± 8.38 a 300 1.46 ± 0.27 c 1.15 ± 0.31 a 0.66 ± 0.21 b 183.10 ± 32.22 b 163.20 ± 28.15 a 15.75 ± 3.42 a 32.25 ± 8.64 a means (n=6) ± sd were compared with duncan test. within each column, values followed by a common letter are not significantly different (p≤0.05). dw: dry weight. fig. 1: leaf fresh weight (fw) in iceplant subjected to different salt conditions. means (n=6) ± sd were compared with duncan test. bars followed by a common letter are not significantly different (p≤0.05). 9 low saline conditions benefited iceplant grown 19 the effect of salinity in leaf mineral concentration of iceplant was also assessed (tab. 4 and 5). despite leaf concentration of c and n was similar between treatments, plants grown with 300 mm nacl had significantly greater concentrations of mg and ca compared to the rest of treatments (tab. 4). on the other hand, the amount of k was lower as salt concentration increased over 100 mm and there were no differences between treatments in the amount of p. with respect to the micronutrients (tab. 5), mn concentration had not a clear trend related to the effect of salinity and there were no dif ferences between treatments in the amount of zn. iceplant that received 100 and 200 mm nacl showed the highest values of fe, and as expected, the accumulation of na was higher as the severity of salt stress increased. discussion high salinity is a critical problem in crop production that results in reduced plant growth and a significant reduction in productivity (tsukagoshi et al., 2015). mesembryanthemum crystallinum (iceplant) is an halophyte that can survive in high salinity soils and switches from c3 photosynthesis to cam under high salinity and drought stress (adams et al., 1998). we have observed that irrigation with 100 mm of nacl produced an improvement in growth, with important effect on parameters affecting edible parts of the crop, such as the leaf area or leaf fresh weight. in addition, the application of higher concentration of salt generated growth reduction. these results are in accordance with the study of winter (1973), in which the maximum growth of iceplant was obtained with a concentration of 100 mm nacl. rodríguez-hernández and garmendia (2021) showed that a moderate concentration of salinity (100 mm nacl) improved the accumulation of nutraceuticals and the production of edible leaves while high saline conditions were detrimental to the plant’s growth. atzori et al. (2017) also described the increase of leaf biomass and area in plants subjected to moderate salinity. in this regard, it should be noted that most halophytes as iceplant require saline conditions to attain optimal growth. according to flowers et al. (1986) m. crystallinum shows optimal growth within 50-250 mm nacl. thus, in this study it was confirmed that m. crystallinum irrigated with 100 mm nacl for two weeks had the highest leaf fw and leaf area. similarly, he et al. (2021) recently showed that 100 mm nacl had the highest shoot fw and largest leaf area compared with those grown at 0 and 250 mm nacl for 15 days. this tab. 2: relative water content (rwc), succulence and root water potential (ψ) in iceplant subjected to different salt conditions. treatment rwc succulence root ψ [nacl] mm (%) (g fw cm-2) (mpa) 0-control 88.59 ± 5.39 b 0.73 ± 0.15 a -0.27 ± 0.08 b 100 94.22 ± 8.59 ab 0.57 ± 0.06 b -0.10 ± 0.01 a 200 97.67 ± 2.38 a 0.53 ± 0.07 b -0.25 ± 0.05 b 300 99.12 ± 1.73 a 0.48 ± 0.04 b -0.15 ± 0.08 a means (n=6) ± sd were compared with duncan test. within each column, values followed by a common letter are not significantly different (p≤0.05). fw: fresh weight. tab. 3: leaf photosynthetic pigment concentration and leaf concentration of starch, total soluble sugars (tss) and proline in iceplant subjected to different salt conditions. treatment chlorophylls carotenoids starch tss proline [nacl] mm (mg g-1 dw) (mg g-1 dw) (mg g-1 dw) (mg g-1 dw) (mmol g-1 dw) 0-control 32.30 ± 7.49 b 5.35 ± 1.10 ab 309.01 ± 90.18 a 189.85 ± 52.17 a 43.66 ± 12.77 c 100 41.60 ± 7.20 a 6.47 ± 1.76 a 84.83 ± 34.52 b 119.70 ± 27.76 b 41.39 ± 11.72 c 200 29.79 ± 3.56 b 4.22 ± 0.64 bc 37.53 ± 13.05 bc 193.60 ± 56.79 a 101.37 ± 30.41 b 300 29.27 ± 7.94 b 3.17 ± 0.88 c 20.06 ± 9.80 c 94.40 ± 25.99 b 203.87 ± 47.56 a means (n=6) ± sd were compared with duncan test. within each column, values followed by a common letter are not significantly different (p≤0.05). dw: dry weight. tab. 4: foliar concentrations of macronutrients in iceplant subjected to different salt conditions. treatment c n mg k p ca [nacl] mm (mg g-1 dw) (mg g-1 dw) (mg g-1 dw) (mg g-1 dw) (mg g-1 dw) (mg g-1 dw) 0-control 431.17 ± 119.95 a 56.39 ± 15.78 a 18.49 ± 3.49 b 43.78 ± 6.29 a 2.70 ± 0.83 a 7.55 ± 1.11 b 100 370.33 ± 57.40 a 45.19 ± 7.31 a 17.98 ± 3.60 b 31.82 ± 4.66 b 2.55 ± 0.66 a 7.98 ± 1.78 b 200 395.40 ± 46.25 a 50.29 ± 4.97 a 22.62 ± 4.86 b 30.69 ± 5.55 b 2.35 ± 0.35 a 8.71 ± 2.24 b 300 362.67 ± 40.93 a 46.41 ± 3.55 a 28.16 ± 3.31 a 33.87 ± 6.80 b 2.45 ± 0.49 a 16.02 ± 2.62 a means (n=6) ± sd were compared with duncan test. within each column, values followed by a common letter are not significantly different (p≤0.05). dw: dry weight. tab. 5: foliar concentrations of micronutrients and sodium in iceplant subjected to different salt conditions. treatment mn zn fe na [nacl] mm (μg g-1 dw) (μg g-1 dw) (μg g-1 dw) (mg g-1 dw) 0-control 483.07 ± 66.04 a 67.06 ± 1.79 a 1666.48 ± 474.77 b 3.41 ± 1.02 c 100 320.47 ± 37.30 b 65.60 ± 5.52 a 2305.05 ± 494.23 a 24.01 ± 7.72 b 200 432.41 ± 63.97 a 70.92 ± 7.34 a 1976.63 ± 454.53 a 30.63 ± 4.38 ab 300 349.84 ± 39.88 b 70.54 ± 8.64 a 900.32 ± 375.47 c 37.18 ± 11.07 a means (n=6) ± sd were compared with duncan test. within each column, values followed by a common letter are not significantly different (p≤0.05). dw: dry weight. 20 m.c. rodríguez-hernández, i. garmendia good performance of the iceplant under low salinity conditions, indicates its potential for saline agriculture. furthermore, an increase in sla was observed in our plants sub jected to saline stress related to the enhancement of leaf area. it can be suggested that the ice plant leaf area was not reduced by salinity because another feature helped in regulating the leaf ion concentration: the epidermal bladder cells, which function as peripheral salt and water reservoirs (luttge et al., 1978; agarie et al., 2007). in addition, it is worth mentioning that the saline treatment applied lasted two weeks. in this short term effect of salinity, concentrations over 100 mm nacl resulted to be detrimental for iceplant growth under pot culture. according to agarie et al. (2007), iceplant subjected to 100 mm nacl for 3 weeks gained a higher dry matter than the same treatment for a week and even at 2 weeks. moreover, only much higher saline treatments (800 mm) showed a decrease in dry biomass as the exposure time increased to 3 weeks. therefore, a longer term treatment of salt stress in iceplant pot culture seems to led to similar results. the increase in rwc of plants irrigated with nacl can be explained by the adaptive mechanisms to the saline stress that m. crystallinum performs, since the bladder cells accumulate water, in addition to salts, to improve ionic and osmotic stress (agarie et al., 2007) and the induction of cam metabolism causes the plant to lose less water related to stomatal closure during the day. due to these adaptations it was expected that the iceplant had greater succulence in salinity treated plants. however, since succulence is calculated as the ratio of fresh leaf weight to the leaf area, the maintenance of high leaf area in saline conditions would explain the decrease in succulence according to our data. on the other hand, the expected response of the root water potential was to decrease with the increase of salinity levels, but our results differed. root ψ in controls was lower than in the 100 and 300 mm nacl treatments. because m. crystallinum is a crass plant, the manipulation and determination of ψ could have hindered using the pressure chamber, which would indicate a possible inaccuracy of these measurements (herppich and herppich, 1997; busso, 2008). in relation to photosynthetic pigments, total leaf chlorophyll concentration was higher in the plants treated with 100 mm nacl. according to herppich et al. (2008), the increase in chlorophyll can be explained by a general physiological stimulation in response to salinity. furthermore, the similar values of chlorophylls in treatments with high salinity levels in relation to the control indicated that in the halophyte m. crystallinum, salinity did not destroy chloroplasts or increase the activity of the chlorophyllase enzyme, which affects the synthesis of chlorophylls (argentel et al., 2006). the concentration of carotenoids was maintained as controls in the treatment of 100 mm nacl, with significant lower values as saline conditions increased. these results did not coincide with those obtained by atzori et al. (2017), where the concentration of carotenoids increased with the increase in salinity. however, redondo-gómez et al. (2010) in arthrocnemum macrostachyum halophilic plant, described a progressive decrease in the concentration of carotenoids in salinity treatments above 100 mm. carotenoids have a membrane stabilizing role (morgan, 2004) and are related to the vaz or xanthophyll cycle. this cycle dissipates energy in the form of heat, protects chloroplasts against photoxidation caused, for example, by saline stress. therefore, similar or increased concentration of photosynthetic pigments in plants subjected to a low salt stress of 100 mm comparing with controls, could presume an increase of the photosynthetic capacity together with greater amount of energy in thermal dissipation over photochemistry (atzori et al., 2017). salinity did not influence negativelly the leaf concentration of c, n, p and zn. as expected, salinity treatments increased the foliar concentration of na in iceplant, demonstrating that m. crystallinum acts as a sequestrant of salts, due to the presence of bladder cells (agarie et al., 2007). ca and mg increased with salinity, reaching the maximum concentration of these minerals with 300 mm nacl. atzori et al. (2017) studied the effect of different levels of salinity on mesembryanthemum and found that none of the salinity treatments negatively affected the biomass production of glacier lettuce and ca leaf concentration increased with increasing salinity. furthermore, the increase in salinity lengthened the vegetative stage, obtaining an additional month of harvest compared to the non-saline treatment. the significant increase of ca and mg concentrations may represent an interesting nutritional improvement achievable in sali nity conditions. calcium is among the main mineral elements lacking in the diet of over two-thirds of the world’s population (white and broadley, 2009). on the other hand, ca and mg are antagonists of na, so in plants not adapted to salinity a decrease in ca or mg concentrations would be expected, due to a displacement by na from the cell membrane binding sites (dodd et al., 2010). but, according to bressan et al. (1998), in iceplant, the increase in ca, as mg, is a cause of adaptation to salinity, since it has a protective effect on membranes under salinity conditions. in relation to the concentration of k, it decreased with salinity, because na competes by the binding sites in the transport system that mediates the taking of k (niu et al., 1995). our results are in the same line of the study by atzori et al. (2017), where na was higher in iceplant irrigated with salt than in the controls, as well as ca and mg, while k decreased with increasing salinity. additionaly, starch leaf concentration of glacier lettuce decreased as salinity increased. similarly, adams et al. (1992) and paramonova et al. (2004), observed the diminishing of starch content in iceplant grown with nacl, probably caused by starch degradation under stress conditions. also, the levels of tss were lower in salt treatments than in control, in accordance with atzori et al. (2017). nevertheless, plants irrigated with 200 mm presented similar values of sugars as the control treatment, while the starch concentration was lower. on the other hand, proline is part of the osmolytes that act in the osmotic adjustment (huang et al., 2000; zobayed et al., 2007). our results showed that proline increased with the amount of salinity in order to retain water in the cells (cvikrová et al., 2013), that probably allowed the increase of rwc in plants subjected to saline conditions. conclusions the analysed growth parameters showed that iceplant is a crop suitable for irrigation with saline water, wich is important in mediterranean region. thus, the implementation of the pot cultivation of m. crystallinum would represent an alternative to the current problem of salinization of arable land and irrigation water, since most traditional crops are sensitive to salinity. furthermore, in this work it has been described that iceplant irrigated with 100 mm nacl during 2 weeks increased important growth parameters as leaf area and its fresh weight. these parameters are especially important in crop yield. also, this treatment of salinity turned out to be the only one capable of maintaining both the shoot and root biomass at values similar to those of the control, while higher salinity conditions were detrimental to growth. on the other hand, aspects highly valued by the consumer in this plant, such as the relative water content and its salty flavor, were enhanced with salinity. despite the halophilic nature of iceplant, salinity treatments for two weeks over 100 mm were not very suitable for edible fresh leaf production under pot culture. acknowledgements the authors wish to thank to susana carrión and mar benavent for their help with biomass analysis and laboratory determinations. conflicts of interest no potential conflict of interest was reported by the authors. low saline conditions benefited iceplant grown 21 funding this work was financed by the 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http://dx.doi.org/10.1042/bj0570508 22 m.c. rodríguez-hernández, i. garmendia zobayed, s.m.a., afreen, f., kozai, t., 2007: phytochemical and phy siological changes in the leaves of st. john’s wort plants under a water stress condition. environ. exp. bot. 59, 109-116. doi: 10.1016/j.envexpbot.2005.10.002 orcid m.c. rodríguez https://orcid.org/0000-0001-6896-8067 idoia garmendia https://orcid.org/0000-0002-8673-6515 address of the corresponding author: m.c. rodríguez-hernández, department of earth and environmental sciences, university of alicante, alicante, po box 99, 03080 spain. e-mail: maricarmen.rodriguez@ua.es © the author(s) 2022. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1016/j.envexpbot.2005.10.002 https://orcid.org/0000-0001-6896-8067 https://orcid.org/0000-0002-8673-6515 angewandte botanik. einfluß verschiedenartiger mineraldüngung. 27 durch diese ergebnisse werden die befunde des vorhergehenden versuches hinsichtlich zunahme der größenverhältnisse und ge schmacksverbesserung bei „volldüngung“ bestätigt. die analytischen untersuchungen dieses versuches sowie von anderen, welche teilweise als ergänzung einiger oben beschriebener zu gelten haben und daher als wiederholungen aus späteren jahren anzusehen sind, waren noch nicht zum abschluß gelangt und sollen in einer weiteren veröffentlichung niedergelegt werden. die indische rundoder rangoonbohne. von e. rost (berlin). seitdem 1884 von davidson und stevenson vergiftungen durch samen von phaseolus lunatus (pois d'achery) beschrieben wurden, sind durch den genuß dieser bohne (mondbohne) zahl reiche vergiftungsfälle") beobachtet worden. 1906 untersuchte diese haricot à acide cyanhydrique eingehend guignard, nachdem 1904 dunstan und henry das blausäureabspaltende glykosid, das phaseolunatin, näher untersucht hatten. in deutschland gaben die von dammann und behrens 1906 beobachteten massenvergif tungen von pferden, rindern und schweinen anlaß zu chemischen untersuchungen dieser bohnenart. neuerdings brachten tages zeitungen die für den fachmann beunruhigende nachricht, es sollten die 50000 t bohnen der ersten lebensmittellieferung der entente an deutschland aus der rangoonbohne bestehen. die rangoonbohne is t die mondbohne, die auch als kra tok-, java-, lima-, duffin-, burma-, paigya-, kidney bohne, fève de kratok, haricot de siève, pois d'achery, amer, adam, portal oder du cap bezeichnet wird. sie ist im tropischen amerika heimisch, wird auf java, in ostindien, im östlichen binnenafrika, auf madagaskar und mauritius angebaut, ist unserer gartenbohne nahe verwandt und kommt mit verschieden *) e . rost, blausäurepflanzen. encyclopäd. jahrb. d. ges. heilkunde, xvi (1909), s. 83. 28 e. rost. die indische rundoder rangoonbohne. farbigem integument vor (schwarz, rotbis blauviolett, braun, gesprenkelt, weiß). die samen der kultivierten sorten sind meist weiß. 4 nach den untersuchungen lange s”) enthielten verschieden farbige sorten 0,12–0,24"/o, nach denen guignards”) 0,08–0,3% blausäure (cnh). die blausäure findet sich im phaseolunatin, dem dextroseäther des acetoncyanhydrins, vor, der unter dem einfluß des in den samen (aber in andren zellen) vorhandenen, auch das amygdalin zerlegénden enzyms bei gegenwart von wasser und bei erhöhter temperatur hydrolytisch gespalten wird: (ch3)2c (cn) o c6 h11o5 + enzym +wasser phaseolunatin = dextroseäther er des azetoncyanhydrins = cnh + (ch3)2co + cs h12 os blausäure azeton zucker durch geeignete anbaubedingungen kann der blausäuregehalt dieser bohne beträchtlich herabgedrückt werden. kohn-abrest fanden in einer aus madagaskar gelieferten bohne etwa 6 mg in 100 g. nach neuen untersuchungen rotheas”) sind im maximum 30 mg cnh in 100 g aufgefunden worden. in frankreich müssen rangoonbohnen mit einem ursprungszeugnis bei der einfuhr versehen sein, die nur bei einem gehalt von weniger als 20 mg cnh in 100 g bohnen gestattet wird. für die ernährung der soldaten, deren ration bohnen 100 g beträgt, waren sie unter keiner bedingung erlaubt. rothea fordert, daß die bohnen, wenn sie für den menschlichen genuß zugelassen werden sollen, eßfertig nicht mehr als 10 mg enthalten. das ist nach den untersuchungen rotheas durchaus möglich. er bestimmte nach dem verfahren von guignard und titrimetrisch die freigemachte blausäure in den bohnen, im aufweichund im kochwasser bei verwendung von 20 g bohnen (29 mg gesamt-cnh in 100 g) und 200 g wasser: bohnen, 12 stunden einbohnen, 24 stunden ein geweicht: geweicht: aufweichwasser: 4,7 mg cnh | aufweichwasser: 12,8 mg cnh bohnen: 14,8 „ „ bohnen: 14,1 „ „ *) w. lange, untersuchungen von samen der mondbohne (phaseolus lunatus l.). arb. reichs-ges.-amt, xxv (1907), s. 478. *) l. guignard, leharicot à acide cyanhydrique. bulletin des sciences pharmacol. 1906. *) rothea, l'utilisation des haricots de birmaniedans l'alimentation hu maine. annal. des falsifications 1918, nr. 121/122, nov.-dez., s. 361. die indische rundoder rangoonbohne. 29 bohnen, 12 stunden einbohnen, 24 stunden ein geweicht, 3 std. gekocht: geweicht, 3 std. gekocht: aufweichwasser: 8,9 mg cnh aufweichwasser: 11,5 mg cnh kochwasser: 15,5 „ „ kochwasser: 10,8 „ „ bohnen: 3,8 „ ** bohnen: 6,7 „ „ 28,2 mg cnh 29,0 mg cnh bohnen, nicht aufgeweicht, in kaltem wasser angesetzt, 3 stunden gekocht: .kochwasser: 13,5 mg cnh bohnen: 7,5 » , alle zahlen beziehen sich auf 100 g bohnen. hieraus ergibt sich, daß erst ein 24-stündiges einweichen die gesamte glykosidmenge spaltet, daß aber so eingeweichte bohnen beim mehrstündigen kochen praktisch blausäurefrei, d. h. entgiftet werden. gleichwohl befürwortet rothea, daß selbst so her gerichtete rangoonbohnen nicht an kranke oder an kinder unter 10 jahren in speisen verabreicht werden. zur entgiftung müssen nach rothea die rangoonbohnen folgende behandlung in der küche erfahren: aufweichen der bohnen während möglichst 24 stunden in viel wasser, waschen mit frischem wasser, ansetzen mit erneutem wasser zum kochen, 3stündiges kochen unter ersatz des verdampfenden wassers, ab gießen des kochwassers. sollten wider erwarten doch rangoonbohnen ins inland ge langen, so müßte erstlich festgestellt werden, ob der ursprüngliche blausäuregehalt der bohnen nicht höher als 30 mg in 100 g ist, ob er sich durch die angegebene küchenmäßige behandlung auf werte unter 10 mg in 100 g herabsetzen läßt und ob die be völkerung über heizmaterialien verfügt, um die rangoonbohnen 3 stunden lang zur beseitigung der freigemachten blausäure zu einem unschädlichen nahrungsmittel zu machen. ob derartige an weisungen jeder haushaltung zugestellt werden können und ob sie auch wirklich befolgt werden, dürfte zu bezweifeln sein. uc1.b3299303_page_045_cut uc1.b3299303_page_046_cut uc1.b3299303_page_047_cut journal of applied botany and food quality 92, 130 137 (2019), doi:10.5073/jabfq.2019.092.018 1department of industrial plant science and technology, college of agricultural, life and environmental sciences, chungbuk national university, republic of korea 2college of agriculture & life sciences, sari, jeju national university, republic of korea 3department of herbal crop research, national institute of horticultural and herbal science, rural development administration, republic of korea influence of ripening stages on phytochemical composition and bioavailability of ginseng berry (panax ginseng c.a. meyer) sora jin1, seung hee eom1, ju-sung kim2, ick-hyun jo3*, tae kyung hyun1* (submitted: march 8, 2019; accepted: may 3, 2019) * corresponding author summary the presence of large amounts of bioactive compounds such as saponins and flavonoids in ginseng (panax ginseng) berry suggests its potential as a functional resource for the food and medical industries, despite the fact that been considered a useless by-products of p. ginseng. in this study, we examined the variations in the antioxidant and anti-melanogenic potential of ginseng berry during the ripening process. we found that fully ripe berry extracts (go-s3) contained the highest level of antioxidant and anti-melanogenic activities. phytochemical screening suggested that alterations in polyphenol contents correlated with the variation in bioactive principles of ginseng berry during the ripening process. furthermore, results obtained by quantitative real-time pcr, western blot, tyrosinase inhibition assay and molecular docking analysis suggested that go-s3 probably inhibits tyrosinase activity by interacting with copper-coordinating histidines and second shell residues of tyrosinase, resulting in the reduction of melanin production in α-msh-stimulated b16f10 cells. taken together, these finding suggest the potential of ginseng berry as a resource for functional applications in the cosmetic industries and demonstrate that fruit ripening stages have profound effects on the pharmaceutical value of ginseng berry. keywords: panax ginseng, antioxidant activity, melanogenesis, anthocyanin, molecular docking. introduction melanin is a pigment found in the skin, hair and eyes; it plays a role of protecting the skin from ultraviolet light damage. however, overproduction of melanin leads to various pigmentation disorders, including melasma, solar lentigo and hyperpigmentation, and to autoimmune disorders such as vitiligo (briganti et al., 2003; niu and aisa, 2017). melanogenesis, the process of melanin synthesis in the melanocytes, begins with l-tyrosine and proceeds through a series of enzymatic and chemical reactions initiated by tyrosinase, an oxidase that is the rate-limiting enzyme for the two initial enzymatic steps in the conversion of tyrosine to melanin (pillaiyar et al., 2017). in addition, tyrosinase related proteins 1 and 2 (trp-1 and trp-2) also contribute to the production of melanin. furthermore, accumulation of reactive oxygen species (ros) and reactive nitrogen species (rns) promotes melanogenesis by increasing the transcription, translation and activities of tyrosinase in melanocytes and/or melanoma cells (liusmith and meyskens, 2016). this suggests a connection between oxidative stress and various pigmentation disorders and suggests that removing ros or the inhibition of oxidative reactions may have promising depigmenting properties. in fact, anti-melanogenic effects of plant-derived antioxidants have been intensively explored (liusmith and meyskens, 2016; kanlayavattanakul and lourith, 2018). the inedible portion of vegetables and some fruits range from 25 to 30% of total production (ajila et al., 2010). despite the fact that by-products or residual biomasses of medicinal plants should be used with care because of undesirable side-effects, by-products of several plants, may be inexpensive and alternative source of antioxidants. therefore, utilization of plant by-products will significantly contribute to economic savings. ginseng (panax ginseng c.a. meyer) is a perennial herb, belonging to the araliaceae family. ginseng root is the most popular medicinal herb in the world, whereas the berry has been considered a useless by-product (hyun and jang, 2017). since it has been reported that the berries contain more ginsenoside re than the roots (attele et al., 2002), recent interest on the part of the food and medical industries has focused on the potential of ginseng berry as a beneficial biomaterial. therefore, in this study, we evaluated the pharmaceutical value of ginseng berry by analyzing its antioxidant activity and antimelanogenic effects and analyzed the variation in bioactivities of ginseng berry during the ripening process. furthermore, to investigate the underlying molecular mechanism by which ginseng berry sup presses melanogenesis, we analyzed the transcription, translation and activity of tyrosinase as well as in silico molecular docking and preformed high performance liquid chromatography (hplc) analysis to determine the existence of various active compounds including ginsenosides and anthocyanins. material and methods plant materials and extraction p. ginseng cultivar (gopoong) was grown at the department of herbal crop research, rural development administration, south korea. ginseng berries were collected from 50 individual plants. berry samples were collected during the three fruit ripening stages: green fruit (unripe stage), red-turning fruit (semi-ripe stage) and dark red fruit (fully-ripe stage) (fig. 1a). the freeze-dried samples were soaked in 70% ethanol (etoh) for 24 h, placed in an ultrasonic bath and sonicated at 55 °c. after filtration, the 70% etoh extracts were evaporated using a rotary vacuum evaporator. determination of total phenolic (tpc), flavonoid (tfc), caro tenoid (tcc), saponin (tsc) and anthocyanin contents (tac) tpc and tfc were determined according to the folin-ciocalteu method and colorimetric method, respectively, as described by hyun et al. (2016). the tpc in the 70% etoh extracts was expressed in milligram gallic acid equivalent (mg gae/g extract) using the equation obtained from the standard gallic acid graph. tfc was determined as milligrams of quercetin equivalents (qe) per gram of extract (mg qe/g extract). characterization of ripening stages of ginseng berry 131 to analyze tcc in 70% ethanol extracts, the absorbance was determined at 490 nm (od480), 510 nm (od510) and 750 nm (od750), respectively, using imarktm microplate reader (bio-rad, hercules, ca, usa). tcc was calculated using following formula, as described by parsons and strickland (1963): total carotenoid (μg/mg of extract) = 7.6 × (od480 od750) 1.49 × (od510 od750) for tac analysis, 10 mg of each extract was dissolved in 1 ml of acidified methanol (1% hcl), and the absorbance was measured at 530 nm (od530) and 657 nm (od657) as described by choi et al. (2018). tac was calculated as follows: total anthocyanin = (od530-0.25 × od675)/ 10 mg of extract tsc was determined by the colorimetric method (lee et al., 2018) using diosgenin as a standard. briefly, 10 mg of each extract was dissolved in 1 ml ethyl acetate, and mixed with 500 μl a reagent (0.5 % (v/v) p-anisaldehyde in ethyl acetate) and 500 μl b reagent (50% (v/v) h2so4 in ethyl acetate). after incubation at 60 °c for 10 min, the absorbance was measured at 430 nm. tsc was calculated as diosgenin equivalent from the equation obtained from the standard diosgenin graph. determination of antioxidant activity to analyze the free radical scavenging activity of each sample, 90 μl of 0.4 mm 1,1-diphenyl-2-picryl-hydrazil (dpph) was mixed with different concentrations (62.5 to 1000 μg/ml) of each test sample. after incubation for 10 min at room temperature, absorbance was measured at 520 nm using a spectrophotometer. the rc50 (50% reduction of dpph radicals) was calculated from a graph of radical scavenging activity vs. extract concentration. the reducing power of each sample was determined according to the method of choi et al. (2018). various concentrations of extracts (100, 200, and 300 μg/ml) were mixed with 0.2 ml of 200 mm sodium phosphate buffer (ph 6.6) and 0.2 ml of 1% potassium ferricyanide. after incubation at 50 ℃ for 20 min, the mixture was mixed with 1 ml of 10% trichloroacetic acid, and centrifuged at 6,500 rpm for 10 min. then 500 μl aliquots of the supernatants were mixed with 500 μl of deionized water and 100 μl of 0.1% ferric chloride, and the absorbance was measured at 750 nm. ascorbic acid was used as a positive control. the oxygen radical antioxidant capacity (orac) assay was con ducted according to choi et al. (2017). a total of 150 μl of 0.08 μm fluorescein diluted in phosphate buffer (75 mm, ph 7.0) was mixed with 25 μl of phosphate buffer (blank), trolox standard (6.25-50 μm), or each extract in separate wells of microplate. after incubation at 37 °c for 10 min in dark, 25 μl of fresh 2,2’-azobis(isobutyramidine) dihydrochloride (0.12 g/ml) was added. the fluorescence intensity was monitored using 485 nm excitation and 525 nm emission wave lengths at 1 min intervals for 90 min in a spectramax gemini em microplate reader (molecular devices, ca, usa). the area under the curve was calculated for each sample by integrating the relative fluorescence curve. orac values were expressed as μm of trolox equivalents (μm te). cell viability assay and determination of melanin synthesis inhibitory activity the b16f10 melanoma cell line was purchased from the korea cell line bank (seoul, korea). cells were cultured in dmem medium supplemented with 10% fetal bovine serum (fbs) and 100 u/ml penicillin in an incubator containing humidified co2 (5%) at 37 °c. cultured b16f10 cells were plated at a density of 1×105 cells/ml in 96-well plates and incubated at 37 °c for 24 h. then, the cells were treated in each concentration of extracts with or without 50 nm of α-melanocyte-stimulation hormone (α-msh). after incubation for 48 h, the medium was replaced with 20 μl of 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (mtt) solution (5 mg/ml in pbs) for 4 h. the formazan crystals were dissolved in dmso, and the absorbance was measured at 520 nm using an imark microplate reader (bio-rad). for the determination of melanin synthesis inhibitory activity, cul tured b16f10 cells were plated at a density of 1×105 cells/ml in 6-well plates and incubated at 37 °c for 24 h. b16f10 cells were treated with each extract and 50 nm α-msh and incubated at 37 °c for 48 h. the cells were washed twice with ice-cold pbs and harvested at fig. 1: the expression pattern of anthocyanin biosynthesis genes at various stages of the ripening process. (a) panax ginseng berry ripening stages considered in this study. (b) the expression levels for each gene were calculated relative to their expression in stage 1. enzyme names were abbreviated as follows: chalcone synthase (chs), flavanone 3-hydroxylase (f3h), dihydroflavonol 4-reductase (dfr) and leucoanthocyanidin dioxygenase (ldox). 132 s. jin, s.h. eom, j.-s. kim, i.-h. jo, t.k. hyun 4,000 rpm for 10 min. the pellets were solubilized in 1 n naoh with 10% dimethyl sulfoxide at 65 °c for 1 h. the amount of melanin was determined by a microplate reader at 490 nm. data are expressed in terms of melanin synthesis inhibitory activity compared to the mock control tyrosinase inhibition assay the tyrosinase inhibitory activity of ginseng fruit extract was measured using tyrosinase inhibition screening kit (biovision, ca, usa) according to the manufacturer’s instructions. after incubation for 30 min at room temperature, tyrosinase activity was determined by a microplate reader at 510 nm. ascorbic acid was used as a positive control. western blot analysis after treatment with various concentrations of extract in the presence of 50 nm α-msh for 48 h, b16f10 cells were analyzed by immunoblotting. the cells were washed with ice-cold pbs and lysed in ripa buffer (50 mm tris-hcl ph 7.5, 150 mm nacl, 1% triton x-100, 0.1% sds, 0.5% sodium deoxylcholate, 1 mm edta, and 10 mm naf). the concentration of protein extracts was determined using piercetm bca protein assay kit (thermo fisher scientific, usa). then, equal amount (20 μg) of proteins was separated by sdspage and transferred to a pvdf membrane (millipore, usa). after blocking with 5% non-fat dried milk, the membranes were hybridized with specific antibodies. the signal was detected and visualized using a chemiluminescence system according to the manufacturer’s instructions. expression analysis using quantitative real-time pcr (qrtpcr) b16f10 cells were treated with α-msh and extracts as described above, and total rna was extracted using trizol reagent (molecular research center, cincinnati, usa). in addition, total rna from ginseng berries was isolated using the favorprep plant total rna purification mini kit (favogen, pingtung, taiwan) according to the manufacturer’s instructions. total rna content and purity were assessed using a denovix ds-11 spectrophotometer (denovix, ul, usa). then, 100 to 250 ng of total rna were reverse-transcribed into cdna using the revertra ace® qpcr rt master mix with qdna remover (toyobo, co., ltd, osaka, japan) in accordance with the manufacturer’s recommendations. qrt-pcr was performed using the sybr® green real-time pcr master mix (toyobo, co., ltd, osaka, japan) in the cfx96tm real-time system (bio-rad) with default parameters. the expression levels of each gene were normalized to actin, and the specific primer pairs used in qrt-pcr are listed in tab. s1. hplc analysis the composition of anthocyanins and ginsenosides in ginseng berries was determined using agilent technologies 1200 series hplc (conquer scientific, ca, usa) with cosmosil 2.5 c18 cholester (2.0id × 50 mm, for anthocyanin analysis) or kinetex 2.6u xb-c18 100a (100 × 4.6 mm, for ginsenoside analysis). for anthocyanin analysis, 10 mg of each extract was dissolved in 1 ml of acidified methanol (1% hcl). the mobile phases consisted of 1% phosphoric acid in water (solvent a) and acetonitrile (solvent b). in the case of ginsenoside analysis, 10 mg of each extract was dissolved in methanol. hplc analytical conditions are described in tab. s2. standard calibration curves of cyanidin-3-glucoside (c3g, sigmaaldrich co., st. louis, mo, usa), delphinidin-3-glucoside (d3g, coresciences, seoul, korea), petunidin-3-glucoside (p3g, sigmaaldrich co., st. louis, mo, usa) and ginseng ginsenosides mix (sigma-aldrich co., st. louis, mo, usa) were constructed. molecular docking molecular blind docking and defined docking were performed using tyrosinase (pdb code, 3nq1), cyanidin-3-glucoside (pubchem cid, 44256715), delphinidin-3-glucoside (pubchem cid, 443650), and petunidin-3-glucoside (pubchem cid, 443651). autodock4 with lamarckian genetic algorithm (morris et al., 2009) was used to calculate the docking results. molecular docking analysis was performed using the protocol described by seo and efferth (2016). for blind docking, grid box was constructed to cover whole residues, and docking parameters were set to 100 runs and 2,500,000 energy evaluations for each cycle. based on blind docking results, grid maps were created for defined docking. docking parameters of defined docking were set to 250 runs and 2,500,000 energy evaluations each time. the lowest binding energies and predicted inhibition constants were obtained from the docking log files. statistical analysis all experiments were conducted with three independent replicates, and duncan’s test was used to determine the significance of dif ferences between the groups. differences at p < 0.05 were considered significant. results and discussion variation in phytochemical contents at the three ripening stages of ginseng berry various environmental and genetic factors, including seasonal variations, growth conditions, stages of maturity and cultivars have great influence on alterations in the accumulation and composition of phytochemicals in medicinal plants (cordenunsi et al., 2002). as a first step, in order to determine the variability in bioactive principles during the fruit ripening process, we compared phytochemical contents, including total phenolic (tpc), flavonoid (tfc), carotenoid (tcc), saponin (tsc) and anthocyanin (tac) contents in the various maturity stages of ginseng berries. tpc, tfc and tac generally increased during the progression of ripening, whereas tcc was higher in extracts from unripe samples (go-s1) than in semi-ripe (go-s2) or fully ripe (go-s3) samples (tab. 1). this suggested that anthocyanins considerably contribute to color differences during the ripening process of ginseng berry. go-s2 contained the highest amount of saponin compounds (9.79 ± 2.98 μg/mg of extract). the content and composition of ginsenosides, pharmacologically active compounds found exclusively in the genus panax of the family araliaceae (pace et al., 2015), exhibit variation among tissues, cultivation ages and environmental conditions (shan et al., 2014; zhang et al., 2014). based on hplc analysis, we detected five ginsenosides: rb2, re, rf, rg1 and rg2 (tab. 2). the amounts of re, rf, rg1 and rg2 decreased during the progress of ripening, whereas the highest amount of ginsenoside rb2 was found in go-s2. it has been reported that the most abundant ginsenosides in ginseng leaf and root are re and rb1, respectively (kim et al., 2014). in the present study, rg1 appeared to be most abundant in ginseng berries (tab. 2). triterpenoid saponins are constitutively synthesized in the leaves, then transported to the roots via the phloem (eom et al., 2017). tissue-specific expression of cytochrome p450s (p450s) and udp-dependent glycosyltransferases (ugts) is crucial for structural diversity and functionalization of triterpenoid saponins (kumar et al., 2012; wang et al., 2018). this suggests that the differential distribution of ginsenosides among tissues might be due to the tissuespecific expression pattern of genes involved in the modification of triterpenoid saponins, including p450s and ugts. characterization of ripening stages of ginseng berry 133 anthocyanin accumulation occurring during fruit ripening is an important physiological process for attracting fruit-eating animals and hence dispersal of seeds (zifkin et al., 2012). in higher plants, the most common types of anthocyanidins are cyanidin, delphinidin, pelargonidin, peonidin, petunidin and malvidin; of these, cyanidin is a major anthocyanidin of berries and red vegetables (khoo et al., 2017). in the case of ginseng berry, delphinidin-3-glucoside (541.8 ± 5.4 μg/g of extract) was the most abundant anthocyanin followed by cyanidin-3-glucoside (462.8 ± 11.7 μg/g of extract) (tab. 2). briefly, biosynthesis of anthocyanin originates from the general phenylpropanoid pathway, followed by the early flavonoid pathway and the anthocyanin-specific pathway (choi et al., 2018). during fruit ripening, the accumulation of anthocyanin is positively associated with the transcription of genes involved in the early flavonoid path way and the anthocyanin-specific pathway (hyun et al., 2014). to determine the relationship between anthocyanin quantities and the expression patterns of anthocyanin biosynthesis-related genes, the expression patterns of chalcone isomerase (chi), flavanone 3-hydroxylase (f3h), dihydroflavonol reductase (dfr) and leuco anthocyanidin dioxygenase (ldox) during fruit ripening were analyzed by qrt-pcr. as shown in fig. 1b, the expression levels of chi1, chi2 and f3h, belonging to the early flavonoid pathway, and dfr and ldox, involved in the anthocyanin-specific pathway, increased during ripening process. this suggests that the accumulation of anthocyanins occurs because of increased transcriptional levels of anthocyanin-related genes in ginseng berry, similar to those described for other plants (hyun et al., 2014; fang et al., 2016). antioxidant activities of ginseng berry at various stages of maturation antioxidants are molecules that protect organisms from the damage caused by unstable molecules known as reactive oxygen species; these have been implicated in the development of aging as well as various diseases including cancer, inflammation, respiratory diseases, cardiovascular diseases, neurodegenerative disorders and digestive diseases (liu et al., 2018). synthetic antioxidants are widely used in the food industry, as well as in other industries, including cosmetics, pharmaceutical and animal nutrition. nevertheless, natural antioxidants, including vitamins, flavonoids and anthocyanins have been increasingly used because of concerns over the long-term safety of synthetic antioxidants (caleja et al., 2017). the variation of these compounds during the ripening process correlated with the antioxidant activity of the fruit (gull et al., 2012). in the case of ginseng berry, the highest dpph free radical scavenging activity was observed in go-s3 (rc50 = 658.7 μg/ml), followed by go-s2 (rc50 = 946.8 μg/ ml) (fig. 2a). to further characterize the variation of antioxidant activity of ginseng berry during the ripening process, the reducing power and orac values of ginseng berry extracts were analyzed. as shown in fig. 2b, the reducing potential of each extract was found to increase in a dose-dependent manner. as expected, go-s3 (od750 value = 0.15) showed the highest reducing power, followed by gos2 (od750 value = 0.11) and go-1 (od750 value = 0.09). similarly, go-s3 at 50 μg/ml exhibited hydrophilic oxygen radical scavenging activity (orac value) of 142.1 μm te and go-s1 displayed the lowest orac value of 112.1 μm te (fig. 2c). to analyze the correlation between the phytochemical content and antioxidant activity of ginseng berry, the pearson correlation coefficient was employed. dpph free radical scavenging activity, reducing power and orac values highly and positively correlated with tfc, tpc and tac, while tcc negatively correlated with antioxidant activities (tab. s3). polyphenol compounds, including anthocyanins, have the potential to prevent various diseases caused by oxidative stress, and possess phenolic hydroxyl groups that are capable of donating hydrogen atom electrons to free radicals and aromatic rings that stabilize and delocalize the unpaired electrons via conjugated aromatic systems (dai and mumper, 2010). this suggests that the antioxidant activity of ginseng berry extracts is due to the reaction of polyphenolic compounds with free radicals, thereby converting them into more stable products. anti-melanogenic potential of ginseng berries to investigate the effect of ginseng berry extracts on melanin production, the inhibitory effects of each extract on α-msh-induced melanin production in b16f10 cells were determined. as shown in fig. 3a, go-s3 (100 μg/ml) significantly inhibited the α-mshinduced melanin production in b16f10 cells, whereas go-s1 and gos2 exhibited no or low inhibitory effect on α-msh-induced melanin synthesis. go-s3 markedly inhibited α-msh-induced melanin synthesis in a dose-dependent manner. go-s3 at 200 μg/ml inhibited melanin production by greater than 50% over the level generated by mock control (fig. 3b). to investigate whether the inhibitory effects on α-msh-induced melanin production were mediated by tab. 1: the effect of various maturity stages of ginseng berry on phytochemical contents. sample remark total flavonoid total phenol total saponin total carotenoid total anthocyanin (mg qe/100 mg)a (mg gae/100 mg)b (μg/mg of extract) (μg/mg of extract) ((od530-0.25*od675)/mg of extract) stage 1 go-s1 0.45±0.01 11.78±3.05 7.04±1.15 0.43±0.09 0.032±0 stage 2 go-s2 0.47±0.01 16.23±2.89 9.79±2.98 0.32±0.07 0.055±0.001 stage 3 go-s3 0.62±0.01 20.28±2.34 7.05±2.52 0.31±0.01 0.200±0.004 a total flavonoid content analyzed as quercetin equivalent (qe) mg/100 mg of extract; values are the average of triplicate experiments b total phenol content analyzed as gallic acid equivalent (gae) mg/100 mg of extract; values are the average of triplicate experiments tab. 2: content of ginsenosides and anthocyanins of ginseng berry in different ripening stage. ginsenosides (μg/g of extract) anthocyanins (μg/g of extract) rb2 re rf rg1 rg2 delphinidin-3-glucoside cyanidin-3-glucoside petunidin-3-glucoside (d3g) (c3g) (pt3g) go-s1 93.36±6.89 110.68±6.09 198.55±7.11 213.67±6.56 180.55±7.95 1.1±0.1 0.5±0.2 go-s2 99.31±7.43 103.52±11 130.33±9.46 150.02±9.08 121.73±7.18 239.6±1.7 233.4±2.0 1.1±0.0 go-s3 83.83±5.1 103.58±10.4 126.24±10.3 139.55±5.4 111.64±7.2 541.8±5.4 462.8±11.7 1.1±0.0 134 s. jin, s.h. eom, j.-s. kim, i.-h. jo, t.k. hyun cell viability, the cytotoxic effect of go-s3 on b16f10 cells was de termined by mtt assay. cell viability at 200 μg/ml of go-s3 was 88.5% that of mock control; however, no statistically significant change in cell viability was observed between mock control and 200 μg/ml of go-s3 (fig. 3c), suggesting that the inhibitory effect of go-s3 on melanin production is not accounted for by cytotoxicity. melanin biosynthesis is catalyzed by three melanocyte-specific enzymes, tyrosinase, trp-1 and trp-2 that are controlled by micro phthalmia-associated transcription factor (mitf) (d’mello et al., 2016). inhibition of mitf-mediated gene expression can serve as a potential mechanism of action of anti-melanogenic agents. however, go-s3 had no effect on the α-msh-induced increases in mitfmediated gene expression (fig. 4a). furthermore, the increased levels of tyrosinase activity in response to α-msh were not suppressed by go-s3 treatment (fig. 4b), suggesting that go-s3 does not regulate the transcriptional and translational machinery related to melanocytespecific enzymes as well as mitf in b16f10 cells. melanin production is regulated by tyrosinase activity, as well as the expression of related genes (slominski et al., 2004). in particular, polyphenols such as quercetin and myricetin have been found to act as competitive inhibitors of oxidation of l-dopa by tyrosinase, thereby exhibiting whitening activity (chang, 2009). therefore, we hypothesized that the anti-melanogenic effect of go-s3 was due to direct inhibition of tyrosinase activity. to test this hypothesis, we performed a tyrosinase inhibition assay in cell-free conditions. as shown in fig. 5, the residual tyrosinase activity was 92.9%, 79.9% and 62.7% of control for 50, 100 and 200 μg/ml of go-3, respectively, suggesting that go-s3 directly inhibited l-dopa oxidation activity of tyrosinase, thereby inhibiting melanin production in α-mshstimulated b16f10 melanoma cells. in silico molecular docking analysis of anthocyanin compounds as tyrosinase inhibitors anthocyanins act as pigments in plants, but have several health benefits, including antioxidative and antimicrobial effects, antiangiogenesis, anti-cancer, anti-diabetes, anti-obesity and neuroprotective effects as well (putta et al., 2018). in the case of ginseng berry, anti-melanogenic activity highly correlated with the contents of polyphenolic compounds, including tac (tab. s3). similarly, anthocyanins and anthocyanin-enriched fractions have been reported to suppress melanin production by inhibiting the enzymatic activity of tyrosinase; because of this property, they have become important sources of functional cosmetics, foods and pharmaceuticals (kubota et al., 2014; jhan et al., 2016). molecular docking has been used as a crucial tool for analyzing the interactions between the target protein and a small molecule, and contributed to discover high-quality enzyme inhibitors that have been advanced to clinical trials (kumalo et al., 2015). to investigate the potential mechanisms of the inhibifig. 2: antioxidant activities of panax ginseng berry extracts were measured by dpph free radical scavenging (a), reducing power (b) and orac (c) assays. dpph radical scavenging activity was calculated as rc50. orac values of each organ extract are expressed as μmol of trolox equivalents per g dry weight. bars represent the mean ± s.e. of three independent experiments. different letters in each column represent significant differences between the ripening stages (p < 0.05). fig. 3: anti-melanogenic activity of panax ginseng berry extracts. (a) effect of panax ginseng berry extracts on melanin production was analyzed in α-msh-stimulated b16f10 cells. (b) dose-dependent anti-melanogenic effects of the extract obtained from fully-ripe berries of panax ginseng (go-s3) in α-msh-stimulated b16f10 cells. (c) effect of go-s3 on cell viability of b16f10 cells. values are the mean ± s.e. of triplicate experiments. bars in the same subfigure with the same lowercase letter are not significantly different (p < 0.05). characterization of ripening stages of ginseng berry 135 tion of tyrosinase activity by anthocyanins, we analyzed the interaction sites and patterns between anthocyanins and tyrosinase using the structure-based molecular docking approach. we first used blind molecular docking to search for the binding site of anthocyanins to tyrosinase (tab. s4). then, the binding energy of d3g, c3g, and pt3g to tyrosinase was predicted by defined molecular docking with a grid laid around tyrosinase residues found by blind docking. c3g (-5.8±0.01 kcal/mol) had higher binding affinity to tyrosinase than did pt3g (-5.68±0.015 kcal/mol) or d3g (-5.23±0.15 kcal/mol) (tab. 3). tyrosinase (pdb code: 3nq1) from bacillus megaterium has a conserved active site containing six copper-coordinating his tidines (his 42, his 60, his 69, his 204, his 208 and his 231) and second shell residues (met 61, met 184, phe 197 and asn 205) involved in coppe uptake and enzyme activity (kanteev et al., 2013). kojic acid (-5.5 kcal/mol), the most commonly used skin-whitening agent, interacts with phe 197, pro 201, asn 205 and arg 209 residues (the entrance to the active site) of b. megaterium tyrosinase (deri et al., 2016). in the case of anthocyanins, d3g, c3g and pt3g exhi bited hydrogen bond interactions with his 42 and hydrophobic interactions with his 60 and his 208 residues in active site and hydrophobic interactions with phe 197 and asn 205 in the second shell residues (tab. 3). these results suggest that anthocyanin, including d3g, c3g and pt3g, directly inhibits tyrosinase activity by interacting with copper-coordinating histidines and second shell residues. conclusions we analyzed variations in antioxidant activity and anti-melanogenic potential during three ripening stages of ginseng berry. during the ripening process, antioxidant and whitening activities mediated by polyphenolic compounds such as anthocyanins increased, suggesting that the maturity of fruits has profound effects on the pharmaceutical value of ginseng berry. in silico molecular docking analysis suggested that c3g, d3g and pt3g inhibit tyrosinase activity, probably by virtue of their ability to bind at active site and second shell residues of the enzyme. nevertheless, several issues, including safety, quality and efficacy of ginseng berry, still need to be addressed. such information will be useful for further studies on ginseng berry for its applications in pharmaceutical industries and phytocosmetics. acknowledgments this study was supported by “cooperative research program for agriculture science & technology development (project no. pj01261303)” rural development administration, republic of korea. fig. 4: effect of panax ginseng berry extract (go-s3) on the levels of melanogenesis-related genes (a) and protein levels of tyrosinase (b) in α-msh-stimulated b16f10 cells. the results shown in (a) were normalized to β-actin mrna levels. (b) total cell lysates were extracted and assayed by western blotting using antibody against tyrosinase. the value for the non-stimulated cells was set to 1.0. values with the same letter are not significantly different as determined by duncan’s multiple range tests. fig. 5: effects of panax ginseng berry extract (go-s3) on tyrosinase activity. values are the average of triplicate experiments and are represented as the mean ± s.e. tab. 3: defined molecular docking of anthocyanins to tyrosinase. compounds lowest binding energy mean binding energy residues involved residues involved in pki (μm)a (kcal/mol) (kcal/mol) hydrogen bond hydrophobic interaction interaction cyanidin-3-5.8±0.01 -4.675±0.035 his 42,val 218 his 60, phe 197, his 204, asn 205, 55.92±0.95 glucoside (c3g) his 208, arg 209, met 215, gly 216, val 217, ala 221 delphinidin-3-5.23±0.15 -3.955±0.125 his 42,val 218 his 60, phe 197, asn 205, his 208, 151.685±38.01 glucoside (d3g) arg 209, met 215, gly 216, val 217, ala 221 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indicates transcriptional regulation of flavonoid metabolism and activation of abscisic acid metabolism. plant physiol. 158, 200-224. doi: 10.1104/pp.111.180950 orcid ick-hyun jo https://orcid.org/0000-0002-5772-8014 address of the corresponding author: dr. ick hyun jo, department of herbal crop research, national institute of horticultural and herbal science, rural development administration, eumseong 369-873, republic of korea e-mail: intron@korea.kr dr. tae kyung hyun, department of industrial plant science and technology, college of agricultural, life and environmental sciences, chungbuk national university, cheongju 28644, republic of korea e-mail: taekyung7708@chungbuk.ac.kr © the author(s) 2019. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1016/j.jpba.2013.10.030 http://dx.doi.org/10.1152/physrev.00044.2003 http://dx.doi.org/10.3835/plantgenome2017.11.0106 http://dx.doi.org/10.3390/molecules191117381 http://dx.doi.org/10.1104/pp.111.180950 i supplementary material supplementary material tab. s1: primer sequences for qreal-time pcr analysis. supplementary material tab. s1: primer sequences for qreal-time pcr analysis. source primer name sequence (5’-3’) mitf-f ggccaaggcagagcaactt mitf-rev gcccatggtggcaagct trp1-f gagtgacatcctgtggctca trp1-rev cgataccctgggaacacttt tyrosinase-f ataggtgcattggcttctgg tyrosinase-rev ccaacgatcccatttttctt �-actin-f cccactcctaagaggaggatg b16f10 cell �-actin-rev agggagaccaaagccttcat pgchi1-f tcaaaccttccaacctggct pgchi1-rev gagacaccatgcttgccaat pgchi2-f tcgttcacttcttccagtcca pgchi2-rev ccacccaggtaccacttctt pgf3h-f ctatcggaggcaatgggtct pgf3h-rev accaacctgatcctgaagca pgdfr-f cgacgaaaccagttggagtg pgdfr-rev gcggcaatgttggcatgata pgldox-f ttgtggccaacttgaatggg pgldox-rev ggcctcttagctccttagca pgactin-f cccgagagaaagtatagtgtatgga panax ginseng pgactin-rev tagagctcttcaacaaccacttttt tab. s2: gradient conditions for hplc analysis. analysis step time (min) solvent a (%) solvent b (%) 0 initial 95a 5b 1 0.30 95a 5b 2 6.00 80a 20b 3 8.00 5a 95b 4 10.00 95a 5b anthocyanin 5 13.00 95a 5b 0 initial 82.0c 18.0b 1 20.0 82.0c 18.0b 2 30.0 70.0c 30.0b saponin 3 50.0 50.0c 50.0b a 1% phosphoric acid in water b acetonitrile c water tab. s2: gradient conditions for hplc analysis. tab. s2: gradient conditions for hplc analysis. analysis step time (min) solvent a (%) solvent b (%) 0 initial 95a 5b 1 0.30 95a 5b 2 6.00 80a 20b 3 8.00 5a 95b 4 10.00 95a 5b anthocyanin 5 13.00 95a 5b 0 initial 82.0c 18.0b 1 20.0 82.0c 18.0b 2 30.0 70.0c 30.0b saponin 3 50.0 50.0c 50.0b a 1% phosphoric acid in water b acetonitrile c water supplementary material ii tab. s3: correlations between the biological activities and phytochemical contents of ginseng berries. tpc tfc tsc tcc tac dpph reducing power orac anti-melanogenic tpc 1 -0.913 0.032 -0.911 0.911 -0.998 0.996 0.996 -0.992 tfc 1 -0.378 -0.664 1.000 -0.886 0.992 0.875 -0.957 tsc 1 -0.441 -0.383 -0.094 -0.260 0.117 0.092 tcc 1 -0.660 0.935 -0.752 -0.943 0.853 tac 1 -0.884 0.992 0.872 -0.955 tab. s4: in silico blind molecular docking of anthocyanins to tyrosinase. compounds lowest binding energy (kcal/mol) mean binding energy (kcal/mol) number of residues involved in hydrophobic interactions pki (µm) cyanidin-3-glucoside (c3g) -5.24 -4.34 13 144.8 delphinidin-3-glucoside (d3g) -2.37 -2.37 7 18230 petunidin-3-glucoside (pt3g) -3.3 -3.3 7 3790 tab. s4: in silico blind molecular docking of anthocyanins to tyrosinase. tab. s3: correlations between the biological activities and phytochemical contents of ginseng berries. art05_oehme.indd journal of applied botany and food quality 84, 151 157 (2011) 1institute for landscape and plant ecology, plant ecology and ecotoxicology, university of hohenheim, stuttgart, germany 2institute of phytomedicine, applied entomology, university of hohenheim, stuttgart, germany response of spring crops and associated aphids to elevated atmospheric co2 concentrations v. oehme 1, p. högy 1, j. franzaring 1, c.p.w. zebitz 2, a. fangmeier 1 (received september 27, 2010) summary having evolved a parasitic relation to their host plants, aphids may serve as indicators of plant responses to environmental changes. the present rise in atmospheric co2 concentrations is expected to alter plant leaf chemistry and may thus alter host plant – aphid relations. we involved a climate chamber system and used bird cherry-oat aphid (rhopalosiphum padi l.) and green peach aphid (myzus persicae s.) and their respective host plants, spring wheat (triticum aestivum l. cv. “triso”) and oilseed rape (brassica napus cv. “campino”), to elucidate the effects of atmospheric co2 enrichment on such bitrophic systems. spring wheat grown at elevated co2 (600 ppm) generally had greater above ground biomass than plants grown at ambient co2 (400 ppm). bird cherry-oat aphid infestation resulted in reduced spring wheat above ground biomass compared to the non-infested control. relative crop growth rate (rgr) was increased by elevated co2. in our study, the relative developmental stage (rds) and intrinsic rate of increase (rm) of the aphids was only slightly and non-signifi cantly increased under elevated atmospheric co2 conditions. the response of aphid weight and rgr to elevated co2 differed, increasing by 24% and 18.2% for bird cherry-oat aphid and decreasing by 12% and 12.5% for green peach aphid, respectively. aphids reared on spring wheat at elevated co2 had a shorter lifespan, whereas the opposite effect was found for aphids reared on oilseed rape. the average number of nymphs of the two pest species showed both an increase under elevated co2. no consistent picture emerges from these fi ndings, and further investigation on host – aphid relations under changing atmospheric conditions such as co2 enrichment appear necessary. introduction atmospheric carbon dioxide (co2) concentration has increased from 290 ppm (parts per million) in 1850 to 375 ppm in 2007 (ipcc, 2007) and will continue to rise in the coming decades due to anthropogenic activities. according to current climate scenarios co2 concentration will increase up to 450-550 ppm at the middle of this century. besides indirect impacts due to climate change co2 enrichment will directly affect both plants and insects (masters et al., 1998; hughes, 2000). several effects of co2 enrichment on plants have been observed such as an increase in photosynthesis rates, leaf area, dry weight and other growth characteristics (owensby et al., 1999). many studies have shown an increase in plant growth in elevated compared to ambient co2 (norby et al., 1999; long et al., 2004; ainsworth and long, 2005). in earlier work when a doubling of atmospheric co2 was considered, cure and acock (1986) reported an increase in yield by 41% on average after assembling yield data for 10 major crops (leaf, grain, tuber and fi ber). corresponding results were obtained by amthor (2001) who estimated an increase in wheat grain mass by 31% on average based on wheat yield data from 50 publications. in more recent work involving face technology (free air carbon dioxide enrichment) at only ca. 200 ppm above ambient instead of doubling, elevated co2 increased aboveground biomass by 12% and grain yield by 10-15% in wheat (kimball et al., 2002a). for oilseed rape, only few data are available on yield and growth response to co2 enrichment. according to franzaring et al. (2008b), shoot biomass of summer oilseed rape tended to be 20% greater and seed output increased by approximately 17% under elevated co2. in this study, plant height and the dry weight of reproductive organs was also signifi cantly increased under elevated co2, indicating a speeding up of plant development. the signifi cant increase in the dry weights of senescent leaves in plant specimens from the elevated co2 treatment strongly suggests that plant phenology is also affected. it was also revealed that elevated co2 infl uences the primary and secondary metabolism of plants (penuelas and estiarte, 1998). many studies have shown changes in foliar sugars, starch and increases in concentrations of carbon based secondary structural compounds due to elevated co2 (penuelas and estiarte, 1998; stiling et al., 1999). the foliar nitrogen content in plants grown under increased co2 was often reported to be reduced by up to 15% (cotrufo, 1998; heagle et al., 2002). the rise in co2 can thus indirectly affect herbivores by biochemically altering the nutritive value of the host plants. the increase in the carbon:nitrogen ratio in host plants generally decreases the nutritive quality for some feeding guilds of pests (e.g. phloemfeeders, leaf miners, xylem-feeders, seed-eaters, whole-cell-feeders and leaf-chewers), leading to an increase in their food consumption rates in order to compensate for the reduced quality (salt et al., 1995; marks and lincoln, 1996; bezemer and jones, 1998). the increased siphoning from phloem-feeders in turn causes a massive reduction in host plant assimilates (watt et al., 1995). the change in the allocation patterns of compounds and the chemical composition of plant tissues indirectly affects the food ecology of phytophagous insects (hunter, 2001). rhodes et al. (1996) have shown that phloem-feeding aphids use amino acids for their protein metabolism, and carbohydrates for energy. the phloem of plants contains high amounts of carbohydrates (0.8-1.8 m), small amounts of amino acids (60-200 mm) and very few lipids (klingauf, 1987; dillwith et al., 1993; sandstöm and moran, 2001; wilkinson and douglas, 2003; douglas, 2006). in order to obtain the necessary amounts of amino acids required for growth, aphids thus consume considerable amounts of carbohydrates from the phloem. improved food quality of a host plant with respect to aphids expresses itself in a higher amino acid to carbohydrate ratio within the phloem (mittler and meikle, 1991). elevated co2 may change the concentrations of some individual amino acids in the phloem sap, thereby affecting the performance of aphids. a study of docherty et al. (1997) proved that reduction of total amino acid concentration in phloem sap was 31% at elevated co2. the reduction in food quality due to elevated co2 also impacts the behaviour and physiology of leaf miner insects (stiling and cornelissen, 2007). many species of herbivorous insects tend to show altered behaviour and characteristics under co2 enrichment. the consequences differ between species and include retarded growth rates, increased nymphal development times and higher mortality rates (lindroth et al., 1993; smith and jones 1998; coviella and truble, 1999; goverde and erhardt, 2003). in 152 v. oehme, p. högy, j. franzaring, c.p.w. zebitz, a. fangmeier contrast, some studies concluded that the development time of phloem-feeding insects may be reduced by 17%, and that adult weight, relative growth rate (rgr) and population size may actually increase due to elevated co2 (bezemer and jones, 1998; newman et al., 2003). in this paper, we investigated the responses of host plants to elevated co2 in order to observe the indirect effects on phloem feeding insects. the experiment was carried out with bird cherry-oat aphids (rhopalosiphum padi l.) on spring wheat (triticum aestivum l. cv. “triso”) and with green peach aphid (myzus persicae s.) on oilseed rape (brassica napus cv. “campino”). determining the effects on these sap-feeding insects is very important for agriculture. myzus persicae causes both direct (leaf curling) and indirect damage of plants (transmission of plant viruses such as lettuce mosaic virus (lmv) and cucumber virus i) (namba and sylvester, 1981). myzus persicae can achieve very high population densities on plant tissue, retarding plant growth rate and thereby causing a perceptible reduction in yield of root and foliage crops (petitt and smilowitz, 1982). rhopalosiphum padi in turn causes a signifi cant decrease in yield on cereal crops via feeding damage, resulting in a reduction of kernel amount and mass. kernel amount was reduced by 36-50% in winter wheat, 24-48% in rye, 41-60% in barley and 41-63% in winter oats. the reduction of thousand kernel weight was 33-65% in winter wheat, 13-26% in rye, 25-47% in winter barley and 43-75% in winter oats (kuroli, 2009). other researchers have conducted experiments on the indirect effects of elevated co2 on myzus persicae feeding on brassica napus (himanen et al., 2008) and on solanum dulcamara (hughes and bazzaz, 2001), but the growth parameters of aphids were not taken into account. review of literature showed that the relative growth rate of aphids may be increased under elevated co2. however, these observations were carried out with other species of aphid as aulacorthum solani (awmack et al., 1997) and sitobion avenae (chen and wu, 2006) on host plants such as vicia faba, where the relative growth rate of sitobion avenae was increased by 33% at 550 ppm co2 and by 74% at 750 ppm co2. unfortunately insect response to elevated co2 differs between host plants and aphid species (bezemer et al., 1998). it is thus necessary to observe specifi c species of aphids on specifi c host plants. for the fi rst time, in this study the development of r. padi on spring wheat and development of m. persicae on oilseed rape from the nymph to the adult stage under elevated co2 was observed, making record of the relative developmental stage, population growth rate and relative growth rate of the aphids. materials and methods cultivation of plants and experimental conditions the experiment was carried out on spring wheat (t. aestivum l. cv. “triso”) from 16 june to 13 august 2008 and on oilseed rape (brassica napus cv. “campino”) from 27 may to 17 august 2009 at the institute for landscape and plant ecology of hohenheim university, germany. a pot experiment was conducted in six controlled-environment chambers (vötsch bioline ®) with two levels of co2 (ambient, 400 ppm and elevated, 600 ppm). seeds of spring wheat and oilseed rape were sown in pots (ø 18 cm) with a mixture of substrate (fruhstorfer erde typ ld 80, industrie-erdenwerk archut, lauterbach, germany) and sand (9:1). germination took place at 22 ± 2oc, 80% relative humidity and 18:6 hour l: d photoperiod. out of the sixteen host plants in each chamber, ten were chosen for aphid infestation and six for plant analysis. plants were grown having a photoperiod of 18 h, photosynthetic photon fl ux density (ppfd) of approximately 520 µmol m-1s-1, a day/night temperature of 22/12oc, irrigated daily with 50 ml water and fertilized weekly using 50 ml of 0.3% nutrient solution (wuxal ®, aglukon gmbh). host plants and climate profi les were rotated weekly between chambers in order to ensure results were not chamber specifi c. further chamber characteristics are given in details in franzaring et al. (2008a). biomass production and plant phenology in order to determine the aboveground biomass of plants at ambient and elevated co2, spring wheat and oilseed rape were harvested at growth stages 12 and 30 (bbch code) according to zadoks et al. (1974) and weber and bleiholder (1990), respectively, dried at 105 oc to constant weight and then weighed on a balance (sartorius analytics a 120 s). subsequently, relative growth rate (rgr, hunt, 1982) of the plants was calculated using equation (1). since in any experiment start weight was similar, we did not refer to start weight as required by hunt (1982). (1) rgr = (1n w2(1n w2(1n w – 1n w1 – 1n w1 – 1n w ) / t2) / t2) / t -t1 where w1w1w is the dry weight (dw) at start of the experiment (t1), w2w2w is the fi nal dw at the end of the experiment (t2t2t ), and t2 t2 t t1 is the time (days) elapsed between the weighing. cultivation of aphids in order to infest the experimental plants with similar aged aphids, synchronized colonies of r. padi and m. persicae were established. a synchronised long-term cultivation was carried out in greenhouse at 20 ± 1°c, relative humidity 60-70%, a lighting duration of 16 h and ppfd of approximately 22.5 µmol m-2 s-1. then the synchronised adult, female apterous aphids were placed on plants, grown in climate chambers under two levels of co2 to produce progeny. petri dishes that had been converted into small plexiglass cages (ø 3.5 cm) and attached with clip on the second leaf of each plant (bbch code 12) were used for aphid rearing. after fi ve hours, female aphids were removed and fi ve newly born nymphs (l1) were allowed to develop until they reach late-nymphal instars in order to determine the relative developmental stages (rds), developmental time and preimaginal mortality. the cages nymphs were observed daily. to assess longevity of adults and reproduction, one of the fi ve aphids per cage after adult moult was put separately in a cage on a young leaf and observed until death. nymphs deposited per female were counted and removed with a paintbrush daily. excess freshly born nymphs and adult pre-reproductive aphids were weighed to determine body size and relative growth rate (rgr). determination of aphids’ growth parameters the development of r. padi and m. persicae was observed and counted daily from start of the experiments until entering the adult stage. to depict any indirect effect of elevated co2 into aphid development, the relative developmental stage (rds), implemented to show the effects if insect growth regulators (zebitz, 1984), was calculated after daily counting and subsequential removal of exuviates of nymphs using equation (2): (2) rds = ∑ (nt st st p sp s •fp•fp•f ) / nt) / nt) / n s where nt st st p sp s is the number of individuals per development stage at time t, fpfpf the multiplication factor of relevant development stage (nymphal stages 1-4, adult stage 5) and ntntn s the total number of s the total number of s individuals per cage. the intrinsic rate of increase (rm, wyatt and white, 1977) of r. padi and m. persicae were calculated from the number of offspring per female after one generation time using the following equation: (3) rm = (0.754 (ln md(0.754 (ln md(0.754 (ln m )) / d where mdmdm is the number of offspring per generation time and d is the generation time (days). crops and related aphids under elevated co2 153 in order to determine the relative growth rate (rgr, howard and dixon, 1995) of r. padi and m. persicae, weights of single adults were measured using a precision balance (sartorius analytic 4504 mp8) and calculated following equation (1). statistical analyses the effects of elevated co2 concentration on growth parameters of r. padi and m. persicae (e.g. nymphs and adult weight as well as the relative growth rate of aphids) were tested using analysis of variance (anova, visual-xsel® 9.0/ doe & weibull). the combined effect of co2 elevation and aphids on plant above ground biomass and relative growth rate were analysed by anova with co2 treatment and aphid infestation as independent variables. treatment means were compared by means of lsd-test. comparison of relative development stages of aphids was done applicating the kruskal-wallis-test. as the fecundity was not normally distributed, treatments were analysed using the non-parametric mann-whitney u-test. the suitable statistical test methods were chosen according to köhler et al. (2002). results plant biomass and phenology in 2008, the phenology of spring wheat was determined from leaf development (9 das, days after sowing) until stem elongation (57 das). the results suggest that plant development was not signifi cantly altered due to elevated co2 during these developmental stages (data not shown). spring wheat grown under elevated co2 signifi cantly increased above ground biomass by 41% as biomass was 7.25 ± 0.24 g dw at 400 ppm and 10.19 ± 0.058 g dw at 600 ppm when the plants were not infested with aphids (tab. 1). this co2-induced increase was even higher (+ 48%) in spring wheat infested with r. padi, above ground biomass being 6.25 ± 0.071 g dw at 400 ppm and 9.27 ± 0.259 g dw at 600 ppm. as expected, the infestation by r. padi impacted plant above ground biomass negatively, reducing it by 14% at 400 ppm co2 and by 9.1% at 600 ppm co2. however, no statistically signifi cant interactions between co2 enrichment and aphid infestation on wheat above ground biomass were detected. the relative growth rate (rgr) of t. aestivum was signifi cantly increased due to elevated co2 (on average by 19%) and signifi cantly reduced when the plants were infested with aphids (on average by 6.1%). there was a slightly higher depression of wheat rgr due to aphid infestation at ambient compared to elevated co2 (7.9 vs. 4.6%), however, these co2 by aphid interactions were below statistical signifi cance. in 2009, the phenology of oilseed rape under co2 enrichments was determined during leaf development (from 12 until 78 das). plant development was not signifi cantly altered due to elevated co2 (data not shown). effects of co2 enrichment and presence of aphids on oilseed rape above ground biomass and rgr were consistently below statistical signifi cance because of large variation between replicates. correspondingly, no signifi cant interactions between co2 enrichment and aphid treatment could be detected. nevertheless, elevated co2 tended to increase rape rgr (on average by 34% across both aphid treatments) (tab. 1). effect of elevated co2 on aphid performance elevated co2 concentrations resulted in several changes of growth parameters of bird cherry-oat and green peach aphids. however, the relative developmental stage (rds) of the aphids remained almost unaffected in enhanced co2 environments (tab. 2). the comparison of average imaginal weight of r. padi and m. tab. 1: above ground biomass [g pot-1] and relative growth rate (rgr) of spring wheat and oilseed rape under ambient or elevated co2 concentration and with or without aphid colonization shortly before stem elongation stage. values represent treatment average ± standard error from three replicate climate chambers, respectively. plant species / ambient co2, elevated co2, ambient co2, elevated co2, signifi cance of treatment effects (f-test) plant trait without aphids without aphids with aphids with aphids co2 aphids co2*aphids wheat biomass 7.25 ± 0.24 a 10.19 ± 0.058 b 6.25 ± 0.071 c 9.27 ± 0.259 d < 0.001 0.001 ns wheat rgr 1.99 ± 0.033 a 2.34 ± 0.008 b 1.84 ± 0.011 c 2.23 ± 0.024 d < 0.001 < 0.001 ns rape biomass 4.66 ± 2.10 a 6.72 ± 1.35 a 5.19 ± 0.642 a 6.07 ± 0.660 a ns ns ns rape rgr 1.32 ± 0.435 a 1.94 ± 0.136 a 1.51 ± 0.003 a 1.85 ± 0.001 a ns ns ns different letters in superscript within one row indicate signifi cantly different treatment means at p < 0.05 (lsd-test), ns is not signifi cant tab. 2: relative development stages of r. padi and m. persicae from fi rst nymphal instar to apterous virgo. columns 1-9 (r. padi) or 1-10 (m. persicae) refer to days after leaving fi ve instar nymphs in the cages. rds of rhopalosiphum padi (from l1 to apterous virgo) [days] co2 treatment 1 2 3 4 5 6 7 8 9 400 ppm 1.0 1.7 2.2 2.7 3.1 3.7 4.3 4.7 5.0 600 ppm 1.0 1.8 2.1 2.6 3.1 3.7 4.4 4.8 5.0 rds of myzus persicae (from l1 to apterous virgo) [days] 1 2 3 4 5 6 7 8 9 10 400 ppm 1.0 1.1 1.7 2.1 2.5 2.9 3.2 3.9 4.3 5.0 600 ppm 1.0 1.2 1.8 2.1 2.5 3.0 3.4 4.0 4.4 5.0 154 v. oehme, p. högy, j. franzaring, c.p.w. zebitz, a. fangmeier persicae before the nymph reproduction and rgr of aphids clearly revealed a co2 effect (tab. 3). average weight of r. padi imago was 570.6 ± 15.8 µg fw at ambient co2 and 707.2 ± 34.0 µg fw at elevated co2 treatment which means a signifi cant increase by 24%. on the other hand, average weight of m. persicae imago decreased signifi cantly from 416.5 ± 17.2 µg at ambient co2 to 366.5 ± 1.1 µg at elevated co2 which corresponds to a decrease by 12% due to elevated co2. the rgr of r. padi feeding on wheat achieved 0.11 ± 0.003 at ambient co2 and 0.13 ± 0.01 at 600 ppm co2. rgr of r. padi was higher than rgr of m. persicae on oilseed rape, the latter which achieved 0.08 ± 0.00 at 400 ppm co2 and 0.07 ± 0.00 at 600 ppm co2. thus, co2 enrichment increased the rgr of r. padi by 18.2%, while it decreased the rgr of m. persicae by 12.5%. r. padi lifespan was slightly shorted under elevated co2 concentration, although this effect was not signifi cant. the lifespan was 39.0 days (ambient) and 39.3 days (elevated co2). in contrast, lifespan of m. persicae was slightly prolonged by 2.1 days. elevated co2 not also affected growth but also reproductive characteristics of aphids. the intrinsic rate of increase (rm) of both aphids was slightly but not signifi cantly higher under elevated co2. the average number of r. padi nymphs per female in plants grown under elevated co2 was increased by 6.0%, although this was not signifi cant. the respective values were 69.2 ± 8.7 nymphs in ambient and 73.3 ± 12.4 nymphs under elevated co2. the average number of m. persicae nymphs per female in plants grown under elevated co2 was increased by 3.5%. the respective values were 59.3 ± 7.8 nymphs in ambient and 61.4 ± 9.5 nymphs under elevated co2. in order to establish the frequency with which the female aphids reproduced under normal and co2 enriched conditions, the daily appearance of nymphs was recorded. during reproduction, the number of r. padi nymphs increased, peaking on day nine. afterwards, it tapered off, the last nymph produced on day 20 (fig. 1). signifi cant co2 effects were found on days 5 to 7 and on days 13 and 14. regarding m. persicae, the number of nymphs increased during the fi rst sixteen days, after which it declined, the last nymph produced on day 32 (fig. 2). a signifi cant co2 effect was found on day 21. discussion according to the current predictions, plants and insects will be infl uenced due to increasing atmospheric co2. the responses of plants and aphids to these changes in our research corresponded partially with predictions. the experiment in controlled-environment chambers was established in order to understand the positive or negative impacts of co2 enrichment on agricultural crops and phloem feeding aphids such as r. padi and m. persicae. our observations showed that the phenology of spring wheat and oilseed rape was not signifi cantly altered due to elevated co2. slafer and rawson (1997) have argued that elevated co2 has no effect on growth and leaf development in wheat. however, franzaring et al. (2008b) suggested that phenological development of oilseed rape was signifi cantly enhanced under elevated co2. slight phenology acceleration under rising co2 was also found by kimball et al., (2002b) on spring wheat. in our experiment, above ground biomass of spring wheat was increased by 41% due to elevated co2. this supports earlier fi ndings on the fertilizing effects of co2 enrichment on c3 plants (e.g. körner, 1991; taylor et al., 1994) and is well in agreement with poorter (1993) who surveyed literature (156 plant species) and found that with a doubling in atmospheric co2 plant biomass during vegetative growth was increased on tab. 3: mean imaginal weight (iw), relative growth rate (rgr), increase rate (rm-values) and mean adult longevity of r. padi and m. persicae under ambient and enhanced co2 conditions. parameters ambient co2 elevated co2 p values (anova) rhopalosiphum padi imaginal weight (iw) [µg] 1 570.6 ± 15.8 707.2 ± 34.0 0.01 relative growth rate (rgr) [µg/µg/day] 1 0.11 ± 0.003 0.13 ± 0.01 0.01 increase rate rm [d-1] 2 0.354 ± 0.01 0.358 ± 0.015 ns duration of life [days] 2 39.3 ± 3.2 39.0 ± 3.5 ns myzus persicae imaginal weight (iw) [µg] 1 416.5 ± 17.2 366.5 ± 1.1 0.001 relative growth rate (rgr) [µg/µg/day] 1 0.08 ± 0.00 0.07 ± 0.00 0.001 increase rate rm [d-1] 2 0.30 ± 0.01 0.31 ± 0.01 ns duration of life [days] 2 39.0 ± 6.5 41.1 ± 9.2 ns 1 n = 50, 2 n = 30, ns, not signifi cant fig. 1: daily average number of rhopalosiphum padi nymphs per treatment (n = 30). asterisks indicate signifi cant co2 effects according to the mann-whitney u-test (* p < 0.05, ** p < 0.01, *** p < 0.001). crops and related aphids under elevated co2 155 average by 37% for c3 crops. correspondingly, the rgr of spring wheat was increased by 17% under elevated co2 in our study. similarly, flynn et al. (2006) investigated potted plants (solanum dulcamara) in glass-topped chambers under two conditions of atmospheric co2 concentration (350/ 750 ppm) and confi rmed enhancement of rgr due to elevated co2. increase of co2 led to signifi cant gain in plant above ground biomass, while the presence of aphids reduced the above ground biomass of spring wheat in both ambient and enriched co2 environments. our results showed that infestation with r. padi caused signifi cant reductions in wheat biomass of 14% and 9.1% at 400 ppm and 600 ppm, respectively. however, no signifi cant effects were found when oilseed rape was infested with m. persicae. hughes and bazzaz (2001) proved that out of fi ve aphid species (acyrthosiphon (2001) proved that out of fi ve aphid species (acyrthosiphon (2001) proved that out of fi ve aphid species ( pisum, aphis nerii, aphis oenotherae, aulacorthum solani and myzus persicae) grown on fi ve host plants (vicia faba, asclepias syriaca, oenothera biennis, nicotiana sylvestris and solanum dulcamara) only aphis nerii had signifi cantly negative effects on the biomass of asclepias syriaca at both ambient and elevated co2. the interaction between co2 and aphid presence on above ground biomass and rgr was insignifi cant for spring wheat and oilseed rape in our study. however, hughes and bazzaz (2001) suggested that there was highly signifi cant interaction between co2 and presence of two species of aphid (myzus persicae and aphis nerii) on above ground biomass of asclepias syriaca and solanum dulcamara. regarding our fi ndings on co2 effects on aphids, r. padi showed an increase in weight of 24% and rgr of 18.2% in the high-co2 treatment. similar results were obtained by bezemer and jones (1998), supporting the theory that insects perform better when feeding on plants grown under co2 enrichment. according to awmack et al. (1997), the aphid aulacorthum solani (homoptera: aphididae) reared on bean (vicia faba) and tansy (tanacetum vulgare) also responded to elevated co2 conditions with increased growth. however, flynn et al. (2006) adduced evidence that co2 did not signifi cantly affect the weight of aphids (macrosiphum euphorbiae thomas). other studies concluded that co2 enrichment can negatively affect insect weight (johns and hughes, 2002; roth and lindroth, 1995) and rgr of leaf-miner pests, reducing rgr by 8.3% (stiling and cornelissen, 2007). in agreement, m. persicae showed decreased aphid weights by 12% and rgr by 12.5% under co2 enrichment in our study. in accordance with bale et al. (2002) the decrease in weights refl ect accelerated plant development due to global climate changes (increase of co2 or temperature), which decrease the amount of feeding time available to the aphids. we observed that the rds and rm of aphids was only slightly and nonsignifi cantly increased under rising atmospheric co2 conditions. our study showed that the fecundities of r. padi and m. persicae feeding on plants grown at elevated co2 increased by 6.0% and 3.5%, respectively. in contrast, traw et al. (1996) reported reduced fecundity of insects. additionally, williams et al. (2003) concluded that elevated co2 has no impact on fecundity of phloem feeding insects. according to lincoln et al. (1993), co2-induced alterations in phytochemical constituents important to insects can potentially alter their behaviours. in our study, the duration of aphids’ life was prolonged by an average of 2.2 days for r. padi and shortened by an average of 0.3 days for m. persicae under elevated co2 concentration. coviella and truble (1999) concluded that aphid’s lifespan is likely to be extended under elevated co2. overall, climate change will impact plants and insects. co2 enrichment can have dramatic consequences for plants due to acceleration of phenological development, changes in phytochemical, biochemical and biosynthetic processes, which in turn may alter future phytophagous insect populations, behaviour, performance and feeding habits. however, from the work published so far, no clear systematic rules on the mode of action and the direction of responses can be derived; rather, experimental results appear to depend on the particular organisms investigated and the experimental conditions applied in the respective studies. thus, further studies in this area are highly recommended. acknowledgements the authors would like to thank to walter damsohn for his excellent practical and technical assistance throughout this work. we also acknowledge dieter oehme for his comments and constructive criticism on an earlier draft of this manuscript. references ainsworth, e.a., long, s.p., 2005: what have we learned from 15 years of free-air co2 enrichment (face)? a meta-analytic review of the responses of photosynthesis, canopy properties and plant production to rising co2. new phytol. 165, 351-372. amthor, j.s., 2001: effects of atmospheric co2 concentration on wheat yield: review of results from experiments using various approaches to control co2 concentration. field crop res. 73, 1-34. awmack, c.s., harrington, r., leather, s.r., 1997: host plant effects on the performance of the aphid aulacorthum solani (homoptera: aphididae) at ambient and elevated co2. glob. change biol. 3, 545549. bale, j.s., masters, g.j., hodkinson, i.d., awmack, c., bezemer, t.m., brown, v.k., butterfield, j., buse, a., coulson, j.c., farrar, j., good, j.e.g., harrington, r., hartley, s., jones, t.h., lindroth, r.l., press, m.c., symrnioudis, i., watt, ad., whittaker, j.b., 2002: herbivory in global climate change research: direct effects of rising temperature on insect herbivores. glob. change biol. 8, 1-16. bezemer, t.m., jones, t.h., 1998: plant-insect herbivore interactions in elevated atmospheric co2: quantitative analyses and guild effects. oikos 82, 212-222. bezemer, t.m., jones, t.h., knight, k.j., 1998: long-term effects of elevated co2 and temperature on populations of the peach potato aphid myzus persicae and its parasitoid aphidium matricariae. oecol. 116, 128-135. chen, f.j., wu, g., 2006: responses of spring wheat to elevated co2 and their effects on sitobion avenae aphid growth, development and reproduction. chin. j. appl. ecol. 17, 91-96. cotrufo, m.f., briones, m.j., ineson, p., 1998: elevated co2 affects fi eld decomposition rate and palatability of tree leaf litter: importance of changes on substrate quality. soil biol. biochem. 30, 1565-1571. coviella, c.e., truble, j.t., 1999: effects of elevated atmospheric carbon fig. 2: daily average number of myzus persicae nymphs per treatment (n = 30). asterisks indicate signifi cant co2 effects according to the mannwhitney u-test (** p < 0.01). 156 v. oehme, p. högy, j. franzaring, c.p.w. zebitz, a. fangmeier dioxide on insect-plant interactions. conserv. biol. 13, 700-712. cure, j.d., acock, b., 1986: crop responses to carbon dioxide doubling. agr. forest meteorol. 38, 127-145. dillwith, j.w., neese, p.a., brigham, d.l., 1993: lipid biochemistry in aphids. in: stanley-samuelson, d.w., nelson, d.r. 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aegypti. entomol. exp. appl. 35, 11-16. address of the authors: viktoriya oehme (corresponding author), dr. petra högy, dr. jürgen franzaring and prof. dr. andreas fangmeier, institute for landscape and plant ecology, plant ecology and ecotoxicology, university of hohenheim, august-von-hartmann-straße 3, d-70599 stuttgart, germany prof. dr. claus p.w. zebitz, institute of phytomedicine, applied entomology, university of hohenheim, otto-sander-straße 5, d-70593 stuttgart, germany art06_15626.indd journal of applied botany and food quality 94, 47 52 (2021), doi:10.5073/jabfq.2021.094.006 1university of novi sad, institute of food technology, novi sad, serbia 2university of belgrade, institute of general and physical chemistry, beograd, serbia 3university of novi sad, faculty of technology, novi sad, serbia shelf-life study of osmodehydrated white cabbage packaged in modified atmosphere: a mathematical approach biljana r. cvetković1*, lato l. pezo2, danijela z. šuput3, biljana lončar3, olivera d. šimurina1, bojana filipčev1, rada c. jevtić-mučibabić1 (submitted: november 29, 2020; accepted: march 2, 2021) * corresponding author summary osmotic treatment (ot) is a process applied for drying of fruits and vegetables where the hypertonic solution is osmotic medium. white cabbage (cultivar „futoški“) shelf-life analysis was conducted after ot in three different hypertonic solutions: a mixture of commercial sucrose and nacl (s1), a mixture of s1 and molasses in the ratio 1:1 (s2) and. pure sugar beet molasses (85.4% dry matter) (s3). after the ot, cabbage samples were packed in high barrier bags in the modified atmosphere packaging (map). the composition of two mix tures of used gas (40:60/co2:n2 and 80:20/co2:n2) was imported into bags. the samples were analysed for l-ascorbic acid content, ph, acidity and a total number of microorganisms and sensorial attributes during 90 days of storage in a refrigerator at 4-8 °c in defined time intervals. during the 90-day storage in the map, microbiological analysis showed that the number of microorganisms decreased during the storage in the map. the highest retention of ascorbic acid (27.35%) was observed in ot cabbage dehydrated in pure molasses solution and 80:20/co2:n2 gas mixture after 90 days of storage. sensory analysis showed that osmodehydrated cabbage for 20 days in s1, and 45 days for ot cabbage in solutions s2 and s3 had acceptable consumable characteristics. key words: cabbage, osmotic treatment, molasses, shelf-life, storage introduction current globalization, growing industrialization and international food trade highlights food safety and product shelf-life extension perhaps as the most important issues of food and equipment manu facturers, stakeholders and consumers (jermann et al., 2015). in terest in the development of novel food solutions capable of keeping food safe and fresh with minimal thermal processing is increased (martynenko et al., 2015). from the ecological aspect, but also from the point of view of achieving the maximum profitability in the food industry, it is desirable to maximize the sustainability of processing with waste reduction. white cabbage (brassica oleracea var. capitata) belong to the brassica family, also has a positive impact on human health by reducing the risk of cardiovascular and cancer diseases (ciska et al., 2016). it is a popular grocery in local markets due to availability, affordability, and consumer preference (šamec et al., 2017). consumption of cabbage in the republic of serbia is 20 kilograms per capita, which is significantly higher than the world average (vlahovic, 2015). white cabbage has high nutritive value due to richness in active phytochemicals, such as vitamins c and e, minerals, dietary fibre, glucosinolates, phenolic acids, flavonoids and carotenoids (liu et al., 2014; podsędek, 2007; williams et al., 2013). the post-harvest stability of cabbage is relatively short, so that minimal processing techniques can be applied to preserve the fresh-like characteristics. one of these mild, non-thermal technologies is osmotic treatment (ot). osmotically treated products are classified as intermediate moisture products, also having a reduced water activity and micro biological stability. the incorporation of the osmotic solutes (carbohydrates, salts, and other solutes of special characteristics) during the osmotic processing generates novel food characteristics of osmo dehydrated food tissue including nutritional, sensory, and functio nally modified characteristics (dermesonlouoglou et al., 2019). osmotic dehydration (od) occurs due to immersion of the food in aqueous solutions of salt or sugar (hypertonic solutions), which results in the incorporation of solids (solute), and reduction of water activity (aw) in food (da costa ribeiro et al., 2016). there are many benefits of osmotic dehydration process in the food industry including improved food quality, energy efficiency, avoiding chemical treatments and stability of the product during storage, packaging, distribution and overall cost reduction (ahmed et al., 2016). usually used hypertonic solutions are concentrated sucrose solution, sodium chloride solutions or combination of these two. the suitability of sugar beet molasses as a hypertonic solution for ot has previously been demonstrated (cvetković et al., 2013; koprivica et al., 2014). sugar beet molasses has a high content of dry matter (over 80%), high content of minerals, antioxidants, vitamins, and other specific bioactive compounds such as betaine and it provides the high os motic pressure in the solution which makes it an excellent medium for ot (šarić et al., 2016). although the effect of osmotic treatment in molasses on the content of mineral and polyphenols in cabbage has already been reported (cvetković et al., 2019), the shelf-life study of osmotically dehydrated cabbage is still missing. packaging in protective atmospheres, where packaging in modified atmosphere (map) has the widest application, is defined as the removal and/or replacement of the atmosphere surrounding the product prior to sealing in the packaging of highly barrier material (workneh and osthoff, 2010). storing food in a modified gaseous atmosphere can maintain quality and extend shelf life, slowing chemical and biochemical spoilage reactions and slowing (or in some cases even preventing) the growth of microorganisms (coles et al., 2003). the product is initially packaged in a mixture of gases (most commonly carbon dioxide, nitrogen and oxygen), which depends on the product, packaging material, expected shelf life and storage conditions (jayas and jeyamkondan, 2002; mcmillin, 2008). in order to maximize the effect of the map, it is desirable to reduce the pro duct storage temperature. the main aim of this study was the evaluation of the shelf-life of osmodehydrated cabbage, packaged in various modified atmospheres in the map at refrigerator temperature. 48 b.r. cvetković, l.l. pezo, d.z. šuput, b. lončar, o.d. šimurina, b. filipčev, r.c. jevtić-mučibabić material and methods plant material cabbage heads, cultivar “futoški”, late fall variety, were harvested in northern serbia (province of vojvodina) in village futog. sugar beet molasses was obtained from the sugar factory pećinci, serbia. overall dry matter content in sugar beet molasses was 85.04%. osmotic treatment (ot) cabbage leaves were cut into square shapes with dimensions of approximately 1 × 1cm. solution s1 was a mixture of sucrose and nacl in the relation of 1.200/350 g/l of distilled water. the second osmotic solution (s2) was a mixture of s1 and molasses in the same quantities with 70% dry matter. sugar beet molasses was used as a third osmotic solution (s3). the cabbage leaves were put in a glass jar with osmotic solutions with a material/solution ratio of 1:5 (w/w). the experiments were conducted at the temperature of 20 °c, under atmospheric pressure, during 5 h. the cabbage samples were stirred for the purpose of solution homogenization on every 15 min. after the osmotic dehydration process, the cabbage samples were washed and gently blotted to remove excessive water. sterilized water was used for washing samples. from the obtained data, water loss (wl) was determined according to the following equation (ganjloo et al., 2011): (1) where mi and mf are the initial and final weight (g) of the samples, respectively; zi and zf are the initial and final mass fraction of water (g water/g sample), respectively. the initial moisture content of fresh cabbage samples was 90.4%, while the final wl for s1, s2 and s3 solutions, after ot, reached the values of 0.191; 0.282 and 0.228, after 5 h of the respectively (cvetković et al., 2019). microbiological analysis microbiological profile of osmodehydrated cabbage was examined by determining a total number of microorganisms (tn) (iso 4833, 1991), yeasts and moulds (iso 21527, 2008), coagulase positive staphylococci (iso 6888-1, 1999), β-glucuronidase positive esche richia coli (iso 16654, 2001), sulphite-induced clostridium (iso 15213, 2003) and salmonella spp. (iso 6579, 2002). determination of acidity and ph the ph of the cabbage was determined by a mobile ph meter (exsticktm, extech instruments, usa), calibrated with buffers ph 4 and 7 before measuring. acidity was determinate by the standard method (srps iso 750, 2003). water activity water activity (aw) of the osmotically treated samples was measured using a water activity measuring device (testo 650, germany) with an accuracy of ±0.001 at 25 ºc. soluble solids content of the osmo tic solutions was measured using an abbe refractometer (carlzeis, jenna) at 20 °c. all analytical measurements were carried out in accordance to aoac (2000) standard methods. hplc analysis of l-ascorbic acid (l-aa) for l-ascorbic acid (aa) extraction 3% metaphosphoric acid in 8% acetic acid solution was used. samples were directly injected (20 μl) in a hplc system after filtration on 0,45 μm filter. hplc system consisted of liquid chromatograph agilent technologies 1100 series, with diode array detector, in column grom_sil 120 ods-5 st (5 μm, 150 × 4 mm). as the mobile phase 100 mm ammonium acetate was used in isocratic flow at a rate of 0.4 ml/min. the temperature of the column was 37 °c, the wavelength was 254 nm. calibration was performed with external standard method. comparison of re tention times and spectra of the ascorbic acid standard were used for l-ascorbic acid identification and quantification in samples. the packaging of ot cabbage in the map after ot in three different solutions, washing and draining, 50 g cabbage samples were packed using laboratory vacuum sealer (audionelektro, swissvac) with teflonized heating areas in polyamide/ polyethylene (pa/pe) bags of 14 × 20 cm size (0.08 cm thickness, <20 g/m2 (24 h, 1 atm) water vapour permeability, and <20 cm3/m2 (24 h, 1 atm) oxygen permeability). after vacuuming the content, the chosen gas mixture was inserted before bag heat sealing. the content of the gas mixture was 40:60/co2:n2 (atmosphere 1) and 80:20/co2:n2 (atmosphere 2). storage of packed samples was con ducted in a refrigerator at 4-8 °c for 90 days for all samples. sensory analysis the methodology of the sensory analysis was carried out in ac cordance with the guidelines for the assessment of food products by methods of scale (iso 4121, 2003). samples were evaluated by 6 trained panelists. samples presented to panelists under room temperature, in individual cabins under a light bulb and in a threedigit code. before the main test, the panelists were defined and clarified particular attributes. samples were assessed for the appearancecolor, odor, taste, aftertaste and texture. the intensity of all sensory impressions was evaluated using a scale from 1 to 6 (1 = does not exist and 6 = extremely expressive). statistical analysis the response surface method (rsm) was applied in order to investigate the primary impacts of the factors (solution type, co2 concentration and storage time), which affect the responses (aa, ph, acidity, tn). the concentration of co2 (x1) (40 and 80%), storage time (x2) (0, 20, 40, 70 and 90 days) and solution type (s1, s2 and s3) were used as the independent variables, while the dependent variables were the ph (y1), acidity (y2), a total number of microorganisms tn (y3) and l-ascorbic acid aa (y4), (tab. 1). the experimental design included 2 × 5 × 3 experiments. a model was fitted to the response surface created by the experimental results, and the models were developed in the form of the second order polynomial (sop). the rsm was performed using statsoft statistical software v.10 (stat soft inc., tulsa, ok, usa). the principal component analysis (pca) has been applied effectively to classify and segregate the different samples. results and discussion tab. 1 displays the studied quality attributes of ot cabbage dehy drated in three solutions packed in different map during 90 days of storage. a slight decrease in total acidity content in all measured samples was detected during the storage. the lowest acidity content after 90 days of storage was observed in cabbage dehydrated in solution s2. some authors previously reported that the map slightly restrained the decrease intitratable acidity (ta) values compared to standard cold storage (sabir et al., 2011). decline in the acidity level can influence consumer’s acceptability so it has been associated with quality loss during postharvest storage (guillén et al., 2006; zapata i i f f i m z m z g wl m g fresh sample = osmodehydrated cabbage shelf-life 49 et al., 2008). other researchers also observed a decrease in the ta of the fruits during storage and they related this acidity reduction due to the hindrance of the ripening process (martínez-romero et al., 2003; moraga et al., 2011). raw futoški cabbage l-ascorbic acid content is 10.76 mg/100 g. ascorbic acid is easily destroyed during processing and storage due its thermo-lability (wolbang et al., 2008) and the effect of aw (corey, et al., 2011). during osmotic dehydration process loss of l-ascorbic acid in the cabbage were about 16% for ot in solution s3 and 19% in solution s1. one of the possible reasons of l-ascorbic acid decrease is chemical degradation by oxidative reactions by enzyme activity such as cytochrome oxidase, ascorbic acid oxidase and peroxidase, aerobic and non-enzymatic anaerobic reactions (martínez-romero et al., 2003; patras et al., 2009; phisut et al., 2013). the results showed the significant decrease of l-ascorbic acid content during the storage (tab. 1). the highest l-ascorbic acid retention during 90 days storage, was achieved in cabbage dehydrated in molasses (s3) and packaged in the map with 80% co2 (27.35% loss). another cause for poor l-ascorbic acid retention is the fact that water soluble compounds leach out from the cell tissues during after osmotic dehydration process (rincon and kerr, 2010). although the amount of ascorbic acid reduces during conventional osmotic dehydration, it seems that higher sugar content may positively affect the retention of ascorbic acid during storage (sharif et al., 2013). microbiological profile of osmodehydrated cabbage samples is ex pressed by a total number of microorganisms as shown in tab. 1. source of microbiological contamination could be the manual preparation of the cabbage (cutting, washing) and present normal microflora. in the beginning, from 20th to 45th day in the map, a total number (tn) of microorganisms decreased probably due to the inaccessibility of oxygen (noseda et al., 2012). at the later storage period re-growth of microorganisms was detected. at the 70th to 90th day of storage an increase in the tn of microorganisms was observed, probably anaerobic microorganisms. the development of yeasts and moulds in the samples during the storage was not observed. pathogenic microorganisms as coagulase positive staphylococci, e. coli, sulphite-induced clostridium and salmonella spp. were not detected ot cabbage samples, which is in line with claims of some other authors (pereira et al., 2004; rodrigues et al., 2006). the gained aw values for samples at the end of the investigation reached the values between 0.84 and 0.85, which ensured that the growth of bacteria is inhibited (barbosa-canovas et al., 2007; šobot et al., 2019). tab. 2 shows the anova calculation of the developed sop models. the analysis showed that the linear and quadratic terms of the solution type were the most dominant factors for ascorbic acid concentration, ph and acidity prediction using the sop model. interestingly, the co2 content in the modified atmosphere was not found as a significant factor to affect any of the studied parameters. the storage time exhibited significant effect only to microbial counts (tn), having linear and quadratic effects. these effects were statistically significant at p<0.05 level. none of these effects was significant at p<0.01, level except for the quadratic term of solution type on total microbial count (tn). the interaction of solution type and storage time was significant at p<0.05 level for the changes in ph and acidity of ot cabbage, whereas co2 content and storage time interaction was significant for acidity at p<0.05 level. the interaction between solution type and co2 content in the map were not significant for the output parameters. tab. 1: experimental data for the shelf-life study of ot cabbage packaged in different map no. map storage solution s1 solution s2 solution s3 sample co2 period aa ph ac tn aa ph ac tn aa ph ac tn content (days) (%) 1 / 0 8.79 5.36 0.590 1700 8.81 6.18 0.440 2400 8.98 6.15 0.48 2000 2 40 20 6.85 5.175 0.505 4650 6.91 6.2 0.440 7600 7.11 6.26 0.505 4750 3 40 40 5.45 5.515 0.435 1450 5.38 5.845 0.485 1400 5.53 6.33 0.490 500 4 40 70 3.59 5.33 0.450 610 3.93 5.9 0.415 1245 3.84 5.715 0.515 990 5 40 90 2.05 5.46 0.395 1250 n.d. 6.16 0.390 2150 2.3 5.715 0.495 1035 6 80 20 6.71 5.265 0.470 5000 6.78 6.25 0.415 7900 7.02 6.15 0.485 8000 7 80 40 5.55 5.425 0.410 3000 5.83 5.935 0.440 675 5.77 6.2 0.470 395 8 80 70 3.92 5.2 0.435 695 3.88 5.87 0.470 840 3.71 5.83 0.535 730 9 80 90 n.d. 5.575 0.430 2650 2.41 6.23 0.380 7250 2.11 5.81 0.565 785 *ym<100 for all samples aa – ascorbic acid (mg/100 g); ac – acidity (%); tn – a total number of microorganisms (cfu/g); n.d. – not detected tab. 2: anova analysis for ascorbic acid (aa), ph, acidity (ac) and microbial counts (tn) during the storage of ot cabbage term df aa ph ac tn sol 1 0.023 1.603* 0.018* 2.8·105 sol2 1 96.746* 0.707* 0.011* 9.7·106** co2 1 1.566 0.001 0.000 4.4·106 t 1 0.668 0.064 0.001 4.0·107* t2 1 0.034 0.056 0.000 6.8·107* sol × co2 1 0.005 0.000 0.001 3.5·104 sol × t 1 0.158 0.315* 0.006* 1.3·106 co2 × t 1 0.298 0.006 0.004* 2.3·105 error 15 6.579 0.397 0.012 3.6·107 r2 0.938 0.874 0.770 0.774 *significant at p<0.05 level, 95% confidence limit sol – osmotic solution; co2-n2 content in the map; t – storage time; aa – ascorbic acid (mg/100 g); ac – acidity (%); tn – a total number of microorganisms (cfu/g); df – degrees of freedom in order to better explain the structure of the exploratory data that would contribute to the comprehension of likenesses and dissimilarities of the ot cabbage samples during storage, pca was applied and the results are presented in fig. 1 the first two pcs explained 69.10% of the total variance in the experimental data. the projection of the factors indicated that co2 concentration, ph and tm contributed mostly to the first principal component pc1 (31.2%, 22.3% and 48,9%, respectively), while the acidity and aa 50 b.r. cvetković, l.l. pezo, d.z. šuput, b. lončar, o.d. šimurina, b. filipčev, r.c. jevtić-mučibabić concentration contributed more to the second principal component pc2 (72.4% and 21.8%, respectively). the separation between samples could be observed from the pca graph, where the highest tn content was noticed for the not-stored samples (sample 1) and the samples packed under lowest co2 concentration in the map (sample 5), regardless of the osmotic solution. the map of pca analysis showed that the first principal component described the differentiation among the samples according to tn content, while the second principal component described the variations in acidity between samples. the original cabbage (ocb, tcb) and salty taste (tsa, ats) are grouped on the right side of the graphic, while the brown-colour samples (acyb, aclybe, acg) with a taste and smell like molasses (tm, tcr, ocr, om) are grouped on the left side of the graphic. the bitter (atsb, atb) and pungent (atp) after taste samples are grouped at the upper side of the graphic, while the acid samples (atsr, atss, tsr) are grouped at the bottom of the graphic. samples with the tough (txtg) and hard (txhd) texture are grouped on the top of the graphic, while chewy texture samples (txcw) are grouped on the bottom of the graphic. pca has led to the useful classification and segregation of the cabbage samples treated with different osmotic solutions (s1, s2, s3). fig. 1: the pca biplot diagram, depicting the relationships among ot cabbage samples treated with different osmotic solutions and packaged in different map during storage. sol − osmotic solution; co2-n2 content in the map; t − storage time; aa − ascorbic acid (mg/100 g); tn − a total number of micro organisms (cfu/g) aa ph acidity tn conc. t sol 2 3 4 5 6 7 8 9 2 3 4 5 6 7 8 9 2 3 4 5 6 7 8 9 -3 -2 -1 0 1 pc 1: 39.62% -2 -1 0 1 2 p c 2 : 2 9. 48 % s1 s2 s3 acly acyb acb ocb oodrs tss tcb tm tp atsb ats atb atp txcw txhd aclyg aclybe om ocr tsa tcr tb atsr tsr atss txtg acly aclygacyb acb acg om ocr ocb oodrs tsw tss tsa tcb tcr tp tb atss atsb ats atb atp txcw txtg txhd aclybe tm atsr tsr 1f 2f 3f 1f40 1f80 2f80 3f80 1f40 1f80 2f80 3f40 3f80 1f40 1f80 2f40 2f80 3f40 3f80 -4 -2 0 2 4 6 pc 1: 34.99% -4 -3 -2 -1 0 1 2 3 4 1f 2f 3f 1f40 2f40 2f80 3f40 3f80 1f40 1f80 122f80 3f80 1f40 1f80 3f40 3f80 -4 -2 0 2 4 6 pc 1: 34.99% -3 -2 -1 0 1 2 3 pc 2 : 1 3. 65 % tsw acg 3f40 1f80 pc 3 : 9 .4 9% 2f80 2f40 2f40 2f40 3f40 s1 s2 s3 s1 s2 s3 after the process of osmotic dehydration of the cabbage samples in solution s1, the sensory panel marked the lightest colour as the most intense property. osmotically dehydrated samples in solutions s2 and s3 are described of yellow-brown colour. no significant change in colour or intensity was observed during the overall storage process in modified atmosphere packaging. cabbage dehydrated in s1 solution retained the odour of fresh cabbage, while the dominant odour of cabbage dehydrated in s2 and s3 solutions was that of molasses and caramel. during the 45-day storage period, a slightly distinctive off odour occurred with cabbage in solution s1 packed in both gas mixtures. in cabbage dehydrated in s2 solution, the taste was rated as sweet-salty and salty with equal intensity. during cabbage storage, after 20 days, bitter taste occurred in all samples dehydrated in s2 and s3 solutions. during storage, the gumminess decreased while the toughness and hardness increased in all observed samples. in order to show the sensory analysis data which could be applied for better understanding of the properties of ot cabbage samples during storage, pca results were shown in fig. 2. the first three pcs clarified 58.13% of the total variance in the experimental data. the projection of the variables in the factor plane indicated that acly, aclyg, om, ocr, tsa, tm, tcr, tp, tb and ats contributed mostly to the first principal component pc1 (5.6%; 6.5%; 6.7%; 5.6%; 8.0%; 8.5%; 7.3%; 5.0%; 5.1% and 6.5%, respectively), while tsw, atss, atsb, ats, atb, atsr, txcw and txtg contributed more to the second principal component pc2 (8.8%; 9.8%; 8.6%, 5.3%, 8.2%, 10.2%, 9.1% and 12.1%, respectively). the third principal component was mostly influenced by: acb, ocb, oodrs, tcb, atb and txhd (8.6%; 5.6%; 20.0%; 5.3%; 6.7% and 21.5%, respectively). the differentiation among samples could be realized from the pca graphic, where the yellowish samples (acly, aclyg), with conclusions the obtained model was able to successfully predict experimental results. the model is easy to implement for osmotic treatment process with desired final quality and could be effectively used for predictive fig. 2: the pca biplot diagram, depicting the sensory analysis of ot cabbage samples treated with different osmotic solutions during storage. acly-appearance-colourlight yellow, aclyg -appearancecolourlight yellow green, aclybe-appearance colour light yellow brown edges, acyb-appearance colour yellow brown, acbappearance colour brown, acg-appearance colour grey, om-odour molasses, ocr-odour caramel, ocb-odour cabbage, oodrs-off odour (rancid, sour), tsw-taste sweet, tss-taste sweet salty, tsataste salty,tcb-taste raw cabbage, tm-taste molasses, tcrtaste caramel, tp-taste pungent, tsr-taste sour, tbtaste bitter atts aftertaste sweet salty, atsbaftertaste sweet bitter, atsaftertaste salty, atb-aftertaste bitter, atsr-aftertaste sour, atpaftertaste pungent, txcw-texture chewiness, txtg-texture toughness, txhdtexture hardness osmodehydrated cabbage shelf-life 51 purposes, modelling and optimization of the osmotic treatment. solution type has a significant influence on ph and acidity, storage period on a total number of microoragisms and gas mixture content has no significant influence on analysed parameters. considering that an impressive amount and wide assortment of information were utilized in the current study to develop the appropriate numerical model, and realizing that the model ended up yielding adequate results, its practical aplication could be expected. acknowledgment these results are part of a project supported by the ministry of education, science and technological development of the republic of serbia, grant number 451-03-68/2020-14/ 200222. conflict of interest no potential conflict of interest was 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https://orcid.org/0000-0003-2994-6871 olivera šimurina https://orcid.org/0000-0002-6532-2612 bojana filipčev https://orcid.org/0000-0001-5878-6227 rada jevtić mučibabić https://orcid.org/0000-0002-8063-2045 address of the corresponding author: biljana cvetković, university of novi sad, institute of food technology, bulevar cara lazara 1, 21000 novi sad, serbia e-mail: biljana.cvetkovic@fins.uns.ac.rs © the author(s) 2021. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1016/j.lwt.2005.07.023 http://dx.doi.org/10.1111/j.1745-4549.2009.00404.x http://dx.doi.org/10.1111/j.1745-4549.2006.00089.x http://dx.doi.org/10.1007/s13197-011-0237-z http://dx.doi.org/10.1007/s11101-016-9454-4 http://dx.doi.org/10.5937/ffr1602135%c5%a0 http://dx.doi.org/10.3311/ppch.13268 http://dx.doi.org/10.1016/j.foodres.2013.03.015 http://dx.doi.org/10.1016/j.ifset.2007.08.001 http://dx.doi.org/10.5897/ajb2010.000-3357 http://dx.doi.org/10.1002/jsfa.3220 art03_erekul.indd journal of applied botany and food quality 85, 17 22 (2012) 1department of crop science, adnan menderes university, aydin, turkey 2department of crop and animal science, humboldt-universität zu berlin, berlin, germany effect of sulphur and nitrogen fertilization on bread-making quality of wheat (triticum aestivum l.) varieties under mediterranean climate conditions o. erekul1, k.-p. götz2, y.o. koca1 (received november 18, 2011) summary turkey has applied for eu-membership, but still faces problems of lacking quality standards for bread wheat. studies on the infl uence of s-fertilization on grain yield and bread-making quality of wheat (triticum aestivum l.) in the region haven’t been carried out until today. this research was conducted for two growing seasons (20082009 and 2009-2010) at adnan menderes university research and experimental farm located in the western turkey (aegean region) at 37º 44’ n 27º 44’ e in order to determine the effects of nitrogen (0, 70, 140, 210 kg ha-1) supplemented with sulphur (0 or 40 kg ha-1) with respect to yield and bread-making quality of the varieties golia and sagittario, grown primarily in western turkey. s-fertilization had positive effects on grain yield and some quality parameters under mediterranean conditions; however, signifi cant differencess were rather rare. particularly the gluten-index and the sedimentation value promoted by s fertilization were among the tested parameters. therefore, s-fertilization in improving bread-making quality of wheat in the region should not be disregarded. grain yield and quality could be promoted simultaneously with increasing n-doses. introduction the area cultivated with wheat (triticum spp. l.) has reduced in the last years in turkey and today covers approximately 8.0 million ha (fao, 2010). about 75% of the wheat produced in turkey is bread wheat type. in the aegean region of western turkey, the wheat yield is mostly higher than the other wheat-cultivated areas (dinc and erekul, 2010). despite the large extent of wheat cultivation in turkey the locally produced wheat hardly achieved suffi cient yield and barely met the standards of food processing industry, thus high quality wheat has to be imported (erekul et al., 2009). yield and bread-making quality is infl uenced by genotype, growth conditions and fertilization regime (johansson et al., 2004). responses of wheat to nitrogen application alone have been well recognized for many varieties under different climates (erekul et al., 2009; tea et al., 2007; guarda et al., 2004; johanson et al., 2004). besides nitrogen, the essential role of sulphur in plant growth and development is well known (zhao et al., 1999a; steinfurth et al., 2012). deficiency of sulphur can decrease grain yield but has a stronger infl uence on bread-making quality (fullington et al., 1987; shahsavani and gholami, 2008), due to the essential role of disulphide bonds in maintaining gluten functionality (zhao et al, 1999b). function of n and s are closely interrelated and an optimal n/s ratio improves bread-making quality. interactions of nitrogen and sulphur fertilization have not been widely investigated (zhao et al., 1999c; tea et al., 2007). although wheat require only a modest amount of sulphur (about 15-30 kg/ha) for optimum growth and grain yield (zhao et al., 1999a; zhao et al., 1999b), defi ciency of sulphur has become increasingly widespread in europe in recent years (schnug, 1991; zhao et al., 1999c). reports on the response of cereals to sulphur nutrition, especially in the mediterranean climate, are not numerous. this research was conducted for two growing seasons, (2008-2009 and 2009-2010) fi rstly to determine to our knowledge in aegean region about the effects of different doses of nitrogen combined with sulphur fertilizing under fi eld conditions with respect to yield and breadmaking quality of two wheat varieties, widely grown in western parts of turkey. materials and methods trials were performed in 2008-2009 and 2009-2010 at adnan menderes university research and experimental farm located in western turkey at 37º 44’ n 27º 44’ e and at 65 m above sea level. mean annual precipitation and temperature (1970-2005) was 648.9 mm and 17.6 ºc, respectively. average monthly temperatures were lower in 2008-2009 than in 2009-2010 with 14.8 ºc and 15.4 ºc, resp. the soil texture is classifi ed as a sandy loam and the soil type is a calcaric fluvisol (fao-classifi cation). soil samples were taken to determine the amount of extractable sulphate-s (tab. 1), which was extracted with 0.016 m kh2po4 and determined with ion chromatography (zhao and grath, 1994). total n was determined by kjeldahl method (bremner and mulvaney, 1982), available p by the method of olsen et al. (1954) and available k was analyzed in 1m nh4oac extract by fl ame emission. the experimental design was a split-split plot with randomized complete block and four replications. wheat (triticum aestivum l.) varieties golia and sagittario were sown between mid-november and the beginning of december (04.12.2008; 26.11.2009) with a rate of 500 seeds m-2. for the phenological growth stages, the bbch-scale was used (bbch monograph, 2001). n treatments (ammonium nitrate) were 0 kg ha-1 n, 70 kg ha-1 n (at sowing), 140 kg ha-1 n (70 kg sowing, + 35 kg beginning of tillering, + 35 kg beginning of shooting) and 210 kg ha-1 n (70 kg sowing, + 70 kg, beginning of tillering, + 70 kg beginning of shooting). sulphur treatment 0 and 40 kg ha-1 s were applied as gypsum betab. 1: chemical properties of soil (ap-horizon) at the study site in aydin soil texture (%) horizon sand silt clay n p k ca mg s1 om2 ph (%) (ppm) (ppm) (ppm) (ppm) (mg/kg) ap 65.2 23.2 11.6 0.13 18.0 383 2897 379 6.3 1.50 8.0 1 available sulphur 2 organic matter 18 o. erekul, k.-p. götz, y.o. koca tween the end of february and the beginning of march (beginning to end of tillering). further fi eld management (p and k fertilization, weed control and pest management) were carried out in accordance to the nutrient state of the soil and crop development. the sampling area for grain yield was 4.8 m2 in the centre of the plot (plot size: 12.0 m2, grain test weight was measured on a 250 g sample and expressed as kg hl-1). grains were milled by the amg (ardic medical gida, ankara, turkey) laboratory mill. grain n-content was determined by kjeldahl method. starch content was determined on a dry weight basis by near infrared refl ectance spectroscopy (nirs), using a perten da-7200 instrument (perten co., huddinge, sweden). bread-making quality analyses were evaluated by the standard method of the international association of cereal chemistry (icc, 1986): wet gluten and gluten index by icc 137, 155 and 158, using a glutomatic 2200 instrument; sedimentation values by zeleny sedimentation test (icc 116); the falling number according to icc 107. the data were analysed by using statistical software spss, version 14.0, univariate model and least signifi cant difference (lsd) among means of cultivars and n and s doses in each year were tested at the p<0.05 level of probability. results and discussion nitrogen and year as main effect showed a signifi cant infl uence on yield (univariate model; data not shown). however, the main effect yield was not different between the cultivars and independend from the sulphur application. the grain yield ranged in 2009 from 2590 kg ha-1 for the control to 4081 kg ha-1 for the highest napplication for golia and for sagittario from 2909 kg ha-1 to 4172 kg ha-1 (tab. 2). the applied sulphur resulted in higher yield up to 4322 and 4379 ha-1 for golia and sagittario, resp., but the increase was not signifi cant. the grain yield was higher in 2010 and ranged from 3015 kg ha-1 for the control to 4698 kg ha-1 for golia and for sagittario from 2963 kg ha-1 to 4627 kg ha-1. no remarkable benefi ts were noticed at a higher yield level in 2010. in aegean region, grain yield average under rain fed conditions is 1,5 times higher than the average of turkey. the difference in effectiveness of nitrogen and sulphur fertilization in the two years was fi rstly and primarily caused by different weather conditions during the growth periods. in this study the varieties remained below their yield potential of about 8000 to 9000 kg ha-1 (dinc and erekul, 2010). intensive rains in 2009 particularly in january and february (total 428.2 mm) caused reducing effects during tillering period (bbch 20bbch 29) and later on ear density (ears m-2; data not shown). also the amount and distribution of rain in aegean region in may can affect the yield formation remarkably. this was more advantageous in may 2010, rain which were higher than 2009, led to remarkably higher yield than 2009. positive effects of sulphur fertilizer on wheat yield have been noticed also in other studies (rasmussen and kresge, 1986; zhao et al., 1999c). although in this study, which is the fi rst study in our region in terms of the effects of nitrogen and sulphur fertilization and its combinations on quality parameters of wheat, gave progressive results, the acquired differences haven’t found to be statistically signifi cant. similar results in terms of yield were mentioned in a study by paulsen (1999). when both years were compared, it can be concluded that sagittario is more susceptible to sulphur fertilization than golia. the measured parameter of the internal grain quality, like grain protein, fl our protein, gluten index, sedimentation value, falling number, with the exeption of the gluten content, were signifi cantly infl uenced by the cultivar, nitrogen application and year. considerably infl uenced by the sulphur application was the protein content in the fl our, the gluten index and the sedimentation value (univariate model; data not shown). as one of the bread making qualities, protein content in both wheat varieties increases in paralel with the increasing nitrogen application. these increases are signifi cant especially between the control (0 kg ha-1 n) and 70/140 kg ha-1 n and 210 kg ha-1 n (tab. 3). protein content ranged between 11.5% and 16.1% and between 12.3% and 15.3 %, in 2009 and 2010, resp., without any distinctive effect of sulphur fertilization. in aegean region decreasing rainfall in the second half of may and increasing temperatures triggers shortening of grain fi lling period (bbch 61-bbch 87), decreasing carbohydrate accumulation, thus increasing the protein content (gooding et al., 2003; erekul et al., 2011). when compared to results of other studies in mediterranean climates (guarda et al., 2004), the protein contents determined in our investigation were either similar or higher. the protein content in fl our is the main quality criterion for wheat, especially for bread making (triboi et al., 2006) and is generally lower than the amount in the grain (zhao et al., 1999b). the protein content in the fl our was about 0.4% to 1.3% lower than the protein content in the whole grain and ranged between 10.8% and 15.3% in 2009 and 2010. effects of increasing nitrogen doses on protein content in fl our have been similar to the effect on protein content in grain. the application of sulphur did not affect the protein content; therefore our results are in accordance with steinfurth et al., 2012 and zhao et al., (1999b), but not with shahsavani and gholami (2008). the effect of sulphure fertilizer on fl our protein content in comparison with the grain protein content was principally the same in sagittario however the differences are not signifi cant. different nitrogen doses led to the known effect on protein content (borghi et al., 1997). sulphur fertilization can have a comparatively low and unstable effect on wheat fl our protein content (järvan et al., 2008). when the protein content in fl our has been examined it can be seen that sulphure application in both wheat varietes is more distinctive tab. 2: effect of nitrogen and sulphur fertilization on grain yield of two wheat varieties. year/ year/ year/ 2009 2010 variety golia sagittario golia sagittario grain yield (kg ha-1) n / s doses 0 40 0 40 0 40 0 40 (kg ha-1) 0 2590 2923 2909 2978 3015 3016 2963 3383 70 3481 3634 3397 3534 4218 4006 3890 4026 140 3854 4242 3846 3955 4540 4280 4240 4196 210 4081 4322 4172 4379 4698 4615 4627 4813 lsd (0.05) = 432 lsd (0.05) = 464 lsd (0.05) = 432 lsd (0.05) = 464 sulphur and nitrogen fertilization of wheat (triticum aestivum l.) performance under mediterranean climate 19 than on grain protein content and the differences in some cases have been recorded signifi cant (tea et al., 2007). the grain quality is a complex of physical and chemical characteristics and its expression depends on their genetic potential and is infl uenced by environmental conditions (johansson, 2002; johansson et al., 2004). in our study, there is found to be no dilution effect between protein content and grain yield (acuna et al., 2005; erekul et al., 2009). in 2009 and 2010 starch content ranged between 60.1-64.8% (data not shown), which is in the range of values for wheat given by gooding and davies (1997) and johansson (2002). it is known that in developed kernels proteins occur fi rst and thereafter starch is produced (sowers et al., 1994). therefore, high crude protein contents were accompanied by lowering starch content in both cereal varieties. protein and starch contents were found to be inversely correlated, which is in line with gooding and davies (1997) and viswanathan and khanna-chopra (2001). wet gluten content ranged between 27% and 33.9 % in 2009 (tab. 4). increasing nitrogen have caused signifi cant increases in wet gluten contents which was also found by pechanek et al. (1997). application of sulphure in 2009 has caused increase in wet gluten content in the doses of 70 kg ha-1 n, 140 kg ha-1 n and 210 kg ha-1 n in golia and in the doses of 140 kg ha-1 n and 210 kg ha-1 n in sagittario. however these increases have not been signifi cant in general. wet gluten content in sagittario was higher than in golia in 2009. these differences are signifi cant in the control (0 kg ha-1 n) with and without sulphur. increase in nitrogen fertilization in 2010 led to higher wet gluten content but not as pronounced as the amount in 2009. s-fertilization resulted in the variety golia in all n levels in higher gluten content, however the differences were not signifi cant. in sagittario variety ultimately the opposite was observed at all nitrogen levels. the decreases occured here have not become in signifi cant levels. in the second year as well, sagittario general produced more wet gluten than golia however in any case, the difference was not signifi cant. tab. 3: effect of nitrogen and sulphur fertilization on protein content in grain and fl our of two wheat varieties year / year / year / 2009 2010 variety golia sagittario golia sagittario grain protein content (%) n / s doses 0 40 0 40 0 40 0 40 (kg ha-1) 0 12.0 11.5 13.1 12.4 12.3 12.7 13.0 12.5 70 14.0 14.3 14.4 14.0 13.8 13.5 13.8 14.2 140 15.2 14.9 15.1 15.7 14.2 14.4 14.0 14.8 210 15.8 15.9 15.9 16.1 14.9 15.1 15.1 15.3 lsd (0.05) = 0.9 lsd (0.05) = 0.9 lsd (0.05) = 0.6 protein content fl our (%) 0 40 0 40 0 40 0 40 0 11.1 10.8 12.2 11.8 11.8 11.9 12.6 11.9 70 13.2 13.3 13.1 13.6 13.0 12.8 13.2 13.7 140 14.2 14.1 14.5 14.8 13.2 13.9 13.2 14.0 210 14.5 15.0 15.0 15.3 13.7 14.3 14.3 14.6 lsd (0.05) = 0.8 lsd (0.05) = 0.8 lsd (0.05) = 0.4 tab. 4: effect of nitrogen and sulphur fertilization on wet gluten content and gluten index of two wheat varieties year / year / year / 2009 2010 variety golia sagittario golia sagittario wet gluten content (%) n / s doses 0 40 0 40 0 40 0 40 (kg ha-1) 0 27.4 27.0 30.7 28.9 28.9 29.0 29.3 29.1 70 30.1 31.8 31.9 30.4 30.2 31.2 31.6 30.9 140 31.6 32.4 32.1 32.8 30.6 31.8 32.4 31.7 210 32.8 33.1 33.5 33.9 31.9 32.5 33.3 33.1 lsd (0.05) = 1.2 lsd (0.05) = 2.0 gluten index (%) 0 40 0 40 0 40 0 40 0 85 86 76 79 86 86 79 80 70 88 88 80 81 88 90 80 85 140 90 90 82 81 91 92 83 83 210 89 90 80 81 90 91 81 82 lsd (0.05) = 1.8 lsd (0.05) = 1.9 year / year / lsd (0.05) = 1.2 lsd (0.05) = 2.0 20 o. erekul, k.-p. götz, y.o. koca generally, we found wet-gluten to be mostly over 28%, which we assume to be between high and very high values (erekul and köhn, 2006). the effect of sulphur fertilizing on wet gluten content has not been so distinctive when both years have been considered but much rather the increases of wet gluten content have been found in golia after sulphur fertilizing. wieser et al. (2004) concluded that the effect of the sulphur fertilizer on gluten proteins in pot experiments were not remarkable. however the results of järvan et al. (2008) are in accordance with our study. gluten index which indicates the quality of the gluten has ranged between 76% and 90% in 2009. the increasing doses of nitrogen in both wheat varieties have caused the gluten index parameters to increase up to 140 kg ha-1 n (tab. 4). these increases are almost signifi cant at all n-levels. application of 210 kg ha-1 n had no infl uence on the gluten index. application of sulphur fertilizer have resulted positively in both wheat varieties, but except for one n application obtained for sagittario, all differences have not been found signifi cant. similar results about the effi ciency of sulphur fertilizer application have been observed also in 2010. the gluten index reached the highest values when 70 kg ha-1 n and 140 kg ha-1 n was applied in 2010. golia achieved signifi cantly higher gluten index values than sagittario in terms of all nitrogen and sulphur doses and in both years. the gluten index has been recently introduced as a better measure of wheat processing quality, rather than wet gluten content (deng et al., 2005). however the gluten index is a new, and yet seldom used parameter for wheat bread-making quality; little information is available on the relationship between the gluten index and other indices, or about the infl uence of environment on the gluten index (johansson et al., 2002). curic et al. (2001) reported optimum values between 75% and 90% for gluten-index of wheat. in our study in both years, the gluten index values have been above 76%. these results are in accordance with johansson et al. (2004). in the same fertilizing doses in both trial years the gluten index values of golia have signifi cantly higher values than sagittario. this implies that the gluten quality and its dough characteristics of golia is higher (kahriman, 2007). it is stated that in many studies done with sulphur fertilizer application, the gluten index among the quality parameters is affected positively at most (schäfer and honermeier, 2007; järvan et al., 2008). the increasing nitrogen doses have brought signifi cant increases of sedimentation values in both wheat varieties and sulphure doses. in general there has been more impact of the application of sulphure fertilizer on sedimentation values in both wheat varieties and years (tab. 5). the application of sulphure fertilizer has resulted in equal or higher sedimentation values in all nitrogen applications in both varieties and years. compared to the gluten index, sagittario has produced signifi cantly higher sedimentation value in all nitrogen doses than golia. impacts of the applications of nitrogen and sulphur on sedimentation values of both wheat varieties have been similar in the second year of the trial. furthermore in the second year of the trial as well, the sagittario variety has produced signifi cantly higher sedimentation values in all nitrogen and sulphur levels than the golia variety. the sedimentation value of the wheat varieties, characterizing the swelling capacity of gluten, exceeded the nominal value (20 ml) for bread-making wheat in both years. in all applications in both years, the minimum limit of 20 ml has been eminently exceeded. the positive effect of the sulphur fertilizer has been seen more on sedimentation value rather than on gluten index parameter. schäfer and honermeier (2007) reported that sulphur application created positive effects on sedimentation value but the results obtained were also not signifi cant. weber at al. (2008) put forward that the effect of additional sulphur application on sedimentation value is extremely limited. the sedimentation value of the sagittario has higher values in both years than the golia and based mainly on genotypic characteristics. increasing nitrogen doses caused the protein contents to increase and due to this, the sedimentation values increase signifi cantly in both varieties and in both years. similar results have also been reported in previous studies (gooding et al., 2003). the falling number has been measured in order to determine impacts of nitrogen and sulphur fertilizer on alpha amylase activity in wheat fl our. the falling number values ranged between 333 s and 396 s in 2009 (tab. 5). in 2009, any distinct impact of nitrogen and sulphur fertilizer has not been determined on the falling number. sagittario showed substantially lower falling number values than the golia variety. in 2010 the falling number values have ranged from 298 s to 351 s and showed lower values than 2009 without impact of nitrogen and sulphur fertilizer. the falling numbers, which allow for conclusions to the enzymatic state of the grain, to the expected baking volume, and to the grade of outgrowth, have an optimum range of 220 to 250 s for winter wheat (diepenbrock et al., 2005). falling number values in both varieties and years have been above tab. 5: effect of nitrogen and sulphur fertilization on sedimentation value and falling number of two wheat varieties year/ year/ year/ 2009 2010 variety golia sagittario golia sagittario sedimentation value (ml) n / s doses 0 40 0 40 0 40 0 40 (kg ha-1) 0 26 28 29 33 27 28 30 32 70 30 30 35 35 30 31 33 35 140 31 33 38 38 32 33 36 36 210 36 37 40 42 34 36 37 39 lsd (0.05) = 1.6 lsd (0.05) = 2.0 falling number (s) 0 40 0 40 0 40 0 40 0 373 352 347 333 324 298 312 323 70 389 348 361 371 347 340 304 329 140 358 381 343 340 329 351 324 311 210 370 396 353 387 341 338 339 333 lsd (0.05) = 17 lsd (0.05) = 24 lsd (0.05) = 1.6 lsd (0.05) = 2.0 sulphur and nitrogen fertilization of wheat (triticum aestivum l.) performance under mediterranean climate 21 the optimum values. falling number values are affected by the climate conditions during the grain fi lling period (bbch 61-bbch 87) and primarily by rainfall more than the fertilizing (gooding et al., 2003). when evaluated from that perspective, decrease in rainfalls and increase in temperatures in grain fi lling period in our region caused the falling number values to increase. this situation has caused enyzme activity of the fl our obtained to decrease. similar results have also been reported by johansson (2002) and gooding et al. (2003). it may also be possible to see differences among genotypes (erekul et al., 2009). in our study, sagittario generally has lower falling number values than golia. no signifi cant effect of nitrogen and sulphur fertilizing applications on falling number values have been found and these fi ndings are in accordance with the results of weber et al. (2008). conclusion quality characters of bread wheat have not adequately been examined on varieties grown in western turkey. due to lack of information, wheat varieties have not been classifi ed into quality groups which cause inability to compare them with international wheat varieties. under more favorable climatic conditions the grain yield could increased up to 210 kg ha-1 n without substantially losses in the grain quality. the results indicate that s-fertilization showed benefi cal effects in grain yield and in some bread-making quality parameters. s-fertilization on protein content was more remarkable in fl our than in grain. the most benefi ted quality parameter by sulphur was the gluten-index and especially the sedimentation value. in this respect, s-fertilization in improving bread making quality of the region should not be disregarded. however, an s fertilization of > 40 kg ha-1 would be advisable to prove more clearly effects on yield and quality parameters. acknowledgements the authors are grateful to dr. r. krause for her help on the quality part at the humboldt university of berlin. references acuna, m.l., savin, r., curá, j.a., slafer, g.a., 2005: grain protein quality in response to changes in pre-anthesis duration in wheats released in 1940, 1964 and 1994. j. agron. crop sci. 191, 226-232. anonymous, 2010: fao data. http://faostat.fao.org/site/567/default.aspx# ancor bbch monograph, 2001: growth stages of monoand dicotyledonous plants. federal biological research centre for agriculture and forestry, edited by u. meier, 2. edition. borghi, b., corbellini, m., minoia, c., palumbo, m., di fonzo, n., perenzin, m., 1997: effects of mediterranean climate on wheat breadmaking quality. eur. j. argon. 6, 145-154. bremner, j.m., mulvaney, c.s., 1982: nitrogen-total. in: page, a.l., millerand, r.h., keeney, d.r. 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late s fertilization. j. plant physiology. 169, 72-77. tea, i., genter, t., naulet, n., lummerzheim, m., kleiber, d., 2007: interaction between nitrogen and sulfur by foliar application and its effects on fl our bread-making quality. j.sci. food agric. 87. 2853-2859. triboi, e., martre, p., girousse, c., ravel, c., triboi-blondel, a.m, 2006: unravelling environmental and genetic relationship between grain yield and nitrogen concentration for wheat. europ. j. agronomy, 25, 108-118. viswanathan, c., khanna-chopra, r., 2001: effect of heat stress on grain growth, starch synthesis and protein synthesis in grains of wheat (triticum aestivum l.) varieties differing in grain weight stability. j. agron. crop sci. 186, 1-7. weber, e.a., koller, w.d., graeff, s., hermann, w., merkt, n., claupein, w., 2008: impact of different nitrogen fertilizers and an additional sulfur supply on grain yield, quality, and the potential of acrylamide formation in winter wheat. j. plant nutr. soil sci. 171, 643655. wieser, h., gutser, r., von tucher, s., 2004: influence of sulphur fertilization on quantities and proportions of gluten protein types in wheat fl our. j. ceral sci. 40, 239-244. zhao, f.j., mcgrath, s.p., 1994: soil extractable sulphate and organic sulphur and their availability to plant. plant soil. 164, 243-250. zhao, f.j., salmon, s.e., withers, p.j., evans, e.j., monaghan, j.m., shewry, p.r., mcgrath, s.p., 1999a: responses of bread-making quality to sulphur in three wheat varieties. j. sci. food agric. 79, 18651874. zhao, f.j., salmon, s.e., withers, p.j., monaghan, j.m., evans, e.j., shewry, p.r., mcgrath, s.p., 1999b: variation in the breadmaking quality and rheological properties of wheat in relation to sulphur nutrition under fi eld conditions. journal of cereal science, 30, 19-31. zhao, f.j., hawkesford, m.j., mcgrath, s.p., 1999c: sulphur assimilation and effects on yield and quality of wheat. j. cereal sci. 30, 1-17. address of the authors: assoc. prof. dr. o. erekul1 (corresponding author; e-mail: oerekul@adu. edu.tr) and dr. y.o. koca1, department of crop science, faculty of agriculture, adnan menderes university, 09100 aydin, turkey; dr. k.p. götz2, department of crop and animal science, faculty of agriculture and horticulture, humboldt-universität zu berlin, albrecht-thaer-weg 5, d-14195 berlin, germany. angewandte botanik. 146 hugo fischer, der gegenwärtige stand der kohlensäurefrage usw. und es käme jemand (ich setze als bekannt voraus, daß solche erfindung nur von der naturwissenschaft ausgehen kann!) zum kriegsminister: „ich bin einer erfindung auf der spur, so und so , welche die ganze kriegführung umgestalten und derjenigen seite, welche in ihrem besitz ist, ungeheure vorteile gewährleisten wird; ich brauche aber zur durchführung der sache ein kleines labora torium mit geeigneter einrichtung, usw.“ und der minister, nach dem e r noch einige fragen gestellt, spräche z u ihm: „schön, was sie brauchen, sollen sie haben! gewinnen sie nur erst mit ihrem „pulver“ ein paar schlachten und erobern sie einige festungen, dann soll die errichtung ihres laboratoriums sofort . . . . . . . in wohlwollende erwägung gezogen werden.“ – so liegt die sache, und e s ist ein schwerer hemmschuh für unsere ganze kulturentwicklung, daß die möglichkeit, einen fruchtbaren gedanken in die tat umzusetzen und ihn der allgemeinheit nutzbar zu machen, von außenbedingungen abhängig ist, die mit seinem wert oder unwert rein gar nichts zu tun haben. die beurteilung des anbauwertes französischer rotkleesaaten. von prof. dr. j. simon-dresden. mit 2 karten. die erste und wichtigste grundlage einer erfolgreichen pflanzenkultur is t die verwendung guten, einwandsfreien saat gutes. die eigenart des heimischen pflanzenbaues, einmal die kulturellen und klimatischen verhältnisse, dann die notwendigkeit des gesteigerten anbaues von getreide und hackfrüchten sowie von futterpflanzen machen es unmöglich, den gesamten bedarf an saatgut für alle pflanzenarten im inlande selbst zu erzeugen: in weitgehendem maße sind und bleiben wir auch für die folge auf den bezug vom auslande angewiesen. ganz besonders behält dies geltung für unsere wichtigste futterpflanze, den rotklee“). *) importiert wurden vor dem kriege jährlich 378000 d z kleesaat. (alves, arbeit. der d . l . g., 1913, bd. 245.) j. simon, die beurteilung des anbauwertes französischer rotkleesaaten. 147 nun is t aber gerade für diesen die anbauwürdigkeit eines saat gutes in hohem grade abhängig von seiner herkunft. praktische erfahrungen und zahlreiche anbauversuche haben längst die tat sache erwiesen, daß die inländischen rotkleesorten und unter diesen besonders die östlichen herkünfte, die schlesischen, ost und westpreußischen a n erster stelle stehen, denen sich als zweifel lo s hochwertig russische und österreichische (böhmen, steiermark), saaten anschließen. bezüglich des wertes der westund süd europäischen provenienzen gehen die meinungen jedoch stark aus einander, und schon bei pfälzer, rheinischen und badischen saaten is t nicht selten über beträchtliche schädigung der kleeschläge in folge der winterkälte im mittleren und östlichen deutschland ge klagt worden. für den rotklee ist demnach die herkunft geradezu eine ausschlaggebende werteigenschaft. für deutsche verhältnisse kann die minderwertigkeit bezw. völlige unbrauchbarkeit der italienischen und der südfranzösischen herkünfte einem zweifel nicht unterliegen. bezüglich der saaten aus dem übrigen frankreich liegen zur sicheren beurteilung ihres anbauwertes jedoch zuverlässige praktische erfahrungen in aus reichendem maße nicht vor: die diesbezüglichen urteile sind meist nur wenig zuverlässig, d a das jeweil verwendete saatgut in seiner herkunft nicht genau genug bekannt war. anders läßt e s sich ja auch nicht erklären, wenn urteile so weit auseinander gehen, daß beispielsweise müller-augustenberg!) auf grund seiner untersuchungen alle französischen herkünfte selbst für das teil weise außerordentlich milde klima badens als nur zu einjähriger nutzung brauchbar erachtet, während störmer-kleine”) auf grund ihrer anbauversuche eine gute nordfranzösische saat un bedingt als für pommersche verhältnisse (wo im rauhen küsten gebiet selbst der petkuser roggen kaum winterhart genug erscheint) sehr gut verwendbar bezeichnen, während sie dem rotklee aus den mittelfranzösischen gebirgsgebieten geringere widerstands fähigkeit beimessen, wie jenem des geographischen norden. nach unseren*) vieljährigen untersuchungen und erfahrungen über dauern die letzteren und natürlich erst recht die provenienzen aus südund westfrankreich selbst die verhältnismäßig milden winter *) untersuchungen über d ie erkennung und den ertrag verschiedener rot kleeherkünfte. berlin, parey 1916. *) deutsche landwirtschaftliche presse 1915, nr. 53. *) jahrbuch für weidewirtschaft, hannover 1919. 10* 148 j. simon, dresdens nicht, während wir den eigentlichen französischen ge birgs-rotklee für widerstandsfähiger erachten. die 1901–1902 durchgeführten umfassenden versuche der d. l. g.) führten hin wieder zur schlußfolgerung, daß ganz allgemein die französischen rotkleesorten für uns als ungeeignet bezeichnet werden müßten; auch die nordwie südfranzösischen saaten waren bei diesen ver suchen durch frost mehrfach erheblich geschädigt worden. um gekehrt fand haselhoff”) zwischen russischem, schlesischem, nord und südfranzösischem, ja sogar italienischem rotklee keinen unter schied hinsichtlich der winterfestigkeit! diese divergenz der meinungen zeigt deutlich, wie weit die so wichtige frage noch von einer klärung entfernt ist. aber auch rein zahlmäßig er scheinen mir die bisher exakt durchgeführten anbauversuche in keinem falle auch nur annähernd zur begründung eines festen urteiles hinreichend: nur in den verschiedensten teilen deutsch lands jahre hindurch fortgeführte vergleichende prüfungen, be i welchen die differenten verhältnisse unserer kontinentalen winter zur vollen einwirkung gelangt sind, können hierzu die notwendigen unterlagen schaffen. dabei ist es keinesfalls angängig, die schluß folgerungen dergestalt z u verallgemeinern, als o b dieselben geltung besäßen gleichermaßen für pommern und sachsen, baden oder posen. vor allem ist dabei aber das den verschiedenen produktions gebieten des mittleren und nördlichen frankreichs einwandsfrei entstammende saatgut getrennt z u prüfen, da dasselbe in abhängig keit von den lokalen erzeugungsbezirken sich ganz anders ver halten wird. der französische samenhandel rechnet aber nach uns zugegangenen direkten mitteilungen das gesamte nördlich d e r linie bordeaux–grenoble liegende gebiet zum französischen norden und handelt die saaten aus dem pariser becken ebenso wie solche aus dem küstengebiet oder aus dem hochland des mittleren frankreichs als „nordfranzösischen rotklee“. für unsere deutschen verhältnisse sind diese provenienzen aber von ganz verschiedenem wert, ja zum teil völlig unbrauchbar: bei neu orientierung der handelsbeziehungen mit unserem west lichen nachbarn muß deshalb jenem irreführenden brauch von vornherein ein riegel vorgeschoben werden dergestalt, daß eine exaktere bezeichnung und zwar nach klimatisch enger umgrenzten gebieten geltung erlangt. 1 ) jahrbuch der deutschen landwirtschafts-gesellschaft 1903, xviii. *) fühlings landwirtschaftliche zeitung 1917. die beurteiluug des anbauwertes französischer rotkleesaaten. 149 die klimaverhältnisse europas, insbesondere wie sie sich unter dem einfluß des golfstromes ganz eigenartig gestalten, habe ich an anderer stelle!) bereits einer kurzen betrachtung unterzogen und dabei ihren einfluß auf die anbauwürdigkeit der dorther stammenden rotkleesaaten angedeutet. um gesagtes nicht wiederholen zu müssen, sei nebenstehend eine z. zt. gebrachte karte wieder gegeben, welche deutlich die gesamten klimaverhält nisse unseres kontinents in großen zügen dartut. auf die speziell französischen verhältnisse möchte ich aber etwas näher eingehen, da deren beachtung die b is jetzt allein zuverlässige und einheit liche grundlage liefert zur beurteilung französischer rotklee provienzen. denn d a diese wichtigste futterpflanze bei uns durch weg z u zweijähriger nutzung angebaut wird, besitzen die notwendige widerstandsfähigkeit nur die aus solchem saatgut erwachsenen pflanzen, welches aus gegenden mit gleichen oder doch ähnlichen klimatischen verhält nissen stammt. die wintertemperaturen bilden hierfür das ausschlaggebende moment. wie aber schon die erwähnte karte erkennen läßt, besitzt das mittlere deutschland im gegensatz zum kalten osten gemäßigte winter, während das westliche frank reich sich milder winter erfreut gleich den warmen küsten des mittelmeeres. anders im mittleren frankreich, wo das rumpf gebirge der centralplatte in einer durchschnittlichen höhe von 800 bis 1000 m ganz anders geartete verhältnisse bedingt: hier im mittelgebirge (vi) climat auvergnat ou limousin, sind die wettererscheinungen reich a n gegensätzen. im großen und ganzen ist das klima rauh, im winter wirft der himmel unmassen von schnee zur erde und überzieht die hochflächen oft mit scharfem frost. im frühling, welcher etwa gleichzeitig wie in mitteldeutschland einsetzt, stürzen gewaltige schmelzwasser durch d ie tief eingeschnittenen flußtäler der loire, des allier, des lot, tarn und ihrer zuflüsse; die nächte sind oft empfindlich kalt und bringen nicht selten den pflanzenkulturen schaden*). mit einem flächenraum von etwa 80.000 qkm umfaßt das gebiet fast den sechsten teil frankreichs, darunter ganz oder zum teil die land schaften auvergne (bis z u 1800 m höhe), lyonnais (bis 1000 m), bourbonnais, marche und limousin (mit einer mittleren höhe von *) illustr. landw. ztg. 1915, nr. 2 3 und mitteil. d.ökon. ges. dresden 1915. *) siehe z u dem folgenden höfer, frankreichs landwirtschaft, frank reichs reichtum, leipzig 1912, sowie scobels geographisches handbuch, 150 j. simon, } ،º // • � º-ź ž ž ;# die beurteilung des anbauwertes französischer rotkleesaaten. 151 500 m), guyenne, u. a. von diesem klimatisch wohl charakteri sierten gebiet unterscheiden sich scharf die vier anderen klima provinzen frankreichs. in diesen kommt der einfluß der meere wie jener der südlichen lage frankreichs – der nördlichste punkt hat die breite von köln und dresden, der südlichste die ca"s ------ii -0"rt ". --von florenz – deutlich zur geltung. das klima der bretagne (i ), climat breton o u armoricain, kennt kaum den frost, in dem stürmereichen land sind die hälfte aller tage regentage, viele anderen trübe und sonnenlos. unter dem einfluß der wärme aber gedeihen hier im freien kamelien, yukkas, araukarien und statt iche feigenbäume. das garonnebecken (ii), climat girondin, besitzt eine mittlere januartemperatur von 4–5° celsius, bedingt 152 j. simon, durch die meeresnähe. die verhältnisse des pariser beckens (iii), climat parisien ou séquanien, sind durch warmes frühjahr, heiße sommer und milde winter mit einer mittleren januar temperatur von 4° celsius charakterisiert. die mediterranen küsten landschaften (provence, languedoc) besitzen mittelmeer-klima (iv), climat méditerranien, mit ganz besonderer eigenart, auf trockene und heiße sommer folgen auffallend milde winter, immer grüne matten und gebüsche zeigen das südeuropäische vegeta tionsgebiet an, der ölbaum is t hier die charakterform der kultur landschaft. ostfrankreich (v), climat vosgien im nördlichen, climat lyonnais ou rhodanien im südlichen teil, hat unter sich annähernd gleiches klima, kontinental-klima, d. h. heiße sommer und verhältnismäßig kalte winter. auf der beigegebenen karte 2 habe ich nach einer skizze im atlas classique von schrader u. gallouédec (paris 1908) diese wohlcharakterisierten 5 klimagebiete frankreichs eingetragen, da z u im gebiete v i jene departements in ihren verwaltungsgrenzen, welche für deutschland geeignete kleesaaten liefern. daneben sind in gleicher blockschrift jene departements, welche für die d . l . g.-versuche saatgut lieferten, angemerkt, endlich in kursiv schrift die anderweit wichtigsten kleesaat liefernden landschaften des landes und seiner nachbargebiete. diese einteilung frankreichs in klima-provinzen erscheint mir nun als die zuverlässigste grundlage zur beurteilung der anbauwürdigkeit französischer rotkleesaaten: nur die aus der klimaprovinz vi, aus dem hochland der auvergne im weitesten sinne, von den sich nach nw und w erstreckenden wellenförmigen abdachungen bis hinauf z u dem steilrand der cevennen stammen den saaten können für den anbau in deutschland zu zweijähriger nutzung in frage kommen. e s sind in erster linie die departe ments cher, allier, creuse, haute-vienne und cantal, ferner in geringerem umfange nièvre, loire, haute loire und corrèze. für das gesamte gebiet dürfte die bezeichnung mittelfranzösisches gebirgsland zutreffen, die aus demselben stammenden rot kleesaaten sind folgerichtig als mittelfranzösischer ge birgsrotklee zu bezeichnen, womit eine scharfe trennung von den herkünften des geographischen norden, westen und süden frankreichs geschaffen ist. daß dabei die aus der bretagne, der normandie und picardie, dem artois, den land schaften bauce, poitou, der touraine, vendée stammenden und die beurteilung des anbauwertes französischer rotkleesaaten. 153 andere in frankreich hochgerühmte herkünfte ausscheiden, ist a u s ihrem für unsere anbauverhältnisse nicht zweifelsfreien wert heraus umso mehr berechtigt, als die ernten bezw. überschüsse des hochlandes auch quantitativ durchaus hinreichen, um die deutsche nachfrage nach französischem rotklee z u decken. unter hervorhebung gleicher oder ähnlicher gesichtspunkte beschäftigen sich mit der herkunftsfrage von rotkleesaaten zwei neuere arbeiten von müller) und oberstein”), welche beide wertvolle beiträge zur klärung des problems bringen. der von müller vorgeschlagenen zusammenziehung aller provenienzen aus dem zwischen dem 45. und 48. breitegrad und der länge 1 " w .– 4 " ö . liegenden bezirke z u einem gemeinsamen herkunftsgebiet vermag ich allerdings nicht zuzustimmen. diese doch wohl z u willkürliche einteilung is t auf einer kartenskizze der arbeit ober steins wiedergegeben, ihr schematischer charakter trägt den klimatischen verhältnissen in nicht genügendem maße rechnung. gewiß decken sich auch die von mir angeführten departements namen, deren bezeichnung und abgrenzungen ja bekanntlich durch d ie revolution von 1789 in rein willkürlicher weise erfolgte, nicht ganz mit der gegebenen klimatischen umgrenzung; so ragen die zur landschaft berry gehörigen nordwestlichen teile des dep. cher bereits in das nordfranzösische tiefland. aber gerade dieser teil is t fü r den kleesamenbau von geringerer bedeutung, so daß man durchaus sagen kann, daß die angeführteu wichtigsten produktions gebiete innerhalb der klimaprovinz liegen, welche infolge ihres gesamtcharakters widerstandsfähige und für den anbau in deutsch land geeignete saaten hervorbringt. die klimatische eigenart dieses mittelfranzösischen gebirgs landes findet in der unkraut-flora des gebietes eine aus wirkung, welche für die herkunftsbestimmung französischer rot kleesaaten von besonderer wichtigkeit ist: die in südund west französischen, sowie auch in den nordwestlichen teilen nordfrank reichs häufigen pflanzen helminthia echioides und torilis nodosa kommen im gebiet nur vereinzelt und zerstreut vor, ihre samen finden sich fast nie, wenn aber, dann höchstens ganz vereinzelt in d e n kleesaaten desselben, völlig fehlen aber die für südliche herkünfte charakteristischen samen von arthrolobium scorpioides *) a . a . o . *) landw. jahrb. l i (1918). „ist die warnung vor rotkleeherkünften m it mediterran-atlantischen charakterbegleitsamen berechtigt?“ 154 j. simon, und centaurea solstitialis sowohl wie die in saaten des küsten gebietes nicht selten vorkommenden eigenartigen muschelfragmente). überhaupt is t e s für den kundigen meist nicht schwierig an de r hand der beischlüsse die spezielle herkunft einer französischen saat mit ausreichender sicherheit festzustellen. wir sind aller dings der meinung, daß gerade bei der beurteilung westländischer oder dieser herkunft verdächtiger kleeprovenienzen nicht auf das vorkommen eines vereinzelten samens, beispielsweise von hel minthia, ein allzu großes gewicht gelegt werden darf. gerade di e genannte pflanze scheint am ehesten neben centaurea solstitialis entwicklungsmöglichkeiten auch bei uns zu finden, ohne sich aller dings dauernd in deutschland einzubürgern: in dresden lieferten auf dem versuchsgelände am botanischen garten aussaaten so wohl a ) in rotklee (dieser wurde je einmal im sommer und herbst geschnitten) wie b) frei ohne solchen üppige, gesunde pflanzen. am 20. oktober 1916 zeigten diese in der gesamter scheinung folgende ungefähren maße: helminthia echiodes höhe 8 0 cm, breite 8 0 cm, centaurea solstitialis 6 0 bzw. 50 cm; beide blühten reich und lieferten reife samen. trotz des kalten winters kam centaurea tadellos durch, im frühjahr erwuchsen auf beiden teilstücken noch mehrere junge pflanzen von beiden arten, zweifellos aus vorjährigem samen stammend. im herbst wurde teilstück b ) umgegraben, a ) nicht (der anstehende rotklee war zweimal geschnitten worden). im frühling 1918 entwickelten sich auf a ) zahlreiche helminthia-pflanzen, zwischen diesen auch 13 kräftige centaurea solstitialis, vorjährige und neue. auch auf der *) diese stammen daher, daß seetange wegen ihres kaligehaltes zum düngen der felder verwendung gefunden haben. in meiner mehrfach erwähnten für den praktischen landwirt bestimmten veröffentlichung hatte ich die hauptsächlich in frage kommende tangart fucus als zu den tiefseepflanzen gehörig bezeichnet, um sie a ls auf dem meeresboden befestigte von den schwimmenden fucoideen der sargassomeere z u trennen. auch unterscheidet man in frankreich für die technik der tanggewinnung und deren verwertung zwischen „tiefseetang“ oder „getrif tetem tang“ und „geschnittenem tang“; für düngungszwecke kommt besonders der erstgenannte in betracht, welcher durch stürme vom meeresboden losgelöst eutweder direkt ans ufer geworfen oder in stillen buchten getriftet wird. an jener trotzdem wissenschaftlich nicht exakten bezeichnung nimmt oberstein anstoß: doch kann ich ihm für seine freundliche belehrung nicht einmal dankbar sein, denn seit dem erscheinen von haeckels klassischen plankton-studien (jena 1890), in welchen derselbe den begriff des littoral-benthos schuf, weiß ich, daß die braunalgen zur photischen region desselben gehören und natürlich keine pflanzen der eigentlichen tiefsee sind und sein können. die beurteilung des anbauwertes französischer rotkleesaaten. 155 umgegrabenen parzelle liefen mehrere helminthia auf, keine centaurea; auf naheliegendem bebautem lande wurden einige exemplare der erstgenannten art gefunden, welche alle reich blühten und fruchteten. an anderer stelle werde ich über die interessanten. resultate dieser versuche, welche sich auch auf arthrolobium scorpioides, torilis nodosa, cephalaria transilvanica, trifolium supinum, ammi majus u. a. m. erstreckten, eingehender berichten. bezüglich der ersterwähnten beiden arten scheinen mir d ie mitgeteilten ergebnisse doch z u einer gewissen vorsicht im urteil beim vorkommen vereinzelter samen derselben in klee saat z u mahnen; e s muß immer wieder betont werden, daß nur das gesamtbild der beischlüsse unter beachtung des fehlenden eine zutreffende herkunftsbeurteilung ermöglichen kann. haben uns die letzten jahre die weitgehende abhängigkeit des heimischen saatenmarktes vom auslande in schmerzlicher weise in erinnerung gebracht, so haben sie unser augenmerk aber auch nachdrücklichst wieder auf die wichtigkeit der inländi schen produktion gelenkt, nicht nur bezüglich des quantitativen ersatzes sondern vor allem auch bezüglich der qualität, der güte des saatgutes. in unserem zeitalter der hochzuchten wird der verwendung angepaßten saatgutes vielfach nicht mehr die gebührende beachtung geschenkt. aber gerade für den rotklee steht die überlegenheit bodenständiger herkünfte a m entstehungsorte außer allem zweifel. ich kann von einem vergleichenden anbau in der unwirtlichen eifel bei 500 m höhe berichten, bei welchem eine selbsterbaute schlecht gereinigte eifeler saat wesentlich bessere erträge lieferte als die schönsten großkörnigen russischen, schlesi schen, böhmischen und steierischen herkünfte. es erscheint dem nach dringend geboten, der erhaltung und verbesserung bodenständiger sorten des rotklee besondere sorgfalt zuzu wenden und die diesbezüglichen bestrebungen auch durch verständ nisvolle anerkennung des höheren wertes einer saat und höhere preisbewilligung z u fördern. uc1.b3299303_page_164_cut uc1.b3299303_page_165_cut uc1.b3299303_page_166_cut uc1.b3299303_page_167_cut uc1.b3299303_page_168_cut uc1.b3299303_page_169_cut uc1.b3299303_page_170_cut uc1.b3299303_page_171_cut uc1.b3299303_page_172_cut uc1.b3299303_page_173_cut art_15981.indd journal of applied botany and food quality 94, 169 175 (2021), doi:10.5073/jabfq.2021.094.020 1ingeniería en agroindustrias, universidad de la costa, oaxaca, méxico 2departamento de fitotecnia, universidad autónoma chapingo, estado de mexico 3departamento de ingeniería agroindustrial, universidad autónoma chapingo, estado de mexico sweetened nopal flakes: a functional snack leidy laura cruz-de la cruz1, rosario garcía-mateos2*, carmen ybarra moncada3, joel corrales-garcía3 (submitted: may 7, 2021; accepted: september 22, 2021) * corresponding author summary nopal (opuntia spp. and nopalea spp. genera) is a crop, recognized for its nutritional and medicinal properties; however, there are some underused species, despite the great genetic diversity in mexico. the genus opuntia spp. is the most consumed nopal, whereas nopalea spp. has low commercial demand, possibly because their nutraceutical attributes are unknown. additionally, the nopal pads or cladodes are little accepted by many consumers, due to their texture and flavor. the study objectives were 1) evaluate the nutraceutical content and antioxidant activity of four nopal cultivars: nopalea cochenillifera cv. texas (nt) and opuntia ficus-indica cv. jade (oj), milpa alta (oma), and atlixco (oa); 2) develop nopal flakes, sweetened with rebaudioside a, from the cultivar with the best nutraceutical quality and sensory acceptability. ascorbic acid, total phenolics, and total flavonoids were determined by spectrophotometric methods, individual flavonoids (quercetin, kaempferol, and isorhamnetin) by hplc, and antioxidant activity by the dpph assay. oa was the cultivar with the best nutraceutical quality. the sweetened nopal flakes of oa, at a concentration of 1.1 mg g-1 rebaudioside a, had the highest sensory acceptability by the panelists in intensity and sweetness preference. the addition of rebaudioside a improved the product’s flavor and contributed to preserve the flavonoids and antioxidant activity. these results will contribute to the chemotaxonomy of o. ficus-indica and n. cochellinifera species, and to the utilization of nopals as functional foods, due to their nutraceutical quality. keywords: antioxidant activity, chemotaxonomy, nopalea, opuntia, nutraceuticals, rebaudioside a. introduction nopal is an endemic plant of america that is distributed as wild or cultivated forms in desert and semi-desert areas of the continent. nopal belongs to the cactaceae family and includes the opuntia and nopalea genera. opuntia spp. is the largest genus with 377 identified species; 104 of them are found in wild-type form in mexico (anayapérez, 2001; stintzing and reinhold, 2005). despite the great diversity of nopal cultivars, few studies have been done to characterize their nutraceutical components, which are metabolites that prevent diseases and maintain the good health of consumers. in the nutraceutical, medicinal, and chemotaxonomical context, the ‘jade’ and ‘texas’ cultivars from the opuntia genus haven been little studied, whereas these properties are unknown in species from the nopalea genus. nopals have been consumed since ancient times for their diverse medicinal properties, such as antidiabetic (shane-mcwhorter, 2009), anticholesterolemic (yang et al., 2008), anticarcinogenic (abou-elella and ali, 2014), anti-inflammatory (park et al., 2001), antiulcerogenic, diuretic and antioxidant (alimi et al., 2010) effects. moreover, nopals have been used for their cicatrizing properties in dermal wounds and gastric ulcers (alimi et al., 2010), which have been attributed to their content of dietary fiber (polysaccharides), mucilage (polysaccharide), amino acids, vitamins, minerals, and phenolic compounds (osuna-martínez et al., 2014). in recent years, steviol glycosides (chemical compounds identified in stevia rebaudiana, a native plant of south america) have obtained relevance in the food, cosmetic, and pharmaceutical industries, since they are non-caloric sweeteners. rebaudioside a is the most abundant steviol glycoside in s. rebaudiana, the most accepted by consumers, and has no bitter flavor. in vitro and in vivo studies have reported that rebaudioside a has no mutagenic, teratogenic, or fer tility effects (aranda-gonzález et al., 2014). the nopal pads or cladodes are little accepted by many consumers, due to its mucilaginous texture, acidity, astringency, and herbal flavor. as an alternative to increase their utilization and consumption, nopal cladodes were transformed into a functional snack by preparation of dehydrated flakes and sweetened with rebaudioside a to improve their texture and flavor, as well as to maintain the nutritional, medicinal, and nutraceutical properties of nopal. in this context, the objectives were to evaluate the ascorbic acid, total phenolic, and flavonoid (quercetin, kaempferol, and isorhamnetin) contents, and the antioxidant activity of four nopal cultivars (nopalea cochenillifera cv. texas and opuntia ficus-indica cv. jade, milpa alta, and atlixco), as well as to develop nopal flakes, sweetened with rebaudioside a, from the cultivar with best nutraceutical quality and sensory acceptability. materials and methods plant material small nopal cladodes (20 days of age) of o. ficus-indica cv. jade (oj), atlixco (oa) and milpa alta (oma), and n. cochellinifera cv. texas (nt) were harvested free from diseases, physical damages, and morphological alterations in campo de experimental la nopalera of universidad autónoma chapingo. samples were freeze-dried (model 7670520, labconco, kansas, usa) at -50 °c and 0.1 mbar for 18 h. each experimental unit consisted of three different plants, and each analysis was done with three replicates. quantification of ascorbic acid the quantification of ascorbic acid was done with the spectro photometric method described by dürüst et al. (1997), which is based on color reduction of the indicator dichloroindophenol. the results were expressed in mg of ascorbic acid per 100 g dry weight (mg 100 g-1 dw). total phenolics freeze-dried nopal (0.3 g) was pulverized and mixed with 10 ml of 80% (v/v) meoh aqueous solution. the mixture was homogenized 170 l.l. cruz-de la cruz, r. garcía-mateos, c. ybarra moncada, j. corrales-garcía by using a vortex and sonicating for 20 min at room temperature (25 ± 2 °c). the extracts were left to rest for 12 h under refrigeration (4 °c) and darkness. afterwards, the extracts were filtered with whatman filter paper no. 1 for the quantification of total phenolics, flavonoids, and antioxidant activity. the content of total phenolics was obtained by the folin-ciocalteu method (gülçin et al., 2004). total phenolics were expressed in mg of gallic acid equivalents per g dw (mg gae g-1 dw). total flavonoids total flavonoids were determined, based on the spectrophotometric method described by gülçin et al. (2011), where 10% (w/v) alcl3 was used as indicator. the results were expressed in mg of quercetin equivalents per g dw (mg qe g-1 dw). antioxidant activity antioxidant activity was measured by the dpph assay (kuskoski et al., 2005). dpph (100 μm) in 80% (v/v) methanol was mixed with the sample, and the reaction was incubated for 60 min. the results were expressed in μmol trolox equivalents per g dw (μmol te g-1 dw). the percentage of inhibited dpph was determined by using the formula %dpph inhibited = (absblank – abssample) / (absblank) *100 (eq. 1) where abs = absorbance and blank = dpph solution at a concentration of 100 μm. identification and quantification of individual flavonoids identification and quantification of individual flavonoids was performed, based on the methodology reported by gray et al. (2006). freeze-dried nopal powder (1.5 g) was blended with 25 ml of etha nol and 10 ml of distilled water. the mixture was sonicated for 30 min at room temperature. thereafter, 4 ml of concentrated hcl were added and the solution was maintained at reflux for 3 h. the volume of the cold mixture was completed to 50 ml with ethanol and was filtered through a millipore nylon membrane (0.22 μm). the extracts were analyzed with a reverse phase hplc system (shimadzu, kyoto, japan), equipped with a diode array detector (dad) and a c18 analytic column (250 × 4.6 mm i.d.: 5 μm) (phenomenex® 110a, germany). the injection volume was 10 μl. as mobile phase, a mixture (50:50, v/v) of methanol and an aqueous solution of phosphoric acid (0.5%) was used in isocratic conditions, with a flow rate of 1.2 ml min-1, and a total elution time of 17 min. column temperature and pressure were 35 °c and 1900-1910 psi, respectively. quercetin, kaempferol, and isorhamnetin were detected at 252 nm and identified by using their respective standards. preparation of sweetened nopal flakes for the preparation of nopal flakes, homogenous nopal cladodes were used with a weight of 187 ± 5.26 g from the oa cultivar, which presented the highest phenolic content and antioxidant activity, compared with the other cultivars. fresh, disinfected nopals were cut transversely into flakes (3 mm thick). the flakes were submerged in different solutions of the sweetener rebaudioside a (2, 4, 6, and 8 g l-1) for one hour in a 1:1.5 proportion (nopal:solution). un sweetened flakes were used as control. previously, the optimal freeze-drying time was determined by drying kinetics and desorption isotherms with three different concentrations of rebaudioside a (0, 5, and 10 g l-1) at a temperature of -50 °c and a pressure of 0.01 mbar. equilibrium moisture content and water activity (aw) of the flakes were measured in 3-h intervals until concluding 21 h. equilibrium moisture content was determined with the equation (geankoplis, 1998) xt = (mi − mss) / mss (eq. 2) where xt = equilibrium moisture content (g of water g-1 dry weight); mi = flake weight (g) per time; mss = flake dry weight (g). moisture content was determined according to aoac method and aw with a aw meter (aqualab, decagon devices, washington, usa). afterwards, both sweetened and unsweetened flakes were frozen in liquid nitrogen and then dehydrated by freeze-drying. the freezedried flakes were covered with aluminum foil and placed in a desiccator to prevent the absorption of moisture. quantification of rebaudioside a sweetened and pulverized flakes (2.0 g) were blended with 10 ml of 70% (v/v) ethanol solution. the mixture was placed in a water bath at 70 °c for 30 min. subsequently, the extracts were filtered with a millipore nylon membrane (0.22 μm). the extracts were analyzed in a reverse phase hplc system (perkin elmer, usa), equipped with a dad and a c18 analytic column (150 × 4.6 mm i.d.: 5 um) (thermo scientific® hipersil ods). injection volume was 10 μl. as mobile phase, acetonitrile and water (80:20) were used, acidified to ph 3 with phosphoric acid, with a flow rate of 1 ml min-1 and a total elution time of 3.5 min. column temperature was room temperature. rebaudioside a was detected at 210 nm and identified by using its respective standard. sensory evaluation sensory evaluation of unsweetened flakes and flakes sweetened with different concentrations of rebaudioside a (2, 4, 6, and 8 g l-1) was done to identify the rebaudioside a concentration with most acceptability by the panelists. the evaluation was done by applying two reference scales: intensity (just about right, jar) and preference (hedonic). the panel was made of 100 non-trained judges (53 men and 47 women) of 15-26 years of age. each panelist evaluated at random the flakes with the five different concentrations of rebaudioside a. statistical analysis the data were analyzed using a randomized complete block design (rcbd), and statistical differences were determined by an analysis of variance (anova), followed by tukey’s mean difference test (p < 0.05). all data were analyzed by using sas software. results and discussion nutraceutical content and antioxidant activity in four nopal cultivars the contents of ascorbic acid, total phenolics, total flavonoids, antioxidant activity, and inhibited dpph after 60 min of reaction are presented in tab. 1 for the cultivars oj, oa, and oma (o. ficusindica) and nt (n. cochellinifera). the ascorbic acid content in the oma and oa cultivars was superior (38.5 and 43.1 mg 100 g-1 dw, respectively) to the same cultivars (19.21 and 25.52 mg 100 g-1 dw, respectively) reported by ramírez-moreno et al. (2013); but medina-torres et al. (2011) found a greater concentration (205.62 mg 100 g-1) for the oma cultivar recollected from different regions to that of the present study. on the other hand, the concentration of phenolic compounds in the studied cultivars (2.9-8.1 mg gae g-1 dw) was within the interval published by guevara-figueroa et al. (2010) for other mexican cultivars of the opuntia genus: tapón (i and ii), blanco, and manso (2.0, 5.2, and 11.7 mg gae g-1 dw, respectively). total flavonoids are not affected by mechanical damages or manipulation of the food, just as it has been reported in other sweetened nopal fl akes 171 phenolic compounds that respond to stress applied to food, such as storage time, refrigeration temperature, and uv light, conditions which cause an increase in the phenolic content as a defense mechanism (de ancos et al., 2009). finally, the cultivars oj, oa, and oma presented an antioxidant activity that was superior to cultivar nt (n. cochellinifera). the oa cultivar excelled for its high content of phenolic compounds and antioxidant activity, reason for which it was selected to prepare the sweetened fl akes. in fig. 1 are shown the chromatograms of the fl avonoid profi le of the four cultivars evaluated in this study. quercetin, kaempferol, and isorhamnetin were identifi ed in all cultivars. these three fl avonoids have been found in several species of the opuntia genus (o. fi cus-indica, o. humifusa, o. monacantha, o. lindheimeri, o. robusta, o. streptacantha, o. undulata, o. rastrera y o. leucotricha) (medina-torres et al., 2011; jun et al., 2013; valente et al., 2007; santos-zea et al., 2011). furthermore, glycosylated fl avonoids have also been identifi ed in the opuntia genus (quercetin-3-o-β-glucopyranoside, quercetin-3-o-rutinoside or rutin, kaempferol-3-o-rutinoside or nicotifl orin, isorhamnetin-3-o-rutinoside or narcissin and isorhamnetin-3-o-glucoside) (guevara-figueroa et al., 2010). however, these metabolites have not been studied in the nopalea genus. the results of the present study could contribute to the chemotaxonomic differences between the species o. fi cus-indica and n. cochellinifera. in addition, the identifi cation of these fl avonoids could explain some of the medicinal properties of nopal. fig. 2 depicts the concentrations of quercetin, kaempferol, and isorhamnetin of the cultivars of this study. isorhamnetin was the fl avonoid that was found in the highest concentration in oma cultivar (3.902 mg g-1 dw), whereas the most prevalent fl avonoid in the oj, oa, and nt cultivars was kaempferol with similar concentrations between them (1.6112.284 mg g-1 dw). the concentrations of the three fl avonoids of all cultivars were inferior to those found by medina-torres et al. (2011) for the same nopal species (o. fi cusindica) (1.995, 2.201, and 4.065 mg g-1 dw, respectively). however, the concentrations of kaempferol and isorhamnetin were superior tab. 1: nutraceutical content and antioxidant activity of four nopal cultivars. cultivar ascorbic acid total phenolic compounds total fl avonoids antioxidant activity dpph inhibited (mg 100 g-1 dw) (mg gae g-1 dw) (mg qe g-1 dw) (μmol te g-1 dw) (%) nt 28.4 ± 1.51 d 4.0 ± 0.51 b 1.9 ± 0.01 bc 5.9 ± 0.58 b 42.6 ± 4.06 b oj 48.9 ± 1.11 a 2.9 ± 0.27 c 2.0 ± 0.14 b 8.1 ± 0.16 a 57.2 ± 1.11 a oa 43.1 ± 0.42 b 8.1 ± 0.46 a 1.7 ± 0.06 c 8.3 ± 0.35 a 58.4 ± 1.18 a oma 38.5 ± 1.51 c 4.6 ± 0.21 b 2.3 ± 0.04 a 8.2 ± 0.17 a 58.9 ± 2.40 a values represent the mean ± standard deviation of 3 replicates. different letters in the same column indicate statistically signifi cant difference by tukey’s test (p < 0.05). abbreviations: nt = nopalea cochellinifera cv. texas; oj, oa, and oma = o. fi cus-indica cv. jade, atlixco, and milpa alta, respectively; gae = gallic acid equivalents; qe = quercetin equivalents; te = trolox equivalents; dpph = free radical 2,2-diphenyl-1-picrylhydrazyl. fig. 1: chromatograms obtained by reverse phase hplc of the fl avonoid profi le identifi ed in four nopal cultivars. a) o. fi cus-indica cv. atlixco; b) o. fi cusindica cv. milpa alta; c) o. fi cus-indica cv. jade; d) n. cochellinifera cv. texas. abbreviations: q = quercetin, k = kaempferol, i = isorhamnetin. fig. 2: flavonoid content in four nopal cultivars. values represent the mean ± standard deviation of 3 replicates. bars with different letters for the same fl avonoid aglycone indicate statistically signifi cant difference by tukey’s test (p < 0.05). abbreviations: dw = dry weight; nt = n. cochellinifera cv. texas; oj, oa, and oma = o. fi cus-indica cv. jade, atlixco, and milpa alta, respectively. quercetine kaempferol isoramnetine flavonoids aglycone oa oma oj nt c o n c e n tr at io n (m g ·g -1 d .w .) 172 l.l. cruz-de la cruz, r. garcía-mateos, c. ybarra moncada, j. corrales-garcía to those reported for the cultivars jalapa and villanueva (o. fi cusindica) (0.119 and 0.474 mg g-1 dw kaempferol, and 0.726 and 0.654 mg g-1 dw isorhamnetin, respectively) by santos-zea et al. (2011). the signifi cant differences observed in the nutraceutical content and antioxidant activity between the cultivars of the present study and the differences found with other studies could be due, as in other foods, to diverse factors: genetic (ramírez-tobías et al., 2012), physiological response to manipulation (de ancos et al., 2009), ripening stage (bakhshi and arakawa, 2006), agronomic practices, environmental conditions (hagen et al., 2007), and postharvest handling (tsao, 2007). in the case of environmental conditions and postharvest handling, plants synthesize fl avonoids and other phenolic compounds as a defense mechanism against uv light, water stress, and predators. however, the chemical composition of a food can also affects the content of some metabolites. villaño et al. (2007) found that the content of phenolic compounds can vary, depending on the composition of the food matrix. in this context, nopal is characterized by a high concentration of mucilage (polysaccharide consisting of galactose, rhamnose, arabinose, and xylose), and its binding to phenolics could explain the low phenolic concentration when only free phenolics are quantifi ed, which could also explain a possible decrease in antioxidant activity (kumar et al., 2006). nopal fl akes from o. fi cus-indica cv. atlixco (oa) drying kinetics and desorption isotherms for the preparation of nopal fl akes, cladodes from the oa cultivar were used, which presented the highest content of phenolic compounds and antioxidant activity compared with the remaining cultivars. the preliminary study of drying kinetics and desorption isotherms, obtained with different rebaudioside a concentrations (0, 5, and 10 g l-1), allowed to determine the optimal drying time, where moisture content and aw of the nopal fl akes were maintained constant (fig. 3). nopal fl akes from oa cultivar, with an initial moisture content of 94.5 ± 0.56% (17.18 gh2o g-1 dw) and aw of 0.987 ± 0.059, were dehydrated until reaching the equilibrium moisture content of 3.36 ± 0.25 % (0.592 gh2o g -1 dw) and aw of 0.217 ± 0.003 at -50 °c and a pressure of 0.01 mbar (fig. 3a and 3b). from 18 h of freeze-drying onwards, the moisture content and aw of the nopal fl akes were maintained constant; therefore, 18 h was chosen as the optimal drying time for the preparation of nopal fl akes, sweetened with rebaudioside a at the concentrations of 0, 2, 4, 6, and 8 g l-1. the concentration of rebaudioside a (0, 5, and 10 g l-1) added to the nopal fl akes did not cause a signifi cant effect over the drying kinetics by freeze-drying. however, the fl akes sweetened with rebaudioside a had a lower aw than unsweetened fl akes (control) (fig. 3b). this could have been due to an interaction between rebaudioside a and the fl akes’ surface, leading to a decrease of available water of the food (shivhare et al., 2004). the desorption isotherms (fig. 3b) correspond to type iii, according to brunauer’s classifi cation (mathlouthi and roge, 2003), characteristic of foods with high content of polysaccharides, such as mucilage of nopal. after 18 h of dehydration, the nopal fl akes had a crispy texture and were stable, and the low aw value is associated with a high level of preservation and shelf-life, since foods with an aw below 0.4 are resistant to microbial deterioration and are less susceptible to enzymatic reactions (igathinathane et al., 2007). sensory evaluation flakes sweetened with 6 g l-1 of rebaudioside a were the best evaluated sensorially (tab. 2), since most panelists (64%) rated their sweettab. 2: sensory evaluation of nopal fl akes of o. fi cus-indica cv. atlixco, sweetened with different concentrations of rebaudioside a. rebaudioside a jar scale hedonic scale (g l-1) response “little sweet” response “adequate” response “very sweet” intensity score preference score % % % 0 95 5 0 1.1 e 3.3 c 2 46 48 6 2.2 d 5.3 b 4 31 55 14 2.7 c 5.4 ab 6 12 64 24 3.2 b 5.9 a 8 5 52 43 3.8 a 5.7 ab values represent the mean ± standard deviation of 100 panelists. different letters in the same column indicate statistically signifi cant difference by tukey’s test (p < 0.05). abbreviations: jar = just about right. fig. 3: drying kinetics (a) and desorption isotherms (b) of nopal fl akes of o. fi cus-indica cv. atlixco, preliminarily sweetened with three concentrations of rebaudioside a (0, 5, and 10 g l-1). values represent the mean ± standard deviation of 3 replicates. abbreviations: xt = equilibrium moisture content, aw = water activity, dw = dry weight. sweetened nopal fl akes 173 ness intensity as “adequate” (jar scale) and were the fl akes that obtained the highest score in preference (hedonic scale) (rothman and parker, 2009). therefore, these fl akes were selected for further analyses. incorporated rebaudioside a hplc analysis allowed to identify and establish for all treatments the real concentration of rebaudioside a incorporated by immersion to the nopal fl akes. fig. 4 shows the chromatograms of rebaudioside a as a standard (fig. 4a) and in sweetened nopal fl akes (fig. 4b). the fl akes that were best scored in the sensory evaluation were those at a concentration of 6 g l-1 of rebaudioside a and presented a concentration determined by hplc of 1.1 ± 0.013mg g-1 of the sweetener (tab. 3). fl akes. the signifi cant differences found between the nutraceutical content of sweetened and unsweetened fl akes could be attributed to leaching of soluble nutraceuticals during immersion in the rebaudioside a solution (naidu, 2003). however, kumar et al. (2006) explained that a decrease in phenolic compounds could be also due to the presence of hydrophilic interactions with components of the food matrix; in this case, an interaction with rebaudioside a. but, despite the loss of phenolic compounds, the antioxidant activity was less affected, probably caused by rebaudioside a (tavari et al., 2015). this phenomenon was observed by carbonell-capella et al. (2013) in papaya, mango, and orange juices, processed with high pressures, where the incorporation of glycosides from s. rebaudiana increased the antioxidant activity of these beverages. fig. 5 depicts the concentrations of quercetin, kaempferol, and isorhamnetin in sweetened and unsweetened fl akes. no signifi cant differences were observed between the fl akes for none of the fl avonoids, but the amounts were inferior to those published by medinatorres et al. (2011) in nopals of o. fi cus-indica (1.99, 2.20, and 4.06 mg g-1, respectively). however, the authors reported that the content of these metabolites was reduced almost entirely after exposure of convective drying at a temperature of 65 °c. the consumption of quercetin (2 mg per kg body weight in humans) has shown signifi cant effects in the reduction of triglycerides, cholesfig. 5: concentration of quercetin, kaempferol, and isorhamnetin in unsweetened fl akes and fl akes sweetened with rebaudioside a (6 g l-1) of nopal o. fi cus-indica cv. atlixco. values represent the mean ± standard deviation of 3 replicates. bars with different letters for the same fl avonoid aglycone indicate statistically signifi cant difference by tukey’s test (p < 0.05). abbreviations: dw = dry weight. tab. 3: concentration of rebaudioside a incorporated to nopal fl akes of o. fi cus-indica cv. atlixco after being submerged in different solutions of rebaudioside a. concentration of real concentration of rebaudioside a rebaudioside a in the immersion solution in the nopal fl akes (g l-1) (mg g-1) 0 nd 2 0.8 ± 0.04 d 4 1.0 ± 0.05 c 6 1.1 ± 0.01 b 8 1.3 ± 0.03 a values represent the mean ± standard deviation of 3 replicates. different letters in the same column indicate statistically signifi cant difference by tukey’s test (p < 0.05). abbreviations: nd = not detected. fig. 4: chromatograms of pure rebaudioside a (a) and nopal fl akes of o. fi cus-indica cv. atlixco, sweetened by immersion in a rebaudioside a solution (6 g l-1) (b). nutraceutical content the nutraceutical content of the most sensorially accepted sweetened fl akes of the oa cultivar (6 g l-1) was evaluated and compared with unsweetened fl akes. tab. 4 shows the levels of ascorbic acid, total phenolics, total fl avonoids, and antioxidant activity of both types of tab. 4: nutraceutical content and antioxidant activity of unsweetened fl akes and fl akes sweetened with rebaudioside a (6 g l-1) of nopal o. fi cusindica cv. atlixco. nutraceutical unsweetened sweetened recovery property fl akes fl akes (%) aa (mg 100 g-1) 38.0 ± 2.21 a 33.9 ± 1.25 b 89.20 tp (mg gae g-1) 5.9 ± 0.05 a 5.0 ± 0.19 b 84.87 tf (mg qe g-1) 2.9 ± 0.22 a 2.9 ± 0.11 a 99.74 aox (μmol te g-1) 9.8 ± 0.09 a 9.0 ± 0.07 b 91.35 (% dpphinhibited) 55.8 ± 0.47 a 51.1 ± 0.39 b 91.71 values represent the mean ± standard deviation of 3 replicates. different letters in the same row indicate statistically signifi cant difference by tukey’s test (p < 0.05). abbreviations: aa = ascorbic acid, tp = total phenolics, tf = total fl avonoids, aox = antioxidant activity, gae = gallic acid equivalents, qe = quercetin equivalents, te = trolox equivalents, dpph = free radical 2,2-diphenyl-1-picrylhydrazyl. quercetine kaempferol isoramnetine flavonoids aglycone hse her c o n c e n tr at io n (m g ·g -1 d .w .) 174 l.l. cruz-de la cruz, r. garcía-mateos, c. ybarra moncada, j. corrales-garcía terol, and glucose in blood, as well as decrease in weight gain (rivera et al., 2008). this dose would be equivalent to eating approximately 130 g of sweetened flakes. furthermore, it has been reported that kaempferol inhibits proliferation of cancer cells and preserves the viability of normal cells (chen and chen, 2013). moreover, recent studies have revealed that isorhamnetin glucosides of o. ficus-indica prevent the development of metabolic abnormalities associated with obesity induced by a high-fat diet and cause anti-inflammatory effects (antunes-ricardo et al., 2015) in mice. finally, steviosides (rebaudioside a and stevioside) provide some benefits to health, such as reduction of blood pressure (hsieh et al., 2003) and can be consumed by patients with diabetes type i and type ii without presenting adverse effects (barriocanal et al., 2008). hence, sweetened nopal flakes could be a functional snack and could possibly be consumed by patients with diabetes. conclusions the nopal cultivars with greater commercial demand o. ficus-indica presented higher antioxidant activity than the underused nt cultivar from n. cochellinifera. the profile of quantified and identified flavonoids in this study could contribute to the chemotaxonomy of both nopal species. the nopal flakes of the oa cultivar (o. ficusindica), sweetened with rebaudioside a (1.118 mg g-1), had a good sensory acceptability. the nutraceutical content of the flakes was decreased during immersion in the rebaudioside a solution; 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accepted: may 3, 2021) * corresponding author summary in this study, the impact of postharvest treatments with calcium chloride (cacl2) in-combination with postharvest coating treatments with plant natural substance arabic gum or cactus mucilage on the qua lity and storage life of tomato fruits during cold storage (10 ± 1 °c, rh 85% ± 3) was evaluated. results of dipping tomato fruits in 6% cacl2 for 10 minutes combined with postharvest coating treatments with either 10% arabic gum or 50% cactus mucilage for 3 minutes showed significant (p<0.05) higher value of fruit firmness, titratable acidity, reduce the percentage of weight loss and percent of decayed fruits. treated fruits took longer to change color from pink to red compared with non-treated fruits. this study showed that dipping tomato fruits in 6% cacl2, or coating with different natural substances alone or in combination with cacl2, enhances tomato fruits’ physical and chemical properties. moreover, preserving tomato fruits can be preserved for a longer time compared with control fruits and maintain the overall fruit quality. keywords: edible coating, arabic gum, cactus mucilage, storage life, physio-chemical properties, firmness, titratable acidity. introduction tomato (solanum lycopersicum l.) of the solanaceae family is one of the most grown and fresh consumed vegetables worldwide (adato et al., 2009). they have a high nutritional value of vitamins, minerals, natural antioxidant compounds and amino acids. in addition, several other beneficial health-related substances were found in tomatoes, such as carotenoids, lycopene and β-carotene, which have been correlated with reducing risk of cancer and some cardiac diseases in humans (borguini and torres, 2009). tomato is classified as a climacteric and highly perishable fruit with a high respiratory peak associated with a high ethylene production rate after harvest (ali et al., 2013), resulting in a short shelf life of usually one to two weeks (kapsiya et al., 2015). during ripening, tomato’s physical properties and chemical composition change dramatically, affecting appearance, color, firmness, flavor, as well as the contents of phenolic, flavonoids and ascorbic acid (garcía et al., 2014; ali et al., 2013). therefore, susceptibility to substantial postharvest qua lity reduction of tomato fruits during handling, transportation, storage and marketing is high (nasrin et al., 2008). several postharvest techniques have been applied to manage fruit ripening by reducing the respiration rate and associated ethylene synthesis, such as controlled atmosphere storage, modified atmospheric gas composition, temperature and humidity control. however, these techniques are expensive (baldwin et al., 2000). edible coatings have been considered as an eco-friendly and costefficient alternative technology to preserve quality and enhance agricultural commodities’ shelf life (hernalsteens, 2020). edible coating of fruits provides several benefits, such as preserving the internal gas composition in tomato, mango, strawberry and guava (zegbe et al., 2015; dhall, 2013). they would form a semi-permeable barrier to gases, and water vapor on fruits surface, thus reducing weight loss, enabling the controlled release of bioactive compounds, enhancing the antioxidant activity, total phenolic contents and leads to preserving the quality attributes of fruits during storage (hernalsteens, 2020; ali et al., 2010). aloe vera, arabic gum, cactus mucilage and guar gum have been used on tomato (ali et al., 2013; ali et al., 2010; al-juhaimi, 2012; garcía et al., 2014). arabic gum is dried gummy exudates from the stem and the branches of acacia senegal (ali et al., 2010) and is composed of a mixture of polysaccharides and glycol proteins. it is used in industries for film-forming and encapsulation (ali et al., 2013). it was also used to delay fruit ripening, extend the shelf life, and reduce fruit browning in guava, strawberry and apple fruits (gurjar et al., 2018; kashif and fahad, 2013; el-anany, 2009). cactus mucilage is a complex hydrophilic polysaccharide found in opuntia ficus-indica pads, that has the potential to create a barrier against water loss and gas exchange. therefore, it could enhance postharvest life of many fruits when applied as an edible coating (gheribi and khwaldia, 2019). according to our best of knowledge, no records are showing the usage of cactus mucilage as a coating substance on tomato fruits so far, while it has been tested on as an edible coating on strawberry (delvalle et al., 2005) and guava fruits (zegbe et al., 2015). it has been demonstrated, that cactus mucilage has a positive impact on extending shelf life, maintaining some quality parameters as firmness and delaying fruit skin color development. postharvest application of calcium chloride on many fruits and vege tables found to delay softening by increasing rigidity of the middle lamella and act as a binding agent in the cell wall to form calcium pectate, which helps conserve the quality and increases the storage life of fruits and make them firmer (mehmet et al., 2016; gao et al., 2020). several researchers reported the benefits of edible coating of fruits with natural substance and calcium chloride (cacl2) treatment (abbasi et al., 2013; senevirathna and daundasekera, 2010), but none of them used arabic gum or cactus mucilage in combination with cacl2 as an edible coating for the preservation and extension of storage life of fresh tomato fruits. the current work aimed to study the effect of several novel combinations of edible coating substances as arabic gum or cactus mucilage and cacl2 postharvest dipping treatments on tomato fruits’ physicochemical properties under cold storage. material and method plant materials and postharvest treatments tomato fruits cultivar izmir were harvested at breaker stage (i.e. the distal end of the fruits just turns yellowish ring) from a commercial farm in tubas-city, the northern part of west bank, palestine. immediately after harvesting, all fruits were transported and careful calcium chloride coating combined with natural substance 101 ly handled to the national agricultural researcher center (narc), ministry of agriculture in qabatya, palestine. only firm and welldeveloped fruits with uniform medium size, free from disease, injures, and bruises, were selected for further use in the experiments. dipping solutions preparation calcium chloride cacl2 6% (w/v) solution was prepared by dissolving 60 g of edible grade cacl2 (wei bang chemical limited company, xiamen ditai chemicals china) in 1000 ml distilled water. the solution was constantly stirred using a magnetic stirrer (model sp 1842026 barnstead thermolyne usa) for 30 min until fully dissolved. arabic gum solution 10% (w/v) was prepared by dissolving 100 g of arabic gum powder (kapadia gum industries pvt ltd. mumbai 400056, india) in 1000 ml distilled water. the solution had been stirred at a constant heat of 40 °c for 60 min on a magnetic stirrer (model sp 18420-26 barnstead thermolyne usa), then filtrated through cheesecloth and filter paper (pore size of 5 μm) to remove any undissolved material. the solution was allowed to cool to room temperature before use (ruelas-chacón et al., 2017). cactus mucilage extract (2/1) (w/w) was freshly prepared from cactus opuntia ficus-indica pads harvested from plants in tubas, palestine. pads were cleaned with 1% chloride, distilled water, then peeled and sliced into 1 cm cubes. thereafter, squeezed by fruit juicers (gje5437, 800wtt model jepas, china) and diluted with distilled water by adding half the weight to obtain cactus mucilage (2:1) (w/w) (zegbe et al., 2015). treatments and experimental work selected fruits of the same size shape and free from visual damage or defects were randomly divided into six treatment groups. each treatment was triple replicated and consisted of 90 fruits per replicate. a separate group of 15 fruits per treatment was used for weight loss assessment. single coated postharvest fruit dipping treatments were applied as; (i) 6% cacl2 for 10 min. (ii) 10% arabic gum (w/v) for 3 min. (iii) cactus mucilage (2/1) (w/w) for 3 min. double coated postharvest fruit dipping treatments were applied as; (i) 6% cacl2 for 10 min, air-dried for 30 min at room temperature, then followed by another dipping in 10% arabic gum (w/v) for 3 min. (ii) 6% cacl2 for 10 min, air-dried for 30 min at room temperature, then followed by another dipping in 50% cactus mucilage (2/1) (w/w) for 3 min., (iii) distilled water for 3 min served as a control. single and doublecoated postharvest treated fruits were left to air-dried for 30 min at room temperature before storage. treated fruits were packed in plastic boxes covered with polyethylene film and stored in a controlled walk-in cold storage room with relative humidity (rh) of 85% ± 3 and temperature at 10 ± 1 °c for 35 days. postharvest fruit quality assessment fruit quality parameters, such as flesh firmness, physical weight loss, total soluble solids (tss) and titratable acidity (ta) were evaluated on the date of the experiment (zero-day) before treatments, and every four days post the experiment first date for 35 days. three fruits were selected randomly from every replicate per treatment used for the assessment. weight loss (%) the same fruit was weighed every four days and the difference in weight loss was expressed in percentage on a fresh weight basis following the standard method; the percent of weight loss of tomatoes fruit sample was calculated using the following formula (pila et al., 2010): percent of weight loss = · 100%. decay percentage fruit decay was determined by visual observation. the various fruits peel spots and rotting development were observed, and the percent of decay was calculated for both treated and non-treated fruits following the formula (pila et al., 2010). percent of decay percentage = · 100%. color development colorimetric measurements of the tomato fruit skin colour were determined for three fruits per replicate per treatment from both sides of each fruit using a chroma meter (konica minolta) hunter. ‘l’, ‘a’ and ‘b’ values measured light reflection and intensity from white to black, green to red and blue to yellow, respectively (voss, 1992). fruit firmness fruit firmness was measured using a digital penetrometers device (lutron fr-5120) with 6 mm steel tip head to measure the average amount of force needed to puncture the fruit’s flesh. the steel cy linder tips were applied to opposite sides of each tomato fruit on an area with the skin removed to expose the fresh flesh (kumah and olympio, 2011). two fruits per replicate per treatment were randomly selected. the results were measured in (n) force unit. total soluble solid (tss) total soluble solid (tss) was measured by a digital refractometer device (model milwaukee ma871) (°brix). tss was calculated for each fruit as an average of two readings of the digital refractometer, and the result was recorded as a degree °brix (pila et al., 2010). the refractometer was calibrated using distilled water before readings were taken. titratable acidity (ta) titratable acidity (ta) in tomato fruit juice samples was quantified by titration method using 0.1 mol l-1 naoh, and the endpoint was ph (8.1). 15 ml of tomato juice were diluted with 85 ml of distilled water; 4 drops of phenolphthalein were added as an indicator for a color change to purple. the tomato fruit juice then titrated with 0.1 naoh to ph (8.1) using the ph meter. the protocol was established by (garner et al., 2003) from uc davis, california, usa. used with modifications to estimate the titratable acidity in the sample. the result was expressed as a citric acid percentage using the following formula: acid % = mill equivalent factor = 0.0064 g statistical analysis all statistical tests were performed using analysis of variance (anova) using the sas (sas institute inc., 1998), duncan multiple range test was used for evaluating mean separation at 5% level of probability, using sigma plot (version 8) in draw figure to show a significant difference. result and discussion weight loss in general, fruit weight loss was normally attributed to fruits senescence or desiccation (nasrin et al., 2008); it was also used as a qua lity index in the postharvest life of fruits. in this study, all samples initial weight final weight initial weight decay fruit initial fruit (mls naoh used)·(0.1 n naoh)·(milliequivelent factor for tomato)·(100) grams of sample 102 f. sati, t. qubbaj showed a gradual loss of weight during storage, starting on day four of the experiment in both treated and non-treated fruits (fig. 1, a). from day 8 until 16, no significant (p<0.05) differences were found between different coating treatments, except coating with 50% cactus mucilage. the control showed a significant (p<0.05) higher weight loss compared to all other coating treatments (fig. 1, a). furthermore, 6% cacl2 in-combination treatments of either arabic gum or cactus mucilage postharvest coating effectively reduced weight loss after 20 days up to the end of the storage period (fig. 1, b). the results are in agreement with previous studies, which reported that weight loss was reduced with natural substance postharvest coating treatment such as arabic gum in tomato (ali et al., 2013), combined coating of arabic gum, chitosan with pectin in mango (chien et al., 2007). furthermore, arabic gum alone or combined with calcium chloride on mango (khaliq et al., 2015). meanwhile, garcía et al. (2014) found coating with pure aqueous extract of aloe vera did not delay the weight loss in tomato. the loss of weight in fruits occurred during the transpiration process through the fruits’ surface, which is a normal metabolic process resulting in shriveling and deterioration of the fruit, depends on the gradient of water vapor pressure between the surrounding atmosphere and the fruit tissue (baldwin et al., 1999). in this study, the reduction in weight loss was probably due to the effects of the coating substance (arabic gum and cactus mucilage) and the cacl2. where, combination treatments act as a barrier against o2, co2, limiting water transfer and solute movement, therefore, reducing respiration, water loss and oxidation reaction rates (baldwin et al., 1999). moreover, cacl2 treatments found to delay ripening and extended storage life (irfan et al., 2013), through reducing the concentration of o2 and increasing the concentration of co2 in which help in delay senescence and ripening by reducing respiration and ethylene production rate in climacteric fruits as tomato fruits (pila et al., 2010). fruit decay during this study, the coating treatments had delayed decay symptoms of fruits upto 20 days post-treatment compared with 12 days in non-treated control. at 24 days of cold storage, fruit decay started in all coating treatments and gradually increased in all fruits with storage time, but not in fruits coated with 6% cacl2-treatments incombination with 10% arabic gum (fig. 2, a). furthermore, the combination treatments of 6% cacl2-treatments in-combination with 10% arabic gum or combination with 50% cactus mucilage showed the lower significant (p<0.05) decayed fruits compared to the alone treatment of each of 6% cacl2, 10% arabic gum and 50% cactus mucilage postharvest treatments at day 28, and at the termination day of the experiment (day 35) (fig. 2, b). fruit decay is a natural phenomenon associated with the ripening process and postharvest diseases (khaliq et al., 2015). the current results clearly indicate a significant role of coating substance and calcium chloride combined treatments in reducing fruit decay. calcium chloride plays a major role in strengthening and thickening the middle lamella of the cell wall, through increased formation and deposition of ca-pectate in the cell wall (garcia and herrera, 1996). during fruit ripening under long-term storage, fungi and bacteria were associated with some enzymatic change processes, which trigger damage to fruits and deteriorate the middle lamella, in particu lar plant enzymes associated with the senescence process (zapata et al., 2008). the current results agreed with sharma et al. (1996), who reported dipping apple fruits in calcium chloride reduced postharvest decay and delayed senescence compared to non-treated fruits. in addition, khaliq et al. (2015) reported, coating mango fruits with arabic gum protected the fruits from pathogen infection and suggested coating forms a film on the mango surface. this film acts as a barrier to protect the fruits. fruit color development tomato fruit color is an important factor in determining fruit quali ty. additionally, it is the first sensory parameter for acceptance by consumers (nasrin et al., 2008). our results indicate a normal pattern of color development in non-treated and treated tomato fruits (fig. 3, a). a significant (p<0.05) development of the red color occurred after 8 days in control (non-treated) fruits compared to other different treated fruits. the ‘a’ value is changed from a negative value (green color) to a positive value (red color) (fig. 3, b). no significant changes in color development were observed in other treatments as fruits were still predominately at the mature breaker stage (pink to light red). during the first 8 days, no significant changes were observed in ‘l’ and ‘b’ values among the treatments. the ‘l’ parameter is a sign of fruit from light to dark; all samples showed decrease ‘l’ values with progressing the storage times. coating substance significantly reserved ‘l’ value compared with control (non-treated) fruits. however, no significant differences were observed among the other treatment at day 35 of storage (fig. 3, a). the ‘b’ value also decreased with increasing the storage time in all fruits (fig. 3, c). coating treatments significantly (p<0.05) delayed color development of tomato fruits. storage time(days) d4 d8 d12 d16 d20 f r w t l o s s (% ) 0 2 4 6 8 10 12 control 6%cacl2 10%arabic gum 50%cactus mucilage 6%cacl2+10%arabic gum 6%cacl2+50%cactus mucilage figure(a) a a bb b b a b c c c c a b c cc c a b c ddd a b c c d d storage time(days) d24 d28 d35 f r w t l o ss (% ) 0 20 40 60 control 6%cacl2 10%arabic gum 50%cactus mucilage 6%cacl2+10%arabic gum 6%cacl2+50%cactus mucilage figure(b) a b b c d d a b c c d d a b c c d d fig. 1 (a, b): effect of natural substance postharvest combination treatments with cacl2 on the fruit weight loss of tomato fruits at cold storage. means followed by the same letter are not significantly different (duncan’s multiple range test, p > 0.05) calcium chloride coating combined with natural substance 103 fig. 2 (a, b): effect of natural substance combination with cacl2 on decay tomatoes fruit during cold storage at (10 °c). means followed by the same letter are not significantly different (duncan’s multiple range test, p > 0.05) storage time (days) d28 d35 d ec y p er ce nt (% ) 0 10 20 30 40 control 6%cacl2 10%arabic gum 50%cactus mucilage 6%cacl2+10%arabic gum 6%cacl2+50%cactus mucilage figure (b)a a d c b e d c c dc b e storage time (days) d12 d16 d20 d24 d ec y p er ce nt (% ) 0 2 4 6 8 10 control 6%cacl2 10%arabic gum 50%cactus mucilage 6%cacl2+10%arabic gum 6%cacl2+50%cactus mucilage figure (a) a a b b c c c a a storage time (days) d0 d4 d8 d12 d16 d20 d24 d28 d35 h un te r s ca le v al ue -15 -10 -5 0 5 10 15 20 control 6%cacl2 10%arabic gum 50%cactus mucilage 6%cacl2+10%arabic gum 6%cacl2+50%cactus mucilage a a a bc ab c a a a dc c a b b a b d c b c d bc c d e a d e c b b dc de c c d d c b dc d d d d storage time (days) d0 d4 d8 d12 d16 d20 d24 d28 d35 h un te r s ca le v al ue 16 18 20 22 24 26 28 a b abab a a a c ac b a b c b b b d a b ab bb a a a a c c bc ab a control 6%cacl2 10%arabic gum 50%cactus mucilage 6%cacl2+10%arabic gum 6%cacl2+50%cactus mucilage a storage time (days) d0 d4 d8 d12 d16 d20 d24 d28 d35 h un te r s ca le v al ue 20 25 30 35 40 45 50 55 control 6%cacl2 10%arabic gum 50%cactus mucilage 6%cacl2+10%arabic gum 6%cacl2+50%cactus mucilage figure (a) l-scale a b a a a d c b a ab bc d e dc a b e d d c a b e d c c a b e d c a b f c d e a d c b a d c b a figure (b) a-scale figure (c) b-scale fig. 3 (a, b, c): effect of natural substance combination with cacl2 on the color development of tomatoes fruit stored at cold storage (10 °c). means followed by the same letter are not significantly different (duncan’s multiple range test, p > 0.05) storage time (days) d0 d4 d8 d12 d16 d20 d24 d28 d35 h un te r s ca le v al ue -15 -10 -5 0 5 10 15 20 control 6%cacl2 10%arabic gum 50%cactus mucilage 6%cacl2+10%arabic gum 6%cacl2+50%cactus mucilage a a a bc ab c a a a dc c a b b a b d c b c d bc c d e a d e c b b dc de c c d d c b dc d d d d storage time (days) d0 d4 d8 d12 d16 d20 d24 d28 d35 h un te r s ca le v al ue 16 18 20 22 24 26 28 a b abab a a a c ac b a b c b b b d a b ab bb a a a a c c bc ab a control 6%cacl2 10%arabic gum 50%cactus mucilage 6%cacl2+10%arabic gum 6%cacl2+50%cactus mucilage a storage time (days) d0 d4 d8 d12 d16 d20 d24 d28 d35 h un te r s ca le v al ue 20 25 30 35 40 45 50 55 control 6%cacl2 10%arabic gum 50%cactus mucilage 6%cacl2+10%arabic gum 6%cacl2+50%cactus mucilage figure (a) l-scale a b a a a d c b a ab bc d e dc a b e d d c a b e d c c a b e d c a b f c d e a d c b a d c b a figure (b) a-scale figure (c) b-scale 104 f. sati, t. qubbaj the number of days required for treated tomato fruits to develop color from pink to full red was up to 16 days from harvest, compared to 8 days in control (non-treated) fruits (fig. 3, b). a clear positive influence of postharvest coating by 6% cacl2-treatments in-combination with 10% arabic gum on delaying fruit ripening, as treated fruits required more time to develop red color (16 days), compared to other treatments which developed the red color stage in 12 days (fig. 3 a, b and c). color development in ripe tomato resulted of the de-novo synthesis of carotenoids, mainly lycopene and β-carotene associated with the change in fruit color from green to red as chloroplasts are transformed to chromoplasts (bathgate et al., 1986). coating substance was found to delay chlorophyll degradation and lycopene synthesis, as well as ripening processes compared to uncoated fruits (yaman and bayoindirli, 2002). however, this delayed fruit color development could be attributed to the modified fruits surrounding atmosphere; reduced o2 concentration and increased co2 concentration. this reduces the respiration and ethylene production rate in climacteric fruits (pila et al., 2010) and, therefore, helps in delay ripening and the associated changes in fruit color development. it was reported that arabic gum coatings delayed colour changes in tomato (ali et al., 2010; al-juhaimi, 2012) and guava (gurjar et al., 2018), while calcium chloride delayed colour in fig fruits (irfan et al., 2013). barman et al. (2011) reported that combined application of putrescine and carnauba wax reduced colour changes and preserved pomegranate quality during storage. in this study, the delay in color changes in tomato fruits treated with 6% cacl2-treatments combined with 10% arabic gum might have been resulted from decreased biosynthesis of carotenoids and preservation of chlorophyll content. firmness tomato fruits’ coating by different natural substance (10% arabic gum and 50% cactus mucilage) in addition to 6% cacl2-treatments showed a positive significant (p<0.05) effect on fruits firmness compared to control (non-treated) fruits (fig. 4 a and b). during the time course of the experiments, high significant (p<0.05) values were found in 6% cacl2-treatment in-combination with 10% arabic gum, or 50% cactus mucilage compared to other solo coating treatments and control. therefore, the in-combination treatments preserved tomatoes fruits’ firmness compared to other treatments (fig. 4 a and b). fruit texture is an important characteristic of fresh horticulture produce. the current results indicted non-treated fruits are softening and lost textural quality rapidly compared to treated fruits. these changes are attributed to deterioration in cell structure, cell wall composition and intracellular components during the ripening process under storage conditions (harold et al., 2007) as it had been reported for a wide range of fruits, including tomato (ruelas-chacon et al., 2017), mango (khaliq et al., 2015; chien et al., 2007) and plums (valer et al., 2013). postharvest application of calcium chloride was found to act as a binding agent in the cell wall to form calcium pectate, which increases the rigidity of the middle lamella and cell wall. it also inhibited cell wall activity degrading enzymes, so the outer membrane for the cell wall becomes more strength and rigid (khaliq et al., 2015; pila et al., 2010). coating substances (arabic gum and cactus mucilage) in-combination treatments also helped create a modified atmosphere surrounding the fruit’s surface. whereas elevating the co2 and decreased the fruit respiration rate (ali et al., 2013), therefore limit the activity of softening related biochemical enzymes activity such as; hydrolases, pectin esterase, and polygalacturonase (arah et al., 2016; ruelas-chacon et al., 2017). therefore, the in-combination treatments of cacl2 and coating substances retained significantly (p≤0.05) a higher firmness over the control fruits may be attributed to the thick coating, which created a modified atmosphere around the fruit surface, as a result, reduced changes in pectin substances and activity of cell wall degrading enzymes (khaliq et al., 2015) in addition to the role of calcium in preserve cell wall structure by interacting with pectin in the cell wall to form calcium pectate complexes and reduce the activity of cell wall degrading enzymes. total soluble solid (°brix) in general, there was a gradual increase in total soluble solid (tss) during the entire storage period (fig. 5). no significant difference (p<0.05) was observed between control (non-treated) fruits and different postharvest coating treatments from zero days (harvest day) until 12 days at cold storage (fig. 5 a). while, after 16 days of storage up to the termination day of the experiments (day 35th), a higher significant (p<0.05) difference in total soluble solid was found in control (non-treated) fruits compared to 6% cacl2-treatments incombination with 10% arabic gum or 50% cactus mucilage coating treatments (fig. 5 b). fruit soluble solid concentration is a good index for determining fruit quality and maturity; it increases with maturity and ripening. changes in the soluble solids content of tomatoes are correlated fig. 4 (a, b): effect of natural substance combination with cacl2 on the firmness (n) of tomato fruits at cold storage (10 °c). means followed by the same letter are not significantly different (duncan’s multiple range test, p > 0.05) storage time (days) 0d 4d 8d 12d 16d f ir m n es s( n ) 0 10 20 30 40 control 6%cacl2 10%arabic gum 50%cactus mucilage 6%cacl2+10%arabic gum 6%cacl2+50%cactus mucilage a a aaa a e a bc e c d a b cc e d b c e c d a c d d a b figure (a) storage time (days) 20d 24d 28d 35d fi rm ne ss (n ) 0 10 20 30 40 control 6%cacl2 10%arabic gum 50%cactus mucilage 6%cacl2+10%arabic gum 6%cacl2+50%cactus mucilage c a b cc d a b c e c d a b c c e d a b dc e c d figure (b) calcium chloride coating combined with natural substance 105 storage time (days) 0d 4d 8d 12d d16 °b ri x 4.1 4.2 4.3 4.4 4.5 4.6 control 6%cacl2 10%arabic gum 50%cactus mucilage 6%cacl2+10%arabic gum 6%cacl2+50%cactus mucilage a a a a a c d figure (a) c ab bc b ab ab c c ab b bc d a storage time (days) d20 d24 d28 d35 °b ri x 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 control 6%cacl2 10%arabic gum 50%cactus mucilage 6%cacl2+10%arabic gum 6%cacl2+50%cactus mucilage a a a d a c b figure (b) ee f e d d d b c b b c c e fig. 5 (a, b): effect of natural substance combination with cacl2 on the total soluble solid (°brix) of tomato fruits at cold storage (10 °c). means followed by the same letter are not significantly different (duncan’s multiple range test, p > 0.05) with hydrolytic changes in polysaccharides (hemicellulose and pectin) during ripening (ruelas-chacon et al., 2017). a similar to the presented tss change pattern was found in tomato fruits coated with arabic gum (ali et al., 2010; al-juhaimi, 2012), or with aloe vera (garcía et al., 2014) and mango fruits coated with arabic gum combined with calcium chloride (khaliq et al., 2015). coating with natural substances can reduce the respiration rate and may slow down the synthesis and use of metabolites resulting in lower tss (ali et al., 2013) that may explain the higher concentration of tss in non-treated fruits. titratable acidity (ta) titratable acidity (ta) is the most essential parameter for evaluating fruits quality during storage. lower values in ta concentration in fruits; faster fruit induced senescence (khaliq et al., 2015). in this study, ta values of coated and non-coated fruit during storage decreased with storage time (fig. 6). after 12 days of storage until the termination day of the experiments (day 35th), the total soluble solid values were significantly higher (p≤0.05) in 6% cacl2-treatments in-combination with 10% arabic gum or with 50% cactus mucilage coating treatments compared to other coating treatments and control (non-treated) fruits (fig. 6 a). while after 20 days a significantly (p≤0.05) higher value was found in 6% cacl2 treatments of, coating with either 10% arabic gum or 50% cactus mucilage compared to control (non-treated) fruits (fig. 6 b). titratable acidity decreased progressively with fruit ripening, and the decline rate significantly affected by the coating treatments with 10% arabic gum or with 50% cactus mucilage coating combined with 6% cacl2. the significant lower level of ta in control fruit compared to coated fruit suggested that, coating delayed ripening by providing modified atmosphere around fruit surface and creating a barrier against o2 and co2, led to reduce respiration rate (rodriguez et al., 2006; ali et al., 2010). preservation of titratable acidity by fruit coating treatments has been reported previously for various fruits as tomato coated with arabic gum (ali et al., 2010; al-juhaimi, 2012) or with aloe vera (garcía et al., 2014) and mango fruits coated with arabic gum combined with calcium chloride (khaliq et al., 2015). conclusion this study indicated that coating tomato fruits with either 10% arabic gum or 50% cactus mucilage combined with pre-dipping in 6% cacl2 positively affect the physical and chemical properties of 0+*"g3(a"m&m.: "+*"(a$"0('&(&k.$"&/3;"+," (+)&(+$4",'83("&("/+.;"4(+'&7$"o!q+ q $'"&'$"*+("437*3h/&*(.5" ;3\ $'$*("o#8*/&*i4")8.0 " storage time (days) d0 d4 d8 d12 d16 ta ( % ) 0.0 0.1 0.2 0.3 0.4 0.5 control 6%cacl2 10%arabic gum 50%cactus mucilage 6%cacl2+10%arabic gum 6%cacl2+50%cactus mucilage a a aaaa a aaaaa a aaa aa aa a b c d b c b b e d figure (a) storage time (days) d20 d24 d28 d35 ta ( % ) 0.0 0.1 0.2 0.3 0.4 0.5 control 6%cacl2 10%arabic gum 50%cactus mucilage 6%cacl2+10%arabic gum 6%cacl2+50%cactus mucilage a e a e b c d e a a b d c d d d bbc bc c b d d figure (b) c fig. 6 (a, b): effect of natural substance combination with cacl2 on the titratable acid of tomatoes fruit at cold storage (10 °c). means followed by the same letter are not significantly different (duncan’s multiple range test, p > 0.05) 106 f. sati, t. qubbaj tomato fruits, thus maintaineing the overall quality of fruits. a significant delay in fruit weight loss, firmness, titratable acidity, soluble solids concentration and color development was found during storage at 10 ± 1 °c compared to uncoated control fruit. further research is needed to investigate the influence of the coating on ripening physio logical processes, postharvest microbes inhibition, and on tomato fruit quality attributes under ambient storage conditions. acknowledgments this research was supported by an-najah national university. the authors gratefully acknowledges the excellent technical assistance by the team of the national agricultural researcher center (narc), ministry of agriculture, qabatya, palestine. conflict of interest no potential conflict of interest was reported by the authors. reference abbasi, n.a., zafar, l., khan, h.a., qureshi, a.a., 2013: effects of naphthalene acetic acid and calcium chloride application on nutrient uptake, growth, yield and postharvest performance of peach fruit. pak. j. bot. 45(5), 1581-1587. adato, a., mandel, t., mintz, s., venger, i., levy, d., yativ, m., domínguez, e., wang, z., vos, r., jetter, r., schreiber, l., heredia, a., rogachev, i., aharoni, a., 2009: fruit-surface flavonoid accumulation in tomato is controlled by a slmyb12-regulated transcriptional network. plos genetics. 5(12), e1000777. doi: 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(1067), 423-427. doi: 10.17660/actahortic.2015.1067.58 orcid tawfiq qubbaj https://orcid.org/0000-0003-2258-1371 address of the corresponding author: tawfiq qubbaj, department of plant production and protection, faculty of agriculture and veterinary medicine, an-najah national university, p.o. box 7, nablus, palestine e-mail: tqubbaj@najah.edu © the author(s) 2021. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1016/j.foodres.2006.04.002 http://dx.doi.org/10.1155/2017/8608304 http://dx.doi.org/10.4038/cjsbs.v39i1.2351 http://dx.doi.org/10.1016/j.postharvbio.2012.10.011 http://dx.doi.org/10.21273/hortsci.27.12.1256 http://dx.doi.org/10.1006/fstl.2001.0827 http://dx.doi.org/10.1002/jsfa.3220 http://dx.doi.org/10.17660/actahortic.2015.1067.58 https://orcid.org/0000-0003-2258-1371 art17_akram.indd journal of applied botany and food quality 85, 105 115 (2012) 1department of botany, university of agriculture, faisalabad, pakistan 2department of botany and microbiology, king saud university, riyadh, saudi arabia 3department of botany, gc university, faisalabad, pakistan regulation in some vital physiological attributes and antioxidative defense system in carrot (daucus carotain carrot (daucus carotain carrot ( l.) under saline stress 1saira bano, 1,2muhammad ashraf, 3*nudrat aisha akram, 2f. al-qurainy (received october 3, 2011) * corresponding author summary regulation of some key metabolic phenomena including antioxidative defense system involved in plant salt tolerance is of great concern. changes in chlorophyll pigments, chlorophyll fl uorescence and leaf gas exchange characteristics, glycinebetaine and proline contents, and enzymatic and non-enzymatic antioxidants was assessed in two carrot (daucus carota l.) cultivars, dc-4 and t-29 under saline stress in a greenhouse study. application of different saline regimes (0, 50, 100 and 150 mm nacl) to the growth medium considerably reduced the shoot and root fresh and dry weights, shoot and root lengths, chlorophyll b contents, leaf water potential (ψw), leaf osmotic potential (ψs), photosynthetic rate (arate (arate ( ), water-use effi ciency, sub-stomatal co2 concentration (ci), stomatal conductance (gs), transpiration rate (e), ci/ci/ci a/ca/c ratio, leaf and root k+ and ca2+ contents, leaf mda, total phenolics, total soluble proteins, and activities of cat, sod and pod enzymes, while a marked increase was observed in leaf turgor potential (ψp), leaf and root na+ and clcontents, leaf proline, glycinebetaine (gb), ascorbic acid (asa) and h2o2 contents in both cultivars. of both carrot cultivars, cultivar t-29 was relatively higher in shoot and root fresh weights, root na+, leaf and root ca2+, leaf proline, mda, total phenolics, soluble proteins and activity of sod enzyme. in contrast, cultivar dc-4 was relatively higher in leaf ψw and ψs, leaf k+, root ca2+ and leaf gb as compared to those in the other cultivar. the relatively better growth of cultivar t-29 was found to be correlated with improved leaf water potential, leaf ca2+, proline, phenolics, and activity of sod enzyme under saline conditions. introduction excessive accumulation of toxic ions such as cland na+ is one of the vital environmental factors, which reduce growth and yield in most salt-affected soils world-wide (raathinasabapathi, 2000; xue et al., 2004; ashraf, 2004, 2009; flowers et al., 2010; kanwal et al., 2011). most crop plants are salt sensitive and they are referred to as glycophytes (xue et al., 2004; parida and das, 2005; munns and tester, 2008), because these ions cause impairment in many physiological and biochemical attributes including water relations, antioxidant defense system, photosynthesis, nutritional balance and metabolism of osmoprotectants (shahbaz et al., 2008; ashraf, 2009; akram and ashraf, 2011; sabir et al., 2011). a number of studies have been conducted to investigate salt tolerance potential in terms of better growth and yield production and the associated mechanisms including leaf fl uorescence, gaseous exchange characteristics, antioxidant capacity, osmoprotectants as well as inorganic nutrient accumulation in various crops, e.g., panicum miliaceum (sabir et al., 2011), sunfl ower (akram and ashraf, 2011), safflower (siddiqi et al., 2011), cucumber (shi et al., 2008), sugar beet (ghoulam et al., 2002), cotton (ashraf, 2002), pea (noreen et al., 2010), eggplant (abbas et al., 2010), rice (habib et al., 2010), maize (ali and ashraf, 2011), wheat (perveen et al., 2010, 2011, 2012), and canola (ashraf and ali, 2008). differential response has been observed in relatively salt tolerant cultivars as compared to that of sensitive ones within the same crop species. for example, recently while working with 10 cultivars of proso millet sabir et al. (2011) and 10 cultivars of saffl ower, siddiqi et al. (2011) have found a differential response under saline conditions in terms of high accumulation of proline and enhanced antioxidant capacity in relatively salt tolerant cultivars as compared to salt susceptible ones. one of the potential adaptations to salt stress is the accumulation of osmoprotectants particularly proline and glycinebetaine in the cytoplasm (kumar et al., 2004; banu et al., 2010). as a consequence, inorganic ions such as na+ and clare sequestered into the vacuole leading to turgor maintenance and osmotic adjustment (bohnert et al., 1995; glenn et al., 1999; ashraf, 2004; banu et al., 2009). in plants, high production of reactive oxygen species (ros) as a result of various abiotic stresses denature dna, carbohydrates, proteins and lipids due to uncontrolled oxidative stress. however, plants possess a very effi cient cascade of enzymatic as well as non-enzymatic antioxidant defense mechanism that shields plants against ros (mittler, 2002). generally, to overcome the uncontrolled oxidation, plants accumulate superoxide dismutase (sod), catalase (cat), monodehydroascorbate reductase (mdhar), glutathione reductase (gr), glutathione-s-transferase (gst), dehydroascorbate reductase (dhar), guaicol peroxidase (gopx), ascorbate peroxidase (apx), and glutathione peroxidase (gpx) as enzymatic antioxidants and apoprotein amino acids, glutathione (gsh), alkaloids, ascorbic acid (asa), tocopherols and phenolics as non-enzymatic antioxidants for scavenging ros (yamaguchi and blumwald, 2005; ashraf, 2009; gill and tuteja, 2010; siddiqi et al., 2011). vegetables generally show high antioxidant metabolism in response to salinity stress as already observed in a number of vegetables such as pea, radish, turnip, caulifl ower and cucumber, etc. (noreen and ashraf, 2009 a,b; volden et al., 2009; colla et al., 2010; neffatti et al., 2011; shahbaz et al., 2012). carrot (daucus carota) is one of the most preferred vegetables worldwide for its edible tubers due to their enriched mineral composition, sugars, phytonutrients, carotene, vitamins a and c, dietary fi bre and high culinary uses (kumar et al., 2004). however, production of carrot is very low due to adaptation to a variety of abiotic stresses including salinity prevalent in many countries particularly falling under arid and semi-arid environments (gonçalves et al., 2010). it is classifi ed as a salt-sensitive plant as it grows best only at 20 mm salinity level. furthermore, 7% growth reduction has been observed for increment of 1 ds/m electrical conductivity, which is mainly attributed to reduced photosynthetic capacity and gaseous exchange disorders (gibberd et al., 2002). in view of the reports presented earlier, we hypothesized that a positive relationship exists between antioxidative defense system and regulation of some key metabolic phenomena and salt tolerance in carrot plants. thus, the present study was conducted to determine the salinity tolerance potential in two carrot cultivars and the pattern of accumulation of chlorophyll regulation, chlorophyll fl uorescence and leaf gas exchange characteristics, accumulation of gb, proline, and enzymatic and non-enzymatic antioxidants due to salt stress in carrot plants. 106 s. bano, m. ashraf, n.a. akram, f. al-qurainy materials and methods a greenhouse experiment was carried-out during november-april, 2010 in the botanical garden, university of agriculture, faisalabad, pakistan. during experimentation, average values of photoperiod, light intensity, relative humidity and temperature were recorded as 12 h, 890 µmol m-2 s-1, 50%, and 26.7-37.9 °c, respectively. seeds of two carrot cultivars (dc-4 and t-29) were supplied by the ayub agricultural research institute, faisalabad, pakistan. a completely randomized design was employed to set-up the experiment. after 10 days of germination, four seedlings were maintained at two-leaf stage per replicate and pots were separated in four sets supplied weekly with hoagland’s nutrient solution supplemented with four varied levels of salt (nacl) [0, 50, 100 and 150 mm]. the salt concentrations increased step-wise per day with 50 mm nacl. the plants were harvested four weeks after the commencement of the saline treatment. the plants were separated into shoots and roots, data recorded for fresh weights and after it, roots and shoots were oven-dried at 70 °c up till constant dry weights achieved. before harvesting, fresh leaves were kept frozen using liquid nitrogen for the estimation of following attributes: chlorophyll contents the fresh leaves (0.5 g) were extracted overnight using 80% acetone. the extract was centrifuged at 10, 000 x g for 5 min and the absorbance of the supernatant read at 645 and 663 nm using a spectrophotometer. chlorophyll a and b were calculated using the formulae proposed by arnon (1949). water relation attributes early in the morning (6.00-8.00 am), a fresh leaf from top was cut and used for the estimation of leaf water potential (ψw) with the help of a scholander type pressure chamber. the same leaf was frozen in a freezer at -80 °c for one week. then cell sap was extracted from the frozen leaf material and directly used for the estimation of osmotic potential (ψs) using an osmometer (wescor, 5500). the difference between ψw and ψs was calculated as leaf turgor potential (ψp). gas exchange characteristics gas exchange attributes including net co2 assimilation rate (aassimilation rate (aassimilation rate ( ), sub-stomatal co2 concentration (ci), stomatal conductance (gs) and transpiration rate (e) were measured on a fresh leaf of each plant using infrared gas analyzer (lca-4 adc portable, analytical development company, hoddesdon, england). chlorophyll fl uorescence maximal quantum yield of psii (fvfvf /fv/fv m/fm/f ), non-photochemical quenching (npq), photochemical quenching (qp) and co-effi cient of non-photochemical quenching (qn) were examined using a os5p modulated fluorometer (adc bioscientifi c ltd. great amwell herts, uk) following strasser et al. (1995). determination of na+, k+ and ca2+ following allen et al. (1985), dried ground leaf or root material (100 mg) was taken in a digestion fl ask and 1 ml digestion mixture (14 g liso4 . 2h2o + 0.42 g se + 350 ml of hydrogen peroxide + 420 ml concentrated sulfuric acid) were added to the fl ask and placed on a hot plate. an increase in temperature was gradual from 50 °c to 200 °c until the mixture turned black. then, perchloric acid (500 µl) was added to the fl asks until the plant material became colorless. the mixture was cooled down, fi ltered and the solution was diluted up to 50 ml using distilled water with the help of volumetric fl ask. the fi ltrate was used for the determination of na+, k+ and ca2+ using a fl ame photometer (model: jenway, pfp-7). for the determination of cl-, the dried ground leaf or root sample (0.1 g) was extracted in 10 ml de-ionized water at 80 °c until the volume became half. again the volume was maintained to 10 ml using deionized water. the clcontent was examined using a chloride analyzer (model 926, sherwood scientifi c ltd., cambridge, uk). leaf proline determination fresh leaf (500 mg) was extracted in freshly prepared 3% sulfosalicylic acid (mol. wt = 254.22) purchased from mp biomedicals, inc. then, the fi ltrate (2.0 ml) was mixed with glacial acetic acid (2.0 ml) and 2.0 ml acid ninhydrin mixture, prepared by mixing ninhydrin (1.25 g), glacial acetic acid (30 ml) and 6m h3po4 (20 ml) in a glass tube. the material was incubated at 100 °c for one h and cooled it. to it, 4.0 ml of toluene were added and mixed well. the optical density (od) of the chromophore containing toluene was read at 520 nm. the proline concentration was estimated following the method described by bates et al. (1973). glycinebetaine (gb) fresh leaf material (1.0 g) was shaken occasionally in 10 ml of 0.5% toluene solution and fi ltered. after fi ltration, 1 ml of the extract was blended with 1 ml of 2n h2so4. then 0.5 ml of this mixture was taken in a glass tube and to it potassium tri-iodide solution (0.2 ml) was added. the contents were shaken and ice-cooled for 90 min. then 2.8 ml of distilled water and 6 ml of 1-2 dichloroethane were added to the mixture. the upper aqueous layer was discarded and od of the organic layer determined at 365 nm.the gb concentration was calculated following grieve and grattan (1983). total phenolics total phenolics were analysed following julkunen-titto (1985). fresh leaf tissue (0.5 g) was homogenized with 80% acetone and centrifuged at 10,000 х g for 10 min. one hundred microliters of the supernatant were mixed with 2 ml of water and 1 ml of folinciocalteau’s phenol reagent and shaken. then 5 ml of 20% sodium carbonate (na2co3) were added and the volume was made up to 10 ml using distilled water. the contents were mixed and od read at 750 nm. h2o2 determination following velikova et al. (2000), fresh leaf (0.5 g) was extracted with 5 ml of 0.1% (w/v) tca. the extract was centrifuged at 12,000 x g for 15 min. to 0.5 ml of the supernatant, 0.5 ml potassium phosphate buffer (ph 7.0) and 1 ml potassium iodide (ki) were added. the mixture was vortexed and its absorbance read at 390 nm. cell membrane injury (cmi) the cell membrane injury was determined as described by yang et al. (1996). the fully expanded youngest leaves of uniform size were excised and placed in test tubes each containing 10 ml deionized distilled water. percent cmi was calculated as: cmi (%) = [ec1-ec0 /ec2-ec0] х 100 activities of antioxidant enzymes the fresh leaf (0.5 g) was ground well in 5 ml cooled phosphate buffer (50 mm; ph 7.8). then the homogenate was mixed with a vortex and centrifuged at 15,000 х g for 15 min at 4 °c. the supernatant was separated and sod activity determined by appraising the photoreduction of nitroblue tetrazolium (nbt) by the enzyme. for this purpose, the standard method as proposed by giannopolitis and ries (1977) was used. three ml of the reaction solution contained 1.3 µm ribofl avin, 50 µm nbt, 75 nm edta, 13 mm methionine, 20 mm phosphate buffer having 7.8 ph. 20 to 50 µl of the enzyme extract were homogenized in a test tube. this physiological and antioxidative regulation in carrot under saline stress 107 solution was irradiated for 15 min under white fl uorescent light. then the od of the solutions was read using a spectrophotometer at 560 nm. the amount of enzyme required to inhibit half of nbt photoreduction was considered equal to one unit of sod activity. peroxidase (pod) and catalase (cat) the protocol of chance and maehly (1955) was followed for the appraisal of peroxidase and catalase activities. three ml of peroxidase reaction solution were mixed with 0.1 ml enzyme extract and then 40 mm h2o2, 20 mm guaiacol and 50 mm phosphate buffer (ph 5 .0) were added to it. the changes in absorbance of reaction solution were recorded at 470 nm after every 30 sec. a change in the absorbance of reaction mixture per min was considered equal to one unit of pod activity. three ml reaction solution used for the determination of catalase activity contained 5.9 mm h2o2, 50 mm phosphate buffer having ph 7.8, and 0.1 ml enzyme extract. for the determination of catalase activity, the reaction was commenced by adding the enzyme extract and the changes in absorbance of the reaction solution after every 20 sec were measured at 240 nm. a change per min in absorbance was considered equal to one unit catalase activity. the activity of each enzyme was calculated and expressed on the basis of total protein measured as described by bradford (1976). malondialdehyde (mda) mda content was estimated with a spectrophotometer (u-2001 hitachi co., tokyo) following carmak and horst (1991). the concentration was calculated using coeffi cient of difference between od at 600 and 532 nm. statistical analysis anova was obtained using jmp v.6.0 provided by sas institute inc., cary, mc, usa. data were presented as the mean ± s.e.; n = 4 for each cultivar and salt treatment. signifi cant differences between the salt levels were observed by a least signifi cance difference test at 0.05% signifi cance level. results analysis of variance of the data for shoot fresh and dry weights of two carrot (daucus carrota l.) cultivars shows that imposition of salt (nacl) stress caused a considerable decrease in shoot fresh and dry weights of both carrot cultivars (tab. 1; fig. 1). of both carrot cultivars, cv. t-29 was relatively more tolerant to salt stress than cv. dc-4. salt stress had a marked reducing effect on root fresh and dry weights of both carrot cultivars. of all salt regimes, 150 mm nacl was highly inhibitory as compared to the other salt treatments. however, the response of both carrot cultivars to salt stress was non-signifi cant in these attributes. imposition of nacl stress induced a substantial decrease in shoot length of both carrot cultivars under test. similarly, a slight reduction in root length was also observed under saline conditions. however, the response of both carrot cultivars to salt stress was inconsistent with respect to these attributes (tab. 1; fig. 1). root medium nacl did not alter the chlorophyll a contents in both carrot cultivars (tab. 1; fig. 1). however, a marked reduction in chlorophyll b contents was observed in both carrot cultivars on exposure to varying salt treatments. in both carrot cultivars, the trend of increase or decrease in both pigments was almost constant (fig. 1). salt stress did not infl uence chlorophyll a/b ratio in both carrot cultivars. both carrot cultivars were almost similar in chlorophyll a/b ratio (fig. 2). application of varying levels of nacl signifi cantly affected all the three leaf water, osmotic and turgor potentials of both carrot cultivars under examination (tab. 1; fig. 2). under saline conditions, a signifi cant decrease (more negative) in leaf water potential as well as leaf osmotic potential (ψs), while a considerable increase in leaf turgor potential of all carrot plants was observed. of both carrot cultivars, cv. t-29 was lower in leaf water and osmotic potentials than cv. dc-4. however, both carrot cultivars did not differ in leaf turgor potential under saline conditions (fig. 2). net co2 assimilation rate (a assimilation rate (a assimilation rate ( ) in both carrot cultivars decreased on exposure to varying saline regimes. however, both carrot cultivars were similar in this gas exchange attribute (tab. 1; fig. 2). under saline conditions, a considerable reduction in transpiration rate in carrot plants was observed. both cultivars were similar in transpiration rate under saline and non-saline conditions (tab. 1; fig. 2). a signifi cant decrease in stomatal conductance was found due to root growing medium salt stress. on the basis of analysis of variance, it is not possible to discriminate the carrot cultivars with respect to stomatal conductance. sub-stomatal co2 concentration was signifi cantly suppressed under saline regimes. both carrot cultivars did not differ signifi cantly under non-saline as well as saline regimes. imposition of nacl stress reduced the ci/ci/ci a/ca/c ratio of both carrot cultivars. of all salt regimes, lowest value of ci/ci/ci a/ca/c was observed at 150 mm nacl. both carrot cultivars showed consistent values of ci/ci/ci a/ca/c ratio under non-saline or saline regimes (tab. 1; fig. 3). wateruse-effi ciency of both carrot cultivars decreased considerably at 150 mm nacl, while in contrast, at 50 and 100 mm nacl levels, a slight increase in wue was observed (tab. 1; fig. 3). both carrot cultivars were almost consistent in water-use-effi ciency. leaf and root na+ concentrations increased signifi cantly with increase in salt concentration. however, higher values of leaf and root na+ were observed in carrot plants at 150 mm nacl and lower under normal as well as at 50 mm nacl in both carrot cultivars. both carrot cultivars were similar in leaf na+ at varying external saline regimes, while root na+ concentration was relatively higher in cv. t-29 than that in cv. dc-4 (tab. 1; fig. 4). under saline conditions, leaf and root potassium (k+) concentrations decreased markedly. lowest values of leaf and root k+ concentrations were observed at 150 mm nacl. both carrot cultivars signifi cantly differed in leaf k+ accumulation, and cv. dc-4 was reasonably higher in leaf k+. a non-signifi cant difference was observed between both carrot cultivars in root k+ concentration (tab. 1; fig. 4). it was observed that leaf and root ca2+ concentrations decreased signifi cantly under saline conditions. leaf ca2+ concentration was almost consistent in both carrot cultivars, while root ca2+ signifi cantly varied and cv. dc-4 was relatively higher in root ca2+ than cv. t-29 under saline regimes (fig. 4). rooting medium salt stress had a marked effect on leaf and root claccumulation in both carrot cultivars (tab. 1; fig. 4). highest value of leaf and root clin carrot plants was observed at 150 mm nacl. the difference between both cultivars was signifi cant and of both carrot cultivars, cv. t-29 was relatively higher than cv. dc-4 in leaf and root claccumulation. leaf free proline accumulation increased signifi cantly in both cultivars under salt stress treatments. the highest proline accumulation was observed at 150 mm nacl (tab. 1; fig. 3). of both carrot cultivars, cv. t-29 was relatively higher in proline content than cv. dc-4 under various salt regimes. a marked increase in glycinebetaine concentration was examined in carrot plants when exposed to varying salt regimes. cv. dc-4 accumulated relatively higher amount of glycinebetaine than cv. t-29 (tab. 1; fig. 3). leaf malondialdehyde (mda) decreased considerably (p ≤ 0.001) in both carrot cultivars under all salt regimes. overall, cv. t-29 had markedly higher concentration of mda than that in cv. dc-4, particularly under saline regimes (tab. 1; fig. 5). leaf hydrogen peroxide (h2o2) in both carrot cultivars increased consistently due to root growing medium salt regimes. leaf h2o2 108 s. bano, m. ashraf, n.a. akram, f. al-qurainy contents were higher at 100 mm nacl in cv. t-29, while in cv. dc-4 at 150 mm. however, both cultivars remained similar in leaf h2o2 contents under saline conditions (tab. 1; fig. 5). leaf total phenolic contents decreased in carrot cv. dc-4, while increased in cv. t-29 under all saline regimes (fig. 5). a marked reduction in leaf total soluble proteins of both carrot cultivars was observed due to root growing medium salt regimes. salt-induced reduction in soluble protein content was signifi cantly higher in cv. dc-4 than that in cv. t-29 (tab. 1; fig. 5). in the present study, the activity of superoxide dismutase (sod), a key antioxidant enzyme, decreased in both carrot cultivars under saline stress. cultivar t-29 had signifi cantly higher activity of sod than that of cv. dc-4 under all salt levels (tab. 1; fig. 5). leaf peroxidase (pod) enzyme activity in both carrot cultivars decreased signifi cantly (p ≤ 0.05) under saline conditions (fig. 5). the salt-induced decrease in pod activity was inconsistent in both carrot cultivars under saline regimes. salt stress had a considerable reducing effect on leaf cat activity in cv. dc-4, whereas in cv. t-29, it remained similar to that in plants grown under non-saline conditions. of both carrot cultivars, cv. t-29 had signifi cantly higher activity of cat than that of cv. dc-4 under all salt regimes (tab. 1; fig. 5). discussion in the present study, there was a considerable decrease in shoot and root fresh and dry weights in both carrot cultivars (dc-4 and t-29) under salt stress. the salt-induced growth reduction in carrot is analogous to what has been earlier reported in different vegetable crops, e.g. turnip (noreen et al., 2010), okra (saleem et al., 2011), chilli (maiti et al., 2010), cucumber (navarro et al., 2006), eggplant (abbas et al., 2010), radish (noreen and ashraf, 2009b), tomato (frary et al., 2010), and pea (noreen and ashraf, 2009a). reduction in plant growth takes place due to salt-induced alteration in various physio-biochemical characteristics (mittler, 2002; shi et al., 2008; ashraf and akram, 2009). of various tab. 1: analyses of variance of data for growth, chlorophyll pigments, photosynthetic attributes, leaf fl uorescence, proline, glycinebetaine, relative membrane permeability, mineral nutrients, enzymatic and non-enzymatic antioxidants of two cultivars of carrot (daucus carota l.) grown for 30 days under varying nacl levels. source df shoot shoot root root shoot root chlorochloroof variation fresh weight dry weight fresh weight dry weight length length phyll a phyll b cultivars (cvs) 1 114.3* 0.018ns 0.368ns 0.123** 0.516ns 1.201ns 0.005*** 0.012** salt stress (s) 3 777.5*** 2.964*** 3.116*** 0.386*** 157.9*** 3.333** 0.0008* 0.005** cvs x s 3 9.91ns 0.045ns 0.227ns 0.068** 9.19ns 0.584ns 0.0004ns 0.0003ns error 24 19.98 0.089 0.295 0.01 7.242 0.465 0.0003 0.001 chloroa e gs cicic cicic /ci/ci a/ca/c wue free proline phyll a/b ratio cultivars (cvs) 1 0.329* 3.983ns 2.949*** 1088.1ns 34871.7*** 0.281*** 11.15** 2048.0*** salt stress (s) 3 0.164* 26.49*** 0.218* 4338.3*** 6127.5*** 0.049*** 9.88** 213.3*** cvs x s 3 0.02ns 0.232ns 0.042ns 1715.4* 3635.4** 0.029** 1.648ns 0.00000008ns error 24 0.043 0.961 0.053 434.7 495.03 0.004 1.313 1.666 leaf gb relative fv/fv/fv m/fm/f npq qp np leaf na+ root na+ membrane permeability cultivars (cvs) 1 0.04** 84.28ns 0.004* 0.011ns 0.0003ns 0.023** 0.781ns 2.53ns salt stress (s) 3 0.34*** 971.0** 0.002ns 0.009ns 0.0033ns 0.003ns 97.66*** 36.26*** cvs x s 3 0.002ns 113.4ns 0.001ns 0.001ns 0.001ns 0.005ns 0.781ns 0.885ns error 24 0.007 146.9 0.001 0.004 0.001 0.003 7.614 2.395 leaf k+ root k+ leaf ca2+ root ca2+ leaf clroot clmda h2o2 cultivars (cvs) 1 73.5* 7.51ns 15.82** 136.1** 140.1ns 0.252ns 202.8*** 2.33ns salt stress (s) 3 169.04*** 34.34* 18.84*** 23.93ns 1451.5*** 72.33*** 40.87* 6.335ns cvs x s 3 21.34ns 3.57ns 3.278ns 4.52ns 1.183ns 1.177ns 18.36ns 4.715ns error 24 15.25 9.75 1.757 13.33 68.32 2.935 9.598 2.178 total sod pod cat soluble proteins cultivars (cvs) 1 18.49*** 29.64*** 0.531ns 18.16*** salt stress (s) 3 6.919*** 1.448ns 1.703** 3.374*** cvs x s 3 1.851*** 0.803ns 0.1ns 2.567*** error 24 0.195 0.623 0.305 0.265 ns = non-signifi cant; *, ** and *** = signifi cant at 0.05, 0.01 and 0.001 levels, respectively. physiological and antioxidative regulation in carrot under saline stress 109 physiological processes, photosynthesis is directly involved in plant growth and development as in this process light energy is converted into usable chemical energy, which is consumed in a variety of plant growth and developmental processes (taiz and zeiger, 2010). in the present study, exposure of carrot plants to saline stress showed considerable alteration in different gas exchange characteristics including photosynthetic rate (aincluding photosynthetic rate (aincluding photosynthetic rate ( ), sub-stomatal co2 concentration (ci), transpiration rate (e) and stomatal conductance (gs). generally, all these gas exchange characteristics decreased due to salt stress. earlier studies reveal that salt-induced plant growth suppression is often correlated with decline in rate of photosynthesis (noreen and ashraf, 2008; siddiqi et al., 2009; akram and ashraf, 2011; saleem et al., 2011). for example, while screening 10 cultivars of saffl ower (carthamus tinctorius l.) for salt tolerance, siddiqi et al. (2009) observed a signifi cant relationship of a with plant biomass under saline stress and suggested photosynthetic capacity as a potential indicator of salinity tolerance in this crop. in contrast, our results did not show such a strong relationship of photosynthetic rate with the growth of both carrot cultivars. so, it means that the differential salt tolerance of the two carrot cultivars is governed by factors other than photosynthesis. salinity impairs the ability of plant cells or tissues to take up water fig. 1: shoot fresh and dry weights, shoot and root lengths and chlorophyll a and b contents of two cultivars of carrot (daucus carota) grown for 30 days under varying nacl levels (mean + s.e; n= 4). ) 110 s. bano, m. ashraf, n.a. akram, f. al-qurainy fig. 2: chlorophyll a/b ratio, water, osmotic and turgor potentials, photosynthetic rate (a ratio, water, osmotic and turgor potentials, photosynthetic rate (a ratio, water, osmotic and turgor potentials, photosynthetic rate ( ), transpiration rate (e), stomatal conductance (gs) and sub-stomatal co2 concentration (ci) of two cultivars of carrot (daucus carota) grown for 30 days under varying nacl levels (mean + s.e; n= 4). from the saline growing medium (munns, 2002; zhu, 2002). this leads to reduced tissue water potential, which in turn, adversely affects the plant growth metabolism (meloni et al., 2001; sabir et al., 2009). in the present study, a signifi cant reduction in leaf water potential (ψw), as well as leaf osmotic potential was observed, which could be one of the factors inducing biomass reduction in both carrot cultivars. similarly, during a study with 18 accessions of proso millet, sabir et al. (2009) found salt-induced reduction in leaf water potential, which ultimately suppressed the growth of proso millet plants. in another study, similar fi ndings were reported by siddiqi and ashraf (2008) that salt stress caused reduction in shoot fresh biomass, which was attributed to suppression in leaf relative water content (rwc) and ψw. plant cells/tissues under stress conditions exhibit different defense mechanisms to protect themselves from the adverse effects of oxidative stress caused due to over-accumulation of reactive oxygen species (ros) (mittler, 2002; ashraf, 2009). to counteract the ros, plants have generated a variety of non-enzymatic and enzymatic detoxifi cation systems and protect cells from oxidative stress (yamaguchi and blumwald, 2005). variation in the transcript and enzyme levels of major antioxidant enzymes during stress is well documented (azooz et al., 2009). the major physiological and antioxidative regulation in carrot under saline stress 111 antioxidant enzymes such as superoxide dismutase (sod), catalase (cat), ascorbate peroxidase (pox) and glutathione reductase (gr) can effectively detoxify ros such as superoxide (o2• -) and hydrogen peroxide (mittler, 2002). recently, azooz et al. (2009) examined the activities of antioxidant enzymes, apx, sod, cat and pod in maize plants and reported that salt-tolerant maize cultivars were higher in antioxidant enzyme activities as compared to those of salt sensitive ones under saline conditions. in addition, they observed a signifi cant relationship between the activities of antioxidant enzymes and plant growth appraised in terms of dry biomass. however, contrarily, noreen et al. (2010) found a nonsignifi cant relationship between growth and the activities of sod, cat and pod in six cultivars of turnip under saline conditions. recently, sabir et al. (2011) while screening 18 accessions of proso millet for salt tolerance found a signifi cant enhancement in the activities of pod, cat and sod under saline conditions, but the relationship between antioxidant enzyme activities and yield of proso millet plants were found to be non-signifi cant. however, in the present study, the activities of sod, pod and cat decreased in both carrot cultivars, but the high biomass producing cv. t-29 had signifi cantly higher activities of cat and sod than those of the other cultivar under all salt regimes. fig. 3: ci/ci/ci a/ca/c ratio, wue, leaf proline, rmp, effi ciency of photosystem-ii (fvfvf /fv/fv m/fm/f ), non-photochemical quenching (npq), photochemical quenching (qp) and co-effi cient of non-photochemical quenching (qn) of two cultivars of carrot (qn) of two cultivars of carrot (qn daucus carota) grown for 30 days under varying nacl levels (mean + s.e; n= 4). 112 s. bano, m. ashraf, n.a. akram, f. al-qurainy 0 2 4 6 8 10 12 14 16 18 20 le af n a+ (m g g1 dr y w t) 0 mm nacl 50 mm nacl 100 mm nacl 150 mm nacl 0 5 10 15 20 r oo t n a+ (m g g1 dr y w t) 0 mm nacl 50 mm nacl 100 mm nacl 150 mm nacl 0 5 10 15 20 25 30 le af k + (m g g1 dr y w t) 0 5 10 15 20 25 r oo t k + (m g g1 dr y w t) 0 2 4 6 8 10 12 14 16 18 le af c a2 + (m g g1 dr y w t) 0 5 10 15 20 25 r oo t c a2 + (m g g1 dr y w t) 0 10 20 30 40 50 60 t-29 dc-4 le af c l (m g g1 dr y w t) 0 2 4 6 8 10 12 t-29 dc-4 r oo t c l (m g g1 dr y w t) fig. 4: leaf and root na+, k+, ca2+ and clconcentrations of two cultivars of carrot (daucus carota) grown for 30 days under varying nacl levels (mean + s.e; n= 4). in addition to enzymatic antioxidants, plants also use non-enzymatic antioxidants such as carotenoids, phenolics, tocopherols, ascorbic acid, and glutathione to scavenge ros. phenolics are antioxidant compounds which are highly soluble in water and have a key role in neutralizing ros by transferring their hydrogen atoms (sakihama et al., 2000; frary et al., 2010). it is believed that plants with high antioxidant levels, whether induced or constitutive, have better resistance to ros (parida and das, 2005; ashraf, 2009). plants show intricate antioxidant response when placed under saline stress, because antioxidant contents are regulated by different qtls/genes under saline conditions (frary et al., 2010). in this study, relatively salt tolerant carrot cultivar t-29 accumulated higher amount of total phenolics, while lower content of leaf mda than the relatively low biomass producing carrot cv. dc-4. these fi ndings are partially parallel to an earlier investigation on wheat in which salt-induced mda content was more in salt sensitive wheat cv. mh-97 as compared to that in salt-tolerant s-24 under saline regimes. osmotic adjustment is one of the major physiological phenomena involved in stress tolerance, which involves high accumulation of organic and inorganic solutes in plant tissues/cells (munns et al., 2006; ashraf and foolad, 2007; abbas et al., 2010). in the present study, leaf proline and gb accumulation increased substantially in both carrot cultivars under saline regimes. these fi ndings are supported by a number of reports which show a signifi cant saltphysiological and antioxidative regulation in carrot under saline stress 113 induced increase in both proline and gb accumulation in different plant species, e.g., proso millet (sabir et al., 2011), okra (saleem et al., 2011), eggplant (abbas et al., 2010), pea (noreen et al., 2010), and turnip (noreen et al., 2010). in the present study, leaf and root k+ levels decreased while na+ and clcontents increased, a general trend of most glycophytes, under saline regimes (munns and tester, 2008). however, of both carrot cultivars, relatively tolerant cv. t-29 was higher in root na+, and leaf and root cl-, while, cv. dc-4 in leaf k+ and root ca2+ under saline conditions. such type of differential inorganic ion accumulation has already been observed in a number of crop plants such as sunfl ower (akram and ashraf, 2011; shahbaz et al., 2011), saffl ower (siddiqi et al., 2011), and turnip (noreen et al., 2010). overall, varying saline regimes considerably reduced the growth, chlorophyll b contents, leaf water potential (ψw), leaf osmotic potential (ψs), net co2 assimilation rate (a assimilation rate (a assimilation rate ( ), water-use effi ciency, stomatal conductance (gs), sub-stomatal co2 concentration (ci), transpiration rate (e), ci/ci/ci a/ca/c ratio, leaf and root k+ and ca2+ contents, leaf mda, total phenolics, total soluble proteins, and activities of cat, sod and pod, while a considerable increase was observed in leaf turgor potential (ψp), leaf and root na+ and clcontents, leaf proline, gb, ascorbic acid (asa), and h2o2 contents in both cultivars. of both carrot cultivars, cv. t-29 was relatively higher in shoot and root fresh weights, leaf and root ca2+, leaf proline, mda, 0 1 2 3 4 5 6 7 le af h 2o 2 (µ m ol g -1 fw t) 0 mm nacl 50 mm nacl 100 mm nacl 150 mm nacl 0 2 4 6 8 10 12 14 16 18 20 m d a (n m ol g -1 fw t) 0 mm nacl 50 mm nacl 100 mm nacl 150 mm nacl 0 1 2 3 4 5 6 7 8 to ta l p he no lic s (m g g -1 fw t.) 0 0,5 1 1,5 2 2,5 3 3,5 4 s o d (u ni ts /m g pr ot ei n) 0 0,5 1 1,5 2 2,5 3 p o d (u ni ts /m g pr ot ei n) 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 c at (u ni ts /m g pr ot ei n) 0 1 2 3 4 5 6 t-29 dc-4 to ta l s ol ub le p ro te in s (µ g g1 fw t) fig. 5: leaf h2o2, mda, total phenolics, activities of sod, pod and cat enzymes and total soluble proteins of two cultivars of carrot (daucus carota) grown for 30 days under varying nacl levels (mean + s.e; n= 4). 114 s. bano, m. ashraf, n.a. akram, f. al-qurainy total phenolics, soluble proteins and activity of sod, while cv. dc-4 was relatively higher in leaf ψw and ψs, leaf k+, root ca2+ and leaf gb as compared to those in the other cultivar. the relatively better growth of cv. t-29 was found to be associated with up-regulation of water relations and higher leaf ca2+, proline, 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developing salt-tolerant crop plants: challenges and opportunities. trends plant sci. 10, 615-620. yang, yang, y g., rhodes, d., joly, r.j., 1996: effects of high temperature on membrane stability and chlorophyll fl uorescence in the glycinebetaine defi cient and glycinebetaine containing maize lines. aust. j. plant physiol. 23, 437-443. zhu, j.k., 2002: salt and drought stress signal transduction in plants. annu. rev. plant biol. 53, 247-273. address of the corresponding author: n.a. akram, e-mail: nudrataauaf@yahoo.com, tel.: +92-41-9201488 journal of applied botany and food quality 94, 192 198 (2021), doi:10.5073/jabfq.2021.094.023 universidade federal do vale do são francisco, centro de ciências agrárias, colegiado de agronomia, petrolina, brazil nitrate reductase activity in the different phenophases of ‘palmer’ mango cultivated in the semiarid alana juliete da silva santos, vespasiano borges de paiva neto*, luciana guimarães sanches, daniel de almeida carreiro, maria poliana martins pereira, monica cristina rezende zuffo borges, stefany emanuella rodrigues dos santos, ítalo herbert lucena cavalcante (submitted: december 30, 2020; accepted: october 6, 2021) * corresponding author summary nitrate reductase is the enzyme that catalyzes the first reduction reaction in the nitrate assimilation process, an important nitrogen (n) source. this element is one of the macronutrients required in greater amounts by mango (mangifera indica l.), exerting a great influence on the growth and development of the plants. in this context, this work aimed to evaluate nitrate reductase activity (nra) throughout the day and characterize it in leaves of 1st and 2nd vegetative flushes and young roots of ‘palmer’ mango cultivated in the brazilian semiarid for each phenophase, leaves and roots were randomly collected from six plants in the orchard. a completely randomized design was employed, with four replications, and the nra quantification was based on the in vivo evaluation method. the enzyme activity was maximum in the period of greatest solar radiation or from 10 a.m. to 11 a.m. the 2nd flush leaves and young the roots constitute the main nitrate assimilation sites in the mango crop in comparation with 1st flush leaves. fertilization with nitrate or potassium sources in the different phenological phases benefit the nra, which remains maximum during reproductive phase than vegetative phase. keywords: phenology; nitrogen; enzymatic activity; mangifera indica l.; floral induction introduction in semiarid conditions, mango cultivation can occur during the entire year as long as adequate plant management is performed to favor flowering (mouco, 2015). however, the crop yield is still relatively low due to factors related to flowering and fruiting, mainly. in this case, the physiological, phytopathological, and nutritional factors related to these events must be observed so that fruit production is satisfactory (coutinho et al., 2016). there are tools for mango flowering management through pruning, nutritional management, and the use of growth regulators (krishna et al., 2017). the synchronization of the canopy vegetation, better performed through pruning in all the branches of the plant, is a necessary first step in the program of flowering management, whose purpose is to leave the branches at the same physiological stage of maturity. therefore, in commercial orchards, after a productive cycle (fruit harvest), the mango plant requires pruning, with the removal of the second branch segment (flush) formed in the previous cycle. this practice encourages the plant to form the next two flushes of the new reproductive cycle. additionally, this practice controls the increase in the canopy diameter of each plant. the next step is the application of paclobutrazol (pbz) in an adequate amount and time (wongsrisakulkaew et al., 2017). this one, by its turn, paralyzes vegetative growth and impedes the distribution of elaborate sap, leading to the accumulation of carbohydrates in the branches, resulting in maturation (taiz et al., 2017). for the well-succeeded stimulation of flowering, nitrate salts must be applied, via foliar spraying, after the mango sprouts reach maturity or sufficient age to overcome any inhibitory influence on the flower ing response (davenport, 2000). potassium nitrate (kno3) induces the nitrate reductase activity (nra), a key enzyme in the nitrate assimilation pathway for the synthesis of amino acids, particularly methionine (anusuya et al., 2018), an intermediary product pre cursor of ethylene, which, by its turn, promotes flowering in the mango plant (sudha, balamohan and soorianathasundaram, 2012; coutinho et al., 2016). nitrate reductase is located in the cellular cytosol and catalyzes the reduction of nitrate to nitrite (no3to no2-), a limiting step in the nitrogen (n) assimilation pathway in amino acids, proteins, and other nitrogen compounds in the cells performing a key role in the response of the plants to deficiency of this element (kaur et al., 2015; taiz et al., 2017). nitrate reductase activity is regulated by several factors, such as the presence of nitrate, that is, its substrate (coutinho et al., 2016; anusuya et al., 2018), light (oliveira et al., 2005), the water status (oliveira et al., 2005), species, growth habit, and habitat, being lower in woody species (rajsz et al., 2018). the regulation of nra is necessary to avoid the accumulation of nitrite, which is toxic for the cell (taiz et al., 2017). furthermore, this regulation has a close relationship with photosynthesis since the step of nitrite reduction to ammonium uses reduced ferredoxin, a product of this process. in this manner, a reduction in photosynthesis could lead to nitrite accumulation in the cellular medium if the enzyme activity was not regulated (lillo et al., 2003). nitrate reduction can occur in the root system or the shoot part of the plants, (taiz et al., 2017), and it may vary among different species (black et al., 2002). oliveira et al. (2005) observed the enzyme activity in leaves and roots of peach palm seedlings (bactris gasipaes) and verified that it was maximum in the leaves, such as observed for the grapevine crop (mesquita et al., 2018). in maize, an alternation in nra was observed between leaves and roots: when the enzyme activity was high in the roots it was low in the leaves, and vice-versa (freitas et al., 2007). in the mango crop (mangifera indica l.) nra was observed in both plant tissues, although it was maximum in the roots (souza et al., 2016), with the study being performed only at the flowering phenophase. in the absence of nra, no3reduction does not occur (kaufholdt et al., 2016), limiting growth, development, and production of pro teins in the plants that assimilate this element (taiz et al., 2017). given the exposed, this work aimed to define the daytime of superior nitrate reductase activity and to characterize the enzyme activity in leaves of 1st and 2nd vegetative flushes and young roots of ‘palmer’ mango plants in the different phenological phases of the crop, aiming to understanding how the plant distributes the nitrogen metabolism in these organs throughout its cycle. nitrate reductase along mango phenophases 193 material and methods the present study was performed between december 2018 and july 2019, in which period the collection of the plant material was performed in two commercial orchards at two nearby farms with ‘palmer’ mango (mangifera indica l.), whose general data concerning the studied areas are found in tab. 1. the vegetal material was obtained from six randomly sampled plants within the mango orchards. from the median portion of each plant, two leaves of the 1st flush (penultimate vegetative flush) and two leaves of the 2nd flush (last vegetative flush) exposed to solar radia tion were collected, totaling 12 leaves of each type, and a portion of thin roots (absorbing) at a depth of 0-10 cm in the irrigated range of the soil. after the collection of the material, laboratory analysis were per formed in the laboratories of analytical chemistry and plant phy siology of the universidade federal do vale do são francisco (univasf), campus of agricultural sciences (cca), located in the city of petrolinape. leaves and roots were stored in plastic bags and aluminum foil, respectively, both stored in a cool box containing ice. the in vivo nitrate reductase activity (nra) was determined according to the method described by majerowicz et al. (2003), with a few modifications. the leaves underwent a washing sequence with running water, 1% detergent, running water, and distilled water, in this order. the roots were washed in a sieve only with running and distilled water to avoid loss of the vegetal material. after drying, 7 mm discs were removed from the leaf blade, forming a compound sample for both leaf types. each simple sample analyzed contained 60 discs and was weighed to obtain the mass. the roots were chopped into small fragments, forming a compound sample. each simple sample contained 0.5 g of roots. the portions of leaves and roots were placed in falcon tubes wrapped in aluminum foil, into which were added 4 ml of a phosphate buffer solution 0.05 m, ph 7.5, potassium nitrate (kno3) 0.05 m, and1% n-propanol. afterward, the plant tissue submerged in the solution was subjected to three vacuum sessions, each during 1-minute (min), with 30-second (s) intervals. the tubes were then properly sealed and incubated for 60 minutes at 30 ºc. after the incubation period, the tubes were subjected to immersion in ice for two minutes. the amount of nitrite (no2-) released into the incubation medium was determined in 2 ml aliquots with the addition of 1 ml of n-naphthyl-ethylene-diamine at 0.2% (m/v) and 1 ml of sulfanilamide at 1% (m/v), reacting for 30 minutes. the samples were analyzed in an even® uv-vis spectrophotometer at 540 nm. the enzyme activity was expressed in μmol of nitrite released per 1 g of fresh leaf per hour of incubation (μmol·no2-.g-1·h-1), calculated based on the linear equation obtained through the nitrite standard curve, previously prepared. a completely randomized design was used in the experiment, with four replications. the obtained data were subjected to analysis of variance by the f test, and the means were compared by tukey’s test with p<0.05 using the r software (r development core team, 2018). the graphics were generated in the sigmaplot software (10.0, systat software, san jose, ca). results nitrate reductase activity (nra) in the ‘palmer’ mango plant was observed throughout the day in mature leaves of 1st and 2nd vegetative flushes and young roots of plants in the floral induction phase, indicating two sites of n reduction (shoot and root) (fig. 1). for the 2nd flush leaves and roots, the nra can be observed from the early morning hours, with a peak at 10 a.m., decreasing from noon. the 1st flush leaves presented a small variation in the enzyme activity in the morning, with no evident activity peak in the monitored hours, although with an equal trend for nra reduction from noon (fig. 1). it is possible to observe that the activity peak coincided with the time of greatest solar radiation (fig. 1). nitrate reductase activity was monitored in the different phenophases of the mango crop, verifying, after harvesting and before pruning (pre-pruning), maximum nitrate reductase activity in the root, statistically differing from the leaves of 1st and 2nd flushes, which tab. 2: collect schedule of mango leaves and roots for analyses of nitrate reductase activity, considering the pruning event as the beginning of the cycle and their respective phenophases, in two farms with similar orchards in age and management. phenophases (bbch-scale*) collect day after pruning sebastião la date da manga bordett pre-pruning [pruning day]** 0 x pruning [15 days after pruning]** 15 x pbz1 [12 days after addition] (329) 97 x pbz 2 [32 days after addition] 117 x branch maturation1,** 125 x pre-induction2 (510) 150 x floral induction3 (510) 157 x flowering (615) 196 x fruiting (703) 249 x *(biologische bundesantalt, bundessortenamt und chemische industrie); **phenophase or event not mentioned in the bbch-scale; 1five days after first foliar spray with 3% potassium sulfate (k2so4); 2one day before first foliar spray with 3% potassium nitrate (kno3); 3five days after first foliar spray with 3% potassium nitrate (kno3); the region climate is classified as semiarid bsh, with a mean annual temperature of 24 °c and annual precipitation under 700 mm (álvares et al., 2013). the climatic data referring to rainfall, solar radiation, temperature, and air relative moisture during the period of the experiment were registered and obtained from the automatic weather station installed at the campus of agrarian sciences (cca/ univasf). cultural practices such as pruning, control of weeds, pests, and diseases were performed according to the described in the technical rules for integrated mango production (lopes et al., 2003). the application of paclobutrazol (pbz) was performed, along with the thinning and dormancy break in the management of floral induction, according to the recommendations of albuquerque et al. (2002). the nutritional management was performed via fertigation according to the analysis of soil and leaves to attend to the demand of the crop (silva et al., 2002). aiming at defining the best time for the collection of the plant material, a daytime characterization of the nra was performed. for that purpose, fully expanded and physiologically mature leaves of 1st and 2nd vegetative flushes and young roots were collected from plants during the floral induction phase, throughout the day every two hours, starting at 8 a.m. and finishing at 4 p.m., a period of free access to the farms. the nra was monitored in the different mango phenological phases, and the collection of the material for this stage was performed at the time of superior activity, defined from daytime characterization. tab. 2 describes the collection site referring to each phenophase. tab. 1: general information regarding the studied areas. orchard farm age (years) spacing (m) size (ha) area 1 sebastião da manga 8 5×8 12.54 area 2 la bordett 7 4×6 2.2 farm 194 a.j. da silva santos, v. borges de paiva neto*, l. guimarães sanches, d. de almeida carreiro, m.p. martins pereira, m.c. rezende zuffo borges, s.e. rodrigues dos santos, í.h. lucena cavalcante did not differ from each other (fig. 2). it can be observed that after pruning the nra was not quantified in the 2nd flush leaves because this management removes this material. it is importante to affirm that these two analyzed flushes were generated in the previous vegetative cycle, while from branch maturation onwards, the analyzed flushes (1st and 2nd flushes) were from the new vegetative cycle. in the phase of plant ‘locking’, an increase in the nra of the leaves was verified to the detriment of the roots. 12 days after the application (daa) of pbz, the superior nra was found in the 1st flush leaves, which was statistically different only from the roots. 2nd flush leaves and roots did not differ from each other (fig. 2). at 32 daa, the enzyme activity was maximum in 1st and 2nd flushes leaves, not differing statistically from each other, although differing from the roots (fig. 2). in the branch maturation phase, the nra was statistically equal in the 2nd flush leaves and roots, being much maximum than in the 1st flush leaves, which differed statistically from the remaining plant tissues (fig. 2). it can be observed that the nra was influenced by the foliar spray ing with kno3 in 2nd flush leaves and roots of mango. the 1st flush leaves exhibited a reduced alteration in enzyme activity in the periods of pre and post floral induction (fig. 2). in pre-induction, the superior nra occurred at the roots, statistically differing from the leaves of 1st and 2nd flush, which were different from each other. after the induction there was a decrease in root nra, equaling the 1st flush leaves, and an increase in enzyme activity in the 2nd flush leaves, making this the main no3reduction site in the mango plant after the exogenous increment of kno3, via foliar spraying. in the flowering phase, the nra found in the 2nd flush leaves was significantly maximum in relation to the 1st flush leaves and roots (fig. 2). although this difference between plant materials does exist, it is observed that the nra begins to decrease in the 2nd flush leaves, in comparison to the previous phase. in the fruiting phase, the nra was maximum in the 2nd flush leaves, followed by roots and 1st flush leaves (fig. 2). although it has been maximum in 2nd flush leaves, the enzyme activity continued to reduce in comparison with the previous phases. in this stage, the remobilization of foliar n for fruit development continues. it can be observed that the cumulative nitrate reductase activity in the analyzed leaves and roots of mango is lower during the vegetative phase when compared with reproductive phase, and in general, the nra variation is small in the leaves of the 2nd flush (fig. 3). time of day (h) 8 10 12 14 16 so la r r ad ia tio n (w .m -2 ) 0 100 200 300 400 500 600 700 m ol o f n o 2.g -1 .h -1 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35solar radiation root leaves of 2nd flush leaves of 1st flush pre -pru nin g pru nin g pb z 1 pb z 2 ma tura tion pre -ind uct ion ind uct ion flo wer ing fru itin g m ol o f n o 2.g -1 .h -1 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 leaves of 2nd flush leaves of 1st flush root b b a aa ab a b a a b a b a bb a a bb a b b a c b phenophases phenophases pre -pru nin g pru nin g pb z 1 pb z 2 ma tura tion pre -ind uct ion ind uct ion flo wer ing fru itin g m ol o f n o 2.g -1 .h -1 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 leaves of 2nd flush leaves of 1st flush root fig. 3: distribution of the nitrate reductase activity between leaves of the 1st and 2nd flushes and root of the ‘palmer’ mango in the different phenophases along the vegetative and reproductive cycle in commercial orchard. pbz 1: 12 days after pbz application; pbz 2: 32 days after pbz application; nra: nitrate reductase activity in μmol of no2· fresh matter-1 · h-1. fig. 1: daytime variation of nitrate reductase activity in leaves of 1st and 2nd vegetative flushes and young roots of ‘palmer’ mango and mean values of solar radiation obtained from the weather station at the campus of agricultural sciences (cca), of the universidade federal do vale do são francisco (univasf) fig. 2: nitrate reductase activity in leaves of the 1st and 2nd vegetative flushes and young roots of ‘palmer” mango along different phenophases. means ± sd followed by the same letters on the bars, into each phenophases singly, do not differ significantly by the tukey test at 5% probability. discussion in the present study, a maximum nra activity was verified at 10 a.m., a similar behavior to that observed by oliveira et al. (2005) in peach palm leaves (bactris gasipaes), in which the maximum nra was also verified at the referred time, three hours after the beginning of the luminous period. furthermore, the authors also observed a decline in the enzyme activity after this time. additionally, for sweet orange (citrus sinensis) cv. valência, the maximum nra was also observed at this same time, corresponding to maximum photosynthesis (dovis et al., 2014). such behavior occurs due to the effect of light on the enzyme activity, which may be either direct, in its activation, or indirect, via photosynthetic process, providing the necessary energy for nitrate assimilation (martuscello et al., 2016). among the climatic factors monitored on the day of the experiment, nitrate reductase along mango phenophases 195 solar radiation seems to be the one that most influenced the activity of the nitrate reductase enzyme. santos et al. (2014) inferred that the enzyme is more active in the maximum intensity hours of global solar radiation. it is possible that in mango, nra presents the same behavior pattern of other species such as tomato (tucker et al., 2004) and annatto (patra et al., 2019), for which it has been shown that nra exhibit circadian rhythm. according to tucker et al. (2004), regulatory mechanisms acting at different levels ranging from transcription to protein degradation. therefore additional investigations to confirm this phenomenon in mango plants are re quired. in that perspective, khouri (2007) reported that the maximum nra values and probably also the maximum absorption of no3occur during the periods of high temperature and evaporative demand. these conditions can increase the influx of water and no3to the leaves, favoring the activity of the enzyme in these tissues, since no3 is translocated to the shoot part via xylem through transpiration flow (taiz et al., 2017), thus increasing the availability of the substrate for the enzyme. given the management that is performed in the mango crop, in the pruning act there is a reduction in the number of flushes, especially the second, making impossible the quantification of nra in its leaves (fig. 2); however, in general, the nitrate absorbed by the roots can be assimilated in the root tissues or in leaf tissues, depending on their availability (oliveira et al., 2011). on the other hand, it is highlighted that the evaluated plants did not receive nitrogen fertilization via fertigation or foliar spraying after harvest (pre-pruning phase), which may be a factor for the low enzyme activity in the leaves of 1st and 2nd flushes. furthermore, the low nra in these tissues might have occurred as a function of the low no3concentrations due to the exportation of n by the fruits harvested in the previous season, approximately two months before the evaluation, since this element is the second most extract macronutrient through harvest in the mango crop (nascimento et al., 2005). it is believed that the maximum enzyme activity in the roots may have occurred as a function of the applications of calcium nitrate, via fertigation, performed during fruit development in the previous cycle, which was not required by the plant, being stored in the root vacuoles for later metabolization. according to prado (2013), fruit crops assimilate no3mainly in the roots, with an increment in the leaves when there is an increase in the availability of this element. the mango plant seems to present great storing capacity of reserve substances, besides presenting a root system that, although being little dense, occupies a large soil volume (castro neto, 1995). likewise, in the grapevine crop, the younger leaves presented high nitrate reductase activity after harvest, which was similar to the accumulation of sucrose and starch (hunter and ruffner, 1997). according to these authors, the increase in the enzyme activity could be related to the annual accumulation of nitrogen in the crop. this difference between the mango and grapevine crops could occur as a function of the n management in the annual cycle; besides, the location of the nitrate assimilation tissue in the plants varies according to the species and may indicate different ecological adaptations (black et al., 2002). at 15 days after pruning, in spite of the plant material analyzed, the nitrate reductase presented a statistically equal activity (fig. 2). in the production pruning, performed just after harvest, the main objectives are to clean out dry and sick branches, besides promoting the central opening of the plant to allow a maximum internal lighting, favoring vegetative growth for the next harvest; in this manner, all resources are directed towards vegetative growth (mouco, 2015). after this cultural practice, nitrogen fertilization was performed, although not observing an increase in the nra of the plant tissues. this may have occurred because the n source provided was ammonium sulfate, which does not contain nitrate (no3-) in its composition, the main substrate for the activation of the enzyme (martuscello et al., 2016). generally, ammonium sulfate and urea are the main n sources provided in this phase for the mango crop. nitrate salts are applied at the dormancy break phase of the buds, via foliar spraying, and during fruit development, via fertigation. with regard to the nra quantification in the post-pbz evaluations, a growth regulator applied to inhibit branch growth and promote bud maturation, increasing the potential of the plant in producing reproductive sprouts (davenport, 2007), in the present study the use of this regulator demonstrated to favor a maximum activity in the leaves (in spite of the flush) than in the roots (fig. 2). pbz has been widely employed in mango farming to restrict vegetative growth, favor carbohydrate accumulation, and promote flowering. it works by blocking the synthesis of gibberellin, which inhibits flowering and stimulates vegetative growth (taiz et al., 2017). the lower activity of the enzyme in the roots, in both evaluated days, may be related to the lower metabolic activity in the root system, since pbz is applied via root system, affecting root growth and leading to a reduction in drainage force, affecting energy consumption and demand, since the energy expenditure for no3reduction in the root tissues is high and roots are considered less efficient assimilation sites than the shoot part (carelli and fahl, 1991), since the leaves are the synthesis center of atp and reducing agents (taiz et al., 2017). the maximum nra in 1st and 2nd flushes leaves may be related to the increase in the carbohydrate content in these sites, a primary energy source, even if most sugars are directed to branch maturation, since the availability of carbohydrates increases the nitrate reduction rate (klepper et al., 1971). although pbz application decreases the root water potential and consequently reduces the transpiration rates, the adequate dose of the product should not decrease the production of photoassimilates (souza et al., 2016), maintaining the plant metabolism in these conditions, what seems to have occurred in the studied plants when observing that the nitrate reductase enzyme maintained its activity, especially considering that the demand for nitrogen compounds should decrease in this locking phase of the plant when there is no generation of new tissues. souza et al. (2016) characterized nitrate reductase activity in leaves and roots of ‘palmer’ mango after pbz application via fertigation and found a maximum activity in the root system, differently from what was observed in the present work. furthermore, they observed that there was no significant effect of the application method and doses of pbz via fertigation on nra. in the branch maturation phase, the nra in the 2nd flush leaves resembled that of the roots (fig. 2) and, in this period, it is common to employ 3% potassium sulfate (k2so4) in two or more foliar spray ings, after leaf maturation, around 60 days after pbz application, with a 7-day interval between them. four k2so4 applications were performed in the studied plants. to the detriment of this result, it is worth noting that potassium (k) is the product component used and, such as n, it is the nutrient that determines gain in the activation of nitrate reductase and, consequently, no3reduction, an already seen fact in cucumber plants (ruiz and romero, 2000). furthermore, this ion interferes with the potassium/nitrogen relation, inhibiting vegetative growth and collaborating with branch maturation in mango, thus improving bud fertility (silva and vilela, 2004). potassium an osmotically active ion involved in the osmoregulation of guard cells, and its concentration in these cells is responsible for the stomatal opening and closing mechanisms (taiz et al., 2017). the increase in the concentration of k in the guard cells reduces their osmotic potential, allowing the inlet of water and, as a function of the high cell turgor, the stomata open (taiz et al., 2017). in this manner, there is an increase in stomatal conductance, and it may favor no3transportation to the shoot part, inducing an elevated nitrate reductase activity in the 2nd flush leaves. according to ruiz and romero (2002), k facilitates the absorption and transportation 196 a.j. da silva santos, v. borges de paiva neto*, l. guimarães sanches, d. de almeida carreiro, m.p. martins pereira, m.c. rezende zuffo borges, s.e. rodrigues dos santos, í.h. lucena cavalcante of no3to the shoot part of the plant, explaining the increase of nra with the increment of this element in the 2nd flush leaves. furthermore, 1st flush leaves presented lower nra, 2nd flush leaves are stronger drains if compared to 1st flush leaves, and, consequently, receiving more substrate, activating nitrate reductase, and favoring its activity. alternatively, the decrease in the enzyme activity with leaf age can be attributed to changes in the general metabolic activity and foliar ontogenesis (black et al., 2002). this fact has been already demonstrated in leaves of the mango variety dasehri, originated from india (ananthanarayanan and chacko, 1983), in which the nra was high in the initial phase of development, with later reduction and stabilization. another management performed in the floral induction process is the reduction of the irrigation water depth along with branch maturation. water deficit decreases nitrate reductase activity due to the decrease in the water flow through the transpiration current and, consequently, the nitrate flow toward the leaves (oliveira et al., 2011), since the enzyme is highly dependent on its substrate. however, this management did not seem to interfere with the nra of the 2nd flush leaves and roots, perhaps because when the analysis was performed the irrigation water depth was reduced in 25% of the total applied, not yet a limiting factor to the activity of the enzyme. besides the foliar spraying with a potassium nitrate, efficient in favoring the activity of the enzyme. dormancy break is performed through the foliar spraying of nitrate salts to stimulate reproductive sprouting (jameel et al., 2018). in the studied plants, 3% potassium nitrate (kno3) was used for this purpose. this element triggers the formation of nitrate reductase, which reduces nitrate and leads to the synthesis of amino acids, especially methionine, which is the precursor of ethylene. this, by its turn, induces flowering (coutinho et al., 2016). in this manner, it is inferred that this processes, mediated by the nra as a function of foliar spraying with kno3, occurs more substantially in the roots and 2nd flush leaves, in the periods of pre and post floral induction, respectively (fig. 2), showing a modulation capacity in the nra by the plant for the benefit of the application of nitrate salts (taiz et al., 2017), since the enzyme is induced by its substrate (martuscello et al., 2016), possibly explaining the activity increase in the 2nd flush leaves after the spraying, in comparison with the roots. according to konishi and yanagisawa (2011), the expression of nr genes is rapidly stimulated in several plants in the presence of no3-. there is a report that in grapevine leaves and roots there was an increase of the nra in the presence of calcium nitrate (ca(no3)2), being evident that the shoot part is the preferential n assimilation site in this crop (mesquita et al., 2018), as well as in peach palm seedlings (bactris gasipaes) (oliveira et al., 2005). as for young coffee plants (coffea arabica l.) irrigated with solutions containing different nitrate concentrations, the nra was maximum in the roots than in the leaves (carelli and fahl, 1991). the preference regarding the nitrate reduction site may vary ac cording to the species, but it is not constant, since it is influenced by environmental and physiological factors (andrews, 1986), such as the irradiance regime, so that in the period of greater photo synthetically active radiation (par) nitrate reduction is maximum in the leaves compared to the roots, and when of the reduction in the par, the nra concentrates in the root tissue (carelli and fahl, 2006). photosynthetic tissues are the most adequate place for nitrate reduction due to their energy consumption, a previously discussed fact. however, there are several reports of nra in root tissues (martuscello et al., 2016), such as in the present study. the decrease in root nra at floral induction and in the subsequent phases (fig. 2) may also be related to the amount of energy made available in these tissues as a function of the energy invested in the formation of panicles and fruits, which are more efficient drains than the roots, in these phenophases (taiz et al., 2017). regarding mango flowering, this is the phase in which there is a demand for an adequate nitrogen supply, which is also related to the age and physiological stage of the plant during the agricultural year. before flowering, there are high values of nitrogen content, while in full bloom and fruit formation there are minor contents (nascimento et al., 2005), which may explain the nra behavior in the referred phenophases. changes in the foliar n content and the photosynthetic capacity during plant development can result in n depletion by drains, such as flowers and fruits (urban et al., 2004), which may be the cause of the beginning of a reduced enzyme activity in the 2nd flush leaves, closer to the inflorescences, acting as a carbohydrate source (energy) for the development of flowers and fruits, consequently reducing nra in this site, since the photosynthetic rates of the plants are fundamental for the increase in the activity of the enzyme (hunter and ruffner, 1997). souza et al. (2016) observed in the ‘palmer’ mango that at the flowering phase there was a decrease in the content of total soluble sugars, reducing sugars, total proteins, and in the content of sucrose, and inferred that this may have occurred due to the energy demand for the formation of inflorescences, source/drain ratio. as previously demonstrated, throughout the evaluated phenophases, in general, there was a reduction in nra, and such a fact can be better observed in the fruiting phase, in which it is inferred that as a function of the growing n demand, the remobilization of foliar n to fruit development continues in this stage and, as a consequence, there is a decrease of this element in the plant organs evaluated in this study. on the other hand, the degradation of foliar proteins generates a greater number of amino acids that act as regulators, decreasing nra probably through the action inhibition of no3transporters in the cell membrane, since circulating amino acids in the phloem can control the no3absorption rate by the roots (imsande and touraine, 1994). this fact can justify the lower nra activity in 1st flush leaves comparing with 2nd flush leaves, whereas those will always be older than these, and, probably with superior degradation of proteins. in the roots, there is little variation in the activity of the enzyme between the phases of floral induction, flowering, and fruiting (fig. 2). it is suggested that this may have occurred due to the soil application of nitrate calcium, performed when the plants presented a defined flowering and early green fruits, acting as an available substrate for the maintenance of nra in the roots and increase in the shoot part during these phenophases, with the 2nd flush leaves being the preferential site for the reduction and assimilation of no3in the mango crop, in these phenological stages. another factor that may have contributed to maintaining nitrate reductase activity in the shoot part at the fruiting phase was the soil fertilization with potassium sulfate and potassium chloride, performed in the orchard during the fruit development stage. according to ruiz and romero (2002), the application of k-based fertilizers facilitates the capture and transportation of no3to the shoot part, since this element is an accompanying cation of no3in the xylem (blevins, 1985). by performing a general nra analysis, an opposed behavior could be verified between the vegetative and reproductive phases when the contribution of each monitored organs was grouped (fig. 3). in the vegetative phase, it is possible to evaluate the consequence of pruning, in which there is no compensation of enzyme activity by the 1st flush leaves and roots as a function of the elimination of the 2nd flush leaves. regarding the increase in nra from 32 days after pbz application, it seems to have occurred as a function of the supply with a potassium source, which favors the absorption and transportation of no3in the plant and, consequently, the activity of the enzyme. the reproductive phase coincided with the foliar and fertigation application of a nitric fertilizer, in which the enzyme activity begins to decrease from flowering, verifying, at the end of fruiting, a similar nitrate reductase along mango phenophases 197 level to that observed at the beginning of the cycle, that is, at prepruning (fig. 3). nutrition can affect the yield of the mango crop. nutrient absorption by the crop varies as a function of the age and phenological stage of the plant. for this reason, it is important to know the dynamic of nutrients in the several parts of the plant, throughout cultivation, aiming to provide subsidies to better adequate fertilization programs for the crop (ordóñez, 2011). conclusions nitrate reductase activity in ‘palmer’ mango plants is influenced by environmental factors, especially solar radiation, and potassium/ nitrate spraying. to obtain a maximum nitrate reductase activity in the leaf and root tissues, the collection of the vegetal material around 10 a.m. is recommended. 2nd flush leaves and roots constitute the main activity sites, whereas in 1st flush leaves there is minimum variation in the nitrate reductase enzyme. the enzyme activity is lower in the vegetative phase than in the reproductive phase. acknowledgement the authors thank the coordination for improvement of maximum education training (capes) for the scholarship granted to alana juliete da silva santos. conflict of interest on behalf of all authors, the corresponding author declares that there is no financial and personal relationships that could inappropriately have influenced our work. references albuquerque, j.a.s., medina, v.d., mouco, m.a.c., 2002: indução floral. in: genú pjc, pinto caq (ed.), a cultura da mangueira, 259-276. embrapa informação tecnológica, brasília. álvares, c.a., stape, j.l., sentelhas, p.c., gonçalves, j.l.m., sparovek, g., 2013: climate classification map for brazil. meteorol. z. 22, 711-728. doi: 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(eds.), mangaprodução integrada, industrialização e comercialização, 321-338. souza, m.a., mesquita, a.c., simões, w.l., ferreira, k.m., araújo, e.f.j., 2016: physiological and biochemical characterization of mango tree with paclobutrazol application via irrigation. pesqui agropecu trop 46, 442-449. sudha, r., balamohan, t., soorianathasundaram, k., 2012: effect of foliar spray of nitrogenous chemicals on flowering, fruit set and yield in mango (mangifera indica l.) cv. alphonso. j. hortic. sci. 7, 190-193. taiz, l., zeiger, e., moller, i., murphy, a., 2017: fisiologia e desenvol vimento vegetal. artmed. tucker, d.e., allen, d.j., ort, d.r., 2004: control of nitrate reductase by circadian and diurnal rhythms in tomato. planta, 219(2), 277-85. doi: 10.1007/s00425-004-1213-x urban, l., lu, p., thibaud, r., 2004: inhibitory effect of flowering and early fruit growth on leaf photosynthesis in mango. tree physiol 24, 387-399. doi: 10.1093/treephys/24.4.387 wongsrisakulkaew, y., boonprakob, u., sethpakdee, r., juntawong, n., 2017: effect of paclobutrazol concentrations and time of foliar application on flowering of ‘namdokmai-sitong’ mango. international journal of geomate 12, 41-45. doi: 10.21660/2017.30.96545 orcid alana juliete da silva santos https://orcid.org/0000-0001-9725-9120 vespasiano borges de paiva neto https://orcid.org/0000-0003-3347-7043 luciana guimarães sanches https://orcid.org/0000-0003-2319-009x daniel de almeida carreiro https://orcid.org/0000-0003-1048-7038 maria poliana martins pereira https://orcid.org/0000-0001-8744-1930 m.c. rezende zuffo-borges https://orcid.org/0000-0002-1013-4710 stefany e. rodrigues dos santos https://orcid.org/0000-0003-2700-0337 ítalo herbert lucena cavalcante https://orcid.org/0000-0003-1610-1546 address of the corresponding author department of agricultural engineering (cca), universidade federal do vale do são francisco, petrolina-pe, brazil. e-mail: vespasiano.paiva@univasf.edu.br © the author(s) 2021. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.21071/az.v65i252.1927 http://dx.doi.org/10.1590/s0103-84782005000300005 http://dx.doi.org/10.1007/s40009-019-00828-8 http://dx.doi.org/10.1093/jpe/rty044 http://dx.doi.org/10.1002/1097-0010(200011)80:14<2069::aid-jsfa749>3.0.co;2-7 http://dx.doi.org/10.1111/j.1744-7348.2002.tb00177.x http://dx.doi.org/10.1590/s0100-204x2014000500008 http://dx.doi.org/10.1007/s00425-004-1213-x http://dx.doi.org/10.1093/treephys/24.4.387 http://dx.doi.org/10.21660/2017.30.96545 https://orcid.org/0000-0001-9725-9120 https://orcid.org/0000-0003-3347-7043 https://orcid.org/0000-0003-2319-009x https://orcid.org/0000-0003-1048-7038 https://orcid.org/0000-0001-8744-1930 https://orcid.org/0000-0002-1013-4710 https://orcid.org/0000-0003-2700-0337 https://orcid.org/0000-0003-1610-1546 journal of applied botany and food quality 92, 25 32 (2019), doi:10.5073/jabfq.2019.092.004 1department of agriculture, payame noor university (pnu), tehran, iran 2department of horticultural science, college of agricultural science, tarbiat modares university (tmu), tehran, iran 3institute of humanities and cultural studies, tehran, iran foliar application of 5-aminolevulinic acid promotes bioactive compounds and nutritional value of purslane, a potential vegetable for the future vahid tavallali1*, samira jandoust2, abazar ashtari mehrjerdi3 (submitted: august 6, 2018; accepted: january 17, 2019) * corresponding author summary decent taste and the salt and drought tolerance of purslane (portulaca oleracea l.) make it a potential vegetable crop for the future. this study investigates the effects of foliar application of 5-aminolevulinic acid (5-ala), a new plant growth regulator, on the growth, mineral composition, and bioactive compounds of purslane. three different levels of 5-ala (0, 25 and 50 mg l-1) were sprayed at 1) two leaved stage and 2) upon the onset of inflorescence appearance on purslane seedlings. results showed that 5-ala application enhanced biomass accumulation in the plant shoot and increased shoot length. concentration of nitrogen, potassium, magnesium, zinc and iron increased in the shoots of 5-ala treated plants, while the calcium concentration remained unaffected. phenolic compounds of the plant were catechin, chlorogenic acid, and ellagic acid, with catechin be ing the main compound. further on, trans-ferulic acid, hesperedin and eugenol were detected in the extract of 5-ala-treated plants. application of 5-ala also increased fatty acids in the plant leaves. total phenolics, ascorbic acid contents and antioxidative activity of shoot were increased in the 5-ala-treated plants. moreover, ph of root exudates of the plants was decreased in 5-ala treated plants. the results revealed that exogenous 5-ala has growth regulatory effects and can enhance the growth, and improve nutritional quality and pharmaceutical properties of p. oleracea. in this regard, the best results were obtained by application of 50 mg 5-ala l-1. keywords: fatty acids, mineral nutrients, organic acids, purslane, phenolic compounds, root exudation. introduction dietary fiber, polyunsaturated fatty acids, and antioxidantive phenols, flavonoids, terpenoids, and vitamins of vegetables are of critical importance for consumer health (barros et al., 2008). purslane (portulaca oleracea l.) is a tolerant species with capability of growing in nutrient-poor soils and harsh environmental conditions such as drought and salt affected soils. the plant is well adopted to hot and dry conditions and can survive under new climate of the globe (fig. 1). in addition to gentle salty/sour taste, it has high nutritious value i.e. high content of linoleic (18:2ω6) and α-linolenic (18:3ω3) acids, and antioxidative compounds (mera-ovando et al., 2014; siriamornpun and suttajit, 2010). based on the usda nutrient database (2018), 100 gr of raw purslane may provide 81% vitamin e, 25% vitamin c, 19% magnesium, 15% iron and 11% potassium of daily requirements for the adults. in sum, these characteristics make purslane a potential food resource for future. the plant is an individual from portulacaceae which contains many species whose number exceeds 120 that are succulent herbs and bushes (hyam and pankhurst, 1995). the aerial organs of these plants are used as a febrifuge, antiseptic, diuretic, vermifuge and antispasmodic (xiang et al., 2005). the plant has antidiabetic effects (sharma et al., 2010) and can be used for reducing triglycerides and cholesterol in blood (zidan et al., 2014). furthermore, antiviral (cai-xia et al., 2010) and antitumoral (zhao et al., 2013) have been noticed in the plant extract. in some regions, especially in west asia, purslane is consumed as a foliar vegetable, too. it is known to have advanced nutritional value than many cultivated vegetables, as a boundless source of ascorbic acid, phenolics and antioxidants (liu et al., 2000). the nutraceutical and bioactive compounds of plant crops can be improved by optimizing growing conditions, cultivation practices and application of biostimulators. in this regard, plant growth regulators are an excellent choice for manipulating plant metabolites and pharmacological effects. 5-aminolevulinic acid (5-ala) is a plant growth regulator (pgr) which can enhance different types of antioxidants in plant tissues (nishihara et al., 2003). it is a precursor of tetrapyrrole compounds such as heme, chlorophylls, and cobalamin (stobart and ameen-bukhari, 1984). in bacteria and plants, prosthetic groups of respiratory enzymes and chlorophylls are provided by this compound (brunham and lascelles, 1963). application of low concentrations of 5-ala (10-300 mg l-1) at early growth stage, have been reported to increase yield of barley, potato, radish, garlic and kidney bean by enhancing growth rates and photosynthesis of these species (hotta fig. 1: purslane is a hardy species with capability to grow in hot and dry environmental conditions. this picture indicates how the plant can survive abiotic challenges such as drought and poor soil. the fleshy nutritious leaves of the plant have decent salty/sour taste. these specifications make it a potential vegetable crop for the future. 26 v. tavallali, s. jandoust, a. ashtari mehrjerdi et al., 1997). watanabe et al. (2006) reported that application of 100 mg l-1 of 5-ala increased the growth and co2 assimilation of grapevine. 5-ala at 10 and 100 mg l-1 enhanced chlorophyll biosynthesis and photosynthesis rate in melon seedlings under low light intensity (wang et al., 2004). this chemical is effective at low concentrations (76.2-762 μm) and seems to be a new pgr. the ameliorative effect of 5-ala on accumulation of simple sugars accompanied by the maintenance of starch content in the leaves and preserving relatively higher leaf mineral contents in different plant species such as rapeseed (liu et al., 2016) and sunflower (akram and ashraf, 2011) have been demonstrated previously. the enhancement of the cell antioxidants is another effect of 5-ala which provides significant protection to plasma membranes against free reactive oxygen radicals (nishihara et al., 2003). therefore, it can be used as a pgr to manipulate secon dary metabolites and promote pharmaceutical properties of medicinal plants. however, there is certainly lack of comprehensive under standing of physiological and biochemical responses of medicinal plants to 5-ala. according to the role of 5-ala on the accumulation of secondary metabolites and bioactive compounds, this study was to evaluate growth, phenolics, antioxidative activity and mineral composition of p. oleracea l. in response to foliar application of 5-ala, in an effort to improve its application for the public consumption. material and methods plant material and growing condition a greenhouse experiment was completed at shiraz, iran. the soil used in this study was a fine topsoil loam taken from 0 to 30 cm of a calcareous soil (typic calcixerepts). physicochemical properties of the soil were as follows: ph: 7.41, ece: 1.2 ds m-1, cec: 10 cmolc kg-1, organic carbon: 8.9 g kg-1 soil, nitrogen (n): 0.07%, phosphorus (p): 13 mg kg-1 soil, potassium (k): 59 mg kg-1 soil, calcium carbonate equivalent (cce): 454 mg kg-1 soil, dtpa-extractable fe: 2.21 mg kg soil-1. nitrogen and phosphorous at the concentration of 50 mg kg-1 soil, and cu, zn and mn at the concentration of 5 mg kg-1 soil were applied in a uniform manner to the soil each as nh4no3, kh2po4, cuso4·5h2o, znso4·7h2o and mnso4·h2o, respectively. 8-liter plastic pots were filled with 7.5 kg of the soil. twenty seeds of purslane (portulaca oleracea l.) were planted in pots and irrigated with deionized water to field capacity twice a week. after fifteen days, at two-leaf stage, the seedlings were thinned to 10 uniform stands in each pot. three concentrations of 5-ala (0, 25 and 50 mg l-1 in the form of δ-aminolevulinic acid hydrochloride) was sprayed on the plants at two stages: after thinning (20 days after planting) and upon the onset of inflorescence appearance (65 days after planting). deionized water was also sprayed as control. the plants were harvested 12 weeks after planting. shoot fresh (fm) and dry mass (dm), and shoot length were measured after twelve weeks. moreover, the following physiological and biochemical traits were analyzed. purslane extracts preparation methanol extracts of p. oleracae l. samples were prepared by the method described in najafian and zahedifar (2015). twenty grams of dried samples were drenched in 250 ml of methanol/water (90:10 v/v) for 48 hours. filtration and concentration of the extracts were done in a rotary evaporator for ten minutes. the desiccated extracts were powdered and by measuring the mass of the fine powders their yields were determined. the powders were maintained at -18 °c before utilization. the needed level of powder in methanol was arranged before measurements, and then total phenol content and the antioxidative activity were evaluated. extraction and hplc analysis of polyphenols extraction of polyphenols was carried out in view of a previous report with a few modifications (justesen et al., 1998). hplc examination was performed on an agilent technologies 1200 series (germany), outfitted with a zorbax eclipse (xdb) c18 (5 μm (id), 4.6 × 150 mm (ft) and a photodiode cluster identifier. at 230 and 280 nm, elution was observed. elution was performed by changing the solvent methanol to formic acid proportion. total phenolics and ascorbic acid contents the total phenolic compounds (tp) in the plant extracts were measured by using the folin-ciocalteu reagent. according to the method described by halicia et al. (2005), 1.2 ml of na2co3 (7.5%, w/v) solution and 1.5 ml of the reagent (diluted 10 times) were added to 300 ml of samples. the mixtures were shaken and left at a dark room for 30 minutes. then the mixture absorbance was measured at 765 nm by a spectrophotometer (varian 220, australia). the measurements were repeated 3 times. the total phenolics’ content was represented as gallic acid (gae) equivalent in 100 g fresh sample. amendments tp content was made by subtracting the ascorbic acid content (aa) from the total phenolics content. ascorbic acid content in the plant leaves was measured using indophenol method according to aoac (1984). 10 g of fresh leaves were homogenized with 48 ml metaphosphoric acetic acid and 2 ml of sodium citrate solution. the samples were filtered using a buchner funnel and suction pump and 10 milliliters of the filtrates were rapidly titrated with the standardized 2,6-dichlorophenolindophenol solution till formation of a permanent pink color. the titrations were repeated 3 times. blank determinations were also carried out by using 10 ml of metaphosphoric acetic acid instead of the filtrate. antioxidative activity measurement for measuring the scavenging capacity of the plant extracts against dpph free radical, the 25 μl of 12-3100 μg ml-1 gallic acid or methanol extracts was blended with 220 μl of 120 mm of dpph in methanol (burits et al., 2001). for thirty minutes, the solutions were retained at ambient temperature. dpph radical restraint was assessed at 515 nm utilizing an el×808 absorbance microplate reader (biotek instruments inc., usa). concentration (mg l-1) needed to restrain dpph radical formation by fifty percent (ic50) were computed from the nonlinear regression between mean % radical-scavenging activity utilizing matlab software (the mathworks inc., usa) and log extract concentration (g ml-1). methanol extract without dpph used as the blank. antioxidant activity was assigned utilizing the following equation: antioxidant activity = 1[(asample-ablank)/acontrol] × 100 where asample and ablank are absorption of the test solution (t = 30 min) and the blank reaction (t = 0 min). control and blank solutions were dpph (without plant extract) and methanol solutions, respectively. fatty acids content fatty acids were determined according to the method described by barros et al. (2008). the fatty acids were methylated with methanol:sulphuric acid:toluene 2:1:1 (v:v), during 12 h at 50 °c. de ionized water was added to the solution to obtain phase separation. after vortexing, the fatty acids methyl ester were recovered with diethyl ether. the upper phase was passed through a micro-column of sodium sulphate anhydrous and then filter by a 0.2 μm nylon filter. the fatty acid profile was analyzed using a gc-ms (agilent 7890 system, agilent technologies inc., usa). the instrument equipped with a split/splitless injector, a flame ionization detector (fid) and a macherey-nagel column (30 m * 0.32 mm id * 0.25 μm df). the effect of 5-ala on bioactive compounds of purslane 27 a a b 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 t p c (g g a e 1 00 g1 ) b a ab 50 55 60 65 70 75 80 85 90 0 25 50 a a (m g 10 0g -1 ) 5-ala (mg l-1) initial temperature of the column was 50 °c, held for 2 min, then a 10 °c/ min ramp to 240 °c and held for 11 min. flow rate of the car rier gas (hydrogen) at 50 °c was 4.0 ml/min (0.61 bar). split injection (1:40) was carried out at 250 °c. one μl of the sample was injected and fatty acid identification was made by comparing the relative retention times from samples with fame peaks (standards). the results were recorded and processed using csw 1.7 software (dataapex 1.7) and expressed in relative percentage of each fatty acid. root exudates analysis the plants’ roots were carefully washed off the soil and used for analyzing organic acids in their exudates according to the method described by czarnota et al. (2001). roots were extracted with methylene chloride acidified with 0.25 % glacial acetic acid, for 30 s. the extract was filtered with a 2.0 μm filter and freeze dried. the concentration of organic acids was determined in freeze dried root exudates. the samples were dissolved in with ethanol (80%). then, 20 microliter of the dissolved extracts were injected into zorbax eclipse (xdb) c18, 4.6 (id) × 150 mm, 5 μm (film thickness) column. the column temperature was 45 °c. agilent hplc 1200 series was used to determine concentration of organic acids in the root exudates. the mobile phase was a mixture of acetonitrile: sulfuric acid: acetic acid (15:4:1) with the flow rate of 1 ml min-1 for 10 minutes. the photodiode array detector was set on 214 nm for quantification of organic acids. for measurement of ph, freeze-dried samples were dissolved in deionized water and ph of the extracts was measured using a ph meter (hanna model hi2210, usa). mineral composition of plant shoot samples were dried for 72 h at 70 °c in an oven. nitrogen concentration of the samples was determined by autotech (model 300). then samples (ca. one gram per replicate) were ashed for 6 h at 550 °c. the white ash powder was added in 5 ml of 2 m hot hydrochloric acid, filtered into a 50 ml volumetric flask, and made up to 50 ml with deionized water. iron, calcium, magnesium, and zinc analyses were done using an atomic absorption spectrophotometer (model varian 220, australia). by using a flame photometer, the concentration of potassium in the plant extracts was determined. statistical analysis the experimental design was a completely randomized design with three treatments. eight replications were considered per treatment and ten seedlings used per replicate. in sum, 240 plants were used in this experiment. statistical analysis was carried out by the statistical software spss (version 20). duncan’s multiple range test (dmrt) at the level of 5% probability was utilized to compare the differences between the means. results plant growth tab. 1 represents the effects of 5-ala on plant growth indices over twelve weeks. the shoot dry mass, fresh mass and mean of shoots’ length of purslane seedlings showed an increasing trend with application of 5-ala. the highest shoot growth parameters of the plants were obtained with application of 50 mg l-1 5-ala. total phenolics and ascorbic acid contents compared to the control, 5-ala applications increased the total polyphenolics (tp) and ascorbic acid (aa) contents in the aerial organs of purslane (fig. 2a, b). no significant differences were observed in tp and aa levels in the leaves of 25 or 50 mg l-1 5-ala treated plants. the highest total phenolics (3.67 g gae 100 g-1) and ascorbic acid content (81.47 mg 100 g-1) was found in plants supplied with 25 mg l-1 5-ala. tp content was positively associated with the antioxidative activity and ascorbic acid content in the plant leaves (fig. 3a, b). phenolic compounds in this study, 3 phenolic compounds (catechin, chlorogenic acid, and ellagic acid) were detected in the control plants (tab. 2). with the exception of chlorogenic acid, 5-ala application had outstandingly positive effects on concentration of these phenolic compounds. in this regard, foliar application of 50 mg l-1 5-ala incited significant higher phenolics in comparison with the 25 mg l-1 treated plants. catechin was the predominant phenolic compound in the control tab. 1: shoot fresh mass (fm) and dry mass (dm) and mean of shoots’ length of portulaca oleracea in response to foliar application of different 5-ala concentrations. measurements 5-ala (mg l-1) 0 25 50 fm (g) 34.53c ± 0.73 49.97b ± 1.00 57.64a ± 1.41 dm (g) 2.42c ± 0.32 3.19b ± 0.31 6.98a ± 0.21 shoot length (cm) 19.44b ± 1.64 21.45b ± 1.75 34.26a ± 1.73 *means followed by the same letter are not significantly different according to dmrt at p≤0.05. data are mean ±standard deviation of eight replications. fig. 2: the effects of different 5-ala treatments on total phenolics (tp) (a) and ascorbic acid (aa) contents (b) in the leaves of portulaca oleracea. means marked by the same letter are not significantly different according to dmrt at p≤0.05. error bars represent standard deviation. 28 v. tavallali, s. jandoust, a. ashtari mehrjerdi purslane plants (0.26 mg g-1) which increased by 200% in 50 mg l-1 5-ala treated plants. ellagic acid content in the control plant extract was 0.02 mg l-1 which was increased by 1100% in the 50 mg l-1 5-ala treated plants. moreover, the 5-ala treatments increased the number of detected phenolics compounds in the plant to six. trans-ferulic acid, hesperedin, and eugenol were the compounds which detected in the 5-ala treated plants. concentration of trans-ferulic acid and eugenol in both 5-ala treatments were the same, however, the 50 mg l-1 5-ala treated plants had higher hesperedin in comparision with the 20 mg l-1 5-ala treated plants. fatty acids concentration two saturated fatty acids (sfa) including palmitic acid (c16:0) and stearic acid (c18:0); and two polyunsaturated fatty acids (pufa) including linoleic acid (18:2ω6) and α-linolenic acid (18:3ω3) were found in the plant leaves. the regression models corresponding to each fatty acid in function of the studied factor are shown in fig. 4. the fits of the regression models have high r2 values (> 0.90) (fig. 4). therefore, r2 indicate a considerable response of fatty acids to the studied factor. the regression model for the concentration of palmitic acid (fig. 4) indicates a decrease from the applied 5-ala. there were 11.8 and 20.4% decrease in palmitic acid for 25 and 50 mg l-1 of applied 5-ala, respectively (fig. 4). the regression model for the concentration of stearic acid (fig. 4) indicates a decrease from the applied 5-ala. there were 19.46 and 32.95% decrease in stearic acid for 25 and 50 mg l-1 of applied 5-ala, respectively (fig. 4). the regression model for the conceny = 11.864x + 34.554 r = 0.96 60 65 70 75 80 85 0 1 2 3 4 a a (m g 10 0g -1 ) tp (g gae 100g-1) y = -1528.4x + 7065.6 r = 0.99 1000 1500 2000 2500 3000 3500 0 1 2 3 4 ic 50 (m g l -1 ) tp (g gae 100 g-1) ba pounds (mg g-1) tab. 2: phenolic compounds in the leaves of portulaca oleracea in response to foliar application of different 5-ala concentrations. phenolic com 5-ala (mg l-1) 0 25 50 catechin 0.26c ± 0.02 0.37b* ± 0.04 0.52a ± 0.03 chlorogenic acid 0.02a ± 0.005 0.01b ± 0.004 0.02a ± 0.006 ellagic acid 0.02c ± 0.004 0.07b ± 0.005 0.22a ± 0.01 trans-ferulic acid nd 0.17a ± 0.02 0.15a ± 0.04 hesperedin nd 0.03b ± 0.004 0.11a ± 0.01 eugenol nd 0.24a ± 0.03 0.24a ± 0.02 *means followed by the same letter are not significantly different according to dmrt at p≤0.05. data are mean ±standard deviation of eight replications. nd: not detected fig. 3: correlations between (a) total phenolics (tp) content and antioxidant activity (ic50) and (b) between total phenolics and ascorbic acid (aa) content in portulaca oleracea. tration of linoleic acid (fig. 4) indicates an increase from the applied 5-ala. there were 20.28 and 37.63% increase in linoleic acid for 25 and 50 mg l-1 of applied 5-ala, respectively (fig. 4). the regression model for the concentration of α-linolenic acid (fig. 4) indicates an increase from the applied 5-ala. there were 3.35 and 5.67% increase in α-linolenic acid for 25 and 50 mg l-1 of applied 5-ala, respectively (fig. 4). mineral composition of the plant regardless of concentration, 5-ala significantly increased fe, zn, k, mg and n contents in purslane shoots (tab. 3). with increasing 5-ala concentration, an increasing trend in the mineral content of the plants was observed. in comparison to the control plants, application of 50 mg l-1 5-ala increased concentration of: fe by 74%, zn by 110%, and n up to 91% in the plant leaves. purslane plants treated with 5-ala accumulated higher mg and k in comparison with the untreated plants. 5-ala at the level of 50 mg l-1 was more effective than 25 mg l-1 treatment. augmentation in shoot mg content up to 10% and 4% and in shoot k content up to 27% and 14% were achieved by 50 and 25 mg l-1 concentrations of 5-ala, respectively. the treatments caused no significant difference in shoot ca concentration. antioxidative activity the antioxidative activity in the plant extract is exhibited in fig. 5. the linear regression equation between ic50 was determined as (y, mg l-1) and 5-ala level (x, % w/v) was determined as y = -683.01x + 3431.7 (r2 = 0.96, p ≤ 0.01) showing that ic50 decreased in response to foliar application of 5-ala. the ic50 for the extract of 5-ala treated plants ranged between 1465.46 ± 6.58 mg l-1 and 2831.49 ± 6.21 mg l-1. the highest antioxidative activity (1465.46 mg l-1) observed in the 50 mg l-1 5-ala treated plants, while the lowest value (2831.49 mg l-1) was found in the control plants. organic acid contents and ph of root exudates without 5-ala application, the purslane root mucilage ph was 7.30 (tab. 4). the 5-ala treatments brought about a significant decrease in the root exudates ph, as the lowest ph was observed at 50 mg l-1 5-ala treatment. various organic acids were detected in the plants roots exudates. the 5-ala treatments influenced concentration of these compounds in the root exudates. higher exudation of organic acids was observed in the 50 mg l-1 5-ala treated plants. after foliar application of 5-ala (50 mg l-1), the highest concentration of organic acids was observed for formic acid (5.84 mg g-1 root dm), effect of 5-ala on bioactive compounds of purslane 29 integrity and reducing root permeability (essa, 2002). minerals are required for osmotic adjustment, protein biosynthesis, and maintenance of root cell membrane integrity (essa, 2002). wei et al. (2012) suggested that exogenous 5-ala promotes growth of young seedlings of brassica campestris ssp. chinensis by increasing formation of amino acids and proteins due to increase in nitrogen absorption and assimilation. the definite effects of 5-ala on improving mineral nutrients absorption and increasing plant growth may be a consequence of enhancement of plant photosynthetic capacity (hotta et al., 1997). the effect of 5-ala on photosynthesis rate enhancement is related to increased stomatal conductance (naeem et al., 2011). in this regard, k plays a key role in controlling stomata and function of photosynthesis apparatus and fe is required for biosynthesis of photosynthetic pigments (marschner, 2011). a b c 9 11 13 15 17 19 st ea ri c ac id (% ) stearic acid = -3.055 (5-ala)+19.737a r2 = 0.99 c b a 15 17 19 21 23 25 27 0 25 50 l in ol ei c ac id (% ) 5-ala (mg l-1) linoleic acid = 3.47 (5-ala)+15.06a r2 = 0.99 b ab a 24 25 26 27 28 29 30 0 25 50 -l in ol ei c ac id (% ) 5-ala (mg l-1) -linoleic acid = 0.77 (5-ala)+26.407a r2 = 0.98 a b c 30 35 40 45 50 p al m it ic a ci d (% ) palmitic acid = -4.885 (5-ala)+52.51a r2 = 0.99 a b c 0 500 1000 1500 2000 2500 3000 3500 0 25 50 ic 50 (m g l -1 ) 5-ala (mg l-1) (mg g-1 dw) tab. 3: mineral composition of portulaca oleracea shoot in response to foliar application of different 5-ala concentrations. minerals 5-ala (mg l-1) 0 25 50 n 69.9c ± 2.06 101.5b ± 2.33 133.8a ± 2.46 k 488c ± 13 559b ± 12 623a ± 14 ca 88.4a ± 0.96 88.7a ± 0.92 87.1a ± 0.91 mg 73.6b ± 0.55 76.8ab ± 0.43 81.3a ± 0.67 zn 0.77c ± 0.14 1.36b ± 0.13 1.62a ± 0.11 fe 9.22b ± 0.99 10.88b ± 1.08 16.12a ± 1.12 *means followed by the same letter are not significantly different according to dmrt at p≤0.05. data are mean ±standard deviation of eight replications. fig. 4: percentage of palmitic (16:0), stearic (18:0), linoleic (18:2ω6), and α-linolenic (18:3ω3) acids of the total fatty acids in leaves of portulaca oleracea l. in response to foliar application of 5-ala. means marked by the same letter are not significantly different according to dmrt at p≤0.05. error bars represent standard deviation. a the fatty acids are expressed in percentages of the total fatty acids; (5-ala) = applied 5-ala (mg l-1); r2 = multiple determination coeffcient. fig. 5: the effects of different 5-ala treatments on antioxidative activity of portulaca oleracea extract measured by dpph assay. means marked by the same letter are not significantly different according to dmrt at p≤0.05. error bars represent standard deviation.followed by malic acid (5.52 mg g-1 root dm), oxalic acid (5.10 mg g-1 root dm), succinic acid (4.71 mg g-1 root dm), and then citric acid (3.83 mg g-1 root dm). glutamic acid had the lowest (0.71 mg g-1 root dm) level among the root exuded organic acids (tab. 4). discussion growth and mineral composition of the plant foliar application of 5-ala is known to enhance plant growth (akram and ashraf, 2011; liu et al., 2016). the present study confirmed that foliar application of 5-ala increases shoots growth of p. oleracea (tab. 1). the effect of 5-ala on enhancing plant growth could be a result of improvement of the plant function in absorption of water and minerals. 5-ala foliar application improved concentrations of essential minerals in the plant shoot. this was an evidence for the role of 5-aminolevulenic acid in reclaiming the cell membrane 30 v. tavallali, s. jandoust, a. ashtari mehrjerdi the significant correlations between the concentrations of minerals in the plant shoot were not necessarily related to synergistic or antagonistic relations among them (wei and zhai, 2010). these correlations could be a subsequence of changes in environmental or physiological factors (wei and zhai, 2010). for instance, the soil used in this study was an alkaline calcareous and the elevated fe and zn uptake from the soil could be a result of the enhanced exudation of organic acids from the roots. the exudations by reducing rhizosphere ph and chelating the fe and zn ions improve their absorption from calcareous soils (marschner, 2011). elevated shoot fe and zn uptake in the 5-ala treated plants might partly be due to increase in n uptake. improved n nutrition status increases the activity and number of fe-transporter proteins on the root cell membranes (murata et al., 2008). furthermore, 5-ala might help fe acquisition by purslane leaves. aminolevulinic acid is absorbed via transport proteins containing amino acid permease 5(aap5), amino acid permease 1 (aap1) and lysine-histidine (lht1) (svennerstam et al., 2007). our results are in agreement with liu et al. (2016) who reported that 5-ala application improved the contents of cu, zn and k in rapeseed. on the contrary, watanabe et al. (2000) showed that 5-ala had no effect on the uptake of ca, mg and k by gossypium l.. in this study, the 5-ala treatments had no significant effects on ca concentration in the plant shoot. this observation can be explained as follows. 1) ca absorption is a passive processand is known to be dependent on environmental conditions e.g. water availability to plant and transpiration rate (marschner, 2011); 2) the abundance of ca in the growing medium could supply high ca to the plant and plants are known to pump out extra ca from their roots. this mechanism allow them to prevent negative effects of high ca concentrations on the normal metabolism of cells (marschner, 2011). did 5-ala affect the bioactive compounds and antioxidative activity in the plant leaves? application of 5-ala significantly increased phenolics in the plant leaves; moreover, new phenolic compounds (trans-ferulic acid, hesperedin, and eugenol) were detected in the leaves of 5-ala treated plants. in agreement, exogenous 5-ala application has been repor ted to increase total phenols in date palm (al-qurashi and awad, 2011) and lettuce (aksakal et al., 2017). extracts of many plants containing phenolics represent effective antioxidative attributes (koleva et al. 2002). other investigations also showed an increase in phenolic compounds of tomato (daneshmand and oloumi, 2014) and fuji apple (xie et al., 2013) in response to 5-ala treatments. 5-ala treatment by promoting phenylalanine ammonia-lyase acti vity, increases the total polyphenol content of plant (wang et al., 2016). xu et al. (2011) demonstrated that 5-ala treatment increased concentrations of flavonoids, anthocyanins, and polyphenolics, and promoted chalcone synthase, phenylalanine ammonia-lyase, and chalcone isomerase activities in ginkgo biloba. in addition to catechin, the predominant phenolic compound in purslane extract, 5-ala treatments brought a large increase in ellagic acid content in the plant extract (tab. 2). both compounds are effective natural ros scavengers in plants. catechin may also act indirectly as antioxidant through its effect on enzyme activities and transcription factors (higdon and frei, 2003). ellagic acid also is an important antiproliferative agent, which prevents the dna binding of carcinogens such as nitrosamines (mandal and stoner, 1990) and polycyclic aromatic hydrocarbons (teel et al., 1986). more importantly, both of these phytochemicals have exhibited antiangiogenesis activity in vitro and in vivo (wang et al., 2012). antiangiogenesis is of important part of the treatment strategies in a variety of neoplasms such as breast, colon, lung, and kidney cancers. in addition to polyphenols, 5-ala significantly increased ascorbic acid (aa) that is synthesized from hexoses through the shikimate and phenylpropanoid pathways (xu et al., 2011). aa has antioxidative activity and has the ability to inhibit cancer and cardiovascular disease (uddin et al., 2014). in parallel with enhancement of shoot polyphenolics and aa content, foliar application of 5-ala increased antioxidative activity in shoots of purslane. our findings are in agreement with akram and ashraf (2011) who illustrated that 5-ala can efficiently enhance the antioxidative activity in helianthus annus l.. akram and ashraf (2013) also demonstrated that application of low levels of 5-ala could considerably increase the levels of nonenzymatic antioxidants like aa and glutathione in leaves of ginkgo. accumulation of phenolics and aa could be the reason of enhancement in the antioxidative activity in purslane leaves (aksakal et al., 2017). the noticeable raises in antioxidant activity of kudzu (pueraria phaseoloides) following 5-ala treatment greatly enhanced the medicinal value of the plant (xu et al., 2010). previous studies also reported that application of low concentration of 5-ala (1 mg l-1) improved antioxidants in brassica napus (naeem et al., 2011) and spinacia oleracea (nishihara et al., 2003). therefore, application of 5-ala to increase content of bioactive compounds could be a useful method to enhance antioxidant capacity and pharma ceutical value of purslane. palmitic acid (37.1-47.9%) was the fatty acid with the highest concentration in purslane leaves. α-linolenic acid (18:3ω3), between 27.1 and 28.7 %, and linoleic acid (18:2ω6), between 18.4 and 25.4% were the other major fatty acids in the plant’s leaves. stearic acid (c18:0), between 10.64 and 16.75%, had the lowest concentration among the fatty acids. these results coincide with those reported by montoya-garcia et al. (2018). meanwhile, mera-ovando et al. (2014) reported three fatty acids in purslane in the following order of concentrations: linoleic > αlinolenic > arachidonic. however, other researchers reported a higher concentration of α-linolenic acid, followed by palmitic and linoleic acids in the plant (siriamornpun and suttajit, 2010). the omega-6/omega-3 ratio in purslane leaves was 0.68 without 5-ala application. this value increased to 0.79 and 0.88 with the application of 25 and 50 mg l-1 5-ala. but, a lower ratio of omega-6/omega-3 fatty acids is more desirable to decrease the risk of chronic diseases, such as cardiovascular, cancer, or inflammatory diseases. sanchez and harwood (2002) stated that the modifications in the fatty acids profile is due to increase or inhibition of oleate desaturase activity during the biosynthesis of triacyl glycerol. tab. 4: organic acid concentrations and ph in root mucilage of portulaca oleracea in response to foliar application of different 5-ala concentrations. 5-ala ph of root concentration of organic acids (mg g-1 root dm) formic acid succinic acid acetic acid malic acid oxalic acid glutamic acid citric acid 0 7.30 ± 0.02a 0.83 ± 0.05c 0.33 ± 0.03c 0.49 ± 0.04c 1.58 ± 0.02c 2.71 ± 0.05c 0.29 ± 0.05c 0.78 ± 0.02c 25 6.90 ± 0.02b 4.21 ± 0.09b 3.58 ± 0.06b 2.12 ± 0.06b 3.56 ± 0.09b 4.66 ± 0.01b 0.54 ± 0.01b 2.87 ± 0.05b 50 6.53 ± 0.04c 5.84 ± 0.05a 4.71 ± 0.09a 3.76 ± 0.05a 5.52 ± 0.05a 5.10 ± 0.02a 0.71 ± 0.01a 3.83 ± 0.09a *means followed by the same letter are not significantly different according to dmrt at p≤0.05. data are mean ±standard deviation of eight replications. (mg l-1) exudates effect of 5-ala on bioactive compounds of purslane 31 conclusion our results showed that foliar application of 5-ala increased shoot fe, zn, n, mg and k contents and improved growth of portulaca oleracea. 5-ala also increased fatty acids, polyphenols content and antioxidant capacity of the plant. therefore, 5-aminolevulinic acid appears to provide an opportunity to increase yield and quality of leafy vegetables. furthermore, it can be used at low concentrations to improve pharmaceutical and nutritional values of medicinal plants. how this information might be used commercially still needs to be resolved. moreover, to realize the mechanism of action of 5-ala specified studies have to be carried out. acknowledgements we would like to thank dr. soheil karimi at the department of horticultural science of university of tehran for his constructive suggestions and revising the paper. we also are thankful to dr. vahid rowshan for his technical assistance in using the 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notulae botanicae horti agrobotanici clujnapoca. 39(1), 41-47. doi: 10.15835/nbha3915880 xu, f., zhu, j., cheng, s., zhang, w., wang, y., 2010: effect of 5-aminolevulinic acid on photosynthesis, yield, nutrition and medicinal values of kudzu (pueraria phaseoloides). tropic. grasslands. 44, 260-265. zhao, r., gao, x., cai, y., shao, x., jia, g., huang, y., qin, x., wang, j., zheng, x., 2013: antitumor activity of portulaca oleracea l. polysaccharides against cervical carcinoma in vitro and in vivo. carbohydr. polym. 96, 376-383. doi: 10.1016/j.carbpol.2013.04.023 zidan, y., bouderbala, s., djellouli, f., lacaille-dubois, m.a., bouchenak, m., 2014: portulaca oleracea reduces triglyceridemia, cholesterolemia, and improves lecithin: cholesterol acyltransferase activity in rats fed enriched-cholesterol diet. phytomed. 21, 1504-1508. doi: 10.1016/j.phymed.2014.07.010. address of the corresponding author: e-mail: vtavallali@gmail.com © the author(s) 2019. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). journal of applied botany and food quality 93, 244 (2020), doi:10.5073/jabfq.2020.093.029 preface special section in memoriam reinhard lieberei on march 5th 2019 professor reinhard lieberei, one of the leading scientists in the field of applied botany, passed away in gorleben at the age of 70. his outstanding research was always characterized by the aspiration to implement results derived from basic scientific studies into practice. in this manner, he worked on a wide array of plant biological topics very successfully. already in very early phases of his scientific career, he exhibited a deep affection towards the various ecological aspects. as a scientist trained in plant physiology and biochemistry, he always used his profound knowledge on metabolic coherences in combination with ecological requirements to elaborate novel concepts, which could be implemented into practice. this trait − in combination with his passion for the tropics − somehow predetermined his scientific work. accordingly, his phytopathological research did not deal with classical, mainstream studies on wheat or potatoes, but was focussed on the south american leaf blight of the rubber tree hevea brasiliensis caused by the ascomycete microcyclus ulei (giesemann et al., 1986). in the course of this very successful research, he re-discovered the strong cyanogenicity of h. brasiliensis (lieberei, 1988). actually, this cognition was the basis to establish a quite novel field of research, i.e., the metabolism of cyanogenic glucosides. by employing novel and unorthodox plant physiological approaches, the various aspects of translocation of cyanogenic glucosides had been studied. as highlight of this innovative research, the linustatin pathway was elucidated (selmar et al., 1988; selmar, 1993). furthermore, reinhard lieberei verified that the injury induced liberation of hcn from the host plant hevea in the course of host-pathogen-interactions does hamper the plants much than the pathogens. these coherences initiated a paradigmic change in ecological biochemistry (lieberei et al., 1989; lieberei et al., 1996). the second tropical crop plant reinhard lieberei was devoted to is cocoa. by pursuing the research of his mentor, böle biehl, on the cocoa fermentation (biehl et al., 1985; biehl et al., 1990), he further elucidated the underlying physiological coherences and intrinsic metabolic processes (niemenak et al., 2006; elwers et al, 2009; voigt and lieberei, 2015), and became a well known and renowned specialist for cocoa fermentation. in this context, together with his former students christina rohsius and silke elwers, he published and actualized the “kakao-atlas”, a standard treatise for all people working within the cocoa business. a further facet of reinhard lieberei’s research is related to plant tissue and organ culture. in this field, he examined the basic physiological and biochemical aspects to optimize related applications, such as mass propagation of various ornamental and medicinal plants (mevenkamp et al., 1994; saare-surminski et al., 2000; hutter et al., 2001). although most of his scientific work was mainly established in the field of applied botany, reinhard lieberei’s research exhibited very strong relations to basic science, especially to the interface between plant physiology, biochemistry and ecology. his contributions to these interdisciplinary research fields were based on his comprehensive knowledge and experience in each branch of plant biology. in this regard, reinhard lieberei was one of the last and rare polymaths in modern plant biology. the broad scientific oeuvre of reinhard lieberei and his important achievements become obvious, when reading the various articles by his scientific scholars and companions, compiled and displayed in this special section. references biehl, b., brunner, e., passern, d., quesnel, v.c., adomako, d., 1985: acidification, proteolysis and flavour potential in fermenting cocoa beans. j. sci. food agric. 36, 583-598. doi: 10.1002/jsfa.2740360710 biehl, b., meyer, b., bin said, m., samarakoddy, r.j., 1990: bean spreading: a method for pulp preconditioning to impair strong nib acidification during cocoa fermentation in malaysia. j. sci. food agric. 36, 583-598. doi: 10.1002/jsfa.2740510105 elwers, s., zambrano, a., rohsius, c., lieberei, r., 2009: differences between the content of phenolic compounds in criollo, forastero and trinitario cocoa seed (theobroma cacao l.). eur. food res. technol. 229, 937-948. doi: 10.1007/s00217-009-1132-y giesemann, a., biehl, b., lieberei, r., 1986: identification of scopoletin as a phytoalexin of the rubber tree hevea brasiliensis. j. phy topathol. 117, 373-376. doi: 10.1111/j.1439-0434.1986.tb04377.x hutter, i., grotkass, c., harnischfeger, g., lieberei, r., feldmann, f., 2001: baptisia tinctoria (l.) r. br.: von der wildsammlung zur in vitro vermehrten kulturpflanze. z. arznei-gewuerzpfl. 6, 35-41. lieberei, r., 1988: relationship of cyanogenic capacity (hcn-c) of the rubber tree hevea brasiliensis to susceptibility to microcyclus ulei, the agent causing south american leaf blight. j. phytopathol. 122, 54-67. doi: 10.1111/j.1439-0434.1988.tb00990.x lieberei, r., biehl, giesemann, a., junqueira, n.t.v., 1989: cyanogenesis inhibits active defense reactions in plants. plant physiol. 90, 33-36. doi: 10.1104/pp.90.1.33 lieberei, r., fock, h.p., biehl, b., 1996: cyanogenesis inhibits active pathogen defence in plants: inhibition by gaseous hcn of photosynthetic co2 fixation and respiration in intact leaves. angew. bot. 70, 230-238 mevenkamp g., lieberei r., harnischfeger g., 1994: baptisia tinctoria (l.) r. brown: micropropagation, in vitro culture and production in direction of pharmaceutically used root biomass. in: bajaj, y.p.s. (eds.), medicinal and aromatic plants vii, 43-55. biotechnology in agriculture and forestry, vol 28. springer, berlin, heidelberg. doi: 10.1007/978-3-662-30369-6_3 niemenak, n., rohsius, c., elwers, s., omokolo ndoumou, d., lieberei, r., 2006: comparative study of different cocoa (theobroma cacao l.) clones in terms of their phenolics and anthocyanins contents. j. food compos. anal. 19, 612-619. doi: 10.1016/j.jfca.2005.02.006 saare-surminski, k., preil, w., know, p.j., lieberei, r., 2000: arabinogalactan proteins in embryogenic and non-embryogenic callus cultures of euphorbia pulcherrima. physiol. plant 108, 180-187. doi: 10.1034/j.1399-3054.2000.108002180.x selmar, d., 1993: transport of cyanogenic glucosides: linustatin uptake by hevea cotyledons. planta 191, 191-199. doi: 10.1007/bf00199749 selmar, d., lieberei, r., biehl, b., 1988: mobilization and utilization of cyanogenic glycosides: the linustatin pathway. plant physiol. 86, 711-716. doi: 10.1104/pp.86.3.711 voigt, j., lieberei, r., 2015: biochemistry of cocoa fermentation. in: schwan, r.f., fleet, g.h, (eds.), cocoa and coffee fermentations, 193-226. crc press, boca raton. dirk selmar institute for plant biology tu braunschweig mendelssohnstraße 4, 38106 braunschweig, germany prof. dr. reinhard lieberei http://dx.doi.org/10.1002/jsfa.2740360710 http://dx.doi.org/10.1002/jsfa.2740510105 http://dx.doi.org/10.1007/s00217-009-1132-y http://dx.doi.org/10.1111/j.1439-0434.1986.tb04377.x http://dx.doi.org/10.1111/j.1439-0434.1988.tb00990.x http://dx.doi.org/10.1104/pp.90.1.33 http://dx.doi.org/10.1007/978-3-662-30369-6_3 http://dx.doi.org/10.1016/j.jfca.2005.02.006 http://dx.doi.org/10.1034/j.1399-3054.2000.108002180.x http://dx.doi.org/10.1007/bf00199749 http://dx.doi.org/10.1104/pp.86.3.711 journal of applied botany and food quality 93, 159 167 (2020), doi:10.5073/jabfq.2020.093.020 1department of biology, college of science, imam abdulrahman bin faisal university, dammam, saudi arabia 2basic and applied scientific research center, imam abdulrahman bin faisal university, dammam, saudi arabia up-modulation of membrane lipid composition and functionality by seed priming under salinity in the hasawi rice variety nabil ben youssef1,2,*, reem mohammed shaher al-moaikal1,2, ghadah hamad saleh al-hawas1,2, farah mustafa alrammah1,2 (submitted: march 14, 2020; accepted: july 28, 2020) * corresponding author summary in this study, we investigated the role of seed priming in mitigating depressive effects of high salinity on membrane lipid composition and membrane functionality in seedlings of hasawi rice cultivar (oryza sativa l). results indicated that seed pre-treatment with hydropriming (hp) and priming with 50 mm nacl solution enhanced germination performance and early seedling growth under salinity condition. priming treatments were found effective in maintaining membrane stability and integrity by increasing total membrane lipid content and reducing membrane damage. concentrations of mono galactosyldiacylglycerol, digalactosyldiacylglycerol and phosphat idylglycerol were greatly increased when seeds were primed. priming also increased phosphatidylcholine pc (lipid forming lamellar structure) and maintained phosphatidylethanolamine pe (non-bilayer-forming lipid) levels resulting in enhanced pc/pe ratio under salinity condition. membrane unsaturation level was also increased, suggesting an improvement in membrane fluidity under salinity conditions. hp and nacl-priming with 50 mm also induced increased contents of total phenolics, total soluble sugar and proline as compared to unprimed seedlings subjected to salinity. it is concluded that hp and priming with 50 mm nacl solution can offer perspectives to improve germination and early growth of hasawi rice under high salinity. this could be achieved through down-regulation of oxidative stress, accumulation of osmoprotectant compounds and improving cell membrane fluidity and integrity. keywords: phosphatidylcholine, linolenic acid, peroxidation, glyco lipids, osmoprtectants. introduction salinity is a common abiotic stress that affects agricultural production and quality. it has been estimated that one-fifth of total cultivated areas and one-third of irrigated agricultural lands are already salt-affected causing huge economic losses. rice (oryza sativa l.), belonging to the poaceae family, is the most important cereal food crops in the world. abiotic stresses alone are responsible for 50% of the total yield loss in rice crops and salinity and drought are the main hindrances to global rice production (ghosh et al., 2016). plants can suffer direct negative impacts from salinity through osmotic effects and direct ion toxicity. excessive salinity increases accumulation of reactive oxygen species (ros), inflicting injury to biomolecules such as lipids, proteins and dna which in turn result in negative effects on metabolism and cellular structures (ibrahim, 2016). biological membranes are generally regarded as one of the main sites of salt injury. lipids as main components of cell membranes play a crucial role in controlling membrane integrity, fluidity and permeability. salt-induced changes in the lipid composition of cell membrane were suggested to contribute to salinity adaptation (guo et al., 2019). physicochemical properties of membranes are strongly influenced by the fatty acid composition and a high level of membrane lipid unsaturation is assumed to maintain the membrane fluidity and functionality. some changes in the fatty acid composition under stress conditions modulate the physiological properties of membranes to better deal with the environmental constraint (guo et al., 2019). the need to develop more salt-tolerant crops has been amplified intensely in recent decades due to increased salinity problems around the world. many approaches have been applied to improve plant salt tolerance; some are labor and time-consuming (e.g., traditional breeding) and others are cost intensive as well as currently intolerable in many countries of the world (e.g genetically modify crops). as alternative, seed priming, which is easily applied and low-cost, is thought to be an effective technique to enhance germination performance under both optimal and adverse environments. it consists of a controlled seed hydration in a specific environment accomplished by the subsequent drying so that the germination processes begins, but radicle emergence is prevented. several priming methods are currently used including hydro-priming, osmo-priming, chemical priming, hormonal-priming, uv radiation priming, biological priming and solid matrix priming (thomas and puthur, 2017; masondo et al., 2018; lingyun et al., 2019; wang et al., 2019). however, these techniques have different properties, effectiveness, and required optimization for each crop species (horii et al., 2007; savvides et al., 2016). evaluating the effectiveness of priming on biological membrane stability and functionality under high salinity, a common condition in the middle east, may represent an approach to deal with the environmental constraint and the problem of limiting production. such studies were not done before with rice in the saudi agricultural backgrounds conditions. therefore, the current study aimed to examine the effects of hydropriming and nacl-priming on the stability of membrane structure and function during early seedling development in hasawi rice. materials and methods plant material and priming 'hasawi' rice variety, used as seed material in our study, is a landrace adapted to the climate of eastern saudi arabia. experiments were carried out in the basic and applied scientific research center of dammam (26°24'23.8'' n, 50°05'04.2'' e, 10 m). seeds with initial moisture content of about 10% (dry weight basis) were surface ste rilized with 0.5% sodium hypochlorite solution for 15 min and washed several times with distilled water to remove the traces of the disinfectant. hydropriming (hp) using double distilled water and nacl-priming with nacl solution (p 50: 50 mm, p 100: 100 mm, p 150: 150 mm) were performed at 20 °c in darkness for 24 h. the seed to solution ratio was 1:4 (w/v). after priming, seeds were washed with distilled water for 2 min, surface-dried using blotting 160 n. ben youssef, r.m.s. al-moaikal, g.h.s. al-hawas, f.m. alrammah paper and then dried back to their initial moisture contents at room temperature. an unprimed dry seeds were maintained as the control treatment for a comparison. seed germination rice primed (hp, p 50, p100 and p 150) and unprimed (up) seeds were incubated in germination chamber set at 25 °c with a 14 h light/10 h dark photoperiod for 7 days in sterile plastic petri dish (90 × 15 mm) containing two sterilized layers of filter paper moistened with distilled water and high salt solution (200 mm nacl). five replicates of 20 seeds per treatment were executed. seeds were considered germinated when 2 mm length radicle protruded through the seed coat. seven days after sowing, length and weight of seedlings (roots and shoots) were measured and seedlings were washed in distilled water and used for biochemical analysis. germinated seeds were recorded daily for 7 days and at the end of experiment, the final germination percentage was calculated. germination index (gi) was calculated according to the method established by the association of official seed analysis (1990). mean germination time (mgt), and seedling vigour were estimated as described by noorhosseini et al. (2018). lipid and fatty acid analysis shoot and root samples were fixed in boiling water for 5 min to stop lipolytic activities. total lipids were extracted in a chloroform/ methanol/water (1/1/1, v/v/v) mixture (bligh and dyer, 1959). lipid classes were separated by thin layer chromatography using the solvent system as described by lepage (1967). after identification by comparison with standards, individual lipids were scraped and then methylated. methyl esters of fatty acids were separated and quantified by gas chromatography using a hewlett–packard chromatograph (4890d) equipped with capillary column (supelcowax-10: 30 m × 0.53 mm × 0.25 lm) and coupled to a flame ionization detector (fid) and injector. fatty acid identification was carried out by comparison of their retention times with those of known standards. to quantify fatty acids, heptadecanoic acid (c17:0) was added as internal standard before methylation. lipid peroxidation lipid peroxidation was evaluated in germinated rice seedlings by malondialdehyde (mda) quantification using the thiobarbituric acid (tba) method. samples were ground in a mortar with 5ml of 0.1% trichloroacetic acid (tca). the homogenate was centrifuged for 5 min at 10 000 g. 250 μl supernatant was mixed with 1 ml of 0.5% tba prepared in tca solution (20%). the mixture was incubated at 95 °c for 30 min and quickly cooled in an ice bath. after centrifugation at 10,000 × g for 10 min, the absorbance of the supernatant was measured at 532 and 600 nm and the value for unspecific turbidity at 600 nm was subtracted. the mda concentration was calculated using an extinction coefficient of 155 mm¯1cm¯1. electrolyte leakage the relative membrane leakage was estimated as electrolyte leakage and determined as described by ghoulam et al. (2002). fresh rice seedlings were excised and immersed in double distilled water at 25 °c for 24 h. following incubation, initial electrical conductivity of solution (ec1) was measured. then, samples were autoclaved at 120 °c for 20 min to release all electrolytes. after cooling, the final electrical conductivity (ec2) was measured and the electrolyte leakage (el) was calculated using the formula: el = (ec1/ec2) × 100. proline content free proline content was estimated according to the method described by bates et al. (1973). samples of rice seedlings were homogenized in 3% aqueous sulfosalicylic acid and centrifuged at 6,000 rpm for 10 min. equal volume of 2.5% ninhydrin solution and glacial acetic acid were added to the supernatant and incubated in water bath at 100 °c for 1 h. after cooling, toluene was added to the reaction mixture and vortexed. absorbance of chromophore containing toluene was measured at 520 nm. proline content was estimated by referring to a standard curve of l-proline. total phenolic content (tpc) total phenolic content (tpc) in rice seedling was estimated according to the folin-ciocalteu method. phenolic compounds were isolated by methalonic extraction (80%). the folin-ciocalteau reagent was added to a suitable aliquot of the extracts, and the absorption of the mixture at 650 nm was measured. total phenolic contents in samples were calculated from the calibration plot and expressed as mg galic acid equivalents per gram of sample. total soluble sugar contents soluble sugar contents were determined according to the anthrone colorimetric method. samples of rice seedling were extracted in ethanol 80%. 3% anthron reagent (sigma aldrich) was added to a suitable aliquot of the extracts. after incubation and the absorption of the mixture in boiling water bath for 1 min, the absorbance was measured at 630 nm. the content of soluble sugar was determined using glucose as standard curve. statistics the experimental design was two factors factorial arranged in a completely randomized design. the first factor was seed priming (up, hp, p 50, p 100 and p 150). salinity was the second factor with 0 and 200 mm nacl. experiments on seed germination consisted of five replications of 20 seeds. seven days after sowing, fresh material from each petri dish were pooled to get a mean value and used as a replication for biochemical studies. lipid peroxidation, electrolyte leakage, proline, total soluble sugar and total phenol were carried out in four replicates. lipid and fatty acid analysis were conducted with three replicates. a two-way analysis of variance (anova) was implemented using ibm spss statistics (version: 20). data were presented as mean ± standard deviation (sd) and the significance of differences was evaluated by duncan’s multiple range test at p = 0.05. results and discussion priming improves seed germination and early seedling growth under high salinity analysis of variance showed high significant effect of salinity on all studied germination parameters (p < 0.001) (tab. 1). severe salinity caused an approximately 42 and 71% reduction in fgp and gi, respectively and delayed germination as indicated by a 17.5% increase in mgt (tab. 2). these results are in agreement with the findings on wheat (bajwa et al., 2018). shereen et al. (2011) reported that rice lines germinated after a delay of 3 days at higher salinity levels. recently, roy et al. (2019) revealed decreased germination of two high yielding rice varieties under salinity condition. in ger mination, imbibition is essential for the restoration of enzymatic activity and both salt induced-poor hydration and ion toxicity delay the imbibition and other physiological processes entail less or delayed germination. further, statistical analysis points out towards the significant effects of priming and its interaction with salinity membrane stability modulation by seed priming 161 tab. 1: two-way anova analysis of priming, salinity, and their interactions for seed germination (fgp: final germination percentage, mgt: mean germination time, gi: germination index) and morphological parameters of rice seedlings (shoot length, root length, biomass and vigour index) parameter source of variance d.f. mean square f sig. fgp salinity (s) 1 8450.000 348.454 0.000*** priming (p) 4 98.250 4.052 0.008** (s) × (p) 4 121.250 5.000 0.002** error 40 24.250 mgt salinity (s) 1 3.923 612.767 0.000*** priming (p) 4 0.412 64.321 0.000*** (s) × (p) 4 0.100 15.545 0.000*** error 40 0.006 gi salinity (s) 1 1576.278 3060.262 0.000*** priming (p) 4 52.099 101.148 0.000*** (s) × (p) 4 4.210 8.173 0.000*** error 40 0.515 shoot length salinity (s) 1 198.403 5376.780 0.000*** priming (p) 4 0.552 14.951 0.000*** (s) × (p) 4 0.663 17.959 0.000*** error 40 0.037 root length salinity (s) 1 265.882 1664.883 0.000*** priming (p) 4 0.340 2.128 0.095ns (s) × (p) 4 0.256 1.602 0.193ns error 40 0.160 biomass salinity (s) 1 31100.180 3344.105 0.000*** priming (p) 4 44.680 4.804 0.003** (s) × (p) 4 195.380 21.009 0.000*** error 40 9.300 sevi salinity (s) 1 9938665.280 3232.360 0.000*** priming (p) 4 12485.350 4.061 0.007** (s) × (p) 4 13729.430 4.465 0.004** error 40 3074.740 *, **, *** and ns denote significance at p < 0.05, 0.01, 0.001 probability level and no significance, respectively tab. 2: effect of priming treatments on the final germination percentage (fgp), mean germination time (mgt), germination index (gi), shoot and root length,, biomass and seedling vigour index of rice seeds germinated under normal and high salinity condition. priming salinity fgp mgt gi shoot lenght root lenght biomass sevi treatment nacl mm (%) (days) (cm) (cm) (mg) up 0 99 a 5.17 a 18.98 a 5.12 a 5.3 a 96.6 a 1031.0 a 200 63 b 6.07 b 5.77 b 0.78 b 0.48 b 32.2 b 79.4 b hp 0 98 a 4.86 c 23.84 c 5.16 a 5.5 a 90.4 c 1045.6 a 200 80 c 5.4 d 12.46 d 1.84 c 1.14 c 46.4 d 283.3 c p 50 0 98 a 4.87 c 23.47 c 5.18 a 5.22 a 88.0 c 1019.9 a 200 77 cd 5.42 d 12.04 de 1.64 c 1.02 bc 45.6 d 205.5 c p 100 0 98 a 4.99 e 21.16 f 5.24 a 5.6 a 86.6 c 1062.8 a 200 71 de 5.39 d 11.17 eg 0.96 b 0.64 bc 39.6 e 114.0 b p 150 0 98 a 5.01 e 20.97 f 5.3 a 5.24 a 90.4 c 1033.4 a 200 70 e 5.43 d 10.84 g 0.86 b 0.52 b 38.8 e 97.0 b up: unprimed, hp: hydroprimed, osp 50: osmoprimed with 50 mm nacl, osp 100: osmoprimed with 100 mm nacl and osp 150: osmoprimed with 150 mm nacl. means in a column with the same letter are not significantly different at p < 0.05 level according to duncan’s multiple range test. (priming × salinity) on fgt, mgt and gi (tab. 1). seed priming was found to be efficient in alleviating the salt-induced adversities on rice germination. maximum effect was observed with hydropriming (hp) and nacl-priming with 50 mm for which fgp are respectively 80 and 77% as compared to the umprimed seeds (63%) under salinity stress. likewise, hp and p 50 induced more uniform and faster germination as indicated by increased gi and decreased mgt (tab. 2). results also showed that priming promoted rapid seedling emergence both under non-salt and salt solutions (fig. 1). primed seeds exhibited, under non-stress condition, higher cumulative germination percentage from the second to the third day of germination, but this difference was equalized to the late days of germination in comparison with controls. however, priming significantly accelerated seed germination under severe salinity condition since the number of days to first germination decreased and the germination percentage increased compared to unprimed seeds (fig. 1). better germination 162 n. ben youssef, r.m.s. al-moaikal, g.h.s. al-hawas, f.m. alrammah performance of primed seeds might be due to priming stimulation of the pre-germination metabolic processes that makes seeds ready for radicle protrusion, repairs membranes and leaches emergence inhibitors (balestrazzi et al., 2011). moreover, a moderate abiotic stress generated by priming treatments during soaking step could be allowing the seeds to cope with environmental stresses during germination (ibrahim, 2016). early seedling growth is a critical stage in the crop growing season and investigation in this phase is still the interest of several researches. our results showed that salinity had highly significant effect (p < 0.001) on shoot length, root length, seedling biomass and seedling vigour index (tab. 1). priming significantly affected shoot length (p < 0.001), seedling biomass (p < 0.05) and seedling vigour index (p < 0.05). a significant two-way interaction (priming and salinity) was found for shoot length (p < 0.001), seedling biomass (p < 0.05) and seedling vigour index (p < 0.05) (tab. 1). in salinity condition, priming significantly reduced the salt negative impact. for shoot length, maximum effects were observed with hp and and p 50 for which reduction rate were about 64 and 68%, respectively compared to stressed control (84%). the same tendency was found in seedling biomass were salt-induced reduction was lower (about 48% for both hp and p 50) than that of stressed control (67%). cell division and cell elongation require turgor pressure that could be reduced by salinity. this will, therefore, decrease the length of the shoots and roots, thus leading to stunted growth. as observed in germination parameters, priming treatments were effective in alleviating the negative impact of salinity on early seedling growth. hp and p 50 appear even more effective than p 100 and p 150 since they have led to significant enhancement in seedling growth as compared to stressed control (tab. 2). these results are supported by ahmadvand et al. (2012) who found that hydropriming and osmopriming with potassium nitrate improved early seedling growth of cotton under saline conditions. also, similar findings were reported by keshavarza and moghadamb (2017) in phaseolus. subramanyam et al. (2019) stated that seed priming with sodium selenate improved growth in rice seedling under high salinity. promoted early seedling growth by hp and p 50 under saline condition might be related to earlier emergence stimulated seedling and the enhancement of cell division and cell enlargement. induction of antioxidative defense system and synthesis of osmoprotectants could also contribute to enhance germination performance and early seedling growth (paul et al, 2017; savvides et al, 2016). effect of priming on membrane lipids membrane lipids are dynamic compounds responsible for the major biological properties of cell membranes which are often considered as the first target of salt injury. variations in membrane lipids are thought to be involved in the salinity adaptation process and might contribute to plant resistance. the present results indicate that total membrane lipids in roots and shoots were significantly affected by salinity (p < 0.001) and priming (p < 0.01) whereas the interaction between these two factors was only significant for lipid content in shoot (p < 0.05) (tab. 3). the high salinity significantly decreased total membrane lipid content both in shoots and roots by 49 and 53% as compared to the respective controls (fig. 2a, b). several investigations have shown the depressive effect of salinity on membranes lipids (chalbi et al., 2013; mansour, 2013). the observed reduction of membrane lipids could result from salt-induced enhancement of lipolytic and peroxidative activities as well as inhibition lipid biosynthesis pathways (guo et al., 2019). our findings confirm these observations and showed, under high salinity condition, enhanced membrane damage. the extent of membrane injury could be assessed by an indirect measurement of electrolyte leakage and lipid peroxidation evaluated by the mda accumulation. these two parameters were significantly affected by salinity and priming and were significantly increased in salt-stressed seedlings as compared with controls (tab. 3; fig. 3a, b). this may reflect the high level of membrane 0 20 40 60 80 100 120 1 2 3 4 5 6 7 c u m u la ti ve g er m in ati o n ( % ) days after sowing p100 0 20 40 60 80 100 120 1 2 3 4 5 6 7 days after sowing p150 0 20 40 60 80 100 120 1 2 3 4 5 6 7 p50 up up+s p p+s 0 20 40 60 80 100 120 1 2 3 4 5 6 7 c u m u la ti ve g er m in ati o n ( % ) hp fig. 1: cumulative seed germination of unprimed and primed rice seeds germinated under high salinity. up: unprimed seeds germinated under no salinity, up+s: unprimed seeds germinated under salinity, p: primed seeds germinated under no salinity, p+s: primed seeds germinated under salinity. hp: hydropriming, p50: nacl-priming with 50 mm nacl, p100: nacl-priming with 100 mm nacl. p150: nacl-priming with 150 mm nacl. membrane stability modulation by seed priming 163 tab. 3: two-way anova analysis of priming, salinity, and their interactions for the biochemical attributes (electrical conductivity, lipid peroxidation, proline content, total phenol, soluble sugar and lipids) of rice seedlings under salinity. parameter source of variance d.f. mean square f sig. mda salinity (s) 1 2155.616 709.707 0.000*** priming (p) 4 87.544 28.823 0.000*** (s) × (p) 4 75.086 24.721 0.000*** error 20 3.037 electrolyte leakage salinity (s) 1 9249.036 199.571 0.000*** priming (p) 4 186.301 4.020 0.015* (s) × (p) 4 187.081 4.037 0.015* error 20 46.345 proline salinity (s) 1 73408.533 981.398 0.000*** priming (p) 4 1411.533 18.871 0.000*** (s) × (p) 4 536.533 7.173 0.001** error 20 74.800 total phenol salinity (s) 1 0.727 21.731 0.000*** priming (p) 4 0.192 5,739 0.003** (s) × (p) 4 0.053 1.575 0.219ns error 20 0.033 soluble sugar salinity (s) 1 789.507 310.666 0.000*** priming (p) 4 28.473 11.204 0.000*** (s) × (p) 4 17.582 6.918 0.001** error 20 2.541 root lipids salinity (s) 1 117.019 199.713 0.000*** priming (p) 4 1.938 3.308 0.031* (s) × (p) 4 0.689 1.177 0.351ns error 20 0.586 shoot lipids salinity (s) 1 282.072 316.147 0.000*** priming (p) 4 5.481 6.143 0.002** (s) × (p) 4 2.678 3.001 0.043* error 20 0.892 *, **, *** and ns denote significance at p < 0.05, 0.01, 0.001 probability level and no significance, respectively injury as a result of oxidative damage. additionally, the observed increased lipid peroxidation could result from the salt-induced overproduction of ros causing peroxidation of unsaturated fatty acids in the biological membranes. this in turn leads to disruption of the physicochemical membrane properties and consequently declined seedling growth (ibrahim, 2016). similar results were found by chalbi et al. (2013) who showed that electrolyte leakage and mda accumulation were negatively correlated with membrane integrity under salinity conditions in hordeum vulgare. our study showed that priming treatments were effective in decreasing salt induced membrane damage, since total lipid contents were significantly increased in both seedling shoots and roots raised from primed seeds as compared to stressed control. hence, 40% and 23% increase in lipid content were observed in shoot seedlings from rice seeds treated with hp and p 50 respectively, as compared to stressed control (fig. 2a). similarly, about 40% increase was seen in root seedlings raised from rice seeds treated with hp as compared to stressed control (fig. 2b). such variations could be expected to promote maintenance of membrane integrity and cellular homeostasis in salt conditions and hence postulating that lipids might be involved in the protection against salinity. this finding was supported by the significant priming-induced decrease in electrolyte leakage and lipid peroxidation observed in stressed seedlings compared to the stressed controls (fig. 3a, b). results showed again that hp and p 50 were more effective than p 100 and p 150 in terms of reducing membrane damage. hence, increases in electrolyte leakage are about 98 and 82% in seedlings from primed seeds with h2o and 50 mm nacl respectively while in stressed control the increase was more marked (175%). furthermore, as much as 139% and 88% lesser mda accumulation can be observed in seedlings from rice seeds treated with hp and p 50 respectively, as compared to the stressed control. these improvements may be related to the induction of antioxidant responses which provide protection against oxidative damage (paul et al., 2017). reorganization of cellular membrane and maintenance of membrane integrity during priming would be responsible for the observed reduction in electrolyte leakage. our observations are well supported by several other reports where hydropriming and osmopriming significantly reduced membrane damage and the mda concentration in salt-stressed cucumis melo and glycine max (farhoudi et al., 2011; yan dai et al., 2017). shoot membrane lipids are mainly composed of monogalactosyldiacylglycerol (mgdg), digalactosyldiacylglycerol (dgdg), and phosphatidylcholine (pc), followed by phosphatidylethanolamine (pe), and phosphatidylglycerol (pg) and a small amount of phosphatidylinositol (pi) and sulfoquinovosyldiacylglycerol (sqdg) (tab. 4). on exposure to high salinity, all lipid classes showed a significant decrease which was dramatic in chloroplast lipids (mgdg, dgdg and pg) as compared to major phospholipids (pc and pe). these changes may negatively affect photosystem stability and activity leading to photosynthesis inhibition (liu et al., 2018). our findings are in line with those obtained by liu et al. (2018) and bejaoui et al. (2016) who revealed a sharped decrease in glycolipids under salinity conditions. salt treatment decreased lipids forming lamellar structure (pc) against a stability of non-bilayer-forming lipids (pe) leading to the reduction of the pc to pe ratio. such modifications impair the cell membrane proprieties and activities (salama and 164 n. ben youssef, r.m.s. al-moaikal, g.h.s. al-hawas, f.m. alrammah fig. 2: membrane lipid content in shoots (a) and roots (b) of early rice seedlings from unprimed and primed seeds grown under normal and high salinity. up: unpriming, hp: hydropriming, p 50: priming with 50 mm nacl, p 100: priming with 100 mm nacl and p 150: priming with 150 mm nacl. bars with different letters are significantly different at p < 0.05 according to duncan’s multiple range test. fig. 3: electrical leakage (a) and mda content (b) of early rice seedlings from unprimed and primed seeds grown under normal and high salinity. up: unpriming, hp: hydropriming, p 50: priming with 50 mm nacl, p 100: priming with 100 mm nacl and p 150: priming with 150 mm nacl. bars with different letters are significantly different at p < 0.05 according to duncan’s multiple range test. fig. 3: electrical leakage (a) and mda content (b) of early rice seedlings from unprimed and primed seeds grown under normal and high salinity. up: unpriming, hp: hydropriming, p 50: priming with 50 mm nacl, p 100: priming with 100 mm nacl and p 150: priming with 150 mm nacl. bars with different letters are significantly different at p<0.05 according to duncan's multiple range test. 0 20 40 60 80 100 up hp p 50 p 100 p 150 a el ec tr ic al c on du ct iv ity (% ) 0 10 20 30 40 up hp p 50 p 100 p 150 b 0 mm 200 mm nacl m d a (n m ol g -1 d w ) b a a a a b c c c a a b c d de a a e a a fig. 2: membrane lipid content in shoots (a) and roots (b) of early rice seedlings from unprimed and primed seeds grown under normal and high salinity. up: unpriming, hp: hydropriming, p 50: priming with 50 mm nacl, p 100: priming with 100 mm nacl and p 150: priming with 150 mm nacl. bars with different letters are significantly different at p<0.05 according to duncan's multiple range test 0 2 4 6 8 10 12 14 16 18 20 22 up hp p 50 p 100 p 150 a 0 mm 200 mm nacl li pi ds (m g. g1 d w ) 0 2 4 6 8 10 12 up hp p 50 p 100 p 150 b li pi ds (m g. g1 d w ) priming a a a a a b bc cd cd d a a a a a b b c bc bc fig. 2: membrane lipid content in shoots (a) and roots (b) of early rice seedlings from unprimed and primed seeds grown under normal and high salinity. up: unpriming, hp: hydropriming, p 50: priming with 50 mm nacl, p 100: priming with 100 mm nacl and p 150: priming with 150 mm nacl. bars with different letters are significantly different at p<0.05 according to duncan's multiple range test 0 2 4 6 8 10 12 14 16 18 20 22 up hp p 50 p 100 p 150 a 0 mm 200 mm nacl li pi ds (m g. g1 d w ) 0 2 4 6 8 10 12 up hp p 50 p 100 p 150 b li pi ds (m g. g1 d w ) priming a a a a a b bc cd cd d a a a a a b b c bc bc fig. 2: membrane lipid content in shoots (a) and roots (b) of early rice seedlings from unprimed and primed seeds grown under normal and high salinity. up: unpriming, hp: hydropriming, p 50: priming with 50 mm nacl, p 100: priming with 100 mm nacl and p 150: priming with 150 mm nacl. bars with different letters are significantly different at p<0.05 according to duncan's multiple range test 0 2 4 6 8 10 12 14 16 18 20 22 up hp p 50 p 100 p 150 a 0 mm 200 mm nacl li pi ds (m g. g1 d w ) 0 2 4 6 8 10 12 up hp p 50 p 100 p 150 b li pi ds (m g. g1 d w ) priming a a a a a b bc cd cd d a a a a a b b c bc bc mansour, 2015). under salinity stress, concentrations of mgdg, dgdg and pg were greatly increased in seedling shoot raised from primed seeds (especially hp and p 50), as compared with stressed controls (tab. 4). such changes could contribute to improving the chloroplast membrane stability and functionality under saline conditions (liu et al., 2018). however, mgdg to dgdg ratio remained unchanged suggesting the upkeep of a relative functionality, even with a reduced membrane structure under salinity (tab. 4). priming also increased bilayer-forming lipids (pc) and maintained pe level resulting in enhanced pc/pe ratio under salinity condition (tab. 4). these results argue for improved membrane stability and functionality required in salt acclimation (salama and mansour, 2015). physicochemical properties of cell membranes are also determined by the fatty acyl composition of membrane lipids. modifications in fatty acid composition are believed to affect membrane fluidity, permeability, and cellular metabolic functions and therefore might be strongly considered in the stress resistance process. our results showed that membrane lipids in untreated shoots had a high level of unsaturated fatty acids (ufas) with 75% of total fatty acids while saturated fatty acids (sfas) accounted only for about 25% (tab. 5). linolenic acid, (c18:3) was the most abundant fatty acid followed by linoleic (c18:2) and stearic (c16:0) acids which have relatively equal proportions. hexadecenoic (c16:1), stearic (c18:0) and oleic (c18:1) acids are faintly represented. the high salinity induced a significant decrease in membrane unsaturation level. this was indicated by the reduced double-bond index and unsaturated-to-saturated fatty acid ratio due to salt-induced decrease in polyunsaturated fatty acid c18:3 in favour of c18:1 and c16:0 (tab. 5). saturated lipids generate liquid-order phases while unsaturated lipids lead to liquiddisordered phases, thereby the presence of fatty acid residues and their state of saturation can directly affect membrane fluidity (guo et al., 2019). hence, decreasing membrane fatty acid unsaturation and unsaturated-to-saturated fatty acid ratio could induce a phase separation in the cell membrane thereby affecting its fluidity and permeability (mansour, 2013; guo et al., 2019). results revealed that priming treatments increased membrane unsaturation level of stressed seedling shoots as indicated by the enhancement of the double-bond index and unsaturated-to-saturated fatty acid ratio in comparison with the stressed controls (tab. 5). such findings suggest a possible priming-induced stimulation of desaturase activity leading accordingly to an improving in membrane fluidity under saline conditions. priming elicits accumulation of proline, soluble sugar and total phenol proline accumulation is recognized as a common metabolic reaction of plants exposed to abiotic stress (ibrahim, 2016). our results supported this observation since it was significantly affected by salin membrane stability modulation by seed priming 165 ity (p < 0.001), priming (p < 0.001) and their interaction (p < 0.05) (tab. 3). salinity stress significantly increased proline concentrations in unprimed seedlings when compared to controls (fig. 4a). such findings were corroborated with previous studies on different species (farhoudi et al., 2011; espanany et al., 2016). interestingly, priming induced further accumulation of proline in early seedlings exposed to high salinity. hp and p 50 appeared once again as the most effective priming treatments as they led to the largest increases under high salinity (fig. 4a). these results are in agreement with those of farhoudi et al. (2011) who showed that seed-nacl priming enhanced proline accumulation in muskmelon seedling under salinity. kubala et al. (2015) reported that the improved germination performance of osmoprimed rape seeds was accompanied by a significant increase in proline content under salinity stress. in addition to its contribution to osmotic adjustment, proline acts as an antioxidant and a stabilizer of subcellular structures and macromolecules under stress condition (hayat et al., 2012; kubala et al., 2015). therefore, the observed increase in proline content in early rice seedlings may provide protection and stability to macromolecules and may reflect the improvement salinity resistance. total phenol content was significantly affected by salinity (p < 0.001) and priming (p < 0.05) whereas the interaction between these two factors was insignificant (tab. 3). our results revealed enhanced accumulation of total phenols in rice seedlings under high salinity condition. however, as observed in proline content, seed pre-treatment by hydropriming induced further accumulation in total phenolics (fig. 4b). several studies have showed increased accumulation in phenolics under various biotic and abiotic stresses (moulick et al., 2016; farooq et al., 2017). du et al. (2019) reported that rice seed priming with sodium selenate induced higher phenol content in seedlings. the protective role of phenolics in plants is renowned by their aromatic structure which stabilizes biomembranes during stress and scavenges ros in cells (taiz et al., 2015). thus, our results suggest that seed pre-treatment, particularly hydropriming, could contribute to membrane stability and antioxidative protection in early seedling development through stimulation of phenol synthesis. sugar are crucial for germination since it is involved in regulating metabolic activities as well as providing carbon source for young seedling growth and contributing to the osmoregulation required for the growth during germination under salinity. as indicated in tab. 3, total soluble sugar was significantly affected by salinity (p < 0.001), priming (p < 0.001) and their interaction (p < 0.05). sever salinity considerably reduced soluble sugar content as compared to control (fig. 4c). previous studies revealed both increase and decrease in sugar content depending on genotypes and stress intensity (paul et al., 2017; bajwa et al., 2018). our data also indicated that all tab. 4: lipid classes in shoot seedlings from unprimed and primed rice seeds grown under normal and high salinity. priming salinity mgdg dgdg pc pe pg pi sqdg pc/pe dgdg/ treatment nacl mm mgdg up 0 6.04 a 2.88 a 3.41 a 1.86 ab 1.49 a 0.87 a 0.37 a 1.83 a 2.1 ab 200 2.4 g 1.19 e 2.35 e 1.87 ab 0.44 f 0.32 e 0.14 e 1.26 b 2.02 ab hp 0 6.01 a 2.85 a 3.45 a 1.86 ab 1.5 a 0.84 a 0.39 a 1.85 a 2.11 ab 200 3.85 d 2.04 c 2.9 c 1.77 b 0.97 d 0.49 cd 0.26 b 1.61 ab 1.89 a p 50 0 5.73 b 2.73 a 3.19 b 1.85 ab 1.42 ab 0.8 a 0.37a 1.72 a 2.1 ab 200 3.40 e 1.66 d 2.58 d 1.55 c 0.86 d 0.45 de 0.25 bc 1.66 ab 2.05 ab p 100 0 5.56 bc 2.52 b 3.15 b 1.9 ab 1.35 bc 0.69 b 0.36 a 1.65 ab 2.21 ab 200 2.79 f 1.25 e 2.53 de 1.58 c 0.58 e 0.38 de 0.20 cd 1.60 ab 2.34 b p 150 0 5.43 c 2.47 b 3.18 b 1.97 a 1.28 c 0.61 bc 0.37 a 1.61 ab 2.2 ab 200 2.54 fg 1.56 e 2.54 de 1.55 c 0.6 e 0.36 e 0.36 de 1.63 ab 2.05 ab up: unpriming, hp: hydropriming, p 50: priming with 50 mm nacl, p 100: priming with 100 mm nacl and p 150: priming with 150 mm nacl. mgdg, monogalactosyldiacylglycerol; dgdg, digalactosyldiacylglycerol; pc, phosphatidylcholine; pg, phosphatidylglycerol; pe, phosphatidylethanolamine; pi, phosphatidylinositol; sqdg, sulfoquinovosyldiacylglycerol; pc/pe, ratio of pc to pe; dgdg/mgdg, ratio dgdg to mgdg. means in a column with the same letter are not significantly different at p<0.05 level according to duncan’s multiple range test. tab. 5: fatty acid composition of total membrane lipids in shoot seedlings from unprimed and primed rice seeds grown under normal and high salinity. fatty acids priming salinity c16:0 c16:1 c18:0 c18:1 c18:2 c18:3 dbi $ ufas : nacl mm sfas* up 0 21.53 ab 1.12 ab 1.77 a 1.89 a 21.52 a 52.17 a 202.6 a 5.33 a 200 25.06 c 1.44 ab 1.60 ab 4.23 b 22.91 a 44.73 b 185.7 b 4.53 b hp 0 21.32 ab 1.22 ab 2.05 abc 2.29 ac 21.93 a 51.20 ac 200.9 a 5.64 ac 200 23.05 abc 0.95 a 2.15 bcd 3 abc 22.06 a 48.8 abc 194.5 ab 5.39 a p 50 0 20.82 a 1.61 b 1.85 ab 2.02 ac 22.28 a 51.43 ac 202.5 a 5.56 ac 200 23.15 abc 1.44 ab 2.02 abc 3.48 abc 22.88 a 46.89 bc 191.4 ab 5.24 a p 100 0 20.88 ab 1.02 ab 2.48 d 2.25 abc 23.28 a 50.1 abc 200.1 a 6.15 c 200 23.03 abc 1.18 ab 2.45 cd 3.41 bc 23.02 a 46.9 bc 191.2 ab 5.68 ac p 150 0 21.75 ab 1.04 a 2.07 bc 2.12 ac 22.85 a 50.17 ac 199.4 ab 5.57 ac 200 23.55 bc 1.35 ab 2.16 bcd 3.38 bc 22.85 a 46.7 bc 190.6 ab 5.31 a up: unpriming, hp: hydropriming, p 50: priming with 50 mm nacl, p 100: priming with 100 mm nacl and p 150: priming with 150 mm nacl. $dbi, double-bond index = ∑(unsaturated fatty acid × number of double bonds), * ufas : sfas, unsaturated–to–saturated fatty acid ratio. means in a column with the same letter are not significantly different at p<0.05 level according to duncan’s multiple range test. 166 n. ben youssef, r.m.s. al-moaikal, g.h.s. al-hawas, f.m. alrammah conclusion the present work showed that high salinity causes severe damage during the germination phase and early seedling growth of hasawi rice. seed pre-treatment with hydropriming and nacl-priming with 50 mm has been revealed to significantly alleviate the harmful effect of salinity. thus, primed seeds became could manage salinity with a better ability to maintain cell membrane integrity and fluidity. in addition, accumulation of phenols and osmoprotectants may reflect a more activated antioxidant system, thus reducing the oxidative stress generated by salinity. these resulted in lower levels of mda and preserve the integrity and stability of biological membranes. on the basis of our experimental data, seed pre-treatment with hp and p 50 reduced the lethal effect of nacl leading to the improvement of germination performance and early seedling growth of hasawi rice. this could be promising for the exploitation of marginalized lands affected by salt. acknowledgments the authors are thankful to the imam abdulrahman bin faisal university for funding this research work (project id 2017-241-sci) conflict of interest no potential conflict of interest was reported by the authors. references ahmadvand, g., soleymani, f., saadatian, b., pouya, m., 2012: effects of seed priming on seed germination and seedling emergence of cotton under salinity stress. world appl. sci. j. 20 (11), 1453-1458. doi: 10.5829/idosi.wasj.2012.20.11.1712 association of official seed analysis, 1990. rules for testing seed. j. seed technol. 12, 1-112. balestrazzi, a., confalonieri, m., macovei, a., carbonera, d., 2011: seed imbibition in medicago truncatula gaertn. expression profiles of dna repair genes in relation to peg-mediated stress. j. plant physiol. 168, 706-713. doi: 10.1016/j.jplph.2010.10.008 bajwa, a., farooq, m., nawaz a., 2018: seed priming with sorghum extracts and benzyl aminopurine improves the tolerance against salt stress in wheat (triticum aestivum l.). physiol. mol. biol. pla. 24(2), 239-249. doi: 10.1007/s12298-018-0512-9 bates, l.; 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10.1016/j.tplants.2015.11.003 shereen, a., ansari, r., raza, s., mumtaz, s., khan, ma., 2011: salinity induced metabolic changes in rice (oryza sativa l.) seeds during germination. pak. j. bot. 43, 1659-1661. subramanyam, k., laing, g.d., els, j. m., damme, v., 2019: sodium selenate treatment using a combination of seed priming and foliar spray alleviates salinity stress in rice. front. plant sci. 10, 116. doi: 10.3389/fpls.2019.00116 taiz, l., zeiger, e., moller, i.m., murphy, a., 2015: plant physiology and development. sixth ed. sinauer associates inc. publishers, massachusetts. thomas, d.t.t., puthur, j.t., 2017: uv radiation priming: a means of amplifying the inherent potential for abiotic stress tolerance in crop plants. environ. exp. bot. 138, 57-66. doi: 10.1016/j.envexpbot.2017.03.003 wang, n., wanga, x., shia, j., liub, x., xua, q., hong, z., meizhen, s., gentu, y., 2019: mepiquat chloride-priming induced salt tolerance during seed germination of cotton (gossypium hirsutum l.) through regulating water transport and k+/na+ homeostasis. environ. exp. bot. 159, 168-178. doi: 10.1016/j.envexpbot.2018.12.024 yan dai, l., zhu, h., yin, k., du, j., zhang, y., 2017: seed priming mitigates the effects of saline-alkali stress in soybean seedlings. chil. j. agr. res. 77(2), 118-125. doi: 10.4067/s0718-58392017000200118 orcid nabil ben youssef https://orcid.org/0000-0003-2597-8067 address of the corresponding author: nabil ben youssef, imam abdulrahman bin faisal university, p.o box 1982, 31441, dammam, saudi arabia e-mail: nabilbtn@gmail.com © the author(s) 2020. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.5897/ajar10.1007 http://dx.doi.org/10.1016/j.plaphy.2016.12.012 http://dx.doi.org/10.4172/2375-4338.1000167 http://dx.doi.org/10.1016/s0098-8472(01)00109-5 http://dx.doi.org/10.3390/ijms20174264 http://dx.doi.org/10.4161/psb.21949 http://dx.doi.org/10.1016/j.biortech.2006.08.030 http://dx.doi.org/10.1016/j.jplph.2015.12.011 http://dx.doi.org/10.1016/j.rhisph.2017.04.010 http://dx.doi.org/10.1016/j.jplph.2015.04.009 http://dx.doi.org/10.1016/j.scienta.2019.05.007 http://dx.doi.org/10.1007/s40626-018-0129-y http://dx.doi.org/10.1007/s10535-012-0144 -9 http://dx.doi.org/10.1016/j.ecoenv.2017.08.017 http://dx.doi.org/10.1016/j.plaphy.2016.11.004 http://dx.doi.org/10.1007/s00344-017-9728-0 http://dx.doi.org/10.1016/j.plgene.2017.04.002 http://dx.doi.org/10.1007/s12298-019-00654-8 http://dx.doi.org/10.1007/s11738-015-1934-4 http://dx.doi.org/10.1016/j.tplants.2015.11.003 http://dx.doi.org/10.3389/fpls.2019.00116 http://dx.doi.org/10.1016/j.envexpbot.2017.03.003 http://dx.doi.org/10.1016/j.envexpbot.2018.12.024 http://dx.doi.org/10.4067/s0718-58392017000200118 art_15408.indd journal of applied botany and food quality 94, 7 14 (2021), doi:10.5073/jabfq.2021.094.002 1department of biology, science and research branch, islamic azad university, tehran, iran 2department of biology, garmsar branch, islamic azad university, garmsar, iran 3antimicrobial resistance research center, bu-ali research institute, mashhad university of medical sciences, mashhad, iran 4department of microbiology and virology, school of medicine, mashhad university of medical sciences, mashhad, iran seed-priming with cold plasma and supplementation of nutrient solution with carbon nanotube enhanced carotenoid contents and the expression of psy and pds in bitter melon (momordica charantia) fereshteh sadat seddighinia1, alireza iranbakhsh1*, zahra oraghi ardebili2, saman soleimanpour 3, 4 (submitted: july 29, 2020; accepted: december 4, 2020) * corresponding author summary recent studies on cold-plasma and nanotechnology in some crop species have shown a potential for application in food, medicine, and crop improvement. here, the behaviours of momordica charantia were evaluated, following supplementation of nutrient solution with multi-walled carbon nanotube (cnt) and seed priming with cold plasma treatments. the ultra-structural study of stems confirmed cnt uptake and symplastic transportation. cnt supplementation and seed-priming with plasma synergistically provoked a drastic increase in the plant’s early growth and performance. quantitative realtime pcr analysis confirmed that the applied treatments mediated variations in transcriptions of the phytoene-synthase gene (mcpsy ) and phytoene desaturase (mcpds). the mcpds and mcpsy genes showed a similar expression trend in which the highest expression levels were observed in cnt50+plasma 60 group. according to hplc analysis, the cnt50+plasma60 treatment was the most effective way to increase concentrations of β-carotene. the applied treatments dependent on dose and treatment method increased zeaxanthin concentration. similarly, cnt50+plasma 60 and cnt100+plasma 60 groups had significantly higher α-carotene levels than the other treatment group. moreover, the statistical analysis confirmed the significant positive correlations between the expression of target genes and concentrations of carotenoids. herein, a theoretical basis was gained to exploit in the food, pharmaceutical, and agricultural industries. keywords: carbon nanotube; cold plasma; carotenoid; gene expression; secondary metabolites; seed-priming introduction momordica charantia l., belonging to the cucurbitaceae family and commonly known as bitter melon, is a medicinal plant that is widely grown in the tropical and subtropical parts of the world such as india, china, and indonesia (jia, 2017). traditionally, bitter melon has been used in the treatment of diabetes and recently many scientific evidence have also verified that it is a promising antidiabetic and therapeutic substance (joseph and jini, 2013). furthermore, bitter melon has some other medicinally useful properties, such as antiviral (pongthanapisith, 2013), antibacterial (costa, 2010), anti sepsis, anticancer (poolperm and jiraungkoorskul, 2017), antihepatotoxic (aparna upadhyay, 2015), antiulcer and antioxidant (gill, 2012). m. charantia contains several different types of primary and secondary metabolites in the whole plant such as phenolic, triterpene, flavonoid, and carotenoid compounds, including alpha and beta-carotene, lycopene, and zeaxanthin (hyun young, 2013; jia, 2017). among them, carotenoids have significant roles in many physiological processes in this plant and may play an important role in the prevention of some diseases (cuong, 2017; ishida, 2004; lee, 2017). for example, carotenoids act as the precursors in the synthesis of abscisic acid, which is mainly involved in the plant development and stress regulations. in addition, they can prevent damaging photooxidative processes (photo-inhibition phenomenon) by absorption of light in photosynthetic membranes. moreover, the associated color in flowers and fruits attracts both pollinators and seed dispersal agents (lee, 2017; tuan, 2011). in humans, the carotenoid derivatives have several medical applications, especially in controlling the chronic and vascular diseases (fiedor, 2014). they are used as a precursor in vitamin a biosynthesis that is essential to prevent blindness and xerophtalmia (tuan, 2011). in addition, carotenoids can significantly reduce the risk of cataracts, photosensitivity disease, lung cancer, prostate cancer and cardiovascular disease. due to the inability of humans to biosynthesize essential carotenoids, it is necessary to be obtained through the food sources. therefore, manipulating the meta bolic pathway of carotenoids to enhance their synthesis can play an important role in increasing the quality and quantity of the product (cuong, 2017; tuan, 2011). in the carotenoid biosynthetic pathway, phytoene synthase (psy) and phytoenedesaturase (pds) are two key enzymes with a close relationship between the associated expression and the level of carotenoids (fig. 1) (cuong, 2017; tuan, 2011). recently non-thermal (cold) plasma technology has been considered as an innovative eco-friendly safe standalone technology, which could be scaled up and exploited in diverse agricultural-related activities (bourke, 2018; iranbakhsh, 2017; iranbakhsh, 2018). during the production of plasma phenomenon, not only uv photons are emitted, but also varieties of active nitrogen and oxygen species, like nitric oxide are generated, which all may individually or simultaneously act as an efficient epigenetic agent to activate key crucial signaling routes, contributed to modulation of plant growth, physio logy, productivity, and protection (bourke, 2018; iranbakhsh, 2017; iranbakhsh, 2018). also, seed pre-treatment with cold plasma as modern eco-agricultural technology could enhance seed germination, seedling growth, however, limited studies are exploring the associated effect on the gene expression (bourke, 2018; iranbakhsh, 2017; iranbakhsh, 2018). to gain insight into diverse nanoproducts, more convincing studies are required to figure out their advantage or toxicity, and elucidate the contributed mechanisms, for further exploitation in the related industries (rajaee behbahani, 2020; sotoodehnia-korani et al., 2020). the different industrial consumptions of carbon nanotubes (cnts), including single-wall (swnts) and multi-wall cnts (mwnts) are rapidly increasing (cano, 2016). based on the recent evidence, cnts are suitable candidates to achieve various agricultural and biotechnological aims, especially as pesticides, nano-encapsulated products, 8 f.s. seddighinia, a. iranbakhsh, z. oraghi ardebili, s. soleimanpour and fertilizers (husen, 2014; kah, 2015; liu, 2015). studies showed that the application of cnts influenced the growth-related traits in triticum aestivum (joshi, 2018), zea mays (yan, 2013), and oryza sativa (yan, 2016). the functionalized carbon nanotubes have been demonstrated to increase the water-retaining capacity, biomass, and fruit yield in plants, which is a significant achievement of nanotechnology in recent years (husen, 2014). however, there is limited evidence, especially at the molecular level and further studies are required. on the other hand, some recent studies on carbon nanotube (cnt) or cold plasma in several crops have shown evidence for increased germination, seedling growth, and physiological activities, including photosynthetic activity, enhancement of the level of key enzymes in the metabolic pathways. these also make positive changes in the gene expression that are essential for cell division and plant development, indicating their potential use in crop improvement [lakshman k. randeniya, 2015 #33;ling, 2014 #34;kole, 2013 #36;husen, 2014 #19;khodakovskaya, 2013 #38(haghighi m, 2014). in this study, early growth and the relationship between carotenoid accumulation and the expression of mcpsy and mcpds, were investigated, using two treatments of cold plasma and carbon nanotube. furthermore, the simultaneous effect of both actions was examined in m. charantia. materials and methods nanomaterial and seeds the uniform seeds of m. charantia, palee f1, were obtained from the east-west seed international ltd, thailand. laboratory and greenhouse experiments were arranged in a completely randomized design with three replications. in this study, mwcnt was purchased from us research nanomaterials, inc (3302 twig leaf lane houston, tx 77084, usa) (tab. 1). plasma experimental apparatus the applied experimental apparatus in this study was dbd (model: ps200, nik fanavaran plasma co., iran). the details and schematic of the applied plasma producing device, and the plasma diagnostic data were represented in our previously published papers (fig. 2a and 2b) (babajani, 2018; iranbakhsh, 2017; iranbakhsh, 2018). plasma at atmospheric pressure is generated between two glass plates as dielectric barriers (the gap between dielectrics: 4 mm) covering the two powered circular plate copper electrodes (radius = 5.5 cm). argon was utilized as a functional gas between dielectrics (flow rate of 2 liters per min (l·min-1)). the dielectric acts as a stabilizing material when the potential across the gap reaches the breakdown vol tage leading to the formation of a large number of micro-discharges. moreover, a modified ac high voltage power supply (mp516, nik plasma tech., iran) was utilized (fig. 2a). the voltage was quantified by a high voltage probe (pintek hvp40) linked to an oscilloscope (tektronix tds1012b). furthermore, the frequency and the voltage of the apparatus were respectively fixed at 13 khz and 10 kv. the instrument power was 80 w, so for 94.98 cm2 plasma treatment areas, the surface power density was equal to 0.84 w/cm2. besides, to provide plasma diagnostics data, the optical emission spectro scopy was carried out (the peaks were compared to the data of the nist atomic spectra database). the temperature was estimated to be between 27-29 °c. the peaks detected in 320-400 nm refer to the presence of uv, oh, and nitrogen related species, whereas the peaks between 700 and 1000 nm related to ar (fig. 2c). fig. 1: carotenoid biosynthesis pathway in plants. the activity of phytoene synthase (psy) and phytoene desaturase (pds) in the biosynthesis of carotenoids. blue colour denotes the carotenoids measured in this study by hplc analysis and red colour indicates enzymatic activities for which gene expression was monitored via real time-pcr. ggdp, geranylgeranyl diphosphate. tab. 1: physical properties of the mwcnt-cooh. mwcnt-cooh young’s modulus (gpa) 1200 tensile strength (gpa) 150 density (g/cm3) 2.6 thermal conductivity (w/m.k) 3000 electrone conductivity (s/m) 105 107 fig. 2: the plasma experimental apparatus: (a) a schematic design of dbd. (b) the plasma experimental apparatus dbd (model: ps200, nik fanavaran plasma co., iran). (c) optical emission spectro scopy-based spectrum to illustrate plasma diagnostic data. treatment of seeds using cnt and cold plasma and greenhouse experiment uniform and healthy seeds of m. charantia were surface-sterilized by submerging in a 1% (v/v) solution of commercial sodium hypochlorite for 15 min and rinsed twice with sterile distilled water. some seeds were soaked in distilled water and the other in mwcnt solutions (50, 100, and 200 mg l-1) for 48 h. after that, the soaked seeds seed priming of bitter melon by cold plasma 9 were treated with cold plasma (above described dbd; surface power density of 0.84 w/cm2) at three different exposure times, including 0 s (control), 60 s, and 120 s. in this experiment, six seeds in three independent replications (three pots; two seeds per pot) were considered for each treatment group. in detail, treatment descriptions are presented in tab. 2. then, the plasma or cnt-primed seeds and the seeds treated by both plasma and mwcnts were planted in pots filled with a cocopeat/perlite (1:1, v/v) near the substrate surface (1-2 cm deep). each treatment was replicated three times. the plants were grown under uniform conditions of temperature (25 ± 2 °c/15 ± 2 °c day/night), relative humidity (60%), and photoperiod (16/8 h light/dark). all pots were irrigated twice a week with hoagland nutrient solution containing different concentrations of mwcnt (0, 50, 100, and 200 mg l-1) and distilled water in intervals (tab. 2). tab. 2: descriptions of treatment groups. group name number treatment description control 12 seeds mwcnt 0 mg l-1 + plasma 0s cnt50 6 seeds mwcnt 50 mg l-1 cnt100 6 seeds mwcnt 100 mg l-1 cnt200 6 seeds mwcnt 200 mg l-1 plasma60 6 seeds plasma 60 s plasma120 6 seeds plasma 120 s cnt50+plasma60 6 seeds mwcnt 50 mg l-1 + plasma 60s cnt100+plasma60 6 seeds mwcnt 100 mg l-1 + plasma 60s cnt200+plasma60 6 seeds mwcnt 200 mg l-1 + plasma 60s cnt50+plasma120 6 seeds mwcnt 50 mg l-1 + plasma 120s cnt100+plasma120 6 seeds mwcnt 100 mg l-1 + plasma 120s cnt120+plasma120 6 seeds mwcnt 200 mg l-1 + plasma 120s field emission scanning electron microscopy (fesem) field emission scanning electron microscopy (fesem; model: tescan mira3-feg, czech. republic) was conducted using gold-plated material to investigate tracing uptake and accumulation of the nanoparticles on the 21-day old samples. cross-sections of stems were prepared. the samples were dehydrated and fixed using a freeze dryer. then, the prepared samples were gold-coated. a portable microscope camera 400x usb (dino lite amh-rut, shenzhen, china; portable, handheld) was used to observe the effects of cnts on imbibition. isolation of rna and cdna synthesis total rna from the leaves of bitter melon were extracted and purified by using the rneasy plant mini kit (qiagen, usa). after extraction, complementary dna (cdna) was synthesized from 1 μg of total rna using quantitect reverse transcription kit (qiagen, usa) according to the manufacturers’ instructions. sequence analysis using sequence data from the sequencing of cdna libraries obtained from bitter melon seedlings. the genes that demonstrated maximum identity and similarity were selected for further study. quantitative real-time pcr analysis of mcpsy and mcpds to design primers for quantitative real-time pcr (qrt-pcr), the bicon designer and primer 3 program (http://frodo.wi.mit.edu/ primer3) was used based on published gene sequences of the m. charantia psy (genbank accession number: ay494789) and pds (genbank accession number: ay494790) cdna sequences. the m. charantia 18s ribosomal rna gene (genbank accession number ay900000.1), as a housekeeping gene, was used as an internal re ference. the sequences of primers for these genes are presented in tab. 3. the level of each gene expression was showed with relative expression, which is the copy number of each gene compared to that of a housekeeping gene. the qrt-pcr was conducted in 0.5 μm of each set of primers in 20 μl final reaction volume of sybr green real-time pcr premix ex taq™ (takara bio inc, japan) on a rotorgene q real-time pcr system (qiagen, usa) under the following conditions: 94 ºc for 5 min followed by 40 cycles of 94 ºc for 15 s, 56 ºc for 15 s, and 72 ºc for 20 s. all experiments were performed in duplicate and three times. relative expression of mcpsy and mcpsd were calculated using equation 2-∆∆ct in which ∆ct was obtained by subtracting the internal control ct value from the ct value of mcpsy and mcpsd (rajaee behbahani et al., 2020). tab. 3: sequences of specific primers used for quantitative real-time pcr. genes primer sequences amplicon size (bp) f gcttcatcgttggttgtctctct r tgctccatttctgcctcttactc f tttgcttggattaccctagacca r tgcaccagcgatcactactttta f ataactcgatggatcgcacgg r tcctccggaatcgaacccta mcpsy 154 mcpds 128 mc18s rrna 136 high-performance liquid chromatography (hplc) analysis of carotenoids carotenoids were extracted from m. charantia leaf samples (0.1 g) in each group with 3 ml of ethanol containing 0.1% ascorbic acid (w/v). this mixture was vortexed for 20 s, and then incubated in a water bath at 85 ºc for 5 min. subsequently, 120 μl of potassium hydroxide (80% w/v) was added to saponify any potentially interfering oils. after vortexing and incubating at 85 ºc for 10 min, the samples were placed on ice and 1.5 ml of cold deionized water and 0.05 ml of β-apo-80-carotenal (12.5 μg ml-1), an internal standard, were added. next, the carotenoids were extracted twice with 1.5 ml of hexane and centrifuged at 1200 g each time to separate the layers. then, the extracts were freeze-dried under a stream of nitrogen gas and resuspended in 50:50 (v/v) dichloromethane/methanol. the extraction method used for carotenoid analysis was similar to that described (howe and tanumihardjo, 2006). for hplc analysis, the carotenoids were separated on an agilent 1100 hplc system with a hector-m c18 column (150 × 4.6 mm, 5 μm, p/n: c18-m51001546) and detected with an array detector at 450 nm. solvent a consisted of methanol/water (92:8 v/v) with 10 mm ammonium acetate. solvent b consisted of 100% methyl tert-butyl ether. the flow rate was maintained at 1 ml/min and samples were eluted with the following gradient: 0 min, 83% a/17% b; 23 min, 70% a/30% b; 29 min, 59% a/41% b; 35 min, 30% a/70% b; 40 min, 30% a/70% b; 44 min, 83% a/17% b; and 55 min, 83% a/17% b. identification and peak assignment of carotenoids were primarily based on comparison of their retention time and uv-visible spectrum data with that of standards, and with guidelines previously presented. statistical analysis the obtained data were subjected to statistical analyses using spss software. all data were expressed as mean ± standard error (se) values of three independent replicates. significant mean differences between the treatments were estimated according to duncan’s multiple range test at the level of p ≤ 0.05. 10 f.s. seddighinia, a. iranbakhsh, z. oraghi ardebili, s. soleimanpour results both cold plasma and cnt treatments synergistically provoked considerable changes in the early growth of the treated plants, especially the combined treatments, among which the simultaneous treatment of 200 mg l-1 cnt and cold plasma for 60 s has shown the best performance (fig. 3). there was a considerable linear relationship between the applied concentrations of the cnt and the growth rate of seedlings. it should be noted that cnt supplementations have not caused any toxic effects. the ultra-structure of stems (by fesem) was photographed to trace the uptake and transportation of mwcnt. cellular uptake and translocation of the cnt were manifested based on the stem ultra-structure (fig. 4). expression levels of the mcpsy and mcpds quantitative real-time pcr analysis was performed to determine the expression levels of mcpsy and mcpds in all treated groups of m. charantia. mcpsy and mcpds expressed in all treated groups, were examined, and shown a similar expression pattern. the mcpsy was expressed at the highest level in the cnt50+ plasma 60 group (26.6fold) and cnt50 group (6.69-fold) (tab. 4). between cnt treated groups, cnt50 had significantly higher expression levels (6.5-fold) than the others (p<0.001). among plasma-treated groups, mcpsy in plasma 60 group (3.65-fold) had significantly higher expression levels (p<0.001) than plasma 120 and control groups (tab. 4). the mcpds expression showed a similar expression trend to mcpsy, and at the highest expression level in the cnt50 group (58.06-fold) and cnt50+ plasma 60 group (37.45-fold) (tab. 4). between cnttreated groups, cnt50 (58.06-fold) had significantly higher levels than the other concentrations (p<0.001). however, there is no significant difference between plasma-treated groups. finally, between the combination groups treated with plasma and cnt, the cnt50+ plasma 60, had significantly higher expression levels (37.45-fold) than the others (p<0.001) (tab. 4). hplc analyses of carotenoids carotenoids (β-carotene, zeaxanthin, α-carotene, and lutein) were identified in different treatment groups by hplc. the carotenoid levels varied in the different treated groups of m. charantia (tab. 4). changes in β-carotene content among plasma-treated groups, the content of β-carotene in plasma 60 was 155.5 μg g-1 fw and had higher levels than plasma 120 and control groups (tab. 4). in addition, in cnt-treated groups, the cnt50 group with 249.3 μg g-1 fw content of β-carotene had significantly higher levels (p<0.001) than cnt100, cnt200, and control groups (tab. 4). totally, between all treated groups, the cnt50 group and cnt50+plasma60 group had significantly higher levels of β-carotene content than the other treated groups (tab. 4). the lowest level of β-carotene was in plasma 120 and cnt200 groups, with no significant difference from the control group (tab. 4). changes in zeaxanthin content the production of zeaxanthin in all groups was less than 9 μg g-1 fw, which is relatively low (tab. 4). in plasma-treated groups, the content of zeaxanthin in plasma 60 was 3.87 μg g-1 fw, with significantly higher levels (p<0.001) than plasma 120 and the control groups (tab. 4). among cnt-treated groups, the cnt50 group with 8.43 μg g-1 fw content of zeaxanthin had significantly higher levels (p<0.001) than the other cnt and control groups (tab. 4). finally, between all treated groups, the cnt50, cnt50+plasma 60 group, and cnt100+plasma 60 group had significantly higher zeaxanthin levels (p<0.001) than the other treated groups (tab. 4). the lowest zeaxanthin level was in plasma 120 and cnt100 groups and there was no significant difference with the control group (tab. 4). changes in α-carotene content the findings show that cnt50+ plasma 60 group had significantly higher α-carotene levels (p<0.001) than the control and other groups. in cnt-treated groups, the cnt50 group had a significantly higher (p<0.001) α-carotene level than the cnt100, cnt200, and control groups (tab. 4). however, there were no statistically significant differences in α-carotene production between plasma-treated groups. among combination groups, treated with plasma and cnt simultaneously, both cnt50+ plasma 60 and cnt100+ plasma 60 groups had significantly higher α-carotene levels than the other treated groups (p<0.001) (tab. 4). meanwhile, the cnt200 group contained the lowest α-carotene level (tab. 4). changes in lutein content tab. 4 shows that the highest lutein level was observed in the cnt50+plasma 60 group with a content of 171.6 μg g-1 fw. moreover, fig. 3: the recorded differences in plant early growth following seed priming with the plasma and the supplementation of carbon nanotubes (cnt), 8 days after the treatments. a-control; b-cnt of 50 mgl-1; c-cnt of 100 mg l-1; d-cnt of 200 mg l-1; e-plasma of 60 s; fplasma of 60 s and cnt of 50 mg l-1; g-plasma of 60 s and cnt of 100 mg l-1; h-plasma of 60 s and cnt of 200 mg l-1; i-plasma of 120 s; j-plasma of 120 s and cnt of 50 mg l-1; k-plasma of 120 s and cnt of 100 mg l-1; l-plasma of 120 s and cnt of 200 mg l-1. seed priming of bitter melon by cold plasma 11 fig. 4: the ultra-structure images are based on the electron microscopy (fesem) for tracing the uptake of cnt in shoots of the seedlings treated by the plasma and cnt. cross-sections of m. charantia main shoots after exposure to (a, b, c, d, and e) control, (f, g, h, i and j) functionalized cnt 200 mgl-1, (k, l, m, n, and o) plasma of 120 s (p, q, r, s, and t) plasma of 120 s + functionalized cnt 200 mgl-1. from left to right panels represent general and detailed information, respectively, concerning selected areas. tab. 4: evaluation of psy and pds genes expression and carotenoid content (μg g-1 dry weight). results expressed as mean ±sd (n=3) in different plasma and cnt treated groups of m. charantia*. psy gene 1±0.001f 6.69±0.19b 0.07±0.03g 3.95±0.14c 4±0.38c 26.62±0.05a 1.12±0.04f 2.21±0.01e 0.36±0.02g 3.03±0.03d 0.14±0.02g 1.1±<0.001f pds gene 1±0.001g 58.06±0.16a 0.43±0.11gh 5.69±0.02d 0.33±0.02gh 37.45±0.81b 0.28±0.09gh 27.09±0.02c 0.73±0.01gh 3.81±0.005e 0.12±0.03h 2.88±0.16f β-carotene 104.8±8.08d 249.3±20.2a 171.6±12.8bc 121.9±8.14cd 155.5±19.3bcd 245±16.14a 202±12.29ab 152.5±20.9cd 99.6±14.42d 209.6±37.1bc 141.3±17.4cd 113.6±4.1cd zeaxanthin 2.31±0.71bc 8.43±1.72a 2.9±0.86bc 3.97±0.32b 3.87±1.17bc 7.44±1.09a 7.32±0.56a 3.02±0.58bc 1.07±0.25c 4.34±1.12bc 3.98±0.87bc 3±0.43bc α-carotene 3.28±0.48e 11.53±1.74abc 10.3±1.9abcd 4.44±0.87de 5.89±0.95cde 14.34±1.93a 13.18±2.28a 10.6±1.32abc 6.3±1.14cde 8.34±1.5bcde 12.45±1.8ab 7.16±1.61cde lutein 39.23±6.42a 123.5±19.3ab 73.6±10.1bcd 69.4±10.38cd 45.67±8.51d 171.6±21.5a 118.6±15.6abc 98.37±9.89bc 64.8±7.31cd 108.5±4.98abc 85.2±5.3bcd 98.3±35bcd *statistical significance of the differences between treated groups was determined using anova followed by paired-group comparisons. the different letters (a, b, c and d) indicate significance at p< 0.05. c on tr ol c n t 50 c n t 10 0 c n t 20 0 p 60 c n t 50 +p 60 c n t 10 0+ p 60 c n t 20 0+ p 60 p 12 0 c n t 50 +p 12 0 c n t 10 0+ p 12 0 c n t 20 0+ p 12 0 12 f.s. seddighinia, a. iranbakhsh, z. oraghi ardebili, s. soleimanpour in cnt-treated groups similar to α-carotene, the cnt50 group had a significantly higher lutein level (p<0.001) than the cnt100, cnt200, and control groups (tab. 4). however, there were no significant differences in lutein levels between plasma-treated groups. meanwhile, the plasma 60 group contained the lowest level of lutein (tab. 4). the correlation between carotenoid contents and the expression of mcpsy and mcpds correlation analysis, inter se mcpsy and carotenoid contents in all groups revealed significant association between the mcpsy with all forms of carotenoid [β-carotene (r=0.602, p< 0.001), zeaxanthin (r=0.539, p< 0.001), α-carotene (r=0.405, p< 0.014) and lutein (r=0.646, p< 0.001)]. also, correlation analysis, inter se the mcpds and carotenoid contents in all groups revealed significant association between this gene with all forms of carotenoid [β-carotene (r=0.643, p< 0.001), zeaxanthin (r=0.568, p< 0.001), α-carotene (r=0.442, p< 0.007), and lutein (r=0.587, p<0.001)]. regarding the coefficients, correlation between the expression of two genes and other variables is significant (p <0.001), showing a direct relationship between them (tab. 5). several genes, like dat in catharanthus roseus (ghasempour, 2019), pal, tat, and ras in salvia verticillate (rahmani, 2020), and hppr, ras, pal, tat genes in satureja khuzistanica (fatemi, 2019). lc-ms analysis revealed that mwcnt supplementation altered total fruit metabolome in tomato crops (mcgehee, 2017). these results manifested that similar to other nanomaterials, cnts can trigger signalling thereby influencing transcriptions of genes and metabolism. apart from cnts, cold plasma is also capable of affecting the transcription of genes. seed priming with cold plasma affected the expression of several genes in diverse plant species, like pal and usp in astragalus fridae (moghanloo, 2019), defenserelated genes in tomato (adhikari, 2020), and wrky1 transcription factor, thcas, oac, cbdas, and ols in hemp (iranbakhsh et al., 2020). in sunflower, the cold plasma priming was also associated with transcriptional responses and substantial variation in phytohormones (mildaziene et al., 2019). the plasma-mediated changes in the transcription of genes can be explained by bioactive signalling agents generated during the plasma generation (babajani, 2018; iranbakhsh, 2017; iranbakhsh, 2018; nasrin safari, 2017; sheteiwy et al., 2018). our results along with these recent reports underline this hypothesis that cold plasma perception and signal transduction can modify transcription of genes, thereby improving plant growth and metabolism. however, more molecular studies are required to fill knowledge gaps and elucidate the potentially involved mechanisms in plant responses to cold plasma and cnts. in the present study, it was shown that these two treatments could have synergistic effects. in this connection, the results of this study also demonstrated that cnt50 + plasma 60 can stimulate the higher expression of mcpsy and mcpds, compared to the other treated groups; however, the other had acceptable expression. thus, it can be concluded that plasma 60 can increase the efficiency of the cnt50. furthermore, the cnt treatments (long-term), especially cnt50, were more efficient than the individual plasma priming (short-term treatment) in affecting the gene expression or carotenoid levels. it should be noted that the significant correlations were found between the mcpsy and mcpds expressions and carotenoids’ concentrations. however, the similar regular linear relationship between the cnt concentrations, gene expression levels, carotenoid contents, and growth performance was not found and the individual or combined treatments of the cnt50 provoked the highest expression of mcpsy and mcpds, as well as terpenoid derivatives. interestingly, the cnt200 treatment led to the best plant growth performance, while cnt50 provoked the highest expression. hence, it can be proposed that cnt can affect diverse signalling pathways, thereby altering growth and metabolism. therefore, it seems that the individual or combined treatments of the cnt and plasma, differentially affect plant cell at diverse growth, physiological, and molecular levels in a dose-dependent manner. to the best of our knowledge, this experiment provides the first molecular evidence on how cold plasma and cnt treatment may synergistically improve carotenoid metabolism which is an important mechanism in terms of photosynthesis performance and protection. the observed molecular variations at the transcriptional level may be explained by modulation in redox homeostasis in response to cnt (fatemi, 2019; rahmani, 2020) and cold plasma (ghasempour, 2019; iranbakhsh et al., 2020; mildaziene et al., 2019; moghanloo, 2019). monitoring the potential variation in cellular redox status following cold plasma and cnts is, therefore, re commended for designing future studies. conclusions in the present article, we addressed the efficacy of cnt and cold plasma treatments to affect the metabolism of carotenoids. according to our results, the cnt and plasma-mediated modification in the mediscussion in the present study, the relationship between carotenoid accumulations and the gene expression of mcpsy and mcpds, under individual and combined treatments of cold plasma and carbon nanotube, were investigated in m. charantia, which could potentially be used as a source of carotenoid in human nutrition. the seed priming with cold plasma and supplementation of nutrient solution with mwcnt in both individual and combined treatments, not only improved plant early growth but also modified the secondary metabolism through changes in expression patterns of two key genes, mcpsy and mcpds, contributed to the synthesis of important terpenoid meta bolites. in our previous report, we provided comprehensive evidence on how cnt and cold plasma treatments were associated with significant modifications in plant growth, biomass accumulation, yield, and differentiation of tissues (especially xylem conducting tissue) (seddighinia, 2020). the individual or combined treatments of cold plasma and mwcnts, especially cnt50+plasma60, were capable of upregulating genes involved in carotenoid metabolism. as it is well known, carotenoids contribute to protecting the photosynthesis apparatus through the xanthophyll cycle. there is limited molecular evidence on transcriptional responses to cnts. in this regard, earlier reports showed that cnt application was associated with changes in growth and development-related genes, including slr1, rtcs, rth1, and rth3 genes in maize (yan et al., 2013) and cycb, ntlrx1, and ntpip1 in tomato (khodakovskaya et al., 2012). moreover, several reports indicated that cnts were associated with upregulation in genes involved in secondary metabolism which our results are consistent with their findings. in line with our results, the cnt treatments mediated changes in secondary metabolism through upregulating tab. 5: correlation coefficients (r) exhibiting relationship between the expression of two evaluated genes (psy and pds) and the measured related carotenoids (lutein, α-carotene, zeaxanthin, and β-carotene). genes lutein α-carotene zeaxanthin β-carotene mcpsy 0.646** 0.405* 0.539** 0.602** mcpds 0.587** 0.442** 0.568** 0.643** *: p≤0.05 **: p≤0.01 seed priming of bitter melon by cold plasma 13 tabolism of carotenoids can be considered as an important mechanism by which these treatments may protect the photosynthesis apparatus against photoinhibition. herein, a pilot experiment is presented for introducing new effective methods, which could be exploited in the food, medicinal, and agricultural industries. acknowledgment authors especially would like to acknowledge of plasma 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2021. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1155/2013/729081 http://dx.doi.org/10.4103/phrev.phrev_52_16 http://dx.doi.org/10.1016/j.plaphy.2020.02.022 http://dx.doi.org/10.1371/journal.pone.0235556 http://dx.doi.org/10.1007/s00344-019-09965-2 http://dx.doi.org/10.1007/s00709-018-1288-z http://dx.doi.org/10.1016/j.envpol.2020.114727 http://dx.doi.org/10.1016/j.foodchem.2010.12.058 http://dx.doi.org/10.1049/iet-nbt.2015.0046 http://dx.doi.org/10.1016/j.jhazmat.2012.12.013 angewandte botanik. kleine mitteilungen. 49 kleine mitteilungen. die alkoholerzeugung aus holz wird in der holzwelt vi (1919) s. 6 besprochen; z. zt. sind 4 brennereien vorhanden, von denen der staat 3 mit einem kostenaufwande von 21,3 millionen mark in mann heim, danzig und stettin gründete, eine vierte in ohdenburg ist in privatbesitz. statt der knappen kartoffeln werden sägespäne als bisher wertlose abfallprodukte verwendet. man erwartet aus 100 kg trockenen sägespänen 6 liter alkohol. die art der gewinnung wird kurz skizziert. – deutschland erzeugt jährlich etwa 600000 t zellstoff. die 12 in deutschland eingerichteten laugenbrennereien können bei vollbetrieb jährlich 116000 hl alkohol liefern, wobei zugrunde gelegt ist, daß 1 t zellstoff 4,5 cbm lauge oder 40,5 liter alkohol entspricht. über den anbau der reismelde (chenopodium quinoa) berichtet bertram kalt in kühn-archiv vii (1918); umfassende anbauversuche hat die pflanzenzuchtstation des landwirtschaftlichen instituts an der universität halle im jahre 1917 ausgeführt. die ergebnisse dieser versuche sind: die reismelde ist danach für den feldbau im deutschen tieflande gänzlich ungeeignet: 1. aus klimatischen gründen wegen ihres hohen wasserbedarfs, wegen ihrer vegetationszeit, wegen ihrer hygroskopischen eigenschaften und der daraus folgenden schwierigen notreife; 2. aus wirtschaftlichen gründen wegen der schwierigkeiten der be stellung, wegen der erforderlichen häufigen hackarbeit, der schwierigkeit der ernte, des drusches und ihres ganz unsicheren reinertrages. sie kann zurzeit auch nicht den kleintierhaltern zum gartenbau empfohlen werden. trennung von forst und weide. diese für die floristische zu sammensetzung und damit für die biologie unserer halbkultur formationen, der weide und der ja bei uns außerhalb der gebirge fast allein bestehenden waldform, der forst, außerordentlich wichtige frage wurde in der jahresversammlung der waadtländischen forstgesellschaft (société vaudoise des forestiers) in lausanne besprochen. im allge meinen sprachen sich die forstlichen fachleute für eine möglichst strenge trennung der nutzungsflächen, die als weide gebraucht werden sollen, von den forstlich zu verwertenden aus, während die landwirt schaftlichen vertreter den gegenteiligen standpunkt vertraten. bei uns in deutschland findet man zumeist den reinen forstbetrieb innerhalb des verwaltungsbereiches der großen forstverwaltungen, während in bauernund gemeindebetrieben vielfach die weidenutzung auf dem waldgelände stattfindet. gerade die letztgenannten formationen be herbergen bekanntlich zahlreiche interessante gewächse. umwandlung von wald in kartoffelland is t in größerem um fange in der schweiz beschlossen worden. auf anregung des eid genössischen ernährungsamtes sollen namentlich geringwertige schachen länder und auenwälder mit leichtem für den kartoffelbau geeignetem boden gerodet werden. ersatz-aufforstungen sollen später mit hilfe bundes im hochgebirge erfolgen. (forstliche mitteilungen ii [1919 eft 4.) angewandte botanik i. 4 50 kleine mitteilungen. gegen die abholzung des haardtwaldes in baden nimmt dr. ratzel-heidelberg in der holzwelt vi (1919) nr. 14 stellung, er führt aus, daß der zu siedelungsland dort neu zu gewinnende boden alter dünensand sei, der in die gefahr des wanderns käme und dadurch fruchtbarer ackerboden mit sand bedeckt würde, daß man relativ schlechten boden gewönne und daß die wasserversorgung der auf das grundwasser der rheinebene angewiesenen städte auch aus den un besiedelten teilen des haardtwaldes hygienisch einwandfreies wasser gewinnen könnte. über flechten als watteersatz wird in der zeitschrift für ab fallverwertung 1917, s. 165 berichtet, daß das aus flechten hergestellte erzeugnis wohl etwas derb ist, daß es aber bei berührung mit einer flüssigkeit fast so weich wird wie echte watte. die verwendung des seetangs in der faserindustrie wird be sprochen in der zeitschr. f. abfallverwertung 1917, s. 23. geheimmittel spielten, wie bei allen weltkatastrophen, so auch im jetzigen weltkriege eine große rolle. oft sind die bestandteile gänzlich indifferent oder doch unschädlich und die hauptrolle spielt neben bewußtem schwindel der aberglaube. letzterer zeigt sich darin, daß die aus kräutern zusammengesetzten rezepte fast stets aus 9 (3 × 3) oder besonders 13 pflanzen gemischt sind. anscheinend recht verbreitet war ein mittel gegen geschlechtskrankheiten, welches unter dem namen „loffa-“ (oder luffa-, hoffa-)kräuter feilgeboten wurde und den ansehnlichen preis von 17,– m. das kilo hatte. auch dieses war aus 13 kräutern zusammengesetzt, neben lignum guajaci, und l. sassafras, radix sarsaparillae und sennesblättern, alle in geringen mengen, war der hauptbestandteil ein gemisch von galeopsis, plantago (blätter und samen), tussilago farfara, achillea millefolium (blätter und blütenköpfe), viola tricolor, arnica (nur die lebhaft rotgelben strahlblätter) und süßholz, lindenund walnußblätter. uber das vorkommen von scopolia carniolica in litauen. während in deutsch-litauen durch die strengen maßnahmen der re gierung gegen die giftmischer scopolia carniolica fast vollkommen aus den gärten verschwunden ist, spielt diese pflanze offenbar in russisch litauen noch eine gewisse rolle. dem botanischen museum zu berlin dahlem sind im laufe des krieges mehrere einschlägige fälle bekannt geworden. so hatten ortsfremde gefangene russen die zur bestimmung eingesandten knollen gefunden und gegessen, worauf sie schwer er krankten und zum teil erblindeten. daß bei den eingeborenen aber die giftigen eigenschaften der pflanze immer noch wohl bekannt sind, beweist ein zweiter fall, in dem eine frau die knollen zu einem selbst mordversuch benutzte. auch aus anderen nachrichten geht hervor, daß dort bei giftmischereien immer noch das „altsitzerkraut“ seine be rüchtigte rolle spielt. vgl. über den gebrauch der pflanze im litauischen sprachgebiet abromeit in königsb. hartung. zeit. 1890, 64; bei po dack in schr. phys.-ök. ges. königsb. 1897, s. 79; ascherson in sitzb. ges. naturf. fr., berlin 1890, s. 59. uc1.b3299303_page_067_cut uc1.b3299303_page_068_cut angewandte botanik. 98 p. graebner, e. medlewska und a. zinz, typha als nutzpflanze. von p. graebner, e. medlewska und a. zinz. (arbeiten der studienkommission für typha-forschung.) (schluß.) 3. zur entwicklung des mechanischen gewebes im blatte der typha angustifolia. 2. von e. medlewska. (schluß) 2. mechanische eigenschaften der rohfaser. nehmen wir als hauptkriterien für den technischen wert der faser den zellu losegehalt (d. h. mangel an leichten oxydationsfähigen substanzen), die reißfestigkeit und elastizität an, dann steht die maifaser in bezug auf den ersten punkt am höchsten (84"/o zellulose). sie läßt sich nur durch sehr vorsichtiges aufschließen (mit/o–% natronlauge) gewinnen, sonst zerfällt sie in die elementarfasern, ein beweis dafür, daß die zwischenzellsubstanz noch sehr wenig widerstandsfähig ist (pentosangehalt 7,8°/o). deswegen, wegen der relativen kürze der zellen (0,54 mm) und der geringen wandungs dicke kann die reißfestigkeit der bündel keine bedeutende sein. dagegen dürfte diese faser den höchsten elastizitätsgrad besitzen, da die verholzung noch sehr gering is t (methylzahl 3,8) und der ca-gehalt nicht so hoch wie später. 4 . festigkeit der typha-faser. untersuchung durch das materialprüfungsamt (21. februar 1918). gewicht der berechneter zugfestigkeit g än durchschnittzugfestig abschnitte licher quermeºrt berechnet keit aus. bruch 650 × 20 schnitt der für den auf 1 qmm | gedrückt ö ö öä geprüften einzelnen faserals dehnung bei 65% luftbät ät querschnitt reißlänge feuchtigkeit adscii11tte g qmm g kg km % 0,0062 0,00413 168 40,7 27,1 | 2,5 0,0072 0,00507 145 28,6 19,1 2,0 0,0089 0,00593 191 32,2 21,5 4,0 0,0080 0,00533 184 34,5 23,0 2,5 0,0080 0,00533 129 24,2 16,1 2,5 0,0062 0,00413 124 30,0 20,0 6,0 0,0072 0,00480 143 29,8 19,9 |3,5 typha als nutzpflanze. 99 5. die kultur von typha. von p. graebner und a. zinz. die nutzbarmachung der größeren typha-arten in erster linie a ls faserpflanzen hat begreiflicherweise bald den wunsch entstehen lassen, nicht nur die natürlichen bestände in ihrem umfange z u erhalten, sondern etwa geeignete gelände mit dem rohrkolben z u bepflanzen und das kolbenschilf anstelle wertloser sumpfgewächse z u setzen. schon die erhaltung der bestände wird vielfach kultur maßnahmen erfordern. auf nur zeitweise überschwemmtem gelände oder auch im flacheren wasser, wird meist schon nach wenigen jahren der boden derart mit grundachsen durchzogen, daß e r fast völlig verfilzt erscheint. die folge ist dann, daß besonders nach trockneren jahren sich sehr reichlich blütenstände, also stengel, entwickeln und daß damit die vegetative vermehrung, die bildung der für die fasergewinnung wertvollen blattriebe zurücktritt. damit wird der bestand entwertet. besonders bei t . latifolia scheint dieses. stadium in der größten mehrzahl der fälle etwa nach 5 bis 8 jahren einzutreten. mit der vegetativen schwächung des bestandes, der dabei zugleich licht wird, siedeln sich meist zunächst größere wiesengräser (phalaris arundinacea, glyceria aquatica u . a.) a n und typha tritt weiter zurück. „ um diesen zustand der überständigkeit z n bekämpfen, scheint das verfahren erfolg zu versprechen, daß in bestimmten zeit abständen, wenn der bestand eine starke dichte erreicht hat, während der ruhemonate durch einen pflug streifen weise diegrund achsen entfernt werden und dadurch der boden an diesen streifen gelockert wird. soweit sich an kleinversuchen bisher fesstellen läßt, wachsen die grundachsen der den streifen benachbarten pflanzen üppig in den gelockerten boden hinein und bereits im herbst zur erntezeit der blätter ist der bestand wieder geschlossen. später, etwa im folgenden jahre kommen dann die stehengebliebenen teile a n die reihe. die kosten des verfahrens werden ganz oder doch zum größten teil dadurch gedeckt, daß die ausgepflügten grund achsen mit ihrem reichlichen stärkegehalt (s . s . 30, 100) gesammelt und verwertet werden. wo ihre technische verwertung nicht möglich ist, geben sie ein gutes viehnamentlich schweinefutter, dessen verwendung!) sich während der kriegszeit vielfach ein *) berichte der deutschen landwirtschaftsges. 1916 (mehrfach). merkbl. bot, gartens u . mus. berlin-dahlem 1. (1917). :: 7 100 p. graebner, e. medlewska und a. zinz, gebürgert hat. wo große massen grundachsen zur verfügung stehen, sodaß ihre technische verarbeitung möglich ist, wird di e sehr zähe faser gewonnen werden können und die stärke mannig fache verwendung finden; schwach geröstet gewinnt sie, nach den versuchen der frau a . zinz, einen kakaoähnlichen geschmack und geruch. es wurden von herrn hofrat prof. dr. loges in pommritz in sachsen im märz und später von herrn geh. reg.-rat prof. dr. thoms-dahlem!) untersuchungen über den nährstoffgehalt angestellt, die erstgenannten ergaben bei den frischen grundachsen 5,92 % rohprotein (mit 2,04 % reinprotein) und 17,49 % kohle hydrate (mit 15,43 % stärke) oder auf die trocknen achsen be rechnet 17,67 % rohprotein und 52,21 % kohlehydrate (mit 46,06% stärke). die untersuchungen des herrn geh. rat thoms ergaben in dem in einer exzelsiormühle gewonnenen pulver, aus dem di e sehr zähen fasern ausgeschieden waren, 29,85% stärkemehl. durch die spätere jahreszeit nach beginn des austreibens war anscheinend schon stärke verloren gegangen. der eigentliche anbau von typha kann in zwei formen vor sich gehen, entweder durch aussaat oder durch pflanzung. w0 wenigstens zeitweise vom wasser verlassenes gelände zur verfügung steht, ist zweifellos die erstere vorzuziehen, weil sie einfach und billig ist. unumgänglich notwendig is t dabei, daß wenigstens di e streifen, in denen aussaat, keimung und erste entwicklung der jungen sämlinge erfolgen soll, gelockert und von etwa konkurrierenden pflanzen befreit wird, daß also lockerer „wunder“ boden geschaffen wird. zwischen anderen dichtstehenden pflanzen, besonders zwischen größeren sauergräsern kommt typha nicht zur entwicklung. die sämlinge erscheinen zwar über dem boden, sterben aber ohne grundachsenentwicklung ab. in wasser gebracht schwimmen die früchte mit ihren haaren anfangs an der oberfläche, durch die wasseraufnahme quellen aber die samen auf, der balg der frucht wird gesprengt und die samen treten heraus; d a sie schwerer sind als wasser, sinken sie z u boden. hier gelangen sie, wenn daswasser ruhig und nicht zu tief (jedenfalls noch in über 3 dm tiefe!) ist, zur keimung, scheinen aber dort sehr schwer wurzel zu fassen und können sich *) vergl. berichte der deutschen pharmaz. gesellsch. xxvi (1916) 179 ff. graebner im merkbl. bot, gartens u. mus. i. (1917). typha als nutzpflanze. 101 auch selbst bei großer vorsicht nur sehr langsam entwickeln. in der freien natur scheinen sie so gut wie stets unter diesen ver hältnissen zugrunde zu gehen, jedenfalls häben zahlreiche von a. zinz u. a. vorgenommene aussaatversuche im wasser nicht das gewünschte resultat ergeben. aussaaten auf dem feuchten ufer krochen leicht und bald ins wasser. die jungen pflänzchen fallen im wasser anscheinend zahlreichen feinden, schnecken usw. zum opfer. auch dem samen müssen tiere nachstellen, denn in der wolle zahlreicher von a. zinz im frühjahr geprüfter halb zerfallener kolben fanden sich an den standorten keine samen.mehr. die aussaat auf nacktem boden erfolgt am besten im märz, a ls dem natürlichen keimungsmonat. wenn es die wasserver hältnisse nicht zulassen, kann auch eine andere zeit gewählt werden, sobald die feuchtigkeit die bodenbearbeitung zuläßt und erfahrungsgemäß die betr. stelle noch einige monate von der über schwemmung freibleibt. die samen von typha bleiben über 2 jahre gut keimfähig. die aussaat darf nicht zu dicht, möglichst ganz weitläufig erfolgen, am besten werden die früchte mit sehr viel feuchtem sande gemischt, d a bei dichtem (rasenartigen) stande der sämlinge diese sich anfangs z u viel konkurrenz machen. die versuche, die bisher angestellt wurden, sind noch z u jung u m positive vorschläge machen zu können. a . zinz hat im jahre 1918 nach den besonders durch herrn oberinspektor peters im dahlemer botanischen garten vorgenommenen vorversuchen a n verschiedenen orten norddeutschlands aussaatversuche im auf trage der deutschen typha-verwertungsgesellschaft anstellen lassen, in erster linie bei alt-borck bei kolberg und bei uhyst in der nieder-lausitz. soweit sich bisher übersehen läßt, is t e s am vorteil haftesten gewesen, bei nicht zu hohem bestande der ursprünglichen gräser resp. sauergräser gräben von über 8 dm breite auszuwerfen v o n (einer je nach dem grundwasserstande wechselnden) 3 bis 4 dm tiefe und den auswurf in der nachbarschaft auszubreiten. je breiter die gräben sind, desto besser. die aussaat geschieht über d ie gesamtfläche des gelockerten bodens, auch über die dämme. die sämlinge faßten dort gut fuß und entwickelten sich im ersten jahre kräftig bis zur normalen höhe der einjährigen pflanze von etwa 5 bis 7 dm. bei einem andern versuche, bei dem der boden nur tief aufgehackt wurde, ging typha ebenfalls ganz dicht auf, e s steht aber noch nicht fest, o b sie dauernd in genügender menge fuß fassen wird; a n den nicht verletzten stellen blieb, wie schon 102 p. graebner, e. medlewska und a. zinz, bemerkt, die saat aus. die kolberger aussaat ging nach freund licher mitteilung des herrn torfinspektor otto durch nachträgliches austrocknen der gräben stark zurück. bei besonders guter kultur erreichten einjährige sämlinge von t. angustifolia eine höhe von über 2 m, wie sie herr ober inspektor peters im botanischen garten erzog. t. latifolia er reichte diese höhe nicht, die höchsten einjährigen pflanzen waren ca. 1,5 m. die bestockungsfähigkeit der jungen pflanzen hängt ganz vom standort und von der saatdichte ab. sehr dicht, wie dichter junger rasen, stehende pflanzen, deren blätter auch im herbst sehr frühzeitig gelb wurden, wurden z. t. nur etwa 3 cm hoch; si e erzeugten keine grundachse, sondern nur ein kleines intra vaginales knöllchen von öfter nur hirsekorngröße. auch a n stand orten mit stark wechselnder feuchtigkeit, die bald durch über rieselndes wasser durchnäßt, bald wieder trocken wurden, zeigte sich ein solches zurückbleiben der pflanzen. bei etwas lockerem stande wurden die jungen pflanzen bis zum herbst etwa 2 dm hoch und am grunde hatte eine kurze meist etwa 2 bis 3 cm lange grundachse die scheiden durchbrochen. bei ganz freiem stande in gleichmäßig nassem bis flachüberschwemmtem boden waren a n den kräftigen pflanzen bis über 6 dm lange grundachsen und zwar bis zu 6 vorhanden, die bereits kräftige triebe über die oberfläche geschickt hatten. zweijährige ungehindert gewachsene pflanzen beider arten bedeckten mehrere quadratmeter mit ihren achsen, in der mitte bereits einen kleinen dichten bestand bildend. wo wegen deswasserstandes, namentlich wegen einerwasser bewegung am ufer usw. eine aussaat keine aussicht auf erfolg bietet, weil die samen und die jungen keimlinge hinund her gespült werden, wird man pflanzung anwenden. hierbei is t d ie hauptvorsicht auf die auswahl des pflanzenmaterials zu verwenden. mit haken oder pflügen werden die grundachsen aus dem boden gerissen, die a n überschwemmten standorten schwimmen. am besten im frühen frühjahr, wenn die triebe noch in ruhe sind, werden aus den grundachsen die verdickten spitzen der letzten ausläufer ausgelesen, am besten solche, die im vorjahre noch gar keine oder doch nur kurze laubblätter getragen haben. diese jungen spitzen fallen schon in der masse der grundachsen durch ihre helle farbe auf. auch später, etwa bis in den mai (oder juni) hinein, kann man die pflanzung vornehmen, indem man dann typha als nutzpflanze. " 103 an den ausgetriebenen sprossen die blätter zurückstutzt; die pflanzen stärken sich dann aber nicht so wie bei der frühlings pflanzung. man macht furchen in den boden des ufers, legt dort die grundachsen wagerecht ein, so daß die spitzen aufwärts schauen und deckt dann die grundachsen zu, möglichst so, daß d ie bedeckende erde etwas über den wasserspiegel hinausragt, damit die grundachsen nicht herausgespült und abgeschwemmt werden können. zur uferfestigung a n größeren flüssen verspricht dies verfahren guten erfolg und soll jetzt z. b . auf veranlassung des bauamtes in großem maßstabe bei der oderregulierung an wendung finden. t . angustifolia wird für diese zwecke die ge eignete art sein. studium über eine brombeerkrankheit von dr. c . hahmann, hamburg. allgemeines. der krebs, der vielfach auf unseren obstbäumen und sträuchern o ft in gefährlichster weise auftritt, is t in seinem ursprung und wesen bei weitem noch nicht so erforscht, wie dieses wünschens wert wäre. nach sorauer wird der krebs als wunde bezeichnet, deren überwallungsränder sich zu wuchernden holzgeschwülsten ausbilden. „der charakter der wucherung liegt in der ausschließ lichen oder überwiegenden bildung von parenchymholz a n stelle der normalen prosenchymatischen holzelemente. die krebsgeschwülste haben für jede gehölzart typische gestalt“). merkwürdig is t e s, daß die krebskrankheiten, mit ausnahme der des weinstockes, lediglich in der familie der rosaceen zu finden sind. nach diesem forscher unterscheiden sich die krebsformen bei den einzelnen gattungen der rosaceen nur „durch die art der reaktion äuf den wundreiz, stimmen aber darin wieder überein, daß si e das auge *) p . sorauer, handb. der pflanzenkrankheiten i, s. 584. uc1.b3299303_page_116_cut uc1.b3299303_page_117_cut uc1.b3299303_page_118_cut uc1.b3299303_page_119_cut uc1.b3299303_page_120_cut uc1.b3299303_page_121_cut art16_ishtiaq.indd journal of applied botany and food quality 84, 223 228 (2011) institute of pure and applied biology, botany division, bahauddin zakariya university multan, pakistan phytotoxicity of nickel and its accumulation in tissues of three vigna species at their early growth stages shabnam ishtiaq, seema mahmood* (received august 2, 2011) * corresponding author summary seedlings of three vigna species; v. cylindrica, v. mungo and v. radiata were investigated for their nickel (ni) tolerance. various growth, photosynthetic attributes and accumulation levels of ni in the roots and shoots were assessed after exposure to 0 (control), 50, 100, 150 mg kg-1 ni. 100 and 150 mg kg-1 ni induced a signifi cant reduction in germination (p<0.001) and fresh biomass (p<0.05) of seedlings. the other growth attributes; root and shoot length, dry biomass, leaf number and leaf area were not much infl uenced by the presence of ni. a drastic decline was observed for the formation of nodules and chlorophyll a and b contents. a steady increase in metal content of the tissues (root and shoot) was observed with an increase in ni levels but the pattern of metal accumulation was the same in all species as bioaccumulation of ni was much profound in the roots (110 mg kg-1) as compared with the shoots (30 mg kg-1). the tolerance indices (ti) of the tissues varied with respect to ni levels. the roots exhibited higher tolerance indices (ti) than the shoots at their respective levels of ni. the results indicated a greater sensitivity of chlorophyll molecules and nodulation to ni. the study depicted inter-specifi c differences in the degree of ni tolerance and its accumulation in the plant tissues. among tested species, v. mungo appeared to be sensitive, while, v. cylindrica showed successful seed emergence, greater dry biomass along with sustainable chlorophyll biosynthesis and nodule formation. a greater root tolerance index, capacity of roots to accumulate appreciable amount of ni in addition to restricted transfer of metal to the aerial tissue may signify a better threshold of the species. v. cylindrica appeared to cope with ni toxicity through an integrated mechanism of metal tolerance which may arise from differential accumulation of ni in the plant parts without damaging the tissue and considerable alteration of important growth parameters. thus, v. cylindrica can be a choice for abandoned soils contaminated with ni. introduction nickel (ni) is among the abundant heavy metals and it constitutes about 0.08% of the earth crust thus it is ubiquitously distributed in soil and water (kupper and kroneck, 2007). ni toxicity is of serious concerns to agriculture, ecosystem and human health (jarup, 2003; pandey and singh, 2011). rapid industrialization and high anthropogenic pressures in the developing countries have encountered excessive amount of ni in the environment. ni originates more frequently from the non-ferrous metal industry, mining, production and disposal of batteries (boularbah et al., 2006). in addition, untreated municipal wastewater, sludge disposal, application of pesticides and phosphate fertilizers are also important contributors of ni pollution (pandey, 2006). like many other developing countries agriculture soils of pakistan are contaminated with heavy metals including nickel (atiq-ur-rehman and iqbal, 2008). ni has been classifi ed among essential micronutrients (brown et al., 1987). it is found associated with some metallo-enzymes which are necessary for various plants processes (giridhara and siddaramappa, 2002). however, like many other essential elements its supra-optimal concentrations are strongly phytotoxic (gautam and pandey, 2008). excessive ni can induce alterations of plant metabolism that leads to the inhibition of germination and growth (khan and khan, 2010). it is known to produce stunted growth, chlorosis and necrosis of leaf which are visible symptoms associated with ni toxicity (seregin and kozhevnikova, 2006). high concentration of ni can inhibit dry matter production and chlorophyll biosynthesis (ahmed et al., 2010). nitrogen fi xation which is typical to leguminous plants depends on the ability of rhizobium spp. to form nodules. but excessive ni has been reported to cause deleterious effects on the genus rhizobia and hence, on nodules formation in a number of leguminous species (vijayarengan, 2000). since, ni is highly mobile therefore it can easily be translocated from the roots to the aerial parts of the plants (jozef et al., 2009). however, its uptake, distribution and bioaccumulation vary considerably in plant species. evidences (edem et al., 2009) show that bioaccumulation of metals in underground and aerial tissues is an indicative of metal tolerance strategy which divides plants into two groups. the fi rst group includes metal excluder species that accumulate and store a signifi cant proportion of heavy metals in their roots while, metal ions are translocated from the roots to the shoots and leaves in the other group called accumulator species (reeves and baker, 2000). changes in growth responses induced by heavy metals are frequently used to assess tolerance indices of plant tissues which serve to suggest metal tolerance sensitivity (ma et al., 2009). both germination and seedling stages are particularly important for successful subsequent development of a crop. the plant species that show heavy metal tolerance at their juvenile stages may produce tolerant adult individuals. thus, exploration of variation at early growth stages may signify overall potential of a crop for its exploitation on contaminated agriculture lands located in the vicinities of large industries of the country. keeping in view the increasing ni toxicity to crop plants and signifi cant importance of pulses as source of low cost vegetable proteins for low income groups in a developing country like pakistan, we assessed relative ni tolerance of three vigna species (v. cylindrica, v. mungo and v. radiata) at their early establishment phases. we investigated changes in germination, various growth and photosynthetic attributes of the species after their exposure to different levels of ni in the soil. the ni content in plant tissues along with tolerance indices for root and shoot growths were also investigated to evaluate tolerance of the species to excessive ni present in the growth medium. 224 s. ishtiaq, s. mahmood materials and methods seeds of three vigna species, vigna cylindrica skeel var. cp-386, vigna mungo l. var. 6036-7 and vigna radiata var. 97003 were obtained from pulse crop division, ayub agriculture research institute, faisalabad, pakistan. air-dried sandy loam soil (ph 7.90) sieved through a 2 mm sieve was thoroughly mixed with nicl2.6h2o (merck, germany) at concentrations of 50, 100 and 150 mg kg-1 substrate singly at the beginning of experiment, while control plants were without ni. since ni salt was hexa-hydrated therefore actual concentration factor of ni was used to maintain ni levels used in the study. thirty-six earthen pots (height 15 cm and internal diameter 12 cm) were fi lled with 1.0 kg of soil appropriately. twenty seeds of each species were sown into these pots. germination was assessed via seedling emergence and seeds were considered to be germinated when seedlings emerged from the soil bearing at least two young leaves. data records were made for 10 days until no germination occurred. for seedling experiment, eight (pre-germinated) 6 days old seedlings were transplanted into each of thirty-six earthen pots (height 30 cm and internal diameter 25 cm) which were fi lled with 3.5 kg of soil containing ni at the rate of 0 (control), 50, 100 and 150 mg kg-1 substrate. seedlings were acclimatized for 8 days then thinned out to four in each pot. the experiments were arranged in a complete randomized manner. to simulate fi eld conditions, plants were grown in a wire netting house under natural conditions (temperature 28 + 5°c and the day-length 12 h). all pots were irrigated with tap water, spraying gently using a spray bottle to avoid leaching. plants were allowed to grow for 4 weeks, when seedlings were about 15-20 cm tall; bearing at least 10-12 leaves were harvested by taking out whole soil from the pot. plants were carefully separated from the soil and washed thoroughly with tap and then with distilled water. the data for various growth attributes were recorded. fresh weights were taken and for dry weight measurements plant material was oven dried at 70°c for 48 h. leaf samples were scanned using deskscan and the images produced were analyzed using delta t-scan leaf image analysis software (delta t devices, uk). 2 g of leaf material from each treatment was extracted in 80% acetone for the determination of chlorophyll a and b. the absorbance of fi ltrates was taken at 645 and 663 nm for chlorophyll a and b respectively following (arnon, 1949) using spectrophotometer (2000 hitachi, japan). metal content in plant tissues were determined by the acid digestion method. the well dried root and shoot samples were ground and a wet digestion was carried out with 3:1 hno3: hclo4 (v/v) (allen et al., 1986). the concentration of nickel was determined using an atomic absorption spectrophotometer (analyst 300, perkin-elmer, germany). the standard was aa ni (camlab, u.k). tolerance indices for plant tissues were calculated following wilkins, 1978 as: root / shoot elongation (cm) at ni treatment ti (%) = x 100 root / shoot elongation (cm) at ni treatment ti (%) = x 100 root / shoot elongation (cm) at ni treatment root / shoot elongation (cm) at control ti (%) = x 100 root / shoot elongation (cm) at control ti (%) = x 100 thus, three series of tolerance indices for root and shoot for each species were obtained. statistical analysis data presented as means (+ s.e) for each parameter. data for germination percentage was arcsin transformed following bliss (1937) then it was subjected to statistical analysis (anova). a twoway analysis of variance was carried out using ms excel 2000 in order to determine signifi cant effects of different ni levels as well as to determine inter-specifi c variability. least signifi cant differences (lsd) between means for species and ni levels were calculated by employing a multiple range test following duncan (1955). results and discussion the results of the study depicted a differential infl uence of various ni levels and variable responses of three vigna species for the attributes studied. the results presented for seedling emergence showed that the maximum number of seedlings emerged at the control for all three species but a gradual decline in seedling emergence was observed with increasing levels of ni. seed germination was severely affected by the highest (150 mg kg-1) ni level. a consistently higher percentage for seedling emergence was observed for v. radiata at all levels of ni except at 100 mg kg-1, where v. cylindrica had the maximum (70%) seedling emergence (fig. 1 a). at low concentration of ni (50 mg kg-1), germination of seeds of all vigna species was greater. with doubling of the ni concentration from 50 to 100 mg kg-1, the percentage of seed germination declined by 10%. germination further declined (more than 50%) when nickel was added at the concentration of 150 mg kg-1. thus, seed germination was severely (p<0.001) affected by the presence of ni and the species also exhibited signifi cantly (p<0.05) variable responses (tab. 1). since, germination is the most crucial stage of plant development. therefore, seed germination is frequently used as an indicator of early response of the plants in a hostile environment (singh et al., 2006; talukdar, 2011). hall (2000) reported that ni inhibits all energy requiring cellular processes during germination thus, slows down emergence of radicles and plumules. in the present study inhibition of seed germination was also observed and consequently seedling failed to emerge owing to the toxicity of ni. ni has caused no inhibitory effect on root elongation and similarly the species have shown invariable responses for root length (tab. 1). however, v. radiata had greater root length at 100 and 150 mg kg-1 ni (fig. 1 b). although various levels of ni had not infl uence shoot elongation but there were present signifi cant interspecifi c differences (tab. 1). v. mungo had consistently the lower shoot length at all ni levels as compared with v. radiata and v. cylindrica (fig. 1 c). 150 mg kg-1 ni had caused a signifi cant reduction (p<0.05) of fresh biomass and species had also shown distinct responses (tab. 1). a consistently greater fresh weight was observed for v. cylindrica at all level of ni (fig. 1 d). the dry weight also exhibited a similar trend as shown by the fresh biomass (fig. 1 e). though, no noticeable infl uence of ni level was observed on dry biomass but species had shown signifi cant (p<0.05) variable response for dry weight production (tab. 1). impact of different abiotic stresses including heavy metals is frequently perceived through dry weight which serves as one of the realistic predictors because it signifi es cumulative effects of various attributes (vijayarengan, 2000). the presence of ni did not induce any drastic decline in dry biomass production of these species (v. cylindrica, v. mungo and v. radiata). no suppression of dry biomass can be ascribed for the enhancement of total organic carbon in leguminous plants in the presence of heavy metals as demonstrated by a number of other workers (jiang et al., 2008; ma et al., 2009). fixation of atmospheric nitrogen, which is a base material for protein, is an important function of root nodule bacteria. moreover, the degree of nodulation is directly related to the nitrogen economy of leguminous plants as it affects their ultimate yield (khan and ti (%) = x 100 phytotoxicity of ni in vigna species 225 khan, 2010). a drastic decline (p<0.01) in the formation of root nodules was observed in the presence of ni. the responses of the species were distinctly (p<0.001) variable (tab. 1). v. cylindrica had shown signifi cantly higher number of nodules whereas, the formation of root nodules was severely affected by the presence of 100 and 150 mg kg-1 ni in v. mungo and v. radiata as the former species had developed no nodules in the presence of higher levels of ni (fig. 1 f). the sensitivity of the genus rhizobium to ni is well documented (rajkumar and freitas, 2008). the poor nodulation in the presence of ni may have hampered nitrogen fi xing system by causing direct toxicity on the rhizobia and/or inhibited legheamoglobin synthesis (jayakumar et al., 2008). however, better growth of v. cylindrica can be correlated with greater number of nodules that carried out more fi xation of atmospheric nitrogen leading to an increase in total protein and in turn greater biomass of the species. thus, our s. ishtiaq and s. mahmood8 8 a 0 10 20 30 40 50 60 70 80 90 100 0 50 100 150 ni l e ve l s (m g k g-1) g er m in at io n pe rc en ta ge v. cylindrica v. mungo v. radiata b 0 5 10 15 20 25 30 0 50 100 150 ni l e ve l s (mg k g-1) r oo t le ng th (c m ) v. cylindrica v. mungo v. radiata c 0 5 10 15 20 25 30 0 50 100 150 ni l e ve l s (m g k g-1) sh oo t le ng th ( cm ) v. cylindrica v. mungo v. radiata d 0 2 4 6 8 10 12 14 0 50 100 150 ni l e ve l s (m g k g-1) f re sh b io m as s/ pl an t (g ) v. cylindrica v. mungo v. radiata e 0 1 2 3 4 5 6 7 0 50 100 150 ni l e ve l s (m g k g-1) d ry b io m as s/ pl an t (g ) v. cylindrica v. mungo v. radiata f 0 5 10 15 20 25 0 50 100 150 ni l e ve l s (mg k g-1) n um be r of n od ul es v. cylindrica v. mungo v. radiata fig. 1: effect of varying levels of ni on various growth attributes: germination percentage (a), root length (b), shoot length (c), fresh biomass/plant (d), dry biomass/plant (e) and number of nodules (f) in three vigna species after 4 weeks growth. values presented are means across three replicates. vertical lines indicate + s.e. fig. 1: effect of varying levels of ni on various growth attributes: germination percentage (a), root length (b), shoot length (c), fresh biomass/plant (d), dry biomass/plant (e) and number of nodules (f) in three vigna species after 4 weeks growth. values presented are means across three replicates. vertical lines indicate + s.e. tab. 1: summary of analysis of variance (anova) for various attributes in three vigna species after 4 weeks growth under varying levels of ni. results are from two way analysis of variance, degree of freedom with df = 3 (levels), df = 2 (species), df = 6 (interaction), lsd by duncan’ s multiple range test (p < 0.05), m.s. = mean square, lsd = least signifi cant difference. *, **, ***, at 0.05, 0.01 and 0.001% level of probability respectively, n.s= non-signifi cant. dw = dry weight attributes m.s. levels signifi cant lsd m.s. species signifi cant lsd germination percentage 4963.19 *** 14.24 793.75 * 11.18 root length (cm) 184.96 n.s 0.00 217.19 n.s 0.00 shoot length (cm) 91.85 n.s 0.00 756.75 *** 6.88 fresh biomass/plant (g) 1.94 * 0.77 37 *** 0.98 dry biomass/plant (g) 0.05 n.s 0.00 0.35 * 0.31 number of leaves 4.93 n.s 0.00 9.36 n.s 0.00 leaf area (cm2) 0.06 n.s 0.00 27.38 *** 0.59 number of nodules 190.84 ** 4.72 355.08 *** 6.02 chlorophyll a (µg g-1 dw) 67790.5 *** 30.50 1024 n.s 0.00 chlorophyll b (µg g-1 dw) 67790.5 *** 25.45 1024 n.s 0.00 root ni content (mg kg-1) 0.00108 *** 0.01 0.00001 n.s 0.00 shoot ni content (mg kg-1) 0.00040 *** 0.005 0.0000021 n.s 0.00 226 s. ishtiaq, s. mahmood observations are consistent with the results obtained by chen et al., (2003). ni levels did not inhibited the leaf number and leaf area (fig. 2 a and b) in these vigna species. however, data presented in fig. 2 c and d showed a decrease in chlorophyll a and b. several leguminous species growing under ni contamination have been shown to produce more leaves despite a decrease in chlorophyll content (gopal et al., 2002; pandey and singh, 2011) thus; our results are in close agreement to the fi nding of these workers. however, decline in photosynthetic green pigments can be attributed to several possible reasons which include oxidative damage of chloroplast membranes, inhibition of chlorophyll biosynthesis and/or replacement of mg ions in a tetrapyrole rings that have been well documented in different groups of plants in response to heavy metals (nagajyothi et al., 2009; talukdar, 2011). accumulation of ni in plant tissues was concentration dependent. a steady increase in metal content in both tissues (root and shoot) was observed with an increase in ni level in the growth medium (fig. 3 a and b). the metal content of roots was considerably higher in all species as compared to shoots. at the highest ni level (150 mg kg-1), 73% of the metal uptake was carried out by the roots in v. cylindrica. however, metal transfer was considerably lower in the shoots as only 20% of the ni was transported from soil to the aerial tissue. thus, metal uptake from soil to roots was higher as compared with its transfer to the shoots. the other two species (v. mungo and v. radiata) also exhibited a similar trend for ni transport from soil to plant tissues but no marked contrast was observed between species for metal content at various levels of ni (tab. 1). hence, the capacity of species to accumulate ni in their tissues vary markedly, metal seems to be restricted in the roots and a limited transfer to the aerial tissues became evident. several other studies (reeves and baker, 2000; qurainy, 2009) also demonstrated differential transport of metals from soil to plant tissues and hyper-accumulators species can accrue far exceeding levels of metals than present in the soil. the hyper-accumulation of ni in the roots can be a manifestation of detoxifi cation mechanism which may involve binding of metal ions with cell wall, pumping of ions to vacuole or formation of non toxic complexes with specifi c metal binding protein without changing cellular metabolism and alteration of membrane structure (mahmood et al., 2007; llamas et al., 2008; qurainy, 2009) thus causing no dramatic decline in growth attributes of the species studied. several workers (li et al., 2003; madhaiyan et al., 2007) used root and shoot tolerance indices to predict the sensitivity of the tissues to stress induced by heavy metals. this study depicted that tolerance indices (tab. 2) were dependant on the concentrations of ni in both tissues (tab. 2). consistently higher tolerance indices were observed for the root tissue in the species at all levels of ni (tab. 2). v. cylindrica had the maximum root tolerance index (ti=97+0.89) followed by v. radiata (ti=89+0.74). however, v. mungo had also shown a better shoot tolerance index. these results indicated that in v. cylindrica and v. radiata the roots demonstrated less sensitive response than the shoots at elevated ni levels. a higher metal sensitivity of the shoot than the roots have also been reported in other species (srivastava et al., 2005). the sensitivity of the shoot can be attributed to higher fraction of cystolic free ni in the shoots than in the roots thus rendering the aerial tissue more sensitive to metal toxicity despite the fact that the roots accumulated more ni (galardi et al., 2007). �������������������������������������� � � � � � � �� �� �� �� � �� ��� ��� �� �� � �� � ������� ���� � � � � �� �� �� �� � � �� ������������� �������� ���������� � � � � � � � � � �� ��� ��� �� �� � �� � ����������� � �� �� � �� � �� �� � � ������������� �������� ���������� � � ��� ��� ��� ��� ��� ��� ��� ��� ��� � �� ��� ��� �� �� � �� � ����������� � � �� �� � � � �� � �� � �� �� �� � � ������������� �������� ���������� � � �� ��� ��� ��� ��� ��� ��� � �� ��� ��� �� �� � �� � ������� ���� � � �� �� � � � �� � �� � �� �� �� � � ������������� �������� ���������� ��������� �������������������������������������������������������������������������������������������������������������������������� ����������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������� ������������� fig. 2: effect of varying levels of ni on various growth attributes: number of leaves (a), leaf area (b), chlorophyll a (c) and chlorophyll b (d) in three vigna species after 4 weeks growth. values presented are means across three replicates. vertical lines indicate + s.e. dw = dry weight. phytotoxicity of ni in vigna species 227��������������������������� �� ���� � �� �� �� �� ��� ��� �� ��� ��� �� �� � �� � ������� ���� �� �� �� � �� � �� � � �� � �� ������������� �������� ���������� ����� � �� �� �� �� ��� ��� �� ��� ��� �� �� � �� � ����������� �� � �� �� � �� � �� � � �� � �� ������������� �������� ���������� ���������������������������������������������������������������������������������������������������� ������������������������������������������������������������������������������������ ������������� fig. 3: bioaccumulation of metal in tissues (root and shoot) of three vigna species. values presented are means across three replicates. vertical lines indicate + s.e. tab. 2: tissue tolerance indices (%) of three vigna species after 4 weeks growth under varying levels of ni. values presented are means across three replicates with + s.e. species root tolerance index (%) shoot tolerance index (%) ni levels (mg kg-1) 50 100 150 50 100 150 v. cylindrica 95 + 0.95 97 + 0.89 89 + 0.91 85 + 0.82 73 + 0.94 61 + 0.52 v. mungo 85 + 0.45 73 + 0.58 76 + 0.26 83 + 0.46 83 + 0.64 77 + 0.96 v. radiata 83 + 0.45 85 + 0.69 89 + 0.74 77 + 0.69 66 + 0.46 53 + 0.55 the study demonstrated that vegetative growth parameters in vigna species did not infl uence by increasing ni concentrations in the soil. however, chlorophyll content and nodulation appeared to be more susceptible in the studied vigna species in response to ni contamination. among the species, v. cylindrica had shown better germination and growth responses, less sensitivity of chlorophyll molecules and nodule formation. a greater root tolerance index, better threshold of the roots to accumulate appreciable amounts of ni along with restricted transfer of metal to the aerial tissue may signify the species as metal excluder. thus, v. cylindrica appeared to possess an integrated mechanism to cope with metal toxicity and can be a potential choice for ni contaminated soils. references ahmed, a., hasnain, a., akhtar, s., hussain, a., abaidullah, yasin, g., wahid, a., mahmood, s., 2010: antioxidant enzymes as biomarkers for copper tolerance in saffl ower (carthamus tinctorius l.). afr. j. biotechnol 9, 5441-5444. allen, s.e., grimshaw, h.m., rowland, a.p., 1986: chemical analysis. in: moor, p.d., champan, s.b. 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(eds.), phytoremediation of toxic metals, 193-229. johnwiley & sons, new york, usa. seregin, i.v., kozhevnikova, a.d., 2006: physiological role of nickel and its toxic effects on higher plants. russ. j. plant physiol. 53, 257-277. singh, p.p., manish, m., singh, j., 2006: impact of fertilizer factory effl uent on seed germination, seedling growth and chlorophyll content of gram (cicer arietinum). j. environ. biol. 27, 153-156. srivastava, s., mishra, s., dwivedi, s., beghel, v.s., verma, s., tandon, p.k., rai, u.n., tripathi, r.d., 2005: nickel phytoremediation potential of broad bean, vicia faba l. and its biochemical responses. bull. environ. contam. toxicol. 74, 715-724. talukdar, d., 2011: effect of arsenic-induced toxicity on morphological traits of trigonella foenum-graecum l. and lathyrus sativus l. during germination and early seedling growth. curr. res. j. biol. sci. 3, 116123. vijayarengan, p., 2000: growth, nodulation and dry matter yield of blackgram cultivars under nickel stress. j. environ. sci. eng. 46, 151158. wilkins, d.a., 1978: the measurement of tolerance to edaphic factors by means of root growth. new phytol. 91, 255-261. address of the author: shabnam ishtiaq, ph. d. scholar, institute of pure and applied biology, botany division, bahauddin zakariya university multan. pakistan p.c. 60800 dr. seema mahmood, professor of botany, institute of pure and applied biology, botany division, bahauddin zakariya university multan. pakistan p.c. 60800, e-mail: drseemapk@gmail.com. journal of applied botany and food quality 92, 320 326 (2019), doi:10.5073/jabfq.2019.092.043 1college of new energy and environment, jilin university, changchun, china 2department of environmental management, kaduna state university, kaduna state, nigeria 3department of biological sciences, bayero university kano, kano state, nigeria mini-review on the efficacy of aquatic macrophytes as mosquito larvicide adamu yunusa ugya1,2, tijjani sabiu imam3, jincai ma1* (submitted: june 12, 2019; accepted: september 25, 2019) * corresponding author summary malaria is a mosquito-borne disease, which is endemic in asia, africa and latin america. vector control is the current strategy used for the eradication and elimination of malaria in these countries, but this control method has not proven to be effective, as malaria continues its increasing trend. although chemical larvicide can also be used to eradicate the malaria vector at the larval stage, preventing the growth of mosquitoes into hematophagous adults, the continuous use of chemical insecticides leads to environmental pollution. it is therefore of paramount importance to identify effective, low-cost, biodegradable and environmentally friendly alternatives to chemical insecticides for the control of mosquito larvae. this mini-review aims to assess the present and future of the use of macrophytes as a mosquito larvicide. we critically analyze the trend of malaria cases in sub-saharan africa and evaluate why botanical larvicides may contribute to the eradication of malaria in the region. the ecological role of macrophytes in the aquatic en vironment and their potential as botanical larvicide are explained in detail. the study illustrates that the macrophytes azolla pinnata, pistia stratiotes, eicchornia crassipes, phragmites australis, nelumbo nucifera, nymphaea lotus, typha latifolia and leucas martinicensis have been effectively used as larvicides against mosquito larvae. it is recommended that additional work be done to purify the biologically active components that are responsible for the larvicidal activity of these macrophytes, and future research should assess the potential of other macrophytes for effective utilization as larvicides. keywords: pistia stratiotes, mosquito larvae, plasmodium falciparum, aquatic ecosystem, anopheles introduction the invasive nature of macrophytes is problematic because they tend to dominate aquatic ecosystems to the detriment of the environment, economy and human health (lamb et al., 2016; ugya, 2015). macrophytes left uncontrolled can cover the entire surface of water bodies, hindering water flow and sunlight penetration, which is detrimental to flora and fauna in the habitat. different control methods have been used against these plants in the past, but recent research has focused on how to utilize the benefits associated with them (chen et al., 2012; hanks et al., 2015; lareo, 1981). these macrophytes largely originate from malaria-endemic places and are regarded as invasive plants that impede economic and ecological improvement (ugya et al., 2019b). a number of researches have shown how these macro phytes can be utilized for wastewater treatment while others have shown how these macrophytes can be used as a botanical larvicide against malaria (ugya et al., 2015a; ugya et al., 2016; ma et al., 2019). malaria is a mosquito-borne disease which is endemic in asia, africa and latin america. this disease has killed approximately 1.5-3 million people and infected 300-500 million (scholte et al., 2003). many affected countries have developed malaria control programs aimed at reducing the transmission of malaria to a level that is no longer a public health problem (deletre et al., 2019). despite this effort, the progress made in malaria elimination and eradication has been minimal due to a lack of public health infrastructure and poor socioeconomic conditions in many affected countries. in addition, the malaria parasite has developed resistance to some anti-malaria drugs making it very difficult to develop an effective vaccine (mwaiswelo et al., 2019; silva et al., 2019). vector control is the current strategy used for the eradication and elimination of malaria. these control measures include the use of insecticide-treated nets and indoor residual spraying. these methods of malaria vector control have not proven to be effective, as malaria continues its increasing trend in endemic countries (baia-da-silva et al., 2019; deletre et al., 2019; mastrantonio et al., 2019). the use of larvicides is critical in malaria vector control, killing mosquitoes in the juvenile stage and preventing their growth into hematophagous adults. larvicides provide an effective control measure because at the larval stage mosquitoes are bound to aquatic habitat, making control strategies and application easier and more efficient (unlu et al., 2019). however, heavy use of chemical insecticides in the control of mosquito larvae has led to environmental pollution which is detrimental to human health. it is therefore paramount to identify effective, low-cost, biodegradable and environmentally friendly alternatives to chemical insecticide for the control of mosquito larvae (pandiyan et al., 2019; pavela et al., 2019; weeks et al., 2019). numerous studies have demonstrated the efficiency of different plants used as larvicides, repellants and ovideterrents with minimal environmental impact (hari and mathew, 2018). macrophytes can be utilized for the production of botanical insecticide, such as larvicides, repellants or ovideterrents, since the plants are abundant due to their fast growing rate (turnipseed et al., 2018). this mini-review aims to assess the present and future use of macrophytes as larvicide against mosquitoes. malaria in sub-saharan africa and the importance of botanical larvicide malaria is a deadly mosquito-borne disease that is caused by a parasite belonging to the genus plasmodium. there are five known species of plasmodium that cause malaria in humans, namely falciparum, vivax, ovale, malariae and knowlesi (walker et al., 2014). the disease is endemic in 87 countries and in 2017 ledto the death of 435.000 people, out of which 404.550 cases are reported to have come from sub-saharan africa (who, 2018). who and the governments of endemic countries have spent an estimated 4 billion usd for malaria control and eradication, but it is unlikely that the goals of the who global technical strategy for malaria 2016-2030 will be achieved based on the trend of death cases shown in fig 1. despite the large sum invested in the program, tanzania has recorded no larvicidal efficacy of aquatic macrophytes 321 decrease in deaths resulting from malaria, while angola, benin and kenya recorded an increase in death cases from 2010 to 2017. the death cases reduction trend is insignificant in countries like cameroon, congo, dr congo, mali, niger, mozambique and sierra leone (fig. 1). countries such as burkina faso, uganda, central africa republic, chad, coted’ivoir, ethiopia, equatorial guinea, ghana and nigeria report a significant reduction in death cases initially, from 2010 to 2012, but from 2014 to 2017, the progress was minimal. vector control has been shown to be the most effective strategy for reducing malaria transmission (pimenta et al., 2015). the two forms of vector control employed in most sub-saharan countries are the use of insecticide-treated mosquito nets and indoor spraying of insecticide. these vector control methods may be effective in the reduction of malaria, but complete eradication will be virtually impossible because the malaria vector is developing resistance genes to insecticide, and the continuous application of insecticides tends to have adverse environmental effects (fullman et al., 2013; n’guessan et al., 2007). the use of larvicides to eradicate mosquito at juvenile stage is signi ficant because mosquito at this stage are confined to particular places of the environment. several synthetic chemical agents have been shown to be effective in killing of mosquito larval but the adverse effect associated with the fate of the pollutants associated with the use of synthetic chemical agent is worrisome. recent studies have focus on how to replace the use of synthetic chemical agents with a more ecological friendly method of controlling mosquito at the larval stage (pandiyan et al., 2019; pavela et al., 2019; weeks et al., 2019). the use of plant material as an alternative larvicide to synthetic chemical agent is promising, effective, easily accessible and environmentally friendly. past research (pavela, 2015) has summarized findings regarding the potential of plant phytochemicals, including steroids, alkaloids, terpenes and phenolic constituents, for use as larvicides. the action of botanical insecticides depends on the plant species, parts extracted, collection site and solvent used for extraction (benelli et al., 2015; stevenson et al., 2017). ecological role of macrophytes in the aquatic environment plants that are able to survive in or around water bodies are referred to as aquatic macrophytes (osti et al., 2018). macrophytes are classified into four major groups, namely emergent (e.g., typha angustifolia), floating-leaved (e.g., nymphaea lotus), submerged (e.g., ceratophyllum submersum) and free-floating (e.g., pistia stratiotes) (pulzatto et al., 2018; ugya et al., 2015b). these plants play a vital role in aquatic ecosystems due to their ability to utilize dissolved nutrients such as nitrogen and phosphorus for metabolic activities (hill, 1979; ugya et al., 2019a). utilizing dissolved nutrients prevents algal blooms and the subsequent death of fish and aquatic mammals that may result due to the toxicity of microcystin produced by toxic algae (chia et al., 2019; turner et al., 2018). macrophytes also indirectly aid the efficiency of nutrient cycling in aquatic environments. they are able to retain solids in their roots and shoots, and their presence enhances suspended solid sedimentation by protecting against wind and waves, thereby reducing current velocity and the rate of erosion (thomaz and cunha, 2010; zhu et al., 2015). macrophytes indirectly enhance the cycling of nitrogen due to the presence of denitrifying bacteria inhabiting their roots and shoots. these denitrifying bacteria aid in the conversion of nitrate in the water into atmospheric nitrogen, thereby depleting the fertility of the water and limiting the growth of toxic algae (hallin et al., 2015). (horppila et al., 2013) demonstrate that macrophytes (phragmites australis) are associated with spatial heterogeneity in aquatic ecosystems. the uneven distribution of species within an aquatic ecosystem resulting from the presence of macrophytes increases the richness of aquatic organisms because spatial heterogeneity enables species to coexist with minimal competition (wang et al., 2016). macrophytes also provide shelter, breeding sites and sources of food for aquatic macro organisms (o’hare et al., 2018). the potential and efficacy of macrophytes as botanical larvicide the use of macrophytes as a tool for the control of mosquito larvae in developing countries is promising. as summarized in tab. 1 and fig. 1: trend of malaria disease in sub-saharan africa 322 a.y. ugya, t.s. imam, j. ma tab. 3, a number of studies have demonstrated the efficacy of macrophytes as mosquito larvicide. the potential of macrophytes has been linked to the presence of phytochemicals (ma et al., 2019), which are bioactive compounds present in plant parts that have long been shown to possess insecticide potential (ghosh et al., 2012). phytochemicals extracted from different plants species have long been used in mosquito control (isman, 1997). however, the discovery of ddt in 1939 overshadowed the use of phytochemical extracts for mosquito control (berenbaum, 1985). macrophytes produce numerous phytochemicals that possess medi cinal and pesticidal properties. a large number of macrophytes have been shown to produce phytochemicals, such as flavonoids, tannins, saponins, alkaloids, resin, glycosides, phenol and phlobatannin, that cause high mortality when used as larvicides against mosquito larvae (ma et al., 2019). flavonoids, also referred to as bioflavonoids, are secondary metabolites that have been shown by researchers such as (huang et al., 2015) to be present in macrophytes. they are produced by the phenylpropanoid metabolic pathway whereby phenylalanine is converted into 4-coumaroyl-coa, which combines with malonyl coa to produce chalcones (a backbone of flavonoids containing two rings). the pathway is then subjected to series of enzymatic actions by anthocyanidin reductase, chalcone isomerase, dihydrokaempfe rol-4-reductase, flavones synthase and others, leading to the production of products such as flavonones, dihydroflavonol, anthocyanins, flavan-3-ols, and proanthocyanidins (gutha et al., 2010; ververidis et al., 2007). anthocyanins, flavonones and dihydroflavonols are the major members of the flavonoids family, and they play a crucial role in the larvicidal potential of macrophytes (batra and sharma, 2013). anthocyanins have been shown (kong et al., 2003) to be produce in response to biotic and abiotic stresses. the strong antioxidant potential of macrophytes’ anthocyanin is due to the presence of positively charged oxygen atoms (sadilova et al., 2006). flavonones are produced as a result of the action of chalcone isomerase on chalcone-like compounds (falcone ferreyra et al., 2012; petrussa et al., 2013), while dihydroflavonols are produced as a result of the action of flavonone-3-dioxygenase, flavonol synthase and dihydroflavonol-4-reductase (tian et al., 2015). the high mortality of mosquito larvae resulting from exposure to extracts of macrophyte leaves recorded by most research is attributed to the larvae feeding on high concentrations of anthocyanin, flavonones and dihydroflavonols (wang et al., 2013). tannins are another phytochemical that have been found in high concentration in macrophytes (yakubu et al., 2017). they are produced in the tannose, which is a chloroplast-derived organelle (brillouet et al., 2013; hillis, 1958) located in the vacuole or surface wax of plants (brillouet et al., 2013). these phytochemicals have a high level of astringency, which could be the reason most plant extracts containing high levels of tannin are highly effective as larvicides tab. 1: macrophytes with larvidal potential sn name of macrophyte mosquito larvae references 1 azolla pinnata aedes aegypti (husna zulkrnin et al., 2018) 2 pistia stratiotes anopheles spp. (ma et al., 2019) 3 eicchornia crassipes culex quinquefasciatus say (jayanthi et al., 2012) 4 phragmites australis culex pipiens (bream et al., 2009) 5 nelumbo nucifera anopheles stephensi (anushree et al., 2014) 6 nymphaea lotus anopheles spp. (yakubu et al., 2017) 7 typha latifolia anopheles spp. (imam and tajuddeen, 2013) 8 leucas martinicensis anopheles spp. (imam and tajuddeen, 2013) tab. 2: characterization of phytochemicals present in different leaves extract of macrophytes sn macrophytes acetone ethanol methanol n-butyl references 1 eicchornia crassipes alkaloid, tannin and phenol, terpenoids, flavonoids, phenol, flavonoids, phenol, (haggag et al., 2017; terpenoids sterols tannins, terpenoids tannins, terpenoids tulika and mala, 2017a) 2 pistia stratiotes alkaloids, flavonoids, steroids, alkaloids, tannins, sterols, (ma et al., 2019; glycosides, terpenoids, flavonoids terpenoids, alkaloids, tulika and mala, photobatannins flavonoids 2017b) 3 potamogeton specie phenol, flavonoids, phenol, flavonoids, (paul et al., 2015; tannins, terpenoids, tannins qais et al., 1998) alkaloids 4 nymphaea species phenol, flavonoids, flavonoids, tannins, phenol, flavonoids, phenols, flavonoids, (afolayan et al., 2013; tannins, saponins, saponins, tarpenoids, tannins, saponins, tannins, glycosides, parimala and shoba, alkaloids, steroids photobatannins, resin alkaloids, steroids saponins, alkaloids, 2013; yakubu et al., steroids 2017) 5 typha latifolia tannins, steroids, photobatannins, resin, alkaloids, tannins, (imam and tajuddeen, saponins, alkaloids, alkaloids, steroids, steroids, phenols, 2013; ramesh et al., glycosides tannins saponins, flavonoids 2013; wangila, 2017) 6 leucas martinicensis flavonoids, tannins, (imam and tajuddeen, steroids, photobatannins, 2013) resin 7 azolla pinnata phenol, steroids phenols, flavonoids, phenol, flavonoids, (thiripurasundari saponins, steroids tannin, saponins and padmini, 2018) larvicidal efficacy of aquatic macrophytes 323 (soares et al., 2012). the efficacy of phytochemicals extracted from macrophytes against mosquito larvae is dependent on the part of macrophyte used, the growth stage of the macrophyte and the solvents used during the preparation of the macrophyte extract, as shown in tab. 2 (daboor and haroon, 2012; haroon and abdelaal, 2016). saponin is a chemical compound that has been identified by (yakubu et al., 2017) as present in macrophytes. the role of this phytochemical is to protect the macrophyte against predators and parasites due to it bitter taste and amphipathic nature. the amphipathic nature of saponin also contributes to the larvicidal potential of macrophyte extracts (lorent et al., 2014). alkaloids are another bioactive compound found in the extract of macrophytes, as shown by (ma et al., 2019). these bioactive compounds contain mostly nitrogen, although other elements such as oxygen, sulphur, chlorine, bromine and phosphorus may sometimes be present in the crude extract of alkaloids (franck, 1967). the extract of alkaloid is an important pharmacological tool used for its antimalarial, anticancer and antibacterial properties (cushnie et al., 2014). alkaloid salts such as nicotine and anabasine have been wide ly used as pesticides, although the high human toxicity associated with these salts discourages their use (duke et al., 2010). the pharmacological activities associated with alkaloids result from alkaloids’ tendency to attack pathogens and prevent them from infecting human hosts. this high toxicity to pathogens and insects could explain why most macrophyte extracts with high or low concentration of alkaloids tend to be associated with high mosquito larvae mortality. resin is another bioactive compound shown by many researchers to be present in extracts of aquatic macrophytes. resin also contributes to the larvicidal potential of extracts due to the presence of terpenes and resin acid, which protect plants against pathogens and insects (klemens and dieter, 2000). the protective role of resin is also part of the reason why macrophyte extracts are highly effective as mosquito larvicide. conclusion many control and prevention measures have been employed to manage the nuisance caused by mosquitoes. inorganic pest control has remained the most effective tool, although it tends to have adverse side effects on humans and other living organisms in the environment. this mini-review demonstrates that extracts of macrophytes are active against mosquito larvae, and hence, these macrophytes could be an effective mosquito larvicide as they have also been found harmless to other aquatic organisms. additional work should be done to further purify the biologically active components that are responsible for the larvicidal activity of these macrophytes, and more research should be done with respect to the potential of other macrophytes for effective utilization and eradication of the malaria vector. references afolayan, a., sharaibi, o., kazeem, m., 2013: phytochemical analysis and in vitro antioxidant activity of nymphaea lotus l. int. j. pharmacol. 9, 297-304. doi: 10.3923/ijp.2013.297.304 anushree, s.r., kuntal, b., aniket, s., goutam, c., 2014: larvicidal activity of nelumbo nucifera gaertn. (nymphaeaceae) against anophe les stephensi (liston 1901) and its effect on non-target organisms. j. mosq. res. doi: 10.5376/jmr.2014.04.0010 baia-da-silva, d.c., brito-sousa, j.d., rodovalho, s.r., peterka, c., moresco, g., lapouble, o.m.m., melo, g.c.d., sampaio, v.d.s., alecrim, m.d.g.c., pimenta, p., lima, j.b.p., lacerda, m.v.g.d., monteiro, w.m., 2019: current vector control challenges in the fight against malaria in brazil. rev. soc. bras. med. trop. 52, e20180542e20180542. doi: 10.1590/0037-8682-0542-2018 batra, p., sharma, a.k., 2013: anti-cancer potential of flavonoids: recent trends and future perspectives. biotech. 3, 439-459. doi: 10.1007/s13205-013-0117-5 benelli, g., murugan, k., panneerselvam, c., madhiyazhagan, p., conti, b., nicoletti, m., 2015: old ingredients for a new recipe? neem cake, a low-cost botanical by-product in the fight against mosquito-borne diseases. parasitol. res. 114, 391-397. doi: 10.1007/s00436-014-4286-x berenbaum, m., 1985: brementown revisited: interactions among allelochemicals in plants. in: cooper-driver, g.a., swain, t., conn, e.e. 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aquatic macrophytes leaves extracts sn macrophytes extract lc50 (ppm) references 1 pistia stratiotes ethanol 63.3 (imam and tajuddeen, 2013) 2 typha latifolia ethanol 68.1 (imam and tajuddeen, 2013) 3 pistia stratiotes ethyl acetate 14.80 (ma et al., 2019) 4 azolla pinnata acetone 1072 (ravi et al., 2018) 5 eicchornia crassipes petroleum ether 71.43 (jayanthi et al., 2012) 6 azolla pinnata 1267 (husna zulkrnin et al., 2018) 7 phragmites australis petroleum ether 60.06 (bream et al., 2009) 8 phragmites australis ethanol 98.72 (bream et al., 2009) 9 nymphaea lotus ethanol 62.8 (yakubu et al., 2017) 10 azolla pinnata methanol 867 (ravi et al., 2018) 11 eicchornia crassipes ethyl acetate 94.68 (jayanthi et al., 2012) 12 eicchornia crassipes ethanol 152.15 (jayanthi et al., 2012) 13 eicchornia crassipes methanol 173.35 (jayanthi et al., 2012) http://dx.doi.org/10.3923/ijp.2013.297.304 http://dx.doi.org/10.5376/jmr.2014.04.0010 http://dx.doi.org/10.1590/0037-8682-0542-2018 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a shallow and large lake (lake taihu, china). plos one 10, e0127915-e0127915. doi: 10.1371/journal.pone.0127915 orcid adamu yunusa ugya https://orcid.org/0000-0002-1490-4479 tijjani sabiu imam https://orcid.org/0000-0002-1926-3076 ma jincai https://orcid.org/0000-0002-0792-0251 address of the corresponding author: jincai ma, college of new energy and environment, jilin university changchun, 130012, china e-mail: jincaima@jlu.edu.cn © the author(s) 2019. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1186/1472-6882-14-397 http://dx.doi.org/10.1002/ps.5118 http://dx.doi.org/10.4314/bajopas.v10i1.91s http://dx.doi.org/10.1371/journal.pone.0127915 https://orcid.org/0000-0002-1490-4479 https://orcid.org/0000-0002-1926-3076 https://orcid.org/0000-0002-0792-0251 art_16353.indd journal of applied botany and food quality 94, 206 212 (2021), doi:10.5073/jabfq.2021.094.025 1julius kühn institute – federal research centre for cultivated plants, institute for breeding research on horticultural crops, quedlinburg, germany 2 kühne jungpflanzen, claus kühne, dresden, germany 3 department of horticultural plant systems, faculty of life sciences, humboldt university berlin, germany spontaneous polyploidisation of interspecific and intersectional pelargonium hybrids during embryo rescue plaschil, s.1*, budahn, h.1, klocke, e.1, wiedemann, m.2, olbricht, k.3 (submitted: august 17, 2021; accepted: november 15, 2021) * corresponding author summary modern pelargonium crispum hybrids (section pelargonium) show low genetic and phenotypic variation due to the domestication effect. species of the sections cortusina, ligularia, and pelargonium are potential breeding partners at the diploid level (2n = 2x = 22). five p. × crispum cultivars were used as seed parents and pollinated with one genotype of p. grandiflorum  (section pelargonium) and three genotypes of p. fulgidum (section ligularia). in both combinations, embryo rescue was necessary. embryos were rescued and cultured on murashige & skoog medium supplemented with phytohormones. after callus and adventitious shoot regeneration 15 viable inter specific hybrids were obtained from crossbreeding with p. grandi florum and 11 intersectional hybrids from crossings with p. fulgidum, respectively. the hybrids were cultivated in the greenhouse until flowering. their hybrid character was evident due to the inter mediate morphological traits. molecular investigations using dp rapd analysis confirmed this. within the f1 population p. × crispum with p. grandiflorum three hybrids and after crossing with p. fulgidum one hybrid possessed larger flowers and fully developed anthers, respectively. their ploidy level was confirmed as tetraploid using flow cytometry. therefore, a spontaneous polyploidisation occurred during in vitro regeneration. the tetraploid f1 hybrids are fertile and could be used for further breeding. key words: genome doubling, flow cytometry, ligularia, pelar gonium crispum, pelargonium fulgidum, pelargonium grandiflorum, ploidy level, somaclonal variation introduction angel or pansy pelargoniums (p. crispum (p.j. bergius) l’hér. hybrids) (section pelargonium (dc.) harv.) are popular ornamental plants already for centuries. their percentage on the production of bedding plants increases continuously. in the 1920s, the documented breeding of angel pelargoniums started in england done by langley-smith (brawner, 2003). however, it is believed that the interspecific hybridisation with p. crispum began much earlier soon after the import of different pelargonium species to europe during the 17th century (brawner, 2003). modern p. crispum hybrids, which should be referred as p. × crispum (olbricht, 2013), show low genetic (plaschil et al., 2017) and phenotypic variation due to crossings within a very narrow gene pool during domestication (brawner, 2003). increase of genetic variability by crosses with wild species could overcome this genetic bottleneck (plaschil et al., 2012, 2015; olbricht, 2013). the genus pelargonium l’hér. with its about 280 species (bakker et al., 1998, 2004, 2005; weng et al., 2012) in 16 sections (bakker et al., 1999a, b, 2000, 2004, 2005; röschenbleck et al., 2014) comprises species of different sections such as cortusina (dc.) harv., ligularia (sweet) harv. and pelargonium, which are potential breeding partners for p. × crispum at the diploid level (2n = 2x = 22). a study of genetic distances of several species of the working collection at the julius kühn institute, quedlinburg provides additional valuable information on potential crossing partners (plaschil et al., 2017). natural diploid hybrids of the section pelargonium are known, e.g. p. cucullatum (l.) l’hér. × p. betulinum (l.) l’hér. and p. scabrum (l.) l’hér. × different diploid species (albers and van der walt, 1984; van der walt et al., 1990; bakker et al., 2004, 2005). in addition, experimental hybrids were created several times. thus p. grandiflorum (andr.) willd. was used as pollen parent for crosses with p. fruticosum (cav.) willd, p. crispum and p. cucullatum and as seed parent for crosses with p. cucullatum, p. glutinosum (jacq.) l’hér., and p. cordifolium (cav.) curtis (horn, 1994). yu (1985) successfully hybridised the species p. crispum with p. grandiflorum and olbricht (2013) p. grandiflorum with p. × crispum, respectively. pelargonium fulgidum (l.) l’hér. (section ligularia), with its very attractive bright red flower colour, was one of the most popular species used for crossings in the early 19th century (albers et al., 1992). together with p. × domesticum l.h. bailey, p. fulgidum is reputed to be an ancestor of the cultivar group uniques, but the pedigrees are not well documented (brawner, 2003). to a small extent uniques are propagated for satisfying the wishes of few enthusiasts in europe and america. however, the extraordinary flower colour of p. fulgidum is still missing in modern p. × crispum assortments. based on these facts one genotype of p.  grandiflorum  (section pelargonium) and three genotypes of p. fulgidum were chosen as pollen donors for crossings with p. × crispum cultivars to enhance the genetic variability. materials and methods plant material five p. × crispum cultivars were used as seed parents (tab. 1). pelargonium grandiflorum and three genotypes of p. fulgidum (11, 123, 48) were the pollen donors. all genotypes, maintained as a clone with at least three plants, were cultivated under greenhouse conditions at the julius kühn institute. the internal standards for flow cytometric investigations, radish (raphanus sativus l.), tomato (solanum lycopersicum l.) ‘stupické’ (doležel et al., 1992) and cauliflower (brassica oleracea l. subsp. capitata convar. botrytis var. botrytis l.) ‘korso’ (plaschil et al., 2020) were cultivated in vitro on murashige and skoog (ms) medium (1962) with 1.07 μm 1-naphtalene acetic acid at 25 °c at 16 h photoperiod. cross combinations and embryo rescue each p. × crispum cultivar was crossed with each pollen donor (tab. 1). for that, flower buds of the seed parents were emasculated polyploidisation of pelargonium hybrids 207 and isolated. the stigmas, when ripen, were pollinated with pollen from at least one anther of the pollen donor. reciprocal crosses were not carried out, because of the low flower number of p. grandiflorum and p. fulgidum. fourteen to thirty-one days after pollination unripe fruits were collected and surface sterilised in a sodium hypochlorite solution (3% active chlorine) (carl roth, karlsruhe, germany) followed by threefold rinsing with autoclaved distilled water. embryos were dissected under sterile conditions and cultivated on ms medium (duchefa, haarlem, the netherlands) supplemented with 2.28 μm zeatin (duchefa), 1.14 μm indole-3-acetic acid (duchefa), and 1.44 mm gibberellic acid 3 (duchefa) in petri dishes (ø 6 cm) at 24 °c in the dark. one week later, a light exposure of 16 h (108 μmol/m-2s-1, provided by cool white fluorescent lamps, philips f17t8/tl741 alto, 17 w, u.s.a.) alternated by 8 h darkness per day was applied. when the cultivated embryos formed adventitious shoots, the shoots were separated, multiplied, and rooted on ms without phytohormones. rooted microplants were transferred into fruhstorfer® sowing and cutting substrate and later potted in a mixture of stender® substrate c420 and sand (1:1). firstly, they were cultivated in a climate chamber. after hardening cultivation took place in the greenhouse until flowering. molecular hybrid identification total dna was isolated using the protocol of porebski et al. (1997) with some modifications: 100 mg fresh leaf material were disrupted in a 1.5 ml tube with 400 μl preheated extraction buffer by a stainless steel grinding ball using a mixer mill mm300 (retsch, haan, germany) two times for 5 minutes with a frequency of 30 s-1. finally, the dna was resuspended in 50 μl te buffer. concentration and purity of the dna were spectrophotometrically determined to adjust all samples to 10 ng μl-1 dna. rapd analysis was performed according to williams et al. (1990) but using a mixture of two de camer primers (dprapd, carl roth, karlsruhe, germany) (budahn et al., 2009) to get more and smaller dna fragments. the amplification products were separated on 1% agarose gels and stained with ethidium bromide solution. size determination was realised by comparison to a 100 bp dna ladder (fischer scientific, carlsbad, ca, usa). flow cytometric and statistical analysis three to seven biological replications of each genotype were analysed with one or two of the three internal standards. radish (2c = 1.11 pg; doležel et al., 1992), tomato ‘stupické’ (2c = 1.96 pg; doležel et al., 1992) and cauliflower ‘korso’ (2c = 1.31 pg; plaschil et al., 2020) were used as internal standards to estimate the dna content and ploidy level of the investigated genotypes. sample preparation, calculation of the 2c values and data analysis were performed as described by plaschil et al. (2020). results fertilisation, embryo rescue and plant regeneration regarding the pollen donor p. grandiflorum 77 flowers of the five p. × crispum cultivars, which were pollinated, resulted in 45 fruits and 88 embryos. between four flowers (‘harlekin’) and twenty-seven flowers (‘soft pink’) were pollinated per cultivar. within the crossing group p. × crispum × p. fulgidum, 319 flowers were pollinated, 173 fruits were harvested and 227 embryos rescued. per cross combination between five flowers (‘harlekin’ × p. fulgidum 11) and 41 flowers (‘merlot’ × p. fulgidum 123) were pollinated. the detailed results of all twenty cross combinations are shown in tab. 2 and 3, respectively. the in vivo embryo development was insufficient so that the hybrid embryos started to abort about 14 days after pollination. at that time, we start to take the embryos in vitro. after a successful fertilisation, 1 3 embryos could be dissected per fruit. the hybrid pelargonium embryos underwent in vitro a callus stage with soft yellow callus followed by formation of adventitious shoots eight to twelve weeks later. on average, 17% of rescued embryos from the crossings p. × crispum × p. grandiflorum and 5% from p. × crispum × p. fulgidum resulted in viable plants, respectively. in both crossing groups the percentage of fruit set per pollinated flowers was nearly similar (58.4% versus 54.2%). nevertheless the cross combinations have a strong influence on the embryo develop ment (tab. 2, 3). a difference in the number of developed embryos per fruits was noticed. whereas in the cross combination p. × crispum × p. grandiflorum predominantly more than one embryo could be dissected this was rarely the case in the crossing group with p. fulgidum. the further development of the embryos was sigtab. 1: pelargonium genotypes used for crossing seed parents (short names) pollen donors* p. × crispum ‘piccola™ harlekin’ (‘harlekin’) p. grandiflorum (deu648pelar038) p. × crispum ‘piccola™ merlot’ (‘merlot’) p. fulgidum 11 (deu648pelar034) p. × crispum ‘piccola™ soft pink’ (‘soft pink’) p. fulgidum 123 (deu648pelar036) p. × crispum ‘piccola™ pink picotee’ (‘pink picotee’) p. fulgidum 48 (deu648pelar035) p. × crispum ‘angeleyes® randy’ (‘randy’) * https://www.bundessortenamt.de/apps6/genbank_zierpfl/public/de tab. 2: regeneration success of hybrids after embryo rescue from crossings p. × crispum × p. grandiflorum explants with in vitro greenhouse adventitious shoots microplants plants pollinated number of rescued seed parents flowers fruits embryos total %1 total %1 total %1 ‘harlekin’ 4 3 7 4 57 4 57 4 57 ‘merlot’ 23 6 9 0 0 0 0 0 0 ‘soft pink’ 27 22 42 14 33 12 29 8 19 ‘pink picotee’ 10 6 9 3 33 2 22 1 11 ‘randy’ 13 8 21 9 43 5 24 2 10 crossing group σ 77 45 88 30 34 23 26 15 17 1percentage relating to the number of cultivated embryos, rounded to whole numbers 208 plaschil, s., budahn, h., klocke, e., wiedemann, m., olbricht, k. nificantly better in the cross combination with p. grandiflorum. concerning the fertilisation and regeneration success, there were also big differences according to the cross combination. in the crossing group p. × crispum × p. grandiflorum, ‘harlekin’ and ‘soft pink’ were the best seed parents. seventy-five percent of the pollinated flowers of ‘harlekin’ and 81.5% of ‘soft pink’ showed fruits and cultivable embryos giving four and eight viable plants in the greenhouse. on the other hand, with ‘merlot’, despite the fruit set, embryo development during in vitro culture was completely absent. in the crossing group with p. fulgidum embryos with seed parents ‘soft pink’ and ‘pink picotee’ did not grow further. in the combination ‘harlekin’ × 48 one rescued embryo could be cultivated to a microplant but died later on. only five of fifteen combinations exclusively with the seed parents ‘merlot’ and ‘randy‘ resulted in viable f1 hybrids. regarding the three pollen donors, the highest number of plants originated from p. fulgidum 123 (seven f1 hybrids what corresponds to 9.6% of pollinated flowers with ‘merlot’ and ‘randy‘) followed by p. fulgidum 48 (three f1 hybrids, 5.4% of the pollinated flowers) and p. fulgidum 11 (one f1 hybrid, 1.5% of the pollinated flowers). hybrid identification and characters for all regenerated hybrids at least three clone plants were cultivated in the greenhouse until flowering. their hybrid character was evident because of the intermediate morphological traits in flower, leaf, and habit (fig. 1 and 2). f1 hybrids of p. × crispum × p. grandiflorum had single rose flowers of intermediate size with fully developed anthers. they always possessed darker marks at the upper petals like p. grandiflorum and in some cases additional marks at the lower petals. the intermediate sized leaves were different lobated, the typical zone from the pollen donor p. grandiflorum was lacking (fig. 1). longitudinal growth was also intermediate. the plants were clearly tab. 3: regeneration success of hybrids after embryo rescue from crossings p. × crispum × p. fulgidum 11, 123 and 48 explants with in vitro greenhouse adventitious shoots microplants plants pollinated number of rescued crossings flowers fruits embryos total (%)1 total (%)1 total (%)1 ‘harlekin’ × 11 6 6 6 0 0 0 0 0 0 ‘merlot’ × 11 40 18 28 2 7 2 7 0 0 ‘soft pink’ × 11 23 10 6 0 0 0 0 0 0 ‘pink picotee’ × 11 17 11 5 0 0 0 0 0 0 ‘randy × 11 28 22 31 2 6 1 3 1 3 pollen donor 11 σ 114 67 76 4 5 3 4 1 1 ‘harlekin’ × 123 5 3 1 0 0 0 0 0 0 ‘merlot’ × 123 41 15 24 7 29 7 29 5 21 ‘soft pink’ × 123 16 10 10 0 0 0 0 0 0 ‘pink picotee’ × 123 15 7 8 0 0 0 0 0 0 ‘randy’ × 123 32 25 33 2 6 2 6 2 6 pollen donor 123 σ 109 62 80 9 11 9 11 7 9 ‘harlekin’ × 48 6 4 5 1 20 1 20 0 0 ‘merlot’ × 48 36 10 25 5 20 4 16 1 4 ‘soft pink’ × 48 19 11 20 0 0 0 0 0 0 ‘pink picotee’ × 48 9 3 1 0 0 0 0 0 0 ‘randy’ × 48 20 16 20 5 25 5 25 2 10 pollen donor 48 σ 96 44 71 11 16 10 14 3 4 crossing group σ 319 173 227 24 11 22 10 11 5 1percentage relating to the number of cultivated embryos, rounded to whole numbers fig. 1: flower and leaf of p. × crispum ‘soft pink’, the hybrids sg2-3.1 and sg2-3.2 and p. grandiflorum (from left to right). fig. 2: flower and leaf of p. × crispum ‘merlot’, the hybrid mf4-2 and p. fulgidum 123 (from left to right). 1 cm 1 cm polyploidisation of pelargonium hybrids 209 more vigorous than those of p. × crispum, but with a poorer branching. three out of eight hybrids from the cross ‘soft pink’ × p. grandiflorum showed distinct larger flowers than their siblings had. in the greenhouse, two of these hybrid clones (sg2-3, sg3-1) consisted of plants with medium (sg2-3.1, sg3-1.1) and plants with large sized flowers (sg2-3.2, sg3-1.2) (fig. 1). f1 hybrids of p. × crispum × p. fulgidum inherited the red flower colour from the pollen donor and the marks from the seed parent (fig. 2). they were male sterile with no or rudimentary anthers. the growth was weaker and plants tended to become succulent. among the five different f1 hybrids of ‘merlot’ × p. fulgidum 123, one genotype possessed larger flowers with fully developed anthers and fruit set after open pollination. in addition, dprapd analysis confirmed the hybrid character of all regenerated f1 plants. specific dna bands of the seed and pollen parents were detected in the f1 plants (fig. 3 and 4). flow cytometry and ploidy levels six genotypes of the crossing group ‘soft pink’ × p. grandiflorum and five genotypes of p. × crispum × p. fulgidum 123, including the assumed tetraploid plants, were proven together with the seed and pollen parents by flow cytometric analysis. the 2c values, calculated with a corresponding internal standard, are given in tab. 4. it was shown, that the assumed tetraploid f1 hybrids of both crossing groups have nearly a doubled 2c value compared to the di ploid hybrids. as expected from different 2c values of the pollen donors of both crossing groups, the mean 2c value of the diploid and tetraploid f1 hybrids, originating from p. fulgidum, was higher than mean 2c value of the diploid and tetraploid f1 hybrids, originating from p. grandiflorum. overall, in the crossing group p. × crispum × p. grandiflorum, spontaneous polyploidisation occurred with a frequency of about 20% and within the crossings p. × crispum × p. fulgidum of about 9%, respectively. fig. 4: dprapd analysis of four f1 hybrids of the crossing ‘merlot’ × p. fulgidum 123 (mf). specific band of ‘merlot’ at about 280 bp (left arrow) and p. fulgidum 123 at about 100, 600 and 1350 bp (right arrows), respectively (m = 100 bp dna ladder). fig. 3: dprapd analysis of eight f1 hybrids of ‘soft pink’ × p. grandiflorum (sg). specific bands of ‘soft pink’ at about 350 and 960 bp (left arrows) and p. grandiflorum at about 490, 590 and 1010 bp (right arrows), respectively (m = two different 100 bp dna ladders). 210 plaschil, s., budahn, h., klocke, e., wiedemann, m., olbricht, k. discussion in the genus pelargonium, only intraand intersubgeneric crossings between genotypes of the same basic chromosome number and ploidy level give a viable, fertile progeny (yu, 1985; horn, 1994). the knowledge of phylogeny, genetic distance, variability, ploidy level, and fertility can support plant breeding and interspecific crosses. although after polyploidisation of p. × crispum the variability was already widened by crossings with p. × domesticum at the tetraploid ploidy level (plaschil et al., 2012, 2015), further interspecific and intersectional hybridisation at the diploid level (2n = 2x = 22) is desirable. reciprocal crosses were omitted, because of fewer flowers of the species compared to the cultivars and the not significant effect on successful fertilisation in pelargonium (yu, 1985). yu (1985) reported about successful interspecific crosses using p. crispum as seed parent and p. grandiflorum as pollen parent. six f1 hybrids regenerated without embryo rescue. it is not known, whether these six hybrids were used for further breeding work. furthermore, olbricht (2013) succeeded in crossing p. grandiflorum with a breeding clone of p. × crispum and backcrosses with p. × crispum until the f3 generation. intersectional hybridisation in pelargonium was stated several times (yu, 1985; horn, 1994). yu (1985) described viable hybrids between p. fulgidum and diploid regal pelargoniums (p. × domesticum, (sect. pelargonium) resulting in cultivars (brawner, 2003) whereas crosses between p. fulgidum and p. rapaceum (l.) l’her. (section hoarea (sweet) de candolle) were not successful. regarding p. × crispum, the successful intersectional hybridisation with p. fulgidum we reported for the first time, because the breeding pedigree of uniques was not documented (brawner, 2003). even though, it is assumed, that the cultivar pac® angeleyes® ‘orange’, which underwent several hybridisation steps, descended from p. fulgidum. one parent of pac® angeleyes® ‘orange’ is the cultivar ‘erin’ (breeder kapac, 1997, usa) and ‘erin’ itself should be originated from crosses with p. fulgidum (hofmann & oblricht, pers. comm.; olbricht, 2013). due to insufficient embryo development after hybridisation, embryo rescue was necessary for both crossing groups as described for other ornamentals (van tuyl et al., 1991; winkelmann et al., 2010; aros et al., 2019). the overall plant regeneration was possible on a low scale of about 3-57% of cultivated embryos. as expected, the rege neration of interspecific p. × crispum × p. grandiflorum hybrids was more successful than that of intersectional p. × crispum × p. fulgidum hybrids, because of the lower genetic distance to the seed parent p. × crispum (plaschil et al., 2017). the regeneration could probably be improved by further investigations concerning the optimal time for fruit harvesting. so far, scemama and raquin (1990) only succeeded in direct embryo culture of a diploid p. × hortorum bailey, when embryos were removed at least 13 days after pollination, whereas they achieved embryo development and germination as early as 5-6 days after pollination using the ovary culture followed by cultivation of the contained embryos with their scarified seed coat. the authors achieved best results with ovary culture 11 days after pollination combined with the embryo culture five to seven days later. in addition, a more subtle adapted mixture of the in vitro medium due to the different genotypes may increase the percentage of viable regenerants (bentvelsen et al., 1990; denis-peixoto et al., 1997; kamlah et al., 2019). through embryo rescue we obtained spontaneous tetraploid f1 hybrids from both, interspecific cross p. × crispum × p. grandiflorum and the intersectional cross p. × crispum × p. fulgidum showing that the in vitro stage is important for the embryo development but it is also an appropriate tool to restore the hybrid fertility and an important prerequisite for further breeding. tab. 4: results of the flow cytometric analysis: mean 2c values of the seed parents, pollen donors and the f1 hybrids of the cross combinations ‘soft pink’ × p. grandiflorum and p. × crispum × p. fulgidum 123 genotype internal standard n 2c value (pg)1 sd (±) ploidy level seed parents ‘merlot’# ‘korso‘/‘stupické’ 6 1.00a 0.05 2x ‘soft pink’# ‘korso‘/‘stupické’ 7 1.01a 0.01 2x ‘randy’# ‘korso‘/‘stupické’ 7 1.01a 0.02 2x pollen donors and their hybrids p. grandiflorum ‘korso’ 3 0.99a 0.01 2x sg2-3.1* ‘korso’ 3 0.98a 0.02 2x sg2-3.2* ‘korso’ 3 2.07d 0.02 4x sg3-1.1* ‘korso’ 4 0.97a 0.01 2x sg3-1.2* ‘korso’ 3 2.09e 0.02 4x sg3-4* ‘korso’ 3 1.01a 0.02 2x sg3-5* ‘korso’ 3 2.14e 0.02 4x p. fulgidum 11 raphanus 3 1.56c 0.09 2x p. fulgidum 123 raphanus 3 1.52c 0.09 2x p. fulgidum 48 raphanus 3 1.55c 0.05 2x mf2-1** ‘stupické’ 4 1.23b 0.03 2x mf3-2** ‘stupické’ 3 2.59f 0.01 4x mf4-2** ‘stupické’ 3 1.19b 0.01 2x rf4-2** ‘stupické’ 3 1.22b 0.02 2x rf7-1** ‘stupické’ 3 1.19b 0.04 2x # data taken from plaschil et al., 2020; *cross combination: ‘soft pink’ × p. grandiflorum (sg); ** seed parents: m = ‘merlot’, r = ‘randy’ and pollen donor f = p. fulgidum 123; n = number of biological replications analysed per genotype; sd = standard deviation; 1 different letters indicate significant differences, tukey’s b-test, α = 5% polyploidisation of pelargonium hybrids 211 chromosomal disturbances up to a whole genome doubling of in vitro plant tissues are a common phenomenon. firstly larkin and scowcroft (1981) summarised these changes under the term somaclonal variation. spontaneous polyploidisation was often reported in in vitro haploid techniques such as microspore, anther or ovule culture, respectively (ahmadi and ebrahimzadeh, 2020; boerman et al., 2020; yuan et al., 2015; campion et al., 1995). the spontaneous chromosome doubling is an important factor for the production of homozygous plants because it is a desirable replacement for colchicine treatment. this phenomenon is described in almost all in vitro techniques such as long term cultures (ziauddin and kasha, 1990), in vitro shoot regeneration from leaves (meyer et al., 2009) or from embryogenic calli (ishigaki et al., 2014). as far as we know, no polyploidisation of pelargonium during in vitro embryo rescue has been described, yet. the influencing factors on somaclonal variation such as exogenous phytohormones or other chemicals in the nutrient medium, the de velopmental stage of the explants, and the duration of the in vitro culture were comprehensively investigated. nevertheless, questions remain open for better use of the process of spontaneous chromosome doubling. obviously, the genetics of the cultivated plants plays a role in induction of the chromosome doubling during the tissue culture. the frequency and regularity of spontaneous polyploidisation during embryo rescue of pelargonium needs further explorations. already larkin and scowcroft (1981) mentioned in vitro pelargonium plants as an example for the presence of somaclonal variations after various in vitro cultures such as genetic instabilities and changes of the ploidy. cassells et al. (1995, 1997) confirmed these findings with other pelargonium accessions applying molecular methods. it could be assumed, that the tendency to genetic changes during the in vitro phase also made the duplication of chromosomes during pelargonium embryo rescue possible. considering the morphological traits of the hybrids, the bright red flower colour of p. fulgidum inherited in crossings with p. × crispum is a promising new feature, but the vigour of the f1 hybrids was reduced. all diploid hybrids were male sterile with further unfavourable morphological traits. their value for further breeding as seed parent has to be proved. probably, the fertility must be restored by polyploidisation. only the fertile tetraploid hybrid is an appropriate crossing partner. in any case, further backcrosses with p. × crispum are necessary to establish cultivars. hybridisation between p. × crispum and p. grandiflorum is supposed to be a promising enhancement of genetic variation in the very narrow gene pool of p. × crispum cultivars. the primary diploid and tetraploid f1 hybrids were vigorous, had attractive flowers with marks and fertile anthers. hence, they could be used for further breeding at two different ploidy levels, e.g. for backcrosses with p. × crispum or crossings with p. × domesticum. the hybridisation success could be increased using embryo rescue. the current results encourage the usage of pelargonium species in breeding programs of p. × crispum combined with the application of embryo rescue. acknowledgement the authors thank dagmar franke for the embryo rescue, martina fuß and anika kunze for molecular analysis, simone abel for flow cytometric measurements as well as annette benecke, eveline kummer and denise brocka for the horticultural assistance. moreover, we thank kühne-jungpflanzen, claus kühne gbr, dresden and günter schumann for supporting this project. conflict of interest no potential conflict of interest was reported by the authors. references ahmadi, b., ebrahimzadeh, h., 2020: in vitro androgenesis: spontaneous vs. artificial genome doubling and characterization of regenerants. plant cell rep. 39, 299-316. doi: 10.1007/s00299-020-02509-z albers, f., gibby, m., austmann, m., 1992: a reappraisal of pelargonium sect. ligularia (geraniaceae). pl. syst. evol. 179, 257-276. doi: 10.1007/bf00937601 albers, f., van der walt, j.j.a., 1984: untersuchungen zur karyologie und mikrosporogenese von pelargonium sect. pelargonium (geraniaceae). pl. syst. evol. 147, 177-188. doi: 10.1007/bf00989382 aros, d., suazo, m., rivas, c., zapata, p., úbeda, c., bridgen, m., 2019: molecular and morphological characterization of new interspecific hybrids of alstroemeria originated from a. caryophylleae scented lines. euphytica 215, 93. doi: 10.1007/s10681-019-2415-4 bakker, f.t., culham, a., daugherty, l.c., gibby, m., 1999a: a trnl-f based phylogeny for species of pelargonium (geraniaceae) with small chromosomes. pl. syst. evol. 216, 309-324. doi: 10.1007/bf01084405 bakker, f.t., culham, a., gibby, m., 1999b: phylogenetics and diversification in pelargonium. in: hollingsworth, p., bateman, r., gornall, r. 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(eds.), plant cell culture: essential methods, 79-95. wiley & blackwell, chichester, uk. yu, s.-n., 1985: untersuchungen zur interspezifischen kompatibilität und biosystematik bei der gattung pelargonium, phd thesis, technische universität münchen, 270 pp. yuan, s., su, y., liu, y., li, z., fang, z., yang, l., zhuang, m., zhang, y., lv, h., sun, p., 2015: chromosome doubling of microspore-derived plants from cabbage (brassica oleracea var. capitata l.) and broccoli (brassica oleracea var. italica l.) frontiers in plant science 6, 1118. doi: 10.3389/fpls.2015.01118 ziauddin, a., kash, k.j., 1990: long-term callus cultures of diploid barley (hordeum vulgare). ii. effect of auxins on chromosomal status of cultures and regeneration of plants. euphytica 48, 279-286. doi: 10.1007/bf00023662 orcid sylvia plaschil https://orcid.org/0000-0001-6689-4113 holger budahn https://orcid.org/0000-0003-1639-8036 evelyn klocke https://orcid.org/0000-0002-3153-1280 klaus olbricht https://orcid.org/0000-0003-1124-2585 address of the corresponding author: julius kühn institute – federal research centre for cultivated plants, in stitute for breeding research on horticultural crops, erwin-baur-straße 27, 06484 quedlinburg, germany e-mail: sylvia.plaschil@julius-kuehn.de © the author(s) 2021. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1111/j.1439-0523.1997.tb02174.x http://dx.doi.org/10.1111/j.1399-3054.1992.tb04764.x http://dx.doi.org/10.1111/j.1439-0523.1994.tb00696.x http://dx.doi.org/10.1111/grs.12040 http://dx.doi.org/10.5073/jabfq.2019.092.007 http://dx.doi.org/10.1007/bf02342540 http://dx.doi.org/10.21273/hortsci.44.7.1957 http://dx.doi.org/10.1111/j.1399-3054.1962.tb08052.x http://dx.doi.org/10.5073/jfk.2020.06.04 http://dx.doi.org/10.17660/actahortic.2015.1087.45 http://dx.doi.org/10.1007/s10722-016-0424-x http://dx.doi.org/10.17660/actahortic.2012.953.21 http://dx.doi.org/10.1007/bf02772108 http://dx.doi.org/10.11646/phytotaxa.159.2.1 http://dx.doi.org/10.1016/s0176-1617(11)80893-x http://dx.doi.org/10.1007/bf00940594 http://dx.doi.org/10.1016/0168-9452(91)90262-7 http://dx.doi.org/10.1016/j.ympev.2012.05.026 http://dx.doi.org/10.1093/nar/18.22.6531 http://dx.doi.org/10.3389/fpls.2015.01118 http://dx.doi.org/10.1007/bf00023662 https://orcid.org/0000-0001-6689-4113 https://orcid.org/0000-0003-1639-8036 https://orcid.org/0000-0002-3153-1280 https://orcid.org/0000-0003-1124-2585 journal of applied botany and food quality 93, 26 33 (2020), doi:10.5073/jabfq.2020.093.004 1department of agriculture, payame noor university (pnu), tehran, iran 2department of horticultural sciences, faculty of agriculture, shiraz university, shiraz, iran 3department of chemistry, payame noor university (pnu), tehran, iran biological and pharmacological activities of essential oils of ocimum basilicum l. grown with zn-salicylic acid nano-complex vahid tavallali1*, hossein gholami2, omid espargham3 (submitted: july 19, 2019; accepted: november 1, 2019) * corresponding author summary a greenhouse study was conducted to investigate the impact of different rates of application of zn-edta, salicylic acid (sa) and zinc-salicylic acid nano-complex (n[zn(sa)2]) on the antioxidant and antimicrobial activities of essential oil (eo) of sweet basil (ocimum basilicum l.). sixty-one compounds were detected in the eos after zn and sa sources were applied to the plants. gc-ms analysis showed that the main components of the eos after the treatment were epi-α-cadinol and trans-α-bergamotene. the highest amount of epiα-cadinol (29.06±1.31%) and trans-α-bergamotene (11.90±1.1%) in the eo were observed at 0.2% n[zn(sa)2] treatment. in general, the application of 0.2% n[zn(sa)2] significantly increased percentages of phenolic and flavonoid compounds of extract. hplc analysis showed that the predominant phenolic compound after treatments with different zn and sa sources were rosmarinic acid and quercetin, respectively. the lowest ic50 values for rns, ros, tbars and h2o2, scavenging activities were obtained in eos of basils which were treated with 0.2% n[zn(sa)2]. zinc-salicylic nano-complex was the most effective treatment to inhibit fungal and bacterial growth. our results are quite encouraging since the eos of n[zn(sa)2] treated basil exhibited potent antioxidant effect, antimicrobial activities comparable with synthetic drugs. keywords: antimicrobial activity, flavonoids, nanoparticle, sali cylic acid, sweet basil, zinc. introduction the use of nanotechnology in all fields, including agriculture is ex panding. one of the most important applications of this technology in agriculture is the use of nanofertilizers for plant nutrition (zhu et al., 2008). nanofertilizers have recently attracted much attention due to their increased absorption by the plant, which is due to its small size and high penetration through cell membranes (panwar et al., 2012). nanoparticles are highly reactivity due to special and higher surfaces, higher density and many reactive regions on the particle surfaces. these characteristics make nanofertilizers and pesticides in nano-scale easy to absorb (chinnamuthu and boopathi, 2009). nanoparticles can bind to specific sites within biomolecules including proteins, nucleic acids and subcellular structures. in this way, they can pass through the cell membrane (krystofova et al., 2013). nanotechnology is gradually moving from the experimental stage to the operational stage, which has led to a more visible presence of this technology in the agricultural sector (baruah and dutta, 2009). in this study, we focus on ocimum basilicum, which is highly diverse in its morphological characteristics and secondary compounds, especially essential oil (telci et al., 2006). basil has been used as herbal medicine to treat headaches, coughs, diarrhea, parasites, warts and kidney diseases (labra et al., 2004). terpenoids constitute a significant proportion of basil essential oil which their type and amount is different in chemotypes, different climatic conditions and stages of plant development (sajjadi, 2006). synthesis of essential oils in plants changes affected by factors such as nutrition, light intensity, photosynthesis, photoperiodic changes, climatic conditions, seasonal variation, plant growth regulators, and environmental stresses such as drought, salinity and temperature (werker et al., 1993). salicylic acid or 2-hydroxybenzoic acid is a phenolic compound in plants that affect growth, respiration, photosynthesis, absorption and ion transport, and create some changes in leaf morphology and chlorophyll structure (popova et al., 2003). the application of salicylic acid can affect a range of different processes in plants such as ger mination of seeds (wang et al., 2006), resistance to pathogens (cag et al., 2009), flowering (koda et al., 1992), exchange and transmission of ions (cag et al., 2009), guard cells function and transpiration regulation (metwally et al., 2003), membrane permeability, photosynthesis and growth rate (khan et al., 2003). zinc is one of the micronutrient elements in the plant that is involved in various plant processes. deficiency of zinc prevents the growth of plants (gurmani et al., 2012). although a plant’s need for zinc is small, zinc deficiency results in severe physiological tensions such as inefficiency of many enzymatic systems and other metabolic actions (sadeghzadeh and rengel, 2006). zinc plays a role in carbohydrate and protein metabolism and is indirectly controlling waterplant relationships. there also is a close relationship between zinc and the amount of auxin in plants. a lack of auxin due to zinc deficiency causes reduced cell wall growth due to high osmotic pressure and limited water absorption by the plant (marschner, 2012). the purpose of this study is to investigate the effect of various zinc and salicylic acid sources on growth characteristics, essential oil yield, antioxidant and antimicrobial properties of sweet basil. materials and methods soil analysis physico-chemical characteristics of the soil were: texturesandy loam, ph 7.1, ece 1.3 ds m-1, cec 11 cmc kg-1, organic matter 8.8 g kg-1, n 0.08%, available k, p, zn and fe 61, 12, 1.5 and 1.01 mg kg-1, respectively. 5 mg kg-1 zn, mn and cu as znso4.7h2o, mnso4. h2o and cuso4.5h2o were applied, respectively. also, 50 mg kg-1 n and p as nh4no3 and kh2po4 were applied to the soil, respectively. plant material the seeds of sweet basil (ocimum basilicum l.) were provided from an herbal garden of bazrco company in tehran, iran. a 2 × 2 × 2 factorial experiment, arranged in a randomized complete design (rcd) with eight replications, was conducted in a greenhouse located at the 29º 31́ n; 52º 31́ e in shiraz, iran (approximately 800-1000 μmol m-2 s-1 par-photosynthetically active radiation, with a 12 h photo essential oils of ocimum basilicum l. grown with zn-salicylic acid nano-complex 27 period and temperature between 22-25 °c.). the experimental units were 7 l plastic pots. treatments were zn-edta (0.1 and 0.2%), sali cylic acid (0.1 and 0.2%), n[zn(sa)2] nano-complex (0.1 and 0.2%) and control (deionized water). salicylic acid and zinc sources were added in two steps. the first one after thinning and the second step was applied previous the flowering inception of plants. basils were collected at full bloom stage. nanoparticles used zn-salicylic acid nanoparticles (n[zn(sa)2]) were purchased from “zist nano fanavaran atiye pajooh” company in fars science and technology park, iran. the size of n[zn(sa)2] nanoparticles were determined by the transmission electron micrographs (tem) (100 kv philips, em208) (fig. 1). time of 30 min the ratio was set on (70:30) which was held isocratic till 40 min. photodiode array detector was set on 280 and 320 nm and chemstation software was used for data analysis. the standard solutions were all dissolved in methanol. the calibration curves with standard phenolic acids and flavonoids were obtained with good correlation. concentrations of phenolic acids and flavonoids are calculated as as milligrams per gram of dry matter (mg g-1 dm). essential oil analysis the experiment was carried out using agilent gas chromatograph series 7890-a with an fid (flame ionization detector) on fused silica capillary hp-5 column (30 m × 0.32 mm i.d.; film thickness 0.25 μm) for quantitation analysis. the injector and detector temperatures were kept at 250 °c and 280 °c, respectively. nitrogen was used as the carrier gas at a flow rate of 1 ml/min; the oven temperature program was 60-210 °c at the rate of 4 °c/min and then programmed to 240 °c at the rate of 20 °c/min and finally held isothermally for 8.5 min; the split ratio was 1:50. gc-ms analysis was carried out for quantification analysis by use of agilent gas chromatograph equipped with fused silica capillary hp-5ms column (30 m × 0.25 mm i.d.; film thickness 0.25 μm) coupled with an agilent mass spectrometer series 5975-c. helium was used as carrier gas. the quadrupole mass spectrometer was scanned over 50-550 amu with an ionizing voltage of 70 ev. ion source and interface temperatures were 230 °c and 280 °c, respectively. mass range was from 45 to 550 amu. the oven temperature program was the same as the gc. the injection volume was 0.1 μl in two mothods. retention indices of eo were determined based on retention times of alkanes (c8-c25) under the same chromatographic conditions. eventually, identification of chemical components of eo was done by correspondence of their retention profiles with those reported in previous studies (adams, 2007). antimicrobial activity of essential oil bacteria and fungi were obtained from the persian type culture collection (ptcc), tehran, iran. those were escherichia coli ptcc 1330 (atcc 8739) and salmonella typhimurium ptcc 1609 (iran isolate) as gram-negative bacteria and staphylococcus aureus ptcc 1112 (atcc 6538) and bacillus subtilis ptcc 1023 (atcc 6633) as gram-positive bacteria. also, the fungi used were fungi candida albicans ptcc 5027 (atcc 10231) and aspergillus niger ptcc 5010 (atcc 9142). the mic (minimum inhibitory concentration) values of the eo were characterized by means of the microdilution method in accordance with the clinical and laboratory standards institute procedures (clsi, 2012). suspended bacteria and fungi strains in luria-bertani (lb) media were tuned to 0.6 mcfarland standards at 640 nm (108 cfu/ml). then, densities were diluted to 105 (cfu/ml) with luria-bertani. the test mixture (600 μl) contained 300 μl of suspensions of bacteria and fungi and 300 μl eo. in the next step, the samples were shaken in incubator (for 24 h at 36 °c). positive control included in ketoconazole, ampicillin and gentamicin for fungi, gram-positive and gram-negative bacteria, respectively. a medium without fungi and bacteria and a medium without essential oils but with bacteria as sterile and growth control were considered, respectively. mic values were used for measuring the antibacterial and antifungal activities by using the formula mic value = [(a640blank a640sample/a640blank] × 100. antioxidant activity of the essential oil antioxidant activities of the eo were evaluated based on ic50, applying nano2, mda, dpph and h2o2 scavenging effects, shown for rns, tbars, ros and h2o2 scavenging activities, respectively (kavoosi and rowshan, 2013; burits et al., 2001). in ros assay, essential oil isolation the aerial parts were air-dried and then hydrodistilled for 3 h using a clevenger type device, according to the method recommended by the british pharmacopoeia (1998). eventually, the samples were then dried over anhydrous na2so4 and kept in sealed vials at low temperature until further analysis. high performance liquid chromatography (hplc) analysis agilent hplc 1200 series was applied to obtain phenolic acids (gallic acid, cinnamic acid, carvacrol, rosmarinic acid and ferulic acid were purchased from sigma-aldrich company for hplc analysis and flavonoids (quercetin, kaempferol, catechin, luteolin and rutin were purchased from sigma-aldrich company for hplc analysis) with the following method; 20 microliter of the dissolved extract was injected to zorbax eclipse (xdb) c18, 4.6 × 5 μm (id) × 150 mm column while column temperature was set on 30 °c. the gradient programming was applied to separate the constituents during 40 min. the mobile phase was a mixture of methanol: formic acid 1% with the flow rate of 1ml min-1, started from (10:90); then it programmed to (25:75) at the time of 10min; changing to (60:40) at 20 min; at the fig. 1: the tem image of zn-salicylic acid nano-complexes. 28 v. tavallali, h. gholami, o. espargham the reaction mixture (260 μl) contained 30 μl eo (0-0.5 mg ml-1 in dmso) and 230 μl dpph (120 mmol l-1 in methanol). in order to measure of rns scavenging effect, to 100 μl of the eo (0-0.5 mg ml-1 in dmso), 250 μl of sodium nitrite (0.02 mg ml-1 in 100 mm sodium citrate) was added. then, the reaction mixture was kept for 120 min at 36 °c. eventually, to this mixture, 600 μl of griess reagent was added. the reaction mixture of 50 μl of the eo (0-0.5 mg ml-1 in dmso) and 50 μl of the mda (0.1 mm in acetic acid ph=4) was used in order to measure of tbars scavenging effect. the, the solutions were kept at a 36 °c for 120 min. after adding one volume of thiobarbituric acid (0.3 mm in acetic acid ph=4), the solutions were incubated at 90 °c for 60 minutes. in h2o2 assay, the reaction mixture (100 μl) contained 50 μl of the eo (0-0.5 mg ml-1 in dmso) and 50 μl of the h2o2 (100 mm in 200 mm phosphate buffer, ph=7.4). the solutions were incubated at 36 °c for 75 minutes. eventually, samples’ absorptions were read with el × 808 absorbance microplate reader (biotek instruments, inc., usa) at wavelengths of 540, 532, 515 and 230 nm for nano2, mda, dpph and h2o2 tests, respectively. the percentage of rns, tbars, h2o2 and ros sca venging were then calculated by using the following formulas: rns scavenging effect (%) = [(a540blank a540sample)/a540blank] ×100 tbars scavenging effect (%) = [(a532blank a532sample)/a532blank] ×100 h2o2 scavenging effect (%) = [(a230blank a230sample)/a230blank] ×100 ros scavenging effect (%) = [(a515sample a515blank)/a515control] ×100 statistical analysis data are presented as mean values ±standard deviation of eight replications. data were statistically analyzed using one-way anova and duncan’s multiple range test (p<0.05) with spss (20.0). results and discussion growth characteristics and eo yield fig. 2a shows that the zn-salicylic acid nano-complex application significantly stimulated most of the essential oil production. the highest eo yields were achieved at 0.2% n[zn(sa)2] addition and lowest in the untreated control. carbon dioxide and glucose are precursors of biosynthesis for monoterpenes. saccharides are the energy source for the synthesis of terpenoids, which due to the role of zinc in the activity of the chloroplasts, photosynthesis, stomatal conductance and saccharides metabolism, the importance of this element in the production and accumulation of essential oil in plants can be significant. also, increasing leaf area due to zinc application, and consequently increasing photosynthesis, co2 stabilization and higher carbohydrate production, is another reason for the increase of secretory glands of essential oil (derakhshani et al., 2011b). zehtab-salmasi et al., 2008, reported that vegetative growth and essential oil yield of peppermint with application of 250 mg l-1 zinc sulfate were significantly higher than control treatment. also, research by grejtovský et al., 2006 showed that the application of 50 mg l-1 of zinc, increases the growth and essential oil yield in chamomile. increasing the essential oil yield after zinc application has also been reported for basil (said-al ahl and mahmoud, 2010). the positive role of spraying salicylic acid in increasing the essential oil yield may be due to increased vegetative growth, nutrient absorption, photosynthetic activity and also the change in the number of secretory glands of essential oil in leaves and flowers. these results are consistent with the findings of another study (gharib, 2007) on the positive role of salicylic acid in increasing the weight percent of essential oil of basil. one reason for increasing the secretory glands of essential oil may be the role of salicylic acid in enhancing the rubisco activity, photosynthesis, chlorophyll content and thereby increasing the performance of dry matter (singh and usha, 2003). the positive effect of zn-salicylic acid nano-complexes spraying on growth and essential oil yield can be attributed to the specific properties such as specific surface area, surface energy and increased surface activity compared to conventional particles (panwar et al., 2012). results indicated that the application of zinc and salicylic acid sour ces have a significant effect on the growth parameters of sweet basil (p ≤ 0.05). the highest plant height and plant weight were found in sweet basil treated with 0.2% and 0.1% n[zn(sa)2], respectively (fig. 2b and c). in this investigation, the use of salicylic acid and zinc in the form of nano-complex had a higher efficiency on growth characteristics than their non-nano forms. probably, the use of zn-salicylic acid nanocomplexes due to small size and their high penetration into cell membranes can justify their positive role (panwar et al., 2012). 581 582 583 584 585 586 b a c b c c d 0 50 100 150 200 250 300 350 pl an t h ei gh t (m m ) b a b d c d c e 0 50 100 150 200 250 300 350 pl an t w ei gh t (g ) c ab a d c cd bc e 0 0,1 0,2 0,3 0,4 0,5 0,6 es se n ti al o il yi el d ( g 10 0g -1 ) a fig. 2: essential oil yield (a), plant height (b) and plant weight (c) of sweet basil under different zinc and salicylic acid sources. values are means ± se (n = 8). bars having different letters are significantly different at the 5% level by duncan’s multiple range test. essential oils of ocimum basilicum l. grown with zn-salicylic acid nano-complex 29 chemical components of eo the quantitative and qualitative compositions of the eos of the sweet basil plants that were supplied with zinc and salicylic acid sources are presented in tab. 1. thirty-eight compounds were identified in the essential oil of basil at control treatment. epi-α-cadinol, eugenol, linalool, and trans-α-bergamotene were the major components in the eo with the value of 24.67, 12.7, 10.85 and 8.36%, respectively. sixty-one compounds were detected in the eos after zn and sa sources were applied to the plants. gc-ms analysis showed that the main components of the eos after treatment of different zn and sa fertilizer sources were epi-α-cadinol and trans-α-bergamotene. the highest amount of epi-α-cadinol (29.06±1.31%) and trans-αbergamotene (11.90±1.1%) were observed in the essential oil of 0.2% n[zn(sa)2] treated plants and the lowest (24.67±1.7 and 8.36±0.86%, respectively) in control plants. the effect of salicylic acid on the variation of chemical composition of essential oils in savory has been reported by haiati and rowshan (2013). hassanpouraghdam et al. (2011) reported that foliar application of zn influences the primary metabolic pathways, which ultimately causes the synthesis of essential oil compounds in basil leaves. in a study on basil, foliar application of zinc chelate improved growth characteristics and essential oil yield, as well as increasing the percentage of linalool and methyl chavicol as the dominant components of essential oil (said-al ahl and mahmoud, 2010). srivastava et al. (2006) observed a significant positive correlation between carbon assimilation pathways and the accumulation of secondary metabolites in turmeric. they consider several internal and external factors to be effective in the production of secondary metabolites, which have identified the most important factor in the supply of micronutrients such as zn; since the zn has been very effective on the biosynthesis of sesquiterpenes. increased zn levels have been associated with increased percentage of propyl 1-propenyl disulfide and reduced percentage of dimethylthiophene in onion (el-tohamy et al., 2009). chand et al., 2007 reported that zn spraying has significantly increased the percentage of essential oil of geranium, and also changes the main components of the essential oil such as rose oxide, linalool, and isomenthone. also, the positive effect of zn on the increase of essential oil yield and methyl chavicol as the domi nant component of anise essential oil has been reported (pirzad et al., 2013). changes in the compounds of the essential oil by zn spraying are related to the effect of this element on divalent cations, enzyme activity and also carbon metabolism. some enzymes that have metal ions in their building play an important role in the biosynthesis of monoterpene compounds (prasad et al., 2008). tavallali et al. (2018) reported changes in the essential oil components of sweet basil after the use of green synthesized zinc-amino nano-complexes. methyl chavicol was identified as the dominant component of the essential oil. phenolic acids and flavonoids profiles identification of phenolic and flavonoid compounds of extract according to their spectral properties was performed out by hplc. calibration curves were prepared by analysis of calibration solutions of investigated compounds in the concentration range from 1 to 500 mg l-1. in this study, carvacrol, rutin, ferulic acid and luteolin were not identified in control treatment. the results showed that the application of zn and sa sources increased the number of phenolic and flavonoid compounds detected in the extract. a total of 10 phenolic compounds, including five phenolic acids and five flavonoids, were identified from the extract of treated plants with zn and sa sources (tab. 2). in general, the application of 0.2% n[zn(sa)2] fertilizer significantly increased percentages of phenolic and flavonoid compounds of extract. hplc analysis showed that the predominant phenolic compound of the extract after treatment of different zn and sa fertilizer sources were rosmarinic acid and quercetin, respectively. in a study by derakhshani et al. (2011a), the application of zinc sulfate increased the phenolic compounds in costmary. an increase in the amount of phenolic compounds may be due to the role of zinc in the expression of genes associated with the biosynthesis of phenolic and flavonoid compounds (karabourniotis and liakopoulos, 2005). song et al. (2015) have shown that zinc inhibits the reduction of vvpal gene expression, thereby increasing the content of phenolic compounds. additionally, zinc with the direct effect on the activation of key enzymes in the biosynthetic pathways of flavonoids, such as chalcone synthase (chs) and chalcone isomerase (chi) increase the production of these compounds in the plants. it also increases the expression of vvchs, vvmybf1 and vvfls4 genes, and stimulates the production of flavonoid compounds in the plant (song et al., 2015). our results showed that the use of zn-salicylic acid nano-complexes has a greater effect than their non-nano form. increasing the transfer velocity, absorption efficiency, and specific surface of nanoparticles compared with conventional particles, can justify the greater effect of these particles (monica and cremonini, 2009). possibly, the entry of nanoparticles in plant cells is done through stomatal opening and natural nanopores which may enhance plant cell metabolic activi ties that lead to higher phenolic compounds production (tarafdar et al., 2014). antioxidant activity the eo from sweet basil aerial parts was investigated for radical scavenging activity using four different assay methods (rns, ros, tbars and h2o2, scavenging activities). the antioxidant activity of samples as milligrams ascorbic acid equivalents per gram of essential oil (mg aae g-1) was calculated and reported in tab. 3. the highest antioxidant activity (22.94±1.7 mg aae g-1) was found in 0.2% n[zn(sa)2] nano-complex treatment followed by 0.1% n[zn(sa)2] nano-complex treatment, which increased the antioxidant activity to 21.44±1.5 mg aae g-1. the lowest amount (8.82±1.2 mg aae g-1) was obtained in untreated plants. on 0.2% n[zn(sa)2] nano-complex treatment, all four different assay methods showed better antioxidant activity than other treatments. therefore, the lowest ic50 values for rns, ros, tbars and h2o2, scavenging activities were obtained in eo of basil plants, which were treated with 0.2% [zn(sa)2] nanocomplex. the comparison of the treatments used in this experiment showed that the use of zinc and salicylic acid in the form of nano has a higher effect than their non-nano form. probably, some of the properties of nanoparticles, such as higher transfer velocities, solubility and greater stability, can justify this high effect (monica and cremonini, 2009). antimicrobial activity in the study, the antimicrobial activities of eo from the aerial parts of sweet basil were tested against gram-negative (salmonella typhimurium and escherichia coli) and gram-positive (staphylococcus aureus and bacillus subtilis) bacteria and fungi (candida albicans and aspergillus niger) growth by minimal inhibitory concentration (mic) method. the results showed that the application of zinc and salicylic acid sources significantly increased the antifungal and antibacterial activity of eo from sweet basil compared to the control treatment (tab. 4). the lowest mic for salmonella typhimu rium, escherichia coli, staphylococcus aureus, bacillus subtilis, candida albicans and aspergillus niger growth were 0.180±0.011, 0.229 ± 0.018, 0.007 ± 0.0005, 0.031 ± 0.003, 0.050 ± 0.005 and 0.068±0.045 mg ml-1 of eo derived from treated basils with 0.2% n[zn(sa)2] nano-complex, respectively. 30 v. tavallali, h. gholami, o. espargham34 v. tavallali, h. gholami, o. espargham tab. 1: phytochemical profile (%) identified in essential oil of sweet basil supplied with diverse zinc and salicylic acid sources. control salicylic salicylic zn-edta zn-edta n[zn(sa)2] n[zn(sa)2] retention compound no. acid 0.2% acid 0.1% 0.2% 0.1% 0.2% 0.1% index ----------0.422±0.05a 0.377±0.03b 0.238±0.02cd 0.212±0.01d 0.251±0.02c 0.258±0.07c 932 α-pinene 1 ----------0.631±0.07a 0.580±0.06b 0.583±0.06b 0.571±0.05b 0.602±0.04b 0.576±0.03b 972 sabinene 2 -----------1.167±0.27a 1.084±0.23a 0.271±0.03b 0.224±0.02c 0.208±0.01c 0.286±0.04b 976 β-pinene 3 -----------3.224±0.59a 3.110±0.52a 0.416±0.03d 0.508±0.04b 0.510±0.03b 0.469±0.02c 990 myrcene 4 3.357±0.43a 0.966±0.07b 0.884±0.07c 0.611±0.05d 3.587±0.45a 0.425±0.03e 0.606±0.04d 999 n-decane 5 -----------0.127±0.02b 0.158±0.03a 0.121±0.03bc 0.091±0.006d 0.132±0.01ab 0.105±0.02cd 1005 α-phellandrene 6 -----------0.167±0.01a 0.092±0.007cd 0.102±0.01bc 0.069±0.005e 0.115±0.02b 0.081±0.007de 1016 α-terpinene 7 -----------0.029±0.004d 0.015±0.002e 0.082±0.005b 0.058±0.006c 0.093±0.007a 0.048±0.003c 1024 p-cymene 8 0.289±0.02b 0.796±0.07a 0.754±0.06a 0.142±0.02cd 0.127±0.02d 0.150±0.01c 0.134±0.01d 1028 limonene 9 3.463±0.66a 1.121±0.31b 1.074±0.20b 0.602±0.05c 3.523±0.50a 0.614±0.05c 1.088±0.11b 1030 1,8-cineole 10 0.397±0.04a 0.125±0.01c 0.101±0.01d 0.145±0.03bc 0.126±0.02c 0.153±0.01b 0.137±0.02bc 1036 (z)-β-ocimene 11 ----------0.039±0.004d 0.018±0.002e 0.085±0.006ab 0.062±0.004c 0.094±0.006a 0.075±0.003b 1041 benzene acetaldehyde 12 -----------0.602±0.07a 0.581±0.05ab 0.561±0.04ab 0.549±0.05b 0.567±0.04ab 0.566±0.03ab 1046 (e)-β-ocimene 13 -----------0.126±0.02b 0.118±0.02b 0.142±0.02a 0.128±0.01b 0.123±0.01b 0.139±0.01a 1057 γ-terpinene 14 ----------0.163±0.03a 0.150±0.02ab 0.145±0.03b 0.137±0.02b 0.120±0.01c 0.151±0. 01ab 1066 cis-sabinene hydrate 15 ----------0.371±0.04a 0.324±0.03bc 0.319±0.04bc 0.281±0.03d 0.332±0.02b 0.309±0.02c 1088 terpinolene 16 10.850±1.89a 5.733±0.51b 5.696±0.45b 0.593±0.05c 0.584±0.06c 0.516±0.04d 0.590±0.04c 1099 linalool 17 -----------0.136±0.02a 0.129±0.02a 0.072±0.007b 0.061±0.004c 0.079±0.005b 0.062±0.004c 1112 1-octen-3-yl acetate 18 -----------0.089±0.009d 0.058±0.006e 0.119±0.01ab 0.102±0.03c 0.128±0.01a 0.114±0.01b 1144 camphor 19 -----------0.220±0.02a 0.190±0.03b 0.161±0.02c 0.132±0.02d 0.198±0.02b 0.170±0.02c 1166 δ-terpineol 20 -----------0.097±0.007a 0.077±0.005b 0.076±0.006b 0.066±0.006c 0.097±0.007a 0.090±0.002a 1177 terpinen-4-ol 21 0.483±0.05f 1.360±0.18b 2.110±0.26a 0.703±0.05d 0.682±0.05de 0.664±0.04e 0.761±0.04c 1190 α-terpineol 22 0.783±0.07a 0.203±0.04e 0.177±0.03e 0.690±0.06b 0.512±0.04c 0.414±0.03d 0.565±0.03c 1199 n-dodecane 23 0.965±0.07a 0.821±0.08b 0.798±0.07b 0.589±0.06c 0.346±0.04d 0.604±0.05c 0.605±0.04c 1212 octanol acetate 24 0.248±0.01a 0.098±0.009c 0.088±0.007c 0.195±0.01b 0.101±0.009c 0.199±0.02b 0.250±0.02a 1222 trans-carveol 25 -----------0.129±0.02b 0.108±0.02b 0.791±0.06a 0.753±0.06a 0.043±0.003c 0.036±0.003c 1256 linalyl acetate 26 6.657±1.03a 1.843±0.28d 2.644±0.36b 1.692±0.23 2.395±0.18c 1.005±0.08f 1.332±0.12e 1286 bornyl acetate 27 0.146±0.01f 0.219±0.03e 0.184±0.03e 0.562±0.05a 0.365±0.03d 0.497±0.03b 0.444±0.03c 1337 δ-elemene 28 0.095±0.007c 0.224±0.03a 0.199±0.04a 0.154±0.02b 0.135±0.02b 0.135±0.01b 0.146±0.01b 1349 α-terpinyl acetate 29 12.704±1.77a 1.140±0.21e 2.288±0.27b 1.711±0.12c 1.389±0.09d 0.403±0.03g 0.741±0.05f 1358 eugenol 30 0.056±0.006d 0.121±0.01b 0.091±0.008c 0.314±0.04a 0.291±0.04a 0.292±0.02a 0.297±0.02a 1375 α-copaene 31 0.084±0.009d 0.130±0.01c 0.060±0.007e 0.214±0.02a 0.130±0.02c 0.222±0.02a 0.168±0.01b 1390 β-cubebene 32 4.894±0.42d 6.117±0.49c 6.083±0.41c 7.520±0.30b 7.245±0.25b 7.970±0.18a 7.877±0.21a 1392 β-elemene 33 -----------0.092±0.009d 0.078±0.008d 0.339±0.04a 0.253±0.03c 0.256±0.01c 0.288±0.02b 1400 n-tetradeane 34 0.330±0.02cd 0.715±0.05a 0.702±0.06a 0.392±0.04b 0.345±0.03c 0.309±0.02d 0.341±0.02c 1405 methyl eugenol 35 -----------0.072±0.007a 0.051±0.006b 0.072±0.005a 0.051±0.004b 0.076±0.005a 0.053±0.006b 1415 cis-α-bergamotene 36 0.196±0.02e 0.338±0.04d 0.308±0.05d 0.597±0.04a 0.469±0.03c 0.563±0.04b 0.587±0.04ab 1418 (e)-caryophyllene 37 -----------0.051±0.005d 0.036±0.004e 0.077±0.005a 0.039±0.003e 0.070±0.004b 0.059±0.005c 1428 β-gurjunene 38 8.369±0.86d 9.222±0.92c 9.093±0.87c 11.150±1.08b 10.920±1.06b 11.903±1.10a 11.838±1.02a 1436 trans-α-bergamotene 39 0.514±0.06d 0.606±0.07c 0.565±0.05c 1.148±0.11a 0.943±0.07b 1.108±0.09a 0.938±0.06b 1438 α-guaiene 40 0.063±0.006e 0.233±0.03a 0.186±0.02c 0.207±0.01abc 0.132±0.01d 0.219±0.02ab 0.196±0.01bc 1443 (z)-β-farnesene 41 1.323±0.12e 2.157±0.15d 1.968±0.17d 2.814±0.19b 2.752±0.18c 2.897±0.18a 2.912±0.17a 1453 α-humulene 42 0.247±0.01e 0.448±0.05c 0.403±0.04d 0.459±0.05bc 0.392±0.04 0.560±0.04a 0.488±0.03b 1457 (e)-β-farnesene 43 0.993±0.11f 1.521±0.17d 1.406±0.14e 1.992±0.12a 1.649±0.09c 1.007±0.07f 1.860±0.08b 1462 allo-aromadendrene 44 4.026±0.47f 6.133±0.60e 6.098±0.64e 7.254±0.55c 7.007±0.52d 7.561±0.41a 7.453±0.19b 1480 germacrene d 45 1.901±0.32d 3.197±0.30c 2.913±0.21c 4.195±0.37b 4.071±0.34b 4.534±0.21a 4.474±0.19a 1496 bicyclogermacrene 46 0.190±0.01d 0.511±0.04a 0.471±0.04ab 0.451±0.04 0.397±0.05c 0.485±0.03ab 0.467±0.03ab 1498 trans-β-guaiene 47 1.191±0.06e 2.010±0.07d 1.927±0.08d 2.682±0.09b 2.519±0.09c 2.795±0.08a 2.565±0.07c 1508 α-bulnesene 48 3.323±0.37f 5.423±0.48de 5.343±0.40e 5.741±0.31c 5.677±0.26cd 6.972±0.16a 6.085±0.15b 1514 γ-cadinene 49 0.859±0.07e 1.214±0.13b 1.146±0.10cd 1.150±0.12c 1.097±0.09d 1.281±0.07a 1.228±0.06b 1521 β-sesquiphellandrene 50 0.486±0.05e 0.612±0.05d 0.651±0.07d 0.698±0.05bc 0.683±0.07cd 0.748±0.06a 0.731±0.04ab 1524 δ-cadinene 51 0.150±0.01e 0.402±0.03d 0.376±0.03d 0.582±0.04a 0.461±0.06c 0.504±0.04b 0.478±0.03bc 1537 α-cadinene 52 0.394±0.02f 0.659±0.05e 0.635±0.06e 0.944±0.07b 0.799±0.06d 0.990±0.07a 0.880±0.06c 1564 (e)-nerolidol 53 -----------0.226±0.01ab 0.197±0.02c 0.223±0.01ab 0.215±0.04bc 0.240±0.02a 0.230±0.01ab 1575 germacrene d-4-ol 54 -----------0.214±0.02ab 0.182±0.02c 0.215±0.02ab 0.190±0.02c 0.228±0.01a 0.195±0.01bc 1577 spathulenol 55 -----------0.523±0.05b 0.494±0.04c 0.529±0.05b 0.485±0.04c 0.556±0.05a 0.516±0.04b 1599 n-hexadecane 56 2.65±0.20d 3.301±0.22b 3.205±0.19bc 3.510±0.22a 3.055±0.17c 3.566±0.10a 3.680±0.11a 1614 1,10-di-epi-cubenol 57 24.679±1.78e 27.520±1.58c 27.402±1.70c 28.510±1.64b 26.620±1.55d 29.062±1.31a 28.737±1.22b 1642 epi-α-cadinol 58 1.039±0.10d 1.663±0.09a 1.611±0.09a 1.404±0.10bc 1.391±0.08c 1.461±0.06b 1.399±0.07c 1649 β-eudesmol 59 1.353±0.10d 1.588±0.09a 1.574±0.11a 1.421±0.09c 1.409±0.09c 1.508±0.06b 1.456±0.07c 1654 α-cadinol 60 0.248±0.01d 0.543±0.03ab 0.544±0.04ab 0.462±0.05c 0.419±0.05c 0.592±0.04a 0.536±0.04b 1685 β-bisabolol 61 data are mean ±standard deviation of eight replications. means followed by the same letter within a row are not significantly different according to duncan’s multiple range test at p< 0.05. 34 v. tavallali, h. gholami, o. espargham tab. 1: phytochemical profile (%) identified in essential oil of sweet basil supplied with diverse zinc and salicylic acid sources. control salicylic salicylic zn-edta zn-edta n[zn(sa)2] n[zn(sa)2] retention compound no. acid 0.2% acid 0.1% 0.2% 0.1% 0.2% 0.1% index ----------0.422±0.05a 0.377±0.03b 0.238±0.02cd 0.212±0.01d 0.251±0.02c 0.258±0.07c 932 α-pinene 1 ----------0.631±0.07a 0.580±0.06b 0.583±0.06b 0.571±0.05b 0.602±0.04b 0.576±0.03b 972 sabinene 2 -----------1.167±0.27a 1.084±0.23a 0.271±0.03b 0.224±0.02c 0.208±0.01c 0.286±0.04b 976 β-pinene 3 -----------3.224±0.59a 3.110±0.52a 0.416±0.03d 0.508±0.04b 0.510±0.03b 0.469±0.02c 990 myrcene 4 3.357±0.43a 0.966±0.07b 0.884±0.07c 0.611±0.05d 3.587±0.45a 0.425±0.03e 0.606±0.04d 999 n-decane 5 -----------0.127±0.02b 0.158±0.03a 0.121±0.03bc 0.091±0.006d 0.132±0.01ab 0.105±0.02cd 1005 α-phellandrene 6 -----------0.167±0.01a 0.092±0.007cd 0.102±0.01bc 0.069±0.005e 0.115±0.02b 0.081±0.007de 1016 α-terpinene 7 -----------0.029±0.004d 0.015±0.002e 0.082±0.005b 0.058±0.006c 0.093±0.007a 0.048±0.003c 1024 p-cymene 8 0.289±0.02b 0.796±0.07a 0.754±0.06a 0.142±0.02cd 0.127±0.02d 0.150±0.01c 0.134±0.01d 1028 limonene 9 3.463±0.66a 1.121±0.31b 1.074±0.20b 0.602±0.05c 3.523±0.50a 0.614±0.05c 1.088±0.11b 1030 1,8-cineole 10 0.397±0.04a 0.125±0.01c 0.101±0.01d 0.145±0.03bc 0.126±0.02c 0.153±0.01b 0.137±0.02bc 1036 (z)-β-ocimene 11 ----------0.039±0.004d 0.018±0.002e 0.085±0.006ab 0.062±0.004c 0.094±0.006a 0.075±0.003b 1041 benzene acetaldehyde 12 -----------0.602±0.07a 0.581±0.05ab 0.561±0.04ab 0.549±0.05b 0.567±0.04ab 0.566±0.03ab 1046 (e)-β-ocimene 13 -----------0.126±0.02b 0.118±0.02b 0.142±0.02a 0.128±0.01b 0.123±0.01b 0.139±0.01a 1057 γ-terpinene 14 ----------0.163±0.03a 0.150±0.02ab 0.145±0.03b 0.137±0.02b 0.120±0.01c 0.151±0. 01ab 1066 cis-sabinene hydrate 15 ----------0.371±0.04a 0.324±0.03bc 0.319±0.04bc 0.281±0.03d 0.332±0.02b 0.309±0.02c 1088 terpinolene 16 10.850±1.89a 5.733±0.51b 5.696±0.45b 0.593±0.05c 0.584±0.06c 0.516±0.04d 0.590±0.04c 1099 linalool 17 -----------0.136±0.02a 0.129±0.02a 0.072±0.007b 0.061±0.004c 0.079±0.005b 0.062±0.004c 1112 1-octen-3-yl acetate 18 -----------0.089±0.009d 0.058±0.006e 0.119±0.01ab 0.102±0.03c 0.128±0.01a 0.114±0.01b 1144 camphor 19 -----------0.220±0.02a 0.190±0.03b 0.161±0.02c 0.132±0.02d 0.198±0.02b 0.170±0.02c 1166 δ-terpineol 20 -----------0.097±0.007a 0.077±0.005b 0.076±0.006b 0.066±0.006c 0.097±0.007a 0.090±0.002a 1177 terpinen-4-ol 21 0.483±0.05f 1.360±0.18b 2.110±0.26a 0.703±0.05d 0.682±0.05de 0.664±0.04e 0.761±0.04c 1190 α-terpineol 22 0.783±0.07a 0.203±0.04e 0.177±0.03e 0.690±0.06b 0.512±0.04c 0.414±0.03d 0.565±0.03c 1199 n-dodecane 23 0.965±0.07a 0.821±0.08b 0.798±0.07b 0.589±0.06c 0.346±0.04d 0.604±0.05c 0.605±0.04c 1212 octanol acetate 24 0.248±0.01a 0.098±0.009c 0.088±0.007c 0.195±0.01b 0.101±0.009c 0.199±0.02b 0.250±0.02a 1222 trans-carveol 25 -----------0.129±0.02b 0.108±0.02b 0.791±0.06a 0.753±0.06a 0.043±0.003c 0.036±0.003c 1256 linalyl acetate 26 6.657±1.03a 1.843±0.28d 2.644±0.36b 1.692±0.23 2.395±0.18c 1.005±0.08f 1.332±0.12e 1286 bornyl acetate 27 0.146±0.01f 0.219±0.03e 0.184±0.03e 0.562±0.05a 0.365±0.03d 0.497±0.03b 0.444±0.03c 1337 δ-elemene 28 0.095±0.007c 0.224±0.03a 0.199±0.04a 0.154±0.02b 0.135±0.02b 0.135±0.01b 0.146±0.01b 1349 α-terpinyl acetate 29 12.704±1.77a 1.140±0.21e 2.288±0.27b 1.711±0.12c 1.389±0.09d 0.403±0.03g 0.741±0.05f 1358 eugenol 30 0.056±0.006d 0.121±0.01b 0.091±0.008c 0.314±0.04a 0.291±0.04a 0.292±0.02a 0.297±0.02a 1375 α-copaene 31 0.084±0.009d 0.130±0.01c 0.060±0.007e 0.214±0.02a 0.130±0.02c 0.222±0.02a 0.168±0.01b 1390 β-cubebene 32 4.894±0.42d 6.117±0.49c 6.083±0.41c 7.520±0.30b 7.245±0.25b 7.970±0.18a 7.877±0.21a 1392 β-elemene 33 -----------0.092±0.009d 0.078±0.008d 0.339±0.04a 0.253±0.03c 0.256±0.01c 0.288±0.02b 1400 n-tetradeane 34 0.330±0.02cd 0.715±0.05a 0.702±0.06a 0.392±0.04b 0.345±0.03c 0.309±0.02d 0.341±0.02c 1405 methyl eugenol 35 -----------0.072±0.007a 0.051±0.006b 0.072±0.005a 0.051±0.004b 0.076±0.005a 0.053±0.006b 1415 cis-α-bergamotene 36 0.196±0.02e 0.338±0.04d 0.308±0.05d 0.597±0.04a 0.469±0.03c 0.563±0.04b 0.587±0.04ab 1418 (e)-caryophyllene 37 -----------0.051±0.005d 0.036±0.004e 0.077±0.005a 0.039±0.003e 0.070±0.004b 0.059±0.005c 1428 β-gurjunene 38 8.369±0.86d 9.222±0.92c 9.093±0.87c 11.150±1.08b 10.920±1.06b 11.903±1.10a 11.838±1.02a 1436 trans-α-bergamotene 39 0.514±0.06d 0.606±0.07c 0.565±0.05c 1.148±0.11a 0.943±0.07b 1.108±0.09a 0.938±0.06b 1438 α-guaiene 40 0.063±0.006e 0.233±0.03a 0.186±0.02c 0.207±0.01abc 0.132±0.01d 0.219±0.02ab 0.196±0.01bc 1443 (z)-β-farnesene 41 1.323±0.12e 2.157±0.15d 1.968±0.17d 2.814±0.19b 2.752±0.18c 2.897±0.18a 2.912±0.17a 1453 α-humulene 42 0.247±0.01e 0.448±0.05c 0.403±0.04d 0.459±0.05bc 0.392±0.04 0.560±0.04a 0.488±0.03b 1457 (e)-β-farnesene 43 0.993±0.11f 1.521±0.17d 1.406±0.14e 1.992±0.12a 1.649±0.09c 1.007±0.07f 1.860±0.08b 1462 allo-aromadendrene 44 4.026±0.47f 6.133±0.60e 6.098±0.64e 7.254±0.55c 7.007±0.52d 7.561±0.41a 7.453±0.19b 1480 germacrene d 45 1.901±0.32d 3.197±0.30c 2.913±0.21c 4.195±0.37b 4.071±0.34b 4.534±0.21a 4.474±0.19a 1496 bicyclogermacrene 46 0.190±0.01d 0.511±0.04a 0.471±0.04ab 0.451±0.04 0.397±0.05c 0.485±0.03ab 0.467±0.03ab 1498 trans-β-guaiene 47 1.191±0.06e 2.010±0.07d 1.927±0.08d 2.682±0.09b 2.519±0.09c 2.795±0.08a 2.565±0.07c 1508 α-bulnesene 48 3.323±0.37f 5.423±0.48de 5.343±0.40e 5.741±0.31c 5.677±0.26cd 6.972±0.16a 6.085±0.15b 1514 γ-cadinene 49 0.859±0.07e 1.214±0.13b 1.146±0.10cd 1.150±0.12c 1.097±0.09d 1.281±0.07a 1.228±0.06b 1521 β-sesquiphellandrene 50 0.486±0.05e 0.612±0.05d 0.651±0.07d 0.698±0.05bc 0.683±0.07cd 0.748±0.06a 0.731±0.04ab 1524 δ-cadinene 51 0.150±0.01e 0.402±0.03d 0.376±0.03d 0.582±0.04a 0.461±0.06c 0.504±0.04b 0.478±0.03bc 1537 α-cadinene 52 0.394±0.02f 0.659±0.05e 0.635±0.06e 0.944±0.07b 0.799±0.06d 0.990±0.07a 0.880±0.06c 1564 (e)-nerolidol 53 -----------0.226±0.01ab 0.197±0.02c 0.223±0.01ab 0.215±0.04bc 0.240±0.02a 0.230±0.01ab 1575 germacrene d-4-ol 54 -----------0.214±0.02ab 0.182±0.02c 0.215±0.02ab 0.190±0.02c 0.228±0.01a 0.195±0.01bc 1577 spathulenol 55 -----------0.523±0.05b 0.494±0.04c 0.529±0.05b 0.485±0.04c 0.556±0.05a 0.516±0.04b 1599 n-hexadecane 56 2.65±0.20d 3.301±0.22b 3.205±0.19bc 3.510±0.22a 3.055±0.17c 3.566±0.10a 3.680±0.11a 1614 1,10-di-epi-cubenol 57 24.679±1.78e 27.520±1.58c 27.402±1.70c 28.510±1.64b 26.620±1.55d 29.062±1.31a 28.737±1.22b 1642 epi-α-cadinol 58 1.039±0.10d 1.663±0.09a 1.611±0.09a 1.404±0.10bc 1.391±0.08c 1.461±0.06b 1.399±0.07c 1649 β-eudesmol 59 1.353±0.10d 1.588±0.09a 1.574±0.11a 1.421±0.09c 1.409±0.09c 1.508±0.06b 1.456±0.07c 1654 α-cadinol 60 0.248±0.01d 0.543±0.03ab 0.544±0.04ab 0.462±0.05c 0.419±0.05c 0.592±0.04a 0.536±0.04b 1685 β-bisabolol 61 data are mean ±standard deviation of eight replications. means followed by the same letter within a row are not significantly different according to duncan’s multiple range test at p< 0.05. 34 v. tavallali, h. gholami, o. espargham tab. 1: phytochemical profile (%) identified in essential oil of sweet basil supplied with diverse zinc and salicylic acid sources. control salicylic salicylic zn-edta zn-edta n[zn(sa)2] n[zn(sa)2] retention compound no. acid 0.2% acid 0.1% 0.2% 0.1% 0.2% 0.1% index ----------0.422±0.05a 0.377±0.03b 0.238±0.02cd 0.212±0.01d 0.251±0.02c 0.258±0.07c 932 α-pinene 1 ----------0.631±0.07a 0.580±0.06b 0.583±0.06b 0.571±0.05b 0.602±0.04b 0.576±0.03b 972 sabinene 2 -----------1.167±0.27a 1.084±0.23a 0.271±0.03b 0.224±0.02c 0.208±0.01c 0.286±0.04b 976 β-pinene 3 -----------3.224±0.59a 3.110±0.52a 0.416±0.03d 0.508±0.04b 0.510±0.03b 0.469±0.02c 990 myrcene 4 3.357±0.43a 0.966±0.07b 0.884±0.07c 0.611±0.05d 3.587±0.45a 0.425±0.03e 0.606±0.04d 999 n-decane 5 -----------0.127±0.02b 0.158±0.03a 0.121±0.03bc 0.091±0.006d 0.132±0.01ab 0.105±0.02cd 1005 α-phellandrene 6 -----------0.167±0.01a 0.092±0.007cd 0.102±0.01bc 0.069±0.005e 0.115±0.02b 0.081±0.007de 1016 α-terpinene 7 -----------0.029±0.004d 0.015±0.002e 0.082±0.005b 0.058±0.006c 0.093±0.007a 0.048±0.003c 1024 p-cymene 8 0.289±0.02b 0.796±0.07a 0.754±0.06a 0.142±0.02cd 0.127±0.02d 0.150±0.01c 0.134±0.01d 1028 limonene 9 3.463±0.66a 1.121±0.31b 1.074±0.20b 0.602±0.05c 3.523±0.50a 0.614±0.05c 1.088±0.11b 1030 1,8-cineole 10 0.397±0.04a 0.125±0.01c 0.101±0.01d 0.145±0.03bc 0.126±0.02c 0.153±0.01b 0.137±0.02bc 1036 (z)-β-ocimene 11 ----------0.039±0.004d 0.018±0.002e 0.085±0.006ab 0.062±0.004c 0.094±0.006a 0.075±0.003b 1041 benzene acetaldehyde 12 -----------0.602±0.07a 0.581±0.05ab 0.561±0.04ab 0.549±0.05b 0.567±0.04ab 0.566±0.03ab 1046 (e)-β-ocimene 13 -----------0.126±0.02b 0.118±0.02b 0.142±0.02a 0.128±0.01b 0.123±0.01b 0.139±0.01a 1057 γ-terpinene 14 ----------0.163±0.03a 0.150±0.02ab 0.145±0.03b 0.137±0.02b 0.120±0.01c 0.151±0. 01ab 1066 cis-sabinene hydrate 15 ----------0.371±0.04a 0.324±0.03bc 0.319±0.04bc 0.281±0.03d 0.332±0.02b 0.309±0.02c 1088 terpinolene 16 10.850±1.89a 5.733±0.51b 5.696±0.45b 0.593±0.05c 0.584±0.06c 0.516±0.04d 0.590±0.04c 1099 linalool 17 -----------0.136±0.02a 0.129±0.02a 0.072±0.007b 0.061±0.004c 0.079±0.005b 0.062±0.004c 1112 1-octen-3-yl acetate 18 -----------0.089±0.009d 0.058±0.006e 0.119±0.01ab 0.102±0.03c 0.128±0.01a 0.114±0.01b 1144 camphor 19 -----------0.220±0.02a 0.190±0.03b 0.161±0.02c 0.132±0.02d 0.198±0.02b 0.170±0.02c 1166 δ-terpineol 20 -----------0.097±0.007a 0.077±0.005b 0.076±0.006b 0.066±0.006c 0.097±0.007a 0.090±0.002a 1177 terpinen-4-ol 21 0.483±0.05f 1.360±0.18b 2.110±0.26a 0.703±0.05d 0.682±0.05de 0.664±0.04e 0.761±0.04c 1190 α-terpineol 22 0.783±0.07a 0.203±0.04e 0.177±0.03e 0.690±0.06b 0.512±0.04c 0.414±0.03d 0.565±0.03c 1199 n-dodecane 23 0.965±0.07a 0.821±0.08b 0.798±0.07b 0.589±0.06c 0.346±0.04d 0.604±0.05c 0.605±0.04c 1212 octanol acetate 24 0.248±0.01a 0.098±0.009c 0.088±0.007c 0.195±0.01b 0.101±0.009c 0.199±0.02b 0.250±0.02a 1222 trans-carveol 25 -----------0.129±0.02b 0.108±0.02b 0.791±0.06a 0.753±0.06a 0.043±0.003c 0.036±0.003c 1256 linalyl acetate 26 6.657±1.03a 1.843±0.28d 2.644±0.36b 1.692±0.23 2.395±0.18c 1.005±0.08f 1.332±0.12e 1286 bornyl acetate 27 0.146±0.01f 0.219±0.03e 0.184±0.03e 0.562±0.05a 0.365±0.03d 0.497±0.03b 0.444±0.03c 1337 δ-elemene 28 0.095±0.007c 0.224±0.03a 0.199±0.04a 0.154±0.02b 0.135±0.02b 0.135±0.01b 0.146±0.01b 1349 α-terpinyl acetate 29 12.704±1.77a 1.140±0.21e 2.288±0.27b 1.711±0.12c 1.389±0.09d 0.403±0.03g 0.741±0.05f 1358 eugenol 30 0.056±0.006d 0.121±0.01b 0.091±0.008c 0.314±0.04a 0.291±0.04a 0.292±0.02a 0.297±0.02a 1375 α-copaene 31 0.084±0.009d 0.130±0.01c 0.060±0.007e 0.214±0.02a 0.130±0.02c 0.222±0.02a 0.168±0.01b 1390 β-cubebene 32 4.894±0.42d 6.117±0.49c 6.083±0.41c 7.520±0.30b 7.245±0.25b 7.970±0.18a 7.877±0.21a 1392 β-elemene 33 -----------0.092±0.009d 0.078±0.008d 0.339±0.04a 0.253±0.03c 0.256±0.01c 0.288±0.02b 1400 n-tetradeane 34 0.330±0.02cd 0.715±0.05a 0.702±0.06a 0.392±0.04b 0.345±0.03c 0.309±0.02d 0.341±0.02c 1405 methyl eugenol 35 -----------0.072±0.007a 0.051±0.006b 0.072±0.005a 0.051±0.004b 0.076±0.005a 0.053±0.006b 1415 cis-α-bergamotene 36 0.196±0.02e 0.338±0.04d 0.308±0.05d 0.597±0.04a 0.469±0.03c 0.563±0.04b 0.587±0.04ab 1418 (e)-caryophyllene 37 -----------0.051±0.005d 0.036±0.004e 0.077±0.005a 0.039±0.003e 0.070±0.004b 0.059±0.005c 1428 β-gurjunene 38 8.369±0.86d 9.222±0.92c 9.093±0.87c 11.150±1.08b 10.920±1.06b 11.903±1.10a 11.838±1.02a 1436 trans-α-bergamotene 39 0.514±0.06d 0.606±0.07c 0.565±0.05c 1.148±0.11a 0.943±0.07b 1.108±0.09a 0.938±0.06b 1438 α-guaiene 40 0.063±0.006e 0.233±0.03a 0.186±0.02c 0.207±0.01abc 0.132±0.01d 0.219±0.02ab 0.196±0.01bc 1443 (z)-β-farnesene 41 1.323±0.12e 2.157±0.15d 1.968±0.17d 2.814±0.19b 2.752±0.18c 2.897±0.18a 2.912±0.17a 1453 α-humulene 42 0.247±0.01e 0.448±0.05c 0.403±0.04d 0.459±0.05bc 0.392±0.04 0.560±0.04a 0.488±0.03b 1457 (e)-β-farnesene 43 0.993±0.11f 1.521±0.17d 1.406±0.14e 1.992±0.12a 1.649±0.09c 1.007±0.07f 1.860±0.08b 1462 allo-aromadendrene 44 4.026±0.47f 6.133±0.60e 6.098±0.64e 7.254±0.55c 7.007±0.52d 7.561±0.41a 7.453±0.19b 1480 germacrene d 45 1.901±0.32d 3.197±0.30c 2.913±0.21c 4.195±0.37b 4.071±0.34b 4.534±0.21a 4.474±0.19a 1496 bicyclogermacrene 46 0.190±0.01d 0.511±0.04a 0.471±0.04ab 0.451±0.04 0.397±0.05c 0.485±0.03ab 0.467±0.03ab 1498 trans-β-guaiene 47 1.191±0.06e 2.010±0.07d 1.927±0.08d 2.682±0.09b 2.519±0.09c 2.795±0.08a 2.565±0.07c 1508 α-bulnesene 48 3.323±0.37f 5.423±0.48de 5.343±0.40e 5.741±0.31c 5.677±0.26cd 6.972±0.16a 6.085±0.15b 1514 γ-cadinene 49 0.859±0.07e 1.214±0.13b 1.146±0.10cd 1.150±0.12c 1.097±0.09d 1.281±0.07a 1.228±0.06b 1521 β-sesquiphellandrene 50 0.486±0.05e 0.612±0.05d 0.651±0.07d 0.698±0.05bc 0.683±0.07cd 0.748±0.06a 0.731±0.04ab 1524 δ-cadinene 51 0.150±0.01e 0.402±0.03d 0.376±0.03d 0.582±0.04a 0.461±0.06c 0.504±0.04b 0.478±0.03bc 1537 α-cadinene 52 0.394±0.02f 0.659±0.05e 0.635±0.06e 0.944±0.07b 0.799±0.06d 0.990±0.07a 0.880±0.06c 1564 (e)-nerolidol 53 -----------0.226±0.01ab 0.197±0.02c 0.223±0.01ab 0.215±0.04bc 0.240±0.02a 0.230±0.01ab 1575 germacrene d-4-ol 54 -----------0.214±0.02ab 0.182±0.02c 0.215±0.02ab 0.190±0.02c 0.228±0.01a 0.195±0.01bc 1577 spathulenol 55 -----------0.523±0.05b 0.494±0.04c 0.529±0.05b 0.485±0.04c 0.556±0.05a 0.516±0.04b 1599 n-hexadecane 56 2.65±0.20d 3.301±0.22b 3.205±0.19bc 3.510±0.22a 3.055±0.17c 3.566±0.10a 3.680±0.11a 1614 1,10-di-epi-cubenol 57 24.679±1.78e 27.520±1.58c 27.402±1.70c 28.510±1.64b 26.620±1.55d 29.062±1.31a 28.737±1.22b 1642 epi-α-cadinol 58 1.039±0.10d 1.663±0.09a 1.611±0.09a 1.404±0.10bc 1.391±0.08c 1.461±0.06b 1.399±0.07c 1649 β-eudesmol 59 1.353±0.10d 1.588±0.09a 1.574±0.11a 1.421±0.09c 1.409±0.09c 1.508±0.06b 1.456±0.07c 1654 α-cadinol 60 0.248±0.01d 0.543±0.03ab 0.544±0.04ab 0.462±0.05c 0.419±0.05c 0.592±0.04a 0.536±0.04b 1685 β-bisabolol 61 data are mean ±standard deviation of eight replications. means followed by the same letter within a row are not significantly different according to duncan’s multiple range test at p< 0.05. 34 v. tavallali, h. gholami, o. espargham tab. 1: phytochemical profile (%) identified in essential oil of sweet basil supplied with diverse zinc and salicylic acid sources. control salicylic salicylic zn-edta zn-edta n[zn(sa)2] n[zn(sa)2] retention compound no. acid 0.2% acid 0.1% 0.2% 0.1% 0.2% 0.1% index ----------0.422±0.05a 0.377±0.03b 0.238±0.02cd 0.212±0.01d 0.251±0.02c 0.258±0.07c 932 α-pinene 1 ----------0.631±0.07a 0.580±0.06b 0.583±0.06b 0.571±0.05b 0.602±0.04b 0.576±0.03b 972 sabinene 2 -----------1.167±0.27a 1.084±0.23a 0.271±0.03b 0.224±0.02c 0.208±0.01c 0.286±0.04b 976 β-pinene 3 -----------3.224±0.59a 3.110±0.52a 0.416±0.03d 0.508±0.04b 0.510±0.03b 0.469±0.02c 990 myrcene 4 3.357±0.43a 0.966±0.07b 0.884±0.07c 0.611±0.05d 3.587±0.45a 0.425±0.03e 0.606±0.04d 999 n-decane 5 -----------0.127±0.02b 0.158±0.03a 0.121±0.03bc 0.091±0.006d 0.132±0.01ab 0.105±0.02cd 1005 α-phellandrene 6 -----------0.167±0.01a 0.092±0.007cd 0.102±0.01bc 0.069±0.005e 0.115±0.02b 0.081±0.007de 1016 α-terpinene 7 -----------0.029±0.004d 0.015±0.002e 0.082±0.005b 0.058±0.006c 0.093±0.007a 0.048±0.003c 1024 p-cymene 8 0.289±0.02b 0.796±0.07a 0.754±0.06a 0.142±0.02cd 0.127±0.02d 0.150±0.01c 0.134±0.01d 1028 limonene 9 3.463±0.66a 1.121±0.31b 1.074±0.20b 0.602±0.05c 3.523±0.50a 0.614±0.05c 1.088±0.11b 1030 1,8-cineole 10 0.397±0.04a 0.125±0.01c 0.101±0.01d 0.145±0.03bc 0.126±0.02c 0.153±0.01b 0.137±0.02bc 1036 (z)-β-ocimene 11 ----------0.039±0.004d 0.018±0.002e 0.085±0.006ab 0.062±0.004c 0.094±0.006a 0.075±0.003b 1041 benzene acetaldehyde 12 -----------0.602±0.07a 0.581±0.05ab 0.561±0.04ab 0.549±0.05b 0.567±0.04ab 0.566±0.03ab 1046 (e)-β-ocimene 13 -----------0.126±0.02b 0.118±0.02b 0.142±0.02a 0.128±0.01b 0.123±0.01b 0.139±0.01a 1057 γ-terpinene 14 ----------0.163±0.03a 0.150±0.02ab 0.145±0.03b 0.137±0.02b 0.120±0.01c 0.151±0. 01ab 1066 cis-sabinene hydrate 15 ----------0.371±0.04a 0.324±0.03bc 0.319±0.04bc 0.281±0.03d 0.332±0.02b 0.309±0.02c 1088 terpinolene 16 10.850±1.89a 5.733±0.51b 5.696±0.45b 0.593±0.05c 0.584±0.06c 0.516±0.04d 0.590±0.04c 1099 linalool 17 -----------0.136±0.02a 0.129±0.02a 0.072±0.007b 0.061±0.004c 0.079±0.005b 0.062±0.004c 1112 1-octen-3-yl acetate 18 -----------0.089±0.009d 0.058±0.006e 0.119±0.01ab 0.102±0.03c 0.128±0.01a 0.114±0.01b 1144 camphor 19 -----------0.220±0.02a 0.190±0.03b 0.161±0.02c 0.132±0.02d 0.198±0.02b 0.170±0.02c 1166 δ-terpineol 20 -----------0.097±0.007a 0.077±0.005b 0.076±0.006b 0.066±0.006c 0.097±0.007a 0.090±0.002a 1177 terpinen-4-ol 21 0.483±0.05f 1.360±0.18b 2.110±0.26a 0.703±0.05d 0.682±0.05de 0.664±0.04e 0.761±0.04c 1190 α-terpineol 22 0.783±0.07a 0.203±0.04e 0.177±0.03e 0.690±0.06b 0.512±0.04c 0.414±0.03d 0.565±0.03c 1199 n-dodecane 23 0.965±0.07a 0.821±0.08b 0.798±0.07b 0.589±0.06c 0.346±0.04d 0.604±0.05c 0.605±0.04c 1212 octanol acetate 24 0.248±0.01a 0.098±0.009c 0.088±0.007c 0.195±0.01b 0.101±0.009c 0.199±0.02b 0.250±0.02a 1222 trans-carveol 25 -----------0.129±0.02b 0.108±0.02b 0.791±0.06a 0.753±0.06a 0.043±0.003c 0.036±0.003c 1256 linalyl acetate 26 6.657±1.03a 1.843±0.28d 2.644±0.36b 1.692±0.23 2.395±0.18c 1.005±0.08f 1.332±0.12e 1286 bornyl acetate 27 0.146±0.01f 0.219±0.03e 0.184±0.03e 0.562±0.05a 0.365±0.03d 0.497±0.03b 0.444±0.03c 1337 δ-elemene 28 0.095±0.007c 0.224±0.03a 0.199±0.04a 0.154±0.02b 0.135±0.02b 0.135±0.01b 0.146±0.01b 1349 α-terpinyl acetate 29 12.704±1.77a 1.140±0.21e 2.288±0.27b 1.711±0.12c 1.389±0.09d 0.403±0.03g 0.741±0.05f 1358 eugenol 30 0.056±0.006d 0.121±0.01b 0.091±0.008c 0.314±0.04a 0.291±0.04a 0.292±0.02a 0.297±0.02a 1375 α-copaene 31 0.084±0.009d 0.130±0.01c 0.060±0.007e 0.214±0.02a 0.130±0.02c 0.222±0.02a 0.168±0.01b 1390 β-cubebene 32 4.894±0.42d 6.117±0.49c 6.083±0.41c 7.520±0.30b 7.245±0.25b 7.970±0.18a 7.877±0.21a 1392 β-elemene 33 -----------0.092±0.009d 0.078±0.008d 0.339±0.04a 0.253±0.03c 0.256±0.01c 0.288±0.02b 1400 n-tetradeane 34 0.330±0.02cd 0.715±0.05a 0.702±0.06a 0.392±0.04b 0.345±0.03c 0.309±0.02d 0.341±0.02c 1405 methyl eugenol 35 -----------0.072±0.007a 0.051±0.006b 0.072±0.005a 0.051±0.004b 0.076±0.005a 0.053±0.006b 1415 cis-α-bergamotene 36 0.196±0.02e 0.338±0.04d 0.308±0.05d 0.597±0.04a 0.469±0.03c 0.563±0.04b 0.587±0.04ab 1418 (e)-caryophyllene 37 -----------0.051±0.005d 0.036±0.004e 0.077±0.005a 0.039±0.003e 0.070±0.004b 0.059±0.005c 1428 β-gurjunene 38 8.369±0.86d 9.222±0.92c 9.093±0.87c 11.150±1.08b 10.920±1.06b 11.903±1.10a 11.838±1.02a 1436 trans-α-bergamotene 39 0.514±0.06d 0.606±0.07c 0.565±0.05c 1.148±0.11a 0.943±0.07b 1.108±0.09a 0.938±0.06b 1438 α-guaiene 40 0.063±0.006e 0.233±0.03a 0.186±0.02c 0.207±0.01abc 0.132±0.01d 0.219±0.02ab 0.196±0.01bc 1443 (z)-β-farnesene 41 1.323±0.12e 2.157±0.15d 1.968±0.17d 2.814±0.19b 2.752±0.18c 2.897±0.18a 2.912±0.17a 1453 α-humulene 42 0.247±0.01e 0.448±0.05c 0.403±0.04d 0.459±0.05bc 0.392±0.04 0.560±0.04a 0.488±0.03b 1457 (e)-β-farnesene 43 0.993±0.11f 1.521±0.17d 1.406±0.14e 1.992±0.12a 1.649±0.09c 1.007±0.07f 1.860±0.08b 1462 allo-aromadendrene 44 4.026±0.47f 6.133±0.60e 6.098±0.64e 7.254±0.55c 7.007±0.52d 7.561±0.41a 7.453±0.19b 1480 germacrene d 45 1.901±0.32d 3.197±0.30c 2.913±0.21c 4.195±0.37b 4.071±0.34b 4.534±0.21a 4.474±0.19a 1496 bicyclogermacrene 46 0.190±0.01d 0.511±0.04a 0.471±0.04ab 0.451±0.04 0.397±0.05c 0.485±0.03ab 0.467±0.03ab 1498 trans-β-guaiene 47 1.191±0.06e 2.010±0.07d 1.927±0.08d 2.682±0.09b 2.519±0.09c 2.795±0.08a 2.565±0.07c 1508 α-bulnesene 48 3.323±0.37f 5.423±0.48de 5.343±0.40e 5.741±0.31c 5.677±0.26cd 6.972±0.16a 6.085±0.15b 1514 γ-cadinene 49 0.859±0.07e 1.214±0.13b 1.146±0.10cd 1.150±0.12c 1.097±0.09d 1.281±0.07a 1.228±0.06b 1521 β-sesquiphellandrene 50 0.486±0.05e 0.612±0.05d 0.651±0.07d 0.698±0.05bc 0.683±0.07cd 0.748±0.06a 0.731±0.04ab 1524 δ-cadinene 51 0.150±0.01e 0.402±0.03d 0.376±0.03d 0.582±0.04a 0.461±0.06c 0.504±0.04b 0.478±0.03bc 1537 α-cadinene 52 0.394±0.02f 0.659±0.05e 0.635±0.06e 0.944±0.07b 0.799±0.06d 0.990±0.07a 0.880±0.06c 1564 (e)-nerolidol 53 -----------0.226±0.01ab 0.197±0.02c 0.223±0.01ab 0.215±0.04bc 0.240±0.02a 0.230±0.01ab 1575 germacrene d-4-ol 54 -----------0.214±0.02ab 0.182±0.02c 0.215±0.02ab 0.190±0.02c 0.228±0.01a 0.195±0.01bc 1577 spathulenol 55 -----------0.523±0.05b 0.494±0.04c 0.529±0.05b 0.485±0.04c 0.556±0.05a 0.516±0.04b 1599 n-hexadecane 56 2.65±0.20d 3.301±0.22b 3.205±0.19bc 3.510±0.22a 3.055±0.17c 3.566±0.10a 3.680±0.11a 1614 1,10-di-epi-cubenol 57 24.679±1.78e 27.520±1.58c 27.402±1.70c 28.510±1.64b 26.620±1.55d 29.062±1.31a 28.737±1.22b 1642 epi-α-cadinol 58 1.039±0.10d 1.663±0.09a 1.611±0.09a 1.404±0.10bc 1.391±0.08c 1.461±0.06b 1.399±0.07c 1649 β-eudesmol 59 1.353±0.10d 1.588±0.09a 1.574±0.11a 1.421±0.09c 1.409±0.09c 1.508±0.06b 1.456±0.07c 1654 α-cadinol 60 0.248±0.01d 0.543±0.03ab 0.544±0.04ab 0.462±0.05c 0.419±0.05c 0.592±0.04a 0.536±0.04b 1685 β-bisabolol 61 data are mean ±standard deviation of eight replications. means followed by the same letter within a row are not significantly different according to duncan’s multiple range test at p< 0.05. 34 v. tavallali, h. gholami, o. espargham tab. 1: phytochemical profile (%) identified in essential oil of sweet basil supplied with diverse zinc and salicylic acid sources. control salicylic salicylic zn-edta zn-edta n[zn(sa)2] n[zn(sa)2] retention compound no. acid 0.2% acid 0.1% 0.2% 0.1% 0.2% 0.1% index ----------0.422±0.05a 0.377±0.03b 0.238±0.02cd 0.212±0.01d 0.251±0.02c 0.258±0.07c 932 α-pinene 1 ----------0.631±0.07a 0.580±0.06b 0.583±0.06b 0.571±0.05b 0.602±0.04b 0.576±0.03b 972 sabinene 2 -----------1.167±0.27a 1.084±0.23a 0.271±0.03b 0.224±0.02c 0.208±0.01c 0.286±0.04b 976 β-pinene 3 -----------3.224±0.59a 3.110±0.52a 0.416±0.03d 0.508±0.04b 0.510±0.03b 0.469±0.02c 990 myrcene 4 3.357±0.43a 0.966±0.07b 0.884±0.07c 0.611±0.05d 3.587±0.45a 0.425±0.03e 0.606±0.04d 999 n-decane 5 -----------0.127±0.02b 0.158±0.03a 0.121±0.03bc 0.091±0.006d 0.132±0.01ab 0.105±0.02cd 1005 α-phellandrene 6 -----------0.167±0.01a 0.092±0.007cd 0.102±0.01bc 0.069±0.005e 0.115±0.02b 0.081±0.007de 1016 α-terpinene 7 -----------0.029±0.004d 0.015±0.002e 0.082±0.005b 0.058±0.006c 0.093±0.007a 0.048±0.003c 1024 p-cymene 8 0.289±0.02b 0.796±0.07a 0.754±0.06a 0.142±0.02cd 0.127±0.02d 0.150±0.01c 0.134±0.01d 1028 limonene 9 3.463±0.66a 1.121±0.31b 1.074±0.20b 0.602±0.05c 3.523±0.50a 0.614±0.05c 1.088±0.11b 1030 1,8-cineole 10 0.397±0.04a 0.125±0.01c 0.101±0.01d 0.145±0.03bc 0.126±0.02c 0.153±0.01b 0.137±0.02bc 1036 (z)-β-ocimene 11 ----------0.039±0.004d 0.018±0.002e 0.085±0.006ab 0.062±0.004c 0.094±0.006a 0.075±0.003b 1041 benzene acetaldehyde 12 -----------0.602±0.07a 0.581±0.05ab 0.561±0.04ab 0.549±0.05b 0.567±0.04ab 0.566±0.03ab 1046 (e)-β-ocimene 13 -----------0.126±0.02b 0.118±0.02b 0.142±0.02a 0.128±0.01b 0.123±0.01b 0.139±0.01a 1057 γ-terpinene 14 ----------0.163±0.03a 0.150±0.02ab 0.145±0.03b 0.137±0.02b 0.120±0.01c 0.151±0. 01ab 1066 cis-sabinene hydrate 15 ----------0.371±0.04a 0.324±0.03bc 0.319±0.04bc 0.281±0.03d 0.332±0.02b 0.309±0.02c 1088 terpinolene 16 10.850±1.89a 5.733±0.51b 5.696±0.45b 0.593±0.05c 0.584±0.06c 0.516±0.04d 0.590±0.04c 1099 linalool 17 -----------0.136±0.02a 0.129±0.02a 0.072±0.007b 0.061±0.004c 0.079±0.005b 0.062±0.004c 1112 1-octen-3-yl acetate 18 -----------0.089±0.009d 0.058±0.006e 0.119±0.01ab 0.102±0.03c 0.128±0.01a 0.114±0.01b 1144 camphor 19 -----------0.220±0.02a 0.190±0.03b 0.161±0.02c 0.132±0.02d 0.198±0.02b 0.170±0.02c 1166 δ-terpineol 20 -----------0.097±0.007a 0.077±0.005b 0.076±0.006b 0.066±0.006c 0.097±0.007a 0.090±0.002a 1177 terpinen-4-ol 21 0.483±0.05f 1.360±0.18b 2.110±0.26a 0.703±0.05d 0.682±0.05de 0.664±0.04e 0.761±0.04c 1190 α-terpineol 22 0.783±0.07a 0.203±0.04e 0.177±0.03e 0.690±0.06b 0.512±0.04c 0.414±0.03d 0.565±0.03c 1199 n-dodecane 23 0.965±0.07a 0.821±0.08b 0.798±0.07b 0.589±0.06c 0.346±0.04d 0.604±0.05c 0.605±0.04c 1212 octanol acetate 24 0.248±0.01a 0.098±0.009c 0.088±0.007c 0.195±0.01b 0.101±0.009c 0.199±0.02b 0.250±0.02a 1222 trans-carveol 25 -----------0.129±0.02b 0.108±0.02b 0.791±0.06a 0.753±0.06a 0.043±0.003c 0.036±0.003c 1256 linalyl acetate 26 6.657±1.03a 1.843±0.28d 2.644±0.36b 1.692±0.23 2.395±0.18c 1.005±0.08f 1.332±0.12e 1286 bornyl acetate 27 0.146±0.01f 0.219±0.03e 0.184±0.03e 0.562±0.05a 0.365±0.03d 0.497±0.03b 0.444±0.03c 1337 δ-elemene 28 0.095±0.007c 0.224±0.03a 0.199±0.04a 0.154±0.02b 0.135±0.02b 0.135±0.01b 0.146±0.01b 1349 α-terpinyl acetate 29 12.704±1.77a 1.140±0.21e 2.288±0.27b 1.711±0.12c 1.389±0.09d 0.403±0.03g 0.741±0.05f 1358 eugenol 30 0.056±0.006d 0.121±0.01b 0.091±0.008c 0.314±0.04a 0.291±0.04a 0.292±0.02a 0.297±0.02a 1375 α-copaene 31 0.084±0.009d 0.130±0.01c 0.060±0.007e 0.214±0.02a 0.130±0.02c 0.222±0.02a 0.168±0.01b 1390 β-cubebene 32 4.894±0.42d 6.117±0.49c 6.083±0.41c 7.520±0.30b 7.245±0.25b 7.970±0.18a 7.877±0.21a 1392 β-elemene 33 -----------0.092±0.009d 0.078±0.008d 0.339±0.04a 0.253±0.03c 0.256±0.01c 0.288±0.02b 1400 n-tetradeane 34 0.330±0.02cd 0.715±0.05a 0.702±0.06a 0.392±0.04b 0.345±0.03c 0.309±0.02d 0.341±0.02c 1405 methyl eugenol 35 -----------0.072±0.007a 0.051±0.006b 0.072±0.005a 0.051±0.004b 0.076±0.005a 0.053±0.006b 1415 cis-α-bergamotene 36 0.196±0.02e 0.338±0.04d 0.308±0.05d 0.597±0.04a 0.469±0.03c 0.563±0.04b 0.587±0.04ab 1418 (e)-caryophyllene 37 -----------0.051±0.005d 0.036±0.004e 0.077±0.005a 0.039±0.003e 0.070±0.004b 0.059±0.005c 1428 β-gurjunene 38 8.369±0.86d 9.222±0.92c 9.093±0.87c 11.150±1.08b 10.920±1.06b 11.903±1.10a 11.838±1.02a 1436 trans-α-bergamotene 39 0.514±0.06d 0.606±0.07c 0.565±0.05c 1.148±0.11a 0.943±0.07b 1.108±0.09a 0.938±0.06b 1438 α-guaiene 40 0.063±0.006e 0.233±0.03a 0.186±0.02c 0.207±0.01abc 0.132±0.01d 0.219±0.02ab 0.196±0.01bc 1443 (z)-β-farnesene 41 1.323±0.12e 2.157±0.15d 1.968±0.17d 2.814±0.19b 2.752±0.18c 2.897±0.18a 2.912±0.17a 1453 α-humulene 42 0.247±0.01e 0.448±0.05c 0.403±0.04d 0.459±0.05bc 0.392±0.04 0.560±0.04a 0.488±0.03b 1457 (e)-β-farnesene 43 0.993±0.11f 1.521±0.17d 1.406±0.14e 1.992±0.12a 1.649±0.09c 1.007±0.07f 1.860±0.08b 1462 allo-aromadendrene 44 4.026±0.47f 6.133±0.60e 6.098±0.64e 7.254±0.55c 7.007±0.52d 7.561±0.41a 7.453±0.19b 1480 germacrene d 45 1.901±0.32d 3.197±0.30c 2.913±0.21c 4.195±0.37b 4.071±0.34b 4.534±0.21a 4.474±0.19a 1496 bicyclogermacrene 46 0.190±0.01d 0.511±0.04a 0.471±0.04ab 0.451±0.04 0.397±0.05c 0.485±0.03ab 0.467±0.03ab 1498 trans-β-guaiene 47 1.191±0.06e 2.010±0.07d 1.927±0.08d 2.682±0.09b 2.519±0.09c 2.795±0.08a 2.565±0.07c 1508 α-bulnesene 48 3.323±0.37f 5.423±0.48de 5.343±0.40e 5.741±0.31c 5.677±0.26cd 6.972±0.16a 6.085±0.15b 1514 γ-cadinene 49 0.859±0.07e 1.214±0.13b 1.146±0.10cd 1.150±0.12c 1.097±0.09d 1.281±0.07a 1.228±0.06b 1521 β-sesquiphellandrene 50 0.486±0.05e 0.612±0.05d 0.651±0.07d 0.698±0.05bc 0.683±0.07cd 0.748±0.06a 0.731±0.04ab 1524 δ-cadinene 51 0.150±0.01e 0.402±0.03d 0.376±0.03d 0.582±0.04a 0.461±0.06c 0.504±0.04b 0.478±0.03bc 1537 α-cadinene 52 0.394±0.02f 0.659±0.05e 0.635±0.06e 0.944±0.07b 0.799±0.06d 0.990±0.07a 0.880±0.06c 1564 (e)-nerolidol 53 -----------0.226±0.01ab 0.197±0.02c 0.223±0.01ab 0.215±0.04bc 0.240±0.02a 0.230±0.01ab 1575 germacrene d-4-ol 54 -----------0.214±0.02ab 0.182±0.02c 0.215±0.02ab 0.190±0.02c 0.228±0.01a 0.195±0.01bc 1577 spathulenol 55 -----------0.523±0.05b 0.494±0.04c 0.529±0.05b 0.485±0.04c 0.556±0.05a 0.516±0.04b 1599 n-hexadecane 56 2.65±0.20d 3.301±0.22b 3.205±0.19bc 3.510±0.22a 3.055±0.17c 3.566±0.10a 3.680±0.11a 1614 1,10-di-epi-cubenol 57 24.679±1.78e 27.520±1.58c 27.402±1.70c 28.510±1.64b 26.620±1.55d 29.062±1.31a 28.737±1.22b 1642 epi-α-cadinol 58 1.039±0.10d 1.663±0.09a 1.611±0.09a 1.404±0.10bc 1.391±0.08c 1.461±0.06b 1.399±0.07c 1649 β-eudesmol 59 1.353±0.10d 1.588±0.09a 1.574±0.11a 1.421±0.09c 1.409±0.09c 1.508±0.06b 1.456±0.07c 1654 α-cadinol 60 0.248±0.01d 0.543±0.03ab 0.544±0.04ab 0.462±0.05c 0.419±0.05c 0.592±0.04a 0.536±0.04b 1685 β-bisabolol 61 data are mean ±standard deviation of eight replications. means followed by the same letter within a row are not significantly different according to duncan’s multiple range test at p< 0.05. 34 v. tavallali, h. gholami, o. espargham tab. 1: phytochemical profile (%) identified in essential oil of sweet basil supplied with diverse zinc and salicylic acid sources. control salicylic salicylic zn-edta zn-edta n[zn(sa)2] n[zn(sa)2] retention compound no. acid 0.2% acid 0.1% 0.2% 0.1% 0.2% 0.1% index ----------0.422±0.05a 0.377±0.03b 0.238±0.02cd 0.212±0.01d 0.251±0.02c 0.258±0.07c 932 α-pinene 1 ----------0.631±0.07a 0.580±0.06b 0.583±0.06b 0.571±0.05b 0.602±0.04b 0.576±0.03b 972 sabinene 2 -----------1.167±0.27a 1.084±0.23a 0.271±0.03b 0.224±0.02c 0.208±0.01c 0.286±0.04b 976 β-pinene 3 -----------3.224±0.59a 3.110±0.52a 0.416±0.03d 0.508±0.04b 0.510±0.03b 0.469±0.02c 990 myrcene 4 3.357±0.43a 0.966±0.07b 0.884±0.07c 0.611±0.05d 3.587±0.45a 0.425±0.03e 0.606±0.04d 999 n-decane 5 -----------0.127±0.02b 0.158±0.03a 0.121±0.03bc 0.091±0.006d 0.132±0.01ab 0.105±0.02cd 1005 α-phellandrene 6 -----------0.167±0.01a 0.092±0.007cd 0.102±0.01bc 0.069±0.005e 0.115±0.02b 0.081±0.007de 1016 α-terpinene 7 -----------0.029±0.004d 0.015±0.002e 0.082±0.005b 0.058±0.006c 0.093±0.007a 0.048±0.003c 1024 p-cymene 8 0.289±0.02b 0.796±0.07a 0.754±0.06a 0.142±0.02cd 0.127±0.02d 0.150±0.01c 0.134±0.01d 1028 limonene 9 3.463±0.66a 1.121±0.31b 1.074±0.20b 0.602±0.05c 3.523±0.50a 0.614±0.05c 1.088±0.11b 1030 1,8-cineole 10 0.397±0.04a 0.125±0.01c 0.101±0.01d 0.145±0.03bc 0.126±0.02c 0.153±0.01b 0.137±0.02bc 1036 (z)-β-ocimene 11 ----------0.039±0.004d 0.018±0.002e 0.085±0.006ab 0.062±0.004c 0.094±0.006a 0.075±0.003b 1041 benzene acetaldehyde 12 -----------0.602±0.07a 0.581±0.05ab 0.561±0.04ab 0.549±0.05b 0.567±0.04ab 0.566±0.03ab 1046 (e)-β-ocimene 13 -----------0.126±0.02b 0.118±0.02b 0.142±0.02a 0.128±0.01b 0.123±0.01b 0.139±0.01a 1057 γ-terpinene 14 ----------0.163±0.03a 0.150±0.02ab 0.145±0.03b 0.137±0.02b 0.120±0.01c 0.151±0. 01ab 1066 cis-sabinene hydrate 15 ----------0.371±0.04a 0.324±0.03bc 0.319±0.04bc 0.281±0.03d 0.332±0.02b 0.309±0.02c 1088 terpinolene 16 10.850±1.89a 5.733±0.51b 5.696±0.45b 0.593±0.05c 0.584±0.06c 0.516±0.04d 0.590±0.04c 1099 linalool 17 -----------0.136±0.02a 0.129±0.02a 0.072±0.007b 0.061±0.004c 0.079±0.005b 0.062±0.004c 1112 1-octen-3-yl acetate 18 -----------0.089±0.009d 0.058±0.006e 0.119±0.01ab 0.102±0.03c 0.128±0.01a 0.114±0.01b 1144 camphor 19 -----------0.220±0.02a 0.190±0.03b 0.161±0.02c 0.132±0.02d 0.198±0.02b 0.170±0.02c 1166 δ-terpineol 20 -----------0.097±0.007a 0.077±0.005b 0.076±0.006b 0.066±0.006c 0.097±0.007a 0.090±0.002a 1177 terpinen-4-ol 21 0.483±0.05f 1.360±0.18b 2.110±0.26a 0.703±0.05d 0.682±0.05de 0.664±0.04e 0.761±0.04c 1190 α-terpineol 22 0.783±0.07a 0.203±0.04e 0.177±0.03e 0.690±0.06b 0.512±0.04c 0.414±0.03d 0.565±0.03c 1199 n-dodecane 23 0.965±0.07a 0.821±0.08b 0.798±0.07b 0.589±0.06c 0.346±0.04d 0.604±0.05c 0.605±0.04c 1212 octanol acetate 24 0.248±0.01a 0.098±0.009c 0.088±0.007c 0.195±0.01b 0.101±0.009c 0.199±0.02b 0.250±0.02a 1222 trans-carveol 25 -----------0.129±0.02b 0.108±0.02b 0.791±0.06a 0.753±0.06a 0.043±0.003c 0.036±0.003c 1256 linalyl acetate 26 6.657±1.03a 1.843±0.28d 2.644±0.36b 1.692±0.23 2.395±0.18c 1.005±0.08f 1.332±0.12e 1286 bornyl acetate 27 0.146±0.01f 0.219±0.03e 0.184±0.03e 0.562±0.05a 0.365±0.03d 0.497±0.03b 0.444±0.03c 1337 δ-elemene 28 0.095±0.007c 0.224±0.03a 0.199±0.04a 0.154±0.02b 0.135±0.02b 0.135±0.01b 0.146±0.01b 1349 α-terpinyl acetate 29 12.704±1.77a 1.140±0.21e 2.288±0.27b 1.711±0.12c 1.389±0.09d 0.403±0.03g 0.741±0.05f 1358 eugenol 30 0.056±0.006d 0.121±0.01b 0.091±0.008c 0.314±0.04a 0.291±0.04a 0.292±0.02a 0.297±0.02a 1375 α-copaene 31 0.084±0.009d 0.130±0.01c 0.060±0.007e 0.214±0.02a 0.130±0.02c 0.222±0.02a 0.168±0.01b 1390 β-cubebene 32 4.894±0.42d 6.117±0.49c 6.083±0.41c 7.520±0.30b 7.245±0.25b 7.970±0.18a 7.877±0.21a 1392 β-elemene 33 -----------0.092±0.009d 0.078±0.008d 0.339±0.04a 0.253±0.03c 0.256±0.01c 0.288±0.02b 1400 n-tetradeane 34 0.330±0.02cd 0.715±0.05a 0.702±0.06a 0.392±0.04b 0.345±0.03c 0.309±0.02d 0.341±0.02c 1405 methyl eugenol 35 -----------0.072±0.007a 0.051±0.006b 0.072±0.005a 0.051±0.004b 0.076±0.005a 0.053±0.006b 1415 cis-α-bergamotene 36 0.196±0.02e 0.338±0.04d 0.308±0.05d 0.597±0.04a 0.469±0.03c 0.563±0.04b 0.587±0.04ab 1418 (e)-caryophyllene 37 -----------0.051±0.005d 0.036±0.004e 0.077±0.005a 0.039±0.003e 0.070±0.004b 0.059±0.005c 1428 β-gurjunene 38 8.369±0.86d 9.222±0.92c 9.093±0.87c 11.150±1.08b 10.920±1.06b 11.903±1.10a 11.838±1.02a 1436 trans-α-bergamotene 39 0.514±0.06d 0.606±0.07c 0.565±0.05c 1.148±0.11a 0.943±0.07b 1.108±0.09a 0.938±0.06b 1438 α-guaiene 40 0.063±0.006e 0.233±0.03a 0.186±0.02c 0.207±0.01abc 0.132±0.01d 0.219±0.02ab 0.196±0.01bc 1443 (z)-β-farnesene 41 1.323±0.12e 2.157±0.15d 1.968±0.17d 2.814±0.19b 2.752±0.18c 2.897±0.18a 2.912±0.17a 1453 α-humulene 42 0.247±0.01e 0.448±0.05c 0.403±0.04d 0.459±0.05bc 0.392±0.04 0.560±0.04a 0.488±0.03b 1457 (e)-β-farnesene 43 0.993±0.11f 1.521±0.17d 1.406±0.14e 1.992±0.12a 1.649±0.09c 1.007±0.07f 1.860±0.08b 1462 allo-aromadendrene 44 4.026±0.47f 6.133±0.60e 6.098±0.64e 7.254±0.55c 7.007±0.52d 7.561±0.41a 7.453±0.19b 1480 germacrene d 45 1.901±0.32d 3.197±0.30c 2.913±0.21c 4.195±0.37b 4.071±0.34b 4.534±0.21a 4.474±0.19a 1496 bicyclogermacrene 46 0.190±0.01d 0.511±0.04a 0.471±0.04ab 0.451±0.04 0.397±0.05c 0.485±0.03ab 0.467±0.03ab 1498 trans-β-guaiene 47 1.191±0.06e 2.010±0.07d 1.927±0.08d 2.682±0.09b 2.519±0.09c 2.795±0.08a 2.565±0.07c 1508 α-bulnesene 48 3.323±0.37f 5.423±0.48de 5.343±0.40e 5.741±0.31c 5.677±0.26cd 6.972±0.16a 6.085±0.15b 1514 γ-cadinene 49 0.859±0.07e 1.214±0.13b 1.146±0.10cd 1.150±0.12c 1.097±0.09d 1.281±0.07a 1.228±0.06b 1521 β-sesquiphellandrene 50 0.486±0.05e 0.612±0.05d 0.651±0.07d 0.698±0.05bc 0.683±0.07cd 0.748±0.06a 0.731±0.04ab 1524 δ-cadinene 51 0.150±0.01e 0.402±0.03d 0.376±0.03d 0.582±0.04a 0.461±0.06c 0.504±0.04b 0.478±0.03bc 1537 α-cadinene 52 0.394±0.02f 0.659±0.05e 0.635±0.06e 0.944±0.07b 0.799±0.06d 0.990±0.07a 0.880±0.06c 1564 (e)-nerolidol 53 -----------0.226±0.01ab 0.197±0.02c 0.223±0.01ab 0.215±0.04bc 0.240±0.02a 0.230±0.01ab 1575 germacrene d-4-ol 54 -----------0.214±0.02ab 0.182±0.02c 0.215±0.02ab 0.190±0.02c 0.228±0.01a 0.195±0.01bc 1577 spathulenol 55 -----------0.523±0.05b 0.494±0.04c 0.529±0.05b 0.485±0.04c 0.556±0.05a 0.516±0.04b 1599 n-hexadecane 56 2.65±0.20d 3.301±0.22b 3.205±0.19bc 3.510±0.22a 3.055±0.17c 3.566±0.10a 3.680±0.11a 1614 1,10-di-epi-cubenol 57 24.679±1.78e 27.520±1.58c 27.402±1.70c 28.510±1.64b 26.620±1.55d 29.062±1.31a 28.737±1.22b 1642 epi-α-cadinol 58 1.039±0.10d 1.663±0.09a 1.611±0.09a 1.404±0.10bc 1.391±0.08c 1.461±0.06b 1.399±0.07c 1649 β-eudesmol 59 1.353±0.10d 1.588±0.09a 1.574±0.11a 1.421±0.09c 1.409±0.09c 1.508±0.06b 1.456±0.07c 1654 α-cadinol 60 0.248±0.01d 0.543±0.03ab 0.544±0.04ab 0.462±0.05c 0.419±0.05c 0.592±0.04a 0.536±0.04b 1685 β-bisabolol 61 data are mean ±standard deviation of eight replications. means followed by the same letter within a row are not significantly different according to duncan’s multiple range test at p< 0.05. 34 v. tavallali, h. gholami, o. espargham tab. 1: phytochemical profile (%) identified in essential oil of sweet basil supplied with diverse zinc and salicylic acid sources. control salicylic salicylic zn-edta zn-edta n[zn(sa)2] n[zn(sa)2] retention compound no. acid 0.2% acid 0.1% 0.2% 0.1% 0.2% 0.1% index ----------0.422±0.05a 0.377±0.03b 0.238±0.02cd 0.212±0.01d 0.251±0.02c 0.258±0.07c 932 α-pinene 1 ----------0.631±0.07a 0.580±0.06b 0.583±0.06b 0.571±0.05b 0.602±0.04b 0.576±0.03b 972 sabinene 2 -----------1.167±0.27a 1.084±0.23a 0.271±0.03b 0.224±0.02c 0.208±0.01c 0.286±0.04b 976 β-pinene 3 -----------3.224±0.59a 3.110±0.52a 0.416±0.03d 0.508±0.04b 0.510±0.03b 0.469±0.02c 990 myrcene 4 3.357±0.43a 0.966±0.07b 0.884±0.07c 0.611±0.05d 3.587±0.45a 0.425±0.03e 0.606±0.04d 999 n-decane 5 -----------0.127±0.02b 0.158±0.03a 0.121±0.03bc 0.091±0.006d 0.132±0.01ab 0.105±0.02cd 1005 α-phellandrene 6 -----------0.167±0.01a 0.092±0.007cd 0.102±0.01bc 0.069±0.005e 0.115±0.02b 0.081±0.007de 1016 α-terpinene 7 -----------0.029±0.004d 0.015±0.002e 0.082±0.005b 0.058±0.006c 0.093±0.007a 0.048±0.003c 1024 p-cymene 8 0.289±0.02b 0.796±0.07a 0.754±0.06a 0.142±0.02cd 0.127±0.02d 0.150±0.01c 0.134±0.01d 1028 limonene 9 3.463±0.66a 1.121±0.31b 1.074±0.20b 0.602±0.05c 3.523±0.50a 0.614±0.05c 1.088±0.11b 1030 1,8-cineole 10 0.397±0.04a 0.125±0.01c 0.101±0.01d 0.145±0.03bc 0.126±0.02c 0.153±0.01b 0.137±0.02bc 1036 (z)-β-ocimene 11 ----------0.039±0.004d 0.018±0.002e 0.085±0.006ab 0.062±0.004c 0.094±0.006a 0.075±0.003b 1041 benzene acetaldehyde 12 -----------0.602±0.07a 0.581±0.05ab 0.561±0.04ab 0.549±0.05b 0.567±0.04ab 0.566±0.03ab 1046 (e)-β-ocimene 13 -----------0.126±0.02b 0.118±0.02b 0.142±0.02a 0.128±0.01b 0.123±0.01b 0.139±0.01a 1057 γ-terpinene 14 ----------0.163±0.03a 0.150±0.02ab 0.145±0.03b 0.137±0.02b 0.120±0.01c 0.151±0. 01ab 1066 cis-sabinene hydrate 15 ----------0.371±0.04a 0.324±0.03bc 0.319±0.04bc 0.281±0.03d 0.332±0.02b 0.309±0.02c 1088 terpinolene 16 10.850±1.89a 5.733±0.51b 5.696±0.45b 0.593±0.05c 0.584±0.06c 0.516±0.04d 0.590±0.04c 1099 linalool 17 -----------0.136±0.02a 0.129±0.02a 0.072±0.007b 0.061±0.004c 0.079±0.005b 0.062±0.004c 1112 1-octen-3-yl acetate 18 -----------0.089±0.009d 0.058±0.006e 0.119±0.01ab 0.102±0.03c 0.128±0.01a 0.114±0.01b 1144 camphor 19 -----------0.220±0.02a 0.190±0.03b 0.161±0.02c 0.132±0.02d 0.198±0.02b 0.170±0.02c 1166 δ-terpineol 20 -----------0.097±0.007a 0.077±0.005b 0.076±0.006b 0.066±0.006c 0.097±0.007a 0.090±0.002a 1177 terpinen-4-ol 21 0.483±0.05f 1.360±0.18b 2.110±0.26a 0.703±0.05d 0.682±0.05de 0.664±0.04e 0.761±0.04c 1190 α-terpineol 22 0.783±0.07a 0.203±0.04e 0.177±0.03e 0.690±0.06b 0.512±0.04c 0.414±0.03d 0.565±0.03c 1199 n-dodecane 23 0.965±0.07a 0.821±0.08b 0.798±0.07b 0.589±0.06c 0.346±0.04d 0.604±0.05c 0.605±0.04c 1212 octanol acetate 24 0.248±0.01a 0.098±0.009c 0.088±0.007c 0.195±0.01b 0.101±0.009c 0.199±0.02b 0.250±0.02a 1222 trans-carveol 25 -----------0.129±0.02b 0.108±0.02b 0.791±0.06a 0.753±0.06a 0.043±0.003c 0.036±0.003c 1256 linalyl acetate 26 6.657±1.03a 1.843±0.28d 2.644±0.36b 1.692±0.23 2.395±0.18c 1.005±0.08f 1.332±0.12e 1286 bornyl acetate 27 0.146±0.01f 0.219±0.03e 0.184±0.03e 0.562±0.05a 0.365±0.03d 0.497±0.03b 0.444±0.03c 1337 δ-elemene 28 0.095±0.007c 0.224±0.03a 0.199±0.04a 0.154±0.02b 0.135±0.02b 0.135±0.01b 0.146±0.01b 1349 α-terpinyl acetate 29 12.704±1.77a 1.140±0.21e 2.288±0.27b 1.711±0.12c 1.389±0.09d 0.403±0.03g 0.741±0.05f 1358 eugenol 30 0.056±0.006d 0.121±0.01b 0.091±0.008c 0.314±0.04a 0.291±0.04a 0.292±0.02a 0.297±0.02a 1375 α-copaene 31 0.084±0.009d 0.130±0.01c 0.060±0.007e 0.214±0.02a 0.130±0.02c 0.222±0.02a 0.168±0.01b 1390 β-cubebene 32 4.894±0.42d 6.117±0.49c 6.083±0.41c 7.520±0.30b 7.245±0.25b 7.970±0.18a 7.877±0.21a 1392 β-elemene 33 -----------0.092±0.009d 0.078±0.008d 0.339±0.04a 0.253±0.03c 0.256±0.01c 0.288±0.02b 1400 n-tetradeane 34 0.330±0.02cd 0.715±0.05a 0.702±0.06a 0.392±0.04b 0.345±0.03c 0.309±0.02d 0.341±0.02c 1405 methyl eugenol 35 -----------0.072±0.007a 0.051±0.006b 0.072±0.005a 0.051±0.004b 0.076±0.005a 0.053±0.006b 1415 cis-α-bergamotene 36 0.196±0.02e 0.338±0.04d 0.308±0.05d 0.597±0.04a 0.469±0.03c 0.563±0.04b 0.587±0.04ab 1418 (e)-caryophyllene 37 -----------0.051±0.005d 0.036±0.004e 0.077±0.005a 0.039±0.003e 0.070±0.004b 0.059±0.005c 1428 β-gurjunene 38 8.369±0.86d 9.222±0.92c 9.093±0.87c 11.150±1.08b 10.920±1.06b 11.903±1.10a 11.838±1.02a 1436 trans-α-bergamotene 39 0.514±0.06d 0.606±0.07c 0.565±0.05c 1.148±0.11a 0.943±0.07b 1.108±0.09a 0.938±0.06b 1438 α-guaiene 40 0.063±0.006e 0.233±0.03a 0.186±0.02c 0.207±0.01abc 0.132±0.01d 0.219±0.02ab 0.196±0.01bc 1443 (z)-β-farnesene 41 1.323±0.12e 2.157±0.15d 1.968±0.17d 2.814±0.19b 2.752±0.18c 2.897±0.18a 2.912±0.17a 1453 α-humulene 42 0.247±0.01e 0.448±0.05c 0.403±0.04d 0.459±0.05bc 0.392±0.04 0.560±0.04a 0.488±0.03b 1457 (e)-β-farnesene 43 0.993±0.11f 1.521±0.17d 1.406±0.14e 1.992±0.12a 1.649±0.09c 1.007±0.07f 1.860±0.08b 1462 allo-aromadendrene 44 4.026±0.47f 6.133±0.60e 6.098±0.64e 7.254±0.55c 7.007±0.52d 7.561±0.41a 7.453±0.19b 1480 germacrene d 45 1.901±0.32d 3.197±0.30c 2.913±0.21c 4.195±0.37b 4.071±0.34b 4.534±0.21a 4.474±0.19a 1496 bicyclogermacrene 46 0.190±0.01d 0.511±0.04a 0.471±0.04ab 0.451±0.04 0.397±0.05c 0.485±0.03ab 0.467±0.03ab 1498 trans-β-guaiene 47 1.191±0.06e 2.010±0.07d 1.927±0.08d 2.682±0.09b 2.519±0.09c 2.795±0.08a 2.565±0.07c 1508 α-bulnesene 48 3.323±0.37f 5.423±0.48de 5.343±0.40e 5.741±0.31c 5.677±0.26cd 6.972±0.16a 6.085±0.15b 1514 γ-cadinene 49 0.859±0.07e 1.214±0.13b 1.146±0.10cd 1.150±0.12c 1.097±0.09d 1.281±0.07a 1.228±0.06b 1521 β-sesquiphellandrene 50 0.486±0.05e 0.612±0.05d 0.651±0.07d 0.698±0.05bc 0.683±0.07cd 0.748±0.06a 0.731±0.04ab 1524 δ-cadinene 51 0.150±0.01e 0.402±0.03d 0.376±0.03d 0.582±0.04a 0.461±0.06c 0.504±0.04b 0.478±0.03bc 1537 α-cadinene 52 0.394±0.02f 0.659±0.05e 0.635±0.06e 0.944±0.07b 0.799±0.06d 0.990±0.07a 0.880±0.06c 1564 (e)-nerolidol 53 -----------0.226±0.01ab 0.197±0.02c 0.223±0.01ab 0.215±0.04bc 0.240±0.02a 0.230±0.01ab 1575 germacrene d-4-ol 54 -----------0.214±0.02ab 0.182±0.02c 0.215±0.02ab 0.190±0.02c 0.228±0.01a 0.195±0.01bc 1577 spathulenol 55 -----------0.523±0.05b 0.494±0.04c 0.529±0.05b 0.485±0.04c 0.556±0.05a 0.516±0.04b 1599 n-hexadecane 56 2.65±0.20d 3.301±0.22b 3.205±0.19bc 3.510±0.22a 3.055±0.17c 3.566±0.10a 3.680±0.11a 1614 1,10-di-epi-cubenol 57 24.679±1.78e 27.520±1.58c 27.402±1.70c 28.510±1.64b 26.620±1.55d 29.062±1.31a 28.737±1.22b 1642 epi-α-cadinol 58 1.039±0.10d 1.663±0.09a 1.611±0.09a 1.404±0.10bc 1.391±0.08c 1.461±0.06b 1.399±0.07c 1649 β-eudesmol 59 1.353±0.10d 1.588±0.09a 1.574±0.11a 1.421±0.09c 1.409±0.09c 1.508±0.06b 1.456±0.07c 1654 α-cadinol 60 0.248±0.01d 0.543±0.03ab 0.544±0.04ab 0.462±0.05c 0.419±0.05c 0.592±0.04a 0.536±0.04b 1685 β-bisabolol 61 data are mean ±standard deviation of eight replications. means followed by the same letter within a row are not significantly different according to duncan’s multiple range test at p< 0.05. 34 v. tavallali, h. gholami, o. espargham tab. 1: phytochemical profile (%) identified in essential oil of sweet basil supplied with diverse zinc and salicylic acid sources. control salicylic salicylic zn-edta zn-edta n[zn(sa)2] n[zn(sa)2] retention compound no. acid 0.2% acid 0.1% 0.2% 0.1% 0.2% 0.1% index ----------0.422±0.05a 0.377±0.03b 0.238±0.02cd 0.212±0.01d 0.251±0.02c 0.258±0.07c 932 α-pinene 1 ----------0.631±0.07a 0.580±0.06b 0.583±0.06b 0.571±0.05b 0.602±0.04b 0.576±0.03b 972 sabinene 2 -----------1.167±0.27a 1.084±0.23a 0.271±0.03b 0.224±0.02c 0.208±0.01c 0.286±0.04b 976 β-pinene 3 -----------3.224±0.59a 3.110±0.52a 0.416±0.03d 0.508±0.04b 0.510±0.03b 0.469±0.02c 990 myrcene 4 3.357±0.43a 0.966±0.07b 0.884±0.07c 0.611±0.05d 3.587±0.45a 0.425±0.03e 0.606±0.04d 999 n-decane 5 -----------0.127±0.02b 0.158±0.03a 0.121±0.03bc 0.091±0.006d 0.132±0.01ab 0.105±0.02cd 1005 α-phellandrene 6 -----------0.167±0.01a 0.092±0.007cd 0.102±0.01bc 0.069±0.005e 0.115±0.02b 0.081±0.007de 1016 α-terpinene 7 -----------0.029±0.004d 0.015±0.002e 0.082±0.005b 0.058±0.006c 0.093±0.007a 0.048±0.003c 1024 p-cymene 8 0.289±0.02b 0.796±0.07a 0.754±0.06a 0.142±0.02cd 0.127±0.02d 0.150±0.01c 0.134±0.01d 1028 limonene 9 3.463±0.66a 1.121±0.31b 1.074±0.20b 0.602±0.05c 3.523±0.50a 0.614±0.05c 1.088±0.11b 1030 1,8-cineole 10 0.397±0.04a 0.125±0.01c 0.101±0.01d 0.145±0.03bc 0.126±0.02c 0.153±0.01b 0.137±0.02bc 1036 (z)-β-ocimene 11 ----------0.039±0.004d 0.018±0.002e 0.085±0.006ab 0.062±0.004c 0.094±0.006a 0.075±0.003b 1041 benzene acetaldehyde 12 -----------0.602±0.07a 0.581±0.05ab 0.561±0.04ab 0.549±0.05b 0.567±0.04ab 0.566±0.03ab 1046 (e)-β-ocimene 13 -----------0.126±0.02b 0.118±0.02b 0.142±0.02a 0.128±0.01b 0.123±0.01b 0.139±0.01a 1057 γ-terpinene 14 ----------0.163±0.03a 0.150±0.02ab 0.145±0.03b 0.137±0.02b 0.120±0.01c 0.151±0. 01ab 1066 cis-sabinene hydrate 15 ----------0.371±0.04a 0.324±0.03bc 0.319±0.04bc 0.281±0.03d 0.332±0.02b 0.309±0.02c 1088 terpinolene 16 10.850±1.89a 5.733±0.51b 5.696±0.45b 0.593±0.05c 0.584±0.06c 0.516±0.04d 0.590±0.04c 1099 linalool 17 -----------0.136±0.02a 0.129±0.02a 0.072±0.007b 0.061±0.004c 0.079±0.005b 0.062±0.004c 1112 1-octen-3-yl acetate 18 -----------0.089±0.009d 0.058±0.006e 0.119±0.01ab 0.102±0.03c 0.128±0.01a 0.114±0.01b 1144 camphor 19 -----------0.220±0.02a 0.190±0.03b 0.161±0.02c 0.132±0.02d 0.198±0.02b 0.170±0.02c 1166 δ-terpineol 20 -----------0.097±0.007a 0.077±0.005b 0.076±0.006b 0.066±0.006c 0.097±0.007a 0.090±0.002a 1177 terpinen-4-ol 21 0.483±0.05f 1.360±0.18b 2.110±0.26a 0.703±0.05d 0.682±0.05de 0.664±0.04e 0.761±0.04c 1190 α-terpineol 22 0.783±0.07a 0.203±0.04e 0.177±0.03e 0.690±0.06b 0.512±0.04c 0.414±0.03d 0.565±0.03c 1199 n-dodecane 23 0.965±0.07a 0.821±0.08b 0.798±0.07b 0.589±0.06c 0.346±0.04d 0.604±0.05c 0.605±0.04c 1212 octanol acetate 24 0.248±0.01a 0.098±0.009c 0.088±0.007c 0.195±0.01b 0.101±0.009c 0.199±0.02b 0.250±0.02a 1222 trans-carveol 25 -----------0.129±0.02b 0.108±0.02b 0.791±0.06a 0.753±0.06a 0.043±0.003c 0.036±0.003c 1256 linalyl acetate 26 6.657±1.03a 1.843±0.28d 2.644±0.36b 1.692±0.23 2.395±0.18c 1.005±0.08f 1.332±0.12e 1286 bornyl acetate 27 0.146±0.01f 0.219±0.03e 0.184±0.03e 0.562±0.05a 0.365±0.03d 0.497±0.03b 0.444±0.03c 1337 δ-elemene 28 0.095±0.007c 0.224±0.03a 0.199±0.04a 0.154±0.02b 0.135±0.02b 0.135±0.01b 0.146±0.01b 1349 α-terpinyl acetate 29 12.704±1.77a 1.140±0.21e 2.288±0.27b 1.711±0.12c 1.389±0.09d 0.403±0.03g 0.741±0.05f 1358 eugenol 30 0.056±0.006d 0.121±0.01b 0.091±0.008c 0.314±0.04a 0.291±0.04a 0.292±0.02a 0.297±0.02a 1375 α-copaene 31 0.084±0.009d 0.130±0.01c 0.060±0.007e 0.214±0.02a 0.130±0.02c 0.222±0.02a 0.168±0.01b 1390 β-cubebene 32 4.894±0.42d 6.117±0.49c 6.083±0.41c 7.520±0.30b 7.245±0.25b 7.970±0.18a 7.877±0.21a 1392 β-elemene 33 -----------0.092±0.009d 0.078±0.008d 0.339±0.04a 0.253±0.03c 0.256±0.01c 0.288±0.02b 1400 n-tetradeane 34 0.330±0.02cd 0.715±0.05a 0.702±0.06a 0.392±0.04b 0.345±0.03c 0.309±0.02d 0.341±0.02c 1405 methyl eugenol 35 -----------0.072±0.007a 0.051±0.006b 0.072±0.005a 0.051±0.004b 0.076±0.005a 0.053±0.006b 1415 cis-α-bergamotene 36 0.196±0.02e 0.338±0.04d 0.308±0.05d 0.597±0.04a 0.469±0.03c 0.563±0.04b 0.587±0.04ab 1418 (e)-caryophyllene 37 -----------0.051±0.005d 0.036±0.004e 0.077±0.005a 0.039±0.003e 0.070±0.004b 0.059±0.005c 1428 β-gurjunene 38 8.369±0.86d 9.222±0.92c 9.093±0.87c 11.150±1.08b 10.920±1.06b 11.903±1.10a 11.838±1.02a 1436 trans-α-bergamotene 39 0.514±0.06d 0.606±0.07c 0.565±0.05c 1.148±0.11a 0.943±0.07b 1.108±0.09a 0.938±0.06b 1438 α-guaiene 40 0.063±0.006e 0.233±0.03a 0.186±0.02c 0.207±0.01abc 0.132±0.01d 0.219±0.02ab 0.196±0.01bc 1443 (z)-β-farnesene 41 1.323±0.12e 2.157±0.15d 1.968±0.17d 2.814±0.19b 2.752±0.18c 2.897±0.18a 2.912±0.17a 1453 α-humulene 42 0.247±0.01e 0.448±0.05c 0.403±0.04d 0.459±0.05bc 0.392±0.04 0.560±0.04a 0.488±0.03b 1457 (e)-β-farnesene 43 0.993±0.11f 1.521±0.17d 1.406±0.14e 1.992±0.12a 1.649±0.09c 1.007±0.07f 1.860±0.08b 1462 allo-aromadendrene 44 4.026±0.47f 6.133±0.60e 6.098±0.64e 7.254±0.55c 7.007±0.52d 7.561±0.41a 7.453±0.19b 1480 germacrene d 45 1.901±0.32d 3.197±0.30c 2.913±0.21c 4.195±0.37b 4.071±0.34b 4.534±0.21a 4.474±0.19a 1496 bicyclogermacrene 46 0.190±0.01d 0.511±0.04a 0.471±0.04ab 0.451±0.04 0.397±0.05c 0.485±0.03ab 0.467±0.03ab 1498 trans-β-guaiene 47 1.191±0.06e 2.010±0.07d 1.927±0.08d 2.682±0.09b 2.519±0.09c 2.795±0.08a 2.565±0.07c 1508 α-bulnesene 48 3.323±0.37f 5.423±0.48de 5.343±0.40e 5.741±0.31c 5.677±0.26cd 6.972±0.16a 6.085±0.15b 1514 γ-cadinene 49 0.859±0.07e 1.214±0.13b 1.146±0.10cd 1.150±0.12c 1.097±0.09d 1.281±0.07a 1.228±0.06b 1521 β-sesquiphellandrene 50 0.486±0.05e 0.612±0.05d 0.651±0.07d 0.698±0.05bc 0.683±0.07cd 0.748±0.06a 0.731±0.04ab 1524 δ-cadinene 51 0.150±0.01e 0.402±0.03d 0.376±0.03d 0.582±0.04a 0.461±0.06c 0.504±0.04b 0.478±0.03bc 1537 α-cadinene 52 0.394±0.02f 0.659±0.05e 0.635±0.06e 0.944±0.07b 0.799±0.06d 0.990±0.07a 0.880±0.06c 1564 (e)-nerolidol 53 -----------0.226±0.01ab 0.197±0.02c 0.223±0.01ab 0.215±0.04bc 0.240±0.02a 0.230±0.01ab 1575 germacrene d-4-ol 54 -----------0.214±0.02ab 0.182±0.02c 0.215±0.02ab 0.190±0.02c 0.228±0.01a 0.195±0.01bc 1577 spathulenol 55 -----------0.523±0.05b 0.494±0.04c 0.529±0.05b 0.485±0.04c 0.556±0.05a 0.516±0.04b 1599 n-hexadecane 56 2.65±0.20d 3.301±0.22b 3.205±0.19bc 3.510±0.22a 3.055±0.17c 3.566±0.10a 3.680±0.11a 1614 1,10-di-epi-cubenol 57 24.679±1.78e 27.520±1.58c 27.402±1.70c 28.510±1.64b 26.620±1.55d 29.062±1.31a 28.737±1.22b 1642 epi-α-cadinol 58 1.039±0.10d 1.663±0.09a 1.611±0.09a 1.404±0.10bc 1.391±0.08c 1.461±0.06b 1.399±0.07c 1649 β-eudesmol 59 1.353±0.10d 1.588±0.09a 1.574±0.11a 1.421±0.09c 1.409±0.09c 1.508±0.06b 1.456±0.07c 1654 α-cadinol 60 0.248±0.01d 0.543±0.03ab 0.544±0.04ab 0.462±0.05c 0.419±0.05c 0.592±0.04a 0.536±0.04b 1685 β-bisabolol 61 data are mean ±standard deviation of eight replications. means followed by the same letter within a row are not significantly different according to duncan’s multiple range test at p< 0.05. 34 v. tavallali, h. gholami, o. espargham tab. 1: phytochemical profile (%) identified in essential oil of sweet basil supplied with diverse zinc and salicylic acid sources. control salicylic salicylic zn-edta zn-edta n[zn(sa)2] n[zn(sa)2] retention compound no. acid 0.2% acid 0.1% 0.2% 0.1% 0.2% 0.1% index ----------0.422±0.05a 0.377±0.03b 0.238±0.02cd 0.212±0.01d 0.251±0.02c 0.258±0.07c 932 α-pinene 1 ----------0.631±0.07a 0.580±0.06b 0.583±0.06b 0.571±0.05b 0.602±0.04b 0.576±0.03b 972 sabinene 2 -----------1.167±0.27a 1.084±0.23a 0.271±0.03b 0.224±0.02c 0.208±0.01c 0.286±0.04b 976 β-pinene 3 -----------3.224±0.59a 3.110±0.52a 0.416±0.03d 0.508±0.04b 0.510±0.03b 0.469±0.02c 990 myrcene 4 3.357±0.43a 0.966±0.07b 0.884±0.07c 0.611±0.05d 3.587±0.45a 0.425±0.03e 0.606±0.04d 999 n-decane 5 -----------0.127±0.02b 0.158±0.03a 0.121±0.03bc 0.091±0.006d 0.132±0.01ab 0.105±0.02cd 1005 α-phellandrene 6 -----------0.167±0.01a 0.092±0.007cd 0.102±0.01bc 0.069±0.005e 0.115±0.02b 0.081±0.007de 1016 α-terpinene 7 -----------0.029±0.004d 0.015±0.002e 0.082±0.005b 0.058±0.006c 0.093±0.007a 0.048±0.003c 1024 p-cymene 8 0.289±0.02b 0.796±0.07a 0.754±0.06a 0.142±0.02cd 0.127±0.02d 0.150±0.01c 0.134±0.01d 1028 limonene 9 3.463±0.66a 1.121±0.31b 1.074±0.20b 0.602±0.05c 3.523±0.50a 0.614±0.05c 1.088±0.11b 1030 1,8-cineole 10 0.397±0.04a 0.125±0.01c 0.101±0.01d 0.145±0.03bc 0.126±0.02c 0.153±0.01b 0.137±0.02bc 1036 (z)-β-ocimene 11 ----------0.039±0.004d 0.018±0.002e 0.085±0.006ab 0.062±0.004c 0.094±0.006a 0.075±0.003b 1041 benzene acetaldehyde 12 -----------0.602±0.07a 0.581±0.05ab 0.561±0.04ab 0.549±0.05b 0.567±0.04ab 0.566±0.03ab 1046 (e)-β-ocimene 13 -----------0.126±0.02b 0.118±0.02b 0.142±0.02a 0.128±0.01b 0.123±0.01b 0.139±0.01a 1057 γ-terpinene 14 ----------0.163±0.03a 0.150±0.02ab 0.145±0.03b 0.137±0.02b 0.120±0.01c 0.151±0. 01ab 1066 cis-sabinene hydrate 15 ----------0.371±0.04a 0.324±0.03bc 0.319±0.04bc 0.281±0.03d 0.332±0.02b 0.309±0.02c 1088 terpinolene 16 10.850±1.89a 5.733±0.51b 5.696±0.45b 0.593±0.05c 0.584±0.06c 0.516±0.04d 0.590±0.04c 1099 linalool 17 -----------0.136±0.02a 0.129±0.02a 0.072±0.007b 0.061±0.004c 0.079±0.005b 0.062±0.004c 1112 1-octen-3-yl acetate 18 -----------0.089±0.009d 0.058±0.006e 0.119±0.01ab 0.102±0.03c 0.128±0.01a 0.114±0.01b 1144 camphor 19 -----------0.220±0.02a 0.190±0.03b 0.161±0.02c 0.132±0.02d 0.198±0.02b 0.170±0.02c 1166 δ-terpineol 20 -----------0.097±0.007a 0.077±0.005b 0.076±0.006b 0.066±0.006c 0.097±0.007a 0.090±0.002a 1177 terpinen-4-ol 21 0.483±0.05f 1.360±0.18b 2.110±0.26a 0.703±0.05d 0.682±0.05de 0.664±0.04e 0.761±0.04c 1190 α-terpineol 22 0.783±0.07a 0.203±0.04e 0.177±0.03e 0.690±0.06b 0.512±0.04c 0.414±0.03d 0.565±0.03c 1199 n-dodecane 23 0.965±0.07a 0.821±0.08b 0.798±0.07b 0.589±0.06c 0.346±0.04d 0.604±0.05c 0.605±0.04c 1212 octanol acetate 24 0.248±0.01a 0.098±0.009c 0.088±0.007c 0.195±0.01b 0.101±0.009c 0.199±0.02b 0.250±0.02a 1222 trans-carveol 25 -----------0.129±0.02b 0.108±0.02b 0.791±0.06a 0.753±0.06a 0.043±0.003c 0.036±0.003c 1256 linalyl acetate 26 6.657±1.03a 1.843±0.28d 2.644±0.36b 1.692±0.23 2.395±0.18c 1.005±0.08f 1.332±0.12e 1286 bornyl acetate 27 0.146±0.01f 0.219±0.03e 0.184±0.03e 0.562±0.05a 0.365±0.03d 0.497±0.03b 0.444±0.03c 1337 δ-elemene 28 0.095±0.007c 0.224±0.03a 0.199±0.04a 0.154±0.02b 0.135±0.02b 0.135±0.01b 0.146±0.01b 1349 α-terpinyl acetate 29 12.704±1.77a 1.140±0.21e 2.288±0.27b 1.711±0.12c 1.389±0.09d 0.403±0.03g 0.741±0.05f 1358 eugenol 30 0.056±0.006d 0.121±0.01b 0.091±0.008c 0.314±0.04a 0.291±0.04a 0.292±0.02a 0.297±0.02a 1375 α-copaene 31 0.084±0.009d 0.130±0.01c 0.060±0.007e 0.214±0.02a 0.130±0.02c 0.222±0.02a 0.168±0.01b 1390 β-cubebene 32 4.894±0.42d 6.117±0.49c 6.083±0.41c 7.520±0.30b 7.245±0.25b 7.970±0.18a 7.877±0.21a 1392 β-elemene 33 -----------0.092±0.009d 0.078±0.008d 0.339±0.04a 0.253±0.03c 0.256±0.01c 0.288±0.02b 1400 n-tetradeane 34 0.330±0.02cd 0.715±0.05a 0.702±0.06a 0.392±0.04b 0.345±0.03c 0.309±0.02d 0.341±0.02c 1405 methyl eugenol 35 -----------0.072±0.007a 0.051±0.006b 0.072±0.005a 0.051±0.004b 0.076±0.005a 0.053±0.006b 1415 cis-α-bergamotene 36 0.196±0.02e 0.338±0.04d 0.308±0.05d 0.597±0.04a 0.469±0.03c 0.563±0.04b 0.587±0.04ab 1418 (e)-caryophyllene 37 -----------0.051±0.005d 0.036±0.004e 0.077±0.005a 0.039±0.003e 0.070±0.004b 0.059±0.005c 1428 β-gurjunene 38 8.369±0.86d 9.222±0.92c 9.093±0.87c 11.150±1.08b 10.920±1.06b 11.903±1.10a 11.838±1.02a 1436 trans-α-bergamotene 39 0.514±0.06d 0.606±0.07c 0.565±0.05c 1.148±0.11a 0.943±0.07b 1.108±0.09a 0.938±0.06b 1438 α-guaiene 40 0.063±0.006e 0.233±0.03a 0.186±0.02c 0.207±0.01abc 0.132±0.01d 0.219±0.02ab 0.196±0.01bc 1443 (z)-β-farnesene 41 1.323±0.12e 2.157±0.15d 1.968±0.17d 2.814±0.19b 2.752±0.18c 2.897±0.18a 2.912±0.17a 1453 α-humulene 42 0.247±0.01e 0.448±0.05c 0.403±0.04d 0.459±0.05bc 0.392±0.04 0.560±0.04a 0.488±0.03b 1457 (e)-β-farnesene 43 0.993±0.11f 1.521±0.17d 1.406±0.14e 1.992±0.12a 1.649±0.09c 1.007±0.07f 1.860±0.08b 1462 allo-aromadendrene 44 4.026±0.47f 6.133±0.60e 6.098±0.64e 7.254±0.55c 7.007±0.52d 7.561±0.41a 7.453±0.19b 1480 germacrene d 45 1.901±0.32d 3.197±0.30c 2.913±0.21c 4.195±0.37b 4.071±0.34b 4.534±0.21a 4.474±0.19a 1496 bicyclogermacrene 46 0.190±0.01d 0.511±0.04a 0.471±0.04ab 0.451±0.04 0.397±0.05c 0.485±0.03ab 0.467±0.03ab 1498 trans-β-guaiene 47 1.191±0.06e 2.010±0.07d 1.927±0.08d 2.682±0.09b 2.519±0.09c 2.795±0.08a 2.565±0.07c 1508 α-bulnesene 48 3.323±0.37f 5.423±0.48de 5.343±0.40e 5.741±0.31c 5.677±0.26cd 6.972±0.16a 6.085±0.15b 1514 γ-cadinene 49 0.859±0.07e 1.214±0.13b 1.146±0.10cd 1.150±0.12c 1.097±0.09d 1.281±0.07a 1.228±0.06b 1521 β-sesquiphellandrene 50 0.486±0.05e 0.612±0.05d 0.651±0.07d 0.698±0.05bc 0.683±0.07cd 0.748±0.06a 0.731±0.04ab 1524 δ-cadinene 51 0.150±0.01e 0.402±0.03d 0.376±0.03d 0.582±0.04a 0.461±0.06c 0.504±0.04b 0.478±0.03bc 1537 α-cadinene 52 0.394±0.02f 0.659±0.05e 0.635±0.06e 0.944±0.07b 0.799±0.06d 0.990±0.07a 0.880±0.06c 1564 (e)-nerolidol 53 -----------0.226±0.01ab 0.197±0.02c 0.223±0.01ab 0.215±0.04bc 0.240±0.02a 0.230±0.01ab 1575 germacrene d-4-ol 54 -----------0.214±0.02ab 0.182±0.02c 0.215±0.02ab 0.190±0.02c 0.228±0.01a 0.195±0.01bc 1577 spathulenol 55 -----------0.523±0.05b 0.494±0.04c 0.529±0.05b 0.485±0.04c 0.556±0.05a 0.516±0.04b 1599 n-hexadecane 56 2.65±0.20d 3.301±0.22b 3.205±0.19bc 3.510±0.22a 3.055±0.17c 3.566±0.10a 3.680±0.11a 1614 1,10-di-epi-cubenol 57 24.679±1.78e 27.520±1.58c 27.402±1.70c 28.510±1.64b 26.620±1.55d 29.062±1.31a 28.737±1.22b 1642 epi-α-cadinol 58 1.039±0.10d 1.663±0.09a 1.611±0.09a 1.404±0.10bc 1.391±0.08c 1.461±0.06b 1.399±0.07c 1649 β-eudesmol 59 1.353±0.10d 1.588±0.09a 1.574±0.11a 1.421±0.09c 1.409±0.09c 1.508±0.06b 1.456±0.07c 1654 α-cadinol 60 0.248±0.01d 0.543±0.03ab 0.544±0.04ab 0.462±0.05c 0.419±0.05c 0.592±0.04a 0.536±0.04b 1685 β-bisabolol 61 data are mean ±standard deviation of eight replications. means followed by the same letter within a row are not significantly different according to duncan’s multiple range test at p< 0.05. 34 v. tavallali, h. gholami, o. espargham tab. 1: phytochemical profile (%) identified in essential oil of sweet basil supplied with diverse zinc and salicylic acid sources. control salicylic salicylic zn-edta zn-edta n[zn(sa)2] n[zn(sa)2] retention compound no. acid 0.2% acid 0.1% 0.2% 0.1% 0.2% 0.1% index ----------0.422±0.05a 0.377±0.03b 0.238±0.02cd 0.212±0.01d 0.251±0.02c 0.258±0.07c 932 α-pinene 1 ----------0.631±0.07a 0.580±0.06b 0.583±0.06b 0.571±0.05b 0.602±0.04b 0.576±0.03b 972 sabinene 2 -----------1.167±0.27a 1.084±0.23a 0.271±0.03b 0.224±0.02c 0.208±0.01c 0.286±0.04b 976 β-pinene 3 -----------3.224±0.59a 3.110±0.52a 0.416±0.03d 0.508±0.04b 0.510±0.03b 0.469±0.02c 990 myrcene 4 3.357±0.43a 0.966±0.07b 0.884±0.07c 0.611±0.05d 3.587±0.45a 0.425±0.03e 0.606±0.04d 999 n-decane 5 -----------0.127±0.02b 0.158±0.03a 0.121±0.03bc 0.091±0.006d 0.132±0.01ab 0.105±0.02cd 1005 α-phellandrene 6 -----------0.167±0.01a 0.092±0.007cd 0.102±0.01bc 0.069±0.005e 0.115±0.02b 0.081±0.007de 1016 α-terpinene 7 -----------0.029±0.004d 0.015±0.002e 0.082±0.005b 0.058±0.006c 0.093±0.007a 0.048±0.003c 1024 p-cymene 8 0.289±0.02b 0.796±0.07a 0.754±0.06a 0.142±0.02cd 0.127±0.02d 0.150±0.01c 0.134±0.01d 1028 limonene 9 3.463±0.66a 1.121±0.31b 1.074±0.20b 0.602±0.05c 3.523±0.50a 0.614±0.05c 1.088±0.11b 1030 1,8-cineole 10 0.397±0.04a 0.125±0.01c 0.101±0.01d 0.145±0.03bc 0.126±0.02c 0.153±0.01b 0.137±0.02bc 1036 (z)-β-ocimene 11 ----------0.039±0.004d 0.018±0.002e 0.085±0.006ab 0.062±0.004c 0.094±0.006a 0.075±0.003b 1041 benzene acetaldehyde 12 -----------0.602±0.07a 0.581±0.05ab 0.561±0.04ab 0.549±0.05b 0.567±0.04ab 0.566±0.03ab 1046 (e)-β-ocimene 13 -----------0.126±0.02b 0.118±0.02b 0.142±0.02a 0.128±0.01b 0.123±0.01b 0.139±0.01a 1057 γ-terpinene 14 ----------0.163±0.03a 0.150±0.02ab 0.145±0.03b 0.137±0.02b 0.120±0.01c 0.151±0. 01ab 1066 cis-sabinene hydrate 15 ----------0.371±0.04a 0.324±0.03bc 0.319±0.04bc 0.281±0.03d 0.332±0.02b 0.309±0.02c 1088 terpinolene 16 10.850±1.89a 5.733±0.51b 5.696±0.45b 0.593±0.05c 0.584±0.06c 0.516±0.04d 0.590±0.04c 1099 linalool 17 -----------0.136±0.02a 0.129±0.02a 0.072±0.007b 0.061±0.004c 0.079±0.005b 0.062±0.004c 1112 1-octen-3-yl acetate 18 -----------0.089±0.009d 0.058±0.006e 0.119±0.01ab 0.102±0.03c 0.128±0.01a 0.114±0.01b 1144 camphor 19 -----------0.220±0.02a 0.190±0.03b 0.161±0.02c 0.132±0.02d 0.198±0.02b 0.170±0.02c 1166 δ-terpineol 20 -----------0.097±0.007a 0.077±0.005b 0.076±0.006b 0.066±0.006c 0.097±0.007a 0.090±0.002a 1177 terpinen-4-ol 21 0.483±0.05f 1.360±0.18b 2.110±0.26a 0.703±0.05d 0.682±0.05de 0.664±0.04e 0.761±0.04c 1190 α-terpineol 22 0.783±0.07a 0.203±0.04e 0.177±0.03e 0.690±0.06b 0.512±0.04c 0.414±0.03d 0.565±0.03c 1199 n-dodecane 23 0.965±0.07a 0.821±0.08b 0.798±0.07b 0.589±0.06c 0.346±0.04d 0.604±0.05c 0.605±0.04c 1212 octanol acetate 24 0.248±0.01a 0.098±0.009c 0.088±0.007c 0.195±0.01b 0.101±0.009c 0.199±0.02b 0.250±0.02a 1222 trans-carveol 25 -----------0.129±0.02b 0.108±0.02b 0.791±0.06a 0.753±0.06a 0.043±0.003c 0.036±0.003c 1256 linalyl acetate 26 6.657±1.03a 1.843±0.28d 2.644±0.36b 1.692±0.23 2.395±0.18c 1.005±0.08f 1.332±0.12e 1286 bornyl acetate 27 0.146±0.01f 0.219±0.03e 0.184±0.03e 0.562±0.05a 0.365±0.03d 0.497±0.03b 0.444±0.03c 1337 δ-elemene 28 0.095±0.007c 0.224±0.03a 0.199±0.04a 0.154±0.02b 0.135±0.02b 0.135±0.01b 0.146±0.01b 1349 α-terpinyl acetate 29 12.704±1.77a 1.140±0.21e 2.288±0.27b 1.711±0.12c 1.389±0.09d 0.403±0.03g 0.741±0.05f 1358 eugenol 30 0.056±0.006d 0.121±0.01b 0.091±0.008c 0.314±0.04a 0.291±0.04a 0.292±0.02a 0.297±0.02a 1375 α-copaene 31 0.084±0.009d 0.130±0.01c 0.060±0.007e 0.214±0.02a 0.130±0.02c 0.222±0.02a 0.168±0.01b 1390 β-cubebene 32 4.894±0.42d 6.117±0.49c 6.083±0.41c 7.520±0.30b 7.245±0.25b 7.970±0.18a 7.877±0.21a 1392 β-elemene 33 -----------0.092±0.009d 0.078±0.008d 0.339±0.04a 0.253±0.03c 0.256±0.01c 0.288±0.02b 1400 n-tetradeane 34 0.330±0.02cd 0.715±0.05a 0.702±0.06a 0.392±0.04b 0.345±0.03c 0.309±0.02d 0.341±0.02c 1405 methyl eugenol 35 -----------0.072±0.007a 0.051±0.006b 0.072±0.005a 0.051±0.004b 0.076±0.005a 0.053±0.006b 1415 cis-α-bergamotene 36 0.196±0.02e 0.338±0.04d 0.308±0.05d 0.597±0.04a 0.469±0.03c 0.563±0.04b 0.587±0.04ab 1418 (e)-caryophyllene 37 -----------0.051±0.005d 0.036±0.004e 0.077±0.005a 0.039±0.003e 0.070±0.004b 0.059±0.005c 1428 β-gurjunene 38 8.369±0.86d 9.222±0.92c 9.093±0.87c 11.150±1.08b 10.920±1.06b 11.903±1.10a 11.838±1.02a 1436 trans-α-bergamotene 39 0.514±0.06d 0.606±0.07c 0.565±0.05c 1.148±0.11a 0.943±0.07b 1.108±0.09a 0.938±0.06b 1438 α-guaiene 40 0.063±0.006e 0.233±0.03a 0.186±0.02c 0.207±0.01abc 0.132±0.01d 0.219±0.02ab 0.196±0.01bc 1443 (z)-β-farnesene 41 1.323±0.12e 2.157±0.15d 1.968±0.17d 2.814±0.19b 2.752±0.18c 2.897±0.18a 2.912±0.17a 1453 α-humulene 42 0.247±0.01e 0.448±0.05c 0.403±0.04d 0.459±0.05bc 0.392±0.04 0.560±0.04a 0.488±0.03b 1457 (e)-β-farnesene 43 0.993±0.11f 1.521±0.17d 1.406±0.14e 1.992±0.12a 1.649±0.09c 1.007±0.07f 1.860±0.08b 1462 allo-aromadendrene 44 4.026±0.47f 6.133±0.60e 6.098±0.64e 7.254±0.55c 7.007±0.52d 7.561±0.41a 7.453±0.19b 1480 germacrene d 45 1.901±0.32d 3.197±0.30c 2.913±0.21c 4.195±0.37b 4.071±0.34b 4.534±0.21a 4.474±0.19a 1496 bicyclogermacrene 46 0.190±0.01d 0.511±0.04a 0.471±0.04ab 0.451±0.04 0.397±0.05c 0.485±0.03ab 0.467±0.03ab 1498 trans-β-guaiene 47 1.191±0.06e 2.010±0.07d 1.927±0.08d 2.682±0.09b 2.519±0.09c 2.795±0.08a 2.565±0.07c 1508 α-bulnesene 48 3.323±0.37f 5.423±0.48de 5.343±0.40e 5.741±0.31c 5.677±0.26cd 6.972±0.16a 6.085±0.15b 1514 γ-cadinene 49 0.859±0.07e 1.214±0.13b 1.146±0.10cd 1.150±0.12c 1.097±0.09d 1.281±0.07a 1.228±0.06b 1521 β-sesquiphellandrene 50 0.486±0.05e 0.612±0.05d 0.651±0.07d 0.698±0.05bc 0.683±0.07cd 0.748±0.06a 0.731±0.04ab 1524 δ-cadinene 51 0.150±0.01e 0.402±0.03d 0.376±0.03d 0.582±0.04a 0.461±0.06c 0.504±0.04b 0.478±0.03bc 1537 α-cadinene 52 0.394±0.02f 0.659±0.05e 0.635±0.06e 0.944±0.07b 0.799±0.06d 0.990±0.07a 0.880±0.06c 1564 (e)-nerolidol 53 -----------0.226±0.01ab 0.197±0.02c 0.223±0.01ab 0.215±0.04bc 0.240±0.02a 0.230±0.01ab 1575 germacrene d-4-ol 54 -----------0.214±0.02ab 0.182±0.02c 0.215±0.02ab 0.190±0.02c 0.228±0.01a 0.195±0.01bc 1577 spathulenol 55 -----------0.523±0.05b 0.494±0.04c 0.529±0.05b 0.485±0.04c 0.556±0.05a 0.516±0.04b 1599 n-hexadecane 56 2.65±0.20d 3.301±0.22b 3.205±0.19bc 3.510±0.22a 3.055±0.17c 3.566±0.10a 3.680±0.11a 1614 1,10-di-epi-cubenol 57 24.679±1.78e 27.520±1.58c 27.402±1.70c 28.510±1.64b 26.620±1.55d 29.062±1.31a 28.737±1.22b 1642 epi-α-cadinol 58 1.039±0.10d 1.663±0.09a 1.611±0.09a 1.404±0.10bc 1.391±0.08c 1.461±0.06b 1.399±0.07c 1649 β-eudesmol 59 1.353±0.10d 1.588±0.09a 1.574±0.11a 1.421±0.09c 1.409±0.09c 1.508±0.06b 1.456±0.07c 1654 α-cadinol 60 0.248±0.01d 0.543±0.03ab 0.544±0.04ab 0.462±0.05c 0.419±0.05c 0.592±0.04a 0.536±0.04b 1685 β-bisabolol 61 data are mean ±standard deviation of eight replications. means followed by the same letter within a row are not significantly different according to duncan’s multiple range test at p< 0.05. 34 v. tavallali, h. gholami, o. espargham tab. 1: phytochemical profile (%) identified in essential oil of sweet basil supplied with diverse zinc and salicylic acid sources. control salicylic salicylic zn-edta zn-edta n[zn(sa)2] n[zn(sa)2] retention compound no. acid 0.2% acid 0.1% 0.2% 0.1% 0.2% 0.1% index ----------0.422±0.05a 0.377±0.03b 0.238±0.02cd 0.212±0.01d 0.251±0.02c 0.258±0.07c 932 α-pinene 1 ----------0.631±0.07a 0.580±0.06b 0.583±0.06b 0.571±0.05b 0.602±0.04b 0.576±0.03b 972 sabinene 2 -----------1.167±0.27a 1.084±0.23a 0.271±0.03b 0.224±0.02c 0.208±0.01c 0.286±0.04b 976 β-pinene 3 -----------3.224±0.59a 3.110±0.52a 0.416±0.03d 0.508±0.04b 0.510±0.03b 0.469±0.02c 990 myrcene 4 3.357±0.43a 0.966±0.07b 0.884±0.07c 0.611±0.05d 3.587±0.45a 0.425±0.03e 0.606±0.04d 999 n-decane 5 -----------0.127±0.02b 0.158±0.03a 0.121±0.03bc 0.091±0.006d 0.132±0.01ab 0.105±0.02cd 1005 α-phellandrene 6 -----------0.167±0.01a 0.092±0.007cd 0.102±0.01bc 0.069±0.005e 0.115±0.02b 0.081±0.007de 1016 α-terpinene 7 -----------0.029±0.004d 0.015±0.002e 0.082±0.005b 0.058±0.006c 0.093±0.007a 0.048±0.003c 1024 p-cymene 8 0.289±0.02b 0.796±0.07a 0.754±0.06a 0.142±0.02cd 0.127±0.02d 0.150±0.01c 0.134±0.01d 1028 limonene 9 3.463±0.66a 1.121±0.31b 1.074±0.20b 0.602±0.05c 3.523±0.50a 0.614±0.05c 1.088±0.11b 1030 1,8-cineole 10 0.397±0.04a 0.125±0.01c 0.101±0.01d 0.145±0.03bc 0.126±0.02c 0.153±0.01b 0.137±0.02bc 1036 (z)-β-ocimene 11 ----------0.039±0.004d 0.018±0.002e 0.085±0.006ab 0.062±0.004c 0.094±0.006a 0.075±0.003b 1041 benzene acetaldehyde 12 -----------0.602±0.07a 0.581±0.05ab 0.561±0.04ab 0.549±0.05b 0.567±0.04ab 0.566±0.03ab 1046 (e)-β-ocimene 13 -----------0.126±0.02b 0.118±0.02b 0.142±0.02a 0.128±0.01b 0.123±0.01b 0.139±0.01a 1057 γ-terpinene 14 ----------0.163±0.03a 0.150±0.02ab 0.145±0.03b 0.137±0.02b 0.120±0.01c 0.151±0. 01ab 1066 cis-sabinene hydrate 15 ----------0.371±0.04a 0.324±0.03bc 0.319±0.04bc 0.281±0.03d 0.332±0.02b 0.309±0.02c 1088 terpinolene 16 10.850±1.89a 5.733±0.51b 5.696±0.45b 0.593±0.05c 0.584±0.06c 0.516±0.04d 0.590±0.04c 1099 linalool 17 -----------0.136±0.02a 0.129±0.02a 0.072±0.007b 0.061±0.004c 0.079±0.005b 0.062±0.004c 1112 1-octen-3-yl acetate 18 -----------0.089±0.009d 0.058±0.006e 0.119±0.01ab 0.102±0.03c 0.128±0.01a 0.114±0.01b 1144 camphor 19 -----------0.220±0.02a 0.190±0.03b 0.161±0.02c 0.132±0.02d 0.198±0.02b 0.170±0.02c 1166 δ-terpineol 20 -----------0.097±0.007a 0.077±0.005b 0.076±0.006b 0.066±0.006c 0.097±0.007a 0.090±0.002a 1177 terpinen-4-ol 21 0.483±0.05f 1.360±0.18b 2.110±0.26a 0.703±0.05d 0.682±0.05de 0.664±0.04e 0.761±0.04c 1190 α-terpineol 22 0.783±0.07a 0.203±0.04e 0.177±0.03e 0.690±0.06b 0.512±0.04c 0.414±0.03d 0.565±0.03c 1199 n-dodecane 23 0.965±0.07a 0.821±0.08b 0.798±0.07b 0.589±0.06c 0.346±0.04d 0.604±0.05c 0.605±0.04c 1212 octanol acetate 24 0.248±0.01a 0.098±0.009c 0.088±0.007c 0.195±0.01b 0.101±0.009c 0.199±0.02b 0.250±0.02a 1222 trans-carveol 25 -----------0.129±0.02b 0.108±0.02b 0.791±0.06a 0.753±0.06a 0.043±0.003c 0.036±0.003c 1256 linalyl acetate 26 6.657±1.03a 1.843±0.28d 2.644±0.36b 1.692±0.23 2.395±0.18c 1.005±0.08f 1.332±0.12e 1286 bornyl acetate 27 0.146±0.01f 0.219±0.03e 0.184±0.03e 0.562±0.05a 0.365±0.03d 0.497±0.03b 0.444±0.03c 1337 δ-elemene 28 0.095±0.007c 0.224±0.03a 0.199±0.04a 0.154±0.02b 0.135±0.02b 0.135±0.01b 0.146±0.01b 1349 α-terpinyl acetate 29 12.704±1.77a 1.140±0.21e 2.288±0.27b 1.711±0.12c 1.389±0.09d 0.403±0.03g 0.741±0.05f 1358 eugenol 30 0.056±0.006d 0.121±0.01b 0.091±0.008c 0.314±0.04a 0.291±0.04a 0.292±0.02a 0.297±0.02a 1375 α-copaene 31 0.084±0.009d 0.130±0.01c 0.060±0.007e 0.214±0.02a 0.130±0.02c 0.222±0.02a 0.168±0.01b 1390 β-cubebene 32 4.894±0.42d 6.117±0.49c 6.083±0.41c 7.520±0.30b 7.245±0.25b 7.970±0.18a 7.877±0.21a 1392 β-elemene 33 -----------0.092±0.009d 0.078±0.008d 0.339±0.04a 0.253±0.03c 0.256±0.01c 0.288±0.02b 1400 n-tetradeane 34 0.330±0.02cd 0.715±0.05a 0.702±0.06a 0.392±0.04b 0.345±0.03c 0.309±0.02d 0.341±0.02c 1405 methyl eugenol 35 -----------0.072±0.007a 0.051±0.006b 0.072±0.005a 0.051±0.004b 0.076±0.005a 0.053±0.006b 1415 cis-α-bergamotene 36 0.196±0.02e 0.338±0.04d 0.308±0.05d 0.597±0.04a 0.469±0.03c 0.563±0.04b 0.587±0.04ab 1418 (e)-caryophyllene 37 -----------0.051±0.005d 0.036±0.004e 0.077±0.005a 0.039±0.003e 0.070±0.004b 0.059±0.005c 1428 β-gurjunene 38 8.369±0.86d 9.222±0.92c 9.093±0.87c 11.150±1.08b 10.920±1.06b 11.903±1.10a 11.838±1.02a 1436 trans-α-bergamotene 39 0.514±0.06d 0.606±0.07c 0.565±0.05c 1.148±0.11a 0.943±0.07b 1.108±0.09a 0.938±0.06b 1438 α-guaiene 40 0.063±0.006e 0.233±0.03a 0.186±0.02c 0.207±0.01abc 0.132±0.01d 0.219±0.02ab 0.196±0.01bc 1443 (z)-β-farnesene 41 1.323±0.12e 2.157±0.15d 1.968±0.17d 2.814±0.19b 2.752±0.18c 2.897±0.18a 2.912±0.17a 1453 α-humulene 42 0.247±0.01e 0.448±0.05c 0.403±0.04d 0.459±0.05bc 0.392±0.04 0.560±0.04a 0.488±0.03b 1457 (e)-β-farnesene 43 0.993±0.11f 1.521±0.17d 1.406±0.14e 1.992±0.12a 1.649±0.09c 1.007±0.07f 1.860±0.08b 1462 allo-aromadendrene 44 4.026±0.47f 6.133±0.60e 6.098±0.64e 7.254±0.55c 7.007±0.52d 7.561±0.41a 7.453±0.19b 1480 germacrene d 45 1.901±0.32d 3.197±0.30c 2.913±0.21c 4.195±0.37b 4.071±0.34b 4.534±0.21a 4.474±0.19a 1496 bicyclogermacrene 46 0.190±0.01d 0.511±0.04a 0.471±0.04ab 0.451±0.04 0.397±0.05c 0.485±0.03ab 0.467±0.03ab 1498 trans-β-guaiene 47 1.191±0.06e 2.010±0.07d 1.927±0.08d 2.682±0.09b 2.519±0.09c 2.795±0.08a 2.565±0.07c 1508 α-bulnesene 48 3.323±0.37f 5.423±0.48de 5.343±0.40e 5.741±0.31c 5.677±0.26cd 6.972±0.16a 6.085±0.15b 1514 γ-cadinene 49 0.859±0.07e 1.214±0.13b 1.146±0.10cd 1.150±0.12c 1.097±0.09d 1.281±0.07a 1.228±0.06b 1521 β-sesquiphellandrene 50 0.486±0.05e 0.612±0.05d 0.651±0.07d 0.698±0.05bc 0.683±0.07cd 0.748±0.06a 0.731±0.04ab 1524 δ-cadinene 51 0.150±0.01e 0.402±0.03d 0.376±0.03d 0.582±0.04a 0.461±0.06c 0.504±0.04b 0.478±0.03bc 1537 α-cadinene 52 0.394±0.02f 0.659±0.05e 0.635±0.06e 0.944±0.07b 0.799±0.06d 0.990±0.07a 0.880±0.06c 1564 (e)-nerolidol 53 -----------0.226±0.01ab 0.197±0.02c 0.223±0.01ab 0.215±0.04bc 0.240±0.02a 0.230±0.01ab 1575 germacrene d-4-ol 54 -----------0.214±0.02ab 0.182±0.02c 0.215±0.02ab 0.190±0.02c 0.228±0.01a 0.195±0.01bc 1577 spathulenol 55 -----------0.523±0.05b 0.494±0.04c 0.529±0.05b 0.485±0.04c 0.556±0.05a 0.516±0.04b 1599 n-hexadecane 56 2.65±0.20d 3.301±0.22b 3.205±0.19bc 3.510±0.22a 3.055±0.17c 3.566±0.10a 3.680±0.11a 1614 1,10-di-epi-cubenol 57 24.679±1.78e 27.520±1.58c 27.402±1.70c 28.510±1.64b 26.620±1.55d 29.062±1.31a 28.737±1.22b 1642 epi-α-cadinol 58 1.039±0.10d 1.663±0.09a 1.611±0.09a 1.404±0.10bc 1.391±0.08c 1.461±0.06b 1.399±0.07c 1649 β-eudesmol 59 1.353±0.10d 1.588±0.09a 1.574±0.11a 1.421±0.09c 1.409±0.09c 1.508±0.06b 1.456±0.07c 1654 α-cadinol 60 0.248±0.01d 0.543±0.03ab 0.544±0.04ab 0.462±0.05c 0.419±0.05c 0.592±0.04a 0.536±0.04b 1685 β-bisabolol 61 data are mean ±standard deviation of eight replications. means followed by the same letter within a row are not significantly different according to duncan’s multiple range test at p< 0.05. 34 v. tavallali, h. gholami, o. espargham tab. 1: phytochemical profile (%) identified in essential oil of sweet basil supplied with diverse zinc and salicylic acid sources. control salicylic salicylic zn-edta zn-edta n[zn(sa)2] n[zn(sa)2] retention compound no. acid 0.2% acid 0.1% 0.2% 0.1% 0.2% 0.1% index ----------0.422±0.05a 0.377±0.03b 0.238±0.02cd 0.212±0.01d 0.251±0.02c 0.258±0.07c 932 α-pinene 1 ----------0.631±0.07a 0.580±0.06b 0.583±0.06b 0.571±0.05b 0.602±0.04b 0.576±0.03b 972 sabinene 2 -----------1.167±0.27a 1.084±0.23a 0.271±0.03b 0.224±0.02c 0.208±0.01c 0.286±0.04b 976 β-pinene 3 -----------3.224±0.59a 3.110±0.52a 0.416±0.03d 0.508±0.04b 0.510±0.03b 0.469±0.02c 990 myrcene 4 3.357±0.43a 0.966±0.07b 0.884±0.07c 0.611±0.05d 3.587±0.45a 0.425±0.03e 0.606±0.04d 999 n-decane 5 -----------0.127±0.02b 0.158±0.03a 0.121±0.03bc 0.091±0.006d 0.132±0.01ab 0.105±0.02cd 1005 α-phellandrene 6 -----------0.167±0.01a 0.092±0.007cd 0.102±0.01bc 0.069±0.005e 0.115±0.02b 0.081±0.007de 1016 α-terpinene 7 -----------0.029±0.004d 0.015±0.002e 0.082±0.005b 0.058±0.006c 0.093±0.007a 0.048±0.003c 1024 p-cymene 8 0.289±0.02b 0.796±0.07a 0.754±0.06a 0.142±0.02cd 0.127±0.02d 0.150±0.01c 0.134±0.01d 1028 limonene 9 3.463±0.66a 1.121±0.31b 1.074±0.20b 0.602±0.05c 3.523±0.50a 0.614±0.05c 1.088±0.11b 1030 1,8-cineole 10 0.397±0.04a 0.125±0.01c 0.101±0.01d 0.145±0.03bc 0.126±0.02c 0.153±0.01b 0.137±0.02bc 1036 (z)-β-ocimene 11 ----------0.039±0.004d 0.018±0.002e 0.085±0.006ab 0.062±0.004c 0.094±0.006a 0.075±0.003b 1041 benzene acetaldehyde 12 -----------0.602±0.07a 0.581±0.05ab 0.561±0.04ab 0.549±0.05b 0.567±0.04ab 0.566±0.03ab 1046 (e)-β-ocimene 13 -----------0.126±0.02b 0.118±0.02b 0.142±0.02a 0.128±0.01b 0.123±0.01b 0.139±0.01a 1057 γ-terpinene 14 ----------0.163±0.03a 0.150±0.02ab 0.145±0.03b 0.137±0.02b 0.120±0.01c 0.151±0. 01ab 1066 cis-sabinene hydrate 15 ----------0.371±0.04a 0.324±0.03bc 0.319±0.04bc 0.281±0.03d 0.332±0.02b 0.309±0.02c 1088 terpinolene 16 10.850±1.89a 5.733±0.51b 5.696±0.45b 0.593±0.05c 0.584±0.06c 0.516±0.04d 0.590±0.04c 1099 linalool 17 -----------0.136±0.02a 0.129±0.02a 0.072±0.007b 0.061±0.004c 0.079±0.005b 0.062±0.004c 1112 1-octen-3-yl acetate 18 -----------0.089±0.009d 0.058±0.006e 0.119±0.01ab 0.102±0.03c 0.128±0.01a 0.114±0.01b 1144 camphor 19 -----------0.220±0.02a 0.190±0.03b 0.161±0.02c 0.132±0.02d 0.198±0.02b 0.170±0.02c 1166 δ-terpineol 20 -----------0.097±0.007a 0.077±0.005b 0.076±0.006b 0.066±0.006c 0.097±0.007a 0.090±0.002a 1177 terpinen-4-ol 21 0.483±0.05f 1.360±0.18b 2.110±0.26a 0.703±0.05d 0.682±0.05de 0.664±0.04e 0.761±0.04c 1190 α-terpineol 22 0.783±0.07a 0.203±0.04e 0.177±0.03e 0.690±0.06b 0.512±0.04c 0.414±0.03d 0.565±0.03c 1199 n-dodecane 23 0.965±0.07a 0.821±0.08b 0.798±0.07b 0.589±0.06c 0.346±0.04d 0.604±0.05c 0.605±0.04c 1212 octanol acetate 24 0.248±0.01a 0.098±0.009c 0.088±0.007c 0.195±0.01b 0.101±0.009c 0.199±0.02b 0.250±0.02a 1222 trans-carveol 25 -----------0.129±0.02b 0.108±0.02b 0.791±0.06a 0.753±0.06a 0.043±0.003c 0.036±0.003c 1256 linalyl acetate 26 6.657±1.03a 1.843±0.28d 2.644±0.36b 1.692±0.23 2.395±0.18c 1.005±0.08f 1.332±0.12e 1286 bornyl acetate 27 0.146±0.01f 0.219±0.03e 0.184±0.03e 0.562±0.05a 0.365±0.03d 0.497±0.03b 0.444±0.03c 1337 δ-elemene 28 0.095±0.007c 0.224±0.03a 0.199±0.04a 0.154±0.02b 0.135±0.02b 0.135±0.01b 0.146±0.01b 1349 α-terpinyl acetate 29 12.704±1.77a 1.140±0.21e 2.288±0.27b 1.711±0.12c 1.389±0.09d 0.403±0.03g 0.741±0.05f 1358 eugenol 30 0.056±0.006d 0.121±0.01b 0.091±0.008c 0.314±0.04a 0.291±0.04a 0.292±0.02a 0.297±0.02a 1375 α-copaene 31 0.084±0.009d 0.130±0.01c 0.060±0.007e 0.214±0.02a 0.130±0.02c 0.222±0.02a 0.168±0.01b 1390 β-cubebene 32 4.894±0.42d 6.117±0.49c 6.083±0.41c 7.520±0.30b 7.245±0.25b 7.970±0.18a 7.877±0.21a 1392 β-elemene 33 -----------0.092±0.009d 0.078±0.008d 0.339±0.04a 0.253±0.03c 0.256±0.01c 0.288±0.02b 1400 n-tetradeane 34 0.330±0.02cd 0.715±0.05a 0.702±0.06a 0.392±0.04b 0.345±0.03c 0.309±0.02d 0.341±0.02c 1405 methyl eugenol 35 -----------0.072±0.007a 0.051±0.006b 0.072±0.005a 0.051±0.004b 0.076±0.005a 0.053±0.006b 1415 cis-α-bergamotene 36 0.196±0.02e 0.338±0.04d 0.308±0.05d 0.597±0.04a 0.469±0.03c 0.563±0.04b 0.587±0.04ab 1418 (e)-caryophyllene 37 -----------0.051±0.005d 0.036±0.004e 0.077±0.005a 0.039±0.003e 0.070±0.004b 0.059±0.005c 1428 β-gurjunene 38 8.369±0.86d 9.222±0.92c 9.093±0.87c 11.150±1.08b 10.920±1.06b 11.903±1.10a 11.838±1.02a 1436 trans-α-bergamotene 39 0.514±0.06d 0.606±0.07c 0.565±0.05c 1.148±0.11a 0.943±0.07b 1.108±0.09a 0.938±0.06b 1438 α-guaiene 40 0.063±0.006e 0.233±0.03a 0.186±0.02c 0.207±0.01abc 0.132±0.01d 0.219±0.02ab 0.196±0.01bc 1443 (z)-β-farnesene 41 1.323±0.12e 2.157±0.15d 1.968±0.17d 2.814±0.19b 2.752±0.18c 2.897±0.18a 2.912±0.17a 1453 α-humulene 42 0.247±0.01e 0.448±0.05c 0.403±0.04d 0.459±0.05bc 0.392±0.04 0.560±0.04a 0.488±0.03b 1457 (e)-β-farnesene 43 0.993±0.11f 1.521±0.17d 1.406±0.14e 1.992±0.12a 1.649±0.09c 1.007±0.07f 1.860±0.08b 1462 allo-aromadendrene 44 4.026±0.47f 6.133±0.60e 6.098±0.64e 7.254±0.55c 7.007±0.52d 7.561±0.41a 7.453±0.19b 1480 germacrene d 45 1.901±0.32d 3.197±0.30c 2.913±0.21c 4.195±0.37b 4.071±0.34b 4.534±0.21a 4.474±0.19a 1496 bicyclogermacrene 46 0.190±0.01d 0.511±0.04a 0.471±0.04ab 0.451±0.04 0.397±0.05c 0.485±0.03ab 0.467±0.03ab 1498 trans-β-guaiene 47 1.191±0.06e 2.010±0.07d 1.927±0.08d 2.682±0.09b 2.519±0.09c 2.795±0.08a 2.565±0.07c 1508 α-bulnesene 48 3.323±0.37f 5.423±0.48de 5.343±0.40e 5.741±0.31c 5.677±0.26cd 6.972±0.16a 6.085±0.15b 1514 γ-cadinene 49 0.859±0.07e 1.214±0.13b 1.146±0.10cd 1.150±0.12c 1.097±0.09d 1.281±0.07a 1.228±0.06b 1521 β-sesquiphellandrene 50 0.486±0.05e 0.612±0.05d 0.651±0.07d 0.698±0.05bc 0.683±0.07cd 0.748±0.06a 0.731±0.04ab 1524 δ-cadinene 51 0.150±0.01e 0.402±0.03d 0.376±0.03d 0.582±0.04a 0.461±0.06c 0.504±0.04b 0.478±0.03bc 1537 α-cadinene 52 0.394±0.02f 0.659±0.05e 0.635±0.06e 0.944±0.07b 0.799±0.06d 0.990±0.07a 0.880±0.06c 1564 (e)-nerolidol 53 -----------0.226±0.01ab 0.197±0.02c 0.223±0.01ab 0.215±0.04bc 0.240±0.02a 0.230±0.01ab 1575 germacrene d-4-ol 54 -----------0.214±0.02ab 0.182±0.02c 0.215±0.02ab 0.190±0.02c 0.228±0.01a 0.195±0.01bc 1577 spathulenol 55 -----------0.523±0.05b 0.494±0.04c 0.529±0.05b 0.485±0.04c 0.556±0.05a 0.516±0.04b 1599 n-hexadecane 56 2.65±0.20d 3.301±0.22b 3.205±0.19bc 3.510±0.22a 3.055±0.17c 3.566±0.10a 3.680±0.11a 1614 1,10-di-epi-cubenol 57 24.679±1.78e 27.520±1.58c 27.402±1.70c 28.510±1.64b 26.620±1.55d 29.062±1.31a 28.737±1.22b 1642 epi-α-cadinol 58 1.039±0.10d 1.663±0.09a 1.611±0.09a 1.404±0.10bc 1.391±0.08c 1.461±0.06b 1.399±0.07c 1649 β-eudesmol 59 1.353±0.10d 1.588±0.09a 1.574±0.11a 1.421±0.09c 1.409±0.09c 1.508±0.06b 1.456±0.07c 1654 α-cadinol 60 0.248±0.01d 0.543±0.03ab 0.544±0.04ab 0.462±0.05c 0.419±0.05c 0.592±0.04a 0.536±0.04b 1685 β-bisabolol 61 data are mean ±standard deviation of eight replications. means followed by the same letter within a row are not significantly different according to duncan’s multiple range test at p< 0.05. essential oils of ocimum basilicum l. grown with zn-salicylic acid nano-complex 31 tab. 2: effect of different zinc and salicylic acid sources on concentrations of phenolic compounds and flavonoids in extract of sweet basil. treatments gallic carvacrol kaempferol rosmarinc rutin catechin cinnamic ferulic luteolin quercetin acid acid acid acid n[zn(sa)2] 3.77±0.18b 2.41±0.10b 3.10±0.21b 5.29±0.23b 2.13±0.09bc 3.51±0.15b 3.71±0.17b 4.06±0.19b 0.91±0.06b 3.97±0.16b 0.1% n[zn(sa)2] 4.37±0.20a 2.65±0.12a 3.81±0.17a 5.70±0.25a 2.74±0.11a 4.22±0.14a 4.04±0.16a 4.54±0.18a 1.13±0.09a 4.21±0.17a 0.2% zn-edta 3.20±0.17cd 2.16±0.12c 2.67±0.19d 4.85±0.21c 1.81±0.10d 3.48±0.11b 3.19±0.20d 4.10±0.16b 0.71±0.08b 3.68±0.19c 0.1% zn-edta 3.83±0.14b 2.38±0.14b 2.92±0.20c 5.10±0.19b 2.25±0.08b 4.05±0.13a 3.41±0.17c 3.43±0.16c 0.83±0.07b 3.97±0.16b 0.2% salicylic acid 3.03±0.18d 2.01±0.11c 2.82±0.16cd 2.88±0.20e 1.56±0.09e 3.50±0.13b 2.81±0.16e 2.11±0.13d nd 3.97±0.16b 0.1% salicylic acid 3.44±0.16c 2.17±0.10c 3.12±0.18b 3.11±0.18d 1.99±0.08cd 3.66±0.12b 3.01±0.17de 2.21±0.14d nd 4.14±0.17a 0.2% control 2.22±0.12e nd 1.54±0.10e 2.11±0.14f nd 2.32±0.10c 1.92±0.09f nd nd 2.87±0.16d nd: not detected *calculated mean amount of the flavonoids and polyphenols (mg g-1 dm) based on the weight of the ground dry plant in eight replicates ± standard deviation. means followed by the same letter within a column are not significantly different according to duncan’s multiple range test at p< 0.05. tab. 3: radical scavenging activity of sweet basil’s essential oils affected by different zinc and salicylic acid sources. properties n[zn(sa)2] n[zn(sa)2] zn-edta zn-edta salicylic acid salicylic acid control 0.1% 0.2% 0.1% 0.2% 0.1% 0.2% antioxidant (mg aae g-1)a 21.44±1.5 22.94±1.7 16.30±1.4 18.78±1.6 15.08±1.3 16.71±1.4 8.82±1.2 ic50 for rns scavenging (mg ml-1)b 1.59±0.1 1.47±0.1 2.62±0.2 2.44±0.3 2.19±0.3 1.94±0.1 5.12±0.5 ic50 for ros scavenging (mg ml-1)c 1.24±0.3 1.03±0.2 1.43±0.3 1.31±0.4 1.76±0.4 1.59±0.3 3.56±0.7 ic50 for tbars scavenging (mg ml-1)d 3.16±0.3 2.87±0.2 4.09±0.3 3.64±0.3 3.84±0.2 3.61±0.3 5.99±0.4 ic50 for h2o2 scavenging (mg ml-1)c 2.33±0.3 2.14±0.2 2.54±0.3 2.41±0.4 3.12±0.3 2.81±0.5 5.43±0.6 adata are presented as milligrams ascorbic acid equivalents per gram of essential oil. bic50 is concentration of essential oil to scavenge rns (reactive nitrogen species) by 50%. cic50 is concentration of essential oil required to scavenge ros (reactive oxygen species) by 50%. dic50 is concentration of essential oil to scavenge tbars (thiobarbituric acid reactive substances) by 50%. eic50 is concentration of essential oil to scavenge h2o2 by 50%. the application of zinc and plant growth regulator such as salicylic acid can affect the antimicrobial activity of the essential oils. due to the complexity of the chemical structure of the essential oils, their volatility and their insolubility in water, investigating their anti microbial activity is complicated. hence, it is difficult to identify the molecular pathways involved in their actions. it is therefore assumed that each of the essential oil components has a separate mechanism for itself. in other words, because of the wide range of essential oil components, their antimicrobial properties do not depend on a single mechanism, but various mechanisms play a role at the molecular level in this regard. one possible mechanism is to irreversibly damage the cell membrane of bacteria, which ultimately causes the leakage of the plasma membrane ions, cytoplasmic material, and energy substrates deficiency, such as glucose. this action will eventually lead to lysis of bacterial cells and death. many researchers focus on the antimicrobial activity of essential oils and test some of the various components of essential oils to find possible synergistic effects (kalemba and kunicka, 2003; bakkali et al., 2008). our results showed that the application of 0.2% n[zn(sa)2] has a significant effect on staphylococcus aureus growth in comparison with other foodborne microbes. in other words, among the studied tab. 4: antimicrobial activity of sweet basil’s essential oils affected by different zinc and salicylic acid sources. mic (mg ml-1) species n[zn(sa)2] 0.1% n[zn(sa)2] 0.2% zn-edta 0.1% zn-edta 0.2% salicylic acid 0.1% salicylic acid 0.2% control b. subtilis 0.053±0.004 0.031±0.003 0.111±0.009 0.095±0.008 0.127±0.011 0.116±0.011 0.149±0.013 s. aureus 0.011±0.002 0.007±0.0005 0.041±0.004 0.030±0.003 0.049±0.003 0.043±0.002 0.069±0.005 e. coli 0.298±0.021 0.229±0.018 0.499±0.025 0.419±0.024 0.536±0.030 0.521±0.031 0.787±0.047 s. typhimurium 0.261±0.015 0.180±0.011 0.453±0.027 0.410±0.025 0.514±0.037 0.488±0.035 0.801±0.052 a. niger 0.079±0.008 0.068±0.045 0.179±0.008 0.154±0.021 0.195±0.028 0.183±0.032 0.230±0.040 c. albicans 0.069±0.005 0.050±0.005 0.140±0.016 0.117±0.011 0.186±0.018 0.159±0.016 0.287±0.022 values are means of mic in eight replicates ± sd (standard deviation). mic: minimal inhibitory concentration. 32 v. tavallali, h. gholami, o. espargham microbes, this bacterium was the most sensitive to the sweet basil essential oil. staphylococcus aureus threatens people’s health through infectious diseases and food poisoning. in addition, it has the abili ty to simultaneously resist multiple antibiotics (dallal et al., 2010; normanno et al., 2007). the results of our study show that essential oil of basils that have been sprayed with zn-salicylic acid nano-complexes have good antimicrobial properties and can therefore be used in the food and pharmaceutical industry. the importance of the phenolic compounds, such as carvacrol, has been proved in improving the antimicrobial properties of essential oils. previous studies have shown that carvacrol inhibits the growth of some bacteria, including e. coli and bacillus cereus (du et al., 2008). in another study by karaman et al. (2001), the strong bacteriostatic effects of thymus revolatus essential oil were shown against staphylococcus aureus and escherichia coli. they expressed cause of these effects the high amount of carvacrol in the essential oil. the mechanisms by which phenolic compounds create toxicity for microorganisms include surface absorption and cell membrane breakdown, interference in the cytoplasmic membrane and membrane proteins function, reaction with enzymes and reduction of metal ions (negi, 2012). in general, it is difficult to compare the reported results about the antibacterial properties of essential oils. the reason for this is the difference in the methods used, their sources of preparation and bacterial strains (basti et al., 2004). conclusion secondary metabolites in plants are of particular importance for food and medical purposes. therefore, any factor that can increase these compounds without changes in the genetic structure of the plant are valuable. in this study, the application of zn and salicylic acid nano-complexes improved growth characteristics, essential oil yield, antimicrobial and antioxidant activities of sweet basil essential oil. improvement of the antimicrobial and antioxidant properties of sweet basil essential oil can increase its application in the food, medical, pharmaceutical, veterinary, cosmetic and sanitary industries. application of nanofertilizers is more advantageous than conventional forms of fertilizers due to stable physical properties, small particle size, higher surface-area-to-volume ratio, high density and high reactivity. in addition, nanofertilizers can be considered as controlling agents for the release of fertilizers in order to produce intelligent nanofertilizers to overcome the technical constraints on the slow and controlled release of elements of nutrition. authors contributions study conception and design: vahid tavallali, acquisition of data: omid espargham, analysis and interpretation of data: vahid tavallali, drafting of manuscript: hossein gholami, critical revision: vahid tavallali. acknowledgements the authors thank dr. vahid rowshan and ms. atefeh bahmanzadegan for the laboratory equipment such as hplc and gc/ms. conflict of interest no potential conflict of interest was reported by the authors. references adams, r.p., 2007: identification of essential oil components by gas chromatography/mass spectrometry. allured publishing, carol stream, il. bakkali, f., averbeck, s., averbeck, d., idaomar, m., 2008: biological effects of essential oils − a review. food chem. toxicol. 46(2), 446-475. doi: 10.1016/j.fct.2007.09.106 baruah, s., dutta, j., 2009: nanotechnology applications in pollution sensing and degradation in agriculture: a review. environ. chem. let. 7(3), 191-204. doi: 10.1007/s10311-009-0228-8 basti, a., razavilar, v., misaghi, a., abbasifar, r., radmehr, b., khalighi sigaroodi, f., 2004: effect of zataria multiflora boiss. essential oil on probability of growth initiation of salmonella typhimurium in a brain heart infusion broth. j. med. plants. 1(9), 85-92. 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(in persian). zhu, h., han, j., xiao, j.q., jin, y., 2008: uptake, translocation, and accumulation of manufactured iron oxide nanoparticles by pumpkin plants. j. environ. monitor. 10(6), 713-717. doi: 10.1039/b805998e orcid vahid tavallali https://orcid.org/0000-0002-6819-2008 address of the corresponding author: vahid tavallali, department of agriculture, payame noor university (pnu), p.o. box: 19395-3697 tehran, iran. e-mail: vtavallali@gmail.com; v.tavalali@pnu.ac.ir © the author(s) 2020. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.2478/v10014-011-0004-x http://dx.doi.org/10.2174/0929867033457719 http://dx.doi.org/10.1016/s0378-8741(01)00238-0 http://dx.doi.org/10.1016/j.foodchem.2012.11.131 http://dx.doi.org/10.1078/0176-1617-00865 http://dx.doi.org/10.1007/bf02115480 http://dx.doi.org/10.3390/ijerph10010047 http://dx.doi.org/10.1016/j.plantsci.2004.04.026 http://dx.doi.org/10.1016/c2009-0-63043-9 http://dx.doi.org/10.1104/pp.102.018457 http://dx.doi.org/10.1080/00087114.2004.10589681 http://dx.doi.org/10.1016/j.ijfoodmicro.2012.03.006 http://dx.doi.org/10.1016/j.ijfoodmicro.2006.10.049 http://dx.doi.org/10.1002/ffj.1869 http://dx.doi.org/10.1023/a:1022556103536 http://dx.doi.org/10.3390/molecules20022536 http://dx.doi.org/10.1007/s40003-014-0113-y http://dx.doi.org/10.1016/j.jclepro.2018.06.078 http://dx.doi.org/10.1016/j.bse.2006.01.009 http://dx.doi.org/10.1016/j.postharvbio.2006.04.010 http://dx.doi.org/10.1006/anbo.1993.1005 http://dx.doi.org/10.1039/b805998e buchbesprechung.indd journal of applied botany and food quality 94, 15 (2021) book review the pomegranate – botany, production and uses ali sarkhosh, alimohammad m. yavari, zabihollah zamani the pomegranate (punica granatum l., family: punicaceae) is one of the oldest known edible fruits and is associated with the ancient civilizations of the middle east. this is the first comprehensive book covering the botany, production, processing, health and industrial uses of the pomegranate. the cultivation of this fruit for fresh consumption, juice production and medicinal purposes has expanded more than tenfold over the past 20 years. presenting a review of pome granate growing, from a scientific and horticultural perspective, this book provides information on how to increase yields and improve shortand medium-term grower profitability and sustainability. it covers: practices to mitigate pests, diseases and abiotic stresses; yield-based nutrition management; cultural practices for cultivars with horticultural traits such as earliness, high yield, improved taste, soft seeds, disease resistance, and low splitting and sunscald rates; increasing crop diversity to aid crop security; and composition, food uses and medicinal applications. this book provides a valuable support not only for farmers and for people involved in the pomegranate industry but it can be recommended without any reservation to plant breeders, plant physiologists, food technologists and chemists to obtain comprehensive information on the current state of knowledge of this important fruit species. the book starts by providing a detailed overview about archaeology, history and symbolism of pomegranate plants. subsequent sections deal mainly with the taxonomy, propagation and breeding of pomegranates. furthermore, the environmental requirements, different harvest methods, various plant diseases and their individual management, postharvest biology, optimal storage conditions and bioactive compounds are discussed in detail on a high scientific level. a number of scientific articles are listed in each annex of the 18 sections, allowing readers to obtain more detailed information on the individual topics. dr. h. schulz e-mail: hs.consulting.map@t-online.de bibliography: ali sarkhosh, alimohammad m. yavari, zabihollah zamani, the pomegranate – botany, production and uses, cab international 2020, 578 pages including bibliographical references and index, price: 176,00 €, isbn: 978-1-78924-076-4 (hardcover). journal of applied botany and food quality 93, 1 10 (2020), doi:10.5073/jabfq.2020.093.001 1department of agricultural and food sciences (distal), food science university campus, university of bologna, italy 2department of analytical chemistry, faculty of sciences, university of granada, spain application of different analytical methods for the determination of phenolics and antioxidant activity in hawthorn (crataegus spp.) bud and sprout herbal extracts federico ferioli1*, elisa giambanelli2, l. filippo d’antuono1 (submitted: november 7, 2018; accepted: october 8, 2019) * corresponding author summary hawthorn (crataegus spp., family: rosaceae) extracts have been used as pharmaceutical preparations owing to positive effects on cardiovascular system. the alcl3-based official method employed for the determination of pharmacologically active compounds was compared with other techniques such as folin-ciocalteau method and hplc-dad. antioxidant activity was determined by abts radical cation assay. methods were applied on extracts from buds and sprouts collected from common hawthorn (c. monogyna jacq., c. laevigata (poir.) dc.) located in northeastern italy. phenolic content determined by alcl3-based method, folin-ciocalteau method, and hplc-dad was in the range 23,534-27,728, 75,284-100,616 and 57,317-58,639 mg kg-1 of dry matter (dm), respectively, in buds, and 17,280-19,330, 27,653-38,590, and 30,635-32,185 mg kg-1 dm, respectively, in sprouts. antioxidant activity ranged from 119,864 to 174,640 and 31,484 to 52,584 mg trolox eq. kg-1 dm in buds and sprouts, respectively. phenolic amount and profile were significantly affected by phenological stage and sampling location. antioxidant activity was related to flavan-3-ol and hydroxycinnamic acid amount, and to non-phenolic substances. alcl3-based method underestimated total phenolic content owing to lack of selectivity to important phenolic classes whereas folin-ciocalteau method was affected by non-phenolic interfering substances. hplc-dad proved to be more effective in determining hawthorn phenolics. keywords: antioxidant activity; hawthorn buds and sprouts; herbal extracts; hplc-dad; phenolics introduction hawthorn (crataegus spp., family: rosaceae) is the name of closely related plant species widely distributed in the northern hemisphere and used since a long time as herbal remedy (liu, 2012). genus crataegus includes between 150 and 1200 species. common hawthorn (c. monogyna jacq.), wild hawthorn (c. laevigata (poir.) dc.) and their hybrids are widespread in middle europe, including italy; c. pentagyna waldst. & kit. ex willd., c. nigra waldst. & kit., and c. azarolus l. are growing in south and southeastern europe, whereas c. pinnatifida bunge and c. scabrifolia (franch.) rehder are common in china (prinz et al., 2007). the first three species play an important role as raw materials for pharmaceutical preparations (prinz et al., 2007; liu et al., 2011a). indeed, c. monogyna, c. laevigata, and their hybrids are allowed by european pharmacopoeia for the preparation of phytomedicines (council of europe, 2004). the biological effects of hawthorn phenolics have been mainly tested employing extracts obtained from leaves, flowers, and fruits by means of ethanol, methanol, water or mixtures of these solvents. hawthorn-based herbal products are nowadays marketed as alternative treatments for a number of diseases such as hypertension, angina, arrhythmia, and the early stages of congestive heart failure (edwards et al., 2012). several studies have demonstrated that extracts from different plant parts show protective effects on the heart and cardiovascular system, improving coronary circulation and endothelium-dependent vasorelaxation and reducing inflammation (kim et al., 2000; schwinger et al., 2000; pittler et al., 2003; quettier-deleu et al., 2003), and possess hypolipidemic effects too (rajendran et al., 1996; zhang et al., 2002; kuo et al., 2009). hypotensive and radical scavenging properties of crataegus spp. have been also investigated (walker et al., 2002; walker et al., 2006; tadić et al., 2008; froehlicher et al., 2009; qiao et al., 2015). the inhibitory effects on human tumor cell growth and a further characterisation of phenolic extracts of hawthorn buds and fruits have been recently carried out (rodrigues et al., 2012). furthermore, the safety of hawthorn fruit and leaf extracts and their suitability for human consumption have been reviewed (daniele et al., 2006). flavone and flavonol glycosides, hydroxycinnamic acids, and b-type procyanidins have been reported as the phenolic compounds exerting the major biological activity (liu et al., 2011a; rodrigues et al., 2012). recent compositional studies of c. monogyna, c. laevigata, c. pentagyna, and c. pinnatifida leaves, flowers, and fruits highlighted relevant differences of the phenolic content and profile among species and plant parts (svedström et al., 2002, 2006; urbonavičiūtė et al., 2006; prinz et al., 2007; liu et al., 2010, 2011a, 2011b). among the methods for the determination of hawthorn phenolic compounds, the most widely used are high performance liquid chromatography (hplc), coupled with diode array detection (dad) and mass spectrometry (ms) and enabling the determination of individual phenolics, and two spectrophotometric procedures: the folinciocalteau (fc) and the aluminum chloride (alcl3)-based methods. fc method is based on an electron-transfer giving rise to the formation of chromogen complexes between specific redox reagents and phenolics with a maximum absorbance at 750 nm (singleton and rossi, 1965). alcl3-based method is adopted by the official italian pharmacopoeia and depends on the capability of alcl3 of forming, in an acid environment, stable chromogen compounds by binding to flavonoids at the carbonyl located at c-4 and hydroxyl groups sited at c-3 or c-5 of the aglycone moiety (smirnova and pervykh, 1998). a research targeted at comparing the effectiveness of these three analytical methods to detect and quantify hawthorn phenolics is still lacking. the present study was aimed at comparing the method adopted by the official italian pharmacopoeia, fc method and hplc-dad in the evaluation of the phenolic content and profile of two common herbal preparations, a glycerinated macerate and a mother tincture, obtained respectively from buds and sprouts of common hawthorn plants. a second objective of this investigation was to verify the effect of the collecting site on the phenolic profile and content of hawthorn buds and sprouts sampled from different locations in the hilly romagna district, northeastern italy. the antioxidant activity of the extracts and its relation to the content of total phenolics and individual phenolic classes were also assessed. 2 f. ferioli, e. giambanelli, l.f. d’antuono materials and methods plant collection and extract preparation the species herein considered belonged to the group of common hawthorn: c. monogyna jacq. and c. laevigata (poir.) dc., with a prevalence of the first one, and many intermediate forms. species identification was carried out on the basis of pignatti (1982), where c. laevigata is reported as c. oxyaxantha l., and confirmed by comparison to vouchers published on flora italica exsiccata. hawthorn plants located in forlì-cesena and ravenna provinces were sampled in march-april at two phenological stages: closed buds (0.5-1 cm length) and sprouts (flowers and leaves, 1.5-2 cm length). buds were collected at rio dei cozzi (province: forlì-cesena; harvest date: 23 march; 44°18´n 11°92´e, 130 m a.s.l.) and brisighella (province: ravenna; harvest date: 24 march; 44°22´n 11°77´e, 107 m a.s.l.). sprouts were collected at magliano (province: forlì-cesena; harvest date: 24 april; 44°17´n 12°10´e, 35 m a.s.l.), rio dei cozzi (harvest date: 24 april), and santa sofia (province: forlì-cesena; harvest date: 30 april; 43°95´n 11°91´e, 257 m a.s.l.). buds and sprouts were harvested during their balsamic period, immediately ground, and then extracted by means of the different maceration processes, commonly adopted in herbal preparation, according to the plant part. in each location buds and sprouts were collected at different sites to allow replication within location. maceration and extraction were carried out in the laboratories of cento fiori s.r.l. (forlì, italy). buds. five hundred grams of fresh buds were soaked in the dark for 30 days at room temperature in 1.10 kg of ethanol and 1.10 kg of glycerol, corresponding to 1.39 and 0.88 l of the two solvents, respectively. the amounts of ethanol and glycerol added to the fresh material were chosen in order to have a final 1/20 (w/w) dry matterto-extracting mixture ratio. food grade ethanol 96.4% was employed. sprouts. five hundred grams of fresh sprouts were soaked in the dark for 30 days at room temperature in 0.70 kg of ethanol and 0.15 kg of water, corresponding to 0.89 and 0.15 l of the two solvents, respectively. the amounts of ethanol and water added to the fresh material were chosen in order to have a final 1/10 (w/w) dry matter-to-extracting mixture ratio and a 60% (v/v) hydroalcoholic mixture as extracting mean. the amount of water to be added was determined taking into account the moisture content of the fresh material. food grade ethanol 96.4% was employed. dry matter was determined gravimetrically as the mass loss of 10 g of fresh material kept at 105 °c until constant weight. it corresponded to 0.22 and 0.25 g g-1 for buds and sprouts, respectively. extracts were daily shaken for 1 min during the soaking period, pressed, filtered in amber glass bottles and kept at 4 °c until analyses. extracts were prepared in duplicate for each sample. each extract was then analysed separately, performing analytical determinations twice for each replication (n = 4). reagents and chemicals all chemicals and solvents were, unless specified, of analytical grade and purchased from sigma-aldrich (st. louis, mo, usa). deionised water was obtained by an elix 10 water purification system from millipore (bedford, ma, usa). chlorogenic acid, (-)-epicatechin, 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (abts), and (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2 carboxylic acid (trolox) standards were from sigma-aldrich. vitexin (apigenin-8-c-glucoside), vitexin-2´´-o-rhamnoside, and hyperoside (quercetin-3-o-galactoside) standards were bought from extrasynthese (genay, france). spectrophotometric determination of total flavonoid content (flav) flav was evaluated according to the official italian pharmacopoeia (official pharmacopoeia of the italian republic, 1998) and smirnova and pervykh (1998). a volume corresponding to 0.600 and 0.800 ml of bud and sprout extracts, respectively, was transferred to a 2-ml polypropylene (pp) microtube and adjusted to a 1/1 (v/v) water-to-organic solvent ratio by the addition of 0.425 and 0.190 ml of water, respectively. extracts were then centrifuged at 21,500 g for 5 min at 10 °c. 0.4 ml of the supernatant fraction were further diluted in a second pp microtube adding 1.6 ml of ethanol/water 1/1 (v/v) before spectrophotometric determination. 0.5 ml of diluted extract, to which were added 5.0 ml of 5% (v/v) acetic acid in methanol and 0.25 ml of 2% (w/v) aluminum chloride hexahydrate (alcl3·6h2o) in methanol, were manually shaken in a 10-ml teflon screw cap glass tube for 10 sec and kept in the dark for 30 min. the absorbance of the solution was then read at 425 nm (25 °c) against methanol in a double beam spectrophotometer (mod. uv-1601) from shimadzu (kyoto, japan). flav was quantified by constructing a calibration curve employing hyperoside as a reference compound. from a stock solution (c = 1.872 mg ml-1) in ethanol/dimethyl sulfoxide 9/1 (v/v), diluted solutions were prepared in ethanol/water 1/1 (v/v) in a concentration range of 0.005-0.494 mg ml-1 (seven calibration points, r > 0.99). each diluted standard solution was analysed in three replications. observed absorbance values were corrected subtracting the absorbance of a blank sample prepared employing 0.5 ml of ethanol/water 1/1 (v/v) in place of hawthorn extract. spectrophotometric determination of total phenolic content (tpc) tpc was determined according to the folin-ciocalteau spectrophotometric method (singleton and rossi, 1965). before analyses bud and sprout extracts underwent the same dilution procedure formerly described for flav determination. 7.5 ml of water, 0.1 ml of the diluted extract and 0.5 ml of folin-ciocalteau reagent were then transferred to a 10-ml teflon screw cap glass tube, manually shaken for 10 sec, added with 2.0 ml of 15% (w/v) sodium carbonate, and finally shaken for further 10 sec. after 2 h in the dark, the absorbance of the solution was read at 750 nm (25 °c) against water in the same double beam spectrophotometer previously described. tpc was assessed by constructing a gallic acid calibration curve. from a stock solution (c = 2.020 mg ml-1) in ethanol, diluted solutions were prepared in ethanol/water 1/1 (v/v) in a concentration range of 0.005-1.010 mg ml-1 (eight calibration points, r > 0.99). each solution was analysed in three replications. observed absorbance values were corrected subtracting the absorbance of a blank sample prepared employing 0.1 ml of ethanol/water 1/1 (v/v) in place of hawthorn extract. determination of phenolics by high performance liquid chromatography coupled with diode array detection (hplc-dad) phenolic extracts were analysed in gradient mode on a hplc system from jasco (tokyo, japan) equipped with two binary pumps (mod. pu-1580), a diode array uv/vis detector (mod. md-1510, quartz flow cell, optical path: 10 mm), and an autosampler (mod. as-2055 plus). the following solvent system was employed: mobile phase a: 0.5% (v/v) formic acid in water/acetonitrile/methanol 95/4/1 (v/v/v); mobile phase b: 0.5% (v/v) formic acid in acetonitrile/methanol 4/1 (v/v). ethanol/water 1/1 (v/v) was used as a cleaning solution for autosampler syringe. solvents were of chromatographic grade. mobile phases and cleaning solution were preliminary filtered through a nylon membrane filter (diameter: 47 mm; pore dimension: 0.45 μm) from gvs filter technology (indianapolis, in, usa), and degassed in an ultrasonic bath for 32 min at room temperature. the gradient program was as follows: 0-35 min, 100 to 74% a; 35-37 min, 74 to 20% a; 37-45 min, 20% a; 45-47 min, 20 to 100% a; 47-57 min, determination of phenolics and antioxidant activity in hawthorn extracts 3 100% a. the flow rate was 1.0 ml min-1 and the injection volume was 5 μl. hplc-dad traces were acquired at 280, 330, 350, and 520 nm, whereas absorption spectra were recorded from 200 to 600 nm. a kinetex 2.6μ c18 100a (75 × 4.6 mm i.d., 2.6 μm particle size) column from phenomenex (torrance, ca, usa) was used for compound separation and maintained at 35 °c during analyses. before injection, 0.600 and 0.800 ml of crude bud and sprout extracts were diluted with 0.425 and 0.190 ml of water, respectively, in a 2-ml pp microtube to reach a 1/1 (v/v) water-to-organic solvent ratio, and centrifuged at 21,500 g for 5 min at 10 °c. one ml of the supernatant fraction was then filtered in a hplc glass vial through a regenerated cellulose (rc) syringe filter (diameter: 13 mm, pore dimension: 0.45 μm) from gvs. hydroxycinnamic and benzoic acids, flavan-3-ols, and flavonols were quantified by external standard mode at 330, 280, and 350 nm, respectively, using as reference compounds chlorogenic acid, (-)-epicatechin, and hyperoside. flavones were quantified at 330 or 350 nm in accordance to the absorption spectrum of each compound. monoglycosyl and unidentified flavones were quantified using vitexin as reference compounds, whereas diglycosyl flavones were quantified by vitexin-2´´-o-rhamnoside. stock solutions were prepared at the following concentrations: chlorogenic acid: 2.056 mg ml-1 in ethanol; (-)-epicatechin: 2.030 mg ml-1 in ethanol; hyperoside: 1.872 mg ml-1 in ethanol/dimethyl sulfoxide 9/1 (v/v); vitexin: 1.140 mg ml-1 in ethanol/dimethyl sulfoxide 8/2 (v/v); vitexin-2´´-o-rhamnoside: 2.560 mg ml-1 in ethanol. diluted solutions for calibration curves were prepared in ethanol/ water 1/1 and analysed in duplicate each. concentration ranges of cali bration curves were 0.005-1.028 (eight calibration points, r > 0.99), 0.005-0.499 (seven calibration points, r > 0.99), 0.005-0.494 (seven calibration points, r > 0.99), 0.002-0.100 (six calibration points, r > 0.99), and 0.005-0.998 mg ml-1 (eight calibration point, r > 0.99) for chlorogenic acid, (-)-epicatechin, hyperoside, vitexin and vitexin-2´´o-rhamnoside, respectively. the limits of detection (lods) and the limits of quantification (loqs) of standard compounds were set at 3 × s/n and 7 × s/n, respectively, where s/n is the signal-to-noise ratio. with regard to phenolic determination in hawthorn buds, lods were at levels of 14, 75, 19, 26, 53, and 27 mg kg-1 dm for chlorogenic acid, (-)-epicatechin, vitexin at 330 nm, vitexin at 350 nm, vitexin-2´´o-rhamnoside (330 nm), and hyperoside, respectively, whereas for the same compounds loqs were at level of 33, 175, 44, 60, 123, and 63 mg kg-1 dm. with regard to phenolic determination in hawthorn sprouts, lods were at levels of 5, 26, 6, 9, 18, and 9 mg kg-1 dm for chlorogenic acid, (-)-epicatechin, vitexin at 330 nm, vitexin at 350 nm, vitexin-2´´-o-rhamnoside (330 nm), and hyperoside, whereas for the same compounds loqs were at levels of 11, 60, 15, 20, 42, and 22 mg kg-1 dm. phenolic identification phenolic identification was carried out comparing peak retention times and uv/vis absorption spectra with those of standard compounds employed in the construction of calibration curves, and by means of a liquid chromatography system hp 1100 series equipped with a diode array detector and coupled with a single quadrupole mass spectrometer (1100 msd series, mod. g1946a) from agilent technologies (santa clara, ca, usa). mass spectra of different peaks were compared to fragmentation patterns and structural information reported in previous works (liu et al., 2011a, b; liu, 2012). other investigations focusing on phenolic determination in green vegetables were useful for a better compound identification and to clarify the order of elution of hydroxycinnamic acids (clifford et al., 2006; lin and harnly, 2009; rodrigues et al., 2012; bonta et al., 2017). compounds reported as unidentified were assigned to the corresponding chemical class on the basis of their uv/vis spectra. the mass spectrometer operated both in positive and negative atmospheric pressure ionisation-electrospray source (api-es) under the following conditions: drying gas (nitrogen) temperature: 350 °c; nebuliser pressure: 35 psig; capillary voltage: 3,000 v; fragmentor voltage: 100 v; mass ranges: 100-700 and 700-1,200 m/z. mobile phases were prepared in 0.1 in place of 0.5% (v/v) formic acid to improve the mass detector response. spectrophotometric evaluation of the antioxidant activity antioxidant activity was evaluated by the disappearance of radical chromogen 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (abts), according to the method proposed by eberhardt et al. (2005), with some modification hereafter reported. briefly, abts radical cation (abts·+) was produced by reacting a 7 mm abts stock solution with 70 mm potassium persulphate overnight at 4 °c, as described by re et al. (1999). abts·+ ethanolic so lution was prepared daily. bud and sprout extracts underwent the same dilution procedure formerly described for flav and tpc determination. before performing abts assay, 0.5 ml of each extracts were transferred to a 2-ml pp microtube and further diluted by adding 1.5 ml of ethanol/water 1/1 (v/v). after the last dilution, 30 μl of solution were added to 3.0 ml of ethanolic abts·+ and shaken in a plastic cuvette. the absorbance was then read at 734 nm (25 °c) in a double beam spectrophotometer over 6 min. absorbance correction was performed by a blank sample prepared employing 30 μl of ethanol/water 1/1 (v/v) in place of hawthorn extract. the anti oxidant activity was referred to as trolox equivalent antioxidant ca pacity (teac) by constructing a trolox calibration curve in a concentration range of 0.005-0.499 mg ml-1 (six calibration points, r > 0.99). trolox diluted solutions were prepared in ethanol/water 1/1 (v/v) from a stock ethanolic solution (c = 1.980 mg ml-1). each solution was analysed in triplicate. statistics data were preliminary processed by analysis of variance (anova), according to a nested design, including the effects: a) plant part (buds, sprouts), and b) location within the same part, since locations were not exactly coincident for the two harvested plant parts. fisher’s least significance difference test (lsd) was used for multiple comparison. anova and lsd tests were also employed to compare the results obtained by the different analytical methods used to determine total phenolic content. correlation analysis was used to investigate the relationships between a) the antioxidant activity, phenolics, and individual phenolic classes and b) phenolic amounts obtained by means of different analytical procedures. the sysyat© 10.0 package (systat software, chicago, il, usa) was used throughout. results and discussion total flavonoid content (flav) flav was significantly affected by phenological stage (tab. 1). buds showed a flavonoid content 38% higher than sprouts. within plant part, the highest flavonoid content was determined in buds from rio dei cozzi and sprouts from magliano. total flavonoid amounts were higher than values amounting to 6,232 and 10,266 mg kg-1 dm and reported by froehlicher et al. (2009) in commercial c. monogyna flowering tops and flowers, respectively. total phenolic content (tpc) on average, tpc was almost three-times higher in buds than in sprouts (+180%), with a different distribution among collection sites, in comparison to total flavonoids (tab. 1). in fact, tpc was higher in buds from brisighella than in sample from rio dei cozzi, whereas 4 f. ferioli, e. giambanelli, l.f. d’antuono santa sofia sample showed the highest tpc content within sprouts. buds and sprouts showed higher and lower tpc values, respec tively, in comparison to commercial c. monogyna flowering tops (56,292 mg gallic acid eq. kg-1 dm) and flowers (49,310 mg gallic acid eq. kg-1 dm) analysed by froehlicher et al. (2009). phenolic composition determined by hplc thirty phenolic compounds were identified by hplc-dad-ms (tab. 2) and quantified by hplc-dad (tab. 3); five hydroxycinnamic acids, two flavones, and two flavonols were not clearly identified but assigned to the corresponding chemical class by the evaluation of their absorption spectra. the total phenolic amount determined by hplc-dad was significantly affected by phenological stage and location (tab. 1). consistently with flav and tpc results, buds showed a phenolic content almost twice higher (+86%) than sprouts. small but significant differences were also noticed between locations, with the highest amounts determined in buds from rio dei cozzi and sprouts from santa sofia. the composition of the phenolic fraction was also affected by plant part and location. (-)-epicatechin was the only identified flavan-3-ol, representing the fourth most relatively abundant compound in buds (tab. 4), whereas in sprouts it amounted on average to less than 0.02 mg mg-1. buds from brisighella showed the highest (-)-epicatechin fraction. no procyanidins were identified in hplc-dad-ms traces, even previously determined in fruits and leaves of crataegus spp. (liu, 2012). in fact, the absolute and individual amount of these compounds has been reported to vary significantly according to plant part and species (svedström et al., 2002; liu et al., 2010). hydroxycinnamic acids accounted for about 0.30 mg mg-1 of phenolics (tab. 4); a significantly higher fraction was observed in buds than in sprouts. within plant part, the relative amount of acids exceeded 0.30 mg mg-1 of phenolics in buds from brisighella and sprouts from santa sofia. chlorogenic acid (ac-6) was prevalent, representing on average the second and the fourth most relatively abundant phenolic compound in buds and sprouts, respectively. flavones were the most abundant phenolic compounds in buds and sprouts, accounting for about half of total phenolics (tab. 5). significant but not relevant differences were determined within sprouts, whereas higher differences were noticed within buds. vitexin-2´´-o-rhamnoside (on-4) and its acylated form vitexin-2´´o-(4´ ´´-o-acetyl)-rhamnoside (on-8) where the dominating compounds, exceeding together 0.40 mg mg-1 of total phenolics. both phenological stage and location affected the relative content of the two main flavones. on-4 was the most abundant phenolic compound, with a relative content range of 0.23-0.28 mg mg-1 of total phenolics. differences among locations were particularly remarkable for on-8. the relative total flavonol amount was highly different between buds and sprouts (tab. 6). five out of eight flavonols were either not detected or determined in traces in buds. location significantly affected flavonol within sprouts. hyperoside (quercetin-3-o-galactoside, ol2) was the dominant flavonol in sprouts, representing the third most relatively abundant phenolic compound. another important flavonol was isoquercitrin (quercetin-3-o-glucoside, ol-4), accounting for over 0.03 mg mg-1 of phenolics in all sprout samples. hplc determination of hawthorn phenolics have been carried out on leaves, flowers and fruits of several crataegus species, as reviewed by edwards et al. (2012). on the whole, buds and sprouts here analysed showed on average a higher total phenolic content in comparison to c. monogyna and c. laevigata samples surveyed by hplcdad in previous investigations and coming from different plant parts (prinz et al., 2007; ringl et al., 2007). in agreement with our results, (-)-epicatechin, chlorogenic acid, vitexin-2´´-o-rhamnoside, vitexin acetyl rhamnoside, and hyperoside were reported as the five tab. 1: effect of plant part, location and analytical method on flavonoid and phenolic content and antioxidant activity. effects1 flav2 tpc3 phentot4 teac5 (mg hyperoside eq. kg-1 dm) (mg gallic acid eq. kg-1 dm) (mg kg-1 dm) (mg trolox eq. kg-1 dm) plant part buds 25,631c 87,950a 57,978b 147,252 sprouts 18,607b 31,439a 31,240a 38,961 significance6 ** ** ** ** lsd7 420 1,356 338 4,399 location (within plant part) buds rio dei cozzi 27,728c 75,284a 58,639b 119,864 brisighella 23,534c 100,616a 57,317b 174,640 significance ** ** ** ** lsd 760 2,453 612 7,958 sprouts magliano 19,330b 27,653a 30,901a 31,484 rio dei cozzi 19,213b 28,075a 30,635a 32,814 santa sofia 17,280c 38,590a 32,185b 52,584 significance ** * ** ** lsd 439 1,416 353 4,595 1 different letters within the same row denote significant difference (p ≤ 0.05) among flav, tpc and phentot values. 2 flav: flavonoid content determined by spectrophotometric alcl3-based method. 3 tpc: total phenolic content determined by folin-ciocalteau spectrophotometric method. 4 phentot: total phenolic content determined by hplc. 5 teac: trolox equivalent antioxidant capacity. 6 ns: not significant; *: p ≤ 0.05; **: p ≤ 0.01. 7 lsd: least significant difference (p = 0.05), referred to column values. determination of phenolics and antioxidant activity in hawthorn extracts 5 ta b. 2 : n am es , a bb re vi at io ns , r et en tio n tim es , m ax im um a bs or pt io n w av el en gt hs a nd m as s sp ec tr al d at a of in di vi du al p he no lic c om po un ds d et er m in ed b y h pl c in h aw th or n bu d an d sp ro ut e xt ra ct s. c om po un d na m e ta g pe ak n o. 1 r t (m in )2 u v λ m ax (n m )3 m w 4 d ia gn os tic fr ag m en t i on s (m /z ) po si tiv e (+ )5 n eg at iv e ()5 h yd ro xy ci nn am ic a ci ds n eo ch lo ro ge ni c ac id a c -1 1 3. 85 23 5, 3 23 35 4 16 3 (1 00 ), 37 7 (4 4) , 3 93 (1 0) 17 9 (1 4) , 1 91 (2 0) , 3 53 (1 00 ) c af fe ic a ci d de ri va tiv e a c -2 2 4. 55 23 5, 3 27 13 5 (1 9) , 1 45 (8 ), 16 3 (1 00 ) 13 5 (1 00 ), 17 9 (4 6) u ni de nt ifi ed h yd ro xy ci nn am ic a ci d a c -3 3 5. 97 23 5, 3 27 3pc ou m ar oy lq ui ni c ac id a c -4 4 6. 27 22 7, 3 07 33 8 11 9 (4 6) , 1 47 (1 00 ), 36 1 (3 5) , 3 77 (6 ) 11 9 (4 ), 16 3 (7 0) , 1 91 (4 ), 33 7 (1 00 ) c af fe ic a ci d a c -5 5 7. 86 23 5, 3 23 18 0 13 5 (3 3) , 1 45 (1 4) , 1 63 (1 00 ), 18 1 (4 ), 21 9 (4 0) 13 5 (6 6) , 1 79 (1 00 ) c hl or og en ic a ci d a c -6 6 8. 29 23 9, 3 23 35 4 16 3 (1 00 ), 35 5 (1 ), 37 7 (5 1) , 3 93 (1 6) 13 5 (7 ), 17 9 (7 ), 19 1 (1 00 ), 35 3 (5 4) u ni de nt ifi ed h yd ro xy ci nn am ic a ci d a c -7 7 9. 97 23 5, 3 23 5pc ou m ar oy lq ui ni c ac id a c -8 9 10 .9 23 5, 3 07 33 8 11 9 (3 8) , 1 47 (1 00 ), 36 1 (2 2) 33 7 (1 00 ) 4pc ou m ar oy lq ui ni c ac id a c -9 10 11 .5 1 23 1, 3 11 33 8 11 9 (4 3) , 1 47 (1 00 ), 36 1 (2 0) 19 1 (8 1) , 3 37 (1 00 ) fe ru lo yl qu in ic a ci d6 a c -1 0 11 13 .2 5 23 1, 3 07 36 8 19 1 (1 00 ), 36 7 (1 8) u ni de nt ifi ed h yd ro xy ci nn am ic a ci d a c -1 1 25 22 .0 5 23 5, 3 27 u ni de nt ifi ed h yd ro xy ci nn am ic a ci d a c -1 2 27 24 .7 5 23 5, 3 27 u ni de nt ifi ed h yd ro xy ci nn am ic a ci d a c -1 3 29 26 .8 7 23 5, 3 23 f la va n3ol s ()e pi ca te ch in e pi 8 10 .4 5 23 1, 2 75 29 0 29 1 (2 1) , 3 13 (1 00 ), 32 9 (1 6) 28 9 (1 00 ) f la vo ne s a pi ge ni n pe nt os id e he xo si de o n -1 12 15 .8 1 23 1, 2 67 , 3 31 56 4 56 3 (1 00 ) u ni de nt ifi ed fl av on e o n -2 13 16 .0 2 23 1, 2 67 , 3 39 v ite xi n (a pi ge ni n8c -g lu co si de ) o n -3 14 17 .6 7 23 5, 2 67 , 3 35 43 2 43 3 (1 00 ), 45 5 (1 4) , 4 71 (4 ) 43 1 (1 00 ) v ite xi n2´ ´o -r ha m no si de o n -4 15 18 .1 5 23 5, 2 67 , 3 39 57 8 57 9 (1 00 ), 60 1 (4 4) , 6 17 (1 4) 43 1 (5 4) , 5 77 (1 00 ) is ov ite xi n2´ ´o -( 4´ ´́o -a ce ty l) -r ha m no si de o n -5 20 19 .9 3 23 5, 2 67 , 3 39 62 0 62 1 (1 00 ), 64 3 (5 8) , 6 59 (4 6) 61 9 (1 00 ) u ni de nt ifi ed fl av on e o n -6 21 20 .7 5 22 7, 2 67 , 3 39 l ut eo lin h ex os id e o n -7 22 21 .2 1 22 7, 3 43 44 8 44 7 (1 00 ) v ite xi n2´ ´o -( 4´ ´́o -a ce ty l) -r ha m no si de o n -8 28 25 .5 3 23 5, 2 67 , 3 39 62 0 62 1 (1 00 ), 64 3 (3 7) , 6 59 (9 ) 61 9 (1 00 ) f la vo no ls q ue rc et in -3 -o -r ha m no sy lg al ac to si de o l -1 16 18 .5 2 25 5, 3 59 61 0 60 9 (1 00 ) h yp er os id e (q ue rc et in -3 -o -g al ac to si de ) o l -2 17 18 .8 7 25 5, 3 51 46 4 30 3 (4 8) , 4 65 (3 ), 48 7 (1 00 ), 50 3 (4 ) 46 3 (1 00 ) r ut in (q ue rc et in -3 -o -r ut in os id e) o l -3 18 19 .2 5 25 5, 3 51 61 0 61 1 (1 0) , 6 33 (1 00 ), 64 9 (1 5) 46 3 (6 5) , 6 09 (1 00 ) is oq ue rc itr in (q ue rc et in -3 -o -g lu co si de ) o l -4 19 19 .5 3 25 5, 3 51 46 4 30 3 (3 9) , 4 65 (5 ), 48 7 (1 00 ), 50 3 (7 ) 46 3 (1 00 ) 8m et ho xy ka em pf er ol -3 -o -g lu co si de o l -5 23 21 .4 1 26 3, 3 47 47 8 47 7 (1 00 ) u ni de nt ifi ed fl av on ol o l -6 24 21 .6 3 25 5, 3 51 8m et ho xy ka em pf er ol -3 -o -p en to si de o l -7 26 22 .6 9 26 3, 3 47 44 8 47 1 (1 00 ), 48 7 (4 ) 44 7 (1 00 ) q ue rc et in 6 o l -8 30 28 .9 7 25 5, 3 67 30 2 30 3 (1 00 ), 32 5 (1 ), 34 1 (1 3) 30 1 (1 00 ) 1 o rd er o f e lu tio n. 2 r t: re te nt io n tim e. 3 λ m ax : m ax im um a bs or pt io n w av el en gt hs . 4 m ol ec ul ar w ei gh t. 5 po la ri ty . f or e ac h fr ag m en t m as sto -c ha rg e (m /z ) r at io a nd th e re la tiv e ab un da nc e (i n br ac ke ts ) h av e be en re po rt ed . 6 c oel ut in g w ith u ni de nt ifi ed c om po un ds . 6 f. ferioli, e. giambanelli, l.f. d’antuono major phenolic compounds in c. monogyna and c. laevigata extracts (svedström et al., 2006; orhan et al., 2007; prinz et al., 2007; ringl et al., 2007; martino et al., 2008; froehlicher et al., 2009). as here observed, total phenolic content and, particularly, phenolic profile were significantly affected by plant part and plant location, with fruits showing the lowest amounts, (orhan et al., 2007; prinz et al., 2007; ringl et al., 2007; froehlicher et al., 2009). comparison of analytical methods for phenolic determination the detected amount of phenolics was significantly affected by the analytical method employed (tab. 1). tpc content of buds, determined by the folin-ciocalteau method, was about three (+243%) and two (+52%) times higher than values obtained by the alcl3-based method (flav) and hplc-dad (phentot), respectively. tpc gave the highest values also for sprouts, without any significant difference with respect to phentot. the lower values obtained by alcl3-based method in comparison to tpc and phentot were expected, owing to the selectivity of this method towards flavonols and flavones (smirnova and pervykh, 1998), and the high relative amounts of other phenolic classes such as flavan-3-ols and hydroxycinnamic acids is hawthorn extracts. in particular, the differences with respect to other methods were higher in buds, where the relative flavonol content was much lower than in sprouts. on the other side, the higher tpc values determined in buds in comparison to phentot and trends observed within bud extracts, could be related to the presence of non-phenolic substances such as ascorbic acid, aromatic amines and sugars that may interfere with the results of this assay, leading to an overestimation of total phenolic content (escarpa and gonzález, 2001; tsao and yang, 2003). the lack of accuracy of folin-ciacalteau method, especially with regard to buds, was confirmed by the negative and significant correlation (r = -0.87, p < 0.01) between tpc and flavtot, whereas in sprouts a positive and significant correlation was verified between the two sets of data (r = 0.86, p < 0.01). the main drawbacks of the folin-ciocalteau procedure and the alcl3-based method seemed to be their sensitivity to the presence of interfering compounds (proteins, sugars) and to the relative amount of different phenolic classes, respectively. in conclusion, hplc-dad proved to be a more reliable and accurate method for total phenolic determination. antioxidant activity (teac) teac was about four times higher in bud than in sprout extracts (tab. 1). within buds, teac was 46% higher in sample from brisighella than in rio dei cozzi. among sprouts, the highest teac value was assessed in the sample from santa sofia. froehlicher et al. (2009) assessed lower antioxidant activities in commercial c. monogyna flowering tops (26,030 mg trolox eq. kg-1 dm) and flowers (36,042 mg trolox eq. kg-1 dm). the antioxidant capacity of hawthorn extracts needs an in-depth investigation, even if it has not been considered until now as the main biological activity associated with these preparations. indeed, teac observed in bud extracts are comparable to values detected in green tea, a popular antioxidantrich and healthy perceived herbal preparation (rusak et al., 2008; komes et al., 2010). correlation of antioxidant activity to total phenolic and phenolic class content antioxidant activity showed the highest correlation to tpc for both buds and sprouts (tab. 7). this result could depend on the effect of the non-phenolic substances, determined by the folin-ciocalteau spectrophotometric method, that can also contribute to antioxidant activity (chun et al., 2003; kim et al., 2003). a significant but lower correlation was observed in sprouts between teac and total phenolics determined by hplc-dad, whereas in buds teac and phentot were negatively related. with regard to single phenolic classes, teac was positively related to the absolute amount of (-)-epicatechin and hydroxicinnamic acid both in buds and sprouts. a low but still significant positive correlation was determined between sprout teac and flavone content, whereas flavone content was negatively related to teac in buds. flavonol content showed no correlation to teac both in buds and sprouts. these findings could partly explain differences in antioxidant activity between locations. buds from brisighella and sprouts from santa sofia showed the highest teac values and also the highest tab. 3: content ranges of phenolic compounds determined by hplc. compound tag1 content range (mg kg-1 dm)2 buds sprouts hydroxycinnamic acids ac-1 2,107-3,160 1,106-1,830 ac-2 nd-18 111-156 ac-3 87-105 229-307 ac-4 78-234 284-525 ac-5 nd-tr tr-34 ac-6 10,248-13,090 3,129-6,198 ac-7 523-760 121-211 ac-8 120-311 151-193 ac-9 202-307 262-702 ac-10 67-138 222-398 ac-11 1,310-1,362 537-911 ac-12 925-1,606 123-173 ac-13 tr 52-383 subtotal (ac-tot) 17,279-19,479 7,626-11,144 flavan-3-ols epi 4,378-11,562 61-878 flavones on-1 254-290 242-254 on-2 86-122 72-85 on-3 1,286-1,930 228-373 on-4 13,303-15,349 6,658-8,501 on-5 813-1,824 327-355 on-6 605-1,129 120-141 on-7 75-98 48-68 on-8 6,288-16,813 4,138-6,076 subtotal (on-tot) 24,778-35,486 13,674-14,609 flavonols ol-1 nd 518-921 ol-2 644-736 4,107-5,913 ol-3 tr 138-215 ol-4 333-381 1,097-1,397 ol-5 nd 109-240 ol-6 nd 132-321 ol-7 427-470 223-469 ol-8 nd 5-165 subtotal (ol-tot) 1,495-1,497 6,488-9,451 total phenolic compounds 57,317-58,639 30,635-32,185 (phen-tot) 1 for compound names see tab. 2. 2 nd: not detectable; tr: traces. determination of phenolics and antioxidant activity in hawthorn extracts 7 tab. 4: effect of plant part and location on (-)-epicatechin, total and individual hydroxycinnamic acid fractions. effects relative amount (mg mg-1 total phenolics)1 epi ac-tot ac-1 ac-2 ac-3 ac-4 ac-5 ac-6 ac-7 ac-8 ac-9 ac-10 ac-11 ac-12 ac-13 plant part buds 0.138 0.317 0.045 < 0.001 0.002 0.003 tr 0.202 0.011 0.004 0.004 0.002 0.023 0.022 tr sprouts 0.011 0.273 0.046 0.004 0.008 0.013 < 0.001 0.135 0.005 0.006 0.014 0.010 0.024 0.005 0.005 significance2 ** ** ** ** ** ** * ** ** ** ** ** ** ** ** lsd3 0.002 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 0.001 < 0.001 0.001 0.001 < 0.001 < 0.001 < 0.001 < 0.001 location (within plant part) buds rio dei cozzi 0.075 0.295 0.054 < 0.001 0.002 0.004 nd 0.175 0.009 0.005 0.005 0.002 0.022 0.016 tr brisighella 0.202 0.340 0.037 tr 0.002 0.001 tr 0.228 0.013 0.002 0.004 0.001 0.024 0.028 tr significance ** ** ** ** ns ** ns ** ** ** ** ** ** ** ns lsd 0.003 0.003 < 0.001 < 0.001 < 0.001 0.001 0.001 0.001 0.001 < 0.001 0.001 < 0.001 sprouts magliano 0.002 0.246 0.036 0.004 0.007 0.009 tr 0.123 0.005 0.006 0.013 0.009 0.029 0.005 0.002 rio dei cozzi 0.004 0.241 0.057 0.004 0.007 0.017 tr 0.098 0.004 0.006 0.008 0.007 0.026 0.004 0.003 santa sofia 0.025 0.333 0.046 0.005 0.009 0.012 0.001 0.182 0.007 0.005 0.021 0.014 0.017 0.006 0.010 significance ** ** ** ns ** ** ** ** ** ** ** ** ** ** ** lsd 0.002 0.002 < 0.001 < 0.001 < 0.001 < 0.001 0.001 < 0.001 0.001 0.001 < 0.001 < 0.001 < 0.001 < 0.001 1 for compound names see table 2; nd: not detectable; tr: traces. 2 ns: not significant; *: p ≤ 0.05; **: p ≤ 0.01. 3 lsd: least significant difference (p = 0.05). tab. 5: effect of plant part and location on total and individual flavone fractions. effects relative amount (mg mg-1 total phenolics)1 on-tot on-1 on-2 on-3 on-4 on-5 on-6 on-7 on-8 plant part buds 0.519 0.005 0.002 0.028 0.247 0.023 0.015 0.001 0.198 sprouts 0.456 0.008 0.002 0.009 0.253 0.011 0.004 0.002 0.167 significance2 ** ** ** ** ** ** ** ** ** lsd3 0.001 < 0.001 < 0.001 < 0.001 0.001 < 0.001 < 0.001 < 0.001 0.001 location (within plant part) buds rio dei cozzi 0.605 0.005 0.002 0.033 0.227 0.031 0.019 0.001 0.287 brisighella 0.432 0.004 0.002 0.022 0.268 0.014 0.011 0.002 0.110 significance ** ** ** ** ** ** ** ** ** lsd 0.002 < 0.001 < 0.001 < 0.001 0.001 < 0.001 0.001 < 0.001 0.001 sprouts magliano 0.447 0.008 0.003 0.007 0.277 0.011 0.005 0.002 0.135 rio dei cozzi 0.484 0.008 0.002 0.012 0.257 0.011 0.004 0.002 0.188 santa sofia 0.438 0.008 0.002 0.009 0.225 0.011 0.004 0.001 0.178 significance ** ** ** ** ** ** ** ** ** lsd 0.001 < 0.001 < 0.001 < 0.001 0.001 < 0.001 < 0.001 < 0.001 0.001 1 for compound names see tab. 2; nd: not detectable; tr: traces. 2 ns: not significant; *: p ≤ 0.05; **: p ≤ 0.01. 3 lsd: least significant difference (p = 0.05). fractions of phenolics such as (-)-epicatechin and hydroxycinnamic acids. furthermore, tpc data may indicate a higher content of nonphenolic substances in these samples, also contributing to antioxidant activity. this result partly agreed with froehlicher et al. (2009) that highlighted a higher free radical scavenging property of (-)-epicatechin in comparison to quercetin glycosides such as rutin and hyperoside. nevertheless, the present investigation pointed out that even hydroxycinnamic acids may positively contribute to the antioxidant capacity of hawthorn preparations. 8 f. ferioli, e. giambanelli, l.f. d’antuono conclusion this study focused on the comparison of the official alcl3-based method with other widespread analytical procedures (folinciocalteu method and hplc-dad) used for the determination of phenolics in extracts obtained from italian hawthorn buds and sprouts. hplc-dad was a more reliable and accurate method for total phenolic determination, in comparison to folin-ciocalteau and alcl3-based spectrophotometric methods, which are known to suffer from the presence of interfering non-phenolic substances and a lack of sensitivity to flavan-3-ols and hydroxycinnamic acids, respectively. phenological stage and geographical origin significantly influenced total phenolic content and the phenolic profile. antioxidant activity was positively related to the amount of (-)-epicatechin and hydroxycinnamic acids whereas no positive correlation was assessed with the content of flavones and flavonols. acknowledgements the authors address special thanks to all the staff of cento fiori s.r.l. (forlì, italy) for sample supplying and processing. they are also grateful to alessandra zaccarelli for the support shown during this project both at cento fiori s.r.l. and at the food science university campus. conflict of interest no potential conflict of interest was reported by the authors. references bonta, r.k., 2017: application of hplc and esi-ms techniques in the analysis of phenolic acids and flavonoids from green leafy vegetables (glvs). j. pharm. anal. 7, 349-364. doi: 10.1016/j.jpha.2017.06.005 chun, o.k., kim, d.o., moon, h.y., kang, h.g., lee, c.y., 2003: contribution of individual polyphenolics to total antioxidant capacity of plums. j. agr. food chem. 51, 7240-7245. doi: 10.1021/jf0343579 clifford, m.n., zheng, w., kuhnert, n., 2006: profiling the chlorogenic acids of aster by hplc–msn. phytochem. analysis 17, 384-393. doi: 10.1002/pca.935 council of europe, 2004: european directorate for the quality of medicines & healthcare (edqm). european pharmacopoeia 5th ed. strasbourg, france, 1712-1715. daniele, c., mazzanti, g., pittler, m.h., ernst, e., 2006: adverse-event profile of crataegus spp. drug safety 29, 523-535. doi: 10.2165/00002018-200629060-00005 eberhardt, m.v., kobira, k., keck, a.s., juvik, j.a., jeffery, e.h., 2005: correlation analyses of phytochemical composition, chemical, and celtab. 6: effect of plant part and location on total and individual flavonol fractions. effects relative amount (mg mg-1 total phenolics)1 ol-tot ol-1 ol-2 ol-3 ol-4 ol-5 ol-6 ol-7 ol-8 plant part buds 0.026 nd 0.012 tr 0.006 nd nd 0.008 nd sprouts 0.260 0.023 0.163 0.006 0.040 0.006 0.008 0.011 0.002 significance2 ** ** ** ** ** ** ** ** ** lsd3 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 0.001 < 0.001 location (within plant part) buds rio dei cozzi 0.025 nd 0.011 tr 0.006 nd nd 0.008 nd brisighella 0.026 nd 0.013 tr 0.006 nd nd 0.007 nd significance ns ns ** ns ** ns ns ns ns lsd 0.001 < 0.001 sprouts magliano 0.305 0.030 0.191 0.007 0.045 0.007 0.010 0.015 < 0.001 rio dei cozzi 0.271 0.024 0.169 0.006 0.041 0.007 0.010 0.013 0.001 santa sofia 0.203 0.016 0.130 0.004 0.034 0.004 0.004 0.007 0.004 significance ** ** ** ** ** ** ** ** ** lsd 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 0.001 < 0.001 1 for compound names see tab. 2; 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jacq.) ethanolic extracts. j. chromatogr. a 1112, 339-344. doi: 10.1016/j.chroma.2006.01.034 walker, a.f., marakis, g., morris, a.p., robinson, p.a., 2002: promising hypotensive effect of hawthorn extract: a randomized double-blind pilot study of mild, essential hypertension. phytother. res. 16, 48-54. doi: 10.1002/ptr.947 walker, a.f., marakis, g., simpson, e., hope, j.l., robinson, p.a., hassanein, m., simpson, h.c.r., 2006: hypotensive effects of hawthorn for patients with diabetes taking prescription drugs: a randomised controlled trial. brit. j. gen. pract. 56, 437-443. zhang, z., ho, w.k.k., huang, y., james, a.e., lam, l.w., chen, z.h., 2002: hawthorn fruit is hypolipidemic in rabbits fed a high cholesterol diet. j. nutr. 132, 5-10. doi: 10.1093/jn/132.1.5 orcid federico ferioli https://orcid.org/0000-0001-8071-1774 elisa giambanelli https://orcid.org/0000-0002-4164-7770 address of the corresponding author: federico ferioli, department of agricultural and food sciences (distal), university of bologna, food science university campus, p.zza goidanich 60, 47521, cesena (fc), italy e-mail: federico.ferioli@unibo.it © the author(s) 2020. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1016/j.chroma.2006.01.034 http://dx.doi.org/10.1002/ptr.947 http://dx.doi.org/10.1093/jn/132.1.5 https://orcid.org/0000-0001-8071-1774 https://orcid.org/0000-0002-4164-77 journal of applied botany and food quality 92, 179 186 (2019), doi:10.5073/jabfq.2019.092.024 1department of agriculture, mediterranea university, reggio calabria italy 2institute of soil & environmental sciences, university of agriculture, faisalabad, pakistan 3burewala sub-campus of university of agriculture faisalabad, burewala, pakistan use of plant growth-promoting rhizobacteria to ameliorate the performance of lentil under salinity adele muscolo1*, maria rosaria panuccio1, zahir zahir2, sajid mahmood2, sajid mohamed nadeem3 (submitted: february 8, 2019; accepted: april 6, 2019) * corresponding author summary in the current agricultural model, the increasing soil salinity, especially in arid and semiarid regions, causes environmental and economic losses. inoculation of plant with growth-promoting rhizobacteria (pgpr) can be a sustainable strategy to increase plant abiotic stress tolerance mainly in the early vulnerable stage of their growth. the efficiency of pgpr inoculation was evaluated in three italian lentil accessions and two pakistan native varieties differing in salinity tolerance. pseudomonas putida (6), pseudomonas fluorescens (6k) and serratia ficaria (w10) were used as bio-inoculants. seedling growth was detected 16 days after nacl treatments. results showed that in absence of salinity, all strains increased differently the growth of lentils compared to the un-inoculated ones. inoculum significantly increased the growth of the most salt sensitive in comparison to the most salt resistant varieties. 6 and 6k were the most effectivegrowth-inducers under salinity stress. a specificity between pgpr and lentil was evident. 6k mostly improved biomass and growth of the italian accessions, while the strain 6 mostly affected the pakistan landraces. keywords: inoculation; lentil; plant growth promoting rhizobacteria; salinity stress introduction the soil salinization constitutes one of the main processes responsible for the degradation and reduced productivity of agricultural lands in arid and semiarid regions (ashraf and sarwar, 2002; munns, 2002). according to an estimate, 1128 million hectare of land are affected worldwide by salinity and sodicity (wicke et al., 2011). most of the legumes are sensitive to salinity and only few can grow on saltaffected soils (ashraf and mcneilly, 2004; morais et al., 2012). lentil (lens culinaris medik.) is the 2nd most important grain legume widely cultivated in semi-arid regions of the world (malik, 2005) throughout indian sub-continent, middle east, northern africa, southern europe, north and south america, australia and western asia (fikiru et al., 2007). the plants are grown for their small lens-shaped edible seeds, which are rich in protein, carbohydrates, calcium, phosphorus, iron and b vitamins. lentil seeds also contain polyphenols, and antioxidants with free radical-scavenging abilities, useful in the prevention of many chronic diseases with consequent beneficial effects on human health (dueńas et al., 2003). lentil is considered a strategic component of the cropping systems in the mediterranean areas. as the majority of legumes, lentil is salt sensi tive and salinity is imposing serious limitations to its productivity, causing yield losses up to 50%. kökten et al. (2010) showed a lentil yield reduction of 20% and 90-100% at an ec of 2 ds/m and 3 ds/m, respectively, suggesting that this crop should not even be grown under slightly saline conditions. even if salinity affects plant growth at all stages of development, germination and seedling emergence represent the most vulnerable stages in plant life cycle because they are the first to be confronted with soil salinity (kitajima and fenner, 2000). it is widely demonstrated, in different crop species (chikpea, barley, wheat, sunflower), that germination capacity de creases markedly increasing salinity (panuccio et al., 2018; kumar et al., 2012; abdoli et al., 2013; wu et al., 2015; alom et al., 2016). rapid, uniform and high germination percentage for legumes is a prerequisite for their successful stand establishment and yield (demir and ermis, 2003) and are considered the screening criteria to select salt tolerant genotypes. high salt concentrations affect plant metabolic processes leading to an increase in ethylene concentration that inhibits root elongation which in turn reduces plant growth and yield (nadeem et al., 2010; panuccio et al., 2014). the pgpr strains, containing acc-deaminase, have the ability to reduce the negative impact of elevated level of ethylene by degrading it into ammonia and α-ketobutyrate (glick et al., 1998). numerous studies (zahran et al., 2012; sobti et al., 2015) showed that the lack of legume productivity in saline soil was due to the reduced survival and proliferation of pgpr in soil (khosravi et al., 2010), with a consequent decline in exopolysaccharides and phytohormones that, binding na+, protect plants from the adverse effects of salinity (upadhyay et al., 2011). on the basis of the above consideration, the development of salt-tolerant symbioses is considered an absolute priority to enable cultivation of leguminous crops in salt-affected soils. the aim of the current research was to relate the extent of a positive association between different strains and lentil varieties with the salt tolerance degree, to find an eco-sustainable and cheap method for improving germination and seedling establishment in salt condition, with the specific objective of extending lentil cultivation in saline and/or salinized area. in this study the efficiency of three salt resistant wheat-growth promoting rhizobacteria, in alleviating salt stress in five lentil varieties has been evaluated and compared by assessing early vegetative growth and phenotypic traits of the inoculated lentils in respect to the un-inoculated ones in saline and non-saline conditions. the existence of specificity between pgpr and lentil variety was also evaluated. materials and methods plant material in this study we analyzed two salt sensitive lentils (masoor 85, mas; punjab masoor 2009, pun) native to, and cultivated in pakistan, two italian accessions ‘ustica’ (ust), and castelluccio (cast) moderate salt stress tolerant landraces, and a canadian commercial variety salt sensitive eston (est) (sidari et al., 2007, 2008; muscolo et al., 2014, 2015). each lentil variety was stored at 20 ± 1 °c and 95% r.u., and selected for size and homogeneity before using. the lentil varieties: masoor 85 (mas) and punjab masoor 2009 (pun), were purchased from pulses section, ayub agriculture research institute, faisalabad, pakistan. ustica was collected in the homonymous island close to sicily (southern italy) and castelluccio was collected in umbria (central italy). 180 a. muscolo, m.r. panuccio, z. zahir, s. mahmood, s.m. nadeem strain selection the pgpr strains (6, 6k and w10) used in this study have been previously and comprehensively evaluated in axenic and field con ditions for their growth promoting activities in wheat under salinity. these strains were shown to have acc-deaminase activity, production of exopolysaccharides, auxin production, p solubilization and root colonization ability (nadeem et al., 2010). growth pouch assay a growth pouch assay was conducted under axenic conditions at three salinity levels (0, 4 and 8 ds m-1) to check the responses of five different lentil varieties to the inoculation with pgpr strains (6, 6k and w10). for each strain, inoculum was prepared in flasks using luria bertani (lb) medium without agar. each flask containing 125 ml of broth was inoculated with a selected strain of bacteria and incubated in a shaking incubator (100 rpm) for 72 h at 28 ± 1 °c. before seed inoculation, an optical density of 0.5 at 535 nm was achieved by getting cell pallets through centrifugation of the bacterial culture and making dilutions in saline solution to maintain uniform cell density (108-109 colony forming units (cfu) ml-1). lentil seeds of each variety were surface sterilized by momentarily dipping them in 95% ethanol and then in 5% sodium hypochlorite solution for 3-4 minutes and a subsequent 5-6 washings with sterilized distilled water. three surface sterilized pre-germinated seeds of each variety were dipped into the inocula for 10 minutes and placed in autoclaved growth pouches (mega international, west st. paul, minnesota, usa). sterilized lb broth was used for the control treatment. the whole procedure of inoculation and sowing was executed in a laminar flow hood to eliminate or reduce the chances of contamination. salinity levels of 4 ds m-1 and 8 ds m-1 were maintained using nacl in sterilized half strength hoagland solution (hoagland and arnon, 1950). each treatment was replicated thrice following completely randomized design. six days after sowing, salinity treatments were applied by irrigating seedlings with saline hoagland (1/2 strength) solution. in the growth chamber, suitable temperature was between 25-30 °c with 10 h light (275 μmol m-2 s-1) and 14 h dark. root and shoot length, fresh and dry biomass, and chlorophyll content were recorded at 16 days after salinity treatment. chlorophyll assay the chlorophyll contents were determined by using photosynthesis measuring system/spad meter (spad-502 plus, konica, minolta inc.). seedlings were harvested and root weight was recorded. statistical analysis data of three replicates were analyzed by using one-way anova and mean comparisons were made with tukey’s test (p<0.05). all data were analyzed using systat 13.0 software (spss inc.). twoway anova was performed to analyze the effects of both salinity and strains and their interaction on shoot and root length, shoot and root fresh and dry biomass and chlorophyll content. all analyses were conducted using systat 13 for windows. significant effects were determined by p = 0.05. results in absence of salinity, for all lentil varieties, root and shoot fresh weights (tab. 1, 2) of inoculated seedlings were significantly higher than un-inoculated ones (controls). however, the degree of lentil growth, depended on the pgpr used for the inoculum. in absence of salinity, ust increased its root and shoot fresh biomass when inoculated with w10, while cas when treated with 6k. in est, mas and pun varieties, the strains that improved shoot biomass were different from those that influenced root growth (tab. 1, 2). in est, shoot biomass was increased by the strain 6, while root growth by the strain w10. shoot biomass of mas increased with w10 while root growth enhanced with 6 and 6k strains. in pun, shoots were mostly increased by 6k, while roots were enhanced by 6. under 4 ds m-1 salinity, 6 and 6k increased the shoot biomass of all lentil varieties except for eston, while w10 mostly improved the root biomass only of cast, est and mas compared to the other treatments. at 8 ds m-1, the inoculation significantly increased the growth of lentils except for pun in which the negative effect of salinity was only slightly reduced by the inocula in comparison to the controls (tab. 1, 2). in absence of salinity, the strains behaved differently in respect to the type of lentil, organ and phenotypic traits. all strains increased shoot elongation in cast, est and mas (fig. 1). all strains, except w10 enhanced shoot length in pun. only tab. 1: shoot fresh weight (mg plant-1) (n = 3) in inoculated lentils ustica (ust), castelluccio (cas), eston (est), masoor 85 (mas), and punjab masoor 2009 (pun) with the different strains (6, 6k, w10) in respect to the un-inoculated one (control), in absence (0 ds m-1) and in presence of nacl (4 and 8 ds m-1) shoot fresh biomass nacl (ds m-1) strain ust cas est mas pun 0 ctr 123.3 ± 0.3c 146.7 ± 0.2d 126.7 ± 0.2c 83.3 ± 0.5c 76.7 ± 0.2d 6 150.0 ± 0.3b 186.7 ± 0.5b 156.7 ± 0.5a 103.3 ± 0.2b 100.0 ± 0.3c 6k 146.7 ± 0.1b 193.3 ± 0.3a 143.3 ± 0.3b 106.7 ± 0.3b 130.0 ± 0.3a w10 156.7 ± 0.3a 153.3 ± 0.2c 130.0 ± 0.1c 110.0 ± 0.1a 116.7 ± 0.2b 4 ctr 101.7 ± 0.6d 100.0 ± 0.3d 126.7 ± 0.1c 73.3 ± 0.3c 66.7 ± 0.2c 6 106.7 ± 0.2c 140.0 ± 0.1b 200.0 ± 0.2a 136.7 ± 0.4a 100.0 ± 0.1b 6k 166.7 ± 0.7a 156.7 ± 0.4a 150.0 ± 0.b 110.0 ± 0.4b 123.3 ± 0.2a w10 146.7 ± 0.3b 120.0 ± 0.1c 196.7 ± 0.2a 133.3 ± 0.3a 96.7 ± 0.2b 8 ctr 76.7 ± 0.3d 83.3 ± 0.3 c 93.3 ± 0.2d 66.7 ± 0.3d 63.3 ± 0.1b 6 143.3 ± 0.3b 160.0 ± 0.4a 146.7 ± 0.3c 80.0 ± 0.1c 73.3 ± 0.2a 6k 136.7 ± 0.3c 160.0 ± 0.1a 210.0 ± 0.1a 123.3 ± 0.2a 66.7 ± 0.3b w10 153.3 ± 0.2a 150.0 ± 0.3b 156.7 ± 0.2b 90.0 ± 0.2b 63.3 ± 0.2b values are the means of three experiments ± se. different letters indicate significant differences (p ≤ 0.05) among different strain treatments within the same cultivar and at the same salt concentration. growth-promoting rhizobacteria improve performance of lentil under salinity 181 6k and w10 stimulated the growth of shoots in ust (fig. 1). under salinity, the inocula increased lentil shoot length compared to the respective controls. at 4 ds m-1 6k and w10 induced the best shoot elongation in ust and cast, 6 and 6k increased shoot length in pun, all strains increased shoot length in mas and est. at 8 ds m-1 all strains increased shoot length in all the varieties in respect to the un-inoculated controls. the effects of strains on root apparatus depended on lentil variety, data evidenced that in absence of salinity only the strain w10 in creased root length in ust and cast accessions. conversely, in presence of salinity all the strains increased root elongation in all the lentils in respect to the own controls, but at different extent (fig. 1). in short, our results highlighted that in the absence of salinity, all strains increased the shoot length more than root length compared to the un-inoculated lentils, except for ust, where both root and shoot lengths were likewise improved. at 8 ds m-1, all strains improved root length. 6 and 6k induced the greatest root elongation in cast and pun, while w10 positively affected est and ust root length. two-way anova showed that salinity and strains individually or in combination, significantly affected lentil growth (tab. 3). shoot length was mainly affected by salinity in ust, cast and mas, while root length was more influenced by strains, except for mas, that was influenced similarly by salinity and strain (f-ratios). shoot and root biomass of lentils were mostly affected by strains except for pun. in short, our results evidenced a specificity between strains and lentils showing that the effects of salt and strain were differentiated and species-specific. the most promising strains for lentil growth in saline conditions were in the following order 6> 6k>w10. at original ec, inoculation increased dry weights (dw) in respect to all the un-inoculated lentils (fig. 2). in presence of salinity, the effect of inoculum was different and depended on the type of strain used and on lentil accession. the dw of the inoculated ust seedlings, under the highest salt condition did not change compared to that of their respective controls at original ec. in cast, under salinity, all inocula induced the highest increase in biomass if compared to the inoculated at original ec. a similar trend was observed in salt affected mas but with a minor increment in biomass. in eston, the seedlings inoculated with the strains 6 and w10 showed the highest dry weight at 4 ds m-1, while at 8 ds m-1 only the strain 6k was able to induce a further dry weight increase. a different behavior was observed for pun, in which salinity decreased the biomass of all inoculated seedlings. root mass ratio (rmr) increased in presence of the inocula but at different extent depending on the affinity of the inoculum with a specific lentil cultivar. rmr increased in ust and mas when inoculated with 6 and w10, in cast and est when treated with 6k and w10, in pun with 6k and w10 but only at a salinity of 4 ds/m. conversely to rmr, shoot mass ratio (smr) decreased in increasing salinity in all lentils and in presence of all the inocula. w10 appeared as the strain the most transversally effective on all the lentils (data not shown). two way anova (tab. 4) evidenced that root and shoot dry weight in cast, ust and est was mainly influenced by strains (f-ratios). conversely, pun shoot and root dry weights were mainly affected by salinity. in mas, root dry weight was influenced by salt, while shoot dry weight was more affected by strain. strains positively influenced also chlorophyll content (fig. 3). the maximum increase in chlorophyll, in the absence of salinity, was observed in cast followed by est and ust, in response to the inoculation with 6k. under 4 ds m-1 salinity, 6k significantly increased chlorophyll amount in ust and pun compared to the respective un-inoculated seedlings. the maximum chlorophyll contents were recorded at 8 ds m-1 in pun and est, inoculated with w10 and 6k, respectively. at the highest salinity (8 ds m-1) also the strain 6 was able to significantly increase the chlorophyll content in all lentil varieties compared to the respective controls. chlorophyll, in all the lentils were more influenced by strains than salinity (f-ratios). in all lentils the combined effect of strain and salinity was less significant than the two factors individually considered, except in pun (fig. 3). discussion because of the contemporary increase in world population and saline lands, nowadays it is of primary importance to maintain soil productivity for a sustainable agriculture mostly in developing countries. microbes and legumes through symbiosis and synergistic coevolution have a great potential for improving productivity playing a key role in inducing abiotic stress resistance in plants (paredes and lebeis, 2016; rosenberg and rosenberg, 2016; agler et al., 2016). tab. 2: root fresh biomass (mg plant-1) (n = 3) in inoculated lentils ustica (ust), castelluccio (cas), eston (est) masoor 85 (mas) and punjab masoor 2009 (pun) with the different strains (6, 6k, w10) in respect to the un-inoculated one (control), in absence (0 ds m-1) and in presence of nacl salinity (4 and 8 ds m-1). root fresh biomass nacl (ds m-1) strain ust cas est mas pun 0 control 50.0 ± 0.1d 39.7 ± 0.2c 20.0 ± 0.1c 26.7 ± 0.5c 30.0 ± 0.1c 6 60.0 ± 0.2c 83.3 ± 0.3b 33.3 ± 0.3b 53.3 ± 0.1a 93.3 ± 0.4a 6k 73.3 ± 0.3b 103.3 ± 0.5a 36.7 ± 0.1b 53.3 ± 0.5a 80.0 ± 0.1b w10 79.7 ± 0.4a 80.0 ± 0.1b 53.3 ± 0.5a 33.3 ± 0.1b 80.0 ± 0.2b 4 control 49.3 ± 0.5c 26.7 ± 0.2d 20.0 ± 0.1d 16.7 ± 0.1d 13.3 ± 0.3d 6 49.7 ± 0.40c 63.3 ± 0.5c 74.0 ± 0.2c 103.3 ± 0.6b 40.0 ± 0.2c 6k 103.3 ± 0.2a 110.0 ± 0.2b 83.3 ± 0.3b 63.3 ± 0.5c 103.3 ± 0.6a w10 80.0 ± 0.1b 116.7 ± 0.1a 113.3 ± 0.3a 166.7 ± 0.2a 90.0 ± 0.2b 8 control 30.0 ± 0.1d 14.0 ± 0.2d 16.7 ± 0.4d 13.3 ± 0.3c 13.3 ± 0.3c 6 93.3 ± 0.2a 133.3 ± 0.5a 60.0 ± 0.2c 146.7 ± 0.1a 16.7 ± 0.4b 6k 60.0 ± 0.1c 120.0 ± 0.2b 116.7 ± 0.2a 136.7 ± 0.2b 13.3 ± 0.6c w10 77.0 ± 0.1b 113.3 ± 0.6c 93.3 ± 0.3b 133.3 ± 0.6b 19.7 ± 0.3a values are the means of three experiments ± se. different letters indicate significant differences (p ≤ 0.05) among different strain treatments within the same cultivar and at the same salt concentration. 182 a. muscolo, m.r. panuccio, z. zahir, s. mahmood, s.m. nadeem interestingly, we found and confirmed that seed inoculation with pgpr improved salinity resistance in lentils, highlighting that salttolerant species of lentils had reduced responsiveness to rhizobia, both in survival and growth. in fact, lentils showing higher levels of tolerance benefitted less from rhizobia inoculation than those with lower salt-tolerance.. these results were in agreement with previous findings of gaballah and gomaa (2005), reporting that the performance of two fava bean varieties improved after bacterial inocu lation in different way. the growth improvement of inoculated lentils was due to the effect of pgpr which having acc-deaminase activity mitigated the inhibitory effects of salinity on root growth, by lowering the ethylene concentration in plant as previous demonstrated by nadeen et al. (2010). it is well known that ethylene inhibits cell expansion in both roots and shoots acting by means of cross-talk with the growth hormone auxin (vanstraelen and benková, 2012). our results evidenced a better root growth in inoculated seedlings under salinity stress compared to the un-inoculated control, confirming that the pgpr contrasted the negative effects of ethylene as demonstrated by nadeen et al. (2010) that used the same our strains. glick et al. (2007) demonstrated that inoculation was able to confer major stress resistance to numerous plants, because of the over-production of exopolysaccharides that protected the plant root from sodium (na) toxi city by decreasing its availability. ilangumaran and smith (2017) in a recent review elucidated the positive impact of exopolysaccharides produced by pgpr in enhancing plant growth in saline environment, suggesting that bacteria promoted plant growth via their growth promoting traits and highlighting that the beneficial effects of pgpr involved key physiological processes such as nutrient upfig. 1: shoot and root length (cm) of ustica (ust), castelluccio (cast), eston (est), masoor 85 (mas) and punjab (pun) lentils, 16 days after the inoculation with 6, 6k and w10 strains under different nacl salinity concentrations (0, 4 and 8 ds m-1). bars represent the mean ± se. different letters indicate significant differences (p ≤ 0.05) shoot length root length ustica e.c. 0 4 8 cm 0 5 10 15 20 25 30 control 6 6 k w10 castelluccio e.c. 0 4 8 cm 0 5 10 15 20 25 30 eston e.c. 0 4 8 cm 0 5 10 15 20 25 30 masoor 85 e.c. 0 4 8 cm 0 5 10 15 20 25 30 control 6 6 k w10 masoor 2009 e.c. 0 4 8 cm 0 5 10 15 20 25 30 control 6 6 k w 10 a ab b c c c cd e e f a abb c c de e f f g a b cc c d d e e e f f abb c ccc d d d e f a b b b b b c cc d c e control 6 6 k w10 control 6 6 k w10 ustica e.c. 0 4 8 cm 0 5 10 15 20 25 30 control 6 6 k w10 castelluccio e.c. 0 4 8 cm 0 5 10 15 20 25 30 control 6 6 k w10 eston e.c. 0 4 8 cm 0 5 10 15 20 25 30 control 6 6 k w 10 masoor 85 e.c. 0 4 8 cm 0 5 10 15 20 25 30 control 6 6 k w 10 masoor 2009 e.c. 0 4 8 cm 0 5 10 15 20 25 30 control 6 6 k w 10 a bcccc d e e e e f a b c cd e f fg gh i ab bcc d e e ef g g a a b c c c cd e e f a ab b c d e ef f g g c growth-promoting rhizobacteria improve performance of lentil under salinity 183 take, photosynthesis, and source-sink relationships that promoted a significant plant growth and development. our results perfectly agree with the above assertions showing that pgpr affected chlorophyll content and/or root growth in a selective way and in respect to lentil variety and salinity level. this results highlighted a specificity between strain and lentil variety. the most promising strains for lentil growth in saline conditions were in the following order 6> 6k> w10. the results obtained showed that the italian local lentils, in absence and also in presence of salinity, had a biomass double than the pakistan accessions, confirming a major productivity also under stress condition. the pakistan varieties, when inoculated, resulted more responsive to treatment showing an increase in biomass in percentage twice higher than that of its own non-inoculated control. conclusion in short, the inoculum with selected wheat-promoting pgpr capable of tolerating saline conditions represents a good practice for impro ving the adaptation of lentils to saline environment. such strains could be very useful for promoting growth of other legumes by itself or in combination with other strains. additionally, the identification of pgpr efficient on both cereal and legumes, may favor a successful use of lentils in crop rotation with maize and other cereals, represen ting a winning strategy to improve or maintain soil fertility under saline conditions with a number of agronomic, economic and environmental benefits compared to monoculture cropping. salt-tolerantpgpr represents a promising way for sustainable lentil production in saline environment. author contribution am and zz designed the research; sm and smn conducted the experiments; am and mrp analyzed the data and wrote the paper. all authors have read and approved the final manuscript. references abdoli, m., saeidi, m., azhand, m., honarmand, s.j., esfandiari, e., shekari, f., 2013: the effects of different levels of salinity and indole3-acetic acid (iaa) on early growth and germination of wheat seedling. j. stress physiol. biochem. 9, 329-338. alom, r., hasan, a., islam, r., wang, q.f., 2016: germination characters and early seedling growth of wheat (triticum aestivum l.) genotypes under salt stress conditions. j. crop sci. biotechn. 19, 383-392. doi: 10.1007/s12892-016-0052-1 andres, j.a., rovera, m., guiñazú, l.b., pastor, s.a., rosas, s.b., 2012: interactions between legumes and rhizobia under stress conditions. in: maheshwari, d. (eds.), bacteria in agrobiology: stress management, 77-94. springer, berlin, heidelberg. doi: 10.1007/978-3-642-23465-1_5 ashraf, m., mcneilly, t., 2004. salinity tolerance in brassica oilseeds. crit. rev. plant sci. 23, 157-174. doi: 10.1080/07352680490433286 ashraf, m.y., sarwar, g., 2002: salt tolerance potential in members of brassicaceae. physiological studies on water relations and mineral contents. in: ahmad, r., malik, k.a. (eds.), prospects for saline agriculture, 237-245. kluwer academic publishers netherlands. doi: 10.1007/978-94-017-0067-2_26 demir, i., ermis, s., 2003: effect of controlled hydration treatment on germination and seedling growth under salt stress during development in tab. 3: analysis of variance of the effects of salinity and strains on shoot and root length (cm plant-1) and shoot and root biomass (mg plant-1) of ustica (ust), castelluccio (cast), eston (est), masoor 85 (mas) and punjab masoor 2009 (pun) lentils. shoot length root length shoot biomass root biomass ust salinity 426.6 *** 198.4 *** 2378.4 *** 2059.3 *** f-ratio strains 336.5 *** 2157.3 *** 12811.6 *** 48785.0 *** salinity × strains 103.2 *** 700.7 *** 2852.2 *** 21125.2 *** 0.995 0.998 1.000 1.000 r cas salinity 308.3 *** 391.9 *** 6286.8 *** 8335.0 *** f-ratio strains 260.6 *** 4618.8 *** 28574.3 *** 92142.7 *** salinity × strains 109.5 *** 1439.5 *** 8378.3 *** 10583.3 *** 0.994 0.999 1.000 1.000 r est salinity 218.1 *** 1612.6 *** 16696.7 *** 67120.8 *** f-ratio strains 510.1 *** 3775.8 *** 37129.9 *** 105785.3 *** salinity × strains 44.9 *** 930.8 *** 18140.8 *** 15412.8 *** 0.999 0.999 1.000 1.000 r mas salinity 201.8 *** 8005.3 *** 2619.8 *** 89068.8 *** f-ratio strains 153.7 *** 7810.5 *** 13987.0 *** 100553.7 *** salinity × strains 34.2 *** 1205.3 *** 7909.2 *** 29643.9 *** 0.989 0.999 1.000 1.000 r pun salinity 48.1 *** 1603.3 *** 5036.1 *** 105114.8 *** f-ratio strains 232.4 *** 6794.4 *** 2187.9 *** 41732.1 *** salinity × strains 12.5 *** 1607.8 *** 543.5 *** 17919.3 *** 0.986 0.999 1.000 1.000 r http://dx.doi.org/10.1007/s12892-016-0052-1 http://dx.doi.org/10.1007/978-3-642-23465-1_5 http://dx.doi.org/10.1080/07352680490433286 http://dx.doi.org/10.1007/978-94-017-0067-2_26 184 a. muscolo, m.r. panuccio, z. zahir, s. mahmood, s.m. nadeem tomato seeds. eur. j. hortic. sci. 68, 53-58. dueńas, m., sun, b., hernández, t., estrella, i., spranger, m.i., 2003: proanthocyanidin composition in the 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agricultural experimental station circufig. 2: total dry weight of ustica (ust), castelluccio (cast), eston (est), masoor 85 (mas) and punjab (pun) lentils, 16 days after the inoculation with 6, 6k and w10 strains in presence of different nacl salinity concentrations (0, 4 and 8 ds m-1). bars represent the mean ± se. different letters indicate significant differences (p ≤ 0.05) tab. 4: analysis of variance of the effects of salinity and strains on shoot and root dry weight (d.w.) of ustica (ust), castelluccio (cast), eston (est), masoor 85 (mas) and punjab masoor 2009 (pun) lentils. ust cast est mas pun root d.w. f-ratio root d.w. f-ratio root d.w. f-ratio root d.w. f-ratio root d.w. f-ratio salt 7.56 ** salt 585.9 *** salt 542.9 *** salt 1369.7 *** salt 517.1 *** strain 75.9 *** strain 569.3 *** strain 655.2 *** strain 965.5 *** strain 245.7 *** salt × strain 25.0 *** salt × strain 123.5 *** salt × strain 226.2 *** salt × strain 327.4 *** salt × strain 108. 9 *** shoot d.w. shoot d.w. shoot d.w. shoot d.w. shoot d.w. salt 7.98 ** salt 542.3 *** salt 255.5 *** salt 127.4 *** salt 788.8 *** strain 187.1 *** strain 602.7 *** strain 428.7 *** strain 228.9 *** strain 276.8 *** salt × strain 81.5 *** salt × strain 47.6 *** salt × strain 212.4 *** salt × strain 46.4 *** salt × strain 43.4 *** ustica c.e. 0 4 8 m g pl an t 1 0 10 20 30 40 control 6 6 k w10 masoor c.e. 0 4 8 m g pl an t-1 0 10 20 30 40 control 6 6 k w 10 d c c b e d a c f c d b e d d d f b c a f b a b castelluccio 0 4 8 m g pl an t-1 0 10 20 30 40 control 6 6 k w 10 e b a f d c d g a a b c punjab e.c. 0 4 8 m g pl an t-1 0 10 20 30 40 control 6 6 k w 10 d b b b e c a c f e e e eston e.c. 0 4 8 m g pl an t-1 0 10 20 30 40 control 6 6 k w10 a d e h b d c g ff e g http://dx.doi.org/10.1021/jf0303215 http://dx.doi.org/10.1007/s10658-007-9162-4 http://dx.doi.org/10.1006/jtbi.1997.0532 growth-promoting rhizobacteria improve performance of lentil under salinity 185 lar no. 347, 1-32. university of california, berkeley. ilangumaran, g., smith, d.i., 2017: plant growth promoting rhizobacteria in amelioration of salinity stress: a systems biology perspective. frontier plant sci. doi: 10.3389/fpls.2017.01768 khosravi, h., yakhchali, b., alikhani, h.a., 2010: potential evaluation of some native rhizobia as plant growth promoting bacteria and their role on decreasing of stress ethylene. iran j. biol. 22(4), 661-671. kökten, k., karaköy, t., bakoğlu, a., akçura, m., 2010: determination of salinity tolerance of some lentil (lens culinaris m.) varieties. j. food agric. environ. 8, 140-143. kumar, r., singh, m.p., kumar, s., 2012: effect of salinity on germination, growth, yield and yield attributes of wheat. int. j. sci. technol. res. 1, 19-23. doi: 10.3923/pjbs.1998.226.227 malik, r., 2005: genetic divergence analysis in lentil (lens culinaris medik). m.sc. thesis, department of agricultural botany, chaudhary charan singh university, meerut (u.p.), india. morais, m.c., panuccio, m.r., muscolo, a., freitas, h., 2012: does salt stress increase the ability of the exotic legume acacia longifolia to compete with native legumes in sand dune ecosystems? environ. exp. bot. 82, 74-79. doi: 10.1016/j.envexpbot.2012.03.012 munns, r., 2002: comparative physiology of salt and water stress. plant cell environ. 2, 239-250. doi: 10.1046/j.0016-8025.2001.00808.x muscolo, a., junker, a., klukas, c., weigelt-fischer, k., riewe, d., altmann, t., 2015: phenotypic and metabolic responses to drought and fig. 3: chlorophyll contents (spad value) detected in leaves of ustica (ust), castelluccio (cast), eston (est), masoor 85 (mas) and punjab (pun) lentils, 16 days after the inoculation with 6, 6k and w10 strains in presence of different nacl salinity concentrations (0, 4 and 8 ds m-1). bars represent the mean ± se. different letters indicate significant differences (p ≤ 0.05) salinity of four contrasting lentil accessions. j. exp. bot. 66, 5467-548. doi: 10.1093/jxb/erv208 muscolo, a., sidari, m., anastasi, u., santonoceto, c., maggio, a., 2014: effect of peg-induced drought stress on germination of four lentil genotypes. j. plant int. 9, 354-363. doi: 10.1080/17429145.2013.835880 nadeem, s.m., zahir, z.a., naveed, m., asghar, h.n., arshad, m., 2010: rhizobacteria capable of producing acc-deaminase may mitigate the salt stress in wheat. soil sci. soc. am. j. 74, 533-542. doi: 10.2136/sssaj2008.0240 panuccio, m.r., chaabani, s., roula, r., muscolo, a., 2018: bio-pri ming mitigates detrimental effects of salinity on maize improving antioxidant defense and preserving photosynthetic efficiency. plant physiol. biochem. 132, 465-474. doi: 10.1016/j.plaphy.2018.09.033 panuccio, m.r., jacobsenm, s.e., akhtar, s.s., muscolo, a., 2014: effect of saline water on seed germination and early seedling growth of the halophyte quinoa. ann. bot. plants 6, plu047. doi: 10.1093/aobpla/plu047 sidari, m., muscolo, a., santonoceto, c., anastasi, u., preiti, g., 2007: response of four genotypes of lentil to salt stress conditions. seed sci. technol. 35, 497-503. doi: 10.15258/sst.2007.35.2.24 sidari, m., santonoceto, c., anastasi, u., preiti, g., muscolo, a., 2008: variations in four genotypes of lentil under nacl-salinity stress. am. j. agric. biol. sci. 3, 410-416. doi: 10.3844/ajabssp.2008.410.416 sobti, a., belhadjb, h.a., djaghoubia, a., 2015: isolation and charac ustica e.c. 0 4 8 sp a d v al ue 0 10 20 30 40 50 control 6 6k w10 castelluccio c.e. 0 4 8 sp a d v al ue 0 10 20 30 40 50 control 6 6k w10 eston e.c. 0 4 8 sp a d v al ue 0 10 20 30 40 50 control 6 6k w10 masoor c.e. 0 4 8 sp a d v al ue 0 10 20 30 40 50 control 6 6k w10 punjab e.c. 0 4 8 sp a d v al ue 0 10 20 30 40 50 control 6 6k w10 a b c d e fg h hil m a b b c c d ef g hh i a b c d e e f g hh i i a b c c d d d e f g g h a ab c c d d de ff g ustica e.c. 0 4 8 sp a d v al ue 0 10 20 30 40 50 control 6 6k w10 castelluccio c.e. 0 4 8 sp a d v al ue 0 10 20 30 40 50 control 6 6k w10 eston e.c. 0 4 8 sp a d v al ue 0 10 20 30 40 50 control 6 6k w10 masoor c.e. 0 4 8 sp a d v al ue 0 10 20 30 40 50 control 6 6k w10 punjab e.c. 0 4 8 sp a d v al ue 0 10 20 30 40 50 control 6 6k w10 a b c d e fg h hil m a b b c c d ef g hh i a b c d e e f g hh i i a b c c d d d e f g g h a ab c c d d de ff g f-ratio salt 5832.3*** strain 7515.4*** salt × strain 3925.8*** f-ratio salt 1455.2*** strain 4043.2*** salt × strain 1293.1*** f-ratio salt 2210.0*** strain 3411.3*** salt × strain 1031.1*** f-ratio salt 932.0*** strain 1921.0*** salt × strain 1484.8*** f-ratio salt 439.3*** strain 2893.8*** salt × strain 319.6*** http://dx.doi.org/10.3389/fpls.2017.01768 http://dx.doi.org/10.3923/pjbs.1998.226.227 http://dx.doi.org/10.1016/j.envexpbot.2012.03.012 http://dx.doi.org/10.1046/j.0016-8025.2001.00808.x http://dx.doi.org/10.1093/jxb/erv208 http://dx.doi.org/10.1080/17429145.2013.835880 http://dx.doi.org/10.2136/sssaj2008.0240 http://dx.doi.org/10.1016/j.plaphy.2018.09.033 http://dx.doi.org/10.1093/aobpla/plu047 http://dx.doi.org/10.15258/sst.2007.35.2.24 http://dx.doi.org/10.3844/ajabssp.2008.410.416 186 a. muscolo, m.r. panuccio, z. zahir, s. mahmood, s.m. nadeem terization of the native rhizobia under hyper-salt edaphic conditions in ouargla (southeast algeria). procedia 74, 1434-1439. doi: 10.1016/j.egypro.2015.07.790 upadhyay, sk., singh, j.s., saxena, a.k., singh, d.p., 2011: impact of pgpr inoculation on growth and antioxidant status of wheat under saline conditions. plant biol. 14, 605-611. doi: 10.1111/j.1438-8677.2011.00533.x vanstraelen, m., benková, e., 2012: hormonal interactions in the regulation of plant development. ann. rev. cell develop. biol. 28, 463-487. doi: 10.1146/annurev-cellbio-101011-155741 wicke, b., smeets, e., dornburg, v., vashev, b., gaiser, t., turkenburg, w., faaij, a., 2011: the global technical and economic potential of bioenergy from salt affected soils. environ. sci. 4, 2669-2681. doi: 10.1039/c1ee01029h wu, g.q., jiao, q., shui, q.z., 2015: effect of salinity on seed germination, seedling growth, and inorganic and organic solutes accumulation in sunflower (helianthus annuus l.). plant soil environ. 61, 220-226. doi: 10.17221/22/2015-pse zahran, h.h., abdel-fattah, m., yasser, m.m., mahmoud, a.m., bedmar, e.j., 2012: diversity and environmental stress responses of rhizobial bacteria from egyptian grain legumes. aust. j. basic appl. sci. 6, 571-583. orcid adele muscolo https://orcid.org/0000-0002-0439-1614 maria rosaria panuccio https://orcid.org/0000-0003-3567-0584 address of the corresponding author: e-mail: adele muscolo, amuscolo@unirc.it © the author(s) 2019. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1016/j.egypro.2015.07.790 http://dx.doi.org/10.1111/j.1438-8677.2011.00533.x http://dx.doi.org/10.1146/annurev-cellbio-101011-155741 http://dx.doi.org/10.1039/c1ee01029h http://dx.doi.org/10.17221/22/2015-pse https://orcid.org/0000-0002-0439-1614 https://orcid.org/0000-0003-3567-0584 journal of applied botany and food quality 92, 33 38 (2019), doi:10.5073/jabfq.2019.092.005 1 department of biotechnology and biosciences, university of milano-bicocca, milano, italy 2 fem2 ambiente srl, milano, italy dna barcoding to trace medicinal and aromatic plants from the field to the food supplement jessica frigerio1, 2, tommaso gorini1, andrea galimberti1, ilaria bruni1, nicola tommasi1, valerio mezzasalma2, massimo labra1* (submitted: october 8, 2018; accepted: january 3, 2019) * corresponding author summary the global market of food supplements is growing, along with consumers demand for high-quality herbal products. nevertheless, substitution fraud, and adulteration cases remain a common safety problem of global concern. in the last years, the dna barcoding approach has been proposed as a valid identification method and it is now commonly used in the authentication of herbal and food pro ducts. the objective of this study was to evaluate whether dna barcoding can be applied to trace the plant species from the starting raw material to the finished commercial products. we selected a panel of 28 phytoextracts obtained through three different extraction methods (i.e., maceration, percolation and sonication) with different solvents (i.e., ethanol, deionized water and glycerol). furthermore, we chose six plant species for which we collected and analysed all the intermediates of the industrial production. we sequenced and analyzed the sequence variability at dna barcoding (psba-trnh, its) and minibarcoding (rbcl 1-b) marker regions. phytoextracts obtained through hydroalcoholic treatment, with the lower per centage of ethanol (<40%), and aqueous processing, at the lowest temperature, had major rate of sequencing and identification success. this study proves that dna barcoding is a useful tool for medicinal and aromatic plants (maps) traceability, which would provide consumers with safe and high-quality herbal products. key words: herbal products, its, maps, minibarcode, psba-trnh, phytoextracts, rbcl introduction medicinal and aromatic plants (maps) and their preparations are products used in medicine, cosmetics and food industry, belonging to plants, fungi, algae or lichens (efsa, 2009). such products are prepared using plants or their parts to exploit their therapeutic and healthy properties (e.g., antioxidant, anti-inflammatory), as well as their flavor or scent (who, 1999). according to a report published by the persistence market research, the global market of herbal supplements had a value of usd 40 billion in 2017 and is expected to reach a market valuation in excess of usd 65 billion by 2025 (persistence market research, 2017). in the last years, the increasing consumption of natural food supplements and the growing awareness of consumers concerning the healthy benefits of these products have been progressively enhancing the market of maps (efsa, 2009). although most of the herbal products used as food ingredients have been available to consumers since decades, the regulation of these products differs greatly among jurisdictions. while some countries consider maps as reliable ingredients for food production, others regulate them as healthy products or medicines. for example, in the european union (eu), most products containing medicinal and aromatic plants are sold as food supplements and regulated under the food law (silano et al., 2011); in australia dietary supplements are considered medicinal products and in canada they are subject to the complex regulation of the natural health pro ducts directorate (nnhpd) of health canada (health can., 2015) as medical products (low et al., 2017). the lack of a clear and shared global regulation and the large market demand of high-quality plantbased items led to safety problems with the increase of substitution, fraud and adulteration cases. anyway, more attention is required to guarantee a high quality level of maps which necessitates a stable raw material and its assurance. as a matter of fact, frequently, valuable plants are substituted with cheaper raw materials, such as the case of saffron substituted with safflower (bosmali et al., 2017). however, adulteration is not necessarily intentional, and herbal pro ducts may be altered due to inadvertent substitution, misidentification or confusion resulting from the use of different vernacular names in the countries of production. according to the world health organization (who), the adulteration of herbal products is a potential threat to consumers’ safety. this condition opens two key issues which refer to the definition of suitable toxicological evaluations to estimate the risks for human health and the setup of an efficient identification system to trace the herbal products from the field to the traded maps items. usually, macroscopic and microscopic examinations are the classic strategies adopted to verify the identity of fresh plants or the origin of plant portions. these tools can be used to trace the herbal products when the plants are processed immediately after being harvested. how ever, when the herbs undergo drying, fragmentation and pulveri zation processes the morphological traits cannot be longer used to reliably assess the botanic source. moreover, many herbal ingredients are obtained by infusion, maceration, distillation or pressing. in these cases, only dedicated chemical analyses of the complex mixtures could permit to achieve a reliable plant identification. in the last years the dna barcoding approach was proposed as a valid molecular identification method to provide species-level reso lution and it is now more and more used in the authentication of taxonomic provenance of herbal and food products (newsmaster et al., 2013; galimberti et al., 2013; mohammed et al., 2017). however, the most important limit of this molecular tool is that it can preferentially be adopted on unprocessed material (e.g., dry, fragmented and shredded plant portions) and several difficulties are encountered when dealing with extracts or with any other process that results in the degradation of the dna. recently, some manuscripts described the efficacy of minibarcode regions (i.e., the analysis of smaller genome portions − 100-150 bp − usually associated to the largest dna barcodes) for the identification of processed plant extracts (raclariu et al., 2017; little, 2014). to date, any study addressed the efficacy of a dna barcoding-based approach to trace herbal products along the entire production chain. in this work, we selected medicinal and aromatic plants in the form of phytoextracts obtained by several industrial companies and subjected to different kind of industrial processes and phytoextraction strategies. the objective of this survey was to evaluate whether or not the dna barcoding approach (using standard barcodes or minibarcode regions) could be applied to trace the plant species from 34 j. frigerio, t. gorini, a. galimberti, il. bruni, n. tommasi, v. mezzasalma, m. labra the field to the finished commercial product in the case of food supplements. therefore, we evaluated which are the industrial processes mostly affecting the efficacy of dna analysis such as sample pretreatment methods, solvents used for extraction and which are the most suitable dna markers to achieve a reliable maps traceability. material and methods study design to test the efficacy of dna traceability at different steps of the industrial production chain of maps, we selected a panel of 28 commercial phytoextracts (tab. 1) sold by three main european com panies. the selected items were obtained starting from 17 plant species (in the initial form of dried raw material) and were processed by the same companies adopting three main extraction procedures, namely maceration, sonication and percolation. maceration consists in the solubilization of the plant material in different solvents like water and alcohol (e.g., ethanol), while percolation involves the slow descent of a solvent through the plant raw material until it absorbs the molecules of interest. both methods rely on liquid filtration and concentration. differently, the sonication provokes cellular cavitation and the release of the phytocomplexes in the solvent used. three different extraction solvents were considered in this study and specifically, ethanol (i.e., alcoholic), deionized water (i.e., aqueous) and glycerol. during maceration the temperature was maintained under the threshold of 55 °c while in percolation higher temperatures (i.e., > 80 °c) were maintained, and sonication was mainly performed at 30-40 °c. after the percolation process, some phytoextracts are dried at very high temperatures (about 200 °c). processing details for each tested phytoextract are shown in tab. 1. to evaluate the efficacy of dna barcoding to trace the intermediates of industrial production after different steps of phytoextraction, we selected a panel of six commercial products obtained only by alcoholic and aqueous extraction procedures (tab. 2). for these samples, we collected and molecularly analysed through dna barcoding, any intermediate of production (fig. 1). dna extraction and dna barcoding analysis all commercial products from tab. 1 were tested for authenticity by sequencing three candidate markers, namely the standard dna barcoding plastidial intergenic spacer psba-trnh (steven and subramanyam, 2009), the nuclear its region (primers its p5-u4, cheng et al., 2016) and the minibarcode region rbcl 1-b (little, 2014). primer details and size of amplified fragments are provided in tab. a.1. a total of 50 mg of dried plant raw material, 150 μl of phytoextract (and intermediate products of phytoextraction, see tab. 2) were treated for dna extraction by using the eurogold plant dna mini kit (euroclone, pero, italy). each commercial phytoextract product was subjected to dna extraction in three replicates. purified dna concentration of each sample was estimated fluorometrically by using nanodrop™ one/onec microvolume uv-vis spectrophoto meter (thermo scientific™). a pcr amplification for each candidate marker was performed using puretaq ready-to-go pcr beads (ge healthcare life sciences, italy) in a 25 μl reaction volume according to the manufacturer’s instructions containing 1 μl 10mm of each primer and up to 3 μl of dna template. pcr cycles consisted of an initial denaturation step for 7 min at 94 °c, followed by 35 cycles of denaturation (45 s at 94 °c), annealing (30 s at different temperatures; see tab. a.1) and extension (1 min at 72 °c), and, hence, a final extension at 72 °c for 7 min. in the case of the intermediates of production listed in tab. 2, we amplified and sequenced only the minibarcode locus rbcl 1b. amplicons occurrence was assessed by electrophoresis on agarose gel using 1.5% agarose tae gel stained with ethidium bromide and amplicon length was measured by comparison against 100 bp ladder. when a sample did not produce any band or showed multiple or nonspecific amplicons, the reaction was repeated increasing the amount of template dna up to 10 μl. purified amplicons were bidirectionally sequenced using an abi 3730xl automated sequencing machine at eurofins genomics (ebersberg, germany). the 3' and 5' terminal portions of each sequence were clipped to generate consensus sequences for each sample. after manual editing, primer removal and pairwise alignment, the obtained sequences for dried raw material were submitted to the international genbank through the embl platform (see tab. a.2 for accession numbers). for all the tested samples (tab. 1 and tab. 2), the reliability of dna barcoding identification was assessed by adopting a standard comparison approach against a genbank database with blastn. each barcode sequence was taxonomically assigned to the plant species with the nearest matches (maximum identity > 99% and query co verage of 100%) according to bruni et al. (2015). we performed the identification separately for the three markers. results and discussion good dna quality (i.e., a260/a230 and a260/a280 absorbance ratios within the range 1.8 2.2) and extraction yield (20-40 ng/μl) were obtained from all the 17 raw material samples. the three canfig. 1: the industrial flowchart of maps production. the numbers indicate the intermediate steps of the industrial process for which the dna barcoding efficacy has been verified (tab. 2). dna barcoding to trace maps from the field to the food supplement 35 sonication 12.94 1.58 × × × etoh < 40% etoh > 40% water glycerol tab. 1: list of the analysed maps samples with details concerning their industrial processing to obtain the final phytoextracts. average yield of dna extraction (with standard deviation) and assessment of positive sequencing of dna barcoding markers (×) are also reported. samples industrial processing dna extraction yield dna barcoding markers phytoextraction solvent value standard psba trnh its rbcl 1 b process ng /μl deviation achillea millefolium l. sonication 12.26 1.55 × echinacea pallida (nutt.) nutt. sonication 16.29 0.97 × harpagophytum procumbens (burch.) dc. ex meisn. melissa officinalis l. percolation 44.63 1.32 × mentha × piperita l. sonication 12.3 0.83 × × × tilia platyphyllos scop. sonication 9.78 1.28 × × × zingiber officinale roscoe sonication 13.16 2.13 × × × arctium lappa l. maceration 1.23 0.2 echinacea angustifolia dc. maceration 2.83 0.5 melissa officinalis l. percolation 1.4 0.44 passiflora incarnata l. maceration 1.74 0.17 taraxacum officinale weber ex f.h. wigg. maceration 2.47 0.54 thymus vulgaris l. maceration 1.78 0.38 arctostaphylos uva-ursi (l.) spreng. percolation 3.1 0.33 cetraria islandica (l.) ach. percolation 2.27 0.94 echinacea purpurea (l.) moench percolation 3.47 0.71 × epilobium angustifolium l. percolation 1.88 0.63 malva sylvestris l. percolation 2.34 0.69 arctostaphylos uva-ursi (l.) spreng. sonication 13.36 1.05 × echinacea purpurea (l.) moench sonication 46.41 0.81 × × × epilobium angustifolium l. sonication 14.78 0.97 × melissa officinalis l. sonication 12,73 0,76 × × × arctium lappa l. maceration 2.69 0.64 echinacea angustifolia dc. maceration 4.73 0.85 × melissa officinalis l. maceration 2.55 0.76 passiflora incarnata l. maceration 2.09 0.6 taraxacum officinale weber ex f.h. wigg. maceration 3.12 0.3 thymus vulgaris l. maceration 2.71 0.8 tab. 2: list of six commercial maps (phytoextracts) traced along their entire production chain. each sample (intermediates of industrial production) was treated for dna extraction and dna barcoding analysis using the minibarcode region rbcl 1-b. numbers indicate the industrial processing step as described in fig. 1. y= correct plant identification by dna barcoding at rbcl 1-b locus, n= dna extraction or amplification failure, = sample was not collected and analysed. plant species solvent steps of the industrial production process 1 2 3 4 5 6 achillea millefolium l. 20% ethanol y y n y y y zingiber officinale roscoe 30% ethanol y y n y y y thymus vulgaris l. 60% ethanol y n y n n melissa officinalis l. 70% ethanol y n y n n echinacea purpurea (l.) moench water y y n y y melissa officinalis l. water y y n y y y 36 j. frigerio, t. gorini, a. galimberti, il. bruni, n. tommasi, v. mezzasalma, m. labra didate genetic markers exhibited high pcr success and the obtained pcr products were successfully sequenced with high-quality bi directional sequences. the blastn analysis suggested that all the obtained sequences corresponded with 100% maximum identity to the species declared by each company. we are aware that multiple cases of 100% maximum identity within the same plant genus could occur, especially concerning the dna minibarcoding rbcl 1b region. for example, this plastid region failed in discriminating echinacea purpurea (l.) moench from conge nerics like echinacea angustifolia dc. or echinacea pallida (nutt.). such events suggest that in some conditions, the main limit of dna minibarcoding relies on the reduced discrimination power among congenerics but it allows to detect plant contaminations when the adulterant/s belong to genera different from the target one. never theless, it should be considered that when dna content is expected to be low (or of low quality), the use of shorter dna barcoding regions offers the best compromise between amplification universality, sequence quality and taxonomic discrimination (little, 2014). concerning the 17 dry raw material samples, our results agree with the assumptions of newmaster and co-workers (2013) who suggested that a dna barcoding approach could be successfully applied to verify the identity of commercial herbal products and to reveal cases of contamination or substitution. therefore, when herbal products are directly used as ingredients of complex food, medicine or cosmetics items, they are subjected to “soft” processing actions such as cleaning, drying and cutting and the dna barcoding (achieved using long barcode fragments > 300 bp) represents a useful tool to trace plant species during the processing (de mattia et al., 2011). as expected, the efficacy of dna extraction and amplification decreased when we analyzed the 28 commercial phytoextracts and their intermediates of industrial processing. overall, the dna amount obtained after extraction processes ranged from 1.5 to more than 40 ng/μl (tab. 1). the extracts obtained through hydroalcoholic treatment, with the lower percentage of ethanol (< 40%), and aqueous processing, at the lowest temperature, contained more dna than the other samples (tab. 1 and tab. 2). in the samples where the dna barcoding analysis worked well, no contamination or adulteration (i.e., the occurrence of dna bacodes of other plant species) were observed. unfortunately, in some groups of extracts, the molecular analysis did not provide reliable dna extraction or high-quality sequences. at technical level, we hypothe size that in general, the high concentration of ethanol used in the industrial processing steps lead to dna precipitation. this was confirmed by the data reported in tab. 2, where samples of thymus vulgaris l. and melissa officinalis l. processed with ethanol at high concentration, showed residual dna in the extraction waste rather than in the phytoextract. for this reason, both the dna extraction and dna barcoding authentication failed when applied to the successive intermediate products of industrial processing and dna was no longer available in the final herbal supplements. in this case, we conclude that for this kind of industrial production, a dna-based approach is not suitable to achieve a reliable traceability of the initial plant raw material. similarly, high temperatures of water du ring aqueous extraction, followed by a drying step (about 200 °c) probably lead to dna fragmentation and degradation (karni et al., 2013) as observed in five of the samples processed with a percolation procedure (tab. 1). conversely, the use of more lukewarm water (i.e., < 55° c) allows to achieve a successful dna extraction, amplifi cation and sequencing of dna barcoding markers (tab. 1). moreover, such conditions also allow the traceability of the intermediate pro-ducts of industrial processing as observed for echinacea purpurea (l.) moench and melissa officinalis l. extracted using water as solvent. concerning glycerol extracts, although this solvent does not act directly on dna molecules, it usually contains ethylhexylglycerin and phenoxyethanol, which are typically used as additives. according to langsrud and co-workers (2016) these antibacterial agents could be responsible for dna loss. for this reason, also the analysis of the dna minibarcode region did not produce amplicons in glycerine extracts (tab. 1). concerning the industrial treatments, the sonication seems to keep the dna of raw materials more intact than the other processes (i.e., maceration and percolation). our results also show that sonicated samples contained higher amounts of dna (i.e. from 9.73 to 44 ng/ μl, tab. 1) compared to the other categories, thus allowing a successful amplification and sequencing of the dna minibarcode marker. concerning the quality of extracted genetic material, the purity of dna is more important than the extraction yield to achieve a good amplification and then a reliable identification (song et al., 2017). it should also be considered that secondary metabolites, like polyphenols and polysaccharides, which are normally extracted along with dna, may interfere with pcr amplification (sahu, thangaraj, and kathiresan, 2012). these molecules could bind dna covalently and make the extraction products impure, with several pro blems for the successive molecular analysis. for example, tannic acids could bind and inactivate taq polymerase (opel, chung, and mccord, 2010). however, in our analysis we hypothesize that the main amplification problem for the phytoextracts is the fragmen tation of dna. in all the tested cases, the dna minibarcode locus rbcl 1-b was most easily amplified and sequenced (tab. 1) than the other two dna barcoding markers. this suggests that the dna obtained from phytoextracts are richer in small dna fragments (80200 bp). such condition is in line with the data reported in recent review articles (mohammed et al., 2017) suggesting that dna barcoding is a reliable and suitable technique only for the herbal product that preserve a good quality dna and with poor fragmentation. in the other cases dna minibarcoding is the most efficient and reliable tool for traceability purposes (song et al., 2017). nowadays, analytical chemistry methods (tlc, hplc) represent the most used tools to verify the quality of maps, however, these approaches are usually directed to define the concentration of specific bioactive molecules or to estimate chemical contaminants (e.g., heavy metals) rather than to identify the occurrence of plant contaminants (sgamma et al., 2017). conversely, the dna barcoding approach is globally recognized as one of the most reliable dnabased approaches to identify species if a well populated reference dataset of dna barcode sequences for the target taxa is available (galimberti et al., 2013). moreover, in the case of contamination (or substitution), dna analyses also allow to simultaneously identify any species (i.e., dna metabarcoding) using high throughput sequencing (hts) sequencing systems (galimberti et al., 2015; mezzasalma et al., 2017). for these reasons, the pharmacopoeia guidelines of some countries such as that of uk (british pharma copoeia commission, 2017) indicate the dna barcoding as one of the official traceability systems in the sector of herbal products. our data support this proposal and the ability of dna minibarcode makers to provide a reliable tracing of the intermediate products of industrial production. however, it is important to underline that some industrial processes demanding high temperatures and the use of solvents, such as a high concentration of ethanol, can induce dna degradation and make this molecular tool less effective. in conclusion, this study leads to two main considerations about the future application of dna barcoding as a quality control tool in the sectors where the medicinal and aromatic plants constitute relevant ingredients (e.g., food, cosmetics and pharmacology). first of all, the current industrial trends promote the adoption of extraction processes from plant raw material, which rely on the reduction of energy consumption (i.e., low temperatures), and on the use of more ‘green’ solvents (e.g., water) to obtain exhausted waste products that can be used in other supply chains (e.g., fertilizers). the adoption and dna barcoding to trace maps from the field to the food supplement 37 spread of this trend should lead to an increased integrity and quality of dna in maps (and related intermediate products) and therefore enhance the success of dna barcoding as a universal traceability system. secondly, the continuous advances in high throughput sequencing and the resulting possibility of exploring multiple short genetic regions simultaneously (i.e. 150-200 bp), could increase the sensi tivity of a dna-based identification.an hts-dna metabarcoding approach would allow to check the presence of several plant contaminants in the same sample, even if occurring at low concentrations (newmaster et al., 2013). sgamma and co-workers (2017) proposed the introduction of dna metabarcoding to evaluate the quality and authenticate herbal drug material in the industrial context. the authors proposed a dedicated dna barcoding flowchart for industrial traceability purposes. our results could be taken into account to improve this flowchart and to also adapt it to the traceability of intermediates of industrial production. interestingly, valentini and co-workers (2017) recently proposed an innovative nanoparticledna barcoding hybrid system called nanotracer that could potentially revolutionize the world of traceability as it allows for rapid and naked-eye molecular traceability of any food and requires limited instrumentation and cost-effective reagents. this and other similar applications (aartse et al., 2017) open the opportunity to really boost the issue of herbal supplements traceability, not only with the industrial actors as the main stakeholders, but also involving a wider cicle of specialists. acknowledgments this work was supported by regione lombardia in the framework of the program ‘accordi per la ricerca e l’innovazione’, pro ject name: food social sensor network food net, grant number: e47f17000020009. the funder had no role in conducting the research and/or during the preparation of the article. fem2-ambiente s.r.l. provided support in the form of a salary for authors jessica frigerio and valerio mezzasalma but did not play a role in the study design, data collection and analysis, decision to 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doi: 10.5402/2012/205049 sgamma, t., lockie-williams, c., kreuzer, m., williams, s., scheyhing, u., koch, e., slater, a., howard, c., 2017: dna barcoding for industrial quality assurance. planta med. 83(14/15), 1117-1129. doi: 10.1055/s-0043-113448 silano, v., coppens, p., larrañaga-guetaria, a., minghetti, p., rothehrang, r., 2011: regulations applicable to plant food supplements and related products in the european union. food funct. 2(12), 710. doi:10.1039/c1fo10105f song, m., dong, g.-q., zhang, y.-q., liu, x., sun, w., 2017: identification 38 j. frigerio, t. gorini, a. galimberti, il. bruni, n. tommasi, v. mezzasalma, m. labra of processed chinese medicinal materials using dna mini-barcoding. chin. j. nat. med. 15(7), 481-486. doi: 10.1016/s1875-5364(17)30073-0 steven g.n., subramanyam, r., 2009: testing plant barcoding in a sister species complex of pantropical acacia (mimosoideae, fabaceae). mol. eco. resour. 9, 172-180. doi: 10.1111/j.1755-0998.2009.02642.x valentini, p., galimberti, a., mezzasalma, v., de mattia, f., casiraghi, m., labra, m., pompa, p.p., 2017: dna barcoding meets nanotechno logy: development of a universal colorimetric test for food authen tication. angew. chem. int. ed. 56(28), 8094-8098. doi: 10.1002/anie.201702120 who, 1999: who monographs on selected medicinal plants. vol. 1, medicinal plants, geneva. http://apps.who.int/medicinedocs/pdf/s2200e/s2200e.pdf address of the corresponding author: massimo labra, department of biotechnology and biosciences, university of milano-bicocca, piazza della scienza 2, 20126 milan, italy e-mail: massimo.labra@unimib.it © the author(s) 2019. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). supplementary material ifrom appendix table a.1: list of primer pairs used for dna barcoding analysis. locus name name 5' 3' sequence tm °c amplified samples size reference rbcl 1 ttggcagcattycgagtaactcc 50 80-200 bp palmieri (2009) rbcl rbcl b aaccytcttcaaaaaggtc 50 psba gttatgcatgaacgtaatgctc 53 300-600 bp steven & subramanyam (2009) psba-trnh trnh cgcgcatggtggattcacaatcc 53 its p5 ccttatcayttagaggaaggag 55 300-750 bp cheng (2016) its its u4 rgtttcttttcctccgctta 55 table a.2: list of all the analysed plant species. the table include the voucher specimens and genbank accession numbers of the raw material samples used to authenticate the phytoextracts and their intermediates of industrial production treated in this study. species type of sample specimen voucher company genbank accession number (rbcl 1-b; psba-trnh; its) achillea millefolium l. dried raw material fem_dbe_001 company 1 ls999840; ls999856; ls999873 arctium lappa l. dried raw material fem_dbe_007 company 3 ls999841; ls999857; ls999874 arctostaphylos uva-ursi (l.) spreng. dried raw material fem_dbe_010 company 2 ls999842; ls999858; ls999875 cetraria islandica (l.) ach. dried raw material fem_dbe_013 company 2 nd; nd; ls999876 echinacea angustifolia dc. dried raw material fem_dbe_015 company 3 ls999843; ls999859; ls999877 echinacea pallida (nutt.) nutt. dried raw material fem_dbe_018 company 1 ls999844; ls999860; ls999878 echinacea purpurea (l.) moench dried raw material fem_dbe_020 company 2 ls999845; ls999861; ls999879 epilobium angustifolium l. dried raw material fem_dbe_026 company 2 ls999846; ls999862; ls999880 harpagophytum procumbens (burch.) dried raw material fem_dbe_029 company 1 ls999847; ls999863; ls999881 malva sylvestris l. dried raw material fem_dbe_031 company 2 ls999848; ls999864, ls999882 melissa officinalis l. dried raw material fem_dbe_033 company 2 ls999849; ls999865; ls999883 mentha x piperita l. dried raw material fem_dbe_045 company 1 ls999850; ls999866; ls99984 passiflora incarnata l. dried raw material fem_dbe_047 company 3 ls999851; ls999867; ls999885 taraxacum officinale weber ex f.h. wigg. dried raw material fem_dbe_050 company 3 ls999852; ls999868; ls999886 thymus vulgaris l. dried raw material fem_dbe_053 company 3 ls999853; ls999869; ls999887 tilia platyphyllos scop. dried raw material fem_dbe_059 company 1 ls999854; ls999870; ls999888 zingiber officinale roscoe dried raw material fem_dbe_061 company 1 ls999855; ls999871; ls999889 supplementary material tab. a.1: list of primer pairs used for dna barcoding analysis. tab. a.2: list of all the analysed plant species. the table include the voucher specimens and genbank accession numbers of the raw material samples used to authenticate the phytoextracts and their intermediates of industrial production treated in this study. from appendix table a.1: list of primer pairs used for dna barcoding analysis. locus name name 5' 3' sequence tm °c amplified samples size reference rbcl 1 ttggcagcattycgagtaactcc 50 80-200 bp palmieri (2009) rbcl rbcl b aaccytcttcaaaaaggtc 50 psba gttatgcatgaacgtaatgctc 53 300-600 bp steven & subramanyam (2009) psba-trnh trnh cgcgcatggtggattcacaatcc 53 its p5 ccttatcayttagaggaaggag 55 300-750 bp cheng (2016) its its u4 rgtttcttttcctccgctta 55 table a.2: list of all the analysed plant species. the table include the voucher specimens and genbank accession numbers of the raw material samples used to authenticate the phytoextracts and their intermediates of industrial production treated in this study. species type of sample specimen voucher company genbank accession number (rbcl 1-b; psba-trnh; its) achillea millefolium l. dried raw material fem_dbe_001 company 1 ls999840; ls999856; ls999873 arctium lappa l. dried raw material fem_dbe_007 company 3 ls999841; ls999857; ls999874 arctostaphylos uva-ursi (l.) spreng. dried raw material fem_dbe_010 company 2 ls999842; ls999858; ls999875 cetraria islandica (l.) ach. dried raw material fem_dbe_013 company 2 nd; nd; ls999876 echinacea angustifolia dc. dried raw material fem_dbe_015 company 3 ls999843; ls999859; ls999877 echinacea pallida (nutt.) nutt. dried raw material fem_dbe_018 company 1 ls999844; ls999860; ls999878 echinacea purpurea (l.) moench dried raw material fem_dbe_020 company 2 ls999845; ls999861; ls999879 epilobium angustifolium l. dried raw material fem_dbe_026 company 2 ls999846; ls999862; ls999880 harpagophytum procumbens (burch.) dried raw material fem_dbe_029 company 1 ls999847; ls999863; ls999881 malva sylvestris l. dried raw material fem_dbe_031 company 2 ls999848; ls999864, ls999882 melissa officinalis l. dried raw material fem_dbe_033 company 2 ls999849; ls999865; ls999883 mentha x piperita l. dried raw material fem_dbe_045 company 1 ls999850; ls999866; ls99984 passiflora incarnata l. dried raw material fem_dbe_047 company 3 ls999851; ls999867; ls999885 taraxacum officinale weber ex f.h. wigg. dried raw material fem_dbe_050 company 3 ls999852; ls999868; ls999886 thymus vulgaris l. dried raw material fem_dbe_053 company 3 ls999853; ls999869; ls999887 tilia platyphyllos scop. dried raw material fem_dbe_059 company 1 ls999854; ls999870; ls999888 zingiber officinale roscoe dried raw material fem_dbe_061 company 1 ls999855; ls999871; ls999889 journal of applied botany and food quality 92, 49 56 (2019), doi:10.5073/jabfq.2019.092.007 1albrecht daniel thaer-institut, humboldt-universität zu berlin 2julius kühn-institut, quedlinburg hybridization between pelargonium acetosum l’hér. and pelargonium ×peltatum r. kamlah1*, i. pinker1, s. plaschil2, k. olbricht1 (submitted: december 11, 2018; accepted: february 7, 2019) * corresponding author summary pelargonium acetosum l’hér. is a wild species from south africa with decorative bluish foliage. only few reports describe crossings between p. acetosum and p. peltatum l’hér. (or p. ×peltatum). therefore, information about hybridization barriers is limited. in this study, two different genotypes of pelargonium acetosum (ac1 and ac2) were crossed with the diploid p. ×peltatum ‘tornado fuchsia’ (ptf). embryos and f1 hybrids from the combination ac1 × ptf were hampered by chlorophyll deficiencies. embryos and seeds of the combination ac2 × ptf were underdeveloped. the reciprocal combination ptf × ac1 did not show any fruit set. the combination ptf × ac2 resulted in low numbers of seeds, which were normally developed. hybrids from seeds were only obtained from the combinations ac1 × ptf and ptf × ac2. embryo rescue of the combinations ac1 × ptf and ac2 × ptf resulted in few but viable hybrids. flowers of all hybrids had shrivelled anthers and proved to be sterile. the occurrence of most hybridization barriers varied strongly between the different combinations and depended on both the genotype and the direction of cross-breeding. the bluish leaf colour did not appear among the f1. to overcome hybrid sterility a polyploidization is suggested. keywords: embryo rescue, hybridization barriers, hybrid variegation, hybrid sterility, incomplete embryo development, interspecific hybridization, wild species introgression introduction in europe and north america, pelargonium cultivars represent a significant fraction of the bedding plant market. in order to maintain that position, breeders regularly have to come up with novelties. interspecific hybridization is one of the main approaches in creating new characteristics, and it counteracts a narrowing gene pool in pelargonium breeding programs (olbricht, 2013). worldwide about 280 pelargonium species (albers and van der walt, 2007) embody valuable genetic resources that have not yet been fully exploited. however, introgression of species is time-consuming and often hampered by reproductive barriers, which depend, amongst other reasons, on the genetical distance between two species. interspecific cross-combinations are usually more successful if both species belong to the same section of pelargonium. other factors influencing interspecific crossability include the ploidy level, the basic chromosome number, and the direction of cross-breeding (horn, 1994). dna-based phylogenetic analyses (bakker et al., 2004; weng et al., 2012; röschenbleck et al., 2014) support a subdivision of the genus into 16 sections and help breeders to recognize possible candidates for introgression. the section ciconium (sweet) harv. contains the horticulturally important species p. inquinans l’hér., p. zonale l’hér. (both ancestors of p. ×hortorum bailey, the ‘zonal geranium’), and p. peltatum l’hér. (the main ancestor of ‘ivy-leaved’ cultivars, usually named p. ×peltatum). in recent decades, several species of this section have been introgressed into p. ×hortorum, such as p. tongaense vorster (esenalieva et al., 2012) and p. quinquelobatum hochst. (denispeixoto et al., 1997; hondo et al., 2015). p. peltatum was formerly placed in the section dibrachya (sweet) harv. but has been included in ciconium (gibby et al., 1990). p. peltatum is characterized by a relatively large genetical distance to other species of the same section (james et al., 2004; weng et al., 2012). in hybridization experiments between various species of section ciconium and p. peltatum, both prezygotic and postzygotic barriers have been observed. an inhibited pollen tube growth or a lack of fertilization (when pollen tubes do grow down the style) represent the main observed prezygotic barriers (coffin and harney, 1978; yu, 1985). known postzygotic barriers include incomplete development of seeds, stunted plant growth, hybrid sterility, chlorophyll deficiencies and hybrid variegation (coffin and harney, 1978; yu, 1985; horn, 1994). the latter is a consequence of the biparental inheritance of plastids in pelargonium: if only one of the two inherited plastid types shows an incompatibility with the nuclear genome, the segregation of plastids often leads to variegated leaves with chlorophyll-deficient sectors (metzlaff et al., 1981, 1982; grieger, 2007; weihe et al., 2009). in the case of an incomplete development or the abortion of the embryo, postzygotic disturbances may be overcome by the use of embryo rescue. in respect to the explant material, embryo rescue techniques can be distinguished into ovary, ovule, and embryo culture (winkelmann et al., 2010). a first study about embryo rescue of p. ×hortorum was published by becker-zens (1983), in which both ovule and embryo culture were performed. scemama and raquin (1990) developed a method to circumvent early embryo abortion using a combination of ovary and embryo culture. bentvelsen et al. (1990) applied embryo culture in various crosses between p. ×peltatum and other species of the section ciconium. all these studies used phytohormone-free nutrient media and aimed at the germination of the embryo, while other studies applied growth regulators to induce callus and adventitious shoots (kato and tokumasu, 1983; kakihara et al., 2012). pelargonium acetosum l’hér. (sect. ciconium) is a species from south africa with 2n=2x=18 chromosomes. it stands out due to its decorative bluish foliage, which is not common among ornamental pelargonium cultivars. despite its distinct appearance, it is closely related to p. zonale (james et al., 2004), and has been successfully introgressed into p. ×hortorum (hondo et al., 2014). a hybridization between p. acetosum and p. ×peltatum using embryo rescue is documented by bentvelsen et al. (1990), resulting in f1 hybrids but no f2 or backcross (bc) generation. yu (1985) described crossings between p. acetosum and p. peltatum as not resulting in viable hybrids, while horn (1994) reported viable but sterile hybrids from the combination p. peltatum × p. acetosum. the objective of this study was to examine hybridization barriers between p. acetosum and p. ×peltatum and to obtain genotypes with a novel variability for further breeding purposes. the long-term bree ding aim is to achieve p. ×peltatum cultivars with bluish foliage. 50 r. kamlah, i. pinker, s. plaschil, k. olbricht materials and methods plant material and cultivation two different genotypes of pelargonium acetosum (ac1 and ac2) and the diploid cultivar p. ×peltatum ‘tornado fuchsia’ (ptf) were used as crossing parents. ac1 was ordered from a nursery (gärtnerei schoebel, germany), ac2 was provided by the julius kühn-institut, quedlinburg. both genotypes were received as adult plants and pro pagated by cuttings. ptf had to be grown from seeds (mail-ordered from mary k’s unique seeds, usa). the cultivation of the plants took place from 08/2016 until 01/2018 in the greenhouse (berlindahlem). plants were potted in a mix of organic substrate (klasmanndeilmann 5) with 10% sand and were fertilized with 0.6% wuxal super every 14 days. assimilation lighting was added using sodium vapour lamps (11 h/d in 2016, 13.5 h/d from 01/2017 until 04/2017, 11 h/d until 07/2017). the temperature ranged between 17 and 35 ºc. pollen viability test pollen of ac1, ac2, and ptf was stained with an aqueous solution of 1% thiazolyl blue tetrazolium bromide (mtt) and 10% sucrose for 30 min at 24 ºc (norton, 1966; firmage and dafni, 2001). from each genotype 3 × 100 pollen grains were evaluated under a transmission light microscope (olympus ix70-s8f2). the number of pollen grains showing a purple colour was determined. pollination flowers were emasculated in the bud stage using forceps (ca. three days before anthesis, when petals became visible at the bud tip). emasculated inflorescences were covered in perforated propylene bags (crispac, 11 × 25 cm), which were sealed with tesa velcro strips. about five days after emasculation, when the stigma lobes had unfolded, anthers of the crossing partner were pressed on the stigma for pollination. pollinated flowers were bagged as described above. the number of pollinated flowers varied in each experiment (see below). observation of embryo development for quantitative analysis of embryo development, unripe fruits were harvested two weeks after pollination and the number of embryos per pollinated flower was determined. in each performed combination (tab. 1) 20 flowers were pollinated. for qualitative examination of embryo development, unripe fruits were harvested two, three, four, and five weeks after pollination (combinations see tab. 1). in each variant (combination × point of time = 24 variants) at least five pollinations were carried out. ovules were taken out of the fruit and were dissected under the stereo microscope (olympus sz-pt) with the help of forceps and a dissecting needle. isolated embryos were documented with a connected camera (olympus uc30) and associated software (cellsens entry 1.6). seed production and sowing the combinations performed in order to achieve seeds are listed in tab. 1. after harvest, seeds were stored in glassine envelopes. before sowing, ca. 0.5 mm of the seed tip was removed with fingernail clippers. with the scarified tip facing down, seeds were sown into multi-cell trays (cell size: 6 × 3 × 3 cm) filled with moistened sowing substrate (klasmann-deilmann 1). seed trays were placed under a foil tunnel in the greenhouse for 14 days. four days after sowing, the front end of the tunnel was opened. during the first two weeks after sowing the temperature ranged between 19 and 30 ºc (22.1 ºc mean). thirteen days after sowing the number of germinated seeds was determined. three weeks after sowing, seedlings were transplanted into multipot trays (5 cm ø per cavity), which were filled with a mix of organic substrate and perlite (klasmann-deilmann stecklingssubstrat). twelve weeks after sowing, plants were potted in a mix of organic substrate (klasmann-deilmann 5) with 10% sand and cultivated as described (see plant material and cultivation). embryo rescue fruits from selected combinations (tab. 1) were harvested two weeks after pollination. they were surface sterilized in 50 ml disinfection solution (3% calcium hypochlorite and one drop of tween20) for 20 min and then rinsed three times in sterile deionized water. under sterile conditions, embryos were excised from fruits using a stereo microscope, forceps, and a dissecting needle. nineteen embryos of ac1 × ac1, 21 embryos of ac1 × ptf, and 20 embryos each of ac2 × ptf and ptf × ptf were placed in glass tubes (10 × 2.5 × 2.5), which were filled with 10 ml of solid embryo-rescuemedium (er-medium, tab. 2). this medium was supplemented with 1 mg l-1 (5.71 μm) indole-3-acetic acid (iaa) and 1 mg l-1 (4.4 μm) 6-benzylaminopurine (bap). for the first two weeks, embryos were cultivated in the dark at 22-24 ºc and subsequently transferred to 10 μmol m-2s-1 photosynthetic active radiation (par). five weeks later (eight weeks later in case of ac2 × ptf), the de veloped callus was divided into smaller portions and then cultivated in glass tubes (10 × 2.5 × 2.5 cm) each filled with 5 ml phytohormone-free active-charcoal-i-medium (ac-i-medium, tab. 2). the glass tubes were placed at 22-24 ºc and 30 μmol m-2s-1 par. once adventitious shoots reached at least 1 cm, they were transferred into culture jars (sigma-aldrich, 66 × 59 × 59 mm, enclosed with magenta b-cap) filled with 20 ml ac-ii-medium. the culture jars were placed at 22-24 ºc and 30 μmol m-2s-1 par for 16 h d-1. rooted and unrooted shoots that had reached at least 2 cm length were planted into 4 cm pots filled with a mix of organic substrate (klasmann-deilmann stecklingssubstrat) and 1/3 perlite. plants were sprayed with water and placed under a transparent cover (to keep air humidity above 60%) at 21 ºc and 60 μmol m-2s-1 par for 16 h d-1. two weeks later plants were transferred into the greenhouse. acclimated plants were cultivated as described (see plant material and cultivation). characterization of plants crossing parents and progeny were morphologically characterized. hybrid status was determined based on the morphology and colour of flowers and leaves. anthers of the hybrids were examined under a stereo microscope. to assess female fertility, stigmas of the hybrids were pollinated with pollen from ptf. tab. 1: performed combinations in each experiment experiment performed combinations quantitative analysis ac1 × ac1, ac1 × ac2, ac1 × ptf of embryo development ac2 × ac2, ac2 × ac1, ac2 × ptf ptf × ptf, ptf × ac1, ptf × ac2 qualitative analysis ac1 × ac1, ac1 × ptf of embryo development ac2 × ac2, ac2 × ptf ptf × ptf, ptf × ac2 seed production ac1 × ac1, ac1 × ptf and sowing ac2 × ptf ptf × ptf, ptf × ac2 embryo rescue ac1 × ac1, ac1 × ptf ac2 × ptf ptf × ptf pelargonium hybridization 51 tab. 2: composition of culture media culture medium macroand growth regulators vitamins other components micronutrients (μm) (mg l-1) (g l-1) er ms 5.71 iaa 2.5 thiamine-hcl 30 sucrose 4.40 bap 0.2 pyridoxine-hcl 7 agar 0.2 biotin 100.0 myo-inositol ac-i ms 2.5 thiamine-hcl 30 sucrose 0.2 pyridoxine-hcl 7 agar 0.2 biotin 3 active charcoal 100.0 myo-inositol ac-ii ms 2.5 thiamine-hcl 30 sucrose 0.2 pyridoxine-hcl 7 agar 0.2 biotin 20 active charcoal 100.0 myo-inositol er = embryo rescue, ac = active charcoal, ms = murashige and skoog (1962), iaa = indole-3-acetic acid, bap = 6-benzylaminopurine statistics statistical analysis was carried out with ibm spss 23. post-hoc comparisons were made using tukey's honestly significant difference (hsd) test. results and discussion characterization of the crossing partners the two genotypes of pelargonium acetosum (ac1 and ac2) were characterized by a matte bluish-green foliage. in contrast to ac1, the leaves of ac2 were more strongly lobed and their bluish colour was more distinct. while the flowers of ac1 had a light salmon-pink colour, the flowers of ac2 were first pale yellow and became white one day after anthesis. both genotypes exhibited a more or less upright growth habit. the cultivar p. ×peltatum ‘tornado fuchsia’ (ptf) was characterized by a trailing growth, glossy green foliage, and single fuchsia flowers. the leaf blade showed a horseshoe-shaped dark zone. both ac1 and ptf were flowering vigorously during this study and proved to be robust. in contrast, ac2 showed a high susceptibility to thrips, which hampered the development of flowers and complicated the use of this genotype. mtt staining (tab. 3) resulted in a low percentage of stained pollen grains in the case of ac1 and a high portion of stained pollen in the case of ac2 and ptf. stained pollen grains were interpreted as able to germinate on a stigma (firmage and dafni, 2001), and the percentage of stained pollen was understood as a measure of ferti lity. based on these assumptions, the male fertility of ac2 and ptf was considered relatively high (plaschil et al., 2017). in contrast, ac1 could only be assessed as partially male-fertile, which makes this genotype less suitable as a pollen parent. interspecific hybridization embryo development fruits from selfings of ac1 (ac1 × ac1) contained a maximum of two embryos and a mean of 0.95 embryos (tab. 4), most likely due to the low fertility of this genotype. two weeks after pollination, embryos were in transition between the torpedo and the cotyledon stage (fig. 1). three weeks after pollination, embryos had already reached their maximum size. the interspecific combination ac1 × ptf resulted in a maximum of four and a mean of two embryos per pollina ted flower, but differences to ac1 × ac1 were not significant (tab. 4). embryos from the combination ac1 × ptf were less green than embryos from the selfings of ac1 (fig. 1). these apparent chlorophyll deficiencies are most likely due to an incompatibility between one of the two plastomes and the nuclear genome (metzlaff et al., 1981, 1982; weihe et al., 2009). embryos of the combination ac1 × ptf often exhibited a slightly irregular morphology. in particular, the coty ledons were often unequally sized (fig. 1). tab. 3: pollen viability of the crossing parents according to the percentage of stained pollen grains after thiazolyl blue tetrazolium bromide (mtt) treatment genotype mean standard deviation stained pollen grains (%)1 (%) ac1 38z 16.09 ac2 86y 4.58 ptf 88y 4.00 1different letters indicate significant differences (tukey-hsd, n = 3 × 100, α = 5%) tab. 4: influence of the combination on the number of embryos, two weeks after pollination combination mean embryo standard total embryo number per deviation number per pollinated flower1 combination ac1 × ac1 0.95 zy 0.83 19 ac1 × ac2 1.65 y 1.46 33 ac1 × ptf 2.00 y 1.38 40 ac2 × ac2 4.05 x 1.10 81 ac2 × ac1 0.00 0.00 0 ac2 × ptf 3.35 x 1.60 67 ptf × ptf 5.45 w 1.57 109 ptf × ac1 0.00 0.00 0 ptf × ac2 0.30 z 0.66 6 1different letters indicate significant differences (tukey-hsd, n = 20, α = 5%) 52 r. kamlah, i. pinker, s. plaschil, k. olbricht selfings of ac2 (ac2 × ac2) resulted in three to five embryos and a mean of 4.05 embryos per pollinated flower (tab. 4). these numbers can be considered normal because usually no more than five ovules are fertilized in a pelargonium flower (yano et al., 1975). the interspecific combination ac2 × ptf resulted in a mean of 3.35 embryos per flower, which was not significantly lower compared to ac2 × ac2 (tab. 4). despite their relatively high number, these hybrid embryos developed poorly. two weeks after pollination the embryos had reached, at most, an early torpedo stadium (fig. 1) and did not develop much further in the following weeks. this delayed and then arrested embryo development might have been caused by an irregular endosperm formation resulting from an incompatibility between the parental genomes in the endosperm (lafon-placette and köhler, 2015). such postzygotic disturbances represent one of the most significant hybridization barriers, which potentially can be overcome by embryo rescue (kuligowska et al., 2016). combinations using ptf as the seed parent contained considerably smaller embryos and seeds than the previously mentioned combinations (fig. 1 and 2). in 11 of 20 fruits deriving from the selfings of ptf (ptf × ptf), the number of five embryos was exceeded because carpels contained ‘twin embryos’. the occurrence of twin embryos in pelargonium was documented by yano et al. (1975) and kubba and tilney-bassett (1980). no fruit development and no embryos were observed in the combination ptf × ac1 (tab. 4), whereas the combination ptf × ac2 resulted in four fruits per 20 pollinated flowers containing 1-2 embryos (a mean of 0.3 embryos per pollinated flower). it appears likely that fertilization was hampered by prezygotic hybridization barriers (and in the case of ptf × ac1 additionally by a low pollen viability). prezygotic barriers are well-known from interspecific crossings using pelargonium peltatum as the seed parent (yu, 1985). however, in order to confirm the occurrence of prezygotic barriers, it would be necessary to monitor the pollen tube growth within the pistil (winkelmann et al., 2010). despite their small number, embryos from the combination ptf × ac2 seemed to develop normally in comparison with ptf × ptf (fig. 1). seed morphology and germination while all the seeds resulting from the selfings of ac1 germinated, the germination percentage of the combination ac1 × ptf was only 44% (tab. 5). seeds from this combination were not as round as those from the selfings of ac1 (fig. 2). the development of most seedlings was hampered by chlorophyll deficiencies. seeds from the combination ac2 × ptf were small and under developed (fig. 2). this result agrees with yu (1985), who reported incompletely developed seeds deriving from the combination p. acefig. 1: morphology of embryos deriving from selfings (control) and interspecific crossings, two and three weeks after pollination (wap), scale bar: 1 mm fig. 2: morphology of seeds deriving from selfings (control) and interspecific crossings, scale bar: 1 mm pelargonium hybridization 53 tosum × p. peltatum. the small and shrivelled seeds from the combination ac2 × ptf (fig. 2) did not germinate (tab. 5). this combination could only be achieved by the application of embryo rescue (see below). out of 96 seeds deriving from the selfings of ptf, 73 germinated (tab. 5). the interspecific combination ptf × ac2 resulted in only six seeds per 20 pollinated flowers (a mean of 0.3 seeds per pollina ted flower). they appeared normal-shaped (fig. 2), and four of them germinated (tab. 5). horn (1994) reported also a low seed set from the combination p. peltatum × p. acetosum (a mean of 0.5 seeds per pollinated flower), but in his case the germination percentage was only 36%. f1 progeny plants from seeds were only obtained from the combinations ac1 × ptf and ptf × ac2. except two plants, all hybrids from the combination ac1 × ptf exhibited chlorophyll deficiencies or hybrid variegation, which severely hampered their development (fig. 3). consequently, only four plants survived the first six months after sowing. severe chlorophyll deficiencies leading to a low survival rate in the f1 also occurred after cross-breeding between p. acetosum and p. ×hortorum (hondo et al., 2014). however, the four hybrids from the combination ptf × ac2 did not show any chlorophyll deficiencies. compared to the plants originating from the selfings of ac1 and ptf, the development of all the interspecific hybrids was at first charac terized by considerably short internodes. then, about 20 weeks after sowing, these hybrids showed elongated internodes and a normal growth habit. however, one hybrid from the combination ptf × ac2 continued growing in a severely stunted manner and did not develop any flowers. hybrids with such stunted growth habit are known from a hybridization between p. peltatum and p. ×hortorum (coffin and harney, 1978) and can be a result of genomic conflict (bomblies and weigel, 2007). hybrids from both interspecific combinations developed sterile flowers. anthers were shrivelled, and no fruit development was observed after pollination with ptf. horn (1994) also reported sterile pro geny from the combination p. peltatum × p. acetosum. hybrid steri lity can be a consequence of either karyotypical differences or genetic incompatibilities (bomblies, 2010). if the hybrid sterility was caused by different karyotypes, a polyploidization could restore fertility and allow further generations on the tetraploid level (sattler et al., 2016). nevertheless, if the sterility was a consequence of genetical incompatibilities, a restoration of fertility would be rather unlikely (rieseberg and willis, 2007). the matte bluish leaf surface of p. acetosum did not appear among hybrids from the combinations ac1 × ptf and ptf × ac2. all plants had leaves with a rather glossy surface comparable to ptf. however, the leaf bases of all hybrids were cordate (unlike the peltate leaf base of ptf), and leaf zonation was not observed. leaf zonation is dominant over zoneless leaves in p. ×hortorum (amoatey and tilneybassett, 1993), but this does not seem to apply here. hybrids from the combination ptf × ac2 showed a high susceptibility to thrips, which was most likely inherited from ac2. information about the inheritance of the waxy leaf surface of p. acetosum is limited. in the hybridization between p. acetosum and p. ×hortorum (hondo et al., 2014), leaves of the f1 generation showed a rather intermediary level of wax bloom, and hybrids with a leaf surface comparable to p. acetosum first appeared in the f2. in tab. 5: influence of the combination on the number of seeds and the germination percentage combination mean seed number standard total seed number number of germination percentage per pollinated flower1 deviation per combination germinated seeds (%) ac1 × ac1 0.80 zy 1.06 16 16 100.00 ac1 × ptf 1.25 y 0.64 25 11 44.00 ac2 × ptf 2.15 x 1.35 43 0 0.00 ptf × ptf 4.80 w 1.15 96 73 76.04 ptf × ac2 0.30 z 0.73 6 4 66.67 1different letters indicate significant differences (tukey-hsd, n = 20, α = 5%) fig. 3: progeny from the combinations ac1 × ptf (left) and ptf × ac2 (right), 14 weeks after sowing. pot diameters: 9 cm, 7 cm, 4 cm (ac1 × ptf), and 9 cm (ptf × ac2). 54 r. kamlah, i. pinker, s. plaschil, k. olbricht fig. 4: shoot regenerates from the interspecific combinations ac1 × ptf (left) and ac2 × ptf (right), cultured on ac-ii-medium (six weeks after transfer). all regenerates within one jar (5.9 cm ø) originated from the same rescued embryo. order to achieve hybrids with bluish leaves from the hybridization between p. acetosum and p. ×peltatum, a further generation would be necessary, which seems only possible through previous polyploidization (see above). hybrid status was determined based on the above-mentioned morphological traits and was additionally supported by the occurrence of hybrid incompatibilities. nevertheless, for an unambiguous confirmation of hybridity, a molecular analysis of the hybrid character should be considered, such as aflp (amplified fragment length polymorphism) or rapd (random-amplified polymorphic dna) fingerprinting (barcaccia et al., 1999; kuligowska et al., 2016). embryo rescue two weeks after the establishment of embryo culture, most embryos started forming callus. in the case of ac2 × ptf, only six of the 20 cultivated embryos formed callus while the rest died off. embryos below the torpedo stage did not survive (scemama and raquin, 1990). a later start of embryo culture may have increased the number of surviving embryos (becker-zens, 1983), as embryos of the combination ac2 × ptf were slightly more developed three weeks after pollination (fig. 1). in contrast, embryos of the other combinations were developed enough to begin embryo culture two weeks after pollination because they were at least in a bent torpedo stage (fig. 1) (bentvelsen et al., 1990; scemama and raquin, 1990). from the fourth week after establishment of culture, countless tiny organoids developed on most samples (except for ac1 × ac1, which showed organoid formation only on less than half of the samples). most samples showed substantial callus proliferation. in the seventh week after establishment, an increasing number of adventitious shoots showed signs of vitrification. these symptoms are known from the micropropagation of p. ×peltatum on culture media containing bap (wojtania and gabryszewska, 2001; wojtania, 2010). some of the samples recovered after transfer to the phytohormone-free culture medium, but only a few adventitious shoots showed substantial elongation. samples of ac1 × ac1 showed a particularly stunted growth and consequently senescence of callus and shoots. after transfer of single shoots into jars, shoots of all samples continued growing slowly, and only a few shoots rooted. a strongly inhibitory effect of bap on shoot elongation of p. ×peltatum was documented by wojtania and gabryszewska (2001). this effect apparently continued a long time after samples were removed from the bap containing medium. as discussed above, embryos and seedlings of the combination ac1 × ptf showed chlorophyll deficiencies most likely due to an incompatibility between one of the two plastomes and the hybrid genome. in vitro, samples of this combination had a strong tendency towards a segregation of intact and chlorophyll-deficient tissue. consequently, many shoot regenerates were completely white and some entirely green (fig. 4). samples of the combination ac2 × ptf exhibited chlorophyll deficiencies only on intercostal fields of the leaf blade (fig. 4). because these symptoms only occurred in vitro, it remains unclear whether these were an effect of genomic conflict or other factors. as a consequence of low shoot qualities, low rooting rates, and chlorophyll deficiencies, very few plants endured the transplantation of shoots into organic substrate and the following acclimatization to the greenhouse. from the combination ac1 × ptf, four genotypes had survived six months later, two of them exhibiting chlorophyll deficiencies and hybrid variegation. from the combination ac2 × ptf, plants of only one genotype was successfully relocated to the greenhouse. these had green and glossy leaves and showed a high susceptibility to thrips, comparable to hybrids from the combination ptf × ac2. all flowering hybrids deriving from embryo rescue exhibited shrivelled anthers. in this regard, they did not differ from hybrids deriving from seeds. morphological traits indicating a spontaneous polyploidization have not been observed. the latter is a frequent result of in vitro regeneration (plaschil et al., 2015). conclusion in the hybridization between p. acetosum and p. ×peltatum, the occurrence of most hybridization barriers varied strongly between the performed combinations and depended on both the genotype and the direction of cross-breeding. the combination ac1 × ptf was hampered by chlorophyll deficiencies and hybrid variegation. viable hybrids were both achieved from seeds and via embryo rescue, but the latter did not considerably increase the number of hybrids. the combination ac2 × ptf was characterized by an incomplete embryo development. in this case, hybrids were only accomplished through the use of embryo rescue. the reciprocal combination ptf × 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(eds.), plant cell culture: essential methods, 79-95. john wiley & sons, london, uk. doi: 10.1002/9780470686522.ch5 wojtania, a., 2010: effect of meta-topolin on in vitro propagation of pelar 56 r. kamlah, i. pinker, s. plaschil, k. olbricht gonium ×hortorum and pelargonium ×hederaefolium cultivars. acta soc. bot. pol. 79(2), 101-106. doi: 10.5586/asbp.2010.013 wojtania, a., gabryszewska, e., 2001: effect of cytokinins and amino acids on multiplication of pelargonium cultivars. acta soc. bot. pol. 70(3), 203-208. doi: 10.5586/asbp.2001.026 yano, f., tokumasu, s., kato, m., 1975: the behaviour of pollen tubes and the developmental process of ovules in pelargonium. euphytica 24(1), 251259. doi: 10.1007/bf00147194 yu, s.-n., 1985: untersuchungen zur interspezifischen kompatibilität und biosystematik bei der gattung pelargonium. phd thesis, technische uni versität münchen, 270 pages. address of the corresponding author: r. kamlah, lausitzer platz 1, 10997 berlin, germany e-mail: rainer.kamlah@web.de © the author(s) 2019. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). angewandte botanik. maßnahmen z. erzielung allgem, rechtzeit. anwend.guter pflanzenschutzmittel 177 durchführbar ist, während wir – der weinbau sei auch hier aus genommen – bisher meist hintendrein kamen und uns darauf be schränken mußten, zu raten, wie es das nächste mal besser gemacht werden könnte. meine aufgabe war es, einmal, nachzuweisen, daß der richtig durchgeführte pflanzenschutz imstande ist, ungeheure werte der heimischen landwirtschaft zu erhalten, und ferner, zu zeigen, wie unsere anstalten bei zweckmäßiger organisation dieser aufgabe gerecht werden können. ist die vertretene auffassung in ihren grundzügen berechtigt, dann werden sich auch mittel und wege finden lassen, sie in die tat umzusetzen. über die bewertung von holzund pflanzenschutzmitteln im laboratorium und über ein neues spritzmittel für den pflanzenschutz. vortrag, gehalten am 4. august auf der diesjährigen tagung der vereinigung für angewandte botanik im mykolog. institut der forstakademie hann.-münden. von dr. richard falck. inhaltsverzeichnis: . allgemeines über den holzschutz. . frühere methoden der schutzmittelprüfung. neue prüfungsmethoden. mykozide wertzahlen; methode 5 gibt höhere werte als methode 2. die bedeutung der löslichkeitsverhältnisse. das resinol m. und seine eigenschaften. die prüfung des resinols als holzschutzmittel. löslichkeit und giftwirkung. . löslichkeit und abwaschbarkeit, prüfung im laboratorium. . klebkraft. . wirksamkeit; prüfungsmethoden. fällungsformen des resinols. . versuchspilze. . vergleichende wertung des resinols und kupferhydroxyds. angewandte botanik i. 12 178 richard falck, 15. germizide und mykozide wertung. 16. löslichkeit des kupfers und resinols unter verschiedenen bedingungen. 17. einfluß der substratzusammensetzung auf die wirkungsintensität. 18. einfluß der reaktion des substrates. 19. das kupfer is t kein spezifisches pilzgift. die germiziden wertzahlen nach methode 2 liegen z u hoch. 20. substratfällung und wasserfällung. 21. feinheit der fällung. einfluß auf wirksamkeit. 22. herstellung der resinolbrühen. a ) die alkalische resinolnatronlösung. b ) die alkaliarme resinolnatronlösung (saures resinolnatron). c ) die resinolkalkbrühe. d ) die resinol-magnesiabrühe. 23. zusammenstellung der resinollösungen und -brühen. 1 . allgemeines über den holzschutz. ich habe ihnen gestern die verschiedenartigen krankheits bilder und zersetzungserscheinungen vorgeführt, denen wir bei der erkrankung des hauses durch den echten hausschwamm begegnen, und habe im anschluß daran die diagnostischen merkmale und di e entwicklungsgeschichte des erregers (demonstration der sporen keimung) erläutert. die vom gesichtspunkt der praktiker wesent lichste frage is t aber d ie der verhütung und bekämpfung de r schwammkrankheiten. in dieser hinsicht hat sich gezeigt, daß die üblichen maßnahmen der trockenhaltung keinen genügenden und dauernden holzschutz gewährleisten, d a das holz während der lagerung, bei der bauausführung und im hause vor feuchtigkeit (feuchter luft) und neu hinzutretenden pilzkeimen nicht hinreichend geschützt werden kann. nur durch geeignete vorbehandlung des holzes mit pilzwidrigen substanzen kann das holz dauernd kon serviert werden, so daß es auch intakt bleibt, wenn e s vorüber gehend feucht wird oder in feuchter luft lagert. 2 . frühere methoden der schutzmittelprüfung. alle billigeren desinfektionsmittel, die wir kennen, sind auch für den holzschutz verwendet und empfohlen worden; selbst koch salzlösungen wurden noch kurz vor dem kriege in steinkohlen bergwerken zur imprägnation des grubenholzes verwendet. dabei muß ich gleich vorausschicken, daß gerade für den schwammschutz des holzes nur die kräftigsten desinfektionsmittel in verhältnis über d ie bewertung von holzund pflanzenschutzmitteln usw. 179 mäßig hohen konzentrationen genügend wirksam sind. um hier weiter z u kommen, war e s erforderlich, für jede der in betracht kommenden substanzen einen genauen wert-maßstab zu finden und festzusetzen, denn e s kommt nicht bloß darauf an, daß wir einen schutz erreichen, sondern daß wir diejenigen mittel herausfinden, die den holzschutz mit geringsten mengen und kosten gewährleisten und auch die übrigen be dingungen möglichst vollkommen erfüllen: haltbarkeit, neutralität gegen die holzfaser, farbvermögen (zur kontrolle), geruchlosigkeit und geringe giftigkeit für mensch und tier (bei verwendung im hause). e s kam also darauf hinaus, brauchbare methoden zur ge naueren wertprüfung der verschiedenen substanzen zur anwendung z u bringen. als ich die versuche vor etwa 15 jahren begann, waren in den großen imprägnationsanstalten für diese prüfungen die sog. schwammkeller in gebrauch. in unterirdischen lichtund luftlosen räumen, deren wände in einzelnen fällen mit wasser berieselt wurden, ließ man pilzbefallenes holz aus häusern – wobei es sich nicht um bestimmte arten handelte –, in möglichst großen mengen auswachsen und brachte die mit dem betreffenden mittel behandelten hölzer damit in unmittelbare berührung. e s wurden hierzu zumeist balken oder schwellenabschnitte verwendet, die monate lang auf dem schwammholz liegen blieben und von den mycelien mehr oder weniger stark angegriffen wurden. es wurden aber auch versuche in noch größerem maßstabe durchgeführt, indem man grubenhölzer imprägnierte und in feuchten bergwerken einbaute oder in die erde vergrub. nach jahren wurden sie dann ausgebaut und beurteilt. daneben waren ganz vereinzelt im labo ratorium versuche mit vergifteten gelatineund agarnährböden in reagenzgläsern schon im gebrauch, der nährboden wurde mit sporen von penizillien geimpft und die entwicklung beobachtet. m it richtig bestimmten reinkulturen holzzerstörender pilze wurde noch nicht gearbeitet. immerhin war man auf diesen wegen schon dazu gelangt. neben weniger wirksamen auch die wirksamsten substanzen zu benutzen, doch waren brauchbare vergleichswerte noch nicht bei der hand, so daß von den größten anstalten noch vor wenigen jahren auch substanzen verwendet wurden, die nur sehr ge ringen desinfektionswert besitzen und die man jetzt nicht mehr in betracht ziehen würde. 12* 180 richard falck, 3. neue prüfungsmethoden. über die von mir verwendeten prüfungsmethoden und di e daraus abgeleiteten wertzahlen habe ich im 7. heft der haus schwammforschungen (gustav fischer, jena, 1913) eingehend be richtet. hierauf wollen wir etwas näher eingehen, weil ich ihnen heute zeigen will, wie sich hieran die methode anschließt, nach der wir auch die pflanzenschutzmittel im laboratorium einer vorprüfung undwertbestimmung zu unterziehen vermögen. nach meiner methode 1 u . 2 wurde vergiftetes bierwürze-agar substrat verwendet und mit reinkulturen holzzerstörender pilze derart geimpft, daß die jedem konzentrationsgrad entsprechende wachstumsgeschwindigkeit mit hilfe der zuwachslängen zuverlässig gemessen werden konnte. als absolute hemmung wurde diejenige konzentration bezeichnet, bei welcher ein myzelzuwachstum nicht mehr zu beobachten ist. zwischen der absoluten hemmung und derjenigen konzen tration, bei der wieder ungehemmtes wachstum (also keine ab nahme der wachstumsgeschwindigkeit) stattfindet, liegen alle ab stufungen in der abnahme der wachstumsgeschwindigkeit (meßbar durch die zuwachslängen) und des hyphenvolums. die methode liefert also auch bei jeder unterhalb der absoluten hemmung ge legenen konzentration (besonders in den zuwachslängen) zahlen werte, die wir der vergleichenden wertung zugrunde legen können. nach meiner methode 3 wurde vom echten hausschwamm durchwachsenes holz mit den lösungen der betreffenden substanz in verschiedener konzentration getränkt und beobachtet, bei welchem gehalt das auswachsen nicht mehr erfolgte. es ist aber holz von gleichwertigem infektionsund virulenzzustand zu verwenden. es hat sich bald gezeigt, daß lösungskonzentrationen, die im agaroder gelatine substrat bereits absolute hemmung bewirken, dem damit getränkten holz noch lange keinen schutz gewähren: daß also die nach methode 1–3 gewonnenen hemmungswerte keineswegs auf die natürlichen ver hältnisse der schwammentwicklung bezogen werden können. das holz wird in der regel in luftfeuchtem zustande befallen, d. h . bei demjenigen feuchtigkeitsgehalt, den es im feuchtigkeits gesättigten raum selbst anzieht. solches holz enthält keine lösungen giftiger substanzen; der giftstoff is t in den zellen und zellwänden ungelöst verteilt, soweit ihn die lösung beim imprägnationsvorgang transportiert hatte. über die bewertung von holzund pflanzenschutzmitteln usw. 181 erst die methode 5, gleichartige holzklötzchen von bestimmter größe mit abgemessenen lösungen bestimmter konzentration zu tränken, zu trocknen und sie dann einem entwickelten schwamm herd von bestimmter art und größe auszusetzen, gibt resultate, d ie wir der beurteilung in der praxis direkt zugrunde legen dürfen. diese methode 5 ist in den letzten jahren weiter aus gebildet worden. wir erzeugen auf künstlichem substrat in kleinstem maßstabe vegetationsverhältnisse, wie sie sich für be stimmte pilze unter natürlichen verhältnissen im großen kaum günstiger darbieten dürften. es würde zu weit führen, wenn ich den weg, den wir bis jetzt zurückgelegt haben, darstellen wollte, nur die wichtigsten auf diesem wege erhaltenen zahlenwerte will ic h hier mitteilen: sie geben uns ein annähernd zutreffendes maß für den mykoziden wert der in betracht kommenden substanzen a ls holzkonservierungsmittel. 4 . mykozide wertzahlen. die in der folgenden zusammenstellung bei jedem körper angegebene wertzahl gibt die gewichtsmenge in grammen an, deren lösung in einem liter wasser dem damit getränkten und dann getrockneten holz einen vollständigen schutz gegen zer setzung durch pilzangriffe gewährt. daneben sind die nach den erstgenannten methoden (hier nach methode 2 ) gewonnenen zahlen vermerkt, die also– in einem liter nährstoffreichen (5% eingedickte bierwürze enthaltenden) agars gelöst – diesen vor dem befall durch holzzerstörende pilze sichern. tabelle. i. mykozide wertzahlen. gewichtsmengen von gleichem wirkungswert bewirken – in 1 liter tränk flüssigkeit gelöst – absolute hemmung des wachstums holzzerstörender pilze.-mmmmmmmmmmmmmmmm nach methode nach methode 2 5 ph dinitro-okresol . . . . .enole 2:4 dinitrophenol . . . . 0,05 1,5 fluoride fluornatrium 1,0 3 0 kieselfluormagnesium . . . 1,0 5,0 sublimat . . . . . . . . 1,0 2.5 schw ? yäznora 5,0 25,0 kupfersulfat . . . . . . 10,0 30,0 182 richard falck, es sind also 3–30mal höhere konzentrationen erforderlich, um holz zu schützen, als nährstoffreiches agaroder gelatine substrat: und zwar erfahren die stärksten gifte die erheblichste reduktion der hemmungswerte, die nitrophenole von 0,05 auf 1,5. nur nach methode 5 können unmittelbar brauchbare wertzahlen erhalten werden; wenn aber die beziehungen zwischen beiden wertreihen einmal bekannt sind, dann können die nach methode 2 gewonnenen zahlen zum mindesten als vorprüfungswerte ein geschätzt werden. die schwermetallsalze – auch das sublimat – müssen hier nach mit rücksicht auf wirkung und preis ausscheiden und kommen als holzschutzmittel kaum noch in betracht. denn es ist auch zu berücksichtigen, daß die starken säuren, an die si e gebunden sind, bei sauerer reaktion der lösungen für die holz faser keineswegs indifferent sind, d a e s sich hier ja um eine ein wirkung während der ganzen lebensdauer des holzes handelt. es bleiben also nur die phenole und die fluorverbindungen übrig. unter den letztgenannten und den anorganischen stoffen überhaupt kommt hiernach für den holzschutz in erster linie das fluornatrium, von organischen körpern das 2:4 dinitro phenol und dinitro-orthok resol in betracht. das fluornatrium ist ein spezifischer giftstoff für die myzelien höherer pilze, während es für andere organismen, ins besondere für menschen und tiere unschädlich ist. es ist un zweifelhaft dasjenige holzschutzmittel, welches in bezug auf spezi fische giftigkeit, haltbarkeit, neutralität usw. den anforderungen am weitesten entgegen kommt. dieses salz is t etwa z u 4% wasserlöslich, völlig neutral und kann daher mit dinitrophenolen gemischt verwendet werden, deren farbkraft eine leichte kontrolle der schutzbehandlung ermöglicht. mischungen von 85 bis 95 ge wichtsteilen fluornatrium mit 5– 15 teilen dinitrophenol oder dinitrokresol-salz sind daher besonders geeignet zur ausübung des holzschutzes. bei der impragnation des holzes tritt allerdings insoweit eine entmischung ein, als das fluornatrium etwas schneller und tiefer in die holzsubstanz eindringt, so daß von einer bestimmten tiefe an nur noch das fluornatrium nach weisbar ist. die kieselfluorwasserstoffsauren salze, unter denen das leicht rein herstellbare magnesiumsalz an erster stelle steht, reagieren über die bewertung von holzund pflanzenschutzmitteln usw. 183 sauer und sind deshalb weniger geeignet, für mischungen mit dinitrophenolen überhaupt nicht verwendbar. wenn sie unsere versuchslisten und die reihe von versuchs klötzchen, von denen ich eine auswahl vorlege, betrachten, dann werden sie sehen, welche summe von einzelversuchen auszuführen war, um zu dieser kurzen zahlenreihe und zu der erkenntnis zu gelangen, daß schließlich nur wenige substanzen für den holz schutz ernstlich in frage kommen. ist das schema einmal vor handen, dann wird es nun leicht sein, neu hinzukommende sub stanzen einzuordnen und zu bewerten. sie brauchen nicht erst in der praxis ausprobiert werden, sondern eine kleine versuchsreihe im laboratorium entscheidet schon über ihren wirkungswert. von unserer größten imprägnationsanstalt, den rütgerswerken, wurden während des krieges auf meinen rat die genannten mischungen von fluornatrium und dinitrophenol!) auch für die imprägnation von eisenbahnschwellen und telegraphenstangen verwendet, solange es noch möglich war, das fluornatrium zu beschaffen. auch beim kyanisieren der telegraphenstangen wurde das sublimat mehr und mehr durch fluornatrium ersetzt. (demonstration einer tele graphenstange, die im untersten besonders gefährdeten teil nach dem nadelverfahren von haltenberger und berdenich d. r. p. 244659 durchlocht und mit dem obigen salzgemisch durchtränkt worden ist; desgleichen volldurchtränkte buchenholzschwellen). 5. die bedeutung der löslichkeitsverhältnisse. die genannten dinitrophenole können frei oder als natrium salze verwendet werden. die bei den bakteriologen allgemein ver breitete annahme, daß nur die freien phenole als desinfektions mittel wirksam seien, trifft für die dinitrophenole in ihrer wirkung gegen fadenpilze keineswegs zu. ich gebe den alkalisalzen sogar den vorzug, weil sie neutral und an der luft, insbesondere mit den wasserdämpfen, nicht flüchtig sind, wie die freien nitround dinitrophenole. die letzteren bieten aber in ihren löslichkeits verhältnissen besondere vorzüge dar, indem sie bei gewöhnlicher temperatur in verhältnismäßig geringem, in der hitze dagegen in erheblich höherem grade wasserlöslich sind. sie lassen sich also bei siedetemperatur in genügend konzentrierter lösung in das *) kommt jetzt unter dem namen „schwammschutz rütgers“ in den handel. 184 richard falck, holz einführen, während die auswaschbarkeit der substanz durch regen usw. bei gewöhnlicher temperatur nur eine geringe is t. deshalb ziehen e s die imprägnationsanstalten vor, die freien dinitrophenole z u verwenden, nachdem sie zusätze (doppelchrom saure salze oder anilin) gefunden haben, ihre zersetzende einwir kung auf die eisenteile der imprägnationsapparaturen aufzuheben, es beträgt beispielsweise die nach einer maßanalytischen methode bestimmte löslichkeit: bei zimmertemperatur bei 100° in % in % 1 . des 2:4 dinitrophenols 0,093 1,5 2 . des-dinitro-o-kresols 0,023 0,2 bei der siedetemperatur des wassers kann also eine 1,5"oige lösung des 2: 4-dinitrophenols in das holz eingeführt werden. die lösung, die der regen auswaschen kann, enthält schlimmsten falls nur 0,09%. ich habe auf diesen punkt hier besonders hinzuweisen, da er dazu geführt hat, nach imprägnationsmitteln zu suchen, die be i hoher giftwirkung möglichst geringe löslichkeit in wasser zeigen. wenn man die patentschriften, welche die verbesserung der im prägnations-methoden in den letzten dezennien betreffen, durch sieht, wird man dieser aufgabenstellung immer wieder begegnen. 6 . das resinol m und seine eigenschaften. die chemische fabrik von f . raschig in ludwigshafen hat durch kondensation aus phenolen und aldehyden ein als „resinol m“ bezeichnetes kunstharz hergestellt und ist dabei von der voraus setzung ausgegangen, daß mit der größe des molekulargewichtes die desinfektionskraft einerseits zunimmt, während die löslichkeit in wasser anderseits sehr stark verringert ist. als phenol ist das harz in alkalilaugen löslich; die in wasser fast unlösliche harzsubstanz kann also leicht in das wasserlös liche natronoder kalisalz übergeführt werden. ammoniak oder organische basen lösen e s aber nicht. die bildung des natriumsalzes erfolgt nach folgender gleichung: r (oh)2 + 2 naoh = r (ona)2 + 2 h2o eine solche wässerige natronlösung des harzes, die etwa 33"/o harz und 13,3°/o natron (naoh) enthält, ist mir zur prüfung ihrer über die bewertung von holzund pflanzenschutzmitteln usw. 185 wirkung als holzkonservierungsmittel übergeben worden. es ist also auch hier wieder der gedanke leitend, daß mit dem harz eine stark konservierende und der auslaugung durch wasser wider stehende substanz zur anwendung gelangt. nach dem impräg nieren des holzes mit der entsprechend verdünnten lösung wird aus dem resinolnatron nämlich durch die allmählich eintretende einwirkung der luft-kohlensäure das harz ausgeschieden und auf d ie holzfaser niedergeschlagen. das harz wird also nachträglich wieder frei und unauswaschbar. (demonstration der reaktionen.) in seiner äußeren beschaffenheit gleicht das harz etwa dem kolophonium, ist aber tiefbraun gefärbt, spezifisch leicht (spez. gewicht 1,135) und von geringer härte, sehr spröde und brüchig, zwischen den fingern leicht verreiblich zu einem die oberfläche rauh machenden, in der wärme klebenden pulver; geschmacklos, aber deutlich nach phenol riechend. die letztgenannten eigen schaften sind auch für alle verbindungen des resinols charakte ristisch. schmelzpunkt 90° (demonstration). (schluß in heft 8.) uc1.b3299303_page_196_cut uc1.b3299303_page_197_cut uc1.b3299303_page_198_cut uc1.b3299303_page_199_cut uc1.b3299303_page_200_cut uc1.b3299303_page_201_cut uc1.b3299303_page_202_cut uc1.b3299303_page_203_cut art05_engert.indd journal of applied botany and food quality 84, 33 39 (2011) institute of agronomy and plant breeding, justus-liebig-university giessen, germany characterization of grain quality and phenolic acids in ancient wheat species (triticum sp.) n. engert, b. honermeier* (received april 28, 2010) summary the matter of this study was to determine the phenolic acid profi le of different ancient wheat, furthermore, to analyze the total phenolic content (tpc) with the folin-ciocalteu assay and the antioxidative capacity with the orac (oxygen radical absorbance capacity) assay. the concentration and composition of free, conjugated, and insoluble bound phenolic acids were analyzed in 20 accessions of wheat (triticum sp.), including 16 ancient wheat and 4 bread wheat samples grown in germany. six phenolic acids were analyzed by hplc, and ferulic acid (fa) was identifi ed to be the abundant phenolic acid. the content of phenolic acids in total was comparable to bread wheat, ranging between 141.0 and 542.0 µg gae/g (gallic acid equivalent/g) whole wheat fl our. in the current study no signifi cant distinction between the analyzed species could be observed. there was no signifi cant impact by the triticum species, neither on the total phenolic content by folin-ciocalteu nor on the antioxidative capacity by orac. correlation analysis between orac values and total phenolic acids demonstrated a positive correlation (r = 0.469, p = 0.05). phenolic acids, tpc and orac values of the analyzed ancient wheat samples were comparable to bread wheat. introduction wheat (triticum aestivum l. ssp. aestivum) is one of the most important agricultural commodities worldwide with 670 million tons in 2009, and it is constantly rising (usda, 2010). the domestication of wheat goes back to 10,000 bc, where wild species were taken into cultivation (feldman, 2001; nesbitt and samuel, 1995). wheat is one of the most important vegetable foods which provides human with basic nutrients (carbohydrates, proteins, vitamins, minerals). besides basic nutrients, wheat caryopses contain secondary plant metabolites, such as carotenoids, fl avonoids and phenolic acids. these compounds can have an infl uence on the utility value and can infl uence the quality characteristics of wheat products. additionally, secondary plant metabolites are bioactive compounds because of their antioxidative capacity (watzl and rechkemmer, 2001; slavin, 2003). einkorn (t. monococcum l.), emmer (t. dicoccum l.) and spelt (t. aestivum l. ssp. spelt) are ancient wheat species with low grain yields and non-threshable grain. wild einkorn, an one-grained wheat, is known as the progenitor of cultivated diploid wheat, which later results in tetraploid wheat (emmer) and hexaploid wheat (spelt) (feldman, 2001). in the last time interest in ancient wheat and a higher variety in nutrition grew in some countries, e. g. germany. for instance, the harvested area of spelt rose from 2003 to 2009 more than 300 % in germany (statistisches bundesamt, 2010). einkorn, emmer and the hexaploid spelt are very robust and modest cereals which can be harvested under moderate environmentally conditions like poor soils and water defi ciency (stagnari et al., 2008). thus, ancient wheat is recommended for organic production, where no synthetic fertilizers or pesticides are used to strengthen the plant. one reason for increasing interest in ancient wheat is the consumer, who claims more biologically grown products because of their environmentally friendlier production (zhao et al., 2007; dangour et al., 2009). another reason is the health benefi cial-aspect of wheat products, especially of whole grain products. investigations report that whole grain wheat products are reducing high blood pressure (behall et al., 2006). ancient wheat products often can be found as whole grain products, where the health aspect is higher than in white fl our products. phenolic acids (pa), such as ferulic, p-cumaric, vanillic, sinapic, syringic, and caffeic acid are valuable antioxidativ ingredients in wheat (mpofu, 2006). it is reported that phenolic acids inhibit lipid oxidation by scavenging free radicals such as hydroxyl radicals (cuppett et al., 1997). the phenolic acid profi le in ancient wheat subjects a high range of variation (mpofu, 2006). in wheat are three different forms of phenolic compounds existing, free phenolic acids, soluble conjugated (e. g. esterifi ed to sugars) and bound phenolic acids (e. g. esterifi ed to cell walls). the predominant phenolic acid is ferulic acid (hatcher and kruger, 1997; sosulski et al., 1982; weidner et al., 1999). the aim of this study is to quantitatively investigate phenolic acids, their composition, and their range of variation in ancient wheat. furthermore, the determination of the antioxidative capacity as a nutritionally positive and health affecting potential in emmer, einkorn and spelt growing in germany is carried out in this study. materials and methods wheat materials in 2006 ancient wheat species were grown in fi eld plots of the experimental station “weilburger grenze” of the institute of agronomy and plant breeding, (degree of longitude: 8° 39’ 16” e degree of latitude: 50° 36’ 12” n, altitude: 158 m). climate conditions were characterized by the annual total precipitation of 600 mm and mean air temperature of 8.5 °c. the experiment was carried out on an alluvial soil which is characterized by a clay content (< 2 µm) of 33 %, and a silt content (2 63 µm) of 58 %. the ph value was 6.3, and the total carbon content (humus) of the soil (soil depth 0 30 cm) was 1.42 %. in total 20 accessions (species and/or cultivars) of the genus triticum (t.) were obtained from the fi eld experiment, including three t.) were obtained from the fi eld experiment, including three t of the section monococcon (diploid), seven of the section dicoccoidea (tetraploid), and 10 of the section triticum (hexaploid) (tab. 1). no plant growth regulators (pgrs) were applied which supposably led to lodging during ripening. hulled wheat was dehulled automatically by a saatmeisterallesdrescher k35 (kurt pelz maschinenbau, germany). the dehulled and freethreshing samples were ground on a cyclotec 1073 sample mill (foss, germany) with a 500 µm sieve to get an whole wheat fl our. the fl our was mixed to ensure homogeneity and was extracted subsequently. chemicals vanillic acid (va), syringic acid (sya), caffeic acid (ca), pcoumaric acid (pca), ferulic acid (fa), as well as sinapic acid (sa) * corresponding author were purchased from sigma-aldrich (taufkirchen, germany). folinciocalteu reagent was purchased from merck (darmstadt, germany). gallic acid, acetonitrile, acetic acid, acetone, ethyl acetate, and methanol were obtained from roth (karlsruhe, germany). water (gradient grade) was acquired by applichem (darmstadt, germany) and ethanol by schmidt (dillenburg, germany). sodium carbonate was purchased from acros organics (new jersey, usa). analytics analysis of quality parameters firstly, thousand grain weight ([g], ista 1999) and the proportion of germinated caryopses [%] were analyzed. according to icc standard methods the following analyses were carried out: total nitrogen (dumas method, icc 167), falling number (hagbert-perten, icc 107/1) and sedimentation test (icc 116/1). extraction of bioactive compounds the extraction of the grain samples was done in three separate fractions ((1) soluble free, (2) soluble conjugated, and (3) bound phenolic acids) as previously described by using the methods of adom and liu (2002) and krygier et al. (1982) with modifications. briefl y: 1 g whole wheat fl our was extracted for one hour with (7:7:6, v/v/v) methanol/acetone/water and centrifuged. the supernatant provided fraction 1 and 2, the residue fraction 3. before extracting all three fractions with ethyl acetate, bound and soluble conjugated phenolic acids had to undergo an alkaline pulping with 5 m naoh. after evaporating the extracts they were resumed in 10 % acetonitrile and stored at -18 °c until analysis. the extracts were used for all further analyses (hplc, folin, orac). analysis of phenolic acids by hplc six phenolic acids were identifi ed by hplc-dad analysis on a c18 column (ec 250 x 4 nucleodur sphinx rp, 5 µm (mn)) (fig. 1). for quantifi cation of the hydroxybenzoic acids the diode array detection recorded at the wavelength of 250 nm and for the hydroxycinnamic acids at 290 nm. the method was modifi ed according to zielinski et al. (2001) and weidner et al. (2000). the temperature for the column was set to 25 °c. the injection volume was 100 µl and elution took place with a gradient mobile system: (a) acetonitrile, (b) acetic acid (0.5 %, ph 4.5) at 1 ml min-1 following the program: 0-14.5 min 8 % a, 15-30 min 10 % a, 31-40 min 80 % a and 40.5-45 min 8 % 6 3 4 5 1 2 fig. 1: hplc chromatogram of a wheat sample with standards. peak 1 vanillic acid (va), 2 syringic acid (sya), 3 caffeic acid (ca), 4 pcoumaric acid (pca), 5 ferulic acid (fa), 6 sinapic acid (sia) tab. 1: wheat material of the eight species with corresponding accessions species section species accession cv. [no.] [no.] 1 monococcon t. boeticum l. subsp. boeticum hausknechtii 1 pseudoreuteri 2 2 monococcon t. monococcum l. subsp. monococcum hohensteinii 1 3 dicoccoidea t. turgidum l. subsp. dicoccoides spontaneovillosum 1 artratum 2 semicanum 3 4 dicoccoidea t. turgidum l. subsp. turgidum griseo buccale 1 centigranum 2 mirabile 3 5 dicoccoidea t. timopheevii zhuk. subsp. timopheevii timopheevii 1 6 triticum t. aestivum l. subsp. spelta albispicatum 1 durhamelianum 2 arduini 3 7 triticum t. aestivum l. subsp. aestivum erythrospermum 1 ferrugineum 2 lutescens 3 miturum 4 8 triticum t. aestivum l. subsp. compactum humboldii 1 erinaceum 2 pseudo-rubiceps 3 34 n. engert, b. honermeier a. the identifi cation was made by retention times and uv/vis spectra compared with commercially available reference compounds. the quantifi cation of all six phenolic acids was done by a fi ve point calibration curve, where the standards were added to a reference fl our before extraction to exclude matrix effects. results were expressed as µg/g and converted in µg gae/g. total phenolic assay by folin-ciocalteu the total phenolic content was determined by using the folinciocalteau micro method (waterhouse, 2001) using gallic acid as a standard. this assay is electron transfer reaction based, which measures the sample’s reducing capacity (huang et al., 2005). folin-ciocalteu reagent consists of phosphotungstic (h3pw12o40) and phosphomolybdic (h3pmo12o40) acids. 40 µl of the sample extract was mixed with 3.16 ml aqua dest. and 200 µl folinciocalteu reagent was added. after 5 min 600 µl saturated sodium carbonate solution was added. the reaction blend was mixed and kept at 40 °c for 30 min in a water bath in darkness. folin-ciocalteu solution was reduced to blue oxides of tungsten and molybdenum and the absorbance was measured spectrophotometrically at 765 nm. the total phenolic content was expressed as gallic acid equivalent (gae). orac (oxygen radical absorbance capacity) assay the determination of the antioxidative capacity with the orac assay, described previously by huang et al. (2002), was operated on the 96-well plate fl uorescence reader fluoroskan (fisher scientifi c). the assay is hydrogen atom transfer reaction based and measures antioxidant capacity towards peroxyl radicals (huang et al., 2005). in general, 150 µl fl uorescein (6-hydroxy-9-(2-carboxyphenyl)(3h)-xanthen-3-on) was mixed with 25 µl of the sample extract h)-xanthen-3-on) was mixed with 25 µl of the sample extract h and incubated at 37 °c for 30 min. to initiate the reaction the ros-generator aaph (2,2’-azobis(2-methylpropionamidine)dihy drochloride) had to be added and the fl uorescence was measured at every 60 s for 90 min (excitation wavelength: 485 nm, emission wavelength: 538 nm). as a standard trolox® (6-hydroxy-2,5,7,8tetramethylchromane-2-carboxylic acid), a vitamin e analogue, was used. the antioxidative capacity was expressed as trolox equivalents (te). statistical analysis results are reported as mean values and the statistical analysis was performed with pasw statistics 18 (spss inc., chicago, il). the following characteristics were statistically tested by the analysis of variance (anova) in dependency on species with a general linear model: total phenolic content (folin-ciocalteu assay), antioxidative capacity (orac assay) and phenolic acid concentration (hplc). correlation analysis was performed by the pearson test, bivariate. results in this study grain quality parameters, concentrations of phenolic acids (tpa), the antioxidant capacity expressed as total phenolic contents (tpc) and orac values were analyzed. despite this, the correlation between the concentration of phenolic acids, total phenolic content and orac values was calculated. grain quality parameters thousand grain weight (tgw) of the ancient wheat species ranged from 24.3 to 49.4 g. the tetraploid species t. timopheevii zhuk. ssp. timopheevii showed highest tgw, therefore the tetraploid species showed in total the highest tgw followed by the hexaploid species. no signifi cant differences were observed in this set of data (fig. 2). the experiment was characterized by a very high germination level of the grain samples, which partly had visible symptoms of germination. t. turgidum l. ssp. turgidum showed in total the highest germination with more than 40 % of the analyzed caryopses. the species seem to have a signifi cant infl uence on germination rate because the tetraploid species showed the highest germination followed by the hexaploid species (fig. 2). in the case of the parameter falling number (fn) the observed values were very low (62 s) because of a high amylase activity by germination (fig. 3). these low falling numbers indicate a very high level of degradation of starch in caryopses. for the quality parameter protein there was no signifi cant difference identifi able. the relative proportion ranged between 12.4 and 20.4 % crude protein of the whole grain (fig. 3). as opposed to crude protein signifi cant differences were observed for the sedimentation test. the results ranged from 8 to 34 with the highest mean value for the tetraploid species, whereas one cultivar of t. aestivum l. ssp. compactum showed the highest value overall (fig. 3). fig. 2: germination rate [%], and thousand grain weight (tgw) [g] of different wheat species (mean ± sd, α=0.05) fig. 3: falling number (fn) [s], crude protein content [%], and sedimentation test of different wheat species (mean ± sd, α=0.05) phenolic acids by hplc the mean value of tpa_total was 334.9 µg gae/g (tab. 2) and the individual concentration of the tpa_total in all accessions varied from minimum 141.0 µg gae/g to maximum 542.0 µg gae/g (not displayed). there was a large species variation with the highest concentration for t. boeticum l. ssp. boeticum. neither between the species nor between the sections signifi cant differences could be found for tpa_total or fa (fig. 4). the value of the tpa consisted phenolic acids in ancient wheat species 35 of six detected phenolic acids: vanillic acid, syringic acid, caffeic acid, p-coumaric acid, ferulic acid, sinapic acid. 70 % of phenolic acids were found as cell wall esterifi ed compounds (tpa 3) followed by 23 % of free esters (tpa 2) and 7 % of free compounds (tpa 1) (tab. 2). the main phenolic acid was ferulic acid with about 65 % (217.9 µg gae/g) (tab. 3) of the phenolic acids (fig. 4). total phenolic assay by folin-ciocalteu the tpc_total of the whole wheat fl our (sum of all three fractions) measured by the folin-ciocalteu assay ranged from 2.3 to 2.6 mg gae/g. fraction 3 (tpc 3) was characterized by the highest total phenolic concentration (40.1 %) with a mean value of 1.0 mg gae/g followed by fraction 1 (36.2 %) with a mean value of 0.9 mg gae/g. the highest concentration of all samples was observed in fraction 1 (tpc 1) for t. turgidum l. ssp. turgidum with a value of 1.1 mg gae/g. no signifi cant difference between the species could be found (fig. 5). a strong correlation between tpa_total and tpc_total values was calculated (r=0.847, p=0.00). there was also a strong relationship between tpa_total and fa_total (r=0.816, p=0.00) (tab. 4). orac (oxygen radical absorbance capacity) assay the antioxidant capacity by the orac assay ranged from 18.2 to 23.8 µmol te/g whole wheat fl our for the sum of all three fractions (orac_total). highest orac values were measured in fraction 1 (orac 1) for the hexaploid wheat species t. aestivum l. ssp. aestivum with 11.1 µmol te/g followed by the diploid t. boeticum l. ssp. boeticum and the tetraploid t. turgidum l. ssp. dicoccoides. the lowest value was observed in t. timopheevii zhuk. ssp. timopheevii in fraction 2 (orac 2) with 3.0 µmol te/g (fig. 6). it was noticeable, that fraction 1 (orac 1) had the highest values holding 46.3 % tab. 2: concentration of phenolic acids (tpa) [µg gae/g] in caryopses of different wheat species [no.] species tpa 1 sd tpa 2 sd tpa 3 sd tpa_total sd 1 t. boeticum l. 27.31 ± 15.66 81.75 ± 76.33 232.43 ± 191.55 341.49 ± 283.54 2 t. monococcum l. 15.38 ± . 98.50 ± . 192.89 ± . 306.76 ± . 3 t. turgidum l. 31.98 ± 13.39 88.75 ± 28.11 247.99 ± 46.86 368.71 ± 75.99 4 t. turgidum l. 14.92 ± 3.95 63.14 ± 10.82 205.20 ± 73.38 283.26 ± 66.34 5 t. timopheevii zhuk. 21.67 ± . 68.11 ± . 315.51 ± . 405.29 ± . 6 t. aestivum l. 18.92 ± 15.06 68.61 ± 22.91 202.98 ± 84.76 312.02 ± 143.90 7 t. aestivum l. 22.76 ± 18.70 61.10 ± 7.69 201.45 ± 95.37 285.31 ± 107.59 8 t. aestivum l. 21.71 ± 5.71 87.58 ± 31.45 288.36 ± 27.47 397.65 ± 36.20 mean 21.83 77.19 235.85 334.87 relative concentration [%] 6.52 23.05 70.43 100.00 p (α= 0.05) 0.823 0.859 0.818 0.915 tab. 3: concentration of ferulic acid (fa) [µg gae/g] in caryopses of different wheat species [no.] species fa 1 sd fa 2 sd fa 3 sd fa_total sd 1 t. boeticum l. 5.09 ± 1.33 25.88 ± 25.05 195.48 ± 173.94 226.44 ± 200.32 2 t. monococcum l. 5.16 ± . 25.73 ± . 153.84 ± . 184.73 ± . 3 t. turgidum l. 5.86 ± 0.03 26.30 ± 6.08 187.20 ± 42.49 219.36 ± 47.41 4 t. turgidum l. 4.54 ± 0.93 26.99 ± 4.45 152.70 ± 39.14 184.23 ± 35.91 5 t. timopheevii zhuk. 5.32 ± . 23.39 ± . 243.54 ± . 272.26 ± . 6 t. aestivum l. 4.86 ± 0.68 28.71 ± 4.68 213.86 ± 24.38 247.43 ± 25.42 7 t. aestivum l. 4.55 ± 1.91 30.02 ± 3.43 178.86 ± 71.98 213.44 ± 73.06 8 t. aestivum l. 2.77 ± 2.52 28.27 ± 2.95 163.89 ± 83.88 194.92 ± 88.21 mean 4.77 26.91 186.17 217.85 relative concentration [%] 2.19 12.35 85.46 100.00 p (α= 0.05) 0.478 0.807 0.943 0.962 tpa_total fig. 4: total phenolic acids (tpa_total) and total ferulic acid (fa_total) values [µg gae/g] in different wheat species (mean ± sd, α=0.05) 36 n. engert, b. honermeier of the orac values in total followed by fraction 3 (orac 3: 33.8 %). orac values were positively correlating with tpc_total on a moderate level (tab. 4). a strong positive relationship between orac_total and fraction 1 of the tpc was observed (r=0.618, p=0.01) and no correlation between orac_total and fraction 2 and 3 of the tpc was found (tab. 4). is necessary to fi nd out more about phenolic compounds and the antioxidative capacity in ancient wheat, as they are an alternative to bread wheat. secondary plant metabolites, like phenolic acids, contribute to health promoting effects of wheat and consumers are increasingly interested in health promoting substances. since the highest proportion of phenolic acids is located in bran and aleurone layer, the analysis of phenolic acids, total phenolic content and antioxidative capacity was carried out in whole wheat fl our (pussayanawin et al., 1988; adom et al., 2005; zhou et al., 2004; anson et al., 2008). the germination rate was relatively high, ranging between 0 and 48 % which resulted in low falling numbers with an average of 62 s. low falling numbers are induced by the high degradation of starch during the process of germination. it can be supposed that humid conditions during grain ripening and a tendency of lodging led to germination before harvesting. the analyzed phenolic acids (tpa_total), which result by addition of the concentrations of the three fractions, differed between 283.3 µg gae/g for t. turgidum l. subsp. turgidum and 405.3 µg gae/g for t. aestivum l. subsp. compactum. einkorn (t. monococcum l. subsp. monococcum) showed the average concentration of 306.8 µg gae/g and emmer (t. turgidum l. subsp. dicoccoides) 368.7 µg gae/g. the phenolic acid concentrations ranged widely and they were comparable to those reported for bread wheat in the literature. for instance, stracke et al. (2009) reported concentrations between 282 µg/g and 1262 µg/g in wheat samples, zhou et al. (2005) analyzed the phenolic acid composition of hard red winter wheat bran extracts with low concentrations between 202.6 µg/g and 244.1 µg/g and li et al. (2008) reported average contents of 615 µg/g (einkorn), 779 µg/g (emmer), and 579 µg/g (spelt). highest concentration of phenolic acids in the used ancient wheat samples showed fraction 3 (tpa 3, bound phenolic acids), which was also in line with literature (stracke et al., 2009; abdel-aal et al., 2001). phenolic acids are bound to hydrolysable tannins, lignins, cellulose and proteins which are mainly structural components of bran, building a protective layer to the seed. phenolics, among phenolic acids, play a role in defending mechanisms against pathogens, parasites and predators (liu, 2004). also, they have antioxidant properties in plants (parr and bolwell, 2000; graf, 1992). during cell elongation ferulic acid is proposed to increase wall extensibility (graf, 1992). in this study about 70 % were bound to cell wall components, which was lower than in other studies, where up to 98 % were bound phenolic components (stracke et al., 2009; li et al., 2008). free phenolic acids showed the smallest contribution to total phenolic acids ranging between 1 % and 2 % (li et al., 2008; abdel-aal et al., 2001). in the present study fraction 1 (tpa 1) contributed 5 to 8 % to tpa_total, which was higher than in the other studies. reasons for the ranging results could be that einkorn and emmer produce more free phenolic acids than bound forms to protect the plant material against antioxidative stress. if it is the case, that ancient wheat have a higher concentration of soluble free phenolic acids, the bioavailability and bioaccessibility could be higher than in bread wheat. e. g. free ferulic acid can be resorbed in the small intestine, whereas bound forms have to be released by bacterial hydrolytic enzymes during fermentation in the large intestine (anson et al., 2009; kroon et al., 1997; zhao et al. 2003). furthermore, different extraction methods are the reason for ranging amounts of phenolic compounds of the analyzed fractions, which leads to the suggestion, that a standardized method should be implemented. ferulic acid was the predominant phenolic acid with about 66 % in wheat, averaging between 185 µg gae/g and 247 µg gae/g. highest concentration of ferulic acid was found in the hexaploid species (t. aestivum). the diploid species (t. boeticum and t. monococcum) and the tetraploid species (t. turgidum and t. timopheevii) could keep fig. 5: total phenolic content (tpc) [mg gae/g] in different wheat species (mean ± sd, α=0.05) fig. 6: antioxidant capacity (orac) [µmol te/g] in different wheat species for fraction 1, 2 and 3 (mean ± sd, α=0.05) tab. 4: pearsons’s correlation coeffi cients (r) between phenolic acids (tpa) and antioxidant capacity (tpc, orac) of different wheat species phenolic acids fraction tpa 1 tpa 2 tpa 3 tpa_total fa_total tpc_total 0.576 ** 0.700 ** 0.814 ** 0.847 ** 0.816 ** orac_total 0.618 ** 0.182 0.116 0.469 * 0.371 * signifi cant at p < 0.05 ** signifi cant at p < 0.01 discussion to our knowledge there are only a few studies published on the comparison of phenolic acid concentrations and the antioxidative capacity of ancient wheat (li et al., 2008; abdel-aal and rabalski, 2008; serpen et al., 2009). for that reason the aim of this study is to evaluate the phenolic acid concentrations of different ancient wheat varieties (accessions) to learn more about those varieties. it phenolic acids in ancient wheat species 37 up with the hexaploid ones. bound ferulic acid (fa 3) contributed the highest concentration of the total ferulic acid in the analyzed ancient wheat samples. ferulic acid is mostly bound to arabinoxylans and other indigestible polysaccharides restricting its release in the small intestine (anson et al., 2009). in the present study the concentrations of ferulic acids were lower compared to those reported previously. li et al. (2008) stated 298 µg/g for einkorn, 476 µg/g for emmer, 365 µg/g for spelt, and 395 µg/g for winter wheat. stracke et al. (2009) found about 85 % (239-1072 µg/g) ferulic acid of the analyzed phenolic acids in total. total phenolic contents of the present ancient wheat samples were reported as gallic acid equivalents in mg/g whole wheat fl our. the highest tpc was found in fraction 3 of the whole grain fl our, where phenolic compounds exist in bound forms (stracke et al., 2009; abdel-aal et al., 2001; adom and liu, 2002). in addition, there were high values of fraction 1. the folin-ciocalteu assay does not only display phenolics, it also reacts with nonphenolic substances such as vitamin c or other organic acids (huang et al., 2005; georgé et al., 2005). that could be an explanation for the high values of fraction 1, where further free phenolic and nonphenolic substances could be present. other studies did not analyze those three fractions, as they divided into fl our and bran. the values of the present study for the total phenolic contents in whole wheat fl our (tpc_total: 2.4 mg gae/g) were higher than in the literature reported for wheat samples. adom et al. (2005) reported 176-195 µmol gae/100 g (0.3-0.4 mg gae/g) for the endosperm fraction of hexaploide wheat. serpen et al. (2008) found total phenolic content values for einkorn with the average of 3.37 µmol/g (0.6 mg gae/g), for emmer with 6.33 µmol/g (1.2 mg gae/g) and for bread wheat with 4.36 µmol/g (0.8 mg gae/g). the widely ranging total phenolic contents may be due to different extracting methods, such as inclusion of soluble free, soluble conjugated and bound phenolic acids. dewanto et al. (2002) and liyana-pathirana and shahidi (2006) drew a similar conclusion. in the current study there were no signifi cant differences in total phenolic contents between the analyzed triticum l. species. einkorn and emmer were at about the same level of total phenolic contents as bread wheat. tpa_total could be attributed to tpc_total because of a strong correlation between the total phenolic acids and total phenolics (r=0.847, p=0.00). comparing the antioxidative capacity of the orac test in whole wheat fl our with the literature was diffi cult because different orac assays and extraction methods had been applied (moore et al., 2005; zhou et al., 2007; liyana-pathirana and shahidi, 2006; okarter et al., 2010). in the present study orac values (orac_total) ranged from 15.1 to 25.2 µmol te/g, which was lower than in most of other investigations of wheat reported in the literature. for instance, orac values of soft wheat cultivars investigated by moore et al. (2005) ranged from 32.9 to 47.7 µmol te/g. slightly higher orac values of 51 to 96 µmol te/g for whole wheat fl ours of different cultivars could be found by orkater et al. (2010). in contrary much higher values of 3406 µmol te/g defatted material were measured by liyana-pathirana and shahidi (2006). the analyzed soft wheat cultivars of zhou et al. (2007) varied between 15.5 µmol te/g and 24.5 µmol te/g, which is in line with the current study. in the current study three separate fractions of the grain samples (soluble free, soluble conjugated, and bound phenolic acids) were used for hplc and orac analysis. it could be observed that orac values in fraction 1 (orac 1, free phenolic acids) contribute 47.7 % to the total orac values and they are even higher than in fraction 3 (orac 3, bound phenolic acids). the bound fraction was reported to contribute about 86 % to the total orac (liyana-pathirana and shahidi, 2006). however, in the present study fraction 1 of the total phenolic acids was correlating with the orac values (r=0.618, p=0.05), therefore the sum of all three tpa fractions were correlating with orac_total (r=0.469, p=0.04). in conclusion, the current study showed, that tpa and tpc values of the applied ancient wheat samples were comparable to the values in the literature reported for bread wheat or spelt wheat. it could be supposed that phenolic acid contents have not been considerably changed during the evolutionary development of wheat although there is a genetic diversity between einkorn, emmer, spelt and bread wheat. therefore, health-benefi cial effects reported for bread wheat seem to be present in ancient wheat, too. the antioxidative capacity of ancient wheat, e.g. einkorn and emmer, is comparable to bread wheat which leads to the suggestion of using those wheat varieties as an alternative to bread wheat. einkorn, emmer and spelt, especially used as whole grain fl ours, could be taken into consideration for human diets, with health benefi ts. but still, the low bioaccessibility of phenolic acids, especially of ferulic acid from cereals, has to be improved. reasons for the wide variation of the phenolic acid concentrations within and between species have to be analyzed further. the effect of the germination rate on phenolic acids, total phenolic content and antioxidative capacity needs to be conducted in further studies. acknowledgements the authors are grateful to the landesbetrieb landwirtschaft hessen, bad hersfeld, germany for dehulling the wheat samples; pd dr. r. pätzold, institute of nutritional science, university of giessen, germany, for scientifi c assistance; r. allerdings, institute of plant production, university of giessen, germany for technical assistance. references abdel-aal, e.-s.m., hucl, p., sosulski, f.w., graf, r., gillott, c., pietrzak, l., 2001: screening spring wheat for midge resistance in relation to ferulic acid content. j. agric. food chem. 49, 3559-3566. abdel-aal, e.-s.m., rabalski, i., 2008: bioactive compounds and their antioxidant capacity in selected primitive and modern wheat species. open agric. j. 2, 7-14. adom, k., liu, r., 2002: antioxidant activity of grains. j. agric. food chem. 50, 6182-6187. adom, k., sorrells, m.e., liu, r.h., 2005: phytochemicals and antioxidant activity of milled fractions of different wheat varieties. j. agric. food chem. 53, 2297-2306. anson, n.m., berg, van den, r., havenaar, r., bast, a., haenen, g.r.m.m., 2008: ferulic acid from aleurone determines the antioxidant potency of wheat grain (triticum aestivum l.). j. agric. food chem. 56, 5589-5594. anson, n.m., berg, van den, r., havenaar, r., bast, a., haenen, g.r.m.m., 2009: bioavailability of ferulic acid is determined by its bioaccessibility. j. cereal sci. 49, 296-300. behall, k.m., scholfield, d.j., hallfrisch, j., 2006: whole-grain diets reduce blood pressure in mildly hypercholesterolemic men and women. j. am. diet. assoc. 106, 1445-1449. cuppett, s., schnepf, m., hall, c., 1997: natural antioxidants – are they a reality? in: shahidi, f. 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ancient wheat species 39 art06_selmar.indd journal of applied botany and food quality 84, 158 161 (2011) 1 institut für pfl anzenbiologie, technische universität braunschweig, braunschweig, germany 2 fachbereich physik, philipps-universität marburg, marburg, germany 3 tem messtechnik gmbh, hannover, germany introducing terahertz technology into plant biology: a novel method to monitor changes in leaf water status björn breitenstein1, maik scheller2, mohammad khaled shakfa2, thomas kinder3, thomas müller-wirts3, martin koch2, dirk selmar1* (received april 21, 2011) * corresponding author summary we present a novel, non-destructive method for determination of changes in leaf water content in the fi eld based on terahertz (thz) technology. in this method, terahertz waves, which are strongly absorbed by water, are generated and detected using a photomixer that converts the optical beat signal of two interfering diode lasers into thz radiation. this allows a coherent detection as basis for the determination of changes in leaf water contents. the reliability of this innovative method was verifi ed by monitoring changes in the leaf water content of young coffee plants in parallel using classical, destructive thermogravimetrical measurements as well as by thz spectroscopy. the broad applicability of this novel device was shown by longand short-term measurements. the changes in leaf water content during drought stress induced dehydration as well as during the course of rapid re-hydration after re-watering vividly highlight the tremendous potential of this novel technique and its high reliability. the fi ndings presented here provide the basis for thz-based in vivo determination of changes in the leaf water content under fi eld conditions. introduction determination of the leaf water content is of high importance for numerous aspects in plant science including basic research and various fi elds of applied plant biology. due to the effects of global climatic changes leading to increasing aridifi cation, there is a crucial need for effective tools to select drought stress resistant plants. accordingly, measurement of plant water stress by remote sensing is actually of high scientifi c interest. in this context, estimation of the leaf water content is inevitable. unfortunately, most techniques, such as the common thermogravimetrical quantifi cation of water content, are destructive. additionally, there are various non-destructive methods for the detection of water that are based on the absorption or refl ection of electromagnetic or radioactive radiation; however, these techniques all have signifi cant drawbacks. for example, during microwave based determination of the leaf water content, the absorbance of the radiation (wavelengths between 3 mm and 1 m, corresponding to frequencies between 100 ghz and 0.3 ghz) is strongly infl uenced by the inorganic salt content of the leaves, which markedly reduces the reliability of this method (ulaby and jedlicka, 1984). furthermore, the resolution of the imaging measurements is limited due to the relatively large wavelength that this method employs. due to the problems related to radioactivity, the application of ß-radiation based sensors is not recommended. indeed, nuclear magnetic resonance (nmr) techniques provide a reliable tool for the determination of leaf water contents, but this approach is not suitable for use in a regular biochemical lab or in fi eld trials due to the size of the nmr-machines. recently, an optimized method for the estimation of leaf water content based on the refl ectance of the visible spectrum was published (zygielbaum et al., 2009). unfortunately, when applying this approach, leaf water contents could only be determined in comparison to other leaves and could not be estimated as real values. the most common methods used for non-destructive quantifi cation of the leaf water content are based on infrared spectroscopy (ir; wavelengths between 0.75 µm and 100 µm, corresponding to frequencies between 400 thz and 3 thz) and employ the refl ectance of infrared radiation (tucker, 1980; hunt et al., 1987; hunt and fig. 1: principle of the thz-based approach for determination of changes in the leaf water content and schematic drawing of the device a) general scheme: two continuous-wave frequency-tunable lasers emitting very similar wavelengths produce an optical beat in the thz range. this beat induces electromagnetic thz radiation in a photomixing emitter (antenna 1). when these thz rays impinge on the detector (antenna 2), an electric current is elicited that varies based on the intensity of the thz radiation. from this data the thz transmission of the sample, e.g. a leaf between the two antennas, can be calculated. as the detector antenna is also irradiated with the original laser generated beat, any phase shifting can be determined (coherent detection). b) schematic drawing of the measurement set-up of the device. x1: beamsplitter; fc1, fc2: fi ber couplers; f1, f2: fi bers; ea: emitting antenna; ra: receiving antenna; el, rl: lenses for thz radiation; sg, tia, lia, sp: components for lock-in amplifi cation and signal recording; t: target, e.g. leaf. thz-based monitoring of changes in leaf water status 159 rock, 1989; eitel et al., 2006; seelig et al., 2008). due to water absorption features in the near(nir) and far-infrared (fir) region, ir-refl ectance spectra have great potential to specify the water content. for example, peñuelas et al. (1997) estimated the plant water content by determining the ratio of the refl ectance at 970 nm to that at 900 nm (r970:r900), while the shortwave infrared water stress index (siwsi) was used by fensholt and sandholt (2003), the three-band ratio indices were applied by pu et al. (2003), and the normalized difference water index (ndwi) was used by gao (1996) and by serrano et al. (2000). to date, many studies have been conducted to defi ne the optimal formulas and wavelengths (fensholt and sandholt, 2003; pu et al., 2003; serrano et al., 2000; sims and gamon, 2003) however, it is diffi cult to accomplish this due to the complex refl ection geometry of the leaves and the vulnerability of the technique to disturbances. accordingly, there has been relatively little validation of the fi eld data to date (claudio et al., 2006). in addition to these remote sensing approaches, analogous conditions also apply to the estimation of changes in the water content of certain vital leaves. although the parameter required in these cases can be specifi ed by solid calibrations, the technique is not sensitive enough to record small differences in the water content of an individual leaf. moreover, because the complex refl ection geometry of leaves changes when the turgor of the cells decreases as a result of water defi cit, nir-based techniques are not suitable for recording small changes in the water content of leaves (hunt et al., 1987). consequently, an alternative is required when such changes should be monitored. hadjiloucas et al. (1999) introduced the measurement of leaf water content using terahertz waves. thz radiation is characterized by wavelengths between 0.1 mm and 3 mm, corresponding to frequencies between 3 thz and 0.1 thz (100 ghz). water strongly absorbs terahertz radiation, while non-polar organic material does not. therefore a transmission measurement setup is very effective for the quantifi cation of the water present in leaves. however, due to its dimensions, the system used by hadjiloucas et al. (1999) is not applicable for fi eld experiments. the set-up presented in this paper (fig. 1) is based on an electromagnetic model of the complex thz-permittivity as a function of the water content of the plant leaf described by jördens et al. (2009) and allows that all optical components can be arranged in a small box for fi eld measurements. material and methods plant material one-year-old coffee plants (coffea arabica l.) with 10 to 12 leaves were used for the experiments. the plants were grown from coffee seeds in green houses under long-day conditions (16 h light / 8 h dark) at about 22 °c in single pots (∅ = 11 cm) under approximately 70% rel. humidity in about 700 ml of standard garden soil. watering was conducted twice a week to maintain the soil water content of approximately 60 % (w/w). to induce drought stress, watering was suspended and the humidity was reduced to 55 % (w/w) for the verifi cation experiments (fig. 2) and to 45 % (w/w) for the monitoring of longand short-term changes in the water content (fig. 3 and 4). during the course of the experiment, the soil water content declined down to 10 % (w/w). in general, all thz measurements were performed in the midmorning. the short term measurements (rehydration) started at 10 am and lasted till 5 pm. thermogravimetrical measurement for the determination of the actual dry weight of the leaves, directly after detaching, the coffee leaves were weighed and subsequently placed in a lab oven for 36 h at 110 °c. after cooling down, the dried leaves were weighed again and the water content was calculated. measurement of thz transmission in this study, terahertz radiation indicates electromagnetic waves with a frequency in the range of 100 ghz to 10 thz (corresponding to a wavelength of 3 mm to 30 µm). in this sparsely explored gap between microwaves and infrared light, only few types of radiation sources are available, which employ both, microwave and optical techniques. for the investigations presented here, the waves were emitted from a small dipole antenna (length 200 µm). by the use of three lenses, the diverging waves were bundled into a beam and fig. 2: verifi cation of thz based determination of the leaf water content coffee leaves still attached to plants that had been subjected to massive drought stress were monitored by periodically determining the thz transmission. at any point of the thz measurement the water content of four individual leaves (from at least two different plants) was determined by classical, destructive thermogravimetrical measurements. each point represents the mean value of at least four different individual samples or leaves, respectively. the mean value of the standard deviation was about 3%. fig. 3: long-term monitoring of dehydration induced changes in leaf water content drought stress related changes in the water content of coffee leaves were induced by suspending watering. every day the same leaf on the experimental plant was measured by analyzing the thz transmission of the intercalary leaf area. to mimic realistic conditions in which plants shall be analyzed, the leaf was removed from the thz system after each measurement. consequently, the appointed area used for the thz measurement may have differed slightly. each point represents the mean value of at least three independent estimations from the same leaf. the mean value of the standard deviation was about 3%. 160 b. breitenstein, m. scheller, m.k. shakfa, t. kinder, t. müller-wirts, m. koch, d. selmar focussed on a small spot with a diameter of approximately 2 mm (see fig. 1b). in turn, a complementary set of lenses collects the rays that emanate from the focus and redirects them to a receiver antenna of the same type. a photomixing effect in the emitting antenna converts the optical beat note of two interfering diode lasers into thz radiation (fig. 1). at the detector antenna, the resulting thz radiation generates an electric current that depends on both the amplitude and the phase of the incident thz wave. because the detector antenna is also irradiated with the original laser generated beat, the phase shifting effects can also be specifi ed. this coherent detection (verghese et al., 1998; wilk et al., 2008) allows the simultaneous determination of both, the absorption of thz radiation (mainly by the water present in the sample), and the changes in the refractive index of the material through which the radiation passes. in contrast to the phase-sensitive set-up employed by hadjiloucas et al. (1999), in our device there is no need to build in an interferometer for microwave or submillimeter wave radiation. all optical components – except for the thz focussing lenses – are standard components for near-infrared light. the major advantage of our set-up is its compactness. all components can be encased in a small box allowing its transfer into the fi eld. moreover, the new principle enables to apply a much larger thz wavelength range. prerequisite for generation of the required effects were two continuous-wave frequency-tunable lasers that have only a small difference in their wavelengths. the laser beams were superimposed in a beamsplitter and then coupled into polarization-maintaining single-mode fi bers. each of the beams obtained after collimation at the fi ber outputs then contains 50% of the light of each laser. this is about 3 to 5 mw per laser per fi ber. as the lasers have different optical frequencies, the interference of the two beam components results in a beat note at the difference frequency, which excites the emitter antenna. by changing either the temperature or the supply current of the laser diodes, the beat frequency could be tuned to arbitrary values between 0 and 2 thz. the measurements presented here were carried out at 200 to 220ghz. it turned out that the measurement results were independent from the frequency throughout this range. the emitted thz electromagnetic power was estimated in the range of several nw. the current detected at the receiver antenna was in the range of some na, which made it necessary to use a lock-in amplifi er for signal recovery. however, the relatively small cost of the laser system and the fact that the thz frequency can be chosen almost arbitrarily in the range 0.1 to 2 thz outweigh the disadvantage of the low thz power. on the other side, the low thz power ensures that the leaves could be measured over a long time period without getting any damages (i.e. necroses, injuries, burnings etc.). the optomechanical laser set-up (including laser-to-fi ber coupling) was mounted on a 15mm thick aluminium board and housed against dust and humidity. so the system could be transported from one institute to another, though slight realignment of the fi ber entrances was sometimes necessarry. therefore, future versions will incorporate an already existing automatic beam alignment system (e.g. fiberlock of tem messtechnik gmbh). the laser set-up can then be hermetically sealed, as it will no longer require any user service. results and discussion verifi cation to validate the suitability of use of the novel terahertz based approach to quantify the leaf water content, approximately 50 individual young coffee plants were subjected to massive drought stress by lowering the soil water content from 50 % to 10 % (w/w). the resulting decrease in the leaf water potential was monitored by periodically determining the thz absorbance and the transmission of leaves still attached to the plants. subsequently, the water content of four individual leaves (from at least two different plants) was determined by classical, destructive thermogravimetrical measurements. comparison of both parameters revealed the same progression, and thus demonstrates the applicability of the novel thz based quantifi cation to estimate the plant water content (fig. 2). although each point of the graphic represents the mean value of at least four different individual leaves, the data match, demonstrating the high reliability of the novel technique presented here. it should be noted that the actual terahertz technique applied only allows determination of the absolute amount of water present in the measuring focus. for a valid quantifi cation of the relative water content, i.e. the percentage of water, the leaf thickness must also be known. because the phase shift between the original beat and the thz wave that passes through the leaf strongly depends on the propagation length, an exact estimation of the leaf thickness can be achieved. in a forthcoming evolution of the terahertz system applied here, this technique will be included while focusing on automatic evaluation and calculation computer programs. these future studies should permit co-estimation of both the absolute water content and the leaf thickness, thereby providing reliable data regarding the actual water concentration of a leaf. applications even without the advanced technique of co-estimation of the water content and leaf thickness, the novel terahertz approach presented here represents a high-end tool for various applications of noncontact online monitoring of changes in the leaf water content such as estimation and recording of its shortor long-term changes. to demonstrate the broad applicability of the novel technique, both long-term and short-term measurements of single plants were performed. long term measurement of a single plant: dehydration the drought stress induced decrease in the leaf water content represents a process that typically lasts for a longer period of time. accordingly, we used the thz system to determine the water content of one leaf of a coffee plant, while watering was suspended for a time period of about three weeks. each day, the water content of the same leaf of this experimental plant was measured by analyzing the thz transmission of the intercalary leaf area. all measurements were performed in the same room in which the plant had been fig. 4: determination of short-term changes in leaf water content during re-hydration the rapid water uptake of drought stressed coffee plants was monitored by quantifying the water content of one leaf. after rewatering, the thz absorbance of one individual coffee leaf was determined every ten minutes without removing the plant from the system. every point represents a single measurement. thz-based monitoring of changes in leaf water status 161 grown. to mimic realistic conditions, the plant was removed from the thz system after each measurement. consequently, the appointed area used for the thz measurement may have differed slightly. despite such putative location-related variations, the data recorded (fig. 3) demonstrate the high performance of the new technique, which indeed enables reliable long term measurements of changes in the leaf water content. short term measurement of a single plant: rehydration in contrast to the slow changes in the leaf water content described above, the increase in the water content after re-watering of a drought stressed plant occurs much more rapidly. accordingly, we quantifi ed this process by measuring the thz transmission of one leaf on a coffee plant every ten minutes without removing the plant from the system. it is interesting to note that the uptake of water could be monitored effi ciently (fig. 4). in contrast to the long term measurements, the area used for thz measurement in the short-term experiment was fi xed throughout the entire experiment. consequently, putative location-related variations as described above are excluded, resulting in a smaller deviation. the data describing the short-term changes in water content underline the high potential of this novel technique and provide the basis for the further development of a portable thz-device to determine in vivo the leaf water content under fi eld conditions. acknowledgements this study was supported by the german federal ministry of food, agriculture and consumer protection (bmelv) as project 2815305707: “terahertz-messung zur in vivo-analyse des trockenstresses bei nutzpfl anzen: optoelektronisches messwerkzeug zur selektiven züchtung und kultivierung”. references claudio, h.c., cheng, y., fuentes, d.a., gamon, j.a., luo, h., oechel, w., qiu, h.l., rahman, a.f., sims, d.a., 2006: monitoring drought effects on vegetation water content and fl uxes in chaparral with the 970 nm water band index. remote sens. environ. 103, 304-311. eitel, j.u.h., gessler, p.e., smith, a.m.s., robberecht, r., 2006: 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from spectral refl ectance: a comparison of indices based on liquid water and chlorophyll absorption features. remote sens. environ. 84, 526-537. tucker, c.j., 1980: remote sensing of leaf water content in the near infrared. remote sens. environ. 10, 23-32. ulaby, f.t., jedlicka, r.p., 1984: microwave dielectric properties of plant materials. ieee trans geosci. remote sens. 22, 406-415. verghese, s., mcintosh, k.a., calawa, s., dinatale, w.f., duerr, e.k., molvar, k.a., 1998: generation and detection of coherent terahertz waves using two photomixers. appl. phys. lett. 73, 3824-3826. wilk, r., breitfeld, f., mikulics, m., koch, m., 2008: a continuous wave thz spectrometer as a non-contact thickness measuring device. appl. opt. 47, 3023-3026. zygielbaum, a.i., gitelson, a.a., arkebauer, t.j., rundquist, c., 2009: non-destructive detection of water stress and estimation of relative water content in maize. geophys. res. lett. 36, l12403. address of the corresponding author: dirk selmar, institut für pfl anzenbiologie, technische universität braunschweig, mendelssohnstraße 4, braunschweig, germany. e-mail: d.selmar@tu-bs.de, tel.: 49-(0)-531-391-5881, fax: 49-(0)-531391-8180 angewandte botanik. kleine mitteilungen. 259 schon sehr tiefgehende, mit starker borke versehene wurzeläste, denen d ie rebläuse keinen großen schaden mehr zufügen konnten, als diese nach mazedonien eingeschleppt wurden. wie sich der verfasser die erziehung baumartiger reben in reblausverseuchten böden vorstellt, ist mir auch nicht klar. frisch ge pflanzte reben gehen doch – auch wenn wir die bodenoberfläche fest stampfen würden – alsbald zugrunde, weil die für die nahrungsauf nahme in betracht kommenden wurzelenden von den rebläusen an gestochen werden und später verfaulen. man is t also gar nicht in der lage, eine baumartige rebe z u erhalten. daß die popoffsche „lösung der phylloxerafrage“ für unsere deutschen verhältnisse vollkommen aussichtslos ist, wird jedem wein bauer sofort klar sein. auch der verfasser sieht ein, daß sich eine baumartige rebenerziehung in deutschland weder mit dem klima noch mit der weinqualität verträgt. e r glaubt aber, man könne auch für deutschland eine erziehungsform finden, die den rebstock üppige ent wicklung gestattet, ohne daß der boden bearbeitet zu werden braucht. e r wird z u diesem vorschlag in deutschen weingebieten wenig zu trauen finden. die winzer werden ihn also nicht aus theoretischen gründen und bedenken ablehnen, sondern weil e r wissenschaftlich noch gar nicht genügend gestützt und praktisch unmöglich ist. k . müller, augustenberg. literatur. d e s raummangels wegen wird in dieser nummer nur eine liste gegeben. die besprechungen wichtigerer arbeiten folgen im nächsten bande. beckenstedt, h., bessere aussichten für die lupinenverwertung. deutsche landw. presse xlvi (1919), nr. 18, s. 587–588. buchka, k . von, das lebensmittelgewerbe. ein handbuch für nahrungsmittelchemiker, vertreter von gewerbe und handel, apo theker, arzte, tierärzte, verwaltungsbeamte und richter. band iv. akademische verlagsgesellschaft m . b . h., leipzig, 1919. 412 seiten mit 2 1 abbildungen. der vorliegende 4 . band des handbuches behandelt in drei ab schnitten die milch und milcherzeugnisse, die süßstoffe und das bier. meyer. das aufbewahren der weintrauben und pflaumen. reichs-gemüse und obstmarkt iv (1919), nr. 108, s. 1. verschiedene konservierungsverfahren von pflaumen und wein trauben für die kleinwirtschaft. my. ehrenberg, p ., hahn-haslinger, e., zyl, j. p. van, vergleich der trocknungskosten für zuckerrüben auf einem trommeltrockner und einer darranlage. landw. jahrb. liii (1919), heft 4 , s . 525–560. nach den angestellten untersuchungen und berechnungen arbeitet der trommeltrockner rationeller und vorteilhafter und ist der darre entschieden vorzuziehen. my. nahrungs mittel. 260 . literatur. genußmittel. etty, m. w., de suikerindustrie in natal. de indische mercur xlii (1919), nr. 43, s. 813–815. verf. bespricht kultur, 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(a. hartlebens chemisch-technische bibliothek, band nr. 360) a. hartlebens verlag, wien u. leipzig, 1919, 428 s. steppes, r., trocknungsverfahren bei getreidegarben. deutsche landw. presse xlvi (1919), nr. 76, s. 574–575, mit 8 abbildungen. bericht über die ernteverfahren in verschiedenen gegenden, den die abbildungen besonders anschaulich gestalten. my. zielstorff, w., uber zusammensetzung und verdaulichkeit von weintrestermehl. ill. landw. ztg. xxxix (1919), nr. 8384, s. 423. untersuchungen und fütterungsversuche mit aufgeschlossenem und nicht aufgeschlossenem weintrestermehl, die seine wertlosigkeit als futtermittel ergaben. my. bernard, ch., 0ver de kieming van de theezaden. mededeelingen van het proefstation vor thee, nr. xliii, batavia 1915, 9 s ., 6 taf. bredemann, g . und schätzlein, chr., über herstellung und zu sammensetzung kleinasiatischer traubensaftkonserven. zeitschr. f. unters. nahrungsu. genußmittel xxxviii (1919), s. 16–24. literatur. 261 den doop, j. e. a., gallobelicus nicotianae konigsberger. bulletin van het deli proefstation medan, sumatra, nr. 12, august 1919. diem, k., bemestingsproeven bij de tabak in het veld. mededeel. van het deli proefstation te medan, sumatra. tweede serie nr. iv, 1919, s. 1–108. hoffmann. der tabakbau. zugleich fünfte, neubearbeitete auflage von a. v. babo, der tabakbau. berlin 1919, paul parey. 181 s., 37 textabbild. und 1 bauplan. auch diese ausgabe, die wesentlich vermehrt worden ist, vor allem durch einschalten des abschnittes „qualität des tabaks“ wird in fachkreisen die ihr zukommende anerkennung finden. ha. leersum, p. van en bernard, ch., over de selectie van de thee plant iii. mededeel. van het proefstation vor thee, nr. xliii, batavia 1915, 22 s. rothenfusser, s., schwarzer tee. – deutscher tee. – deutscher schwarzer tee. zeitschr. f. öffentl. chemie xxv, s. 111–121, 127–139. angabe der wichtigsten verordnungen, der teeersatzmittel und ihrer bestandteile und der vorschriften für ihren verkauf. ha. tabakfermentation im kleinen. land u. frau iii (1919), nr. 34, s. 265. einige fermentierverfahren für tabak in kleinen mengen. my. die tafeltraubenkultur in belgien. wein und rebe i (1919), heft 1, s. 59–62. anlage, zucht, arten und erfolge der weintraubenkultur in belgien. my. alpers, k., die bedeutung der obstkernsammlung und die her stellung von obstkernöl im kleinbetriebe. pharm. ztg. lxiii (1918), s. 354–355. auszüge und mitteilungen. tropenpfl. 1919, nr. 8, s. 262. piassavafett wird als ein dem kokosfett ähnliches fett aus den piassavapalmnüssen gewonnen. ha. cavel, ä die gewinnung von kienöl (holzterpentinöl) aus den wurzelstöcken (stumpen, stubben usw.). farbe und lack 1919, s. 44, 52, 61, 68. besprechung amerikanischer patente. schilderung einer auf arbeitung der wurzelstöcke zu kolophonium und terpentinöl in oster reich-ungarn. beschreibung der stockholzextraktion mit unbeweglichem extraktionsgut und beweglicher extraktionsfläche in bosnien. ha. der ölgehalt der mandschurischen sojabohne. allg. drogisten ztg. 1919, nr. 4, s. 14. bei chemischen analysen der mandschurischen sojabohne sind niemals 20% öl festgestellt worden, aber olmühlen in dalny behaupten 20% gewonnen zu haben. daneben is t festgestellt worden, daß der olgehalt je nach der güte der ernte sehr verschieden ist. er schwankt, soweit mit sicherheft bekannt ist, zwischen 16,94 und 1822% öl. p . graebner, jun. engländer, p., ergebnis der verwendung inländischer leinölersätze º der lack-, firnisund kitterzeugung. ölu. fettindustrie i, 3 , s , 1–3. ein vollwertiger ersatz für leinöl is t bisher noch nicht gefunden worden. zur verfügung standen an rohstoffen nur cumaronharz und mineralöl in reichlicheren mengen. ha. fette. 262 literatur. engländer, p., etwas zur wirtschaftlichkeit der fetthefefabrikation. brennereiztg. 1919, nr. 1347. da der fettpilz nur auf der oberfläche gedeiht, sind zur wirt schaftlichen gewinnung des pilzes sehr flache schalen von großen ab messungen nötig. ha. fordyoe, l. und torrance, d. m., analyse von pflaumenkernen. chem. news 118, s. 242–243, cornell college. pflaumenkerne mit äther extrahiert liefern 42% öl. außerdem sind 2,47"o n und 37,42% zucker (fruktose und glukose, vielleicht auch rohrzucker) nachzuweisen. das öl besteht aus einem demkokosöl und einem dem kakaoöl ähnlichen ol. für das erstere geben die ver fasser an: e.– 5", d. 0,9055, v. z. 239,8, für das letztere: d. 0,9119, v. z. 207,4. beide öle sind nicht flüchtig. ha. griffiths-jones, e., agyptisches lattichöl. reports and notes of th e public health laboratories, cairo 1918, 1 ; analyst 44, s . 170. der olgehalt des von lactuca scariola oleifera stammenden samens beträgt 35,7% (niedrigster) bis 36,3% (höchster wert). das o l is t goldgelb, selbst bei 0 " klar bleibend und gehört zu den halbtrocknen den ölen. (vergl. ref. s . 203) ha. grün, ad., die fettchemie und fettindustrie in den jahren 1914 bis 1918. chem. ztg. xliii (1919), nr. 127, s. 717–724; nr. 130, s . 737–739. verf. stellt in einer literaturübersicht die über fettchemie und fettindustrie von 1914–1918 handelnden abhandlungen zusammen. einleitend wird ein wirtschaftlicher überblick gegeben. – in fort setzung seiner literaturübersicht über die fettchemie und fettindustrie von 1914–1918 bespricht verf, die untersuchungen über bestandteile der fette und wachse. ha. heuß, r., teeröl als brennstoff. zeitschr. d . bayr. revisionsvereins xxiii, s. 1 ; allgem. zeitschr. f. bierbrauerei u . malzfabr. xlvii, s . 99–100. .. knorr, fr., über die zusammensetzung einiger speisefette und speiseöle. seifensieder-ztg. und revue über die harz-, fettund olindustrie mit dem beiblatt der „chemisch-technische fabrikant“ xlvi (1919), nr. 24, s. 521–522. .verf. gibt in tabellenform die untersuchungsergebnisse an fetten und olen, die für genußzwecke bestimmt sind, an. die untersuchungen wurden schon vor dem kriege vorgenommen. ha. mineralölversorgungsgesellschaft, schmieröle aus urteer. tägl. ber. über d . petroleumind. 1919, nr. 26; braunkohle xviii, s. 82. moore, r . j. und egloff, g., fette und fettsäuren aus petroleum. chem. metallurg. engineering xviii (1918), s. 308–311. 0drich, w., 0lsaaten und ole in niederländisch-indien. seifenfabr. xxxix (1919), s. 125–126. rothéa, beitrag zum studium des traubenkernföles, des johannis beerkernöles, des tomatenkernöles, sowie der kuchen, die bei der herstellung hinterbleiben. bull. sciences pharmacol. xxvi, s . 105– 110. laboratoire d e l'inspection technique des substances angaben der bestandteile der einzelnen kernarten. ha. thurston, a., maisöl. middl. drugg. and pharm. rev. lii (1918), s . 155–156. ohio state university. beschreibung und herstellung des maisöles. ha. literatur. 263 thurston, a., sesamöl. middl: drugg. and pharm. rev. lii (1918), s. 252–255. ohio state university. beschreibung und herstellung des sesamöls. ha. thurston, a... sojabohnenöl. middl. drugg. and pharm. rev. lii (1918), s. 202–203. ohio state university. beschreibung und herstellung des sojabohnenöles. .. ha. weis, aug., die verarbeitung des maises auf keime und 0l. seifen sieder-ztg xlvi, s. 116, 138–139. verf. schildert die maisölfabrikation. ha. andés, l. e., die gewinnung von harz in mitteleuropa. seife iii, s. 684–687, 702–704. -übliche verfahren zur coniferenharzgewinnung in österreich ungarn. ha. andés, l. e., ein neues produkt aus dem neuseeländischen kauri harz. neueste erfindungen xlvi, s. 60–62 das durch kondensation des beim schmelzen des kauriharzes gewonnene flüchtige ol enthält ein nebenprodukt, das benzin und benzol ersetzen und als ol in der lackindustrie verwandt werden kann. ha. andés. l. e., uber geigenharz. neueste erfindungen xlvi, s. 198 bis 199. schilderung der herstellungsrezepte und aufzählung der im handel gebräuchlichen marken. ha. cavel, l ., über den antiseptischen wert einiger ätherischer öle. chem.-ztg. 1918, s . 453. goldschmidt, f . and weiß, g., deutsche harze und ihre eignung für die seifenfabrikation. seifenfabr. xxxix (1919), s , 69–73; ztschr. angew. chem. xxxii (1919), i, s. 33–36. gschwender, g., die rosenölerzeugung bulgariens. seifenfabr. xxxviii (1918), s. 213–214. hwr., die wichtigsten ausländischen gummikulturpflanzen und ihre beziehungen zur maschinenindustrie. der weltmarkt vii (1919), nr. 22, s . 432. es wird kurz auf die wichtigsten ausländischen gummikultur pflanzen hingewiesen, die für die deutsche gummiund maschinen industrie von bedeutung sind. als solche werden angeführt: der kautschukbaum, die gummiliane, der indische feigenbaum und der guttaperchabaum ha. h ., kirschgummi. reichs-gemüseu. obstmarkt iv (1919), nr. 117, s. 2. kleiner gemeinverständlicher aufsatz über auftreten, bestandteile, entstehung und verwertung des kirschgummis oder kirschharzes. ha. klimburg, h . v ., die harze. seife iii, s. 802–804. beschreibung der harze. ha. leiningen, graf zu, gewinnung von kolophonium und terpentinöl in deutschland. seife iii, s. 600–601, 619–621. ausführliche beschreibung der verfahren zur gewinnung der coniferenharze. ha. merz, j., verfahren zur unmittelbaren sonderung und rein darstellung der in harzhaltigen rohstoffen enthaltenen festen und öligen produkte. d . r . p . 302 442, kl. 22h vom 31. 8. 16 aus gegeben 20. 5 . 1919. ha. harze usw. 264 i. iteratur. kautschuk usw. farbstoffe. salvaterra, h., extraktionsharze aus fichtenscharrharz, i. mitt. chem-ztg. xliii (1919), nr. 130, s. 739. tschirch. a., entstehung der harze. chem.-zentralbl. ii , s . 673. phytosterinhyperbolie entsteht durch tiefgreifende verletzungen der pflanzen. aus den phytosterinen entstehen die resinole, die beim abbau die terpene ergeben. durch luftund lichteinwirkung bilden sich die resinotannole aus den resinolen. ha. walbaum, h., zur kenntnis des japanischen pfefferminzöls. journ, prakt. chem. xcvi (1918), s. 245–250. wallach, 0 . und mitarbeiter, zur kenntnis der terpene und der ätherischen 0le. lieb. ann. chem. bd. 418 (1919), s . 36–69. wilson, c . p . und young, c . 0., eine methode zur bestimmung des gehalts der schalen der agrumenfrüchte an flüchtigem 0l. journ. ind. eng. chem. ix (1917), s. 959–961. zur bestimmung des „lgehalts der schalen der agrumenfrüchte eignet sich am besten die destillation mit wasserdampf, wozu eigens seitens des verfassers eine flasche mit engem, tariertem hals her estellt wurde, die als auffanggefäß dient. ha.wj, f. e., die kristallisation des menthols. journ. amer. chem. soc. xxxix (1917), s. 1515. boutaric, j., zusammenfassung einer studie über verschiedene madagaskar-kautschuksorten und ihre mischung mit guayule und balata. cautchouc e t guttapercha xvi, s. 9893–9900. hillen, g . h., arbeiten über kautschuk und guttapercha. ztschr. für angewandte chemie xxxii. aufsatzteil i (1919), s. 301–304. nr. 78, s . 309–312. verf. setzt seine auf seite 279 begonnene literaturübersicht über kautschuk und guttapercha fort und führt die literatur über vulkan sation, regeneration und fabrikation von kautschukund guttapercha waren und analytik an. ha. vries, 0 . de, bereiding e n eigenschappen van plantagerubber. uit gave van d e vereenigung „centraal rubberstation“, 1919. cross, c . f ., greenwood, c . v . und lamb, m . c., kolloidale gerb stoffverbindungen und deren anwendung. journ. soc. dyers colourists xxxv, s. 62–68, london, technical college of leather sellers company. farbwerke vorm. meister lucius und brüning, höchst a. m . wer fahren zur darstellung von leukoverbindungen der indigogelb reihe. d . r . p . 312601, kl. 12p, vom 30. 12. 1915, ausgegeben 28 ). 1919. feigl, fr., studien über die anfärbbarkeit anorganischer körper. quantitativer teil gemeinsam mit brauch, a . österr. chem. ztg. xxii, s. 36–37, 42–44. wien. jalade, e., einige gerberinden aus französisch-guyana: deren verwendbarkeit in der gerberei. bull. sciences pharmacol. xxvi. s . 115–124. keine dieser rinden besitzt einen für die herstellung von gerº stoffauszügen oder eine sonstige wirtschaftliche ausbeutung" und fü r eine einfuhr nach europa genügenden gehalt a n gerbstoff. ha. literatur. 265 kryz, f .. uber das verhalten des farbstoffes der beeren des wilden efeus gegen reagenzien. osterreich. chem.-ztg. 1919, s . 92. wahrscheinlich gehört der farbstoff der beeren des wilden efeus (hedera helix) z u den anthocyanfarbstoffen und zwar in dieselbe gruppe wie der rotweinfarbstoff. ha. levinstein, h., die wichtigkeit der englischen farbstoffindustrie für den staat. vortrag, gehalten in der jahresversammlung der society o f chemical industry in london 17. 7 . 1919. ztschr. f. angew. chemie xxxii (1919), nr. 63, wirtschaftl. teil ii , s . 509. verf. schildert die lage wie folgt: 1. e s wäre nach abschluß d e s krieges ein wahnsinn uns nicht wirtschaftlich von deutschland unabhängig z u machen. 2 . wahnsinn wäre e s, deutschland im alleinigen besitz von fabriken zu lassen, die diesem ermöglichten, diesen krieg 3 jahre und länger zu führen. 3 . e s is t unmöglich, unsere handels vormachtstellung z u behaupten, wenn wir die einzige chance, das er findungstalent unserer chemiker zu organisieren, fortwerfen. ha. 0eman, e., verfahren zur herstellung von azofarbstoffen. d . r . p . 312495, kl. 22a vom 14. 12. 1916, ausgegeben 26. 5. 1919. driesen, p . a., etwas über die einwirkung von kupfersalzen, die der faser entwickelt werden. chem. weekblad xvi, s. 628–632. eiden. heyking, wann und wie sollen wir unser rohr ernten? il l. landw. ztg. xxxix (1919), nr. 6970. s. 347–348 mit einer abbildung. nachdem über die ernte und verwertung des rohres in schweden berichtet ist, werden angaben über zweckmäßige aberntung des rohres in deutschland gemacht. my. loewenthal, r., neuerungen in der chemischen technologie der spinnfasern. chem.-ztg xliii (1919), nr. 129, s. 729–731. in seiner abhandlung über die neuerungen in der chemischen technologie der spinnfasern führt verfasser auch die literatur über färberei, echtheit der färbungen und theorie des färbeprozesses an. ha. marquart-landsberg, die bedeutung des hanfes für deutschland. mitteilungen der landesstelle für spinnpflanzen nr. 10, s . 74–76. e s handelt sich um eine große zahl von einund ausfuhrtabellen. p . g. jun. marquart, benno, die erträge des hanfes. mitteilungen der landes stelle für spinnpflanzen 1919, 7 , n . 48–49. in den ländern, in denen der hanfbau schon seit vielen jahren betrieben wird, sind seine erträge natürlich auch am größten. s o sind in italien ernten von 13–15 dz rohfaser pro hektar keine seltenheit. im durchschnitt wurden in den jahren 1912–13 9–11 dz geerntet. in ungarn betrug der durchschnitt nur 8–10 dz rohfasern, während e r in kroatien und slovenien mit 6 d z und in rußland sogar mit 5 d z weit zurückbleibt. trotzdem in deutschland der flachsanbau vor dem kriege fast vollständig vergessen war, konnten doch schon ernten erzielt werden, die sich den italienischen gleichwertig zur seite stellen können. p . g. jun. schmidt, 0 ., die stellung der spinnpflanzen im landwirtschafts ºbe. mitteil. d . landesstelle f. spinnpflanzen 1919, nr. 7 , s . 4 9 is 51. angewandte botanik i. 18 fasern. 266 literatur. hölzer. pflanzenbau. neger, f. w., die nadelhölzer (koniferen) und übrigen gymno spermen. sammlung göschen nr. 355, 2. verbesserte auflage. g. j. göschensche verlagshandlung, berlin und leipzig, 1919. 156 seiten mit 81 textabbildungen, 5 tabellen und 4 karten. allendorf und ehrenberg, die aufgaben des sonderausschusses für zuckerrübenbau. mitteilungen der d. l. g. xxxiv (1919), stück 40 . programmatische aufzählung der zur förderung des zuckerrüben baus z u unternehmenden versuche und untersuchungen. rabanus. allendorf und ehrenberg, das beizen des saatgutes nach den neuesten erfahrungen. flugblatt nr. 17 der versuchsstation fü r pflanzenkrankheiten der landwirtschaftskammer für die provinz sachsen. boshart, k., der anbau des baldrians. heilund gewürzpflanzen ii i (1919), heft 3 , s . 57–66. böttner, j., gartenbuch für anfänger. unterweisung im anlegen, bepflanzen, pflegen des hausgartens, im obstbau, gemüsebau und in der blumenzucht. 13. auflage. trowitzsch & sohn, frankfurt an der oder, 1919. 572 seiten mit 627 abbildungen. filter, p., die herkunftsermittelung der leinsaaten des handels. die landw. versuchsstationen. xciii (1919), heft v/vi, s. 221–246 enthält die feststellung des für die einzelnen provenienzen (argentinien, nordamerika, ostindien, nordund südrußland, türkei, persien, marokko, china, japan) charakteristischen unkrautbesatzes, fettand wassergehaltes, tausendkorngewichts und der reinheit. r . fischer, h., die kohlenstoffernährung der kulturpflanzen. garten flora (deutsche gartenbau-gesellschaft) lxviii (1919), s. 165–168 (vergl. auch diese zeitschrift s . 138ff.). harreveld, j. van, statistiek van de verbreiding en de productie der rietsoorten in oogst 1916. meded. v. het proefstat. voor de java-suikerindustrie. landbouwkundige serie 1919, nr. 4 . in tabellenform gehaltener bericht über die verbreitung und produktion von rohrzuckersorten auf java im herbst 1919. . my. heinrich, m., beiträge über die keimung bespelzter und nackter timothyfrüchte. die landw. versuchsstationen xciii (1919), heft v/vi, s. 259–276. herpers, h., zum anbau des wintergemüses. land und frau ii i (1919), nr. 41. beiblatt zur deutschen landw. presse. hiltner, kartoffelernteschätzungen. praktische blätter für pflanzen bauund pflanzenschutz xvii (1919), heft 78, s. 87–96. koerner, w. f., feldmäßiger buschbohnenanbau zur samen gewinnung. ääääg presse xlvi (1919), nr. 80.lang, h., kann man die saat gegen vogelund mäusefraß schützen württemb. wochenbl. f. landwirtschaft 1919, nr. 39. die behandlung mit corbin wird empfohlen. r . reckert, j. , winterhafer. deutsche landw. presse xlvi (1919, nr. 72, s . 543–544. ergebnis der letztjährigen aussaat sowie allgemeine angaben über bodenbearbeitung, düngung und aussaatmenge von winterhafer. m y rippel, a., die wachstumskurve der pflanzen und ihre mathe matische behandlung durch robertson und mitscherlich. fühlings landw. zeitung lxviii (1919), heft 11/12, s. 201–214. ? literatur. 267 siebert, a., kürbis-anbau und -verwertung. land und frau iii (1919), nr. 41. (beiblatt zur deutschen landw. presse.) störmer, uber die ernte der lupinen. ill. landw. ztg. xxxix (1919), nr. 7576, s. 381–382. verschiedene erntemethoden für die lupine und ihrwert. 1. das ernten in windhaufen, 2. das ernten in puppen nach kühn, 3. das reutern, 4. das ernten mit dem bindemäher. (vgl. ref. s. 212.) my. u. rigolen und rodenverbesserungsmittel. der kleingarten vä9) heft 10, s. 153–156, mit zwei abbildungen. ausführliche beschreibung des rigolens und gleichzeitigen ein bringens von bodenverbesserungsmitteln wie asche und torfmehl und besprechung ihres wertes. my. wangenheim, f., über die ernte der lupinen. ill. landw. ztg. xxxix (1919), nr. 7778, s. 395. aus der praxis gewonnene erfahrungen. my. werner, h., der kartoffelbau nach seinem jetzigen rationellen stand punkte (thaer-bibliothek). achte, neubearbeitete auflage von prof. dr. c. v. eckenbrecher. " parey, berlin, 1919. 190 seiten mit 29 textabbildungen. works polaks frutal, pfefferminzkultur in holland. deutsche parfümerie-ztg. v, s. 32. – c. 1919, ii, s. 380. zschokke, heranzucht von veredelungsunterlagen im inlande. schweiz. zeitschr. f. obstund weinbau xxviii (1919), nr. 23. anleitung zur zucht von veredelungsunterlagen aus samen. r . behr, m., neue erdbeersorten. blätter für die deutsche hausfrau (wochenbeilage zur ill. landw. ztg.) nr. 36 (1919), s. 101, mit 2abb. aufzählung neuer, wenig bekannter erdbeersorten und erörterung ihrer vorzüge. .. my. 0berstein, otto, über das vorkommen echter knospenvariationen bei pommerschen und anderen kartoffelsorten. deutsche landw. presse xlvi (1919), nr. 74. bail, ungeziefervertilgung mittels blausäuregas. gesundheitsing. xlii (1919), s. 33. baunacke, w., wühlmausbekämpfung. mitteil. d. d . l . g . xxxiv (1919), stück 43, s . 561–562. empfohlen wird die massenbekämpfung mit den üblichenma bethel, e., puccinia subnitens and it s aecial hosts. phytopathology vii (1919), nr. 2. briosi, g., sopra una nuova mallattia dei bambu. atti r . acc. lincei xxv (1916). brucker, von der bekämpfung des amerikanischen stachelbeer mehltaues. badisches landw. wochenblatt 1919, nr. 34. chivers, a . h., the injurious effects o f tarvia fumes on the vege tation. phytopathology vii (1917), nr. 1. dern, der stand der reblausbekämpfung in franken. vortrag, ge halten im fränkischen weinbau-verein. weinbau und weinhandel xxxvii (1919), nr. 34 u. 36. pflanzenzucht. pflanzen krankheiten. 18* 268 literatur. duysen, f., uber den roggenstengelbrand (urocystis occulta). mitt. der d. l. g. xxxiv (1919), stüsk 44, s. 569–570. beschreibung des schädlings und angabe von bekämpfungs maßnahmen (beizen). r. duysen, f., einiges über das vorkommen von botrytis cinerea auf raps. mitteil. der deutschen landw. gesellsch. 1919, nr. 34. ehrenberg, paul, vorschläge für die wirksame bekämpfung de s steinbrandes beim winterweizen auf kleinen besitzungen zur aussaat im herbst 1919. hannoversche landund forstwirtschaft liche zeitung lxxii (1919), nr. 31 u. 32. florin, r., 0m äppleträdens skorvsjuka och dess bekämpande. sveriges. pomol. fören. arsskr. 1918. führer, bekämpfung des unkrautes. mein sonntagsblatt 1919, s. 53 . neben vorbeugungsmitteln werden direkte bekämpfungsmittel angeführt. spezielle angaben finden sich über hedrich, ackersenf, raute, kresse, disteln, löwenzahn, wegwart und quecke. ha. gaul, kupfervitriol als saatgutbeizmittel. deutsche landw. presse xlvi (1919), nr. 83, s. 628. verf. empfiehlt rückkehr zur alten knpfervitriolbeize, da nach den erfahrungen in seinem dienstbereiche uspulun und weizenfusario versagt haben. gescher, cl., die feinde des sauerwurms. weinbau u . weinhandel xxxvii (1919), nr. 39. güssow, h . t., the pathogenic action o f rhizoctonia o n potato. phytopathology vii (1917), nr. 3. hall, c. j. j. van, ziekten en plagen der cultuurgewassen in nederlandsch-indie, in 1918. mededeel. 3 6 van het laboratorium voor plantenziekten te buitenzorg. der durch die im jahre 1918 aufgetretenen krankheiten ver ursachte schaden war geringer als in den vorjahren. e s folgt eine aufzählung der aufgetretenen krankheiten. ha. hasse, m., der gummifluß der steinobstbäume. erfurter führer in obstu . gartenbau, s . 34. higgins, b . b., a disease of pecan catcins. phytopathology vi i (1917), nr. 1. jegen, g., die frostspannerbekämpfung. schweiz. zeitschr. für obst u . weinbau xxviii (1919), nr. 22, s. 361–363. v. schilderung der lebensgeschichte des schädlings und der b e kämpfung mit klebringen. r . jegen, g., die schädlingsbekämpfung im winter. schweiz. zeitschr. f. obst u . weinbau xxviii (1919), nr. 23. anleitung zur sachgemäßen winterbekämpfung der obstbaum schädlinge. r. koerner, willi f ., die moniliakrankheit der kirschbäume. lan und frau iii (1919), nr. 43. beiblatt der deutschen landw. presse geschichte des schädlings und bekämpfungsmaßnahmen. r . lek, van der, h . a . a., 0ver de z. g . „verwelkingsziekten“ in he bizonder die, welke door verticillum alboatrum veeroorzaak worden. tijdschr. over plantenziekten xxv (1919), s. 20–52,2 taf die krankheitszeichen der „verwelkungskrankheiten“ (verticill osen), sind nur wenig feststehend. e s werden die verticilliosen be tomaten, gurken, melonen und kartoffeln angeführt. die bekämpfung is t auf die anzucht widerstandsfähiger sorten beschränkt. ha. literatur. 269 lindner, h., zur verhütung der schwarzbeinigkeit junger kohl pflanzen. der praktische ratgeber im obstund gartenbau 1919, nr. 13, s. 100. die ursache der krankheit sucht verf. in der zu dichten saat, in zu reichlicher bewässerung und in der schlechten durchlüftung. zu warm gehaltene saatbeete ergeben dünnbeinige und wenig widerstands fähige pflanzen. ha. mc cubbin, w. a., does cronartium viticola winter on the currant? phytopathology vii (1917), nr. 1. mc cubbin, w. a., contributions to our knowledge of the white pine blister-rust. phytopathology vii (1917), nr. 2. müller, h. c. und molz, e., kupfervitriol als saatgutbeizmittel. deutsche landw. presse xlvi (1919), nr. 78, s. 590. vor der anwendung des cuso als beizmittel gegen steinbrand wird gewarnt. r. pape, h., brennesselschädlinge. deutsche landw. presse xlvi (1919), nr. 70, s. 528–529, mit 7 abbildungen. reckendorfer, f., der rotbrenner. allgem. weinzeitung xxxvi (1919), nr. 36. enthält die lebensgeschichte, den schaden und die bekämpfungs weise der krankheit. r. reiling, h., zur frage derwundkorkbildung der kartoffelknollen. fühlings landw. zeitung lxviii (1919), heft 9/10, s. 190. roark, r. c., als insektenvertilger verwandte pflanzen. americ. journ. pharm. xci, s. 25–37, jan.; s. 91–107, febr. insecticide and fungicide laboratory miscellaneous division bureau of chemistry dep. of agriculture. washington. verf. gibt eine übersicht über die pflanzen, deren giftige wirkung auf insekten bisher bekannt ist. ha. s ., bekämpfung der obstbaumschädlinge. schweiz. zeitschrift für obstu . weinbau xxviii (1919), nr. 22, s 369–371. die obstbäume sollen im herbste abgekratzt und mit einem kalk anstrich versehen werden. r. schädlinge der obstbäume und deren bekämpfung. der badische obstzüchter xiv (1919), nr. 9 u. 10. schöppach, das vermehrte auftreten des steinbrandes. deutsche landw. presse xlvi (1919), nr. 77. verlangt mehr aufklärung bei den landwirten über die gefahr des brandes und seine bekämpfungsmaßnahmen. r. schoevers, t . a . c., nieuwe ziekten, waarop gelet moet warden. tijdschr. over plantenziekten xxv (1919), s. 95–98. verf. beschreibt eine neue wurzelerkrankung der spinatpflanzen, die an wurzelbrand erinnert. ihre ursache ist noch nicht bekannt. ha. seelhorst, c . v ., die zwergmaus. illustr. landw. ztg. xxxix (1919), nr. 6768, s . 337–338. in der gegend von göttingen und minden trat dieser schädiger des getreides (abfressen der ähren) reichlich auf. verf. läßt eine be schreibung des tieres folgen. ha.ä c. d., buckeye-rot of tomato fruit. phytopathology vii917), nr. 2. smith, ci. 0., sour rot o f lemon in california. phytopathology vii (1917), nr. 1. 270 literatur. b0den. gärung. snell, karl, kindelbildung im innern einer knolle. leutsche landw. presse xlvi (1919), nr. 86, s. 654. stanford, e. e. andwolf, f. h., studies on bacterium solanaceanum. phytopathology (vii) (1917), nr. 3. stevens, n. e. and hawkins, l. a., some changes produced in strawberryfruits by rhizopus nigricans. phytopathology vii (1917), nr. 3. stift, feinde und krankheiten der zuckerrübe. blätt. rübenb. xxvi (1919), s. 75. (vgl. s. 128 oben.) verhoeven, w. b. l., zaai graanontsmetting. tijdschr. over planten ziekten 1919, xxv. beiblatt, s. 5–10. beschreibung der üblichen beizverfahren, die gegen die ver schiedenen brandkrankheiten und gegen den keimschimmel bei weizen, hafer, gerste und roggen anwendung finden. ha. voges, e., das diesjährige verhalten der schädlinge. deutsche landw. presse xlvi (1919), nr. 73. weier, j. r., sparassis radicata, an undescribed fungus on the roots conifers. phytopathology vii (1917), nr. 3. welten, h., pflanzenkrankheiten. leipzig, ph. reclam, 76 text abbildungen, 2 bunte und 3 schwarze tafeln. werth, das mutterkorn des getreides und anderer gräser. deutsche landw. presse 1919, s. 53 mit einer farbigen kunstdruckbeilage. beschreibung der krankheit und besprechung der für den schäd ling in betracht kommenden bekämpfungsmittel. ha. zimmermann, h., rübenschäden. illustr. landwirtschaftl. zeitung xxxix (1919), 61/62, s. 298–299. kurze mitteilungen über die in mecklenburg beobachteten rüben schäden, ihre bekämpfung, und über die entwicklung der rübenbestände. ha. barthel, chr., beitrag zur frage der nitrifikation des stallmist stickstoffes in der ackererde. centralblatt für bakteriologie usw. abt. ii , xlix (1919), s. 382–392. heinrich, r . und nolte, 0., dünger und düngen. anleitung zur praktischen verwendung von stallund kunstdünger. 7 . auflage. paul parey, berlin, 1918. my lemmermann, 0 . und einecke, a., über den stickstoffhaushalt der böden und die wirkung von stroh und zucker. die landw. wer suchsstationen xciii (1919), heft vvi, s. 209–220. mitscherlich, e . a., saucken, s. v ., iffland, f. , vegetationsversuche mit verschiedenen kalidüngesalzen und zur phosphorsäure-kalk düngung. landw. jahrbücher liii (1919), heft 4, s. 501–514 mit 4 tafeln. my. pfeiffer, th. und rippel, a., über den einfluß der steine im boden auf das wachstum der pflanzen. ii. die landw. versuchsstationen xciii (1919), heft v/vi, s. 277–284. barthel, chr. und sandberg, e ., weitere versuche über das kasein spaltende vermögen von zur gruppe streptococcus lactis gehören den milchsäurebakterien. centralblatt f. bakteriologie usw. ii . abt.: xlix (1919), s. 392–412. osterwalder, a., die selbstheranzucht von reinhefe. schweizerische zeitschr. für obstund weinbau xxviii (1919), nr. 18 , s . 297–300. literatur. 271 grevillius, a. y., zur mikroskopie des schilfmehls. (arundo phrag mites l.) die landw. versuchsstationen xciii (1919), heft v/vi, s. 195–208 mit 1 tafel. hessdörfer, praktisches taschenbuch für gartenfreunde. 4. auflage, parey, berlin, 1918. lange, w., webers illustrierte gartenbibliothek. band i, garten gestaltung der neuzeit von willy lange und otto stahn. 4. auflage. j. j. weber, leipzig, 1919. 463 seiten mit 309 ab bildungen und 16 bunten tafeln. mededeelingen van het deli proefstation te medan-sumatra. ii., serie nr. 5. verslag over 1. juli 1918 bis 30. juni 1919. personalnachrichten. dr. friedr. tobler, bisher außerordentlicher professor an der universität münster i. w., wurde vom kuratorium des forschungs institutes sorau zum direktor dieser anstalt gewählt und hat diewahl angenommen als nachfolger des nach dresden übersiedelten prof. dr. a.herzog. oberforstmeister riebel, langjähriger mitschriftleiter der zeit schrift für forstund jagdwesen und direktor der forstakademie hann.-münden, gestorben. max heßdörfer, herausgeber der gartenwelt, starb in strauß berg am 7. januar 1920. (* 10. dezember 1863 in fulda.) nachruf von siebert in gartenwelt 1920, nr. 5. b dr. s. h. koorders am 15. november 1919 in buitenzorg ge st0rben. geh. oberregierungsrat prof. dr. j. behrens, direktor der bio logischen reichsanstalt für landund forstwirtschaft in berlin-dahlem trat in den ruhestand. geh. regierungsrat prof. dr. o. appel an der biologischen reichsanstalt in berlin-dahlem wurde zum direktor dieser anstalt etmännt. techn. mikr. verschiedenes. uc1.b3299303_page_277_cut uc1.b3299303_page_278_cut uc1.b3299303_page_279_cut uc1.b3299303_page_280_cut section 9 uc1.b3299303_page_281_cut uc1.b3299303_page_282_cut uc1.b3299303_page_283_cut uc1.b3299303_page_284_cut uc1.b3299303_page_285_cut uc1.b3299303_page_286_cut uc1.b3299303_page_287_cut uc1.b3299303_page_288_cut uc1.b3299303_page_289_cut journal of applied botany and food quality 93, 11 19 (2020), doi:10.5073/jabfq.2020.093.002 1department of environmental sciences, the university of lahore-lahore, pakistan 2department of agronomy, university of agriculture, faisalabad, pakistan 3nuclear institute for agriculture & biology, faisalabad, pakistan 4soil and water testing laboratory for research, bahawalpur, pakistan 5institute of soil and environmental sciences, university of agriculture, faisalabad, pakistan 6sub-campus depalpur, university of agriculture faisalabad, okara, pakistan zinc nutrition application augments morpho-physiological attributes, productivity and grain zinc bioavailability of paddy rice qamar uz zaman*1,2, zubair aslam*,2, muhammad rashid3, ambreen aslam1, nusrat ehsan1, umair riaz4, rana nadeem abbas2, asif iqbal2, muhammad shahbaz naeem2, safdar bashir5, 6, zulfiqar ahmad saqib5, muhammad yaseen5 (submitted: may 13, 2019; accepted: december 3, 2019) * corresponding author summary zinc (zn) deficiency is the most important micronutrient disorders affecting plants and human health. present study evaluated the potential of various zn application methods in improving the perfor mance of selected rice genotypes and zn bioavailability in grains. pre-selected zn application methods through pot experiments were validated in the field. harvested grains were fed to albino rats for zn bioavailability. results revealed that soil + foliar application of zn was effective in improving the seedling growth of rice genotypes by modulating the agronomic, water related and biochemical attributes. the rats gained more body weight fed with rice genotype accession-164 (high zn accumulator) compared with the minimum for super basmati (low zn accumulator) feed. in crux, soil application of zn at 15 kg ha-1 followed by foliar application of 0.25% znso4.7h2o solution at tillering and heading stages produced the highest grain yield (26.25%, 29.11%) with maximum bioavailable zn (21.02%, 22.50%) during both years, respectively, in the grains for combating malnutrition in the tested rats. keywords: application methods, genotypes, growth, micronutrient, rice, zinc. introduction human beings need at least 22 mineral elements for fitness and proper growth (welch and graham, 2004). nutrient demand of human body is met through balanced food. recent studies indicate that of the total global population, about 60, 30 and 20% of people are deficient in iron, iodine and zinc, respectively (graham et al., 2007). apart from unbalanced food intakes, such minerals deficiencies also owe substantially to growing food crops in soils, which are extremely low in phyto-availability of minerals (graham et al., 2007). zn deficiency is common in soils of various countries including pakistan (kumssa et al., 2015). cereals are more prone to the zn deficiency as compared with legumes, and often result in reduction of yield and nutritional quality of the former (kumssa et al., 2015). malnutrition in human beings regarding zn can be rectified through nutritional diversification, food fortification by zn, or by escalating zn concentrations in staple crops (biofortification). biofortification of cereal crops using the inorganic fertilizers and cultivation of such verities having greater capability of absorbing nutrients is the best approach for both improving the yield and nutritional value of staple crops. out of different practical approached, agronomic biofortification through the foliar application is economically sustainable and practically adoptable solution to overcome the zn deficiency issue in rice (zaman et al., 2017). zinc is the major micronutrient limiting rice growth and yield in many of the rice growing areas in pakistan and elsewhere (quijanoguerta et al., 2002). reduced tillering, increased spikelet sterility and extension in the maturity period of the rice crop are major symptoms of zn deficiency and playing major role in yield and quality reduction (lopez-millan et al., 2005). zn is often applied for improving zn availability in the soil and its bioavailability for plants. zinc can be applied via the soil, seeds, leaves and roots (esfandiari et al., 2016; rehman et al., 2017). the efficiency of zn can be evaluated in terms of the ratio of shoot dry matter or grain yield produced between the treatments with or without zn application. in the sub soils, the zn deficiency problem can be solved by the foliar spray (hussain et al., 2013). generally, the zn applied through the foliar and soil application improved the yield and quality status of rice using znso4 (zaman et al., 2018) or by using the seed treatment and foliar application of zn (farooq et al., 2018). proper availability at the peak periods of crop demand and the costs involved in zn fertili zation are crucial for harvesting good rice yields and making final recommendations regarding the method of its application under field conditions. keeping in view the importance of zn for plant growth, the severity of its deficiency in soils and plants, and the significance of its bioavailability, an attempt has been made to optimize the most suitable method of zn application in rice under field conditions. a field appraisal of selected zn application methods was carried out for enhancing productivity, quality, and zn biofortification of rice grains. such zn-biofortified rice grains were also assessed for zn bioavailability in albino rats. materials and methods to evaluate the performance of different zn application methods for rice crop, the experiments were conducted at glass-house and field conditions and later on tested for zn bioavailability in albino rats. a − pot experiment study site and experimental design a glass-house experiment was conducted at nuclear institute for agriculture and biology, faisalabad, pakistan in 2014. experiment was designed in a completely randomized design with two factors (5 rice genotypes and 15 zn application methods) and each treatment was replicated thrice. experimental treatments: five zn efficient basmati genotypes of rice viz., super basmati, accession-126, accession-154, acces12 q. zaman, z. aslam, m. rashid, a. aslam, n. ehsan, u. riaz, r.n. abbas, a. iqbal, m.s. naeem, s. bashir, z.a. saqib, m. yaseen sion-164 and accession-175 selected from a screening experiment containing 183 accessions/genotypes on the basis of grain zn contents by following the protocols jones and case (1990) and on yield potential (data not shown) was used in this study. seeds were obtained from the plant genetic resource institute (pgri), national agricultural research centre (narc), islamabad, pakistan. the experiment comprised of 15 treatments for zn application using znso4.7h2o as a zn source: control (nil zn), seed priming (0.5% solution for 10 h), soil application (15 kg ha-1 at the time of sowing), foliar application (0.25% solution at 30 das), root dipping (0.5% solution for five minutes at the time of transplanting), seed priming + soil application, seed priming + foliar application, seed priming + root dipping, soil application + foliar application, soil application + root dipping, foliar application + root dipping, seed priming + soil application + foliar application, seed priming + soil application + root dipping, soil application + foliar application + root dipping, and seed priming+ soil application + foliar application + root dipping. crop husbandry: ten seeds of each genotype (with around 12% moisture content) were sown in each earthen pot (45 cm depth × 30 cm diameter) filled with 10 kg soil. for priming, 50 g seeds were soaked in well aerated znso4.7h2o solution for 10 h at 25 ± 2 °c, and dried back to their initial weight. aquarium pump was used for aeration purpose. for seedling root dipping, seedlings raised in a nursery were uprooted 15 days after sowing (das) and dipped in the znso4.7h2o solution for 5 minutes, and were planted in the pots. foliar application was done 30 das. for soil application, all the zn was applied in pots at the time of sowing. basal doses of urea, di-ammonium phosphate and potassium sulphate were applied at the rates of 24 (n), 40 (p), and 62 (k) gram per pot, respectively. the plants were harvested at 45 das for subsequent determinations. data collection: three randomly selected plants were tagged for measuring plant height. at harvesting, the tagged plants from each pot were counted for number of tillers. for chlorophyll contents, fresh leaves were cut into 0.5 cm segments and extracted overnight with 80% acetone at -10 °c after 30 das. the extract was centrifuged at 14,000 rpm for 5 min at 25 °c and absorbance of supernatant was read at 645 and 663 nm using a spectrophotometer (t60 u spectrophotometer pg instruments, limited, usa). the readings were taken according to nagata and yamashita (1992). for relative leaf water content (rwc), fresh leaves (0.5 g) were weighed and were floated on water for 4 h, and saturated weight (ws) was measured there after. these leaves were oven-dried for 24 h at 85 °c to determine dry weight (wd) and readings were taken by following the method of barrs and weatherly (1962). to determine membrane permeability, leaf electrolyte leakage was measured following the protocol of blum and ebercon (1981). six leaf segments of similar size were lightly washed with distilled water and immersed in a test tube having 6 ml distilled water for 12 h at room temperature. then electrical conductivity (ec1) of solution was measured with a conductivity meter (model dds-11a, shanghai leici instrument inc., shanghai, china). samples were then heated in boiling water for 20 min and cooled to room temperature. the conductivity of killed tissues (ec2) was again measured. electrolyte leakage was measured as the ratio of ec1 to ec2 and expressed in percentage. for shoot zn concentration, a diacid mixture (hno3:hclo4 ratio of 2:1) method was used by following the protocols of jones and case (1990). b − field experiments study site and experimental design the field experiments were conducted at research farm of nuclear institute for agriculture & biology (niab), faisalabad, pakistan during kharif, 2014 and 2015. the experiments were laid out in a randomized complete block design with factorial arrangement of treatments and were replicated thrice having net plot size 2.0 m × 6.0 m. experimental treatments: the same set of rice genotypes was used as in the pot experiments. there were four zn (znso4.7h2o) treatments including control (non-zn application), soil application (sa) at 15 kg ha-1, foliar application (fa) at 0.25% (at tillering and heading) and soil application followed by two foliar applications as in previous treatments. crop husbandry: rice seeds of selected genotypes were sown on 20th of june 2014 and 25th june of 2015 using hand drill in a nursery. seedlings (30 day-old) were manually transplanted in puddled fields by maintaining r × r and p × p distance of 22.5 cm. at transplantation, uniform application of fertilizer in the form of 100 n, 67 p and 60 k kg ha-1 was done by applying urea, di-ammonium phosphate and potassium sulphate, respectively. at 25 days after transplantation, second dose of n at 60 kg ha-1 was applied. all the zn by soil application method was applied at the time of sowing while foliar application of zn was performed at tillering and heading stages in both years. to maintain the submerged soil conditions during the crop growth canal water was used. irrigation water was maintained at a depth of 3-4 cm at transplantation, and one week afterwards at a depth of 5-6 cm throughout the growing season till one week before harvesting. for weed control, mixture of ethoxy sulphuran and phenoxyprop-p-ethyle at 200 g and 370 ml ha-1, respectively was applied at specific intervals. carbofuran was broadcasted at 25 kg ha-1 to shield the crop from the insects. crop was harvested manually on 15th and 21st november, during 2014 and 2015, respectively. each plot was harvested and threshed separately. data collection: to determine plant water relations, three penultimate leaves from five selected rice plants of each treatment were harvested at tillering (75 days after sowing). pressure chamber was used to determine the leaf water potential. after the determination of water potential, leaf tissues were frozen for 48 h and thawed. the sap was extracted by centrifuging at 5000 × g and osmotic potential was determined (digital osmometer, wescor, logan, ut, usa). chlorophyll contents, relative water contents, and electrolyte leakage were determined as described in the pot experiment by getting the samples from the selected plants. net assimilation rate (nar) was estimated following hunt (1978). stomatal conductance (gs), photosynthetic rate (a), and transpiration rate (e) of 3rd leaf from top of each selected plant were recorded by using a photosynthetic system (lca-4; analytical development company, hoddesdon, england). all the gas exchange attributes were recorded at the tillering stage between 10:00 am to 01:00 pm. during data recording, leaf chamber molar gas flow rate was 248 μmol s-1, ambient co2 conc. (cref) was 352 μmol mol-1, temperature of leaf chamber (tch) varied from 36.1 to 40.4 °c, ambient pressure (p) 98.01 kpa, molar flow of air/leaf area 221.06 mol m-2 s-1. par was maximum up to 1050 μmol m-2 s-1 and leaf chamber volume gas flow rate (v) was 380 ml min-1. a meter rod was used to measure plant height at maturity starting from the base of plant up to the tip of flag leaf. unit area was selected randomly from each plot for counting the number of tillers. panicle length was measured by measuring tape from 20 randomly selected panicles from each plot. after harvesting and threshing of crop, five random samples were taken from each treatment for 1000-kernel weight on electronic balance. each plot was harvested and threshed separately and clean paddy rice was air-dried. yield of each plot was weighed and values were adjusted to 12% moisture and expressed in t ha-1. micro-jheldahl digestion and ammonia distillation process was used to determine the protein contents of the kernels which were then converted to protein contents by multiplying with the factor 5.95. milled rice kernels were grinded in a grinding mill for determination of ker zinc bioavailability in rice 13 nel amylase contents (juliano, 1971). kernel dimension (length) was taken on 100 normal kernels from each treatment with the help of a digital vernier caliper. c − animal feeding experiment study site and experimental design this experiment was carried out at institute of pharmacy, physiology and pharmacology, university of agriculture, faisalabad, pakistan during winter, 2014 and 2015 and was laid out in completely randomized design (crd). experimental treatments: zinc biofortified rice kernels of five selected genotypes (super basmati, accession-154, accession-175, accession-164 and accession-126) were produced as described above in both years in field experiments. after harvesting, kernels of all five genotypes of best treatment (best one from the four zn treatments “soil + foliar application of znso4” for rat experiment) was analyzed and grouped into five different categories of grains on the basis of having different levels of zn (i.e. 27, 45, 50, 55 and 59 mg kg-1 dry weight, respectively. kernels of rice genotype g1 (super basmati) were considered as control. experimental conditions for animals: forty male wister strain albino rats having initial body weight of 100 ± 10 g were purchased from the same department. the rats were acclimated for five days prior to experiment with an evaluation of their health status in both years. animals were kept in metallic cages and maintained in a controlled room 22 ± 2 °c with relative humidity of air 55 ± 5%, and having the optimum lightening in a daily cycle, (i.e. 12 h light: dark periods). the study was reviewed and approved by an animal care and use committee prior to the commencement of the research and the experiment was performed according to the rules and regulation protocols accepted by local ethical commission for investigation on animals. rats were maintained in collective cages (4 rats per cage) as treatment plan. rat feed was made by following ain standards and during the whole period (one month) of experiment, all rats were supplied with deionized water to eliminate additional zn sources. after one month feeding of zn biofortified kernels to male albino rats, their body weight was recorded in both years. statistical analysis the collected data from each experiment were analyzed using statistics 8.1 software. highest significant difference (hsd) test at 5% probability level was used to compare the treatment means. correlation coefficients among different traits were calculated using a computer assisted program minitab 14. results pot experiment all zn application methods significantly improved plant water-relations, and agronomic as well as biochemical attributes of rice genotypes, compared with control at 45 das (tab. 1). soil + foliar application of znso4.7h2o at 15 kg ha-1 and 0.25% solution, respectively tab. 1: agronomic, water and biochemical traits of rice genotypes influenced by different zn application methods in a pot experiment till 45 das factor agronomic trait water related trait biochemical trait ph (cm) t (per plant) rwc (%) chl a chl b t chl s zn el (mg l-1) (mg l-1) (mg l-1) (mg kg-1) (%) genotype (g) g1 (super basmati) 51.79 a 4.42 a 55.42 a 3.37 b 1.74 c 5.12 d 52.29 d 15.27 c g2 (accession-126) 46.65 cd 3.35 d 46.99 c 3.06 d 4.08 b 7.15 c 68.19 a 20.41 a g3 (accession-154) 44.52 d 3.13 d 44.54 d 3.26 c 0.61 e 3.87 e 59.36 c 21.07 a g4 (accession-164) 48.69 bc 3.71 c 53.10 b 0.52 e 7.22 a 7.75 b 67.80 a 17.59 b g5 (accession-175) 50.12 ab 4.08 b 53.45 ab 7.10 a 0.95 d 8.06 a 63.60 b 17.81 b application method (m) control 37.16 hi 1.73 h 44.94 gh 3.28 hi 2.78 ij 6.06 kl 40.81 j 22.95 ab seed priming (sp) 40.62 gh 2.33 g 46.62 fh 3.33 g-1 2.81 h-j 6.14 jk 43.86 ij 21.96 bc soil application (sa) 47.23 ef 2.86 fg 48.44 d-g 3.41 e-g 2.86 f-i 6.27 h-j 47.86 g-j 19.99 cd foliar application (fa) 49.96 de 3.33 ef 49.27 d-g 3.43 d-g 2.89 e-h 6.33 g-i 50.03 g-i 19.37 c-e root dipping (rd) 43.44 fg 2.66 g 47.73 e-g 3.37 f-h 2.83 g-j 6.20 i-j 46.01 h-j 20.89 b-e sp + sa 54.51 cd 4.66 d 53.26 a-d 3.55 a-d 2.98 b-d 6.53 c-e 52.78 f-h 16.15 f-h sp + fa 58.06 bc 5.00 cd 54.37 a-c 3.57 a-c 3.01 a-d 6.58 b-d 55.97 d-g 15.46 g-i sp + rd 52.77 d 4.53 d 52.04 b-e 3.52 b-e 2.96 c-e 6.48 d-f 53.05 e-h 17.29 e-g sa + fa 64.99 a 6.20 a 57.53 a 3.64 a 3.09 a 6.74 a 61.72 d 13.36 i sa + rd 61.16 ab 5.33 bc 55.40 a-c 3.59 a-c 3.03 a-c 6.62 a-c 58.48 d-f 14.55 hi fa + rd 62.28 ab 5.66 ab 56.53 ab 3.62 ab 3.07 ab 6.69 ab 60.95 de 13.96 hi sp + sa + fa 39.46 g-1 3.53 e 50.83 c-f 3.49 c-e 2.92 d-g 6.42 e-g 88.16 b 18.40 d-f sp + sa + rd 38.99 g-1 3.33 ef 50.85 c-f 3.48 c-f 2.93 d-f 6.42 e-g 76.56 c 18.43 d-f sa + fa + rd 39.16 g-1 3.53 e 50.83 c-f 3.47 d-f 2.92 d-g 6.40 f-h 94.75 ab 18.49 d-f sp + sa + fa + rd 35.56 i 1.40 h 41.86 h 3.24 i 2.75 j 5.99 l 102.81 a 25.19 a hsd (g) (p ≤ 0.05) 2.18 0.25 2.29 0.05 0.04 0.06 3.76 1.21 hsd (m) (p ≤ 0.05) 4.71 0.55 4.94 0.11 0.09 0.13 8.11 2.61 g × m (p ≤ 0.05) ns ns ns ns ns ns ns ns ph = plant height; t = tillers; el = electrolyte leakage; rwc = relative water contents; s zn = shoot zn contents; chl a = chlorophyll a; chl b = chlorophyll b; t chl = total chlorophyll contents; hsd = highest significant difference; sp = seed priming; sa = soil application; fa = foliar application; rd = root dipping. any two means within a column followed by same letter are not significantly different at p ≤ 0.05, ns = non-significant, n = 3. 14 q. zaman, z. aslam, m. rashid, a. aslam, n. ehsan, u. riaz, r.n. abbas, a. iqbal, m.s. naeem, s. bashir, z.a. saqib, m. yaseen recorded the maximum plant height (64.9 cm), number of tillers (6.2 per plant), rwc (57.5%), chlorophyll a (3.6 mg l-1), chlorophyll b (3.1 mg l-1), total chlorophyll contents (6.7 mg l-1) and minimum electrolyte leakage (13.4%). root dipping (0.5% znso4.7h2o) followed by the foliar application (0.25% znso4.7h2o) was the second best treatment for these attributes. the maximum shoot zn contents (102.8 mg kg-1) were observed in the treatment ‘seed priming + soil application + foliar application + seedling root dipping’, which showed toxic and detrimental effects on seedling growth. among various rice genotypes, super basmati performed best for agronomic and water related traits. however, chlorophyll contents and shoot zn contents were higher in accession-175 and accession-126, respectively, compared with other genotypes. the minimum electrolyte leakage was observed in accession-154 (tab. 1). interaction between rice genotypes and zn application methods was not significant for all agronomic, water related and biochemical attributes. correlation analysis revealed that electrolyte leakage had negative relationship with plant height, rwc, shoot zn contents, tillers per plant and total chlorophyll contents (tab. 2). however, total chlorophyll contents and tillers per plant were positively correlated with shoot zn contents, rwc and plant height. likewise, rwc was positively linked with plant height and chlorophyll b, however, chlorophyll a was negatively related to chlorophyll b (tab. 2). field experiment zinc application methods and rice genotypes significantly influenced the plant water-relations in both years (tab. 3). however, there was non significant interaction between genotypes and application methods. zn application treatment ‘foliar + soil application’ significantly enhanced the water potential, osmotic potential, and rwc of rice genotypes in both years, compared with the non-zn application control. foliar zn application was found to be more effective than soil zn application for all of these attributes. zn application significantly improved chlorophyll contents of rice genotypes in both years, compared with control (tab. 3). total chlorophyll contents were the highest in ‘soil + foliar application’ treatment followed by foliar zn application alone, and soil zn application alone (tab. 3). in different genotypes, zn-induced increase in water related traits and chlorophyll contents in rice genotypes followed the order of super basmati > accession-175 > accession-164 > accession-126 > accession-154 (tab. 3). electrolyte leakage in rice genotypes was significantly decreased by different zn application methods, compared with the control. however, soil + foliar application of zn recorded the maximum reduction in the electrolyte leakage in both years. the zn-induced reductions in electrolyte leakage of different rice genotypes followed the order of super basmati < accession-175 < accession-164 < accession-126 < accession-154 (tab. 3). tab. 2: correlation matrix of agronomic, water related and biochemical attributes of rice genotypes influenced by different zn application methods in a pot experiment variable chl a chl b el ph rwc s zn t chl chl b -0.754** el -0.066 ns -0.074 ns ph 0.099 ns 0.039 ns -0.691** rwc 0.110 ns 0.136* -0.837** 0.572** s zn -0.059 ns 0.184** 0.153* 0.330** -0.078 ns t chl 0.148** 0.537** -0.196** 0.187** 0.347** 0.202** t 0.148** -0.001ns -0.851** 0.845** 0.743** 0.145* 0.189** ph = plant height; el = electrolyte leakage; rwc = relative water contents; s zn = shoot zn contents; t = tillers; chl a = chlorophyll a; chl b = chlorophyll b; t chl = total chlorophyll contents; * = significant at p ≤ 0.05; ** = significant at p ≤ 0.01; ns = non-significant tab. 3: influence of zn applications on water-related and biochemical traits of five rice genotypes for two growth seasons in a field experiment factor water related trait biochemical trait water potential osmotic potential relative water total chlorophyll electrolyte leakage (mpa) (mpa) contents (cm) contents (mg l-1) (%) 2014 2015 2014 2015 2014 2015 2014 2015 2014 2015 genotype (g) g1 (super basmati) 2.07 a 2.18 a 2.51 a 2.59 a 57.03 a 58.22 a 5.23 d 5.57 d 14.92 c 14.38 d g2 (accession-126) 1.61 d 1.66 d 1.87 c 1.96 d 47.77 b 48.67 b 7.29 c 7.42 c 19.92 a 19.83 b g3 (accession-154) 1.23 e 1.33 e 1.72 d 1.84 e 44.40 c 46.56 b 4.06 e 4.10 e 21.20 a 21.02 a g4 (accession-164) 1.76 c 1.86 c 1.96 bc 2.04 c 53.88 a 54.89 a 7.95 b 8.03 b 17.82 b 17.72 c g5 (accession-175) 1.95 a 2.06 b 2.04 b 2.14 b 55.57 a 57.20 a 8.28 a 8.38 a 17.87 b 17.79 c application method (m) control 1.51 c 1.70 d 1.78 c 1.99 d 45.25 d 46.50 d 6.38 d 6.51 d 23.10 a 22.97 a soil application (sa) 1.59 b 1.79 c 1.87 b 2.08 c 50.43 c 51.32 c 6.51 c 6.64 c 20.23 b 20.16 b foliar application (fa) 1.61 b 1.85 b 1.92 b 2.15 b 53.72 b 54.90 b 6.62 b 6.75 b 17.02 c 16.92 c sa × fa 1.70 a 1.92 a 2.02 a 2.24 a 57.53 a 59.71 a 6.74 a 6.90 a 13.04 d 12.90 d hsd (g) (p ≤ 0.05) 0.13 0.06 0.13 0.07 3.24 3.38 0.11 0.09 1.38 1.17 hsd (m) (p ≤ 0.05) 0.07 0.05 0.07 0.06 2.72 2.83 0.09 0.07 1.15 0.98 g × m (p ≤ 0.05) ns ns ns ns ns ns ns ns 3.66 ** any two means within a column followed by same letters are not significantly different at p ≤ 0.05, ns = non-significant, n = 3. zinc bioavailability in rice 15 ph ot os yn th et ic ra te (µ m ol m -2 s1 ) 0 5 10 15 20 25 s. basmati acc-126 acc-154 acc-164 acc-175 t ra ns pi ra tio n ra te (m m ol m -2 s1 ) 0 2 4 6 8 10 12 14 st om at al c on du ct an ce (m ol m -2 s1 ) 0.0 0.2 0.4 0.6 0.8 control sa fa sa + fa n et a ss im ila tio n ra te (g m -2 d ay -1 ) 0 1 2 3 4 5 control sa fa sa + fa 2014 2015 fig. 1: effect of zn fertilization treatments on physiological and growth attributes of rice genotypes. vertical bars above mean denote the standard error of three replicates. zinc application methods and rice genotypes significantly (p ≤ 0.05) affected the physiological growth attributes (fig. 1). maximum transpiration rate, stomatal conductance, photosynthetic rate and net assimilation rate were observed in soil + foliar application followed by foliar zn application alone and soil zn application alone in both years. among different rice genotypes, the highest values of these attributes were recorded in super basmati, followed by accession-175, accession-164, accession-126, and accession-154 (fig. 1). all the zn application methods and rice genotypes significantly (p ≤ 0.05) affected the plant height, number of tillers, panicle length, 1000-kernels weight and kernel yield in both years at field levels (tab. 4). the interactive effect between the both factors was also sig16 q. zaman, z. aslam, m. rashid, a. aslam, n. ehsan, u. riaz, r.n. abbas, a. iqbal, m.s. naeem, s. bashir, z.a. saqib, m. yaseen nificant for total tillers for first year, 1000-kernels weight and kernel yield during both years. soil + foliar application of zn had the maximum plant height, number of tillers, panicle length, 1000-kernels weight and kernel yield in both years (tab. 4), followed by foliar application and soil application treatments. among genotypes, the maximum plant height was recorded for accession-164, while for the values of all yield related traits were in order of super basmati > accession-175 > accession-164 > accession-126 > accession-154. zinc application methods significantly (p ≤ 0.05) affected the shoot zn and kernel zn contents of rice in both years, while kernel protein contents varied significantly (p ≤ 0.05) in the second year of the study only. there was no significant effect on kernel amylose contents and kernel length by zn application methods in both years (tab. 5). maximum shoot zn and kernel zn contents were recorded in the treatment ‘soil + foliar application of zn’ followed by foliar zn application alone, and soil zn application alone in both years. all the grain quality attributes varied significantly among different rice genotypes. shoot zn content, kernel zn content, kernel protein content, and kernel amylose content in different rice genotypes followed the order of accession-126 > accession-164 > accession-175 > accession-154 > super basmati, while kernel length was in the order of super basmati > accession-175 > accession-154 > accession-164 > accession-126 (tab. 5). correlation analysis indicated negative relationship between electrolyte leakage and plant height, panicle length, photosynthetic rate, total tillers, grain yield and 1000-kernels weight, respectively (tab. 6). grain zn contents were also negatively correlated with 1000-kernels weight and grain yield. however, total chlorophyll contents and 1000-kernels weight were positively linked with total tillers, photosynthetic rate, plant height and panicle length. tab. 4: influence of zn application on agronomic and yield-related traits of five rice genotypes for two growth seasons in a field experiment factor plant height total tillers panicle length 1000-kernels weight kernel yield (cm) (m-2) (cm) (g) (t ha-1) 2014 2015 2014 2015 2014 2015 2014 2015 2014 2015 genotype (g) g1 (super basmati) 141.58 b 140.77 b 448.50 a 454.33 a 26.60 a 26.90 a 24.03 a 24.09 a 4.50 a 4.60 a g2 (accession-126) 139.28 b 137.58 b 411.08 c 419.50 c 22.97 c 23.19 cd 20.55 d 20.61 d 3.23 d 3.31 d g3 (accession-154) 110.48 c 110.50 c 398.00 d 404.33 d 21.53 d 21.65 d 19.24 e 19.28 e 3.02 e 3.11 e g4 (accession-164) 150.08 a 148.40 a 432.17 b 438.92 b 23.89 bc 23.75 bc 21.59 c 21.59 c 3.52 c 3.60 c g5 (accession-175) 137.78 b 136.18 b 444.67 a 449.33 a 25.09 b 25.21 b 22.77 b 22.80 b 4.09 b 4.20 b application method (m) control 130.66 c 128.49 b 414.13 c 423.93 c 21.74 d 21.83 c 20.48 c 20.49 c 3.39 c 3.58 c soil application (sa) 133.60 c 132.47 b 419.33 b 430.40 b 23.29 c 23.34 b 21.11 bc 21.17 b 3.46 bc 3.71 b foliar application (fa) 137.71 b 137.10 a 436.33 a 435.87 b 24.64 b 24.68 b 21.75 b 21.79 b 3.55 b 3.80 b sa × fa 141.39 a 140.69 a 437.73 a 442.93 a 26.38 a 26.72 a 23.21 a 23.25 a 4.28 a 4.37 a hsd (g) (p ≤ 0.05) 3.82 4.98 4.30 7.41 1.22 1.67 0.81 0.79 0.15 0.10 hsd (m) (p ≤ 0.05) 3.20 4.18 3.61 6.22 1.02 1.40 0.68 0.66 0.13 0.09 g × m (p ≤ 0.05) ns ns 11.41 ns ns ns 2.16 2.11 0.41 0.28 any two means within a column followed by same letters are not significantly different at p ≤ 0.05, ns = non-significant, n = 3. tab. 5: influence of zn application on kernel quality and grain zn biofortification of five rice genotypes for two growing seasons in a field experiment factor shoot zn contents kernel zn contents kernel protein contents kernel amylose contents kernel length (mg kg-1) (mg kg-1 ) (%) (%) (mm) 2014 2015 2014 2015 2014 2015 2014 2015 2014 2015 genotype (g) g1 (super basmati) 42.34 d 42.24 d 27.75 e 27.70 e 7.02 c 7.05 c 24.23 e 24.30 e 10.92 a 10.91 a g2 (accession-126) 62.28 a 62.33 a 59.08 a 59.16 a 7.31 a 7.37 a 27.27 a 27.30 a 10.58 c 10.58 d g3 (accession-154) 50.42 c 50.89 c 45.16 d 45.23 d 7.08 c 7.10 c 25.23 c 25.28 c 10.67 c 10.66 c g4 (accession-164) 60.60 a 60.78 a 55.20 b 55.32 b 7.21 b 7.23 b 27.23 a 27.33 a 10.61 c 10.61 c g5 (accession-175) 56.19 b 56.37 b 50.51 c 50.64 c 7.16 b 7.17 b 26.12 b 26.20 b 10.75 b 10.74 b application method (m) control 45.20 d 45.23 d 43.16 d 43.10 d 7.11 7.09 c 25.46 25.90 10.71 10.72 soil application (sa) 52.69 c 52.78 c 46.24 c 46.55 c 7.16 7.14 b 26.64 26.70 10.68 10.67 foliar application (fa) 57.70 b 57.82 b 48.10 b 40.94 b 7.16 7.16 b 26.90 26.93 10.66 10.66 sa × fa 61.72 a 61.78 a 52.67 a 52.80 a 7.17 7.19 a 27.12 27.22 10.70 10.71 hsd (g) (p ≤ 0.05) 3.76 3.23 1.35 1.67 0.09 0.08 0.69 0.54 0.09 0.07 hsd (m) (p ≤ 0.05) 3.15 3.49 1.13 1.27 ns 0.04 ns ns ns ns g × m (p ≤ 0.05) ns ns 3.59 ns ns ns ns ns ns ns any two means within a column followed by same letters are not significantly different at p ≤ 0.05, ns = non-significant, n = 3. zinc bioavailability in rice 17 animal feeding experiment feeding of zn biofortified rice to rats significantly (p ≤ 0.05) increased their body weight in all groups except the last group assessed weekly (fig. 2) in both years. compared to initial body weight, the maximum increase in body weight of rats was observed for rats fed with kernels of accession-164 (treated with 55 mg kg-1 of zn) at 1st, 2nd, 3rd and 4th week in 2014 (35.22%, 52.28%, 68.22% and 80.59%) and 2015 (36.57%, 54.56%, 71.00% and 83.73%), respectively (fig. 2). however, body weight of rats was not improved after feeding of kernels of accession-126 regardless high kernel-zn concentration in this group. the increase in the body weight of male wister rats was in the order of g4> g3> g5> g2> g1 in both years. discussion different zn application methods considerably improved the crop stand establishment, water relations, photosynthetic pigments, tillering, kernel quality and biofortification of the tested rice genotypes. application of zn in the zn-deficient calcareous soil followed by foliar spray significantly (p ≤ 0.05) improved the agronomic traits. cakmak (2008) reviewed a number of studies and documented the positive effects of zn application on plant growth and productivity in zn-deficient calcareous soils. increasing doses of zn improved crop water relations that might be ascribed to better root growth and increased the water uptake by plants (zhao et al., 2016). better root development enabled the plants to extract water efficiently, and hence showed healthy water relations (tabs. 1 and 3). rwc increased gradually with the increasing zn concentration and achieved the highest (27.13% and 27.33%) during both years under field study where 15 kg ha-1 zn was applied in soil followed by foliar application of 0.25% zn solution. nonetheless, higher concentration of zn gradually decreased rwc of rice leaves, which might be due to disturbed osmotic relations at higher levels of zn. decline of water uptake rate or decrease of hydraulic conducti vity of root at higher zn levels might also have resulted in lower rwc of the leaves (chen et al., 2008). there are also reports showing disturbed water relations of the leaves exposed to zn-deficient environments (zhao et al., 2016). the minimum electrolyte leakage in rice leaves was observed for soil + foliar application of zn which might also be related with higher leaf rwc, which also corresponded to higher gs (fig. 1). similar responses have been reported for various filed crops (tas and tas, 2007). as zn has major role in the membrane system. zinc has a unique property of existing in a divalent state, without any redox cycling, and is thereby stable in biological medium in which oxido-reductive potential is subjected to continuous flux. as a result of these properties, membrane lipid packing is protected from ros per-oxidation, which in turn prevents ion leakage from ion channels (samreen et al., 2013). gas exchange characteristics like stomatal conductance (gs), photosynthetic rate (a) and transpiration rate (e) showed significant increase with increase in the zn application. wang and jin (2005) reported that deficiency of zn in the soil depressed photosynthetic activity because of decrease in gs and increase in substomatal co2 concentration (ci). the reduction in photosynthetic activity under control plots (0.31 mg kg-1 zn) of rice genotypes might be due to low chlorophyll contents. zinc also provides building material for leaf chlorophyll contents. soil + foliar application of zn improved the chlorophyll a, b and total chlorophyll contents in all rice genotab. 6: correlation matrix of agronomic, physiological and biochemical attributes of rice genotypes influenced by different zn application methods (means of both years used in the analysis) variable el g zn kw ky t chl tt pr ph g zn 0.03ns kw 0.74** 0.26* gy 0.66** 0.30* 0.87** t chl 0.14ns 0.61** 0.26* 0.14ns tt 0.73** 0.22ns 0.88** 0.77** 0.41** pr 0.85** 0.17ns 0.89** 0.75** 0.27* 0.86** ph 0.52** 0.20ns 0.59** 0.45** 0.71** 0.69** 0.57** pl 0.87** 0.16ns 0.85** 0.74** 0.22 ** 0.82** 0.88** 0.58** ph = plant height; el = electrolyte leakage; g zn = grain zn contents; tt = total tillers; t chl = total chlorophyll contents; kw = 1000 kernel weight; ky = kernels yield; pr = photosynthetic rate; pl = panicle length; * = significant at p ≤ 0.05; ** = significant at p ≤ 0.01; ns = non-significant fig. 2: percent increase in body weight of groups of male rats after feeding with zn biofortified rice of both years. vertical bars above mean denote the standard error of three replicates. 18 q. zaman, z. aslam, m. rashid, a. aslam, n. ehsan, u. riaz, r.n. abbas, a. iqbal, m.s. naeem, s. bashir, z.a. saqib, m. yaseen types showing involvement of zn in chlorophyll synthesis (abadi and sepehri, 2016). application of zn in soil followed by foliar spray had positive effect on chlorophyll formation which ultimately promoted the photosynthetic rate. there was a linear increase in transpiration rate (e) of rice genotypes that might be linked to optimum zn nutrition. similar finding has been given by tariq et al. (2014) who reported that application of zn increased the photosynthesis and chlorophyll production. marschner (2012) reported an instant decline in transpiration efficiency of leaves exposed to deficiency of zn. our findings suggested that balanced application of zn in zn-deficient soils improved gas exchange characteristics of the rice genotypes. similar trend, as in e, with varying zn application methods was also observed for gs. zinc deficiency was associated with lower gs, which is consistent with the findings of khan et al. (2013). zinc application in soil followed by foliar application substantially increased the gs. zn presumably involved in regulation of stomata due to its role in maintaining integrity of the membranes (khan et al., 2013). our data showed that zn deficiency not only resulted in poor water uptake, but also depicted poor transpiration efficiency. any potential benefit of lower gs on water relations appears to be negated by a reduction in net carbon fixation. in the present study, application of zn increased the height of rice plants (8 and 9%), total tillers (5 and 8%), panicle length (21 and 21.55%) and kernel yield (26.25%, 29.11%) during both years respectively, over control. zn has been reported to be involved in cell elongation and/or improved cell division rates (cakmak, 2000b; ramesh et al., 2014), and meristematic growth (abaid-ullah et al., 2015). chang et al. (2005) also opined that zn is necessary for cell division and elongation as the fundamental events of growth. such basic event of growth as enhanced cell division and/or cell elongation, better enzymatic activity and auxin metabolism might have improved morphological traits of rice with zn supply in the present study. zn application increased its availability and resulted in reduced spikelet sterility and increased number of panicle bearing tillers (abaid-ullah et al., 2015). our findings also supported this phenomenon. khan et al. (2013) reported maximum panicle length, kernel per panicle and grain yield with the application of zn at 9 kg ha-1. increase in kernels per panicle and 1000-grain weight was due to the involvement of zn in grain partitioning (gomez-coronado et al., 2016). shoot zn concentration ranged from 40.8 to 102.8 mg kg-1 in various zn application treatments (tab. 1). usually a concentration greater than 50 mg kg-1 zn in rice plant is considered a threshold level for achieving a positive impact in the grain zn concentration that ultimately brings a positive impact on human health for combating malnutrition (cakmak, 2008). application of zn by all methods simultaneously proved to be toxic and suppressed rice seed germination and its seedling growth. higher zn concentration may restrict the seedling growth by suppressing the cell division (gomez-coronado et al., 2016). moreover, higher concentration of zn may diminish the root and leaf development owing to substantial decrease in nadph (nicotinamide adenine dinucleotide phosphate) production in chloroplasts (mousavi, 2011). nonetheless, zn concentration in shoots of rice plants grown in field experiments ranged from 42 to 62 mg kg-1 under different zn applications. phattarakul et al. (2012) reported that zn concentration in brown rice was increased by 25% and 32% by foliar and foliar + soil zn applications as compared with control, respectively. soil application of zn followed by foliar sprays significantly increased zn concentration (22.03 and 22.50%) during both years respectively in rice kernels. foliar applied zn is easily absorbed and transported through phloem (haslett et al., 2005). increase in zn concentration of all rice genotypes would provide significant nutritional benefits to rice consumers, particularly for those who have limited access to zn from other food sources. high grain zn has also important agronomic benefits for plants grown under low zn supply. it has been reported that nearly 2800 proteins require zn for their structural and functional integrity (hippler et al., 2015), and hence suggesting a high need for zn during root and coleoptiles development for active protein synthesis and/or other related functions. application of zn to rice not only improved the crop stand establishment, water relations, photosynthetic pigments, tillering ability, kernel yield but also significantly improved the kernel length, zn content, amylose and protein contents. rashid et al. (2004) also reported that deterioration of the kernel quality of rice was associated with zn deficiency. feeding of zn excessive and deficient rice kernel diets resulted in poor body and organ weight gain of albino rats. these results are in accordance with della lucia et al. (2014) who stated that enhanced zn dietary intake would result in an increase in the weight gain of test animals but the dose above optimal concentration cause severe complications including growth retardation, poor appetite, impaired immunity and diabetes that might be the reasons for the reduction in the body weight (prasad, 2014; maares and haase, 2016). another report concluded that the food conversion efficiency of zn-sufficient animals was higher than zn-deficient animals (lazarte et al., 2015). gain in body weight of rats might be associated with increased feed intake per day. conclusion present study concluded that soil application of 15 kg ha-1 znso4.7h2o followed by foliar application at 0.25% znso4.7h2o solution at tillering and heading stage of rice improved the crop stand establishment, water relations, membrane permeability, photosynthetic pigments, tillering ability, kernel length, kernel yield, kernel zn, amylose and protein contents, and was effective in kernel biofortification. these promising genotypes (accession-175 > accession-164 > accession-126 > accession-154) may be further utilized in breeding programs for genetic potential to transfer the desirable gene(s) to improve zn as well as yield potential of evolving genotypes and may help in developing zn biofortified rice which is the need of the hour. nonetheless, various minerals have been reported to interact and influence the uptake of zn from roots to developing grains (fageria 2001; lopez et al., 2002). future studies aimed at biofortifying rice kernels need to look for such interactive effects of other essential mineral nutrients. moreover, specific role of zn in stomatal regulation requires further investigation. acknowledgements the financial support from higher education commission pakistan (hec) under project no. 20-2014/nrpu/r&d/12/4188 is highly acknowledged. conflict of interest no potential conflict of interest was reported by the authors. references abadi, v.a.j.m., sepehri, m., 2016: effect of piriformospora indica and azotobacter chroococcum on mitigation of zinc deficiency stress in wheat (triticum aestivum l.). symbiosis. 69, 9-19. abaid-ullah, m., hassan, m.n., jamil, m., brader, g., shah, m.k.n., sessitsch, a., hafeez, f.y., 2015: plant growth promoting rhizobacteria: an alternate way to improve yield and quality of wheat (triticum aestivum). int. j. agri. biol. 17, 51-60. barras, h.d., weatherley, p.e., 1962: a re-examination of relative tur gidity technique for estimation of water deficit in leaves. australian j. biol. sci. 15, 418-428. zinc bioavailability in rice 19 blum, a., ebercon, a., 1981: cell membrane stability as a measure of drought and heat tolerance in wheat. crop sci. 21, 43-47. cakmak, i., 2000b: possible roles of zinc in protecting plant cells from damage by reactive oxygen species. new phytol. 146, 185-205. cakmak, i., 2008: enrichment of cereal grains with zinc: agronomic or genetic biofortification? 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prasad, a.s., 2014: impact of the discovery of human zinc deficiency on health. j. trace elem. med. biol. 28, 357-363. quijano-guerta, c., kirk, g.j.d., portugal, a.m., bartolome, v.i., mclaren, g.c., 2002: tolerance of rice germplasm to zinc deficiency. field crop res. 76, 123-130. rashid, m., khalil, s., ayub, n., alam, s., latif, f., 2004: organic acids production and phosphate solubilization by phosphate solubilizing microorganisms (psm) under in vitro conditions. pak. j. biol. sci. 7, 187-196. ramesh, a., sharma, s.k., sharma, m.p., yadav, n., joshi, o.p., 2014: inoculation of zinc solubilizing bacillus aryabhattai strains for improved growth, mobilization and biofortification of zinc in soybean and wheat cultivated in vertisols of central india. app. soil ecol. 73, 87-96. rehman, a., farooq, m., ozturk, l., asif, m., siddique, k.h.m., 2017: zinc nutrition in wheat-based cropping systems. plant soil 422(1-2), 283-315. samreen, t., shah, h.u., ullah, s., javid, m., 2013: zinc effect on growth rate, chlorophyll, protein and mineral contents of hydroponically grown mung beans plant (vigna radiata). arabian j. chem. 7, 145-152. tariq, m., hameed, s., malik, k.a., hafeez, f.y., 2007: plant root associated bacteria for zn mobilization in rice. pak. j. bot. 39, 245-253. tas, s., tas, b., 2007: some physiological responses of drought stress in wheat genotypes with different ploidity in turkiye. world j. agri. sci. 3, 178-183. wang, h., jin, j.y., 2005: photosynthetic rate, chlorophyll fluorescence parameters and lipid peroxidation of maize leaves as affected by zinc deficiency. photosynthetica. 43, 591-596. welch, r.m., graham, r.d., 2004: breeding for micronutrients in staple food crops from a human nutrition perspective. j. exp. bot. 55, 353-364. zhao, a.q., tian, x.h., chen, y.l., li, s., 2016: application of znso4 or zn-edta fertilizer to a calcareous soil: zn diffusion in soil and its uptake by wheat plants. j. sci. food agri. 96, 1484-1491. zaman, q.u., aslam, z., yaseen, m., ihsan, m.z., khaliq, a., fahad, s., bashir, s., ramzani, p.m.a., naeem, m., 2017: zinc biofortification in rice: leveraging agriculture to moderate hidden hunger in developing countries. arch. agron. soil sci. 64, 140-161. zaman, q.u., aslam, z., rashid, m., khaliq, a., yaseen, m., 2018: in fluence of zinc fertilization on morpho-physiological attributes, growth, productivity and hematic appraisal of paddy rice. j. anim. plant sci. 28(3), 778-790. address of the corresponding author: e-mail: qamar.zaman1@envs.uol.edu.pk; zauaf@hotmail.com © the author(s) 2020. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). art14_akram.indd journal of applied botany and food quality 85, 91 96 (2012) 1department of botany, university of agriculture, faisalabad, pakistan 2department of botany and microbiology, king saud university, riyadh, saudi arabia 3department of botany, gc university, faisalabad, pakistan salt-induced regulation of photosynthetic capacity and ion accumulation in some genetically diverse cultivars of radish (raphanus sativus l.) 1 zahra noreen, 1,2 muhammad ashraf, *3 nudrat aisha akram (received april 28, 2011) * corresponding author summary salt-induced changes in growth, various gas exchange characteristics, and ion accumulation were examined during a greenhouse experiment on six radish (raphanus sativus l.) cultivars i.e., radish red neck, radish lal pari, radish mino japani, radish 40 days, mannu early and desi. varying levels of salt (0, 80, and 160 mm 160 mm 160 m nacl) of the growth medium markedly decreased the shoot and root dry weights, relative water contents, osmotic potential, photosynthetic and transpiration rates, stomatal conductance, substomatal co2 concentration, ci /ca/ca/c ratio, water use effi ciency, leaf and root k+ and ca2+, while increasing the leaf and root na+ and clof all six radish cultivars. of all cultivars, mannu early and desi were higher in shoot and root dry weights than the other cultivars, and thus, they were ranked as relatively salt tolerant. however, none of the earlier mentioned physiological attributes was found to be an effective criterion in discriminating the six radish cultivars. overall, the response of each cultivar to salt stress appraised using various physiological attributes was specifi c. introduction it is now well established that high salt concentrations in water or soil adversely affect various biochemical and physiological processes leading to poor plant vigor and yield in most plant species (ashraf, 2004; munns and tester, 2008). salt-induced osmotic stress and ion toxicity are the major causes of plant growth reduction (zhu, 2001; sairam and tyagi, 2004). a number of mechanisms involved in plant salt tolerance, ion homeostasis, and differential regulation of biochemical and physiological processes have gained a signifi cant importance (ashraf, 2004; munns, 2005; genc et al., 2007). previously, it was found that growth suppression may be a nonspecifi c effect of salts, depending more on the total concentration of soluble salts than on specifi c ions (maas and nieman, 1978). it is well documented that the accumulation of na+ and ciions in the leaves is the most important factor which causes considerable injury to ultra-structure of different organelles of plants subjected to salt stress (martinez-barroso and alvarez, 1997). it is suggested that generally crop cultivars which show more tolerance to salt stress, accumulate ciin their roots and as a whole prevent the negative effects of salt on plant growth (saied et al., 2003). cellular k+/na+ and ca2+/na+ ratios also can affect salt tolerance of plants to a varying extent (yasar, 2007; yildiz et al., 2008). salt stress also adversely affects plant photosynthesis (dubey, 2005; arfan et al., 2007; noreen and ashraf, 2008). however, it is diffi cult to assess whether a reduced rate of photosynthesis is the cause of growth reduction, or merely the consequence of growth reduction (munns and tester, 2008). for instance, salt-induced reduction in growth of barley (fricke et al., 2004) and maize (cramer and bowman, 1991) has been due to rapid change in leaf expansion rate resulting into a buildup of unused photosynthates in growing tissues (munns, 1993; munns et al., 2000). in view of paul and foyer (2001) accumulation of unused photosynthates under saline conditions may generate feedback signals to down-regulate photosynthesis to compensate the reduced demand arising from growth inhibition. however, at high salt stress, excessive accumulation of salts in the cytoplasm or chloroplast of mesophyll cells inhibits photosynthetic enzymes thereby reducing the photosynthetic rate (munns, 1993; dubey, 2005). the reduction in photosynthesis under salt stress can also be attributed to a decrease in stomatal closure because higher stomatal conductance is known to increase co2 diffusion into leaves thereby favoring higher photosynthetic effi ciency (downton, 1977; seemann and critchley, 1985). during a greenhouse experiment on radish plants, salt stress (90 and 240 mm nacl) considerably decreased the photosynthetic activity resulting in reduced plant growth, leaf area, chlorophyll contents and chlorophyll fl uorescence (jamil et al., 2007). radish (raphanus sativus l.), being a potential vegetable, is utilized in a variety of ways, e.g., fresh, pickled, dried, cooked as well as a fodder. it is an important source of medicinal foods and one of the most recalcitrant crop plants in nature (curtis, 2003). generally, radish is categorized as moderately sensitive to salinity (maas and hoffman, 1977), while sonneveld (1988) reported a low sensitivity of radish to salt stress. despite of being a potential vegetable crop world-over, little is known about its mechanism of salt tolerance. thus, the premier objectives of this study were to observe the effect of varying levels of salt on some key physiological processes such as photosynthetic capacity and mineral nutrient accumulation in six genetically diverse radish cultivars. it was also examined whether photosynthetic capacity and pattern of accumulation of nutrients could be used as effective selection criteria for salt tolerance in radish. materials and methods to examine changes in growth, gas exchange characteristics, some water relation attributes and nutrient accumulation in six cultivars/ lines of radish (raphanus sativus l.) i.e., radish red neck, radish lal pari, radish mino japani, radish 40 days, mannu early and desi, a greenhouse experiment was conducted at the botanical garden, university of agriculture, faisalabad. the seeds of all cultivars were obtained from the ayub agricultural research institute, faisalabad, pakistan. five seeds of each cultivar were sown per plastic pot (23.5 cm diameter and 29 cm deep) fi lled with 10 kg dry river sand. the plants were thinned to two plants per pot after 14 d of growth. three nacl treatments (0, 80, and 160 mm) in hoagland’s nutrient solution were applied to three week-old plants. the nacl concentration was increased step-wise in aliquots of 40 mm every day until the appropriate concentration attained. after 20 days of salt treatment, two plants from each pot were uprooted carefully and separated into shoots and roots. the plant samples were oven dried at 65 °c and dry weights recorded. before harvesting the plants, the data for the following parameters were recorded: 92 z. noreen, m. ashraf, n.a. akram photosynthetic capacity and ion accumulation in radish leaf osmotic potential one fully expanded youngest leaf from each plant was excised and frozen in a freezer below -20 oc for more than seven days after which time the frozen leaf material was thawed and the sap extracted by pressing the material with a glass rod. the sap was used directly for the determination of osmotic potential in a vapor pressure osmometer (vapro, 5520). relative water content (rwc) relative water content was determined following jones and turner (1978) by using a leaf of uniform size from each replicate. gas exchange characteristics to measure different gas exchange parameters such as net co2 assimilation rate (a), transpiration (e), stomatal conductance (gs) and sub-stomatal co2 concentration (ci), an infra-red gas analyzer (analytical development company, hoddesdon, england) was used. all measurements were made on a fully expanded youngest leaf of each plant from 10.00 to 12.00 h with the following specifi cations/ adjustments of the leaf chamber: atmospheric pressure 99.9 kpa, water vapor pressure into the chamber ranged from 6.0 to 8.9 mbar, temperature of leaf ranged from 28.4 to 32.4 oc, ambient temperature ranged from 22.4 to 27.9 oc, molar fl ow of air per unit leaf area 403.3 mmol m-2 s-1, par at leaf surface was maximum up to 918 µmol m-2 s-1, and ambient co2 concentration was 352 ppm. determination of mineral elements in plant tissues the dried ground plant leaf or root material (0.1 g) was taken in a digestion fl ask and to this digestion fl ask, 1 ml of digestion mixture (0.42 g of se and 14 g of liso4 . 2h2o to 350 ml of h2o2, mixed well and 420 ml of conc. h2so4 were added slowly to it keeping it in an ice bath) was added and placed the fl ask on a hot plate. the temperature was increased gradually from 50 °c to 200 °c. when the mixture turned black, 0.5 ml of hclo4 was added to the sample, and heated again until the material became colorless (allen et al., 1986). the fl asks were removed from the hot plate and cooled down. the solution was diluted up to 50 ml in a volumetric fl ask and fi ltered. the fi ltrate was used for the determination of k+, ca2+ and na+. statistical analysis analysis of variance of all parameters was computed using the mstat computer package (mstat development team, 1989). standard errors of means were also calculated to observe intra-mean variation. results salt stress markedly suppressed the shoot and root dry weights of all radish cultivars, though radish cultivars differed signifi cantly in response to varying nacl concentrations of the growth medium (fig. 1). from the mean data, it is apparent that lines mannu early and desi had greater shoot and root dry biomass than the other cultivars at all salt regimes. leaf osmotic potential in all radish cultivars signifi cantly decreased (p ≤ 0.001) with increase in nacl concentration in the growth medium. cultivar desi followed by radish red neck was the lowest in leaf osmotic potential of all radish cultivars at the highest salt regime, whereas desi followed by radish mino japani was lower than the other cultivars at 80 mm nacl. fig. 1: shoot and root dry weights, leaf osmotic potential, relative water contents and shoot and root na+ concentration of six radish (raphanus sativus l.) cultivars subjected to different concentrations of nacl (means ±s.e; n = 4). values showing mean squares from analysis of variance of data for each variable. *, ** and *** signifi cant at 0.05, 0.01 and 0.001, respectively; ns = non-signifi cant; s = salt stress; cvs = cultivars. fig. 1 shoot and root dry weights, leaf osmotic potential, relative water contents and shoot and root na+ concentration of six radish (raphanus sativus l.) cultivars subjected to different concentrations of nacl (±s.e; n =4). values showing mean squares from analysis of variance of data for each variable. *, ** and *** significant at 0.05, 0.01 and 0.001, respectively; ns= non-significant; s = salt stress; cvs = cultivars. 0 0.5 1 1.5 2 2.5 3 s h o o t d ry w t (g /p la n t) salt stress 0 mm salt stress 80 mm salt stress 160 mm s,�5.26***;�cvs,�0.75***;�s�x�cvs,�0.06ns 0 0.2 0.4 0.6 0.8 r o o t d ry w t (g /p la n t) salt stress 0 mm salt stress 80 mm salt stress 160 mm s,�0.586***;�cvs,�0.051**;�s�x�cvs,�0.006ns 0 0.5 1 1.5 2 2.5 l e a f o s m o ti c p o te n ti a l (m p a ) s,�2.85***;�cvs,�0.175***;�s�x�cvs,�0.058* 0 20 40 60 80 100 r w c ( % ) s,�282.4***;�cvs,�137.7***;�s�x�cvs,�11.74ns 0 25 50 75 100 n l e a f n a + ( m g g -1 d .w t) s,�16373.4***;�cvs,�12.58ns;�s�x�cvs,�108.8ns 0 2 4 6 8 10 n r o o t n a + ( m g g -1 d .w t) s,�71.34***;�cvs,�2.75***;�s�x�cvs,�1.028* r ad is h re d ne ck r ad is h re d ne ck r ad is h la l pa ri r ad is h la l pa ri r ad is h la l pa ri r ad is h la l pa ri r ad is h la l pa ri r ad is h m in o r ad is h m in o shoot and root dry weights, leaf osmotic potential, relative water contents and shoot and root na r ad is h m in o shoot and root dry weights, leaf osmotic potential, relative water contents and shoot and root na r ad is h m in o r ad is h m in o r ad is h m in o j ap an i j ap an i shoot and root dry weights, leaf osmotic potential, relative water contents and shoot and root na j ap an i shoot and root dry weights, leaf osmotic potential, relative water contents and shoot and root na j ap an i j ap an i j ap an i r ad is h 40 d ay s r ad is h 40 d ay s shoot and root dry weights, leaf osmotic potential, relative water contents and shoot and root na r ad is h 40 d ay s shoot and root dry weights, leaf osmotic potential, relative water contents and shoot and root na cultivars subjected to different concentrations of nacl (means ±s.e; r ad is h 40 d ay s cultivars subjected to different concentrations of nacl (means ±s.e; r ad is h 40 d ay s r ad is h 40 d ay s r ad is h 40 d ay s m an nu e ar ly m an nu e ar ly shoot and root dry weights, leaf osmotic potential, relative water contents and shoot and root na m an nu e ar ly shoot and root dry weights, leaf osmotic potential, relative water contents and shoot and root na cultivars subjected to different concentrations of nacl (means ±s.e; m an nu e ar ly cultivars subjected to different concentrations of nacl (means ±s.e; variable. *, ** and *** signifi cant at 0.05, 0.01 and 0.001, respectively; ns = non-signifi cant; s = salt stress; cvs = cultivars. m an nu e ar ly variable. *, ** and *** signifi cant at 0.05, 0.01 and 0.001, respectively; ns = non-signifi cant; s = salt stress; cvs = cultivars. m an nu e ar ly m an nu e ar ly m an nu e ar ly d es i d es i shoot and root dry weights, leaf osmotic potential, relative water contents and shoot and root na d es i shoot and root dry weights, leaf osmotic potential, relative water contents and shoot and root na cultivars subjected to different concentrations of nacl (means ±s.e; d es i cultivars subjected to different concentrations of nacl (means ±s.e; variable. *, ** and *** signifi cant at 0.05, 0.01 and 0.001, respectively; ns = non-signifi cant; s = salt stress; cvs = cultivars. d es i variable. *, ** and *** signifi cant at 0.05, 0.01 and 0.001, respectively; ns = non-signifi cant; s = salt stress; cvs = cultivars. d es i d es i d es i r ad is h re d ne ck r ad is h re d ne ck r ad is h la l pa ri r ad is h la l pa ri r ad is h la l pa ri r ad is h la l pa ri r ad is h la l pa ri r ad is h m in o r ad is h m in o concentration of six radish ( r ad is h m in o concentration of six radish ( r ad is h m in o r ad is h m in o r ad is h m in o j ap an i j ap an i concentration of six radish ( j ap an i concentration of six radish ( j ap an i j ap an i j ap an i r ad is h 40 d ay s r ad is h 40 d ay s concentration of six radish ( r ad is h 40 d ay s concentration of six radish ( = 4). values showing mean squares from analysis of variance of data for each r ad is h 40 d ay s = 4). values showing mean squares from analysis of variance of data for each r ad is h 40 d ay s r ad is h 40 d ay s r ad is h 40 d ay s m an nu e ar ly m an nu e ar ly concentration of six radish ( m an nu e ar ly concentration of six radish ( = 4). values showing mean squares from analysis of variance of data for each m an nu e ar ly = 4). values showing mean squares from analysis of variance of data for each variable. *, ** and *** signifi cant at 0.05, 0.01 and 0.001, respectively; ns = non-signifi cant; s = salt stress; cvs = cultivars. m an nu e ar ly variable. *, ** and *** signifi cant at 0.05, 0.01 and 0.001, respectively; ns = non-signifi cant; s = salt stress; cvs = cultivars. m an nu e ar ly m an nu e ar ly m an nu e ar ly d es i d es i raphanus sativus d es i raphanus sativus = 4). values showing mean squares from analysis of variance of data for each d es i = 4). values showing mean squares from analysis of variance of data for each d es i d es i d es i photosynthetic capacity and ion accumulation in radish 93 leaf relative water content (rwc) of all radish cultivars signifi cantly decreased with increase in nacl of the growth medium (fig. 1). the cultivars also differed signifi cantly in this water relation attribute. leaf rwc was found to be highest in radish red neck followed by radish lal pari at the highest (160 mm) nacl level (fig. 1). addition of salt to the rooting medium caused a signifi cant reduction in all gas exchange attributes (net co2 assimilation rate, transpiration rate, stomatal conductance, sub-stomatal co2, ci /ca/ca/c ratio, water use effi ciency) (fig. 2) and the cultivars did not differ signifi cantly in all these gas exchange attributes except in transpiration rate. however, radish lal pari followed by radish red neck and radish mino japani had greater transpiration rate than the other cultivars at the highest salt regime. na+ concentrations in the leaves and roots of the six radish cultivars increased signifi cantly with increase in salt level of the rooting medium (fig. 1). cultivar radish 40 days had considerably higher leaf na+ concentration than the other cultivars at the highest salt concentration. in contrast, root na+ concentration was the highest in cv. desi of all cultivars at the highest salt stress. while, at 120 mm nacl, cvs. desi and mannu early were higher in root na+ as compared with the other cultivars. leaf and root clconcentrations increased signifi cantly in the six radish cultivars with the addition of nacl to the growth medium, and the cultivars also differed signifi cantly in these physiological attributes. cultivars radish red neck, radish mino japani and desi had greater leaf clthan the other cultivars at the highest salt regime, while root clwas higher in radish red neck and radish 40 days compared with the other cultivars at 120 mm nacl (fig. 3). a marked reduction in k+ accumulation in the leaves and roots of the six radish cultivars was observed due to imposition of salt stress to the growth medium, particularly at the highest salt regime (fig. 3). a maximum leaf k+ was observed in radish lal pari followed by radish mino japani and desi at the highest salinity level, whereas the same was true for root k+ in mannu early and desi (fig. 3). leaf and root ca2+ of the six radish cultivars decreased signifi cantly with increase in salt level of the rooting medium (fig. 3). the cultivars also differed signifi cantly in accumulation of ca2+ in the leaves or roots. leaf ca2+ was higher in radish red neck and radish lal pari compared with the other cultivars at 120 mm nacl. however, cultivar radish red neck followed by radish mino japani had greater accumulation of ca2+ in the roots at the highest salt regime. discussion in the present study, varying levels of salt caused a marked reduction in the shoot and root dry weights in the six radish cultivars. salt-induced reduction in the radish cultivars is analogous to what has earlier been observed in a number of plant species including rice (alam et al., 2004), wheat (arfan et al., 2007; shahbaz et al., 2008), spinach, cucumber and pepper (kaya et al., 2001), sunfl ower (akram et al., 2009; noreen et al., 2009), tomato (satti and al-yahyai, 1995), cotton (leidi and saiz, 1997) etc. the present study also revealed considerable inter-cultivar variation in salt tolerance in the set of six genetically diverse cultivars of radish. earlier, a great magnitude of inter-cultivar (intra-specifi c) variation has been observed in many crop plants, e.g., barley and wheat fig. 2: different gas exchange characteristics of six radish (raphanus sativus l.) cultivars subjected to different concentrations of nacl (mean ±s.e; n = 4). values showing mean squares from analysis of variance of data for each variable. * and *** signifi cant at 0.05 and 0.001, respectively; ns = non-signifi cant; s = salt stress; cvs = cultivars. fig. 2 different gas exchange characteristics of six radish (raphanus sativus l.) cultivars subjected to different concentrations of nacl (±s.e; n =4). values showing mean squares from analysis of variance of data for each variable. * and *** significant at 0.05 and 0.001, respectively; ns = non-significant; s = salt stress; cvs = cultivars. 0 5 10 15 20 a ( µ m o l c o 2 m -2 s -1 ) salt stress 0 mm salt stress 80 mm salt stress 160 mm s,�549.5***;�cvs,�2.89ns;�s�x�cvs,�3.031ns 0 1 2 3 4 5 e ( m m o l h 2 o m -2 s -1 ) salt stress 0 mm salt stress 80 mm salt stress 160 mm s,�27.75***;�cvs,�0.249*;�s�x�cvs,�0.355*** 0 50 100 150 200 250 300 350 g s ( m m o l m -2 s -1 ) s,�166840.3***;�cvs,�972.3ns;�s�x�cvs,�1356.2ns 0 50 100 150 200 250 300 350 c i (µ m o l m o l-1 ) s,�40413.6***;�cvs,�329.3ns;�s�x�cvs,�538.9ns 0 0.2 0.4 0.6 0.8 1 n a o a y c i/c a r a ti o s,�0.326***;�cvs,�0.003ns;�s�x�cvs,�0.004ns 0 1.5 3 4.5 6 w u e ( µ m o l c o 2 /m m o l h 2 o ) s,�12.18***;�cvs,�0.728ns;�s�x�cvs,�0.847ns r ad is h re d ne ck r ad is h re d ne ck r ad is h la l pa ri r ad is h la l pa ri r ad is h la l pa ri r ad is h la l pa ri r ad is h la l pa ri r ad is h m in o r ad is h m in o r ad is h m in o r ad is h m in o r ad is h m in o j ap an i j ap an i different gas exchange characteristics of six radish ( ja pa ni different gas exchange characteristics of six radish ( ja pa ni j ap an i j ap an i r ad is h 40 d ay s r ad is h 40 d ay s different gas exchange characteristics of six radish ( r ad is h 40 d ay s different gas exchange characteristics of six radish ( = 4). values showing mean squares from analysis of variance of data for each variable. * and *** signifi cant at 0.05 and 0.001, respectively; ns = r ad is h 40 d ay s = 4). values showing mean squares from analysis of variance of data for each variable. * and *** signifi cant at 0.05 and 0.001, respectively; ns = r ad is h 40 d ay s r ad is h 40 d ay s r ad is h 40 d ay s m an nu e ar ly m an nu e ar ly different gas exchange characteristics of six radish ( m an nu e ar ly different gas exchange characteristics of six radish ( = 4). values showing mean squares from analysis of variance of data for each variable. * and *** signifi cant at 0.05 and 0.001, respectively; ns = m an nu e ar ly = 4). values showing mean squares from analysis of variance of data for each variable. * and *** signifi cant at 0.05 and 0.001, respectively; ns = m an nu e ar ly m an nu e ar ly m an nu e ar ly d es i d es i raphanus sativus d es i raphanus sativus = 4). values showing mean squares from analysis of variance of data for each variable. * and *** signifi cant at 0.05 and 0.001, respectively; ns = d es i = 4). values showing mean squares from analysis of variance of data for each variable. * and *** signifi cant at 0.05 and 0.001, respectively; ns = d es i d es i d es i r ad is h re d ne ck r ad is h re d ne ck r ad is h la l pa ri r ad is h la l pa ri r ad is h la l pa ri r ad is h la l pa ri r ad is h la l pa ri r ad is h m in o r ad is h m in o r ad is h m in o r ad is h m in o r ad is h m in o j ap an i j ap an i l.) cultivars subjected to different concentrations of nacl (mean ±s.e; j ap an i l.) cultivars subjected to different concentrations of nacl (mean ±s.e; j ap an i j ap an i j ap an i r ad is h 40 d ay s r ad is h 40 d ay s l.) cultivars subjected to different concentrations of nacl (mean ±s.e; r ad is h 40 d ay s l.) cultivars subjected to different concentrations of nacl (mean ±s.e; r ad is h 40 d ay s r ad is h 40 d ay s r ad is h 40 d ay s m an nu e ar ly m an nu e ar ly l.) cultivars subjected to different concentrations of nacl (mean ±s.e; m an nu e ar ly l.) cultivars subjected to different concentrations of nacl (mean ±s.e; = 4). values showing mean squares from analysis of variance of data for each variable. * and *** signifi cant at 0.05 and 0.001, respectively; ns = m an nu e ar ly = 4). values showing mean squares from analysis of variance of data for each variable. * and *** signifi cant at 0.05 and 0.001, respectively; ns = m an nu e ar ly m an nu e ar ly m an nu e ar ly d es i d es i l.) cultivars subjected to different concentrations of nacl (mean ±s.e; d es i l.) cultivars subjected to different concentrations of nacl (mean ±s.e; = 4). values showing mean squares from analysis of variance of data for each variable. * and *** signifi cant at 0.05 and 0.001, respectively; ns = d es i = 4). values showing mean squares from analysis of variance of data for each variable. * and *** signifi cant at 0.05 and 0.001, respectively; ns = d es i d es i d es i 94 z. noreen, m. ashraf, n.a. akram photosynthetic capacity and ion accumulation in radish (richards et al., 1987), rice (akbar and yabuno, 1974), canola (ulfat et al., 2007; athar et al., 2009), and tomato (foolad, 1996; perez-alfocea et al., 1996). the growth reduction in the radish cultivars might have been due to a limited supply of metabolites to young growing tissues, and regulation of a number of biochemical or physiological processes including photosynthesis, leaf water relations and nutrient imbalance (maas and nieman, 1978; munns and james, 2003; ashraf, 2004; juan et al., 2005). in the present study, photosynthetic and transpiration rates, stomatal conductance (gs), sub-stomatal co2 (ci), and relative intercellular co2 (ci /ca/ca/c ) of all the radish cultivars were reduced linearly with increase in salt concentration of the growth medium. the reduction in photosynthesis under salinity stress can also be attributed to a decrease in stomatal closure, because higher stomatal conductance is known to increase co2 diffusion into leaves thereby favoring higher photosynthetic activity (seemann and critchley, 1985; dubey, 2005). in the present study, net co2 assimilation rate (a assimilation rate (a assimilation rate ( ) had a positive relationship with all these gas exchange variables (a a positive relationship with all these gas exchange variables (a a positive relationship with all these gas exchange variables ( vs gs or ci or ci/ci/ci a/ca/c or e, r = 0.740***; 0.666***; 0.665***; 0.794***; and gs vs ci r = 0.681***). these results indicate that salt-induced reduction in growth in all six radish cultivars was due to decline in photosynthesis, and decline in net photosynthesis under salt stress occurred principally due to stomatal closure, which is in agreement with a number of earlier studies (brugnoli and bjorkman, 1992; dionisio-sese and tobita, 1998; raza et al., 2006; ulfat et al., 2007). however, it was not possible to discriminate among the radish cultivars on the basis of these gas exchange attributes. for instance, salt tolerant cultivars cvs. mannu early and desi were lower in gs compared to the salt sensitive cultivars (fig. 1), but they were similar in photosynthetic capacity. these results support the argument that gs is not always associated with a. this has earlier been observed in different crops such as sunfl ower (rawson and constable, 1980), and andropogon glomeratus (bowman, 1987). these fi ndings suggest that cultivar variation for salt tolerance in radish was not due to differences in stomatal conductance and thus it cannot be used as an effective selection criterion for salt tolerance in radish. in view of some earlier reports it is evident that a positive relationship between photosynthetic rate and crop growth or yield under saline conditions exists in different crops such as spinacia oleracea (robinson et al., 1983), asparagus offi cinalis (faville et al., 1999), six brassica diploid and amphiploid species (nazir et al., 2001; ashraf, 2001), wheat (raza et al., 2006), and 34 canola cultivars (ulfat et al., 2007). all these reports suggest that the rate of photosynthesis can be used as a selection criterion for salt tolerance, particularly where a close relationship between photosynthesis and growth under salt stress is found (qasim et al., 2003; ashraf, 2004). if we draw relationships between shoot dry biomass and net co2 assimilation rate of six radish cultivars differing in salt tolerance, growth in terms of dry biomass of all cultivars was positively associated with net co2 assimilation rate (a assimilation rate (a assimilation rate ( vs shoot dry weight r= 0.591***). however, such a relationship was not found when individual radish cultivars differing in salt tolerance were compared with respect to their rate of photosynthesis. for example, salt tolerant cvs. mannu early and desi higher in growth were similar to the salt sensitive radish lal pari in net co2 assimilation rate, at different salt concentrations of the growth medium. these results are in close conformity with some earlier fi ndings in different crops in which a non-signifi cant relationship between growth and photosynthetic rate was observed such as in diplachne fusca (myers et al., 1990), trifolium repens (rogers and noble, 1992), and spring wheat fig. 3: shoot and root k+, cland ca2+ concentrations of six radish (raphanus sativus l.) cultivars subjected to different concentrations of nacl (mean ±s.e; n = 4). values showing mean squares from analysis of variance of data for each variable. *, ** and *** signifi cant at 0.05, 0.01 and 0.001, respectively; ns = non-signifi cant; s = salt stress; cvs = cultivars. fig. 3 shoot and root k , cland ca concentrations of six radish (raphanus sativus l.) cultivars subjected to different concentrations of nacl (±s.e; n =4). values showing mean squares from analysis of variance of data for each variable. *, ** and *** significant at 0.05, 0.01 and 0.001, respectively; ns non-significant; s = salt stress; cvs = cultivars. 0 5 10 15 20 l e a f k + ( m g g -1 d .w t) salt stress 0 mm salt stress 80 mm salt stress 160 mm s,�703.7***;�cvs,�13.87**;�s�x�cvs,�3.64ns 0 5 10 15 r o o t k + ( m g g -1 d .w t) salt stress 0 mm salt stress 80 mm salt stress 160 mm s,�223.9***;�cvs,�17.74***;�s�x�cvs,�5.756*** 0 20 40 60 80 l e a f c l (m g g -1 d .w t) s,�2070.0***;�cvs,�179.2***;�s�x�cvs,�27.29ns 0 5 10 15 20 25 30 35 r o o t c l (m g g -1 d .w t) s,�876.6***;�cvs,�55.31**;�s�x�cvs,�12.92ns 0 2.5 5 7.5 10 p n d l e a f c a 2 + (m g g -1 d .w t) s,�28.45***;�cvs,�2.663*;�s�x�cvs,�0.234ns 0 2.5 5 7.5 10 12.5 r o o t c a 2 + ( m g g -1 d .w t) s,�106.3***;�cvs,�2.13ns;�s�x�cvs,�2.684* r ad is h re d ne ck r ad is h re d ne ck r ad is h la l pa ri r ad is h la l pa ri shoot and root k r ad is h la l pa ri shoot and root k r ad is h la l pa ri r ad is h la l pa ri r ad is h la l pa ri r ad is h m in o r ad is h m in o and ca r ad is h m in o and ca r ad is h m in o r ad is h m in o r ad is h m in o j ap an i j ap an i and ca j ap an i and ca2+ j ap an i 2+ values showing mean squares from analysis of variance of data for each variable. *, ** and *** signifi cant at 0.05, 0.01 and 0.001, respectively; ns = ja pa ni values showing mean squares from analysis of variance of data for each variable. *, ** and *** signifi cant at 0.05, 0.01 and 0.001, respectively; ns = ja pa ni j ap an i j ap an i r ad is h 40 d ay s r ad is h 40 d ay s concentrations of six radish ( r ad is h 40 d ay s concentrations of six radish ( values showing mean squares from analysis of variance of data for each variable. *, ** and *** signifi cant at 0.05, 0.01 and 0.001, respectively; ns = r ad is h 40 d ay s values showing mean squares from analysis of variance of data for each variable. *, ** and *** signifi cant at 0.05, 0.01 and 0.001, respectively; ns = non-signifi cant; s = salt stress; cvs = cultivars. r ad is h 40 d ay s non-signifi cant; s = salt stress; cvs = cultivars. r ad is h 40 d ay s r ad is h 40 d ay s r ad is h 40 d ay s m an nu e ar ly m an nu e ar ly concentrations of six radish ( m an nu e ar ly concentrations of six radish ( values showing mean squares from analysis of variance of data for each variable. *, ** and *** signifi cant at 0.05, 0.01 and 0.001, respectively; ns = m an nu e ar ly values showing mean squares from analysis of variance of data for each variable. *, ** and *** signifi cant at 0.05, 0.01 and 0.001, respectively; ns = non-signifi cant; s = salt stress; cvs = cultivars. m an nu e ar ly non-signifi cant; s = salt stress; cvs = cultivars. m an nu e ar ly m an nu e ar ly m an nu e ar ly d es i d es i concentrations of six radish ( d es i concentrations of six radish (raphanus sativus d es i raphanus sativus values showing mean squares from analysis of variance of data for each variable. *, ** and *** signifi cant at 0.05, 0.01 and 0.001, respectively; ns = d es i values showing mean squares from analysis of variance of data for each variable. *, ** and *** signifi cant at 0.05, 0.01 and 0.001, respectively; ns = d es i d es i d es i r ad is h re d ne ck r ad is h re d ne ck r ad is h la l pa ri r ad is h la l pa ri l.) cultivars subjected to different concentrations of nacl (mean ±s.e; r ad is h la l pa ri l.) cultivars subjected to different concentrations of nacl (mean ±s.e; r ad is h la l pa ri r ad is h la l pa ri r ad is h la l pa ri r ad is h m in o r ad is h m in o l.) cultivars subjected to different concentrations of nacl (mean ±s.e; r ad is h m in o l.) cultivars subjected to different concentrations of nacl (mean ±s.e; r ad is h m in o r ad is h m in o r ad is h m in o j ap an i j ap an i l.) cultivars subjected to different concentrations of nacl (mean ±s.e; j ap an i l.) cultivars subjected to different concentrations of nacl (mean ±s.e; values showing mean squares from analysis of variance of data for each variable. *, ** and *** signifi cant at 0.05, 0.01 and 0.001, respectively; ns = ja pa ni values showing mean squares from analysis of variance of data for each variable. *, ** and *** signifi cant at 0.05, 0.01 and 0.001, respectively; ns = ja pa ni j ap an i j ap an i r ad is h 40 d ay s r ad is h 40 d ay s l.) cultivars subjected to different concentrations of nacl (mean ±s.e; r ad is h 40 d ay s l.) cultivars subjected to different concentrations of nacl (mean ±s.e; values showing mean squares from analysis of variance of data for each variable. *, ** and *** signifi cant at 0.05, 0.01 and 0.001, respectively; ns = r ad is h 40 d ay s values showing mean squares from analysis of variance of data for each variable. *, ** and *** signifi cant at 0.05, 0.01 and 0.001, respectively; ns = r ad is h 40 d ay s r ad is h 40 d ay s r ad is h 40 d ay s m an nu e ar ly m an nu e ar ly l.) cultivars subjected to different concentrations of nacl (mean ±s.e; m an nu e ar ly l.) cultivars subjected to different concentrations of nacl (mean ±s.e; values showing mean squares from analysis of variance of data for each variable. *, ** and *** signifi cant at 0.05, 0.01 and 0.001, respectively; ns = m an nu e ar ly values showing mean squares from analysis of variance of data for each variable. *, ** and *** signifi cant at 0.05, 0.01 and 0.001, respectively; ns = m an nu e ar ly m an nu e ar ly m an nu e ar ly d es i d es i l.) cultivars subjected to different concentrations of nacl (mean ±s.e; d es i l.) cultivars subjected to different concentrations of nacl (mean ±s.e; values showing mean squares from analysis of variance of data for each variable. *, ** and *** signifi cant at 0.05, 0.01 and 0.001, respectively; ns = d es i values showing mean squares from analysis of variance of data for each variable. *, ** and *** signifi cant at 0.05, 0.01 and 0.001, respectively; ns = d es i d es i d es i photosynthetic capacity and ion accumulation in radish 95 (hawkins and lewis, 1993; ashraf and o’leary, 1996). thus, these results show that photosynthetic rate cannot be used as a potential selection criterion for salt tolerance in radish. in the present study, leaf osmotic potential or rwc was also not associated with the growth of six radish cultivars. these results are supported by the conclusive statement of munns (1993) in a comprehensive review that relationship between leaf turgor and salt tolerance occurs occasionally i.e. maintenance of higher plant water status is not associated with salt tolerance. thus, the differential growth of radish cultivars under salt stress may have been due to factors other than water relations. the most prominent effect of salinity on plant growth is the excessive accumulation of na+ and clin the leaves resulting in ionic imbalance, specifi c ion effects, and nutrient-defi ciency symptoms in plants (grattan and grieve, 1999; zhang and blumwald, 2001; munns, 2002; ashraf, 2004). thus, it is imperative to assess pattern of ion accumulation of toxic ions in different plant parts of a crop species to understand as to whether the species uses partial exclusion or inclusion mechanism for tolerating toxic ions present in its growth medium. in the present study, the radish cultivars differed signifi cantly in accumulation of na+ k+, ca2+ and clin the leaves and roots. for example, the relatively salt tolerant cvs. mannu early and desi accumulated relatively higher concentrations of na+ in their roots thereby checking the uptake of this toxic ion to the shoot. such kind of mechanism has already been observed in different crops such as hordeum vulgare (carden et al., 2003), tomato (foolad, 1996), wheat (wyn jones et al., 1984; munns and james, 2003), and trifolium alexandrinum (ashraf et al., 1986). overall, the decline in the growth of all six radish cultivars examined in the present study was due to reduction in photosynthetic capacity and ion accumulation. however, a positive correlation was observed between shoot dry weight and photosynthetic rate as well as reduction in photosynthesis was found to be closely associated with decreased stomatal conductance. however, none of the physiological attributes determined in the present study was found to be effective in discriminating the radish cultivars for salt tolerance. acknowledgment the work presented in this manuscript is a part of research work conducted by ph.d scholar dr. zahra noreen, (pin no. 041212343b-049) whose study was funded by the higher education commission through the indigenous ph.d scheme. references akbar, m., yabuno, t., 1974: breeding for saline resistant varieties of rice. i-comparative performance of some rice varieties to salinity during early development stages. japan. j. breed. 24, 176-181. akram, m.s., ashraf, m., akram, n.a., 2009: effectiveness of potassium sulfate in mitigating salt-induced adverse effects on different physiobiochemical attributes in sunfl ower (helianthus annuus l.). flora 204, 471-483. alam, m.z., stuchbury, t., naylor, r.e.l., rashid, m.a., 2004: effect 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(eds.), salinity tolerance in plants, 189205. wiley, new york. yasar, f., 2007: effect of salt stress on ion and lipid peroxidation content in green beans genotypes. asian j. chem. 19, 1165-1169. yildiz, k., uzal, ö., yilmaz, h., 2008: consequences of naci salinity on growth and on accumulation in selected strawberry cultivars. eur. j. hortic. sci. 73, 69-72. zhang, h.x., blumwald, e., 2001: transgenic salt-tolerant tomato plants accumulate salt in foliage but not in fruit. nat. biotechnol. 19, 765-768. zhu, j.-k., 2001: plant salt tolerance. trends plant sci. 6, 66-71. address of the corresponding author: nudrat aisha akram, e-mail: nudrataauaf@yahoo.com, tel.: +92-41-9201488, fax: +92-41-92001488 journal of applied botany and food quality 95, 94 99 (2022), doi:10.5073/jabfq.2022.095.012 university of alicante age of plant influences the effect of salinity in yield and mineral content of ice plant m.c. rodríguez-hernández*, i. garmendia (submitted: february 21, 2022; accepted: may 18, 2022) * corresponding author summary the use of salinity-tolerant plants represents a response to the problem of the expansion of salinized soils, making coastal and salt affected areas productive. furthermore, limited fresh water resour ces may increasingly constrain the use of low-quality irrigation water. therefore, intensified use of halotolerant crop plants will be necessary. mesembryanthemum crystallinum is a salinity-tolerant plant widely distributed and currently with a great gastronomic interest because is considered a functional food. the objective of this work was to evaluate the differential effect of a moderate salinity treatment imposed in ice plants of 40 or 55 days after transplanting. thus, m. crystallinum of 40 and 55 days were grown under 0 and 100 mm nacl during two weeks. the results showed that the effect of salinity depended of the age of plants. growth parameters as shoot biomass or shoot height decreased in plants of 40 days after transplanting (dat) subjected to salinity while no differences were found in 55 dat plants. also, salinity improved important yield para meters as leaf fresh mass and area when the treatment was applied in 55 dat plants and caused higher sla and chlorophylls content in both groups of plants. ice plant can be intentionally cultivated 55 dat under moderate salinity conditions to enhance crop yield which could contribute to a more extensive use of its edible leaves as functional and alternative food. keywords: mesembryanthemum crystallinum, plant age, glacier lettuce, saline conditions, plant production. introduction recent models of global change have predicted a dramatic increase in desertification (schneider et al., 2007), saline and arid conditions, and a rise in sea levels, therefore, arable land will be increasingly affected (atzori et al., 2017). furthermore, the need for irri gation will be unavoidable in these locations, limiting freshwater resources and the use of quality irrigation water (kundzewicz et al., 2007). on the other hand, it must be taken into account that most conventional crops are sensitive to salt (koyro et al., 2011), resulting in yield reductions due to water deficit stress, ionic toxicity, nutritional disorders and genotoxicity (munns, 2002). therefore, the use of salinity-tolerant crops represents a response to the problem of decreased freshwater availability and the expansion of salinized soils, making coastal and salt-affected areas productive (atzori et al., 2017). in this way, greater sustainable food production would be achieved. halophytic species, tolerant to salinity and native to marshes and saline sites, have the characteristic of being able to grow and reproduce in saline soils, where approximately 99% of other species could not (flowers and colmer, 2008). an alternative is to implement the cultivation of halophytic or sali nity-tolerant plants such as mesembryanthemum crystallinum or ice plant. mesembryanthemum crystallinum is a succulent plant, which has been commonly studied for displaying c3 and cam photosynthesis in response to environmental stresses such as high salinity (abd el-gawad and shehata, 2014). it is native to the nambian desert in southern africa and is widely distributed and naturalized in western australia, southwestern us, the pacific coast of mexico and chile (bohnert and cushman, 2000). during the 18th and 19th centuries m. crystallinum was used in medicine, in bladder problems, gallbladder, whooping cough, tuberculosis or urinary problems, as it acted as a diuretic (raak et al., 2014). in this sense, it is already being consumed as a vegetable crop in several countries such as india, california, australia and new zealand and in some countries of europe (agarie et al., 2009), i.e. in germany (herppich et al., 2008) and in the netherlands (atzori et al., 2017). on the other hand, is well known for its antioxidant activity (ibdah et al., 2002; agarie et al., 2009) and the ability to rapidly accumulate phytochemicals and secondary metabolites, such as beta-carotene, in a cell-specific manner (weeplian et al., 2018). furthermore, ice plant was classified as a highly functional food due to high concentration of polyols. previous work carried out by our group have demonstrated that ice plant cultivated under different salt levels respond in a different way. thus, high level of salinity improved the concentration of bioactive compounds, including its antioxidant properties, but this was in de triment of plant growth. however, irrigation with 100 mm of nacl improved both the production of edible leaves and the accumulation of nutraceuticals (rodríguez-hernández and garmendia, 2022a). in this sense, controlled deficit irrigation has been proposed as an efficient strategy in terms of agronomic management to reduce the use of this scarce resource and improve the organoleptic and functional quality of tomato (martí-renau et al., 2018). similarly, it would be interesting to analyze how the timing of salinity conditions affects plant growth and quality. therefore, we sought to study if the plant age influences the effect of salinity in ice plant. thus, the objective of this work was to evaluate the distinguishing effect of a moderate salinity level on m. crystallinum depending on plant age, focusing on the yield and nutritional quality of its edible leaves. materials and methods plant material and growth conditions seeds of mesembryanthemum crystallinum l. were collected from wild plants in alicante (se spain), disinfected with 0.5% naclo during 2 h and pre-hydrated with aerated, deionized water for 22 h. seeds were germinated in vermiculite hydrated with deionized water and maintained in a growth chamber set to 24 °c air temperature (t) and 70% relative air humidity (rh), day and night (d/n) (thomas et al., 1998). photosynthetically active photon flux rate in the chamber was approx. 400 μmol m-2 s-1 (at a 16/8 h light-dark cycle) supplied by a combination of fluorescent tubes (philips tld 36w/83 germany and silvana f36w/gro, usa). after 20 days, seedlings were transplanted to 1 l plastic pots and transferred to a greenhouse under semi-controlled conditions of t d/n: 25/18 °c; rh d/n: 60/80% and received natural daylight (mean photosynthetic photon flux rate of 400 μmol m-2 s-1) (agarie et al., 2007). pots with vermi culite were watered twice a week with a total of 300 ml of hoagland solution. after 40 days, plants were grown under 0 and 100 mm nacl. then irrigation with hoagland solution was increased to age influences the effect of salinity in ice plant 95 500 ml per week (divided into two irrigation times) in the whole of plants to supply increasing watering demand. and after more 15 days (55 days after transplanting (dat)) another group of plants were grown under the same two conditions described previously. to avoid an osmotic shock, the concentration of nacl was increased gradually during the first week of the salinity treatment to reach the desired nacl concentration, and maintained for one additional week. after two weeks of salinity conditions, plants were harvested and the different determinations were performed (fig. 1). lichtenthaler (1987) were used to calculate the concentration of chlorophylls and carotenoids. for mineral analysis, leaf samples (0.5 g dm) were dry-ashed and dissolved in hcl according to duque (1971). magnesium, potassium, phosphorus, calcium, sodium, manganese, zinc and iron concentrations were determined using a perkin elmer optima 4300 inductively coupled plasma optical emission spectroscopy (icp-oes) (perkin elmer, usa). the operating parameters of the icp-oes were: radio frequency power 1300 w, nebulizer flow 0.85 l min-1, nebulizer pressure 206.84 kpa, auxiliary gas flow 0.2 l min-1, sample introduction 1 ml min-1 and three replicates per sample. total carbon and nitrogen were quantified after combustion (950 °c) of leaf dm with pure oxygen and an elemental analyzer provided with a thermal conductivity detector (truspec cn, leco, usa). statistics the results were analyzed with two-way analysis of variance (anova) by the statistical program spss v.26 (ibm corp., usa). the variance was related to the main factors (salinity treatment and age of plants when salinity was imposed) and to the interaction between them. the means ± standard deviation (sd) were calculated and, when the f ratio was significant (p<0.05), least significant differences were evaluated by the duncan test. significance levels were always set at 5%. results regarding to the growth parameters, differences were observed in the analyzed variables (fig. 2). salinity did not influence shoot dm when salt condition was imposed 55 days after transplanting, while it reduced shoot growth in 40 dat plants (salinity * age, p<0.001). otherwise, salinity did not influence root dm or its length independently of the age of plants (salinity, p>0.05). however, both para meters were lower in 40 dat plants (age, p<0.001). the effect of salinity on shoot to root ratio depended of the age of plants (salinity * age, p<0.001). in fact, it only decreased in plants treated with 100 mm nacl at 40 dat. furthermore, 55 dat plants had lower shoot to root relation than 40 dat control plants (age, p<0.001). concerning shoot height of the plants, it was significantly lower in 40 dat treatment subjected to salinity. the results in tab. 1 indicated that salinity increased the leaf fm production in 55 dat plants (salinity * age, p<0.001), while it did not influence in 40 dat mesembryanthemum plants. as expected, 40 dat plants had a lower leaf fm than the 55 dat plants (age, p<0.001). regarding to the leaf area and sla, salinity influenced both parameters (salinity, p<0.001), with the highest values being found in the saline treatment with 55 dat plants. on the other hand, leaf rwc in 40 dat salt-treated plants was lower than that of controls of the same age and both treatments in 55 dat plants (age, p<0.05). and leaf succulence was significantly higher in 55 dat controls than in salt-grown plants of the same age (salinity * age, p<0.01). in relation to photosynthetic pigments (fig. 3), both the concentration of chlorophylls and carotenoids was higher in the saline treatments, especially in 55 dat plants (salinity, p<0.05; age, p<0.01). macronutrients (tab. 2) and micronutrients (tab. 3) showed, in general, significant differences between treatments. specifically, although the main factors studied did not have significant effect on the foliar c concentration, the amount of foliar n was reduced due to salt conditions (salinity, p<0.01). the concentration of p was not influenced by salinity, although it was affected by the age of the plant (age, p<0.001), with the lowest values presented in 55 dat plants. something similar happened with the foliar k content, which presented the lowest values in the saline treatment of 55 dat plants and did growth parameters plant dry mass (dm) was determined after drying fresh matter at 80 °c in an oven until constant mass which was reached after minimum one week. in addition, shoot and root length were measured. leaf production and water status production of edible part of the plant was measured as leaf fresh mass (fm), and leaf area which was estimated by the app “easy leaf area free” (easlon and bloom, 2014). specific leaf area (sla) was calculated as the ratio of the leaf area and the dry mass of leaves. the leaf relative water content (rwc) was recorded according to weatherley’s method (1950), using the following formula rwc (%): (fm-dm) / (tm-dm) × 100, being fm: fresh mass, tm: turgid mass, and dm: dry mass of the tissue, respectively. foliar succulence was measured according to atzori et al. (2017) by the ratio of the fm of the leaves and the foliar area. biochemical and mineral analysis the analyses were performed on the youngest full-mature leaves harvested at midday, frozen in liquid nitrogen and stored at -20 °c for later quantifications. the concentration of foliar photosynthetic pigments was determined according to sesták et al. (1971). the samples (20 mg fm) were included in 5 ml of 96% ethanol at 80 °c for 10 min to extract the pigments. the absorbance of the extracts was spectrophotometrically measured and the equations reported by fig. 1: illustrative diagram of the experimental design. 96 rodríguez-hernández, m.c., garmendia, i. not show differences between treatments in 40 dat plants (salinity * age, p<0.05). the content of ca and mg was similar in 55 dat treatments and the 40 dat plants presented a lower concentration of these nutrients than 55 dat plants (age, p<0.001). a very marked reduction of ca was observed in 40 dat plants grown with salinity (salinity * age, p<0.01; salinity, p<0.05) and without significant differences between the saline treatments in the case of mg (salinity, p>0.05). the concentration of mn (tab. 3) did not show a clearly established pattern, since the salinity reduced its concentration in 55 dat plants, while its concentration was increased in 40 dat ones (salinity * age, p<0.001). the opposite occurred in concentration of fe, with a significant interaction between the main factors studied (salinity * age, p<0.001). in relation to the zn concentration, no significant differences were observed between treatments in 55 dat plants while in 40 dat plants, there was a decrease of the zn concentration with the salinity. as expected, salinity increased the foliar na concentration in both groups of plants (salinity, p<0.001), fig. 3: leaf photosynthetic pigment concentration in ice plant grown under non-saline (0 mm nacl) and saline (100 mm nacl) conditions, 40 or 55 days after transplanting. means (n=6) ± sd were compared with duncan test. within each variable, values followed by a common letter are not significantly different (p≤0.05). within the graph the explanation for the symbols of anova are: not significant at p>0.05 (ns), significant at p< 0.05 (*), p< 0.01 (**) and p< 0.001 (***). dat: days after transplanting. dm: dry mass. fig. 2: shoot and root dry mass (a), shoot to root ratio (b) and shoot and root length (c) in ice plant grown under non-saline (0 mm nacl) and saline (100 mm nacl) conditions, 40 or 55 days after transplanting. means (n=6) ± sd were compared with duncan test. within each parameter, values followed by a common letter are not significantly different (p≤0.05). within each graph the explanations for the symbols of anova are: not significant at p>0.05 (ns), significant at p< 0.05 (*), p< 0.01 (**) and p< 0.001 (***). dat: days after transplanting. dm: dry mass. tab. 1: leaf fresh weight (fm), leaf area, specific leaf area (sla), relative water content (rwc) and succulence in ice plants grown under non-saline (0 mm nacl) and saline (100 mm nacl) conditions, 40 or 55 days after transplanting. nacl age leaf fm leaf area sla rwc succulence (mm) (dat) (g) (cm2) (cm2 g-1) (%) (g fm cm-2) 0 40 66.53 ± 16.19 c1 133.67 ± 19.13 b 72.47 ± 16.95 c 88.18 ± 7.48 ab 0.51 ± 0.04 b 0 55 99.87 ± 7.16 b 139.12 ± 20.00 b 96.47 ± 12.95 c 88.59 ± 5.39 ab 0.73 ± 0.15 a 100 40 62.58 ± 15.65 c 110.83 ± 23.51 c 125.88 ± 19.58 b 82.41 ± 7.33 b 0.56 ± 0.05 b 100 55 138.09 ± 16.23 a 243.34 ± 16.47 a 178.97 ± 53.94 a 94.22 ± 8.59 a 0.57 ± 0.06 b salinity **2 *** *** ns ns age *** *** ** * *** salinity * age *** *** ns ns ** 1means (n=6) ± sd were compared with duncan test. within each column, values followed by a common letter are not significantly different (p≤0.05). 2means ±sd (n= 6) significance at p< 0.05 according to anova test, not significant at p>0.05 (ns), significant at p< 0.05 (*), p< 0.01 (**) and p< 0.001 (***). dat: days after transplanting c age influences the effect of salinity in ice plant 97 and the plants that presented a higher na concentration were the 40 dat plants treated with salt (age, p<0.001; salinity*age, p<0.001). discussion in response to the hypothesis raised in this work, we assume that the effect attributed to salinity depended in a significant way on the age of the plant according to our results. salinity affected differently shoot dm and shoot height of mesembryanthemum, which had no effect in the 55 dat plants, while in 40 dat ones, 100 mm saline treatment reduced these growth parameters with respect to its controls. this could be due to the fact that 55 dat plants are better adapted to salinity than 40 dat plants according to herppich et al., (1992). however, salinity did not produce differences in root dm or root length despite the fact that, as we expected, both root dm and length were lower in 40 dat plants. thus, root growth parameters indicated that salinity would not have indirect effects on decreased water and nutrient absorption in m. crystallinum. in terms of fresh leaf biomass and leaf area, a moderate salinity was beneficial for the 55 dat ice plant, since the maximum fm and area values of the leaves were obtained with the irrigations with 100 mm nacl according to previous work (rodríguez-hernández and garmendia, 2022b). however, in the 40 dat plants, this effect was not significant in the case of the leaf fm and salinity decreased leaf area. our results in leaf fresh production are in accordance with herppich et al. (2008) who reported that a moderate saline treatment affected m. crystallinum growth only in plants older than 105 days and did not affect younger plants. this different behaviour depending on the age of the plants could be partially explained by the increase in rwc in 55 dat plants irrigated with nacl, although it was not significant and did not occur in the case of the 40 dat plants. similarly, herppich et al. (2008) described that a moderate saline treatment did not significantly affect mean leaf water content between plants of different age. the results can be explained by the adaptative mechanisms to the saline stress that mesembryanthemum crystallinum has, since the bladder cells accumulate water, in addition to salts, to improve ionic and osmotic stress (agarie et al., 2007) and the cam metabolism causes the plant to lose less water, due to stomatal closure during the day (herppick et al., 2008). in the same line were the results of atzori et al. (2017), in which more mature ice plants increased their biomass and area when they were irrigated with moderate salinity, suggesting an extension of vegetative period due to salinity. also, it was observed a significant increase in sla with salinity, this may be due to the increase in leaf area under saline conditions in 55 dat plants, although of the fact that 40 dat plants with greater area were the controls. on the contrary, atzori et al. (2017) found no significant differences in this parameter. in any case, salinity produced an improvement in growth in 55 dat plants, in important variables for the productivity of glacier lettuce, such as the fresh leaf biomass or area, when the salt concentration used was 100 mm nacl. herppich et al. (2008) indicated that the tab. 2: foliar concentrations of macronutrients in ice plant grown under non-saline (0 mm nacl) and saline (100 mm nacl) conditions, 40 or 55 days after transplanting. nacl age c n p k ca mg (mm) (dat) (mg g-1 dm) (mg g-1 dm) (mg g-1 dm) (mg g-1 dm) (mg g-1 dm) (mg g-1 dm) 0 40 373.91 ± 35.75 ab1 64.18 ± 3.79 a 4.20 ± 0.46 a 75.05 ± 13.50 a 4.84 ± 0.85 b 13.26 ± 1.32 b 0 55 431.17 ± 119.95 a 56.39 ± 15.78 ab 2.70 ± 0.83 b 43.78 ± 6.29 b 7.55 ± 1.11 a 18.49 ± 3.49 a 100 40 322.22 ± 53.30 b 52.66 ± 9.68 b 4.29 ± 0.90 a 81.28 ± 9.49 a 2.53 ± 0.47 c 10.90 ± 1.20 b 100 55 370.33 ± 57.40 ab 45.19 ± 7.31 b 2.55 ± 0.66 b 31.82 ± 4.66 c 7.98 ± 1.78 a 17.98 ± 3.60 a salinity ns2 ** ns ns * ns age ns ns *** *** *** *** salinity * age ns ns ns * ** ns 1means (n=6) ± sd were compared with duncan test. within each column, values followed by a common letter are not significantly different (p≤0.05). 2means ±sd (n= 6) significance at p< 0.05 according to anova test, not significant at p>0.05 (ns), significant at p< 0.05 (*), p< 0.01 (**) and p< 0.001 (***). dat: days after transplanting dm: dry mass tab. 3: foliar concentrations of micronutrients in ice plant grown under non-saline (0 mm nacl) and saline (100 mm nacl) conditions, 40 or 55 days after transplanting. nacl age mn zn fe na (mm) (dat) (μg g-1 dm) (μg g-1 dm) (μg g-1 dm) (mg g-1 dm) 0 40 107.91 ± 29.18 d1 64.72 ± 16.44 a 1509.37 ± 339.51 b 4.04 ± 0.46 c 0 55 483.07 ± 66.04 a 67.06 ± 1.79 a 1666.48 ± 474.77 b 3.41 ± 1.02 c 100 40 194.63 ± 10.26 c 49.78 ± 14.45 b 471.32 ± 294.84 c 75.82 ± 6.32 a 100 55 320.47 ± 37.30 b 65.60 ± 5.52 a 2305.05 ± 494.23 a 24.01 ± 7.72 b salinity *2 ns * *** age *** ns *** *** salinity * age *** ns *** *** 1means (n=6) ± sd were compared with duncan test. within each column, values followed by a common letter are not significantly different (p≤0.05). 2means ±sd (n= 6) significance at p< 0.05 according to anova test, not significant at p>0.05 (ns), significant at p< 0.05 (*), p< 0.01 (**) and p< 0.001 (***). dat: days after transplanting dm: dry mass 98 rodríguez-hernández, m.c., garmendia, i. repetitive irrigation with salinity did not negatively affect the growth of the plant, as in winter (1973), in which the maximum growth was obtained with a concentration of 100 mm nacl. in fact, atzori et al. (2017) observed growth reduction only in the youngest stage of plants and with high salinity level. similarly, herppich et al. (1992) obtained that in 10 dat plants, still in development, salinity caused a delay in their phenology. it was suggested that the adaptive mechanisms that make glacier lettuce tolerant against salinity, bladder cells and c3-cam facultative metabolism, are not fully developed in younger plants, so they are more sensitive to salinity. our results corroborated that at the beginning of the cultivation of the ice plant, the concentration of salts in the irrigation water should be adjusted, being able to increase salinity in stages over two months after plant transplanting. due to the adaptations to salinity developed in m. crystallinum, it was expected that the plant had greater succulence under salini ty treatment. on the contrary, in this work and in discordance with atzori et al. (2017), 55 dat ice plants had greater succulence in the control. since succulence is calculated as the ratio of leaf fm between the leaf area, the increase in leaf area caused by saline conditions in 55 dat plants would explain a decrease in succulence. however, in 40 dat plants there were no significant differences in water status between the control plants and those treated with nacl. adams et al. (1998) found that juvenile ice plants have a smaller number and size of bladder cells, which would explain that the results are different depending on the age of the plant. in relation to photosynthetic pigments, salinity had no detrimental effects on the concentration of chlorophylls, and even in both 40 and 55 dat plants, was higher in the plants treated with salinity. ac cording to argentel et al. (2006), the halophilic plant m. crystallinum increased the activity of the chlorophyllase enzyme, which affects the synthesis of chlorophylls under saline conditions. herppich et al. (2008) explained the increase in chlorophyll by a general physio logical stimulation in response to salinity. furthermore, barker et al. (2004) observed that salinity did not decrease the concentration of carotenoids as in our work, since in both 40 and 55 dat plants the concentration of carotenoids was slightly higher in plants irrigated with 100 mm nacl than in the controls. these results coincide with those obtained by atzori et al. (2017) where the concentration of carotenoids increased with the increase in salinity. salinity did not influence the concentration of c, p and mg independently of the date when the saline condition was imposed. otherwise, as expected, the addition of nacl increased the foliar concentration of na in ice plants, demonstrating that m. crystallinum acts as a sequestrant of salts, due to the presence of bladder cells (agarie et al., 2007). moreover, adams et al. (1998) found that in mature leaves more na is concentrated than in young and that young leaves have a smaller number and size of bladder cells. this research suggests less compartmentalization of salts in bladder cells of young leaves, which would cause toxic effects to the plant and explain the worst results in growth parameters as leaf fm with salinity in 40 dat plants. our results showed that leaves of 55 dat plants accumulated less na, probably related to a dilution effect associated to the increase of their leaf biomass production. potasium, ca and mg are antagonists of na, thus in plants not adapted to salinity a decrease in their concentration would be expected, due to a displacement by na from the cell membrane binding sites (niu et al., 1995; dodd et al., 2010). however, ca and mg remained in variable with salinity in 55 dat plants and k in 40 dat ones, while the concentration of k decreased with salinity in 55 dat plants and ca in 40 dat plants. in the study by atzori et al. (2017) na was accumulated in ice plants irrigated with salt, while k decreased with increasing salinity. these results are in the same line as those obtained in our study in 55 dat plants. in addition, 40 dat plants subjected to 100 mm nacl, the foliar mn concentration increased and n, ca, zn and fe decreased. perhaps the decrease of some of these minerals under saline conditions, was another factor that caused the depletion of some growth parameters in 40 dat plants. conclusions the favourable results in the growth parameters such as greater fresh foliar mass and area in 55 dat plants, indicate that ice plant can not only be a suitable crop for breeding under moderately saline conditions (100 mm), but also stimulate the development of its edible part. furthermore, parameters as sla, rwc, pigments and nutrients as c, n, p, ca, mg, zn and fe remained unchanged or enhanced in 55 dat plants treated with salinity, showing that these plants maintain or increase its nutritional quality under these conditions. however, in 40 dat plants the adaptive mechanisms seem to be not fully developed, so they show more sensitive to salinity. according to our results, the implementation of the cultivation of glacier lettuce under greenhouse conditions could be performed with moderately salinized water resources, especially in plants over 8 weeks of growth. acknowledgments the authors wish to thank to susana carrión and mar benavent for their help with biomass analysis and laboratory determinations. conflicts of interest no potential conflict of interest was reported by the authors. funding this work was financed by the valencian community – spain (gv/2018/068). references abd el-gawad, a.m., shehata, h.s., 2014: ecology and development of mesembryanthemum crystallinum l. in the deltaic mediterranean coast of egypt. j. basic appl. sci. 1(1), 29-37. doi: 10.1016/j.ejbas.2014.02.003 adams, p., nelson, d., yamada, s., chmara, w., jensen, r.g., bonhert, h.j., griffiths, h., 1998: growth and 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t., 2014: mesembryanthemum crystallinum l. as a dermatologically effec tive medicinal plant − first results from 3 pilot studies. forsch. kom plementmed. 21, 366-373. doi: 10.1159/000369909 rodríguez-hernández, m.c., garmendia, i., 2022a: optimum growth and quality of the edible ice plant under saline conditions. j. sci. food agric. 102(7), 2686-2692. doi: 10.1002/jsfa.11608 rodríguez-hernández, m.c., garmendia, i., 2022b: only low saline conditions benefited yield and quality of ice plant grown in pot culture. j. appl. bot. food qual. 95, 17-22. doi: 10.5073/jabfq.2022.095.003 schneider, s.h., semenov, s., patwardhan, a., burton, i., magadza, c.h.d., oppenheimer, m., pittock, a.b., rahman, a., smith, j.b., suarez, a., yamin, f., 2007: assesing key vulnerabilities and the risk from climate change. in: parry, m.l., canziani, o.f., palutikof, j.p., van der linden, p.j., hanson, c.e., (eds.), climate change 2007: impacts, adaptation and vulnerability. contribution of working group ii to the fourth assesment report of the intergovernmental panel on climate change. cambridge university press, 20. sesták, z., càtsky, j., jarvis, p., 1971: plant photosynthetic production: manual of methods (1st ed.). dr. w. junk publishers, 818. thomas, j., malick, f., endreszl, c., davies, e., murray, k., 1998: distinct responses to copper stress in the halophyte mesembryanthemum crystallinum. physiol plant. 102, 360-368. doi: 10.1034/j.1399-3054.1998.1020304.x weatherley, p.e., 1950: studies in the water relations of the cotton plant: the field measurements of water deficits in leaves. new phytol. 49, 81-89. doi: 10.1111/j.1469-8137.1950.tb05146.x winter, k., 1973: co2 fixation reactions in the salt plant mesembryanthemum crystallinum under varied external conditions. planta. 114, 75-85. orcid m.c. rodríguez https://orcid.org/0000-0001-6896-8067 idoia garmendia https://orcid.org/0000-0002-8673-6515 address of the authors: department of earth and environmental sciences, university of alicante, alicante, po box 99, 03080 spain e-mail: maricarmen.rodriguez@ua.es © the author(s) 2022. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1007/s003440000033 http://dx.doi.org/10.1007/s11104-009-0196-6 http://dx.doi.org/10.3732/apps.1400033 http://dx.doi.org/10.1111/j.1469-8137.2008.02531.x http://dx.doi.org/10.1111/j.1438-8677.1992.tb00264.x http://dx.doi.org/10.1046/j.1365-3040.2002.00895.x http://dx.doi.org/10.1016/0076-6879(87)48036-1 http://dx.doi.org/10.1016/j.foodchem.2018.01.098 http://dx.doi.org/10.1046/j.0016-8025.2001.00808.x http://dx.doi.org/10.1104/pp.109.3.735 http://dx.doi.org/10.1159/000369909 http://dx.doi.org/10.1002/jsfa.11608 http://dx.doi.org/10.5073/jabfq.2022.095.003 http://dx.doi.org/10.1034/j.1399-3054.1998.1020304.x http://dx.doi.org/10.1111/j.1469-8137.1950.tb05146.x https://orcid.org/0000-0001-6896-8067 https://orcid.org/0000-0002-8673-6515 art18_nemeth.indd journal of applied botany and food quality 85, 116 119 (2012) 1department of medicinal and aromatic plants, corvinus university of budapest, hungary connection of frost tolerance and alkaloid accumulation potential in poppy (papaver somniferum l.) c. jászberényi1, é. németh1 (received december 22, 2011) summary in the central-european region, a growing interest for winter poppy varieties both for industrial and culinary purposes can be observed. in connection with breeding, we studied if winter hardiness and alkaloid accumulation capacity are linked in some way. experimental plots sown in autumn and thinned (selected) due to winter frosts were compared with spring sown ones of the same genotypes during 2009-2010. we found, that the overwintered populations exhibited signifi cantly lower total alkaloid contents (1.065%) than the ones from spring sowing (1.479%). this tendency was proved for each of the individual alkaloid compounds morphine, codeine, thebaine and narcotine, too. at the same time, maximum values in some individual samples of the overwintered plants reached similar or higher contents than the spring sown ones. therefore it is supposed that even if gene frequency in direction for enhanced alkaloid synthesis might be larger in frost sensitive ecotypes, the potential for breeding high alkaloid accumulating cultivars exists also among frost tolerant genotypes. introduction in hungary, poppy (papaver somniferum l.) is an important industrial crop which is cultivated on large scale thorough the country, on more than ten thousand hectares. besides, poppy is an essential culinary crop producing delicious, fatty oil containing seeds both for food industry and the households. in the central-european region, especially in hungary and austria, poppy cultivation may take place either as winter or a spring crop, similarly to cereals. winter poppy ecotypes possess enhanced frost tolerance in leaf rosette stage, while spring poppy is generally killed or severely damaged by frosts under average winter conditions. cultivation of winter poppy in our region obtained enhanced signifi cance in the last decade, because of the higher and more stable yields among the changing and varying climatic conditions. until recently, breeding has not been focused on this aspect. in the hungarian list of varieties, only fi ve cultivars belong to the winter poppy group while 14 ones are registered as spring poppy cultivars. breeding has now started to establish new, frost tolerant genotypes which possess also high capsule and seed yields and can be used either as industrial varieties of high alkaloid contents or as culinary ones with very low alkaloid contents in coincidence with the 162/ 2003. (x. 16.) government decree of hungary. unfortunately, there is practically no scientifi c background information on the genetical determination of frost tolerance in poppy. not even the physiological or morphological protection mechanisms against low temperatures are fully ascertained. our recent results suggested, that proline and soluble sugars (glucose, fructose, sucrose) might contribute to the osmotic protection mechanism against cold stress, however, presumably it is not the only and main factor in it (jászberényi and németh, 2011a). dobos (2010) observed that winter poppy is a morphologically adapted genotype with greyish coloured and horizontally laying leaf rosette, violet coloured petals and shorter vegetation cycle compared with spring types. however, these traits may only marginally stay in real connection with winter hardiness. information on the inheritance of opiate alkaloids is much more abundant (bernáth and németh, 2009; németh et al., 2011). however, it has been proved, that accumulation of individual alkaloids is regulated in a complex, metabolon like system including multigene effects, enzymatic interactions and compartmentation. therefore, till now practical results in breeding have mainly been achieved by individual selection. in the practice it seems to be accepted that winter tolerant genotypes are not able to accumulate high levels of alkaloids. however, as no scientifi c information and experimental results exist on the linkage of the winter hardiness and alkaloid content, it can be presumed as a statement based on limited information and experience. therefore, in our work we studied the question if there is any well established connection between alkaloid accumulation and the overwintering potential of poppy genotypes. using several breeding strains – because of the varieties of original spring ecotype usually killed by frost – and sowing them both in autumn and spring, we examined if the selection by winter frosts resulted in any shift in alkaloid accumulation level and/or pattern of the capsules of the same genotypes. materials and methods our examination was carried out at the research fi eld of the department of medicinal and aromatic plants of corvinus university of budapest, in 2009-2010. the experimental area is located in the south-eastern part of budapest, at a geographical elevation of 100-150m. the soil is light sand, the organic matter content is low (0.2-0.4%), humus content is 1.5%, nitrogen and phosphorous levels medium, potassium levels show good supply. fig. 1 and 2. illustrate the precipitation and the temperature values during the examination period. different crossed f2 and f3 breeding strains were used for this experiment. its reason was that based on the earlier experiences the varieties belonged to spring ecotype are often damaged or killed by frost in winter, contrary to the winter poppy varieties which are able to overwinter. the experimental population propagated by selfi ngs afterwards (jászberényi and németh, 2011b). the parental combinations were as follows: ‘medea’ x ‘no.67’, ‘minoán’ x ‘kozmosz’, ‘medea’ x ‘kozmosz’, ‘korona’ x ‘no.67’, ‘korona’ x ‘kozmosz’, ‘ametiszt’ x ‘leila’, ‘no.1/172’ x ‘leila’. the names in italics show registered varieties, while the ones indicated by numbers were selected and stabilized breeding strains. they included both high and low alkaloid accumulating genotypes. reciprocal crossings were handled jointly. each progeny represented the full genotypic constitution without previous selection into any direction. sowing of each seed plot was carried out at the end of september 2009 and at the end of march 2010 by hand into open fi eld. the plots were 10m2 in 3 replications. during the vegetation period, the usual poppy agrotechnics had been applied. to avoid the effects of metaxenia (bernáth et al., 2003), isolation of individual fl owers was carried out before anthesis with removing the isolation after ecotypes of poppy 117 fertilisation. the capsules were harvested on 7th july (autumn sown plots) and the 13th of july (spring sown plots). analysis of alkaloids was carried out by tlc method in 12 replications per population (németh et al., 2011). after extraction of pulverized capsules in chloroformmethanol (4:1) solvent in soxhlet apparatus, for separation horizontal tlc chambers (htrennkammer desaga nr. 120150) were used. evaluation was carried out by densitometric analysis (chr-scan tr-541 equipment with a labchromtm ried out by densitometric analysis (chr-scan tr-541 equipment tm ried out by densitometric analysis (chr-scan tr-541 equipment chromatographic data processing system version 5.2). the densitometric scanning profi les of four alkaloids (morphine, codeine, thebaine and narcotine) were calibrated against the corresponding standards. the accuracy of the measurements is the level of 0.0001%, coeffi cient of variation of parallel measurements is 3.19%. in the following, alkaloid data refer to the content of ripe capsules in % dry mass. biometric evaluation of data (descriptive statistics, distributions fi tting, t-test) was carried out by pasw statistics 18 software. results based on fi eld evaluation at the end of november and beginning of april, density of the overwintered f2 and f3 populations decreased by 30-50%. it proves the intended natural selection by winter frosts and shows in general a medium level frost tolerance. alkaloid content of the experimental populations because of the different levels, the evaluation of the total alkaloid content of the experimental population were at fi rst carried out in two groups: combinations of low alkaloid containing varieties and that of high alkaloid containing cultivars. in both categories, samples from autumn sown plots are inferior (0.37% and 1.27%, respectively) compared to the spring sown ones (0.57% and 1.76%, respectively). standard deviations of data in autumn sown and spring sown plants are practically the same (0.66; 0.68) in the high alkaloid group, while it is somewhat different in the low alkaloid group (0.26; 0.48). in fig. 3. distributions of the studied samples are illustrated together with the fi tted normal equations whose peaks show the mean values of the populations. the negative shift in the alkaloid content of the overwintered plots is obvious. evaluation has been carried out also according hybrid strains, separately. comparing the total alkaloid values of the same genotypes originating from autumn or spring sowing, it could be established that in the majority of cases there are signifi cant deviations between them (tab. 1). the presence of this difference seems to be independent of the genetic combination or of the fact if it is an f2 or f3 generation. not only means but also the marginal values show the difference among the experimental populations. except the combinations ‘korona’ x ‘kozmosz’, ‘ametiszt’ x ‘leila’, ‘no.1/172’ x ‘leila’, the minimum values of the strains are lower in autumn sown plots than in the spring sown ones in each case. the lowest individual value in the populations is 0.10% both in case of autumn hybrids or the population not being selected by frost (tab. 1.). the maximum values are even more interesting. existence of some single plants in the frost selected population reaching similar values than the ones in the non-selected ones may indicate the potential of winter hardy genotypes for high alkaloid accumulation. in the recent study both similar (‘medea’ x no.67 f2, ‘medea’ x ‘kozmosz’ f3) and higher (‘minoán’ x ‘kozmosz’ f2, ‘korona’ x no. 67 f3) individual values had been found in the autumn sown plots compared 0,0 2,0 4,0 6,0 8,0 10,0 12,0 09 .09 .3r d 10 .09 .1s t 10 .09 .2n d 10 .09 .3r d 11 .09 .1s t 11 .09 .2n d 11 .09 .3n d 12 .09 .1n d 12 .09 .2n d 12 .09 .3n d 01 .10 .1n d 01 .10 .2n d 01 .10 .3n d 02 .10 .1s t 02 .10 .2n d 02 .10 .3r d 03 .10 .1s t 03 .10 .2n d 03 .10 .3r d 04 .10 .1s t 04 .10 .2n d 04 .10 .3r d 05 .10 .1s t 05 .10 .2n d 05 .10 .3r d 06 .10 .1s t 06 .10 .2n d 06 .10 .3r d 07 .10 .1s t 07 .10 .2n d mm precipitation -15,0 -10,0 -5,0 0,0 5,0 10,0 15,0 20,0 25,0 30,0 35,0 09 .09 .3r d 10 .09 .1s t 10 .09 .2n d 10 .09 .3r d 11 .09 .1s t 11 .09 .2n d 11 .09 .3n d 12 .09 .1n d 12 .09 .2n d 12 .09 .3n d 01 .10 .1n d 01 .10 .2n d 01 .10 .3n d 02 .10 .1s t 02 .10 .2n d 02 .10 .3r d 03 .10 .1s t 03 .10 .2n d 03 .10 .3r d 04 .10 .1s t 04 .10 .2n d 04 .10 .3r d 05 .10 .1s t 05 .10 .2n d 05 .10 .3r d 06 .10 .1s t 06 .10 .2n d 06 .10 .3r d 07 .10 .1s t 07 .10 .2n d °c mean min max fig. 2: the temperature values at the experimental fi eld in 2009/2010 from the time of sowing to harvesting by decadal (oc) fig. 1: precipitation amount at the experimental fi eld in 2009/2010 from the time of sowing to harvesting by decadal (mm) 118 c. jászberényi, é. németh with the spring sown ones. nevertheless, in majority of cases, the populations’ maximum values were higher in the non-frost selected, spring sown plots (1.00%-4.20%) (tab. 1.). the total mean standard deviation of the tested combinations in autumn and spring sowing plots are 0.70 and 0.82 respectively. it indicates similar order of magnitude and no obvious connection with the experimental treatments. alkaloid spectrum of the experimental combinations fig. 4 shows the levels of four measured alkaloid compounds: morphine, codeine, thebaine and narcotine, separately. (as any other alkaloids are present only in traces, the total values are identical with the mean values of the total alkaloid levels in tab. 1.). it seems that in the majority of strains each alkaloid compound was reduced in the samples from overwintered plants, although a signifi cant difference could be proved only for thebaine. opposite tendencies were found only in two cases: for codeine content in f2 population of the cross ‘medea’x no.67 and for narcotine content in f3 population of the cross ‘korona’ x no. 67. mean values for morphine, codeine, thebaine and narcotine in samples of overwintered plants are 0.68, 0.06, 0.16 and 0.16%, respectively. the same compounds in samples of spring sown plants were 0.84, 0.10, 0.34 and 0.21%, respectively. maximum values of alkaloids in the overwintered populations were 1.12, 0.29, 0.50 and 0.53 for morphine, codeine, thebaine and narcotine, respectively, while they reached 1.26, 0.49, 0.93 and 0.60% for these four compounds, respectively. discussion the study focused on the question if alkaloid accumulation may be limited to winter hardy genotypes of poppy as frequently anticipated in the practice. fig. 3: distributions of experimental data from autumn sown (dark) and spring sown (light) plots. left: high alkaloid containing group; right: low alkaloid containing group of hybrids. tab. 1: total alkaloid content values of the experimental populations in autumn and spring sown (selected by frosts) plots total alkaloid content (%) autumn sowing spring sowing min. max. mean std. dev. min. max. mean std. dev. f2 ’medea’ x no. 67 0.10 2.90 1.54 a 0.94 0.60 2.90 1.90 a 0.63 ’minoán’ x ’kozmosz’ 0.15 2.20 1.24 a 0.48 0.70 2.05 1.39 a 0.47 ’medea’ x ’kozmosz’ 0.40 2.30 1.43 a 0.61 1.15 2.60 1.91 b 0.46 ’korona’ x no. 67 0.40 2.20 1.28 a 0.54 1.10 4.20 2.18 b 0.90 ’korona’ x ’kozmosz’ 0.30 1.70 0.88 a 0.44 0.30 2.50 1.59 b 0.80 ’ametiszt’ x ’leila’ 0.05 1.05 0.48 a 0.30 0.00 1.20 0.51 a 0.41 ’no. 1/172’ x ’leila’ 0.00 0.85 0.36 a 0.22 0.00 2.30 0.58 a 0.72 mean 0.27 2.26 1.04 a 0.69 0.77 2.85 1.43 b 0.88 f3 ’medea’ x no. 67 1.05 2.40 1.73 a 0.43 1.70 3.20 2.37 b 0.44 ’minoán’ x ’kozmosz’ 0.20 2.30 1.26 a 0.64 0.85 2.95 1.84 b 0.64 ’medea’ x ’kozmosz’ 0.00 2.50 1.23 a 0.60 0.60 2.60 1.56 a 0.54 ’korona’ x no. 67 0.25 3.10 1.33 a 0.90 0.55 2.10 1.36 a 0.47 ’korona’ x ’kozmosz’ 0.10 1.65 0.78 a 0.38 0.50 2.70 1.52 b 0.74 ’ametiszt’ x ’leila’ 0.00 0.75 0.28 a 0.23 0.25 1.00 0.61 b 0.25 mean 0.32 2.39 1.10 a 0.72 0.84 2.71 1.54 b 0.74 total mean 1.07 a 0.70 1.48 b 0.82 ecotypes of poppy 119 the experimental plots thinned due to winter frosts exhibited signifi cantly lower total alkaloid contents than the samples from spring sown plots of the same populations. although in open fi eld, weather conditions – especially summer rain – may contribute to alkaloid accumulation and eventual losses (bernáth and tétényi, 1981), our fi ndings ascertained the old practical observations. the lower level in overwintered plants was proved for each of the individual alkaloid compounds morphine, codeine, thebaine and narcotine, too. the genes which might be in linkage with those regulating frost tolerance seem to be more likely present at the primarly level of alkaloid biosynthesis. these regulate the synthesis of general precursors and not the characteristic opiate alkaloid compounds of the poppy capsule formed at the fi nal stages of biosynthesis (ziegler et al., 2009). clarifi cation of this question needs further research. however, at the same time, maximum values in some individual samples of the overwintered plants reached similar or higher contents than the spring sown ones. therefore it is supposed that even if gene frequency in direction for high alkaloid level might be larger in frost sensitive ecotype, the potential for breeding high alkaloid accumulating cultivars exists also among frost tolerant genotypes. acknowledgement the work was supported by otka grant no. k 62732 and támop4.2.1/b-09/1/kmr-2010-0005 grant. references bernáth, j., tétényi, p., 1981: the effect of environmental factors on growth, development and alkaloid production of poppy (papaver somniferum l.) ii. interaction of light and temperature. biochem. physiol. pfl . 176, 599-605. bernáth, j., németh, é., 2009: breeding of poppy. in: vollmann, j., rajcan, i. (eds), oil crops, 449-468. springer series „handbook of plant breeding” springer sciences + business media, dordrecht. bernáth, j., németh, é., petheő, f., 2003: alkaloid accumulation in capsules of the selfed and cross-pollinated poppy. plant breeding 122, 263-267. dobos, g., 2010: personal communication government regulation 162/ 2003. (x. 16.) on the order of growing, traffi cking and using plants suitable for producing drugs. jászberényi, c., németh, é., 2011a: frost tolerance of spring and winter ecotypes of poppy (papaver somniferum l.). international symposium on papaver, book of abstracts, 7-11, february, lucknow, india. jászberényi, c., németh, é., 2011b: observation on the inheritance of some morphological characteristics of poppy (papaver somniferum l.). kertgazdaság 43, 53-62. németh-zámbori, é., jászberényi, cs., rajhárt, p., bernáth, j., 2011: evaluation of alkaloid profi les in hybrid generations of different poppy (papaver somniferum l.) genotypes. industrial crop. prod. 33, 690-696. ziegler, j., facchini, p.j., geissler, r., schmidt, j., ammer, c., kramell, r., voigtlander, s., gesell, a., pienkny, s., brandt, w., 2009: evolution of morphine biosynthesis in opium poppy. phytochem. 70, 1696-1707. address of the authors: csilla jászberényi (corresponding author) and éva németh, department of medicinal and aromatic plants, corvinus university of budapest, villányi str. 35-43., h-1118 budapest, hungary fig. 4: alkaloid spectrum of the experimental populations in autumn (’a’) and spring (’s’) sown (selected by frosts) plots 0,0 0,5 1,0 1,5 2,0 2,5 f2 _m ex 67 _a f2 _m ex 67 _s f2 _m i x ko _a f2 _m i x ko _s f2 _m ex ko _a f2 _m ex ko _s f2 _k or x6 7_ a f2 _k or x6 7_ s f2 _k or xk o_ a f2 _k or xk o_ s f2 _a m xl e_ a f2 _a m xl e_ s f2 _1 /17 2 x le _a f2 _1 /17 2 x le _s f3 _m ex 67 _a f3 _m ex 67 _s f3 _m i x ko _a f3 _m i x ko _s f3 _m ex ko _a f3 _m ex ko _s f3 _k or x6 7_ a f3 _k or x6 7_ s f3 _k or xk o_ a f3 _k or xk o_ s f3 _a m xl e_ a f3 _a m xl e_ s al ka lo id co nt en t( % ) morphine codeine thebaine narcotine art02_oz_neu.indd journal of applied botany and food quality 84, 125 133 (2011) 1 engineering faculty, department of food engineering, university of osmaniye korkut ata, karacaoglan campus 80000 osmaniye,turkey 2 science and arts faculty, department of biology, university of osmaniye korkut ata, karacaoglan campus 80000 osmaniye,turkey effects of 1-methylcylopropene (1-mcp) and modifed atmopshere packing (map) on postharvest browning and microbial growth of loquat fruit a.t. oz1*, z. ulukanli2 (received october 20, 2011) * corresponding author summary "champagne de grasse" loquat fruits were initially treated with 0 (control), 312.5 ppb and 625 ppb of 1-methylcylopropene (1-mcp) for 6 h at 20°c. after 6 h, treated fruits were divided into the two main groups: within the fi rst main group, 1-mcp treated fruits were placed in modifed atmopshere packing (map) and stored for 12 days at 5°c with 90% of relative humidity (rh). control fruits without exposure to 1-mcp were placed in map and maintained under the identical storage conditions. within the second main group, 1-mcp treated fruits were subjected to two different storage temperatures. the fi rst were placed in trays and then kept for 12 days of storage at 5°c with 90% of rh and the second group was stored for 9 days at 20°c with 60% of rh. similarly, the control not subjected to 1mcp treatment was maintained under the same condition. treatment groups and control were examined for the following parameters: the fl esh fruit fi rmness, total soluble solid (tss) content, titratable acidity (ta), color value (l and chroma), browning index and microbial quality. unpacked 1-mcp treatments (312.5 and 625 ppb) improved the storage quality of loquat fruits, but, the most effi cient treatment was achieved with 625 ppb of 1-mcp. however, the combination of 1-mcp and map revealed a better effect on maintaining the fruit quality as indicated by the delay of softening, deterioration rates, ta, browning rate and the signifi cant inhibition of total aerobic mesophiles, psychrotrophic aerobic bacteria, yeast and molds during the whole storage period. the results indicated that the most effi cient application in all treatments was obtained using the combination of 1-mcp (625 ppb) with map at 5°c storage. introduction loquat (eriobotrya japonica lindl.) is a member of the rosaceae family that is widely cultivated in the subtropical regions of china, japan, india, israel and the mediterranean area (shaw, 1980). turkey is one of the largest loquat producers known, dating back to over 30 years. approximately 96% of the total production of loquats is primarily concentrated in the two provinces of mediterranean region: antalya and mersin. a large proportion of fruits produced in turkey are consumed mainly as fresh fruit and are also exported to kuwait, saudi arabia, russia, jordan, germany and other european countries (karadeniz et al., 2009). most fruits such as loquat fruits are highly perishable commodities, susceptible to decay, mechanical damage, moisture losses and microbial decay during the postharvest life. consumer interest worldwide in the quality of fresh products has increased in recent years. product quality is a complex issue, since it includes visual characteristics, physical properties such as texture, mineral and vitamin content, fl avor, and other organoleptic characteristics. once fruits are harvested, postharvest handling practices do not usually improve on the quality attained in the fi eld; they only may slow the rate at which deterioration occurs (fallik, 2008). packaging is one of the most important processes to maintain the quality of food products for storage, transportation and end-use (han, 2005). modifi ed atmosphere packaging extends the shelf life of food products and prevents (or at least retards) any undesirable changes in the wholesomeness, safety, sensory characteristics, and nutritive value of foods. 1-methylcyclopropene (1-mcp) is an inhibitor of ethylene perception that binds irreversibly to the ethylene binding protein. it is currently known to have commercial applications for maintaining the storage life of horticultural products, with highly effective, with nontoxic and odorless properties (sisler and serek, 1997). although ethylene-independence was observed during the ripening of some non-climacteric fruits, several studies revealed that ethylene has extended the storage life of some non climacteric fruits including cherries, citrus and strawberries (shaw, 1980). the effi cacy of 1-mcp treatment or map storage has been previously evaluated for their ability to maintain loquat fruit quality (ding et al., 1998; 2002; cai et al., 2000). however, literature surveys indicated that the combined effect of 1-mcp and map on quality attributes and shelf life of loquat fruits has not been investigated yet. therefore, the purpose of this study was to determine the combined effect of 1-mcp and map on changes in fruit quality (fi rmness, skin color, tss, ph, ta, browning rate), and shelf life of ‘champagne de grasse’ loquat fruit during refrigerator storage at 5°c and room temperature at 20°c. materials and methods fruit, treatment and storage "champagne de grasse" loquat fruits were hand-harvested at ripe stage according to fruit skin color (fully yellow). the loquats were obtained from an orchard in district erdemli (mersin) and held in a cardboard box and alveolar trays during transport to the laboratory and analyzed within 3 h of harvest. loquats were graded according to visual defects and uniformity of shape, size and color. fruits were placed in l m3 containers before the beginning of the 1-mcp treatment. commercially available 1-mcp tablets were obtained from agrofresh inc./rohm and haas company and the desired concentration of 1-mcp was prepared according to the manufacturer’s instruction (ethylbloc, bio-technologies for horticulture, walterboro, sc). briefl y, each individual 1-mcp tablet including 312.5 ppb of 1-mcp was added into 15 ml of solution. the recommended dose of two tablets for upper concentration was also used in this investigation. afterwards, the tube containing 1-mcp tablet plus solvent was shaken thoroughly and immediately transferred to the polyethylene container. the ventilation process for each 1-mcp treatment in the polyethylene container including airtight lid were carried out for 6 h at 20°c and 60% relative humidity. upon exposure to 1-mcp treatments, one set of fruits was divided and maintained under the following conditions: i) 312.5 ppb of 1-mcp+unpacked state; ii) 625.5 ppb of 1-mcp+unpacked state; iii) not treatment with 1-mcp (control)+unpacked state. after treatment, all unpacked loquats (treated and control) were stored for 12 days at 5°c with 90% rh and for 9 days at 20°c with 60% of rh. unless otherwise stated, analyses of unpacked 1-mcp and control fruits were examined on 126 a.t. oz, z. ulukanli the day of 0 and after 3, 6, and 9 days of storage at 20°c. in addition, all fruits stored at 5°c were examined on the day of 0 and after 4, 8, and 12 days of storage. the other set of loquats subjected to the same 1-mcp treatment as mentioned above were divided and maintained under the modifi ed atmosphere packaging (also called polyethylene bulk liner, 30µ /p-plus antimist, 700x700 in bag size) : i) 312.5 ppb of 1-mcp+map; ii) 625.5 ppb of 1-mcp+map; iii) not treatment with 1-mcp (control) + map. 1-mcp+map and control fruits were stored for 12 days at 5°c with 90% rh. the fruits were taken and examined on the day of 0, 4, 8, and 12 in order to determine and compare the effects of mcp treatments+map and control stored at 5°c. fruit fi rmness and total soluble solids fruit fi rmness was determined by selecting 10 fruits per treatment at intervals during storage period. fruit fi rmness was measured using a fhr-1 (1 kg) fi rmness tester (nippon optical works co. ltd.tokyo-japan) equipped with a cylinder type probe (5-mm diameter). measurements were made on two opposite sides of each fruit after removal of a small piece of peel. the total soluble solids (tss) content (%) of loquat juice was measured with a hand held refractometer (krüss-germany). the results were expressed as % at 20°c. titratable acidity loquat pulp (40g) was homogenized in 50 ml of distilled water. titratable acid (ta) content was determined by titration with 0.1 n naoh to ph 8.1, and the results were expressed as percentage of malic acid (mirdehghan, 2007). color value fruit skin color of loquat fruit was measured by two readings on the two different symmetrical faces of the fruit in each replicate, using a konica minolta (cr-400) chroma meter and colorimeter calibrated with a white standard tile. results were expressed as a skin color as described chroma value from (a*²/b*²)1⁄2 value. browning index browning index was observed every 2 day for 12 days for 4°c storage by measuring the extent of browning area as described by akhtar et al. (2010). using 30 fruits on the following scale: using 30 fruits on the following scale: 0 = no browning; 1 = less than 1⁄4, 2 = 1⁄4 to 1⁄2 browning; 3 = 1⁄2 to 3⁄4 browning; 4 = more than 3⁄4 browning; the browning index was calculated using the following formula: browning index= [1 x n1 + 2 x n2 + 3 x n3 + 4 x n4] x 100 microbiological analyses total aerobic mesophiles, psychrotrophic aerobic bacteria, yeast and mold counts were enumerated to determine the combined effect of unpacked 1-mcp and 1-mcp+map treatments. for microbiological analysis, aseptically deseeded loquat fruit (25g) was homogenized for 2 min in 225 ml of sterile peptone water (0.1%, w/v) with a stomacher (interscience). the homogenized samples were subjected to ten fold dilution using the same diluent and pipetted into a sterile petri dish from each dilution, then appropriate melted agar poured in and mixed with the sample. the total aerobic mesophilic count was determined on plate count agar (30°c for 3 days). the psychrotrophic count was enumerated on plate count agar (5°c for 10 days). the yeast and mold count was enumerated on potato dextrose agar (25°c ph 3.5, for 5 days). three samples per treatment were aseptically taken and analyzed during the storage at intervals. the results were then reported as log cfu/g. statistical analysis experiments were performed using a completely randomized design with 10 replicates per treatment. three kg fruit was used in each map packed. all statistical analyses were performed with spss 13.0 software for windows. the results were obtained using glm multivariate procedures which provide analysis of variance by one or more factor variables. means were compared by the least signifi cant difference tested at signifi cance levels of (p < 0.05). results and discussion fruit fi rmness application of 1-mcp (312.5 and 625 ppb) treatments delayed the softening of loquat fruits when they were kept both unpacked and in map storage for the fi rst eight day of storage. additionally, map without 1-mcp treatment had the same effect as the ones mentioned above. the effect of 1-mcp treatment in combination with map on the fi rmness of loquat fruit during 5°c storage was statistically different compared to the control. as shown in fig. 1 and fig. 2, the strongest effect on fruit fi rmness was obtained by 1-mcp (625 ppb)+map at 5°c storage when compared to unpacked 1-mcp (312.5 ppb) and control stored at 5°c and 20°c. fruit fi rmness in the present work softened much more rapidly in unpacked control than in unpacked 1-mcp treated fruits at 5°c and 20°c, and 1mcp+map treated fruits at 5°c storage. the results in this work are in accordance with the results of tian et al. (2000), jiang et al. (2001) and ding et al. (2002), who observed a signifi cant delay of senescence associated with the inhibition of ripening rate and the loss of fruit fl esh fi rmness when used with 1mcp+map at low storage temperature. fig. 1: changes in fruit fl esh fi rmness of loquat fruit stored at 5 °c for 12 d after harvest. bars indicate ± s.e. 1-mcp and map treatment on loquat 127 soluble solids content tss content was determined using the control fruits, and after 1mcp (312.5 and 625 ppb) treatment on fruits for 6 h at 20ºc. at the initial day, the lowest tss content was obtained by 625 ppb of 1-mcp treatment among all applications (fig. 3). treatment of 312.5 ppb of 1-mcp+map, 312.5 and 625 ppb of 1-mcp+ unpacked and control fruits at 5ºc increased its tss content for the fi rst eight days but then decreased sharply. as shown in fig. 3, 625 ppb of 1-mcp+map for 12 days at 5ºc signifi cantly lowered the tss content of loquats when compared to unpacked 625 ppb of 1-mcp and other treatments applied. in contrast to the effects of 625 ppb of 1-mcp+map treatment at 5ºc, higher tss content was obtained from 625 ppb of 1-mcp treatment at 20ºc (fig. 4). tss content of unpacked 312.5 and 625 ppb of 1-mcp treatments and control stored at 20ºc increased continuously up to 6 days of storage. however, this continuous increase declined slightly in unpacked 312.5 ppb of 1-mcp treatment and control. high tss content in fruits is affected by several factors such as dehydration of fruits, treatment conditions, packaging material type and/or maturity stage at harvest, as suggested by cai et al. (2006). in previous studies, it was also indicated that tss value varied from fruit to fruit, treatment and/ or storage conditions. for example, bregoli et al. (2005) found the low tss value of 1-mcp treated nectarine fruit stored at low temperature, while watkins (2006) reported variable results of tss content in relation to 1-mcp treated and untreated fresh fruits. in addition to those studies, cai et al. (2006) also observed that tss contents increased initially and then decreased in the control, 0.5 and 50 µl/l 1-mcp treated fruits stored at 20ºc for 8 days. the results of the present study did not differ from the fi ndings of bregoli et al. (2005) and cai et al. (2006). titratable acidity the primary organic acid in loquat fruit is malic acid, which is the principal fl avor component. malic acid accounts for approximately 90% of the total acid contents in loquat fruit. at the initial day, 1mcp (312.5 and 625 ppb) treated fruits after 6 h and the control were examined for titratable acidity (ta). ta of 625 ppb 1-mcp treated fruits was higher than 312.5 ppb 1-mcp treated fruits and control. whereas, ta content of 312.5 ppb 1-mcp treated fruits + map and control at 5oc increased up to the fi rst fourth day of storage, then, fig. 2: changes in fruit fl esh fi rmness (n) of loquat fruit stored at 20°c for 9 d after harvest. bars indicate ± s.e. fig. 3: changes in fruit total soluble solids of loquat fruit stored at 5°c for 12 d d after harvest. bars indicate ± s.e. fig. 4: changes in fruit total soluble solids of loquat fruit stored at 20°c for 9 d after harvest. bars indicate ± s.e. 128 a.t. oz, z. ulukanli this content decreased in control except 312.5 ppb 1-mcp treated fruits + map (fig. 5). for the fi rst six days of 20oc storage, ta content of 1-mcp (312.5 and 625 ppb) treated fruits decreased signifi cantly and was less than that of the control, but then increased with the storage time (fig. 6). the effect of 1-mcp, map and storage temperature and the combination of those factors on ta value was statistically different. the present result indicated that 1-mcp (625 ppb)+map effectively reduces the loss of ta, as shown in fig 5. it has been previously suggested that the decrease of ta value in some fruits treated with 1-mcp depends on the concentration of organic acids present in fruit, conversion of acids to sugars in fruits during respiration. the decrease in ta value in the present work may be due to these factors as stated by previous authors (ding et al., 1998; bregoli et al., 2005; watkins, 2006). fruit color changes changes in color intensity and quality are important indicators of maturity and quality for fresh loquat. brightness (l) value, red intensity, and the yellow intensity were measured using a minolta chromameter (l, a, b) and these values were used to calculate the chromaticity parameters: fruit lightness (l), chroma values. fruit l* and chroma values increased in 1-mcp (312.5 and 625 ppb)+map and control+map at 5°c up to 8 days and results were expressed in fig. 7 and fig. 8. in contrast, those values in all treatments decreased signifi cantly with the ripening at 5ºc after 8 days of storage. 1mcp (625 ppb)+map kept fruit chroma values lower than 1-mcp (312.5 ppb) and control at 5°c. at 20°c, l value of unpacked 1mcp (312.5 ppb) and control decreased until 3 days of storage. while this value started to increase towards the end of storage. unpacked 1-mcp (625 ppb) treatment in all applications and storage temperatures made the fruit lightness color value higher and/ or longer. unpacked 1-mcp (625 ppb) treatment on chroma value up to 6 days at 20°c appeared to be higher than the results obtained by unpacked 1-mcp (312.5 ppb) treatment and control (fig. 9). towards the end of storage, treatments of 1-mcp (312.5 and 625 ppb) and control groups showed approximate values as shown in fig. 9. ding et al. (2002) reported that map storage inhibited fruit color changes slightly. but the result of 1-mcp treatment with or without map showed no change on fruit skin color. towards the end of storage, it appeared that all results revealed nearly approximate values. in a similar study by aguayo et al. (2006), it was reported that skin luminosity (l*) increased in correlation with the time of storage. 1-mcp treatment during storage was found to be signifi cant for both the a/b ratio and l* color parameters. similar results were also reported in other fruits such as apple, pineapple. (budu and joyce, 2003). browning index the effects of 1-mcp treatment and map storage on browning index at 5°c and 20°c storage are presented in fig. 11 and fig. 12. there were signifi cant differences between the combination of 1mcp (625 ppb) with map and other treatments. the present study showed that value of browning of unpacked loquat fruit increased during the storage at 5°c for 12 d and 20°c for 9 d (fig. 11 and fig. 12). but, browning index seemed to be higher at 20°c. the best result for browning index in the present study was obtained in 625 ppb of 1-mcp treated fruits at 5°c for 12 d compared to other treatments. the browning index value was statistically signifi cant fig. 5: changes in fruit ta value of loquat fruit stored at 5°c for 12 d after harvest. bars indicate ± s.e. fig. 6: changes in fruit ta value of loquat fruit stored at 20°c for 9 d after harvest. bars indicate ± s.e. 1-mcp and map treatment on loquat 129 showed an accordance with the study of cai et al. (2006), who also reported that 0.5 and 5 µl /l 1-mcp treatment had a reducing effect on fruit browning index. in an another study by ding et al. (1998), it was reported that loquat fruit retained its initial quality and chemical fig. 9: changes in fruit chroma color value of loquat fruit stored at 5°c for 12 d. bars indicate ± s.e fig. 7: changes in fruit l value of loquat fruit stored at 5°c for 12 d after harvest. bars indicate ± s.e. fig. 8: changes in fruit l color value of loquat fruit stored at 20°c for 9d after harvest. bars indicate ± s.e. at 1-mcp and/or map storage and the interaction between factors during storage at 5°c and 20°c. two concentrations of 1-mcp and map reduced the browning index and additionally improved the visual quality maintenance of loquat during storage. this fi ndings 130 a.t. oz, z. ulukanli fig. 10: changes in fruit chroma color value of loquat fruit stored at 20ºc for 9d. bars indicate ± s.e components in 0.15% perforated polyethylene fi lm packaging at 1°c and 5°c, which showed an accordance with fi nding of the present study. on the other hand, guelfat-reich (1970) indicated that polyethylene wraps increased internal browning and postharvest rotting of loquat fruit. previous studies suggested that browning index value showed some kind of changes according to fruit variety, modifi ed atmosphere packing material thickness, treatment and/or storage conditions. fig.11: changes in fruit browning index of loquat fruit stored at 5°c for 12 d after harvest. bars indicate ± s.e. fig. 12: changes in fruit browning index of loquat fruit stored at 20°c for 9 d after harvest. bars indicate ± s.e. for instance, the effect of map on the browning tendency of loquat during 5ºc storage was observed by examining the hunter l value in the report of ding et al. (2002). and the results showed that there was a rapid decrease when hunter l value was measured. the result of the present study was similar to the fi ndings of ding et al. (2002). besides that, in the present study, l values revealed some differences between treatments during storage, but, it gave very close values for all treatments towards the end of storage. 1-mcp and map treatment on loquat 131 microbial count total aerobic mesophilic count (tmc) for the map and unpacked loquat stored at 5°c for 12 days and 20°c for 9 days, respectively were shown in fig. 13 and fig. 14. tmc (log cfu/g) of unpacked loquat fruits at 5 and 20°c signifi cantly increased during the storage (fig. 13 and fig. 14). combination of 1-mcp (625 ppb) + map suppressed the total aerobic mesophiles up to the four days of storage at 5°c in comparison to 1-mcp (312.5 ppb) treatment and the control. map signifi cantly suppressed the growth of total aerobic mesophiles than unpacked one at 5°c total yeast and mold count (log cfu/g) of unpacked loquat stored at 5°c for 12 days and 20°c for 9 days increased signifi cantly as shown in fig. 15 and fig.16. however, total yeast and mold count of 1-mcp (625 ppb) + map loquat fruit stored at 5°c for 12 days remained negligible compared to 1-mcp (312.5) + map and control + map (fig. 15). in addition, unpacked 1-mcp (625 ppb) at 20°c during the 9 days of storage inhibited signifi cantly the growth of yeast and molds when compared to unpacked 1-mcp (312.5 ppb) and control group (fig. 16). fig. 13: changes in total aerobic mesophilic count (log cfu/g) of loquat fruit stored at 5°c for 12 d. bars indicate ± se fig. 15: changes in total yeast and mold count (log cfu/g) of loquat fruit stored at 5°c for 12 d after harvest. bars indicate ± se. fig. 14: changes in total aerobic mesophilic count (log cfu/g) of loquat fruit stored at at 20°c for 9 days. bars indicate ± se 132 a.t. oz, z. ulukanli fig.16: changes in total yeast and mold count (log cfu/g) of loquat fruit stored at 20°c for 9 d after harvest. bars indicate ± se. total psychrotrophic bacterial count in unpacked samples increased during 12 days of storage at 5°c (fig. 17). 1-mcp + map and unpacked 1-mcp treatments signifi cantly suppressed the growth of psychrotrophs (fig. 17). 1-mcp+map treatment (especially 625 ppb) stored at 5°c for 12 days signifi cantly decreased psychrotrophic aerobic bacteria count (log cfu/g) of loquat fruit compared to control+map treatment. 1-mcp (312.5 and 625 ppb) + map reduced the growth of psychrotrophic aerobic bacteria for 4 days after treatment, whereas, population of psychrotrophic aerobic bacteria of map control and unpacked control showed an increase during all the storage. specifi cally, the combination of 1-mcp (625 ppb)+map appeared to be more effective in inhibiting the growth of microorganisms compared to other treatment. in earlier studies, 1-mcp, mcp+map and /or other applications on the growth of microorganism varied with several application parameters including concentration, time, temperature and the nature of commodity (de ell et al., 2001; gong et al., 2002). for example, two different research groups indicated that 1-mcp treatment has no benefi cial effect in inhibiting the growth of microorganisms on pineapple slices (budu and joyce, 2003; rocculi et al., 2009). nonetheless, rupasinghe et al. (2005) observed the benefi cial effect of 1-mcp (1 µl l-1 for 24 h) treatment on empire apples and apple slices, and they reported that the growth of total microorganisms decreased when apple/apple slices were exposed to 1-mcp. the benefi cial effect of 1-mcp (for 24 h at 5°c + cacl2) and the combination of 1-mcp + cacl2 and ca on fresh-cut strawberries was also observed by aguayo et al. (2006) who reported that all fig. 17: changes in psychrotrophic aerobic bacteria count (log cfu/g) of loquat fruit stored at 5°c for 12 d after harvest. bars indicate ± se. treatments slowed down the microbial (psychrotrophic and yeast) growth. moreover, the synergistic effect of 1-mcp and modifi ed atmosphere packaging (map) on various fruits such as cantaloupe, honeydew melon, mango and pineapple has been also shown to reduce the growth of molds, mesophiles and psychrotrophic microorganisms (portela and cantwell, 1998; qi et al., 1999; rattanapanone et al., 2001; liu et al., 2007). the present results appeared to be in parallel with the fi ndings of portela and cantwell (1998); qi et al. (1999); rattanapanone et al. (2001); rupasinghe et al. (2005) aguayo et al. (2006), liu et al. (2007). conclusion what the present study suggests is that the combination of 1-mcp and map in accordance with low storage temperature delayed senescence processes and at the same time it kept fruit fl esh fi rmness within acceptable range. furthermore, 1-mcp (625 ppb) treatment with map reduced the fruit ripening, browning rate, growth of total aerobic mesophiles, yeast/mold and psychrotrophic aerobic bacteria. acknowledgements authors thanks amcor flexibles packaging company (ledbury herefordshire, uk) for donating the packaging materials, the sources of 1-mcp for agrofresh inc./rohm and haas company and sources of fruit materials for alata horticultural research institute. this work was also supported by osmaniye korkut ata university scientifi c research project (oku-bap, osmaniye-turkey). references aguayo, e., jansasithorn, r., kader, a.a., 2006: combined effects of 1-methylcyclopropene, calcium chloride dip, and/or atmospheric modifi cation on quality changes in fresh-cut strawberries. postharvest biol. tec. 40, 269-278. akhtar, a., abbasi, n.a., hussain, a., 2010: effect of calcium chloride treatment on quality characteristics of loquat fruit during storage. pak. j. bot. 42, 181-188. bregoli, a.m., ziosi, v., biondi, s., rasori, a., ciccioni, m., costa, g., torrigiani, p., 2005: postharvest 1-methylcyclopropene application in ripening control of ‘stark red gold’ nectarines:temperature-dependent effects on ethylene production and biosynthetic gene expression, fruit quality, and polyamine levels. postharvest biol. tec. 37, 111-121. budu, a.s., joyce, d.c., 2003: effect of 1-methylcyclopropene on the quality of minimally processed pineapple fruit. aust. j. exp. agr. 43, 1-mcp and map treatment on loquat 133 177-184. cai, c., chen, k., xu, w., zhang, w., li, x., ferguson, i., 2006: effect of 1-mcp on postharvest quality of loquat fruit. postharvest biol. tec. 40, 155-162. de ell, j.r., khanizadeh, s., saad, f., ferree, d.c., 2001: factors affecting apple fruit fi rmness – a review. j. am. pomol. soc. 55, 8-27. ding, c., chachin, k., ueda, y., imahori, y., wang, c.y., 2002: modifi ed atmosphere packaging maintains postharvest quality of loquat fruit. postharvest biol. tec. 24, 341-348. ding, c.k., chachin, k., hamauzu, y., ueda, y., imahori, y., 1998: effects of storage temperatures on physiology and quality of loquat fruit. postharvest. biol. tec. 14, 309-315. fallik, e., 2008: postharvest treatments affecting sensory quality of fresh and fresh-cut products. in: paliyath, g., murr, d.p., handa, a.k., lurie, s. (eds.), postharvest biology and technology of fruits, vegetables, and flowers, 301. wiley-blackwell publishing, iowa. gong, y.p., fan, x.t., mattheis, j.p., 2002: responses of ‘bing’ and ‘rainier’ sweet cherries to ethylene and 1-methylcyclopropene. j. amer. soc. hort. sci. 127, 831-835. guelfat-reich, s., 1970: storage of loquat. fruits d'outre mer. 25, 169173. han, j.h., 2005: new technologies in food packaging: overview. in: han, j.h. (ed.), innovations in food packaging, 1. elsevier science & technology books. floras, j.d., matsos, k.l., 2005: introduction to modifi ed atmosphere packaging. in: han, j.h. (ed.), innovations in food packaging, 161. elsevier science & technology books. karadeniz, t., yarilgaç, t., bostan, s.z., 2009: situation of loquat (eriobotrya japonica lindl) growing in turkey. acta hortic. 825, 137139. liu, c.l., hsu, c.k., hsu, m.m., 2007: improving the quality of freshcut pineapples with ascorbic acid/sucrose pretreatment and modifi ed atmosphere packaging. packag. technol. sci. 20, 337-343. mirdehghan, s.h., rahemi, m., castillo, s., martinez-romero, d., m. serrano, valero, d., 2007: pre-storage application of polyamines by pressure or immersion improves shelf-life of pomegranate stored at chilling temperatures by increaseding endogenous polyamine levels. postharvest biol. tec. 44, 26-33. jiang, y., joyce, d.c., terry, l.a., 2001: 1-methylcyclopropene treatment affects strawberry fruit decay. postharvest biol. tec. 23, 227-232. portela, s.i., cantwell, m.i., 1998: quality changes of minimally processed honeydew melons stored in air or controlled atmosphere. postharvest. biol. tec. 14, 315-357. rattanapanone, n., lee, y., wu, t., watada, a.e., 2001: quality and microbial changes of fresh-cut mango cubes held in controlled atmosphere. hortscience 36, 1091-1095. rocculi, p., cocci, e., romani, s., sacchetti, g., dalla rosa, m., 2009: effect of 1 mcp treatment and n2o map on physiological and quality changes of fresh cut pineapple. postharvest biol. tec. 51, 371377. rupasinghe, h.p.v., murr, d.p., deell, j.r., odumeru, j., 2005: infl uence of 1-methylcyclopropene and natureseal on the quality of fresh-cut ‘empire’ and ‘crispin’ apples. j. food quality 28, 289-307. shaw, p.e., 1980: loquat. in: nagy, s., shaw, p.e. (eds.), tropical and subtropical fruits, 480-481. westport, ct: avi. sisler, e.c., serek, m., 1997: inhibitors of ethylene responses in plants at the receptor level. recent developments. physiol. plant 100, 577-582. tian, m.s., prakash, s., elgar, h.j., young, h., burmeister, d.m. ross, g.s., 2000: reponses of strawberry fruit to 1-methylcyclopropene (1-mcp) and ethylene. plant growth regul. 32, 83-90. qi, l., wu, t., watada, a.e., 1999: quality changes of fresh-cut honeydew melons during controlled atmosphere storage. j. food quality 22, 513521. watkins, b.c., 2006: the use of 1-methylcyclopropene (1-mcp) on fruits and vegetables. biotechnol. adv. 24, 389-409. address of the authors: engineering faculty, department of food engineering, university of osmaniye korkut ata, karacaoglan campus 80000 osmaniye, turkey e-mail: aysetulinoz@osmaniye.edu.tr tel.: +903288251818/3603, fax:+903288251866 science and arts faculty, department of biology, university of osmaniye korkut ata, karacaoglan campus 80000 osmaniye, turkey e-mail: zeynepulukanli@osmaniye.edu.tr tel.: +903288251818/2530, fax:+903288251866 art05_lange.indd journal of applied botany and food quality 85, 34 40 (2012) 1 julius kühn-institut (jki), federal research centre for cultivated plants 2 university of applied science ostwestfalen-lippe effects of steam and vacuum administration during decontamination on essential oil content in herbal medicines h. lange1*, k. schwarzer 2, a. dammann2, u. müller 2, k.r. richert-pöggeler1, h. krüger1 (received march 16, 2012) * corresponding author summary saturated steam decontamination is an application for elimination of microorganisms from the surface of different materials. this technique has been optimized for the treatment of dried spices or pharmaceuticals, which could have been contaminated with microorganisms during cultivation, processing, storage or transport. the described saturated steam decontamination is based on the lemgo process. this method does not kill microorganisms, but removes them physically from the surface. our investigation focused on measuring the effects of steam temperatures at 120 °c and 100 °c, respectively, for 20 s with a subsequent fl ash vacuum of 20 s. applications of fl ash vacuum as well as saturated steam heated to 120 °c were also tested separately. the impact of these parameters on the essential oil content and on the surface of different medicinal plants such as marjoram, oregano, fennel and eucalyptus was analysed using gas chromatography and scanning electron microscopy. especially in herbal drugs with glandular trichomes such as marjoram and oregano severe surface destruction was visible accompanied by high losses of essential oil from 93 % in marjoram tissue to 59 % in oregano tissue. for fennel and eucalyptus that possess protected essential oil storage cells only minor or no reduction of volatiles has been observed during exposure to saturated steam. the experiments show clearly a positive correlation between stability of essential oil cavities and essential oil content preservation. introduction essential oils are stored in oil storage cavities that can be found distributed in tissues of blossoms, leaves, seeds, pericarps, roots, resins, barks or wood. several genera of lamiaceae accumulate essential oil in glandular hairs. in this case, the oil is protected solely by a cuticle layer with a resinous fi lm (guenther, 1949). these essential oils play an important role at chemical-ecological interactions of plants and their environment. numerous monoterpenes protect the plant from direct or indirect attack by herbivores and microorganisms. on the other hand, some monoterpenes serve as attractant for insects (hallahan et al., 2000; harborne et al., 1991; langenheim et al., 1994; pickett et al., 1991; wise et al., 1999). glandular hairs can be further divided into the group of peltate glandular hairs and the group of capitate glandular hairs. both types of glandular hairs are present on leaf surfaces of oregano and marjoram belonging to the lamiaceae family. these groups differ in anatomy and mode of secretion (burbott and loomis, 1969). peltate hairs consist of a basal cell, a broad and short stalk cell with cutinized outer walls, and a round broad head of secretory cells. secretory cells of peltate hairs show different positioning depending on the leaf surface morphology. as shown for marjoram in the right panel of fig. 1 secretory cells can be exposed or as shown for oregano in the left panel secretory cells can be sunken in a pit formed by epidermal tissue (bruni and modenesi, 1983). the second large group of hairs is represented by capitate hairs. they consist of a basal cell, a stalk cell and a unior bi-cellular head (schnepf et al., 1972; heinrich et al., 1973; amelinxen et al., 1965). capitate hairs are much smaller than peltate hairs (werker et al., 1985(a); werker et al., 1985(b)) and start very early in development with secretion. at this stage peltate glandular hairs are not yet fully developed (fairbanks et al., 1971) and functioning. saturated steam decontamination is the preferred method for decontamination of herbs and spices in germany (weber, 2003). due to the intensive heat transfer and moisturization of herbal surfaces, this process leads to an effi cient and well applicable thermal decontamination of microorganism containing material. for plants with spore-formers on their surface, an extended treatment time up to 20 min is required to kill them, as spores are highly resistant to heat (kabelitz, 1996). the lemgo process, that has been developed at the university of applied science ostwestfalen-lippe, germany, uses a reduced vaporisation time (20 s) followed by a subsequent fl ash evaporation for removing microbes (müller et al., 2002). this so-called fl ash-effect is caused by an extremely rapid evacuation of the treatment chamber and requires special technology for achieving appropriate conditions. this mechanical decontamination method is characterised by a minimum saturated steam temperature of 80 °c, which is needed for the decontamination effect. the physical forces during fl ash evaporation are strong enough for overcoming adhesion of microbes to herbal surfaces resulting in decontamination (lilie, 2009; lilie et al., 2006). previous decontamination studies were conducted in a 700 ml laboratory equipment for inoculated model systems (lilie et al., 2009) as well as naturally contaminated plant material such as pepper (piper nigrum)(lilie et al., 2007; lilie et al., 2004) and camomile (matricaria chamomilla) (rumke et al., 2006). the decontamination of camomile using the lemgo process resulted in a reduction up to 5 decades of total microbial count using a steam temperature of 120 °c for 10 to 20 s. though the material was humidifi ed up to 35 %, the concentration of essential oil had not been affected (rumke et al., 2006). in further investigations it was clearly demonstrated that the process also reduces bacterial spores quickly (müller, 2010). this study describes the individual volatiles in selected aromatic plants using gas chromatography and it refl ects morphological changes of essential oil cells using scanning electron fig. 1: scanning electron microscopy (sem) images of peltate glandular hairs of oregano left (origanum vulgare) and marjoram right (majorana hortensis). steam and vacuum on herbal medicines 35 microscopy. furthermore, the impact of the applied decontamination method on the essential oil concentration is measured and observed losses of volatiles are correlated with differences in overall leaf structure. materials und methods plant material the plant material for marjoram (majorana hortensis l., lamiaceae), oregano (origanum vulgare, lamiaceae) and fennel (foeniculum vulgare, apiaceae) was provided by majoranwerke aschersleben and was cultivated and harvested in 2009. eucalyptus leaves (eucalyptus grandis, myrtaceae) were purchased from the company “martin bauer” (vestenbergsgreuth, germany) in 2009. all plant materials were air-dried before saturated steam treatment (lemgo process) and all results of essential oil composition refer to air-dry mass. lemgo process the decontamination tests were conducted in a laboratory decontamination facility, which comprises a 0.7 l treatment chamber and an 8 l vacuum buffer tank. both repositories are cylindric and made of stainless steel. the connection of the single devices is carried out with pipelines with an inner diameter of 1.27 cm. the record of temperature and pressure is provided by a data acquisition system. steam treatment and evacuation periods are regulated by electronically controlled pneumatic angle valves before and behind the treatment chamber. the evacuation of the treatment chamber is conducted by a water ring compressor with gas emitter. a tube bundle heat exchanger between vacuum buffer tank and water ring compressor encourages pressure reduction and provides the condensation of steams extracted from the treatment chamber. the heat exchanger is working with water and comprises a capacity of 0.4 l (lilie et al., 2007). decontamination treatment of marjoram, oregano, fennel and eucalyptus only a maximum of 5 g of the appropriate plant material could be decontaminated in the small treatment room. in the course of saturated steam decontamination, a steaming with 100 °c and 120 °c heated vapor was conducted with the accordant equilibrium pressure and lasted 20 s. for achieving saturated steam conditions, a prevacuum of 20 s was generated. the period of the fl ash-vacuum, which is critical for decontamination, was also 20 s. to investigate how “steam” and “vacuum” individually affected the plant material the following treatments (see tab. 1) were performed: the herbal drugs were treated with saturated steam (treatment 3). after 20 s of steam the chamber was opened slowly to ambient atmosphere. compressed air, instead of steam, was introduced into the treatment chamber to identify only the impact of vacuum on herbal drug tissue (treatment 4). tab. 1: the parameters of applied treatments to study effects of steam decontamination treatment steam steam air preand type temperature time pressure postvacuum time treatment 1 120 °c 20 s 20 s treatment 2 100 °c 20 s 20 s treatment 3 120 °c 20 s treatment 4 2.5 bar 20 s determination of microbial count microbial analyses were conducted following the european pharmacopoeia (6th edition). the total plate count results are stated as colony forming units per g (cfu/g). the quantitative detection limit was 10² cfu/g. essential oil analysis 100-200 mg of dried plant material (marjoram, oregano, fennel and eucalyptus) were weighed into a 100 ml centrifuge tube and homogenized in iso-octane with an ultra-turrax. this mixture had been low speed centrifuged at 3000 rpm in a table top centrifuge and the supernatant was analyzed by gas chromatography (hewlett packard hp 5890 series ii gc). the essential oil content in ml/100 g (air dried mass) is defi ned as the total value of the specifi c individual components determined by gas chromatography (krüger et al., 1998). scanning electron microscope analysis the lower and upper leaf surface of steam treated material has been applied for scanning electron microscopy. the samples were mounted accordingly with double-sided, non conductive sticky tape on aluminium stubs and coated with gold. the chamber of the sputter coater was fi lled with argon during deposition of heavy metal. images were collected using the scanning electron microscope quanta 250 (fei worldwide corporate headquarters) equipped with a tungsten cathode. the acceleration voltage was 10 kv. on average 20 gland scales were analysed and representative images selected. images were adjusted in brightness and contrast using adobe photoshop cs4. statistical analysis each saturated steam treatment was carried out three times and was analysed twice for essential oil (n=3). statistical evaluation was accomplished with systat software inc sigmaplot 11. for testing signifi cance, a one-way anova was carried out. a direct comparison of groups was performed with a tukey-test. results changes in content of ingredients and leaf surface structures changes of essential oil content and surface structure modifi cations were investigated under four different treatment conditions (tab. 1) in marjoram, oregano, fennel and eucalyptus. marjoram the control sample was characterised by an initial value of essential oil of 0.91 ml/100 g dry product (tab. 2). marjoram is an herbal drug with high sensitivity against decontamination methods with saturated steam. figure 2, a0 shows that the peltate glandular hairs of untreated marjoram are not embedded in the leaf matrix, but exposed with a sphere-like appearance. exposing marjoram to 120 °c saturated steam for 20 s with a subsequent 20 s vacuum results in a loss of 93 % of essential oil content. as depicted in fig. 2, a1 this is due to severe damages of glandular scales during the decontamination procedure. the cuticle of the glandular trichome becomes perforated releasing essential oil that most likely is evaporated with the water layer. the loss of content is visible by severe shrinkage from the outer area that leads to almost complete fl attening in the middle region of the original sphere-like appearance of the cell. steam temperatures of 100 °c do not have as dramatic effects as observed after treatment 1 conditions (tab. 1). the peltate glandular hairs in fig. 2, a2 show less shrinkage and smaller disruptions in its cuticle resulting in reduced essential oil loss of 86 %. comparisons of the parameters “steam” and “vacuum” provided evidence that the observed 36 h. lange, k. schwarzer, a. dammann, u. müller, k.r. richert-pöggeler, h. krüger damages in marjoram glandular scales and accompanied ingredient losses were more pronounced by steam rather than vacuum exposure (compare fig. 2, a3 and a4). as fig. 2, a3 illustrates incubation of leaf tissue, with steam heated to 120 °c causing isolated surface disruptions of the peltate glandular hairs. in contrast application of compressed air showed for the majority of visible cells deformations in peltate glandular hairs shape leading to more fl attened appearance but did not show perforations of the cuticle surface. the measured essential oil content of 0.84 ml/100 g was similar to untreated leaves which contained 0.91 ml/100 g (tab. 2). the saturated steam decontamination shows only for treatment 1 a reduction of two decades of total plate count. treatments 2 and 3 reduce the microorganisms on marjoram very sparsely and the last treatment 4 provides no reduction. tab. 2: comparison of essential oil content of marjoram in ml/100 g and total plate count (colony forming units, cfu/g) after the lemgo process with four different treatment parameters. same superscript means no signifi cant difference between groups. marjoram treatment essential loss of see panel parameter oil content essential oil in fig. 2 cfu/g (compare tab. 1) mean ± std [%] control 0.91 ± 0.04 a a0 5.1 x 105 treatment 1 0.07 ± 0.04 d 93 a1 2.2 x 103 treatment 2 0.13 ± 0.04 c 86 a2 4.8 x 105 treatment 3 0.07 ± 0.04 d 93 a3 1.2 x 105 treatment 4 0.84 ± 0.04 a 0 a4 5.3 x 105 oregano among the investigated lamiaceae species oregano presented the highest essential oil content of 1.82 ml per 100 g dry mass. in contrast to marjoram, the peltate glandular hairs are not exposed but embedded into the leaf matrix as revealed by scanning electron microscopy (fig. 1 and 2, b1). thus, peltate glandular hairs of oregano seem to be less exposed to destruction by external forces, which may account for the observed lower loss of essential oil after the decontamination treatments. deformation of peltate glandular hairs occurs naturally as indicated in the control (fig. 2, b0). the different steam temperatures during decontamination had a signifi cant effect on essential oil content as shown for marjoram. treatment with 120 °c or 100 °c steam and vacuum caused a loss of 59 % and 43 %, respectively (tab. 3). most peltate glandular hairs are deformed by saturated steam treatments as revealed in scanning electron microscopy (fig. 2, b1-b3). images collected from leaves exposed to treatment 3 and 4 confi rmed the destructive forces by steam rather than vacuum on oregano peltate glandular hairs. in the picture for saturated steam treatment without vacuum (fig. 2, b3), the treatment/application resulted in multiple broken, some intact, but also semi-broken peltate glandular hair cells. apparently the sunken appearance of oregano secretory cells protected superior against external forces. only half of essential oil content was lost during decontamination in contrast to marjoram tissue which showed an almost complete loss of terpenes during decontamination. the microbial reduction results of oregano are similar to marjoram. treatment 1 is the only treatment variation which decreases the total plate count of oregano by about 3 decades. for methods 2, 3 and 4, no appreciable degradations of the colony forming units of oregano are found. tab. 3: comparison of essential oil content of oregano in ml/100 g and total plate count (colony forming units, cfu/g) after the lemgo process with four different treatment parameters. same superscript means no signifi cant difference between groups. oregano treatment essential loss of see panel parameter oil content essential oil in fig.2 cfu/g (compare tab. 1) mean ± std [%] control 1.82 ± 0.09 a b0 6.1 x 106 treatment 1 0.75 ± 0.09 c 59 b1 2.5 x 103 treatment 2 1.05 ± 0.10 b 43 b2 3.5 x 106 treatment 3 0.74 ± 0.04 c 59 b3 9.7 x 105 treatment 4 1.92 ± 0.11 a 0 b4 7.1 x 106 fennel fennel seeds accumulate the valuable essential oils in a number of channels inside the fruit as shown in fig. 3, c0-c4. the channel structure allows an increase in storage capacity 4-9 fold magnitude compared to the sphere-like peltate glandular hairs. each compart fig. 2: scanning electron microscopy of marjoram and oregano. a0 = on the surface-fi tting intact peltate glandular hair, a1 = collapsed peltate glandular hair with large holes in cuticle after, a2 = collapsed peltate glandular hair with minor holes, a3 = collapsed peltate glandular hair with large hole in cuticle, a4 = intact peltate glandular hair / b0 = peltate glandular hairs embedded in leaf surface on the control sample of oregano, b1 – b3 = many collapsed peltate glandular hairs, b4 = two intact and one collapsed peltate glandular hairs. steam and vacuum on herbal medicines 37 ment is embedded in several cell layers derived from the fruit coat. they are spaced throughout the upper and lower fruit coat surrounding the endosperm. in addition to the essential oil channel cell wall (fig. 3, c4) such protective shell offers optimal protection against environmental infl uences like sun and rain. in comparison with the control (8.79 ml/100 g), fennel had an essential oil value of 8.21 ml/100 g after the treatment with 120 °c saturated steam for 20 s and a subsequent vacuum of 20 s, this means a loss of 7 % (tab. 4). from all parameters tested a minor, signifi cant reduction in essential oil content was only evident in treatments comprising 120 °c steam temperatures (tab. 4). no effect on gland scale anatomy was visible in treated samples (fig. 3, c1-c4) compared to the unexposed control tissue (fig. 3, c0). in contrast to marjoram and oregano, treatment 1 reduces the total plate count below the detection limit. in treatment 2 and 4 the number of microorganisms does not change. treatment 3 reduces the total plate count by about 0.5 decades. tab. 4: comparison of essential oil content of fennel in ml/100 g and total plate count (colony forming units, cfu/g) after the lemgo process with four different treatment parameters. same superscript means no signifi cant difference between groups. fennel treatment essential loss of see panel parameter oil content essential oil in fig.2 cfu/g (compare tab. 1) mean±std [%] control 8.79 ± 0.15a c0 5.3 x 105 treatment 1 8.21 ± 0.14b 7 c1 1.0 x 102 treatment 2 8.51 ± 016a 3 c2 3.5 x 105 treatment 3 8.14 ± 0.13b 8 c3 8.7 x 104 treatment 4 8.62 ± 0.12a 0 c4 5.1 x 105 eucalyptus eucalyptus lacking gland scales (carr and carr, 1970) possesses essential oil containing cells that are located in the leaf mesophyll (king et al., 2006). thus the positioning of storage cells is subdermal in contrast to the external structure of gland scales among lamiaceae species. the oil content of eucalyptus is 1.54 ml/100 g (tab. 5), which is similar to concentrations found in the leaves of studied lamiaceae. in eucalyptus, preservation of essential oil has been found in all treatments. the images in fig. 3, d0-d3 display gland openings which do not show morphological changes after treatments with saturated steam. the lemgo process is a superfi cial treatment and thus hardly affects the amount of essential oil, which is located inside of the phytopharmaceutical. eucalyptus holds a uniformly developed spicular wax fi lm on the leaf surface (louro, 2002) which had been distorted after hot steam exposure. independently of treatment type 1, 2 or 3 (tab. 1) the spicules were accumulated forming discrete clusters on the leaf surface. only after application of vacuum alone, the wax spicules were retained in their original form (fig. 3, d4). the microorganism reduction outcomes are identical to marjoram. treatment 1 reduces the total plate count below the detection limit and treatment 3 decreases the germs by one decade. the other reduction methods 2 and 4 show no decrease. tab. 5: comparison of essential oil content of eucalyptus in ml/100 g and total plate count (colony forming units, cfu/g) after the lemgo process with four different treatment parameters. same superscript means no signifi cant difference between groups. eucalyptus treatment essential loss of see panel parameter oil content essential oil in fig. 2 cfu/g (compare tab. 1) mean±std [%] control 1.54 ± 0.06 a d0 2.6 x 106 treatment 1 1.48 ± 0.08 a 0 d1 1.0 x 102 treatment 2 1.43 ± 0.07 a 0 d2 4.7 x 105 treatment 3 1.54 ± 0.08 a 0 d3 1.3 x 105 treatment 4 1.52 ± 0.02 a 0 d4 1.8 x 106 fig. 3: scanning electron microscopy of fennel and eucalyptus: c0 c1= cross-sectional fennel fruit with oil channels, c3= cross-sectional fennel fruit with three oil channels, c2 c4 = oil channel with thick channel wall and partition of fennel fruit / d0 = three oil gland vents with intact wax layer on the control sample of eucalyptus, d1 = one oil gland vent with cumulated wax layer around it, d2 = close cumulated wax layer of eucalyptus after treatment 2, d3 = cumulated wax layer, d4 = normal wax layer. 38 h. lange, k. schwarzer, a. dammann, u. müller, k.r. richert-pöggeler, h. krüger discussion indeed the observed essential oil losses within species of the lamiaceae family are larger than with those of investigated members from other plant families of myrtaceae and apiaceae. the obtained data on plant morphology provide clear evidence that structure and position of essential oil storage cells are mainly accountable for the observed differences in response to heat exposure. herbal drugs which belong to the family of lamiaceae store essential oils in peltate glandular hairs, where the storage compartment is protected by an elevated cuticle (fig. 1) covered by a thin wax layer (guenther et al., 1949; croteau and wildung, 2005). it is known that essential oils evaporate at a very low rate as long as the wax coated cuticle is intact. less than 10 % essential oil loss per year has been observed in dried leaf material when stored at room temperature (burbott and loomis, 1969). furthermore it has been demonstrated that peltate glandular hairs of peppermint kept their essential oil for several years when freeze-dried (maffei, chialva et al., 1989). however, the wax-coated cuticle of lamiaceae can be broken by mechanical forces or by high temperatures (guenther, 1949). the holes visible in peltate glandular hairs of marjoram after exposure to 100 °c and 120 °c heated steam (fig. 2, a0-a2) provide direct evidence that the wax-coated cuticle has been destroyed. we assume that also the severe shrinkage of peltate glandular hairs after such heat treatment (fig. 2, b1-b3) resulted in distortion of the protective cell layer promoting release of essential oil. the temperature effects on deformation of thick wax layers were clearly illustrated on the eucalyptus leaf surface, a member of the myrtaceae (fig. 3, d1-d3). eucalyptus, however, showed no essential oil loss. this is due to the different leaf structure harboring internal oil glands (james and bell, 1999) that are not modified during heat exposure (fig. 4 c). this is also true for the tested member of apiaceae fennel. the protective layers surrounding the oil storage cells within the fennel grain prevent major oil losses (fig. 4 d). interestingly the degree of essential oil loss after steam decontamination was not equal among the members of lamiaceae. this indicates that interactions between plant surfaces and steam during the decontamination process are rather complex. whereas in oregano more than half of essential oil content from the glandular hairs is preserved during the decontamination process, marjoram shows almost a complete loss of essential oils. the anatomy of the peltate glandular hairs of the different lamiaceae species alone does not provide a suffi cient explanation for this phenomenon. it has been shown that the structure and size of the peltate glandular hairs of oregano (origanum vulgare) and marjoram (majorana hortensis l. in fig. 1) are identical (bosabalidis et al., 1997). the analysis of leaf context surrounding the glandular scale and its positioning on the leaf revealed differences among the investigated lamiaceae and those most likely contributed mainly to the broad range in observed losses. the function of epidermal cells that are arranged around the basal cell of the peltate glandular hairs of oregano is distinct (stahl-biskup et al., 2002; bosabalidis et al.,1997; bosabalidis et al., 2002) from those in marjoram. adjacent cells of oregano form an accessory of the peltate glandular hair, having a role in the volatile oil secretion. depending on cell-distribution, -size, -shape, -vacuolization and -density they are involved in transport of photosynthesis products from the mesophyll to the basal cells of the peltate glandular hairs (bosabalidis et al., 2002). the positioning of peltate glandular hairs among the investigated lamiaceae is distinct and thus the susceptibility to external forces. whereas the peltate glandular hairs of marjoram are exposed on the leaf surface, they are immersed in the epidermal cell layer in case of oregano (fig. 1). therefore they provide less target space for the steam. in consequence fewer damage of the top cell layer occurs, illustrated by reduced essential oil loss. in fig. 4 a model for the steam-plant surface interactions is exemplifi ed for exposed and immersed scales. due to the different positioning of the upper trichome cell with regard to the leaf surface the size of formed condensate layer during steam deposition phase is much larger in case of treated marjoram leaves compared to treated oregano leaves (fig. 4 upper panel). consequently the vapor-accessible surface in exposed scales is larger, allowing a higher number of thermal as well as physical interactions leading to additive effects. for example the essential oils within the marjoram scales may heat up faster and the wax layer is broken easier. the immersed peltate glandular hair of oregano (fig. 4, b) is only superfi cially touched by the steam. at the moment we cannot exclude that, perhaps, differences in cell wall composition are also contributing to the noticed variability in sensitivity to applied incubations during decontamination. further investigations will have to address such interactions in greater detail, for example, the chemical composition of the wax layer or its deformation during saturated steam treatment. furthermore, ultra structural studies of peltate glandular hairs as well as surrounding leaf tissue before and after treatments will help to identify the most sensitive parts. such knowledge will be seminal for designing procedures that guarantee effi cient decontamination by a minimum loss of valuable ingredients even for heat sensitive material. fig. 4: upper panel = peltate glandular hair of marjoram with epidermis in a plane (a), sunken peltate glandular hair of oregano (b), oil containing cell and oil gland of eucalyptus (c) and oil channel of fennel (d) before the steam deposition phase in which the wax layer is fully functional. lower panel = (a), (b), (c) and (d) during steam deposing phase when the condensate layer is formed. steam and vacuum on herbal medicines 39 analysis of the total plate count indicated that effi cient removal of microbes depended rather on physical forces applied than texture and shape of biological material (tab. 2 5). only joined application of vacuum and hot steam as provided in treatment 1 affected the total plate count negatively (increase of about 2 3 decades). the other three treatment variations decreased the total plate count only about a half decade. most likely a temperature of 100 °c for 20 s and subsequently sudden vacuum (treatment 2) is too low for a fl ash effect which would rip the microorganisms off the plant surface and the application time is too short for a thermal killing of the germs. the latter is probably also true for applied parameters during treatment 3 (lilie, 2009; kessler, 1996). as revealed in treatment 4 application of vacuum alone does not lead to removal of bacteria (tab. 2 5). in summary our studies provided clear evidence that in case of plants with peltate glandular hairs (marjoram, oregano) further improvement of the applied decontamination technology is necessary. whereas the benefi ts regarding microbial decontamination are obvious the accompanied essential oil losses are intolerable and need to be minimized. acknowledgements the research project (aif-rp-no. 15547 bg) was supported from the budget of the federal ministry of economic affairs through the arbeitsgemeinschaft industrieller forschungsvereinigungen “otto von guericke” e.v. (aif) (association of industrial research organisations). we would like to thank all funding organizations. we are very grateful to elke zimmermann for excellent technical assistance in sample preparation for sem analysis and to christine langanke as well as bärbel zeiger for great reprocessing and analytic measurement of the plant materials. literature bosabalidis, a.m., kokkini s., 1997: intraspecific variation of leaf anatomy in origanum vulgare grown wild in greece, bot. j. linn. soc. 123, 353-362. bosabalidis, a.m., 2002: structural features of origanum sp.. in: spiridon e. kintzios (ed.), oregano: the genera origanum and lippia, 1st edition (august 29, 2002). london, crc press. bruni, a., modenesi, p., 1983: development, oil storage and dehiscence of peltate trichomes in thymus vulgaris (lamiaceae). nord. j. bot. 3, 245-251. burbott, a.j., loomis, w.d., 1969: evidence for metabolic turnover of monoterpenes in peppermint. plant physiol. 44, 173-179. carr, d.j., carr, s.g.m., 1970: oil glands and ducts in eucalyptus l’hérit. ii. development and structure of oil glands in the embryo. aust. j. bot. 18, 191-212. europäisches arzneibuch, 6. aufl age, grundwerk 2008, 2.6.12, mikrobiologische prüfung nicht steriler produkte: zählung der gesamten vermehrungsfähigen keime. stuttgart und eschborn: deutscher apotheker verlag und govi-verlag – pharmazeutischer verlag gmbh. 210220. fairbanks, g., steck t.l., wallach, d.f.h., 1971: electrophoretic analysis of the major polypeptides of the human erythrocyte membran. biochemistry 10, 2606-2617. guenther, e., 1949: the essentials oils of the plant families rutaceae and labiatae, vol. 3, van nostrand reinhold, new york. hallahan, d.l., 2000: monoterpenoid biosynthesis in glandular trichomes of labiate plants. adv. bot. res. 31, 77-120. harborne, j.b., 1973: recent advances in the ecological chemistry of plant terpenoids. in: harborne, j.b., tomas-barberan, f.a. 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(eds.), ecology, chemistry and tomas-barberan, f.a. (eds.), ecology, chemistry and t biochemistry of plant terpenoids, 297-313, oxford university press, oxford. rumke, t., lilie, m., wilhelm, p., müller, u., 2006: schonende keimzahlreduktion bei kamillenblüten (chamomilla recutica (l.) rauschert). z. arzneigewurzpfl a. 11, 142-144. schnepf, e., 1972: tubuläres endoplasmatisches reticulum in drüsen mit lipophilen ausscheidungen von ficus, ledum und salvia. biochem. physiol. pfl anz. 163, 113-125. stahl-biskup, e., 2002: essential oil chemistry of the genus thymus – a global view. in: stahl-biskup, e., sáez, f. 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(ed.), comprehensive natural products chemistry, vol 2, isoprenoids including carotenoids and steroids, 97-153, elsevier, oxford. 40 h. lange, k. schwarzer, a. dammann, u. müller, k.r. richert-pöggeler, h. krüger addresses of authors: heinz lange1*, hans krüger, julius kühn-institut (jki), federal research centre for cultivated plants, institute for ecological chemistry, plant analysis and stored product protection, erwin-baur-strasse 27, 06484 quedlinburg, e-mail: heinz.lange@jki.bund.de katja r. richert-pöggeler, julius kühn-institut (jki), federal research centre for cultivated plants, institute for epidemiology and pathogen diagnostics, messeweg 11/12, 38104 braunschweig e-mail: katja.richert-poeggeler@jki.bund.de anna dammann, knut schwarzer, ulrich müller, university of applied science ostwestfalen-lippe, fb4, labor verfahrenstechnik, liebigstraße 87, 32657 lemgo, e-mail: fb4vt@hs-owl.de journal of applied botany and food quality 92, 327 335 (2019), doi:10.5073/jabfq.2019.092.044 1institute of human nutrition sciences, warsaw university of life sciences-sggw, warsaw, poland 2department of food science and technology, bogor agricultural university, ipb, bogor, indonesia the therapeutic properties of lemon balm (melissa officinalis l.): reviewing novel findings and medical indications katarzyna świąder1*, katarzyna startek, christofora hanny wijaya2 (submitted: september 11, 2019; accepted: october 17, 2019) * corresponding author summary melissa officinalis, a perennial plant in the family lamiacae, known as lemon balm, is a popular herb with multiple therapeutic properties. significant content of active compounds (including rosmarinic acid) is reported to be responsible for the broad health effects of lemon balm. melissa officinalis has antioxidant, anti-inflammatory, anti microbial and antidepressant activities. it can be used in the treatment of sleep disorders, neurodegenerative diseases and obesity as well as in ophthalmology, gynaecology, oncology, gastroenterology and cardiology. this review includes literature on the chemical composition of melissa officinalis and the possibilities of its use in medicine as well as functional food. furthermore, the side effects and the contraindications to the usage of this herb were summarized. keywords: lemon balm, melissa officinalis l., therapeutic, herb, health usage introduction melissa officinalis l. (fig. 1) is a perennial plant in the family lamiacae. it occurs naturally in the mediterranean and west asia. moreover, it is cultivated widely in europe and north america. melissa is known for its characteristic lemon taste and aroma (moradkhani et al., 2010). this herb is also known under other names as lemon balm, balm, common balm or balm mint. notes about lemon balm come from the antiquity when it was described by among others -hippocrates and dioskurides. in the middle ages, avicenna (9801037) recommended it to strengthen the heart, and paracelsus (14931541) prepared “the elixirs of life” containing the lemon balm. melissa officinalis has been traditionally known for being able to restore youth, support weakened people and prevent baldness. in herbal medicine, leaf, herb and essential oil of lemon balm are used (senderski, 2009). melissa officinalis due to its numerous healthpromoting properties, apart from medical use, is also increasingly used as an ingredient in supplements and functional foods. a compilation of the latest data on the therapeutic properties of lemon balm will facilitate its application also in food products. the chemical composition fresh herbs contain phenolic compounds, l-ascorbic acid, carotenoids, flavonoids and terpenoids. lemon balm leaves are rich in flavonoids (0.5 % dry weight) consist of quercitrin (a derivative of quercetin), ramnocitrin, luteolin and its derivatives (luteolin 7-o-βd-glucuronopyranoside, luteolin 3’-o-β-d-glucuronopyranoside, apigenin 7-o-β-d-glucopyranoside, and luteolin 7-o-β-d-glucopyranoside-3’-o-β-d-glucuronopyranoside). the major components among terpenoids are neral. geranyl acetate, ursolic acid and tannins (moradkhani et al., 2010; miraj et al., 2017). 0.087 g/100 g of caffeic acid and 21.15 g/100 g of rosmarinic acid were detected in the hydroethanolic extract of lemon balm leaves and phenolic compounds constituted 33.97% (ożarowski et al., 2016). the lemon balm essential oil, secreted by glandular trichomes, has a pale-yellow colour with lemony aroma (moradkhani et al., 2010). depending on the study, the amount of essential oil strongly varied, i.e. 0.02-0.30% of the plant weight (moradkhani et al., 2010), 0.1% of herb (sadraei et al., 2003), 0.34% of dry leaf mass (abdellatif et al., 2014) or 0.01-0.35% (kittler et al., 2018). the main components of lemon balm essential oil are listed in tab. 1. the therapeutic properties on the basis of the literature review, the results indicating the healthpromoting properties of lemon balm were collected. individual stu dies were conducted in vitro and in vivo, including human studies (tab. 2). antioxidant properties − mostly the therapeutic properties of lemon balm based on antioxidant activities due to the high content of flavonoids, melissa officinalis has antioxidant properties. in vitro studies confirmed that 100-500 μg/ml of hydroethanolic extract from the herb had a cytoprotective activity against the toxic effects of hydrogen peroxide on human umbilical vein endothelial cells. the extract also reduced amount of hydrogen peroxide in intraand extracellular fluids. this may be important in the prevention of cardiovascular diseases (safaeian et al., 2016). antioxidative activity was examined by three methods: the abts radical cation (abts·+), the dpph free radical (dpph) and the ferric reducing antioxidant power (frap). the activity of herb lemon fig. 1: melissa officinalis l. https://commons.wikimedia.org/wiki/melissa_officinalis#/media/ file:melissa_officinalis_001.jpg 328 k. świąder, k. startek, c.h. wijaya balm was comparable to respectively 10.6, 36.1 and 61.8 μm trolox/100 g dry matter – trolox is a water-soluble derivative of vita min e. such antioxidant activity of lemon balm is average in comparison to other herbs that were analyzed (wojdyło et al., 2007). the antioxidant properties were also confirmed in the study involving radiology staff exposed to long-term low-dose ionizing radiation. they consumed lemon balm infusion (1.5 g dried leaves) twice a day for a period of 30 days. there was a significant improvement in the activity of catalase, superoxide dismutase and glutathione peroxidase in plasma as well as a reduction of dna damage, lipid peroxidation and myeloperoxidase activity. lemon balm supplementation may provide adequate protection against low-dose radiation (zeraatpishe et al., 2011). anti-inflammatory properties the anti-inflammatory properties of the melissa officinalis leaf oil were studied in rats with paw oedema. an oral dose of 200 or 400 mg/kg of essential oil or 10 mg/kg of indomethacin, a drug from the group of non-steroidal anti-inflammatory drugs, was used. it was observed that both doses of lemon balm oil significantly reduced swelling and the effect was similar to that of a standard medicine (bounihi et al., 2013). antimicrobial properties in in vitro tests, the essential oil of melissa officinalis leaves had an inhibitory effect on the growth of gram-positive (staphylococcus aureus, bacillus subtilis, listeria monocytogenes) and gram-negative (pseudomonas aeruginosa, escherichia coli, klebsiella pneumoniae, salmonella enterica) bacteria, yeasts (candida albicans, saccharomyces cerevisiae) and fungi (fusarium oxysporum spp., mucor ramannianus) (abdellatif et al., 2014). the water extract of lemon balm leaves and rosmarinic acid also have antiviral activity. in in vitro studies, it inhibited the binding of hsv-1 (herpes simplex virus 1) to host cells and virus penetration in cells (astani et al., 2014). the inhibitory effect of concentrated methanolic lemon balm leaf extract on enterovirus 71 (ev71) has also been demonstrated (chen et al., 2017). anxiolytic and anti-depressant properties lemon balm leaf is a frequently used herbal drug with anxiolytic and anti-depressant effect. it lowers the level of anxiety, facilitates falling asleep, prevents gastrointestinal disorders on the mental background and has sedative properties. it is usually recommended to consume infusions from 2-3 g of leaves 3 times a day (nowak, 2009). mice subjected to chronic stress receiving aqueous extracts of melissa officinalis and passiflora caerulea (200 mg/kg) had lower levels of corticosterone and higher blood glucose (feliú-hemmelmann et al., 2013). in the rat study, however, it was shown that only chronic use of lemon balm (30, 100, 300 mg/kg of ethanolic extract) had an anxiolytic effect. the same doses did not give a satisfactory effect in a single portion (taiwo et al., 2012). it was also found that the antidepressant effect of lemon balm can occur by regulating 5-ht turnover in brain regions associated with serotoninergic neurotransmission (lin et al., 2015). in the human study, it was shown that the best concentration was achieved after using a single dose of 600 mg lemon balm leaf extract, a feeling of calm after eating 300 mg, however, it was necessary to consume as much as 900 mg to reduce the sense of “vigilance” (kennedy et al., 2003). on the other hand, 600 mg of a product containing lemon balm leaf extracts and valerian root weakened the negative symptoms of anxiety (kennedy et al., 2006). lemon balm used in beverages or yoghurts can also have an effect that improves well-being and cognitive abilities. consumption of a drink containing 0.3 g of lemon balm leaf extract (containing more than 6% of rosmarinic acid) and additionally a natural sweetener reduced the level of anxiety in the subjects and improved the memory up to 3 hours after consumption. however, this did not affect psychomotor skills. the intake of a higher dose (0.6 g) improved mathe matical and psychomotor skills. regardless of the dose, the drink with melissa officinalis reduced the level of cortisol among the subjects. the results of lemon balm leaf extract in yogurt were partially different lower doses of lemon balm improved alertness, remembering and mathematical abilities, but the use of a higher dose (0.6 g) was associated with a significant feeling of tiredness (scholey et al., 2014). application in sleep disorders melissa officinalis is a frequently used sleeping remedy for the elderly (lutomski, 2000). in a 30-day study in healthy subjects, 360 mg of valeriana officinalis root extract and 240 mg of melissa officinalis extract consumed 0.5 h before bedtime showed to improve its quality. however, this did not affect the general well-being of the subjects (cerny and schmid, 1999). a 15-day pilot study showed that in the stressed group, 600 mg of lemon balm leaf extract (300 mg for breakfast and 300 mg for lunch) reduced anxiety (15-18%) and insomnia related to anxiety (42%). in tab. 1: the content of the main components of lemon balm essential oil (%) based on the results of various researches. components sadraei et al., 2003 abdellatif et al., 2014 meftahizade et al., 2010 miraj et al., 2017 kittler et al., 2018 (z)-citral (neral) 24.5 30.2 43.8 0.8-45.7 (e)-citral (geranial) 35.3 44.2 5.2 0.5-34.3 citronellal 12.9 6.3 0.01 14.4 1.3-60 β-caryophyllene 4.9 1.3 13.5 8.1 1.2-18.6 β-caryophyllene oxide 2.7 1.3 0.3 11.0 0.4-54.1 linalool 0.9 0.3 citronellol 6.2 geraniol 0.7 0.6 5.3 isogeraniol 6.4 nerol acetate 5.1 geraniol acetate 7.1 2.3 10.2 germacrene d 0-13.6 the therapeutic properties of lemon balm (melissa officinalis l.): review 329 tab. 2: summary of human studies on using melissa officinalis tested properties applied dose time of use observed effect test group study antioxidant properties 2 × 1,5 g of leaf infusion 30 days increase in catalase, superoxide n=55 zeraatpishe dismutase, glutathione peroxidase et al., 2011 activity; reduction of dna damage, lipid peroxidation, myeloperoxidase activity application 1200 mg of extract 3 months reduction of pms symptoms severity n=50 akbarzadeh et al., in gynecology 2015 1200 mg of essence 7 days/month reduction of pms psychosomatic n=50 heydari et al., 2018 for 3 months symptoms, anxiety, depression, sleep problems and social problems 3 × 330 mg 3 days/month reduction of systemic symptoms n=50 mirabi et al., 2018 of herb extract for 2 months (lethargy and fatigue) in dysmenorrhea 2 × 1000 mg 4 weeks improvement in women with n=19 darvish-mofrad of aqueous extract hypoactive sexual desire disorder kashani et al., 2018 of the dried leaves anxiolytic and anti300 mg of leaf extract 1 day feeling of calmness n=20 kennedy et al., 2003 600 mg extract of 1 day reduction of anxiety symptoms n=24 kennedy et al., 2006 melissa officinalis leaves and valeriana officinalis root 0,3 g leaf extract 1 day reduction of anxiety symptoms n=5 scholey et al., 2014 (in a drink) 2 × 300 mg of leaf extract 15 days reduction of anxiety, aggression n=20 cases et al., 2011 and hyperexcitation 2 × 500 mg of leaf extract 14 days reduction of anxiety symptoms n=28 alijaniha, 2015 per day 60 drops of melissa 16 weeks reduction of agitation among n=42 akhondzadeh et al., officinalis extract per day patients with mild to moderate 2003 (1:1 in 45% alcohol; alzheimer’s disease at least 500 μg citral/ml) application 240 mg of melissa 30 days improvement of the sleep quality n=66 cerny and schmid, in sleep disorders officinalis leaves extract 1999 and 360 mg of valeriana officinalis root extract 2 × 300 mg of leaf extract 15 days reduction in insomnia frequency n=20 cases et al., 2011 2 × 160 mg of valeriana 1 day improvement of the sleep quality n=50 taavoni et al., 2013 officinalis extract and in women during menopause 80 mg of melissa officinalis extract properties supporting 600 mg of leaf extract 1 day improvement of the concentration n=20 kennedy et al., 2003 0,3 g leaf extract 1 day improvement of the cognitive abilities n=5 scholey et al., 2014 (in drink) 0,6 g leaf extract 1 day improvement of the mathematical n=5 scholey et al., 2014 (in drink) and psychomotor skills 60 drops of melissa 16 weeks better outcome on cognitive function n=42 akhondzadeh et al., officinalis extract per day among patients with mild to moderate 2003 (1:1 in 45% alcohol; at alzheimer’s disease least 500 μg citral/ml) application 3 × 1 g of herb extract 2 months increased ejection fraction, higher n=35 javid et al., 2018 in cardiology nitric oxide concentration, lower lactate dehydrogenase level and reduced systolic and diastolic blood pressure in patients with chronic stable angina 2 × 500 mg of leaf extract 14 days reduction of heart rate palpitations n=28 alijaniha, 2015 by 36.8% 3 × 1000 mg of 2 months reduction of aspartate transaminase n=28 jandaghi et al., 2016 dried leaves activity depressant properties the functioning of the nervous system and cognitive functions 330 k. świąder, k. startek, c.h. wijaya addition, aggression and hyperexcitation decreased. reductions in other symptoms such as eating problems, guilt and tiredness, which were considered to be the cause of fear, were also observed (cases et al., 2011). sleep disorders in women during menopause were also examined. it was shown that two doses of 160 mg of valeriana officinalis extract and 80 mg of melissa officinalis extract allows to reduce the symptoms of sleep disorders (taavoni et al., 2013). in the mouse study, it was found that lemon balm extract (400, 800 mg/kg) significantly reduced the time of falling asleep and extended sleep time. the effect correlated positively with the dose, and the dose of 800 mg/kg had almost identical effect to that of diazepam − a psychotropic drug. it was also shown that the mixture with lavender extract (lavandula angustifolia) had a synergistic effect in terms of length of falling asleep and the sleep itself, which may be important in the treatment of insomnia (hajhashemi and safaei, 2015). effect on memory and neurodegenerative diseases numerous studies are directed to the action of melissa officinalis in the prevention of memory disorders and neurodegenerative diseases. melissa officinalis and rosmarinic acid can help patients with alzheimer’s disease via inflammatory and neuroprotective effects as well as inhibition of the acetylcholine esterase activity and β-amyloid plaque formation in brain (mahboubi, 2019). the rats received intraperitoneally 50-400 mg/kg lemon balm leaf extract. some of them were additionally treated with scopolamine, which is toxic to the nervous system. the 200 mg/kg extract significantly improved the ability to learn and remember and diminished the effect of scopolamine. in addition, inhibition of acetylcholine esterase activity was observed (soodi et al., 2014). in another study, in which rats were given a hydroethanolic extract of lemon balm leaves (200 mg/kg) for 28 days, an improvement in long-term memory was observed. at the same time, the level of acetylcholine esterase mrna decreased in the cerebral cortex and the β-secretase mrna transcription decreased, which may be important in the prevention of alzheimer’s disease (ożarowski et al., 2016). in traditional iranian medicine, a mixture of lemon balm herb and boswellia serrata oleo gum resin extract is used to improve the ability to remember. the use of 200 or 400 mg extracts from both plants in adult rats for 4 weeks had a protective effect against the toxic action of scopolamine on neurons. a better effect in memory tests could be related to antioxidant and anti-inflammatory effects (mahboubi et al., 2016). in vitro tests have also shown that 15% aqueous leaf extract (10 μg/ ml) protects hippocampal neurons against mdma-induced apoptosis (3,4-methylenedioxymethamphetamine) and lowers caspase-3 activity (hassanzadeh et al., 2010). the extract of the same concentration also had a protective effect on hypoxia in nerve cells. the level of caspase-3 and malondialdehyde decreased. in addition, lemon balm reduced the expression of the hif-1α gene (hypoxia induced factor 1α). melissa officinalis can thus be a neuroprotective factor in diseases related to oxidative stress (bayat et al., 2012). it was found that the acid fraction of the ethanolic extract of lemon balm leaves (1:10) had a significant protective effect in the case of neurotoxicity caused by β-amyloid plaques and oxidative stress. probably it was the action of polyphenols and triterpenoids through antioxidant mechanisms or stimulation of nicotinic receptors, and this fraction contains the highest concentration of rosmarinic acid. the use of 10 μg/ml extract or 1 μg/ml acid fraction reversed the effects of toxicity and increased cell viability to its original state (sepand et al., 2013). the efficacy of lemon balm has also been studied in people with mild to moderate alzheimer’s disease. twenty patients received 60 drops of leaf extract a day (1:1 with 45% alcohol, 500 μg citral/ ml) for 16 weeks and fifteen for placebo. already from the fourth week of therapy, significantly better results in the cognitive assessment were observed, and from the 8th week also in the assessment of the severity of dementia. in addition, fewer patients experienced agitation (psychomotor anxiety) in the group using lemon balm than placebo (akhondzadeh et al., 2003). application in ophthalmology macular degeneration is a common disease among older people. the main cause of this disease is the action of free oxygen radicals. the influence of hydroethanolic extract from lemon balm leaves alsl1023 (6.25-200 μg/ml) on oxidative stress in human cells of the retinal pigment layer was investigated in vitro. the extract significantly increased cell viability and decreased apoptosis caused by oxi dative stress. this involved the inhibition of caspase-3/7 and parp, which are the stimulators of apoptosis. the protective effect of lemon balm extract was mediated by the protein kinase b signal transduction pathway. it can be concluded that the lemon balm extract effectively protects against oxidative stress induced by hydrogen peroxide 3 × 1 g of herb extract 2 months reduction of anxiety, stress, n=35 haybar et al., 2018 depression and sleep in patients with stable angina 3 × 500 mg 1 week improvement of the sleep quality and n=80 soltanpour et al., of dried leaf powder the anxiety in patients underwent 2019 coronary artery bypass surgery 700 mg/d 12 weeks improvement of lipids, apo a-i, n=31 asadi et al., 2018 of hydroalcoholic extract apo b/apo a-i ratios in patients with type ii diabetes 700 mg/d 12 weeks improvement in glycemic control, n=31 asadi et al., 2019 of hydroalcoholic extract lipid profile, inflammation and systolic blood pressure application in infusion made of 1 month reduction in body mass, bmi, n=18 jafari et al., 2018 obesity therapy 2 g of lemon balm diastolic blood pressure twice a day application 20% tincture 30 days no reduction in muscle activity n=12 bortoletto, 2016 in bruxism (2 × 15 drops/d) 1 drop of homeopathic 30 days reduction of tooth grinding n=39 tavares-silva et al., preparations in children with bruxism 2019 per year of age the therapeutic properties of lemon balm (melissa officinalis l.): review 331 through antiapoptotic and antioxidant effects (jeung et al., 2016). diabetic retinopathy is another condition where medicinal plants are considered as a potential treatment. due to capability to modu late blood sugar and lipid levels, melissa officinalis can be used for preventing diabetes complications. rosmarinic acid also shows beneficial suppressing retinal neovascularization by inhibition of retinal endothelial cells proliferation and angiogenic tube formation (parveen et al., 2018). application in gynecology lemon balm is used in menstrual disorders, such as lack of or irregular menstruation, in menstrual pain and during excessive bleeding (kapczyński, 2000). the use of 1,200 mg of lemon balm extract among high school students significantly reduced the severity of premenstrual syndrome symptoms during the 3-month treatment. the observed positive effect concerned psychological, physiological and social symptoms. the regulation of the gaba-ergic system may be a likely mechanism for the action of melissa officinalis (akbarzadeh et al., 2015). these results were confirmed by similar research in which female adolescents were treated with 1,200 mg of lemon balm essence during first 7 days of menstruation for three cycles. there was observed an improvement in psychosomatic symptoms, anxiety, sleep disturbances, depression and social dysfunction (heydari et al., 2018). lemon balm seems to be helpful in reduction of systemic symp toms that are associated with dysmenorrhea. fifty students with dysmenorrhea received 330 mg of lemon balm herb extract three times for three days during two menstrual cycles. trial showed a significant decrease in severity of lethargy and fatigue (mirabi et al., 2018). another indication of lemon balm is hypoactive sexual desire disorder − the most common sexual dysfunction in women. in a randomized, double-blind placebo-controlled study a group of 19 women (18 to 50 years old) consumed twice a day two capsules of melissa officinalis (containing 500 mg of aqueous extract of the dried leaves) for four weeks. the treatment led to the increase in frequency of sexual intercourse and had beneficial effects in every domain of this sexual dysfunction (arousal, orgasm, satisfaction, pain, lubrication, desire). improvement can be connected with anti-depressive and anti-anxiety effects, as well as a role of increased norepinephrine and cholinergic activity in sexual desire and arousal (darvish-mofrad-kashani et al., 2018). application in oncology glioblastoma multiforme is one of the most common and most fatal cancers of the central nervous system. the in vitro effects of aqueous and ethanolic extracts of lemon balm and rosmarinic acid on c6 cell lines of this tumour were investigated in rats. in each case, cytotoxic effects and suppression of proliferation were observed, however, rosmarinic acid showed the weakest effect, and its higher doses (≥ 200 μm) had pro-oxidative properties and initiated cell death through necrosis. in various ways, lemon balm can be a neuroprotective factor in the treatment of glioma (ramanauskiene et al., 2016). an ethanol lemon balm leaf extract of 5 μg/ml significantly reduces the proliferation of hct-116 cells (human colon cancer cells), and at a concentration of 1,000 μg/ml significantly reduces their via bility (encalada et al., 2011). the effect of hydroethanolic extract of lemon balm leaf extract (5-1,000 μg/ml) on several different tumour cell lines: a549 (lung cancer cells), mcf-7 (breast cancer), skov3 (ovarian cancer cells) and pc-3 (prostatic adenocarcinoma). even the lowest concentrations of the extract significantly reduced cell viability and inhibited their growth (jahanban-esfahlan et al., 2015; jahanban-esfahlan et al., 2017). melissa officinalis extract was also examined as a potential treatment against human pancreatic cancer cells. however, this plant extracts did not show efficient as antitumoral agent (mouhid et al., 2018). doxorubicin (dox) is a cytostatic drug with high activity in various types of cancer, however, it also has a cardiotoxic effect. it was found that lemon balm herb extract reduced the oxidative stress caused by dox − decreased lipid peroxidation, protein oxidation and increased antioxidant capacity. it also inhibited inflammatory reactions by lowering the expression of the nuclear factor κβ (nf-κβ), tumour necrosis factor-α (tnf-α) and cyclooxygenase-2 and myeloperoxidase activity. incorporation of lemon balm herb extract into doxorubicin may provide a safer cancer treatment protocol (hamza et al., 2016). effect on muscle activity bruxism is a disorder that occurs in 13.5-33% of children, and is characterized by repetitive muscle activity, e.g. clenching and rubbing of teeth during sleep. the effect of 20% tincture from lemon balm (2 × 15 drops/d, 30 days) on muscle activity was studied in 12 children aged 6-10 years. however, no reduction in muscle activity was observed in children (bortoletto, 2016). another research with outcomes measured by a visual analogue scale of children’s teeth showed beneficial effects on bruxism. 39 children (3-12 years old) were treated with homeopathic preparations of m. officinalis, phytolacca decandra, a combination of both or placebo (1 drop per year of age for 30 days). the study showed that the use of lemon balm can reduce tooth grinding in children with bruxism (tavares-silva et al., 2019). application in gastroenterology lemon balm can be used in the irritable bowel syndrome. in an in vitro study, it was demonstrated that essential oil and citral (one of the main components of the oil) have the effect of relieving intestinal spasms caused by potassium chloride, serotonin and acetylcholine (sadraei et al., 2003). the possibility of modulating the visceral hypersensitivity by the hydroalcoholic lemon balm extract in the irritable bowel syndrome in rats is also indicated (dolatabadi et al., 2018). it has also been shown that the methanolic leaf extract (150, 300 and 450 mg/kg) in single dose may have a protective effect on stomach ulcers in rats. the potential mechanism was to increase the activity of superoxide dismutase and glutathione peroxidase and inhibition of lipid peroxidation in cell membranes − reduction of malondialdehyde production (saberi et al., 2016). in the study involving patients with infantile colic there was a positive effect of the supply of chamomile (m. chamomilla l.), lemon balm and l. acidophilus (ha122). the group, that was administered 2 ml of solution (18 mg chamomile, 130 mg lemon balm and 2 × 109 lactic bacteria) in two doses over 28 days, showed a significant ly lower incidence of crying in comparison to infants who received simethicone (martinelli et al., 2017). application in cardiology melissa officinalis is considered as safe alternative to pharmacotherapy in mild palpitations. the use of a dry leaf extract (2 × 500 mg/d, 14 days) among 28 people resulted in a reduction in the rate of palpitations by 36.8%. in addition, a decrease in anxiety symptoms was observed (alijaniha, 2015). in a rat study, it was found that the supply of lemon balm herb extract for one week (50, 100, 200 mg/kg) was associated with changes in electrocardiographic results. the effects were similar to anti-arrhythmic drugs (joukar and asadipour, 2015). in addition, lemon balm may support hyperlipidaemia therapy. 332 k. świąder, k. startek, c.h. wijaya in the study, various doses of ethanolic extract (25, 50 and 75 mg/ kg) were given to hypercholesterolemic rats. it was found that irrespective of the dose, lemon balm reduced the activity of hepatic enzymes: alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase, as well as decreased the level of cholesterol in the blood. the effect of lemon balm was the same as atorvastatin − a drug commonly used to treat hyperlipidaemia (zarei et al., 2014). a similar result was obtained in a study involving people with hyper lipidaemia. a two-month treatment with powdered lemon balm leaves (3 × 1,000 mg/d) resulted in a decrease in the mean ldl level and aspartate aminotransferase (jandaghi et al., 2016). melissa officinalis was also found to improve lipids and apo a-i, apo b/apo a-i ratio in patients with type ii diabetes who received 700 mg/d hydroalcoholic extract of lemon balm for 12 weeks. these effects can be useful in preventing cardiovascular diseases in this group of patients (asadi et al., 2018). this treatment resulted also in a positive changes in glycemic control (hba1c, activity of β-cells, fasting blood sugar), lipid profile (triglycerides, hdl-c), inflammation (high-sensitivity c-reactive protein) and systolic blood pressure (asadi et al., 2019). medical lemon balm was also tested for the use in stable angina. in a double-blind study, 80 patients with this disorder were given 3 g of lemon balm herb extract per day or placebo. the study lasted three months. after this time, a higher cardiac ejection fraction, higher nitric oxide concentration, lower lactate dehydrogenase concentration and reduced systolic and diastolic blood pressure were found in the study group compared to control (javid et al., 2018). there was also a significant reduction of anxiety, stress, depression and sleep problems that are common in patients with stable angina (haybar et al., 2018). it has been shown that lemon balm can be administered to patients who underwent coronary artery bypass surgery due to its beneficial effects on anxiety and sleep disorders that are common in this group of patients. for a week, eighty people three times a day took 500 mg of lemon balm dried leaf powder or placebo. the treatment improved the sleep quality and reduced the anxiety in patients after coronary artery bypass surgery (soltanpour et al., 2019). application in obesity therapy the effectiveness of lemon balm in the treatment of obesity was also examined. it was used in genetically obese mice of the ob-x mix (0.5 mg per day for 5 days), which consisted of morus alba, melissa officinalis and artemisia capillaris. it was observed that weight gains slowed down and visceral fat content decreased without affecting other organs. probably these components act specifically on adipose tissue (yoon and kim, 2011). als-l1023 (made from lemon balm leaves by extraction with ethyl acetate) was administered to obese rats at 0.4 or 0.8% of the diet mass. the preparation inhibited the growth of body fat, significantly reduced its mass and protected against weight gain. inhibition of angiogenesis and mmp activity was also found. als may be a potential method in the prevention and treatment of obesity (park et al., 2015). als-l1023 was also administered to rats on a high-fat diet. it was observed that in comparison with the group on a high-fat diet without supplementation there was a decrease in body weight (without affecting the calorie intake of food), alanine aminotransferase and aspartate aminotransferase as well as suppression of steatosis, inflammatory cell infiltration and collagen accumulation in the liver. medical lemon balm may have a potentially positive effect in the prevention and treatment of obesityinduced human nafld (kim et al., 2017). in the study involving twenty welders, two monthly interventions were used with the use of green tea or lemon balm infusions (2 × 2 g/d). in both cases, a similar decrease in body weight, bmi index and diastolic blood pressure was observed (jafari et al., 2018). safety of use there was no negative effect of a single dose of lemon balm extract (containing 100, 250 or 500 mg of rosmarinic acid) on the overall health and morphological and biochemical results of blood (noguchi shinohara et al., 2015). in one study, the only side-effect of using lemon balm (1,000 mg dry extract/day) was increased appetite (alijaniha, 2015). one of the cases described in the literature indicates the risk of withdrawal syndrome with such symptoms as anxie ty, irritability, decreased appetite, concentration and sleep. however, the described patient consumed up to 4 cups of lemon balm infusion per day for about 2 months, and symptoms appeared two days after cessation (demirci et al., 2015). no adverse reactions are observed in healthy adults after a recommended intake of up to 30 days or in amounts contained in food. melissa officinalis has the gras (generally regarded as safe) status with a maximum level of 0.5% in baked products. there are, however, risk statements when consumed by pregnant women, nursing women and paediatric patients, since genotoxic properties have been demonstrated in in vitro studies. lemon balm should also not be used in people with thyroid disorders or those who use tranquilizers (abudayyak et al., 2015; miraj et al., 2017). the panel on food additives and nutrient sources added to food (ans) concludes that due to the lack of an appropriate dossier supporting the use of oregano and lemon balm extracts as additives, the safety of lemon balm extracts at the proposed uses in eight food cate gories and use levels respectively 2.0 mg/kg bw/day for women and 2.3 mg/kg bw/day for men cannot be assessed (efsa, 2010). conclusions lemon balm can be used for both prevention and treatment. melissa officinalis indicates antioxidant, anti-inflammatory, antimicrobial and antidepressant properties, as well as a wide range of applications in the treatment of sleep disorders, neurodegenerative diseases and obesity as well as in ophthalmology, gynaecology, oncology, gastroenterology and cardiology. most of the therapeutic properties of lemon balm are based on antioxidant activities. this plant as quite safe and can be widely used in many fields of medicine, as well as a component of supplements and functional food. references abdellatif, f., boudjella, h., zitouni, a., hassani, a., 2014: chemical composition and antimicrobial activity of the essential oil from leaves of algerian melissa officinalis l. excli j. 13, 772-781. doi: 10.17877/de290r-6927 abudayyak, m., özdemir 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https://orcid.org/0000-0002-2938-7662 © the author(s) 2019. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). address of the corresponding author: katarzyna świąder, institute of human nutrition sciences, warsaw uni versity of life sciences-sggw, warsaw, poland http://dx.doi.org/10.1177/0748233710383889 angewandte botanik. über einemikroskopisch-graphische methode derbestimmung des fasergehaltes von gespinstpflanzen. (mit 2 figuren auf tafel i. ) von prof. dr. a . herzog. aus den arbeiten der forschungsstelle für bastfasern in sorau n.-l. die feindliche absicht, unsere altgewohnten röhstoffquellen abzuschneiden, und insbesondere unsere blühende textilindustrie lahmzulegen und z u vernichten, hat natürlich unsererseits dazu geführt, für die bisher aus dem auslande in großen mengen be zogenen faserstoffe, wie baumwolle, jute, schafwolle und seide ersatz zu schaffen. wohl haben einige der auf diesem gebiete gemachten zahl reichen vorschläge z u praktisch brauchbaren ergebnissen geführt, so daß z u erwarten steht, daß sie auch in friedenszeiten ihren wert behalten werden, allein von der überwiegenden mehrzahl aller fälle kann dies sicherlich nicht behauptet werden! leider muß auch festgestellt werden, daß vielfach unter dem fadenscheinigen deckmantel vaterländischer gesinnung, faserpflanzen und faser gewinnungsverfahren inmarktschreierischer weise angeboten wurden, d ie sich bei näherer prüfung für die allgemeinheit als gänzlich wertlos erwiesen haben. auch beweist es eine vollständige unkenntnis der sachlage oder eine absichtliche täuschung der öffentlichkeit, wenn in einem atem mit der rettung des vaterlandes aus fasernot an gekündigt wurde, daß die angepriesenen faserstoffe imstande sein würden, baumwolle, jute, flachs usw. auch nach dem kriege voll ständig z u ersetzen bzw. z u verdrängen. man vergaß eben auf der suche nach geeigneten gespinst pflanzen sehr häufig, daß keine höhere pflanze völlig frei von faser strängen ist. letzteres ist auch selbstverständlich, da die „fasern“ angewandte botanik i. 5 66 a. herzog, nach den klassischen untersuchungen schwendeners!) neben der nährstoffleitung auch die besondere aufgabe haben, der lebenden pflanze die zum gedeihen unbedingt nötige festigkeit und wider standsfähigkeit gegen äußere einflüsse zu verleihen. unter diesen umständen is t e s daher nicht wesentlich, daß eine pflanze „fasern“ überhaupt enthält, als vielmehr, daß diese genügend fest, leicht gewinnbar und in den betreffenden organen der pflanze so stark angereichert sind, daß ihre gewinnung im großen wirtschaftlich wird. gerade hier fehlt es aber auch dem in der textilindustrie tätigen a n dem unbedingt nötigen vergleichsmaßstab, d . h . an einem brauchbaren bestimmungsverfahren für den technisch in erster linie maßgebenden gesamtfasergehalt. die bekannten technischen methoden der faserstoffgewinnung bieten durchaus nicht immer die möglichkeit, den absoluten und relativen fasergehalt auch nureiniger maßen einwandfrei z u ermitteln. wohl reichten einige von ihnen hin, vergleichende untersuchungen von organen ein und derselben pflanzenart, z. b . des flachses, auszuführen, aber die verhältnisse ändern sich sofort, sobald man nach dem gleichen verfahren eine andere faserpflanze z u bereiten versucht. so eignet sich z . b . die wasserröste sehr gut zur bereitung bzw. zur bestimmung des faser gehaltes von flachs und hanf, nicht aber auch zu der des ginsters, der typha und des weidenröschens. leider sind auch die zahlreichen chemischen verfahren zur bestimmung des „cellulosegehaltes“ nicht ohne weiteres auf die hier vorliegenden verhältnisse über tragbar, da sie in der regel keine sichere trennung der fasern von den anhängenden zellgeweben zulassen und weil ferner der chemische begriff „cellulose“ durchaus nicht immer mit dem der technischen „faser“ zusammenfällt. so ist denn leicht z u verstehen, daß selbst in solchen kreisen, die sich mit der eingehenden prüfung von ersatzfaserpflanzen be schäftigen, unklarheit über die in den bestimmten fällen vorliegenden fasergehalte herrscht. wenn z. b . von einer seite der bastgehalt der wildwachsenden brennessel z u 2 5 und mehr °/o angegeben wurde, während der tatsächliche nur 5–8°/o beträgt, so bedeutet dies eine völlig willkürliche verschiebung des technologischen begriffes „faser“, die durch nichts gerechtfertigt ist. leider hat auch die tagesund fachpresse durch kritiklose verbreitung derartiger halt loser angaben völlig falsche vorstellungen verbreitet und hoffnungen *) s . schwendener, das mechanische prinzip im bau der monocotylen. leipzig 1874. mikrosk-graph. methode d. bestimm. d. fasergehaltes v. gespinstpflanzen. 67 erweckt, die sich in wirklichkeit niemals werden erfüllen lassen. ferner muß vorläufig noch dahingestellt bleiben, ob die in einigen fällen vorgeschlagene „inkulturnahme“ solcher pflanzen zu einem praktisch brauchbaren ergebnis führen wird, insbesondere, ob diese imstande sein wird, den schon vorhandenen, längst bewährten heimischen textilpflanzen, dem flachse und hanfe, die auf eine viel tausendjährige kultur zurückblicken können, den rang abzulaufen. letzteres müßte aber doch auch angestrebt und sicherlich erreicht werden, wenn anders es einen sinn haben soll, an stelle eines ver stärkten anbaues von flachs und hanf, der aufnahme neuer faser pflanzen in den landwirtschaftlichen betrieb daswort zu reden. im übrigen bleibt auch noch sehr zu berücksichtigen, daß die schwierig keiten nicht so sehr im anbau und in der ernte der pflanze liegen, als vielmehr in deren technischer aufschließung und mechanischer ausarbeitung. und gerade in dieser hinsicht haben die bisherigen praktischen erfahrungen in überreichem maße gelehrt, daß keine der vorgeschlagenen gespinstpflanzen dem flachse gegenüber irgend welche vorzüge besitzt, ja diesen auch nur einigermaßen erreicht! im verlaufe zahlreicher untersuchungen und begutachtungen von ersatzfaserpflanzen bin ic h z u der überzeugung gekommen, daß dasmikroskop noch die beste möglichkeit bietet, über die in einem pflanzenorgan vorhandene relative und absolute fasermenge auf schluß z u geben. im hinblick auf die oben auseinandergesetzte wichtigkeit dieser angelegenheit, sei es mir im folgenden gestattet, das von mir gewählte verfahren a n der hand von einigen original lichtbildern zu erläutern. die zur untersuchung auf fasergehalt vorliegenden pflanzen werden sorgfältig sortiert und eine oder mehrere pflanzen von durch schnittlicher beschaffenheit z u den nachfolgenden arbeiten aus gewählt. diese vorsicht ist genau zu beachten, d a bekanntlich dickere organe bei sonst gleichen verhältnissen relativ faserärmer sind als dünnere. nunmehr wird aus dermitte des zu untersuchenden organes (stengel, blatt) ein der länge nach genau bestimmtes stück (etwa 1 0 cm) herausgeschnitten und, falls es nicht schon trocken sein sollte, durch mehrtägiges lagern a n der luft getrocknet und auf einer empfindlichen wage ausgewogen. zur bestimmung der trockensubstanz wird sodann bei 110° vollständig ausgetrocknet und wieder gewogen. dieses gewicht ist der späteren berechnung d e s fasergehaltes zugrunde z u legen. von einem der beiden stengel 0der blattreste wird nunmehr, unmittelbar a n die schon vorhandene 5* 68 a. herzog, schnittfläche anschließend, ein etwa 1 cm langes stückchen behutsam abgeschnitten und nach der in der botanischen histologie allgemein üblichen härtung mit alkohol und einbettung in paraffin zur herstellung feiner mikroskopischer querschnitte verwendet. in manchen fälllen ist die paraffineinbettung vollständig zu umgehen, vorausgesetzt, daß der zu präparierende gegenstand freihändig oder mittels des mikrotomes gut geschnitten werden kann. als beobachtungsflüssigkeit empfiehlt sich die anwendung konzentrierten glyzerins, da dieses vorzüglich aufhellt und auch keinerlei der nach folgenden untersuchung nachteilige veränderungen der größen verhältnisse der t'flanzengewebe durch quellung usw. bewirkt. der zu prüfende querschnitt wird natürlich selbst bei benutzung voll kommener schneideeinrichtungen und völliger beherrschung der in frage kommenden technik nicht immer die gesamte schnittfläche in tadelloser beschaffenheit umfassen. dies ist jedoch kein erheblicher mangel, da das gleiche prüfungsergebnis auch mit einem nur teil weise erhaltenen schnitt erzielt werden kann, sofern die im folgenden gegebenenwinke genau beachtet werden. auf vollkommen einwand freie schnitte, die als schaupräparate natürlich sonst erwünscht sind, kann also hier verzichtet werden. die experimentelle bestimmung des gesamtfasergehaltes läuft nun darauf hinaus, die von den faseranteilen des organquerschnittes gedeckten flächen graphisch auszumessen und hieraus unter be rücksichtigung des mittleren spezifischen gewichtes der cellulose . (1,5 g) die in der oben gewählten längeneinheit (10 cm) enthaltene fasermenge zu berechnen. die praktische ausführung gestaltet sich nun wie folgt: liegt, was in der regel der fall sein wird, ein nicht voll ständiger schnitt vor, so is t vor allem für eine möglichst genaue zentrierung des präparates sorge zu tragen. dies gelingt sehr leicht, wenn ein nach den angaben heims von den optischen werken e . leitz in wetzlar hergestelltes zählplättchen mit kon zentrischen ringen und 8 sektoren zur verfügung steht (vergl. fig. 1) . es läßt sich ebenso wie ein gewöhnliches mikrometer plättchen auf d ie sehfeldblende des okulares legen und mit hilfe der augenlinse des letzteren scharf einstellen. es versteht sich von selbst, daß das zentrieren nur bei schwachen vergrößerungen vorgenommen werden kann, d a der schnitt, zum mindesten bei stengelund wurzelstücken, vollständig zu überblicken sein muß. sehr zweckmäßig erweisen sich hierbei die von verschiedenen optischen mikrosk-graph. methode d. bestimm. d. fasergehaltes v. gespinstpflanzen. 69 firmen gelieferten objektive mit veränderlicher eigenvergrößerung (z . b . a * von c . zeiss-jena oder g von winkel-göttingen). über die mit dem besonders geeigneten winkelschen objektiv und ver schiedenen okularen erzielbaren vergrößerungen gibt die folgende zahlentafel aufschluss. stellung vergrößerungen mit den huyghensschen und des index komplanatischen okularen. am objektiv g 1 2 3 4 5 0 5 6 10 12 20 1 6 8 12 16 24 2 7 9 15 18 30 3 9 10 18 . 20 36 4 10 12 20 24 40 5 11 13 22 26 44 6 12 15 24 30 48 7 13 16 –26 32 52 8 14 18 28 36 . 56 9 15 19 30 z8 60 10 16 20 32 40 64 bei dem verhältnismäßig großen objektabstand dieser systeme empfiehlt sich die verwendung eines besonderen präpariermikroskops, etwa des nach den angaben p . culmanns") von der firma zeiss-jena gebauten monokularen, bildaufrichtenden prismenmikro skops. wo vorhanden, ist auch daswinkelsche zeichenmikroskop nach behrens”) zu derartigen arbeiten mit großem vorteil brauchbar, d a e s bei benutzung der mitgelieferten lupen und objektive eine reihe von bequem abstufbaren vergrößerungen zuläßt (2–38) und in verbindung mit dem zeichenapparat nr. 1 dieser firma in aus gezeichneter weise auch zum zeichnen der schnitte benutzt werden kann. selbstverständlich sind auch die binokularen präpariermikro skope sehr gut brauchbar, nur muß beim zeichnen die zeichenfläche entsprechend geneigt werden, um verzerrungen der bilder zu ver meiden. am besten geschieht dies mittels des verstellbaren zeichen tisches nach bernhard*), der heute von verschiedenen optischen werkstätten geliefert wird. bei querschnitten von breiten blättern tritt insofern eine änderung in der vorbereitung der auszumessenden fläche ein, als e s genügt, die zahl der auf dem gesamtquerschnitt *) zeitschr. f. wiss. mikr. 20, 416–420, 1903. *) katalog nr. 5 2 von r .winkel, göttingen, 86–88. *) ztschr. f. wiss. mikr. 9 , 439–445, 1892 u . 11, 298–301, 1894. 70 a. herzog, vorhandenen gefäßbündel und einfachen baststränge bezw. deren einzelfasern zu ermitteln und ihre fläche nach dem im folgenden angegebenen verfahren zu bestimmen. hierbei ist es angezeigt, ein netzmikrometerplättchen in das okular einzulegen, um irrtümer in der zählung zu vermeiden. die zählung kann auch bei dikotylen stengeln platz greifen, sofern neben dem primären bast kein sekundärer vorhanden ist, da in diesem falle keine nennenswerten unterschiede in der ausbildung der bastzellen vorhanden sind. (vergl. fig. 1–2). nach erfolgter zentrierung des schnittes wird ein vollständig erhaltener, tunlichst großer sektor (*/4 oder */2) des präparates entsprechend markiert. es geschieht dies am einfachsten so, daß die grenzen durch kleine tuschepunkte, die man mit einer feinen borste aufträgt, kenntlich gemacht werden. allgemein gültige regeln lassen sich hier nicht geben, indessen lehrt die erfahrung sehr bald das richtige treffen. innerhalb des begrenzten sektors werden nunmehr die faseranteile, die in der regel als bündel auftreten (vergl. lichtbild 2 ), mit hilfe eines mikroskopischen zeichenapparates genau abgezeichnet, wobei e s jedoch genügt, die äußere begrenzung der einzelbündel wiederzugeben. da diezeich nung in mittlerer vergrößerung (etwa 300–500) auszuführen ist, muß selbstverständlich das präparat systematisch so lange ver schoben werden, bis sämtliche vorhandene faseranteile ins gesichts feld gelangt und abgezeichnet sind. in solchen fällen, wo die fasern auf dem schnitte mehr oder weniger getrennt voneinander auftreten, wie z . b . beim brennesselund ramiestengel usw., kann insofern eine vereinfachung des meßverfahrens eintreten, als an die stelle der zeichnung die einfache zählung der im begrenzten sektor enthaltenen einzelfasern tritt. selbstverständlich muß aber auch hier die durchschnittliche querschnittsfläche in der noch anzugebenden weise bestimmt werden. die im ersten falle erhaltenen zeichnungen der faserbündel werden nunmehr nach einer der bekannten methoden ihrer fläche nach ausgemessen. am genauesten geschieht dies mit hilfe eines polarplanimeters oder nach der von ambronn angegebenen methode, bei welcher das gezeichnete flächenstück ausgeschnitten und auf einer genauen wage gewogen wird. aus dem gleichzeitig ermittelten flächen einheitsgewicht des benutzten zeichenpapiers läßt sich die dem ausgeschnittenen stücke entsprechende fläche leicht berechnen. zur not kann die bestimmung auch mit einem in qmm mikrosk-graph. methode d. bestimm. d. fasergehaltes v. gespinstpflanzen. 71 geteilten, auf die zeichnung aufgelegten pauspapier bezw. einer entsprechenden glasoder zellhorntafel vorgenommen werden. um die wahre größe der von den faseranteilen gedeckten fläche zu érhalten, is t natürlich das erhaltene snmmarische ergebnis noch durch die ein für allemal genau bestimmte quadratische vergrößerung der zeichnung zu dividieren, wie leicht einzusehen, is t die so gefundene fläche, die natürlich äuf den gesamten querschnitt umzurechnen ist, noch um den von den zellkanälen der fasern gedeckten flächenanteil zu verringern, da es lediglich auf die von den zellwänden gedeckte fläche ankommt. zu diesem zweck wird ein dem durchschnitt entsprechendes bündel in sehr starker ver größerung (1000–2000 linear) abgezeichnet, wobei aber nunmehr auch die innenbegrenzung der einzelzellen sorgfältig wieder zugeben ist. naturgemäß wird diese zeichnung aus mehreren teil stücken bestehen, d a die starke vergrößerung immer nur einen teil des bündels zu überblicken gestattet. durch planimetrische ausmessung wird nunmehr das relative verhältnis der von der zellwand und vom zellkanal gedeckten querschnittsfläche er mittelt und der endgültigen berechnung der wirklichen ge samtfläche der von den faserwandungen gedeckten gesamtfläche zugrunde gelegt. in gleicher weise wird bei den oben er wähnten isolierten fasern vorgegangen, nur mit dem unterschied, daß die berechnung durch mültiplikation der von einer faser wandung gedeckten fläche mit der zahl der früher ermittelten einzelfasern, die auf den gesamten organschnitt entfallen, erfolgt. stellen die zu bestimmenden „fasern“ neben einfachen baststrängen auch gefäßbündel dar, wie dies z . b . bei den schäften und blättern der monocotylen der fall ist, dann ist das gewählte verfahren gleichfalls anwendbar, nur mit dem unterschied, daß bei den ge fäßbündeln auch die anteile der parenchymund holzzellen in rechnung gezogen werden müssen, was natürlich die arbeit ziem lich umständlich macht. in der regel wird es aber auch hier genügen, nur die von den bastbelägen allein gedeckten flächen z u ermitteln, da erfahrungsgemäß die bastzellen wegen ihrer zähigkeit und starken wandverdickung die technisch allein wert vollen teile des bündels ausmachen. der gesamtfasergehalt des in untersuchung gezogenen organes läßt sich nun rechnerisch leicht aus -der gefundenen querschnittsfläche, dem durchschnitt lichen spezifischen gewicht der fasersubstanz (s = 1,5 g ) und dem eingangs bestimmten trockensubstanzgewicht eines 1 0 cm 72 a. herzog, langen organteils ermitteln. bedeuten q die von den wandungen der fasern gedeckte gesamtquerschnittsfläche in qmm und s das spezifische gewicht der faser (s = 1,5 g), so ergibt sich das in mg ausgedrückte gewicht g des in einem 10 cm langen organ stück enthaltenen bastes zu: g = 100 q 1,5 der fasergehalt f in % berechnet sich hieraus und dem in mg ausgedrückten gewichte g des 10 cm langen organstückes zu: _ 100 g r=-gt: es ist einleuchtend, daß die absolute querschnittsfläche der einzelbastzelle auch zur kennzeichnung verschiedener eigenschaften der technischen faser herangezogen werden kann. insbesondere gilt dies von der feinheit, die nach meinen erfahrungen besser durch die metrische nummer im sinne der textilindustrie, als durch angaben von breitenund dickenwerten ausgedrückt wird. es geht dies ju . a . aus verschiedenen zusammenstellungen der von mir gefundenen nummerund meßwerte verschiedener fasern deutlich hervor!). auch zur beurteilung der festigkeitsverhältnisse einer faser erweist sich die kenntnis der querschnittsfläche sehr nützlich, d a nur die von der wandung eingenommene fläche als tragender querschnitt in frage kommt. wenngleich die vorbeschriebene methode nur bei genügender vertrautheit mit mikroskopischen arbeiten zum ziel führt, und naturgemäß umständlicher ist als ein rein chemisches bestimmungs verfahren, so bietet si e dafür den nicht z u unterschätzenden vor teil, in allen fällen, d. h . unabhängig von der schwierigkeit und art irgend eines technischen aufschließungsund ausarbeitungs verfahrens, anwendbar z u sein. auch die möglichkeit, die faserigen anteile allein, also ohne zellige anhängsel, in beliebigen pflanzen teilen bestimmen zu können, muß als besonderer vorteil bezeichnet werden. wie ich aus zahlreichen prüfungen dieser art entnehmen konnte, bieten die gefundenen werte nicht nur ein gutes vergleich bares material hinsichtlich des relativen gesamtfasergehaltes ver schiedener pflanzen, si e lassen sich auch mit vorteil zur kenn *) a . herzog, mikrophotogr. atlas der technisch wichtigen faserstoffe. münchen 1908. mikrosk-graph. methode d. bestimm. d. fasergehaltes v. gespinstpflanzen. 73 zeichnung der für die verfrachtung der pflanzen so wichtigen volumsverhältnisse heranziehen. wie nämlich die einfache über legung ergibt, stellt der prozentuale anteil der von den fasern auf dem organquerschnitt gedeckten fläche gleichzeitig auch die volumsprozente des bastes dar, da auf verhältnismäßig kurzen organlängen keine nennenswerten änderungen in der verteilung und mächtigkeit der ausbildung der faserigen elemente zu ver zeichnen sind. handelt es sich um genaue ermittlungen, dann is t e s natürlich erforderlich, die vorerwähnte prüfung auf verschiedene zonen des betreffenden organes auszudehnen und aus den so erhaltenen prüfungsergebnissen den zugehörigen durchschnitt z u ermitteln. selbstverständlich muß in diesem falle der gesamtquerschnitt d e s betreffenden organstückes vorher ermittelt werden, was am besten gelegentlich der zentrierung des schnittes durch mikrosko pisches zeichnen des organumrisses und durch auswertung der dargestellten fläche in sehr rascher weise ausführbar ist. ein sehr einfaches verfahren zur herstellung tadelloser übersichts querschnitte, die u . a . auch z u photographischen aufnahmen ge eignet sind, habe ich kürzlich eingehend beschrieben!). selbst verständlich läßt sich auch auf derart hergestellten bildern der anteil der einzelnen gewebe und des etwa im innern des organs befindlichen lufterfüllten hohlraumes am gesamtvolumen leicht und rasch ermitteln. (vergl. tafel i fig. 1.) erklärung der tafel i.“ fig. 1 . übersichtsquerschnitt eines flachstengels (linum usitatissimum) nach zentrierung mittels der heimschen bakterienzählplatte. vergr. 40. fig. 2 . randstück eines stengelquerschnittes des leindotters (camelina sativa). . in de r sekundären rinde ein aus mehreren mäßig verdickten bastfasern zu sammengesetztes bündel sichtbar. vergr. 420. *) zeitschr. f. wiss. mikr. 34, 241–244, 1918. uc1.b3299303_page_083_cut uc1.b3299303_page_084_cut uc1.b3299303_page_085_cut uc1.b3299303_page_086_cut uc1.b3299303_page_087_cut uc1.b3299303_page_088_cut uc1.b3299303_page_089_cut uc1.b3299303_page_090_cut uc1.b3299303_page_091_cut journal of applied botany and food quality 94, 176 181 (2021), doi:10.5073/jabfq.2021.094.021 1department of pharmacy, faculty of medicine, university of nis, serbia 2department of mechanic, faculty of mechanic, university of nis, serbia 3department of physiology, faculty of medicine, university of nis, serbia impact of covid pandemic on attitude and prevalence of plant-based food products consumption in serbia dragana pavlovic1*, jelena matejic1, ivan pavlovic2, milica veljkovic3 (submitted: june 10, 2021; accepted: october 25, 2021) * corresponding author summary covid pandemic influence on eating behavior and dietary habits with respect to various plant-based foods in serbia were estimated by an anonymous questionnaire. most examinees agree that fruits and vegetables contribute to strengthening immunity and that herbal medicines and natural products have beneficial effects on health. around 55% of examinees consider their diet balanced, and 4% have started to drink herbal teas more often when the pandemic started. garlic and ginger were the most frequently reported newly included plants in examinees’ diet. the attitudes toward plant-based food products are not strongly dependent on the education level. the age and previous dietary habits of examinees have great influence in the current frequency of consuming fruits, vegetables, herbal teas, spices, and dietary supplements. there was a significant shift toward greater use of herbal teas and dietary supplements, especially among the population that has already consumed them occasionally. somewhat concerning is the fact that around half of respondents use dietary supplements without the recommendation of an expert. people with good dietary habits and the elderly were most prone to improving their diet. roughly 15% of all respondents now have the same habits as before the pandemic, although they improved their diets temporarily at the beginning of covid pandemic. keywords: dietary style, fresh and processed, plant-based products, winter food, self-reported data. introduction the covid (sars-cov-2 coronavirus) pandemic presents a global issue – a great threat to global public health that affects almost every segment of human life. apart from the quarantine and self-isolation of many individuals that promoted unhealthy behavior, pandemic has affected dietary habits and eating behavior alongside personal attitudes and food preferences (ruiz-roso et al., 2020; sidor and rzymski, 2020; chaari et al., 2020). a balanced, optimal diet supports and enhances the immune system function both by providing an effective response against pathogens and by resolving infections in a shorter time, avoiding any further complications (childs et al., 2019). the intake of plant-based foods may also enhance the diversity of nutrients that reach the gut and support the gut microbiome (childs et al., 2019). nutrition and proper diet can promote the functioning of the immune system as a preventive measure by reducing both inflammation and oxidative stress, as well ensuring that the body is in the strongest possible state to battle the virus (chaari et al., 2020; aman et al., 2020). world health organization (who) especially encourages consumption of fresh fruits and vegetables through general nutrition advice for adults during the covid outbreak: to eat fresh and unprocessed foods, moderate amounts of fat and oil, less salt and sugar, and to drink enough water every day (who, 2021). thus, one of the main solutions to promote health should be through healthy eating (shi and yan, 2020). some people are prone to including herbal teas, spices, and dietary supplements both as a replacement or in addition to their regular diet (hamulka et al., 2020). traditionally, pickled vegetables have played a significant role in the dietary habits of the european population, especially during the winter (jeločnik et al., 2019). processed fruits and vegetables are convenient because they are available throughout the year regard less of the season and could supply consumers’ needs for all nutrients since they have a similar concentration of those compounds as fresh equivalents. in some cases, the nutrients from canned food are more readily digestible than those of the fresh equivalents. also, processed plant-based food can be stored for extended periods of time without the need for refrigeration and presents useful pantry food to stock in case people cannot go grocery shopping for various reasons (featherstone, 2016). the last fact was very important during the quarantine and self-isolation due to the covid-19 pandemic. in the light of these considerations and the general impressions of dietary changes during the past period, this study aims to highlight the influence of the covid-19 pandemic on: i) eating behavior and dietary habits changes with respect to various plant-based foods; ii) frequency of consumption of herbal teas and dietary supplements; iii) quantity of traditionally pickled plant-based food prepared for winter period, and iiii) the general attitude toward diet, plant-based food, natural products, and dietary supplements. materials and methods a cross-sectional study was performed using an anonymous online questionnaire that contained 16 open-ended and closed-ended ques tions. in addition to fresh, frozen, and processed fruits and vegetab les, herbal spices and herbal teas are also considered plant-based food products for the purposes of this research. the questions in the survey were related to the general attitude towards these products, and the frequency of their use before and during the covid pandemic in the region of serbia. most of the questions were multiple-choice, allowing respondents to select a pre-specified answer. self-perception on diet and lifestyle changes from the beginning of the pandemic was also recorded, and in the last question, examinees had to specify how much they agree/disagree with some claims. data collection was carried out through a structured questionnaire in serbian created in microsoft forms (microsoft 365). the original questionnaire in serbian, the english translation and the results are available as data set at pavlovic et al. (2021). the questionnaire was modeled based on our previous survey on attitude and prevalence of bee product usage in pediatric pulmonology patients (živanović et al., 2019). the study was conducted by the ethical principles of the helsinki declaration. ethical committee of the faculty of medicine, university of nis, serbia, approved the investigation. the invitation to participate in the survey was shared by e-mail, viber, or by social plant-based food consumption and covid 177 networks such as facebook, and instagram. the first few invitations were given to the people authors knew personally, with a request to share this questionnaire with wider potential respondents. the survey was available for completion in january 2021. participation was completed anonymously, voluntarily, and individual responses were not linked to a specific participant. data obtained by filling out the questionnaire were statistically analyzed by mathlab r2017 software. the data were shown in frequency distribution tables expressed as percentages, which were further analyzed using pearson’s chi-square test. a probability value of p < 0.05 was considered of significance. results and discussion the covid-19 pandemic is impacting not only people’s lives and longevity but also global food trade, food supply chains, and food security and consequently people’s nutrition (fao, 2021). the containment measures adopted by national governments to limit the spread of the coronavirus have caused harmful disruptions through out all elements of the supply chain, from production to processing to distribution (fao, 2021). the current coronavirus pandemic is special compared to the other infectious disease epidemics: since its beginning people had to stay at home or work at home for an extended periods, schools, restaurants, stores, and markets were closed for months, and public transport was restricted. thus, the changes in eating habits could not be attributed only to the knowledge of strengthening immune defenses but also must be perceived in terms of all factors related to the coronavirus pandemic. the government of serbia has enacted series of laws, decrees, orders, decisions, and solutions from the beginning of the pandemic till today (ministry of health and institute of public health of serbia, 2021). the most severe measurement against the global pandemic was lockdown that was 52 days in duration (from 3/15/2020 till 5/6/2020). our anonymous online questionnaire estimated the covid-19 pandemic influence on eating behavior and dietary habits in serbia, alongside preference and personal attitudes toward fruits, vegetables, herbal teas, and spices. dietary supplements were also considered through 3 questions since they have been the focus of public interest since last year (aman et al., 2020; shi and yan, 2020; hamulka et al., 2020). the complete answers of the questionnaires are available as data set at pavlovic et al. (2021). the total number of completed question naires was 408. the average age of the respondents is 27.5±6.6 years, most of them are employed (n=261), followed by students (n=64), unemployed (n=62), and retired citizens (n=21). around a fifth of respondents were males (20.1%). the highest level of education was college/postgraduate training and high school, 278 and 128, respectively. only 2 reported basic level of education. change in eating behavior and dietary habits toward fruits, vegetables, herbal teas, and spices previously published data reported insufficient fruit and vegetable consumption in serbia (rodić-trmčić et al., 2015). almost all examinees in our survey agreed that fruits and vegetables contribute to strengthening immunity although 24% and 14.5% of them, included greater amounts of fruit and vegetables, respectively, into their diet compared to the amount before the covid pandemic. education level influenced the tendency to use the same amounts of fruits or vegetables in combination with dietary supplements (p=0.0163, and p=0.0117, respectively), while the most used vegetables were roots and tubers (potatoes, carrots, beets), followed by vegetables from legume (beans, peas, green beans), cabbage (cabbage, cauliflower, broccoli) and onion family (onion, garlic). regarding fruits, the most popular are southern fruits (oranges, lemons, bananas), followed by seasonally grown (apples, pears, plums) and wild seasonal fruits (wild strawberries, raspberries, blackberries). the increased intake of vitamin c could be an explanation for such agrums popularity. on the other hand, nutritional value of wild-growing fruits was mentioned in 2% of responses, overall interest in edible wild plants seems low. the study conducted in central italy shows that knowledge of wild plants use is principally concentrated in the elderly, alongside the lack of interest of younger generations for such kind of information (ranfa and bodesmo, 2017). recent work of matejić et al. (2020) documents that the use of medicinal plants for eating, cooking, and compote in addition to treating health complaints is widespread in some parts of serbia. fruits and vegetables that are most frequently newly included in examinees’ diet since the beginning of the pandemic were: garlic, beets, horseradish, celery, pomelo, avocados, mango, and pineapple. ginger, thyme, ribwort plantain, parsley, nettle, turmeric, and wild oregano were reported as recently introduced medicinal plants/spices. garlic and ginger were highest ranked from all listed plants. considering the growing popularity of garlic, who pointed out that, while garlic is a healthy food that may have some antimicrobial properties, there is no evidence from the current outbreak that eating garlic has provided protection from the new coronavirus (who, 2021). garlic antimicrobial properties have been well documented in many references in literature and the committee on herbal medicinal products of the european medicines agency (ema/hmpc) recommend it as a traditional herbal medicinal product used for the relief of the symptoms of the common cold (ema/hmpc/7686/2013). according to ema/hmpc ginger rhizome (zingiber officinale roscoe) is a herbal medicinal product for the prevention of nausea and vomiting in motion sickness, and a traditional herbal medicinal product for the symptomatic treatment of mild, spasmodic gastro intestinal complaints including bloating and flatulence (ema/ hmpc/577856/2010). still aqueous and organic extracts of ginger root have repeatedly been shown to possess broad anti-infectious (antibacterial, antifungal, antiviral, and anthelmintic) properties, and ginger is one of the favorite folk remedies for infectious diseases (ema/hmpc/749154/2010). according to a survey conducted among pediatric pulmological patients’ parents/guardians, one of the most popular homemade bee product blends applied to the child for respiratory problems consisted of honey and ginger (živanović et al., 2019). change in the frequency of herbal teas and dietary supplement consumption significant number of respondents of lower education level (completed elementary and high school) tend to use a smaller quantity of fruits and vegetables in combination with dietary supplements (p=0.0235, and p=0.0234, respectively). compared to women, men were prone to use the same quantity of fruits now as before the pandemic (p=0.0335), while women use the same quantities of fruits and vegetables but with the addition of dietary supplements (p=0.0180, and p=0.0042, respectively). around one-third of all respondents reported regular use of dietary supplements since the beginning of the pandemic, although only 5% and 2.5% of them admitted to lower amounts of fruits and vegetables in the diet, respectively (fig. 1). the sales of dietary supplements on the global market had increased dynamically in the wake of covid-19 in most of the countries, and at the beginning of the pandemic, some types of supplements recorded even triple-digit growth rates (hamulka et al., 2020). over the counter (otc) market in serbia has increased by 15% compared to the prior (pre-pandemic) year with additional 43 m eur in sales. the highest contributor of the increase was vitamins and minerals, along with other dietary supplements. in retail, otc segment of the serbian pharma market represented 34% of sales. regardless of the 178 d. pavlovic, j. matejic, i. pavlovic, m. veljkovic otc market‘s constant annual growth (for example, there was an increase from 2018 to 2019 by about 12%), the exceptional impact of covid on the otc market cannot be ignored. in 2020 vitamins and minerals overtook the leading market position from cough and cold products on the serbian otc market (chamber of commerce of serbia, 2021). around half of our respondents use dietary supplements without the recommendation of an expert. bearing in mind potential interaction, overdosage, and side effects of prolonged uncontrolled dietary supplements application (ronis et al., 2018), especially when polypharmacy takes place, it is worrisome that a great number of consumers do not discuss the use of supplements with healthcare professionals. our survey revealed that 43% of respondents support the statement that vitamins, minerals, and dietary supplements are necessary for maintaining good health in current times while around 38% disagree with that conclusion. according to the opinion of most respondents (87%), herbal medicines and natural products (teas, balms, bee products, etc.) have a beneficial effect on health. such a high percentage was not a sur prise since almost every home in serbia possesses a copy of „lečenje biljem“ (herbal therapy, 1973). this book (written by a university professor, dr. jovan tucakov) systematizes ethnopharmacological knowledge herbal products and their specific uses, and self-medication practice based on the book’s contents or related ethnopharmacological experience within the family is very common (stojanović et al., 2017). herbal teas are the most popular type of tea and could be noticed that individuals that used to drink herbal tea occasionally moved toward the regular use with the covid-19 pandemic (tab. 1). yet only 4% of examinees went from no use to regular consumption of herbal teas with the start of the pandemic. the influence of education level on the frequency of tea consumption was not statistically significant. female gender and advanced age (50+) significantly increase the probability of regular and frequent tea usage (p=0.0291, and p=0.0041, respectively). as it could be seen from tab. 1, both herbal teas and dietary supplements gained popularity from the beginning of the pandemic, although a greater shift toward more frequent use is achieved in supplement application. dietary supplements should not and cannot be used to treat the problems caused by an unhealthy diet (shi and yan, 2020). the caution towards supplement use, especially among vulnerable populations (e.g., people with chronic conditions), is a wise approach (shi and yan, 2020). variation in the quantity of traditional plant-based food left for winter food preparation is a strong part of the serbian family tradition. seasonal vegetables are traditionally fermented, and pickles generally consumed during the wintertime (ubiparip samek et. al. 2021). the most consumed processed vegetables in serbia are ajvar, cab bage, and turshiya, while the fruit is processed to different sweet preserves. they are often made at home in the autumn, even in cities, but are also sold in supermarkets and served in restaurants. turshiya is made with green peppers, green tomatoes, carrots, cauliflower, cabbage, and celery. the vegetables are put in a large, covered container, pressed down with some twigs and a stone, and a marinade made of salt, vinegar, and water is poured over. the pickles are left to ferment and are commonly consumed till early summer. pickled cabbage (sauerkraut) is made separately in a barrel with whole cab bages and marinade of only with salt and water afterward. pickled cabbage is used both as a winter salad and as an important ingredient of some dishes (jelocnik et al. 2019). ajvar (pepper-based pate made from red bell peppers) and its variations (which also include carrots, eggplant, onion, garlic, and tomatoes) are a regular part of the table spread during the winter. sweet fruit preserves such as jams, jellies, marmalades, and compote (matejić et al., 2020) are often eaten for breakfast as spread on bread or as an ingredient of a pastry or dessert. the winter food left for 2019/2020 winter was an excellent source of processed vegetables and fruits during the lockdown in spring of 2020. recommendations of the institute of public health on proper nutrition during covid-19 pandemics among others are: „to re duce the number of trips to the market or supermarket, in addition to fresh, you can also buy frozen or canned fruits and vegetables that also contain vitamins and minerals.“ (institute of public health niš, 2020). accordingly, one of the questions of our survey dealt with the quantity of „winter food“ prepared for the pandemic 2020/2021 winter. compared to last year, many respondents reported the same amount of frozen and processed fruits and vegetables prepared for winter (73%); significantly greater or greater amounts were declared by 11%, somewhat or much smaller amounts by around 10% and 5%, respectively, while 1% of examinees did not prepare at all. these results could either be caused by fear of new quarantine and food shortage or by the worsening economic situation. besides the constant growth of fruits and vegetable prices in recent years (institute of public health of serbia 2020), covid-19 has magnified social inequalities and made the poorest families struggling to put food on the table (fao, 2020). the general attitude toward diet, plant-based food, natural products, dietary supplements, and teas although most respondents in our survey have the attitude that fruits and vegetables contribute to strengthening immunity, 3% of exa fig. 1: comparison of the amounts of (fresh, frozen, and processed) fruit/ vegetable respondents declared to eat now vs. before the covid-19 pandemic. * denotes the significant difference in fruit and vegetable consumption in the selected category. tab. 1: frequency of use of herbal teas and dietary supplements before and during covid pandemic. frequency of use herbal teas (%) dietary supplements (%) before during before during covid covid covid covid regularly 20.6 24.8 8.8 22.1 often 15.9 21.6 7.1 15.4 periodically 35 31.1 32.1 27 when i am ill 9.6 6.4 9.1 6.1 really rare 11.3 10 18.9 12.3 only in winter 3.2 1.2 2.2 1 never 4.4 4.9 21.8 16.1 plant-based food consumption and covid 179 minees do not believe in the health benefits of everyday fruit and vegetable consumption. regarding the quality of plant-based food, around 70% of respondents connect the excellent appearance of vegetables and fruits (seemingly the most appealing ones) with overtreatment with different chemicals, and more than half of examinees prefer moderate price and appearance of herbal food. accordingly, fruits and vegetables are most commonly bought at the open market place, followed by supermarkets and groceries although similar numbers of respondents declared each of these three places for purchase. it is interesting to mention a fast reaction of a new generation of producers and consumers that have made a facebook group in response to the closing of marketplaces during the lockdown and the need of citizens to supply food. the largest such group in the capital of south serbia (niškafriška – eng. fresh in niš) has grown to an online platform that provides complete technical support to both customers and manufacturers. the group offers fresh and processed fruits and vegetables, honey, dairy products, nuts, etc. (niskafriska market, 2021). the conclusions of some recent studies (ruiz-roso et al., 2020; sidor and rzymski, 2020) pointed out diet impairment due to the covid-19 pandemic while some others reported its improvement (rodríguez-pérez et al., 2020). according to surveys conducted in spain, italy, brazil, colombia, and chile, the covid-19 pandemic modified dietary trends of adolescents, seemingly because families had more time to cook and improve eating habits by increasing legume, fruit, and vegetable intake. this change did not increase the overall diet quality (ruiz-roso et al., 2020). this group of authors pointed out also that females living in europe and those who reported a higher maternal education had the highest rates of adherence to the weekly food intake recommendation (ruiz-roso et al., 2020). our finding that people with good dietary habits are most prone to improve eating behavior is following the conclusions of sidor and rzymski (2020). they concluded that overweight and obese subjects are most prone to quarantine-related adverse dietary modification resulting in lower daily frequency of fruit, vegetable, and legume consumption, increased food consumption and snacking, and the grea test tendency to consume meat, sweets, salty snacks, and fast foods daily (sidor and rzymski, 2020). a cross-sectional study on the eating behavior changes of the adult population due to the covid-19 confinement pointed out a significantly higher adherence to the mediterranean diet during the confinement across all 16 european countries included. also, stricter contingency restrictions seemed to lead to a significantly higher increase in adherence to a healthy diet (rodríguez-pérez et al., 2020). although there were no statistically significant differences between fruits, vegetables, and herbal tea consumption before and during the covid-19 pandemic, a great number of respondents in our study noted a shift toward greater use of plant-based food (fig. 1). indeed, around half of the examinees pointed out that their diet slightly moved toward a „healthy“ one since the beginning of the pandemic (among them near 14% significantly approved their dietary practice) while 4% reported a negative impact of the pandemic on their diet style. 15% of all respondents now have the same habits as before the pandemic although they improved their diets at the beginning of the covid-19 issue (fig. 2). old habits appear hard to change even when motivation and some initial improvement exist. according to self-assessment more than a quarter (26.2%) of respondents abso lutely consider their diet balanced with a sufficient intake of fruits and vegetables, and around 30% consider it strongly balanced. yet, 10% admitted to poor dietary habits. there was a significant shift toward greater use of herbal teas and dietary supplements (with p values 0.0391 and 0.0002, respectively) especially among the population that had already used them occasionally. the number of respondents who regularly take dietary supplements and herbal teas significantly increased while the ones that use them never or very rarely significantly decreased (pearson’s χ2, p < 0.05). the strongest statistical significance was noticed for the shift toward regular dietary supplements intake (p=0.00000017, χ2=27.3689). according to our results, the attitudes toward plantbased food are not dependent on the education level while the age of examinees and their previous dietary habits influence greatly the frequency of consuming fruits, vegetables, herbal teas, and dietary supplements. the older respondents are, the they are more likely to improve their lifestyle according to self-evaluation. our survey has shown that pandemic did not much affect dietary habits except greater vitamins, minerals, and supplements usage and moderate shift toward larger consumption of different plant-based food products. yet this cross-sectional study has given indications that a modest shift toward greater use of plant-based food in everyday diet and awareness of its importance in the covid pandemic exist. strengths and limitations of the study our study group included respondents that filled out an online ques tionnaire in serbian language during the month of january 2021. since examinees from different age groups and from the different geographical locations were invited to participate in the survey, we can say that the sample of 408 completed questionnaires could be adequate in size for this type of study and deduced conclusions. only a quarter of the examinees were male, since women are more likely to participate in various questionnaires. this is the first study conducted on the use of plant-based food products and dietary supplements before and during the covid pandemic in the serbianspeaking area. one of the limitations of this study is the self-reported data, where one can only rely on the memory from the time of fruits, vegetables, teas, spices, and dietary supplements consumption before the pandemic and its readiness to honestly report the usage of such products. conclusion having in mind that the current pandemic impacted, among other things, eating behavior and dietary habits, dietary changes from the beginning of the pandemic are in the focus of today’s research. the covid-19 pandemic influenced consumption and attitude toward fruits, vegetables, teas, spices, and dietary supplements in serbia. the self-perception of personal diet changes revealed shift toward a healthier lifestyle in around half of respondents, although 4% reported worsening dietary habits in comparison to the period before fig. 2: self-perception on diet change since the beginning of covid pandemic. 180 d. pavlovic, j. matejic, i. pavlovic, m. veljkovic the new corona sars-cov-2 pandemic. an interesting observation is that 15% of all respondents improved their diets at first but have since returned to the same habits as before the pandemic. also, a greater use of herbal teas and dietary supplements was noticed especially among the people who already have used them from time to time. yet, dietary supplements are often used without consultation with health professionals. since plant-based food besides nutritive possesses numerous health-promoting effects, its consumption should be constantly pointed to the global population, especially in the time of highly processed food in which we are living. acknowledgments this work is supported by the ministry of education, science and technological development of the republic of serbia (grant no: 451-03-9/2021-14/200113) and an internal scientific project (no. 15) of the faculty of medicine, university of niš. conflict of interest no potential conflict of interest was reported by the authors. references aman, f., masood, s., 2020: how nutrition can help to fight against covid-19 pandemic. pak. j. med. sci. 36 (covid19-4), covid19 121-123. doi: 10.12669/pjms.36.covid19-s4.2776 chaari, a., bendriss, g., zakaria, d., mcveigh, c., 2020: importance of dietary changes during the coronavirus pandemic: 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is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). https://orcid.org/0000-0001-6048-4834 https://orcid.org/0000-0001-6410-4296 https://orcid.org/0000-0003-0062-4342 https://orcid.org/0000-0001-6705-4706 journal of applied botany and food quality 92, 24 (2019) buchbesprechung gewürze und küchenkräuter – gewinnung, inhaltsstoffe, wirkungen, verwendung eberhard teuscher, mit beiträgen von monika werner in der 2. auflage seines naturwissenschaftlich orientierten stan dardwerks nimmt der autor den leser wieder mit auf eine faszi nierende entdeckungsreise durch die welt der gewürze und kräuter. in 84 reich bebilderten portraits erfährt er vieles über botanik, anbau und ernte, gewinnung und handelsformen, inhaltsstoffe, qualitätsmerkmale und wirkungen der bekanntesten gewürzpflanzen sowie deren verwendung in küche und hausmedizin. in anhängen werden 200 weitere, in europa seltener genutzte gewürze sowie 150 gewürzmischungen beschrieben. vom stinkenden asant über den kostbaren safran bis zum duftenden zimt stellt der autor ins gesamt 300 pflanzen sowie die daraus gewonnenen gewürze vor allem aus naturwissenschaftlicher, pharmazeutischer und medizi nischer sicht vor. dabei wird auch auf die jeweilige kultivierung, die gewinnung der gewürzdrogen, deren unterschiedliche handelsformen, sowie auf die chemie und analytik der wichtigsten pflanzeninhaltsstoffe näher eingegangen. darüber hinaus finden sich auch hinweise auf die physiologische wirksamkeit und ihre toxikologie. vielen monographien von gewürzdrogen sind rezepturen beigefügt, die zum eigenen experimentieren und probieren mit den oftmals exotischen düften einladen sollen. in einem allgemeinen teil werden zunächst generelle aspekte behandelt, die ein besseres grundverständnis für das gebiet der gewürze und gewürzmischungen vermitteln sollen (z.b. wertbestimmende inhaltsstoffe, pharmakologische, toxikologische, konservierende und sensorische eigen schaften, züchtung, anbau, ernte und aufbereitung, analytik). die neue auflage liefert in den einzelnen monographien deutlich mehr hinweise auf die einzelnen wirk-mechanismen und ermöglicht es dem leser dadurch, selbst eine bessere bewertung publizierter versuchsergebnisse vornehmen zu können. so werden z.b. vermehrt angaben zur versuchsmethodik und zur dosis der zur wirkungs untersuchung durchgeführten experimente geliefert. viele mono graphien enthalten auch kurze abschnitte, in denen zur ver vollständigung diejenigen gewürzdrogen beschrieben sind, die in ländern mitteleuropas bisher nur selten oder gar nicht eingesetzt werden, die jedoch in anderen ländern verbreitet anwendung finden. jeder monographie ist ein individueller literaturteil zugeordnet, der neben wissenschaftlichen arbeiten zur jeweiligen pflanzenart auch spezifische literatur aus dem internet enthält. darüber hinaus steht im anhang des buches ein kapitelüberschreitendes verzeichnis von fachbüchern und monographien zur verfügung. das vorliegende standardwerk richtet sich in erster linie an apotheker, ärzte, biologen sowie studenten der genannten fachrichtungen, ist aber durchaus auch lebensmittelchemikern, die sich auf den bereich der medizinal und gewürzpflanzen fokussiert haben, dringend zu empfehlen. auch für interessierte laien ist das buch als nachschlagewerk durchaus geeignet, da es neben den zahlreichen informationen auch ausge zeichnete farbaufnahmen liefert, die den leser mit dem aussehen der jeweiligen pflanzen und der gewürzdrogen vertraut machen. dr. h. schulz e-mail: hartwig.schulz@julius-kuehn.de bibliografie: eberhard teuscher, gewürze und küchenkräuter – gewinnung, inhaltsstoffe, wirkungen, verwendung, deutscher apotheker verlag, stuttgart, mit beiträgen von monika werner, 2. überarbeitete und erweiterte auflage 2018, 639 seiten, 38 s/w und 192 farbige abbildungen, 75 formelkästen mit 600 formelzeichnungen, preis: 185,€, isbn: 978-3-8047-3306-0 journal of applied botany and food quality 93, 66 75 (2020), doi:10.5073/jabfq.2020.093.009 1departamento de agronomía, facultad de ciencias agrarias, universidad nacional de colombia, bogotá, colombia 2laboratorio de fisiología y bioquímica vegetal, departamento de biología, facultad de ciencias, universidad nacional de colombia, bogotá, colombia 3departamento de ingeniería civil y agrícola, facultad de ingeniería, universidad nacional de colombia, bogotá, colombia growth, development and quality of passiflora tripartita var. mollissima fruits under two environmental tropical conditions mildred mayorga1, gerhard fischer1*, luz marina melgarejo2*, alfonso parra-coronado3 (submitted: october 23, 2019; accepted: march 1, 2020) * corresponding author summary the curuba (passiflora tripartita var. mollissima) is an important andean fruit in bioprospecting industries because of its pleasant taste and aroma, antioxidant potential and sedative action. the ob jective of this study was to evaluate the development of curuba plants and the physicochemical characteristics of fruits under two environmental tropical altitudinal conditions. crops were established in a low zone (2,006 m.a.s.l.) and a high zone (2,498 m.a.s.l.) in the municipality of pasca (cundinamarca, colombia). phenological monitoring was carried out in the principal growth stages. the weight, length, diameter, color, firmness, ph, total soluble solids, total titratable acidity and organic acid content were measured in the fruits. climatic parameters were monitored during the crop cycle, and base temperatures and thermal times were estimated. the temperature and photosynthetically active radiation (par) were the climatic factors that had the greatest effect on plant development. the base temperatures of growth of the primary branches, floral buds and fruits were 4.3 °c, 3.1 °c and 0.01 °c, respectively. in the lower zone, the plants accumulated more growing degree days than in the upper zone. the fruits in the upper zone presented a higher weight, total titratable acidity and ascorbic acid content. the plants presented a marked response to the differential agroecological conditions of the two sites. introduction the curuba or banana passionfruit (passiflora tripartita var. mollissima) is a semi-perennial fruit plant with a climbing habit that is native to the andean zone and belongs to the passifloraceae family, passiflora genus and tacsonia subgenus (primot et al., 2005; campos and quintero, 2012). the species p. tripartita var. mollissima is grown commercially in colombia along the andes mountain range (segura et al., 2005) between 2,000 and 3,200 m.a.s.l. curuba fruits are desired for their organoleptic properties (yockteng et al., 2011), providing a source of vitamins a, b, c and niacin, calcium, iron, phosphorus, potassium, zinc, and fiber (leterme et al., 2006; chaparro-rojas et al., 2014; conde-martínez et al., 2014). in addition, it has a high antioxidant activity and a high content of phenolic compounds (vasco et al., 2008; simirgiotis et al., 2013), especially tannins, flavonoids and phenolic acids (rojano et al., 2012; chaparro-rojas et al., 2014). according to larcher (2003), “phenology is the study of the times of recurring phenomena of life history of plants and animals in relation to climate” and is one of the simplest ways to assess changes during life cycles (menzel, 2002). phenological observations are important in agriculture for evaluating the good development of plants and making agronomic management decisions related to the date of carrying out crop tasks; they are also used in agrometeorological studies to analyze the relationships between crop development and climate (chmielewski, 2013). one method widely used to predict growth and plant development is growing degree days (gdd), which express the amount of energy that a species requires to successfully complete a certain phenological stage or crop cycle (nunes et al., 2016) and consist of the cumulative difference between the average ambient temperature and the base temperature, below which the plant cannot develop (pedro-júnior and sentelhas, 2003). for this reason, the base temperature of each phenological stage of a species is a tool that allows better management and control of a crop (madakadze et al., 2003). physical (light, photoperiod, temperature, precipitation), edaphic and biotic factors continuously affect the physiology of plants and are determinants of plant growth (menzel, 2002; gallé et al., 2015), fruit quality (kullaj, 2016) and post-harvest life because they modify the physiology, morphology and chemical composition (ahmad and siddiqui, 2015). understanding how the phenology of a plant responds to climatic variations is important for predicting adaptive changes in response to climate change (miller-rushing et al., 2007); however, these responses differ not only between individuals but also depend on developmental adaptation to regionally differing factors (doi et al., 2010); therefore, it is important to conduct comparative studies between zones for the same species. information on the growth and phenological development of p. tri partita var. mollissima is scarce, as is knowledge on the effect of climatic factors on its development. some studies have been carried out on other passion fruits of economic interest, such as sweet granadilla (passiflora ligularis juss.) (rodríguez-león et al., 2015), yellow passion fruit (passiflora edulis sims f. flavicarpa degener) (menzel et al., 1987; gómez et al., 1999; freitas et al., 2015; santos et al., 2016) and gulupa passiflora edulis sims (lederman and gazit, 1993; carvajal et al., 2012; flórez et al., 2012; franco et al., 2014; rodríguez et al., 2019). furthermore, studies evaluating the characteristics of fruits produced under different environmental tropical conditions have been carried out in fruit trees, such as purple passionfruit (flórez et al., 2012), sweet granadilla (espinosa et al., 2015; espinosa et al., 2018), cape gooseberry (fischer et al., 2007), guava (solarte et al., 2014) and grapevine (greer and weedon, 2012; martínez-lüscher et al., 2015). there is little research on the physicochemical characterization of curuba fruits, and the few studies there are have focused on evaluating the effect of different postharvest treatments (téllez et al., 2007; botia-niño et al., 2008), which is very important to improving quality conservation and commercialization of the fruit; however, knowledge on how environmental growing conditions affect fruits is important to generating agronomic management practices aimed at improving quality and production characteristics. accordingly, this research aimed to determine the base temperature for four phenological stages of the curuba, its duration in terms of thermal time, and the physicochemical characteristics of the fruits under two different altitudinal tropical conditions. characterization of passiflora tripartite grown under different tropical conditions 67 material and methods plant material and growth conditions in two areas in the municipality of pasca (cundinamarca, colombia), a high zone and a low zone, three-month-old curuba seedlings (p. tripartita var. mollissima), which had the first tendril emitted, were planted. the plants were obtained from a commercial nursery in the region. in the high zone, the crop was established in the santa teresita village on the “el refugio” farm, located at 4°16´ 20.9´́ n 74°19´ 21.7´́ w, at an altitude of 2,498 m.a.s.l. in the low zone, the crop was established in the san pablo village on the “bellavista” farm, located at 4°18´ 45.52´́ n 74°19´ 21.7´́ w, at an altitude of 2,006 m.a.s.l. in order to estimate the base temperatures for the main stages of development of the curuba, plantings were made at different times. in the high zone, a crop was established in december 2014 and another in may 2015; while in the low area, the planting was done in july, 2015. in each area, the plants were established with distances of 3 m between rows and 4 m between plants in a 600 m2 area, for a total of 50 plants and a planting density of 834 plants/ha. the soil in the upper zone was characterized by a sandy-loam texture, with a ph of 4.7; cation exchange capacity of 4.53 meq/100 g; 0.82% of n; 2.1 meq/100 g of ca; 0.51 meq/100 g of k; 0.48 meq/10 g of mg; 33.9 mg/kg of p; 11 mg/kg of s; 1.19 mg/kg of cu; 103 mg/kg of fe; 1.53 mg/kg of mn; 2.05 mg/kg of zn; and <0.12 mg/kg of b. the soil of the low zone was characterized as having a clayey texture, with a ph of 4.5; cation exchange capacity of 10.96 meq/100 g; 0.24% of n; 8.74 meq/100 g of ca; 0.31 meq/100 g of k; 2.02 meq/100 g of mg; 0.34 mg/kg of p; 24.56 mg/kg of s; 0.033 mg/kg of cu; 2.51 mg/ kg of fe; 0.18 mg/kg of mn; 0.17 mg/kg of zn; and 0.14 mg/kg of b. the climatic conditions of the two zones are described in tab. 1. a tutored system was used, which consisted of the installation of two horizontal wires for the conduction of the primary branches, one located at 1.5 m and the other at 2 m above the ground level. when the plants reached the second wire (2 m), a pinch was made in order to break the apical dominance and promote growth of the primary branches. with the pruning management, four primary branches were left on each plant: two at the level of each wire on opposite sides. when the primary branches reached the length of 1.8 m, they were pruned to promote the growth of secondary branches, which in turn were pruned when they reached a length of 0.75 m. weather stations (coltein ltda., bogota, colombia) with dataloggers (coltein ltda., bogotá and hobo u12-006, onset computer cor poration, bourne, ma, usa) were installed in each of the two study areas for recording temperature, relative humidity (sensors vp-3, decagon devices, usa) and photosynthetically active radiation (par) (sensors li 190 b, li-cor inc. lincoln, ne, usa). the data logging was configured for 15 min. intervals. the vapor pressure deficit (vpd) was calculated from the air temperature and relative humidity data with the method proposed by allen et al. (1998). the daily light integral (dli) was calculated from the average daily par during daylight hours and expressed in mol m-2 day-1. phenological monitoring the phenological monitoring of the plants was done from december 2014 to june 2016. the measurements were done weekly, using the phenological stages described by rodríguez-león et al. (2015) for passiflora ligularis because there is no defined phenological scale for curuba. to carry out the monitoring, 15 plants were randomly selected in each study area, considering that fournier-origgi and charpantier-esquivel (1975) recommended a minimum sample size of 10 plants for phenological studies on tropical trees. during the growth of the main stem (bbch-scale 3), the length of the stem and the number of knots were evaluated, until more than half of the sampled plants reached the final length of the stem (2 m). in the evaluation of the branch development (bbch-scale 2), 15 primary branches, 3 cm in length, were marked, and the length and number of nodes of each marked branch were recorded until reaching the final preassigned length of 1.8 m. subsequently, when the secondary branches were developed, 15 branches, 2 cm in length, were marked, and length and number of nodes were evaluated weekly until they reached the final length of 0.75 m. during the development of the floral bud (bbch-scale 5), 10 buds of approximately 1 cm in length in the apical part of the growing secondary branches were marked on eight plants, for a total of 80 floral buds per zone. with each marked bud, the length and diameter were recorded until the opening of the flower. during the fruit development (bbch-scale 7), five fresh fruits of approximately 2 cm in length were marked on the secondary branches of eight plants, for a total of 40 fruits per zone. in each marked fruit, the length and diameter were recorded until the fruits reached harvest maturity, characterized by growth cessation and a slight loss of the firmness of the fruit. determination of base temperatures and thermal times the base temperatures (bt) and thermal times (growing degree days, gdd) of four phenological stages of the curuba were estimated: stem and primary branch growth, secondary branch growth, flower average temperature (°c) relative humidity (%) 620 680 vapor pressure deficit (vpd; kpa) tab. 1: average climatic conditions of the two cultivation areas in the municipality of pasca (cundinamarca, colombia): high zone (2,498 m.a.s.l.) from december 2014 to april 2016 and low zone (2,006 m.a.s.l.) from july 2015 to june 2016. climate variable low zone high zone daytime (6:00-18:00 h) 19.4 14.9 nighttime (18:01-5:59 h) 16.1 12.9 average low temperature (°c) 11.5 8.6 average high temperature (°c) 26.7 23.8 daytime (6:00-18:00 h) 80.5 90.2 nighttime (18:01-5:59 h) 87.3 90.3 par in hours of maximum radiation (μmol m-2 s-1 of photons 10:00 h) daily light integral (dli, mol m-2 day-1) 15.2 17.5 daytime (6:00-18:00 h) 0.68 0.41 nighttime (18:01-5:59 h) 0.52 0.38 precipitation during growth (mm) 681 696 68 m. mayorga, g. fischer, l.m. melgarejo, a. parra-coronado development, and fruit growth, using the methodology described by parra-coronado et al. (2015). the base temperature (bt) for each of the phenological stages was determined from the sum of the average daily temperatures, recorded in each locality. the coefficient of variation (cv) of the estimated thermal times (gde) was then minimized, considering a temperature range between 0 and 12 °c with increments of 0.1 °c, for each of the phenological stages. the bt for each phenological stage was obtained using the solver tool for excel® with a quadratic regression model, and corresponded to that temperature for which the lowest cv for the gde was obtained (parra-coronado et al., 2015). the duration of each phenological stage in terms of thermal time (gdd) was determined from the estimated bt values for each stage. the gdds were calculated as the daily sum of the difference between the average temperature and the bt for each stage (equation 1). (1) where, gdd is the thermal time (°c) accumulated during the n days of duration of the phenological stage, ti is the average daily temperature (°c) for day i and bt is the base temperature (°c). gdds were calculated taking into account the following considerations presented as (2), (3), (4) (parra-coronado et al., 2015): tmax + tmin ti = (2) 2 ti > bt, gddi = ti bt (3) ti < bt, gddi = 0 (4) where, tmax and tmin are the maximum and minimum temperature for day i, respectively. physicochemical characterization of fruits in each study area, 40 fruits were collected in the harvest maturity stage (firmness 61-63 n corresponding to 6.2-6.4 kg/f), maintained at room temperature (18 °c) and transported to the plant physiology and biochemical laboratory of the department of biology of the universidad nacional de colombia, bogotá, to take the different physicochemical measurements. the length and diameter of the fruits were measured using a digital calibrator (fischer scientific 0-150 mm); the fresh weight was measured on an analytical balance (mettler ab204); and the firmness was measured with a penetrometer (0-13 kg) using a 2 mm head, with which force was applied in three points of the central part of each fruit. the colorimetric coordinates l*a*b* were evaluated at three points in the equatorial zone of the fruit exocarp, using a tristimulus colorimeter (hunter associates laboratory, mini scan xe plus, reston, va, usa), and the coordinate values were esti mated, l*c*h° (l*: brightness, c*: chroma or saturation, and h°: color circle tone), as indicated by sui et al. (2016) and barrera et al. (2008). subsequently, the fruit juice was extracted, and the total soluble solids (tss) measurements were taken with the use of a digital refractometer (hanna instruments, woonsocket, usa). the total titratable acidity (tta), expressed as meq-g of oxalic acid in 100 g of pulp, was determined by titration with 0.1 n naoh, as indicated by hernandez et al. (2010). the content of organic acids (malic, citric, oxalic and ascorbic) of the fruit juice was determined as described by solarte et al. (2014), for which 2 g of juice were placed in a 15 ml falcon tube covered with aluminum foil, and 12 ml of 5 mm phosphoric acid were added. the mixture was homogenized with vortexing and then centrifuged at a centrifugal force of 2,817 × g at 12 °c for 30 min; after which, a 2 ml sample of the supernatant was taken, filtered through 0.2 μm drum filters, and placed in a vial. the samples were analyzed in hplc system equipped with a uv-visible detector (waters, milford, ma, usa) with an hplc roa organic acid h+ column of 30 cm × 7.8 mm. to obtain the calibration curves, a standard mixture of ascorbic acid, citric acid, malic acid, and oxalic acid, at five concentration levels between 0.01-1 mg·ml-1, were used. chromatogram peaks were identified by comparing retention times of the samples with external standards (hplc grade, sigma, usa). statistical analysis for each major evaluated phenological stage, the trend of growth was adjusted to seven different mathematical models: linear, polynomial of the second degree, exponential of two parameters, exponential of three parameters, gompertz, logistics of three parameters and monomolecular; based on studies by paine et al. (2012), who de monstrated that nonlinear models are appropriate for modelling plant growth. the linear model has an absolute growth rate that is constant (paine et al., 2012); in the polynomial model, growth follows a smooth curve, potentially with great complexity (poorter, 1989; heinen, 1999). the logistic model is characterized as having a sigmoidal shape and results from the combination of the exponential and monomolecular models, separated by an inflection point (franco et al., 2014). the exponential model is useful for describing continuous growth or decreases when growth conditions remain favorable (rojas-lara et al., 2008). the monomolecular model has no inflection point and its shape is concave downwards, the growth with this model is characterized by being fast at the beginning and, later, slow (paine et al., 2012). in the gompertz model, growth declines exponentially over time (heinen, 1999) and differs from the logistic model in that the inflection point is first presented (paine et al., 2012). in each case, the model that presented the highest coefficient of determination (r2) and the lowest root mean square error (rsme) was selected using statistical software statistix 9.0®. the parameterization of the gompertz model was carried out as indicated by winsor (1932), with the polynomial model by rodríguez and leihner (2006), and the monomolecular model by seber and wild (1989). in order to establish the differences between the bt of each phe nological phase to estimate the gdd and to compare means, a tukey test was used with the statistical package spss v.17 (spss inc., chicago, il, usa). additionally, spearman’s correlation coefficients between the daily growth rates of each evaluated structure and the climatic variables for each zone were estimated. in relation to the physicochemical characteristics of the fruits, the assumptions of normality and homocedasticity were checked, and t-student tests (p≤0.05) were carried out with sas® software version 9.2. results climatic conditions of study areas fig. 1 shows that the high zone was characterized by a higher relative humidity and par, and the lower zone was characterized by a higher temperature and vpd. in both zones, the months of march and june had higher and lower temperatures, respectively. while the month of may had the highest relative humidity, and the month of september had the lowest relative humidity, the opposite of the vpd. in the lower zone, the highest par occurred in the month of september and, in the upper zone, in the month of january. the rainfall regime in the municipality of pasca is bimodal, presenting two rainy seasons: the first in the months of april and may and the second in the months of october and november. 𝑛𝑛𝑛𝑛 𝑖𝑖𝑖𝑖𝑖𝑖 𝑛𝑛𝑛𝑛 𝑖𝑖𝑖𝑖𝑖𝑖 𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺 = � (𝑇𝑇𝑇𝑇𝑖𝑖𝑖𝑖 − 𝐵𝐵𝐵𝐵𝑇𝑇𝑇𝑇) = � 𝑇𝑇𝑇𝑇𝑖𝑖𝑖𝑖 − 𝑛𝑛𝑛𝑛𝐵𝐵𝐵𝐵𝑇𝑇𝑇𝑇 characterization of passiflora tripartite grown under different tropical conditions 69 base temperature (bt) the bt was different in each of the evaluated phenological stages. the growth of the primary branches was the stage that had the highest bt (4.3 °c), accumulating 1,204±69 growing degree days (gdd). the bt for the growth of the secondary branches was 1.5 °c, and the thermal time was 1,449±253 gdd. the develop ment of a floral bud had a bt of 3.1 °c and an accumulation of 532±11 gdd, while the bt for fruit growth was 0.01 °c and the thermal time was 1,456±224 gdd. for the estimation of the thermal time in the growth stage of the main stem, the same bt was used as for the development of the primary branches (4.3 °c) since both structures have similar development processes (vegetative stage) and, probably, do not directly support the development of fruits (reproductive stage), contrary to the secondary branches where the fruits are developing. vegetative phase development the increase in the length and number of nodes of the main stem as a function of the thermal time presented good adjustment to the logistic model of three parameters. in the low zone, the plants accumulated 1,799 gdd until reaching the final height of the stem, developing a total of 47 nodes; while in the high zone, the plants reached this height and the same number of nodes with 1,398 gdd, that is, 22% less gdd than in the lower zone (fig. 2a and 2b). this difference in thermal time does not indicate that the development of the stem in the upper zone occurred in a shorter calendar time; on the contrary, in the lower zone, the elongation and development of the stem nodes were more accelerated (data not shown), but the accumulation of gdd was greater because of the higher average temperature of this site, as explained in the second paragraph of the discussion chapter. the increase in length and development of the nodes in the primary and secondary branches presented a good fit to the polynomial model of second degree. the primary branches in the low zone reached the final length with 1,084 gdd, and, in the high zone, the final length was reached with 1,177 gdd, indicating that, in spite of the lower temperature in the high zone, the plants there accumulated 8.6% more gdd in this stage (fig. 2c), taking a longer calendar time. in both zones, the primary branches developed 31 nodes when they reached the final length (fig. 2d). in the development of secondary branches, in the high zone, they reached a length of 75 cm, accumulating 1,353 gdd; while in the low zone, they accumulated 1,798 gdd, 33.9% more gdd than in the high zone (fig. 2e). as with the development of the main stem, the secondary branches developed faster in the lower zone, possibly because of the higher average temperature. in both zones, the secondary branches presented 21 nodes when they reached the length of 75 cm (fig. 2f). reproductive phase development the growth in length of the floral buds as a function of thermal time was adjusted to an exponential model of three parameters. the growth of the floral buds had a similar pattern and thermal times in the two zones. in the upper zone, the buds accumulated 517 gdd until reaching the floral opening, and, in the lower zone, they accumulated 537 gdd (fig. 3a). the emergence of the perianth from inside the bracts that encapsulate the fruit occurred in the low zone with 212 gdd, and, in the high zone, they emerged with 222 gdd (fig. 3b), when the floral bud was 2.5 cm long. the increase in length and diameter of the fruits as a function of thermal time was adjusted to the monomolecular model. in the low zone, the fruits completed their development when they accumulated 1,222 gdd, reaching a length of 8.4 cm and a diameter of 3.8 cm; in the high zone, the fruits accumulated 1,414 gdd to reach the point of harvest maturity (15.7% more gdd than in the low zone). however, the fruits in the high zone reached a greater length and diameter than low zone, 10.8 and 4.2 cm, respectively (fig. 3c and 3d). effect of climatic conditions on phenology in the plants in the lower zone, the growth rate in diameter and length of the fruits showed an inverse and significant correlation with the dli; while fruit diameter had a direct correlation with daytime and nighttime temperature (tab. 2). in the plants grown in the upper zone, the rate of development of node numbers in the secondary branches showed a significant and inverse correlation with the daytime temperature and dli. the fruit growth, both length and diameter, showed a significant and direct correlation with the dli, but an inverse correlation with the nighttime temperature (tab. 2). fig. 1: monthly behavior of climatic variables of the two cultivation areas in the municipality of pasca (cundinamarca, colombia): high zone (2,498 m.a.s.l.), and low zone (2,006 m.a.s.l.). 70 m. mayorga, g. fischer, l.m. melgarejo, a. parra-coronado fig. 2: node length, growth and development of node number as a function of thermal time in the main stem (a and b), primary branches (c and d) and secondary branches (e and f) of p. tripartita var. mollissima in a high zone (2,498 m.a.s.l.) and a low zone (2,006 m.a.s.l.) in the municipality of pasca (cundinamarca, colombia). vertical bars indicate the standard error (n = 15). fig. 3: growth in length (a) and diameter (b) of flower buds; length (c) and diameter of fruits (d) as a function of the thermal time of p. tripartita var. mollissima in a high zone (2,498 m.a.s.l.) and a low zone (2,006 m.a.s.l.) in the municipality of pasca (cundinamarca, colombia). the vertical bars indicate the standard error (n=80 floral buds and n=40 fruits). characterization of passiflora tripartite grown under different tropical conditions 71 the responses of fruit development to the environmental conditions were contrary in the two zones, possibly because of the adaptation process of the plants since, in the site with a lower average temperature (high zone), high nocturnal temperatures negatively affected the development of the fruits, while, in the area with least dli (low zone), high levels of dli negatively affected development. physicochemical characteristics of fruits the fruits produced in the high zone presented a greater fresh weight, length and diameter than the fruits in the low zone (tab. 3), suggesting that the differential climatic conditions during the growth and development of the two crops influenced the final size of the fruits. the color of the fruits in the low zone presented greater luminosity (l) and saturation (chroma) than those in the high zone, and their tone (ºh) corresponded to an intermediate shade between green and yellow, with a greater tendency towards yellow (tab. 3). the firmness of the fruits at the point of harvest maturity did not show significant differences between the two zones. in the low zone, the fruits presented a higher tss content, and those of the higher zone had a higher tta (tab. 3). the predominant organic acid in the curuba fruits was oxalic acid, with levels above 8,000 mg/100 g, followed by citric, ascorbic and malic acids (tab. 3). the fruits in the upper zone had a higher con tent of ascorbic and citric acids than the fruits in the lower zone; while the fruits in the lower zone had a higher content of malic and oxalic acids than those in the upper zone (tab. 3). discussion in various studies on the phenology of fruit species, including passion fruits, thermal times have been determined using a standard base temperature of 10 °c; however, in some research on other tropical altitudinal commercial fruit plants such as feijoa (acca sellowiana) and cape gooseberry (physalis peruviana), variations of the bt according to the species and the phenological stage (parra-coronado et al., 2015; salazar et al., 2008) have been reported. these findings coincided with the present study, in which the vegetative development phase had a higher bt than the phase of reproductive development, and the fruit development stage had the lowest bt (0.01 °c). this response could have been due to the fact that the fruits that started their growth were acclimated at low temperatures at the zone of growth (parra-coronado et al., 2015), such as the cape gooseberry, which has a bt of 1.9 °c for its fruit growth (salazar et al., 2008). in addition, the curuba can tolerate temperatures up to -5 °c and is cultivated at altitudes up to 3,400 m in peru (national research council, 1989). also, in strawberry plants, a bt as low as 0 °c was reported for leaf insertion (rosa et al., 2011). temperature is one of the climatic variables that have the greatest influence on the growth and development of crops and, therefore, on their different phenological stages, whose duration is a good indicator of potential crop growth. calendar time (days) has often been used to predict the beginning and end of phenological stages without considering the physiological aspects of plants for their development; however, its physiology is affected by the predominant temperature in the crop (parra-coronado et al., 2015). methods have been used for a better prediction of the onset and end of phenological stages, which consider the effect of temperature on the development of plants (salazar-gutierrez et al., 2013). one of the more widely used methods is the accumulation of average daily temperature above a base temperature (bt), which is known as thermal time or growth degree-days (gdd). gdds are defined as the number of day degrees needed to complete a phenological stage or a specific development process and are used to estimate the dates of appearance of knots, leaves, inflorescences, and fruit set, as well tab. 2: matrix of correlations between phenological development indicators and climatic variables evaluated in p. tripartita var. mollissima in a high zone (2,498 m.a.s.l.) and a low zone (2,006 m.a.s.l.) in the municipality of pasca (cundinamarca, colombia). the number in each box indicates spearman’s correlation coefficient and * the level of significance: * p≤0.05, ** p<0.01. development indicator low zone high zone temperature temperature vpd dli temperature temperature vpd dli nighttime daytime daytime nighttime daytime daytime length of main stem (cm/day) 0.02 -0.003 -0.17 -0.09 -0.45 -0.05 -0.001 -0.04 nodes of main stem (nodes/day) 0.20 0.17 0.15 0.27 -0.23 -0.06 -0.27 -0.08 length of primary branches (cm/day) 0.44 0.59 0.35 0.47 0.12 0.25 0.41 0.17 nodes of primary branches (nodes/day) 0.23 0.37 0.23 0.28 0.01 0.30 0.29 0.38 length of secondary branches (cm/day) 0.07 0.42 0.53 0.41 -0.16 -0.42 -0.31 -0.49 nodes of secondary branches (nodes/day) 0.21 0.46 0.51 0.28 -0.18 -0.56* -0.36 -0.52* floral bud length (cm/day) -0.27 -0.24 -0.18 -0.24 0.17 0.33 -0.09 0.04 fruit length (cm/day) 0.58 0.68 0.36 -0.71* -0.94** 0.23 0.78 0.88* fruit diameter (cm/day) 0.70* 0.79* 0.34 -0.81* -0.97** 0.11 0.75 0.83* tab. 3: physicochemical characteristics of p. tripartita var. mollissima fruits in a high zone (2,498 m.a.s.l.) and a low zone (2,006 m.a.s.l.) in the municipality of pasca (cundinamarca, colombia). the values indicate the means ± the standard error (n=40). the * indicates significant differences according to the t-student test (p≤0.05), ns: not significant. variable high zone low zone significance fresh weight (g) 109.4±2.2 94.8±1.5 * length(cm) 11.1±0.1 9.5±0.1 * diameter (cm) 4.4±0.03 4.0±0.03 * tone (°h) 115.6±0.7 111.9±0.6 * brightness 36.4±0.6 44.8±0.8 * saturation (chroma) 14.9±0.4 20.5±0.6 * firmness (n) 63.1±3.8 60.74±3.16 ns tss (°brix) 8.3±0.1 9.65±0.17 * ph 3.2±0.02 3.44±0.01 * total titratable acidity (%) 1.7±0.03 0.94±0.04 * citric acid (mg/100 g) 445±14 120±10 * malic acid (mg/100 g) 20±1 37±1 * oxalic acid (mg/100 g) 8525±439 17365±465 * ascorbic acid (mg/100 g) 189±18 37±6 * 72 m. mayorga, g. fischer, l.m. melgarejo, a. parra-coronado as to estimate the growth of fruits, harvest date and potential crop production (parra-coronado et al., 2015). longer thermal times and fewer calendar days to complete each phenological stage in areas with higher average temperature (low zone) result from acceleration in phenology as a response to in creases in temperature (menzel et al., 2006; miller rushing et al., 2007) because of the accumulation of heat units, as has been reported in other high zone fruit trees such as acca sellowiana (parra-coronado et al., 2015) and for climbing habits, as in vitis vinifera (martínez et al., 2015) and passiflora sp., in which high temperature conditions increase the production of knots and the elongation of internodes (menzel et al., 1987). the temperature and dli were the only climatic variables that had an effect on the daily growth rates; however, the response was different and even inverse, depending on the stage of growth and the zone. bahuguna et al. (2015) indicated that plants can alter their response to changes in temperature in the shortand long-term, depending on the phenological stage, the type of tissue and the metabolic composition. the correlation coefficients between the temperature (daytime and nighttime) and the daily growth rate in the different phenological stages were in most cases positive and higher in the lower zone, which had the highest temperature and the greatest daily and annual temperature variation. menzel et al. (2006) indicated that phenophases exhibit a stronger response to temperature in warmer regions than in colder regions. additionally, it is possible that, because of greater fluctuations in the climatic conditions in the low zone, the plants have developed different strategies of adaptation to this variability (doi et al., 2010). during the development of the main stem and primary branches, the growth was slower in the plants in the high zone, where a greater par was seen. additionally, in the upper zone, the development of node numbers of secondary branches showed a negative correlation with the dli. a short stem length under high solar radiation conditions may be due to an inhibitory effect of uv light on the synthesis of auxins (fischer, 2000). in passiflora alata, vegetative growth has been stimulated with the use of a shading nets that decreased the incidence of light and uv rays (freitas et al., 2015). the growth model during the development of the main stem in p. tripartita var. mollissima was similar to that reported for p. ligularis, which had a good fit to the logistic model of three parameters in the growth in length and nodes of the main stem, primary branches and secondary branches (rodríguez-león et al., 2015). the growth in curuba floral bud length was adjusted to the exponential model, while in sweet granadilla it was adjusted to the three-parameter logistic model (rodríguez-león et al., 2015). the differential response between the two species may result from the morphology of the floral bud, where the curuba features a bud with an elongated hypanthium, and the granadilla has a rounded bud. on the other hand, the growth of the curuba fruits was adjusted to the monomolecular model, while, in comparable studies, the fruits of sweet granadilla and yellow passionfruit have been adjusted to logistic models (rodríguez-león et al., 2015; gómez et al., 1999). and purple passionfruits have been adjusted to logistic and monomolecular models (lederman and gazit, 1993; carvajal et al., 2012; flórez et al., 2012; franco et al., 2014). in all cases, these models are asymptotic, where growth rates are initially rapid and then gradually decrease as the fruits reach their final size. although the phenological development was more accelerated in the low zone, the fruits produced there were smaller, an effect that may have been due to the fact that, under high temperature conditions, allocation to fruits is reduced as a result of the shorter duration of the crop cycle, where photosynthesis rates do not compensate for reduced development times (parent and tardieu, 2014). in addition, there may be limited transport of sucrose and starch metabolism because the genes involved in sucrose synthesis, sucrose transport, sucrose breakdown and starch synthesis are suppressed (bahuguna and jagadish, 2015). v. vinifera fruits have also shown less weight in plants grown in greenhouses under high temperature conditions (28/18 °c), as compared to fruits in plants grown under ambient temperature conditions (24/14 °c) (martínez et al., 2015). in the low zone, the smaller fruit size was also associated with the lower par of the zone, which is reflected in the negative correlation between the dli and the length and diameter of the fruits since luminosity is the fundamental factor for photosynthesis and production of photoassimilates that support fruit development (martínez-vega et al., 2008). the greater weight and size of fruits in the high zone is an indicator of better quality according to the colombian technical standard for the curuba (ntc 1262), which established first quality fruits as those that exceed 11 cm in length, 4.5 cm in diameter and a 100 g weight. second quality fruits have a length between 8 and 11 cm, diameter between 3.8 and 4.5 cm and weight between 70 and 100 g, characteristics of an average fruit produced in the low zone. for fruit color, the tone of the fruits from the low zone was closer to yellow than those of the high zone, which could indicate a lower chlorophyll content that occurs in low light conditions as a result of a lower synthesis of chlorophylls (kullaj, 2016) and in high temperature conditions because fruit ripening processes that include degradation or unmasking of chlorophylls are accelerated (hornero-méndez and mínguez-mosquera, 2002; barrera et al., 2008). the higher content of tss in the curuba fruits from the lower zone was similar to that reported for v. vinifera fruits, which presented high rates of maturation and accumulation of tss related to high temperatures during late stages of fruit development (greer and weedon, 2012), probably because of higher hydrolysis of starch, giving rise to soluble sugars (sucrose, fructose and glucose) (téllez et al., 2007). the lower tta in the fruits of this same area can be associated with the fact that, under high temperature conditions, the respiration rates of curuba fruits increase (téllez et al., 2007), a process that uses organic acids as a substrate (botia-niño et al., 2008; batista-silva et al., 2018). additionally, fruit ripening is accelerated, in which organic acids are converted into sugars through the gluconeogenesis process (prassana et al., 2007; famiani et al., 2015). the lack of studies on the biochemical characterization of curuba fruits has led to the assumption that citric acid predominates in this fruit, as seen in other passionfruits (hernández and fischer, 2009). however, the results of this study indicate a predominance of oxalic acid in curuba fruits. the content of ascorbic acid in the fruits of the upper zone was much higher than that reported by téllez et al. (2007) and by vasco et al. (2008), while the fruits of the lower zone had a lower ascorbic acid content than those reported by these authors. the ascorbate pooling in fruit crops is affected by abiotic factors, such as light or temperature, because of its role in the antioxidant cellular system (fenech et al., 2019). this, the higher content of ascorbic acid in fruits in the high zone, may be due to the conditions that included higher par and lower temperature. high light in particular is translated into a ros burst caused by an increased photoreduction and photorespiration, which, in turn, leads to increased ascorbate biosynthesis in order to detoxify these ros (asada, 1999). the higher content of ascorbic acid in the high zone fruits is a favorable characteristic for the commercialization of the product, given the nutritional importance of this compound (vitamin c) be cause of its antioxidant activity and its function as cofactor in many enzymes, such as enzymes involved in the synthesis and stability of collagen and in the synthesis of carnitine (fenech et al., 2019). the response of the curuba plants under the two evaluated climatic characterization of passiflora tripartite grown under different tropical conditions 73 conditions and the effect of environmental factors on the quality and organoleptic characteristics of the fruits suggested agronomic management alternatives for changing climate environment scena rios, such as water management, pruning and radiation, in order to maintain good physiological performance. conclusions the curuba plants showed a phenotypic response to adjusting their growth and development to the climatic conditions of each zone. the plants grown in the zone with a higher temperature (lower zone) presented a greater accumulation of growing degree days and a more accelerated phenological development; however, the fruits produced there were smaller because of the shorter accumulation time of photoassimilates and lower daylight integral. the temperature and dli were the two climatic factors that had the greatest effect on plant phenology. the fruits produced in the two zones showed marked differences in their physicochemical properties, suggesting that the contrasting climatic conditions had an important effect on the quality of the fruits, with the fruits from the upper zone having a greater size, weight and content of ascorbic acid, conditions that infer better quality. acknowledgments the authors thank colciencias for financing this study within the framework of the project “ecophysiology, mineral nutrition and integrated management of pests and diseases in avocado, banana passion fruit, purple passion fruit and tree tomato, geared towards agronomic management, as a raw material for the development of products of commercial interest” led by l.m. melgarejo, within the framework of the “national network for bioprospecting tropical fruits-rifrutbio”, contract rc no. 0459-2013; the government of cundinamarca; the ceiba foundation for financing m. mayorga’s master’s thesis; carolina rueda and carlos patiño for allowing the establishment of crops on their farms; and stephany hurtado for helping with data collection in the field. conflict of interest no potential conflict of interest was reported by the authors. references ahmad, m.s., siddiqui, m.w., 2015: factors affecting postharvest quality of fresh fruits. in: ahmad, m.s., siddiqui, m.w. 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temperaturas. rev. colomb. cienc. hortic. 1/1, 67-80. doi: 10.17584/rcch.2007v1i1.1146 vasco, c., ruales, j., kamal-eldin, a., 2008: total phenolic compounds and antioxidant capacities of major fruits from ecuador. food chem. 111/4, 816-823. doi: 10.1016/j.foodchem.2008.04.054 winsor, c.p., 1932: the gompertz curve as a growth curve. proc. natl. acad. sci. 18/1, 1-8. yockteng, r., coppens, g., souza-chies, t., 2011: wild crop relatives: genomic and breeding resources: tropical and subtropical fruits. springer science + business media. orcid mildred mayorga: https://orcid.org/0000-0002-4467-829x gerhard fischer: https://orcid.org/0000-0001-8101-0507 luz marina melgarejo: https://orcid.org/0000-0003-3148-1911 alfonso parra-coronado: https://orcid.org/0000-0001-9045-2083 address of the corresponding authors: prof. dr. gerhard fischer (r), departamento de agronomía, facultad de ciencias agrarias, universidad nacional de colombia, a.a. 14490, bogotá (colombia). e-mail: gfischer@unal.edu.co prof. dr. luz marina melgarejo, laboratorio de fisiología y bioquímica vegetal, departamento de biología, facultad de ciencias, universidad nacional de colombia, a.a. 14490, bogotá (colombia). e-mail: lmmelgarejom@unal.edu.co © the author(s) 2020. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1007/s10722-005-2255-z http://dx.doi.org/10.1016/j.foodchem.2015.07.021 http://dx.doi.org/10.17584/rcch.2007v1i1.1146 http://dx.doi.org/10.1016/j.foodchem.2008.04.054 i supplementary material supplementary material tab. s1: growth adjustment equations of p. tripartita var. mollissima structures in a high zone (2,498 m.a.s.l.) and a low zone (2,006 m.a.s.l.) in the municipality of pasca (cundinamarca, colombia). parameter low zone high zone length of main stem 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝑛𝑛𝑛𝑛𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿ℎ (𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐) = 308.39/(1 + 𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸(2.7110 − 0.00179 ∗ 𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺)) 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝑛𝑛𝑛𝑛𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿ℎ (𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐) = 256.62 / (1 + 𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸(2.4362 − 0.00251 ∗ 𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺)) number of nodes, main stem 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝐿𝐿𝐿𝐿𝑁𝑁𝑁𝑁 = 64.672 / (1 + 𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸(1.4049 − 0.00133 ∗ 𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺)) 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝐿𝐿𝐿𝐿𝑁𝑁𝑁𝑁 = 71.876 / (1 + 𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸(1.4811 − 0.00150 ∗ 𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺)) length of primary branches 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝑛𝑛𝑛𝑛𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿ℎ (𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐) = 1.996 + 0.0505 ∗ 𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺 + 0.0000973 ∗ 𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺 2 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝑛𝑛𝑛𝑛𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿ℎ (𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐) = −7.943 + 0.1627 ∗ 𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺 + 0.00000335 ∗ 𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺2 number of nodes, primary branches 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝐿𝐿𝐿𝐿𝑁𝑁𝑁𝑁 = 2.5417 + 0.0173 ∗ 𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺 + 0.00000674 ∗ 𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺 2 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝐿𝐿𝐿𝐿𝑁𝑁𝑁𝑁 = 0.9674 + 0.0279 ∗ 𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺 + 0.00000151 ∗ 𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺2 length of secondary branches 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝑛𝑛𝑛𝑛𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿ℎ (𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐) = −0.401 + 0.0626 ∗ 𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺 − 0.000011 ∗ 𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺2 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝑛𝑛𝑛𝑛𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿ℎ (𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐) = −4.1602 + 0.0739 ∗ 𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺 − 0.00000894 ∗ 𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺2 number of nodes, secondary branches 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝐿𝐿𝐿𝐿𝑁𝑁𝑁𝑁 = 3.0797 + 0.0143 ∗ 𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺 − 0.00000212 ∗ 𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺2 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝐿𝐿𝐿𝐿𝑁𝑁𝑁𝑁 = 1.5248 + 0.0206 ∗ 𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺 − 0.00000435 ∗ 𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺2 length of flower buds 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝑛𝑛𝑛𝑛𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿ℎ (𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐) = 0.4242 + 0.5291 ∗ 𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸(0.0056 ∗ 𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺) 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝑛𝑛𝑛𝑛𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿ℎ (𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐) = 0.3097 + 0.0986 ∗ 𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸(0.0088 ∗ 𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺) diameter of flower buds 𝐺𝐺𝐺𝐺𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝑐𝑐𝑐𝑐𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐷𝐷𝐷𝐷 (𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐) = 2.463 ∗ 𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸(−𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸(2.416 − 0.007 ∗ 𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺)) 𝐺𝐺𝐺𝐺𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝑐𝑐𝑐𝑐𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐷𝐷𝐷𝐷 (𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐) = 3.617 ∗ 𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸(−𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸(2.523 − 0.0058 ∗ 𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺)) fruit length 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝑛𝑛𝑛𝑛𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿ℎ (𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐) = 8.719 ∗ (1 − exp�−0.0025 ∗ (gdd + 86.988)�) 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝑛𝑛𝑛𝑛𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿ℎ (𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐) = 10.793 ∗ (1 − exp�−0.0026 ∗ (gdd + 86.691)�) fruit diameter 𝐺𝐺𝐺𝐺𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝑐𝑐𝑐𝑐𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐷𝐷𝐷𝐷 (𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐) = 4.189 ∗ (1 − exp�−0.002 ∗ (gdd + 78.144)�) 𝐺𝐺𝐺𝐺𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝑐𝑐𝑐𝑐𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐷𝐷𝐷𝐷 (𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐) = 4.398 ∗ (1 − exp�0.0022 ∗ (gdd + 69.928)�) supplementary material tab. s1: growth adjustment equations of p. tripartita var. mollissima structures in a high zone (2,498 m.a.s.l.) and a low zone (2,006 m.a.s.l.) in the muni cipality of pasca (cundinamarca, colombia). journal of applied botany and food quality 92, 172 178 (2019), doi:10.5073/jabfq.2019.092.023 1school of applied biosciences, kyungpook national university, daegu, korea 2department of botany, abdul wali khan university mardan, pakistan 3department of agriculture, abdul wali khan university mardan, pakistan bacillus subtilis jw1 enhances plant growth and nutrient uptake of chinese cabbage through gibberellins secretion sang-mo kang1, muhammad hamayun2, muhammad aaqil khan1, amjad iqbal3, in-jung lee1* (submitted: august 10, 2018; accepted: july 16, 2019) * corresponding author summary in the present study, we have isolated rhizospheric bacteria jw1 from rice paddy in andong, south korea. the culture filtrate (cf) analysis of jw1 showed higher contents of gibberellins ga1, ga4, ga7, organic acids, fatty acids and tricalcium phosphates. the 16s rdna gene sequencing and phylogentic analysis revealed that the strain jw1 has a 99% homology with bacillus subtilis sequences from blast search.the growth promotion capability of the strain jw1 was initially assessed on waito-c and whayoung-beyo rice cultivars, which improved the growth attributes of the rice cultivars. similarly, a significant increase in plant height, biomass, chlorophyll contents and nutrient uptake have been noticed, when the chinese cabbage was treated with jw1 strain. from the results, it is concluded that the integrative use of b. subtilis jw1 can promote plant growth by secreting bioactive compounds. therefore, b. subtilis jw1 may be utilized as an eco-friendly bio-fertilizer in the agricultural fields after successful field trials. keywords: bacillus subtilis jw1; bioactive gas; phosphatesolubilization; nutrient uptake; growth promotion introduction over the last few years, the multifaceted interactions of plant growth promoting microrganisms have been extensively explored as a productive source of novel bioactive natural products (bilal et al., 2018; hamayun et al., 2017; hussain et al., 2018; ikram et al., 2018; mehmood et al., 2019). in fact, the growth and development of the host plant under stress conditions can be improved by the production of microbial secondary metabolites (ismail et al., 2019; ismail et al., 2018; jan et al., 2019; joo et al., 2009; mehmood et al., 2018; nusrat et al., 2019). moreover, beneficial rhizospheric bacteria or plant growth promoting rhizospheric bacteria (pgpr) can also promote the growth of host plant under normal as well as stress conditions through the release of bioactive secondary metabolites (souza et al., 2015). these bioactive compounds, include indole acetic acid (iaa), cytokines (ck), jasmonic acid (ja), gibberellins (ga) and abscisic acid (aba). in other words, these bioactive compounds are known as plant hormones or growth regulators (marques et al., 2010). similarly, pgpr can fix biological nitrogen, solubilize the insoluble/non-available phosphorus (jeon et al., 2003), and alleviate stress through changes in acc deaminase expression (souza et al., 2015). furthermore, various pgpr have shownhostile activity against phyto-pathogenic microorganisms by releasing antipathogenic compounds (lucy et al., 2004). during the last few decades, researchers have concentrated on the role of soil microorganisms that convert insoluble phosphate to soluble forms (ordoñez et al., 2016; ruangsanka, 2014). most of rhizospheric bacteria that solubilize inorganic phosphate belongs to pseudomonas, enterobacter, bacillus and some soil fungi, such as aspergillus (osorio vega, 2007; patgiri and bezbaruah, 1990; rao, 1982; whitelaw, 1999). pgpr are also reported to produce organic acids, including gluconic, acetic, malic, citric, lactic, oxalic, formic, and 2-keto-gluconicacids. these microrganisms also re lease protons into the soil (osorio vega, 2007), thus converting the non-available form of phosphorus into available forms. phosphate solubilizing and siderophore producing microbes, such as acinetobacter rhizosphere, burkholderia cepacia, streptomyces tendae and serratia marcescensare considered as important pgpr because of their proven activity inenhancingcrop production (ben farhat et al., 2009; dimkpa et al., 2009; gulati et al., 2010; song et al., 2008; vejan et al., 2016). bacillus species have also shown support towards the optimal growth of the host plants by protecting them against the enimies and/or by secreting phytohormones and organic acids (tahir et al., 2017; yi et al., 2018). previously, various bacterial strains have been reported for cabbage growth promotion under stress conditions (hussein et al., 2016; liu et al., 2016). chinese cabbage (brassica rapa l.) is grown for vegetable purpose and for their active nutritional value. due to its nutritional value, high yield varieties were introduced, which increases its production, although it requires large quantities of fertilizers and chemicals. for sustainable cultivation of chinese cabbage in an eco-friendly manner, and less dependence on synthetic fertilizers and agrochemicals, utilizing native microflora in the form of pgpr can be an ideal strategy. the current study was designed to identify the novel rhizospheric bacteria from the paddy fields in andong, south korea; to screen the isolated bacteria for plant growth promotion abilities (like phosphate solubilizing, siderophore formation and phytohormones production) in chinese cabbage. material and methods isolation of rhizospheric bacteria rhizospheric soil collected from the rice paddies in andong was screened for bacterial isolates capable for plant growth promotion. plant samples with rhizospheric soil were packed in sterilized polyethylene zip-bags. the bags were then stored in an ice box and the samples were transported to the laboratory. from the roots of each plant 1 g of soil was dissolved in saline water (0.85%) and marked as a stock solution. a serial dilution (10-1 to 10-9) was initially setup and finally 10-4 dilution was selected for further analysis on the basis of results (kang et al., 2009). lb agar media were used for bacterial isolation by direct plating method and the plates were incubated at 28 °c till the emergence of bacterial colonies. pure colonies were obtained by picking a single colony and re-plated on lb agar media. pure colonies were identified by observing the size, color, shape and growth pattern (barillot et al., 2013; garcía-salamanca et al., 2013). bacillus subtilis a plant growth promoter 173 phosphate solubilization bacterial isolate jw1 was inoculated on 0.5% ofnational botanical research institute’s phosphate (nbrip) media plates (generously provided by national botanical research institute) (nautiyal, 1999). the plates were then incubated 30 °c for 3-days. the formation of halos by the inoculated bacteria indicates phosphate solubilization. the process of phosphate solubilization was monitored by checking the ph of liquid nbrip medium after every 12 h till the end of the experiment. organic acids production millipore filter (0.22 μm) was used to obtain bacterial culture filtrate. the filtered cultures (10 μl) were then injected into a high performance liquid chromatography (hplc; model: waters 600e) for further analysis. the hplc was equipped with a refractive index detector (model: waters 410) and rspak kc-811 (8.0 × 300 mm) column. the conditions for the analysis were: eluent = 0.1% h3po4/ h2o, flow rate = 1.0 ml/min, temperature = 40 °c. the concentration of each organic acid was determined through comparison with their respective standards (retention times and peak areas). succinic acid (sigma-aldrich, usa), lactic acid, malic acid, and butyric acid (supelco, usa) were used as standards (kang et al., 2012). screening of isolate jw1 on rice to assess the growth activity of the bacterial isolate jw1, waito-c (dwarf and gas mutant rice cultivar) and whayoung-beyo (normal gas producing rice cultivar) were used as test plants. initially, seeds of the tested cultivars were surface sterilized with sodium hypochlorite and then thoroughly washed with sterilized distilled water. the clean seeds were spread in a plate containing sterilized distilled h2o for germination. germinated seedlings were transplanted in magenta box, containing 90 ml of water agar media (0.8%). the box wastransferred to thepresetgrowth chamber (light intensity = 1,000 μmol m-2 s-1 + temperature = 35 °c for 16 h as a day and 20 °c for 8 h as a night). when the tested rice cultivars reached to two-leaved stage, a 10 μl of lyophilized bacterial filtrate suspension was applied to the tip of apical meristem.the growth attributes of both rice cultivars were analyzed after 10 days of treatment. quantification of bacterial secreted gibberellins the bacterial isolate jw1 was inoculated into nutrient broth (120 ml) for 7 days at 30 °c (shaking incubator-120 rpm) as described earlier (kang et al., 2009). the culture medium and bacterial cells were separated through centrifugation (2500 × g at 4 °c for 15 min). the culture medium (50 ml) was used to extract and purify the gas as described by kang et al. (2009). before purification, deuterated ga internal standards (20 ng; [17, 17-2 h2] ga1, ga4, ga7), obtained from prof. lewis n. mander, australian national university, canberra, australia, were added to the cf. the cf was subjected to chromatographic and mass spectroscopy techniques for identification and quantification of gas. the data were calculated in ng/ml and the analysis was repeated three times. molecular identification of isolate jw1 the genomic dna of isolate jw1 was isolated following the standard protocol of sambrook (2001). the 16s rrna gene was amplified and sequenced using the 27f (5´agagtttgatc (c/a) tggctcag-3´) and 1492r (5´-cgg (t/c) taccttgttacgactt-3´) universal primers (khan et al., 2014). the blast search program of ncbi genbank database/ eztaxon was used to determine the nucleotide sequence homology of bacterial isolate jw1. to conduct phylogenetic analysis, neighbour joining method was used with the help of mega v. 6.1 (tamura et al., 2013). the closely related sequences with highest homology, query coverage, and lowest e-values were used for alignment with clustalw. the isolate jw116s rrna gene sequence was submitted to genbank. isolate jw1 bioassay on chinese cabbage healthy and disease free seeds of chinese cabbage were obtained from seminis korea co. (korea) with a 95% germination rate. these seeds were surface sterilized using 2.5% sodium hypochlorite so lution for 20 minutes and washed twice with autoclave distilled water. the seeds were then subjected to germination assay in plastic trays under greenhouse conditions with the day/night cycle: 14 h at 28 °c/10 h at 25 °c; relative humidity 60-70%. uniform size seedling were transplanted into pots (15 seedlings per treatment) filled with autoclave horticulture soil. the composition of horticultural soil was as follows: peat moss (10-15%), perlite (35-40%), coco peat (45-50%), zeolite (6-8%), and nh+4~ 0.09 mg/g, no−3~ 0.205 mg/g, p2o5 ~ 0.35 mg/g, and k2o ~ 0.1 mg/g to prepare the microbe-free condition. the experimental treatments comprised, (1) control (2) isolate jw1 treated plants and (3) inoculation of chinese cabbage with bacterial free cf (kang et al., 2009). a 25 ml of bacterial cells and cell free extracts were applied to plants, twice consecutively, in a week. after 2 weeks of such treatment, the experiment was harvested and analyzed for shoot length, root length, fresh and dry biomass, chlorophyll contents (spad-502 minolta, tokyo, japan). for dry weight data, respective plants were randomly collected and dried in oven at 70 °c for 72 h. nutrient uptake by chinese cabbage minerals like calcium, potassium, magnesium, phosphate and sulfate were extracted from dried plant samples and determined by inductively coupled plasma mass spectroscopy (vg elemental, plasma quad 3, perkin elmer, united states). the quantity of minerals was estimated using known standard values. statistical analysis the data were subjected to analysis of variance using sas-9.1 software (sas institute, cary, nc). mean values among treatments were compared by duncan’s multiple range test (dmrt) test at p ≤ 0.05 significance level. results phosphate solubilization by bacillus subtilis jw1 the halos formation shows the tricalcium phosphate solubilization capacity of bacterial isolate jw1, which reached to its maximum after 60 h. the phosphate solubilization potential of the isolate jw1 was cross checked by monitoring the ph of the media after every 12 h, which showed a steady decline up to 48 h (fig. 1). the ph of the broth was decreased in response to phosphate solubilizing activity of the isolate jw1, accompanied by a significant drop from an initial ph of 7.0 to ph 4.1 after 72 h. organic acid production by isolate jw1 in lb media current study shows that the cf of b. subtilis jw1 contained citric acid, butyric acid, lactic acid, malic acid, and succinic acid. the organic acid analysis showed that jw1 strains have the ability to produce significantly higher quantities of lactic acid (28.7 μg/ml) in lb medium, followed by succinic acid (10.7 μg/ml), butyric acid and malic acid (1.85 μg/ml) respectively (fig. 2). 174 s.-m. kang, m. hamayun, m.a. khan, a. iqbal, i.-j. lee bioactive gas detected in the culture filtrate of isolate jw-1 the cf of isolate jw-1 was evaluated for the existence of bio active gibberellins. our results confirmed the existence of bioactive gas, i.e. ga4, ga1 and ga7. the quantities of bioactive gas were, ga4 (2.26 ng/ml), ga1 (0.12 ng/ml) and ga7 (0.08 ng/ml) as given in fig. 3. screening of isolate jw1 on rice we assessed the effect of cf of isolate jw1 on shoot length and plant fresh biomass of waito-c and whayoung-beyorice seedlings after seven days of incubation. it was observed that application of isolate jw1 on waito-c significantly promoted the shoot length (20.86%) and fresh plant weight (31.75%) as compared to control. a similar growth promotion was also observed in whayoung-beyo (tab. 1; fig. 4). fig. 1: (a) the rate of halos formation of b. subtilis jw1 in liquid nbrip medium and ph of the medium. values given are means of three replicates and error bars indicate standard deviation (b) the corresponding b. subtilis jw1on nbrip growth media plates after 72 hours of incubation at 30 °c. fig. 2: organic acids production of bacillus subtilis jw1 after five days of incubation at 28 oc. fig. 3: bioactive gas detected in the cf of bacillus subtilis jw1. fig. 4: influence of b. subtilis jw1 culture filtrates (10 μl) on growth attributes of waito-c and whayoung-beyoseedlings after seven days of incubation. tab. 1: effects of bacillus subtilis jw1 on growth attributes of waito-c and whayoung-beyo waito-c whayoung-beyo treatment shoot length fresh biomass shoot length fresh biomass (cm) (g/plant) (cm) (g/plant) control 12.94±0.38b 0.19±0.01b 12.10±0.44b 0.13±0.01b isolate jw1 15.64±0.39a 0.25±0.01a 15.06±0.36a 0.18±0.01a each value in the table are mean ± se of three replicates. values in columns followed by different letters significantly different at p > 0.05 based on dmrt analysis. molecular identification and phylogenetic analysis the isolate jw1 was identified by relating the amplified sequences of 16s rrna region using universal primers, with the associated bacillus subtilis a plant growth promoter 175 sequences existing in the genbank database of ncbi (http://www. ncbi.nlm.nih.gov/blast/). the closely related sequences were re covered from genbank and subjected to phylogenetic analysis (neighbor joining method) by using mega 6.1. the closely related sequences with highest homology, query coverage, and lowest e-values were used for alignment with clustalw. the jw1 isolate was identified as bacillus subtilis (fig. 5). the newly identified isolate b. subtilis jw1 16s rrna gene sequence was submitted to genbank and was allotted accession no. km 264401. isolate jw1 promotes growth of chinese cabbage the current study revealed that application of isolate jw1 and cell free cf significantly promoted plant growth attributes including, shoot and root length, fresh/dry weight, and chlorophyll contents as compared to control (tab. 2; fig. 6). the shoot length of chinese cabbage was significantly increased by cell free cf (12.65%) followed by isolate jw1 (3.16%), as compared to control. the same trend was noted for the plant fresh and dry biomass (tab. 2). higher chlorophyll contents were detected in chinese cabbage when treated with cell free cf and isolate jw. b. subtilis jw1 promotes nutrients uptake of chinese cabbage chinese cabbage inoculated with cell free cf and with isolate jw1 significantly enhanced p, k, mg and s contents of the treated plants as compared to the control (tab. 3). inoculation of plants with isolate jw1 significantly increases the nutrient content of k (6.48%) and s (45.91%) as compared to cell free cf (s-2.85%; k-32.29%) and control treatments. however, no significant differences in mg contents were observed in different treatments. however, ca and p contents were significantly higher in plants treated with bacterial cell free cf i.e. ca (11.53%) and p (73.30%), and jw1 i.e. ca (4.17%) and p (50.54%) as compared to control (tab. 3). tab. 2: effect of b. subtilis jw1 and cell free cf of b. subtilis jw1 on growth attributes and chlorophyll contents of chinese cabbage treatments shoot length root length fresh biomass dry biomass chlorophyll (cm/plant) (cm/plant) (g/plant) (g/plant) (spad) control 11.38±0.33b 6.36±0.07c 2.14±0.08c 0.21±0.01c 29.32±0.35c b. subtilis jw1 11.74±0.22ab 7.22±0.13b 2.40±0.04b 0.24±0.01b 31.48±0.42b cells-free extract 12.82±0.21a 7.84±0.13a 2.82±0.10a 0.31±0.01a 33.92±0.50a each value in the table are mean ± se of three replicatesand subsequent comparisons were conducted using duncan’s multiple range tests at p < 0.05. fig. 5: the evolutionary history was inferred by using the maximum likelihood method based on the tamura-nei model. evolutionary analysis were conducted in mega v. 6.1. the phylogenetic analysis was performed by constructing neighbour joining (nj) tree using 16s rdna gene sequences from isolate jw1and related bacterial strains. 176 s.-m. kang, m. hamayun, m.a. khan, a. iqbal, i.-j. lee discussion the area that surrounds the plant roots (rhizosphere) is a multi farious system in which pgprs interact with the host plants for development and higher production. the exact mechanism of pgpr on plant growth promotion is still unknown. many researchers have previously reported a significant improvement in crops yield in response to microbial inoculations under normal as well as stress conditions. in the current study, we have also observed an increase in plant height and dry biomass in the jw1 associated plants that might be due to the availability of soluble p in the presence of phosphate solubilizing rhizospheric bacteria. higher halos formation with respect to the time indicated towards low ph and thus the transformation of phosphate from non-available in an available form. the ph of the media has been monitored for every 12 h, a decline in ph reflected the release of organic acids (most probably gluconic acid and 2-ketogluconic acid) that indicates the conversion of insoluble phosphate into soluble phosphate. current results confirmed the findings of nautiyal (1999), who has demonstrated the efficiency of bacterial strains nbri0603, nbri2601, nbri3246 and nbri4003 to solubilize phosphorous and make it available for the plants to achieve normal growth. similarly, li et al. (2018) has demonstrated that bacillus subtilis strain sem-9 has the ability to release p from inorganic phosphate sources, including calcium hydrogen phosphate, aluminum phosphate, calcium phosphate and ferric phosphate. also, the strain b. subtilis jw1 has produced optimum amounts of organic acids (malic acid, succinic acid, lactic acid and butyric acid) after five days of incubation at 28 oc. the production of these organic acids by our isolate b. subtilis jw1 might be associated with the digestion of soil organic matter, i.e. through the oxidation of carbonaceous matter in the rhizosphere. the production of organic acids by the bacillus sp. makes the ph of the soil low that make the p soluble and available to the plants (li et al., 2018). furthermore, the optimum availability of minerals could enhance soil fertility and prolong micro-organisms survival in the soil (vejan et al., 2016; yang et al., 2011). in the current study, the higher nutrient uptake in plant inoculated with b. subtilis jw1 suggests an increased availability of soluble nutrients in the rhizosphere of chinese cabbage. bacillus megaterium strain tv-91c and bacillus subtilis strain tv-17c has been reported to improve the macro and micronutrients uptake by cabbage seedlings (turan et al., 2014). indeed, plant can take up the minerals from the rhizosphere, where the pgpr can interact with host roots and help them to absorb optimum amounts of mineral nutrients from the soil (turan et al., 2012). moreover, we have also observed that the cf of b. subtilis jw1 promoted the growth attributes (such as shoot and root lengths, biomass and chlorophyll contents) of chinese cabbage, which can be attributed to its potential to release bioactive ga in the culture medium. ga is well known phytohormone that can promote plant growth even under stressful conditions (khan et al., 2018; kim et al., 2009). it has been demonstrated previously that some of the plant growth promoting microorganisms, asprgillus fumigatus ts1 and fusarium proliferatum brl1can be utilized as a plant growth promoter as it secretes gas. in addition, the isolates have intensively colonized host roots and significantly enhance endogenous ga, thus promoted host plant growth (bilal et al., 2018). similarly, higher root and shoot growth and ga contents have been recorded in cabbage seedlings inoculated with bacillus megaterium strain tv-91c, pantoeaagglomerans strain rk-92, and bacillus subtilis strain tv17c (turan et al., 2014). in the light of these findings, it is obvious that using such useful gas producing pgpr as eco-friendly bio fertilizer might enhance the growth of economically important crop plants to sustain agriculture. conclusion from the growth promoting capabilities of our isolate b. subtilis jw1, it is concluded that the current strain secreted higher amounts of exogenous gibberellins and have the ability to metabolize phosfig. 6: influence of b. subtilis jw1 and cell free cf on growth attributes of chinese cabbage. tab. 3: effect of isolate jw1 and bacterial cell free cf on nutrient uptake of chinese cabbage treatments k ca mg p s control 10.49±0.01c 31.12±0.01c 5.35±0.02b 4.57±0.01c 2.57±0.03c b. subtilis jw1 11.17±0.02a 32.42±0.03b 5.38±0.02b 6.88±0.01b 3.75±0.01a cell-free extract 10.79±0.02b 34.71±0.03a 5.69±0.02a 7.92±0.06a 3.40±0.02b each value in the table are mean ± se of three replicatesand subsequent comparisons were conducted using duncan’s multiple range tests at p < 0.05. bacillus subtilis a plant growth promoter 177 phate into a form that is readily available to the plant. further studies are needed to isolate the genes coding for gibberellin production in b. subtilis jw1 and for determining how to use the new knowledge under field conditions. author contributions formal analysis, muhammad hamayun and amjadiqbal; funding acquisition, in-jung lee; investigation, sang-mo kang, muhammad hamayun and muhammad aaqil khan; project administration, in-jung lee; resources, in-jung 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ijlee@knu.ac.kr dr. amjad iqbal: e-mail: amjadiqbal@awkum.edu.pk http://dx.doi.org/10.1016/j.plaphy.2018.11.029 http://dx.doi.org/10.1111/j.1574-6968.1999.tb13383.x http://dx.doi.org/10.1016/j.plaphy.2019.04.019 http://dx.doi.org/10.1371/journal.pone.0154438 http://dx.doi.org/10.1007/s11104-017-3528-y http://dx.doi.org/10.1590/s1517-838220080001000030 http://dx.doi.org/10.1590/s1415-475738420150053 http://dx.doi.org/10.3389/fmicb.2017.00171 http://dx.doi.org/10.1093/molbev/mst197 http://dx.doi.org/10.1002/jpln.201200054 http://dx.doi.org/10.3390/molecules21050573 http://dx.doi.org/10.1007/s00374-010-0527-z http://dx.doi.org/10.1111/1462-2920.14305 angewandte botanik. typha als nutzpflanze. " 103 an den ausgetriebenen sprossen die blätter zurückstutzt; die pflanzen stärken sich dann aber nicht so wie bei der frühlings pflanzung. man macht furchen in den boden des ufers, legt dort die grundachsen wagerecht ein, so daß die spitzen aufwärts schauen und deckt dann die grundachsen zu, möglichst so, daß d ie bedeckende erde etwas über den wasserspiegel hinausragt, damit die grundachsen nicht herausgespült und abgeschwemmt werden können. zur uferfestigung a n größeren flüssen verspricht dies verfahren guten erfolg und soll jetzt z. b . auf veranlassung des bauamtes in großem maßstabe bei der oderregulierung an wendung finden. t . angustifolia wird für diese zwecke die ge eignete art sein. studium über eine brombeerkrankheit von dr. c . hahmann, hamburg. allgemeines. der krebs, der vielfach auf unseren obstbäumen und sträuchern o ft in gefährlichster weise auftritt, is t in seinem ursprung und wesen bei weitem noch nicht so erforscht, wie dieses wünschens wert wäre. nach sorauer wird der krebs als wunde bezeichnet, deren überwallungsränder sich zu wuchernden holzgeschwülsten ausbilden. „der charakter der wucherung liegt in der ausschließ lichen oder überwiegenden bildung von parenchymholz a n stelle der normalen prosenchymatischen holzelemente. die krebsgeschwülste haben für jede gehölzart typische gestalt“). merkwürdig is t e s, daß die krebskrankheiten, mit ausnahme der des weinstockes, lediglich in der familie der rosaceen zu finden sind. nach diesem forscher unterscheiden sich die krebsformen bei den einzelnen gattungen der rosaceen nur „durch die art der reaktion äuf den wundreiz, stimmen aber darin wieder überein, daß si e das auge *) p . sorauer, handb. der pflanzenkrankheiten i, s. 584. 104 c. hahmann, und dessen nächste umgebung als entstehungsort bevorzugen. der grund dafür ist in der lockerung des achsenkörpers an der ansatz stelle einer knospe zu suchen. hier ist stets der holzring schmaler und wird schließlich von der parenchymatischen markbrücke quer durchsetzt“!). der beginn der erscheinung fällt in das zeitige frühjahr. eine kleine rißwunde bildet den ausgangspunkt der krankheit, die dann sehr schnell mit reichlicher kallusbildung weiter fortschreitet und gewaltige dimensionen annehmen kann. nach wulff*) dagegen entsteht die krankheit an der stammbasis, sehr oft in der nähe des wurzelhalses und zeigt dort auch die beste entwicklung. allmählich schreitet sie nath oben fort, die augen dabei oft völlig krebsfrei lassend, und erreicht schließlich die spitzen der stämme. nach wulff is t keine „wunde“ als primäre ursache und kéin wundreiz vorhanden*). das parenchymatische kallus gewebe, das sehr empfindlich gegen witterungseinflüsse, vor allem gegen frost ist, kann durch geringe kältegrade verletzt, zur bildung neuen wuchergewebes veranlaßt werden, d a infolge der parenchy matischen natur des gewebekomplexes in der vorangegangenen vegetationsperiode reservestoffe (stärke) sehr reichlich gebildet und aufgespeichert worden sind“). bei spiraea hingegen soll die ursache der krebsbildung in der stauung von plastischem material liegen"). beim weinstockkrebs werden einerseits frostwirkungen als ursache der krankheit angesprochen"), andererseits wird die entstehung der geschwülste auf stauung des plastischen materials, infolge z u kurzen schnittes, zurückgeführt?). durch entdeckungen anderer forscher, die parasiten als urheber der kallusbildungen hinstellen, werden diesewidersprüche in der entstehung der krebs krankheiten noch vermehrt. s o hat güssow") bei einer rosen krankheit in england einen pilz (coniothyrium fuckelii saec.) als krankheitserreger angegeben. *) p . sorauer, i. a. a. o . s . 605. *) th. wulff, 1908, studium über heteroplastische gewebewucherungen am himbeerund stachelbeerstrauch: arkiv för botanik, vii, nr. 14, s. a. s. 4. *) th. wulff, 1908, a. a. o . s . 14. *) p . sorauer i, a. a. o . s . 605. *) p . sorauer i, a. a. o . s . 599. *) p . sorauer i, a. a. o . s . 595. ') blankenhorn und mühlhäuser, p . sorauer i, s. 596. *) h . t . güssow, parasitic rose canker in journal o f the royal horti cultural society, nov. 1908, london. studium über eine brombeerkrankheit. 105 dieselbe krankheit und denselben urheber haben auch köck) und laubert*) festgestellt. für eine brombeerkrankheit h a t güssow”) ebenfalls einen pilz (coniothyrium tumaefaciens güssow sp. n.) gefunden. sorauer und andere schreiben dagegen den parasiten nur sekundären charakter zu. die an brombeersträuchern auftretende krebskrankheit. der auf brombeersträuchern auftretende krebs ist schon seit längerer zeit bekannt. sorauer beobachtete ihn in vier fällen a n wilden brombeersträuchern*). die kalluswülste haben nach ihm ihren ursprung a n zentimeterlangen, durch spannungsdifferenzén entstandenen rißstellen. dicht a n der außenseite der hartbast stränge beginnt eine reiche parenchymwucherung, die e r ihrer natur nach als typische überwallungsränder ansieht. e r bemerkte schon eine voranlage zur krebsbildung. an den betreffenden stellen, die später die kalluswülste aufweisen, ist der aus hartbast strängen und ihren derbwandigen verbindungselementen gebildete mechanische ring durch feinwandiges parenchymgewebe unter brochen. als ursache für die krankheit gibt sorauer frost schäden an. e r kann jedoch seine vermutung nicht durch beweise stützen. bei seinen künstlich angestellten versuchen hat e r in keinem falle derartige luxuriierende gewebewucherungen erzielen können. ob die künstlichen versuche, wenn sie früher als sorauer e s tat, angesetzt werden, anders verlaufen, muß abgewartet werden. auch güssow") hat die entstehung des brombeerkrebses unter sucht. e r fand als ursache einen pilz, den e r infolge des unter schiedes seiner sporengröße von den anderen coniothyrium-arten a ls comiothyrium tumaefaciens güssow s p . n . bezeichnete. daß güssows versuche einwandfrei waren, davon konnte sich wulff a n dessen eigenem material überzeugen"). nun tritt seit dem *) köck, ein für österreich neuer rosenschädling. zeitschrift f. d. landw. versuchswesen in österreich 1905, s . 660/666. *) laubert, ref. zeitschr. pfl. krankh. xvii, s. 252. (köck, ein für österreich neuer rosenschädling.) *) güssow, 1908 a. a. o . s . 229 f. *) p . sorauer i. s. 604; ii. s. 227; iii. s. 28. *) güssow 1908, a. a. o . s . 229. *) th. wulff, weitere studien über die kalluskrankheit des himbeer strauches in arkiv för botanik viii, nr. 15. (mitgeteilt am 10. märz 1909 durch a . g . nathorst und j. eriksson, s. 3). 106 c. hahmann, jahre 1914 eine brombeerkrankheit auch in rißen bei blankenese (hamburg) in einem garten auf. diese krankheit hat die größte ähnlichkeit mit den bisher beschriebenen krebskrankheiten der brombeersträucher. es handelt sich um die sorte „theodor reimers“. vor dem eintritt der krankheit war der ertrag außer ordentlich gut. bei den befallenen pflanzen wird die blütenbildung verzögert, früchte entwickeln sich daran nicht, sodaß dadurch der ertrag gänzlich herabgesetzt wird. der station für pflanzenschutz in hamburg wurde das material, welches ich untersuchte, zugesandt. ich überzeugte mich auch selbst an ort und stelle von dem stand der krankheit. das resultat der untersuchung sei im folgenden kurz mitgeteilt. die krankheit tritt hauptsächlich direkt über der erde, also direkt über dem wurzelhalz auf, wie dieses fig. 1 deutlich erkennen läßt!). fig. 1 stellt ein stammstück dar, das unmittelbar über dem erdboden abgeschlagen ist. die kalluswülste sind hier besonders groß und stark; sie erreichen eine höhe von 50/70 cm von der erde aus gemessen, eine dicke von 10/12 cm im durchmesser. dabei verdickt sich der stamm oft um das 2–3fache”). aber auch andere stellen der sprosse werden von der krankheit befallen. so sieht man die wülste bis zum gipfel der pflanzen dringen. (vergl. fig. 2, wo ein mittleres sproßstück dargestellt ist.) be sonders bevorzugte stellen, als welche sorauer”) die augen ansieht, konnten nicht festgestellt werden. die krankheit trat sowohl an hauptwie an nebensprossen auf. an den augen zeigte sie sich sogar nur vereinzelt. auch die blühenden seitenzweige wurden oft von ihr heimgesucht. an keiner stelle jedoch war ihre ent wicklung so stark, wie in der unteren stammpartie oberhalb des wurzelhalses. solange die wucherungen noch jung und wachstums fähig sind, sind sie hart und von „straffer konsistenz“*). ihre farbe ist grau-weiß bis weißlich-gelb. dieser ton rührt von den großen luftgefüllten interzellularräumen her. anfangs sind die wucherungen ganz winzig, (vergl. fig. 4, anfangsstadien der gewebewucherungen darstellend), erreichen aber bald eine größe von 1–2 cm, ja von 3–5 cm durchmesser. *) vergl. auch fig. 3, die die krebsbildung auch unmittelbar über dem wurzelhals zeigt, darunter die ausgebildeten wurzeln. *) th. wulff, 1908, a. a. o. s. 4. *) sorauer i, s. 605. “) th. wulff, 1908, a. a. 0. s. 4. • studium über eine brombeerkrankheit. 107 sie erscheinen anfangs wie kleine warzen, die zunächst ver einzelt auftreten, sich aber später zu großen haufen vereinigen. s ie bekommen dadurch ein blumenkohlähnliches aussehen!). auf dem stamme können die wülste teils mit schmaler, teils mit breiter fig. 1. fig. 2 . fig. 3. basis aufsitzen*). oft können auch mehrere warzen zusammen verschmelzen. bald aber wechseln sie ihre farbe. diese wird dunkler und geht schließlich ins bräunliche bis braune resp. dunkel braune bis schwarzbraune über. jetzt fangen die wucherungen a nweicher z u werden, bis sie endlich gänzlich der fäulnis verfallen. *) th. wulff, 1908, s. 4. *) th. wulff, 1908, s. 4f. und taf. 3 , fig. h . 108 c. hahmann, als ursache hierfür sindwitterungseinflüsse, sowie die einwirkungen von pilzen und bakterien anzusprechen. doch auch insekten und würmer wirken hierbei in ihrer weise. betrachten wir nun die entstehung der krankheit mikros kopisch, so zeigt sie in ihrem verlauf große ähnlichkeit mit dem von güssow!) untersuchten „parasitic rose canker“. auf den sprossen beobachtet man kleine erhebungen, die rundliche oder mehr noch länglich-runde gestalt haben können. anfangs sind fig. 4. diese noch von der rinde bedeckt, später dagegen durchbrechen sie dieselbe mit einem längsriß. es treten schwarze, runde körperchen aus ihnen heraus, die sich bei stärkerer vergrößerung als pilzkörper darstellen. die umgebung der erhebungen fällt schon makroskopisch durch eine verfärbung ins dunkelrote bis rotbraune auf. mikroskopisch erweisen sich diese zellpartien als abgestorben. solche verfärbte stellen sind überall auf der rinde bemerkbar. der pilz entwickelt sich also unter der epidermis und bricht später nur durch diese durch, um seine sporen nach außen *) güssow, 1908, a. a. o., s. 222 ff . studium über eine brombeerkrankheit. 109 gelangen zu lassen. die fruchtkörper haben rundliche form, oben in eine spitze zulaufend, und sind dunkelbis schwarzbraun gefärbt, ihre oberfläche von einer maschenartigen beschaffenheit. beim durchschneiden dieser fruchtkörper stößt man auf die auf 30–35 u langen, durchschnittlich 3u breiten sporenträgern sitzenden sporen. die fruchtkörper erreichen eine größe von 290–330 u im durch messer, die sporen, die sehr klein, einzellig, rundlich, länglichrund b is eiförmig und von grau-grünlicher bis schmutzig-grünlicher farbe sind, eine größe von 4,5–6,5 u (lang) und 3–4,5 u (dick). e s handelt sich um eine art der gattung coniothyrium. güssow nannte den pilz coniothyrium tumaefaciens güssow sp . n ., d a die sporengröße von den anderen coniothyrium-arten verschieden ist!). meine messungen weichen von den messungen güssows nur wenig ab. vereinigen wir in der beschreibung des pilzes beide maßangaben, so erscheint mir der von güssow neu gewählte name für diesen pilz berechtigt. zwar ist uns die gattung coniothyrium hauptsächlich als saprophytisch lebend bekannt, doch haben wir auch beispiele ihrer parasitischen lebensweise”). e s sei hier nur a n coniothyrium diplodiella (speg.) sacc. a n reben und con. concentricum (desm.) sacc. auf yucca-blättern u. a. erinnert*). wie ist es jedoch dem pilz möglich in das gewebe des brombeer strauches einzudringen? ein schwach vergrößerter brombeersproß zeigt allerwärts kleine „wunden“, deren entstehung in der haupt sache dem wind zugeschrieben werden muß. der wind weht die sprosse . hin und her, wobei die dornen die haut der nachbar sprosse ritzen. was hierbei die dornen ausrichten können, können z . b . auch die den sprossen als stütze dienenden spaliere oder auch kleine, scharfkantige sandkörnchen, die vom winde gegen d ie epidermis geweht werden. dem letzteren umstand is t e s, neben dem frost, auch wohl vor allem zuzuschreiben, daß der haupt infektionsherd direkt über dem erdboden auftritt. von hier aus is t e s den dort liegenden sporen durch den wind.am leichtesten möglich in die pflanzen z u gelangen. normalerweise versuchen die pflanzen diese kleinen wunden z u heilen, e s entstehen winzige erhebungen. die aber vom pilz *) güssow, 1908, s. 229 f. *) sorauer ii, 2. aufl., s. 385. * th. wulff, einige botrytis-krankheiten der ribes-arten in arkiv för botanik 1908. 110 c. hahmann, befallenen stellen verhalten sich anders. der pilz dringt in das gewebe ein und breitet sich in den der wunde benachbarten zellen aus, seine nahrung diesen entziehend. die befallenen zellen sterben bald, unter verfärbung ins dunkelrote bis rotbraune, ab. nun bildet der pilz seine fruchtkörper, die wenn sie reif sind, hart werden und schließlich, wiewir sahen, mit ihrer spitze die epidermis durchbrechen, ihre sporen zu entlassen. das durch den pilz ab getötete, unelastische gewebe setzt sich dem wachstum des sprosses entgegen. es entstehen risse oder spalten in der epidermis, durch bildung von wundgewebe versucht die pflanze diesem schaden abzuhelfen, was ihr ja auch unter normalen bedingungen möglich ist. die von beiden seiten des risses kommenden kallus gewebe treffen in der mitte aufeinander und vereinigen sich. der pilz hindert aber dieses ausheilen. ein querschnitt durch einen derartigen riß mit wundgewebe zeigt am rande des kullusgewebes kleine schwarze stellen. hier hat sich der pilz schon wieder ein genistet. es ist ihm ja auch ein leichtes in die wundgewebezellen, deren zellwände zart und fein sind, deren epidermis wenig wider standsfest ist, einzudringen. das nun vom pilz neuerdings ab getötete gewebe sucht die pflanze durch weitere kallusbildung zu ersetzen. so wogt der kampf zwischen pilz und pflanze hin und her. das dünnwandige, zarte kallusgewebe ist aber noch anderen einflüssen ausgesetzt. hier ist es vor allem der frost, der die pflanzen in schwerster weise schädigen kann. schon geringe frostgrade töten die empfindlichen parenchymzellen ab, die pflanze bildet neues wundgewebe, sodaß jetzt ein kampf zwischen frost und pflanze entbrennt. schließlich kommt es zu kallusbildungen rings um den ganzen stamm, sodaß die oberen pflanzenteile voll ständig von den unteren partien abgetrennt dastehen. daß dies das ende der pflanze bedeutet, is t klar ersichtlich. von solchen durch den frost geschädigten pflanzenteilen hat sich der pilz völlig zurückgezogen!). aus güssows und aus den vorliegenden unter suchungen erhellt aber deutlich, daß den ersten anstoß zu dieser krankheit zunächst der pilz gibt, er ist deren erreger. erst in zweiter linie treten die einwirkungen des frostes in die er scheinung. künstliche infektionsversuche, auch an wilden brombeeren, konnten bisher leider noch nicht ausgeführt werden. ein umstand jedoch spricht meines erachtens für eine infektion auch nach dieser ) güssow, 1908, s. 227 f. studium über eine brombeerkrankheit. 111 seite. die befallenen sprosse wurden von dem besitzer abgeschnitten und in eine abseits gelegene wilde brombeerhecke geworfen. die bisher krebsfreien wilden brombeersprosse wiesen im nächsten jahre viele solche auswüchse auf. wie ist dieser krankheit entgegen zu treten? güssow!) schreibt für seine rosenkrankheit größte aufmerksamkeit auf die ersten krankheitsanzeichen und bestreichen dieser stellen und ihrer umgebung mit „creosoted wood tar“ vor. durch diese behandlung werden die sporen des pilzes getötet und die ausbreitung der krankheit wird verhindert. vorgeschrittene stadien sind auszu schneiden und die wunden ebenfalls mit holzteer oder wachs zu bestreichen. „badly cankered twigs“ sind abzuschneiden und zu verbrennen. daß größte obacht auf die krankheitsanfänge gegeben werden muß, is t nach der geschilderten entwicklung des krebserregers selbstverständlich. ich schlage vor, bei vorgeschritteneren stadien nicht holzteer, sondern steinkohlenteer zu verwenden. holzteer dringt z u tief in die gewebe ein*). die verkrebsten stellen sind m it einem scharfen messer b is auf das gesunde holz auszuschneiden und mit einem glühenden eisen auszubrennen. nach einiger zeit, meist nach wenigen tagen, wenn die wunde etwas abgetrocknet is t, wird steinkohlenteer mit einem pinsel auf die wunde aufge strichen*). bei dieser behandlung wird man alle pilzsporen abtöten können. vorsichtshalber kann man diewunde im nächsten jahre nochmals überteeren. auf dieseweise kann man die pflanzen wohl in den meisten fällen vor dem sicheren absterben retten. das im steinkohlenteer wirksame element, das kreosot, bringt neben dem pilz, auch die obere holzschicht zum absterben, wodurch der holzfäule vorgebeugt wird. für das ausschneiden is t mai und juni, eine zeit, wo die pflanze in voller vegetation steht, am günstigsten, d a dann die vernarbung am besten vor sich geht“). wie die erscheinung nach dieser behandlung verläuft, wird später mitgeteilt werden. stark verkrebste stämme werden abgeschnitten und verbrannt. *) güssow, 1908, s. 229. *) w. breitwieser (m. d. g . 1908, s . 22, ref. zeitschrift f. d . landw. versuchsw. in österreich xi, 1908, s. 514 f.) . *) um das abfließen des teers zu verhindern, schlägt breitwieser vor, d ie betreffenden stehlen mit trockener holzasche z u bestreuen. *) w. breitwieser, 1908, s . 514 f. uc1.b3299303_page_121_cut uc1.b3299303_page_122_cut uc1.b3299303_page_123_cut uc1.b3299303_page_124_cut uc1.b3299303_page_125_cut uc1.b3299303_page_126_cut uc1.b3299303_page_127_cut uc1.b3299303_page_128_cut uc1.b3299303_page_129_cut journal of applied botany and food quality 94, 1 6 (2021), doi:10.5073/jabfq.2021.094.001 justus liebig university, institute of botany, giessen, germany common garden versus common practice – phenotypic changes in trifolium pratense l. in response to repeated mowing thomas gross*#, christina magdalena müller#, annette becker, birgit gemeinholzer, volker wissemann (submitted: june 15, 2020; accepted: november 30, 2020) summary plants react to various biotic and abiotic factors by adjusting their growth pattern. this process, called phenotypic plasticity, can also be observed in the agronomical important fodder plant trifolium pratense l. (red clover) which is grown worldwide for its high protein content and soil improving ability. after cutting it changes its growth pattern due to phenotypic plasticity and initiates a second growth phase. here, we analyze the regrowth dynamics of red clover in response to grazing or mowing by recording the plant’s architecture, leaf morphology, and growth performance in different cutting experiments. as previous studies were limited in the number of individuals and carried out via common garden experiments, we analyzed the regrowth reaction of t. pratense under standard agricultural field conditions. t. pratense forms smaller and rounder leaflets after repeated cutting, compensated by the production of more biomass. nitrogen contents were subjected to seasonal changes, rather than by changes through cutting. as middle to late cut plants had higher regrowth capacities and regrew more biomass than early cut plants, an optimal time point for biomass harvest can be suggested to maximize the total yield of red clover biomass. keywords: red clover, mowing, phenotypic plasticity, leaf roundness, biomass production introduction plants are exposed to various biotic and abiotic factors, which they cannot evade due to their sessile nature. therefore, they adapt their growth behaviour to local environmental constraints leading to phenotypic variation owing to their open blueprint (domagalska and leyser, 2011; teichmann and muhr, 2015; gastal and lemaire, 2015; prins and verkaar, 1992). detailed phenotypic plasticity responses due to environmental perturbations were observed in many different plant species like trifolium repens l., populus trichocarpa hook., and pisum sativum l. (stafstrom, 1995; goulas et al., 2002; ryle et al., 1985; wu and stettler, 1998). a major factor inducing phenotypic plasticity is the loss of biomass through herbivory or cutting. the fabaceae t. repens reacts to cutting by producing more but smaller leaves compared to uncut plants (goulas et al., 2002). as t. repens is a common fodder plant this effect might be of agronomical importance, as repeated cutting could reduce the amount of harvested biomass (gastal and lemaire, 2015; prins and verkaar, 1992; briske and richards, 1995). a close relative to t. repens, trifolium pratense l. (red clover), is used in many parts of the world as high-quality fodder plant due to its naturally high protein content, deep root systems, and high yield which give t. pratense preference over t. repens. it is grown in monocultures or in a grass mixture where it increases the nitrogen content of the surrounding plants (eriksen et al., 2012; fernandez and warembourg, 1987; beecher et al., 2015; dewhurst, 2013; kleen et al., 2011). in addition, red clover produces high amounts of the enzyme polyphenol peroxidase which increases both nitrogen uptake and polyunsaturated fatty acids by livestock (lee et al., 2006; winters and minchin, 2005) and reviewed in van ranst et al. (2011). furthermore, digestibility analyses have shown the advantages of re-grown red clover biomass in terms of protein content and digestible fiber content. in addition to varying growing conditions throughout the season, the cutting regime of red clover has a great influence on the harvested biomass, with respect to number of cutting events and their time points (wiersma et al., 1998). however, both factors are also dependent on the respective red clover cultivar and their perseverance (marshall et al., 2017; wiersma et al., 1998). in a previous study, phenotypic plasticity of red clover during regrowth after cutting/mowing was analysed in a common garden experiment (herbert et al., 2018). after cutting, red clover produces, similar to t. repens, more leaves than uncut plants but they are of the same size than before cutting. only the length of newly formed petioles is reduced when compared to uncut plants. taken together, cutting triggers a second growth phase in red clover and could thereby contribute to yield increase. however, this analysis was performed under artificial conditions as a common garden experiment in a greenhouse using only a limited number of plants and these were cut only once. we were interested in the growth changes of t. pratense grown under field conditions with multiple cutting events and how they differ from the changes observed in the common garden. in particular, it is interesting to see whether the second growth phase after the cut also contributes to increased yields in field trials. here, we investigated the native response of locally adapted wild red clover plants as used in herbert et al. (2018) to cutting and how different mowing regimes influence leaf morphology. we hypothesized that field grown red clover plants will only partially reflect the reaction shown by the common garden greenhouse experiment from herbert et al. (2018) as they are more prone to abiotic and biotic stresses. thus, the questions arise (i) whether and how plants compensate cutting losses during regrowth and by this if the experiment of herbert et al. (2018) can be validated under common practice conditions in a field experiment, (ii) if different mowing regimes influence the amount of harvested biomass of the natural population red clover, and (iii) whether nitrogen content, as a marker for protein content and fodder quality, differs in the leaflets after mowing. material and methods the experimental fields were located at the agricultural research and developmental farm of the justus-liebig-university giessen in rauischholzhausen/germany (50°45’55.9008’’n, 8°52’1.2648’’e). the red clover seeds are derived from the same wild populations in saxony, saxony-anhalt, thuringia, thuringian-forest, and ucker * corresponding author # these authors contributed equally to this work 2 t. gross, c.m. müller, a. becker, b. gemeinholzer, v. wissemann marck as described in herbert et al. (2018), (rieger hofmann seed company; blaufelden, germany). we sowed the red clover in autumn 2016, the mowing started in 2017. the plots were 2 m wide and 3 m long. twelve different mowing regimes (tab. 1), with 3 plots per regime, were applied. for further analysis plots 1, 3, 4, 6, 7, 9, 10, and 12 were considered, especially plots 4, 7, and 10 as those were mown three times. each mowing regime was carried out three times on randomly distributed plots (fig. 1, tab. 1). the measurements were taken 18 20 days after mowing to evade the first stress responses. for each treatment, plant height, number of inflorescences, length and width of five leaflets per plant of ten randomly chosen plants were measured from three plots, totalling 30 plants per treatment and 150 leaflets. to avoid boundary effects in morphological measurements only plants internal of a 50 cm strip around the edges of each tested plot were analysed. the following characters were recorded: weight of the fresh material (fm), height of the plants, width and length of one leaflet, from the latter two characters we determined the roundness and the leaflet area. to calculate the roundness we used leaflet length / leaflet width, the leaflet area was determined by (leaf length / 2) * (leaf width / 2) * π. for nitrogen content measurements, the harvested t. pratense material was dried after mowing at 90 °c. the nitrogen content was measured with cn analyser vario max (elementar analysensysteme gmbh, hanau, germany) (data was uploaded to the bexis database and is accessible under https://doi. org/10.25829/bexis.26586-5, https://doi.org/10.25829/bexis.26606-1, and https://doi.org/10.25829/bexis.26666-1). for the questioning and the statistical calculation, we grouped the different plots in classes together after the time of mowing (tab. 1): early means the time between 12.6 12.09, middle: 03.07 02.10, late 14.07 13.10 and later 31.07 17.10, regardless of time: all plot s̓ which are mown three times (4, 7, and 10). plot 1 is the unmown control field. the statistical tests and graphics were conducted in r (r core team, 2013) with the following packages pmcmr (pohlert, 2014) and ggplot2 (wickham, 2009). we tested for normality with the shapiro-wilk test and variance homogeneity was tested with bartlett test, if both tests were true, we applied a one-way analysis of variance (anova). the tukey hsd post hoc test was used to identify significant anova results. if the normality and/or the variance homogeneity was not true, the kruskal-wallis test was applied and the posthoc-kruskal-dunn test to further analyse the significant results of the kruskal-wallis test. additionally we tested 16 different generalized linear mixed models (glmms) using template model builder (tmb) (brooks et al., 2017), for the impact of classes (early, middle, late, and later) the cut systems (1/2/3/no) and the time in the year (supplement tab. 1). the final model was chosen by the akaike information criterion (aic; supplement tab. 1). after first model validation, we transformed the data for leaflet roundness (standardized) and leaflet area (log) (supplement fig. 1, 2 and 3). tab. 1: mowing regime and dates of measurement for each plot plot classes mowing regime 1st mowing date 2nd mowing date 3rd mowing date of measurement of measurement of measurement 1 (a-c) not mown every time every time every time 2 (a-c) early 1× mown middle of june 30.06.2017 12.06.2017 3 (a-c) early 2× mown middle of june 30.06.2017 middle of august 29.08.2017 12.06.2017 14.08.2017 4 (a-c) early 3× mown middle of june 30.06.2017 middle of august 29.08.2017 middle of september 03.10.2017 12.06.2017 14.08.2017 12.09.2017 5 (a-c) middle 1× mown beginning of july 20.07.2017 03.07.2017 6 (a-c) middle 2× mown beginning of july 20.07.2017 end of august 19.09.2017 03.07.2017 31.08.2017 7 (a-c) middle 3× mown beginning of july 20.07.2017 end of august 19.09.2017 beginning of october 17.10.2017 03.07.2017 31.08.2017 02.10.2017 8 (a-c) late 1× mown middle of july 31.07.2017 14.07.2017 9 (a-c) late 2× mown middle of july 31.07.2017 middle of september 03.10.2017 14.07.2017 12.09.2017 10 (a-c) late 3× mown middle of july 31.07.2017 middle of september 03.10.2017 middle of october 31.10.2017 14.07.2017 12.09.2017 13.10.2017 11 (a-c) later 1× mown end of july 18.08.2017 31.07.2017 12 (a-c) later 2× mown end of july 18.08.2017 beginning of october 17.10.2017 31.07.2017 02.10.2017 fig. 1: structure of the test field of trifolium pratense. 1-12 the different mowing regimes, a-c the randomly distributed plots. reaction of t. pratense to repeated mowing 3 results and discussion for plant height and the number of inflorescences, we could show that with increasing mowing frequencies the plant height and the number of inflorescences decreased compared to the not mown control group (suppl. fig. 4 a and b). while these results show nicely the cutting effect on the regrowth ability of the red clover plants, our investigation concentrated on leaflet roundness and the potential increase in leaflet area associated with mass build-up in response to repeated cutting. for this, we identified the effect of different mowing frequencies (no cut, 1. cut, 2. cut, 3. cut) and time of mowing in the year (classes: early, middle, late, later) on leaflet roundness, leaflet area and the amount of harvested biomass. for all plots, which were mown at least two times (3, 4, 6, 7, 9, 10, and 12), the roundness of leaflets increased after the 2nd cut p < 0.001 (no normality distribution and no variance homogeneity p < 0.001), compared to the unmown red clover plants (control plants, plot 1) only the 2nd cut had significantly increased leaflet roundness. the 1st cut and the control plants showed no changes in leaflet roundness (p = 0.82; suppl. fig. 5 a). for the roundness of leaflets after three cuts regardless of the classes in the plots 4, 7, and 10 the data are not normally distributed (p = 0.003) and there is no variance homogeneity (p = 0.018; fig. 2 a), the plots within the different cuts were compared. the kruskal-wallis test detected significance in the roundness of leaflets (p < 0.001) after cutting and the posthoc-kruskal-dunn test identified that there are significant differences between all three cuts with p < 0.001. in the three plots 4, 7 and 10, differences were detected between plot 4 and the two other plots (7 and 10) in leaflet roundness (p < 0.001; fig. 2 a). compared to the control group (plot 1) there are no significant differences between the plots in the 1st cut and the control group (p = 0.129) in leaflet roundness. after the 1st cut, the roundness of the leaflets varied between 0.4 0.6, 2th cut 0.57 0.77 and 3rd cut 0.57 0.87 (with 1 being round; fig. 2 a). the leaflet roundness increased with each cutting. especially the plots 7 and 10 showed a great increase after the 2nd and 3rd cut (p = 0.01, fig. 2 a and 3 b, c). considering the four mowing classes (early, middle, late, and later), we observed a significant (p < 0.001) increase in leaflet roundness in all of our classes, regardless of the time period of mowing (fig. 3 a-d). for plot 4 the roundness of leaflets after mowing differed significantly (p < 0.001) from the plots 7 and 10 (fig. 2 a). we detected a significant difference in the roundness of leaflets in plot 4 between the 1st / 2nd (p = 0.002) and 1st / 3rd (p < 0.001) mowing (fig. 3 a). between the 2nd and 3rd mowing there were no differences detectable in plot 4 (p = 0.49; fig. 3 a). our model (lm5) and the data show that the production of rounder leaflets after mowing is enhanced through multiple mowing events (cut and date; chi < 0.001, p < 0.001) and independent of the season. as herbert et al. (2018) mowed red clover only once, this increase in roundness was not detectable. leaf shape is often subjected to phenotypic plasticity depending on nutrient or water supply, and on temperature (li et al., 2019; dorken and barrett, 2004; royer et al., 2009). however, leaflet roundness increases regardless of the classes, indicating a low or no contribution of these abiotic factors to the change in roundness. interestingly, leaf shape is also dependent on the age of the plant, and in glycine max (l.) merr. older plants form less round leaflets (yoshikawa et al., 2013). hence, we can propose that cutting rejuvenates t. pratense and early regrowth produces juvenile, rounder, leaflets. the leaflet area did not change for plots mown twice (3, 4, 6, 7, 9, 10, and 12) p = 0.160 (no normality distribution and no variance homogeneity p < 0.001) and only compared to the control plants the 1st and 2nd cut showed a significant decrease in the leaflet area (p < 0.001; suppl. fig. 1 b). after three cuts, regardless of the classes in the plots 4, 7, and 10, the leaflet area data is not normally distributed (p < 0.001) and there is no variance homogeneity (p = 0.026; fig. 2 b). significance was detected by the kruskal-wallis test in the leaf fig. 2: development of (a) roundness of leaflets after mowing three times (up to 1 for round), (b) leaflet area [cm²] after mowing three times, (c) weight [kg_fm/m2] of the harvested plots after mowing three times in the year regardless of the season and (d) the nitrogen content [%] in the leaves after mowing. red dots = outlier; plot: 1 = not mown, 4 = early, 7 = middle, 10 = late. 4 t. gross, c.m. müller, a. becker, b. gemeinholzer, v. wissemann let area (p < 0.001) after cutting and the posthoc-kruskal-dunn test identified significant differences between all three cuts in terms of leaflet area with p < 0.001. differences in leaflet area were detected between plot 4 and the two other plots (7 and 10, p < 0.001; fig. 2 b). compared to the control group (plot 1) there are no significant differences between no cut and 2nd cut (p = 0.399). after first mowing, the leaflet area varied between 4 5 cm² and remained stable after the second mowing (fig. 2 b). only after the third cut, the leaflet area of t. pratense decreases (fig. 2 b). the model lm4 shows a significant influence of mowing frequencies (chi < 0.001, p < 0.001) and the time of mowing (chi < 0.001, p < 0.001) for leaf let area. considering the four treatment classes (early, middle, late, and later), we observed an increase of leaflet area after the 2nd cut and a decrease after the 3rd cut in which it returns to the size of the 1st cut (early p < 0.001) or below (middle, late, later p < 0.001; fig. 2 b). for plot 4 the leaflet area after mowing differed significantly (p < 0.001) from the plots 7 and 10 between the second and third mowing. the leaflet area in plots 7 and 10 decreased slightly after the 3rd cut (fig. 2b), the leaflet area in plot 4 is relatively stable during the three times of mowing. thus, the leaflet area of t. pratense remains rather stable even after two mowing events. this is in contrast to the reaction described in herbert et al. (2018) and in t. repens (goulas et al., 2002), in which the leaflet area decreased already after the first cut. red clover shows greater regrowth capabilities under field conditions than in common garden greenhouse conditions. for all plots mown twice (3, 4, 6, 7, 9, 10, and 12), we observed a significant increase in weight with 46.02 % (p < 0.001) (suppl. fig. 5 c). the biomass weight over three cuts regardless of the season was normally distributed (p = 0.083) and variance homogeneity was detected (p = 0.206). an anova showed highly significant weight differences between the biomasses of different mowing regimes and the tukey hsd post hoc test showed highly significant weight differences among / between all three cuttings (fig. 2 c) but not among the three plots (4, 7 and 10; p = 0.997). after the first cut, 8.18 kg fresh material (fm)/m² were harvested in total (fig. 2 c, tab. 2). through the second time of mowing 11.97 kg fm/m² were harvested (fig. 2 c, tab. 2), thus the plants produced 46.38 % more above ground biomass after the first cut, a result similar to observations by eriksen et al. (2012) in a ryegrass / clover mixture. eriksen et al. (2012) also showed that a third cut reduced yield / weight of red clover. these results described here from the field experiments are in concordance with the common garden greenhouse experiments of herbert et al. (2018) after which red clover initiates a new growth phase after cutting to compensate the loss of biomass. similarly, this was also observed for the relative t. repens, which compensates the leaf loss tab. 2: weight development after three times of mowing regardless of the classes (only aspect the three cuts) and dependent on the class (early, middle, late, and later). weight measure in kg fresh material (fm) per m². mowing plots weight difference % kg_fm/m² in weight kg_fm/m² regardless of time 1. cut 4, 7, 10 8.18 2. cut 4, 7, 10 11.97 3.79 46.38 3. cut 4, 7, 10 1.81 -10.16 -84.87 early 1. cut 4 3.05 2. cut 4 3.14 0.09 3.01 3. cut 4 1.01 -2.13 -67.90 middle 1. cut 7 2.68 2. cut 7 4.09 1.42 52.96 3. cut 7 0.59 -3.50 -85.54 late 1. cut 10 2.45 2. cut 10 4.73 2.28 93.20 3. cut 10 0.21 -4.52 -95.56 later 1. cut 12 2.38 2. cut 12 3.45 1.08 45.26 fig. 3: development of leaflet roundness (up to 1 for round) (a) after early, (b) middle, (c) late and (d) later mowing in the year. ** p < 0.01, *** p < 0.001 reaction of t. pratense to repeated mowing 5 after defoliation in 5-9 days (ryle et al., 1985; black et al., 2009). however, repeated cutting seems to decrease the regeneration abili ty of red clover (eriksen et al., 2012). these results are based only on the “three times mowing per year” regime regardless of mowing date. however, analysing the dates of mowing more thoroughly (tab. 2, fig. 3), suggests that mowing twice in the middle and late vegetation period is more beneficial for the gain of biomass weight, similar results for different cutting regimes were shown by wiersma et al. (1998). an increase of weight of 50% 90% was observed after the second cut, but a reduction in biomass of -84.87% as compared to the first cutting after the third cutting. the later cutting regime (only two cuts) improved yield to 45.26% (tab. 2). however, it should be noted that there were 60 days between the 1st and 2nd cut and only 30 days between the 2nd and 3rd cut. however, the daily growth rate between the 1st and 2nd cut is 0.02 kg fm/m²/day, 0.073 times higher (early, middle, late) than between the 2nd and 3rd cut, thus we can assume that even with 60 days between the 2nd and 3rd cut, the amount of harvested biomass would have been less than after the 1st cut. for the early mowing the harvested biomass weight remained the same between the 1st and 2nd cut (p = 0.921) and decreased after the 3rd cut, in the other three classes the weight increased after the 1st cut and decreased after the 2nd cut (fig. 3 a d). temporal analysis of plot 4 revealed that an early mowing is less suitable for increasing growth (fig. 2 c, 3 a), similar results for an early mowing in the season were shown by wiersma et al. (1998) which resulted in an reduced yield. we detected a change in nitrogen content over the year in the different mowing regimes 2nd cut – 3rd cut (p < 0.001). in all three plots, the nitrogen content increased during the season. plot 4 had the biggest increase of the nitrogen content from 3.76% to 5.39% (fig. 3 d), while the increases in plots 7 and 10 were lower, 3.46% to 4.18% and 3.77% to 4.46%, respectively. based on the differences between plot 4 to 7 (p = 0.03; fig. 3 d), mowing neither increased nor decreased the nitrogen content in our red clover plants. dahlin and mårtensson (2008) could show a similar effect for t. repens after mowing. however, they used potted plants and mowed only once, thus the seasonal effect is missing. nevertheless, even repeated cutting did not reduce the nitrogen content, such that high protein content and quality of the harvested material remains stable. the perseverance of red clover was not a primary target of analysis in this study, but we found the absence of cutting negatively affecting the survival of red clover plants. after the eighth month, the red clover had died on the unmown control plots while mown red clover plots were healthy (suppl. fig. 6 and https://doi.org/10.25829/ bexis.26586-5). conclusion in summary, we could validate the findings of the common garden greenhouse experiment from herbert et al. (2018) into the field. t. pratense compensates the losses after cutting through starting a new growth phase, especially when the cutting was in the middle of june leading to fresh weight gain. different mowing regimes influence the harvested biomass: mowing at a later time in the year and after two cuts plants produce the highest biomass in the field. how ever, the timing of the mowing had no influence on the n-content. mowing early in the season does not significantly increase the biomass but mowing in the middle or later in the vegetation period (beginning of june) increases the biomass of the harvested red clover and by this an increased total nitrogen content was achieved. however, even very late mowing (end of july) is more profitable than mowing three times early in the season (tab. 2). acknowledgements we like to thank prof. rod snowdon and lothar behle-schalk from the field station rauischholzhausen and their team for technical support with the field work. the help of prof. hans-werner koyro and nicol strasilla with the determination of the n-content is gratefully acknowledged. also, we like to thank the students dennis oderwald, larissa witzel, and julian garrecht for assisting with the field measurements. this project was funded by the german research foundation (dfg wi2028/8-2, be2547/12-2, and ge1242/14-2). conflict of interest no potential conflict of interest was reported by the authors. references beecher, m., hennessy, d., boland, t.m., mcevoy, m., o’donovan, m., lewis, e., 2015: the variation in morphology of perennial ryegrass cultivars throughout the grazing season and effects on organic matter digestibility. grass forage sci. 70, 19-29. doi: 10.1111/gfs.12081 black, a., laidlaw, a., moot, d., o’kiely, p., 2009: comparative growth and management of white and red clovers. ir. j. agric. food res. 48, 149-166. briske, d., richards, j., 1995: plant responses to defoliation: a physiolo gical, morphological and demographic evaluation. in: bedunah, d.j., sosebee, r.e. 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b: after transformation with standardize of leaflet roundness; c: without transformation of leaflet area. d: transformed log leaflet area. supplementary material supplement tab. 1: model selection after aci. roundness of leaflets~ (selected model orange), leaflet area ~ (selected model green), df degrees of freedom, aic akaike information criterion, loglik log-likelihood. supplement fig. 1: plotted residual and fitted values from model lm5 leaflet roundness upper row and leaflet area lower row. a: without transformation of leaflet roundness; b: after transformation with standardize of leaflet roundness; c: without transformation of leaflet area; d: transformed log leaf let area. supplementary material ii supplement fig. 2: model lm5 validation after transformation of leaflet roundness. a: residuals vs. cutting events; b: residuals vs. experimental day (days of mowing events); c: residuals vs. classes (early, middle, late, later, and no~control); d: roundness vs. residuals in cut events with smoothed regression line; e: roundness vs. residuals in classes with smoothed regression line. supplement fig. 3: model lm4 validation after transformation of leaflet area. a: residuals vs. cutting events; b: residuals vs. experimental day (days of mowing events); c: residuals vs. classes (early, middle, late, later and no~control); d: roundness vs. residuals in cut events with smoothed regression line; e: roundness vs. residuals in classes with smoothed regression line. iii supplementary material supplement figure 3: model lm4 validation after transformation of leaflet area. a: residuals vs. cutting events; b: residuals vs. experimental day (days of mowing events); c: residuals vs. classes (early, middle, late, later and no~control); d: roundness vs. residuals in cut events with smoothed regression line, e roundness vs. residuals in classes with smoothed regression line. supplement figure 4: trifolium pratense after the different mowing regimes. a: height of plants; b: number of inflorescences. supplement fig. 4: trifolium pratense after the different mowing regimes. a: height of plants; b: number of inflorescences. supplement figure 5: trifollium pratense after two times of mowing (regardless of the time) and the not mown control group. a: roundness of leaflets in the different plots; b: leaflet area in the different plots; c: weight of the harvested biomass of the plots. supplement figure 6: survival rate of the control red clover plants during the field experiment. supplement fig. 5: trifolium pratense after two times of mowing (regardless of the time) and the not mown control group. a: roundness of leaflets in the different plots; b: leaflet area in the different plots; c: weight of the harvested biomass of the plots. supplement figure 5: trifollium pratense after two times of mowing (regardless of the time) and the not mown control group. a: roundness of leaflets in the different plots; b: leaflet area in the different plots; c: weight of the harvested biomass of the plots. supplement figure 6: survival rate of the control red clover plants during the field experiment. supplement fig. 6: survival rate of the control red clover plants during the field experiment. supplementary material iv journal of applied botany and food quality 95, 1 5 (2022), doi:10.5073/jabfq.2022.095.001 1instituto politécnico naiconal, centro de investigación en biotecnología aplicada, carretera estatal santa inés tecuexcomactepetitla, km 1.5, tepetitla de lardizábal, tlaxcala c.p. 90700, méxico 2universidad politécnica de tlaxcala, av. universidad politécnica no.1 san pedro xalcatzinco, 90180 tepeyanco, tlaxcala, méxico comparative study of anthocyanin extraction methods in dahlia pinnata petals sulem yali granados-balbuena1, vanesa chicatto-gasperín1, lucía aztatzi-rugerio1, ericka santacruz-juárez2, raúl rene robles-de la torre1, erik ocaranza-sánchez1*, maría reyna robles-lópez1* (submitted: september 19, 2021; accepted: january 8, 2022) * corresponding author summary anthocyanins are phenolic compounds responsible for the color of numerous plant sources. in literature, there is little information about the dahlia flower as a potential source of anthocyanins. this study aimed to develop a procedure for anthocyanins extraction from black dahlia petals using fresh and dehydrated material. a three-stages nested design was used to develop the methodology, 3 solid-liquidratios and 6 dissolvents nested in 3 methods. the highest yields were obtained with the homogenization assisted maceration technique, citric acid solvent (2, 4, 6%), and a ratio of 1:30 with dry petals. the results of this study show the opportunity to obtain a high antho cyanin content from black dahlia and open the possibility to use it as another important source of this pigment. keywords: dahlia, anthocyanins, sonication, maceration, homogeni zation, extraction. introduction the colorants used in the food industry are generally of synthetic origin and are used to improve visual perception or when the natural color has been affected during processing, storage, or distribution (coultate and blackburn, 2018). however, concerns about health and restriction in the use of synthetic colorants in several countries have led to a tendency to replace artificial colorants with natural pigments (mcgill, 2009). anthocyanins are a representative group of natural pigments; since of their wide distribution, water solubility, and health benefits such as antioxidant, anti-cancer, anti-diabetic, and visual health improvement effects (khoo et al., 2017). in addition, they have a wide range of colors ranging from red, blue, purple, magenta, and orange. this latter feature added to their health benefits has allowed its application in the food industry and led to the constant search for sources of this pigment such as grape skin, blueberry, cranberry, raspberry, blackberry, radish, cherry, rosella, purple cabbage, among others (leong et al., 2018). dahlia flower with red, purple, and black shades owes its hue to the presence of anthocyanins (lara-cortés et al., 2014; deguchi et al., 2016) this flower is native to mexico, belongs to the asteraceae family, and is part of the heliantheae tribe, which the aztecs called cocoxochitl because of its hollow stem. in pre-hispanic times it had a dominant culinary and decorative use. in addition, its petals were used to extract pigment and color cotton (luna monterrojo, 2008; laracortés et al., 2014). in literature, information about dahlia flower as a source of anthocyanins for food application is scarce, so the study of pigment content is a fundamental factor in defining its potential; therefore, it is essential to develop extraction processes that help to know the conditions that lead to its optimization. in most cases, extraction of anthocyanins for their application in food is carried out with solvents such as water and ethanol, which are usually acidified with weak acids such as acetic and citric (belwal et al., 2018). the subject of this work was dahlia pinnata, and the objective was to perform a comparative study of anthocyanin extractions in fresh and dried petals, so three different extraction techniques were used with variations in solvents and solid-liquid ratio. materials and methods dahlia flowers were collected in the municipality of huamantla, located in the eastern part of the state of tlaxcala, mexico (coordinates: 19.3420758, -97.91872470000001). the dahlia petals were manually removed from flowers, 13 kg of this material were obtained; 3 kg were used for fresh experiments and the remainder was dried at a temperature of 30 °c in a drying oven (sev® s/n), up to a constant weight. dehydrated petals were vacuum-packed and stored in darkness at room temperature until use. extraction procedure for anthocyanins since dahlia blooming is annual, it is considered important to study in three extraction conditions of fresh and dehydrated material. the extraction was performed by maceration which involves the simple diffusion of the pigment towards the liquid phase, using three solid ratios (fresh or dehydrated), with six different types of solvent (liquid phase) and for the extraction three conditions of the solid material (simple maceration, assisted maceration with homogenization and assisted maceration with sonication). in all of them, the ratio (s/l) or solid/solvent was 1:10, 1:20 and 1:30, using distilled water, acidified at 2, 4 and 6% with citric acid, 2% ethanol, and 2% acetic acid. after each extraction was finished, the liquid extract was separated from the solid residue through a line cloth filter and subsequently with 0.25 μm whatman member filter twice. all experiments were performed in triplicate using amber material and in the absence of illumination. simple maceration this conventional extraction was carried out in a 100 ml flask for 24 hours at 25 ºc. dahlia petals (1.5 g) were mixed with the different solvents to give the ratios 1:10, 1:20 and 1:30. this method was called maceration. homogenization assisted maceration about 1.5 g of plant material was extracted with the different solvents at the indicated rates, by mechanical way. this method was called homogenization. sonication assisted maceration an ultrasound-assisted extraction process was carried out with 1.5 g of fresh and/or dry material. later, different solvents were added 2 s.y. granados-balbuena, v. chicatto-gasperín, l. aztatzi-rugerio, e. santacruz-juárez, r.r. robles-de la torre, e. ocaranza-sánchez, m.r. robles-lópez to give the respective mass liquid ratios. samples were treated for 60 minutes using a branson 3800h ultrasound bath (110 w, 40 khz) at 25 ºc. this experiment was called sonication. total anthocyanin content the total monomeric anthocyanin was quantified through the diffe rential ph method (hutabarat et al., 2019). values were expressed in mg of cyanidin-3-glucoside and calculated as follows: anthocyanin extraction yield (mg/100 g) = where: mw: molecular weight of cyanidin-3-glucoside (449.2 g/ mol), df: extract dilution factor, v = volume of extract (l), 1000 = conversion factor from g to mg, e = molar extinction coefficient of cyanidin-3-glucoside (26900 l/mol.cm) l = length of the cuvette (cm), m = weight of the sample (g) in dried basis. finally, the results were expressed as mg of cyanidin-3-glucoside per 100 g. in any case, the result of the pigment has been adjusted to the solid content in the material. scanning electron microscope (sem) material observations before and after extraction were performed by scanning electron microscopy; for this purpose, samples of dry petals with the highest and lowest anthocyanin yield obtained were selected. therefore, the micrographs were: homogenization with 2% citric acid, simple maceration with 2% acetic acid, sonication with 4% citric acid, simple maceration with 2% acetic acid, and maceration with 2% citric acid, all of them with the solid-liquid ratio at 1:30, finally the control it was the petal with no treatment. after extraction, the samples were dried to constant weight and stored in vacuumsealed flasks. subsequently, they were gold-plated in a denton vacu um desk v equipment, and the images were obtained through the jsm 66101v electron microscope. statistical analysis and graphs the experimental design was a nested design type (fig. 1), in which the treatments were: homogenization, maceration, and sonication. the factors were the solid-liquid ratio (levels: 1:10, 1:20, and 1:30) and the solvent (levels: water, citric acid 2%, 4%, and 6%, 2% ethanol, and 2% acetic acid). the statistical software minitab 16 (minitab inc.) was used to perform the variance analysis (anova) followed by tukey’s multiple comparison test. the confidence level used for the variance analysis was 95%. extraction graphics were performed with graphpad prism version 8.4.0 program. results and discussion fresh material tab. 1 summarizes the reported anthocyanin content in mg/100 g (dry base) extracted under different extraction conditions. the method that influenced obtaining the highest yields was maceration followed by grinding and sonication. it was not possible to extract the pigment in maceration using water as a solvent, because immediately extract turned brown showing its oxidation. the same phenomenon happened with sonication using water, it is deduced that prolonged time could induce pigment degradation. previous studies indicate that the extraction of anthocyanins using ultrasound leads to oxidation reactions and it has also been reported that prolonged sonication times influence the degradation of phenolic compounds such as anthocyanins (mane et al., 2015; dailey and vuong, 2015). fig. 2 shows the solvent that contributed to high yields (6.63-13.67 mg/ 100 g) was generally 2% acetic acid (p <0.05), followed by acidified water with 6% citric acid at intervals of (1.50-8.23 mg/100 g), unlike distilled water, which was the solvent with the lowest yields (1.20-2.42 mg/100 g) and in some cases not detected. it is therefore clear that an acid medium is needed to provide stability to anthocyanins. acidified solvents are more efficient in extracting anthocyanins than those that are neutral, since at different ph values different anthocyanin structures predominate, for example, at ph values below 4 the predominant form is the flavillium cation, whose structure is more stable than the others (quinoidal base, carbinol pseudo base, and chalcone (azwanida, 2015; ngamwonglumlert et al., 2017). the combination of factors that led to obtaining the highest yield corresponds to maceration using 2% acetic acid with the ratio of 1:30 (13.67-5.85 mg/100 g of anthocyanin), unlike the lower yields that were tested with sonication, using water with 2% citric acid with the same solid-liquid ratio (0.580.25 mg/100 g anthocyanin). finally, statistical analysis will conclude that the different solid-liquid ratios proposed in this work (1:10, 1:20, and 1:30); do not affect the extraction when petals are fresh. dried material the homogenization method showed the highest extraction yields, followed by maceration and sonication in dehydrated dahlia petals (tab. 2). these results can be attributed to the fact that homogenization, compared to maceration, leads to cell lysis through shear forces that release the pigment to the medium, resulting in a faster dif fusion process. another factor to consider is extraction time, which, in the case of maceration ends when a balance is reached between the concentration of metabolites in the extract and petals, so it can take hours and even days to obtain higher yields. these results cofig. 1: experimental design of extraction treatments the black-spot shapes indicate repeated experiment abs × mw × df × v × 1000 *100 ( × l × m) anthocyanin extraction methods in dahlia pinnata petals 3 tab. 1: anthocyanin yields in fresh dahlia petal extracts anthocyanins mg/100 g disolvent ratio water 2% citric acid 4% citric acid 6% citric acid 2% etanol 2% acetic acid homogenization r 1:10 n.d. 6.30 ± 0.24 6.03 ± 0.39 8.23 ± 1.03 3.21 ± 0.43 *7.95 ± 0.88 r 1:20 n.d. 6.31 ± 1.88 5.22 ± 0.78 5.73 ± 0.32 3.09 ± 0.26 *10.78 ± 0.53 r: 1:30 n.d. 5.24 ± 1.07 5.26 ± 1.33 6.41 ± 0.44 2.88 ± 0.00 *6.53 ± 0.56 maceration r 1:10 1.20 ± 0.19 4.87 ± 0.43 2.21 ± 0.37 4.38 ± 0.06 4.13 ± 0.54 *8.97 ± 0.76 r 1:20 2.34 ± 0.44 4.87 ± 0.36 4.29 ± 0.42 3.76 ± 0.22 4.63 ± 0.84 *10.78 ± 0.04 r: 1:30 2.42 ± 0.18 5.05 ± 028 5.03 ± 0.56 4.61 ± 0.90 3.99 ± 0.22 *13.67 ± 5.85 sonication r 1:10 n.d 0.96 ± 0.10 0.94 ± 0.15 1.25 ± 0.30 5.65 ± 0.43 *6.63 ± 0.80 r 1:20 n.d. 0.70 ± 0.46 0.97 ± 0.21 5.20 ± 0.34 5.20 ± 0.39 *8.29 ± 0.90 r: 1:30 n.d. 0.58 ± 0.25 0.97 ± 0.47 1.50 ± 0.66 4.55 ± 0.64 *7.83 ± 1.32 the * symbol indicates significant differences (p <0.05) fig. 2: anthocyanin yields in fresh dahlia petal extracts tab. 2: anthocyanin yields in dried dahlia petal extracts anthocyanins mg/100 g ratio water 2% citric acid 4% citric acid 6% citric acid 2% ethanol 2% acetic acid homogenization r 1:10 12.3 ± 1.4 *27.07 ± 9.40 *26.87 ± 4.04 *33.19 ± 1.86 1.23 ± 0.43 4.86 ± 1.66 r 1:20 16.63 ± 2.10 *35.21 ± 3.88 *38.27 ± 4.66 *29.50 ± 0.64 1.22 ± 0.21 2.64 ± 1.27 r: 1:30 16.68 ± 2.08 *36.53 ± 2.37 *24.42 ± 15.04 *30.48 ± 3.45 1.01 ± 0.19 2.17 ± 0.42 maceration r 1:10 3.39 ± 0.65 6.39 ± 0.33 3.60 ± 0.88 7.33 ± 0.73 0.87 ± 0.35 4.00 ± 0.34 r 1:20 4.90 ± 0.28 12.58 ± 3.34 12.26 ± 1.95 11.29 ± 1.06 1.06 ± 0.15 5.33 ± 0.61 r: 1:30 6.91 ± 0.56 19.79 ± 3.56 17.87 ± 1.96 26.86 ± 8.40 1.02 ± 0.17 4.49 ± 0.99 sonication r 1:10 2.93 ± 0.80 1.81 ± 0.22 3.73 ± 1.48 4.46 ± 0.54 1.05 ± 0.17 3.01 ± 0.17 r 1:20 3.83 ± 0.79 0.47 ± 0.01 4.76 ± 2.22 1.36 ± 1.11 1.36 ± 0.24 3.27 ± 0.38 r: 1:30 6.18 ± 1.49 0.07 ± 0.01 12.94 ± 5.67 10.46 ± 3.20 1.77 ± 0.19 2.40 ± 0.91 the * symbol indicates significant differences (p <0.05) incide with other research published and carried out on other biological materials (sarker et al., 2006; rostagno and prado, 2013; azwanida, 2015). on the other hand, the difference that led to homogenization is superior to sonication (although both are methods of cellular disruption) is due firstly to the fact that homogenization is less aggressive and secondly to the fact that the time spent in sonication was probably very long. solvents with higher yields were citric acid at different concentrations, with no significant differences (p <0.05), followed by water, 2% ethanol, and 2% acetic acid. as mentioned above, acidified systems promote the stability of anthocyanins and it is also known that an acidic ph promotes the cleavage of phenols bound to proteins and carbohydrate polymers since phenols are protonated at low ph values 4 s.y. granados-balbuena, v. chicatto-gasperín, l. aztatzi-rugerio, e. santacruz-juárez, r.r. robles-de la torre, e. ocaranza-sánchez, m.r. robles-lópez and adopt a hydrophobic nature that allows them to penetrate the micelles with a surfactant effect (chaves et al., 2020). regarding the solid-liquid relationships raised, there were significant differences (p <0.05), being the 1:30 relationship the one that contri buted to obtaining the highest yields in the three different methods. fig. 3 shows the values obtained. as noted, homogenization along with acidified water were the factors that led to higher extraction yields, however, no significant differences(p <0.05) were found between the use of citric acid at 2, 4, and 6%, therefore the use of the 2% is recommended since a smaller amount of the acidulant is required. finally, the solid/liquid ratio 1:30 showed a yield interval between 24.42 and 35.53 mg/100 g of pigment. in summary, extraction yields in dry petals were higher than fresh material, with a content of up to 35.53 mg/100 g, compared to the fresh samples where the maximum yields were 13.67 mg/100 g. sem analyses fig. 4 shows sem images of cellular morphological changes in the epidermis caused by maceration, sonication and homogenization methods on dry petals. the control (fig. 4a) is a surface structure of cells of the papillose type, freely arranged and with large intercellular spaces. in the maceration process with 2% citric acid (fig. 4b), moderate changes in morphology were observed, some agglomerations of apparently empty cavities indicate that there was an extraction process. in the sample extracted with homogenization in the presence of acetic acid (fig. 4c), it is observed that the structure of the cones is preserved, only more agglomerates are observed, so the extraction could not be as efficient. the homogenization carried out with 2% citric acid (fig. 4d) led to the petal having notable morphological changes; as the formation of cavities due to transferring the cellular content to the solvent and slight fragmentation of the structures. this fragmentation increases the surface, which increases the mass transfer and extraction efficiency. for the petals with a sonication process in 2% citric acid (fig. 4e) and with 2% acetic acid (fig. 4f), more drastic morphological changes are shown, presenting a fragmentation of structures and erosion processes, probably caused by the implosion of bubble cavities on the surface of the petals, causing a release of cellular contents to the extraction medium (chemat et al., 2017; bailes and glover, 2018). conclusions based on a comparative study of extraction methods in dried dahlia petals, the factors that led to achieving the highest yields of 24 to 38 mg/100 g were through homogenization using water acidified with citric acid 2-6% with a ratio of 1:30. for fresh petal yields, the fig. 4: a) control. b) maceration with 2% citric acid. c) homogenization with 2% acetic acid. d) homogenization with 2% citric acid. e) sonication with 4% citric acid. f) sonication with 2% acetic acid. fig. 3: anthocyanin yields in dried dahlia petal extracts anthocyanin extraction methods in dahlia pinnata petals 5 highest values were obtained by maceration with 2% acetic acid in a solid-liquid ratio of 1:30. the images of the sem showed that there were changes in the cellular morphology of the epidermis of the dried petals with every method of extraction; homogenization, maceration, and sonication. the most notable change occurred with sonication since fragmentations were observed in the cellular structure of the epidermis. finally, due to the results obtained show a high content of anthocyanins, it is concluded that this flower has potential for application in food, however, it is suggested to carry out safety tests as well as to evaluate the stability of the pigment. acknowledgements the authors thank consejo nacional de ciencia y tecnología (conacyt) for the financial support. conflicts of interest no potential conflict of interest was reported by the authors. references azwanida, n.n., 2015: a review on the extraction methods use in medicinal plants, principle, strength and limitation. med. aromat. plants 4. doi: 10.4172/2167-0412.1000196 bailes, e.j., glover, b.j., 2018: intraspecific variation in the petal epidermal cell morphology of vicia faba l. 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s.m., 2017: anthocyanidins and anthocyanins: colored pigments as food, pharmaceutical ingredients, and the potential health benefits. food nutr. res. 61, 1361779. doi: 10.1080/16546628.2017.1361779 lara-cortés, e., martín-belloso, o., osorio-díaz, p., barrera-necha, l.l., sánches-lópez, j.a., bautista-baños, s., 2014: antioxidant capacity, nutritional and functional composition of edible dahlia flowers. rev. chapingo ser. hortic. 20, 101-116. doi: 10.5154/r.rchsh.2013.07.024 leong, h.y., show, p.l., lim, m.h., ooi, c.w., ling, t.c., 2018: natural red pigments from plants and their health benefits: a review. food rev. int. 34, 463-482. doi: 10.1080/87559129.2017.1326935 luna-monterrojo, v., 2008: 50 aniversario de la dalia como la flor nacional de méxico. retrieved 6th march 2020, from http://www.inecol. mx/inecol/index.php/es/2017-06-26-16-35-48/17-ciencia-hoy/244-50aniversario-de-la-dalia-como-la-flor-nacional-de-mexico. mane, s., bremner, d.h., tziboula-clarke, a., lemos, m.a., 2015: effect of ultrasound on the extraction of total anthocyanins from purple majesty potato. ultrason sonochem. 27, 509-514. doi: 10.1016/j.ultsonch.2015.06.021 mcgill, a.e.j., 2009: the potential effects of demands for natural and safe foods on global food security. trends food sci. technol. 20, 402-406. doi: 10.1016/j.tifs.2009.01.051 ngamwonglumlert, l., devahastin, s., chiewchan, n., 2017: natural colorants: pigment stability and extraction yield enhancement via utilization of appropriate pretreatment and extraction methods. crit. rev. food sci. nutr. 57, 3243-3259. doi: 10.1080/10408398.2015.1109498 rostagno, m., prado, j., 2013: natural product extraction − principles and applications. the royal society of chemistry. sarker. s., latif z., gray a., 2006: natural products isolation, second ed. humana press. orcids sulem yali granados-balbuena https://orcid.org/0000-0002-6273-7329 vanesa chicatto-gasperín https://orcid.org/0000-0002-9826-6768 lucía aztatzi-rugerio https://orcid.org/0000-0002-8066-9964 ericka santacruz-juárez https://orcid.org/0000-0002-1090-1879 raúl rene robles-de la torre https://orcid.org/0000-0001-8696-4326 erik ocaranza-sánchez https://orcid.org/0000-0001-6778-7248 maría reyna robles-lópez https://orcid.org/0000-0003-0220-0340 addresses of the corresponding authors: instituto politécnico naiconal, centro de investigación en biotecnología aplicada, carretera estatal santa inés tecuexcomac-tepetitla, km 1.5, tepetitla de lardizábal, tlaxcala c.p. 90700, méxico e-mail: erik ocaranza sánchez eocaranza@ipn.mx maría reyna robles lópez mrobles@ipn.mx http://dx.doi.org/10.4172/2167-0412.1000196 http://dx.doi.org/10.1016/j.flora.2018.06.005 http://dx.doi.org/10.1016/j.trac.2017.12.018 http://dx.doi.org/10.3389/fchem.2020.507887 http://dx.doi.org/10.1016/j.ultsonch.2016.06.035 http://dx.doi.org/10.1111/cote.12334 http://dx.doi.org/10.3390/technologies3040302 http://dx.doi.org/10.2503/hortj.mi-121 http://dx.doi.org/10.1155/2019/6806790 http://dx.doi.org/10.1080/16546628.2017.1361779 http://dx.doi.org/10.5154/r.rchsh.2013.07.024 http://dx.doi.org/10.1080/87559129.2017.1326935 http://dx.doi.org/10.1016/j.ultsonch.2015.06.021 http://dx.doi.org/10.1016/j.tifs.2009.01.051 http://dx.doi.org/10.1080/10408398.2015.1109498 https://orcid.org/0000-0002-6273-7329 https://orcid.org/0000-0002-9826-6768 https://orcid.org/0000-0002-8066-9964 https://orcid.org/0000-0002-1090-1879 https://orcid.org/0000-0001-8696-4326 https://orcid.org/0000-0001-6778-7248 https://orcid.org/0000-0003-0220-0340 journal of applied botany and food quality 95, 85 93 (2022), doi:10.5073/jabfq.2022.095.011 division of quality and sensory of plant products, georg-august-universität göttingen, göttingen, germany influence of urban gardening conditions on the concentration of antioxidant secondary plant metabolites in kale marie bayer, susanne neugart, tobias pöhnl* (submitted: november 4, 2021; accepted: march 11, 2022) * corresponding author summary during the covid-19 pandemic urban gardening became popular across the globe. leafy vegetables supplement the daily diet and contribute to consumers health. within the last decade kale (brassica oleracea var. sabellica l.) gained popularity in urban gardening. however, shading due to unfavourable cardinal directions may reduce plant growth and accumulation of health-promoting secondary plant metabolites such as polyphenols, carotenoids and glucosinolates in kale. we compared authentic urban gardening conditions for kale grown in all four cardinal directions of a residential building. the overall concentration of carotenoids did benefit from sun exposed growing locations, including indoor cultivation behind uv light filtering glass windows, while concentrations of nutritionally important lutein did not differ among the locations and their altered growth conditions regarding abiotic stressors such as sun exposure, temperature, and water consumption. total concentration of phenolics profited the most from direct sunlight but is severely reduced behind glass windows. overall, satisfying growth rates of kale were achieved under all applied conditions, encouraging outdoor urban gardening with kale plants even in shaded locations. keywords: urban gardening, kale, brassica oleracea var. sabellica l., flavonoids, carotenoids, glucosinolates introduction urban gardening became a global trend not only in one‘s own balcony or garden, but also community gardens, guerrilla gardening projects, self-harvesting fields or solidarity agriculture in urban and suburban areas (kalantari et al., 2018). singapore, being highly urbanized, wants to grow 30% of its food within the city by 2030 (montesclaros and teng, 2019). within the european union, urban gardening is promoted, highlighting its positive contribution to eu’s economic, social, and environmental goals. in long-term, growing plants as part of urban gardening can have positive impact on environment and climate. the green spaces that are created can, for example, provide habitats and food for pollinating insects such as bees, contributing to urban biodiversity (matteson et al., 2008). furthermore, urban gardening involves a more diverse range of va rieties contributing to agro-biodiversity (galluzzi et al., 2010). in cities urban gardening causes less transportation related emissions and urban gardens can reduce the „heat island effect“ of largely ur banized areas and provide shading (wolf and robbins, 2015). during the covid-19 pandemic people had to stay at home and consequently invested in their gardens which lead to a large increase of urban gardening activities worldwide (mullins et al., 2021). positive impacts of urban gardening during the covid-19 pandemic have been focused in several recent publications. under lockdown conditions long stretched supply chains, spanning across borders were suddenly interrupted while demand for fresh foods increased up to 44% in germany (pulighe and lupia, 2020). meanwhile consumers started to grow their own vegetables at home. in canada 17.4% of home gardeners started during the covid-19 pandemic, which further improved self-rated physical, emotional, and mental health with older (> 70 years of age) people (corley et al., 2021; mullins et al., 2021). abiotic stress such as high temperatures, water deficit or direct sun light does enhance the accumulation of health promoting secondary plant metabolites such as phenolic compounds and carotenoids. for most urban gardeners, location is an unchangeable factor but influences plant growth decisively. shading in northern-located and lack off (uv) radiation indoors are widely believed to be unsuitable for cultivation. kale (brassica oleracea var. sabellica l.) belongs to the brassicaceae family, and is regarded a “superfood”, rich in health beneficial calcium, folate, riboflavin, vitamin k, vitamin a, lutein, and glucosinolates (miękus et al., 2020). kale is predominantly cultivated in the northern hemisphere, and its cultivation in northern germany has a long tradition. depending on selected variety and weather conditions, kale can tolerate temperatures as low as -8 °c, being also suitable for late cultivation. in germany 18.500 tons of kale were harvested in 2020, accounting for no significant change within the last 10 years (statistisches bundesamt, 2021). kale, is considered a rich source of phenolic compounds as high as 300 mg per 100 g fresh matter (schmidt et al., 2010). flavonoids in kale are predominantly kaempferol and quercetin glycosides, with quercetin being the more effective antioxidant (mageney et al., 2017). flavonoids possess a variety of biological activities that protect against cardiovascular diseases or cancer (dabeek and marra, 2019). within plant tissue flavonoids are important for plant defence against abiotic stressors such as uv radiation (d’amelia et al., 2018). analogously, carotenoids are health-beneficial lipophilic compounds and β-carotene and lutein are important quality traits of kale (lee et al., 2018). furthermore, kale is a rich source of lutein (lee et al., 2018) which is important for eye and skin health. their concentration is largely effected by irradiation and 4-fold longer photoperiods may increase β-carotene and lutein concentrations by more than 50% to 10.4 ± 0.4 and 13.5 ± 0.6 mg per 100 g fresh weight (lefsrud et al., 2006). glucosinolates are predominantly found in brassicaceae vegetables, including kale. among the 120 different glucosinolates, in kale predominantly glucoiberin, gluconapin, progoitrin, glucoraphanin, gluconasturtiin, and glucobrassicin are reported (kusznierewicz et al., 2013; hahn et al., 2016). for the plant glucosinolates are of great importance in herbivore defense upon enzymatic breakdown by myrosinase (ec 3.2.1.147) and the release of toxic nitriles, isothiocyanates, epithio nitriles, and thiocyanates (kusznierewicz et al., 2013). for human nutrition their pungent aroma is characteristic, being accompanied by their antimicrobial and anticancerogenic properties (miękus et al., 2020). consequently, it is of great interest for urban farmers to cultivate vegetables rich in diverse secondary plant metabolites to boost their immune system. with high food prices adequate nutrition and supply with essential vitamins and minerals becomes challenging for people 86 m. bayer, s. neugart, t. pöhnl with low income and limited capacities for self-supply. therefore, it is important for this group to use the limited available space to supplement their diet with healthy food and reduce food costs. vegetables are an important source of vitamins, secondary plant metabolites, and minerals that can supplement caloric staple foods for a healthy lifestyle. due to the orientation of balconies and other shading sources light conditions might be unfavorable throughout growth in urban gardening. therefore, the aim of the study was to evaluate the concentration of health-promoting secondary plant metabolites in kale under realistic urban gardening conditions by comparing plants grown in all four cardinal directions of a residential house and considering indoor and outdoor growing. a step towards sustainable development goal 2 “zero hunger” the findings should be beneficial for not only low income consumers from developing countries, but also encourage urban gardeners worldwide to grow healthy food. materials and method experimental site and plant growth kale plants were grown from 20.05.2020 (sowing) to 01.07.2020 (harvest) inside and outside of a residential building in mecklenburg lake district (germany). plants were placed outside in the four car dinal directions (north, east, west, and south) and inside in southern and eastern location of the building. noteworthy, the building was rotated 20° westwards. outdoor plants were placed on a small balcony like structure approximately 70 cm above ground level directly attached to the building. indoor grown plants were placed directly behind windows. each of the six repetitions consisted of 10 individual plants. for cultivation 10 pots were filled with fruhstorfer soil (hawita, vechta, germany). for germination a thin layer of sowing soil (evergreen garden care, mainz, germany) was added, into which two kale seeds cv. winterbor f1 (bejo, warmenhuizen, netherlands) per pot were sown with a sowing depth of 0.5 cm and placed after 3 days of vernalization at their destined location. after one week the plants were separated and the weaker of the two plants was removed. throughout the experiment plants were supplied with water according to their needs. applied water doses were recorded daily and plant highs in were documented weekly, using the highest part of the plant. local precipitation for outdoor grown plants was recorded with a precipitation gauge. plant development was evaluated according to the bbch scale (meier, 2018). for harvest, three times three plants were pooled and five youngest fully developed leaves were combined, resulting in three subsamples per treatment and a total of 18 samples of six locations. whole leaves were immediately frozen and transferred to freeze drying and further analysis. extraction of phenols the method for identification and quantitation of was based on a previously established method from neugart et al. (2014). in brief, 10 mg of above-mentioned freeze-dried samples were extracted in duplicate with 600 μl of 60% methanol (v/v) in a thermoshaker (eppendorf, hamburg, germany) at 1400 rpm and 20 °c for 40 min. subsequently, samples were centrifuged at 4500 rpm and 20 °c for 10 min. the supernatant was collected in a new reaction vessel and the pellet was redissolved in 300 μl 60% meoh and reextracted at 1400 rpm and 20 °c for 15 min, before centrifuging again. this procedure was repeated once, resulting in a total of three extraction steps. the combined supernatants were evaporated to total dryness in an rvc 2-25 cd plus vacuum centrifuge (christ, osterode am harz, germany). the residue was redissolved in 250 μl 10% methanol and clarified in spin-x/filter tubes with a 0.22 μm cellulose acetate membrane (corning costar spin-x, sigma aldrich, st. louis, mi, usa) at 3000 rpm and 20 °c for 5 min. the filtrate was used for subsequent hplc analyses and antioxidative assays. extraction of carotenoids for identification and quantitation of carotenoids from kale, 10 mg of above-mentioned freeze-dried sample was extracted in duplicate in 500 μl of a 1:1 methanol/tetrahydrofurane (thf) mixture (v/v). the sample was vortexed and mixed at 1400 rpm and 20 °c for 10 min. subsequently, samples were centrifuged at 4500 rpm and 20 °c for 5 minutes and the supernatant was collected. this procedure was repeated twice, resulting in a total of 1.5 ml extracted sample. all samples were evaporated to total dryness in above-mentioned vacuum evaporator. 100 μl of methyl tert-butyl ether (mtbe) was added to the dry residue to re-dissolve the sample. to each test tube addi tional 150 μl methanol (meoh 100%) was added, thus resulting in a total volume of 250 μl. subsequently, samples were passed through polytetrafluoroethylene filters with a pore size of 0.2 μm (chromafil xtra, macherey-nagel, düren, germany) and the filtrate was used for further hplc measurements and antioxidative assays. extraction of glucosinolates for the identification and quantitation of glucosinolates in kale a modified method according to grosser and van dam (2017) was applied in duplicate. 20 mg of above-mentioned freeze-dried sample was weighed in before adding 750 μl of 70% hot methanol (70 °c). half of the samples were also spiked with 100 μl of a sinigrin internal standard solution. differences between spiked and unspiked samples was used to determine internal standard area and unspiked samples were subsequently used for sinigrin determination, while other glucosinolates were analyzed in duplicate. samples were subsequently shaken at 1400 rpm in the abovementioned thermoshaker for 10 min. afterwards, the mixture was centrifuged at 4500 rpm for 5 min and the supernatant was collected, while the pallet was reextracted twice with 500 μl hot 70% methanol under the same conditions. for solid phase extraction (spe), syringes were filled with a thin layer of glass wool, to which 500 μl of a deae sephadex (cytiva, marlborough, ma, usa) suspension was pipetted. the spe column was pre-conditioned twice with 1 ml of imidazole solution and washed twice with 1 ml ultrapure water. the above-mentioned extract was then added to the column. afterwards, each test tube was rinsed twice with ultrapure water to ensure transfer of all residues. subsequently, absorber columns were rinsed twice with 1 ml of sodium acetate buffer (ph 4.3). 75 μl of purified arylsulfatase (ec 3.1.6.8) (merck, darmstadt, germany) was added and for at least 16 h, enzymatic desulphurisa tion was conducted. desulfoglucosinolates were eluted twice with 500 μl of ultrapure water and transferred to above-mentioned spin-x filters. these were centrifuged at 4000 rpm at 20 °c for two min before hplc measurement. antioxidant teac and dpph assays the antioxidant activity was either measured in phenolic or in carotenoid extracts. the trolox equivalent antioxidant capacity (teac) assay and well as the 2,2-diphenyl-1-picrylhydrazyl-hydrate (dpph) assay were performed based on a slightly modified 96 microwell plate method. both assays used individual calibration series of trolox on each plate. for the teac assay, 10 μl sample or trolox standard solution were mixed with 150 μl working solution in a 96 microwell plate and measured in a synergy htx microwell plate reader (biotek instruments, bad friedrichshall, germany) at a wavelength of 734 nm after six min. for the dpph assay 20 μl sample or trolox standard solution were mixed with 180 μl dpph solution. after incubation for 30 min under darkness plates were measured at a wavelength of 515 nm. influence of urban gardening on secondary plant metabolites 87 hplc measurement of phenols phenolic compounds including hydroxycinnamic acid derivatives and flavonoids glycosides were quantitated based on a method by neugart et al. (2014) using a shimadzu prominence hplc (shimadzu, kyoto, japan) equipped with a dgu-20a5 degasser, lc-20at pump, sil-20ac autosampler, cto-10as column oven, and spd-m20a photodiode array detector. an ascentis express f5 column (150 × 4.6 mm i.d., 5 μm particle size, merck, darmstadt, germany) protected by an ascentis f5 guard column (5 × 4.6 mm i.d., 5 μm particle size, merck) was used at 25 °c. eluent a was 0.5% acetic acid, and eluent b was 100% acetonitrile. the gradient (eluent b) was 5-12% (0-3 min), 12-25% (3-46 min), 25-90% (46-49.5 min), 90% isocratic (49.5-52 min), 90-5% (52-52.7 min), and 5% isocratic (52.7-59 min) at a flow rate of 0.85 ml * min-1. phenolic compounds were measured at wavelengths of, 320 nm for hydroxycinnamic acid derivatives, 330 nm for acylated flavonoid glycosides, and 370 nm for non-acylated flavonoid glycosides. for quantitation authentic standards of quercetin-3-glucoside, kaempferol-3-glucoside, and chlorogenic acid were used (roth, karlsruhe, germany). hplc measurement of carotenoids and chlorophylls carotenoids and chlorophylls were quantitated using the above mentioned shimadzu prominence hplc system with a c30 caro tenoid column (250 × 4.6 mm i.d.; 5 μm particle size) (ymc, kyoto, japan) protected by a ymc c30 guard cartridge (10 × 4.6 mm i.d.; 5 μm particle size) (ymc). eluents a and b consisted of methanol, methyl tert-butyl ether (mtbe), and water (80:18:2, v/v/v, eluent a; 8:90:2, v/v/v, eluent b (schex et al., 2018). the gradient used (eluent a) was 90-40% (0-30 min), 40-0% (30-35 min), isocratic at 0% (35-37 min), 0-90% (37-40 min), followed by an isocratic step at 90% (40-45 min) at a flow rate of 0.6 ml * min-1 at an oven temperature of 30 °c. carotenoids were detected at a wavelength of 454 nm and chlorophyll a and b were measured at 663 and 647 nm, respectively. furthermore, uv/vis spectra were recorded between 200 and 600 nm. for identification and quantitation authentic standards of β-carotene (roth), lutein, zeaxanthin (extrasynthese, lyon, france), chlorophyll a, and chlorophyll b (sigma-aldrich) were used. furthermore, spectra of carotenoids and chlorophylls were compared to those reported previously (schex et al., 2018). hplc measurement of glucosinolates glucosinolates were identified and quantitated based on a previously published method by grosser and van dam (2017), using refence factors and calculations, suggested by clarke (2010). for separation a jasco 4000 series hplc, equipped with a pu-4185 pump, as-4250 autosampler, uv-4070 uv/vis detector, and co-4060 column oven was used. a nucleodur 100 (125 × 2 mm i.d.; 5 μm particle size; macherey-nagel, düren, germany) column was used for separation. at a flow rate of 0.6 ml * min-1. eluents were ultrapure water (eluent a) and acetonitrile (eluent b). the applied gradient (eluent b) was 120% (0-20 min), isocratic 20% (20-25 min), 201% (25-27 min), and isocratic 1% (27-35 min). detection wavelength was 229 nm. for identification retention times were compared to those of authentic standards of sinigrin, progoitrin, glucobrassicanapin, and glucobrassicin (phytolab, vestenbergsgreuth, germany). statistical analysis statistical analysis of the results was performed using r statistical software, version 4.1.0. analysis of variance (anova) with post-hoc tukey-test was performed across all growth locations. all analytical measurements were conducted in duplicate. values are means ± standard deviation of three biological repetitions. for this investigation, a significance level of α ≤ 0.05 was applied. results temperature average morning temperatures measured inside and in eastern location were highest, reaching 20.0 to 20.7 °c due to indoor heat storage and early solar radiation exposure in the east. at noon temperatures were highest inside in southern and (28.9 °c) and eastern (26.6 °c) locations as well as southern location outside (23.7 °c), following the suns position. during evening hours location in southern (inside), northern, and western location were warmest, reaching 21.6 to 23.6 °c. changing sun exposure throughout the day lead to the described pattern and further, indoor locations favored heat storage. in addition to the daily documented temperature data, meteorological data from within 15 km of the experimental site was used for independent temperature data collection (appendix). overall temperature increased from an average of 12.9 °c in the first week to 19.6 °c in week 6 of the experiment. analogously, minimum temperature increased from only 3.5 °c to above 10 °c from week 4 onwards. hot days were absent during the experiment, reaching a maximum of 28.3 °c. water supply of the plants and plant growth water supply of plants was ensured by natural precipitation and additional watering according to necessity. particularly the first half of the growing season was dry, in total there were only 15.6 litres of precipitation per square meter in the first three weeks, which was less than observed at the nearby weather station (31.1 litres per m2). watering ranged from 30 ml to 175 ml per day and increased throughout the experiment. in southern located plants (outside) most supplemental water, accounting for a total of 1550 ml, was needed, followed by the southern indoor location with 1025 ml over the experimental period. the northern (405 ml) and western (455 ml) locations required the least amount of supplemental water. plants grown inside (southern and eastern location) were in average 83 and 12% higher than their outside grown counterparts (south and east, respectively) during the first two weeks of cultivation. during growth, differences between location vanished until week 4. however, at harvest shortest plants were observed in outside grown plants in the south while being uniform in between other locations (tab. 1). analogously, leaf development of the main shoot was ahead at early stages for indoor grown plants, as well as for plants grown in southern location. in the third week of the experiment, east (outside) and west showed a fully unfolded leaf on average (bbch-code: 11), while the inner sites already had two leaves (bbch-code: 12), and in southern location (outside) there was an average of 1.6 deciduous leaves. until the fifth week, plants grown outside in southern location were the most developed while plants grown in northern and eastern location (outside), which were lagging behind up to third week, have almost caught up. meanwhile, plants grown in western location still lagged behind in their development. at the end of the experiment all plants grown outside were comparably developed, ranging between an bbch index of 6.2 and 6.8, while indoor grown plants were most developed, reaching 7.2 and 7.8. phenolics kale samples contained a variety of different quercetin and kaempferol glycosides including acylated and non-acylated flavonoids. overall, the sum of flavonoid glycosides was lowest in plants grown inside (southern and eastern location) and in western location (outside), ranging from 28.0 ± 3.4 to 33.2 ± 7.1 mg per 100 g fresh weight (fw) (tab. 2). highest concentrations were found outside in eastern, and southern location, ranging between 265.6 ± 25.7 and 272.8 ± 65.4 mg per 100 g fw. highest concentrations were found of quercetin3,7,4’-o-triglucoside, kaempferol-3-o-caffeoyl-sophoroside-7-o88 m. bayer, s. neugart, t. pöhnl glucoside, and kaempferol-3-o-sinapoyl-sophoroside-7-o-glucoside in southern and eastern outside locations, respectively. noteworthy, quercetin-3-o-sinapoyl-sophoroside-7-o-glucoside concentrations were highest in northern location. furthermore, concentrations of the quercetin glycosides quercetin-3-o-caffeoyl-sophoroside-7-oglucoside, and quercetin-3-o-sophoroside-sinapoyl-7-o-diglucoside were highest in northern located plants. kaempferol-3-o-sinapoylsophoroside-7-o-diglucoside represented 8 to 10% of all flavonoids in locations with high total flavonoid concentrations, namely northern, eastern, and southern (outside) located plants, while their con centration was especially low in plants grown in all other locations. the lowest concentrations for almost all flavonoid glycosides are found in eastern inside (12% when being compared to the southern outside location) and southern inside (10% when being compared of the southern outside location) locations. in western located plants quercetin-3,7,4’-o-triglucoside represented a quarter of all flavonoids. hydroxycinnamic acids were comparably distributed to the flavonoid glycosides, also being lower in their total concentration, ranging between 12.3 ± 0.4 and 36.4 ± 5.4 mg per 100 g fw. highest concentrations were measured in northern, eastern (outside), and southern (outside) locations, while concentrations were lowest in eastern (inside), southern (inside), and western locations. neochlorogenic acid and disinapoyl-gentiobiose were highest in their concentration, accounting for approximately 65% of total hydroxycinnamic acids, regardless of their growth location. carotenoids and chlorophylls characteristic carotenoids of kale such as lutein, violaxanthin, neoxanthin, zeaxanthin, and β-carotene were found in samples gathered from all locations. total concentrations of carotenoids were highest in eastern and southern located samples, regardless of their position tab. 1: average height of kale plants in cm, grown between 31.05.2020 and 01.07.2020. different letters indicate significant differences of means according to tukey’s test (p ≤ 0.05). 05/31/2020 06/05/2020 06/11/2020 06/16/2020 06/22/2020 06/28/2020 07/01/2020 north (outside) 2.3 ± 0.2b 4.1 ± 0.2d 7.2 ± 1.3d 13.5 ± 1.3a 17.9 ± 1.9ab 23.4 ± 2.3ab 23.4 ± 1.6ab east (inside) 4.5 ± 0.8a 6.5 ± 0.6a 8.1 ± 1.7a 14.6 ± 0.9a 19.7 ± 1.7a 23.1 ± 2.0ab 28.3 ± 2.1ab east (outside) 2.4 ± 0.2b 4.3 ± 0.4d 7.9 ± 1.0d 14.9 ± 0.8a 18.8 ± 1.4a 22.5 ± 1.5ab 22.9 ± 2.2ab south (inside) 4.3 ± 0.5a 6.0 ± 1.1ab 8.3 ± 1.7ab 14.7 ± 1.9a 18.3 ± 2.2a 20.7 ± 2.0b 27.8 ± 4.1a south (outside) 2.4 ± 0.2b 5.3 ± 0.6bc 8.1 ± 1.1bc 15.3 ± 1.7a 18.9 ± 2a 25.4 ± 3.5a 20.8 ± 1.4b west (outside) 2.5 ± 0.2b 4.5 ± 0.5cd 6.2 ± 1.4cd 14.4 ± 1.4a 15.7 ± 1.7b 24.0 ± 2.8a 25.0 ± 2.8a tab. 2: flavonoid glucosides [mg * 100 g-1 fw] in kale leaves harvested after 7 weeks of growth under urban gardening conditions in northern, eastern, western, and southern locations, including 2 indoor locations (east and south). different letters indicate significant differences of means according to tukey’s test (p ≤ 0.05). north east east south south west (outside) (outside) (inside) (outside) (inside) (outside) k-3-o-cou-soph-7-o-glc 11.6 ± 2.5ab 14.8 ± 1.3a 2.0 ± 0.3c 14.5 ± 7.0a 1.9 ± 0.1c 4.9 ± 0.6bc k-3-o-caf-soph-7-o-glc 35.2 ± 3.1a 38.0 ± 5.3a 3.3 ± 0.5b 27.8 ± 11.0a 3.8 ± 0.9b 10.8 ± 3.9b k-3-o-caf-soph-7-o-diglc 1.7 ± 1.2a 0.0 ± 0.0b 0.2 ± 0.0ab 0.8 ± 0.7ab 0.2 ± 0.0ab 0.3 ± 0.2ab q-3-o-fer-soph-7-o-glc 1.0 ± 0.2a 1.0 ± 0.3a 0.2 ± 0.1b 1.2 ± 0.5a 0.2 ± 0.1b 0.3 ± 0.0b k-3-o-fer-soph-7-o-glc 9.4 ± 2.6ab 15.8 ± 4.2a 2.1 ± 0.5b 15.1 ± 7.8a 1.9 ± 0.1b 2.2 ± 1.1b k-3-o-fer-soph-7-o-diglc 13.2 ± 1.9ab 20.6 ± 1.8a 3.7 ± 1.6b 25.8 ± 6.5a 2.5 ± 1.3b 6.5 ± 0.8b k-3-o-hfer-soph-7-o-glc 10.8 ± 1.0a 12.5 ± 1.1a 2.1 ± 0.2b 11.8 ± 2.8a 2.3 ± 0.1b 4.9 ± 1.4b k-3-o-hfer-soph-7-o-diglc 4.1 ± 0.4c 10.7 ± 1.5b 0.6 ± 0.3c 17.3 ± 3.7a 0.5 ± 0.4c 1.6 ± 0.2c q-3-o-sin-soph-7-o-glc 7.7 ± 2.6a 1.8 ± 0.7b 0.7 ± 0.1b 0.0 ± 0.0b 0.7 ± 0.0b 0.7 ± 0.2b k-3-o-sin-soph-7-o-glc 9.8 ± 2.8b 25.7 ± 4.9a 1.0 ± 0.3b 35.7 ± 8.0a 0.8 ± 0.3b 2.6 ± 0.3b k-3-o-sin-soph-7-o-diglc 14.2 ± 4.7b 26.8 ± 3.6a 0.6 ± 0.1c 28.0 ± 7.2a 0.7 ± 0.2c 3.2 ± 0.7c q-3-o-soph-sin-7-o-diglc 17.9 ± 4.3a 14.9 ± 5.4ab 4.2 ± 1.6b 10.4 ± 8.2ab 3.1 ± 1.0b 2.8 ± 1.3b q-3,7-di-o-glc 9.5 ± 1.7a 7.9 ± 3.0ab 3.4 ± 1.2ab 5.3 ± 5.3ab 2.2 ± 0.4b 3.2 ± 0.1ab k-3-o-soph-7-o-glc 8.9 ± 1.2c 16.4 ± 1.2b 1.3 ± 0.2d 29.0 ± 3.4a 1.2 ± 0.2d 3.1 ± 1.1d q-3-o-soph-7-o-glc 6.0 ± 1.3b 15.5 ± 1.7a 0.5 ± 0.1b 19.1 ± 5.5a 0.5 ± 0.1b 1.3 ± 0.2b q-3,7,4‘-triglc 20.2 ± 2.3bc 39.7 ± 4.0a 6.7 ± 1.2cd 27.6 ± 11.9ab 5.0 ± 0.8d 17.6 ± 3.8bcd i-glc 2.6 ± 0.4b 3.6 ± 1.1a 0.6 ± 0.1b 3.5 ± 11.7a 0.6 ± 0.1b 0.8 ± 0.1b total flavonoids 183.7 ± 30.7b 265.6 ± 25.7ab 33.2 ± 7.1c 272.8 ± 65.4a 28.0 ± 3.4c 66.6 ± 12.8c *abbreviations: q: quercetin, k: kaempferol, i: isorhamnetin, cou: coumaroyl, caf: caffeoyl, fer: feruloyl, hfer: hydroxyferuloyl, sin: sinapoyl, glc: glucoside, soph: sophoroside, n.d.: not detectable. influence of urban gardening on secondary plant metabolites 89 tab. 3: concentrations of hydroxycinnamic acids [mg * 100 g-1 fw] in kale samples harvested after 7 weeks of growth under urban gardening conditions in northern, eastern, western, and southern locations, including 2 indoor locations (east and south). different letters indicate significant differences of means according to tukey’s test (p ≤ 0.05). north east east south south west (outside) (outside) (inside) (outside) (inside) (outside) neochlorogenic acid 13.9 ± 1.2ab 17.4 ± 3.6a 5.8 ± 0.8c 16.3 ± 5.8a 4.5 ± 0.2c 6.3 ± 0.9bc disinapoyl-gentiobiose 6.3 ± 0.7ab 8.3 ± 0.8a 4.8 ± 0.7bc 6.6 ± 2.1ab 3.4 ± 0.3c 2.9 ± 0.5c sinapoylferuloyl-gentiobiose 2.4 ± 0.6ab 2.6 ± 0.1a 2.1 ± 0.2ab 2.9 ± 0.9a 1.3 ± 0.1b 1.3 ± 0.1b trisinapoyl-gentiobiose 3.9 ± 0.6ab 4.6 ± 0.5a 2.4 ± 0.2bc 4.7 ± 1.4a 1.9 ± 0.1c 2.0 ± 0.3c disinapoylferuloyl-gentiobiose 2.5 ± 0.3ab 3.5 ± 0.9a 1.6 ± 0.1bc 3.4 ± 0.6a 1.2 ± 0.1c 1.4 ± 0.1bc total hydroxycinnamic acid glycosides 29.2 ± 3.2ab 36.4 ± 5.4a 16.6 ± 1.9bc 33.9 ± 10.3a 12.3 ± 0.4c 14.0 ± 1.9c tab. 4: concentration of carotenoids and chlorophyll a and b [mg * 100 g-1 fw] in kale samples harvested after 7 weeks of growth under urban gardening conditions in northern, southern, western, and eastern locations, including 2 indoor locations (east and south). different letters indicate significant differences of means according to tukey’s test (p ≤ 0.05). north east east south south west (outside) (outside) (inside) (outside) (inside) (outside) lutein 3.8 ± 0.1a 4.8 ± 0.5a 4.8 ± 0.5a 4.6 ± 0.2a 4.4 ± 0.3a 3.9 ± 0.7a violaxanthin 0.7 ± 0.3a 0.6 ± 0.0a 0.7 ± 0.1a 0.5 ± 0.1a 0.7 ± 0.2a 0.5 ± 0.1a neoxanthin 0.5 ± 0.2c 0.4 ± 0.1c 2.8 ± 0.6a 0.5 ± 0.1c 2.3 ± 1.1ab 0.8 ± 0.5bc (all-e)-β-carotene 1.0 ± 0.1a 1.4 ± 0.1a 1.4 ± 0.3a 1.3 ± 0.1a 1.2 ± 0.1a 1.1 ± 0.2a (9z)-β-carotene 0.3 ± 0.2a 0.3 ± 0.0a 0.2 ± 0.0a 0.3 ± 0.0a 0.2 ± 0.0a 0.4 ± 0.4a zeaxanthin 0.1 ± 0.0abc 0.2 ± 0.0ab 0.0 ± 0.0c 0.3 ± 0.0a 0.1 ± 0.1bc 0.1 ± 0.0bc total carotenoids 6.4 ± 0.4c 7.6 ± 0.7abc 9.8 ± 0.3a 7.5 ± 0.3abc 8.9 ± 1.1ab 6.8 ± 1.6bc chlorophyll a] 26.0 ± 1.6a 33.3 ± 2.2a 41.4 ± 17.7a 28.6 ± 6.0a 34.6 ± 14a 48.7 ± 2.9a chlorophyll b 14.9 ± 0.5ab 16.3 ± 1.1a 9.8 ± 2.8b 15.1 ± 1.2ab 10.0 ± 2.9b 11.5 ± 2.1ab total chlorophylls 40.9 ± 1.9a 49.6 ± 3.3a 51.2 ± 20.5a 43.7 ± 7.2a 44.6 ± 16.3a 60.2 ± 3.9a tab. 5: concentration of glucosinolates progoitrin, epiprogiotrin, sinigrin, glucobrassicanapin, glucobrassicin, and total glucosinolates [mg * 100 g-1 fw] in kale samples harvested after 7 weeks of growth under urban gardening conditions in northern, southern, western, and eastern locations, including 2 indoor locations (east and south). different letters indicate significant differences of means according to tukey’s test (p ≤ 0.05). north east east south south west (outside) (outside) (inside) (outside) (inside) (outside) progiotrin 0.6 ± 0.3ab 0.5 ± 0.2ab 0.2 ± 0.1ab 0.6 ± 0.1a 0.1 ± 0.0b 0.4 ± 0.2ab epiprogiotrin 0.8 ± 0.2a 0.3 ± 0.0bc 0.2 ± 0.1c 0.6 ± 0.1ab 0.3 ± 0.0bc 0.2 ± 0.2c sinigrin 3.8 ± 0.8bc 5.3 ± 0.6ab 1.5 ± 0.7d 5.9 ± 0.9a 2.6 ± 0.6cd 2.7 ± 0.8cd glucobrassicanapin 0.5 ± 0.4b 0.5 ± 0.1b 0.3 ± 0.0b 2.3 ± 0.7a 0.3 ± 0.1b 1.1 ± 0.2b glucobrassicin 11.7 ± 3.2b 8.9 ± 2.1b 5.8 ± 0.6b 34.5 ± 10.6a 4.9 ± 2.4b 17.6 ± 3.0b total glucosinolates 17.5 ± 4.5b 15.5 ± 1.9b 8.0 ± 0.8b 43.9 ± 11.6a 8.1 ± 3.2b 21.9 ± 2.4b tab. 6: antioxidant activity in phenolic and carotenoid extracts [mmol trolox equivalent * 100 g-1 fw] in kale samples harvested after 7 weeks of growth under urban different letters indicate significant differences of means according to tukey’s test (p ≤ 0.05). north east east south south west (outside) (outside) (inside) (outside) (inside) (outside) dpph phenolics 3.7 ± 0.8b 4.2 ± 0.2ab 1.0 ± 0.2c 4.9 ± 0.3a 0.8 ± 0.2c 1.2 ± 0.2c teac phenolics 6.0 ± 0.6c 8.0 ± 0.4b 2.0 ± 0.4d 9.3 ± 0.4a 1.8 ± 0.4d 2.9 ± 0.2d dpph carotenoids 6.1 ± 1.1bc 8.7 ± 1.7ab 3.9 ± 0.4c 10.6 ± 2.3a 2.4 ± 0.9c 3.4 ± 1.1c teac carotenoids 8.9 ± 0.7bc 10.8 ± 1.9ab 4.7 ± 0.6d 13.3 ± 2.2a 5.1 ± 1.0d 6.1 ± 0.2cd inside or outside, ranging between 7.5 ± 0.3 and 9.8 ± 0.3 mg per 100 g fw. neoxanthin concentration was strongly increased to 2.8 ± 0.6 and 2.3 ± 1.1 mg per 100 g fw at both inside locations in southern and eastern location, compared to those concentrations measured outside in northern, eastern, and southern locations. zeaxanthin was present only in small concentrations and was only found at minimum concentrations under indoor conditions. lutein was found at high concentrations in all locations, ranging from 3.8 ± 0.1 to 4.8 ± 0.5 mg per 100 g fw. β-carotene concentration was between 1.0 ± 0.1 and 1.4 ± 0.3 mg per 100 g fw in all samples. 90 m. bayer, s. neugart, t. pöhnl total chlorophyll concentrations did not differ between any of the chosen locations ranging between 40.0 ± 1.9 and 60.2 ± 3.9 mg per 100 g fw. furthermore, concentrations of chlorophyll a were not different, but tended to be elevated inside (east and south) and western locations. however, the ratio of chlorophyll a to chlorophyll b was highest in inside locations (east and south) and in western location, being in clear contrast to the other three locations (south, north, and east). glucosinolates glucosinolates progiotrin, epiprogoitrin, sinigrin, glucobrassica napin, and glucobrassicin were found in all kale samples of our study. highest overall concentrations of 43.9 ± 11.6 mg per 100 g fw were found in samples grown in southern (outside) location, reaching twofold the concentration of glucosinolates in the location with the second highest concentrations (west; 21.9 ± 2.4 mg per 100 g fw). concentrations were highest for glucobrassicin and sinigrin, comprising for approximately 90% of all found glucosinolates, regardless of their location. glucobrassicin concentration was highest in southern outside location, reaching 34.5 ± 10.6 mg per 100 g fw, while other locations were between 4.9 ± 2.4 and 17.6 ± 3.0 mg per 100 g fw. noteworthy, all five reported glucosinolates were highest in southern (outside) location and the surplus in comparison to other locations was especially pronounced for glucobrassicin (148% higher than average). furthermore, kale grown indoors tended to have the lowest glucosinolate concentration, also not being significantly lower than other outdoor grown kale samples (northern, eastern, and western locations). antioxidant activity antioxidant activity, measured by the dpph assay in methanolic extracts for determination of phenolic compounds was highest for samples grown at outside locations oriented towards the south and east, reaching 4.9 ± 0.3 and 4.2 ± 0.2 mmol trolox equivalent (te) per 100 g fw. meanwhile, antioxidant activity was between 0.8 ± 0.2 and 3.7 ± 0.8 mmol te per 100 g fw in indoor locations (south and east), as well as in western located plants. differences reached a 4 to 5-fold difference between highest and lowest recorded concentrations. analogously, teac assay was highest in southern located and outside grown plants (9.3 ± 0.4 mmol te per 100 g fw), while plants grown inside and in western location were lowest (1.8 ± 0.4 to 2.9 ± 0.2 mmol te per 100 g fw), also reaching a 5-fold difference between the extrema. in lipophilic extracts being used for the determination of carotenoids and chlorophylls highest antioxidant activity was found in southern and eastern located plants, reaching 8.7 ± 1.7 to 10.6 ± 2.3 mmol te per 100 g fw in the dpph assay and 10.8 ± 1.9 to 13.3 ± 2.2 mmol te per 100 g in the teac assy. meanwhile, lowest antioxidant activity was found at locations inside in both antioxidant assays. discussion growth conditions and adaption growth conditions were sufficient to grow kale at all urban gardening locations that were part of our investigation. non-surprisingly, indoor cultivated plants had a clear advantage during early stages of growth when outside temperatures were still below 10 °c during night-time. regarding overall plant height there was no difference after 7 weeks of cultivation, suggesting that all applied conditions are suitable to grow kale in urban gardening. kale is a known frost tolerant vegetable (heyduck et al., 2020) and therefore not prone to cold night-time temperatures, as present in spring and early summer. furthermore, kale is known to grow also under unfavourable light conditions. these attributes make kale a typical german winter vegetable that is also suitable for cultivation in northern locations with extensive shading, as being part of our study, or late autumn and winter harvests. the relatively short growth period of 7 weeks resulted in substantial leaf material for consumption, especially considering trending uses as baby leaf salad and is in agreement with previous reports (heyduck et al., 2020). however, while kale cultivation is stagnating in germany, the us marked saw a boost in kale growth within the last decade (kirby and granatstein, 2018; statistisches bundesamt, 2021). during our study irrigation, but no fertilization, was applied to provide optimum growth condition as it is achievable in residential urban gardening. however, highest water consumption in southern outside location clearly shows that a sufficient supply with water must be provided to heat exposed plants as water reservoirs on balconies are limited. although short-term drought stress in kale resulted in an increase in phenolics and glucosinolates (barickman et al., 2020), this was not the subject of this research. phenolic compounds in kale highest concentrations of total phenolic compounds were found in our southern located cultivation side due to most extensive sun exposure (majer et al., 2014). however, plants grown in eastern and, most surprising, northern location were almost equally rich in phenolic compounds. meanwhile, plants grown indoor behind glass protection or in the shaded western location side were drastically lower in their phenolic content (flavonoids and hydroxycinnamic acids), containing only 14 to 17% of the maximum concentration found in southern located plants. comparable observations were made with the same cultivar under artificial light conditions, where lowest par light intensities did not reduce kaempferol-o-hydroxyferuloyl-sophorosideo-glucoside concentrations, while highest concentrations under poor light conditions were observed (neugart et al., 2013). noteworthy, our concentrations were approx. 50% of previously reported concentrations. concentrations of other flavonoids were also comparable to those reported in this study, also variations due to our different locations were much more pronounced than those based solely on par light differences between 200 and 800 mmol m-2 s-1 the accumulation of phenolic compounds is known to be largely light dependent. especially uv radiation exposure leads to the increase of phenolic compounds in kale (neugart et al., 2014; neugart et al., 2016). window glass is blocking a substantial share of uv radiation and especially uvb radiation, responsible for the upregulation of phenol biosynthesis (neugart et al., 2014). thus, the growth outside and at sun exposed locations has a clear advantage regarding the dietary phenol intake and possible health benefits thereof. the distribution of flavonoid glycosides in shaded locations with a known low concentration in total phenolics favored quercetin glycosides which represented 42 to 47% of all flavonoid glycosides, while in sun exposed locations their share decreased to 23 to 39% and a higher share of acylated kaempferol glycosides. the shift of mono-hydroxylated to di-hydroxylated b-ring compounds or increase of acylation is a well-known response to uv radiation (agati et al., 2011). for human consumption and urban gardeners the higher share of quercetin glycosides may result in a higher antioxidant activity and better radical oxygen species (ros) scavenging potential (fiol et al., 2012). nevertheless, kaempferol glycosides contribute to the antioxidant activity in kale, dependent on the acylated hydroxycinnamic acid (fiol et al., 2012). consequently, kaempferol-3-o-sinapoyl-sophoroside-7-o-diglucoside, and kaempferol-3-o-caffeoyl-sophoroside7-o-glucoside were drastically elevated under full sunlight in the southern outside location. however, in contrast to previous findings we could not find a decrease in kaempferol glycosides but a more pronounced increase in quercetin glycosides (neugart et al., 2016). both kaempferol and quercetin are abundantly available in the influence of urban gardening on secondary plant metabolites 91 human diet and known for their health beneficial effects (dabeek and marra, 2019; pan et al., 2010). one of the characteristics of urban gardening is shading due to buildings or other plants comparable to a canopy (martínez-lüscher et al., 2019). consequently, phenolic concentration and composition could be affected by different shading conditions in urban gardeing. carotenoids kale (cv. ‘winterbor’) was found to be an excellent source for lutein also concentrations were 50% lower than reported by becerramoreno et al. (2014). concentrations of carotenoids β-carotene and violaxanthin were also 20 to 50% lower than reported in the same study. those differences can be due to the young age of our leaves. the concentration of carotenoids depended on the location of the cultivation site of kale plants and was not influenced by indoor or outdoor cultivation except for neoxanthin. locations with limited sun light availability, i.e. northern and western located plants, were lowest in total carotenoids. previous studies showed that longer photoperiods increase lutein and β-carotene concentrations and red light (640 nm) stimulates the accumulation of lutein in kale (lefsrud et al., 2008). concentrations of lutein were independent of their growth location during our study. in agreement to our results lefsrud et al. (2006) found no influence on lutein, β-carotene, and chlorophyll a concentrations at low irradiance doses in growth chambers between 125 and 460 μmol * m-2 * s-1. irradiance doses of 620 μmol * m-2 * s-1 resulted in slightly higher concentrations of lutein, β-carotene, and chlorophyll a. in urban gardening shading depends on the actual growth location and time of year, which are unchangeable factors. nevertheless, extended exposure to sun light might be beneficial to achieve higher carotenoid concentrations but effects are limited and also shaded plants provide a considerable supply of health beneficial carotenoids. this observation is especially interesting for consumers as lutein is of great nutritional value due to its antioxidant activity, eye protective potential against uv radiation and its contribution to brain development (ochoa becerra et al., 2020). due to the limited stability of lutein its application in the food industry is limited and fresh sources are valuable for consumers. noteworthy, neoxanthin concentrations were highest in indoor grown plants. even though the actual function of neoxanthin is still disputed (giossi et al., 2020) our results indicate that it has a role in the uv response in outdoor conditions but not in indoor conditions. glucosinolates glucosinolates were not affected by growth conditions, except for 50% higher concentration in southern located plants. those elevated glucosinolate concentrations can be caused by abiotic stresses, such as drought (schreiner et al., 2009) or elevated temperature (bohinc and trdan, 2012). mild drought events in our southern locations might be evident as sun exposure and water consumption were highest. furthermore, the strongly elevated concentrations of indolic glucobrassicin might be a result of high temperatures as shown by bohinc and trdan (2012). composition of glucosinolates in all samples was dominated by glucobrassicin and sinigrin that were previously found in substantial amounts in kale by hahn et al. (2016) and kusznierewicz et al. (2013). highest concentrations of glucobrassicin and substantial concentrations of sinigrin are characteristic for cv. ‘winterbor’ under sufficient sulfur supply (kopsell et al., 2003). furthermore, total glucosinolates, sinigrin, and glucobrassicin ranked lowest when being grown indoor, indicating that light conditions might influence their accumulation and consequently taste. modification of aforementioned glucosinolates has recently been achieved by white, blue, and red leds in brassica juncea sprouts (park et al., 2020). for consumers and urban gardeners with might be of particular interest since glucosinolates do contribute to healthy secondary plant metabolites in kale (miękus et al., 2020) but also have characteristic pungent flavor that can lead to rejection, especially by children. based on our results there is no clear explanation for the strongly elevated concentrations in southern located plants, but overall results suggest that all chosen locations were suitable for urban gardening and no location is favorable, when aiming for especially mild kale leaves, also indoor cultivation might be favorable with lowest overall concentrations. antioxidant activity due to the large contribution of phenolics to the antioxidant activi ty their occurrence explains the high overall antioxidant activity of samples grown in southern and eastern located plants. overall concentrations of phenolic compounds showed a clear differentiation between highly sun exposed locations without reduction of par or uv radiation provided by glass windows. such an effect was previously found in linden leaves where sun exposed leaves had a higher singlet oxygen neutralizing capacity compared to shade leaves (majer et al., 2014) and is known in many other species (shahidi and ambigaipalan, 2015). concentrations in carotenoids, including β-carotene and lutein, did not differ to the same extent. noteworthy, concentrations of phenolic compounds were surprisingly high in our samples grown in northern located with no direct sunlight but were lowest in their carotenoid concentration. western located plants were lowest in their content of phenolic compounds as well as carotenoids. nevertheless, carotenoids contribute to the antioxidant activity in the plants and humans even though their bioavailability follows a different mechanism and requires some fat as vehicle. conclusion cultivation of kale was feasible under all applied conditions, having no significant reduction in growth at locations with expected lower light intensities. regarding the nutritional value it can be concluded that all samples contain healthy secondary plant metabolites, including phenolics, glucosinolates, and carotenoids. kale is known for its, in general, high carotenoid concentration and therefore, its beneficial effects on human vision. especially when aiming at lutein supply the location of cultivation site was wildly irrelevant while phenolic compounds, rather known for their general antioxidant and anti-inflammatory effects, are triggered by direct sunlight. glucosinolates known for their anticarcinogenic effects, but also a pungent cabbagelike aroma, are low in kale and mainly not affected by the growing conditions in urban farming. however, indoor farming can presumably shield insects and light and preventing intensive glucosinolate accumulation. in summary, urban gardening offers a great way to provide consumers with fresh and healthy, fast-growing vegetables, especially for use as baby leaf salads. acknowledgements eike stefan dobers (university of applied sciences neubrandenburg) provided us with meteorological data from trollenhagen. further more, we thank ulf jaeger and christian petri for technical support with the secondary metabolite analysis and layla engelhardt for support with our antioxidant assays. conflict of interest no potential conflict of interest was reported by the authors. note by the editor this publication is part of the 2022 special section “applied 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http://dx.doi.org/10.3390/horticulturae6040077 http://dx.doi.org/10.3390/su12125012 http://dx.doi.org/10.1016/j.foodres.2017.11.041 http://dx.doi.org/10.1016/j.foodchem.2009.09.004 http://dx.doi.org/10.1021/jf901076h http://dx.doi.org/10.1016/j.jff.2015.06.018 http://dx.doi.org/10.1289/ehp.1408216 https://orcid.org/0000-0002-4574-6733 https://orcid.org/0000-0003-4314-6926 i supplementary material fig. 2: exemplary chromatograms of northern located kale (brassica olera cea var. sabellica l.) samples, gathered after 6 weeks of growth. a: phenolic compounds measured at 330 nm (black line) and 370 nm (grey line); 1: neochlorogenic acid, 2: q-3,7,4’-triglc, 3: k-3-osoph-7-o-glc, 4: q-3-o-soph-7-o-glc, 5: k-3-o-hfer-soph-7-oglc, 6: k-3-o-hfer-soph-7-o-diglc; 6*: q-3,7-di-o-glc (shoulder peak at low concentration) 7: k-3-o-caf-soph-7-o-glc 8: q-3-osin-soph-7-o-glc, 8*: k-3-o-caf-soph-7-o-diglc (shoulder peak at low concentration), 9: q-3-o-soph-sin-7-o-diglc, 10: k-3-o-sinsoph-7-o-glc, 10*: q-3-o-fer-soph-7-o-glc (small shoulder peak), 11: k-3-o-sin-soph-7-o-diglc, 12: k-3-o-fer-soph-7-o-glc, 13: k-3-o-fer-soph-7-o-diglc, 14: k-3-o-cou-soph-7-o-glc, 15: i-glc, 16: disinapoyl-gentiobiose, 17: sinapoylferuloyl-gentiobiose, 18: trisinapoyl-gentiobiose, 19: disinapoylferuloyl-gentiobiose; b: carotenoids and chlorophylls measured at 450 nm (grey line) and 660 nm (black line). abbreviations referrer to footnotes explained in tab. 2.; 1: violaxanthin, 2: neoxanthin, 3: chlorophyll b, 4: lutein, 5: zeaxanthin, 6: chlorophyll b, 7: (all-e)-β-carotene, 8: (9z)-βcarotene. c: glucosinolates measured at 259 nm: 1: progoitin, 2: epirogoitin, 3: sinigrin, 4: glucobrassicanapin, 5: glucobrassicin. appendix fig. 1: minimum, maximum and mean temperature measured at the trollenhagen weather station (university of applied sciences neubrandenburg) during the experiment. journal of applied botany and food quality 92, 193 198 (2019), doi:10.5073/jabfq.2019.092.027 1julius kühn institute, federal research centre for cultivated plants, institute for ecological chemistry, plant analysis and stored product protection, berlin, germany 2julius kühn institute, federal research centre for cultivated plants, information centre and library, quedlinburg, germany otto appel and his contributions to food quality and safety at the beginning of the 20th century hartwig schulz1*, heike riegler2 (submitted: june 18, 2019; accepted: july 11, 2019) summary otto appel (1867-1952) is best known for his research in the field of phytopathology, in particular for his discoveries on bacterial and fungal diseases of crops such as potato and cereals. his work ranged from fundamental research, like the discovery and description of pathogens and their ways to affect their host plants, to applied research on cultivation practices and storage methods. he published numerous scientific articles as well as practical recommendations for farmers in form of flyers and pocket books with the aim to improve yield and quality and to reduce losses and, thus, securing the supply with plant based food and materials. his commitment to applied research was also reflected in his long-term membership in the board of the association for applied botany and later on in the awarding of honorary presidency of the association. as director of the federal biological research centre for agriculture and forestry in berlin, he was the key player in setting up an efficient and well-organized plant protection service in germany. otto appel’s achievements significantly influenced agricultural practices and generally enhanced food quality and safety in germany and beyond. they are still remembered today, when the deutsche phytomedizinische gesellschaft awards the otto appel medal to outstanding researchers in phytomedicine every two years. early years being born on may 19th, 1867 in coburg, germany, into a merchant family, friedrich carl lois otto appel developed a strong interest in botany and plant systematics in his youth. intended to pursue a career with a scientific background, he became a pharmacy assistant. in his free time, he continued to collect and describe the morphology of the species carex from the cyperaceae family, which led to his first scientific publication of caricological notes from the hercynian territory (appel, 1890). he was involved with several learned societies such as the geographical society of thuringia to jena, the botanical association of entire thuringia, the botanical society of switzerland and the botanical association of copenhagen. after his university studies in pharmacy in breslau, he took over a pharmacy but continued his botanical studies on the side. further publications and talks on cyperaceae and the flora of the black forest and the swabian alb (appel, 1891, 1893) gained him the attention of professor dr. julius sachs, a pioneer in experimental plant physio logy, which eventually led to otto appel pursuing a doctorate. in his doctoral thesis “über phytound zoomorphosen (pflanzengallen)” (appel, 1899) he examined both the causes of plant galls and their origin in various parts of the plant. in the following two years, he deepened his knowledge in bacterio logy at the institute for hygiene and bacteriology of the university of würzburg and as assistant at the bacteriological institute of the agricultural institute of the albertus university of königsberg. in 1899, at the age of 32, appel followed the call of prof. carl von tubeuf to the newly established biological department of agriculture and forestry of the imperial health department in berlin. discovering the connection of potato diseases in the field and potato storage in berlin, appel investigated fungal and bacterial infections of potato and cereals, which were the most important crops for people’s nutrition at that time. compared to cereals, potatoes produced nearly the same amount of protein and even the double content of carbohydrates per unit area. thus, he focused on potatoes first, where in his opinion, the greatest losses occurred and the fastest successes could be expected. in 1899, appel started with his studies to optimize the storage of potatoes at the field station in berlin-dahlem, which resulted in a detailed recommendation on the construction of potato clamps (appel, 1902a). from his experiments, he concluded that many of the diseases had their origin in the field. he stated: in these investigations, as well as in a series of occasional observations, i became convinced that the diseases to be observed in the preservation of potatoes are at least partly related to the symptoms of the disease, which also occur during the growing season... (translated from german original appel, 1903). it quickly became clear that he had to examine the whole cycle of the pathogen carefully, starting from storage to the subsequent culture, in order to prevent and interrupt further spreading of the disease. in 1901, appel identified bacillus phytophthorus (= pectobacterium atrosepticum) in potatoes and, two years later, provided a detailed description: the bacillus phytophthorus is a fairly thick, gram-negative rod. its length is very variable; in rotting potatoes and also in the diseased tissue of the plant it occurs predominantly as a very short rod with an approximate length of 1.2 1.5 μ. ...mobility is extraordinarily large, especially in young cul tures (translated from german original, appel, 1903). appel noted that, in the case of blackleg potato disease, the infection always emanated from an affected tuber, and the bacteria spread through the conducting vessels into other plant tissues (fig. 1). this was confirmed, when spraying the foliage several times with a bac terial suspension did not lead to an infection. at the experimental field in berlin-dahlem, appel planted pre-infected tubers of the varieties cv. ‘white rose’ and ‘dabersche’ to further analyse transmission of the bacterial infection. at the time of harvest, crop failure by blackleg disease in cv ‘white rose’ amounted to 62.11%, but in uninfected tubers it was only 4.29%. in contrast to that, cv. ‘dabersche’ showed a rapid wound healing after infection treatment and, as a consequence, suppression of the infection. losses due to blackleg disease in the pre-infected culture amounted to only 9.4% for this variety. according to the observation of otto appel, potatoes with a thick skin, higher starch content, and generally varieties with a late harvest date were more resistant than the others were. his studies corroborate impressively how important the choice of the right variety is in the fight against blackleg potato disease and how signi ficantly resistance breeding can support the control of this pest. in further field trials, appel also proved that infection of potato plants via the soil is possible. as a consequence, he recommended that potatoes should not be cultivated on polluted areas to avoid accumulation of the pathogen in the soil (appel, 1903). journal of applied botany 194 h. schulz, h. riegler appel’s findings on blackleg disease have been summarized as follows in a yearly report on advances in phytomedicine (hollrung, 1903): • it is not possible to combat blackleg in the field. • for cultivation of potatoes, avoid fields where, one or two years before, a disease had appeared, as well as those on which beans, lupins, carrots, teltow turnips or cucumbers were infected by the same pest. • do not use sliced potato tubers for planting, just whole tubers. • avoid strong nitrogen fertilization. • an infection can be prevented by using new, healthy seeds. variable as was previously thought, and that it is quite possible to distinguish the individual species sufficiently, without placing particular emphasis on the very uncertain feature of the substrate (paraphrased from german original: appel and wollenweber, 1910). when searching for specific media for the differentiation of the broadest possible spectrum of fusarium, appel and wollenweber (1910) tested, among others: stems of potato (old mature stems), pea, apple (two-year shoots), rye, lupine, bean (vicia faba), fruits of apple, pear, melon, banana, potato tubers, carrot roots, rye seeds and leaves of peas, potatoes and apple (fig. 3). artificial media based on nutrient salts and defined carbohydrate and protein sources have been investigated, too. on these standardized nutrient media, appel and wollenweber cultivated the fusaria they had, described their properties and rearranged them. furthermore, they assigned many hithero unknown pathogenic fungi (tab. 1). they summarized the most important results in the standard work “grundlagen einer monographie der gattung fusarium (link)” (= fundamentals of a monograph of the genus fusarium (link)) as follows (translated from german original appel and wollenweber, 1910): • it has been proven that a better differentiation of fusarium species is possible by using features that have not been previously applied or not sufficiently appreciated. • a culture method was found for the cultivation of normal conidia. the criteria of the normal concept in conidia were derived from form, construction, colour, changes in content, and age of the individual conidia. • as a result, cooked vegetables were able to produce normal forms of fusarium species. • the conidia were mainly detected on stems, but several exceptions occurred. • for fruit types, conidia and chlamydospores were found in most species, the latter not necessarily, the former always in addition to numerous fusarium species, otto appel also described various other phytopathogenic fungi over the years (tab. 1). among them are several species obtained from the former german colonies cameroon and samoa, especially those collected from cocoa (appel and strunk, 1904; appel and laubert, 1906). he also collected infestation symptoms of various pathogens, preserved them in formalin, and thus created an extensive wet collection of phytopathological features for research and teaching purposes (fig. 4). this collection still exists today at the julius kühn institute, the successor institution of the biological department of agriculture and forestry, and a digital catalog can be found at https://phytopath-sammlung. julius-kuehn.de/. fig. 1: blackleg disease on potato. left: sprouting potato with symptoms of blackleg disease after cultivation in infected soil, specimen collected by otto appel and preserved in formalin (phytopathological collection of the julius kühn institute; image by manfred gräfe und djavad taghizadhe, stadtmuseum berlin). right: illustration in the pocket atlas of potato diseases, drawn by august dressel (appel, 1926). investigations on fusarium until the mid-19th century, phytopathogenic fungi were not regarded as the cause of plant diseases, but as their consequence. this view was corrected by anton de bary, who showed that it is not the plant that produces the fungus, but that the fungus infests the plant and causes the disease (de bary, 1861). julius kühn and other researchers came to a similar conclusion almost at the same time. the fact that pests were now accepted as independent organisms opened the doors for their targeted control. when appel was promoted to laboratory director at the biological department of agriculture and forestry of the imperial health department in 1903, his most intense period of research began (fig. 2). from all over germany, reports of the occurrence of fusarium species in various cultures arrived at otto appel’s laboratory, accom panied by numerous submissions of infested plant material. appel and his student georg schikorra focused their research activities on infections on legumes and pea in particular (schikorra, 1907). they produced pure cultures from isolated fusarium, carried out retransmission experiments on pea and could prove the pathogenicity of this fungal pathogen. appel and his colleague hans wilhelm wollenweber extended these studies by including several other fusarium species. together they managed to prove that the fusaria are not as fig. 2: otto appel (fourth from the left) and co-workers at the botanical laboratory in berlin, about 1908 (image: archive of the julius kühn institute). https://phytopath-sammlung.julius-kuehn.de/ https://phytopath-sammlung.julius-kuehn.de/ otto appel’s contributions to food quality and safety 195 the control of smut fungi aside from the genus fusarium, the biological department of agriculture and forestry in berlin also focused on methods to control the spread of smut fungi. these activities were further reinforced when in 1905 smut fungi appeared so strongly in the province hanover that, according to a report, up to 50% of the wheat ears were destroyed (appel and riehm, 1911a). appel tried to develop a method for combating the two most important smut fungi species, ustilago hordei and ustilago tritici. the special challenge here was that the fungus infects the embryo inside the seed and not the outside of the kernel, as with other smut fungi. therefore, surface treatment of seeds with copper vitriol or 0.1% formalin solution or various hot water treatments proved to be ineffective (appel and gassner, 1907a). a production of smut fungi-free seeds was not possible in practice, since there were no pathogen free fields available. only the use of less susceptible varieties proved to be a promising approach to control the further spread of smut fungus. it was found that varieties with a short flowering phase or only slightly opened flowers were much less affected (appel and riehm, 1911b). by breeding appro priate genotypes, the infestation pressure could thus be reduced. however, this is a long-term approach, while otto appel was looking for a suitable solution with a shorter time frame. in literature, appel came across the hitherto unregarded work of jensen (1891), who was able to create smut fungi free plants from previously infected seeds. based on the assumption that germinating spores or seeds react more sensitively to external influences than resting spores or seeds, it was now necessary to determine which external conditions would be optimal to reduce the spores without harming the embryo within the seed. in this context, appel gained deeper insights into the biology of airborne pathogens, in particular at which temperature or grain moisture the fungus germinated, at what time the germination took place and at which temperature the pathogen could be killed safely. as a result, he recommended the following sequence for a hot water treatment to combat the pathogen (appel and gassner 1906, 1907a, 1907b): 1) activation of the fungal mycelium by soaking of the seeds for 4 h in 27 °c water, 2) killing the mycelium by a hot water treatment (5 min in 52 °c water), 3) drying the seeds the importance of resistance breeding otto appel valued resistance breeding as a very useful tool for maintaining the health of crops. at that time, breeding was still performed by the selection of plants with desired traits from the available pool of crop plants, as it had been done for hundreds of years, but not by goal-oriented crossing. although appel had only little experience in plant breeding, he highlighted the enormous potential of this approach and repeatedly made this topic the subject of discussions. in a review published in “science”, he provided a detailed description of necessary breeding activities in order to receive disease-resistant varieties (appel, 1915). here, he mentioned that the basis of resistance breeding is the existence of suitable resistant plant material, either in already existing varieties or in closely related wild plants. the transfer of the valuable features can be obtained via grafting or crossing. as possible aims of such breeding approaches he proposed: 1) wheat varieties with closed flowers to control loose smut, 2) short flowering time in cereals for controlling ergot, 3) small, hairy leaves in potato, which dry faster, to combat phytophthora, 4) a few small lenticels in potato tubers to combat soft rot and 5), rapid cork formation in potato tubers to combat fusarium (appel, 1915). feeding experiments with harmful feed in addition to phytopathological investigations, otto appel also performed studies to answer the question whether crops affected by pests can be safely used as animal feed. while previous work mainly focused on food intake, appel was also interested in whether pest infected feed could possibly cause changes to the digestive system, the associated glands and other internal organs, that led to a disease of the animals. together with the veterinarian franz koske, he conducted feeding experiments on pigs, poultry and pigeons with wheat infested with common bunt (tilletia caries) as well as tests on pigs and cattle with potatoes infested with potato blight (phytophthora infestans) and tuber wet rot (pectobacterium atrosepticum) (appel and koske, 1907). in a subsequent autopsy, no changes were re cognized in the internal organs of any of the animals, although the fig. 4: specimen of infected potato tubers preserved in formalin (collected and prepared by otto appel) − left to right: dying radicles caused by rhizoctonia solani, silver scurf caused by spondylocladium atrovirens (= helminthosporium solani), two examples of fusarium infested tubers (phytopathological collection of the julius kühn institute; images by manfred gräfe und djavad taghizadhe, stadtmuseum berlin). fig. 3: several fusarium species after four weeks of different culture media (image taken from appel and wollenweber, 1910). 1. fusarium solani, 2. f. orthoceras, 3a. f. subulatum on agar no. 42 with orange colored conidia on pale aerial mycelium, 3b. f. sub ultum on agar no. 46 with dark red thallus depicted from below, 4. f. willkommii, 5. f. subulatum, juvenile stage of the culture from 3b, 6. f. coeruleum, 7. f. discolor, 8a. f. metachroum on agar no. 42 with orange colored conidia, 8b. f. metachroum on agar no. 46 with dark red submerged thallus, 9. f. finely powdered, dar ker colored conidia on ochre colored, wrinkled thallus. 196 h. schulz, h. riegler discharged feces during the time of feeding had consisted mostly of spores. appel and koske (1907) concluded that, in the case of an unfavorable feeding effect, the detection of fungal spores is not sufficient to explain a possible harmfulness of a feed. faintness and diarrhea were observed in pigs and cattle for a short time during the experiments with dry and wet rotten potatoes, but these symptoms quickly disappeared. disease symptoms did not occur, but the weight gain of treated animals was slightly lower than that of the control group. in addition, the texture of fat in the treated pigs was less firm. however, the appearance and taste of the meat was not affected in pigs or cattle (appel and koske, 1907). reorganisation of the plant protection service in germany a major outbreak of phytophthora infestans halved the german potato harvest in 1916 and, complemented by halted trade due to world war one, led to mass starvation in germany known as the turnip winter. this made the importance of an effective crop protection clear once more. in 1919, otto appel held a talk on the future of plant protection in germany at the meeting of the association for applied botany. it was subsequently published as the very first article in the journal angewandte botanik (nowadays journal of applied botany and food quality), the new central publishing organ of the association with paul graebner, ernst gilg and karl müller as its first editors (appel, 1919). in this talk, he emphasized the necessity of a centrally organized plant protection service. he called for reliable statistics on the occurrence of plant diseases and for standardized efficacy testing of plant protection agents as well as for the application equipment. he argued that both, scientists and the practicians in plant protection would benefit from a closer collaboration and demanded that specia lized professorships for phytomedicine should be created instead of treating this subject as a minor side interest. when otto appel became director of his institute, now called im perial biological research centre for agriculture and forestry (biologische reichsanstalt für landund forstwirtschaft) in 1920, he used his newly gained responsibility to reorganize the institution, established new branches and field offices throughout germany, and reactivated the plant protection service, making it powerful and sustainable. at that time he had no longer time for his own scientific research, but was focused mainly on the creation of new positions in the field of crop protection and thus to continuously expand the research work of his institute. aside from plant protection, he also emphasized the importance of harvest protection as a national challenge (appel, 1933a) and the impact of pest control on food products (appel, 1932a). he suggested measures to increase agricultural production (appel, 1932b) and contributed to a reformation of the training of technical assistants in plant protection (appel, 1941). he referred to the enormous possibilities of resistance breeding for modern crop protection, repeatedly (appel, 1915, 1927a, b, c, 1930). he was co-editor of several books such as “handbuch der pflanzenkrankheiten” (appel, 1937), and of the journals “deutsche landwirtschaftliche rundschau” and “der biologe”. the “pocket atlases” published by appel were received with great interest by farmers and quickly became standard works for the most important diseases and pests of crops and their control (appel, 1925, 1926a, 1926b, 1928a, b, 1929a, 1929b, 1931, 1933b, tab. 1: phytopathogenic fungi identified by otto appel and colleagues. where applicable, revised names according to index fungorum (www.indexfungorum. org, accessed 5th july, 2019) have been added. name given by otto appel reference revised name acremonium sclerotiniarum appel & laubert (appel and laubert, 1906) colletotrichum theobromae appel & strunk (appel and strunk, 1904) corymbomyces appel & strunk (appel and strunk, 1904) sphaerostilbella (henn.) sacc. & d. sacc. corymbomyces albus appel & strunk (appel and strunk, 1904) diplodina corticola appel & strunk (appel and strunk, 1904) discella cacaoicola appel & strunk (appel and strunk, 1904) fusarium colorans de jonge, appel & wollenw. (appel and wollenweber, 1913) fusarium discolor appel & wollenw. (appel and wollenweber, 1913) fusarium elegans appel & wollenw. (appel and wollenweber, 1913) fusarium gibbosum appel & wollenw. (appel and wollenweber, 1913) fusarium martii appel & wollenw. (appel and wollenweber, 1913) fusarium solani (mart.) sacc. fusarium metachroum appel & wollenw. (appel and wollenweber, 1913) fusarium orthoceras appel & wollenw. (appel and wollenweber, 1913) fusarium oxysporum schltdl. fusarium rostratum appel & wollenw. (appel and wollenweber, 1913) fusarium rubiginosum appel & wollenw. (appel and wollenweber, 1913) fusarium subulatum appel & wollenw. (appel and wollenweber, 1913) fusarium theobromae appel & strunk (appel and strunk, 1904) fusarium ventricosum appel & wollenw. (appel and wollenweber, 1913) rectifusarium ventricosum (appel & wollenw.) l. lombard & crous lasiodiplodia nigra appel & laubert (appel and laubert, 1907) pyricularia caudata appel & strunk (appel and strunk, 1904) trichoconis caudata (appel & strunk) clem. rhabdospora ramealis var. macrospora appel & laubert (appel and laubert, 1907) rhabdospora theobromae appel & strunk (appel and strunk, 1904) typhula stricta appel (appel and laubert, 1907) typhula intermedia appel & laubert (appel and laubert, 1907) typhula variabilis riess ustilago dura appel & gassner (appel , 1912) http://www.indexfungorum.org http://www.indexfungorum.org otto appel’s contributions to food quality and safety 197 1934, 1944). in 1933, he retired from his position as director of the imperial biological research centre for agriculture and forestry at the age of 66. his achievements were honored with honorary degrees from the universities of vienna, sofia and berlin. after being active in many learned societies in his field and being member of the academy of sciences leopoldina, he was also honorary president of the vereinigung für angewandte botanik (association for applied botany) in germany and honorary chairman of the vereinigung deutscher pflanzenärzte (association of german plant medics, which has later been re-organized into the german phytomedical society). on his 85th birthday on may 19, 1952, he became the first phytopathologist to be awarded the german grand federal cross of merit (bundesverdienstkreuz, fig. 5). shortly thereafter, he passed away on november 10, 1952. otto appel has met the challenges in phytomedicine of his time with perspicacity and enthusiasm, offered numerous solutions and revolutionized plant protection in many areas. his work on plant protection has made him known far beyond the borders of germany. today he is regarded as one of the most important phytopathologists in the first half of the 20th century and in remembrance, the deutsche phytomedizinische gesellschaft awards the otto appel medal to outstanding researchers in phytomedicine. abdruck aus den schriften der physikalisch-ökonomisch. gesellschaft zu königsberg i.pr., jahrgang xxxix. verlag: königsberg leupold. appel, o., 1902a: untersuchungen über das einmieten der kartoffeln. arbeiten aus der biologischen abtheilung des kaiserlichen gesundheitsamtes 2 (3), 373-436. appel, o., 1903: untersuchungen über die schwarzbeinigkeit und die durch bakterien hervorgerufene knollenfäule der kartoffel. arbeiten aus der biologischen abtheilung für landund forstwirtschaft am kaiserlichen gesundheitsamt 3 (4), 364-434. appel, o., 1912: krankheiten der futterpflanzen unter besonderer berücksichtigung der gräser und kleearten, beitr. pflanzenzucht 2, 32-64 appel, o., 1915: disease resistance in plants. science 41, 773-782. appel, o., 1919: die zukunft des pflanzenschutzes in deutschland, angewandte botanik 1, 1-15. appel, o., 1925: kartoffelkrankheiten. 1. teil knollenkrankheiten (pareys taschenatlanten nr. 1). parey, berlin. appel, o., 1926a: taschenatlas der kartoffelkrankheiten, 2. teil: staudenkrankheiten (pareys taschenatlanten nr. 2). parey, berlin. appel, o., 1926b: taschenatlas der krankheiten der zuckerrübe. parey, berlin. appel, o., 1927a: einige neuere fragen der kartoffelzüchtung. mitteilungen der deutschen landwirtschafts-gesellschaft 27, 1-4. appel, o., 1927b: beitrag zur frage der immunitätszüchtung der kartoffel. pflanzenbau 4, 51-56. appel, o., 1927c: die züchtung krankheitswiderstandsfähiger sorten. illustrierte landwirtschaftliche zeitung 47, 464-466. appel, o., 1928a: taschenatlas der krankheiten des kernund steinobstes. 1. teil: kernobst (pareys taschenatlanten nr. 4). parey, berlin. appel, o., 1928b: taschenatlas der krankheiten des kernund steinobstes. 2. teil: steinobst (pareys taschenatlanten nr. 5). parey, berlin. appel, o., 1929a: taschenatlas der krankheiten des beerenund schalen obstes (pareys taschenatlanten nr. 6). parey, berlin. appel, o., 1929b: krebsfeste kartoffelsorten und die häufig mit ihnen verwechselten anfälligen sorten (pareys taschenatlanten nr. 7), parey, berlin. appel, o., 1930: pflanzenpathologie und pflanzenzüchtung. der züchter 11, 309-312. appel, o., 1931: taschenatlas der getreidekrankheiten (pareys taschenatlanten nr. 10). parey, berlin. appel, o., 1932a: schädlingsbekämpfung und ihre auswirkung auf die deutsche volksernährung. friedrichswerther monatsberichte 22, 85-86. appel, o., 1932b: maßnahmen zur steigerung und verbilligung der landwirtschaftlichen produktion. die deutsche landwirtschaft 50, 72-744. appel, o., 1933a: erntesicherung, eine nationale notwendigkeit. landbau und technik, 2 seiten. appel, o., 1933b: taschenatlas der gemüsekrankheiten (pareys taschenatlanten nr. 11). parey, berlin. appel, o., 1934: taschenatlas der krankheiten des weinstockes (pareys taschenatlanten nr. 12). parey, berlin. appel, o., 1937: handbuch der pflanzenkrankheiten, 6. band: pflanzenschutz. verlag paul parey, berlin. appel, o., 1941: die technische assistentin im künftigen pflanzenschutzdienst. berlin. appel, o., 1944: taschenatlas der schädigung in futterpflanzen 1. teil lupine, serradella und luzerne (pareys taschenatlanten nr. 14 ). parey, berlin. appel, o. gassner, g., 1906: der brand des hafers und seine bekämpfung. flugblatt nr. 38. kaiserliche biologische anstalt für landund forstwirtschaft. appel, o., gassner, g.,1907a: der derzeitige stand unserer kenntnisse von den flugbrandarten des getreides und ein neuer apparat zur einfachen durchführung der heißwasserbehandlung des saatgutes. arbeiten aus der kaiserlichen biologischen anstalt für landund forstwirtschaft 3, 1-20. appel, o., gassner, g., 1907b: untersuchungen über den brand, insbesonacknowledgements we would like to thank johannes hallmann, cordula gattermann and georg f. backhaus, upon whose german articles on otto appel this work is based (hallmann, 2018; gattermann and backhaus, 2018). references appel, o., 1890: caricologische notizen aus dem herzynischen gebiet. mitteilungen der geographischen gesellschaft für thüringen zu jena, 8, 41-44 appel, o., 1891: bemerkungen über einige arten der gattung carex. berichte der bayerischen botanischen gesellschaft 1, 72-77. appel, o., 1893: vergleich der flora der baar mit der des benachbarten schaffhausen. mitteilungen des badischen botanischen vereins 106, 53-59. appel, o., 1899: über phytound zoomorphosen (pflanzengallen). sonderfig. 5: prof. dr. dr. h.c.mult. otto appel, honored with the german grand federal cross of merit in 1952 (bundesverdienstkreuz). 198 h. schulz, h. riegler dere den flugbrand des getreides. arbeiten aus der biologischen anstalt für landund forstwirtschaft 4, 9-13. appel, o., koske, f., 1907: versuche über die wirkung einiger als schädlich verdächtiger futtermittel. arbeiten aus der kaiserlichen biologischen anstalt für landund forstwirtschaft 5, 361-376. appel, o., laubert, r., 1907: bemerkenswerte pilze. i. arbeiten aus der kaiserlichen biologischen anstalt für landund forstwirtschaft 5, 147154. appel, o., riehm, e., 1911a: die bekämpfung des flugbrandes von weizen und gerste. arbeiten aus der kaiserlichen biologischen anstalt für landund forstwirtschaft 8, 343-426. appel, o., riehm, e., 1911b: untersuchungen über die brandkrankheiten des getreides. mitteilungen aus der kaiserlichen biologischen anstalt für landund forstwirtschaft 11, 9-12. appel, o., strunk, h.f., 1904: über einige in kamerun auf theobroma cacao beobachtete pilze. zentralblatt für bakteriologie und parasitenkunde 2 (11), 551-557, 632-637. appel, o., wollenweber, h.w., 1913: grundlagen einer monographie der gattung fusarium (link). arbeiten aus der kaiserlichen biologischen anstalt für landund forstwirtschaft 8, 1-207. de bary, a., 1861: die gegenwärtig herrschende kartoffelkrankheit, ihre ursache und ihre verhütung: eine pflanzenphysiologische untersuchung. förstner’sch buchhandlung, leipzig 1861, pp. 75. gattermann, c., backhaus, g.f., 2018: otto appel – ein leben im dienst des pflanzenschutzes, journal für kulturpflanzen 70 (1), 1-11. doi: 10.1399/jki.2018.01.01 hallmann, j. 2018: die phytomedizinischen herausforderungen im leben von otto appel: ein überblick seiner wissenschaftlichen arbeiten anlässlich seines 150. geburtstages, journal für kulturpflanzen 70 (1), 1224. doi: 10.1399/jfk.2018.01.02 hollrung, m., 1903: 3. krankheiten der wurzelfrüchte, b) kartoffel. jahresbericht über die neuerungen und leistungen auf dem gebiete der pflanzenkrankheiten 6, 129-130. jensen, j.l., 1891: et par meddelelser om bortskaffelsen af vaarsaedens brand. meddelelser til deltagerne i faellesindkjebet af undersogt markfrø i foraaret 1890, kopenhagen, 22. schikorra, g., 1907: fusarium-krankheiten der leguminosen. arbeiten aus der kaiserlichen biologischen anstalt für landund forstwirtschaft 5, 157-183. orcid heike riegler https://orcid.org/0000-0002-1302-4533 address of the corresponding author: hartwig schulz, julius kühn institute, federal research centre, cultivated plants, institute for ecological chemistry, plant analysis and stored product protection, königin-luise-straße 19, 14195 berlin, germany e-mail: hartwig.schulz@julius-kuehn.de © the author(s) 2019. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1399/jki.2018.01.01 http://dx.doi.org/10.1399/jfk.2018.01.02 journal of applied botany and food quality 93, 168 176 (2020), doi:10.5073/jabfq.2020.093.021 1laboratory of plant productivity and environmental constraint, lr18es04, biology department, faculty of sciences, university tunis el manar, tunisia 2laboratory of dry land farming and oasis cropping, ira, university of gabes, tunisia oxidative stability of olive oil enriched with oleaster leaves under accelerated storage conditions amina ayadi1, yassine yahia2, khadija ben othman2, hédia hannachi1,* (submitted: march 22, 2020; accepted: august 25, 2020) * corresponding author summary olive oil is rich in natural antioxidants that conserve its quality under storage conditions. however, there is a growing need to improve the quality of olive oil under storage conditions using phenol-enriched olive oil. in the present study, olive oil from the chemlali cultivar was enriched with wild olive tree leaves or oleaster. the oil composition was analyzed before and after accelerated storage conditions using a schaal test. standard oil parameters, including free acidity; peroxide value; iodine value; specific extinction k232 and k270; fatty acid profile, and polyphenolic, chlorophyll, and carotenoid content, were evaluated for the control olive oil (coo) and the enriched olive oil (eoo). polyphenolic compounds were identified for coo, eoo, and oleaster leaf extracts (ole) using high-performance liquid chromatography. antioxidant activity was analyzed using 2,2-diphenyl-1-picrylhydrazyl (dpph) and 2,2’ azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (abts) tests. the results showed that enriching the olive oil quantitatively and qualitatively improved the polyphenolic composition, pigment contents, and the antioxidant activity. the eoo was more resistant to oxidation under accelerated storage conditions. the addition of wild olive leaves also significantly improved the resistance of the olive oil to oxidation and can, therefore, be used as a source of natural antioxidants to improve the oxidative stability of edible oils. key-words: olive oil, oleaster leaves, oxidative stability, polyphenolic compounds, fatty acids introduction olive oil is an important component of the mediterranean diet and is known to provide protection against cardiovascular diseases and cancer due to its fatty acid profile and minor components, such as polyphenolic constituents (gharby et al., 2011). lipid oxidation is the most important cause of olive oil quality deterioration during storage. the primary lipid oxidation products are hydroperoxides. hydroperoxides are highly unstable and react to form secondary products, such as hydrocarbons and alcohols, which are oxidized to carboxylic acids (bešter et al., 2008). recent studies have suggested that the polyphenolic compounds that are naturally present in virgin olive oil improve its resistance to oxidative deterioration (frankel, 1998). polyphenolic antioxidants interrupt the initiation and propagation stages of the oxidative chain reaction by reacting with lipid radicals to form more stable products (gordon et al., 2001). the oxidation process can also be delayed using endogenous antioxidants that enhance oxidative stability; in addition, synthetic antioxidants can be used to prolong the stability of oils under storage. however, toxicologists and nutritionists have sought to ascertain the noxious effects associated with the use of certain synthetic antioxidants, such as butylated hydroxytoluene (bht) and butylated hydroxyanisole (bha) (barlow, 1990). in recent times, interest in sources of natural antioxidants that can be used to enrich oils to reduce lipid oxidation has increased. for example, olive oil has been enriched with polyphenolic compounds obtained from olive cake (suárez et al., 2010). the addition of olive leaves prior to the extraction process has been shown to improve the organoleptic quality and stability of olive oil (di giovacchino et al., 1995). olive leaves also provide a rich source of natural antioxidants (salah et al., 2012; hannachi et al., 2019), including a diversity of polyphenolic compounds that may act as an effective defense system against free radical attacks (bouaziz et al., 2008). the aim of this study is to study the evolution of the oxidation process of olive oil enriched with oleaster leaves under accelerated storage conditions. standard olive oil parameters, including the free acidity (fa), peroxide value (pv), iodine value (iv), specific extinction k232 and k270, fatty acid profile, polyphenolic profile, and pigment contents, were evaluated for the control olive oil (coo) and the enriched olive oil (eoo) before and after accelerated storage conditions. antioxidant activity was determined for both the coo and eoo using the abts and dpph radical scavenging activity. materials and methods materials olive oil of the chemlali cultivar (olea europaea l. var. europaea) was obtained from oil mills located in the limaoua area of the gabes region in southern tunisia and was kept in amber-colored glass bottles in the dark at an ambient temperature. the leaves of wild olive trees or oleasters (olea europaea l. var. sylvestris) were collected from tounine (a natural ecosystem) from gabes in southern tunisia. the collected oleaster leaves were washed, air dried at room temperature, powdered using a laboratory mill (type fw135), and stored in a dry and dark location. methods enrichment of olive oil by oleaster leaves the enrichment procedure involved immersing the oleaster leaf powder in olive oil. the maceration process was conducted with 100 ml olive oil and 10 g powdered oleaster leaves under agitation at 25 °c for 24 h. after maceration, the samples were filtered and kept in glass bottles in the dark at an ambient temperature. ac celerated oxidation stability testing was conducted on the eoo and the coo to evaluate their resistance to oxidation. accelerated oxidation experiment the effect of storage on the oxidative stability of the oils was conducted using the schaal oven method as simplest accelerated test. the samples were stored in the dark at a temperature of 65 °c in closed glass bottles for 24 days (yang et al., 2013). the oxidative stability testing was conducted by evaluating standard olive oil parameters every three days. the fa, pv, iv, and specific extinction coefficients at 232 and 270 nm (k232 and k270) were improvement of olive oil stability 169 determined according to the methods described in the commission of the european communities (cec, 2003). all parameters were evaluated for the eoo and the coo. extraction of polyphenolic compounds extraction of polyphenolic compounds from olive oil oil samples of 7.5 g were mixed with 5 ml methanol/water (80:20 v/v) solution and vigorously homogenized with a vortex. the two phases were separated by centrifugation at 3800 rpm for 15 minutes, and the hydroalcoholic phase was collected. the centrifuge (labofuge 200) was equipped with an integrated angle rotor (12 × 15 cm), and 3030 × g was used as the relative centrifugal force (sedimentation coefficient). the extraction was repeated with 5 ml methanol/water (80:20 v/v) solution three times. the hydroalco holic extracts were then combined and conserved at 4 °c in the dark (servili at al., 1999). extraction of polyphenolic compounds from oleaster leaves the extraction process of the oleaster leaf powder was performed in an ultrasonic bath (clean 120 hd) at 20 °c for 40 minutes. powdered oleaster leaves (5 g) were sonicated in 50 ml methanol hplc grade. the oleaster leaf extract was subsequently filtered and centrifuged at 3800 rpm for 20 minutes. the obtained extracts were kept at 4 °c in the dark for analysis. determination of total polyphenolic content the total polyphenolic content was determined using the folinciocalteau method (benavente-garcia et al., 2000). a total of 0.5 ml folin-ciocalteau (prolabo) reagent was added to 0.1 ml polyphenolic extract obtained from olive oil and the diluted oleaster leaf extract. after 5 minutes, 4 ml sodium carbonate solution (1 m) was added. after 90 minutes of incubation at room temperature in the dark, the absorbance was measured at 760 nm using a t60 uvvisible spectrophotometer. gallic acid was used to produce a calibration curve. the total polyphenolic content was expressed as mg gallic acid equivalent per kilogram of oil (ppm) and as mg gae/kg dried matter (ppm) for the olive oil and oleaster leaf extract, respectively. hplc analysis of polyphenolic compounds olive oil (20 μl) and oleaster leaf methanol extracts were injected into an hplc unit ajilent technologies 1200 series equipped with a reverse phase dc18 column (5 μm, 4.6 × 250 mm) and a uv detector at 280 nm and integrator. the flow rate was 0.9 ml/min using water/ acetic acid (v/v) as solvent a and acetonitrile as solvent b at 0.9 ml/ min. elution proceeded for around 40 minutes. the peaks of the polyphenolic compounds were identified by matching the retention time and the uv spectra of the standard peaks (benavente-garcia et al., 2000). quantification of the polyphenolic compounds was conducted by calibration curves relative to external standards. the levels of polyphenolic compounds detected were expressed as ppm. standard olive oil parameters the fa, pv, k232, k270 and fatty acid composition were determined according to official methods described in the commission of the european communities (cec, 2003). the iv and other para meters were based on international standards or reports in the litera ture. fa was expressed as oleic acid percentage and was determined by acid-base titration with a 0.177 n sodium hydroxide solution of free fatty acids in the olive oil samples previously dissolved in neutralized ethanol using phenolphthalein as an indicator. the fa was calculated as follows: (v × n × m ) fa = m × 10 v: volume (ml) of potassium hydroxide solution used in titration n: concentration (0.1775 mole/l) of potassium hydroxide solution used in titration m: molar weight (g/mole) of oleic acid (282.47 g/mole) m: weight in grams of the sample. the pv was determined by dissolving one gram of the olive oil samples in 15 ml acetic acid and 10 ml chloroform, to which 1 ml potassium iodide solution was subsequently added. the obtained solution was kept in the dark for five minutes at 25 °c to react with the saturated potassium iodine solution. a total of 75 ml distilled water was added. the liberated iodine was titrated with a 0.01 n sodium thiosulfate (na2s2o3) standard solution using starch solution as an indicator. white titration (white test) was conducted under the same conditions. the pv was expressed as milliequivalents of active oxygen per kilogram of olive oil (meq o2/kg oil): (v v0) × 1000 × tip (meqo2 / kg = w v: volume (ml) of standardized sodium thiosulphate solution used for sample titration v0: volume (ml) of standardized sodium thiosulphate solution used for the white titration t: sodium thiosulfate (na2s2o3) normality w: olive oil weight (g). the specific extinction coefficients k232 and k270 at 232 and 270 nm were measured using a t60 uv-visible spectrophotometer for 0.1 g olive oil samples dissolved in 10 ml cyclohexane using a quartz cuvette with a path length of 1 cm. eλkλ = (c × s) kλ: specific extinction at wavelength λ (232 and 270 nm) eλ: extinction measured at wavelength λ (232 and 270 nm) c: concentration of olive oil solution (g/100 ml) s: path length of the quartz cell (1 cm) the iv was assessed according to the analytical method described by the association of official analytical chemists (aoac, 1980) by titration with 0.1 n na2s2o3 of 0.3 g olive oil sample previously dissolved in 10 ml chloroform in the presence of the wijs reactive and 10% ki. the iv was expressed as g iodine per 100 g olive oil (gi/100g): (v0 v) × 1.27iv (gi / 100 g) = w v0: volume (ml) of standardized sodium thiosulphate solution used for the white titration v: volume (ml) of standardized sodium thiosulphate solution used for the sample titration w: olive oil weight (g) pigment contents pigment contents were determined by dissolving 7.5 g olive oil samples in hexane (25 ml), and the absorbance at 470 and 670 nm was measured to determine the carotenoid and chlorophyll contents, respectively (minguez-mosquera, 1991). the chlorophyll and caro tenoid contents were expressed as mg of pheophytin “a” and lutein per kg of oil (ppm), respectively. 170 a. ayadi, y. yahia, k.ben othman, h. hannachi (a670 × 106)chlorophylls (ppm) = (613 × 100 × d) (a470 × 106)carotenoids (ppm) = (2000 × 100 × d) a670: absorbance at 670 nm a470: absorbance at 470 nm d: optical pathway (1 cm). fatty acid composition using gc/ms fatty acid composition was carried out following their transformation on fatty acid methyl-ester (fames) after cold alkaline transesterification with a methanolic potassium hydroxide solution. fames were prepared by dissolving 0.2 g olive oil in 3 ml of hexane and adding 0.4 ml 2 n methanolic potassium hydroxide solution. after vigorous shaking for 30 seconds, the solution was leaved to stratify until the upper solution became clear. the upper layer containing the methyl esters was subsequently decanted (cec, 2003). the obtained fames (1 μl) were analyzed on an agilent hp series gc 6890 coupled with an hp 5973 ms detector fitted with a cpsil-88 column (30 m × 0.25 mm i.d., film thickness 0.20 μm). helium was used as a carrier gas at 1.2 ml/min. the oven temperature was held at 60 °c for 1 minute, increased to 220 °c at 10 °c/min for 10 minutes, then to 220 °c at 15 °c/min for 15 minutes. the injector and flame ionization detector temperatures were maintained at 250 °c. the methyl ester peaks of the samples were identification via a comparison with those of the methyl esters reference mixtures. antioxidant activity dpph radical scavenging activity a total of 20 μl olive oil extract was mixed with 200 μl 2,2-diphenyl-1-picrylhydrazyl (dpph) methanolic solution (0.2 mm). the mixture was shaken and left for 30 minutes in the dark at 25 °c. the absorbance of the solution was measured at 517 nm to determine the concentration of the remaining dpph. the radical scavenging activity on dpph radicals was determined by the following formula: abs t0 -abs tiabsorbance reduction at 517 nm = × 100 abs t0 with abs t0: control absorbance in presence of dpph and abs ti: absorbance measured in the presence of a related concentration of trolox with dpph. the curve absorbance reduction at a 517 nm function of the trolox concentration (mm) was used to determine the dpph radical scavenging activity of the samples. the radical scavenging activity was expressed as mg trolox equivalent antioxidant capacity per kilogram of oil (mg teac/kg oil) for the olive oil extract and as mg teac/kg dry weight for the oleaster leaf extract. abts+ radical scavenging activity the abts.+ radical was generated by mixing 7 mm 2,20-azinobis3-ethylbenzothiazoline-6-sulphonate abts solution with 2.45 mm potassium persulfate (k2s2o8). the mixture was kept in the dark at room temperature for 16 h before use. following this, the abts.+ solution was diluted with ethanol to produce an absorbance of 0.700 ± 0.02 at 734 nm. a quantity of 20 μl extract was added to 180 μl abts+ solution. the absorbance was measured at 734 nm after 5 minutes of incubation (djeridiane et al., 2006). the radical scavenging activity on the abts radical was determined by the following formula: abs t0 -abs tiabsorbance reduction at 734 nm = × 100 abs t0 with abs t0: control absorbance in presence of abts and abs ti: absorbance measured in the presence of a related concentration of trolox with abts. the curve absorbance reduction at a 734 nm function of the trolox concentration (mm) was used to determine the abts radical scavenging activity of the samples. the radical scavenging activity was expressed as mg trolox equivalent antioxidant capacity per kilogram of oil (mg teac/kg oil) for the olive oil extract and as mg teac/kg dry weight for the oleaster leaf extract. statistical analysis all experiments were performed in triplicate on three independent samples, and the results were presented as mean ± standard deviation. an analysis of variance (anova) and a duncan’s multiple range test were performed using xlstat software (www.xlstat.com) to evaluate the significance of the difference between the control and enriched olive oil at p < 0.05. the freely available heatmapper web server (http://www.heatmapper.ca; babicki et al., 2016) was used to generate a heat map to interactively visualize the data (i.e., the polyphenolic compounds, antioxidant activity, fatty acids, standard olive oil parameters) of the coo and eoo. results composition of eoo the values of the studied parameters of the coo and eoo are provided in tab. 1 and 2. the quality parameters were in accordance with extra virgin olive oil quality standards established by the international olive council (ioc, 2019). the fa, pv, specific extinctions, and iv did not change significantly after enrichment of the olive oil with oleaster leaves. the enrichment process did not change the fatty acid composition, which showed a high percentage of oleic acid (75.91 for coo and 77.28 for eoo). however, the enrichment process improved the pigment and polyphenolic contents, and the antioxidant capacity differed significantly between the coo and eoo. the oleaster leaves increased the level of chlorophyll from 1.35 ± 0.04 ppm in the coo to 7.37 ± 0.12 ppm in the eoo. the carotenoid content showed an average of 1.75 ± 0.03 ppm in the eoo and 0.58 ± 0.02 ppm in the coo. the total polyphenolic content was 123.80 ± 3.03 ppm in the coo and 441.61 ± 5.28 ppm in the eoo. therefore, the process of enriching the olive oil with oleaster leaves improved the antioxidant activity of the eoo. the ddph test showed that the antioxidant activity increased from 76.53 (coo) to 500.00 (eoo) mg teac/kg oil (eoo). the abts test showed that the antioxidant capacity of the eoo (928.88 mg teac/kg oil) improved compared with the coo (160.00 mg teac/kg oil; see tab. 2). tab. 3 provides a list of the main phenolic acids and flavonoid compounds found in the eoo, coo, and ole. luteolin-7-o-glucoside (2979 ± 18.66 ppm) was the major compound in the ole, followed by rutin (2684.05 ± 21.89) and quercetin3-o-rhamonoside (1541 ± 39.18 ppm). meanwhile, acacetin, apigenin, p-coumaric acid, naringenin, and kaempferol were the major compounds in the coo. the enrichment process increased the levels of polyphenolic compounds detected in the eoo. for example, the level of rutin in the coo was 0.31 ± 0.03 ppm, while 21.78 ± 0.57 ppm was recorded in the eoo. the level of p-coumaric acid increased from 0.28 ± 0.03 (coo) to 0.89 ± 0.02 ppm (eoo). 4-o-caffeoylquinic and caffeic acids were only detected in ole and eoo. based on the polyphenolic compounds and antioxidant activities visualized by the heatmap (see fig. 1), the eoo was distinguished by its polyphenolic richness and high antioxidant activity compared with the coo. improvement of olive oil stability 171 tab. 1: standard quality indices and fatty acid composition of studied olive oils. coo eoo norma of extra virgin olive oil (ioc, 2019) fa 1.20 ± 0.15 a 1.22 ± 0.17 a < 2.0 pv (meq o2/kg) 6.98 ± 1.20 a 7.01 ± 1.17 a < 20 iv (gi/100g) 88.87 ± 3.12 a 87.56 ± 3.21 a k232 2.49 ± 0.52 a 2.47 ± 0.90 a < 2.50 k270 0.20 ± 0.05 a 0.23 ± 0.09 a < 0.22 c16:0 (%) 11.44 ± 1.92 a 10.24 ± 1.00 a 7.5 20.00 c16:1(%) 0.90 ± 0.08 a 1.08 ± 0.05 a 0.30 3.50 c18:0 (%) 2.61 ± 0.05 a 2.97 ± 0.04 a 0.5 5.00 c18:1 (%) 75.91 ± 5.76 a 77.28 ± 4.59 a 55.00 83.00 c18:2 (%) 7.86 ± 1.12 a 6.71 ± 1.10 a 2.50 21.00 c20:0 (%) 0.27 ± 0.02 a 0.25 ± 0.02 a ≤ 0.60 c20:1 (%) 0.23 ± 0.06 a 0.20 ± 0.05 a ≤ 0.40 each value is the mean ± standard deviation; fa: free acidity; pv: peroxide value; iv: iodine value; k232: specific extinction at 232nm; k270: specific extinction at 270 nm; coo: control olive oil; eoo: enriched olive oil with oleaster leaves; means in the same line with different letters differ significantly (p < 0.05). tab. 2: pigments, polyphenols, and antioxidant activity of oleaster leaves and studied olive oils extracts. ole coo eoo chlorophylls (ppm) 8.76 ± 0.74 1.35 ± 0.04 b 7.37 ± 0.12 a carotenoids (ppm) 2.46 ± 0.62 0.58 ± 0.02 b 1.75 ± 0.03 a polyphenols (ppm) 689.80 ± 30.75 123.80 ± 3.03 b 441.61 ± 5.28 a dpph (mg teac/kg) 781.18 ± 2.71 76.53 ± 2.71 b 500.00 ± 9.54 a abts (mg teac/kg) 1160.43 ± 4.63 160.00 ± 4.63 b 928.88 ± 11.03 a each value is the mean ± standard deviation; teac: trolox equivalent antioxidant capacity; coo: control olive oil; eoo: enriched olive oil with oleaster leaves; ole: oleaster leaves extract. mg teac/kg dry weight for ole and mg teac/kg oil for coo and eoo; means in the same line of studied olive oils (coo and eoo) with different letters differ significantly (p < 0.05). tab. 3: polyphenolic compounds of olive oils and oleaster leaf extracts. polyphenolic compounds brut formula [m-h] m/z rt (min) ole coo eoo quinic acid c7h12o6 191 2.032 645.43 ± 10.87 0.2 ± 0.01 b 7.00 ± 0.05 a 4-o-caffeoylquinic acid c16h18o9 353 12.483 29.32 ± 0.94 nd 0.30 ± 0.00 chlorogenic acid c16h18o9 353 9.779 27.68 ± 0.14 nd nd caffeic acid c9h8o4 197 14.445 4.65 ± 1.81 nd 0.10 ± 0.00 4,5-di-o-caffeoylquinic acid c25h24o12 515 26.839 142.38 ± 1.54 0.06 ± 0.00 b 1.34 ± 0.01 a p-coumaric acid c9h8o3 163 20.873 25.29 ± 0.97 0.28 ± 0.03 b 0.89 ± 0.02 a rutin c27h30o16 609 23.804 2684.05 ± 21.89 0.31 ± 0.00 b 21.78 ± 0.57 a luteolin-7-o-glucoside c21h20o11 447 24.449 2979.38 ± 18.66 0.50 ± 0.02 b 23.93 ± 0.2 a quercetrin (quercetin-3-o-rhamnoside) c21h20o11 447 26.811 1541.16 ± 39.18 0.26 ± 0.00 b 12.46 ± 0.14 a naringin c27h32o14 579 25.849 95.86 ± 1.44 0.02 ± 0.00 b 0.76 ± 0.01 a apegenin-7-o-glucoside c15h10o5 431 26.733 123.29 ± 0.57 0.04 ± 0.00 b 1.08 ± 0.01 a quercetin (quercetin-3-o-rhamonoside) c15h10o7 447 26.802 153.7 ± 0.24 0.07 ± 0.00 b 1.23 ± 0.01 a kaempferol c15h10o6 285 31.768 55.85 ± 0.31 0.32 ± 0.00 b 0.79 ± 0.01 a naringenin c15h12o5 271 33.777 2.79 ± 0.03 0.12 ± 0.00 a 0.15 ± 0.00 a apigenin c15h10o5 269 34.353 6.55 ± 0.07 0.24 ± 0.00 a 0.29 ± 0.01 a acacetin c16h12o5 283 40.087 nd 0.01 ± 0.00 a 0.01 ± 0.00 a each value is the mean ± standard deviation; coo: control olive oil; eoo: enriched olive oil with oleaster leaves; ole: oleaster leaves extract; nd: not determined. means in the same line of studied olive oils (coo and eoo) with different letters differ significantly (p < 0.05). 172 a. ayadi, y. yahia, k.ben othman, h. hannachi olive oil criteria under accelerated storage conditions standard oil parameters the fa increased from 1.20 ± 0.15 to 2.81 ± 0.20 for the coo and to 2.40 ± 02.0 for the eoo. therefore, the enrichment of olive oil with oleaster leaves appeared to improve its oxidative stability (see fig. 2a). the initial pv value was 6.98 ± 1.20 and 7.01 ± 1.17 meq o2/kg for the coo and eoo, respectively. after 24 days under accelerated storage conditions, the pv of the coo and eoo increased to 29.17 ± 2.15 and 21.74 ± 2.48 meq o2/kg, respectively. during the first six days, the peroxide values showed a slight increase. after 15 days under accelerated storage conditions, a substantial increase in the pv to a level of 11.98 ± 2.18 meq (o2)/kg in the eoo and 19.94 ± 2.11 meq (o2)/kg coo was noted (see fig. 2b). under accelerated storage conditions, the iv decreased for the both olive oils. after 12 days, there was a clear difference in the iv of the coo (71.30 ± 3.23 gi/100 g) and the eoo (77.30 ± 2.17 gi/100 g). a lower value indicates a lower degree of unsaturation, which suggested that the double bonds in the eoo were more protected against autoxidation during accelerated storage conditions than the coo (see fig. 2c). specific extinctions provided information on the oil quality under accelerated storage conditions. the k232 and k270 of the both the coo and eoo increased. the increase of k232 of coo and eoo was greater after nine days (see fig. 3a). however, the increase in the k270 values was greater after 12 days (see fig. 3b) for the both oils. a heatmap analysis showed that after accelerate storage conditions, the eoo was characterized by a lower fa and pv and high iv, suggesting that the eoo was more resistant to oxidation (see fig. 1). pigments contents the enrichment process increased the pigment contents (see fig. 1, tab. 2). under accelerated storage conditions, the pigments contents decreased in the coo and eoo. after 24 days of accelerated storage condition, the eoo was richer in chlorophylls (3.10 ± 0.51 ppm) and carotenoids (0.26 ± 0.09 ppm) than the coo, with an amount of 0.24 ± 0.04 and 0.08 ± 0.01 ppm detected for chlorophylls and carotenoids, respectively (see fig. 4). fatty acids composition the results showed that, under accelerated storage conditions, a slight modification was noted in the fatty acid profiles of the coo and eoo (see tab. 4). the reduction in the linoleic acid content was higher compared with the other fatty acids in both the eoo and coo. after 24 days under accelerated storage conditions, the decrease in linoleic acid in the coo was higher compared with the eoo. the eoo had a higher content of active antioxidant compounds, which protect unsaturated fatty acids against deterioration under accelerated storage conditions (see fig. 1). discussion the ole demonstrated a higher polyphenolic content (689.80 ± 30.75 ppm) than the coo (123.80 ± 3.03 ppm), which agrees with the results of previous studies (dabbou et al., 2011; djenane et al., 2019; hannachi et al., 2019, 2020). the variation in the polyphenolic content can be explained by factors such as climatic conditions, extraction method, and genotype. the level of polyphenols has been shown to positively correlate with biotic and abiotic stress in plants (boscaiu et al., 2010). the oleaster trees utilized in the present study originated from a natural ecosystem and did not benefit from the same cultivation techniques as cultivated olive trees. as such, the oleaster trees had higher exposure to environmental stress than cultivated olive trees (hannachi et al., 2008). the use of synthetic food additives, such as bht and bha, has decreased because of their potential risks to human health demonstrated in many experimental animals (farag et al., 2006). therefore, identifying natural sources of antioxidants that can substitute for these synthetic compounds is of great interest. olive leaves represent a potential source of natural antioxidants. in recent times, olive leaves have been used as additives to olives (2-3%) before processing to produce oils with an improved flavor and higher resistance to oxi polyphenolic compounds and antioxidants activity (abts and dpph) fa (free acidity); pv (peroxide value); iv (iodine value); k232, k270 (specific extinction) fatty acids composition fig. 1: heatmap of olive oil parameters, polyphenolic compounds profile, and antioxidants activities (dpph and abts) of the coo and eoo. the mean values refer to colors from the minimum displayed in blue to the maximum represented with yellow (i: initial value before accelerate storage condition; f: final value after accelerate storage condition). improvement of olive oil stability 173 fig. 4: chlorophylls (a) and carotenoids (b) contents of the coo and eoo under accelerated storage. fig. 2: fa (a), pv (b) and iv (c) of the coo and eoo under accelerated storage. dation (ranalli et al., 2006). it has been reported that the addition of olive leaves during the olive oil extraction process can improve the quality of the oil (malheiro et al., 2013; hannachi and elfalleh, 2020). polyphenols extracted from olive leaves have been used to increase the nutritional value of table olives (lalas et al., 2011). in addition, the enrichment of various edible vegetable oils with olive leaf extracts has been found to improve their oxidative stability (bouaziz et al., 2008). olive leaf extracts exhibit high antioxidant activity refig. 3: specific extinctions k232 (a) et k270 (b) of the coo and eoo under accelerated storage. 174 a. ayadi, y. yahia, k.ben othman, h. hannachi lating to the synergism of their compounds and may be more suitable as a functional food ingredient than olive fruit extracts (hannachi et al., 2020). the number and location of the hydroxyl group in the polyphenolic compounds influences their antioxidant activity against various radicals (xie et al., 2015). the present study noted an improvement in the oxidative stability of olive oil enriched with oleaster leaves. this enrichment quantitatively and qualitatively improved the polyphenolic composition, which explained the improvement in the oxidative stability of the eoo under accelerated storage conditions. it has been reported that polyphenolic compounds are responsible for approximately 50% of the stability of olive oil (gutiérrez et al., 2001) and show higher antioxidant activity than tocopherols in oils when exposed to high temperatures (jiménez et al., 2017). polyphenols show antioxidant potential through their ability to donate hydrogen atoms to lipid radicals, thereby producing lipid derivatives and antioxidant radicals (shahidi and ambigaipalan, 2015). paiva-martins (2007) reported a better score in taste quality after enriching oil with a polyphenol leaf extract and found insignificant differences between the flavor of oil before and after enrichment. elsewhere, the potential bioaccesibility of the polyphenol components of enriched oils compared with a control has been demonstrated based on an in vitro gastrointestinal digestion model (gastric and duodenal steps; suárez et al., 2010). various other studies have focused on the enrichment of edible oils with olive leaves, such olive, palm, and sunflower oils (bouaziz et al., 2008; japon-lujan et al., 2008; hannachi and elfalleh, 2020). oleaster leaves have been used as a food supplement added to raw minced beef as a means of enhancing safety and prolonging shelf life (djenane et al., 2019). the results of the oil standard parameters, such the pv, which measures the level of hydroperoxide products formed in the initial stages of lipid oxidation and is an indicator of the primary oxidation state of oils, showed that the eoo was more stable against lipid oxidation. the pv is one of the most frequently evaluated quality parameters during olive oil production, storage, and marketing (nouros et al., 1999). the absorptions at 232 and 270 nm are of special importance and provide information on the oxidative stability of virgin olive oil (cec, 2003). a conjugated dienic system constitutes around 90% of the hydroperoxides formed by lipoperoxidation that are absorbed in the uv range (232 nm). a conjugated triene system absorbed in the uv range of 270 nm reflects the extent of oxidation (laguerre et al., 2007). the increase of absorption at 232 nm and 270 nm due to the formation of conjugated dienes and trienes is proportional to the uptake of oxygen and the formation of peroxides during the early stages of oxidation. it has been shown that the polyphenolic compounds of hydrolysate olive leaf extract decreased the formation of lipid hydroperoxides and conjugated dienes and trienes formation in refined oils during pan-frying (bouaziz et al., 2008), confirming the results of the present study, which found that the improvement of the eoo could be explained by its higher content of active antioxidant compounds afforded by the enrichment process. hence, the enrichment of olive oil with oleaster leaves may retard the formation of oxidation products. these results agree with the results noted by aydeniz and yilmaz (2012), who observed the effect of polyphenolic compounds on the acidity of frying oils. the chlorophyll and carotenoid content of olive oil interfere with its oxidative stability and can act as antioxidants in the dark and as prooxidants in light (gutierrez et al., 1992). in the present study, enrichment increased the pigment content in eoo, which explained the improvement in oxidative stability. all samples showed reduced chlorophyll contents at the end of storage, which indicated that the compounds acted as antioxidants, as the accelerated storage condition test was conducted in the dark. chlorophylls and carotenoids provide a more intense green pigmentation and confer a higher nutritional quality (malheiro et al., 2013). a significant decrease in linoleic acid c18:2 of both the coo and eoo from 7.86 ± 1.12 to 1.43 ± 0.11 (coo) and 3.37 ± 0.17% (eoo) was noted in the present study. linoleic and palmitic acids are usually used as indicators of the extent of fat deterioration because lino leic acid is more susceptible to oxidation than palmitic acid, which is more stable (de marco et al., 2007). unsaturated fatty acids are more susceptible to oxidation and are the major cause of the deterioration of lipids (shahidi and zhong, 2010). conclusion olive oil enriched with oleaster leaves showed improved polyphenolic and chlorophyll contents, which contributed to the increase in radical scavenging activity. the application of accelerated storage conditions based on a schaal test affected the quality of both the coo and eoo, though the coo was more affected than the eoo. standard oil parameters and fatty acid composition showed that the olive oil enriched with oleaster leaves was more resistant to oxidation. the oleaster leaves protected the oil against oxidative deterioration and, therefore, constitute a natural source of antioxidants that can be used to improve the shelf life of foods. conflict of interest no potential conflict of interest was reported by the authors. references aoac, 1980: official methods of analysis of the association of official analytical chemists, 13th ed. official analytical chemists’ eds., washington, usa. tab. 4: fatty acid composition of the coo and eoo before and after accelerated storage. coo eoo accelerated storage initial final initial final c16:0 11.44 ± 1.92 a 12.35 ± 0.94 a 10.24 ± 1.00 a 10.85 ± 0.57 a c16:1 0.90 ± 0.08 a 0.99 ± 0.01 a 1.08 ± 0.05 a 1.03 ± 0.01 a c18:0 2.61 ± 0.05 a 4.20 ± 1.39 b 2.97 ± 0.04 a 3.01 ± 0.43 b c18:1 75.91 ± 5.76 a 73.62 ± 0.77 a 77.28 ± 4.59 a 73.06 ± 2.81 a c18:2 7.86 ± 1.12 a 1.43 ± 0.11 b 6.71 ± 1.10 a 3.37 ± 0.17 b c20:0 0.27 ± 0.02 a 0.36 ± 0.03 b 0.25 ± 0.02 a 0.29 ± 0.05 b c20:1 0.23 ± 0.06 a 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commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). orcid yassine yahia: https://orcid.org/0000-0002-1088-3287 hédia hannachi: https:/orcid.org/0000-0002-7550-2182 address of corresponding author: hédia hannachi, laboratory of plant productivity and environmental constraint, lr18es04, biology department, faculty of sciences, university tunis el manar, 2092, tunisia hannachi_hedia@yahoo.fr http://dx.doi.org/10.4172/2161-0444.1000124 http://dx.doi.org/10.1021/jf102203x http://dx.doi.org/10.1016/j.jff.2015.05.005 http://dx.doi.org/10.1016/j.indcrop.2015.11.044 journal of applied botany and food quality 93, 330 336 (2020), doi:10.5073/jabfq.2020.093.040 1school of agriculture, food & wine, the university of adelaide, australia 2department of soil and environmental sciences, university of barisal, bangladesh influence of the order in which low and high c/n residues on soil nutrient availability and wheat nutrient uptake shamima sabreen1, hasinur rahman1,2, petra marschner1* (submitted: august 11, 2020; accepted: february 3, 2021) * corresponding author summary it is well-known that the c/n ratio of plant residues can influence soil nutrient availability, but the effect of repeated addition of plant residues with different c/n ratio is less explored. in previous stu dies, we showed that nutrient availability and soil respiration after the second residue addition is influenced not only by the c/n ratio of that residue, but also by the c/n ratio of the previously added residue. these experiments were carried out without plants and it was unclear how the legacy effect would influence plant growth and nutrient uptake. the aim of this experiment was to assess plant growth, nutrient uptake and soil nutrient availability after the second residue addition with different length of time between the first and second residue addition where the first and second residue had the same or a different c/n ratio. high (h) or low c/n (l) residue was added at the start of the experiment, the second residue with either the same or a different c/n ratio was added on days 7, 14, 21 or 28 with a total residue addition of 20 g kg-1 giving four residue treatments: hh, ll, lh and hl. wheat was planted immediately after the second residue addition and grown for 28 days. n and p availability were measured on days 7, 14, 21 and 28 and at plant harvest. soil n and p availability after the second residue addition were in the order hh<lh<hl<ll. wheat biomass generally did not differ between ll, hl and lh, but wheat in hl and lh had a lower shoot/root ratio than in ll suggesting that in hl and lh the plants were able to compensate the lower nutrient availability by increased root growth. in conclusion, the c/n ratio of the previous residue addition influenced nutrient availability after the second residue addition, but plant growth did not differ between hl, lh and l because plants in the former developed a more extensive root system and could therefore access the nutrients released during decomposition of l even in treatments where both h and l were present in the soil. keywords: legacy effect; n availability, p availability; residue c/n ratio; shoot/root ratio; wheat introduction nutrients in organic amendments are an important source of nutrients for crops (flavel and murphy, 2006; quilty and cattle, 2011). however, nutrient release cannot be predicted reliably because decomposition of organic amendments and concomitant nutrient release are influenced by environmental factors such as temperature and rainfall which influence microbial activity, soil properties such as nutrient binding capacity and by residue properties, particularly the c to nutrient ratio (quilty and cattle, 2011). nevertheless organic amendments are likely to used more frequently in the future as inorganic fertilizer prices increase (conyers and moody, 2009; cordell et al., 2009; hargreaves et al., 2008; quilty and cattle, 2011). decomposition rate and nutrient release are influenced by the chemical composition of organic soil amendments (tian et al., 1992b; vanlauwe et al., 1996a). water-soluble organic c is quickly decomposed (hadas et al., 2004), whereas decomposition of lignin is slow (nwoke et al., 2004). nutrient concentration of the organic amendment influences not only decomposition rate but also nutrient availability. for example, organic materials with c/n > 20 and c/p > 200 are decomposed more slowly than those with lower nutrient ratios and result in at least temporary net immobilisation as microbes take up n or p from the soil to satisfy their n and p demand (enwezor, 1976). these well-known principles are used to predict nutrient release from organic amendments. however, quite often the actual amount and timing of nutrient release differs from that predicted (freschet et al., 2012; rashid et al., 2013; strickland et al., 2009). this suggests that other factors also influence decomposition rate and nutrient release. one such factor is previous management history of the soil, which has been shown to influence soil physical properties, organic matter content, nutrient availability and microbial community composition (lewis et al., 2014). (carrillo et al., 2012) found that in the three weeks after the new litter addition, available n concentrations were influenced by both the previous and new litter properties. in two recent studies, we investigated the legacy effect of the previous plant residue c/nutrient ratio on the microbial activity, biomass and nutrient availability after addition of a second residue with the same or different c/nutrient ratio (marschner et al., 2015; nguyen et al., 2016). in this context, the term legacy effect refers to the influence of the first residue on decomposition and nutrient release after the second residue is added. when the second residue was added three weeks after the first, available n and p concentrations 21 days after the second residue addition decreased in the following order: low following low c/n residue > low following high c/n residue > high following low c/n residue > high following high c/n residue (marschner et al., 2015). thus, the previous high c/n residue reduced available n concentrations after low c/n residue addition compared to low c/n after low c/n residue. on the other hand, the previous low c/n residue resulted in greater available n concentrations after high c/n residue addition compared to high c/n following high c/n residue. nguyen et al. (2016) found that when low c/n was added after high c/n residue, the available n concentration after the second residue addition was lower when the second residue was added 10 days after the first than when the interval between residue addition was 30 days. this indicated that the extent by which the legacy effect influences nutrient availability is dependent on time between residue additions. our previous studies on the legacy effect were conducted without plants. therefore it was not clear if the legacy effect influences plant growth and nutrient uptake and if this changes with time between residue additions. the aim of the present study was to assess plant growth, nutrient uptake and soil n and p availability after the second residue addition with different length of time between the first and influence of residue c/n ratio on nutrient availability 331 second residue addition. the hypotheses were: (1) n and p availabi lity after the second residue is influenced by the legacy effect, i.e., is lower when low c/n residue follows high c/n residue than if low c/n residue follows low c/n residue; (2) the legacy effect on n and p availability will decrease with time between residue additions; (3) plant growth and n and p concentrations after the second residue will be influenced by the legacy effect, i.e., will be greater if high c/n residue follows low c/n residue than if high c/n residue follows high c/n residue, and will be greater with low following high c/n residue than with high following low c/n residue. materials and methods silt loam soil was collected from 0 15 cm at waite campus, the university of adelaide (34°58’s, 138°37’e). the area is in a semiarid region and has a mediterranean climate with cool, wet winters, and hot and dry summers. the soil is classified as chromosol in australian soil classification (isbell, 2002), and a rhodoxeralf in us soil taxonomy (staff, 2014). the soil was managed for over 80 years in the waite long-term rotation trial as permanent pasture. it has the following properties: sand 70%, silt 20% and clay 10%, ph (1:5 soil: water) 7.3, electrical conductivity (ec 1:5) 742 μs cm-1, total n 134 mg kg-1 and total p 461 mg kg-1, total organic c (toc) 15 g kg-1, available n 15 mg kg-1, available p 32 mg kg-1, maximum water holding capacity (whc) 327 g kg-1 and bulk density 1.3 g cm-3. after collection, the soil was dried at 40 °c in a fan-forced oven. in summer, daytime temperatures often exceed 40 °c in the top soil and soils are air-dry for several weeks, therefore this treatment is not unnatural. after drying, visible plant debris was removed and the soil sieved to < 2 mm. before the start of the experiment, the soil was pre-incubated for 10 days at 20 °c at 50% of whc to reactivate the microbes and to stabilise their activity after rewetting. this water content was chosen because setia et al. (2011) found that microbial activity of a soil of this texture was maximal at 50% whc which was confirmed in our recent study (marschner et al., 2015). after pre-incubation, 400 g moist soil was filled into plastic pots lined with plastic bags. throughout the experiment the soil water content was maintained at 50% water holding capacity by weighing the pots daily and adjusting the weight by adding reverse osmosis (ro) water if necessary. in the period of rapid plant growth, soil moisture was adjusted twice daily. the removal of soil for analysis was considered when adjusting the water content. shoots of mature faba bean (vicia faba l.) with c/n 60 were used as high c/n residue, shoots of young kikuyu grass (pennisetum clan destinum l.) with c/n 20 as low c/n residue (tab. 1). after collec tion, the shoots were oven dried at 40 °c, ground and sieved to particle size 0.25 2 mm. residues added to pre-incubated soil at a total residue addition rate of 20 g kg-1. at the start of the experiment, either high (h) or low c/n residue (l) was added (tab. 2). in treatments where only one residue type was added (h0, h7, h14, h21, h28, l0, l7, l14, l21 and l28) 20 g kg-1 of h or l residue was added on day 0. in hl0, a 1:1 mixture of h and l was added on day 0. in the hl or lh treatments, the combination of letters indicates the order in which the residues were added, i.e. h then l or l then h. in these treatments, 10 g kg-1 residue was added on day 0 and 10 g kg-1 of residue with the other c/n ratio was added on based on the day on which the second residue was added, i.e. rd7, rd14, rd21 and rd28 when the second residue was added on days 7, 14, 21 and 28. in rd0, the second residue was added two hours after the first. at each addition, the residues were thoroughly mixed into the soil. after addition of the second residue, pre-germinated wheat seeds (triticum aestivum l. cv krichauff) were planted in all treatments of the group (h, l, hl and lh) and thinned to four plants per pot after three days. pots were kept in a glasshouse with natural light in randomized block design and watered regularly by weight. during plant growth in rd0 and rd7, temperatures in the glasshouse exceeded 35 °c during the day. although the pots were watered twice a day, the soil dried out between watering events. in the other treatment groups temperatures were lower and the soil water content could be maintained. in all treatments, the plants were harvested 28 days after planting. except for rd0, available n and p were determined every seven days between the first and second residue addition and at plant harvest. on sampling days where residue application coincided with soil samp ling, e.g. day 7 in h7 or l7, soil was sampled approximately three hours after residue addition. for a given treatment, the last soil samp ling was conducted on the day the plants were harvested, 28 days after planting. soil texture was determined using the hydrometer method (bowman et al., 2002). soil maximum water holding capacity was measured using a sintered glass funnel (haines, 1930). soil ph and ec were measured in a 1:5 (w/v) soil to reverse osmosis (ro) water ratio after 1 h end-over-end shaking. total organic carbon of soil and plant residues was determined after (walkley and black, 1934). available n was extracted in 2 m kcl. nitrate-n was measured in the filtered extract as described in miranda et al. (2001), nh4-n was determined according to willis et al. (1996). available n is the sum of no3-n and nh4-n. available p was determined by the anion exchange method of kouno et al. (1995). at harvest, plants were separated into shoots and roots which were washed and then dried at 65 °c. for total n and p in soil and shoots, the material was digested with h2so4 and a mixture of hno3 and hclo4, respectively. total n was measured by a modified kjeldahl method (vanlauwe et al., 1996b). total p in the digest was measured by the phosphovanado-molybdate method according to (hanson, 1950). shoot n and p uptake per pot was calculated by multiplying n and p concentration by shoot dry weight. shoot n and p uptake per g tab. 1: properties of low c/n (young kikuyu grass shoots) and high c/n (mature faba bean shoots) residues (n=3 ± standard error). low c/n high c/n total organic c (g kg-1) 361±5 372±3 total n (g kg-1) 17.7±0.4 6.3±0.1 total p (g kg-1) 3.7±0.1 0.2±0.0 c/n ratio 20±1 60±1 c/p ratio 97±4 1585±87 tab. 2: treatments and plant growth period, where h represents high c/n ratio residue and l low c/n residues. shaded areas indicate plant growth period. *group names are based on the day on which the second residue was added. 12 table 2. treatments and plant growth period, where h represents high c/n ratio residue and l low c/n residues. 369 shaded areas indicate plant growth period. 370 371 period (days) 0-7 8-14 15-21 22-28 29-35 36-42 43-49 treatment group* h7 rd7 h h14 rd14 h h21 rd21 h h28 rd28 h hl7 rd7 h l hl14 rd14 h l hl21 rd21 h l hl28 rd28 h l l7 rd7 l l14 rd14 l l21 rd21 l l28 rd28 l lh7 rd7 l h lh14 rd14 l h lh21 rd21 l h lh28 rd28 l h *group names are based on the day on which the second residue was added. 372 373 374 332 s. sabreen, h. rahman, p. marschner 0 h 0 20 40 60 80 100 7 h h l hl lh 7 14 h 7 14 21 h a va ila bl e n (m g kg -1 ) 0 20 40 60 80 100 7 14 21 28 h sampling day (a) rd 0 (e) rd 28(d) rd 21 (b) rd 7 (c) rd 14 f a d bc c a a b a bab e c a d b f aab e f aa f b d a c d d b a e c b a f a c a f a c a e c b a e a c f a d a a d a f e c aa pp p p p root was calculated as mg shoot n or p uptake divided by g root dry weight. statistical analysis the experiment was arranged in a randomized block design with four replicates per treatment. data was tested for normality and all data was normally distributed. data of available n, p and ph was analysed by one-way repeated measures anova for each group se parately. when the interaction between treatment and sampling time was significant, post-hoc tukey test was carried out on the treatment × sampling time interaction (p ≤ 0.05). additionally, soil data for a given sampling day was compared by one-way anova across treatment groups. plant data (root, shoot weight, shoot/root ratio, shoot n and p concentration etc.) was analysed by one-way anova followed by tukey test. results available n and p in treatments where high c/n residue (h) was added at 20 g kg-1 on day 0 (h0, h7, h14, h21 and h28), available n was low and did not change over time (fig. 1). however, it changed in the other treatments. in rd0 (residues added and wheat planted on day 0), available n after residue addition and at harvest of wheat (day 28) in l0 and hl0 (20 g kg-1 as only l or 1:1 mixture of h and l) was ten-fold higher in l0 and four-fold higher in hl0 than in h0 (fig. 1a). in treatment group rd7 (second residue added and wheat planted on day 7, harvest of wheat on day 35), available n at harvest was highest in l7 and lowest in h7 (fig. 1b). it did not differ between hl7 and lh7 where it was about 50% lower than in l7. available n decreased over time in treatments with l. when the second residue added and wheat planted on day 14 (harvest of wheat on day 42, treatment group rd14), changes in available n over time differed between l14, lh14 and hl14 (fig. 1c). in l14, which had higher available n than the other treatments on day 14 and at harvest, available n increased from day 7 to day 14 about two-fold, but then decreased by 75% to harvest. in hl14, changes in available n over time were similar as in l14, but at a much lower level (about 50% lower). in lh14, available n was highest on day 7 and decreased over time. in treatment group rd21 (second residue added and wheat planted on day 21, harvest of wheat on day 49), available n did not change between days 7 and 14 and was about 5-fold greater in l21 and lh21 than in h21 and hl21 (fig. 1d). on day 21 (after addition of the second residue in hl and lh) available n more than two-fold in l21 and eight-fold in hl21 than on day 14, but then decreased again to harvest by about 50%. in lh21, available n decreased from day 14 to harvest. in treatment group rd28 (second residue added and wheat planted on day 28, harvest of wheat on day 56), available n did not differ between days 7, 14 and 21 and was higher in l28 and lh28 than in h28 and hl28 (fig. 1e). compared to day 21, available n on day 28 (i.e. after the second residue addition in hl and lh) was two-fold higher in l28 and four-fold higher in hl28, but then decreased again to harvest with a greater relative decrease in lh28 than in l28 (by 30% compared to 10%). in lh28, available n decreased by about 50% from day 21 to day 28, but then remained unchanged until harvest. in all treatments and groups, available p was highest at harvest (fig. 2). in rd0, available p n increased about five-fold from day 0 to harvest and was highest in l0 and lowest in h0 (fig. 2a). at harvest, but not on day 0, available p was higher in hl0 than h0. in all other treatment groups, available p did not differ among treatments on day 7 (fig. 2). in rd7 at harvest, when available p was up to 10-fold higher than on day 7, it was two-fold higher in l7, hl7 and lh7 than in h7 (fig. 2b). in rd14, available p on day 14 (after the second residue addition in hl and lh) compared to day 7 was four-fold higher in l14, threefold higher in hl14 and two-fold higher in lh14 (fig. 2c). on day 14 and at harvest available p was highest in l14 and lowest in h14. available p was similar in hl and lh and about 30% lower than in l14. fig. 1: available n concentration on days 7, 14, 21, 28 and at plant harvest 28 days after planting (indicated by p) in treatments with only high c/n residue added (h), only low c/n residue added (l), high followed by low c/n (hl) or low followed by high (lh) where the second residue was added the same day as the first (a) or 7 (b), 14 (c), 21 (d) or 28 (e) days after the first (rd0, rd7, rd14, rd21, rd28) 7 (a), 14 (b), 21 (c) or 28 (d) days after the first (rd7, rd14, rd21, rd28) (n=4 ± standard error). columns within a treatment group with different letters are significantly different (p≤ 0.05). influence of residue c/n ratio on nutrient availability 333 available p in rd 21 was generally highest in l21 and lowest in h21 (fig. 2d). on day 14, it was about 50% lower in hl21 than lh21, but after the second residue addition in hl and lh on day 21, later available p differed little between the two treatments where it was at least two-fold higher than in h21. in rd28, available p form day 14 was highest in l28 (fig. 2e). on day 14, available p was lower in hl28 than in lh28, but the reverse was true on day 28 (after addition of the second residue) and at harvest. in hl28 and lh28 available p was about 30% lower than l28 and two-fold higher than in h28. the soil ph ranged between 6 and 6.8 and changed little over time (data not shown). it tended to be highest after addition of h and lowest after l amendment. wheat growth and nutrient uptake treatment differences were more pronounced in shoot dry weight than total plant dry weight (tab. 3). shoot dry weight was always lowest with only h added and highest with only l added. compared to the treatment with only l added, shoot dry weight was 20-30% lower in the two treatments with both l and h added (lh and hl). shoot dry weight was similar in hl and lh, except for rd28 where it was higher in lh28 than hl28. root dry weight did not differ among treatments in rd0, rd7 and rd14 (tab. 3). but in rd21 and rd28, root dry weight was lower in treatments with only h and l than in hl and lh. total plant dry weight was lowest in the treatments with only h added and highest with only l added (tab. 3). it was similar in lh and fig. 2: available p concentration on days 7, 14, 21, 28 and at plant harvest (indicated by p) in treatments with only high c/n residue added (h), only low c/n residue added (l), high followed by low c/n (hl) or low followed by high (lh) where the second residue was added the same day as the first (a) or 7 (b), 14 (c), 21 (d) or 28 (e) days after the first (rd0, rd7, rd14, rd21, rd28) (n=4 ± standard error). columns within a treatment group with different letters are significantly different (p≤ 0.05). tab. 3: shoot, root and total dry weight (g pot-1) of wheat 28 days after the second residue addition in treatments with only high c/n residue added (h), only low c/n residue added (l), high followed by low c/n (hl) or low followed by high (lh) where the second residue was added 7, 14, 21 or 28 days after the first (rd7, rd14, rd21, rd28) (n=4 ± standard error). values followed by different letters are significantly different (p≤ 0.05). shoot root total shoot/root ratio dry weight (g pot-1) rd7 h7 0.09 ±0.01 a 0.17 ±0.01 abcd 0.25 ±0.02 a 0.51 ±0.04 a l7 0.52 ±0.01 gh 0.20 ±0.01 abcde 0.71 ±0.02 fg 2.66 ±0.14 h hl7 0.30 ±0.02 b 0.20 ±0.02 bcde 0.50 ±0.04 cd 1.52 ±0.07 cde lh7 0.29 ±0.02 b 0.21 ±0.02 bcde 0.49 ±0.04 bc 1.41 ±0.07 cd rd14 h14 0.21 ±0.10 a 0.15 ±0.01 abc 0.36 ±0.11 a 1.38 ±0.63 ab l14 0.44 ±0.11 efg 0.13 ±0.04 abcde 0.57 ±0.16 fg 2.63 ±0.69 i hl14 0.45 ±0.03 efg 0.19 ±0.01 abcde 0.63 ±0.04 cdefg 2.38 ±0.08 h lh14 0.43 ±0.03 ef 0.21 ±0.02 bcde 0.64 ±0.05 cdefg 2.09 ±0.11 fgh rd21 h21 0.10 ±0.01 a 0.13 ±0.01 a 0.23 ±0.01 a 0.79 ±0.06 ab l21 0.64 ±0.01 i 0.14 ±0.00 ab 0.78 ±0.01 g 4.47 ±0.18 j hl21 0.50 ±0.02 gh 0.22 ±0.02 cde 0.72 ±0.04 fg 2.31 ±0.11 gh lh21 0.47 ±0.01 fgh 0.24 ±0.02 e 0.71 ±0.02 fg 2.00 ±0.14 fgh rd28 h28 0.10 ±0.01 a 0.15 ±0.01 abc 0.25 ±0.02 a 0.67 ±0.02 ab l28 0.50 ±0.05 gh 0.16 ±0.02 abcd 0.66 ±0.06 defg 3.25 ±0.10 i hl28 0.37 ±0.01 cde 0.22 ±0.01 de 0.59 ±0.01 cdef 1.67 ±0.09 def lh28 0.45 ±0.01 efg 0.24 ±0.01 e 0.69 ±0.02 defg 1.91 ±0.09 efg 0 h 0 20 40 60 80 100 120 7 h h l hl lh 7 14 h 7 14 21 h a va ila bl e p (m g kg -1 ) 0 20 40 60 80 100 120 7 14 21 28 h (a) rd 0 (e) rd 28(d) rd 21 (b) rd 7 (c) rd 14 sampling day a b a a e bc d ab d cd cd ab cd f e e d a d b d c a ab c d e a ab ab f e d bc h g f e ab d a e c a bc b bc b a b p p p p p 334 s. sabreen, h. rahman, p. marschner hl and about two-fold than with h only. total plant dry weight in hl and lh was about 25% lower than l in rd0 and rd7, but did not differ among these treatments when the second residue was added 14 or more days after the first (rd14, rd21 and rd28). the shoot/root dry weight ratio was lowest when only h residue was added and highest with only l residue (tab. 3). in hl and lh, the ratio was lower than with only l added, but more than twice as high than with only h amendment. the shoot n and p concentration with only h residue added was about 30% lower than in the other treatments (tab. 4). shoot n concentration was similar in treatments with l residue (l, hl and lh), except in rd0 where it was higher in the mixed treatments than with only l added. shoot p concentration did not differ among treatments with l addition (l, hl and lh), except in rd14 and rd21 where it was about 25% lower in hl and lh than in l. shoot n and p uptake (mg pot-1) was lowest with only h amendment where it was four to six-fold lower than in the treatments with l added (l, lh and hl) (tab. 4). shoot n uptake was about 25% lower in hl and lh than in l except in rd0 and rd14 where it did not differ among treatments with l. shoot p uptake was 30-50% lower in hl and lh than in l, the only exception was rd28 where shoot p uptake did not differ between l and lh. shoot n and p uptake per g root was about five-fold lower when only h was added compared to treatments amended with l (tab. 5). shoot n uptake per g root was highest with only l amendment where it was 25-30% higher than in hl and lh which did not differ in shoot n uptake per g root. compared to hl and lh, shoot p uptake per g root was 0.3 to two-fold higher with only l amendment. discussion this study confirmed the existence of a legacy effect of the previous residue addition on nutrient availability after the second addition (marschner et al., 2015; nguyen et al., 2016), but also showed that in treatments where both low and high c/n residues are present (hl and lh) plants compensate lower n and p availability compared to l only by developing a more extensive root system. available n and p the first hypothesis (n and p availability after the second residue are influenced by the legacy effect, i.e., is lower when low c/n residue follows high c/n residue than if low c/n residue follows low c/n residue) can be confirmed. tab. 4: shoot and root n and p concentration (g kg-1) and uptake (mg pot-1) of wheat 28 days after the second residue addition in treatments with only high c/n residue added (h), only low c/n residue added (l), high followed by low c/n (hl) or low followed by high (lh) where the second residue was added 7, 14, 21 or 28 days after the first (rd7, rd14, rd21, rd28) (n=4 ± standard error). values followed by different letters are significantly different (p≤ 0.05). shoot n concentration p concentration n uptake p uptake group treatment g n kg-1 mg p kg-1 mg n pot-1 mg p pot-1 rd7 h7 19.11 ±2.62 b 3.50 ±0.16 b 1.56 ±0.10 a 0.30 ±0.03 a l7 33.18 ±1.08 efgh 6.12 ±0.15 h 17.07 ±0.62 f 3.15 ±0.06 h hl7 34.66 ±1.40 fgh 5.28 ±0.28 efgh 10.40 ±0.49 cd 1.59 ±0.11 bcde lh7 31.57 ±2.41 efgh 4.70 ±0.33 cdefg 8.84 ±0.23 bcd 1.32 ±0.06 bc rd14 h14 21.97 ±1.84 ab 4.22 ±0.52 bc 5.24 ±3.02 a 1.05 ±0.65 a l14 20.13 ±6.11 bcde 4.22 ±1.25 fgh 11.69 ±2.89 ef 2.44 ±0.61 gh hl14 28.09 ±2.36 efgh 4.37 ±0.26 bcde 12.30 ±0.15 de 1.92 ±0.03 cdef lh14 27.62 ±1.91 cdef 3.98 ±0.12 bcd 11.76 ±0.15 de 1.71 ±0.09 cdef rd21 h21 21.82 ±0.26 bcd 3.84 ±0.08 bcd 2.18 ±0.14 a 0.38 ±0.03 a l21 36.16 ±0.21 h 4.94 ±0.11 defg 22.96 ±0.36 g 3.13 ±0.05 h hl21 35.46 ±0.57 gh 4.17 ±0.24 bcde 17.81 ±0.75 f 2.08 ±0.06 def lh21 31.62 ±1.02 efgh 3.63 ±0.15 bc 14.78 ±0.58 ef 1.69 ±0.05 cdef rd28 h28 22.90 ±0.55 bcd 2.25 ±0.07 a 2.29 ±0.16 a 0.22 ±0.02 a l28 33.94 ±1.41 efgh 4.59 ±0.44 bcdefg 17.15 ±2.05 f 2.37 ±0.48 fg hl28 30.69 ±1.23 efgh 3.84 ±0.15 bcd 11.38 ±0.70 de 1.43 ±0.09 bcd lh28 35.21 ±1.21 fgh 4.18 ±0.31 bcde 15.84 ±0.62 f 1.88 ±0.14 cdef tab. 5: shoot n and p uptake per g root of wheat 28 days after the second residue addition in treatments with only high c/n residue added (h), only low c/n residue added (l), high followed by low c/n (hl) or low followed by high (lh) where the second residue was added 7, 14, 21 or 28 days after the first (rd7, rd14, rd21, rd28) or full residue amount added on day 0 (rd0)(n=4 ± standard error). values followed by different letters are significantly different (p≤ 0.05). group treatment n uptake p uptake mg pot-1 rd0 h0 4.96 ±0.30 a 1.83 ±0.17 ab l0 43.37 ±4.06 bc 12.54 ±0.48 fg hl0 39.83 ±0.66 b 5.51 ±0.26 bcd rd7 h7 9.59 ±0.94 a 1.78 ±0.10 ab l7 87.99 ±2.91 f 16.32 ±1.09 gh hl7 52.52 ±2.81 bcd 8.02 ±0.63 de lh7 44.68 ±4.78 bc 6.66 ±0.69 cde rd14 h14 15.70 ±1.09 a 2.83 ±0.11 abc l14 87.29 ±4.58 f 18.28 ±1.57 hi hl14 66.60 ±5.10 de 10.37 ±0.58 ef lh14 57.92 ±6.27 bcd 8.34 ±0.65 de rd21 h21 17.25 ±1.10 a 3.03 ±0.19 abc l21 161.75 ±6.76 h 22.07 ±0.84 i hl21 81.99 ±4.21 ef 9.72 ±0.95 ef lh21 63.51 ±6.19 cde 7.28 ±0.71 de rd28 h28 15.40 ±0.64 a 1.51 ±0.07 a l28 110.12 ±3.26 g 14.91 ±1.41 gh hl28 51.60 ±4.76 bcd 6.44 ±0.52 cde lh28 66.99 ±1.81 de 8.04 ±0.92 de influence of residue c/n ratio on nutrient availability 335 before the second residue addition, available n and p were as expected from many previous studies (kwabiah et al., 2003; tian et al., 1992a). they followed the order h=hl < lh=l. immediately after the second residue addition (days 7, 14, 21 and 28 in rd7, rd14, rd21 and rd28, respectively), available n and p were influenced by the legacy effect because they were affected by both c/n ratio of the second residue and the c/n ratio of the previously added residue. they were in the order h < lh < hl < l. thus in lh, presence of l in soil when h was added increased available n and p compared to h whereas addition of l into a soil containing h (hl) reduced nutrient availability compared to l. but at harvest of wheat (28 days later), differences in available n and p between hl and lh were smaller or there were no differences between the two treatments. in previous studies, we also found that differences in available n and p between hl and lh decreased with time after the second residue addition (marschner et al., 2015; nguyen et al., 2016). this can be explained by decreasing residue decomposition rates due to depletion of easily decomposable compounds (marschner et al., 2014; wessels perelo and munch, 2005), and in this study, plant n and p uptake. compared to immediately after the second residue addition, available n was lower at harvest, which indicates that plant n uptake exceeded n net mineralisation. in contrast, available p was higher at harvest than immediately after the second residue addition, suggesting than p mineralisation was greater than p uptake, probably because root density was not very high and p is poorly mobile in soil (e.g. bertrand et al., 2003). the lower ph in soil amended with l compared to h residue is likely due to greater nitrification with the former due to its higher n concentration (xu et al., 2006). the second hypothesis (the legacy effect on n and p availability will decrease with time between residue additions) can be confirmed for available n, but not for available p. n availability did not differ between hl and lh when the second residue was added seven days after the first (rd7) and the differences were smaller when there were 14 days between first and second addition (rd14) than in treatments where the second residue was added 21 or 28 days after the first (rd21, rd28). the lack of difference between hl and lh when there were two weeks or less between residue additions may be because relatively large amounts of the previously added residue was still in the soil when the second residue was added. with longer time between residue additions, the amount of the previously added residue would be low and nutrient availability mainly affected by the second residue added. there was no clear pattern in available p immediately after the second residue addition with time between residue additions. available p did not differ among treatments in rd7 and rd21, but in rd14 and rd28, the available p concentration was greater in hl than in lh. wheat growth and nutrient uptake plant growth in rd0 and rd7 was lower compared to the other treatment groups. this is likely to be due to the much higher temperature in the glasshouse during this time (> 35 °c during the day) compared to the other treatment groups (20 25 °c during the day). this tempe rature difference may have confounded any effect of time between residue additions on plant growth. to minimise this environmental effect in future experiments, plant growth should occur in all treatments at the same time. when only h was added, shoot and total plant dry weight as well as shoot n and p concentrations were low compared to the other treatments which can be explained by the low soil available n and p concentrations. shoot dry weight in h was about five-fold lower than in l and the shoot/root dry weight ratio was also lower in h. thus the plants responded to the low nutrient availability in h by a growing a more extensive root system, which is a common response in plants to the low nutrient availability (marschner, 2012). however, even with the greater root system, shoot and total plant growth and shoot n and p concentrations were lower in h than in the other treatments. wheat in the treatment with only l added had the highest shoot and total dry weight and up to two-fold higher shoot n and p concentrations than plants with only h added which can be explained by the greater available n and p concentrations in the former. since root dry weight did not differ between the two treatments, the shoot/root ratio was lower in l than in h. a relatively small root system compared to the shoots is a common plant response to high nutrient availability (marschner, 2012). shoot n concentrations were higher in lh than in h, which can be explained by the higher soil n availability in the former. however, shoot n concentrations did not differ between hl and l although n availability was lower in the former. however, hl had a lower shoot/ root ratio than l. thus relative to the shoots, plants in hl had more roots than those in l which allowed them to take up more n than would be expected based on available n concentrations. the third hypothesis (plant growth and n and p concentrations after the second residue will be influenced by the legacy effect, i.e., will be greater if high c/n residue follows low c/n residue than if high c/n residue follows high c/n residue, and will be greater with low following high c/n residue than with high following low c/n residue) can only be partly confirmed. plant growth and shoot n and p concentrations after the second residue addition were influenced by the legacy effect, i.e., were greater if high c/n residue followed low c/n residue (lh) than only high c/n residue was added. however, plant growth and n and p concentrations did not differ between hl and lh. it was expected that plant growth and shoot n and p concentrations would be lower in lh the hl because available n and p concentrations immediately after the second residue addition were lower in the former. however, there were no differences in shoot, root dry weight or shoot n and p concentrations between the two treatments. this indicates that the plants were able to compensate differences in available n and p concentrations in the soil by an extensive root system. time between the first and second residue influenced soil n availability, but not the differences in total plant dry weight between l and hl or hl and lh. this too indicates that wheat compensated lower nutrient availability by growing more roots. conclusion the study showed that plants can compensate for legacy-induced differences in nutrient availability by a larger root system. this larger root system also over-rode the effect of time between residue additions on soil nutrient availability. thus the impact of the legacy effect on crop growth may be smaller than expected from soil nutrient availability. this indicates for plants with an extensive root system, the order in which h and l are added and the time between residue additions is not important. however, in soils where root growth is restricted or in plants with inherently small root systems, the influence of the legacy effect on plant growth may be 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berlin, division urban plant ecophysiology, section quality dynamics / postharvest physiology, berlin, germany 2leibniz-institute for agricultural engineering potsdam-bornim e.v., potsdam, germany impact of postharvest uv-c and ozone treatment on textural properties of white asparagus (asparagus offi cinalisof white asparagus (asparagus offi cinalisof white asparagus ( l.)* s. huyskens-keil1**, k. hassenberg2, w.b. herppich2 (received september 1, 2011) summary optimization of postharvest treatments and storage requirements to reduce microbiological spoilage is essential for the food supply chain of asparagus. in this context, generally recognized as safe (gras) treatments such as uv-irradiation and washing with ozonated water gain more and more importance. information on uv-c and ozone as postharvest treatment for quality assurance of white asparagus is scanty. in the present study, asparagus spears were harvested and exposed to the above mentioned treatments and their combination. the infl uence of both postharvest treatments on biomechanical and biochemical textural related cell wall metabolism was investigated. uv-c-irradiation and washing with ozonated water resulted in a slight reduced respiration in white asparagus spears, but increase in spear tissue toughness. total cell wall compounds were only tendentiously reduced after 4 days of shelf-life at 20 °c by application of aqueous ozone and uv-c. however, the dosages used in this experiment were relatively low and, hence, did not have pronounced effects. furthermore, the possible mechanism of uv-c and ozone mediated changes in textural related enzyme activities of white asparagus spears have to be investigated in more detail. introduction the demand for white asparagus (asparagus offi cinalisthe demand for white asparagus (asparagus offi cinalisthe demand for white asparagus ( l.) has greatly increased during recent years. this is not only due to its unique fl avour; asparagus is also considered a highly nutritional vegetable containing important antioxidant and anticancerogenic compounds. the fl eshy spears of white asparagus are developmental immature and rapidly growing subterraneous shoots (o’donoghue and somerfield, 1998). after harvest, they retain their physiological activity and even continue growth at high rates leading to a rapid decline of respiratory substrates and drastic changes in textural related cell wall metabolism (herppich et al., 2005). the unaltered continuation of shoot differentiation also includes thickening and lignifi cations of cell walls of both sclerenchyma sheath cells and vascular bundles (waldron and selvendran, 1990). the spears become fi brous and though being highly undesired in horticulture. thus, toughness of asparagus spears is a major factor in determining their quality. as asparagus spears are purchased not only as fresh commodity but increasingly as convenience product (i.e. sliced, fresh-cut), quality assurance has to focus on one hand on the retardation of metabolic processes accompanied by undesired textural changes and, on the other hand, on the avoidance of microorganism development in postharvest, thus meeting hygienic requirements. in food industry, losses due to microbiological spoilage (e.g. pectobacterium carotovorum, phythophthora infestans, escherichia coli etc.) have been estimated as being as high as 30 %. therefore and due to new food safety regulations (haccp concept, traceability), the optimization of postharvest treatments and storage requirements is an essential tool for the food supply chain management of asparagus. postharvest treatments targeted to sanitation purposes may include physical treatments (e.g. uv-irradiation, gamma-irradiation) or fumigation treatments with generally recognized as safe (gras) compounds such as ozone (jamieson et al., 2009). sanitation treatments with chlorine or methylbromide are forbidden by law. thus, uv-irradiation and ozone gain more and more importance. numerous studies have already been focused on the bactericidal and fungicidal capacity of ozone and uv-c treatments, but information on product quality attributes and physiological responses is limited. ozone is a strong oxidizing agent that acts on carbon residues dissolved in the washing water as well as on the product surface, and thus, it is very effective in destroying microorganisms. the advantage of using ozone is the effi cacy at low concentrations and short contact times as well as the absence of detectable residues in/on treated food due to the quick auto-decomposition to oxygen (jamieson et al., 2009). it can be applied as gas or dissolved in water and has been used to sanitizing surfaces or for water desinfection being also implemented into excisting washing processes (e.g. hassenberg et al., 2007; artés et al., 2009), and, thus, extend storage time of perishable food. due to its effi cient bactericidal and fungicidal properties, ozone is applied to several food products such as tomato, strawberry, grape and plum (tzortzakis et al., 2007a; rodoni et al., 2010), carrot (hassenberg et al., 2008), apple, strawberry, lettuce (hassenberg et al., 2007) and cantaloupe (rodgers et al., 2004). however, rather less attention has been paid to the impacts of postharvest ozone application on quality attributes, nutritional or health promoting composition of horticultural products. skog and chu (2001) and salvador et al. (2006) reported that ozone signifi cantly improved quality and storage life of broccoli, cucumber and persimmon, respectively. keutgen and pawelzik (2008), tzortzakis et al. (2007b), and aguayo et al. (2006) reported similar fi ndings, demonstrating an inhibitory effect of ozone on undesired changes of anthocyanins, phenols, ascorbic acids in some strawberry cultivars and of carbohydrates, carotenoids and phenolic compounds in tomato fruits. however, application of low ozone might even enhance secondary plant metabolites such as phenolic compounds (artés-hernández et al., 2007) assumingly due to plant stress responses. moreover, ozone treatments may also result in loss of ascorbic acid (quiang et al., 2005), anthocyanin content and inhibition of desired volatile compounds (perez et al., 1999). in terms of ozone-mediated changes in textural quality, only few and contradictory studies have been performed. those studies revealed either an ozone-induced delay in fruit softening as shown for strawberry and tomato (rodoni et al., 2010; perez et al., 1999), the inhibition of undesired textural related cell wall modifi cations i.e. in green asparagus (an et al., 2007), or negative effects on membrane stability in carrots (liew and prange, 1994) and enzyme inactivation associated with the loss of crispness in lettuce (artés et al., 2009). in contrast to the limited knowledge on the impact of ozone on * this paper was presented at the 46th meeting of the „deutsche gesellschaft für qualitätsforschung (pfl anzliche nahrungsmittel)“ dgq e.v. ** corresponding author 230 s. huyskens-keil, k. hassenberg, w.b. herppich characteristic quality attributes of fruit and vegetables during storage, a comprehensive view of uv-c effects in postharvest is present. the use of uv-c irradiation in the wavelength range of 190-280 nm is primarily effective for surface decontamination of perishables. uv-c light can directly damage microbial dna (artés et al., 2009) or act by inducing the synthesis of plant secondary metabolites that effectively block or slow spore germination in plant tissue (perkins-veazie et al., 2008). application of uv-c offers several advantages as it does not leave any residue, it is lethal to most types of microorganisms, and it does not require extensive safety equipment to be implemented (artés et al., 2009). many studies have reported the advantageous effects of low or sublethal doses of uv-c (0.2-14 kj m-2) to reduce microbial growth in e.g. broccoli (lemoine et al., 2007), strawberry (marquenie et al., 2002; pan et al., 2004), carrots (mercier et al., 2000), grapefruit (d’hallewin et al., 2000), processed food (bintsis et al., 2000), peach (el ghaouth et al., 2003) and tomatoes (liu et al., 1993). however, uv-c is also known to delay ripening-associated and postharvest senescence processes (barka et al., 2000; costa et al., 2006; gonzalez-aguilar et al., 2007; poubol et al., 2010), to reduce chilling injury in pepper (vicente et al., 2005) and to improve fi rmness retention (maharaj et al., 1999; barka et al., 2000; lamikanra et al., 2005; pombo et al., 2009). contradicitive fi ndings are reported by allende et al. (2006) and artés et al. (2009), who found an uv-c induced tissue softening and browning in lettuce and caulifower, while no effect of uv-c on green asparagus spear texture was found (poubol et al., 2010). as demonstrated, uv-c contributes to alleviate and reduce tissue colonization by pathogen (e.g. pan et al., 2004) and on the other hand, uv-c affects several physiological processes, i.e. acting indirectly by stimulating plant defence mechanism in fruit and vegetables, leading to an accelerated synthesis of secondary plant metabolites and thus, is able to enhance nutraceutical content (cisneros-zevallos, 2003; perkins-veazie et al., 2008). nevertheless, information on physiological responses and textural properties of white asparagus as affected by ultraviolet-c (uv-c) radiation and ozonated water is scant. moreover, the mechanism underlying the mode of action of these physical and chemical postharvest treatments on texture related cell wall metabolism is poorly understood. hence, the aim of our investigation was to evaluate the plant responses to uv-c and ozone in terms of quality-related textural properties of asparagus spears during shelf-life. material and methods plant material and experimental setup freshly harvested from a commercial fi eld (spargelhof nottebohm gbr, kartzow, germany), white asparagus spears of the cultivar gijnlim were transported to the laboratory, washed, sorted according to ec quality standard class i, cut to a length of 22 cm (mean spear diameter: 1.8 ± 0.2 cm) and randomly separated into 27 batches of approximately 500 g. thereafter, batches of spears were subjected to the following treatments a) uv-c (254 nm) at 1 kj m-2 for 8 min using a 12 w vl-6c uv-c light source (6 w-254 nm tube, vilber lourmat, marne-la-vallee, france) or b) submerged in ozonated water (3 ppm) for 30 s or c) combined treatment of uv-c (1 kj m-2 for 8 min) and ozonated water (3 ppm) for 30 s. ozonated water was generated using a “bewazon 1“ ozone generator (0.02 g o3 min-1; bwt water technology ltd., schriesheim, germany). for all o3-treatments, water temperature was set to 10 °c using a thermostat 45 (haake, karlsruhe, germay). ozone concentration was measured with a complementary chlorine/ozone cuvette test applying a lasa® 2plus photometer (dr. bruno lange gmbh & co., düsseldorf, germany). before treatments as well as after 2 and 4 d of storage, treated and untreated (control) spears were shock-frozen in liquid nitrogen and kept at -25 °c for further analysis of cell wall composition (pectic substances, cellulose, hemicellulose, lignin). during storage, each batch of treated and control spears was placed loosely in a plastic container (30 x 40 x 5 cm2) and fully covered with cloths, which was carefully soaked with demineralised water. in this water vapour saturated atmosphere, spears were stored at temperatures of 20 °c for up to four days. the experiment was conducted with three replicates per treatment and repeated three times. determination of respiration and biomechanical properties on the initial day of the experiment (day 0), 12 spears were used for measurements to evaluate the biological variability. on days 2 and 4 of the experiment, six spears per treatment (2 of each batch) were randomly taken out of storage and equilibrated to room temperature (approximately 21.4 ± 0.9 °c) in water vapour saturated atmosphere for 1 h. then, co2 release of mixed samples of three asparagus shoots was measured at 20 °c in closed perspex® cylinders with infrared sensors (fya600co2, ahlborn messund regeltechnik gmbh, holzkirchen, germany). from the increase in co2 concentration over time and the spears’ dry mass, potential respiration activity was calculated as mmol g-1 d-1. afterwards, fresh mass (fm, electronic balance bp 210 s, sartorius ag, göttingen, germany), total length, and the diameters at positions 2.5 cm, 7.5 cm, 12.5 cm and 18 cm from the base (electronic sliding calliper) were determined for each spear. thereafter, the spears were sliced with a stainless steel microtome blade (feather s35, 0.26 mm total thickness) adapted to a zwicki 1120 material testing machine (zwick, ulm, germany; crosshead speed 600 mm min-1) to obtain tissue strength e was calculated from the length of deformation at a force of 2 n (atkins and vincent, 1984; billau et al., 1990; herppich et al., 2005). mean cutting force over the entire spear diameter (fcut) and the actual cutting length (lcut) was used to calculate the cutting energy (ecut = fcut / (lcut * π/4), which is closely related to spear toughness and fi brousness (vincent, 1990). from the sections, fresh mass (fm) was obtained. all sections were dried in an oven (85 °c) to constant weight for determination of the spear dry mass (dm). dry mass related water content was calculated as wcdm = (fm – dm)/dm. analysis of biochemical cell wall properties approximately 300 g of shock-frozen asparagus spears of each treatment were freeze-dried (christ alpha 1-4, christ; osterode, germany), and thereafter subjected for further analysis of cell wall content of proteins, pectic substances, cellulose, hemicellulose and lignin which in total are presented in fig. 3 as total cell wall compounds. the alcohol insoluble fraction (aif) was used to determine the cell wall protein content modifi ed according to the method described by bradford (1976). fifty mg of the dried ground material was dispersed in 1 ml phosphate buffer with a vortex mixer (3 x 10 s), and afterwards centrifuged (11400 rpm, 15 min) at 4 °c. in a reaction tube, 100 µl of the supernatant were diluted with 900 µl phosphate buffer, 1 ml coomassie blue added and the carefully mixed solution measured photometrically (595 nm) after 20 min (pu 8730, philips, kassel, germany). a calibration series (10 to 50 µg) was obtained from phosphate buffer and bovine serum albumin. cell wall extraction for the determination of pectic substances (water soluble pectin, edta-soluble pectin and water insoluble pectin) was conducted according to blumenkrantz and asboehansen (1973) modified by huyskens (1991). the colorimetric uv-c and ozonated water treatment of white asparagus 231 determination of the pectin fractions was conducted using metahydroxybiphenyl (mhdp, sigma h 6527, sigma-aldrich chemie gmbh, münchen, germany) as a colour reagent and following the method described by mccomb and mccready (1952). in each fraction, the amount of galacturonic acid was measured photometrically (pu 8730, philips, kassel, germany) at 520 nm. analyses were performed with three replications for each treatment. the content of pectic substances was expressed as mg galacturonic acid g-1 dry mass. cellulose and lignin were analysed according to the methods of goering and van soest (1972) and aoac (1999). one gram freeze-dried sample was extracted with 100 ml acid detergent fibre (adf) reagent (n-cetyl-n, n,n-trimethyl-ammonium bromide dissolved with 96 % h2so4) using a fibertec system (m 1020, tecator, sweden). thereafter, the solution was vacuum fi ltered, washed with boiled double distilled water until removal of the acidity and again washed with 90 % acetone. the residue was dried at 105 °c for 24 h, weighed, ash-dried at 500 °c for 24 h and weighed again to calculate adf. the dried adf residue was used for acid detergent lignin (adl) determination. cellulose content was calculated as the difference between adf and adl. the content of lignin and cellulose, respectively, were expressed as mg g-1 dry mass. with the neutral detergent fiber (ndl) approach (van soest and goering, 1963), one gram of freeze-dried material was cooked in 100 ml of ndl mixture (titriplex iii, disodium borate, dodecyl hydrogen sulphate sodium, ethylene glycol monoethyl ether) to determine the hemicellulosic cell wall fraction. afterwards, the solution was vacuum fi ltered, washed with demineralized water and with 90 % acetone. the insoluble residue was dried at 105 °c for 24 h, weighed, ash-dried at 500 °c for 24 h and weighed again to calculate ndf. the hemicellulose content was obtained by subtracting adf from ndf and given as mg g-1 dry mass. statistical analysis all data were statistically analysed (anova) with spss 13.0 (spss inc., usa). signifi cant differences were determined by tukey test (p < 0.05). in fi gures, the mean variability of data was indicated by the standard deviation. results and discussion asparagus spears are known for their high postharvest metabolic activity which leads to a rapid decline of nutritionally valuable respiratory substrates such as sugars or organic acids. moreover, the unaltered shoot differentiation also includes thickening and lignifi cations of cell walls in the sclerenchyma ring and in vascular bundles. these processes rapidly result in the undesired toughening of spears. in practice, these changes in postharvest spear quality are predominantly controlled by ice-cooling of harvested spears and cold storage. on the other hand, uv-c irradiation or washing with ozonated water, which may be applied for fresh produce sanitation in various european and non-european countries, are known to potentially affect texture properties of treated produce. however, studies on the impact of these treatments on spear textural properties are rare (an et al., 2006; an et al., 2007; poubol et al., 2010) and information yet published on possible responses of plants to these treatments is equivocal. therefore, this study elaborates the effects of uv-c and ozone treatment on physiological activity of white asparagus spears and focused on their impact on the texture related biochemical and biophysical properties of spears. in the presented investigation, respiration, an indicator of overall physiological activity, continuously declined during storage. the observed changes were, however, only slightly infl uenced by uv-c or ozone or the combined application of both (fig. 1). it could be observed that both treatments tended to reduce the physiological activity of asparagus spears during shelf-life, i.e. they alleviated respirational carbon losses. nevertheless, this effect was only limited and not signifi cant in comparison to control spears. there are some reports that washing with ozonated water, also in combination with uv-c, do not reveal a pronouncedly retard respiration and ethylene production, e.g. in tomatoes (tzortzakis et al., 2007b) and brassica spp. (martinez-sánchez et al., 2008). signifi cant inhibition of microorganism development by uv-c and ozone was demonstrated for many horticultural commodities (e.g. tzortzakis et al., 2007a; hassenberg et al., 2008; rodoni et al., 2010): ozone and uv-c treatments, on the other hand, have been reported to accelerate physiological processes, thus, reducing overall produce quality. in this context, enhanced respiration may indicate metabolic disturbance due to a rising energy and substrate demand for repair processes of ozone or uv-c mediated physiological injuries (liew and prange, 1994). for ozonation, this increase in respiration, being possibly associated with a rise in ethylene production, has been reported to be accompanied by decrease tissue and membrane stability (liew and prange, 1994). this was obviously not the case in white asparagus spears. in green asparagus spears, uv-c irradiation was found to accelerate respiration; this response was also accompanied by changes in spear crispness (poubol et al., 2010). in the present study, there was a signifi cant increase in cutting energy and, thus, in tissue toughness of all asparagus spears within four days of shelf-life (fig. 2). this increase could not signifi cantly be prevented by the postharvest treatments applied. nevertheless, washing with ozone tendentiously retarded the increase in spears toughness during prolonged storage. in contrast, uv-c irradiation and its combination with ozone resulted in an enhanced cutting energy compared to control spears on day 4 of storage. this may refl ect the fi ndings of poubol et al. (2010); it may point to more general shoot response to increased uv irradiation. in addition, spear stiffness declined during the entire storage period, i.e. spears became more elastic irrespective of the treatments (data not shown). results reported on the effects of washing with low ozone concentrations (0.05 1.0 µmol mol-1) as well as low uv-c doses (0.5 4.0 kj m-2) on elastic properties of fruits are highly equivocal. low-level ozone (0.005 1.0 µmol mol-1) as well as low uv-c (0.5 4.0 kj m-2) applications were reported to have no pronounced effect on fruit fi rmness of tomato (tzortzakis et al., 2007b) and blueberry (perkins-veazie et al., 2008), while in contrast, uv-c irradiation even induced tissue deterioration in fig. 1: respiration rate (mg co2 kg h-1) of asparagus spears during storage (4 days at 20 °c in water saturated air) as affected by postharvest treatments of uv-c (1 kj m-2 for 8 min), ozonated water (3 ppm for 30 s) and combined uv-c and ozonated water. data are means ±sd (n=6). 232 s. huyskens-keil, k. hassenberg, w.b. herppich lettuce and caulifower (allende et al., 2006; artés et al., 2009). the uv-c effect, however, obviously dependent on the irradiation dosage applied. in green asparagus, low uv-c doses of up to 2.4 kj m-2 prevented asparagus spear toughening for 4 days of storage; in contrast application of uv-c above 3.8 kj m-2 did not show any effect on textural properties (poubol et al., 2010). this is not consistent with our results. even the very low uv-c doses (1 kj m-2) applied signifi cantly enhanced tissue toughness in white asparagus spears. the observed changes in white asparagus spear toughness were not due to any variation in tissue water status (data not shown; herppich et al., 2005). in contrast, they have been shown to be largely associated with changes in structural cell wall components (herppich and huyskens-keil, 2008). in the presented study, however, the general increase in the cell wall components (cellulose, hemicelluloses, pectic substances, lignin and protein) in control spears (fig. 3), was even retarded by aqueous ozone, uvc and combined ozone/uv-c applications. this was attributed to changes in the composition of the structural major cell wall components. whereas cellulose and hemicellulose were not infl uenced by the treatments (data not shown), the general increase in lignin (fig. 4) and pectin (fig. 5) appeared to be slightly inhibited by uvc irradiation and the combined uv-c/ozone treatment after 4 days of shelf-life. at the early storage, lignin incorporation was even slightly accelerated by uv-c and ozone in comparison to control spears. uv-c irradiation was shown to activate the synthesis of compounds of the phenylpropanoid pathway, such as lignin and phenolic compounds by stimulating the activity of the key enzyme phenylalanine ammonia-lyase (pal) (charles et al., 2008). this lignifi cation is assumed to mainly cause reinforcement of the cell wall. on the other hand, several studies demonstrated that uv-c and ozone treatments may reduce the activity of texture-related enzymes generally enhanced during postharvest life of fruits and vegetables (e.g. costa et al., 2006). in green asparagus, aqueous ozone inhibited pal activity and thus, the increase in phenolic cell wall compounds (an et al., 2006). furthermore, uv-c irradiation may reduce activity of cell wall degrading enzymes (e.g. pg, pme), thus delaying the disintegration of membrane and the loss of tissue fi rmness (e.g. barka et al., 2000; pombo et al., 2009). the degradation of cell wall components is related to the enzymatic activity of several cell wall proteins (brummell and harpster, 2001). in the presented study, cell wall protein content of asparagus spears nearly linearly decreased during the experimental period. fig. 2: cutting energy (j m-2) of asparagus spears during storage (4 days at 20 °c in water saturated air) as affected by postharvest treatments of uv-c (1 kj m-2 for 8 min), ozonated water (3 ppm for 30 s) and combined uv-c and ozonated water. data are means ±sd (n=6). fig. 3: changes in total content of structural cell wall compounds (mg g dm-1) of asparagus spears during storage (4 days at 20 °c in water saturated air) as affected by postharvest treatments of uv-c (1 kj m-2 for 8 min), ozonated water (3 ppm for 30 s) and combined uv-c and ozonated water. data are means ±sd (n=6). fig. 4: changes in cell-wall lignin (mg g dm-1) of asparagus spears during storage (4 days at 20 °c in water saturated air) as affected by postharvest treatments of uv-c (1 kj m-2 for 8 min), ozonated water (3 ppm for 30 s) and combined uv-c and ozonated water. data are means ±sd (n=6). fig. 5: changes in cell-wall pectic substances (mg g dm-1) of asparagus spears during storage (4 days at 20 °c in water saturated air) as affected by postharvest treatments of uv-c (1 kj m-2 for 8 min), ozonated water (3 ppm for 30 s) and combined uv-c and ozonated water. data are means ±sd (n=6). uv-c and ozonated water treatment of white asparagus 233 this effect was signifi cantly accelerated by all postharvest treatments, specifi cally after 4 days of storage (fig. 6). the pronounced reduction of cell wall protein content by uv-c and ozone treatments might refl ect the inhibition of pal activity as found for green asparagus (an et al., 2007). on the other hand, it might be an indication of stress mediated responses (kucera et al., 2003) associated with the loss of integrity and functionality of membranes and degradation of solubilised cell wall protein. in this context, the topic certainly requires additional systematic investigations. allende, a., mcevoy j., luo, y., artés, f., wang, c., 2006: effectiveness of two-sided uv-c treatments in inhibiting natural microfl ora and extending the shelf-life of minimally processed ‘red oak leaf’ lettuce. food microbiol. 23, 241-249. an, j., zhang, m., lu, q., 2006: effects of pretreated ozone and modified atmosphere packaging on the quality of fresh-cut green asparagus. int. agrophysics 20, 113-119. an, j., zhang, m., lu, q., 2007: changes in some quality indexes in freshcut green asparagus pretreated with aqueous ozone and subsequent modifi ed atmosphere packaging. j. food eng. 78, 340-344. aoac, 1999: official methods of analysis (14th edn). association of offi cial analytical chemists, washington dc, usa. artés, f., gómez, p., aguayo, e., escalona, v., artés-hernández, f., 2009: sustainable sanitation techniques for keeping quality and safety of fresh-cut plant commodities. postharvest biol. technol. 51, 287-296. artés-hernández, f., aguayo, e., artes, f., tomás-barberán, f., 2007: enriched ozone atmosphere enhances bioactive phenolics in seedless table grapes after prolonged shelf-life. j. sci. food agric. 87, 824-831. atkins, a.g., vincent, j.f.v., 1984: an instrumented microtome for improved histological section and the measurement of fracture toughness. j. mater. sci. lett. 3, 310-312. barka, e.a., barka, j., kalantari, j., makhlouf, arul, j., 2000: impact of uv-c illumination on the cell wall-degrading enzymes during ripening of tomato (lycopersicon esculentum l.) fruit. j. agric. food chem. 48, 667-671. billau, w., buchloh, g., hartmann, h.d., 1990: the influence of temperature, cultivar, soil-type and stalk-diameter on the lignifi cation of white asparagus. acta hort. 271, 173-184. bintsis, t., litopoulou-tzanetaki, e., robinson, r.k., 2000: existing and potential applications of ultraviolet light in the food industry − a critical review. j. sci. food agric. 80, 637-645. blumenkrantz, n., asboe-hansen, g., 1973: new method for quantitative determination of uronic acids. anal. biochem. 54, 484-489. bradford, m.m., 1976: a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. anal. biochem. 72, 248-254. brummell, d.a., harpster, m.h., 2001: cell wall metabolism in fruit softening and quality and its manipulation in transgenic plants. plant mol. biol. 47, 311-340. charles, m.t., goulet, a., arul, j., 2008: physiological basis of uv-c induced resistance to botrytis cinerea in tomato fruit iv. biochemical modifi cation of structural barriers. postharvest biol. technol. 47, 4153. cisneros-zevallos, i., 2003: the use of controlled postharvest abiotic stresses as a tool for enhancing the nutraceutical content and addingvalue of fresh fruits and vegetables. j. food sci. 68, 1560-1565. costa, l., vicente, a.r., civello, p.m., chaves, a., martinez, g.a., 2006: uv-c treatment delays postharvest senescence in broccolo fl orets. postharvest biol. technol. 39, 204-210. d`hallewin, g., schirra, m., pala, m., ben-yehoshua, s., 2000: ultraviolet-c irradiation at 0.5 kjm-2 reduces decay without causing damage or affecting postharvest quality of star ruby grapefruit (c. paradisi macf.). j. agric. food chem. 48, 4571-4575. el ghaouth, a., wilson, c.l., callahan, a., 2003: induction of chitinase, beta-1,3 glucanase and phenylalanine ammonia lyase in peach fruit by uv-c treatment. phytopathol. 93, 349-355. goering, h.k., van soest, p.j., 1972: forage fibre analyses. agric. handbook 379. washington, usa. gonzález-aguilar, g.a., zavaleta-gatica, r., tiznado-hernández, m.e., 2007: improving postharvest quality of mango ‘haden’ by uv-c treatment. postharvest biol. technol. 45, 108-116. hassenberg, k., idler, c., molloy, e., geyer, m., pöchl, m., barnes, j., 2007: use of ozone in a lettuce washing process – an industrial trial. j. sci. food agric. 87, 914-919. conclusion plant reaction to hormic uv-c and ozone application is different in terms of their alarm-signalling processes. these processes serve to modify metabolism. the capacity of the antioxidative defence system, however, is often increased in response to the above treatments. as a result, radical production may exceed scavenging fi nally leading to disruption of metabolism as indicated e.g. by accelerated gas exchange, loss of tissue integrity and cell wall modifi cations. thus, the impact of ozone and uv-c on white asparagus spears resulted in a slight reduced respiration, but increase in spear tissue toughness. total cell wall compounds were only tendentiously reduced, while cell wall protein was strongly affected after 4 days of shelf-life at 20 °c by application of aqueous ozone and uv-c. the degradation of solubilised cell wall protein might indicate stress mediated responses associated with the loss of integrity and functionality of membranes. further emphasis will be placed on the possible mechanism of uv-c and ozone mediated changes in textural related enzyme activities of white asparagus spears in more detail. acknowledgements the authors gratefully acknowledge the laboratory staff susanne meier of the section quality dynamics/postharvest physiology, and gabriele wegner and corinna rolleczek of the department of horticultural engineering. references aguayo, e., escalona, v.h., artés, f., 2006: 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17, 235-287. vicente, a.r., pineda, c., lemoine, l., civello, p.m., martinez, g.a., chaves, a.r., 2005: uv-c treatments reduce decay, keep quality and alleviate chilling injury in pepper. postharvest biol. technol. 35, 6978. waldron, k.w., selvendran, r.r., 1990: effect of maturation and storage on asparagus (asparagus offi cinalisstorage on asparagus (asparagus offi cinalisstorage on asparagus ( ) cell wall composition. physiol. plant. 80, 576-583. adresses of the authors: dr. susanne huyskens-keil, humboldt-universität zu berlin, division urban plant ecophysiology, section quality dynamics / postharvest physiology, lentzeallee 55/57, d-14195 berlin, germany e-mail: susanne.huyskens@agrar.hu-berlin.de dr. werner b. herppich, dr. karin hassenberg, leibniz-institute for agricultural engineering potsdam-bornim e.v., department horticultural engineering, max-eyth-allee 100, d-14469 potsdam, germany art15_özcakmak.indd journal of applied botany and food quality 85, 97 99 (2012) 1 department of food processing, terme vocational school, ondokuz mayis university, terme, samsun, turkey the effects of heracleum platytaenium boiss essential oil on the growth of ochratoxigenic penicillium verrucosum (d-99756) isolated from kashar cheese sibel özçakmak (received july 13, 2011) summary the antifungal effects of essential oil isolated from heracleum platytaenium boiss on the growth of ochratoxigenic penicillium verrucosum (d-99756) isolated from kashar cheese were investigated. minimal inhibitory concentration (mic) and minimal lethal concentration (mlc) tests were performed by using broth dilution method. controls were prepared with three groups; with pure essential oils (eo) (positive control), methanol and without eos. the serial doubling dilution of oil was prepared in methanol with concentrations ranging from 500 to 31µl/ml. applied incubation periods were 25 oc for 10 days for mic and 48 hours at the same temperature for mlc. after the incubation period, the tubes were controlled for either mycelia growth or turbidity and sediments. the maximal inhibitory dilution (the minimal inhibitory concentration), exhibiting any growth, was regarded as mic. tubes containing penicillium verrucosum which did not display any growth were subcultured to determine if the inhibition was reversible or permanent. the eo dilution, which is the not visible growth of typical p. verrucosum colonies on the plates, was accepted as lethal effect. mlc was the lowest concentration of antifungal to completely inhibit fungal growth. the mic and mfc values were determined as 31 and 125 µl/ml, respectively. introduction the presence and growth of fungi in food may cause spoilage and result in a reduction in quality and quantity. growth of commonly occurring fi lamentous fungi in foods may result in production of toxins known as mycototoxins, which can cause a variety of ill effects which has a range of biological activities including acute toxicity, teratogenicity, mutagenicity and carcinogenicity in humans, beginning from allergic responses to immunosuppression and cancer. the most important mycotoxins are afl atoxins, ochratoxin-a, fumonisins, trichothecenes and zearalenone. ochratoxin-a produced by aspergillus ochraceus, a. niger, a. carbonarius, penicillium verrucosum and p. viridicatum (pitt, 1987; abarca et al., 1994; varga et al., 1996) is probably carcinogenic and may cause urinary tract cancer and kidney disease (krogh et al., 1974; pitt, 2000). penicillium species are frequent contaminants of different food products and known as producers of a variety of secondary metabolites (frisvad and fitenborg, 1983). p. verucosum is primarily encountered on cereals and cheeses (larsen et al., 2001). various species of heracleum have been reported to possess antibacterial and antifungal properties (ciesla et al., 2008). heracleum sp. is a member of umbelliferae family whose essential oils (eos) have antibacterial and antifungal effects (i̇şcan et al., 2004). the fruits and the leaves of this genus are used as fl avouring agent and spice for food and also antiseptic, carminative, digestive and analgesic effect (firuzi et al., 2010). chemical composition of the eos of some heracleum species was investigated by some researches. the major compounds of different heracleum species were reported as octyl acetate by işcan et al. (2004), 1-octanol, octylbutyrate, octanol by janssen et al. (1986) and kürkçüoğlu et al. (1995) and myristicin, e-anethole, octyl isobutanoate, hexyl butanoate, octyl acetate and elemicin by firuzi et al. (2010). materials and methods materials the essential oil of the plant heracleum platytaenium boiss was obtained from biological science department, faculty of science and literature in samsun-turkey. preparation of the essential oil dilutions the serial doubling dilution of each oil (pinto et al., 2007) was prepared in methanol (rasooli and abyaneh, 2004) with concentrations ranging from 500 to 31 µl ml-1. fungal strain and preparation of spore suspension the fungal strain used in this study was penicillium verrucosum dierck (d-99756), obtained from faculty of food engineering in istanbul technical university, istanbul-turkey. the strain was cultured from frozen stocks and maintained on malt extracted agar (mea, 2% malt, 0.1% peptone, 1.5% agar) slants at 25 oc in the dark for 7-10 days. the strain was cultivated on mea slants at 25 oc in the dark for 7-10 days (cabanas et al., 2009). spores were harvested by adding 10 ml of sterile saline solution (nacl, 0.85%) containing tween 80 (0.1% v/v). the spore suspension was adjusted to approximately 106 spores ml-1 using haemocytometer (elias et al., 2006). test organism was enumerated in growing broth media by serial dilution method (özkan et al., 2010). the viability and inoculums size of the strains were checked using quantitive colony counts. the plates were incubated for 72 h at 25 oc in the dark (tavares et al., 2008). growth media and chemicals yeast extract sucrose (yes) agar, yes broth, methanol, tween-80, sodium chlorid (nacl) were obtained from merck, germany. yes culture media was used to determine inhibitory concentrations and yes agar was used to determine if the inhibition was reversible or permanent (bragulat et al., 2001). 5 ml sterile yes broth tubes containing 106 spores ml-1 were dispensed into sterile tubes using for broth dilution method. petri dishes (90 mm) were fi lled with 1520 ml of yes agar for fungicidal effect. controls with and without methanol (negative controls) and using pure eos (positive controls) incubated under the same conditions. antifungal activity of essential oils with macrodilution method a macrodilution broth method was used to determine the minimal inhibitory concentrations (mic) and minimal lethal concentrations 98 s. özçakmak (mlc) (lass-flörl et al., 2003; tavares et al., 2008). fifty micro liters from four eos dilutions at various concentrations (500, 250, 125, 62, 31, µl ml-1) were added in 5 ml sterile yes broth tubes containing 106 spores ml-1 and these solutions mixed gently. the tubes were incubated at 25 oc for 7-10 days on an incubator shaker as to evenly disperse the oils throughout the broth in tubes. the fungal growth was indicated by the turbidity. the highest dilution (lowest concentration), showing no visible growth compared with eo-free controls, was regarded as mic. the oil dilutions supplying inhibitory effect were tested for lethal effect as well. the inoculations of 1 ml for pour plate and 30 µl for three point inoculation methods from the tubes were subcultured on yes agar plates and than incubated at 25 oc for 72 hours. the essential oil dilution, which is not visible growth of typical p. verrucosum colonies on the plates, was accepted as lethal effect. the mlc was defi ned as the lowest eo concentration at which 99% of the inoculum was killed. all the experiments were performed in duplicate and three independent experiments were run with concordant results. results the results of experimental design in this research are presented in tab. 1, fig. 1 and fig. 2a 2b. the mycelial growth was visible in negative control tube (fig. 1b) while any fungal growth wasn’t seen in positive control tubes (fig. 1a). the highest dilution concentration (31 µl ml-1) was mic value (fig. 1c). the growth of p. verrucosum was inhibited 100% at mic value. heracleum species has just been investigated for anticandidal activity by naeini et al. (2009), i̇şcan et al. (2003 and 2004) and kuljanabhagavad et al. (2010). the researches reported that different heracleum spp. had notable anticandidal effect. in this study, it was determined that the mycelial formation of p. verrucosum could be prevented using h. platytaenium essential oil. tab. 1: mic/mfc values of heracleum platytaenium eo against ochratoxigenic p. verrucosum strain. the inhibitory and lethal effects of h. platytaenium eo dilutions (µl ml-1) on p. verrucosum. positive controls negative controls 500 250 125 62 31 mic + + + + + + mlc + + + + +: inhibition positive -: inhibition negative fig. 1: the inhibitory effects of h. platytaenium eo dilutions on p. verrucosum after 9 days of incubation at 25°c. the left tube represents complete inhibition at pure form of eo, the right tube represents complete inhibition at 31 µl ml-1 and the middle tube represents negative control tube. 2a 2b fig. 2: mfc results after 72 hours of incubation at 25 °c using three point inoculation (fig. 2a) and pour plate mehods (fig. 2b). the left petri dishes for non-additive and the right petri dishes for additive with h. platytaenium eo dilutions represent subcultured inoculations from mic tubes. 1a 1b 1c the lethal effect was assayed using two methods. the results both were the same. oil required for mlc value is more than mic. the inoculations subcultured on yes agar plates could be killed at 125 µl ml-1 oil dilutions (fig. 2a 2b). effects of heracleum platytaenium boiss essential oil on the growth of ochratoxigenic penicillium verrucosum 99 discussion in this study, it was demonstrated that h. platytaenium had inhibitory and toxic effects against p. verrucosum. in general, the cytotoxic activity of eos is mostly due to the presence of aldehydes, alcohols, methylene dioxy compounds and phenols (kuljanabhagavad et al., 2010). firuzi et al. (2010) reported that heracleum species had the cytotoxic activity mostly due to the presence of myristicin, elemicin and (e)-anethole. heracleum species has just been investigated for anticandidal activity by naeini et al. (2009), i̇şcan et al. (2003 and 2004) and kuljanabhagavad et al. (2010). this survey is the fi rst report that the inhibitory effect of h. platytaenium against ochratoxigenic p. verrucosum. acknowledgements the present research was funded by the scientifi c research programme of ondokuz mayis university. the authors thank also terme vocational school for their support. references abarca, m.l., bragulat, m.r., castella, g., cabanes, f.j., 1994: ochratoxin a production by strains of aspergillus niger var. niger. appl. environ. microbiol. 60, 2650-2652. bragulat, m.r., abarca, m.l., cabanes, f.j., 2001: an easy screening method for fungi producing ochratoxin a in pure culture. int. j. food microbiol. 71, (2-3), 139-144. cabanas, r., abarca, m.l., bragulat, m.r., cabanes, f.j., 2009: in vitro activity of imazalil against penicillium expansum: comparison of the clsi m38-a broth microdilution method with traditional techniques. int. j. food microbiol. 129, 26-29. ciesla, ł., bogucka-kockab, a., hajnosc, m., petruczynika, a., waksmundzka-hajnosa, m., 2008: two-dimensional thin-layer chromatography with adsorbent gradient as a method of chromatographic fi ngerprinting of furanocoumarins for distinguishing selected varieties and forms of heracleum spp. j. chromatogr. a. 1207, (1-2), 160 168. elias, b.c., said, s., albuquerque, s., pupo, m.t., 2006: the influence of culture conditions on the biosynthesis of secondary metabolites by penicillium verrucosum dierck. microbiol. res. 161, 273-280. firuzi, o., asadollahi, m., gholami, m., javidnia, k., 2010: composition and biological activities of essential oils from four heracleum species. food chem. 122, 117-122. frisvad, filtenborg, o., 1983: classification of terverticillate penicillia based on profi les of mycotoxins and other secondary meta-bolites. appl. environ. microbiol. 46, 1301-1310. i̇şcan, g., demirci, f., kurkcuoglu, m., kivanc, m., baser, k.h.c., 2003: the bioactive essential oils of heracleum sphondylium l. subsp. ternatum (velen.). brummitt. z. naturforsch. c. 58, 195-200. i̇şcan, g., ozek, t., ozek, g., duran, a., baser, k.h.c., 2004: essential oils of three species of heracleum anticandidal activity. chem. nat compd. 40, 544-547. janssen, a.m., chin, n.l.j., scheffer, j.j.c., baerheim, s.a., 1986: sci. pharm. weekbl. 8, 289-292. krogh, p., hald, b., englund, p., rutqvist, l., swahn, o., 1974: contamination of swedish cereals with ochratoxin a. acta pathol. microbiol. scand. [b] 82, 301-302. kuljanabhagavad, t., sriubolmas, n., ruangrungsi, n., 2010: chemical composition and antımicrobial activity of the essentıal oil from heracleum sıamıcum. j. health res. 24, 55-60. kürkcüoglu, m., ozek, t., baser, k.h.c., malyer, h., 1995: composition of the essential oil of heracleum platytaenium boiss. from turkey. j. ess. oil res. 7, 69-70. lass-flörl, c., speth, c., kofler, g., dierch, m.p., gunsilius, e., würzner, r., 2003: effect of increasing inoculum sizes of aspergillus hypae on mics and mfcs of antifungal agents by broth microdilution method. int. j. antimicrob. agents 21, 229-233. larsen, t.o., svendsen, a., smedsgaard, j., 2001: biochemical characterization of ochratoxin a producing strains of the genus penicillium. appl. environ. microbiol. 67, 3630-3635. naeini, a., khosravi, a.r., chitsaz, m., shokri, h., kamlnejad, m., 2009: anti-candida albicansactivity of some iranian plants used in traditional medicine. j. mycol. med. 19, 168-172. özkan, g., sagdic, o., gokturk, r.s., unal, o., albayrak, s., 2010: study on chemical composition and biological activities of essential oil and extract from salvia pisidica. lwt food sci. technol. 43, 186190. pinto, e., salgueiro, r., cavaleiro, c., palmeira, a., gonçalves, m.j., 2007: in vitro susceptibility of some species of yeasts and filamentous fungi to essential oils of salvia offi cinalis. ind. crop prod. 26, 135-141. pitt, j.i., 1987: penicillium vindicatum, pemcillium verrucosum and production of ochratoxin a. appl. environ. microbiol. 53, 266-269. pitt, j.i., 2000: toxigenic fungi and mycotoxins. brit. med. bull. 56, 184192. rasooli, i., abyaneh, m.r., 2004: inhibitory effects of thyme oils on growth and afl atoxin production by aspergillus parasiticus. food control. 15, 479-483. tavares, a.c., gonçalves, m.j., cavaleiro, c., cruz, m.t., lopez, m.c., cahoto, j., salgueir, l.r., 2008: essential oil of daucus carota subsp. halophilus: composition, antifungal and cytotoxicity. j. ethnopharmacol. 119, 129-134. varga, j., keveii, e., rinyu, e., teren, j., kozakiewicz, z., 1996: ochratoxin production by aspergillus species. appl. environ. microbiol. 62, 4461-4464. address of the author: ph. dr. sibel özçakmak, tel.: +90 362 8761318 / 114, fax: +90 362 8761317, e-mail: sibelo@omu.edu.tr journal of applied botany and food quality 92, 123 129 (2019), doi:10.5073/jabfq.2019.092.017 department of applied biology, school of basic medical and biological sciences, soochow university, suzhou, pr china rapid analysis of the bioactive components in saxifraga stolonifera, an edible and medicinal herb with anti-tumor effects, by hplc-dad, esi/msn meng zhang, dong liu, yu-qing zhang* (submitted: november 13, 2018; accepted: april 10, 2019) * corresponding author summary saxifraga stolonifera is an edible and herbaceous plant, which has been demonstrated to have anti-tumor effects in vivo and in vitro. the aim of this paper is to determine the main bioactive components in s. stolonifera, and their distribution in different parts of s. stoloni fera and in s. stolonifera that was cultivated in different places in china using a high-performance liquid chromatography-diode array detector and electrospray ionization/ion trap mass spectrometry (hplc-dad-esi/msn). four main components were identified and three were quantified. the contents of gallic acid, protocatechuic acid and bergenin had significant differences not only between the roots and stems-leaves of the plant, but also among different cultivated varieties of s. stolonifera. the experiment showed that the method used here exhibited good repeatability and recovery. therefore, the results provide reliable data for research and development in the future on the level and distribution of the three bioactive components of s. stolonifera. keywords: gallic acid; protocatechuic acid; bergenin; hplc-dad; saxifraga stolonifera introduction saxifraga stolonifera curtis is an herb that belong to the saxifraga ceae family. this family is nearly cosmopolitan in distribution. the vast majority of the genera and species are found in the northern hemisphere, particularly in mountainous areas, with centres of diversity in western north america, east asia and the himalayas and europe (soltis, 2007). in east asia, s. stolonifera is generally considered a decorative horticultural plant, and it is sometimes used as a medicine. in china, s. stolonifera is used as traditional medicine to treat many diseases, such as tympanitis, haemorrhoids and phthisis bulbi. recently, s. stolonifera has also been found to have pharmacological benefits, including anti-inflammatory effects, beneficial effects for prostate hyperplasia, liver function protection, and anticancer effects. it was found that saxifragin isolated from s. stolonifera has antiinflammatory effects via the inhibition of nf-κb involved caspase-1 activation (cheon et al., 2015). researchers found that saxifragin can suppress the lipopolysaccharide (lps)-induced production level of nitrous oxide (no), pge2, and pro-inflammatory cytokines, such as tnfα, il-1β and il-6, in vitro and in vivo. in addition, injec tions, tablets, and suppositories made from 100% of the whole plant s. stolonifera have positive effects on prostate hyperplasia (chen et al., 2003). nakagiri et al. (2001) demonstrated that s. stolonifera or isolation of s. stolonifera protects liver function. their group found that increased level of alanine aminotransferase (aat) and glutamic oxaloacetic transaminase (got) in animals that were treated with lps were reduced after treatment with s. stolonifera. it was demonstrated that quercetin isolated from s. stolonifera exhibited a strong inhibitory effect on human gastric cancer cell line (bgc-823) cells in a timeand dose-dependent manner (chen et al., 2008). moreover, ju found that extracts of s. stolonifera have positive effects on benign thyroid tumours (longtao, 2008). in addition, the intake of s. stolonifera by drinking a decoction of dried leaves had antitumor effects on gastric cancers (nagata et al., 2016). the structures of gallic acid, protocatechuic acid, and bergenin are identified (chen et al., 2008) and presented in fig. 2. gallic acid, protocatechuic acid, and bergenin are phenolic acids, and bergenin is a gallic acid derivative as well as a c-glycoside, having the simplest structure in nature. in recent years, some reports have clarified that s. stolonifera has many biologically active components, including quercetin, quercetin-3-o-rhamnoside (luo et al., 1988), saxifragin (morita et al., 1974), kaempferol, bergenin (luo et al., 1988), gallic acid (luo et al., 1988), protocatechuic acid (luo et al., 1988) and chlorogenic acid (aoyagi et al., 1995). gallic acid has many important biological activities such as anti-inflammatory effects (luo et al., 1988), antitumour effects (kawada et al., 2001), apoptosis-inducing effect (ohno et al., 1999), antibacterial activities (chanwitheesuk et al., 2007), anti-melanogenic and antioxidant properties (kim, 2007), anticancer effects (faried et al., 2007), and cardioprotective effects (priscilla et al., 2009). further, protocatechuic acid has biological properties such as anti-ageing effects (zhang et al., 2011), anti-inflammatory effects (min et al., 2010), antioxidant properties (shi et al., 2006), antihepatotoxicity (liu et al., 2002), antitumour effects (tseng et al., 1998, 2000) and anti-neurotoxicity (an et al., 2006). bergenin’s biological activities include antioxidant properties (nazir et al., 2011), antihepatotoxic activity (kim et al., 2000), antiinflammatory effects (swarnalakshmi et al., 1984), antimicrobial activities (prithiviraj et al., 1997), electrocatalytical properties (zhuang et al., 2008) and neuroprotective effects (takahashi et al., 2003). therefore, it is meaningful to determine the composition and pharmacological activities of s. stolonifera and to find more rational uses of s. stolonifera as a source of crude drugs to provide medicines to alleviate and treat many diseases and conditions. despite the many studies that have analysed the structure and pharmacological activity of the components that have been isolated from s. stolonifera, the distributions and differences in gallic acid, protocatechuic acid and bergenin contents between s. stolonifera stem-leaves and roots have rarely been examined. the aim of this study was to evaluate the gallic acid, protocatechuic acid and berge nin contents in s. stolonifera and to establish a method for deter mining the gallic acid, protocatechuic acid and bergenin contents in s. stolonifera. the gallic acid, protocatechuic acid and bergenin contents in the samples was assayed by reversed-phase highperformance liquid chromatography (hplc) with dad detection. materials and methods chemicals and materials the gallic acid standard was purchased from targetmol (targetmol. co. ltd, usa, cas:149-91-7). both the protocatechuic acid and bergenin standards were purchased from tokyo chemical industry (tokyo chemical industry co. ltd., tokyo, japan, cas: 99-503,477-90-7). the solvents used for the extraction of s. stolonifera samples were analytical-grade anhydrous alcohol (qiangsheng 124 m. zhang, d. liu, y.-q. zhang chemical industry, jiangsu, china) and milli-q distilled water (millipore australia pty. ltd., north ryde, new south wales, australia), whereas those used for the hplc analysis were hplcgrade acetonitrile (merck kgaa, darmstadt, germany) and milli-q distilled water. equipment the shimadzu hplc system (shimadzu inc., lc-20a, japan) consists of a computer-controlled system with upgraded lc-20a software and an scl-10a vp system controller. chromatographic separation was achieved using a j&k chemica hplc-c18 column (2.1 69 × 150 mm, 5 μm). other accessories include two lc-20at shimadzu liquid chromatography pumps, an rf-20a high-sensitivity fluorescence detector, an spd m20a diode array detector, and a cto-20a column oven. hplc chromatographic conditions the mobile phase consisted of acetonitrile (a) and 0.4% glacial acetic acid (b). the gradient program was as follows: a. 0-30.0 min, 5%-20%, b. 0-30.0 min, 95%-80%. the column temperature was maintained at 30 °c. the flow rate was 1.0 ml/min, and the injection volume was 20 μl. preparation of the standard sample solution standard samples of gallic acid 1 mg, protocatechuic acid 1 mg, and bergenin 2 mg were dissolved in a 1 ml 70% methanol solution, respectively (i.e., 1.0 mg/ml, 1.0 mg/ml and 2 mg/ml, respectively). for uv spectroscopy analysis, the standard gallic acid, proto catechuic acid and bergenin solution were all diluted with 70% me thanol solution into five concentrations as follows: standard gallic acid: 10, 200, 300, 400, and 450 μg/ml; standard protocatechuic acid: 2, 50, 100, 150, and 180 μg/ml; and standard bergenin: 100, 300, 600, 800, and 1,860 μg/ml. all the standard samples were measured in the 200-400 nm uv absorption spectra range. sample preparation eight sample groups were used in this experiment, and they included the entire s. stolonifera, the above-ground part (the leaves and branches of s. stolonifera) and the below-ground part (the roots of s. stolonifera). the s. stolonifera herb that was cultivated in suzhou (china) was purchased from a guangliangji chinese medicine store, and the other four s. stolonifera herbs cultivated in different places in china were purchased from sichuan, fujian, suqian, and shuyang. both the above-ground part and the below-ground part were washed with water and then placed in a vacuum freeze-drying machine for 3 days, after which they were ground into fine powder. the dried s. stolonifera powder was filtered by a sieve. all the samples were then stored in polyethylene bags in the freezer at -20 °c for further investigation. preparation of the sample solution each sample (200 mg) was added into a 5 ml 70% methanol solution (the other 30% was ultrapure water), and the solutions were then extracted by ultrasound for 90 min with the temperature set at 75 °c. finally, all the extracts were filtered through a 0.45-μm membrane for the hplc analysis. analytic conditions the sample solution was analysed by hplc using a shimadzu lc20a hplc-dad with a c18 reversed phase column and a metaguard column (4.6 mm metasil aq 5u c18 120a). the sample solution was filtered through a 0.45-μm nylon membrane filter (millipore), and 20 μl was injected into the liquid chromatography column. the flow rate was set at 1.0 ml·min-1. the temperature of the column oven was set at room temperature. the mobile phase consisted of solvent b (acetonitrile) and solvent a (milli-q distilled water) at 70/30 (v/v). the spectra were recorded in the 200 to 400 nm range, and the absorbance of the effluent was monitored at 275 nm. electrospray ionization/ion trap mass spectrometry mass spectrometric analysis for the determination of gallic acid, protocatechuic acid and bergenin was performed using a tsq quantum ultra-triple-quadrupole mass spectrometer (thermo fisher scientific inc., waltham, ma, usa), which was equipped with an electro-spray ionization (esi) interface in the negative mode. the following are the parameters of the mass spectrometer: sheath gas flow rate of 40 (arbitrary units); auxiliary gas flow rate of 10 (arbitrary units); spray voltage of 2500 v; vaporizer temperature of 350 °c; and capillary temperature of 350 °c. helium was used as the collision gas for collision-induced dissociation (cid). quantification method the quantification of gallic acid, protocatechuic acid and bergenin was performed using the external standard method. three standard samples of gallic acid, protocatechuic acid and bergenin at various concentration levels were injected into the hplc-dad system, and the peak areas corresponding to the standard sample concentrations were observed, from which the calibration curves were created. the levels of the three main components in s. stolonifera were calculated with the calibration curves. results and discussion chromatographic and spectral characteristics the hplc chromatogram and tic of the entire plant were identified by hplc-dad-esi/msn, which are presented in fig. 1a and b. as shown in fig. 1 and tab. 1, there are four main components in s. stolonifera. according to their molecular weight and ms/ms spectra of these four components (fig. 1c), we could identity the potential compounds. the molecular weight of these four components (1-4) are 170, 314, 154 and 328, and, the molecular formula are c7h6o5, c13h16o9, c7h6o4, and c14h16o9, respectively, which may be gallic acid, norbergenin, protocatechuic acid, and bergenin, respectively. the standard curve and of gallic acid, protocatechuic acid and bergenin according to the four main active components identified by mass spectrometry, three of the main components gallic acid, protocate chuic acid and bergenin were qualitatively and quantitatively analysed in this study by hplc-dad. as shown in fig. 2, the hplc-dad spectra of the three standard samples gallic acid, protocatechuic acid and bergenin were compared in detail with the retention time and uv spectra of three main components in s. stolonifera (fig. 2a). the results showed that the peak times of the three standard compounds on hplc-dad were 9.258, 15.317 and 19.582 min, respectively, and there were three corresponding absorption peaks on the hplc-dad spectra of s. stolonifera, the retention time of these absorption peaks was about the same as that of standard compounds. fig. 2b showed the comparison of uv spectra of three standard samples with the three peaks of s. stolonifera. it could be found that the retention times and uv absorption spectra of the three peaks were the same as that of the rapid analysis of the bioactives in s. stolonifera 125 tab. 1: ms/ms analysis of methanol extract of s. stolonifera. peak rt [m-h]molecular weight tentative identification molecular formula 1 3.185 169.0 170.0 gallic acid c7h6o5 2 3.859 313.2 314.2 norbergenin c13h16o9 3 4.498 152.9 154.02 protocatechuic acid c7h6o4 4 4.888 327.2 328.07 bergenin c14h16o9 3.859 fig. 1: chromatography profile (275 nm) (a), the total ion chromatography (tic) (b) and mass spectrometry (c) of s. stolonifera by hplcdad-ms/es .̄ in fig. 1 a, peak 1, 2, 3 and 4 respresent four components in s. stolonifera. ms/es¯ spectra (a, b, c and d) of the four components in s. stolonifera are corresponding to 1, 2, 3 and 4 in fig. 1 a and b, respectively. a b c a b c d three standard samples. this fully proved the identification results of hplc-dad-esi/msn, the three absorption peaks with retention time at 9.258, 15.317 and 19.582 min are gallic acid, protocatechuic acid and bergenin, respectively (fig. 2c). in order to quantitatively analyze the three main active components in s. stolonifera, standard solutions of the three compounds were injected into the hplc column (fig. 3) with the concentrations as follows. gallic acid: 10, 200, 300, 400, and 450 μg/ml; protocatechuic acid: 2, 50, 100, 150, and 180 μg/ml; and bergenin: 100, 300, 600, 800, and 1860 μg/ml. the regression equations and correlation coefficients (r) were calculated by plotting the peak-area (y) vs. concentration (x, μg/ml) as follows: the correlation coefficients (r) of gallic acid were y = 0.10139 + 0.04775x, r = 0.99734. the correlation coefficients (r) of protocatechuic acid were y = 0.08249 + 0.03626x, r = 0.9955. the correlation coefficients (r) of bergenin were y = 0.95934 + 0.00596x, r = 0.98215 (x: sample concentration; y: peak area). the reproducibility of the hplc for the determination of gallic acid, protocatechuic acid and bergenin in s. stolonifera the sample extraction solution of s. stolonifera was measured by hplc-dad five times, and the content of gallic acid, protocatechuic acid, and bergenin was calculated. as shown in tab. 2, the values of the repeated measurements of gallic acid were 0.339, 0.357, 0.336, 0.339, and 0.337 mg/g, with an average of 0.342 mg/g (±0.009 mg/g) and an rsd = 2.570%. in addition, the values of repeated measurements of protocatechuic acid were 0.077, 0.87, 0.078, 0.078, and 0.087 mg/g, with an average of 0.081 mg/g (±0.005 mg/g) and an rsd = 6.037%. furthermore, the values of repeated measurements of bergenin were 2.260, 2.701, 2.204, 2.294, and 2.581 mg/g, with an average of 2.409 mg/g (±0.218 mg/g) and an rsd = 9.052%. these results indicate that the hplc determination of the gallic acid, protocatechuic acid and bergenin in s. stolonifera shows good repeatability. extraction recovery of gallic acid, protocatechuic acid and bergenin in s. stolonifera to determine the accuracy of the hplc results, an ethanol extraction of s. stolonifera was used as a test sample solution. the sample solution was divided into three parts, and the content of gallic acid, protocatechuic acid, and bergenin in these samples were determined. the content of gallic acid, protocatechuic acid, and bergenin were calculated by the linear equation produced by the standard samples; 70, 50, and 1500 μg of gallic acid, protocatechuic acid, and bergenin standard samples were added to the test sample solution before blending as part of the recovery study. the experimental results showed that the extraction recovery rate for gallic acid, protocate chuic acid, and bergenin in s. stolonifera were 102.491%±6.516%, 99.685%±10.485%, and 105.050%±10.197%, respectively (tab. 3). these results show high recovery considering the complexity of the analyses. the recovery tests of gallic acid, protocatechuic acid and bergenin in s. stolonifera exhibit very high precision. the difference in gallic acid, protocatechuic acid and bergenin contents between cultivated s. stolonifera varieties this experiment tested the five varieties of s. stolonifera cultivated in different locations in china. the gallic acid, protocatechuic acid and bergenin content in different varieties of cultivated s. stolonifera were investigated by longitudinal comparison. the results are shown in tab. 4 and fig. 4. here it can be seen that there is a significant difference (p < 0.05) in gallic acid and protocatechuic acid content and no significant difference in bergenin content between the five cultivated varieties. on a dry-weight basis, s. stolonifera that was cultivated in fujian, suzhou, and suqian contained the highest concentrations of gallic acid, protocatechuic acid, and bergenin (0.538, 0.616, and 15.314 mg/g, respectively). meanwhile, s. stoloni fera cultivated in suqian contained the lowest concentration of gallic 126 m. zhang, d. liu, y.-q. zhang a c b fig. 2: hplc-dad patterns of three standards (ga, pa and bergenin) and the main components in s. stolonifera (a). uv spectra (b) and their molecular structures (c) of three standards and three peaks in s. stolonifera. rt: retention time of three peaks in hplc-dad. acid (0.191 mg/g), and s. stolonifera cultivated in shuyang con tained the lowest concentration of protocatechuic acid (0.171 mg/g) and bergenin (9.605 mg/g). the results therefore indicate that s. stolonifera cultivated in fujian, suzhou, and suqian have the greatest potential for the commercial extraction of gallic acid, protocatechuic acid and bergenin, respectively. the distribution of gallic acid, protocatechuic acid and bergenin in the s. stolonifera cultivated in suzhou the roots, stem-leaves and whole plant of s. stolonifera that was cultivated in suzhou were extracted using 70% anhydrous alcohol to analyse the distribution of gallic acid, protocatechuic acid and bergenin in the stem-leaves and roots. the extracts were analysed using hplc and dad, and the results are shown in fig. 5. there were significant differences (p < 0.05) in gallic acid, protocatechuic acid and bergenin content between the different parts of s. stolonifera. the gallic acid level in the roots was as high as 0.199 mg/g, whereas the stem-leaves contained much less gallic acid (less than one-fifth of the content in the roots). conversely, the content of protocatechuic acid and bergenin in the roots was as high as 0.090 mg/g and 1.799 mg/g, respectively, whereas stem-leaves contained 0.554 mg/g and 14.463 mg/g, respectively. conclusion our research demonstrated that there are four main components in s. stolonifera, gallic acid, norbergenin, protocatechuic acid and bergenin. three main components were quantitatively analysed, and the results showed that the contents of gallic acid, protocatechuic acid and bergenin in the stem-leaves and roots of s. stolonifera have obvious differences. the content of gallic acid in the roots (0.199 mg/g) was much higher than that in the stem-leaves (0.037 mg/g). in addi tion, the content of protocatechuic acid and bergenin in the roots (0.090 mg/g and 1.799 mg/g, respectively) were much lower than those in the stem-leaves (0.554 mg/g and14.463 mg/g, respectively). rapid analysis of the bioactives in s. stolonifera 127 furthermore, s. stolonifera that was cultivated in different locations in china has differences in the content of these three bioactive com ponents. s. stolonifera cultivated in suqian contained the lowest concentration of gallic acid (0.191 mg/g). s. stolonifera that was culti vated in shuyang contained the lowest concentration of protocatechuic acid (0.171 mg/g) and bergenin (9.605 mg/g). moreover, the method used in this study for determining the gallic acid, protocate chuic acid, and bergenin content exhibited good repeatability (rsd 2.570%, 6.037% and 9.052%) and recovery (102.491%, 99.685% and 105.050%, respectively). the results provide reliable data for research and development in the future on the level and distribution of gallic acid, protocatechuic acid and bergenin in s. stolonifera. acknowledgements this work was supported by the earmarked fund (cars-18-zj0502) for china agriculture research system (cars) and by a project funded by the priority academic program development of jiangsu higher education institutions, p. r. china. author contributions yu-qing zhang conceived this study; meng zhang and dong liu constructed the database and performed the statistical analysis and wrote the paper. yu-qing zhang revised the paper. references an, l.j., guan, s., shi, g.f., bao, y.m., duan, y.l., jiang, b., 2006: protocatechuic acid from alpinia oxyphylla against mpp+-induced neurotoxicity in pc12 cells. food chem. toxicol. 44, 436-443. doi: 10.1016/j.fct.2005.08.017 aoyagi, y., kasuga, a., fujihara, s., sugahara, t., 1995: isolation of antioxidative compounds from saxifraga stolonifera. j. jpn. soc. food sci. 42, 1027-1030. doi: 10.3136/nskkk.42.1027 chanwitheesuk, a., teerawutgulrag, a., kilburn, j.d., rakari yatham, n., 2007: antimicrobial gallic acid from caesalpinia mimo soides lamk. food chem. 100, 1044-1048. doi: 10.1016/j.foodchem.2005.11.008 chen, a., wang, s., 2003: cn patent. 1393229 a. chen, z., liu, y.m., yang, s., song, b.a., xu, g.f., bhadury, p.s., jin, l.h., hu, d.y., liu, f., xue, w., zhou, x., 2008: studies on the chemical constituents and anticancer activity of saxifraga stolonifera (l.) meeb. bioorgan. med. chem. 16, 1337-1344. doi: 10.1016/j.bmc.2007.10.072 cheon, s.y., chung, k.s., jeon, e., nugroho, a., park, h.j., an, h.j., 2015: anti-inflammatory activity of saxifragin via inhibition of nfkappa b involves caspase-1 activation. j. nat. prod. 78, 1579-1585. doi: 10.1021/acs.jnatprod.5b00145 faried, a., kurnia, d., faried, l.s., usman, n., miyazaki, t., kato, h., kuwano, h., 2007: anticancer effects of gallic acid isolated from indonesian herbal medicine, phaleria macrocarpa (scheff.) boerl, on human cancer cell lines. int. j. oncol. 30, 605-613. doi: 10.3892/ijo.30.3.605 kawada, m., ohno, y., ri, y., ikoma, t., yuugetu, h., asai, t., watanabe, m., yasuda, n., akao, s., takemura, g., minatoguchi, s., gotoh, k., fujiwara, h., fukuda, k., 2001: anti-tumor effect of gallic acid on ll-2 lung cancer cells transplanted in mice. anti-cancer drug 12, 847fig. 3: standard curves of samples concentration and their absorption at 275 nm. a, b and c represent the curves of standard gallic acid, protocatechuic acid and bergenin, respectively. tab. 2: reproducibility of gallic acid, protocatechuic acid, and bergenin of the hplc analysis. sample values of repeated measurements (n = 5, mg/g) mean sd rsd gallic acid 0.339 0.357 0.336 0.339 0.337 0.342 0.009 2.570 protocatechuic acid 0.077 0.087 0.078 0.078 0.087 0.081 0.005 6.037 bergenin 2.260 2.701 2.204 2.294 2.581 2.409 0.218 9.052 http://dx.doi.org/10.1016/j.fct.2005.08.017 http://dx.doi.org/10.3136/nskkk.42.1027 http://dx.doi.org/10.1016/j.foodchem.2005.11.008 http://dx.doi.org/10.1016/j.bmc.2007.10.072 http://dx.doi.org/10.1021/acs.jnatprod.5b00145 http://dx.doi.org/10.3892/ijo.30.3.605 128 m. zhang, d. liu, y.-q. zhang a b fig. 5: (a) the distribution of gallic aicd, protocatechuic acid and bergenin in different parts of s. stolonifera (n = 8). (b) hplc chromatogram of the standard sample, alcohol extract of the stem-leaves, roots of s. stolonifera and the whole herb. the superscript letters of the same column of data are completely different, indicating that the difference is significant (p < 0.05), and any of the same letters indicates that the difference is not significant (p < 0.05). tab. 3: the recoveries of gallic acid, protocatechuic acid and bergenin in s. stolonifera. sample sample content dosage theoretical value measured value recovery sd rsd (μg) (μg) (μg) (μg) (%) (%) gallic acid 136.668±0.009 70 206.668 211.92±0.040 102.491% 0.067 6.516 protocatechuic acid 32.532±0.005 50 82.532 82.32±0.023 99.685% 0.105 10.485 bergenin 963.776±0.218 1500 2463.776 2590.992±0.768 105.050% 0.107 10.197 values represent means ± sd (n = 5). tab. 4: the difference in gallic acid, protocatechuic acid and bergenin content between s. stolonifera varieties. sample gallic acid protocatechuic acid bergenin resource area (mg/g) ±sd (mg/g) ±sd (mg/g) ±sd sichuan in western china 0.347±0.014b 0.422±0.056b 11.381±0.373c shuyang in eastern china 0.287±0.014c 0.171±0.008e 9.605±0.099d suqian in eastern china 0.191±0.016e 0.362±0.056c 15.314±0.832a fujian in southern china 0.538±0.046a 0.217±0.014d 13.841±0.515b suzhou in eastern china 0.212±0.011d 0.616±0.035a 14.137±0.805ab values represent means ± sd (n = 8). the superscript letters of the same column of data are completely different, indicating that the difference is significant (p < 0.05), and any of the same letters indicates that the difference is not significant (p > 0.05). fig. 4: the difference in content and hplc pattern of ga, pa and bergenin in s. stolonifera cultivated in five different locations in china. a b rapid analysis of the bioactives in s. stolonifera 129 852. doi: 10.1097/00001813-200111000-00009 kim, h.s., lim, h.k., chung, m.w., kim, y.c., 2000: antihepatotoxic activity of bergenin, the major constituent of mallotus japonicus, on carbon tetrachloride-intoxicated hepatocytes. j. ethnopharmacol. 69, 79-83. doi: 10.1016/s0378-8741(99)00137-3 kim, y.j., 2007: antimelanogenic and antioxidant properties of gallic acid. biol. pharm. bull. 30, 1052-1055. doi: 10.1248/bpb.30.1052 liu, c.l., wang, j.m., chu, c.y., cheng, m.t., tseng, t.h., 2002: in vivo protective effect of protocatechuic acid on tert-butyl hydroperoxideinduced rat hepatotoxicity. food chem. toxicol. 40, 635-641. doi: 10.1016/s0278-6915(02)00002-9 longtao, j., 2008: cn patent. cn 101444566 b. luo, h., wu, b., chen, j., 1988: research of active compounds in saxifraga. journal of china pharmacology university 19, 1-1. min, s.w., ryu, s.n., kim, d.h., 2010: anti-inflammatory effects of black rice, cyanidin-3-o-beta-d-glycoside, and its metabolites, cyanidin and protocatechuic acid. international immunopharmacology 10, 959-966. doi: 10.1016/j.intimp.2010.05.009 morita, n., shimizu, m., arisawa, m., koshi, m., 1974: studies on the medicinal resources. xxxvi. the constituents of the leaves of saxifraga stolonifera meerburg (saxifragaceae). chem. pharm. bull. (tokyo) 22, 1487-1489 nakagiri, r., kamiya, t., hashizume, e., 2001: us patent. 1213027 a2. nazir, n., koul, s., qurishi, m.a., najar, m.h., zargar, m.i., 2011: evaluation of antioxidant and antimicrobial activities of bergenin and its derivatives obtained by chemoenzymatic synthesis. eur. j. med. chem. 46, 2415-2420. doi: 10.1016/j.ejmech.2011.03.025 ohno, y., fukuda, k., takemura, g., toyota, m., watanabe, m., yasuda, n., qiu, x.b., maruyama, r., akao, s., gotou, k., fujiwara, t., fujiwara, h., 1999: induction of apoptosis by gallic acid in lung cancer cells. anti-cancer drug 10, 845-851. doi: 10.1097/00001813-199910000-00008 priscilla, d.h., prince, p.s.m., 2009: cardioprotective effect of gallic acid on cardiac troponin-t, cardiac marker enzymes, lipid peroxidation pro ducts and antioxidants in experimentally induced myocardial infarction in wistar rats. chem.-biol. interact. 179, 118-124. doi: 10.1016/j.cbi.2008.12.012 prithiviraj, b., singh, u.p., manickam, m., srivastava, j.s., ray, a.b., 1997: antifungal activity of bergenin, a constituent of flueggea microcarpa. plant pathol. 46, 224-228. doi: 10.1046/j.1365-3059.1997.d01-220.x shi, g.f., an, l.j., jiang, b., guan, s., bao, y.m., 2006: alpinia protocatechuic acid protects against oxidative damage in vitro and reduces oxidative stress in vivo. neurosci. lett. 403, 206-210. doi: 10.1016/j.neulet.2006.02.057 swarnalakshmi, t., sethuraman, m.g., sulochana, n., arivudai nambi, r., 1984: a note on the antiinflammatory activity of bergenin. curr. sci. india 53, 917-917. takahashi, h., kosaka, m., watanabe, y., nakade, k., fukuyama, y., 2003: synthesis and neuroprotective activity of bergenin derivatives with antioxidant activity. bioorgan. med. chem. 11, 1781-1788. doi: 10.1016/s0968-0896(02)00666-1 tseng, t.h., hsu, j.d., lo, m.h., chu, c.y., chou, f.p., huang, c.l., wang, c.j., 1998: inhibitory effect of hibiscus protocatechuic acid on tumor promotion in mouse skin. cancer lett. 126, 199-207. doi: 10.1016/s0304-3835(98)00010-x tseng, t.h., kao, t.w., chu, c.y., chou, f.p., lin, w.l., wang, c.j., 2000: induction of apoptosis by hibiscus protocatechuic acid in human leukemia cells via reduction of retinoblastoma (rb) phosphorylation and bcl-2 expression. biochem. pharmacol. 60, 307-315. doi: 10.1016/s0006-2952(00)00322-1 zhang, x.l., shi, g.f., liu, x.z., an, l.j., guan, s., 2011: anti-ageing effects of protocatechuic acid from alpinia on spleen and liver antioxidative system of senescent mice. cell. biochem. funct. 29, 342347. doi: 10.1002/cbf.1757 zhuang, q., chen, j.h., chen, j., lin, x.h., 2008: electrocatalytical properties of bergenin on a multi-wall carbon nanotubes modified carbon paste electrode and its determination in tablets. sensor actuat b-chem. 128, 500-506. doi: 10.1016/j.snb.2007.07.040 orcid yu-qing zhang https://orcid.org/0000-0001-7670-386x meng zhang https://orcid.org/0000-0002-9689-7865 address of the corresponding author: professor yu-qing zhang, department of applied biology, school of basic medical and biological sciences, soochow university; rm702-2303, renai road no. 199, dushuhu higher edu. town, suzhou 215123, pr china e-mail: sericult@suda.edu.cn © the author(s) 2019. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1097/00001813-200111000-00009 http://dx.doi.org/10.1016/s0378-8741(99)00137-3 http://dx.doi.org/10.1248/bpb.30.1052 http://dx.doi.org/10.1016/s0278-6915(02)00002-9 http://dx.doi.org/10.1016/j.intimp.2010.05.009 http://dx.doi.org/10.1016/j.ejmech.2011.03.025 http://dx.doi.org/10.1097/00001813-199910000-00008 http://dx.doi.org/10.1016/j.cbi.2008.12.012 http://dx.doi.org/10.1046/j.1365-3059.1997.d01-220.x http://dx.doi.org/10.1016/j.neulet.2006.02.057 http://dx.doi.org/10.1016/s0968-0896(02)00666-1 http://dx.doi.org/10.1016/s0304-3835(98)00010-x http://dx.doi.org/10.1016/s0006-2952(00)00322-1 http://dx.doi.org/10.1002/cbf.1757 http://dx.doi.org/10.1016/j.snb.2007.07.040 https://orcid.org/0000-0001-7670-386x https://orcid.org/0000-0002-9689-7865 art10 journal of applied botany and food quality 82, 55 59 (2008) 1 institute for plant biology, technical university braunschweig, germany 2 institute for plant cultivation, schnega, germany 3 institute for crop and soil science, julius-kühn-institute (jki), federal research centre for cultivated plants, braunschweig, germany experimental field cultivation of in vitro propagated high-yield varieties of tropaeolum majus l. maik kleinwächter1, imke hutter2, carolin schneider2, ewald schnug3, dirk selmar1* (received may 10, 2008) summary about 10,000 mass propagated clonal progenies of the medicinal plant tropaeolum majus l. had been cultivated in an experimental field trial to analyze the large scale cultivation of nasturtium-plants for pharmaceutical utilization. the glucotropaeolin contents of the eight tropaeolum-clones, which had been established and propagated by in vitro-culture techniques, had been monitored and compared with unselected plants from commercial seed mixtures (sm-plants). whereas the intra-clonal variation of the glucosinolate levels was significantly lower than the variability of the sm-plants, the glucotropaeolin content in the clonal progenies was markedly lower than in both, in the clonal mother plants as well as in the sm-plants. the proposed explanation for this phenomenon is based on the fact that the genetically identical cloned plants reveal only a very narrow phenotypical amplitude, which accordingly resulted in designated glucosinolate levels due to the certain environmental situations. however, under changing conditions, the corresponding glucotropaeolin content might be much lower. in contrast, the sm-plants reveal – due to the strong genetic heterogeneity – a much broader phenotypical amplitude of their physiological characteristics. consequently, under changing growth conditions various individual plants may accumulate high amounts of glucotropaeolin. these coherences explain both, firstly, the finding that the clonal mother plants revealed very high glucotropaeolin levels under the certain – maybe spatial limited cultivation conditions – whereas their progenies accumulate far less glucosinolates; and secondly, that the average content in the sm-plants is higher than the mean content of the clonal progenies. these data suggest that the much cheaper growing of nasturtium plants from seeds should be favoured over the more sophisticated in vitro-propagation techniques. anyhow, for industrial farming there is one great advantage for the usage of in vitro generated tropaeolum plants: the selected, high glucosinolate-nasturtium clones all reveal a compact growth with short tendrils. therefore, the mechanical harvest of the corresponding clonal progenies, is quite unproblematic in comparison to the difficile harvest of sm-plants, most exhibiting tendrils of several meters. introduction nasturtium (tropaeolum majus l.), a traditional medicinal plant containing high amounts of glucotropaeolin is used to treat infections of the urinary tract (hoffmann-bohm and koch, 1994). in contrast to most other glucosinolate plants t. majus contains just one type of glucosinolate (kjær et al., 1978), i.e. the benzylglucosinolate, also named glucotropaeolin. in the course of any decompartmentation, e.g. during herbivore feeding or pathogen attack, glucotropaeolin is hydrolyzed by myrosinases to yield the unstable thiohydroximateo-sulphonates. depending on the reaction conditions (i.e. ph, the presence of fe2+-ions, or the abundance of epithiospecifier proteins) these compounds react to a wide array of further products, the socalled mustard oils, comprising of isothiocyanates, nitriles or thiocyanates (for review bones and rossiter, 2006; halkier and gershenzon, 2006; selmar, 2008), revealing important functions within the various ecological interactions, e.g. as protective agent (for review see wittstock et al., 2003; wheat et al., 2007). when tropaeolum leaves are consumed, the mustard oils produced are absorbed in the intestine and finally excreted into the urine (mennicke et al., 1988; vermeulen et al., 2003). due to the antimicrobial activity of the benzyl isothiocyanate, the growth of bacteria causing the inflammations is inhibited and the infection of the urinary tract (e.g. cystitis) is cured (hoffmann-bohm and koch, 1994). recently, various efforts had been made to use dried nasturtium leaves as modern phytopharmacon (kleinwächter 2008; kleinwächter et al., 2008). for this purpose, a sufficient high glucotropaeolin concentration in the dried drug is required, i.e. a very high glucosinolate concentration in the fresh leaves as well as a minimal loss whilst drying. a corresponding screening of several hundreds individual nasturtium plants resulted in the selection of eight promising clones from which in vitro-plants had been generated and micro-propagated (kleinwächter et al., 2008). corresponding progenies had been transferred into soil and were cultivated in a comprehensive field trial (fig. 1). in this paper, the glucotropaeolin contents of these nasturtium plants are presented and compared with those of the mother plants in order to create a solid basis for the large scale cultivation of tropaeolum-plants for pharmaceutical utilization. materials and methods plant material from the plants selected by an extensive screening described by kleinwächter et al. (2008) in vitro-plants plants have been generated and propagated on 0.8 % agar with ms-media (murashige and skoog, 1962), containing 0.1 ppm tdz (1-phenyl-3-(1,2,3thiadiazol-5-yl-urea) and 0.5 ppm iaa (3-indole acetic acid); for details see matallana et al. (2006). after mass propagation the in vitro-plants (fig. 1a) had been transferred into soil. after eight weeks of acclimatization in a green house (fig. 1b), over 10,000 progenies were planted on an experimental field with about 60 cm spacing (fig. 1c). for fertilization, 100 kg n / ha (as calcium/ammonia nitrate) and 100 kg s / ha was applied as elemental sulphur (kumulus, basf). in total, an experimental area of nearly one half hectare was used for cultivation of tropaeolum (fig. 1d). three months later leaves were harvested. the plants derived from the commercially available seed mixture (sm) were sowed by applying six seeds per hole about three weeks before the in vitro-plants were transferred in the field as described by kleinwächter et al. (2008). sample preparation tropaeolum leaves were harvested while the plants were flowering. for quantification of glucosinolates in the fresh leaves, directly after* corresponding author. 56 maik kleinwächter, imke hutter, carolin schneider, ewald schnug, dirk selmar fig. 1: mass propagation of tropaeolum majus. a) large-scale in vitro-propagation of the eight selected varieties in the growth chamber. b) acclimatization of the progenies in the green house. c) field trial of fl 37: like the other clones, also this variety exhibits a compact growth with short tendrils. d) aerial view of the entire field trial 2005. detaching the leaves – still in the field – the plant material was shock frozen in liquid nitrogen. to exclude any intra-individual variations, in each case 15 leaves of each plant were pooled as one sample. the material was homogenized with mortar and pestle in liquid nitrogen, and subsequently freeze dried. 20 mg aliquots of the lyophilized materials were used for the glucotropaeolin analyses. each quantification was performed as independent double estimation. quantification of glucotropaeolin quantitative analyses of glucotropaeolin were performed by hplc: 160 µl arbutin (10 mm) were added as internal standard to each 20 mg-aliquot. the samples were extracted three times with 1 ml of meoh (80 %, containing 8.5 mm ammonia acetate). extraction was boosted using ultrasonification (10 min at 50°c). after centrifugation (15 min at 10.000 x g), the supernatants were pooled and concentrated by evaporation to final volumes of about 200 to 300 µl. after the estimation of the exact volumes, water was added to yield exactly 600 µl of aqueous samples. then 1280 µl ammonia acetate (43.5 mm) and 120 µl meoh were added to obtain a final volume of exactly 2 ml and to achieve the composition of the hplc eluent a (see below). hplc analysis was performed using a rp 18 column (250 x 4 mm). elution (1 ml / min) was achieved by applying a one step gradient 8 min eluent a followed by 3 min eluent b (a: 6 % meoh, 40 mm ammonia acetate, b: 14 % meoh, 40 mm ammonia acetate). for the detection of glucotropaeolin and arbutin absorbance was recorded at 230 nm. in order to eliminate all undesired substances from the column, after each chromatography, a rinse cycle with 80 % meoh (10 min) and a re-equilibration step (25 min, eluent a) were introduced. based on the peak areas of glucotropaeolin and the internal standard, the amounts of glucotropaeolin were calculated. results and discussion variation of glucotropaeolin content of nasturtium plants in order to screen for glucotropaeolin-rich tropaeolum-varieties exploitable for pharmaceutical usage more than 200 individual plants had been analyzed by kleinwächter et al. (2008). this screening mass propagation of glucosinolate-rich nasturtium plants 57 revealed that the glucosinolate content in the leaves varied drastically from less than 40 µmol/g d.w. (~ 16 mg /g d.w.) to more than 130 µmol/g d.w. (~ 50 mg /g d.w.), corresponding to an average value of 60 µmol/g d.w. (fig. 2). for the selection of suitable plants, special attention was paid to high glucotropaeolin content in the fresh leaves, to minimal losses of glucotropaeolin whilst drying and to compact growth characteristics (kleinwächter et al., 2008). from the most promising plants eight defined clones had been established by in vitro-culture techniques. the question arose, if the glucosinolate levels of the genetically identical progenies of each clone would be similar to that of their mother plants, and if the variability of the glucotropaeolin contents might be less than that of the original, unselected nasturtium population. as expected, the intra-clonal variation was significantly lower than the variability of the unselected plants: in contrast to the high standard deviation of 32.4% (based on the average glucotropaeolin content for the original population of 60 µmol/g d.w., fig. 2), it was markedly lower for the clonal progenies, being in the range from 8.8 to 15.2 % with an average value of only 12.0 % (fig. 3). the same outcome is achieved when the highest divergences from the mean value are compared: this value is far over 110 % for the unselected mother plants and thereby nearly five times higher than for the clonal progenies. when the mean values of the glucotropaeolin contents of the progenies are compared with those of the mother plants, it becomes obvious that the clones reveal quite lower glucosinolate levels than their genetically identical ancestors (fig. 4). yet, in this context it has to be considered that – due to the limited lifetime of the mother plants – the glucosinolate analyses pertain to plants which were grown in different, succeeding years. as the weather conditions had been quite different in the summers of the years 2004 and 2005, it can be assumed that either higher mean temperatures or less rainfall influenced markedly the growth as well as the glucosinolate content of the nasturtium plants; yet, these variables are not subject of this study and will be considered elsewhere. similar effects were described by jensen et al. (1996), who found that the soil water potential influences the glucosinolate levels in rape seeds, and by rosa (1997), who reported an corresponding impact of the temperature. fig. 2: variation of glucotropaeolin contents of 60 individual nasturtium plants. for the determination of the glucotropaeolin content, plants had been shock-frozen still in the field. quantification was performed by hplc. all values are based on at least two independent estimations, bearing maximal methodological variations of less than 1.5 %, average value was about 1 %. in order to illustrate the entire plasticity of the glucosinolate levels, the standard deviations as well as the maximal differences from the mean value ( ø ) are mentioned. 1 µmol glucotropaeolin per g dry weight (d.w.) corresponds to about 0,4 mg/g d.w. 80 60 140 100 120 40 20 0 ø = 60.0 ± 19.4 (32.3 %) g lu co tr o p ae o lin [ m o l / g d .w . ] 117.0 % 46.7 % fig. 3: variation of glucotropaeolin contents of four nasturtium clones. for the determination of the glucotropaeolin content, plants had been shock-frozen still in the field. quantification was performed by hplc. all values are based on at least two independent estimations, bearing maximal methodological variations of less than 1.5 %, average value was about 1 %. in order to illustrate the plasticity of the glucosinolate levels in each clone, the standard deviations as well as the maximal differences from the mean value ( ø ) are mentioned. * the progenies of bg 26 revealed two quite different phenotypes: whereas ca. 55% of the bg 26-plants showed a normal growth, about 45% exhibited a reduced, dwarf-like phenotype. g lu c o tr o p a e o li n [ m o l / g d .w .] bg 26 0 20 40 60 80 100 fl 37 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 bg 08 fl 31 15.7 % 10.2 % ø = 79.9 ± 7.0 (8.8 %) 15.5 15.9 ø = 65.8 ± 7.6 (11.6 %) 24.6 24.2 ø = 62.4 ± 9.5 (15.2 %) normal growth dwarf-growth* 15.0 % 21.8 % ø = 63.2 ± 7.9 (12.5 %) g lu c o tr o p a e o li n [ m o l / g d .w .] g lu c o tr o p a e o li n [ m o l / g d .w .] g lu c o tr o p a e o li n [ m o l / g d .w .] % % % % 58 maik kleinwächter, imke hutter, carolin schneider, ewald schnug, dirk selmar fig. 4: the glucosinolate levels in mother plants, their clonal progenies and plants derived from seedlings. for the determination of the glucotropaeolin content, plants had been shock-frozen still in the field. quantification was performed by hplc. in the case of the clonal progenies, each value corresponds to the mean value of 10 individual plants; each single determination is based on at least two independent estimations, bearing maximal methodical variations of less than 1.5%. standard deviations are referred to the individual, clonal variability. in the case of the plants derived from the commercial seed mixture, 60 (2004), 50 (2005) and 15 individuals plants (2006), respectively, had been analyzed. bg 08 bg 26 bg 81 fl 31 fl 37 bg 75 mother plants (2004) progenies (2005) seed mixture progenies (2006) seed mixture, garden (2004) seed mixture, field (2006) seed mixture, garden (2005) 80 60 140 100 120 40 20 0 g lu c o tr o p a e o li n [ m o l / g d .w . ] the assumption that the weather conditions indeed strongly influence the glucosinolate level also in tropaeolum is underlined by the finding that 2004 also the glucotropaeolin content in the plants derived from the commercial seed mixture (sm-plants, grown in the botanical garden) is significantly lower than in the year 2005 (fig. 4). unfortunately, no corresponding data for the year 2006 are available. an analogous influence on the content of secondary plant products by various climatic factors had been reported frequently; in this context, water shortage and corresponding drought stress responses represent important parameters, known to modulate the content of glucosinolates (e.g. mailer and cornish, 1987; jensen et al., 1996) and other natural products (for review see selmar, 2008). surprisingly, the mean glucotropaeolin-levels of the sm-plants (grown in the same experimental field as the clonal progenies) are markedly higher than that of the clonal progenies originated from the high yield mother plants (fig. 4). these findings demonstrate that beside the weather conditions also other factors strongly effect the glucotropaeolin content. one possible explanation for this phenomenon is based on the fact that the genetically identical cloned plants reveal only a very narrow phenotypical amplitude, which accordingly – in a certain environmental situation – results in a designated high glucosinolate level. however, under other conditions, the corresponding glucotropaeolin content might be much lower. in contrast, the sm-plants reveal – due to the strong genetic heterogeneity – a much broader phenotypical amplitude and the physiological reactions might be very diverging. hence it is very likely, that under different growth conditions, various individual plants respond with the accumulation of high amounts of glucotropaeolin. these coherences might explain both, firstly, the finding that the clonal mother plants revealed very high glucotropaeolin levels under the certain – maybe spatial limited cultivation conditions – whereas their progenies accumulate far less glucosinolates, and secondly, that the average content in the sm-plants is higher than that of the clonal progenies. a further explanation for the observed differences in the glucosinolate levels of mother plants and their clonal progenies might be related to different basic growth characteristics: plants which had been developed from in vitro-plants reveal a different growth pattern than seedlings. even after the required acclimatization of the propagated in vitro-plants for eight weeks in the greenhouse – compared to normal seedlings – the progenies showed an reduced growth for several weeks, when transferred into the field. in this context, the development of the root system could play an important role. a corresponding observation was made for the related in vitro plants: only under enhanced sulphur concentration in the medium, they reveal the same glucosinolate levels as the soil grown mother plants (matallana et al., 2006), although the overall amount of sulphur available in the media is quite sufficient and in the same range as in the soil. from this it was deduced that the root system of in vitroplants is not as effective as that of soil grown plants (matallana et al., 2006). however, a corresponding initial difference in the root system of in vitro-plants and seedlings should be levelled during the course of the plant growth and development in the field. conclusion the data presented in this paper demonstrate that the range of variation in glucosinolate content is far lower in the clonal progenies than in the plants originated from the standard seed mixture (smplants). however the mean glucosinolate contents are significantly lower than that of the sm-plants. thus, the less cost-intensive growing of nasturtium plants from seeds should be favoured over the more sophisticated in vitro-propagation techniques. anyhow, for industrial farming there might be one great advantage for the usage of in vitro generated tropaeolum-plants. beside the high glucosinolate levels, the nasturtium clones had also been selected for a compact growth, generating only very short tendrils. therefore, the mechanical harvest of the corresponding clonal progenies, is quite unproblematic in comparison to the difficile harvest of plants exhibiting tendrils of several meters, which are frequently present in the sm-plants. acknowledgements this study was supported in part by the fachagentur für nachwachsende rohstoffe e.v. (fkz: 22018101). the authors appreciate mass propagation of glucosinolate-rich nasturtium plants 59 the organization of the field trial by juliane thiele, and wish to thank dr. anja brauer for providing the photo of the aerial view of the field trial 2005. references bones, a.m., rossiter, j.t., 2006: the enzymic and chemically induced decomposition of glucosinolates. phytochemistry 67, 1053-1067. halkier, b.a., gershenzon, j., 2006: biology and biochemistry of glucosinolates. annual plant reviews of plant biology 57, 303-33. hoffmann-bohm, k., koch, h.p., 1994: tropaeolum. in: hänsel, r., keller, k., rimpler, h. and schneider, g. (eds.), hagers handbuch der pharmazeutischen praxis 6, 1004-1013. springer verlag, berlin. jensen, c.r., mogensen, v.o., mortensen, g., fieldsend, j.k., milford, g.f.j., andersen, m.n., thage, j.h., 1996: seed glucosinolate, oil and protein contents of field-grown rape (brassica napus l.) affected by soil drying and evaporative demand. field crops research 47, 93-105. kjær, a., øgaard madsen, j., maeda, y., 1978: seed volatiles within the family tropaeolaceae. phytochemistry 17, 1285-1287. kleinwächter, m., 2008: pflanzenbiologisch-biochemische grundlagen zur pharmazeutischen nutzung der kapuzinerkresse (tropaeolum majus l). dissertation an der fakultät für lebenswissenschaften der tu braunschweig kleinwächter, m.s., schnug, e., selmar, d., 2008: the glucosinolatemyrosinase-system in nasturtium (tropaeolum majus l.) – variability of biochemical parameters and screening for clones feasible for pharmaceutical utilization. journal of agricultural and food chemistry, in prep. mailer, r.j., cornish, p.s., 1987: effects of water stress on glucosinolate and oil concentrations in the seeds of rape (brassica napus l.) and turnip rape (brassica rapa l. var. silvestris [lam.] briggs). aust. j. exp. agric. 27, 707-711. matallana, l., kleinwächter, m., selmar, d., 2006: sulfur is limiting the glucosinolate accumulation in nasturtium in vitro-plants (tropaeolum majus l.). j. appl. bot. 80, 1-5. mennicke, w.h., görler, k., krumbiegel, g., lorenz, d., rittmann, n., 1988: studies on the metabolism and excretion of benzyl isothiocyanate in man. xenobiotica 18, 441-447. murashige, t., skoog, f., 1962: revised medium for growth and bioassay with tobacco tissue cultures. physiol. plant. 15, 473-497. rosa, e.a.s., 1997: daily variation in glucosinolate concentrations in the leaves and roots of cabbage seedlings in two constant temperature regimes. j. sci. food agric. 73, 364-368. selmar, d., 2008: biosynthesis of cyanogenic glycosides, glucosinolates and nonprotein amino acids. in: wink, m. (ed.), annual plant reviews: biochemistry of plant secondary metabolism. wiley, in press. vermeulen, m., van rooijen, h.j.m., vaes, w.h.j., 2003: analysis of isothiocyanate mercapturic acids in urine: a biomarker for cruciferous vegetable intake. j. agric. food chem. 51, 3554-3559. wheat, c.w., vogel, h., wittstock, u., braby, m.f., underwood, d., mitchell-olds, t., 2007: the genetic basis of a plant-insect coevolutionary key innovation. pnas 104, 20427-20431. wittstock, u., kliebenstein, d.j., lambrix, v., reichelt, m., gershenzon, j., 2003: glucosinolate hydrolysis and its impact on generalist and specialist insect herbivores in recent advances. phytochemistry: integrative phytochemistry. in: romeo, j.t. (ed.), ethnobotany to molecular ecology, pergamon press, oxford, 101-125. address of the author: dirk selmar, institute for plant biology, technische universität braunschweig, mendelssohnstraße 4, 38106 braunschweig, germany; tel.: 49-(0)531-3915881; fax: 49-(0)531-391-8180; e-mail: d.selmar@tu-bs.de << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 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/pagesize [907.087 680.315] >> setpagedevice angewandte botanik. w angewandte botanik zeitschrift für erforschung der nutzpflanzen organ der vereinigung für angewandte botanik herausgegeben von prof. dr. p. graebner prof. dr. e. gilg botanischer garten der universität berlin; dahlem ° botanisches museum der universität berlin; dahlem und dr. k. müller landw. versuchsanstalt augustenberg i. baden i. schriftführer der vereinigung für angewandte botanik erster band (1919) berlin verlag von gebrüder born traeger w 85 schöneberger ufer 12a 1919 alle rechte, insbesondere das recht der übersetzung in fremde sprachen, vorbehalten druck von e. buchbinder (h. duske) in neuruppin inhaltsverzeichnis 1. 0riginalarbeiten seite appel, 0. die zukunft des pflanzenschutzes in deutschland . . . . 3 falck, richard. über die bewertung von holzund pflanzenschutzmitteln im laboratorium und über ein neues spritzmittel für den pflanzen schutz . . . . . . . . . . . . . . . . . . . . 177, 225 fischer, hugo. der gegenwärtige stand der kohlensäurefrage für pflanzenkulturen . . . . . . . . . . . . . . . . . . 138 graebner, p ., medlewska, e . und zinz, a . typha als nutzpflanze . 30, 9 8 hah mann, c . studium über eine brombeerkrankheit . . . . . . . 103 herzog, a . über eine mikroskopisch-graphische methode der bestimmung des fasergehalts von gespinstpflanzen . . . . 65 kochs, j. untersuchungen über den einfluß verschiedenartiger mineral düngung auf die zusammensetzung von obstdauerwaren . . . . 15 lakon, georg. über die bezahnung der kiele der vorspelze bei lolium perenne l . und l . multiflorum lmk. . . . . . . . . . . 250 lang, wilh. welche maßnahmen sind geeignet, die anwendung der vor handenen guten pflanzenschutzmittel zu allgemeiner und rechtzeitiger durchführung zu bringen . . . 156 medlewska, e . typha als nutzpflanze (siehe graebner) . . . . . 38, 9 8 müller, k . bericht über die 15. hauptversammlung der vereinigung für angewandte botanik (4./5. viii. 1919) . . . . . . . . . . 186 neger, f. w. ein neues, untrügliches merkmal für rauchschäden bei laubhölzern 129 rippel, august. der biologische abbau der pflanzlichen zellmembranen 78 rost, e . die indische rundoder rangoonbohne . . . . . . . . . 2 7 saba litschka, th. verbreitung falscher ansichten über den wert pflanz licher nahrungsmittel im volke . . . . . . . . . 74 simon, j. die beurteilung des anbauwertes französischer rotkleesaaten 146 zinz, a . typha als nutzpflanze (siehe graebner) . . . . . . . . 9 9 z4 769 iv literaturverzeichnis ii . literaturverzeichnis 1 . pflanzliche nahrungsund futter mittel beckenstedt, lupinenverwertung 259 buchka, lebensmittelgewerbe 259 dittrich, vergiftungen durch pilze 51 döderlein, wegweiser für pilzfreunde 51 ehrenberg, hahn, zyl, trocknungs kosten für zuckerrüben 259 –, winterfutter 193 einecke, farbenänderungen der kar toffelblüte 113 etty, suikerindustrie in natal 261 fischmann, brennessel 260 frank, kartoffel 51 samen 193 –, futtermittelhandel 260 gaßner, entwicklungsrhythmus des wintergetreides 51 gerlach, entbitterung der lupine 193 gerum, ausmahlungsgrad der mehle 114 –, stärkegehalt der haferflocken 113 gramberg, wildgemüse usw. 193 grevillius, mikroskopie des schilf mehles 271 groß, haselnußernte 1917 51 haberlandt, zellwandverdauung 193 hansen, gräserbestimmungen 194 henkel, grünfutter im winter 194 hermann, trockenpilze 114 –, pilzkochbuch 51 herter, schimmelpilze des brotes 51 –, fornet, schimmelpilze des brotes 114 hiltner, heimischen pflanzenwelt 51 fº ) hoffmann, einsäuerungsmethoden 114 hueppe, unser täglich brot in krieg und frieden 52 janson, sprengstoffe im obstbau 194 kaufmann, verwertung von pilzen und wildfrüchten 52 futtergewinnung aus der kiehl, erfahrungen eines rübenbauers 52 kinzel, futtermittelkontrolle 114, 194 –, verderben von futtermitteln 114 kirchner, stoppelfruchtbau zurfutter gewinnung 195 kling, kriegsfuttermittel 53 kochs, einfluß von mineraldüngung auf obstdauerwaren 15 –, hauhechel und bingelkraut 195 kole, garnalenen zeesterrenmeel 114 kroemer, wurzelentwicklung der ge müsepflanzen 5 3 lapicque, meeralgen als pferdefutter 53 lauterbach, kartoffeltrocknung 260 gabriel, entbittern der reismelde magnus, strohaufschließung 195 manfeld, bericht der untersuchungs anstalt für nahrungsund genuß mittel 114 markgraf, artischocken u. kardi 260 –, wintergemüse 2 maurizio, nahrungsmittel aus ge treide 195 mohs, rübenmehl 260 molisch, zucker aus ahornbäumen 53 neubauer, feuchtigkeitsgehalt der futtermittel beim mahlen 195 –, spelzengehalt und futterwert der müllereiabfälle 195 pfeiffer, simmermacher, gehalt der haferpflanzen a n stickstoff, phosphorsäure und kali 223 pfeiler und engelhardt, rizin in futtermitteln 260 pringsheim, strohaufschluß 196 richert, pilzverwertung 260 rieken,vademecum für pilzfreunde 196 r täöst, rangoonbohne 27 sabalitschka, lupinenverwertung 115 –,wert pflanzlicher nahrungsmittel 74 salkowski, kohlehydratgehalt der flechten 115 scherer, lebensmittel und die ersatz stoffe 260 literaturverzeichnis v schindler, mikroskopische scheidung alpwirtschaftlich wich tiger gräserarten 196 schnegg, edelpilzzucht (champignon kultur) 54 –, speisepilze 53 schultze, zucker holländisch-indiens 199 schütze, wildpflanzen-lexikon 196 schweinfurth, brotbacken mit zu satz von flechten in ägypten 196 senft, untersuchung der wichtigsten nahrungsund genußmittel 197 snell, vermehrung der kartoffel 197, 212 steppes, trocknungsverfahren bei ge treidegarben 260 stoffert, obstund gemüsegut der neuzeit 54 thoms, entbitterung der lupine 197 trenkle, gemüsesamenbau 54 unter hasterlik, reiz-undrauschmittel 199 hoepner, schalengehalt in kakao waentig, holzaufschließung zu futter zwecken 54 wagner, schöler, strohstoff und seine verdaulichkeit 197 v. wenckstern, süßpreßfutterver fahren 197 weydemann, erdbeeren 198 winterstein, rohrzucker aus pflanz lichen objekten 115 wischo, stärke aus roßkastanien zade, haferbau 54 zander und schätzlein, heilund gewürzpflanzen als honigspender 54 zielstorff, weintrestermehl 260 115 2. genußmittel (zucker, obst und gemüse vgl. 1. nahrungsmittel) beitter, kaffee-ersatzstoffe 54 bernard, theezaden 260 beythien, gewürze und gewürz-ersatz 199 braun, tabak 54 bredemann, traubensaftkonserven 260 coene, rauchtabak 54 den doop, gallobelicus nicotianae 260 diem, tabak 261 erzeugnissen 115 hoffmann, tabakbau 55, 261 joachimowitz, bilsenkrautsamen enthaltender mohn 115 kißling, tabakkunde, tabakbau 115 knapp, estimation of cacao 115 kochs, einfluß von mineraldüngung auf obstdauer waren 15 kuráz, safran und seine kultur 57 lamberger, tabakbau 55 –, tabakfermentation 55 leersum, bernard, theeplant 261 liehr, mohn 199 manfeld, bericht der untersuchungs anstalt für nahrungsund genuß mittel 114 preißecker, brezina, wenusch,tabak streckung und tabakersatz 55 roß, heil-, gewürzu. teepflanzen 199 rothenfusser, tee 261 schmidt, einfuhr von heilund ge würzpflanzen 117 schnegg, edelpilzzucht (champignon kultur) 54 schönfelde, selbstgezogener tabak 55 schröter, tabakbau, kunstu. kau tabak 55 schulz, p. f. f., tabakpflanze und ihre schädlinge 54 senft, untersuchung der wichtigsten nahrungsund genußmittel 197 steppes, deutscher tabakbau 55 zander, heilund gewürzpflanzen als honigspender 54 3. arzneimittel aron, kardobenediktenkrautöl 199 blüm ml, stammbuch des apothekers mergenthaler 115 bohn, heilwerte heimischer pflanzen 55 buschmann, chemische bestandteile von bulbus scillae 115 cocx, valeriana officinalis 115 dezani, ricerche farmacognostiche sulla „catha edulis“ 55 vi literaturverzeichnis fritsche, selen im pflanzlichen und tierischen organismus? 115 fühner, scopoliawurzel als gift und heilmittel 200 grimme, capsella bursa pastoris 200 –, manihotsamen 200 hangseth, einsammeln und anbau medizinischer pflanzen 115 kofler, capita papaveris als ver fälschung von opium 56 –, mit brechweinstein enzianpulver 56 holmes, belladonna 116 –, strophanthus semina 116 hoyer, verfälschung von foliasennae mit folia sennae „palthe“ 55 joachimowitz, sennesblätter 56 kuráz, safran und seine kultur 57 lauffer, la mandragore 57 linde, radix violae odoratae und radix violae tricoloris 116 lingelsheim, stammpflanze der eschenmanna 116 meyerhoff, bazar der drogen und wohlgerüche in kairo 201 orta, coltivazione delle piante medi cinali 116 osterwalder, pharmazeutisch wich tige gentiana-wurzeln 116 roß, heil-, gewürzund teepflanzen 199 schmidt, einfuhr von heilund ge würzpflanzen 117 schrötter, arzneipflanzenkulturen 57 schulz, arzneipflanzen 58 schütze, wildpflanzenlexikon 196 semmel, chinesischer rhabarber 58 sieburg, rudolf kobert 117 tschirch, offizinelle rhabarberarten 202 tun mann, unterscheidung von rha pontic und rheum 58 wasicky, arzneiwarenerzeugung 117 –, arzneipflanzenkultur 117 –, verfälschungen von drogen 59 –, hoyer, substitution von catechu 117 vermischtes wimmer, untersuchung von rha pontik und rheum 118 winterstein, jod in pflanzen 118 – und weinhagen, arekaalkaloide 118 zander, heilund gewürzpflanzen als honigspender 34 4. fette alpers, obstkernsammlung und obst kernöl 261 aron, konstitution einiger öle 59 –, kardobenediktenkrautöl 199 bücher, fickendey, schildkröten ölpalme 202 engelhardt, seifenfabrikation 202 engländer, leinölersatz 261 –, fetthefefabrikation 262 fordyce und torrance, pflaumen kerne 262 griffiths-jones, lattichöl 262 groß, haselnußernte 1917 51 grün, fettchemie.u. fettindustrie 262 heiduschka, lüff, öl der nacht kerze 118 heuß, teeröl 262 kleeberger, düngungsversuche mit raps 60 knorr, speisefette 262 lier, mohn 199 moore, egloff, fette äus petroleum 262 odrich, ölsaaten 262 prasch, obstkernöle 203 rothéa, traubenusw. kernöl 262 schelenz, bucheckernöl 118 thurston, maisöl 262 –, sesamöl 263 –, sojabohnenöl 263 weis, maisölfabrikation 263 5. ätherische öle, harze, gummi andés, harzgewinnung 263 –, geigenharz 263 –, neues produkt aus kauriharz 263 chiej -gamacchio, coltivazione et lavorazione dellamenta da essenza 59 literaturverzeichnis vii cavel, antiseptischer wert einiger öle 263 goldschmidt u. weiß, seifenfabri kation aus deutschen harzen 263 grimme, manihotsamen 201 gschwender, rosenölerzeugung 263 hwr., gummikulturpflanzen 263 h., kirschgummi 265 klimburg, harze 263 leiningen, kolophonium u. terpen tinöl 263 merz, reindarstellung öliger produkte 263 salvaterra, extraktionsharze aus fichtenscharrharz 264 tschirch, entstehung der harze 264 walbaum, japanisches pfefferminz öl 264 wallach, terpene und ätherische öle 264 wilson, young, agrumenfrüchte-öl 264 wright, krystallisation des menthols 264 6. kautschuk und guttapercha boutaric, madagaskar-kautschuk sorten 264 grimme, manihotsamen 201 hillen, kautschuk u. guttapercha 264 vries, plantagerubber 264 7. gerbund farbstoffe cross, greenwood, lamb, kolloidale gerbstoffverbindungen 264 kryz, farbstoff der beeren deswilden efeu 265 feigl, anfärbbarkeit anorganischer körper 264 hollborn, teerfarbstoffe 118 jalade, gerberinden 264 levinstein, englische farbstoff industrie 265 ocman, azofarbstoffe 265 schmidt, gerbstoffhaltige rinden 60 8. faserstoffe barfuß, brennesselfaser 203 beckenstedt, lupinen verwertung 259 collin, sind einheimische spinnfasern jetzt überflüssig 118 driesen, kupfersalzwirkung auf fasern 265 fischmann, brennessel 260 graebner, kolbenschilf als faser pflanze 60 –, medlewka, zinz, typha als nutz pflanze 30 haase -ullersdorf, rasenröste des flachses 118 . haller, nachweis der typhafaser 204 heerberger, juteersatz 60 herzog, anatomischer bau der teich binse 118 –, bastfasern des flachsstengels 118 –, bestimmung des fasergehalts von gespinstpflanzen 65 –, flachsstengel 60 heyking, rohrernte 255 v. hippel, rohstoffversorgung d. deut schen textilindustrie 60 –, deutsche rohstoff-, insbesondere spinnfaserversorgung 60 van itersen jr., vezelstoffen 61 koller, heygny, zeitner, weinreben faser 119 kruse, feldmäßiger anbau der nessel 203 kuhnert blankenese, hanfanbau 203 kuhnow, hanf und seine entwicklung 119 leykum, hopfenfaser 203 –, lupinenfaser 119 –, typha als faser 119 –, schilfkultur 60 löwenthal, technologie der spinn fasern 265 marquart, fruchtfolge und ausdehnung des hanfbaus 122 –, hanf 265 marschik, melilotusklee 120 mayer, nesselanbauversuche in bayern 204 viii literaturverzeichnis medlewska, typha 38, 98 netolitzki, buchenschwamm 60 rasser, nesselfaser 60 reinhard, hopfenfasern 120 sabalitschka, lupinenverwertung 115 schmidt, o., spinnpflanzen im land wirtschaftsbetriebe 265 schürhoff, faserforschung 204 –, verbaumwollung von fasern 61 –, verbaumwollung von pflanzen fasern 120 –, lupine als faserpflanze 120 schütze, wildpflanzen-lexikon 196 schwiers, zellstoff 61 sellengren, faserstoffe aus torfmasse 204 süvern, torffaser 204 ulbrich, ist baumwolle in deutsch land anbaufähig? 120 –, heimische faserpflanzen 120 –, seegras als textilfaser 120 –, waldwolle als spinnfaser 120 –, blumenbinse als faserpflanze 121 –, besenginster als faserpflanze 61 –, wurzeln heimischer gräser als faserstoffe 61 volpato, behandlung von stroh 61 (volpato), strohfaser in der textil industrie 205 warburg, weltvorräte 121 zinz, typha 99 9. hölzer abeles, weichholzhandel (fichte und tanne) 61 flatscher, holzwirtschaft in deutsch österreich 61 flemming, in der tischlerwerkstatt vernachlässigte hölzer 61 freund, korkersatz 121 fritsche, anbaumethode der fichte 205 großmann, weymouthskiefer 61 huerre, la distillation sèche du bois de juniperus oxycedrus 121 keßler, aus schwedens holzindustrie 62 moll, janssonius, mikrographie des holzes der auf java vorkommenden baumarten 205 neger, nadelhölzer 262 pfeifer, holzhandel und holzindustrie ostpreußens 62 schüpfer, forstwissenschaft 121 waentig,holzaufschließung zu futter zwecken 54 wimmer, erträge des deutschen waldes 62 10. pflanzenbau, physiologie der nutz pflanzen allendorf, ehrenberg, rübenbau 266 becker, serologische untersuchungen über pflanzenbau und pflanzenzucht 122 boskart, baldriananbau 266 bokorny, pflanzendüngung mit menschlichem harn u. sulfitlauge 64 boerger, erschließung der deutschen moorböden 207 böttner, gartenbuch für anfänger 266 brick, widerstandsfähigkeit gegen parasiten 207 bruns,gründüngung imgartenbau 207 chiej-gamacchio, coltivazione et lavorazione dellamenta da essenza 54 coene, anbau guten rauchtabaks 54 deiner ittendorf, düngungsver suche in der buschobstpflanzung 121 duysen, keimkraftdauer einigerwich tiger samen 213 zucker filter, leinsaaten 266 hedler, holzversorgung zur kriegs zeit 62 heinrichs, holz als sparsamer bau stoff 6? fischer, kohlensäurefrage 138 –, kohlenstoffernährung der kultur pflanzen 266 –, w., kalkempfindlichkeit des leins 215 fritsche, anbaumethode d. fichte 205 fru wirth, unkraut auf dem acker land 62 literaturverzeichnis ix gentner, feldund pfeilkresse als kuhnert blankenese, ackerunkräuter 208 grosser, wirkung der uspulunbeize auf die keimfähigkeit 208 hagelberg, plantagenbau im mexikan. tieflande 208 hammerstein, landwirtschaft eingeborenen afrikas 208 hangseth, einsammeln und anbau medizinischer pflanzen 115 harre veld, statistiek van de riet soorten 266 hayunga-weener, schlick als pflanzenschutzmittel 62 heinrich, keimung der timothy früchte 266 herpers, wintergemüse 266 herrmann, keimungsenergie des kiefernsamens 63 hessdörfer, taschenbuch für garten freunde 271 hiltner, düngerbedürfnis der acker böden und wiesen 208 –, kartoffelernteschätzungen 266 hoffmann, tabakbau 52, 261 honcamp, steigerung der landwirt schaftlichen produktion 209 janson, sprengstoffe im obstbau 194 kiehl, erfahrung eines rübenbauers 52 –, umänderung von fruchtfolgen 209 kinzel, durchfrieren von samen 214 kirchner, stoppelfruchtbau z. futter gewinnung 195 kißling, tabakkunde, tabakbau 115 kleeberger, düngungsversuche mit raps 60 –, kulturund düngungsversuche mit lein 209 kochs, einfluß von mineraldüngung auf obstdauerwaren 15 körner, buschbohnenanbau 266 krömer, wurzelentwicklung der ge müsepflanzen 53 kruse, feldmäßiger anbau der nessel 203 kryz, ermittlung des spezifischen ge wichts für technische kartoffel prüfung 209 der hanfanbau 203 kuhnow, hanf und seine entwicklung 119 kuráz, safran und seine kultur 57 lamberger, tabakbau 55 lang, saatschutz gegen fraß 266 lange, webers illustr. gartenbiblio thek 271 leikum, schilfkultur 60 marquart, liehr, mohn und sein anbau 199 fruchtfolge und aus dehnung des hanfbaus 122 mayer, nesselanbauversuche in bayern 204 mitscherlich, standweite von kultur pflanzen 122 –, standraumweite 209 –, feldversuche in der landwirtschaft lichen praxis 209 molisch, pflanzenphysiologie in der gärtnerei 62 müller, k., lösung der phylloxera frage 258 –, pflanzenschutz in baden (1915–18) 121 nolte, wirkung der kaliendlaugen auf boden und pflanze 64 nowacki, kleegrasbau 210 orta, coltivazione della piante medi cinali 116 paulig, moorkultivierung 210 pfeiffer, vegetationsversuch 62 plaut, periodische erscheinungen an wurzeln 62 poenicke, zwergobstbau 210 reinhardt, serradella-bau 211 reinau, kohlensäure u. pflanzen 211 reckert, winterhafer 266 rippel, pflanzliche zellmembranen 78 –, wachstumskurve der pflanzen 266 rüllmann, flachsbau in bayern 211 v. rümker, leidner, sortenanbau versuche 211 schönfelder, selbstgezogener tabak 55 schroeter, tabakbau, kunstu. kau tabak 55 x literaturverzeichnis schrötter, arzneipflanzenkulturen 57 schwede, keimungsverhältnisse der nesselsamen 211 v. seelhorst, düngungsfragen 212 siebert, kronenlichtnelke 212 –, kürbisanbau 267 –, lachenalia tricolor 212 simon, französische rotkleesaaten 146 snell, vermehrung d.kartoffel 197,212 spahr, stickstoffdüngemittel 212 stepper, deutscher tabakbau 55 stoffert, obstund gemüsegarten der neuzeit 54 störmer, lupine 212 störmer, lupinenernte 267 trenkle, gemüsesamenbau 54 tschirsch, anbau offizineller rha barberarten 202 u, bodenverbesserungsmittel 267 ulbrich, ist baumwolle in deutsch land anbaufähig? 120 wangenheim, lupinenernte 267 wasicky, arzneipflanzenkultur 58, 117 weber, kultur der nessel 212 wehsarg, ackerunkräuter in deutsch land 213 werner, kartoffelanbau 267 wiesmann, einfluß des lichtes auf das wachstum des hafers 122 wittmack, treiben derzierpflanzen 62 zade, haferbau 54 zschokke, veredlungsunterlagen 267 ii . pflanzenzüchtung becker, serologische untersuchungen über pflanzenbau und pflanzenzucht 122 behr, neue erdbeersorten 267 duysen, keimkraftdauer einiger wich tiger samen 213 einecke, farbenänderungen der kar toffelblüte 113 fruhwirth, umzüchtung von winter in sommergetreide 213 gassner, entwicklungsrythmus des wintergetreides 51 killer, umzüchtung von winterin sommerweizen 213 lebedinski, darwins zuchtwahl 63 molz, züchtung widerstandsfähiger rebsorten 123 oberstein, knospenvariationen bei kartoffeln 267 prinz, sortenelend im deutschen obst bau 123 sommer, kartoffelzüchtung v . ubisch, gerstenkreuzungen 124 12. samenkunde duysen, keimkraftdauer einiger wich tiger samen 213 filter, leinsaaten herkunft 266 gabriel, entbittern der reismelde samen 193 grosser, wirkung der uspulumbeize auf die keimfähigkeit 208 heinrich, timothyfrüchte 266 herrmann, keimungsenergie des kiefernsamens 6 3 kinzel, durchfrieren von samen 214 schwede, keimungsverhältnisse der nesselsamen 211 trenkle, gemüsesamenbau 54 vogt, ratgeber bei samenanerkennun gen 124 wittmack, gemüsesamenbau 214 –, samenbau im kleingarten 63 13. pflanzenkrankheiten adank, verhütung von frostschäden an reben 124 appel, pflanzenschutz in deutschland 3 bail, ungeziefervertilgung 267 baunacke, wühlmausbekämpfung 267 bethel, puccinia subnitens 267 boltjes, oortwyn, jets over het kwee ken van ziektevry pootgoed b ij aardappeln 215 börner, blunk, rapsglanzkäfer, rapsund kohlerdflöhe 214 briosi, nuova mallatia dei bambu 267 brick, schwarzfleckigkeit der tomaten 125 –, widerstandsfähigkeit gegen para siten 207 brucker, stachelbeermehltau 267 literaturverzeichnis xi burckhardt, bekämpfung des korn –, schädlingsbekämpfung im winterkäfers 63 –, otiorrhynchus rotundatus 125 chivers, the injurions effects of tar via fumes on the vegetation 267 dern, hagelbeschädigte reben 125 –, reblausbekämpfung 267 dewitz, die immunsande 215 duysen, roggenstengelbrand 268 ehrenberg, beizung des winter weizens gegen steinbrand 215 –, steinbrandbekämpfung 268 esmarch, blattrollkranke kartoffeln 125 –, kartoffelkrankheiten 63 falck, bewertung von pflanzenschutz mitteln im laboratorium 177 fischer, ed., biologie d. uredineen 215 florin, om äppletradens skorvsjuka och dess bekämpande 268 friedrichs, plocaederus obesus, feind des kapokbaumes 125 führer, unkrautbekämpfung 268 fulmek, sonderbarer kartoffelfeind (lecanium corni beté) 215 gaßner, sortenempfänglichkeit von getreide gegen rostpilze 215 gaul, kupfervitriol al s saatgutbeiz mittel 268 gescher, feinde des sauerwurms 268 güssow, pathogenic action o f rhizoc tonia on potato 268 haber, krebs 216 hahmann, brombeerkrankheit 103 hall, ziekten en plagen der cultuur gewassen 268 hayunga-weener, schlick als pflan zenschutzmittel 82 herrmann, bekämpfung des obst wicklers 125 hasse, gummifluß der steinobstbäume 268 higgins, a disease of pecan eatkins 268 hiltner, blattrollkrankheit der kar toffel 63, 215 hollrung, „kälken“ welzens 216 des sommer jegen, frostspannerbekämpfung 268 268 jordi, blattrollkrankheit der kartoffel 216 klein, aaskäfer auf rübenblättern 126 kniep, antherenbrand (ustilago vio lacea) 217 knischewsky, voss, erdflöhe 126 lakon, köck, neuer schädling auf picea pun gens 63 korff, pfefferminzrost 126 körner, moniliakrankheit 268 kunkel, wart of potatoes 217 entomophtoreen (insekten feind) 126 lang, anwendung von pflanzenschutz mitteln 156 lauritzen, relation of temperature and humidity to infection b y certain fungi 217 lek, verwelkingsziekten 268 lengerken, otiorrhynchus rotundatus 126 lerch, parasitism of puccinia gra minis 217 lindner, schwarzbeinigkeit junger kohlpflanzen 269 linsbauer, bekämpfung der kohl weißlinge 217 lüstner, krankheiten der obstbäume 126 –, bekämpfung des oidiums 126 mc. cublin, does cromartium viticola winter on the currant 269 –, white pine blister rust 269 mac millan, fusarium-blight of po tatoes 217 mac rostic, inheritance of anthrac nose resistance a s indicated" by a cross between a resistant and a succeptible bean 219 melhuß, studies on the crownrost 217 müller, k., arsenbrühen, ersatz für nikotinbrühen 127 –, bekämpfung der rebenperonospora 217 –, inkubationskalender 64 xii literaturverzeichnis müller, lösung d. phylloxerafrage 258 –, rebenperonospora 64 –, pflanzenschutz in baden 1915–18 121 –, rebschädlinge 63 müller, molz, kupfervitriol als saat gutbeizmittel 269 müller-thurgau und osterwalder, bekämpfung der kohlhernie 126 –, bordeauxbrühe 127 naumann, monilia-gefahr 219 –, azaleen-schädling (gracilaria za. chrysa) 219 –, stachelbeerrost 219 neger, apfelbaumkrebs 219 –, blattrollkrankheit der kartoffel 217 –, krankheiten unserer waldbäume. und gartengehölze 218 –, bedeutung des habitusbildes für die diagnostik von pflanzenkrank heiten 219 –, rauchschäden 129 o., kräuselkrankheit desweinstocks 125 opitz und oberstein, steinbrand bekämpfung 127 osterwalder, hexenbesen 127 pape, brennesselschädlinge 268 –, pflanzliche schädlinge unserer öl gewächse 219 popoff, lösung der phylloxorafrage 127 reckendorfer, der rotbrenner 269 reh, hemoeserna nebulella als sonnen blumenschädling 127 reiling, wundkorkbildung an kar toffelknollen 269 roark, als insektenvertilger wandte pflanzen 268 rose, blistercanker of apple-trees 219 schaffnit, pflanzenschutzdienst in der rheinprovinz 219 scheidter, tannensterben im franken walde 128 schellenberg, gelbsüchtige reben 219 schilling, nikotin-seifenbrühe zur bekämpfung des heuund sauer wurms 219 schmitthenner, reblauswiderstands fähigkeit amerikanischer reben 220 schöppach, steinbrand 269 schoevers, nieuwe ziekten 269 schröder-hall, beizbehandlung des saatguts 220 schulte, bekämpfung von schädlingen und krankheiten des weinstocks (1918) 124 schulz, p. f. f., tabakpflanze und ihre schädlinge 54 schwartz, nacktschneckenplage nordfrankreich (1916) 219 seelhorst, zwergmaus 268 sherbakoff, buckeye-rot of tomato fruit 268 shinbo, japanische pflanzengallen 220 smith, sour rot of lemon 269 snell, kindelbildung im innern einer knolle 270 stanford, wolf, bacterium solanace arum 270 stevens u. hawkins, rhizopus ni gricans 270 steyer, pflanzenschutzstelle 220 stift, feinde der zuckerrübe 128, 240 tisdale, physoderma disease of corn 220 in lübeck themas, seed disinfection by formal durch reformierung der rebenkultur dehyde vapor 220 verhoeven, zaai graamontsmetting270 voges, diesjähriges verhalten der schädlinge 270 vogt, ratgeber bei samenanerkennun voss, ver gen 124 lüstner, krankheiten der kulturpflanzen in der rheinprovinz 1916–17 124 –, rapsglanzkäfer und rapsverborgen rüßler 128 wehnert, bespritzungsversuche an kartoffeln 220 weier, sparassis radicata 270 welten, pflanzenkrankheiten 270 werth, mutterkorn 270 wittmack, gemüsesamenbau 219 –, samenbau im kleingarten 68 literaturverzeichnis xiii wöber, kupferkalkbrühe 220 zacher, schädlinge der kartoffel 221 –,weißährigkeit der wiesengräser 220 zimmermann, erdraupe (agrotis segetum) 221 –, rübenschäden 270 14. bodenbakteriologie u. a. arnd, nitrifikation der moorböden 64 barthel, nitrifikation des stallmistes 270 bokorny, pflanzendüngung mit menschlichem harn u. sulfitlauge 64 boerger, erschließung der deutschen moorböden 207 bruns, gründüngung im gartenbau 207 dein er itt e ndorf, düngungs versuche in der buschobstpflanzung 121 ehrenberg, bodenkolloide 64 friederichs, rapsglanzkäfer als schädling 215 gehring, düngungswirkung des gua nols 221 greve, künstliche düngemittel 221 heinrich und nolte, düngen 270 hiltner, düngebedürfnis der acker böden und wiesen 208 kleeberger, düngungsversuche mit raps 60 –, kulturund düngungsversuche mit lein 209 lemmermann und einecke, stick stoffhaushalt der böden 270 mitscherlich, saucken, iffland, düngungsversuche 270 nolte, denitrifikation bei gegenwart schwer zersetzlicher organischersub stanzen 128 –, kunstdünger 221 –, wirkung der kali-endlaugen auf boden und pflanze 64 pfeiffer, rippel, einfluß der steine auf das wachstum 270 seelhorst, düngungsfragen 212 spahn, stickstoffdüngemittel der zu kunft 212 u. bodenverbesserungsmittel 261 15. gärungsorganismen usw. barthel, sandberg, versuche über das " kaseinspaltende vermögen von zur gruppe streptococcus lactis gehören den milchsäurebakterien 270 osterwalder, selbstheranzucht von reinhefe 270 salkowski, kohlehydratgehalt der flechten und einfluß der chloride auf alkoholgärung 115 1 6 . technische mikroskopie (vgl. auch die mikr. arbeiten unter 1 , 2 , 3 , 8 , 9 , 1 3 ff.) grevillius, mikroskopie des schilf mehls 271 moll, j., mikrographie des holzes der auf java vorkommenden holzarten 205 s ch indler, mikroskopische unter suchungen alpwirtschaftlich wich tiger gräserarten 196 17. verschiedenes bode, gärtnerische betriebslehre 223 braun, edler, dade, deutsche land wirtschaft nach dem kriege 6 4 christoph, landwirtschaft und in dustrie 64 hansen, landwirtschaftliches unter richtswesen 223 hessdörfer, taschenbuch für garten freunde 271 koenig, gärtnerberuf 64 lange, webers illustrierte garten bibliothek 271 m am m e n -brandstein, zwischen feld und wald 64 schelenz, rudolf kobert 117 sieburg, rudolf kobert 117 soskin, französischer sudan und die schibutter industrie 223 stichel, argentinien 223 vir, zu stephan romers gedenken 117 kampf xiv kleine mitteilungen – personalnachrichten ill. kleine mitteilungen alkoholerzeugung aus holz anbau der reismelde (kalt) trennung von forst und weide umwandlung von wald in kartoffelland abholzung des haardtwaldes in baden (ratzel) flechten als watteersatz verwendung des seetangs in der faserindustrie geheimmittel (loffa-kräuter) . . . . . . . . vorkommen von scopolia carniolica in littauen . j. schuster. zur geschichte der quassia amara w. herter. die krankheit des „fadenziehenden brotes“ und seine ver hütung – gips im brot – neue billige pilzbücher . . k. müller-augustenburg. die lösung der phylloxerafrage durch refor mierung der rebenkultur . . . . iw. personalnachrichten (siehe sachregister s. 273 ff.). seite 128, 224, 271 title page uc1.b3299303_page_008_cut.pdf table of contents uc1.b3299303_page_011_cut.pdf section 1 uc1.b3299303_page_015_cut.pdf section 2 uc1.b3299303_page_017_cut.pdf section 3 art_15702.indd journal of applied botany and food quality 94, 132 139 (2021), doi:10.5073/jabfq.2021.094.016 1institute of genetics and physiology, hebei academy of agriculture and forestry sciences, shijiazhuang, china 2hebei plant genetic engineering center, shijiazhuang, china the influence of 1-mcp on the fruit quality and flesh browning of ‘red fuji’ apple after long-term cold storage jingang he1,2, yunxiao feng1,2, yudou cheng1,2, shen wang1, yaxiong fu1, junfeng guan1,2* (submitted: january 25, 2021; accepted: june 28, 2021) * corresponding author summary this study assessed the influence of 1-mcp treatment on the fruit quality and flesh browning of ‘red fuji’ apple at shelf life after long-term cold storage. the ‘red fuji’ fruit were stored at 0±0.5 °c for 270 days after treating with 1.0 μl l-1 1-methylcyclopropylene (1-mcp). fruit quality, browning rate of stem-end flesh, chloro genic acid content, polyphenol oxidase (ppo) activity were analyzed at shelf-life under 20±0.5 °c, the expression profile of ethylene re ceptors (mders1), phenylalnine ammonia lyase genes (mdpal1, mdpal2), quinate hydroxycinnamoyl/hydrxycinnamoyl coa shi kimate gene (mdhct3), polyphenol oxidase genes (mdppo1, mdppo5) and lipoxygenase gene (mdlox) were measured by realtime quantitative pcr. 1-mcp treatment improved the fruit storage quality, decreased stem-end flesh tissue browning, and fruit decay. in addition, the fruit respiration rate and ethylene production rate increased at shelflife, but this increase could be inhibited by 1-mcp. the same rule was observed in the changes of chlorogenic acid content and ppo activity, the expression of mders1, mdpal1, mdppo1 and mdlox were inhibited by 1-mcp as well in the stem-end flesh. thus, 1-mcp treatment improves the fruit quality of ‘red fuji’ apple at shelf-life after long-term cold storage, and inhibits the browning of stem-end flesh by decreasing the chlorogenic acid content and ppo activity. mdpal1, mdhct3, mdppo1 and mdlox participate in the flesh browning progress. keywords: malus × domestica; 1-methylcyclopropylene; storage quality; chlorogenic acid; polyphenol oxidase introduction 1-methylcyclopropene (1-mcp), an inhibitor of ethylene perception, delays fruit ripening and extends the storage and shelf life by impeding binding of ethylene to its receptors (sisler et al., 2003). 1-mcp based technology is widely used by apple industries around the world to help maintain fruit quality, especially texture (watkins, 2008; mattheis et al., 2008). apples are characterized by valuable nutritional and taste qualities, and among the most popular fruit species in china. ‘red fuji’ apple (malus × domestica cv. red fuji) accounts for about 72% of the total apple yield in china. low-temperature storage is generally used as commercial application to prolong the storage period. however, a number of physiological disorders limit its quality in cold storage and shelf life, including flesh browning and superficial scald (he et al., 2016). flesh browning is a result of membrane damage and it is usually associated with enzymatic oxidation of phenolic compounds by polyphenol oxidase (ppo) to o-quinones, which polymerize nonenzymatically to produce heterogeneous black, brown or red pigments commonly called melanins (tomás-barberán et al., 2010). the occurrence of browning is mainly due to disruption of cellular compartmentalization, allowing the enzymatic oxidation of phenolic substrates in the vacuole by ppo located in the cytoplasm. this oxidation generates o-quinones that react with other compounds and result in polymerization to produce the browning appearance (franck et al., 2007; sun et al., 2011; lin et al., 2014). chlorogenic acid is an important substrate for ppo enzymatic browning reaction (tomás-barberán et al., 2010). the biosynthetic pathway of chlorogenic acid is initiated from the deamination of phenylalanine to cinnamic acid by phenylalanine ammonia lyase (pal), followed by hydroxylation and methylation of cinnamic acid by cinnamate 4-hydroxylase (c4h) and 4-hydroxycinnamoylcoa ligase (4cl), respectively. thus ρ-coumaric acid is produced, then hydroxycinnamoyl-coa shikimate/quinate hydroxycinnamoyl transferase (hct/hqt) pathway begins, and chlorogenic acid is synthesized finally (niggeweg et al., 2004; mahesh et al., 2007; zhao et al., 2013). furthermore, pal, 4cl and hct/hqt are considered as key regulators related to chlorogenic acid biosynthesis (lallemand et al., 2012; escamilla-treviño et al., 2014; yuan et al., 2014). damage of membrane integrity is considered the primary event that ultimately leads to the development of browning (kou et al., 2015). previous studies have demonstrated that the membrane lipid metabolism is correlated to lipoxygenase (lox) (mao et al., 2007). lox is potentially involved in senescence, membrane alter-actions and lipid degradation in plants under stress (gao et al., 2015). lox catalyzes peroxidation of plasma membrane lipids, increases lipid unsaturation, and thus changes membrane fluidity (wang, 2001), resulting in the loss of membrane integrity and increased membrane permeability (aghdam et al., 2013). it has been proven that normal tissue does not develop browning. in fruit suffering from environment stress, membrane lipid composition, the structure and function of cell membranes are all altered, thereby cellular compartmentalization is broken down, which results in the release of enzymes to contact with their substrates, and then promoting browning (pongprasert et al., 2011; lin et al., 2016). the effects of 1-mcp on the incidence of physiological disorders of apples have been variable (watkins, 2007). our previous research found that 1-mcp can effectively inhibit the flesh softening and reduce the flesh browning in ‘red fuji’ apple (he et al., 2016). the objective of the current study was to investigate the effects of 1-mcp treatment on fruit quality, flesh browning and its regulation mechanism of flesh browning in ‘red fuji’ apple during cold storage and shelf life. with regard to ‘red fuji’ apples, it is important to discuss the potential relationship of browning, the metabolism of phenols and membrane lipid during cold storage and shelf life. our specific aim was to elucidate the role of ethylene, chlorogenic acid and membrane lipid metabolism in stem-end browning. to achieve the objective, we assessed the activity of key enzyme ppo and its gene expression, and investigated the gene expression pattens in chlorogenic acid biosynthesis pathway and membrane lipid peroxidation gene. postharvest 1-mcp treatment reduced apple flesh browning 133 materials and methods materials and 1-mcp treatment ‘red fuji’ apples were harvested from a commercial orchard during the optimum commercial harvest period in hebei province of china (n38°23 4́1.92 e114°27´21.66). the apple fruit (average single fruit weight of 237.73 ± 20.42 g) were randomly divided into 2 groups of 48 kg fruit each. fruit of each group were divided into 3 equal parts and carefully put into 3 sealed plastic boxes (60 l), then were exposed to 0, and 1.0 μl l-1 1-mcp (dow chemical co., midland, mi, usa) at 25 ± 2 °c for 24 h. the group without 1-mcp (0 μl l-1) was set as control (he et al., 2016). after treatment, the fruit were packed with microporous plastic film (15 μm in thickness) and packed into paper box. each box that contained 30 fruits was stored at 0 ± 0.5 °c with 85% 95% relative humidity. the fruit were stored at 20 ± 0.5 °c as shelf life after 270 days of cold storage. the respiration rate and ethylene production rate were measured at 1, 3, 5 and 7 days of shelf life, and the fruit quality was measured on the harvest day (0 d), the end of cold storage (270 d), 1 d (270 + 1 d) and 7 d (270 + 7 d) at shelf life. fruit quality fruit firmness was measured at two equidistant points on the equa torial region with the skin removed, using a digital fruit hardness meter (model: gy-4, top instruments co., ltd., hangzhou, china) with a pressure head 8 mm in diameter. the firmness was auto matically calculated and expressed in kg cm-2. the flesh juice was squeezed from two equidistant points and measured for soluble solids content (ssc) by a pal-1 pocket digital refractometer (atago co., ltd.,tokyo, japan). titratable content (ta) was determined by acid base titration with 0.01 mol l-1 naoh up to ph 8.1, and the result was calculated as malic acid (%). browning rate of stem-end flesh was calculated as the percentage of browning fruit number in the total fruit number. fruit decay rate was calculated as the percentage of rotten fruit in the total fruit number. three replicates of 10 fruit each were measured for each treatment at every sampling time. sampling for each treatment, three replicates were set and there were 10 fruits in each replicate at every sampling time (0 d, 270 + 1 d, 270 + 7 d). after firmness and ssc measurement, we cut flesh of 0.5 cm wide and 1.0 cm deep, about 1 cm from the stem of each fruit being tested, and the stem-end flesh was frozen and ground into powder with liquid nitrogen immediately, then stored at -80 °c for chlorogenic acid content, ppo activity analysis and rna isolation. respiration rate and ethylene production rate respiration rate was measured by hfy-1a co2 infrared analyzer (kexi instrument co., ltd., jiangsu, china) after the fruits were sealed in the container for 1 h, 10 ml of gas was withdrawn from the container and injected to the analyzer, respiration rate was expressed as co2 release rate (μl kg-1 h-1). for ethylene measurements, 1 ml of gas was withdrawn from the container after the fruits were sealed for 3 h, and then injected into a gas chromatograph (model: gc9790ii, fuli instruments technology co., ltd., wenling, china) equipped with a gdx-502 column and a flame ionization detector (fid). the temperatures of column, vaporization oven and fid were 78 °c, 120 °c and 200 °c, respectively. n2 was used as the carrier gas with a rate of 40 ml min-1. ethylene production rate was expressed as μl kg-1 h-1. 10 fruits were sealed in each container, and three replicates were measured for each treatment. chlorogenic acid content and ppo activity chlorogenic acid was extracted from 2 g frozen powder of stemend flesh and its content was measured via high-performance liquid chromatography (hplc) according to the method of he et al. (2017). with a hitachi l2000 hplc system (hitachi, tokyo, japan), which consisted of a hitachi pump l-2130, a hitachi automatic sample injector l-2200, and a hitachi uv detector l-2400 and equipped with a lachrom c18 column (250 mm × 4.6 mm, 5 μm), hplc was performed in a mobile phase of 5% acetic acid (solvent a), h2o (solvent b) and 100% acetonitrile (solvent c) at a flow rate of 1 ml min-1 and a temperature of 30 °c, and monitored at a wave length of 280 nm with injection of 5 μl samples. samples were tested using external standard method, which used retention time to determine the quality and peak area for quantification. the analysis was performed in triplicates for each sample to obtain the mean value and standard deviation. for ppo (ec 1.14.18.1) extraction and activity measurement, 1 g of frozen stem-end flesh tissue was powdered in liquid nitrogen with a mortar and blended in 5 ml of 100 mmol l-1 phosphate buffer (ph 7.0) containing 0.3 g pvp. next, the solution was centrifuged at 12 000 × g for 15 min at 4 °c, after which the supernatant was collected as the crude enzyme extract. the reaction mixture con tained 100 mmol l-1 catechol in 50 mmol l-1 phosphate buffer (ph 6.0) (cheng et al., 2015). changes in the absorbance at 420 nm were measured by using a spectrophotometer (model: uv-2100, unico instrument, dayton, usa). the ppo active unit (u) was determined as the change value of absorbance increased by 0.01 per minute per gram of flesh sample and the result was expressed as u g-1 fw. rna isolation and real-time quantitative pcr analysis total rnas were isolated from fruit stem-end flesh, using improved hexadecyltrimethylammonium bromide (ctab) method (gasic et al., 2004). genomic dnas were eliminated with primescript rt reagent kit with gdna eraser (perfect real time) (takara bio inc., japan) and first strand of cdna was generated by reverse transcription. primers for real-time quantitative pcr (qrt-pcr) were designed according to apple (malus × domestica) nucleotide sequences re gistered in genbank of ncbi by dnaman 6.0 software. the sequence of primers was shown in tab. 1. qrt-pcr analysis was performed on abi 7500 real-time system using tb green™ premix ex taq ii quantitative pcr kit (takara bio inc., japan). the relative gene expression amount was calculated with the formula 2-δδct by excel 2007 (livak and schmittgen, 2001). the mdactin gene (genbank id: xm_029089583.1) was used as internal reference and the amount of gene expression in the stem-end flesh at 0 d was defined as 1.0. statistical analysis all statistical data analyses were done by spss (18.0). the data were analyzed by one-way analysis of variance (anova). means were separated using duncan method at p<0.05. data are shown as means ± standard errors. fig. 1-8 were generated by graphpad prism 8.0, and different letters in fig. 1-8 represented significant difference at p<0.05. results fruit quality, browning rate and decay rate the fruit firmness, ssc and ta content decreased after 270 days of cold storage, and 1-mcp treatment could maintain higher firmness, ssc and ta, but there was no significant difference in ssc at 270 + 7 d (fig. 1). this indicated that 1-mcp effectively delayed fruit 134 j. he, y. feng, y. cheng, s. wang, y. f1, j. guan after 270 d cold storage, the mdpal1 expression level was nearly 200 times higher than the initial storage fruit, and then downregulated at shelf life. 1-mcp treatment significantly decreased mdpal1 expression except that there was no significant difference with the control at 270 + 7 d (fig. 6a). different with mdpal1, the mdpal2 expression was down-regulated during cold storage, and was gradually up-regulated at shelf life. however, both the downregulation and up-regulation were alleviated by 1-mcp treatment (fig. 6b). the expression level of md4cl2 increased during cold storage, and then decreased during the shelf-life, but the expression of md4cl2 was inhibited by 1-mcp treatment especially at the end of shelflife (fig. 6c). the expression level of mdhct3 was significantly up-regulated during storage and was nearly 100 times higher at 270 + 7 d than that at the initial storage, and 1-mcp exhibited an obviously inhibition at the hct3 expression (fig. 6d). ppo activity and the expression level of mdppos ppo activity increased during cold storage and shelf-life, which was significantly inhibited by 1-mcp (fig. 7). the expression of mdppo1 reached the peak value at the end of cold storage, decreased at 270 + 1 d and increased again at 270 + 7 d in shelf life (fig. 8a). there was no obvious change of mdppo5 expression during cold storage and earlier shelf-life, but a dramatical up-regulation was observed at 270 + 7 d in shelf-life (fig. 8b). it is worth noting that the tab. 1: sequence of primers for qrt-pcr gene genbank id primer sequences (5’-3’) mdactin xm_029089583.1 5’-tgaccgaatgagcaaggaaattact-3’ 5’-tactcagctttggcaatccacatc-3’ mders1 nm_001328757.1 5’-ggagatctcgttggacgcaa-3’ 5’-ggatgactctgaccactggc-3’ mdetr2 xm_008358087.3 5’-atgttatgtggcagcgcagg-3’ 5’-ggcacacaattctgccaaca-3’ mdpal1 xm_008357397.2 5’-gtgtttaattaggcggggga-3’ 5’-aaattgattgcctcatatcacagat-3’ mdpal2 xm_029105821.1 5’-agcaccacctctttccattcc-3’ 5’-gccgggaaaaactaagtggg-3’ md4cl2 xm_008364603.2 5’-ctcgatcgatgcgtgttgac-3’ 5’-tgcctttgatccccgacttc-3’ mdhct3 xm_008344601.3 5’-tcaccatttccttgcgcctc-3’ 5’-acgttgactccctcacggta-3’ mdppo1 nm_001319261.1 5’-gagctcatgaaggccctacc-3’ 5’-tttggagctcgagttctggg-3’ mdppo5 jq388482.1 5’-tcaaatcgacgccaacctca-3’ 5’-gtttcgattgaaccggccc-3’ mdlox nm_001294093.1 5’-agcgacaagaaagaagaacc-3’ 5’-tactgaccgtagttgattgc-3’ fig. 1: fruit firmness (a), ssc (b) and ta content (c) of control and 1-mcp treated ‘red fuji’ apple at shelf life after cold storage. data are shown as means ± standard errors, different letters represent significant difference at p<0.05. softening and maintained fruit quality at shelf life in ‘red fuji’ apple. in addition, the browning rate of stem-end flesh was as high as 46.7% in the control fruit at 270 d, while the value is 0 in the 1-mcp treatment fruit. the browning became more serious during the shelf life, but 1-mcp significantly delayed and reduced the browning rate (fig. 2a). meanwhile, 1-mcp effectively reduced fruit decay (fig. 2b). respiration rate, ethylene production rate and the expression of ethylene receptor gene (mders1) in the stem-end flesh during the shelf life, the respiration rate of fruit increased at first, reached the peak value at 270 + 5 d and then decreased. the respiration rate was significantly lowered by 1-mcp (fig. 3a). the ethylene production rate of the control fruit displayed an obvious increase and reached the peak value at 27 + 3 d, but kept at a very low level in 1-mcp treatment fruit during shelf life (fig. 3b). the ethylene production and the expression of mders1 was effectively inhibited by 1-mcp treatment (fig. 4). chlorogenic acid content and biosynthesis related genes ex pression compared with the initial stage of storage, the chlorogenic acid content of stem-end flesh increased gradually with the extension of storage time, which was markedly inhibited by 1-mcp (fig. 5). postharvest 1-mcp treatment reduced apple flesh browning 135 up-regulation of mdppo1 and mdppo5 was significantly inhibited by 1-mcp treatment. the expression level of mdlox gene significantly increased during cold storage, and subsequently decreased. in the control fruit, the expression of mdlox showed an increasing trend. however, 1-mcp treatment effectively inhibited the expression and kept it at a very low level (fig. 9). correlation analysis according to the correlation analysis (tab. 2), the browning rate of stem-end flesh is significantly correlated with chlorogenic acid content (r=0.962**), ppo activity (r=0.915**), the expression of mdpal1 (r=0.572**), mdhct3 (r=0.926**), mdppo1 (r=0.743**) and mdlox (r=0.595**). the chlorogenic acid content was sig nificantly correlated with ppo activity (r=0.865**), the expression amounts of mdpal1 (r=0.500*), mdhct3 (r=0.904**), ppo activity and mdppo1 expression (r=0.841**). discussions postharvest treatment with 1-mcp, an inhibitor of ethylene action, has been shown to improve quality characteristics of apples, includ ing reduced ethylene production and respiration, as well as improved firmness and acidity retention (watkins, 2007; deell et al., 2007). in our study, 1-mcp could effectively improve the quality of ‘red fuji’ apple fruit after long-term cold storage, and significantly reduce the respiration rate and ethylene production rate during cold storage and shelf life (fig. 3), which was the physiological basis of 1-mcp function in delaying fruit senescence. therefore, the fruit treated with 1-mcp kept higher fruit firmness and ta content, while maintaining a higher ssc (fig. 1). 1-mcp treatment also significantly reduced fig. 5: chlorogenic acid content of stem-end flesh tissue in ‘red fuji’ apple at shelf life after cold storage. data are shown as means ± standard errors, different letters represent significant difference at p<0.05. fig. 3: fruit respiration rate (a) and ethylene production rate (b) of ‘red fuji’ apple at shelf life after cold storage. data are shown as means ± standard errors, different letters represent significant difference at p<0.05. fig. 4: mders1 expression level of stem-end flesh tissue in ‘red fuji’ apple at shelf life after cold storage. data are shown as means ± standard deviations, different letters represent significant difference at p<0.05. fig. 2: browning rate (a) of stem-end flesh and decay rate (b) of ‘red fuji’ apple at shelf life after cold storage. data are shown as means ± standard errors, different letters represent significant difference at p<0.05. 136 j. he, y. feng, y. cheng, s. wang, y. f1, j. guan fruit decay and stem-end flesh browning (fig. 2). the physiological effect of ethylene begins with the recognition of ethylene signal by ethylene receptors (etr) located in the endoplasmic reticulum (alonso et al., 2003), and 1-mcp can delay and inhibit a series of fruit ripening and senescence processes caused by ethylene through the competition with ethylene binding sites on the receptors (blankenship and dole, 2003). ireland et al. (2012) found that 100 μl l-1 ethylene in apples could up-regulate the expression of ers1 gene, while in our study, 1-mcp significantly inhibited the expression of mders1 (fig. 4), so it was speculated that mders1 was involved in the flesh browning caused by ethylene signal. the development of fruit browning depends on the enzymatic action of the ppo. the apple’s susceptibility to flesh browning is thought to be the result of complex interplay between the ppo enzyme and fig. 6: the expression level of mdpal1 (a), mdpal2 (b), md4cl2 (c) and mdhct3 (d) of stem-end flesh tissue in ‘red fuji’ apple at shelf life after cold storage. data are shown as means ± standard deviations, different letters represent significant difference at p<0.05. fig. 7: ppo activity of stem-end flesh tissue in ‘red fuji’ apple at shelf life after cold storage. data are shown as means ± standard errors, different letters represent significant difference at p<0.05. fig. 8: mdppo1 (a) and mdppo5 (b) expression level of stem-end flesh tissue in ‘red fuji’ apple at shelf life after cold storage. data are shown as means ± standard deviations, different letters represent significant difference at p<0.05. fig. 9: mdlox expression level of stem-end flesh tissue in ‘red fuji’ apple at shelf life after cold storage. data are shown as means ± standard deviations, different letters represent significant difference at p<0.05. postharvest 1-mcp treatment reduced apple flesh browning 137 polyphenol content (di guardo et al., 2014). however, the effects of 1-mcp on the incidence of physiological disorders of apples have been variable (watkins, 2007). ripening-related disorders such as senescent breakdown are greatly inhibited by 1-mcp (moran et al., 2005; deell et al., 2007), as are disorders such as superficial scald that is associated with ethylene production (rupasinghe et al., 2000; tsantili et al., 2007). the present results showed that the content of chlorogenic acid and ppo activity in the stem-end flesh increased after cold storage (fig. 5, fig. 7), which was positively correlated with the degree of flesh browning (fig. 2, tab. 2). 1-mcp significantly reduced the chlorogenic acid content and ppo activity, which was the biochemical basis of 1-mcp delaying the occurrence of flesh browning. however, it has been previously shown that 1-mcp treatment can make ‘empire’ apples more sensitive to co2 concentration, and exacerbate flesh browning because of co2 injury (jung and watkins, 2011; lee et al., 2012). therefor, we deduced that the stem-end flesh browning in ‘red fuji’ apple is the consequence of senescence caused by ethylene, and 1-mcp could effectively inhibit the browning. pal, 4cl and hct are involved in chlorogenic acid biosynthesis pathway in apple and pear fruit, and pal is the enzyme that catalyzes the first step reaction of phenylpropane metabolic pathway (cheng et al., 2015; di guardo et al., 2014; both et al., 2018). this study demonstrated that 1-mcp treatment significantly decreased the expression of mdpal1 (fig. 6a), mdhct3 (fig. 6d) and mdppo1 (fig. 8a), and displayed a lower chlorogenic acid content (fig. 5) and ppo activity (fig. 7) in the stem-end flesh. however, the expres sion trends of mdppo5 (fig. 8b), mdpal2 (fig. 6b) and md4cl2 (fig. 6c) were not consistent with the changes of chlorogenic acid content and flesh browning. lox are the crucial enzymes of membrane lipid metabolism, and might induce degradation of phospholipids and unsaturated fatty acids (wang, 2011; aghdam and bodbodak, 2014). high levels of unsaturated fatty acids could maintain normal cellular function and prevent the interaction of ppo with phenol substrates resulting in enzymatic browning (liu et al., 2006). it has been proven that lox activity was significantly higher and the gene expression increased when tissue browning occurred (sheng et al., 2016). in our experiment, 1-mcp treatment reduced the mdlox gene expression level in the stem-end flesh (fig. 9) which was consistent with the obvious browning inhibition. this implied that 1-mcp plays an important role in the integrity maintaining of membrane lipid in the stem-end flesh of ‘red fuji’ apple. conclusion postharvest 1-mcp (1.0 μl l-1) treatment can effectively improve the quality of ‘red fuji’ apple after long-term cold storage, and keep a low respiration rate and minimum ethylene production rate. moreover, 1-mcp treatment markedly reduce the stem-end flesh browning by down-regulating the expression of mdpal1, mdhct3, mdppo1 and mdlox, and then inhibiting the increase of chlorogenic acid content and ppo activity. in conclusion, 1-mcp based technology shows positive effects on quality control and reducing storage browning in ‘red fuji’ apple. acknowledgements this research was funded by the haafs agriculture science and technology innovation project (2019-2-1), modern agricultural industry technology research system of hebei province (hbct2018100207), and the young talents fund of hebei pro 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http://dx.doi.org/10.1007/s10658-007-9208-7 postharvest 1-mcp treatment reduced apple flesh browning 139 yuan, y., wang, z.y., jiang, c., wang, x.m., huang, l.q., 2014: exploiting genes and functional diversity of chlorogenic acid and luteolin biosyntheses in lonicera japonica and their substitutes. gene 534(2), 408-416. doi: 10.1016/j.gene.2012.09.051 zhao, s.c., tuan, p.a., li, x.h., kim, y.b., kim, h., park, c.g., yang, j.l., li, c.h., park, s.u., 2013: identification of phenylpropanoid biosynthetic genes and phenylpropanoid accumulation by transcriptome analysis of lycium chinense. bmc genomics. 14(1), 802. doi: 10.1186/1471-2164-14-802 orcid jingang he https://orcid.org/0000-0001-9986-8720 yunxiao feng https://orcid.org/0000-0003-0117-7062 yudou cheng https://orcid.org/0000-0002-5990-0041 shen wang https://orcid.org/0000-0002-1932-4641 junfeng guan https://orcid.org/0000-0001-8570-9219 address of the corresponding author: junfeng guan, institute of genetics and physiology, hebei academy of agriculture and forestry sciences, shijiazhuang, china e-mail: junfeng-guan@263.net © the author(s) 2021. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1016/j.gene.2012.09.051 http://dx.doi.org/10.1186/1471-2164-14-802 https://orcid.org/0000-0001-9986-8720 https://orcid.org/0000-0003-0117-7062 https://orcid.org/0000-0002-5990-0041 https://orcid.org/0000-0002-1932-4641 https://orcid.org/0000-0001-8570-9219 journal of applied botany and food quality 92, 117 122 (2019), doi:10.5073/jabfq.2019.092.016 1comsats university islamabad, abbottabad campus, department of environmental sciences, abbottabad, pakistan 2saline agriculture research centre, university of agriculture, faisalabad, pakistan 3institute of plant nutrition and soil science, kiel university, kiel, germany sodium in the leaf apoplast does not affect growth of maize (zea mays l.) under saline field conditions muhammad shahzad1, haris usman1, rafiq ahmad1, sabaz ali khan1, zulfiqar a. saqib2, karl-hermann mühling3* (submitted: october 18, 2018; accepted: april 9, 2019) * corresponding author summary studies dealing with leaf apoplastic na+ concentration of monocots, such as maize, under actual saline soils are scarce. therefore, the current study was aimed to investigate the growth, total ions and leaf apoplastic na+ concentration of salt sensitive maize plants growing in saline soils. plants were subjected to salt stress with an electrical conductivity (ec) of 3, 8 10 and 14 ds m-1 using completely randomized design (crd) for 3 weeks. shoot fresh weight, plant height, leaf area and leaf length of maize plants drastically decreased when plants were exposed to increasing salt stress. we found that maize could display a steep increase in na+ concentration in the total shoot biomass with maximum 82.3 μmol g-1 fw, when plants were subjected to highest soil salinity at 14 ds m-1. as expected, other cations i.e., k+, ca2+ and mg2+ decreased with increasing ec of the soil compared to na+. surprisingly, a maximum of 17 mm na+ were found in the leaf apoplast of maize grown under very high soil sali nity at ec 14 ds m-1. considering this lower leaf apoplastic na+ concentration at such a high ec level in maize plants, current study does not corroborate that surplus sodium in the leaf apoplast can result in dehydration and cell death under salt stress. keywords: soil salinity, sodium, apoplast, growth, zea mays l. introduction salinity is considered as the major environmental hazard especially for the agricultural crops like maize in which rapid growth reduction has been observed already in the initial phase of salinity stress (zörb et al., 2015). maize is the third most important cereal crop after wheat and rice that is utilized as a staple food in many parts of the world. in pakistan maize was cultivated on about 1.13 million hectares during the year 2010-2015 (gop, 2015). in the year 2013-17, average maize production in pakistan was 5.476 million tons (fao, 2019). however, maize yield in pakistan is considered still very low compared to the remaining maize producing countries. the reduced production of maize is a result of high soil ph, soil salinity and shortage of good quality of water for irrigation purposes. plant growth is generally affected by salt stress in three different ways i.e. osmotic stress, ionic imbalance in cells and ultimately ionic toxicity. moreover, soluble salt concentration and duration of exposure to salt stress decides the severity of plant growth reduction (tavakkoli et al., 2011). reduction in the growth is characterized by the rapid response due to decreased soil water potential. widerange metabolic and osmotic problems of plants can be triggered by the high absorption of na+ ions in shoots under salt stress. the accumulation of na+ ions in shoots is greater than in roots; therefore, shoots are more susceptible to na+ (munns and tester, 2008). as already shown by fortmeier and schubert (1995), na+ ions do significantly affect maize growth compared to clions. sodium ions get stored in higher amounts in shoots in contrast to roots, for this reason leaves are much susceptible towards sodium ions (tester and devenport, 2003). therefore, current study focuses on the investigation of na+ concentration in the leaf apoplast of maize plants. oertli (1968) proposed that high level of na+ accumulation in the apoplast leads to turgor loss, dehydration and finally death of leaves. flowers et al., (1991) and speer and kaiser (1991) measured 600 mm and 87 mm na+ concentrations in the leaf apoplast of rice and salt-sensitive pea, respectively under salt stress, whereas later study also found low apoplastic na+ concentration (7 mm) in saltresistant spinach. in contrast, mühling and läuchli (2002) found extremely low na+ accumulation in the leaf apoplast of maize in a hydroponic experiment under salinity. the aim of the current investigation was to study growth reduction of salt-sensitive maize plants as a result of na+ accumulation in the leaf apoplast under actual saline environment, as studies under saline field conditions are missing to clarify whether na+ in the leaf apoplast is causing a decline in leaf growth. materials and methods soil analysis and maize cultivation saline soils were collected from four different areas of district faisalabad, from up to 0-6 and 6-12 cm depths with the help of auger. there were total twenty soil samples which were dried in the air and sieved to remove any stone and sand particles and further treated for soil analysis i.e., electrical conductivity (ec), ph, saturation percentage (sp) and textural class which are given in tab. 1. according to international soil classification system soil textural class was analyzed with the help of hydrometer method as mentioned by moodie et al. (1959). to determine the saturation percentage the soil paste was dried to a constant weight at 105 °c and was later calculated with the help of following formula. mass of wet soil – mass of oven dry soil saturation % = × 100 mass of oven dry soil ph meter was used to measure ph of the saturated soil paste. electrical conductivity was measured by using a digital conductivity meter. for this purpose, an extract of saturated soil paste was used for the analysis. maize seeds of rmw8 * psev3-157.5.4.2 were sown in saline soils in seed lab in comsats, abbottabad with controlled environment under optimum light energy. at first six seeds were sown per pot, which were later reduced to four plants for further experimentation. deionised water was used for irrigation throughout the growing period. maize plants were harvested during the vegetative growth without salt injury symptoms after three weeks. 118 m. shahzad, h. usman, r. ahmad, s.a. khan, z.a. saqib, k.-h. mühling collection of apoplastic washing fluid infiltration − centrifugation technique was used for the collection of apoplastic washing fluid (lohaus et al., 2001; mühling and sattelmacher, 1995). leaves were removed from plants with the help of a razor blade and were cautiously washed with deionised water. later leaves were cut in segments of about 5.5 cm and with the help of tissue paper, the leaf segments were carefully dried and weighed before infiltration. the leaf segments were then put in empty plastic syringes (60 ml). the syringes were then filled with deionized water up to 40 ml. tip of the syringe was covered with silicon cork and a pressure of almost 20 kpa was produced on the leaf segments by pulling plunger of the syringe. then plunger was released and pushed so that leaves get infiltrated (lohaus et al., 2001). after that, the infiltrated leaf segments were dried carefully with tissue paper and weighed again to get approximate volume of infiltrated water. leaf segments were placed in a plastic vessel of 10 ml, which was then placed in a 50-ml falcon tube. the falcon tube containing intact leaves was placed in a centrifugation tube, which was adjusted at 100 × g at 5 °c for 5 minutes. samples of apoplastic washing fluid were pipetted into 1.5 ml eppendorf tube and stored at -20 °c for further analysis. growth parameters fresh weight of the plants was obtained soon after the harvest with the help of an analytical balance having precision of 0.1 mg. later, plant shoot was placed in an oven at 60 ºc for three days to obtain the dry weight. digital images of the plants and leaves were taken after harvesting. later on, area and length of each leaf and shoot height of the harvested plants was calculated with the help of imagej 1.49 software by setting a scale against specific number of pixels. leaf area and length were demarcated, thereby obtaining leaf area and length measurements. determination of total ion absorption in shoot of maize plants the dried leaves samples were crushed manually with the help of ceramic mortar and pestle. crushed samples were weighed, then, put in ceramic pots to be placed in an oven at 520 °c for about 4 hours to obtain ash of the samples. cations were investigated in this ash of dried plant matter. 2 ml of 4 m hno3 was added to each ash sample and was stirred gently after every half an hour up to a total of three hours to get suspension. then 8 ml distilled water was added to make 10 ml final volume. whatman filter paper no. 21 was used to filter the suspension into vials. after that, ions concentration was determined in the filtrate for na+, ca2+, k+ and mg2+ through aas i.e atomic absorption spectrophotometer (aanalyst 700, perkin elmer, usa). the unit ‘μmol g-1 fw’ was used for the calculation of concentration of the ions in total shoot samples. determination of total ion concentration in apoplastic washing fluid collected apoplastic washing fluid of each sample was first dried in an oven at 80 °c for 4 hours. 3 ml of 4n hno3 was added and was stirred gently after every half an hour up to a total of three hours to get suspension. later 7 ml deionised water was added and a final volume of 10 ml was achieved. atomic absorption spectrometer was used to analyse the na+, k+, ca2+ and mg2+. the unit ‘mm’ was used to demonstrate the amount of cations in leaf apoplast of maize plants. statistical analysis data were normally distributed and significant variances at p≤0.05 among treatments were determined by means of the general linear model with a tukey test using spss statistics 17.0 (statistical product and service solutions, chicago, il, usa). the significant differences were indicated with the help of small alphabets at top of each bar. the mean standard error (s.e.) was indicated by error bars on the bars of figures. results agronomic traits affected under salt stress fresh shoot biomass of maize plants was maximum (6.1 g) when treated with soil of ec 3 ds m-1. in contrast to plants treated with ec 3 ds m-1, maize plants growing in soil with an ec of 14 ds m-1 showed significant damaging effect with 68% decreased shoot biomass. an inverse relation was observed between plant growth and ec of the soil samples, as with increasing concentration of salt in the soil i.e., ec 8 ds m-1, 10 ds m-1 and 14 ds m-1 resulted in shoot biomass of 2.9 g, 2.3 g, and 2.0 g, respectively (fig. 1a). when considering the dry shoot biomass maximum value was 0.61 g when treated with soil 1 however, this dry weight was significantly reduced to 0.17 g when plants were treated with soil 4 i.e., ec of 14 ds m-1. in com parison to ec 3 ds m-1 (soil 1) dry shoot biomass was reduced by 56%, 67%, and 72% when plants were treated with soil having an ec of 8 ds m-1 (soil 2), 10 ds m-1 (soil 3), and 14 ds m-1(soil 4), respectively. maximum shoot height of maize plant was 37 cm when ec of soil was 3 ds m-1 i.e., soil 1. the shoot height of maize plants was significantly reduced to 12 cm when treated with soil 4 having an ec of 14 ds m-1, which is 67% reduction when compare to the shoot height in soil 1. with an increase in soil salinity shoot height was reduced i.e., 23 cm and 19 cm, with an ec of 8 ds m-1 (soil 2) and 10 ds m-1 (soil 3), respectively. leaf length was 28 cm when ec of the collected soil sample was 3 ds m-1 (soil 1) and at highest ec level i.e., 14 ds m-1 (soil 4) the leaf length was (7.18 cm) significantly reduced by 74%. the findings related to leaf length under salt stress further revealed that it was considerably decreased with increasing ec levels, as with an ec of 8 ds m-1 (soil 2) and 10 ds m-1 (soil 3) tab. 1: location, depth, ec, ph, sp% and textural class of the collected soil samples from district faisalabad, pakistan (n = 5). sample name location depth ec(ds m-1) ph sp % textural class soil 1 (chak no. 26 gb) 31°15’36.4”n 73°17’40.1”e 6-12 cm 3 7.88 41.95% loamy sand soil 2 (chak no. 73 gb) 31°18’19.6”n 73°11’41.6”e 6-12 cm 8 7.9 40.19% loamy sand soil 3 (chak no. 73 gb) 31°18’19.6”n 73°11’41.6”e 0-6 cm 10 8.42 31.96% loamy sand soil 4 (chak no. 33 gb) 31°14’12.2”n 73°09’23.5”e 0-6 cm 14 7.89 35.10% sandy clay loam apoplastic na+ in maize leaves under salt stress 119 the leaf length of maize plant was 16 cm and 11 cm, respectively. fig. 1(d) displays the relationship of leaf area of maize plants with respect to ec of the soil. in soil 1 the leaf area of maize plant was 0.701 cm2. in contrast, a significant reduction of 75% in the leaf area (0.174 cm2) of the maize plants was observed in soil 4. similar to above mentioned growth parameters leaf area of the maize plants was found to be decreased with an increase in the soil salinity. salt stress induced significant ionic changes in total shoot of the maize plant maximum na+ concentration in the total shoot of maize plants was 82 μmol g-1 fw when they were subjected to 14 ds m-1 (soil 4), while minimum was 10 μmol g-1 fw when treated with 3 ds m-1 (soil 1). an absolute increase in the na+ concentration was observed starting from ec 8 ds m-1 (soil 2) to 14 ds m-1 (soil 4). however, when compared to ec 3 ds m-1 (soil 1), increase in na+ concentration in the total shoot of maize plants was 523%, 631%, and 695% when treated with soil samples having an ec of 8 ds m-1 (soil 2), 10 ds m-1 (soil 3), and 14 ds m-1 (soil 4), respectively (fig. 2a). an inverse relation was observed between k+ concentration and ec of the soil samples. with an increase in soil salinity level i.e., ec 8 ds m-1 (soil 2), 10 ds m-1 (soil 3) and 14 ds m-1 (soil 4), k+ concentrations in maize shoots were decreased by 15%, 17%, and 23%, respectively, when compared to k+ concentration in soil 1. maximum k+ concentration in the total shoot of maize plant was 78 μmol g-1 fw, when they were subjected to soil 1, while minimum was 60 μmol g-1 fw when treated soil 4 (fig. 2b). decrease in the ca2+ concentration was observed starting from ec 3 ds m-1 to 14 ds m-1. maximum ca2+ concentration was 29 μmol g-1 fw, when ec of soil was 3 ds m-1 (soil 1). ca+2 concentration was reduced to 13 μmol g-1 fw, when plants were treated with soil 4. similarly, when soil had an ec of 8 ds m-1 (soil 2) and 10 ds m-1 (soil 3), the ca2+ concentration was reduced by 33% and 36%, respectively when compared to ec 3 ds m-1 (soil 1) (fig. 2c). maximum mg2+ concentration was 8.09 μmol g-1 fw in soil 1. however, mg2+ concentration was 7.43 μmol g-1 fw in soil 4 which was 8% reduction compared to the mg2+ concentration in soil 1. similarly, mg2+ concentration was, 7.95 μmol g-1 fw, and 7.93 μmol g-1 fw when maize plants were treated with soil that had an ec of 8 ds m-1 (soil 2), and 10 ds m-1 (soil 3), respectively (fig. 2d). ionic pattern of the extracted leaf apoplastic washing fluid in maize under salt stress an increase in the na+ concentration was noticed starting from ec 3 ds m-1 (soil 1) to 14 ds m-1 (soil 4). maximum na+ concentration in the extracted apoplastic washing fluid of maize leaves was 17 mm when they were subjected to soil 4, while minimum was 5 mm, when treated with soil 1. however, noticeably when compared to ec 3 ds m-1 (soil 1), increase in na+ concentration in the extracted apoplastic washing fluid of maize leaves was 64%, 164%, and 229% when treated with soil samples having an ec of 8 ds m-1 (soil 2), 10 ds m-1 (soil 3), and 14 ds m-1 (soil 4), respectively (fig. 3a). k+ concentration in the extracted apoplastic washing fluid of maize leaves was 4.5 mm when treated with soil having an ec of 3 ds m-1 (soil 1) however, this k+ concentration was significantly reduced to 0.1 mm when plants were treated with soil 4 with an ec of 14 ds m-1. in comparison to ec 3 ds m-1 (soil 1), k+ concentration was reduced by 36%, 66%, and 97% when plants were treated with soil having an ec of 8 ds m-1 (soil 2), 10 ds m-1 (soil 3), and 14 ds m-1 (soil 4), respectively (fig. 3b). ca2+ concentration in the extracted apoplastic washing fluid of the maize leaves was 5.24 mm when treated with soil 1. ca2+ concentration was 1 mm, when ec of the soil was 14 ds m-1 (soil 4). decrease in the ca2+concentration was observed starting from ec 3 ds m-1 (soil 1) to 14 ds m-1 (soil 4). however, compared to ec 3 ds m-1 (soil 1), when treated soil had an ec of 8 ds m-1 (soil 2), 10 ds m-1 (soil 3) and 14 ds m-1 (soil 4) then ca2+ concentration was significantly reduced by 49%, 58%, and 81%, respectively (fig. 3c). mg2+ concentration was found maximum with 26 mm in soil 1 (i.e., ec 3 ds m-1). this mg2+ concentration was 3 mm when ec of the soil was 14 ds m-1 (soil 4), which is 89% reduction when compared to the mg2+ concentration at ec 3 ds m-1 (soil 1). in comparison to mg2+ concentration of ec 3 ds m-1 (soil 1), soil samples with an ec of 8 ds m-1 (soil 2), 10 ds m-1 (soil 3), and 14 ds m-1 (soil 4) showed a mg2+ concentration of 19 mm, 11 mm, and 3 mm, res pectively (fig. 3d). the ratios between k+ and na+, ca2+ and na+ and mg2+ and na+ decreased in total shoot and leaf apoplast of maize plants under salt stress. maximum ratios between k+:na+, ca2+:na+ and mg2+:na+ were found at ec 3 ds m-1 (soil 1) in both total shoot and in the leaf apoplast of maize plants. however, minimum ratios were found at ec 14 ds m-1 (soil 1) (tab. 2). fig. 1: fresh shoot biomass g (a), shoot height cm (b), leaf length cm (c) and leaf area cm2 (d) of maize plants (on y axis) as affected by varying ec level of the soil samples (on x axis). different letters indicate significant differences between the ec levels at p < 0.05 (n ≥ 3). fig. 2: effect of various ec level of the soil samples (on x axis) on na+ concentration μmol g-1 fw (a), k+ concentration μmol g-1 fw (b), ca2+ concentration μmol g-1 fw (c), and mg2+ concentration μmol g-1 fw (d), in the total shoot of maize plants. different letters indicate significant differences between the treatments at p < 0.05 (n ≥ 3). 120 m. shahzad, h. usman, r. ahmad, s.a. khan, z.a. saqib, k.-h. mühling discussion effect of soil salinity on cation composition in leaves of maize salinity is the major environmental problem damaging crop plants by their growth restriction, ultimately reducing agricultural yield throughout the world (manivannan et al., 2007; shrivastava 2015). numerous crops e.g. barley wheat, rice, cotton, bean and maize have been mentioned for their yield reduction under salt stress (demiral et al., 2005; jaleel et al., 2008; basal 2010; shahzad et al., 2012). in current study, when compared to soil with an ec 3 ds m-1, maize growing in soil possessing an ec of 14 ds m-1 resulted in significant damaging influence with 68% decreased shoot bio mass during salt stress (fig. 1a). increased shoot and root osmolality can be resulted through increased ion concentration in plant tissues under salinity stress. however, sudden reduction in the growth of the roots and leaves is an immediate consequence of the osmotic effect (munns, 2002; munns and tester, 2008; wang et al., 2012). reduced growth under higher saline conditions have been attributed to the inefficient plants capability to osmotic balance which can be a result of saturated solute uptake, or increased energy demands of the system (munns, 1988; gale and zeroni, 1984). according to slabu et al. (2009) and tester and davenport (2003) and fortmeier and schubert (1995) na+ causes more damage in saline conditions as compared to any other ion e.g cl-. sümer et al. (2004) mentioned that na+ toxicity can also occur in the initial phase of salinity stress. in the present investigation, decrease in fresh biomass of plant shoots and leaf area (fig. 1a and 1d) was found inversely proportional to increase in salinity level. in contrast to plants subjected to soil with ec 3 ds m-1, results related to leaf area showed a significant decline (75%) in maize growing in soil with an ec of 14 ds m-1. it has been reported that continuous exposure to increased level of salinity in the rooting medium progressively reduces the size of the leaf over time (munns et al., 1988). when compared to the shoot height at ec 3 ds m-1, 67% reduction was noticed when plants were treated with soil having an ec of 14 ds m-1. moreover, in the present study, in comparison to ec 3 ds m-1, leaf length was also significantly reduced by 74% when treated with soil having an ec of 14 ds m-1, which is in line with previous findings where reduced growth of the younger leaves compared to the older leaves suggested that leaf elongation is restricted immediately when the salt is applied to the maize roots (cramer, 1992; shahzad et al., 2012). according to tester and davenport (2003) k+ is involved in the activation of numerous enzymes in plants however, higher na+ contents compete with k+ for binding sites as a consequence leads to enzymes inactivation which disturbs the important cellular functions. in the current study compared to ec 3 ds m-1, increase in na+ concentration in the total shoot of maize plant was 695% when treated with soil samples having an ec of 14 ds m-1 (fig. 2a). moreover, an inverse relation was observed between k+ concentration and ec of the soil samples as increase in soil salinity resulted in decreased k+ concentrations (fig. 2b). this is in conformity with our result as the k+ content significantly decreased by 23% in the presence of ec 14 ds m-1 (fig. 2b). low k+:na+, ca2+:na+ and mg2+:na+ ratios under salinity stress (tab. 1 and 2) disturb plant expansion and eventually prove to be damaging (schachtman and liu, 1999). ca2+ may also get replaced from the membranes of root cells due to high concentration of na+, thus leading to a decrease in the k+:na+ selectivity (murata et al., 2000). enhancement in ca2+ regulated membrane stability, consistently results in decline of k+ loss from the root zone and a more suitable root k+ proportion (cachorro et al., 1994; navari-izzo et al., 1993). ca2+ normally plays a regulatory role in the metabolic processes. ca2+ provide shield to cell membrane against harmful saline impact, as ca2+ compete with na+ for membrane binding spots (zidan et al., 1990; abdel, 2011). it has been observed that ca2+ contents declined in tomato, wheat and barley leaf by raising nacl content in the nutrient solution (navarro et al., 2000; cuartero et al., 2006; ehret et al., 1990). ebert et al. (2002) stated that cationic associations in the shoot cells such as ca2+:na+ exhibit a major impact on salt adaptability than entire sodium contents. in the present study ca2+ concentration was reduced to 55% at ec 14 d sm-1 in comparison to ec 3 d sm-1. our finding confirms the decline in ca2+ uptake in maize under nacl application (cramer, 2002; hu et al., 2007). high salt deposition is a probable reason for selectivity of nutrient absorbance because excessive na+ and clmay obstruct the uptake of other ions (k+, ca2+ and mg2+) in the root and their passage through the xylem into the aerial parts, ultimately leading to nutritional scarcity in the tissue (murillo-amador et al., 2006). when com pared to ec 3 ds m-1 mg2+ was reduced by 8% when treated with soil possessing an ec of 14 ds m-1. the proportions of na+:ca2+, na+:k+ and na+:mg2+ under saline conditions noticed in the total plant revealed that k+, ca2+ and mg2+ transfer is hindered by na+ under salt treatment and may disrupt plant metabolic processes and affect plant development. fig. 3: na+ concentration mm (a), k+ concentration mm (b), ca2+ concentration mm (c), and mg2+ concentration mm (d) as influenced by the increasing ec level of the soil samples, different letters indicate significant differences between the treatments at p < 0.05 (n ≥ 3). tab. 2: influence of different ec levels on the k+/na+, ca2+/na+ and mg2+/na+ ratios in total shoot and apoplast of maize plants. different letters indicate significant differences between the treatments at p < 0.05 (n ≥ 3), ±se. total shoot apoplast total shoot apoplast total shoot apoplast ec (ds m-1) k+/na+ k+/na+ ca2+/na+ ca2+/na+ mg2+/na+ mg2+/na+ 3 7.80 ± (0.95)a 0.89 ± (0.01)a 2.90 ± (0.40)a 1.04 ± (0.04)a 0.81 ± (0.10)a 5.21 ± (0.20)a 8 1.05 ± (0.10)b 0.35 ± (0.01)b 0.30 ± (0.03)b 0.32 ± (0.01)b 0.12 ± (0.01)b 2.34 ± (0.03)b 10 0.86 ± (0.06)b 0.12 ± (0.004)c 0.24 ± (0.02)b 0.17 ± (0.01)c 0.10 ± (0.001)b 0.87 ± (0.01)c 14 0.73 ± (0.04)b 0.08 ± (0.005)d 0.16 ± (0.01)b 0.06 ± (0.002)d 0.09 ± (0.003)b 0.41 ± (0.04)c apoplastic na+ in maize leaves under salt stress 121 does the apoplastic na+ concentration affect leaf growth under saline field conditions? studies dealing within the leaf apoplast of monocots, such as maize, to determine the cations and anions under actual saline soils are scarce. many of the deleterious effects of na+ seem to be related to the structural and functional integrity of membranes (kurth et al., 1986). volkmar et al. (1998) suggested that when plants are continuously exposed to salinity stress, na+ can get accumulated either in the cytoplasm or in the apoplast of cell. earlier studies suggested that reduction in shoot growth can possibly be attributed to dehydration of leaf cell, when xylem na+ entered the leaf apoplast that induces osmotic stress (flowers et al., 1991; oertli, 1968). we are reporting here for the first time the apoplastic na+ (fig. 3a) accumulation under the influence of saline soils with various ec levels that were collected from district faisalabad. our results showed that when maize plants are treated with soil having an ec of 14 ds m-1, it results in (3.3 fold) increase of apoplastic na+ concentration compared to ec 3 ds m-1. however, maximum na+ concentration in apoplastic fluids (awf) remained too low (17 mm) under short term salt stress when compared to absolute na+ concentration in total shoot of maize plants (fig. 3a). these findings do not support oertli’s hypothesis that leaves dehydration and turgor loss can be a consequence of high levels of salt accumulation in the apoplast of salt-sensitive plants. in addition, we confirm the earlier study with maize by mühling and läuchli (2002a,b) which were performed in hydroponics. possible mechanism of low apoplastic na+ concentration in leaf tissue during continuous exposure to excessive salt could be through na+ transport via h+/na+ antiporter into leaf vacuoles (pitann et al., 2009). conclusions maize growth was significantly reduced under increased ec salinity levels ranged from 3 ds m-1 to 14 ds m-1 (slightly to highly saline soils). na+ concentration in total shoot significantly increased with higher ec levels, which has been suggested by numerous scientists as a reason for decreased maize growth at high salinity levels. however, in this study low na+ concentration in the subcellular compartment i.e., leaf apoplast of maize was found under the influence of actual saline soil, which has not been reported earlier. acknowledgments authors acknowledge higher education commission (hec), pakistan for the award of a research grant (no: pd-ipfp/hrd/ hec/2013/1975). the authors thank dr. kiramat khan for his excellent technical assistance. the authors also want to thank fawad kaleem and sanjeet kumar for their assistance. conflict of interest the authors declare that they have no conflict of interest. references abdel, l., 2011: ameliorative effect of calcium chloride on growth, anti oxidant enzymes, protein patterns and some metabolic activities of canola (brassica napus l.) under seawater stress. j. plant nutr. 34, 13031320. doi: 10.1080/01904167.2011.580817 basal, h., 2010: response of cotton (gossypium hirsutum l.) genotypes to salt stress. pak. j. bot. 42, 505-511. cachorro, p., ortiz, a., cerda, a., 1994: implications of calcium nutrition on the response of phaseolus vulgaris l. to salinity. plant soil 159, 205212. doi: 10.1007/bf00009282 cramer, g.r., 1992: kinetics of maize leaf elongation ii responses of a na+ excluding cultivar and a na-including cultivar to varying na+/ca2+ salinities. j. exp. bot. 43, 857-864. cramer, g.r., 2002: sodium-calcium interactions under salinity stress. in: läuchli, a., lüttge, u. 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doi: 10.5829/idosi.mejsr.2012.12.1.1660 wani, a.s., ahmad, a., hayat, s., fariduddin, q., 2013: salt-induced modulation in growth, photosynthesis and antioxidant system in two varieties of brassica juncea. saudi j. biol. sci. 20, 183-193. doi: 10.1016/j.sjbs.2013.01.006 zidan, i., azaizeh, h., neumann, p.m., 1990: does salinity reduce growth in maize root epidermal cells by inhibiting their capacity for cell wall acidification. plant physiol. 93, 7-11. zörb, c., mühling, k.h., kutschera, u., geilfus, c.m., 2015: salinity stiffens the epidermal cell walls of salt-stressed maize leaves: is the epidermis growth-restricting? plos one 10, 1-15. doi: 10.1371/journal.pone.0118406 orcid zulfiqar a. saqib https://orcid.org/0000-0002-7718-873x karl-hermann mühling https://orcid.org/0000-0002-9922-6581 address of corresponding author: prof. dr. karl-hermann mühling, institute of plant nutrition and soil science, kiel university, hermann rodewald str. 2, 24118 kiel, germany e-mail: khmuehling@plantnutrition.uni-kiel.de © the author(s) 2019. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1016/j.scienta.2006.02.010 http://dx.doi.org/10.1016/j.plantsci.2009.01.002 http://dx.doi.org/10.1016/s1360-1385(99)01428-4 http://dx.doi.org/10.1111/j.1439-037x.2011.00487.x http://dx.doi.org/10.1016/j.sjbs.2014.12.001 http://dx.doi.org/10.1002/jpln.200900052 http://dx.doi.org/10.1093/jxb/erq422 http://dx.doi.org/10.4141/p97-020 http://dx.doi.org/10.5829/idosi.mejsr.2012.12.1.1660 http://dx.doi.org/10.1016/j.sjbs.2013.01.006 http://dx.doi.org/10.1371/journal.pone.0118406 https://orcid.org/0000-0002-7718-873x https://orcid.org/0000-0002-9922-6581 journal of applied botany and food quality 92, 336 342 (2019), doi:10.5073/jabfq.2019.092.045 1centre of applied studies for the sustainable management and protection of mountain areas (crc ge.s.di.mont.), university of milan, edolo (bs), italy 2department of health, animal science and food safety (vespa), university of milan, milan, italy 3department of sciences and technological innovation (disit), università degli studi del piemonte orientale, alessandria, italy 4department of agriculture and forestry science (dafne), università degli studi della tuscia, viterbo, italy 5department of agricultural and environmental sciences production, landscape and agroenergy (disaa), university of milan, milan, italy quality traits of saffron produced in italy: geographical area effect and good practices luca giupponi1, giulia ceciliani1, valeria leoni1,*, sara panseri2, radmila pavlovic1,2, guido lingua3, alfredo di filippo4, annamaria giorgi1,5 (submitted: july 23, 2019; accepted: october 3, 2019) * corresponding author summary saffron (crocus sativus l.) is the most expensive spice in the world and is used in food, cosmetic and dyeing industries. considering that the production of saffron is increasingly widespread in medium-small italian farms as well as the scarceness of information and studies regarding the quality of the saffron produced in italy, the principal aim of this study was to investigate the quality of italian saffron. qualitative analysis was conducted in accordance with iso 3632 1,2:20102011 considering 484 samples collected over four years (2015-2018). in particular, moisture content, aroma strength (safranal), colouring strength (crocin) and flavour strength (picrocrocin) were assessed for each sample, and whether spice quality varied according to the geographical area where the spice was produced was also investigated. qualitative analysis showed that the majority (84-93%) of the samples analysed are of the first quality category, regardless of the year of production. moisture content and colouring strength are the factors that influence the quality of the spice most. principal component analysis showed that quality is not influenced by the geographical area where the spice was produced. finally, some best agricultural practices to obtain a high quality saffron spice are reported. keywords: crocus sativus, picrocrocin, crocin, safranal, iso 3632, food quality introduction dried stigmas of saffron (crocus sativus l., iridaceae) are considered the most expensive spice by weight worldwide (winterhalter and straubinger, 2000). crocus sativus is an autumn-flowering geophyte extensively grown in the near east and the mediterranean basin since the late bronze age (zohary and hopf, 1994; negbi, 1999). saffron is a male-sterile triploid lineage that ever since its origin has been propagated vegetatively only. more precisely, it has been demonstrated that crocus sativus is an autotriploid that evolved in attica by combining two different genotypes of crocus cartwrightianus herb., a species occurring in southeastern mainland of greece and on the aegean islands (nemati et al., 2019). triploid sterility and vegetative propagation prevented afterwards segregation of the favorable traits of c. sativus, resulting in worldwide cultivation of a unique clonal lineage (nemati et al., 2019). its long scarlet stigmas are highly valued for flavouring foods and for textiles and cosmetic dyeing industries (basker and negbi, 1983). in addition, several studies have demonstrated that the chemical compounds of this spice act as anti-ulcer agent (tamaddonfard et al., 2019), play a role as anti-cancerogens (milajerdi et al., 2016), reduce artherosclerosis (ghaffari and roshanravan, 2019) and have antidiabetic potential (yaribeygi et al., 2019), making saffron an interesting matter not only as a spice but also as functional food or as herbal product. in recent years demand for this spice has increased due to asian popu lation growth (arslanalp et al., 2019) and to the spread of asian cooking worldwide. today, world saffron production is estimated at about 200 tons per year (fernandez, 2004), and the principal producers and exporting countries (iran, afghanistan and india) are located in asia (oec, 2019). nevertheless, also in europe there are some countries that produce this spice and spain, italy and greece are the most important of these. however, in the last century, european saffron cultivation areas have decreased production: in spain, saffron dropped from 6,000 ha in 1971 to 200 ha in 2004 (fernandez, 2004), in greece from 1,600 ha in 1982 to 860 ha according to the most recent information (fernandez, 2004), and in central italy they reduced from 300 ha in 1910 to 60 ha some years ago (gresta et al., 2008). on the contrary, an enormous increase has been regis tered in iran and in other asian countries (yasmin et al., 2018). it has been estimated that iran produces 76% of total world saffron production annually (milajerdi et al., 2016). iran has the widest area cultivated (47.000 ha), most of which is located in the khorasan province (ehsanzadeh et al., 2004). in italy, the traditional cultivation areas of saffron are mostly concentrated in sardinia (about 25 ha) and abruzzo (about 6 ha) (gresta et al., 2008) and, although cultivated areas have decreased compared to past centuries in recent years, there has been renewed interest in saffron cultivation which has increased recently also in northern italy (giorgi et al., 2015a; giorgi et al., 2017; manzo et al., 2015) in the alpine macro-region (eusalp). nowadays, italy produce about 400 kg of saffron per year and is one of major saffron-importing countries together with spain, france, sweden, germany, the united kingdom and switzerland (oec, 2019). the main reason why these developed countries produce a small amount of saffron is due to the high requirement of meticulous manual operations for its production and the high cost of labor. in fact, the flowers of c. sativus must be picked during the early hours of the day, tepals must be carefully separated from stigmas and dried slowly at a low temperature. furthermore, this work is concentrated on a few days a year and on a few hours a day, and all the other activities (field preparation, corm planting, weeding, etc.) are performed by hand (husaini et al., 2010). no irrigation, chemical fertilization or chemical weed treatments are applied in some geographical areas in which it is cultivated, making it a very interesting option for farms working according to an organic regime, which, in italy and in europe, are in continuous expansion (willer and lernoud, 2017). due to its biological, physiological and agronomic traits, saffron can exploit quality traits of saffron produced in italy 337 marginal land and can be included in low input cropping systems, representing a viable alternative crop for sustainable agriculture in fragile agroecosystems such as those in rural and mountain areas (gresta et al., 2008). italy has many abandoned rural areas, mostly in mountain areas, where c. sativus is and can be grown (giorgi et al., 2015a; giorgi et al., 2017). moreover, it is a country of im portant food traditions, and has many high quality foods that are appealing for the development of tourism (corigliano, 2003). some italian traditional dishes are prepared with saffron (such as “risotto alla milanese” and “bagoss” cheese (giorgi et al., 2015a; giorgi et al., 2017; manzo et al., 2015) but currently there are few and fragmentary data about the quality of saffron produced in italy today, despite the fact that this aspect is of fundamental importance for the startup and promotion of unique and successful local agro-food chains (giupponi et al., 2018a). for this reason, the aims of this research are: • to evaluate the quality of saffron produced in italy during the last four years (2015-2018) according to the procedures established by iso 3632 1,2:2010-2011 that evaluate the concentration of the three main metabolites responsible for saffron colour, flavour and aroma: crocin (c44h64o24), picrocrocin (c16h26o7) and safranal (c10h15o) respectively. these analyses were conducted considering about 100 samples for each year as well as their geographical area of production in order to highlight possible qualitative differences (parizad et al., 2019). • to estimate the average amount of spice produced by italian farms in 2018 in order to show how much farmers invest in saffron production nowadays. • to suggest advice and/or best practices useful for farmers to improve saffron quality. this research was carried out in collaboration with many italian farms, farmer associations and three italian universities. materials and methods saffron samples during the four production seasons 2015-2018, 484 saffron samples were collected throughout italy. in detail, 88 samples were collected in 2015, 126 in 2016, 127 in 2017 and 143 in 2018 (fig. 1). each sample, consisting of about 1.5 2 g of dry stigmas, was stored in the laboratory in the dark and at 20 °c, inside a sterile airtight container, in order to avoid altering its chemical characteristics, before being analysed. the geographic coordinates (latitude and longitude) and the altitude of each sampling field were known. the saffron producers from whom the 2018 samples were collected were also asked to report, on a voluntary basis and using an online questionnaire created using google form, the annual quantity of spice produced in order to estimate the average saffron production of the italian farms involved in this study. quality evaluation in order to determine the quality of saffron produced in italy the samples collected were pulverized using mm400 vibrational mill (frequency: 30 hz; time: 1 minute), then moisture content and the amount of picrocrocin (flavour strength), safranal (aroma strength) and crocin (colouring strength) were measured according to procedures established by iso 3632 1,2:2010-2011. these analyses were performed each year (from 2015 to 2018) two months after the collection of the flowers and drying of the stigmas. moisture content was determined by weighing 500 mg of dried powder saffron and incubating it in an oven for 16 hours at 103 ± 2 °c. each sample was later weighed and moisture content (wmv) was calculated by the following formula: wmv = m0−m×100m0% where m0 is the mass, in grams, of the test portion before incubation and m1 is the mass, in grams, of the dry residue after incubation. amounts of picrocrocin, safranal and crocin were determined by uv-vis spectrophotometric analyses. 500 mg of the powdered saffron was transferred into a 1000 ml volumetric flask and 900 ml of distilled water was added. after stirring with an electromagnetic agitator (falc 60) for 1 hour at room temperature (20 °c), the solution was made up to 1000 ml with distilled water and filtered. the extract was diluted (1:10) with distilled water. extracts were directly analyzed by spectrophotometer (varian cary 50 uv-vis) to determine the amount of picrocrocin, crocin and safranal expressed as the absorbance of a 1% aqueous solution of dried saffron at 257, 330 and 440 nm respectively, using a 1 cm pathway quartz cell. picrocrocin, safranal and crocins determination [a1%1cm (λ max)] of each sample was calculated using the following formula: a1%1cm (λ max) = d × 10000/m × (100 wmv) where d is the specific absorbance; m is the mass, in grams, of the test portion; wmv is the moisture expressed as percentage mass fraction of the sample. all analytical steps were conducted in the dark to keep the saffron solution away from all light. spectrophotometric analyses were performed in duplicate. values of moisture content, picrocrocin, safranal and crocin were used to evaluate the quality category of saffron samples according to the quality category limits established by iso 3632 (tab. 1). fig. 1: geographical origin of the saffron samples (purple dots) divided by year of production/analysis. orange line shows the borders of italy. 338 l. giupponi, g. ceciliani, v. leoni, s. panseri, r. pavlovic, g. lingua, a. di filippo, a. giorgi statistical analysis boxplots were used to visualize the data regarding moisture content, picrocrocin, safranal and crocin amounts of samples according to different years of production. in addition, one-way analysis of variance (anova) was performed considering moisture content, picrocrocin, safranal and crocin as dependent variables and year of production as the independent variable. the saffron samples were also sorted according to principal component analysis (pca) in order to highlight the main variables that differentiated the samples. pca was performed considering moisture content, picrocrocin, safranal and crocin amount of each sample and the data referring to their geographical area of production: latitude, longitude and altitude. statistical analyses were performed using “ggplot2” package (wickham, 2016) of r 3.5.2 software (r development core team, 2019). results the quality analysis of saffron samples collected from 2015 to 2018 shows that over 80% of samples are in the first quality category (fig. 2). in particular, 2018 first category samples increased by over 90% and those of the second quality category are in the lower percentage (3-11%) over the four-year period. in all four years the percentage of third category samples is low (1-2%) while the percentage of samples which could not be classified in any quality category increased (3-8%). considering aroma strength (fig. 3) and flavour strength (fig. 4) most samples are in the first category range (tab. 1) and there are no significant differences between years as shown by the anova results (tab. 2). instead, when taking into consideration moisture content (fig. 5) and colouring strength (fig. 6), a larger number of samples are outside the first category limit in all years considered. in fact, as reported in figure 7, the 64 samples (12.8%) ≥70 ≥55 ≥40 20-50 20-50 20-50 ≥200 ≥170 ≥120 tab. 1: quality category limits for saffron in filaments according to iso 3632 1,2:2010-2011. characteristics specifications categories i ii iii moisture and volatile matter content (%) ≤12 ≤12 ≤12 flavour strength (picrocrocin) a11%cm 257 nm aroma strength (safranal) a11%cm 330 nm colouring strength (crocin) a11%cm 440 nm fig. 2: quality of italian saffron produced from 2015 to 2018 according to iso 3632 1,2:2010-2011. key: i, first quality category; ii, second quality category; iii, third quality category; nc, not classifiable. fig. 3: boxplots of aroma strength (safranal) of the saffron samples collected/analyzed during the four years (2015-2018). red broken lines show the limits of the three quality categories (iso 3632 1,2:20102011). for each boxplot, mean (black broken line) and median (black line) were highlighted. not assigned to the first category have low colouring strength and/or high moisture content. pca biplot (fig. 8) shows that the samples are arranged along the first axis (pc1) according to quality category, in particular, from the left to the right of the pc1 the quality of the spice increases, as do flavor strength and colouring strength, while moisture and aroma strength decrease. the elevation of the areas where the saffron samples analyzed were produced (which varies from 0 to 1500 m a.s.l.), as well as latitude and longitude, do not influence the quality category (fig. 8). according to the 53 data items collected from questionnaires filled out by farmers in the year 2018 it turns out that, on average, italian farmers produce small amounts of saffron: 144.6 ± 166 g. discussion according to the results obtained in the four-year period (2015-2018), the quality of saffron produced on italian farms is, mostly, of the first category of quality according to iso 3632. samples that are not first quality have, in most cases, high moisture and/or low colouring strength. these are therefore the two main variables that influence the quality of saffron produced in italy and to which particular attention must be paid. drying of the stigmas is a particularly delicate and quality traits of saffron produced in italy 339 subjective step since farmers perform the procedure using different methods and tools (wood embers, stove, oven, microwave, dehydrator etc.) which can be equally effective but not all easy to use. the fact is that, regardless of the drying method used, saffron must have a moisture content below 12% (tab. 1), to be classified into one of the three iso 3632 quality categories. although the results of this research did not reveal qualitative differences related to the four production years and differences due to the latitude, longitude and altitude of the area where the saffron was produced, these aspects could be better investigated using new approaches and innovative systems for saffron quality assessment (kiani et al., 2018) and/or by analyzing the volatile organic composition (vocs) of saffron (maggi et al., 2010). in fact, plant secondary metabolites vary, in quantitative and qualitative terms, in relation to the environmental conditions of growth (giorgi et al., 2015b; giupponi et al., 2018b; pavlovic at al., 2019) and italy is a country that, due to its geographical characteristics, presents fig. 4: boxplots of flavour strength (picrocrocin) of the saffron samples collected/analyzed during the four years (2015-2018). red broken lines show the minimum limit of the first quality category (iso 3632 1,2:2010-2011). for each boxplot, mean (black broken line) and median (black line) were highlighted. tab. 2: one-way anova results. the effect of year of production on moisture, crocin, picrocrocin and safranal is shown. key: ns, not significant (p > 0.05). source degrees of sum of mean of f-value p-value of variance freedom squares squares moisture 3 21.6 7.186 2.203 0.087 ns crocin 3 5477 1826 1.441 0.230 ns picrocrocin 3 752 250.7 1.411 0.239 ns safranal 3 32 10.75 0.349 0.790 ns fig. 5: boxplots of moisture content in the saffron samples collected/ analyzed during the four years (2015-2018). red broken line shows the maximum limit (12%) of the three quality categories (iso 3632 1,2:2010-2011). for each boxplot, mean (black broken line) and median (black line) were highlighted. fig. 6: boxplots of coloring strength (crocin) of the saffron samples collected/analyzed during the four years (2015-2018). red broken line shows the minimum limit of the first quality category (iso 3632 1,2:2010-2011). for each boxplot, mean (black broken line) and median (black line) were highlighted. fig. 7: moisture content and colouring strength of saffron samples (black dots) not belonging to the first quality category. the broken lines and the background colors define the limits of the three quality categories according to iso 3632 1,2:2010-2011. 340 l. giupponi, g. ceciliani, v. leoni, s. panseri, r. pavlovic, g. lingua, a. di filippo, a. giorgi areas with somewhat different climatic and environmental conditions (blasi et al., 2014). the variation of some metabolites produced by plants in relation to the environment was also found for saffron as regards safranal (lage and cantrell, 2009) but it would be interesting to investigate whether other chemical compounds that make up the aroma profile of saffron vary with the variation of the cultivation environment and spice processing methods. in fact the aroma profile of saffron is complex: as well as safranal, 3,5,5-trimethyl-2-cyclohexen-1-one (isophorone), 2,6,6-trimethyl-2-cyclohexene-1,4-dione (4-ketoisophorone), 3,5,5-trimethyl-3-cyclohexen-1-one (an isomer of isophorone), 2,6,6-trimethyl-1,4-cyclohexadiene-1-carboxaldehyde (an isomer of safranal), 2,2,6-trimethyl-1,4-cyclohexanedione,4-hydroxy-2,6,6-trimethyl-1-cyclohexen-1-carboxaldehyde (htcc) and 2-hydroxy-4,4,6-trimethyl-2,5-cyclohexadien-1-one are also responsible for saffron aroma (winterhalter and straubinger, 2000; maggi et al., 2010). it has been already demonstrated that italian saffron differs significantly from the same spice from other regions of the world, using different techniques and approaches: ptr-tof-ms and hplc analysis showed a sharp separation between iranian and italian samples and a higher content of total crocins and safranal in italian saffron (masi et al., 2016). the analysis of vocs showed also to be a potential tool to discriminate saffron samples with different geographical origins (d’auria et al., 2004; anastasaki et al., 2009). also, by a basic analysis as the iso3632:2011 it is possible to determine if edaphoclimatic and cultivation conditions significantly influence the quality of the spice (parizad et al., 2019). the results of this research are comparable with the ones analysed by the research team for the only italian alpine region (giorgi et al., 2017) and no significative difference in the quality parameters was found according the area in the italian peninsula. pca results (fig. 8) showed that most of the low quality samples are those that have high moisture content and/or those that have low colouring strength, low flavour strength and high aroma strength. while the high moisture content is due to poor drying of the stigmas and/or poor storage, the low colouring strength may be due to prolonged exposure of the stigmas to light during the collection phase and/or that of storage. in fact, crocin, the digentiobiosyl ester of crocetin, has high instability to light (tsimidou and tsatsaroni, 1993). furthermore, the high aroma strength of some poor quality samples may be related to their low flavour strength. in fact, picrocrocin is a monoterpene glycoside precursor of safranal: ß-glucosidase action on picrocrocin liberates aglycone (htcc) which is then transformed to safranal by dehydration during the drying process of the stigmas (caballero-ortega et al., 2007). therefore, drying and correct storage of the spice are of fundamental importance for saffron qua lity. this is also confirmed by bolandi et al. (2008) according to whom, after 12 weeks of storage, the colour strength of saffron had a noticeable decrease and bitterness also decreased whereas the aroma increased especially in storage conditions with high temperature and humidity. given that, based on the results obtained, good quali ty saffron can be produced in italy throughout the peninsula and at very variable altitudes (from 0 to 1500 m a.s.l.), this spice could be a resource for rural and/or mountain areas (giorgi and scheurer, 2015) and to develop profitable agricultural systems with low environmental impact. the sustainable development of marginalized areas is essential to rediscover the relationship with nature, with territorial knowledge and consequently with land conservation. the low environmental impact that this cultivation produces is in agreement with european regulations about organic agriculture (regulation 2018/848) and the recent 17 sustainable development goals (sdg) established by the united fig. 8: pca order biplot of saffron samples associated with geographical (latitude, longitude and elevation) and chemical-physical variables (moisture, flavour strength, color strength and aroma strength). the dots were colored according to the quality category of the samples: i, first quality category; ii, second quality category; iii, third quality category; nc, not classifiable. the first two principal components (pcs) explain 55.14% of the total variance in the dataset. quality traits of saffron produced in italy 341 nations for the global agenda 2030 (united nations, 2015). in addition, from the perspective of a circular economy, saffron could be a resource because tepals of crocus sativus (the main waste product during the production phases of saffron) can be used not only as a fertilizer for the soil or as ornamentation of dishes/products based on saffron, but also as a raw material for the cosmetic and pharmaceutical industry since saffron petals have substances (flavonoids and anthocyanins) with antioxidant, anti-inflammatory and antidepressant activity (akhondzadeh basti et al., 2007; hosseinzadeh et al., 2007; babaei et al., 2014). from the analysis of the data referring to the quantity of spice that the italian farms involved in this research produced in 2018, it emerged that on average they produce small quantities (145 g) even though some companies produce up to 700 g. the low production of spice per farm is justified by the fact that saffron production involves meticulous and almost exclusively manual work. to encourage greater production in developed countries such as italy where labour costs are high, it would be appropriate improve the mechanization of different production steps, as husaini et al. (2017) report in their study in the kashmir area. furthermore, scientific research should be encouraged so that it is possible to find new low-cost methodologies with low environmental impact to improve production processes (aghaei et al., 2018). but above all to maximize flower yield per bulb (without altering the quality). this objective could be achieved with the selection of healthy and productive bulbs or with the use of arbuscular mycorrhizal fungi as recently experimented by caser et al. (2019). it would also be interesting to develop saffron-based functional cookies as proposed by bhat et al. (2018), perhaps combined with other typical food products of high nutritional value such as, for italy, “mais nero spinoso” (cassani et al., 2017) and “grano siberiano valtellinese” (giupponi et al., 2019) recently characterized. this would allow the creation of unique, quality and low environmental impact food chains that are in line with the european directives for smart, sustainable and inclusive growth (eu commission, 2019). considering that most of the italian farmers are small producers, to valorise the quality of italian saffron it could be convenient to organize associations and/or consortiums to better increase the visibility of this precious cultivation. some associations are already working on this such as the national association “zafferano italiano”, and the local associations “oltre la goggia” and “zafferano terra di lavoro”. another innovative way to “network” can be the use of social networks like the recent facebook group (https://www.facebook.com/groups/zafferanounimont/) created to give visibility to the various farmers but also to allow sharing of ideas and information among farmers, researchers, agronomists, restaurateurs and others interested in the saffron chain. conclusion this research assessed the quality of saffron produced in italy and implemented the scarce data currently available on this topic. a large proportion of the samples produced and analysed over the four years of study were found to be of good quality, regardless of the environment/geographical area of cultivation. nevertheless, some good practices to improve the quality of saffron are listed below: • to remove the basal parts of the stigmas, which are often white or less coloured, in order to obtain a spice with high colouring strength; • to dry the stigmas carefully (moisture must be less than 12% according to iso 3632) and at temperatures below 55 °c (giorgi et al., 2015a) to avoid alteration of the thermolabile molecules; • to preserve the spice in a cool, dark and dry place, inside a sterile airtight container, in order to avoid altering its chemical characteristics (tsimidou and tsatsaroni, 1993; bolandi et al., 2008). these suggestions may be useful not only for italian farmers but also for all those who produce “red golden saffron” and want to improve its quality since to produce food of high nutritional quality must be a strategic objective for intelligent growth not only in italy and europe (eu commission, 2019) but also throughout the whole world. acknowledgements we wish to thank all the farmers and associations that have contributed to this study. this research was supported by “italian mountain lab” project and “dara crc ge.s.di.mont.” agreement. references aghaei, z., jafari, s.m., dehnad, d., ghorbani, m., hemmati, k., 2018: refractance-window as an innovative approach for the drying of saffron petals and stigma. j. food process eng. 41, e12863. doi: 10.1111/jfpe.12863 akhondzadeh, b.a., moshiri, 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leoni, centre of applied studies for the sustainable management and protection of mountain areas (crc ge.s.di.mont.), university of milan, via morino 8, 25048 edolo (bs), italy e-mail: valeria.leoni@unimi.it © the author(s) 2019. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). https://doi.org/10.17660/actahortic.2004.650.18 https://www.sciencedirect.com/science/article/pii/s0753332218335753 https://www.sciencedirect.com/science/article/pii/s0753332218335753 https://doi.org/10.1016/j.biopha.2018.10.031 https://doi.org/10.1080/14786419.2014.997725 https://doi.org/10.1515/opag-2017-0005 http://dx.doi.org/10.1659/mrd-journal-d-15-00061 http://dx.doi.org/10.1007/s10722-019-00755-z http://dx.doi.org/10.1659/mrd-journal-d-17-00013.1 https://doi.org/10.1080/11263504.2017.1306003 http://dx.doi.org/10.1051/agro:2007030 http://dx.doi.org/10.1016/j.jfoodeng.2017.06.022 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ehteshamul-haque, rajput m. tariq, mohammad athar (received august 25, 2010) summary with the rising popularity of organic farming, due to adverse effect of chemicals, the seaweed fertilizer industry is growing rapidly worldwide. seaweeds act as natural plant growth stimulator and enable the plants to withstand drought, disease or frost. root diseases of tomato and sunfl ower caused by root rotting fungi, fusarium spp., rhizoctonia solani and macrophomina phaseolina, and root knot nematode, meloidogyne spp., are the major constraints in tomato and sunfl ower production. in our studies, ethanol and water extracts of several seaweeds showed signifi cant nematicidal activity against meloidogyne javanica. in this study, effi cacy of three seaweeds spatoglossum variabile, melanothamnus afaqhusainii and halimeda tuna was compared with a fungicide topsin-m and a nematicide carbofuran both in screen house and under fi eld condition. seaweed and pesticides showed more or similar suppressive effect on root pathogens of tomato and sunfl ower by reducing fungal root infection and nematode’s galls on roots and nematode’s penetration in roots. however, mixed application of s. variabile with carbofuran caused maximum reduction in nematode’s penetration in roots and produced greater fresh shoot weight, root length and maximum yield of tomato under fi eld condition. seaweeds offer a non-chemical means of disease control, which would also protect our environment from the use of hazardous chemicals. introduction global utilization of seaweed is a multi-billion dollar industry. research and utilization of marine algae have increased markedly from last several decades (jimenez-escrig and sanchez-muniz, 2000). a number of products based on algae have been developed and applied in many fi elds like foods, pharmaceuticals, cosmetics and nutritional supplements (smit, 2004). much of this is based on farming of edible species or on the production of agar, carrageen and alginate. perhaps the longest established, most widespread and most proven effective use of seaweed is as a fertilizer (chapman and chapman, 1980; hattori, 1999). the high fi ber content of seaweed acts as a soil conditioner and assists moisture retention, while the mineral content is a useful fertilizer and source of trace elements (mat-atko, 1992). they also contain bio-control properties and contain many organic compounds and growth regulators such as auxins, gibberellins and precursor of ethylene and betaine which affect plant growth. seaweed extracts have been reported to increase plant resistance to pests and diseases, plant growth, yield and quality (jolivet et al., 1991; mat-atko, 1992; shyamali et al., 1982). management of soil-borne plant pathogens, including parasitic nematodes, is one of the single greatest challenges facing modern agriculture worldwide. the importance of soil-borne pathogens in modern agriculture systems is made especially clear by the current rush worldwide to fi nd alternatives to methyl bromide for pre-plant treatment of soils used to produce certain high-value crops. losses caused by plant parasitic nematodes are estimated about us $100 billion annually (saifullah et al., 2007), whereas fungal pathogens including species of fusarium, phytophthora, rhizoctonia, pythium, verticillium and macrophomina phaseolina caused billions dollar losses each year. most soilborne pathogens are diffi cult to control by conventional strategies such as the use of resistant cultivars and synthetic fungicides (weller et al., 2002). moreover the effect of synthetic fungicides on the environment and on human health are leading to signifi cant changes in the use of fungicides for the management of plant disease (perkins and patterson, 1997). as a consequence there is an increased emphasis on ways to minimize the use of pesticides (maas and galletta, 1997). in our previous study, several seaweeds collected from karachi coast have shown effective control of root rotting fungi like macrophomina phaseolina, rhizoctonia solani, fusarium species and root knot nematode (meloidogyne spp.,) on various crops (sultana et al., 2007; 2008; 2009). in this study, effi cacy of some seaweeds was examined alone or with common pesticides topsin-m (fungicide) and carbofuran (nematicide) both in screen house and under fi eld condition in suppressing the soilborne diseases and growth of sunfl ower (helianthus annuus l.) and tomato (lycopersicon esculentum mill.). materials and methods seaweeds melanothamnus afaqhusainii, spatoglossum variabile and halimeda tuna collected from buleji beach, karachi were used in these experiments individually or with commercial pesticides, topsin-m (fungicide) and carbofuran (nematicide) to compare the effi cacy of these seaweeds with common commercial pesticides. the experiments were conducted both under screen house and fi eld conditions using sunfl ower and tomato as test plants. screen house experiment dry powder of seaweeds was mixed with sandy loam soil (ph 8.1) to give a concentration of 1.0% w/w. the soil was naturally infested with 3-7 sclerotia of macrophomina phaseolina g-1 of soil as determined by wet sieving and dilution plating (sheikh and ghaffar, 1975), 2-6% colonization of rhizoctonia solani on sorghum seeds used as baits (wilhelm, 1955) and 3,000 cfu g-1 of soil (cfu = colony forming units) of a mixed population of fusarium oxysporum and f. solani as determined by soil dilution (nash and snyder, 1962). one kg of amended soil was transferred to clay pots (12 cm diam.). the pots were watered daily to allow the decomposition of the organic substrate. after three weeks, four equal size seedlings of tomato (lycopersicon esculentum mill.) variety roma, raised in steam sterilized soil were transplanted after root treatment with aqueous suspension of topsin-m (200 ppm) for 15 minutes, whereas carbofuran (at 0.05 g/kg soil) was applied after nematode inoculation. a set of seaweeds and pesticides separately were also kept for comparison. the pots without seaweeds and /or pesticides served as control. the pots were kept randomized on a screen house bench at 50% whc (water holding capacity) with four replicates of each treatment (keen and raczkowski, 1921). the seedlings were inoculated with meloidogyne javanica eggs/ juveniles at 2000 eggs/second stage juveniles per pot after one week seaweeds as an alternative to chemical pesticides 163 of seedlings establishment. whereas in case of sunfl ower six seeds were sown in each pots and 20 ml of topsin-m (200 ppm) suspension was drenched per pots. after one week of germination only four seedlings were kept and excess were removed and inoculated with nematode suspension similar to tomato experiment. to determine the effi cacy of seaweeds and pesticides on the root pathogens and plant growth, plants were uprooted after six weeks of nematode inoculation. observations were made on plant height, fresh shoot weight, root length and root weight. nematode infection was determined by counting the numbers of galls per root system (taylor and sasser, 1978). to determine nematode penetration and infection by root-infecting fungi, roots from each plant were cut into 1-cm long pieces and fi ve pieces of tap roots from each plant were used for assessment of fungal infection. the remaining roots were mixed thoroughly, and 1-gram sub-sample was wrapped in muslin cloth and dipped in boiling 0.25% acid fuchsin stain for 3-5 minutes. roots were left in the stain to cool, and then washed under tap water to remove excess stain. roots were transferred to vials containing glycerol and water (1:1 v:v) with a few drops of lactic acid. roots were macerated in an electric blender for 45 seconds and the resulting suspension was suspended in 50 ml water. numbers of juveniles and females in fi ve 5 ml sub samples were counted with the aid of dissecting microscope and numbers of nematode/g root were calculated (siddiqui and ehteshamulhaque, 2001). to determine the incidence of fungal infection, 1-cm long root pieces from tap roots (fi ve pieces from each plant) were surface disinfested with 1% ca (ocl)2 solution and plated onto potato dextrose agar amended with penicillin (100,000 units/l) and streptomycin (0.2 g/l). after incubation for 5 days at 28 oc, colonies of macrophomina phaseolina, rhizoctonia solani and species of fusarium were recorded. the experiment was conducted twice. data were subjected to analysis of variance (anova) and means were separated using the least signifi cant difference (lsd) according to gomez and gomez, 1984). field experiments dry powder of seaweeds m. afaqhusainii, s. variabile and h. tuna were mixed in sandy loam soil at 70 g per two meter rows and watered 2-3 days interval to allow the organic matters to decompose. the soil had a natural infestation of 6-18 sclerotia/g of soil of macrophomina phaseolina, 5-12% colonization of rhizoctonia solani on sorghum seeds used as baits and 2800cfu/gm of soil of mixed population of fusarium oxysporum and f. solani. after two weeks of seaweed decomposition, three-week-old tomato seedlings of equal size, raised in steam sterilized soil were transplanted in each row at 12 seedlings per row. in another set, topsin-m (200 ppm) at 200 ml/ 2 meter row and carbofuran (1 g/ 2 m row) were applied in seaweed amended and non-amended soil. after one week of seedling transplantation, each row was inoculated with aqueous suspension of meloidogyne javanica eggs/juveniles at 4000/ two meter row. each treatment was replicated four times and plants were watered 2-3 days intervals depending upon requirement of plants. effect of seaweed and pesticides on soilborne pathogens, growth and yield of tomato were recorded after 10 weeks of nematode inoculation. effect of seaweeds on root rotting fungi and root knot nematode was determined as described above except addition of data on nematode’s population in soil. nematode’s population in soil around roots was determined by baermann funnel method (schindler, 1961). in case of sunfl ower, 30 seeds were sown in each row and seedlings were inoculated with nematode suspension after one week of germination. observations were recorded after 6 weeks of nematode inoculation. results screen house experiment sunfl ower on sunfl ower application of s. variabile alone or h. tuna with topsin-m or carbofuran caused complete inhibition of macrophomina phaseolina infection on sunfl ower roots. seaweeds alone or with pesticides also signifi cantly reduced fusarium solani infection (tab. 1). application of seaweeds with pesticides also caused a suppressive effect on nematode by reducing the nematode’s penetration in roots (tab. 1). seaweed soil amendment showed a positive effect on plant growth by enhancing the plant height (tab. 2). tab. 1: effect of green (halimeda tuna), brown (spatoglossum variabile) and red (melanothamnus afaqhusainii) seaweeds on the infection of macrophomina phaseolina, rhizoctonia solani and fusarium solani and meloidogyne javanica on sunfl ower roots under screen house condition. treatments m. phaseolina r. solani f. solani no.of knots / juveniles / females / root system g root infection % control topsin-m carbofuran h. tuna at 1% w/w (a) s. variabile at 1% w/w (b) m. afaqhusainii at 1% w/w (c) topsin-m + a carbofuran + a topsin-m + b carbofuran + b topsin-m + c carbofuran + c lsd0.05 for fungi = treatments = 11.91, pathogens = 5.92 ns 10.6 1mean values in column showing differences greater than lsd values are signifi cantly different at p< 0.05. 2mean values in rows for fungi showing differences greater than lsd values are signifi cantly different at p< 0.05. 31.2 0 12.5 12.5 0 18.7 0 0 6.2 6.2 12.5 12.5 25 12.5 12.5 31.2 12.5 18.7 6.2 43.7 6.2 43.7 25 12.5 43.7 18.7 12.5 25 18.7 25 6.2 37.5 18.7 12.5 25 37.5 15.5 4.9 10.5 8.7 8.5 3.8 9.8 7.9 7.6 7.4 9.1 7.4 13.2 1.6 4.9 8.2 3.3 4.9 2.7 1.6 3.3 3.3 1.6 1.6 164 v. sultana, g.n. baloch, j. ara, s. ehteshamul-haque, r.m. tariq, m. athar tomato application of seaweeds alone and or with topsin-m and carbofuran also showed a suppressive effect on root rotting fungi rhizoctonia solani and fusarium solani. halimeda tuna with topsin-m and s. variabile with carbofuran caused complete suppression of m. phaseolina, r. solani and f. solani on tomato roots (tab. 3). seaweeds alone and or with pesticides also caused a suppressive effect on nematode by reducing the number of galls on roots. application of h. tuna with topsin-m and carbofuran caused better protection of roots from nematode invasion than applied separately (tab. 3). greater plant height and fresh shoot weight was achieved by the mixed application of s. variabile and topsin-m (tab. 4). whereas h. tuna produced maximum root length. field experiment sunfl ower seaweed alone and/or with pesticides, topsin-m and carbofuran caused a suppressive effect on root rotting fungi of sunfl ower by reducing the infection of macrophomina phaseolina, fusarium solani and rhizoctoni solani (tab. 5). seaweed application alone or with topsin-m and carbofuran also produced an adverse effect tab. 2: effect of green (halimeda tuna), brown (spatoglossum variabile) and red (melanothamnus afaqhusainii) seaweeds on the growth of sunfl ower under screen house condition. treatments plant height fresh shoot weight root length fresh root weight (cm) (g) (cm) (g) control topsin-m carbofuran h. tuna at 1% w/w (a) s. variabile at 1% w/w (b) m. afaqhusainii at 1% w/w (c) topsin-m + a carbofuran + a topsin-m + b carbofuran + b topsin-m + c carbofuran + c lsd0.05 5.11 ns ns 0.391 1mean values in column showing differences greater than lsd values are signifi cantly different at p < 0.05. ns = non-signifi cant 20.2 23.4 26.3 28.1 27.3 25.9 25.7 28.8 25.0 31.7 27.5 26.2 3.8 2.9 3.5 3.5 4.6 3.2 3.6 3.7 3.6 3.7 4.1 3.3 12.1 12.5 13.1 9.1 12.4 10.1 9.3 8.5 9.7 11.4 12.0 9.9 0.48 0.52 0.69 0.63 1.02 0.74 0.66 0.99 0.51 0.48 0.70 0.68 6.2 6.2 0 6.2 0 0 0 0 0 0 6.2 0 25 6.2 0 6.2 6.2 6.2 0 6.2 6.2 0 6.2 0 37.5 18.7 12.5 12.5 12.5 31.2 0 25 18.7 0 12.5 12.5 71.5 32.9 33.4 28.7 18.5 41.5 21.2 19.1 18.3 30.7 22.3 10.8 398 381 261 389 246 294 149 166 258 297 253 259 tab. 3: effect of green (halimeda tuna), brown (spatoglossum variabile) and red (melanothamnus afaqhusainii) seaweeds on the infection of macrophomina phaseolina, rhizoctonia solani, fusarium solani and meloidogyne javanica on tomato roots under screen house condition. treatments m. phaseolina r. solani f. solani no.of knots / juveniles / females / root system g root infection % control topsin-m carbofuran h. tuna at 1% w/w (a) s. variabile at 1% w/w (b) m. afaqhusainii at 1% w/w (c) topsin-m + a carbofuran + a topsin-m + b carbofuran + b topsin-m + c carbofuran + c lsd0.05 for fungi = treatments = 8.71, pathogens = 4.32 42.32 1922 1mean values in column showing differences greater than lsd values are signifi cantly different at p< 0.05. 2mean values in rows for fungi showing differences greater than lsd values are signifi cantly different at p< 0.05. seaweeds as an alternative to chemical pesticides 165 on root knot disease (tab. 5). maximum reduction in nematode’s penetration in roots was achieved by the mixed application of m. afaqhusainii and carbofuran. maximum fresh shoot weight was produced by s. variabile used alone (tab. 6). tomato seaweed soil amendment, alone or with topsin-m and carbofuran caused a suppressive effect on root rotting fungi rhizoctonia solani and fusarium solani infecting tomato roots. no infection of f. solani was observed where s. variabile and m. afaqhusainii used alone or where m. afaqhusainii used with topsin-m and h. tuna applied tab. 4: effect of green (halimeda tuna), brown (spatoglossum variabile) and red (melanothamnus afaqhusainii) seaweeds on the growth of tomato under screen house condition. treatments plant height fresh shoot weight root length fresh root weight (cm) (g) (cm) (g) control topsin-m carbofuran h. tuna at 1% w/w (a) s. variabile at 1% w/w (b) m. afaqhusainii at 1% w/w (c) topsin-m +a carbofuran +a topsin-m + b carbofuran + b topsin-m + c carbofuran + c lsd0.05 5.011 1.661 7.851 1.821 1mean values in column showing differences greater than lsd values are signifi cantly different at p< 0.05. 19.65 17.87 19.67 21.14 26.9 21.47 23.2 24.1 27.7 24.5 22.87 19.12 3.56 3.19 3.41 3.6 5.92 5 5 3.87 6.9 6.75 4.9 3.9 9.65 5.6 18.9 21.1 19.6 6.4 5 5.5 5.2 12.1 5.6 6.2 1.95 1.92 2.05 2.2 4.2 3.4 2.73 2.36 4.4 3.6 2.9 2.3 tab. 5: effect of green (halimeda tuna), brown (spatoglossum variabile) and red (melanothamnus afaqhusainii) seaweeds on the infection of macrophomina phaseolina, rhizoctonia solani, fusarium solani and meloidogyne javanica on sunfl ower roots under fi eld condition. treatments m. phaseolina r. solani f. solani no. of knots / juveniles / nematodes / root system females / g root 100 g soil infection % control topsin-m carbofuran h. tuna at 1% w/w (a) s. variabile at 1% w/w (b) m. afaqhusainii at 1% w/w (c) topsin-m + a carbofuran + a topsin-m + b carbofuran + b topsin-m + c carbofuran + c lsd0.05 for fungi = treatments = 11.91, pathogens = 5.92 30.81 8.81 ns 1mean values in column showing differences greater than lsd values are signifi cantly different at p< 0.05. 2mean values in rows showing differences greater than lsd values are signifi cantly different at p< 0.05. 18.7 0 6.2 0 0 18.7 6.2 0 6.2 6.2 0 0 12.5 6.2 12.5 0 18.7 12.5 12.5 18.7 0 6.2 0 0 12.5 0 0 0 0 25 0 12.5 0 6.2 0 0 59.6 35.2 38.6 12.0 47.8 10.6 27.3 17.7 16.5 27.4 19.2 21.3 14 11.5 7 9 6 7 8 14 10 9 8 4 30.5 20 32 28 20 40 40 16 36 32 20 28 with carbofuran (tab. 7). seaweed also showed inhibitory effect on meloidogyne javanica. highest reduction in neamtode’s penetration in roots was achieved by the mixed application of s. variabile and carbofuran (tab. 7). soil amendment with seaweeds alone or with pesticides caused a positive effect on plant growth by enhancing the plant height, fresh shoot weight, root length and yield. maximum plant height was achieved where s. variabile was used with topsinm, whereas with carbofuran s. variabile produced greater fresh shoot weight and root length. spatoglossum variabile also produced highest yield (in terms of number and by weight of fruits) of tomato used either with topsin-m or carbofuran (tab. 8). 166 v. sultana, g.n. baloch, j. ara, s. ehteshamul-haque, r.m. tariq, m. athar discussion marine biosphere is an untapped reservoir of agrochemically potent compounds. seaweed has been used as a fertilizer for many years and are the most widely used biostimulant in agriculture management (hattori, 1999). in this study, seaweeds like spatoglossum variabile, halimeda tuna, and melanothamnus afaqhusainii, showed more or less similar suppressive effect on root rotting fungi and root knot nematode to chemical fungicides (topsin-m) and nematicide (carbofuran). considerable evidence has been accumulated in recent years to support and identify the benefi ts associated with the use of tab. 6: effect of green (halimeda tuna), brown (spatoglossum variabile) and red (melanothamnus afaqhusainii) seaweeds on the growth of sunfl ower under fi eld condition. treatments plant height fresh shoot weight root length fresh root weight (cm) (g) (cm) (g) control topsin-m carbofuran h. tuna at 1% w/w (a) s.variabile at 1% w/w (b) m. afaqhusanii at 1% w/w (c) topsin-m + a carbofuran + a topsin-m + b carbofuran + b topsin-m + c carbofuran + c lsd0.05 28.11 50.41 ns 11.11 1mean values in column showing differences greater than lsd values are signifi cantly different at p< 0.05. ns = non-signifi cant 71.7 101 67.9 58.2 87.7 62.1 72.0 76.3 67.9 71.0 91.1 75.1 48.0 90.8 55.9 58.8 101.1 46.0 58.2 77.3 69.0 37.8 85.7 74.5 10.9 9.4 10.4 13.7 12.4 10.6 14.1 11.6 11.7 10.2 8.9 11.9 10.1 8.5 8.5 8.1 19.7 7.0 20.3 6.3 5.7 7.3 5.5 8.1 6.2 0 6.2 6.2 0 0 12.5 0 6.2 12.5 6.2 0 56.2 6.2 18.7 12.5 12.5 12.5 31.2 12.5 18.7 31.2 6.2 12.5 43.7 12.5 6.2 25 0 0 12.5 0 6.2 18.7 0 6.2 19.7 9.6 13.3 8.6 9.2 16 6.8 11.6 12.3 8.7 12.1 11.2 85.8 59.9 114.8 29.9 63.1 79.7 53.2 71.2 38.1 14.1 46.4 31.3 44.8 21.4 9.9 8.3 8.9 9.9 9.9 9.9 12.4 9.1 4.1 11.6 tab. 7: effect of green (halimeda tuna), brown (spatoglossum variabile) and red (melanothamnus afaqhusainii) seaweeds on the infection of macrophomina phaseolina, rhizoctonia solani, fusarium solani and meloidogyne javanica on tomato roots under fi eld condition. treatments m. phaseolina r. solani f. solani no. of knots / juveniles / females / nematodes / root system g root 100 g infection % infection % control topsin-m carbofuran h. tuna at 1% w/w (a) s. variabile at 1% w/w (b) m. afaqhusainii at 1% w/w (c) topsin-m + a carbofuran + a topsin-m + b carbofuran + b topsin-m + c carbofuran + c lsd0.05 for fungi = treatments = 12.41, pathogens = 6.22 9.21 54.41 ns 1mean values in column showing differences greater than lsd values are signifi cantly different at p< 0.05. 2mean values in rows showing differences greater than lsd values are signifi cantly different at p< 0.05. seaweed in crop production systems. seaweed extracts have been reported to increase plant resistance to pests and diseases, plant growth, yield and quality (jolvet et al, 1991; pardee et al.,, 2004; verkleij, 1992). application of seaweed to plants can result in decreased levels of nematode attack (ara et al., 1997; wu et al., 1997; 1998). seaweeds contain elaborate secondary metabolites that play a signifi cant role in the defense of the host against predators and parasites which offer a potential novel approach to control population of plant parasitic nematodes (paracer et al., seaweeds as an alternative to chemical pesticides 167 tab. 8: effect of green (halimeda tuna), brown (spatoglossum variabile) and red (melanothamnus afaqhusainii) seaweeds on the growth and yield of tomato under fi eld condition. treatments plant height fresh shoot weight root length fresh root weight no. of fruits / weight of fruits / (cm) (g) (cm) (g) 4 plant 4 plant (g) control topsin-m carbofuran h. tuna at 1% w/w (a) s. variabile at 1% w/w (b) m. afaqhusainii at 1% w/w (c) topsin-m + a carbofuran + a topsin-m + b carbofuran + b topsin-m + c carbofuran + c lsd0.05 7.51 25.61 5.91 ns 9.61 201.21 1mean values in column showing differences greater than lsd values are signifi cantly different at p< 0.05. ns = non-signifi cant 34.8 42.0 35.4 42.4 39.3 38.8 33.7 39.0 46.4 43.5 43.3 40.1 28.4 33.7 18.7 43.1 22.3 28.4 30.9 56.2 62.4 89.3 43.7 55.6 23.8 24.2 21.5 22.4 25.2 22.3 19.0 27.0 27.0 30.3 29.4 25.6 3.5 4.8 3.9 5.0 9.4 4.9 3.8 7.5 7.5 8.1 6.6 6.2 13.7 20.0 14.7 21.5 22.5 20.5 13.0 23.2 28.0 28.5 22.5 22.5 152.5 308.7 292.5 317.5 407.5 348.7 175 447.5 490 523 412.5 361.2 1987; jacobs et al., 2003). in a large number of marine algae, antimicrobial activities are attributed to the presence of acrylic acid. seaweeds contain 1-aminocyclopropane-1-carboxylic acid (acc), which has antimicrobial activity (nelson and van standen, 1985). whereas wu et al, (1997) suggested that betains of the brown alga ascophyllum nodosum caused a reduction of m. javanica and m. incognita infection on tomato. in this study, seaweeds showed more or less similar suppressive effect on root rotting fungi and root knot nematode like chemical pesticides, topsin-m (fungicides) and carbofuran (nematicide). however, mixed application of s. variabile with carbofuran caused maximum reduction in nematode’s penetration in roots and produced greater fresh shoot weight, root length and maximum yield of tomato under fi eld condition, which is in agreement with our previous study (sultana et al., 2009). acknowledgement financial support provided by the pakistan agricultural research council, islamabad under the agricultural linkages program (alp) with usa is sincerely acknowledged. references ara, j., ehteshamul-haque, s., sultana, v., ghaffar, a, qasim, r., 1997: use of sargassum species for the control of meloidogyne javanica in okra. nematol. medit 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sultana, v., ehteshamul-haque, s., ara, j., 2007: management of root diseases of soybean and tomato with seaweed application. phytopath. 97, 112 (abstr.). sultana, v., ara, j., ehteshamul-haque, s., 2008: suppression of root rotting fungi and root knot nematode of chili by seaweed and pseudomonas aeruginosa. j. phytopath. 156, 390-395. sultana, v., ehteshamul-haque, s., ara, j., athar, m., 2009: effect of brown seaweeds and pesticides on root rotting fungi and root-knot nematode infecting tomato roots. j. appl. bot. food qual. 83, 50-53. taylor, a.l., sasser, j.n., 1978: biology, identifi cation of root-knot nematodes (meloidogyne species). north carolina state university raleigh, graphics, usa. verkleji, f.n., 1992: seaweed extracts in agriculture and horticulture: a review. biol. agric. hort. 8, 309-324. weller, d.m., raaijimakers, j.m., gardener, b.b.m., thomashow, l.s., 2002: microbial population responsible for specifi c soil suppressiveness to plant pathogens. annu. rev. phytopathol. 40, 309-348. wilhelm, s., 1955: longevity of the verticillium wilt fungus in the laboratory and fi eld. phytopath. 45, 180-181. wu, y., jenkins, t., blunden, g., whapham, c., hankins, s.d., 1997: the role of betains in alkaline extracts of ascophyllum nodosum in reduction of meloidogyne javanica and m. incognit infestations of m. incognit infestations of m. incognit tomato plants. fund. appl. nematol. 20, 99-102. wu, y., jenkins, t., blunden, g., von mende, n., hankins, s.d., 1998: suppression of fecundity of the root-knot nematode, meloidogyne javanica, in monoxenic cultures of arabidopsis thaliana treated with an alkaline extracts of ascophyllum nodosum j. appl. phycol. 10, 91-94. address of the author: prof. dr. viqar sultana, biotechnology and drug development laboratory, department of biochemistry, university of karachi, karachi-75270, pakistan. mr. ghulam nabi baloch, agricultural biotechnology and phytopathology laboratory, department of botany, university of karachi, karachi-75270, pakistan. prof. dr. jehan ara, post harvest technology and food biochemistry laboratory, department of food science and technology, university of karachi, karachi-75270, pakistan. prof. dr. syed ehteshamul-haque, agricultural biotechnology and phytopathology laboratory, department of botany, university of karachi, karachi-75270, pakistan. dr. rajput m. tariq, m.a.h. qadri biological research centre, university of karachi, karachi-75270, pakistan. dr. mohammad athar, pest detection and emergency projects, california department of food and agriculture, 3288 meadowview road, sacramento, ca 95832, usa. e-mail: atariq@cdfa.ca.gov art19_athar.indd journal of applied botany and food quality 85, 120 125 (2012) department of botany, university of karachi, karachi, pakistan biomonitoring of heavy metal contamination in pongamia pinnata and peltophorum pterocarpum growing in the polluted environment of karachi, pakistan muhammad shafi q, muhammad zafar iqbal, m. saeed arayne, mohammad athar* (received may 23, 2011) * corresponding author summary determination of some of the important heavy metals like lead and cadmium was carried out in the city environment of karachi. foliage parts of two roadside trees, pongamia pinnata (l.) merrill and peltophorum pterocarpum d.c. backer ex k. were used to carry out such investigation. five roadside points were selected for the study in different parts of the city. the investigations showed that high level of lead and cadmium was found in the foliage of p. pinnata and p. pterocarpum, which were growing along the busy roads of the city. the level of pb and cd in the foliage of the above mentioned trees was quite high at m.a. jinnah road as compared to shahrahe-faisal, nazimabad, gulshan-e-iqbal and karachi university campus. low traffi c activities at the university campus showed lowest lead and cadmium levels in the foliage of both tree species than the other point of the city. in this study, p. pinnata showed more accumulations of lead and cadmium than p. pterocarpum. this difference might be due to large surface area of the foliage in p. pinnata that is available for exposure to any pollutants as compared to p. pterocarpum. p. pinnata is a useful plant species in removing different heavy metals from the urban environment of the city. it is therefore suggested, that p. pinnata should be given more preference for future plantation in the areas, particularly along the busy roads and highways. introduction in recent year’s concern over the air pollution problem has increased tremendously in the wake of population explosion, industrialization, urbanization, transportation and other human activities (shah et al., 2004). trace elements are known to be essential for all plants. a feature of the physiology of these elements is that although many are essential for growth, they can also have toxic effects on cells at higher concentration (berber et al., 2010). contamination of vegetation by airborne trace metals is particularly massive in urban areas, notably along highways (aksoy and ozturk, 1996). emissions from autovehicular activities contribute most of air pollution problems and affect the growth of plants. atmospheric trace metal levels have been reported to exhibit great variations due to automobile activities. for the last 40 years road transport emissions were considered to be the principal source of lead in the urban environment (akbar et al., 2006). there are about 50 metals that are of special interest with respect to the toxicological importance to human health, plants and animals (burhan et al., 2001). lead and cadmium are the toxic elements of primary importance in ecotoxicology (breckle and kahile, 1992). high level of lead was found in roadside plants. an enhancement of lead was found in roadside soil and vegetation due to combustion of leaded petrol by automobile exhaust in baghdad city (khalid et al., 1981). palm leaves, which were collected from 25 different sites of baghdad city, showed a relative increase of pb deposition due to vehicles using leaded petrol (hanna and al-bassam, 1983). increased fallout of different types of metals from vehicles in karachi city is increasing day by day. yousafzai (1991) found high level of pb (810-4527 ppm) and cd (0.2-4.5 ppm) in the street dust of metropolitan city of karachi. considerable higher levels of pb, cu and zn have been recorded in soil of the stands where suaeda fruticosa and salsola baryosma have been associated with prosopis julifl ora along the super highways (iqbal et al., 1998). shafiq et al. (2011) found highest levels of pb (95 ppm) and cd (2.96 ppm) in alstonia scholaris and high levels of pb (89 ppm) and cd 1.76 ppm) in cassia siamea from karachi. traffi c emissions on roads are the main cause of heavy metal accumulation on the surrounding environment including vegetation, which might have an ecological effect on them. casselles (1998) found that some heavy metals such as pb decrease in the leaves of plants with increasing distance from the road. high lead concentrations that were attributed to vehicle emission sources were reported in amman city center (jaradat et al., 1999). there are several direct and indirect methods of estimating the trace metal levels in urban environment. plant materials such as tree bark, tree rings and leaves of higher plants have been used to detect the deposition, accumulation and distribution of metal pollution for many years (aksoy and sahln, 1999). to study airborne dust particles deposited in the polluted areas, the most suitable method is leaf collection (freer-smith et al., 1997). karachi is a densely populated city with major industrial units of the country. a high rate of economic growth during the last few decades has resulted in mass-scale urban mobility. large scale urban mobility demands large transport system to carry people and goods from one place to another. the transport system in the city emits toxic materials such as carbon particles, unburned and partially burned hydrocarbons, fuels, tar materials, lead compounds and other toxic elements in the environment. the city presents heavy vehicular activities and high concentration of trace metals in the air. pollutant like so2 (100-134 µg m-3), co (7-5 mg m-3), no2 (38-63 µg m-3) (ghauri et al., 1988) and pb (4527 ppm), cd (4.5 ppm), zn (2215 ppm) and cu (275 ppm) (yousufzai, 1991) from these sources are considered to be important in deteriorating air quality of the city. the aim of the present research was to determine the heavy metals content (lead and cadmium) in the foliage of some important plants (pongamia pinnata and peltophorum pterocarpum) growing in different areas of the city of karachi, pakistan. materials and methods site description karachi is situated on the coast along the arabian sea at a latitude of 24° 48’ n and longitude of 66° 55’ e. the soil is calcareous, marine in origin and belongs to upper tertiary period. moving away from the coast, the ground rises gently forming a large plain to the north and east on which the city is built. the city is between 1.5 and 37 m above sea level. chaudhry (1961) has characterized the climate of karachi as subtropical maritime desert. average wind biomonitoring of heavy metals in trees of karachi 121 velocity is 12 m s-1 during june and july and 3.5 m s-1 from january to march. during the southwest monsoon season winds blow from the sea towards the coast, whereas during the northeast monsoon their direction is reversed. therefore, pollutants are pushed inland during the southwest monsoon season and are blown out to sea during the northeast monsoons (unep, 1992). pollution and traffi c density data and estimated pollutant level has been observed (tab. 1-2). the hot and humid rainy season, which is variable, lasts from june to september. minimum rainfall is 1 mm in the month of october whereas, the maximum rainfall (85 mm) occurs in the month of july. the winter season is very short lasting from middle of november to middle of february. temperature is mild with no frost. dew formation is quite common, the relative humidity is high. the climatic conditions at the control site (karachi university campus) are not different from other sites of the city. selection of sites the site in urban area is disturbed mainly by autovehicular activities, includes all main traffi c network (gulshan-e-iqbal, nazimabad, shahrah-e-faisal and m.a. jinnah road) whereas, karachi university is relatively a clean area (shafiq et al., 2011). brief description of the study area is given below (fig.1): a. m.a. jinnah road: this site is the most congested site along the quaid-e-azam tomb. the density of traffi c from gulshan-e-iqbal, nazimabad, liaquatabad and shahrah-e-faisal passes through this road. multistoryed buildings are common at this site. the slow movement of traffi c starts building up toxic pollutants in the area. b. shahrah-e-faisal: shahrah-e-faisal has many multistoryed buildings. this site is 15 km from the eastern site of the quaid-eazam tomb and heavily infl uenced by traffi c activities. c. nazimabad: nazimabad handles a large traffi c volume, moving from north to west of the city. this site is about 10 km away from the north of quaid-e-azam tomb. d. gulshan-e-iqbal: gulshan-e-iqbal is located about 8 km north east of the quaid-e-azam tomb. this place is comparatively open with low traffi c. however, traffi c congestion in the morning hours is common due to trade activities at old vegetable and fruit market. e. karachi university campus: karachi university campus is situated at the outskirts of the city. this site is relatively free from the autovehicular activities as compared to other sites of the city. fig. 1: map of the study area (a = karachi university, b = gulshan-e-iqbal, c = nazimabad, d = shahrah-e-faisal, e = m.a. jinnah road). tab. 2: estimated emission of pollutant from vehicles in pakistan, 1996, 1997 and 2000) tons x 103 pollutants 1996 1997 2000 source co 6000 6500 8000 qureshi (2000) hc 1250 1340 1650 nox 440 460 540 so2 69 72 85 pm10 125 135 160 total no. of vehicles 3276000 3515000 4291000 tab. 1: pollution level of karachi city pollutants max. min. ave. source so2 (µg m-3) 134 25 61.3 ghauri et al., a report of sparco (1988) no2 (µgm-3) 544 38 104 ghauri et al., a report of sparco (1988) o3 (µg m-3) 50 36 43 ghauri et al., a report of sparco (1988) sulphur (ppm) 565 91 266 iqbal, tropical ecology (1988) pb (ppm) 2677 4 1488 yousufzai, pak. j. sci. & ind. res. (1991) cd (ppm) 1.2 0.2 0.8 yousufzai, pak. j. sci. & ind. res. (1991) cu (ppm) 187 3 134 yousufzai, pak. j. sci. & ind. res. (1991) zn (ppm) 638 6 285 yousufzai, pak. j. sci. & ind. res. (1991) mn (ppm) 305 4 173 yousufzai, pak. j. sci. & ind. res. (1991) co (ppm) 10 2 6 ziaurrehman, environmental news (1993) co2 (ppm) 350 170 260 ziaurrehman, environmental news (1993) hc (vol. %) 2.3 0.4 1.3 ziaurrehman, environmental news (1993) pm (µg cm-3) 566 67 316 ziaurrehman, environmental news (1993) note: no2, o3, co, co2, hc, pm are in the air. other values were of street dust. in organic chemistry, a hydrocarbon (hc) is an organic compound consisting entirely of hydrogen and carbon (wikipedia, 2012). 122 m. shafi q, m.z. iqbal, m.s. arayne, m. athar karachi university campus is clean and 20 km away from quaide-azam tomb. pongamia pinnata (l.) merrill (charr) is a medium sized, almost evergreen tree and spreading shady crown. it is indigenous to the foothills of the himalayas, but cultivated in plains for its ornamental value. peltophorum pterocarpum d.c. backer ex k. heyne is native to ceylon and north australia, commonly cultivated as roadside tree in gardens and in plains of pakistan. the leaf samples of common roadside trees like pongamia pinnata and peltophorum pterocarpum having uniform growth and diameter breast height (dbh) were chosen from each site. the samples affected by traffi c were obtained from road edge at a distance of one meter. leaf samples of three individual of each species were randomly collected from each area at two-meter height throughout the plant canopy to give representative average sample. unwashed leaf samples were oven dried at 80° c for 24 hours and made in powdered form. 0.5 g powdered leaf sample was taken in 100 ml pyrex beaker and 10 ml concentrated nitric acid was added. the beaker was partially covered with watch glass to avoid any loss of acid. later, the beaker was kept on the hot plate in fume chamber for digestion. the sample was digested till a clear solution resulted. the watch glass was removed from the top of the beaker and heating continued till the volume of content was reduced to 1-2 ml. evaporation was allowed but not to dryness. later the content was cooled down. the content was dissolved in 0.1n hcl, fi ltered through whatman fi lter paper no. 44 and the volume was made up to 50 ml in volumetric fl ask. digested plant material solution was analyzed for metal contents on the atomic absorption spectrophotometer (perkin elmer 3100) using appropriate cathode. a series of standard solutions for each element in the range of absorbance noted for unknown samples were simultaneously run on atomic absorption spectrophotometer. the calibration curves obtained for absorbance versus concentration data were statistically analyzed using fi tting of straight line by least square method. three replicates were used in this analysis. concentration of elements is expressed as µg g-1. all reagents were of analar grade. all glassware’s were carefully cleaned with double distilled water and later rinsed with deionized water. the analyses of lead and cadmium were performed at wavelengths 283.3 nm and 228.8 nm, respectively. to confi rm the validity of data, a comparison from control and between the means of treatment was done by duncan’s multiple range tests. the data collected was statistically analyzed by analysis of variance techniques on personnel computer software package, co-stat version 3. nomenclature of the plant species was followed according to stewart (1972). results the analysis showed high levels of lead and cadmium in the leaves of pongamia pinnata and peltophorum pterocarpum collected from the polluted as compared to less polluted sites of the city (fig. 2 3). a signifi cant (p<0.05) difference was found in bp content in the leaves collected from the polluted and less polluted areas. the values were found higher in the city environment than at karachi university campus. highest concentration of lead detected in the leaves of p. pinnata was 106 µg g-1 at m. a. jinnah road, while, the lowest level of 18 µg g-1 was recorded in leaves collected from the karachi university campus. at shahrah-e-faisal, the concentration of lead in p. pinnata leaves was 71 µg g-1, while, an average level of lead content was recorded from nazimabad (54 µg g-1) and gulshane-iqbal (43 µg g-1). level of lead in the leaves of p. pterocarpum ranged between 7-81 µg g-1. lowest level of lead (7 µg g-1) was found in leaf samples collected from karachi university campus, while highest level of lead (81 µg g-1) was recorded at m.a. jinnah road. level of lead in p. pterocarpum leaves was found at gulshan-e-iqbal (14 µg g-1), nazimabad (36 µg g-1) and shahrah-e-faisal (57 µg g-1) sites, respectively. cadmium analyzed in leaves of p. pinnata and p. pterocarpum showed low levels as compared to lead. the leaves of both the tree species showed high level of cadmium in samples collected from the city area as compared to karachi university campus. cadmium level in leaves of both tree species was found in the range of 0.262.10 µg g-1). cadmium was highest (2.10 µg g-1) in the leaves of p. pinnata at m.a. jinnah road whereas, the lowest level of cadmium (0.26 µg g-1) was recorded at karachi university campus. cadmium was found higher at shahrah-e-faisal (1.16 µg g-1), while at nazimbad and gulshan-e-iqbal, the concentration of cadmium in leaves of the same species was 0.63 µg g-1 and 0.43 µg g-1, respectively. different level of cadmium was also observed in the foliage of p. pterocarpum collected from both polluted and less polluted sites of the city. the level of cadmium in the leaves of p. pterocarpum was in the range of 0.46-1.53 µg g-1. highest cadmium level (1.53 µg g-1) was detected fig. 2: level of lead and cadmium µg g-1 in the foliage of pongamia pinnata. all values are signifi cantly (p<0.05) different according to duncan’s multiple range test. sites a = m.a. jinnah road, b = shahrah-e-faisal, c = nazimabad, d = gulshan-e-iqbal, e = karachi university campus content-sing. – only one type of letters in the title biomonitoring of heavy metals in trees of karachi 123 at m.a. jinnah road, while the lowest was recorded at karachi university campus (0.46 µg g-1). cadmium level at shahrah-efaisal (1.06 µg g-1), nazimabad (0.70 µg g-1) and gulshan-e-iqbal (0.60 µg g-1) was in between m.a. jinnah road and karachi university campus. discussion there has been increasing a continuing interest in the study of heavy metal content of roadside trees. current research reports found high levels of lead and cadmium contents in the foliage of plants species growing in the urban environment of city. the pb content in this study was found in the range of 7-106 µg g-1 with great differences among sites and plant species. in the present study the concentration of lead in leaves sample of p. pinnata and p. pterocarpum was found high at the polluted site as compared to karachi university campus. this high level of lead at polluted site might be due to presence of high lead additive compounds used in petrol. the concentration of pb in its petrol reported in 1991 was the highest (1.5-2.0 g pb l-1) of all produced by the various asian countries and far exceeded who guide lines of of 0.15 g pb l-1 (parekh et al., 2002). a complete monitoring data on both metals is sparse. however, it should be noted that lead content of petrol are quite high (1.5-2.0 µg-l), which are above the guideline of world health organization (0.5-1.0 µg-l) (unep, 1992). furthermore, the lead-based anti-knock substance (tetraethyl lead) added to the petrol often being released into the atmosphere is deposited along the road-sides (kazmi et al., 2002), thereby raising the level of lead in plants. air borne pb is closely associated with the density and congestion of motor vehicle traffi c. it is therefore not surprising to see that pb concentrations in the leaf of trees growing at m.a. jinnah road was high as compared to leaf samples collected from other less polluted sites (shahrah-e-faisal, nazimabad, gulshan-e-iqbal and karachi university campus) of the city. accumulation and deposition of metals on the surface of leaves can increase the metal concentrations. the higher level of pb in the leaf of p. pinnata (107 µg g-1) was recorded at m.a. jinnah road, which might be due to the large surface area that is available for exposure to any pollutant. aksoy et al. (2000) found high level of pb (15.98177 µg g-1) in unwashed leaves sample of robinia pseudo-acacia l. growing along roadside. high concentration of pb in leaves sample of p. pterocarpum was also found at m.a. jinnah road, where the traffi c density was highest as compared to karachi university campus. the amount of lead potentially available to plants in any given locality obviously depends on the density of vehicles. low vehicular traffi c activities at the campus showed lowest lead contents for p. pinnata and p. pterocarpum. the cadmium level in the air is also an important component leading to the problems of environmental pollution. lagerwerff and specht (1970) have suggested that cadmium is found in lubricating oils as a part of many additives. the concentration of cadmium in leaf sample of tree species collected from m.a. jinnah road, shahrah-efaisal, nazimabad, gulshan-e-iqbal and karachi university campus showed a distribution similar to those obtained for lead. the data emphasized that motor vehicles traffi c load is a major cause of high level of cd content in leaves p. pinnata and p. pterocarpum collected from the polluted areas of the city. the fl ow of traffi c at m.a. jinnah road near quaid-e-azam tomb is quite slow due to the presence of traffi c signals. the area is rather congested with the result that pollutants emitted from the motor vehicles remained suspended in the atmosphere for some time and ultimately deposited on the surface of leaves. level of cadmium was found in the range of 2.960.26 µg g-1. maximum level of cadmium (2.96 µg g-1) was found in the foliage of p. pinnata at m.a. jinnah road, which is highly polluted site of the city. similarly aksoy and sahln (1999) had investigated high level of cadmium (1.38 µg g-1) in unwashed leaves of eleagnus angustifolia l. collected from the crowded parts of city center in kayseri (turkey). ara et al. (1996) had also found cd content in the leaves of ficus religiosa (0.04 ppm) and eucalyptus sp., (0.03 ppm) from some other polluted area of the city. these results indicate that metal aerosols after deposition on the leaf surface are responsible for increase in the level of cadmium in the city samples as compared to karachi university campus. moreover, in the present study it was observed that p. pinnata leaf sample collected from the polluted sites showed a tendency of higher concentration of lead and cadmium than p. pterocarpum. these atmospheric pollutants can play an important part in growth and development of plants growing in the polluted environment. the quantifi cation of such contaminants has come to a priority issues for the betterment of the environment. it might be also helpful in understanding the current level of available pollutant in order to raise the alarm about excessive atmospheric contaminants levels increasing due to automobile activities. the results reported here demonstrate that leaves of both tree investigated served as a good bioindicator and can be used in elemental air pollution monitoring studies in urban sites. the use of plants as a fig. 3: level of lead and cadmium µg g-1 in the foliage of peltophorum pterocarpum. all values are signifi cantly (p<0.05) different according to duncan’s multiple range test. sites a = m.a. jinnah road, b = shahrah-e-faisal, c = nazimabad, d = gulshan-e-iqbal, e = karachi university campus 124 m. shafi q, m.z. iqbal, m.s. arayne, m. athar complementary tool to traditional (instrumental) methods of studying atmospheric pollution from anthropogenic and natural sources became an established technique in the past 30-40 years because of the development of powerful analytical techniques (berlizov et al., 2007). a comprehensive review on the use of plants for airmonitoring purposes was given by mulggrew and williams (2000). bioindicators can be defi ned as organisms (a part of an organism or a population of organisms) which are able to give information’s on the quality (of a part) of its environment (figueiredo et al., 2007). heavy metals from vehicular emissions have continuously added to the pool of contaminants in the environment (hashisho and elfadel, 2004). in present investigation, a comparative study of the capabilities of both investigated tree species support that leaves of both tree species can be used for air pollution monitoring in urban sites, where severe environmental conditions are resulting due to automobile activities. in conclusion, the present study brings out the clear picture about the levels of trace metals present in the biological material collected from the different site of the city. the relatively higher levels of both metals are indicative of the fact that the local atmosphere is undergoing to an alarming situation for the plant health due to automobile activities. it is high time to evolve an air pollution abatement strategy to ward off people against the hazardous effects arising from elevated trace metal levels (shah and shaheen, 2007). metal contamination from automobile activities sources is an important environmental concern around the world and in pakistan. due to rapid urbanization and industrial development in recent years, atmospheric pollution has caused serious deterioration of the terrestrial environment in many countries. the result could be used as preliminary baseline data for trace elements concentrations in the ecosystems for future assessment and monitoring. environment friendly organization in public and governmental sectors, environment management worker, policymakers, scientists, educators and researcher are providing informative data on the biomonitoring of various types of pollutants. it is based on the sampling and analysis of the materials collected from the contaminated site. plant, soil, water and air samples are most commonly measured. this technique helps in investigating the level of contamination of pollutants in any given environment. the results of these measurements provide information and guide about the current level of pollutants in the environment. it is logical to conclude that heavy metals, which are discharged into the atmosphere through motor vehicles exhaust, are deposited on and penetrate into leaves. leaf at its various stages of development serves as a good indicator to air pollutants. pollutants derived from the auto-emission can directly affect the foliage of plants by entering the leaf, destroying individual cells, and reducing the plant ability to produced food. reduction in leaf length, width, area and dry weight of roadside plants was the witness of bad effects of the city environment. it is found that the plants growing close to the busy road of the city are highly affected by autoemission. the inhibitory effects on the growth of plants are due to the presence of toxic material in the auto emission. overall study reveals that, high level of lead and cadmium was found in the foliage of plants growing in the polluted city environment. however, high level of metals was detected in p. pinnata as compared to p. pterocarpum. it is therefore, suggested that p. pinnta should be given more preference over p. pterocarpum for further plantation in the city, particularly along the busy roads to lessen the burden of pollutants from the urban environment. continuous monitoring and measurements of heavy metal are required from plants growing adjacent to roadways. references akbar, k.f., headley, a.d., hall, w.h.g., athar, m., 2006: heavy metal contamination of roadside soils of northern england. soil and water res. 1, 158-163. aksoy, a., sahln, u., 1999: elaeagnus angustifolia l. as a biomonitor of heavy metal pollution. turk. j. bot. 23, 83-87. aksoy, a., ozturk, m., 1996: phoenix dactylifera l., as a biomonitor of heavy metal pollution in turkey. j. trace microprobe t. 14, 605-614. aksoy, a., sahln, u., duman, f., 2000: robinia psueo-acacia l. as a 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baghdad. water air soil poll. 19, 3-11. hashisho, z., el-fadel, m. 2004: impacts of traffi c induced lead emissions on air, soil and blood lead levels in beirut. environ. monitor. assess. 93, 185-202. iqbal, m.z., sherwani, a.k., shafiq, m., 1998: vegetation characteristics and trace metals (cu, zn and pb) in soils along the super highways near karachi, pakistan. stud. bot. hung. 29, 79-86. jaradat, q., momani, k., jiries, a., el-alala, a., batarseh, m., sabri, t.g., 1999: chemical composition of urban wet deposition in amman jordan. water air soil poll. 112, 55-65. kazmi, s.m.a., shaukat, s.s., khan, d., 2002: some physical and biochemical effects of automobile exhaust pollution on common roadside plants in karachi, pakistan. hamdard medicus xvl, 38-43. khalid, b.y., salih, b.m., issac, m.w., 1981: lead contamination of soil in baghdad city, iraq. bull. environ. contam. toxicol. 27, 634-638. lagerwerff, j.w., specht, a.w., 1970: contamination of roadside and vegetation with cadmium, nickel, lead and zinc. environ. sci. technol. 4, 583-586. mulgrew, a., williams, p., 2000: biomonitoring of air quality using plants, air hygiene report no 10, berlin, germany, february 2000, who cc. parekh, p.p., khwaja, h.a., khan, a.r., naqvi, r.r., malik, a., khan, k., hussain, g., 2002: lead content of petrol and diesel and its assessment in an urban environment. environ. monit. assess. 74, 255-262. biomonitoring of heavy metals in trees of karachi 125 qureshi, i.h., 2000: air pollution and human health. proc. pakistan acad. sci. 37, 109-117. shah, m.h., shaheen, n., 2007: statistical analysis of atmospheric trace metals and particulate fractions in islamabad, pakistan. j. hazard. mat. 147, 759-767. shah, m.h., shaheen, n., jaffar, m., saqib, m., 2004: distribution of lead in relation to size of airborne particulate matter in islamabad, pakistan. j. environ. manage. 70, 95-100. shafiq, m., iqbal, m.z., arayne, m.s., athar, m., 2011: alstonia scholaris r. br. and cassia siamea lamk as possible biomonitors of lead and cadmium in the polluted environment of karachi, pakistan. j. appl. bot. food qual. 84. in press. stewart, r.r., 1972: flora of west pakistan. in: nasir e. and ali s.i. (eds.), an annotated catalogue of the vascular plants of west pakistan and kashmir. fakhri printing press, karachi. unep, united nation environmental program, 1992: urban air pollution in mega cities of the world. blackwell publishers, oxford, uk wikipedia 2012: hydrocarbon. http://en.wikipedia.org/wiki/hydrocarbon #cite_note-0. visited on 31 jul. 2012 yousaf, z., alousufzaii, a.h.k., 1991: lead and heavy metals in the street dust of metropolitan city of karachi. pakistan j sci. ind. res. 34, 167-172. address of the author: dr. muhammad shafi q, department of botany, university of karachi, karachi-75270, pakistan prof. dr. muhammad zafar iqbal, department of botany, university of karachi, karachi-75270, pakistan prof. dr. m. saeed arayne, department of chemistry, university of karachi, karachi-75270, pakistan dr. mohammad athar, california department of food and agriculture, 3288 meadowview road, sacramento, ca 95832, usa current address: department of food science and technology, university of karachi-75270 pakistan e-mail: atariq@cdfa.ca.gov) art02_jansen.indd journal of applied botany and food quality 84, 6 10 (2011) 1julius kühn-institut, bundesforschungsinstitut für kulturpfl anzen, institut für resistenzforschung und stresstoleranz, quedlinburg, germany 2dieckmann gmbh & co. kg, nienstädt, germany rapid methods for selecting single kernels of waxy barley g. jansen1, f. ordon1, e. knopf 2, k. dieckmann2 (received may 21, 2010) summary the rapid estimation of the amylose content in waxy barley is of particular importance in breeding new varieties with a low amylose content and in controlling the amylose content in the industrial production process, e.g. milling companies. therefore, an amylose test measuring the quotient of the absorbance of the starch-iodine complex in the absorption maximum for amylose and for amylopectin was modifi ed and adapted for the determination in single kernels of commercial and waxy barley. to achieve this, single kernel were crushed, dissolved with 1n sodium hydroxide solution and stained with iodine-potassium iodide solution. next, the amylose iodine and the amylopectin iodine complex were measured colorimetrically in wells of microtitre plates (96 samples). the modifi ed two-wavelength iodine binding procedure was used as a standard chemical analysis for developing a non destructive near infrared (nir) method to distinguish single kernel of commercial barley and waxy barley. the calibration was calculated using the transmission spectra and the modifi ed partial least squares (mpls)-method. spectral transmission in a wavelength range between 900 nm and 1100 nm were measured in a cuvette holding 23 single kernels. the quality of the calibration was controlled by cross validation (coeffi cient of cross validation 1-vr = 0.936; standard error of cross validation secv = 0.020) and a prediction (coeffi cient of prediction rsq = 0.883; standard error of prediction sep [c] = 0.028) in the range of r-values between 0.63 and 0.86. applying this calibration facilitates an unequivocal differentiation between commercial and waxy barley genotypes on the single kernel level. introduction starch is one of the major polysaccharides in cereal grains. barley starch, like other cereal grains, comprises the components amylose and amylopectin. out of these, amylose is the minor component consisting of linear long chains of α-(1→4) linked d-glucose residues. in contrast, amylopectin is a highly branched polymer consisting of many α-(1→6) linked d-glucose side chains attached to the α-(1→4) polymer (mac gregor and bhathy, 1993). grains of barley normally contain about 20-30 % amylose (mac gregor and morgan, 1984; morrison et al., 1984). waxy barley, consisting nearly exclusively of amylopectin, contains according to banks (1970) only 0.4-13 % and according to morrison et al. (1984) 2-8 % amylose. many different procedures have been published for the determination of the amylose in starch using differential scanning calometry (mestres et al., 1996; moorthy et al., 2006; creek et al., 2007), capillary electrophoresis (herrero-hartinez et al., 2004), thermogravimetric analysis (stawki, 2008), high performance size exclusion chromatography (cheu and bergmann, 2007), potentiometric titration (schwank et al., 1990), and amperometric titration (larsen et al., 1953). a lot of these procedures use colorimetric methods (mahmood et al., 2007), such as “blue-value” (sowbhagya and mysore, 1971; knutson and grove, 1994; anaro et al., 2009), and “r-value” (hovenkamp-hermelink et al., 1988; hovenkamp-hermelink et al., 1988; hovenkamp-hermelink haase, 1993). the blue iodine-amylose-complex is measured at an absorption maximum at approximately 620 nm (stark and yin, 1986). the amylopectin reacts weakly with iodine at about 530-550 nm and gives a red coloured complex (mac gregor and bhathy, 1993). most of the described methods are very time consuming and it is necessary to isolate the starch or to mill the grains for the determination of amylose. with the release of the waxy barley “waxyma” in europe in 2008 the determination of amylose content in barley became an important issue for breeders and industries. the aim of this work is therefore to develop a new simple and rapid method for the determination of amylose and amylopectin in single barley grains based on a non destructive nir-technique. material and methods plant material analyses were carried out using the waxy barley cultivar “waxyma”, new lines of waxy barley as well as the varieties 'lomerit', 'action' and 'malwinta' supplied by dieckmann gmbh & co. kg nienstädt, germany. additional barley cultivars (nearly twenty varieties) were used to develop the nir calibration. these cultivars were grown under fi eld conditions in groß lüsewitz, germany, in 2008 and 2009 applying cultural management practices common for the region. whole meal sample preparation barley awns were removed and the grains were ground in a falling number mill (laboratory mill 3100, perten) to pass a 0.8 mm sieve (flamme et al., 1999). starch sample preparation in laboratory scale the starch was prepared using a glutomatic system (perten instruments) combined with a fl ow stream channel, described previously (flamme et al., 1999). isolated starches had a high purity with lower than 0.3 % raw protein. amylose measurement using amperometric titration amperometric titration method was used as the standard method to determine the iodine binding capacity of isolated barley starches mainly according to larson et al. (1953) and richter et al. (1968). the procedure was modifi ed as follows: 40 mg of isolated starch was put into a volume of 1.0 ml of distilled water to swell the starch granules over night. next 1.0 ml of 2n sodium hydroxide solution was added while stirring constantly. the starch was extracted for 2h in a refrigerator and then the solution was neutralized with 1n hydrochloric acid and fi lled to a volume of 25 ml with distilled water. this polysaccharide solution was used for the titration with iodinepotassium iodate solution (1.78 g kio3 + 19.36 g ki + 0.84 g nahco3 were dissolved in 1 l of distilled water, titration solution: tenfold dilution of the above solution). selection of waxy barley 7 for amperometric titration an automated titrator (orion 960) equipped with a special beaker and a double platinum electrode for karl-fischer-titration was used. ten ml of the starch solution were taken into the beaker and dissolved with 30 ml distilled water. adding of the titration solution was carried out in steps of 0.2 ml. after fi nishing the titration, an automatic evaluation was performed using the fi rst derivation of the titration curve. then the amylose content was calculated with the iodine binding capacity of amylose of 19.5 g of iodine per 100 g polysaccharide (banks et al., 1974). amylose measurement in single barley grains using a twowavelength colorimetric method 1. modifi ed method with perchloric acid as solvent: barley grains were sliced in two halfes with a razor blade. the half without the embryo was milled in a swing mill (retsch). then a spatula tip of meal was extracted in perchloric acid according to hovenkamp-hermelink (1988). after staining with ihovenkamp-hermelink (1988). after staining with ihovenkamp-hermelink 2-ki solution the absorbance at 620 and 550 nm was measured. the ratios of the absorbencies (r-value) were used to differentiate between waxy and non waxy barley grains. 2. modifi ed method with sodium hydroxide as solvent: according to hovenkamp-hermelink (1988) and hovenkamp-hermelink (1988) and hovenkamp-hermelink haase (1993) the ratio of absorption at two wavelengths was used to estimate the amylose content. the method was modifi ed as follows: barley grains were divided into two halves with a razor blade. the half without the embryo was crushed and extracted over night at room temperature with 2 ml of 1n sodium hydroxid solution. samples were permanently stirred in glass tubes using a magnetic stirrer. after dissolving with distilled water and neutralisation with 1n hydrochloric acid the starch containing solution was fi lled to a volume of 100 ml. then 0.1 ml of the starch solution was stained with 0.1 ml of iodine solution (5.08 g ki and 2.54 g i dissolved in 1l distilled water, fourfold dilution of the above solution). staining was carried out in microtitre plates in a 96well formate (hu et al., 2010) and the measuring of the absorbance at 620 and 550 nm was conducted in a microtitre plate photometer (anthos ht iii). the knowledge of the r-value enables us to differentiate between waxy (r-value ~ 0.7) and non waxy barley grains (r-value ~ 0.9). amylose measurement using nir for nir measurement awns were removed and transmission spectra were measured with an infratec 1255 (fa. foss) using a wavelength range between 900 nm and 1100 nm. the device was equipped with a special cuvette. in this cuvette it was possible to analyse 23 single kernels in one rotation of the cuvette. near infrared transmittance spectra were collected from 1104 single barley kernels of different genotypes with normal and low amylose content. as standard chemical method, the modifi ed two-wavelength colorimetric method for single kernels in microtitre plates was used as described above. the spectra and analytical data were separated into 108 samples (calibration set) and into 996 samples (prediction set). every tenth sample was selected for the calibration set. using the mpls-method the calibration was calculated between spectra data and data of chemical analysis. statistical analysis all tests were repeated twice. the error between the duplicates was lower than three percent. all statistical computations were made using sas version 9.2 (sas institute, inc., cary, nci, usa). correlation analysis was conducted using pearson's correlation test. differences between methods were assessed using the tukey-test. results the amperometric method was performed to measure the amylose content in conventional and in waxy barley. titration curves of different barley varieties are shown in fig. 1. fig. 1: amperometric titration curves and fi rst derivative of the waxy barley 'waxyma' and the standard barley 'lomerit'. the consumption of ki-kio3-solution was measured by using the fi rst derivative of titration curve. amylose contents were calculated on the basis of the iodine binding capacity of amylose. different amylose contents and starch contents of barley varieties are given in tab. 1. whereas 'waxyma' showed an amylose content of approximately 5 %, the amylose content of conventional barley varieties varied between 20.3 % and 24.1 % amylose. in tendency, the starch content of cultivar 'waxyma' is a little bit lower in comparison to the starch content in the conventional variety 'lomerit'. several methods have been tested to extract and dissolve the starch of whole barley grains using the two-wavelength iodine binding procedure. out of these the extraction medium perchloric acid was not suited, because whole grains can not be dissolved (fig. 2) resulting in very low extinctions at 550 nm (amylopectin) and 620 nm (amylose). also the use of the 1n sodium hydroxide was not suited without stirring the solution. the best result was achieved, when whole grains were stirred over night (16 h) in 1n sodium hydroxide at room temperature, although the highest r-value was achieved, when using perchloric acid as extraction medium to dissolve the starch (tab. 2). when using perchloric acid as a solvent the barley grains have to be milled. nevertheless, both established two-wavelength colorimetric methods, as described above, had the ability to clearly separate waxy from non waxy grains. there was a signifi cant correlation (pearson correlation coeffi cient, r²= 0.79, n = 12) between r-values ����� ����� ����� ����� ���� ���� ���� ���� � � ��� � ��� � ��� � iod-addition [ml] el e k tr o d e v o lt a g e [m v ] ���� � ��� ���� ���� ���� fi rs t d e ri v at iv e ��������� ����� �������� ��������� ����� ��������� ����� ���������� �������� ����� ���������� ��������� tab. 1: starch content and amylose content of different barley varieties. variety starch content amylose content [% in dm] [%] 'waxyma' (harvest 2007) 59.17 4.6 'waxyma' (harvest 2008, altfeld) 67.42 5.5 'waxyma' (harvest 2008, bördegrün) 65.39 5.3 'lomerit' (harvest 2008) 70.22 23.3 'malwinta' (harvest 2008) 65.42 20.3 8 g. jansen, f. ordon, e. knopf , k. dieckmann determined with perchloric acid as solvent and the procedure with sodium hydroxide as solvent. r-values, determined with the solvent perchloric acid were signifi cantly higher (p = 0.0011) than r-values determined with the solvent sodium hydroxide (tab. 2). in breeding it is very useful to have a rapid non-destructive test to characterize waxy and non waxy barley. near infrared spectroscopy provides an alternative non-destructive technique for measuring the amylose content. results of calibration and prediction are given in fig. 3. mpls-method was conducted by correlating the spectra of the calibration set and the r-value determined by the two-wavelength colorimetic method. different mathematical pre-treatments of the raw spectra were tested and the calibration with the highest correlation coeffi cient (r²) and the lowest standard error of calibration (sec) was selected. an r² of 0.945 with a (sec) of 0.019 was found, when using non pre-treated grains. the quality of the calibration was then controlled by a cross validation resulting in an r² of 0.936 and the secv of 0.020. the calibration provided acceptable results for predicting the r-value in barley, showing a r² of 0.883 and a sep [c] of 0.028 in the prediction set. the r-value ranged between 0.63 and 0.86. the developed method can be used for the identifi cation of waxy and non waxy barley types by analysing single barley grain. however, for a further classifi cation within different waxy genotypes and non waxy genotypes this method is not well suited. discussion there are many different procedures to measure the amylose content in starch derived from plants resulting in problems of standardizing these methods for quantifying the amylose content. fitzgerard et al. (2009) highlighted the need to standardize the way amylose is measured in rice, because fi ve different versions of the iodine binding method are in use. the repeatability was high within laboratories but reproducibility between laboratories was low. zhu et al. (2008) compared various amylose determination methods from different starch sources (corn, rice, wheat, and potato) to conclude, that each amylose determination method has its benefi ts and limitations. a two-wavelength iodine binding procedure seemed to be the most precise and generally applicable method. in a study of hu et al. (2010) the colorimetric iodine-potassium-iodide and the enzyme-based megazyme methods (gibson et al., 1997) for amylose-detection in cereal grains were compared. amylose detection ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� ���� 550 nm 620 nm e x ti n c ti o n �� ���� ������� �������� �� ���� ���� �������� ��� �� �� ���������� ���� fig. 2: extinction of the amylose iodine and the amylopectin iodine complex at 620 and 550 nm using different solvents for whole grains. tab. 2: r-values of barley lines (harvest 2009), determined with perchloric acid and sodium hydroxide as grain solvent. line r-value r-value (solvent perchloric acid) (solvent sodium hydroxide) 1 1.213 0.837 2 1.226 0.844 3 0.697 0.647 4 0.771 0.644 5 0.766 0.649 6 1.084 0.660 7 0.736 0.654 8 0.778 0.648 9 0.755 0.665 10 0.790 0.662 11 0.715 0.642 12 0.739 0.644 fig. 3: a: correlation between nit-spectra data and chemical analysis data (r-values) determined by the two-wavelength colorimetric method in single kernels. b: scatter plots of the analytical determined and predicted r-values for the mpls-model using nir. � � � � � � � � � ������ ������ ������ ������ ������ ������ ������ ������ ������ ��������������������� � ������������ ������������������ ������ ������ ������ ������ ������ ������ �� ��������������������� selection of waxy barley 9 accuracy, using the iodine-potassium method, was comparable with the megazyme method in samples with a low to medium amylose content. another problem in amylose determination is the dissolution of the starch components. mahmood et al. (2007) discussed that the solvent may also affect the r-values. they came to the conclusion that the cold sodium hydroxide method is an accurate method for a preliminary screen of a large number of samples. therefore, in this study the dissolution of samples with sodium hydroxide and the dual wavelength iodine binding technique was chosen and modifi ed in order to use it as a standard method to determine the amylose content in single kernels of waxy and normal barley grains. based on this standard method a non destructive nir-method was developed. delwiche and graybosch (2002) used near infrared spectroscopy to identify waxy wheat, and differentiate them from non-waxy, partial waxy and wild-type phenotypes, but a further differentiation among the non-waxy types was diffi cult. delwiche and graybosch (2007) used ground material and scanned different lines in nir refl ectance. ground material was also used by lee et al. (2007) for rapid prediction of amylose content of polished rice by fourier transform near-infrared spectroscopy. near infrared transmittance (nit) technique for predicting maize seed composition in single kernel was described by baye et al. (2006) for the determination of the protein, oil and starch content. up to now the prediction of the amylose content in whole grain maize (campbell et al., 1997) was only applicable to a sample containing many grains, but not to a single grain. dowell et al. (2009) used an automated single kernel near-infrared sorting system to separate single waxy wheat kernels from segregating breeding lines. as shown in this paper the nit-technique can also be successfully used in the classifi cation of single kernels of waxy and non-waxy barley varieties, thereby facilitating selection for the amylose content in large numbers of grain samples as this has been demonstrated already for maize (campbell et al., 1999) and wheat (dowell et al., 2009). besides this, this method is well suited to detect contamination of normal and waxy genotypes during transport and storage. however, the developed method is too vague for a further differentiation of single kernels within waxy and non waxy barley genotypes. acknowledgements the authors thank m. tobien for technical assistance. this work was supported by the federal ministry of food, agriculture and consumer protection via the agency for renewable resources (research project fkz: 22019308) references avaro, 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cereal chemists, st. paul. macgregor, a.w., morgan, j.e., 1984: structure of amylopectins isolated from large and small starch granules of normal and waxy barley. cereal cem. 61, 222-228. mahmood, t., turner, m.a., stoddard, f.l., 2007: comparison of methods for colorimetric amylose determination in cereal grains. starch/stärke 59, 357-365. mestres, c., matencio, f., pons, b., yajid, m., fliedel, g., 1996: a rapid method for the determination of amylose content by using differentialscanning calorimetry. stärke 48, 2-6. moorthy, s.n., andersson, l., eliasson, a.-c., santacruz, s., ruales, j., 2006. determination of amylose content in different starches using modulated differential scanning calorimetry. starch/stärke 58, 209-214. morrison, w.r., milligan, t.p., azudin, m.n., 1984: a relationship between the amylose and lipid contents of starches from diploid cereals. 10 g. jansen, f. ordon, e. knopf , k. dieckmann j. cereal sci. 2, 257-271. richter, m., augustat, s., schierbaum, f., 1968: ausgewählte methoden der stärkechemie. fachbuchverlag leipzig. schwank, u., jacobi, a., drawert, f., fischbeck, g., 1990: halbkornmethode zur bestimmung des amylosegehalts in gerste. starch/stärke 42, 86-89. sowbhagya, c.m., bhattacharya, k.r., 1971: a simplified colorimetric method for determination of amylose content in rice. die stärke 23, 53-56. stark, j.r., yin, x.s., 1986: the effect of physical damage on large and small barley starch granules. starch/stärke 38, 369-374. stawski, d., 2008: new determination method of amylose content in potato research. food chem. 110, 777-781. zhu, t., jackson, d.s., wehling, r.l., geera, b., 2008: comparison of amylose determination methods and the development of a dual wavelength iodine binding technique. cereal chem. 85, 51-58. address of the authors: gisela jansen, julius kühn-institut, bundesforschungsinstitut für kulturpfl anzen, institut für resistenzforschung und stresstoleranz, ot groß lüsewitz, rudolf-schick-platz 3, 18190 sanitz, germany dr. frank ordon, julius kühn-institut, bundesforschungsinstitut für kulturpfl anzen, institut für resistenzforschung und stresstoleranz, erwin-baurstraße 27, 06484 quedlinburg, germany dr. erich knopf, dieckmann gmbh & co. kg, kirchhorster straße 16, 31688 nienstädt, germany karin dieckmann, dieckmann gmbh & co. kg, kirchhorster straße 16, 31688 nienstädt, germany art17 journal of applied botany and food quality 82, 99 102 (2008) 1humboldt university berlin, institute for horticultural science, berlin germany, 1asection of urban horticulture, berlin, germany 1b section of tree production and nursery, 2landesforstanstalt eberswalde, section of forest development/monitoring, eberswalde, germany influence of the season on the salicylate and phenolic glycoside contents in the bark of salix daphnoides, salix pentandra, and salix purpurea n. förster1a, c. ulrichs1a, m. zander1b, r. kätzel2, i. mewis1a (received june 15, 2008) summary due to the benefits of herbal medicine and their wide range of application for human health, the usage of natural drug products, such as willow bark extract, has increased in the last few years. the principle active compounds of the drugs comprised primarily of willow bark are phenolic glycosides like salicylates. phenolic glycoside profiles of bark vary among species and between the seasons. to identify and preserve willow clones with high salicylate content for possible commercial usage at a later stage, we have screened three salix sp. in respect to their chemical profiles. the willow species analysed were: salix daphnoides, salix pentandra, and salix purpurea. these species had distinct phenolic glycoside profiles. the major salicylate of s. daphnoides and s. purpurea clones was salicortin, whereas the main compound of s. pentandra was 2’o-acetylsalicortin. according to the chemical profiles of 140 clones, seven independent clones of s. daphnoides and s. purpurea as well as four clones of s. pentandra with high phenolic glycoside contents were picked to study seasonal changes in bark chemistry. overall, the clones of s. daphnoides showed the highest mean salicylate and phenolic glycoside contents, followed by s. pupurea and s. pentandra. the secondary metabolite content of willow bark clones decreased during the vegetative season from march to june 2007 and further from june to july 2007. our study revealed that for optimum yield of phenolic glycosides the species, the clone, and the time of harvest during the season have to be taken in consideration. introduction herbal medicine products are dietary supplements that people take to improve their health. many herbs have been used for a long time because of their claimed health benefits. in recent years herbal medicine gained more and more interest, even within the scientific community. at the end of the 20th century, many medical studies published proved biotic effects of willow bark extracts. certain phenolic glycosides of salix bark, particulary salicin and its esters like tremulacin or salicortin, have been shown to relieve rheumatic disturbances, infections, and headache (biegert et al., 2004; chrubasik et al., 2000; chrubasik et al., 2001; lardos et al., 2004). these glycosides have non-inflammable, temperature-reducing, and pain-alleviating effects. adverse effects, like a negative influence on the aggregation of the thrombocytes or local lesions of the gastric mucosa caused by the synthetic medicine product acetylsalicylic acid (aspirin), have not been reported for salicin. phenolic glycosides present in bark vary among salix species (thieme, 1965; thieme, 1971; meier et al., 1985a). commercial extraction of phenolic glycosides from willow bark is only economical when the content reaches a minimum of 1% total salicin, according to the „european scientific cooperative on phytotherapy“ (escop). furthermore, it has to be taken into consideration that the phenolic glycosides content varies with the seasons (thieme, 1965; thieme, 1971). to identify and preserve willow clones with high salicylate content for possible recovery of certain phenolic compounds at a later stage, we have screened clones of salix daphnoides, salix purpurea, and salix pentandra in respect to their chemical profiles. a second object of the study was to analyse the seasonal variability of salicylate and phenolic glycoside contents in the salix species to identify the optimum harvest time for secondary metabolites. materials and methods origin and selection of plant material osier stacks of different willow clones were collected in the northeast of germany and the north-west of poland in april and may 2006 and stock planted in zepernick (berlin). bark samples of 140 collected clones were taken in december 2006. according to their chemical profiles seven independent clones of s. daphnoides (da7, da40, da52, da56, da58, da61, da64) and s. purpurea (pu9, pu10, pu11, pu12, pu15, pu24, pu25) as well as four clones of s. pentandra (pe11, pe12, pe13, pe23) with high phenolic glycoside contents were picked for further studies. to determine changes within phenolic glycosides within the beginning vegetation period, willow bark for chemical analysis was collected in march, june, and july 2007. extraction procedure and analysis the peeled bark was lyophilised in a christ alpha 14 at exerted pressure of 0.034 bars for two days up to 20°c. after milling of probes (retsch mm 301, total samples 12 g) for 1.5 min at 20000 hz the phenolic glycoside samples, 50 mg each, were extracted with 750 µl methanol. 100 µl of standard solution (resorcinol) was added to the first extraction. after conditioning the probes for 10 min in an ultrasonic bath (bandelin sonorex super rk 513) and centrifugation for 5 min and 6000 rpm (eppendorf centrifuge 5416) the supernatant was collected. the pellet was used for reextraction with methanol (500 µl, 80 %) twice more. the combined supernatants were concentrated in a vacuum concentrator (speed vac sc110, savant instruments) until a volume of 150 µl was remaining. the extracts were filled up to 1 ml with ultra pure water (milliq quality) and filtered by using spin-x tubes (corning, the netherlands). the obtained extracts were analysed by hplc. 10 µl of willow bark extracts were injected in a dionex p680a hplc system equipped with an asi-100 auto sampler and a pda-100 photodiode array detector. phenolic glycosides were separated on a narrow bore column (acclaim polar advantage c16: 3 µm, 120 å, 2.1 x 150 mm, dionex). eluents used for hplc analysis were: a) 2% tetrahydrofuran and 0.5% phosphoric acid and b) 100% methanol. the eluent programme was: 0% b (0 5 min), 15% b (10 min), 25% b (20 min), 35% b (30 min), 50% b (35 min), 100% b (40 42 min) and 0% b (44 49 min). the flow rate was 0.35 ml/min and the eluent was detected at 270 nm. qualitative identification of the phenolic glycosides was carried out by using standards and via specific uvspectra (shao, 1991). the quantitative analysis based on peak area 100 n. förster, c. ulrichs, m. zander, r. kätzel, i. mewis relative to the standard resorcinol by using respective response factors. for peak evaluation the software chromeleon version 6.0 was used. data were analysed for significant differences by using analysis of variance following the mean comparison test tukey’s hsd for honest significant difference with systat 12.0. results and discussion the chemical compounds identified in willow species were the salicylates salicin, salicortin, 2’-o-acetylsalicin, 2’-o-acetylsalicortin, and tremulacin and the other phenolic glycosides syrengin, picein, catechin, ampelopsin, vimalin, purpurein, and naringenin-5glycoside. furthermore, a not-yet identified salicin derivate (elution at circa 35 min) and an unknown phenolic glycoside (elution at about 18 min) could be detected. clones of s. daphnoides showed the highest mean salicylate and phenolic glycoside content, followed by s. pupurea and s. pentandra. every salix species had a characteristic phenolic glycoside profile. the major salicylate present in s. daphnoides and s. purpurea was found to be salicortin, whereas the main compound of s. pentandra was 2’-o-acetylsalicortin. type-chromatograms for clones of the three analysed willow species are given in fig. 1 3. initial total phenolic glycoside content determined in march for s. daphnoides was 8.24%, for s. purpurea 5.67%, and 3.25% for s. pentandra. the clone variability of secondary metabolites in willow species was found to be different with highest variability in s. daphnoides, followed by s. purpurea and s. pentandra (tab. 1). the secondary metabolites generally decreased from march to june 2007 and further from june to july 2007, whereby the the total phenolic glycosides decreased significantly in all species already from march to june (fig. 4). furthermore, the salicylate content of all species was significantly lower in july compared to march 2007. significant differences were found more frequently between march and june than between june and july. the salicylate and phenolic glycoside contents in clones of s. daphnoides, s. pentandra, and s. purpurea decreased about 30% from march to june (tab. 1). the salicylate contents of s. daphnoides and s. purpurea decreased further from 4.71% and 3.27%, respectively around 18% from june to july. also from june to july, the total phenolic glycoside contents decreased in s. daphnoides and s. purpurea around 22% and 14% from initially 5.14% and 4.07%. an increase of 8% from 2.15% phenolic glycosides content was detected in s. pentandra from june to july. but the salicylic content remained unchanged in these months in s. pentandra. in general it could be seen that the secondary metabolite contents decreased from march to june with more intensity than from june to july. our data collected for the seasonal variability are proof of the results reported by meier et al. (1985a, 1985b). they found that the salicylate and phenolic glycoside content decreased from winter to summer. furthermore, our results show the same trend as documented by thieme (1965, 1971). he found that the maximum content of phenolic fig. 3: chromatogram of salix purpurea bark (clone 9, march 2007) 0,0 2,5 5,0 7,5 10,0 12,5 15,0 17,5 20,0 22,5 25,0 27,5 30,0 32,5 35,0 37,5 40,0 -50 500 1.000 1.500 30.04.2007 #58 n201 s. purpurea pu9 (2) uv_vis_1 mau min s a lic in r e s o rc in s y re n g in 2 'o -a ce ty ls a lic in s a lic o rt in p h e n o lg lu c o s id a m p e lo p si n v im a lin 2 '-o -a c e ty ls a lic o rt in p u rp u re in t re m u la ci n s a lic in d e ri v a t w vl:270 nm fig. 2: chromatogram of salix pentandra bark (clone 11, march 2007) 0,0 2,5 5,0 7,5 10,0 12,5 15,0 17,5 20,0 22,5 25,0 27,5 30,0 32,5 35,0 37,5 40,0 -50 500 1.000 30.04.2007 #38 n181 s. pentandra pe11 (2) uv_vis_1 mau min s a lic in r e so rc in s yr e n g in 2 'o -a ce ty ls a lic in c a te ch in s a lic o rt in p h e n o lg lu co si d a m p e lo p si n v im a lin 2 'o -a ce ty ls a lic o rt in p u rp u re in wvl:270 nm fig. 1: chromatogram of salix daphnoides bark (clone 52, march 2007) 0,0 2,5 5,0 7,5 10,0 12,5 15,0 17,5 20,0 22,5 25,0 27,5 30,0 32,5 35,0 37,5 40,0 -50 500 1.300 30.04.2007 #12 n155 s. daphnoides da52 (1 uv_vis_1 mau min s a lic in r e so rc in s yr e n g in 2 'o -a c e ty ls a lic in c a te c h in s a lic o rt in p h e n o lg lu co si d a m p e lo p s in v im a lin 2 'o -a ce ty ls a lic o rt in n a ri n g e n in -5 -g lu co si d p u rp u re in t re m u la ci n s a lic in d e ri v a t wvl:270 nm phenolic glycosides in salix bark 101 0 1 2 3 4 5 6 7 8 9 s. daphnoides s. pentandra s. purpurea c o n te n t [% ] sc march sc june sc july pgc march pgc june pgc july a a b a b b a b b a b b a a b a b b glycosides could be detected during spring, in march. the content of phenolic glycosides decreased from spring over summer to come to a minimum in september/october. in autumn and winter, an accumulation of these compounds was found by thieme (1965, 1971). further research in our institute about the seasonality of phenolic glycoside contents of willow bark in autumn and winter is ongoing. several compounds in the three willow species restrained differently in their seasonal development. from march to june most of the compounds in s. daphnoides decreased, especially purpurein, tremulacin, ampelopsin, catechin, syringin, salicin, 2’-o-acetylsalicortin. in some clones, a few compounds like tremulacin, catechin or purpurein could not be detected anymore. naringenin-5-glycoside showed an increase from march to june but a decrease from june to july. another decreasing compound was salicortin. syrengin, ampelopsin, purpurein, tremulacin, and 2’-o-acetylsalicortin increased from march to july. in s. pentandra from march to june decreasing concentrations of purpurein, syrengin, 2’-o-acetylsalicin, 2’-o-acetylsalicortin, as well as vimalin and increasing concentrations of salicin, picein, and salicortin could be detected. from june to july some compounds like 2’-o-acetylsalicortin, syrengin, vimalin, purpurein increased, and some compounds like picein, salicortin, and ampelopsin decreased. in s. purpurea the content of syrengin, picein, 2’-o-acetylsalicin, ampelopsin, vimalin, purpurein, and tremulacin declined from march to june, whereas the content of catechin and 2’-o-acetylsalicortin increased. from june to july decreasing concentrations of 2’-o-acetylsalicortin, purpurein, and salicortin and increasing concentrations of vimalin and tremulacin could be found. a general, clear trend could not be found. only some compounds like syrengin, 2’-o-acetylsalicin, ampelopsin, vimalin, purpurein, and tremulacin decreased in all three willow species from march to june. from june to july an increasing content of syrengin, vimalin, and tremulacin and a decreasing content of salicortin could be detected. the production of willow bark extracts is very expensive. therefore, the use of willow species and clones with highest amounts of salicylates is obligatory. furthermore, we had to select individual clones from which maximum harvests per hectare can be gained, e. g. with fast growth performance (riipi et al., 2002). besides the bark, other plant organs of willow, like the leaves contain phenolic glycosides (julkunen-tiitto, 1992; thieme 1965). usage of these parts for phenolic glycoside production could be also considered. there are other factors which influence secondary metabolite content in willow bark, such as the species, the clone (julkunen-tiitto, 1992), the climate (meier et al., 1985b; julkunen-tiitto, 1985), the nutrient supply of the plant (hakulinen, 1998; julkunen-tiitto et al., 1993), the physiological age (julkunen-tiitto, 1992; thieme, 1965; fig. 4: seasonal variability of salicylates and phenolic glycosides in the bark of willow species (different small letters indicate seasonal significant differences of salicylate contents within the species and large letters indicate differences within levels of phenolic glycosides, tukey‘s hsd test, p < 0.05; salicylate: sc and phenolic glycoside: pgc). tab. 1: salicylate (sc) and phenolic glycoside (pgc) contents in salix daphnoides, salix pentandra, and salix purpurea bark in march, june, and july salicylate and phenolic glycoside content [%] salix daphnoides salix pentandra salix purpurea sc pgc sc pgc sc pgc march 6.41 8.24 2.41 3.25 4.69 5.67 ± 1.71 ± 1.96 ± 0.27 ± 0.14 ± 0.66 ± 0.68 june 4.71 5.14 1.84 2.15 3.27 4.07 ± 1.31 ± 1.48 ± 0.27 ± 0.35 ± 0.74 ± 0.77 july 3.85 4.01 1.80 2.32 2.67 3.52 ± 0.68 ± 0.84 ± 0.16 ± 0.18 ± 0.53 ± 0.49 102 n. förster, c. ulrichs, m. zander, r. kätzel, i. mewis thieme, 1971; sulima et al., 2006), the time of day (thieme, 1971), and the gender (thieme, 1965). all parameters need to be considered for effective commercial recovery of phenolic glycosides. acknowledgements the project was funded by ble (bundesanstalt für landwirtschaft und ernährung). references biegert, c., wagner, i., lüdtke, r., kötter, i., lohmüller, c., günaydin, i., taxis, k., heide, l., 2004: efficacy and safety of willow bark extract in the treatment of osteoarthritis and rheumatoid arthritis: results of 2 randomized double-blind controlled trials. the journal of rheumatology 31, 2121-2130. chrubasik, s., eisenberg, e., balan, e., weinberger, t., luzzati, r., conradt, c., 2000: treatment of low back pain exacerbations with willow bark extract: a randomized doubleblind study. the american journal of medicine 109, 9-14. chrubasik, s., künzel, o., black, a., conradt, c., kerschbaumer, f., 2001: potential economic impact of using a proprietary willow bark extract in outpatient treatment of low back pain: an open non-randomized study. phytomedicine 8, 241-251. hakulinen, j., 1998: nitrogen-induced reduction in leaf phenolic level is not accompanied by increased rust frequency in a compatible willow (salix myrsinifolia)melampsora rust interaction. physilogia plantarium 102, 101-110. julkunen-tiitto, r., 1992: analyse und verteilung phenolischer glycoside (salicinderivate) in weiden (salicaceae). die holzzucht 46, 2-5. julkunen-tiitto, r., 1985: chemotaxonomical screening of phenolic glycosides in northern willow twigs by capillary gas chromatography. journal of chromatography 324, 129-139. julkunen-tiitto, r., tahvanainen j., silvola j., 1993: increased co2 and nutrient status changes affect phytomass and the production of plant defensive secondary chemicals in salix myrsinifolia (salisb.). oecologia 95, 495-498. lardos, a., schmidlin, c.b., fischer, m., ferlas-chlodny, e., loniewski, i., samochowiec, l., musial, h.d., 2004: wirksamkeit und verträglichkeit eines wässrig ausgezogenen weidenrindenextraktes bei patienten mit hüftund kniearthrose. zeitschrift für phytotherapie 25, 275-281. meier, b., lehmann, d., sticher, o., bettschart, a., 1985a: identifikation und bestimmung von je acht phenolglykosiden in salix purpurea und salix daphnoides mit moderner hplc. pharmaceutica acta helvetiae 60, 269-275. meier, b., sticher, o., bettschart, a., 1985b: weidenrinden-qualität. gesamtsalicinbestimmung in weidenrinden und weidenpräparaten mit hplc. deutsche apotheker zeitung 125, 341-347. riipi, m., ossipov, v., lempa, k., haukioja, e., koricheva, j., ossipova, s., pihlaja, k., 2002: seasonal changes in birch leaf chemistry: are there trade-offs between leaf growth and accumulation of phenolics? oecologia 130, 380-390. shao, y., 1991: phytochemischer atlas der schweizer weiden. zürich 1991, diss. eth nr. 9532. sulima, p., przyborowski, j.a., wiwart, m., 2006: willow bark – herbal raw material harvested from plants cultivated on arable lands. herba polonica 52 18-25. thieme, h., 1965: die phenolgykoside der salicaceen 5. mitteilung: untersuchungen über die gykosidspektren und den glykosidgehalt der mitteldeutschen salixarten. pharmazie 20, 570-574. thieme, h., 1971: vorkommen und verbreitung von phenolglucosiden in der familie der salicaceen. herba polonica 17, 248-257. address of the authors: dr. inga mewis, prof. dr. dr. christian ulrichs and nadja förster, institute for horticultural science, section urban horticulture, humboldt university berlin, lentzeallee 55-57, d-14195 berlin, germany. dr. matthias zander, institute for horticultural science, section tree production and nursery, humboldt university berlin, lentzeallee 75, d-14195 berlin, germany. dr. ralf kätzel, eberswalde forestry research institute of the state of brandenburg, section forest development/monitoring, landesforstanstalt eberswalde, alfred-möller-straße 1, d-16225 eberswalde, germany. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 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/pagesize [907.087 680.315] >> setpagedevice art03 journal of applied botany and food quality 82, 15 20 (2008) 1institute of botany, academy of sciences of the czech republic, pruhonice, czech republic 2institute for environmental studies, faculty of science of the charles university, czech republic different effect of mycorrhizal inoculation in direct and indirect reclamation of spoil banks j. rydlová1, d. püschel1, m. vosátka1, k. charvátová2 (received april 24, 2008) summary spoil banks generated during coal mining are usually reclaimed by layering of fertile soil over original barren clay (co called indirect reclamation). this well-proven method is effective from the aspect of vegetation establishment and production, but it is very expensive. direct reclamation of spoil bank clay promises much cheaper approach, yet its success is uncertain and the process might be rather long-term. this two-year field study aimed to assess the effect of application of commercially produced inoculum of arbuscular mycorrhizal fungi (amf) symbivit® on growth of two plant species commonly used for reclamation (lotus corniculatus and arrhenatherum elatius) sown on three different substrates: organic substrate (mixture of papermill waste, tree-bark and compost) and loess (both substrates typical for indirect reclamation) and original spoil bank clays (simulation of direct reclamation). on organic substrate and loess, a. elatius outcompeted the legume and established 100 % cover in all treatments. the effect of mycorrhizal inoculation was not observed. in contrast, on clay both species established successfully. the produced biomass and cover were, however, substantially lower compared to organic substrate and loess. in clay the positive effect of introduced amf on plant was observed. mycorrhizal inoculation was useful for supporting plant growth at direct reclamation. direct reclamation in itself seems suitable for small-scale application, i.e. in patches where indirect reclamation is inconvenient or more diverse vegetation is required. key words: arbuscular mycorrhizal fungi; inoculum; clay; papermill waste; loess; arrhenatherum elatius; lotus corniculatus introduction large areas of spoil banks are created as a consequence of the extensive open-cast coal mining in the northern part of the czech republic. spoil banks consist mostly of grey miocene clays, which had to be put aside in order to access coal. while the majority of spoil banks created in past decades have already been successfully reclaimed, new spoil banks are still created as the mining continues. natural spontaneous revegetation is not only surprisingly swift, but the established ecosystems are more "close to the nature" (in terms of species composition and general look of the landscape) than the sites reclaimed by human (prach, 2003; hodacová and prach, 2003). although the reclamation procedures could, therefore, seem unnecessary, two reasons justify it. firstly, a legislative obligation requires the mining companies to reclaim the areas disturbed by their mining activity within certain time. secondly, the mining-affected sites differ in their characteristics, e.g. soil properties. while some substrates enable fast establishment of plant cover, other substrates are less suitable for spontaneous revegetation. some of spoil bank miocene clays have low ph, fertility and drainage ability and high vulnerability to erosion. therefore, spontaneous plant succession is limited on these sites. for both reasons, the reclamation of spoil banks will probably remain an important restoration procedure in mining regions. importantly, reclamation of spoil banks is quite costly process. the current reclamation practice usually avoids direct reclamation (i.e. reclamation of original spoil bank clays). before the standard planting of trees or agricultural management is performed, the original substrates are usually covered by a layer of organic matter (a mixture of lignocellulose papermill waste, tree-bark and compost). this material is not only suitable for plants in terms of nutrients availability, but it provides also anti-erosion stabilisation of slopes due to its physical structure. alternatively, loess is applied as a topsoil horizon on original clays. loess is soil of relatively high quality, which was stockpiled when the coal seams were exposed for mining and which could be later re-applied to the sites when the spoil banks are re-claimed. despite the majority of spoil bank surface is treated as demonstrated, marginal sites and non-reclaimed patches of original clays are exposed to the process of natural plant succession. spontaneous plant succession on various mining-affected sites, including coal mine spoil banks, has been monitored for decades (prach, 1987; skousen et al., 1994; jochimsen, 1996; wiegleb and felinks, 2001). the first plant colonizers can be found already one year after spoil bank formation. after approximately 12 years, the annuals or biennials (belonging mostly to the families chenopodiaceae, brassicaceae, polygonaceae and asteraceae) are replaced by perennial communities with dominant forbs. later, communities with dominant grasses are established (prach, 1987). such progress of plant succession on spoil banks could be significantly affected by amf – arbuscular mycorrhizal fungi (allen and allen, 1980; rydlová and vosátka, 2001), which can provide important benefits for survivorship and growth of plants, mostly due to the increased uptake of nutrients (smith and read, 1997). furthermore, specific composition of amf can affect the structure of plant communities on spoil banks (püschel et al., 2007). initially, the population of amf on fresh spoil banks or other disturbed ecosystems is very low (allen and allen, 1980; waaland and allen, 1987; mott and zuberer, 1987). the speed of subsequent development of amf is probably determined by multiple factors, e.g. by the distance of the sources of amf propagules and possible means of their dispersion (wind, animals etc.), by the climate and by the presence of suitable host plants. the development of amf and progress of plant succession are likely contingent on each other. it can be generalised that plant species of later stages of succession can profit from mycorrhizal symbiosis in contrast with certain species of the early stages (mostly of the nonmycorrhizal families chenopodiaceae and brassicaceae). in the consequence of the development of amf in the soil, non-mycorrhizal plants are out-competed by mycorrhizal plants that pre-dominate the site (allen and allen, 1984). a two-year field experiment was designed to verify the hypothesis whether mycorrhizal inoculation or application of slow-release biologic fertiliser guarantees sufficient survivorship and growth of sown plants during direct reclamation of spoil banks. if this practice turned to be successful, it could become an alternative approach to o 16 j. rydlová, d. püschel, m. vosátka, k. charvátová standard costly indirect reclamation, when huge quantities of soil or organic material are transported and applied on spoil banks. material and methods the two-year field experiment on the coal-mine spoil bank vršany (north-bohemian coal basin, the czech republic) was set up in september 2004 just after the technical phase of reclamation was finished. the experimental area was divided into three equal blocks with different substrates. while the first block was left with original spoil bank miocene clay, the second and third block were covered by 20 cm thick layer of either organic substrate (a mixture of lignocellulose papermill waste, tree-bark and compost) or loess, respectively. chemical characteristics of the substrates are given in tab. 1. each) in each plot. in each sampling square, plants were cut 2 cm above the soil surface, dried at 70 °c to constant weight and weighed. however, this methodology could not be applied for the third block (original clay). due to extremely poor growth of both sown plant species in clay in the first year, the total plant biomass of each plot had to be harvested in order to obtain at least basic data (though statistically untreatable). in the second year, plant growth performance on clay improved, but the vegetation was very heterogeneous and it was still impossible to use the method of randomly distributed sampling squares. for that reason the plots on clay block were divided into 36 square "sub-plots" (1 m2 each) and the biomass was completely harvested from each sub-plot and statistically treated as replicates. cover vegetation cover was visually estimated either for the whole plot (for both years in blocks layered with loess or organic substrate, for 2005 in block with original clay), or the cover was individually scored for each of 36 sub-plots (on clay block in 2006). mycorrhizal colonisation for assessment of mycorrhizal colonisation of plants on organic substrate and loess, one mixed root sample (consisting of roots of five plants) was obtained for each of five sampling squares per plot. on clay, 5 root samples for both a. elatius and l. corniculatus were collected from each plot. the diversified character of vegetation in 2006 allowed more complex sampling methodology: the two species were sampled not only when growing individually (in patches isolated from each other) but also when growing together in close communities. in the laboratory, the roots were carefully washed from the soil, stained with 0.05 % trypan blue in lactoglycerol (koske and gemma, 1989) and the percent of root length colonised with amf was evaluated using a segment method under the compound microscope (giovannetti and mosse, 1980). mycorrhizal inoculation potential at the beginning of the experiment, all three studied substrates were sampled for assessment of initial mycorrhizal inoculation potential – mip (defined as the ability of soil to induce mycorrhizal colonisation of plant roots). then the sampling for mip was performed during each harvest. for organic substrate and loess, the samples of soil were obtained from the rhizosphere of plants in analogous process described for mycorrhizal colonisation of roots (see above). for clay, 25 soil samples per plot were randomly collected from the rhizosphere of established plants in 2005. diversified character of vegetation on clay in 2006 allowed evaluating possible effects of different types of vegetation on mip. therefore, in 2006 soil samples (5 samples per each plot) were collected from the rhizosphere of a. elatius and l. corniculatus growing separately, from the community of these plant species and from bare spaces without vegetation. the mip bioassays were performed on zea mays as the host plant with five replicates in each treatment. plants were grown in 180-ml pots in a greenhouse, watered daily with deionised water and supplemented with light from 400 w metal halide lamps for 12 hours a day. plants were harvested after four weeks, the roots were washed from the soil and stained with 0.05 % trypan blue in lactoglycerol (koske and gemma, 1989). the percentage of mycorrhizal colonisation of roots was evaluated under the stereomicroscope according to the gridline intersect method (giovannetti and mosse, 1980). tab. 1: chemical characteristics of the substrates in the field experiment. clay – original miocene clay from spoil bank, organic substrate – consists of lignocellulose papermill waste, tree-bark and compost, loess – original overburden soil. clay organic substrate loess ph (h2o) 6.25 7.32 7.92 ph (kcl) 6.13 7.15 7.64 c total [%] 2.58 6.80 12.48 n [%] 0.06 0.12 0.40 c/n 42 58 31 a p [mg·kg-1] 9.7 56.1 17.2 b mg [mg·kg-1] 778 992 775 b ca [mg·kg-1] 1 804 8 004 5 820 b k [mg·kg-1] 238 403 161 a olsen (1954) b mehlich (1978) on each of three blocks, four plots (36 m2 each) with different amendment treatments were established: 1) fertilised with 0.69 kg·m-2 of a long-term, slow release complete natural fertilizer conafer®, which is composed mostly of extracts of sea organisms, natural humates, ground minerals and rocks (producer symbio-m ltd., czech republic); 2) inoculated with amf in the form of 1.7 l·m-2 of symbivit® (producer symbio-m ltd., czech republic) containing reproductive particles (spores, mycelium and colonised root fragments) of 6 different glomus species on an inert carrier (mixture of slate, zeolite and expanded clay), 3) inert carrier lacking propagules of amf was applied in the same dose as in the previous plot (this amendment was used in order to distinguish the net effect of amf on the development of the plant cover and the effect of abiotic components of the inoculum), 4) unamended control plot. all plots were sown with seeds of the grass arrhenatherum elatius (l.) presl, and the legume lotus corniculatus l. at the dose of 2000 seeds of either species per one m2. the experiment was sampled and harvested in september 2005 and september 2006. the following parameters were examined: plant biomass in both years, vegetation growing in blocks covered either with organic substrate or with loess was very dense. the vegetation was represented only by the grass a. elatius, which out-competed l. corniculatus (see results). due to the dense cover, the plants were harvested in five randomly distributed sampling squares (0.5×0.5 m mycorrhizal inoculation in spoil bank reclamation 17 statistical analysis data showed gamma distribution (biomass) or binomial distribution (colonisation, mip) and were analysed using generalised linear models (s-plus 2000; mathsoft inc., usa) with substrate and amendment as independent variables. for evaluation of main effects of factors on mycorrhizal colonisation, only the data representing colonisation of a. elatius (as a plant species present on all three substrates) were included into the analysis. for evaluation of main effects of factors on mip, the data of vegetation-free spots were excluded from the analysis, because the presence of host plants is necessary precondition for amf development. results plant biomass although the seeds of both species germinated successfully already in the autumn 2004, no plants of l. corniculatus were found on organic substrate or loess at either of the two harvests (2005 and 2006). because a. elatius developed very dense cover already during the 1st growing season, l. corniculatus was out-competed by the fully engaged grass cover. at the end of the first growing season (2005), there was no effect of substrate or amendment on plant biomass; due to extremely low biomass, the plots in clay block were excluded from the analysis (tab. 2). in organic substrate and loess a. elatius developed 100 % cover and produced high amount of biomass (the average shoot dry weight was 653 g·m-2 and 853 g·m-2, respectively). in contrast, only isolated individual plants of a. elatius and l. corniculatus (mostly less than 5 % cover) with very low biomass grew on clay plots. the total biomass in clay ranged from 5 g·m-2 (unamended plot) to 59 g·m-2 (amendment with symbivit). in 2006, grass on organic substrate and loess formed similar biomass (average values 576 g·m-2 and 705 g·m-2, respectively), which was significantly higher as compared to clay (44 g·m-2). there was also a significant effect of interaction between substrate and amendment on biomass production (tab. 2). although the effect of amendment on plant biomass was not significant when evaluated for all 3 substrates together, for clay alone a strong significant effect of amendment on plant biomass and vegetation cover was found (chi square 211.87, p<0.001 and 86.21, p<0.001, respectively). both these parameters were highest after symbivit application and reached low values in unamended treatment (fig. 1a, b). mycorhizal colonisation mycorrhizal colonisation was significantly affected by substrate, by amendment and also by interaction of these two factors in both years; because l. corniculatus was present only in clay, the data of this legume were not included into the evaluation of the effect of factors (tab. 3). in 2005, root colonisation in organic substrate was negligible to zero. in loess and clay in 2005 and in all three substrates in 2006, colonised roots of plants were observed in all treatments, i.e. also in those plots that were not inoculated with symbivit. none the less, the inoculation with symbivit still generally increased mycorrhizal colonisation of a. elatius. in contrast, colonisation of l. corniculatus was not positively affected by symbivit amendment (tab. 3). in carrier-amended treatment on clay, the colonisation level of both a. elatius and l. corniculatus was significantly increased if those species grew in engaged community, as compared to isolated patches of individual plant species. mycorrhizal inoculation potential initial mip of the substrates, i.e. before the substrates were amended and sown with plants, was zero for organic substrate and clay and 4 % (of colonised roots in bioassay) for loess. during the experiment, mip was significantly affected by substrate, by amendment and by interaction of these two factors in both years (tab. 4). generally, organic substrate showed the lowest mip in both years, while the highest mip was found in clay. there was no clear trend in the effect of amendments on mip (tab. 4) due to strong interaction of factors. tab. 2: effect of the substrate (original clay, organic substrate or loess) and its amendment (natural slow-release fertiliser conafer®, commercial mycorrhizal inoculum symbivit®, carrier of mycorrhizal inoculum without living propagules of amf, unamended) on plant biomass according to generalised linear models. for year 2005, only the results for organic substrate and loess were included into statistic analysis due to poor growth of plants on clay (see text). chi-square values and probability levels are presented (*** p<0.001, ns – nonsignificant effect). year substrate (a) amendment (b) a × b chi square value 2005 0.48 0.46 0.45 significance ns ns ns 2006 255.07 254.3 212.9 *** ns *** 0 20 40 60 80 100 conafer symbivit carrier control s h o o t b io m a ss [g .m -2 ] bc a b c 0 5 10 15 20 25 30 35 conafer symbivit carrier control v e g e ta tio n co ve r [% ] b a b c a) b) fig. 1: effect of amendment (natural slow-release fertiliser conafer®, commercial mycorrhizal inoculum symbivit®, carrier of mycorrhizal inoculum without living propagules of amf, unamended control) on shoot biomass (a) and vegetation cover (b) of plants growing in clay at the end of the second growing season. columns marked with the same letter are not significantly different according to chi square test (p<0.05). data are means of 36 replicates. 18 j. rydlová, d. püschel, m. vosátka, k. charvátová tab. 3: effect of treatment (natural slow-release fertiliser conafer®, commercial mycorrhizal inoculum symbivit®, carrier of mycorrhizal inoculum without living propagules of amf, unamended) on mycorrhizal colonisation of plants growing on different substrates. for the evaluation of effect of factors, the data for l. corniculatus in clay were excluded, because this species was not present in organic substrate and loess. values in columns within each substrate or plant marked with the same letter are not significantly different according to chi square test (p<0.05). effect of factors according to generalised linear models (*** p<0.001). data are means of five replicates. the empty boxes mean that the data were not available due to the absence of plants on relevant sites. substrate plant amendment mycorrhizal colonisation [%] year 2005 year 2006 organic substrate a. elatius conafer 2 a 8 b symbivit 0 b 33 a carrier 0 b 7 bc unamended 0 b 5 c loess a. elatius conafer 9 b 74 b symbivit 33 a 81 a carrier 8 c 49 c unamended 4 d 78 ab clay a. elatius conafer 19 b 61 b symbivit 31 a 78 a carrier 12 c 32 c unamended 5 d 57 b a. elatius conafer 53 c (community) symbivit 76 b carrier 94 a unamended l. corniculatus conafer 36 c 93 a symbivit 27 c 84 ab carrier 56 b 56 c unamended 85 a 76 b l. corniculatus conafer 80 b (community) symbivit 88 ab carrier 90 a unamended factor chi square value/ substrate (a) 845.66 1086.45 *** *** significance amendment (b) 486.49 859.08 *** *** a×b 429.76 610.06 *** *** the structure of plant cover significantly influenced mip of clay substrate (chi square 1536.41, p<0.001). in most treatments, presence of l. corniculatus, either alone or as a component of a community, increased mip of the substrate in comparison either with the patches of a. elatius alone, or with the bare areas without vegetation. a. elatius even negatively affected mip in plot with inert carrier amendment and in unamended plot. discussion relatively high values of mycorrhizal colonisation and mip in treatments without application of amf, especially on clay, are indicative of surprisingly rapid natural dispersion of amf propagules to freshly created spoil banks. wind, soil erosion and small mammals are the possible dispersal agents that enable the amf to invade new areas (warner et al., 1987; allen and allen, 1984). the transport by wind was probably the most important way by which amf propagules dispersed on the experimental site. strong winds are very frequent on spoil banks and small soil particles containing amf propagules can be easily moved through the air from surrounding areas with established plant cover, especially during dry weather season. moreover, despite the experimental area was fenced to prevent access of deer and rabbits, smaller mammals (or insect) could migrate across the plots and transport soil particles containing spores or mycelium of amf not only from outside the plots, but also between the treatments. this factor, unfortunately, cannot be effectively eliminated in the field experiment. mycorrhizal inoculation in spoil bank reclamation 19 despite rapid natural colonisation of the study site by amf, the application of symbivit inoculum containing selected strains of amf significantly increased mycorrhizal colonisation of grass roots in all substrates. the introduced fungi were probably more infective as concerns the grass roots. however, quite different data were obtained for the legume, which was extensively colonised regardless of inoculation (i.e. amf naturally invading clay substrate successfully colonised roots of this plant species). yet, the effectiveness of the introduced amf was probably still higher as shown by significantly highest biomass and plant cover on clay plots treated with symbivit. application of the inert inoculum carrier and the fertilizer conafer also increased plant growth parameters on clay, but to a lower extend as compared to symbivit. inoculum carrier consisting of slate, zeolite and expanded clay might probably improve physical properties of the clay substrate, especially its aeration, while the fertilizer could contribute the to better plant performance due to its content of nutrients. organic substrate and loess showed substantially higher concentration of nutrients (especially p and n) than clay, thus the application of the fertilizer or inoculation with symbivit had no effect on plant growth. the amount of nutrients was probably sufficient to cover plant demands, thus mycorrhizal symbiosis was not beneficial for plants (bethlenfalvay et al., 1983). at the same time, mycorrhizal colonisation of a. elatius in organic substrate was very low and might have negligible effect on plant growth. the development of amf in organic substrate was restricted, which was also obvious from a low level of mycorrhizal inoculation potential. it is well documented that when applied in high doses, organic additives (such as compost, manure or sewage sludge) can be harmful to amf (sáinz et al., 1998; jensen and jakobsen, 1980; thorne et al., 1998), possibly due to toxic components contained in the organic matter (lambert and weidensaul, 1991). moreover, the establishment of mycorrhizal association and the development of amf could be also restricted by high concentrations of available p in this substrate (douds and schenck, 1990; vivekanandan and fixen, 1991). both of these explanations are probably applicable for our study, where plants were seeded and amf inoculum subsequently introduced directly into the layer of organic substrate. in other studies where positive effects of organic amendments on development of mycorrhizal symbiosis were detected (muthukumar and udaiyan, 2000, 2002; mäder et al., 2000; palenzuela et al., 2002), much lower (by one order) doses of organic matter were applied as compared to the dose used for reclamation of our spoil bank site. the initial mip of loess in our study, though very low, could be probably ascribed to the presence of infective amf propagules in this soil already before it was layered on the spoil bank. the amf can colonise loess during its storage in stockpiles, which are often overgrown with vegetation. in association with suitable host plants amf can proliferate in loess during this stockpiling stage and consequently can be dispersed on spoil banks. in both years, the lowest mip was detected in the organic substrate, where the development of amf was suppressed. in contrast, the highest mip was found in clay where any amendment probably led to amf proliferation and subsequent increase of mip. surprisingly, the application of symbivit had not always result in the highest mip in relevant plots. this could be probably related to rapid dispersion of amf propagules to the spoil bank from its surroundings (see above; püschel et al., unpublished results), which was reflected in increased ability of soil to induce mycorrhizae. however, the total number of prapagules is probably not the only important factor affecting mip of soil. based on results of a long-term field trial comparing organic and conventional farming systems, mäder et al. (2000) summarised that also soil properties such as nutrient content, biological activity, soil structure etc. are important factors, which can substantially affect the capacity of soil to initiate am symbiosis. there was a strong effect of plant cover on mip. presence of l. tab. 4: effect of the substrate (original clay, organic substrate or loess) and its amendment (natural slow-release fertiliser conafer®, commercial mycorrhizal inoculum symbivit®, carrier of mycorrhizal inoculum without living propagules of amf, unamended) on mycorrhizal inoculation potential (mip). mip is expressed as percentage of mycorrhizal colonisation of roots of bioassay plants. values in columns within each substrate marked with the same letter are not significantly different according to chi square test (p<0.05). effect of factors according to generalised linear models (*** p<0.001). data are means of five replicates. substrate amendment mip [%] year 2005 year 2006 organic substrate conafer 33 a 11 c symbivit 13 b 27 a carrier 0 d 2 d unamended 3 c 14 b loess conafer 40 b 60 a symbivit 47 a 45 b carrier 23 c 37 c unamended 10 d 45 b clay conafer 90 a 53 c symbivit 79 ab 68 a carrier 81 b 61 b unamended 34 c 29 d factor substrate (a) 4327.87 2955.26 chi square value *** *** / significance amendment (b) 2211.85 2670.64 *** *** a×b 999.46 2014.38 *** *** 0 10 20 30 40 50 60 70 80 90 conafer symbivit carrier control m ip (% ) d b c a k l m n p p q r x y z fig. 2: effect of different vegetation cover on mycorrhizal inoculation potential (mip) of clay substrate in different amendments (natural slow-release fertiliser conafer®, commercial mycorrhizal inoculum symbivit®, carrier of mycorrhizal inoculum without living propagules of amf, unamended control) at the end of the second growing season. mip is expressed as percentage of mycorrhizal colonisation of roots of bioassay plants. white columns – bare places with no plants, light grey columns – arrhenatherum elatius, dark grey columns – lotus corniculatus, black columns – community of a. elatius and l. corniculatus. in the control treatment, the two plant species grew separately and did not form a community. columns marked with the same letter are not significantly different within each amendment according to chi square test (p<0.05). data are means of five replicates. 20 j. rydlová, d. püschel, m. vosátka, k. charvátová corniculatus positively influenced the development of amf as compared to a. elatius. the different influence of the two plant species was probably caused by higher mycorrhizal dependency (van der heijden, 2003) of the legume. both substrates commonly used for spoil bank reclamation, loess and the organic substrate, are rich in nutrients, which provide suitable conditions for very rapid establishment of plant cover and formation of large plant biomass. however, plants with high competition abilities are likely to dominate such sites. sowing a mixture of several plant species can thus result in monoculture plant cover. direct reclamation of miocene clays cannot guarantee comparable high yields, regardless the application of amf inoculum. on the other hand, more diverse vegetation cover establishes on clay and the positive effect of inoculation on plant growth is evident in this substrate. while the results of this study would not change the prevalent trend of indirect reclamation into direct one, they indicate that, at some circumstances, the examined method of mycorrhizal inoculation can be applied in small-scale. in the patches of spoil banks, where layering of loess or organic substrate is inconvenient, the inoculation with amf will help to establish the cover of vegetation, which will probably have more diverse character compared to surrounding reclaimed sites. acknowledgement financial support for this study was provided by the ministry of education, youth and sports of the czech republic (grant no. 1m6798593901) and by the academy of sciences of the czech republic (grant no. av0z60050516). references allen, e.b., allen, m.f., 1980: natural re-establishment of vesiculararbuscular mycorrhizae following stripmine reclamation in wyoming. j. appl. ecol. 17, 139-147. allen, e.b., allen, m.f., 1984: competition between plants of different successional stages: mycorrhizae as regulators. can. j. bot. 62, 26252629. bethlenfalvay, g.j., bayne, h.g., pacovsky, r.s., 1983: parasitic and mutualistic associations between a mycorrhizal fungus and soybean: the effects of phosphorus on host plant-endophyte interactions. physiol. plant. 57, 543-548. douds, d.d., schenck, n.c., 1990: increased sporulation of vesiculararbuscular mycorrhizal fungi by manipulation of nutrient regimes. appl. environ. microbiol. 56, 413-418. giovanetti, m., mosse, b., 1980: an evaluation of techniques for measuring vesicular-arbuscular mycorrhizal infection in roots. new phytol. 84, 489500. hodacová, d., prach, k., 2003: spoil heaps from brown coal mining: technical reclamation versus 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revegetation of 15 abandoned mine land sites in west-virginia. j. environ. qual. 23, 1224-1230. smith, s.e., read, d.j., 1997: mycorrhizal symbiosis. academic press, london. thorne, m.e., zamora, b.a., kennedy, a.c., 1998: sewage sludge and mycorrhizal effects on secar bluebunch wheatgrass in mine spoil. j. environ. qual. 27, 1228-1233. van der heijden, m.g.a., 2003: arbuscular mycorrhizal fungi as a determinant of plant diversity: in search of underlying mechanisms and general principles. in: van der heijden, m.g.a., sanders, i.r. (eds.), mycorrhizal ecology, 225-242. springer, berlin. vivekanandan, m., fixen, p.e., 1991: cropping systems effects on mycorrhizal colonisation, early growth, and phosphorus uptake of corn. soil sci. soc. am. j. 55, 136-140. waaland, m.e., allen, e.b., 1987: relationships between va mycorrhizal fungi and plant cover following surface mining in wyoming. j. range manage. 40, 271-276. warner, n.j., allen, m.f., macmahon, j.a., 1987: dispersal agents of vesicular-arbuscular mycorrhizal fungi in a disturbed arid ecosystem. mycologia 79, 721-730. wiegleb, g., felinks, b., 2001: predictability of early stages of primary succession in post-mining landscapes of lower lusatia, germany. appl. veg. sci. 4, 5-18. address of the authors: dr. j. rydlová, dr. d. püschel, dr. m. vosátka, institute of botany, academy of sciences of the czech republic, 252 43 pruhonice, czech republic. bc. k. charvátová, institute for environmental studies, faculty of science of the charles university, benátská 2, 128 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/pagesize [907.087 680.315] >> setpagedevice art06_krueger.indd journal of applied botany and food quality 84, 40 46 (2011) 1department of pomology, geisenheim research center, geisenheim, germany 2department of wine analysis and beverage technology, geisenheim research center, geisenheim, germany effects of cultivar, yield, berry weight, temperature and ripening stage on bioactive compounds of black currants e. krüger1, h. dietrich2, m. hey2, c.-d. patz2 (received april 28, 2010) summary total anthocyanin, total phenols, ascorbic acid, antioxidant capacity (teac) were quantifi ed in fruit juice of 23 black currants cultivars grown at geisenheim (lat. 49.6°), germany, in the years 2003-2005. in addition, yield and 100-berry weight were recorded. there were remarkable variations in the phytochemical contents within the cultivars: 2.1-fold for teac and total phenols, 3.4-fold for total anthocyanins and even 4.4-fold for ascorbic acid. these differences are genetically determined but also signifi cantly modifi ed by plant physiology and environmental factors. the analysed compounds were negatively correlated with yield and 100 berry weight (teac: r = -0.34 and -0.43; total phenols: r = -0.34 and -0.48) while anthocyanins showed a signifi cant negative relationship only with the 100berry weight (r = -0.38). in contrast, teac, total phenol and total anthocyanin were signifi cantly positive correlated with the temperature during the fruit developing phase, expressed as degree days (teac: r = 0.36, total phenols: r = 0.33 and total anthocyanins: r = 0.51, respectively). the ascorbic acid content was neither infl uenced by the plant parameters yield and 100-berry weight nor by degree days. there were also remarkable yearly changes of the analysed parameters within individual cultivars caused by low yield in 2004. introduction in the last decade researchers got an increased awareness on health benefi cial effects of a plant-based diet in the prevention of various diseases associated with oxidative stress and some degenerative diseases. recent studies summarized the current knowledge on antioxidants and their possible benefi cial role on human health from epidemiological, in vitro and in vivo studies in general (among others beattie et al., 2005; manach et al., 2005; williamson and manach, 2005; duthie, 2007; mcghie and walton, 2007) and of black currants in particular (lister et al., 2002; karjalainen et al., 2009). black currants (ribes nigrum l.) are rich in antioxidants when compared to other fruit species (deighton et al., 2002; halverson et al., 2002). the major components are the ascorbic acid, a micronutrient which is essential for human health, and polyphenols with anthocyanins as the most abundant (kähkönen et al., 2001). within the anthocyanins, cyanidin-3-rutinoside and delphinidin3-rutinoside predominate, followed by cyanidin-3-glucoside and delphinidin-3-glucoside. the fl avonol glycosides of quercetin, myricetin and kaempferol are only present in much lower concentrations (herrmann, 2001). black currants are mainly grown for industrial use like making juices and nectars, jams and other products and only small amounts are consumed freshly. being native to central and northern europe and northern asia (brennan, 1996), main production area is still in this part of the world. for many fruit species it has been demonstrated, that there is a great infl uence of cultivar on antioxidant capacity and phenolic content, whilst for black currant only limited information is available, especially for a series of cultivars grown under the same growing condition. the study of moyer et al. (2002) reported on the phytochemical content of 32 black currant cultivars grown in oregon. likewise, giné bordonaba and terry (2008) analysed 17 cultivars or selections only from the scotch black currant breeding program. in both studies results are based on fruit matrix. only a limit number of cultivars were evaluated in most other studies. therefore, a comparison of the health benefi cial components in black currants between cultivars is diffi cult due different growing conditions and varying analytic methods. furthermore, there is limited knowledge on how environmental factors such as temperature and physiological characteristics like fruit size and yield may affect health functional compounds of black currants. a better knowledge of these factors would rise up the possibility to cultivate fruits with high levels of bioactive compounds benefi cial for human health. the aim of our study was therefore to analyse antioxidative capacity (teac), phenolics, anthocyanins and ascorbic acid in the juice of 23 black currant cultivars grown under the same conditions and to link the analytic data with environmental and plant parameters. material and methods plants 23 varieties of black currants (listed in tab. 2) were grown in an orchard at geisenheim research station (lat. 49.6°n) in rows with a plant distance of 1.50 x 3.00 m. fertilization, weed control, pest and disease management were done to the common standard of the region. irrigation was provided when necessary and soil moisture tension exceeded -350 hpa. fruit harvest and processing in 2003 to 2005, fruits from 5 to 8 plants per cultivar were picked when the majority of the berries per plant were sound, fully ripe and regarded as commercially ripe. overripe fruits were avoided by the natural fruit drop of dull black fruits. all fruits per plot were weighted. weight of 100 berries was determined as a surrogate for fruit size. after harvest, all fruits per variety were stored in plastic bags at -20 °c. for juice processing (after 2-3 month of storage), up to 5.0 kg fruits per cultivar were crushed in a disc-mill and heated to 50 °c. mash enzyme was added for a better degradation of pectin. after standing for 2 h, the mash was pressed (hp-2h, hafi co, fischer maschinenfabrik, neuss), fi lled into 330 ml brown glass bottles and heated to 90 °c for pasteurising. ripening stage for evaluating the infl uence of ripeness on fruit quality parameters, fruits of three cultivars were harvested at three different ripening stages from the same bushes: semi-ripe (one fourth of the berry surface still red-green), ripe (all berries black and fully ripe) and slightly overripe (some berries are shrivelling or nearly dropping down). for each sampling date, 1.0 to 1.5 kg were harvested per variety in 2005 and 2006. after harvest, fruits were treated as described before. analytical methods total phenols were analyzed with the folin-ciocalteu method (singleton and rossi, 1965) modifi ed by ritter (1994). results are expressed as (+)-catechin equivalents. the antioxidative capacity (teac) was determined according to re et al. (1999). data are given as trolox equivalent in mmol/l. the anthocyanins were analyzed after membrane fi ltration with hplc (dionex, p680 pump, autosampler asi-100, pda-100 photodiode array detector) on a lichrospher 100 rp 18 column (250 x 3 mm, 5 µm) with 4 mm precolumn. flow rate 0.5 ml/min, 25°c, 20µl injection volume. gradient elution was water/acetonitile/ phosphoric acid (94/4/2 v/v and 48/50/2 v/v), respectively. the anthocyanins were calculated as cyanidin-3-glucoside (extrasynthèse, france). ascorbic acid was analyzed potentiometrically according to ifu method no. 17 (anonym, 1964). weather and calculation of degree days the weather conditions at ripening time were quite different in the three years examined. especially, average temperature between may and july was lower in 2004 in comparison to the other two years (tab. 1). for analysing the infl uence of temperature in different years on the content of bioactive compounds in the fruit, temperature was transformed into thermal units according to lantin (1986). in short: a scheme gives two values for every daily minimum and maximum temperature, which have to be summarized to the daily thermal unit. an example for a given day: the minimum temperature of 7° c becomes a value of 1.3 and the maximum temperature of 18° c is transformed to 1.8. both values result in a thermal unit of 3.1. a calculation of degree days was made for the time between start of fruit colouring until harvest. vars, respectively. data of teac, total phenols, total anthocyanins, ascorbic acid, yield, berry weight and degree days were subjected to variance component analyses. missing values (3 for total anthocyanins and 7 for berry weight) were replaced by the calculated average of the individual cultivar for pca calculation. results and discussion infl uence of cultivar mean antioxidative capacity, total phenolics, total anthocyanins and ascorbic acid for all cultivars harvested in 2003 to 2005 are shown in tab. 2. cultivars are listed with increasing values of mean teac value. the ranking in mean total polyphenols among the cultivars were similar to those of teac, whereas greater rank changes occurred for total anthocyanins and ascorbic acid. for each parameter, changes were great between the cultivars with the highest and those with the lowest average content: 2.1-fold for teac value and total phenolics, 3.4-fold for total anthocyanins and even 4.4-fold for ascorbic acid. the infl uence of cultivars was signifi cant for all traits refl ecting the described differences between cultivars. the range of values found for antioxidant capacity, phenolic content and ascorbic acid in this study were similar to a previous study with the same sample preparation and juice processing method but with different cultivars (dietrich et al., 2000). the average value for teac (53.0 mmol/l) as an average of all cultivars and years exceeded widely the average of the fi rst study (32.4 mmol/l) showing that newly bred cultivars may exhibit higher antioxidative capacity. average phenolic (5534 mg/l) and ascorbic acid content (1307 mg/l) in this study was slightly lower than in the previous study (6622 mg/l and 1449 mg/l, respectively). due to different analysing methods and different analysing matrices such as juice or berries, our results are not always comparable to the literature. in addition, data for anthocyanin content of the juices may be reduced by aging when the monomeric anthocyanins of fresh processed juices changed to polyphenols (eder, 1996; iversen, 1999; rubinskiene et al., 2005). this might be one reason for changes in the anthocyanin content between the different years, whilst cultivar variation could not be explained because the analysis of juices from one year were always done at the same time for all cultivars. big variation for antioxidative capacity among cultivars was also described by deighton et al. (2002) and moyer et al. (2002). also, genotypic effects on content of polyphenols and anthocyanins of black currants are well known in the literature (moyer et al., 2002; rubinskiene et al., 2006; giné bordonaba and terry, 2008). the great differences of ascorbic acid within the cultivars examined are in good agreement with viola et al. (2000) who also described a threefold variation of ascorbic acid content among 6 different cultivars. in our study, there were also partially remarkable variations of the different traits within individual cultivars from year to year (see standard deviation). for teac the greatest changes were found for cvs. kristin and ometa and total phenols varied highly within cvs. farleigh, kristin, ben lomond and pc 96. very big differences between the years were found for total anthocyanins, especially within cvs. farleigh, pc 95, foxendown and pc 96. for ascorbic acid the greatest variation were found within cvs. pc 110, tifon, narwe viking, and ben lomond, respectively. these changes of the different parameters within individual cultivars resulted in signifi cant differences between the years (tab. 4). the lowest value for teac and total phenols as an average of all cultivars was found in 2003 while the content of anthocyanins was lowest in 2005. the content of ascorbic acid was not altered by yearly infl uences. signifi cant differences were also present for yield per plant and berry weight between cultivars (tab. 3). the highest average yield was recorded for cv. tsema and the lowest for cv. titania. average berry weight was high for cv. intercontinental and low for cv. triton. tab. 1: temperature during may to august in 2003, 2004 and 2005 average temperature per decade (° c) 2002 2003 2004 2005 may fi rst decade 16.1 11.5 12.6 second decade 13.1 15.2 12.1 third decade 18.3 13.3 18.5 june fi rst decade 22.2 18.2 15.3 second decade 21.2 16.3 18.4 third decade 21.6 17.7 23.5 july fi rst decade 18.2 18.4 16.8 18.1 second decade 18.4 23.0 18.4 22.3 third decade 19.8 20.6 20.4 august fi rst decade 18.2 27.9 23.9 second decade 20.9 23.4 19.6 third decade 19.3 18.6 16.6 time from beginning of fruit colouring until harvest of all cultivars 2003: 27 may to 8 july 2004: 21 may to 21 july 2005: 24 may to 14 july statistical analyses all statistics were performed using spps for windows, version 17.0. analyses of variance were done with cultivars and ripening stage as fi xed and years as random effects. duncan-test was applied to verify statistical differences for cultivars and ripening stage at p ≤ 0.05. phenotypic correlations were estimated between teac, total phenolics, total anthocyanins and ascorbic acid to yield, berry weight and degree days for the whole data set and individual cultieffects of cultivar, yield, berry weight, temperature and ripening stage on bioactive compounds of black currants 41 the results in tab. 4 also demonstrate the remarkable differences in yield and berry weight as an average of all cultivars in the three years examined. the highest yield was obtained in 2003 and the lowest in 2004. berry weight was highest in 2005. the low yields in 2004 refl ect the bad growing conditions during mid july until the end of august 2003, where the daily average temperature rose up to 29° c and hampered fl ower initiation (tab. 1). plants were in a very bad condition at the time and photosynthetic activity was strongly reduced which might be also a result of abiotic stress. normally it should be expected that a reduced fruit set leads to an increased fruit size. but, in 2004, fruit size was substantially reduced for some cultivars, which might be a result of this stress, also. correlations of phytochemicals antioxidative capacity was strongly related to the phenolic content (r = 0.84) whereas only moderate correlations exist to the anthocyanin content and among total phenolic content and total anthocyanin content (tab. 5). moyer et al (2002) mentioned for 32 black currants a much lower correlation among antioxidative capacity measured as orac (oxygen radical absorbance capacity) and total phenols (r = 0.44, p < 0.005) and between orac and anthocyanins (r = 0.38, p < 0.005) while they observed a much higher correlation between total phenols to anthocyanins (r = 0.63, p < 0.005) than we did. it should be noted that the mechanism of teac and orac assay are quite different in measuring antioxidative capacity. however, anthocyanins are the most abundant polyphenols in black currants and phenolic acids and other phenolic compounds are of minor importance, both for the content of polyphenols and for the antioxidant level of black currant (kähkönen et al., 2001). applying hplc-analysis dietrich et al. (2002) found a total phenol content of 2791 mg/l (1805-4126 mg/l) in 9 black currant cultivars, which was dominated by an anthocyanin content of 2600 mg/l (1688-3846 mg/l). in contrast, giné bordonaba and terry (2008) found no correlation between total phenols and anthocyanins. beside the fact that ascorbic acid is a powerful antioxidant, no correlation between teac and ascorbic acid could be found in our study (tab. 5). deighton et al. (2002) showed that vitamin c is the major contributor to the antioxidant capacity of black currants. in contrast to this result, dietrich et al., (2002) reported on a vitamin c ratio of 22 % on the teac value of black currants. correlations to yield, berry weight and degree days slight negative, but highly signifi cant correlations were determined between yield or berry weight and teac-value or total phenolic content, respectively (tab. 6). no correlation was found between yield and anthocyanin content, but a negative infl uence existed between berry size and anthocyanin content. fig. 1 demonstrates the correlation between yield and teac and total phenols within the whole data set, all 5 varieties with the lowest average yield over the three years examined exhibit high teac and total phenol values and are mainly concentrated for each of the three years on the left side of the graph while all 5 varieties with the highest average yield had low values and are located more on the right side. however, some exceptions occur, for example for cvs. ben loyal, tiben and tsema in one of the three years when yield was extremely low for cv. ben loyal in 2004, or teac values were higher than in the other two years for cv. tiben in 2004 and for cv. tsema in 2005. there were also low negative but highly signifi cant correlations between berry weight and teac tab. 2: antioxidative capacity (teac), total phenols (folin), total anthocyanins (sum hplc) and ascorbic acid of 23 black currant cultivars harvested in 20032005 (cultivars are arranged in increasing order of teac-value) teac (mmol/l ) total phenols (mg/l) total anthocyanins (mg/l) ascorbic acid (mg/l) ben loyal 34.1 ± 3.2 a 3726 ± 299 a 493 ± 55 a 970 ± 208 abc ben connan 38.6 ± 5.9 ab 4069 ± 460 ab 720 ± 79 ab 1098 ± 303 abcde pc 106 39.2 ± 7.3 ab 3789 ± 454 a 871 ± 228 ab 821 ± 249 ab bona 40.5 ± 6.8 abc 4481 ± 759 abc 1357 ± 377 bc 1027 ± 325 abcd pc 110 43.0 ± 6.0 abcd 4223 ± 47 ab 847 ± 292 ab 1289 ± 405 bcdef farleigh 46.1 ± 9.0 abcde 4424 ± 1071 abc 1038 ± 550 abc 616 ± 44 a intercontinental 46.6 ± 9.8 abcdef 4425 ± 602 abc 1004 ± 437 abc 1117 ± 215 abcde polar polar polar 48.4 ± 6.6 abcdef 5012 ± 527 abcd 1452 ± 407 bc 1168 ± 198 bcdef triton 49.4 ± 9.9 abcdef 5171 ± 401 bcd 1188 ± 281 abc 1355 ± 286 cdef titania 50.6 ± 7.0 abcdefj 5305 ± 260 bcde 1100 ± 261 abc 1330 ± 284 bcdef andega 53.3 ± 6.2 abcdefg 4859 ± 136 abc 1077 ± 334 abc 997 ± 163 abc tisel 53.6 ± 4.3 bcdefg 6151 ± 586 defg 755 ± 278 ab 2718 ± 243 g pc 95 53.7 ± 9.3 bcdefg 5587 ± 928 cdef 968 ± 579 abc 2563 ± 256 g kristin 54.8 ± 20.7 bcdefg 5675 ± 1078 cdefg 1149 ± 269 abc 836 ± 233 abc ben tron 56.6 ± 11.2 bcdefg 6476 ± 640 efg 1243 ± 141 bc 1318 ± 170 bcdef foxendown 59.6 ± 1.6 cdefg 6696 ± 428 fgh 1291 ± 493 bc 1562 ± 238 ef tifon 61.4 ± 5.7 defg 6213 ± 544 defg 1070 ± 64 abc 1243 ± 364 bcdef narwe viking 61.8 ± 11.9 defg 6711 ± 622 fgh 1442 ± 200 bc 1274 ± 438 bcdef tiben 62.4 ± 8.3 defg 6475 ± 960 efg 1683 ± 433 c 940 ± 285 abc ben lomond 64.1 ± 6.7 defg 6287 ± 1024 defg 1455 ± 365 bc 1646 ± 362 f ometa 65.8 ± 21.0 efg 6904 ± 516 gh 1375 ± 353 bc 1105 ± 85 abcde tsema 66.1 ± 13.2 fg 6868 ± 401 fgh 1323 ± 273 bc 1534 ± 197 def pc 96 70.0 ± 11.6 g 7765 ± 1095 h 1690 ± 715 c 1521 ± 125 def mean 53.0 5534 1156 1307 the values represent means of three years ± standard deviation; p = < 0.05 42 e. krüger, h. dietrich, m. hey, c.-d. patz tab. 3: yield and 100-berry weight of 23 black currant cultivars harvested in 2003-2005 (cultivars are arranged in increasing order of teacvalue) yield per plant (g) 100-berry-weight (g) ben loyal 1712 ± 736 cd 84.2 ± 6.9 fg ben connan 1523 ± 367 abcd 68.7 ± 0.2 cdef pc 106 1480 ± 521 abcd 78.0 ± 7.9 efg bona 1684 ± 571 bcd 88.0 ± 1.5 gh pc 110 1240 ± 488 abcd 80.9 ± 8.7 efg farleigh 865 ± 409 abc 69.7 ± 9.8 def intercontinental 1168 ± 635 abcd 101.6 ± 13.9 h polar 861 ± 278 abc 51.0 ± 2.1 abcd triton 1088 ± 282 abcd 46.5 ± 4.3 a titania 680 ± 198 a 64.9 ± 13.5 abcde andega 959 ± 387 abcd 53.6 ± 1.3 abcd tisel 1015 ± 588 abcd 64.0 ± 4.8 abcde pc 95 1064 ± 810 abcd 64.0 ± 12.4 abcde kristin 697 ± 320 a 62.2 ± 15.2 abcde ben tron 1073 ± 392 abcd 61.9 ± 9.0 abcde foxendown 694 ± 329 a 49.2 ± 11.1 abc tifon 1239 ± 390 abcd 47.4 ± 3.8 ab narwe viking 719 ± 329 a 56.7 ± 4.8 abcd tiben 1763 ± 413 d 66.2 ± 3.3 bcdef ben lomond 890 ± 100 abcd 69.0 ± 3.9 def ometa 1279 ± 255 abcd 52.4 ± 1.1 abcd tsema 1719 ± 323 cd 66.2 ± 19.8 bcdef pc 96 821 ± 294 ab 53.1 ± 4.1 abcd mean 1141 65.2 the values represent means of three years ± standard deviation; p = < 0.05 tab. 4: yearly means of antioxidative capacity (teac), total phenols (folin), total anthocyanins (sum hplc) and ascorbic acid, yield and 100berry weight of 23 black currant cultivars 2003 2004 2005 teac (mmol/l) 46.3 a 59.0 b 53.9 b total phenols (mg/l) 5019 a 5772 b 5813 b total anthocyanins (mg/l) 1172 ab 1322 b 985 a ascorbic acid (mg/l) 1278 a 1429 a 1213 a yield per plant (g) 1533 c 818 a 1132 b 100-berry-weight (g) 62.8 ab 61.5 a 72.4 b the values represent means of all cultivars; means followed by the same letter in a row do not differ signifi cantly; p = < 0.05 tab. 5: correlations coeffi cient within teac-value, total phenols, total anthocyanins and ascorbic acid in black currant juice based on 23 cultivars teac total total ascorbic phenols anthocyanins acid teac 1 0.84** 0.56* ns total phenols 1 0.52** total anthocyanins 1 correlation after pearson, bivariat; * p ≥ 0.05; ** p ≥ 0.01, ns = not signifi cant tab. 6: correlation coeffi cients between teac value, total phenols, total anthocyanins and ascorbic acid with yield, berry weight and temperature units yield 100-berry-weight (g/plant) (g) degree days teac -0.34** -0.43** 0.36** total phenols -0.34** -0.48** 0.33* total anthocyanins ns -0.38** 0.51** ascorbic acid ns ns ns correlation after pearson, bivariat; * p ≥ 0.05; ** p ≥ 0.01 degree days: calculated temperature units from beginning of fruit colouring until harvest fig. 1: correlation of yield with antioxidant activity (teac) and total phenol content of 23 black currant cultivars. value, total phenolic and anthocyanin content, respectively (tab. 6). as shown in fig. 2 for all traits, cultivars with the lowest average berry size are again more present on the left side whereas those with the highest average berry size are more concentrated on the right side of the graphs. neither yield nor berry weight had any infl uence on the content of ascorbic acid. to the best of our knowledge, there is no information available on the infl uence of yield on bioactive components of berry fruits. the observed negative infl uence of berry weight, as a surrogate of berry size, on the examined biochemical compounds are in agreement with other studies. while anthocyanins are mainly localized in the skin of black currant fruit, an increase of fruit size will decrease the surface area : volume ratio and consequently the concentrations of anthocyanins. anthocyanins are an important fraction of total phenolics and both are substantial contributors to the antioxidant capacity. effects of cultivar, yield, berry weight, temperature and ripening stage on bioactive compounds of black currants 43 therefore, part of the variability of the teac values and the anthocyanin and phenolic contents among the cultivars might be attributed to increased berry size of individual cultivars. moyer et al. (2002) showed for different cultivars of black currants and highbush blueberries, which are similar in shape to black currants, a signifi cant impact of berry size on anthocyanins content. connor et al. (2002) reported for blueberry of inverse signifi cant to high signifi cant correlations between berry weight (fresh berries) and antioxidative capacity (r = -0.28), total phenolics (r = -0.44) and anthocyanin content (r = -0.41). the observed phenomenon illustrates that, beside the cultivar infl uence on the accumulation of biochemical components, the genetically based variations in yield and berry weight among cultivars also account for some of the observed variation in teac value and total phenolic content and, in addition, berry weight also for anthocyanin content. this suggests that breeding for higher yield and greater berry size might concomitantly limit the values for antioxidative capacity, anthocyanin and phenolic content in black currant. however, when considering the relationship between yield and teac value or total phenolic content for individual cultivars during the three years of the study (tab. 7), it could be noted, that 60.8 % and 65.2 % of all cultivars showed a close negative relationship between both parameters (r = > 0.5 to 1.0) while yield affected negatively the anthocyanin content only by 40 % (r = > 0.5 to 1.0). the frequency of moderate to very high negative correlation coeffi cients between berry weight and teac-value, total phenolic or anthocyanin content raised up to 37.5 % and 43.8.0 % (r = > 0.5 to 1.0), giving variation of berry size within a cultivar a less pronounced effect on the examined traits than yield. this demonstrates that, within a cultivar, other factors than genetics may infl uence teac value and total phenolic content as well. it is well known, that the yield potential can be modifi ed by unfavourable conditions such as temperatures during fl ower initiation or nutrition and carbohydrate status of the plant. in our study, the high temperatures at fl ower initiation in 2003 reduced the yield potential in 2004 and thus seem to promote an accumulation of the teac value and the phenolic content. similar results are reported from tree fruits: stopar et al. (2002) found an increased polyphenol level both in apple skin and cortex with decreasing fruit load and just recently, andreotti et al. (2010) demonstrated also for nectarine that a reduced fruit load increased the content of polyphenols in the pulp. positive correlations were observed between degree days and anthocyanins (r = 0.51) and to a much lesser degree to phenolics and teac (tab. 6). when looking at individual cultivars, degree days also affected in 60 % of all cultivars the concentration of anthocyanin in black currant fruit (tab. 7). degree days might be depending on: a) differences in daily temperature from the beginning of fruit colouring to harvest of each cultivar; this was the case in our study, when temperature during fruit ripening was highest in 2003 and lowest in 2004 (tab. 1); b)variation in the visual rating of the beginning of fruit colouring; this might be one factor infl uencing the value of the degree day, but beginning of fruit colouring is far away (around 3-4 weeks) from harvest. however, from a physiological point of view, conditions around the potential harvest date seem to be more important. c) decision when to harvest the individual cultivar and thus by the maturity of the berries. as was shown by a sub trial conducted in 2005-2006, teac value und total phenolic content increased in tendency and anthocyanin level signifi cantly from the semiripe (one fourth of the berry surface still red-green), to ripe stage (all berries black and fully ripe) whereas ascorbic acid content declined with proceeding ripening (tab. 8). after delayed harvest, (slightly overripe berries, which were already shrivelling or nearly dropping down) teac values and phenolic and anthocyanin contents remained nearly unaffected when compared to ripe berries while ascorbic acid level was still decreasing. however, differences of teac value and total phenol and anthocyanin content between the different ripening stages were less pronounced compared to the observed variations within individual cultivars in the years 2003 to 2005. only anthocyanin level increased substantially during fruit ripening, which is in agreement with results reported by toldam-anderson and hansen (1997) or rubinskiene and viškellis (2002). however, berries of the main trial from 2003 to 2005 were never harvested in a semi-ripe stage but after visual survey of colouring (black and fully ripe) and softening of the berries and their affi nity to pre-harvest fruit drop. thus, the data of our study may be an indication that the weather conditions, mainly temperature, during ripening time of an individual cultivar determines to a much higher degree the accumulation of these parameters than a delayed harvest can do. fig. 2: correlation of 100-berry weight with antioxidant activity (teac), total phenol and total anthocyanin content of 23 black currant cultivars. 44 e. krüger, h. dietrich, m. hey, c.-d. patz an impact of environmental pre-harvest factors such as temperature on these phytochemicals is shown by wang and zheng (2001) who found for strawberries a more pronounced accumulation of anthocyanins, phenolics and antioxidative capacity under warm night/day temperature compared to low day/night temperatures. in addition, at constant day temperature, the accumulation of fl avonoids in strawberries was favoured by higher night temperature in comparison to low night temperature (wang et al., 2003). davik et al. (2006) de-davik et al. (2006) de-davik monstrated that antioxidative capacity of strawberries was negatively correlated with rainfall and positively to minimum temperature on the day prior to harvest. the observed decline of ascorbic acid during maturity (tab. 7) is in accordance to fi ndings of rubisnkiene et al. (2006) and viola et al. (2000). the results of viola et al. (2000) indicate that the genotypic ranking of ascorbic acid content is already manifested at the berry expansion phase, before the fruits start to get coloured. this might explain why no correlation was found between ascorbic acid content and degree days calculated from fruit colouring until harvest in our study. however, lee and kader (2000) and atkinson et al. (2006) reported of higher ascorbic acid content with increasing intensity of radiation. redalen (1993) showed that the ascorbic acid content of black currants was reduced by elevated temperature when grown in controlled growth chambers. comparable results are reported by wang and camp (2000) for strawberries. total variance (70.7 %), respectively (tab. 9). the fi rst pc was mainly dominated by the total phenols and teac value and, less important, total anthocyanins and berry weight (vector loadings are 0.899, 0.876, 0.693 and -0.6635, respectively). the parameter cultivar loaded to only -0.437. this is surprising low, but underlined, that in this data set other factors than only genetic determines the accumulation of the phytochemicals. the second pc is mainly attributed to ascorbic acid and its infl uencing factor degree days (vector loadings -0.719 and 0.705) while pc 3 is defi ned by cultivar and the genetically based yield (scores -0.724 and 0.642). tab. 7: frequency of correlation coeffi cient between teac value, total phenols, total anthocyanins and ascorbic acid with yield, berry weight and temperature units based on single cultivar calculation negative correlation positive correlation frequency of coeffi cent frequency of coeffi cent frequency of coeffi cent % frequency of coeffi cent frequency of coeffi cent frequency of coeffi cent % n < 0.5 >0.5-0.7 >0.7-1.0 ∑ >0.5 -1.0 <0.5 >0.5-0.7 >0.7-1.0 ∑ >0.5-10 teac yield 23 4 3 11 60.8 4 1 4.3 berry weight 16 3 2 4 37.5 1 1 5 37.5 degree days 23 2 3 13.0 7 3 8 47.8 phenols yield 23 4 3 12 65.2 3 1 4.3 berry weight 16 1 3 4 43.8 2 2 4 37.5 degree days 23 5 1 3 17.4 4 3 7 43.5 anthocyanins yield 20 3 4 4 40.0 3 1 5 30.0 berry weight 13 3 5 38.5 4 1 7.7 degree days 20 2 10.0 6 1 11 60.0 tab. 8: teac-value, total phenols, total anthocyanins and ascorbic acid of black currant affected by ripening stage (average of the cvs. pc 73, pc 106 and tsema in 2005 and 2006) teac total total ascorbic phenols anthocyanins acid (mmol/l) (mg/l) (mg/l) (mg/l) ripening stage semi-ripe 49.8 5041.5 133.2 a 1919.3 a ripe 59.0 5919.3 818.0 b 1471.3 ab slightly overripe 61.1 5638.3 891.3 b 1135.2 a signifi cance ns ns *** * ns = non signifi cant, * signifi cant at p<0.05, *** signifi cant at p<0.001 tab. 9: principal component analyses of ratings for selected phytochemicals, yield, berry weight and degree days of black currant juices of 23 cultivars principal component item 1 2 3 cultivar -0.437 0.263 -0.724 ascorbic acid 0.347 -0.719 0.228 total phenols 0.899 -0.021 -0.028 total anthocyanins 0.693 0.432 -0.002 teac 0.876 0.098 -0.098 yield -0.448 0.305 0.642 berry weight -0.635 0.272 0.124 degree days 0,436 0.705 0.191 variance explained by pc, % 39.5 18.1 13.1 pc-analysis to simplify the infl uencing factors and for easier interpretation, all data were subjected to principle component analyses. three signifi cant pcs were derived accounting for 39.5, 18.1 and 13.1 % of the conclusion the study shows the great genetical variation in content of anthocyanins, total phenols, and ascorbic acid, and in the level of teac within different cultivars, giving the possibility to enhance their level of health functional phytochemicals by breeding. also fruit size is often regarded as an important breeding aim. however, while anthocyanins are mainly localized in the skin of black currant fruit, big fruit size might imply the risk that the bioactive compounds will be reduced when the ratio between surface area and fruit volume becomes too wide. the study also showed that environmental factors like temperature during the fruit period and fruit load may infl uence the accumulation of health functional phytochemicals in black currant fruit. this underlines that investigation on the phytochemical properties of fruits and effects of cultivar, yield, berry weight, temperature and ripening stage on bioactive compounds of black currants 45 the comparison of different cultivars have to be done for more than one year to minimize infl uences of external factors such as weather parameters, fruit load, etc.. finally, objective tools to determine the harvest date of the berries seem to be helpful ensuring to get fruits with high contents of bioactive compounds. references andreotti, a., ravaglia, d., costa, g., 2010: effects of fruit load and refl ective mulch on phenolic compounds accumulation in nectarine fruit. europ. j. hort. sci. 75, 53-59. anonym, 1964: ifu methode no 17: http://www.ifu-fruitjuice.com/ifumethods. atkinson, c.j., doods, p.a.a., ford, y.y., lemiere, j., taylor, j.m., blake p.s., paul, p., 2006: effects of cultivar, fruit number and reflected photosynthetically active radiation on fragaria x ananassa productivity and fruit ellagic acid and ascorbic acid concentrations. ann. bot. 97, 429-441. beattie, j., crozier, a., duthie, g.g., 2005: potential health benefits of berries. current nutr. & food sci. 1, 71-86. brennan, r., 1996: currants and gooseberries. in: janick, j., moore, j.n. 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stopar, a., bolcina, u., vanzo, a., vrhovsek, u., 2002: lower crop load for cv. jonagold aplles (malus x domestica borkh.) increases polyphenol content and fruit quality. j. agric. food chem. 50, 1643-1646. toldam-anderson, t.b., hansen, p., 1997: growth and development in black currant (ribes nigrum). iii: seasonal changes in sugars, organic cids, chlorophyll and anthocyanins and their possible metabolic background. j. hort. sci. 72, 155-169. viola, r., brennan, r.m., davies, h.v., sommerville, l., 2000: l-ascorbic acid accumulation in berries of ribes nigrum l.. j. horticultural sci. biotechnology, 75, 409-412. williamson, g., manach, c., 2005: bioavailability and bioefficacy of polyphenols in humans. ii. review of 93 intervention studies. am. j. clin. nutr. 81, 243-255. wang s.y., zeng, w., 2001: effect of plant temperature on antioxidants capacity in strawberry. j. agric. food chem. 49, 4977-4982. wang, s.y., camp, m.j., 2000: temperature after bloom affects plant growth and fruit quality of strawberry. sci. hort. 85, 183-199. wang, s.y., zheng, w., maas, j.l., 2003: high plant growth temperatures increases antioxidant capacity in strawberry fruit. acta hort. 626, 57-63. address of the authors: 1department of pomology, geisenheim research center, von-lade-straße 1, 65366 geisenheim, germany 2department of wine analysis and beverage technology, geisenheim research center, von-lade-straße 1, 65366 geisenheim, germany 46 e. krüger, h. dietrich, m. hey, c.-d. patz art_15479.indd journal of applied botany and food quality 94, 26 38 (2021), doi:10.5073/jabfq.2021.094.004 1 state research institute for viticulture and pomiculture, weinsberg, germany 2 university of hohenheim, institute of crop science, quality of plant products (340e), stuttgart, germany near-infrared spectroscopy in process control and quality management of fruits and wine jana gehlken1,2, martin pour nikfardjam1, melissa kleb2, christian zörb2* (submitted: september 27, 2020; accepted: january 27, 2021) * corresponding author summary recently, rapid quality assessment of food is an increasingly important topic. the rising demand of consumers for high quality products generates a need to establish fast and suitable analytical methods. near-infrared (nir) spectroscopy has turned out to be a time-saving, cheap, easy-to-use and environmentally friendly technique, which can be applied for the determination of manifold quality attributes in various kinds of food matrices. this article gives overview of the basic principles of near-infrared measurements and describes the immense field of applications, with the main focus on fruits, grapes and wine and the evaluation of wine aroma. keywords: near-infrared spectroscopy, nir, process control, quali ty management, fruit analysis, grape and wine analysis, wine aroma introduction the safety and accurate quality assessment of food is becoming increasingly important in the last decades. various affairs in food safety and quality (e.g. bse, horse meat in ready meals, fipronil in eggs) (european food safety authority; world health organization) trigger development and continuous improvement of control systems for food safety and food quality with consumer protection as a main goal. a set of methods has been evolved in the last decades including improved classical wet-lab methods or quick methods. most classical wet-lab methods give precise and accurate results, but assets and drawbacks may come along. high-performance liquid chromatography (hplc) or gas chromatography (gc), often coupled with mass spectrometry (ms), require experienced users, expensive technical equipment and ultrapure, high priced chemicals, which additionally hold the risk of harming the user’s health and/or may affect the environment if not used adequately. another main shortcoming is the time requirement for those methods, which may take several hours or even days to produce results. nir spectroscopy increasingly gains attention, because it is easy to use, cheap, provides results quickly and direct implementation in production processes is possible. thus, the delay of time caused by sampling, analysis and evaluation can be avoided, so immediate intervention in case of irregularities is possible, which reduces the waste of resources and economic losses. furthermore, the consumer’s awareness for quality characteristics of food (presence/absence of various ingredients, origin of the product, cultivation technique or manufacturing process) is growing. the use of nir spectroscopy enables continuous monitoring of these attributes to meet consumer’s expectations and prevent food fraud. development of instrumentation and industrial applications of nir spectroscopy began to increase in the second half of the 20th century. first applications to agricultural products were done in 1964 by norris who recognized the potential of this technique already at the early stages of development (siesler, 2002). since then, the field of application has expanded (> 700,000 results for nir analysis of food, google scholar, july 2020) just as the diversity of instrumentation, for instance on-line measurements and portable devices (aleixandretudo et al., 2019; amuah et al., 2019; fernández-novales et al., 2019; beć et al., 2020). the objective of this article is (i) to review the basic principles of near-infrared measurements and (ii) to give a detailed overview of applications with the main focus on fruit, grape and wine quality control and the option to evaluate wine aroma by the use of nir spectroscopy. basic principles of near-infrared measurements near-infrared radiation was discovered by friedrich wilhelm herschel in the year 1800 (herschel, 1800) and covers the range between mid-infrared and visible light in the electromagnetic spectrum (780 to 2500 nm). when near-infrared radiation hits a sample, the radiation is absorbed, transmitted or reflected, which in each of the three cases depends on the chemical composition and the physical properties of the sample (nicolaï et al., 2007). the near-infrared spectrum is dominated by overtone and combination bands of ch, oh and nh functionalities and, in case of moist samples, water, which shows two very strong and broad absorption bands in the near-infrared region (siesler, 2002; burns and ciurczak, 2008; beć and huck, 2019). the absorption bands often overlap, which results in very low structural selectivity and impedes the interpretation of spectra and especially qualitative evaluation (næs et al., 2004). for this reason, it is necessary to use multivariate statistical techniques (often called chemometrics) to extract the information linked directly to the desired compounds for the development of calibration models. additionally, irrelevant information has to be suppressed in the spectra, which can be achieved by various preprocessing techiques (nicolaï et al., 2007). even within the same matrix, the use of different preprocessing techniques can result in varying effects on the resulting calibration models (purwanto et al., 2015; khodabakhshian et al., 2016; xu et al., 2017) just as the use of different calibration techniques (giovenzana et al., 2015; altieri et al., 2017; malegori et al., 2017). the validation of calibration models is essential to assure their prediction accuracy and can either be performed internally (cross-validation) or with an independent set of samples. statistic values, which are often used to describe the accuracy of a model, are the root mean square error (rmse), the correlation coefficient (r) or the multiple correlation coefficient (r2) (næs et al., 2004). applications of near-infrared spectroscopy applications in process control and quality management of fruit and wine industry for the successful application of nir spectroscopy, a number of conditions has to be fulfilled. first, the compound or property has to have an influence on the nir spectrum. in case of such an influence nir spectroscopy in process and quality control 27 mostly a relationship exists between intensity of absorption bands and concentrations. relationships can also be indirect, when a compound or property is correlated with another one, which is directly related to the spectrum. the samples for building the model must be selected according to the concentration range of the examined compound and must cover all sources of variability, e.g. different seasons or cultivars. furthermore, the sources of error have to be identified to evaluate and to improve the precision of the model. finally, the validity over time needs to be given by regular maintenance (macho and larrechi, 2002). qualitative analysis is based on grouping samples according to selected properties and is mainly used for identification purposes and quality judgement. classification by nir spectroscopy can be more precise than by mid-infrared (mir) spectroscopy, but appropriate mathematical operations are needed for every model to minimize misclassifications. for quantitative analysis, lambert-beer’s law is applied, which describes a linear relationship between concentration and absorbance at a given wavelength (siesler, 2002). quantitative analysis often shows very good and reliable performance for compounds present in high amounts, but often decreases for lower concentrations (nicolaï et al., 2007). in general, the detection limit is about 0.1% (m/m), however, lower values can be reached in some cases, depending on the application and the characteristics of sample and analyte matrix (pasquini, 2003). the main areas of application of nir spectroscopy are petrochemistry, environmental analysis, the clinical and pharmaceutical sector and the agricultural food sector (blanco and villarroya, 2002). the food sector represents a very extensive field of raw materials, intermediates and products. therefore, the establishment of a uniform procedure for quality assessment would be of great interest for producers and consumers. for instance, grain and grain products belong to the earliest applications of nir spectroscopy for quality control in the food sector (williams et al., 1985). most applications to grain and grain products such as flour focus on the determination of compounds like protein content, moisture, ash (manley et al., 2013) or dietary fiber content (kays et al., 1996; archibald and kays, 2000; kays and barton, 2002; zörb et al., 2018), which all represent important quality parameters. nowadays, for the determination of the protein content, a portable nir spectrometer can be used (bhandari et al., 2004). many but not all quality characteris tics of bread are correlated with the protein content (zörb et al., 2018). moreover, the suitability of nir spectroscopy as a determination method for contaminants in grains was shown for ergot bodies in cereals (vermeulen et al., 2013). other application examples for meat, milk and their derived pro ducts as well as for vegetables are described elsewhere (huang et al., 2008; qu et al., 2015). applications for fruit analysis the nir spectroscopy is a suitable method for the determination of fruit maturity, scheduling of harvesting time, quality assessment of fruit products or the detection of possible contaminations. tab. 1 gives an overview of current applications of nir spectroscopy in fruit analysis. the application of nir spectroscopy may start at the field for monitoring plant nutrient status, for instance the leaf nitrogen status in pear orchards (wang et al., 2017). it was also possible to monitor the infestation of two different species of larvae, which can affect apples by nir spectroscopy (siegwart et al., 2015). in affected fruits, two larva species could be differentiated. the method can help to reduce pesticides and might serve for the development of alternative crop protection methods. even fruits grown at the same site, under similar conditions and harvested similarly can vary in size, color, shape and biochemical composition, which may result in a different inner and outer quality. to avoid harvesting of fruits, which have not reached criteria for maturity, adequate parameter determination of the best time for harvesting can be achieved quickly and simultaneously by nir spectroscopy. some of those measurements in the field were available using special portable devices, e.g. for blueberries (riberafonseca et al., 2016b). to protect the post-harvest quality of fruits within a possible storage or transport time, it is important to know the optimum conditions for storage and for transport (kienzle et al., 2011). post-harvest quality has to be controlled constantly depending on fruit characteristics, for example when fruits are climacteric, or in case of differing marketing destination, such as direct or later sale, food processing or export. besides visible changes like shape or color, changes in biochemical composition can occur, which may also affect consumer’s acceptance. fruits for immediate sale must be free of damages and spoilage. in various studies (lorente et al., 2015; folch-fortuny et al., 2016; wedding et al., 2019), damages and infections were identified by nir spectroscopy even if barely visible. for example, based on nir measurements, peaches were classified due to different susceptibility to brown rot (spadoni et al., 2016). moreover, the number of days before the decay of peaches could be determined (huang et al., 2017). therefore, recommendations for the best time to eat the fruit can be provided based on the results of nir measurements. furthermore, the results can help to optimize storage conditions and to decide on marketing destinations to avoid economic losses and minimize food waste, e.g. in case of damages or short shelf-life. besides outside appearance, taste plays a key role in the consumer’s acceptance. the contents of sugar and acid often determine whether the taste is conceived as pleasant. in addition, the sugar/acid ratio and the brima (brix minus acids) value (jordan et al., 2001) can serve as flavor indices (ncama et al., 2017). fruits with high contents of dry matter are often preferred by the consumers, which has been observed in sensory evaluation of mangos (dos santos neto et al., 2018) and pears (serra et al., 2019) classified by their dry matter content using nir spectroscopy. due to the increasing interest in foods with health benefits, the consumers may pay more attention to the content of various compounds, which are claimed to have positive health effects. therefore, it is favorable to implement rapid and precise methods for non-destructive analysis of those valuable substances. despite the low concentration of several of these interesting compounds, good or excellent results could be achieved by the use of nir spectroscopy to monitor the quality of fruits and fruit products. climate change aspects and environmental pollution may further intensify the consumer’s awareness for food derived from sustainable cultivation or of regional origin. simultaneously, this trend may enhance the risk of food fraud further making it necessary to establish a fast and easy method for supervision. in several studies, e.g. origin discrimination of chinese quince fruits (shao et al., 2017), discrimination between different farming systems for strawberries (amodio et al., 2017) and pineapples (amuah et al., 2019) and discrimination between naturally and artificially ripened mangos (lakade et al., 2019) was possible with high accuracies by the use of nir spectroscopy. despite these examples, it might further be difficult to determine the origin of production of diverse plant products and one has to be further critical with these reports. as shown in tab. 1, nir spectroscopy is widely used in fruit and fruit product analysis and numerous studies on manifold quality attributes are available. however, in many cases the calibration needs to be further validated by larger sets of samples to stabilize the calibration and improve prediction accuracy. additionally, a larger variety of properties should be examined to check, in which cases global or separated calibration models are needed for analysis. specific spectroscopy-based devices, such as the da-meter, show the potential of nir technique for the food sector. the da-meter is a portable spectrometer, which measures the index of absorbance difference (iad). 28 j. gehlken, m. pour nikfardjam, m. kleb, c. zörb f ru it c om po un ds f ru it m at ur it y f ru it q ua lit y tab. 1: applications of nir spectroscopy in fruit analysis fruit parameters/properties r2 reference acerolaa titratable acid 0.66 – 0.74 malegori et al., 2017 ascorbic acid 0.40 – 0.65 avocado moisture content 0.91 – 0.95 olarewaju et al., 2016 dry matter 0.91 – 0.95 mesocarp oil content 0.67 – 0.71 mangoa total soluble solids 0.92, 0.87 nascimento marques et al., 2016; dry matter 0.67, 0.84 dos santos neto et al., 2017 titratable acid 0.50, firmness 0.72, peacha flesh firmness 0.80 uwadaira et al., 2018 water soluble pectin 0.82 pomegranate total soluble solids 0.93 (r) khodabakhshian et al., 2017b titratable acid 0.94 (r) ph value 0.84 (r) avocado bruises 60 – 100b wedding et al., 2019 rot 65 – 84b citrus fruits decay lesions 90 – 96b folch-fortuny et al., 2016 fungal infections 98b lorente et al., 2015 date fruit total soluble solids 0.97 alhamdan and atia, 2017 moisture content 0.94 colour (b*) 0.64 kiwifruit total soluble solids 0.58 – 0.83 li et al., 2017 flesh firmness 0.30 – 0.60 mangoa dry matter 0.75 dos santos neto et al., 2018 internal browning 83.1b gabriëls et al., 2020 peach days before decay 82.26b huang et al., 2017 pear maturity stage 97.2b xu et al., 2017 persimmon soluble tannins 0.92 cortés et al., 2017 strawberry storage time 97.4b shen et al., 2018 açai anthocyanins 0.74 de almeida teixeira et al., 2015 apple (peel/flesh) phenolic compounds 0.91/0.84 pissard et al., 2018 berry fruits phenolic compounds 0.98 gajdoš kljusurić et al., 2016 antioxidant activity 0.99 blackberry carotenoids 0.71 toledo-martín et al., 2018 phenolic compounds 0.65 castanhola anthocyanins 0.80 câmara costa et al., 2017 phenolic compounds 0.82 juçara anthocyanins 0.73 de almeida teixeira et al., 2015 jujube total sugar 0.90 guo et al., 2016 total acid 0.98 phenolic compounds 0.94 antioxidant activity 0.96 mango soluble solids content 0.68 purwanto et al., 2015 titratable acid 0.69 persimmon colour index 0.99 altieri et al., 2017 firmness 0.99 soluble solids content 0.98 titratable acid 0.98 soluble tannins 0.99 pineapplea total soluble solids 0.85 amuah et al., 2019 pomegranate firmness 0.94; khodabakhshian et al., 2016; total soluble solids 0.94; 0.97 khodabakhshian et al., 2017a titratable acid -; 0.93 ph value 0.88; 0.94 strawberry soluble solids content 0.83 mancini et al., 2020 firmness 0.54 nir spectroscopy in process and quality control 29 watermelon total soluble solids 0.71 tamburini et al., 2017 lycopene 0.81 β-carotene 0.74 wax jambu anthocyanins 0.98 viegas et al., 2016 phenolic compounds 0.94 apple juice z. rouxii contamination 0.99 niu et al., 2018 cashew apple nectar total sugar 0.71 caramês et al., 2017 total soluble solids 0.62 titratable acid 0.75 ph value 0.75 ascorbic acid 0.84 guava nectar total sugar 0.82 caramês et al., 2017 total soluble solids 0.88 titratable acid 0.79 ph value 0.76 ascorbic acid 0.86 guava pulp total sugar 0.72 alamar et al., 2016 total soluble solids 0.66 titratable acid 0.79 moisture 0.94 ascorbic acid 0.85 kiwi juice z. rouxii contamination 0.99 niu et al., 2018 strawberry juice soluble solids content 0.98 wlodarska et al., 2019 phenolic compounds 0.84 strawberry powder freshness of fruits 97b wang et al., 2019 various fruit juices saccharin adulteration 98b mabood et al., 2018 yellow passion fruit pulp total sugar 0.90 alamar et al., 2016 total soluble solids 0.54 titratable acid 0.59 ph value 0.61 moisture 0.93 a portable nir spectrometer used b classification rate [%] f ru it p ro du ct q ua lit y the iad describes the difference in chlorophyll absorbance at 670 nm and 720 nm and can be used to pre-estimate fruit ripeness (ziosi et al., 2008). applications for grape and wine analysis as well as for other fruits, nir spectroscopy may be used for monitoring grape quality and the following steps of winemaking. tab. 2 shows various applications in viticulture, grape quality and wine analysis. in vineyards, water availability is an important aspect for grapevine growth. due to climate change, irrigation becomes increasingly important in viticulture to avoid negative effects of water stress for plant vigor and the resulting wine quality. to measure plant water status, nir spectroscopy can be used directly in the vineyard (giovenzana et al., 2018). nir spectroscopy was used as an on-the-go spectral measurement to obtain information about the leaf water status of vines (diago et al., 2018). the spectrometer with an operation wavelength from 1100 2100 nm was mounted on an all-terrain vehicle with a motion speed of 5 km/h in the vineyard. to prove the data set from the nir spectrometer, the stem water potential was used for correlation purpose. this study showed two absorption peaks at about 1450 nm, which corresponded to the first overtone of the symmetric and asymmetric hydroxyl (oh) bond stretching and/or combination bands and around 1940 nm, which can be assigned to the combination of the oh stretching and bending bands of water (diago et al., 2018). those stretching, bending and combinations are vibrational reactions of the organic groups provoked by the electromagnetic excitation of the nir spectroscopy. due to the fact that leaves have high water content, the prevalence of the oh group absorbance and the corresponding nir spectra is proven (nicolaï et al., 2007). other literature shows water-related bands at 975, 1200, 1470, 1930 and 2500 nm (clevers et al., 2010; cao et al., 2013). the results may contribute to the development of better irrigation scheduling to reach higher irrigation efficiency. in addition to water, the grapevine requires a number of mineral elements to grow, flower and to produce fruit (white, 2015). for instance, the concentration of nitrogen, sulfur and the c/n-ratio in vine leaves and grape berries was determined by nir spectroscopy (cuq et al., 2020). however, calibration models with high accuracy were reported for nitrogen (r2 = 0.95) and for the c/n-ratio (r2 = 0.87) in leaves and berries. based on these results, sustainable fertilization strategies for plant nutrients might be improvable. grape maturity estimation is mainly based on the development of sugar and acid concentrations, which both can be determined by nir spectroscopy as demonstrated in various studies. the sugar concentration decides on the potential alcoholic strength while ph value and titratable acid help to control wine quality, e.g. red wine color (nogales-bueno et al., 2014). further maturity parameters (maturity index, various organic acids) were examined by nir spectroscopy in grape berries at different stages of development (fernándeznovales et al., 2009a; musingarabwi et al., 2016). in addition to these technological ripening parameters such as sugar and acid content, the phenolic maturity of the grapes may play an important role for the resulting wine quality due to its influence on organoleptic properties (ferrer-gallego et al., 2011). the anthocyanin contents were examined in grapes of different ripening times by the use of nir spectroscopy. although the contents could be determined re30 j. gehlken, m. pour nikfardjam, m. kleb, c. zörb liably, a separation for the different ripening times only based on the anthocyanin content was not possible, probably due to different varieties examined (hernández-hierro et al., 2013). portable devices for infield measurements can save time, because it is still a huge effort to sample enough grapes to achieve representative results for the whole vineyard (fernández-novales et al., 2019). total soluble solids, anthocyanins and total polyphenols were examined using a device mounted on an all-terrain-vehicle (fernández-novales et al., 2019). the results show that the method has great potential for mapping the grape composition in the vineyard and harvest scheduling based on these results. besides the technological and phenolic maturity, the phytosanitary status of the grapes is of particular importance for the quality of the resulting wine. one of the most common grape diseases is grape rot caused by botrytis cinerea which negatively affects taste and color of the resulting wine (morales-valle et al., 2011) (except of “noble rot“) and can also affect food safety (serra et al., 2005). both in the vineyard and at delivery at the winery the phytosanitary status of the grapes is estimated visually, which makes the results subjective. nir spectroscopy was shown to be suitable to quantify botrytis bunch rot, although further calibration is needed (hill et al., 2014). direct application of nir technology in the vineyard would be advantageous, such as a device mounted on a grape harvester for sorting purposes. a real-time analysis of crushed grapes upon delivery at wineries was executed for the determination of ten different parameters (porep et al., 2015a). the prediction accuracy of a global calibration model was insufficient for quantitative purposes (0.2 < r2 < 0.7), but could be improved by the development of separate calibration models for different grape colors, vintages and wineries. commonly, glycerol, gluconic acid and acetic acid contents are used to receive information about the phytosanitary status of the grape (sturm, 2009). ergosterol, a sterol occurring in fungi but not in plant or animal tissue, was determined in grape mashes to prove the suitability of nir spectroscopy for the detection of grape rot (porep et al., 2014). good results were achieved (r2 = 0.841 for cross-validation), which allow at least a semi-quantitative determination. in-line determination under industrial conditions confirmed the results (porep et al., 2015b). a real-time objective method for wineries may further improve wine quality and safety and moreover enables fair pricing of delivered grapes due to their phytosanitary status. wine fermentation is a very complex process, which requires detailed knowledge about the chemical composition of the fermenting mixture. for a non-viscous fermentation process, the content of yeast assimilable nitrogen plays an important role in avoiding sluggish or stuck fermentations (gardner et al., 2002). the grape must or mash undergoes many changes during fermentation, e.g. degradation of sugar and formation of ethanol. nir spectroscopy can be used in white grape must to pre-estimate the potential alcoholic strength and the wine quality due to the contents of sugar and acid (wu et al., 2010). time-related changes in the fermentation process can be monitored, which enables to classify different fermentation stages (cozzolino et al., 2006a) and to predict the stage of fermentation due to the concentration of different compounds such as sugars, alcohols and secondary metabolites (di egidio et al., 2010). the whole fermentation process can be monitored and critical points can be identified (buratti et al., 2011), also regarding further treatment of the wine, such as the calcium content in must and ground wine for sparkling wine production (véstia et al., 2019). during wine production some by-products remain, such as grape pomace, which is rich in phenolic compounds (kammerer et al., 2004). nir spectroscopy can be used to easily determine its content, so grape pomace may be used as a natural source for extracting phenolic compounds for further use i.e. in bioeconomy instead of disposing of it directly (páscoa et al., 2015). for the resulting wines, the chemical composition can be examined similarly. in an extensive study 15 parameters in red, white and rosé wines of different ages and from different origins were determined (urbano-cuadrado et al., 2004). with the intention to facilitate the quality evaluation of wines, different types of wines were measured in their bottles (cozzolino et al., 2007). even though no exact quantitative evaluation was possible, however, the technique is suitable for qualitative evaluation and screening. an important aspect for the control of wine quality is the verification of the geographic origin, which is closely linked to climate, fertilization and soil cha racteristics affecting quality and taste of the wine (fischer et al., 1999). by the use of nir spectroscopy, wines from different countries could be differentiated with higher accuracy than wines from different regions in the same country, which may be caused by more similar growing conditions within the same country (riovanto et al., 2011). the contents of stable isotopes of mineral elements can serve as indicators for authenticity, but cannot be determined by nir spectroscopy because no specific isotope absorption is present in the nir spectrum. indirect estimation might only be possible due to association with organic functional groups (cozzolino et al., 2008c). furthermore, the variety of a wine is an important aspect since it is linked to sensory and chemical characteristics. grape varieties could successfully be differentiated by nir spectroscopy (cozzolino et al., 2003). moreover, wines made from a single variety may be distinguished from blends. for the determination of the single varieties in blended wines difficulties may occur for low contents and with an increasing number of varieties in the blend. wine aroma and flavor are very complex issues, which are influenced by a large number of aspects, among them the selected yeast strain and bacteria (boido et al., 2009). different oenococcus oeni strains for malolactic fermentation in red and white wines could be differentiated by nir spectroscopy (cozzolino et al., 2012). depending on the bacteria strain, classification rates up to 100% could be obtained. this way, potentially useful commercial wine strains can be identified, which cause different aroma and flavor profiles. in grape and wine analysis, nir spectroscopy is widely used with promising results. main compounds like ethanol or sugar content can be determined with very high accuracy. for components being present in lower or trace amounts, such as polyphenolic compounds, it is difficult to obtain exact results, because their concentration is probably too small to affect the nir spectrum significantly. indirect determination is possible with good results due to correlation with compounds in higher concentration, but model robustness might be low for different batches (nicolaï et al., 2007). problems may also occur due to interferences (martelo-vidal and vázquez, 2014b, 2015). furthermore, differentiation between various attributes showed difficulties in some cases with nir spectroscopy giving only medium results. applications in the evaluation of wine aroma the decision to buy a wine is based on outer influences, e.g. design of the bottle, name, variety, marketing or recommendations. for the motivation to buy the same wine again, the most important aspect is the taste and the aroma. the personal impression always remains subjective, although individual wines are closely linked to certain taste and flavor attributes depending on different characteristics like grape variety or wine style (cayuela et al., 2017). the large number of compounds contributing to wine aroma and their low concentrations require very complex analyses. the most current method for the analysis of aroma compounds is gas-chromatography, often coupled with mass spectrometry as a detector (gc-ms). this way, many different classes of compounds can be analyzed precisely even if they only occur in trace amounts. tab. 3 gives an overview of the applications in grape and wine aroma evaluation using nir spectroscopy. to obtain an objective opinion on nir spectroscopy in process and quality control 31 tab. 2: applications of nir spectroscopy in grape and wine analysis parameters r2 reference soluble solids contenta 0.94 herrera et al., 2003 reducing sugar content 0.98 fernández-novales et al., 2009b total soluble solidsa 0.91 urraca et al., 2016 titratable acida 0.86 chauchard et al., 2004 soluble solids contenta (berries/bunches) 0.66/0.65 giovenzana et al., 2015 titratable acida 0.85 sugar concentration 0.99 nogales-bueno et al., 2014 titratable acid 0.98 ph value 0.94 total phenolic content 0.89 soluble solids contenta 0.92 ribera-fonseca et al., 2016a titratable acida 0.87 firmnessa 0.89 total anthocyaninsa 0.94 monomeric anthocyaninsa 0.68 – 0.96 dry matter 0.88 cozzolino et al., 2008b condensed tannins 0.81 total anthocyanins 0.90 janik et al., 2007 total phenolic content (grapes/grape skins) 0.98/0.98 ferrer-gallego et al., 2011 flavonols (grapes/grape skins) 0.93/0.99 flavanols (grapes/grape skins) 0.97/0.99 phenolic acids (grapes/grape skins) 0.75/0.94 anthocyanins (grapes/grape skins) 0.95/0.97 total anthocyanins 0.86 hernández-hierro et al., 2013 non-acylated anthocyanins 0.86 total phenolic content (grape seeds) 0.53 – 0.78 rolle et al., 2012 extractability 0.50 extractable total phenolic content 0.82 nogales-bueno et al., 2015 extractable flavanols 0.82 extractable anthocyanins 0.79 total and individual anthocyanins 0.35 – 0.90 diago et al., 2016 mcp tannins 0.67 – 0.83 aleixandre-tudo et al., 2019 anthocyanins 0.50 – 0.93 total phenols 0.61 – 0.90 colour density 0.59 – 0.94 soluble solids content (grape juice) 0.98 wu et al., 2010 ph value (grape juice) 0.95 total yeast assimilable nitrogen 0.96 petrovic et al., 2020 free amino nitrogen 0.91 ammonia 0.88 volumic mass 0.99 fernández-novales et al., 2008 reducing sugars 0.99 volumic mass 0.94 fernández-novales et al., 2011b total polyphenol index 0.98 colour intensity 0.98 volumic mass 0.98 fernández-novales et al., 2011a malvidin-3-glucoside 0.81 – 0.94 cozzolino et al., 2004 pigmented polymers 0.82 – 0.94 tannins 0.70 – 0.90 total phenolic content (grape pomace, raw/milled) 0.93/0.96 páscoa et al., 2015 total antioxidant capacity (grape pomace, raw/milled) 0.91/0.97 ethanol 0.67 cozzolino et al., 2007 ph value 0.50 free so2 0.83 total so2 0.70 polyphenolic compounds 0.41 – 1 martelo-vidal and vázquez, 2014a g ra pe p ar am et er s f or d et er m in in g m at ur it y w in e f er m en ta ti on w in e c om po un ds 32 j. gehlken, m. pour nikfardjam, m. kleb, c. zörb sodium 0.92; 0.77 sauvage et al., 2002 potassium 0.89; 0.89 cozzolino et al., 2008c magnesium 0.92; 0.73 calcium 0.91; 0.90 phosphorus -; 0.79 sulfur -; 0.70 iron -; 0.86 manganese -; 0.78 boron -; 0.81 grape variety 100/96b cozzolino et al., 2003 geographic origin 93.5b liu et al., 2006 78b liu et al., 2008 76b cozzolino et al., 2011 60b riovanto et al., 2011 60.4b teixeira dos santos et al., 2017 95/87/84b hu et al., 2019 a portable nir spectrometer used b classification rate [%] sensory characteristics of wines, evaluation needs to be carried out by a sensory panel consisting of specially trained experts. sensory evaluations are time-consuming and despite the skills of the judging persons the results remain subjective. nir spectroscopy has been tested as an alternative method. it could be shown that some relationship exists between the spectra and the sensory properties of a wine, even without knowledge about chemical compounds (cozzolino et al., 2005). the results also showed that nir spectroscopy is a suitable method in addition to sensory evaluation (cayuela et al., 2017). however, total substitution of sensory evaluation by instrumental analysis is not possible because the concentration of aroma compounds does not necessarily correlate with their contribution to the overall aroma, a factor, which is not sensed by instruments. apart from sensory characteristics, classes or single aroma compounds in wines can be analyzed by nir spectroscopy. the detection and determination of desired aroma compounds as well as of those causing off-flavors have been examined. despite their low concentrations, high prediction accuracies were reached in various studies (cynkar et al., 2007; smyth et al., 2008; genisheva et al., 2018; fuentes et al., 2019). in some cases, the prediction accuracy of the calibration models was good to excellent while in the validation it was considerably worse, but were improved by the separation of the sample set (lorenzo et al., 2009; garde-cerdán et al., 2010; garde-cerdán et al., 2012). nonetheless, nir spectroscopy showed the potential to be used at least as a screening method. most of the evaluations in tab. 3 have been done for sensory attributes or aroma compounds in wine. few examinations were done regarding the aroma of grapes intended for winemaking (cynkar et al., 2007; boido et al., 2013), which has a great influence on the aroma of the resulting wine and therefore represents an important quality prediction parameter. many volatile compounds, which contribute to the aroma of the resulting wine are accumulated in the grapes as non-volatile glycosylated compounds but are released during winemaking and storage (aroma potential) (wirth et al., 2001). an early determination of the aroma potential can be helpful to decide about the best treatment of the grapes during winemaking. although glycosylated compounds are important, they only repre sent a part of the grape aroma and its role in the resulting wine aroma. the determination of the whole aroma profile of grapes by nir spectroscopy would provide a helpful tool to sort grapes before winemaking depending based on their aroma quality. moreover, all these studies have been carried out under laboratory conditions, tab. 3: applications of nir spectroscopy in the evaluation of grape and wine aroma samples compounds/attributes r2 reference chardonnay and riesling wines sensory attributes 0.68 – 0.86 (r) cozzolino et al., 2005 overall flavor 0.56 (r) sweetness 0.60 (r) riesling wines sensory attributes 0.38 – 0.89 (r) cozzolino et al., 2006b red and white wines sensory attributes 0.73 – 0.93 (r) cayuela et al., 2017 red wines wine scores 0.65 (r) cozzolino et al., 2008a tannat grapes glycosylated compounds 0.27 – 0.82 boido et al., 2013 white grape juice glycosylated compounds 0.73 – 0.94 cynkar et al., 2007 riesling wines esters 0.74 smyth et al., 2008 short-chain fatty acids 0.80 monoterpenes 0.90 white wines alcohols / esters 0.94 – 0.97 genisheva et al., 2018 aged red wines alcohols / esters / short chain fatty acids 0.29 – 0.94 lorenzo et al., 2009 barrel-aged red wines oak volatile compounds / ethylphenols 0.27 – 0.84 garde-cerdán et al., 2010 red and white grapes and wines guaiacol glycoconjugates 0.97 (r) fuentes et al., 2019 barrel-aged red wines haloanisols / halophenols 0.22 – 0.99 garde-cerdán et al., 2012 nir spectroscopy in process and quality control 33 which may cause a loss of time in practical application due to additional sampling time. the development of an in-line nir spectroscopy measurement of grape aroma compounds would be a great progress for the evaluation of the aroma quality of grapes directly on arrival at the winery. conclusions the suitability of nir spectroscopy has been examined and shown in numerous studies on fruits, grapes, wine, and wine aroma. miscellaneous compounds and attributes can be obtained from the nir spectrum in various kinds of matrices. with an adequate calibration, the nir spectroscopy provides a wide range of applications in process control and quality management (fig. 1). the calibration represents the most important and the most time-consuming part in establishing nir spectroscopy as an analytical method. dates quality at different stages of maturity utilising near-infrared (nir) spectroscopy. int. j. food prop. 20, 2950-2959. doi: 10.1080/10942912.2017.1387794 a ltieri, g., genovese, f., tauriello, a., di renzo, g.c., 2017: models to improve the non-destructive analysis of persimmon fruit properties by vis/nir spectrometry. j. sci. food agric. 97, 5302-5310. doi: 10.1002/jsfa.8416 a modio, m.l., ceglie, f., chaudhry, m.m.a., piazzolla, f., colelli, g., 2017: potential of nir spectroscopy for predicting internal quality and discriminating among strawberry fruits from different production systems. postharvest biol. technol. 125, 112-121. doi: 10.1016/j.postharvbio.2016.11.013 a muah, c.l.y., teye, e., lamptey, f.p., nyandey, k., opoku-ansah, j., adueming, p.o.-w., 2019: feasibility study of the use of handheld nir spectrometer for simultaneous authentication and quantifi cation of quality parameters in intact pineapple fruits. j. spectrosc. doi: 10.1155/2019/5975461 a rchibald, d.d., kays, s.e., 2000: determination of total dietary fiber of intact cereal food products by near-infrared refl ectance. j. agric. food chem. 48, 4477-4486. doi: 10.1021/jf000206j b eć, k.b., grabska, j., huck, c.w., 2020: principles and applications of miniaturized near-infrared (nir) spectrometers. chem. eur. j. 26, 1-20. doi: 10.1002/chem.202002838 b eć, k.b., huck, c.w., 2019: breakthrough potential in near-infrared spectroscopy: spectra simulation. a review of recent developments. front. chem. 7, 48-69. doi: 10.3389/fchem.2019.00048 b handari, d.g., millar, s.j., richmond, j.c., 2004: prediction of grain protein content through portable ft-nir measurement of developing wheat. in: lafi andra, d., masci, s., dʼovidio, r. 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infrared spectroscopies combined with electronic nose and electronic tongue. anal. chim. acta 697, 67-74. doi: 10.1016/j.aca.2011.04.020 b urns, d.a., ciurczak, e.w., 2008: handbook of near-infrared analysis. 3rd edition, boca raton, taylor & francis group. c âmara costa, r., uchida, v.h., bezerra veríssimo miguel, t., duarte, m.m.l., gomes de lima, k.m., 2017: quantifi cation of quality parameters in castanhola fruits by nirs for the development of prediction models using pls and variable selection algorithms on a laboratory scale. anal. methods 9, 352-357. doi: 10.1039/c6ay02454h c ao, q., zhegalova, n.g., wang, s.t., akers, w.j., berezin, m.y., 2013: multispectral imaging in the extended near-infrared window based on endogenous chromophores. j. biomed. opt. 18, 101318. doi: 10.1117/1.jbo.18.10.101318 c aramês, e.t.s., alamar, p.d., poppi, r.j., pallone, j.a.l., 2017: quality control of cashew apple and guava nectar by near infrared spectroscopy. j. food compos. anal. 56, 41-46. doi: 10.1016/j.jfca.2016.12.002 c ayuela, j.a., puertas, b., cantos-villar, e., 2017: assessing wine sensory attributes using vis/nir. eur. food res. technol. 243, 941-953. doi: 10.1007/s00217-016-2807-9 f ig. 1: field of applications of nir spectroscopy on-line determination provides similar results compared to measurements under laboratory conditions (aleixandre-tudo et al., 2019), so nir spectrometers can be integrated directly in the process. however, the potential of nir spectroscopy sometimes reaches its limits. for compounds, which are present in small amounts, semiquantitative determination or screening is possible (porep et al., 2014; nogales-bueno et al., 2015). nonetheless, several studies on compounds occurring in small amounts resulted in high prediction accuracy (ferrer-gallego et al., 2011; genisheva et al., 2018). classifi cation of samples can be improved by the use of mir spectroscopy or the combination of both techniques (riovanto et al., 2011; teixeira dos santos et al., 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weinsberg, germany e-mail: jana.gehlken@lvwo.bwl.de martin pour nikfardjam, state research institute for viticulture and pomiculture, traubenplatz 5, 74189 weinsberg, germany e-mail: martin.pourn@lvwo.bwl.de melissa kleb, institute of crop science, quality of plant products (340e), university of hohenheim, schloss westflügel, 70599 stuttgart, germany e-mail: melissa.kleb@uni-hohenheim.de christian zörb, institute of crop science, quality of plant products (340e), university of hohenheim, schloss westflügel, 70599 stuttgart, germany e-mail: christian.zoerb@uni-hohenheim.de © the author(s) 2021. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1016/j.tplants.2018.08.012 journal of applied botany and food quality 92, 204 215 (2019), doi:10.5073/jabfq.2019.092.029 institute of plant nutrition and soil science, kiel university plant-derived sulfur containing natural products produced as a response to biotic and abiotic stresses: a review of their structural diversity and medicinal importance muna ali abdalla*, karl h. mühling* (submitted: april 16, 2019; accepted: august 14, 2019) * corresponding author summary plant-derived sulfur-containing secondary metabolites constitute a small group of low-molecular weight natural products, which play a vital role in plant-pest interactions in numerous plant families and represent major defense molecules in the asteraceae, alliaceae, and brassicaceae families. in this review we highlight the crucial role of environmental stress factors in the production of s-containing secondary metabolites. furthermore, we describe a serendipitous variety of plant-derived sulfur-containing natural products produced or induced under biotic and abiotic stress and their structural diversity, promising pharmacological properties for use by humans, and beneficial effects for plants. specifically, cruciferous phytoalexins are known as elicit plant defense molecules. glucosinolates are candidates for tumorpreventive effects. cysteine sulfoxides found in garlic are considered as profound antimicrobial agents. in this review, we discuss types of s bonds in the molecules and their relevance for the medicinal effect as well as the biological activities of sulfur-containing secondary metabolites and possible future avenues. keywords: phytoalexins, glucosinolates, isothiocyanates, allicin, bioactivity, sulphur introduction sulfur is found in important metabolic molecules such as common sulfur-containing amino acids (methionine and cysteine), glutathione, proteins and sulpholipids (abdallah et al., 2010), as well as glucosinolates and allyl cys sulfoxides (saito, 2004). subsequently, these molecules play important roles in the plant lifecycle and the protection of plants from different environmental stresses and pathogens (lee et al., 2011). additionally, protein synthesis is reduced under sulfur deficiency conditions, in addition to the accumulation of soluble inorganic and organic nitrogenous compounds, e.g., as paragine (mortensen et al., 1992). the symptoms of sulfur defi ciency resemble that of nitrogen deficiency but are visible in younger leaves, as described by schnug and haneklaus (2005). moreover, without an adequate sulfur supply, plants cannot make efficient use of nitrogen and other nutrients and do not reach their full growth potential, in addition to an increased susceptibility to diseases (rausch and wachter, 2005). therefore, sulfur nutrition is known to have a potential effect on plant health (bloem et al., 2007), which can be maintained by a significant homeostasis between nitrogen and sulfur, as indicated by gerendás et al. (2008a) and maathuis (2009). for instance, several experimental studies have suggested that glucosinolate concentrations are influenced by higher sulfur fertility levels (kopsell et al., 2003; aries et al., 2006; malhi et al., 2007; schonhof et al., 2007). plants regulate the use of available sulfur, which is required for plant growth and development, in addition to resistance to stress (hawkesford, 2012). for synthesis of cysteine, a key component and a direct/indirect precursor of thiol-containing peptides, inclu ding glutathione (gsh), phytochelatins (pcs), and metallothioneins (mts) (davidian and kopriva 2010), inorganic sulfate is taken up by the roots from the rhizosphere and activated to adenosine 5'-phosphosulfate (aps) by atp sulfurylase. consequently, aps reductase catalyzed the reduction of aps into sulfite, which is reduced to sulfide and ultimately incorporated into o-acetylserine to form cysteine (reviewed by kopriva et al., 2012). on the other hand, aps is phosphorylated by aps kinase to obtain 3-phosphoadenosine 5-phosphosulfate (paps), which participates in the synthesis of other s-containing tryptophan-derived (indolic) and methionine-derived (aliphatic) natural products including glucosinolates and phytoalexines (frerigmann and gigolashvili, 2014). the sulfur assimilation pathways including enzymes involved in cysteine biosynthesis, and an overview of cysteine-rich peptides and s-containing secon dary metabolites biosynthesis are shown in fig. 1. both primary and secondary s-containing compounds play an important role in plant health and regulatory mechanism under stresses (rausch and wachter, 2005). in this regard, glutathione (gsh) is considered as the most important antioxidant and protector of plants under oxidative stress and the major non-protein sulfur source in plants (bloem et al., 2007; haneklaus et al., 2007, 2009; rennenberg and herschbach, 2012). it is very important to note that over the past decade, the important roles of secondary s-containing compounds in oxidative stress signaling and responses have received more attention (chan et al., 2019). for instance, del carmen martínez-ballesta et al. (2013) found that both foliar and root glucosinolate pool was increased in several brassica species under abiotic stresses including drought and salt stress. moreover, the hydrolysis products of glucosinolates might be implicated in the oxidative stress responses (chan et al., 2019). the regulation of primary and secondary sulfur metabolism has been investigated by using genomic, biochemical, and cellular studies. in this context, mugford et al. (2011) indicated that over-expression of aps reductase did not affect glucosinolate levels but increased the accumulation of thiols. consequently, both glucosinolate and thiols levels were not affected in mutants lacking the apr2 isoform of this enzyme. until now, the biosynthesis of s-containing secondary metabolites, the regulatory mechanisms involved in their production in response to biotic and abiotic stress, is still not fully understood. the main objective of this review is to emphasize the importance of environmental stress factors in the induction of s-containing secon dary metabolites. this could be used as a tool to manipulate the biosynthesis and induction of novel s-containing natural products with potential biological activities. in this review, we discuss plant-derived s-containing natural pro ducts isolated or induced under biotic and abiotic stresses, in addition to their medicinal significance. plant-derived sulfur containing natural products produced under biotic and abiotic stresses sulfur-containing phytoalexins from brassicaceae naturally occurring cruciferous phytoalexins are only produced in journal of applied botany importance of sulfur containing natural products 205 soil so4-2 glucosinolates phytoalexins high cys level rich thiol-reactive peptides s-containing secondary metabolites ppi adenosine5ʹ-phosphosulfate (aps) aps reductase so32 sulfite reductase s2 o-acetylserine thiolyase cys atp 2 gsh gssg amp 6 fd.red 6 fd.oxi sulfite sulfide o-acetylserine ch3cooh aps kinase paps involved in plant abiotic stress tolerance high gsh level involved in plant biotic and abiotic stress atp sulfurylase ! phytochelatins (pcs) ! metallothioneins (mts) ! glutathione (gsh; γ glutamyl-cysteinyl-glycine) plant so4-2 " plant growth regulators " antioxidants " antibiotics " metal chelators small quantities in damaged tissue and as complex mixtures inside plant tissue, and following exposure to pathogenic microbes. therefore, their isolation requires multiple chromatographic steps owing to their chemical stability under extraction as well as isolation steps, which afford small quantities. approximately 44 phytoalexins are known and their structures have mostly been confirmed by chemical synthesis. brassinin (1), 1-methoxybrassinin (3), and cyclobrassinin (6), were obtained from the inoculation of chinese cabbage heads brassica campestris with the bacterium pseudomonas cichorii. additionally, ultraviolet irradiation or inoculation with the bacterium erwinia carotovora enhanced the production of these molecules. their structures were identified by spectroscopic analysis and confirmed by synthesis (takasugi et al., 1986). three brassinin-related stress metabolites, named brassitin (2), 1-methoxyspirobrassinol (22), and (2r,3r)-1-methoxyspirobrassinol methyl ether (23), were obtained from the japanese radish “daikon” after inoculation with pseudomonas cichorii. the occurrence of 1-methoxyspirobrassinol (22), and 1-methoxyspirobrassinol methyl ether (23) suggests the in volvement of oxidized intermediates in the biosynthesis from brassinin to spirobrassinin (monde et al., 1995). 4-methoxybrassinin (4) was obtained from the inoculation of white cabbage heads b. oleracea with pseudomonas cichorii (monde et al., 1990). 1-methoxybrassitin (5) was obtained from the chinese cabbage brassica cam pestris (cruciferae) in addition to brassinin (1), 1-methoxybrassinin (3), cyclobrassinin (6) (takasugi et al., 1988). cyclobrassinin sulfoxide (7) was found in elicited leaves of brassica juncea (devys et al., 1990). sinalbin a (8) was produced by white mustard sinapis alba as a result of treatment with biotic and abiotic elicitors. additionally, sinalbin b (9) was found in extracts from elicited plants, and not in non-elicited controls. the structures of compounds 8 and 9 were confirmed by chemical synthesis (pedras and zaharia, 2000). phytoalexins 4-methoxycyclobrassinin (10), rutalexin (11), dehydrocyclobrassinin (12), 4-methoxydehydrocyclobrassinin (13), spirobrassinin (20), brassicanate a (27), brassicanal a (28), caulilexin a (30), brassilexin (31), in addition to 1-methoxybrassinin (3) and 4 methoxybrassinin (4), were detected in the nonpolar extract of roots of canola (brassica napus) infected with phytopathogenic plasmo diophora brassicae (clubroot). quantitative analysis of the compounds was performed by using hplc (dad and lc-esi-ms) ana lysis (pedras et al., 2008). surprisingly, the concentration of many of the induced compounds collected from the infected roots of canola was significantly increased after several weeks (tab. 1). cabbage tissue brassica oleracea inoculated with pseudomonas cichorii delivered 1-methoxybrassenins a and b (14 and 15) (monde et al., 1991). brassicanal c (29) was produced in florets of cauliflower (brassica oleracea) under abiotic conditions (uv light), along with isalexin, spirobrassinin (20), 1-methoxybrassitin (5), and caulilexins a (30) and b (pedras et al., 2006a). wasalexins a (16) and b (17) were obtained from the foliar tissue of wasabi under elicitation with p. lingam, p. wasabiae or cuc12 (pedras et al., 1999). uv irradiation of chopped stem tubers of kohlrabi (brassica oleracea) and subsequent incubation for 4 days resulted in the isolation of (r)-1-methoxy-spirobrassinin (21) along with cyclobrassinone, and spirobrassinin (20). additionally, the production of these indole phytoalexins was also enhanced by elicitation with cucl2 (gross et al., 1994). however, the authors did not determine the yields of compounds 20 and 21, which were induced in response to uv light and cucl2 conditions. erucalexin (24) was found in leaves of dog mustard erucastrum gallicum in response to biotic stress such as that elicited with s. sclerotiorum and abiotic stress elicited with cucl2 (pedras et al., 2006). uv radiation, nacl irrigation, or cucl2 spray induced the production of wasalexins a (16) and b (17) in thellun fig. 1.: sulfur assimilation pathway. the initial step of sulfur assimilation pathway is catalyzed by atp-sulfurylase. role of atp-sulfurylase in plant abiotic and biotic stress tolerance through different rich thiol-reactive peptides including (cys, gsh, and mts) and s-containing secondary metabolites is listed. positive and negative regulation of atp-sulfurylase is indicated by arrows and blunt ends, respectively (redrawn from saito 2004; hirai and saito 2008). 206 m.a. abdalla, k.h. mühling giella halophila; biswasalexins a1 (18) and a2 (19) were obtained from head-to-tail photodimerization of wasalexin a (16) (pedras et al., 2009). sinalexin (32) was found in white mustard (sinapis alba) under biotic or abiotic stress (pedras and smith, 1997). brussalexin a (33) was produced in brussels sprouts irradiated under uv light (pedras et al., 2007a). furthermore, camalexin (34) and 6-methoxycamalexin (35) were produced by camelina sativa leaves as a response to fungus altenmia brassme elicitation (browne et al., 1991). 1-methyl-camalexin (36) was obtained from the leaves of capsella bursa-pastoris elicited by alternaria brassicae (jimenez et al., 1997). regarding compounds 34, 35 and 36, which were obtained from different plant species in very low quantities, the authors did not mention exactly how much compounds were isolated in total from the crude material. hence, quantitative data is very important to understand how much of the compounds was induced in how many days after inoculation. canola plants brassica rapa delivered rapalexins a (37) and b (38) as a response to biotic stress when infected with the oomycete albugo candida and abiotic stress by uv (pedras et al., 2007b). isolated or induced s-containing natural products under biotic and abiotic stress, their plant sources and yield data are listed in tab. 1. from the findings mentioned above, it is very important to note that the elicited compounds have different incubation periods, which are required for maximum production. consequently, the induced compounds can be detected within hours up to weeks depending on type of stress and the particular species as reviewed by (pedras et al., 2011). for instance, brassicanal c (29) was detected after 48 hours and reached its maximum production after approximately three days upon elicitation with uv light (pedras et al., 2006a). however, the yield was relatively lower after 5 days. rapalexins a (37) and b (38) were induced after 8 and 5 days, respectively, and reached their maximum production after 9 and 8 days, respectively. the yield of both compounds was not increased after 10 days (pedras et al., 2007b). although the phytoalexins above have been identified by mass spectrometry, nmr, ir, and uv spectroscopy, hplc-dadms represent the most reliable technique to identify and quantify them in plant extracts hence most of these compounds were produced in low quantities (pedras et al., 2006). organosulfur compounds obtained from garlic cysteine sulfoxides found in garlic (allium sativum l. family lilia ceae) and other allium species are synthesized by the enzyme alliinase in crushed plant material (kusterer and keusgen 2010). allicin (40), a typical cysteine sulfoxide of garlic, is synthesized from non-proteinogenic amino acid alliin (39), as a first primary product (kusterer and keusgen 2010). rearrangement of compound 40 delivers diallyl sulfides (such as compounds 41 and 44), dithiins (45 and 46), ajoene (42) (amagase et al., 2001). furthermore, thiacremonone (47) was isolated from garlic (kim et al., 2012). importantly, allicin (40) is known as a defence compound with a wide range of pharmacological properties (borlinghaus et al., 2014). for instance, several garlic preparations have been produced to prevent stroke and arteriosclerosis. in this regard, allicin (40) and other organosulfur compounds were suggested to be the main metabolites responsible for the entire activity (krest and keusgen 1999). glucosinolates glucosinolates are known as sulfur-containing molecules present in cruciferous vegetables including arabidopsis thaliana and brassica crop species (ahuja et al., 2016). more than 200 different naturally occurring glucosinolates have been identified (koprivova and kopriva 2016). the general structure of glucosinolates is shown in fig. 6; more than 200 side-groups (r) were found in the literature (franco et al., 2016). different side-groups (r) of some glucosinolates are shown in fig. 7. glucosinolates form the essential component of the dual glucosinolate-myrosinase system, which consists of glucosinolates and their hydrolytic enzymes, myrosinases, which catalyze the breakdown of glucosinolate into various bioactive compounds such as isothiocyanates because of tissue disruption or insect attack (barth et al., 2006; pitann et al., 2017). in addition to isothiocyanates, thiocyanates and nitriles are considered as important breakdown products of glucosinolates, which played a pivotal role in plant defense against various pathogenic microbes and herbivores (huseby et al., 2013). compound name r r1 r2 1 brassinin h h s 2 brassitin h h o 3 1-methoxybrassinin och3 h s 4 4-methoxybrassinin h och3 s 5 1-methoxybrassitin och3 h o fig. 2: brassinin (1), brassitin (2), 1-methoxybrassinin (3), 4-methoxybrassinin (4) and 1-methoxybrassitin (5) compound name r r1 r2 6 cyclobrassinin h h sch3 7 cyclobrassinin sulfoxide h h s(o)ch3 8 sinalbina och3 h s(o)ch3 9 sinalbinb och3 h sch3 10 4-methoxycyclobrassinin h och3 sch3 fig. 3: cyclobrassinin (6), cyclobrassinin sulfoxide (7), sinalbins a and b (8 and 9) and 4-methoxycyclobrassinin (10) importance of sulfur containing natural products 207 34: camalexin r=r1=h 35: 6-methoxycamalexin, r= h, r1= och3 36: 1-methylcamalexin, r=ch3, r1=h 37: rapalexin a, r=h 38: rapalexin b, r=oh 1 fig. 4. structures of sulfur-containing phytoalexins (11– 38) 2 3 4 5 organosulfur compounds obtained from garlic 6 cysteine sulfoxides found in garlic (allium sativum l. family liliaceae) and other allium 7 species are synthesized by the enzyme alliinase in crushed plant material (kusterer and 8 keusgen 2010). allicin (40), a typical cysteine sulfoxide of garlic, is synthesized from 9 non-proteinogenic amino acid alliin (39), as a first primary product (kusterer and 10 keusgen 2010). rearrangement of compound 40 delivers diallyl sulfides (such as 11 compounds 41 and 44), dithiins (45 and 46), ajoene (42) (amagase et al., 2001). 12 furthermore, thiacremonone (47) was isolated from garlic (kim et al., 2012). importantly, 13 allicin (40) is known as a defence compound with a wide range of pharmacological 14 properties (borlinghaus et al., 2014). for instance, several garlic preparations have been 15 produced to prevent stroke and arteriosclerosis. in this regard, allicin (40) and other 16 organosulfur compounds were suggested to be the main metabolites responsible for the 17 entire activity (krest and keusgen 1999). 18 19 20 21 39: alliin 40: allicin 41: diallyl disulfide (dds) 42: ajoene 43: s-allyl cysteine (sac) 44: diallyl trisulfide (dts) 45: 2-vinyl-4 -h-1,3-dithiin 46: 3-vinyl-4-h-1,2-dithiin 47: thiacremonone fig. 5. structures of organosulfur compounds found in garlic (39– 47) 22 23 24 25 26 11: rutalexin 12: dehydrocyclobrassinin r=h 13: 4-methoxydehydrocyclobrassi nin r=och3 14: 1-methoxybrassenin a r=h2 15 1-methoxybrass-enin b r=o 16: wasalexin a 17: wasalexin b 18: biswasalexin a1 19: biswasalexin a2 20: (s)-spirobrassinin 21: (r)-1-methoxyspirobrassinin 22: 1 methoxyspirobrassin ol 23: (2r,3r)-1-methoxy spirobrassinol methyl ether 24: erucalexin 25: dioxibrassinin 26: brassicanal b 27: brassicanate a r=cooch3 28: brassicanal a r=cho 29: brassicanal c 30: caulilexin a 31: brassilexin r =h 32: sinalexin r=och3 33: brussalexin a fig. 4: structures of sulfur-containing phytoalexins (11-38) sinigrin (48) is hydrolyzed by myrosinase upon mechanical injuries of the plant tissue, which produces compounds named allyl isothiocyanate, ally thiocyanate, allyl cyanide, and 1-cyano-2,3-epi thiopropane (gerendás et al., 2009; saladino et al., 2016). the biosynthesis of glucosinolates can be manipulated by several environmental stress factors including light, nutrients, fungal infection, wounding, and insect damage (gerendás et al., 2008b; radojcic redovnikovic et al., 2008). for instance, kim et al. (2018) indicated that kale grown under treatments with nacl, na2seo3, or both would enhance the biosynthesis of glucosinolates including sinigrin (48) as well as isothiocyanates. in a recent study, geilfus et al. (2016) found that the total glucosinolates content in chinese cabbage (brassica rapa l. ssp. pekinensis) increased with higher n and s treatment. however, the ratios among individual glucosinolates remained unchanged. additionally, the authors observed that the addition of 0.3 g sulfur per pot significantly enhanced the whole shoot biomass compared with the 0 g sulfur control (geilfus et al., 2016). a previous report indicated that the infection of chinese cabbage (brassica 208 m.a. abdalla, k.h. mühling campestris sp. pekinensis) with plasmodiophora brassicae changed the levels of various glucosinolates. additionally, significant diffe rences in the glucosinolate content was observed in susceptible and tolerant varieties (ludwig-müller et al., 1997). subsequently, the authors reported that tolerant varieties induced the production of aromatic glucosinolates between 14 and 30 days following the infection, whereas susceptible varieties enhanced the biosynthesis of aliphatic and indole glucosinolates. other reports indicate that the level of indole glucosinolates increased in oilseed rape and chinese cabbage in response to alternaria brassicae infection (doughty et al., 1991; rostás et al., 2002). furthermore, the biosynthesis of indole gluco sinolate was significantly increased by elicitation of arabidopsis with erwinia carotovora (brader et al., 2001). a recent study indicated that additional sulfur supply in the nutrient medium enhanced the content of aliphatic glucosinolates in eruca sativa (kastell et al., 2018). moreira-rodríguez et al. (2017) studied the effect of uva and uvb light and methyl jasmonate on the biosynthesis of glucosinolates and other metabolites in broccoli sprouts. the results revealed that treatments with uva + methyl jasmonate and uvb + methyl jasmonate induced the level of total glucosinolate by ~154% and ~148%, respectively. methyl jasmonate (mj) stimulated the biosynthesis of indole glucosinolates such as neoglucobrassicin (61) (~538%). furthermore, uvb increased the level of aliphatic and indole glucosinolates, including glucoraphanin (51) (~78%) and 4-methoxy-glucobrassicin (60) (~177%) (moreira-rodríguez et al., 2017). isolated or induced compounds under stress, their plant sources and yield data are listed in tab. 1. types of s bonds in the molecules and their relevance for the medicinal effect structural diversity of plant-derived sulfur containing natural pro ducts is not only means the potential chemical structures (1-53) for drug development, however the more interesting feature of these different compounds, which might be responsible for the selective and specific biological activities. different sulfur bonds in the molecules may have relevance for the medicinal effect of s-containing natural products. for instance, disulfide bonds have gained significant attention in various fields including pharmaceutical, biochemical and biotechnological fields. they have important properties such as the capability to break into a reduced form of glutathione in a thiol disulfide exchange reaction, they are stabile in human body, and have no physiological toxicity (wang et al., 2016). disulfide bonds participate in the biological activity of several s-containing secondary metabolites, as reported by feng et al. (2016), who indicated that the disulfide bond is important for the antimicrobial activity of ajoene (42). moreover, thiosulfinates including ajoene (42) demonstrated antimicrobial potential owing to the presence of a disulfide bond, which reacts with the thiol groups of cellular proteins. because ajoene (42) contains a sulfinyl group, which has been found to be res ponsible for the antibacterial activity of allicin (40), its antimicrobial activity has been attributed to the presence of a disulfide bond and the sulfinyl (feng et al., 2016). moreover, allicin (40) is known as a reactive sulfur species (gruhlke and slusarenko, 2012) with oxidizing characteristics. for instance, it can oxidize thiols in cells, including glutathione and cysteine residues in proteins, through disulfide bond formation. consequently, redox-stimulated structural changes in proteins result in a net change of loss or gain function, which is known for the plant protein npr1, a key protein in pathogen-triggered immunity (tada et al., 2008). pedras et al. (2006a) reported that among phytoalexins elicited in floret of cauliflower under abiotic stress (uv light), caulilexin a (30), which has a disulfide bridge, demonstrated a complete growth inhibition of rhizoctonia solani at 5.0 × 10-4 m and sclerotinia sclerotiorum at 1.0 × 10-4 m. moreover, compound (30) exhibited complete growth inhibition at 1.0 × 10-8 m in a tlc bioassay against cladosporium cucumerinum, whereas all other tested phytoalexins inhibi ted the mycelial growth at 1.0 × 10-6 m, which is 100 times higher concentration. moreover, allium species, which contain a vinyldithiinsin group with exo and endo double bonds in a ring system, have demonstrated remarkable bioactivities. for instance, 3,4-dihydro-3-vinyl-1,2-dithiin (46) was reported to have higher antioxidative activity for human ldl than aliphatic dialk(en)yl disulfides and trisulfides. this can be attributed to the conjugation of a double bond to a nonbonding electron on the sulfur in a ring system, as reported by higuchi et al. (2003) biological activities of plant-derived sulfur containing metabolites produced under biotic and abiotic stresses antimicrobial activity in principle plants have three main approaches to fight pathogenic microbes including strengthening the cell wall, inhibition of microbial enzymes by apoplastic defense and synthesis of toxic natural products such as phytoalexins (bloem et al., 2015). phytoalexins 1-38 have shown promising antifungal activities against a wide range of plant pathogenic fungi in addition to antibacfig. 6: general structure of glucosinolates 39: alliin 40: allicin 41: diallyl disulfide (dds) 42: ajoene 43: s-allyl cysteine (sac) 44: diallyl trisulfide (dts) 45: 2-vinyl-4 -h-1,3-dithiin 46: 3-vinyl-4-h-1,2-dithiin 47: thiacremonone fig. 5: structures of organosulfur compounds found in garlic (39-47) importance of sulfur containing natural products 209 terial activity (reviewed by pedras and yaya, 2010). for instance, the phytoalexins sinalbins a (8) and b (9) demonstrated antifungal activity against plant pathogenic fungi leptosphaeria maculans. at a concentration of 5 × 10-4 m, sinalbin a (8) exhibited complete inhibition of spore germination (ed50 2 × 10-4 m at 48 h) for the duration of the assay, which was 7 days. additionally, sinalbin b (9) showed moderate activity (ed50 7 × 10-4 m at 48 h) at a similar concentration (5 × 10-4 m), which was approximately 30% germination inhibition in comparison to controls, after 48 h (pedras and zaharia, 2000). the antimicrobial potential of allium species and their organosulfur compounds including allicin (40) thiosulphinates, and their transformation products has been previously reported (samadi and keusgen, 2013). for instance, the minimal concentration of diallyl disulfide (41) and allicin (40) to inhibit the growth of escherichia coli and staphylococcus aureus was 6.15 and 0.17 mm, respectively, which was 35× more than allicin (40) alone (koch and lawson, 1996). additionally, pure allicin (40) extracted from garlic showed remarkable antibacterial activity towards bacillus spp., salmonella typhimurium, and vibrio cholerae with complete growth inhibition at a concentration of 80 μm (borlinghaus et al., 2014). strehlow et al. (2016) reported that sample solutions contain 3 mg/ ml racemic alliin and 0.2 mg/ml alliinase did not exhibit antibacterial activity, on the other side the combination of racemic alliin and alliinase, which might be contain 0.75 mg/ml allicin (40) de monstrated inhibitory activity against e. coli. it is very important to mention the substantial achievement of strehlow et al. (2016), who studied the stabilization of both alliin (39) and alliinase in lactose microspheres obtained by previously described spray-drying formulation. the authors indicated for the first time that allicin (40) synthesized in situ could be readily used for the treatment of pulmonary microbial infections (strehlow et al., 2016). a previous study indicated that several studies have indicated the antibacterial potential of glucosinolates and their hydrolysis pro ducts, in addition to their activity against molds and yeasts (saladino et al., 2016). sinigrin (48) had weak antimcrobial activity, whereas its hydrolysis products exhibited a promising effect on the inhibition of microbial growth (brabban and edwards, 1995; pitann et al., 2017). anticancer potential indole phytoalexins are known to have anti-cancer, chemopreventive, and antiproliferative activity (chripkova et al., 2016). furthermore, many phytoalexins such as brassinin (1), cyclobrassinin (6), and spirobrassinin (20), exhibit a cytotoxic effect (sabol et al., 2000). additionally, 1-methoxybrassinin (3) (pilatova et al., 2005), 1-methoxyspirobrassinol (22), and 1-methoxyspirobrassinol methyl ether (23) have demonstrated antiproliferative activity (monde et al., 2005). diallyl sulfides, such as diallyl disulfide (41) and diallyl trisulfide (44) obtained from garlic, have anticancer effects (lai et al., 2012). the cytotoxicity of compound 44 showed great effects on the production of reactive oxygen species (ros); compound 44 is known as key mediator in the apoptotic signaling pathway and induces the ros-dependent caspase pathway in u937 leukemia cells (choi and park, 2012). compound (44) was found to activate apoptosis against a diverse group of human cancer cell lines in vitro in addition to providing better protection towards tumor growth in animal models in vivo such as colorectal cancer (yu et al., 2012). s-allylcysteine (43) exhibited an in vitro cancer chemopreventive effect. it was suggested to be an interesting therapeutic agent for prostate cancer (liu fig. 7: different side groups (r) of some glucosinolates (48-64) no. name r no. name r 48 sinigrin 57 glucoerucin 49 gluconapin 58 glucosquerellin 50 glucoiberin 59 glucobrassicin 51 glucoraphanin 60 4-methoxygluco-brassicin 52 glucoalyssin 61 neoglucobrassicin 53 glucohesperin 62 4-hydroxygluco-brassicin 54 glucoibarin 63 glucotropaeolin 55 glucohirsutin 64 gluconastrutiin 56 glucoibervirin 210 m.a. abdalla, k.h. mühling ta b. 1 : pl an t s pe ci es , i nv es tig at ed p la nt p ar t, ty pe o f s tr es s, is ol at ed o r i nd uc ed c om po un ds o f i nt er es t a nd y ie ld d at a p la nt s pe ci es in ve st ig at ed k in d of is ol at ed o r in du ce d co m po un ds y ie ld d at a r ef er en ce s pl an t p ar t st re ss b ra ss ic a ca m pe st ri s l . c ab ba ge b io tic a nd b ra ss in in (1 ), 1m et ho xy br as si ni n (3 ), b ra ss in in (1 ) ( 8 m g) , 1 -m et ho xy br as si ni n (3 ) ( 39 m g) , a nd (t a k a su g i e t a l., ss p. p ek in en si s he ad s ab io tic an d cy cl ob ra ss in in (6 ), cy cl ob ra ss in in (6 ) ( 20 m g) 19 86 ) r ap ha nu s sa tiv us r oo ts b io tic b ra ss iti n (2 ), 1m et ho xy sp ir ob ra ss in ol (2 2) , b ra ss iti n (2 ) ( 6 m g) , 1 -m et ho xy sp ir ob ra ss in ol (2 2) (6 m g) , a nd ( m o n d e e t a l., 1 99 5) va r. ho rt en si s an d (2 r ,3 r )1m et ho xy sp ir ob ra ss in ol (2 r ,3 r )1m et ho xy sp ir ob ra ss in ol m et hy l e th er (2 3) (1 6 m g) m et hy l e th er (2 3) b . o le ra ce a l . c ab ba ge b io tic 4m et ho xy br as si ni n (4 ) c om po un d 4 (6 m g) ( m o n d e e t a l., 1 99 0) va r. ca pi ta ta he ad s b ra ss ic a ca m pe st ri s l . c ab ba ge b io tic 1m et ho xy br as si tin (5 ) c om po un d 5 (1 5. 9 m g) ( t a k a su g i e t a l., 1 98 8) ss p. p ek in en si s he ad s b ra ss ic a ju nc ea le av es b io tic c yc lo br as si ni n su lf ox id e (7 ) n ot d et er m in ed ( d e v y s et a l., 1 99 0) si na pi s al ba l ea f a nd b io tic a nd si na lb in s a (8 ) a nd b (9 ) si na lb in a (8 ) ( 3. 5 m g) a nd s in al bi n b (9 ) ( 5. 5 m g) ( p e d r a s an d z a h a r ia , st em ti ss ue s ab io tic 20 00 ) b ra ss ic a na pu s in fe ct ed b io tic 4m et ho xy cy cl ob ra ss in in (1 0) , r ut al ex in (1 1) , t he c on ce nt ra tio n of c om po un d 10 (3 2 nm ol /g fr es h w ei gh t) . c om po un d 11 ( p e d r a s et a l., 2 00 8) cv . w es ta r ro ot s de hy dr oc yc lo -b ra ss in in (1 2) , 4 -m et ho xy (8 .5 a nd 9 .6 n m ol /g fr es h w ei gh t) in ro ot s co lle ct ed fo ur a nd fi ve to s ix w ee ks de hy dr oc yc lo br as si ni n (1 3) , s pi ro br as si ni n af te r i no cu la tio n, re sp ec tiv el y. c om po un d 12 a nd 1 3 w er e in du ce d af te r fi ve (2 0) , b ra ss ile xi n (3 1) w ee ks in th e in fe ct ed ro ot s bu t n ot in th e co nt ro l r oo ts . t he c on ce nt ra tio n of co m po un ds 2 0 an d 31 w as 2 126 a nd 5 -7 n m ol /g fr es h w ei gh t, re sp ec tiv el y. t he y w er e de te ct ed o nl y in th e in fe ct ed ro ot s co lle ct ed a ft er fi ve a nd s ix w ee ks . b ra ss ic a ol er ac ea c ab ba ge b io tic 1m et ho xy br as se ni ns a (1 4) a nd b (1 5) c om po un d 14 (6 .1 m g) a nd 1 5 (1 20 m g) ( m o n d e e t a l., 1 99 1) va r. c ap ita ta tis su e b ra ss ic a ol er ac ea fl or et s a bi ot ic b ra ss ic an al c (2 9) t he c on ce nt ra tio n of c om po un d 29 w as 0 .2 2± 0. 05 μ m ol /1 00 g fr es h flo re t t is su e, ( p e d r a s et a l., 2 00 6a ) va r. bo tr yt is 0. 49 ±0 .0 9 μm ol /1 00 g fr es h flo re t t is su e, 0 .4 3± 0. 09 μ m ol /1 00 g fr es h flo re t t is su e, an d 0. 30 ±0 .0 9 μm ol /1 00 g fr es h flo re t t is su e af te r 4 8, 7 2, 9 6 an d 12 0 ho ur s, re sp ec tiv el y. w as ab ia ja po ni ca , fo lia r b io tic a nd w as al ex in s a (1 6) a nd b (1 7) , w as al ex in s a (1 6) a nd b (1 7) w er e pr od uc ed in re la tiv el y lo w a m ou nt s un de r ( p e d r a s et a l., 2 00 9) sy n. e ut re m a w as ab i tis su e ab io tic an d bi sw as al ex in s a 1 (1 8) a nd a 2 (1 9) n ac l c on di tio ns . c om po un d 18 (6 0 nm ol /g fr es h w ei gh t) a nd 1 9 (1 5 nm ol /g fr es h w ei gh t) in u v -i rr ad ia te d pl an ts . t he y ie ld w as d ec re as ed fo r 1 8 an d 19 in p la nt s sp ra ye d w ith c uc l 2 , in w hi ch th e co nc en tr at io n of c om po un d 18 w as a pp ro xi m at el y 10 n m ol /g fr es h w ei gh t an d 19 w as n ot d et ec te d b ra ss ic a ol er ac ea st em a bi ot ic (r )1m et ho xy -s pi ro br as si ni n (2 1) no t d et er m in ed ( g r o ss e t a l., 1 99 4) va r. go ng yl od es tu be rs e ru ca st ru m g al lic um l ea ve s b io tic a nd e ru ca le xi n (2 4) t he y ie ld o f c om po un d 24 w as 2 .2 m g. t he is ol at io n st ep s w er e re pe at ed fo ur ti m es ( p e d r a s et a l., 2 00 6) ab io tic to o bt ai n a su ffi ci en t a m ou nt fo r s tr uc tu re e lu ci da tio n an d bi oa ct iv ity . (1 2 m g w as is ol at ed in to ta l f ro m 2 00 p la nt s an d 6 g of e xt ra ct ). si na pi s al ba l ea ve s b io tic o r si na le xi n (3 2) 1. 4 m g ( p e d r a s an d sm it h , ab io tic 19 97 ) b . o le ra ce a sp ro ut s a bi ot ic b ru ss al ex in a (3 3) 2 m g of c om po un d 33 w as o bt ai ne d fr om 3 .9 k g of fr es h tis su e. ( p e d r a s et a l., 2 00 7a ) va r. ge m m ife ra c am el in a sa tiv a l ea ve s b io tic c am al ex in (3 4) a nd 6 -m et ho xy ca m al ex in (3 5) n ot d et er m in ed (b r o w n e e t a l., 1 99 1) c ap se lla b ur sa -p as to ri s l ea ve s b io tic 1m et hy lca m al ex in (3 6) n ot d et er m in ed ( j im e n e z e t a l., 1 99 7) b ra ss ic a ra pa b io tic r ap al ex in s a (3 7) a nd b (3 8) fo r r ap al ex in a (3 7) , t he 0 .4 -0 6, 0 .7 -1 .1 , a nd 0 .7 -0 .9 n m ol g -1 fr es h le av es (p e d r a s et a l., 2 00 7b ) w er e pr od uc ed a ft er 8 , 9 , 1 0 da ys , r es pe ct iv el y fr om in oc ul at io n. h ow ev er , th e fo r c om po un d 38 , 2 .2 -3 .3 , 4 .1 -8 .1 , 4 .3 -8 .3 . 5 .3 -1 4. 7, 3 .9 -9 .7 , a nd 7 .5 -9 .1 n m ol g -1 fr es h le av es w er e pr od uc ed a ft er 5 , 6 , 7 , 8 , 9 , a nd 1 0 da ys , r es pe ct iv el y fr om in oc ul at io n. c om po un d 37 w as o nl y de te ct ed a ft er 8 d ay s b ra ss ic a ol er ac ea r oo ts a bi ot ic si ni gr in (4 8) t he c on ce nt ra tio n in cr ea se d by th e ad di tio n of n a 2 se o 3 a lo ne (k im e t a l., 2 01 8) or in c om bi na tio n w ith n ac l. b ra ss ic a ol er ac ea b ro cc ol i a bi ot ic g lu co ra ph an in (5 1) u v b in cr ea se d th e co nt en t t o 23 .6 ±2 .1 m m ol /k g dr y w ei gh t ( m o r e ir a -r o d r íg u e z va r. ita lic a sp ro ut s et a l., 2 01 7) b ra ss ic a ol er ac ea b ro cc ol i a bi ot ic 4m et ho xy -g lu co br as si ci n (6 0) u v b in cr ea se d th e co nt en t t o 12 .7 ±0 .5 m m ol /k g dr y w ei gh t ( m o r e ir a -r o d r íg u e z va r. ita lic a sp ro ut s et a l., 2 01 7) b ra ss ic a ol er ac ea b ro cc ol i a bi ot ic n eo gl uc ob ra ss ic in (6 1) in cr ea se d sy ne rg is tic al ly b y 96 .4 ±1 .5 a nd 9 2. 8± 6 m m ol /k g dr y w ei gh t ( m o r e ir a -r o d r íg u e z va r. ita lic a sp ro ut s un de r u va + m j an d u v b + m j tr ea tm en t, re sp ec tiv el y. et a l., 2 01 7) importance of sulfur containing natural products 211 et al., 2012). moreover, allicin (40), has been reported to be a pro mising candidate as a tumor suppressor in human colon cancers (bar-chen et al., 2010). more and above, several studies have indicated that brassicaceae species possess chemoprevention towards different types of cancers in humans owing to their high glucosinolates content (merah, 2015). anti-inflammatory activity in a recent study, garlic, allicin (40), and the commonly used drug praziquantel were used to treat six groups of mice infected with schistosoma mansoni cercariae. the results showed that the treatment decreased the worm burden in addition to the reduction of serum concentrations of liver fibrosis markers and proinflammatory cytokines. nonetheless, praziquantel exhibited the most inhibitory activity for the reduction of the number of worms (metwally et al., 2018). the study of lee et al. (2012) indicated that two isomers (z and e) of ajoene (42) and their oxidized sulfonyl derivatives de monstrated anti-inflammatory activity by suppressing the production of nitric oxide and prostaglandin e2 (pge2) in addition to the expression of the pro-inflammatory cytokines including tumor necrosis factor α (tnfα), interleukin-1 β (il-1β), and interleukin-6 in lipopolysaccharide (lps)-stimulated macrophages. the study of vo et al., (2013) indicated that aromatic glucosinolates had a moderate anti-inflammatory effect. the authors indicated that because naturally occurring glucosinolates in plant material were present as a mixture of various aromatic and aliphatic compounds, these metabolites might act synergistically during consumption of a normal diet. they also suggested that these compounds can be hydrolyzed by the enzyme myrosinase, which is present in various brassica species. the hydrolysis products include isothiocyanates and glucoraphanin (51) and sulforaphane in the case of the aliphatic metabolite. fahey et al. (1997) suggested that these metabolites have better bioactivity than the entire glucosinolates. neuroprotective properties among glucosinolates, glucoraphanin (51), sulforaphane, and isothiocyanates were found to be the most fascinating compounds as modulators of various systems associated with the pathogenic mechanism of various neurological diseases including oxidative stress, apoptosis, and inflammation (venditti and bianco, 2018). additionally, garlic compounds demonstrated a neuroprotection effect, which was attributed to their antioxidant capacity, modulation of apoptosis mediators and reduction of the formation of amyloid protein (venditti and bianco, 2018). plant protection phytoalexins are serendipidious plant metabolites, owing to their amazing role in protecting plants against a wide range of microbial pathogenic microorganisms. elevated levels of such defensive natural products in plants might increase their resistance to disease (pedras and yaya, 2010; bloem et al., 2015). curtis et al. (2004) studied the antifungal activity of allicin (40) in fresh garlic juice by using a plate-diffusion method using sporeseeded agar and found that allicin (40) demonstrated strong in vitro activity against many plant-pathogenic fungi such as magnaporthe grisea, botrytis cinerea, plectospherella cucumerina, and alterna ria brassicicola. furthermore, nematicidal activities of diallyl disulfide (41), and diallyl trisulfide (44) against the pine wood nematode (bursaphelenchus xylophilus) have been reported; diallyl trisulfide (44) exhibited more than 10-fold lower lc50 than diallyl disulfide (41) (cetintas and yarba, 2010). distilled garlic oil significantly inhibited root galling after inoculation of tomato roots with root-knot nematode (meloidogyne incognita) (danquah et al., 2011). the insecticidal and acute toxicity effect of diallyl disulfide (41) and diallyl trisulfide (44), against callosobruchus chinensis (found in bean weevil) were studied; diallyl trisulfide (44) possessed stronger toxicity than diallyl disulfide (41) and crude garlic oil. it has been reported that diallyl trisulfide (44) demonstrated promising effect against rice weevil sitophilus oryzae, red flour beetle (tribolium castaneum), and the maize weevil (sitophilus zeamais) (huang et al., 2000; mikhaiel, 2011; koul, 2004). diallyl trisulfide (44) was the strongest fumigant ingredient in garlic oil and showed higher activity than diallyl disulfide (41) against the pine wood nematode (bursaphelenchus xylophilus) (koul, 2004; park et al., 2006; nowsad et al., 2009; mikhaiel, 2011). several garlic-based products are on the market today for use in different agricultural and horticultural practices. however, many of these products have not been approved by the fda (us food and drug administration) (anwar et al., 2017). glucosinolates hydrolysis products have demonstrated potential antimicrobial activity against different plant pathogenic microorganisms (bloem et al., 2015; hanschen et al., 2018). accordingly, they could be used as alternatives to commercial chemicals to control phytopathogenic microorganisms. glucosinolates are mainly present in young leaves, seeds, and siliques; additionally, their intermediates are found in leaves, stems, and roots (agneta et al., 2014). aghajanzadehdivaei (2015) indicated, that the higher content of indole glucosinolates and subsequently their hydrolysis products, which were detected in roots, could be attributed to their better stability in the soil than in air. furthermore, ludwig-mueller et al. (1999) reported that these metabolites might limit the development of root disease, which has been raised by plasmodiophora brassicae. vola tile molecules obtained from macerated brassicae root tissue decreased the fungal infection of wheat, gaeumannomyces graminis (angus et al., 1994). rhizospheric strains of fusarium possessed a potential effect on lepidium sativum against pythium ultimum. subsequently, these strains enhanced the production of benzyl isothiocyanate, and of its precursor glucotropaeolin in the roots of brassicaceae plants. moreover, the assemblage of isothiocyanate in roots enhanced the resistance of l. sativum against pythium ultimum (ishimoto et al., 2004). moreover, cauliflower plants (brassica oleracea var. botrytis) were infected by peronospora parasitica to evaluate the relationship between glucosinolates and resistance against downy mildew. the authors reported that a higher amount of sinigrin (48) was detected in resistant varieties in comparison to susceptible ones (ménard et al., 1999). different biological activities of plant-derived s-containing secon dary metabolites were emphasized to alert researchers of the importance of biotic and abiotic stress factors as a potential tool to produce and enhance the synthesis of s-containing secondary metabolites with remarkable bioactivities. as reported in several studies, it is clear that sulfated phytoalexins and glucosinolates constitute an important part of the plant defense repertoire owing to their fascinating antimicrobial activity, which is induced by stress. less research has been performed on in vivo studies of bioactive phytoalexins, glucosinolates and their hydrolysis products, as most of the reviewed bioassay studies have been performed in vitro. accordingly, in vivo experiments in animal models are very important to confirm the investigated antimicrobial, anticancer, anti-inflammatory, antiviral, antioxidant and neuroprotective activities of the mentioned s-containing compounds. owing to the increase in drug resistance against many pathogenic fungi and bacteria around the world, there is a need to improve testing methods to identify bioactive s-containing natural products against clinically pathogenic microbes. in vitro bioassays are very necessary to gain valuable insights into s-containing natural pro ducts susceptibility testing. 212 m.a. abdalla, k.h. mühling conclusions sulfur-containing natural products have fascinating biological activities and an important role in response to biotic and abiotic stress. phytoalexins are an example of a defense system produced by plants against pests and pathogens. garlic and its compounds possess health-promoting properties, as reported in many studies. the phy siologically active compounds present in garlic exhibit potential pharmacological benefits. moreover, glucosinolates hydrolysis pro ducts have a biocidal effect. currently, sulfur research has occupied the forefront of interest in plant science and involves various plant species. specifically, sulfur assimilation pathway enzymes including atp-sulfurylase are major targets of recent plant nutrition research, which could offer big bene fits including improved productivity and quality of crops in addition to their resistance to multiple stress conditions. moreover, an in-depth investigation of the regulation of sulfur metabolism will allow a better understanding on how deeply sulfur metabolism is involved in the biosynthesis of plant primary and secondary metabolites. the examples reported in this review suggest that the use of a sulfur stress or other environmental biotic or abiotic stresses could be another tool to manipulate the biosynthetic machinery of sulfurcontaining natural products to induce new and bioactive compounds with interesting bioactivities for human and plant health. author contributions maa and khm conceptualized the idea, maa wrote, and edited the manuscript and khm provided input during preparation, edited, and 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creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1126/science.1156970 http://dx.doi.org/10.1039/c39860001077 http://dx.doi.org/10.1246/bcsj.61.285 http://dx.doi.org/10.2174/0929867325666180912105036 http://dx.doi.org/10.2174/1385272820666151207194002 http://dx.doi.org/10.3892/or.2012.1882 angewandte botanik. einführung die erfahrungen des weltkrieges haben gelehrt, daß die erfolgreiche und für deutschland so bezeichnende verbindung von wissenschaft und praxis, wie sie in der physik und chemie besteht und zu einer glänzenden entwicklung geführt hat, auf dem gebiete der biologie bei verschiedenen zweigen noch nicht so eng geknüpft ist. die nachteile dieses zustandes haben sich oft sehr empfind lich fühlbar gemacht und zersplitterung von kräften und geld mitteln veranlaßt. der so schnell eingetretene mangel an vielen wichtigen, der industrie unentbehrlichen rohstoffen aus dem pflanzen reich und die notwendigkeit, ersatz dafür zu finden, haben in viel fach überraschender weise gezeigt, wie lückenhaft die botanische kenntnis zahlreicher bei uns in deutschland einheimischer oder gar ausländischer pflanzen ist. es hat sich herausgestellt, daß die verwertungsmöglichkeiten mancher gewächse am ende des 1 8 . jahrhunderts besser bekannt waren a ls vor dem kriege, weil die technik im letzten jahrhundert sich vielfach an käufliche fremde rohstoffe gewöhnt hatte und daher der anreiz fehlte, sich hier im lande eingehender mit ihnen zu beschäftigen. die folge war, daß die botanische erforschung zahlloser nutzpflanzen arg in den hintergrund trat. die chemische zu sammensetzung oder die physikalischen eigenschaften der rohstoffe waren oft besser bekannt, als die lebensgeschichte ihrer stamm pflanzen, nicht nur bei tropischen arten, sondern auch selbst bei der flora der engsten heimat. die botanik wurde daher meist lediglich als eine hilfswissenschaft der übrigen naturwissenschaften betrachtet. ungeheuere summen sind vergeudet worden, weil eine botanische zentrale fehlte, in der alles wichtige über nutzpflanzen zusammenkam; unzählige untersuchungen wurden unnütz wieder holt und ergaben infolge zersplitterung und unzureichender wissen schaftlicher mittel der einzelnen untersuchungsstellen oft schlechte oder ganz falsche resultate. die kosten der irrtümer trug die praxis. angewandte botanik i. 1 2 einführung. . in verbindung mit den reichen pflanzenschätzen und wissen schaftlichen materialien des dahlemer botanischen gartens und museums, mit unterstützung des hamburger instituts für angewandte botanik und vertrauend auf die mitarbeit der vielen im versuchswesen tätigen fachgenossen, will die „an gewandte botanik“ diese lücken ausfüllen und gemeinsam mit der vereinigung für angewandte botanik, die seit 15 jahren denselben zielen zustrebt, der wissenschaftlichen botanik den platz unter den der praxis, der volkswirtschaft und technik dienenden wissen schaften erstreiten, den sie zweifellos im interesse der gedeihlichen weiterentwicklung oder neuentwicklung unserer volkskraft verdient. alles was von pflanzen stammt, muß auch von wissenschaftlich botanischer seite und nicht allein vom systematischen standpunkte, sondern besonders vom biologischen untersucht werden! berlin-dahlem, hamburg, augustenberg i. baden, den 22. märz 1919 p. graebner, e. giig, a. voigt, k. müller. arbeitsgebiete der angewandten botanik. pflanzliche nahrungsu. futterpflanzenbau; physiologie der mittel; nutzpflanzen genußmittel" pflanzenzüchtung arzneimittel samenkunde fette pflanzenkrankheiten ätherische öle, harze, gummi bodenbakteriologie u. ä. kautschuck und guttapercha gärungsorganismen gerbu. farbstoffe technische mikroskopie faserstoffe verschiedenes hölzer journal of applied botany and food quality 91, 260 (2018) buchbesprechung keimarme ernährung – bei immunschwäche und immunsuppression, in der schwangerschaft und im alter heribert keweloh und uta reinecke das buch stellt die ernährung derjenigen menschen in den mittelpunkt, die über ein geschwächtes immunsystem verfügen und daher dem angriff von infektionserregern in besonderem maße ausgesetzt sind. dies sind nach aussage der autoren insbesondere patienten mit immunsuppression, deren zahl stetig zunimmt. empfehlungen zur keimarmen ernährung unterscheiden sich mitunter erheblich und sind oftmals nicht wissenschaftlich belegt. es kann sogar vorkommen, dass die lebensqualität durch eine rigorose keimarme kost drastisch eingeschränkt wird. im ersten teil des buches stellen die autoren sowohl die schädlichen als auch die nützlichen in lebensmitteln vorkommenden mikroorganismen vor und führen dabei an, welche gesundheitlichen risiken bei aufnahme bestimmter erreger ggf. bestehen können. in diesem zusammenhang wird neben den in enger symbiose mit dem menschen lebenden mikroorganismen auch auf verderbniserreger (z.b. toxinbildner), lebensmittel fermentierende bakterien und pilze, probiotische bakterien sowie krankheitserreger näher eingegangen. im zweiten teil wird dann vor allem auf die menschlichen abwehrreaktionen hinsichtlich verschiedener krankheitserreger sowie die konstellation unterschiedlicher personengruppen mit geschwächtem immunsystem bezug genommen. hierbei wird nicht nur die mögliche immunsuppression durch medikamente, sondern insbesondere auch die nach chemotherapie und organtransplantation einhergehende schwächung des menschlichen immunsystems behandelt. ebenso finden die gegen eigene körperzellen gerichteten autoimmunkrankheiten und allergien erwähnung, da bei diesen krankheiten ebenfalls immunsuppressiva eingesetzt werden. im dritten teil erfährt der leser, welche hygienischen vorkehrungen im umgang mit lebensmitteln getroffen werden können, um speisen sicher zubereiten zu können. es wird darauf hingewiesen, dass letztendlich eine optimale lebensmittel-hygiene bereits beim einkauf beginnt und sich über die sorgfältige einhaltung der kühlkette, die richtige lagerung sowie die zubereitung in der küche entsprechend fortsetzt. in dem vierten teil gehen die autoren schließlich auf die mikrobiologisch-hygienischen eigenschaften von lebensmitteln näher ein. hier erhält der leser wichtige informationen zum richtigen umgang mit den einzelnen lebensmittelgruppen (obst, gemüse, kartoffeln, nüssen, getreide, milch, fleisch, eier, fisch sowie deren erzeugnisse. das buch kann als spannende und gleichzeitig lehrreiche lektüre einer breiten leserschicht uneingeschränkt empfohlen werden. es gibt nicht nur berufsgruppen wie ärzten, pflegepersonal, ernährungstherapeuten, diätassistenten und köchen eine wertvolle unterstützung bei ihrer arbeit, sondern liefert auch medizinischen laien eine ausgezeichnete möglichkeit, umfassende informationen über den aktuellen kenntnisstand einer keim armen ernährung zu erhalten. im anhang sind einige wissenschaftliche fachartikel zu der behandelten thematik aufgeführt, sodass es den lesern möglich ist, in einzelfällen mehr hintergrundinforma tionen zu den jeweiligen aspekten zu erfahren. dr. h. schulz e-mail: hartwig.schulz@julius-kuehn.de bibliografie: heribert keweloh und uta reinecke, keimarme ernährung – bei immunschwäche und immunsuppression, in der schwangerschaft und im alter, wissenschaftliche verlagsgesellschaft mbh, stuttgart, 1. auflage 2018, 185 seiten mit 88 abbildungen und 11 tabellen, preis: 24,80 €, isbn 978-3-8047-3687-0 (print), isbn 978-3-80473873-7 (e-book, pdf) journal of applied botany and food quality 92, 232 239 (2019), doi:10.5073/jabfq.2019.092.032 1institute of food science and human nutrition, leibniz university hannover, hannover, germany 2institute of botany, leibniz university hannover, hannover, germany watercress – cultivation methods and health effects jan philipp schuchardt1, andreas hahn1, theresa greupner1, paulina wasserfurth1, maría rosales-lópez2, johann hornbacher2, jutta papenbrock2* (submitted: may 18, 2019; accepted: july 20, 2019) * corresponding author summary watercress, nasturtium officinale r. br., is a native water or semiaquatic plant that has a high nutrient density. physiologically relevant are the various glucosinolates, which possess positive health effects in form of their thioand isothiocyanates. in an interdisciplinary project, we aim to develop a hydroponic, and finally an aquaponic, circulatory cultivation system and to study the health effects of watercress. in humans, there is a lack of data-based knowledge on potential beneficial health effects of watercress. growth of watercress was followed during one season in an open-door hydroponic system. watercress was also cultivated in the greenhouse in diffe rent substrates with different concentrations of nutrients and salt. the biomass production is strongly dependent on the temperature. the glucosinolate contents differ significantly during the growing season, especially during flowering. watercress naturally grows in nutrientrich fresh waters, however, when cultivated at nacl concentrations of up to 120 mm the gain in biomass is still high. in a human proofof-concept study, indications for antioxidant and anti-inflammatory effects of fresh watercress were observed already after a single dose intake of fresh watercress (85 g). further in vivo and in vitro studies are planned to study health beneficial effects of watercress and its metabolic activity. keywords: anti-inflammatory, antioxidative, gluconasturtiin, glucosinolates, hydroponic cultivation, peitc. introduction watercress (nasturtium officinale r. br.), a member of the brassi caceae, is a perennial aquatic or semi-aquatic plant species native to europe and asia. watercress grows in nutrient-rich, streaming freshwater (kopsell et al., 2007). watercress is traditionally used as winter salad as it grows in flowing water even at cool temperatures as long as the water is not frozen. due to its special demands, the cultivation of watercress declined although nutritionally valuable metabolites have been identified. usually, watercress is cultivated in sophisticated held back streaming waters, but also grows well in moist soil or hydroponic cultures. when commercially grown, watercress cuttings or seedlings are planted into beds with a mixture of soil and gravel, leveled out to ensure even water flow through the beds. upper parts of the watercress are harvested several times per growing season, leaving enough stem to ensure new growth (tab. 1). the species needs low amounts of nitrogen and phosphate in comparison to other plant species while producing large amounts of biomass (kopsell et al., 2007). as it is quite low in energy, watercress has a high nutrient density for vitamins b1, b2, b3, b6, e, c, polyphenols (flavonoids, phenolic acids, proanthocyanidins) as well as terpenes (inclu ding carotenoids) (klimek-szczykutowicz et al., 2018). like all members of the brassicaceae plant species watercress contains mustard oil glycosides or glucosinolates (gls). these nitrogen and sulfur containing secondary metabolites are derived from amino acids and are synthesized by the plant to cope with biotic stressors. glucosinolates and thioglucosidases (ec 3.2.1.147) are usually stored in different cells or cell compartments, but get together once the plant tissues are disrupted (ahuja et al., 2016). thioglucosidases then hydrolyze the gls leaving an unstable aglucone behind, which further reacts to thiocyanates, isothiocyanates and nitriles depen ding on ph, metal ions and present specifier proteins (chen et al., 2019). in the case of watercress, the eponymous gl gluconasturtiin predominates, a precursor of the breakdown product phenethyl isothiocyanate (peitc). isothiocyanates and thiocyanates are very reactive substances leading to numerous conjugates with thiol containing compounds like n-acetylcysteine, glutathione, cysteine and many more, forming stable dithiocarbamates (müller et al., 2018). several health beneficial effects have been postulated for watercress. these include antioxidant, anti-inflammatory, immunomodulating, anti-diabetic, anti-allergic, antibacterial, hypolipemic, cardioprotective and anticancer effects as well as beneficial effects on the reproductive system (summarized in tab. 2-4). most of these effects have been observed in vitro (tab. 2) and in animal studies (tab. 3), while only a few human intervention studies have been carried out (tab. 4). however, findings from in vitro studies do not necessarily apply in vivo, especially when looking at antioxidant effects of compounds (berger et al., 2012). although some studies analyzed the administration of isolated peitc (yuan et al., 2016), which is the main isothiocyanate of watercress, the investigation of single compounds does not necessarily allow drawing conclusions from the effects of whole watercress – an edible green with other known health-promo ting ingredients. overall, the data on the nutritional effects of watercress is very limi ted. the few human studies that administered watercress focused on tab. 1: cultivation methods of watercress. cultivation method place substrate used reference beds with flowing water germany (erfurt) soil mixed with gravel pink, 1993 beds with flowing water great britain (dorset, hampshire) soil mixed with gravel casey & smith, 1994; crisp, 1970 beds with flowing water usa (california, hawaii, florida) soil or sand fennell, 2006 hydroponics or overhead spray lines australia (brisbane, sydney, melbourne) nutrient solution fennell, 2006 journal of applied botany hydroponic cultivation and health effects of watercress 233 antioxidant (fogarty et al., 2013; gill et al., 2007) and anticancer effects (hoffmann et al., 2009), while anti-inflammatory effects have not been investigated in humans so far. in an interdisciplinary research project, we aim to optimize the cultivation of watercress in aquaponic circulatory systems. another aim of the project is to study the health effects of freshly harvested watercress in vivo. a proof-of-concept study was conducted to prove the applicability of a specific study design to investigate antioxidative and anti-inflammatory effects of watercress in humans. the effect of a single watercress dose on markers of oxidative stress/lipid peroxidation (malondialdehyde, mda) and inflammation (il-6, tnfα and il-10) was investigated in subjects who had to complete a highintensity training to induce oxidative stress and a pro-inflammatory condition. tab. 2: health effects of watercress – in vitro studies. effect dosage form cell line/ experimental model results/mechanism(s) reference anticancer extract of watercress human mda-mb-231 • suppression of invasive potential rose et al., 2005 breast cancer cells • inhibition of metallo-proteinase 9 anticancer extract of watercress human ht115 colon cancer cells • suppression of invasive potential boyd et al., 2006 anticancer peitc biliary tract cancer cells • reduction in cisplatin resistance li et al., 2016 • increased rate of apoptosis of cancer cells • inhibited xenograft tumor growth anticancer extract of watercress human colon cancer cells • watercress extract proved to be effective boyd et al., 2006 (ht29 cells) against tumor initiation, proliferation and metastasis:  inhibition of dna damage (initiation)  accumulation of cells in the s phase of the cell cycle (proliferation)  inhibition of invasion through matrigel (metastasis) antioxidant, extracts of watercress rat liver homogenate • reduced serum alanine aminotransferase and bahramikia and hypolipidemic aspartate aminotransferase levels compared to yazdanparast, 2010 and cardio high-fat diet groups protective • reducing power in a ferric reducing antioxidant power assay • concentration-dependent scavenging ability on 2,2-azinobis 3-ethylbenzothiazoline-6-sulfonate, 1,1-diphenyl-2-picrylhydrazyl, nitric oxide radicals, and hydrogen peroxide • chelating ability on ferrous ions • prevention of thiobarbituric acid reactive substances formation in ferrous ion/ascorbate induced lipid peroxidation in a dose dependent manner antioxidant and watercress juice digestive enzymes: • inhibition of α-glucosidase, α-amylase and lipase spínola et al., 2017 antidiabetic α-glucosidase, α -amylase and lipase antioxidant extract of watercress direct measurement • improved total antioxidant activity, reducing ozen, 2009 (aqueous and ethanolic) of antioxidant capacity power, dpph* radicals and superoxide of watercress extract anion radicals scavenging activities antioxidant and extract of watercress human pbmc • increased gene expression of detoxification hofmann et al., 2009 anticancer & peitc enzymes (gp×1 and sod2) • increased sod2 activity antipeitc murine raw 264.7 macrophages • inhibition of no production → decreased tsai et al., 2010 inflammatory production of tnfα and il-10 by activated macrophages • increased no clearance antiallergic extract of watercress rat peritoneal mast cells and • inhibition of histamine release hoshino et al., 1998 (ethanol) rat basophilic leukemia cells (rbl-2h3) antibacterial extract of watercress gramnegative bacteria • antibacterial activity for all bacterial strains zafar et al., 2017 (methanol) (e.g. escherichia coli, klebsiella • highest inhibitory activity against pneumoniae) and grampositive bacillus cereus and escherichia coli bacteria (e.g. enterococcus faecalis and bacillus cereus) antibacterial extract of watercress mycobacterium tuberculosis • inhibitory activity against mycobacterium quezada-lázaro et al., (chloroform) h37rv bacteria tuberculosis h37rv bacteria 2016 dpph, 2,2-diphenyl-1-picrylhydrazyl; gp×1, glutathione peroxidase 1; il-10, interleukin 10; no, nitric oxide; peitc, phenethyl isothiocyanate; sod, superoxide dismutase; tnfα, tumor necrosis factor α. 234 j.p. schuchardt, a. hahn, t. greupner, p. wasserfurth, m. rosales-lópez, j. hornbacher, j. papenbrock materials and methods plant material the watercress cultivar was originally obtained from the nursery fischer, erfurt (http://erfurt-hochheim.de/gewerbe_und_handel/handel/ ?id=75). it was further propagated on the trout farm of the family göckemeyer in poggenhagen (http://www.edelkrebs-niedersachsen. de/forellen/). there, cuttings have been taken for propagation in the greenhouse at the institute of botany, leibniz university hannover. sampling and cultivation experiments growth of watercress was followed during one growth season in an outdoor hydroponic system and samples were collected every two tab. 3: health effects of watercress – animal studies. effect dosage form species results/mechanism(s) reference anticancer aqueous solution swiss mice • suppression of ehrlich tumor growth de souza et al., 2016 of watercress anti-inflammatory extract of rats • inhibition of carrageenan-induced paw edema sadeghi et al., 2014 watercress • activity against formalin-evoked paw edema • decreased swelling and tissue damage induced by carrageenan or tpa antioxidative, extract of (hypercholesterolaemic) • decrease of hepatic mda, gr and gpx activities yazdanparast et al., 2008 hypolipemic and watercress rats • reduced total cholesterol, triglycerides and cardioprotective low-density lipoprotein • increased levels of blood high-density lipoprotein cholesterol antioxidant and extract of rats • protection against increase in ros, gsh, lpo and shahani et al., 2017 anti-inflammatory watercress pco in gentamicin-induced nephrotoxicity • protection against increase in no and tnfα in gentamicin-induced nephrotoxicity antioxidant and extract (diabetic) rats • improvement of antioxidant status: sod, gr, gpx, fenton-navarro et al., 2018 antidiabetic of watercress mda in plasma and different tissues, total antioxidant status • hypoglycemic effect of aqueous watercress extract was 76.6 % higher than that of insulin; glucose levels were normalized on the third week up to the eighth week antidiabetic different extracts (diabetic) rats • decrease of blood glucose after 1 week and 2 months hoseini et al., 2009 of watercress (ethyl acetate, methanol and aqueous) antidiabetic and extract of (diabetic) rats • decrease of serum glucose, total cholesterol hadjzadeh et al., 2015 hypolipidemic watercress and ldl-cholesterol (hydro-alcoholic) antioxidant extract rats • attenuation of vancomycin-induced nephrotoxicity karami et al., 2018 of watercress • mda levels decreased compared to control (hydro-alcoholic) antioxidant watercress juice mice • enhancement of superoxide dismutase activity casanova et al., 2017 in erythrocytes • improved glutathione balance • diminished lipid oxidation in all matrices antioxidant extract rats • decreased lipid peroxidation in liver, brain and kidney ozen, 2009 of watercress (ethanolic) antioxidant watercress oil rabbits • improved sod activity and gsh concentrations alagawany et al., 2018 immuno-modulating extract rainbow trout • enhancement of hematological and immunological asadi et al., 2012 of watercress parameters (including hb and mchc, lysozyme and complement activities, total protein and globulin levels) reproductive extract of (diabetic) rats • increased levels of testosterone, lh, fsh, and mohammadi et al., 2017 system watercress fast-motility sperm (hydro-alcoholic) fsh, follicle stimulating hormone; gpx, glutathione peroxidase; gr, glutathione reductase; gsh, glutathione; hb, hemoglobin; ldl, low density lipoprotein; lh, lh luteinizing hormone; lpo, lipid peroxidation; mchc, mean corpuscular hemoglobin concentration; mda, malondialdehyde; no, nitric oxide; pco, protein carbonyl; peitc, phenethyl isothiocyanate; ros, reactive oxygen species; sod, superoxide dismutase; tnfα, tumor necrosis factor α, tpa, 12-o-tetradecanoylphorbol-13-acetate. hydroponic cultivation and health effects of watercress 235 weeks from april 2017 till november 2017. cultivation of watercress was established by testing different substrates (soil, soil/sand mixtures, water) and with different concentrations of nutrients (nitrogen and phosphate) and nacl. glucosinolate measurements by hplc / lc-ms gls were analyzed by hplc according to boestfleisch et al. (2017) with modifications. all standard substances were checked for identity by lc-ms. the gl contents in the watercress samples were measured in triplicates. human study design and subjects in a cross-over study, 4 subjects consumed a single dose of 85 g of fresh watercress (along with 50 ml salad dressing, 50 g iceberg lettuce and 50 g cucumber) at breakfast. this amount has been selected since initial human studies also administered 85 g of raw watercress daily (gill et al., 2007; fogarty et al., 2013). the diet was compared to a control breakfast (two buns with butter, cheese and 50 g iceberg lettuce). the study was conducted with healthy, untrained human subjects (1 male, 3 females, mean age: 28±6; mean bmi: 22.7±3.5 kg/m2; mean weight: 66±18 kg) who had to complete a high-intensity training session to induce oxidative stress and a proinflammatory condition. the subjects were asked to refrain from consuming foods rich in polyphenols, vitamin e and vitamin c (especially berries, grapes, nuts), 7 days before the start of the study. on each of the 2 study days, fasting blood was taken from the subjects in the morning (baseline), followed by the consumption of the test breakfast (watercress or control). 120 minutes after breakfast, the subjects completed a 30-minute continuous running based endurance workout combined with bodyweight strength-endurance exercises (burpees, push-ups, squats and sit-ups) at 80% of their maximal heart rate (hrmax). participants were instructed to perform 10 reps of each exercise in between replicated 400 m track runs. throughout the workout, the hr was continuously recorded using a polar a300 fitness and activity tracker with a h7 bluetooth heart rate sensor (polar electro oy, kempele, finland). five minutes after the training session, the second blood draw was taken (5 min post-exercise). the subjects were allowed to drink water ad libitum. concentrations of inflammatory cytokines and mda were determined at baseline and 5 min post-exercise. blood samples were obtained by venipuncture of an arm vein using multifly needles (sarstedt, nümbrecht, germany) into heparin plasma monovettes (sarstedt, nümbrecht, germany). mda was analyzed in heparin plasma using the tbars assay kit from cayman chemical (biomol, hamburg, germany) according to the manufactures instructions. il-10, il-6 and tnfα were analyzed in whole blood cultures after ex vivo immune cell stimulation via lipopolysaccharide (lps). briefly, heparinized blood samples were diluted 1:4 with rpmi 1640 including hepes and l-glutamine (sigma-aldrich, hamburg, germany) and added antibiotics (100 u/ml penicillin and 100 μg/ml streptomycin, sigma-aldrich, hamburg, germany). the blood was seeded into 12-well microtiter plates and stimulated with 10 ng/ml (final concentration) of lps (sigma-aldrich, hamburg, germany) or medium alone in duplicates. blood was incubated at 37 °c for 24 h. after incubation, il-10, il-6 and tnfα were simultaneously quantified in whole blood culture supernatant using a bio-plex multiplex immunoassay including a bio-plex magpix™ multiplex reader. results growth of plants in 2017, water and plant samples were taken from the outdoor culture system in poggenhagen (fig. 1a) every one to two weeks to analyze the nutrient requirements and plant constituents of watercress (data not shown). the biomass production is strongly dependent on the temperature and plants accumulate significantly higher contents of calcium and potassium as the season progresses with peaks reached in mid-summer. to be able to work under controlled conditions, cuttings have been prepared and rooted for propagation in the greenhouse. in the greenhouse, the watercress can be cultivated all year-round with constant biomass growth in 1/2-hoagland nutrient solution (fig. 1b). it can also be cultivated on different substrates (mixture of soil and sand or sand), but then the cultivation requires more care. the watercress is relatively salt-tolerant, even at about 120 mm nacl in the nutrient solution, the plants are vital and show biomass growth (data not shown). glucosinolate levels gls were analyzed by lc-ms to identify all gls. afterwards an hplc method was applied using detection at 229 nm to be able to analyze many samples in a cost-efficient way. a typical chromatogram is shown in fig. 2a. the main gl found in watercress is gluconasturtiin (fig. 2b). the gl contents differ significantly during the growing season, especially during flowering. plants grown outside show similar contents of gluconasturtiin throughout the growing season with elevated levels in flowering plants and plants with developed pods when compared to plants early in the season which are not flowering. contents of glucobrassicin, neoglucobrassicin and glucoarabishirsutain are lower in plants with developed pods later in the season compared to non-flowering plants early in the season. the contents of the gl 4-methoxyglucobrassicin on the other hand, show an increase in flowering plants with pods compared to plants with flower buds. overall, the total content of indolic and aliphatic gls drops in the course of the season, whereas the content of the aromatic gls elevates slightly (tab. 5). flowering had even smaller effects on the gl levels and composition when plants cultivated in the greenhouse (data not shown). tab. 4: health effects of watercress – human studies. effect dosage form results/mechanism(s) reference anticancer and fresh watercress • reduced lymphocyte dna damage gill et al., 2007 antioxidant • altered blood antioxidant status anticancer fresh watercress • genotype dependent increase in gpx and sod enzyme activity hofmann et al., 2009 in red blood cells in gstm1*0, but not in gstm1*1 anticancer and fresh watercress • decrease of exercise induced dna damage and lipid peroxidation fogarty et al., 2013 antioxidant anticancer peitc • inhibition of metabolic activation and lung carcinogenicity of tobacco yuan et al., 2016b gpx, glutathione peroxidase; gstm1, glutathion s-transferase m1; peitc, phenethyl isothiocyanate. 236 j.p. schuchardt, a. hahn, t. greupner, p. wasserfurth, m. rosales-lópez, j. hornbacher, j. papenbrock to analyze the degradation of gls after cutting the plants, freshly harvested watercress material was stored at 4 °c in the refrigerator in an inflated plastic bag for 1, 2, 3 and 5 d. no significant changes in the gluconasturtiin content occurred (data not shown). anti-inflammatory effects of watercress in humans a trend for increasing concentrations of il-10, il-6, and tnfα in lps-treated whole blood and mda in plasma was observed 5 min post exercise after the control breakfast suggesting that the exercise protocol was effective in inducing a pro-inflammatory condition and oxidative stress, respectively (fig. 3). compared to the control breakfast, the increase of inflammatory cytokines and mda concentrations 5 min post exercise after the watercress breakfast was lower. discussion successful cultivation of watercress in the greenhouse it was demonstrated that watercress can be cultivated all year round in a greenhouse on either sandy substrate or in hydroponic nutrient solution. therefore, it is not necessary to invent a system with running water. even without constant aeration watercress produces large amounts of biomass, even at high nutrient or salt concentrations, which is in agreement with the literature (kaddour et al., 2013; fernàndez et al., 2016). the gain in biomass was strictly temperature-dependent. therefore, for an all-year-round cultivation a greenhouse would be a prerequisite for controlled hydroponic and aquaponic cultivation. the content of secondary metabolites changes during the season. especially flowering has strong effects on the contents of gl. therefore, the development of a watercress cultivation system avoid fig. 1: cultivation of watercress in a) poggenhagen in the field and in the b) greenhouse. a) b) fig. 2: a) hplc chromatogram of a watercress extract. b) gluconasturtiin (phenethyl glucosinolate, chem spider id 7827641) is the most abundant glucosinolate in the eponymous watercress nasturtium officinale. a) b) hydroponic cultivation and health effects of watercress 237 fig. 3: effect of acute watercress consumption on blood markers of inflammation (il-10, il-6, tnfα) and oxidative stress/lipid peroxidation (mda) after high-intensity training in untrained subjects (n=4). mda levels were measured in heparin plasma. inflammatory cytokines were measured in ex vivo lps-stimulated whole blood cultures. ing flowering at all would be preferable. temperature probably also contributes to changes in gl content and pattern, since contents seem to be influenced not only by daytime temperature, but also by the difference between daytime and nighttime temperature (engeleneigels et al., 2006). anti-inflammatory effects of watercress in humans − a new approach anti-inflammatory effects of watercress have not yet been investi gated in humans. isothiocyanates in general have been shown to possess anti-inflammatory properties through the regulation of cytokines of the tnf family primarily via activation of the nf-κb pathway (heiss et al., 2001; wierinckx et al., 2005; dey et al., 2006; karmakar et al., 2006). also, studies with knockout mice showed that the nuclear factor erythroid 2-related factor 2 (nrf2) plays an important role in the anti-inflammatory and antioxidative effects of peitc (boyanapalli et al., 2014). although, the differences of il-10, il-6, tnfα and mda concentrations between control and watercress were not statistically significant due to high interindividual differences and a low case-number, the results of the present pilot study can be interpreted as a trend towards an anti-inflammatory and antioxidant effect of fresh watercress even at a single dose. measurable effects after a single dose of watercress were also shown by fogarty et al. (2013). in particular, the applied model for the induction of oxidative stress and inflammation in combination with cytokine analysis in whole blood cultures after ex vivo immune cell stimulation appears applicable to the relevant question. to consolidate these preliminary results, we plan to apply the outlined study protocol with a larger sample size over a longer intervention time in future studies. in addition, studies with patients suffering from diseases associated/accompanied with increased oxidative stress and inflammation (e.g. asthma) are planned. in these studies, additional biomarkers are necessary to examine the beneficial effects of watercress on human health. however, long-term stu dies with fresh watercress are difficult to realize due to its sharp taste, tab. 5: mean concentration of different glucosinolates (gls) (μmol g dw-1) in watercress in above ground plant material collected at different time points. plants were growing in an outdoor aquaponic system and harvested to analyze changes in gl contents in the course of the season and developmental stage of the plants. the standard deviation represents the values for three biological replicates. sampling date 4-methoxyglucobrassicin neoglucogluconasturtiin glucobrassicin glucobrassicin arabishirsutain 12.5.2017 0.144 ± 0.060 0.123 ± 0.012 0.063 ± 0.011 0.659 ± 0.117 5.028 ± 0.606 19.5.2017 0.128 ± 0.007 0.075 ± 0.009 0.04 ± 0.003 0.457 ± 0.111 5.427 ± 0.232 26.5.2017* 0.122 ± 0.018 0.109 ± 0.026 0.041 ± 0.110 0.390 ± 0.127 4.882 ± 0.782 09.6.2017** 0.176 ± 0.022 0.09 ± 0.016 0.041 ± 0.017 0.369 ± 0.106 6.017 ± 0.310 16.6.2017 0.187 ± 0.035 0.072 ± 0.010 0.043 ± 0.027 0.298 ± 0.146 6.418 ± 0.229 23.6.2017*** 0.167 ± 0.079 0.038 ± 0.028 0.031 ± 0.011 0.219 ± 0.075 5.101 ± 1.691 *first flowers, **many flowers, ***no flowers anymore. 238 j.p. schuchardt, a. hahn, t. greupner, p. wasserfurth, m. rosales-lópez, j. hornbacher, j. papenbrock gastric discomfort causing effects (two subjects complained about slight gastric discomfort after the watercress consumption), limited shelf life, and elaborate logistics in the daily distribution. conclusion/outlook as demonstrated, watercress can be successfully cultivated all year round in the greenhouse without a reduction in its valuable contents. to optimize the utilization of nutrients, the hydroponic culture will be combined with the cultivation of fish in one greenhouse in the near future. to circumvent the problem of limited shelf life other methods will be developed to provide humans with the valuable metabolites of watercress. the human pilot study indicates that fresh watercress has positive effects on oxidative stress and inflammatory markers under induced pro-oxidative and pro-inflammatory conditions. future long-term intervention studies with larger collectives must be carried out to confirm these results. in the ongoing project we aim to develop watercress dosage forms to overcome these obstacles. processing technologies like gentle freeze-drying, grinding, subsequent encapsulation and gastric juice resistant coatings appear proper to ensure high tolerability, preservation of valuable ingredients (primarily gluconasturtiin and myrosinase), high bioavailability and compliance in long-term intervention studies. in future clinical studies with watercress extracts, the dose and duration must be carefully considered. a recent study observed an effect of peitc in high concentrations on accumulation of reactive oxygen species and cytoskeletal changes, resulting as a consequence of cytotoxicity (dayalan naidu et al., 2018). authors’ contributions jps, ah and jp conceived and designed the experiments. jps, pw, mrl and jh performed the experiments. jps, ah, tg and jp analyzed the data and prepared the manuscript. all authors read and approved the manuscript. acknowledgments we would like to thank stefan göckemeyer, poggenhagen, who drew our attention on this traditional vegetable and supported us 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(phila) 9, 396-405. doi: 10.1158/1940-6207.capr-15-0380 zafar, r., zahoor, m., shah, a.b., majid, f., 2017: determination of antioxidants and antibacterial activities, total phenolic, polyphenol and pigment contents in nasturtium officinale. pharmacologyonline 1, 11-18. orcid theresa greupner 0000-0001-7510-4654 juttta papenbrock 0000-0003-0942-4072 address of the corresponding author: jutta papenbrock, institute of botany, leibniz university hannover, herren häuser str. 2, 30419 hannover, germany e-mail: papenbrock@botanik.uni-hannover.de © the author(s) 2019. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1074/jbc.m104794200 http://dx.doi.org/10.1007/s00394-009-0039-5 http://dx.doi.org/10.1007/s11738-013-1377-8 http://dx.doi.org/10.5812/jjnpp.67178 http://dx.doi.org/10.1016/j.neuroscience.2006.04.075 http://dx.doi.org/10.1016/j.fitote.2018.05.031 http://dx.doi.org/10.1021/jf072793f http://dx.doi.org/10.18632/oncotarget.7171 http://dx.doi.org/10.1007/s10886-018-1016-3 http://dx.doi.org/10.4314/ajtcam.v13i2.3 http://dx.doi.org/10.1016/j.taap.2005.04.010 http://dx.doi.org/10.3109/13880209.2013.821138 http://dx.doi.org/10.1080/15376516.2016.1258748 http://dx.doi.org/10.1111/jfbc.12335 http://dx.doi.org/10.1155/2010/293642 http://dx.doi.org/10.1016/j.jneuroim.2005.05.013 http://dx.doi.org/10.1016/j.cbi.2008.01.006 http://dx.doi.org/10.1158/1940-6207.capr-16-0032 http://dx.doi.org/10.1158/1940-6207.capr-15-0380 journal of applied botany and food quality 92, 226 231 (2019), doi:10.5073/jabfq.2019.092.031 1institut für pflanzenkultur e. k., schnega, germany commercial micropropagation in germany imke hutter 1*, carolin schneider 1 (submitted: june 17, 2019; accepted: august 30, 2019) * corresponding author summary since the first experiments of gottlieb haberlandt (1854-1945) in the early 1900 on the in vitro cultivation of plant tissue, the fields of application have expanded from research of plant physiology to applications in breeding, molecular and microbiology and it became also an important tool for commercial plant production (laimer and rücker, 2003). different fields of application and their perspectives will be discussed. numbers of commercial micropropagation in germany will be presented from 2004 to 2017 and possible reasons for changes investigated. keywords: in vitro culture; plant tissue culture; commercial micropropagation introduction gottlieb haberlandt wanted to study the mutual influence of cells as the smallest “living units” within plant tissue using sterile in vitro conditions. scientists had already shown before him, that whole plants could be generated from isolated embryos, but it was haberlandt who was able to generate plants from already differentiated tissue by adding plant growth regulators like cytokinins and auxins externally to nutrient media. today in vitro culture of plants is commonly used as a tool in research for plant physiology and breeding, molecular and microbiology and secondary metabolite production. it is a main pillar for the conservation of important culture varieties or endangered species in genebanks, when storage of vegetative plant material is necessary (f. e. allium spp., mentha spp., musa spp.). in the 1980s it also became an important technology for commercial micropropagation, which made it possible to produce a high number of genetically identical and morphologically homogeneous plants. sterile conditions eased the production of pathogen-free plants. another advantage is the possibility to produce independently of seasonal changes in northern europe. in 1963, the international association of plant tissue culture (iaptc) was founded as a platform for this fast evolving research field. the major objective of the iaptc was “to promote the interest of plant tissue culture workers”. with increasing fields of application, the association grew to over 4500 members from 123 countries around the world in 1995-1996. after 25 years, it needed to reflect the increasing role and impact of plant biotechnology and was renamed into international association of plant tissue culture & biotechnology (iaptc&b, 1998). since 2006 it supports as international association of plant biotechnology (iapb) the plant tissue culture and biotechnology around the world, by publishing the journal “in vitro cellular and developmental biology – plant” together with the society for in vitro biology (sivb) and holding a worldwide recognised conference every fourth year (upcoming conference in south korea in 2022) (iapbhome.com, 2019). in 1985, the arbeitskreis deutsche in vitro kulturen adivk (association of german in vitro culture laboratories) was founded as a network for scientific institutions and commercial laboratories to further study and communicate micropropagation techniques and protocols for new genera. winkelmann et al. reported in 2006 about the state-of-the-art of commercial micropropagation in germany, analysing data of the annual statistics of the commercial adivk members, which were collected each year on a voluntary basis, making no claim to be complete. winkelmann et al. (2006) focused on the development of the number of companies, plants and plant genera since its foundation in 1985 to 2004. ever since then commercial adivk members continued to report their annual production numbers, so that after another 15 years we want to highlight the development of the different application fields of in vitro culture technology and the development of plant production for that period. we extracted data of winkelmann et al. (2006) from the years 1985 and 1995 and continued with the data of 2005 (10 year steps), followed by 2010 and 2015 (5 year steps) and the latest data from 2017. development of production capacities in other countries will be only marginally touched, where they have an impact on the german market. micropropagation as a tool for mass propagation in vitro culture of plants is established by using all parts of plant tissue (meristems, buds, leaves, stems), though for each plant species the optimal explant type must be investigated. zygotic embryos can be used to induce somatic embryogenesis. explants are surface steri lized and transferred to nutrient media containing macroand microelements, vitamins, plant growth regulators and sugar. for each plant species the composition of the nutrient media must be optimized. the most common nutrient media are modified after murashige and skoog (1962). the usage of the plant growth regulators cytokinins and auxins makes controlled shoot or root growth possible. micropropagation is carried out by successive cutting of shoots, followed by rooting and later hardening of plantlets in greenhouses. a challenge remains the optimization of the process from establishment of in vitro culture to micropropagation at large scale and greenhouse production. evaluation of these process steps results in a production success, which is calculated as the number of plantlets, that need to be produced in vitro to produce one sellable plant. this production process is the clue for cost calculations and can show a large range from sometimes less than 30% to 90% (quambusch et al., 2017). still customers’s need for uniform appearance and performance of plants make micropropagation a perfect tool for the production of clonal plant material. adivk commercial members state “mass propagation”, “stock plant propagation”, “pathogen elimination” and “in vitro storage” as the most important applications. large numbers of identical disease-free plants can be produced, meeting market’s and customer’s demands to get standardised plants of consistent healthy quality. in the beginning mainly ornamentals and small fruits were produced, with orchids and strawberrys as the most important plant genera resp. journal of applied botany commercial micropropagation in germany 227 species. during the following decades a diversification of micropropagation techniques opened new fields of applications but still the above mentioned are the most important ones for commercial laboratories (winkelmann et al., 2006). during the last 15 years since the first analysis of the adivk statistics by winkelmann et al. (2006) the market for plant production has undergone major changes that all had an impact on the development of companies and plant production. aim of this paper is to analyse possible reasons and evaluate the state of the art. since the foundation of the adivk the number of commercial members has risen from 12 in 1985 to 28 in 1995 and remains more or less stable until to date (27 in 2017). the number of plants reported by these members has risen from 5 million plants per year to an immense increase of 49 million plants in 2005 and dropped down to 28 million in 2017 (fig. 1). the statistics of the adivk distinguish different categories of plants according to their market. for the group of ornamentals the orchids are shown separately due to their outstanding development and high production numbers. small fruits have a remaining importance as well as woody plants. micropropagation of agricultural species and vegetables as well as medicinal plants remains a niche application (fig. 2). the production of orchids is the most important commercial application related to plant numbers (46% or 13 million in 2017). when interpreting the numbers it has to be considered that the leading company of orchid production in germany does not report its production numbers since 2005, but states an annual production of 50 million plants alone in 2017 (mitschka and büchler, 2018). the increase in orchid production was realised due to higher market prices in the early 2000s, followed by a decrease due to shifts of production capacities to low labour cost countries, a process which is still going on. today the market in germany and europe is almost saturated, so that companies seek new markets for example in the usa (mitschka and büchler, 2018). nevertheless the number of orchids sold in germany remains high with a market share of 34% of all ornamentals in 2017 (statista.com), phalaenopsis being the most important genus (95%, followed by cypripedium, cambria, oncidium and cattleya species). in general the market for all ornamentals (cut flowers, indoor flowers and flowers for balcony, beds and borders) is highly fragmented and internationalised with breeders, growers and retailers, who all participate in the added value-chain. micropropagation laboratories provide mass propagated plant material to gardeners and nurseries, as well as mother stocks to breeders. in germany production value of ornamentals reached altogether 1.1 billion euro, produced by more than 3,000 companies in 2017 (statista.com). for comparison the biggest european auctioneer in the netherlands retailed ornamentals of 4.7 billion euro in 2017, with decreasing numbers (11.7 billion plants) but increasing prices (taspo.de). most ornamentals are produced outside of europe in kenia, ethiopia or israel and imported back into europe (mitschka and büchler, 2018). germany remains a big market for ornamentals but adivk members produce a relatively small share of 5 million ornamentals per year. micropropagation of ornamentals underlies not only production capacities but also fashions. during the last decades production numbers of different ornamental species has undergone severe changes that are not only due to shifts to low labour cost countries but also to popularity of plant species and the demand for innovations (fig. 3). micropropagation of ornamentals started with the most popular genera in the 1980s namely anthurium, spatiphyllum and gerbera with a peak in production of those species in 1995 (total number almost fig. 2: plant production numbers of different categories of plants from 1985 to 2017 (stated by adivk members, voluntary basis). fig. 1: plant production via micropropagation in germany from 1985 to 2017. number of companies and number of plants (stated by adivk members, voluntary basis). 228 i. hutter, c. schneider 6 million plants). they were replaced by gentiana and dendranthema until 2005, followed by helleborus which since 2010 shows a stable production. altogether more than 130 plant genera are reported by adivk members, resulting in a share of almost 18% of all plants produced. the production of small fruit plants remains an important share with 6 million plants or 21% of the micropropagation per year (fig. 4). inside the group the highest numbers are generated with fragaria and rubus due to their importance in fruit production. here the pricing pressure from low labour cost countries resulted in a decrease in fragaria production, but the numbers remain more or less stable since 2015 as there is a demand for disease-free plant material. since 2010, an increasing number of vaccinium is produced as it is a more and more popular fruit in germany and growers have a demand for productive, vital plants that produce large and tasty fruits which results in selection efforts (pers. communication). woody plants include ornamental shrubs like rhododendron, prunus, syringa, rosa and others and broad leave trees like prunus avium, robinia pseudoacacia, betula spp., populus spp., juglans spp. for forest tree production. in 2017, a total number of 1.44 million plants (5%) was produced (fig. 5). for ornamental trees and shrubs rhododendron and syringa have the biggest importance for garden and landscaping. micropropagation of rhododendron increased from 0 in 1985 to 1.4 million within 10 years. a severe decrease from 2005 to 2010 is mainly due to a shift of production to low labour cost countries especially to asia. since then the production is stable with 250,000 plants in 2017. micropropagation of rosa spp. had its peak in 1995 with 500,000 million plants and is not longer reported today for germany, whereas syringa spp. shows increasing production numbers with 426,000 per year in 2017. broad leave tree species for timber production are mainly found within the genera of prunus, robinia, betula and populus but are not distinguished from ornamental shrubs in this figure. micropropagation of forest trees is estimated to be less than 250,000 plants per year (pers. communication). high hopes lay on the somatic embryogenesis of conifer trees with many scientific and commercial institutions investigating the optimisation for different conifer tree species, like pseudotsuga menziesii and picea abies for timber production and abies nordmanniana for christmas tree production in europe. only from a swedish consortium it is known that the production process can be considered as semi-commercial with a production of somatic embryos in temporary immersion system, sorting of embryos by camera and mechanical pricking and planting. fig. 3: production of selected ornamentals from 1985 to 2017 (stated by adivk members, voluntary basis). fig. 4: plant production numbers of small fruit species from 1985 to 2017 (stated by adivk members, voluntary basis). commercial micropropagation in germany 229 agricultural plant species are mainly produced via micropropagation to maintain culture collections, produce virus-free mother stocks and induce double haploids for breeding purposes. main species are solanum tuberosum, beta vulgaris and hordeum vulgare each with 0.5 million plants per year which sums up to 2.2 million plants per year for all reported 12 genera (2017). micropropagation of medicinal plants remains a niche production where baptisia tinctoria (175,000 plants until 2018) makes the biggest share of 92 % of all 15 genera reported. other applications another application, which was not mentioned above, is the production of secondary metabolites via liquid cell cultures. the most prominent example is the production of taxol® via cell cultures of taxus baccata in aerated stirred tanks. defined cell lines are upscaled in defined nutrient media and the active ingredient can be extracted and further processed (phytonbiotech.com). other approaches for the production of taxol® investigate metabolic pathways of endophytic fungi (souvik, 2014) or genetically transformed yeasts (anand, 2013) but without commercial applications until now. new developments and future perspectives micropropagation of plants was also meant as a method to produce sterile plant material, free of bacterial or fungal pathogens. since the work of müller and döring (2009) on bacterial populations in in vitro cultures of eleutherococcus spp., investigations on diffe rent plant species have shown, that bacterial and fungal endophytes can live inside plant material, only being detected by molecular techniques (quambusch et al., 2014; quambusch and winkelmann, 2018). these bacteria and fungi must not be phytopathogenic but can stay latent in the plant material without causing any diseases or even function as beneficial endophytes that provide nutrients to plants. the most commonly used examples are symbionts like rhizobia sp. or mycorrhizal fungi. as a consequence, the european union funded the cost action “endophytes in biotechnology and agriculture” (fa1103, 2011-2015) to let scientists discuss the new challenges and perspectives of these findings and the possibilities of the use of endophytes to create a beneficial microbiome for breeding purposes. it was followed by cost action fa1405 “using three-way interactions between plants, microbes and arthropods to enhance crop protection and production” which takes additionally the influence of arthropods into account. all these studies and future applications are not possible without micropropagation techniques. discussion today micropropagation of plant material is a methodology that does not seem to bear any more secrets. developed techniques provide reliable tools, which are used worldwide. what are the future challenges? we see different intersections that influence a forecast for developments in commercial application and research (fig. 6). intersections differ for companies, who take micropropagation as a reliable method in their production chain and scientists (commercial and institutional), who use it as a tool for their research. commercial micropropagation laboratories often originate from family-owned or owner-managed nurseries, which saw the advantage to implement the new technology into their production to produce large numbers of homogeneous, pathogen-free plants. other applications followed with a continuous fragmentation and internationalisation of the market. today about 60% of adivk members are still family-owned or owner-managed. family-owned companies see their advantage in a sustainable entrepreneurship with high flexibility, individual personnel management, regional bonds, and innovative power. they shine with exceptional services, personal interactions with their customers and in niches. as weaknesses, they mention unsettled succession plans with potential conflicts between family members and hindered access to capi tal resulting in less vigour. next generations will struggle with the balancing act between pricing pressure, the need for innovation and the shortage of skilled personnel (müller, 2012). in the following we want to highlight a few of the intersections specified in fig. 6. for companies the reduction of labour costs to meet pricing pressure remains the most important issue. winkelmann et al. (2006) fig. 5: plant production numbers of tree species and shrubs from 1985 to 2017 (stated by adivk members, voluntary basis). 230 i. hutter, c. schneider had discussed, that new technological innovations such as temporary immersion system (tis) were needed to reduce labour costs. this forecast did not come true: tis is not used in commercial plant production to a large extent due to problems with hyperhydricity and contaminations in larger vessels. reduction of labour costs is mainly achieved by shifting manual labour to countries with lower salary level like east europe, africa, asia or india. but when we report here a shift of production capacities to low labour cost countries it does not necessarily mean, that companies from those regions took over market shares but, that german companies also founded sister companies or built strategic partnerships with companies from abroad. this enables them to install quality standards that still meet their customer’s demands, but the shift of production numbers from germany to those partners is not shown in the annual statistics due to adivk’s regulations. the highly fragmented and internationalised market for ornamentals poses additional challenges for companies. as soon as a market is saturated, as it seems to be for orchids in europe, they must explore and capture new markets, with the risk of failure. germany’s largest orchid producer expanded with production facilities to the united states to hold and increase his market share of impressing 25%. today development of new micropropagation techniques is less important than logistics and interaction with customers. for example, barcoding of culture vessels as a safety system for calculation of propagation cycles, tracking of vessels or online analysis of stock numbers guarantees the utmost reliability of the production. an intensive quality management assures customers that mother stocks are virus-free, clonal plants are not mixed and that each partner of the added value-chain is aware of the standardised process steps he is responsible for (mitschka and blücher, 2018). winkelmann et al. (2006) also proposed that new protocols for the propagation of woody plants should be developed as there seemed to be a willingness of foresters to pay higher prices. more protocols have been developed but still foresters are not easily convinced to invest more capital in optimised plant material. the development of protocols only seems to be economically interesting for rare and highly valuable tree species such as wavy grain acer or juglans hybrids. high expectations lay on the development of fully mechanised production systems for conifer trees like norway spruce or douglas fir via somatic embryogenesis. thus, until now those efforts still need investment in technological innovations to produce trees in profitable numbers. for agricultural crops and vegetables, the application of in vitro culture cannot be a methodology for mass propagation as the added value is too low in comparison to seeds, but it is an important tool in research and breeding to optimise specific characteristics. a new challenge is the breeding of beneficial endophytic microbiomes for agricultural crops. still a lot of research is needed in this field which results in two conferences to be held in 2019: the austrian symposium “micrope: microbe-assisted crop production – op portunities, challenges & needs” and the 2nd eucarpia workshop on implementing plant-microbe interactions in plant breeding (eu carpia, european association for research on plant breeding). conclusions micropropagation of plant material is no longer an own research field but will remain as a technique for companies to produce a large number of plants for the international market and at the same time as an irreplaceable tool in breeding and research. the different plant groups have different challenges for micropropagation companies. still the stable number of companies since 1995 shows that they can persist in a more and more globalised market finding their application field or market niche. what will guarantee their future is an innovative production chain in combination with breeding and selection for new fashionable varieties (orchids, ornamentals), an enhanced quality (woody plants, small fruit plants, medicinal plants) or improved microbiome (agricultural crops and vegetables). acknowledgments we thank the arbeitskreis deutsche in vitro kulturen for the long lasting efforts to contribute to research and development in micropropagation of plants and its members for the willingness to contribute to the statistics. andreas meier-dinkel undertook the task to generate the annual data from the questionnaires provided by commercial laboratories. we dedicate this article to the late prof. dr. reinhard lieberei, who encouraged us to find our own way. author contributions: imke hutter wrote the manuscript and extracted data of the adivk statistics. carolin schneider provided critical feedback and helped shaping the manuscript. references anand, d., 2013: metabolic engineering of taxol biosynthesis in yeast saccharomyces cerevisiae using a yeast artificial chromosome (yac). degree projects in molecular biology, retrieved 18.06.2019 from http:// lup.lub.lu.se/student-papers/record/3812527 friedrich, g., 2018: royal flora holland macht 4,7 milliarden euro umsatz, retrieved 14.06.2019 from https://taspo.de/gartenmarkt/royalfloraholland-macht-47-milliarden-euro-umsatz/ laimer, m., rücker, w., 2003: plant tissue culture − 100 years since gottlieb haberlandt, springer-verlag wien. doi: 10.1007/978-3-7091-6040-4 mitschka, y., büchler, j., 2018: hark – wachstumsstrategie für die orchideenzucht: wird das geschäft auch in den usa florieren? 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(eds.), plant cell culture protocols. methods in molecular biology, vol. 1815. humana press, new york, ny. doi: 10.1007/978-1-4939-8594-4_4 souvik, k., satpal, s., chelliah, j., 2014: rethinking production of taxol® (paclitaxel) using endophyte biotechnology. trends biotechnol. 32, 304-311. doi: 10.1016/j.tibtech.2014.03.011 statista, 2019: umsatz im gesamtmarkt blumen und pflanzen in deutschland in den jahren von 2005 bis 2018 (in milliarden euro). retrieved 18.06.2019 from https://de.statista.com/statistik/daten/studie/206256/umfrage/umsatz-mit-blumen-und-pflanzen/ vasil, i.k., 2008: history and evolution of the international association for plant biotechnology (iapb), 1963-2008. in vitro cell dev.-pl. 44 (5), 365-372. doi: 10.1007/s11627-008-9148-8 winkelmann, t., geier, t., preil, w., 2006: commercial in vitro plant production in germany in 1985-2004. plant cell, tiss. org. 86 (2), 319-327. doi: 10.1007/s11240-006-9125-z address of the author: imke hutter, institut für pflanzenkultur e. k., solkau 2, 29465 schnega e-mail: hutter@pflanzenkultur.de © the author(s) 2019. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1007/s11240-009-9536-8 http://dx.doi.org/10.1111/j.1399-3054.1962.tb08052.x http://dx.doi.org/10.1016/j.scienta.2017.03.020 http://dx.doi.org/10.1093/treephys/tpu027 http://dx.doi.org/10.1007/978-1-4939-8594-4_4 http://dx.doi.org/10.1016/j.tibtech.2014.03.011 http://dx.doi.org/10.1007/s11627-008-9148-8 http://dx.doi.org/10.1007/s11240-006-9125-z art03_shahbaz.indd response of blue panic grass to water defi cit conditions 135 journal of applied botany and food quality 84, 134 141 (2011) department of botany, university of agriculture, faisalabad, pakistan response of differently adapted populations of blue panic grass (panicum antidotale retz.) to water defi cit conditions m. shahbaz*, m. iqbal, m. ashraf (received january 18, 2011) * corresponding author summary to explore plant species that could tolerate harsh environment, six ecotypes of panicum antidotale were collected from different habitats varying in water availability, salt content and agricultural practices within the faisalabad city. all six ecotypes were grown under normal growth conditions for six months, after which time they were subjected to three drought levels (control (normal irrigation), 60% and 30% fi eld capacity). imposition of drought caused a marked reduction in shoot and root fresh and dry biomass, shoot length, chlorophyll b, a/b ratio, net co2 assimilation rate, transpiration rate, and water use effi ciency in all six populations. while shoot p, root n, p and ca2+ were remained unaffected due to application of drought stress conditions. of the six ecotypes, population collected from sludge of disposal channel and that from the along the botanical garden produced higher quantity of plant biomass and net co2 assimilation rate as compared to the others. introduction the genus panicum comprises over 500 species distributed mostly in tropical and subtropical regions of the world. panicum antidotale, blue panic or giant panic is a native of southeast asia. it is a robust and shortly rhizomatous perennial grass that grows up to 1.5 m with very deep root system (jacobs and wall, 1993). the fl owers of panicum antidotale are hermaphrodite (have both male and female organs) and are cross-pollinated through wind. reproduction of this grass is either through seeds or by vegetative structures like rhizome (encycloweedia, 2002). it is an excellent sand binder and prefers arid and semi-arid conditions (cope, 1982). blue panic has ability to withstand a variety of climatic conditions including severe environmental stresses like drought and salt stress (ashraf, 2004; ahmad et al., 2010). it can tolerate salinity up to 15,000 mg l-1 and drought, using almost 50% less water than alfalfa (bokhari et al., 1988). blue panic is an ideal fodder grass because of its high protein contents (15-18%) (bokhari et al., 1988). due to high tolerance against a multitude of stressful environments, blue panic is widely distributed in a variety of climatic conditions including the whole indo-pakistan region (ahmad et al., 2010). it extensively occurs in arid and semi-arid region where plants usually experience drought stress (cope, 1982). plants exhibit resistance to various stresses including drought by under-going morphological changes and alterations in genetic and biochemical attributes (levitt, 1972). alteration in various mechanisms by plants to produce resistance against drought, tolerance at the cellular level is essential, because the cellular processes are most sensitive due to change in cell turgor under drought (taiz and zeiger, 2006). reduced cell turgor may lead to impaired growth in most plant species (coleman, 2008). drought-induced decrease in plant biomass has been extensively reported in the literature in many crops like wheat, maize, rice grasses etc. (manabendra et al., 1998; akram et al., 2008; kamran et al., 2009). drought stress also has a negative effect on net co2 assimilation rate, transpiration rate and stomatal conductance (ares et al., 2000). mineral composition of soil is also changed under drought stress which causes poor absorption of minerals (garg et al., 2004; samarah et al., 2004). the responses of plants to drought depend on the severity of drought, i.e., intensity and duration of drought. it also depends on the plant species and developmental growth stage (chaves et al., 2003). of morphological traits required to resist to early drought stress conditions, a deep and dense root system is probably the most important one (gregory, 1989; robertson et al., 1993). high root growth by diverting assimilates from shoots towards roots is also a strategy of plants to cope with the adverse effects of drought stress, and resulting in to high root/shoot ratio (o’tool and bland, 1987). ecotypes of panicum antidotale are found cultivated on ecologically distinct habitats in the faisalabad region (gelani, 2000). due to the distribution of this grass on a wide range of habitats, it is expected that all these ecotypes are potentially adapted to a variety of environmental stresses like drought, salinity, toxic nutrients etc. it is also hypothesized that ecotypes inhabiting a particular habitat since a long period must have genetically adapted on this environment. thus, the main objective of the present study was to examine variation in drought tolerance of differently adapted blue panic grass ecotypes using some key physiological attributes. materials and methods an experiment was conducted in a wire-house in the botanical garden, university of agriculture, faisalabad (uaf), pakistan (latitude 31°30 n, longitude 73°10 e and altitude 213 m). six populations of blue panic (panicum antidotale) grass were collected from different habitats from the suburbs of faisalabad city. brief description of the habitats is presented in tab. 1. the habitats were selected on the basis of water availability, salt content, soil texture, physico-chemical characteristics and environmental conditions. soil analysis the soil adhering to the roots was used to analyze various physicochemical characteristics. the soil ph and ece were determined by using ph and ec meter respectively. analysis of soil was carried out following the specifi c methods described in hand book no.60 (us salinity laboratory staff, 1954). experimental detail: a total of 90 to 100 plants of each population were collected from each habitat and all plants were established in normal soil under natural conditions for six months in the botanical garden of the university of agriculture, faisalabad-pakistan. young tillers of uniform size from each population were collected and then transplanted in plastic pots (30 cm diameter; at a rate of 6 tillers per response of blue panic grass to water defi cit conditions 135 pot) each having 8 kg dry sandy soil. soil with ph = 7.4, ece = 2.8 ds m-1, and saturation percentage = 27.7 was used for this study. when the plants grown in pots attained a height of about 60 cm, their shoots were clipped at the height of 30 cm and then subjected to three levels of drought (control = normal irrigation, 60% fi eld capacity, and 30% fi eld capacity) in four replications. a total 72 pots were used in this study. after four weeks of plant growth under drought, all plants were harvested from each pot carefully, washed with distilled water and data for the following attributes were recorded: 1. shoot and root fresh and dry weights (g): four plants from each pot were used for appraising shoot and root fresh and dry biomass, while the remaining two used for quantifying chlorophyll contents and mineral nutrients. both parts, i.e., shoots and roots were weighed separately and then oven-dried at 65 °c for one week. 2. chlorophyll pigments: the chlorophyll contents were determined from green leaves following arnon (1949). fresh leaf material (0.2 g) was extracted in 80 % acetone and centrifuged at 10,000 x g for 5 minutes. absorbance of the supernatants of all samples was measured at 663 and 645 nm using uv-visible spectrophotometer (hitachi-u2001, tokyo, japan). 3. gas exchange parameters: net co2 assimilation rate and other gas exchange characteristics were measured using an open system portable infrared gas analyzer (lca-4; analytical development company, hoddesdon, england). these measurements were made on a fully expanded youngest leaf from each plant from 10:15 to 12:45 hours with the following adjustments of leaf chamber:ambient co2 concentration (cref) 353 µmol mol-1, molar fl ow of air per unit leaf area (us) 221.04 mol m-2 s-1, temperature of leaf chamber varied from 31.4 to 37.9 oc, leaf surface area 11.35 cm2, ambient pressure 99.3 kpa, water vapor pressure into the chamber ranged from 0.0006 to 0.00089 mpa, leaf chamber gas fl ow rate (v) 252 µmol s-1, par (qleaf) at the leaf surface was maximum up to 1048 µmol m-2 s-1. 4. determination of mineral elements in plant tissues the dried ground plant material (shoot or root) (0.1 g) was digested with a mixture of sulphuric acid and hydrogen peroxide following wolf (1982). the digested samples were analyzed for potassium and calcium using a fl ame photometer (pfp7; gransmore green, dunmow uk). phosphorus was determined spectrophotometrically (hitachi-u2001, tokyo, japan) following the method of jackson (1962) and n by titration method following allen et al. (1998). statistical analysis data for different parameters were analyzed statistically by adopting two-way analysis of variance technique based on completely randomized design with four replications according to steel and torrie (1997). the least signifi cance difference test (lsd) was used for appraising the signifi cant difference between the mean values (snedecor and cochran, 1980). results drought stress signifi cantly reduced shoot fresh and dry weights of all populations of panicum antidotale (tab. 2; fig. 1a and b). since the populations differed signifi cantly in control values, so it is not legitimate to interpret the results on mean fresh and dry weights basis. thus, the populations were compared on percent of control basis (in fig. 1 values presented in parenthesis are percent of control). at 60 and 30 % fi eld capacity, populations 2 and 6 were higher in percent shoot fresh and dry weights as compared to the other populations (fig. 1), while the population 1 was the lowest of all six populations. root fresh and dry weights also decreased signifi cantly under water defi cit conditions. of the six diverse populations collected from different habitats, populations 3 and 4 were better as compared to the others under well watered or water defi cit conditions. populations 2 and 6 were lower in root fresh and dry weights than those of the other populations of p. antidotale (tab. 2; fig. 1c and d). shoot length of all populations was signifi cantly reduced under water defi cit conditions (tab. 2; fig. 1e). of different populations, populations 2 and 3 were higher and populations 5 and 6 lower in shoot length than the other populations at 60% and 30% fi eld capacities, respectively. drought stress signifi cantly affected chlorophyll a and b contents of all populations of p. antidotale (tab. 2; fig. 1f and g), while the populations did not differ signifi cantly in both chlorophyll a and b pigments under non-stressed or drought stress conditions. in chlorophyll a/b ratio (fig. 1h), population behavior was different. populations 2 and 3 showed an increase in chlorophyll a/b ratio at tab. 1: habitats details and physico-chemical characteristics of soils from where panicum antidotale ecotypes were collected. characteristics population 1 population 2 population 3 population 4 population 5 population 6 sites of collection along a on sludge of a dry shady along roadside, a derelict fi eld, botanical garden, water channel, disposal channel, condition, uaf uaf uaf uaf uaf uaf habitat description roots are soft textured very hard dry rain-fed undisturbed land moist shady exposed to soil textured salineloamy soil loamy soil highly saline sodic soil water k+ (mg kg-1 dry soil) 90 70 175 40 115 60 ca2+ (mg kg-1 dry soil) 44 19 20 13 12 16 n (%) 4.70 7.90 0.52 1.86 1.24 0.86 p (%) 0.06 0.04 0.06 0.04 0.03 0.05 saturation percentage 40 35 23 29 27 22.2 ph 6.6 7.35 6.8 8 7.65 6.70 ece (ds m-1) 19.8 2.5 17.4 1.5 1.4 2.9 136 m. shahbaz, m. iqbal, m. ashraf response of blue panic grass to water defi cit conditions 137 60% fi eld capacity, while all other populations showed a decrease in this attribute. maximum reduction in chlorophyll a/b ratio was observed in population 6 at 30 % fi eld capacity. imposition of different water regimes caused a signifi cant reduction in net co2 assimilation rate, transpiration rate and stomatal conductance (tab. 2; fig. 2a, b and c). this reduction was maximum at 30% fi eld capacity. populations 4, 5, and 6 were lower in photosynthetic rate than the others at 30 or 60% fi eld capacity. however, populations 2 and 5 had signifi cantly lower transpiration rate than the other populations under water defi cit conditions. stomatal conductance was lower in all populations except populations 3 and 4 at both 30 and 60% fi eld capacities and population 1 had high gs value at 60% fi eld capacity (fig. 2). populations 1, 2 and 5 were higher in water use effi ciency (a / e) as compared to the other populations under normal or drought stress conditions (tab. 2; fig. 2e). data for sub-stomatal co2 concentration and ci/ca showed that neither the drought caused a signifi cant effect nor populations differed signifi cantly for this attribute (tab. 2; fig. 2d and f). all six populations of p. antidotale differed signifi cantly for shoot n. shoot n was high in populations 2, 3 and 5 at 30% fi eld capacity while in others the difference in shoot n was not discernable (tab. 3; fig. 3a). however, maximum root n contents were observed in population 4 at 30% fi eld capacity (fig. 3b). imposition of water defi cit conditions had no signifi cant effect on shoot p (tab. 3; fig. 3c). of various populations, an increase in shoot p was observed in population 4 and a decrease in populations 5 and 6 at 60% and 30% fi eld capacities. population 5 was the lowest in root p of all populations under drought stress conditions, while in the others the difference was not signifi cant (fig. 3d). water defi cit conditions signifi cantly increased shoot k+ concentration of all populations, but for root k+, the pattern of increase or decrease was not consistent (tab. 3; fig. 3e and f). water defi cit conditions had no signifi cant effect on shoot ca2+ or root ca2+ concentrations. populations also did not differ signifi cantly in both these attributes (tab. 3; fig. 3g and h). discussion water scarcity is known to directly reduce the plant productivity (waseem et al., 2006). however, responses of plants to water defi cit conditions are very complex. of these, adaptive changes are of importance (chaves et al., 2002) like decrease in leaf area which further causes reduction in net co2 assimilation rate and growth (pereira and chaves, 1993). decrease in growth is common in all crop plants under drought stress. plants tend to cope this adverse effect by adopting various strategies. thus, there is a need to explore such mechanisms or criteria adopted by the plants which might differ on locality basis in even a single species. availability of water is an effective selective pressure which is an important factor in driving the evolution of natural plant populations tab. 2: mean squares from analyses of variance of data for growth attributes and gas exchange characteristics of panicum antidotale when plants were subjected to control or water defi cit conditions for three weeks (n = 4) source of variation degrees of freedom shoot fresh weight shoot dry weight root fresh weight drought (d) 2 17367.7*** 3933.6*** 2837.1*** populations (p) 5 518.21*** 133.61*** 259.37*** d x p 10 452.66*** 114.70*** 139.47*** error 54 70.968 20.380 29.651 source of variation degrees of freedom root dry weight shoot length drought (d) 2 1030.2*** 7278.9*** populations (p) 5 123.63** 230.72** d x p 10 69.783*** 233.01*** error 54 21.064 51.279 source of variation degrees of freedom chlorophyll a chlorophyll b chlorophyll a/b ratio drought (d) 2 0.592*** 3.314*** 0.010*** populations (p) 5 0.035ns 0.078ns 0.041*** d x p 10 0.058*** 0.160** 0.013*** error 54 0.015 0.047 0.00011 source of variation degrees of freedom a e gs drought (d) 2 4259.5*** 44.252ns 76834.1*** populations (p) 5 320.72*** 16.67ns 105407.2*** d x p 10 213.25*** 32.694ns 13381.1*** error 54 52.53 18.52 19.85 source of variation degrees of freedom cicic wue cicic /ci/ci a/ca/c drought (d) 2 177589.6ns 420.86ns 0.012ns populations (p) 5 47355.7ns 705.34* 0.043ns d x p 10 165651.3** 243.97ns 0.040** error 54 61140.6 207.12 0.015 *, **, *** = signifi cant at 0.05, 0.01, and 0.001 levels, respectively. ns = non-signifi cant source of variation drought (d) populations (p) d x p error source of variation populations (p) d x p error source of variation populations (p) d x p error source of variation populations (p) d x p error source of variation populations (p) d x p error 136 m. shahbaz, m. iqbal, m. ashraf response of blue panic grass to water defi cit conditions 137 fig. 1: growth attributes and chlorophyll contents of panicum antidotale retz when plants were subjected to various water defi cit conditions for three weeks (figs. in parenthesis are percent of control) (p1 to p6 = populations 1 to 6), fc = field capacity for drought tolerance (bennington and mcgraw, 1995; dudley, 1996a, b; heschel et al., 2002). however, natural populations evolve a multitude of morpho-anatomical and physiological characteristics that enable the plants to thrive well under harsh environmental conditions including drought stress. in present study, water defi cit conditions reduced plant biomass (both fresh and dry biomass) of all six ecotypes of blue panic grass. reduction in biomass under water defi cit conditions has already been observed in many crops, e.g., grasses (ashraf and yasmin, 1995), wheat (peschke et al., 1997; ashraf et al., 1998), maize (abrechit and carberry, 1993) and rice (manabendra et al., 1998). however, reduction in plant biomass differed among different ecotypes of p. antidotale. populations collected from sludge of disposal channel and that from the botanical garden were superior in shoot biomass production as compared to the other populations. one of important factor involved in plant growth is photosynthesis. 138 m. shahbaz, m. iqbal, m. ashraf response of blue panic grass to water defi cit conditions 139 fig. 2: gas exchange of panicum antidotale retz when plants were subjected to various water defi cit conditions for three weeks (p1 to 6 = populations 1 to 6), fc = field capacity high biomass in above mentioned populations might due to high photosynthetic rate. inter-cultivar difference has already been observed in barley (blum, 1996; martin et al., 1991), blackgram (ashraf and karim, 1991), lentil and indian mustard (wright et al., 2001), and brassica carinata (ashraf and sharif, 1998). growth and photosynthesis are two important aspects interlinked with each other. ratio of photosynthetic rate and transpiration rate is known as water use effi ciency (wue). under drought stress plants adapt themselves by improving water use effi ciency and decreasing stomatal conductance (dudley, 1996a; nativ et al., 1999; ares et al., 2000). improvement in water use effi ciency might be due to reduced in leaf size under drought stress as a result of which plants reduce water loss from leaves (givnish, 1979) and stomatal conductance also decreases under water shortage (zangerl and bazzaz, 1984). in the present study, a marked reduction in stomatal conductance has been observed in all ecotypes collected from various habitats. the reduced stomatal conductance has positively enhanced the water use effi ciency in all populations but on the other hand, reduction in stomatal conductance resulted in decreased net co2 assimilation rate and it may happen even at moderate level of drought stress (athar and ashraf, 2005). in most of the species reduction in growth is correlated well with reduced photosynthetic activity but this is not true for all species (athar and ashraf, 2005). in the present study, plant growth was positively correlated with net co2 assimilation rate (r = 0.719***) and thus the droughtinduced reduction in biomass production could have been due to reduced photosynthetic rate under drought conditions. plants require mineral nutrients for their proper growth and development, but under drought stress the soil mineral composition is also impaired (marschner, 1995). decrease in water availability under drought generally results in reduced total nutrient uptake and frequently reduces the concentrations of mineral nutrients in crop plants (marschner, 1995; baligar et al., 2001). generally, plants showing small reduction in absorption of nutrients are considered 138 m. shahbaz, m. iqbal, m. ashraf response of blue panic grass to water defi cit conditions 139 as drought resistant (gunes et al., 2006) but plant species and even genotypes within species may vary in their response to mineral uptake under water stress (garg, 2003). in the present study, all six p. antidotale ecotypes collected from different habitats showed a signifi cant difference in shoot n, p, k+ and root k+. likewise, a large amount of variation in n, p and k+ uptake has already been reported in a set of 20 chickpea genotypes (gallani et al., 2003). furthermore, shoot k+ and ca2+ concentrations were higher in all ecotypes under drought stress as compared to those in plants experienced normal watering. high ca2+ concentration under drought stress has already been reported in pearl millet (ashraf et al., 2001). both k+ and ca2+ are very important for plant growth in terms of stomatal regulation and membrane stability, respectively (mengel and kirkby, 2001). effi cient plants are active in uptake of n, p, and k+ as compared to drought sensitive plants under drought stress (rengel, 2001). similarly, drought tolerant cultivars accumulated more n, p, k+, and ca2+ as compared to sensitive as observed in chickpea (gunes et al., 2006), soybean (samarah et al., 2004), and grasses (akram et al., 2008). in shoot k+, populations collected from sludge of disposal channel and from dry shady conditions showed higher concentration of shoot k+ as compared to the other ecotypes, but the difference in populations with respect to shoot or root ca2+ was not prominent. in conclusion, the ecotypes of p. antidotale collected from different habitats showed a signifi cant reduction in plant fresh and dry biomass, chlorophyll pigments, photosynthetic and transpiration rates, while an increase in shoot n, p, k+ and root k+ was observed under water defi cit conditions. of all six ecotypes, one collected from disposable channel sludge and the botanical garden showed higher plant biomass in comparison with the others. increase in plant biomass was found to be associated with higher photosynthetic rate in all six ecotypes. in other physiological attributes like net co2 assimilation rate, water use effi ciency and shoot n the populations collected from habitats containing with high amount of sludge and that from dry shady conditions performed better performance as compared to the other populations. references abrechit, d.g., carberry, p.s., 1993: the infl uence of water defi cit prior to tassel initiation in maize growth, development and yield. field crop res. 31, 55-69. ahmad, m.s.a., ashraf, m., ali, q., 2010: soil salinity as a selection pressure is a key determinant for the evolution of salt tolerance in blue panicgrass (panicum antidotale retz.). flora 205, 37-45. akram, n.a., shahbaz, m., ashraf, m., 2008: nutrient acquisition in differentially adapted populations of cynodon dactylon (l.) pers. and cenchrus ciliaris l. under drought stress. pak. j. bot. 40, 1433-1440. allen, j.f., holmes, n.g., 1986: electron transport and redox titration. in: hipkins, m.f., baker, n.r. 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exus and abutilon theophrasti. ecology 65, 207-217. address of the author: corresponding author’s email: shahbazmuaf@yahoo.com art06_perveen.indd journal of applied botany and food quality 85, 41 48 (2012) 1department of botany, university of agriculture, faisalabad-pakistan 2dept. of botany and microbilogy, king saud university riyadh, riyadh, saudi arabia is pre-sowing seed treatment with triacontanol effective in improving some physiological and biochemical attributes of wheat (triticum aestivum l.) under salt stress? shagufta perveen1, muhammad shahbaz1*, muhammad ashraf 1, 2 (received january 24, 2012) * corresponding author summary in this study, seed-priming of two wheat cultivars s-24 and mh-97 was carried out with three triacontanol (tria) levels (0, 10 and 20 µm20 µm20 µ ) for 12 h. seeds pre-treated with tria were allowed to m) for 12 h. seeds pre-treated with tria were allowed to m grow for 24 days in full strength hoagland’s nutrient solution in a greenhouse, thereafter two salt treatments (0 and 150 mm greenhouse, thereafter two salt treatments (0 and 150 mm greenhouse, thereafter two salt treatments (0 and 150 m nacl) were applied and after 21 days of salt application, changes in growth attributes such as shoot and root dry weights, shoot and root lengths and total leaf area, leaf water relations such as water potential (ψw), osmotic potential (ψs), turgor potential (ψp) and relative water contents (%), membrane permeability (%), total free amino acids, free proline, glycinebetaine and total phenolic contents were determined. yield attributes such as 100-seed weight, total grain yield, number of grains and number of fertile tillers per plant were recorded at crop maturity. salinity stress of 150 mm nacl mm nacl mm signifi cantly caused reduction in all growth and yield attributes, leaf water relations except leaf turgor potential, while increased membrane permeability (%), leaf free proline, glycinebetaine and total free amino acids in both cultivars. total phenolics and relative water contents (%) remained unaffected under salt stress. pre-sowing seed treatment with tria did not mitigated the adverse effects of salt stress on wheat and thus could not prove to be effective to promote signifi cantly its growth, yield and other physiological and biochemical attributes (except leaf water potential of cultivar mh97) under stress and non-stress conditions. overall, performance of cultivar s-24 was better in growth, leaf water relations and free proline contents as compared to mh-97 under both non-saline and saline conditions. introduction plant hormones are involved in rapid induction of plant responses to external environmental cues (pedranzani et al., 2003). abiotic stresses alter the levels of phytohormones that result in reduced plant growth (morgan, 1990). however, exogenous application of plant hormones is an attractive approach that has been reported to increase salt tolerance in crop plants by regulating various growth processes (barakat, 2011; zeid, 2011; hussain et al., 2011). shekoofeh and shahla (2012) found that treatment with plant hormones enhanced salt stress tolerance in crops by reducing the level of h2o2 (oxidative stress), decreasing membrane damages and increasing the accumulation of organic solutes for growth processes. triacontanol (tria) is also known as a plant hormone since 1977 as it effi ciently involved in promoting growth and yield of several crop species like rice, wheat, maize, tomato, greengram, hyacinth bean, coffee senna and wild mint (mamat et al., 1983; ries et al., 1977; ries, 1985, 1991; kumaravelu et al., 2000; khan et al., 2009; naeem et al., 2009, 2010, 2011). the stimulatory effects of tria on water and nutrients uptake, photosynthesis, enzyme activities, membranes stability and gene regulation have made this plant growth regulator interesting for many researchers working for improving crop productivity under abiotic stress conditions including salt stress (muthuchelian et al., 1996; chen et al., 2002, 2003; kilic et al., 2010, 2011; perveen et al., 2010, 2011). as an effective growth regulator, tria has been used for enhancing shoot and root growth and producing secondary metabolites in different plant species (reddy et al., 2002; giridhar et al., 2005; malabadi et al., 2005; malabadi and nataraja, 2007; parimalan et al., 2009). it inhibits enzymatic and non-enzymatic peroxidative breakdown of membrane lipids (ramanarayan et al., 2000). for example, seed treatment with tria has been reported to increase the activity of a key antioxidant enzyme peroxidase that plays a role in reducing the level of h2o2 (a reactive oxygen species) (perveen et al. 2011). soil salinity is one of the major abiotic stresses that contribute to crop yield losses worldwide (grewal, 2010). salt stress exerts negative effects on growth, yield and water status of wheat (akbari ghogdi et al., 2012). salt-induced damage to plasma membrane results in increased cell permeability that causes electrolyte leakage (blum and ebercon, 1980). however, plants escape osmotic stress by sequestering toxic na+ and clions into vacuoles and accumulation of some non-toxic compatible solutes such as free amino acids, proline and glycinebetaine in cytoplasm to balance decreased water potential through osmotic adjustment (di martino et al., 2003; heidari, 2012; geng et al., 2012). among various strategies to cope the adverse effects of salt stress on agricultural crops, several shot-gun approaches are in vogue these days. seed-priming (seed-soaking in the solution of some organic/ inorganic solutes, plant growth regulators or plant hormones etc.) is one of these approaches that have gained much more importance due to being simple, easy to apply, and with low risk and low cost. keeping in view the role of tria, the objectives of the present study were to assess whether or not pre-sowing seed treatment with tria could be effective in reducing the adverse effects of salt stress on growth and yield of wheat crop and whether tria could modulate various physiological and biochemical attributes like free amino acids, proline, glycinebetaine, total phenolics, membrane permeability (%) and leaf water relations under saline conditions. materials and methods an experiment was conducted in the wire-house of the botanical garden (altitude 213 m, latitude 31°30’n and longitude 73°10’e), to examine the plausible role of tria applied as a pre-sowing seed treatment on wheat under salt stress during spring, 2010. the climatic conditions were: mean day and night temperature cycle of 20 and 6°c, 10 and 14 h light and dark period (photoperiod with ppfd 825-1150 µmol m-2 s-1), and rh 54 ± 5%. seed of two spring wheat cultivars namely s-24 (salt tolerant) and mh-97 (moderately salt sensitive), was obtained from the botany department, university of agriculture, faisalabad, pakistan and ayub agricultural research institute, faisalabad, pakistan, respectively. before the start of the experiment, surface sterilization of the seed of both cultivars was done using sodium hypochlorite solution (5%) for 5 min, rinsed with sterilized water and air-dried. then seeds (one hundred of 42 s. perveen, m. shahbaz, m. ashraf each cultivar) were soaked in varying concentrations of triacontanol solutions (0, 10, and 20 µmsolutions (0, 10, and 20 µmsolutions (0, 10, and 20 µ ) prepared in hot distilled water at 90°c m) prepared in hot distilled water at 90°c m along with 0.1% tween-20 (cited in chen et al., 2002). after 12 h of soaking, the seeds were re-dried to original weight with forced air under shade. ten seeds per pot were allowed to germinate in thoroughly washed sand. after 10 days of seed germination, six plants were maintained by thinning in each pot/replicate. twenty four day-old plants were treated with saline stress for further 21 days. there were two salt (nacl) levels, i.e., 0 mmdays. there were two salt (nacl) levels, i.e., 0 mmdays. there were two salt (nacl) levels, i.e., 0 m (control) and m (control) and m 150 mm150 mm150 m . every week hoagland’s nutrient solution (full strength) was applied @ two litters per pot. for attaining the desired level of salt, nacl in hoagland’s nutrient solution was added in an aliquot of 50 mmof 50 mmof 50 m solution to each pot every day. every week salt level m solution to each pot every day. every week salt level m (150 mm(150 mm(150 m nacl) was applied in hoagland’s nutrient medium until the m nacl) was applied in hoagland’s nutrient medium until the m end of the experiment. the sand moisture content was maintained every day by watering 200 ml h2o to each pot. there were four replicates of each treatment, and all experimental units arranged in a completely randomized design. from each pot two plants were harvested when plants were 45 days old. after taking fresh weights, plants were oven-dried at 65oc for one week and their dry biomass recorded (shoot and root dry weights). in addition, data for other growth attributes, e.g., shoot and root length and leaf area per plant, was also recorded. before harvesting, data for various physiological/ biochemical attributes were recorded. total free amino acids total free amino acids were determined according to moore and stein (1957). fresh leaves (0.5 g) were ground in 10 ml of citrate buffer (ph 5.0) and the mixture was centrifuged at 15000 x g for 10 min. the extraction samples were further processed with ninhydrin solution and the optical density of the solution read at 570 nm using a spectrophotometer (irmeco u2020). total phenolics the amount of total phenolics was determined following the method of julkenen-titto (1985). fresh leaves (0.1 g) were ground properly in 2 ml of 80% acetone, centrifuged at 10,000 x g, for 15 min and the supernatant collected in a microfuge tube. to 100 µl of the supernatant 2.0 ml of distilled water, 0.5 ml of folinciocalteau’s phenol reagent, and 2.5 ml of 20% na2co3 solution were added and raised the fi nal volume to 5 ml by adding distilled h2o. after 20 min of vortexing, the absorbance was read at 750 nm using a spectrophotometer (irmeco u2020). leaf free proline estimation of free proline in the leaf tissues was performed following bates et al. (1973). toluene was used as a blank and absorbance read at 520 nm on a spectrophotometer. using a standard curve the concentration of free proline was determined and calculated on the basis of fresh weight as follows: µg proline ml-1× ml of toluene/115.5 µmole proline g-1 fresh weight= µg proline ml fresh weight= µg proline ml × ml of toluene/115.5 fresh weight= × ml of toluene/115.5 g of sample µmole proline g g of sample µmole proline g fresh weight= g of sample fresh weight= glycinebetaine content glycinebetaine was determined according to the method of grieve and grattan (1983). fresh leaf (0.5 g) was homogenized with 10 ml distilled water. whatman no. 2 fi lter paper was used for homogenate fi ltration. the extract (1 ml) was mixed with 1 ml of 2 n h2so4. to 0.5 ml of the above mixture, potassium tri-iodide solution (0.2 ml) was added and then the mixture was cooled in an ice bath for 90 min. to the sample mixture, 2.8 ml of chilled distilled water and 6 ml of 1-2-dichloromethane were added. from two distinct layers, the absorbance of the lower organic layer was read at 365 nm using a uv-visible spectrophotometer (irmeco u2020). relative membrane permeability (%) fresh leaves (0.5 g) were chopped and placed in 10 ml of distilled water. then samples were vortexed for 5s and the electrical conductivity (eco) determined. the test tubes were kept at 4°c for overnight and the ec1 measured. then the samples were autoclaved for 1 h and ec2 measured after cooling the solution to room temperature. relative membrane permeability was measured using the following formula: rmp (%) = (ec1-eco/ec2eco) x 100 leaf water potential: leaf water potential of the second leaf from top was determined with a scholander type pressure chamber (arimad-2-japan) following the method of scholander et al. (1964). leaf osmotic potential: osmotic potential of the leaf used for water potential determination was determined using an osmometer (vapro, vapor pressure osmometer, model 5520, usa). turgor potential: leaf turgor potential was determined as the difference between water potential and osmotic potential values (nobel, 1991). relative water content (rwc): relative water content was calculated following the method of jones and turner (1978). fresh leaf samples were weighed (fw), soaked in deionized water in the dark for 24 h to re-hydrate and their turgid weight (tw) recorded. after that, the samples were oven-dried at 80°c for 48 h to get their dry weight (dw). relative water content was calculated using the following formula: rwc = [(fw tw)/(fw dw)] ×100 yield attributes: yield parameters i.e., grain yield and number of grains per plant, 100-grain weight, and number of fertile tillers, were determined at maturity. statistical data analysis: the data for each variable was analyzed by a costat computer package (cohort software, berkeley, ca). the least signifi cance difference test was used to compare mean values (snedecor and cochran, 1980). results salt stress (150 mmsalt stress (150 mmsalt stress (150 m nacl) signifi cantly reduced all the growth and m nacl) signifi cantly reduced all the growth and m yield attributes such as shoot and root dry weight, shoot and root length, total leaf area, grain yield and number of grains per plant, 100-grain weight and number of fertile tillers per plant of the two wheat cultivars, s-24 and mh-97 (tab. 1; fig. 1). cultivar s-24 was higher in shoot dry weight, and shoot and root lengths as compared to mh-97, while mh-97 was better in root dry weight. in yield attributes both cultivars did not show any signifi cant difference except that number of tillers was more in cultivar mh-97 than s-24 (tab. 1; fig. 1). pre-sowing tria treatment increased shoot dry weight and total leaf area in cultivar s-24 under non-saline conditions, while in mh-97 under both saline and non-saline conditions and increase fresh weight= treatment of wheat seeds with triacontanol under salt stress 43 tab. 1: growth and yield attributes, contents of total free amino acids, proline, glycinebetain, total phenolics, membrane permeability (%) and leaf water relations of salt-stressed and non-stressed wheat (triticum aestivum l.) plants raised from seed treated with triacontanol for 12 h. source of variation df shoot dry root dry total leaf area shoot length root length grain yield wt. wt. per plant cultivars (cvs) 1 0.183* 0.004** 110.78ns 608.27*** 29.34*** 5.294ns salinity (s) 1 9.001*** 0.107*** 224486*** 1241.38*** 108.51*** 22.96** triacontanol (tria) 2 0.033ns 0.001ns 653.28 ns 0.2986ns 1.590ns 0.099ns cvs x s 1 0.014ns 0.002* 216.46ns 21.78** 9.507** 0.505ns cvs x tria 2 0.078ns 0.003** 1773.79ns 13.69** 5.090** 1.291ns s x tria 2 0.089ns 0.0005ns 3133.43* 2.146ns 7.132*** 0.836ns cvs x s x tria 2 0.030ns 0.004** 1040.74ns 6.424ns 4.882** 1.032 error 24 0.027 0.0004 893.97 2.014 2.014 2.077 source of variation df 100-seed number of number of amino acids proline gb wt. grains fertile tillers per plant per plant cultivars (cvs) 1 0.0003ns 3079ns 7.260** 0.288ns 0.015* 0.0002ns salinity (s) 1 3.8025*** 5470.8* 5.575** 36.98* 0.070*** 0.936*** triacontanol (tria) 2 0.1733ns 27.16ns 0.385ns 2.459ns 0.007ns 0.051ns cvs x s 1 0.2336ns 0.012ns 0.340ns 1.708ns 0.008ns 0.006ns cvs x tria 2 0.0477ns 617.90ns 0.802ns 4.292ns 0.003ns 0.010ns s x tria 2 0.0133ns 716.85ns 2.478* 3.175ns 0.0004ns 0.008ns cvs x s x tria 2 0.4811ns 208.78ns 0.271ns 3.401ns 0.00006ns 0.018ns error 24 0.1625 1144.97 0.561 8.629 0.003 0.023 source of variation df total mp (%) water osmotic turgor rwc (%) phenolics potential potential potential cultivars (cvs) 1 0.622ns 68.555* 0.481*** 0.086** 0.160** 72.74* salinity (s) 1 5.530ns 257.498*** 0.368*** 0.256*** 0.010ns 38.73ns triacontanol (tria) 2 0.359ns 12.037ns 0.040* 0.009ns 0.036ns 39.08ns cvs x s 1 3.644ns 4.674ns 0.022ns 7.640ns 0.030ns 2.451ns cvs x tria 2 2.729ns 1.379ns 0.006ns 0.036* 0.055ns 2.387ns s x tria 2 0.492ns 5.393ns 0.003ns 0.010ns 0.021ns 8.812ns cvs x s x tria 2 4.513ns 21.528ns 0.007ns 0.001ns 0.008ns 2.220ns error 24 1.441 11.5576 0.008 0.009 0.017 16.13 *, **, and *** = signifi cant at 0.05, 0.01, and 0.001levels, respectively; ns = non-signifi cant; df = degrees of freedom; gb = glycinebetaine; mp = membrane permeability; rwc = relative water content in shoot growth was more at 20 µmin shoot growth was more at 20 µmin shoot growth was more at 20 µ of tria under non-saline m of tria under non-saline m conditions (tab. 1; fig. 1). root dry weight increased in cultivar mh-97 under saline conditions, while in s-24 under non-saline conditions by application of tria as seed treatment (tab. 1; fig. 1). root length was signifi cantly infl uenced by tria application and 10 µm10 µm10 µ tria was proved to be more effective in enhancing root m tria was proved to be more effective in enhancing root m length under non-saline conditions, while under saline conditions the pre-sowing treatment with tria imposed signifi cant effects on root length in s-24 only. pre-sowing seed treatment with tria only increased number of fertile tillers in cultivar mh-97 under nonsaline conditions, while its effect was non-signifi cant on all other yield attributes (tab. 1; fig. 1). total free amino acids slightly increased under saline conditions in both wheat cultivars i.e. s-24 and mh-97 (tab. 1; fig. 1). the presowing tria application did not signifi cantly alter total free amino acids contents in both wheat cultivars (tab. 1; fig. 1). root medium salinity markedly increased proline and glycinebetaine contents in both wheat cultivars (tab. 1; fig. 2). cultivar s-24 accumulated higher amount of proline as compared to that of mh-97 especially under saline conditions, while in glycinebetaine the cultivars did not show a signifi cant difference (tab. 1; fig. 2). the pre-sowing tria application did not signifi cantly alter the proline and glycinebetaine contents in both wheat cultivars (tab. 1; fig. 2). sodium chloride induced salinity stress did not affect total phenolics in both wheat cultivars (tab. 1; fig. 2). the cultivars did not show any difference in total phenolics under both stress and non-stress conditions. the pre-sowing treatment of tria was also found to be non-effective in altering total phenolics in both wheat cultivars (tab. 1; fig. 2). membrane permeability (%) increased signifi cantly in both s-24 and mh-97 wheat cultivars under saline conditions. cultivar mh-97 was higher in membrane permeability than cv. s-24 under either saline or non-saline conditions (tab. 1; fig. 2). the pre-sowing application of tria decreased membrane permeability (%) under non-saline conditions in both wheat cultivars (tab. 1; fig. 2). leaf water relation attributes like ψw and ψs signifi cantly decreased in both cultivars under saline conditions (tab. 1; fig. 2). cultivar s-24 was higher in ψw and ψs than mh-97 under both prevailing conditions. the pre-sowing treatment of tria at 10 µm level signifi cantly increased leaf ψw, while decreased leaf ψs of salt44 s. perveen, m. shahbaz, m. ashraf fig. 1: growth and yield attributes and total free amino acids of salt-stressed and non-stressed wheat (triticum aestivum l.) when 24-day old plants subjected for 21 days to saline or non-saline conditions (seed priming for 12 h). sensitive wheat cultivar mh-97 (tab. 1; fig. 2). salinity stress did not alter the leaf turgor potential signifi cantly; however, the two cultivars differed signifi cantly in leaf turgor potential as cultivar s24 was higher in this attribute than mh-97 under both normal and salt stress conditions. the pre-sowing application of tria did not signifi cantly affect the leaf turgor potential; however, in mh-97 the leaf turgor potential value was higher at the level of 10 µmleaf turgor potential value was higher at the level of 10 µmleaf turgor potential value was higher at the level of 10 µ under m under m non-saline or saline conditions (tab. 1; fig. 2). the relative water content (%) remained unchanged in both wheat cultivars both under salinity stress and tria treatment. however, water contents were higher in s-24 than those in mh-97 both under prevailing conditions (tab. 1; fig. 2). statistical data analysis: the data for each variable was analyzed by a costat computer package (cohort software, berkeley, ca). the least signifi cance difference test was used to compare mean values (snedecor and cochran, 1980). treatment of wheat seeds with triacontanol under salt stress 45 fig. 2: contents of free proline, glycinebetaine, total phenolics, membrane permeability (%) and leaf water relations of wheat (triticum aestivum l.) when 24-day old plants subjected for 21 days to saline or non-saline conditions (seed priming for 12 h). discussion due to their role in regulating various physiological and biochemical processes, new plant growth regulators are being extensively used these days in improving abiotic stress tolerance in crop plants (peleg and blumwald, 2011). plant growth regulators are also being used as seed priming agents to reduce the adverse effects of salt stress in different studies (iqbal and ashraf, 2007). triacontanol is a growth promoter that naturally occurs in plant epicuticular waxes (ries et al., 1977). it is reported that exogenous application of tria stimulates the induction of a secondary messenger (9-β-l (+)-adenosine) which moves rapidly throughout the plant body and regulates various physiological and biochemical processes and thereby increasing plant growth and yield (ries and houtz, 1983; ries, 1985, 1991; ries et al., 1990, 1993). in our study, salinity stress (150 mmin our study, salinity stress (150 mmin our study, salinity stress (150 m nacl) exerted a negative m nacl) exerted a negative m 46 s. perveen, m. shahbaz, m. ashraf effect on all growth and yield attributes like shoot and root dry weight, total leaf area, shoot and root lengths, grain yield and number of grains per plant, 100-grain weight, and fertile tillers of both wheat cultivars, s-24 and mh-97. this is analogous to what has been observed in a number of earlier studies (shahbaz et al., 2008, 2011; akram et al., 2011; chaabane et al., 2011). in our fi ndings, although improvement in some growth attributes (root dry weight under saline and total leaf area under non-saline conditions in mh-97, while shoot and root length and number of fertile tillers under both stress and non-stress conditions in both wheat cultivars) was observed due to tria application. however, overall effect of pre-sowing seed treatment with tria proved non-signifi cant on all growth and yield attributes. these results are in agreement with some other studies on various crop species. for example, bittenbender et al. (1978) found a non-signifi cant effect of tria on photosynthesis and fi nal yield of rice seedling under dark. in another report by bole and dubetz (1978) tria did not improved yield and yield components of water stressed wheat plants when applied as a soil supplement. charlton et al. (1980) reported that leeds durum wheat seeds treated with tria and its derivatives did not promote germination and growth. in tissue culture studies, germination and seedling growth of various weed and horticultural crops (lettuce, oat, soybean and wheat etc.) remained unaffected by tria application (marcelle and chrominski, 1978; hoagland, 1980; eriksen et al., 1982). non-signifi cant effect of tria as pre-sowing seed treatment on fresh biomass of wheat plants has also been reported earlier (perveen et al., 2010). in our experiment, total phenolic contents remained unaffected, while total free amino acids increased signifi cantly in both wheat cultivars under saline conditions. similarly, tammam et al. (2008) reported an increase in free amino acid content in wheat under saline conditions. salama et al. (1994) reported that under salinity stress, crop plants accumulate free amino acids which help maintain the osmotic balance by decreasing the cellular osmotic potential in plants under saline conditions. in this study, pre-sowing application of tria did not alter the total free amino acids or total phenolics signifi cantly in both wheat cultivars under control or saline conditions. contrarily, application of tria signifi cantly enhanced the accumulation of free amino acids and phenols in green gram under normal growth conditions (kumaravelu et al., 2000). proline plays a signifi cant role as an endogenous osmotic regulator in wheat, and its accumulation in plant tissues indicates plant ability to tolerate salt stress (munns et al., 2006). in our experiment, salt stress increased total free proline and glycinebetaine in both wheat cultivars. s-24 accumulated higher proline compared to that in mh97. as a negative correlation was found between leaf free proline or glycinbetaine contents, and leaf water potential (ψw), which became more negative due to increased osmotic potential, so it could be suggested that these two osmotica play a central role in osmotic adjustment under salt stress (shao et al., 2006; moghaieb et al., 2004). pre-sowing tria application proved ineffective in changing the contents of proline and glycinebetaine under normal or saline conditions. borowski and blamowski (2009) reported the ameliorating effect of tria on reducing the chilling stress in ocimum basilicum l. plants by decreasing free proline content, while increasing leaf area and activity of catalase. contrarily, krishnan and kumari (2008) reported that proline content decreased in triatreated soybean plants under salt stress. there are several reports which show that tria reduces the level of membrane damages due to its action as an antioxidant compound to inhibit peroxidation of membrane lipids (ramanarayan et al., 2000; grzegorezyk et al., 2006; khan et al., 2009). in the present study, application of tria as seed treatment decreased membrane permeability (%) of the two wheat cultivars only under normal conditions. salinity has been known to disturb the water relations of plants as a consequence of decreased leaf osmotic potential in external soil solution (munns, 2005). in our study, leaf ψw and ψs decreased in the two wheat cultivars under saline regime. however, leaf turgor potential and relative water contents (rwc) remained unaffected under salinity stress. early responses to salinity included decrease in water potential and rwc (gucci et al., 1997; alarcon et al., 1993). pre-sowing tria application (10 µm) increased the leaf water potential, while decreased osmotic potential of cv. mh-97 both under control and salt stress. these fi ndings are in agreement with those of krishnan and kumari (2008) who reported that tria could improve leaf water relations by decreasing leaf osmotic potential in soybean under salinity stress. non-signifi cant effect of tria on most of growth and yield attributes, free amino acids, free proline, glycinebetaine, total phenolics, leaf turgor potential and relative water contents (%) under stress or non-stress conditions could be attributed due to its lack of effective penetration through seed coat as earlier reported by hoagland (1980) or due to the formation of some inhibitory compounds by trace amounts of other aliphatic alcohols, esters or chemicals (laughlin et al., 1983; jones et al., 1979; ries et al., 1983, 1984). from the present study, it could be concluded that salinity stress signifi cantly decreased shoot and root dry weight, total leaf area, shoot and root lengths, leaf water relations except turgor pressure, and yield attributes, i.e., grain yield per plant, 100-seed weight, number of grains and number of fertile tillers per plant, while increased membrane permeability (%), contents of total free amino acids, leaf proline and glycinebetaine in both wheat cultivars. furthermore, treatment of 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treatments at 45ºc, 50ºc or 55ºc for 2 or 3 min after harvest and kept at 0°c for 45 days and additional 4 days at 20°c to determine the effects of hot water dip treatments on storage and shelf life of big top nectarine fruits. the effects of hot water dip treatments on quality parameters (weight loss, skin color, flesh firmness, total soluble solids and titratable acidity) and incidence of chilling injury (ci) and fungal decay were assessed after 45 days of storage at 0°c and subsequent 2 days and 4 days at 20°c following cold storage. hot water dip treatment at 45°c for 2 min reduced weight loss and ci, delayed color development and maintained fruit lightness, but insufficient to delay fruit softening. incidence of fungal decay was low during storage and shelf life period. our results indicated that hot water dip treatments had potential to maintain postharvest quality for big top nectarines. introduction postharvest fungicide treatments are required to control postharvest diseases in peaches and nectarines during storage. however, health and environment concerns lead to government regulations restricted postharvest use of fungicides in several fruits and vegetables. iprodine was not labeled for peaches and nectarines in europe and usa since 1996. some fungicides (e.g. dichloran) are also ineffective in control of common postharvest diseases of peach and nectarines (karabulut et al., 2002). postharvest hot water treatments have been studied to control postharvest decay in peaches and nectarines (wells, 1971; margosan et al., 1997; karabulut et al., 2002; karabulut and baykal, 2004). peaches subjected to 46°c hot water treatment for 2-8 min were found to have substantially decreased their disease susceptibility (margosan et al., 1997). postharvest life of nectarines is also limited by chilling injury (ci) or internal breakdown. most nectarine cultivars develop ci if they are held for more than 2-3 weeks below 8°c (lill et al., 1989). storage life of most nectarine cultivars with low susceptibility to chilling injury varied from 4 to 6 weeks at 0°c. with ci-susceptible cultivars, the marketing life was reduced to 2-3 weeks at 0°c (crisosto et al., 1999). onset of ci symptoms such as internal and external browning, flesh breakdown, woolliness, reddish discoloration, loss of ability to ripen and increased incidence of decay (lurie and crisosto, 2005) determine storage/shipping potential because their development reduces consumer acceptance (crisosto et al., 1999). hot water treatments have been shown effective in reducing the ci susceptibility in citrus (gonzalez-aguilar et al., 1997; schirra and d’hallewin, 1997; porat et al., 2000; özdemir and dündar, 2001), persimmon (lay-yee at al., 1997) and plum (abu-kpawoh et al., 2002). our previous study with big top nectarine (çelik et al., 2006) showed that storage life of this cultivar is limited to 30 days with 2 days of shelf life due susceptibility of this cultivar to ci. ci also increased the incidence of decay. in this study, we aimed to extend storage life of big top nectarines by hot water dip treatments; we focused on the effects of hot water dip treatments on incidence of chilling injury and fungal decay and on some quality indices of big top nectarines after storage and shelf life. materials and methods plant material nectarine cv. big top fruits were obtained from a commercial orchard in mersin, turkey. big top®-zaitabo originates from zaiger’s inc. (ca, usa) and was introduced into europe around 1989. fruit has intense red color on skin, firm yellow flesh and high levels of sugars but little acidity and aroma (lavilla, 2002). fruits were harvested from trees grafted on gf-677 rootstocks at firm-ripe stage and immediately transported via ventilated truck to cold storage facilities of department of horticulture, faculty of agriculture, mustafa kemal university where they were sorted and selected for medium size (5865 mm), uniform maturity and appearance and freedom from defects. hot water dip treatments fruits were dipped in hot water at 45ºc, 50ºc or 55ºc for 2 or 3 min. control fruits were dipped in water at 20°c for 2 or 3 min. treatments were carried out in a tank fitted with heating elements (0-90ºc, 2 x 2000 watt), an electronic recirculation pump (400 watt). the tank contained 375 litres of water. even water circulation, and temperature, within the baths was achieved by pumping water through perforated pvc tubing (25.4 mm i.d.). during each treatment, bath temperature was constantly maintained within ± 0.5°c of the required temperature by means of an electronic thermostat. following treatment, the fruits were allowed to dry for about 6 h at room temperature, then packed into 10-12 kg commercial plastic boxes (52 cm x 36 cm x 18 cm) and kept at 0°c (±0.5) in 85-90% (±5%) relative humidity for 45 days. each treatment was repeated three times using 5 fruits per replication that had been randomly selected from the same lot of fruit. three replicates per treatment were removed from cold storage after 45 days and subsequently held at 20°c (±1.0) and 65-70% (±5%) relative humidity for 4 days to simulate shelf life. postharvest quality evaluation postharvest quality of fruits was assessed after 45 days of storage and additional 2 and 4 days at 20°c following cold storage. fruits were numbered and individually weighted to determine weight loss. weight loss was calculated as percentage loss of initial weight. flesh firmness was measured on two opposite sides of each fruit at the equatorial region, after the removal of a 1 mm thick disk of skin from each side of the fruit and the force in kg required to insert an effegi penetrometer (model ft 327) fitted with an 8 mm diameter probe was recorded and expressed as newton (n). total soluble solids (tss) content and titratable acidity (ta) were assessed in juice obtained from five fruits per replicate. tss content was determined with a refractometer (atago model atc-1e) and ta by titration of 5 ml of fruit juice with 0.1 n naoh to ph 8.1 and expressed as g malic acid per 100 ml juice. skin color was determined with a minolta chroma meter cr-300 (osaka, japan). color measurements were recorded using the cie l*a*b* color space. from these values, hue angle was calculated as, h° = tan“1 (b*/a*). color values for each fruit were computed as means of two measurements taken from opposite sides at the equatorial region of the fruit. incidence of fungal decay was determined by counting the number of decayed fruit in each replicate of treatments on the day removal from storage and after 2 and 4 days at 20°c. fruits were halved and examined visually for different manifestations of ci or internal breakdown such as lack of juiciness (mealiness or woolliness), flesh browning, flesh bleeding, and flesh translucency (gel breakdown) immediately after 45 days of storage and after 2 and 4 days at 20°c following 45 days of storage. the severity of ci as was assessed as described by fernandez-trujillo and artes (1997) on a scale (1 to 5) where 1 = none, 2 = very slight, 3 = slight, 4 = moderately severe and 5 = severe. statistical analysis the data were analyzed as a factorial experiment in a completely randomized block design by anova using sas software of sas institute, cary, n.c. (sas, 1990). each treatment, consists of five fruits were replicated three times. mean separation was performed by fisher’s least significance test at p<0.05 level using sas’s proc glm procedure. data for percent weight loss and incidence of chilling injury were arcsine transformed and analyzed by anova and back transformed for reporting. results and discussion weight loss weight loss reached to about 11% after 45 days of storage (tab. 1). an additional weight loss of about 5% and 9% occurred during 2 and 4 days at 20°c following 45 days of cold storage, respectively (tab. 2). 45°c-2 min treatment resulted in lower weight loss than control and other treatments after 45 days of storage at 0°c (tab. 1) and after additional 2 days and 4 days at 20°c following 45 days of cold storage (tab. 2). in previous studies with peaches and nectarines, hot water treatments had no effect (malakou and nanos, 2005) or slight effect (zhou et al., 2002) on weight loss. however, fallik (2004) suggested that recrystallization or „melting“ of the wax layer due to how water treatments sealed barely visible cracks in the cuticle through which water could escape and this sealing of cracks or natural openings significantly reduced weight loss. consistent with our results, hot water treatments reduced weight loss in some fruits (garcia et al., 1995; özdemir and dündar, 2001; vicente et al., 2002). flesh firmness fruit softening occurred during 45 days of storage at 0°c (tab. 1). after 45 days of storage at 0°c, flesh firmness was at about 57 n. fruit softening continued during shelf life period following cold storage (tab. 2). flesh firmness was about 54 n and 14 n after additional 2 days and 4 days at 20°c following 45 days of storage, respectively. flesh firmness of fruits from 2-min hot water dip treatments was lower or similar to that of control fruits during 45 day of storage at 0°c (tab. 1). fruits from 3-min hot water dip treatments were firmer than control fruits during 45 days of storage at 0°c (tab. 1). hot air /hot water treatments have been showed to delay fruit softening during storage in peaches and nectarines (anthony et al., 1989; zhou et al., 2002; malakou and nanos, 2005). in other studies with peaches and nectarines, flesh firmness of fruits treated with hot air/ hot water was similar to control fruit (obenland and aung, 1997; karabulut and baykal, 2004; zhang et al., 2007). the delay of fruit softening might be due to inactivation of cell wall hydrolytic enzymes, mainly polygalacturonase (malakou and nanos, 2005) or associated to less ethylene production by heated fruits than control fruits (budde et al., 2006). 50°c-3min treatment resulted in firmer fruits than other 3-min hot water dip treatments during 45 days of storage at 0°c (tab. 1). similarly, obenland and aung (1997) reported that nectarine fruits treated with 50°c of hot water were firmer than those treated with 46°c of hot water which soften to the same extent as non-heated fruits. the little or no effect of hot water tab. 1: effects of hot water treatments on postharvest quality, incidence of chilling injury (ci) and fungal decay in big top nectarine cultivars after 45 days of storage at 0°c. treatments weight flesh tss ta skin color ci severity decay loss (%) firmness (n) (%) (%) l* h° (%) of ciz (%) at harvest 71.69(a) y 14.20(a) 0.56(a) 38.46(a) 36.10(a) 45 days at 0°c control-2min 11.87ab x 57.24bc 13.60 0.27b 36.08de 25.93f 13.33a 2.00bc 0.00a 45°c-2min 8.00e 55.44d 13.03 0.26b 43.25a 36.31a 0.00b 1.00c 0.00a 50°c-2min 11.51bcd 56.62c 13.10 0.30a 37.99cd 29.58cd 33.33a 3.67a 0.00a 55°c-2min 12.47a 53.84e 13.47 0.30a 37.84cd 28.58de 26.67a 2.33b 13.33a control-3min 11.71abc 55.57d 12.13 0.26b 40.44b 31.29c 0.00b 1.00c 0.00a 45°c-3min 10.85cd 57.57 b 13.00 0.27b 39.45bc 33.25b 0.00b 1.00c 0.00a 50°c-3min 10.65d 58.91a 13.73 0.31a 36.58de 26.83ef 13.33ab 2.00bc 13.33a 55°c-3min 11.97ab 56.72c 13.00 0.27b 35.78e 26.83ef 20.00ab 2.00bc 0.00a mean 11.13 56.49(b) 13.13(b) 0.28(b) 38.43(a) 29.82(b) 13.33 1.75 3.33 x mean separation was performed by fisher’s least significance test. treatment means (n=3) followed by same letter within column are not significantly different at p<0.005. yletters in parenthesis indicates comparison of means of storage time. values represents mean of all treatments for each storage time. zseverity of ci as was assessed on a scale (1 to 5) where 1 = none, 2 = very slight, 3 = slight, 4 = moderately severe and 5 = severe. hot water dips and nectarines 137 138 elif ertürk çandir, fatma temizyürek, ahmet erhan özdemir dip treatments was observed on flesh firmness during additional 2 days and 4 days at 20°c after cold storage (tab. 2) which was consisted with the findings of margosan et al. (1997) on several peaches and nectarines cultivars kept at 20°c for 4 days following 2 weeks at 1°c. budde et al. (2006) reported that when ethylene production is already triggered, heat treatments have no influence on fruit firmness, as evidenced in peaches harvested in a more advanced maturity, where heated and control fruit softened at the same rate during 3-day shelf life period at 20°c. total soluble solid content total soluble solid (tss) content was not affected by hot water dip treatments during cold storage (tab. 1) and during shelf life period after cold storage (tab. 2) in agreement with previous reports on nectarines and peaches (margosan et al., 1997; obenland et al., 1999; zhou et al., 2002). tss content decreased significantly after 45 days storage at 0°c compared to tss content at harvest (tab. 1). tss content did not change significantly during shelf life period (tab. 2 and 3). titratable acidity titratable acidity (ta) declined significantly after 45 days storage at 0°c (tab. 1). previous studies showed that hot air / hot water treatments had no significant effect on ta in peaches and nectarines (zhou et al., 2002; malakou and nanos, 2005), mandarins (schirra and d’hallewin, 1997) and apples (fallik et al., 2001) or reduced ta in nectarines (lay-yee and rose, 1994), strawberries (garcia et al., 1995; vicente et al., 2002) and apples (klein and lurie, 1990) and oranges (özdemir and dündar, 2006) during storage and shelf life period. fruits from 50°c-2min, 55°c-2min and 50°c-3min treatments maintained higher ta than control fruits during 45 days of storage (tab. 1) in contrast to previous studies (schirra and d’hallewin, 1997; fallik et al., 2001; zhou et al., 2002; malakou and nanos, 2005). during shelf life period, hot water treated fruits and control fruits had similar ta (tab. 2) in agreement with previous studies (klein and lurie, 1990; vicente et al., 2002; özdemir and dündar, 2006; zhang et al., 2007). skin color skin color parameters of l* (lightness) value did not significantly change, but hue angle (h°) value decreased after 45 days of storage at 0°c compared to initial values (tab. 1). during shelf life period, skin color becomes darker (lower l*value) with slight reduction in h° values (tab. 2). the effects of hot water dip treatments on skin color parameters were significant during storage, but these effects was not observed after 2 days and 4 days at 20°c following 45 days of cold storage. 45°c2min and 45°c-3min treated and control-3min fruits showed higher l* values than fruits from other treatments after 45 days of cold storage. lower l* values observed in fruits from other treatments might indicate skin browning associated with heat damage and chilling injury and combination of thereof (tab. 1). lay-lee and rose (1994) reported that one of the main disorders associated with tab. 2: effects of hot water treatments on postharvest quality, incidence of chilling injury (ci) and fungal decay in big top nectarine cultivars after 45 days of storage at 0°c and subsequent 2 days and 4 days at 20°c. treatments weight flesh tss ta skin color ci severity decay loss (%) firmness (n) (%) (%) l* h° (%) of ciz (%) 45 days at 0°c+ 2 days at 20°c control-2min 5.72bc x 53.09a 15.07a 0.25a 37.85a 27.56a 0.00b 1.00b 0.00c 45°c-2min 2.75e 52.07a 14.13a 0.27a 38.68a 31.26a 0.00b 1.00b 0.00c 50°c-2min 3.76d 54.98a 14.80a 0.30a 36.41a 29.28a 26.67a 3.00ab 20.00ab 55°c-2min 3.89d 54.59a 15.40a 0.31a 36.20a 27.46a 13.33ab 1.67ab 0.00c control-3min 6.78ab 50.05a 15.80a 0.28a 35.57a 28.55a 26.67a 2.67ab 26.67a 45°c-3min 4.61cd 53.05a 14.40a 0.31a 39.01a 32.77a 0.00b 1.00b 0.00c 50°c-3min 4.79cd 54.59a 16.20a 0.27a 37.71a 30.20a 13.33ab 2.00ab 0.00c 55°c-3min 7.17a 55.80a 16.27a 0.27a 36.20a 29.74a 0.00b 1.00a 6.67bc mean 4.82(b) y 53.53(a) 15.26(a) 0.28(b) 37.20(a) 29.60(a) 10.00(b) 1.67(a) 6.67(a) 45 days at 0°c+ 4 days at 20°c control-2min 10.97a 11.70a 15.67a 0.30a 30.32a 27.80a 70.00a 2.33ab 26.67ab 45°c-2min 6.13d 14.19a 14.00a 0.30a 37.40a 31.49a 6.67c 0.67b 0.00c 50°c-2min 8.15c 19.12a 16.07a 0.30a 35.64a 28.07a 26.67b 2.33ab 13.33bc 55°c-2min 7.33cd 10.92a 14.33a 0.32a 34.30a 25.47a 26.67b 2.00ab 26.67ab control-3min 9.95ab 14.32a 14.13a 0.29a 37.78a 30.88a 80.00a 3.33a 26.67ab 45°c-3min 8.97bc 13.89a 15.53a 0.31a 36.13a 29.29a 6.67c 0.67b 0.00c 50°c-3min 8.82bc 13.66a 15.27a 0.26a 35.43a 27.55a 33.33b 2.67a 0.00c 55°c-3min 10.53ab 17.16a 15.20a 0.30a 35.27a 32.73a 33.33b 1.67ab 46.67a mean 8.86(a) 14.37(b) 15.03(a) 0.30(a) 35.28(b) 29.16(a) 35.42(a) 1.96(a) 17.50(a) x mean separation was performed by fisher’s least significance test. treatment means (n=3) followed by same letter within column are not significantly different at p<0.005. treatment means was compared separately for each shelf life period. yletters in parenthesis indicates comparison of means of shelf life period. values represents mean of all treatments for each shelf life period. zseverity of ci as was assessed on a scale (1 to 5) where 1 = none, 2 = very slight, 3 = slight, 4 = moderately severe and 5 = severe. hot water dips and nectarines 139 heat treatment at 41-46°c for 24-48 hours was scald (external browning) in fantasia nectarines. the incidence of scald increased with increasing temperature and the length of treatment during storage at 0°c for 3 weeks. hot water treatment at about 45-50°c was not injurious to a number of nectarine and peach cultivars tested (wells, 1971; margosan et al., 1997; obenland and aung, 1997; malakou and nanos, 2005). our data showed that the hot water dip treatment at a temperature of >45°c for >2 min might result in heat damage on skin of big top nectarines as indexed by lower l* values. control-2min fruits showing ci symptoms (tab. 1) had also darker skin color (lower l* value). chilling injured peach and nectarine fruits show darker skin due to development of scald on skin along with flesh scald (fernandez-trujillo et al., 2000; lurie and crisosto, 2005). 45°c-2min and 45°c-3min treatment resulted in higher h° values than other treatments after 45 days of cold storage. higher h° values might indicate the delay of red color development by 45°c hot water dip treatment. previous studies showed that there is no or slight effect of hot air/hot water treatments on skin color of peaches and nectarines during storage and shelf life period (margosan et al., 1997; obenland et al., 1999; obenland et al., 2005; budde et al., 2006). in peach and nectarine fruits, red color development is a result of anthocyanin accumulation (tomas-barberan et al., 2001). there is no detailed study investigating the effect of hot air/hot water treatments on skin color in peaches and nectarines. with other fruits, delay of color development by hot air/hot water treatments was attributed to a diminution of pal activity which brought to reduction of anthocyanin synthesis (civello et al., 1997; vincente et al., 2002). incidence of chilling injury and fungal decay incidence of chilling injury (ci) was examined in fruits after removal from 45 days of cold storage and after 2 days and 4 days at 20°c following cold storage. a cultivar was determined to have reached the end of market life when >25% of fruit became mealy or leathery, had flesh browning or severe flesh bleeding (nanos and mitchell, 1991). as in commercial practice, only moderate and severe levels were considered as losses, representing the intensity of ci (fernandez-trujillo and artes, 1997). ci symptoms in susceptible peach and nectarine cultivars mainly develop during fruit ripening after cold storage (crisosto et al., 1999). therefore, the data from fruits kept at 4 days at 20°c following 45 days of storage might explain better the effects of hot water dip treatments on incidence of ci. control fruits showed high incidence of ci with moderate and severe symptoms after 4 days at 20°c following 45 days of storage. 45°c-2min and 45°c-3min treatments with none or very slight ci symptoms on fruits reduced incidence of ci after additional 2 and 4 days at 20°c following cold storage (tab. 2). hot air/hot water treatments have been reported to alleviate chilling injury in peaches and nectarines (kerbel et al., 1985; li and han, 1998; murray et al., 2007). 50°c and 55°c treatments did not reduce incidence of ci. fruits dipped in hot water at 50°c or and 55°c for 2 min or 3 min showed moderate or severe ci symptoms more than 25% of fruits after 4 days at 20°c following 45 days of storage (tab. 2). high-temperature forced-air treatment at about 50°c acted to accelerate development of mealiness, a major ci symptom in peach and nectarine fruits (obenland and carroll, 2000). authors suggested that too severe of a pre-stress may act as a damaging factor and overwhelm any positive benefit that is conferred by the heat treatment. incidence of fungal decay was low during cold storage (tab. 1) and shelf life period following cold storage (tab. 2). we observed infections caused by botrytis cinerea, penicillium expansum, rhizopus stolonifer, monilinia fructicola on fruits. fruits from 45°c-2min and 45°c-3min treatments showed no visible fungal growth during storage and shelf life period following cold storage. post-harvest hot water treatments have been shown to control postharvest decay in peaches and nectarines (wells, 1971; margosan et al., 1997; karabulut et al., 2002). however, it is not possible to reach conclusion for effects of hot water on fungal decay in big top nectarine fruits due to lower incidence of fungal decay. acknowledgements the authors wish to thank the uni-tarim coop. for supplying the fruits and the mustafa kemal university research foundation (project no: 04 m 0104) for their financial support during the course of this research. references abu-kpawoh, j.c., xi, y.f., zhang, y.z., jin, y.f., 2002: polyamine accumulation following hot-water dips influences chilling injury and decay in ‘friar’ plum fruit. j. food sci. 67, 2649-2653. anthony, b.r., phillips, d.j., badr, s., aharoni, y., 1989: decay control and quality maintenance after most air heat treatment 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peaches and plums. j. agri. food chem. 49, 4748-4760. vicente, a.r., martinez, g.a., civello, p.m., chavesa, a.r., 2002: quality of heat-treated strawberry fruit during refrigerated storage. postharvest biol. tech. 25, 59-71. wells, j., 1971: postharvest hot-water and fungicide treatments for reduction of decay of california peaches plums and nectarines. usda ars marketing research report no, 908. zhang, h., zheng, x., yu, t., 2007: biological control of postharvest diseases of peach with cryptococcus laurentii. food control 18, 287-291. zhou, t., xu, s., sun, d., wanga, z., 2002: effects of heat treatment on postharvest quality of peaches. postharvest biol. tech. 54, 17-22. address of the author: assist. prof. dr. elif ertürk çandir (corresponding author), fatma temizyürek, and assist. prof. dr. ahmet erhan özdemir, mustafa kemal university, faculty of agriculture, department of horticulture, 31034 antakya, hatay, turkey. << /ascii85encodepages false /allowtransparency false 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/pagesize [907.087 680.315] >> setpagedevice journal of applied botany and food quality 95, 100 104 (2022), doi:10.5073/jabfq.2022.095.013 1department of horticulture, faculty of agriculture, çukurova university, adana, turkey effects of temperature on pollen viability and in vivo pollen tube growth in citrus sinensis şenay karabıyık (submitted: november 11, 2021; accepted: june 27, 2022) summary temperature is the major factor in global warming and strongly influences the success of plant sexual reproduction. therefore, there is an urgent need to assess the vulnerability of species for breeding and planning effective actions. in this study, effects of different temperatures and storage duration on pollen viability and in vivo pollen tube growth were analysed in citrus sinensis ‘kozan yerlisi’. the pollens were obtained before anthesis and stored at 10 °c, 20 °c and 30 °c for 1, 2 or 3 days before analysis. the pollen viability decreased with increasing temperatures and storage duration from 61.3% to 5.8%. in vivo pollen germination results showed that for one day storage, the pollen growth rates were similar to the control treatment while storing pollens two or three days differed from the control. pollen germination after storage at 10 °c was lower and not efficient, while after storage at 30 °c no pollen tube germination at all was seen on the stigma. at the same time, storing pollen in low temperature caused delay in pollen tube germination and pollen tube growth, suggesting that pollen that are stored in low temperatures pass into a dormancy process for three to five days. in conclusion, this study indicated that pollen storage in 30 °c for a long time causes loss of pollination ability while storage in 10 °c causes dormancy in pollen. keywords: citrus sinensis, local variety, temperature, dormancy, pollen tube, progamic phase introduction the cultivated land in several regions of the world has been affected by environmental stresses, which causes a substantial amount of reductions in crop productivity (mahajan and tuteja, 2005). it is predicted that environmental stresses will become more intense and frequent with climate change, especially by global warming. the world population is estimated to be 10 billion in 2050, which will require 70% more food. crop tolerance to heat stress has to be considered for planning effective actions (aronne et al., 2021) and tolerant crops should be developed (thomas et al., 2020) for feed the increasing population. the reproductive phase of plants is much more sensitive than the vegetative phase to the effects of global warming (hedhly et al., 2008) and must be studied urgently especially for native species. for sexual reproductive success of plants pollen is crucial, which must survive and interact with the environment throughout the period from pollen release until ovule fertilization (mulcahy, 1979; aronne et al., 2021). within this scope, new knowledge is needed about the effects of low and high temperature in the progamic phase to optimise breeding programmes in order to obtain new varieties to adapt climate change scenarios. researchers should determine the tolerances of plants to extreme temperatures in terms of reproductive biology to provide generation continuity. the progamic phase, the time from pollination to fertilization, is one of the most critical phases during the sexual reproduction process in plants. in this period, specific interactions occur between the male gametophyte and pistil (montalt et al., 2019), which include stigmatic receptivity, pollen grain germination, pollen tube growth and ovule degeneration (hedhly, 2011). these phases depend not only on genotype but also on the environmental conditions during flowering and pollination (distefano et al., 2012). although temperature has a clear effect and high temperature accelerates pollen germination in vitro and pollen tube growth in the style, it also accelerates the loss of stigmatic receptivity (hedhly et al., 2005) and pollen germination capacity (distefano et al., 2012; akhoundnejad et al., 2020). although citrus is grown in various regions of the world currently, the global climate change is predicted to lead to an increase in ave rage temperatures in the future. this could limit plant cultivation in some areas and affect citrus growing areas (montalt et al., (2019). therefore, determining pollen performance and temperature sensitivity is especially relevant for citrus (distefano et al., 2018). in turkey, average temperatures in the flowering period of citrus are generally between 15-20 °c, with a gradual increase of up to 30 °c in daytime and decrease to less than 10 °c at night. the cultivation and breeding programmes in turkey are located in the mediterranean region in warm conditions; in the aegean region with moderate temperatures and at the eastern black sea and western marmara region characterized by colder temperatures, where both frost and high temperature risks exists to some extent (çimen and yeşiloğlu, 2016; yeşiloğlu et al., 2018). the results of this study may be useful for planning breeding programmes with regard to choosing the most favourable time and location to perform pollination depending on temperature forecasts. this research aimed to study the effects of pollen storage at dif ferent temperatures and duration periods on pollen viability and in vivo pollen tube growth in the progamic phase of citrus sinensis ‘kozan yerlisi’. materials and methods this study was conducted in the citrus research fields of cukurova university, adana/turkey in april 2019. in the present work the local orange variety citrus sinensis ‘kozan yerlisi’ was used. kozan yerlisi is a seeded, self-compatible and locally produced cultivar in turkey with a high manner. pollen storage in different temperatures and durations to obtain fresh pollen, lots of flowers were collected one day before anthesis. anthers were removed from the flowers and left for dehiscence at room temperature overnight. obtained fresh pollens were separated into 10 cups. pollen cups were placed in silica gel cups for 2 hours for removing humidity for 5 hours. one of the cups was left for fresh pollen studies and each 3 pollen cups of remaining 9 cups were placed to different temperature regime at 10 oc, 20 oc and 30 oc. each stored pollen cup was removed at 1st, 2nd and 3rd day of the storage. temperature affects pollen tube growth of citrus sinensis 101 pollen viability tests for each storage temperature (10 oc, 20 oc and 30 oc) and storage duration (1, 2 and 3 days), pollens were immediately tested with 1% 2,3,5 triphenyl tetrazolium chloride (ttc) solution according to norton (1966). the pollen viability of fresh pollens was determined using the same method. pollination studies sixty unopened flower buds were carefully emasculated with forceps to avoid any injury of the stigma just prior to their opening. all previously opened flowers and small immature buds were removed from selected branches. the emasculated flowers were covered with cotton bags to avoid free pollination. one day later, at anthesis, the emasculated flowers were pollinated with fresh pollen by hand using a small brush and covered with cotton bags immediately. then, 180 flowers (60 for each temperature regime) were emasculated and were hand-pollinated one day later with pollen, stored for 1 day at 10 °c, 20 °c and 30 °c. the experiment was repeated with pollen, which was stored for 2 and 3 days, respectively. the day of pollination was accepted as the beginning of anthesis and day count was started after this day. pollinated flower samples were collected in 2 days intervals from 1 dap (days after pollination) to 15 dap separately for each temperature and pollen storage period. five pistils were collected for each sampling date and immediately fixed in fpa 70 (stösser et al., 1985; karabiyik and eti, 2020). in vivo pollen tube growth rate pollen tube growth rates on pollinated pistils were evaluated from the samples collected and fixed from pollination studies. pollen tubes in the stigma and style, as well as ovules were monitored on squash preparations of pistils, previously softened in 8n sodium hydroxide for 5-7 h, stained with 0.1% aniline blue in 0.1 n k3po4 (kho and baer, 1970; preil, 1970) and observed under a fluorescence microscope (olympus bx51, tokyo, japan) equipped with a u-mwu filter (olympus, tokyo, japan). pollen tube growth was determined as percentage of the style traversed by the longest pollen tube in each pistil by a digital micrograph system (olympus dp72 camera, tokyo, japan). statistical analysis the pollen viability studies were conducted in completely randomised design with two factors and three replications. jmp 8.0.1 was used for anova test and the results were compared with lsd test. arc-sin transformation was used in percent values before analysis. results pollen viability level effect of storage temperatures and duration days on pollen viabili ty level is expressed as percentage in tab. 1. the statistical analysis revealed significant differences between different temperatures, storage duration and temperature-duration interaction. the viability rate of fresh pollens derived just after anthesis was 61.3% (not shown in table). in terms of average values of different temperatures, the highest viability was obtained from 10 oc with an average of 41.9% tab. 1: pollen viability levels of ‘kozan yerlisi’ orange stored in different temperatures and different durations (%) temperature duration temperatureaverage 1 2 3 10 °c 54.7 a1 38.6 b 32.4 c 41.9 a 20 °c 51.2 a 34.3 c 13.4 d 33.0 b 30 °c 40.9 b 27.6 c 5.8 e 24.8 c durationaverage 48.9 a 33.5 b 17.2 c lsddurat= 2.785*** lsdtemp= 2.785*** lsddurat × temp= 4.823*** 1differences between averages showed by different letters are statistically important ***p ≤ 0.001. fig. 1: pollen tube growth rate in control and different temperatures during 1-3 days. a. pollen tube growth rate of control treatment. b. pollen tube growth rate in 10 oc. c. pollen tube growth rate in 20 oc. d. pollen tube growth rate in 30 oc. (tt: ovule) 102 ş. karabıyık and is followed by 20 oc (33.0%) and 30 oc (24.8%). the pollen viability level decreased with longer storage periods. duration of storage also affected the pollen viability levels (tab. 1). in terms of duration × temperature values, the highest viability levels were obtained from 1 day-10 oc (54.7%) and 1 day-20 oc (51.2%) within the same statistical group. these values were followed by 1 day-30 oc (40.9%) and 2 days-10 oc (38.6%). the lowest results were obtained from third day as 5.8%, 13.4% and 32.4%, from 30 oc, 20 oc and 10 oc, respectively. in vivo pollen germination the effect of temperature on pollen tube growth in citrus sinensis ‘kozan yerlisi’ was evaluated with the pistil squash method. the results of control and different storage temperature regimes are shown in fig. 1 as line graphs. fig. 1a shows that in control treatments, pollen tubes germinated between the parenchyma cells of the stigmoid tissue reaching the stylar canals in 1 dap (day after pollination). then, the pollen tubes expeditiously preceded inside the stylar canals and first pollen tubes were reached to the ovules at 7 dap. pollens which were stored at 10 °c for 1 day had slightly slower pollen tube growth than control treatment until 5 dap and hereupon they started to elongate normally penetrating to the ovule at 9 dap (fig. 1b). the 2 days and 3 days storage in 10 °c were slower than both control and 1 day10 °c conditions. at the same time, pollen tubes did not germinate efficiently on the stigma in both treatments and the 3 days storage in 10 °c treatment caused delay in pollen germination on stigma (fig. 1b). in 2 and 3 days storage, pollen tubes penetrated to the ovules at 11 and 13 dap, respectively. the pollen tubes reached to the ovule were not as plenty as control treatments in none of the pollens which were stored in 10 °c. pollen germination and tube elongation after storage at 20 °c for 1 day was similar to the control treatment. however, storing pollens 2 days and 3 days at 20 oc caused retardation of pollen tubes in penetration to the ovules (fig. 1c). moreover, after 3 days storage, the pollen tubes slowed down especially after 3 dap. this retardation and late reaching to the ovules may be due to low pollen viability capacity. after 30 °c storage, pollens which were stored for 1 day germinated on the stigma and reached the style until 3dap. in this treatment, pollen tubes inside the stylar canals were not as much as in 20 °c and 10 °c treatments. however, no pollen germination was detected after 2 and 3 days storage showing that the pollens lost their germination capacity in long storage at 30 °c (fig. 1d). discussion various abiotic stresses including global warming are negatively affecting the plant productivity worldwide. therefore, it is necessary to know the response of plants, especially the local cultivars, in order to cope with this upcoming problem. citrus is grown in many regions in the world (montalt et al., 2019). however, temperature limits the geographical distribution of plant species (hedhly et al., 2005) especially for subtropical fruit trees like citrus. it has been proven before that reproductive phases such as male and female formation (distefano et al., 2018), pollen properties (hedhly et al., 2004; hedhly et al., 2005; distefano et al., 2012; distefano et al., 2018), progamic phase (distefano et al., 2018; montalt et al., 2019) and embryo formation (sanzol and herrero, 2001) are the most temperate-vulnerable stages. this study aimed to analyse temperature effects on pollens, which were stored in different temperatures and different duration span, on pollen viability level and in vivo pollen tube growth rates in local cultivar citrus sinensis ‘kozan yerlisi’. the results demonstrate that extreme temperatures and prolonged duration of pollen storage in high temperatures can cause loss of pollen viability and hence fruit or seed set ratio. in a previous study, pollen viability level of different citrus species differed between 12% and 98.6% while viability of c. sinensis ‘hamlin’ pollens was 85.3% (yamamoto et al., 2006). in another study conducted in citrus reticulata ‘shogua’, researchers reported that pollen storage for 0, 3, 6, 9, 24 and 48 hours at 20 °c decreased the pollen viability level from 96.47% to 17.13% (chelong and sdbodee, 2012). distefano et al. (2018), who have studied the effects of temperature on in vitro pollen germination, detected the highest pollen germination level in 25 °c (96%), while it was lower in 15 °c (11%), 20 °c (40%) and 30 °c (25%). researchers also reported that the incubation temperature affected the germination capacity of the pollen. bennici et al. (2019) reported parallel results in clementine as 25 °c gave better germination results than 30 °c. for in vivo pollen germination and pollen tube growth rate studies, the pistil squash method was used herein, which enables analysing the daily progression of pollen tubes inside the stigma, style and ovary by fixing the samples 2 days intervals from pollination until 15 days after pollination. this way, the effect of temperature and duration could be investigated more effectively. in vivo pollen germination after 1 day storage in all temperatures had quite similar pollen tube growth rates and ovule penetration periods compared to the control treatment. this indicates that there was a decrease in pollen viability level and pollen could conserve germination ability for 1 day without any important effects on germination in field conditions. at the same time, in vivo pollen germination capacity, which is expressed as intensity of germinated pollen on the stigma and inside the style (distefano et al., 2012), was decreased by storing pollens during 2 or 3 days in these temperatures. this study is the first to show the differences in pollination capa city of pollens stored for 1, 2 or 3 days at different temperatures. in recent studies, only effects of temperature on in vivo pollen germination were studied in citrus and reported that high temperatures were accelerated while low temperatures were decelerated the pollen tube growth in stylar canals (distefano et al., 2012; montalt et al., 2019). hedhly et al. (2004) in sweet cherry and hedhly et al. (2005) obtained the same results in peaches. sanzol and herrero fig. 2: pollen tube growth in ‘kozan yerlisi’ orange cultivar. a. normally germinated and dormant pollens on stigma. b. pollen tube elongation inside the stylar canals. scale bar = 100 μm. c. pollen tube penetration to the ovule. ca b temperature affects pollen tube growth of citrus sinensis 103 (2001) also indicated that the temperature has an important effect on pollen tube elongation influencing effective pollination period of species. in this study, storing pollens in 10 °c caused a delay in pollen germination suggesting that pollen storage in low temperature during 2 or 3 days caused dormancy in pollen germination and tube elongation for citrus sinensis “kozan yerlisi”. this was reported by hedhly et al. (2005) as peaches grown in 10 °c have been showed a delay in pollen germination compared with 20 °c and 30 °c. these results also suggested that, in order to take more actual results for pollen storage studies, the researchers should store their pollens in normal conditions for a while before testing or using it for pollination, instead of using pollens immediately after storage. on the other hand, aloisi et al (2020) reported the pollens stored in 15 °c broke down self-incompatibility by inhibiting t-gase activity resulting with pollen tube growth. by this opinion, pollination in cold stress in self-incompatible cultivars was studied by montalt et al. (2022) for breaking down self-incompatibility and the researchers were obtained a few viable seeds. the recent studies and this study showed that this procedure might be useful in breeding studies for alleviating incompatibility effects. although other researchers used in vitro germination tests to show viability of pollens, in this study pollen viability test was used and pollen germination was examined as in vivo. when both viability and in vivo germination studies were taken into account, it can be seen that the results give parallel results which shows this test was also usable. in general, pollen viability, in vitro pollen germination and in vivo pollen tube growth tests show parallel results (karabiyik and eti, 2016; karabiyik et al., 2016; karabiyik and eti, 2020). moreover, since in vitro pollen germination can be influenced by the media and environmental conditions, pollen viability and in vivo pollen tube growth is not. this situation is originated from pollen viability tests like ttc reflects the activity of dehydrogenase enzymes involved in the respiratory activity of living tissues which is also associated with its germination capacity (derin and eti, 2001). at the same time, by ttc test if there will be any dormancy for pollen tube formation like in this study, pollens would show their actual viability on the stigma without detecting the accurate germination time and germination medium for in vitro studies. similarly, the in vivo pollen germination and in vivo pollen tube growth studies give optimal results for pollen germination because it shows the real germination performance of pollen on the plant. conclusion in conclusion, this study showed that the temperature influences the pollen viability and pollen tube growth rate in citrus sinensis ‘kozan yerlisi’. pollens storing at 30 °c for a long time lost their pollination ability while storage at 10 °c caused a slower pollen tube growth but also slower pollen aging. as a result, pollen stored in low tempe rature conditions showed dormancy which could be broken in field conditions in 3 days. moreover, pollen incubation in 30 °c caused a strict decrease in pollen viability and no pollen tube growth in stigma and style. in future researches, it would be relevant to investigate the effect of temperature to male and female organ development and in vivo pollen germination rate and intensity by simulating low and high temperatures in laboratory conditions. conflict of interest no potential conflict of interest was reported by the author. references aloisi, i., distefano, g., antognoni, f., potente, g., parrotta, l., faleri, c., del duca, s., 2020: temperature-dependent compatible and incompatible pollen-style interactions in citrus clementina hort. ex tan. show different transglutaminase features and polyamine pattern. front. plant sci. 1018. doi: 10.3389/fpls.2020.01018 akhoundnejad, y., daşgan, y. h., karabiyik, ş., 2020: pollen quality, pollen production and yield of some tomato 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eti, s., 1985: bahçe bitkilerinde döllenme biyolojisi uygulamalı kurs notları. 18-22 mart 1985. adana (yayınlanmamış). thomas, s., ramakrishnan, r.s., kumar, a., sharma, r., tiwari, m., pathak, n., 2020: putrescine as a poliamines and its role in abiotic stress tolerance: a review. j. pharmacogn. phytochem. 9(1), 815-820. yamamoto, m., kubo, t., tominaga, s., 2006: self and cross incompati bility of various citrus accessions. j. japan. soc. hortic. sci. 75(5), 372378. doi: 10.2503/jjshs.75.372 © the author(s) 2022. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). yeşiloğlu, t., yilmaz, b., i̇ncesu, m., çimen, b., 2018: the turkish citrus industry. xxx. international horticultural congress. 12-16 august 2018, i̇stanbul/turkey. orcid s. karabıyık https://orcid.org/0000-0001-8579-6228 address of the author: şenay karabıyık, department of horticulture, faculty of agriculture, çukurova university, 01330, adana/turkey e-mail: senaybehlul@gmail.com http://dx.doi.org/10.1126/science.206.4414.20 http://dx.doi.org/10.1016/s0304-4238(00)00252-1 http://dx.doi.org/10.2503/jjshs.75.372 https://orcid.org/0000-0001-8579-6228 art01_kutschera_gs.indd journal of applied botany and food quality 85, 1 5 (2012) 1institut für biologie, abteilung pfl anzenphysiologie und evolutionsbiologie, universität kassel, kassel, germany 2arbeitsgruppe biologiedidaktik, biologisch-pharmazeutische fakultät, universität jena, jena, germany physiological phytopathology: origin and evolution of a scientifi c discipline u. kutschera1*, u. hossfeld2 (received november 28, 2011) summary in 1860, the german biologist anton de bary (1831-1888) elucidated the life cycle of the pathogenic oomycete phytophthora infestans, which causes late blight in potatoes and was responsible for severe famines during the 1840s. in a book on this topic published 150 years ago, de bary (1861) established the scientifi c discipline of physiological plant pathology. here we summarize the life and scientifi c achievements of anton de bary, who coined the terms “symbiosis” and “parasitism”, with reference to charles darwin’s (1809-1882) principle of descent with modifi cation by means of natural selection. then, we outline de bary’s discovery of the cause of the wheat stem rust disease, which is attributable to infections with the fungus puccinia graminis. since ongoing pathogen-host plant co-evolution is well documented in nature, we conclude that “nothing in phytopathology makes sense except in the light of darwinian evolution”. finally, we describe the value of basic research in the plant sciences with reference to practical applications, such as the maintenance and enhancement of crop yields and food quality. introduction one century ago, the american phytopathological society published the fi rst issue of a journal that persists to the present day. for the fi rst page of this new periodical, the distinguished botanist and microbiologist erwin f. smith (1854-1927) was asked to write a tribute. to the surprise of some of his north american colleagues, in phytopathology volume 1 (no. 1), a german biologist was honoured in the following words: “of all the personalities contributing to the advancement of plant pathology from its crude beginnings to the present time, none has been more interesting than that of de bary, none more productive of important results. de bary cleared the way for all that has followed in plant pathology and we must ever think of him with that reverence due a great master” (smith, 1911). in a subsequent biography of erwin f. smith, anton de bary was characterized as an excellent biologist “gifted with brilliance and the instincts of a cautious experimental scientist, one who refused to admit or advance any truth as fact until proved by strict technical procedures” (rodgers, 1952). anton de bary (1831-1888) was one of several outstanding 19th century scientists who remains “in the shadow” of the famous british naturalist charles darwin (1809-1882). this is to a large extent due to the fact that he studied “lower organisms”, such as plasmodial slime molds, fungi, and plants (hoppe and kutschera, 2010). in only one of the many books authored by de bary references are made to “higher animals” (vertebrates) or humans (de bary, 1885). moreover, it is rarely appreciated that it was anton de bary (1878), who defi ned the terms “symbiosis” and “parasitism” with reference to plants and darwin’s principle of natural selection (kutschera and niklas, 2004; 2005). one hundred and fi fty years ago, a little-known monograph on the potato late blight was published that inaugurated the scientifi c discipline of experimental plant pathology (de bary, 1861). in this article, the achievements of anton de bary are outlined. then, the origin and evolution of experimental plant pathology is discussed with respect to pathogenic fungi and bacteria. finally, we describe theoretical and applied aspects of these international research agendas with reference to darwin’s origin of species (1859, 1872). the plant and fungi collector evolves into a laboratory scientist anton de bary (fig. 1) was born on the 26th of january 1831 in frankfurt-main, germany. from 1849 to 1853 he studied medicine at the universities of heidelberg, marburg and berlin, where he earned his academic degree with an unpublished dissertation on a botanical subject. the title of this work of 1853, de plantarum generatione sexuali, indicates the primary interest of the young physician: plants and related sessile organisms. at the same time, the 22-yearold junior scientist, who was already an experienced plant and fungi collector, published his fi rst monograph. in these researches on the * corresponding author fig. 1: photograph of the founding father of experimental plant pathology and his research object, potato plants (solanum tuberosum), infected by the oomycete phytophthora infestans. the name “infectious plant destroyer” for this pathogenic fungus was coined by anton de bary in 1876. 2 u. kutschera, u. hossfeld physiological phytopathology smut fungi and the diseases that they cause in plants, with regards to cereals and other crop species, de bary (1853) summarized the current knowledge on this topic and postulated that only by means of proper experiments the true causes of these devastating plant diseases may be elucidated some time in the future. in 1854, de bary became a lecturer (privatdozent) for botany at the university of tuebingen. only one year later (1855), the specialist for fungi and plants received an appointment as professor of botany at the university of freiburg im breisgau. at the botanical institute in freiburg i. br., which later became one of the leading centres for plant research in germany (briggs, 2010), the biologist published his seminal monograph on the potato blight (de bary, 1861). after twelve years of hard work, the famous scientist left freiburg to accept a more attractive position at the university of halle, where he stayed from 1867 to 1872. he spent his fi nal and most productive years at the newly constituted university of strassburg, where de bary served as the fi rst rector. the biologist died in strassburg on the 19th of january 1888 as a result of a tumour infection (sparrow, 1978; horsfall and wilhelm, 1982). anton de bary started his career as a fi eld naturalist, who collected plants and fungi in the nearby country side. although he was formally trained as a surgeon, his interest in medicine was overshadowed by his drive to study plants, fungi and other “lower organisms” such as myxomycetes (de bary, 1859, 1864, 1866, 1884). he established sophisticated laboratory techniques to analyze the life histories of plant parasites, myxomycetes and other “primitive” living beings. hence, anton de bary was a botanist, plant physiologist, myxomycetologist and phytopathologist (hoppe and kutschera, 2010). moreover, with the publication of his lectures on bacteria (de bary, 1885), the laboratory scientist became one of the founding fathers of modern bacteriology. potato blight and the origin of physiological phytopathology in a recent article entitled “genome evolution in plant pathogens”, fungus-like parasites (oomycetes), such as the potato blight pathogen phytophthora infestans, and their infection strategies are described (dodds, 2010). however, the author of this popular summary of potato blight research did not mention that, 150 years before his paper was published, the discipline of physiological plant pathology was established. at that time, the life cycle of p. infestans was elucidated. this discovery, which was summarized in a seminal book, was published in november 1861 under the title the currently spreading potato disease, its cause and its prevention. a study based on the principles of plant physiology (fig. 2). herein the author inaugurated the discipline of experimental phytopathology, i.e., the scientifi c study of plant diseases (de bary, 1861). in february 1861, the german botanist anton de bary documented in detail how the vegetative body (mycelium) of p. infestans spreads through the leaf tissue of infected potato (solanum tuberosum) plants (fig. 1). in a series of elegant experiments, de bary recorded the symptomatic progression of the plant disease ‘late blight’ against the developmental stages of p. infestans. based on these studies, which demonstrated a positive correlation between the life cycle of p. infestans and stages of plant disease progression, de bary concluded that the oomycete is the causal agent of the disease of the potato plant. in this monograph (fig. 2), which was primarily based on his own research, anton de bary provided experimental evidence that potato tubers are infected by the fungus via the brown, blighted leaves and described the spread of the disease in the fi eld. specifi cally, de bary (1861) provided detailed drawings of cross sections through the leaves of infected potato plants, showing the vegetative structure (mycelium) spreading via the intercellular spaces of its host organism (fig. 3 a). he also monitored the development of the conidiophores as they emerge from the pores of the stomata (fig. 3 b) and the germination of isolated spores of p. infestans (fig. 3 c). based on fig. 3: the discovery of the life cycle of the late blight oomycete phytophthora infestans by anton de bary. the mycelium of p. infestans in a leaf of a potato (solanum tuberosum) plant (a), spore germination in liquid culture (c), and a conidiophore (with sporecontaining conidia, arrows) that emerges from a stomatum on the underside of a leaf (b). cp = conidiophore, ep = epidermis, sp = spore, st = stomatum (adapted from de bary, 1861). fig. 2: title page of anton de bary’s monograph on the potato late blight. this book also contains recommendations how to prevent the spread of this devastating plant disease. physiological phytopathology 3 these and other observations carried out according to the principles of experimental plant physiology, a scientifi c discipline established in germany during the 1850s by julius sachs (1832-1897) (kutschera and briggs, 2009, 2012), the author concluded that “the disease of the leaves, stems and fruits is caused by a pathogenic fungus, p. infestans, and the disease of the tubers occurs via infection from the leaves” (de bary, 1861). moreover, the scientist, who was working at that time at the university of freiburg i. br., concluded that “it will never be possible to drive the parasite p. infestans to extinction … however, a careful selection of uninfected tubers for agriculture will be suffi cient to prevent large-scale spreads of this devastating plant disease” (de bary, 1861). at the end of the text, he informed agriculturalists how to prevent the re-occurrence of another, devastating potato blight epidemic, such as that in ireland from 1845 to 1848, which caused crop losses and famine (andrivon, 1996; reader, 2009; matta, 2010; skelsey et al., 2010). spontaneous generation of lower organisms? it should be noted that de bary used his empirical proof for the “fungal theory”, which states that p. infestans is the causal organism of potato blight, to refute the doctrine of spontaneous generation. in 1861, many scientists still believed in the emergence of “lower” or “primitive” organisms, such as p. infestans, from dead material under present-day environmental conditions. the doyen of phytopathology provided experimental data documenting that the oomycete developed only from its own spores and never appears de novo. with the publication of these evidence-based conclusions, which supported the corresponding “microbe-experiments” of louis pasteur (18221895), the belief in “generation without parents”, which had never been supported by unequivocal evidence, disappeared forever from the scientifi c literature (matta, 2010). hence, the origin of plant pathology 150 years ago led to the demise of a dogma that had been the topic of endless debates among naturalists and philosophers. in his book on the origin of species, darwin (1859, 1872) ignored the “spontaneous generation − debate”, because, in his view, the evidence for this concept had always been weak and controversial. wheat stem rust: infection experiments and their consequences in 1865, the 33-year old german botanist julius sachs published his famous book entitled experimental physiology of plants. in this seminal work (sachs, 1865), the junior professor summarized the state of the art of a young scientifi c discipline that later evolved into plant physiology, i.e., the systematic study of the processes which go on in living, green, chloroplast-bearing sessile organisms. it is well known that anton de bary was heavily infl uenced by the work of sachs, who established, notably in his masterpiece lectures on the physiology of plants (sachs, 1882), high standards concerning how to perform reproducible experiments under controlled laboratory conditions (kutschera and briggs, 2009, 2012; kutschera and niklas, 2009). in the “shadow of sachs (1865)”, de bary published another major work wherein he used his experimental protocols from his potato blight studies in an even more sophisticated way. with reference to his earlier, descriptive work on the “brand fungi”, i.e., pathogenic organisms associated with the rust disease in wheat (de bary, 1853), he turned his attention to the life cycles of these pathogens. in a major research paper that appeared in a little-known journal, de bary (1865) elucidated the complex development and the different spore forms on two unrelated host plants (a phenomenon known as heteroecism), in several species of rust fungi. with a focus on wheat (triticum aestivum), he described the formation of fig. 4: healthy wheat (triticum aestivum) plants in the fi eld (a) and a dying wheat leaf (b) that is infected by the pathogenic fungus puccinia graminis, which causes this plant disease (stemor cereal rust). diagram of the life cycle of p. graminis via uredinia-telia (c). these propagules spread asexually on the infected triticum plants (circle), release basidiospores that infect barberry (berberis vulgaris) plants, where they reproduce sexually via pycnia-aecia, and fi nally re-infect wheat plants via aeciospores. anton de bary, who elucidated this grass (triticum) host 1alternate (berberis) host 2cycle, recommended removing barberry plants in the vicinity of wheat fi elds to prevent the spread of the cereal rust. 4 u. kutschera, u. hossfeld physiological phytopathology the brownish uredia and uredospores of the stem rust, which is caused by the fungus puccinia graminis (fig. 4 a c). based on de bary’s seminal work of 1865 and subsequent studies, the complex life cycle, with alternation of generations on different host plants, has been elucidated in detail. due to the elegant studies of de bary (1865) we know that the common barberry (berberis vulgaris), as well as a grass species, is required for the stem-, blackor cereal rust (puccinia graminis) to complete its life cycle (drews, 2001). during the spring and early summer, stem rust infections on wheat and other cereal species (fig. 4 a, b) produce dikaryotic urediniospores. these propagules, which are produced within the uredinia, are distributed by the wind to nearby conspecifi cs. here they germinate on the stems or leaves and then infect their new host plant through the stomata. this asexual summer circle, which spreads the infection over wide areas, is indicated in fig. 4 c as a circle. at the end of the growing season, the cereal rust produces dikaryotic teliospores, which, during the next spring, develop into basidiospores. these propagules can not infect cereal plants. however, they are carried by the wind to a second host plant, barberry (berberis vulgaris) and related species. there, the basidiospores infect young leaves via the penetration of the epidermal cells. the resulting infection structures (pycnia or spermagonia), which represent the sexual stage of the life cycle, form, after fertilisation, so-called aecia. these structures produce aeciospores, which are carried by the wind to cereal plants. after infection, the aeciospores develop into uredinia, and thus the next life cycle of the pathogenic fungus p. graminis begins (fig. 4 c). the elucidation of this “sophisticated” (i.e., evolved) life cycle of a plant pathogen, that still causes severe problems in africa today (singh et al., 2008), originated with the work of de bary (1865). these insights were based on careful infection experiments and the use of different host plants and spores (drews, 2001). one practical consequence rapidly emerged from this elegant work: the systematic removal of barberry plants close to crop fi elds. it should be noted that anton de bary’s research was based on the principles of experimental plant physiology (see the sub-titles of the monographs de bary, 1861 and 1863), a scientifi c discipline that was still in its infancy when the botanist-mycologist carried out his seminal work. conclusions: a de baryian view of phytophathology the german biologist anton de bary (fig. 1) was one of the fi rst to employ the emerging principles of experimental botany to study the causes of diseases in major crop species, such as potato and wheat. based on detailed analyses, he elucidated the life cycles of pathogenic fungi, concluded that these eukaryotic microbes are the causative agents of disease development, and hence became the founding father of experimental plant pathology. this scientifi c discipline originated 150 years ago with the publication of a monograph on potato blight (de bary, 1861) (fig. 2). in his famous publication on symbioses and pathogens in plants, de bary (1878) pointed out that only a darwinian perspective yields meaningful conclusions – in other words, “nothing in plant pathology makes sense except in the light of darwinian (adaptive) evolution”. anton de bary’s studies on potato blight and wheat stem rust highlight the practical value of basic research with respect to the maintenance of stable crop yields and food quality (orbach, 2011; kutschera and wang, 2012). his insights concerning the mode of infection and the spread of plant pathogens in the fi eld led to measures to prevent these epidemics from occuring in the future. moreover, de bary (1861, 1865) explicitly pointed out that it will never be possible to completely eliminate pathogenic micro-organisms such as phytophtora or puccinia, because these living beings will always fi nd ways to adapt to new environmental conditions via rapid microevolutionary processes. this classical “darwinian view” of phytopathology was correct. today we know that dynamic plant-microbe co-evolutionary events occur (kutschera and niklas, 2004; kutschera, 2009). hence, new pathogens, such as the recently described modifi ed stem rust strain puccinia graminis race ug99, which has a devastating impact on wheat production in africa (singh et al., 2008; dodds, 2010; mcclung, 2011), can not simply be eliminated. these pests will continue to cause severe crop losses in those regions of the earth where food production is steadily threatened by plant diseases, insect calamities, droughts, and civil wars. acknowledgements we thank two anonymous reviewers for helpful comments on the manuscript. this work was supported by the alexander von humboldtstiftung, bonn, germany (avh-fellowship stanford 2010/2011 to u. k.). references andrivon, d., 1996: the origin of phytophthora infestans populations present in europe in the 1840s: a critical review of historical and scientifi c evidence. plant pathol. 45, 1027-1035. briggs, w.r., 2010: a wandering pathway in plant biology: from wildflowers to phototropins to bacterial virulence. annu. rev. plant biol. 61, 1-20. darwin, c., 1859: on the 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vorlesungen über pflanzen-physiologie. wilhelm engelmann, leipzig. singh, r.p., hodson, d.p., huerta-espino, j., jin, y., njau, p., wanyera, r., herrera-foessel, s.a., ward, r.w., 2008: will stem rust destroy the world’s wheat crop? adv. agron. 98, 271-309. skelsey, p., rossing, w.a.h., kessel, g.j.t., van der werf, w., 2010: invasion of phytophthora infestans at the landscape level: how do spatial scale and weather modulate the consequences of spatial heterogeneity in host resistance? phytopathology 100, 1146-1161. smith, e.f., 1911: anton de bary. phytopathology 1, 1-2. sparrow, f.k., 1978: professor anton de bary. mycologia 70, 222-252. address of the corresponding author: u. kutschera, institute of biology, university of kassel, heinrich-plett-str. 40, d-34132 kassel, germany / department of plant biology, carnegie institution for science, stanford, california 94305, usa. e-mail: kut@uni-kassel.de journal of applied botany and food quality 92, 100 105 (2019), doi:10.5073/jabfq.2019.092.014 department of medicinal and aromatic plants, szent istván university, budapest, hungary variability of thujone content in essential oil due to plant development and organs from artemisia absinthium l. and salvia officinalis l. huong thi nguyen*, péter radácsi, péter rajhárt, éva zámboriné németh (submitted: march 4, 2019; accepted: march 22, 2019) * corresponding author summary the study compared changes in essential oil content and its thujone ratio in two popular herbs (artemisia absinthium l. and salvia offi cinalis l.), pertaining to plant development and plant organs. both species were harvested in 2018 at the vegetative, floral budding, flowering and after flowering phases; flowers and leaves were sampled separately. the essential content is always higher in the flowers than in the leaves at the same phenophase in both species we exa mined. decreased essential oil content in both organs during the developmental phases was also common to both species. in s. offi cinalis, both leaf and flower oils showed quantitatively different compositional profiles. during plant development, the main component α-thujone decreased significantly in leaf oils, while both thujone isomers demonstrated statistically stable values in flower oils. in a. absinthium, leaf and flower oils exhibited similar thujone ratios. during plant development, neither of the thujone isomers changed significantly in leaf oils but the ratio of α-thujone increased in the flowers. it was established, that only the distribution and dynamics of total volatiles showed common features in the two species, while the variability of thujone ratios represents differences specific to each target species. for the praxis, in s. officinalis timing of harvest seems to be more important while in a. absinthium the ratio of organs may play a more significant role in reaching lower thujone levels of the drug. keywords: absinthe, medicinal plants, ontogenesis, flowering, chemotype, chemical variability introduction the monoterpene ketones, αand β-thujones, are natural substances found in plants, commonly used for flavoring of foods and beverages (lachenmeier and uebelacker, 2010). the most well-known product containing thujone is certainly absinthe, produced from wormwood (artemisia absinthium l.). for bitter spirit drinks, such as absinthe, a maximum limit of 35 mg/kg thujone was indicated by the eu council directive 88/388/ec. based on a typical recipe, 2.5 kg of a. absinthium yields 100 litres of absinthe (max, 1990). this is equivalent to 4.4 mg a. absinthium oil or 2 mg to 4 mg thujone per drink, which would be far below the level at which acute toxicity effects were observed. subsequently lachenmeier and uebelacker (2010) provided evidence that the current eu limits ensure sufficient protection for consumers. according to the recent regulation of the european parliament and council (2008), thujone in chemically pure form is not allowed to be added to foods, but it may be introduced indirectly into foods by using plants containing thujone. the ema (european medicines agency) suggests in its wormwood monograph a daily limit of 3.0 mg thujone/day/person for thujone in absinthii herba for a maximum duration of use of 2 weeks (ema/ hmpc, 2008a). in the case of salvia officinalis, plant material with low thujone content should be preferred and a daily limit set at 5.0 mg thujone/day/person for a maximum duration of use of 2 weeks (ema/hmpc, 2008b). although both αand β-thujones are also found naturally in considerable concentrations in other essential oils (eo) such as tansy (tanacetum vulgare), white-cedar (thuja occidentalis), and other artemisia and achillea species (pelkonen et al., 2013), the above mentioned a. absinthium and s. officinalis species are the most characteristic ones as well as being found in numerous preparations frequently used by consumers. the mean concentration of αand β-thujones in a. absinthium oils are 5.8% and 12.5% respectively, as calculated from a review of 24 references (lachenmeier and nathan-maister, 2007). in the case of s. officinalis, the ratio of α-thujone varied from 1.2 to 45.8% and that of β-thujone between 1.0% and 40.1%) as summarized in kintzios (2003). just as chemosyndromes of the plants may vary according to plant parts and ontogenetic phases (németh-zámbori, 2015), the thujone content may vary on a large scale, too. in a. absinthium, ariño et al. (1999) reported qualitatively similar but quantitatively diffe rent eo profiles from leaves and flowers. judzentiene and budiene (2010) determined higher ratios of thujone in flowers (5.3-10.4% of the oil) than in leaves (0.0-8.9% of the oil). on the other hand, in s. officinalis samples collected from a wild region in china, the percentage of β-thujone was higher in leaves (14.86%) than in the flower oil (6.05%) while the ratio of α-thujone was three times higher in the flowers than in the leaf oil (li et al., 2015). in the case of tansy (tanacetum vulgaris), most leaf oils of twenty samples showed lower concentrations of thujone than those detectable in the inflorescence oils (judzentiene and mockute, 2005). during the plant development of a. absinthium, the ratio of the main compound β-thujone reached its highest ratio (51.99%) in the flower oil at floral budding; however, in the leaf oil the highest ratio (63.41%) was measured at the vegetative stage (nguyen et al., 2018). ben farhat et al. (2016) reported that in s. officinalis, the proportion of thujone varied depending on the ontogenetic phase and showed a peak at the fruiting stage. in tansy (tanacetum vulgaris), the maximum proportion of α-thujone (44%) was detected in two stages: in the leaf rosette phase and at the beginning of fruit setting (goudarzi et al., 2015). while thujone is receiving more and more attention from researchers and producers, reliable data on its variability are both scarce and contradictory. therefore, the aim of our study was to detect the influencing factors on the variation of thujone-containing eo depending on plant organ and harvesting time (plant development). we asked whether the rows of chemosyndromes could be generalized or whether they are specific for the species, and our study is a comparision of two important thujone-containing species (a. absinthium and s. officinalis). materials and methods plant material and growth conditions the plant material used in this study consisted of wormwood (artemisia absinthium l.) and sage (salvia officinalis l.). the ac characterisation of thujone content in wormwood and sage 101 cession of a. absinthium originated from gatersleben genebank (germany) with the denomination “belgien”, where thujone chemotype individuals were identified during our previous work (nguyen et al., 2017). for s. officinalis, we investigated an accession of un known origin (sage016) maintained in the gene bank of the depart ment of medicinal and aromatic plants, szent istván university. the plants were grown in open field plots at the experimental station of szent istván university, in budapest. the soil is sandy-loam, ph 7.8, humus content 1.2%. the plants were cultivated without addi tional fertilization; mechanical weed control and, in dry periods, irrigation was performed. the experiment was carried out in 2018 with four harvesting times from a 3-year-old cultivated population, based on the phenological stages of each species (tab. 1). chemical analyses essential oil extraction in both species, samples were collected in four phenological stages from perennial, cultivated plantations. each time, 10 randomly cho sen individual plants were harvested and the plant material was dried at room temperature (20-25 °c) in shade for two weeks. after drying, the harvested individual samples were mixed creating one bulk sample representative for the population, for each harvest time. then, leaves and flowers were separated from stems and each of them were used in three replicates for eo distillation and gc-ms analysis. 50 g dried materials from each sample was hydro-distilled in a clevenger-type apparatus using 500 ml of water for 2.5 hours according to the method recommended by the vii. hungarian pharmacopoeia. the oils were collected, and traces of water removed with anhydrous sodium sulphate. then, the extracts were separated with a syringe filter and stored in an airtight vial in a refrigerator at 4 °c prior to analysis. gas chromatography-mass spectrometry analysis the gc-ms analyses were carried out using an agilent technologies 6890n instrument equipped with hp-5ms capillary column (5% phenyl, 95% dimethyl polysiloxane, length: 30 m, film thickness: 0.25 nm, i.d. 0.25 mm) and an agilent technologies ms 5975 inert mass selective detector. the temperature program was the following: initial temperature 60 °c, then by a rate of 3 °c/min up to 240 °c; the final temperature was kept for 5 min. carrier gas was helium (1 ml min−1), injector and detector temperatures were 250 °c. split ratio: 30:1. 10 μl of eo has been diluted by n-hexane to 1 ml and from this the injected quantity was 0.2 μl. ionization energy was 70 ev. the ms was recorded in full scan mode that revealed the total ion current (tic) chromatograms (mass range m/z 50-550 μma). components were identified by comparison of their linear retention indices, which were calculated using the generalized equation of van den dool and dec. kratz (1963) with literature data (tab. 3; 4); and by matching their recorded mass spectra with those in mass spectra library references (nist ms search 2.0 library, wiley 275). statistical analysis spss version 23 was used to analyse the data. the two-way anova test was conducted on both species to compare the eo content of leaves and flowers harvested at different phenological stages (vegetative, floral budding, flowering and after flowering). for evaluating changes of the concentrations of thujone isomers among the phenophases, one-way anova was used for both the leaf and flower samples. homogeneity of variances was checked with levene’s test and the normality of variances was checked using the kolmogorovsmirnov method. results and discussion variability of essential oil content according to the two-way anova test of between-subjects effects, significant differences between both species could be detected in both flower and leaf samples harvested at different phenological stages. significant differences were detected also between leaf and flower samples collected at the same phenological phases, with a single exception (in s. officinalis, after flowering stage). the results are summarized in tab. 2. the highest eo content of s. officinalis was obtained from flowers in floral budding phenophase (2.82 ml/100 g) and the lowest one was also detected in flowers at after flowering stage (0.73 ml/100 g). flowers always contained higher levels of volatiles compared with the leaves at the same developmental stage. in both organs, the actab. 1: harvesting times and developmental phases of the experimental species species harvesting time bbch code description denomination (hess et al., 1997) of phase a. absinthium 08.06.2018 s. officinalis 25.04.2018 a. absinthium 12.07.2018 s. officinalis 09.05.2018 a. absinthium 08.08.2018 s. officinalis 12.06.2018 a. absinthium 10.09.2018 s. officinalis 12.07.2018 40 vegetative 55 first individual flowers visible (still closed) floral budding 65 full flowering: 50% of flowers open, first petals may have fallen flowering 67 flowering finishing: majority of petals fallen or dry after flowering harvestable vegetative plant parts or vegetative propagated organs begin to develop tab. 2: changes in essential oil content of the leaves and flowers of experimental species during different growth stages species phenological essential oil content (ml/100 g) stage leaves flowers vegetative 2.08 c floral budding 1.30 ab 2.82 bc flowering 1.15 abc 1.30 bb after flowering 0.91 aa 0.73 aa vegetative 1.72 c floral budding 0.90 ab 1.86 bb flowering 0.61 aa 1.69 bb after flowering 0.26 a lower case letters in columns of each species represent significant differences between phenological phases of the same organ and capital letters in rows represent significant differences between leaves and flowers at the same phenological phase, according to the games-howell test at p=0,05. s. officinalis a. absinthium 102 h.t. nguyen, p. radácsi, p. rajhárt, é. zámboriné németh cumulation level of volatiles decreased continually during the plant development. hossein mirjalili et al. (2006) also reported that eo content of iranian s. officinalis reached the highest level at floral budding (0.9%), then decreased until fruiting. in the case of a. absinthium, the results are similar. highest oil content was found in flowers at the floral budding stage (1.86 ml/100 g) and the lowest values were obtained from flowers after flowering (0.26 ml/100 g). the eo content of the flowers was significantly higher than that of the leaves and its tendency to decrease during the plant development was also detected. in the case of a. absinthium the present results corroborate previous data on the tendency to reduce the concentration of volatile compounds during the vegetation period (nguyen et al., 2018). in several other species a similar phenomenon has been established: the accumulation of volatile compounds reaches a peak at the beginning of flowering and decreases sharply after flowering and further on, during seed ripening (mohammadi et al., 2015; németh, 2005). this distribution and the dynamics of oil accumulation may be in connection with the ecological role of the volatile molecules both as defense molecules and/or attractants (guitton et al., 2010; németh, 2001). variability of thujones in essential oil salvia officinalis l. the qualitative and quantitative results on the composition of s. officinalis eo distilled from flowers and leaves are listed in tab. 3. in total, ten constituents which were higher than 1% of the gc area were identified, representing 91,5-94.1% of the total area. leaf oils and flower oils showed different profiles. α-thujone (16.724.7%) is the major component of leaves while it is found only in lower concentrations in the flower oils where viridiflorol was detected as the main component (20.9-28.6%). β-thujone was present both in leaves and flowers and varied from 2.9% (in flowers) to 8.6% (in leaves). both isomers of thujone always showed higher ratios in leaves than did in flowers at the same time. similar to this finding, perry et al. (1999) also found lower thujone levels in flowers (16%) compared with leaves (31%). further, a study by santos-gomes and fernandes-ferreira (2001) indicated that α-thujone content in the oil of stems, leaves and flowers of s. officinalis in portugal represented about 55, 30, and 18% of the total oil, respectively. during the sampled phenophases, in the leaf samples the highest level of α-thujone accumulation was registered at the first two developmental phases (23.15-24.70%), and it decreased significantly du ring the flowering period. in flowers, the same dynamics of α-thujone ratio were detected with a peak at the floral budding stage (14.2%), although the differences were not significant. the ratio of β-thujone in the leaves fluctuated, with the highest values after flowering and the lowest ones in floral budding. in flower oils the concentration of β-thujone was statistically stable. considering the sum of both isomers together, a significant decrease could be ascertained in the case of the leaf oils, with a maximum at floral budding (29.1%) and a minimum after flowering (25.3%), while in the flowers the variation was not significant. decreasing ratios of α-thujone in the eo of s. officinalis during plant development were reported by santos-gomes and fernandesferreira (2001). similarly, ben farhat et al. (2016) detected a decreasing tendency of β-thujone accumulation during consecutive phenophases. however, contrary to our findings as well as the above mentioned studies, the fruiting stage was determined to be peak accumulation stage for α-thujone in spain (ben farhat et al., 2016) and for both thujone isomers in iran (mirjalili et al., 2006). nevertheless, we have to add that neither of these studies dealt with samples from individual plant organs, but rather investigated the whole shoots. in parallel with the decrease of thujone content, the other skeleton type compounds − except bornane class − also tended to decrease during plant development, while the ratios of sesquiterpenes (mostly that of viridiflorol and biformene) were increasing. this indicates gene expression or enzyme activity changes at the starting phases of terpene biosynthesis (tab. 3). tab. 3: eo composition1 (gc area %) of s. officinalis obtained from leaves and flowers at different phenological stages leaf flower compounds rt lri2 lri3 v fb f af fb f af 40 55 65 67 55 65 67 β-pinene 6.45 981 979 4 2.1 1.8 0.7 5.7 9.9 5.1 4.7 1,8-cineol 8.07 1034 1035 4 8.1 7.9 6.3 7.7 11.3 10.4 3.9 α-thujone 10.68 1105 1105 23.2 b 24.7 b 20.5 ab 16.7 a 14.2 a 12.4 a 9.8 a β-thujone 11.11 1113 1112 5.7 ab 4.4 a 5.1 a 8.6 b 3.0 a 3.4 a 2.9 a camphor 12.19 1144 1146 11.8 8.8 8.6 5.8 3.9 2.5 3.1 borneol 13.09 1162 1169 4 1.8 4.0 3.0 5.9 4.7 7.1 6.0 β-caryophyllene 23.31 1420 1420 10.1 9.9 10.2 6.4 8.3 5.9 8.2 α-humulene 24.67 1454 1457 4 11.3 8.8 9.4 4.5 6.4 4.8 5.8 viridiflorol 30.39 1598 1495 4 14.3 18.3 19.6 21.6 20.9 24.6 28.6 biformene 45.59 2022 2026 5 4.9 3.5 8.4 8.8 10.7 18.1 20.0 total monoterpenes (%) 52.59 51.6 44.2 50.3 47.0 40.8 30.5 total sesquiterpenes (%) 40.61 40.6 47.6 41.2 46.4 53.4 61.0 total identified percentage 93.20 92.2 91.7 91.5 93.4 94.1 91.5 v: vegetative; fb: floral budding; f: flowering; af: after flowering letters in rows represent significant differences between phenological phases of the same organ 1 components reaching 1% of gc area are listed 2 linear retention indices calculated relative to the elution ranking of n-alkanes (c9-c20) on hp-5ms column 3 linear retention indices according to adams (2007) 4 linear retention indices from literature ben farhat et al. (2016) on hp-5ms column 5 linear retention indices from literature petrović et al. (2006) on hp-5ms column characterisation of thujone content in wormwood and sage 103 artemisia absinthium l. the total area of the identified components in a. absinthium eo varied between 86.7% and 97.6%. during evaluation of the components which are higher than 1% of gc area, 12 compounds were identified and listed in tab. 4. both leaves and flowers accumulated β-thujone as the main volatile component and α-thujone was the second largest compound. it was observed that the ‘α’ isomer always showed higher ratios in leaves than in flowers at the same sampling time. in case of the ‘β’ isomer higher ratios in the flowers were registered only until flowering. during plant development, in the leaf samples the ratio of both isomeric forms of thujone reached the highest percentages at the floral budding stage: 25.4% of α-thujone and 66.8% of β-thujone were measured. however, the fluctuation that we noted does not reflect significant differences. in the flowers, the maximum concentration of α-thujone appeared at the flowering stage (24.5%), where this peak value is significant, while β-thujone reached the highest ratios at the after flowering stage, with no statistical difference compared to the former stages (60.9%). the sum of both thujone isomers in leaves did not show significant differences among the developmental phases we studied. however, in the case of flowers a significant change was registered with increasing percentages in the later phenophases from 68.3% (at floral budding) to 81.3% (after flowering). carnat et al. (1992) reported that the content of both thujone iso mers decreased during the harvesting periods from july to novem ber; however, these data refer to the whole aerial parts of a. ab sinthium. no reference was found in this study, either for the plant organs separately or for exact information regarding the pheno phases. our previous work showed that the ratio of the main compound β-thujone reached the highest ratio in the flowers at the floral budding stage and decreased significantly after that; while in the leaves, the highest value of this compound was measured at the vegetative stage and fluctuated after that (nguyen et al., 2018). the changes of α-thujone are less characteristic and do not follow the pattern of the other thujone isomer. during the different developmental stages and for both organs, a. absinthium oils were dominated by the monoterpene fraction (73.794.4%) while sesquiterpenes were present in relatively lower percentages (3.2-15.6%) both in leaf and flower oils. the highest concentration of monoterpene compounds was detected in the leaf oils at the floral budding stage (94.4%) and in flower oils at the after flowering stage (77.2%), which were in harmony with the accumulation dynamics of β-thujone. in parallel with this, the ratio of sesquiterpenes increased following the after flowering phase and reached 15.6% as highest ratio (in leaves). as changing tendencies were different for each skeleton class of monoterpenes and sesquiterpenes, they might be resulted by changes at the level of terpene synthases. conclusion the accumulation of volatile compounds as eo showed similar organic distribution and accumulation dynamics in both species of our study. the eo content of the flowers was always superior com pared with the leaves at the same phenological phase both in a. ab sinthium and s. officinalis. the highest level of eo content was found in leaves at the vegetative stage (2.08 ml/100 g in s. officinalis samples and 1.72 ml/100 g in a. absinthium samples); and in flowers at the floral budding stage (2.82 ml/100 g in s. officinalis samples and 1.86 ml/100 g in a. absinthium samples). after the peaks, the concentrations decreased during the development of plants. between the two species, the distribution of the thujone isomers is a characteristic difference. our results confirmed former descriptions (nguyen et al., 2017): the accumulation level of β-thujone was higher than that of α-thujone in each sample of a. absinthium. on the other hand, s. officinalis oils contained higher α-thujone percentab. 4: eo composition1 (gc area %) of a. absinthium obtained from leaves and flowers at different phenological stages leaf flower compounds rt lri 2 lri3 v fb f fb f af 40 55 65 55 65 67 sabinene 6.36 976 969 1.5 0.3 0.2 1.7 1.4 1,8-cineol 8.07 1034 1030 4 0.9 0.5 0.2 1.2 0.7 0.2 α-thujone 10.68 1105 1105 4 23.0 a 25.4 a 23.0 a 15.9 a 24.5 b 20.3 b β-thujone 11.11 1113 1112 61.7 a 66.8 a 49.5 a 52.4 a 51.5 a 60.9 a lavandulol 13.18 1166 1165 1.1 1.4 0.8 2.6 0.8 0.3 β-caryophyllene 23.31 1420 1417 1.1 0.7 1.9 1.4 1.0 1.2 β-selinene 25.98 1486 1489 1.8 1.5 3.2 3.1 1.4 2.9 neryl-isobutanoate 26.26 1492 1490 1.4 1.4 1.3 neryl-isovalerate 29.54 1583 1582 1.6 3.3 geranyl 2-methyl butanoate selin-11-en-4-α-ol 32.56 1661 1658 0.9 2.8 1.1 chamazulene 35.10 1733 1730 2.5 0.1 0.1 7.0 0.9 0.9 total monoterpenes (%) 88.3 94.4 73.7 73.7 78.8 81.7 total sesquiterpenes (%) 5.6 3.2 15.6 13.0 14.3 13.4 total identified percentage 93.8 97.6 89.3 86.7 93.1 95.1 v: vegetative; fb: floral budding; f: flowering; af: after flowering letters in rows represent significant differences between phenological phases of the same organ 1 components reaching 1% of gc area are listed 2 linear retention indices calculated relative to the elution ranking of n-alkanes (c9-c20) on hp-5ms column 3 linear retention indices according to adams (2007) 4 linear retention indices from sharopov et al. (2012) on hp-5ms column 29.80 1591 30.25 3.4 4.7 104 h.t. nguyen, p. radácsi, p. rajhárt, é. zámboriné németh tages compared with the other isomer, which coincides with previous data (kintzios, 2003; ben farhat et al., 2016). at the same time, in leaves of both species and in the flowers of a. absinthium the above mentioned characteristic thujone isomer is the major component of the eo; however, in s. officinalis the flowers accumulated viridiflorol in the highest concentrations. the ratios of both thujone isomers in leaves show higher percentages than in flowers at the same harvesting time, which is a common feature for both a. absinthium and s. officinalis in the study. differences between the species were nevertheless registered according to the dynamics and variations of the percentages during the study phases. in s. officinalis, the leaf oil presented significant changes concerning both αand β-thujones as well as their sum, while these ratios were statistically stable in the flower oil. to the contrary, a. absinthium leaf oil proved to be more stable, while the ratio of both thujones and their sum increased in the flower oil during plant development. these findings may show that the accumulation tendency of total volatiles is a more common feature of these species and − according to the references − of many other eo bearing ones. however, the proportional changes of thujone isomers in the eo exhibit characteristic differences between s. officinalis and a. absinthium, which might represent the different ecological roles of these monoterpenes for the study species. for the praxis, in s. officinalis timing of harvest seems to be more important while in a. absinthium the ratio of organs may play a more significant role in reaching lower thujone levels of the drug. according to this, a later harvest of s. officinalis, preferably in flowering stage would be advantageous and a. absinthium herb should contain more leaves than flowers. acknowledgement this research 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10.1021/jf001102b sharopov, f.s., sulaimonova, v.a., setzer, w.n., 2012: composition of the essential oil of artemisia absinthium from tajikistan. rec. nat. prod. 6, 127-134. van den dool, h., dec. kratz, p., 1963: a generalization of the retention index system including linear temperature programmed gas − liquid partition chromatography. j. chromatography a 11, 463-471. doi: 10.1016/s0021-9673(01)80947-x orcid huong thi nguyen https://orcid.org/0000-0003-0400-7309 péter radácsi https://orcid.org/0000-0003-1562-4708 éva zámboriné németh https://orcid.org/0000-0003-4359-0728 address of corresponding author: huong thi nguyen, department of medicinal and aromatic plants, szent istván university, 1518 budapest, p.o. box 53, hungary e-mail: nguyenhuongnimm@gmail.com / nguyen.thi.huong@kertk.szie.hu © the author(s) 2019. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1016/j.yrtph.2012.11.002 http://dx.doi.org/10.1002/ffj.1570 http://dx.doi.org/10.1021/jf001102b http://dx.doi.org/10.1016/s0021-9673(01)80947-x https://orcid.org/0000-0003-0400-7309 https://orcid.org/0000-0003-1562-4708 https://orcid.org/0000-0003-4359-0728 mailto:nguyenhuongnimm@gmail.com mailto:nguyen.thi.huong@kertk.szie.hu journal of applied botany and food quality 92, 57 63 (2019), doi:10.5073/jabfq.2019.092.008 1college of food science and engineering, south china university of technology, guangzhou, china 2aijia biotechnology co. ltd, changsha, hunan, china in vitro assessment of anti-diabetic potential of four kinds of dark tea (camellia sinensis l.) protein hydrolysates keying su1, xinliang mao1, liping ai2, xuewu zhang1* (submitted: august 23, 2018; accepted: january 30, 2019) * corresponding author summary the contributions of four kinds of dark tea (camellia sinensis l.) proteins and their hydrolysates to hypoglycemic activity were investigated in vitro. four kinds of water-extracted dark tea proteins were hydrolyzed with trypsin and alcalase, respectively. the complete proteins had α-amylase inhibitory activity with half maximal inhi bitory concentration (ic50) values ranging from 1.27 to 2.78 mg/ml. most of the dark tea proteins and hydrolysates significantly inhibited α-glucosidase and dipeptidyl peptidase (dpp-iv), with ic50 values in the range of 0.0103 -1.3114 mg/ml and 0.1000 -1.3364 mg/ml, respectively. in general, heimaojian (hmj) and qianliang (ql) hydrolysates displayed high α-glucosidase inhibitory activity, while hmj, fuzhuan (fz), and heizhuan (hz) hydrolysates exhibited a strong ability to inhibit dpp-iv. this study demonstrates the potential of dark tea proteins and their hydrolysates as a source of functional food and medicine for the control of type 2 diabetes. key words: dark tea; hydrolysis; dpp-iv; α-glucosidase; α-amylase introduction type 2 diabetes is one of the leading public health problems in modern society. this chronic disease increasingly exists in the aging population, especially in developed countries. by 2030, it will affect 438 million people, with 70% of cases occurring in lowto middleincome families (yu et al., 2012). people who suffer from type 2 diabetes are strongly predisposed to atherosclerotic cardiovascular disease (cvd) (harnedy and fitzgerald, 2013), which is a major cause of morbidity and mortality. some synthetic medicines are used to control type 2 diabetes. one therapeutic approach is to suppress the absorption of glucose by inhibiting carbohydrate-hydrolyzing enzymes such as α-amylase, which acts on long-chain carbohydrates, and α-glucosidase, which catalyzes the cleavage of glucose from disaccharides (lebovitz et al., 1997). acarbose and voglibose are widely known as inhibitors of α-amylase and α-glucosidase. another mechanism is the inhibition of dipeptidyl peptidase-iv (dpp-iv) activity. dpp-iv rapidly meta bolizes glucagon-like peptide 1 (glp-1) and glucose-dependent insulinotropic polypeptide (gip), two insulinotropic incretin hormones that enhance glucose-dependent insulin secretion and regulate postprandial blood glucose levels (green et al., 2004). many dpp-iv inhibitors are used, including vildagliptin, saxagliptin, and sitagliptin. tea (camellia sinensis l.) is regarded as one of the most popular beverage plants consumed worldwide. it has been reported that 78% of the total amount of tea produced and consumed around the world is dark tea (full-fermented), 20% is green tea (non-fermented), and less than 2% is yellow or oolong tea (semi-fermented) (xiao et al., 2011; li et al., 2012). one of the most important dark tea-production areas in china is anhua county in hunan province, which is famous for fuzhuan brick tea, qianliang tea, and heimaojian tea. tea has many pharmacological and therapeutic properties, inclu ding anti-diabetic effects. stote and baer (2008) indicated that tea consumption may affect glucose metabolism and insulin signaling by enhancing insulin sensitivity and endothelial function. many studies have verified that tea extracts significantly inhibit the activity of both α-amylase and α-glucosidase (miao et al., 2015; peng et al., 2015; koh et al., 2010), enzymes that play key roles in carbohydrate digestion and have been recognized as therapeutic targets for modulating postprandial hyperglycemia. gao et al. (2012) demonstrated that tea polyphenols can exert anti-oxidative and hypolipidemic effects in rats with streptozotocin-induced diabetes. huang et al. (2013) stated that 95% ethanol precipitate from an aqueous extract of pu-erh tea exhi bited a remarkable inhibitory effect against α-glucosidase in vitro, as well as a significant (p < 0.05) effect on postprandial hyperglycemia in diabetic mice. recently, chen and guo (2017) investigated the effects of polysaccharides and polyphenolic fractions of zijuan tea on α-glucosidase activity and blood glucose levels. the results indicated that the polysaccharide or theaflavin fractions inhibited α-glucosidase at a greater rate than acarbose (positive control). zhou et al. (2018) reported that tea polyphenols can alter autophagy levels to improve glucose and lipid metabolism in diabetic rats with cardiomyopathy. however, the anti-diabetic effects of dark tea proteins have not yet been investigated. this study focused on four kinds of representative dark teas from hunan province: heimaojian (hmj), fuzhuan tea (fz), heizhuan tea (hz), and qianliang tea (ql). the objective of this work was to examine the inhibitory effects of their protein extracts and hydro lysates on the activity of α-amylase, α-glucosidase, and dpp-iv. materials and methods materials dried dark tea leaves for all four kinds of tea were provided by aijia biotechnology co. (hunan province, china). α-glucosidase from saccharomyces cerevisiae (≥10 u/mg protein), α-amylase from bacillus licheniformis liquid (cas: a4862), dpp-iv inhibitor screening kit (mak203-1kt), 4-nitrophenyl α-d-glucopyranoside (pnpg) (cas: 3767-28-0), and soluble starch were purchased from sigma chemical co. (st. louis, mo, usa). bradford protein assay kit (p0006) was purchased from beyotime (haimen, china). acarbose hydrate was purchased from aladdin. the alcalase enzyme (with a claimed activity of ≥200 u/mg) was purchased from aoboxing bio-tech co. (beijing, china), and trypsin (250 u/mg) was purchased from huaqisheng bio-tech co. (guangzhou, china). 2,4,6-trinitrobenzenesulfonic acid (tnbs, 5% in h2o) reagent was purchased from ark pharm (usa). sodium dodecyl sulphate was purchased from biofroxx. all other reagents were of analytical grade. preparation and purification of dark tea protein extracts dark tea leaves were milled, sieved (20 mesh), accurately weighed (25.0 g), and extracted in an ultrapure water bath (500 ml) for 30 min 58 k. su, x. mao, l. ai, x. zhang at 95 ℃. after filtration with gauze and quantitative filter papers, the filtrates were concentrated by rotary vaporization under reduced pressure at 50 ℃ to obtain the syrup extracts. small amounts of water were then used to dilute the syrup extracts. finally, the extracts were lyophilized, and the residues (test samples) were collected for subsequent analysis. to purify the dark tea protein, portions of the test samples were dissolved in ultrapure water at a concentration of 5 mg/ml and placed in well-prepared dialysis membranes (500 da). ultrapure water (250 times the volume of the samples) at room temperature was placed on the opposite side of the membrane and replaced three times (every 12 hours). after dialysis, 5% (w/v) activated carbon was added at 45 ℃ for 30 min. after filtration with quantitative filter papers, saturated ammonia sulfate solution was added (vammonia sulfate:vsample = 4:1), and the protein was allowed to precipitate at 4 ℃ for 12 hours. the subsidence was obtained by centrifugation at 6000 × g for 20 min at 4 ℃, and then redissolved in ultrapure water before dialysis at room temperature for 24 hours to eliminate micromolecules. enzymatic hydrolysis of dark tea proteins dark tea proteins were enzymatically treated with trypsin and alca lase following the method described previously (harnedy and fitzgerald, 2013; wang and zhang, 2017), with some modifications. briefly, solutions of the crude proteins were preheated to 50 ℃ and adjusted to ph 8.0 with 2.0 m naoh. either alcalase or trypsin was added at an enzyme/substrate (e/s) ratio of 1.5% (w/w). the reaction mixtures were kept in a water bath with shaker at 50 ℃ for six hours, and 2.0 m naoh was used to maintain the ph value as monitored by a ph meter (fe30, mettler toledo, switzerland). the enzymes were inactivated by heating at 90 ℃ for 20 min. hydrolysate samples were subsequently freeze-dried. determination of proteins and degree of hydrolysis proteins were determined using a bradford protein assay kit (p0006). briefly, 5-μl test extract solutions (2 mg/ml) were mixed with 250 μl coomassie brilliant blue (g-250) dye in a 96-well plate. absorbance at 595 nm was measured within 2 hours using a micro plate reader (sunrise v1.05, tecan, switzerland). to obtain a standard curve, bsa protein standard solutions of varying concentrations (0 mg/ml, 0.125 mg/ml, 0.25 mg/ml, 0.5 mg/ml, 0.75 mg/ml, 1.0 mg/ml, and 1.5 mg/ml) were used. degree of hydrolysis (dh) was monitored using the tnbs method described by spellman et al. (2003), with some modifications. du ring the course of the test, 0.5 m naoh was used to keep the reaction at ph 7.0 as monitored by a ph meter. 0.125 ml of the test samples (2 mg/ml) were added to test tubes and mixed with 1.0 ml of sodium phosphate buffer (0.2125 m, ph 8.2). tnbs reagent (1 ml, 0.1% w/v) was then added to each tube, followed by incubation at 50 ℃ for 60 min in a water bath (protected from light). furthermore, the reaction was stopped by adding 2.0 ml hcl (0.1 mol/l). samples were then allowed to cool to room temperature, and absorbance values were measured at 340 nm using a microplate reader (sunrise v1.05, tecan, switzerland). l-leucine (0-2 mm) was used to generate a standard curve. dh values were calculated using the following formula: where an1 is the amino nitrogen content of the protein substrate before hydrolysis (mg/g protein), an2 is the amino nitrogen content of the protein substrate after hydrolysis (mg/g protein), and npb is the nitrogen content of the peptide bonds in the protein substrate (mg/g protein). reversed-phase high-performance liquid chromatography (rphplc) analysis the peptide compositions of dark tea protein hydrolysates were analyzed by reversed-phase high-performance liquid chromatography (rp-hplc) using an agilent 1260 hplc system with a uv detector. briefly, samples were diluted to 1 mg/ml in ultrapure water, filtered through a 0.22-μm syringe filter, and then injected into an analytical c18 column with a length of 250 mm, inner diameter of 4.6 mm, and particle size of 5 μm (c18-120-5 4e, shodex, japan). samples were eluted at a total flow rate of 0.5 ml/min at 30 ℃, with both water (solvent a) and acetonitrile (solvent b) as follows: 15% b at 0-2 min; 15%-20% b at 2-10 min; 20%-25% b at 10-20 min; 25%-80% b at 20-30 min. elution was monitored at 215 nm with an ultravioletvisible (uv-vis) detector. α-amylase inhibition assay the α-amylase inhibitory assay was performed according to the method previously described by yu et al. (2012), with slight modifications. a 1% starch solution was preheated at 95 ℃ for 5 min. 10 μl of α-amylase solution (1 u/ml in ultrapure water) was premixed with 20 μl of test sample solutions at different concentrations (0.25 mg/ml, 0.5 mg/ml, 1 mg/ml, 1.5 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml) in ultrapure water. following incubation at 37 ℃ for 15 min, 500 μl of 1% starch solution (in 0.2 m sodium phosphate buffer, ph 6.9) was added to start the reaction. the reaction was carried out at 37 ℃ for 5 min and terminated by adding 600 μl of dns reagent (1% 3,5-dinitrosalicylic acid, 12% na-k tartrate in 0.4 mol/l na2co3). the reaction mixture was placed in a boiling water bath for 15 min. after the samples cooled down to room temperature, absorbance at 540 nm was determined by a microplate reader (yu et al., 2011a; yu et al., 2011b). acarbose was used as a positive control. sodium phosphate buffer (ph 6.9) was used in place of the test samples for the blank. for all tests, the inhibition assay was performed in quadruplicate. the inhibition of enzyme activity was calculated as follows: inhibition (%) = [1 − (asample /ablank)] × 100 α-glucosidase inhibition assay the α-glucosidase inhibitory activity was assayed according to a previous study by lin et al. (2015), with modifications and optimization. 20 μl of test extract solutions at different concentrations (0.01 mg/ ml, 0.02 mg/ml, 0.05 mg/ml, 0.1 mg/ml, 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml, and 2 mg/ml) and of acarbose (positive control, 0.125 mg/ ml, 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml, and 2 mg/ml) were premixed with 10 μl of α-glucosidase (0.2 u/ml) at 37 ℃ for 20 min. next, 40 μl of pnpg (10 mm, in 0.2 m phosphate buffer, ph 6.8) and 50 μl of 0.2 m phosphate buffer were added and incubated at 37 ℃ for 30 min. the reaction was stopped by adding 100 μl of 0.2 m sodium carbonate. phosphate buffer was used in place of the test samples for the blank. to determine sample background interference, each sample was added to the phosphate buffer during reactions without the enzyme solution. each inhibition assay was performed in quadruplicate. absorbance at 405 nm was measured using a microplate reader. the α-glucosidase inhibition (%) was calculated as follows: inhibition (%) = 1 − [(asample − abackground)/ablank] × 100 dpp-iv inhibition assay the dpp-iv inhibitory activity assay was performed using a dppiv inhibitor screening kit (mak203). the effectiveness of the test inhibitors was compared with a known dpp-iv inhibitor, sitagliptin. before use, reagents were allowed to come to room temperature and slightly centrifuged to maintain integrity. assays were performed as an2 an1 dh% = 100 npb anti-diabetic potential of dark tea 59 specified by the product technical bulletin. after all reagents were diluted to the required concentrations, 50 μl of enzyme solution and 25 μl of test sample solution were premixed and incubated at 37 ℃ for 10 min; then 25 μl of dpp-iv substrate was added. after waiting 15-30 min to allow all reagents to mix, fluorescence (flu, λex = 360 nm, λem = 460 nm) was measured once per minute at 37 ℃ using a cytation 5 imaging reader (biotek, usa). a control assay was also prepared by using dpp-iv assay buffer in place of the inhi bitor samples. to remove background interference, buffer was added instead of enzyme solution. the relative dpp-iv inhibition (%) was calculated as follows: relative inhibition (%) =1− [(slopesample – slopebackground)/slopecontrol] × 100% where slope = (flu2 – flu1)/(t2 – t1) = flu/minute statistical analysis results are presented as the mean of triplicate determinations ± sds. ibm spss 22.0 (spss, inc., chicago, il, usa) was used to identify significant differences (p < 0.05) between data. results and discussion extraction and purification of dark tea proteins the method of preparing dark tea proteins under ordinary pressure is not fully described in the existing scientific literature, but plant derived proteins are usually extracted by one of three types of solvents: aqueous, alkaline, or organic (harnedy and fitzgerald, 2013; lin et al., 2015; siow et al., 2016). in consideration of people’s drinking habits and environmental protection concerns, aqueous solvent was used in this study. the concentration of protein in tea leaves is around 0.20 g/g dry matter. in this study, the yield ranged from 7.39% ± 0.87% (for the complete ql protein) to 20.3% ± 1.19% (for the hmj hydrolysate treated with alcalase). the presence of fiber and phenolic compounds, as well as protein glycosylation, all have inhibitory effects on protein hydrolysis, which cause misleading results in enzyme inhibitory assays. phenolic and flavone compounds also inhibit the activity of many enzymes. puri fication of proteins was therefore a critical step in removing some of the inhibitory agents and increasing the accuracy of the study. after the purification treatment, the concentration of protein increased dramatically (shown in fig. 1). the trypsin-generated ql fraction realized the largest increase at 404.8% (from 0.1080 g/g extract to 0.5452 g/g extract), followed by the complete ql protein at 321.65% (from 0.0739 g/g extract to 0.3116 g/g extract) and the complete hmj protein at 247.16% (from 0.1767 g/g extract to 0.6134 g/g extract). the average increase in protein content for 12 protein samples was 195.11%. enzymatic hydrolysis of protein fractions the primary sequence of the protein substrate and the specificity of the enzyme(s) used determined the types of bioactive peptides gene rated from a particular protein. trypsin and alcalase have been widely used on plant proteins to produce bioactive peptides (harnedy and fitzgerald, 2013; kalyankar et al., 2013). in this study, the tnba assay was used to quantify the degree of hydrolysis (dh) of four kinds of dark tea protein hydrolysates. fig. 2 illustrates that the dh of hmj, fz, and ql were higher when trypsin was used compared to alcalase, with the exception that hz reached a higher dh when treated with alcalase. according to researchers, alcalase is a proteolytic preparation derived from bacillus licheniformis and consists mainly of subtilisin endoproteinase, with a minor glutamyl endopeptidase activity (kalyankar et al., 2013). subtilisin acts as a relatively nonspecific proteinase but preferentially cleaves peptide bonds after large non-β-branched hydrophobic residues (gron et al., 1992), while trypsin specifically targets diaminocaproic acid and arginine. this may indicate that lower concentrations of diaminocaproic acid and arginine exist in hz. moreover, when hydrolyzed by trypsin at a ph of 8.0 for 6 hours at 50 ℃, fz reached the highest dh at 49% ± 0.52%, followed by hmj (41.39% ± 1.02%), ql (35.96% ± 0.145%), and hz (34.53% ± 0.52%). these values were higher than those obtained in previous studies; for instance, spirulina platensis hydrolyzed by trypsin had a dh of 18.26%-20.2% (fan et al., 2018; wang and zhang, 2016). the dh for proteins treated with alcalase followed a slightly different order than for those treated with trypsin, as follows: fz (43.98% ± 0.6 3% ) > hz (36.93% ± 1.2%) > hmj (33.86% ± 0.73%) > ql (22.84% ± 0.92%). these values were similar to those found in the literature; for instance, 20.8%-24.4% for spirulina platensis hydrolyzed by alcalase (fan et al., 2018; wang and zhang, 2016). the peptide compositions of dark teas were analyzed by rp-hplc. this study displays partial profiles during min 0-10, since no signal was obtained in the last 10 min (not included in fig. 3). in min 0-10, obvious differences were seen between the complete protein extracts fig. 1: protein concentrations of four kinds of dark tea extracts and their hydrolysates before and after purification treatments. mean ± sd (n=3). bars with different letters are significantly different at p < 0.05. fig. 2: degree of hydrolysis (dh, %) for four kinds of dark tea generated by trypsin and alcalase and their complete protein extracts. mean ± sd (n=4). bars with different letters are significantly different at p < 0.05. 60 k. su, x. mao, l. ai, x. zhang fig. 3: rp-hplc absorbance profiles at 215 nm for complete proteins and hydrolysates of hmj (a), fz (b), hz (c), and ql (d). and the trypsinand alcalase-generated hydrolysates. for example, the hmj protein mainly had one wide peak at around 6 min, whereas after hydrolysis, there were two high peptide peaks. the trypsin hydrolysates displayed narrower and higher peaks than the alcalase hydrolysates. all four kinds of dark tea hydrolysates generated by trypsin displayed similar, moderate peptide profiles in which the first peak was eluted out earlier, at around 5 min. the order of elution time was fz < hmj < hz ≈ ql (fig. 3), which was also consistent with the order of their dh values. this suggests that trypsin released peptides from the various dark teas at different rates. in addition, for both complete proteins and hydrolysates, a narrow high peak appeared at a retention time of 12.5 min, with a nearly undetectable signal at 280 nm (not included in fig. 3), the absorbance of peptide bound. the strongest signal was at 215 nm, suggesting the presence of a flavanoid. although for an individual protease, higher dh might cause more bioactive peptides to be released from proteins, the ultimate and maximal inhibition activity of the hydrolysates were determined by the enzyme’s structure, especially the nature of the peptides released in different hydrolysates. this explains the lack of correlation between the dh and the inhibitory activity of the samples. in vitro assessment of biological activity α-amylase inhibition activity several plant proteins and peptides have been found to inhibit αamylase activity in vitro (siow et al., 2017; sintsova et al., 2018; natashya et al., 2018). however, to date, no reports are available on the α-amylase inhibitory activity of dark tea protein hydrolysates. the ic50 values of the four kinds of dark tea hydrolysates generated by trypsin and alcalase were as follows: 2.78 ± 0.04 mg/ml for hmj, 1.38 ± 0.03 mg/ml for fz, 1.95 ± 0.06 mg/ml for hz, and 1.27 ± 0.03 mg/ml for ql. the ic50 value of acarbose was 0.56 ± 0.06 mg/ml. it is possible that compounds with α-amylase inhibitory activity in the complete protein may be degraded during hydrolysis by trypsin and alcalase. a similar phenomenon was observed by harnedy and fitzgerald (2013), in which the palmaria palmata protein exhibited higher renin inhibitory activity than its hydrolysate generated by alcalase. fig. 4 indicates the dose-response curves of four kinds of complete proteins. inhibitory activity reached nearly its highest stage at a sample concentration of 4 mg/ml, in the following order: ql (64.94% ± 1.25%) > fz (61.08% ± 0.76%) > hz (59.09% ± 1.10%) > hmj (56.24% ± 0.90%). interestingly, the α-amylase inhibitory activity of all four kinds of dark tea was largely diminished after hydrolysis, and none of the hydrolysate fractions demonstrated significant inhibitory activity at a concentration range of 1-4 mg/ml (indicated in tab. 1). most of the protein hydrolysates of hmj, hz, and ql negatively inhibited α-amylase. low inhibitory activity was found in the hydrolysates of fz; the highest value occurred at a concentration of 1 mg/ml, (21.91% ± 0.02% for the trypsin-generated hydrolysate and 29.48% ± 0.04% for the alcalase-generated hydrolysate). no regular doseresponse relationship was found (see tab. 1). the reason for this is unknown; perhaps α-amylase’s acting sites are related to the cleavage sites of trypsin and alcalase, whereas fz has a different protein composition than hmj, hz, and ql. anti-diabetic potential of dark tea 61 α-glucosidase inhibition activity as an important carbohydrate-hydrolyzing enzyme, α-glucosidase plays a key role in carbohydrate digestion. the inhibition of α glucosidase helps to delay carbohydrate digestion and prolongs overall carbohydrate digestion time, reducing the glucose absorption rate and consequently blunting the postprandial plasma glucose rise (bhandari et al., 2008; kim et al., 2011). several previous studies have reported the inhibitory activities of plants. for example, zhou et al. (2017) reported that flavanone compounds in dark tea had an ic50 value of 10.2 μm. liu et al. (2013) reported that aqueous extracts prepared from nelumbo nucifera leaves achieved an ic50 value of 1.86 ± 0.018 mg/ml. as indicated in tab. 2, nearly all the proteins and their hydrolysates exhibited higher α-glucosidase inhibitory activity than acarbose (ic50 = 0.7265 ± 0.058 mg/ml). ql exhibited high activity, with an ic50 value of 0.0103 ± 0.025 mg/ml before hydrolysis, and 0.0349 ± 0.0025 mg/ml and 0.0233 ± 0.0024 mg/ml after treatment with trypsin and alcalase, respectively. hmj showed lower inhibitory capacity after being hydrolyzed, suggesting that some proteins with α-glucosidase inhibition activity may have been cleaved. moreover, no significant differences were detected among the complete hz proteins and the two enzymatic hydrolysates. the lowest inhibitory activity was observed in the fz hydrolysate diges ted by trypsin, with an ic50 value of 1.3114 ± 0.0174 mg/ml. dpp-iv inhibition activity the dpp-iv inhibitory activities of hmj, fz, hz, and ql protein hydrolysates obtained by trypsin and alcalase, respectively, were investigated. in general, fairly high dpp-iv relative inhibitory activity was observed in the protein fractions, with hmj having the largest dpp-iv relative inhibitory ability, followed by fz and hz; ql had the lowest dpp-iv relative inhibitory activity. in a study by harnedy and fitzgerald (2013), an ic50 value of 2.52 ± 0.05 mg/ml was obtained for palmaria palmata aqueous extracts generated by alcalase, which is much higher than the value in the present study. the most effective protein fraction was hmj hydrolyzed with alcalase, which recorded an ic50 value of 0.1000 ± 0.0266 mg/ml. ic50 values of 0.1794 ± 0.0204 mg/ml and 0.1315 ± 0.0017 mg/ml were found in the complete hmj protein and the trypsin-treated hmj fraction. the relative inhibitory ability of fz and hz increased dramatically after hydrolysis (tab. 2), suggesting that some effective bioactive peptides were obtained. ql appeared to have the least effective relative inhibitory activity but still had ic50 values of 1.0900 ± 0.027 mg/ml (control), 1.3364 ± 0.0056 mg/ml (trypsin-generated hydrolysate), and 1.0281 ± 0.016 mg/ml (alcalase-generated hydrolysate). the results of this in vitro study clearly suggest that dark tea proteins and their hydrolysates have the potential to control hyperglycemia. the α-amylase, α-glucosidase, and dpp-iv inhibitory activities obfig. 4: inhibitory activity against α-amylase of different concentrations of four kinds of complete black tea proteins. mean ± sd (n=4). tab. 1: inhibitory activity (%) of four kinds of dark tea hydrolysates generated by trypsin(a) and alcalase(b) against α-amylase at concentrations of 1 mg/ml, 2 mg/ml, and 4 mg/ml. concentrations of 4–8 mg/ml were also investigated in the study but not included in this paper. mean ± sd (n=3). n.a. = no inhibition detected. values with different letters are significantly different at p < 0.05. enzyme sample inhibitory activity (%) 4 mg/ml 2 mg/ml 1 mg/ml trypsin hmj -30.97 ± 0.06 n.a. -29.20 ± 0.04 n.a. -34.16 ± 0.06 n.a. fz 7.88 ± 0.01 c 18.81 ± 0.02 b 21.91 ± 0.02 a hz -26.44 ± 0.11 n.a. -4.66 ± 0.10 n.a. 2.36 ± 0.05 d ql -5.91 ± 0.02 n.a. -10.45 ± 0.03 n.a. -2.65 ± 0.05 n.a. alcalase hmj -30.51 ± 0.04 n.a. -28.81 ± 0.03 n.a. -20.49 ± 0.08 n.a. fz 1.68 ± 0.03 c 14.84 ± 0.13 b 29.48 ± 0.04 a hz -54.44 ± 0.08 n.a. -51.27 ± 0.07 n.a. -24.21 ± 0.02 n.a. ql -7.96 ± 0.05 n.a. -13.30 ± 0.01 n.a. -3.10 ± 0.01 n.a. tab. 2: ic50 (concentration that inhibits enzyme activity by 50%) of four kinds of dark tea and their hydrolysates generated by trypsin and alcalase against α-glucosidase and dpp-ⅳ. mean ± sd (n=3). ic50 with different letters for each of the activities are significantly dif ferent at p < 0.05. sample hydrolysate ic50 (mg/ml) for α-glucosidase complete trypsin alcalase hmj 0.0942 ± 0.0023 g 0.2161 ± 0.0036 f 0.2036 ± 0.0022 f fz 0.5401 ± 0.0042 d 1.3114 ± 0.0174 a 0.6588 ± 0.0045 b hz 0.5478 ± 0.0128 d 0.6231 ± 0.0355 c 0.5069 ± 0.0011 e ql 0.0103 ± 0.0025 h 0.0349 ± 0.0026 h 0.0233 ± 0.0024 h sample hydrolysate ic50 (mg/ml) for dpp-ⅳ complete trypsin alcalase hmj 0.1794 ± 0.0204 ghi 0.1315 ± 0.0017 hi 0.1000 ± 0.0266 i fz 0.9124 ± 0.0216 d 0.1011 ± 0.0190 i 0.1998 ± 0.0176 gh hz 0.8467 ± 0.0035 e 0.3993 ± 0.0178 f 0.2477 ± 0.0278 g ql 1.0900 ± 0.0270 b 1.3364 ± 0.0056 a 1.0281 ± 0.0160 c 62 k. su, x. mao, l. ai, x. zhang doi: 10.1016/j.foodchem.2012.08.038 kim, j.s., hyun, t.k., kim, m.j., 2011: the inhibitory effects of ethanol extracts from sorghum, foxtail millet and proso millet on α-glucosidase and α-amylase activities. food chem. 124 (4), 1647-1651. doi: 10.1016/j.foodchem.2010.08.020 koh, l.w., wong, l.l., loo, y.y., kasapis, s., huang, d., 2010: evaluation of different teas against starch digestibility by mammalian glycosidases, j. agric. food chem. 58(1), 148-154. doi: 10.1021/jf903011g lebovitz, h.e., 1997: alpha-glucosidase inhibitors. endocrinol. metab. clin. north am. 26, 539-551. liu, s., li, d., huang, b., chen, y., lu, x., 2013: inhibition of pancreatic lipase, α-glucosidase, α-amylase, and hypolipidemic effects of the total flavonoids from nelumbo nucifera leaves. j. ethnopharmacol. 149, 263269. lin, y.s., chen, c.r., wu, w.h., wen, c.l., chang, c.i., hou, w.c., 2015: anti-α-glucosidase and anti-dipeptidyl peptidase-iv activities of extracts and purified compounds from vitis thunbergii var. taiwaniana. j. agric. food chem. 63(28), 6393-6401. doi: 10.1021/acs.jafc.5b02069 miao, m., jiang, b., jiang, h., zhang, t., li, x.. 2015: interaction mechanism between green tea extract and human alpha-amylase for reducing starch digestion, food chem. 186, 20-25. doi: 10.1016/j.foodchem.2015.02.049 peng, s., xue, l., leng, x., yang, r., zhang, g., hamaker, b.r., 2015: slow digestion property of octenyl succinic anhydride modified waxy maize starch in the presence of tea polyphenols. j. agric. food chem. 63 (10), 2820-2829. li, s., lo, c.y., pan, m.h., lai, c.s., ho, c.t., 2012: black tea: chemical analysis and stability. food funct. 4(1), 10-18. doi: 10.1039/c2fo30093a sintsova, o., gladkikh, i., chausova, v., monastyrnaya, m., anastyuk, s., chernikov, o., yurchenko, e., aminin, d., isaeva, m., leychenko, e., kozlovskaya, e., 2018: peptide fingerprinting of the sea anemone heteractis magnifica mucus revealed neurotoxins, kunitz-type proteinase inhibitors and a new β-defensin α-amylase inhibitor. j. proteomics. 173, 12-21. doi: 10.1016/j.jprot.2017.11.019 siow, h.l., gan, c.y., 2016: extraction, identification, and structure-activity relationships of antioxidative and α-amylase inhibitory peptides from cumin seeds (cuminum cyminum). j. funct. foods 22, 1-12. doi: 10.1016/j.jff.2016.01.011 siow, h., lim, t.s., gan, c., 2017: development of a workflow for screening and identification of α-amylase inhibitory peptides from food source using an integrated bioinformatics-phage display approach: case study − cumin seed. food chem. 214, 67-76. doi: 10.1016/j.foodchem.2016.07.069 spellman, d., mcevoy, e., fitzgerald, r.j., 2003: proteinase and exo peptidase hydrolysis of whey protein: comparison of the tnbs, opa and ph stat methods for quantification of degree of hydrolysis. int. dairy j. 13(6), 447-453. doi: 10.1016/s0958-6946(03)00053-0 stote, k.s., baer, d.j., 2008: tea consumption may improve biomarkers of insulin sensitivity and risk factors for diabetes. j. nutr. 138(8), 15841588. wang, z.j., zhang, x.w., 2016: inhibitory effects of small molecular peptides from spirulina (arthrospira) platensis on cancer cell growth. food funct. 7, 781-788. wang, z.j., zhang, x.w., 2017: isolation and identification of anti-proli ferative peptides from spirulina platensis using three-step hydrolysis. j. sci. food agric. 97, 918-922. xiao, j., huo, j., jiang, h., yang, f., 2011: chemical compositions and bioactivities of crude polysaccharides from tea leaves beyond their useful date. int. j. biol. macromol. 49(5), 1143-1151. yu, z., yin, y., zhao, w., wang, f., yu, y., liu, b., liu, j., chen, f., 2011a: characterization of ace-inhibitory peptide associated with antioxidant and anticoagulation properties. j. food sci. 76(8), 1149-1155. doi: 10.1111/j.1750-3841.2011.02367.x yu, z., yin, y., zhao, w., yu, y., liu, b., liu, j., chen, f., 2011b: novel served were different for hmj, fz, hz, and ql proteins and their hydrolysates generated by trypsin and alcalase. generally, all four kinds of dark tea protein extracts demonstrated moderate inhibitory activity on α-amylase (ic50 values ranged from 1.27 to 2.78 mg/ml), but inhibitory activity was nearly undetectable after hydrolysis. all of the proteins and hydrolysates displayed good inhibitory activity on α-glucosidase (ic50 values ranged from 0.0103 to 1.3114 mg/ml) and dpp-iv (ic50 values ranged from 0.1000 to 1.3364 mg/ml). the hmj and ql extracts were better α-glucosidase inhibitors, while the hmj extracts, fz hydrolysates generated with trypsin and alcalase, and hz hydrolysates generated with trypsin and alcalase recorded better inhibitory activity on dpp-iv than other fractions. it is noted that the protein concentrations in dark tea under typical drinking conditions (steeped at 85 ℃ for 10 min) detected by bradford assay were as follows: 0.03595 mg/ml (hmj), 0.04758 mg/ml (fz), 0.01493 mg/ml (hz), and 0.08977 mg/ml (ql). thus, theoretically, the protein concentrations in tea as it is typically drunk is close to the ic50 value for α-glucosidase, but 10and 100-fold concentrations of the tea, respectively, would be required to achieve the ic50 values for dpp-iv and α-amylase. in addition, due to the degradation of protein or peptides in the gastrointestinal tract, doses higher than the theoretical values are needed; hence, more effective proteinextraction techniques deserve further study in the future. references bhandari, m.r., nilubon, j.a., hong, g., kawabata, j., 2008: α glucosidase and α-amylase inhibitory activities of nepalese medicinal 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10.1111/j.1750-3841.2012.02688.x green, b.d., gault, v.a., o’harte, f.p., flatt, p.f., 2004: structurally modified analogues of glucagon-like peptide-1 (glp-1) and glucosedependent insulinotropic polypeptide (gip) as future antidiabetic agents. curr pharm des. 10, 3651-3662. gron, h., meldal, m., breddam, k., 1992: extensive comparison of the substrate preferences of two subtilisins as determined with peptide substrates which are based on the principal of intramolecular quenching. biochem. 31, 6011-6018. harnedy, p.a., fitzgerald, r.j., 2013: in vitro assessment of the cardioprotective, anti-diabetic and antioxidant potential of palmaria palmata protein hydrolysates. j. appl. phycol. 25(6), 1793-1803. doi: 10.1007/s10811-013-0017-4 huang, q., chen, s., chen, h., wang, y., wang, y., hochstetter, d., xu, p., 2013: studies on the bioactivity of aqueous extract of pu-erh tea and its fractions: in vitro antioxidant activity and α-glycosidase inhibitory property, and their effect on postprandial hyperglycemia in diabetic mice. food chem. toxicol. 53, 75-83. doi: 10.1016/j.fct.2012.11.039 kalyankar, p., zhu, y., o’keeffe, m., o’cuinn, g., fitzgerald, r.j., 2013: substrate specificity of glutamyl endopeptidase (ge): hydrolysis studies with a bovine α-casein preparation. food chem. 136, 501-512. anti-diabetic potential of dark tea 63 peptides derived from egg white protein inhibiting alpha-glucosidase. food chem. 129(4), 1376-1382. doi: 10.1016/j.foodchem.2011.05.067 yu, z., yin, y., zhao, w., 2012: anti-diabetic activity peptides from albumin against α-glucosidase and α-amylase. food chem. 135(3), 2078-2085. doi: 10.1016/j.foodchem.2012.06.088 zhou, h., lia, h., yang, m., 2017: c-geranylated flavanones from yingde dark tea and their antioxidant and α-glucosidase inhibition activities. food chem. 235, 227-233. address of the corresponding author: prof. x.w. zhang, college of food science and engineering, south china university of technology, 381 wushan road, guangzhou 510640 e-mail: snow_dance@sina.com © the author(s) 2019. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). journal of applied botany and food quality 93, 122 129 (2020), doi:10.5073/jabfq.2020.093.016 1szent istván university, department of refrigeration and livestock product technology, budapest, hungary 2 institute of biotechnology and food technology, industrial university of ho chi minh city, ho chi minh, vietnam 3szent istván university, department of postharvest science and sensory evaluation, budapest, hungary 4szent istván university, department of physics and control, budapest, hungary comparison of 1-mcp treatment on four melon cultivars using different temperatures lien phuong le nguyen1,2*, tamás zsom3, mai sao dam2, lászló baranyai*4, géza hitka3 (submitted: september 9, 2019; accepted: february 14, 2020) * corresponding author summary the aim of this work was to evaluate the effect of 1-methylcyclopropene (1-mcp) treatment on muskmelons (cucumis melo l. var. reticulates naudin) using different temperatures during the treatments. three application temperatures (5, 10 and 20 °c) and four melon cultivars (‘centro’, ‘lillo’, ‘donatello’ and ‘celestial’) were investigated. three groups of each cultivar were treated with 625-650 nl l-1 gaseous 1-mcp for 24 h at 5, 10 and 20 °c. during the 24 h long gaseous 1-mcp treatment, the control group was kept at 5 °c. after treatment, all samples were stored at ambient temperature (20 °c) for 10 d. the results showed that 1-mcp treated melons released less ethylene and co2 compared to controls. moreover, 1-mcp application could slow the softening as well as the color change of melon throughout shelf-life in comparison to controls. no significant differences were observed among 1-mcp treatment temperatures for four melon cultivars. 1-mcp did not reduce disease severity of treated melons. keywords: 1-methylcyclopropene, cantaloupe, storage, shelf-life, cucumis melo introduction cantaloupe (cucumis melo l. var. reticulates naudin) is one of the most common melon varieties due to its attractive orange flesh, unique flavor and nutritional value (aguayo et al., 2007; silveira et al., 2008). however, short-term shelf-life due to microbial growth and deterioration in appearance and nutritional value result in losing market value (fallik et al., 2000). thus, the time for transport, sale and consumption of melon is limited and this produce is used primarily for fresh-market consumption (abadias et al., 2014). 1-methylcyclopropene (1-mcp) is an ethylene action inhibitor (sisler, 2006). 1-mcp successfully controls the ripening of fruit and vegetable during storage and transport by preventing negative ethylene effects (sisler, 2006). recently, many studies were published about the effects of 1-mcp in delaying ripening and maintaining texture, firmness, taste and appearance of fruit (zhao et al., 2018; cheng et al., 2019). responses of melon to 1-mcp were also reported. 1-mcp had strong effect on ‘galia’ melon at intermediate to advanced stages of ripening (ergun et al., 2005). those authors found that the change of surface color was dramatically delayed by 1-mcp. 1-mcp retained the firmness of intact and fresh-cut melon compared to control (gal et al., 2006; ergun et al., 2007). in addition, the symptom of water soaking in fresh-cut melon was also postponed by 1-mcp (ergun et al., 2007). fresh-cut ‘charentais’ melon treated with 1-mcp accumulated higher values of aroma volatiles than untreated samples (amaro et al., 2012). the benefit of 1-mcp in extending the shelflife of muskmelon was to retain firmness and soluble solid content, suppress the overripe flavor development (agehara et al., 2018). in literature, most of the studies focused on the effect of 1-mcp concentration (alves et al., 2005; ergun et al., 2005; de melo et al., 2008), different treatment duration (gal et al., 2006) or application form including gaseous 1-mcp and aqueous 1-mcp (amaro et al., 2012; shi et al., 2014; bai et al., 2014; agehara et al., 2018). in addition, 1-mcp treatment on melon was mainly carried out at ambient temperature within a day after harvest (alves et al., 2005; ergun et al., 2005; ergun et al., 2007; gal et al., 2006; shi et al., 2014; agehara et al., 2018). the 1-mcp treatment is commonly performed at 20-25 °c for 12-24 h in experiments to reach desired effects on crops (blankenship and dole, 2003). 1-mcp may reach ethylene receptors more easily at higher temperature (watkins, 2016). for example, 1-mcp treatment affected broccoli (brassica oleracea) at both 5 and 20 °c, however, it showed better effective ness at 20 °c (ku and wills, 1999). in case of penstemon (penstemon hartwegii), 1-mcp treatment at 2 °c was not effective whereas ap plication at 20 °c completely controlled ripening (serek et al., 1995). for coriander (coriandrum sativum), 1-mcp did not yield benefit at low application temperature (5-10 °c) because of low affinity to the ethylene receptors (jiang et al., 2002). nevertheless, in commercial practice, it is not easy to carry out the 1-mcp treatment at the harvest day due to transport or occasional lack of the air tight storage room (watkins and nock, 2005). in commercial facilities, fruit are amassed in storage rooms at cold temperature after harvest. the 1-mcp treatment is carried out when rooms are filled to desired level. therefore, commercial utilization of 1-mcp is usually conducted at cold temperature (watkins and nock, 2005). moreover, melon has a high rate of respiration and senescence when storage temperature is above 5 °c after harvest (edwards and blennerhassett, 1994). therefore, precooling followed by 1-mcp application at cold temperature is the most suitable. although there are several studies published about 1-mcp application on melon, information about response to different treatment temperature is still limited. the aim of this study was to investigate the effect of 1-mcp treatment temperature (using 5, 10 and 20 °c) on four melon cultivars (‘lillo’, ‘centro’, ‘donatello’ and ‘celestial’) during shelf-life at 20 °c. the results of this work provide useful information for commercial application of 1-mcp. materials and methods materials fruit of four cultivars (‘lillo’, ‘centro’, ‘celestial’ and ‘donatello’) of the muskmelon variant (cucumis melo l. var. reticulates naudin) were harvested at the half slip stage in july 2017 and 2018. cultivars were selected according to their popularity and high production quantity in hungary. fruit were selected for uniformity of size, shape and absence of external damage. small and large diameters of melons were 12.0 ± 0.3 cm and 14.0 ± 0.2 cm, respectively. harvested response of melon cultivars to 1-mcp treatment temperature 123 fruit were transported to the laboratory (budapest, hungary) and treatments were applied within 24 h of harvest. fruit temperature was 20 ± 1 °c during transportation. for each cultivar, melons were randomly divided into four groups (15 fruit per group). fruit of three groups were subjected to 1-mcp treatment at different temperatures and the others were stored as controls. the shelf-life was monitored and quality measurements were performed at 20 °c. the 1-mcp (0.14 % 1-mcp tablet, agrofresh, usa) as an application of the smartfresh® system was provided by rohm and haas polska sp.z.o.o. (warsaw, poland). 1-mcp application melons were kept at 5, 10 and 20 °c, respectively, for 24 h before treatment. three groups were treated with 625-650 nl l-1 gaseous 1-mcp in an air-tight plastic box of 500 l at 5, 10 and 20 °c, respectively, on the 1st day after harvest for 24 h. the control group (untreated) was kept at 5 °c during this period. after treatment, the samples of the four groups were stored at room temperature (20 °c) and at relative humidity (rh) of 55%. measurements measurements of stiffness, ethylene production, respiration rate, rind color, chlorophyll fluorescence and disease severity were performed according to nguyen et al. (2019). all parameters except disease severity were measured at the initial time of 0 d, followed by 5 d and 10 d. disease severity was assessed at 4 d, 7 d and 10 d. during the experiment, non-destructive tests were performed with 15 randomly selected fruit each interval. respiration was evaluated in triplicates due to limitation of resources. stiffness fruit firmness was estimated using the acoustic vibration method (chen and de baerdemaeker, 1993) and expressed as the parameter stiffness (s, g2/3 s-2). stiffness of the samples was determined at 2 points on the exterior circumference of each fruit, using an afs dtf v0.0.0.105 acoustic firmness instrument (aweta, nootdorp, the netherlands). ethylene production ethylene production was determined by an ica-56 handheld ethylene analyzer (international controlled atmosphere ltd., tonbridge, uk). one melon was placed in a hermetically closed plastic container of 4 l for 1 h before measurement was performed. measurement was repeated in triplicates. results were expressed in microliter gas produced per kilogram of fruit fresh mass in one hour (μl kg-1 h-1). respiration rate carbon dioxide production was measured in a closed respiratory system for 1 h. the system was built with hermetically closed acrylic sheet containers equipped with fy a600-co2h carbon dioxide sensors (ahlborn mess-und regelungstechnik gmbh, holzkirchen, germany). measurement was repeated in triplicates. changes in co2 concentration was recorded with an almemo 3290-8 data logger (ahlborn mess-und regelungstechnik gmbh, holzkirchen, germany) and results were expressed as ml kg-1 h-1. surface color melon rind color was measured with a portable minolta chroma meter cr-400 (minolta corporation, osaka, japan). standard cie l*, a* and b* color characteristics were determined at three points on the external circumference of each fruit. the hue angle value was calculated as arctangent of b*/a*. chlorophyll fluorescence chlorophyll fluorescence was determined at three equidistant points on the equator of each fruit by a pam wincontrol-3 controlled monipam multi-channel chlorophyll fluorometer (heinz walz gmbh, effeltrich, germany). from the recorded minimal and maximal chlorophyll fluorescence signals (f0, fm) the potential maximum quantum yield of the photosystem ii (fv/fm) was calculated. disease severity melons were examined for mold growth on the rind or stem during storage. infection was expressed on the scale of 1-3, where 1 = good, fruit without decay; 2 = fair, fruit with moderate decay; 3 = bad, fruit with severe decay. decay was visually assessed according to the total area of infection on rind or stem. disease severity was calculated as an average score of all melon within a group (yang et al., 2003). statistical analysis all data were subjected to statistical analysis with ibm spss version 22 (ibm corp, new york, usa) using analysis of variance (anova). the effects of cultivar, treatment temperature and storage time were evaluated. the anova f value was used to compare effects to natural variability of readings. tukey’s method was used as post-hoc test to compare groups with p<0.05. on figures, results are reported as means and standard deviations. results ethylene and co2 production the initial ethylene concentrations largely varied among fruit of the four cultivars, ranging from 36 μl kg-1 h-1 to 46 μl kg-1 h-1 (fig. 1). fruit of ‘lillo’ had the lowest ethylene production followed by those of ‘donatello’, ‘celestial’ and ‘centro’. ethylene production of both untreated and treated fruit decreased during storage, but at different rates. rates of ethylene release of controls gradually declined during storage (fig. 1), while that of treated fruit pronouncedly decreased after treatment, obviously reflecting a direct response to 1-mcp. observed differences in rates of ethylene release and its production kinetics between fruit of the four cultivars were significant (p<0.001; tab. 1). according to the observed drop in ethylene production, fruit of cultivars ‘centro’ and ‘donatello’ responded more sensitively to 1-mcp than the others. there were no significant differences between the effects of treatment temperature on ethylene production of fruit of the four cultivars. the initial carbon dioxide release rates (approximately 28 ml kg-1 h-1) were almost the same for fruit of the four melon cultivars (fig. 2). however, during the experiment, the carbon dioxide production of ‘lillo’ fruit decreased more sharply than that of other cultivars (fig. 2). according to anova, cultivar but not treatment temperature affected respiration significantly (p<0.001; tab. 2). again, 1-mcp treatment but not temperature affected co2 production of treated fruit compared to that of controls. in this context, sensitivity of ethylene production to 1-mcp treatment was 4.4 times higher than that of co2 production. ethylene and co2 production followed similar tendency, their values strongly correlated during the experiment (r = 0.920). stiffness the stiffness of all melons declined throughout storage. however, softening of 1-mcp treated fruit was pronouncedly lower than that of untreated samples (fig. 3). this clearly indicated that 1-mcp suppressed softening of fruit. 124 l.p.l. nguyen, t. zsom, m.s. dam, l. baranyai, g. hitka fig. 1: ethylene production of fruit of four melon cultivars during shelf-life at 20 °c. data are shown as means ± sd. tab. 1: statistical evaluation of ethylene production (anova) interaction effectfactors for ethylene main effect × temperature × cultivar time *** – *** treatment *** – *** temperature – – – cultivar *** – – *** p<0.001, ** p<0.01, * p<0.05, n = 144 tab. 2: statistical evaluation of co2 respiration (anova) interaction effect factors for co2 main effect × temperature × cultivar time *** – *** treatment *** – ** temperature – – – cultivar *** – – *** p<0.001, ** p<0.01, * p<0.05, n = 144 fig. 2: carbon dioxide production of fruit of four melon cultivars during shelf-life at 20 °c. data are shown as means ± sd. 10 20 30 40 50 0 5 10 5°c 10°c 20°c control e th yl en e (µ l /k g. h) storage time in days celestial 10 20 30 40 50 0 5 10 5°c 10°c 20°c control e th yl en e (µ l /k g. h) storage time in days donatello 10 20 30 40 50 0 5 10 5°c 10°c 20°c control e th yl en e (µ l /k g. h) storage time in days celestial 10 20 30 40 50 0 5 10 5°c 10°c 20°c control e th yl en e (µ l /k g. h) storage time in days donatello response of melon cultivars to 1-mcp treatment temperature 125 according to the anova (tab. 3), effects on fruit stiffness of 1-mcp treatment and cultivar but not of temperature were significant (p<0.001). only fruit of ‘donatello’ responded significantly to changes in temperature (p<0.05) but this was less than 1% of the effect of 1-mcp treatment (anova f=3.3 << 370.1). stiffness values correlated strongly with maximal chlorophyll fluorescence (fm, r = 0.976) and efficiency of photosystem ii (fv/fm, r = 0.938). chlorophyll fluorescence parameters during storage, treated melons retained higher fv/fm than controls (fig. 4). the results showed that 1-mcp treatment clearly affected fv/fm (p<0.001). additionally, cultivar was found to have only weak interaction effect with 1-mcp treatment (anova f = 5.3, p<0.01). according to the statistical analysis, primarily storage time and 1-mcp treatment contributed to changes in chlorophyll fluorescence (p<0.001; tab. 4). besides the strong relationship with stiffness, fv/fm obtained significant correlation with hue angle (r = 0.871; p<0.001). this agreement between surface color and green pigment measurements was expected. at the end of the experiment, controls of all cultivars had the lowest f0 and fm (data not shown). obviously, chlorophylls of untreated samples were more rapidly degraded than that of treated fruit, indicating the retardation of ripeness and/or senescence. tab. 3: statistical evaluation of stiffness (anova) interaction effect factors for stiffness main effect × temperature × cultivar time *** ** *** treatment *** – ** temperature * – – cultivar *** – – *** p<0.001, ** p<0.01, * p<0.05, n = 720 tab 4: statistical evaluation of fluorescence fv/fm (anova) interaction effect factors for fv/fm main effect × temperature × cultivar time *** – – treatment *** – ** temperature – – – cultivar – – – *** p<0.001, ** p<0.01, * p<0.05, n = 720 fig. 3: stiffness of melon during shelf-life at 20 °c. data are shown as means ± sd hue angle value the hue angles of all samples decreased during shelf-life at 20 °c (fig. 5). the color of the rind of controls turned to yellow more rapidly than that of treated samples. thus, this color change is often used as an indicator of ripening (dong et al., 2002). this is also supported by our results as hue angle significantly correlated with chlorophyll fluorescence and stiffness (r = 0.898; p<0.001). both 1-mcp treatment and cultivar but not temperature significantly affected (p<0.001) hue angles (tab. 5). temperature had only minor interactive effect with cultivar (anova f = 2.4, p<0.05). disease severity ripe fruit can be more sensitive to microbial infection due to softer texture and changing compounds, therefore 1-mcp treatment might affect disease severity, too. in the presented experiment, 1-mcp treatment showed smaller effect on disease severity (anova f = 7.2, p<0.01) compared to storage time and cultivar (tab. 6). severity of disease of all fruit increased with increasing treatment temperature during prolonged storage (c.f. days 7 and 10; tab. 7). severity of disease was always highest in the controls of all cultivars and difference among groups were not significant. nevertheless, the susceptibility of melons depended on the cultivar. ‘lillo’ and ‘donatello’ fruit better retained appearance than those of ‘celestial’ 126 l.p.l. nguyen, t. zsom, m.s. dam, l. baranyai, g. hitka tab. 5: statistical evaluation of hue angle (anova) interaction effect factors for hue angle main effect × temperature × cultivar time *** – *** treatment *** – * temperature – – * cultivar *** – – *** p<0.001, ** p<0.01, * p<0.05, n = 720 fig. 4: fv/fm of fruit of the four melon cultivars during shelf-life at 20 °c. data are shown as means ± sd fig. 5: hue angle value of four melon cultivars during shelf-life at 20 °c. data are shown as means ± sd tab. 6: statistical evaluation of disease severity (anova) interaction effect factors for disease severity main effect × temperature × cultivar time *** – – treatment ** – – temperature – – – cultivar *** – – *** p<0.001, ** p<0.01, * p<0.05, n = 720 response of melon cultivars to 1-mcp treatment temperature 127 and ‘centro’ after 10 d of shelf-life. at the end of the experiment, disease severity was high for all samples. it might be the result of initial infection from soil and lack of surface cleaning. microorganisms present on rind surface can develop rapidly during transport and storage (bastos et al., 2005), especially when temperature promotes microbial growth (yang et al., 2003). (blankenship and dole, 2003). application of gaseous 1-mcp at high concentration was also reported to induce desired effect, such as 1 ml l-1 for pear (gao et al., 2015). the effect of 1-mcp also depends on factors such as cultivar (wei et al., 2019), stage of maturity, concentration, temperature and treatment time (watkins, 2006). in case of apple, deell et al. (2002) reported a relationship between temperature and time. the effective exposure time of apple at 3 °c was 9 h, while the equivalent time required to reach the same effect at 23 °c was only 6 h. similarly, a 6 h 1-mcp treatment of apple at 20 °c had the same effectiveness as a 24 h treatment at 0 °c and 5 °c (dauny and joyce, 2002). however, watkins and nock (2005) did not find difference between 24 h 1-mcp treatment of warm and of cold fruit of four apple cultivars. in the presented study, 1-mcp treatments markedly reduced the ethylene and carbon dioxide production in fruit of all four melon cultivars. this observation is in agreement with a prior study on melons (alves et al., 2005). similar results were also reported for other muskmelon varieties, and ‘galia’ (ergun et al., 2007; gal et al., 2006) and ‘charentais’ melons (du chatenet et al., 2000). in addition, application of 1-mcp maintained the firmness of melon. the efficiency of 1-mcp in retaining the firmness was found for other fruits including tomatoes (wills and ku, 2002), pears (kubo et al., 2003; gao et al., 2015), apples (milinkovic et al., 2018), apricots and plums (dong et al., 2002). pulp softening, respiration and ethylene production of muskmelon ‘solar king’ was affected by 1-mcp treatment, but did not affect ph, total titratable acidity, total soluble solid content, total soluble sugar content, mass loss and external appearance (de lima et al., 2004). an earlier research also indicated that 1-mcp retards the decline in fluorescence parameters of apple during storage (mir et al., 2001), in agreement with the present study. our data show that 1-mcp delayed the ripening of four melon cultivars, but 1-mcp treatment did not inhibit the microbial development on the melon surface. however, another report found that the combination of 1-mcp and cold storage decreased rot of ‘orange flesh’ melons (alves et al., 2005). another study also indicated that 1-mcp could decrease the decay by inhibiting the fruit softening of ‘new queen’ melon. delaying the fruit softening could slow down the development of microorganisms in the fruit flesh, thus decrease the decay incidence of melon (zhang et al., 2019). it was concluded in that study that fruit softening is favorable for the reproduction and spreading of microorganisms. results of this study on the four melon cultivars clearly indicate that treatment temperature did not affect the efficacy of 1-mcp. presented results differ from a previous report (perzenlan et al., 2014), which concluded that application of 1-mcp at 20 °c was more effective in reducing softening of ‘galia’ melon than at 5 °c or 10 °c. this might be due to varying responsivity of the different melon species, varieties and cultivars to 1-mcp. a prior experiment with two cultivars in one season showed no effect of 1-mcp treatment temperature in the range of 5-20 °c (nguyen et al., 2018). the statistical analysis of the obtained results determined significant contribution of the melon type to the responses to 1-mcp exposure. this suggests that it is not possible to draw a simple general conclusion, and findings may be limited to certain groups of melon types. conclusions application of 625-650 nl l-1 gaseous 1-mcp at 5, 10 and 20 °c prolonged the shelf-life of fruit of four melon cultivars. the treatment with 1-mcp delayed the ripening of melons during 10 d of shelf-life. in addition, fruit of the cultivars ‘celestial’, ‘centro’, ‘donatello’ and ‘lillo’ showed similar patterns of quality changes as analyzed by variations in ethylene release, respiration, stiffness, rind color and chlorophyll fluorescence. treatment temperatures did not affect significantly any of these parameters and their changes during storage. tab. 7: disease severity of melon cultivars during shelf-life at 20 °c cultivars sample 4th day 7th day 10th day lillo 1-mcp5°c 1.0 1.33 a 2.27 a 1-mcp10°c 1.0 1.40 a 2.33 a 1-mcp20°c 1.0 1.53 a 2.47 a untreated 1.0 1.60 a 2.53 a donatello 1-mcp5°c 1.0 1.33 a 2.33 a 1-mcp10°c 1.0 1.47 a 2.33 a 1-mcp20°c 1.0 1.47 a 2.40 a untreated 1.0 1.53 a 2.47 a centro 1-mcp5°c 1.0 1.53 a 2.53 a 1-mcp10°c 1.0 1.60 a 2.60 a 1-mcp20°c 1.0 1.67 a 2.60 a untreated 1.0 1.90 a 2.70 a celestial 1-mcp5°c 1.0 1.53 a 2.53 a 1-mcp10°c 1.0 1.67 a 2.47 a 1-mcp20°c 1.0 1.67 a 2.60 a untreated 1.0 1.80 a 2.70 a subscripts indicate 1-mcp treatment temperature. means followed by the same letters in columns are not significantly different, p<0.05. discussion our results showed that 1-mcp reduced the respiration rate, ethy lene production and pulp softening. it was coincident with earlier report for ‘galia’ melon (de lima et al., 2004; gal et al., 2006). those authors found that fruit treated with 1-mcp for 12 h at 25 °c was firmer and lower in carbon dioxide production compared to control. however, mass loss, total titratable acidity, ph, soluble solids content, total soluble sugars content were not affected by 1-mcp. similar results were observed in our study. at the end of measurement, the mass loss of melon was about 6% (data not shown), and the soluble solid content around 7.5% (data not shown) for all samples. nevertheless, the effect of 1-mcp on mass loss, soluble solid content also depends on cultivar. for example, there was a difference in mass loss between treated and untreated fruit for ‘orange flesh’ and ‘charantais’ melon, but not for ‘galia’ melon (alves et al., 2005; de melo et al., 2008). similarly, 1-mcp treatment decreased the soluble solid content for ‘orange flesh’ melon, while 1-mcp had no effect on soluble solid content for ‘galia’ and ‘charantais’ melon (alves et al., 2005). in our work, there was no difference between three different treatment temperatures for all cultivars. studies of 1-mcp application on melon were usually carried out at ambient temperature (alves et al., 2005; du chatenet et al., 2000; ergun et al., 2007; gal et al., 2006; shi et al., 2014). earlier publications reported that 1-mcp treatment at cold temperature had less effect than at warm temperature. the reason was found to be the lower affinity of ethylene receptor to 1-mcp at cold temperature (blankenship and dole, 2003). 1-mcp delays the ripening by occupying ethylene receptors, so that ethylene is unable to elicit its action. the application time of 12-24 h is generally considered long enough to reach full response 128 l.p.l. nguyen, t. zsom, m.s. dam, l. baranyai, g. hitka this finding is in contrast with earlier reports obtained with ‘galia’ melons. this discrepancy and our results suggest that different melon varieties, types and cultivars may differ in their response to 1-mcp. acknowledgements the project is supported by the european union and co-financed by the european social fund (grant agreement no. efop-3.6.3 vekop-16-2017-00005). the project is supported by the euro pean structural and investment funds (grant agreement no. vekop 2.3.3-15-2017-00022). this research was supported by the ministry for 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w., jin, x., li, j., 2019: calcium chloride and 1-methylcyclopropene treatments delay postharvest and reduce decay of new queen melon. sci. rep. 9(1). doi: 10.1038/s41598-019-49820-8 zhao, j., xie, x., dai, w., zhang, l., wang, y., fang, c., 2018: effects of precooling time and 1-mcp treatment on ‘bartlett’ fruit quality during the cold storage. sci. hortic. 240, 387-396. doi: 10.1016/j.scienta.2018.06.049 orcid lien phuong le nguyen https://orcid.org/0000-0002-5975-9906 mai sao dam https://orcid.org/0000-0002-3170-0785 lászló baranyai https://orcid.org/0000-0001-6177-7364 géza hitka https://orcid.org/0000-0003-0942-8102 adress of the corresponding author: e-mail: baranyai.laszlo@etk.szie.hu © the author(s) 2020. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1007/bf00040512 http://dx.doi.org/10.1177/1082013214520786 http://dx.doi.org/10.1111/j.1750-3841.2008.00939.x http://dx.doi.org/10.1016/j.biotechadv.2006.01.002 http://dx.doi.org/10.21273/hortsci.40.7.2096 http://dx.doi.org/10.1016/j.biotechadv.2006.01.005 http://dx.doi.org/10.17660/actahortic.2016.1120.1 http://dx.doi.org/10.5073/jabfq.2019.092.019 http://dx.doi.org/10.1016/s0925-5214(01)00201-0 http://dx.doi.org/10.1016/s0925-5214(03)00104-2 http://dx.doi.org/10.1038/s41598-019-49820-8 http://dx.doi.org/10.1016/j.scienta.2018.06.049 art20_buchbesprechung.indd journal of applied botany and food quality 85, 126 (2012) buchbesprechung handbuch des arzneiund gewürzpfl anzenanbaus (band 2), grundlagen des arzneiund gewürzpfl anzenanbaus ii das von dem verein für arzneiund gewürzpfl anzen saluplanta e.v. (bernburg, deutschland) herausgegebene 5bändige werk „handbuch des arzneiund gewürzpfl anzenanbaus“ beschreibt umfassend alle aspekte dieses interessanten themenkomplexes und behandelt dabei insbesondere die grundlagen des arzneiund gewürzpfl anzenbaus, auftretende krankheiten und schädigungen der pfl anzen und liefert darüber hinaus zahlreiche anbauanleitungen. bei der erstellung des handbuchs haben insgesamt 110 autoren aus unterschiedlichen wissenschafts-disziplinen mitgearbeitet. nachdem im november 2009 der erste band des handbuchs erschienen ist, folgte bereits im august 2010 der zweite band, der nahtlos mit kapiteln zum integrierten und ökologischen anbau, zu nachernteprozessen, verarbeitung, qualitätssicherung, vermarktung und marketing an den ersten band anknüpft. an diesem band haben 56 führende fachleute der branche unter redaktioneller leitung von bernd hoppe mitgearbeitet. in dem ersten kapitel wird zunächst sehr detailliert auf die individuellen standortanforderungen sowie auf die bedeutung von fruchtfolge, bodenbearbeitung und düngung eingegangen. auch auf die bewertung der saatgutqualität bzw. die behandlung von saatund pfl anzgut wird hier speziell bezug genommen. schließlich werden auch möglichkeiten zur beregnung sowie die aspekte der unkrautbekämpfung und des pfl anzenschutzes ausführlich erörtert. ein weiteres kapitel widmet sich am beispiel ausgewählter arzneiund gewürzpfl anzen dem nachernteverhalten und den unterschiedlichen verarbeitungstechniken wie trocknung, wasserdampfdestillation, gefriertrocknung, frischpfl anzenextraktion, der herstellung von frischpfl anzenpresssäften sowie dem in der parfümerie eingesetzten enfl eurage-verfahren. es schließen sich zwei kapitel an, die auf unterschiedliche qualitätssicherungssysteme und die in diesem zusammenhang eingesetzten analytischen techniken näher eingehen. hierbei wird auch auf die mit qs-systemen in verbindung stehenden regelwerke, normen und richtlinien bezug genommen. sehr ausführlich wird die durchführung einer gefahrenanalyse entsprechend einem haccpkonzept beschrieben. in diesem zusammenhang werden auch zahlreiche beispiele für checklisten, mit denen die einzelnen prozesse hinsichtlich möglicher risiken überwacht werden können, vorgestellt. auch die analytischen methoden, die im rahmen der qualitätskontrolle zur anwendung kommen, werden ausführlich behandelt. das buch schließt mit einem kapitel, das auf den einkauf und die vermarktung von arzneiund gewürzpfl anzen fokussiert ist. hier wird der leser mit den in der branche üblichen beschaffungswegen (vertragsanbau, exklusivanbau, werksanbau und wildsammlungen) konfrontiert und erfährt dabei auch die vorund nachteile dieser unterschiedlichen strategien. das handbuch versteht sich als anleitung und nachschlagewerk und kann in diesem zusammenhang nicht nur praktikern im bereich des arzneiund gewürzpfl anzenanbaus, sondern ebenso studenten, naturwissenschaftlern in der forschung und in der industrie un-bedingt empfohlen werden. dr. h. schulz e-mail: hartwig.schulz@jki.bund.de bibliografi e handbuch des arzneiund gewürzpfl anzenanbaus, band 2: „grundlagen des arzneiund gewürzpfl anzenbaus teil ii“, herausgeber: saluplanta e.v. bernburg, verein für arzneiund gewürzpfl anzen (www.saluplanta.de), 1. aufl age; 768 seiten, 140 farbfotos, 269 grafi ken, 236 tabellen. erschienen august 2010. isbn 978-3-935971-55-3, preis: 79,€ zzgl. mwst und versandkosten. journal of applied botany and food quality 85, 127 (2012) buchbesprechung arzneibuch der chinesischen medizin erich a. stöger und fritz friedl das interesse an der chinesischen medizin ist in den westlichen ländern während der letzen jahre deutlich gewachsen und entsprechend wird dieser heilmethode im zunehmenden maße aufmerksamkeit entgegengebracht. das aus dem chinesischen übersetzte werk folgt diesem trend und schließt damit eine lücke, die bislang im deutschsprachigen raum bestanden hat. um den praxiswert vorhandener tcm-monographien weiter zu verbessern und den nutzern konkrete hilfen anzubieten, haben die autoren neben den bereits in der 1. ausgabe vorhandenen mikroskopischen zeichnungen der arzneidrogen bei 35 monographien zusätzlich auch noch farbfotos von dünnschichtchromatogrammen aufgenommen. dabei werden stets zwei unterschiedliche handelsübliche fertigplatten verwendet, um die variabilität der stationären phasen auf das trennergebnis zu demonstrieren. entsprechend den vorgaben des europäischen arzneibuchs wurde dabei auch darauf geachtet, für toxische lösemittel wie benzol und chloroform geeignete alternativen zu fi nden. als vorlage für die zweite, überarbeitete ausgabe der loseblattsammlung dient das amtliche arzneibuch der volksrepublik china in seinen ausgaben der jahre 2000, 2005 und 2010; darüber hinaus wurden die einzelnen monographien anhand von fachpublikationen weiter ergänzt und aktualisiert. die 330 arzneidrogen sind alphabetisch nach ihrer gattungsbezeichnung sortiert; die deutsche drogenbezeichnung ist dabei jeweils als untertitel genannt. an den drogentitel schließt sich eine kurze beschreibung an, die hinsichtlich des verwendeten pfl anzenteils, der erntezeit sowie der verarbeitung (z.b. reinigung, zerkleinerung, trocknung) informationen liefert. in den abschnitten „morphologie“ und „prüfung auf identität“ sind die individuellen makround mikroskopischen beschreibungen sowie chemische und physikalische testverfahren zur qualitativen bzw. quantitativen charakterisierung der drogen zu fi nden. darüber hinaus wird in einem abschnitt jeweils auf die wirkcharakteristik im rahmen der chinesischen medizintheorie eingegangen. in einem weiteren abschnitt werden darüber hinaus die teilweise erforderlichen vorbehandlungsverfahren der rohdrogen näher beschrieben. darüber hinaus werden bei einzelnen arzneipfl anzen warnhinweise hinsichtlich nebenund wechselwirkungen mit anderen pfl anzlichen drogen geliefert, die allerdings keinen anspruch auf vollständigkeit erheben. im vorspann wird eine kurze einführung in die chinesische medizin gegeben. hier kann sich der leser über den spezifi schen denkansatz, die grundzüge der chinesischen diagnostik, die krankheitslehre der tcm sowie über diverse rechtliche aspekte informieren. der anhang beinhaltet die einzelnen pharmazeutischen verarbeitungshinweise, allgemeine chemische und physikalische nachweismethoden sowie ein umfangreiches reagenzienverzeichnis. in dem gesamtregister der sammlung sind die chinesischen pfl anzennamen mit chinesischen schriftzeichen sowie das synonymaverzeichnis enthalten. derzeitig gibt es kein vergleichbares werk im deutschsprachigen raum. die monographie-sammlung sollte daher in keiner apotheke fehlen, die mit dem verkauf und der herstellung chinesischer arzneimittel befasst ist. dr. h. schulz e-mail: hartwig.schulz@jki.bund.de bibliografi e: erich a. stöger und fritz friedl, arzneibuch der chinesischen medizin – monographien des arzneibuches der volksrepublik china 2000, 2005 und 2010, deutscher apotheker verlag, stuttgart, aus dem chinesischen übersetzt, erweitert und kommentiert von erich a. stöger, mit makroskopischen und mikroskopischen zeichnungen der drogencharakteristika nach lou zhicen, zhao dawen, shen yuan sowie dünnschichtchromatogrammen von xie peishan, 2 ringordner, loseblattausgabe der 2. aufl age mit 13. aktualisierungslieferung 2012 (fortsetzungswerk), 1806 seiten, 200 s/w abbildungen, 125 farbige abbildungen, preis: 138,€, isbn: 978-3-7692-5641-3 journal of applied botany and food quality 85, 128 (2012) buchbesprechung praktische diätetik – grundlagen, ziele und umsetzung der ernährungstherapie elisabeth höfl er und petra sprengart das praxisorientierte lehrbuch soll diätassistentinnen während ihrer ausbildung die notwendigen kenntnisse und fähigkeiten zur diätetischen versorgung von patienten insbesondere in krankenhäusern und rehabilitationszentren vermitteln. nach einer kurzen generellen einführung in die gesunde ernährung von erwachsenen gehen die beiden autorinnen zunächst auf die zur prävention geeignete ernährung, differenziert nach alter und lebenssituation, näher ein. darüber hinaus wird auch kurz der kulturelle hintergrund christlicher, jüdischer und muslimischer speisegesetze beleuchtet. der hauptteil des buches widmet sich dann den unterschiedlichen kostformen in der ernährungstherapie (vollkost, leichte vollkost, vegetarische kostformen, ernährung bei demenzerkrankungen, spezielle gastroenterologische diäten, energiedefi nierte, proteindefi nierte und elektrolytdefi nierte diäten, ernährungsempfehlungen bei onkologischen und fettdefi nierten erkrankungen sowie lebensmittelallergien). hier fi nden sich lösungen für die meisten ernährungsprobleme unserer zeit: übergewicht/adipositas, stoffwechselerkrankungen, herz-kreislauf-erkrankungen sowie mangelernährung und deren folgen. auch zur behandlung gastroenterologischer, nephrologischer und onkologischer erkrankungen werden diätetische behandlungsmöglichkeiten vorgestellt. ein eigener abschnitt ist den allergischen lebensmittel-hypersensibilitäten gewidmet; hierbei wird auf die durchführung einer spezifi schen eliminationsdiät (bei verdacht auf bestimmte, allergen wirksame lebensmittel) bzw. einer oligoallergenen basisdiät (wenn die allergische symptomatik nicht einem bestimmten lebensmittel zugeordnet werden kann) näher bezug genommen. die einzelnen kostformen werden zunächst hinsichtlich ihrer grundlagen und ihres diätetischen prinzips näher beschrieben; anschließend werden anhand umfangreicher tabellen die einzelnen pfl anzlichen und tierischen lebensmittel sowie deren zubereitungsformen hinsichtlich ihres jeweiligen eignungswertes klassifi ziert. die inhaltlichen schwerpunkte wurden mit der arbeitsgemeinschaft leitender lehrkräfte an diätschulen abgestimmt und basieren auf leitlinien und anerkannten empfehlungen der fachgesellschaften. alle kapitel sind nach einer einheitlichen systematik aufgebaut und beinhalten begriffsdefi nitionen, wissenschaftliches grundlagenwissen, das jeweilige diätetische prinzip und dessen durchführung. auf diese weise erhält der leser schnell einen überblick über die jeweils beschriebene therapieform. die diätetischen möglichkeiten sind insgesamt anschaulich präsentiert und erleichtern anhand vieler kochund küchentechnischer ratschläge die umsetzung im praktischen alltag. das buch liefert insgesamt eine wertvolle unterstützung bei der anwendung wissenschaftlich anerkannter erkenntnisse in die praxis. zu jedem thematischen abschnitt fi nden sich übungsaufgaben, die dazu dienen sollen, das erworbene wissen zu festigen. außerdem wird für weiterführende studien zu jedem buchkapitel spezifi sche fachliteratur angeboten. bei einer neuaufl age würde man sich allerdings wünschen, dass die grundlagen der lebensmittelkunde in einem eigenen, vorangestellten abschnitt zusammengefasst werden und nicht in unterschiedlichen kapiteln angeführt werden. für das selbststudium wäre es bestimmt auch nützlich, die ergebnisse der zahlreichen übungsaufgaben am ende des buches nachschlagen zu können. das buch liefert nicht nur eine wertvolle hilfe bei der ausbildung von diätassistenten, sondern kann auch lesern anderer, angrenzenden berufsgruppen wie z.b. ökotrophologen als ein willkommenes nachschlagewerk für die alltägliche praktische arbeit dringend empfohlen werden. dr. h. schulz e-mail: hartwig.schulz@jki.bund.de bibliografi e: elisabeth höfl er und petra sprengart, praktische diätetik – grundlagen, ziele und umsetzung der ernährungstherapie, wissenschaftliche verlagsgesellschaft mbh, stuttgart, 1. aufl age 2012, 763 seiten mit 33 farbigen abbildungen, 205 tabellen sowie 141 übungsaufgaben, preis: 39,€, isbn: 978-3-8047-2943-8 journal of applied botany and food quality 93, 321 329 (2020), doi:10.5073/jabfq.2020.093.039 1laboratory of plant physiology and biochemistry, department of biological science, higher teacher training college, university of yaoundé i, cameroon 2applied biotechnology engineering laboratory, department of agriculture and agropastoral, higher technical teacher training college, university of yaoundé i, cameroon 3 faculty of agronomy and agricultural science, university of dschang, cameroon 4 university of hamburg, department of chemistry, hamburg school of food science, division of food microbiology and biotechnology, institute of plant sciences and microbiology, hamburg, germany 5crop plant museum gorleben, cpmg, germany assessment of the profile of free amino acids and reducing sugars of cacao beans from local cameroonian trinitario (snk varieties) and forastero (tiko varieties) using fermentation-like incubation nicolas niemenak1*, jos victor evina eyamo1,2, stéphanie astride mouafi djabou1,3, audrey germaine ngouambe tchouatcheu1, clemens bernhardt4, reinhard lieberei4,5, bernward bisping4 (submitted: december 7, 2020; accepted: january 3, 2021) * corresponding author summary this study investigated the profile of cacao beans from the local cameroonian trinitario (snk) and forastero (tiko) in terms of aroma precursors (amino acids and reducing sugars) through fermentation-like incubations. treatments consisted of incubating beans in acetic acid, 100 mmol/l for two days followed by 200 mmol/l of acetic acid for three days (treatment t1) and in 100 mmol/l of lactic acid for two days followed by 200 mmol/l of acetic acid for three days (treatment t2). both treatments resulted in an increase of free amino acids content by 1.5 2.5 times in snk and tiko varieties. the ratio of the hydrophobic amino acids over the rest of amino acids showed the preponderance of t1 on the hydrophobic amino acids released in tiko while in the snk, some varieties displayed the highest ratio in t2. glucose and fructose content in tiko and snk beans increased 2 to 3 times during incubation. galactose and raffinose were found in unfermented beans. after incubation, raffinose was missing while at the same time a raise of galactose content could be seen. these results highlighted that acidification remains the factor inducing the releasing of free hydrophobic amino acids, the genotype being less involved. key words: theobroma cacao; snk and tiko varieties; amino acids; sugars; cacao beans. introduction the unique taste of chocolate attributed to fermented cacao beans after roasting is due to aroma compounds generated during maillard reaction. these non-enzymatic reactions involve oligopeptides, free amino acids and reducing sugars which constitute the main ingredients of aroma compounds (voigt et al., 2016; adedeye et al., 2010). therefore, free amino acids and reducing sugars are usually called cacao aroma precursors or flavor precursors (biehl et al., 1985; amin et al., 1997; santander et al., 2019). they are released from storage proteins and sucrose respectively during the fermentation process which remains the key step to obtain high-quality cacao beans. cacao fermentation consists of two main steps: external and internal fermentation. external fermentation consists of microbial degradation of mucilaginous pulp rich in sugar. a succession of yeasts, lactic acid and acetic acid bacteria on mucilaginous pulp results in ethanol, lactic and acetic acids along with pulp depectinization and liquefaction. firstly, the alcoholic fermentation takes place and slowly increases the ph (initially below 4 due to the presence of citric acid) and quickly creates ideal conditions for the growth of lactic acid bacteria (lab). the labs consume glucose for lactic acid biosynthesis and also enhance the temperature to a range of 35 40 °c. after two or three days, the manual fermentation medium stirring and the disappearance of mucilage create aeration allowing acetic acid bacteria (aab) to grow. through aab activity, the ethanol previously released is oxidized (or converted) into acetic acid. this high exothermic reaction increases both the temperature up to 45 °c or more and the ph to a range of 5 5.5. simultaneous to external fermentation, the second phase of fermentation is also running. the lactic and acetic acids released, penetrate the cotyledons at a first stage through hilum, later, the testa also is getting permeable (rohsius, 2007). the migration of these two organic acids gradually lowers the internal ph of the cacao beans from 6.5 to 4.5 approximately, causes the death of the embryo, disintegrates the cell compartments, releases endogenous enzymes and finally induces degradation of phenolic compounds, sucrose into reducing sugars and storage proteins into oligopeptides and amino acids (hansen et al., 1988; thompson et al., 2001; schwan and wheals, 2004; jespersen et al., 2005; camu et al., 2007; nielsen, 2007; afoakwa et al., 2013). it is well established that among storage proteins found in cacao beans, only from globulin namely vicilin-class (7s) and from albumin, oligopeptides and free amino acids are generated through the enzyma tic process (voigt et al., 1994; voigt and biehl, 1995; caligiani et al., 2016; marseglia et al., 2014; kumari et al., 2016). this biochemical reaction involves two specific endogenous enzymes, aspartic endoprotease and carboxypeptidase, and requires favourable and specific conditions for each one. though more research remains to be done for a deep understanding of the mechanism of albumin degradation, nevertheless biehl and ziegleder (2003) and d’souza et al. (2008) argued that albumin is essentially a source of oligopeptides. however, some authors proved that in criollo genotypes albumin is the source of free amino acids (marseglia et al., 2014). regarding globulin, it has been demonstrated that aspartic endoprotease and carboxypeptidase induce proteolysis of vicilin-class globulin (7s) of all cacao beans varieties tested (voigt et al., 1994; voigt and biehl, 1995). finding from voigt et al. (1994) highlighted the mechanism and revealed the necessary cooperation between both enzymes for the release of cacao-specific aroma precursors. firstly, through aspartic endoprotease activity, few amino acids and hydrophobic peptides are generated. thanks to carboxypeptidase, hydrophobic peptides are degraded into hydrophobic amino acids and hydrophilic 322 n. niemenak, j.v.e. eyamo, s.a.m. djabou, a.g.n. tchouatcheu, c. bernhardt, r. lieberei, b. bisping peptides. the mixture of these two compounds displayed the typical flavor of cacao during roasting in the presence of reducing sugars (spencer and hoge, 1992; voigt et al., 1993; voigt et al., 1994a; kratzer et al., 2008). reducing sugars are also an essential ingre dient of cacao aroma (cerbulis et al., 1955; barisic et al., 2019). the main reducing sugars found in fermented cacao beans are glucose and fructose. they are issued from the enzymatic hydrolysis of sucrose, which involves invertase (caligiani et al., 2016; mayorgagross et al., 2016). considering the specific conditions required by each endogenous enzyme involved in the production of amino acids and reducing sugars for their optimal activity, it seems obvious that obtaining a specific cacao flavour lay on the physico-chemical parameters observed during the fermentation process and on the genotype. these two aspects determine not only the variability of the amino acids and reducing sugars produced but also their amount. three main cacao genetic groups (forastero, criollo and trinitario) have been defined to classified cacao based on morphological traits and geographical origins. criollo was first domesticated in central america more than 2000 years ago, while lower amazon forastero variety (amelonado type) was domesticated in brazil. forastero group also includes many other populations from all the amazonian (upper amazon forastero, upa) and orinoco regions and presents a high diversity, as revealed by many genetic studies (loor solorzano et al., 2012). trinitario group is hybrid between criollo and forastero. in cameroon, the 5th largest cacao producer (icco, 2019), cacao varieties from the three major group are cultivated. however, trinitario variety is largely constituted by local genotypes developed since the 1950s commonly called snk: “selection of nkoemvone” (efombagn et al., 2006; nyasse et al., 2007; efombagn et al., 2013). this variety was widely spread in cameroonian cacao farms and today with german cacao constitutes the main varieties (efombagn et al., 2006). the term “german cacao” was given by farmer for traditional cacao, known for their resistance to black pod disease and because cacao was partly introduced in cameroon during the german colonization period. the german cacao presents a high level of admixture of diverse genes (stoll et al., 2017). genes of lower amazon cacao (54%) are predominant followed by upper amazon cacao (33%) and criollo (7%). only a low content of trinitario is found (efombagn et al., 2008). contrary to snk varieties, forastero varieties named tiko’s are less known by farmers. there are no data concerning their behaviour during fermentation, biochemical profile and agronomic traits. yet, these varieties are present in cacao farms. to enhance cameroonian cacao, the characterization of these two subgroups in terms of their aroma precursor release is required. ngouambe et al. (2019) showed that the purple and brown beans in cameroon raw cacao contain a high level of amino acids. interestingly, the group of hydrophobic and acidic amino acids accounted for 61.70% and 30.17% in brown cacao beans and 60.70% and 37.51% in the purple. as natural fermentation is hard to control, lab-scale fermentation also known as fermentation-like incubation have been optimized to appreciate the changes occurring inside the cacao beans through degradation of different storage compounds induced by the penetration of lactic acid and acetic acid (rohsius et al., 2006; kadow et al., 2015; eyamo et al., 2016; john et al., 2016). in view of the interesting results obtained through this technique, it could also be used to collect information on the profile of cacao beans subjected to optimal fermentation conditions. the present work aimed to assess the profile of free amino acids and reducing sugars of cacao beans from the local varieties (snk and tiko) of cameroon using fermentation-like incubation. the impact of the successive or simultaneous presence of lactic acid and acetic acid at the start of the incubation process on the cacao beans aroma precursors released from the local cameroonian varieties is highlighted. materials and methods materials cacao beans collection the fruits analysed were harvested in september october 2016 from field grown plants of each genotype conserved in cacao germplasm collection at nkoemvone (snk collection) research station of the institute for agricultural research for development (irad) in the south region of cameroon. the collection was established in the 1950s, after random selection by farmers and the breeders in the field, based on yield and assigned accession numbers prior to their transfer and their vegetative propagation in the nurseries on-station (efombagn et al., 2009ab). pods are from seven local trinitario varieties: snk10, snk15, snk16, snk48, snk64, snk377, snk450; two f1 hybrid local varieties: snk620 and snk624 resulting from introduced trinitario variety crossed with upper amazonian variety ics84 × upa 337 (nyasse et al., 2006 ; efombagn et al., 2013); two upper amazo nian varieties which are the introduced forastero regularly found in cameroonian cacao farm: imc67 and t60/877 and two local forastero: tiko31 and tiko32. the name ‘‘tiko’’ is referred to the locality (in south west region of cameroon) were these varie ties were selected and recognised for their tolerance to phytophthora megakarya and their productivity. for each variety, about twenty full ripe (based on their characteristic color change) undamaged pods were harvested, transported to the laboratory and fermentation-like incubation was directly applied. germinated seeds were not encountered in the pods. reagents and standards unless otherwise specified, all the chemicals and solvents used were analytical grade purchased from merck (darmstadt, germany). water was purified in a milli-q water purification system (millipore, bedford, ma, usa). methods fermentation-like incubation as presented in tab. 1, two different treatments were applied to thirteen cacao varieties, according to bahmann (2013) and eyamo et al. (2016) with slight modifications. six to seven intact fruits were harvested at the institute for agriculture research for development at nkoemvone station (south cameroon) and transported to the laboratory. three independent repetitions were made (not at the same day) for each treatment and five (05) fruits were used for each fermentation-like incubation repetition. then, at the end of the experimentation, about 20 intact fruits were managed for each variety. for fermentation-like incubation, cacao pods were washed twice for 15 minutes in sodium hypochlorite solution (3 ml/l; 0.3%) and then rinsed three times with sterilized water. after natural drying under a laminar flow hood, pods were sprayed with ethanol, flamed, and opened. each cacao pod contained ca. 30-50 beans depending on the tab. 1: experimental design of fermentation-like incubations. days of incubation treatments day 1 day 2 day 3 day 4 day 5 30 ºc 40 ºc 45 ºc 50 ºc 50 ºc t1 acetic acid 100 mm; ph 4 acetic acid 200 mm; ph 5 3 repetitions of 5 fruits 3 repetitions of 5 fruits (25 seeds), n=3 (25 seeds), n=3 t2 lactic acid 100 mm; ph 4 acetic acid 200 mm; ph 5 3 repetitions of 5 fruits 3 repetitions of 5 fruits (25 seeds), n=3 (25 seeds), n=3 amino acids and reducing sugars in cacao beans 323 variety. twenty-five beans randomly selected from the five opened cacao pods were put into a sterilized glass bottle containing 150 ml incubation medium (100 mmol/l acetic acid or 100 mmol/l lactic acid, and adjusted to ph 4 with 1 mmol/l naoh; (tab. 1). after two days of incubation, the beans were transferred under the hood to a second sterilized glass bottle containing the same volume of 200 mmol/l acetic acid ph 5 and incubated for three more days. during this second phase, glass bottles were constantly shaken under laminar flow hood to increase oxygen in the media. the temperature was controlled throughout the experiments by incubation the bottles in a 30-50 °c water bath (tab. 1). after incubation, the cacao beans were sun-dried between 8 a.m. and 5 p.m. for 7 days with stirr ing every three hours (final water content: 6.9%). the dried beans were stored in black plastic bags and transported to the university of hamburg for biochemical analysis. biochemical analysis seventy five dried beans from the three incubations, resulting from each treatment and variety, were mixed and cut lengthwise with a cacao guillotine (type magra, swiss company teserba). the shells and radicles were removed. cotyledons were coarsely crushed with a blender and the particles were stored at 20 °c. for defatting, two grams of crushed cotyledon were milled with 10 ml n-hexane in a ball mill (typ mm200 from retsch, germany) by shaking for 10 minutes at a frequency of 25/s. the homogenate was rinsed out of the ball mill with about 75 ml of petroleum ether (bp 40-60 °c), then filtered and dried in a vacuum drying oven. defatted fine cotyledon powder was stored in -20 °c for free amino acids and sugar analysis. free amino acids analysis free amino acid contents were analysed according to rohsius et al. (2006) with slight modifications. 0.5 g of defatted ground cacao powder was stirred at < 4 °c for 1 hour with 1.4 g of polyvinyl-polypyrrolidone (pvpp) and 50 ml of distilled (or deionized) water. the ph was adjusted to 2.5 with 50% of aqueous solution of trifluoroacetic acid (1/1; v/v). the solution was centrifuged at 2,800 g (centrifuge heraeus thermoscientific megafuge 11r, hanau, germany) for 10 minutes at 5000 rpm. the clear supernatant solution was filtered through a 0.45 μm filter (multoclear, cs-chromatography). 30 μl of each sample was lyophilized (1h; -20 °c; 0.05 mbar, christ alpha 1-2 ldplus, martin christ gefriertrocknungsanlagen gmbh, osterode am harz, germany) directly in the vial and stored at -20 °c until analysis. free amino acids extraction was repeated twice (technical repetition for extraction). free amino acids were separated with reverse-phase hplc apparatus (hplc pump 64 from knauer; degaser, degassex dg-4400 from phenomenex) after they had been converted to o-phthalaldehyde (opa) derivatives. the chromatographic system consists of precolumn (licchrocart 250-4 (merck, darmstadt, germany)); separating column (lichrospher 100 rp-18; 5 μm); an autosampler (merckhitachi as-4000) and a fluorescence photometer detector (hitachi f-1050). eluents were: a; containing 1.6 l sodium acetate solution/ glacial acetic acid (50 mmol/l; ph 6.2), 50 ml meoh (lichrosolv r, gradient grade), 20 ml tetra hydrofuran (lichrosolv r; gradient grade) and b; containing 200 ml sodium acetate solution/glacial acetic acid (50 mmol/l; ph 6.2), 800 ml meoh (lichrosolv r, gradient grade). the column was equilibrated with eluent a and elution gradient was (a+b=100%; v/v): (1) 2 min 100-95% a, (2) 10 min, 95-85% a, (3) 8 min, 85-60% a, (4) 5 min, 60-50% a, (5) 15 min, 50-0% a, (6) 10 min, constant 0% a, (7) 5 min, 0-100% a, (8) 20 min, constant 100% a. the conditions of elution were as follows: temperature 30 °c; flow rate. 1.3 ml/min. for the measurement by hplc, 800 μl of borate buffer (200 mmol/l of boric acid; adjusted to ph 9.5 with concentrated koh, boiled for 5 min) was added to each lyophilized sample. before injection of 400 μl of opa-reagent was added to convert the amino acids into o-phthalaldehyde derivatives. 20 μl of this mixture was finally injected into the column. after injection, the derivatization was stopped 2 min later by passing the eluent through the column. for each amino acid extraction, two injections were done (technical repetition for hplc). the opa reagent was prepared 24 hours before being used. the preparation of the opa-reagent consisted of a mixture of 100 mg of ophtaldialdehyde (merck, no. 11452) previously dissolved in 2.5 ml of meoh (lichrosolv r, gradient grade) with 22.4 ml of borate buffer (ph 9.5) and 100 μl of 2-mercaptoethanol (merck, n° 15433). the amino acids of each sample were identified based on the retention time of commercial standards derivatized amino acids while, they were quantitated via a peak surface of chromatogram using the calibration curve from standard mixtures containing 1-10 pmol/μl of each amino acid. the standard deviation of reference substances was equal to ± 2.0% excepted for glutamic acid, glutamine, alanine, tryptophan (± 2.9 3.8%); arginine, asparagine, threonine and serine (± 5.7 7.3%); glycine (± 9.2%) and lysine (± 9.6%). reducing sugar analysis sugars were extracted and quantitated in duplicate (two technical replicates) according to rohsius (2007) with some modifications. 100 mg of defatted powder was mixed with 1 ml of ultrapure water (type i, elga purelab, high wycombe, united kingdom) for the extraction of sugars. the solution was homogenized on a laboratory vortex for 20 s and incubated on a thermomixer comfort (eppen dorf, hamburg, germany) for 1 hour at 1300 rpm and 80 °c followed by centrifugation for 10 minutes at 16060 g (biofuge pico, heraeus, hanau, germany). 300 μl of the supernatant was collected and diluted with 900 μl of ultrapure water. the solution was filtered through a 0.2 μm syringe membrane filter (pes perfect flow, wicom, heppenheim, germany) and subjected to hplc analysis. the sugars were separated by ligand exchange chromatography in an isocratic mode. 10 μl of each sample was injected onto the column rezex rcm-monosaccharide ca2+ column with 8% crosslinking (phenomenex, torrance, usa) established at 85 °c using a flow rate of 0.6 ml/min of ultrapure water. the chromatographic system consisted of as-2000a autosampler; l-6200 smart pump; l-7350d column oven; l-7490 refractive index detector (all merck hitachi, darmstadt, germany) and erc 3512 degasser (erma, tokyo). d-7000 hplc-system-management hsm version 4.1 software was used to record the data. the identification of each sugar was based on retention times of commercial standards used. the retention times of the various sugars were determined by recording the refractive index (ri). sugars were quantitated with the peak area of each sugar in samples and calibration curve of standard solutions. extract injection was repeated twice (technical replicate for hplc). statistical analysis all data were analysed and graphics drawn using r-studio version 0.96.122. statistical significance was assessed by analysis of variance (anova). the least significant difference (lsd) was used to separate and compare the means and significance was accepted at 5% level (p < 0.05). results and discussion amino acid contents in unfermented beans (treatment 0; t0), the total contents of free amino acids varied among varieties (fig. 1). they ranged between 4684.98 mg/kg fat free dry material (ffdm) (snk 10) and 7234.08 324 n. niemenak, j.v.e. eyamo, s.a.m. djabou, a.g.n. tchouatcheu, c. bernhardt, r. lieberei, b. bisping mg/kg ffdm (tiko31). at this stage, it was interesting to note that no significant difference was observed between snk and tiko as well as imc67 and t60/877 varieties (fig. 1). it is well known that unfermented cacao beans contain free amino acids (kirchhoff et al., 1989), their content varies between 2000 to 4000 mg/kg ffdm (rohsius et al., 2006). however, our results showed that the quantities found in all the varieties used were relatively high. nevertheless, it cannot be used to determine their potential for future operations because it is well established that unfermented cacao beans do not present any aromatic compound during roasting and do not generate any cacao flavor (lopez, 1986; puziah et al., 1998; hurst et al., 2011). in addition, the percentage of the hydrophobic amino acid group was relativity lower than the acidic group (fig. 2). according to kirchoff’s classification, the average ratio of hydrophobic/acidic/ other amino acids is 31% / 59% / 10%. the ratio obtained showed the predominance of free acidic amino acids group unlike those reported by puziah et al. (1998) where the third group (other amino acids) exhibited the highest content (30% / 18% / 52%); or those reported by rohan (1964) in malaysian unfermented cacao beans where the hydrophobic are predominant (41% / 26% / 33%). our results showed that tiko varieties displayed highest acidic amino acids percentage 62% against 58% and 60% in snk and imc67 + t60/877 respectively (fig. 2). in some particular varieties; imc67, snk10, snk16, snk40, snk48 and tiko31, the percentage of the acidic amino acids was 2 times higher than hydrophobic amino acids (fig. 2). this difference could be attributed to the genetic background of each variety since analysis was done in unfermented beans. glutamic acid was the most represented amino acid. its content represents at least 60% of the total content of acidic amino acids in all the varieties. for hydrophobic amino acids, alanine was the most abundant amino acid. in snk450 for example, its content represents about 50% of the total hydrophobic group (1205.41 mg/kg ffdm over 2495.19 mg/ kg ffdm) (fig. 2). after five days of incubation in acetic acid only (treatment 1; t1), the content of free amino acids considerably increased compared to the contents recorded in unfermented beans (t0). the total amino acid concentration doubled in all the forastero and tiko varieties; by 2.84; 2.94; 2.26; 2.78 for imc67, t60/887, tiko31 and tiko32 respectively (fig. 1). according to the total concentration, the snk varieties can be divided into two groups: the varieties which doubled their total concentration namely snk15 (10324.30 versus 4438.92 mg/kg ffdm), snk48 (13080.60 versus 6502.49 mg/kg ffdm), snk624 (10776.26 versus 5198.05 mg/kg ffdm) and snk64 (15122.72 versus 5415.46 mg/kg ffdm); and those which registered a simple increase namely snk10 (+ 5%), snk16 (+ 30%), snk377 (+ 48%) and snk450 (+ 8%) (fig. 2). the abundance of free amino acids observed is mainly due to the content of the hydrophobic amino acids which considerably increased during incubation (fig. 2). the average ratio of hydrophobic / acidic / other was 43% / 21% / 36%. in addition, the varieties showing an exponential increase also displayed a three to four times higher content of hydrophobic amino acids. their concentrations in leu and phe particularly increased in all the treatments compared to those of unfermented beans (t0). leu was almost 10-fold higher in snk10 (1338.15 against 137.48 mg/kg ffdm), snk48 (1778 against 164.92 mg/kg ffdm), snk64 (2087.51 against 217.01 mg/kg ffdm); snk624 (1382.02 against 125.86 mg/kg ffdm) and snk16 (1068.03 against 182.86 mg/kg ffdm). phe was 4 8-fold higher in snk15 (999.52 versus 194.83 mg/kg ffdm); snk450 (953.07 versus 165.89 mg/kg ffdm); snk48 (1335.51 versus 323.25 18 a a a b ab a a a a ab a a a b a b b b a c a b c c b c a b a a a a bc a a b b a b 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000 t ot al fr ee a m in o ac id s (m g/ kg ff dm ) varieties t0 t1 t2 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000 t0 t1 t2 t0 t1 t2 t0 t1 t2 t0 t1 t2 t0 t1 t2 t0 t1 t2 t0 t1 t2 t0 t1 t2 imc67 snk10 snk15 snk16 snk377 snk450 snk48 snk620 f re e am in o ac id s (m g/ kg ff dm ) treatments and varieties other amino acids (mg/kg ffdm) acidic amino acids (mg/kg ffdm) hydrophobic amino acids (mg/kg ffdm) 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000 t0 t1 t2 t0 t1 t2 t0 t1 t2 t0 t1 t2 t0 t1 t2 snk624 snk64 t60/887 tiko31 tiko32 f re e am in o ac id s (m g/ kg ff dm ) treaments and varieties fig. 2: distribution of hydrophobic amino acids, acidic amino acids and other amino acids in unfermented (t0) and incubated (t1 and t2) cacao beans from different cacao varieties. t1 treatment consisted of sterile incubation of 25 cacao beans of five individual fruits in 100 mmol/l acetic acid ph 4 for two days following their transfer in 200 mmol/l acetic acid ph 5 for three days and subsequent sun drying. t2 treatment consisted of sterile incubation of 25 cacao beans of five individual fruits in 100 mmol/l lactic acid ph 4 for two days following their transfer in 200 mmol/l acetic acid ph 5 for three days and subsequent sun drying. values are means of n = 3 (three biological replicates based on two technical replicates of extraction). means with different letters were significantly different by tukey (p < 0.05). γ-amino butyric acid (gaba) was detected in all cacao samples but was excluded in the analysis. fig. 1: total free amino acids content in unfermented (t0) and incubated (t1 and t2) cacao beans from different cacao varieties. t1 treatment consisted of sterile incubation of 25 cacao beans of five individual fruits in 100 mmol/l acetic acid ph 4 for two days following their transfer in 200 mmol/l acetic acid ph 5 for three days and subsequent sun drying. t2 treatment consisted of sterile incubation of 25 cacao beans of five individual fruits in 100 mmol/l lactic acid ph 4 for two days following their transfer in 200 mmol/l acetic acid ph 5 for three days and subsequent sun drying. values are means of n = 3 (three biological replicates based on two technical replicates of extraction). means with different letters were significantly different by tukey (p < 0.05). γ-amino butyric acid (gaba) was detected in all cacao samples but was excluded in the analysis. amino acids and reducing sugars in cacao beans 325 mg/kg ffdm); snk624 (1116.31 versus 255.01 mg/kg ffdm) and snk64 (1691.42 versus 208.22 mg/kg ffdm). the same trends was observed between the tiko varieties where the hydrophobic amino acids were three and four times more abundant; 7267.80 in t1 versus 1651.21 mg/kg ffdm in t0 and 6251.20 in t1 versus 1585.24 mg/kg ffdm in t0 for tiko31 and tiko32 respectively. leu and phe were also particularly abundant; 17 and 13 times for leu; 7 and 6 times for phe in tiko31 and tiko32 respectively (supplement 2). as observed in the hydrophobic amino acids, incubation in acetic acid (t1) resulted in an exponential increase in the total content of the third group of amino acids (named other amino acids). in snk15 and imc67 particularly, this increase was 18-fold greater than (t0); 5979.29 mg/kg ffdm versus 318.73 mg/kg ffdm and 3941.47 versus 215.69 mg/kg ffdm respectively. tiko32 and tiko31 showed a similar evolution with an increase of 13 and 5-fold respectively (supplement 1 and 2). interestingly, the relative abundance of other amino acids increased compared to t0: 38% versus 4% (snk15); 35% versus 7% (tiko32); and 33% versus 7% (snk16); 32% versus 7% (snk620). although this quantitative leap was induced by all the amino acids belonging to this group, trp, arg and lys amino acids have a significant contribution. the latter represent approximately 63%, 79%, 75% and 78% of the total amount recorded in this third group in snk15, snk16, snk620 and tiko32 respectively. in addition, it should be noted that trp, thr and lys which were not detected in unfermented cacao beans (t0) in imc67, snk10, snk15, snk16, snk450, snk620, snk64 and tiko32 were present after incubation (supplement 2). after beans incubation in lactic acid for two days followed by three days in acetic acid (treatment 2; t2), half of the varieties showed a lower amount of total free amino acids compared to unfermented cacao (fig. 1). these latter varieties belong exclusively to snk subgroup (snk15, snk16, snk377, snk450, snk620, and snk624). for the rest of the varieties, the increase recorded ranged between 1.08-fold (tiko 31; 7883.85 versus 7234.08 mg/kg ffdm) – 2.16fold (snk10; 4684.98 versus 10129.96 mg/kg ffdm) higher than t0. the average ratio hydrophobic/ acidic/ other was 42% / 28% / 30% (fig. 2). the concentration of hydrophobic amino acids increased in all varieties excepted in some snk varieties: snk16 (-12.55%), snk377 (-13%) and snk624 (-10%) compared to t0. this reduction is mostly due to a lower content of leu and phe in these varieties. the amount of the other free amino acid group increased during incubation compared to t0; +20% (30% in t2 versus 10% in t0) and slightly decreased compared to t1 -6% (30% in t2 versus 36% in t1). as in t1, the content of all the amino acids belonging to this third group increased. the appearance of lys, trp and thr mostly contributed to this increase. arg also contributed significantly, its concentration was approximately 11 times higher in snk10 in treatment 2 after incubation (1364.96 versus 124.23 mg/kg ffdm) (supplement 3). compared to t0, the percentage of the acidic amino acid decreased by half (28% in t2 versus 59% in t0). however, this residual rate remains relatively high compared to t1 (28% versus 21%). it is important to note that the respective contents of all the amino acids in this group dropped during this incubation. moreover, as found in unfermented cacao beans, glutamine does not appear in imc67, snk15, snk16, snk377, snk620 and snk624 varieties (supplement 3). the changes in relative concentration of free amino acids were observed in both incubations. the free amino acids found originated from those initially present in unfermented cacao beans and mostly from proteolysis during incubation. thus, the increase observed in incubated beans compared to unfermented beans is exclusively due to the penetration of the acids. biehl and passern (1982) demonstrated that only the acidification (with acetic acid) can promote proteolysis in cotyledons of cacao beans. moreover, more studies have well demonstrated that the acidification of the cotyledons creates adequate conditions for the endoprotease activities of cacao beans. the amount of free amino acids depends on the ph within the cotyledons and therefore on the flow of acid entering (voigt et al., 2018). thus, the variations in total amounts of free amino acids observed between the two treatments in the same variety may be entirely due to the type and the flow of the organic acid in the cacao beans during incubation. we can assume that the two types of chronology of the acids used (treatment 1 and treatment 2) do not allow the same flow and consequently the acidification results in each case does not lead to the same pattern of proteolysis. andersson et al. (2006) investigated the transport characteristics of the seed coat of theobroma cacao and found that certain structures strongly influence the course of transport processes in the mature seed. these authors suggested that flavour precursor development in seeds is dependent to transport kinetics of water and solutes into the seeds during fermentation process. our results showed that, incubation with acetic acid from the two first days (t1) would promote better proteolysis with a net increase of +67% compared to the average content of free amino acids released in t2. however, only the hydrophobic ratio on the rest of the amino acids is decisive. the total amounts of amino acids recorded in incubated beans were close to those reported by puziah et al. (1998) through natural fermentation and kirchhoff et al. (1989) using fermentation-like incubation. these similarities highlight the fact that the experimental design well-mimicked natural fermentation and remains a useful tool of deep-understanding for the main role of lactic and/or acetic acid on biochemical reactions occurring inside the beans during fermentation process. the ratio of hydrophobic on the rest of the amino acids (acidic and other amino acids) recorded ranged between 0.29 (tiko31; treatment 0) and 0.96 (snk15; treatment 2). excepted snk337, and t60/887 with 0.66 and 0.54 respectively, all the unfermented cacao beans (treatment 0) exhibited a ratio below 0.5. for t1 and t2, the ratio ranged between 0.5 1. however, the varieties used do not show a similar hierarchy between ratio in t1 (rt1) and t2 (rt2). excepted for snk624, the rest of snk varieties showed higher ratio value in t2 than t1. on the other hand, tiko, imc67 and t60/877 varieties recorded highest value in t1 (fig. 3). the difference observed between rt0 and rt1 and between rt0 and rt2 inside the varieties clearly revealed the impact of the fermentation process on hydrophobic free amino acids released and the acidic amino acids decreasing. moreover, it highlighted the crucial role of acidification on aroma precursor’s behavior during incubation or fermentation. the accumulation of hydrophobic free amino acids is explained by the cleavage characteristics of two proteases of cacao beans. the aspartic endoprotease (e.c. 3.4.23) attacks the storage proteins pre ferentially at sites of hydrophobic amino acids and the carboxypeptidase (e.c. 3.4.16) releases single hydrophobic amino acids (biehl et al., 1993; voigt et al., 1994). as a result of the different ph and temperature optima of these enzymes, proteolysis primarily depends on the fermentation conditions: duration and intensity of acidification, temperature and aeration (ziegleder and biehl, 1988). thus, the total amount of free amino acids and oligopeptides liberated during hydrolysis varies considerably (de witt, 1957; biehl and passern, 1982; biehl et al., 1985; rohsius et al., 2006). the fact that rt1 is relatively lower than rt2 in some snk varieties is mostly due to the low rate of acidic amino acids disappearance observed in t1. the data collected revealed that t1 released more hydrophobic amino acids but few acidic amino acids are lost. the hydrophobic amino acids are the only free amino acids that contribute to chocolate flavours during roasting (mulono et al., 2016). thus, the ratio expressed may indicate the useful free amino acids fraction of marketed cacao samples. according to the latter results, it is obvious that for snk and tiko varieties, the early production of acetic acid during fermentation process is necessary for a better yielding of amino acids 326 n. niemenak, j.v.e. eyamo, s.a.m. djabou, a.g.n. tchouatcheu, c. bernhardt, r. lieberei, b. bisping especially free hydrophobic amino acids. therefore, acidification of cacao beans cells by acetic acid and its driving force in lowering the cell ph impact strongly the protease activity, the free amino acid and oligopeptides release during the fermentation process. sugars content in unfermented cacao beans, the concentration of sucrose ranged between 53.20 mg/g ffdm (tiko31) – 35.56 mg/g ffdm (t60/887). these initial concentrations showed a high variability among snk varieties. snk620, snk624 and snk450 displayed higher values with 49.23 mg/g ffdm, 52.34 mg/g ffdm and 49.26 mg/g ffdm respectively. after incubation, the concentration of sucrose dropped drastically. according to the residual concentration of sucrose, the varie ties studied can be divided into three groups. the first group, constituted by imc67, tiko31, t60/887 and snk377 where the sucrose was completely degraded during incubation in both treatments. the second group which includes snk64 and tiko32, only incubation in t1 allowed 100% of degradation. it is important to note that in this group the degradation of sucrose induced by t2 was above 90%. the last group was exclusively constituted by snk varieties (snk10, snk16 snk620, snk624, snk450 and snk15), where the residual content of sucrose was found in both treatments. nevertheless, the value recorded was below 10 mg/g ffdm in both treatments (fig. 4). it is well established that sucrose is the main disaccharide found in unfermented cacao beans. data from snk and tiko varieties com pared to those reported by reccenius et al. (1972) were two to three times higher (53.20 mg/g ffdm against 18.50 mg/g ffdm). the difference displayed among varieties could be attributed to their genetic background. since all the varieties were subjected to the same treatments, the percentage of degradation reflected the impact of treatments applied. unlike the tiko, imc67 and t60/877 varieties, in the snk varieties, the degradation of sucrose was not completed with regards to the treatment. thus, this latter required proper fermentation practice to release more reducing sugars from sucrose. limiting factor for the production of aroma relevant maillard reactions being reducing sugars (rohsius, 2007). before subjected to incubation, a small content of glucose was exclusively found in tiko32 (1.93 mg/g ffdm), snk16 (3.30 mg/g ffdm) and snk15 (2.49 mg/g ffdm). after incubation, the concentration of glucose exponentially increased in all the varieties. in t1, it ranged between 8.16 mg/g ffdm (snk624) – 5.40 mg/g ffdm (snk10) and between 3.09 mg/g ffdm (snk624) – 11.39 mg/g ffdm (snk48) in t2. excepted in snk620 and snk624, the rest of the varieties recorded their highest concentration in t2 (fig. 5a). in contrast to glucose, fructose was found in unfermented cacao beans in all the varieties. its initial content ranged between 0.90 mg/g ffdm (imc67) to 6.64 mg/g ffdm (snk16). after incubation, as observed with the glucose released, the concentration of fructose exponentially increased in both treatments. in t1, it ranged between 6.93 mg/g ffdm (snk377) – 9.85 mg/g ffdm (snk64) and between 1.74 mg/g ffdm (snk24) – 12.46 mg/g ffdm (snk48) in t2. in tiko varie ties (tiko31 and tiko32), t2 displayed higher value than t1. after t2 treatment, the concentrations found were 8 times higher than those reported in unfermented cacao beans, while those recorded in t1 were only 5 times higher. as in glucose, t2 released more fructose than t1 in tiko varieties and in some snk varieties (snk16, snk620, snk624, snk337 and snk15) (fig. 5b). glucose and fructose are the main reducing sugars found in fermented cacao beans. in fermented traded cacao beans, rohsius et al. (2010) reported a distribution of 0.45 mg/g ffdm : 0.67 mg/g ffdm : fig. 2: ratio of hydrophobic amino acids group / acidic group + other amino acids group. t0 t1 t2 fig. 3: ratio of hydrophobic amino acids / acidic amino acids + other amino acids in unfermented (t0) and incubated (t1 and t2) cacao beans from different cacao varieties. t1 treatment consisted of sterile incubation of 25 cacao beans of five individual fruits in 100 mmol/l acetic acid ph 4 for two days following their transfer in 200 mmol/l acetic acid ph 5 for three days and subsequent sun drying. t2 treatment consisted of sterile incubation of 25 cacao beans in 100 mmol/l lactic acid ph 4 for two days following their transfer in 200 mmol/l acetic acid ph 5 for three days and subsequent sun drying. values are means of n = 3 (three biological replicates based on two technical replicates of extraction). means with different letters were significantly different by tukey (p < 0.05). γ-amino butyric acid (gaba) was detected in all cacao samples but was excluded in the analysis. 0 10 20 30 40 50 60 sa cc ha ro se (m g/ g ff dm ) varieties t0 t1 t2 a b b b b b b b b b b b b b a a a a a a a a a a a a a a fig. 4: sucrose content in unfermented (t0) and incubated (t1 and t2) cacao beans from different cacao varieties. t1 treatment consist ed of sterile incubation of 25 cacao beans of five individual fruits in 100 mmol/l acetic acid ph 4 for two days following their transfer in 200 mmol/l acetic acid ph 5 for three days and subsequent sun drying. t2 treatment consisted of sterile incubation of 25 cacao beans in 100 mmol/l lactic acid ph 4 for two days following their transfer in 200 mmol/l acetic acid ph 5 for three days and subsequent sun drying. values are means of n = 3 (three biological replicates based on two technical replicates of extraction). means with different letters were significantly different by tukey (p < 0.05). amino acids and reducing sugars in cacao beans 327 0.04 mg/g ffdm for sucrose : glucose : fructose. they are indispensable aroma precursors. their carbonyl group is involved in streckertype reaction in maillard reaction during the roasting process. our results showed the preponderance of t1 over t2 in tiko, imc67 and t60/877 varieties. however, all the snk varieties did not show the same trends. surprisingly, the trend was the same as observed in the amount of amino acids released. snk16, snk620, snk624, snk377, snk15 released more reducing sugars in t1 while snk64, snk10, snk450 and snk48 released more in t2. however, the content recorded did not correspond to those expected coming from sucrose degradation. this was due to exudation into the medium which occurs during incubation or fermentation. galactose and raffinose were also found in unfermented cacao beans. the concentration of raffinose was higher than galactose in all the varieties. it ranged between 12.31 mg/g ffdm (tiko31) – 9.66 mg/g ffdm (imc67) while galactose ranged between 4.72 mg/g ffdm (snk624) – 1.68 mg/g ffdm (t60/887) (fig. 6a). contrary to the monosaccharides regularly found in cacao (glucose and fructose), the concentration of galactose did not exponentially increase after the incubation. in both treatments, the concentration recorded in each variety was barely two times higher than those obtained in unfermented cacao beans (against eight times higher in fructose). with the exception in snk16, snk620 and snk624, raffinose disappeared entirely during incubation in the rest of varieties (fig. 6b). raffinose is a trisaccharide containing galactose linked by α-(16) bond to the glucose unit of sucrose (švejstil et al., 2013). this non-reducing sugar is found in many fruits and cereals (jovanovicmalinovska et al., 2013; svejstil et al., 2015). cerubulis (1955) is the first author who reported the presence of raffinose in the cacao beans followed by reccenius et al. (1972). in the varieties used for this study, the content of raffinose was two to three times lower than sucrose. their entire disappearance after incubation was due to enzymatic and/or non-enzymatic degradation into sucrose and galactose. this may justify not only the slight increase of galactose observed in some varieties after incubation but also the lower residual sucrose content found in snk varieties. conclusion the fermentation-like incubation which well-mimicked the fermentation process allows predicting the behavior of local cameroonian cacao varieties (snk and tiko). the high content of free hydrophobic amino acids and reducing sugars generated in both treatments carried out demonstrated that these varieties have an irrecusable potential of aroma precursors. thus, our data provide additional knowledge to be considered in the promotion of cameroonian cacao. therefore, it will be interesting to set up appropriate natural fermentations according to specific agro-ecological conditions. otherwise, the results obtained from both treatments showed that the beginning bc b 0 5 10 15 20 f ru ct os e (m g/ g ff dm ) varieties t0 t1 t2 0 5 10 15 g lu co se (m g/ g ff dm ) varieties t0 t1 t2b a b b b b b b b b bc b bc bc b b b b bc b b b b b b a a a a a a a a a a a a a a b b b b b b ab ab b b b b b b b b b b b b b b b b b b b a a fig. 5: fructose (a) and glucose (b) contents in unfermented (t0) and incubated (t1 and t2) cacao beans from different cacao varieties. t1 treatment consisted of sterile incubation of 25 cacao beans of five individual fruits in 100 mmol/l acetic acid ph 4 for two days following their transfer in 200 mmol/l acetic acid ph 5 for three days and subsequent sun drying. t2 treatment consisted of sterile incubation of 25 cacao beans in 100 mmol/l lactic acid ph 4 for two days following their transfer in 200 mmol/l acetic acid ph 5 for three days and subsequent sun drying. values are means of n = 3 (three biological replicates based on two technical replicates of ex traction). means with different letters were significantly different by tukey (p < 0.05). 0 5 10 15 20 25 g al ac to se (m g/ g ff dm ) varieties t0 t1 t2a 0 5 10 15 20 r af fi no se (m g/ g ff dm ) varieties t0 t1 t2b fig. 6: galactose (a) and raffinose (b) contents in unfermented (t0) and incubated (t1 and t2) cacao beans from different cacao varieties. t1 treatment consisted of sterile incubation of 25 cacao beans of five individual fruits in 100 mmol/l acetic acid ph 4 for two days following their transfer in 200 mmol/l acetic acid ph 5 for three days and subsequent sun drying. t2 treatment consisted of sterile incubation of 25 cacao beans in 100 mmol/l lactic acid ph 4 for two days following their transfer in 200 mmol/l acetic acid ph 5 for three days and subsequent sun drying. values are means of n = 3 (three biological replicates based on two technical replicates of extraction). means with different letters were significantly different by tukey (p < 0.05). 328 n. niemenak, j.v.e. eyamo, s.a.m. djabou, a.g.n. tchouatcheu, c. bernhardt, r. lieberei, b. bisping of fermentation (presence or absence of acetic acid, treatment 1 and treatment 2 respectively) slightly influences the result in snk and tiko varieties. therefore, fermentation process allowing acidification of beans with acetic acid at the early stage is necessary for free amino acid release. moreover, investigation on the special flavors generated from these varieties during the roasting process and also on the dynamics of the accumulation of these aroma precursors during the fermentation process will be helpful to valorize these widely spread varieties in cameroonian cacao farms. acknowledgment the study was supported by the alexander-von-humboldt stiftung (www.humboldt-stiftung.de) via grant to nicolas niemenak (3.4cmr/1115305 stp). this publication was prepared in memory of prof. dr. reinhard lieberei who paved and guided our way in cacao research through his profound 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(eds.), analysis of nonalcoholic beverages, 321-393. modern methods of plant analysis, volume 8, springer-verlag, berlin, heidelberg. orcid nicolas niemenak https://orcid.org/0000-0003-0044-7294 stéphanie astride mouafi djabou https://orcid.org/0000-0001-9215-3239 reinhard lieberei https://orcid.org/0000-0001-7326-0469 bernward bisping https://orcid.org/0000-0003-1027-1106 address of the corresponding author: nicolas niemenak, laboratory of plant physiology and biochemistry, de partment of biological science, higher teacher training college, university of yaoundé i, p.o. box 47 yaoundé, cameroon e-mail: niemenak@yahoo.com © the author(s) 2021. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1016/j.fm.2011.12.021 http://dx.doi.org/10.1371/journal.pone.0048438 http://dx.doi.org/10.1016/j.foodres.2016.04.017 http://dx.doi.org/10.18805/ajdfr.v35i2.10724 http://dx.doi.org/10.1016/j.ijfoodmicro.2006.09.010 http://dx.doi.org/10.1016/j.jfca.2005.02.006 http://dx.doi.org/10.1016/j.cropro.2006.03.015 http://dx.doi.org/10.1016/j.foodres.2013.10.032 http://dx.doi.org/10.1002/(sici)1097-0010(199812)78:4<543::aid-jsfa152>3.0.co;2-2 http://dx.doi.org/10.1021/jf60180a033 http://dx.doi.org/10.1080/10408690490464104 http://dx.doi.org/10.1007/s00217-005-0130-y http://dx.doi.org/10.5073/jabfq.2017.090.034 http://dx.doi.org/10.1515/sab-2015-0019 http://dx.doi.org/10.1007/s13197-019-03749-y http://dx.doi.org/10.1016/0308-8146(94)90155-4 http://dx.doi.org/10.1016/j.foodchem.2015.07.068 http://dx.doi.org/10.1007/bf01202506 i supplementary material su pp le m en t 1 : h yd ro ph ob ic , a ci di c an d to ta l f re e am in o ac id s co nt en ts in u nf er m en te d ca ca o be an s (t 0) o f d iff er en t c lo ne s. su pp le m en t 1 : h yd ro ph ob ic , a ci di c an d to ta l f re e am in o ac id s co nt en ts in u nf er m en te d co co a be an s (t 0) o f d if fe re nt c lo ne s. a a /c lo ne s im c 6 7 sn k 10 sn k 15 sn k 16 sn k 37 7 sn k 45 0 sn k 48 sn k 62 0 sn k 62 4 sn k 64 t 60 /8 87 t ik o 31 t ik o 32 l eu 21 4. 92 ±5 .0 0 11 4. 41 ±0 .5 1 13 7. 48 ±8 .0 6 18 2. 86 ±1 2. 42 33 6. 92 ±2 .5 4 23 5. 62 ±1 .2 5 16 4. 92 ±4 .0 4 14 2. 79 ±5 .6 8 15 2. 86 ±3 .0 4 21 7. 01 ±1 6. 61 17 7. 24 ±1 4. 54 12 8. 68 ±5 .7 1 14 5. 77 ±5 .0 8 a la 30 6. 29 ±1 2. 18 34 7. 22 ±5 .3 8 40 5. 47 ±2 9. 47 46 4. 47 ±5 .0 8 77 7. 09 ±1 .6 4 12 05 .4 1± 11 .2 9 48 5. 11 ±7 .1 4 72 7. 29 ±4 0. 59 32 6. 22 ±7 .5 1 38 0. 42 ±2 7. 31 54 2. 71 ±4 0. 94 48 1. 39 ±1 1. 10 61 6. 73 ±1 0. 90 ph e 24 6. 65 ±1 .7 3 22 2. 96 ±1 .0 1 19 4. 83 ±1 5. 28 30 6. 08 ±0 .0 2 53 7. 99 ±0 .5 7 16 5. 89 ±2 .2 6 32 3. 25 ±3 .9 7 38 0. 39 ±1 5. 57 25 5. 01 ±2 .3 4 20 8. 22 ±1 6. 59 28 9. 08 ±1 8. 24 22 9. 81 ±3 .4 0 21 8. 22 ±1 7. 89 t yr 27 8. 07 ±3 .6 6 34 0. 33 ±5 .0 1 31 5. 93 ±2 6. 76 45 8. 59 ±8 .3 9 68 5. 90 ±1 .0 8 28 1. 03 ±4 .6 6 41 8. 62 ±3 .4 0 58 9. 93 35 1. 21 ±3 .1 9 27 6. 99 ±1 9. 14 41 9. 15 ±2 8. 76 52 1. 63 ±1 .1 2 32 0. 65 ±5 .5 8 v al 31 8. 09 ±5 .9 0 12 6. 36 ±1 .3 7 20 1. 09 ±1 4. 18 22 8. 98 ±3 .0 1 36 1. 46 ±0 .1 4 31 8. 63 ±0 .4 4 20 3. 39 ±0 .9 8 21 2. 14 ±1 0. 24 26 5. 59 ±3 .1 6 32 9. 61 ±2 3. 61 24 8. 44 ±1 5. 87 14 5. 75 ±3 .9 1 14 2. 94 ±9 .6 4 il e 28 6. 92 ±5 .2 6 18 3. 01 ±0 .2 2 19 7. 33 ±1 3. 94 18 9. 65 ±2 .2 3 35 2. 14 ±0 .2 0 28 8. 59 ±1 .0 0 20 4. 73 ±1 .8 8 19 9. 66 ±7 .5 7 23 9. 23 ±3 .1 6 30 5. 47 ±2 3. 04 22 6. 15 ±1 4. 41 14 3. 92 ±3 .1 7 14 0. 90 t ot al h yd ro ph ob ic 16 50 .9 7± 38 .4 7 13 34 .3 3± 10 1. 90 14 52 .1 4± 99 .0 0 18 30 .6 6± 12 8. 86 30 51 .5 2± 18 9. 73 24 95 .1 9± 39 0. 43 18 00 .0 4± 13 0. 79 22 52 .2 2± 23 7. 28 15 90 .1 5± 70 .0 2 17 17 .7 5± 66 .4 6 19 02 .7 9± 13 7. 58 16 51 .2 1± 17 9. 31 15 85 .2 4± 18 6. 28 a sp 18 5. 48 ±5 .6 8 22 2. 98 ±2 .9 6 29 5. 99 ±2 4. 66 28 8. 31 ±1 .5 4 92 .1 7± 0. 57 28 3. 87 ±3 .4 6 19 9. 46 ±5 .2 1 14 8. 46 ±6 .8 1 17 3. 02 ±0 .5 6 24 8. 90 ±1 5. 66 14 3. 27 ±8 .8 7 28 2. 05 ±9 .0 4 13 1. 67 ±0 .9 5 g lu 20 90 .7 2± 11 9. 16 14 34 .3 8± 80 .6 0 17 51 .6 3± 14 8. 47 16 55 .0 1± 20 .6 6 10 90 .2 0± 3. 67 21 32 .3 0± 20 .5 7 23 63 .1 9± 28 .5 1 20 95 .5 7± 11 5. 99 16 93 .4 2± 8. 98 17 89 .8 9± 12 8. 06 12 87 .6 1± 90 .5 7 14 83 .6 4± 11 .4 2 16 36 .0 1± 1. 24 a sn 99 1. 48 ±1 6. 04 67 2. 14 ±7 .3 3 72 3. 46 ±5 5. 99 16 76 .6 7± 21 .9 0 11 68 .8 9± 5. 56 31 54 .1 6± 2. 92 12 72 .3 6± 14 .5 8 15 29 .7 9± 96 .2 6 78 3. 82 ±9 .2 3 88 0. 15 ±7 5. 09 11 41 .7 6± 66 .4 5 24 41 .6 5± 7. 34 10 04 .5 3± 4. 42 h is 31 7. 81 ±8 .6 9 33 7. 76 ±3 .7 2 34 0. 41 ±5 .7 4 64 3. 03 ±8 7. 96 33 6. 30 ±7 .9 5 43 3. 65 ±1 8. 25 33 2. 94 ±5 .0 0 28 5. 27 35 3. 55 ±3 4. 58 39 7. 46 ±9 .1 0 29 8. 99 ±2 4. 47 t ot al a ci di c 35 85 .5 1± 87 0. 94 26 67 .2 8± 54 6. 03 27 71 .0 9± 74 8. 19 39 60 .4 1± 78 0. 61 29 94 .3 0± 49 5. 10 55 70 .3 4± 14 54 .8 4 41 71 .3 2± 10 01 .0 8 42 07 .4 8± 91 5. 80 29 83 .2 1± 68 2. 63 32 04 .2 2± 71 9. 94 29 26 .2 0± 56 7. 58 46 04 .8 0± 10 16 .4 1 30 71 .2 2± 69 1. 44 t hr 15 6. 17 ±0 .3 57 12 5. 64 ±3 .4 5 86 .8 1 12 8. 08 t rp 35 6. 88 ±1 5. 22 53 2. 19 ±2 .5 1 33 4. 29 ±9 .8 9 g ly 10 2. 97 ±1 2. 75 85 .3 9± 2. 27 10 3. 02 ±4 .9 2 12 1. 38 ±3 .4 3 14 1. 42 ±5 .5 5 16 9. 88 ±5 .4 9 12 5. 35 ±6 .4 8 10 3. 62 ±7 .1 1 10 7. 91 ±3 .4 3 11 7. 95 ±3 .4 8 13 0. 53 ±1 7. 77 10 8. 41 ±1 4. 99 12 0. 56 ±6 .9 6 a rg 12 1. 60 ±4 .1 4 12 4. 23 ±0 .2 6 11 2. 67 ±7 .7 1 21 0. 27 ±3 .2 3 40 8. 26 ±8 .5 0 24 0. 11 ±1 .8 6 17 5. 84 ±4 .8 1 16 0. 06 ±8 .4 2 10 9. 67 ±0 .8 2 13 7. 68 ±6 .8 4 14 2. 55 ±1 2. 17 25 9. 33 ±3 .9 3 14 9. 38 ±2 .3 8 se r 94 .1 5± 15 .7 8 11 6. 85 11 2. 30 ±6 .4 3 12 9. 44 ±1 .4 3 13 5. 43 ±7 .7 4 10 2. 69 ±0 .5 8 10 2. 79 ±7 .2 4 10 7. 59 ±4 7. 12 11 2. 26 ±4 .4 9 10 2. 49 ±2 9. 09 11 0. 70 ±2 2. 02 94 .0 2± 7. 53 ly s 20 3. 71 ±0 .1 8 16 7. 27 ±5 .8 4 13 0. 41 ±0 .1 0 17 1. 41 12 5. 57 16 0. 79 ±9 .8 3 16 5. 32 ±3 8. 82 t ot al o th er 31 8. 73 ±1 4. 01 68 3. 37 ±1 25 .1 6 21 5. 69 ±6 .8 20 44 3. 97 ±5 4. 13 15 71 .2 2± 16 7. 99 83 8. 35 ±4 4. 88 62 1. 12 ±3 3. 79 36 6. 48 ±3 2. 83 62 4. 67 ±2 7. 35 49 3. 48 ±1 0. 99 53 6. 38 ±2 4. 46 97 8. 06 ±9 8. 77 36 3. 98 ±2 7. 68 t ot al fr ee a a 55 55 .2 3± 92 3. 42 46 84 .9 8± 77 3. 10 44 38 .9 2± 85 4. 01 62 35 .0 5± 96 3. 60 76 17 .0 5± 85 2. 82 89 03 .8 9± 18 90 .1 5 65 92 .4 9± 11 65 .6 6 68 26 .1 9± 11 85 .9 1 51 98 .0 5± 78 0. 01 54 15 .4 6± 79 7. 39 53 65 .3 8± 72 9. 63 72 34 .0 8± 12 94 .5 0 50 20 .4 5± 90 5. 41 % a a h yd ro ph ob es 29 .7 1 28 .4 8 32 .7 1 29 .3 6 40 .6 6 28 .0 2 27 .3 0 33 .0 0 30 .5 9 31 .7 1 35 .4 6 22 .8 2 31 .5 7 % a a a ci di c 64 .5 56 .9 3 62 .4 2 63 .5 1 39 .3 1 62 .5 1 63 .2 7 61 .6 3 57 .3 9 59 .1 6 54 .5 3 63 .6 5 61 .1 7 % a a o th er s 5. 75 14 .5 9 4. 87 7. 13 20 .6 3 9. 42 9. 43 5. 37 12 .0 2 9. 13 10 .0 1 13 .5 3 7. 26 supplementary material ii su pp le m en t 2 : h yd ro ph ob ic , a ci di c an d to ta l f re e am in o ac id s co nt en ts in c ac ao b ea ns o f d iff er en t c lo ne s af te r 5 d ay s of fe rm en ta tio nlik e in cu ba tio n in tr ea tm en t 1 . su pp le m en t 2 . h yd ro ph ob ic , a ci di c an d to ta l f re e am in o ac id s co nt en ts in c oc oa b ea ns o f d if fe re nt c lo ne s af te r 5 d ay s of fe rm en ta tio nlik e in cu ba tio n in tr ea tm en t 1 . t 1 a a /c lo ne s im c 6 7 sn k 10 sn k 15 sn k 16 sn k 37 7 sn k 45 0 sn k 48 sn k 62 0 sn k 62 4 sn k 64 t 60 /8 87 t ik o 31 t ik o 32 l eu 21 65 .3 1± 11 2. 66 70 3. 92 ±2 0. 14 13 88 .1 5± 28 .5 21 10 68 .0 3± 18 .8 2 14 74 .7 6± 88 .1 6 12 39 .4 0± 25 .1 6 17 78 .0 0± 90 .4 9 78 4. 65 ±0 .3 0 13 82 .0 2± 1. 57 20 87 .5 1± 32 .8 0 21 86 .0 7± 6. 73 22 78 .0 3± 88 .8 4 20 16 .2 0± 39 .8 4 a la 64 6. 51 ±0 .6 6 28 3. 60 ±1 3. 69 48 1. 99 ±9 .3 8 31 8. 23 ±4 .8 7 48 2. 79 ±4 .6 2 51 5. 00 ±0 .6 20 66 9. 64 ±1 3. 19 31 4. 50 ±2 .6 7 47 7. 00 ±4 .0 6 75 7. 20 ±1 0. 80 77 5. 88 ±0 .7 3 91 6. 86 ±2 4. 05 78 2. 72 ±4 .5 2 ph e 17 08 .0 2± 41 .5 3 43 5. 80 ±1 3. 26 99 9. 52 ±2 7. 18 84 9. 24 ±2 0. 69 11 64 .8 1± 26 .7 0 95 3. 07 ±5 .2 1 13 35 .5 1± 25 .2 3 66 4. 93 ±1 1. 49 11 16 .3 1± 4. 89 16 91 .4 2± 35 .4 9 16 43 .1 5± 5. 99 17 34 .2 4± 64 .9 7 14 78 .4 8± 5. 43 t yr 10 68 .8 8± 0. 11 33 9. 99 ±1 2. 58 69 6. 83 ±1 8. 50 59 4. 78 ±1 3. 55 80 8. 39 ±1 0. 43 64 5. 41 ±1 .0 1 85 6. 06 ±2 3. 81 50 4. 22 ±2 5. 92 78 8. 71 ±6 .1 8 10 04 .1 4± 13 .4 5 10 18 .2 4± 5. 41 10 29 .6 7± 35 .1 6 89 9. 07 ±2 .2 5 v al 77 3. 63 ±2 2. 25 26 9. 64 ±9 .4 5 46 9. 64 ±8 .3 9 40 0. 80 ±9 .6 5 49 5. 01 ±9 .1 5 48 4. 33 ±4 .8 9 63 6. 57 ±1 1. 22 35 7. 26 ±3 .7 9 60 2. 25 ±2 .9 4 71 9. 99 ±1 6. 44 73 6. 42 ±2 .8 7 78 5. 05 ±2 9. 72 64 0. 36 ±3 .0 6 il e 54 2. 25 ±2 2. 76 16 9. 43 ±3 .4 7 31 0. 80 ±7 .1 3 28 6. 00 ±2 .2 1 33 1. 59 ±1 2. 70 33 4. 42 ±4 .4 1 43 9. 97 ±1 2. 64 24 6. 98 ±0 .5 7 42 4. 44 ±2 .9 0 50 3. 61 ±6 .7 3 51 0. 05 ±4 .4 6 52 3. 94 ±2 0. 71 43 4. 35 ±2 .4 1 t ot al h yd ro ph ob ic 69 04 .6 2± 65 0. 12 22 02 .3 9± 18 6. 78 43 46 .9 7± 40 2. 92 35 17 .1 0± 31 5. 50 47 57 .3 9± 44 7. 48 41 71 .6 5± 33 8. 30 57 15 .7 6± 50 5. 95 28 72 .5 6± 21 1. 82 47 90 .7 5± 38 0. 36 67 63 .8 9± 62 4. 06 68 69 .8 3± 64 0. 99 72 67 .8 0± 66 1. 09 62 51 .2 0± 59 2. 92 a sp 25 9. 22 ±0 .2 5 97 .6 7± 5. 11 17 7. 68 ±2 .8 7 15 6. 60 ±7 .6 4 19 1. 98 ±7 .1 8 21 7. 63 ±1 .0 6 25 7. 15 ±8 .1 1 17 1. 28 ±2 .3 8 18 1. 99 ±9 .1 4 25 3. 30 ±7 .2 8 26 1. 57 ±0 .5 1 27 1. 33 ±1 .7 8 23 1. 51 ±7 .5 1 g lu 10 80 .8 6± 20 .0 0 54 9. 63 ±1 4. 35 81 3. 27 ±4 0. 99 65 6. 03 ±1 4. 60 79 1. 16 ±5 9. 87 87 7. 13 ±3 .6 71 11 28 .7 3± 11 .1 3 99 9. 00 ±1 2. 51 89 7. 27 ±1 1. 00 94 6. 03 ±2 9. 57 10 20 .1 0± 52 .1 4 11 08 .3 2± 10 .9 1 95 9. 40 ±2 7. 79 a sn 90 0. 86 ±6 .9 8 30 4. 74 ±1 1. 29 62 5. 18 ±9 .2 60 46 1. 03 ±4 .3 5 65 8. 10 ±8 .2 1 64 4. 73 ±8 .6 0 10 71 .0 9± 34 .0 5 71 7. 25 ±2 0. 05 81 7. 15 ±0 .4 6 12 31 .3 7± 19 .3 88 87 8. 37 ±1 9. 07 11 96 .4 8± 10 .8 9 98 5. 75 ±1 1. 46 g ln 70 4. 35 ±2 1. 62 41 9. 71 ±1 3. 21 27 3. 87 ±4 .7 9 45 6. 61 ±4 .5 0 37 8. 21 ±1 .5 7 42 6. 93 ±2 1. 76 41 0. 13 ±1 0. 71 57 8. 84 ±4 8. 45 69 2. 05 ±2 .3 7 69 6. 96 ±1 6. 88 60 2. 20 ±4 5. 15 h is 30 7. 91 23 1. 76 t ot al a ci di c 29 45 .3 0± 35 3. 28 95 2. 05 ±2 26 .2 4 20 35 .8 5± 27 3. 14 18 55 .4 6± 19 2. 82 20 97 .8 7± 26 0. 85 21 17 .7 2± 29 1. 13 6 28 83 .9 1± 44 3. 63 18 87 .5 4± 42 0. 83 23 06 .5 6± 33 8. 68 30 09 .5 5± 42 6. 66 28 52 .1 0± 32 9. 58 35 04 .8 7± 45 1. 72 6 27 78 .8 8± 35 4. 90 t hr 36 3. 25 ±5 .0 3 10 0. 64 ±5 .3 2 42 9. 43 ±2 83 .6 8 15 3. 78 ±0 .3 0 23 9. 61 ±2 .1 6 20 6. 91 ±0 .3 7 30 0. 59 ±3 .7 6 14 5. 20 ±6 .8 2 25 0. 09 ±6 .6 1 33 0. 12 ±6 .0 1 36 6. 65 ±3 .0 5 37 2. 77 ±2 1. 59 32 9. 84 ±1 .1 6 t rp 97 4. 49 ±1 24 .8 6 58 0. 53 40 9. 67 ±3 27 .0 1 55 9. 77 ±2 9. 35 73 2. 40 ±1 3. 89 63 0. 94 ±2 1. 43 72 4. 44 ±3 9. 50 58 2. 41 ±6 2. 34 63 1. 98 ±2 2. 89 80 8. 71 ±8 .6 2 87 4. 37 ±5 .1 0 79 3. 28 ±8 2. 81 74 1. 69 ±2 1. 03 g ly 20 2. 03 ±1 .3 1 90 .4 2± 5. 43 68 4. 94 ±7 44 .0 9 12 4. 14 ±0 .2 9 15 9. 45 ±6 .1 5 13 6. 42 ±2 .8 5 19 7. 51 ±0 .7 3 11 1. 75 ±0 .8 2 16 1. 74 ±0 .0 7 19 9. 24 ±0 .5 0 21 6. 35 ±4 .6 4 23 0. 62 ±1 2. 56 21 0. 43 ±1 .7 3 a rg 21 37 .2 4± 25 .0 7 45 6. 30 ±2 2. 19 13 72 .3 0± 28 .3 8 85 0. 88 ±1 5. 10 15 95 .3 9± 20 .3 0 11 41 .2 4± 6. 86 14 20 .8 3± 16 .3 6 62 6. 24 ±5 .2 1 12 52 .6 9± 15 .5 9 18 63 .6 4± 41 .8 8 20 83 .8 7± 16 .9 6 18 17 .8 4± 70 .8 0 16 18 .0 3± 19 .6 2 se r 40 0. 17 ±3 .7 8 10 7. 28 ±1 5. 19 23 6. 58 ±2 7. 56 16 6. 16 ±1 3. 72 23 6. 34 ±6 .0 38 23 0. 54 ±1 8. 38 27 7. 89 ±9 .5 4 13 5. 66 ±5 .8 1 24 7. 65 ±5 .8 1 35 4. 99 ±1 6. 16 44 9. 34 ±9 .3 8 43 7. 21 ±3 2. 01 37 2. 13 ±2 .2 7 l ys 17 81 .3 8± 58 .6 3 44 7. 73 ±8 .2 6 70 0. 69 ±6 49 .7 4 72 8. 90 ±3 8. 31 12 90 .7 8± 3. 56 94 3. 97 ±1 6. 49 13 04 .6 6± 3. 68 49 3. 19 ±1 7. 35 10 46 .1 7± 5. 68 15 90 .7 8± 22 .8 9 19 62 .0 8± 31 .3 1 18 07 .4 9± 80 .7 1 15 18 .6 7± 9. 30 m et 12 0. 70 ±2 7. 26 10 7. 82 ±4 .6 0 10 6. 13 ±7 .9 1 12 7. 84 ±1 3. 40 70 .7 7± 4. 08 25 4. 96 ±4 7. 04 16 5. 38 88 .5 9± 2. 12 20 1. 77 ±3 7. 36 10 9. 09 ±1 .5 1 14 9. 61 ±6 2. 02 13 8. 95 ±4 7. 16 t ot al o th er 59 79 .2 9± 80 9. 40 17 82 .9 2± 22 1. 67 39 41 .4 7± 41 7. 10 26 89 .7 8± 31 9. 64 43 81 .8 6± 59 9. 99 33 60 .8 2± 42 7. 70 44 80 .9 1± 52 4. 24 22 59 .8 5± 23 2. 52 36 78 .9 47 ±4 63 .2 3 53 49 .2 7± 69 3. 46 60 61 .7 8± 82 6. 90 56 08 .8 5± 72 0. 23 49 29 .7 7± 62 0. 96 t ot al fr ee a a 15 82 9. 22 ±1 81 2. 82 49 37 .3 7± 63 4. 70 10 32 4. 30 ±1 09 3. 18 80 62 .3 5± 82 7. 97 11 23 7. 13 ±1 30 8. 32 96 50 .2 0± 10 57 .1 4 13 08 0. 60 ±1 47 3. 83 70 19 .9 6± 86 5. 17 10 77 6. 26 ±1 18 2. 28 15 12 2. 72 ±1 74 4. 19 15 78 3. 71 ±1 79 7. 48 16 38 1. 53 ±1 83 3. 05 13 95 9. 86 ±1 56 8. 79 % h yd ro ph ob ic a a 43 .6 1 44 .6 0 42 .1 0 43 .6 1 42 .3 3 43 .2 2 43 .2 2 40 .9 1 43 .2 2 44 .7 2 43 .5 2 43 .1 8 44 .7 8 % a ci di c a a 18 .6 0 19 .2 8 19 .7 1 23 .0 1 18 .4 6 21 .9 4 22 .0 4 26 .8 8 21 .4 0 19 .9 0 18 .0 6 21 .3 9 19 .9 0 % o th er s a a 37 .7 9 44 .6 38 .1 9 33 .3 8 39 .2 1 34 .8 4 34 .7 4 32 .2 1 35 .3 8 35 .5 8 38 .4 2 35 .4 3 35 .3 2 su pp le m en t 3 : h yd ro ph ob ic , a ci di c an d to ta l f re e am in o ac id s co nt en ts in c ac ao b ea ns o f d iff er en t c lo ne s af te r 5 d ay s of fe rm en ta tio nlik e in cu ba tio n in tr ea tm en t 2 . su pp le m en t 3 : h yd ro ph ob ic , a ci di c an d to ta l f re e am in o ac id s co nt en ts in c oc oa b ea ns o f d if fe re nt c lo ne s af te r 5 d ay s of fe rm en ta tio nlik e in cu ba tio n in tr ea tm en t 2 . a a /c lo ne s im c 6 7 sn k 10 sn k 15 sn k 16 sn k 37 7 sn k 45 0 sn k 48 sn k 62 0 sn k 62 4 sn k 64 t 60 /8 87 t ik o 31 t ik o 32 l eu 82 7. 99 ±0 .1 1 13 26 .4 1± 59 .0 1 62 6. 05 ±0 .4 4 42 4. 90 ±9 .9 1 75 5. 21 ±2 4. 23 10 87 .5 2± 59 .4 8 13 86 .4 0± 73 .7 7 73 1. 12 ±1 .4 5 27 3. 61 ±1 3. 06 87 5. 18 ±5 5. 71 11 72 .6 6± 35 .1 2 99 0. 29 ±4 4. 80 91 7. 63 ±1 9. 37 a la 42 9. 75 ±1 .0 9 40 1. 13 ±1 2. 98 31 3. 66 ±0 .1 8 21 9. 47 ±0 .3 8 39 0. 16 3± 9. 22 49 2. 18 ±4 .7 3 55 4. 20 ±0 .5 0 31 1. 54 ±0 .2 1 26 2. 14 ±1 2. 39 50 8. 38 ±6 .3 7 46 6. 85 ±1 2. 02 52 1. 99 ±1 0. 56 61 2. 05 ±1 6. 66 ph e 58 8. 10 ±7 .8 6 10 42 .8 5± 22 .8 5 42 6. 60 ±3 .9 1 29 1. 97 ±3 .7 5 57 1. 94 ±1 7. 37 84 6. 29 ±1 0. 82 10 49 .1 0± 11 .7 6 64 5. 95 ±4 .0 2 24 1. 48 ±1 0. 38 59 0. 47 ±1 6. 81 92 7. 01 ±3 3. 36 66 7. 58 ±9 .6 9 64 3. 89 ±1 9. 33 t yr 40 2. 64 ±5 .5 0 73 8. 96 ±1 9. 77 34 1. 51 ±2 .8 2 30 8. 10 ±3 .7 0 42 2. 22 ±9 .6 3 57 5. 58 ±3 .9 8 71 3. 61 ±6 .6 9 47 8. 59 ±3 .9 0 28 9. 23 ±1 1. 01 41 8. 72 ±5 .4 7 63 2. 04 ±1 3. 24 50 1. 19 ±3 .1 8 51 2. 49 ±1 2. 99 v al 35 8. 76 ±1 .7 9 45 3. 84 ±9 .1 8 25 1. 45 ±2 .1 4 19 8. 05 ±1 .5 1 31 7. 04 ±8 .6 8 39 0. 58 ±4 .9 5 47 6. 68 ±1 .4 3 34 4. 42 ±3 .0 5 19 2. 05 ±8 .9 9 35 8. 19 ±1 2. 78 42 6. 65 ±1 3. 72 39 3. 55 ±3 .4 8 37 9. 88 ±9 .8 3 il e 22 0. 24 ±0 .6 2 30 4. 57 0± 8. 67 15 6. 89 ±1 .0 8 15 8. 40 ±0 .6 8 19 4. 53 ±5 .3 5 26 7. 67 ±8 .2 3 34 7. 27 ±7 .5 6 24 2. 40 ±3 .6 7 17 2. 05 ±8 .4 5 24 1. 06 ±9 .8 9 27 2. 82 ±7 .1 6 24 9. 64 ±8 .2 2 23 5. 47 ±4 .2 7 t ot al h yd ro ph ob ic 28 27 .5 0± 21 1. 19 42 67 .7 8± 40 4. 22 21 16 .1 8± 16 1. 50 16 00 .9 17 ±9 5. 97 26 51 .1 2± 19 7. 44 36 59 .8 4± 30 4. 71 45 27 .2 8± 39 2. 65 27 54 .0 4± 19 5. 53 14 30 .5 9± 46 .7 8 29 92 .0 2± 22 0. 34 38 98 .0 5± 33 9. 38 33 24 .2 7± 25 5. 12 33 01 .4 4± 23 5. 40 a sp 11 0. 90 ±0 .8 0 19 9. 47 ±2 .4 2 90 .3 4± 2. 11 79 .3 3± 0. 07 11 3. 94 ±2 .0 0 15 7. 51 ±1 .8 8 20 6. 78 ±2 .3 9 14 1. 34 ±0 .2 4 61 .4 1± 2. 92 12 2. 74 ±2 .1 6 15 1. 76 ±1 .8 1 15 1. 09 ±3 .9 4 15 2. 12 ±4 .0 5 g lu 72 7. 29 ±3 5. 92 78 9. 48 ±2 5. 44 66 1. 84 ±3 .9 6 69 2. 42 ±3 1. 69 71 9. 36 ±6 3. 17 80 5. 04 ±5 4. 27 97 8. 62 ±2 5. 88 96 0. 48 ±3 7. 10 92 3. 34 ±2 1. 51 84 8. 09 ±2 7. 13 69 8. 00 ±1 5. 73 10 06 .7 5± 0. 30 13 27 .9 2± 41 .0 3 a sn 64 4. 19 ±0 .7 9 54 0. 75 ±1 .9 6 32 3. 33 ±7 .8 3 34 7. 73 ±3 .8 7 44 1. 21 ±1 5. 80 66 2. 60 ±2 1. 02 80 7. 10 ±9 .9 3 57 0. 90 ±4 .1 9 33 8. 45 ±2 3. 68 66 3. 53 ±1 7. 95 63 5. 09 ±2 1. 27 62 7. 42 ±4 .2 6 10 84 .1 2± 38 .3 8 g ln 39 6. 96 ±1 8. 68 33 9. 70 ±1 .0 7 34 1. 08 ±2 5. 94 24 1. 81 39 6. 33 ±0 .0 0 28 8. 29 ±4 .6 5 28 2. 77 ±6 .6 9 t ot al a ci di c 14 82 .3 9± 33 4. 47 19 26 .6 7± 24 8. 36 10 75 .5 2± 28 7. 36 11 19 .5 0± 30 7. 33 12 74 .5 2± 30 3. 04 19 64 .8 7± 29 5. 62 23 33 .6 0± 36 8. 23 16 72 .7 3± 40 9. 73 13 23 .2 2± 44 0. 03 18 76 .1 9± 34 3. 05 18 81 .2 0± 24 8. 95 20 73 .5 6± 38 2. 20 28 46 .9 5± 58 1. 81 t hr 11 2. 39 ±0 .5 3 21 0. 42 ±7 .9 0 92 .0 8± 1. 45 11 9. 71 ±0 .4 1 17 7. 67 ±0 .8 4 22 9. 87 ±6 .6 6 14 1. 71 ±3 .4 2 17 7. 07 13 3. 56 ±3 .4 6 19 7. 98 ±1 0. 89 16 5. 79 ±8 .0 3 14 2. 06 ±5 .4 0 g ly 10 6. 64 ±6 .5 0 14 4. 28 ±0 .9 6 89 .6 7± 5. 64 79 .8 5± 2. 40 11 2. 01 ±2 .1 2 14 8. 13 ±2 .3 0 17 1. 46 ±2 .2 5 12 5. 31 ±9 .7 3 83 .7 4± 3. 39 12 1. 29 ±1 .7 8 16 2. 89 ±6 .5 9 15 0. 80 ±1 0. 83 13 4. 54 ±3 .8 7 se r 13 7. 88 ±6 .6 2 23 3. 13 ±8 .0 6 89 .5 6 81 .2 5 10 4. 04 ±4 .7 0 16 9. 38 ±0 .3 1 20 2. 59 ±0 .6 5 13 1. 04 ±1 .0 2 16 9. 60 12 9. 86 ±0 .7 5 22 2. 42 ±1 3. 88 19 6. 24 ±3 1. 94 16 9. 39 ±4 .6 0 a rg 53 4. 55 ±0 .3 3 13 64 .9 6± 47 .1 1 44 5. 21 ±1 .0 9 25 7. 87 ±0 .5 9 58 0. 10 ±1 6. 48 10 13 .7 7± 3. 32 11 19 .8 7± 2. 74 58 6. 17 ±3 .9 0 19 1. 09 ±7 .7 9 52 8. 76 ±1 1. 32 12 16 .7 3± 54 .1 6 73 7. 23 ±5 .1 3 56 8. 11 ±1 4. 10 l ys 53 9. 77 ±4 2. 59 11 81 .1 7± 15 .4 9 41 0. 69 ±2 2. 20 22 6. 93 ±9 .3 9 48 8. 19 ±1 2. 65 84 0. 34 ±5 9. 69 99 6. 02 ±7 .2 6 44 8. 03 ±1 5. 95 15 2. 87 ±1 2. 38 49 1. 71 ±4 .2 4 10 92 .4 9± 13 .2 0 82 1. 63 ±9 3. 63 54 9. 17 ±1 5. 60 t rp 34 5. 06 67 3. 10 40 1. 23 ±1 3. 38 54 7. 39 ±2 6. 63 61 1. 99 ±1 8. 31 60 8. 23 ±2 9. 63 32 8. 52 ±6 .6 07 39 3. 54 ±1 1. 80 51 6. 64 ±1 0. 54 41 4. 30 ±1 0. 43 41 2. 37 ±1 9. 31 m et 12 8. 40 ±3 4. 00 69 .3 3± 0. 47 18 6. 95 ±1 4. 28 67 .1 5 t ot al o th er 17 76 .3 1± 20 6. 54 39 35 .5 0± 52 1. 97 11 27 .2 3± 18 5. 26 64 5. 92 ±9 4. 29 18 05 .3 1± 21 4. 64 29 66 .0 3± 37 9. 43 35 18 .7 9± 41 0. 30 20 40 .5 1± 23 3. 79 11 02 .9 1± 80 .2 6 17 98 .7 5± 19 3. 09 34 76 .3 3± 47 1. 55 24 86 .0 0± 29 9. 64 19 75 .6 6± 20 5. 34 t ot al fr ee a a 60 86 .2 1± 75 2. 21 10 12 9. 96 ±1 17 4. 55 43 18 .9 5± 63 4. 14 33 66 .3 4± 49 7. 60 57 30 .9 6± 71 5. 13 85 90 .7 5± 97 9. 77 10 37 9. 68 ±1 17 1. 20 64 67 .3 0± 83 9. 05 38 56 .7 2± 56 7. 08 66 66 .9 7± 75 6. 49 92 55 .5 9± 10 59 .9 0 78 83 .8 5± 93 6. 97 81 24 .0 5± 10 22 .5 6 % h yd ro ph ob ic 46 .4 5 42 .1 3 24 .9 1 47 .5 6 46 .2 5 42 .6 0 43 .6 1 42 .5 8 37 .1 0 44 .8 8 42 .1 2 42 .1 7 40 .6 3 % a ci di c 24 .3 5 19 .0 2 48 .9 9 32 .2 6 22 .2 4 22 .8 7 24 .4 8 25 .8 7 34 .3 1 28 .1 4 20 .3 2 26 .3 0 35 .0 4 % o th er s 29 .1 8 38 .8 5 26 .1 0 19 .1 8 31 .5 1 34 .8 0 33 .9 1 31 .5 5 28 .5 9 26 .9 8 37 .5 6 31 .5 3 24 .3 1 iii supplementary material journal of applied botany and food quality 94, 220 228 (2021), doi:10.5073/jabfq.2021.094.027 1school of food technology nutrition and bioengineering, makerere university, kampala, uganda 2institute of nutritional sciences, justus-liebig university, giessen, germany morphological characteristics, bioactive compounds content, and antioxidant activity of different accessions of african eggplant (solanum anguivi lam.) aisha musaazi sebunya nakitto1, 2*, yusuf byenkya byaruhanga1, anika e. wagner2, john h. muyonga1 (submitted: july 7, 2021; accepted: december 10, 2021) * corresponding author summary african eggplant (solanum anguivi lam.) fruits reportedly exhibit antidiabetic properties, possibly due to the presence of bioactive compounds. this study aimed to assess the bioactive compounds content (bcc) and antioxidant activity (aa) in the fruits of fourteen african eggplant accessions. the relationship between the fruit bcc and aa, and the plant (leaf, stem and fruit) morphological characteristics was determined. morphological traits for the plant accessions were characterized based on existing solanum species descriptors. total phenolics, flavonoids, saponins, vitamin c and aa were determined by spectrophotometry, while total alkaloids were detected by gravimetry. hplc was used for the quantification of phenolic compounds. morphological characteristics, bcc and aa differed among the accessions. the fruit’s accessions contained total phenolics (8.0-12.4 mg gallic acid equivalent/g dry weight (dw)), saponins (51.1-124.8 mg diosgenin equivalent/g dw), alkaloids (81.4-127.7 mg/g dw), vitamin c (3.6-6.4 mg ascorbic acid equi valent/g dw), and flavonoids (0.9-2.1 mg quercetin equivalent (qe)/ g dw) and exhibited a high aa (1.2-4.6 mg qe/g dw). amongst the quantified phenolic compounds, chlorogenic acid (21.4-301.3 μg/ g dw) had the highest content. cluster analyses showed that mor phological characteristics might be useful to predict accessions with similar bcc and aa. accessions with high total phenolics provided the highest aa, and, therefore, may mediate health benefits. keywords: solanum anguivi, antioxidant capacity, bioactive com pounds, morphological characteristics, accession. introduction fruits and vegetables are particularly protective against diseases associated with oxidative stress due to possessing bioactive compounds such as phenolics, flavonoids and saponins (zhang et al., 2015). an imbalance between overproduction or accumulation of free radicals in the body and the potential of a biological system to detoxify the reactive substances results in oxidative stress (dhalaria et al., 2020). oxidative stress leads to the damage of large biomolecules such as lipids, dna, and proteins, resulting in the pathogenesis of non communicable diseases (ncds) such as type 2 diabetes mellitus (t2dm), as evidenced in several clinical and experimental studies (maritim et al., 2003; zhou et al., 2003; zhang et al., 2015). collectively, ncds (cardiovascular diseases, cancer, diabetes, and chronic respiratory disease) are responsible for 71% of all deaths worldwide (who, 2018). plant bioactive compounds with antioxidant pro perties play a vital part in reducing oxidative stress by inhibiting free radical inhibitors, which ultimately prevents and/ or treats ncds (dhalaria et al., 2020) solanum anguivi lam. fruits (salf) may alleviate diseases such as hypertension, atherosclerosis, and diabetes (elekofehinti et al., 2013a). this may be attributed to bioactive compounds present in the fruits such as phenolics, flavonoids, saponins, and alkaloids (elekofehinti et al., 2013b; oyeyemi et al., 2015). solanum anguivi lam. belongs to the family solanaceae and genus solanum l. (bukenya and carasco, 1995; united states department of agriculture, 2021). it is native to africa and has also been reported to occur in asia and australia (bukenya and carasco, 1995; jayanthy et al., 2016; nakitto et al., 2021). it is commonly known as “forest bitter berry” or “african eggplant”, although the latter is also used in reference to solanum aethiopicum (s. aethiopicum) and solanum macropcarpon (s. macropcarpon) (elekofehinti et al., 2013a). solanum anguivi lam. (s. anguivi is henceforth used in reference to the whole plant) plants are consumed as leafy and/ or fruit vegetables (denton and nwangburuk, 2011). the bioactive compounds content (bcc) and morphological characteristics of some solanum fruits have been reported to vary among their accessions (hanson et al., 2006; sseremba et al., 2017; tembe et al., 2020). although the bcc and antioxidant capacity (aa) of african eggplants have been reported (elekofehinti et al., 2013b; oyeyemi et al., 2015), there is paucity of data on their variations among the accessions. some studies (osei et al., 2010; sseremba et al., 2017; tembe et al., 2020) have reported variations in some morphological (flower, stem, fruit, and leaf) characteristics in s. anguivi accessions. however, data on variations in other morphological characteristics among s. anguivi accessions as well as the relationship between morphological characteristics and, bcc and aa are lacking. this information would equip researchers and consumers with the knowledge to identify s. anguivi accessions better, and to possibly predict the accessions with similar bcc and aa based on their morphological characteristics. this study sought to assess the bcc and aa of fruits of s. anguivi accessions and to determine whether these were related to the plant morphological characteristics. additionally, the study determined the relationship between the bcc and aa of the fruits. regression models were derived to determine the bcc that may mostly affect the aa of the fruits. materials and methods chemicals and reagents the reference standards (quercetin, gallic acid, l-ascorbic acid, diosgenin), hplc grade -methanol, ethanol, sulphuric acid, acetic acid, ammonium hydroxide; and analytical grade -thiourea, trichloroacetic acid, folin-ciocalteu reagent, 1-diphenyl-2-picrylhydrazyl (dpph), vanillin, dinitrophenyl hydrazine, hydrated copper (ii) sulphate, potassium acetate, sodium bicarbonate, and aluminium chloride, were all from sigma-aldrich (munich, germany). plant collection a preliminary survey was carried out to determine the s. anguivi accessions in mukono district (uganda), where they have been documented to grow (stedje and bukenya-ziraba, 2003). nabiyagi morphological characteristics and bioactive compounds content of solanum anguivi accessions 221 village (gps 0.472336, 32.802484) was randomly selected from mukono district as the study area, and a quadrat random sampling technique was used to identify the occurrence of s. anguivi accessions. fourteen s. anguivi accessions were available in the study area. identification of the accessions was carried out with reference to the documented phenotypic characteristics of s. anguivi. (bukenya and carasco, 1995), while authentication was carried out by an expert taxonomist at the department of botany, makerere university (uganda). samples were obtained from 12 plants per accession in may 2018. branches from each accession were plucked, and the morphological characteristics were then assessed on the same day. unripe fruits were collected from 12 plants per accession, pooled, and then dried to form flour as described under “sample preparation for chemical analysis”. this entire process was replicated after two weeks to obtain second flour samples for each accession, and thus each accession had two flours collected independently. morphological characterisation morphological characterisation was based on the descriptors of tomatoes and eggplants (ecpgr, 2008), with modifications. accessions in the present study were given codes (based on their morphological characteristics) for identification purposes. the accessions were characterized based on the amount and colour of pubescence on the leaves and stems and the colours of the leaves, stems, and fruits. fruits were further characterized based on size/diameter (very small = < 0.8 cm, small = > 0.8 1.2 cm, intermediate = >1.2 1.8 cm, large = > 1.8 cm), shape, top (style scar area) appearance, venations, and also the average number of fruits per twig. sample preparation for chemical analysis the fruits were sorted to remove those with damages on the pericarp. the stalks were then plucked off and the fruits were washed with distilled water and patted dry with a cotton cloth. mature fruits (50) of similar sizes obtained from 12 plants for each accession were cut into four parts and those infested with pests were discarded. the sliced samples were dried in an oven [infrared food oven gl-2a, guangzhou itop kitchen equipment co, ltd. guangdong, china (mainland)] at 40 °c for 16 hr. the samples were then milled (wonder mill, pocatello, idaho) at a “pastry” setting to obtain fine flour. the flours were stored in sealed plastic bottles at -20 °c until analysis. this entire process was replicated to get a second independent set of flours per accession from the fruits that were collected the second time. extraction and quantification of total bioactive compounds content and antioxidant activity of salf extraction was carried out using 80% methanol (kim et al., 2003) as described by makkar (2003), with slight modifications. the dried and powdered fruits of 0.2 g was extracted in 20 ml 80% methanol three times for 10 min each time. the extracts were then pooled together for bcc and aa analyses. each accession had two extracts from the two independent flour samples. the bcc and aa analyses were carried out in triplicates for each extract. all absorbances were measured using an ultraviolet spectrophoto meter (perkin-elmer 3100, artisan technology group 101e mercury drive, champaign, il, usa) and 80% methanol was used as blank. all quantities were expressed on a dry weight basis (dwb). the total phenolic content (tpc) was measured using the folin-ciocalteu reagent (fcr) method (singleton et al., 1998), estimated from a gallic acid (≥ 98%, sigma-aldrich, germany) standard curve (0.1 mg stock solution, 0.02 -1.0 μl) and expressed as gallic acid equivalent (gae). total flavonoids content (tfc) was determined as described by kumar et al. (2012), estimated from a quercetin (≥ 95%, sigmaaldrich, germany) standard curve (0.1mg stock solution, 0.02-1.0 μl), and expressed as quercetin equivalent (qe). total saponin content (tsc) was determined using the method of hiai et al. (1976), estimated from a diosgenin (≥ 93%, sigma-aldrich, germany) standard curve (10 100 μg/ml) and expressed as diosgenin equivalent (de). vitamin c content was determined using the method of omaye et al. (1979), estimated from an ascorbic acid (≥ 99.7%, sigma-aldrich, germany) standard curve (0.02 mg/ml stock solution, 0.063 to 0.5 ml), and expressed as ascorbic acid equivalent (aae). total alkaloid content (tal) was determined using the method by harborne (1973) and computed as mg/g. the antioxidant activity (aa) was determined by free radical scavenging capacity (frsc) and total antioxidant capacity (tac). the sample extracts were allowed to react with the stable free radical 1,1-diphenyl-2-picrylhydrazyl (dpph) (≥ 90%, merck, germany) assay according to brand-williams et al. (1995). briefly, to 3.9 ml of 6×10-5 mol/l (24 mg/l) dpph in methanol, 0.1 ml sample extract was added. the mixture was vortexed and kept in the dark for 30 min. the frsc was calculated as: eq. (1) a quercetin standard curve was prepared with frsc (%) against concentration (1-10 μg/ml), and the total antioxidant capacity (tac) of the samples was subsequently estimated from the quercetin standard curve and expressed as qe. hplc quantification of phenolic compounds hplc quantification was carried out to assess the variation of phenolic compounds in salf accessions. these included gallic acid, chlorogenic acid, caffeic acid, rutin, and quercetin, as reported by elekofehinti et al. (2013b). hplc analysis was carried out for all accessions except for gp1, which was excluded due to the small sample quantity. the fruit powders (0.5 g) were extracted in 5 ml methanol according to elekofehinti et al. (2013b) for 30 min under constant shaking (ika vibrax vxr, ika-labortechnik, staufen i.br., germany). the extracts were then filtered through whatman filter papers (grade 597 ½, diameter 125 mm) (ge healthcare-uk limited, chalfont st. giles, uk), which were then dried (speedvac plus sc11a, farmingdale, ny, usa). the dried filtrate was redissolved in 1 ml methanol, and filtered through a 0.45 μm syringe filter (carl roth). hplc analysis was performed on an agilent lc 1100-system as described by de araújo et al. (2014) with slight modifications to reflect the instrument specifications and sample volume. an eclipse plus c18 column (4.6 × 250 mm, 5 μm) with matching guard was used with solvent a (10% methanol, 0.9% trifluoroacetic acid, tfa), solvent b (75% methanol, 0.25% tfa), and a flow rate of 0.5 ml/min. the gradient programme set-points were 15-30%b (11 min); 30-99%b (11 min, 8 min steady), 99-10%b (3 min), return to initial values (2 min); all relevant peaks were eluted between minutes 8 and 30. for uv detection, the wavelength was set to detect the different phenolic compounds in one run. thus, 310 nm was used, except for the time of 8-12 min (280 nm) and from 24 onwards (250 nm). to quantify the phenolic and flavonoid compounds, 5 calibration curves (gallic acid, chlorogenic acid, caffeic acid, rutin, quercetin, with rt = 8.5, 17.4, 21.0, 25.2 and 28.2 min, respectively) with 7 concentrations (r²>99.99) were applied. all samples were run in duplicate (injection volume 5 μl). statistical analysis statistical analyses were carried out using ibm spss for windows version 21 (armonk, ny, usa). the bcc and aa data were proven for normality of distribution (kolmogorov-smirnov, and shapirofree radical scavenging capacity (%) = ((absorbance blank − absorbance of sample) × 100)/(absorbance of blank) eq. (1) 222 a.m.s. nakitto, y.b. byaruhanga, a.e. wagner, j.h. muyonga wilk). one-way anova was carried out to determine statistically significant differences in sample means, and the post-hoc duncan test was used to separate the means at p < 0.05. the results were expressed as mean ± standard error of the mean (sem). no statistical analyses were carried out for the hplc results. pearson’s correlation was carried out to determine linear relationships between aa and bcc. equations for the prediction of tac were derived using multilinear regression. a principal component analysis (pca) was applied for bcc and aa, to determine the contribution of different variables (per component) to the variance of data. categorisation of accessions based on chemical and morphological data was carried out using hierarchical (ward’s method) and two-step cluster analyses, respectively. results morphological characteristics the morphological characteristics varied among the accessions (tab. 1 and 2). the typical morphological features are shown in fig. 1 and the morphological characteristics for leaves and stems of the fourteen accessions are summarised in tab. 1. all accessions had green leaves (not shown) while 64.3% had green stems. the leaves in this study had sparse to dense pubescence, that was mostly purple (71.4%) in leaves, and white and dense (57.1%) in stems. half of the accessions had fruits with two colours (a combination of either light green, dark green, white, or cream) with each colour occupying half of the fruit from the scars (tab. 2 and fig. 2). the size of the fruits in the present study ranged from very small (< 0.8 cm) to large (> 1.8 cm), with none to intermediate venations. the fruits were mostly small (64.3%), spherical (92.7%), and flat at the top (style scar area) (64.3%). dark or very dark green fruits had sparse parallel venation, while white or light greenish cream fruits had no venation. the twigs had an average of eight fruits. bioactive compounds content and antioxidant activities of salf accessions the bcc significantly differed among accessions (tab. 3). the total phenolics (8.0 -12.4 mg gae/g dw) were highest in accession gp1 and least in wp1, while the total flavonoids (0.9 -2.1 mg qe/g dw) were highest in gv2, gv3, and gp1 and least in cp1 and wp1. the total saponins (51.1 -124.8 mg de/g dw) were highest in gc2 and gv3, and least in gc5 and gv2. the total alkaloids (81.4 -127.7 mg/g dw) were high and not significantly different among 10 accessions, with the least content in cp1 and gc1. vitamin c (3.6 -6.4 mg aae/g dw) was highest in gv3 and gc2, and least in gc3 and lv1 accessions. hplc results showed that chlorogenic acid had the highest content of the analyzed phenolic compounds in most of the salf accessions (tab. 4). the hplc results suggested variation of the phenolic compound contents amongst the salf accessions. the frsc (3.4 -11.5%) and tac (1.2 4.6 mg qe/g dw) significantly differed among salf accessions (tab. 5). accession gp1 had the highest frsc and tac, followed by gv1 while wp1 had the least. antioxidant activity and its relation with the content of the bioactive compounds tac and frsc had similar statistically significant positive correlations with tpc (r = 0.74, p = 0.000), tfc (r = 0.23, p = 0.038) and tsc (r = 0.25, p = 0.028) (tab. 6). vitamin c had a statistically significant negative correlation with frsc (r = -0.22, p = 0.048), while its negative correlation with tac was not statistically significant. principal component analysis (pca) grouped the data into three components which cumulatively explained 78.8% variation in the data (fig. 3). component 1 (frsc, tac and tpc), 2 (tsc and tal) and 3 (tfc and vitamin c content) accounted for 39.8%, 21.9% and 17.1% variance in the data, respectively. fig. 1: photographs of morphological characteristics of the leaves and stems of the solanum anguivi lam. accessions showing variation in pubescence density − sparse (a), intermediate (b), dense (c) and very dense (d); pubescence colour − purple (d), white (d) and purple and white (e); and stem colour − green (d), and green and purple (a). sparse pubescence morphological characteristics and bioactive compounds content of solanum anguivi accessions 223 tab. 1: morphological characterisation of leaves and stems of fourteen solanum anguivi lam. accessions. accession code leaf stem pub. distr. pub. colour pub. distr. pub. colour colour lv1 dense white dense white green and purple gv1 sparse white sparse white green and purple gv4 intermediate purple and white dense purple and white green cp1 intermediate purple and white dense purple and white green gc1 very dense purple and white very dense purple and white green gc2 dense purple and white dense purple and white green gc3 dense purple and white dense purple and white green and purple gc4 very dense purple and white very dense purple green and purple gc5 sparse purple and white intermediate purple and white green wp1 very dense purple and white very dense purple green gv2 intermediate white dense white green gv3 sparse white sparse white green and purple gp1 intermediate purple and white dense purple and white green gc6 dense purple and white dense purple and white green pub. distr. = pubescence distribution, pub. colour = pubescence colour tab. 2: morphological characterisation of fruits of fourteen solanum anguivi lam. accessions. accession code colour size shape fruit top venation av. twig lv1 light green small spherical flat dense, reticulate 9 gv1 very dark green very small spherical cuspidate sparse, parallel 8 gv4 dark with light green very small spherical flat intermediate, reticulate 8 cp1 white small spherical flat none 12 gc1 light green with white small spherical cuspidate sparse, parallel 12 gc2 dark green with white small spherical cuspidate sparse, parallel 12 gc3 dark with light green small spherical flat intermediate, reticulate 8 gc4 light green with white small spherical flat intermediate, reticulate 8 gc5 dark green with cream intermediate ovoid elliptic intermediate, reticulate 8 wp1 white small spherical flat none 5 gv2 dark green large spherical flat sparse, parallel 1 gv3 dark green small spherical flat sparse, parallel 6 gp1 light greenish cream small spherical conical none 8 gc6 light green with white intermediate spherical flat intermediate, reticulate 10 size= fruit size diameter (very small = < 0.8 cm, small = > 0.8 1.2 cm, intermediate = >1.2 1.8 cm, large = > 1.8 cm); av. twig = average number of fruits per twig; fruit top = style scar area. regression models were derived to determine the bcc that affected salf aa the most. model 1 was for tac mg qe/g dw (eq. 2), and model 2 for frsc (%) (eq. 3). i) model 1 eq. (2) where, r = 0.784, r2 = 0.615, adjusted r2 = 0.600 and the unstandardized coefficients of the constant, phenolics and vitamin c were statistically significant at p < .001, .001 and .005 respectively. ii) model 2 eq. (3) where r = 0.788, r2 = 0.621, adjusted r2 = 0.607 and the unstandardized coefficients of the constant, phenolics and vitamin c were statistically significant at p < .005, .001 and .005 respectively. the regression models showed that total phenolic and vitamin c contents mostly affected the tac and frsc of salf. the models (eq. 2 and eq. 3) derived in this study were good as indicated by their moderately high adjusted r2. the models may thus explain 60% variance of the salf aa. cluster analysis cluster analysis based on morphological characteristics revealed that leaf and stem pubescence colours and fruit colour were the most important for the categorisation of s. anguivi accessions (fig. 4). cluster analysis based on the chemical compositions categorized the s. anguivi accessions into four clusters (fig. 5). cluster 1 accessions had low contents of tfc and tpc and tac, with moderate to low vitamin c and tsc. cluster 2 accessions had high tfc, tpc, tsc, and tal, with low vitamin c. cluster 3 accessions had low tpc, tsc, tal, vitamin c, and tac; with moderate tfc. cluster 4 accessions had the highest tfc, tsc, tal, and vitamin c; moderate levels of tpc, and the lowest tac. tac (mg qe/g)dw = − 3.087 + 0.661[tpc(mg gae/g dw)] − 0.230[vitamin c (mg a ae/g dw) frsc % = − 6.356 + 1.561[tpc(mg gae/g dw)] − 0.608[vitamin c (mg a ae/g dw)] 224 a.m.s. nakitto, y.b. byaruhanga, a.e. wagner, j.h. muyonga gc1 gc2 gc3 gc4 gc5 wp1 gv2 gv3 gp1 gc6 fig. 2: photographs of fourteen accessions of solanum anguivi lam. fruits. photographs are not to scale. the accessions with one colour included lv1 (small, light green), gv1 (very small, very dark green), cp1 (small and white), wp1 (small, white), gv2 (large, dark green), and gv3 (small, dark green). accessions with two colours included gv4 (very small, dark with light green), gc1 (small, light green with white), gc2 (small, dark green with white), gc3 (small, dark with light green), gc4 (small, light green with white), gc5 (intermediate size, dark green with cream), gp1 (small, light greenish cream) and gc6 (intermediate size, light green with white). comparison between cluster analysis based on morphological cha racteristics (fig. 4) and that based on chemical compositions (fig. 5) showed that 35.7% of the accessions, that is, 5 (gv4, gc3, cp1, gc4, and gc6) out of the 14, appeared together in both analyses. discussion the morphological characteristics of s. anguivi plant accessions have been previously reported (osei et al., 2010; sseremba et al., 2017; tembe et al., 2020); however, the data was limited. osei et al. (2010) only reported that the salf were small with venations, however, they did not define the small size and the venations of the salf. additionally, sseremba et al. (2017) and tembe et al. (2020) did not report the fruit colours and shapes and the leaf and stem pubescence distribution of salf accessions. they only gave generalized characteristics of the solanum species in their respective studies. simultaneously, data on the appearance of the fruit top (style scar area), the average number of fruits per twig, and the amount and type of venations on the fruits are scarce and have been described in the present study (tab. 2 and fig. 2). similar to our results, osei et al. (2010) reported a round shape for salf. contrary to our findings (tab. 1), osei et al. (2010) observed an absence of pubescence on the leaves of s. anguivi accessions, and an absence to sparse pubescence in the leaves of s. aethiopicum and s. macrocarpon accessions. furthermore, the salf in the study by osei et al. (2010) were small size, unlike the present study where the salf accessions ranged from very small to large size (tab. 2). the morphological variations in the present study may be due to genetic differences in the accessions. the analysis of molecular variance (amova) by stedje and bukenyaziraba (2003) showed that the variance within s. anguivi accessions based on dna data was great (90.42%). therefore, the present study adds further information to the knowledge regarding morphological variations of s. anguivi accessions. the differences in tpc among the accessions in the current study morphological characteristics and bioactive compounds content of solanum anguivi accessions 225 tab. 3: bioactive compounds content of solanum anguivi lam. fruit accessions. accession code tpc tfc tsc tal vitamin c (mg gae/g) (mg qe/g) (mg de/g) (mg/g) (mg aae/g) lv1 10.30±0.32 c 1.58±0.05 b, c 101.17±1.64 c 108.04±1.81 a, b, c 3.56±0.19 g gv1 11.13±0.18 b 1.73±0.04 b 115.43±1.68 b 123.94±9.96 a 4.29±0.40 f gv4 10.52±0.17 b, c 1.28±0.05 c, d 87.91±3.30 e 105.46±1.71 a, b, c 4.27±0.21 f cp1 9.60±0.27 d, e, f 0.89±0.16 e 114.64±1.91 b 91.42±3.66 c, d 5.01±0.24 c, d, e gc1 9.55±0.08 d, e, f 1.56±0.09 b, c 76.02±5.80 f 81.38±2.58 d 4.31±0.21 f gc2 10.0±0.17 c, d, e 1.53±0.07 b, c 124.83±0.83 a 111.81±6.20 a, b 6.05±0.13 a, b gc3 9.59±0.17 d, e, f 1.69±0.05 b 104.48±1.41 c 113.51±12.46 a, b 4.17±0.07 f, g gc4 9.33±0.03 e, f 1.55±0.05 b, c 91.63±3.22 d, e 122.55±7.97 a 4.38±0.13 e, f gc5 9.46±0.20 d, e, f 1.72±0.22 b 51.13±1.48 g 97.77±5.84 b, c, d 4.31±0.12 f wp1 8.04±0.28 g 1.00±0.06 d, e 77.15±3.45 f 112.57±4.08 a, b 4.69±0.15 d, e, f gv2 10.10±0.19 c, d 2.07±0.09 a 58.51±1.81 g 92.26±7.68 c, d 5.20±0.18 c, d gv3 10.15±0.26 c, d 2.06±0.15 a 121.92±2.62 a, b 118.24±8.67 a, b 6.40±0.11 a solanum anguivi lam. fruits were obtained from 12 plants per accession. the fruit batches were obtained twice (two weeks apart) to obtain two independent flour samples per accession. values are the mean ± sem of two independent experiments measured in triplicates. the means were computed on a dry weight basis. means within the same column with different superscripts are significantly different at p < 0.05. tpc = total phenolic content, gae = gallic acid equivalent, tfc= total flavonoid content, qe = quercetin equivalent, tsc = total saponin content, de = diosgenin equivalent, tal= total alkaloid content, aae = ascorbic acid equivalent. tab. 4: hplc quantification of phenolic compounds in solanum anguivi lam. fruit accessions. accession code gallic acid chlorogenic acid caffeic acid rutin quercetin (μg/g) (μg/g) (μg/g) (μg/g) (μg/g) lv1 53.93 21.37 15.06 36.69 35.11 gv1 21.16 244.28 19.54 65.22 28.11 gv4 46.08 30.61 12.92 15.45 10.71 cp1 39.79 103.10 19.28 36.32 35.34 gc1 23.71 192.96 14.61 28.93 10.77 gc2 55.29 22.93 14.22 27.26 28.04 gc3 20.60 58.84 6.64 13.81 30.57 gc4 37.21 16.00 16.18 10.43 36.09 gc5 49.00 156.45 24.56 72.02 10.93 wp1 44.37 111.83 15.36 20.25 27.99 gv2 50.57 304.33 36.95 15.29 14.47 gv3 70.00 120.95 28.37 28.75 11.75 gc6 58.68 59.77 20.87 20.83 11.72 solanum anguivi lam. fruits were obtained from 12 plants per accession. fruits for each accession were dried and one extract was obtained for each accession. values are the means of duplicate analyses from one extract per accession. the means were computed on a dry weight basis. statistical analysis was not carried out on the data. may have been due to variations in the proportions of phenolic compounds contained in the different accessions as similarly reported for s. melongena genotypes by stommel and whitaker (2003). chlorogenic acid content was the highest of the quantified phenolic compounds in the salf accessions, and accessions with the highest chlorogenic acid had the highest tpc. the total flavonoid contents of the salf accessions in this study may also have been due to variations in the contents of the flavonoid compounds such as rutin and quercetin. as observed in the tpc and tfc, the saponin and alkaloid compounds may have varied among the accessions and thus influenced the accessions’ total saponin and alkaloid contents. since the accessions in this study were obtained from the same location, the differences in the bcc may be attributed to differences in the genotypes of the accessions. in agreement with our findings, s. melongena accessions have been reported to significantly differ in tpc and vitamin c contents (hanson et al., 2006), while eggplant cultivars have been reported to significantly differ in total flavonoids (kaur et al., 2014), saponins and alkaloids (agoreyo et al., 2012). to assess whether the contents of the bioactive compounds in salf were substantial for potential health benefits, we compared the salf bcc with foods that have been documented as rich sources. plums and apricots have been documented as among the top 100 most rich polyphenol sources (pérez-jiménez et al., 2010). the tpc range of salf accessions in this study was higher than for plums (5.6 mg gae/g dw) (miletić et al., 2014) and apricots (4.7 gae/g dw) (miletić et al., 2014), which suggested that all salf accessions in this study were rich in phenolics. flavonoids are reportedly rich in onions (kozłowska and szostak-węgierek, 2018) with 1.3 -2.1 mg qe/g dw (sharma et al., 2014), similar to the values in the pre sent study. the salf accessions were also possibly rich in saponins and alkaloids due to their higher contents as compared to saponin-rich fenugreek genotypes (9.0 -17.0 mg de/g dw) (arivalagan et al., 2013) and alkaloid-rich s. torvum (1.2 mg/g dw) (pérez-amador et al., 2007). the vitamin c content of the salf accessions falls within the range for tomatoes (2.0 -5.2 mg/g dw) (hallmann, 2012), which are reported as rich sources (lykkesfeldt et al., 2014). therefore, all salf accessions in the present study are potentially rich sources of total phenolics, flavonoids, saponins, alkaloids and vitamin c. 226 a.m.s. nakitto, y.b. byaruhanga, a.e. wagner, j.h. muyonga active compounds protect the body against several diseases such as cancer, type 2 diabetes, inflammatory disorders, and other cardiovascular diseases (dhalaria et al., 2020). in the present study, accession gp1 had the highest tac and frsc and may therefore possess the highest health benefits. similar to the present study, okmen et al. (2009) reported significant differences in the tac of eggplant cultivars. the positive correlation between tsc and aa in this study tab. 5: antioxidant activity of fruits from solanum anguivi lam. accessions. accession total free code antioxidant capacity radical scavenging capacity (mg qe/g dw) (%) lv1 2.68±0.14 c, d 7.29±0.36 c gv1 3.69±0.10 b 9.35±0.21 b gv4 2.43±0.04 d, e 6.4±0.09 d, e cp1 2.85±0.07 c 7.2±0.18 c, d gc1 2.31±0.07 e, f 6.19±0.16 e gc2 1.60±0.07 h 4.4±0.16 g gc3 2.60±0.11 c, d, e 6.78±0.23 c, d, e gc4 2.63±0.16 c, d, e 6.96±0.39 c, d, e gc5 1.91±0.05 g 5.24±0.12 f wp1 1.20±0.03 i 3.37±0.06 h gv2 2.05±0.08 f, g 5.29±0.17 f gv3 1.44±0.14 h 3.98±0.34 g gp1 4.58±0.14 a 11.51±0.33 a gc6 2.00±0.15 g 5.36±0.37 f solanum anguivi lam. fruits were obtained from 12 plants per accession. the fruit batches were obtained twice, two weeks apart to obtain two independent flour samples per accession. values are the mean ± sem of two independent experiments measured in triplicates. the means were computed on a dry weight basis. means within the same column with different superscripts are significantly different at p < 0.05. qe = quercetin equivalent, dw = dry weight. fig. 3: components derived from principal component analysis of solanum anguivi lam. fruits based on their bioactive compounds content and antioxidant activities. tac = total antioxidant capacity, frsc = free radical scavenging capacity, phenolics = total phenolic content, flavonoids = total flavonoid content, saponins = total saponin content, alkaloids = total alkaloid content, vit_c = vitamin c content. component 1 = free radical scavenging capacity, total antioxidant capacity, and total phenolic content; component 2 = total saponin content and total alkaloids; and component 3 = total flavonoid content and vitamin c content. free radicals are a major cause of the propagation stage of the oxidation process (elekofehinti et al., 2013b) that may lead to oxidative stress. bioactive compounds in fruits have been shown to suppress free-radical development, which further reduces the oxidative stress created in the body (dhalaria et al., 2020). consequently, the bio fig. 5: a dendrogram representing diversity and clusters of fourteen solanum anguivi lam. fruit accessions based on their bioactive compounds content and total antioxidant capacity, using ward’s minimum variance in hierarchical cluster analysis. cluster 1 accessions had low total flavonoids, phenolics and antioxidant activity, with moderate to low vitamin c and saponin contents. cluster 2 accessions had high total flavonoids, phenolics, saponins and alkaloids, with low vitamin c content. cluster 3 accessions had low total phenolics, saponins, alkaloids, vitamin c and antioxidant capacity, with moderate total flavonoid contents. cluster 4 accessions had the highest total flavonoids, saponins, alkaloids and vitamin c, with moderate levels of total phenolics, and the lowest antioxidant capacity. fig. 4: two-step cluster analysis of fourteen accessions of solanum anguivi lam. based on their morphological characteristics. rescaled distance cluster combine morphological characteristics and bioactive compounds content of solanum anguivi accessions 227 agrees with findings obtained by elekofehinti et al. (2013a), who showed that salf saponins had dose-dependent frsc. similar to our results, positive correlations between tpc and frsc of s. melon gena cultivars (okmen et al., 2009) and s. melongena accessions (hanson et al., 2006) have been reported. the negative correlation between vitamin c and frsc in the present study may show pro oxidant properties of vitamin c in salf. similarly, peyrat-maillard et al. (2001) reported that adding vitamin c to malt rootlet extracts led to an antagonist effect on their tac. vitamin c pro-oxidant properties are reported to be more evident under some circumstances, such as the presence of metal ions (iron and copper) and adequate ph (alkali) conditions (arrigoni and de tullio, 2002). ghislaine et al. (2014) and oyeyemi et al. (2015) reported the presence of iron (467.7 and 22.2 mg/100 g dw, respectively) and copper (0.1 and 1.4 mg/ 100 g dw, respectively) in salf. further studies investigating the antagonistic effect of vitamin c in salf and its possible relationship with iron and copper contents may be suitable. cluster analysis was carried out to assess whether the morphological characteristics of s. anguivi may be used to predict the salf accessions with similar bcc and aa. the clusters formed based on chemical compositions (fig. 5) agree with the correlation results and the aa regression models in this study. cluster 1 was characterized by low phenolics and flavonoids, which may explain their low tac. furthermore, the accessions that had moderate vcc and low saponin contents had the least tac, and those with low vcc and moderate tsc had significantly higher tac than the former. cluster 2 accessions had chemical compositions similar to the correlation results obtained in this study: high phenolic, high flavonoid, high saponin, and low vcc, and high tac. although cluster 3 accessions were characterized by low vitamin c, and high flavonoid contents, the low phenolic and saponin contents may have resulted in the low tac. the low tac in cluster 4 may be due to the high vcc, whose negative effect may have suppressed the positive contribution on tac by the mode rate and high amounts of phenolics and saponins, respectively. bcc and tac may therefore be good predictors for variation among salf accessions given that the results from the cluster analysis were cohe rent with the correlation and regression analyses. due to some similarities between the clusters formed based on morphological characteristics and based on chemical composition (35.7% of the accessions were grouped together in both cluster analyses), morphological features may be used to predict some accessions with similar chemical compositions. conclusion the morphological characteristics of s. anguivi varied among the accessions. all accessions were rich in phenolics, flavonoids, saponins, alkaloids, and vitamin c. however, the bcc and aa significantly differed among the salf accessions. chlorogenic acid had the highest content of the analyzed phenolic compounds in the fruits. high amounts of total phenolic, flavonoid, or saponin contents may lead to high aa, while high vitamin c content may negatively affect the aa of salf. therefore, the results guide which accession one may consume to benefit from a given bioactive compound. the association between the morphological characteristics and the chemical compositions for some salf accessions suggested that the morphological characteristics of s. anguivi lam. may be used to predict accessions with similar bcc and aa. acknowledgment the german academic exchange service (daad) and food technology and business incubation centre (ftbic) (makerere university) funded the study. the funding sources were not involved in the conduction of the research and/or preparation of 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(solanaceae) in uganda. euphytica 131, 293-297. doi: 10.1023/a:1024079208879 stommel, j.r., whitaker, b.d., 2003: phenolic acid content and composition of eggplant fruit in a germplasm core subset. j. am. soc. hortic. sci. 128, 704-710. tembe, k., lagat, s., ambuko, j., chemining’wa, g., owino, w., 2020: variation in morphological and agronomic traits of selected african eggplant accessions. j. med. act. plants 9, 34. doi: 10.7275/1fy5-dy82 united states department of agriculture, 2021: plants profile for solanum anguivi [www document]. nat. resour. conserv. serv. solanum anguivi lam. url https://plants.usda.gov/core/profile?symbol=soan8 (accessed 3.25.20). who, 2018: non-communicable diseases. url https://www.who.int/newsroom/fact-sheets/detail/noncommunicable-diseases (accessed 4.8.20). zhang, y.j., gan, r.y., li, s., zhou, y., li, a.n., xu, d.p., li, h. bin, kitts, d.d., 2015: antioxidant phytochemicals for the prevention and treatment of chronic diseases. molecules 20, 21138-21156. doi: 10.3390/molecules201219753 zhou, j.f., chen, p., zhou, y.h., zhang, l., chen, h.h., 2003: 3,4-methylenedioxymethamphetamine (mdma) abuse may cause oxidative stress and potential free radical damage. free radic. res. 37, 491-497. doi:10.1080/1071576031000076286 orcid aisha musaazi sebunya nakitto https://orcid.org/0000-0002-4986-5713 yusuf b. byaruhanga https://orcid.org/0000-0001-8283-1681 anika e. wagner https://orcid.org/0000-0003-3047-7128 john h. muyonga https://orcid.org/0000-0002-5598-1462 address of the corresponding author: aisha musaazi sebunya nakitto, school of food technology nutrition and bioengineering, makerere university, p.o. box 7062 kampala, uganda. e-mail: aishasebunya@gmail.com; aisha.nakitto70@students.mak.ac.ug © the author(s) 2021. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1007/978-94-017-0273-7 http://dx.doi.org/10.1515/ijfe-2017-0302 http://dx.doi.org/10.1002/jsfa.5617 http://dx.doi.org/10.1016/j.jfca.2006.03.001 http://dx.doi.org/10.1007/978-94-009-5921-7 http://dx.doi.org/10.1055/s-0028-1097639 http://dx.doi.org/10.5958/0974-4150.2016.00022.5 http://dx.doi.org/10.1016/j.foodres.2013.09.049 http://dx.doi.org/10.1021/jf0343074 http://dx.doi.org/10.1007/978-3-319-54528-8_54-1 http://dx.doi.org/10.1016/s2221-1691(12)60019-7 http://dx.doi.org/10.3945/an.113.005157 http://dx.doi.org/10.1002/jbt.10058 http://dx.doi.org/10.17221/166/2013-cjfs http://dx.doi.org/10.3390/molecules26072044 http://dx.doi.org/10.1080/10942910801992942 http://dx.doi.org/10.1016/0076-6879(79)62181-x http://dx.doi.org/10.1038/ejcn.2010.221 http://dx.doi.org/10.1006/fstl.2001.0752 http://dx.doi.org/10.1002/jsfa.6450 http://dx.doi.org/10.1016/s0076-6879(99)99017-1 http://dx.doi.org/10.5897/jpbcs2017.0663 http://dx.doi.org/10.1023/a:1024079208879 http://dx.doi.org/10.7275/1fy5-dy82 http://dx.doi.org/10.3390/molecules201219753 http://dx.doi.org/10.1080/1071576031000076286 https://orcid.org/0000-0002-4986-5713 https://orcid.org/0000-0001-8283-1681 https://orcid.org/0000-0003-3047-7128 https://orcid.org/0000-0002-5598-1462 journal of applied botany and food quality 95, 23 30 (2022), doi:10.5073/jabfq.2022.095.004 1laboratory of legumes and sustainable agrosystems, centre of biotechnology of borj-cedria, hammam-lif, tunisia 2faculty of sciences of gabès, university of gabes, gabes, tunisia 3laboratory of extremophiles plants, centre of biotechnology of borj-cedria, hammam-lif, tunisia effect of increasing zinc levels on trigonella foenum-graecum growth and photosynthesis activity fadwa melki1, 2, ons talbi zribi3, sabrine jeder1, 2, faten louati1, issam nouairi1, haythem mhadhbi1, kais zribi1* (submitted: september 21, 2021; accepted: january 17, 2022) * corresponding author summary zinc is an indispensable element for the plant growth and the cellu lar metabolism. however, this mineral element becomes harmful at high quantities. the effects of high zinc supply on different physio logical parameters were investigated in fenugreek. seedlings were grown in plastic pots filled with inert sand under five znso4 treatments (c: control :1.5 μm zn; 1 mm, 2 mm, 3 mm and 4 mm znso4). results showed a decrease of 56% to 75% in shoot dry weight and a decrease of 65% to 90% in roots dry weight, relatively to the control. in addition we showed a significant reduction in photosynthetic para meters, with the highest value of co2 assimilation under 1 mm zn (3.3 μmol co2, m-2·s-1) and a lower value under 4 mm zn (0.5 μmol co2, m-2·s-1). the concentration of zinc in plant shoot was around two folds the control under 1, 2 and 3 mm zn and about four folds under the maximal concentration, 4 mm zn. in roots, we showed a progressive increase of zinc content. increasing zinc concentration induced a significant decrease of phosphorus concentration in shoot. fenugreek was mainly affected by zinc excess greater than 1 mm znso4, however at the highest concentration, fenugreek plants ex hibited different adaptation strategies. key words: trigonella foenum-graecum, heavy metals toxicity, soil contamination, mineral nutrition, plant physiology introduction zinc (zn) is an essential micronutrient that is needed by plants for growth and varied biochemical and physiological pathways (dinesh et al., 2018). it plays a vital role as a constituent of metalloenzyme and a cofactor for some enzymes such as dehydrogenases, oxidases and peroxidases (broadley et al., 2007). it is involved also in the synthesis of chlorophyll and carotenoids, auxin, nucleic acids and proteins. furthermore, it plays an important role in photosynthesis and in the regulation of cytoplasmic concentration of nutrients, structural stability of cell membranes and proteins, as well as protection of biomembranes against oxidative damage (cakmak, 2000; paunov et al., 2018; dos santos et al., 2019). nevertheless, high levels of this element can induce toxic effects to sensitive plants. indeed, frequent zn contamination of surface soils originating from prolonged use of zn fertilizers, input from industrial pollution, and mining activities may lead to zn toxicity in plants (martens and smolders, 2013). broadley et al., (2007) pointed out that high levels of zn could inhibit plant growth and evolution by inducing a perturbation in the absorption and the translocation of nutrients or by interfering with metabolic processes and antioxidant defense system. indeed, excess zn induce generally functional disorders in plants such as plasma membrane permeability damage and photosynthesis inhibition, as well as interference with phosphorous, magnesium, and manganese uptake (sagardoy et al., 2009; paunov et al., 2018; khan et al., 2019; szopiński et al., 2019; dobrikova et al., 2021). zn toxicity symptoms include stunted growth, leaf chlorosis, necro tic spots, root system damage and disturbance in water balance (sagardoy et al., 2010). plants differ in their ability to tolerate ele vated concentrations of zn in the soil (rascio and navari-izzo, 2011; andresson et al., 2018). similarly, to other countries, tunisia faces the problem of zn toxicity, notably in soils around open-cast mining. the culture of legumes in marginal and moderately contaminated soils could be beneficial in double ways. in fact, legumes with their specific characteristic to fix symbiotically the nitrogen in association with rhizobia can contribute to the rehabilitation and the decontamination of these soils. in the other way, moderately contaminated soils could be additional areas for the culture of some heavy metals-tolerated legumes. to verify this hypothesis, the selection of legumes species according to their zn tolerance and metal accumulation potential was a subject of several researches. zribi et al. (2015) showed that medicago sativa under nitrogen fixing symbiosis could tolerate 2 mm zn and accumulate zn in their roots and promoting the phytostabilisation process. a study carried out on the behaviour of trifolium repens growing in a field polluted with cd, pb and zn showed that the metals were preferentially accumulated in the roots than in the aerial part and exposed the plants to the oxidative stress (bidar et al., 2007). fenugreek (trigonella foenum-graecum l.) belongs to the family of fabaceae and it is cultivated in many countries such as india, pakistan, egypt, france, yemen, spain, turkey, morocco, china and tunisia. india is the largest producer in the world (zandi et al., 2015). fenugreek can be grown easily in low-input, marginal environment. it is a precious aromatic and medicinal plant used for its healthy and nutritious benefits, as it contains a considerable number of vitamins, proteins and minerals (zandi et al., 2015; ahmad et al., 2016). by contrast, these virtues of fenugreek cannot deny that this plant is somehow known by its considerable ability to stock enormous amounts of particular heavy metals (pattnaik and reddy, 2011). the aim of this study was to investigate the behavior of fenugreek under zn excess in term of growth, photosynthesis activity and nutrition and to verify if this legumes species is appropriate to be cultivated in zn contaminated soils. materials and methods plant material and germination conditions seeds of fenugreek plant (trigonella foenum-graecum l.) were disinfected and sterilized with ethanol (70%) for 1 minute, then they are rinsed several times with distilled water. the seeds were soaked in distilled water for 3 hours and germinated in darkness at 25 °c in in petri dishes on filter paper moistened with distilled water. threeday-old seedlings were then transferred for 52 days into plastic pots (0.5 l) filled with sterile sand (three seeds were replicated in every pot and before the start of treatments only the performant seedling was retained). the experiment was carried out in controlled greenhouse (25 °c/19 °c, 16 h photoperiod, and 60% relative humidity). 24 f. melki, o.t. zribi, s. jeder, f. louati, i. nouair, h. mhadhbi, k. zribi plants were irrigated with nutrient solutions (vadez et al., 1996) added by different zn concentrations. the nutrient solution contained the following concentrations of macro and micro-elements: kh2po4 (0.36 mm); k2so4 (0.7 mm); mgso4, 7h2o (1 mm); cacl2 (1.65 mm); hbo3 (4μm); mnso4, h2o (6.6 μm); znso4, 7h2o (1.55 μm); cuso4, 5h2o (1.56 μm); na2moo4, 7h2o (0.12 μm); coso4, 7h2o (0.12 μm) and fe (1.26 mg·l-1). the ph of the nutrient solution was adjusted to 7. a concentration of 1.55 μm znso4 was used as control (c), and the excess zn treatments were 1 mm (zn1), 2 mm (zn2), 3 mm (zn3) and 4 mm znso4 (zn4). the experiment was set up in a completely randomized design with five replications (5 pots per treatment) and the irrigation was performed homogeneously with 40 ml of the nutritive solution in each pot (estimated after field capacity calculation), generally two times per week. growth parameters fifty-two days old plants were harvested and separated into shoots and roots. roots were rinsed three times with cold distilled water and blotted with filter paper. the fresh weight (fw) was immediately determined, while the dry weight (dw) was determined after drying in an oven at 60 °c until constant weight. the length of the primary root was also measured at the final harvest with a ruler. tolerance index (ti) was calculated as the ratio between whole plant dry weights of plants cultivated in presence of zn and the whole plant dry weight of control plants (ullah et al., 2020). water relations tissue water content (twc) was determined using the following equation: tw (ml·g-1 dw) = (fw-dw)/dw where fw is fresh weight determined 2 h after harvest, and dw is dry weight obtained after drying at 60 °c to constant weight. pigment content and gas exchange leaf chlorophyll and carotenoid concentrations (mg·g-1 fw) were determined spectrophotometrically according to arnon (1949) and mc kinney (1941). five ml of acetone 80% were added to fresh leaf samples. chlorophyll (a, b) and carotenoid concentrations were measured at 645, 663 and 460 nm respectively according to the equations reported by mc kinney (1941). co2 assimilation rate (a), stomatal conductance (gs), transpiration rate (e) and water use efficiency [wue = a/e] were determined on fenugreek leaves just before harvest by using a portable infrared co2/h2o gas exchange system (lcpro+, uk). measurements were carried out between 10:00 and 13:00 h on the youngest fully emerged leaf (n = 3 leaf samples taken from three different plants per treat ment). data were automatically collected every minute after the photosynthesis rate had stabilised. ions content desiccated shoot and root samples were ground to a fine powder using porcelain mortar and pestle, and then ions digestion was achieved in 4/1 (v/v) hno3/hclo4 mixture (somer and unlu, 2006). zn, magnesium (mg) and iron (fe) concentrations were determined by atomic absorption spectroscopy (aas) (perkin elmer analyst 300) and phosphorus was assayed using the vanado-molybdate method (fleury and leclerc, 1943). translocation factor (tf) was calculated as the ratio of the metal concentration in shoots to metal concentration in roots (zhou et al., 2013). proline content proline content was determined according to bates et al. (1973). samples were homogenized in 3% (w/v) sulfosalicylic acid and centrifuged for 15 min at 14000 g. the supernatants added with ninhydrin and glacial acetic acid were then incubated for 1 h in boiling water. after cooling in an ice bath, toluene was added and proline was assayed with a spectrophotometer at 520 nm. proline content was calculated against standard proline. statistical analyses data were analysed using the statistical software xl stat (anova i). the number of repetitions was three for ions content and gas exchange parameters and five for other parameters. significant differences between means were separated using the duncan test (p = 0.05). results zinc effects on plant growth and water content a significant reduction in shoot dw was observed in fenugreek plants grown in sand culture supplemented with 1, 2, 3 and 4 mm znso4 (56%, 68%, 73% and 75% relatives to control, respectively) (fig. 1a). root dw was more sensitive to zn toxicity than shoot dw. indeed, root biomass decreased by 65, 73, 77 and 90% in presence of 1, 2, 3 and 4 mm znso4 respectively as compared to control plants (fig. 1b). shoot and root lengths are similarly affected by zn supply (fig. 1c and 1d). indeed, these parameters significantly decreased by 29, 39, 45 and 53% in presence of 1, 2, 3 and 4 mm znso4 respectively as compared to control plants. thus, it appears that biomass accumulation is more susceptible to zn toxicity than length. root/shoot (r/s) dw ratio decreased by 17, 20, 32 and 68% in plants grown with 1, 2, 3 and 4 mm znso4 in the culture medium respectively when compared to controls. interestingly, zn supply has no significant effect on both shoot and root water content of fenugreek plants as compared to control plants (fig. 2). leaf gas exchange and pigment concentration variations in main gas exchange parameters were similar to those observed for plant biomass production. control plants displayed the highest values of net co2 assimilation rate (a) (fig. 3a), stomatal fig. 1: shoot (a) and root dry weight (b) and shoot (c) and root length (d) of fenugreek plants grown with different znso4 concentrations for 52 days. values on the error bars of a and b correspond to the root:shoot dw ratio, values (means ± se of five replicates) followed by the same letter are not significantly different (duncan test, p = 0.05). zinc effect on physiology of fenugreek 25 conductance (gs), (fig. 3b) and leaf transpiration rate: e (fig. 3c). with the increase in soil zn content, a, gs, and e in leaves decreased significantly. co2 assimilation rate and transpiration rate decreased by approximately 34%, 52%, 69% and 74% in presence of 1, 2, 3 and 4 mm znso4 respectively as compared to control plants. nevertheless, stomatal conductance decreased by 50, 72, 84 and 85% in zn1, zn2, zn3 and zn4 treatments respectively comparing to treated plants. it is worth mentioning that zn supply has no significant effect on wue in fenugreek plants (fig. 3d). the changes in pigments contents showed similar trends, decreasing with the increase of soil zn content compared to control plants (tab. 1). chl a, b and total content decreased significantly by approximately 43, 75, 79 and 84% in presence of 1, 2, 3 and 4 mm znso4 respectively. total carotenoid content decreased also by 57, 78, 85 and 89% in zn1, zn2, zn3 and zn4 plants respectively as compared to control ones. the chl a/b ratio decreased by almost 18% in all treated plants. however, the caro/chl ratio decreased by 32% in presence of 1, 3 and 4 mm znso4 as compared to control plants. nutrition status root and shoot zn concentrations of fenugreek plants increased generally in response to increasing zn concentrations in the growth medium. root zn concentrations increased by 1.9; 2.2; 2.9 and 3.3 fold in presence of 1, 2, 3 and 4 mm znso4 respectively. however, shoot zn concentrations increased by 2 -fold in zn1 and zn2 treatments and by 2.3 and 3.7-fold in zn3 and zn4 treatments respectively comparing to treated plants. it is worthy to indicate that under control conditions as well as in presence of 1, 2, and 4 mm znso4 the accumulation of zn in fenugreek plants was approximately the same between roots and shoots (fig. 4). the rate of zn translocation from roots to shoots was about 0.90, 0.95 and 1.0 in control and zn1 and zn4 treatments. it is worth mentioning that there is no significant difference in this parameter between control and zntreated plants. phosphorus (p) concentrations decreased significantly by 65% in shoots of fenugreek plants cultivated in presence of 3 and 4 mm znso4 but did not change significantly in roots (fig. 5). c 1 2 3 4 znso4 (mm) a a a a a 0 2 4 6 8 10 12 sh oo t w at er c on te nt (m l. g1 d w ) a a a a a r oo tw at er c on te nt (m l. g1 d w ) c 1 2 3 4 znso4 (mm) 0 2 4 6 8 10 12 c 1 2 3 4 znso4 (mm) a a a a a 0 2 4 6 8 10 12 sh oo t w at er c on te nt (m l. -1 d w ) a a a a a r oo tw at er c on te nt (m l. g1 d w ) c 1 2 3 4 znso4 (mm) 0 2 4 6 8 10 12 24 a b bc c c 0 2 4 6 8 a ( m ol c o 2. m -2 s1 ) cc bc b a 0 0.1 0.2 0.3 0.4 gs (m m ol h 2o . m -2 s1 ) dd c b a 0 0.5 1.0 1.5 2.0 e ( m ol . m -2 s1 ) 0 a a a a b 2 4 6 w u e (a /e ) c 1 2 3 4 znso4 (mm) c 1 2 3 4 znso4 (mm) a b bc c c 0 2 4 6 8 a ( m ol c o 2. m -2 s1 ) cc bc b a 0 0.1 0.2 0.3 0.4 gs (m m ol h 2o . m -2 s1 ) dd c b a 0 0.5 1.0 1.5 2.0 e ( m ol . m -2 s1 ) 0 a a a a b 2 4 6 w u e (a /e ) c 1 2 3 4 znso4 (mm) c 1 2 3 4 znso4 (mm) c bc b a a r oo t z n co nc en tr at io n (m g. g -1 d w ) a b cbc d 0 0.5 1.0 1.5 2.0 2.5 sh oo t z n co nc en tr at io n (m g. g -1 d w ) 0 0.5 1.0 1.5 2.0 2.5 ab ab bc c a 0 0.3 0.6 0.9 1.2 1.5 z n tr an sl oc at io n fr om ro ot s to s ho ot s c 1 2 3 4 znso4 (mm) (a) (b) (c) c bc b a a r oo t z n co nc en tr at io n (m g. g -1 d w ) a b cbc d 0 0.5 1.0 1.5 2.0 2.5 sh oo t z n co nc en tr at io n (m g. g -1 d w ) 0 0.5 1.0 1.5 2.0 2.5 ab ab bc c a ab ab bc c a 0 0.3 0.6 0.9 1.2 1.5 z n tr an sl oc at io n fr om ro ot s to s ho ot s c 1 2 3 4 znso4 (mm) (a) (b) (c) fig. 2: shoot and root water content of fenugreek plants grown with dif ferent znso4 concentrations for 52 days. values (means ± se of five replicates) followed by the same letter are not significantly different (duncan test, p = 0.05). fig. 3: co2 assimilation rate (a), stomatal conductance (b), transpiration rate (c) and water use efficiency (d) of fenugreek plants grown with different znso4 concentrations for 52 days. values (means ± se of five replicates) followed by the same letter are not significantly different (duncan test, p = 0.05). tab. 1: effect of zn supply on leaf pigment content of fenugreek plants grown with different zn concentrations for 52 days. values (means se of three replicates) followed by the same letter are not significantly different (duncan test, p=0.05). c 1 mm 2 mm 3 mm 4 mm chla (mg.g-1 fw) 7.99 a 4.54 b 1.97 c 1.61 c 1.21 c chlb (mg.g-1 fw) 2.80 a 1.96 b 0.79 c 0.70 c 0.53 c chlt (mg.g-1 fw) 10.7 a 6.50 b 2.76 c 2.31 c 1.75 c carot (mg.g-1 fw) 1.62 a 0.69 b 0.35 c 0.23 c 0.17 c chl a/b 2.87 a 2.48 b 2.31bc 2.29 bc 2.25 c car/chl 0.15 a 0.10 b 0.13 a 0.10 b 0.10 b fig. 4: shoot zn concentration (a), root zn concentration (b) and zn translocation factor (c) of fenugreek plants grown with different znso4 concentrations for 52 days. values (means ± se of three replicates) followed by the same letter are not significantly different (duncan test, p = 0.05). 26 f. melki, o.t. zribi, s. jeder, f. louati, i. nouair, h. mhadhbi, k. zribi proline content our results showed that increasing zn concentration has no significant effect on both shoot and root proline content in fenugreek plants (fig. 6). symplastic transport. in the present study, fenugreek plants cultivated during 52 days in sand culture supplied with 1 mm znso4 showed 56% reduction in shoot dw compared to control plants. however, al khateeb and al-qwasemeh (2014) showed a 70% reduction in relative fresh weight of two solanum species: solanum lycoper sicum and solanum nigrum grown in vitro under 1 mm znso4. furthermore, the tolerance index calculated on the basis of whole plant dw of fenugreek plants cultivated in presence of 1 mm znso4 was 0.38 (tab. 2) which suggest relative tolerance of this species to zn toxicity when cultivated under 1mm znso4. indeed, lux et al. (2004) pointed out that plants with tolerance index between 0.35 and 0.6 are considered as plants with medium tolerance. however, in presence of a severe zn stress (4 mm znso4), the tolerance index decreased substantially. besides zn vacuolar compartmentalization, cited as a potential mechanism for zn detoxification, its retention by linking to the molecule of phytic acid in non-vacuolated tissues may also play a key role in zn detoxification (andressen et al., 2018). 0 5 10 15 20 25 30 p ro lin e co nt en t ( m ol . g -1 d w ) a a a a a shoot root a a a a a c 1 2 3 4 znso4 (mm) 0 5 10 15 20 25 30 p ro lin e co nt en t ( m ol . g -1 d w ) a a a a a shoot root a a a a a c 1 2 3 4 znso4 (mm) fig. 6: shoot and root proline content of fenugreek plants grown with different znso4 concentrations for 52 days. values (means ± se of three replicates) followed by the same letter are not significantly different (duncan test, p = 0.05). fig. 5: shoot and root phosphorus content of fenugreek plants grown with different znso4 concentrations for 52 days. values (means ± se of three replicates) followed by the same letter are not significantly different (duncan test, p = 0.05). discussion the present study revealed that the increase of znso4 concentration in the medium leads to the reduction of fenugreek growth features. this may be due to the toxic effect of zn that damages plant growth (paunov et al., 2018). indeed, lower levels of znso4 reduced dry weight, shoot length and root length slightly compared to higher levels (fig. 1). reduction of growth under excess zn have already been described for other species and it varies in plants according to their tolerance level and experimental conditions such as concentration of the metal and the duration of the stress (sinisha and puthur, 2018). our results showed that the decrease in root biomass of fenugreek plants cultivated under zn excess was slightly higher than in shoot biomass and r/s dw ratios decreased significantly with increasing zn supply. these findings suggest that zn had more inhibitory effects on the root than on the shoot. similar results were observed by sagardoy et al. (2009). glińska et al. (2016) pointed out that excess zn decreased shoot and root growth of triticum aestivum seedlings likely by disturbing cell division and/or elongation. li et al. (2013) revealed that the reduction of root growth in triticum aestivum seedlings exposed to zn excess is linked to a substantial loss of cell viability in the root tips and to an increased level of lignification. furthermore feigl et al. (2015) demonstrated that high zn concentrations caused significant deposition of callose in root apical meristem of b. juncea and b. napus, which may contribute to growth inhibition because it lowers cell wall loosening and hampers tab. 2: tolerance index of fenugreek cultivated during 52 days in presence of increasing concentrations of znso4 zn1 zn2 zn3 zn4 tolerance index 0.38 0.29 0.25 0.16 our results showed also that shoot and root length are similarly affected by zn supply. in contrast, feigl et al. (2016) showed no significant effect of zn supply on root length in brassica napus plants cultivated during fourteen days under 50, 150 and 300 μm znso4. marichali et al., (2016) reported that the repression of root elongation of nigella sativa l. exposed to zn excess was explained by the inhibition of cell proliferation and subsequent elongation. kaur and garg (2021) pointed out that the decrease in growth under zn excess is a non-specifc manifestation of alterations in physio-biochemical traits which can result from direct effects (toxicity due to accumulation in tissues) and/or from indirect effects. indirect factors include disturbances in photosynthetic activity, limitation of minerals and water acquisition as well as the induction of oxidative stress through overproduction of ros. zhang et al. (2020) reported that excess zn damages the organization of mitochondria and leads to decrease in nicotinamide levels, and consequently reduced the energy metabolism, which may explain for the diminution in overall plant growth. remarkably, we conclude in this research that increasing znso4 concentration has no significant effect in both shoot and root water content. these results suggest that osmotic adjustment in fenugreek subjected to zn excess is efficient and that this cultivar may regulate cell osmotic potential to reduce toxic effects of zn. contrarily, many studies showed a decrease in both, shoot and root water content of many species under zn excess exposure (sagardoy et al., 2009; rucinska-sobkowiak, 2016). by maintaining the water content, fenugreek seems capable to avoid the reduction in root and shoot hydraulic conductivity and the reduction in aquaporin activity, which are described as principal causes of water content decrease (kaur and garg, 2021). these results suggest also that fenugreek may regulate cell osmotic potential to maintain stable water status under zn stress exposure. the reduction of growth by zn toxicity in fenugreek plants may be the consequence of the decrease in photosynthesis. indeed, it was show that the plant growth is closely, related to the quantity of assimilated co2. similarly, it has been shown that photosynthesis activity depend to zn supply (sagardoy et al., 2009 and 2010). indeed, according to van assche and clijsters (1986), high zn concentrations can affect rubisco activity. the decrease in photosynthesis under zn excess exposure could be due to many factors such as the decrease in chlorophyll biosynthesis, the inhibition of the activities zinc effect on physiology of fenugreek 27 of key enzymes of the calvin cycle, the reduction in chlorophyll a fluorescence and the inhibition of photosynthetic electron transport (yang et al., 2020). our data showed also that stomatal conductance and transpiration rate decreased significantly with increasing zn supply. this may be interpreted as a water saving mechanism, which correlates with changes in shoot water content. furthermore, the decrease in gs may be ascribed to stomatal closure or decrease of the stomatal aperture size. it was also demonstrated that the decrease in stomatal conductance under zn excess may be related to an alternation in the k+/ ca2+ ratio in the guard cells and/or to the abscisic acid concentration, which controls the stomatal movement (marschner, 1995). another factor that is important to consider in plants under zn toxicity is water use efficiency (wue). we showed in this experiment that increasing zn concentrations in the culture medium up to 3 mm znso4 has no significant effect in wue in fenugreek plants. leaf chlorophyll content is an important physiological index directly related to photosynthesis in plants. chlorophylls and carotenoids are involved primarily in light harvesting and a balance in their amounts is imperious for optimum light energy capture in photosynthesis (wahid and ghazanfar, 2006; polivka and frank, 2010). in the present experiment, we noted a gradual decrease in the concentrations of chlorophyll a, b total and carotenoids with the increase in znso4 concentrations in the soil. furthermore, paunov et al. (2018) reported a 55% decrease in chl a content and 24% reduce in chloro phyll b content in leaves of durum wheat after 7 days treatment with only 600 μm zn. nevertheless, zhang et al. (2020) showed no significant effect of zn stress in the content of chl and car in tobacco leaves cultivated during 10 days in presence of only 200 μm zn. the decline in chlorophyll content in the plants exposed to zn toxicity is believed to be probably due to (i) inhibition of important enzymes, such as 6-aminolevulinic acid dehydratase (aladehydratase) (padmaja et al., 1990) and protochlorophyllide reductase (van assche and clijsters, 1990) associated with chlorophyll biosynthesis; and/or (ii) impairment of the supply of mg2+ and fe2+ (marschner, 1995). however, in this experiment, we showed that shoot mg and iron concentration of fenugreek plants was not significantly affected by zn supply (fig. 7) which suggest that the decrease in chlorophyll content of fenugreek leaves was mainly due to the decrease of activities of enzymes related to chlorophyll biosynthesis. furthermore, many studies suggest that heavy metal ions could interfere with chl biosynthesis through central mg ion substitution, which therefore impairs the functioning of light harvesting complex ii: lhcii (cenkci et al., 2010). in this study, fenugreek plants cultivated under znso4 showed a significant decrease in chl a/b ratio as compared to control plants, suggesting that chl a was degraded at a higher rate than chl b. similar findings are observed in phasoelus vulgaris plants cultivated under zn excess (vassilev et al., 2011). reduction of the chlorophyll a/b ratio indicate that psi is degraded faster than psii (andersson et al., 2004) or that lhcii remains intact longer than the reaction centres (moy et al., 2015). similar to chlorophyll, carotenoids content also significantly decreased in response to increasing soil zn concentrations. giannakoula et al., (2021) reported that the reduction in carotenoid content observed in many plant species during metal toxicity may be due to a protective mechanism that preserves chlorophyll pools at the expense of carotenoids, due to overproduction of reactive oxygen species. broadley et al. (2007) reported that high levels of zn could decrease plant growth via the induction of a perturbation of the absorption and the repartition of nutrients or by interfering with metabolic processes and antioxidant defence system. in the present study, control plants showed statistically significantly lower amounts of zn in both shoots and roots compared to all zn-treated plants. furthermore, the strong positive correlation between zn in plant roots and zn in the culture medium indicates that the zn content in roots strongly depend on its concentration in the soil. marschner (1995) reported that relatively to the highly mobile elements such as k or p and the immobile element ca, zn has an intermediate mobility. our results showed that under control conditions as well as in pre sence of znso4, the accumulation of zn in fenugreek plants was approximately the same between roots and shoots. rascio and navariizzo (2011) pointed out that zn repartition and translocation in plants is influenced by the level of zn amount and plant species. at high exogenous quantities of zn, the tolerance of plants is expressed by an accumulation of this metal in the root and the leaves. pearson and rengel (1995) have suggested that the transpiration stream may be a driving force in translocation of zn and its accumulation in leaves. furthermore, fenugreek plants cultivated in presence of znso4 accumulate high shoot zn concentrations. it seems that fenugreek is likely characterized by an efficient zn transport from roots to shoots. indeed, plants tolerant to zn toxicity can reduce the metal damage and grow optimally under zn excess (mateos-naranjo et al., 2014). however, in the present study, we found that plant biomass production was more closely related to co2 assimilation rate, stomatal conductance and pigment contents than to translocation factor. indeed, a weak correlation was found between translocation factor and whole plant dw (data not shown). thus, our findings suggest that the increasing accumulation of zn in plant leaves subjected mainly to 4 mm znso4 may be attributed to the inability of fenugreek plant to chelate zn with organic and inorganic acids to make it insoluble and limit its transport from roots to leaves. accumulated zn may affect the structure of the thylakoid membranes in chloroplasts and lead to a decrease of electron transport rates. therefore, the effects of zn stress on plant growth and biomass production were likely related to the accumulation of zn in leaves at first and then reduced photosynthesis. huang et al. (2019) observed similar results in zelkova schneideriana plants cultivated during 15 days in presence of increasing concentrations of zn. several research accomplished in different plant types showed the presence of a reversed relationship between phosphorus (p) and zn accumulation in plants (khan et al., 2019). furthermore, zn excess is known to interfere with fe, p, mg and mn uptake by competing with these ions for binding at numerous sites, such as principal absorption region or loading region of roots (tewari et al., 2008). bazihizina et al. (2014) pointed out that changes in nutrient contents under zn excess exposure may also be due to alteration in the functioning of membrane transporters and ion channels and to membrane depolari zation. in this experiment, shoot fe content of fenugreek plants was not affected by zn excess exposure. similarly, yang et al. (2011) reported that vitis vinifera leaves retained high level of fe under zn stress, which was attributed to enhanced translocation of this element from the root to shoots. in our present work, we found that increasing zn amount has no ab ab b ab a 0 1 2 3 sh oo t f e (m g. g -1 d w ) znso4 (mm) c 1 2 3 4 ab b b b a 0 10 20 30 40 50 znso4 (mm) c 1 2 3 4 sh oo t m g (m g. g -1 d w ) fig. 7: shoot content of mg and fe of fenugreek plants grown with different znso4 concentrations for 52 days. values (means ± se of three replicates) followed by the same letter are not significantly different (duncan test, p = 0.05). ab ab b ab a 0 1 2 3 sh oo t f e (m g. g -1 d w ) znso4 (mm) c 1 2 3 4 ab b b b a 0 10 20 30 40 50 znso4 (mm) c 1 2 3 4 sh oo t m g (m g. g -1 d w ) 28 f. melki, o.t. zribi, s. jeder, f. louati, i. nouair, h. mhadhbi, k. zribi substantial consequence on root p concentrations. however, shoot p concentrations decreased significantly under zn excess. sagardoy et al. (2009) showed that increasing zn supply up to 300 μm zn increased significantly leaf p content and it has no significant effect on root p content of sugar beet plants. proline prevents membrane damage and had a protective role in lipid peroxidation induced by metals (thounaojam et al., 2012). in the present study, we showed that both shoot and root proline content in fenugreek plants did not increase significantly as zn levels increased. contrarily, al khateeb and al-qwasemeh (2014) showed a significant increase in proline content in both solanum lycopersicum and solanum nigrum grown under different levels of cuso4, znso4 and cdcl2. parlak and yilmaz (2012) reported also that proline content increased under zn toxicity in three tested plants. conclusion in conclusion, excess zn in fenugreek plants caused an array of effects related to the zn levels in the culture medium. lower levels of znso4 reduced growth and physiological parameters slightly compared to higher levels. these effects 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https://orcid.org/0000-0002-1004-3691 ons talbi zribi https://orcid.org/0000-0003-4902-6925 sabrine jeder https://orcid.org/0000-0001-6101-3292 issam nouairi https://orcid.org/0000-0003-1850-1164 haythem mhadhbi https://orcid.org/0000-0003-0786-4269 kais zribi https://orcid.org/0000-0001-8856-9573 http://dx.doi.org/10.1111/j.0031-9317.2004.0275.x http://dx.doi.org/10.1021/acs.jafc.6b00274 http://dx.doi.org/10.1016/b978-0-12-473542-2.x5000-7 http://dx.doi.org/10.1007/978-94-007-4470-7 http://dx.doi.org/10.1016/s0021-9258(18)51320-x http://dx.doi.org/10.1111/ppl.12331 http://dx.doi.org/19910743084 http://dx.doi.org/10.1016/j.ecoenv.2012.08.023 http://dx.doi.org/10.3390/ijms19030787 http://dx.doi.org/10.1021/ar100030m http://dx.doi.org/10.1016/j.plantsci.2010.08.016 http://dx.doi.org/10.1007/s11738-016-2277-5 http://dx.doi.org/10.1111/j.1438-8677.2008.00153.x http://dx.doi.org/10.1111/j.1469-8137.2010.03241.x http://dx.doi.org/10.1007/s12892-018-0042-0 http://dx.doi.org/10.3389/fpls.2019.00748 http://dx.doi.org/10.1016/j.plaphy.2012.01.006 http://dx.doi.org/10.1002/jpln.200700222 http://dx.doi.org/10.3390/plants9030310 http://dx.doi.org/10.1016/s0176-1617(86)80157-2 http://dx.doi.org/10.1111/j.1365-3040.1990.tb01304.x http://dx.doi.org/10.3390/ijms19030787 http://dx.doi.org/10.1016/j.jplph.2005.07.007 http://dx.doi.org/10.1007/s11738-010-0687-3 http://dx.doi.org/10.1371/journal.pone.0228563 http://dx.doi.org/10.1007/s11738-014-1714-6 http://dx.doi.org/10.1016/j.ecoenv.2020.110856 http://dx.doi.org/10.1002/etc.2389 http://dx.doi.org/10.1080/15226514.2013.828017 https://orcid.org/0000-0002-1004-3691 https://orcid.org/0000-0003-4902-6925 https://orcid.org/0000-0001-6101-3292 https://orcid.org/0000-0003-1850-1164 https://orcid.org/0000-0003-0786-4269 https://orcid.org/0000-0001-8856-9573 30 f. melki, o.t. zribi, s. jeder, f. louati, i. nouair, h. mhadhbi, k. zribi address of the corresponding author: dr. kais zribi, laboratory of legumes and sustainable agrosystems, centre of biotechnology of borj-cedria, bp 901, 2050 hammam-lif, tunisia. e-mail: kais.zribi@cbbc.rnrt.tn © the author(s) 2022. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). journal of applied botany and food quality 92, 94 99 (2019), doi:10.5073/jabfq.2019.092.013 1laboratory for plant breeding and conservation of genetic resources, centro de bioplantas, universidad de ciego de ávila, ciego de ávila, cuba 2escuela superior politécnica agropecuaria de manabí manuel félix lópez (espammfl), campus politécnico el limón, carrera de ingeniería agrícola, calceta, manabí, ecuador 3school of life sciences, university of kwazulu-natal, durban, south africa cryopreservation of sorghum seeds modifies germination and seedling growth but not field performance of adult plants ariel villalobos1, melissa arguedas1, doris escalante1, julia martínez1, byron e. zevallos2, inaudis cejas1, lourdes yabor1, marcos edel martínez-montero1, sershen3, josé carlos lorenzo1 (submitted: november 22, 2018; accepted: february 7, 2019) summary climate change poses risks to both wild and crop plant biodiversity, which can be mitigated by cryopreservation (usually at -196 °c in liquid nitrogen [ln]) of crop germplasm. cryopreservation is widely regarded as a reliable method for the ex situ conservation of plant genetic resources but its effects on subsequent field performance of popular crop species such as sorghum are largely unknown. this hampers the large-scale implementation (i.e. germplasm banks) of cryostorage for such species. this short communication describes the early stages of germination and field performance of plants derived from cryopreserved sorghum seed. compared with the control, cryopreservation significantly increased seed electrolyte leakage and from 24 to 120 hours, percentage of germination of the control was ~2.6 folds higher than cryopreserved seeds. at 0 days, chlorophyll a/b rate was ~1.7 folds higher in the control and at 7 and 14 days, chlorophyll a level (~1.5 folds) and chlorophyll a/b rate (~1.8-1.9 folds) were higher in the control. contrastingly, at 7 days, seedlings derived from cryopreserved seeds (treatment seedlings) showed ~1.5 folds more superoxide dismutase activity and ~1.9 folds more peroxidase activity. in contrast, treatment and con trol adult plants were statistically comparable in terms of chlorophylls, proteins, superoxide and peroxidase activities, plant architecture, and yield components. the fact that differences in biochemical indicators observed between control and treatment seedlings did not persist in adult plants validates the use of seed cryopreservation for the conservation of sorghum genetic resources. keywords: field performance; germplasm preservation; liquid nitrogen; sorghum bicolor (l.) moench. introduction sorghum, an african grass related to sugarcane and maize, is the fifth most important cereal crop globally and is known to tolerate lownutrient soils and drought. cultivated varieties are phenotypically diverse (kayodé et al., 2006; labeyrie et al., 2016) and are grown for food, feed, fiber and fuel due to the high soluble sugar content of their stems (breeze, 2018; manzelli et al., 2006; mathur et al., 2017; paterson et al., 2009). in 2016, the world area harvested for sorghum reached 44 771 056 ha and the world production, 63 930 558 t (food and agriculture organization of the united nations statistics (faostat, 2017)). access to sorghum seed is crucial for farming and shortfalls are common in many countries. farmer-farmer exchange is important for providing locally-adapted seed to fill this gap but access varies considerably among households, affecting quantities supplied and terms of exchange (mcguire, 2008; otieno et al., 2018; smale et al., 2018). in the context of climate change with risks to lose diversity, cryopreservation of plant materials in liquid nitrogen (ln) has been described as a suitable technology to conserve genetic re sources of several species (panis, 2018; berjak et al., 2010), such as actinidia deliciosa (mathew et al., 2018), solanum betaceum cav. (graça et al., 2018), elaeis guineensis jacq. (beulé et al., 2018) and lilium (pan et al., 2018). however, the potential effects of cryostsorage of explants and seeds on subsequent plant growth in the field must be established before large-scale implementation in cryobanks (engelmann and ramanatha, 2012). in this regard, studies have shown that cryostorage of seed-derived germplasm can compromise the vigor of recovered plants (berjak et al., 2010). studies have also shown that recovery after cryopreservation of excised embryonic axes also seldom results in the production of trueto-type plants (harding, 2004; mycock, 1999; steinmacher et al., 2007; wesley-smith et al., 2001). it has been known for some time now that exposure to different types of stress can alter subsequent plant responses (bruce et al., 2007), but there is little understanding of how stresses imposed at the embryonic stage for example, are translated or manifested during subsequent plant growth. a few reports suggest that there exists with in cryopreserved plant materials some ‘memory’-based mechanism that senses environmental signals (forsyth and staden, 1983; kvaalen and johnsen, 2008), which in turn influence adaptive traits in the seedlings they give rise to. with this background, this short communication describes the early stages of germination, seedling growth and field performance of plants produced from cryopreserved sorghum seed. materials and methods plant material harvested sorghum seeds (cv. ciap2) were air dried at room temperature from 15% to 6% moisture content and then stored for 4 months at 4 °c in the dark in hermetically sealed containers. seeds with 6% moisture content (fresh weight basis) (ista, 2005) were used in subsequent experiments. seed cryopreservation and recovery one batch of the seeds was placed in cryo-vials (volume: 2 ml; 5 seeds per cryo-vial) and directly plunged in ln for 24 h (referred to hereafter as treated/cryopreserved seeds). recovery of seeds from ln was performed according to stanwood and bass (1981). another batch of seeds was stored at 4 °c until further use (referred to hereafter as control seeds). cryopreservation of sorghum seeds 95 seed electrolyte leakage, germination and seedling growth from 0 to 144 hours electrolyte leakage from seeds was measured as recommended by martínez-montero (2002) immediately after seed recovery from ln. percentage of seed germination (0-144 hours) and seedling weight (0-72 hours) were also evaluated by incubating seeds on filter papers in petri dishes (ø: 10 cm; 15 ml distilled water; five replicates of 10 seeds/dish). these parameters were also measured for control seeds. studies of seed and seedlings from 0 to 14 days a second group of control and treated seeds was set out to germinate as described above (five replicates of 10 seeds/dish) and evaluated at 0, 7 and 14 days for chlorophyll pigments (porra, 2002), total proteins (bradford, 1976), and activities of superoxide dismutase (kumutha et al., 2009) and peroxide oxidase (sagiv and barakiva, 1972) were evaluated in the seeds (0 day) or the primary leaves (7 and 14 days). hypocotyl length, primary leaf length, radicle length, total plantlet fresh weight, and total plantlet dry weight were also recorded. growth of adult plants in a plant bed until harvest at 110 days ninety treated and control seeds were randomly selected and sown in a plant bed as described in fig. 1. technical instructions pro vided by the cuban ministry for agriculture to cultivate sorghum were applied. border plants, which had more space to grow, were not considered. levels of chlorophyll pigments (porra, 2002), total proteins (bradford, 1976), and activities of superoxide dismutase (kumutha et al., 2009) and peroxide oxidase (sagiv and barakiva, 1972) were evaluated in middle-aged leaves at 62 days after planting on soil (anthesis). additionally, the following agricultural traits were evaluated according to peacock (1990) at 62 days of growth: plant height, number of leaves per plant, middle-aged leaf length and width, number of stems per plant, and fresh and dry weight of plants. all plants were harvested after 110 days of growth and the following traits were recorded: panicula length and width, number of branches per panicula, number of grain per panicula branch, number of grains per panicula, fresh weight of 1000 grains and dry weight of 1000 grains. statistical analysis spss (version 17.0 for windows, spss inc.) was used to compare growth and physiological parameters between control and treated plants/seeds using a students t-test (p≤0.05). results seed electrolyte leakage, germination and seedling growth from 0 to 144 hours compared with the control, cryopreservation significantly increased electrolyte leakage from seeds (~2 folds: 19.1% / 9.4%, fig. 2a) immediately after recovery from ln. although percentage of germination was similar at 144 hours, from 24 to 120 hours, percentage of germination of the control was ~2.6 folds higher (average, fig. 2b). seedling weight of control group was also higher (average ~1.2 folds, fig. 2c). studies of seed and seedlings from 0 to 14 days tab. 1 shows the effects of cryopreservation on early stages of germination (0, 7, 14 days). except for chlorophyll a + b on 0 day, statistically significant differences were observed on all sampling fig. 1: superior view of the useful area of the plant bed, made of ferralyticred soil and filter-cake-sugarcane ashes (1:1, v:v). dots symbolize seeds (70 cm × 25 cm apart). the broken arrow in the middle of the plant bed represents the microject irrigation system, which watered the plants for 5 min every 8 h. the plant bed was 50 cm high and its bottom contained a 10 cm-high stones layer to facilitate drainage. days (tab. 1): at 0 day, chlorophyll a/b rate was ~1.7 folds higher in the control treatment (1.13 / 0.65); at 7 days the level of chlorophyll a was ~1.5 folds higher in the control (21.37 μg g-1 fresh weight / 14.02 μg g-1 fresh weight); and chlorophyll a/b rate was ~1.9 folds higher in the control (1.31 / 0.71). contrastingly, at 7 days, plantlets derived from cryopreserved seeds showed ~1.5 folds (0.42 u mg-1 proteins / 0.27 u mg-1 proteins) more specific superoxide dismutase activity and ~1.9 folds (0.71 u mg-1 proteins / 0.37 u mg-1 proteins) more specific peroxidase activity. at 14 days, plantlet chlorophyll a levels was ~1.5 folds higher in the control (25.31 μg g-1 fresh weight / 17.13 μg g-1 fresh weight) and chlorophyll a/b rate was ~1.8 folds (1.38 / 0.75) higher in this group (tab. 1). contrastingly, the specific peroxidase activity was ~1.7 folds higher in cryopreserved seed-derived seedlings (0.60 u mg-1 proteins / 0.36 u mg-1 proteins). 96 a. villalobos, m. arguedas, d. escalante, j. martínez, b.e. zevallos, i. cejas, l. yabor, m.e. martínez-montero, sershen, j.c. lorenzo growth of adult plants in a plant bed until harvest at 110 days the data shown in tab. 2 indicates that seed cryopreservation has no significant effects on adult plants relative to the control. interes tingly, ex vitro, 100% of seed germination was recorded in both treat ments. differences in biochemical indicators between treatment and control seedlings reported above (tab. 1) did not persist in adult plants (tab. 2), e.g. levels of chlorophylls and proteins, and superoxide dismutase and peroxide oxidase activities. discussion cryopreservation imposes a series of stresses on plant material both during storage and upon recovery, and this can induce modifications in plants produced from cryopreserved explants/seeds (benson, 2008a; benson, 2008b). for example, partial dehydration and cryopreservation reduced the number of amaryllis belladonna excised zygotic embryos that produced seedlings, as well as the subsequent biomass of these seedlings relative to non-cryopreserved embryos (berjak et al., 2010). those authors showed that a. belladonna seedling produced from cryopreserved explants also exhibited lower co2-assimilation rates and stomatal density, abnormal root growth, damage to the photosynthetic apparatus and less efficient adjustment of leaf water potential relative to control seedlings. other studies have also reported phenotypic variation in in vitro recovery times, plant heights and modes of development (steinmacher et al., 2007) and regeneration in plants recovered from cryopreserved germplasm (harding, 2010). similarly, our results for sorghum suggest that cryopreservation increased electrolyte leakage from seeds (fig. 1a), and delayed germination (fig. 1b) and seedling growth (fig. 1c). cell membranes are one of the main targets of numerous stress events, including cryopreservation (berjak et al., 2010; close, 1996, 1997; harding, 2010; sangwan et al., 2002; uemura and steponkus, 1994). previously, our group has studied the effects of ln on the subsequent germination and growth of common bean, tomato, tobacco, maize and teramnus labialis (l.f.) spreng seeds. in brief, these studies showed that cryopreservation induced some morphological, physio logical and biochemical (e.g. chlorophyll, carotenoids, proteins, malondialdehyde, other aldehydes, soluble and cell wall-linked phenolics, and peroxidase and superoxide dismutase activity) changes b c a fig. 2: effect of seed cryopreservation on seed electrolyte leakage (a), germination (b) and seedling growth (c) from 0 to 144 hours. in each moment of evaluation, results with the same letters are not statistically different (t-test, p>0,05). vertical bars represent se. cryopreservation of sorghum seeds 97 that gradually disappeared as the plants grew (acosta et al., 2018; arguedas et al., 2018a; arguedas et al., 2018b; cejas et al., 2012; pérez-rodríguez et al., 2017; zevallos et al., 2014). this supports our present findings for sorghum where cryopreservation-induced declines in seedling chlorophyll a levels and consequently chlorophyll a/b rate, and enhanced superoxide dismutase and peroxidase activities did not persist in adult plants. various markers, including electrolyte efflux, peroxide and superoxide oxidations, reflect the structural and functional integrity status of cell membranes after exposure to such stressful events (dumet and benson, 2000). various abiotic stresses decrease the chlorophyll content in plants (ahmad et al., 2012) and this decline is believed to be due to inhibition of important enzymes, such as δ-aminolevulinic acid dehydratase and protochlorophyllide reductase associated with chlorophyll biosynthesis (van assche and clijsters, 1990). however, a major finding of the present study is that cryopreservation did not appear to affect the phenotype of adult sorghum plants in terms of growth and performance significantly. this report therefore demonstrates the value of seed cryopreservation to conserve sorghum genetic resources, although in large-scale field experiments, the reduced germination speed, seedling weight and the increase of enzyme activities may result in inhomogeneous field establishment and lower yield. author contribution: av, ma, de, jm, bez, ic, ly, memm, s and jcl designed the research; av, ma, de and jm conducted the experiment; av, bez, ic, ly, memm, s and jcl analyzed data; s and jcl wrote the paper; jcl had primary responsibility for the final content. all authors have read and approved the final manuscript. acknowledgements this research was supported by the university of ciego de avila (cuba), the escuela superior politécnica agropecuaria de manabí manuel félix lópez (ecuador), and the university of kwazulu-natal (south africa). references acosta, y., hernández, l., mazorra, c., quintana, n., zevallos, b.e., cejas, i., sershen, lorenzo, j.c., martínez-montero, m.e., fontes, d., 2018: seed cryostorage enhances subsequent plant productivity in the forage species teramnus labialis (l.f.) spreng. cryoletters (in press). ahmad, p., kumar, a., gupta, a., hu, x., azooz, m.m., sharma, s., 2012: polyamines: role in plants under abiotic stress. crop production for agricultural improvement. springer. arguedas, m., gómez, d., hernández, l., engelmann, f., garramone, r., cejas, i., yabor, l., martínez-montero, m.e., lorenzo, j.c., 2018a: maize seed cryo-storage modifies chlorophyll, carotenoid, pro tein, aldehyde and phenolics levels during early stages of germination. acta physiol. plant. 40, 118. doi: 10.1007/s11738-018-2695-7 arguedas, m., villalobos, a., gómez, d., hernández, l., zevallos, b., cejas, i., yabor, l., martínez-montero, m.e., lorenzo, j.c., 2018b: field performance of cryopreserved seed-derived maize plants. cryoletters 39 (6), 366-370. benson, e., 2008a: cryopreservation of phytodiversity: a critical appraisal of theory & practice. critic. rev. plant sci. 27, 141-219. doi: 10.1080/07352680802202034 benson, e.e., 2008b: cryopreservation theory. plant cryopreservation: a practical guide, 15-32. berjak, p., bartels, p., benson, e., harding, k., mycock, d., pammenter, n., sershen, w., 2010: cryoconservation of south african plant genetic diversity. in vitro cell. dev. biol.plant 47, 65-81. doi: 10.1007/s11627-010-9317-4 beulé, t., ilbert, p., adeoti, k., durand-gasselin, t., dumet, d., engelmann, f., morcillo, f., 2018: recovery of oil palm (elaeis tab. 1: studies of seed and seedlings from 0 to 14 days. in each moment of evaluation (0, 7, 14 days after exposure to ln), results with the same letter are not statistically different (t-test, p>0.05). intervals represent average ± se. 0 days 7 days 14 days (seeds were evaluated) (primary leaves were evaluated) (primary leaves were evaluated) noncryopreserved noncryopreserved noncryopreserved cryopreserved seeds cryopreserved seeds cryopreserved seeds seeds seeds seeds chlorophyll a (μg · g-1 fresh weight) 16.85 ± 0.39 a 12.37 ± 0.27 b 21.37 ± 0.28 a 14.02 ± 0.18 b 25.31 ± 0.25 a 17.13 ± 0.30 b chlorophyll b (μg · g-1 fresh weight) 14.97 ± 0.30 b 19.17 ± 0.40 a 16.39 ± 0.31 b 19.95 ± 0.47 a 18.36 ± 0.36 b 22.93 ± 0.57 a chlorophyll a + b (μg · g-1 fresh weight) 31.82 ± 0.49 a 31.53 ± 0.43 a 37.76 ± 0.34 a 33.97 ± 0.58 b 43.66 ± 0.40 a 40.06 ± 0.56 b chlorophyll a/b 1.13 ± 0.03 a 0.65 ± 0.02 b 1.31 ± 0.04 a 0.71 ± 0.02 b 1.38 ± 0.03 a 0.75 ± 0.03 b protein content 5.06 ± 0.16 a 4.13 ± 0.17 b 6.76 ± 0.22 a 5.12 ± 0.15 b 7.51 ± 0.17 a 6.23 ± 0.16 b (mg proteins · g-1 fresh weight) superoxide dismutase activity 1.70 ± 0.04 b 1.88 ± 0.02 a 1.83 ± 0.02 b 2.12 ± 0.02 a 1.88 ± 0.00 b 2.15 ± 0.01 a (mg · g-1 fresh weight) specific superoxide dismutase activity 0.34 ± 0.02 b 0.46 ± 0.02 a 0.27 ± 0.01 b 0.42 ± 0.01 a 0.25 ± 0.01 b 0.35 ± 0.01 a (u mg-1 proteins) peroxidase activity 2.43 ± 0.08 b 2.79 ± 0.07 a 2.49 ± 0.07 b 3.60 ± 0.05 a 2.71 ± 0.06 b 3.71 ± 0.03 a (mg · g-1 fresh weight) specific peroxidase activity 0.48 ± 0.01 b 0.68 ± 0.02 a 0.37 ± 0.01 b 0.71 ± 0.02 a 0.36 ± 0.01 b 0.60 ± 0.01 a (u mg-1 proteins) hypocotyl length (cm) ----2.79 ± 0.01 a 2.46 ± 0.04 b 6.93 ± 0.04 a 6.44 ± 0.04 b primary leaf length (cm) ----2.91 ± 0.02 a 2.71 ± 0.03 b 7.26 ± 0.06 a 6.69 ± 0.03 b radicle length (cm) ----4.98 ± 0.06 a 4.61 ± 0.12 b 8.00 ± 0.10 b 8.19 ± 0.13 a total plantlet fresh weight (mg) ----0.24 ± 0.00 a 0.20 ± 0.00 b 0.51 ± 0.00 a 0.41 ± 0.00 b total plantlet dry weight (mg) ----0.08 ± 0.00 a 0.07 ± 0.00 b 0.17 ± 0.00 a 0.13 ± 0.00 b 98 a. villalobos, m. arguedas, d. escalante, j. martínez, b.e. zevallos, i. cejas, l. yabor, m.e. martínez-montero, sershen, j.c. lorenzo guineensis jacq.) somatic embryos cryostored for 20 years. cryoletters 39, 60-66. berjak, p., pammenter, n.w., 2010: effects of cryopreservation of recal citrant amaryllis belladonna zygotic embryos on vigor of recovered seedlings: a case of stress ‘hangover’? physiol. plant. 139, 205-219. doi: 10.1111/j.1399-3054.2010.01358.x bradford, m., 1976: a rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. analytical biochemical 72, 248-254. doi: 10.1016/0003-2697(76)90527-3 breeze, e., 2018: sweet and juicy: identification and origins of the dry alleles in sorghum. the plant cell. doi: 10.1105/tpc.18.00748 bruce, t.j., matthes, m.c., napier, j.a., pickett, j.a., 2007: stressful “memories” of plants: evidence and possible mechanisms. plant science 173, 603-608. doi: 10.1016/j.plantsci.2007.09.002 cejas, i., vives, k., laudat, t., gonzález-olmedo, j., engelmann, f., martínez-montero, m.e., lorenzo, j.c., 2012: effects of cryo preservation of phaseolus vulgaris l. seeds on early stages of ger mination. plant cell rep. 31, 2065-2073. doi: 10.1007/s00299-012-1317-x close, t.j., 1996: dehydrins: emergence of a biochemical role of a family of plant dehydration proteins. physiol. plant. 97, 795-803. doi: 10.1111/j.1399-3054.1996.tb00546.x close, t.j., 1997: dehydrins: a commonalty in the response of plants to dehydration and low temperature. physiol. plant. 100, 291-296. doi: 10.1111/j.1399-3054.1997.tb04785.x dumet, d., benson, e.e., 2000: the use of physical and biochemical studies to elucidate and reduce cryopreservation induced damage in hydrated/desiccated plant germplasm. in: engelmann, f., takagi, h. (eds.), cryopreservation of tropical plant germplasm: current research progress and application. jircas/ipgri, tsukuba/rome. engelmann, f., ramanatha, r., 2012: major research challenges and directions for future research. in: normah, m.n.,chin, h.f., reed, b.m. (eds.), conservation of tropical plant species. springer verlag, berlin. faostat, 2017: fao cereal supply and demand brief. retrieved from forsyth, c., staden, j.v., 1983: germination of tagetes minuta li temperature effects. ann. bot. 52, 659-666. doi: 10.1093/oxfordjournals.aob.a086622 graça, d., correia, s., ozudogru, e., lambardi, m., canhoto, j., 2018: cryopreservation of tamarillo (solanum betaceum cav.) embryogenic cultures. in: jain, s., gupta, p. (eds.), step wise protocols for somatic embryogenesis of important woody plants. forestry sciences. springer. harding, k., 2004: genetic integrity of cryopreserved plant cells: a review. cryoletters 25, 3-22. harding, k., 2010: plant and algal cryopreservation: issues in genetic integrity, concepts in cryobionomics and current applications in cryo biology. j. mol. biol. biotech. 18, 151-154. ista, 2005: international rules for seed testing. international seed testing association, bassersdorf, switzerland. kayodé, a.p., linnemann, a.r., nout, m.r., hounhouigan, j.d., stomph, t.j., smulders, m.j., 2006: diversity and food quality pro perties of farmers’ varieties of sorghum from bénin. j. sci. food agric. 86, 1032-1039. doi: 10.1002/jsfa.2451 kumutha, d., ezhilmathi, k., sairam, r., srivastava, g., deshmukh, p., tab. 2: growth of adult plants in a plant bed until harvest at 110 days. statistically significant differences were not recorded (t-test, p>0.05). intervals represent average ± se. days after plantings and traits measured non-cryopreserved seeds cryopreserved seeds traits evaluated in middle-aged leaves at 62 days after planting on soil (anthesis). chlorophyll a (μg · g-1 fresh weight) 85.17 ± 0.37 85.63 ± 0.44 chlorophyll b (μg · g-1 fresh weight) 56.10 ± 0.31 55.63 ± 0.44 chlorophyll a + b (μg · g-1 fresh weight) 141.27 ± 0.41 141.26 ± 0.88 chlorophyll a/b 1.52 ± 0.01 1.54 ± 0.00 protein content (mg proteins · g-1 fresh weight) 8.97 ± 0.03 9.05 ± 0.03 superoxide dismutase activity (mg · g-1 fresh weight) 3.36 ± 0.07 3.39 ± 0.07 specific superoxide dismutase activity (u mg-1 proteins) 0.38 ± 0.01 0.37 ± 0.01 peroxidase activity (mg · g-1 fresh weight) 15.03 ± 0.19 15.11 ± 0.18 specific peroxidase activity (u mg-1 proteins) 1.68 ± 0.02 1.67 ± 0.02 agricultural traits evaluated at 62 days after planting on soil (anthesis). plant height (cm) 168.75 ± 0.53 169.15 ± 0.49 number of leaves per plant 10.38 ± 0.15 10.25 ± 0.15 middle-aged leaf length (cm) 97.99 ± 0.24 97.79 ± 0.18 middle-aged leaf width (cm) 7.72 ± 0.03 7.70 ± 0.01 number of stems per plant 1.00 ± 0.00 1.00 ± 0.00 stem diameter (cm) 5.89 ± 0.02 5.88 ± 0.02 fresh weight of plants (g) 1.10 ± 0.02 1.09 ± 0.03 dry weight of plants (g) 0.27 ± 0.01 0.28 ± 0.01 agricultural traits evaluated at at 110 days after planting on soil (harvest). panicula length (cm) 17.91 ± 0.03 17.88 ± 0.02 panicula width (cm) 6.07 ± 0.02 6.06 ± 0.01 number of branches per panicula 29.80 ± 0.15 29.83 ± 0.09 number of grains per panicula branch 37.43 ± 0.77 37.93 ± 0.67 number of grains per panicula 1119.15 ± 27.93 1131.58 ± 20.96 fresh weight of 1000 grains (g) 27.98 ± 0.70 28.29 ± 0.52 dry weight of 1000 grains (g) 5.13 ± 0.07 5.15 ± 0.07 cryopreservation of sorghum seeds 99 meena, r., 2009: waterlogging induced oxidative stress and antioxidant activity in pigeonpea genotypes. biologia plantarum 53, 75-84. doi: 10.1007/s10535-009-0011-5 kvaalen, h., johnsen, ø., 2008: timing of bud set in picea abies is 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cold, antioxidant and osmotic pre-treatments maintain the structural integrity of meristematic cells and improve plant regeneration in cryopreserved kiwifruit shoot tips. protoplasma 255, 1065-1077. doi: 10.1007/s00709-018-1215-3 mathur, s., umakanth, a., tonapi, v., sharma, r., sharma, m.k., 2017: sweet sorghum as biofuel feedstock: recent advances and available resources. biotechnol. biof. 10, 146. doi: 10.1186/s13068-017-0834-9 mcguire, s.j., 2008: securing access to seed: social relations and sorghum seed exchange in eastern ethiopia. hum. ecol. 36, 217-229. doi: 10.1007/s10745-007-9143-4 mycock, d., 1999: addition of calcium and magnesium to a glycerol and sucrose cryoprotectant solution improves the quality of plant embryo recovery from cryostorage. cryoletters 20(2), 77-82. otieno, g., lacasse, h., adokorach, j., mulumba, j.w., recha, j.w., reynolds, t.w., fadda, c., 2018: social seed networks for climate change adaptation in uganda: strategies to improve access to genetic 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martis, m., narechania, a., otillar, r.p., penning, b.w., salamov, a.a., wang, y., zhang, l., carpita, n.c., freeling, m., gingle, a.r., hash, c.t., keller, b., klein, p., kresovich, s., mccann, m.c., ming, r., peterson, d.g., mehboob ur, r., ware, d., westhoff, p., mayer, k.f.x., messing, j., rokhsar, d.s., 2009: the sorghum bicolor genome and the diversification of grasses. nature 457, 551. doi: 10.1038/nature07723 peacock, j., 1990: investigación del icrisat sobre sorgo en los trópicos semiáridos. icrisat 17. pérez-rodríguez, j.l., escriba, r.c.r., gonzález, g.y.l., olmedo, j.l.g., martínez-montero, m.e., 2017: effect of desiccation on phy siological and biochemical indicators associated with the germination and vigor of cryopreserved seeds of nicotiana tabacum l. cv. sancti spíritus 96. in vitro cell. dev. biol.-plant, 1-9. doi: 10.1007/s11627-017-9857-y. porra, r., 2002: the chequered history of the development and use of simultaneous equations for the accurate determination of chlorophylls a and b. photosynth. res. 73, 149-156. doi: 10.1023/a:1020470224740 sagiv, j., bar-akiva, a., 1972: visual demonstration of differences in per oxidase activity in iron and manganese deficient citrus leaves. experien tia 28, 645-646. doi: 10.1007/bf01944952 sangwan, v., örvar, b.l., dhindsa, r.s., 2002: early events during low temperature signaling. plant cold hardiness. springer. smale, m., assima, a., kergna, a., thériault, v., weltzien, e., 2018: farm family effects of adopting improved and hybrid sorghum seed in the sudan savanna of west africa. food pol. 74, 162-171. doi: 10.1016/j.foodpol.2018.01.001 stanwood, p., bass, l., 1981: seed germplasm preservation using liquid nitrogen. seed sci. technol. 9, 423. steinmacher, d.a., saldanha, c.w., clement, c.r., guerra, m.p., 2007: cryopreservation of peach palm zygotic embryos. cryoletters 28, 13-22. uemura, m., steponkus, p.l., 1994: a contrast of the plasma membrane lipid composition of oat and rye leaves in relation to 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genetic resources, centro de bioplantas, universidad de ciego de ávila, ciego de ávila 69450, cuba e-mail: jclorenzo@bioplantas.cu byron e. zevallos, escuela superior politécnica agropecuaria de manabí manuel félix lópez (espammfl), campus politécnico el limón, carrera de ingeniería agrícola, calceta, manabí, ecuador sershen, school of life sciences, university of kwazulu-natal, durban, 4001, south africa © the author(s) 2019. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). vorgabe neu journal of applied botany and food quality 81, 15 20 (2007) 1) institute of grassland science, ningxia university, ningxia, china 2) abteilung ökologie und geobotanik, johann wolfgang goethe-universität, frankfurt, deutschland biomass and grazing potential of the stipa loess steppes in ningxia (northern china) in relation to grazing intensity xie, y.1), wittig, r.2) (received november 6, 2006) summary the mere amount of biomass is not a suitable measuring unit for the economic value of a plant community, because not all species are equally appreciated by cattle and sheep and some are even unpalatable. that is why we summarised palatability and appreciation to a feeding value. together with the biomass this feeding value was used to calculate the grazing potential of the plant communities investigated. in the stipa loess steppes of ningxia (northern china) the two characteristic feather grasses dominating in ungrazed or slightly grazed areas are estimated as species of very high feeding value. those species, which show increasing biomass parallel to increasing grazing intensity are of lower feeding value. except of overgrazed areas, the stipa grandis steppes have a higher grazing potential than the stipa bungeana communities. a comparison of biomass and grazing potential shows that the relative differences between the grazing potential of the communities existing at different grazing levels are much higher than the relative differences in biomass. introduction steppe represents the main vegetation type of large areas of northern and western china, stretching from the mongolian highlands across the loess plateau to the middle of the tibetan upland. in the autonomous region of ningxia and its surrounding 70% of the surface area is classified as steppe and used for pasture-farming (national research council, 1992; national bureau of statistics of china, 1998). the dry steppe representing the dominant vegetation type in the autonomous region of ningxia hui with about 60 % of the whole surface area (guo et al., 1988) has suffered extreme degradation over the past 30 years. in previous papers we have shown the effects of different levels of grazing on soil parameters of stipa loess steppes in ningxia hui (xie and wittig, 2003), and we have identified indicator species of different grazing levels (xie and wittig, 2004). in the following we will deal with biomass and grazing potential of the same steppe types in relation to grazing intensity. area under investigation the area under investigation is a dry steppe landscape located in the southern autonomous region of ningxia hui. in ningxia almost natural steppe vegetation (i.e. ungrazed ore only slightly grazed) exists only in the yunwushan dry steppe nature reserve, which is situated in the southern part of the autonomous region. therefore this nature reserve was investigated especially intensively. the area is approximately 800 km2 in size and lies between 1.650 m and 2.148 m above sea level (an average elevation of about 1.780 m). yinchuan, the capital of the autonomous region, is approximately 300 kilometres away by road. as in all other areas of ningxia (see ma, 1996) the most significant sources of income are arable farming and animal husbandry. about 54 % of the area are used for grazing. the yunwushan area was declared a nature reserve in 1982. arable farming and animal husbandry have been prohibited since then. the climate of the area of investigation represents a typical continental climate of the northern hemisphere, slightly influenced by the pacific summer monsoon. summers are relatively humid and warm; winters are rather cold, dry and windy. low precipitation and a comparatively strong rate of evaporation lead to high aridity. the potential evaporation occurs at a rate almost 4 times as high as the annual rainfall. as the area is located in the western part of the north shanxi-loess plateau, the soil is a direct product of the mighty layer of loess. at an altitude of less than 2000 m a rich castanozem can be found, above 2000 m a mountain phaeozem is the characteristic type of soil. some more information on these two prevailing types of soil as well as some climate data are given by xie and wittig (2004). methods grazing intensity. the intensity of grazing activities was defined according to monitoring data collected (number and breed of livestock grazing per grazing area unit, frequency of grazing activities) and interviews with responsible officials of the nature reserve and with farmers in the area. the grazing intensity (g) was broken down into the following 5 levels, which are explained in detail by xie and wittig (2003, 2004): • g0: not or very slightly grazed; • g1: slightly grazed; • g2: moderately grazed; • g3: intensively grazed; • g4: over-grazed. measuring the aboveground biomass. in the stipa bungeana and the stipa grandis steppe three plots of 5 m x 5 m were chosen from each level of grazing intensity. at the end of the vegetation period (end of september) of the year 1999 five samples (1 m2 each) of the above ground biomass (separated for species) were collected from equidistant points along the two diagonals in each plot. the harvested material was dried and the twelve most important species were weighted separately each. the remaining species were weighted together. for each plot the total biomass and the percentage of the twelve species was calculated. calculation of the grazing potential. not all species are equally appreciated by cattle and sheep and some are even unpalatable. therefore the mere amount of biomass alone does not represent an adequate parameter for the economic value of a grazed area or a plant community. that is why we attributed a feeding value, ranging from 0 to 4, to all those species which together represent 80 to 90 % of the biomass of the stipa steppes of the investigated areas. this feeding value is based on literature (guo et al., 1988; wu, 1997) and on interviews with local farmers. together with its biomass the feeding value of a species allows to characterise its grazing potential. from the grazing potential of all species the total grazing potential of a plant community can be calculated (see formula 1 and 2). formula 1: grazing potential (pi) of a species pi = wi x bi bi : average biomass of a species; wi : feeding value of a species formula 2: total grazing potential (ptot) of a plant community ptot = σ pi results biomass. different grazing intensities clearly lead to differences in the above ground biomass (fig. 1 and tab. 1). only between g0 and g1 the difference is rather low and not significant. regarding all other grazing levels the biomass differs significantly (t-test: p=0.01). generally one can observe a steady but not linear decrease of biomass from g0 to g4. particularly evident is the reduction of the biomass when regarding the two highest grazing levels: at level 3 only 55 %, at level 4 only 25 % of the biomass present in g0 were measured for the stipa bungeana steppe. regarding the stipa grandis steppe, this reduction is even stronger, particularly at level g4 (only 19 % of g0). comparing the two types of stipa steppes at levels g0 to g3 the stipa grandis steppe is more productive than the stipa bungeana steppe. at the level of overgrazing (g4), the stipa bungeana steppe has a higher biomass than the stipa grandis steppe. however, this difference is not significant. relatively the decrease of biomass with increasing grazing intensity is at all grazing levels higher in the stipa grandis steppe than in the stipa bungeana steppe. in g0 the stipa species represent the highest percentage of the total biomass (see fig. 2 and 3). with increasing intensity the biomass of the stipa species decreases more rapidly than the total biomass. comparing the two species, s. grandis reacts more sensitive than s. bungeana and is often totally absent at grazing intensity g4. similarly, but on a lower level, reacts koeleria cristata. adenophora potaninii and serratula centaureoides only occur at levels g0 to g2. a second group of species shows increasing biomass parallel to increasing grazing intensity, however, decreases at level g4. this group consists of agropyron cristatum, carex stenophylloides, artemisia sacrorum, artemisia frigida and thymus mongolicum. particularly evident is the high biomass of artemisia frigida at level g2 (and a little bit lower at g3) of the stipa grandis community and that of artemisa sacrorum in the analogous grazing levels of the stipa bungeana community. a third group of species (convolvulus ammannii, potentilla acaulis and stellera chamaejasme) shows a continuing increase of its biomass with increasing grazing intensity. feeding value and grazing potential. the two characteristic feather grasses dominating at intensity g0 and g1 are estimated as species fig. 1: aboveground biomass of the stipa steppes on loess soils in the yunwushan area (ningxia, pr china), in relation to grazing intensity fig. 2: relative biomass (percentage of the total biomass) of important species of the stipa grandis steppes on loess soils in the yunwushan area (ningxia, pr china), in relation to grazing intensity 16 xie, y., wittig, r. tab. 1: aboveground biomass of 16 important plant species of stipa steppes on loess soils in the yunwushan area (ningxia, pr china), in relation to grazing intensity1) stipa grandis steppe mean biomass (mb) and standard deviation (sd) life form g0 g1 g2 g3 g4 mb2) sd mb2) sd mb2) sd mb2) sd mb2) sd total biomass 262.8 245.3 187.6 127.9 50.1 % of g0 100 93.3 71.4 48.3 19.1 stipa bungeana graminoid 41.6 2.06 42.5 3.95 38.6 4.66 31.4 5.12 8.6 1.44 stipa grandis graminoid 144.6 4.11 136.8 12.33 51.5 6.42 23.3 2.63 5.3 0.21 koeleria cristata graminoid 7.6 0.14 4.1 0.86 2.3 0.18 cleistogenes squarrosa graminoid 3.6 0.21 3.9 0.66 5.7 0.26 6.3 0.45 6.2 0.91 agropyron cristatum graminoid 5.4 0.85 6.4 1.14 7.6 0.66 1.7 0.21 carex stenophylloides graminoid 1.6 0.31 2.4 0.32 2.7 0.42 2.4 0.31 0.7 0.11 artemisia sacrorum subshrub 6.4 1.21 6.5 0.85 12.3 2.11 10.2 2.01 2.6 0.36 artemisia frigida subshrub 9.3 1.12 11.5 1.54 48.6 5.77 31.2 2.76 2.9 0.32 thymus mongolicum subshrub 2.3 0.81 2.6 0.33 3.8 1.20 4.1 0.39 0.7 0.10 convolvulus ammannii forb 1.7 0.25 2.4 0.74 7.2 2.51 potentilla acaulis forb 1.6 0.21 1.4 0.34 2.7 0.66 3.3 0.46 6.4 0.88 stellera chamaejasme forb 0.6 0.22 1.5 0.32 2.1 0.19 6.7 1.14 astragalus adsurgens forb 1.5 0.13 3.2 0.98 1.3 0.18 bupleurum chinense forb 0.2 0.16 0.2 0.09 0.1 0.04 adenophora potaninii forb 0.7 0.17 0.4 0.11 serratula centauroides forb 2.6 0.56 1.7 0.23 0.1 0.06 sum 229.0 8.54 224.2 5.64 180.5 8.63 118.4 9.74 47.3 7.13 % of the total biomass3) 87.00 91.00 96.00 92.00 94.00 stipa bungeana steppe mean biomass (mb) and standard deviation (sd) life form g0 g1 g2 g3 g4 mb2) sd mb2) sd mb2) sd mb2) sd mb2) sd total biomass 238.6 232.4 177.8 122.1 58.7 % of g0 100 97.4 74.5 51.2 24.6 stipa bungeana graminoid 151.4 6.22 148.4 7.69 63.5 3.91 28.1 3.11 13.7 1.52 stipa grandis graminoid 5.6 0.74 4.7 0.63 2.8 0.32 2.1 0.31 koeleria cristata graminoid 8.3 1.14 7.4 0.65 1.8 0.23 cleistogenes squarrosa graminoid 2.6 0.31 2.3 0.24 3.7 0.56 3.9 0.45 4.3 0.57 agropyron cristatum graminoid 4.8 0.66 7.6 1.14 6.9 0.77 2.1 0.24 carex stenophylloides graminoid 1.8 0.32 2.2 0.32 3.2 0.42 2.5 0.31 0.4 0.08 artemisia sacrorum subshrub 9.3 0.54 12.4 1.24 36.1 3.01 41.4 2.89 8.5 0.62 artemisia frigida subshrub 5.3 0.63 4.4 0.62 7.4 0.98 6.1 0.83 3.4 0.28 thymus mongolicum subshrub 3.4 0.22 3.8 0.31 8.3 0.96 11.2 2.11 4.7 0.37 convolvulus ammannii forb 1.9 0.26 2.1 0.31 2.3 0.44 6.7 0.86 potentilla acaulis forb 1.3 0.21 1.4 0.34 3.1 0.48 3.6 0.29 4.1 0.67 stellera chamaejasme forb 1.6 0.31 2.2 0.61 4.3 0.58 7.4 0.86 astragalus adsurgens forb 3.6 0.22 3.9 0.54 1.7 0.31 bupleurum chinense forb 2.3 0.22 2.1 0.38 0.6 0.11 adenophora potaninii forb 0.2 0.07 0.1 0.06 serratula centauroides forb 0.60 0.11 0.20 0.07 0.10 0.04 sum 200.5 11.31 204.4 7.64 143.5 6.86 107.6 8.61 53.2 6.57 % of the total biomass3) 84.00 88.00 80.00 88.00 92.00 1) 15 repetitions per grazing intensity 2) [g/m2] 3) see fig. 1 stipa loess steppes in northern china 17 of high feeding value (tab. 2). those species, which show increasing biomass parallel to increasing grazing intensity, are of lower feeding value. except of overgrazed areas, the stipa grandis-steppes have a higher grazing potential than the stipa bungeana communities (fig. 4). a comparison of fig. 4 and fig. 2 shows that the relative differences between the grazing potential of the communities established at different grazing levels are much higher than the relative differences in biomass (fig. 2). discussion composition and cover of species in grazed ecosystems result from two processes running contrarily: disturbance and regeneration (numata, 1969; willms et al., 1990). fineto medium-scale disturbance leads to local succession, whereas on a larger scale a dynamic equilibrium often exists (herben et al, 1993a, b). disturbance is caused by grazing and trampling, and it leads to a reduced growth and reproduction. nevertheless those species, which are less appreciated by grazing animals than others (i.e. species possessing a low feeding value), obtain a relative advantage (risser, 1988; goldstein et al., 1990; pieper, 1994; chen, 1997; bork et al., 1998). less appreciated are in particular hairy and thorny species, species with a high amount of sclerenchym as wells as bad tasting ones. totally avoided are toxic species. thus species composition and cover as well as the grazing value of the community mirror the grazing intensity (bell, 1971; austin et al., 1981; cheng and zhang, 1992; archer and stokes, 2000; mané-bielfeldt, 2000). our results confirm these expectations: increasing grazing intensity leads to a change in species composition and to a reduction of the grazing potential of the community. with increasing grazing level, fig. 3: relative biomass (percentage of the total biomass) of important species of the stipa bungeana steppes on loess soils in the yunwushan area (ningxia, pr china), in relation to grazing intensity fig. 4: grazing potential of the stipa steppes on loess soils in the yunwushan area (ningxia, pr china), in relation to grazing intensity tab. 2: grazing value of important members of the stipa steppes growing on loess soils in the yunwushan area (ningxia, pr china) species feeding attributes feeding value carex stenophyloides, cleistogenes squarrosa, koeleria cristata very valuable: appreciated and preferently eaten agropyron cristatum, astragalus adsurgens, stipa bungeana, valuable: stipa grandis main fodder, regularly eaten artemisia frigida, convolvulus ammannii, potentilla acaulis, suitable: thymus mongolicum important fodder substitute when main fodder plants have decreased artemisia sacrorum, bupleurum chinense little suitable: only used during hunger periods stellera chamaejasme not suitable: avoided 0 4 3 2 1 18 xie, y., wittig, r. species of high feeding value more and more disappear, leaving the cattle and sheep no other choice than to feed on the less appreciated species. not only in the area of investigation but also in other holarctic steppes particularly artemisia species are favoured by high grazing intensity (see e.g. li, 1989; peer et al., 2001). these species are hairy, highly sclerenchymatic and taste badly. however, the artemisia species of the area under investigation are not unpalatable and therefore have not been attributed with the lowest grazing value. when the level shifts from “highly grazed” to “overgrazed”, unpalatable and toxic species become dominant resulting in a steppe community of a very low grazing potential. it is common knowledge that grassland communities of humid areas show a higher productivity and consist of less sclerenchymatic and badly tasting species than those of dry areas. as the stipa grandis steppe grows at an altitudinal belt, where the climate is not as dry as that of the belt of the stipa bungeana community, it is no wonder that, at all grazing levels except of g4, the stipa grandis steppe shows a higher grazing potential than the stipa bungeana steppe. in semiarid regions overgrazing generally causes desertification. that is why one can conclude that at least some desert species are less vulnerable of overgrazing than species of non-desert areas. thus we have an explanation for the exception observed at level g4: as stipa bungeana and the most characteristic members of its community are central asiatic species (ma and liu, 1986, 1988), they are better adapted to desert conditions than the mongolian stipa grandis and its characteristic companions. when regarding the results, it should not be forgotten that our investigation represents a freeze frame recording, not a monitoring. the productivity of a grazed ecosystem, however, is not only depending upon the intensity of grazing, but also on the climate (shipley, 1942; stoddart, 1975; brown, 1995; shen, 1995; zhang, 1998). thus different absolute results will be obtained in different years. however, one may assume that the interannual climate variation will only slightly influence the relative differences between different grazing levels. freeze frame recording also means that our results do not refer to the annual biomass but to the biomass of the harvest date. but again this investigation gap does not influence the validity of the relative results. conclusions our results confirm that the mere amount of biomass is not a suitable measuring unit for the economic value of a plant community. the grazing potential calculated from biomass and feeding value of the different species of the community, decreases much more rapidly with increasing grazing intensity than the biomass. thus the calculation of sustainable densities of animal husbandry should not be based on biomass but on the grazing potential of the grazed communities. acknowledgements the authors would like to thank their colleague jörg steinbach (gießen) for valuable comments, dick byer for the improvement of the english, and cornelia anken for the careful handling of the manuscript. references archer, s., stokes, c., 2000: stress, disturbance and change in rangeland ecosystems. in: arnalds, o., archer, s. (eds.), rangeland desertification, 17-38. kluwer academic publishers, dordrecht. austin, m.p., williams, d.b., belbin, l., 1981: grassland dynamics under sheep grazing in australian mediterranean climate. vegetatio 40, 201211. bork, e.w., west, n.e., walker, j.w., 1998: cover components on longterm seasonal sheep grazing treatments in three-tip sagebrush steppe. j. range management 51, 293-300. brown, r.w., 1995: the water relations of range plants: adaptations to water deficits. in: bedunah, d.j., sosebee, r. (eds.), wildland plants: physiological ecology and developmental morphology, 291-413. society for range management, denver, co. chen, h., 1997: the recovery pathway of desertified grassland in mid-basin valley of one river and its two tributaries in tibet. (chinese, engl. summary). pratacultural science 14, 1-4. cheng, j., zhang, w., 1992: grassland quality evaluation in hilly regions of loess plateau using topsis method. 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(chinese). ningxia people’s publishing house, yinchuan. herben, t., krahulec, f., hadincová, v., skàlovà, h., 1993a: smallscale variability as a mechanism for large-scale stability in mountain grasslands. j. veg. sci. 4, 163-170. herben, t., krahulec, f., hadincová, v., kovàrovà, m., 1993b: smallscale spatial dynamics of plant species in a grassland community over six years. j. veg. sci. 4, 171-178. li, y.-h., 1989: impact of grazing on aneurolepidium chinense steppe and stipa grandis steppe. acta oecologica 10, 31-46. ma, k., 1996: die futterressourcen ningxias und ihre nutzung. eine simulationsstudie zur nachhaltigen landnutzung im nordwesten chinas. shaker verlag, aachen. ma, d., liu, h., 1986: flora ningxiaensis, tomus i. (chinese) ningxia people’s publishing house, yinchuan. ma, d., liu, h., 1988: flora ningxiaensis, tomus ii. (chinese) ningxia people’s publishing house, yinchuan. mané-bielfeldt, a., 2000: untersuchungen zum futterangebot der steppenweide in ningxia (nordwest-china) und zur nutzung durch lokale kleine wiederkäuer. cuvillier verlag, göttingen. national bureau of statistics of china, 1998: china statistical year book 1998. china statistics press, beijing. national research council, 1992: grasslands and grassland sciences in northern china. national academy press, washington, dc. numata, m., 1969: progressive and retrogressive gradient of grassland vegetation measured by degree of succession, ecological judgement of grassland condition and trend (iv). vegetatio 19, 96-127. peer, t., millinger, a., gruber, j.p., hussain, f., 2001: vegetation and altitudinal zonation in relation to the impact of grazing in the steppe lands of the hindu kush range (n-palkistan). phytocoenologia 31, 477498. pieper, r.d., 1994: ecological implications of livestock grazing. in: vavra, m., laycock, w.a., pieper, r.d. (eds.), ecological implications of livestock herbivory in the west. society for range management, 177-211. denver, co. risser, p.g., 1988: diversity in and among grasslands. in: wilson, e.o. (ed.), biodiversity, 176-180. national academy press, washington, dc. shen, y., 1995: grassland succession and improvement in yanchi semi-desert area. (chinese, engl. summary) j. arid land resources environ. 9, 6269. shipley, m.a., 1942: estimating the value of range forage for grazing use by means of animal-unit-month factor table. university of nevada reno, nevada. stoddart, l.a., 1975: range management. mcgrawhill, new york. willms, w.d., smoliak, s., dormaar, j.f., 1990: vegetation response to time-controlled grazing on mixed and fescue prairies. j. range managestipa loess steppes in northern china 19 ment 43, 513-517. wu, n., 1997: ecological situation of high-frigid rangeland and its sustainability: a case study on the constraints and approaches in pastoral western sichuan, china. dietrich remier verlag, berlin. xie, y., wittig, r., 2003: growth parameters of characteristic species of stipa steppes in northern china as indicators of the grazing intensity. j. applied botany 77, 68-74. xie, y., wittig, r., 2004: the impact of grazing intensity on soil characteristics of stipa grandis and stipa bungeana steppe in northern china (autonomous region of ningxia). acta oecologica 25, 197-204. zhang, w., 1998: changes in species diversity and canopy cover in steppe vegetation in inner mongolia under protection from grazing. biodiversity and conservation 7, 1365-1381. addresses of the authors: 1) institute of grassland science, ningxia university, helanshan west road 489, 750021 yinshuan ningxia, china. 2) abteilung ökologie und geobotanik, institut für ökologie, evolution und diversität der johann wolfgang goethe-universität, siesmayerstraße 70, d-60323 frankfurt. 20 xie, y., wittig, r. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true 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<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> >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice art13 journal of applied botany and food quality 82, 103 107 (2009) leibniz institute of plant genetics and crop plant research (ipk), gatersleben, germany variability of alkaloid content in papaver somniferum l. a. dittbrenner, h.-p. mock, a. börner, u. lohwasser (received august 11, 2008) summary a total of 300 accessions of opium poppy (papaver somniferum l., papaveraceae) of the ipk genebank collection from nearly all over the world were cultivated under field conditions in gatersleben for morphological and phytochemical characterisation. altogether 35 morphological and agronomic characters were collected for all accessions. determination of chromosome numbers with flow cytometry showed that the accessions of subspecies setigerum are tetraploid whereas all accessions of the other subspecies are diploid. composition and content of the five main alkaloids (morphine, codeine, thebaine, papaverine and noscapine) were analysed by high performance liquid chromatography (hplc). total alkaloid content varied between 683.32 and 25,034.84 µg/g dry matter (first year) and 1,799.49 and 25,338.55 µg/g dry matter in the second year of cultivation. there is a highly significant correlation between total content of alkaloids and morphine in both years (r=0.926/p=0.000; r=0.918/p=0.000). in contrast, the other four main alkaloids show less or no correlation with each other or the total alkaloid content. this analysis demonstrated that the amount and composition of the main alkaloids are highly variable. additionally, there is no important correlation between morphological characters and alkaloid content. so it is not possible to use these characters as a prediction tool of alkaloid content during breeding process. introduction papaver somniferum l. is one of the few species which has been used since the neolithic. its worldwide distribution results from its long history in cultivation. the use of poppy alkaloids for medicinal purpose is proved for the middle ages, when it was an ingredient of the tincture called ‘theriak’. later in the 16th century paracelsus developed another famous tincture called ‘laudanum’ (seefelder, 1996). opium poppy contains more than 40 different alkaloids. the five main alkaloids of p. somniferum are morphine, codeine, thebaine, papaverine and noscapine (dingermann et al., 2004). morphine is the dominant alkaloid and a strong naturally occurring pain reliever. an industrial synthesis of this substance is possible but with very low yield, e.g. 10% in fuchs-synthesis. codeine and noscapine are used as cough suppressant and papaverine is a smooth muscle relaxant. thebaine is not directly used therapeutically but is industrially converted into other pain relievers. during the long time of cultivation a lot of different forms (landraces, cultivars, varieties and chemical types) which belong to one species have been bred and cultivated. the infraspecific classifications of danert (1958) and hammer (1981) as well as hanelt and hammer (1987) are based on a few morphological characters such as capsule dehiscence, shape of stigmatic lobes and colour of flowers and seeds. it is differentiated between three subspecies (setigerum (dc.) corb., somniferum and songaricum basil.), four convarieties and a total of 52 botanical varieties. the subsp. setigerum, separated from the other two subspecies according to the hairiness of buds, is supposed to be the wild relative of the cultivated subspecies (hammer & fritsch, 1977; kadereit, 1986). actually there exist two contrary breeding aims: the first breeding for lines with a very high content of alkaloids for medicinal purpose and the opposite aim are lines with very low alkaloid content for food production (prajapati et al., 2002). the aims of this work were at first to investigate the variability of morphological characters as well as the variability of alkaloid content. the second point is to find out if there is a correlation between morphological and phytochemical data and if it is possible to use special morphological characters as a prediction tool of alkaloid content during breeding process. materials and methods plant material and morphological characterisation in total 300 accessions of p. somniferum from nearly all over the world were cultivated under field conditions in gatersleben in 2005, 2006 and 2007. the morphological characterisation was done according to a descriptor (dittbrenner et al., 2008). this descriptor was developed to standardise the characterisation and contains chromosome number as well as agronomic and morphological characters including flowering date, plant height, number and size of capsules, shape and hairiness of leaves, as well as flower and seed colour. during the whole time of field cultivation weather conditions for example temperature, amount of rainfall and solar radiation were measured. the amount of rainfall in the period of time from flowering until the end of maturation was higher in 2005 with 159.9 mm compared to 123.1 mm in 2006 and 247.4 mm in 2007. these values were accompanied by lower temperatures in 2005. in this year an average of 23.53 °c was measured 2 m above ground compared to 25.62 °c in 2006 and 24.22 °c in 2007. phytochemical analyses ripe and dried capsules from the first flower of a plant (primary capsule) were harvested in the field from all 300 accessions in the years 2005 and 2006. in 2007 only a selection of 40 accessions which represented 10 accessions with very low alkaloid content, 10 with the highest and 20 accessions from the middle were measured to validate results from the other two years. seeds, stigmatic disc and stalk were removed. nine capsules per accession were ground to powder. for methanol extraction (75% meoh) 50 mg of a mixture of three capsules were used. in total three pooled samples per accession were analysed. extraction took place in an ultrasonic bath at 40 °c. the extracts were resolved by reversed phase hplc (agilent system). columns were 150 x 2.00 mm packed with 5 µm porous silica gel (c18 gemini; phenomenex). a gradient analysis (tab. 1) was carried out with the following solvents: a mixture of 0.1 n nh3 solution and 0.1 n nh4cl solution with a ph of 8.8 (a) and 100% acetonitrile (b). the flow rate was 0.2 ml/min and injection volume 10 µl. for peak identification the retention time as well as the uvvis spectrum in the range of 200-400 nm of each substance were used. the content of the five main alkaloids (morphine, codeine, 104 a. dittbrenner, h.-p. mock, a. börner, u. lohwasser in the year 2005, from 978.10 to 22,575.20 µg/g dry matter in 2006 and from 573.30 to 13,554.95 µg/g dry matter in 2007 (tab. 2). there was a highly significant correlation between total content of alkaloids and morphine in both years (2005: r=0.926/p=0.000; 2006 (tab. 4): r=0.918/p=0.000). in 2007 a higher correlation coefficient between total content of alkaloids and morphine (r=0.963/p=0.000) could be detected within the 40 accessions. in nearly all accessions morphine was the main alkaloid. in 2005 there were just six accessions with a different main alkaloid: two with papaverine, two with codeine, one with thebaine and one with noscapine as the most abundant alkaloid. in the second year three accessions showed a higher content of papaverine than morphine. within the analysis of the 40 accessions of 2007 there were again three accessions with higher papaverine content than morphine and one accession in which noscapine was the most abundant alkaloid. the bar diagram (fig. 1) demonstrates total alkaloid content in both years for a selection of 10 accessions which represents a cross section through the profile of all 300 accessions. some accessions like m408, m670 and m691 had a clearly increased content in 2006 compared with 2005 while m238 and m755 had lower values in 2006. a similar picture could be shown for comparison of morphine content in both years (fig. 2). the composition of the main alkaloids varied from accession to accession and between the years 2005 and 2006 (fig. 3, 4). some lines like m408 and m691 had a clearly increased content of noscapine in 2006 compared to 2005, while the codeine content of m238 is decreased in the second year of cultivation compared to 2005. the ttest showed that within all 300 accessions values for morphine, noscapine and thebaine are significantly different and in most cases higher in the second year of cultivation compared to 2005. this behaviour could not be detected for codeine and papaverine, respectively. tab. 1: hplc gradient for separation of alkaloids. (solvent a: a mixture of 0.1 n nh3 solution and 0.1 n nh4cl solution; solvent b: acetonitrile) minutes % a % b 0 76 24 11 76 24 14 64 36 33 29 71 36 14 86 46 0 100 50 0 100 60 76 24 75 76 24 thebaine, papaverine and noscapine) was calculated in µg/g dry matter (dm) in reference to the calibration curve of each main alkaloid at 280 nm. the empower software was used for data acquisition and analysis. statistical analysis was done with spss 10.0 (1999). for metric/non metric data pearson’s/spearman’s correlations coefficient were used for correlation analyses between morphological characters and content of alkaloids determined in 2005 and 2006. results phytochemical analyses a high variation could be detected with respect to the quantitave composition of the main alkaloids within all 300 accessions. the morphine content ranged from 363.00 to 17,749.05 µg/g dry matter tab. 3: spearman’s correlations coefficient between content of main alkaloids and some morphological characters. (c-codeine, m-morphine, n-noscapine, p-papaverine, t-thebaine) c m n p t total content plant colour corr. coeff. 0.262 0.228 0.225 0.456 0.171 0.348 sig. (2-tailed) 0.000 0.000 0.000 0.000 0.003 0.000 anthocyanins corr. coeff. -0.137 -0.076 -0.144 -0.417 -0.046 -0.191 in buds sig. (2-tailed) 0.018 0.191 0.013 0.000 0.428 0.001 flower colour corr. coeff. 0.140 0.179 0.180 0.474 0.084 0.291 sig. (2-tailed) 0.017 0.002 0.002 0.000 0.153 0.000 colour corr. coeff. 0.096 -0.088 -0.012 0.323 -0.081 0.005 of filaments sig. (2-tailed) 0.100 0.134 0.836 0.000 0.165 0.935 capsule corr. coeff. 0.007 0.229 0.111 0.458 -0.010 0.270 dehiscence sig. (2-tailed) 0.902 0.000 0.061 0.000 0.872 0.000 tab. 2: range of main alkaloids and total alkaloid content measured during three years. (dm dry matter). content 2005 (µg/g dm) content 2006 (µg/g dm) content 2007 (µg/g dm) alkaloids minimum maximum minimum maximum minimum maximum codeine 6.01 3,595.13 3.00 2,718.39 37.82 2,827.96 morphine 363.00 17,749.05 978.10 22,575.20 573.30 13,554.95 noscapine 0.00 5,947.52 0.00 6,641.89 0.00 6,637.92 papaverine 0.00 3,151.21 0.00 2,817.48 3.79 3,607.48 thebaine 0.02 2,797.36 0.35 2,942.29 1.27 2,574.87 total 683.32 25,034.84 1,609.49 25,338.55 1,126.21 18,749.14 alkaloid variability of p. somniferum 105 morphological characterisation and correlation with phytochemical data according to the descriptor, 35 morphological and agronomic characters were collected for all 300 accessions in 2005, 2006 and 2007. most notably, it proved difficult to distinguish between round and angular stigmatic lobes and dehiscent or indehiscent capsules in 2005 as well as 2007. in those years some accessions showed half open capsules possibly due to a long period of rain during maturity. another problem was determining seed colour; in some cases there were different seed colours in the capsules of one plant. determination of chromosome numbers with flow cytometry showed that the accessions of subspecies setigerum are tetraploid whereas all accessions of the other subspecies are diploid. tab. 4: pearson’s correlations coefficient between content of main alkaloids and two morphological characters. (c-codeine, m-morphine, n-noscapine, ppapaverine, t-thebaine) c m n p t total content codeine corr. coeff. 1 0.231 0.232 0.284 0.480 0.432 sig. (2-tailed) 0.000 0.000 0.000 0.000 0.000 morphine corr. coeff. 0.231 1 0.208 0.163 0.207 0.918 sig. (2-tailed) 0.000 0.000 0.005 0.000 0.000 noscapine corr. coeff. 0.232 0.208 1 0.368 0.031 0.506 sig. (2-tailed) 0.000 0.000 0.000 0.588 0.000 papaverine corr. coeff. 0.284 0.163 0.368 1 0.108 0.407 sig. (2-tailed) 0.000 0.005 0.000 0.063 0.000 thebaine corr. coeff. 0.480 0.207 0.031 0.108 1 0.342 sig. (2-tailed) 0.000 0.000 0.588 0.063 0.000 total content corr. coeff. 0.432 0.918 0.506 0.407 0.342 1 sig. (2-tailed) 0.000 0.000 0.000 0.000 0.000 plant height corr. coeff. -0.178 -0.117 -0.088 -0.399 -0.025 -0.193 sig. (2-tailed) 0.002 0.043 0.130 0.000 0.672 0.001 seed yield corr. coeff. -0.163 -0.195 -0.207 -0.459 -0.096 -0.304 per capsule sig. (2-tailed) 0.005 0.001 0.000 0.000 0.097 0.000 number of corr. coeff. -0.056 0.088 0.008 -0.159 0.013 0.046 stigmatic rays sig. (2-tailed) 0.337 0.130 0.884 0.006 0.820 0.432 total content of alkaloids 0 4000 8000 12000 16000 20000 24000 28000 32000 36000 m408 m670 m691 m152 m156 m238 m263 m755 m86 m567 accessions µ g /g d m 2005 2006 fig. 1: bar diagram of total content of alkaloids in 2005 and 2006 with standard deviation. (dm – dry matter) morphine 0 4000 8000 12000 16000 20000 24000 28000 32000 m408 m670 m691 m152 m156 m238 m263 m755 m86 m567 accessions c o n te n t (µ g /g d m ) 2005 2006 fig. 2: bar diagram of morphine content in 2005 and 2006 with standard deviation. (dm – dry matter) m 4 0 8 m 6 7 0 m 6 9 1 m 1 5 2 m 1 5 6 m 2 3 8 m 2 6 3 m 7 5 5 m 0 8 6 m 5 6 7 codeine thebaine papaverine noscapine 5,947.52 µg/g dm 0 500 1000 1500 2000 2500 c o n te n t (µ g /g d m ) accession 2005 fig. 3: distribution pattern of codeine, thebaine, papaverine and noscapine in the year 2005. 106 a. dittbrenner, h.-p. mock, a. börner, u. lohwasser ç only weak correlations (with correlation coefficients smaller than 0.480) were detected between any morphological characters for example plant, flower and filament colour, plant height, seed yield per capsules as well as number of stigmatic rays and the content of different alkaloids or total alkaloid content in both years. in tab. 3, four values for 2006 are listed as an example. furthermore, even no pattern was recognisable in the composition of alkaloids which was based on the existing infraspecific classification. the only exception was that tetraploid accessions of subsp. setigerum showed significant higher papaverine content than the two other subspecies. discussion the qualitative and quantitative composition of the main alkaloids of all 300 accessions was highly variable, but with morphine as the main alkaloid in nearly all accessions. shukla et al. (2006) observed within 98 germplasm lines morphine content as major constituent followed by noscapine, codeine, thebaine and papaverine. our results showed no such clear behavior. in general, accessions had high accumulations of morphine. the position of the other main alkaloids changed comparing all accessions in all three years of cultivation. in addition, we detected several accessions with noscapine, papaverine or codeine as major constituent in all three years. similar results are documented by gümüsçü et al. (2008) who found two germplasm lines out of 99 which had more codeine and noscapine compared to morphine, respectively. one possible explanation of an accumulation of noscapine or papaverine could be an increased enzymatic activity in appropriate parts of the biosynthetic pathway that results in larger amounts of these alkaloids. codeine is a precursor of morphine. less enzymatic activity involved at a particular conversion step causing partial blocking of morphine pathway (codeine to morphine) could induce an accumulation of codeine. tookey et al. (1976) reported about increased codeine to morphine ratio caused by lancing the capsules during maturation under controlled environmental conditions. this induced not an increase in production of codeine but rather a translocation from stem to capsules. so codeine constituent could be explained in two ways genotypic, block in alkaloid biosynthesis, or environmental. due to the fact that codeine as major constituent was only detected within two accessions in 2005 most likely in our case is the environmental influence on its accumulation. strong winds in 2005 could have caused damages on capsules as well as lower temperatures during capsule maturation could have influenced enzymatic activity. during the maturation period rainfall and low temperatures might reduce alkaloid accumulation. according to hofman and menary (1979) three mechanisms could cause lower alkaloid content during wet weather: leaching, fungal activity and the changed activity of capsule enzymes. the amount of rainfall in the period of time from flowering until the end of maturation was higher in 2005 with 159.9 mm compared to 123.1 mm in 2006. these values were accompanied by lower temperatures in 2005. high rainfall and relative humidity permit heavy fungal growth. their results showed that staked capsules were relatively undamaged by fungal growth compared to unstaked control capsules. staking treatment reduced interplant abrasion and produced capsules with a comparatively intact waxy bloom (hofman and menary, 1984). a damaged and dewaxed capsule is more susceptible to leaching by rain. according to hofman and menary (1984) up to 60 and 80% of morphine and codeine content were removed from the mature capsules by leaching. in contrast, the loss of thebaine averaged only 40%. bernáth and tétényi (1979, 1981) demonstrated that higher temperatures as well as light intensity influenced alkaloid biosynthesis positively. the total amount of alkaloids reached a maximum value in a „high temperature“ programme under the 3.2x104 lux illumination. within this programme maximum temperatures of 26.0/16.0 °c (day/night) are used in phytotron compared to 18.5/11.5 °c (day/night) in „low temperature“ programme. it was not possible to detect strong correlations between morphological characters like flower colour, plant height and content of major alkaloids as it was reported in bajpai et al. (2000). they investigated 210 opium poppy lines according to morphological characters and alkaloid content. the conclusion of their work was that high morphine yield is related to large size of capsules and peduncles, small plant height, absence of pigmentation in flowers and low level of seed production. however, the mentioned correlations between morphological characters and phytochemical data showed correlation coefficients with values lower than 0.500. these data are comparable to the correlations obtained in this study in which less than 25 % of the total variance is explained by that statistical correlation. khanna and singh (1975) had done correlation studies in p. somniferum within five varieties and three strains. as one result of their work they showed that plant dry weight, capsule number and stigmatic rays are significantly correlated with opium, morphine and seed yield. between the observed characters within our investigations for example capsule numbers per plant or stigmatic rays and content of alkaloids exist no significant correlations. within all 300 accessions only tetraploid subsp. setigerum could be separated according the hairiness of buds. additionally, most of these lines showed a higher content of papaverine. but this alkaloid content was not distinct enough to use it as a separation character within all 300 accessions. all in all there is no important correlation between morphological characters and alkaloid content. so it is not possible to use any of these characters as a prediction tool of alkaloid content during breeding process. for this purpose it is necessary to continue measuring the alkaloid content directly. acknowledgements we thank the leibniz institute of plant genetics and crop plant research (ipk), gatersleben for financial support. we express our gratitude to silke peterek as well as renate kurch and her group for excellent assistance. references bajpai, s., gupta, a.p., gupta, m.m., sharma, s., govil, c.m., kumar, s., 2000: inter-relation between descriptors and morphine yield in asian fig. 4: distribution pattern of codeine, thebaine, papaverine and noscapine in the year 2006. (dm – dry matter) m 4 0 8 m 6 7 0 m 6 9 1 m 1 5 2 m 1 5 6 m 2 3 8 m 2 6 3 m 7 5 5 m 0 8 6 m 5 6 7 codeine thebaine papaverine noscapine 6,641.89 µg/g dm 0 500 1000 1500 2000 2500 c o n te n t (µ g /g d m ) accession 2006 alkaloid variability of p. somniferum 107 ç germplasm of opium poppy papaver somniferum. genet. resour. crop evol. 47, 315-322. bernáth, j., tétényi, p., 1979: the effect of environmental factors on growth. development and alkaloid production of poppy (papaver somniferum l.) i. responses to day-length and light intensity. biochem. physiol. pflanzen 174, 468-478. bernáth, j., tétényi, p., 1981: the effect of environmental factors on growth. development and alkaloid production of poppy (papaver somniferum l.) ii. interaction of light and temperature. biochem. physiol. pflanzen 176, 599-605. danert, s., 1958: zur systematik von papaver somniferum l. kulturpflanze 6, 61-88. dittbrenner, a., lohwasser, u., mock, h.-p., börner, a., 2008: molecular and phytochemical studies of papaver somniferum l. in the context of infraspecific classification. acta hort., in press. dingermann, t., hiller, k., schneider, g., zündorf, i., 2004: schneider. arzneidrogen. 5. aufl., elsevier, münchen. gümüsçü, a., arslan, n., sarihan, e.o., 2008: evaluation of selected poppy (papaver somniferum l.) lines by their morphine and other alkaloids content. eur. food res. technol. 226, 1213-1220. hammer, k., fritsch, r., 1977: zur frage nach der ursprungsart des kulturmohns (papaver somniferum l.). kulturpflanze 25, 113-124. hammer, k., 1981: problems of papaver somniferum-classification and some remarks on recently collected european poppy land-races. kulturpflanze 29, 287-296. hanelt, p., hammer, k., 1987: einige infraspezifische umkombinationen und neubeschreibungen bei kultursippen von brassica l. und papaver l. feddes repert. 98, 553-555. hofman, p.j., menary, r.c., 1979: variations in morphine, codeine and thebaine in the capsules of papaver somniferum l. during maturation. aust. j. agric. res. 31, 313-326. hofman, p.j., menary, r.c., 1984: losses, by leaching, of alkaloids from the capsule of the poppy (papaver somniferum l.) during maturation. aust. j. agric. res. 35, 235-261. kadereit, j.w., 1986: papaver somniferum l. (papaveraceae): a triploid hybrid? bot. jahrb. syst. 106, 221-244. khanna, k.r., singh, u.p., 1975: correlation studies in papaver somniferum and their bearing on yield improvement. planta med. 28, 92-96. prajapati, s., bajpai, s., singh, d., luthra, r., gupta, m.m., kumar, s., 2002: alkaloid profiles of the indian land races of the opium poppy papaver somniferum l. genet. resour. crop evol. 49, 183-188. seefelder, m., 1996: opium: eine kulturgeschichte. 3. überarb. aufl., ecomed, landsberg. shukla, s., singh, s.p., yadav, h.k., chatterjee, a., 2006: alkaloid spectrum of different germplasm lines in opium poppy (papaver somniferum l.). genet. resour. crop evol. 53, 533-540. spss for windows, 1999: spss inc., chicago. tookey, h.l., spencer, g.f., grove, m.d., kwolek, w.f., 1976: codeine and morphine in papaver somniferum grown in a controlled environment. planta med. 30, 340-348. address of the authors: anke dittbrenner, dr. ulrike lohwasser, priv. doz. dr. hans-peter mock and priv. 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/pagesize [907.087 680.315] >> setpagedevice journal of applied botany and food quality 93, 217 224 (2020), doi:10.5073/jabfq.2020.093.026 1department of horticulture, agrifood research and technology centre of aragon (cita), zaragoza, spain 2agrifood institute of aragon – ia2 (cita-university of zaragoza), zaragoza, spain sugar content and organic acid profiles of local apple cultivars recovered from mountain zones lourdes castel1, ana pina1,2, patricia irisarri1, pilar errea1,2* (submitted: april 6, 2020; accepted: october 2, 2020) * corresponding author summary ancient apple cultivars grown in local areas have so far been largely unexplored and may attract a large share of consumers oriented towards natural foods evoking ancient flavors. in this work, 34 traditional apple cultivars of the pyrenees were analyzed in terms of sugar and acidity profile and their relation, as alternative fruits for a growing demand in society regarding quality and nutritional properties. the results show a wide range between cultivars of analyzed variables, especially in terms of acid, ph and soluble solids, with high standard deviation values. the results were higher than in other similar studies of commercial apple cultivars, as well as higher values related to other local accessions. the large differences between cultivars can be attributed to the origin of the plant material, since all cultivars were grown under the same geographical conditions and with the same applied agronomic practices. the associations found in this study provide information about the nutritional content of the analyzed apples and their organoleptic and physicochemical qualities, and could be useful in targeting specific consumer requirements. in conclusion, this study highlights the suitability of these local accessions recovered in the pyrenees as key genetic resources to be used in future breeding programs, as well as its potential use for different products which would open the door to new varieties and new consumption alternatives. key words: apple cultivars, ancient varieties, sugars, organic acids, pca introduction the genetic diversity of apple cultivars represents an unique and invaluable natural resource (vanderzande et al., 2017). in mountainous areas from aragon (north-eastern spain), generations of farmers performing selection processes in their orchards and home gardens have given rise to a wide diversity of quality fruit-plant material that constitutes an extraordinary genetic heritage of germplasm. in the past, apple production was based on local varieties, but the great varietal reconversion in the 1960s led to the abandonment of many of these varieties that had an important significance in local food. currently, the world production of about 70 million metric tons/year is based on only a small number of cultivars: only seven apple cultivars account for more than 50% of the production in europe (van nocker et al., 2012). the massive use of few related commercial cultivars has dramatically reduced the genetic diversity of local apple varieties, and many interesting and well-adapted traditional and local varieties, which are known to contain many desirable attributes, have been lost. the reduction in genetic variability is an important limitation of the ability to respond to new needs and challenges. the selection programmes for new apple varieties have resulted in a decrease in the diversity of apple genetic background (cornille et al., 2012). but the development of new apple cultivars with desirable characteristics and a wide genetic base is dependent upon breeders having access to as much genetic diversity as possible. and now, there is a growing demand in society regarding quality and health, which is increasing the interest in varieties encoding for fruit quality and disease resistance traits that offer new opportunities for cultivation and use as raw material for breeding. this local germplasm is cultivated mainly in marginal areas, is well adapted to the local environment and is resistant to biotic and abiotic stress. the study of the species and fruit varieties that have survived this abandonment, their potential in relation to other more widespread species and the understanding of the physiological characteristics that make them worthy of this survival constitute a fundamental aspect, not only of the preservation of this richness and diversity genetics, but also of the value of the potential development of these species in these areas of possible fruit regeneration. local apple accessions from these mountain zones were collected and recovered and their genetic identity and variability analysed (pina et al., 2014). an important range of genetic variability was detected, suggesting interesting differences in the physical and chemical composition and nutritional properties of the fruit, which could increase the value of these local accessions. the chemical composition of apples is very complex. among the most important constituents are sugars and organic acids (lanzerstorfer et al., 2014). their contents and especially their ratio greatly affect the desirability of apple for direct consumption and their suitability for processing (petkovsek et al., 2007). the sugar profile of the fruit pulp has an effect on the organoleptic properties and nutritional value of these fruits. the most significant sugars are fructose, glucose, sucrose and sorbitol (zhang et al., 2010; larsen et al., 2019). the organic acids in apples include malic, citric, shikimic, fumaric, and quinic acids (mapson, 1970).these make an important contribution to the flavor of the fruit and also to their stability, colour, nutrition, acceptability and keeping qualities. the balance between sweetness and acidity is also responsible for the taste and flavor of apples (persic et al., 2017; petkovsek et al., 2007; hecke et al., 2006). several studies have addressed the chemical composition of different apple cultivars, but most have focused on a limited number of cultivars from among those most widely consumed and appreciated (keenan et al., 2012; guan et al., 2015). as a result, very few data are available in the literature on the nutritional properties of geographically limited area, particularly traditional apple cultivars (jakobek and barron, 2016; larsen et al., 2017). such a lack of information prevents the use of these germplasms for the genetic improvement of new cultivars and re-evaluating local food products, something which may attract a large number of consumers looking for natural foods that evoke ancient flavors. to the best of our knowledge, the sugar and organic acid compositions of local apples recovered from the mountainous areas of the pyrenees have not been investigated until now. the main objectives of this paper were to characterize local apple cultivars from mountainous areas by analyzing their sugar and acid contents, total soluble solids (tss, °brix) and ph, as well as the study of the relationships established between the various characteristics in order to increase 218 l. castel, a. pina, p. irisarri, p. errea the value of this unique local material, especially as far as its chemical composition is concerned. material and methods plant material thirty-four apple accessions (malus domestica borkh) and one reference cultivar 'golden delicious' were analyzed. these accessions belong to the cita germplasm collection and were recovered on various prospecting missions to mountainous areas of aragon (north-eastern spain) since 2001. they were collected in the pyrenean localities in aragon and included samples from old, abandoned trees and small farms and were shown to be unique genotypes by ssr markers (pina et al., 2014).these trees were propagated vegetatively and were established in collections located in bescós de la garcipollera (huesca, aragon) at an altitude of 900 m. apple fruits were collected between august and october at the ripening period of each cultivar. fifteen fruits were selected from each accession for analysis. morphological data harvest time, fruit weight and firmness of flesh were recorded for each cultivar according to upov (1974), ibpgr (watkins and smith, 1983) and royo diaz et al. (2017) guidelines. fruits were hand-picked at technological maturity by a single person to ensure consistency of the maturity grade. the harvest date decision was based on development in fruit removal force, sensory evaluation (color, softness, smell, and taste) and supported by the determination of the starch-iodine test ctifl scale (platon, 1995) reached at least an index of 6. firmness was measured using a digital fruit firmness teste (model tr turoni) with a 11 mm plunger tip. 'golden delicious' was used as a reference cultivar, which matured in the first two weeks of october. in this way, five different groups were classified from the collection dates (1 – very early, 3 – early, 5 – medium, 7 – late or very late) determination of total soluble solids, acid content and ph soluble solid content (ssc) were determined from the juice of the samples analysed in each batch. the juice was obtained by liquefying apple pieces with skin and pulp of at least 10 fruits. juice was filtered through a paper funnel (filter paper circles-folded, ø 185 mm, chmlab group, barcelona, spain), to separate the solid phase from the liquid one. once the juice was obtained, the ssc were measured at 20 °c using a refractometer (pocket refractometer pal-1, atago, tokyo, japan) and expressed as °brix. the acid content and ph were determined at 20 °c, using a digital ph meter calibrated with ph 4 and ph 7 buffers measured with a model760 basic titrino (785 dmp titrino, metrohm, herisau, switzerland). in addition, 25 ml of the filtered juice was placed in a beaker. the sample was taken to the valorimeter (760-sample change, metrohm, herisau, switzerland) and the juice was titrated with 0.1 n naoh. the results obtained were: ph and total acid (ta), and malic and citric acid contents. determination of sugars (sucrose, glucose, fructose and xylose) individual sugar contents (sucrose, glucose, fructose and xylose) were determined according to the method described by blanco et al. (1988) using high-performance liquid chromatography (hplc icp activa horiva jobin yvon, kyoto, japan). juice (1 ml) was weighed and diluted with milli-q water to 25 ml. the samples were purified and filtered through a methanol and polyvinylidene difluoride-activated sep-pack cartridge (waters cromatography, cerdanyola del vallés, spain) with a 0.45 μm filter. then, 20 μl of the treated sample was injected into the chromatographic system. the separation was performed using a waters sugar-pak i (waters cromatography, cerdanyola del vallés, spain) (300 × 6.0 mm) 10 μm column, at 90 °c, using as a mobile phase a solution of 50 mg/l na2ca edta at a flow rate of 0.44 ml/min. statistical analysis all traits were measured, or scored, for each genotype separately over the two-year period, and the two-year means were calculated. all the statistical analyses were performed using spss statistics version 21.0 (ibm, new york, u.s.a). the minimum and maximum values, standard deviation, and variance were calculated for each accession studied. the normality of the data was analysed using the shapirowilk test, and traits showing normal distribution were analysed by one-way analysis of variance (anova), considering genotype and year as independent factors. traits that did not meet the normality criteria were analysed with the kruskal-wallis nonparametric test. in addition, correlations between and within traits for each year were analysed using pearson coefficients. correlations were calculated using the raw data from the two-year period, according to pearson’s test at p ≤ 0.01 and p ≤ 0.05. the results for the individual sugars were compared using one-way anova and the differences determined tested with the kruskalwallis test at a significance level of 0.05. data for each genotype were averaged and mean values used as estimated genotypic values. multidimensional scaling (proxscal algorithm) analysis was applied to the studied accessions as an attempt to identify how the studied components are distributed based on their nutritional compound content and organoleptic quality. applying multidimensional scaling (mds) generated a spatial map that facilitated an intuitive understanding of the relationships between the objects represented on the map. the distances between the objects indicate their (dis) similarities: similar objects are represented by closely spaced points and dissimilar objects by points that are further apart. the dimensions are factors with real meaning that make it possible to explain the differences between groups. the component matrix was evaluated and orthogonal factors rotated using variance maximising (varimax). finally, a dendrogram was constructed to evaluate the homogeneity of the clusters using the inter-group linkage method. results and discussion harvest time and morphological characterization in the local apple accessions harvest times showed five different groups using 'golden delicious' as reference cultivar: very early, early, medium, late and very late (tab. 1). the time varied from 21 august for the earliest cultivar (accession 29) to 15 november for the latest cultivars (accessions 5, 7, 18 and 21). most of the local accessions matured in the early and medium harvest periods. in spain, 87% of apple production is limited to four cultivars: 'golden delicious' (more than 50% of production), 'gala', 'red delicious' and 'fuji' (iglesias et al., 2016), which are harvested in this region between mid-august and mid-october. local varieties were severely displaced by more modern ones from other countries, and this limited variability also in traits of interest, included a wide range of collection times found in traditional cultivars. other morphological observations (tab. 1) showed different ranges of weight and firmness. total weight showed values between 48.84 and 276.18 g. cultivars producing smaller apples were accession 16. the firmness varied between 5.40 in accession 30 to 9.88 kg/cm² in accession 7, showing great variability as well as other studies related to local germplasm (ramos-cabrer et al., 2007). sugars and organic acids of mountain apple cultivars 219 ph, total acid (ta), malic, citric and soluble solid content tab. 2 shows the analytical results for ph, acid composition and ssc (°brix) for the 34 local apple accessions. a great variability was found in terms of ph, ta and ssc. ta contents varied from 0.334 g/kg in accession 10 to 12.055 g/kg in accession 18. these data are higher than those recorded in other research carried out on granny smith and other local apple cultivars (begic-akagi et al., 2014), which showed values between 0.62 and 15.36 g/kg, and those reported by hecke et al. (2006), who reported ta values of commercial cultivars between 6.26 and 14.12 g/kg. some studies have determined that ta had a negative correlation with sensory acceptability indicating it had a negative impact on panellist acceptability (keenan et al., 2012). in general, many consumers determine apple quality based on their balance of sweetness and acidity, and good balance between acidity and sweetness should be a good criterion in selecting apples for processing applications. the main acids involved in the acidity of the fruit are organic. these compounds, along with the sugars, influence the organoleptic perception of sweetness and the aroma of the apple (harker et al., 2006; aprea et al., 2017). the content of organic acids in fruit juices not only influences their flavour but also their stability, nutrition, acceptability and keeping quality. besides their importance in flavour, acids are important in gelling product processing because they affect the gelling properties of pectin. also, the non-volatile malic, citric and quinic acids mediate the sour-to-astringent taste of apples (chapman and horvat, tab. 1: different ranges of harvest time, fruit weight (gr), firmness (kg/cm²), ground colour, relative area of over colour, intensity of over colour and general shape of the 34 cultivars used in this study. descriptors u_56 u_24 u_52 u_35 u_36 u_37 u_38 u_28 upov cultivar harvest fruit firmness ground relative area hue of intensity of time weight (g) (kg/cm²) colour of over colour over colour over colour general shape 1 very late 93.51 8.20 whitish green not visible not visible oblong intermediate-conical 2 early 212.82 8.15 yellow-green not visible red not visible short-globose conical 3 early 158.53 6.95 yellow-green very large orange medium flat-globose 4 late 248.71 9.13 whitish yellow two colours medium flat-globose 5 very late 129.19 9.55 not visible not visible pink not visible flat-globose 6 very early 165.71 7.53 whitish yellow two colours red light flat-globose 7 late 248.75 9.88 yellow-green two colours orange dark oblong-conical 8 medium 178.27 7.28 yellow-green two colours light short-globose conical 9 medium 194.37 8.95 yellow-green not visible not visible flat-globulose 10 early 276.18 7.93 whitish yellow not visible orange not visible flat 11 late 224.99 8.75 yellow-green two colours light short-globose conical 12 very early 181.77 9.30 yellow-green not visible red not visible flat 13 early 155.18 7.50 yellow-green very small medium globose 14 early 221.07 8.25 green not visible reddish brown not visible globose intermediate conical 15 early 86.52 7.80 green two colours reddish brown light oblong-conical 16 very late 48.84 9.13 yellow-green very small red dark globose intermediate conical 17 medium 244.05 6.48 yellow-green very large pink dark flat-globulose 18 medium 151.06 6.20 yellow very small light short-globose conical 19 late 66.45 7.58 yellow-green not visible not visible flat 20 early 80.79 6.60 yellow not visible red not visible flat cronical-trunk 21 early 247.19 8.13 yellow very large red medium conical 22 medium 240.90 6.90 yellow very large dark flat cronical-trunk 23 early 213.86 7.35 green not visible red not visible flat cronical-trunk 24 early 265.33 7.45 yellow-green very large red dark flat cronical-trunk 25 early 199.47 9.63 not visible very large pink dark globose intermediate conical 26 very early 260.00 8.10 yellow-green very small light flat-globose 27 early 167.11 6.08 yellow not visible not visible oval cronical-trunk 28 very early 123.67 6.60 whitish yellow not visible red not visible flat-globose 29 very early 182.43 7.23 whitish yellow very large dark short-globose conical 30 early 155.05 5.40 whitish yellow not visible pink not visible flat-globose 31 early 133.75 6.85 yellow very small pink dark short-globose conical 32 very early 147.10 6.25 yellow very large orange dark flat-globose 33 medium 198.78 7.75 green very small orange light short-globose conical 34 medium 162.64 8.45 yellow-green two colours medium flat-globose 220 l. castel, a. pina, p. irisarri, p. errea 1989). the regular consumption of fruit acids is helpful in preventing illness and mild metabolic disorders in the human body. for example, fruit acids work as ‘natural’ tooth cleaners. the ssc expressed as °brix is commonly used as an estimate of fruit sweetness and is included in assessments of the postharvest quality of apples (guan et al., 2015). in the set of accessions studied, ssc ranged between 10.5 and 19.60 °brix (tab. 2). these results were higher than in other similar studies of commercial apple cultivars, which recorded values of 9.5 to 15.8 (aprea et al., 2017), 11.8 to 14 (vieira et al., 2009) 10.48 to 14.68 (wu et al., 2007), or 11.4 to 16.1 °brix (thompson-witrick et al., 2014), as well as higher values related to other local accessions from southern italy, with values of between 11.1 and 16.1 °brix (panzella et al., 2013) or non-commercial old varieties of apples cultivated in upper austria, of between 8 and 18.9 °brix (lanzerstorfer et al., 2014). the ph values of the apple cultivars were also very variable, from 2.97 to 8.19. this range of values is higher than others reported previously. in an apple germplasm collection from southern brazil (vieira et al., 2009), the ph of the apple cultivars varied from 3.90 ('imperatriz') to 4.27 ('fred hough'), and for apple cultivars grown in china (wu et al., 2007) the ph values varied between 3.59 and 4.16. in other studies carried out on commercial apple varieties, it was observed that the highest ph was between 3.46 and 4.21, and that these values were inversely correlated with the ta values (keenan et al., 2012). in general, there was wide-ranging variation among the apple cultivars in terms of ta content, ph and ssc, as well as harvesting time (tab. 1, 2) with high standard deviation values. these parameters also varied greatly in the aforementioned studies and may be due to the genotype, the specific geography of the different areas, or the various agronomic practices used. since all the local apple cultivars used in this study were grown in the same location using similar horticultural practices, the variation in physical and chemical characteristics can be attributed to genetic variability, which was previously analysed for these genotypes (pina et al., 2014). the striking compound variability found in these local accessions provides information on the strong added value they represent. sugar content: sucrose, glucose, fructose, xylose, total sugars, sucrose between glucose and glucose between fructose. another characteristic related to fruit quality is sugar content. tab. 3 displays the sugar distribution range for these 34 local accessions. in general, the total population of local accessions presents a wide range of values in the variables such as the ratio glucose/fructose, total sugars, sucrose and glucose. the total sugar content of the apple cultivars in the study ranged between 79.8 and 253.6 g/l with an average concentration of 150.8 g/l. this agrees with the values reported by begic-akagic et al. (2014) for traditional cultivars from bosnia-herzegovina, with values of between 80.06 and 119.7 g kg-1. for 62 heritage apple cultivars, wang (2010) reported total sugar content ranging between 92.2 and 164.9 g/l. for commercial cultivars, these values were found to range between 115 and 183 g/kg (hecke, 2006), but also in another study on commercial cultivars (vieira, 2009), the total sugar content (100 g/l) ranged from 11.54 ('imperatriz') to 14.78 ('fuji suprema'). the sugar composition showed a proportion similar to other related studies. the amount of sucrose, fructose and glucose ranged from 0.27 g/100 ml to 8.13 g/100 ml, from 4.68 g/100 ml to 14.7 g/100 ml and from 0.96 g/100 ml to 7.74 g/100 ml, respectively. xylose, a major component of xyloglucans, represented only a small fraction of the measured sugars, ranging between nondetectable and 1.26 g/100 ml. similar levels have been reported in previous investigations (zhang et al., 2010; celik et al., 2018). as expected, the levels of sugar composition were fructose > glucose > sucrose, similar to other works. an investigation of pomaces from 26 cultivars observed proportions of 18-31% fructose, 3.4-24% sucrose, and 2.5-12.4% glucose per absolute weight of dry apple pomace (queji et al., 2010). another study of 11 cultivars measured average contents of 12.7% glucose, 17.9% fructose, and 7.0% sucrose (sato et al., 2010). these results correspond with other reports analysing commercial cultivars, which showed average levels of 53.9% fructose, 33.8% glucose and 24% sucrose (average of eight commercial cultivars) (wu et al., 2007) or 52.39 and 33.41 g/kg for fructose and glucose (begic-akagi et al., 2014). however, other results, also from commercial cultivars, the analysis of sugar composition showed sucrose (41.8%) and fructose (39.1%) as the most abundant sugars, with few exceptions, followed by glucose (18.3%) (aprea et al., 2017). the majority of cultivars studied contained more fructose and less glucose, a fact that is an advantage for diabetes patients, since it helps to keep the blood sugar level constant (begic-akagi et al., 2014). the sugar content of apples differs depending on the weather conditions, cultivar, culturing technology, position and exposure of the fruits in the crown. some local accessions with low levels of fructose could be of particular interest to consumers. several studies have examined total fruit sugar and the increasing levels of fructose, glucose and sucrose at advanced stages of fruit maturity (łata, 2008; veberic et al., 2005). it is widely known that the sugar profile of pulp flesh is an important component of chemical composition and provides valuable information on the authenticity of fruit juices related to juice production and the organoleptic properties and nutritional value of apples (waldbauer et al., 2017). it also affects the organoleptic properties and nutritional value of fruit products, although other aspects of the chemical composition should be considered when evaluating the quality of apples. even though the sugar and organic acid contents can be influenced by environmental factors and horticultural practices in an orchard (colaric et al., 2007), they depend mainly on plant genotype (persic et al., 2017). regarding entab. 2: descriptive statistics for the variables of ta (total acid), malic acid, citric acid ph, and ssc (soluble solids content ° brix) of the 34 local accessions studied. for each trait, minimum, maximum and mean values and standard deviation (sd) are presented. %cv percent coefficient of variation min max mean sd %cv % ta 0.33 12.06 1.72 2.32 1.35 malic acid (g/l) 1.87 60.27 8.97 11.57 1.29 citric acid (g/l) 1.79 57.57 8.57 11.06 1.29 ph 2.97 8.19 4.19 1.20 0.29 ssc (°brix) 10.5 19.6 14.83 1.84 0.12 tab. 3: descriptive statistics for the variables of sucrose, glucose, xylose, fructose, total sugars, sucrose between glucose and glucose between fructose of the 34 local accessions studied. for each trait, minimum, maximum and mean values and standard deviation (sd) are presented. min max mean sd sucrose (g/100 ml) 0.27 8.13 3.48 2.02 glucose (g/100 ml) 0.96 7.74 3.07 1.51 xylose (g/100 ml) 0.00 1.26 0.38 0.28 fructose (g/100 ml) 4.68 14.70 8.15 2.34 total sugars (g/100 ml) 7.98 25.36 15.08 4.49 sucrose/glucose 0.09 4.54 1.33 0.98 glucose/fructose 0.13 0.85 0.38 0.14 * total sugars: sum of the values of glucose, xylose and fructose. sugars and organic acids of mountain apple cultivars 221 vironmental conditions, it has been reported that fruit characteristics of different cultivars cultivated in mountain areas are characterised by higher sugar content than those of the same cultivar cultivated in flood plain areas (iglesias, 2012). these differences might be attributed to the fact that they are better adapted to conditions of increased radiation and the influence of altitude, which may affect the hormonal balance as well as the nature and quantity of compound synthesis. pearson correlation coefficients for the different linked fruit quality traits evaluated are shown in tab. 4 at p < 0.01 and p < 0.05. ta is associated with fruit maturity and should be evaluated according to the relationships between the soluble solid and total organic acid contents. the local accessions studied did not show a significant correlation at the 0.01 positive level (p < 0.01) between ta and malic and citric acid contents. therefore, fruits with higher ta do not display more citric and malic acid. in addition, ph and ssc showed a significant correlation at the 0.05 positive levels (p < 0.05) with a value of the pearson correlation coefficient r of 0.389. related to sugars, fructose was positively correlated with glucose (r = 0.701), and we also found a positive correlation between sucrose, glucose and fructose content and total sugar content (r = 0.615; r = 0.775 and r = 0.862, respectively). although the higher content of fructose with respect to the other sugars was in agreement with most studies on apple cultivars, the positive correlation between glucose and total sugars, as well as negative correlations was not found in other studies (larsen et al., 2019). effect of fruit maturity classification on sugar content and ssc to evaluate the effect of maturity stage on sugar content and ssc, the kruskal-wallis nonparametric test was used to compare the mean values. simple linear and logarithmic correlation analysis was used to indicate a measure of the correlation and the strength of the relationship between these variables. discriminant analysis (da) is based on the extraction of linear discriminant functions of the independent variables by means of a qualitative dependent variable and several quantitative independent variables. two processes were applied in da: (1) stepwise da, which selected the quantitative variables that enhance discrimination of the groups established by the dependent variable; and (2) introduction of all independent variables. the objective of this process was to avoid information loss, although the system obtained was more complex. in our results, there was a significant relationship between sucrose and ssc and between fructose and glucose. they did not show a significant relationship between the stage of maturity of the fruit and the variables of fructose and °brix, and other variables studied. for fructose and ssc, whereas the local accessions with an early maturity stage present higher contents of fructose and lower ssc, those with a later ripeness stage have a lower fructose content and higher ssc. however, no significant differences in the ripeness stage for these two variables were found. as well, although the distribution indicates that the early ripening accessions have different sugar contents, no significant differences were observed in the distribution of glucose and sucrose related to the ripeness stage (p > 0.005). analysis of harvest time of the collection studied related to the rest of the variables studied. the local accessions have been clustered in five groups according to the time of harvest (fig. 1). the dendrograms derived from hierarchical cluster analysis reveal five figures, each consisting of different number of accessions (fig. 1). these results indicated that the local apples studied have a wide range of harvest times, characteristic of the ripening of apples in mountain areas and also linked to the biodiversity of genotypes present in the collection of local accessions. fig. 1a shows how to group the accessions harvested very early, giving rise to three different groups, accession 26 being the most differentiated of the group, which stands out for having content of two colours and the lowest total sugar values of the whole studied collection. the second group is formed by accession 29, noted for being very attractive to the consumer, since its epidermis is dark red with high wax content. one of the characteristics of this accession is that it is the first to be harvested, more than 15 days different from the rest of the group. the first group is characterized by having low values of total sugars, with accession 32 being the lowest. fig. 1b shows the dendrogram that groups the accessions of ‘early’ collection time into four groups. in group i, accession 13 stands out tab. 4: pearson’s correlation coefficients (r) between fruit weight, firmness, harvest time, and physico-chemical parameters of the 34 local accessions studied. correlation with a p <0.05 are marked in bold. fruit firmharvest malic citric total sucrose/ glucose/ weight ness time ph acid acid ssc sucrose glucose fructose xylose sugars glucose fructose %ta 0.353* -0.119 0.022 -0.093 -0.069 -0.069 -0.148 0.059 -0.185 -0.148 -0.108 -0.120 0.151 -0.150 fruit weight 1 0.173 -0.156 -0.253 0.010 0.010 -0.250 -0.150 -0.195 -0.132 0.074 -0.197 0.091 -0.209 firmness 1 0.421* 0.018 -0.270 -0.270 -0.134 0.231 0.123 -0.021 0.066 0.138 0.076 0.173 harvest time 1 0.171 -0.187 -0.187 -0.085 0.241 0.129 -0.079 -0.253 0.095 0.018 0.234 ph 1 -0.089 -0.089 0.389* 0.374* 0.338 0.334 -0.082 0.451** 0.001 0.121 malic acid 1 1,000** -0.058 0.049 -0.165 -0.142 -0.137 -0.116 0.129 -0.119 citric acid 1 -0.058 0.049 -0.165 -0.142 -0.138 -0.116 0.129 -0.119 ssc 1 0.488** 0.038 0.303 0.284 0.408* 0.325 -0.137 sucrose 1 0.172 0.198 0.083 0.615** 0.657** 0.115 glucose 1 0.701** -0.055 0.775** -0.426* 0.686** fructose 1 0.263 0.862** -0.208 0.010 xylose 1 0.218 0.217 -0.334 total sugars 1 0.057 0.267 sucrose/glucose 1 -0.399* glucose/fructose 1 ** the correlation is significant at the 0.01 level (bilateral). * the correlation is significant at the 0.05 level (bilateral). 222 l. castel, a. pina, p. irisarri, p. errea for being the one with the lowest values of xylose, sucrose and relationship between sucrose and glucose, and accession 30 for having the highest value of ssc (19.6 °brix) and the lowest value of firmness. group iii consists of accession 23, which has the lowest value of ssc (10.5 °brix) of the entire collection. within this group is accession 25, which is the one with the highest value of xylose. group iv consists of three accessions, 10, 24 and 21. they stand out for having very extreme values: the highest in glucose and fructose in accession 21 and the highest values in weight and lowest in ta in accession 10. this accession group is very interesting for a market entry towards consumers who like sweet and low acid apples. fig. 1c shows the accession harvested at ‘medium’ time. that includes 7 accessions, with ranges of ssc, sugars and acids in the middle of the values. the accessions collected ‘late’ (1d), correspond to local varieties that have the highest values of total sugars and lower but above average levels of malic and citric acids: specifically, accession 16 in group ii. this does not mean that they are unpleasant in the face of consumer taste, since the very high levels of sugars and very low levels of organoleptic acidity complement each other. the smallest group (3 accessions) is harvested ‘very late’ (1e). multidimensional scaling analysis principal component analysis (pca) was performed to provide easy visualisation of the complete data set in a reduced dimension plot. pca reduces the number of variables and improves the visualisation of the data structure. using pca allowed us to visualise technological groups of the varieties based on their acidity and sugar profiles and identify their potential use for manufacturing processed apple products. the first two principal components (pcs) accounted for 51% of the total variance, with 32% explained by pc1 and 19% by pc2 (fig. 2). the representation of the relationships between the variables analysed when considering dimension 1 (nutritional content) and dimension 2 (organoleptic qualities that influence consumer taste and the transformation processes for elaborating transformed products) allowed the studied variables to be clearly differentiated into five groups. if dimension 1 (pc1) is the nutritional content, the distribution obtained suggests that genotype is the main factor determining the variability of the sugar compounds in apples and may serve in the future to select apple genotypes with a better nutritional quality or those that are most suitable for the processing industry, as has been seen in other research studies (vieira et al., 2009). the grouping of variables in dimension 2 (pc2) includes all the organoleptic qualities that influence consumer taste and the transformation processes for elaborating transformed products, such as the acidity; this, and the sugar/acid ratio are responsible for the taste and flavour of apples and affect the organoleptic properties and nutritional value of fruit products (wu et al., 2007). the results obtained in this work show that local accessions have lower sucrose content and a lower sucrose-glucose relationship than reported in other similar works. this could have an impact on the acceptance of these measures, be recommendable for consumers who are intolerant to various sugar compounds, and be useful for making mixtures in processing industries. with regard to ssc, xylose and ph, it was observed that ssc has a proportional relationship with the various types of acidity, grouped differently in the two dimensions. when the acidity increases, the ssc decreases. the acidity together with the sweetness, are the main characteristics responsible for the taste of the fruits and their acceptance by consumers. at present, one of the objectives of the apple breeding programmes is fig. 1: dendograms that group the variables studied according to harvesting time. dendograms using average linkage (between groups) rescaled distance cluster combine. a) very early b) early c) medium d) late e) very late sugars and organic acids of mountain apple cultivars 223 to improve the marketing calendar, to obtain either earlier or later varieties. as well, both ta and ssc are important considerations when selecting eating apples, as many consumers determine apple quality based on the balance of sweetness and acidity. all the accessions analysed are suitable for direct consumption, since they contain low levels of acidity, high sugar ratios and a wide variation in collection times. in addition, combinations of these different accessions could be used in the processing industry; for example, for producing cider, jams or juices. the results obtained in this work reveal the high potential of local cultivars. the analysis of compounds carried out within this genetic diversity has revealed that there is a striking variability of analyzed variables (20 in total) especially in terms of acid, ph and ssc in genetically different local accessions. likewise, this collection has shown a higher ta and ssc content, a lower sucrose content and lower sucrose-glucose relationship in comparison to other accessions (begic-akagi et al., 2014; aprea et al., 2017). this study shows the importance of appreciating and conserving local varieties that may represent a genetic reservoir, which could be introduced into improvement programmes for new hybrids. a more detailed knowledge of the variability of morphological and fruit quality traits of these local apple cultivars will be of benefit in the future selection of apple genotypes with improved nutritional quality and suitable processing characteristics for the manufacturing of apple products. this study opens the door to genetic studies of specific food value characteristics, such as bioactive properties, sugar/acid balance, as well as to their suitability for processing industries that could be useful for breeding programs. acknowledgments this research was supported by the grants rta2015-00052-c02-00 from the institute for agriculture and food research and technology (inia) and group a12 from aragon government (spain). conflict of interest no potential conflict of interest was reported by the authors. references aprea, e., 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https://www.bioversityinternational.org/e-library/publications/detail/apple-descriptors/ wu, j., gao, h., zhao, l., liao, x., chen, f., wang, z., hu, x., 2007: chemical compositional characterization of some apple cultivars. food chem. 103(1), 88-93 doi: 10.1016/j.foodchem.2006.07.030 zhang, y., li, p., cheng, l., 2010: developmental changes of carbohydrates, organic acids, amino acids, and phenolic compounds in 'honeycrisp' apple flesh. food chem. 123(4), 1013-1018. doi: 10.1016/j.foodchem.2010.05.053 orcid lourdes castel – no orcid ana pina https://orcid.org/0000-0002-4988-9467 patricia irisarri https://orcid.org/0000-0003-0773-5133 pilar errea https://orcid.org/0000-0002-2889-1728 address of the corresponding author: department of horticulturae, agrifood research and technology centre of aragon (cita). instituto agroalimentario de aragón – ia2 (citauniversidad de zaragoza). avda. montañana, 930, 50059, zaragoza, spain e-mail: perrea@aragon.es © the author(s) 2020. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1016/j.foodchem.2013.02.121 http://dx.doi.org/10.1016/j.lwt.2017.04.017 http://dx.doi.org/10.1016/j.scienta.2007.05.004 http://dx.doi.org/10.1016/j.scienta.2014.04.037 http://dx.doi.org/10.1080/14620316.2007.11512227 http://dx.doi.org/10.1021/jf503379t http://dx.doi.org/10.1007/s10681-011-0579-7 http://dx.doi.org/10.1007/s11295-017-1206-0 http://dx.doi.org/10.1002/jsfa.2113 http://dx.doi.org/10.1016/j.scienta.2009.06.012 http://dx.doi.org/10.1016/j.foodchem.2010.05.053 http://dx.doi.org/10.1055/s-0043-111898 http://dx.doi.org/10.1016/j.foodchem.2006.07.030 vorgabe neu journal of applied botany and food quality 81, 45 48 (2007) leibniz institut für gewässerökologie und binnenfischerei, berlin reduction in germination rate and elevation of peroxidase activity in zea mays seedlings due to exposure to different microcystin analogues and toxic cell free cyanobacterial crude extract stephan pflugmacher (received february 23, 2007) summary agricultural crop plants may come into contact with cyanobacterial toxins via spray irrigation with water contaminated with cyanobacteria/ cyanobacterial toxins. many of the bloom forming cyanobacteria are known to produce toxins amongst those the group of the microcystins, cyclic heptapeptides are the best known once. in this study the germination of zea mays under exposure to different microcystins and cell free cyanobacterial crude extract containing microcystin-lr was investigated. the concentration used for all microcystins in this study was 5.0 µg l-1 which is well in the environmental range. the inhibition of germination was shown as well as the inhibition of root and shoot length by toxin exposure. as a sign for the generation of oxidative stress promoted by the toxins taken up, guajacol peroxidase was measured showing in most toxin exposures an elevation of peroxidase activity. this study showed that there is a potential concern a reduction in crop yield and also to human health if agriculturally important crop plants were exposed to cyanobacterial toxins via spray irrigation. introduction modern agriculture is dependent on spray irrigation of the crops to ensure the crop yield necessary. in many countries, especially in semiarid and arid regions this water is taken from water bodies in which bloom-forming cyanobacteria might be present. from many of these cyanobacteria it is well known that they can produce a variety of different secondary metabolites, so called cyanobacterial toxins (cyanotoxins) and it was estimated that 25 75 % of these cyanobacterial blooms are toxic (lawton and codd, 1991). under these toxins, hepatotoxins, neurotoxins and cytotoxins can be found. the microcystins, cyclic heptapeptides of five relatively invariant d-amino acids and two most variable l-amino acids, are the most well known group from which about 60 analogues were known (sivonen and jones, 1999). spray irrigation with water contaminated with cyanobacteria respectively their toxins showed to have negative effects on terrestrial plants. in most cases this studies were performed in hydroponic culture using the most common hepatotoxin, microcystinlr. effects monitored were decrease in root length (kos et al., 1995; mcelhiney et al., 2001; kurki-helasmo and meriluoto, 1998), inhibition of photosynthesis (abe et al., 1996), inhibition of sucrose transport (siegl et al., 1990), inhibition of protein phosphatases (mckintosh et al., 1990), indicating the uptake of toxin via the root system. a previous study showed the uptake of two microcystins (mclr and mc-lf) in different agricultural important crop plants, amongst those corn (zea mays) by peuthert et al. (in press) giving toxin values in roots of z. mays of 12.7 (mc-lr), 14.5 (mc-lf) and shoots of z. mays of 26.5 (mc-lr), 18.0 (mc-lf) after 24 h exposure time. from cyanobacterial toxins it is known that they can generate oxidative stress after being taken up by plant cells (gehringer et al., 2003; pflugmacher, 2004; pflugmacher et al., 2006). oxidative stress is always generated from a rapid and transient production of high quantities of reactive oxygen species (ros). unless the production of ros in plants is a natural phenomenon via the photosynthesis in the chloroplasts, excessive ros formation could also be triggered by quite a number of external factors amongst those exposures to xenobiotics or cyanobacterial toxins (cossu et al., 1997; pflugmacher et al., 2006). oxidative damage has been found in dna, proteins, carbohydrates and lipids (cadenas, 1995). to prevent oxidative damage from ros a protective system has been evolved based on small molecular antioxidants such as glutathione (gsh), ascorbate or tocopherols and antioxidative enzymes such as superoxide dismutase, catalase, peroxidase, glutathione s-transferase and glutathione reductase. aim of this study was to examine the response of corn seedlings when exposed to different microcystin variants and furthermore to cell-free cyanobacterial crude extract containing microcystin-lr. materials and methods plant material and exposure experiments corn (zea mays l. cv badischer gelber) seeds were purchased from kiepenkerls (norken, germany). seeds were soaked in running tap water overnight. the seeds were germinated non-aseptically on the surface of wet filter papers (whatman no. 1, roth, karlsruhe, germany) in the dark at 13 °c in plastic plates with 24 wells. in each well one seed was placed in exposure medium (1 ml). for each toxin treatment five plastic plates were used which are 100 seeds per treatment. concentration of the different microcystins in the exposure medium was 5.0 µg l-1 and in the used cyanobacterial cell-free crude extract was equivalent to a microcystin-lr concentration of 5.0 µg l-1. microcystin variant and cyanobacterial crude extract the different microcystin analogues (tab. 1) were purchased from axxora (lörach, germany) and in case of mchcyr was a gift from prof. dr. g.a. codd (university of dundee). the cyanobacterial bloom material (mainly microcystis aeruginosa and aphanezomenon flosaqua) was collected in june 2000 from the shore of lake müggelsee, berlin (germany). to obtain the cell-free crude extract, 20 g dry weight of bloom material was suspended in 500 ml milli-q water and stirred on ice for 15 min. after ultrasonication on ice, centrifugation of the resulting slurry was done at 22,000 x g for 15 min. supernatant was collected and stored on ice. the pellet was reprocessed in the same manner as described above five times and the extracts were combined thereafter. the extract was stored in the deep freezer (-80°c) before use. toxin analysis of the crude extract revealed the presence of microcystin-lr (mc-lr) and not quantifiable traces of microcystinrr (mc-rr). the extract was diluted to a final concentration of 5.0 µg l-1 mc-lr. determination of cyanobacterial secondary metabolites analyses were performed as described in (pflugmacher et al., 2006) using a waters hplc system (waters, eschborn, germany) with photodiode array detector (waters 2996) detection. separation was carried out on a symmetry 5 µm c18 column (3.9 x 150 mm). the mobile phase consisted of solvent a: milli-q water and solvent b: acetonitrile (rathburn, walkerburn, uk) both containing 0.1 % (v/v) trifluoracetic acid. solvent b was linearly increased from 30 % to 45 % over 10 min at a flow rate of 1 ml min-1. column temperature was maintained at 40 °c and the injection volume was set to 80 µl. enzyme preparation enzyme preparation from z. mays seedlings roots and shoots was carried out according to (pflugmacher et al., 2006). shoots and roots were cut of with a scalpel and were frozen in liquid nitrogen, ground to a powder with mortar and pestle and suspended in ice-cold 0.1 m sodium phosphate buffer ph 6.5 containing 14 mm dithioerythritol and 1 mm ethylenediaminetetraacetic acid. after removing cell debris at 5000 x g, the supernatant was centrifuged at 100,000 x g for 60 min to collect the microsomal fraction. the supernatant (soluble fraction) was precipitated twice with solid ammonium sulphate (0-30 % and 30-80 % saturation). the pellet from the last precipitation step was suspended in 20 mm sodium phosphate buffer (ph 7.0) and the resulting extract (soluble fraction) was desalted using nap-10 columns (amersham pharmacia, uppsala, sweden). enzymatic measurements photometric determination of peroxidase (pod, e.c. 1.11.1.9) activity was done according to (putter, 1965) using guajacol as a substrate. the assay contained 01.mm sodium phosphate buffer ph 6.0, 0.3 mm guajacol, 0.12 mm h 2 o 2 , 40 µl of protein extract to a total volume of 1200 µl and increase in absorption was measured at 436 nm. enzyme activities are given in nkat mg-1 protein (si unit: katal = conversion rate of one mol substrate per second). protein content of the samples was determined according to (bradford, 1976) using bovine serum albumin fraction v as calibration standard at 595 nm. analysis of data the statistical significance was calculated by one-way analysis of variance (anova) followed by newman-keuls test, p < 0.05 (spss 9.0 for windows, chicago, usa). results seeds from z. mays were exposed to different microcystin analogues in a concentration of 0.5 µg l-1 for 7 days. tab. 2 showed the germination rate of z. mays seeds. the highest reduction in germination rate was seen using the cell free cyanobacterial crude extract (90 %) and mc-la (88 %). for all other microcystin variants the inhibition tab. 1: microcystin variants used in this study and their structural differences concerning the amino acid composition and the molecular weight. analogue structure molecular weight reference microcystin-lr cyclo (-d-ala-l-leu-d-measp-l-arg-adda-d-glu-mdha-) 994 botes et al. (1985) microcystin-rr cyclo (-d-ala-l-arg-d-measp-l-arg-adda-d-glu-mdha-) 1037 kusumi et al. (1987) microcystin-yr cyclo (-d-ala-l-tyr-d-measp-l-arg-adda-d-glu-mdha-) 1044 botes et al. (1985) microcystin-lf cyclo (-d-ala-l-leu-d-measp-l-phe-adda-d-glu-mdha-) 985 azevedo et al. (1994) microcystin-la cyclo (-d-ala-l-leu-d-measp-l-ala-adda-d-glu-mdha-) 909 botes et al. (1982) microcystin-lw cyclo (-d-ala-l-leu-d-measp-l-trp-adda-d-glu-mdha-) 1024 lawton et al. (1994) [d-asp3, (z)-dhb7]microcystin-htyr cyclo (-d-ala-l-hty-d-asp-l-arg-adda-d-glu-(z)-dhb-) 1044 sano et al. (1998) tab. 2: germination rate of z. mays seeds exposed to different microcystin variants. treatment germinated seeds reduction in germination (n = 100) [%] control 100 0 mc-lr 68 32 mc-rr 93 17 mc-yr 73 27 mc-lf 74 26 mc-la 12 88 mc-lw 89 11 mchcyr 72 28 crude extract (mc-lr) 10 90 46 stephan pflugmacher fig. 1: z. mays seeds exposed to different microcystin variants a) control, b) mc-lr, c) mc-rr, d) mc-yr, e)mc-lf, f) mc-la, g) mc-lw, h) htyr-mc, i) crude extract of germination was between 17 32 % compared to control seeds. the exposure to microcystins and to cell free cyanobacterial crude extract had significant overall effects on primary root length and shoot length (fig. 1). the shoot length of the z. mays seeds were significantly reduced in all exposures but highest reduction was detected in exposures using the mc-lr containing crude extract (fig. 2a). a similar picture was obtained looking at the root length. also here all exposures reduced the primary root length significantly from control up to a factor of 18 (crude extract containing mc-lr) compared to controls (fig. 2b). activity of the pod in the shoots showed, that in most seeds exposed to microcystin analogues a significant elevation of enzyme activity was measured, which was highest in exposures using mc-lf, mclw or mc-lr. the elevation of pod activity was not significant in exposures using mc-rr. in two exposures (mc-la and ce) the activity of the pod was decreased under control levels (fig. 3a). a similar activity pattern was measured in the roots with the exception of mc-rr exposures all pod activity were significantly different from control. in most cases an elevation was seen also in the shoots. in exposures using mc-la and ce a decrease of pod activity was determined (fig. 3b). discussion during spray irrigation events, water contaminated with cyanobacterial toxins could be transferred to the field and the crop plants growing there. in this experiment germination rate of the z. mays seeds were inhibited by exposure to the different microcystin analogues. the highest inhibition was detected using the mc-la one of the more lipophilic microcystin analogues and cyanobacterial cell-free crude extract containing mc-lr. comparing the crude extract with the pure toxin a big difference in the potency to reduce germination is visible. whereas the crude extract inhibits the germination more or less completely (90 %) the purified toxin gives only a reduction of 32 % from control. this led to the hypothesis that more bioactive compounds are present in the crude extract leading to such an effect as also suggested by pietsch et al. (2001). inhibition of germination by cyanobacterial toxins was also shown by metcalf et al. (2004) in pollen of nicotiana tabacum exposed to cylindrospermopsin, another cyanobacterial toxin produced by cyanobacteria such as cylindrospermopsis raciborskii, umezakia natans, aphanizomenon ovalisporum or rhaphidiopsis curvata. obermeyer et al. (1998) correlated the inhibition of pollen tube growth in lilium longiorum with the inhibition of protein phosphatases by okadaic acid. also microcystins are known to inhibit the activity of protein phosphatases (mckintosh et al., 1990) and therefore interfere with plant growth. previous work showed, that the growth and lateral root development of sinapis alba was inhibited by exposure to the cyanobacterial toxin mc-lr in a concentration of 3.0 µg l-1. a complete inhibition of root formation was reported by mckintosh et al. (1990) in s. alba exposed to 5 mg l-1 mc-lr. also in medicago sativa a significant reduction in root growth was monitored when plants were exposed to two different microcystins (mc-lr, mc-lw) and to cyanobacterial crude extract containing mc-lr in a concentration of 5.0 µg l-1 (pflugmacher et al., 2006). in this experiment the growth inhibition was more effective in the shoots of z. mays than in the roots. highest 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 co nt ro l m clr m c -r r m cyr m clf m c -l a m clw m ch cy r ce (m clr ) p e ro xi d a se a ct iv ity [µ ka t m g p ro te in ] -1 a b fig. 3: peroxidase activity in shoots (a) and roots (b) of z. mays seedlings after exposure to different microcystin variants. fig. 2: shoot length (a) and root length (b) of z. mays seedlings after exposure to different microcystin variants. 0.0 50.0 100.0 150.0 200.0 250.0 300.0 350.0 0.0 25.0 50.0 75.0 100.0 125.0 150.0 175.0 200.0 co nt ro l m clr m crr m cyr m clf m cla m clw m ch cy r ce (m clr ) ro o t le n g th [m m ] sh o o t le n g th [m m ] a b effects of microcystins in zea mays 47 inhibition was achieved using the cell free crude extract containing mc-lr again pointing out, that there might be more compounds included counting for the measured effect. the generation was oxidative stress by cyanobacterial toxins was shown in lepidium sativum and medicago sativa yielding to an elevation of antioxidative enzymes such as glutathione peroxidase, catalase or glutathione s-transferase (gehringer et al., 2003; pflugmacher et al., 2006). activity determination of the guajacol peroxidase in z. mays root and shoots showed also an elevation of this enzyme by exposure to the different microcystin analogues. with mc-la and the crude extract containing mc-lr a decrease of pod activity was measured. a possible explanations would be; an inhibition of the protein (enzyme) by an excessive amount of reactive oxygen species as known for protein phosphatases (huber, 2007). these experiments have shown that z. mays seedlings are sensitive to exposure of cyanobacterial toxins such as microcystins. the contact of z. mays to these toxins might come via spray irrigation. after uptake of the toxin in the seeds germination, root and shoot development were negatively affected. this might lead to a decrease in crop yield in two ways: first by the reduced germination of the seeds and second by morphological damages leading to e. g. smaller plants. because of the uptake of toxin in the plants (järvenpää et al., 2007; peuthert et al., in press) this could pose a possible risk for human consumption after such practices too. references abe, t., lawson, t., weyers, j.d.b., codd, g.a., 1996: microcystin-lr inhibits photosynthesis of phaseolus vulgaris primary leaves: implications for current spray irrigation practice. new phytol. 133, 651-658. bradford, m.m., 1976: a rapid and sensitive method for the quantification of microgram quantities of proteins utilizing the principle of protein-dye binding. anal. biochem. 72, 248-254. cadenas, e., 1995: mechanisms of oxygen activation and reactive oxygen species detoxification. in: ahmad, s. (ed.), oxidative stress and antioxidant defences in biology, 1-61. chapman & hall, london. cossu, c., doyotte, a., jacquin, m.c., babut, m., exinger, a., vasseur, p., 1997: glutathione reductase, selenium-dependent glutathione peroxidase, glutathione levels and lipid peroxidation in freshwater bivalves unio tumidus as biomarkers of aquatic contamination in field studies. ecotoxicol. environ. safety 38, 122-131. gehringer, m.m., kewada, v., coates, n., downing, t.g., 2003: the use of lepidium sativum in a plant bioassay system for the detection of microcystin-lr. toxicon 41, 871-876. huber, s., 2007: exploring the role of protein phosphorylation in plants: from signalling to metabolism. biochem. soc. transact. 35, 28-32. järvenpää, s., lundberg-niinistö, c., spoof, l., sjövall, o., tyystjärvi, e., meriluoto, j., 2007: effects of microcystins on broccoli and mustard, and analysis of accumulated toxin by liquid chromatography-mass spectrometry. toxicon (in press). kos, p., gorzo, g., suranyi, g., borbely, g., 1995: simple and efficient method for isolation and measurement of cyanobacterial hepatotoxins by plant tests (sinapis alba l.). anal. biochem. 225, 49-53. kurki-helasmo, k., meriluoto, j., 1998: microcystin uptake inhibits growth and protein phosphatase activity in mustard (sinapis alba l.) seedlings. toxicon 36, 1921-1926. lawton, l., codd, g.a., 1991: cyanobacterial (blue-green algal) toxins and their significance in uk and europeanwaters. j. j.w.e.m. 5, 460-465. mackintosh, c., beattie, k.a., klumpp, s., cohen, p., codd, g.a., 1990: cyanobacterial microcystin-lr is a potent and specific inhi-bitor of protein phosphatases 1 and 2a from both mammals and higher plants. febs letters 264, 187-192. mcelhiney, j., lawton, l.a., leifert, c., 2001: investigations into the inhibitory effects of microcystins on plant growth, and the toxicity of plant tissues following exposure. toxicon 39, 1411-1420. obermeyer, g., klaushofer, h., nagl, m., höftberger, m., bentrup, f.w., 1998: in vitro germination and growth of lily pollen tubes is affected by protein phosphatase inhibitors. planta 207, 303-312. peuthert, a., chakrabarti, s., pflugmacher, s., 2007: uptake of microcystins-lr and -lf (cyanobacterial toxins) in seedlings of several important agricultural plant species and the correlation with cellular damage (lipid peroxidation). environ. toxicol. (in press). pflugmacher, s., 2004: promotion of oxidative stress in c. demersum due to exposure to cyanobacterial toxin. aquatic tox. 3, 169-178. pflugmacher, s., jung, k., lundvall, l., neumann, s., peuthert, a., 2006: effects of cyanobacterial toxins and cyanobacterial cell-free crude extract on germination of alfalfa (medicago sativa) and induction of oxidative stress. environ. toxicol. chem. 25, 2381-2387. pietsch, c., wiegand, c., ame, m.v., nicklisch, a., wunderlin, d., pflugmacher, s., 2001: the effects of a cyanobacterial crude extract on different aquatic organisms: evidence for cyanobacterial toxin modulating factors. environ. toxicol. 16, 535-542. putter, j., 1965: peroxidases. in: bergmeyer, h.u. (ed.), methoden der enzymatischen analyse, vol. 2, 685-690. academic press, new york. siegl, g., mackintosh, c., stitt, m., 1990: sucrose-phosphate synthetase is dephosphorylated by protein phosphatase 2a in spinach leaves. febs lett. 270, 198-202. sivonen, k., jones, g., 1999: cyanobacterial toxins. in: chorus, i., bartram, j. (ed.), toxic cyanobacteria in water. a guide to their public health consequences, monitoring and management, 41-111. e. & fn. spon on behalf of who, london. address of the author: pd dr. stephan pflugmacher, leibniz institute of freshwater ecology and inland fisheries, ag biochemical regulation, müggelseedamm 301, d-12587 berlin email: pflugmacher@igb-berlin.de 48 stephan pflugmacher << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true 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<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> >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice art28 journal of applied botany and food quality 82, 170 178 (2009) 1 humboldt-universität zu berlin, institute for horticultural sciences, section quality dynamics/postharvest physiology 2 landesumweltamt brandenburg, eberswalde and vern e.v. (verein zur erhaltung und rekultivierung von nutzpflanzen in brandenburg, greiffenberg) development of a network for the on-farm conservation of crop genetic resources: first results of a pilot project for the re-introduction of old lactuca varieties to the market c. lehmann1, g. lissek-wolf 1, r.vögel2, s. huyskens-keil1 (received october 8, 2008) summary in a pilot project, we examined the chance of maintaining plant genetic resources by commercial utilization of old varieties using lactuca sativa as a model plant. nine market gardens in the region of berlin and brandenburg cultivated 18 old varieties during four cultivation periods to test field performance. they supplied the products to the market in their customary manner to analyse marketing success. seven of the market gardens practice organic horticulture. in a complementary field trial at humboldt-universität zu berlin, we established data concerning the field performance of the varieties, analysed dry matter contents, nitrate and phenol concentrations, and observed shelf life for two days under simulated retail conditions (18°c, 80% rel. air humidity). generally, yield was acceptable for market purposes. however, cultivation in autumn failed because of the cold climate. biotic and abiotic factors like slugs or hail caused non-specific damages. specific problems of particular varieties were less important. based on the results of 2007, the varieties can be put preliminarily into three categories: suitable for on-farm conservation, suitable for home gardens, and varieties with contrasting results depending on the respective market garden. the nitrate concentrations of all varieties were clearly below the eu acceptable limit of 2500 mg/kg fresh weight of lettuce grown in the field. the phenol concentrations varied from 3.3 to 17.2 mg gae/g dry weight. generally, the cultivars had a reasonable shelf life of one to two days, however three varieties showed a better storability whereas four other cultivars deteriorated rapidly. marketing success was good in berlin city but poor in the countryside of brandenburg. the regular customers of the market gardens in berlin who prefer organic food are a promising target group for further stimulation of interest to buy rare crop varieties. the on-farm conservation of old varieties in market gardens requires relatively large quantities of seeds of good quality. however there might arise problems in seed supply as the vern e.v. was confronted with bottleneck problems. therefore, we organised a network of interested market gardens who take on maintenance and propagation of individual varieties. the network will be developed in co-operation with the vern e.v. who will also process the seed as well as organise the exchange of the various varieties within the network. further, the network will deal with problems concerning maintenance breeding and seed quality. introduction within the past decades, the diversity of cultivated plants decreased seriously (fao, 1996 a). a major part of crop plant diversity is used neither in market nor in home gardening because the demand of intensive production relies on a relative small number of elite varieties. further, plant breeders concentrate to meet the demand of intensive market gardening exclusively. the loss of agro-biodiversity puts plant genetic resources at risk and jeopardizes the cultural heritage of crop plant diversity as well as traditional knowledge and use (gladis, 2001). two main complementary methods have been developed to conserve crop genetic resources: ex-situ in gene banks and in-situ in farmer’s fields or in the natural environment (gepts, 2006). in 1996 the fao adopted the global plan of action for the conservation and sustainable utilization of plant genetic resources (fao, 1996 b) which emphasized in-situ conservation and proposed a catalogue of measures promotion. on-farm management is particular important because it allows conservation of genetic resources by using them economically. in industrialised countries on-farm management aims to re-introduce old varieties into the market (efken, 2005). therewith a broader range of varieties will be made available for farmers and consumers. a further advantage is a dynamic use of crop plant genetic resources (cgr) in contrast to gene bank conservation. however, only those old varieties with an acceptable field performance are suitable for an effective on-farm management. the successful commercialization of old varieties is crucial for a sustainable on-farm management. the marketing concepts have to be concerned with regard to the complete value chain and the customer focus (efken, 2005). public relations work is required to encourage public awareness about cgr and to stimulate customer’s interest for rare crop varieties. in 2002, the german federal ministry of food, agriculture and consumer protection (bmelv) published the national programme for the conservation and sustainable use of plant genetic resources (bmelv, 2002) to implement the global plan of action of leipzig (fao, 1996 b). in this context, pilot projects are promoted that add to the conservation and to the innovative sustainable use of biodiversity. in a pilot project, lactuca sativa was chosen as a model plant because in comparison to other crops it is easy to grow and has a short period of cultivation. furthermore, l. sativa stands out due to a remarkable number of distinct forms (helm, 1954; de vries, 1997) and a broad range of old varieties (rodenburg, 1960). altogether, at humboldtuniversität zu berlin (hu) 57 old lactuca varieties were tested for field performance and suitability for the market as a requirement of an on-farm management (lehmann et al., 2008). in 2007 a set of 18 candidate varieties was examined in practice. we aimed to develop a collection of old varieties and to make it available to local market gardens as a niche product for an extensive horticultural production. furthermore, rarities which are more suitable for home gardening will be added to an expanded collection. the complete value chain from seed supply to cultivation and marketing was tested in a pilot project to analyse stimulating as well as inhibiting factors of an on-farm management. the objective is to demonstrate criteria for a promising and sustainable on-farm management under market conditions. tab. 1: location and marketing of nine market gardens that 2007 participated in the pilot project. market location coordinates marketing (special features) garden no. 1 ahrenzhain / brandenburg 51°40’n 13°30’o weekly market in berlin (organic horticulture) 2 barenthin / brandenburg 52°55’n 12°14’o weekly market in berlin, direct delivery, (organic horticulture) 3 oderberg / brandenburg 52°52’n 14°00’o direct delivery, retail trade in oderberg (conventional horticulture) 4 dahlem / berlin 52°28’n 13°17’o farmer’s shop in berlin (organic horticulture) 5 bornow / brandenburg 52°10’n 14°11’o weekly market in berlin (organic horticulture) 6 fürstenhof / mecklenburg 53°56’n 12°45’o direct delivery, food coop, school canteen, (organic horticulture) 7 bastorf / mecklenburg 54°07’n 11°41’o weekly market and health food shop in wismar (organic horticulture) 8 friesack / brandenburg 52°43’n 12°37’o farmer’s shop, canteen (adult education, conventional horticulture) 9 pinnow / brandenburg 53°02’n 14°00’o farmer’s shop, weekly market in angermünde, canteen (institution for handicaped persons, organic horticulture) tab. 2: evaluation and pilot tests of 18 old lettuce varieties in four cultivation periods in 2007. form variety (gene bank accession or origin) characteristic early1 spring2 summer3 autum4 butter head amphore (rijk zwaan) – reference variety red coloured leaves x x x 9 butter head frühlingsgruß (ipk lac 89) green, small compact head x lettuce cabbage (ipk lac 76) green, small compact head x bunte forelle (ipk lac 81) green with red speckled pattern x stuttgarter sommer (ipk lac 17) green, great loose head x gigant (ipk lac 03) green, cultivar from quedlinburg (1955) x brunetta (ipk lac 68) reddish brown, cultivar from quedlinburg (1954) x goldforelle (ipk lac 38) yellowy green, with red speckled pattern x x brauner sommer (ipk lac 89) green, with brown painted leaf margins x hitzkopf (ipk lac 95) grenn, medium sized, loose head x 4 leaf lettuce früher gelber krausblättriger (ipk lac 101) green, lobed leaves, plant funnel-shaped x struwwelpeter (ipk lac 233) green, characteristic name x hohlblättriger butter (ipk lac 104) green, very delicate leaves x ochsenzunge (unknown) green, elongated, very delicate leaves x 3 romana-type wiener maidivi (ipk lac 312) green, outwardly curved leaf margins x x trianon (ipk lac 122) green, open romana-type x romaine red cos (ipk lac 315) red romana-type, delicate leaves x x 1 latin-type rehzunge (unknown) dark green, reminiscent of a spinach plant x 1 stem lettuce chinesische keule (dreschflegel) consumption of the stem or shoot x x x 1 sowing end of february, planting end of march, harvest middle of may 2 sowing end of march, planting beginning of may, harvest beginning of june 3 sowing end of may, planting in the second half of june, harvest beginning of august 4 sowing beginning of august, planting beginning of september, no harvest material and methods field evaluation and pilot tests in 2007, a field evaluation at the experimental station of humboldtuniversität zu berlin (hu) and pilot tests in nine market gardens were simultaneously carried out in four cultivation periods (tab. 1). at hu, the experimental design was a complete randomized block with 15 plots per block and three replications. 12 plants of a variety were planted per plot with a distance of 30 x 30 cm. the plots were fertilized with 50 g/m² organic manure comprising of 14% organic n with planting. during the first two cultivation periods the plots were covered with fleece, during the next two cultivation periods nets were stretched over the plots to protect for birds and rabbits. the soil was prepared by hoeing and weeds were removed two times between planting and harvest. the plots were watered to demand. during the growing period the following data were collected: germination in percent, number of not harvestable plants in percent, fresh weight at harvest after removing the outer leaves as „market weight“. furthermore, diseases and pests were recorded. nine selected market gardens joined in the pilot project, seven in the region of berlin and brandenburg and two in mecklenburg-western pomerania (tab. 1). as the basic requirement to participate in the pilot project, the market gardens were asked to cultivate five old lactuca varieties in two cultivation periods (see tab. 2) in two sets each of at least 40 plants and collect data relevant for practice (see above). the market gardens carried out the field tests in their common practice. furthermore, the market gardens had to assess the varieties on-farm management and marketing potential of old lactuca varieties 171 172 c. lehmann, g. lissek-wolf , r.vögel, s. huyskens-keil according to the following criteria: suitability of the varieties to the respective site, yield, suitability for commercialization, personal estimation of the varieties. climate in 2007 the temperature means per months at the experimental station in berlin-dahlem exceeded the long term records of the years 19712000: from march to june by 2.0 3.7°c, temperature in july was average, while in august and september the temperature was 0.2 0.7°c cooler than the average (chmielewski, 2008). in april 2007 precipitation was very low with 4 mm compared to the long term record of 34 mm (1971 2000). in mai rainfall was three times higher (169.1 mm) than the long term record (50.6 mm), and from june till september precipitation was twice as much as the average (chmielewski, 2008). thus, april was hot and dry, mai was hot and rainy, and the summer months were rainy and cool. test sets based on preliminary field tests at hu (hartkopf, 2006; schuller, 2008) sets of 5 7 varieties were put together for four cultivation periods (early, spring, summer and autumn; see tab. 2). originally, these varieties except three varieties were accessions from the gene bank in gatersleben and some of them are listed in the „catalogue for rare crops“ (vern, 2008). altogether, 18 lactuca varieties were evaluated. the current commercial cultivar ‘amphore’ (rijk zwaan) suitable for all cultivation periods served as a reference. however, this cultivar was not available until the second cultivation period. each test set comprised varieties that were distinct from the usual commercial lettuce selection as well distinguishable from each other (tab. 2) to present the customers a clear offer with regard to test for marketing success. the varieties were chosen to represent conspicuously various lactuca forms. lactuca forms not customary in germany (like the latin group type ‘rehzunge’ or the stem lettuce ‘chinesische keule’) were included to contribute to more diversity. stem lettuce is common in some regions of asia. the stems (or shoots) of this lactuca form are eaten raw or cooked. chemical analyses two samples per plot were taken from each variety of the field experiment at hu for chemical analyses. dry matter was determined after 48 h at 104°c drying. nitrate concentrations were analysed after extraction in potassium aluminium sulphate with an ionic sensitive elektrode (künsch et al., 1981). assay of total phenols was performed by means of the folin-ciocaltrau-method with gallic acid as the reference (jennings, 1981). the adsorption was determined at a wavelength of 765 nm using a spectrophotometer (lkb novaspek iii, pharmacia, freiburg) and the phenol concentrations calculated as mg gallic acid-equivalent (gae) per g dry matter. three samples per variety served to test shelf life for two days at 18°c and 80% rel. air humidity. customer’s motivation with questionnaires we examined the knowledge of the customers about old varieties at two different locations of market stands and in a farmer shop in berlin. furthermore, we examined the willingness of customers to contribute to the conservation of old varieties with their purchasing behaviour. results the cultivation for the field experiment evaluation and the pilot tests succeeded in the first three cultivation periods, however failed in autumn because the plants developed very poorly due to the cold and rainy weather. at hu, cultivation in spring was slightly impaired because the plantation took place at the beginning of may when a heat-wave started. germination the germination rates varied between the varieties as well as between the market gardens (tab. 3). even the germination rate of the commercial cultivar ‘amphore’ varied between 14 100%, their average germination rate in most of the market gardens was high. in the first cultivation period two varieties could not be grown in one market garden (market garden no. 4) because they germinated too poorly (tab. 4). varietal uniformity in the evaluation experiment as well as in the pilot tests, it was found that some individuals of old varieties not anymore being subject of breeding work deviated from their variety specific pattern. for example, the colour of the leaf edges of ‘brunetta’ varied from red to brown and in varieties like ‘gigant’ or ‘stuttgarter sommer’ divergent heads were formed. in contrast, the commercial cultivar ‘amphore’ was uniform. field performance the percentage of harvestable plants varied considerably between varieties as well as between market gardens (tab. 4). predominantly, abiotic and biotic factors caused non-specific losses or damages. for example hail, snails or wireworms induced some major damages in the market gardens. fungal pathogens were rather location-dependent, e.g. the soil borne fungus sclerotinia sclerotiorum caused some losses in the field evaluation at hu and in market garden no. 4. slight infestations of septoria leaf spot occurred in market garden no. 2 and medium to strong infestation of grey mould in market garden no. 3 where several varieties were infested at a time. specific problems with internal tipburn were noticed in ‘stuttgarter sommer’ in all market gardens. internal tipburn was further detected in ‘frühlingsgruß’, ‘früher gelber krausblättriger’, ‘struwwelpeter’, ‘hohlblättriger butter’ and ‘lettuce cabbage’. in addition, ‘hohlblättriger butter’ bolted very fast in the spring cultivation period. yield tab. 5 shows the mean „market weight“ of the varieties. the weight of particular varieties varied between the market gardens and also between the two sets of a cultivation period within a market garden. thus, the varieties demonstrated remarkable ranges when grown in various market gardens. in the field evaluation at hu the fresh weight of the varieties was below average in spring cultivation because the plants developed poorly during the hot weather period in may. within a cultivation period some varieties tended to have a higher fresh weight, while other varieties revealed a lower weight. ‘goldforelle’ had the lowest weight. generally, romana types weighed more than 300 g on average and were heavier than butterhead or loose leaf types. on-farm management and marketing potential of old lactuca varieties 173 tab. 3: germination rates (%) of old lettuce varieties examined in the field evaluation and in the pilot tests of three market gardens. variety field evaluation garden no. 2 garden no. 3 garden no. 4 garden no. 5 mean hu 1. set 2. set 1. set 2. set 1. set 2. set 1. set 2. set early cultivation frühlingsgruß 82 78 74 46 46 12 62 no early 57 lettuce cabbage 49 28 28 16 10 26 12 cultivation for 24 früher gelber krausblättriger 43 48 69 28 12 44 10 internal reasons 36 wiener maidivi 53 55 18 44 38 62 54 46 rehzunge 80 63 58 58 96 92 68 74 spring cultivation amphore (reference) 100 46 14 92 98 100 100 94 42 76 bunte forelle 92 26 45 76 57 40 15 63 47 51 stuttgarter sommer 96 69 72 79 67 50 50 73 41 66 struwwelpeter 54 52 61 71 69 50 50 54 53 57 hohlblättriger butter 82 78 83 78 80 35 35 71 46 65 romaine red cos 86 69 46 84 84 50 20 64 78 65 chinesische keule 94 not cultivated not cultivated not cultiv. 60 not cultivated 77 summer cultivation amphore (reference) 98 86 84 70 100 no summer 68 48 79 gigant 89 74 75 24 67 cultivation for 16 32 54 brunetta 96 52 22 40 72 internal reasons 28 36 49 goldforellen 87 85 93 20 51 53 60 64 ochsenzunge 83 59 69 16 63 30 40 51 trianon 82 70 50 40 82 54 32 59 chinesische keule 77 76 54 not cultivated not cultivated 69 tab. 4: percentage of not harvestable plants (losses in %) variety field evaluation garden no. 2 garden no. 3 garden no. 4 garden no. 5 mean hu 1. set 2. set 1. set 2. set 1. set 2. set 1. set 2. set early cultivation frühlingsgruß 2.8 23.1 21.6 0.0 4.3 0.0 0.0 no early 8.6 lettuce cabbage 0.0 40.0 61.5 12.5 20.0 0.0 poor gercultivation for 24.0 früher gelber krausblättriger 0.0 33.3 66.7 0.0 16.6 0.0 mination internal reasons 34.5 wiener maidivi 0.0 16.7 17.2 0.0 0.0 0.0 10.8 7.5 rehzunge 0.0 100.0 80.7 7.1 0.0 36.8 0.0 37.4 spring cultivation amphore (reference) 13.9 36.5 28.6 10 0.0 1.4 4.0 0.0 0.0 13.3 bunte forelle 5.6 35.1 23.1 25.0 27.5 15.0 20.0 0.0 4.3 19.5 stuttgarter sommer 8.3 50.0 36.0 22.5 32.5 6.0 12.0 1.4 0.0 20.9 struwwelpeter 8.3 28.6 39.4 10.0 7.5 2.0 2.0 0.0 5.7 12.4 hohlblättriger butter 2.8 28.6 22.2 32.5 40.0 100 100 5.6 23.9 39.5 romaine red cos 5.6 29.7 7.7 15.0 17.5 0.0 5.0 0.0 0.0 9.6 chinesische keule 2.8 not cultivated not cultivated not cult. 8.3 not cultivated summer cultivation amphore (reference) 8.3 20.9 11.9 5.7 0.0 no summer no data 9.4 gigant 16.7 10.0 9.8 12.5 25.0 cultivation for 62.5 68.8 29.3 brunetta 13.9 10.7 25.0 20.0 10.0 internal 28.6 72.2 27.8 goldforellen 2.8 17.4 4.0 5.0 30.0 reasons 0.0 66.7 18.0 ochsenzunge 13.8 6.3 10.8 25.0 7.5 0.0 75.0 20.8 trianon 19.4 10.5 11.1 7.5 0.0 0.0 65.6 16.3 chinesische keule 2.8 2.4 6.9 not cultivated not cultivated 4.0 174 c. lehmann, g. lissek-wolf , r.vögel, s. huyskens-keil tab. 5: fresh weight of plants at harvest after removing the outer leaves („market weight“ in g*) in the field evaluation at humboldt-universität zu berlin and in the pilot tests of three market gardens (means ± sd, min. – max. in parentheses) variety field evaluation garden no. 2 garden no. 3 garden no. 4 garden no. 5 mean hu 1. set 2. set 1. set 2. set 1. set 2. set 1. set 2. set early cultivation frühlingsgruß 301.12 ± 38.56 not established 289 ± 21 402 ± 23 244 ± 29 234 ± 40 no early cultivation 294 (273.36 345.15) (251 321) (369 430) (200 325) (150 305) for internal reasons lettuce cabbage 272.81 ± 19.78 not established 285 ± 50 340 ± 74 200 ± 34 poor ger274 (258.27 295.33) (230 380) (275 425) (140 245) mination früher gelber 351.61 ± 36.35 not established 260 ± 17 310 ± 31 260 ± 46 poor ger295 krausblättriger (326.30 393.27) (230 280) (275 356) (200 300) mination wiener maidivi 437.31 ± 54.09 not established 303 ± 298 403 ± 42 311 ± 43 313 ± 56 353 (380.62 488.36) (276 345) (340 430) (240-430) (205 425) rehzunge 352.24 ± 16.04 not established 293 ± 18 424 ± 48 268 ± 27 225 ± 44 312 (333.72 61.88) (270 326) (360 492) (220 320) (150 335) spring cultivation amphore 84.63 ± 14.04 158 ± 13 159 ± 15 258 ± 19 285 ± 22 298 ± 40 254 ± 46 214 ± 65 260 ± 51 219 (reference) (72.49 100.00) (140 180) (130 175) (231 284) (250 320) (240 350) (200 330) (95 280) (185 320) bunte forelle 129.51 ± 21.20 241 ± 14 247 ± 19 224 ± 42 230 ± 28 207 ± 36 202 ± 54 216 ± 66 327 ± 35 225 (107.96 150.35) (215 265) (210 275) (139 311) (202 300) (135 295) (145 295) (110 330) (270 370) stuttgarter sommer 130.73 ± 28.83 234 ± 33 218 ± 42 255 ± 33 377 ± 74 234 ± 28 273 ± 67 243 ± 64 350 ± 59 257 (97.49 148.95) (185 270) (170275) (213 307) (244 500) (190 290) (135 405) (110 350) (285 435) struwwelpeter 160.47 ± 1.73 327 ± 32 324 ± 30 371 ± 25 380 ± 51 267 ± 45 274 ± 46 272 ± 69 288 ± 83 296 (158.54 161.88) (290 385) (285 385) (340 412) (275 425) (180 365) (200 395) (140 370) (185 385) hohlblättriger 165.91 ± 21.97 248 ± 8 245 ± 16 423 ± 17 388 ± 41 no harvest 198 ± 35 296 ± 78 281 butter (151.63 191.21) (235 260) (215 265) (390 444) (314 415) bolted (135 270) (180 395) romaine red cos 159.71 ± 30.57 250 ± 18 243 ± 27 538 ± 30 545 ± 53 366 ± 43 325 ± 53 307 ± 43 306 ± 18 338 (132.38 192.72) (220 270) (190 280) (498 589) (488 629) (295 445) (275 425) (195 365) (265 325) chinesische keule 188.37 ± 17.75 not cultivated not cultivated not 191 ± 55 not cultivated 190 (170.38 205.93) cultivated (105 300) summer cultivation amphore 310.50 ± 43.14 156 ± 9 152 ± 12 250 ± 32 251 ± 30 no summer cultivation not established 224 (reference) (281.95 360.12) (140 170) (135 170) (203 291) (210 293) for internal reasons gigant 276.61 ± 11.20 290 ± 17 295 ± 16 340 ± 62 386 ± 116 200 ± 66 177 ± 50 281 (264.23 286.04) (260 315) (265 320) (240 427) (247 591) (80 290) (90 260) brunetta 234.51 ± 6.19 229 ± 14 233 ± 10 274 ± 28 232 ± 47 156 ± 38 153 ± 44 216 (227.70 239.78) (200 250) (215 245) (232 306) (129 293) (95 220) (90 228) goldforellen 173.05 ± 42.35 220 ± 15 218 ± 17 193 ± 22 200 ± 28 103 ± 23 108 ± 19 174 (129.12 213.61) (190 240) (185 245) (165 230) (165 260) (65 135) 85 140 ochsenzunge 334.13 ± 64.13 375 ± 24 353 ± 17 453 ± 61 408 ± 61 344 ± 89 194 ± 101 (274.13 401.69) (320 410) (295 395) (343 549) (324 511) (230 450) (60 390) trianon 345.21 ± 88.27 335 ± 20 335 ± 25 359 ± 39 361 ± 90 388 ± 118 299 ±86 346 (249.72 423.82) (295 360) (300 355) (285 411) (236 530) (240 600) (160 410) chinesische keule 271.95 ± 19.76 345 ± 26 339 ± 23 not cultivated not cultivated 319 (255.30 293.79) (290 375) (290 370) * fresh weights were determined with a precision of 0.1 g at hu and with a precision of 5 g in the market gardens. chemical analyses and shelf life the results of the quality analyses of material from the field evaluation at hu are shown in tab. 6. with regard to undesirable compounds the nitrate content varied considerably in all tested lettuce varieties from 28 1192 mg/kg fresh weight. the varieties ‘goldforelle’, ‘struwwelpeter’, ‘frühlingsgruß’ and ‘amphore’ tended to have the lowest nitrate contents. generally, the nitrate contents meet the values reported for endogen levels of butterhead lettuce (herrmann, 2001), in any case were clearly below the acceptable limit of 2500 mg/kg (eu-vo 466/2001) of lettuce grown in the field. the climate mediated variation of nitrate contents became apparent also in 2007: the undesirable highest contents were determined in the early and first cultivation period (march/april) and in summer (end of june on-farm management and marketing potential of old lactuca varieties 175 beginning of august) when it was cool and rainy. the lowest contents were analysed in the warm months of the spring cultivation period (beginning of may/beginning of june). the health promoting compounds, e.g. total phenols varied from 3.3 to 17.2 mg gae/g dry matter (tab. 6). the phenol contents were tendentiously higher in plants cultivated in spring where the average temperature and irradiation were higher in comparison to the other cultivation periods. the phenol contents were in general comparatively high for lettuce cultivars and suggest a high nutritional value of the old varieties; however this has to be examined in more detail. the shelf life of the lettuce varieties under simulated retail conditions (18°c, 80% rel. humidity, 2 days) can generally be classified as medium (score 2) (tab. 6). the varieties ‘lettuce cabbage’, ‘wiener maidivi’ and ‘trianon’ were better suitable for a short term storage than other varieties, however dry matter changes were rather high compared with commercially available lettuce. the varieties ‘früher tab. 6: content of dry mass, nitrate, total phenols and changes of dry mass after two days of storage (18°c, 80% rel. air humidity) of old lettuce varieties (means ± sd, min. – max. in parentheses), and shelf life evaluation (score; medians, min. – max. in parentheses) variety dry mass nitrate total phenol shelf life (%) 1 (mg/kg fw)-1 (mg gae/g dm) 1 change of dm (%) score* early cultivation frühlingsgruß 4.16 ± 0.52 1191.67± 259.92 4.32 ± 0.31 21.29 ± 1.61 ab 2 a (3.69 4.72) (892.00 1356.00) (3.98 4.58) (19.86 23.04) (2 3) lettuce cabbage 4.82 ± 0.77 704.33 ± 216.25 3.83 ± 0.17 20.47 ± 0.38 ab 1 ab (4.03 5.57) (576.00 954.00) (3.66 4.00) (20.21 20.74) (1 2) früher gelber 4.99 ± 0.29 712.33 ± 170.02 6.17 ± 0.41 26.62 ± 1.85 a 3 a krausblättriger (4.66 5.21) (528.00 863.00) (5.73 6.53) (24.68 28.38) (3 3) wiener maidivi 5.66± 0.96 419.67 ± 93.09 6.17 ± 0.64 17.36 ± 2.63 b 1 b (4.80 6.71) (361.00 527.00) (5.63 6.87) (14.40 19.43) (1 1) rehzunge 4.43 ± 0.29 630.33 ± 178.03 5.30 ± 0.71 not analysed (4.19 4.75) (428.00 763.00) (4.49 5.85) spring cultivaltion amphore 7.75 ± 0.43 53.62 ± 20.3 17.24 ± 1.46 14.45 ± 2.28 a 3 a (reference) (7.36 8.21) (34.16 74.77) (15.66 18.53) (12.30 16.84) (3 3) bunte forelle 6.88 ± 0.55 146.90 ± 96.72 10.99 ± 2.76 12.70 ± 1.26 a 3 a (6.27 7.35) (35.43 208.59) (9.36 14.18) (11.41 13.91) (3 3) stuttgarter sommer 7.06 ± 0.45 83.74 ± 49.30 12.13 ± 3.80 14.83 ± 2.39 a 2 a (6.80 7.58) (27.28 118.28) (9.37 16.46) (12.91 17.39) (2 3) struwwelpeter 7.85 ± 0.57 28.18 ± 1.84 10.76 ± 0.52 15.15 ± 2.54 a 3 a (7.40 8.49) (26.30 29.97) (10.24 11.27) (13.35 18.05) (3 3) hohlblättriger butter 7.36 ± 0.12 73.50 ± 39.80 16.45 ± 2.38 9.39 ± 2.51 a 3 a (7.26 7.49) (45.51 119.07) (14.67 19.15) (7.62 11.17) (3 3) summer cultivation amphore 4.77 ± 0.19 393.67 ± 226.19 5.69 ± 0.74 15.90 ± 0.84 ab 2 a (reference) (4.59 4.96) (158.00 609.00) (4.84 6.13) (14.94 16.45) (2 2) gigant 7.21 ± 0.50 623.67 ± 67.93 8.33 ± 0.47 19.76 ± 2.55 ab 2 a (6.68 7.68) (546.00 672.00) (8.05 8.87) (17.33 22.41) (2 2) brunetta 6.66 ± 0.48 531.33 ± 505.85 7.83 ± 1.15 14.73 ± 1.34 ab 2 a (6.10 7.00) (220.25 1115.71) (6.81 9.07) (13.46 16.13) (2 2) goldforellen 6.56 ± 0.27 325.74 ± 50.75 5.82 ± 1.35 29.34 ± 3.58 a 2 a (6.29 6.82) (279.06 396.29) (4.98 7.38) (25.57 32.69) (2 2) ochsenzunge 6.65 ± 0.82 484.33 ± 397.59 10.97 ± 0.35 13.17 ± 0.56 ab 2 a (5.82 7.45) (208.00 940.00) (10.67 11.35) (12.52 13.57) (2 2) trianon 8.50 ± 1.02 136.33 ± 63.58 13.22 ± 1.50 9.27 ± 0.51 b 1 a (7.38 9.36) (71.00 198.00) (11.50 14.23) (8.89 9.85) (1 1) *score: 1 = marketable, 2 = slightly wilted, 3 = strongly wilted, not marketable; medians 1 the friedman test (α = 0.05) did not reveal any significant differences for dry mass, nitrate and total phenol contents. within each cultivation period, means, respectively medians sharing the same superscript letter within one column are not significantly different (nemenyitest, α = 0.05). 176 c. lehmann, g. lissek-wolf , r.vögel, s. huyskens-keil gelber krausblättriger’, ‘amphore’, ‘struwwelpeter’ and ‘hohlblättriger butter’ could be stored for a relatively short time, i.e. they can be recommended solely for direct marketing. estimation of varieties by market gardens based on the estimation of the market gardens after the first year of the pilot tests the varieties may be classified in three categories: generally suitable for on-farm conservation in market gardens, not suitable for market gardening, and differently estimated varieties. only two butterhead types (‘gigant’ and ‘bunte forelle’) were generally suitable, all other butter head lettuces were estimated differently by the respective market gardens. however, none of the butter head lettuces was evaluated as not suitable by all market gardeners. the three romana lettuce types (‘wiener maidivi’, ‘romaine red cos’ and ‘trianon’) proved generally to be suitable. two of the leaf lettuces (‘struwwelpeter’ and ‘ochsenzunge’) were assessed positively; the other two (‘hohlblättriger butter’, ‘früher gelber krausblättriger’) were evaluated rather suitable for home gardens. the latin group type was estimated useful for market gardening in most cases. only two market gardens tested the stem lettuce and achieved good results in the field. marketing was more of a problem because the customers need special information how to use it. customer’s motivation the customers in the country side of brandenburg did not accept the old varieties with an unfamiliar appearance instead preferred ‘normal’ green butterhead type. however, in the city of berlin the old varieties were easy to sell because these customers like unusual and new offers. with questionnaires we examined the customer’s motivation at market stands on two weekly markets and in a farmer’s shop in berlin (fig. 1). the involved gardeners practice organic horticulture and are direct marketers with a lot of regular customers. only 19% of the respondents knew the correct answer that it means a variety not any more registered. 51% had wrong or confuse ideas. further enquiries revealed that the majority confused wild plants with the term historical variety. when the customers were asked for their reasons for buying an old variety most of them agreed with all possible options with the emphasis on supporting the diversity of vegetable varieties. the analysis of the questionnaires showed that it has priority for those customers to buy organic vegetables. they support the diversity of varieties as a further positive effect if such an offer is available. all customers questioned considered the conservation of old varieties an important issue and 85% would pay a higher price. discussion suitability for on-farm conservation the pilot tests were successful except for cultivation in autumn and provided an insight into problems as well as chances of success for the re-introduction of old varieties into the market. basically, the pilot tests confirmed the opinion that re-introducing old varieties into the market is in the interest of both, growers and consumers (hardon and van hintum, 1994). on the one hand, market gardens expand their assortments of goods and on the other hand, customers gain more choice. however, the lack of acceptance by rural customers takes a constrictive effect. obviously, a lot of public relations work is necessary to attract greater attention and raise awareness for the conservation of agro-biodiversity (kleinhüttelkotten et al., 2006). the results of 2007 preliminarily allow to classify the varieties with regard to their suitability for onfarm conservation in which some varieties like ‘bunte forelle’, ‘wiener maidivi’ or ‘struwwelpeter’ were well suited, however other varieties like ‘früher gelber krausblättriger’ or ‘hohlblättriger butter’ are more adequate for home or hobby gardens. generally, we can establish that predominantly nonspecific factors raised problems like snails, wireworms or hail that affected not only individual varieties. only some varieties had specific problems with tipburn like ‘stuttgarter sommer’ or ‘hohlblättriger butter’. marketing success and potential target groups of consumers in the city of berlin marketing was very successful in contrast to the rural region of brandenburg. therefore it is more promising to focus the re-introduction of old varieties on the city where potential customers are available. the analysis of the interviews made clear that the regular customers of the direct marketers contribute essentially to the sales success because the strong customer loyalty helped to accept old varieties. in conclusion, customers who prefer organic food are frequently disposed to make a contribution to the conservation and promotion of agro-biodiversity. therefore, these customers are a promising target group for further information campaigns and public relations concerning genetic resources as well as stimulation of interest for rare crop varieties. fig. 1: questioning of customers at two locations of weekly market stands and in a farmer’s shop in berlin (june/july 2007), n= 48 questionnaires, respectively short interviews. what is your understanding of the term ’historical variety’? 0 20 40 60 80 100 other i have no idea answer 1+2 2: varieties that are not any more registered 1: plants, not manipulated by plant breeding % why do you buy a historical variety? multiple answers were allowed 0 20 40 60 80 100 other reasons, e. g. curiosity to support the diversity of varieties to support a regional product higher nutritional value more attractive than the usual offer % how important is the conservation of old varieties in your opinion? 0 20 40 60 80 100 important very important % is the price reasonable? 0 20 40 60 80 100 i don't know no yes % would you pay a higher price for an old variety (to contribute to conservation)? 0 20 40 60 80 100 i don't know no yes % on-farm management and marketing potential of old lactuca varieties 177 the need for a network for the on-farm management of old varieties in the context of the pilot project it turned out that the marked gardens demand relatively large seed quantities of individual varieties exceeding the magnitude a seed initiative like the vern e.v. can regularly provide. the vern e.v. maintains a multitude of old crop varieties, demonstrates the diversity of crops in show gardens for the public, and provides hobby gardeners with seeds in small quantities via the „catalogue for rare crops“ (vern, 2008). the available capacity of the vern e. v. is not designed to additionally provide sufficient seed quantities of particular varieties for the on-farm management in market gardens. in face of this bottleneck problem it became essential to find new ways to produce seed. the arche noah seed network in austria sets an important example in the conservation and propagation of old varieties. under the auspices of arche noah maintainers who take on responsibility for particular varieties form a network (arche noah, 2008) thereby achieve a great capacity of work. a further example that sheds light on the aspect of maintenance breeding is the approach of vern e.v. which pursues the conservation of old potato varieties in cooperation with a commercial breeder. in this way a limited number of special attractive varieties are better commercially available. in the context of the pilot project, we took first steps to form an onfarm network. market gardens participating in the project since 2007 were won over to take on maintenance and propagation of particular varieties from 2008. vern e.v. will purify and process the seed. the network will exchange the various old varieties via vern e.v. furthermore, the network will work on breeding aspects. since sometimes off-types appeared in the pilot tests it will be necessary to eliminate off-types from seed bearing plants to maintain the respective variety identity. based on the results of the field trials we establish or verify the respective variety descriptions because they are a prerequisite for such selections. in defining the respective varietals identities it must be taken into account what may be required for uniformity of an old variety in the context of an on-farm network. from our point of view uniformity may be less strictly determined than required by law (german seed act: saatg and sortg) because we intend the conservation of old varieties as genetic resources. in this context, a certain variability may be tolerated as long as the varietals identity is recognizable. on-farm conservation as a dynamic method leads to the question if it is desirable to improve special characteristics of individual old varieties by breeding. the participants of the network may deal with the details from case to case and find adequate solutions when they practice participative breeding. moreover, the pilot tests revealed that action must be taken to improve seed quality. the heterogeneous germination rates (tab. 3) indicate that various causes influenced the germination success. on the one hand, conditions varied between the respective market gardens and on the other hand, the seed of the old varieties was not produced following the standards of current commercial cultivars. the vern e.v. produces seed outdoors which may result in loss of seed quality because lettuce is quite sensitive to rainy weather during flowering and seed ripening. further, seed vigour depends on the degree of ripeness (bremer, 1962). with the help of the network we search for appropriate solutions that can be realized by the involved maintainers according to their equipments. we develop a co-operation model for the emerging network with the aim to produce adequate quantities of seed in good quality, ensure technical support for the maintainers, facilitate exchange between maintainers concerning seed propagation and breeding work, and satisfy the legal requirements because seed exchange will be restricted to the network under the auspices of the vern e.v. moreover, interchange between network members may help to optimize cultivation of the old varieties and stimulate new marketing activities. acknowledgement the pilot project is funded by bundesministerium für ernährung, landwirtschaft und verbraucherschutz (bmvel/ble) fkz 05bm007/2. we thank the seed company rijk zwaan for gratuitously providing the seed of the reference variety ‘amphore’, thus supporting the project. we thank inge dressel for technical assistance and tobias schober for supporting the field trials. we are grateful to ruth kleinöder who conducted pre-tests in the field at the domäne dahlem and helped with many valuable suggestions and a lot of advice. references bremer, a.h., 1962: salat, lactuca sativa l.. in: kappert, h., rudorf, w. (hrsg.), handbuch der pflanzenzüchtung, band 6, züchtung von gemüse, obst, reben und forstpflanzen, 253-270. paul parey berlin, hamburg. bundesministerium für ernährung, landwirtschaft und verbraucherschutz (bmelv), 2002: nationales fachprogramm zur erhaltung und nachhaltigen nutzung pflanzengenetischer ressourcen landwirtschaftlicher und gartenbaulicher kulturpflanzen. [online] http: //www.genres.de/pgr/nationales_fachprogramm/ (cited 19 may 2008). chmielewski, 2008: wetterbeobachtungen [online] http://www.agrar.huberlin.de/struktur/institute/pfb/struktur/agrarmet/service/wb (cited 19 may 2008). de vries, i.m., 1997: origin and domestication of lactuca sativa l. genetic resources and crop evolution 44, 165-174. efken, j., 2005: on-farm-management in deutschland – funktion, gestaltung und strategie. schriften zu genetischen ressourcen 25, 14-22 [online] http://www.genres.de/infos/pdfs/bd25/25-09.pdf (cited 6 may 2008). fao, 1996a: the state of the world’s plant genetic resources for food and agriculture, fao rome, [online] http://193.43.36.103/ag/agp/agps/ pgrfa/pdf/swrfull2.pdf (cited 6 may 2008). food and agriculture organization, rome. fao, 1996b: global plan of action for the conservation and sustainable utilization of plant genetic resources for food and agriculture and the leipzig declaration adopted by the international technical conference on plant genetic resources, leipzig, germany 17-23 june 1996 [online] www.fao.org/ag/agp/agps/pgrfa/pdf/gpaeng.pdf (cited 6 may 2008). food and agricultire organization, rome. gepts, p., 2006: plant genetic resources conservation and utilizaton: the accomplishments and future of a social insurance policy. crop science 46, 2278-2292. gladis, t., 2001: wertevielfalt, biodiversität und genetische ressourcen. schriften zu genetischen ressourcen 16, 14-22 [online] http://www. genres.de/infos/rei-bd16.htm (cited 6 may 2008). hardon, j.j., van hintum, t.j.l., 1994: integrated approaches to ex-situ and in-situ conservation. in: begemann, f., hammer, k. (eds.), integration of conservation strategies of plant genetic resources in europe, 176-180. proceedings of an international symposium on plant genetic resources held in gatersleben, germany, 6-8 december 1993. zadi and ipk. hartkopf, e., 2006: evaluierung eines sortiments alter salatsorten, lactuca sativa l. bachelor-arbeit, institut für gartenbauwissenschaften, fachgebiet pflanzenzüchtung, landwirtschaftlich-gärtnerische fakultät der humboldt-universität zu berlin. helm, j., 1954: lactuca sativa l. in morphologisch-systematischer sicht. die kulturpflanze 2, 72-129. 178 c. lehmann, g. lissek-wolf , r.vögel, s. huyskens-keil herrmann, k., 2001: inhaltsstoffe von obst und gemüse. verlag eugen ulmer gmbh &co., stuttgart. jennings, a.c., 1981: the determination of dihydroxy phenolic compounds in extracts of plant tissues. analytical biochemistry 188, 396-398. kleinhückelkotten, s., wippermann, c., behrendt, d., fiedrich. g., schürzer de magalhaes, i., klär, k., wippermann, k., 2006: kommunikation zur agro-biodiversität. voraussetzungen für und anforderungen an eine integrierte kommunikationsstrategie zu biologischer vielfalt und genetischen ressourcen in der land-, forst-, fischereiund ernährungswirtschaft (einschließlich gartenbau). ecolog-institut/sinus sociovision, hannover/heidelberg. künsch, u., schärer, h., temperli, a., 1981: eine schnellmethode zur bestimmung von nitrat in frischgemüsen mit hilfe der ionensensitiven elektrode. mitteilungen der eidgenössischen forschungsanstalt für obst-, weinund gartenbau, wädenswil, flugschrift 106. lehmann, c., lissek-wolf, g., vögel, r., huyskens-keil, s., 2008: striking a new path for conservation of crop genetic resources: first results of a pilot project to re-introduce old lactuca varieties into the market. acta horticulturae, in press. rodenburg, c.m., 1960: salatsorten: eine internationale monographie. sortenbeschreibungen nr. 3, instituut voor de veredeling van tuinbouwgewassen, wageningen, holland, 260. schuller, ch., 2008: anbaueignung und qualitätsmerkmale ausgewählter historischer salatsorten (lactuca sativa l.), bachelor-arbeit, institut für gartenbauwissenschaften, arbeitsgruppe produktqualität/qualitätssicherung, landwirtschaftlich-gärtnerische fakultät der humboldtuniversität zu berlin. vern, 2008: katalog für seltene kulturpflanzen (catalogue for rare crop varieties) [online] http://www.genres.de/infos/vern/kataloguebersicht/ sortenliste.htm (cited 19 may 2008). addresses of the authors: dr. cornelia lehmann, gunilla lissek-wolf, dr. susanne huyskens-keil, humboldt-universität zu berlin, institute for horticultural sciences, section quality dynamics/postharvest physiology, lentzeallee 75, 14195 berlin e-mail: cornelia.lehmann@agrar.hu-berlin.de rudolf vögel, landesumweltamt brandenburg, tramper chaussee 2, 16225 eberswalde and vern e.v., verein zur erhaltung und rekultivierung von nutzpflanzen in brandenburg, burgstraße 20, 6278 angermünde/ot greiffenberg << 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/pagesize [907.087 680.315] >> setpagedevice art04 journal of applied botany and food quality 82, 21 25 (2008) agricultural research council (cra) – cat research unit, italy uptake and distribution of lead in tobacco (nicotiana tabacum l.) l. del piano, m. abet, c. sorrentino, l. barbato, m. sicignano, e. cozzolino, a. cuciniello (received february 18, 2008) summary six nicotiana tabacum varieties were in vitro cultured and experimentally exposed to lead in order to estimate lead uptake and distribution in tobacco plantlets and to observe possible differences depending on variety. fifty-day-old plantlets were exposed to four pb rates ranging from 0 to 200 mg dm-3. no statistically significant effect of lead on dry matter weight was observed for any variety or plant part. tissue lead concentration was determined on upper leaves, lower leaves, stems and roots. lead accumulation in the plant positively correlated with lead exposure level. lead concentration in the different plant parts decreased as follows: root, lower leaves, stem and upper leaves. all tobacco varieties showed similar behaviour with respect to lead treatment level and revealed the same distribution pattern of lead concentration in the different plant part. the highest values of tissue lead concentration were found in tobacco variety pr61, while the lowest in g94-2. introduction in nature lead is a non-essential element found in all environmental media and in all components of the biosphere. in the earth's crust the average natural concentration of pb is 16 mg kg-1 (nriagu, 1978). in the last sixty years large amounts of lead have been extracted, concentrated, used, and re-emitted into the environment, resulting in a much higher lead concentration than it used to be. indeed lead is one of the most frequently encountered heavy metals in polluted environments. in soil, in rural, urban and industrial (smelters) areas pb concentrations (in the upper layer) range from 5 to 60, from 50 to 300 and from 300 to 20,000 mg kg-1 respectively (ewers and schlipköter, 1991). moreover lead is one of the most persistent metals being estimated to have a soil retention time of 150-5,000 years (shaw, 1990). lead is a hazardous element with a long biological half-life and accumulates in bones and teeth (steenhout, 1982). in humans it induces diverse physiological and biochemical disorders, as well as mutagenic and carcinogenic effects (gerber, 1980, ewers and schlipköter, 1991). in addition to food, beverage and inhaled air, cigarette smoking may be a substantial source of lead and other heavy metal intake (chiba and masironi, 1992; ewers and schlipköter, 1991). these elements pass from tobacco to the smoke as they are only partially retained by cigarette filters, therefore smokers have higher lead levels than non-smokers (grasmick, 1985; hunter, 1986; quinn and delves, 1987; watanabe et al., 1985). moreover, cigarette smoke is a source of environmental pollution not only from the smoke exhaled by the smokers, but also from sidestream smoke emitted by burning cigarettes. sidestream smoke, which is inhaled by nonsmokers via passive smoking, usually contains relatively high concentrations of many noxious substances including heavy metals (chiba and masironi, 1992). passive smoking plays an important role in the exposure of children to lead. parental smoking has a significantly greater influence on blood lead levels than other environmental or dietary factors (andren et al., 1988). nicotiana tabacum l. is recognized to be an accumulator of metals from the soil (clarke and brennan, 1983; mench et al., 1989; wagner and yergan, 1986; wagner et al., 1988; doroszewska and bebec, 2004). the concentration of metals in tobacco leaves may be influenced by soil type, ph, agricultural practices (adamu et al., 1989a; adamu et al., 1989b; bell et al., 1988; bell et al., 1992; tsadilas et al., 2005) and genotype (wagner and yergan, 1986; wagner et al., 1988). it is reported that tobacco leaves may have a lead content varying from 0 to 200 mg kg-1 dry weight (tso, 1991). little information is available concerning lead tissue distribution and varietal variation in tobacco. the present study aims at evaluating lead uptake and distribution within plantlets of n. tabacum l. and revealing possible differences depending on variety. the use of a method based on culture solution was preferred as a model system before extending the trial to soil at the greenhouse and field levels. material and methods plant culture seeds of six n. tabacum l. varieties, belonging to the tobacco institute of scafati collection, burley ist bu23, burley ist g94-2, bright ist g19, bright ist g165, perustitza ist pr61, and perustitza ist p2b, were aseptically germinated in petri dishes on solidified (agar 8 g dm-3) murashige and skoog medium (ms) (murashige and skoog, 1962). after two weeks, the plantlets were transferred into solidified ms (agar 4 g dm-3) in glass vessels and cultured for three weeks. the plantlets were then transferred into liquid ms, in other glass vessels, containing a perforated plastic raft to hold up the plantlets, and cultured for two more weeks. at this stage, the plants were utilized for lead exposure experiments. the following growing conditions were used: 26 °c, 4000 lux (philips tl 33 40 w), and 16/8 hours light/dark cycle. lead exposure experiments the ms culture medium was removed from the vessels and the roots of the plantlets were rinsed three times with sterile distilled water. then plantlets, selected for uniformity, were exposed for 10 days to pb, at the concentration of 0, 2, 20 and 200 mg dm-3, supplied with aqueous solutions of lead nitrate or potassium nitrate, calculated to contain the same amount of nitrate as in each lead rate. at the end of the experiment, plants were thoroughly washed in distilled water, placed on filter paper and divided into four parts: upper leaves, lower leaves, stem and roots. samples for each plant part were prepared by collecting ten plants into one sample and dried in a forced ventilated oven at 60 °c for 24 hours and then at 100 °c for three hours. dry matter of each plant part was determined. chemical analysis of lead dried samples were powdered and wet digested with nitric acid, using a microwave oven (cem, mod. mds 2000). lead content was 22 l. del piano, m. abet, c. sorrentino, l. barbato, m. sicignano, e. cozzolino, a. cuciniello measured by atomic absorption spectrophotometry (varian mod. spectra aa20), equipped with graphite furnace. statistical analysis analysis of variance (anova) was performed using statistica software (version 5.0 for windows, 1996, statsoft, inc. tulsa, usa). the lead concentrations in plant tissue were log transformed to normalize the frequency distributions. post hoc comparisons to determine the significant differences between means were performed by newman-keuls test. results plantlets exposed to lead nitrate as well as potassium nitrate showed no evident sign of suffering and had the same appearance as before being exposed. for each plant part and each tobacco variety no fig. 1: tissue pb concentration [mg kg-1 (d.m.)] in different plant parts of six tobacco varieties exposed to four pb rates for 10 days. values represent mean (+ s.e.) of six replicates, each collected from 10 plants. black column: 0 mg dm-3 pb; white column: 2 mg dm-3 pb, striped column: 20 mg dm-3 pb, grey column: 200 mg dm-3 pb. tab. 1: effect of lead rate (a), plant part (b) and variety (c) on tissue pb concentration [mg kg-1 (d.m.)] in tobacco plantlets exposed to four pb rates for ten days. values are the mean of six replicates, each collected from ten plants. values shown are the back-transformed concentrations calculated from log-transformed data. values within columns followed by the same letter were not significantly different at p= 0.01 according to the newman-keuls test. (a) lead rate (b) plant part (c) variety (mg dm-3) 0 21d upper leaves 63d bu23 697a 2 265c lower leaves 877b g94-2 369b 20 1919b stem 304c g19 614a 200 12972a root 8072a g165 705a pr61 728a p2b 622a statistically significant effect of lead nitrate as well as potassium nitrate rates on dry matter weight was revealed. for all varieties, lead concentration was determined for the upper leaves, lower leaves, stem, and root of plants exposed to the four lead rates. for all the examined varieties, tissue pb concentration increased as the lead exposure rate increased, for all plant parts. for all treatments, the highest lead concentrations were revealed in the root, the lowest in the upper leaves (fig. 1). the different rates of lead application had significant effects on the lead concentration determined in various plant parts and varieties (p<0.001). lead accumulation in the plant positively correlated with the lead treatment level; moreover, significant differences between the mean values at each lead rate were revealed (tab. 1a). the comparison of mean values for the lead concentration in the various plant parts showed that the differences were statistically significant. the lead accumulation in the different plant parts decreased as follows: root, lower leaves, stem and upper leaves (tab. 1b). in tab. 2: effect of plant part (a) and variety (b), for each lead rate, on tissue pb concentration [mg kg-1 (d.m.)] in tobacco plantlets exposed to four pb rates for ten days. values are the mean of six replicates, each collected from ten plants. values shown are the back-transformed concentrations calculated from log-transformed data. values within columns followed by the same letter were not significantly different at p= 0.01 according to the newman-keuls test. lead rate (mg dm-3) 0 2 20 200 (a) plant part upper leaves 5 d 25 d 144 d 872 d lower leaves 16 b 351 b 4279 b 24412 b stem 9 c 128 c 702 c 11179 c root 258 a 4398 a 31477 a 119591 a (b) variety bu23 25 a 277 a 2387 ab 14488 ab g94-2 24 a 99 b 790 d 9804 c g19 11 c 353 a 2601 ab 13829 ab g165 27 a 355 a 2171 b 12156 bc pr61 15 b 349 a 3139 a 16885 a p2b 29 a 291 a 1505 c 11912 bc lead uptake in tobacco 23 particular, the pb concentration in the roots was about one and two orders of magnitude higher than in the lower and upper leaves respectively; intermediate values between those of lower and upper leaves were observed in the stem. tobacco variety perustitza ist pr61 had the highest values of pb concentration and burley ist g942 the lowest. the comparison of mean values between the tobacco varieties showed that the differences were statistically significant for burley ist g94-2 only (tab. 1c). in order to have more detailed information on the behaviour of tobacco plant with respect to lead exposure statistical analysis was also performed on each lead rate, each plant part and each variety separately. for each lead exposure level, significant effects of plant part and variety (p < 0.001) were revealed. at each lead rate the pb concentration in the different plant parts decreased as follows: root, lower leaves, stem and upper leaves (tab. 2a) as occurred when the mean values over all lead rates were compared (tab. 1b). it is worth noting the presence of lead in each part of control plants, especially in the roots where an amount of about 250 mg kg-1 dry matter was detected. these findings can be explained by the uptake of lead present as a contaminant in the ms culture medium during the growth stages of the plantlets. interestingly, the distribution of lead in the different parts of control plants was the same as that of the other treatments (tab. 2a). for each lead rate the tobacco varieties were ranked in a different order (tab. 2b). however with the increase in lead exposure rate for perustitza ist pr61 pb concentration was trending upwards, while for burley ist g94-2 it was the lowest for each lead rate. for each plant part significant effects of lead rate and variety on pb concentration (p<0.001) were observed. the comparisons of means showed that the differences between each lead rate were statistically significant (tab. 3a), as occurred when the mean values over all plant parts were compared (tab. 1a). for each plant part, tobacco varieties were ranked in a different order (tab. 3b). however, it is worth noting that burley ist g94-2 revealed the lowest pb concentrations in each plant part. for each variety significant effects of lead rate and plant part on lead concentration (p < 0.001) were revealed. all tobacco varieties showed similar behaviour with respect to lead treatment level with significant differences between the mean values at each lead rate (tab. 4a) and revealed the same distribution pattern of pb concentration in the different plant part (tab. 4b). discussion for this research an in vitro culture procedure, based on the use of three different ms culture media (solid, semi-solid and liquid) during the growth, was developed in order to obtain vigorous tobacco plantlets without any agar residues or breaking in the root system. indeed, 50-day-old plantlets could not be taken from solid medium without damaging the roots and young tobacco seedlings exhibited signs of suffering when cultured directly in liquid medium. tab. 3: effect of lead rate (a) and variety (b), for each plant part, on tissue pb concentration [mg kg-1 (d.m.)] in tobacco plantlets exposed to four pb rates for ten days. values are the mean of six replicates, each collected from ten plants. values shown are the back-transformed concentrations calculated from log-transformed data. values within columns followed by the same letter were not significantly different at p= 0.01 according to the newman-keuls test. plant part upper lower stem root leaves leaves (a) lead rate (mg dm-3) 0 5 d 16 d 9 d 258 d 2 25 c 351 c 128 c 4398 c 20 144 b 4279 b 702 b 31477 b 200 872 a 24412 a 11179 a 119591 a (b) variety bu23 82 a 938 b 348 a 8888 ab g94-2 48 bc 421 c 183 b 5105 c g19 71 a 862 b 274 a 8529 ab g165 62 ab 1283 a 390 a 8041 b pr61 88 a 893 b 341 a 10486 a p2b 43 c 1174 a 346 a 8564 ab tab. 4: effect of lead rate (a) and plant part (b), for each tobacco variety, on tissue pb concentration [mg kg-1 (d.m.)] in tobacco plantlets exposed to four pb rates for ten days. values are the mean of six replicates, each collected from ten plants. values shown are the back-transformed concentrations calculated from log-transformed data. values within columns followed by the same letter were not significantly different at p= 0.01 according to the newman-keuls test. variety bu23 g94-2 g19 g165 pr 61 p2b (a) lead rate (mg dm-3) 0 25 d 24 d 11 d 27 d 15 d 29 d 2 277 c 99 c 353 c 355 c 349 c 291 c 20 2387 b 790 b 2601 b 2171 b 3139 b 1505 b 200 14488 a 9804 a 13829 a 12156 a 16885 a 11912 a (b) plant part upper leaves 82 d 48 d 71 d 62 d 88 d 43 d lower leaves 938 b 421 b 862 b 1283 b 893 b 1174 b stem 348 c 183 c 274 c 390 c 341 c 346 c root 8888 a 5105 a 8529 a 8041 a 10486 a 8564 a 24 l. del piano, m. abet, c. sorrentino, l. barbato, m. sicignano, e. cozzolino, a. cuciniello lead exposure experiments were performed utilizing water solutions containing lead nitrate only, because lead ions cannot be added to the culture medium as they form insoluble salts with many of the anions present in the medium, such as phosphate and sulphate. the results of this study revealed no significant effect of lead on dry matter of each plant part for all the tobacco varieties in vitro exposed to pb from 2 to 200 mg dm-3. in research studies previously performed, a general reduction and delay in growth was observed in nicotiana tabacum when 28 day-old plantlets had been treated with lead (basile et al., 1992; del piano et al., 1995), and more than 60 % inhibition of root elongation was shown in seeds exposed to 2 mg dm-3 pb rate (del piano et al., 1996). it is reasonable to suppose that the growth stage has affected the plant's response to lead, as 50 day-old plants were used instead of younger plants or seedlings. although a decrease in plant dry weight caused by lead treatment has been generally reported, also for other species, exposed to comparable lead rates, no variation or even an increase in dry matter, has been found (jones, 1973; wierzbicka, 1999). as regards lead uptake in tobacco plants results showed pb accumulation mainly in the roots in agreement with the findings widely reported on other species (fodor, 1998; huang and cunningham, 1996; kabata-pendias, 2001; kumar et al., 1995; zimdahl, 1976). in this research, 50 day-old plantlets, instead of seedlings, as generally reported in other studies performed in culture solution (fodor, 1998; huang and cunningham, 1996; kumar et al., 1995; wierzbicka, 1995; wierzbicka, 1999) were utilized and roots, stem and leaves, divided in upper and lower leaves, were examined. thus more information about the distribution of lead in the different parts of the plant was obtained, despite using an in vitro model system. for all n. tabacum varieties examined, results indicated that lead content in each plant part increased with the increasing lead exposure rate. these findings are consistent with those reported for lolium perenne (jones, 1973), brassica juncea (kumar et al., 1995; jiang et al., 2000) and sesbania drummondii (sahi et al., 2002). a concentration-dependent pb accumulation has also been found both in roots and shoots of zea mays, but not in the shoots of ambrosia artemisiifolia, a weed common on pb-contaminated sites (huang and cunningham, 1996). for all exposure rates and for all varieties the lead accumulation in the different plant parts decreased as follows: root, lower leaves, stem and upper leaves. it is worth noting that a similar distribution pattern of lead was found also in control plants, owing to the uptake of lead present as a contaminant of the culture media salts (despite using only analytically pure reagents), demonstrating a similar response of tobacco plantlets when exposed to lead in trace or higher rates. root lead content in the nicotiana tabacum varieties, at the 2 mg dm-3 pb rate, ranged from 2,000 to 8,000 mg kg-1, most often about 5,000 mg kg-1 (fig. 1). in a study on pb uptake, performed at the same lead rate in culture medium, a pb amount of about 3,900 and 2,100 mg kg-1 dry matter was reported, in the roots of hordeum vulgare and zea mays, respectively (wierzibcka and antosiewicz, 1993). root lead uptake, for most of the tobacco varieties, was slightly higher than that of hordeum vulgare, except for g94-2 which was similar to that of zea mays. moreover, the results indicated that, at the pb exposure rate of 2 mg dm-3, tobacco plants concentrated pb in their roots, on average, by 2,200 fold (tab. 2a). the concentration factor (the ratio between tissue metal concentration and medium metal concentration) decreased as the lead rate increased and it was 600 at 200 mg dm-3, indicating that a possible saturation of the root pb binding sites occurred. it is interesting to note that the high potential of root pb uptake in tobacco plants is confirmed by the large amount of metal (250 mg kg-1) found in control plants (tab. 2). the presence of high pb values in the roots of control plants, coming from the contamination of the reagents utilized in culture media has been also reported for allium cepa (wierzbicka, 1999). as regards the aerial parts of plant, at the lowest lead exposure rate (2 mg dm-3) results revealed a pb content of 350, 25 and 128 mg kg-1 for lower leaves, upper leaves and stem respectively, with a corresponding concentration factor of about 170, 10 and 60. at the highest lead exposure rate (200 mg kg-1) pb concentration factors decreased, always being higher than one unit, also for upper leaves. also kumar et al. (1995), comparing the ability of brassica species and other plants to accumulate pb, reported for tobacco seedlings (17 day-old) exposed to 625 mg of pb/g dw sand-perlite mixture, a lead content of about 800 mg kg-1 for shoots and a concentration factor higher than one unit. all tobacco plants examined showed the same response with respect to lead exposure and a similar pattern of lead distribution within the plantlet. as regards tissue pb concentration, tobacco variety g94-2 showed the lowest values for each exposure rate and for each plant part. it is worth pointing out that g94-2 50-day-old plantlets had higher values of total plant dry matter than other varieties grown under same conditions. moreover roots had a larger diameter and a higher percentage of dry matter (25 %) on total plant than the other tobacco plants (10-20 %). the dilution effect, caused by the higher root weight, and the larger dimensions of the roots might explain the low pb concentrations found in variety g94-2. the role of root quality (large and fine) has also been proposed for cd accumulation traits in nicotiana tabacum and nicotiana rustica (wagner and yeargan, 1986). the results of this research highlight the great potential of nicotiana tabacum for lead uptake by the roots, and suggest that, although the transport of lead from root to the aerial parts of the plant is limited, it can be accumulated in the leaves. it is worth noting that the in vitro model system used in this study allows tobacco genotypes to be tested in a relatively short time as regards heavy metal uptake, in the absence of environmental interferences and avoiding complicating soil processes. however, although the in vitro technique may be a useful investigative tool, it might not accurately reflect metal uptake and distribution occurring in soil. thus investigations should be extended to greenhouse and field cultivation. acknowledgments this research was carried out with financial support from the commission of the european community, tobacco and information and research fund, project 96/t/35 "monitoring and minimizing heavy metal contents in tobacco". it does not necessarily reflect the views of the commission and in no way anticipates its future in this area. references adamu, c.a., bell, p.f., mulchi, c.l., chaney, r.l., 1989a: residual metal concentrations in soils and leaf accumulations in tobacco a decade following farmland application of municipal sludge. environ. pollut. 56, 113-126. adamu, c.a., mulchi, c.l., bell, p.f., 1989b: relationships between soil ph, clay, organic matter and cec and heavy metal concentration in soils and tobacco. tob. sci. 33, 96-100. andren, p., schutz, a., wahter, m., attewell, r., johansson, l., willers, s., skerfving, s., 1988: environmental exposure to lead and arsenic among children living near a glass-works. sci. total environ. 77, 25-34. basile, a., del piano, l., abet, m., sorrentino, c., cafiero, g., 1992: effects and localization of lead in nicotiana tabacum l. 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(supplement 1), 423429. zimdahl, r.l., 1976: entry and movement in vegetation of lead derived from air and soil sources. j. air pollut. control assoc. 26, 655-660. address of the authors: luisa del piano (corresponding author), massimo abet, ciro sorrentino, loredana barbato, mariarosaria sicignano, eugenio cozzolino, antonio cuciniello. agricultural research council (cra) – cat research unit, via p. vitiello n. 108, 84018 – scafati (sa) – italy << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /colorconversionstrategy 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/pagesize [907.087 680.315] >> setpagedevice journal of applied botany and food quality 92, 313 319 (2019), doi:10.5073/jabfq.2019.092.042 1department of agricultural chemistry, institute of environmentally-friendly agriculture (iefa), college of agriculture and life sciences, chonnam national university, gwangju, korea 2department of horticulture, college of agriculture and life sciences, chonnam national university, gwangju, korea 3asian pear research institute, chonnam national university, gwangju, korea changes in activity and isozyme patterns of peroxidase and chitinase in kiwifruit pollen yong-su song1,#, sang-hyun lee2,3,#, jung-an jo3, seung-hee choi1, dong-jun seo1, yong-kyu lee3, ung yang3, woo-jin jung1,* (submitted: july 24, 2019; accepted: september 25, 2019) summary in this study, changes in activity and isozyme patterns of peroxidase (pod) and chitinase in kiwifruit (actinidia chinensis) pollen were investigated under different storage conditions. although residual activity was detected in heat-treated pollen, changes in pod activity were observed due to difference in storage conditions as revealed by preliminary studies in which pollen germination varied with dif ferent storage conditions. pod activity of kiwifruit pollen increased as proportions of viable pollen increased, indicating a positive correlation (r2=0.993) between pollen viability and pod activity. there was a detectable difference in the relative activity of pod enzyme between heat-treated and viable pollen. decoloration of congo red was observed in germination medium which fresh pollen was cultured. the activity of individual chitinase isozymes present in kiwifruit pollen differed depending on storage conditions, which had a direct impact on pollen vigor. although direct evidence showing that chitinase isozymes are implicated in pollen vigor is still uncertain, distinction of isozymes may facilitate more precise identification of viable pollen which possesses germination potential from nonviable pollen. taken together, these results suggest that monitoring the activity of pod and chitinase can be an attractive alternative to evaluate pollen vigor in kiwifruit. keywords: actinidia chinensis, chitinase isozymes, kiwifruit pollen, monitoring, pod activity, viability introduction kiwifruit (actinidia chinensis planch) is a dioecious species and mainly wind-pollinated, but artificial pollination of kiwifruit is becoming a desirable alternative to ensure a satisfactory crop yield. it is necessary to determine the viability of stored pollen before its use in artificial pollination, since pollen viability that affected pollination efficiency may be reduced during storage period. thus, developing a robust method that can be used routinely for verifying pollen vigor is important to assure successful pollination. the longevity of pollens varies greatly with different plant species and is known to be affected by storage conditions such as temperature and humidity (dafni and firmage, 2000). to find optimal conditions for long-term preservation of pollen used in artificial pollination, effects of pollen storage under different temperature conditions on pollen viability have been studied in several plant species including caladium (deng and harbaugh, 2004), phalaenopsis (yuan et al., 2018), hazel (novara et al., 2017), date palm (mesnoua et al., 2018), and kiwifruit (borghezan et al., 2011). the presence of peroxidases has been reported in the pollen wall of several plant species including lily (dashek et al., 1979), poplar (ashford and knox, 1980), and kiwifruit (speranza et al., 2012). changes in peroxidase activity and isozyme pattern during pollen maturation and germination are well understood (malik and gupta, 1976; parui et al., 1998; dai et al., 2007), although pollen has relatively low peroxidase activity as well as low isozyme polymorphism compared to other plant tissues (malik and gupta, 1976; bredemeijer, 1984). the majority of prior researches on peroxidase activities have focused to how they contribute to physiological processes associated with pistil-pollen interaction (bredemeijer, 1984). interestingly, peroxidase activity in cross-pollinated tobacco styles increases steadily during pollen tube growth (bredemeijer, 1974) while peroxidase activity in pistils pollinated with non-viable pollen was shown to increases irrespective of the penetration of pollen tube in pyrostegia venusta (chaudhary et al., 2010). this indicates their role in defense response (pandey et al., 2017) rather than being implicated to stigma receptivity (dafni and maués, 1998). indeed, among proteins induced during plant defense, class iii plant peroxidases are known to be implicated in cell wall rigidification through cross-linking of several compounds such as lignin, suberin, extensins and polysaccharide-linked ferulates (pandey et al., 2017). most plant peroxidases belong to class iii heme-containing peroxidases (ec 1.11.1.7) which catalyze reactions that involve not only h2o2 decomposition but also hydroxyl radical (·oh) generation with a superoxide anion (o2·-) and h2o2 as substrates (schopfer et al., 2001). experimental evidence suggests that ·oh is involved in pollen wall loosening (at the germination aperture) whereas h2o2 strengthens the wall through peroxidase-mediated ferulate oxidation in tobacco pollen (smirnova et al., 2014). mentioned above, several studies on the activity of peroxidases implicated in physiological processes of pollen had been described, the involvement in the regulation of cell wall extensibility during germination, and its increased activity observed during maturation suggests a crucial role of peroxidase in pollen development. thus, monitoring changes in peroxidase activity can provides information on germinability of pollen. however, there is little known the effect of difference in storage conditions affecting pollen viability on the activity of peroxidase in kiwifruit pollen. it is known that hydrolytic enzymes which diffuse from pollen after landing on the receptive stigma could function in pollen tube growth and penetration (knox, 1984). some morphogenetic changes that occur during plant development require the disruption of existing plant tissues and it may occur during pollen tube growth through stylar transmitting tissue (neale et al., 1990). accumulation of chitinases in bean abscission zone and their presence in anthers and pistils imply that the involvement of chitinases in many plant processes requiring cell wall disruptions in rapidly growing tissues such as young leaves, stems, flowers, and pollen (del campillo and lewis, 1992). these observations are further supported by evidence that a high expression of poplar win6 gene (wound-inducible chitinase) was noted # these authors contributed equally to this work. * corresponding author 314 y.-s. song, s.-h. lee, j.-a. jo, s.-h. choi, d.-j. seo, y.-k. lee, u. yang, w.-j. jung in unwounded young leaves, and a sharp increase in win6 expression in pollen coincided with the onset of anther dehiscence (clarke et al., 1994). however, little is known the major function of chitinase on the viability of kiwifruit pollen, and it remains unclear whether difference in enzyme activity and isozyme patterns is due to changes in pollen viability. thus, the objective of this study was to examine the activity and isozyme patterns of peroxidase and chitinase to establish a reliable method for evaluating pollen vigor in kiwifruit. to achieve this objective, we designed the following experiments: 1) under different storage conditions, in vitro germination of kiwifruit pollen was tested; 2) through various experimental approaches (in vitro germination, active staining, and congo red decoloration), whether difference in storage conditions that affected pollen viability was associated with changes in activity of peroxidase was investigated; 3) changes in chitinase activity and isozyme pattern were also investigated under different storage conditions. materials and methods materials kiwifruit pollen grains, actinidia chinensis (matua i) and a. chinensis (matua ii) were purchased from local market in naju, jeonnam, korea and stored in tightly sealed containers with desiccant at -20 °c until further use. germination of kiwifruit pollen unless otherwise noted, kiwifruit pollen cultivar matua i was used for in vitro germination. prior to germination, stored pollen was rehydrated for 12 h at 4 °c in a sealed container lined with moist filter paper. the pollen was scattered uniformly on a medium containing 10% sucrose (w/v) and 1% agar (w/v) and then incubated in the dark at 25 °c with a relative humidity (rh) of 80-90%. to determine difference in pollen viability by storage conditions, pollen was subjected to the following storage conditions: heat-treated frozen storage (hf), fresh frozen storage (ff), moisture-treated frozen storage (mf), moisture-treated cold storage (mc), and moisture-treated rt storage (mr). for hf preparation, pollen was heated at a temperature of 100 °c using a moisture analyzer (mb23, ohaus co., usa) with infrared heat source for 10 min and then kept at -20 °c for 24 h. for ff preparation, pollen was kept at -20 °c and no treatment was applied until use. for mf, mc, and mr preparations, pollen was placed in a sealed container lined with moist filter paper and stored at 4 °c for 12 h. after that, pollen was subdivided into three groups and each group was kept at -20 °c or 4 °c or 20 °c, respectively, for 24 h. to investigate the effect of duration of room temperature (rt, 20 °c, rh 50~55%) exposure on pollen germination, pollen was collected at various time periods (6, 12, 18, and 24 h) after exposure to rt. subsequently, germination was measured every 1 h during incubation. in some experiments, either fresh or heat-treated pollen were incubated on a medium supplemented with 0.01% (w/v) congo red (sigma, st. louis, mo, usa). in other experiments, different contents (0, 10, and 20 nmol) of n-acetyl glucosamine (glcnac) were added to the germination medium. for pollen germination analysis, digital images of randomly chosen fields were acquired with a video camera (axiocam icc1, carl zeiss, germany) connected to axiostar plus (carl zeiss, germany). the percentage of germination was determined by scoring at least 50 pollen grains per treatment from digital images. pollen grains with tube length equal to or exceeding their diameter were considered to have germinated. extraction of proteins proteins were extracted from kiwifruit pollen (10 mg) according to the method described by persia et al. (2008) with some modifications. briefly, pollen was suspended in 200 μl of wash buffer (50 mm tris-hcl, ph 8.5, 3 mm egta, 3 mm edta, 2 mm mgcl2, and 2 mm dithiothreitol) and incubated at 4 °c for 30 min with occasional shaking. the suspension was centrifuged at 10000 × g for 5 min at 4 °c, and the resulting supernatant was collected and kept at -20 °c until use. the pellet was resuspended in 200 μl lysis buffer (50 mm tris hcl at ph 8.5, 3 mm egta, 3 mm edta, and 2 mm mgcl2) and homogenized for 2 min on ice using a grinding pestle (biomasher-ii, nippi inc., tokyo, japan). the homogenate was kept at 4 °c for 30 min and centrifuged at 10000 × g for 5 min at 4 °c, and the resulting supernatant was collected. crude enzymes obtained from extraction by using wash buffer and lysis buffer, respectively, were used in active staining of chitinase. for pod activity assay, 10 mg of pollen was homogenized in 200 μl of lysis buffer (50 mm tris-hcl, ph 8.5, 3 mm egta, 3 mm edta, and 2 mm mgcl2) for 2 min on ice using a grinding pestle (biomasher-ii, nippi inc., tokyo, japan). following incubation at 4 °c for 30 min, the homo genate was centrifuged at 10000 × g for 5 min at 4 °c. the resulting supernatant was collected and used as crude extract to determine pod activity. protein concentration in crude extracts was determined according to the method of bradford (1976) with bovine serum albumin as standard. assay of peroxidase activity pod activity was assayed based on the method described by chance and maehly (1955). the reaction mixture contained 50 μl of 20 mm guaiacol, 2.87 ml of 10 mm phosphate buffer (ph 7.0), and 25 μl of crude extract. the reaction was initiated by adding 20 μl of 40 mm h2o2. increase in absorbance at 470 nm was measured at 1 min intervals using a uv-visible spectrophotometer (optizen 3220uv, mecasys co., korea). one enzyme unit was defined as the amount of enzyme capable of producing 1 μmol of tetraguaiacol per minute at 25 °c. active staining of peroxidase for in-gel peroxidase activity assay, native polyacrylamide gel electrophoresis (page) was performed according to the method described by ornstein (1964). duplicate sets of protein samples (30 ug/lane) (heat-treated matua i, moisture-treated matua i, fresh matua i, and fresh matua ii) were loaded onto each half of a 10% (w/v) gel and resolved by native page. after electrophoresis, gel was divided in half longitudinally and one half was stained with 0.12% (w/v) coomassie brilliant blue (cbb) r-250. the other half was equilibrated with 50 mm tris buffer (ph 6.8) for 10 min. the gel was then incubated in 50 mm tris buffer (ph 6.8) containing 0.46% (v/v) guaiacol and 13 mm h2o2 until reddish-brown bands appeared, after which it was fixed in a mixture of water/methanol/acetic acid (6.5:2.5:1, v/v) as described previously (caruso et al., 1999). active staining of chitinase chitinase active staining was performed according to the method described by trudel and asselin (1989). crude proteins (5 ug) were loaded into each lane of two different 10% (w/v) gels with or without 0.01% (w/v) glycol chitin embedded, and two gels were resolved in parallel by sds-page. following electrophoresis, one gel was stained with 0.12% (w/v) cbb r-250. the other gel was incubated with 100 mm sodium acetate buffer (ph 5.0) containing 1% (v/v) triton x-100 and 1% (w/v) skim milk at 37 °c for 2 h with reciprocal shaking. this was followed by overnight incubation under the same conditions except that there was no skim milk in the buffer solution. subsequently, the gel was immersed in 500 mm tris-hcl (ph 8.9) solution containing 0.01% (w/v) calcofluor white m2r (sigma, st. peroxidase and chitinase in kiwifruit pollen 315 louis, mo, usa) for 7 min, and lysed zones were visualized and photographed using a uv transilluminator (wgd-30; daihan sci. co., korea). statistical analysis data were compared using tukey’s studentized range (hsd) test, with p ≤ 0.05 indicating statistical significance. all data were analyzed using statistical analysis system 9.1 and are presented as mean ± standard error (se). results and discussion germination of kiwifruit pollen at various storage conditions to identify change in pollen performance after storage under different conditions, germinations of kiwifruit pollen subjected to five storage conditions mentioned above were evaluated. as shown in fig. 1a, a germination of 72.3% was observed in ff treatment, while little (0.9%) or no germination was observed in hf treatment. the germination of mf or mc treatment was similar to that of ff treatment, while that of mr treatment exhibited a 2.3-fold decrease. these results indicate that storage at high temperature has a negative impact on pollen germination while moisture pre-treatment has no effect on pollen germination. a previous report has shown that prolonged exposure to high humidity (95% rh for 5 h) at 38 °c does not affect the viability of tobacco pollen as assessed by fluorochromatic reaction, although it significantly reduces pollen germination in vitro (shivanna and cresti, 1989). when pollen grains were subjected to both high rh and high temperature simultaneously, their germination was delayed compared to that of pollen exposed to high rh at ambient temperature vitro (shivanna and cresti, 1989). this is in agreement with our observations. in the present study, we found that pollen germination of mr treatment was significantly lower than that of moisture treated-group (mf or mc) though their temperature conditions were more favorable for germination than those tested by shivanna and cresti (1989). the effect of temperature was also evident when germination of kiwifruit pollen was examined after rt exposure for various durations (6, 12, 18, and 24 h). we found that germinations were similar when kiwifruit pollen was exposed to rt ranging from 6 to 18 h. however, when exposure was more than 18 h, pollen germination reduced to <40% (fig. 1b). these results indicate that duration of rt exposure above 18 h has adverse effect on pollen germination. deng and harbaugh (2004) have investigated the effect of storage temperature (rt, 4 °c, and -20 °c) over a period of 6 days on caladium pollen viability. their results showed that only 5% of pollen stored at rt for 1 day remained viable. more than 99% of pollen lost their viability after being stored at -20 °c for 1 day. in contrast, storage temperature of 4 °c seemed to slow down the decline of germination and it has resulted in 32.5% viability after 1 day. the decrease in pollen viability under rt storage has also been described elsewhere (borghezan et al., 2011; novara et al., 2017; mesnoua et al., 2018; yuan et al., 2018), showing that pollen stored at subzero temperature is effective for long-term preservation. peroxidase activity of kiwifruit pollen we have previously observed that the germination of kiwifruit pollen varied with storage conditions. we also confirmed that pollen germination in mr treatment decreased by less than a half compared to those in other conditions except hf treatment. to determine whether changes in activity and isozyme patterns of pod and chitinase were due to difference in storage conditions affecting viability of kiwifruit pollen, the following experiments were conducted under the same storage conditions tested above. first, we investigated pod activity in kiwifruit pollen using various experimental approaches. as shown in fig. 2a, pod activity of kiwifruit pollen increased when proportions of viable pollen were increased. there was a positive correlation between pollen viability and pod activity (r2=0.993), suggesting that pod activity could be used as a valuable index to predict pollen viability. a comparison of pod activity in kiwifruit pollen under different storage conditions showed that pod activity was relatively lower in hf treatment than that in any other group (fig. 2b). in hf treatment, pod activity was observed. however, pollen grains were non-viable. this is consistent with our previous result showing high level of pod activity in hf-treated pear pollen (song et al., 2018). this might be partially attributed to residual activities caused by the presence of pod enzymes in non-viable pollen (rodriguez-riano and dafni, 2000). although the reduction in pod activity was not drastic in other treatments except for hf treatment, these results indicate that it is most suitable to store kiwifruit pollen under ff treatment to maintain the pollen vigor. pod activities of two kiwifruit cultivars (matua i and ii) showed similar levels (fig. 2c). pod activity of kiwifruit pollen was also confirmed by active fig. 1: effects of different storage conditions on in vitro germination of kiwifruit pollen. pollen germination was observed following five storage conditions (a) or after various durations of rt exposure (b). hf, heat-treated frozen storage; ff, fresh frozen storage; mf, moisture-treated frozen storage; mc, moisture-treated cold storage; mr, moisture-treated rt storage. different letters on the error bars indicate significant differences based on tukey’s studentized range at p ≤ 0.05. error bars represent the se of mean values (n = 6). 316 y.-s. song, s.-h. lee, j.-a. jo, s.-h. choi, d.-j. seo, y.-k. lee, u. yang, w.-j. jung staining (fig. 3). pod enzymes were seen as yellow to dark yellow bands on a clear background (fig. 3b). two isoform bands at around 66 kda were found, and there was a detectable difference in the relative activity of these two isoforms among treatments. the staining intensity of moisture-treated pollen was slightly lower than those of fresh pollens (fig. 3b, compare lane b with lanes c and d). in the case of heat-treated pollen, there was a very faint band that was easily distinguishable compared with those of other treatments (fig. 3b, lane a). peroxidase isozyme profiles of pollen collected before and after anthesis have been investigated (parui et al., 1998). considerable variation in the enzyme profiles was observed, with individual isoforms showing a tendency to increase in their staining intensity upon maturing of anther. the increase in activity of peroxidase isoenzymes with maturity of pollen can be explained by the fact that peroxidases play an important role in cell wall biosynthesis (passardi et al., 2004). furthermore, observed changes in the activity of peroxidase isozymes during pollen germination have been confirmed in a previous study of malik and gupta (1976). dai et al. (2007) have screened 186 protein spots differentially expressed in mature and germinated rice pollen using 2-de-based proteomic approaches. their study revealed that wall metabolismfig. 2: changes in peroxidase (pod) activity of kiwifruit pollen. the activity of pod was examined based on proportions of viable pollen grains (a), under different storage conditions (b), and between kiwifruit cultivars (matua i and ii) (c). hf, heat-treated frozen storage; ff, fresh frozen storage; mf, moisture-treated frozen storage; mc, moisture-treated cold storage; mr, moisture-treated rt storage; hm, heat-treated matua i; fm(i), fresh matua i; fm(ii), fresh matua ii. different letters on error bars indicate significant differences based on tukey’s studentized range at p ≤ 0.05. error bars represent the se of mean values (n = 3). fig. 3: identification of pod activity in kiwifruit pollen. proteins were loaded to 10% (w/v) gel and resolved by native page. after electrophoresis, protein bands were visualized by cbb staining (a), and pod activities were detected by in-gel activity assay (b). pod activity was also confirmed by decoloration of germination medium supplemented with 0.01% (w/v) congo red (c). m, size marker; a, heat-treated matua i; b, moisture-treated matua i; c, fresh matua i; d, fresh matua ii. arrows indicate two pod isoforms. peroxidase and chitinase in kiwifruit pollen 317 related proteins such as udp-glucose pyrophosphorylase, polygalacturonase, class iii peroxidases, and β-expansins were up-regulated on germination, might be mainly responsible for pollen tube growth. this study also found that there were multiple isoforms in differentially expressed proteins and different expression patterns between isoforms during the developmental switch from mature pollen to germination and tube growth. in the case of β-expansin and class iii peroxidase, some isoforms were up-regulated and others were down-regulated, suggesting that they involved differently in pollen germination. as mentioned in the introduction, plant peroxidases catalyze reactions involving not only h2o2 decomposition but also .oh generation with superoxide anion (o2 .-) and h2o2 as substrates (schopfer et al., 2001). nitrogen double bonds that are characteristic of azo dyes (such as congo red) may be broken via oxidation by .oh generated from peroxidase, thereby resulting in the loss of color (cripps et al., 1990). interestingly, during the early stage of kiwifruit pollen germination, extracellular h2o2 accumulation in the culture medium by germinating pollen has been reported (speranza et al., 2012). thus one can assume, based on the results presented by speranza et al. (2012), that it is possible to judge pollen vigor depending on the degree of decoloration of the medium through pod activity when kiwifruit pollen is cultured in a germination medium containing congo red. to determine whether the degree of decoloration might be different depending on pollen vigor, fresh or heat-treated pollen were incubated on a medium supplemented with congo red. as shown in fig. 3c, a measurable decoloration (more than half of the dia meter) was observed in germination medium with which fresh pollen was cultured. however, no decoloration was observed in the germination medium with which heat-treated pollen was cultured. some metabolites such as .oh generated by fresh pollen harboring pod activity during germination might have contributed to the observed decoloration in germination medium. fungal lignin peroxidase and plant horseradish peroxidase are known to be involved in the decolo ration of azo dyes (cripps et al., 1990). our finding, together with previously discussed results (that is, change of pod activity with different storage conditions, correlation between pollen viability and pod activity, active staining of pod enzyme, and the decoloration of germination medium), suggest that monitoring pod activity can help verify pollen performance in kiwifruit. chitinase activity of kiwifruit pollen to evaluate the potential of chitinase enzyme as an indicator for pollen vigor, chitinase activity in kiwifruit pollen was examined under different storage conditions (hf, ff, mf, and mr). as shown in fig. 4b, seven isozymes (ch1-ch7) were detected, and the relative molecular weights of these enzymes were found to differ, ranging from 15 to 90 kda through active staining. for some isozymes, there were detectable differences in the relative activity among treatments. staining intensities of ch6 and ch7 were quite higher in ff treatment than those in other treatments. interestingly, ch3 isoform was detected only in ff treatment among all seven isoforms. we found that some isozymes such as ch2, ch4, and ch5 were detected in all treatments. in contrast, isozyme ch6 or ch7 was not detected in hf treatment. these results indicate that activities of individual chitinase isozymes present in the kiwifruit pollen can differ depending on storage conditions which have a direct impact on pollen vigor. although direct evidence showing that chitinase isozymes are implicated in pollen vigor is still uncertain, monitoring ch3 isoform may help verify the performance of kiwifruit pollen. plant chitinases (ec 3.2.1.14) are known to protect plants against fungal pathogens by inhibiting mycelial growth or by degrading chitin, a linear homopolymer of β-1,4-linked n-acetyl glucosamine (glcnac) residue (collinge et al., 1993). the possible involvement of chitinase has been elucidated in non-defensive roles such as flowering, reproduction, seed germination, somatic embryogenesis, and plant growth (patil and widholm, 1997). furthermore, differential regulation of individual chitinase isoforms in response to pathogen attacks and abiotic stresses has been reported in peas, barley, tobacco, and peanut (collinge et al., 1993). these findings motivated us to further investigate the potential involvement of chitinase in pollen germination. hence, we studied whether pollen germination was affected by in vitro supplement of glcnac. to assess the effect of glcnac, kiwifruit pollen was incubated in germination medium supplemented with either 0 (control), 10, or 20 nmol glcnac. as shown in fig. 5, treatment with 10 or 20 nmol of glcnac gave rise to pollen germination of 77.1% or 74.5%, respectively, compared to germination of 60.3% in the non-treated group. although the addition of glcnac did not show a dose-dependent effect, our results implied a possible involvement of chitinase in kiwifruit pollen germination. endogenous substrate for plant chitinases has not yet been found. fig. 4: changes in activity and isozyme pattern of chitinase in kiwifruit pollen. crude proteins were extracted by using wash buffer or lysis buffer and loaded into each lane of two different 10% (w/v) gels with or without 0.01% (w/v) glycol chitin embedded. following electrophoresis, protein bands were visualized by cbb staining (a), and individual chitinase isozymes were detected by chitinase active staining (b). m, size marker; hf, heat-treated frozen storage; ff, fresh frozen storage; mf, moisture-treated frozen storage; mr, moisture-treated rt storage. arrows indicate seven chitinase isoforms. 318 y.-s. song, s.-h. lee, j.-a. jo, s.-h. choi, d.-j. seo, y.-k. lee, u. yang, w.-j. jung however, there is strong evidence that these enzymes can catalyze hydrolytic decomposition of arabinogalactan proteins (agps) present in plant (van hengel et al., 2001). van hengel et al. (2001) have shown that immature carrot seed agps that can promote somatic embryogenesis contain glcnac and glucosamine residues. in addition, they are sensitive to endochitinase cleavage. agps are commonly found in stigma exudates, transmitting tissues, pollen grains, and pollen tubes in many species. they have been implicated in pollen formation and hydration as well as in pollen tube growth and guidance (nguema-ona et al., 2012). in tobacco, stylar transmitting tissue-specific glycoproteins belonging to agp family are implicated in promoting pollen tube growth and affecting pollen tube guidance, and its sugar moieties may be a source of nutrients for these biological activities (cheung et al., 1995; wu et al., 1995). thus, it can be inferred that chitinases present in pollen may also participate in pollen tube growth through hydrolytic action on endogenous substrate. conclusion this study shows that the viability of kiwifruit pollen differs depending on storage conditions and that storing pollen in ff treatment is the most desirable for maintaining pollen vigor. pod activity was found to be closely related to pollen viability. although residual pod activity was detected in heat-treated pollen, pod activity was also altered by different storage conditions as revealed by preliminary studies in which pollen viability was affected depending on storage conditions. these results (i.e., detectable difference in relative activity of pod enzymes between heat-treated and viable pollen, and decoloration of congo red caused by fresh pollen possessing pod activity) demonstrate that monitoring pod activity can help verify pollen performance in kiwifruit. furthermore, distinction of chi tinase isoforms may facilitate more precise identification of viable pollen which possesses germination potential from non-viable pollen. taken together, monitoring the activity of pod and chitinase can be an attractive alternative to evaluate pollen vigor in kiwifruit. acknowledgements this 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doi:10.5073/jabfq.2020.093.025 1department of horticulture and food technology, university of applied sciences weihenstephan-triesdorf, freising, germany 2department of applied artificial intelligence, deggendorf institute of technology, technology campus grafenau, grafenau, germany 3division urban plant ecophysiology, faculty of life sciences, humboldt-universität zu berlin, berlin, germany evaluating the practicability of commercial food-scanners for non-destructive quality assessment of tomato fruit simon goisser 1, 3, michael fernandes2, sabine wittmann1, christian ulrichs3, heike mempel1* (submitted: june 18, 2020; accepted: october 15, 2020) summary the assessment of tomato fruit quality depends on a variety of extrinsic and intrinsic quality parameters such as color, firmness and sugar content. conventional measurement methods of these quality parameters are time consuming, require various measurement devices, and in case of intrinsic quality, involve destructive measurements. latest research focused on the non-destructive determination of these parameters by using spectroscopic measurements. the goal of this study was to evaluate the capability of three commercial ly available portable and miniaturized vis/nir spectrometers, so called food-scanners, in predicting various tomato quality attributes in a non-destructive way. additionally, this study evaluated the software provided by manufacturers for building of prediction models by comparing the results derived from those software tools to state-ofthe-art software for multivariate analysis. evaluation of food-scanner spectra resulted in prediction models of high accuracy (r² > 0.90) for tomato fruit firmness, dry matter, total soluble solids and color values l*, a* and h°. prediction models computed with manufacturer’s software showed similar accuracy to those derived from state-of-the-art evaluation software. results of this study illustrate the great potential of commercial food-scanners for non-destructive quality measurement. further important features of food-scanners with respect to the application along the fresh produce supply chain are addressed. keywords: nir spectroscopy; tomato; quality control; non-destructive measurement; food-scanner introduction fresh tomato fruit (solanum lycopersicum l.) is the vegetable species most consumed in germany in terms of quantity with an average amount of 2.3 mio t per year (statista, 2019). when it comes to cultivation, distribution and marketing of tomatoes, various commercial quality parameters are of high relevance. these commercial quality attributes differ depending on the position in the supply chain. producer prioritize qualities beneficial for an unproblematic cultivation of plants such as disease resistance, cultivation work, fruit weight, fruit size and appearance. in order to meet the requirements of a demand driven supply chain, additional postharvest characteristics such as uniformity, shape, color and firmness as well as shelf life are of importance (folta and klee, 2016). from the perspective of the consumer, taste and flavor are relevant quality attributes, and lack of flavor was identified as the primary reason for consumer dissatisfaction for tomatoes (bruhn et al., 1991). a recent study on consumer acceptance of tomatoes cultivated for fresh consumption attested the importance of sensory traits like juiciness, sweetness and taste intensity, which influence consumer purchase preference (casals et al., 2019). additionally, tomato firmness and color are vital quality parameters which influence acceptability and marketability (batu, 2004). some of the above mentioned quality parameters of tomato fruit must meet certain standards within the european union regarding the marketing and commercial quality control (ble, 2019). besides these standards required by law, retail companies impose additional requirements which oftentimes exceed those statutory provisions. these provisions regarding quality relate primarily to extrinsic attributes that are important for marketing and trading, e.g. appearance or absence of damage and deterioration. firmness is used as an indicator for the classification of tomatoes into three classes (extra, class i, class ii), yet the quoted indications „firm, reasonably firm, slightly less firm than class i“ and severity of defects (unece, 2018) are prone to subjectivity. visual and haptical impressions dominate quality assessment along the fresh fruit supply chain, therefore the evaluation of these traits highly depends on experience of staff in quality control (goisser et al., 2020a). the application of instrumental measurements for these commercially important traits could help to reduce the variation between individual controller, offer higher precision and supply a standardized language of fruit quality among industry, consumers and research (abbott, 1999). furthermore, the measurement of intrinsic quality parameters such as sugar content (°brix) as well as acidity, which relate to tomato flavor and sweetness (causse et al., 2007), could allow the estimation of sensory traits such as tomato taste. traditional instrumental measurement methods of extrinsic and intrinsic quality parameters are often time consuming and require trained personal in combination with various measurement devices. the determination of firmness via penetrometer and sugar content per refractometer demands destructive measurement methods, in the case of acidity handling of chemicals is necessary for titration. besides these destructive measurement methods of internal quality parameters, optical measurement methods like visible and near infrared (vis/nir) spectroscopy have been successful in determining important quality parameters of agricultural and horticultural pro ducts. nir spectroscopy can be applied for qualitative applications, such as identification and classification of samples, as well as quantitative applications in order to determine major constituents in samples such as fruit and vegetables (pasquini, 2003). as emphasized in the recent guidelines on objective tests for fruit and vegetables (oecd, 2018), nir spectroscopy comprises the ability to simultaneously predict multiple quality attributes using only a single spectrum, therefore serving as multidimensional predictor of fruit quality. a summary provided by dos santos et al. (2013) highlights successful reported applications for analysis of various quality attributes along a variety of fruit and vegetable species using portable spectrometers instead of traditional laboratory nir devices. ongoing technological developments promoted the miniaturization and commercialization of nir sensors, so called food-scanners. some of these devices are particularly designed for end-consumers and should enable them to identify macronutrients, allergens, calories or food contaminants (rateni et al., 2017). first studies on the predictability of individual tomato quality para* corresponding author non-destructive quality assessment using food-scanners 205 meters such as sugar content and lycopene (sheng et al., 2019; goisser et al., 2020b) using portable handheld nir spectrometers indicate the potential of these miniaturized devices for non-destructive quality evaluation. commercially manufactured portable nir instruments are relatively new and previous research either compared the performance of various instruments for the prediction of single quality traits such as dry matter (kaur et al., 2017) or used individual devices for the prediction of multiple quality attributes within selected fruit such as kiwi (li et al., 2018) or avocado (ncama et al., 2018). the results of these studies illustrate the potential of food-scanners for fast and non-destructive quality measurement. with regard to the practical use of these devices along the fresh produce supply chain, a recent study identified important requirements as stated by supply chain actors, e.g., ease of use and robustness of devices and reliability and accuracy of prediction models (goisser et al., 2020a). the aim of the study is to examine the assessment of the most important quality attributes of tomato fruit by using three commercially available portable food-scanners. contrary to previous research, which focused on prediction accuracy and spectral pre-processing, this study investigates user friendliness and potential of an application in practice along the fresh fruit supply chain. therefore, this study used manufacturer’s software for building of prediction models as well as state-of-the-art software for multivariate analysis. by comparing the results derived from both software tools, the accuracy and reliability of prediction models computed with manufacturer’s software can be assessed. for the evaluation of the full potential of each food-scanner, the full available spectral range was used during building of nir prediction models. in order to generate a high variability for different quality parameters, two different experimental approaches were used in this study. in one experiment, different ripening stages from one tomato cultivar were examined, whereas the second experiment analyzed tomatoes of uniform ripening levels but different cultivars and origins. these different approaches highlight the fact that users have to adhere to certain requirements in order to create good prediction models. further important aspects with respect to usability and applicability by non-experts are discussed. materials and methods experimental setup and sample material all research was conducted at facilities of the university of applied sciences weihenstephan-triesdorf. in order to examine tomato fruit quality in its entirety the experiment comprised two separate parts. within the first part, quality of one tomato variety (solanum lyco persicum cv. avalantino) was monitored from fruit ripening during cultivation throughout harvest up to postharvest storage. tomatoes were cultivated during summer 2019 in a greenhouse at the univer sity of applied sciences weihenstephan-triesdorf (48°24’12’’n, 11°43’50’’e) under commercial growth conditions. a hydroponic recirculating drip irrigation system was utilized for tomato cultivation. the average air temperature during cultivation within the greenhouse was 20.1 ± 8.1 °c and relative humidity was 74.5 ± 17.7%. in total, 245 tomatoes were used for the first part of the experiment. evaluation of tomato quality during ripening was conducted from the middle of july until start of august 2019 on five days with two to five days in between. five different ripening stages were determined for harvest by using the usda color chart (usda, 1975) as orientation: 1) green (tomato surface of full green color) 2) breaker (shift in color from green to yellow) 3) turning (break in color from yellow to orange) 4) light red (change in color from orange to light red) 5) red (tomato surface of full red color). on any measurement day five tomatoes of every ripening stage were randomly selected and harvested, resulting in batches of 25 tomatoes each day and 125 fruit total. calyx was removed and samples were numbered for recording of spectra and subsequent reference measurements. in order to monitor fruit quality during postharvest storage, 120 tomatoes of the ripening stage (5) were randomly selected and harvested from the greenhouse in the fourth week of august, 2019. after numbering the tomatoes were positioned on a metal grid to allow full air circulation at room temperature of 22.4 ± 1.7 °c and relative humidity of 67.4 ± 3.0% for the storage period of 22 days. measurement of fruit quality was conducted on the day of harvest and 8, 13, 17, 20, and 22 days after harvest in batches of 20 randomly selected samples. for the second part, 200 tomatoes consisting of eight different varieties à 25 fruit were purchased at four different supermarkets in freising, germany, from january to march, 2019. only plasticwrapped tomatoes of commercial grade one were selected. in order to generate variability, various tomato types (roma, salad, cherry) from different countries of origin were analyzed (tab. 1). not all fruit cultivars could be identified due to missing indications on packaging labels. all spectral and related reference measurements in both parts of the experiment were conducted on a single fruit basis. prior to spectroscopic and reference analysis fruit was kept at room temperature to allow acclimatization. temperature of fruit surface was measured immediately before recording of spectra using an infrared meter (amir 7834, ahlborn meßtechnik gmbh, holzkirchen, germany) and averaged 19.1 ± 0.4 °c. tab. 1: sample composition of supermarket tomatoes. tomato type variety n origin salad n/a 25 the netherlands salad n/a 25 the netherlands salad prince 25 germany roma n/a 25 spain miniature roma aromatica 25 the netherlands miniature roma sunstream 25 the netherlands cherry romantic 25 spain cherry n/a 25 germany acquisition of spectra using portable nir devices fruit spectra of intact tomato fruit were recorded with three different portable and commercially available food-scanners (fig. 1). at first, two spectra for each tomato were acquired on opposite sides of the fruit equator using the f-750 produce quality meter (firmware v.1.2.0 build 7041, felix instruments, portland, usa). during each recording of a new measurement, the f-750 uses a reference shutter for normalization of the spectrometer output and accounts for dark current and ambient light by recording dark scans. after recording of spectra data was transferred from the sd-card to a computer for subsequent analysis. in a next step, the portable nir spectrometer h-100f (sunforest, incheon, korea) was used for recording of spectra on same two spots on opposite sides of the fruit equator as the f-750. to ensure scan quality, the white standard attached in the protective cap of the measurement head was used for referencing at the beginning and after every 20 measurements. raw spectra was saved to a computer by linking the h-100f via usb and running the sunforest h-100 laboratory program (v 1.1, sunforest, incheon, korea). at last, spectra of each tomato was recorded using the scio™ molecular sensor (scio™ version 1.2, consumer physics, hod hasharon, israel). the scio™ applies a led light source which illuminates a considerably smaller surface area compared to the halogen lamps of the f-750 and h-100f. in order to depict an appropriate average fruit spectra, four equidistant scan points (approximately 90° apart) around the fruit equator were deemed more suitable, covering the two measurement points used with the f-750 and h-100f. the device 206 s. goisser, m. fernandes, s. wittmann, c. ulrichs, h. mempel possesses a white standard within its protective plastic case, which was used for referencing at the start and after every 20 measurements. the technical features of all three devices have been elaborated in several previous works (kaur et al., 2017; li et al., 2018). depending on the respective device, two to four spectra were recorded for each fruit. for each device, all spectra belonging to one tomato fruit were averaged and the averaged spectra used for correlation with quality parameters and subsequent model building. measurement of fruit quality parameters measurements of external and internal quality parameters were performed immediately after recording of spectra and followed a defined operating procedure (fig. 2). at first, fruit color was measured around the fruit equator with a colorimeter (pce-csm 2, pce instruments, meschede, germany) consistent with the spots of nir spectra acquisition. color readings were automatically averaged by the colorimeter and the averaged values used for subsequent reference. firmness of each fruit was recorded in n as an average of two measuring points using a penetrometer (fruit texture analyser, guess manufacturing ltd., cape town, south africa). the applied probe head was 1 cm² in surface and used a measure ment speed of 5 mm/s and test depth of 7.5 mm. after acquisition of external quality parameters fruit were randomly selected for dry matter (dm) measurement. within the first part of the experiment, dry matter of 50 tomatoes was analyzed during the ripening period, consisting of two tomatoes of each of the five ripening stages on five measurement days. during analysis of stored fruit, five tomatoes were selected randomly for dm measurement on each of the six days of measurement, resulting in 30 tomatoes. in total, 80 samples were analyzed for dm within the first part. in the second part, ten tomatoes within the batch of 25 fruit of each of the eight varieties were drafted randomly for dm measurement, resulting in 80 samples in total. tomatoes were cut into four parts, put in small aluminum containers and placed in a dry oven for 48 h at 80 °c until constant dry weight was reached. afterwards the ratio of dry weight to initial fresh weight was used to calculate % dm. the remaining tomatoes within each part of the experiment were blended using a conventional smoothie mixer (wmf kult x mix&go 300 watt, wmf group gmbh, geislingen/steige, germany). the purée was filtered using filtering paper. 2-3 drops of stirred filtrate were used for measurement of total soluble solids (tss) using a digital refractometer (hi 96801, hanna instruments, woonsocket, usa). 10 ml stirred filtrate was used for the determination of titratable acidity with a titrator (hi 902 potentiometric titrator and hi 921 autosampler, hanna instruments, woonsocket, usa). results were expressed as g/l citric acid in fresh weight. tss concentration and amount of titratable acidity were used to calculate brix/acid ratio as stipulated by oecd guidelines (oecd, 2018). statistical and chemometrical analysis general data analysis such as calculation of average values and standard deviations (sd) was performed with microsoft® excel® 2016. the evaluation of a linear relationship between recorded spectra and tomato quality attributes was performed with the respective analysis software tools from the device manufacturer for the scio™ and f-750. in order to evaluate the performance of the manufacturer’s software and to carry out a neutral overall evaluation of food-scanner predictability of quality parameters, raw spectra of both devices was additionally analyzed with the multivariate data analysis software the unscrambler® software (version 10.5.1, camo, oslo, norway). in comparison, the h-100f is not provided with an end-user tool for creating prediction models, therefore spectra was only analyzed with the unscrambler® software. software such as the unscrambler® allow the computation of the correlation coefficient of both calibration fig. 1: commercially available portable nir food-scanner: (left) scio™ molecular sensor with protective plastic case, (middle) f-750 produce quality meter, (right) h-100f portable spectrometer with protective plastic cap. fig. 2: illustration of the measurement procedure and measuring points for nir spectra as well as quality parameters within this experiment. non-destructive quality assessment using food-scanners 207 (r ²c) and cross-validation model (r ²cv) as well as the corresponding calibration and cross-validation root mean square error (rmsec, rmsecv). these factors are used to evaluate the performance of the respective prediction model. additionally, the software offers numerous possibilities for data pre-processing and further analysis. outliers detected with the unscrambler®, which indicated errors during spectra collection, were omitted from evaluation. after recording of spectra with the scio™, the spectra are uploaded to a cloud-based browser application (the lab, consumer physics, hod hasharon, israel) which allows building of prediction models. the default pre-processing settings within the cloud-application were used in order to build prediction models, namely logarithm, averaging all four scans per sample, first derivative (window of 35 and polynom degree of 2), selecting the full wavelength range from 740-1070 nm and subtracting the average. every tenth spectra was used for crossvalidation. the algorithm was set to partial least square regression (plsr) and outlier detection as well as additional filters were disabled. raw spectra were then exported from the cloud-based samples library to an excel-file and analyzed with the unscrambler®, applying the same pre-processing settings as set in the cloud-application as well as plsr, using 20 random segments for cross-validation. the f-750 comes with a free software tool for analysis of spectra and building of prediction models. spectra recorded with the f-750 was loaded into the most recent model builder software (version 1.3.0.192 beta, felix instruments, portland, usa). the software uses nonlinear iterative partial least squares (nipals) regression and leaveone-out method for cross-validation. spectra in second derivative form, which is used by default by the software, was transferred from the model builder software to an excel-file and loaded into the unscrambler® for additional evaluation. the whole wavelength range from 477-1059 nm was utilized for building prediction models. both spectra per sample were averaged and no further pre-processing was applied. plsr algorithm was utilized for building of prediction models, using 20 random segments for cross-validation. for the h-100f, raw spectra in second derivative form was exported from the sunforest h-100 laboratory program in the wavelength range from 650-950 nm. after averaging the two scans per sample no further pre-processing was applied. 20 random segments were used for cross-validation. the scio™ application as well as the f-750 model builder software allow the selection of a specific spectral range for evaluation. within this study, the full wavelength range of all food-scanners was selected in order to evaluate the full potential of these devices. since both software programs of the f-750 and h-100f do not allow access to raw spectra, the automatically computed second derivative of spectra was used. gathering of information on usability characteristics a qualitative study regarding the application of food-scanners along the fresh produce supply chain identified several important requirements for food-scanners to allow the utilization in daily quali ty control processes, e.g., high prediction accuracy, convenience in handling and robustness of food-scanners (goisser et al., 2020a). based on these results as well as personal experience of the authors through collaborations with supply chain actors, characteristics of high importance with respect to the usability and applicability of food-scanners were identified within four categories, namely device independency, data handling, practical handling and available accessories. manufacturer’s specifications were used to evaluate device independency and available accessories, whereas personal assessments of the authors were used besides manufacturer’s specifications for the evaluation of data handling and practical handling of foodscanners. in order to determine a practice-oriented scan speed for each device, 30 consecutive measurements on various tomatoes were conducted with each food-scanner and measured using a stop watch. based on these measurements, arithmetic mean as well as standard deviation of scan speed for each device were calculated. results distribution of tomato fruit quality for both parts of the ex periment value ranges, arithmetic mean and standard deviation (sd) for all quality parameters measured in both parts of the experiment are shown in tab. 2. as indicated by the value range, variance within the first part of the experiment was greater for quality parameters l*, a*, c*, h°, firmness and brix/acid ratio. vice versa, quality attributes such as dm, tss and titratable acidity showed higher variability within the second part of the experiment. variance of color value b* differed only slightly between both parts of the experiment. food-scanner spectra vs. quality parameters (manufacturer’s software) the model builder software allocated by felix instruments for the f-750 provides the user with information on model linearity of the calibration set (r ²c) as well as the correlation coefficient after performing a leave-on-out cross-validation (r ²cv). root mean square errors for both calculations are also provided (rmsec, rmsecv). for the purpose of this study, these values were chosen as denominator of performance (tab. 3). the cloud application provided by consumer physics for the evaluation of scio™ spectra with respect to its correlation to reference values does not differentiate between calibration and validation models and therefore only displays a single r² and rmse value (tab. 3). evaluation of spectra collected with the f-750 in the first part of the experiment yielded models of high prediction accuracy (r ²cv > 0.90) tab. 2: distribution of reference values of quality parameters within each part of the experiment. l*, a*, b*, c*, h°: colorimetric values; dm: dry matter; tss: total soluble solids; sd: standard deviation parameter experiment n range mean sd part l* 1 245 29.13 51.36 36.03 5.40 2 120 32.33 41.88 36.12 2.11 a* 1 245 -7.97 31.69 17.08 13.19 2 120 11.55 28.46 22.29 2.87 b* 1 245 18.01 39.27 27.65 3.34 2 120 16.49 27.99 21.16 2.57 c* 1 245 21.49 49.26 34.74 6.11 2 120 23.66 39.59 30.85 2.79 h° 1 245 40.87 106.54 62.07 22.50 2 120 35.18 63.39 43.56 5.02 firmness (n) 1 245 6.14 90.75 30.53 19.26 2 120 12.67 42.24 24.43 7.21 dm (%) 1 80 5.50 7.58 6.09 0.38 2 80 4.02 9.69 6.72 1.74 tss (%) 1 165 4.50 7.30 5.54 0.59 2 120 3.1 9.00 5.78 1.84 acidity (g/l) 1 165 2.43 6.02 3.73 0.77 2 120 2.40 9.21 5.08 1.32 brix/acid ratio 1 165 8.63 27.21 15.60 3.98 2 120 7.38 18.22 11.42 2.39 208 s. goisser, m. fernandes, s. wittmann, c. ulrichs, h. mempel for color values l*, a*, h° as well as firmness. prediction models of moderate performance were obtained for chroma c* (r ²cv = 0.72) as well as tss (r ²cv = 0.56), whereas color value b* and brix/acid ratio yielded predictions of poor performance. no feasible calibration and cross-validation could be acquired for dm (r ²cv = 0.03) and titratable acidity (r ²cv = 0.02). within the second part of the experiment, prediction models computed for dm and tss showed high predictive capabilities (r ²cv > 0.90). models of moderate performance were acquired for color value l* (r ²cv = 0.52) and b* (r ²cv = 0.65), whereas predictions of color value a*, chroma c*, hue h°, firmness, titratable acidity and brix/acid ratio displayed poor accuracy (r ²cv < 0.50). prediction models of high accuracy (r ²p > 0.90) computed for the scio™ device in the first experiment part were obtained for color value a*, hue h° and firmness, while models for l*, chroma c* and tss were of good performance with r ²p of 0.89, 0.80 and 0.71, respectively. models of poor predictive capability were acquired for color value b* (r ²p = 0.42) and brix/acid ratio (r ²p = 0.49), whereas the prediction of dm and titratable acidity was not feasible. analysis of spectra gathered in the second part of the experiment resulted in prediction models of high accuracy (r ²p = 0.97) for dm and tss. moderate r ²p were obtained for color value b*, chroma c*, titratable acidity and brix/acid ratio, however the prediction models computed for color values l*, a*, hue h° as well as firmness showed low performance. food-scanner spectra vs. quality parameters (the unscrambler®) the evaluation of raw spectra of all three commercially available food-scanners using the unscrambler® software showed great differences in the accuracy of quantitative prediction of certain tomato quality attributes (tab. 4). within the first part of the experiment, tomato fruit quality was monitored from fruit ripening during cultivation up to postharvest storage. the evaluation of all three portable food-scanners within this part of the experiment resulted in cross-validation models of high accuracy (r ²cv > 0.90) for color values l* and a*, hue h° as well as firmness (fig. 3a). validated models of color value b* showed low to moderate accuracy for the f-750 (r ²cv = 0.29), scio™ (r ²cv = 0.55) and h-100f (r ²cv = 0.48) instrument. prediction models of chroma c* yielded good results for all three devices, ranging from r ²cv = 0.73 to 0.80 and 0.83 for f-750, h-100f and scio™ respectively. unlike the second part of the experiment, calibration models for dry matter indicated only small predictive capability for all three instruments, and subsequent cross-validation resulted in a significant drop in r ², which overall led to prediction models of poor performance. the validation of models for tss achieved predictions of moderate (f-750 and h-100f) to good (scio™) performance, whereas crossvalidation of prediction models for titratable acidity only yielded models of moderate accuracy for all three food-scanners. prediction models for the brix/acid ratio ranged from r ²cv = 0.69 over 0.70 to 0.74 for the f-750, h-100f and scio™, respectively, indicating moderate to good predictability. in the second part of the experiment, fruit quality of eight different tomato varieties was determined. the evaluation of prediction models computed with food-scanner scans and reference measurements yielded models of high accuracy (r ²cv > 0.90) for both dry matter and tss (fig. 3). additionally, all three sensors obtained cross-validated prediction models of moderate accuracy (r ²cv ranging from 0.54 to 0.75) for color values l* and b*, hue h°, firmness, acidity as well as the brix/acid ratio for all three devices. low to moderate predictability was achieved for color value a*, where r ²cv ranged from 0.48 over 0.49 to 0.54 for f-750, scio™ and h-100f, respectively. prediction of chroma c* was poor for the f-750 (r ²cv = tab. 3: prediction of tomato quality parameters of the f-750 produce quality meter and scio™ molecular sensor using the respective manufacturer’s software. l*, a*, b*, c*, h°: colorimetric values; tss: total soluble solids; r²c: r² of calibration; rmsec: root mean square error of calibration; r²cv: r² of cross-validation; rmsecv: root mean square error of cross validation; pc: principal components f-750 scio™ (λ = 477 1059 nm) (λ = 740 1070 nm) parameter experiment part n r²c rmsec r²cv rmsecv pc r²p sep pc l* 1 245 0.90 1.68 0.90 1.70 3 0.89 1.83 9 2 120 0.54 1.44 0.52 1.46 3 0.47 1.54 3 a* 1 245 0.95 2.83 0.95 2.88 4 0.92 3.81 12 2 120 0.21 2.54 0.19 2.58 3 0.37 2.28 3 b* 1 245 0.27 2.85 0.25 2.89 3 0.42 2.56 5 2 120 0.66 1.49 0.65 1.52 3 0.72 1.36 4 c* 1 245 0.74 3.15 0.72 3.24 4 0.80 2.76 7 2 120 0.41 2.14 0.39 2.17 2 0.62 1.73 4 h° 1 245 0.96 4.60 0.96 4.71 4 0.91 6.95 12 2 120 0.42 3.84 0.40 3.90 3 0.39 3.91 2 firmness (n) 1 245 0.92 5.40 0.92 5.53 4 0.91 5.84 10 2 120 0.49 5.16 0.47 5.25 3 0.46 5.29 6 dm (%) 1 80 0.08 0.59 0.03 0.61 1 0.07 0.37 1 2 80 0.94 0.41 0.93 0.47 6 0.97 0.31 8 tss (%) 1 165 0.56 0.39 0.56 0.39 2 0.71 0.32 7 2 120 0.93 0.47 0.92 0.51 6 0.97 0.30 8 acidity (g/l) 1 165 0.03 2.00 0.02 2.01 2 0.00 2.00 1 2 120 0.51 0.92 0.49 0.94 3 0.66 0.77 6 brix/acid ratio 1 165 0.43 3.09 0.42 3.13 2 0.49 2.92 4 2 120 0.46 1.76 0.46 1.77 2 0.51 1.68 3 non-destructive quality assessment using food-scanners 209 0.42) as well as the h-100f (r ²cv = 0.49), whereas the evaluation of the scio™ device yielded moderate results (r ²cv = 0.65). differences in usability characteristics important features with respect to device independency, data handl ing, practical handling and available accessories of food-scanners derived from manufacturer’s specifications and personal assessment of the authors are compiled in tab. 5. with respect to the independent use of these devices without addi tional equipment, the necessity of internet access and additional devices during scanning as well as the need for external whitereferencing of these spectrometers was evaluated. the investigation showed that the f-750 and h-100f can be used as stand-alone and offline instruments that require no additional devices for scanning or displaying of results. both instruments are equipped with on-board prediction models for evaluation of nir spectra and displays for the illustration of the respective measurement results. in contrast, the scio™ requires constant internet access during scanning as well as an additional mobile device. for the scio™, an additional device (e.g., smartphone, tablet) is mandatory, works as an intermediate link between the miniaturized nir spectrometer and the cloud database, and serves as display for measurement results. the scio™ as well as the h-100f provide protective plastic caps, which on the one hand serve as protection during transportation and on the other hand contain the respective white standard for regular referencing of the spectrometer. contrary to this, the f-750 uses a shutter with gold-coated foil as its reference standard, which is calculated for every sample measurement, therefore an external white reference is not needed. as a second aspect, the handling of the generated data was examined. spectra collected with the f-750 can be accessed either by removing the sd card on which spectra is stored and transferring the data to a computer or using the wi-fi function of the sd card. although a special file format is used for the spectra, the transfer to other multivariate analysis software is easily possible due to the free software and open format. scio™ spectra is only accessible through an online database by using a web browser or the cor responding scio™ mobile app. furthermore, the scio™ requires a separate development license for the access of raw spectra, which can be downloaded in a simple file format. spectral data stored on the h-100f can either be transferred via usb to a computer or accessed via bluetooth using a smartphone. spectra provided by the h-100f can either be accessed with special software for multivariate data analysis, e.g., the unscrambler®, or by using data analysis software such as r or python combined with syntax analysis of the respective file format. the ability of building own prediction models and the knowledge necessary for doing so differs significantly between the three devices. the manufacturer of the f-750 provides a free software tool for analysis of spectra and building of own prediction models and supports users with a systematic instruction on how to build custom tailored models. by following these instructions step by step, unexperienced users are able to build own prediction models without specialized knowledge in nir spectroscopy. as for the scio™, the manufacturer provides a cloud-based browser application that allows the building and evaluation of prediction models. however, the utilization of these self-developed prediction models for future predictions requires the programming of separate mobile applications. in contrast to this, building of own prediction models by end-users is not possible with the h-100f. additional differences were identified with respect to features in practical handling of the three food-scanners. regular handling of tab. 4: prediction of tomato quality parameters of three commercially available nir devices using the unscrambler® software for evaluation. l*, a*, b*, c*, h°: colorimetric values; tss: total soluble solids; r²c: r² of calibration; rmsec: root mean square error of calibration; r²cv: r² of cross-validation; rmsecv: root mean square error of cross validation; pc: principal components f-750 scio™ h-100f (λ = 477 1059 nm) (λ = 740 1070 nm) (λ = 650 950 nm) parameter experiment n r²c rmsec r²cv rmsecv pc r²c rmsec r²cv rmsecv pc r²c rmsec r²cv rmsecv pc part l* 1 245 0.90 1.70 0.90 1.74 1 0.91 1.61 0.90 1.74 9 0.93 1.39 0.93 1.44 3 2 120 0.71 1.14 0.62 1.32 6 0.62 1.31 0.57 1.40 4 0.75 1.05 0.70 1.16 6 a* 1 245 0.97 2.44 0.96 2.53 4 0.93 3.41 0.92 3.71 9 0.96 2.65 0.96 2.74 3 2 120 0.61 1.78 0.48 2.08 6 0.55 1.91 0.49 2.05 5 0.63 1.74 0.54 1.95 7 b* 1 245 0.31 2.78 0.29 2.83 3 0.60 2.12 0.55 2.26 8 0.53 2.30 0.48 2.43 5 2 120 0.68 1.45 0.66 1.51 3 0.76 1.25 0.73 1.35 4 0.78 1.22 0.72 1.38 7 c* 1 245 0.75 3.04 0.73 3.20 4 0.85 2.36 0.83 2.54 8 0.82 2.58 0.80 2.71 5 2 120 0.43 2.10 0.42 2.14 1 0.69 1.56 0.65 1.67 5 0.60 1.78 0.49 2.00 7 h° 1 245 0.96 4.52 0.96 4.70 3 0.93 6.04 0.92 6.50 9 0.95 4.86 0.95 4.95 2 2 120 0.72 2.66 0.64 3.03 6 0.61 3.15 0.54 3.45 5 0.78 2.37 0.73 2.64 6 firmness (n) 1 245 0.93 5.17 0.93 5.31 3 0.90 6.07 0.89 6.55 9 0.92 5.35 0.92 5.46 2 2 120 0.73 3.77 0.64 4.37 6 0.66 4.19 0.54 4.95 11 0.72 3.80 0.67 4.18 6 dm (%) 1 80 0.47 1.58 0.18 1.99 7 0.54 0.28 0.32 0.33 6 0.33 0.32 0.16 0.36 5 2 80 0.96 0.33 0.94 0.43 7 0.98 0.25 0.97 0.32 8 0.96 0.35 0.94 0.42 7 tss (%) 1 165 0.80 0.26 0.69 0.32 8 0.85 0.23 0.81 0.26 10 0.77 0.28 0.67 0.34 11 2 120 0.94 0.46 0.92 0.51 5 0.97 0.32 0.96 0.35 6 0.97 0.32 0.96 0.38 7 acidity (g/l) 1 165 0.60 0.49 0.51 0.54 6 0.63 0.46 0.57 0.51 8 0.68 0.43 0.63 0.47 5 2 120 0.74 0.67 0.64 0.79 6 0.71 0.71 0.66 0.78 6 0.82 0.57 0.75 0.67 8 brix/acid ratio 1 165 0.74 2.11 0.69 2.30 6 0.75 2.06 0.70 2.25 7 0.77 1.96 0.74 2.10 6 2 120 0.62 1.47 0.54 1.64 5 0.77 1.15 0.65 1.42 12 0.71 1.29 0.64 1.45 7 210 s. goisser, m. fernandes, s. wittmann, c. ulrichs, h. mempel fruit and vegetables leads to light soiling of food-scanners, e.g., through adhering water and dirt or particles of rough and hairy fruit skins. therefore, lens and light source of the scio™ and h-100f device have to be cleaned regularly to guarantee undistorted measurements. due to the design of the f-750, the lens protects the light source, therefore only the lens requires regular cleaning. according to manufacturer’s specifications, the f-750 battery lasts over 1600 scans and the h-100f battery over 2000 scans. results of our own measurements estimate the battery life of the scio™ at 250 scans. own measurements identified differences in scan speed, ranging from 4.9 s (h-100f), 8.2 s (scio™) to 14.3 s (f-750). it should be noted that the whole time interval from the beginning of one to the beginning of the next possible scan was recorded instead of just measuring the time until the measured value was displayed. due to the built-in technology and the focus on different end-user groups, there are also differences in the weight and dimensions of the three foodscanners. the f-750 is robustly designed for the utilization within the orchard through farmers and producers, whereas the scio™’s small and lightweight design was initially targeted for end-consumer use. the h-100f has similar dimensions to the f-750, but since plastic is used for the casing instead of metal, it weighs significantly less. all three devices provide accessories for the measurement of smaller objects and fruit. in addition, the h-100f features a built-in radiofrequency identification (rfid) reader for the identification of individual tagged trees or areas, whereas the f-750 has a gps sensor for online mapping of non-destructive quality readings. manufacturers offer supplementary accessories for additional areas of application such as liquids (f-750, scio™) and powders (f-750). discussion prediction results of food-scanner spectra by using two different experimental designs (tomatoes during ripening and different tomato cultivars from the supermarket) we were able to generate great variance for almost every quality parameter examined. color values l*, a*, c*, h° and firmness showed great variation in reference values in the first part of the experiment due to ripening-related changes during cultivation and storage. the combination of different tomato cultivars in the second part of the experiment resulted in a wide range of reference values for dm and tss. the evaluation of nir prediction models using food-scanners yielded a higher coefficient of determination (r²) for the respective part of the experiment with a wide range in fruit quality of the samples used in calibration and validation sets (tab. 2). these findings are in line with su et al. (2014) and highlight the importance of the variability of samples for building a feasible nir prediction model of the quality trait of interest. this wide range of reference values also makes prediction models more robust, since both bad and good qua lities are integrated. thus a realistic range of values is represented. when creating future prediction models during practical application of food-scanners along the fresh fruit supply chain, attention should be paid to a large variance of the desired quality trait of interest. in order to guarantee this variance, users responsible for building prediction models must have extensive knowledge of fruit quality. for the following comparison of our results to scientific literature, results computed with the independent software the unscrambler® are used as reference. the models with the best accuracy due to the wide range of reference values are used for comparison (tab. 3). utilizing nir spectra for the determination of tomato firmness during cultivation and storage resulted in good prediction models (r²cv > 0.89) for all three food-scanners. these results are superior to findings achieved on intact tomatoes using portable and laboratory nir instruments reaching from r²cv = 0.78 (ecarnot et al., 2013) to r²cv = 0.82 (he et al., 2005). similar results for firmness prediction (r ² = 0.89) on intact tomatoes were achieved by kusumiyati et al. (2008) using portable nir spectroscopy. tomato dry matter content varies greatly depending on the tomato cultivar (ercolano et al., 2008). screening of one tomato cultivar from cultivation to storage in our study showed a smaller range of dry matter distribution (2.08%) compared to screening multiple tomato cultivars (5.67%). prediction of dry matter with only one tomato cultivar yielded no feasible models (r²cv = 0.16 0.32). these findings are in line with poor prediction results (r²cv = 0.39 0.49) for one tomato cultivar using a laboratory nir instrument (torres et al., 2015). contrary to this, nir prediction models of high accuracy (r²cv > 0.90) could be created for the model containing multiple cultivars with all three food-scanners. prediction of dry matter in apple, kiwifruit and stone fruit applying the same food-scanners we used showed similar predictive capabilities of this fruit trait (kaur et al., 2017). models containing only one cultivar yielded moderate predictions of tss (r²cv = 0.67 0.81), which is in range of previous studies using only one tomato cultivar and laboratory nir instruments (flores et al., 2009; torres et al., 2015; clément et al., 2008). building of tss prediction models using multiple tomato cultivars in our study yielded accurate results (r²cv > 0.90). previous studies using labofig. 3: correlation between measured and predicted firmness (a), tss (b), and dm (c) of cross validated models built with the unscrambler® of all three food-scanners used in this study. the black line symbolizes the line of best fit. 3 4 5 6 7 8 9 10 11 3 4 5 6 7 8 9 10 11 d m n ir p re di ct io n (% ) dm reference measurement (%) c 2 3 4 5 6 7 8 9 2 3 4 5 6 7 8 9 t s s n ir p re di ct io n (% ) tss reference measurement (%) b 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100 f irm ne ss n ir p re di ct io n (n ) firmness reference measurement (n) a f-750 h-100f scio™ non-destructive quality assessment using food-scanners 211 ratory nir instruments (he et al., 2005; saad et al., 2016) and a miniaturized nir sensor (sheng et al., 2019) obtained similar prediction models. in the first part of the experiment all three food-scanners yielded prediction models of high accuracy for lightness l* and color value a* (r²cv > 0.90, tab. 3), which is in the range of findings from previous work using benchtop uv-vis-nir spectrometer and port able nir instrument (clément et al., 2008; kusumiyati et al., 2008). prediction of color value b* yielded best results in the second part of the experiment for all three food-scanners (r²cv = 0.66 0.73). however, previous studies demonstrated better correlations of r² = 0.82 0.92 (kusumiyati et al., 2008; clément et al., 2008). correlation of food-scanner spectra to chroma c* in our study obtained moderate to good results (r²cv = 0.73 0.83) in the first part of the experiment. various previous research using portable nir spectrometer yielded slightly better prediction models of r² = 0.90 0.87 (ecarnot et al., 2013; kusumiyati et al., 2008). evaluation of hue h° yielded prediction models of high accuracy (r²cv > 0.90), which is similar to findings in the literature on portable nir instrument performance (ecarnot et al., 2013; kusumiyati et al., 2008). previous studies applying laboratory nir instruments demonstrated the difficulty of predicting acidity in intact tomato fruit (flores et al., 2009; oliveira et al., 2014) and yielded prediction accuracies comparable to our results. as elaborated by oliveira et al. (2014), tomatoes generally show low concentration and a heterogeneous composition of tss and acidity. compared to other fruit species, tomatoes have no homogeneous composition of flesh since they are divided into different loculi. this structure can cause interference when nir radiation is penetrating fruit and could be the reason for these moderate correlations in both parts of the experiment (r²cv = 0.51 0.75). this moderate predictability of acidity combined with the mathe matical relation yielded prediction models for brix/acid ratio of likewise moderate accuracy (r²cv = 0.54 0.74) in both experiments. no literature reports could be found regarding prediction models for brix/acid ratio in intact tomatoes. however, previous research on tomato juice using a laboratory nir instrument obtained slightly superior accuracies (r² = 0.74 0.86) for comparable wavelength regions (jha and matsuoka, 2004). all three food-scanners had similar accuracies for the prediction of each quality trait within each part of the experiment (tab. 3). the slight differences in model accuracy can be explained by the differences in wavelength ranges of the respective scanners. kaur et al. (2017) evaluated the spectral features of all three food-scanners used in this study and found strong correspondence to the water absorbance bands at 760 nm, 840 nm and 960 nm, with slight variations between instruments. as demonstrated by mcglone and kawano (1998) using kiwifruit, the water and carbohydrate absorbance bands at 840 nm and 960 nm strongly correlate to prediction models de veloped for tss and dm. the good correlation of food-scanner spectra and tomato tss and dry matter in our study are in line with these findings. with respect to the determination of color values, the f-750 covers almost the whole visible spectrum up to near infrared (477 -1059 nm), whereas the h-100f comprises the red wavelength range of the visible spectrum (650 950 nm). contrary to this, the scio™ covers a small range of the far-red end of the visible spectrum. performance of manufacturer’s software compared to the unscrambler® various previous studies tried to determine the performance of the three food-scanners used in this study for the prediction of internal quality traits of selected fruit and vegetables. however, most studies only used the portable nir spectrometers for collection of spectra and utilized the corresponding software tools provided by manufacturers for data extraction. in many scientific studies, the subsequent multivariate data analysis was performed by using specialized software for multivariate data analysis such as matlab (kaur et al., 2017; teye et al., 2019) or the unscrambler® (ncama et al., 2018). to the best of our knowledge, a direct comparison of these provided software tools to specialized software has only been done by li et al. tab. 5: compilation of food-scanner characteristics with respect to usability and applicability features device f-750 scio™ h-100f device independency necessity of internet access during scanning no yes no additional devices needed for scanning none smartphone / tablet none internal / external white-referencing internal external external data handling access of spectra free, sd-card licenced, online database free, usb, bluetooth ability for building own prediction models free software available programming of own apps necessary no specialized knowledge necessary for model building no yes yes practical handling instrument maintenance lens cleaning lens and led cleaning lens and light source cleaning running costs none none none durability of battery life > 1600 scansa > 250 scansb ~ 2000 scansa scan speed (mean and standard deviation) 14.3 ± 0.1 sb 8.2 ± 0.7 sb 4.9 ± 0.1 sb device weight 1.05 kg 36 g 440 g device dimension 18 × 13.5 × 6 cm 5.5 × 3.7 × 1.4 cm 19 × 17 × 10.7 cm available accessories provided accessories small fruit adaptor, gps small object holder rubber mold for small fruit, built-in rfid reader purchasable accessories liquids and powders kit liquid accessory n/s aaccording to manufacturer’s specifications b results of own measurements 212 s. goisser, m. fernandes, s. wittmann, c. ulrichs, h. mempel highlighted in previous research (fan et al., 2019; wedding et al., 2013), additional integration of biological and seasonal variability of fruit can contribute to build more reliable and robust prediction models and thereby unlock potential of non-destructive quality assessment via nir food-scanners in practical applications. comparison of usability characteristics a direct comparison of the three food-scanners used in this study shows clear differences with regard to usability and applicability characteristics (tab. 5). both f-750 and h-100f are not reliant on internet access during scanning and can therefore be used independently at all points of the fresh produce supply chain, from orchards to incoming goods control in warehouses to retail stores. in contrast, the scio™ requires constant internet access during scanning as well as an additional mobile device (e.g., smartphone, tablet). this finding is contrary to statements on the scio™ website, where offline scanning is advertised (consumer physics, 2020). however, personal experience as well as communication with scio™’s manufacturer revealed that offline scanning is not possible (consumer physics, 2017). therefore, the scio™ can not be used as an independent measuring device. additionally, the device can only be used effectively in places with a stable internet connection, which is not always guaranteed in large warehouses or remote orchards. data handling of the f-750 can be described as very user-friendly. on the one hand, spectra are provided freely, on the other hand the gratis software and instructions for model building allows non-experts of nir spectroscopy the building of new prediction models. with respect to the practical use of food-scanners for fresh produce quality control, new quality parameters could be developed by users along the supply chain themselves. in contrast, users of the scio™ device need to purchase an additional license to access raw spectra for building of own prediction models. to enable the use of these models for future predictions, further know-how in app programm ing is required. for the h-100f, spectra are also freely accessible, but the manufacturer does not provide software for building of own prediction models. according to the manufacturer, a short form of model building program was provided in the past, but deemed not useful. in order to analyze the spectra and respective reference data and produce calibration models in a suitable data format with the h-100f, model building programs such as the unscrambler® can be used (sunforest, 2018). however, these specialized programs are not suitable for many users in day-to-day practice, since on the one hand they are very expensive and on the other hand they require a lot of training. the evaluation of the practical handling of all three food-scanners showed comparable effort in device maintenance. additionally, none of these devices requires further running costs. both f-750 and h-100f are characterized by very long battery runtimes and can therefore be used for several days in practical applications. the battery of the scio™ lasts for approximately 250 measurements. when used continuously, e.g., during quality assessment in the orchard or incoming goods control, this number can be reached within a short period of time. theoretically, the battery life of the scio™ can be extended by using a mobile powerbank and charging the device parallel to scanning. however, an additional device and thus another dependency would be necessary. an important aspect in practical handling is the scan each device requires for one measurement, since it serves as indication for labor efficiency. previous work identified rapid measurements as important feature of portable food-scanners (goisser et al., 2020a). our measurement results for scan speed (tab. 5) surpass those reported in previous studies (kaur et al., 2017) as well as manufacturer’s specifications (sunforest, 2020; consumer physics, 2020). it should be noted that in contrast to these (2018) for the scio™ device with three kiwi quality parameters during a quality estimation trial. the authors obtained similar predictive performance for dry matter, tss and firmness using scio™ lab as well as the unscrambler®. however, such a comparison has not yet been conducted for the f-750, which also provides its own software for evaluation and building of prediction models. in order to evaluate the potential application of these portable food-scanners in practice along the fresh fruit supply chain, this study used manufacturer’s software as well as the unscrambler® software for building of prediction models for a variety of tomato fruit quality traits. by comparing the results derived from both software tools, the accuracy and reliability of prediction models computed with manufacturer’s software can be assessed. a direct comparison of the prediction models created with the un scrambler® (tab. 3) to models developed using manufacturer’s software (tab. 4) indicates similar predictive performances for models of high accuracy (r²cv > 0.90) for both devices. the small variations in model performance was most likely due to slightly deviating numbers of principal components (pc), which are selected automatically and individually by each respective software. additionally, the variability in sampling during cross-validation could cause these minor dif ferences. some prediction models of poor to moderate accuracy (0.40 < r²cv < 0.80) achieved similar results for both the unscrambler® and manufacturer’s software (e.g., color values b* and c* for the f-750 as well as color value c* for scio™ within both parts of the experiment). however, most prediction models within this area of accuracy computed with the unscrambler® surpass those derived from f-750 or scio™ manufacturer’s evaluation software. whereas the evaluation of dm and acidity with the unscrambler® within the first part of the experiment indicated merely moderate predictive capabilities, neither f-750 or scio™ software obtained any form of linear correlation between food-scanner spectra and reference values. it should be noted that the evaluation of spectra using manufacturer’s software yielded similar results to evaluations applying the unscrambler® software for prediction models of high accuracy (r²cv > 0.90). in particular, these models of high accuracy are relevant when it comes to the practical application of food-scanners. this study therefore was able to demonstrate that existing food-scanners, including the software supplied, are suitable for creating feasible prediction models for fruit quality traits. therefore, actors along the fresh fruit supply chain can use these devices independently and are not reliant on additional professional software for data evaluation. these results are in line with findings of li et al. (2018), who found similar model accuracies for kiwi quality attributes using both the unscrambler® and scio™ software. the results of this study demon strate the great predictive potential and informative value of commercially available portable food-scanners. by combining multiple prediction models of high accuracy of various quality traits within one global prediction model, portable food-scanners can be used as multidimensional and non-destructive measurement tools of fruit quality. therefore, future users can gain a comprehensive picture of fruit quality using only one single scan. this study used two different experimental approaches in order to generate high variability of tomato fruit quality parameters. the utilization of fruit during ripening and storage resulted in a larger range of reference values l*, a*, b*, c*, h° and firmness compared to fruit from retail stores. vice versa, differences in variety and origin within the second experiment increased variability of reference values dm, tss and acidity. with the exception of color value b*, nir prediction models yielded higher correlation for the experiment with a larger range of reference values. when it comes to the de velopment of new prediction models by users in daily practice, a broad range of fruit qualities and respective reference values must be taken into account in order to obtain good prediction models. as non-destructive quality assessment using food-scanners 213 references, we recorded the whole time interval from the start of one scan to the beginning of the next possible scan. for the scio™, qua lity and stability of the internet connection is essential with regard to scan speed. both f-750 and h-100f display the scan result a few seconds before the next scan is possible, which most likely explains these deviations from manufacturer’s specifications. however, the scan speed measured in our study is much more practice-oriented. assuming a fixed timeframe for the application of food-scanners in quality control processes, different numbers of scans can be performed with each device. depending on the device, different numbers of measuring points would be available for quality assessment. due to the built-in technology, weight and dimension differs notably between devices. these features have to be considered when the devices are used portable in the orchard or warehouse and have to be physically carried around by the users. all manufacturers provide accessories that enable the measurement of fruit and vegetables of various sizes. therefore, users along the fresh produce supply chain are not limited in their use of these de vices due to fruit size. however, additional fruit properties such as the color and thickness of fruit skin have to be considered when applying nir spectroscopy, since these properties could greatly influence the accuracy and reliability of nir prediction models. the f-750 provides a gps function, which enables the measurement values to be displayed in a map view. by using rfid tags in orchards in combination with the built-in rfid reader, the h-100f takes a similar approach. on the one hand, fruit ripeness can be monitored in orchards, on the other hand, this function can also be used to verify the origin of produce as well as quality of origin by measurements at production companies. additional accessories for the measurement of liquids can be purchased for the scio™, the f-750 provides a purchasable liquids and powders kit. users are therefore able to use these portable nir instruments for testing various substances within the food industry (e.g., fruit juices, beverages, dried herbs) or completely different areas of application in other industries. conclusion the predictability of various tomato quality parameters using three commercially available portable food-scanners was analyzed and the software for building of own prediction models provided by manufacturers of these devices evaluated through comparison to state-ofthe-art software for multivariate analysis. the results highlight the capability of these devices in predicting various internal fruit quality parameters in a non-destructive way. our findings are consistent with previous research, which in many cases used laboratory nir instruments compared to portable and miniaturized nir devices. quality control of fruit and vegetable along the fresh produce supply chain is often limited to optical inspections and in some cases not performed at all. through the combination of a multitude of internal quality measurements within a single scan, food-scanners provide a comprehensive and objective picture of fruit quality. food-scanners can be used on various points along the fresh produce supply chain and replace traditional time-consuming and elaborate destructive measurement methods of internal fruit quality. therefore, foodscanners can be an important tool in making fruit quality along the whole fresh produce supply chain more transparent and comprehensible. the comparison of software provided by manufacturer to state-ofthe-art software shows that the provided software delivers good results, especially for models of high prediction accuracy. applied to the practical application of these devices in day-to-day processes of quality control this means that special expertise in the field of nir spectroscopy and model building is not necessary. since some manufacturers provide basic software solutions in combination with operating instructions, users are enabled to build their own prediction models. as highlighted in this study, variation in fruit quality is of great importance in order to build accurate prediction models. the variation of the respective fruit quality parameter can be influenced in various ways, e.g., through different ripening stages, variation in origin and cultivars or seasonal differences. with regard to the practical use and long term application of food-scanners along the fresh produce supply chain, it is vital that users of these devices are aware of the importance of the variability of reference values when it comes to creating new, reliable and robust prediction models. based on previous studies and personal experience with supply chain actors, an exemplary framework evaluating food-scanner characteristics with respect to usability and applicability was designed. the evaluation of these usability characteristics showed various differences between the three devices used in this study, especially with regard to the independent use of these devices and handling of gene rated data. potential users of food-scanners must inform themselves in advance and make sure that the respective device meets their company’s requirements, especially in day-to-day work processes of fruit quality control. for a further spread and effective use of these de vices in practice, manufacturers must pay attention to user-friendliness and simple model building solutions. acknowledgements the research was financed by the bavarian ministry of food, agri culture and forestry as part of the alliance “wir retten lebensmittel” [we save foodstuffs] as well as the qs science funds in fruit, vegetables and potatoes. the authors address special thanks to step systems gmbh from nuremberg, germany for their loan of two food-scanner devices during the course of this experiment. conflict of interest no potential conflict of interest was reported by the authors. references abbott, j.a., 1999: quality measurement of fruits and vegetables. post harvest biol. tech. 15, 207-225. doi: 10.1016/s0925-5214(98)00086-6 batu, a., 2004: determination of acceptable firmness and colour values of tomatoes. j. food eng. 61, 471-475. doi: 10.1016/s0260-8774(03)00141-9 ble, 2019: obst und gemüse von a bis z. vermarktungsnormen frisches obst und gemüse. retrieved from https://www.ble.de/de/themen/ er naeh r ung-l ebensm it tel / ver ma rk t ungsnor men /obstgemuese/ vermarktungsnormen-hilfen-zur-anwendung/obstundgemuesea_z. html?nn=8904900. 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http://dx.doi.org/10.1016/j.postharvbio.2015.04.004 art_15772.indd journal of applied botany and food quality 94, 124 131 (2021), doi:10.5073/jabfq.2021.094.015 1division of food and nutrition sciences, ohio university, usa 2 fox paw farm, llc., adams county, ohio, usa a comparative analysis of p awpaw (asimina triloba) quality and nutritional data robert g. brannan1*, emily e. anderson1, ronald l. powell2, maria n. coyle1 (submitted: february 14, 2021; accepted: june 1, 2021) * corresponding author summary the north american pawpaw (asimina triloba [l.] dunal) from sixteen varieties was analyzed for size, ph, obrix, fi rmness, and pulp and skin color. a varietal composite was used to determine nutrient composition. data from the current study was compared to previous literature values, and all results fi t well with previous literature. the average weight of the fruits across all varieties was 194 g and ranged from 122 to 292 g, the ph of the fruits ranged from 5.4 to 6.3, the average obrix ranged from 18.2% to 26.1%, and fi rmness ranged from 0.391 kg to 0.727 kg. the color of the skin and the pulp differed by variety but was well-within previously reported values. pawpaw pulp nutritional values verifi ed the nutritional values previously reported for pawpaw pulp from fruit harvested in korea and will be used as the basis for inclusion of pawpaw in the usda national nutrient database for standard reference. several specifi c recommendations are made: 1) fi rmness of 0.2 to 0.7 kg of force can be used to describe ripe pawpaw fruit; 2) the serving size for raw pawpaw pulp is onehalf cup (120 g); and 3) pawpaw pulp nutrition compares favorably to that of banana or mango. introduction the north american pawpaw (asimina triloba [l.] dunal), hereafter pawpaw, is the largest edible fruit native to the united states (layne, 1996). the pawpaw is in the annonaceae family, of which 14 of the 2300 species are native to the united states (cronquist, 1981), including nine species of asimina (callaway, 1993). the pawpaw grows on fruit-bearing trees about 5 to 10 meters tall and often found in clusters (pomper et al., 2003; pomper and layne, 2005). the leaves are distinctive compared to leaves of other asmina species because they are long, egg-shaped, and membranous in texture (kral, 1960; layne, 1996). the buds are dark maroon in color (pomper and layne, 2005) and the fl owers are unable to selfpollinate, requiring hand pollination using a genetically different tree (bois, 2001; layne, 1996; willson and schemske, 1980) or pollinators such as fl ies, beetles, and other nocturnal insects, which are unreliable for proper pollination (layne, 1996; pomper et al., 2008; willson and schemske, 1980). these pawpaw structures are shown in fig. 1. more detailed information about the botanical characteristics of the pawpaw can be found in the literature (pomper and layne, 2005). the fruit may grow alone or in clusters and are botanically categorized as a pulpy berry, probably an aggregate berry (jones and layne, 1997; pomper and layne, 2005). the fruit’s edible fl esh can range in color from creamy white to bright yellow to orange, has a custard-like texture, and has 12-20 bean-shaped seeds embedded in the pulp in two rows (layne, 1996; peterson, 2003; pomper and layne, 2005). the pawpaw is a climacteric fruit and ethylene and respiratory peaks are seen within three days after the fruit is harvested (archbold and pomper, 2003), causing the fruit to become undesirably soft and brown within 5 days of harvest at ambient temperature (galli et al., 2008). refrigeration at 4 °c can delay the softening of the fruit for several weeks, but does not prevent skin browning (galli et al., 2008; pomper and layne, 2005). food quality research on the pawpaw fruit pulp appearance in the literature has been gradual, with our lab and others reporting on a variety of quality characteristics. pulp browning and the activity of polyphenol oxidase, the enzyme responsible for pulp browning has been characterized, (brannan, 2016; zhang et al., 2017), as has the identifi cation of cell wall components and phytochemicals (brannan et al., 2019, 2015). the utilization of pulp and seed components, especially phytochemicals and antioxidants, as potentially valueadded food ingredients has been reported (brannan et al., 2018; brannan and salabak, 2009; harris and brannan, 2009; kobayashi et al., 2008; nam et al., 2019, 2017). sensory quality of the pulp (brannan et al., 2012; mcgrath and karahadian, 1994) and the application of processing techniques to extend shelf life (brannan et al., 2019; brannan and wang, 2017; zhang et al., 2017) also has been reported. the totality of research about the nutrient composition of pawpaw pulp includes two studies, one that reported nutritional information for whole pawpaws including the pawpaw skin, which is rarely consumed and often considered inedible (peterson et al., 1982), and a more recent study about pawpaw fruit grown in korea (nam et al., 2018). many of these food quality studies sought to differentiate quality parameters by pawpaw variety (brannan, 2016; brannan et al., 2015; greenawalt et al., 2019) or pulp ripeness (harris and brannan, 2009; kobayashi et al., 2008; nam et al., 2019). the objective of the present study was to investigate the quality, nutritional, and compositional characteristics of pawpaw pulp from different varieties and compare and contextualize the results to similar characteristics reported previously. fig. 1: artists rendition of the parts of the pawpaw (asimina triloba) plant. a) mature fruit on the tree; b) mature fruit cut lengthwise to expose the seeds; c) branch with bud; d) blossom. (artwork © by author m.n. coyle) comparative analysis of pawpaw nutrition 125 materials and methods pawpaw collection and processing pawpaw fruit were collected from fox paw farm, l.l.c. in adams county, ohio (38°39’n, 83°41’w, 273 m above sea level) in september 2019. located in usda hardiness zone 6, the climate for adams county, ohio shows average rainfall of 1133 mm, snowfall of 445 mm, 126 days of precipitation, average january temperature of -6.6 °c, and average july temperature of 29.6 °c. the farm is a rural 3.5-acre plot that contains pawpaw trees planted from 20032006 in three blocks. at the time of data collection, the north block contained trees in east-to-west rows with 2.5 m between trees. the east block contained rows running north-south with trees 2.5 m apart. the west block contained nine rows running north-south with 3 m between trees. in all three blocks, rows were planted 4.5 m apart. sixteen varieties with at least four fruits from each variety were harvested for this study. the varieties were selected at the time of harvest to be sure all fruits were of similar ripeness and were harvested by hand to ensure there was no damage or bruising to the fruits being used in the analysis. the sixteen varieties were 'estill', 'green river belle', 'ixl', 'ksu atwood™', 'lynn’s favorite', 'mango', 'mitchell', 'nc-1', 'overleese', 'pickle', 'potomac™', 'quakers delight', 'saa-zimmerman', 'sab overleese', 'shenandoah™', and 'wabash™'. the harvested fruits were then transferred to the food innovation laboratory at ohio university on ice. fruit weight, size, skin and pulp color, fi rmness of the fruit, and ph of the pulp were recorded for each individual fruit before processing. the skin color (l*, a*, b*) was measured in three places on each fruit using a using a konica bc-10 colorimeter (konica minolta sensing americas inc., ramsey, nj), after which the hue angle was calculated according to previous work (brannan et al., 2015). a ~25 mm circle of skin was removed in three spots on the exterior of the fruit to assess pulp color and fruit fi rmness on the exposed pulp. the fi rmness of the exposed pulp, reported as kg of force, was measured by penetrometry on the fruit situated on a solid platform with a 10-mm diameter cylindrical probe with a crosshead speed of 5 mm/s to a depth of 10 mm using a ta-xt2i texture analyzer (texture technologies corp., scarsdale ny/stable micro systems, godalming, surrey, uk). varietal composites for nutritional analysis were prepared according to the following scheme. one whole fruit from each of the sixteen varieties was randomly assigned into one of four groups. pulp from each variety was not pooled before being assigned to a group, rather each individual whole fruit assigned to a group was processed by removing the skin and seeds from the pulp by hand, after which an equal weight of that pulp was used to create a composite from the one fruit of each variety in each group. thus, the four composites were compositionally identical except that the source of the pulp from each variety was an individual fruit, not a composite of fruits from each variety. after each composite was mixed thoroughly, pulp from the composites was distributed into foodsaver® bags, which were sealed without any attempt to exclude oxygen from the bags and held at -20 °c. a total of nine of the composite samples, two duplicates from three of the composites and three triplicates from the one remaining composite, were assigned three-digit random codes before being transported on dry ice to the laboratories for analysis. nutrient analysis nutrient analysis was performed by q laboratories (cincinnati, ohio) except for total dietary fi ber and vitamin d, which were performed by medallion labs (minneapolis, minnesota). analysis for each analyte was performed in triplicate from each of the nine randomly-coded bags of pawpaw pulp (described above), according to standard aoac international methods (aoac international, 2016) except for metals (ca, fe, k, na), which were quantifi ed using inductively coupled plasma optical emission spectroscopy (icpoes) after acid digestion of the pulp. the aoac methods used for each nutrient are as follows: ash (aoac 923.03), cholesterol (aoac 994.10), lipid (aoac 922.06), moisture (aoac 934.06), fatty acids (monounsaturated, polyunsaturated, saturated and trans) (aoac 996.06), total dietary fi ber (aoac 991.43), vitamin d (aoac 2011.11). data analysis means and standard deviations were generated for the response variables. inferential statistics were generated to compare varieties using analysis of variance and means separation was achieved using duncan’s post-hoc analysis using ibm spss statistics software. signifi cance was determined at p<0.05. results and discussion of the sixteen varieties of pawpaw analyzed and reported in this study, there was existing literature data for nine of the varieties, which was used for comparative analysis of the attributes for which whole fruit or pulp was not pooled, i.e. fruit size and weight, texture, obrix, ph, and pulp and skin color. the sixteen varieties were pooled into varietal composites for nutritional analysis. fruit size by variety and serving size determination tab. 1 shows the mean weight, length, and width for each of the varieties of pawpaws used for this study. the average weight of the fruits across all varieties was 194 grams and ranged from 122 grams for variety 'pickle' to 292 grams for variety 'ksu-atwood'. although there were signifi cant differences in average fruit weight by variety (p<0.001), with the six heaviest varieties signifi cantly heavier than the ten least heavy varieties (tab. 1), post-hoc means separations did not elucidate other differences. a recent study that examined average fruit weight from all fruit of 52 varieties collected during 8 harvests from 2005 to 2012 at three sites including fox paw farm, the location of fruit collected for this study, determined that fruit weight is normally distributed across all varieties and the average fruit weight by variety ranged from 72-244 g (greenawalt et al., 2019). results of the much smaller sample from this study fi t well with those results. of the 9 varieties of which comparisons to previous research can be made (tab. 1), 8 of the varieties from this study, all but 'lynn’s favorite', weighed more and all nine varieties had larger dimensions than was reported previously and (brannan et al., 2015) even though they came from the same location. the average weight of the fruit of the 9 varieties from the comparative study was 136 g, compared to 203 g for the same varieties from the current study. because the intention of the current study was not to collect all fruit but rather to create composites for nutritional analysis, it is likely that larger fruit were selected. the average fruit weights were used as a basis to determine the serving size of the pawpaw because currently there is none. after some experimentation, the registered dietitian on the project team (author e.e. anderson) determined that one half cup (120 g) was an appropriate serving size. this determination was based on the knowledge that 120 g is similar to the serving size of other popular fruits and can be obtained from the pulp of one large (~240 g) or two small (~120 g) whole pawpaw fruits, assuming a 50% yield of pulp from the skin and seeds. fruit ph, obrix, fi rmness, and color by variety as shown in tab. 2, the ph of the fruits from each variety ranged from 5.4 to 6.3, which categorizes pawpaw as a low acid fruit along 126 r.g. brannan, e.e. anderson, r.l. powell, m.n. coyle tab. 1: mean and standard error (s.e.) for size (weight, length, width) of sixteen varieties of whole pawpaws (asimina triloba), nine of which can be compared directly to a previous study (brannan et al., 2015). (different superscript letters within a column indicate significant differences at p<0.05.) variety study weight (g) s.e. length (cm) s.e. width (cm) s.e. varieties for which comparative analysis can be made green river belle current 187 abcde 6.2 16.6 abc 0.2 5.8 a 0.4 previous 152 10.5 5.7 ixl current 213 bcdef 63.6 15.6 ab 3.4 8.6 bcde 0.9 previous 127 9.5 5.4 ksu-atwood current 292 f 22.7 18.3 bc 0.5 11.2 g 0.5 previous 162 11.1 5.2 lynn’s favorite current 130 ab 10.6 13.8 a 0.7 8.6 bcde 0.4 previous 179 11.5 5.6 nc1 current 217 cdef 19.5 16.3 abc 0.9 9.3 cdef 0.8 previous 204 10.5 6.4 overleese current 223 def 38.0 14.2 a 0.8 10.4 fg 0.7 previous 130 9.1 5.8 quaker’s delight current 186 abcde 22.3 16.2 abc 1.0 7.8 abc 0.2 previous 84 7.9 4.8 saa-zimmerman current 247 def 28.6 19.0 c 0.7 8.3 abcd 0.5 previous 130 9.8 5.5 shenandoah current 133 abc 11.3 13.8 a 0.6 6.8 a 0.3 previous 123 8.6 5.6 varieties for which comparative analysis cannot be made estill current 188 abcde 24.3 15.0 ab 1.1 9.8 ef 0.4 mango current 175 abcd 17.8 14.4 a 1.1 8.0 abcd 0.3 mitchell current 195 abcde 32.4 16.3 abc 1.4 9.0 bcdef 0.4 pickle current 122 a 10.7 14.3 a 0.6 8.5 bcde 0.2 potomac current 267 ef 34.1 15.8 abc 0.9 11.2 g 0.5 sab overleese current 197 abcde 29.1 14.8 a 0.9 9.3 def 0.4 wabash current 194 abcde 25.0 15.5 ab 1.2 7.5 ab 0.6 tab. 2: mean and standard error (s.e.) for obrix, firmness, and ph of sixteen varieties of whole pawpaws (asimina triloba), nine of which can be compared directly to a previous study (brannan et al., 2015) for all values except ph which can be compared to a different study (brannan, 2016). (different superscript letters within a column indicate significant differences at p<0.05.) variety study °brix s.e. firmness s.e. firmness s.e. ph s.e. (kg) (n) varieties for which comparative analysis can be made green river belle current 21.6 defg 0.3 0.610 bc 0.083 5.17 0.74 6.1 fg 0.0 previous 25.1 0.419 6.8 ixl current 22.6 fg 0.5 0.410 ab 0.037 4.10 0.43 5.5 ab 0.1 previous 22.9 0.643 6.5 ksu-atwood current 22.3 efg 0.4 0.603 abc 0.096 4.92 0.81 6.1 fg 0.7 previous 27.1 0.206 6.3 lynn’s favorite current 20.6 cde 0.7 0.551 abc 0.056 5.47 0.55 5.4 a 0.1 previous 28.0 0.232 6.2 nc1 current 21.2 defg 0.5 0.464 ab 0.083 4.55 0.82 5.4 a 0.1 previous 25.7 0.248 6.1 overleese current 20.6 cde 0.3 0.347 a 0.039 3.43 0.38 5.8 cde 0.0 previous 25.1 0.198 6.5 quaker’s delight current 17.1 a 0.3 0.507 abc 0.057 5.04 0.52 5.5 a 0.0 previous 20.9 0.499 6.5 saa-zimmerman current 19.2 bc 0.5 0.580 abc 0.054 5.81 0.53 6.3 g 0.0 previous 19.9 0.551 6.5 shenandoah current 22.3 efg 0.6 0.727 c 0.172 7.24 1.68 5.8 cde 0.0 previous 21.2 0.432 6.7 varieties for which comparative analysis cannot be made estill current 24.7 h 0.6 0.391 ab 0.048 3.94 0.45 5.8 cde 0.1 mango current 18.2 abc 0.3 0.421 ab 0.075 4.18 0.78 5.6 abc 0.1 mitchell current 20.4 cd 0.4 0.490 abc 0.061 4.86 0.57 5.5 ab 0.1 pickle current 21.7 defg 0.2 0.391 ab 0.049 3.91 0.52 6.0 ef 0.1 potomac current 21.0 def 0.7 0.623 bc 0.048 6.11 0.47 5.7 cd 0.1 sab overleese current 26.1 h 0.6 0.429 ab 0.046 4.22 0.45 5.8 cde 0.0 wabash current 23.0 g 1.1 0.590 abc 0.101 5.79 0.99 5.7 cde 0.1 comparative analysis of pawpaw nutrition 127 with such fruits as avocado, most melons, mango, papaya, and others. these values are in agreement with previously reported values (brannan, 2016). significant differences (p<0.001) for ph among varieties were found in the current study (tab. 2) but not in the comparative study. the practical impact of the ph of pawpaw relates to the polyphenol oxidase (ppo), the enzyme responsible for skin and tissue browning. all of the varieties in this and previous studies exhibited pulp ph in the range in which pawpaw ppo has been shown to exhibit its highest activity (fang et al., 2007), suggesting that none of these varieties would be more resistant than any other to ppo activity. the obrix in the whole fruits were measured using a refractometer calibrated for direct reading of percent sugar and should not be confused with the percent sugar from the composites reported later in this paper. the average obrix of the varieties ranged from 18.2% to 26.1%, again showing good agreement with previously reported values (tab. 2). variation in percent sugar of pawpaw fruit likely is due to an increase in sugars during climacteric ripening, although there is no research to support this hypothesis. in this study, varieties 'estill' and 'sab overleese' exhibited significantly higher obrix values than the other 14 varieties (p<0.001). the firmness of pawpaw is directly related to the ripeness of the fruit. there is no benchmark firmness value that defines a ripe pawpaw fruit and the current best practice for harvest is to squeeze each fruit by hand, looking for a small amount of give in the skin, an unreliable indicator. the least firm variety at the time of harvest was 'estill' with a firmness of 0.391 kg, and the firmest variety was 'shenandoah' with a firmness of 0.727 kg. although there was a significant difference between these two varieties (p<0.022), post-hoc analysis did not reveal other differences of note (tab. 2). we have shown that certain cell wall xyloglucans, pectins, and arabinogalactins degrade during refrigerated storage of pawpaw pulp (brannan et al., 2019) and it is likely that compositional degradation of the cell wall causes loss of turgor during ripening and storage. whole fruit firmness from nine varieties in the current study compares well to the firmness of fruits from similar varieties measured previously (tab. 2). one conclusion that can be drawn from this comparison is the certainty that firmness values between 0.2 and 0.7 kg of force using the method described herein would describe ripe fruit because all of the fruits from the comparison studies were in this range and were considered ripe as determined by hand. the cie color (l*, a*, b*) and hue angle for the skin and pulp of the varieties sampled in this study are shown in tab. 2 and 3, respective ly. there were significant differences (p<0.001) between varieties for all of these values, however, the interpretation of these results tends to be descriptive rather than inferential because certain varieties have skin and pulp that tends to be lighter or more yellow than others. the skin color range for all of the varieties (tab. 3) exhibited a* values (-6.0 to -14.0), b* values (37.0 to 49.3), and hue angles (99.0 to 109.1) indicating that the skin is yellow-green or green-yellow. this range of skin color by variety has led to the conclusion that skin color is not a reliable indicator of fruit ripeness (mcgrath and karahadian, 1994). the pulp color range for all of the varieties (tab. 4) exhibited a* values (1.0 to 11.6), b* values (32.8 to 51.2), and hue angles (76.2 to 88.3) indicating that the pulp is orange to yellow. these results are in strong agreement with previous research (tab. 4). nutritional analysis of pawpaw the proximates, vitamins, minerals, and essential and non-essential amino acids for varietal composites of pawpaw pulp from this and comparative studies (nam et al., 2018; peterson et al., 1982) are tab. 3: mean and standard error (s.e.) for cie l*, a*, b* and hue angle (hue) of the skin from sixteen varieties of whole pawpaws (asimina triloba), eight of which can be compared directly to a previous study (brannan et al., 2015) and variety shenandoah to a different study (zhang et al., 2017). (different superscript letters within a column indicate significant differences at p<0.05.) variety study l* s.e. a* s.e. b* s.e. hue s.e. varieties for which comparative analysis can be made green river belle current 62.9 ce 0.8 -7.8 def 0.7 43.5 efgc 1.0 100.2 ab 1.0 previous 65.8 -3.1 34.2 95.1 ixl current 65.4 de 0.8 -12.7 ab 0.5 49.3 h 1.0 104.5 cde 0.7 previous 63.4 -9.4 39.5 103.3 ksu-atwood current 59.7 ab 0.6 -12.8 ab 0.3 37.4 ab 1.2 108.9 f 0.4 previous 61.4 -8.6 24.6 109.2 lynn’s favorite current 59.5 ab 0.8 -12.5 ab 0.8 44.5 fg 1.2 105.7 de 1.2 previous 63.9 -3.8 35.8 96.1 nc1 current 61.8 abc 0.6 -12.6 ab 0.5 39.5 abcd 1.2 107.6 ef 0.7 previous 62.9 -4.8 30.2 99.0 overleese current 59.9 ab 0.7 -14.0 a 0.7 40.1 abcde 0.8 109.2 f 0.8 previous 65.1 -8.8 33.0 104.9 quaker’s delight current 67.4 e 0.8 -13.0 ab 0.5 45.1 fg 1.0 106.1 def 0.7 previous 69.3 -6.8 41.3 99.3 saa-zimmerman current 67.7 e 0.7 -11.0 bc 0.4 41.9 cdef 1.5 104.7 cde 0.8 previous 65.3 -10.2 36.9 105.4 shenandoah current 60.1 ab 0.6 -11.3 bc 0.8 39.8 abcde 1.1 105.9 de 1.0 previous 61.8 -8.0 31.9 104.0 varieties for which comparative analysis cannot be made estill current 59.3 ab 0.5 -8.0 def 0.5 37.5 ab 1.1 102.3 bc 0.9 mango current 62.1 bc 0.4 -9.1 cde 0.8 38.4 abc 0.9 103.5 bcd 1.2 mitchell current 64.4 cd 0.8 -8.2 def 1.4 43.2 defg 1.2 100.9 ab 13.8 pickle current 64.5 cd 0.5 -6.0 f 0.7 37.0 a 0.9 99.0 a 1.0 potomac current 59.8 ab 0.7 -7.6 ef 0.8 41.5 cdef 1.5 100.5 ab 1.1 sab overleese current 59.2 a 1.9 -8.1 def 1.1 41.1 bcdef 1.9 101.1 ab 12.2 wabash current 65.2 de 0.7 -10.1 cd 0.9 46.2 gh 1.2 102.4 bc 1.2 128 r.g. brannan, e.e. anderson, r.l. powell, m.n. coyle tab. 4: mean and standard error (s.e.) for cie l*, a*, b* and hue angle (hue) of the pulp from sixteen varieties of whole pawpaws (asimina triloba), eight of which can be compared directly to a previous study (brannan et al., 2015) and variety shenandoah to a different study (zhang et al., 2017). (different superscript letters within a column indicate significant differences at p<0.05.) variety study l* s.e. a* s.e. b* s.e. hue s.e. varieties for which comparative analysis can be made green river belle current 70.8 bc 3.5 7.0 de 0.3 45.6 cd 1.1 81.2 cd 0.5 previous 73.9 10.0 46.2 77.7 ixl current 82.4 g 1.3 2.4 ab 0.3 32.8 a 1.2 85.9 f 0.4 previous 77.1 6.3 45.1 82.0 ksu-atwood current 78.3 defg 1.0 5.0 cd 0.5 42.6 c 1.3 83.3 de 0.5 previous 75.0 5.6 44.8 82.8 lynn’s favorite current 75.8 cdefg 4.1 4.5 c 1.1 36.4 ab 2.6 82.9 de 1.4 previous 72.6 11.1 42.9 75.4 nc1 current 78.2 defg 1.3 4.3 bc 0.5 37.9 b 1.6 83.5 e 0.5 previous 77.1 10.1 45.9 77.0 overleese current 72.5 bcde 2.1 6.5 cde 0.7 47.3 cde 1.3 82.2 de 0.7 previous 79.3 2.1 34.6 86.5 quaker’s delight current 82.6 g 1.0 1.0 a 0.2 32.9 a 1.2 88.3 g 0.3 previous 77.8 6.5 46.1 81.9 saa-zimmerman current 80.3 fg 0.8 7.0 de 0.5 49.9 de 1.0 82.1 de 0.5 previous 79.8 4.4 44.2 84.3 shenandoah current 74.4 bcdef 0.8 9.3 fg 1.0 49.4 de 1.4 79.3 bc 0.8 previous 64.3 6.3 39.8 81.0 varieties for which comparative analysis cannot be made estill current 68.4 ab 2.0 10.6 gh 0.8 51.2 e 2.9 74.3 ab 2.3 mango current 75.9 cdefg 2.7 6.2 cde 1.0 44.7 cd 2.3 82.4 de 1.0 mitchell current 63.0 a 2.4 11.3 gh 0.8 46.0 cde 1.7 76.3 a 0.6 pickle current 71.8 bcd 1.0 11.3 gh 0.4 48.7 de 0.7 77.0 a 0.4 potomac current 73.9 bcdef 1.8 11.7 h 1.0 49.6 de 1.3 76.9 a 0.8 sab overleese current 76.2 cdefg 3.2 7.4 ef 0.5 48.5 de 1.5 81.3 cde 0.6 wabash current 79.4 efg 1.8 5.7 cde 1.2 36.8 ab 2.7 81.9 de 1.2 shown in tab. 5. we were able to locate historical pawpaw nutritional information from the 1963 usda agriculture handbook #8 (watt and merrill, 1963), which includes information for the proximates (water, protein, fat, carbohydrate, and ash) and calories. it is unclear whether pawpaw nutritional values were listed in the database before this time. in 1982, a nutritional analysis of pawpaw pulp with skin was reported (peterson et al., 1982), however, many consider the skin undesirable and only consume the pulp. nonetheless, including the nutritional information for the pulp with skin in the comparison has value for those who do consume the skin. recently, korean researchers published a detailed nutritional analysis of pawpaw pulp (nam et al., 2018) but did not note the variety of pawpaw used. the comparison in tab. 5 revealed more similarities than differen ces between the composite from this study and the korean study. kilocalories calculated from the proximates were nearly identical (85 v 84) for the two studies. there were similarities in total lipid, ash, dietary fiber, potassium, and iron between the two studies. nutrient analysis from the current study indicated less moisture and protein but more carbohydrates, especially glucose, vitamin c, and calcium than the korean study. pawpaw nutritional quality compared to common fruits to place the nutritional quality of the pawpaw in perspective, a weight-to-weight comparison of pawpaw nutrition information from this study to 100 g of seven common fruits (us department of agriculture, 2020) is shown in tab. 6. on a per weight basis, pawpaw has 33-67% more lipid, 4-68% more carbohydrates, 42-69% more dietary fiber, and 16-70% more sugars than the seven other fruit. on the other hand, pawpaw has 2 to 12-fold less vitamin c than the other fruits, except apple. prior to this research, which establishes one-half cup (120 g) as a standard serving size for pawpaw, no standard serving size of pawpaw existed. serving size is an important measure because the ubiquitous nutrition facts labels on food products in the u.s. are based on serving size. tab. 7 shows a nutritional comparison of pawpaw to the same seven fruits based on serving size. one serving of the seven other fruits ranges from 118 g (banana) to 182 g (medium apple). on a per serving basis, the pawpaw nutrient composition most resembles the nutritional content provided by a banana or mango. pawpaw is very similar in calories and carbohydrates, has similar potassium as mango, but has much less vitamin c than both fruit. pawpaw does have more fat and fiber than banana and mango. it is an interesting coincidence that the pawpaw’s closest nutritional comparisons on a per serving basis are the banana and mango because research has shown that banana and mango are the predominant flavors of pawpaw pulp (brannan et al., 2012; mcgrath and karahadian, 1994). benefits of up-to-date pawpaw nutritional information it is our intention to use the information from this and the korean study to petition the usda for inclusion of pawpaw in the usda’s national nutrient database for standard reference. the database will be a boon for disseminating pawpaw’s nutrition information to growers, the food industry, and consumers interested in learning more about the pawpaw. up-to-date nutritional data that reflects only the edible portion of the fruit can also be used to generate nutrition facts panels for food labels. although raw fruits and certain low volume small businesses are exempt from having the nutrition facts labels, other small businesses and/or foods for sale that make nutrient claims (e.g. “gluten free”, “low fat”, etc.) are required to have nutrition facts labeling, even if they are exempt from the comparative analysis of pawpaw nutrition 129 tab. 5: pawpaw (asimina triloba) nutritional information for 100 g of pulp, one serving of pulp (1/2 cup, 120 g), and 100 g of pulp with skin. (“n.d.” indicates that the nutrient was not included in the analysis. the “<” symbol indicates the nutrient was analyzed but could not be detected at or above the threshold level.) pulp (without skin) pulp and skin current study nam et al., 2018 peterson et al., 1982 nutrient unit 100 g 1 serving 100 g 1 serving 100 g proximates calories kcal 85 102 841 101 80 calories kj 357 428 353 423 335 moisture g 74.5 89.4 79.1 94.9 73.2 protein g 0.7 0.9 1.5 1.8 1.2 total lipid g 0.6 0.7 0.4 1.7 1.2 mufa g 0.05 0.06 0.06 0.07 n.d. pufa g < < 0.06 0.07 n.d. saturated fa g < < 0.05 0.06 n.d. trans fa g < < n.d. n.d. n.d. cholesterol mg < < n.d. n.d. n.d. ash g 0.4 0.5 0.4 0.5 0.6 carbohydrates (by difference) g 23.8 28.6 18.6 22.3 18.8 dietary fiber g 4.5 5.4 5.8 7.0 2.6 total sugars (calculated) g 16.3 19.5 13.1 15.7 n.d. sucrose g 11.4 13.7 9.3 11.2 n.d. glucose g 2.7 3.2 2.1 2.5 n.d. fructose g 2.2 2.6 1.7 2.0 n.d. lactose g < < n.d. n.d. n.d. maltose g < < n.d. n.d. n.d. vitamins vitamin a iu n.d. n.d. 82 98 87 vitamin c mg 4.92 5.9 1.0 1.2 18.3 vitamin d iu < < n.d. n.d. n/a thiamin mg n.d. n.d. n.d. n.d. 0.01 riboflavin mg n.d. n.d. n.d. n.d. 0.09 niacin mg n.d. n.d. n.d. n.d. 1.1 minerals calcium mg 13 16 8 10 63 copper mg n.d. n.d. n.d. n.d. 0.5 iron mg 0.2 0.2 0.3 0.4 7 magnesium mg n.d. n.d. 10 12 113 manganese mg n.d. n.d. n.d. n.d. 2.6 phosphorus mg n.d. n.d. n.d. n.d. 47 potassium mg 201 241 239 287 345 sodium mg 1.0 1.2 n.d. n.d. n.d. sulfur mg n.d. n.d. n.d. n.d. 70 zinc mg n.d. n.d. 0.5 0.6 0.9 essential amino acids cystine mg n.d. n.d. n.d. n.d. 4 histidine mg n.d. n.d. 44 53 21 isoleucine mg n.d. n.d. 13 15 70 leucine mg n.d. n.d. 38 45 81 lysine mg n.d. n.d. 30 36 60 methionine mg n.d. n.d. n.d. n.d. 15 phenylalanine mg n.d. n.d. 28 33 51 threonine mg n.d. n.d. 24 29 46 tryptophan mg n.d. n.d. n.d. n.d. 9 valine mg n.d. n.d. 24 29 58 non-essential amino acids alanine mg n.d. n.d. 67 81 n.d. aspartic acid mg n.d. n.d. 47 57 n.d. glutamic acid mg n.d. n.d. 58 69 n.d. glycine mg n.d. n.d. 29 38 n.d. proline mg n.d. n.d. 163 196 n.d. serine mg n.d. n.d. 35 42 n.d. tyrosine mg n.d. n.d. 16 20 25 1kcal calculated from proximate analysis (moisture, lipid, protein, carbohydrate) 2vitamin c value was not determined in this study. it was from a previous study (harris and brannan, 2009). 130 r.g. brannan, e.e. anderson, r.l. powell, m.n. coyle label for other reasons. most food companies provide nutrition facts on their labels whether they are required to or not because it provides a layer of transparency for customers. as the popularity of the fruit grows, demand for up-to-date nutritional information likely will increase and the information will be beneficial for health clinicians recommending pawpaw to increase fiber intake, for example. conclusions results from this study confirm that the size, ph, obrix, firmness, color of the skin and pulp, and nutritional information fits well with previous literature. pawpaw ph is in the high range for ppo activity, so strategies to reduce pawpaw browning that rely on inhibition of ppo activity should take this into account. variations in firmness of the pawpaw pulp observed in this study are likely are due to differential degradation of cell wall polysaccharides. pawpaw skin and pulp color fall well-within the previously established values. pawpaw pulp nutritional values compare to the values from the korean study and will be used as the basis for inclusion of pawpaw in the usda’s national nutrient database for standard reference. data from this study was used as the basis for several specific recommendations: 1) firmness values between 0.2 and 0.7 kg of force using the method described herein describe ripe pawpaw fruit; 2) the serving size for raw pawpaw pulp is one-half cup (120 g); and 3) pawpaw pulp nutrition should be compared favorably to that of a banana or mango. acknowledgements funding provided by generous grants from the northern nut growers association and the national pawpaw foundation. conflict of interest no potential conflict of interest was reported by the authors. references aoac international, 2016: official methods of analysis of aoac international, 20th ed. aoac international, rockville, md. archbold, d.d., pomper, k.w., 2003: ripening pawpaw fruit exhibit respiratory and ethylene climacterics. postharvest biol. tech. 30, 99-103. doi: 10.1016/s0925-5214(03)00135-2 bois, j., 2001: anachronistic relationships. bioscience 51, 318-320. doi: 10.1641/0006-3568 brannan, r.g., 2016: polyphenol oxidase in pawpaw (asimina triloba [l.] dunal) fruit pulp from different varieties. j. food res. 5, 33-39. doi: 10.5539/jfr.v5n1p33 brannan, r.g., faik, a., goelz, r., pattathil, s., 2019: identification and tab. 6: comparison of pawpaw pulp nutritional information from this study (except where noted) to existing nutritional information for seven common fruits on a 100-gram basis. pawpaw apple banana blueberry mango papaya pineapple strawberry 100 g 100 g 100 g 100 g 100 g 100 g 100 g 100 g calories (kcal) 85 52 89 57 60 43 50 32 protein (g) 0.7 0.3 1.1 0.7 0.8 0.5 0.5 0.7 total fat (g) 0.6 0.2 0.3 0.3 0.4 0.3 0.1 0.3 carbohydrate (g) 23.8 13.8 22.8 14.5 15.0 10.8 13.1 7.7 dietary fiber (g) 4.5 2.4 2.6 2.4 1.6 1.7 1.4 2.0 total sugar (g) 16.3 10.4 12.2 10.0 13.7 7.8 9.8 4.9 1vitamin c (mg) 4.9 4.6 8.7 9.7 36.4 60.9 47.8 58.8 calcium (mg) 13 6 5 6 11 20 13 16 iron (mg) 0.2 0.1 0.3 0.3 0.2 0.3 0.3 0.4 potassium (mg) 201 107 358 77 168 182 109 153 sodium (mg) 1 1 1 1 1 8 1 1 1vitamin c value was not determined in this study. it was from a previous study (harris and brannan, 2009) tab. 7: comparison of pawpaw nutritional information from this study (except where noted) to existing nutritional information (us department of agri culture, 2020) for seven common fruits per serving. pawpaw apple banana blueberry mango papaya pineapple strawberry 1/2 cup, pulp 1 medium fruit 1 medium fruit 1 cup, fruit 1 cup, pieces 1 cup, pieces 1 cup, chunks 1 cup, halves (120 g) (182 g) (118 g) (148 g) (165 g) (145 g) (165 g) (152 g) calories (kcal) 102 95 105 84 99 62 83 49 protein (g) 0.8 0.5 1.3 1.0 1.3 0.7 0.8 1.1 total fat (g) 0.7 0.4 0.4 0.4 0.7 0.4 0.2 0.5 carbohydrate (g) 28.6 25.1 26.9 21.5 24.8 15.7 21.6 11.7 dietary fiber (g) 5.4 4.4 3.1 3.6 2.6 2.5 2.3 3.0 total sugar (g) 19.6 18.9 14.4 14.8 22.6 11.3 16.2 7.4 1vitamin c (mg) 5.9 8.4 10.3 14.4 60.1 88.3 78.9 89.4 calcium (mg) 16 11 6 9 18 29 21 24 iron (mg) 0.2 0.2 0.4 0.4 0.3 0.4 0.5 0.6 potassium (mg) 241 195 422 114 277 264 180 233 sodium (mg) 1 2 1 1 2 12 2 2 1vitamin c value was not determined in this study. it was from a previous study (harris and brannan, 2009) http://dx.doi.org/10.1016/s0925-5214(03)00135-2 http://dx.doi.org/10.1641/0006-3568 http://dx.doi.org/10.5539/jfr.v5n1p33 comparative analysis of pawpaw nutrition 131 analysis of cell wall glycan epitopes and polyphenol oxidase in pawpaw (asimina triloba [l.] dunal) fruit pulp as affected by high pressure processing and refrigerated storage. food sci. tec. int. 25, 711-722. doi: 10.1177/1082013219856769 brannan, r.g., peters, t., talcott, s.t., 2015: phytochemical analysis of ten varieties of pawpaw (asimina triloba [l.] dunal) fruit pulp. food chem. 168, 656-661. doi: 10.1016/j.foodchem.2014.07.018 brannan, r.g., peters, t.e., black, k.j., kukor, b.j., 2018: valorization of underutilized north american pawpaw (asimina triloba): investiga tion as a lipid oxidation inhibitor in turkey homogenate model system. j. sci. food agr. 98, 2210-2214. doi: 10.1002/jsfa.8706 brannan, r.g., salabak, d.e., 2009: ability of methanolic seed extracts of pawpaw (asimina triloba) to inhibit n-3 fatty acid oxidation initiated by peroxyl radicals and reactive oxygen, nitrogen, and sulfur. food chem. 114, 453-458. doi: 10.1016/j.foodchem.2008.09.071 brannan, r.g., salabak, d.e., holben, d.h., 2012: sensory analysis of pawpaw (asimina triloba) pulp puree: consumer appraisal and descrip tive lexicon. j. food res. 1, 179-192. doi: 10.5539/jfr.v1n1p179 brannan, r.g., wang, g., 2017: effect of frozen storage on polyphenol oxidase, antioxidant content, and color of pawpaw (asimina triloba [l.] dunal) fruit pulp. j. food res. 6, 93-101. doi: 10.1016/j.lwt.2017.07.023 callaway, m.b., 1993: pawpaw (asimina triloba): a “tropical” fruit for temperate climates. in: janick, j., simon, j.e. (eds.), new crops, 505-515. cronquist, a., 1981: an integrated classification of flowering plants. columbia university press. fang, c., wang, c., xiong, y.l., pomper, k.w., 2007: extraction and characterization of polyphenol oxidase in pawpaw (asimina triloba) fruit. j. food biochem. 31, 603-620. doi: 10.1111/j.1745-4514.2007.00133.x galli, f., archbold, d.d., pomper, k.w., 2008: loss of ripening capacity of pawpaw fruit with extended cold storage. j. agri. food chem. 56, 10683-10688. doi: 10.1021/jf801857g greenawalt, l.g., powell, r., brannan, r.g., 2019: comparative analysis of pawpaw production data from 2005-2012. j. amer. pomolog. soc. 73, 2-11. harris, g.g., brannan, r.g., 2009: a preliminary evaluation of antioxi dant compounds, reducing potential, and radical scavenging of pawpaw (asimina triloba) fruit pulp from different stages of ripeness. lwt-food sci. tec. 42, 275-279. doi: 10.1016/j.lwt.2008.05.006 jones, s.c., layne, d.r., 1997: pawpaw description and nutritional infor mation. kysu extension bulletin. kobayashi, h., wang, c., pomper, k.w., 2008: phenolic content and antioxidant capacity of pawpaw fruit (asimina triloba l.) at different ripening stages. hortscience 43, 268-270. doi: 10.21273/hortsci.43.1.268 kral, r., 1960: a revision of asimina and deeringothamnus (annonaceae). brittonia 12, 233-278. doi: 10.2307/2805119 layne, d.r., 1996: the pawpaw [asiminia triloba (l.) dunal]: a new fruit crop for kentucky and the united states. hortscience 31, 777-784. doi: 10.21273/hortsci.31.5.777 mcgrath, m., karahadian, c., 1994: evaluation of physical, chemical, and sensory properties of pawpaw fruit (asimina triloba) as indicators of ripeness. j. agr. food chem. 42, 968-974. doi: 10.1021/jf00040a025 nam, j.-s., jang, h.-l., rhee, y.h., 2018: nutritional compositions in roots, twigs, leaves, fruit pulp, and seeds from pawpaw (asimina triloba [l.] dunal) grown in korea. j. appl. bot. food qual. 91, 47-55. doi: 10.5073/jabfq.2018.091.007 nam, j.-s., jang, h.-l., rhee, y.h., 2017: antioxidant activities and phenolic compounds of several tissues of pawpaw (asimina triloba [l.] dunal) grown in korea. j. food sci. 82, 1827-1833. doi: 10.1111/1750-3841.13806 nam, j.-s., park, s.-y., oh, h.-j., jang, h.-l., rhee, y.h., 2019: phenolic profiles, antioxidant and antimicrobial activities of pawpaw pulp (asimina triloba [l.] dunal) at different ripening stages. j. food sci. 84, 174-182. doi: 10.1111/1750-3841.14414 peterson, r.n., 2003: pawpaw variety development: a history and future prospects. horttechnology 13, 449-454. doi: 10.21273/horttech.13.3.0449 peterson, r.n., cherry, j.p., simmons, j.g., 1982: composition of paw paw (asimina triloba) fruit. annual report northern nut growers association 73, 97-107. pomper, k.w., crabtree, s.b., layne, d.r., peterson, r.n., masabni, j., wolfe, d., 2008: the kentucky pawpaw regional variety trial. j. amer. pomolog. soc. 62, 58-69. pomper, k.w., layne, d.r., 2005: the north american pawpaw: botany and horticulture. in: janick, j. (ed.), horticultural reviews, 349-382. john wiley & sons, inc., hoboken, new jersey. pomper, k.w., layne, d.r., peterson, r.n., wolfe, d., 2003: the pawpaw regional variety trial: background and early data. horttechnology 13, 412-417. doi: 10.21273/horttech.13.3.0412 us department of agriculture, 2020: fooddata central [www docu ment]. url https://fdc.nal.usda.gov/ watt, b., merrill, a., 1963: agriculture handbook no 8: composition of foods – raw, processed, prepared. consumer and food economics research division united states department of agriculture, washington, dc. willson, m.f., schemske, d.w., 1980: pollinator limitation, fruit production, and floral display in pawpaw (asimina triloba). bulletin of the torrey botanical club 107, 401-408. doi: 10.2307/2484160 zhang, l., dai, s., brannan, r.g., 2017: effect of high-pressure processing, browning treatments, and refrigerated storage on sensory analysis, color, and polyphenol oxidase activity in pawpaw (asimina triloba l.) pulp. lwt − food sci. tec. 86, 49-54. doi: 10.1016/j.lwt.2017.07.023 orcid robert g. brannan https://orcid.org/0000-0003-0240-6373 address of the corresponding author: robert g. brannan, division of food and nutrition sciences, e170 grover center, athens, oh 45701, usa e-mail: brannan@ohio.edu © the author(s) 2021. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1177/1082013219856769 http://dx.doi.org/10.1016/j.foodchem.2014.07.018 http://dx.doi.org/10.1002/jsfa.8706 http://dx.doi.org/10.1016/j.foodchem.2008.09.071 http://dx.doi.org/10.5539/jfr.v1n1p179 http://dx.doi.org/10.1016/j.lwt.2017.07.023 http://dx.doi.org/10.1111/j.1745-4514.2007.00133.x http://dx.doi.org/10.1021/jf801857g http://dx.doi.org/10.1016/j.lwt.2008.05.006 http://dx.doi.org/10.21273/hortsci.43.1.268 http://dx.doi.org/10.2307/2805119 http://dx.doi.org/10.21273/hortsci.31.5.777 http://dx.doi.org/10.1021/jf00040a025 http://dx.doi.org/10.5073/jabfq.2018.091.007 http://dx.doi.org/10.1111/1750-3841.13806 http://dx.doi.org/10.1111/1750-3841.14414 http://dx.doi.org/10.21273/horttech.13.3.0449 http://dx.doi.org/10.21273/horttech.13.3.0412 http://dx.doi.org/10.2307/2484160 http://dx.doi.org/10.1016/j.lwt.2017.07.023 https://orcid.org/0000-0003-0240-6373 journal of applied botany and food quality 93, 44 53 (2020), doi:10.5073/jabfq.2020.093.006 1institute of landscape and plant ecology, university of hohenheim, stuttgart, germany 2institute of crop science, university of hohenheim, stuttgart, germany 3institute of soil science and land evaluation, university of hohenheim, stuttgart, germany effects of soil warming and altered precipitation patterns on photosynthesis, biomass production and yield of barley ireen drebenstedt1*, iris schmid1,2, christian poll3, sven marhan3, robert kahle3, ellen kandeler3, petra högy1 (submitted: october 25, 2019; accepted: february 2, 2020) * corresponding author summary crop productivity and plant physiology are affected by rising temperatures and altered precipitation patterns due to climate change. we studied the impacts of an increase in soil temperature of 2.5 °c, a decrease in summer precipitation amount of 25%, a reduction in summer precipitation frequency of 50%, and their interactions on photosynthesis, biomass production, and yield of spring barley (hordeum vulgare l. cv. rgt planet) in a temperate agricultural ecosystem near stuttgart (germany). leaf gas exchange of barley appeared to be affected mainly by drought in the form of reduced precipitation frequency or by a combination of changes in soil temperature and precipitation patterns. in contrast, biomass production and yield parameters were more affected under soil warming alone. in addition, biomass of roots increased under soil warming at stem elongation. stable grain yield was observed under reduced precipitation amount and also under increased evaporation through soil warming. these findings provide additional evidence that barley is relatively drought tolerant, which should be taken into consideration in the context of appropriate crop selection under climate change. keywords: soil warming; altered precipitation patterns; climate change; barley introduction temperature and precipitation are two important climate factors controlling crop production (richardson et al., 2009; hatfield et al., 2011). an increase in temperature and change in precipitation patterns can negatively affect crop development and crop yield (damatta et al., 2010). however, other aspects of predicted climate change are an increase of atmospheric carbon dioxide (co2) concentration and of tropospheric ozone (o3) concentration, which can occur simultaneously with changes in temperature and precipitation during crop growth (damatta et al., 2010). in germany, average air temperature increased by 1.4 °c from 1881 to 2016 (dwd, 2017). according to climate predictions, mean air temperature will continue to increase by 1.2 5.3 °c until 2100, as compared to 1971-2000 (dwd, 2017). closely related to a rise in air temperature is an increase in soil temperature (zheng et al., 1993). in addition, precipitation is expected to change as precipitation events become less frequent. during summer months, average precipitation amount is expected to decrease up to 9% until 2100, with few regional differences, compared to 1961-1990 (dwd, 2017). predicting effects of elevated soil temperature due to global warming is more complex than corresponding changes in air temperature because soil temperature is additionally influenced by other factors such as soil moisture and texture, vegetation, or season (gray and brady, 2016). it is known that crop growth and development are stimulated by an increase in soil temperature, especially during early growth stages, resulting in earlier flowering times (patil et al., 2010; gavito et al., 2001). in addition, uptake of water and nutrients is accelerated under warmer soil temperatures in temperate climates (bowen, 1991). an increase in soil temperature directly affects root development (gray and brady, 2016), which can lead to an increase in root biomass (clark and reinhard, 1991). understanding reactions of root growth in crops under global warming is crucial due to the essential role of root systems in water and nutrient uptake. accordingly, traits such as abiotic stress tolerance or water use efficiency (wue; biomass produced per unit of transpiration), which are linked to crop performance under future climate conditions, are closely related to root structure in the soil (nagel et al., 2009). it is known that rising air temperatures can impact plant physiological processes, including photosynthesis, which can lead to shortened life cycle, reduced plant productivity, and reduced crop yield (conroy et al., 1994). however, impacts of elevated soil temperature on cereal physiology are not well understood. warm periods often occur in combination with reduced water availability. under elevated temperatures plant water status is critical, because only well-watered plants tend to maintain stable tissue water status (machado and paulsen, 2001; wahid et al., 2007). low water availability is known to decrease plant growth and to delay plant development. it can also result in crop yield reduction by limit ing plant organ growth and final size (blum, 1996). the magnitude of agricultural yield losses is tightly linked to the developmental stage at which crops experience water stress (gray and brady, 2016). physiological processes such as photosynthesis are also limited by water limitation, mainly due to reduced stomatal conductance (gs), or by metabolic impairment, leading to lower co2 assimilation (flexas and medrano, 2002). often warming and drought occur in the field simultaneously, but their effects on crop performance are often analysed separately (shah and paulsen, 2003; gray and brady, 2016). however, the combination of multiple abiotic stresses can result in climate change effects that differ strongly from those observed in single-factor experiments (gray and brady, 2016) and often result in more adverse impacts on plant development and crop yield than under a single stressor (barnabás et al., 2008). to date, little data is available from climate manipulation experiments done in agricultural ecosystems. the cultivation of barley (hordeum vulgare l.) is expected to increase in the future due to its relative drought tolerance, which is an important trait with respect to food security (richardson et al., 2009; högy et al., 2013). however, barley is vulnerable to reduced water availability during flowering and ear formation, because water shortage can shorten the grain filling period and therefore have negative impacts on barley grain weight and size (sánchez-díaz et al., 2002; gonzález et al., 2007; samarah et al., 2009). spring barley is used as feedstock for animal feed and malt production. with regard to the effect of air temperature increase on barley grain yield, previous studies have shown a reduction in yield (savin et al., 1997; alemayehu et al., 2014). effects of soil warming and altered precipitation on barley 45 the aim of the present study was to investigate the interactive effects of soil warming and altered precipitation amount and frequency on photosynthesis, crop development, and yield of spring barley in an arable field near stuttgart (germany). we hypothesized (i) that soil warming accelerates barley development during spring but not during later developmental stages, when the soil is dryer due to higher air temperatures and less precipitation in comparison to the period of spring. thus, an elevation in soil temperatures during later growth stages would decrease soil water amount additionally, which limits plant growth. furthermore, (ii) we expected a greater influence on photosynthesis from elevated soil temperature than from altered precipitation amount and frequency, because this physiological process is well known to be highly sensitive to temperature changes. we hypothesized further (iii) that reduced precipitation amount or frequency during summer months will decrease biomass production and grain yield. finally, (iv) we expected an additive negative effect of the three climate factors − soil warming, reduced precipitation amount, and reduced precipitation frequency − on ecophysiology of barley. to test these hypotheses, we used the hohenheim climate change (hocc) experiment where since 2008 an increase in soil warming (+ 2.5 °c) and during summer a reduction in precipitation amount (-25%) and frequency (-50%) is simulated under field conditions. we collected data on plant physiological responses and plant performance. photosynthesis was measured at stem elongation and flowering. plant development was monitored over the entire growing period. biomass and yield data were collected at stem elongation, flowering, and maturity. materials and methods site description the hohenheim climate change (hocc) experiment is located at the research station heidfeldhof at the university of hohenheim (stuttgart) (48°43´n, 9°13´e, 401 m a.s.l.), and was established in 2008. the soil is a loess-derived stagnic luvisol with ph 7.0, organic carbon content of 12.1 g kg−1, and texture of 9.4% sand, 68.1% silt, and 22.6% clay. annual mean air temperature and precipitation at the site (1961-1990) were 8.7 °c and 679 mm, respectively (dwd, 2019). in 2016, the annual mean air temperature and precipitation were 10.1 °c and 595.4 mm, respectively (weather station “hohen heim”, agricultural technology centre (ltz) augustenberg, 2018). during the growing season of spring barley, from april until august 2016, average air temperature was 15.7 °c and total precipitation was 312 mm (fig. 1), which is in the range of the long term average air temperature and total precipitation of 15.6 °c and 377.4 mm, respectively (1961-1990, agricultural technology centre (ltz) augustenberg, 2018). experimental design within the hocc experiment, future climate conditions, i.e., soil temperature (t), precipitation amount (a), and frequency (f) were simulated based on climate change predictions to 2100 for southwest germany (umweltbundesamt, 2006). since 2008, soil temperature has been manipulated during the entire year and precipitation patterns have been manipulated during summer months (june to august). in 2016, precipitation manipulation began on 04 june 2016 and was conducted until the final harvest of barley: in the ambient soil temperature treatments this date was 02 august 2016 while in the elevated soil temperature treatment harvest date was 27 july 2016. treatments are set up in a split-plot-design replicated in four blocks. each block consists of two plots (each 1 m × 4 m), one with ambient and one with elevated soil temperature. soil temperature is elevated by 2.5 °c (te) at 4 cm depth and is achieved by heating cables installed on the soil surface (rs 611-7918, rs components gmbh). dummy cables on ambient soil temperature plots (ta) account for effects of the presence of heating cables on the soil, such as retention of water from precipitation. each plot consists of four 1 m × 1 m subplots, each having a different combination of the two precipitation factors; amount (a) and frequency (f). the surface area of the subplots (1 m × 1 m) is lower than that normally used in field experiments, but was considered suitable as the plant density was comparable to other field experiments studying effects of soil warming or low water availability on cereals (gonzález et al., 2010; patil et al., 2010) and allowed a high number of treatment replicates. roofs are used to protect the plots from precipitation (folitec uv 5 foil, folitec agrarfolien-vertriebs gmbh). the height of the roofs is between 2 and 2.4 m at the lowest and highest point, respectively. precipitation is collected in rain barrels and subplots are manually watered, making it possible to precisely control precipitation amount on the plots. in the manipulated plots precipitation amount fig. 1: average daily air temperature (2 m), ambient and elevated daily soil temperature at the experimental site during the growing season from 01 april until 31 august 2016. harvest dates are labelled as follows: h1: harvest 1 at stem elongation (dc 31); h2: harvest 2 at flowering (dc 65); h3: harvest 3 at maturity (dc 92); ta: ambient temperature; te: elevated temperature. the harvest of plants grown under ambient soil temperature was about one week after plants grown under elevated soil temperature. harvests dates: h1-ta at 02 june 2016; h1-te at 25 may 2016; h2-ta at 01 july 2016; h2-te at 23 june 2016; h3-ta at 02 august 2016; h3-te at 27 july 2016 (see tab. 1) (a). daily precipitation and the amount of daily precipitation reduced by 25 % (named as reduced daily precipitation) as well as soil moisture measured in different treatments (ambient; at 2.5 °c elevated soil temperature over the whole growing period; at -25 % reduced precipitation amount from 04 june 2016 until final harvest (b). temperature and precipitation data are from the weather station “hohenheim” of the agricultural technology centre (ltz) augustenberg, germany. soil moisture data are from tdr probes installed in 0-15 cm depth at every subplot at the hocc experiment. 46 i. drebenstedt, i. schmid, c. poll, s. marhan, r. kahle, e. kandeler, p. högy was reduced by 25% (ar) compared to ambient precipitation (aa). precipitation frequency simulated longer dry periods by reducing the number of rainy days by 50% (fr), i.e. the cumulative precipitation amount of two events was delivered as one event compared to ambient precipitation frequencies (fa). pvc barriers around each subplot impede lateral water movement. in addition to the roofed plots (rr), each block includes two subplots without roofs (roof-control: rc) to control for any roof effect on plant development. precipitation patterns are not manipulated in the roof-control subplots. in every subplot, soil temperature is recorded using temperature probes at 4 cm depth and soil moisture is measured in a range of 0-15 cm depth using tdr probes (cs630/cs635, campbell scientific ltd.). additional information about the experimental setup is given in poll et al. (2013). plant cultivation and biomass harvest since 2008, within the hocc experiment, wheat, barley, and oilseed rape have been cultivated in a crop rotation. this study deals with spring barley (hordeum vulgare l. cv. rgt planet, rubin® tt stained), which was sown on 05 april 2016 (0 days after sowing, 0 das) at a density of 400 plants m-2 and adjusted to a final density of 290 plants m-2 on 06 may 2016. plants were fertilised with 60 kg n ha-1 using calcium ammonium nitrate (29 april 2016). on 06 june 2016, 2.5 l ha-1 fungicide osiris was applied. three harvests were made at specific plant developmental stages. the first harvest took place at the beginning of stem elongation (dc 31; bbch code (meier, 2001), while the second harvest was at full flowering (dc 65) (tab. 1). at the first and second harvests, two representative plants per subplot were cut one cm above the soil surface. the numbers of green and senescent leaves, stems, and ears were counted and fresh weight was determined. as plants cultivated on subplots with elevated soil temperature grew faster and reached the specific dc stage earlier than plants on subplots with ambient soil temperature, plants on heated subplots were harvested approximately one week before the non-heated plants (tab. 1). at the final harvest (dc 92), all plants in a square of 0.5 m × 0.5 m in the centre of each subplot were cut one cm above the soil surface and treated identically to the plants taken at the first and second harvests. leaves and stems were dried at 60 °c and ears at 30 °c to constant weight. ears were threshed to separate grains. grain yield was measured and thousand grain weight (tgw) was determined using a seed counter (condator “e”, pfeuffer, germany). grains were then separated into grain size classes (gsc: >2.8 mm; 2.8 2.5 mm; 2.5 2.2 mm; <2.2 mm) using a sortimat (type k, pfeuffer, germany). biomass of roots were sampled with a cylinder (20 cm length, 4.5 cm ø), taking a soil core containing roots of two barley plants on 01 june 2016 (das 57, dc 31), 27 june 2016 (das 83, dc 65) and 19 july 2016 (das 105, dc 92), which were near the three harvest dates of the aboveground biomass (tab. 1). because of the severe soil disturbance, sampling of barley roots was not possible in all subplots and was done only at roof-control subplots with ambient and elevated soil temperature, meaning that no effects of changes in precipitation patterns on biomass of roots could be tested. roots were washed over a sieve (mesh size 1 mm) and dried at 40 °c for 2 days to determine the root dry weight per plant. measurement of plant-related parameters five plants in the center of each subplot were labelled with rings around the stems. these plants were monitored for all crop development parameters. plant phenology was measured weekly using the bbch decimal codes (meier, 2001). greenness index of the pen ultimate leaf was measured from 62 das onwards using a spad meter (konica minolta optics inc., japan) to detect possible differences in leaf senescence during the growing period between all treatments. the spad measurements were performed at three different positions at the central part of the leaf. from these three values a mean value was calculated. water use efficiency of the biomass (wueb) was calculated by dividing total aboveground biomass per plant by total water use per plant until final harvest. additionally, the ratio between grain yield per plant and total water use per plant until final harvest was calculated for the water use efficiency of grain yield (wuey). total water use per plant was calculated by dividing precipitation amount per m² (from sowing until final harvest) by the number of plants per m² of each subplot. precipitation amount data were taken by the weather station “hohenheim” (agricultural technology centre (ltz) augustenberg, 2018). precipitation amount per m² was higher in subplots with ambient than elevated soil tempe rature, because final harvest of barley under ambient soil temperature conditions was approximately one week later. leaf gas exchange on each subplot one plant was labelled and used only for gas exchange measurements. the youngest fully expanded leaf was chosen for the measurement, resulting in a total of one measurement per plant at each measurement date. gas exchange was measured during two different time periods: (1) one week before and one week after the first harvest (stem elongation) and (2) one week before and one week after the second harvest (flowering) with a li-cor open photosynthesis system (li-6400). measurements during stem elongation were taken on 20 may, 01 june, and 07 june 2016; those during flowering were taken on 22 june, 27 june, and 04 july 2016 between 09:30 and 13:30 each. before each measurement, the spad value of the leaf used for gas exchange measurement was measured three times to calculate an average spad value. then the leaf was fixed in the chamber head and the in-chamber leaf area was calculated using a ruler. afterwards, the in-chamber conditions were adjusted and the leaf adapted for 10 minutes to the conditions inside the chamber. in-chamber conditions were as follows: reference co2 (co2r) was set to 400 μmol co2 mol-1 and light intensity in the leaf chamber (parin) was set to 1500 μmol m-2 s-1. flow rate to the leaf chamber was adjusted to 400 μmol s-1. also, relative humidity (rh) in the leaf chamber, leaf temperature (tleaf), and vapour pressure deficit at the leaf surface (vpdl) were controlled: leaf temperature reflected the mean midday temperatures of each time period. for time period 1, rh was adjusted to 57.8 ± 4.7%, tleaf was set to 21.2 ± 3.1 °c, and vpdl was 1.2 ± 0.2. tair outside the leaf chamber was on average 19.7 ± 3.6 °c. during time period 2, the parameters were as follows: rh 52.2 ± 9.0%, tleaf 30.0 ± 0.03 °c, vpdl 1.9 ± 0.3, and tair 30.8 ± 1.6 °c. the means of each gas exchange parameter for time periods 1 and 2 were then calculated. net photosynthesis (anet), stomatal conductance (gs), and transpiration (e) were derived from the gas exchange measurements. instantaneous water use efficiency of photosynthesis (wuep) was calculated using the formula anet/e. tab. 1: harvest dates of the aboveground biomass. plants on plots with ambient and elevated soil temperature were sown on the same day (05 april 2016) but harvested on different dates (ta: 02 august 2016 and te: 27 july 2016). harvest date harvest development stage ambient elevated soil temperature soil temperature first dc 31, stem elongation 02 june 2016 25 may 2016 second dc 65, full flowering 01 july 2016 23 june 2016 final dc 92, maturity 02 august 2016 27 july 2016 effects of soil warming and altered precipitation on barley 47 statistical tests each variable was analysed by a linear mixed-effects model. fixed factors were “soil temperature” (ta and te), “precipitation amount” (aa and ar), and “precipitation frequency” (fa and fr). random factors were block, plot and subplot. data were analysed separately for each measurement date and were checked for outliers using the grubb’s test (grubbs, 1950). outliers were eliminated from the data set. an analysis of variance (anova) was applied to the model to detect significant main and interaction effects of the fixed factors soil temperature (t), precipitation amount (a), and precipitation frequency (f) on each variable (e.g., plant height). data were ln transformed prior to analysis if heterogeneity of variance was identified by levene’s test. a level of probability of p ≤ 0.05 was set as statistically significant. least significant difference (lsd) post-hoc tests were performed. the data were analysed with the statistical software r (version 3.4.2 for windows, r foundation for statistical computing, vienna, at). the lme function of the r 3.4.0 nlme package provided the linear mixed-effects model. for the grubb’s test the r package “outliers” was applied and the levene’s test was done with the levenetest function of the r package “car”. the lsd test was done with the r package “agricolae”. results environmental conditions warming increased soil temperature in 4 cm depth over the entire growing period by on average 1.51 ± 0.49 °c in roofed plots and 1.94 ± 0.35 °c in roof-control plots. plants grown under ambient soil temperature developed more slowly than those in the elevated soil temperature treatment and therefore were finally harvested six days later than plants under soil warming (tab. 1). as a consequence, the precipitation amount and the number of rain events varied between subplots with ambient and elevated soil temperature. in ambient soil temperature plots, precipitation amount was 139.7 mm in the control and 104.8 mm in the reduced treatment, meaning a reduction in precipitation amount by 25% (34.9 mm). under soil warming, the precipitation amount was reduced by 25% (33.6 mm) from 134.3 mm in the control to 100.7 mm in the reduced treatment. the number of rainy days was decreased by 50% from 26 to 13 and from 24 to 12 days, for ambient and elevated soil temperature subplots, respectively. soil warming and a reduction in precipitation amount decreased soil moisture compared to control subplots (fig. 1), but not significantly maybe due to variability in the soil moisture measurements. plant development plants under soil warming developed faster with the beginning of stem elongation, which led to accelerated formation of the first node (dc 31) by seven days (tab. 2). accordingly, the first harvest at stem elongation had to be conducted earlier on elevated soil temperature subplots than on ambient soil temperature subplots. under soil warming conditions, plants also reached full flowering (dc 65) and fully ripe (dc 89) stages seven and five days earlier, respectively. the final harvest of hard grains (dc 92) of barley grown under elevated soil temperature was six days before that grown under ambient soil temperature conditions. from the beginning of plant development measurements (24 das) until the last measurement date (111 das), elevated soil temperature increased plant height (fig. 2). roof effects on barley height were limited to das 38 and were less pronounced under ambient (+8%) than under elevated soil temperature (+30%) (data not shown). spad values of the penultimate leaf, measured on five monitored plants per subplot, were increased due to elevated soil temperature on average from 38.9 to 46.0 at 70 das and from 42.8 to 46.9 at 77 das (fig. 3). after plants under elevated soil temperature reached full flowering stage (dc 65) at das 84, spad values at the warmed plots approximated the values at the control plots. a reduction in precipitation amount and frequency had no significant effect on spad values over the entire vegetation period. leaf gas exchange during stem elongation, leaf gas exchange was measured on leaves of plants with similar spad values (between 40.3 and 43.1) over all treatments (data not shown). thus, differences in gs and e were not due to differences in spad values. during flowering, the youngest fully expanded leaf showed no differences between spad values over all treatments. however, spad values at flowering were lower than at stem elongation, falling between 30.0 and 39.6. during stem elongation, longer dry periods as consequence of reduced precipitation frequency reduced gs by 33% (fig. 4). a reduction in precipitation amount increased gs and e by 30% and 20% retab. 2: duration of growth stages from sowing until final harvest of spring barley. decimal code (dc) was used to quantify the growth stages (meier, 2001). sowing date: 05 april 2016. final harvest of plants grown under ambient and elevated soil temperature were on 02 august 2016 and 27 july 2016, respectively. date of reaching a specific development stage duration from sowing to achieve each stage (days) development stage ambient soil elevated soil ambient soil elevated soil (dc stadiums) temperature temperature temperature temperature first leaf unfolded (11) 29 april 2016 29 april 2016 24 24 first tiller detectable (21) 14 may 2016 14 may 2016 39 39 first node at least 1 cm above tillering node (31) 01 june 2016 25 may 2016 57 50 flag leaf unrolled, ligule just visible (39) 09 june 2016 03 june 2016 65 59 first awn visible (49) 14 june 2016 06 june 2016 70 62 end of heading (59)1 03 july 2016 23 june 2016 89 79 full flowering: 50% of anthers mature (65) 28 june 2016 21 june 2016 84 77 late milk (77) 14 july 2016 03 july 2016 100 89 fully ripe (89) 22 july 2016 17 july 2016 108 103 hard grain harvest (92) 02 august 2016 27 july 2016 119 113 1a high number of plants entered the full flowering stage (dc 65) before all plants completed the bbch stage end of heading (dc 59). therefore, the dc 59 stage was completed on ambient and elevated soil temperature subplots after the dc 65 stage was finished. 48 i. drebenstedt, i. schmid, c. poll, s. marhan, r. kahle, e. kandeler, p. högy yield parameter at maturity, soil warming increased the number of ears per plant by 36% (fig. 6) and tended to increase the biomass of ears by 51% (p = 0.057, tab. 3) as well as grain yield by 54% (p = 0.057, fig. 6). barley grown under reduced precipitation frequency had 6% less tgw compared to controls (tab. 3). harvest index was not significantly affected by the climate factors soil warming, precipitation amount, and precipitation frequency. the wuey of barley increased by 13% under reduced precipitation amount. all grain size classes (gsc) were affected by reduction in precipitation amount (tab. 3). thus, reduced precipitation amount led to a 9% increase in grains >2.8 mm, whereas gsc 2.8-2.5 mm, gsc 2.5-2.2 mm, and gsc <2.2.mm decreased by 11%, 8%, and 2%, respectively. a reduction in precipitation frequency increased gsc 2.5-2.2 mm by 43%. barley tended to produce 1% more grains >2.5 mm under reduced precipitation amount (p = 0.053, data not shown). roofing increased gsc >2.8 mm by 19%, and decreased gsc 2.8-2.5 mm by 19% (data not shown). discussion plant development elevated soil temperature accelerated barley development over the entire growing period to maturity, resulting in about one week earlier fig. 2: plant height measured between 24 das and 111 das at ambient (ta) and elevated soil temperature (te). asterisks indicate significant differences between plants under ambient and elevated soil temperatures (*p ≤ 0.05; **p ≤ 0.01); n = 4. fig. 3: spad values of the penultimate leaf, measured under ambient (ta) and elevated soil temperature (te). spad values are averages of five plants of each subplot, used as monitor plants. asterisks indicate significant differences between plants under ambient and elevated soil temperatures (*p ≤ 0.05; **p ≤ 0.01); n = 4. fig. 4: reactions of net photosynthesis (anet) (a), stomatal conductance (gs) (b), and transpiration (e) (c) to changes in soil temperature (ta, ambient; te, elevated), precipitation amount (aa, ambient; ar, reduced) and precipitation frequency (fa, ambient; fr, reduced). measurements were performed at stem elongation and flowering. means and sds are shown, asterisks indicate significance (*p ≤ 0.05, **p ≤ 0.01) tested by three-way anova applied to a mixed-effects model; n = 3. different letters indicate significant differences between treatments (lsd test, p ≤ 0.05). spectively under ambient soil temperature. however, this effect was opposite that under elevated soil temperature, where reduced precipitation amount decreased gs and e by 20% and 16%, respectively. wuep of barley was reduced by 13% under reduced precipitation amount among plants grown under ambient soil temperature (tab. 3). but under elevated soil temperature, the wuep increased by 16% if the precipitation amount was reduced. at flowering, the spad values of plant leaves used for leaf gas exchange measurements were similar (between 30.0 and 39.6) for all treatments (data not shown). the gas exchange parameters anet, gs and e were not significantly affected by any of the three climate factors (fig. 4). however, values of gs and e were considerably lower at flowering than at stem elongation, resulting in lower rates of anet in all treatments. no treatment effect on wuep could be detected at flowering. biomass production at stem elongation, biomass of senescent leaves was 71% higher under ambient than elevated soil temperature, whereas biomass of green leaves and total aboveground biomass remained unaffected (tab. 3). at flowering, soil warming increased aboveground biomass production by 6% and increased biomass of green leaves and stems by 135% respective 26%. if soil warming and reduced precipitation frequency occurred at the same time, there was an increase in aboveground biomass (+18%), biomass of senescent leaves (+35%), and ears (+21%). at maturity, biomass of stems increased by 46% due to soil warming. barley had a 13% higher wueb under reduced precipitation amount. moreover, wueb increased tendentially by 60% under elevated soil temperature (p = 0.067, tab. 3). root biomass increased by 80% under elevated soil temperature at stem elongation, whereas at flowering or maturity no effects on root biomass could be detected (fig. 5). effects of soil warming and altered precipitation on barley 49 ta b. 3 : b io m as s pr od uc tio n, y ie ld p ar am et er s, a nd w at er -u se e ffi ci en cy o f ba rl ey . p la nt s w er e gr ow n un de r am bi en t ( t a ) or e le va te d (t e) s oi l t em pe ra tu re in c om bi na tio n w ith th e fo llo w in g pr ec ip ita tio n pa tte rn s: am bi en t ( a a) o r r ed uc ed (a r) pr ec ip ita tio n am ou nt a nd a m bi en t ( f a ) o r r ed uc ed (f r) pr ec ip ita tio n fr eq ue nc y. a t a t e t hr ee -w ay a n o va b a a a r a a a r m ai n ef fe ct s in te ra ct io ns f a f r f a f r f a f r f a f r t a f t ×a t ×f a ×f t× a ×f b io m as s pr od uc tio n [g d w p la nt -1 ] f ir st h ar ve st (s te m e lo ng at io n) a bo ve gr ou nd 0. 68 ± 0 .2 4a 0. 67 ± 0 .0 7a 0. 63 ± 0 .2 2a 0. 56 ± 0 .2 0a 0. 53 ± 0 .2 9a 0. 48 ± 0 .1 7a 0. 52 ± 0 .1 2a 0. 45 ± 0 .0 5a ns ns ns ns ns ns ns g re en le av es 0. 34 ± 0 .1 3a 0. 35 ± 0 .0 5a 0. 34 ± 0 .1 1a 0. 30 ± 0 .1 0a 0. 32 ± 0 .1 6a 0. 29 ± 0 .1 0a 0. 31 ± 0 .0 5a 0. 26 ± 0 .0 4a ns ns ns ns ns ns ns se ne s. le av es 0. 01 4± 0. 00 8a b 0. 01 0± 0. 00 6a bc 0. 01 3± 0. 00 9a 0. 01 0± 0. 00 7a bc 0. 00 4± 0. 00 5c 0. 00 2± 0. 00 0a b 0. 00 7± 0. 00 0b c 0. 01 0± 0. 00 3a bc 0. 03 5 0. 06 2 ns ns ns ns ns st em s 0. 33 ± 0 .1 1c de 0. 31 ± 0 .0 4d e 0. 28 ± 0 .1 1b cd 0. 25 ± 0 .1 1e 0. 20 ± 0 .1 3a bc 0. 18 ± 0 .0 7a 0. 20 ± 0 .0 6a b 0. 18 ± 0 .0 1a bc 0. 06 5 ns ns ns ns ns ns se co nd h ar ve st (fl ow er in g) a bo ve gr ou nd 3. 84 ±0 .6 4b cd 3. 09 ±0 .5 4c d 3. 94 ±0 .9 7b cd 2. 86 ±0 .1 2d 4. 05 ±0 .1 2a bc 5. 56 ±1 .3 5a 5. 07 ±1 .1 0a b 5. 22 ±1 .7 4a b 0. 01 4 ns ns ns 0. 01 0 ns ns g re en le av es 0. 26 ± 0 .0 7b 0. 26 ± 0 .0 6b 0. 25 ± 0 .1 2b 0. 23 ± 0 .0 4b 0. 61 ± 0 .1 2a 0. 59 ± 0 .1 7a 0. 57 ± 0 .1 6a 0. 51 ± 0 .1 6a 0. 00 3 ns ns ns ns ns ns se ne s. le av es 0. 20 ±0 .0 4a b 0. 15 ±0 .0 4a bc 0. 21 ±0 .0 6a 0. 15 ±0 .0 4a bc 0. 09 ±0 .0 3c 0. 17 ±0 .0 5a b 0. 14 ±0 .0 5b c 0. 16 ±0 .0 6a bc ns ns ns ns 0. 00 3 ns ns st em s 1. 90 ±0 .3 7b cd 1. 68 ±0 .3 8c d 2. 17 ±0 .6 1a bc 1. 54 ±0 .0 5d 2. 39 ±0 .1 2a b 2. 89 ±0 .7 4a 2. 76 ±0 .4 9a 2. 50 ±0 .3 1a b 0. 02 0 ns ns ns 0. 08 2 ns ns e ar s 1. 48 ±0 .3 0a b 1. 01 ±0 .1 7b c 1. 31 ±0 .3 0a bc 0. 95 ±0 .1 1c 0. 96 ±0 .0 9c 1. 57 ±0 .3 0a 1. 60 ±0 .5 3a 1. 53 ±0 .5 4a ns ns ns ns 0. 01 0 ns ns f in al h ar ve st (m at ur ity ) a bo ve gr ou nd 2. 34 ± 0 .3 8a 2. 55 ± 0 .6 1a 2. 71 ± 0 .4 6a 2. 61 ± 0 5 0a 3. 47 ± 0 75 a 3. 71 ± 0 .9 4a 4. 09 ± 0 .7 9a 3. 63 ± 1 .1 9a 0. 06 2 ns ns ns ns ns ns se ne s. le av es 0. 18 ± 0 .0 4c 0. 20 ± 0 .0 4b c 0. 22 ± 0 .0 5a bc 0. 19 ± 0 .0 5b c 0. 25 ± 0 .0 6a bc 0. 26 ± 0 .0 6a b 0. 30 ± 0 .0 5a 0. 27 ± 0 .0 8a b 0. 07 6 0. 08 9 ns ns ns ns ns st em s 0. 68 ±0 .1 3d 0. 76 ±0 .1 8c d 0. 81 ±0 .1 2b cd 0. 74 ±0 .1 5c d 0. 99 ±0 .2 0a bc 1. 06 ±0 .2 1a b 1. 15 ±0 .1 9a 1. 08 ±0 .2 8a b 0. 04 8 ns ns ns ns ns ns e ar s 1. 48 ±0 .2 1d 1. 59 ±0 .3 9c d 1. 68 ±0 .3 0b cd 1. 68 ±0 .3 1b cd 2. 23 ±0 .4 9a bc 2. 39 ±0 .6 ab 2. 64 ±0 .5 6a 2. 28 ±0 .8 3a bc 0. 05 7 ns ns ns ns ns ns yi el d pa ra m et er s f in al h ar ve st (m at ur ity ) g ra in s iz e cl as se s [% g ra in s] >2 .8 m m 52 .1 ± 4 .6 bc 44 .8 ± 6 .3 c 56 .6 ± 3 .4 bc 53 .4 ± 1 0. 4b c 65 .2 ± 9 .9 ab 63 .9 ± 9 .2 ab 71 .2 ± 1 7. 0a 73 .8 ± 1 2. 56 a 0. 07 8 0. 02 3 ns ns ns ns ns 2. 82. 5 m m 34 .0 ± 6 .0 a 36 .0 ± 3 .9 a 30 .4 ± 3 .6 ab 33 .3 ± 8 .1 a 22 .9 ± 4 .1 bc 23 .9 ± 2 .9 bc 18 .7 ± 7 .0 c 20 .1 ± 8 .1 c 0. 03 0 0. 03 5 ns ns ns ns ns 2. 52. 2 m m 9. 6 ± 1. 8a b 13 .7 ± 2 .9 a 8. 8 ± 1. 3b 9. 6 ± 2. 1a b 7. 2 ± 3. 2b 7. 5 ± 4. 2b 6. 1 ± 5. 0b 5. 3 ± 3. 7b ns 0. 03 0 0. 04 8 ns ns ns ns <2 .2 m m 4. 3 ± 2. 3a b 5. 5 ± 1. 3a 4. 2 ± 1. 9a b 3. 6 ± 1. 7a b 4. 7 ± 2. 7a 4. 7 ± 2. 9a 1. 3± 0 .2 b 3. 3± 2 .3 ab ns 0. 01 5 ns ns ns ns 0. 06 3 yi el d pa ra m et er s f in al h ar ve st (m at ur ity ) h ar ve st in de x 0. 54 6± 0. 02 a 0. 53 6 ±0 .0 3a 0. 53 1 ±0 .0 3a 0. 55 3 ±0 .0 2a 0. 56 1± 0. 01 a 0. 55 2 ±0 .0 4a 0. 55 7 ±0 .0 2a 0. 53 7 ±0 .0 4a ns ns ns ns ns ns ns t g w 47 .1 ± 1 .7 ab 44 .4 ± 1 .1 b 47 .5 ± 1 .8 ab 47 .5 ± 2 .8 ab 48 .3 ± 2 .1 a 48 .0 ± 1 .9 a 50 .3 ± 6 .2 a 50 .2 ± 4 .4 ab ns ns 0. 05 0 ns ns ns 0. 07 4 [g 1 00 0 gr ai ns -1 ] w at er -u se f in al h ar ve st (m at ur ity ) to ta l w at er u se 0. 8 ± 0. 2a 0. 8 ± 0. 1a 0. 8 ± 0. 1a 0. 7 ± 0. 2a 0. 7 ± 0. 1a 0. 8 ± 0. 0a 0. 7 ± 0. 05 a 0. 7 ± 0. 1a ns ns ns ns ns 0. 09 1 ns [l p la nt -1 ] w at er u se e ffi ci en cy f ir st h ar ve st (s te m e lo ng at io n) w u e p 4. 2 ± 1. 1a bc 5. 2 ± 1. 1a 4. 5 ± 1. 0a b 3. 7 ± 0. 7b c 3. 3 ± 0. 7c 3. 8 ± 0. 6b c 4. 0 ± 0. 8a bc 4. 2 ± 0. 8a b ns ns ns 0. 02 0 ns ns ns [μ m ol m m ol -1 ] se co nd h ar ve st (fl ow er in g) w u e p 3. 2 ± 1. 5a 4. 8 ± 0. 8a 4. 0 ± 0. 4a 3. 8 ± 0. 7a 3. 0 ± 0. 5a 3. 5 ± 0. 8a 3. 2 ± 1. 5a 3. 3 ± 0. 7a ns ns ns ns ns ns ns [μ m ol m m ol -1 ] f in al h ar ve st (m at ur ity ) w u e b [g l1 ] 3. 0 ± 0. 2d 3. 1 ± 0. 5d 3. 4 ± 0. 8c d 3. 8 ± 1. 1b cd 4. 8 ± 1. 4a bc 5. 0 ± 0. 9a bc 5. 6 ± 1. 2a 5. 6 ± 2. 3a b 0. 06 7 0. 01 7 ns ns ns ns ns w u e y [g l1 ] 1. 6 ± 0. 1c 1. 7 ± 0. 3c 1. 8 ± 0. 5b c 2. 1 ± 0. 7a bc 2. 7 ± 0. 8a b 2. 9 ± 0. 5a b 3. 1 ± 0. 8a 3. 1 ± 1. 5a b 0. 06 2 0. 04 8 ns ns ns ns ns a d at a ar e m ea ns ± s ta nd ar d de vi at io ns a cr os s fo ur re pl ic at es (n = 4 ) a nd w er e te st ed b y th re ew ay a n o va fo r m ai n ef fe ct s or in te ra ct io n ef fe ct s of th e fix ed fa ct or s t, a , a nd f . l sd p os tho c re su lts in di ca te s ta tis tic al ly s ig ni fic an t di ff er en ce s at p < 0 .0 5 le ve l o f p ro ba bi lit y an d ar e la be lle d by d iff er en t l et te rs a bo ve th e st an da rd d ev ia tio ns . b ns = n ot s ig ni fic an t ( p > 0. 05 ); b ol d nu m be rs in di ca te s ig ni fic an t m ai n or in te ra ct io n ef fe ct s of t , a , f (* p ≤ 0. 05 , * *p ≤ 0. 01 ) a nd n um be rs in it al ic s in di ca te tr en d (0 .1 ≥ p > 0 .0 5) . a bb re vi at io ns : s en es . l ea ve s = se ne sc en t l ea ve s; d w = d ry w ei gh t; t g w = th ou sa nd g ra in w ei gh t; w u e = w at er u se e ffi ci en cy . 50 i. drebenstedt, i. schmid, c. poll, s. marhan, r. kahle, e. kandeler, p. högy flowering and final harvest. similarly, the rate of peanut development was also accelerated under elevated soil temperature in a greenhouse experiment (prasad et al., 2006). in contrast, plant development of winter wheat, which was also more rapid under elevated soil temperature, declined after stem elongation (patil et al., 2010). in the present study, the height of barley was significantly higher under elevated soil temperature over the entire growing period. this effect on canopy height was also reported for winter rapeseed grown under elevated soil temperature within the hocc experiment in 2014 (bamminger et al., 2016). our hypothesis, that elevated soil temperature accelerated plant development during spring was supported by these results. in plots with soil warming the evaporation rate was most likely increased, but the soil was still moist due to continuous precipitation events during spring 2016. in accordance, during spring there was no water scarcity and barley growth seemed to be stimulated due to soil warming. however, in contrast to our hypothesis we also found a more rapid plant development during later growth stages and at maturity. these findings are supported by a relatively wet summer with high precipitation amounts in the end of may and during june 2016. thus, different than expected, the soil was relatively wet after spring and an additional evaporation due to soil warming was most likely not strong enough to limit plant growth. in addition, also the wueb tended to increase in plots with soil warming. this can be an indication that barley did not experience water stress after spring under elevated soil temperature despite less total water use per plant due to higher evaporation compared to control group. leaf gas exchange photosynthesis is known as one of the most vulnerable physiological processes to warming in crops. in the present study, an increase in soil temperature showed no significant impact at stem elongation on anet, gs, or e, suggesting (1) crop photosynthesis reacts differently to changes in air and soil temperature, which has also been reported for grain yield in many studies (stone et al., 1999; gavito et al., 2001; patil et al., 2010) and (2) the soil temperature increase in this study may have been too small to prompt physiological changes. this is in agreement with findings of gavito et al. (2001) in winter wheat, who increased soil temperature by 5°c in chambers with a separate control of air and soil temperature, and who detected no effect of ele vated soil temperature on anet. however, the effect of a reduction in precipitation amount on gs and e seemed to depend on soil temperature: gs and e increased under ambient and decreased under elevated soil temperature if precipitation amount was reduced. these findings support observations from other studies, demonstrating that multiple factor experiments can identify new and more adverse effects of climate change on plant physiology than single factor experiments can do. this also confirmed our hypothesis that the simultaneous occurrence of multiple climate factors results in an additive negative effect on barley ecophysiology. in addition, longer dry periods as consequence of reduced precipitation frequency decreased gs at stem elongation, but e and anet were unaffected. in former studies with barley grown in growth chambers, gs decreased as a consequence of reduced water amount (schmid et al., 2016; gonzález et al., 2010). a simultaneous occurrence of reduced precipitation amount and soil warming decreased wuep, which is in agreement with the observed reactions of droughtand temperature-stressed wheat plants grown in a greenhouse (shah and paulsen, 2003). at flowering, the youngest fully developed plant leaves were still green, with spad values above 30 during gas exchange measurements. values of anet, gs, and e were lower than at stem elonga tion but without significant effects due to the three climate factors. similarly, jensen et al. (1996) measured gas exchange in oilseed rape at tleaf of 23-30 °c and also detected higher gs (and anet) values before flowering and a decrease in those parameters during and after flowering. it has also been reported for wheat that anet and gs in 16 genotypes were on average higher during stem elongation than during flowering (reynolds et al., 2000). overall, we hypothesized a greater impact of elevated soil temperature than of changes in precipitation patterns on photosynthesis, given that photosynthesis is a temperature sensitive process. this hypothesis could not be confirmed, since reduced precipitation frequency surrounding the stem elongation period significantly affected gas exchange by reducing gs. soil warming had a significant impact on gs and e only when it simultaneously occurred in combination with reduced precipitation amount. this was perhaps due to the fact that the effects of air and soil temperature on crop photosynthesis are different: an increase in air temperature directly affects leaf gas exchange, whereas elevated soil temperature indirectly affects crop physiology through effects on root growth and plant water and nutrient availability. biomass production at the early developmental stage (stem elongation), barley leaves senesced more under ambient than elevated soil temperature conditions. other studies have reported that biomass of senescent leaves typically increased under warming, as this is a symptom of heat stress (bita and gerats, 2013), but we could not detect this in the present study. in contrast to the study of patil et al. (2010) of winter wheat, aboveground biomass of barley did not increase under soil warming; it remained unaffected. but we observed an increase in root biomass fig. 6: effects of soil temperature (ta, ambient; te, elevated), precipitation amount (aa, ambient; ar, reduced), and precipitation frequency (fa, ambient; fr, reduced) on (a) ear number per plant and (b) grain yield per plant. measurements were performed at plant maturity. means and sds are shown, asterisk indicates significance (*p ≤ 0.05, tested by three-way anova applied to a mixed-effects model); n = 4. different letters indicate significant differences between treatments (lsd test, p ≤ 0.05). fig. 5: effects of elevated temperature (te, dark grey) compared to ambient temperature conditions (ta, light grey) on root dry weight (dw) of barley. harvests were done at stem elongation, flowering, and plant maturity. means and sds are shown, asterisk indicates significance (*p ≤ 0.05, tested by a mixed-effects model); n = 4. effects of soil warming and altered precipitation on barley 51 under soil warming, possibly because root growth is stimulated up to a species-specific temperature optimum (gray and brady, 2016). this could have led to an increase in the nutritive value of barley or have mitigated negative impacts of water loss through transpiration under elevated soil temperature on barley biomass production. at flowering, plants grown under soil warming conditions produced greater biomass of green leaves and stems, leading to an increase in aboveground biomass. gavito et al. (2001) also observed an increase in leaf and stem biomass under elevated soil temperature in climate chamber grown winter wheat which was harvested one week after the beginning of flowering. an increase in aboveground biomass of winter wheat was also reported by patil et al. (2010) under elevated soil temperature. in our experiment, a combination of warming and reduced precipitation frequency increased aboveground biomass and biomass of ears. this is similar to a study of winter wheat in which a higher total aboveground biomass also occurred at flowering under the condition of soil warming and reduced precipitation frequency interaction (patil et al., 2010). no effect of soil warming on root biomass was observed in our study at this stage. this was likely due to the completion of root growth before the beginning of flowering, providing the plant with more energy for the grain filling period. this may also explain our result that at maturity no soil warming effect was detected in root biomass. at maturity, elevated soil temperature increased biomass of stems. similarly, the aboveground biomass of field-grown maize in a cooltemperate climate increased under elevated soil temperature (stone et al., 1999). however, a former study at the same experimental area (hocc experiment) in 2010 found no significant effect of elevated soil temperature on aboveground biomass of spring barley (h. vulgare cv. quench) (högy et al., 2013). we hypothesized that we would detect a decrease in biomass production through reduced precipitation amount and frequency during summer months. we cannot confirm this by the results of the present study, as changes in precipitation patterns from the beginning of june to beginning of august did not appear to adversely affect biomass production of spring barley. some possible explanations for this result are: (1) barley is relatively tolerant to water scarcity and therefore the simulated precipitation changes were too moderate to harm biomass production, or (2) the relatively wet conditions during june 2016 mitigated negative effects of reduced precipitation amount and frequency on biomass production. yield components the final harvest of barley grown under elevated soil temperature occurred one week before plants under ambient soil temperature, however, no yield losses were detected in all treatments. under elevated soil temperature, barley experienced a two-day longer grain filling period compared to plants under ambient soil temperature, but this period occurred earlier than that of those grown under ambient conditions. under soil warming plants needed in total 26 days from full flowering (das 77) to full ripeness (das 103), whereas control plants needed 24 days. a lengthening in grain filling duration under soil warming is in contrast to a previous study with wheat and elevated air temperature, where a decrease in the length of the grain filling period was observed (sofield et al., 1977). in our study, these additional two days could explain the observed tendency toward grain yield increase under soil warming, meaning plants had more time to acquire carbohydrates for grain growth. these results are hard to compare with literature values, since only a few experiments with cereals grown under manipulated moderate soil warming in an arable field have been conducted to date. however, in a similar study at the same experimental site, no effect on spring barley grain yield was observed by högy et al. (2013) and also in a lysimeter experiment with winter wheat, soil warming of 5 °c showed no effect on grain yield (patil et al., 2010). in studies in which air temperature was increased, inducing heat stress on cereals, reductions in grain yield under warming have been reported (alemayehu et al., 2014; savin et al., 1997), whereas in our experiment a soil temperature increase of about 2 °c did not exceed the temperature optimum of barley and therefore grain yield was resilient and tended to increase. this may have been due to (1) sufficient water availability during the growing period as a consequence of moderate and relatively high ambient precipitation amounts during spring and june 2016, or to (2) stimulated root growth at stem elongation through an enhanced supply of water and nutrients. contrary to our hypothesis, changes in precipitation patterns had no effect on grain yield, possibly due to an increase in wuey under reduced precipitation amount. because the barley cultivar rgt planet is preferred as malting barley, their grain size is important for brewers and malt houses because it positively correlates with the amount of malt extract that can be obtained (schwarz and li, 2011). in our study, soil warming led to the formation of more ears per plant, but had only a minor impact on grain size: only the second biggest gsc, 2.8-2.5 mm, decreased under elevated soil temperature, as högy et al. (2013) found in the same experiment with spring barley in 2010. mostly reduced precipitation amount affected grain size due to shifting grain size patterns: barley produced more grains >2.8 mm and fewer grains smaller than 2.8 mm. therefore, grains >2.5 mm, which are relevant for the brewing industry, tended to increase under reduced precipitation amount. we also found that a reduction in precipitation frequency, unlike our observations under reduced precipitation amount, induced barley to produce more grains of smaller size, 2.5-2.2 mm, which was reflected by a reduction in tgw. overall, spring barley was shown to be tolerant of an absolute water shortage resulting from lower precipitation amount: grain yield was shown to be stable due to a shift in grain size patterns by the formation of more bigger grains and fewer smaller grains. in addition, the increase in biomass of roots at stem elongation under soil warming possibly mitigated negative impacts of reduced water availability on aboveground biomass and grain yield. conclusions the results of the present study indicate that with constant soil warm ing and a reduction in precipitation amount and frequency during summer months, barley produces stable biomass and yield with changes in ear number, grain size classes and biomass of roots. overall, barley development and biomass production were more strongly affected by elevation in soil temperature than by altered precipitation patterns. knowledge about climate change effects on barley production can help farmers to select appropriate crop varie ties under future climate conditions. however, a further interaction with an increasing atmospheric co2 concentration have to be investigated as well under field conditions, since effects of elevated soil temperature and altered precipitation patterns on barley ecophysiology, growth and yield can be different under atmospheric co2 enrichment. acknowledgements many thanks to gina gensheimer, zorica kauf, christiane scherzer and many students for their help, especially during the final harvest. we also thank herbert stelz for helping to maintain the hocc experiment. this work was kindly supported by the german federal environmental foundation (dbu) through a phd scholarship for ireen drebenstedt and financial support for field and laboratory work (grant number 20016/416). 52 i. drebenstedt, i. schmid, c. poll, s. marhan, r. kahle, e. kandeler, p. högy author contributions cp, ph and ek designed the study. is, cp, sm, ek, ph provided critical feedback of the manuscript. root biomass data originate from rk. id performed the experiments and wrote the manuscript. all authors read and approved the final manuscript. conflict of interest no potential conflict of interest was reported by the authors. references agricultural technology centre (ltz) augustenberg, 2018: agrarmeteorologie baden-württemberg, station hohenheim. retrieved 2nd september 2019, from http://www.wetter-bw.de/. alemayehu, f.r., frenck, g., van der linden, l., mikkelsen, t.n., jørgensen, r.b., 2014: can barley (hordeum vulgare l. s.l.) adapt to fast climate changes? a controlled selection experiment. genet. resour. crop evol. 61, 151-161. bamminger, c., poll, c., sixt, c., högy, p., wüst, d., kandeler, e., marhan, s., 2016: short-term response of soil microorganisms to biochar addition in a temperate agroecosystem under soil warming. agric. ecosyst. environ. 233, 308-317. doi: 10.1016/j.agee.2016.09.016 barnabás, b., jäger, k., fehér, a., 2008: the effect of drought and heat stress on reproductive processes in cereals. plant cell environ. 31, 11-38. doi: 10.1111/j.1365-3040.2007.01727.x bita, c.e., gerats, t., 2013: plant tolerance to high temperature in a changing environment. scientific fundamentals and production of heat stresstolerant crops. front. plant sc. 4, 1-18. doi: 10.3389/fpls.2013.00273 blum, a., 1996: crop responses to drought and the interpretation of adaptation. plant growth regul. 20, 135-148. doi: 10.1007/978-94-017-1299-6_8 bowen, g.d., 1991: soil temperature, root growth and plant function. in: waisel, y., eshel, a., kafkaki, u. 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http://dx.doi.org/10.1023/a:1022683210334 http://dx.doi.org/10.1071/a96064 http://dx.doi.org/10.1111/jac.12127 effects of soil warming and altered precipitation on barley 53 shah, n.h., paulsen, g.m., 2003: interaction of drought and high temperature on photosynthesis and grain-filling of wheat. plant soil 257, 219226. doi: 10.1023/a:1026237816578 sofield, i., evans, l.t., cook, m.g., wardlaw, i.f., 1977: factors influ encing the rate and duration of grain filling in wheat. aust. j. plant physio. 4, 785-797. doi: 10.1071/pp9770785 stone, p.j., sorensen, i.b., jamieson, p.d., 1999: effect of soil tempera ture on phenology, canopy development, biomass and yield of maize in a cool-temperate climate. field crops res. 63, 169-178. doi: 10.1016/s0378-4290(99)00033-7 umweltbundesamt, 2006: künftige klimaänderungen in deutschland regionale projektionen für das 21. jahrhundert. dessau. wahid, a., gelani, s., ashraf, m., fooland, m., 2007: heat tolerance in plants. an overview. environ. exp. bot. 61, 199-223. doi: 10.1016/j.envexpbot.2007.05.011 zheng, d., hunt, e.r. jr., running, s.w., 1993: a daily soil temperature model based on air temperature and precipitation for continental applications. clim. res. 2, 183-191. orcid christian poll https://orcid.org/0000-0002-9674-4447 sven marhan https://orcid.org/0000-0002-8954-5968 petra högy https://orcid.org/0000-0002-4833-1381 address of the authors: ireen drebenstedt1*, iris schmid1,2, christian poll3, sven marhan3, robert kahle3, ellen kandeler3, petra högy1 1institute of landscape and plant ecology (320), plant ecology and ecotoxicology, university of hohenheim, august-von-hartmann straße 3, 70599 stuttgart, germany 2institute of crop science (340), fertilization and soil matter dynamics, university of hohenheim, fruwirthstraße 20, 70599 stuttgart, germany 3institute of soil science and land evaluation (310), soil biology, university of hohenheim, emil-wolff-straße 27, 70599 stuttgart, germany *corresponding author: ireen.drebenstedt@uni-hohenheim.de © the author(s) 2020. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1023/a:1026237816578 http://dx.doi.org/10.1071/pp9770785 http://dx.doi.org/10.1016/s0378-4290(99)00033-7 http://dx.doi.org/10.1016/j.envexpbot.2007.05.011 art05 journal of applied botany and food quality 81, 132 135 (2007) lehrstuhl für gemüsebau – qualität pflanzlicher nahrungsmittel, wissenschaftszentrum weihenstephan, technische universität münchen essential oils as antioxidants of different coloured carrot cultivars (daucus carota l. ssp. sativus hoffm.)* ruth habegger, w.h. schnitzler (received may 23, 2007) summary eight different coloured carrot cultivars, in orange, white, yellow, purple with orange or yellow and cream core, and red colour, were examined for their content and composition of essential oils and for their antioxidant capacity. for this purpose, two biochemical test systems were chosen (abts-test, mda-test for lipid peroxidation). white and yellow carrot cultivars contained higher amounts of essential oils than orange cultivars. ‘nutri red’ had very high content of essential oils. there were significant differences between the cultivars in composition of their essential oils as shown by nine monoterpenes and two sesquiterpenes. in both test systems essential oils of carrots showed antioxidant activity. the essential oils of purple and yellow coloured carrots had higher teac values than the essential oils of orange, white and red carrots. introduction the presence of antioxidants in natural plants has received much attention as a disease preventive substance. several herbs and spices have been investigated for the antioxidative activities of their essential oils, for example salvia, eucalyptus, basil, and thyme leaves. some of the major aroma compounds found in these volatile extracts presented varying amounts of antioxidant effect, comparable to the known antioxidant α-tocopherol (fellah et al., 2006; lee and shibamoto, 2001; lee et al., 2005). essential oil from pinus mugo and some of its components showed antioxidative activity in several biochemical test systems (grassmann et al., 2003). there are vegetables like carrot, celery, celeriac, and parsnip, in which the essential oils serve as aroma relevant substances. up to now, nutritional value of these plants was based on their content of minerals, vitamins, and dietary fibre. but furthermore their nutritional and healthy value may be extended by antioxidant capacity of secondary plant products. the objective of this study was to analyse new carrot cultivars in comparison with orange carrots for the amount and antioxidant capacity of essential oils. materials and methods in 2005 and 2006 eight different coloured carrot cultivars were cultivated in a research field of the chair of vegetable science – quality of vegetal foodstuff at dürnast, freising, according to good agricultural practice: ‘napoli’ f1 (orange; bejo), ‘bolero’ f1 (orange, nickerson zwaan), ‘white satin’ f1 (white; bejo), ‘mello yello’ f1 (yellow; bejo), ‘purple haze’ f1 (purple with orange core; bejo), ‘purple rain’ f1 and ‘deep purple’ f1 (purple with yellow or whitecream core; bejo), ‘nutri red’ (red, seminis). carrots were harvested at optimal ripening stage. after washing, roots were sorted according to the european union external quality standard. a representative sample of at least 50 pieces was taken and deep frozen. at analysis date the deep frozen material was crushed and mixed. in that way losses of the volatiles were reduced. the essential oils were extracted by simultaneous steam distillation and pentane extraction (sde) in likens-nickerson-apparatus. in the further text these volatiles were called essential oils, whereby they were no essential oils in closer sense. the amount of the essential oil was determined gravimetrically. separation and identification of components was carried out as described earlier (habegger and schnitzler, 2005). 110 of about 400 detected components were identified (habegger and schnitzler, 2000). levels of separated components were calculated as area percent. analyses were done with 4 replications. for detection of antioxidant capacity a 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (abts) decoloration assay was used (cano et al., 2000; yu and ong, 1999). abts was oxidized by peroxidase (pod) and hydrogen peroxide (h2o2) to the green abts radical cation. by addition of antioxidants, here essential oils dissolved in ethanol, the abts-radical will be reduced to the original not coloured component. the reaction can be followed photometrically (wavelength 734 nm). the difference of absorption between 2 and 6 minutes is a measure of antioxidant capacity and is calculated into trolox equivalent antioxidant capacity (teac) value. increasing values of teac correspond to a higher antioxidant capacity. antioxidant capacity of the essential carrot oil was measured in a further model system for lipid peroxidation, the malondialdehyde (mda) test. lipid peroxidation of cell membranes has been simulated by α-linolenic acid; oxidation has been induced by copper (cu(ii)) and h2o2. as an end product of lipid peroxidation malondialdehyde is quantified by adduct formation with 1-methyl-2-phenylindol giving a red to purple substance which is quantified photometrically at 586 nm. values for inhibition of lipid peroxidation were calculated based on total oil amount of 100 g fresh matter (fm). inhibition was compared to the control without addition of essential oil (grassmann et al., 2001, 2002). analysis of variance was calculated by sas, multiple mean comparison by tukey-test at 95 % probability. equation of regression was calculated by excel 2000. results and discussion total amount of essential oil the carrot cultivars differ in the total amounts of essential oil content (tab. 1). cultivars with purple colour have about the same amount as the orange cultivars. ‘white satin’ and ‘mello yello’ have significantly higher contents of essential oils than orange and purple cultivars. alasalvar et al. (2001) reported highest total volatiles in white and lowest in yellow carrots. but names of carrot cultivars were not published. in 2005 the oil content of ‘nutri red’ was significantly the highest; in 2006 it was similar to ‘napoli’. ‘napoli’ tends to produce higher amounts of essential oil than ‘bolero’ which shows the differences between orange coloured cultivars in general. * the paper was presented at the 42th meeting of the „deutsche gesellschaft für qualitätsforschung (pflanzliche nahrungsmittel) dgq e.v.“ antioxidant capacity of essential oils essential oil of carrots has proved an antioxidant activity in abtstest. but the cultivars differ in their antioxidant capacity measured as trolox equivalents (fig. 1). purple coloured cultivars have the highest values of teac per mg essential oil. ‘white satin’ has the lowest trolox equivalents. the orange cultivars and ‘nutri red’ are similar in the antioxidant capacity of their essential oil. because of the high differences in total oil content, values for antioxidant capacity are calculated in teac in oil of 100 g fresh matter (fig. 2). because of the high antioxidant capacity in 1 mg oil, ‘purple rain’ and ‘deep purple’ have the highest teac despite their lower total oil content. ‘mello yello’, ‘purple haze’ and ‘nutri red’ are very similar and have some higher teac than ‘napoli’, ‘bolero’ and ‘white satin’. lipid peroxidation is inhibited by essential carrot oils (mda-test); but there are no differences between the tested cultivars. the inhibition by essential oils of ‘bolero’, ‘mello yello’ and ‘nutri red’ cultivated in 2003, is about 54 %, 57 % and 55 % compared to the control (völker, 2004). composition of the essential oils the essential oil of carrots is mainly composed of monoand sesquiterpenes. monoterpenes are for example: α-pinene, β-pinene, sabinene, β-myrcene, α-terpinene, limonene, γ-terpinene, p-cymene, α-terpinolene. sesquiterpenes are for example: β-caryophyllene, trans-γ-bisabolene (tentatively). trans-γ-bisabolene has been confirmed as an important carrot volatile in literature (toth-markus and takacs-hajos, 2001). these 9 monoterpenes and 2 sesquiterpenes amount to 70-90 % of total peak area (tab 2). additionally there are further components with a lower area percentage in the gas chromatogram. the orange carrot cultivars differ in their quantitative composition of the essential oils. in general, the main monoterpene is α-terpinolene which corresponds to results of alasalvar et al. (2001). the main sesquiterpene is β-caryophyllene. in 2005 and 2006 the essential oil of ‘napoli’ has higher area percentage of sabinene and β-myrcene than ‘bolero’. the essential oil of ‘white satin’ is characterized by high area percentage of β-caryophyllene, α-terpinolene and sabinene. the range of the last two components depends on cultivation conditions (habegger and schnitzler, 2005). the area percentage of β-caryophyllene in essential oil of ‘mello yello’ is very low. the main substance is α-terpinolene, followed by sabinene in 2006. but this seems to be an exception because the results in 2003 and 2004 were similar to 2005 (habegger and schnitzler, 2005). area of trans-γ-bisabolene is about 10 percentage. alasalvar et al. (2001) published, that terpinolene (24 %), sabinene (23 %), β-caryophyllene (14 %) and γ-bisabolene (14 %) are the main terpenoids in yellow carrots. in the gas chromatogram of the essential oil of ‘purple haze’ βcaryophyllene, trans-γ-bisabolene and α-terpinolene are the main terpenoids. the next one on the range is α-pinene, which is not usually for orange carrot cultivars. many similarities in composition of the essential oils are found for ‘purple rain’ and ‘deep purple’. the area of trans-γ-bisabolene amounts to 20 percentage of total gas chromatogram area. alphaterpinolene and β-caryophyllene are the next components on the range. the essential oil of ‘nutri red’ is dominated by α-terpinolene with about 40 to 50 area percentage. trans-γ-bisabolene is a ‘smaller’ component like in the orange carrot cultivar ‘napoli’. between antioxidant capacity and the components of essential carrot oils there seems to be only one positive and high correlation between the teac values and the area percentage of trans-γ-bisabolene. equation for regression are as following: in 2005 y = 0.620x + 2.738 (r2 = 0.696) and in 2006 y = 0.877x 0.361 (r2 = 0.932). there is no improvement in the correlation when adding area percentage of one of the other ten monoand sesquiterpenes. the antioxidant capacity of the different components of essential oils has to be measured to explain the antioxidant effect of the total oils. tab. 1: essential oil content of different carrot cultivars (mg/100 g fresh matter) cultivar 2005 2006 napoli f1 2.27 ± 0.19 3.56 ± 0.24 bolero f1 1.87 ± 0.20 2.27 ± 0.14 white satin f1 3.50 ± 0.31 4.84 ± 0.32 mello yello f1 3.27 ± 0.21 4.27 ± 0.40 purple haze f1 2.11 ± 0.17 1.73 ± 0.21 purple rain f1 2.32 ± 0.22 2.81 ± 0.24 deep purple f1 2.58 ± 0.08 2.24 ± 0.20 nutri red 4.44 ± 0.62 3.63 ± 0.20 fig. 1: antioxidative potential of essential oil of different carrot cultivars in two years (2005 column left; 2006 column right). 0 5 10 15 20 25 30 35 40 n ap ol i f 1 b ol er o f 1 w hi te s at in f 1 m el lo y el lo f 1 p ur pl e h az e f 1 p ur pl e r ai n f 1 d ee p p ur pl e f 1 n ut ri r ed t e a c µ m o l/ 1 m g o il fig. 2: antioxidative potential of essential oil of different carrot cultivars calculated in teac (µmol/100 g fresh matter) in two years (2005 column left; 2006 column right). 0 10 20 30 40 50 60 70 80 90 100 n ap ol i f 1 b ol er o f 1 w hi te s at in f 1 m el lo y el lo f 1 p ur pl e h az e f 1 p ur pl e r ai n f 1 d ee p p ur pl e f 1 n ut ri r ed t e a c ( µ m o l/ 1 0 0 g f m ) essential oils as antioxidants of different coloured carrot cultivars 133 134 ruth habegger, w.h. schnitzler because of its higher antioxidant capacity of essential carrot oil the nutritional and health value of the purple and yellow cultivars has increased compared to the orange cultivars. references alasalvar, c., grigor, j.m., zhang, d., quantick, p.c., shahidi, f., 2001: comparison of volatiles, phenolics, sugars, antioxidant vitamins, and sensory quality of different colored carrot varieties. j. agric. food chem. 49, 1410-1416. tab. 2: levels of compounds in essential oil of different carrot cultivars (gc peak area %). 2005 compound napoli bolero white satin mello yello purple haze purple rain deep purple nutri red f1 f1 f1 f1 f1 f1 f1 α-pinene 2.80 1.20 3.15 0.91 7.88 2.70 0.86 9.88 β-pinene 1.94 0.78 1.84 2.50 2.17 1.61 1.48 6.20 sabinene 10.11 4.50 18.38 9.56 0.62 0.59 0.09 0.27 β-myrcene 9.80 4.73 6.72 4.40 3.22 3.47 7.54 2.29 α-terpinene 0.16 0.10 0.22 0.19 0.06 0.06 0.05 0.08 limonene 2.38 2.18 4.19 2.20 1.43 1.63 2.21 3.28 γ-terpinene 2.08 2.24 2.18 5.10 3.48 5.91 4.80 6.19 p-cymene 0.45 0.45 0.46 1.47 0.48 1.23 1.22 1.07 α-terpinolene 18.87 19.86 8.55 32.31 13.00 19.12 17.48 43.66 β-caryophyllene 13.72 35.54 29.24 3.78 24.03 13.09 9.02 0.84 γ-bisabolene 8.37 3.74 0.23 14.92 22.23 23.14 21.01 8.45 sum of further identified 1.65 1.23 2.05 1.38 2.00 2.18 2.67 4.93 monoterpenes sum of further identified 3.16 5.39 5.40 3.28 3.99 3.29 3.68 1.34 sesquiterpenes myristicin 0 0.013 0 0 0.10 0.022 0 1.08 sum of total identified 75.50 81.95 82.60 81.99 84.70 78.04 72.11 89.56 compounds 2006 compound napoli bolero white satin mello yello purple haze purple rain deep purple nutri red f1 f1 f1 f1 f1 f1 f1 α-pinene 3.50 1.59 4.42 1.27 14.10 3.19 0.77 7.12 β-pinene 2.54 0.60 2.23 1.83 1.58 0.85 0.60 4.75 sabinene 23.31 9.51 13.88 25.45 0.36 0.13 0.09 0.15 β-myrcene 16.86 4.73 4.40 10.31 4.94 4.68 7.01 3.09 α-terpinene 0.15 0.14 0.12 0.24 0.04 0.05 0.04 0.05 limonene 1.99 1.81 3.10 1.68 1.38 1.37 2.04 2.34 γ-terpinene 1.91 3.70 1.67 5.43 4.39 7.26 4.35 4.81 p-cymene 0.25 0.49 0.25 0.83 0.37 0.82 0.91 0.65 α-terpinolene 23.44 28.42 20.28 33.42 20.66 26.07 25.55 57.53 β-caryophyllene 10.77 32.03 31.20 1.96 19.74 15.64 11.31 1.06 γ-bisabolene 5.68 2.19 0.11 7.48 16.35 22.14 18.03 6.75 sum of further identified 1.09 0.99 0.88 0.84 1.13 1.53 1.86 2.48 monoterpenes sum of further identified 1.56 2.85 3.73 1.53 1.93 2.56 3.18 0.88 sesquiterpenes myristicin 0 0.66 0 0 0.55 0.13 0 0.61 sum of total identified 93.05 89.71 86.27 92.27 87.52 86.41 75.75 92.78 compounds cano, a., acosta, m., arnao, m.b., 2000: a method to measure antioxidant activity in organic media. application to lipophilic vitamins. redox rep. 5, 356-370. fellah, s., diouf, p.n., petrissans, m., perrin, d., romdhane, m., abderrabba, m., 2006: chemical composition and antioxidant properties of salvia officinalis l. oil from two culture sites in tunisia. j. essent. oil res. 18, 553-556. grassmann, j., schneider, d., weiser, d., elstner, e.f., 2001: antioxidative effects of lemon oil and its components on copper induced oxidation of low-density lipoprotein. drug res. 51, 799-805. grassmann, j., hippeli, s., elstner, e.f., 2002: plant’s defence and its benefits essential oils as antioxidants of different coloured carrot cultivars 135 for animals and medicine: role of phenolics and terpenoids in avoiding oxygen stress. plant physiol. biochem. 40, 471-478. grassmann, j., hippeli, s., vollmann, r., elstner, e.f., 2003: antioxidative properties of the essential oil from pinus mugo. j. agric. food chem. 51, 7576-7582. habegger, r., schnitzler, w.h., 2000: aroma compounds in the essential oil of carrot (daucus carota l. ssp. sativus). 1. leaves in comparison with roots. j. appl. bot. 74, 220-223. habegger, r., schnitzler, w.h., 2005: aroma compounds of coloured carrots (daucus carota l. ssp. sativus hoffm.). j. appl. bot. food qual. 79, 130135. lee, k.g., shibamoto, t., 2001: antioxidant activities of volatile components isolated from eucalyptus species. j. sci. food agric. 81, 1573-1579. lee, s.j., umano, k., shibamoto, t., lee, k.g., 2005: identification of volatile components in basil (ocimum basilicum l.) and thyme leaves (thymus vulgaris l.) and their antioxidant properties. food chemistry 91, 131137. toth-markus, m., takacs-hajos, m., 2001: flavour substances of carrot cultivars. acta alimentaria 30, 205-218. völker, d., 2004: einfluss der sorte auf zusammensetzung und antioxidative wirkung ätherischer öle von möhren (daucus carota ssp. sativus). diplomarbeit, lehrstuhl für gemüsebau, technische universität münchen. yu, t.w., ong, c.n., 1999: lag-time measurement of antioxidant capacity using myoglobin and 2,2’azino-bis(3-ethylbenzthiazoline-6-sulfonic acid): rationale, application, and limitation. anal. biochem. 275, 217-223. address of the authors: dr. ruth habegger, professor dr. w.h. schnitzler, lehrstuhl für gemüsebau – qualität pflanzlicher nahrungsmittel, wissenschaftszentrum weihenstephan, technische universität münchen, d-85350 freising << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true 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/pagesize [907.087 680.315] >> setpagedevice journal of applied botany and food quality 95, 60 66 (2022), doi:10.5073/jabfq.2022.095.008 department of agrotechnology, college of abouraihan, university of tehran, iran simultaneous measurement of rice grain friction coefficient and angle of repose using rotating cylinders jafar massah*, javad khazaei (submitted: december 25, 2021; accepted: march 16, 2022) * corresponding author summary several methods have been developed to measure the friction coefficient of grains and seeds as well as their angle of repose, independently. however, it seems that one single method capable of simultaneous measurement of these two important parameters of grain products is of importance. in this study, simultaneous measurement of brown rice grain friction coefficient and angle of repose is performed by using rotating cylinders method. the effects of rotational speed, cylinder diameter and filling degree on rice grains, also lower and higher angle of repose along with their friction properties were considered. results showed that proper speed for reaching slumping and rolling motions depends on cylinder diameter and filling degree varying from 0.5 to 9.7 rpm and 2 to 34.6 rpm, respectively. an insignificant difference in the range of 1° to 4° was observed between lower and upper angles of repose which indicates the reliability of the proposed method to determine the dynamic angle of repose. results also revealed that rotational speed of cylinders did not significantly affect the lower and upper angles of repose; however, they were not affected by filling degree. finally, the friction coefficient of rice grains was affected by cylinder diameter and filling degree. the findings of this study may be applied in the design and mathematical modeling of grains’ motions in rotary dryers, mixers, and silos. keywords: rotating cylinders, friction coefficient, angle of repose, filling degree. introduction rice is one of the most important agricultural products around the world having an central role in human nutrition. storage, handling, and processing of rice grains are challenging, especially when con sidering minimization of product loss and maintaining grain quality during the storage. rotary drums with internal flights are widely used in food, mineral, and pharmaceutical industries for drying, mixing, coating, or chemical reaction processes (zhang et al., 2018; he et al., 2019). using rotary cylinder is a rather new method to measure both static and dynamic angles of repose and wall friction coefficient. two other angles, i.e., lower and upper angles of repose can also be determined through this method. measurements of these angles along with internal friction coefficient of grains and friction coefficient between grains and wall are useful in mathematical modeling of grains’ motions in rotary dryers, mixers and silos (helder et al., 2006). with a rotating drum half-filled with uniform sediment grains, three different angles of repose are clearly identified. they are the upper angle of repose formed at the inception of an avalanche, the lower angle of repose at the end of an avalanche, and the dynamic angle of repose characterized with continuous sediment transport down the slope. the study suggested that the different angles of repose should be treated with caution when applying in investigations of bedload transport, dune migration and local scour development (cheng et al., 2017). research indicates that angles of repose of grains are related to the slipping and rolling properties, specific weight, size and shape of the grains. these angles increase by increasing the slipping and rolling friction coefficient and decreasing the grains sphericity. however, they decrease when the size of the grains exceed a threshold value. the results of these research also show that under the present simulation conditions, the angle of repose is significantly affected by the sliding and rolling frictions, particle size and container thickness, and is not sensitive to density, poisson’s ratio, damping coefficient and young’s modulus. increasing rolling or sliding friction coefficient increases the angle of repose, while increasing particle size or container thickness decreases the angle of repose. based on the numerical results, em pirical equations are formulated for engineering application. the proposed simulation technique and equations are validated by com paring the physical and numerical experiments, where focus is given to the effects of particle size and container thickness (helder et al., 2006; zhou et al., 2002). several studies have been carried out to determine the static and dynamic angles of repose, lower and upper angles of repose, and wall friction coefficient for industrial materials (spurling, 2000; mellmann, 2001; aranson and tsimring, 2006). however, search reviews in bibliographic databases reveal that the simultaneous measurement of static and dynamic angles of repose, wall friction co efficient, and lower and upper angles of repose via rotating cylinders for agricultural grains may not be fully studied by researchers especially for a grain similar to brown rice. the objective of this study was: (a) to simultaneous measurement of brown rice grain friction coefficient and angles of repose via rotating cylinders, and (b) to investigate the effects of cylinder diameter, rotational speed and filling degree on friction coefficient, static, dynamic, lower, and upper angles of repose of the grains. according to the literature, two types of angles of repose have been investigated, i.e., static and dynamic angles of repose which can be measured using several methods. in the most common method, which is called piling method, grains flow onto a circular plate from a certain height. static slope angle (φd) is measured using eq. (1) φd = arctan (2 h / dh) (1) where h is the height in m, and dh is the diameter of the cone surface in m (joshi et al., 1993; kingsly et al., 2006). in another method which is called emptying method, grains are poured out through an outlet at the side of a container. the gradient of the grains inside the container is considered as the angle of repose (khazaei and ghanbari, 2010; jain and bal, 1997; karababa, 2006). a conventional method to determine the friction coefficient is the use of materials on an adjustable tilting surface. in this method, a hollow cylinder, open at both ends, is filled with the testing material and is placed on an adjustable tilting surface. the angle of the surface gradually increases until the materials inside the cylinder start to slip measurement of rice grain friction coefficient and angle of repose 61 down the surface. the static friction coefficient equals to the tangent of the surface angle (khazaei and ghanbari, 2010). in rotary cylinders, to measure the angle of repose and friction properties, the material is poured (up to the half) into the cylinder. by increasing the rotational speed, several types of movement are identified, namely, slipping, slumping, rolling, cascading, cataracting and centrifuging (fig. 1) (mellmann, 2001). the material behavior in rotary cylinder depends on various parameters, such as cylinder rotational speed, cylinder diameter, filling degree, particle size and grain shape (henein et al., 1983; mellmann, 2001; spurling, 2000). an undesirable behavior. in this motion, materials move as fluid inside the cylinder with no particle mixing. this motion mode should be prevented by rough walls or attaching sandpaper to the wall. in general, measuring the angles of repose for solid materials in rotating cylinders occurs in the slumping motion (mellmann, 2001). in this motion mode, the materials are lifted by the rotating cylinder wall to reach the upper angle of repose (β) at which grains are began to slide down and inclined to the horizontal by the lower angle of repose (α) (lie et al., 2005a, 2005b). α is also called natural angle of repose (aranson and tsimring, 2006). a detailed analysis of the α is brought by mellmann (2001) and liu et al. (2005b) who studied α in slumping motion. as a result, they found that friction coefficient of grains to the wall (µ) can be calculated by eq. (4): μ = tan α (4) in slumping motion, the material bed is lifted until it reaches β angle. at the upper point, particles begin to slide down in the form of an avalanche inclined to the horizontal to reach α (fig. 2a). by increasing the cylinder rotation speed, time intervals between two avalanches decreases and a continuous flowing happens which is called rolling motion. according to mellmann et al. (2004) and liu et al. (2006), the slope of the bed surface is remained nearly constant which is called dynamic angle of repose (q ) (fig. 2b). dynamic angle of repose (q ) is defined using eq. (5). α + β q = (5) 2 by increasing the speed, cascading motion occurs. the transition from rolling to cascading depends on both the rotational speed and particle size (spurling, 2000). slumping mode occurs in rotational speeds lower than 3% of standard speed in which the centrifuging motion happens. however, rolling and cascading modes often occur in moderate speeds, between 3% and 30% of the speed where centrifuging motion happens (henein et al., 1983). according to mellmann (2001), standard speed in which the centrifuging mode occurs (nc) can be calculated by eq. (6). (6) began to slide down and inclined to the horizontal by the lower angle of repose (α) (lie et al., 2005a, 2005b). α is also called natural angle of repose (aranson and tsimring, 2006). a detailed analysis of the α is brought by mellmann (2001) and liu et al. (2005b) who studied α in slumping motion. as a result, they found that friction coefficient of grains to the wall (µ) can be calculated by eq. (4): in slumping motion, the material bed is lifted until it reaches β angle. at the upper point, particles begin to slide down in the form of an avalanche inclined to the horizontal to reach α (figure 2a). by increasing the cylinder rotation speed, time intervals between two avalanches decreases and a continuous flowing happens which is called rolling motion. according to mellmann et al. (2004) and liu et al. (2006), the slope of the bed surface is remained nearly constant which is called dynamic angle of repose (θ ) (figure 2b). dynamic angle of repose (θ ) is defined using eq. (5). by increasing the speed, cascading motion occurs. the transition from rolling to cascading depends on both the rotational speed and particle size (spurling, 2000). slumping mode occurs in rotational speeds lower than 3% of standard speed in which the centrifuging motion happens. however, rolling and cascading modes often occur in moderate speeds, between 3% and 30% of the speed where centrifuging motion happens (henein et al., 1983). according to mellmann (2001), standard speed in which the centrifuging mode occurs (nc) can be calculated by eq. (6). (4) αµ tan= (5) 2 βα θ + = (6) r g nc ⎟ ⎠ ⎞ ⎜ ⎝ ⎛ = π 30 6 fig. 1: transverse motion modes of materials in a rotating cylinder (khazaei and ghanbari, 2010). two principal criteria for evaluating the material modes of operation are characterized in rotating cylinders: filling degree (eq. 2) and froude’s number (eq. 3) (ingram et al., 2005; liu et al., 2006). f = (1/π ) × (ε − sin ε cos ε) (2) fr = ω2 r / g (3) where ƒ is the filling degree of cylinder, ε is the filling angle which corresponds to the half bed angle of the circular segment occupied with material, fr is froude’s number, ω is the angular speed of cylinder in rad/s; r is the cylinder radius in m, and g is the gravitational constant in m/s2. transition from one mode to another occurs due to the changes in cylinder rotational speed and cylinder filling degree. in low speeds or lower filling degrees, slipping mode occurs which is fig. 2: (a) material bed motion in slumping mode, and (b) material bed motion in rolling mode. (a) (b) where r is the cylinder radius in m, and g is the gravitational constant in m/s2. centrifuging motion often occurs in speeds more than nc. in this mode, grains on the outer paths begin to adhere to the wall as a uniform film. therefore, friction coefficient and angles of repose are studied in slumping and rolling motion modes (henein et al., 1983). 62 j. massah, j. khazaei materials and methods materials rice grains were grown and harvested in a research farm in guilan (a province of iran), in 2019. the grains were cleaned manually to remove all foreign material and broken grains. the initial moisture content of the grains was 7.5% (w.b.). samples with higher moisture content were prepared by adding distilled water to the grains, ac cording to the equation reported by kingsley et al. (2006). the pre pared samples were held in polyethylene bags and stored at 5° c in a refrigerator for 48h before using them for the experiments. the physical dimensions of the rice grains were determined by taking 200 grains randomly and measuring the grains linear dimensions (length, width, and thickness) using a micrometer reading to 0.01 mm. for each grain, the geometric mean diameter, sphericity, volume, and surface area were determined using the equations reported by al mahasneh and rababah (2007) and baryeh and mangope (2003). the bulk density, true density, porosity and 1000 grainmass were also determined according to the methods proposed by kingsly et al. (2006). the experimental setup, simultaneously, determined the μ and angles of repose of the rice grains via rotating cylinders. a device with three horizontal rotating cylinders with diameters equal to 15, 20, and 30 cm was used for the experiments (fig. 3). each cylinder was made of polyethylene having a transparent glass plate at the front for visual observation. rotational speed of the cylinders was adjustable varying from 0.50 to 100 rpm and the filling degree of cylinders ranged from 7 to 46% of the total volume of cylinder. to avoid unwanted sliding motion, the inner walls of the cylinders were glued with sandpaper (spurling, 2000; ingram et al., 2005). during the experiments, material behavior was recorded by a 100-fps digital canon-pc1210 camera with 10 megapixel resolutions which was installed perpendicularly to the plane of the glass plate. physical dimensions including length, width and thickness were measured for randomly selected 100 grain samples with a 0.01 mm accuracy micrometer. other physical dimensions were calculated using the equations available in mohsenin (1986), baryeh and mangope (2002), and kingsly et al. (2006). obtained values are brought in tab. 1. in this study, the results of angles of repose obtained in cylinder rotating approach were compared with those obtained by piling and emptying methods, described in section 2. for the piling method, grains were flowed onto a horizontal surface through a funnel. then, d and h of the formed cone were measured and φd was calculated using eq. (1). in the emptying method, grains were poured out through a 25 × 25 × 20 cm outlet in front of a container. the gradient of the grains inside the container was determined as the static angle of repose (jain and bal, 1997; khazaei and ghanbari, 2010; karababa, 2006). furthermore, the friction coefficient measured in this study was compared to the result obtained by the tilting method described in section 2. in the tilting method, grains were poured into a hollow cylinder with a length of 80 cm and 50 cm in diameter and open at both ends which was placed on an adjustable tilting surface. the method of measurement is brought in section 2. fig. 3: the device used in this study which was established by ghanbari (2008). methods in the beginning of the experiments, each of the partially filled cylinders was rotated at 0.5 rpm. then, the speed gradually increased to reach the slumping or rolling motion. this process was recorded by the camera and then, images were extracted from video by windows movie maker software. images were used for the direct measurement of α and β in slumping mode and q in rolling mode using eq. (5). meanwhile, μ of rice grains was calculated using eq. (4). the experiment was carried out for all three cylinders with four replications. since both α and the motion behavior of the grains depend on their physical properties including size, shape and density. therefore, tab. 1: dimensional characteristics of rice grains dimension mean standard deviation length (mm) 7.94 0.46 width (mm) 1.89 0.10 thickness (mm) 1.72 0.09 geometric mean diameter (mm) 2.96 0.90 sphericity 0.37 0.01 surface area (mm2) 27.60 1.82 volume (mm3) 13.66 1.33 1000 grain mass (g) 20.31 0.004 density (g/mm3) 0.72 0.13 results and discussion fig. 4 depicts filling degrees at different rotating speeds in slumping, rolling, and cascading modes for cylinders with diameters of 15, 20, 30 cm. this figure shows that the slumping and rolling modes occurred in the range of 0.5 to 3 rpm with fr = 4.27 × 10-5 and 2 to 30 rpm with fr = 0.68 – (1.53 × 10-3), respectively in the largest cylinder. these parameters were in the range of 0.5 to 2.6 rpm with fr = 2.51 × 10-5 and 2 to 38 rpm with fr = 4.02 – (6.79 × 10-4) for the cylinder with 20 cm diameter. and for the smallest cylinder, they ranged from 0.5 to 9.7 rpm with fr = 2.06 × 10-5 and 8.6 to 34.6 rpm with fr = 4.89 – (7.77 ×10-3). as a result, by increasing the cylinder diameter for a constant filling percentage of grains, the rotation speed for transition from slumping to rolling motion effectively increased. similar results have been reported by liu et al. (2005a) and cristo et al. (2006) for coffee beans, grains gravel, sand and limestone grains. in fact, the transition from slumping to rolling mode depends on cylinder rotational speed, friction coefficient of grains to wall, cylinder filling percentage and materials physical properties (mellmann, 2001). the effects of the filling degree on β is reported for rice grains during slumping motion in fig. 5. as discussed in section 3, it is necessary to use the slumping motion to determine angles of repose. accordingly, it is essential to determine the rotational speed and per centage of filling degree for achieving slumping and rolling motion for rice grains. based on mellmann (2001), aranson and tsimring (2006), and liu et al., (2005a), α equals to static angle of repose and measurement of rice grain friction coefficient and angle of repose 63 fig. 4: filling degree at different rotating speeds in slumping, rolling, and cascading modes for cylinders with diameters equal to (a) 15 cm, (b) 20 cm, and (c) 30 cm. fig. 5: effect of filling degree on upper angle of repose for cylinders with diameters equal to 15, 20, and 30 cm. also, tangent of this angle equals to grains to wall friction coefficient. according to the figure, in the smallest cylinder, filling percentage had slight effect on β. however, for both the medium and the largest cylinders, this effect was higher. α and β ranged from 40° to 46° and 44° to 48°, respectively. liu et al. (2006) determined that β for steel balls and fertilizer pellets was 34.97° and 40.7° degrees, respectively. henein et al. (1983) obtained β for 15 industrial materials and reported values more than 32°. khazaei and ghanbari (2010) studied the effects of rotary cylinder parameters on α and β of wheat grains and showed that these angles vary from 27° to 33° and 33° to 37°, respectively. according to fig. 6, cylinder rotational speed had effective effects on α and β. also, the effect of different filling degrees and cylinder diameters on these parameters appeared to be effective, ranged from 1° to 4°. similar results are reported by liu et al. (2005b) for industrial materials. also, davidson et al. (2000) reported the range of 2° to 4° for α and β in sand with mean diameter of 4 mm. fig. 7 shows that the calculated θ for rice grains using eq. (5) were in the range of 42° to 47° at different cylinder diameters and filling degrees. according to the figure, filling degree has decreasing effects on θ in cylinders with 20 and 30 cm diameter; however, θ increases by increasing the filling degree for cylinder with 15 cm diameter. meanwhile, the diameter of the cylinder has also effective effects on θ. using prevalent methods for wheat grains, tabatabaeefar (2003) found that increasing the moisture content of 8-22% increased θ from 37° to 45°. results showed that both measured and calculated dynamic angle of repose (θ) in three different methods were effectively different (tab. 2). the difference between the values from rotating cylinders and emptying method was between 16-17°. in addition, the difference between the values from rotating cylinders and emptying methods 64 j. massah, j. khazaei with piling method were 1-15°. the difference between methods may be due to difference in grain mass and method apparatus. based on the results of this study, friction coefficient of rice grains to a wall surface covered with sandpaper, depends on filling degree and cylinder diameters (fig. 8). according to this figure, for cylinders with 30 and 20 cm diameters, filling degree had decreasing effect on wall friction coefficient; however, friction coefficient increased by increasing the filling degree for the cylinder with 15 cm diameter. results from these experiments showed effective differences for the average friction coefficient obtained from rotating cylinder and tilting method (tab. 3). the results in tab. 4 demonstrate dynamic angle of repose in rolling mode for rice grains. these results suggest that the eq. (5) is reliable enough to measure θ by using α and β in slumping motion. also, the results in tab. 5 have been obtained by measuring the friction coefficient in rolling mode and its calculation using eq. (4). the results show that eq. (4) is reliable enough to calculate the friction coefficient during the experiments. tab. 2: dynamic angle of repose obtained by three different methods. method average calculated θ (eq. 5), (°) average measured θ (°) rotating cylinder 44.04 43.91 piling 29.00 emptying 27.41 fig. 6: the differences between lower and upper angles of repose in rice grains due to different filling degrees and cylinder diameters ranges from 1° to 4°. fig. 7: effects of filling degree on calculated dynamic angle of repose using eq. (5) for cylinders with diameters equal to 15, 20, and 30 cm. fig. 8: effects of filling degree on friction coefficient of rice grains for cylinders with diameters equal to 15, 20, and 30 cm. tab. 3: friction coefficient of rice grains using two different methods. method friction coefficient rotating cylinder 0.92 tilting 0.64 tab. 4: dynamic angle of repose in rolling mode for rice grains. cylinder diameter filling degree measured β measured α measured θ calculated θ (cm) (%) (°) (°) (°) (°) 15 18.0 44 43 46 43.5 25.7 46 43 47 44.5 33.8 45 43 46 44 46.6 46 44 47 45 20 16.5 48 46 44 47 25.9 44 41 45 42.5 36.0 47 44 44.5 45 43.0 45 43 45 44 30 7.7 45 42 39 43.5 12.2 44 40 41 42 19.0 45 43 43 44 28.4 44 40 41 42 measurement of rice grain friction coefficient and angle of repose 65 conclusions rotating cylinders approach is a fast and reliable method to determine the friction coefficient and angles of repose of grain materials. the transition from one mode to another mode is specified as a function of the cylinder rotation speed and filling degree. the slumping-rolling transition speed depends on the cylinder diameter and filling degree in the range of 5.0 to 7.9 and 2 to 6.34 rpm, respectively. the effects of rotational speed on α and β for rice grains were significant. with increasing cylinder diameter, the differences in between α and β for rice grains increased ranging from 1 to 4°. cylinder diameter and filling degree had significant effects on friction coefficient. in this study, it was possible to simultaneously determine the α, β, and friction coefficient of rice grains. simultaneous determinations of these properties are important in fast and cost-effective studies to model grains’ motions in rotary dryers, mixers and silos. the results of this research can be used in agricultural machines such as combine harvesters, food industry machines, and all equipment related to granular materials. for an estimate of auger friction force, rotating cylinders is a fast and reliable method to determine the friction coefficient and angle of repose of grain materials. based on the results of this research, cylinder diameter and filling degree had significant effects on the friction coefficient. the effect of rotational speed on the lower angle of repose (α) and upper angle of repose (β) of grains were significant. conflict of interest no potential conflict of interest was reported by the authors. references al-mahasneh, m.a., rababah, t.m., 2007: effect of moisture content on some physical properties of green wheat. j. food eng. 79(4), 1467-1473. doi: 10.1016/j.jfoodeng.2006.04.045 aranson, i.s., tsimring, l.s., 2006: patterns and collective behavior in granular media: theoretical concepts. rev. mod. phys. 78(2), 641. doi: 10.1103/revmodphys.78.641 baryeh, e.a., mangope, b.k., 2003: some physical properties of qp-38 variety pigeon pea. j. food eng. 56(1), 59-65. doi: 10.1016/s0260-8774(02)00148-6 cheng, n.s., zhao, k., 2017: difference between static and 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10.1016/s0032-5910(01)00520-4 address of the corresponding author: jafar massah, department of agrotechnology, college of abouraihan, university of tehran, iran. e-mail: jmassah@ut.ac.ir tab. 5: friction coefficient in rolling mode for rice grains and calculated values using eq. (4). cylinder diameter filling degree calculated friction (cm) (%) coefficient 15 18.0 0.931804 25.7 0.931804 33.8 0.931804 46.6 0.964937 20 16.5 1.034687 25.9 0.86865 36.0 0.964937 43.0 0.931804 30 7.7 0.899731 12.2 0.86865 19.0 0.931804 28.4 0.838497 http://dx.doi.org/10.1016/j.jfoodeng.2006.04.045 http://dx.doi.org/10.1103/revmodphys.78.641 http://dx.doi.org/10.1016/s0260-8774(02)00148-6 http://dx.doi.org/10.1016/j.ijsrc.2016.09.001 http://dx.doi.org/10.1016/j.jfoodeng.2005.04.010 http://dx.doi.org/10.14356/kona.2000021 http://dx.doi.org/10.1016/j.powtec.2019.05.083 http://dx.doi.org/10.1016/j.jfoodeng.2005.04.010 http://dx.doi.org/10.1007/bf02661016 http://dx.doi.org/10.1016/j.powtec.2005.04.030 http://dx.doi.org/10.1006/jaer.1996.0119 http://dx.doi.org/10.1006/jaer.1993.1016 http://dx.doi.org/10.1016/j.jfoodeng.2004.11.028 http://dx.doi.org/10.1016/j.jfoodeng.2005.04.033 http://dx.doi.org/10.1016/j.cep.2005.10.009 http://dx.doi.org/10.1016/j.powtec.2005.04.040 http://dx.doi.org/10.1016/j.ces.2005.02.020 http://dx.doi.org/10.1016/s0032-5910(00)00402-2 http://dx.doi.org/10.1002/aic.10266 http://dx.doi.org/10.1016/j.powtec.2018.08.057 http://dx.doi.org/10.1016/s0032-5910(01)00520-4 66 j. massah, j. khazaei © the author(s) 2022. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). art08_nudrat.indd journal of applied botany and food quality 84, 169 177 (2011) 1department of botany, university of agriculture, faisalabad, pakistan *2second affi liation: department of botany and microbiology, king saud university, riyadh, saudi arabia does exogenous application of salicylic acid improve growth and some key physiological attributes in sunfl ower plants subjected to salt stress? sibgha noreen1, muhammad ashraf 1,2, nudrat aisha akram*1 (received september 2, 2010) * corresponding author summary to appraise the effect of foliar-applied salicylic acid (sa) on growth and some key physiological attributes in sunfl ower plants under salt stress, a greenhouse experiment was conducted. two sunfl ower lines (hysun-33 and sf-187) were subjected to non-saline (control) and saline regimes (120 mm nacl). after 14 days of initiation of salt treatment plants of both sunfl ower lines were supplied with varying levels (100, 200 and 300 mg l-1) of sa applied foliarly to all sunfl ower plants exposed to normal or saline substrates. after 21 days of sa application, data for growth (shoot biomass), photosynthetic pigments (chlorophyll a and b), water relation components, and accumulation of proline and mineral nutrients were recorded. salt stress adversely affected the growth, chlorophyll pigments, water relations and contents of some key mineral nutrients, while increased the amount of proline and leaf and root na+ as well as clcontents in both sunfl ower lines. foliar-applied sa improved growth, chlorophyll a and b pigments, leaf turgor potential, and leaf and root ca2+ concentrations. of all salicylic acid levels used in the present study, 200 and 300 mg l-1 were found to be relatively more effective than the other levels in improving chlorophyll a and b pigments, leaf turgor potential, leaf and root ca2+ concentration, while the other attributes remained unaffected due to sa application. of both sunfl ower lines, hysun-33 had higher amounts of photosynthetic pigments and essential nutrients than did sf-187. introduction plants exposed to saline environments experience four basic problems such as reduced water potential, accumulation of toxic ions (na+, cl-) and their harmful effects on various physiological and biochemical processes of the plant, thereby decreasing the absorption of some essential mineral nutrients such as k+ and ca2+, hormone imbalance, and production of reactive oxygen species (ros) (munns, 2005; ashraf, 2009; ashraf et al., 2010). availability of nutrients to plants depends on the functioning of membrane transporters that mediate the translocation of nutrients from the soil into the plant thereby compartmentalizing them into the cells, tissue and organs (tester and davenport, 2003; epstein and bloom, 2005). for example, ca2+-atpases (ca2+-pumps) regulate low cytoplasmic ca2+ levels in plants (geisler et al., 2000). salt tolerant cultivars absorb toxic ions to a lesser magnitude as compared to salt susceptible ones. the uptake of ions in higher quantities results in burning symptoms of leaves and ultimately leading to plant death (munns et al., 1983; flowers and flowers, 2005; akram et al., 2009, 2010). in view of a number of reports it is now evident that soil ph, composition and concentration of elements, climatic conditions, soil textural class, quality of irrigation waters and type of plant species affect the nutrient dynamics and their absorption by plants under saline conditions (grattan and grieve, 1999; zhu, 2003; munns, 2005; akram et al., 2010). for instance, high soil na+ impairs the ca2+ activity in the external medium, thereby restricting its availability in plants e.g., in celosia argentea (carter et al., 2006), setaria verticillata (horst et al., 2006), and maize (suarez and grieve, 1988). it is well established that mineral nutrients acting synergistically or antagonistically may imbalance the nutrition of plants under saline environments. the defi ciencies of most of the nutrients commonly occur due to higher absorption of na+ and clby plant tissues (grattan and grieve, 1999; subbarao et al., 2003; hu et al., 2005; ashraf et al., 2010). in view of these reports, it is likely that reduction in uptake of mineral nutrients under saline conditions may occur due to na+-induced blockage or reduced activity of membrane ion transporters. similarly, potassium uptake is perturbed by salinity thereby resulting in a reduced k+/na+ ratio (izzo et al., 1991; subbarao et al., 1990). however, high k+/na+ ratio in plants under saline conditions has been suggested as an important selection criterion for salt tolerance (shah et al., 1987; reynolds et al., 2005). plant hormones affect plant growth in multifarious ways affecting a number of physiological/biochemical processes in plants subjected to biotic and abiotic stresses (reymond and farmer, 1998; steduto et al., 2000; ashraf et al., 2008, 2010). salicylic acid (sa) is one such plant growth regulators, which participate in the regulation of a number of physiological events taking place in the plant (dolatabadian et al., 2008; ashraf et al., 2010). sa regulates some key plant functions such as stomatal functioning (aldesuquy et al., 1998), ion uptake and transport (glass, 1975; kaydan et al., 2007), photosynthesis (noreen and ashraf, 2008), water relations (barkosky and einhellig, 1993), production rate and content of anthocyanin and chlorophyll (khurana and maheshwari, 1980). it also increases growth (arfan et al., 2007; ashraf et al., 2010), and up-regulates antioxidative system (noreen et al., 2009). all these functions have a signifi cant role in plant tolerance to salinity (noreen and ashraf, 2008; noreen et al., 2009; dolatabadian et al., 2008; abreu and munne-bosch, 2009; ashraf et al., 2010). furthermore, sa can greatly perturb the trans-membrane electrochemical potential of mitochondria and the atp-dependent proton gradient of tonoplast-enriched vesicles (macri et al., 1986). in view of all the above reports, the principal objective of the present study was to appraise whether varying levels of exogenously applied sa could alleviate the adverse effects of salt stress on photosynthetic pigments, water relations and ion accumulation in sunfl ower plants. furthermore, some key biochemical processes infl uenced by sa were identifi ed that could be used as selection criteria in sunfl ower under saline conditions. materials and methods to assess the infl uence of foliar-applied salicylic acid to offset the salt-induced adverse effects on chlorophyll pigments, water relation components and accumulation of some key inorganic nutrients in sunfl ower (helianthus annuus l.), an experiment was conducted at the botanical garden of the university of agriculture, faisalabad, pakistan under the growth conditions as described earlier (noreen and ashraf, 2008). two sunfl ower lines (hysun-33 and sf-187) 170 s. noreen, m. ashraf, n.a. akram infl uence of exogenously applied salicylic acid on sunfl ower were subjected to two saline regimes i.e., 0 and 120 mm nacl prepared in the modifi ed full strength hoagland’s nutrient solution. different concentrations (100, 200 and 300 mg l-1) of salicylic acid (c7h6o3) were prepared and ph of the solutions was adjusted at 5.5. salicylic acid was applied exogenously in combination with 0.1 percent (v/v) tween-20 as a surfactant to ensure spreading of the applied solution on the leaf surface so as to attain maximal penetration into the leaf tissues. the plants were harvested 21 days after the foliar application of sa at the vegetative stage and data for following attributes were recorded: photosynthetic pigments chlorophylls a and b were appraised following arnon (1949). the leaf samples (0.2 g each) were prepared by cutting the leaves into small pieces having 0.5 cm size. the extraction of the leaf samples was done in 80% acetone at -4 °c for 24 h. the extracted material was centrifuged at 10, 000 x g for 5 min. the absorbance of the supernatant was recorded by using a uv-visible spectrophotometer (model hitachi-u 2001, tokyo, japan) at 645 and 663 nm against a blank containing 80% acetone. leaf water potential (ψw) a scholander type water potential measuring system (cook and sons, birmingham, england) was used to measure leaf water potential. a fully expanded youngest leaf from the top was cut from each plant and measurements were made following scholander et al. (1965). leaf osmotic potential (ψs) the leaf osmotic potential was measured following wilson et al. (1980). the leaf material was kept in polypropylene micro-centrifuge tubes containing capacity of 2 cm3. the tubes were frozen at -45 °c for one week. the material was thawed and homogenized with a tissue grinder. then the homogenized material was centrifuged at 8000 x g for 4 min. a 10 µl was used in a wescor-5500 vapor pressure osmometer for measuring osmotic potential. leaf turgor potential (ψp) the leaf turgor pressure was determined using the following equation: ψp = ψw – ψs relative water content (rwc) the fully expanded leaves were collected randomly from each plant from each replicate. after washing the samples were weighed and these values referred to as initial readings. then, the leaf samples were placed in distilled water for 3 h in the dark at room temperature. the turgid leaves were blotted dry and weighed. after weighing, the material was oven-dried at 80 °c for 24 h. rwc of the leaves was appraised as described by jones and turner (1980) using the following formula: rwc (%) = [(f. wt. – d. wt.)/(t. wt. – d. wt.)] x 100 where f.wt, d.wt, and t.wt are the fresh, oven dry, and turgid weights, respectively. free proline content free proline was determined following bates et al. (1973). leaf tissue (0.5 g) was ground with 10 ml of 3% (w/v) sulfo-salicylic acid solution. the sample fi ltrate containing 2.0 ml was reacted with 2.0 ml acid ninhydrin solution (1.25 g ninhydrin mixed with 30 ml glacial acetic acid), 20 ml (6m orthophosphoric acid) and 2.0 ml m orthophosphoric acid) and 2.0 ml m glacial acetic acid. the reaction was done at 100 °c, and terminated in an ice bath. a continuous stream of air was passed through the reaction mixture added with 4 ml toluene. the absorbance of the supernatant was read at 520 nm using a spectrophotometer (model hitachi u 2001 tokyo, japan). proline concentration was worked out using the following formula: free proline (µmol g-1 fresh weight) = proline (µg ml-1) x volume of toluene (ml)/(mol. wt. of proline (g mol-1) x leaf tissue fresh weight (g) analysis of nutrients the oven-dried plant material (0.1 g) was ground and passed through a 40-mesh screen for chemical analysis. a digestion mixture consisting of h2so4 and h2o2 was used to digest the material. the chemical analysis of different ions (k+, ca2+, na+) was carried out following wolf (1982). determination of na+, k+ and ca2+ different concentrations of standards for sodium (na+), potassium (k+) and calcium (ca2+) were prepared. the standard curves of these ions were drawn using a fl ame photometer (jenway-pfp7, ele instrument co, ltd. england). the samples were run for the determination of na+, k+ and ca2+ and the values of the ions in the unknown samples were worked out using the appropriate standard curves. determination of chloride (cl-) the chloride content was determined by extracting plant material (0.5 g) with 10 ml distilled de-ionized water. the material was heated at 80 °c till the volume was reduced to half. the volume of the solution was again made to 10 ml with distilled water. chloride analyzer (model 926, sherwood scientifi c ltd, cambridge, uk) was used to determine the chloride content in the extracted samples. statistical analysis of data analysis of variance (anova) of the data for all attributes was worked out using the costat computer package (cohort, berkely, california). the least signifi cance difference test was employed following snedecor and cochran (1980) to assess whether mean values differed signifi cantly. results data presented in tab. 1 for percent inhibition in shoot fresh and dry biomass showed that although salt stress caused a marked reduction in shoot biomass, foliar-applied salicylic acid reduced the saltinduced inhibition in these growth attributes in both hybrid lines of sunfl ower. of all sa levels, 200 and 300 mg l-1 were relatively more effective in improving growth measured in terms of shoot biomass. addition of salt (nacl) to the root growing medium markedly reduced chlorophyll a in both hybrid lines. however, values of chlorophyll a were found similar in both hybrid lines (tab. 2). the foliar spray of 200 or 300 mg l-1 sa caused a signifi cant improvement in chlorophyll a content in both sunfl ower hybrid lines grown under saline and non-saline regimes (fig. 1). however, under salt treatment, foliar applied 300 mg l-1 sa signifi cantly improved chlorophyll a in line sf-187, while, 200 mg l-1 of sa in hybrid line hysun-33 (fig. 1). salt stress signifi cantly reduced chlorophyll b in both hybrid lines (tab. 2; fig. 1). line hysun-33 maintained greater infl uence of exogenously applied salicylic acid on sunfl ower 171 content of chlorophyll b than that in sf-187 under saline conditions. salicylic acid improved chlorophyll b. of all sa levels, 300 mg l-1 was found better than the others. the chlorophyll a/b ratio of the sunfl ower hybrid lines was not infl uenced by salinity. however, there were signifi cant differences between the hybrid lines in terms of chlorophyll a/b ratio. leaf water potential (ψw) of both sunfl ower lines was signifi cantly reduced due to salinity, and more marked being in line sf-187 (tab. 2; fig. 1). exogenous application of varying levels of sa did not change the leaf water potential of sunfl ower hybrid lines under non-stressed conditions, whereas, under salinity stress, foliar application of all varying levels of sa caused a signifi cant decrease (more negative increase) in leaf water potential, particularly, in line hysun-33 (fig. 1). imposition of salinity had a signifi cant reducing effect on leaf osmotic potential (ψs) of both sunfl ower lines (tab. 2; fig. 1). lines differed only under saline conditions. line sf-187 had lower leaf osmotic potential than that in line hysun-33 under salt-stressed conditions. foliar spray of sa did not affect leaf osmotic potential of the hybrid lines under non-stress conditions, whereas under saline regimes, all sa levels caused a decrease in leaf osmotic potential but only in line sf-187. leaf turgor potential (ψp) remained almost unaltered due to imposition of salt stress (tab. 2; fig. 1). both hybrid lines did not signifi cantly differ in leaf turgor pressure. foliar-applied 200 or 300 mg l-1 sa resulted in enhanced leaf turgor potential of salt stressed plants of line sf-187, whereas the reverse was true in line hysun-33. relative water content (rwc) of sunfl ower plants was signifi cantly decreased because of raising salinity of the growth medium (p ≤ 0.05) (tab. 2; fig. 2). moreover, both lines also differed signifi cantly in this parameter. foliar-applied sa did not signifi cantly alter leaf rwc in the salt-stressed or non-stressed plants of both lines. values for rwc did not vary signifi cantly due to exogenous application of sa under salt-treated and untreated plants. proline content differed signifi cantly due to different salt and sa treatments. however, both sunfl ower lines did not differ signifi cantly in proline content. moreover, there were non-signifi cant interactions between salinity, sa or hybrid lines. proline content increased with increasing levels of sa under saline and non-saline conditions. line hysun-33 maintained higher content of proline compared to that in line sf-187. both lines, hysun-33 and sf-187, contained higher content of proline under salt stress than that under normal conditions (tab. 2; fig. 2). concentrations of na+ in the leaves and roots increased signifi cantly due to imposition of salt stress in the growth medium (tab. 2). both lines did not show difference in leaf na+, whereas they differed signifi cantly in root na+. exogenous application of different levels of sa had a slight decreasing effect on na+ content in the leaf tissues of salt stressed plants of both lines, particularly after foliar application of 300 mg l-1 sa (fig. 2). on the other hand, foliar-applied sa at different concentrations produced inconsistent pattern of na+ accumulation by the roots of sunfl ower hybrid lines (fig. 2). the addition of nacl to the growth medium resulted in a signifi cant reduction in root k+ content of the hybrid lines. both sunfl ower lines also differed signifi cantly in this physiological attribute (p ≤ 0.05). the accumulation of k+ by the leaf or root tissues was not affected signifi cantly due to foliar spray of sa. however, a slight improvement in this ion content was observed in both hybrid lines under saline and non-saline regimes. the sa application resulted in a slight increase in the uptake of k+ by the root tissues in line hysun-33 as compared to that in line sf-187 under nacl treatment (fig. 2). the concentration of ca2+ in the leaves of hysun-33 was higher than that in line sf-187 grown under non-saline conditions. contrarily, the pattern of accumulation of ca2+ in the leaves of both lines was inconsistent under salinity stress (tab. 2; fig. 3). however, accumulation of ca2+ in the roots of line sf-187 was elevated as compared to that in line hysun-33 under salt-stressed and nonstressed conditions. foliar application of 100 mg l-1 sa resulted in increased uptake of ca2+ by leaf tissues of line hysun-33 under saltstressed conditions. furthermore, foliar spray of 200 and 300 mg l-1 sa resulted in a maximal increase in root ca2+ in the sunfl ower hybrid lines under salt stress and non-stress substrates (fig. 3). the plants grown under nacl-treated regime showed a signifi cant effect on the uptake of clby leaves and roots of the sunfl ower hybrid lines under salt-stressed and non-stress regimes (tab. 2). there was no signifi cant difference in both lines in leaf or root clcontent (fig. 3). furthermore, accumulation of clby leaf or root tissues was little affected by foliar application of sa under saline or non-saline tab. 1: percent inhibition in shoot biomass due to salt stress (120 mm nacl) and percent improvement due to foliar-applied varying levels of salicylic acid of two lines of sunfl ower. salicylic acid (sa) percent (%) inhibition under saline conditions shoot fresh weight shoot dry weight sf-187 hysun-33 sf-187 hysun-33 0 mg l-1 39.47 50 24.4 42.1 100 mg l-1 40.8 49.9 27.3 45.4 200 mg l-1 41.4 46.5 47.9 41.0 300 mg l-1 38.6 44.08 36.2 37.6 percent (%) improvement due to foliar-applied sa under normal and saline conditions shoot fresh weight shoot dry weight sf-187 hysun-33 sf-187 hysun-33 control saline control saline control saline control saline 100 mg l-1 12 9.47 4.44 4.75 9.5 13.2 8.2 1.9 200 mg l-1 26.13 22.08 4.62 11.9 15.7 20.6 11.4 9.8 300 mg l-1 14.24 15.93 10.5 23.6 2.53 13.7 16.8 25.9 172 s. noreen, m. ashraf, n.a. akram infl uence of exogenously applied salicylic acid on sunfl ower tab. 2: analyses of variance of the data (mean squares) for chlorophyll pigments, water relation components, free proline and concentrations of inorganic nutrients in two sunfl ower (helianthus annuus l.) hybrid lines when varying levels of salicylic acid were applied as a foliar spray to 24-day old plants subjected to normal or saline conditions. sources of variation df chlorophyll a chlorophyll b chlorophyll leaf water leaf a/b ratio potential osmotic potential salt (s) 1 3.21*** 0.82*** 0.284ns 2.024*** 2.043*** hybrid lines (hbl) 1 0.095ns 0.027* 0.825* 0.007ns 0.157** salicylic acid (sa) 3 0.246* 0.009ns 0.394ns 0.035*** 0.018ns s x hbl 1 0.054ns 0.006ns 0.002ns 0.073*** 0.288*** s x sa 3 0.021ns 0.009ns 0.065ns 0.007ns 0.023ns hbl x sa 3 0.06ns 0.003ns 0.087ns 0.064*** 0.018ns salt x hbl x sa 3 0.084ns 0.002ns 0.148ns 0.005ns 0.003ns error 48 0.068 0.005 0.184 0.005 0.016 leaf turgor relative water leaf free leaf na+ root na+ potential content proline salt (s) 1 0.0004ns 2635.4*** 51.12*** 2053.2*** 4856.3*** hybrid lines (hbl) 1 0.099* 141.6** 0.004ns 3.376ns 481.3** salicylic acid (sa) 3 0.039ns 26.0ns 2.416** 8.471ns 77.61ns s x hbl 1 0.071ns 1.28ns 1.062ns 0.083ns 549.3** s x sa 3 0.013ns 13.52ns 0.08ns 6.74ns 104.8ns hbl x sa 3 0.095* 29.43ns 0.064ns 5.83ns 47.41ns salt x hbl x sa 3 0.01ns 16.18ns 0.527ns 1.365ns 25.84ns error 48 0.023 14.91 0.561 3.757 63.42 leaf k+ root k+ leaf ca2+ root ca2+ leaf clsalt (s) 1 7255.8*** 4863.7*** 73.68*** 454.9*** 4728.3*** hybrid lines (hbl) 1 24.47ns 111.2* 2.939ns 44.76** 7.548ns salicylic acid (sa) 3 108.7* 28.22ns 2.925ns 65.48*** 13.52ns s x hbl 1 16.72ns 3.478ns 1.67ns 11.41ns 27.59ns s x sa 3 12.48ns 5.03ns 11.81*** 1.767ns 15.33ns hbl x sa 3 2.898ns 10.0ns 2.574ns 7.422ns 7.317ns salt x hbl x sa 3 10.7ns 6.70ns 9.32** 3.299ns 1.453ns error 48 30.05 23.68 1.462 5.13 24.96 root clleaf root leaf root k+/na+/na+ + k+/na+/na+ + ca2+/na2+/na2+ + ca2+/na2+/na2+ + salt (s) 1 2499.0*** 782.4*** 57.87*** 23.87*** 7.49*** hybrid lines (hbl) 1 2.074ns 1.64ns 6.54*** 0.226ns 2.02*** salicylic acid (sa) 3 8.56ns 4.44ns 0.234ns 0.232ns 0.26** s x hbl 1 25.6ns 2.99ns 9.37*** 0.097ns 1.387*** s x sa 3 5.67ns 2.026ns 0.154ns 0.317ns 0.038ns hbl x sa 3 5.1ns 2.756ns 0.232ns 0.123ns 0.05ns salt x hbl x sa 3 3.18ns 1.503ns 0.127ns 0.199ns 0.069ns error 48 8.55 1.647 0.296 0.132 0.053 *, **, *** = signifi cant at 0.05, 0.01 and 0.001 levels. ns = non-signifi cant regimes. salt stress had a signifi cant decreasing effect on leaf and root k+/na+ ratios of both sunfl ower lines. in contrast, under saline conditions, at 200 mg l-1 of sa a slight increase in k+/na+ ratio was observed in cv. sf-187. foliar-applied varying levels of salicylic acid showed inconsistent results for these ionic ratios (tab. 2; fig. 4). leaf and root ca2+/na+ ratios were signifi cantly (p ≤ 0.001) infl uenced by salt stress. a non-signifi cant effect of exogenously applied salicylic acid was observed on leaf ca2+/na+ ratio, while root ca2+/na+ was increased due to sa application. of all sa levels, 300 mg l-1 was highly effective for improving tissue ca2+/na+ ratios in both sunfl ower hybrids. of both sunfl ower hybrids, sf-187 was better than hysun-33 in this attribute (tab. 2; fig. 4). discussion salicylic acid (sa) being a vital plant growth regulator as well as an antioxidant (raskin et al., 1992) has a potential to allay the saltinfl uence of exogenously applied salicylic acid on sunfl ower 173 fig. 1: chlorophyll a and b contents, chlorophyll a / b ratio, and leaf water, osmotic and turgor potentials of two sunfl ower (helianthus annuus l.) hybrid lines when varying levels of salicylic acid were applied as a foliar spray to 24 day-old plants subjected to normal or saline conditions (mean ± s.e.; n = 4). fig. 2: relative water content, proline accumulation, and leaf and root na+ and k+ contents of two sunfl ower (helianthus annuus l.) hybrid lines when varying levels of salicylic acid were applied as a foliar spray to 24 day-old plants subjected to normal or saline conditions (mean ± s.e.; n = 4). 174 s. noreen, m. ashraf, n.a. akram infl uence of exogenously applied salicylic acid on sunfl ower induced harmful effects on crop growth and development (el-tayeb, 2005; arfan et al., 2007; ashraf et al., 2010). photosynthetic pigments such as chlorophyll a and b are chief components of photosystems driving the mechanism of photosynthesis and hence growth in terms of biomass production or seed yield. in the present study, the contents of photosynthetic pigments was slightly improved with sa application. these fi ndings are similar to those of ghai et al. (2002) who showed a considerable improvement in chlorophyll contents due to foliar applied sa (200 mg l-1). similarly, fariduddin et al. (2003) reported that brassica juncea plants sprayed with fig. 3: leaf and root ca2+ and claccumulation in two sunfl ower (helianthus annuus l.) hybrid lines when varying levels of salicylic acid were applied as a foliar spray to 24 day-old plants subjected to normal or saline conditions (mean ± s.e.; n = 4). fig. 4: leaf and root k+/ na+ and ca2+/ na+ ratios of two sunfl ower (helianthus annuus l.) hybrid lines when varying levels of salicylic acid were applied as a foliar spray to 24 day-old plants subjected to normal or saline conditions (mean ± s.e.; n = 4). infl uence of exogenously applied salicylic acid on sunfl ower 175 10-5 m of sa showed 20 percent higher chlorophyll than those sprayed with water only. by contrast, no change in chlorophyll content was observed in corn and soybean plants exogenously supplied with acetyl salicylic acid (khan et al., 2003). in the present study, a positive correlation of photosynthetic effi ciency (astudy, a positive correlation of photosynthetic effi ciency (astudy, a positive correlation of photosynthetic effi ciency ( ) with chlorophyll a or chlorophyll b (r = 0.416**; 0.436**, respectively) r = 0.416**; 0.436**, respectively) r has been observed. such relationships between a and chlorophyll contents have earlier been reported by fariduddin et al. (2003). they found that exogenous sa application caused an improvement in photosynthetic capacity in salt stressed brassica juncea plants in association with improved chlorophyll content. the reduction in chlorophyll contents may occur due to acceleration in chlorophyll degradation or reduction in chlorophyll synthesis. however, it has been reported that imposition of salt stress causes deterioration in the structure of chloroplast e.g., thylakoid membranes and plastids due to direct na+ toxicity and cellular oxidative damage (mittler, 2002). in the present study, a strong negative correlation between leaf na+ and each of leaf chlorophyll a, chlorophyll b, or a (r = -0.610***; r = -0.610***; r -0.804*** and -0.527*** respectively) has been observed. maintenance of plant water status or osmoregulation is a vital physiological process for maintaining optimal plant growth (taiz and zeiger, 2006). in the present study, leaf water relation parameters, leaf turgor, water and osmotic potentials, and relative water content of both sunfl ower lines were adversely affected by salt stress. application of 200 or 300 mg l-1 sa improved leaf relative water content (rwc), but decreased leaf osmotic potential and leaf ψw. moreover, leaf rwc and leaf ψp of the salt stressed plants of line sf-187 were higher than those in the leaves of line hysun-33. leaf osmotic potential of the salt stressed plants of sf187 was also reduced due to sa application. these fi ndings are in agreement with an earlier study (ashraf, 1989) in which leaf turgor potential increased with a concomitant decrease in osmotic potential in blackgram (vigna mungo) under salt stress. decrease in osmotic potential may occur due to accumulation of inorganic and/or organic solutes. of various organic solutes, glycinebetaine, amino acids mainly proline, and soluble sugars play important roles in osmotic adjustment (hasegawa et al., 2000; ashraf and foolad, 2005, 2007). among inorganic solutes, k+ and na+ are considered very important because they also play a vital role in osmoregulation, but na+ is potentially damaging when compared either with k+ or organic solutes (subbarao et al., 2001; tester and davenport, 2003). however, exogenous application of sa decreased leaf na+ content in the salt stressed plants of both sunfl ower lines, but leaf k+ remained almost unaffected in both shoots and roots of both lines due to foliar-applied sa application. if relationships are worked out between leaf ψs and each of proline, leaf na+, or leaf k+ (leaf op vs leaf proline; leaf op vs leaf na+; leaf op vs leaf k+, r = 0.625***; 0.652***; -0.649***), it is amply clear that leaf r = 0.625***; 0.652***; -0.649***), it is amply clear that leaf r na+ and leaf proline had a marked contribution in osmoregulation. increased accumulation of proline in the salt stressed plants of both sunfl ower lines is similar to the fi ndings of shakirova et al. (2003) who reported that sa-treated wheat seedlings showed enhanced accumulation of free proline. this can be further supported by the inference drawn by ashraf and harris (2004) that under salt stress, higher accumulation of proline is one of the vital strategies of plants for causing osmotic adjustment, particularly in the presence of high cytosolic na+. in view of the results from the present study and all these reports, it can be suggested that although exogenous sa reduced accumulation of na+ in the leaves, its concentration is high enough to cause toxic effects, so increased accumulation of proline in the salt stressed sunfl ower plants reduced the toxic effect of na+ during osmotic adjustment. the results for accumulation of different mineral ions in the root and shoot tissues of both sunfl ower lines showed enhanced accumulation of na+ and claccompanied with a decline in k+ and ca2+ in both sunfl ower lines under saline substrate. these results support the viewpoint that plants subjected to saline substrate are prone to specifi c ion toxicity, ionic imbalance, and nutrient defi ciency (ashraf, 1994, 2004). however, exogenously applied sa reduced leaf na+ of both sunfl ower lines. these results support the earlier fi ndings of el-tayeb (2005) in which sa application resulted in reduced na+ in the leaves of barley seedlings under salt stress. in contrast, k+ accumulation remained almost unchanged due to sa application. however, root or leaf ca2+ content of salt stressed plants of both sunfl ower lines increased due to exogenously applied sa. these results support the earlier fi ndings of kawano and muto (2000) who observed that sa enhanced the cytosolic ca2+ level in tobacco cell suspension culture. in view of these results and some published reports available in the literature, it is suggested that exogenous sa application might have elevated cytosolic ca2+ that acted as a second messenger taking part in a multitude of physiological responses including expression of osmotic responsive genes (pardo et al., 1998) and antioxidant enzymes (chen and li, 2001; agarwal et al., 2005). to appraise salt tolerance, k+/na+ and ca2+/na+ ratios are considered as potential selection criteria, however, in the present study salt stress signifi cantly decreased the leaf and root k+/na+ ratios, while ca2+/ na+ ratios were not infl uenced in sunfl ower plants and foliar-applied salicylic acid showed inconsistent results for these ionic ratios. in conclusion, salt stress adversely affected the growth, chlorophyll pigments, water relations and contents of some key mineral nutrients, while increased the amount of proline and leaf and root na+ as well as clcontents in both sunfl ower lines. of both sunfl ower lines, hysun-33 had higher amounts of photosynthetic pigments and essential nutrients than did sf-187. foliar-applied sa improved growth, chlorophyll a and b pigments, leaf turgor potential, and leaf and root ca2+ concentrations. of all salicylic acid levels, 200 and 300 mg l-1 were found to be relatively more effective than the other levels in improving chlorophyll a and b pigments, leaf turgor potential, leaf and root ca2+ concentration, while the other attributes remained unaffected due to sa application. references abreu, m.e., munne-bosch, s., 2009: salicylic acid defi ciency in nahg transgenic lines and sid2 mutants increases seed yield in the annual plant arabidopsis thaliana. j. exp. bot. 60, 1261-1271. agarwal, s., sairam, r.k., srivastava, g.c., tyagi, a., meena, r.c., 2005: role of aba, salicylic acid, calcium and hydrogen peroxide on antioxidant enzymes induction in wheat seedlings. plant sci. 169, 559570. akram, m.s., ashraf, m., akram, n.a., 2009: effectiveness of potassium sulfate in mitigating salt-induced adverse effects on different physiobiochemical attributes in sunfl ower (helianthus annuus l.). flora 204, 471-483. akram, m.s., ashraf, m., shahbaz, m., akram, n.a., 2009: growth and photosynthesis of salt-stressed sunfl ower (helianthus annuus) plants as affected by foliar-applied different potassium salts. j. plant nutr. soil sci. 172, 884-893. aldesuquy, h.s., mankarios, a.t., awad, h.a., 1998: effect of some antitranspirants on growth, metabolism and productivity of saline-treated wheat plants: induction of stomatal closure, inhibition of transpiration and improvement of leaf turgidity. acta bot. hung. 41, 1-10. arfan, m., athar, h.r., ashraf, m., 2007: does exogenous application of salicylic acid through the rooting medium modulate growth and photosynthetic capacity in differently adapted spring wheat cultivated under salt stress? 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(flutriafol, prothioconazole, tebuconazole), the benzimidazole fuberidazole, imidazoles (imazalil, prochloraz), and the strobilurin fluoxastrobin on barley (hordeum vulgare) was investigated using commercial seed dressings also including pyrimethanil (anilinopyrimidine) and triazoxide (benzotriazine), under controlled conditions. irrespective of temperature or soil water content (swc) triazole-containing seed treatments had a significant effect on the time and rate of plant emergence. both triadimenolcontaining products significantly reduced the length of subcrown internodes and resulted in reduced shoot length three weeks after sowing. growth suppression was stronger under optimal environmental conditions (17 to 19 °c, 60 % swc). under suboptimal conditions – 9 to 10 °c and 40 % swc, respectively – no differences in shoot length were detected five weeks after sowing, whereas under optimal conditions plant growth retardation was still significant. the flutriafol-containing product partly inhibited shoot elongation, but never affected dry mass accumulation and root growth. the strobilurin-containing seed dressing had no marked plant growth activities on seedling emergence, shoot length and subcrown internode, but slightly stimulated root growth under all environmental conditions. the results indicate varying pgr activities of triazole seed dressings in response to mixing partner and growth conditions and suggest an increased stress tolerance of seedlings treated with triadimenol, enabling barley to better cope with suboptimal environmental conditions. introduction seeds of crops are regularly dressed with fungicides to control seedand soil-borne plant pathogens for uniform and healthy early plant development and ultimately, to maximize crop yield. demethylation inhibitors (dmis) of fungal sterol biosynthesis, particularly triazoles, imidazoles and pyrimidines, represent the most important group of modern systemic fungicides used for seed dressings (kuck et al., 1995). in addition to fungicidal compounds, triazoles (e.g. uniconazole, paclobutrazole) are used also for plant growth regulation (pgr) (buchenauer, 1995). similarly numerous morphological and physiological side-effects in plants have been described for fungicidal triazole-derivatives in addition to their primary effect on sterol biosynthesis of fungi, primarily based on the inhibition of cytochrome p450-dependent enzyme activities (förster et al., 1980a; fletcher et al., 1986; gao et al., 1987; siefert and grossmann, 1996). triazoles registered for their fungicidal activity in cereals exhibit growth retardation activity, e.g. tebuconazole and metconazole in oil seed rape (gilgenberg-hartung, 1999; kruse and verreet, 2005). triazole seed treatments affect many aspects of plant growth and development. triadimefon and triadimenol, early commercialized dmis, result in growth inhibition of roots, shoots and coleoptiles of barley seedlings (förster et al., 1980a). retardation of primary leaf elongation has been reported in several crops (buchenauer and röhner, 1981; buchenauer et al., 1984). pgr effects of triadimenol and triticonazole in wheat seedlings include reductions in the length of coleoptiles, primary leaves and subcrown internodes (anderson, 1989; cavariani et al., 1994; montfort et al., 1996). tebuconazole showed pgr activities in linseed and wheat seedlings (cavariani et al., 1994; hack, 1994). seed treatments with propiconazole and diclobutrazole increased frost hardiness and tolerance of plants to drought and salt stress (buchenauer et al., 1981). the sterol biosynthesis inhibitor imazalil restricted elongation of the subcrown internode and inhibited tillering of wheat seedlings (chinn et al., 1980). the pyrimidine derivatives triarimol, nuarimol and fenarimol also exhibit pgr activities (shive and sisler, 1976; buchenauer and röhner, 1977; konstantinidou-doltsini et al., 1987). the primary effect of fungicide seed treatments is restricted to early stages of plant growth, but side-effects on plant development may also influence yield formation in later growth stages. furthermore, these physiological and morphological side-effects could improve the tolerance of barley subjected to various environmental stress conditions during seedling growth and development, because triazole applications gave an increased stress tolerance in various crops (fletcher et al., 2000). as the expansion of cropping area and climate change are associated with an increase in unfavourable conditions – temperature and soil conditions – in early growth stages, information is required whether fungicidal seed dressings including triazoles are suitable to promote crop development under suboptimal conditions. therefore, it was investigated whether the triadimenolcontaining seed dressing mixtures (+ imazalil + fuberidazole; baytan ufb®; + prothioconazole + triazoxide; baytan²®) exhibit stronger morphological and physiological side-effects on barley seedlings than a flutriafol-containing combination (+ prochloraz + pyrimethanil; rubin®) or the mixture of the triazoles prothioconazole and tebuconazole combined with fluoxastrobin and triazoxide (efa®). the environmental factors temperature, sowing depth and soil water content (swc) were varied to assess their influence on incidence and magnitude of pgr effects. materials and methods seed and seed treatment pathogen-free seed of winter barley (hordeum vulgare l., cultivar fee) was treated with commercial fungicides as summarized in tab. 1. commercial products were used to guarantee optimum formulation of the fungicidal ingredients. they were applied at recommended application rates. plant cultivation barley kernels were sown in plastic containers (36 x 42 cm, height 18 cm) in four rows (row distance 10 cm) and a distance of 2.5 cm journal of applied botany and food quality 82, 60 68 (2008) 1 institute of crop science and resource conservation (inres –phytomedicine), university of bonn, germany 2 bayer cropscience, langenfeld, germany effect of environmental conditions on plant growth regulator activity of fungicidal seed treatments of barley andreas görtz1, erich-christian oerke1, thomas puhl2, ulrike steiner1 (received march 30, 2008) within the rows, resulting in 48 seeds per container. in experiments on the effect of growth temperature and sowing depth sand (0 2 mm) was used as substrate; a mixture of sand and sieved c-horizon (< 0.6 cm) (ratio 1 : 1) was utilized in experiments on the influence of soil water content. plants were grown in an univariate experimental design (i) at 17 19 °c and 9 10 °c (60 % swc, 5 cm sowing depth (sd)), (ii) at 40 % swc and 60 % swc (17 19 °c, 5 cm sd), and (iii) with 3 cm sd and 5 cm sd (17 19 °c, 60 % swc) under controlled conditions for 35 days. plants were illuminated with at least 300 µmol par m-2 s-1, 14 hours per day. each treatment consisted out of four replicates of 24 plants (half container) and the containers with different treatments were distributed randomly under the respective growth conditions. plants were fertilized with a solution containing (nh4)2so4 (0.566 g kg-1 soil), k2so4 (0.3 g kg-1), kh2po4 (0.193 g kg-1) and mgso4 (0.507 g kg-1). each pot also received 30 ml of each of the following micronutrients: mnso4, 12.3 g l-1, znso4, 17.6 g l-1, cuso4, 15.72 g l-1, h3bo3, 5.72 g l -1, (nh4)6mo7o24, 1.84 g l -1, and feso4, 99.55 g l-1. fertilization was split into three applications ten, fifteen and twenty days after sowing. control of soil water content (swc) plants were irrigated as necessary to maintain optimal plant development. for experiments using specific swcs, watermark water potential sensors (spectrum technologies, inc., plainsfield, usa) were used for swc measurements. sensors were placed into the centre of each container during filling with the soil matrix which was adjusted with water to the designated swc. before and during the experiments, electrical resistance of each container was measured with an ohmmeter (pontavi wh 2, hartmann and braun, frankfurt, germany) at least once a day and compared with resistance values received from a calibration line of different swcs in the lab. assessment of morphological and physiological parameters the number of emerged seedlings was counted every day from the first sign of emergence until all germinated seedlings had emerged. mean emergence was calculated by counting the emerged plants in each of all sowing rows of every treatment. for the assessment of the following parameters 5 plants per replicate were selected randomly (n = 20). shoot length, i.e. the distance between soil-surface and the tip of the longest leaf was measured 21 and 35 days after sowing. the length of the subcrown internode was determined 35 days after sowing. the chlorophyll concentration of primary leaves was determined using a minolta spad 502® chlorophyll meter (minolta, munich, germany). the spad-meter (soil plant analysis development) measures the greenness of leaf tissue (ratio 650 nm to 940 nm). meter readings are given in spad-values indicating the relative chlorophyll concentration. spad-values were measured as an average of 5 leaves per replicate at the same date (n = 20). for dry matter determination 5 shoots per replicate were harvested 35 days after sowing by cutting the seedlings above the soil surface. shoots were dried first at room temperature and then at 105 °c for 24 h (n = 20). to determine the root dry mass after 35 days of plant growth, at least 15 intact root systems were removed from the soil by washing. root systems were dried first at room temperature and then at 105 °c for 24 h. root characteristics were determined using the winrhizo system (regent instruments, quebec, canada). root systems images of single plants were generated using a digital flatbed scanner (agfa snapscan 1236) according to the manufacturer’s instructions. root samples were placed in clear plastic trays, filled with water on the scanner bed. the scanner was equipped with a transmitted light source in the lid, so light came from above the roots during the scanning process. images were analyzed using the root tab. 1: composition and application rate of four seed dressing products. no. active ingredient(s) chemical group a.i. product application rate a [g/l] per 100 kg [mg a.i./kg] 1 triadimenol triazoles 75 baytan ufb® 400 ml 300 imazalil imidazoles 10 40 fuberidazole benzimidazoles 9 36 2 triadimenol triazoles 187 .5 baytan²® 200 ml 375 prothioconazole triazoles 25 50 triazoxide benzotriazines 10 20 3 flutriafol triazoles 16 .7 rubin® 250 ml 42 prochloraz imidazoles 38 .5 96 pyrimethanil anilinopyrimidines 42 105 4 prothioconazole triazoles 20 efa® 200 ml 40 tebuconazole triazoles 3 .75 8 fluoxastrobin strobilurins 37 .5 75 triazoxide benzotriazines 10 20 a on barley seed plant growth regulator activity of seed treatments 61 62 andreas görtz, erich-christian oerke, thomas puhl, ulrike steiner analysis software winrhizo. it calculated morphological root measurements based on tennant’s statistical line intersect method (tennant, 1975). root samples were analyzed for root length, average diameter and volume. statistical analysis the statistical analysis was performed using the software programme spss (vers. 12, munich, germany). one-way anova (p < 0.05) was used to detect differences between means. in case of significant differences among treatments, the tukey test was used to differentiate treatments means. in figures, significant differences among treatments are shown by different letters. results effect of temperature on plant growth regulator activity plant emergence at 17 to 19 °c, the optimal growth temperature, seeds treated with triadimenol-containing products as well as the mixture of triazoles and strobilurin gave emergence rates similar to untreated seeds; only the flutriafol-containing treatment had a marked effect on the emergence rate (tab. 2). at lower temperature, only the strobilurincontaining mixture had no significant effect on plant emergence. all seed treatments, however, delayed the time of emergence by at least one day; the combination triadimenol + fuberidazole as well as flutriafol + prochloraz delayed emergence by two days (fig. 1). at 17 to 19 °c, only the dressings including triadimenol and flutriafol delayed emergence, the other triazoles had no significant effect under optimal temperatures. shoot length both triadimenol seed dressings significantly retarded shoot length of 21 day-old seedlings grown at 17 to 19 °c and 9 to 10 °c, respectively, whereas the two other mixtures had no significant effect on plant height (fig. 2). differences between untreated and triadimenol-treated seedlings were less pronounced at 9 to 10 °c than at 17 to 19 °c. after growth for five weeks under suboptimal conditions, retardation effects on shoot length could not be detected any longer for any seed dressing (fig. 2). barley treated with triadimenol + prothioconazole was even significantly taller than that treated with the strobilurin-containing product. at 17 to 19 °c, shoots of 35 dayold barley treated with triadimenol dressings still were significantly shorter than the other treatments except for plants treated with the flutriafol-containing mixture. shoot dry matter production after 35 days at 9 to 10 °c, no significant differences in shoot dry mass were detected among treated and untreated seedlings (fig. 2). compared to untreated barley, the treatment including fluoxastrobin significantly increased shoot dry mass production under optimal temperature. seed treatments with triadimenol and flutriafol had no marked effect at 17 to 19 °c. tab. 2: influence of environmental conditions and seed treatments on mean emergence of barley seedlings per seed row (max. 12). environment seed dressing untreated triadimenol triadimenol prothiocon. flutriafol control + imazalil + prothioconazole + tebucon. + prochloraz + fuberidazole + triazoxide + fluoxastr. + pyrimeth. + triazoxide temperature 9 10 °c 10.6 a 7.1 b 7.6 b 9.6 a 7.4 b 17 19 °c 10.6 a 9.6 ab 9.9 ab 10.4 a 8.8 b soil water 40 % 10.1 a 8.3 b 8.8 b 10.6 a 8.8 b content 60 % 10.6 a 8.3 c 9.4 bc 9.8 ab 8.8 bc sowing 3 cm 11.1 a 10.0 a 9.9 a 11.0 a 10.4 a depth 5 cm 11.9 a 9.5 b 9.1 b 10.3 b 9.6 b same letters in a row indicate values not significantly different (p < 0.05; tukey test) c um ul at iv e em er ge n ce ra te [% ] 0 10 20 30 40 50 60 70 80 90 100 9 10 11 12 13 14 15 16 17 18 19 6 7 8 9 10 11 12 13 14 days after sowing 0 10 20 30 40 50 60 70 80 90 100 9 10 11 12 13 14 15 16 17 18 19 6 7 8 9 10 11 12 13 149 10 11 12 13 14 15 16 17 18 19 6 7 8 9 10 11 12 13 14 days after sowing untreated triadimenol+imazalil+fuberidazole triadimenol+prothioconazole+triazoxide prothioconazole+tebuconazole +fluoxastrobin+triazoxide flutriafol+prochloraz+pyrimethanil a b c um ul at iv e em er ge n ce ra te [% ] fig. 1: effect of growth temperature on the emergence of barley following fungicidal seed treatments. a, 9 10 °c; b, 17 19 °c (dotted lines indicate 50 % and 90 % emergence, respectively). plant growth regulator activity of seed treatments 63 length of subcrown internode independent of temperature conditions, both triadimenol-containing seed dressings substantially inhibited the elongation of subcrown internodes as compared to the other treatments. at 17 to 19 °c, flutriafol + prochloraz reduced the length of subcrown internodes significantly when compared to control plants. nevertheless, the elongation was affected to a much lesser extent than by the triadimenol-containing seed dressings (fig. 2). effect of soil water content on plant growth regulator activity of fungicides plant emergence all seed treatments containing dmi ingredients significantly reduced the rate of seedling emergence under both swcs, except for the strobilurin-containing dressing which had no marked effect as compared to untreated barley (tab. 2). with 40 % swc, seed treatments significantly delayed barley emergence, except for the strobilurin-containing product (fig. 3). in later stages, all treated seeds came up to similar emergence rates, not significantly different from that of untreated barley. with optimal swc, barley emergence was faster from untreated seeds than from dressed seeds. triadimenol + imazalil + fuberidazole and the fluoxastrobin product delayed emergence similarly, whereas the other two treatments slowed down the rate of emergence to a greater extent (fig. 3). shoot length three weeks after sowing, barley dressed with triadimenol-containing products and grown at 60 % swc was severely stunted (fig. 4). shoot elongation of the other treatments was affected to a much lesser extent. triadimenol + prothioconazole significantly restricted shoot elongation also under dry soil conditions. after five weeks of growth in dry soil no significant differences were observed among treatments (fig. 4). when grown at optimal swc of 60 % barley plants treated with triadimenol + prothioconazole or flutriafol + prochloraz were significantly shorter than untreated plants. the other seed dressings resulted in low growth suppression when compared to untreated plants. shoot dry matter production in all treatments, low swc (40%) caused a marked reduction in shoot dry mass compared to 60 % swc; the difference was more pronounced than for shoot length (fig. 4). five weeks after seeding no significant differences were detected among treatments. in contrast, barley treated with triadimenol-containing seed dressings and grown under optimal swc produced significantly less dry matter than untreated ones. shoot dry matter of barley treated with triadimenol + prothioconazole was also significantly lower than that of plants grown from seeds dressed with flutriafol + prochloraz and prothioconazole + tebuconazole + fluoxastrobin, respectively. length of subcrown internode under both swcs, triadimenol-containing seed dressings significantly inhibited elongation of the subcrown internode. the length of subcrown internodes of seedlings developing from the other treatments was similar to that of untreated plants (fig. 4). untreated triadimenol+imazalil+fuberidazole triadimenol+prothioconazole+triazoxide prothioconazole+tebuconazole+fluoxastrobin + triazoxide flutriafol+prochloraz+pyrimethanil a a b b a ab b c c a a a b b a ab b c c 9 10 ˚c 17 – 19 ˚c ca 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 s h o o td ry m as s [m g] s h o o tl en gt h [c m ] a b b a b a b a a a b b a b a b a b a b a a a b b b a a a a a a b b b b bb 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 0 9 10 ˚c 21 days after s owing 35 days after s owing 17 19 ˚c 9 10 ˚c 17 19 ˚c 9 10 ˚c 17 19 ˚c l en gt h o f su b cr ow n in te rn o de [m m ] 0 5 10 15 20 25 30 35 40 45 0 5 10 15 20 25 30 35 40 45 a a b b a ab b c c a a a b b a ab b c c 9 10 ˚c 17 – 19 ˚c ca 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 s h o o td ry m as s [m g] s h o o tl en gt h [c m ] a b b a b a b a a a b b a b a b a b a b a a a b b b a a a a a a b b b b fig. 2: effect of growth temperature on plant growth regulator activities of fungicidal seed dressings on shoot length (a), dry matter production (b) and length of subcrown internodes (c) of barley. values with same letters are not significantly different (p < 0.05, tukey test). untreated triadimenol+imazalil+fuberidazole triadimenol+prothioconazole+triazoxide prothioconazole+tebuconazole +fluoxastrobin+triazoxide flutriafol+prochloraz+pyrimethanil c u m u la ti v e em er g en ce ra te [ % ] 0 10 20 30 40 50 60 70 80 90 100 8 9 10 11 12 13 14 15 16 17 a days after sowing 7 7 8 9 10 11 12 13 14 15 16 17 b 0 10 20 30 40 50 60 70 80 90 100 days after sowing c u m u la ti v e em er g en ce ra te [ % ] 0 10 20 30 40 50 60 70 80 fig. 3: effect of soil water content (swc) on the emergence of barley dressed with various fungicides. a, 40 % swc; b, 60 % swc (dotted lines indicate 50 % and 90 % emergence, respectively). 64 andreas görtz, erich-christian oerke, thomas puhl, ulrike steiner effect of sowing depth on plant growth regulator activity of fungicides plant emergence with 3 cm sowing depth (sd), no fungicide treatment had a marked effect on the rate of seedling emergence. sowing at 5 cm depth, however, resulted in a significant reduction of barley emergence for all treatments (tab. 2). both triadimenol-containing treatments delayed emergence of barley sown at 3 cm depth at least by one day. seedlings of the other treatments emerged slightly faster, but never reached the time of emergence of untreated barley. with 5 cm sd, seed treatments led to a significant delay in emergence. especially triadimenol + imazalil + fuberidazole, but also the flutriafolcontaining and the other triadimenol-containing dressings delayed seedling emergence stronger than the strobilurin-containing treatment (fig. 5). shoot length with 3 cm sowing depth, all treatments except for the flutriafolcontaining product resulted in a significant reduction of shoot length 21 days after seeding (fig. 6). inhibition of shoot length from the combination triadimenol + prothioconazole was even significantly stronger than from the strobilurin-containing mixture. with 5 cm sowing depth, barley dressed with triadimenol and flutriafol products, respectively, was significantly shorter than plants from the other treatments. five weeks after sowing at 3 cm depth no significant differences among untreated and treated barley could be detected any longer (fig. 6). shoot length of barley dressed with triadimenolcontaining products planted at 5 cm depth was still significantly reduced 35 days after seeding. shoot dry matter production with both sowing depths, no seed treatment significantly influenced dry matter production of barley seedlings (fig. 6). shallow seeding slightly favoured dry matter accumulation in all treatments. length of subcrown internode independent of sd, triadimenol-containing products markedly reduced the elongation of subcrown internodes as compared to the other treatments (fig. 6). deeper seeding resulted in a subcrown internode generally 15 mm longer than for barley from seeds placed at 3 cm depth. effect of environmental conditions and fungicide seed treatments on chlorophyll content due to large plant-to-plant variability significant differences in chlorophyll concentration between untreated and fungicide-treated seedlings were not detected, except for triadimenol-containing seed treatments which caused either a significant or slight increase in chlorophyll content when compared to untreated plants (tab. 3). a marked effect of environmental conditions on the side-effect of seed treatments on chlorophyll concentration could not be detected. effect of environmental conditions and fungicides on morphology and biomass of roots root length no seed treatment exhibited a significant plant growth activity on root length (tab. 4). at 9 to 10 °c, root length of seedlings treated with triadimenol and flutriafol, respectively, was significantly shorter untreated triadimenol+imazalil+fuberidazole triadimenol+prothioconazole+triazoxide prothioconazole+tebuconazole+fluoxastrobin + triazoxide flutriafol+prochloraz+pyrimethanil 0 50 100 150 200 250 300 a a a a a 0 50 100 150 200 250 300 s h oo td ry m as s [m g] a a a a a 40 % 21 35 60 % 40 % 60 % 40 % 60 % 0 5 10 15 20 25 30 35 40 45 a a a b b a a a b b l en g th o f su b cr o w n in te rn o d e [m m ] 40 % 60 % cb 0 5 10 15 20 25 30 35 40 s ho o tl en g th [c m ] a b a a a a a a a 21 35 days after sowing21 days after sowing a c b c a b c a b a ba ba b a ba b a b a b a c a bb c a b soil water content soil water content fig. 4: effect of soil water content (swc) on plant growth regulator activities of fungicidal seed treatments on shoot length (a), dry matter production (b) and length of subcrown internode (c) of barley. values with same letters are not significantly different (p < 0.05, tukey test). untreated triadimenol+imazalil+fuberidazole triadimenol+prothioconazole+triazoxide prothioconazole+tebuconazole +fluoxastrobin+triazoxide flutriafol+prochloraz+pyrimethanil 0 10 20 30 40 50 60 70 80 90 100 6 7 8 9 10 11 12 13 14 15 16 7 8 9 10 11 12 13 14 15 16 17 18 days after sowing 0 10 20 30 40 50 60 70 80 90 100 6 8 c um ul at iv e em er ge nc e ra te [ % ] a days after sowing b fig. 5: effect of sowing depth on the emergence of barley dressed with various fungicides. a, 3 cm sowing depth; b, 5 cm sowing depth (dotted lines indicate 50 % and 90 % emergence, respectively). plant growth regulator activity of seed treatments 65 than that of the strobilurin-containing treatment. except for 60 % swc, seed treatments with this product slightly stimulated root growth. diameter of roots no fungicide seed dressing had a significant side-effect on average root diameter. except for 40 % swc, roots of treated and untreated tab. 3: effect of environmental conditions and seed treatments on relative chlorophyll concentration of barley leaves measured by spad-meter readings 21 and 35 days after sowing. seed dressing environmental condition 21 days after sowing 35 days after sowing temperature: 9 10 °c 17 19 °c 9 10 °c 17 19 °c untreated control 41.6 a 42 a 47.8 a 37.8 a triadimenol + imazalil + fuberidazole 41.9 a 44 a 48.1 a 41.7 a triadimenol + prothiocon. + triazoxide 43.8 a 43.3 a 48.5 a 41.4 a prothiocon. + tebucon. + fluoxastr. + triaz. 40.8 a 43.1 a 49.2 a 38.7 a flutriafol + prochloraz + pyrimethanil 40.5 a 43.2 a 47.9 a 40.4 a soil water content: 40 % 60 % 40 % 60 % untreated control 38.1 ab 42.1 a 41.7 b 40.9 ab triadimenol + imazalil + fuberidazole 41.7 a 42.3 a 44.8 a 41.5 ab triadimenol + prothiocon. + triazoxide 38.4 ab 42.4 a 41.5 b 41.1 ab prothiocon. + tebucon. + fluoxastr. + triaz. 37.7 ab 44.8 a 42.9 ab 42.1 a flutriafol + prochloraz + pyrimethanil 35.9 b 41.9 a 41.6 b 39.2 b sowing depth: 3 cm 5 cm 3 cm 5 cm untreated control 44.8 a 43.6 a 39.4 cd 40 b triadimenol + imazalil + fuberidazole 47.3 a 47 a 44.9 a 44.7 a triadimenol + prothiocon. + triazoxide 44.7 a 47.6 a 43.6 ab 44.8 a prothiocon. + tebucon. + fluoxastr. + triaz. 44.5 a 46.1 a 38.9 d 41.3 b flutriafol + prochloraz + pyrimethanil 45.5 a 44.7 a 41.9 bc 40.7 b same letters in a column indicate values not significantly different (p < 0.05; tukey test) barley seedlings had similar diameters 35 days after sowing. under dry soil conditions, roots of barley dressed with strobilurin or flutriafol-containing products were significantly thicker than roots of untreated and triadimenol + prothioconazole-treated plants, respectively. significant differences between the root diameters of treated seedlings occurred also for plants grown at 9 to 10 °c and 17 to 19 °c, respectively. untreated triadimenol+imazalil+fuberidazole triadimenol+prothioconazole+triazoxide prothioconazole+tebuconazole+fluoxastrobin + triazoxide flutriafol+prochloraz+pyrimethanil 0 5 10 15 20 25 30 a b c c b a a a b d c d b c a b aa b b a b a a c b c a b c s h o ot d ry m as s[ m g ] s h o ot d ry m as s[ m g ] 3 cm 5 cm 3 cm 5 cm 3 cm 5 cm l en g th o f s u bc ro w n in te rn o d e [m m ] 0 5 10 15 20 25 30 35 40 45 a a a b b a a a b b 3 cm 5 cm ca b 0 10 20 30 40 50 60 a a a a a a a a a a 0 10 20 30 40 50 60 a a a a a a a a 0 5 10 15 20 25 30 s h oo tl en g th [c m ] a bc c b a a ab d cd bc ab a ab b ab a a c bc abc a b c c b a a a b d c d b c a b aa b b a b a a c b c a b c s h o ot d ry m as s[ m g ] s h o ot d ry m as s[ m g ] 3 cm 5 cm 3 cm 5 cm 21 days after sowing 35 days after sowing 3 cm 5 cm l en g th o f s u bc ro w n in te rn o d e [m m ] 0 5 10 15 20 25 30 35 40 45 a a a b b a a a b b 3 cm 5 cm c sowing depth sowing depth fig. 6: effect of sowing depth on plant growth regulator activities of fungicidal seed dressings on shoot length (a), dry matter production (b) and length of subcrown internodes (c) of barley. values with same letters are not significantly different (p < 0.05, tukey test). 66 andreas görtz, erich-christian oerke, thomas puhl, ulrike steiner root biomass under both temperature conditions and sowing depths, seedlings treated with the strobilurin-containing product produced the highest root dry matter (tab. 4). at 17 to 19 °c, the dry matter was significantly higher than that of all other treatments. at low temperature, no significant differences could be detected between untreated and fungicide-treated barley seedlings; root dry matter of barley grown from seeds treated with the strobilurin product, however, was significantly higher than that of triadimenoland flutriafol-treated plants. for both, sd and swc, no significant differences could be detected among treatments. discussion composite seed dressing products containing triadimenol had similar pgr activities as described for the use of triadimenol alone. reduction and delay of plant emergence, inhibition of shoot growth and the reduction in subcrown internode length are commonly observed morphological side-effects following triadimenol applications (frohberger, 1978, förster et al., 1980a, cavariani et al., 1994; montfort et al., 1996). the strong inhibition of root growth described by förster et al. (1980a) could not be confirmed, probably because of the different stages of plant development in which the root length were examined in both studies. a compensation of the initial growth inhibition in later development stages would be plausible. an increase in leaf thickness in response to a reduction of leaf area from triazole derivatives has been reported by benton and cobb (1995). additional layers of palisade mesophyll cells per unit area described for triadimenol-treated wheat (gao et al., 1987) may be involved in these effects and may also explain the slight increase in chlorophyll concentration of barley plants. the results revealed that shoot biomass of seedlings was affected less than shoot elongation, maybe due to increased width and thickness of leaves, often reported as a side-effect of triazole applications (buchenauer and röhner, 1981; förster et al., 1980b; buchenauer et al., 1984; gao et al., 1987; benton and cobb, 1995). lower pgr activities of seed dressings containing flutriafol and prochloraz and prothioconazole and tebuconazole, respectively, may be attributed to a lower affinity of the different triazoleand imidazole-derivatives to the cytochrome p-450 dependent enzymes involved in gibberellin biosynthesis. shoot length and subcrown internode length of seedlings from kernels treated with these products were reduced only slightly. these plant growth parameters support the previous assumption that also other triazoles and imidazoles retard early plant growth. the concentration of active ingredients in seed treatments as well as in planta and their affinity to the cytochrome p-450 dependent enzymes, however, may have an impact on the magnitude of plant growth activities. cavariani et al. (1994) reported that plant growth retardation from tebuconazole was lower than that from triadimenol at the same concentration. tab. 4: effect of environmental conditions and seed treatments on root morphology and root dry weight of barley 35 days after sowing. seed treatment environmental conditions growth temperature sowing depth soil water content 9 -10 °c 17-19 °c 3 cm 5 cm 40 % 60 % root length [cm] untreated control 175.1 ab 266.9 a 286.6 a 284.3 a 473.1 a 510.8 a triadimenol + imazalil + fuberid. 161.1 ab 273.4 a 272.8 a 290.4 a 538.6 a 436.3 a triadimenol + prothioc. + triaz. 139.1 b 289.0 a 263.9 a 255.6 a 507.7 a 429.2 a prothioc. + tebuc. + fluoxa. + triaz. 200.8 a 335.0 a 339.8 a 351.4 a 556.0 a 502.0 a flutriafol + prochloraz + pyrimeth. 145.2 b 309.3 a 301.3 a 322.4 a 494.7 a 447.5 a root dry-matter [mg] untreated control 11.7 ab 9.8 b 39.0 a 37.3 a 30.6 a 39.9 a triadimenol + imazalil + fuberid. 11.3 ab 10.0 b 34.5 a 33.1 a 30.6 a 29.2 a triadimenol + prothioc. + triaz. 10.6 b 11.2 b 35.3 a 35.1 a 32.6 a 24.0 a prothioc. + tebuc. + fluoxa. + triaz. 12.5 a 14.2 a 39.5 a 39.1 a 30.2 a 38.2 a flutriafol + prochloraz + pyrimeth. 9.9 b 11.2 b 38.3 a 35.8 a 29.1 a 25.1 a root diameter [mm] untreated control 0.23 ab 0.29 ab 0.35 a 0.37 a 0.24 c 0.26 a triadimenol + imazalil + fuberid. 0.23 b 0.31 a 0.36 a 0.38 a 0.26 abc 0.29 a triadimenol + prothioc. + triaz. 0.28 a 0.29 ab 0.37 a 0.37 a 0.25 bc 0.29 a prothioc. + tebuc. + fluoxa. + triaz. 0.23 b 0.26 b 0.34 a 0.36 a 0.28 a 0.28 a flutriafol + prochloraz + pyrimeth. 0.24 ab 0.30 a 0.33 a 0.35 a 0.27 ab 0.29 a same letters in a column indicate values not significantly different (p < 0.05; tukey test) plant growth regulator activity of seed treatments 67 montfort et al. (1996) attributed the reduced emergence of azoletreated wheat seedlings to the reduction of subcrown internode length, the results, however, revealed that flutriafol + prochloraz significantly reduced the emergence rate without affecting the length of subcrown internodes. reduced coleoptile elongation particularly described as an effect of triadimenol treatments (frohberger 1977, 1978), probably had a stronger effect on seedling emergence of barley dressed with triadimenol than on plants treated with flutriafol + prochloraz. the emergence of primary leaves, produced from retarded coleoptiles and additionally exposed to suboptimal environmental conditions is more complicated. this assumption is in accordance with lower emergence of seeds treated with triadimenol and flutriafol products at lower sowing depth or grown at low temperature. lindstrom et al. (1976) and dejong and best (1979) concluded that the rate of coleoptile elongation before seedling emergence is the most important determinant of crop establishment. factors that reduce the rate and magnitude of coleoptile elongation increase the risk of stand failure (allan et al., 1962; burleigh et al., 1965; lindstrom et al., 1976; sunderman, 1964). a disturbed geotropism may also contribute to a reduced emergence rate (förster et al., 1980b). primary leaves of seedlings from triadimenol-treated kernels sometimes emerged some days later than the majority, exhibiting the shape of a horseshoe, in which the tip of the primary leaves still remained reversed in the soil. environmental conditions, particularly suitable sowing conditions like moderate swc and ambient temperature, enhanced and extended pgr activities of fungicidal seed treatments. shoot growth regulating activities of both triadimenol seed treatments were markedly stronger than at lower temperature or low swc. only under optimal growth conditions the effect was still detectable 35 days after sowing. higher transpiration rates at 60 % swc and 17 to 19 °c, respectively, based on a high air to leaf vapour pressure deficit, are likely to enhance uptake and transfer of azoles into stems and leaves. in contrast, low transpiration under suboptimal conditions resulted in a lower uptake and transfer of active ingredients, which influenced shoot development to a much lesser degree. the length of subcrown internodes indicated that temperature and soil water content have no effect on the magnitude of pgr activity of triadimenol-containing seed dressings and that both factors have no marked effect on crown depth. shallow sowing, however, decreased the length of subcrown internodes, and triadimenolcontaining seed treatments significantly reduced the distance even more. huang and taylor (1993) reported that the formation of subcrown internodes in wheat seedlings is markedly influenced by sowing depth, whereas webb and stephens (1936) and dofing and schmidt (1984) concluded that the crown position is primarily affected by soil temperature. our results indicate that, in addition to sowing depth fungicidal seed dressings exhibit a strong effect on the length of subcrown internodes. the determination of the crown position takes place early after seed germination and plant emergence, respectively (taylor and mccall, 1936). uptake of high triadimenol amounts by the roots within the first days of plant development lead to a strong inhibition of the subcrown internode length. in addition to their fungicidal and plant growth retardation effects, triazoles have been reported to enhance tolerance of plants to environmental stress factors (fletcher et al., 2000). the smaller growth inhibitory effects of triazole-containing seed treatments, observed under less favourable growth conditions (low temperature, low swc), may be also explained as improved stress response of treated barley seedlings. partial closure of stomata and a transient rise in abscisic acid levels reported for triadimefon-treated beans (asare-boamah et al., 1986) as well as the maintenance of increased antioxidant activity by uniconazole in maize (li et al., 1998) are physiological effects of triazoles which may improve growth under stress conditions. sairam et al. (1995) reported that triadimefon increased tolerance of wheat to drought stress by reducing transpiration and increasing the relative water content as well as membrane stability. as shorter barley seedlings with low levels of gibberellins were highly tolerant to stress, sarkar et al. (2004) concluded that reduced gibberellin levels coupled with reduction in height are important for the induction of stress tolerance. shorter seedlings due to triadimenol treatments most probably possess higher tolerance to environmental stresses. consequently, triadimenol-treated seedlings may show increased survival during periods of drought or low temperatures, but, due to the lesser inhibition of gibberellic acid biosynthesis, stress tolerance induced by fungicidal seed treatments is likely to be lower than from commercial pgr like paclobutrazole. this study indicates that triadimenol-containing seed treatments possess stronger pgr activities than seed treatments containing other triazoles or imidazoles. strobilurins also known to influence plant development and yield formation of cereals are reported to interfere with pgr metabolism and to have an anti-stress activity (grossmann and retzlaff, 1997). as part of composite seed dressings they seem to initiate other metabolic modifications than triazoles resulting in a kind of compensation. the intensity of pgr is modified by environmental conditions the seedlings are subjected to in early growth stages. in addition to their fungicidal activities, triadimenolcontaining seed treatments could contribute to an increased tolerance to abiotic stress factors enabling barley seedlings to better cope with suboptimal growth conditions. references allan, r.e., vogel, o.a., peterson, c.j., 1962: seedling emergence rate of fall sown wheat and its association with plant height and coleoptile length. agronomy journal 54, 347-350. anderson, h.m., 1989: effect of triadimenol seed treatment on vegetative growth in winter wheat. crop research 29, 29-36. asare-boamah, n.k., hofstra, g., fletcher, r.a., dumbroff, e.b., 1986: triadimefon protects bean plants from water stress through its effect on abscisic acid. plant cell physiology 27, 383-390. benton, j.m., cobb, a.h., 1995: the plant growth regulator activity of the fungicide, epoxiconazole, on galium aparine l. (cleavers). plant growth regulation 17, 149-155. buchenauer, h., 1995: dmi-fungicides – side effects on the plant and problems of resistance. in: lyr, h. (ed.), modern selective fungicides properties, applications, mechanisms of action, 259-290. gustav fischer verlag, new york. buchenauer, h., kohts, t., roos, h., 1981: zur wirkungsweise von propiconazol, cga 64251 und dichlobutrazol in pilzen und gerstenkeimlingen. mitteilungen biologischen bundesanstalt landu. forstwirtschaft berlin-dahlem 202, 310-311. buchenauer, h., kutzner, b., kohts, t., 1984: effect of various triazole fungicides on growth of cereal seedlings and tomato plants as well as on gibberellin contents and lipid metabolism in barley seedlings. journal of plant diseases and protection 91, 506-524. buchenauer, h., röhner, e., 1977: einfluß von nuarimol, fenarimol, triadimefon und imazalil auf den gibberellinund lipidstoffwechsel in jungen gerstenund weizenpflanzen. mitteilungen der biologischen bundesanstalt landu. forstwirtschaft berlin dahlem 178, 158. buchenauer, h., röhner, e., 1981: effect of triadimefon and triadimenol on growth of various plant species as well as on gibberellin content and sterol metabolism in shoots of barley seedlings. pesticide biochemistry and physiology 15, 58-70. burleigh, j.r., allan, r.e., vogel, o.a., 1965: varietal differences in seedling emergence of winter wheats as influenced by temperature and 68 andreas görtz, erich-christian oerke, thomas puhl, ulrike steiner depth of plants. agronomy journal 57, 195-198. cavariani, c., velini, e.d., bicudo, s.j., nakagawa, j., 1994: avaliação dos efeitos de doses de triadimenol e de tebuconazole sobre o crescimento do mesocótilo em plântulas de trigo. pesq agropec bras 29, 1035-1039. chinn, s.h.f., verma, p.r., spurr, d.t., 1980: effects of imazalil seedtreatment on subcrown internode lengths and coleoptile-node tillering in wheat. canadian journal of plant science 60, 1467-1472. dejong, r., best, k.f., 1979: the effect of soil water potential, temperature and seeding depth on seedling emergence of wheat. canadian journal of soil science 59, 259-264. dofing, s.m., schmidt, j.w., 1984: inheritance of subcrown internode length in a winter barley cross. crop science 24, 692-694. fletcher, r.a., hofstra, g., gao, j., 1986: comparative fungitoxic and plant growth regulating properties of triazole derivatives. plant cell physiology 27, 367-371. fletcher, r.a., gilley, a., sankhla, n., davis, t.d., 2000: triazoles as plant growth regulators and stress protectants. horticulture revue 24, 55-138. förster, h., buchenauer, h., grossmann, k., 1980a: nebenwirkungen der systemischen fungizide triadimefon und triadimenol auf gerstenpflanzen. i. beeinflussung von wachstum und ertrag. journal of plant diseases and protection 87, 473-492. förster, h., buchenauer, h., grossmann, k., 1980b: nebenwirkungen der systemischen fungizide triadimefon und triadimenol auf gerstenpflanzen. ii. cytokinin-artige effekte. journal of plant diseases and protection 87, 640-653. frohberger, p.e., 1977: triadimenol, ein neues systemisches breitbandfungizid mit besonderer eignung für die getreidebeizung. mitteilungen der biologischen bundesanstalt landu. forstwirtschaft berlin-dahlem 178, 150-151. frohberger, p.e., 1978: baytan, a new systemic broad spectrum fungicide especially suitable for cereal seed treatments. pflanzenschutz-nachrichten bayer 31, 11-24. gao, j., hofstra, g., fletcher, r.a., 1987: anatomical changes induced by triazoles in wheat seedlings. canadian journal of botany 66, 11781185. gilgenberg-hartung, a., 1999: metconazolea new fungicide to control leafand ear diseases in cereals and oilseed rape. gesunde pflanzen 51, 55-57. grossmann, k., retzlaff, g., 1997: bioregulatory effects of the fungicidal strobilurin kresoxim-methyl in wheat (triticum aestivum). pesticide science 50, 11-20. hack, c., 1994: fungicidal and plant growth regulatory effects of tebuconazole on linseed. annals of applied biology 124, 22-23. huang, b., taylor, h.m., 1993: morphological development and anatomical features of wheat seedlings as influenced by temperature and seedling depth. crop science 33, 1269-1273. konstantinidou-doltsini, s., buchenauer, h., grossmann, f., 1987: influence of the systemic fungicides nuarimol and imazalil on growth and metabolism of wheat plants. journal of plant diseases and protection 94, 369-379. kruse, t., verreet, j.-a., 2005: epidemiological studies on winter oilseed rape (brassica napus l. var. napus) infected by phoma lingam (teleomorph leptosphaeria maculans) and the effects of different fungicides applications with folicur. journal of plant diseases and protection 112, 17-41. kuck, k.h., scheinpflug, h., pontzen, r.,1995: dmi fungicides. in: lyr, h. (ed.), modern selective fungicides properties, applications, mechanisms of action, 259-290. gustav fischer verlag, new york. li, l., van staden, j., jager, a.k., 1998: effects of plant growth regulators on the antioxidant system in seedlings of two maize cultivars subjected to water stress. plant growth regulation 25, 81-87. lindstrom, m.j., papendick, r.i., kohler, f.e., 1976: a model to predict winter wheat emergence as affected by soil temperature, water potential and depth of planting. agronomy journal 68, 137-141. montfort, f., klepper, b.l., smiley, r.w., 1996: effects of two triazole seed treatments, triticonazole and triadimenol, on growth and development of wheat. pesticide science 46, 315-322. sarkar, s., perras, m.r., falk, d.e., zhang, r., pharis r.p., fletcher, r.a., 2004: relationship between gibberellins, height and stress tolerance in barley (hordeum vulgare l.) seedlings. plant growth regulation 42, 125-135. sairam, r.k., shukla, d.s., deskmukh, p.s., 1995: effect of triazoletriadimefon on tolerance to moisture stress in wheat (triticum aestivum). indian journal of agricultural science 65, 483-489. siefert, f., grossmann, k., 1996: non-fungicidal, secondary effects of triazole compounds, as exemplified by the triazole fungicide epoxiconazole. gesunde pflanzen 48, 224-231. shive, j.b., sisler, h.d., 1976: effects of ancymidol (a growth retardant) and triarimol (a fungicide) on growth, sterols and gibberellins of phaseolus vulgaris (l.). plant physiology 57, 640-644. sunderman, d.w., 1964: seedling emergence of winter wheat and ist association with depth of sowing, coleoptile length under various conditions and plant height. agronomy journal 56, 23-25. taylor, j.w., mccall, m.a., 1936: influence of temperature and other factors on the morphology of the wheat seedlings. journal of agricultural research 52, 557-568. tennant, d., 1975: a test of the modified line intersect method for estimating root length. journal of ecology 63, 995-1001. webb, r.b., stephens, d.e., 1936: crown and root development in wheat varieties. j. agricultural research 52, 569-583. addresses of authors: andreas görtz (corresponding author), dr. erich-christian oerke and dr. ulrike steiner, institute for crop science and resource conservation (inres – phytomedicine), university of bonn, nussallee 9, d-53115 bonn, germany. dr. thomas puhl, bayer cropscience gmbh, elisabeth-selbert-str. 4a, 40764langenfeld, germany << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /colorconversionstrategy 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/pagesize [907.087 680.315] >> setpagedevice journal of applied botany and food quality 91, 232 236 (2018), doi:10.5073/jabfq.2018.091.031 friedrich-alexander-universität erlangen-nürnberg, lehrstuhl für pharmazeutische biologie, erlangen, germany development and validation of a quick assay for the total glucosinolate content in horseradish (armoracia rusticana) using glucose strips and a blood glucose meter nadine meitinger, wolfgang kreis* (submitted: august 28, 2018; accepted: october 7, 2018) * corresponding author summary a quick assay to determine the total glucosinolate content of fresh horseradish roots in less than 10 minutes is described. the method involves the following steps: 1. maceration of horseradish root with 4% phosphoric acid to avoid enzymatic degradation of endogenous glucosinolates, 2. neutralization of the extract and determination of free glucose using a commercial blood glucose meter, 3. enzymatic hydrolysis of the glucosinolates by exogenous myrosinase, 4. de tection of released glucose, again using a blood glucose meter, and 5. calculation of the glucosinolate content on the basis of the difference between the two glucose values determined. the newly developed assay (‘itc quick test’) was compared with a standard high-performance liquid chromatographic method for glucosinolate analysis. keywords: armoracia rusticana, glucosinolates, allyl isothio cyanate, sinigrin, glucose introduction the root of armoracia rusticana p. gaertn., b. mey. & scherb. (horseradish) is extensively used as a spice, usually grated fresh as a condiment or as a food additive for its pungent flavor. both taste and quality of horseradish as well as of other cruciferous vegetables are significantly determined by the glucosinolate content. glucosinolates are anionic and sulfur-rich natural products, which primarily occur in plants of the brassicaceae family. the characteristic flavor mainly originates from the breakdown of water-soluble glucosinolates, stored in the plant vacuole, into volatile isothiocyanates (sampliner and miller, 2009; agneta et al., 2012) another feature of these breakdown products is their antimicrobial, antifungal and chemopreventive action (glenn et al., 1988; brabban and edwards, 1995; talalay and zhang, 1996). disruption of glucosinolatecontaining plant material by herbivore attack, chopping or chewing not only results in the release of isothiocyanates, thiocyanates and nitriles but also of glucose and sulfate in amounts equimolar to the amount of glucosinolate precursor present. the conversion of the glucosinolates is initiated by the action of a thioglucosidase termed myrosinase. several methods for glucosinolate analysis determining the total glucosinolate content as well as individual glucosinolates and their breakdown products are already available (moreno et al., 2006). the method most commonly used for glucosinolate determination is the quantitative analysis of desulfoglucosinolates by reversedphase hplc (en iso 9167). this method, which was developed by fenwick et al. (1982), uses an on-column enzymatic desulfation treatment of plant extracts followed by hplc detection of the resulting desulfoglucosinolates. many of the other described methods resulted from various attempts that have been made to simplify and accelerate the glucosinolate quantification. still, most of the available methods include time-consuming sample preparation – especially those involving a column purification step (smith and dacombe, 1987; heaney et al., 1988; doorn et al. 1999; gallaher et al., 2012). not in all cases validation of the described methods have been reported and, frequently, rapeseed samples were analyzed. there is thus still a demand for rapid and reliable methods for glucosinolate quantification. such a fast and simple method could be applied in the field or used for rapid screening of incoming or stored plant material and goods. tholen et al. (1993) proposed the use of glucose test strips to test for glucose released by myrosinase action to estimate glucosinolates. these attempts were also restricted to rapeseed analysis. interestingly, this very simple and rapid method has been ignored in recent reports and reviews and has not been developed further. we here adopted the idea of tholen et al. (1993) and combined a quick and simple extraction procedure with the determination of the total glucosinolate content by measuring the glucose released following glucosinolate hydrolysis with exogenous myrosinase. the released glucose was then determined with a commercial blood glucose meter and the appropriate test strips. we present a simple, fast and reliable method for estimating the total glucosinolate content in horseradish roots. this method may turn out an alternative to more sophisticated, costly and rather time-consuming hplc, gc and/or spectrophotometric methods (fig. 1). + so4 2hplc glucose meter myrosinase o hoh o o h oh s n o s o o – o n cs oh o o h oh o h o h sinigrin allylisothiocyanate glucose h2o fig. 1: rapid assay (‘itc quick test’) for estimating the glucosinolate content of horseradish. exogenous myrosinase is added to allow for the complete degradation of endogenous glucosinolates. equimolar amounts of glucose, sulfate and isothiocyanate are released. glucose is determined using a commercial glucose strip/meter system. materials and methods materials sinigrin monohydrate was purchased from phytoplan diehm & neuberger gmbh (heidelberg, germany). fresh horseradish roots were provided by schamel meerrettich gmbh & co.kg (baiersdorf, germany). sinapis alba l. seeds were purchased in a local store. all other chemicals were used in p.a. quality and purchased from carl roth gmbh & co.kg (karlsruhe, germany). quick asasy for glucosinolate determination 233 glucosinolate extraction and determination using the glucose strip/glucose meter method (‘itc quick test’) a 1 cm thick slice was cut from a horseradish root. from this, two pieces were carved with a cylindrical plunge borer with an inner diameter of 8 mm. the horseradish pieces were homogenized with 1 ml of 4% v/v phosphoric acid using a mortar and a pestle. the slurry was diluted with 4 ml of distilled water and filtered through a disposable syringe filled with cotton wool. the extract was neutralized by adding 165 μl of 5 n koh. to determine the glucosinolate content together with the endogenous glucose content, 50 μl of the extract were mixed with 100 μl of exogenous myrosinase solution (minimum activity 10 u/ml) on a piece of parafilm m®. to determine the endogenous glucose content only, 50 μl of the extract were mixed with 100 μl of distilled water as a blank control. after 5 min the glucose content of the sample and the blank was measured with an accu-check aviva® blood glucose meter (roche diagnostics, mannheim, germany). the glucosinolate content of the sample was calculated with the formula: gls (mm) = 0.079 × (sample [mg/dl] blank [mg/dl] × 0.82) the unit mg/dl (which is us standard) was used here since this value can directly be read from the blood glucose meter. the standard curve was prepared by dissolving sinigrin in horseradish extract. the concentrations of the prepared standard solutions ranged from 0.5 mm to 12 mm. standard solutions were measured as described above. analyzing the pure horseradish extract (without additional sinigrin) served to eliminate the glucosinolate and endogenous glucose concentration present in the extract. the factor which takes into account that the blank consists of a mixture of extract and water while in the sample extract is mixed with myrosinase solution was determined by comparing blanks prepared with 100 μl of double distilled water to blanks prepared with 100 μl of heat-denatured myrosinase solution. additionally, extracts spiked with glucose in three different concentrations were analyzed with water and heat-denatured myrosinase addition. hplc and gc-ms hplc: 1 ml of the horseradish extract was mixed with 150 μl of 0.5 m barium acetate. subsequent desulfation of the glucosinolates was performed following the protocol of li et al. (2004). a 20 μl fraction of the desulfoglucosinolate-fraction (water eluate) was in jected onto a hplc system (binary hplc pump 1525, dual λ absorbance detector 2487, autosampler 717plus; waters gmbh, eschborn, germany) equipped with a reprosil fluosil 100 pfp column (250 × 4.6 mm; 5 μm) (dr. maisch hplc gmbh, ammerbuchentringen, germany). the following gradient of acetonitrile (b) and water (a) was used at a flow rate of 1 ml/min: 0 5 min: 0% b; 5 30 min: 30% b, 30 32 min: 40% b, 32 36 min: 40% b; 36 40 min: 100% b; 40 45 min: 100% b. desulfoglucosinolates were identified with a uv detector at 229 nm. sinigrin concentration was estimated by comparison of hplc retention time with that of an authentic sinigrin standard after on-column desulfation as described above. gc-ms: isothiocyanates (itc) in the extracts were analyzed by gcms. 1 ml of the horseradish solution was extracted with 1 ml of dichloromethane by vortexing for 30 s. the mixture was centrifuged for 5 min at 13000 × g (heraeus fresco 17, heraeus deutschland gmbh & co., hanau germany) before the organic phase was sepa rated. gc-ms analysis of the organic phase was performed using gc-2010 equipped with the mass spectrometer qp-2010s (shimadzu deutschland gmbh, duisburg, germany) using the following con ditions: db-5ms (agilent technologies deutschland gmbh, wald bronn, germany) column (30 m × 0.25 mm, film 0.25 μm); column oven temperature: 60 °c; injector temperature: 230 °c; injection volume: 1 μl; split ratio: 1:100; ion source temperature: 200 °c; interface temperature: 210 °c; ms in scan mode: 35 m/z 200 m/z. the flowing temperature gradient with helium as gas carrier was implemented at a flow rate of 46.3 cm/s. the temperature was kept constant for three min at 60 °c before it was increased to 210 °c at a rate of 10 °c per min. isolation of myrosinase and myrosinase assay myrosinase was extracted from sinapsis alba (mustard) seeds. 10 g seeds were ground in a commercial electric coffee grinder and homogenized with double distilled water in a 1:10 ratio (w/v) for 30 min at 4 °c. the homogenate was filtered through 3 layers of miracloth® and centrifuged for 20 min at 20000 × g for 4 °c (ss34 rotor, sorvall rc5b plus; kendro laboratory products gmbh, langenselbold, germany). (nh4)2so4 was slowly added to the extract as a fine powder while stirring for 30 min to achieve a 50% salt saturation. after centrifugation at 20000 × g for 20 min the pellet was discarded. the (nh4)2so4 concentration in the supernatant was increased to 100% before the solution was treated as above. the harvested pellet was dissolved in 10 ml of double distilled water and centrifuged again. the protein solution was desalted and concentrated to an activity of at least 10 u/ml using amicon ultra-15 (10 kda nmgg) centrifugation filters (merck millipore, darmstadt, germany). myrosinase activity was measured following the protocol of li et al. (2004) with minor modification. 900 μl of sodium phosphate buffer (20 mm, ph 6.5) and 100 μl of a sinigrin solution (1 mm in water) were mixed in a quartz cuvette and pre-incubated at 37 °c for 1 min. the reaction was started by adding 100 μl of protein extract previously diluted 1:40 with water and the decline as result of sinigrin hydrolysis was measured at 227 nm for 180 s using a uv-vis spectrophotometer. method validation for validation we compared the ‘itc quick test’ with an established hplc method (li et al., 2004). to determine recovery, sinigrin was dissolved in 4% phosphoric acid to yield concentrations between 1.0 10 mm sinigrin. these spiked phosphoric acid solutions were used to prepare the horseradish extracts as described above. the glucosinolate content of the pre pared extracts was determined as outlined above and compared with the corresponding non-spiked extracts. results and discussion developing a rapid method for glucosinolate determination (‘itc quick test’) hydrolysis of total glucosinolates with myrosinase yields equimolar amounts of glucose. therefore, glucosinolates were here quantified by measuring the amount of glucose released from them after complete enzymatic hydrolysis. glucose was determined using a commercial blood glucose meter and the appropriate analytic strips. as mentioned before, tholen at al. (1993) already used commercial glucose test strips typically used for the semi-quantitive determination of glucose in urine. others (smith and dacombe, 1987; heaney et al., 1988) describe the use of a glucose/peroxidase assay for glucose quantification as a surrogate for glucosinolates present in a given extract. whenever glucosinolates are quantified based on enzymatically released glucose it has to be taken into account that plant extracts also contain free glucose and glucosides others than glucosinolates. therefore, an on-column purification step to remove endogenous glucose (heaney et al., 1988) or the removal of endo genous glucose from the extract by using glucose oxidase before glucosinolates were hydrolyzed by exogenous myrosinase (doorn 234 n. meitinger, w. kreis et al., 1999) is described. the protocol of smith and dacombe (1986) involves the parallel preparation of two extracts, one with water as extractant to achieve glucosinolate hydrolysis utilizing endogenous myrosinase, the other with acidified methanol (40%) to inhibit endogenous myrosinase and thus to determine the amount of endogenous glucose present in the extract. the method of tholen et al. (1993) hydrolyzes glucosinolates at ph 9.0 which was supposed to prevent the liberation of glucose from sources other than glucosinolates. this approach was justified by the fact that myrosinase is quite active at this ph whereas most other glucosidases are inactive at ph values higher than ph 8.0. however, the amount of endogenous glucose was not directly considered in their method and can therefore not be used for plant materials others than rapeseed which contain negligible amounts of free glucose only. we here aimed at avoiding the time-consuming removal of endogenous glucose from the extract. at the same time the method should allow for the estimation of endogenous free glucose in a given plant material. glucosinolates were extracted from ca. 1 g of a fresh horseradish root using 1 ml of phosphoric acid. sampling was standardized by withdrawing two pieces of horseradish with a cylindrical plunge borer with an inner diameter of 8 mm (1.04 ± 0.01 g; n = 10). before use the horseradish extract was diluted with 4 ml of water and filtered through a syringe partly filled with cotton wool to remove any resi dual plant tissue. glucosinolate extraction from the horseradish roots was a first critical step because glucosinolates are immediately hydrolyzed upon homogenization of the plant material by endogenous myrosinase. extraction with hot water or methanol is therefore used in many protocols in order to avoid enzymatic degradation (gallaher et al., 2012; heaney et al., 1988; li and kushad, 2004). the use of boiling solvents is, however, not suitable for a quick glucosinolate assay, that can be used in the field. if organic solvents are used to inhibit the endogenous myrosinase during extraction, the solvent would have to be removed before the glucosinolates can be hydrolyzed by the addition of myrosinase solution. glucosinolate extraction using 4% phosphoric acid according to doorn et al. (1998) at room temperature turned out to be a fast and reliable method. no myrosinase activity was detected in horseradish root extracts under these conditions. the activity was also not detectable after the extracts had been neutralized with koh which indicated that the myrosinase was irreversibly inhibited. in contrast myrosinase activities of 0.17 ± 0.04 u/ml were found in horseradish preparations derived from extraction of horseradish roots with double-distilled water. each of the different horseradish extracts was also analyzed for the presence of isothiocyanates by gc/ms (fig. 2). in the extracts prepared with phosphoric acid, no isothiocyanates were detected, not even after the extracts were neutralized with koh showing that glucosinolates were not hydrolyzed during extraction. when water was used for horseradish extraction, aitc (allyl isothiocyanate) as well as peitc (phenethyl isothiocyanate) were present demonstrating the glucosinolate breakdown. sinigrin, the major glucosinolate of horseradish (about 80%), is stable in the acidic extraction medium (doorn et al., 1998). after neutralization of the extract the glucosinolates were hydrolyzed by the addition of exogenous mustard myrosinase which was prepared from sinapsis alba seeds (see material and methods). neutralization of the extract was necessary to optimize the conditions for the addition of the exogenous mustard myrosinase. the addition of 165 μl 5 n koh neutralized the extract (ph 7.01 ± 0.01; n = 4). endogenous free glucose was determined after adding water instead of myrosinase solution to the extract. the difference of sample and blank then corresponded to the amount of glucosinolates present in the extract. the myrosinase solution applied for hydrolysis had to be desalted before use since it was found that high salt concentrations resulted in an error reading of the glucose meter. the desalted enzyme solution could be stored at -20 °c for several weeks without a significant loss of activity. complete glucosinolate hydrolysis was achieved after 5 min of incbubation when 1 u/ml of myrosinase was added to 50 μl of horseradish extract. this assay was linear up to 16 mm sinigrin. influence of the sample matrix on glucose determination we used a commercial blood glucose meter for quantifying the glucose content in horseradish extracts. this technique allows glucose quantification within seconds. blood glucose meters are used to determine the glucose content of blood samples. therefore, we had to verify its accuracy for measuring glucose in horseradish root extracts. a linear relationship between the actual glucose concentration and the values measured with the glucose meter was proven using three different matrices (fig. 3). the measured glucose values strongly depended on the sample matrix. when glucose was dissolved in pure water the measured glucose values displayed on the glucose meter were twice as high as predicted by the glucose meter. on the other hand, when glucose was dissolved in a horseradish extract/myrosinase solution the measured glucose concentration matched the amount of glucose present in the assay. to allow for this matrix effect a standard curve was generated with different amounts of sinigrin dissolved in horseradish extract. the glucose release was then measured after addition of myrosinase and incubation using the blood glucose meter. the values were each corrected by subtracting the glucose content of the crude extract (fig. 4). linearity was provided up to 15 mm sinigrin. the factor derived from the slope of the line was used for calculating the glucosinolate content of the extract. the slope of the curve was 1.4 times higher when sinigrin was dissolved in water instead of horseradish extract. this fact corresponds well the matrix effect mentioned above. we also accounted for matrix effects for other constellations (stop solutions, controls) and finally calculated the total glucosinolate content using the following formula: gls (mm) = 0.079 × (sample [mg/dl] blank [mg/dl] × 0.82) where ‘gls’ is glucosinolates, ‘sample’ is the read of the sample after myrosinase action and ‘blank’ is the read when water instead of myrosinase solution was added. x 100.000 5 4 3 2 1 0 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 time (min) in te ns ity (a u ) fig. 2: gc-ms analysis of horseradish extracts prepared with different extracting agents. a – aqueous extract; b – 4% phosphoric acid extract; c – 4% phosphoric acid extract after neutralization. aitc – allyl isothiocyanate; peitc – phenethyl isothiocyanate) quick asasy for glucosinolate determination 235 method validation to check the recovery in the ‘itc quick test’, horseradish roots were extracted with 4% phosphoric acid spiked with sinigrin in the concentration range between 1.0 – 10.0 mm sinigrin (n = 10). the recovery assays were compared with non-spiked extracts. the recovery rate was 90 ± 7%. in order to challenge accuracy and reproducibility of the developed quick test the glucosinolate contents of four different horseradish roots were analyzed in quadruplicate. all extracts were analyzed using the ‘itc quick test’ as well as by hplc (li and kushad, 2004) and the results were compared (fig. 4). for the calculations it has to be considered that sinigrin represents about 80% of the total glucosinolate content (li and kushad, 2004; uematsu et al., 2002; kübler, 2010; own spot samples; fig. 5). the ‘itc quick test’ therefore shows values about 20% higher than the respective hplc values for sinigrin. comparison of the ‘itc quick test’ results with those obtained by hplc showed that there was reasonable good agreement within the two methods. both methods showed similar standard deviations, which justified the use of the quick assay using commercial glucose strips. the ‘itc quick test’ has been developed for the estimation of glucosinolates in fresh horseradish roots but it is also applicable for estimating glucosinolates in other cruciferous vegetables. for example, we also estimated the glucosinolate content in garden cress (2.3 mm), radish (0.3 mm) and broccoli (0.5 mm) using this method. it is advised, however, to calibrate the method for each type of vegetable to address the matrix effects (see above). 0 10 20 30 40 50 0 20 40 60 80 100 120 glucose added (mg/dl) g lu co se d et er m in ed (m g/ dl ) a b c fig. 3: influence of the sample matrix on the glucose concentration values measured with a blood glucose meter. a – glucose dissolved in pure water (y = 2.0 x; r2 = 0.99); b – glucose dissolved in water and treated with myrosinase solution (y = 1.6 x; r2 = 0.99); c – glucose dissolved in horseradish extract and treated with myrosinase solution. the glucose concentration originating from the extract is eliminated by subtraction. (y = 1.1 x; r2 = 0.99). 0 2 4 6 8 10 12 14 16 0 50 100 150 200 250 300 sinigrin concentration (mm) g lu c o s e c o n c e n tr a ti o n ( m g /d l) a b fig. 4: calibration curves prepared with sinigrin as standard. a – calibration curve prepared with sinigrin dissolved in extract (y = 12.7 x; r2 = 0.99). this fact takes the “matrix effect” into account. the glucose concentration originating from the extract is eliminated by subtraction. b – calibration curve prepared with aqueous sinigrin solutions (y = 18.3 x; r2 = 0.99). conclusion we here have picked up the idea of tholen at al. (1993) to use commercial glucose test strips to quantify glucosinolates. we have developed a very simple, fast, reliable and robust technique to de termine the glucosinolate content in fresh horseradish roots. our assay can be carried out in less than 10 min and requires very simple and small equipment. therefore, this method suits well for the screening of horseradish and other glucosinolate-containing plants, either in the field or in quality control of incoming and stored goods. acknowledgements we thank barbara white for linguistic advice. we also thank the students steffen baier, melike yazici and marie krenauer for technical assistance. we thank schamel meerrettich gmbh & co. kg for their financial support. references agneta, r., rivelli, a.r., ventrella, e., lelario, f., sarli, g., bufo, s.a., 2012: investigation of glucosinolate profile and qualitative aspects in sprouts and roots of horseradish (armoracia rusticana) using lcesi-hybrid linear ion trap with fourier transform ion cyclotron resonance mass spectrometry and infrared multiphoton dissociation. j. agric. food chem. 60, 7474-7482. doi: 10.1021/jf301294h brabban, a.d., edwards, c., 1995: the effects of glucosinolates and their hydrolysis products on microbial growth. j. appl. bacteriol. 79, 171-177. doorn, h.e., van der kruk, g.c., van holst, g.j., 1999: large scale determination of glucosinolates in brussels sprouts samples after de 0 2 4 6 8 0 1 2 3 4 5 6 7 glucose strip/meter method (mm glucose released) h p l c ( m m s in ig ri n ) r2 = 0.90 fig. 5: correlation between the amount of sinigrin determined by hplc and glucose released from the same samples using the ‘itc quick test’ developed here. 236 n. meitinger, w. kreis gradation of endogenous glucose. j. agric. food chem. 47, 1029-1034. doi: 10.1021/jf9808170 doorn, h.e., van holst, g.j., van der kruk, g.c., raaijmakers-ruijs, n.c., postma, e., 1998: quantitative determination of the glucosinolates sinigrin and progoitrin by specific antibody elisa assays in brussels sprouts. j. agric. food chem. 46, 793-800. doi: 10.1021/jf970523z fenwick, g.r., heaney, r.k., mullin, w.j., van etten, c.h., 1982: glucosinolates and their breakdown products in food and food plants. food sci. nutr. 18, 123-201. doi: 10.1080/10408398209527361 gallaher, c.m., gallaher, d.d., peterson, s., 2012: development and validation of a spectrophotometric method for quantification of total glucosinolates in cruciferous vegetables. j. agric. food chem. 60, 13581362. doi: 10.1021/jf2041142 glenn, m.g., chew, f.s., williams, p.h., 1988: influence of glucosinolate content of brassica (cruciferae) roots on growth of vesicular-arbuscular mycorrhizal fungi. new phytol. 110, 217-225. heaney, r.k., spinks, e.a., fenwick, g.r., 1988: improved method for the determination of the total glucosinolate content of rapeseed by determination of enzymically released glucose. analyst 113, 1515-1518. doi: 10.1039/an9881301515 li, x., kushad, m.m., 2004: correlation of glucosinolate content to myrosinase activity in horseradish (armoracia rusticana). j. agric. food chem. 52, 6950-6955. doi: 10.1021/jf0401827 kübler, k., 2010: analyse von glucosinolaten und isothiocyanaten mittels flüssigkeitschromatographiebzw. gaschromatographie-massenspek trometrie, dissertation, justus-liebig-university, gießen. moreno, d.a., carvajal, m., lópez-berenguer, c., garcía-viguera, c., 2006: chemical and biological characterization of nutraceutical compounds of broccoli. j. phar. biomed. anal. 41, 1508-1522. doi: 10.1016/j.jpba.2006.04.003 sampliner, d., miller, a., 2009: ethnobotany of horseradish (armoracia rusticana, brassicaceae) and its wild relatives (armoracia spp.): reproductive biology and local uses in their native ranges. econ. bot. 63, 303-313. doi: 10.1007/s12231-009-9088-1 smith, c.a., dacombe, c., 1987: rapid method for determining total glucosinolates in rapeseed by measurement of enzymatically released glucose. j. sci. food agric. 38, 141-150. doi: 10.1002/jsfa.2740380206 talalay, p., zhang, y., 1996: chemoprotection against cancer by isothiocyanates and glucosinolates. biochem. soc. trans. 24, 806-810. doi: 10.1042/bst0240806 tholen, j.t., buzza, g., mcgregor, d.i., truscoir, r.j.w., 1993: measurement of the glucosinolate content in rapeseed using the trubluglu meter. plant breed. 110, 137-143. doi: 10.1111/j.1439-0523.1993.tb01225.x uematsu, y., hirata, k., suzuki, k., iida, k., ueta, t., kamata, k., 2002: determination of isothiocyanates and related compounds in mustard extract and horseradish extract used as natural food additives. shokuhin eiseigaku zasshi. 43, 10-17. doi: 10.3358/shokueishi.43.10 address of the corresponding authors: e-mail: wolfgang.kreis@fau.de © the author(s) 2018. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). art05a journal of applied botany and food quality 80, 36 39 (2006) norwegian university of life sciences, 1 department of plant and environmental sciences, 2 department of mathematical sciences and technology, n-1432 aas, norway an approach towards rapid optical measurements of antioxidant activity in blueberry cultivars* siv f. remberg1, anne-berit wold1, knut kvaal2, maigull appelgren1, karin haffner1 (received january 2, 2006) summary blueberries are well known for their high antioxidant levels. compared to bilberries (v. myrtillus) with higher antioxidant activity and more intensive blue colour throughout the whole berry, highbush blueberries have the blue pigments concentrated in the skin. highbush blueberry skin is found to contain a very high content of phenolic compounds. to measure the total antioxidant activity in blueberries, several methods, mostly destructive, including the frap assay, have been used. this work is an initial approach towards a simple and rapid method, combining optical and antioxidant activity measurements. highbush blueberry (v. corymbosum) cultivars ‘bluecrop’, ‘hardyblue’, ‘patriot’, and lowbush cultivars ‘putte’ (a hybrid originated from v. angustifolium) and ‘aron’ (v. corymbosum x v. uliginosum) were grown at the norwegian university of life sciences (59º 40’n). berries were harvested at commercial blue-ripe stage of maturity. fresh berries were cut horizontally and placed on a scanner in order to examine berry size and skin thickness. berries were weighed, and analysed for antioxidant activity using the frap (ferric reducing ability of plasma) assay. the frap assay is a non-specific method based on absorption changes following a reduction of a ferricto a ferrous-complex in the presence of antioxidants. own previous results have shown that antioxidant activity and berry weight varied between cultivars (remberg et al., 2003). small berries had higher antioxidant activity compared to larger berries. in this follow-up project, skin thickness and berry diameter were measured by using an imageprocessing program. berry and skin cross-section areas were correlated with the antioxidant activity. introduction blueberries are good sources for antioxidants (halvorsen et al., 2002), and the antioxidant activity has been found to correlate well with the total phenolic and anthocyanin content of the fruits (ehlenfeldt and prior, 2001). the skin of highbush blueberries contains a very high content of phenolic compounds, and in highbush blueberries, pigments are concentrated in the skin (allanwojtas et al., 2001; kalt et al., 2001). since pigments and phenolic compounds in cultivated blueberries are confined primarily to the skin (lee and wrolstad, 2004), berry size is expected to be an important factor as related to antioxidant activity. examining highbush (v. corymbosum) and lowbush (v. angustifolium) blueberry cultivars, kalt et al. (2001) found that the smaller lowbush blueberries were consistently higher in anthocyanins, total phenolics, and antioxidant activity. the aim of the present investigation was to predict antioxidant activity by measuring physical attributes in blueberry cultivars. ‘bluecrop’, ‘hardyblue’ and ‘patriot’, being important cultivars in norway, are mainly produced for the fresh marked. the swedish cultivar ‘putte’ and the finnish cultivar ‘aron’ are considered to be especially winter hardy, and therefore suitable for scandinavia. the berries of these two cultivars are rather small with a dark blue colour. in previous studies, three methods have been used to assess total antioxidant activity in plants. as concluded in halvorsen et al. (2002), the frap assay was also chosen in these experiments. materials and methods plant material highbush blueberry cultivars ‘bluecrop’, ‘hardyblue’, ‘patriot’ (v. corymbosum), and lowbush cultivars ‘putte’ (a hybrid originated from v. angustifolium) and ‘aron’ (v. corymbosum x v. uliginosum) were grown at the norwegian university of life sciences (59º 40’n) as a randomized block trial with four replicates of each cultivar. the field was planted in 1990 (‘putte’ in 1996). the berries were harvested by hand in the season 2001 at commercial blue-ripe stage of maturity. berry weight, diameter, skin thickness and antioxidant activity (frap) were determined. scanning the scanner combined with statistics and image analysis makes an excellent toolbox to measure interesting physical quality attributes on berries (haffner et al., 2000), and is applied here on highbush blueberries. fresh berries were cut horizontally and placed on a scanner (agfa snapscan 1212u) and scanned using scanwise software (scanwise 1.04). berry and flesh diameter (fig. 1) were measured at two positions on each berry using an image-processing program (adobe photoshop 7.0). of each cultivar, a total of 10 berries in three replicates were measured. these measurements were used to calculate skin thickness, berry and skin cross-section area of each cultivar. frap assay the plant material was homogenised, and 3 g homogenate was dissolved in 30 ml methanol. bottles were flushed with nitrogen before closing, and the samples were mixed and sonicated on a waterbath at 0 °c for 15 min. the extracts were stored at –20 ºc. samples of 1.5 ml were centrifuged at 12.000 x g for 2 min at 4 ºc. the concentration of antioxidants in the supernatant was measured in triplicate. frap values were determined in extracts (benzie and strain, 1996), with the exception that the samples were not diluted with water (halvorsen et al., 2002). a technicon ra 1000 system was used for the measurements of absorption changes at 600 nm that appear when the tptz-fe3+ complex is reduced to the tptzfe2+ form in the presence of antioxidants. an aqueous solution of 500 mm feso4 x 7 h2o was used for calibration of the instrument. data analysis one-way analysis of variance (anova) was applied to test differences between the cultivars on the different physical measurements, berry weight and antioxidant activity. to make a prediction model of antioxidant activity based on physical measurements, a pls (partial least square)-model was applied using the unscrambler 9.0 by camo. * this publication was presented as a poster on the cost action 924 working group meeting; „non-destructive methods for detecting health-promoting compounds“ (remberg et al., 2005). results and discussion the most important result, as evaluated by means of partial least square regression (plsr) (martens and næs, 1989), is shown in fig. 2, where ‘patriot’ and ‘bluecrop’ have larger skin area and lower frap values compared to ‘hardyblue’ and ‘putte’. the other variables (berry weight, berry diameter, flesh diameter, berry circumference and berry cross-section area) are clustered at the left side in the pls plot. this indicates that these are highly correlated. ‘aron’ did not respond as a significant variable in the pls model. this is implied by the fact that the variables measured and calculated did not correlate significantly compared to the other cultivars, with the exception of skin thickness and frap-values. the anova showed a great variation between the different cultivars (tab. 1). all the cultivars were significantly different regarding berry weight. ‘patriot’ had the largest berries, followed by ‘bluecrop’ and ‘hardyblue’, while ‘putte’ and ‘aron’ had the smallest. tab. 2 shows correlations between antioxidant activity and physical measurements. berry weight, diameter, circumference and cross-section area had significant negative correlations to antioxidant activity. a relationship between berry weight and frap-value was calculated to r = -0.664, indicating that smaller berries have higher frap-values. a schematic outline between berry weight and antioxidant activity is shown in fig. 3. antioxidant activity (solid bars) and berry weight (lines) are negatively correlated, with a higher antioxidant activity in small berries. concerning the antioxidant activity, ‘putte’ had highest values, followed by ‘hardyblue’, ‘aron’, ‘patriot’ and ‘bluecrop’. this confirms the results by remberg et al. (2003), where ‘putte’, ‘hardyblue’ and ‘aron’ were found to have significantly higher frap-values than ‘bluecrop’. fig. 2: pls plots presenting variables and samples. pa = ‘patriot’, bl = ‘bluecrop’, hb = ‘hardyblue’, pu =‘putte’, numbers indicate cultivar replicates. variables clustered together are indicated by a filled circle (berry weight, berry diameter, flesh diameter, berry circumference and berry cross-section area). fig. 1: cultivated blueberries ‘putte’ (a) and ‘patriot’ (b) cut horizontally, placed on a scanner and measured at two positions (marked with a black cross in picture b on the top left). antioxidant activity in blueberries 37 a b a b tab. 2: correlations between antioxidant activity (frap) and berry weight, berry diameter, berry circumference, skin thickness, berry and skin cross-section area. antioxidant activity (frap) r p-value berry weight (g) -0.664 0.007 berry diameter (mm) -0.582 0.023 berry circumference (mm) -0.582 0.023 skin thickness (mm) 0.439 0.101 berry cross-section area (mm2) -0.615 0.015 skin cross-section area (mm2) -0.452 0.090 tab. 1: effects of blueberry cultivar on berry weight, physical variables in berry cross-sections and antioxidant activity. cultivar berry berry flesh berry skin berry skin frap weight diameter diameter circumference thickness area area (mmol/100g fw) (g) (mm) (mm) (mm) (mm) (mm2) (mm2) aron 0.77e 11.72c 11.31c 36.81c 0.62 108.85c 7.48b 3.03abc bluecrop 2.94b 19.88a 19.52a 62.41a 0.54 312.17a 11.14ab 2.28c hardyblue 1.57c 15.52b 15.13b 48.74b 0.60 190.76b 9.61ab 3.40ab patriot 3.34a 20.70a 20.30a 64.98a 0.60 337.31a 12.89a 2.81bc putte 1.08d 14.36b 13.97b 45.09b 0.59 163.31b 8.79b 3.94a mean 1.94 16.44 16.04 51.61 0.59 222.48 9.98 3.09 level of significance *** *** *** *** ns *** *** *** *** = p < 0.001, ns = non significant numbers with different letters are significantly different for most of the physical measurements, ‘patriot’ had the highest, while ‘putte’ and ‘aron’ had the lowest values. ‘aron’ had slightly thicker skin compared to the other cultivars analyzed. the skin thickness was measured in this experiment using a scanner and an image-processing program. using light microscopy to study cell walls and epidermal pigment of three v. corymbosum cultivars, allanwojtas et al. (2001) found differences between cultivars on pigment distribution. while the epidermis of ‘burlington’ and ‘elliot’ consisted of two layers with pigments, ‘coville’ epidermis consisted of three layers. ‘aron’ and ‘putte’ had significantly smaller cross-section skin area than ‘bluecrop’ and ‘patriot’ due to lower berry diameter. no literature was found discussing physical measurements in blueberries and antioxidant activity. kalt et al. (2001) analyzed 80 highbush and 135 lowbush blueberry clones for berry weight, anthocyanins and total phenolics, and found no relationship between fruit weight and anthocyanin content. berry weight and size are in the literature used synonymously (eck, 1988), and blueberry shape varies between cultivars (keipert, 1981). predicting a spherical berry shape, this work confirms that berry size measured as diameter is highly correlated with berry weight (r = 0.981). lee and wrolstad (2004) found highest antioxidant activity measured as frap and orac (oxygen radical absorbing capacity) in blueberry skin compared to flesh and seeds. in our experiments, skin thickness had no influence on antioxidant activity, but high correlations between berry size/weight and frap values were found. scanning was an excellent tool to confirm our findings. 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 aron bluecrop hardyblue patriot putte f r a p ( m m o l/ 1 0 0 g f w ) 0,00 0,50 1,00 1,50 2,00 2,50 3,00 3,50 4,00 b e rr y w e ig h t (g ) frap (mmol/100g fw) berry weight (g) fig. 3: relationship between berry weight and antioxidant activity (frap). conclusions for spherical shaped blueberries, berry weight and/or diameter measurements can be used to estimate antioxidant activity, measured as frap. this work indicates that non-destructive measurements can be used to predict health-promoting compounds in cultivated blueberries. acknowledgements the authors wish to thank signe hansen and kari grønnerød for technical assistance, sigbjørn vestrheim for carefully reviewing the manuscript, and the norwegian research council for financing the work. references allan-wojtas, p.m., forney, c.f., carbyn, s.e., nicholas, k.u.k.g., 2001: microstructural indicators of quality-related characteristics of blueberries – an integrated approach. lebensm.-wiss. u.-technol. 34, 23-32. benzie, i.f.f., strain, j.j., 1996: the ferric reducing ability of plasma (frap) as a measure of „antioxidant power“: the frap assay. anal. biochem. 239, 70-76. eck, p., 1988: blueberry science. rutgers university press, new brunswick, nj. 38 siv f. remberg, anne-berit wold, knut kvaal, maigull appelgren, karin haffner ehlenfeldt, m.k., prior, r.r., 2001: oxygen radical absorbance capacity (orac) and phenolic and anthocyanin concentrations in fruit and leaf tissue of highbush blueberry. j. agric. food chem. 49, 2222-2227. haffner, k., wang, l., munkeby, m., sparre, h., 2000: der scanner – ein nützliches instrument im labor: messungen an himbeeren. gartenbauwissenschaft 65, 6-8. halvorsen, b.l., holte, k., myhrstad, m.c.w., barikmo, i., hvattum, e., remberg, s.f., wold, a.-b., haffner, k., baugerød, h., andersen, l.f., moskaug, j.ø., jacobs jr, d.r., blomhoff, r., 2002: a systematic screening of total antioxidants in dietary plants. j. nutr. 132, 461-471. kalt, w., ryan, d.a.j., duy, j.c., prior, r.l., ehlenfeldt, m.k., vander kloet, s.p., 2001: interspecific variation in anthocyanins, phenolics, and antioxidant capacity among genotypes of highbush and lowbush blueberries (vaccinium section cyanococcus spp.). j. agric. food chem. 49, 4761-4767. keipert, k., 1981: beerenobst. ulmer verlag, stuttgart. lee, j., wrolstad, r.e., 2004: extraction of anthocyanins and polyphenolics from blueberry processing waste. j. food sci. 69, 564-573. martens, h., næs, t., 1989: multivariate calibration. wiley, chichester. remberg, s.f., blomhoff, r., haffner, k., 2003: total antioxidant capacity and other quality criteria in blueberries cvs ‘bluecrop’, ‘hardyblue’, ‘patiot’, ‘putte’ and ‘aron’ after storage in cold store and controlled atmosphere. acta hort. 600, 595-598. remberg, s.f., wold, a.-b., kvaal, k., appelgren, m., haffner, k., 2005: an approach towards rapid optical measurements of antioxidant activity in cultivated blueberry cultivars. non-destructive methods for detecting health-promoting compounds – cost action 924 working group meeting. bornimer agrartechnische berichte 54, 82. addresses of the authors: siv fagertun remberg1, dr. anne-berit wold, assoc. prof. dr. maigull appelgren, prof. dr. karin haffner, norwegian university of life sciences, department of plant and environmental sciences, p.o.box 5003, n-1432 aas. assoc. prof. knut kvaal, norwegian university of life sciences, department of mathematical sciences and technology, p.o.box 5003, n-1432 aas. 1corresponding author: siv.remberg@umb.no antioxidant activity in blueberries 39 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /colorconversionstrategy 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<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> >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice vorgabe neu journal of applied botany and food quality 80, 100 110 (2006) technical university of cartagena. etsia and plant biotechnology institute department of agricultural and food engineering1. department of agricultural production2 ultrastructure of the onset of chilling injury in cucumber fruit j.p. fernández-trujillo1*, j.a. martínez2 (received april 3, 2006) summary the onset of the symptoms of disorders provoked by low temperature storage or chilling injury (ci) in pickling cucumber fruit (cucumis sativus l. cv. 'trópico' and in the cv. 'perichán 121') and associated rot were monitored by cryoscanning electron microscopy. fruit were stored at 4oc for different times (4 to 12 d) and samples were transferred to 20oc every 2-3 d (cv. trópico). in a second experiment, fruit cv. 'perichán 121' were stored at 6oc. macroscopic ci symptoms included small-flattened areas with sunken but externally sound tissue. later, the damage was manifested as pitting and decay due to the presence of necrotrophic fungi (pleospora herbarum, alternaria sp.) on the surface of the broken areas, and botrytis cinerea in cv. 'perichán 121'. the period of induction of ci becoming apparent lasted about 4 d at 4oc followed by a phase of slow increase of around 4-5 d prior to an exponential increase in ci. micro fractures of 45-250 µm length developed in ci tissue around the stomata of 20 µm ø. the first response to chilling was the sinking of stomata accompanied by 10 µm ø fractures with collapse of hypodermal cells. these small fractures expanded into a sink of around 40 µm ø and more than 50 µm depth. refrigerated tissue had visibly collapsed in 4-6 d, starting with a small brown area indicating collapse of parenchymatous cells, followed by sinking and collapse of epidermal cells, particularly around the micro fractures. the pitted tissue showed flattening of the cell walls, plasmalemma and middle lamella region, with severity increasing with increasing depth in the parenchymatous tissue. translucent water soaked areas were also located depth in the mesocarp tissue with similar cell damages. 1. introduction below 7-13oc cucumber fruit is very sensitive to chilling injury (ci), which is the main physiological disorder that limits the use of refrigeration to extend its shelf-life. the breakdown of the tissue provides a suitable environment for the growth of saprophytic pathogens that colonize chilling injured fruit (hakim et al., 1999; kang et al., 2002). the main symptoms of ci are tissue collapse, pitting, translucent water-soaked spots and water-soaked areas in the mesocarp (fukushima et al., 1977; hakim et al., 1999; kang et al., 2002). extensive decay (i.e. alternaria sp.) is known to develop when chilled cucumbers are removed from low-temperature storage (abe, 1990). cucumbers chilled at 0oc have vertical fine wrinkles and/or shallow pitting, while fruit at 5oc have deep pitting and/or surface depressions (fukushima et al., 1977). fukushima and tsugiyama (1977) reported plasmalemma leakage in cucumber fruit stored at 0oc and membrane breakage in fruit stored at 5oc. the severity of pitting is also inversely related to the relative humidity of the storage atmosphere and the growing temperatures (abe, 1990; hakim et al., 1999; kang et al., 2002), although the primary effect of low temperature on pitting is not a dehydration dependent process (jackman et al., 1989). cryogenic scanning electron microscopy (cryo-sem) avoids typical problems that arise with other methods in high water content tissues, such as extraction by chemical fixation or solubilization of fats and carbohydrate, and structure collapse, shrinkage and distortion, (echlin, 1992). for this reason cryo-sem is a suitable tool to observe ci symptoms (rhee and iwata, 1982; tatsumi et al., 1987), sound epicarp fruit tissue, fungal infection processes, surface organisms, and disorders (echlin, 1992). pitting in cucumber fruit is associated with a combination of factors such as epidermal cracks, the collapse of inner tissues including the hypodermal tissue (parenchyma), and the deposition of mucilage (tatsumi et al., 1987). the cracks in the cuticle and the sinking of epidermal cells near the stomata lead to an enhanced transpiration rate (tatsumi et al., 1987). as regards tissue destruction, abe (1990) classified pitting as type i (affecting parenchymatous cells), or type ii (affecting both epidermal and hypodermal cells). typical symptoms of ci in different fruit tissue including cucumber have been characterized previously (abe, 1990; jackman et al., 1989; lurie et al., 1997; rhee and iwata, 1982; tatsumi et al., 1987). the first goal of the present study was to visualize the development of ci of the epicarp tissue and cells (including cell wall and plasmalemma) of cucumber fruit using cryo-sem. different storage conditions and two cultivars were used to provoke several ci symptoms. 2. material and methods 2.1. greenhouse and plant material two sets of experiments with different cucumber (cucumis sativus l.) cultivars were carried out. these cultivars used for fresh consumption in spain and russia were parthenocarpic, pickling types with very soft-spined fruits. in the first experiment perlite-grown cucumbers cv. 'trópico f1' were used. in the second, cucumber fruit of cv. 'perichán 121', which had been grown in similar conditions, were purchased from a local supermarket when fruit remained refrigerated less than 1 d at 5-8oc. in the first experiment, cucumber fruit were grown in a glasshouse during the winter and spring seasons in cartagena (murcia, se spain) as described by gómez et al. (2003). the two successive crops considered in the first study were transplanted in january and april (referred to as winter and spring seasons respectively). the cucumber canopy cv. 'trópico' f1 (nunhem seeds, the netherlands), in rows, covered about 140 m2 of ground, with a plant density of 2.08 plants · m-2. the plants were grown on perlite (type k-13) in polyethylene bags using an average of 8 dm3 per plant. the plants were irrigated with a nutrient standard solution (ph = 5.5 to 6.0, ec = 2.2 ms · cm -1) of the following composition (in mmol · l-1): 13 no3-, 1.7 h2po 4-, 0.5 nh4+, 7.5 k+, 3.5 ca2+, 2.0 mg2+. micronutrients used were 110 mg : l-1 of a commercial micronutrient complex containing 5.77 ppm fe, 0.055 ppm co, 0.66 ppm cu, 0.44 ppm b, 1.87 ppm mn, 0.66 ppm zn and 0.055 ppm mo. the open-drainage rate for programming the fertigation was about 30% of the solution used (i.e. we assumed that 70% is used for the plant). the crops were managed as usual in commercial greenhouses under fertigation, pruning and climate control as described by gómez et al. (2003). 2.2. harvesting and chilling injury studies in the first experiment, the fruit development periods, corresponding to the time taken to reach the commercial maturity stages, ranged from 12 to 28 d after anthesis, although it usually lasted 12-15 d in the two harvest dates considered in both seasons. fruit were packed in polyethylene bags to reduce dehydration, transported within 20 min to the laboratory, and stored for 1-2 h at 6oc ± 1oc in a domestic refrigerator. then fruit with an equatorial caliber of 140160 mm and minimum color of 125-128o (hue angle) and 31-34% lightness were selected and randomly divided into bags of five fruits each (n = 5 replicates). these were sealed in 20 ± 2 µm thick, nonoriented macroperforated (32 holes of 1.2 mm diameter per square dm) cast polypropylene film (plásticos del segura, murcia, spain). these bags increase the relative humidity (rh) around the fruit up to 95-99% without changing the gaseous atmosphere, which is useful for differentiating ci effects from moderate to severe dehydration symptoms. in this first experiment, three replicates of five fruit each harvested in the winter were stored for up to 4 d at 4 ± 0.2oc, then transferred without opening the perforated bags to 20 ± 1oc and 75 ± 5% rh for 4-8 d in order to allow chilling injury to develop. before examination they were stored at 4oc for 1-2 d more. this experiment was designed to visualize the severe ci that can be delayed or masked by refrigeration according to the results found by previous authors (artés et al., 1998; fukushima et al., 1977; hakim et al., 1999). this experiment was also conducted to optimize the cryo-sem techniques for ci studies. in the spring season three replicates of five fruit per bag were stored for up to 12 d at 4 ± 0.5oc and 95% rh before examination to avoid the appearance of severe ci symptoms. plastic bags remained inside a 360 l cabinet with a continuous humidified air flow of 250 l:h-1 supplied by bubbling air in water using a membrane compressor (schego optimal, germany). this experiment uses modified atmosphere packaging for retail display, because rh levels surrounding the fruit are higher and fluctuate less than in macroperforated bags (fernándeztrujillo and artés, 1998). in the second experiment, cucumbers cv. 'perichán 121', a winter line of cucumber developed by agrícola perichán sat (mazarrón, murcia, se spain) were used. the fruit were purchased packed in macroperforated polypropylene (20 µm thickness, 6 perforations of 5 mm ø per square dm) bags of three fruits each within a molded plastic tray to avoid mechanical damage. the perforations were only on the adaxial face of the package. the fruit were stored for 0, 9, 16 and 23 d at 6 ± 0.2oc and 98% rh. the rh and temperature used represent the subsequent storage of cucumber fruit in domestic refrigerators after purchase. weight loss was evaluated immediately after storage or after the additional shelf-life period of 4 d at 20oc. ci symptoms evaluated visually on a 0 to 4 scale according to the mean diameter of the pitted areas (0 = sound; 1-2, very slight to slight, ø between 0 and 5.5 mm or 5.5 < ø < 15 mm; 34, moderate to severe, 15 < ø < 25 mm or ø >25 mm). a similar scale was used for scoring shriveling symptoms. the fungi were identified according to conventional methodology (artés et al., 1998). 2.3. cryo scanning electron microscopy cryo-sem, also known as low temperature sem (echlin, 1992), was used to study epidermal disorders and microscopic ci symptoms. cucumbers were examined with a magnifying glass to select areas of interest. control fresh fruit (sound, non-refrigerated) or refrigerated fruit were allowed to equilibrate at room temperature before cryofixation to avoid water condensation. samples of the fruit epidermis were obtained for sem by dissecting sections (3-5 mm2 by 1-1.5 mm thick) from different epicarp areas (the distal and proximal areas, and the middle, depending on the ci symptom studied) with a razor blade. tissue leakage was removed by blotting. the cucumber tissue samples were placed on a flat specimen holder with a 1:1 mixture of colloidal graphite g303 (agar scientific ltd., essex, england) and tissue-tek 4583 glue (sakura, zoeterwoude, the netherlands), skin (with or without ground tissue) side up. the fresh samples were frozen with nitrogen slush. a nitrogen gas flow was used to remove possible water vapour inside the equipment (craig and beaton, 1996). once frozen, the sample was transferred to the preparation chamber (polarprep 2000 cryo transfer system) under vacuum with a polaron pp7480 polarprep control unit and a pp7483 polaron gas control unit (quorum technologies, east sussex, uk). here the superficial ice was removed from the surface of the specimen by sublimation when heating the sample with a resistor from (-140ºc) to -85ºc for 5 min (vacuum pressure around 0.08 mm hg). then the sample was cooled again to -140ºc and coated with a goldpalladium alloy (80:20) in a polaron polarprep 2000 during 180 s at 10 ma in order to improve the specimen conductivity. cooling was used to avoid the accumulation of electrical surface charges and therefore increasing the quality of the final image. the coating thickness (8-10 nanometers) was monitored by polaron ff7690 film thickness monitor. the valve gate between the preparation chamber and the microscope chamber was opened and the sample holder was mounted for observation on the cold stage inside the hitachi s-3500 n microscope chamber (hitachi, ratingen, germany). to ensure the correct insertion of the holder in the chamber an infrared chamberscope was used. the accelerating voltage for sample observation ranged from 2 to 15 kv with a cold stage at -140ºc and a cold trap at -190ºc to avoid contamination by aerial particles (both temperatures were maintained with liquid n2). levels of magnification ranged from x40 to x6000 (usually less than x1300). working distances ranged from 3.9 to 30 mm, depending on the detail required. generally the working distance was close to 10 ± 3 mm. three samples were mounted per holder. images were exported to jpg format at 640 x 480 pixels and 256 colours (black & white image) or bmp format (2560 x 1920 pixels). 3. results and discussion 3.1. onset of weight loss and macroscopic ci symptoms and associated decay during cold storage weight loss increased linearly in winter harvested 'trópico' cucumber fruit from the first experiment by around 0.1% (w/w) every day (r2 = 0.98) reaching, for example, around 0.56 ± 0.1% after 6 d at 4oc (data not shown). the 4 d shelf-life periods increased weight loss by about 1.2% (before 6 d of storage) or more (around 2%) above this period probably because of the onset of ci. in spring harvested 'trópico' cucumber fruit from the first experiment, dehydration mainly affected the fruit with slight symptoms (pulp whitening and pulp dryness) with dehydration indices of around 25%, 40% and 60% after 4, 9 or 12 d at 4oc plus the additional shelf-life periods. the first visible chilling injury symptoms at 4oc developed after 6-9 d (in fruit from the first experiment harvested in the spring) and were represented by slight pitting and a burst in decay, which showed a broad range depending on many factors (0% to 45% fruit). decay was mostly due to alternaria sp., which usually accompanies ci (artés et al., 1998), though species of pleospora herbarum (perfect state teleomorph of stemphylium botryosum) also developed. in cv. 'perichán 121', botrytis sp emerged from the hypodermal tissue and sometimes used stomata breakage. this indicated a very short period of induction for the first visible ci to be seen after 4-5 d at 4oc under the macroperforated film (i.e. high chilling injury in cucumber fruit 101 fig. 1: first chilling injury symptoms on the epidermis of cucumber fruit cv. 'trópico'. a. macroscopic aspect of chilling injured fruit. b. sound epidermal tissue including stomata covered by epicuticular wax (wx), active stomata (s) and broken trichome covered by continuous epicuticular wax (t). c. epidermal depression surrounding sunken stomata. a c b rh). additionally, the shelf-life period used in winter harvested fruit from the first experiment favored better observation of the symptoms that usually develop slowly at low temperatures (artés et al., 1998). in contrast, the colonization of the pitting by decay was very rapid, 102 j.p. fernández-trujillo, j.a. martínez fig. 2: chilling injury symptoms on the epidermis of cucumber fruit cv. 'trópico'. a. epidermal cells of sound tissue with epicuticular wax. b. micro fracture with some conidia inside c. crack with a half-moon shape. a b c reaching almost 100% of the pitted fruit, which also agrees with the onset of ci in tomato fruit (artés et al., 1998). this period might be as short as 3 d at 5oc (tatsumi et al., 1987) or longer. in this experiment micro cracks and depressed stomata appeared after 4-5 d at 4oc plus the additional shelf-life period. the more evident macroscopic ci symptoms include sunken but intact tissue, flattened tissue as a form of pitting, and alternaria sp. decay on flattened or pitted areas. chilling injury in cucumber fruit 103 fig. 3: a. sound cucumber fruit cv 'trópico' tissue with stomata seen from the surface covered by epicuticular wax, stomata and broken trichome covered by continuous epicuticular wax. b. massive sinking (craters) of epidermal tissue surrounding stomata. a b 3.2. development of chilling injury as monitored by cryo-sem the first symptoms of epidermal deterioration, which are not observed visually, were cracks in the cuticle and sinking of the hypodermal and epidermal cells near the stomata. microscopic symptoms of the extent and severity of the different disorders detected are described below, irrespective of the experiment considered. 3.2.1. onset of ci by observing depressed epidermal area including micro cracks and sunken trichomes sound fruit had homogeneous surface (fig. 1 b), with stomata frequency depending on the maturity stage of the tissue (fig. 1 b). epidermal cells close to the stomata were elevated and covered with a smooth wax layer resembling an extinct volcano structure of 40104 j.p. fernández-trujillo, j.a. martínez fig. 4: chilling injury on the distal zone of cucumber fruit cv. 'perichán 121' stored in macroperforated films. a. cross-section with light brown epidermis and pitting after 9 d at 6oc. b. cross section of fresh and sound epidermal tissue. epidermal cells (ep), compact parenchyma (cp) and parenchyma (p). c. pitted hypodermis after 23 d at 6oc. a b c ° ep cp p 60 µm ø (fig. 1 b). the characteristic trichomes of cucumbers popularly known as spines were not observed in physiological mature cucumber fruit from the cultivars studied, although the fruit showed the characteristic crater of less than 350-400 µm diameter when trichomes were detached. a top view of the epidermal tissue with very slight ci symptoms (fig. 1 a) pointed to structural breakdown with void cells underneath the stomata. stomata started to sink as a result of low temperature storage (fig. 1 c). sound tissue (fig. 2 a) rarely had the small holes of 10 µm ø that expanded to become a sink of around 40 µm ø and more than 50 µm deep (fig. 2 b), although cracks and micro cracking are common in stored fruit (lurie et al., 1997; artés et al., 1998). chilling injury in cucumber fruit 105 fig. 5: details of chilling injury in cv. 'perichán 121' after 9 d at 6oc. a. hypodermal tissue with chilling injured plasmalemma. b. detail of parenchymatous cell deterioration caused by pitting. intercellular space (is). c. cell wall deterioration. cell wall (cw), middle lamella region (mlr). d. chilling injured plasmalemma. e. aspect of a water-soaked cell of the spongy mesocarp (5 mm below the fruit surface). is p mlr cw a cb d e the limit of pore diameter for the visual detection of pitting in sunken epidermal areas was established by tatsumi et al. (1987) at 70-130 µm ø, while micro cracks that appeared in epidermis suffering ci had 45-250 µm length. then, the irregular shapes (waning moon, mussel) of the micro cracks can be detected sometimes by optical microscopy. these micro cracks were frequently associated to stomata collapse (fig. 2 c), and were more evident in cv. 'trópico' than in cv. 'perichán 121' as a result of the shelf-life periods used 106 j.p. fernández-trujillo, j.a. martínez fig. 6: collapse of parenchymatous cells of cucumber fruit cv. 'trópico'. a. top and cross section of sound tissue. b. top and cross-section of with arrows indicating epidermal pitting and collapsed parenchymatous cells. c. macroscopic symptoms (black arrow). d. detail of pitted tissue. c d b a ° chilling injury in cucumber fruit 107 fig. 7: necrotic tissue of cucumber fruit cv. 'trópico'. a. surface view of different degrees of necrotic tissue in the distal area of cucumber fruit. b. external aspect of the necrotic tissue. c. detail of necrotic tissue by cryo-sem, with epidermal and parenchyma cells affected. a b c after refrigeration in the former cultivar. sometimes fruit epidermis can develop epidermal cracking as a result of a sudden increase in the water content of the soil, atmospheric humidity or temperature (opara et al., 1997); in some cases, the cracks may be lignified. smaller micro cracks were or not near to each other depending on nearby stomata densities. an extensive collapse joining nearby cracks (fig. 2 b) in one was generally the result of hypodermal chilling injury. these results matched with the initial ci symptoms around the stomata observed in cross-sections of parenchyma cells of pitted cucumber (rhee and iwata, 1982), and in cold stored mango and avocado (bower et al., 2003). the final step of pitting included massive sinking of the fresh sound epidermal tissue (fig. 3 a) that led to irregular craters including a group of stomata and cells beside the stomata of 300 µm ø or more, and up to 660 µm in length (fig. 3 b). however, this was not very common in these experiments, in contrast with the results obtained by fukushima et al. (1977) at 5oc, indicating a probable cultivar effect on pitting. another ci symptom was the sinking of the basal cells of epidermal tissue from which trichomes (spines) of cucumber cv. 'trópico' were dislodge. the sinking were observed visually even in non-refrigerated tissues due to dehydration. craters with a neat circular shape of 350-400 µm ø included collapsed and empty epidermal cells, and ci expanded this diameter up to 500 µm (data not shown). more probably, this was a result of the combined effect of ci and severe dehydration in this area (tatsumi et al., 1987). 108 j.p. fernández-trujillo, j.a. martínez 3.2.2. onset of ci as monitored by cryo-sem in the cross-section of the fruit in sound tissue, cell walls plus plasmalemma were clearly differentiated from the internal content and no damages were observed in the cultivar 'perichán 121' (fig. 4 b). pitted tissue (fig. 4 a) resulted from the flattening of the cell wall, middle lamella region and plasmalemma (fig. 4 c, 5 a to 5 c). the deeper the hypodermal cells, the higher the severity of the disorder. in the cv. 'perichán 121' stored for 9 d at 6oc, the fruit surface presented pitted tissue, and the first injured cells and areas of collapsed tissue respectively were 85250 µm or 200-350 µm below the fruit surface (fig. 4 c). ci was more severe in fruits stored 23 d, and botrytis sp. developed during the first day of shelf-life at 20oc. after 23 d at 6oc, ci tissues had injured cells even in the first layer of hypodermal cells (30-50 µm below the epidermal cells) or massively collapsed tissue. the appearance of chilling injured cells several layers below the fruit surface has been reported in cucumber (fukushima et al., 1977) and eggplant (abe, 1990) pitting. in fresh sound cucumber tissue cv. 'trópico', the overall appearance of the parenchyma tissue is that of a compacted mass of turgid of empty cells (fig. 6 a), depending on the cutting method or the tissue leakage that may remain on the cuts after blotting the tissue before the cryo-sem analysis. healthy hypodermal cells just beneath the epidermal cells were flattened, while the typical cells deep within the parenchyma tissue were bigger and rounded. however, parenchymatous cells suffering ci collapsed about 100-200 µm below the epidermis, causing a subsequent epidermal depression (fig. 6 d) and later a massive epidermal collapse (around 20-30 µm deep) (fig. 6 b). the kind of pitting found here, resulting in destruction of both epidermal and parenchymatous cells (fig. 6 b and 7 a), has been named as type ii (abe, 1990), in contrast with type i pitting, when only epidermal cells are destroyed. when epidermal ci was evident and severe in a small-flattened area (fig. 7 a), epidermal cells beneath the epidermis were also collapsed (fig. 7 a and 7 c), as occurred in tomato fruit (lurie et al., 1997). the flattened areas were characterized by a shapeless mass of parenchyma cells compared with sound tissues (fig. 7 b), and included the epidermis of 6-20 layers and the fruit surface (fig. 7 c). the flattened tissue was frequent in the skin of the cucumber apex (fig. 6 c). and was subsequent to pitting, or the appearance of translucent water-soaked areas that advanced from the mesocarp to the cucumber fruit surface and resulted in a massive tissue damages (water soaked blemishes), as reported in melons (xu et al., 1990) and cucumbers (kang et al., 2002). mesocarp tissue (spongy endocarp with a translucent aspect located about 5 mm below the fruit surface) observed by cryosem in the cultivar 'perichán 121' had tissues showing a damaged cell wall, middle lamella and plasmalemma (fig. 5 d and 5 e), which is affecting these areas more severely than layers above the cross-sections. water evaporation of intercellular water and transpiration, though limited in this experiment by the macroperforated packaging used, would raise the tonicity of the remaining solution and reduce the rupture of the protoplasts, inducing water-soaking at an early stage of ci (tatsumi et al., 1987). 4. conclusions the visualization and extent of ci symptoms can be monitored by cryo-sem. the breakage of the plasmalemma, cell wall and middle lamella zone in hypodermal cells preceded the collapse of hypodermal tissues and the appearance and expansion of epidermal micro cracks. the severity of ci increased with increasing depth in the parenchymatous tissue, leading to translucent water soaked areas when affected the mesocarp tissue. the sinking of epidermal tissues, particularly around the stomata and trichomes, and ci-associated rots were later events in the development of the disorder. packing cucumbers in perforated films at higher rh seems to induce less severe ci symptoms as observed by decreased tissue collapse and better cell wall integrity. however, possible cultivarand seasonrelated effects on the period required for inducing the first irreversible ci symptoms cannot be ruled out. acknowledgement this work was supported by ministry of education & science (spain) and feder (agl2000-0450) and fundación séneca de la región de murcia (pb/22/fs/02). thanks are due to a. alcolea (saitupct), j.m. mercader, m.c. garcía-romero, c. miranda, c. liuzzo and m.d. gómez-lópez for technical assistance, and to plásticos del segura sl for supplying the films. references abe, k., 1990: ultrastructural changes during chilling stress. in: wang, c.y. (ed.), chilling injury of horticultural crops, 71-84. crc press, boca raton, florida, usa. artés, f., garcía, f., marquina, j., cano, a., fernández-trujillo, j.p., 1998: physiological responses of tomato fruit to cyclic intermittent temperature regimes. postharvest biol. technol. 14, 283-296. bower, j.p., dennison, m.t., fowler, k., 2003: avocado and mango cold storage damage as related to water loss control. acta hortic. (ishs) 628, 401-406. craig, s., beaton, c.d., 1996: a simple cryo-sem method for delicate plant tissues. j. microscopy 182, 102-105. echlin, p., 1992: low-temperature scanning electron microscopy. in: echlin, p. (ed.), low-temperature microscopy and analysis, 349-411. plenum pub. co., ny, usa. fernández-trujillo, j.p., artés, f., 1998: intermittent warming storage of peaches packed in perforated polypropylene. leb. wiss. technol. 31, 38-43. fukushima, t., tsugiyama, t., 1977: chilling-injury in cucumber fruits. 2. chemical analyses of leakage substances and anatomical observation of symptoms. sci. hortic. (amster.) 6, 199-206. fukushima, t., yamazaki, m., tsugiyama, t., 1977: chilling-injury in cucumber fruits. 1. effects of storage temperature on symptoms and physiological changes. sci. hortic. (amster.) 6, 185-197. gómez, m.d., baille, a., gonzález-real, m.m., mercader, j.m., 2003: dry matter partitioning of greenhouse cucumber crops as affected by fruit load. acta hortic. (ishs) 614, 573-578. hakim, a., purvis, a.c., mullinix, b.g., 1999: differences in chilling sensitivity of cucumber varieties depends on storage temperature and the physiological dysfunction evaluated. postharvest biol. technol. 17, 97-104. jackman, r.l., yada, r.y., marangoni, a.g., parkin, k.l., stanley, d.w., 1989: chilling injury. a review of quality aspects. j. food qual. 11, 253-278. kang, h.m., park, k.w., saltveit, m.e., 2002: elevated growing temperatures during the day improve the postharvest chilling tolerance of greenhouse-grown cucumber (cucumis sativus) fruit. postharvest biol. technol. 24, 49-57. lurie, s., laamim, m., lapsker, z., fallik, e., 1997: heat treatments to decrease chilling injury in tomato fruit. effects on lipids, pericarp lesions and fungal growth. physiol. plant. 100, 297-302. opara, l.u., studman, c.j., banks, n.h., 1997: fruit skin splitting and cracking. hort. rev. 19, 217-262. rhee, j., iwata, m., 1982: histological observations on the chilling injury of cucumber fruit during cold storage. j. jap. soc. hort. sci. 51, 231-236. tatsumi, y., maeda, k., murata, t., 1987: morphological changes in chilling injury in cucumber fruit 109 cucumber fruit surfaces associated with chilling injury. j. jap. soc. hort. sci. 56, 187-192. xu, l., zhang, w.y., tian, y.w., 1990: effects of chilling injury on the morphology and cell structure of hami melon fruits. acta bot. sinica 32, 772-776. address of the authors: technical university of cartagena. campus paseo alfonso xiii, etsia and plant biotechnology institute. 30203 cartagena (murcia), spain. department of agricultural and food engineering. (e-mail: jf68es@terra.es or juanp.fdez@upct.es) 1 department of agricultural production. 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<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> >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice journal of applied botany and food quality 94, 92 98 (2021), doi:10.5073/jabfq.2021.094.011 1department of analytical development and research, section phytochemical research, wala heilmittel gmbh, bad boll/eckwälden, germany 2department of plant systems biology, hohenheim university, stuttgart, germany morphology and phytochemistry of sanguisorba officinalis l. seeds (rosaceae) marek bunse1,2, florian conrad stintzing1, dietmar rolf kammerer1,* (submitted: october 15, 2020; accepted: april 29, 2021) * corresponding author summary great burnet (sanguisorba officinalis) has been used as medicinal plant for more than 2000 years. however, little is known about the morphology and the secondary metabolites of its seeds. the inves tigations reported here focus on the morphology and the characteri zation of phenolics and fatty acids in s. officinalis seeds. for this purpose, dried seeds were investigated using scanning electron microscopy to clarify their compartment structures. furthermore, the seeds were extracted with ch2cl2 and meoh to characterize the fatty acids and to assess the secondary metabolite profile. the seed structure consists of a floral cup, a brown pericarp with calcium oxalate crystals, a fibre layer and the seed kernel with its seed coat. individual compounds were characterized by high performance liquid chromatography and gas chromatography coupled with mass spectrometric detection (hplc-dad-msn and gc/ms). ch2cl2 extraction and gc investigations revealed the occurrence of fatty acids (29 % of the seed dry weight), with linoleic acid, linolenic acid, and oleic acid as major compounds. in addition, meoh extracts were analyzed by lc/msn, which revealed the occurrence of flavonoids (quercetin, catechin, epicatechin), ellagitannins and caffeic acid derivatives. introduction members of the rosaceae family are generally subdivided into three subfamilies: rosoideae, amygdaloideae, dryadoideae. this family of flowering plants includes about 4,828 known species in 91 genera, which may grow as trees, shrubs, or herbaceous plants. this family is characterized by exceptional horticultural significance with eco nomically important fruit-bearing plants. the fruits occur in many varieties and were once considered the main characters for the definition of the aforementioned subfamilies. the fruits are also characterized by great diversity and may occur as follicles, capsules, nuts, achenes, drupes and accessory fruits, like the pome of an apple, or the hip of a rose. well-known examples of the three subfamilies are as follows: amygdaloideae: plums, cherries, peaches, apricots, almonds, apples, pears, quince, ornamental shrubs such as exochorda, sorbaria and physocarpus; dryadoideae: cercocarpus, chamaebatia, dryas, purshia; rosoideae: strawberries, blackberries and raspberries, roses and other ornamental and medicinal plants such as geum, potentilla, alchemilla and sanguisorba (challice, 1974; mcneill, 2012; simpson, 2018). the genus sanguisorba is comprised of approximately 18 to 34 species and subspecies, which are widely distributed in the northern hemisphere of eurasia and north america (gbif secretariat, 2019; kurtto, 2009). the common name of sanguisorba in western countries is burnet, and the plant is known to have hemostatic properties. the plants are perennial herbs with leaf rosettes, the stems of which grow 10 to 200 cm high, with further leaves arranged alternately up the stem. the leaves are pinnate with serrated margins. flowers are small, tetramerous or trimerous and often unisexual, and they lack petals (wang et al., 2020). the stamina have long filaments, and gynoecia consist of a single carpel topped with a feathery style (kalkman, 2004). the flowers are small, dense clusters or spikes with a length of 1 to 7 cm (blaschek et al., 2018; uchida and ohara, 2018). the flowering stage ranges from june to september and the fruit phases are usually from august to november. the fruits of sanguisorba species belong to the nuts. the nuts of great burnet (s. officinalis) are narrowly winged (fig. 1), with smooth surfaces, oval or broadly triangular in cross-section; not heteromorphic. the size ranges from 2.6 × 1.4 × 1.4 mm to 3.5 × 2.2 × 2.1 mm (verband botanischer gärten, 2020). s. officinalis is a typical traditional chinese medicinal plant. especially the roots and herbal parts have been used to treat burns, hematemesis, asthma, intestinal infections and dermatitis (lee et al., 2010; yang et al., 2015; yokozawa et al., 2002; yu et al., 2011; zhang et al., 2018). the primary biologically active constituents belong to the terpenoids, tannins and flavonoids, which are associated with antioxidant, anti-inflammatory, antiviral, antifungal, hemostatic and cytotoxic activities (cai et al., 2012; kim et al., 2008; liang et al., 2013; sun et al., 2012; zhang et al., 2013; zhang et al., 2012). there is an increasing interest of finding essential fatty oils needed for human health and well-being, for pharmaceuticals, nutritional foods or cosmetics. as an example, the seed oil of borago officinalis l. is rich in gamma-linolenic acid and used as dietary food supplement and ingredient for cosmetic preparations (asadi-samani et al., 2014). many traditional medi cinal plants contain phenolic fatty oil in their seeds, which could be of interest for exactly these purposes. but there are only a few studies on almost forgotten medicinal plants and their primary and secondary metabolites of the seeds. since studies on s. officinalis seeds are still rare, the present study aimed at a profound characteri zation of the phenolic compounds and reports the detailed structure of the seeds for the first time. materials and methods seeds of sanguisorba officinalis l. (year of harvest: 2015; location: germany; voucher number: hoh-022713; thousand-seed weight (tsw) = 2.04082 g), sanguisorba minor scop. (year of harvest: unknown; location: germany; voucher number: hoh-022757; tsw = 8.33333 g), sanguisorba tenuifolia fisch. ex link (year of harvest: unknown; location: germany; voucher number: hoh-022755; tsw = 1.33333 g), sanguisorba parviflora takeda (s. tenuifolia var. parviflora; year of harvest: unknown; location: germany; voucher number: hoh-022756; tsw = 1.0989 g) were acquired from jelitto perenial seeds gmbh (schwarmstedt, germany). the species were identified by dr. r. duque-thüs and voucher specimens were deposited with the herbarium of the institute of botany, hohenheim university. seeds of sanguisorba officinalis l. 93 dry seeds were cut with a razor blade and were photographed with a sony alpha 6000 (sigma 70 mm 1:2.8 dg macro + macro lens). seeds of s. officinalis were mounted on adhesive carbon tabs on aluminium stubs and sputter-coated with gold-palladium (20/80; 14-15 ma; 0.1 0.15 mbar; scd 040, balzer union, wallruf, germany) and investigated using a scanning electron microscope dsm 940 (zeiss, oberkochen, germany) at 5 kv (lorenz et al., 2018). moreover, seeds of s. officinalis were photographed at different developmental stages, i.e. after pre-soak in water (5 days), after germination (8 days) and as seedling (14 days). furthermore, seeds of s. officinalis (20.0 g) were immersed in ch2cl2 (180 ml) and comminuted by ultra-turrax treatment (3 min; 21000 rpm, ika werke gmbh & co. kg, staufen, germany), under external ice cooling. after mace ration overnight at 4 °c the seeds were filtered off over celite by vacuum suction and extracted a second time in the same manner. an oil fraction (5.74 g; 28.7% of the seed weight) was recovered from the combined ch2cl2 extracts by vacuum rotoevaporation of the solvent. subsequently, the degreased seeds were extracted twice with meoh (180 ml) overnight (+ 4 °c), filtered off and meoh was removed in vacuo to yield a crude extract. for gc analyses, fatty acid methyl esters were prepared by on-column derivatization with trimethylsulfonium hydroxide (tmsh, 0.25 m in meoh). briefly, 10 mg of viscous oil sample (ch2cl2 residue) were dissolved in 2000 μl tbme. aliquots of 10 μl of this test solution were mixed with 170 μl of tbme followed by 60 μl of tmsh. subsequently, the mixture was directly injected into the gc/ms system (perkinelmer clarus 500; (heinrich et al., 2017)). for phenolic compound analysis, the previously mentioned meoh crude extract (3-5 mg/ml) was redissolved in meoh/h2o (50/50, v/v). liquid chromatographic analyses were carried out on an agilent 1200 hplc system (agilent technologies, inc., palo alto, usa), equipped with a binary pump, a micro vacuum degasser, an auto sampler, a thermostatic column compartment and a uv/vis diode array detector. a kinetex® c18 reversed-phase column (2.6 μm particle size, 150 × 2.1 mm i.d., phenomenex ltd., aschaffenburg, germany) was used for chromatographic separation. the lc system was coupled to an hctultra ion trap mass spectrometer (bruker daltonik gmbh, bremen, germany) with an esi source operating in the negative ion mode (bunse et al., 2020). for characterizing the compound profiles, three biological replicates (n = 3) of all samples were prepared. in addition, technical replicates were prepared to obtain the graphs, which allowed the calculation of standard deviations. results seed morphology the nuts of sanguisorba reveal species-dependent variations. naturally, also nuts within a species reveal variations of their individual appearance (fig. 1). fig. 2 shows seeds and their cross sections of s. officinalis (a), s. minor (b), s. tenuifolia (c), and s. parviflora (d). the species a, c, d are characterized by similar narrowly winged seed shapes and contained only one seed kernel (achene), whereas the nuts of s. minor (b) usually contained 1-3 seed kernels per nut, and the hypanthium showed serrated netting strips. for a more detailed investigation of the seed morphology, sem images of the seed structures of s. officinalis were taken. the dried fruit (seed) of s. officinalis consists of several layers (fig. 3). the floral cup or hypanthium forms the outer layer, the surface of the fruit. the pericarp is located below the perigynous hypanthium and encloses the inner seed kernel. the pericarp shows an outer layer with cells containing crystal structures. the cells located inside show a fibrous structure with thickened inner walls and extended cells. the pericarp converges at the apex of the seed and forms the stylus, which ends with the stigma in the flowering stage. the seed kernel is located inside the pericarp. it consists of the embryo with its two cotyledons, the epicotyl, the hypocotyl and the radicle covered by the seed coat. beside the aforementioned morphological structures, the longitudinal section of pre-swollen seeds (5 days) of great burnet (fig. 4, a) showed the white endosperm of the cotyledons. furthermore, sem analyses revealed the epicotyl (e) of the embryo with the base of plumule. fig. 4 b shows the primary root of a seedling 8 days after germination with a piece of seed coat at its tip. after 6 further days, a seedling of s. officinalis (fig. 4, c) has developed a root (r), an elongated hypocotyl (h), green cotyledons (co), and the epicotyl shows primary leaves (pl, leaf bud) at an early developmental stage. lipid constituents for investigating the lipid constituents, a ch2cl2 extract from dried seeds of s. officinalis was prepared to yield a fatty oil (29% of dry weight of the seeds). gc/ms analyses revealed a complex peak profile. by comparing the mass spectra of individual components with those of reference compounds, a number of fatty acids with carbonchain lengths of c16 c22 were assigned (fig. 5). unsaturated fatty acids: 39% linolenic acid (c18:3), 36% linoleic acid (c18:2), 17% fig. 1: overview of seed shapes and their morphological variations of s. officinalis (a), s. minor (b), s. tenuifolia (c) and s. parviflora (d). scale bare = 1 mm. 94 m. bunse, f.c. stintzing, d.r. kammerer a b c d 238 fig. 3: sem micrographs of the fruit (seed) of sanguisorba officinalis. a, longitudinal section. b, longitudinal 239 section of the perygynous hypanthium and the pericarp with calcium oxalate crystals. c, inner layer of the pericarp 240 with elongated cells. d, surface of the fruit. e, surface of the seed kernel (testa, seed coat). e: epicotyl; h: hypocotyl; 241 r: radicle. scale bars: a, d = 500 µm, b = 20 µm, c = 50 µm, e = 200 µm. 242 243 hypanthium pericarp stylus embryo cotyledons h r e testa hypanthium pericarp calcium oxalate a b c d e fig. 3: sem micrographs of the fruit (seed) of sanguisorba officinalis. a, longitudinal section. b, longitudinal section of the perygynous hypanthium and the pericarp with calcium oxalate crystals. c, inner layer of the pericarp with elongated cells. d, surface of the fruit. e, surface of the seed kernel (testa, seed coat). e: epicotyl; h: hypocotyl; r: radicle. scale bars: a, d = 500 μm, b = 20 μm, c = 50 μm, e = 200 μm. fig. 2: seed shapes and cross sections of s. officinalis (a), s. minor (b), s. tenuifolia (c) and s. parviflora (d). scale bare = 2 mm. 244 fig. 4: a, longitudinal section of a s. officinalis seed after pre-soak in water for 5 days. b, germinated seed 245 (s. officinalis) with primary root (r) after 8 days. c, seedling 14 days after germination. 246 co: cotyledons, e: epicotyl, h: hypocotyl, pl: primary leaves, r: radicle/ root. scale bars = 1 mm. 247 248 a c testa cotyledons h r e pericarp hypanthium b r co e h r pl fig. 4: a, longitudinal section of a s. officinalis seed after pre-soak in water for 5 days. b, germinated seed (s. officinalis) with primary root (r) after 8 days. c, seedling 14 days after germination. co: cotyledons, e: epicotyl, h: hypocotyl, pl: primary leaves, r: radicle/ root. scale bars = 1 mm. oleic acid (c18:1) and < 0.5% eicosenoic acid (c20:1); saturated fatty acids: 4% palmitic acid (c16:0), 2% stearic acid (c18:0), < 2% arachidic acid (c20:0) and behenic acid (c22:0). thus, linolenic acid, linoleic acid and oleic acid were the predominant fatty acids. phenolic constituents methanolic crude extracts of the previously defatted seeds of s. officinalis were investigated by lc/msn. in summary, 18 compounds were tentatively assigned in this fraction based on their retention time (tr), uv spectra, mass-to-charge ratios (negative ionization mode), as well as their specific fragmentation patterns and corresponding bibliographic references. the chromatogram (fig. 6) and mass spectra revealed the occurrence of compounds belonging to different substance classes. among these, hydroxybenzoic acids (methyl gallate-hexoside), tannins (ellagic acid), flavonoids (quercetin-pentoside, catechin, procyanidins) were assigned, but also l-tryptophan, being an aromatic representative of amino acids (tab. 1). furthermore, the relative abundance [%] of the assigned compound is shown in fig. 7. a b c d a b c d seeds of sanguisorba officinalis l. 95 249 fig. 5: fatty acid composition of s. officinalis seeds illustrated as mean value of the relative abundance of individual 250 compounds [%] including standard deviation (n=3). palmitic acid (c16:0), stearic acid (c18:0), oleic acid (c18:1), 251 linoleic acid (c18:2), linolenic acid (c18:3), arachidic acid (c20:0), eicosanoic acid (c20:1), behenic acid (c22:0). 252 253 254 255 256 fig. 6: lc/msn chromatogram (uv detection at 280 nm) of phenolic compounds in a meoh seed extract of 257 sanguisorba officinalis. for compound characterization, cf. table 1. 258 259 0 10 20 30 40 50 re la ti ve a bu nd an ce [% ] uv 280 nm 0 5 10 15 20 25 in te ns iti y [m a u ] 20 30 40 50 time [min]0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 249 fig. 5: fatty acid composition of s. officinalis seeds illustrated as mean value of the relative abundance of individual 250 compounds [%] including standard deviation (n=3). palmitic acid (c16:0), stearic acid (c18:0), oleic acid (c18:1), 251 linoleic acid (c18:2), linolenic acid (c18:3), arachidic acid (c20:0), eicosanoic acid (c20:1), behenic acid (c22:0). 252 253 254 255 256 fig. 6: lc/msn chromatogram (uv detection at 280 nm) of phenolic compounds in a meoh seed extract of 257 sanguisorba officinalis. for compound characterization, cf. table 1. 258 259 0 10 20 30 40 50 re la ti ve a bu nd an ce [% ] uv 280 nm 0 5 10 15 20 25 in te ns iti y [m a u ] 20 30 40 50 time [min]0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 fig. 5: fatty acid composition of s. officinalis seeds illustrated as mean value of the relative abundance of individual compounds [%] including standard deviation (n=3). palmitic acid (c16:0), stearic acid (c18:0), oleic acid (c18:1), linoleic acid (c18:2), linolenic acid (c18:3), arachidic acid (c20:0), eicosenoic acid (c20:1), behenic acid (c22:0). fig. 6: lc/msn chromatogram (uv detection at 280 nm) of phenolic compounds in a meoh seed extract of sanguisorba officinalis. for compound characterization, cf. tab. 1. 260 fig. 7: phenolic composition of s. officinalis seeds illustrated as mean value of the relative abundance of individual 261 compounds [%] (n=2). for compound assignment, see table 1. 262 263 tables 264 265 tab. 1: compound assignment of metabolites detected in meoh extracts of s. officinalis seeds using hplc-dad-266 esi-msn (negative ionization mode) 267 268 no.a constituent tr [min] llmax, uv [nm] [m-h][m/z] fragmentation ions [m/z] reference 1 l-tryptophan 20.8 218, 280 203 186, 159, 142, 116 b 2 1-o-caffeoylquinic acid 24.2 324, 374 353 345, 323, 289, 191, 179, 171, 135, 127 (plazonić et al., 2009) 3 methyl-gallate-hexoside 26.1 214, 270 345 183, 168, 124 (abu-reidah et al., 2015) 4 procyanidin b1/b2 27.9 324, 374 578 559, 425, 407, 289, 285, 257 (bunse et al., 2020) 5 trans-3-p-coumaroylquinic acid 29.2 204, 230sh, 312 337 311,289, 191, 163, 119 b(makita et al., 2017) 3.2 % 0.5 % 7.0 % 0.1 % 1.7 % 33.5 % 14.9 % 1.1 % 5.6 % 0.4 % 7.6 % 6.1 % 4.0 % 0.3 % 2.9 % 1.6 % 0.7 % 1.2 % 2.5 % 5.1 % 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 fig. 7: phenolic composition of s. officinalis seeds illustrated as mean value of the relative abundance of individual compounds [%] (n=2). for compound assignment, see tab. 1. discussion the dry fruits of sanguisorba form achenes, which persist in dense spikes until late autumn when they shatter, scattering most seeds within a 1 m2 area around the plant. when harvested, a dry, papery calyx hull surrounds the achenes (holloway and matheke, 2003). the fruits of s. officinalis, s. tenuifolia, and s. parviflora (fig. 1) are ellipsoid to globose, 4-angled, 4-winged with smooth surfaces and one achene. in contrast, the fruits of s. minor have serrated netting strips. the serrated surface of s. minor appears to be an adaption aiming at the distribution of seeds via the fur of mammals. large numbers of seeds are distributed by passive attachment to the fur of mammals. as mammals walk through the vegetation, seeds of different sizes, rough surfaces and distributions of hooks are dislodged from the parent plants and attached to the fur (stiles and fenner, 2000). s. officinalis has short, erect botryoids with several single flowers on it. the gynoecium, the female part of a flower, is unicarpellate and includes a stigma, a stylus and a unilocular ovary. it is surrounded by a perigynous hypanthium. within the locule, a single anatropous unitegmic ovule is formed. after successful fertilization of an inflorescence, the seed (embryo) is formed inside the gynoecium. during that process the inflorescence begins to wither, the bracts and the stamina at the apex of the hypanthium and the stigma dry out and fall off. the fruit (seed, nut) is formed with the dried hypanthium still serving as protection. the gynoecium wall becomes the pericarp during fructification. the inner epidermis of the pericarp is called the endocarp, the outer epidermis is referred to as exocarp, with the mesocarp in between. in the case of nuts, these three layers are equally lignified. the outer layer of s. officinalis pericarp, the exocarp, appears to be less lignified, but at the same time containing calcium oxalate crystals (weddellit, ca(c2o4) · 2 h2o, (hartl et al., 2007)). calcium oxalate widely occurs in plants and may account for 3-80% of plant dry weight (libert and franceschi, 1987). as much as 90% of total calcium in a plant may be found as its oxalate salt (gallaber, 1975). calcium oxalate is generally considered an end product, and thus its formation reduces the availability of calcium, although some studies have also shown calcium oxalate to be a reversible product (franceschi and horner, 1980; franceschi, 1989; huang et al., 2015). the occurrence of calcium oxalate crystals in flowering plants is more or less widespread in different plant parts, such as leaves, stems, roots, floral parts, fruits, seeds and pericarps (mukherjee and jana, 2014). calcium oxalate plays an important role in plant defense. among others, it is an effective deterrent to herbivores. the embryo is surrounded by a thin seed coat, the testa. the two cotyledons serve as nutritional tissue for germination. to monitor seed germination and development into a seedling, nuts of s. officinalis were pre-swollen in water and scattered on soil. after 5 days the cotyledons of the embryo seemed to bulge, and the epicotyl, hypocotyl and radicle started to be developed. after 8 days the radicle of the first seedlings broke off the seed coat, and 14 days after germination small seedlings were developed. seeds of great burnet (s. officinalis) germinate most rapidly at ca. 25 °c after 6 months of dry storage at 4 °c. the cold period is necessary to break seed dormancy. the seeds also germinate without stratification, but then only very sporadically and over a longer period of time (holloway and matheke, 2003). starch, sugars and fats of the cotyledons provide the energy for germination. the seeds were analyzed by gc/ms to assess their fatty acid profile revealing an average composition typical of members of the rosaceae family. the study of matthaus and özcan (2014) showed that oil contents of 26 varieties of rosaceae seeds ranged from 3.5 to 46.2 g/100 g. these oils were composed of 3.25-9.17% palmitic, 1.19-4.27% stearic, 6.50-67.11% oleic, 22.08-68.62% lino leic and 0.10-61.59% eicosenoic acids (matthaus and özcan, 2014). based on these wide ranges it is not surprising that the fatty acid profile of s. officinalis seeds is within the aforementioned margins (unsaturated fatty acids: 39% linolenic acid (c18:3), 36% lino leic acid (c18:2), 17% oleic acid (c18:1) and < 0.5% eicosenoic acid 96 m. bunse, f.c. stintzing, d.r. kammerer tab. 1: compound assignment of metabolites detected in meoh extracts of s. officinalis seeds using hplc-dad-esi-msn (negative ionization mode). no.a constituent tr λmax, uv [m-h]fragmentation reference [min] [nm] [m/z] ions [m/z] 1 l-tryptophan 20.8 218, 280 203 186, 159, 142, 116 b 2 1-o-caffeoylquinic acid 24.2 324, 374 353 345, 323, 289, 191, (plazonić et al., 2009) 179, 171, 135, 127 3 methyl-gallate-hexoside 26.1 214, 270 345 183, 168, 124 (abu-reidah et al., 2015) 4 procyanidin b1/b2 27.9 324, 374 578 559, 425, 407, 289, (bunse et al., 2020) 285, 257 5 trans-3-p-coumaroylquinic acid 29.2 204, 230sh, 312 337 311, 289, 191, b(makita et al., 2017) 163, 119 6 procyanidin 30.3 202, 230sh, 280 577 559, 451, 425, 407, (bunse et al., 2020) 389, 289, 285, 257, 213 7 catechin / epicatechin 31.9 204, 230sh, 280 289 245, 227, 203, 187, (zhao et al., 2013) 175, 161 8 caffeoylquinic acid 32.4 202, 232, 324 353 289, 250, 191, 173, b 127, 85 9 procyanidin trimer 33.1 202, 230, 280 865 847, 739, 695, 677, (rockenbach et al., 2012) 577 543, 525, 391 10 5,6,7-trihydroxy-2,3-dihydrocyclopenta[b] 36.5 280, 358, 522 291 247, 219, 203, 191, (fraternale et al., 2015) chromene-1,9-dione-3-carboxylic acid 175 11 p-coumaroylquinic acid 39.3 232, 312 337 191, 173, 155, 127, (bunse et al., 2020) 111, 93, 85 12 procyanidin dimer 40.7 202, 230, 282 577 451, 425, 407, 389, (bunse et al., 2020) 289, 285, 257, 213 13 unidentified gallic acid derivative 42.9 202, 224, 278 497 465, 345, 183, 168, (hofmann et al., 2016) 124 14 trigalloyl hexose 43.9 280, 360 635 465, 313, 285, 241, (abu-reidah et al., 2015) 221, 193, 169 15 unidentified gallic acid derivative 45.3 234, 274, 364 497 463, 345, 313, 183, (hofmann et al., 2016) 168, 124 16 unidentified 46.0 280, 374 537 519, 339, 195, 179, 161, 143, 119, 89 17 unidentified 51.7 234, 280, 314 662 644, 602, 516, 456, 438, 324, 307, 248, 205, 163, 145 18 quercetin-3-o-arabinoside / dxyloside 53.1 240, 258, 362 433 345, 301, 257, 229, (respect for phytochemicals, 185 2020.000z) 19 ellagic acid 54.7 254, 362 301 284, 257, 245, 229, b 201, 185, 165 20 isoquercetrin 56.5 238, 268, 360 463 301, 271, 255, 229, (ibrahim et al., 2015) 193, 179, 151, 107 a for peak assignment see fig. 5. b reference spectra (mona, 2020). (c20:1); saturated fatty acids: 4% palmitic acid (c16:0), 2% stearic acid (c18:0), < 2% arachidic acid (c20:0) and behenic acid (c22:0)). in addition, the phenolic profile of s. officinalis seeds is typical of the rose family (fahad al juhaimi et al., 2016). hydroxybenzoic acids, tannins and flavonoids belong to the main classes of phenols detected in great burnet herb, roots and flowers (bunse et al., 2020). most of these phenolics are bioactive constituents and are exploited for pharmacological purposes as mentioned before. in contrast, the role of these compounds in seeds of s. officinalis is still largely unknown and needs further elucidation. however, based on their activity profile, protection of the seeds from bacterial or fungal growth and rot appears conclusive. conclusion this study aimed at a profound characterization of the morphology of sanguisorba officinalis seeds and of their primary and secondary metabolites. for the first time the structure of great burnet seeds was elucidated by scanning electron microscopy. the seed is enclosed by a thin seed coat, which is surrounded by the pericarp and the floral seeds of sanguisorba officinalis l. 97 cup. as a special feature, calcium oxalate crystals were discovered in the outer layer of the lignified pericarp. the occurrence of calcium oxalate crystals in flowering plants is more or less widespread in different plant parts and plays an important role in plant defense. in addition to these morphological studies, the fatty acid and phenolic compound profiles of s. officinalis seeds were characterized for the first time. the genus sanguisorba is an important member of the rosaceae family, the natural habitat of which is becoming increasingly rare. this short study provides a first insight into the seed structure of great burnet and complements previous studies on the flower deve lopment of different sanguisorba species (wang et al., 2020). in the future, further sanguisorba species should be investigated regarding their morphology and phytochemistry to allow direct comparison of taxonomically related species. furthermore, more detailed information on different compartment structures also considering their specific secondary metabolites may help to deduce the functionality of the latter in the plant. this may also contribute to a better chemotaxonomic differentiation between closely related species or hybrids. in addition, new sources of healthy fatty oils, especially needed for human health and well-being, but also cosmetic applications, are increasingly important. for example, they can supply the body with essential fatty acids or support it in healing processes, e.g. inflammatory skin regions. in the future there could be a greater focus on indigenous medicinal plants cultivated locally, that contain highquality oils as deduced from their compound profile being abundant in unsaturated fatty acids and revealing the occurence of biologically active phenolics. acknowledgement the authors are grateful to prof. dr. waltraud x. schulze (department of plant systems biology, hohenheim university) for the support in creating the manuscript. the authors also gratefully acknow ledge dr. rhinaixa duque-thüs (institute of botany, hohenheim university) for identification of the plant specimens and we would like to thank dr. annerose heller and her team (institute of botany, hohenheim university) for the great support in teaching microscopy and botany over the last years. conflict of interest the authors reported no potential conflict of interest. references abu-reidah, i.m., ali-shtayeh, m.s., jamous, r.m., arráez-román, d., segura-carretero, a., 2015: hplc–dad–esi-ms/ms screening of bioactive components from rhus coriaria l. 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10.1093/chromsci/bms138 orcid marek bunse https://orcid.org/0000-0003-2563-6534 dietmar rolf kammerer https://orcid.org/0000-0003-1126-3431 florian conrad stintzing https://orcid.org/0000-0001-7006-6804 address of the corresponding authors: prof. dr. dietmar rolf kammerer, department of analytical development and research, section phytochemical research, wala heilmittel gmbh, dorfstraße 1, 73087 bad boll/eckwälden, germany e-mail: dietmar.kammerer@wala.de © the author(s) 2021. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1186/s40529-014-0048-4 http://dx.doi.org/10.3390/molecules14072466 http://dx.doi.org/10.1016/j.foodres.2012.07.001 http://dx.doi.org/10.3390/molecules17077629 http://dx.doi.org/10.1111/1442-1984.12195 http://dx.doi.org/10.1093/botlinnean/boaa009 http://dx.doi.org/10.1016/j.phymed.2015.09.006 http://dx.doi.org/10.1016/s0006-2952(01)00930-3 http://dx.doi.org/10.1016/j.jep.2010.08.060 http://dx.doi.org/10.1016/j.ijbiomac.2018.01.214 http://dx.doi.org/10.1016/s1995-7645(13)60117-0 http://dx.doi.org/10.3390/molecules171213917 http://dx.doi.org/10.1093/chromsci/bms138 https://orcid.org/0000-0003-2563-6534 https://orcid.org/0000-0001-7006-6804 https://orcid.org/0000-0003-1126-3431 art02 journal of applied botany and food quality 80, 6 13 (2006) institute of agricultural and environmental sciences, estonian agricultural university, tartu, estonia, and department of environmental sciences, nova scotia agricultural college, truro, nova scotia, canada observations on the gynoecial pathway for pollen tube growth in sweet lowbush blueberry (vaccinium angustifolium ait.) merrit noormets, a. randall olson (received august 17, 2005) summary gynoecial structure in sweet lowbush blueberry, vaccinium angustifolium ait., was investigated in order to characterize the pollen tube pathway in order to provide a framework for further studies on pollination and fungal infection. closed flower buds and pollinated open flowers were collected from managed lowbush blueberry fields in colchester county, nova scotia, canada. following chemical fixation, the tissue samples were examined histologically using light and scanning electron microscopy. the continuous pathway was characterized by a fluted, exudate-filled stylar canal that connects the wet stigmatic surface with the exudate covered surface of the ovarian placentae. following pollen deposition and germination, tubes growing along the pathway eventually arrive at the micropyles of the anatropous ovules; ovule penetration by pollen tubes and fertilization of the female gametophytes ensue. the pollen tube pathway of this taxon conforms to the general pattern reported from other ericalean taxa. introduction among the ericaceae, vaccinium is a highly polymorphic genus containing species that are typically acidophilic, predominantly outcrossing, and distributed throughout many regions of the arctic, temperate and tropical zones (camp, 1942, 1945; vander kloet, 1988). in eastern north america, the wild blueberry industry depends on the successful management of the sweet lowbush blueberry, vaccinium angustifolium ait. this economically important woody perennial species spreads vegetatively by the formation of rametes. when sexually mature, erect shoots produce numerous inverted pedunculate, racemose inflorescences (camp, 1945). each inflorescence bears several complete, actinomorphic, pentacyclic flowers characterized by an urceolate perianth configuration (palser, 1961). during anthesis, the sympetalous corolla will recurve at the tips to expose the papillate stigma and the porocidal tips of the mature anthers (vander kloet, 1988). the floral morphology, nectar production and pendulous orientation of the blossoms of sweet lowbush blueberry are consistent with other entomophilous species of this genus (wood, 1961; wood, 1962; hall et al., 1971). the syncarpous, pentacarpellate gynoecium typical of the genus vaccinium consists of a single stigma attached to an elongate, hollow style subtended by a pentalocular ovary with axile placentation and numerous ovules (bell and burchill, 1955; munoz and lyrene, 1985). the gynoecium is usually surrounded by ten stamens attached to the base of the corolla near the nectariferous tissue located on the upper ovarian wall; this configuration in conjunction with the position of the poricidal anther tips in relation to the stigma indicate mechanical pollen transfer by insect sonication (mcgregor, 1976; jacquemart and thompson, 1996). when released from the anthers, vaccinium pollen is of the dry type, covered with pollenkitt and dispersed in tetrads held together by viscin threads (heslopharrison and shivanna, 1977; pacini, 2000). each individual pollen grain is bicellular and tricolporate with pollen germination occurring two to three hours following deposition on a receptive wet stigma (munoz and lyrene, 1985; vander kloet, 1988). following successful pollination, pollen tube growth through the various gynoecial tissues may take three to four days before reaching the ovule-bearing placentae within the ovary (bell, 1957). typically in angiosperms, determination of pollen compatibility and the nutritional support of pollen tube growth are mediated by the gynoecial tissues (herrero and hormaza, 1996). in addition, recent research summarized by lord and russell (2002) indicates that pollen tubes in some way become predisposed to receive and follow molecular signals originating from inside of the ovules during their passage through the stigma and style. thus, an essential pathway for pollen tube growth and guidance is established extending from the stigmatic surface to the ovular micropyle within the ovary. certain environmental stresses such as frost, however, may interfere with compatible pollen tube growth in sweet lowbush blueberry due to differences in tissue sensitivities along this same gynoecial pathway. even before anthesis, ovule bearing placentae may sustain damage severe enough to interfere with normal fruit set of this economically important wild species (olson and eaton, 2001). in addition, gynoecial tissues of sweet lowbush blueberry are susceptible to infection by the fungus monilinia vaccinii-corymbosi which transforms a normally fleshy fruit into a pseudosclerotium called a mummy berry (hildebrand and braun, 1991). fungal access to the inner ovarian tissues is achieved when fungal infection hyphae from germinating conidia on the stigma grow in conjunction with pollen tubes down the style and enter the ovary; in effect the pollen tube pathway functions as the gynoecial infection pathway for this economically important disease (shinners and olson, 1996). given the economic importance of this species, there is relatively little information available concerning the basic micromorphology of compatible pollen tube growth so critical for fruit production. this study, therefore, uses light and scanning electron microscopy to describe the gynoecial pathway for pollen tube growth in the sweet lowbush blueberry from eastern canada. materials and methods flower buds of sweet lowbush blueberry, vaccinium angustifolium ait., were collected in may and june in 2000 and in 2001 in fields in kemptown and debert, colchester county, n.s., canada. for convenience, the following developmental stages were determined and followed during the study: (1) young bud – very little of the corolla is visible; (2) petal elongation – the petals elongate but stay tightly closed; (3) petal spread – elongation is completed and the flower bud is just opening at the tips of the petals; (4) open flower – the flower is at anthesis and the petals are open and distinctively recurved with the stigma protruding and exposed; (5) petal drop – the corolla and stamens have senesced and dropped off. the flower buds were chemically field fixed using either 3% ga (glutaraldehyde) or faa (formalin-acetic acid-alcohol) for both light (lm) and scanning electron microscopy (sem). selected gynoecial tissue samples were dissected and placed in a fresh fixative. for sem the fixed tissue samples were washed several times in phosphate buffer (ph 6.8) and then postfixed in 2% osmium tetroxide followed by three washes in phosphate buffer. then tissue samples were dehydrated in an ethanol series to anhydrous ethanol. dehydrated specimens were critical point dried and coated with platinum/gold using a samsputter 2a sputter coater. the observations were conducted using a bausch and lomb nanolab 2000 sem at a voltage of 15 kv. for lm histological examination the dehydrated specimens were subjected to a propylene oxide series, infiltrated with spurr's resin (spurr, 1969) and sectioned on a sorvall mt 6000 ultramicrotome between 0.5-1.0 µm. for general observation, certain sections were mounted on gelatin-coated slides and stained with azure ii (1%) methylene blue (1%). to determine the presence of insoluble polysaccharides in the selected plastic sections staining with periodic acid-schiff's reagent was carried out. selected sections were subjected to a saturated solution of dnph (2,4 dinitrophenyl-hydrozine) and 1% periodic acid prior to usage of schiff's reagent (o'brien and mccully, 1981). then slides were counter stained in 1% aniline blue black to identify the proteins (fisher, 1968). additional plastic sections were stained with 1% sudan black b solution to detect lipids (bronner, 1975). the stained sections were then mounted with sp15500 permount (fisher scientific) (berlyn and miksche, 1976; o'brien and mccully, 1981). observations were carried out using a leitz diaplan photomicroscope. for aniline blue-uv induced fluorescence microscopy to identify callose associated with pollen tube walls, certain specimens of the field fixed plant material were selected and hand sectioned. sectioned tissue samples were stained in a solution of 0.05% aniline blue in 0.15 m k2hpo4 at ph 8.6. observations were made using an olympus bh2-rfl photomicroscope with uvfl objectives and barrier filter l-435. results the essentially capitate stigma of vaccinium angustifolium is of the wet type, having five lobes separated by five main grooves radiating outward from a central depression at the center of the stigma; the distal end of each groove terminates with a short bifurcation (fig. 1a). all five lobes appear slightly raised and rounded near the outer rim of the stigma giving the surface an overall convex contour. although histologically the stigma is composed of parenchymatous ground tissue, the surface is characterized by low papillate cells that become inundated with an exudate before anthesis (fig. 1b, d). post-fixation, plastic embedded sections of the papillate surface stained with sudan black b demonstrate a lipidic component to the exudate. below the papillate surface are several layers of elongated cells characterized by large interstitial spaces also filled with lipidic exudate (fig. 1c). the intercellular spaces of these cells lead directly into the fluted, hollow canal in the center of the style. following successful pollination, pollen tetrads deposited on the stigma appear embedded in the lipidic exudate (fig. 2a, b). individual pollen grains in each tetrad are tricolporate and two celled. upon germination, pollen tubes emerge from adjacent grains in the tetrad and grow down through the exudate in between the cells of the stigma (fig. 2c). the numerous pollen tubes pass through the exudate filled intercellular spaces of the stigma cells and converge near the entrance to the upper stylar canal (fig. 2d). the five grooves separating the lobes of the stigma are continuous with five hollow clefts of the stylar canal that appear overall starshaped in cross section (fig. 3a). the hollow canal is lined by an epithelial transmitting tissue and is filled with a mucilaginous secretion. the parenchymatous ground tissue that makes up the bulk of the style is vascularized by the five bundles. each of these vascular bundles is situated adjacent to the tip of the cleft of the canal. the junction between the stylar canal and upper ovary is continuous; each hollow cleft of the canal is directly connected to an ovarian locule through a thin cleft forming a conduit between adjacent locular septae in the upper ovary (fig. 3b). the pollen tubes enter the mucilage filled clefts of the stylar canal and proceed to grow downward toward the base of the stylar canal (fig. 3a); pollen tubes from each canal cleft will enter an ovarian locule through the associated conduit (fig. 3b). the parenchymatous ovary is characterized by the five locules with axile placentation and numerous ovules. the upper region of each placenta has a small cleft that is continuous with the cleft in between the associated upper ovarian septae. the placental cleft opens onto the surface of the placenta. the surface of each ovule-bearing placenta is composed of cells that are also papillate in appearance (fig. 4a, b). the surface of placenta is covered by a mucilaginous secretion that appears similar to and continuous with that observed in the stylar canal. the micropyle of each anatropous ovule is oriented toward the placental surface (fig. 4a, b). the mature ovules are unitegmic and contain a polygonum-type female gametophyte (fig. 5a). once in the locule, pollen tube growth proceeds across the placental surface toward the anatropous ovules (fig. 4c). the numerous pollen tubes eventually form a tortuous mat on the surface of each placenta (fig. 4d). the pollen tubes grow among the ovular micropyles; only one pollen tube will penetrate a micropyle of a single ovule in order to facilitate the fertilization events (fig. 5b). discussion although there is a great deal of variation in the morphological details, members of the ericales have gynoecia composed of a clearly defined stigma, style and ovary (cronquist, 1981). according to the characteristics of the receptive surface for pollen capture, heslopharrison and shivanna (1977) categorize ericalean stigmas as belonging to the wet-type producing an exudate on a receptive surface with or without low to medium papillate cells. the overall appearance of the stigma from the closely related monotropaceae, pyrolaceae and ericaceae varies and may be described as funnel-like, truncate, obtuse or capitate with lobes (cronquist, 1981; palser et al., 1992; olson, 1991, 1994). among the ericaceae, stigma structure of the genus rhododendron is the most thoroughly documented (williams et al., 1982; palser et al., 1992). in rhododendron, each truncate stigma is characterized by an overall lobed appearance created by several main grooves that radiate outward from a central depression; each of the grooves, however, is further subdivided by a network of numerous laterally branching short grooves (palser et al., 1992). although the wet receptive surface is nonpapillated, the numerous surface grooves reflect a system of exudate filled convoluted furrows that extend to the stylar canal. the overall shape of the stigma in the ericaceous vaccinium angustifolium is similar to that in rhododendron; the stigmas of both species are essentially convexly capitate and lobed. a contrast to the numerous surface grooves of rhododendron, however, there are only five large grooves that radiate from a central depression and each of these terminates at its distal end in a bifurcation. although the receptive surface of the stigma in both genera is covered with an exudate, rhododendron is nonpapillate in contrast to the low papillate cells observed in v. angustifolium. in addition, the stigmatic cells below the receptive surface of v. angustifolium separate from one pollen tube pathway in sweet lowbush blueberry 7 fig. 1: stigmas of vaccinium angustifolium. a. sem of a stage 1 stigma with arrow indicating the bifurcation at the distal end of a groove. x 382. b. sem of a stage 2 stigma and the appearance of the exudate. x 428. c. resin-embedded longitudinal section through a stage 3 stigma. arrow indicates the lipidic exudate filling the space between papillated cells (stained with azur ii/methyl blue). x 307. d. sem of a stage 3 stigma. stigmatic surface is covered with copious exudate. x 615. figure abbreviations: pe, petal; st, stigma; sy, style; ex, exudate; po, pollen tetrad; ol, ovary locule; ov, ovule; sc, stylar canal; pl, placental tissue; ow, ovary wall. another forming a network of exudate filled interstitial spaces that may be described as an transmitting tract. stigmatic exudates are presumably involved with pollen adhesion, hydration, germination and the eventual penetration of the gynoecial tissues by the pollen tubes (knox, 1984; herrero and hormaza, 1996; lord and russell, 2002). in vaccinium angustifolium, noormets and olson (2002) conducted receptivity tests on fresh stigmata using a technique adapted and modified from dafni (1992) based on the detection of peroxidase activity. the first appearance of the exudate coincides with detectable peroxidase activity in stage one (young bud) of individual flower development. although the stigmatic exudate of vaccinium angustifolium was not analyzed chemically in the present study, basic histochemical staining identifies lipids as a major constituent. wolters-art et al. (1998) investigated 8 merrit noormets, a. randall olson fig. 2: v. angustifolium stage 4 stigmas with pollen tetrads. a. sem of a stigma with pollen tetrads embedded in the surface exudate. arrows indicate pollen tetrads. x 1800. b. sem of a stigma showing the emergent pollen tubes penetrating the exudate. arrows indicate the pollen tubes. x 2995. c. resin-embedded longitudinal section trough a pollinated stigma. pollen tetrads embedded in the exudate. arrows indicate the pollen tubes growing in between the stigmatic surface cells. x 410. d. aniline blue fluorescent micrograph of a longitudinal free-hand section through the stigma and stylar canal. arrows indicate the numerous pollen tubes. x 165. pollen tube pathway in sweet lowbush blueberry 9 the possible role lipids play in the complex pollen-stigma interactions. their experiments and careful analyses indicate that lipids are a crucial factor in establishing an external water gradient required for water uptake by both the pollen grains and the pollen tubes. it seems reasonable to suggest that lipids on the stigmatic surface of v. angustifolium may also help direct pollen tube penetration and growth in the gynoecial tissues for similar reasons. previous observations on the stylar structure in certain members of the ericales reveal a common pattern; the stylar canal is hollow, fluted and filled with a mucilaginous substance presumably derived from an epithelial transmitting tissue that lines the canal. among those members of the pyrolaceae and monotropaceae that were studied, the number of mucilage filled clefts in the fluted canal is five and reflects the number of ovarian locules (pyykkö, 1968; olson, 1991, 1994). in the ericaceae, the number of mucilage filled stylar clefts varies from five to ten among the various species of rhododendron but also reflects the number of ovarian locules (williams et al., 1982; palser et al., 1992). in vaccinium elliotii, v. corymbosum and their hybrids, there are five stylar clefts that become filled with a mucilaginous secretion (munoz and lyrene, 1985). as expected, the observations of the style in v. angustifolium conforms to the previously established pattern characterized by five mucilage filled stylar clefts that are aligned with a corresponding ovarian locule. at the style-ovary junction in members of the ericales, each cleft of the stylar canal is restricted to a narrow cleft between the upper ovarian septae that forms a conduit into each ovarian locule. these septal clefts are continuous with an associated cleft in the placental tissues bearing the ovules. in various genera studied from the ericaceae, monotropaceae and pyrolaceae, the clefted placental tissue is relatively massive and covered with a mucilaginous secretion; each locular region has the appearance of containing two protruding, ovule bearing placental lobes (pyykkö, 1968, 1969; palser, 1992; olson, 1991, 1994). in vaccinium angustifolium, the ovary is clearly divided into five locules separated by complete septae with axile placentation throughout the ovary. there is also a short cleft in the upper placenta that is aligned with the corresponding conduit connecting the stylar canal with the upper locule. the opening into each locule of v. angustifolium, therefore, leads directly to the secretion-coated surface of the ovule bearing placenta. herein lies the significance of the structural alignment between the stylar canal and the ovarian locules in all ericalean taxa studied including v. angustifolium. observations on the gynoecial pathway for pollen tube growth in vaccinium angustifolium are in accordance with previous descriptions from other ericalean species (williams et al., 1982; munoz and lyrene, 1985; palser et al., 1992; olson, 1991, 1994). in the aforementioned studies, the pollen tubes follow an unobstructed pathway characterized by continuous secretions along the entire journey from the stigma to the ovules. the numerous pollen tubes growing from the stigmatic surface, through the stylar canals, into the locules and along the placental surfaces in v. angustifolium remain in direct contact with gynoecial secretions until they penetrate the ovular micropyles. fig. 3: v. angustifolium. a. aniline blue fluorescent micrograph of a free-hand cross section through a stage 4 stylar canal. arrow indicates the pollen tubes. x 206. b. aniline blue fluorescent micrograph of a free-hand cross section through the junction between the stylar canal and upper ovary at stage 5. arrow indicates the connecting conduit. x 206. 10 merrit noormets, a. randall olson fig. 4: v. angustifolium. a. sem of anatropous ovules at stage 4. x 1270. b. sem of ovule and papillated placental surface at stage 4. arrow indicates the micropylar opening near the placental surface. exudate is removed during the chemical preparation. x 2408. c. sem of the ovules and pollen tubes on a placental surface at stage 5. arrow indicates a tortous mat of pollen tubes. x 505. d. sem of the pollen tubes at stage 5 with arrow indicating collapsed pollen tubes. x 2535. pollen tube pathway in sweet lowbush blueberry 11 acknowledgements the authors thank ms. sandra fisk for help with manuscript preparation and dr. ping li from the faculty of science electron microscopy suite of dalhousie university. this research was supported in part by the canadian international development agency (cida) as part of the requirements for a doctoral degree at the estonian agricultural university. references bell, h.p., 1957: the development of the blueberry seed. can. j. bot. 35, 139-153. bell, h.p., burchill, j., 1955: flower development in the lowbush blueberry. can. j. bot. 33, 251-258. berlyn, g.p., miksche, j.p., 1976: botanical microtechnique and cytochemistry. iowa state university press, ames, ia. bronner, r., 1975: simultaneous demonstration of lipids and starch in plant tissues. stain technol. 50, 1-4. camp, w.h., 1942: on the structure of populations in the genus vaccinium. brittonia 4, 189-204. camp, w.h., 1945: the north american blueberries with notes on other groups of vaccinaceae. brittonia 5, 203-220. cronquist, a., 1981: an integrated system of classification of flowering plants. columbia university press, new york. dafni, a., 1992: pollination ecology – a practical approach. oxford university press, new york. fisher, d.b., 1968: protein staining of ribboned epon sections for light microscopy. histochemie 16, 92-96. hall, i.v., forsyth, f.r., lightfoot, h.j., boch, r., 1971: volatiles of lowbush blueberry nectar. hortscience 6, 493-494. herrero, j., hormanza, j.i., 1996: pistil strategies controlling pollen tube growth. sex plant reprod. 9,343-347. heslop-harrison, y., shivanna, k.r., 1977: the receptive surface of the angiosperm stigma. annals bot. 41,1233-1258. hildebrand, p.d., braun, p.g., 1991: factors affecting infection of lowbush blueberry by ascospores of monilinia vaccinii-corymbosi. can. j. plant pathol. 13, 232-240. jacquemart, a.l., thompson, j.d., 1996: floral and pollination biology of three sympatric vaccinium (ericaceae) species in the upper adrennes, belgium. can. j. bot. 74, 210-221. knox, r.b., 1984: pollen-pistil interactions. in: linskens, h.f., heslopharrison, j. 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<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> >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice journal of applied botany and food quality 92, 258 266 (2019), doi:10.5073/jabfq.2019.092.036 1botany department, faculty of science, suez canal university, ismailia, egypt 2biology department, college of science, jouf university, saudi arabia 3biology department, faculty of science, taibah university, al‑sharm, yanbu, saudi arabia 4botany department, faculty of agriculture, suez canal university, ismailia, egypt 5botany and microbiology department, faculty of science, cairo university, giza, egypt 6department of agriculture, university of swabi, khyber pakhtunkhwa, pakistan calcium availability regulates antioxidant system, physio-biochemical activities and alleviates salinity stress mediated oxidative damage in soybean seedlings amr a. elkelish1, taghreed s. alnusaire2, mona h. soliman3*, salah gowayed4, hoda h. senousy5, shah fahad6 (submitted: april 17, 2019; accepted: august 16, 2019) * corresponding author summary salinity is considered as one of the devastating abiotic stress fac‑ tors and global climate change has further worsened the situation. present experiments were aimed to evaluate the role of calcium (ca) availability on growth and salinity tolerance mechanisms in soybean. seedlings were grown with (2 mm ca) and without ca supplemen‑ tation and modulation in key physiological and biochemical para‑ meters were studied. salinity (100 mm nacl) stress resulted in growth reduction in terms of height and biomass accumulation, which was more pronounced in ca-deficient plants. relative to control (ca defi‑ cient) and nacl stressed plants, ca supplemented seedlings exhibited higher relative water content, pigment synthesis and the photosyn‑ thetic efficiency. ca availability affected the synthesis of proline, glycine betaine and soluble sugars under normal and saline growth conditions. optimal ca supplementation up‑regulated the activities of antioxidant enzymes assayed and the contents of non‑enzymatic antioxidants (ascorbate, glutathione, and tocopherol) thereby reflect‑ ing in amelioration of nacl induced oxidative damage. moreover, in‑ creased accumulation of phenols due to ca supplementation and the amelioration of nacl mediated decline if nitrate reductase activity was observed. more importantly, ca availability reduced the accu‑ mulation of na under control and nacl stressed conditions restrict‑ ing the damging effects on metabolism. availability of optimal ca potentially regulates the salinity tolerance mechanisms in soybean by maintaining osmoregulation and antioxidant metabolism. keywords: calcium, antioxidant system, osmolytes, lipid peroxi‑ dation, glycine max l. introduction increasing salinity in soils has become one of the serious threats to distribution, survival, and productivity of major crop plants (ashraf and harris, 2004). increased salinity hampers key growth and meta‑ bolic processes including photosynthesis, ion transport their assimi‑ lation and distribution, enzyme activity and osmolyte metabolism (iqbal et al. 2015). salinity interferes with carbon and nitrogen me‑ tabolism in plants leading to alterations in the physiological processes responsible for growth and developmental regulation (ahanger and agarwal, 2017; elkelish et al., 2019). presence of high concen‑ tration of toxic ions like sodium in the soil hampers the uptake of important nutrients including nitrogen, sulfur, calcium, and potas‑ sium (reda et al., 2011). salinity stress imposes hyper‑osmotic and hyper‑ionic stress in plants leading to membrane disorganization and restriction of functioning in chloroplast and mitochondria. higher salinity declines the soil osmotic potential thereby prevent the uptake of water into the plant ultimately due to occurance of physiological drought often reflected as reduced growth and development (ozden et al., 2009). it has been reported that salinity stress increases the generation of toxic reactive oxygen species (ros) and plants have an antioxidant system comprised of enzymatic and non‑enzymatic components to neutralize ros and safeguard the cellular function‑ ing (noctor and foyer, 2016). increased generation of ros due to stress affects the macromole cules like proteins, nucleic acids etc. (ahanger and agarwal, 2017) rendering plant cells less efficient. it has been reported that salini ty generated ros reduce the rate of photosynthesis, metabolism of nitrogen and carbohydrates often reflected in reduced growth rate and yield (ahanger and agarwal, 2017; soliman et al., 2018). more importantly, salinity influences the expression of proteins regu‑ lating the structural and functional integrity of organelles like chlo‑ roplast and mitochondria (omidbakhshfard et al., 2012). therefore, for maximally averting the negative effects of salinity stress key mechanisms are initiated to reduce the impact on the growth and productivity of the plant. salt exclusion through channel proteins, up‑ regulation of ros scavenging system and accumulation of osmolytes are included among the basic mechanisms leading to enhanced pro‑ tection to structure and function of cellular organelles by regulating ros and stress signaling (suo et al., 2017). therefore, strengthening the tolerance mechanisms and understanding their implications on the overall growth performance is requisite for achieving sustainable productivity. like other beneficial nutrients, interaction between available cal‑ cium (ca) with the salinity plays an important role in initiating and strengthening of the tolerance mechanism. salinity reduces uptake of ca inducing oxidative damage through greater accumulation of ros. ca deficiency limits root growth, induces leaf necrosis and curling, affects cellular signaling and can result in blossom end rot, bitter pit and fruit cracking, affect cellular signaling and sensing of other mineral elements like potassium (wang et al., 2018). once na+ is taken up in excess, leakage of k+ and ca2+ follows resulting in im‑ balance of the cellular ion homeostasis ultimately causing oxidative stress, growth retardation and programmed cell death (cabot et al., 2014). the role of optimal ca availability in growth regulation has not been worked so far. we hypothesized that availability of opti‑ mal ca can ameliorate the deleterious effects of salinity stress by strengthening the key tolerance mechanisms. therefore, experiments were conducted using one of the important legume crop, soybean, to investigate the role of ca availability in salinity stress mitigation by evaluating its involvement in the (a) regulation of the antioxidant system and osmolytes, (b) secondary metabolite accumulation and the (c) mineral nutrition. calcium availability alleviates salinity stress‑mediated oxidative damage in soybean seedlings 259 materials and methods experimental design and treatment a greenhouse experiment was conducted at the department of botany of the faculty of science, suez canal university, ismailia, egypt, during november and december 2018. soybean (glycine max l.) seeds were sterilized with 3% sodium hypochlorite and sown in earthen pots filled with an equal quantity of reconstituted soil con‑ taining peat, compost, and sand in the ratio of 3:1:1. concentration of available n, p, k and ca in soil substrate was 53.21, 20.03, 56.28 and 17.33 mg kg‑1 soil respectively. pots were wetted by applying 200 ml of full‑strength nutrient solution. soon after the germination num‑ ber of seedlings per pot was maintained five. pots were grouped into two categories where one group was irrigated with nutrient solution (100 ml per pot) with ca (in the form of 2 mm ca (no3)2) and an‑ other group without ca. 100 mm nacl was supplemented in the form of modified hoagland solution to induce salinity stress. the compo‑ sition of nutrient solution used was 3 mm kno3, 2 mm ca(no3)2, 2 mm mgso4, 1 mm nh4h3po4, 50 μm kcl, 25 μm h3bo4, 2 μm mncl2, 20 μm znso4, 0.5 μm cuso4, 0.5 μm (nh4)6mo7o24, and 20 μm na2fe‑edta (per et al., 2016). therefore, we have following set of treatments: (a) control (hoagland solution without ca). (b) ca (full strength hoagland solution). (c) salt stressed (hoagland solution without ca + 100 nacl). (d) salt stress + ca (full strength hoagland solution + 100 nacl). plants were irrigated on every alternate day. thirty days old plants were harvested and analyzed for photosynthetic attributes, osmo‑ regulatory components, oxidative stress markers, and antioxidants. during the entire experimental time, period pots were kept in a greenhouse and arranged in a complete randomized block design with five replicates for each treatment. measurement of photosynthetic pigments, stomatal conductance, and photosynthetic efficiency for extraction of chlorophyll and carotenoids, fresh leaf tissue was homogenized in pestle and mortar using 80% acetone (arnon, 1949). the homogenate was centrifuged, and an optical density of supernatant was taken at 480, 645 and 663 nm against 80% acetone. for measurement of photosynthetic efficiency and stomatal conduc‑ tance, fully expanded leaf was analyzed using the infrared gas ana‑ lyzer (cid‑340, photosynthesis system, bio‑science, usa). determination of leaf water content, soluble sugars, proline, and glycine betaine content relative water content (rwc) of leaves was determined by punching an equal number of leaf discs from each treatment with a sharp cork borer, and their fresh weight (fw) was taken. thereafter the same leaf discs were floated in petri dishes containing distilled water for 1 hr for measuring the turgid weight (tw). the same discs were oven dried at 80 oc for 24 hrs for the dry weight (dw) measurement (smart and bingham, 1974). rwc was calculated by the following formula: a method described by bates et al. (1973) was used to estimate the proline content. dry (500 mg) leaf sample was homogenized in 3% sulphosalicylic acid and subjected to centrifugation for 20 minutes at 3000 g. the supernatant (2 ml) was mixed with 2 ml glacial acetic acid and 2 ml ninhydrin reagent at 100 oc in a water bath for 1 hour. after that samples were cooled on an ice bath and proline was sepa‑ rated using toluene. absorbance was read spectrophotometrically at 520 nm. calculation was done from standard curve of proline. for measurement of soluble sugar, content oven dried samples were macerated in 80% ethanol and after centrifugation supernatant was reacted with anthrone reagent following method of shields and burnett (1960). calculation was done from standard curve of glu‑ cose. for the determination of glycine betaine (gb) method of grieve and grattan (1983) was followed. dried plant samples were extracted in distilled water and filtered. the filtrate was diluted by h2so4 (2 n) and cold ki‑i2 reagent was added to an appropriate volume of the aliquot. samples were centrifuged at 10,000 g for 15 minutes. the supernatant was aspirated, and per‑iodide crystals were dissolved in 1,2‑dichloroethane and after two hours absorbance was taken at 365 nm. the calculation was done using a standard of glycine betaine. lipid peroxidation and hydrogen peroxide estimation for the measurement of lipid peroxidation, the formation of malon‑ aldehyde (mda) content was estimated. fresh leaves were homo‑ genized in 1% trichloroacetic acid (tca) and the homogenate was centrifuged at 10,000 g for 5 min. thereafter, 1.0 ml supernatant was mixed with 4 ml 0.5% thiobarbituric acid and mixture was boiled for thirty minutes at 95 oc. tubes were cooled on an ice bath and centrifuged again for 5 min at 5000 g for clarification. the opti‑ cal density of supernatant was measured at 532 and 600 nm (heath and packer, 1968). hydrogen peroxide was estimated by macerating 0.5 gm fresh leaf tissue in 5 ml of 0.1% tca and the homogenate was centrifuged for 15 minutes at 12,000 g. supernatant (0.5 ml) was mixed with 0.5 ml potassium phosphate buffer (ph 7.0) and potassium iodide (1 ml). tubes were thoroughly shaken by vortexing and the absorbance was taken at 390 nm (velikova et al., 2000). determination of nitrate reductase fresh 500 mg tissue was extracted in ice‑cold mortar using 100 mm potassium phosphate buffer (ph 7.5) containing 5 mm cysteine, 1 mm edta and 0.5% pvp. the homogenate was centrifuged at 15,000 g for 20 min and the supernatant was used for the deter‑ mination of nr activity. the reaction mixture consisted 100 mm potassium phosphate buffer (ph 7.5), 7 mm kno3, 10 mm mgcl2, 0.14 mm nadh and the enzyme extract. the reaction was initi‑ ated by addition of nadh and after incubation for 30 minutes at 27 oc, the reaction was terminated by addition of 100 μl zinc ace tate (0.5m) followed by centrifugation for 10 minutes at 3000 g and subsequent addition of 1% sulfanilamide and 0.01% naphthalene‑ di‑amine‑dihydrochloride (ned). after 20 minutes the absorbance was read at 540 nm (debouba et al., 2006). assay of antioxidant enzymes antioxidant enzymes were extracted by homogenizing 5.0 gm of fresh leaf tissue in pre‑chilled pestle and mortar in phosphate buffer (50 mm, ph 7.0) supplemented with 1% polyvinyl pyrrolidone and 1 mm edta. the homogenate centrifuged at 15,000 g for 20 min at 4 oc and supernatant was used as an enzyme source. protein content in the supernatant was determined by lowry et al. (1951) method. the assay of superoxide dismutase (sod, ec 1.15.1.1) was carried by measuring the ability of enzyme extract to inhibit the photochemical reduction of nitroblue tetrazolium chloride (nbt) at 560 nm (beyer and fridovich, 1987). the reaction mixture contained 50 mm sodi‑ um phosphate buffer (ph 7.5), 0.1 μm edta, l-methionine (13 mm), 75 μm of each nbt and riboflavin, and 100 μl enzyme extract in a final volume of 1.5 ml. samples were shaken and illuminated for 15 min and the reaction was terminated by switching off the light. the absorbance of the enzyme containing illuminated samples was recorded against their respective non‑illuminated blank at 560 nm. fw ‑ dw rwc × 100 tw ‑ dw 260 a.a. elkelish, t.s. alnusaire, m.h. soliman, s. gowayed, h.h. senousy, s. fahd the amount of enzyme causing 50% inhibition in nbt photoreduc‑ tion was considered as one unit of sod and activity was expressed as eu mg‑1 protein. for measuring the activity of ascorbate peroxidase (apx, ec 1.11.1.11) decrease in absorbance was monitored at 290 nm for 3 min in a reaction mixture containing 50 mm potassium phosphate buffer (ph 7.0), ascorbic acid (0.5 mm), hydrogen peroxide (0.1 mm) and 100 μl of enzyme extract in final volume of 1 ml (nakano and asada, 1981). the extinction coefficient of 2.8 mm‑1 cm‑1 was used for calculation. for the assay of glutathione reductase (gr; ec 1.6.4.2) method of foyer and halliwell (1976) was adopted. briefly, assay mix‑ ture (1.0 ml) contained sodium phosphate buffer (50 mm, ph 7.8), 0.12 mm nicotinamide adenine dinucleotide phosphate (nadph), 0.5 mm oxidized glutathione (gssg) and 100 μl enzyme extract. change in absorbance at 340 nm was followed for 2 min. activity was expressed as l mol nadph oxidized min‑1 (units mg‑1 protein) and extinction coefficient for of 6.2 mm‑1 cm‑1 was used for calcula‑ tion. determination of ascorbate, tocopherol and reduced glutathione for the determination of ascorbate (asa) content 400 mg fresh plant material of each treatment was extracted in 5 ml of 6% tca fol‑ lowed by centrifugation at 5000 g for 10 min. to 1 ml supernatant was added 2% dinitrophenylhydrazine (prepared in 9 m h2so4) and 10% thiourea. the mixture was boiled in a water bath for 15 min and cooled followed by the addition of 5 ml of cooled 80% h2so4. thereafter the optical density was measured at 530 nm (mukherjee and choudhuri, 1983) and calculation was done from a standard curve of ascorbate. the method described by (baker et al., 1980) was followed for the estimation of tocopherol. 100 mg fresh leaf material was extracted with 5 ml of ethanol and petroleum ether (1.6:2) and an extract was centrifuged at 12,000 g for 10 minutes. 1.0 ml supernatant was mixed with 0.2 ml of 2, 2‑dipyridyl (2%, prepared in ethanol) and left for 5 min in dark for color (red) development. thereafter 4 ml of distilled water was added and the absorbance was measured at 520 nm. reduced glutathione (gsh) was estimated by extracting fresh 100 mg leaf tissue in phosphate buffer (ph 8.0) and an extract was centri‑ fuged for 15 minutes at 3000 g. thereafter 500 μl of supernatant was reacted with 5, 5‑dithiobis‑2‑nitrobenzoic acid and allowed to stand for 10 minutes. optical density was measured at 412 nm (ellman, 1959) and the concentration of gsh was determined from a standard graph of gsh. estimation of total phenols dry (500 mg) plant samples were extracted in 80% ethanol and an extract was centrifuged at 10,000 g for 10 minutes. the supernatant was mixed with folin and ciocalteau’s phenol reagent in alkaline medium and optical density of the mixture was recorded at 750 nm (slinkard and singleton, 1977). the computation was done from the standard curve of catechin. estimation of ions for estimation of na and ca, one gm oven dried samples were acid digested using h2so4 and hclo4. digested samples were diluted to 100 ml using distilled water and were read on flame photometer. statistical analysis data are mean (±se) of three replicates and duncan’s multiple range test was performed using one way anova for determining the least significant difference (lsd) at p <0.05. results ca supplementation protects plant growth and biomass production under salinity stress results showing the impact of ca availability on the height and biomass accumulation under salinity and normal conditions are pre‑ sented in fig. 1. relative to control, ca improved height, fresh and dry weight by 19.30, 19.19 and 19.58% respectively. salinity (100 mm nacl) reduced the height (37.67%), fresh (25.79%), and dry (38.81) biomass accumulation over the control plants. application of ca sig‑ nificantly reduced the negative effects of nacl and ameliorated the decline by 17.90, 12.78 and 9.23% over the nacl stressed counter‑ parts (fig. 1). fig. 1: effect of salinity (100 mm nacl) on (a) shoot length, (b) fresh and (c) dry weight of soybean (glycine max l.) with and without cal‑ cium supplementation. data is mean (±se) of three replicates and bars with different letters denote significant difference at p ≤ 0.05 ca supplementation improves photosynthetic efficiency and the pigment synthesis supplementation of ca resulted in significant enhancement in the pig ment synthesis (chlorophylls and carotenoids) and also ameliorated the nacl induced decline. relative to control, total chlorophylls, ca‑ rotenoids, photosynthetic rate, and stomatal conductance increased by 19.97, 17.25, 20.15 and 14.44% in ca treated seedlings. however, nacl stress reduced chlorophylls (45.18%), carotenoids (30.72%), photosynthetic rate (41.88%) and stomatal conductance (35.98%) over the control plants. the reduction was mitigated by supplemen‑ tation of ca with an amelioration of 29.64% in total chlorophylls, calcium availability alleviates salinity stress‑mediated oxidative damage in soybean seedlings 261 14.10% in carotenoids, 22.33% photosynthetic rate and 15.54% in stomatal conductance over the nacl stressed plants (fig. 2a‑d). ca availability improves rwc, and proline, glycine betaine and sugar accumulation under salinity stress in normally grown seedlings, ca increased the accumulation of pro‑ line (pro), glycine betaine (gb) and soluble sugar content significant‑ ly over the ca deficient control plants. an increment of 22.63% in pro,14.28% in gb and 26.53% sugar content was observed over the control plants. percent increase in pro, gb, and sugars due to nacl stress was 31.01, 28.96, and 37.43% respectively over the control. the maximal increase in pro (38.39%), gb (39.49%) and sugar (42.14%) content was observed in nacl + ca treated plants over the control counterparts (fig. 3a‑c). a reduction of 27.85% due to salinity was observed in rwc over the control seedlings, however, ca was effec‑ tive in improving and also mitigating the negative effects of nacl on rwc. an increase of 7.16% in rwc was observed due to ca over control and caused an amelioration of 14.57% in seedlings treated with nacl + ca over the nacl stressed plants (fig. 3d). fig. 3: effect of salinity stress (100 mm nacl) on content of (a) proline, (b) glycine betaine, (c) soluble sugars and (d) relative water content (rwc) in soybean (glycine max l.) with and without calcium supplementation. data is mean (±se) of three replicates and bars with different letters denote significant difference at p ≤ 0.05 fig. 2: effect of salinity stress (100 mm nacl) on (a) total chlorophylls, (b) carotenoids, (c) photosynthetic rate and (d) stomatal conductance in soybean (glycine max l.) with and without calcium supplementation. data is mean (±se) of three replicates and bars with different letters denote significant difference at p ≤ 0.05 262 a.a. elkelish, t.s. alnusaire, m.h. soliman, s. gowayed, h.h. senousy, s. fahd antioxidant system was up-regulated due to ca supplementation supplementation of ca resulted in a significant increase in the ac‑ tivities of sod, apx, and gr, and the contents of asa, gsh, and tocopherol (fig. 5 and 6). relative to control, an increase of 21.37% in sod, 19.73% in apx and 9.46% in gr were observed due to supplementation of ca. nacl stress increased the activities of sod (36.54%), apx (40.00%) and gr (33.40%) over the control, however application of ca further enhanced the activities. relative to con‑ trol, an increase of 44.98, 45.37 and 39.26% in sod, apx and gr were observed due to an application of ca to nacl stressed plants (fig. 5a‑c). contents of asa, gsh, and tocopherol increased by 8.89, 9.91 and 21.97% in ca treated plants over the ca deficient con‑ trols (fig. 6a‑c). the maximal increase in gsh (30.06%) and toco‑ pherol (35.36%) was observed in seedlings treated with nacl + ca over the control. salinity reduced asa (15.57%) while as increased gsh (26.55%) and tocopherol (14.48%) over the control. reduction in asa was ameliorated by application of ca and an amelioration of 4.67% in asa content was observed in nacl + ca treated seedlings over the nacl stressed plants (fig. 6a). ca reduces oxidative damage by reducing the formation of hydrogen peroxide and lipid peroxidation it was observed that ca supplementation reduced the generation of h2o2 and lipid peroxidation significantly over the control as well as nacl stressed plants (fig. 4 a and b). accumulation of h2o2 in‑ creased by 37.98% in nacl stressed plants causing 25.15% increase in lipid peroxidation over the control plants. supplementation of op‑ timal ca reduced the generation of h2o2 by 23.29% over the control leading to a declined lipid peroxidation by 13.38% in them. in com‑ parison to nacl stressed plants amelioration of 17.56% in h2o2 and 11.59% in lipid peroxidation was observed in seedlings treated with nacl + ca (fig. 4 a and b). ca improves the synthesis of total phenols and enhances nitrate reductase activity ca supplemented seedlings exhibited an apparent increase in the ac‑ cumulation of total phenols over the ca deficient normal and salinity stressed plants. relative to control, ca supplemented plants showed an increase of 22.39% in total phenol contents which was reduced by 18.33 % due to nacl stress (fig. 7). relative to control, treatment of 100 mm nacl reduced the activity of nitrate reductase (nr) by 43.25%. however, an amelioration of 11.96% was observed in nacl + ca over the nacl stressed plants. under normal growth conditions, nr activity increased (11.89%) significantly due to ca availability (fig. 8). ca availability reduces na uptake and reduces the na/k ratio supplementation of ca reduced the uptake of na to upper parts (leaf) significantly (tab. 1). relative to ca deficient control, ca decreased by 35.13% in leaf and 31.75% in root due to nacl stress, however, accumulated na in leaf (68.54%) and root (65.98%) more than the control. relative to control, ca supplemented seedlings exhibited an increase in the leaf ca (24.38%), while a decrease in na content of leaf (28.53%) and root (13.15%). however, in nacl stressed plants ca application reduced the na accumulation by 42.26% and 32.65% in leaf and root tissues (tab. 1). discussion increased soil salinity is considered a critical threat to optimal crop productivity. such problems have arisen mainly due to the introduc‑ tion of agricultural malpractices like the use of salt‑rich and polluted fig. 5: effect of salinity stress (100 mm nacl) on activity of (a) superoxide dismutase (b) ascorbate peroxidase and (c) glutathione reductase in soybean (glycine max l.) with and without calcium supplementa‑ tion. data is mean (±se) of three replicates and bars with different letters denote significant difference at p ≤ 0.05 fig. 4: effect of salinity stress (100 mm nacl) on (a) hydrogen peroxide and (b) lipid peroxidation (mda) in soybean (glycine max l.) with and without calcium supplementation. data is mean (±se) of three replicates and bars with different letters denote significant difference at p ≤ 0.05 calcium availability alleviates salinity stress‑mediated oxidative damage in soybean seedlings 263 water for irrigations, which has resulted in continuous accumulation of salts to toxic levels making the growth and development of grow‑ ing crops difficult. for ensuring better crop productivity under such growth conditions, efficient and robust management practices need to be introduced. in this context, we investigated the potential of soil ca availability in mitigating the adverse effects of salinity. the indigenous concentration of ca in nutrient solution proved ef‑ fective in ameliorating the nacl induced growth decline. nacl re‑ duces the cellular growth and division by reducing the activity of cyclin‑dependent kinase and cycb1;2 promoters resulting in the production of smaller meristems (west, 2004). salinity stress‑medi‑ ated decline in meristem is due to the reduced cellular water content (mahmoodzadeh et al., 2007). in the present study, ca application effectively regulated the growth of soybean by improving biomass production and growth under salinity stress. ca is important for cell cycle progression under stress and is an important secondary mes‑ senger. the role of ca2+ mediated signaling in cell cycle progres‑ sion has been established (machaca, 2010) availability of optimal ca concentration in the present study may have prevented cell cycle arrest besides regulating the cell proliferation (kahl and means, 2003). as a signaling molecule ca ion acts as a convergence point of many signaling pathways thereby helping plant cells to repro‑ gram their cellular set up in response to stress signal (tuteja and mahajan, 2007). ca helps in stress signal perception by membrane receptors, secondary messenger generation, protein phosphorylation and dephosphorylation targeting transcription factors resulting in regulation of gene expression ultimately imparting tolerance to stress (tuteja and mahajan, 2007). presence of optimal ca in solution resulted in significant improve‑ ment in the synthesis of chlorophylls and carotenoids resulting in greater photosynthetic efficiency and stomatal conductance. earlier xu et al. (2013) have also demonstrated that the exogenous appli‑ cation of cacl2 ameliorated the negative effects of drought stress on growth by improving the chlorophyll pigment synthesis and the chlorophyll fluorescence and photosynthetic rate. ca deficiency in‑ tensified the reduction in photosynthetic pigment and performance fig. 8: effect of salinity stress (100 mm nacl) on content the activity of nitrate reductase in soybean (glycine max l.) with and without cal‑ cium supplementation. data is mean (±se) of three replicates and bars with different letters denote significant difference at p ≤ 0.05 fig. 6: effect of salinity stress (100 mm nacl) on content of (a) ascorbic acid, (b) reduced glutathione and (c) tocopherol in soybean (glycine max l.) with and without calcium supplementation. data is mean (±se) of three replicates and bars with different letters denote significant difference at p ≤ 0.05 fig. 7: effect of salinity stress (100 mm nacl) on content of total phenols in soybean (glycine max l.) with and without calcium supplementa‑ tion. data is mean (±se) of three replicates and bars with different letters denote significant difference at p ≤ 0.05 264 a.a. elkelish, t.s. alnusaire, m.h. soliman, s. gowayed, h.h. senousy, s. fahd under salinity conditions. reduced photosynthetic efficiency due to salinity stress results from the increased chlorophyll degradation re‑ flected in increased chlorophyllase activity and reduced synthesis of chlorophyll intermediates. also, reduced photosynthetic efficiency due to salinity stress results from ros induced photoinhibition pri‑ marily through damage to the structure of the pigment‑protein com‑ plex (ahanger et al., 2017). optimal supplementation of ca has ade‑ quately protected the pigment proteins and also have a visible impact on the allocation of key mineral ions like mg and n for chlorophyll and rubisco synthesis. further research is needed to unravel the actual mechanism. ca2+ regulates photosynthetic electron transport and light‑dependent me‑ tabolism through interplay with thylakoid acidification and redox homeostasis. reduced nr activity due to salinity has been reported by others as well in crops like triticum aestivum (ahanger and agarwal, 2017) and vigna radiata (shahi and srivastava, 2018), however, ca mediated regulation of nr activity under salinity has not been worked. ca considerably ameliorated the salinity mediated decline in nr activity depicting its beneficial role in the protection of enzyme activity. ca‑mediated growth and photosynthetic improve‑ ment can be attributed to reduced na uptake in them. na is toxic to plants at higher concentrations and optimal ca supplementation mediated decline in its accumulation directly shows its influence on the transport proteins controlling the uptake and the partitioning of na within the cells mainly na+/ca2+ exchanger (dreval et al., 2005). salinity affects the transport and homeostasis of ca in plants by inhibiting ca2+‑atpase (geisler et al., 2000). also, ca availabil‑ ity may have changed the expression of other membrane proteins like tonoplast h+‑atpase and na+/h+ antiport reducing the accumulation of excess levels of na+. increased presence of ca in cells is recog‑ nized by ca sensors or ca2+ binding proteins and leads to activation of protein kinases (tuteja and mahajan, 2007). increased nacl concentration in soil solution reduces the tissue growth by affecting the water content significantly and in confirma‑ tion to our results are the findings of (polash et al., 2018). higher accumulation of sugars, pro and gb in ca supplemented seedlings resulted in enhanced rwc and also ameliorating the ill effects of sa‑ linity on cellular functioning. calcium regulates the growth of plants by affecting mineral transport and accumulation of metabolites leading to the maintenance of water content (mozafari, 2013). ca‑ mediated enhancement in the accumulation of pro, sugars, and gb resulted in higher rwc preventing the decline in photosynthesis and the hyper‑osmotic effects of salinity on the protein structure (munns and tester, 2008). benhassaini et al. (2012) have also demon‑ strated salinity mediated enhancement in pro in pistacia atlantica. pro and gb have a role in protecting the protein turnover machinery and improving the expression of stress proteins for greater tolerance. greater accumulation of compatible osmolytes like pro, gb, sug‑ ars improves plant performance under stressful conditions through increased rwc. increased gb accumulation in ca supplemented plants may have prevented salt mediated photo‑inhibition by protect‑ ing the thylakoid membrane structure and the atp synthesis lead‑ ing to greater photosynthetic efficiency in them (wang et al., 2018). sivakumar et al (2000) has demonstrated increased carboxylase activity of rubisco in plants accumulating higher contents of osmo‑ lytes. pro and gb prevent the salinity mediated ros initiated lipid peroxidation, deformation, and degradation of a nucleus, chromatin condensation and pcd. ca supplementation enhanced the synthesis of pro, sugars, and gb resulting in enhanced rwc, photosynthetic efficiency, and amelioration of oxidative damage. the osmolytes have been believed to strengthen the ros scavenging systems and stress signaling mechanisms (ahanger et al., 2018). salinity stress increased the generation of ros like h2o2 causing peroxidation of membranes, however, ca supplementation reduced the generation of h2o2 under normal conditions and also declined the nacl induced ros levels. recently, ahanger et al. (2018) have also demonstrated reduced membrane stability due to salinity medi‑ ated accumulation of h2o2. stresses affect the polyunsaturated fatty acids significantly leading to greater membrane instability. the re‑ duction in h2o2 and mediated by ca have been previously reported to impart a positive impact on the photosynthetic efficiency of plants (sakhonwasee and phingkasan, 2017). optimal supplementation of ca may have reduced the activity of lipoxygenase leading to pre‑ vention of any damage to membrane fatty acids besides its appar‑ ent effect on the efficiency of the antioxidant system. in vicia faba, siddiqui et al. (2018) have also demonstrated up‑regulation of the antioxidant system due to ca supplementation under cadmium stress. in the present study, it was observed that ca up‑regulated antioxi‑ dant defense system by up-regulating sod, apx and gr concomi‑ tant with an increase in the content of gsh, tocopherols, and asa. strengthening the antioxidant system prevents cellular oxidative damage by quick elimination of ros. ca-induced enhancement in an antioxidant system precludes any damage to chloroplast and mi‑ tochondrial electron transport chain by maintaining optimal h2o2 levels so that their beneficial functions continue (mittler, 2017). in‑ creased sod activity in ca supplemented seedlings prevent hydroxyl (oh‑) radical formation resulting in enhanced cellular functioning. earlier ahmad et al. (2015) have demonstrated up‑regulation of the antioxidant system in mustard due to calcium supplementation. in‑ creased apx and gr activity in ca supplemented plants and main‑ tenance of higher concentrations of non‑enzymatic components lead to protection of cells from the oxidative burst through the support of cellular redox state. antioxidants including low molecular weight antioxidants like asa, gsh, and tocopherol are rich redox buffers, which interact with numerous cellular components besides other vi‑ tal roles in defense and as enzyme cofactors influencing the growth and development of plants by modulating processes from mitosis and cell elongation to senescence and death (potters et al., 2004). ca supplementation strengthens the evidence that redox homeosta‑ sis involves precise ros antioxidant interaction as an interface for signals derived from the metabolism and the environment (foyer and noctor, 2005). the antioxidant system was further strength‑ ened by the accumulation of phenols in ca supplemented seedlings. therefore, it is inferred from the present study that ca availability potentially affects the growth and tolerance mechanisms in soybean and could be exploited for enhancing the yield productivity. tab. 1: effect of salinity stress (100 mm nacl) on the uptake of sodium and calcium (mg g‑1 dw) in soybean (glycine max l.) with and without calcium supplementation. data is mean (±se) of three replicates and data followed by different letters denote significant difference at p ≤ 0.05 na ca leaf root leaf root control 3.866 ± 0.132c 4.493 ± 0.469c 5.908 ± 0.646b 3.420 ± 0.287b nacl 12.29 ± 1.119a 13.209 ± 0.339a 3.832 ± 0.139d 2.334 ± 0.141d ca 2.763 ± 195d 3.902 ± 0.105cd 7.813 ± 0.331a 4.990 ± 0.200a nacl + ca 7.096 ± 0.193b 8.895 ± 0.573b 4.986 ± 0.126c 3.196 ± 0.176bc calcium availability alleviates salinity stress‑mediated oxidative damage in soybean seedlings 265 conclusion it can be said that application of optimal ca prevents the structural and functional integrity of soybean to counteract the salinity stress mediated growth restrictions. optimal ca availability benefited soy‑ bean under salinity stress by restricting the uptake of sodium and in‑ creasing the synthesis of photosynthetic pigments and photosynthetic functioning. ca supplementation strengthened the indigenous tole‑ rance mechanisms like the synthesis of compatible solutes and up‑ regulation of antioxidant system for preventing damage to cellular macromolecules. nacl-mediated ros-generated oxidative damage effects were averted by ca availability by improving the redox com‑ ponents. by up‑regulating the antioxidant system and the synthesis of osmolytes ca availability can be exploited for improving the crop productivity. further studies for unraveling the molecular mecha‑ nisms and the probable crosstalk of ca with other key molecules in salinity stress mitigation can be handy. references ahanger, m.a., agarwal, r.m., 2017a: salinity stress induced alterations in antioxidant metabolism and nitrogen assimilation in wheat (triticum aestivum l) as influenced by potassium supplementation. plant phys. biochem. 115, 449‑460. doi: 10.1016/j.plaphy.2017.04.017 ahanger, m.a., alyemeni, m.n., wijaya, l., alamri, s.a., alam, p., ashraf, m., ahmad, p., 2018: potential of exogenously sourced kinetin in protecting solanum lycopersicum from nacl‑induced oxidative stress through up‑regulation of the antioxidant system, ascorbate‑glutathione cycle and glyoxalase system. plos one, 13/9, e0202175. doi: 10.1371/journal.pone.0202175 ahanger, m.a., tomar, n.s., tittal, m., argal, s., agarwal, r.m., 2017: plant growth under water/salt stress: ros production; antioxidants and significance of added potassium under such conditions. physiol. mol. biol. pla. 23/4, 731‑744. doi: 10.1007/s12298‑017‑0462‑7 ahmad, p., sarwat, m., bhat, n.a., wani, m.r., kazi, a.g., tran, l.-s.p., 2015: alleviation of cadmium toxicity in brassica juncea l. 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x., zhang, l., 2013: the effect of calcium chloride on growth, photosynthesis, and antioxidant responses of zoysia japonica under drought conditions. plos one, 8/7: e68214. doi: 10.1371/journal.pone.0068214 orcid amr elkelish https://orcid.org/0000‑0002‑1683‑8436 mona soliman https://orcid.org/0000‑0002‑9102‑4790 address of the corresponding author: mona h. soliman: biology department, faculty of science, taibah univer‑ sity, al‑sharm, yanbu el‑bahr, 46429, kingdom of saudi arabia e‑mail: monahsh1@gmail.com © the author(s) 2019. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creative‑ commons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1007/s11099-016-0205-y http://dx.doi.org/10.22271/tpr.2018.v5.i2.029 http://dx.doi.org/10.1104/pp.103.033548 https://doi.org/10.1016/j.plantsci.2011.02.006 http://dx.doi.org/10.1007/s13580-017-0194-1 http://dx.doi.org/10.1080/01904167.2018.1454470 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different tomato (lycopersicon esculentum mill.) cultivars under defined heat stress (hst) conditions were investigated. plants were grown under two day/night temperature regimes (26/20 ºc and 37/27 ºc, respectively) in growth chambers at the department of vegetable crops, institute for horticultural sciences, faculty of agriculture and horticulture, humboldt university of berlin. the experiments were conducted twice and were set up in a randomized design with five replicates. the reproductive processes in tomato were more sensitive to high temperatures than the vegetative ones. the number of pollen grains, number of fruits and fruit fresh masses produced by the heat tolerant cultivars were higher than those of the heat sensitive cultivars. however, hs pretreatments had no positive effects on tomato growth and development. introduction tomato (lycopersicon esculentum mill.) is usually produced during winter in sudan. it is grown throughout the country where irrigation water and arable land are available and is mainly grown by small holders who use relatively poor crop management practices. heat stress (hst) is one of the most important constraints on crop production that adversely affects the vegetative and reproductive processes of tomato and ultimately reduces yield and fruit quality (abdul-baki, 1991; gruda, 2005). plants respond to hst by changing their metabolic pathways to acclimatize to high temperature. under hst, synthesis of many proteins is repressed and some of them, which are called heat shock proteins (hsps), start to be synthesized (vierling, 1991). hsps synthesis is induced by a rapid rise in temperature of approximately 10 °c or more above the optimal growth temperature (nover and scharf, 1997). the authors reported that hsps plays a major role in mitigating the deleterious effects of heat-induced protein denaturation. moreover, physiological responses of plants to hst, such as the damage of structure and the disorder of physiological metabolism, have been documented (vierling, 1991; blum et al., 2001). although the damage and death of cells are caused by extreme hst, many plants can survive in otherwise lethal high-temperature regimes if they are first subjected to a pretreatment at non-lethal high temperatures (vierling, 1991). exposure of plants to elevated temperatures for short term, heat shock (hs), results in a complex set of gene expressions selective translation of mrna-encoding hs proteins, thereby enhancing thermotolerance and improving cellular survival to subsequent hst (nover et al., 1989; gong et al., 2001). heat shock can be used as alternative to chemical control of vegetable seeds diseases and in the post harvest to improve the quality of vegetables (loaiza-velarde and saltveit, 2001; loaiza-velarde et al., 1997). moreover, yarwood (1961) demonstrated that leaves subjected to high temperatures (50 °c) for short periods (15-30 s) tolerated high temperatures (55 °c) longer than untreated leaves. in addition, lin et al. (1984) reported that soybean seedlings exposed to 40 °c for 2 h produced hsps and tolerate temperature of 45 °c. however, plants transferred directly from 28 to 45 °c did not produce hsps. chen et al. (1982) mentioned that tomato leaf tissues of plants grown in temperature regimes below 30 °c were killed in about 15 min at 50 °c, while tomato plants increased significant tolerance when exposed to temperatures above 30 °c for 24 h. the results of the above researchers led to the assumption that hs treatments on tomato plants would be of benefit for tomato production under high temperature conditions. thus, this study was carried out to investigate whether or not any positive effects of hs on the vegetative growth and productive development in tomato plants cultivated under high temperature occur in order to mitigate the effect of hst conditions. on this basis, the production of tomatoes in arid tropic areas should be possible even during the summer. materials and methods two heat tolerant and one heat sensitive cultivars of different origin were selected for this study, namely: ‘drd85 f 1 ’, ‘kervic f 1 ’ and ‘uc 82-b’, respectively. the plants were grown in the greenhouse of the department of vegetable crops, institute for horticultural sciences, faculty of agriculture and horticulture, humboldt university of berlin (latitude 52° 30` n, longitude 13° 25` e). for more details concerning the plant cultivation, see abdelmageed et al. (2003). 35 days after sowing the transplants were subjected to hs treatments by immersing the shoot system in a hot-water bath at 50 °c for 30 s. another set from each cultivar was left as control (without hs treatment). thereafter, the plants were divided into two sets. one set was transferred in one plant growth chamber under normal temperature (nt), 26/20 °c for 13/11 h (day/night). another set was transferred in a second plant growth chamber under hst conditions, 37/27 °c for 13/11 h (day/night). during the day, 550 µmol m-2 s-1 irradiance from a combination of fluorescent and incandescent lights were provided for each set, on the top of the plants. temperature and relative humidity were continuously recorded using hygrothermographs (belfort instrument, baltimore, md). the experiments were conducted twice and were set up in a randomized design with five replicates. plants were rotated within the plant growth chamber every week to avoid any potential positional effects. the following parameters were recorded: leaf area (cm2), measured with an electronic leaf area meter, type li-cor model 3100 (lincoln, ne-usa), fresh and dry mass (g plant-1) of different plant parts, number of fruits and fruit fresh mass (g plant-1). number of pollen grains per flower was recorded according to sato et al. (2000) and * the results of this paper were partly presented at the international conference on tropical and subtropical agricultural research for development, „deutscher tropentag, 2003“, october 8-10, göttingen. aloni et al. (2001). leaf area ratio (lar), specific leaf area (sla), and leaf weight ratio (lwr) were calculated according to radford (1967). statistical analysis collected data were analysed using the statistical software spss version 10.0. one-way analysis of variance (anova) was used to determine the significance of variation among the different treatments. mean separation was done by duncan’s multiple range test. a combined analysis of variance was performed. the same conclusions were drawn from each experiment and the data are presented as mean values of 10 replicates across the two experiments. results systematic and consistent differences between the plants subjected or not subjected to hs pretreatment at both temperature regimes were noticed. however, no positive effects of hs pretreatment on tomato plants under both temperatures regimes were shown. leaf area was generally reduced for the plants that were subjected to hs pretreatment compared to that not pretreated (tab. 1). similar results were found for leaf fresh and dry mass as well as stem fresh and dry mass (data not shown). at both temperature regimes, there were significant differences among the cultivars when subjected or not subjected to hs pretreatment. ‘kervic f 1 ’ and ‘drd85 f 1 ’ showed the better results in all plant parameters measured. moreover, there was no significant difference in lar, sla and lwr (data not shown) among the different cultivars when the plants were subjected or not to hs pretreatment at both temperature regimes except for the cultivar ‘uc 82-b’ (tab. 1). numbers of pollen grains produced and released by the plants under nt regime were higher than that produced and released under hst conditions. however, among the cultivars at hst conditions there were no significant differences when the plants were not subjected to hs pretreatment. ‘kervic f 1 ’ had the highest number of pollen grains when the plants were subjected to hs pretreatment. at nt there were significant differences among the cultivars when not subjected to hs pretreatment, while ‘uc 82-b’ produced the lowest number of pollen grains per flower. the number of fruits per plant, and fruits fresh mass per plant showed the same trend as described above for the number of pollen grains (tab. 1). discussion high temperatures affected the vegetative and reproductive organs and tissues of tomato plants for all investigated cultivars. ‘kervic f 1 ’ and ‘drd85 f 1 ’ were more tolerant to high temperatures than ‘uc 82-b’. this confirms earlier findings of abdul-baki (1991) and peet et al. (1997) who reported the adverse effect of hst on the vegetative and reproductive development in tomato plants. the effect of hst on reproductive development was more pronounced than on vegetative growth. reduction in pollen production is an example of this in all cultivars at hst conditions. kuo et al. (1986) suggested as mechanism that proline accumulation in tomato leaf tissue at high temperature leads to the depletion in the reproductive tissue, thereby seriously reducing pollen formation or viability. in agreement with the results of sato et al. (2000) the number of fruits per plant and fruits fresh mass in tomatoes were also reduced at high temperature regimes (tab. 1). flower abortion and delay of growth of newly formed fruits acted as a feedback control mechanism to prevent too generative growth of tomatoes due to a high sinksource ratio, influenced by high air temperature (de koning, 1989; gruda, 2005). yarwood (1961) and lin et al. (1984) reported positive effects of heat shock treatments on the plants that later on exposed for a short period to higher temperature. heat shock response has been extensively studied in different plants (vierling, 1991). it has been known that plants induced thermotolerance and can survive under a normally lethal high temperature if they are preconditioned by mild heat shock treatment (hong and vierling, 2000). although heat shock response has been extensively studied in plants, most of the studies have focused on the response at the whole plant level. however, the heat shock affects the development of each plant organ differently. hong and vierling (2000) reported that seedling development during the very early stage shows stronger thermotolerance than the late stage. heat shock treatment in the present study had no positive effect on the vegetative growth and reproductive development and the hope that heat shock treatment would be beneficial for tomato plants, particularly for the reproductive development at high temperatures, was not fulfilled. on the other hand, this is in agreement with the results of abdul-baki (1991), who suggested that heat shock proteins have little to do with fruit set. frova et al. (1991) reported that relative to the heat shock response in vegetative tissues, the response in pollen is weak, the subset of hsps made are present in low amounts and mature pollen seems incapable of synthesizing hsps under high tab. 1: influence of heat shock pretreatment on some plant and physiological parameters of tomatoes grown under controlled conditions parameters leaf area (cm2) lar (cm2 g-1) sla (cm2 g-1) number of pollen number of fruits fruit fresh mass grains flower-1 plant-1 (g plant-1) treatment / temp. (°c) 37/27 26/20 37/27 26/20 37/27 26/20 37/27 26/20 37/27 26/20 37/27 26/20 kervic f 1 (cont.) 851.3 bc 1516.6 a 66.6 c 101.4 a 100.6 c 149.3 a 57.0 a 75.0 a 2.0 ab 7.0 a 14.5 a 46.3 a kervic f 1 838.9 bc 1126.6 b 92.7 b 79.1 bc 143.5 b 113.8 b 69.0 a 72.0 ab 2.5 a 7.5 a 17.8 a 50.0 a drd85 f 1 (cont.) 910.1 b 1077.5 b 74.5 c 72.3 c 118.3 c 108.7 b 61.0 a 68.0 b 1.8 ab 6.0 a 10.7 b 40.5 ab drd85 f 1 678.1 c 1046.9 b 63.1 c 86.3 abc 98.9 c 129.9 ab 60.0 a 74.0 ab 2.3 a 6.5 a 11.5 ab 37.5 ab uc82-b (cont.) 1266.2 a 1212.8 b 130.4 a 83.5 bc 171.7 a 111.7 b 37.0 c 57.0 c 0.0 b 6.5 a 0.0 b 22.0 c uc 82-b 862.8 bc 1238.9 b 90.7 b 92.4 ab 118.5 c 122.3 b 48.0 b 60.0 c 0.0 b 6.0 a 0.0 b 30.7 bc mean 901.2 1203.2 86.4 85.9 125.3 122.6 55.3 67.7 1.4 6.58 9.08 37.8 s.e. 35.68 31.17 5.03 2.75 5.95 4.04 2.05 1.38 0.21 0.18 1.36 1.87 cont. = control (without heat shock pretreatment), lar = leaf area ratio, sla = specific leaf area. means followed by the same letter(s) within each column are not significantly different at p < 0.05, according to duncan multiple range test. n = 10 plants influence of heat stress on tomatoes 27 temperature conditions. in addition, the plants in the present study were well irrigated. kimpel and key (1985) reported that hsps in soybean might accumulate under hot field conditions for plants subjected to drought but not for irrigated plants. under field conditions in sudan other factors, such as low relative humidity, insect and virus diseases as well as soil properties have to be considered as well. optimization of microclimate could be very important to ensure a good performance of new tolerant varieties cultivated during the summer in sudan. acknowledgments the german academic exchange services (daad) is acknowledged for providing a scholarship for the first author. references abdelmageed, a.h.a., gruda, n., geyer, b., 2003: effect of high temperature and heat shock on tomato (lycopersicon esculentum mill.) genotypes under controlled conditions. 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101-111. radford, p.j., 1967: growth analysis formulaetheir use and abuse. crop sci. 7, 171-175. sato, s., peet, m.m., thomas, j.f., 2000: physiological factors limit fruit set of tomato (lycopersicon esculentum mill.) under chronic, mild heat stress. plant cell environ. 23, 719-726. vierling, e., 1991: the roles of heat shock proteins in plants. annu. rev. plant physiol. plant mol. biol. 42, 579-620. yarwood, c.e., 1961: acquired tolerance of leaves to heat. science 134, 941-942. address of the authors: dr. adil h.a. abdelmageed, department of horticulture, faculty of agriculture, university of khartoum, 13314 shambat, p.o. box 32, khartoum north, sudan. email: aadilhassan@hotmail.com dr. habil. n. gruda, institute for horticultural sciences, faculty of agriculture and horticulture, humboldt university of berlin, d-14163 berlin, germany. email: nazim.gruda@rz.hu-berlin.de 28 a.h.a. abdelmageed, n. gruda << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true 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<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> >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice vorgabe neu journal of applied botany and food quality 80, 165 170 (2006) institute of fruit science, vegetable science and viticulture, university of hohenheim, germany changes of temperature exotherms and soluble sugars in grapevine (vitis vinifera l.) buds during winter radu badulescu, michael ernst (received july 5, 2006) summary nonstructural carbohydrates (nsc), including water soluble carbohydrates (wsc) are thought to serve an important role in freezing tolerance of many plants. raffinose family oligosaccharides (rfos) are α-galactosyl derivatives of sucrose. the most common rfos are the trisaccharide raffinose, the tetrasaccharide stachyose, and the pentasaccharide verbascose. rfos are nearly ubiquitous in the plant kingdom and are found in a large variety of seeds from many different families. severely cold winter temperatures can significantly impact grapevine productivity through tissue and organ destruction caused by freeze injury. crop loss and the need to retrain vines after bud, cane, and trunk injury mean financial loss, often for one or more years. buds of grapevine (vitis vinifera l.), grown at the vineyard of the institute of fruit science, vegetable science and viticulture, university of hohenheim, germany, were sampled during winter and analyzed for their concentration of soluble sugars (i.e. glucose, fructose, sucrose, raffinose and stachyose) and thermal analysis was performed to determine their freezing points. freezing of extracellular water was recorded from -5 to -16 °c with a minimum at the beginning of january; freezing of intra-cellular water was recorded from -11 to -24 °c. apical buds are very important organs as they determine further growth and development of tree species. bud physiological state, including saccharide metabolism, determines their growth activity. the concentration of soluble sugars was highest by the end of december. sugar concentrations in basal buds were significant higher than in buds from intermediate and apical shoot sections. a significant correlation could be proofed between sugar concentrations (i.e. raffinose and stachyose) and air temperature before sampling. but there was no correlation between freezing temperature of extra-cellular or intra-cellular water and soluble sugars in bud tissues. introduction plants have evolved a variety of mechanisms for resisting cold temperatures (a review is given by e.g. burke et al., 1976). temperature is expected to affect all plant processes. consistent with this, expression of a large number of specific mrnas and proteins is up-regulated during cold-acclimation. their possible functions are outlined, encompassing a wide range of processes, some possibly related to other winter stresses besides cold. some of the coldresponsive sequences are associated with constitutive or stress-related metabolism, others are probably protective, some may influence freezing, and many are of as yet unknown function (pearce, 1999). soluble carbohydrates are strongly correlated with freezing tolerance of plants. sucrose is often the main sugar associated with cold hardiness, but not for all species (gusta et al., 1996). fructans accumulate in grasses (smith, 1968) and raffinose is the main sugar in e.g. ajuga reptans (bachmann et al., 1994). sugars are known to protect cells and membranes (e.g. levitt, 1980) and soluble carbohydrates in the apoplast interfere with ice crystal growth and reduced mechanical injury associated with freezing (olien, 1967). the resistance of dormant grapevine buds against cold temperatures is based on supercooling (burke et al., 1976; andrews et al., 1984). the freezing temperature of supercooled tissues can be monitored by the detection of evolved (exothermic) heat caused by freezing. in grapevine buds two temperature exotherms are normally registered. the high-temperature exotherm (hte) indicates freezing of extracellular water and the low-temperature exotherm (lte) indicates freezing of intra-cellular water (wolf and cook, 1994). the exotherms showing up as temperature peaks during freezing of supercooled water are generated by the dissipation of latent heat. the peak area should thus correspond to the amount of freezing water, whereas the form of the peak is influenced by the degree of supercooling and by the freezing speed which depends on the structure of the tissue. the following investigations were conducted to characterize freezing events and changes in soluble carbohydrates of grapevine (vitis vinifera l.) buds during winter months. materials and methods plant material vitis vinifera l. (cv. bacchus) was grown at the vineyard of hohenheim university, germany. bud samples were collected from basal, intermediate, and apical sections of the shoots. sampling took place after natural defoliation at the following dates: 1998/11/12, 1998/ 11/26, 1998/12/10, 1998/12/23, 1999/01/07, 1999/01/21, 1999/02/ 25, 1999/03/11, 1999/03/25, 1999/04/08 and 1999/04/16. sampling was repeated in the following winter season (1999/2000). methods thermal analysis was performed by using an exotherm-measuringinstrument (kryoscan 4.0, manufactured by r. blaich, university of hohenheim, germany). the computerized system is capable of controlling the freezing rate and collecting, storing, and analyzing data to determine their freezing point using temperature exotherm analysis. the principle of this analysis is described by wample et al. (1990). with the multi-channel data registration 16 buds were measured simultaneously with a cooling rate of 8 °k/h and a temperature precision of 0.2 °k. temperature measurements on complete cold adapted latent buds during freezing usually revealed one or two high temperature exotherms (htes) at temperatures between -5 and -10°c and one to three low temperature exotherms at temperatures (ltes) down to -25°c, depending on the frost adaptation of the buds. dry matter (dm) content from buds and according shoot sections was determined. for carbohydrate analysis, the lyophilized samples were ground. 60 mg of dried tissue was extracted twice for 15 min in 7 ml boiling h 2 o dest . the two extracts were centrifuged, and the combined supernatant (14 ml) was passed through a 0.22 µm membrane filter. soluble sugars were quantified using high-performance anion exchange chromatography (hpae) combined with pulsed amperometric detection (pad) in a dionex® dx300 ion chromatograph fitted with a carbopac® pa-1 column (chatterton et al., 1989). flow rate was 1 ml*min-1 at about 1900 psi. carbohydrate elution was effected under alkaline conditions (150 mm naoh). the high ph (12-13) of the eluant converts hydroxyl groups of the oligosaccharides into oxyanions. the degree of oxyanion interaction with the anion exchange resin determines carbohydrate retention times. adding a competing ion such as acetate (0-500 mm naoac) to the eluant reduces the retention times. sugars in the extract were identified by comparing their hplc-pad retention times on various gradients with those of known sugars (i.e. glucose, fructose, sucrose, raffinose, stachyose) and by spiking the extracts with known pure sugars. known pure sugars were used for calculation of the sugar concentration in extracts. statistical analysis was undertaken with sas 6.12 (sas institute inc., cary, nc/usa) using proc anova/glm procedures and tukey’s studentized range test for analysis of variance and proc corr procedures and pearson’s correlation coefficient for analysis of correlation. results temperature exotherms the average weight of buds was around 30 mg, about 40-60 % were contributed by the bud scales and the wool. the greatest part of living tissue consists of a woody bud pad (subtending nodal tissue) which carries the shoot primordia. histologically it belongs to the cane and its size depends among others on the experience and skill of the person removing the bud. the freezing of the apoplastic water of the bud always produces a relatively large hte. only in 60 % of all buds at least one distinct lte of the primary shoot primordium (primary bud) was obtained and only in few cases the ltes of the small secondary buds could be detected. evidently the water content of these shoot primordia, in particular of the secondary and tertiary buds is at the limit of the resolution of the measuring device which is further diminished by the (then frozen) apoplastic water of the bud pad which separates the freezing shoot primordia from the sensor plate. high-temperature exotherms (htes) indicating the freezing of extracellular water were recorded from -5 to -16 °c with the minimum at the beginning of january (fig. 1). low-temperature exotherms (lte) indicating the freezing of intra-cellular water were recorded from -11 to -24 °c. low-temperature exotherms of basal buds were significant higher than exotherms of intermediate and apical buds. temperature exotherms were neither correlated to air temperature measured during the days before sampling nor to any of the soluble sugar concentrations (tab. 1). in our case it could be clearly shown, that an hte caused no cell damage in the buds (fig. 3), while after the appearence of the lte(s) a coagulation of protoplasts in primary and secondary buds could be found (fig. 4). soluble sugars soluble sugars were detected as glucose, fructose, sucrose, raffinose and stachyose with highest concentrations in basal buds and lowest concentrations in apical buds (fig. 2). sucrose was the dominant soluble carbohydrate and the reducing sugars (i.e. glucose, fructose) reached also rather high concentrations while raffinose and stachyose was detectable only in small concentrations. from november to late december/early january total soluble sugars increased from about 80 mg/g dm to almost 150 mg/g dm. the lowest concentration of total soluble sugars was registered at the end of march with about 35 mg/g dm followed by a slow increase of the sugar concentration in the bud tissue. differences in sugar concentrations of buds according to bud position were significant between basal/intermediate and basal/apical for the following fractions: total soluble sugars, fructose and sucrose. the concentration of glucose was significantly different only for basal and terminal buds. differences in sugar concentrations between intermediate and apical buds were never significant. for apical and intermediate buds a similar pattern of significant changes during sampling in total soluble sugars was notable. between sugar concentrations and air temperature of the days before sampling (3, 5, 7, 9 days before sampling) exists a significant correlation (tab. 1). the pearson’s correlation coefficient is regularly higher for intermediate and apical buds than for basal buds. the correlation coefficient was highest for raffinose and also stachyose showed a relatively high correlation coefficient. from the dominating sugars, the correlation coefficient from sucrose concentration with air temperature before sampling was lowest while the correlation coefficient of glucose concentration with air temperature before sampling was highest. generally the correlation coefficient between sugar concentrations and air temperature before sampling increased from three days before sampling to seven days before sampling and decreased with longer time periods (tab. 1). -25 -20 -15 -10 -5 0 12 .1 1. 19 98 26 .1 1. 19 98 10 .1 2. 19 98 24 .1 2. 19 98 07 .0 1. 19 99 21 .0 1. 19 99 04 .0 2. 19 99 18 .0 2. 19 99 04 .0 3. 19 99 18 .0 3. 19 99 01 .0 4. 19 99 15 .0 4. 19 99 t e m p e ra tu re [ °c ] hte lte -25 -20 -15 -10 -5 0 12 .1 1. 19 98 26 .1 1. 19 98 10 .1 2. 19 98 24 .1 2. 19 98 07 .0 1. 19 99 21 .0 1. 19 99 04 .0 2. 19 99 18 .0 2. 19 99 04 .0 3. 19 99 18 .0 3. 19 99 01 .0 4. 19 99 15 .0 4. 19 99 t e m p e ra tu re [ °c ] hte lte intermediate -25 -20 -15 -10 -5 0 12 .1 1. 19 98 26 .1 1. 19 98 10 .1 2. 19 98 24 .1 2. 19 98 07 .0 1. 19 99 21 .0 1. 19 99 04 .0 2. 19 99 18 .0 2. 19 99 04 .0 3. 19 99 18 .0 3. 19 99 01 .0 4. 19 99 15 .0 4. 19 99 t e m p e ra tu re [ °c ] hte lte apical intermediate basal fig. 1: high-temperature exotherms (hte) and low-temperature exotherms (lte) of grapevine buds of different sections of the shoot. 166 radu badulescu, michael ernst discussion overwintering canes of woody plants may freeze at temperatures below -10 °c without damage. it is generally assumed that in this case ice crystals are formed in the xylem or in the apoplast but not within the cells of pith rays or meristems which freeze only at considerable lower temperatures (generally below -30°c) if the plants are cold adapted. a special case are winter buds. while the base of the bud behaves like the cane tissue, the eyes of frost hardened woody plants freeze at -20°c or below. therefore superchilling of such buds usually produces so called high temperature exotherms (htes) caused by freezing of apoplastic water and one ore more low temperature exotherms (lte) resulting from the freezing of cells. the number of ltes depends on the complexity of the bud. there is a large number of papers on these phenomena in different plants: vitis (andrews et al., 1984), tomato (anderson and ashworth, 1985), prunus spp. (quamme, 1974), cherry (andrews and proebsting, 1987; callan, 1990), peach (quamme, 1978, 1983; andrews et al., 1986; ashworth and davis, 1984, 1987; rajashekar, 1989), apple (quamme, 1976), persimmon (kang et al., 1997), blackberry tab. 1: correlation analysis (pearson’s correlation coefficient) between mean air temperature recorded for different time periods (1-9 days, indicated as t 1-3 to t 1-9 ) before sampling, temperature exothermes (hte = high-temperature exotherme, lte = low-temperature exotherme) and sugar concentrations of grapevine buds. basal buds glucose fructose sucrose raffinose stachyose hte r = 0.01 -0.01 -0.05 -0.14 0.05 p(r) = 0.944 0.953 0.771 0.422 0.782 lte r = -0.03 -0.22 -0.54 0.33 0.03 p(r) = 0.895 0.342 0.011 0.149 0.891 t 1-3 r = -0.54 -0.30 -0.19 -0.65 -0.50 p(r) = 0.001 0.051 0.220 0.001 0.001 t 1-5 r = -0.57 -0.32 -0.19 -0.75 -0.53 p(r) = 0.001 0.032 0.228 0.001 0.001 t 1-7 r = -0.59 -0.35 -0.20 -0.77 -0.55 p(r) = 0.001 0.019 0.185 0.001 0.001 t 1-9 r = -0.58 -0.33 -0.17 -0.72 -0.55 p(r) = 0.001 0.027 0.259 0.001 0.001 intermediate buds glucose fructose sucrose raffinose stachyose hte r = 0.01 0.02 0.05 0.03 0.12 p(r) = 0.956 0.923 0.765 0.845 0.488 lte r = -0.26 -0.22 -0.12 -0.08 -0.07 p(r) = 0.226 0.308 0.578 0.728 0.759 t 1-3 r = -0.65 -0.52 -0.43 -0.70 -0.55 p(r) = 0.001 0.001 0.004 0.001 0.001 t 1-5 r = -0.67 -0.55 -0.49 -0.77 -0.57 p(r) = 0.001 0.001 0.001 0.001 0.001 t 1-7 r = -0.69 -0.56 -0.51 -0.76 -0.57 p(r) = 0.001 0.001 0.001 0.001 0.001 t 1-9 r = -0.67 -0.57 -0.51 -0.72 -0.l56 p(r) = 0.001 0.001 0.001 0.001 0.001 apical buds glucose fructose sucrose raffinose stachyose hte r = 0.05 -0.06 -0.24 -0.21 -0.14 p(r) = 0.782 0.729 0.181 0.247 0.432 lte r = -0.25 -0.24 -0.11 -0.11 -0.13 p(r) = 0.308 0.334 0.693 0.650 0.595 t 1-3 r = -0.62 -0.50 -0.43 -0.67 -0.49 p(r) = 0.001 0.001 0.004 0.001 0.001 t 1-5 r = -0.62 0.50 -0.45 -0.71 -0.52 p(r) = 0.001 -0.001 0.002 0.001 0.001 t 1-7 r = -0.63 -0.52 -0.48 -0.72 -0.54 p(r) = 0.001 0.001 0.001 0.001 0.001 t 1-9 r = -0.63 -0.54 -0.51 -0.71 -0.56 p(r) = 0.001 0.001 0.001 0.001 0.001 changes of soluble sugars in grapevine during winter 167 (warmund et al., 1992), blueberry (biermann et al., 1979; flinn and ashworth, 1994 a, b), strawberry (hummel and moore, 1997) forsythia (flinn and ashworth, 1995), azalea (graham and mullin, 1976 a, b), rhododendron (kaku et al., 1981), conifers (ide et al., 1998). it is generally assumed that a high frost resistence depends on a gradual cold acclimatisation, the mechanisms of which are, however, far from being known. important factors are desiccation, change of cell wall structures (in wood: wisniewsky, 1993), biochemical processes such as deposition of specific proteins (marentes et al., 1993; hon et al., 1995 in graminees; salzmann et al., 1996 in grapevine), proline (ait barka and audran, 1997) or the conversion of polysaccharides to monosaccharides (kliewer, 1965). andrews et al. (1983, 1984, 1986), wolf and pool (1986, 1987), and wolf and cook (1994) compared varieties on the basis of exotherm measurements since it had been shown by pierquet and stushnoff (1977), pierquet et al. (1980) and quamme (1986) that ltes were reliable indicators for the death of grapevine buds. however, flinn and ashworth (1994) concluded from experiments with blueberries and forsythia that this is not necessarily true for flower buds of other plants which are anatomically different although kadir and proebsting (1994) had used exotherm measurements to test sweet cherry cultivars for dormant bud hardiness. temperature exotherms in our investigations showed a systematic pattern for apical, intermediate and basal buds. with the exception of one measuring date, freezing of extraand intra-cellular water was at the lowest temperature at the end of december and the beginning of january. there are several factors affecting the thermal analysis of grapevine buds (wolf and cook, 1994). research on soluble sugars in grapevine is focussed on berries. glucose, fructose and sucrose constitute the major sugars in the grape plant and also in buds (wample and bary, 1992), according to our findings. the presence of raffinose and stachyose is also reported in grape plants (not specifically for buds), detected with means of paper chromatography (kliewer, 1965). our results show, that raffinose and stachyose were present in grapevine buds throughout the whole sampling period, but only in low concentrations. in grapevine buds, koussa et al. (1998) found generally lower concentrations of total soluble sugars compared to internodes. in their experiments, the accumulation of raffinose and sucrose, starting in october, was related to the decrease of daily average temperatures and also wample and bary (1992) and hamman et al. (1996) found a relation between soluble carbohydrates and air temperature. koussa et al. (1998) see the role of soluble carbohydrates as cryoprotectors during winter confirmed and the presence of carbohydrate mediated frost protection of buds of norway spruce (lipavská, et al., 2000). imanishi et al. (1997) found in their experiments, that the raffinose family of the oligosaccharides is involved in the freezing tolerance of plants, they are potential cryoprotectants in cold-acclimated plant cells. this is confirmed by our results, however, the correlation coefficient of glucose, fructose and stachyose with air temperature before sampfig. 3: section through primary vegetation cone of a compound bud after occurrence of a hte. no damage is visible. fig. 4: vegetation cone after appearance of lte: due to freezing the cytoplasm has coagulated and forms small clots within the cell. 168 radu badulescu, michael ernst 0 50 100 150 200 250 12 .1 1. 98 26 .1 1. 98 10 .1 2. 98 23 .1 2. 98 07 .0 1. 99 21 .0 1. 99 25 .0 2. 99 11 .0 3. 99 25 .0 3. 99 08 .0 4. 99 16 .0 4. 99 s o lu b le s u g a rs [ m g /g d m ] stachyose raffinose sucrose fructose glucose 0 50 100 150 200 250 12 .1 1. 98 26 .1 1. 98 10 .1 2. 98 23 .1 2. 98 07 .0 1. 99 21 .0 1. 99 25 .0 2. 99 11 .0 3. 99 25 .0 3. 99 08 .0 4. 99 16 .0 4. 99 s o lu b le s u g a rs [ m g /g d m ] stachyose raffinose sucrose fructose glucose 0 50 100 150 200 250 12 .1 1. 98 26 .1 1. 98 10 .1 2. 98 23 .1 2. 98 07 .0 1. 99 21 .0 1. 99 25 .0 2. 99 11 .0 3. 99 25 .0 3. 99 08 .0 4. 99 16 .0 4. 99 s o lu b le s u g a rs [ m g /g d m ] stachyose raffinose sucrose fructose glucose apical intermediate basal fig. 2: concentration of soluble carbohydrates in grapevine buds of different sections of the shoot. (dm = dry matter) ling is higher than the correlation coefficient of sucrose with air temperature. but in both investigations, concentration of raffinose in grapevine buds is well correlated to air temperature before sampling. we anticipate that further investigations will better define the role of soluble carbohydrates as cryoprotectors. references ait barka, e., audran, j.c., 1997: response of champenoise grapevine to low temperatures: changes of shoot and bud proline concentrations in response to low temperatures and correlations with freezing tolerance. j. hort. sci. 72, 577-582. anderson, j.a., ashworth, e.n., 1985: ice nucleation in tomato plants. j. amer. soc. hort. sci. 110, 291-296. andrews, p.k., proebsting, jr. e.l., 1987: effects of temperature on the deep supercooling characteristics of dormant and deacclimating sweet cherry flower buds. j. amer. soc. hort. sci. 112, 334-340. andrews, p.k., proebsting, e.l., campbell, g.s., 1983: an exotherm sensor for measuring the cold 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1983: relationship of air temperature to water content and supercooling of overwintering peach flower buds. j. amer. soc. hort. sci. 108, 697-701. quamme, h.a., 1986: use of thermal analysis to measure the freezing resistance of grape buds. can. j. plant sci. 66, 945-952. rajashekar, c.b., 1989: supercooling characteristics of isolated peach flower bud primordial. plant physiol. 89, 1031-1034. salzman, r.a., bressan, r.a., hasegawa, p.m., ashworth, e.n., bordelon, b.p., 1996: programmed accumulation of lea-like proteins during desiccation and cold acclimation of overwintering grape buds. plant, cell and environment 19, 713-720. smith, d., 1968: carbohydrates in grasses. iv. influence of temperature on the sugar and fructosan composition of timothy (phleum pratense) plant parts at anthesis. crop sci. 8, 331-334. wample, r.l., bary, a., 1992: harvest date as a factor in carbohydrate storage and cold hardiness of carbernet sauvignon grapevines. j. amer. soc. hort. sci. 117, 32-36. wample, r.l., reisenauer, g., bary, a., schuetze, f., 1990: microcomputer-controlled freezing data acquisition and analysis system for cold hardiness evaluation. hort. sci. 25, 973-976. warmund, m.r., takeda, f., davis, g.a., 1992: supercooling and extracellular ice formation in differentiating buds of eastern thornless blackberry. j. amer. soc. hort. sci. 117, 941-945. wolf, t.k., cook, m.k., 1994: cold hardiness of dormant buds of grape cultivars: comparison of thermal analysis and field survival. hort. sci. 29, 1453-1455. wolf, t.k., pool, r.m., 1986: microcomputer-based differential thermal analysis of grapevine dormant buds. hort. sci. 21, 1447-1448. wolf, t.k., pool, r.m., 1987: factors affecting exotherm detection in the differential thermal analysis of grapevine dormant buds. j. amer. soc. hort. sci. 112, 520-525. address of authors: radu badulescu, 1020 w. pecan st., brea, california 92821, usa (raduva@ uni-hohenheim.de) michael ernst, staatsschule für gartenbau und landwirtschaft, 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<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> >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice vorgabe neu journal of applied botany and food quality 80, 138 144 (2006) 1arc infruitec-nietvoorbij, stellenbosch, south africa 2department of food science, stellenbosch university, stellenbosch, south africa 3federal centre for breeding research on cultivated plants, institute of plant analysis, quedlinburg, germany production and quality aspects of rooibos tea and related products. a review elizabeth joubert1, 2, hartwig schulz3 (received june 19, 2006) summary use of the herbal tea, rooibos, made from the indigenous south african fynbos plant, aspalathus linearis spp. linearis, has shown tremendous growth on the international markets since the 1990s. from a small beginning in 1904, solely depended on wild harvesting, the industry has developed out of the selected and cultivated nortier type, leading to improved quality. traditional rooibos is processed, entailing an oxidation („fermentation“) step, essential to develop the characteristic sweetish flavour and red-brown colour. higher antioxidant levels for unfermented rooibos resulted in the development of green rooibos and extracts enriched in aspalathin, a potent antioxidant unique to rooibos. major markets for rooibos extracts are ready-to-drink iced teas and cosmetic products. introduction the use of rooibos tea (aspalathus linearis) spans at least more than 230 years and is still enjoyed by an ever-increasing market. in 1772, the botanist, carl thurnberg, during a visit to africa, reported that indigenous people of the cape, the khoi, used the rooibos plant to make a beverage. the plant is indigenous to south africa and forms part of the fynbos biome, one of six floral kingdoms. in the early 1900s the tea came to the attention of benjamin ginsberg, a merchant of clanwilliam. he bought the tea for marketing from descendants of the khoi in the region who, during the summer months, harvested the rooibos plants growing wild in the mountains. in the early 1930s the first experiments with cultivation of rooibos took place. by the second world war, the demand for rooibos tea had escalated due to a shortage of oriental tea. however, after the war, the rooibos tea market collapsed; poor and inconsistent quality tea, and overproduction made rooibos as crop uneconomical by 1953/ 54. these factors prompted the establishment of the „rooibos tea control board“ to stabilize the industry through orderly marketing and standardization of the tea quality. this one-channel marketing system was abolished in 1997, which opened the way for several marketing companies to enter the rooibos industry. the worldwide demand for rooibos tea grew from 524 tons in 1955 to 10,600 tons in 2003, with exports comprising 6,400 tons. most of the tea is exported to germany (previously west germany), where it was first sold in 1961 as „rotbuschtee“ or „massai-tee“. some tea was also exported to the usa and sold under the trade name ‘kaffree tea’ (morton, 1983). since 1984 rooibos was sold in japan where its „anti-ageing“ properties were an important selling aspect. recently, the opportunities, provided by a changing herbal tea market, stimulated the development of unfermented („green“) rooibos. most of its production is sold as herbal tea or is used to prepare extracts for the food, beverage and cosmetic industries. botanical classification the rooibos tea plant, aspalathus linearis (brum.f.) dahlg., is unique to south africa. aspalathus linearis ssp. linearis is the most common of the subspecies and includes those types used for tea production. the plants are erect or prostrate shrubs, growing up to 2 m tall in nature, with yellow flowers and needle-like leaves. dahlgren (1968) first described the variation in this species, giving a detailed account of the morphology and geographical distribution of the various wild forms and different types. the size, density of branching, development of short shoots, leaf size and flowering time of the species a. linearis vary considerably. it is only the red type, divided into the selected and improved nortier type (cultivated), and the wild-growing cederberg type, with its broader and coarser leaves (morton, 1983) that is normally used for processing. other types, i.e. the grey, black and red-brown types, were in the early years harvested in the wild for tea processing, and also today a small quantity of wild tea finds its way to the processors. marketing of the grey and black types was discontinued by 1966 due to their poor quality (anon., 1967). the black type gives an infusion that is not typically red-brown, nor is the flavour characteristic (coetzee et al., 1953). cultivation commercial cultivation of the nortier type rooibos occurs mainly in the cederberg mountain region, but some tea is also cultivated in areas as far as darling and nieuwoudtville. the plants grow in deep, well-drained, sandy, acid soil (nolte, 1968). being a legume, the plant has a well-nodulated root system for fixing elemental nitrogen from soil water. in vitro propagation was investigated for producing large numbers of homogenous plants (le roux et al., 1992), but due to poor survival when planted out, plants are still propagated from seeds. recent attempts to propagate rooibos from cuttings were also not successful (j. brand, rooibos ltd, clanwilliam, pers. comm., 2006). seedlings, 100 to 150 mm high, are planted between june and august at approximately 8,000 to 10,000 plants per hectare. branching is stimulated by topping the plant after about 8 months to a height of 30 cm. the first harvest takes place after 18 months during summer, but full production is only realized after 3 years. a fully-grown bush yields about 70 to 125 g of dry tea (j. brand, rooibos ltd, pers. comm., 2006). cheney and scholz (1963) documented that the best harvest occurs in the fourth and fifth years after planting and that some of the bushes start to die after the seventh harvest. however, the present situation has changed drastically, with bushes already dying after the second harvest. this problem is caused amongst other by the two fungus species, diaporthe phaseolorum and neocosmospora vasinfecta (smit and knox-davies, 1989). insects that could cause considerable damage to the plants, if not effectively controlled, are the clearwing moth (sesiidae), looper (isturgia exerraria) and leafhopper (molopoterus theae theron). chemical spaying is used to control these insects, but the demand for organically produced tea has prompted research into development of an integrated pest management programme. biological control mechanisms such as the use of pheromone for the clearwing moth are investigated (j. brand, rooibos ltd, pers. comm., 2006). for the production of traditional rooibos („fermented“ tea) harvesting takes place in the summer, from december, until early autumn. tea is harvested mechanically or manually with sickles by cutting the whole bush a few centimeter above the topping height. no or little flowers should be present since it gives an unpleasant flavour to rooibos tea. after harvesting the branches are bound in bundles for transport to the processing yard. processing the processing of traditional rooibos tea entails the following: tea shoots are shredded into 3 to 4 mm lengths, placed in a „fermentation“ heap and bruised by late afternoon, followed by addition of water, further bruising and mixing. bruising and addition of water are necessary to accelerate „fermentation“, which is initiated during shredding. the water also serves to extract polyphenols, acting to colour the stems when absorbed. fermentation takes place during the night and the heap is spread open to dry in the sun the following morning. fermentation can take from 8 to 24 h, with an average fermentation time of 12 to 14 h, depending on the climatic conditions, the composition of the plant material and processing conditions. during fermentation the aroma of the wet tea changes from resinous, haylike and grassy to sweet, apple-like or honey-caramel to sour depending on the stage of fermentation. as soon as the characteristic sweet, honey-like aroma and red-brown colour of the tea are fully developed, the product is spread open in a thin layer (15-20 mm) to dry in the sun. the development of a sour aroma is indicative of over-fermentation. brushing is done to break up any lumps and to accelerate drying, but excessive quantities of tea dust are formed which have to be separated later on. before packaging the tea is sieved to obtain the required fine cut, which is then steam-pasteurized to ensure that the tea meets the required microbial standards. since no control over individual processing parameters is possible with the traditional open-air fermentation method, controlled fermentation and drying of rooibos have been investigated as alternatives to ensure optimum and consistent quality (joubert and de villiers, 1997). however, this progress is resisted by industry due to cost implications in terms of capital expenditure and energy requirements. research showing unfermented rooibos to have higher antioxidant capacity than its traditionally processed counterpart (von gadow et al., 1997a; standley et al., 2001), stimulated the development of green rooibos (de beer and joubert, 2002) as an alternative rooibos product for the international market. with the processing of green rooibos, the oxidative changes should be kept to a minimum to obtain tea with a green colour. this is achieved, either by inactivation of enzymes by subjecting the tea to a steaming process or drying whole shoots at low temperatures and air humidity to a critical moisture content before shredding, followed by further drying to the required moisture content. other processing includes the preparation of extracts and powders. first developed in the 1980s (joubert, 1988, 1990), it is only the past few years that aqueous extracts and extract powders, prepared from fermented tea, find application in the food and beverage industries (anon., 2005a,b). a large selection of ready-to-drink beverages, either natural or flavoured, is offered on the market in south africa, and has recently been introduced also to the european market. other applications include candies, flavouring of a carobbased „chocolate“ and coating of honeybush tea leaves (cyclopia spp.) with a rooibos extract to enhance its antioxidant activity. tab. 1: comparison of the antioxidant activity of aqueous soluble solids of green and fermented rooibos assay endpoint green rooibos fermented rooibos reference(s) dpph • % scavenginga 86.6 83.4 von gadow et al., 1997b % scavengingb 87.3 83.0 joubert et al., 2004 ec50 c 2.33 3.62 adapted from standley et al., 2001 ec50 c 3.24 3.87 joubert et al., 2004 rate of scavengingd 8.3 x 10-4 7.35 x 10-4 winterton, 1999 o2 -• ic50 e 44.4 60.5 adapted from standley et al., 2001 ic50 e 69.4 78.3 joubert et al., 2004 linoleic acid emulsion % inhibition (cd) f 28.6 28.0 joubert et al., 2005 β-carotene-linoleic acid oxidation aacg 557 607 von gadow et al., 1997b sunflower oil-in-water emulsion % inhibition (peroxides)h 90.0 80.9 winterton, 1999 induction time (pv) i 35 31 % inhibition (cd) j 58.1 54.5 methyl linoleate micelles % inhibition of tbarsk 22.8 30.3 winterton, 1999 a scavenging (%) of dpph • (6 x 10-5 m) after 2 h b scavenging (%) of dpph • (3.04 x 10-5 m) after 20 min c effective concentration of soluble solids (mg) per ml reaction mixture to scavenge 50% of dpph • d dpph • rate of scavenging (s-1), calculated during unsteady state conditions (time 0 – 3 min), expressed as the change in the absorbance at 515 nm over time. e concentration of soluble solids (mg) per ml reaction mixture required to inhibit 50% of nbt reduction f inhibition (%) of conjugated diene (cd) formation after 21 h incubation at 40°c g antioxidant activity h inhibition of peroxides after 35 days incubation at 30°c i time required for oxidation to reach a peroxide value (pv) of 10 meq/kg oil with incubation at 30°c j inhibition of cd formation after 31 days incubation at 30°c k inhibition of the formation of thiobarbituric reactive substances (tbars) after 16 h incubation at 37°c production and quality aspects of rooibos tea and related products 139 extracts from green rooibos (tiedtke and marks, 2002; otto et al., 2003), containing high levels of the active principle, aspalathin, are produced for the nutraceutical and cosmetic industries. green rooibos is preferably used because of its higher antioxidant activity (tab. 1). selective extraction of fermented rooibos also provides a product with enhanced antioxidant activity (von gadow et al., 1997b; joubert et al., 2004), but the brownish dried extract is not suitable for the cosmetic industry. quality standards and control apart from quality standards pertaining to microbial contamination and pesticide residue levels (anon., 2000), no regulatory quality standards or requirements relating to composition, active compounds or antioxidant activity exist for traditional and green rooibos. nevertheless, there is an increasing interest to offer green rooibos extracts with high levels of aspalathin for the cosmetic and functional food markets (b. weinreich, raps & co, kulmbach, germany, pers. comm., 2003). however, the aspalathin content of green rooibos is not routinely determined by processors and presently visual inspection of colour serves as the only quality control parameter. the leaves should have a light green colour, giving a light yellowish to orange infusion. the development of a red-brown leaf colour is indicative of undesirable oxidation, and thus degradation of aspalathin in green rooibos. usually less than 7% of the aspalathin content is retained after fermentation (joubert, 1996). the lack of a rapid screening method for quantification of aspalathin content of green rooibos prompted the development of nir (steuer et al., 2000; schulz et al., 2003; manley et al., 2006), as well as raman spectroscopy (unpublished data) prediction models. their advantage is that measurement of aspalathin can be done directly on the dried, ground plant material. manufacturers of extracts have introduced product specifications such as minimum total polyphenol (tp) content and total antioxidant activity (taa) values. green rooibos extract, standardized at 15% aspalathin content, and a fermented rooibos extract, standardized in terms of orientin content, have recently been introduced for the functional food market. another factor worth considering is authenticity. the increasing demand for organic rooibos has sparked renewed interest from farmers in rooibos growing in the wild. some samples of wild rooibos, obtained from a processing yard, showed atypical hplc profiles compared to the cultivated type (fig. 1). sample 1 contains rutin as major compound with little or no aspalathin whereas in sample 2 an unidentified dihydrochalcone or flavanone has been detected by hplc-dad as major compound. van heerden et al. (2003) demonstrated large phenolic variation in wild populations of rooibos. chemical composition rooibos tea is caffeine-free with a low tannin content compared to black tea (blommaert and steenkamp, 1978). exhaustive extraction of the water extract with an organic solvent showed that it contains ca. 50% complex tannin-like substances (ferreira et al., 1998). the isolated tannin was shown to be an irregular heteropolymer of the procyanidin type with (+)-catechin and (-)-epicatechin as chain-extending units and only catechin as the terminal flavanol (ferreira et al., 1998; marais et al., 1998). other condensed tannintype compounds are procyanidin b3 and the profisinidin triflavanoid, bis-fisetinidol(4β,6:4βb,8)-catechin which are present only at very low concentration (ferreira et al., 1995). apart from the two major dihydrochalcones, aspalathin (structure elucidation by koeppen and roux, 1966) and nothofagin (joubert, 1996), the flavones orientin, isoorientin (fig. 2) (koeppen and roux, 1965) vitexin, isovitexin, chrysoeriol (rabe et al., 1994) and luteolin (snyckers and salemi, 1974) have been detected in rooibos, as well as the flavonols rutin, isoquercitrin (koeppen et al., 1962), quercetin (snyckers and salemi, 1974), hyperoside (bramati et al., 2002), luteolin-7-o-glucoside (kazuno et al., 2005) and the flavanol (+)catechin (ferreira et al., 1995) have been found in the plant material. a biomimetic study of the fermentation process showed oxidative conversion of aspalathin into the flavanones, (s)and (r)eriodictyol-6-c-β-d-glucopyranoside (marais et al., 2000). traces of the flavanones, dihydroorientin and dihydro-isoorientin in fermented rooibos were noted by bramati et al. (2002). the presence of the novel compound, 5,7-dihydroxy-6-c-β-d-glucopyranosylchromone, suggests oxidative conversion of dihydro-isoorientin during fermentation (ferreira et al., 1995). the phenolic acids, isolated from fermented rooibos comprise the benzoic acids, phydroxybenzoic acid, protocatechuic acid, vanillic acid and syringic acid, and the cinnamic acids, p-coumaric acid, ferulic acid and caffeic acid (rabe et al., 1994). whereas most of the flavonoids occur ubiquitously in the plant kingdom, until now aspalathin was only detected in rooibos, contributing to its novelty value. analysis of 97 samples of rooibos, 0 0.02 0.04 0.06 0.08 0.1 0.12 0 10 20 30 40 50 60 70 80 90 100 110 retention time (min) a u ( 2 8 0 n m ) 0 0.002 0.004 0.006 0.008 0.01 0.012 0.014 0 10 20 30 40 50 60 70 80 90 100 110 retention time (min) a u ( 3 2 0 n m ) cultivated rooibos (seedling) wild rooibos (sample 1) aspalathin 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0 10 20 30 40 50 60 70 80 90 100 110 retention time (min) a u ( 2 8 0 n m ) wild rooibos (sample 2) aspalathin unknown rutin fig. 1: hplc chromatogrammes of a rooibos seedling (sampled in the seed bed) and two samples of wild rooibos bushes obtained from a drying yard. hplc analysis was performed according to the method described by joubert et al. (2005) 140 elizabeth joubert, hartwig schulz harvested from different bushes located in 24 plantations over the production area during winter time, showed large variation in the aspalathin and nothofagin content. large variation was also noticed in the aspalathin content of 3-year-old bushes, planted on the same farm. this is mainly attributed to genetic variation of individual plants. the processed tea retains comparatively low quantities of aspalathin and nothofagin (tab. 2), yet aspalathin, together with rutin, was shown to be one of the major flavonoids of an infusion prepared from fermented rooibos (bramati et al., 2002). quercetin, luteolin and chrysoeriol are often present in very low quantities in a cup of tea (toyoda et al., 1997). the major compounds in tea powders prepared from fermented rooibos with a large percentage of stems comprised aspalathin, nothofagin, orientin, vitexin, isoquercitrin and rutin (tab. 3). natural tea flavour and flavoured tea products the aroma of green rooibos tea is grassy, hay-like, whereas the fermented tea has a sweet and sometimes pleasant caramel note. the typical flavour of fermented rooibos tea is influenced by numerous volatile constituents of different chemical groups such as hydrocarbons, alcohols, aldehydes, ketones, lactones, acids, esters, imides, phenols and furans (habu et al., 1985; kawakami et al., 1993). model experiments applying dichloromethane extraction and simultaneous steam distillation and extraction (sde) were carried out and the volatile substances were tentatively identified by gc-ms. the main components in the sde extract were guaiacol, 2-phenylethanol, various ketones, β-damascenone and 6,10,14-trimethyl-2-pentadecanone, as well as acids such as acetic acid, 3-methylbutanoic acid, hexanoic acid and octanoic acid. while the dichloromethane tea extracts were found to contain many kinds of lactones such as 4-butanolide and dihydroactinidiolide, the sde extracts lacked these aroma substances (kawakami et al., 1993). in spite of the fact that more than 120 volatile substances have been identified in rooibos tea extracts, no olfactory studies have been done to identify aroma substances responsible for the aroma profile of rooibos tea infusions. present available flavourings of rooibos tea are limited mainly to the aroma types „vanilla“ and „lemon“. relating to the latest wellness trend, mixtures with some herbs such as rosemary or ginger are also offered. beside this, rooibos tea flavoured with honey aroma is consumed in south africa. this product should not be confused with honeybush tea, which is a herbal tea prepared from cyclopia species. health value the discovery by annetjie theron that rooibos tea contributes considerably to the convalescence of her 14-month-old, colicky baby, started off rooibos tea as a healthy drink. snyckers and salemi (1974) attributed the anti-allergic effect of rooibos when administered to babies to the antispasmodic properties of quercetin and luteolin that would have a calming effect on the stomach. the indication that topical application of aqueous rooibos extract helps for skin problems such as eczema and nappy rash has resulted in development of special skin creams for babies and other natural cosmetic products. previously, these products were limited to the south african market, but are now shipped worldwide (levy, 2004). with the interest in natural antioxidants, attention also turned to rooibos. antioxidant activity has been demonstrated for rooibos in a variety of in vitro test systems. scavenging of the biologically relevant reactive oxygen species (ros), o 2 -•, •oh and h 2 o 2 (yoshikawa et al., 1990; standley et al., 2001; lee and jang, 2004; joubert et al., 2004), inhibition of lipid peroxidation (von gadow et al., 1997a, b; hitomi et al., 1999; winterton, 1999; joubert et al., 2005) and scavenging of dpph• and abts+• (bramati et al., 2003; schulz et al., 2003; joubert et al., 2004; manley et al., 2006) have been demonstrated. von gadow et al. (1997a) showed that green tea (camellia sinensis) is more potent than green or fermented rooibos, applying the dpph• and carotene-linoleic acid assays. this was confirmed by richards et al. (2001), using the abts+• and frap assays, taking the total polyphenol content of the aqueous extract into account. bramati et al. (2003) showed that taa of green rooibos leaves is ca. 50% of that of green and black tea. the taa of green rooibos extracts correlates with their aspalathin content (botha, 2005). schulz et al. (2003) find the apparent contribution of aspalathin to the taa of green rooibos between 22 to 57%. the relative antioxidant potency of aspalathin as the major flavonoid (von gadow et al., 1997c) is of special interest to the extract manufacturer. fig. 3 summerises the relative potency of this dihydrochalcone compared to other rooibos flavonoids as determined in the dpph• and o 2 -• assays. its flavone analogues, orientin and isoorientin (joubert et al., 2004), which are present in comparable quantities in fermented rooibos, as well as nothofagin (snijman et al., 2003) are less potent. aspalathin was found to be one of the major contributors to pro-oxidant activity of rooibos extracts (joubert et al., 2005). however, it is postulated that ros are key mediators of apoptosis which eliminates cancer cells (salganik, 2001). the oh oh o oh r o oh h oh oh oh oh oh oh o oh oh o oh h oh oh oh o a, b d c oh oh o oh oh o o oh oh oh oh fig. 2: structures of aspalathin (r = oh) and nothofagin (r = h) [a,b], orientin [c] and iso-orientin [d] production and quality aspects of rooibos tea and related products 141 oxidative status prevailing within a cell would therefore determine whether the biological response would be beneficial or deleterious (klaunig and kamendulis, 2004). presently, a study is in progress to investigate the bio-availability of aspalathin. sufficient knowledge of its absorption and biotransformation in the human body would be necessary to evaluate the biological significance of this compound. references anonymous, 1967: rooibos tea control board 12th annual report. clanwilliam, south africa. anonymous, 2000: agricultural product standards act. act no. 119 of 1990, regulation no. 1177 of 24 november 2000. pretoria, south africa. anonymous, 2005a: unique functional rooibos ingredient. sa food review 32, 21. anonymous, 2005b: cognis offers rooibos tea extract, expands plant line. foodnavigator electronic newsletter, 1 dec 2005. blommaert, k.l.j., steenkamp, j., 1978: tannienen moontlike kafeïeninhoud van rooibostee, aspalathus (subgen. nortiera) linearis (burm. fil) r. dahlgr. agroplantae 10, 49. botha, m., 2005: use of near infrared spectroscopy (nirs) and spectrophotometric methods in quality control of green rooibos (aspalathus linearis) and honeybush (cyclopia genistoides). m.sc. thesis, stellenbosch university, stellenbosch, south africa. bramati, l., minoggio, m., gardana, c., simonetti, p., mauri, p., pietta, p., 2002: quantitative characterization of flavonoid compounds in rooibos tea (aspalathus linearis) by lc-uv-dad. j. agric. food chem. 50, 5513-5519. bramati, l., aquilano, f., pietta, p., 2003: unfermented rooibos tea: quantitative characterization of flavonoids by hplc-uv and detertab. 3: the total polyphenol and major flavonoid content of fermented rooibos compound content content content (mg / g powder)1 (mg / 150 ml cup of tea)2 (mg / 150 ml cup of tea)3 aspalathin 3.50 ± 1.33 1.05 3.09 nothofagin 1.10 ± 0.18 0.33 nd4 orientin 1.61 ± 0.16 0.48 2.51 isoorientin 0.52 ± 0.09 0.16 2.08 vitexin 2.36 ± 0.10 0.71 0.83 isovitexin 0.89 ± 0.25 0.27 0.66 isoquercitrin + rutin5 3.15 ± 0.87 0.95 _ isoquercitrin + hyperoside6 _ _ 1.07 rutin _ _ 3.17 luteolin trace trace 0.07 quercetin trace trace 0.27 chrysoeriol trace trace 0.06 total polyphenols7 273 ± 28 82 888 1 rooibos extract powder prepared by aqueous extraction of waste material containing a high percentage of stems, followed by concentration and spraydrying. number of analysed production batches: 9. 2 based on reconstituted tea powder (300 mg/150 ml). 3 data adapted from bramati et al. (2002); data converted to represent the flavonoid content given by a 2.5 g teabag/150 ml (infused for 10 min). 4 not determined 5 co-eluted; expressed in terms of quercetin 6 co-eluted; expressed in terms of isoquercitrin 7 total polyphenol content expressed as gallic acid equivalents (determined with folin-ciocalteu reagent) 8 data adapated from bramati et al. (2003). 142 elizabeth joubert, hartwig schulz fig. 3: comparison of the dpph• and o 2 -• scavenging ability of aspalathin with other rooibos flavonoids 0 20 40 60 80 100 chrysoeriol vitexin rutin iso-orientin isoquercitrin aspalathin luteolin orientin quercetin % scavenging of radical o2 dpph • • tab. 2: aspalathin and nothofagin content of unfermented (green) and fermented (traditional) rooibos tea tea parameter aspalathin nothofagin (g/100g) (g/100g) unfermented1 range 3.84-9.66 0.2-1.24 average (n = 97) 6.62 0.67 fermented range 0.02-1.16 0-0.40 average (n = 89) 0.26 0.12 1samples prepared by drying whole shoots at 40°c in a forced air circulation drying tunnel mination of the total antioxidant activity. j. agric. food chem. 51, 74727474. de beer, s.w., joubert, e., 2002: preparation of tea-like beverages. sa patent nr. 2002/2802. cheney, r.h., scholtz, e., 1963: rooibos tea, a south african contribution to world beverages. econ. bot. 17, 186-194. coetzee, w.h.k., van der merwe, h.c., burger, i.j., 1953: die samestelling van rooibostee. boerdery in suid-afrika 28, 95-99. dahlgren, r., 1968: revision of the genus aspalathus. ii. the species with ericoid and pinoid leaflets. 7. subgenus nortieria. with remarks on rooibos tea cultivation. bot. notiser 121, 165-208. ferreira, d., marais, c., steenkamp, j.a., joubert, e., 1995: rooibos tea as a likely health food supplement. in: proceedings of recent development of technologies on fundamental foods for health, 73-88. korean society of food science and technology, seoul, korea. ferreira, d., malan, e., marais, s.s., joubert, e., 1998: isolation and identification of water-soluble tannin compounds found in rooibos tea. project it270016 progress report. arc-fruit, vine and wine research institute, stellenbosch, south africa. habu, t., flath, r.a., mon, t.r., morton, j.f., 1985: volatile components of rooibos tea (aspalathus linearis). j. agric. food chem. 33, 249254. hitomi, e., tamura, s., tsurumoto, y., tsuda, t., nakano, m., 1999: antioxidant activity of rooibos tea (aspalathus linearis). j. jap. soc. food sci. technol. 46, 779-785 (cab abstract). joubert, 1988: effect of agglomeration on the properties of spray-dried rooibos tea. intern. j. food sci. technol. 23, 203-207. joubert, 1990: chemical and sensory analyses of sprayand freeze-dried extracts of rooibos tea (aspalathus linearis). intern. j. food sci. technol. 25, 344-349. joubert, e., 1996: hplc quantification of the dihydrochalcones, aspalathin and nothofagin in rooibos tea (aspalathus linearis) as affected by processing. food chem. 55, 403-411. joubert, e., de villiers, o.t., 1997: effect of fermentation and drying conditions on the quality of rooibos tea. intern. j. food sci. technol. 32, 127-134. joubert, e., winterton, p., britz, t.j., ferreira, d., 2004: superoxide anion radical and α,α-diphenyl-β-picrylhydrazyl radical scavenging capacity of rooibos (aspalathus linearis) aqueous extracts, crude phenolic fractions, tannin and flavonoids. food res. intern. 37, 133-138. joubert, e., winterton, p., britz, t.j., gelderblom, w.c.a, 2005: antioxidant and pro-oxidant activities of aqueous extracts and crude polyphenolic fractions of rooibos (aspalathus linearis). j. agric. food chem. 53, 10260-10267.. kawakami, m., kobayashi, a., kator, k., 1993: volatile constituents of rooibos tea (aspalathus linearis) as effected by extraction process. j. agric. food chem. 41, 633-636. kazuno, s., yanagida, m., shindo, n., murayama, k., 2005: mass spectrometric identification and quantification of glycosyl flavonoids, including dihydrochalcones with neutral loss scan mode. anal. biochem. 347, 182-192. klaunig, j.e., kamendulis, l.m., 2004: the role of oxidative stress in carcinogenesis. ann. rev. pharmacol. toxicol. 44, 239-267. koeppen, b.h., roux, d.g., 1965: c-glycosylflavonoids. the chemistry of orientin and iso-orientin. biochem. j. 97, 444-448. koeppen, b.h., roux, d.g., 1966: c-glycosylflavonoids. the chemistry of aspalathin. biochem. j. 99, 604-609. koeppen, b.h., smit, c.j.b., roux, d.g., 1962: the flavone c-glycosides and the flavanol o-glycosides of aspalathus acuminatus (rooibos tea). biochem. j. 83, 507-511. lee, e.-j., jang, h-d., 2004: antioxidant activity and protective effect on dna strand scission of rooibos tea (aspalathus linearis). biofactors 21, 285-292. le roux, j.j., brown, n.a.c., leivers, s., 1992: micropropagation of aspalathus linearis through bud multiplication. plant cell tissue organ culture 28, 225-227. levy, a.c., 2004. beyond black: tea products for health. tea & coffee trade j. 176, 68-71. manley, m., joubert, e., botha, m. 2006: quantification of the major phenolic compounds, soluble solid content and total antioxidant activity of green rooibos (aspalathus linearis) by means of near infrared spectroscopy. j. near infrared spectrosc. (in press). marais, c., janse van rensburg, w., ferreira, d., steenkamp, j.a., 2000: (s)and (r)eriodictyol-6-c-β-d-glucopyranoside, novel keys to the fermentation of rooibos (aspalathus linearis). phytochemistry 55, 4349. marais, s.s., marais, c., steenkamp, j.a., malan, e., ferreira, d., 1998: progress in the investigation of rooibos extractives. in: gross, g.g., hemingway, r.w., yoshida, t. (eds.), abstracts of the 3rd tannin conference, 129-130. bend, oregon. morton, j.f., 1983: rooibos tea, aspalathus linearis, a caffeineless, lowtannin beverage. econ. bot. 37, 164-173. nolte, l.e., 1968: die produksie van rooitee. boerdery in suid-afrika 44, 17-22. otto, f., grüner, s., weinreich, b., 2003: extract from „green“ rooibos – a new raw material for the cosmetic industry. euro cosmetics 9, 20-23. rabe, c., steenkamp, j.a., joubert, e., burger, j.f.w., ferreira, d., 1994: phenolic metabolites from rooibos tea (aspalathus linearis). phytochemistry 34, 1559-1565. richards, e.s., joubert, e., gelderblom, w.c.a., manley, m, 2001: relative antioxidant potencies of the aqueous extracts of honeybush (cyclopia species), rooibos (aspalathus linearis) and camellia sinensis teas. in: abstracts of iubmb/sasbmb special meeting: the biochemical & molecular basis of disease, 19-23 november 2001, cape town, south africa, 121. salganik, r.i., 2001: the benefits and hazards of antioxidants: controlling apoptosis and other protective mechanisms in cancer patients and the human population. j. amer. coll. nutr. 20, 464-472. schulz, h., joubert, e., schütze, w., 2003: quantification of quality parameters for reliable evaluation of green rooibos (aspalathus linearis). eur. food res. technol. 216, 539-543. smit, w.a., knox-davies, p.s., 1989: elimination of diaporthe phaseolorum and neocosmospora vasinfecta from rooibos tea seeds by hot-water treatment and acid scarification. phytophylactica 21, 297-299. snijman, p., gelderblom, w.c.a., joubert, e., green, i., 2003: biological activity of selected flavonoids of rooibos (aspalathus linearis). in: proceedings of the 1st international conference on polyphenols and health (abstracts), vichy, france, 358. clermond-ferrand, inra, france. snyckers, f.o., salemi, g., 1974: studies on south african medicinal plants. part i. quercetin as the major in vitro compound of rooibos tea. j. sa chem. inst. 27, 5-7. standley, l., winterton, p., marnewick, j.l., gelderblom, w.c.a., joubert, e., britz, t.j., 2001: influence of the processing stages on antimutagenic and antioxidant potentials of rooibos tea. j. agric. food chem. 49, 114-117. steuer, b., schütze, w., schulz, h., joubert, e., 2000: analytische charakterisierung des fermentationsprozesses von rotbuschtee (aspalathus linearis). deutsche gesellschaft für qualitätsforschung (pflanzliche nahrungsmittel) e.v., vortragstagung in karlsruhe, tagungsband, 257260. tiedtke, j., marks, o., 2002: rooibos – the new „white tea“ for hair and skin care. euro cosmetics 6, 16-19. toyoda, m., tanaka, k., hoshino, k., akiyama, h., tanimura, a., saito, y., 1997: profiles of potentially antiallergic flavonoids in 27 kinds of health tea and green tea infusions. j. agric. food chem. 45, 2561-2564. van heerden, f.r., van wyk, b.-e., viljoen, a.m., steenkamp, p.a., 2003: phenolic variation in wild populations of aspalathus linearis (rooibos tea). biochem. sys. ecol. 31, 885-895. von gadow, a., joubert, e., hansmann, c.f., 1997a: comparison of the antioxidant activity of rooibos tea (aspalathus linearis) with green, production and quality aspects of rooibos tea and related products 143 oolong and black tea. food chem. 60, 73-77. von gadow, a., joubert, e., hansmann, c.f., 1997b: effect of extraction time and additional heating on the antioxidant activity of rooibos tea (aspalathus linearis) extracts. j. agric. food chem. 45, 1370-1374. von gadow, a., joubert, e., hansmann, c.f., 1997c: comparison of the antioxidant activity of aspalathin with that of other plant phenols of rooibos tea (aspalathus linearis), alpha-tocopherol, bht and bha. j. agric. food chem. 45, 632-638. winterton, p., 1999: antioxidant activity of rooibos tea (aspalathus linearis) in model lipid and radical generating sytems. m. sc. thesis, university of stellenbosch, stellenbosch, south africa. addresses of the authors: e. joubert, arc infruitec-nietvoorbij, private bag x5026, 7599 stellenbosch, south africa, e-mail: joubertl@arc.agric.za h. schulz, federal centre for breeding research on cultivated plants, institute of plant analysis, neuer weg 22-23, d-06484 quedlinburg, germany, email: h.schulz@bafz.de (corresponding author) 144 elizabeth joubert, hartwig schulz << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false 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<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> >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice art23 journal of applied botany and food quality 82, 141 147 (2009) humboldt university of berlin, institute for horticultural sciences do soilless culture systems have an influence on product quality of vegetables? n. gruda (received march 28, 2008) summary in the horticulture industry, the focus has traditionally been on yield. however, consumers’ interest worldwide in the quality of horticultural products has increased in the recent past and will become the driving force in the future. soilless culture systems (scss), the most intensive production method in today’s horticulture industry, are based on environmentally friendly technology, which can result in higher yields, even in areas with adverse growing conditions. however, using scss does not automatically result in the production of high-quality vegetables. numerous studies confirm that a scs enables growers to produce vegetables without quality losses compared to soil cultivation. an adaptation of cultural management to the specific cultural system, as well as crop demand, can further result in the improvement of the quality of horticultural products. introduction the significance of the nutritional quality of vegetables is increasingly important for greenhouse growers who want to meet the everincreasing demand of consumers in the highly competitive fresh vegetable market (fanasca et al., 2006). product quality is a complex issue, and it depends on several factors. a recent comprehensive review of product quality for greenhouse vegetables can be found in gruda (2005). it is necessary to differentiate between objective, measurable traits, and subjective quality parameters, for instance, taste. different countries worldwide establish their set of objective criteria for the quality of fresh fruits and vegetables. these official quality grades and standards for classification and evaluation are based on the codex alimentarius of the fao of the united nations and the world health organization (kader, 2001; gruda 2005). process quality, often found in the literature under the term, quality management, is another very important characteristic. while the product quality answers the questions of what has been produced, what extent this product fulfills the quality standards, and how the product corresponds to our quality expectations, the process quality answers the questions of how the product has been produced and which processes have been used during production. however, the dependence on the natural environmental conditions (stochastic influence variables) greatly complicates the control of production. the spatial and temporal heterogeneity for a plant’s development condition requires extraordinarily high precision of the processing control (schmidt, unpublished). the processes of greenhouse production have greatly advanced in the last several decades. this development has usually been accompanied with the development of scss, which is the most intensive and effective production method in today’s agriculture industry (morard, 1995; dorais et al., 2001a; grillas et al., 2001). the term „soilless culture“ is defined as the cultivation of plants in systems without soil „in situ“. in recent years, a multitude of innovative cultivation procedures using bags, mats, and containers, in addition to nutrient solutions, have been developed. these cultivation methods include systems without a solid medium, as well as aggregate systems, in which inorganic or organic substrates are used. scss guarantee flexibility and intensification and provide high crop yield and high-quality products, even in areas with adverse growing conditions (morard, 1995; grillas et al., 2001). they offer significant advantages: a reduction of soil-borne diseases and complete control over water and nutrient supplies. therefore, control over the concentration and composition of the nutrient solution, the common issue of scss, is more precise and the desiderated ratio of nutrients more exact without the interference of organic matter or cation exchange capacity in soil. however, consumers often criticize such production systems. this negative attitude toward scs-grown products is the result of greater ecological and health consciousness (keiser-gloor, 1990). controversy arises because of supposedly unnatural hydroponics production techniques, which is the result of artificial growth and low internal quality, such as taste and nutrient value, as compared to so-called healthier products of plants grown in soil. it concerns scss such as nutrient film technique (nft), plant plane hydroponics (pph), and rock wool (rw) more than those with organic substrates such as peat, wood fiber (wf), bark, and other organic materials, as well as vegetables more than ornamentals (schnitzler and gruda, 2002). furthermore, it is important to mention that in this complex combination of „soilless culture“ and „product quality“, not only the interests of the producers, but also an ideological background could be found. soil has the highest value in the production chain for the supporters of soil products. however, one may forget that although the development of a scs over the last twenty years began primarily for economic reasons (rivière and caron, 2001; benoit and ceustermans, 2004), there was also an important ecological advantage conferred from the use of methylbromide (vanachter, 1983). moreover, the recirculation of drain water changed from a recommended guideline into a widely accepted ecological option by the public (benoit and ceustermans, 2004), so that scss today are recognized as environmentally friendly cultivation procedures. lately, there have been some efforts to bring both positions a little closer, e.g., by use of organic fertilizers in scss (liedl et al., 2004; peet et al., 2004; succop and newman, 2004; brentlinger, 2007). microgreens are also being grown hydroponically and organically (brentlinger, 2007). indeed, vegetables grown in soilless culture can be certified as organic in some countries, for instance, in the u.s. aside from some general pieces of information about scss (schnitzler and gruda, 2002; savvas, 2003), no recent review exists concerning the product quality of horticultural products cultivated in these systems. the objective of this paper is, therefore, to present a brief review, regarding the influence that scss may have on the quality of these products. quality of product from soil and scss a principle in applied research is to use control treatment which, in this case, prompts the question: what control treatment should be compared with a scs? simply, the cultivation in soil. but, in fact, they are two different systems that are not really comparable. from this point-of-view, we can only speak for a system comparison, and this is not very easy. similar to a comparison between organic and 142 n. gruda conventional produce, a comparison between a scs and a conventional cultivation system, could be not explained convincingly due to poor methodology (giles, 2004) and the high degree of interactions. indeed, the only reliable way to compare soil with soilless systems is to place both systems in the most optimal growing conditions. however, for consumers and legislators, the main concerns are safety and product quality with scss. following this statement, it is also clear that an influence of scss on the product quality would be expected. one could suppose, in this case, that the results could have several different outcomes: (i) a soilless product is equal, (ii) better or (iii) worse in quality than a product grown in soil. among all freshly produced vegetables, the tomato ranks as the highest in consumer preference worldwide. in europe, canada, and in the large horticultural industry complexes in the u.s., 95% of greenhouse tomatoes are produced in scss (peet and welles, 2005). as shown in tab. 1, all three hypotheses regarding the quality of tomatoes produced in scss may be possible. the contradictory results can sometimes be attributed to incomparable data, resulting from differences in growth factors, such as types of fertilizers, climates, plant cultivars, and variations in experimental design (künsch et al., 1994a; schnitzler and gruda, 2002). furthermore, the physiological state of ripeness is important, e.g., firmness and composition (schnitzler et al., 1994; petersen et al., 1998). most of the recent literature indicates that there are no objective differences between quality properties of tomato fruits produced in conventional or a scs (first column of tab. 1). künsch et al. (1994a) compared the quality of soilless (rw culture) and conventional tab. 1: quality properties of tomato fruits – a comparison between soil and soilless culture, according to different authors.* properties no differences between soil and soilless culture systems (+) soil culture (+) soilless culture systems uniformity weight massantini, 1962; yuxian et al., 1997; massantini et al., 1988 size abak and celikel, 1994; alan et al., 1994 massantini, 1962; gül and sevgican, 1992; gül and sevgican, 1994 alan et al., 1994; yuxian et al., 1997 consistency, texture gül and sevgican, 1992; gül and sevgican, massantini, 1962; benoit and ceustermans, 1994; thybo et al., 2005 1987; massantini et al., 1988; osvald and petrovic, 1997; gilinger pankotai et al., 1998 dry matter granges, 1980; simitchiev et al., 1983; bævre, massantini, 1962; garan’ko, 1968; bævre, granges, 1980; 1985; abak and celikel, 1994; gilinger 1985; mauromicale et al., 1996; özçelik and noguera et al., 1988 pankotai et al., 1998; thybo et al., 2005 akilli, 1999; yuxian et al., 1997 sugar simitchiev et al., 1983; bævre, 1985; buret massantini, 1962; garan’ko, 1968; granges, and duprat, 1985; mars et al., 1985; 1980; bævre, 1985; lãcãtus et al., 1995; schnitzler et al., 1994**; gilinger pankotai mauromicale et al., 1996 et al., 1998 massantini, 1962 fiber soluble solids buret and duprat, 1985; mars et al., 1985; benoit and ceustermans, 1987; alan et al., gül and sevgican, 1992; gül and sevgican, 1994; mauromicale et al., 1996; yuxian et al., 1994; alan et al., 1994; çelikel, 1999a; thybo 1997; özçelik and akilli, 1999 et al., 2005 vitamins granges, 1980; simitchiev et al., 1983; gül benoit and ceustermans, 1987; lãcãtus et al., and sevgican, 1994; abak and celikel, 1994; 1995; mauromicale et al., 1996; özçelik and alan et al., 1994; gilinger pankotai et al., akilli, 1999 1998; çelikel, 1999a carotenoids lãcãtus et al., 1995 granges, 1980 acidity simitchiev et al., 1983; buret and duprat, benoit and ceustermans, 1987; alan et al., granges, 1980; 1985; mars et al., 1985; gül and sevgican, 1994; özçelik and akilli, 1999 thybo et al., 2005 1994; alan et al., 1994; gilinger pankotai et al., 1998; gül and sevgican, 1992; schnitzler et al. 1994; abak and celikel, 1994; çelikel 1999a minerals simitchiev et al., 1983; benoit and ceustermans, 1987; gilinger pankotai et al., 1998 taste künsch et al., 1994b; osvald and petrovic, osvald and petrovic, 1997; massantini et al., 1997; auerswald et al., 1996; granges et al., 1988, yuxian et al., 1997 2000; thybo et al., 2005 *based on santamaria and valenzano (2001), modified and supplemented with results from other studies. ** results for sugar/acid ratio. + = system advantages for different properties soilless culture and product quality of vegetables 143 tomatoes produced in the same variety. although the sugar/acid ratio was significantly higher in soilless produced tomatoes, no sensory and few qualitative differences were observed between fruit produced by these two methods. also, abak and celikel (1994) found no differences between organic substrates and rw or soil with respect to fruit size, acidity, dry matter, and vitamin c content of tomatoes. furthermore, no differences were found between the cultivation in different systems in the greenhouse, such as aeroponics, nft, rw, and soil during two growth periods (gysi et al., 1997). sometimes, the properties in product quality were altered under the influence of different cultivation methods. according to auerswald et al. (1996), the content of reducing sugars, titratable acids, ascorbic acid, and carotenoids in tomatoes alternated under the influence of three cultivation methods, nft, pph, and cultivation in the soil, during the course of one year. however, sensory tests revealed no differences in taste or consistency due to the methods of cultivation. recently, thybo et al. (2005) reported that for most tomato fruit sensory characteristics, the greatest variation was due to differences in variety, followed by maturity, harvest time, and electric conductivity (ec), while the type of growth medium (soil or rw) had little or no effect. in addition, no significant differences are found in literature for other products: lettuce (künsch et al., 1996; siomos et al., 2001), paprika (lãcãtus et al., 1995), melon (güler et al., 1995), or strawberry (paraskevopoulou-paroussi and paroussis, 1995; fernandez et al., 2006) harvested from the scss and soil. only a few studies have indicated that better tomato fruit quality can be obtained from plants cultivated in soils than in a scs (tab. 1). in some instances, the same author showed a similar or better characteristic, e.g., dry matter between treatments (granges, 1980). this result can also be found in literature for other products, e.g., lettuce (siomos et al., 2001). the indispensable requirement that a scs does not negatively affect the product quality assumes the proper use of scs procedures and an adapted culture technology. for example, it is well known that scss represent a finite buffer capacity regarding water and fertilizer supply, as well as ph-value of the nutritive solution, due to relatively small and restrictive root areas. consequently, insufficient water supply and nutrient imbalance could induce blossom end-rot (ber) of greenhouse tomatoes. failure to provide plants with irrigation during noon hours in the summer could result in quality losses and potentially plant death in scss. plants that are cultivated in the soil have a better chance of recovering without visible quality deficiencies. despite indications that the quality of scs crops may be worse than those grown in soil, one should keep in mind that plant abnormalities are not necessarily „soilless culture-specific“, but caused by deficiency of water supply at high temperatures. according to adams (2002), the most common cause of ber is a combination of both high temperature and water stress, regardless of production system. for properties, such as uniform weight, size, and consistency, scs tomatoes present a better fruit quality than tomatoes grown in soil. according to granges (1980), tomatoes grown in greenhouse soil have higher acidity and less reduced sugars than those grown in nft systems. benoit and ceustermans (1987) found that tomatoes produced in a nft-system were firmer and richer in vitamin c than the soil-grown plants. tomato fruit also contains more sugar, acid, and sodium, which result in a more distinctive taste. moreover, the nitrate content of nft-grown fruits was considerably lower, while the phosphorus, potassium, calcium, and magnesium concentrations were practically identical to those in the soil grown plants. rouphael et al. (2004) reported that no differences were observed in dry matter or total protein content, while carbohydrate concentration (glucose, fructose, sucrose, and starch) was higher in scss zucchini (cucurbita pepo l.) cv. ‘afrodite’ over soil culture. for several good reasons, there are far fewer reports comparing the product quality of ornamentals and nursery shrubs in scss and soil. generally, (i) scss have proven to be very suitable for the production of flowers, (ii) substrate cultures for pot or container plants have become the normal technique, and (iii) these systems are regarded by consumers as more environmentally friendly for ornamentals and nursery plants than for vegetable production (schnitzler and gruda, 2002). however, using scss does not automatically always result in high-quality products, although it has been shown that it is possible to produce soilless crops without quality losses. additionally, there are a lot of references (e.g. for tomatoes, second column of tab. 1) that indicate that by using scss, the product quality could be improved. comparing different systems requires primarily optimizing them. only under these circumstances, will it be possible to make a direct and accurate comparison. since many scss use a solid growing medium, the comparison between the substrates could be also applicable. product quality for different growing media as soon as substrates began to be environmentally hazardous in the 1980s, a strong emphasis was placed on research for modern horticultural techniques to comply with ecological mandates (benoit and ceustermans, 2004). consequently, a lot of new organic growing media, based on renewable raw materials, were and continue to be investigated. nowadays, the utilization, nature of materials used for scss, and growing media are diverse (gruda et al., 2005). as in the above example, the large spectrum of substrates offered in horticulture along with the importance of the cultural guidelines should be emphasized. only adapted cultural guidelines will confer the advantages of substrates and culture procedures for successful cultivation. physical, chemical, and biological characteristics of the substrates must correlate with water and fertilizer supply, climate conditions, and plant needs (gruda and schnitzler, 2000a, 2000b, 2004a, 2004b, 2006). these differences between growing media have to be considered. for example, in a comparison between peat and its substitutes, such as bark, wood fiber substrate, paper and straw substrates, the activity of microorganisms must be evaluated. in order to build up their own body protein components, these microorganisms need mineral nitrogen (n), which they gain from the available n content in the substrate. consequently, n would not be readily available for the plants. this effect is one of the most important factors leading to potential quality losses (gruda et al., 2000). it is possible that given equal n tomato transplants grown in a (wood fiber substrate), wfs will not grow as well (e.g., more slowly and with n-deficiency symptoms) as one produced in a peat substrate. thus, transplant producers may determine that using wfs has a negative effect on the quality of their product (= transplants). however, it was also shown that this growth-retarding effect, due to n immobilization in wf, can be eliminated by n-impregnation of the substrates and additional n fertilization during cultivation. thus tomato transplants, which were grown on impregnated wfs substrate, showed the same quality as plants that were cultivated in peat (gruda et al., 2000). in conclusion, each substrate requires its own optimum growing technology. the quality of the product reflects the adherence of different guidelines to each substrate. if we focus on peat, a highquality product could be expected (and not only quality). a similar case is if we focus on peat-free substrates. usually in optimal culture guidance for each substrate and there is no impact of substrate per se on product quality (abak and celikel, 1994; özeker et al., 1999; özçelik et al., 1999; schnitzler and gruda, 2002; angelis et al., 2001; lopez et al., 2004; parks et al., 2004). recently, rodriguez et al. (2006) investigated different combinations of media (coarse perlite, medium perlite, and pine bark) and containers (polyethylene 144 n. gruda bags and plastic pots) for hydroponic production of ‘galia’ muskmelons (cucumis melo l.) and found that fruit yield and fruit quality were not affected by any combination of media and containers. the average content of soluble solids was generally greater than 10 degrees brix. possible ways to improve the product quality due to scss climate conditions could have enormous influences on the product quality of greenhouse fresh vegetables. they do not only affect the physiological processes and lead to differences in appearance of vegetable products, but they also influence the internal quality of vegetables. sensory ingredients such as sugars, acids, and flavor substances as well as vitamins and secondary plant compounds can be affected by changing the climate condition in the greenhouse (gruda, 2005). however, according to ho (2004) the key to the future of glasshouse production will be the further development of scss. the use of a scs not only makes it possible to have better control over water and nutrient supplies, but such a system is ideal for holistic control of crop development, crop yield, and product quality (ho, 2004). moreover, the improvement of yield will not be the sole driving force for the horticultural industry in the future. more importantly, qualities such as taste and health value for vegetables will become the criteria for the consumer’s choice (ho, 2004). several properties of the nutrient solution can effectively modify produced quality, for instance, ec or nutrient concentration, chemical forms of the elements, nutrient management, temperature of the nutrient solution, ph, etc. the following examples will illustrate the possibility of an improvement of vegetables product quality due to a precise amount of nutrients in scss: a) proper management of the salt concentration of the nutrient solution can provide an effective tool to improve the vegetable quality. many investigations have shown that using solutions with moderate electrical conductivity, achieved by adding nacl or nutrients, can improve the tomato fruit quality in terms of organic acidity and soluble solid (mizrahi and pasternak, 1985; sonneveld and welles, 1988; adams, 1989, 1991; cornish, 1992; petersen et al., 1998; adams and ho, 1989; elia et al., 2001; de pascale et al., 2001). moreover, an increase in the dry matter of tomato fruits occurred due to an active osmotic adjustment of plants to guarantee further water uptake under high saline conditions (hasegawa et al., 2000; plaut et al., 2004). a detailed review of these effects is presented in dorais et al. (2001b). furthermore, sato et al. (2006) found an increase not only in sugar content, but also in the organic and some amino acids that may contribute to a better taste (nelson, 2002) of tomato fruits, due to a nacl application in the nutrient solution. recently, consumers’ awareness increased concerning healthpromoting compounds and properties such as antioxidant capacity and nutritional value in vegetables (d’amico et al., 2003; dumas et al., 2003). krauss et al. (2006) investigated the influence of three different salt levels (ec = 3, 6.5, and 10 ds m-1) in scss grown tomatoes. rising ec-values of the nutrient solution increased vitamin c, lycopene and ß-carotene (the precursor to vitamin a) in fresh fruits up to 35%. the phenol concentration was tendentiously enhanced, and the antioxidative capacity of phenols and carotenoids increased on a fresh weight basis. additionally, the higher ec values caused an increase of total soluble solids and organic acids, parameters determining the taste of tomatoes. an enhancement of some of these health-promoting substances was also found in earlier investigations by petersen et al. (1998) affirming the hypothesis that saline water may increase desirable compounds in tomatoes. similar results were also obtained for sweet pepper and cucumber (sonneveld and van der burg, 1991; trajkova et al., 2006), eggplant (savvas and lenz, 1994), celery (pardossi et al., 1999), as well as watermelon (colla et al., 2006). rouphael et al. (2006) reported that increasing salinity from 2.0 to 4.1 ds m-1 improved fruit quality of zucchini squash with regard to a higher content of dry matter, reduced sugars, starch, total carbohydrates, and vitamin c. however, at some point increases in salinity limit marketable yield, increase the incidence of ber, and reduce the fruit size. manipulating the indoor climate, such as humidity, temperature, and ambient co2 level, may offset the negative effect of high salinity on yield and fruit quality (dorais et al., 2001b). an unequal ec achieved with a split root system was suggested for growing tomato plants in hydroponics, in order to avoid or mitigate these high salinity issues, and as a consequence, to improve both yield and fruit quality (tabatabaie et al., 2004). b) fanasca et al. (2006) investigated the effect of cationic proportions (k/ca/mg) in the nutrient solution on fruit quality (quality attributes and antioxidant content) of tomatoes grown in soilless culture and demonstrated that a high proportion of k in the nutrient solution increased the attributes and antioxidants’ content (especially lycopene) of tomato fruit, whereas a high proportion of mg in the solution improved the total antioxidant activity of tomato, cv. ‘lunarossa’. antioxidants are believed to be important in the prevention of diseases such as cancer and cardiovascular disease. lycopene is one of the main antioxidants found in fresh tomatoes and processed tomato products. the lycopene content also accounts for the redness of the fruit, which is one of its main qualities that interests the industry and consumers (dumas et al., 2003). c) different strategies are developed to maintain the nitrate limit set by the authority, although recent literature shows that methaemoglobinaemia has been linked to endogenous nitrite production (leifert and golden, 2000; trewavas, 2004; gruda, 2005). closed scss or the use of only small volumes of substrate is regarded as a possibility to control nitrate contents during critical periods (shinohara and suzuki, 1988; künsch et al., 1996; matthäus, 1996; schnitzler and gruda, 2002). gonnella et al. (2004) reported that the replacement of the nutrient solution with rain water three days before harvesting resulted in one third of the nitrate reduction in leaves. these results are in agreement with the findings of martignon et al., 1994 for endive and celery. by eliminating 90% of n in the nutrient solution one week prior to harvest, the nitrate content of endive (chicorium endivia l. var. crispum hegi) leaves was halved and decreased by 56% and 32%, in leaf blades and in the ribs of celery, respectively (martignon et al., 1994). environment concerns effective greenhouse production means dealing with environmental conditions. location properties relate to components such as: climate (irradiation, temperature, the length of the day, water balance, etc.), edaphic (structure, chemical and biological soil properties, water and air content at different water tensions), and management (cultivation measures) factors. a recent trend shows that protected cultivation has become more common in areas with mild climates, but not in areas with severe climates where it originated (tognoni et al., 1999; gruda, 2005). however, sometimes, although the climate conditions for plant cultivation are adequate, the lack of soil properties, such as salinization, erosion, poor soils structure, infertility, contamination, etc., makes a profitable greenhouse production in those areas impossible. an approach seems to be the development of scss. in particular, these systems are widely used in countries, such as israel or those in the mediterranean region, where favorable climate conditions and problematic soil properties exist. since this paper referred only to the quality of vegetables, some important general aspects regarding the application of a scs have yet to be considered. through the application of scss, an increase soilless culture and product quality of vegetables 145 in the efficiency of fertilizers and water use should be emphasized. moreover, due to the maintenance of hygienic conditions and an integrated pest management, a reduction of pesticides rather than in conventional greenhouse production could be realized. conclusion in conclusion, horticultural production through scss is environmentally friendly and enables application of specific quality management. however, using scss does not automatically result in the production of high-quality vegetables. the adaptation of cultural management to the specific conditions of the system or substrate as well as crop demands can improve their product quality. high yields do not automatically imply high quality, therefore, a compromise between both needs to be established. literature abak, k., celikel, g., 1994: comparison of some turkish originated organic and inorganic substrates for tomato soilless culture. acta hort. 366, 423427. adams, p., 2002: nutritional control in hydroponics. in: savvas, d., passam, h.c. 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maturity. j. sci. food and agri. 85, 2289-2296. vanachter, a., 1983: an evaluation of the soil disinfection practices in belgium and problems involved with efficiency, safety and impact on the environment. acta hort. 152, 41-48. yuxian, x., xiufeng, w., papadopoulus, a.p., 1997: a multiplayer soilless system for greenhouse tomato production pioneered in shandong province, people’s republic of china. horttechn. 7, 169-176. address of the author: dr. habil. n. gruda, humboldt university of berlin, institute for horticultural sciences, lentzeallee 55/57, 14195 berlin, germany e-mail: nazim.gruda@rz.hu-berlin.de << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true 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/pagesize [907.087 680.315] >> setpagedevice art07_fischer.indd journal of applied botany and food quality 85, 49 54 (2012) 1universidad pedagógica y tecnológica de colombia, facultad de ciencias agropecuarias, grupo de ecofi siología vegetal, tunja, colombia 2universidad nacional de colombia, facultad de agronomía, departamento de agronomía, bogotá, colombia 3universidad de córdoba, facultad de ciencias agrícolas, montería, colombia 4universidad pedagógica y tecnológica de colombia, facultad de ciencias agropecuarias, grupo de investigaciones agrícolas, tunja, colombia physicochemical behavior of riesling x silvaner grapevine fruit under the high altitude conditions of colombia (south america) p.j. almanza-merchán1, g. fischer2, a. herrera-arévalo2, a. jarma-orozco3, h.e. balaguera-lópez4 (received august 30, 2011) 1 in this article the maturation stage also includes the ripening of the berry on the plant summary the valle del sol (sun valley) of the boyacá department is a zone with temperate tropical climate conditions (2,500 m above sea level) that is suitable for the production of grapes for quality wine. the objective of this investigation was to study the physical and chemical behavior during growth and development of the grapevine fruit var. riesling x silvaner, produced for winemaking, in the municipality of corrales (boyacá, colombia). to determine the physical and chemical characteristics of the fruit starting at 28 days after anthesis (daa), 14 weekly samplings were carried out, in each of which three clusters were taken from randomly selected plants. the development of the berry lasted 119 daa in which three stages were defi ned: herbaceous, veraison and maturation1. the herbaceous stage ended at 63 daa, the veraison period lasted 14 days and ended at 77 daa, whereas the maturation and ripening stage lasted 42 days; no period of overmaturity was observed. the behavior of the fresh mass, dry mass and diameter of the fruit followed a double sigmoid curve. during berry development, total soluble solids (from 5.03 to 23.73 °brix at the harvest point), ph (from 2.88 to 3.71) and technological maturity index (from 2.27 to 21.84) all increased, whereas total titratable acidity decreased from 3.96 to 1.11%. introduction vinifera grapevines were introduced during the last 450 years to north and south america, south africa, south east asia and australia; and as a result, about 50 years ago, a grapevine industry developed in the tropics with entirely different climatic and growing conditions than those in the “traditional” subtropical and temperate regions (lavee, 2000). it is well known that the tropics are characterized by a uniform annual photoperiod and uniform temperature conditions without marked temperature seasons in all altitudes (lauer, 1986). phenology is the study of phases, physiological events or periodic and repetitive activities of the life cycle of plants that occur seasonally throughout the year in response to the climate (mantovani et al., 2003; gris et al., 2010). lambers et al. (1998) defi ned growth as an increment in dry mass, volume, length or area, which in many cases involves the division, expansion, and differentiation of cells. according to the above, knowing the phenology of a species, we can determine the potential of a region to produce a crop within the limits of its climate pattern (morlat and bodin, 2006; webb et al., 2007). and with studies of growth and development, we can establish how a plant or organ, in this case the fruit, behaves under certain conditions, which may be useful, among other factors, for scheduling cultural practices such as fertilization, irrigation, plant protection and time of harvest (mullins et al., 1992). the growth of the grapevine fruit (vitis vinifera l.) consists of two successive sigmoid cycles, each with distinct characteristics (coombe, 1992). the fi rst cycle of the berry formation starts with increased cell division in the pericarp tissues, this process gradually changes to cell elongation, which slows at the end of the fi rst sigmoid cycle. in this state, the berry is green and grows slowly, but malic acid accumulation in the pericarp occurs. the second cycle begins with the accumulation of sugar, softening and coloring of the berry and increase in size (coombe, 1992; hidalgo, 2002; salazar and melgarejo, 2005). according to conde et al. (2007), in many grape cultivars, the fi rst growth phase is followed by a phase where growth is temporarily stopped. the duration of this phase is specifi c for each cultivar and ends with the completion of the herbaceous berry stage. after this period of non-growth, a second stage takes place, veraison, in which the skin of the berry starts changing color, indicating the onset of maturation. the most representative changes of the composition of the grape fruit occur during this maturation stage. conde et al. (2007) mention that the berry passes from a state where it is small, hard and acidic, with low sugar content to a state where the fruit becomes larger, softer and sweeter, with a lower acid content, and with a characteristic fl avor and color particular to the variety. during fruit development of the grape, major changes occur at the biochemical level that lead to fruit ripening and determine its quality, of which, the most representative are the accumulation of total soluble solids (mainly sugars), increased ph and technical maturity index, decreased acidity (gris et al. 2010; almanza-merchán and balaguera-lopez, 2009; almanza et al., 2010), degradation of chlorophyll, accumulation of pigments in the epidermis, the synthesis of aromatic substances and fl avor changes (conde et al., 2007; ali et al., 2011). these features are important to the monitoring of the development and ripening of the berry, mainly in areas where new varieties of grapes are to be introduced (gris et al., 2010) in order to determine its productive potential. the valle del sol (sun valley) of the boyacá department (colombia) is characterized by a cold tropical climate zone suitable for the cultivation of grapevine for the production of quality wines (quijano, 2004). early studies in these agro-ecological conditions were performed with the pinot noir variety, which identifi ed four phenological stages, herbaceous, ripening, maturation and overripening (almanza-merchán and balaguera-lopez, 2009), and showed that the development of the fruit until harvest lasted 126 days after anthesis (daa), during this lapse 826.2 growing degree days accumulated (almanza et al., 2010). because the physicochemical behavior of some important varieties for winemaking under conditions of high tropics is not known, the objective of this research was to study the behavior during growth and fruit development of grapevine ‘riesling x silvaner’. materials and methods this research was conducted between july and november of 2009, in a commercial vineyard in the municipality of corrales (boyacácolombia), situated at 5°48’30 n and 72°58’35” w at an altitude of 50 p.j. almanza-merchán, g. fischer, a. herrera-arévalo, a. jarma-orozco, h.e. balaguera-lópez riesling x silvaner grapevine in the high altitude of colombia 2,450 m. the climate conditions during the experiment are shown in tab. 1, where the average temperature value was 16.7 °c (maximum and minimum temperatures 22.3 °c and 10.4 °c, respectively), the relative humidity was 86.3% and the total rain fall was 243.11 mm. an average of 4.2 h d-1 of direct sunshine (tab. 1) and a high solar insolation, with a mean value of 476 cal cm-2 d-1, were recorded. the soils are stony and a light-textured, sandy loam type with a low natural fertility. the evaluated variety corresponds to the clonal selection of vitis vinifera l., ‘riesling x silvaner’, also known as riesling becker, provided by the vineyard “loma de puntalarga” in nobsa (boyacá). the crop was established 8 years ago and set at a distance of 1.2 x 0.8 m between rows and plants, respectively, using the guyot simple pruning type at a three-wire trellis. for the experiment, 42 clusters were randomly selected from an equal number of plants. each week three clusters were harvested randomly. from each cluster, 10 fruits were taken to determine the physical characteristics and 10 fruits for the chemical characteristics. fourteen samples were taken from the time the fruit size allowed for the taking of these measurements (28 daa). the fruit variables measured were: fresh and dry mass (balance, 001 g precision; dry mass after subjecting the fruit to 75 °c for 48 h), equatorial diameter and length, absolute growth rate (according to hunt, 1990), total titratable acidity (tta; aoac, 1990; expressed as percentage of tartaric acid), total soluble solids (tss; digital refractometer hanna instruments hi 96801, 0-85 °brix; clarkson laboratory & supply inc., chula vista, ca), and ph (potentiometer previously calibrated with buffer solutions of ph 7.0 and 4.0). the technological maturity index (tmi) was obtained through the relationship between tss and tta. each cluster corresponded to one repetition in each sample. the data were analyzed using descriptive statistics and the average and standard error were calculated. in addition, statistical models with the greatest adjustment were determined. for the data analysis, sas v. 8.1e (cary, nc) program was used. results and discussion phenological stages of the grapevine fruit the results indicate that the ‘riesling x silvaner’ fruit presented three well-defi ned stages of development: herbaceous, veraison and maturation and ripening (fig. 1). the herbaceous stage lasted from fruit set to 63 daa, representing the longest stage. agustí (2008) and reynier (1995) indicate that in this period cell division is predominant which favors rapid growth of the fruit; and the berry begins its evolution and has a green color with a high photosynthetic capacity and consistency. the veraison stage ended at 77 daa with a period of 14 days. some authors state that this stage marks the onset of maturation; the berries lose their fi rmness and change color (ali et al., 2011; conde et al., 2007). however, with riesling x silvaner, a cultivar with “white” fruits in this stage, the berries take on a translucent appearance. according to mullins et al. (1992), at this stage the seed reaches physiological maturity, while respiration, photosynthesis and chlorophyll content decrease; the fruit is covered with pruina (waxy epicuticular coating of the grape) and herbaceous aromas decline (hidalgo, 2002; reynier, 1995). the maturation stage culminated at 119 dda, registering a span of 42 days. during this period, the berry has a quick gain of biomass (fig. 1) and an accelerated evolution of biochemical components (hidalgo, 2002; salazar and melgarejo, 2005). contrary to that reported by other authors for ‘pinot noir’ (almanza-merchán and balaguera-lópez, 2009; almanza et al., 2010), with ‘riesling x silvaner’ there wasn’t an over ripeness stage, but the onset and duration of the other periods coincide with those found by almanza-merchán and balaguera-lópez (2009) and differs only in duration of maturation which was observed by almanza et al. (2010) for ‘pinot noir’ as lasting 35 days. these results confi rm that the duration of the phenological stages can vary representatively between varieties, climate and geographical location (bodin and morlat, 2006; webb et al., 2007; gris et al., 2010). knowing the duration of the phenological phases in specifi c tab. 1: environmental conditions of the corrales municipality (boyacá, colombia) during the study period (july-november, 2009). days after antesis precipitation mean temp. mean max. temp. mean min. temp. sun shine relative humidity (daa) (mm) (°c) (°c) (°c) (h d-1) (%) 7 2.8 16.7 21.6 10.5 3.8 87.4 14 14.3 16.8 21.2 10.7 4.4 89.5 21 2.31 16.6 21.6 10.0 5.5 90.2 28 5.93 17.1 23.5 10.8 5.5 89.8 35 28.11 16.3 21.1 10.4 4.3 90.4 42 0.61 16.6 21.6 10,4 5.3 86.6 49 2.2 16.7 22.4 9.8 5.2 87.0 56 40.9 16.1 21.6 10.0 2.9 87.6 63 0.7 16.9 21.8 9.7 4.8 85.6 70 2.2 16.8 21.5 9.5 4.1 86.5 77 0.0 16.9 23.2 10.2 4.4 85.1 84 22.0 16.3 22.3 10.1 3.1 79.1 91 48.71 16.8 23.4 11.0 2.1 88.4 98 11.6 17.0 22.3 11.1 2.5 87.0 105 28.72 17.1 23.4 11.8 3.0 87.3 112 0.0 17.2 22.8 10.7 5.2 85.5 119 32.02 17.5 23.8 11.0 6.8 74.5 mean value 243.11* 16.7 22.3 10.4 4.2 86.3 *accumulated precipitation during the study period riesling x silvaner grapevine in the high altitude of colombia 51 agro-ecological conditions is essential in viticulture, as it facilitates the selection of crop management practices. physical fruit characteristics the accumulation of dry and fresh weight of fruits conformed to a logistic growth model and had a typical behavior of a double sigmoid curve (fig. 1), characteristic for this species (almanza et al., 2010; opara, 2000; coombe, 1976). the dry mass of the fruit presented lower values until 63 daa, when the fi rst sigmoid phase was completed, and coincided with the herbaceous stage, characterized by active cell division (hernandez, 2000). in this period, the absolute growth rate (agr) was also low, but then the dry mass showed a continuous rise until the harvest at which point 0.33 ± 0.019 g was accumulated by the berry. the mass gain was more pronounced between 77 and 84 daa, and from day 105 on (fig. 1a). the absolute growth rate for dry mass had maxima at 49 and 84 daa and showed a dramatic increase from 112 daa until harvest, when it reached its maximum value (0.0129 g d-1). this behavior also indicated that the dry mass did not follow an asymptotic curve (fig. 1a and c), possibly due to the continuous accumulation of photoassimilates in the last stage of fruit growth (grange, 1996). a low gain of fresh mass up to 42 daa was observed, after that, a rapid increase ending at 49 daa followed by a slowing until 63 daa was observed. therefore, agr peaked at 49 daa (0.04 g d-1) and then declined. subsequently, increases were observed for fresh mass, mostly between 70 and 77 daa and after 112 daa, meaning agr showed high values in the same periods. at harvest, the fresh mass and agr were 1.94 ± 0.05 g and 0.03 g d-1, respectively (fig. 1b y c). conde et al. (2007) mentioned that berry size from veraison to harvest almost doubles. at this stage, the cell elongation is the predominant process, infl uenced by the plasticity of the cell walls and the turgor pressure of the cells responsible for the sharp increase in volume and mass (coombe, 1960). it is possible that the increase in the concentration of sugars during ripening causes a decrease in osmotic potential and therefore an increase in fruit fresh mass produced by an increase in water holding capacity (coombe, 1960). longitudinal and equatorial diameters were adjusted to a cubic model and had a double sigmoid trend. the completion of the fi rst sigmoid stage (herbaceous stage) was at 63 daa similar to the behavior of fresh and dry weight of the fruit, but with the difference that diameter had a asymptotic growth at the end of the second sigmoid stage. a similar behavior was reported in the grape by dokoozlian (2000). the longitudinal diameter was greater than the equatorial one during the whole development of the fruit and at harvest the obtained values were 1,49±0,03 cm and 1,42±0,04 cm, respectively (fig. 2), which indicates that these fruits are bigger than those of the variety ‘cabernet sauvignon‘ which presents an average of 12 mm. � � �� �� � � � �� � � ��������� ���������� � �� � � �� � � � �� � � �������� ���������� � � � �� �� � � �� � � � �� � �� �� � �������������������������� �� �� �� �� �� �� �� �� �� �� �� ��� ��� ��� � � � �� �� �� � � � �� � �� �� � �������������� ������������ � ������� ������� ���������������� � �� �� ���� ������� ��� ��� � �������� ���������������������� � ���� ���� ����������� � ���������� �������� ���������� ���� ���� ���� ���� ���� ���� ���� ���� ���� ��� ��� ��� ��� ��� ��� ��� ��� ��� ��� ��� ���� ���� ���� ���� ���� ����� ����� ����� ����� ����� ����� ����� ����� ������������������������������������������������������������������������������������������� ���������� ���������� �� ���������� ������ ������� ������� ���� ������������ ������ ����� ��������� ������������������������������������������������������������������������������������������������ ������������������� �� �������������������������� �� �� �� �� �� �� �� �� �� �� �� ��� ��� ��� � �� � � �� �� �� � � ����������������������� ������������������������ ��������������������� ���������������������� ������ ��������������������� � �� � ������ � ���������������� ������ ������������������ � �� � ������� � ��������������� ���������� �������� ���������� ��� ��� ��� ��� ��� ��� ����� ��������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������������� ����������������������������� �������������������������������������� ���� �������������� ��� ��������� ����������� ��� ���������� ��� ���� �������� ���� ��������� ��� ���� ����������� ����������������������������������������������������������������������� �������� ������������������������������������������������������������������������������������������ ��������������������������������������������������������������������������������������� ������������������� ������ ������������ ��������������� ������������� ����������� ��������������� ����������������������������������������������������������������������������������������������� ������������������������������������������������������������������������������������������ �������������������������������������������������������������������������������������������� ������ ��� ���� ������ ��� �������� ��� ������� ����� ������ ��� ���� ������������������� ������ fig. 1: behavior of dry mass (a), fresh mass (b) and absolute growth rate (agr) (c) of the grapevine ‘riesling x silvaner’ fruit during growth and development under high altitude tropical conditions. vertical bars indicate the standard error of each mean (n = 3). rsme, root square mean error. fig. 2: behavior of the diameter of the grapevine ‘riesling x silvaner’ fruit during growth and development under high altitude tropical conditions. vertical bars indicate the standard error of each mean (n = 3). 52 p.j. almanza-merchán, g. fischer, a. herrera-arévalo, a. jarma-orozco, h.e. balaguera-lópez riesling x silvaner grapevine in the high altitude of colombia chemical characteristics of the fruit the concentration of chemical substances is important to the quality and quantity in the berries of the grape for growth and yield regulation (gil, 2004). the increase of the tss during the grape fruit development was described by a third degree polynomial. between 28 and 42 daa, 63 and 77 daa (veraison stage) and 98 and 112 daa, the tss remained almost constant, at other times there was a representative increase. at the end of the herbaceous stage, veraison and harvest, the fruit of ‘riesling x silvaner’ presented 14.53±0.35, 15.4±0.3 and 23.73±0.49 °brix, respectively (fig. 3a). accordingly, dokoozlian (2000) affirms that the grapes accumulate more sugars during maturation than many other fruits. the tss content found in our study at harvest is higher than those of the cabernet-sauvignon grape (giribaldi et al., 2010) and the superior cultivar (grangeiro et al., 2002), but lower than those in ‘pinot noir’ berries cultivated in a cold tropical climate (almanza-merchán and balagueralópez, 2009; almanza et al., 2010). 80 to 95 % of the total soluble solids are composed of sugars and the content is associated with the sugars dissolved in the cellular juice (osterloh et al., 1996), to a lesser extent they also contain organic acids, proteins, fats and several minerals. the sugar necessary for the growth and maturation of the berries must be imported principally from the leaves and to a lesser extent from the trunk and roots (dokoozlian, 2000), because sucrose is the principal sugar transported to the fruits, although it seems that a high activity of the acid invertase enzyme exists in the grape fruit (fillion et al., 1999) because the sugars with the highest concentrations are fructose and glucose (ali et al., 2011; herrmann, 2001). gil (2004) also reports that the principal cause of the increase of tss is the transport of sucrose from the leaves and reserve sites of the vascular and peripheral system, with a rate of 27 to 30 cm h-1. conde et al. (2007) mention that after the veraison period a continuous amount of glucose and fructose is accumulated in the vacuoles of the mesocarp cells, which possibly explains the increase of the tss of the ‘riesling x silvaner’ fruits. also, dokoozlian (2000) affi rms that the sugars also come from skeletons of carbon generated by organic acids and amino acids. there was a quadratic decrease of the tta as the fruit development increased; in the beginning of the herbaceous period, the acidic contents were high with values of about 4 %, nevertheless, in the herbaceous stage the tta remained almost constant with values about 2 %, whereas at harvest it presented the lowest concentrations with 1,09±0,06 % (fig. 3b), a value that can be considered to be a high as compared to the contents found in cabernet franc, merlot, sangiovese and syrah varieties (gris et al., 2010), but it is similar to the one found in ‘pinot noir’ (almanza-merchán and balaguera-lópez, 2009) under high altitude tropical conditions, which confi rms those mentioned by bluske (2008), that is, in high altitudes the values of acidity are high because there is minor respiration of malic acid compared with that of tartaric acid, and consequently produce wines that are too smooth. nevertheless, herrmann (2001) states that during grape maturation there is a greater decrease in malic acid than in tartaric acid. also, the continuous precipitations that appeared during the development of the berries could increase the taa, as was found by jubilee et al. (2010) in fruits of ‘cabernet sauvignon’, produced in the off-season. the berries synthesize only a small part of the acids (hardy, 1968), most of them are translocated from the leaves, as a consequence of the photosynthetic activity which is closely involved to its synthesis (gil, 2004). tartaric acid is accumulated during the initial stage of the berry development and its concentration is higher in the periphery of the developing berry. on the contrary, malic acid is stored in the cells of the pulp at the fi nal stage of the fi rst phase of growth (conde et al., 2007; de bolt et al., 2006). during maturation, this acid is metabolized, transformed into sugars and used as a source of energy during the maturation phase (conde et al., 2007), also it can be diluted by the water that accumulates in the fruit (almanzamerchán and balaguera-lópez, 2009) (processes that would explain its decrease [fig. 3b]). these acids give the acidity to the wine and in part determine its quality (conde et al., 2007). dokoozlian (2000) mentions that malic and tartaric acids make up approximately 90 % of the whole acidity. the remaining percentage in the berries depends on other acids such as citric (up to 5 % [herrmann, 2001]), succinic, lactic and acetic acids that are present principally in the maturation stage (conde et al., 2007). the fl avor of the grape fruit is principally the result of the acid/sugar ratio and the synthesis of aromatic compounds, or of precursors that take place at the same moment. the development of these characteristics will determine to a great extent the quality of the fi nal product (boss and davies, 2001). in congruity, the technological maturity index (tmi) of ‘riesling x silvaner’ increased rapidly up to harvest; conforming to a quadratic function, with the exception of the herbaceous stage, where the tmi remained almost constant. at harvest, the tmi was 21,84±1,27 (fig. 4a), this value is considered to be low as compared to indices found by grangeiro et al. (2002); gris et al (2010) and almanza et al. (2010). this might be explained by the high acid content (fig. 3b), since the measured tss �� � � � �� �� �� � � � � �� �� �� �� �� �������� ��������� ������ ��������������������� � �� � ����� � ������� �������������������������� �� �� �� �� �� �� �� �� �� �� �� ��� ��� ��� � � � �� � � �������� ��������� ������ ����������������� � � � ��� � ����� � � ���������� �������� ���������� ��� ��� ��� ��� ��� ��� ��� ��� ��� ������������������������������������������������������������������������������������������������ ��� ���� ���������� ���������� �� ���������� ������ ������� ������� ���� ������������ ������ ����� ����������������������������������������������������������������������������������������������� fig. 3: behavior of the total soluble solids (tss) (a) and the total titratable acids (tta) (b) of the grapevine ‘riesling x silvaner’ fruit during growth and development under high altitude tropical conditions. vertical bars indicate the standard error of each mean (n = 3). riesling x silvaner grapevine in the high altitude of colombia 53 was considered high (fig. 3a). jubileu et al. (2010) mention that the use of the maturation index must be done carefully, since an increase of sugar does not always represent a decrease of acidity and one cannot compare the different varieties of grape (rizzon and miéle, 2002), but it might be useful to compare the behavior of the same variety under different growing conditions. meanwhile, blouin and guimberteau (2004) affi rm that this index serves as a reference for an ideal harvest time from the wine point of view. nevertheless, jubileu et al. (2010) and gonçalves et al. (2002) coincide in affirming that the tss and the tta contents are of fundamental importance in monitoring the harvest point of grape fruits, making a better control of the quality of the raw material for wine making possible. the fruit development with values near to 2.5, and then it increases gradually during maturation due to the decrease of malic acid. this behavior aligns with dokoozlian (2000) who affi rms that the ph is a measurement of the concentration of hydrogen ions in the berry and is generally related to the acidity of the juice. the measured ph at the crop harvest was 3.71±0.01 (fig. 4b), an advisable value for securing quality wines, since, in accordance with rizzon and miéle (2002), and values of ph lower than 3.3 can negatively affect wine quality. conclusions under the agroecological conditions of the corrales municipality in boyacá (colombia), the fruit development of the ‘riesling x silvaner’ grape was 119 days from anthesis to harvest and its development could be explained effi ciently by a double sigmoid curve, with three defi nite stages: herbaceous, veraison and maturation. the herbaceous stage fi nished at 63 daa and was the longest period. the veraison period was the shortest (14 days) and fi nished at 77 daa, whereas the maturation stage lasted 42 days. during the development of the berries, tss increased, as did ph and tmi, but taa decreased. references agustí, m., 2008: crecimiento y maduración del fruto. in: azcón-bieto, j., talón, m. (eds.), fundamentos de fi siología vegetal, 519-535. 2nd edition. mcgraw-hill interamericana, madrid. ali, k., maltese, f., fortes, a.m., pais, m.s., choi, y.h., verpoorte, r., 2011: monitoring biochemical changes during grape berry development in portuguese cultivars by nmr spectroscopy. food chem. 124, 1760-1769. almanza, p.j., quijano-rico, m.a., fischer, g., chaves, b., balagueralópez, h.e., 2010: physicochemical characterization of ‘pinot noir’ grapevine (vitis vinifera l.) fruit during its growth and development under high altitude tropical conditions. agron. colomb. 28, 173-180. almanza-merchán, p., balaguera-lópez, h.e., 2009: determinación de los estadios fenológicos del fruto de vitis vinifera l. bajo condiciones del altiplano tropical en boyacá. rev. udca actual. divulg. cient. 12, 141-150. aoac, 1990: offi cial methods of analysis. 15th edition. association of offi cial analytical chemists, arlington, va. blouin, j., guimberteau, g., 2004: maduración y madurez de la uva. ediciones mundi-prensa, madrid. bluske, i., 2008: los vinos de altura. in: http://vinosdealtura.es/img/pdf/ producción.pdf; consulted: january, 2010. bodin, f., morlat, r., 2006: characterization of viticultural terroirs using a simple fi eld model i. validation of the water supply regime, phenology and vine vigour in anjou vineyard (france). plant soil 281, 37-54. boss, p.k., davies, c., 2001: molecular biology of sugar and anthocyanin accumulation in grape berries. in: roubelakis-angelakis, k.a. (ed.), molecular biology and biotechnology of grapevine, 1-33. kluwer academic publishers, dordrecht. conde, c., agasse, a., glissant, d., tavares, r., gerós, h., delrot, s., 2006: pathways of glucose regulation of monosaccharide transport in grape cells. plant physiol. 141, 1563-1577. conde, c., silva, p., fontes, n., dias, a.c.p., tavares, r.m., sousa, m.j., agasse, a., delrot, s., gerós, h., 2007: biochemical changes throughout grape berry development and fruit and wine quality. food 1, 1-22. coombe, b.g., 1992: research on development and ripening of the grape berry. amer. j. enol. viticult. 43, 101-110. coombe, b.g., 1976: the development of fleshy fruits. annu. rev. plant physiol. 27, 207-228. �� � � �� �� � � �� � � � � � �� �� �� �� �������� ��������� �������������������������� �� �� �� �� �� �� �� �� �� �� �� ��� ��� ��� � � �������� ��������� � � ������ ������������������ � � � ��� � ����� ������ ��������������������� � �� � ������ � ������ ���������� �������� ���������� ��� ��� ��� ��� ��� ��� ��� ����� �������������������������������������������������������������������������������������� ���������� ���������� �� ���������� ������ ������� ������� ���� ������������ ������ ����� ��������� ������������������������������������������������������������������������������������� fig. 4: behavior of the technological maturity index (tmi) (a) and the ph value (b) of the grapevine ‘riesling x silvaner’ fruit during growth and development under high altitude tropical conditions. vertical bars indicate the standard error of each mean (n = 3). the ph conformed to a third degree polynomial, which was characterized by a slight decrease at the beginning of the herbaceous stage and a rapid increase towards the end of the same period, whereas in the veraison phase it remained stable and again it increased up to 112 daa to remain stable up to the harvest (fig. 4b). in congruity, dokoozlian (2000) affi rms that the ph is a measurement of the concentration of ions hydrogen in the berry and is generally related to the acidity of the juice. in accordance with the latter author, the ph is relatively constant during the fi rst stage of 54 p.j. almanza-merchán, g. fischer, a. herrera-arévalo, a. jarma-orozco, h.e. balaguera-lópez coombe, b.g., 1960: relationship of growth and development to changes in sugars, auxins and gibberellins in fruit of seeded and seedless varieties of vitis vinifera. plant physiol. 35, 241-250. de bolt, s., cook, d.r., ford, c.m., 2006: l-tartaric acid synthesis from vitamin-c in higher plants. proc. natl. acad. sci. usa 103, 5608-5613. dokoozlian, n.k., 2000: grape berry growth and development. in: christenson, l.p. (ed.), raisin production manual, 30-37. university of california, agricultural and natural resources publication 3393, oakland, ca. fillion, l., ageorges, a., picaud, s., coutos-thevenot, p., lemoine, r., romieu, c., delrot, s., 1999: cloning and expression of a hexose transporter gene expressed during the ripening of grape berry. plant physiol. 120, 1083-1093. gil, g.f., 2004: fruticultura: madurez de la fruta y manejo poscosecha. ediciones universidad católica de chile, santiago. giribaldi, m., hartung, w., schubert, a., 2010: the effects of abscisic acid on grape berry ripening are affected by the timing of treatment. j. int. sci. vigne vin (special issue macrowine), 9-15. gonçalves, c.a.a., lima, l.c.o., chalfun, n.n.j., regina, m.a., alvarenga, a.a., souza, m.t., 2002: fenologia e qualidade do mosto de videiras ‘folha de figo’ sobre diferentes porta-enxertos, em caldas, sul de minas gerais. cienc. agrotec. 26, 1178-1184. grange, r., 1996: crecimiento del fruto. in: azcón-bieto, j., talón, m. (eds.), fisiología y bioquímica vegetal, 449-462. interamericanamcgraw-hill, madrid. grangeiro, l.c., leão, p.c.s., soares, j.m., 2002: caracterização fenológica e produtiva da variedade de uva superior seedless cultivada no vale do são francisco. rev. bras. frutic. 24, 552-554. gris, e.f., burin, v.m., brighenti, e., vieira, h., bordignon-luiz, m.t., 2010: phenology and ripening of vitis vinifera l. grape varieties in são joaquim, southern brazil: a new south american wine growing region. cien. inv. agr. 37, 61-75. hardy, p.j., 1968: metabolism of sugars and organic acids in immature grape berries. plant physiol. 43, 224-228. herrmann, k., 2001: inhaltsstoffe von obst und gemüse. ulmer verlag, stuttgart. hernández, a., 2000: introducción al vino de chile. colección en agricultura de la facultad de agronomía e ingeniería forestal. pontifi cia universidad católica de chile, santiago. hidalgo, l., 2002: tratado de viticultura general. ediciones mundi-prensa, madrid. hunt, r., 1990: basic growth analysis. plant growth analysis for beginners. unwin hyman, boston, ma. jubileu, b. da s., sato, a.j., roberto, s.r., 2010: caracterização fenológica e produtiva das videiras ‘cabernet sauvignon’ e ‘alicante’ (vitis vinifera l.) produzidas fora de época, no norte do paraná. rev. bras. frutic. 32, 451-462. lambers, h., chapin iii, f.s., pons, t.l., 1998: plant physiological ecology. springer-verlag, new york. lauer, w., 1986: das klima der tropen und subtropen. in: rehm, s. (hrsg.), grundlagen des pfl anzenbaues in den tropen und subtropen, 26-45. verlag ulmer, stuttgart. lavee, s., 2000: grapevine (vitis vinifera) growth and performance in warm climates. in: erez, a. (ed.), temperate fruit crops in warm climates, 343-366. kluwer academic publishers, dordrecht. mantovani, m., ruschel, a.r., sedrez dos reis, m., puchalski, a., nodari, r.o., 2003: fenologia reprodutiva de espécies arbóreas em uma formação secundáriada fl oresta atlântica. rev. árvore 27, 451-458. morlat, r., bodin, f., 2006: characterization of viticultural terroirs using a simple fi eld model based on soil depth – ii. validation of the grape yield and berry quality in the anjou vineyard (france). plant soil 281, 55-69. mullins, m.g., bouquet, a., williams, l.e., 1992: biology of the grapevine. cambridge university press, new york. opara, l.u., 2000: fruit growth measurement and analysis. hortic. rev. 24, 373-431. osterloh, a., ebert, g., held, w.h., schulz, h., urban, e., 1996: lagerung von obst und südfrüchten. verlag ulmer, stuttgart. quijano, m., 2004: ecología de una conexión solar. de la adoración del sol al desarrollo vitivinícola regional. cultura científi ca 2, 5-9. reynier, a., 1995: manual de viticultura. 5th edition. ediciones mundiprensa, madrid. rizzon, l.a., miéle, a., 2002: avaliação da cv. cabernet sauvignon para elaboração de vinho tinto. ciênc.tecnol. aliment. 22, 192-198. salazar, d.m., melgarejo, p., 2005: viticultura. técnicas del cultivo de la vid, calidad de la uva y atributos de los vinos. ediciones mundiprensa, madrid. santos, c.e., roberto, s.r., sato, a.j., jubileu, b.s., 2007: caracterização da fenologia e da demanda térmica das videiras ‘cabernet sauvignon’ e ‘tannat’ para a região norte do paraná. acta sci. 29, 1-366. taiz, l., zeiger, e., 2006: plant physiology. 4th edition. sinauer associates inc. publishers, sunderland, ma. webb, l.b., whetton, p.h., barlow, e.w.r., 2007: modelled impact of future climate change on the phenology of winegrapes in australia. aust. j. grape wine res. 13, 165-175. address of the authors: prof. dr. pedro josé almanza-merchán, universidad pedagógica y tecnológica de colombia, facultad de ciencias agropecuarias, grupo de ecofi siología vegetal, tunja, colombia e-mail: ppcalma@gmail.com prof. dr. gerhard fischer and prof. dr. aníbal herrera-arévalo, universidad nacional de colombia, facultad de agronomía, departamento de agronomía, bogotá, colombia prof. dr. alfredo jarma-orozco, universidad de córdoba, facultad de ciencias agrícolas, montería, colombia helber enrique balaguera-lópez, universidad pedagógica y tecnológica de colombia, facultad de ciencias agropecuarias, grupo de investigaciones agrícolas, tunja, colombia art10 journal of applied botany and food quality 81, 158 164 (2007) 1unidad de fruticultura, cita-dga, zaragoza, spain. 2departamento de pomología, eead-csic, zaragoza, spain 3estación experimental la mayora, csic, málaga, spain warm temperatures at bloom reduce fruit set in sweet cherry a. hedhly1,2, j.i. hormaza3, m. herrero1,2 (received june 5, 2007) summary warm springs have often been assumed as a prelude of a good fruit set in temperate fruit tree species. however, recently, evidences have accumulated on erratic fruit set under apparently good and warm springs in mediterranean conditions. the fact that these observations mainly occurred in sweet cherry (prunus avium), a species adapted to high latitudes and cold climates raised the question of whether warm temperatures at flowering could have a detrimental effect on fruit set. to evaluate this hypothesis two different sweet cherry cultivars were subjected under field conditions to a slight increase in temperature at bloom over two different years. while the minimum temperature remained stable, the maximum temperature increased 5-7ºc, resulting in a moderate increase of the average temperature of 1-3ºc. this was sufficient to drastically reduce fruit set in both years and cultivars. to know the vulnerable phase to warm temperatures the process was timed back: final fruit set differences were established in the first three weeks following pollination, but the onset of fruiting – when these differences appeared – was tracked back to one week after pollination. the process from pollination to fertilization was examined under both conditions. fertilization occurred six days after pollination. higher temperatures accelerated pollen tube growth rate but also reduced the number of growing pollen tubes along the style. in the ovary, the warm treatment accelerated ovule degeneration. these findings alert on the potential negative effect of even slight increases in temperature during cherry blooming, which nowadays – due to global warming trends – is a plausible and realistic scenario under mediterranean climatic conditions. introduction climatic conditions at flowering are known to have an effect on the subsequent fruit set and cold and rainy springs may jeopardize pollination. likewise cold temperatures at flowering reduce the speed of pollen tube growth and shorten the effective pollination period (sanzol and herrero, 2001). however, relatively warm springs have often been assumed as a prelude of a good fruit set in several temperate fruit tree species. nevertheless, in the last years casuistry has accumulated on erratic fruit set in fruit tree species under apparently “good ” and warm springs in mediterranean regions. the fact that these observations mainly occurred in sweet cherry (prunus avium l.), a species adapted to high latitudes and cold climates raised the question of whether warm temperatures at flowering could have a detrimental effect on the subsequent fruit set. in fact, the reproductive phase is one of the most sensitive plant developmental stages to heat stress (hall, 1992) and both high and low temperatures at blooming time are known to have a detrimental effect on subsequent fruit set. different works have studied the effect of high temperatures on fruit set by applying temperature stress during the whole reproductive cycle in a range of different species (monterroso and wien, 1990; hall, 1992; gross and kigel, 1994; peet et al., 1998; prasad et al., 2002, 2003). although the results obtained have revealed a negative effect on fruit set, such experimental procedures could not disentangle the relative importance of the different developmental stages occurring from the flower to the whole plant levels. alternatively, the effect of temperature on particular processes has been evaluated. high temperature, alone or in combination with other environmental stresses, has been shown to affect both pollen and pistil functions (erickson and markhart, 2002; young et al., 2004; koti et al., 2005). however the sensitive stages seem to be species specific and depended on the type of stress. in sweet cherry, previous works have revealed a negative effect of high temperatures on the length of stigmatic receptivity (hedhly et al., 2003) and ovule viability (postweiler et al., 1985). temperature also affects pollen tube kinetics and population census along the style (hedhly et al., 2004) with a strong genotype-temperature interaction that could reflect the geographic origin of the pollen donor and its adaptation to the prevailing environmental conditions (hedhly et al., 2005a). however, field results and laboratory work are not always easy to merge. the main objective of this work is to evaluate whether moderately high temperatures at bloom affect fruit set in sweet cherry, and to characterize its effect on the reproductive phase. for this purpose, we applied an increase of temperature at flowering time for the first two weeks after anthesis under orchard conditions. we characterized the pattern of flower and fruit drop, ovary and ovule growth following pollination, and the progamic phase spanning from pollination to fertilization. materials and methods plant material and experimental procedure the experiments were carried out in a sweet cherry collection located at the campus of aula dei in zaragoza, spain. the cultivars used were ‘vignola’ and ‘sunburst’ as pistil donors, and ‘napoleon’ and ‘burlat’ as pollen donors. the trees were 12 years old at the beginning of the trials, grafted on ‘santa lucia 64’ rootstocks, pruned as open center and with a plantation frame of 5 x 4 m. the increase of temperature in the warm treatment was obtained by covering the trees of the pistil donors in the field with a polyethylene cage (metallic structure covered with 0.178 mm polyethylene). the control trees were under the same metallic structure just covered with an insect proof to avoid bee pollination. this procedure revealed to be efficient to increase temperature without affecting other parameters (rodrigo and herrero, 2002a). the experiments were carried out over two years, one and three trees per treatment were used in the first and second year of the experiments, respectively. temperature was monitored every 5 min outside (control treatment) and inside (warm treatment) the plastic cage with a ‘data logger’ (testostor 175-3, testo, lenzkirch, germany) placed at 60 cm above the soil surface, protected from the sun and oriented to the north. the plastic cage was maintained for up to two weeks in both years. pollination techniques two different crosses were carried out: ‘vignola’ x ‘napoleon’ in the first year and ‘sunburst’ x ‘burlat’ in the second year. to avoid the potential interference of flower emasculation on fruit set (hedhly, 2003), evaluation was carried out in non-emasculated flowers. several branches were randomly chosen from both the control and warm treatments the day prior to anthesis and, to have a synchronized population of flowers of the same age, only flowers at balloon stage (baggiolini, 1952) were left in the tree, whereas all the others were removed. after this process all the selected branches presented at least 80 flowers/branch. this flower population was distributed randomly between treatments with a minimum of 250 flowers/ treatment. in the second year, a second experiment was performed with flowers treated similarly to evaluate the reproductive process and a number of reproductive biology parameters (see below). to obtain pollen, flowers from the pollen donors were collected at balloon stage from 6 trees/pollen donor; anthers were separated from their filaments and left to dehisce on a piece of paper at room temperature during 24-48 h. pollen was then sieved through a 0.26 µm mesh, and frozen at -20ºc. pollination was carried out on the day of flower anthesis and prior to anther dehiscence to avoid self pollination. evaluation of the onset of fruiting and flower and fruit drop flower and fruit drop were monitored in the two experiments, the control and the warm treatments, by weekly counts from anthesis to fruit ripening. with the aim of establishing the times of more intense dropping, we calculated the relative drop as the percentage of flower/ fruit dropped each week in relation to the initial number of flowers at anthesis. proportion comparisons were analyzed using yates’ chisquare goodness of fit test in 2x2 contingency tables testing for association between temperature and both initial and final fruit set as well as relative fruit drop. microscopic preparations in the second year of the experiment, a batch of 10 flowers/treatment was daily fixed in faa (formalin: acetic acid: 70% ethanol, 1 : 1 : 18 v/v; johansen, 1940) during 10 days after anthesis from the ‘sunburst’ x ‘burlat’ cross. microscopic observations were made of squashed stigma-styles washed three times in water, one hour each, autoclaved for 10 min at 1 kg/cm2 in 5% sodium sulfite (jefferies and blecher, 1974), and stained with 0.1% aniline blue in 0.1 n k3po4 (linskens and esser, 1957). the ovaries were separated in the washing step and maintained in distilled water overnight. the next day, the ovary area was measured under a binocular microscope (wild m8) and using an image analysis system (quantiment 570, leica, cambridge, uk) connected to a camera (cohu 8310 rgb colour camera, san diego, calif., usa), we measured ovary area. then, the obturators and ovules were dissected under the binocular microscope, and the ovule area measured before squashing and staining with aniline blue. preparations were examined under an ortholux ii microscope equipped with uv epifluorescence with a band pass 355-425 exciter filter and an lp 460 barrier filter. ovules were scored for pollen tube penetration and also for ovule viability through the presence of callose deposits in the chalaza of degenerating ovules (anvari and stösser, 1978). proportion comparisons were analyzed using yates’ chi-square goodness of fit test in 2x2 contingency tables testing for association between temperature and ovule viability. for the percentage of flowers with pollen tube at style base, fisher’s exact test was used. analyses of variance were carried out for ovule and ovary growth, pollen tube growth along the style and the number of pollen tubes at the stylar base. the spss package (v 11.0.1, spss inc., chicago, illinois) was used for all analysis. results fruit set the plastic cage induced an appreciable increase in temperature (tab. 1). while the average minimum temperature was practically unaffected by the plastic cage, the average maximum temperature increased 5.8ºc the first year and 6.9ºc the second year. this resulted in an average increase in the mean temperature of 1.4ºc and 3ºc, respectively. this moderate increase in temperature drastically reduced fruit set in both experiments (fig. 1a and b), from 18% to 5% in ‘vignola’ and from 25% to 4% in ‘sunburst’. in both cultivars, a highly significant effect of temperature was revealed for the initial 4 weeks after anthesis (‘vignola’ n = 533, yates’ χ2 = 27.870, p < 0.000; ‘sunburst’: n = 452, yates’ χ2 = 50.533, p < 0.000), and final fruit set (‘vignola’ n = 533, yates’ χ2 = 20.743, p < 0.000; ‘sunburst’: n = 452, yates’ χ2 = 40.862, p < 0.000). tab. 1: average temperatures (ºc) registered in the control and warm treatments for two weeks following pollination in the two years of experiments. maximum minimum average control warm control warm control warm first year 23.7 29.5 7.6 6.7 14.7 16.1 second year 24.1 31.0 7.3 8.4 15.2 18.2 the analysis of flower/fruit drop revealed that the final fruit set was established during the first three to four weeks following pollination. chi-square analysis revealed no significant association between temperature and fruit set after the forth week in both cultivars (‘vignola’ n = 92, yates’ χ2 = 0.161, p = 0.688; ‘sunburst’: n = 76, yates’ χ2 = 0.011, p = 0.91). indeed, flower drop in both conditions was concentrated between the second and the fourth week (fig. 1c and d), and in the warm treatment this flower drop was significantly higher during the third week for both years (1999: n = 497, yates’ χ2 = 52.411, p < 0.000; 2001: n = 391, yates’ χ2 = 22.624, p < 0.000). these results indicate that the effect of temperature occurred before the third week following anthesis. the onset of fruiting the comparison of ovary growth in the warm and control treatments shows similar growth patterns. although the process appeared somehow accelerated under the warm treatment, no significant effect was found for the nine days following anthesis (fig. 2). ovary growth started five days after pollination and was apparent by 7 days after pollination. to evaluate if this ovary growth was related to ovule growth, ovule area was also measured. in cherry, from the two ovules present in an ovary, usually only one, the primary ovule, develops into a seed; the other, the secondary ovule usually aborts. sequential examination of the ovules showed that, indeed, the secondary ovule did not grow and remained with a stable area of 0.48 mm2 and eventually shriveled. on the contrary, the primary ovule started to grow 4 days after pollination and growth was most conspicuous 7 days after pollination. thus, ovule growth encompassed ovary growth. as for ovary growth, temperature did not affect significantly ovule growth. interestingly, for both parameters, growth did not occur in all ovaries or ovules and a population of flowers, presumably those that will eventually drop, did not continue growing. the separation of these two populations of flowers occurred as early as seven days after pollination suggesting that the reproductive phase might be the main candidate for the effect of temperature. warm stress and fruit set 159 160 a. hedhly, j.i. hormaza, m. herrero within 24 hours after pollination (fig. 3a), and penetrated the transmitting tissue in the style (fig. 3b) reaching the base of the style 2 to 4 days after pollination (fig. 3c). within the ovary, the pollen tubes grew on the surface of the obturator to reach the ovule. by this time the secondary ovule had already started degeneration, shown by the deposition of callose at the chalaza that spreads over the nucellus and integuments (fig. 3d). the pollen tubes entered the micropyle and traversed the nucellus to achieve fertilization (fig. 3e) 6 days after pollination; and the first embryos were soon apparent (fig. 3f). this process was also studied in the warm treatment. temperature accelerated significantly pollen tube growth only during the first 2 days after pollination (f = 5.888, p = 0.026) (fig. 4a). the number of pollen tubes reaching the stylar base slightly increased during the first days after pollination but, at higher temperatures, a significant lower number of pollen tubes were recorded once the process was established (fig. 4c). thus, while an average of 3.7 pollen tubes per flower was observed in the control treatment, only 2.0 pollen tubes were recorded in the warm treatment. anova analysis on pooled data over the six last days, when the number of pollen tubes at style base stabilized and normally no further pollen tube growth takes place in the style, revealed a significant reduction in the number of pollen tubes at the base of the style in the warm treatment (f = 6.543, p = 0.012) in the ovary, the first pollen tubes on the obturator were observed in the control treatment 3 days after pollination, but the warm treatment advanced this step by one day. most flowers had pollen tubes at this structure 4 days after pollination in both treatments. but the proportion of pistils showing pollen tubes in the obturator was 68% in the warm treatment compared to 88% in the control. this proportion was reduced again along the rest of the ovary pathway and only 12% and 25% of the flowers got fertilized in the warm and the control treatments, respectively. interestingly, the fertilization level registered in the control corresponded to the final fruit set registered in the same experiment. ovule degeneration to evaluate to which extent ovule degeneration may be responsible for these reduced fertilization levels, ovule viability has been evaluated in the same samples (tab. 2). degeneration of the secondary ovule was already apparent in 30% of the flowers at anthesis and occurred in 100% of the flowers after 3 days in the warm treatment and 4 days in the control, although one day after anthesis 70% of the flowers had the secondary ovule degenerated in both treatments. also, in both conditions, 20% of the flowers presented both ovules degenerated at anthesis. while the warm treatment accelerated the degeneration of both first and secondary ovules, the final percentage of flowers with two degenerated ovules was unexpectedly the same (60%) under both conditions. yates’ chi-square test revealed no significant effect of temperature on ovule viability over the pooled data through the 5-10 days after anthesis. thus, around 40% of flowers presented at least one viable ovule, which represents the theoretical maximum fertilization rate that could be achieved. discussion a small increase in temperature at flowering time drastically reduced fruit set in two different sweet cherry cultivars and in two different years. while the increase in mean temperature was low, 1.4ºc and 3ºc, and the average minimum temperatures were similar in both treatments, an average increase of 6 -7ºc in the maximum temperatures was registered, which could potentially explain this effect. a negative effect of high temperatures at flowering on the subse0 20 40 60 80 100 0 1 2 3 4 5 6 7 8 weeks after anthes is control warm b 0 20 40 60 80 100 0 1 2 3 4 5 6 7 8 weeks after anthes is control warm d 0 20 40 60 80 100 0 1 2 3 4 5 6 7 weeks after anthes is control warm a 0 20 40 60 80 100 0 1 2 3 4 5 6 7 8 weeks after anthes is control warm c % fl o w er s/ fr ui ts % f lo w er s/ fr u it s fig. 1: effect of temperature on flower/fruits drop pattern (a, b) and times of maximum flower/fruit drop (c, d) under control and warm conditions in the crosses ’vignola’ x ‘napoleon’ (a, c) and ‘sunburst’ x ‘burlat’ (b, d). the reproductive phase to evaluate the events that occurred within this frame of time, the progamic phase, spanning from pollination to fertilization (linskens, 1986), was characterized under field conditions. pollen germinated ovary ovule c on tr ol w ar m a re a (m m 2 ) days alter anthesis 0 1 2 3 4 5 6 1 3 5 7 9 0 5 10 15 20 25 30 1 3 5 7 9 0 1 2 3 4 5 6 1 3 5 7 9 0 5 10 15 20 25 30 1 3 5 7 9 fig. 2: ovary and ovule growth following pollination under control and warm conditions in the cross ‘sunburst’ x ‘burlat’. warm stress and fruit set 161 quent fruit set has been reported in other fruit tree species as apricot (burgos et al., 1991), olive (cuevas et al., 1994), and peach (kozai et al., 2004). the analysis of the relative flower/fruit drop revealed that final fruit set is defined during the first 3-4 weeks following anthesis. this time period agrees with the time recorded for sour cherry (bradbury, 1929; lech and tylus, 1983), apricot (rodrigo and herrero, 2002b) and other fruit trees (sedgley and griffin, 1989). moreover, ovary and ovule growth shows that around seven days after pollination, a population of flowers starts to grow, while others remain unchanged and eventually drop. this timing points to the progamic phase as the main vulnerable process to warm temperatures. an effect of temperature on the reproductive phase has been observed. subjecting pollinated flowers to higher temperatures accelerated pollen tube growth rate in the pistil. however, the number of pollen tubes reaching the stylar base was reduced. the accelerating effect of higher temperatures on pollen tube growth was first reported in oenothera (lewis, 1942) and appears to be a general feature in different plant species. it has been widely reported in fruit trees (pear: mellenthin et al., 1972; lombard et al., 1972; almond: socías i company et al., 1976; plum: jefferies et al., 1982; apple: williams et al., 1984; sour cherry: cerovic and ruzic, 1992a; apricot: austin et al., 1998; sweet cherry: hedhly et al., 2004; peach: hedhly et al., 2005b), and in herbaceous plants (ryegrass: elgersma et al., 1989; alfalfa: katepa-mupondwa et al., 1996; groundnut: kakani et al., 2002). however, few works have dealt with pollen tube dynamics expressed as the variation in the census of pollen tubes in the style. the results obtained in this work in the field agree with recent findings, under laboratory conditions, reporting a negative effect of high temperatures on pollen dynamics of several sweet cherry cultivars (hedhly et al., 2005a) although in some peach cultivars a positive effect was registered (hedhly et al., 2005b). these discrepancies could reflect the different adaptation of these two species to temperature since sweet cherry is adapted to regions with lower temperatures than peach. furthermore, within a given species, genotypic differences were recorded in this parameter reflecting the adaptation to temperature of the pollen donor (hedhly et al., 2004). the occurrence of degenerated ovules early at anthesis registered in this work seems to be a common feature in sweet and sour cherry (eaton, 1959; furukawa and bukovac, 1989; thompson, 1996; cerovic and micic, 1999). anomalies in ovule and embryo sac development have also been registered in other fruit trees such as apricot (eaton and jamon, 1965), pistachio (grundwag and fahn, 1969), avocado (tomer and gottreich, 1976), olive (rallo et al., 1981), or almond (pimienta and polito, 1983), and these anomalies have been related to fruit set. in sweet cherry, it has been put forward that short ovule and embryo sac longevity could potentially explain the level of fruit set obtained (eaton, 1959), mainly when a delay in pollination occurs (stösser and anvari, 1982, 1983). likewise, ovules are considered to be highly vulnerable structures to increasing temperatures (williams, 1970; stösser and anvari, 1982; postweiler et al., 1985; cerovic and ruzic, 1992b; cerovic et al., 2000). while at anthesis both ovules were degenerated in a small proportion of the flowers, this proportion increased to 60% at fertilization time. in this work, although high temperature accelerated ovule degeneration, surprisingly the final percentage of flowers with at least one fig. 3: the reproductive process. (a) pollen grains germinate and traverse the stigmatic surface within 24 hours after pollination. bar = 70 µm. (b) pollen tube growth along the transmitting tissue 2 days after pollination. bar = 200 µm. (c) one pollen tube at the base of style 3 days after pollination. bar = 20 µm. (d) a degenerated ovule: callose deposits located in the chalaza spread over the nucellus and some parts of integuments 4 days after pollination. bar = 150 µm. (e) pollen tube penetrating the nucellus to carry out embryo sac fertilization 6 days after pollination. bar = 70 µm. (f) fertilised ovule showing a young embryo 8 days after pollination. bar = 70 µm. 162 a. hedhly, j.i. hormaza, m. herrero viable ovule, or with both ovules degenerated, was similar in the warm and control treatments. early ovule degeneration accounts in part for reduced fruit set, but cannot fully explain the percentage of fertilized ovules, since in spite of having 40% of the flowers with viable ovules at the time of fertilization only 25% got fertilized in the control, which correspond to the final fruit set. in the warm treatment this proportion was reduced to 12% that yielded a 4% final fruit set. thus, warm temperature could also affect pollen tube growth in the ovary in this species. differences between fertilization rate and actual fruit set in the warm treatment could be, however, also explained by an effect on early embryo development and/or by fruit drop. the experimental procedure used herein applying the warm treatment specifically at flowering time together to the results obtained along the reproductive phase underline the consequences that slight increases in temperature at this time may have on the subsequent fruit set. the results obtained in this work together to those previously obtained on the shortening of stigmatic receptivity with increasing temperatures (hedhly et al., 2003) show that an increase in the mean temperature as low as 1.4 to 3ºc during the first 2 weeks after anthesis has a direct effect on fruit set and could explain the erratic bearing recorded in some years with apparent “good” and warm springs. due to climate change, these situations of warm springs could be more frequent in the future; in fact, the evaluation of temperature increase in time series shows that increases are most conspicuous at this time of the year (easterling et al., 1997; sparks and menzel, 2002). anyhow these temperature increases can be common in mediterranean areas, and the results obtained in this work will contribute to explain the erratic fruit set registered in this and other temperate fruit tree species. acknowledgments the authors thank a. escota and r. lopez for technical assistance. a.h. was supported by an aeci, sia-dga, csic fellowship. financial support for this work was provided by project grants inia-rta 01-103, cicyt agl2003-05318 and agl2006-13529 and by the “grupo de investigación consolidado de aragón: a-02, a-43”. references anvari, s.f., stösser, r., 1978: eine neue fluoreszenz-mikroskopische methode zur beurteilung der befruchtungsfähigkeit der samenanlagen bei prunus. z. pflanzenzüch. 81, 333-336. austin, p.t., hewett, e.w., noiton, d., plummer, j.a., 1998: self incompatibility and temperature affect pollen tube growth in ‘sundrop’ apricot (prunus armeniaca l.). j. hort. sci. biotech. 73, 375-386. baggiolini, m., 1952: les stades repérés des arbres fruitiers à noyau. revue romande d’agriculture, viticulture et d’arboriculture 8. bradbury, d., 1929: a comparative study of the developing and aborting fruits of prunus cerasus. am. j. bot. 16, 525-542. burgos, l., egea, j., dicenta, f., 1991: effective pollination period in apricot fig. 4: pollen tube growth in the style up to ten days after pollination in the cross ‘sunburst’ x ‘burlat’ under control and warm conditions expressed as (a) the percentage of style traveled by the longest pollen tube, (b) the percentage of flowers with pollen tubes at the stylar base, and (c) the number of pollen tubes at the stylar base. vertical bars represent standard errors. 0 2 4 6 0 2 4 6 8 10 days after anthes is control warm c 0 20 40 60 80 100 0 2 4 6 8 10 a 0 20 40 60 80 100 0 2 4 6 8 10 b % o f s ty le % o f fl o w er n um b er o f p o ll en tu b es at st y le b as e tab. 2: effect of temperature on ovule longevity expressed as the percentage of flowers with one (secondary), and two (primary and secondary) degenerated ovules under warm and control conditions in the cross ‘sunburst’ x ‘burlat’. days after anthesis 0 1 2 3 4 average 5-10 flowers with one degenerated ovule (%) control 30 70 60 70 100 100 warm 30 70 90 100 100 100 flowers with two degenerated ovules (%) control 20 10 20 20 60 60 warm 20 20 30 60 60 59 warm stress and fruit set 163 number of seeds per pod. can. j. plant sci. 76, 259-262. koti, s., reddy, r.k., reddy, v.r., kakani, v.g., zhao, d., 2005: interactive effects of carbon dioxide, temperature, and ultraviolet-b radiation on soybean (glycine max l.) flower and pollen morphology, pollen production, germination, and tube lengths. j. exp. bot. 56, 725-736. kozai, n., beppu, k., mochioka, r., boonprakob, u., subhadrabandhu, s., kataoka, i., 2004: adverse effects of high temperature on the development of reproductive organs in ‘hakuho’ peach trees. j. hortic. sci. biotech. 79, 533-537. lech, w., tylus, k., 1983: pollination, fertilization and fruit setting of some sour cherry varieties. acta hortic. 139, 33-39. lewis, d., 1942: the physiology of incompatibility in plants. i. effect of temperature. proc. royal soc. 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and fruit set. in: webster, a.d., looney, n.e. (eds.), cherries: crop physiology, production and uses, 223241. cab international, oxford. tomer, e., gottreich, m., 1976: defectives ovules in avocado cultivars. j. am. soc. hortic. sci. 101, 620-623. williams, r.r., 1970: iiifactors affecting pollination in fruit trees. in: luckwill, l.c., cutting, c.v. (eds.), physiology of tree crops, 193-207. academic press, london. williams, r.r., brain, p., church, r.m., flook, v.a., 1984: flower receptivity, pollen transfer and fruit set variations during a single flowering period of cox’s orange pippin apple. j. hortic. sci. 59, 337-347. young, l.w. wilen, r.w., bonham-smith, p.c., 2004: high temperature stress of brassica napus during fowering reduces microand megagametophyte fertility, induces fruit abortion, and disrupts seed production. j. exp. bot. 55, 485-495. addresses of the authors: 1estación experimental de aula dei, csic. apartado 202, 50080, zaragoza, spain. 3 estación experimental la mayora, csic. 29750, algarrobo-costa, málaga, spain. correspondence: e-mail: ahedhly@eead.csic.es << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb 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/pagesize [907.087 680.315] >> setpagedevice journal of applied botany and food quality 92, 378 387 (2019), doi:10.5073/jabfq.2019.092.050 1laboratory for analytical chemistry and industrial analysis, faculty of chemistry and chemical engineering, university of maribor, slovenia 2department of agrochemistry and brewing, slovenian institute of hop research and brewing, žalec, slovenia 3department of chemistry and biochemistry, faculty of chemistry and chemical technology, university of ljubljana, slovenia response surface methodology: an optimal design applied for maximum ultrasound-assisted extraction efficiency of phenolic acids from coriandrum sativum l. milena ivanović1, maša islamčević razboršek1, iztok jože košir2, mitja kolar3* (submitted: may 20, 2019; accepted: november 15, 2019) * corresponding author summary in this study, a combined three-factors-three-level box-behnken design with a response surface methodology was used to optimize the ultrasound-assisted extraction of bound phenolic acids from coriander fruits. temperature (x1, 20-60 °c), sonication time (x2, 15-45 min) and naoh concentration (x3, 2-4 m) were studied as independent variables in order to obtain the optimal extraction conditions. for this purpose, a two-step analytical procedure was applied: first, alkaline hydrolysis and extraction under the influence of ultrasound was performed followed by a clean-up step using solidphase extraction method. after derivatisation, the extracted phenolic acids were analysed using gc-ms. the interrelationship between the dependent and operational variables were well fitted (r2 >0.90) to the quadratic term models. the results obtained in this study confirmed that studied factors had a significant influence on phenolic acids extraction recovery. in favour of maximum extraction yields, the following experimental conditions are suggested: a sonication time of 17.4 min at 35.3°c and with a naoh concentration of 2.02 m. these results can be utilized for further isolation of active phenolic compounds from other parts of coriander plant as well as for phenolic acids study over various plant materials from the apiaceae family. keywords: coriander, phenolic acids, ultrasound-assisted extraction, hydrolysis, experimental design. introduction polyphenols represent a large class of chemical compounds, divided into sub-groups they include: phenolic alcohols, phenolic acids, phenylpropanoids, flavonoids, flavones, glycoflavonones, flavonones and biflavonyls, isoflavones, xanthones, stilbenes, tannins and quinines (ferrazzano et al., 2011). the contents of those compounds were determined in different plant materials and plant products, such as: berries (ćujić et al., 2016), aromatic plants (roby et al., 2013; kocak et al., 2016), tee plants (nováková et al., 2010; corbin et al., 2015), as well as beers, juices and wine (abad-garcía et al., 2009; ivanova-petropulos et al., 2015; moura-nunes et al., 2016). in nature, polyphenols occur in free and conjugated forms, with one or more sugar residues linked to hydroxyl groups, whereby direct linkages of the sugar (polysaccharide or monosaccharide) to an aromatic carbon exist. however, free forms of phenolic acids are very rare in nature. acidic, basic and enzymatic hydrolysis are the most commonly used methods for the extraction of phenolic acids from natural materials (ross et al., 2009; ahmad et al., 2016). despite current trends in sample handling, which focus on the development of faster, safer and more environmental friendly extraction techniques, both liquid-liquid extraction (lle) and solid-phase extraction (spe) are still useful and widely accepted techniques for the exhaustive extraction of polyphenols. the mentioned extraction techniques, with different organic solvents (methanol, ethanol, propanol, ethylacetate and acetone) and solvents mixtures, were developed for isolation of polyphenols from natural sources (minuti et al., 2006; irakli et al., 2012; wang et al., 2013; gültekin-özgüven et al., 2015). additionally, the efficiency of those extractions can be significantly improved by using different external effects; for example, a modern method is microwave-assisted extraction (mae), which uses frequencies from 0.3 to 300 ghz and can be more suitable for the extraction of polyphenols as compared to traditional extraction methods. recent studies also proved that the use of ultrasound can enhance the extraction efficiency through acoustic cavitation and mechanical effects (carrera et al., 2012; reboredo-rodríguez et al., 2014; corbin et al., 2015; oniszczuk et al., 2015). some of the ultrasound-assisted extraction (uae) advantages for the extraction of plant bioactive components are shortened extraction time, lower solvent consumption and increased extraction yields (oniszczuk et al., 2016). coriander (coriandrum sativum l.) is a medicinal and aromatic plant belonging to the apiaceae family, widely grown in north africa and the middle east, which has gained an increasing interest in western europe (barros et al., 2012). several authors have reported on the health benefits of consuming different parts of the coriander plant (flowers, leaves, stems and roots) (randall et al., 2013; duarte et al., 2016). the content of the important pharmaceutical potential compounds in coriander was reported in a review by sahib et al. (2012). methanolic, ethanolic and acetone coriander extracts were investigated by some authors, but polyphenol fractions have not been thoroughly studied (kaiser et al., 2013; msaada et al., 2013; martins et al., 2016; zeković et al., 2016). to the best of our knowledge, this is the first study regarding the bound phenolic acids content in coriander fruits, obtained after optimized alkaline hydrolysis. the aim of the study was to examine the characterization of different polyphenols in coriander fruits. first, the total phenolic content (tpc) and total flavonoid content (tfc) in methanolic extracts of the coriander fruits were determined according to the standard spectrometric methods. then, the box-behnken design (bbd) combined with a response surface experimental design methodology (rsm) was performed. in this way, the alkaline hydrolysis in combination with the uae was optimized for the extraction of the trans-cinnamic acid, vanillic acid, syringic acid, p-coumaric acid, ferulic acid and caffeic acid from the coriander fruits. bbd was used to test the influence of three different factors (sonication time, temperature and concentration of naoh) on the hydrolysis process and to determine the maximum phenolic acid yields. the extraction recoveries of active compounds, according to the uae and hydrolysis conditions, were also taken into account. phenolic composition of coriander fruit 379 materials and methods reagents folin-ciocalteu phenol reagent (2 n), sodium acetate (ch3coona), standard compounds; trans-caffeic acid (99%), vanillic acid (97%), syringic acid (97%), trans-p-coumaric acid (98%), trans-o-coumaric acid (98%), trans-ferulic acid (98%) and trans-cinnamic acid and solvents; tetrahydrofuran-thf (99.5%) and pyridine (99.9%) were supplied by merck (germany). the thf was distilled before use. the derivatisation reagent n-methyl-n-(trimethylsilyl)trifluoroacetamide (mstfa) as well as protocatechuic acid (99%), rutin (99%), hplc-grade methanol (meoh), sodium hydroxide (naoh, 99%), aluminium chloride (alcl3) and sodium carbonate (na2co3) were purchased from sigma (usa). gallic acid, gc-grade toluene (99.5%) and hydrochloric acid (hcl, 36.5%) were purchased from carlo erba (italy). dichloromethane (dcm) was purchased from jt baker (germany), l-ascorbic acid (99.7%) was purchased from alkaloid (macedonia) and edta was purchased from kemika (croatia). water (resistivity above 18 mω cm) used was obtained from a milliq water purification system. methanolic extraction of polyphenols from coriander coriander samples (cultivated in romania) were purchased from the local supermarket in maribor, slovenia. the fruits were milled in an electric blender (gorenje, slovenia), packaged in glass vessels and kept in a dark place at room temperature before analysis. for the uae, 1.00 g of homogenized sample was weighed into centrifuge tube. the sample was extracted separately three times by sonication with 10 ml of 80% or 100% meoh for different lengths of time (15, 30 and 45 min). after each extraction, the extracts were centrifuged for 10 min at 6,000 rpm, and the supernatants were combined and evaporated to absolute dryness by rotary evaporation (büchi, r-100). the dry extracts were kept at 4 °c until analysis. beside the uae, a conventional extraction technique (ce) was also performed, and the results were compared. for the ce, 1.00 g of the homogenised sample was extracted with 80% or 100% meoh in the way that homogenate was stirred with a magnetic stirrer at 900 rpm at room temperature for 30 min or for 24 h. extractions were performed in triplicates. total phenolic content (tpc) the tpc in the methanolic extracts of coriander fruits was determined according to the standard spectrometric method, with some modifications (jiménez et al., 2015). briefly, 40 μl of properly diluted methanolic extract was mixed with 3.160 ml of ultra-pure water and 200 μl of folin-ciocalteu s̓ phenol reagent. after 6 min, 600 μl of na2co3 (0.2 g ml-1) was added. the tubes were allowed to stand for 2 h in a dark place at room temperature. standard solutions of gallic acid in the concentration range of 50-500 mg l-1 were prepared in the 80% methanol. for construction of the calibration curve, absorbances were measured at the wavelength of 765 nm (shimadzu uv-vis spectrophotometer, kyoto, japan). samples were measured under the same conditions. the tpc in the coriander fruits was expressed as milligrams of gallic acid equivalents per gram of dry weight (mg gae g-1 dw). all samples were analysed in duplicates. total flavonoid content (tfc) the tfc was determined using the aluminium chloride colorimetric method (milan, 2011). 500 μl of properly diluted crude methanolic extract was added to 1.5 ml of pure methanol and mixed well. after that, 0.1 ml of alcl3 (10 %), 0.1 ml of ch3coona (1 m) and 2.8 ml of ultra pure water was added. standard solutions of rutin in the concentration range of 10-100 mg l-1 were prepared in the same way. the tubes were allowed to stand for 30 min in a dark place at room temperature, and the absorbances were measured at the 415 nm against a blank. the tfc was expressed as milligrams of rutin per gram of dry weight (mg rut g-1 dw). all samples were analysed in duplicates. identification and quantification of phenolic acids by gas chromatography-mass spectrometry (gc-ms) experimental design in order to optimize the hydrolysis conditions for the extraction of the target phenolic acids from the coriander fruits, an experimental design was applied. design-expert software (design expert 10) was used for the experimental design and statistical analysis of the data. a three-level (-1, 0 and +1) three-factor box-behnken design (bbd) combined with a response surface methodology (rsm) was conducted to the design experimental project. the influence of three major factors: temperature (x1), sonication time (x2) and naoh concentration (x3) were tested as independent variables. the temperature extraction was evaluated in the range 20-60 ºc, sonication time covered the range of 15 to 45 min and naoh concentration was evaluated between 2 m and 4 m. the actual and coded values of the operational variables are shown in tab. 1. a total of fifteen experiments were carried out; of these, three were replications of the central point with different combinations of the temperature, sonication time and naoh concentration. the extraction yields of caffeic acid (y1), ferulic acid (y2), vanillic acid (y3), p-coumaric acid (y4) and syringic acid (y5) as well as the extraction recoveries (%) of caffeic acid (y6), p-coumaric acid (y7), ferulic acid (y8) and trans-cinnamic acid (y9) were selected as dependent variables. the experimental data were fitted to a secondorder polynomial model to obtain the regression coefficients. the generalized second-order polynomial model used in the response surface analysis was as follows: (1.) where y is the response variable, xi and xj are the independent variables and k is the number of tested variables (k = 3). the regression coefficient is defined as β0 for the intercept, βi for the linear, βii for the quadratic and βij for the cross product term. hydrolysis and extraction of phenolic acids the powdered sample (1.00 g) of coriander fruits was mixed with 20 ml of naoh (which contained 1% l-ascorbic acid and 10 mm edta as stabilizers) at three different concentrations (2, 3 and 4 m) in a 100 ml round-bottom flask. these samples were mixed properly for 1–2 min and kept in an ultrasound bath (model-lwb 106d, 6 methodology (rsm) was conducted to the design experimental project. the influence of three 155 major factors: temperature (x1), sonication time (x2) and naoh concentration (x3) were tested 156 as independent variables. the temperature extraction was evaluated in the range 20 – 60 ºc, 157 sonication time covered the range of 15 to 45 min and naoh concentration was evaluated 158 between 2 m and 4 m. the actual and coded values of the operational variables are shown in 159 tab. 1. a total of fifteen experiments were carried out; of these, three were replications of the 160 central point with different combinations of the temperature, sonication time and naoh 161 concentration. the extraction yields of caffeic acid (y1), ferulic acid (y2), vanillic acid (y3), p-162 coumaric acid (y4) and syringic acid (y5) as well as the extraction recoveries (%) of caffeic 163 acid (y6), p-coumaric acid (y7), ferulic acid (y8) and trans-cinnamic acid (y9) were selected 164 as dependent variables. the experimental data were fitted to a second-order polynomial model 165 to obtain the regression coefficients. the generalized second-order polynomial model used in 166 the response surface analysis was as follows: 167 𝑌𝑌𝑌𝑌 = 𝛽𝛽𝛽𝛽0 + �𝛽𝛽𝛽𝛽𝑖𝑖𝑖𝑖𝑋𝑋𝑋𝑋𝑖𝑖𝑖𝑖 𝑘𝑘𝑘𝑘 𝑖𝑖𝑖𝑖=1 + �𝛽𝛽𝛽𝛽𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑋𝑋𝑋𝑋𝑖𝑖𝑖𝑖 2 𝑘𝑘𝑘𝑘 𝑖𝑖𝑖𝑖=1 + �𝛽𝛽𝛽𝛽𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑋𝑋𝑋𝑋𝑖𝑖𝑖𝑖𝑋𝑋𝑋𝑋𝑖𝑖𝑖𝑖 𝑘𝑘𝑘𝑘 𝑖𝑖𝑖𝑖=1 (1. ) 168 where y is the response variable, xi and xj are the independent variables and k is the number 169 of tested variables (k = 3). the regression coefficient is defined as β0 for the intercept, βi for the 170 linear, βii for the quadratic and βij for the cross product term. 171 tab. 1: 172 hydrolysis and extraction of phenolic acids 173 the powdered sample (1.00 g) of coriander fruits was mixed with 20 ml of naoh (which 174 contained 1% l-ascorbic acid and 10 mm edta as stabilizers) at three different concentrations 175 (2, 3 and 4 m) in a 100 ml round-bottom flask. these samples were mixed properly for 1–2 min 176 and kept in an ultrasound bath (model-lwb 106d, daihan labtech co. ltd, korea) for varying 177 lengths of time (15, 30 and 45 min) at various temperatures (20, 40 and 60 °c). the temperature, 178 sonication time and naoh concentration were based on the experimental design (tab. 2). after 179 the hydrolysis, the samples were acidified to a ph of 2 using 6 m hcl. prepared samples were 180 added to pre-conditioned (2 x 3 ml of meoh, and 2 x 3 ml of acidified water [ph=2]) hlb 181 supelco® spe cartridges (ivanović et al., 2016). cartridges were washed with ultra pure 182 water (2 x 3 ml) to remove sugars and other polar compounds. the free phenolic acids fraction 183 was eluted with 2 x 2 ml of thf. the eluate was collected and dried in a rotary evaporator (at 184 tab. 1: experimental variables (factors and respective levels). independent variable unit symbol values (uncoded and coded) temperature °c x1 20 (-1) 40 (0) 60 (1) sonication time min x2 15 (-1) 30 (0) 45 (1) naoh concentration mol l-1 (m) x3 2 (-1) 3 (0) 4 (1) 380 m. ivanović, m.i. razboršek, i.j. košir, m. kolar daihan labtech co. ltd, korea) for varying lengths of time (15, 30 and 45 min) at various temperatures (20, 40 and 60 °c). the temperature, sonication time and naoh concentration were based on the experimental design (tab. 2). after the hydrolysis, the samples were acidified to a ph of 2 using 6 m hcl. prepared samples were added to pre-conditioned (2 × 3 ml of meoh, and 2 × 3 ml of acidified water [ph=2]) hlb supelco® spe cartridges (ivanović et al., 2016). cartridges were washed with ultra pure water (2 × 3 ml) to remove sugars and other polar compounds. the free phenolic acids fraction was eluted with 2 × 2 ml of thf. the eluate was collected and dried in a rotary evaporator (at 40 °c) to absolute dryness. then, the sample was derivatised by adding 100 μl of mstfa and 50 μl pyridine, heated at 80 °c for 1h, diluted with toluene up to 1 ml and analysed using gc-ms. the analyses were carried out in duplicates. at the same time, the extraction recovery for every performed experiment was determined. for this purpose, 1.00 g of the powdered sample was spiked with a standard compounds mixture of an exact ly known concentration. the spiked samples were exposed to the same conditions as the unspiked samples; thus, spiked and unspiked samples were used for the extraction recovery determination (%). preparation of calibration curves standard stock solutions of caffeic acid, ferulic acid, p-coumaric acid, vanillic acid, syringic acid and o-coumaric acid (internal standard-istd) were prepared by accurately weighing 10 mg of each into a 10 ml volumetric flask. then, the standards were dissolved in thf. working calibration solutions were prepared by combining various volumes (from 10 μl to 100 μl) of phenolic acids stock solutions with 50 μl of istd in 50 ml conical glass flasks. each solution was derivatised by adding of 100 μl of mstfa and 50 μl of pyridine for 1 h at 80 °c in a sand bath. after the derivatisation was finished, tms derivatives were quantitatively transferred into 1 ml flasks and filled up to the mark with toluene. five working solutions in concentrations ranging from 1 to 100 mg l-1 were injected in triplicates. the calibration curves were constructed by linear regression of the peak-area ratio of the individual phenolic acid (pa) standard to the istd (y), versus the concentration (mg l-1) (x). the working solutions were prepared fresh daily. gc-ms parameters tms derivatives of pas were analysed with a varian 3900 gas chromatograph, coupled to an ms/ms saturn 2100 ion trap mass spectrometer. gc separation was performed using an agilent technologies, j&w scientific capillary column db-5 (30 m × 0.25 mm × 0.25 μm). 1 μl of the sample was injected in split mode (split ratio 1:10). carrier gas was he (6.0 uhp) at a flow rate of 1.0 ml min-1. the initial oven temperature was 40 °c, held for 1 min, and then the temperature was raised up to 320 °c at a rate of 10 °c min-1, held for 3 min (ivanović et al., 2016). total run time was 32 min. mass spectra were recorded in scan or sim mode in a range from 50 to 650 m/z using electron ionization energy at 70 ev. peak identification was done by comparing retention times (tr) and spectral properties with those of standard compounds and by library matching from nist ms library containing the mass spectra of tms derivatives of phenolic acids (ivanović et al., 2016). results and discussions determination of the tpc and tfc in the first step of this study, two extraction methods (conventionalce or ultrasound assisted extraction-uae) were used to extract total polyphenols from the coriander fruits under the previously described conditions. fig. 1 represents the extraction yields of the two mentioned extraction techniques for total phenolic (tpc) and total flavonoid content (tfc). extraction techniques were compared regarding two influencing variables, namely the type of extraction solvent (80% aqueous meoh solution and 100% meoh) and sonication time (20, 30 and 45 min). the results showed that the obtained extraction rates were affected by the type of the extraction as well as by the solvent used (fig. 1). in general, the highest yields in total phenolics and total flavonoids from the coriander fruits were obtained by extraction using 80% meoh under the 24 h long stirring and by 45 min of sonication using 100% meoh, respectively. compared to the uae, the ce method resulted in significantly lower contents of tfc, but this not was case for the tpc. these results are in agreement with the results, reported for other plants (metrouhamir et al., 2015). in the case of coriander fruits, it was confirmed that the uae was not effective for tpc recovery. this can be explained by the fact that longer extraction time can lead to the degradation of unstable and sensitive natural compounds like phenolics (da porto et al., 2013; qiao et al., 2013; qu et al., 2017). conversely, when compared to the ce, application of uae may contribute to a lower solvent consumption and shorter extraction time. in this study by the 45 min long uae at least 90% of tpc were extracted with 24 h stirring ce technique. for the tfc, the 45 min sonication leads to the extraction of sig nificantly higher concentration when compared with the 24h ce. the values for the tpc and tfc in the coriander fruits were varied among the extracts and ranged from 800 to 2900 mg gae g-1 dw and from 540 to 850 mg rut g-1 dw, respectively. fig. 1: effect of the extraction solvent and extraction time on the tpc (a) and on the tfc (b) in coriandrum sativum l. fruits. phenolic composition of coriander fruit 381 identification and quantification of bound phenolic acids by gc-ms as previously mentioned, phenolic acids in the plant materials can be present in free or bound form. free phenolic acid are extractable using different organic solvents, or their mixtures. conversely, bound phenolic acids can be extracted after being released by alkaline, acidic and enzymatic hydrolysis. from the literature, it is known that the efficiency of a hydrolysis reaction can be affected by: the concentration of a hydrolysis reagent, temperature, reaction time, mass of analysed samples, application of microwaves or ultrasound, etc. (cheng et al., 2014). therefore, concentractions of identified phenolic compounds from similar samples in final extracts can be very different. in this study, six different phenolic acids (caffeic acid, ferulic acid, p-coumaric acid, vanillic acid, syringic acid and protocatechuic acid), including their geometrics isomers, were identified in the coriander fruits (fig. 2). box-behnken experimental design in this section, a box-behnken design (bbd) combined with a response surface methodology (rsm) was used to optimize the alkaline hydrolysis and extraction conditions of the previously identified bound phenolic acids (fig. 2). for optimization, the experiments were conducted by a 23 full factorial central composite design. all of the experimental data obtained from the 15-run experiments are shown in tab. 2. the yields of caffeic acid (y1), ferulic acid (y2), vanillic acid (y3), p-coumaric acid (y4) and syringic acid (y5) as well as the extraction recoveries of caffeic acid fig. 2: gc chromatograms of: a) silylated standard mixture of phenolic acids (1. vanillic acid; 2. o-coumaric acid (istd); 3. protocatehuic acid; 4. syringic acid 5. cis-ferulic acid; 6. trans-p coumaric acid; 7. trans-cinnamic acid; 8. cis-caffeic acid; 9. trans-ferulic acid; 10. trans-caffeic acid); b) silylated phenolic acids present in coriandrum sativum l. extract obtained after spe (1. vanillic acid; 2. o-coumaric acid (istd); 3. protocatehuic acid; 4. syringic acid 5. cis-ferulic acid; 6. trans-p coumaric acid; 7. l-ascorbic acid (stabilizer); 8. cis-caffeic acid; 9. trans-ferulic acid; 10. trans-caffeic acid). tab. 2: mean responses obtained for investigated phenolic acids from the experimental design. run temperature sonication naoh con. caffeic ferulic vanillic p-coumaric syringic caffeic ferulic p-coumaric trans (°c) time (min) (mol l-1) acid acid acid acid acid acid acid acid cinnamic unit mg g-1 mg g-1 mg g-1 mg g-1 mg g-1 % % % % x1 x2 x3 y1 y2 y3 y4 y5 y6 y7 y8 y9 1 40 (0) 30 (0) 3 (0) 1.94 0.55 0.25 0.38 5.13 ·10-3 55 70 72 71 2 40 (0) 45 (+1) 4 (+1) 1.43 0.54 0.15 0.27 nq 70 61 69 62 3 60 (+1) 45 (+1) 3 (0) 1.66 0.63 0.17 0.32 2.13 ·10-3 82 81 83 65 4 60 (+1) 30 (0) 4 (+1) 1.68 0.47 0.15 0.12 nq 83 82 95 61 5 20 (-1) 30 (0) 2 (-1) 1.53 0.24 0.16 0.05 nq 86 102 101 87 6 20 (-1) 45 (+1) 3 (0) 1.51 0.38 0.19 0.21 1.33 ·10-3 81 83 77 78 7 60 (+1) 30 (0) 2 (-1) 1.41 0.60 0.19 0.30 nq 89 80 96 84 8 40 (0) 15 (-1) 2 (-1) 1.43 0.37 0.17 0.11 nq 99 102 88 95 9 40 (0) 30 (0) 3 (0) 1.94 0.56 0.25 0.38 5.13 ·10-3 55 70 72 71 10 40 (0) 45 (+1) 2 (-1) 1.72 0.47 0.18 0.29 nq 77 91 93 89 11 40 (0) 15 (-1) 4 (+1) 1.68 0.47 0.15 0.15 nq 90 91 95 75 12 40 (0) 30 (0) 3 (0) 1.94 0.56 0.25 0.38 5.13 ·10-3 55 70 72 71 13 20 (-1) 30 (0) 4 (+1) 1.47 0.58 0.14 0.15 nq 92 79 88 67 14 20 (-1) 15 (0) 3 (0) 1.52 0.32 0.17 0.12 4.8 ·10-4 100 105 102 76 15 60 (+1) 15 (-1) 3 (0) 1.40 0.52 0.19 0.27 nq 94 96 97 74 nq-not quantified 382 m. ivanović, m.i. razboršek, i.j. košir, m. kolar (y6), ferulic acid (y7), p-coumaric acid (y8) and trans-cinnamic acid (y9) were determined as the dependent variables. the concentration of protochatechuic acid in all obtained extracts was below loq and therefore, not used for method optimization. the p-value and f-test were used to determine the significance of each coefficient. the high f-value and the small p-value mean signi ficant corresponding variables. the results, values of ‘‘prob > f’’ less than 0.05, indicates that the model terms are significant. the optimized conditions were validated for the maximum phenolic acids yield and extraction recovery based on the values obtained using rsm. the experimental values were compared with predicted values based on cv% in order to determine the validity of the model. for the graphical presentation of the influence of tested conditions on the extraction yields, the three dimensional (3-d) surface response plots were generated by varying two variables within the experimental range and by holding one variable constant at the central point. conversely, contour plots were generated for the graphical description of the influence of tested variables on the extraction recoveries. the test of statistical significance was based on the total error criteria with confidence levels of 95.0%, 99.0% and 99.9%. effect of the independent variables on the phenolic acids yield to study the interactive effects of the operational parameters on the extraction yields, the three-dimensional (3d) profiles of multiple non-linear regression models were depicted in fig. 3. the profiles present the interaction of two process factors (sonication time and temperature), while the third factor (naoh concentration) was fixed at its middle level (3m). optimization of the extraction process for the phenolic acids yield was carried out by applying second-order polynomial equation. the analysis of variance (anova) showed that this model adequately fig. 3: 3d plots of ultrasound hydrolysis and extraction of bound phenolic acids (mg g-1 dw) from coriandrum sativum l., varying sonication time and temperature (the concentration of naoh is constant in the central point). (a) caffeic acid, (b) ferulic acid, (c) vanillic acid, (d) p-coumaric acid and (e) syringic acid. phenolic composition of coriander fruit 383 tab. 3: analysis of variance (anova) of response surface second order polynomial models for phenolic acids yield. variables responses (mg g-1 dw) caffeic acid ferulic acid mean of square f value p value mean of square f value p value model 0.04 18.00 0.003** 0.07 36.02 <0.001*** x1 8.76 ·10-5 0.39 0.559 0.23 116.20 <0.001*** x2 7.59 ·10-4 3.39 0.125 0.04 20.83 0.006** x3 2.90 ·10-4 1.30 0.307 0.09 48.22 0.001*** x1x2 1.55 ·10-3 6.90 0.047* 1.92 ·10-4 0.10 0.766 x1x3 2.23 ·10-3 9.95 0.025* 0.20 102.14 <0.001*** x2x3 5.40 ·10-3 24.13 0.004** 2.68 ·10-4 1.38 0.293 x1x1 0.01 57.38 < 0.001*** 0.05 23.01 0.005** x2x2 8.46 ·10-3 37.77 0.002** 0.01 7.28 0.043* x3x3 8.59 ·10-3 38.35 0.002** 0.02 9.89 0.026* r2 0.9701 0.9848 adjusted r2 0.9162 0.9575 adeq. precision 12.162 21.713 tab. 3: continued. varibales responses (mg g-1 dw) vanillic acid p-coumaric acid syringic acid mean f value p value mean f value p value mean f value p value of square of square of square model 0.06 53.60 <0.001*** 0.10 26.04 >0.001** 6.80·10-4 75.46 < 0.001*** x1 0.02 13.96 0.014* 0.19 48.08 0.001*** 1.64·10-5 1.82 0.236 x2 1.16 ·10-3 1.10 0.343 0.12 31.37 0.003** 2.87·10-5 3.18 0.135 x3 0.05 46.33 0.001*** 3.38 ·10-3 0.87 0.393 8.67·10-19 9.62·10-14 1.000 x1x2 0.02 15.40 0.011* 7.04 ·10-3 1.82 0.235 3.22·10-5 3.57 0.117 x1x3 8.71·10-4 0.82 0.406 0.19 49.18 <0.001*** 8.67·10-19 9.62·10-14 1.000 x2x3 2.20 ·10-3 2.07 0.210 5.11·10-3 1.32 0.302 0.00 0.00 1.000 x1x1 0.10 97.12 <0.001*** 0.16 41.93 >0.001** 2.08·10-3 230.88 < 0.001*** x2x2 0.09 81.57 <0.001*** 5.23 ·10-3 1.36 0.297 2.08·10-3 230.88 < 0.001*** x3x3 0.29 277.26 <0.001*** 0.26 67.32 <0.001*** 2.80·10-3 310.97 < 0.000*** r2 0.9897 0.9791 0.9927 adjusted r2 0.9713 0.9415 0.9795 adeq. precision 20.904 17.059 21.697 level of significance *p < 0.05, **p < 0.01, ***p < 0.001 represented the experimental data. the coefficient of multiple determination (r2) for phenolic acid yields (caffeic acid, ferulic acid, p-coumaric acid, syringic acid and vanillic acid) was 0.92; 0.96; 0.94; 0.98 and 0.97, respectively. analyses of variance for the response surface polynomial models are compiled in tab. 3. for caffeic acid, (y1), x1x2, x1x3, x2x3, x12, x22 and x32 were significant model terms (p < 0.05) (tab. 3). it means that only combinations of two factors influenced the extraction yield of caffeic acid significantly. non-significant variables were removed and the following second-order polynomial equation was found to represent the extraction yield adequately: y1=0.29+0.02x1x2+0.02x1x3−0.04x2x3−0.06x12−0.05x22−0.05x32 (2.) the highest positive effect on the extraction yield of caffeic acid showed interactions between x1x3 and x1x2 with the maximums achieved when the factors were controlled to the ultrasound influence for 30 min at 40 °c and naoh concentration of 3m. caffeic acid was the most abundant among the phenolic acids presented in the coriander fruits with concentrations ranging from 1.4 to 1.9 mg g-1 dw. the f-value of 36.02 for ferulic acid indicated that the model was significant. x1, x2, x3, x12, x22 and x32 were significant model terms (p < 0.05). it means that all tested parameters had a significant effect on the extraction yield of ferulic acid. meanwhile, the p value of x1x3 was lower than 0.05, the interaction of the temperature and naoh concentration also had a significant influence on the extraction yield of ferulic acid. the reduced second-order polynomail equation for ferulic acid was: y2=1.34−0.17x1−0.07x2−0.11x3+0.69·10−3x1x2+0.11x12+0.06x22 +0.07x32 (3.) the maximum yield of ferulic acid (0.634 mg g-1 dw) was achieved when the factors were adjusted to the central point values: temperature-60 °c; sonication time-45 min and naoh concentration3m. the developed model was significant for vanillic acid, with the f value of 53.60. there is only a 0.02% chance that an f-value this large could occur due to noise. in this case, x1, x3, x1x2, x12, x22 and x32 were significant model terms, with probability values of less than 0.05. these results indicated that the sonication time only influences the extraction yield of vanillic acid when combined with temperature 384 m. ivanović, m.i. razboršek, i.j. košir, m. kolar (p<0.001). the following is a reduced second-order equation that represents the extraction yield of vanillic acid: y3=−1.39+0.04x1+0.08x3−0.06x1x2−0.17x12−0.15x22−0.28x32 (4.) the f-value of 26.04 for p-coumaric acid implies the model was significant. in this case, x1, x2, x1x3, x12 and x32 were significant model terms (equation 5): y4=−0.42+0.15x1+0.12x2−0.22x1x3−0.21x12−0.27x32 (5.) the optimal conditions for the maximum p-coumaric acid extraction yield were those from the bbd central point (0.38 mg g-1 dw). the high f-value (75.46) for the syringic acid implies the significance of the model. there is only a 0.01% chance that an f-value this large could occur due to noise. in this case, the influence of significant model terms (x12, x22 and x32) can be represented by the following reduced second-order polynomail equation: y5=−0.40−5.93·10−5x12−1.06·10−4x22−0.03x32 (6.) effect of the independent variables on the phenolic acids ex traction recovery contour plots (fig. 4), which are the graphical representations of the quadratic polynomial regression equation, illustrate the significant (p < 0.05) interaction effects of the sonication time and naoh concentration on the recovery of the investigated compounds, with the temperature fixed at 45 °c (middle level). the results of this study also confirm that the studied conditions have a significant influence on the phenolic acids extraction recoveries, especially those from the hydroxycinnamic acid derivative group (qu et al., 2017). trans-cinnamic acid was not identified in the tested coriander samples, but it has also been a subject of study. the coefficient of multiple determination (r2) of caffeic acid, ferulic acid, p-coumaric acid and trans-cinnamic acid were 0.97, 0.93, 0.95 and 0.96, respectively (tab. 4). the model showed high significant (p < 0.001) values with the experimental data for all tested responses. the analysis of variance (anova) showed a significant (p < 0.001) negative linear (x2) effect on the extraction recoveries (tab. 4). it can be explained by the fact that the extended application of the ultrasound to the same matrix causes the degradation of phenolic acids with double-bounds in their structures (da porto et al., 2013). the f-value of 52.43 implies the model was significant for the ex traction recovery of caffeic acid. there is only a 0.02% chance that an f-value this large could occur due to noise. in this case, x2, x12, x22 and x32 are significant model terms and can be represented by the following equation: y6=7.42−0.52x2+1.14x12+0.93x22+0.80x32 (7.) represented equation means that only the sonication time (p < 0.001) has a significant influence on the extraction recovery of the caffeic acid. the negative influence of the naoh concentration on the sta bility of caffeic acid was probably eliminated by adding l-ascorbic acid and edta as stabilisers (narita et al., 2013). an adequate precision ratio greater than 4 is desirable. in this case, the value of 22.15 indicates an adequate signal, and this model can be used to navigate the design space. based on the regression coefficient (β) values, the influence of the ultrasound (x2) had a significantly negative effect on the extraction recovery of ferulic acid followed by naoh concentration (x3), interaction (x1x3), and temperature (x1). after removing the non significant variables, the following second-order polynomial equa fig. 4: contour plots of extraction recoveries (%) for bound phenolic acids from coriandrum sativum l., varying sonication time and naoh concentration (the temperature is constant in the central point-45 °c). (a) caffeic acid, (b) ferulic acid, (c) p-coumaric acid and (d) trans-cinnamic acid. phenolic composition of coriander fruit 385 tab. 4: analysis of variance (anova) of response surface second order polynomial models for phenolic acids recovery. variables responses (%) caffeic acid ferulic acid mean of square f value p value mean of square f value p value model 1.28 52.43 <0.001*** 271.38 22.61 >0.001** x1 0.07 3.03 0.142 112.50 9.38 0.028* x2 2.14 87.63 <0.001*** 760.50 63.38 <0.001*** x3 0.10 3.94 0.104 480.50 40.04 >0.001** x1x2 0.08 3.20 0.134 12.25 1.02 0.359 x1x3 0.10 4.22 0.095 156.25 3.02 0.015* x2x3 7.47·10-4 0.03 0.868 90.25 7.52 0.041* x1x1 4.79 196.72 <0.001*** 397.44 33.12 0.002** x2x2 3.20 131.49 <0.001*** 436.67 36.39 0.002** x3x3 2.34 96.20 <0.001*** 106.67 8.89 0.031* r2 0.9895 0.9760 adjusted r2 0.9706 0.9329 adeq. precision 22.150 21.713 tab. 4: continued. variables responses (%) p-coumaric acid trans-cinnamic acid mean of square f value p value mean of square f value p value model 0.03 32.93 <0.001*** 0.03 40.67 <0.001*** x1 3.19·10-4 0.36 0.573 0.02 22.62 0.005** x2 0.06 69.91 <0.001*** 0.02 23.48 0.005** x3 0.02 19.47 0.006** 0.18 269.21 <0.001*** x1x2 3.93·10-3 4.46 0.088 6.06·10-3 9.15 0.029* x1x3 4.05·10-3 4.60 0.085 1.12·10-3 1.69 0.250 x2x3 0.04 39.96 0.002** 3.51·10-3 5.30 0.070 x1x1 0.09 105.48 <0.001*** 1.50·10-3 2.27 0.193 x2x2 0.01 12.86 0.016* 8.85·10-3 13.36 0.015* x3x3 0.05 57.88 <0.001*** 0.01 19.08 0.007** r2 0.9834 0.9865 adjusted r2 0.9536 0.9623 adeq. precision 16.626 21.044 level of significance *p < 0.05, **p < 0.01, ***p < 0.001 tion was found to represent the extraction recovery of ferulic acid adequately: y7=70−3.75x1−9.75x2−7.75x3+6.25x1x3−4.75x2x3+10.38x12+ 10.88x22+5.37x32 (8.) for p-coumaric acid, the f-value was 32.93. there is only a 0.06% chance that an f-value this large could occur due to noise. it was found that the major negative influence on this phenolic acid recovery has an interaction (x2x3) followed by the influence of the ultrasound (x1) and naoh concentration (x3). those interactions can be represented by following reduced equation: y8=4.28−0.09x2−0.05x3−0.09x2x3+0.16x12+0.06x22+0.12x32 (9.) trans-cinnamic acid has also been a focus of this research. this study proved that stability of trans-cinnamic acid was also affected by the tested conditions of temperature, sonication time and naoh concentration. the model was significant with an f-value of 40.67. the equation that described the influence of the independent variables on the trans-cinnamic acid recovery can be written as: y9=4.26−0.04x1−0.04x2−0.15x3−0.04x1x2+0.05x22+0.06x32 (10.) choosing the best hydrolysis and extraction conditions must take into account both, phenolic acids yield and phenolic acids extraction recovery. thus, the optimum conditions applied were 35.3 ºc, 2.02 m concentration of naoh and a sonication time of 17.4 minutes. conclusions this paper represents our contribution to the understanding of coriandrum sativum l. polyphenol composition. to that end, the total phenolic content (tpc) and total flavonoid content (tfc) in the methanolic extract of coriander fruits were firstly determined. the maximum measured values for the tpc and tfc were 2900 mg gae g-1 dw and 850 mg rut g-1 dw, respectively. additionally, a simple and fast ultrasound-assisted extraction (uae) method involving gc-ms analysis for isolation and quantitative determination of total (free and bound) phenolic acids together with their geometric isomers was used for the first time. a response surface methodology and box-behnken design were successfully applied for the optimization of the most important uae parameters (temperature, sonication time and naoh concentration). due to the satisfactory statistical parameters (r2 and cv) and analysis of variance (anova), it could be concluded that the developed second-order polynomial models 386 m. ivanović, m.i. razboršek, i.j. košir, m. kolar provided an adequate mathematical description of the uae extraction yields. furthermore, according to the results obtained, it could be concluded that all tested parameters had a significant impact on the extraction yields of phenolic acids, but sonication time was the most critical factor. with the aim to maximize the extraction yields of all independent variables, the following extraction conditions were determined to be the most optimal: sonication time of 17.4 min at 35.3 °c and naoh concentration of 2.02 m. finally, it can be concluded that coriander fruit is rich in polyphenols, including phenolic acids and can represent a potential natural source of antioxidants for food and pharmaceutical industries. acknowledgments authors thank financial support provided by the slovenian research agency (arrs), programmes p1-0153 and p2-006. m. ivanović also acknowledge the financial support for phd study provided by the erasmus mundus joineu-see penta project. references abad-garcía, b., berrueta, l.a., garmón-lobato, s., 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sativum l.): a potential source of high-value components for functional foods and nutraceuticals a review. phyther. res. 1456, n/a-n/a. doi: 10.1002/ptr.4897 wang, l., yao, y., he, z., wang, d., liu, a., zhang, y., 2013: determination of phenolic acid concentrations in wheat flours produced at different extraction rates. j. cereal sci. 57, 67-72. doi: 10.1016/j.jcs.2012.09.013 zeković, z., kaplan, m., pavlić, b., olgun, e.o., vladić, j., canli, o., vidović, s., 2016: chemical characterization of polyphenols and volatile fraction of coriander (coriandrum sativum l.) extracts obtained by subcritical water extraction. ind. crops prod. 87, 54-63. doi: 10.1016/j.indcrop.2016.04.024 orcid: iztok jože košir https://orcid.org/0000-0002-2829-1335 address of the authors: milena ivanović; maša islamčević razboršek, laboratory for analytical chemistry and industrial analysis, faculty of chemistry and chemical engineering, university of maribor, smetanova ulica 17, si-2000 maribor, slovenia iztok jože košir, slovenian institute of hop research and brewing, department of agrochemistry and brewing, cesta žalskega tabora 2, si3310 žalec, slovenia mitja kolar, department of chemistry and biochemistry, faculty of chemistry and chemical technology, university of ljubljana, večna pot 113, si-1000 ljubljana, slovenia e-mail: mitja.kolar@fkkt.uni-lj.si © the author(s) 2019. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1016/j.indcrop.2016.01.015 http://dx.doi.org/10.1016/j.ultsonch.2012.12.007 http://dx.doi.org/10.1016/j.ultsonch.2017.05.034 http://dx.doi.org/10.1016/j.cbpb.2013.07.004 http://dx.doi.org/10.1016/j.foodchem.2013.10.157 http://dx.doi.org/10.1016/j.indcrop.2012.08.029 http://dx.doi.org/10.1016/j.foodchem.2008.07.064 http://dx.doi.org/10.1002/ptr.4897 http://dx.doi.org/10.1016/j.jcs.2012.09.013 http://dx.doi.org/10.1016/j.indcrop.2016.04.024 mailto:mitja.kolar@fkkt.uni-lj.si journal of applied botany and food quality 93, 300 312 (2020), doi:10.5073/jabfq.2020.093.037 runder tisch kakao hamburg, berlin, germany the biochemistry of cocoa flavor – a holistic analysis of its development along the processing chain daniel kadow (submitted: august 11, 2020; accepted: december 10, 2020) summary the seeds of the cacao tree theobroma cacao l. are the key ingredient of chocolate (codex alimentarius, 1981). they have a unique and complex flavor profile. up to 87 descriptors can be distinguished (januszewska et al., 2018). beside the typical chocolate aroma, floral, fruity and nutty aroma notes may occur. regarding taste attributes, typical cocoa bitterness and acidity are of key importance. a pleasant, mouth-filling astringency does complete the typical flavor characteristics. these attributes can be more or less intense, or even absent as in the case of floral, fruity and nutty notes, resulting in a great diversity in the flavor profile between countries, regions and even plantations (sukha et al., 2007). this diversity depends on a network of multiple interacting factors along the processing chain, from the genetic background of the cacao trees, seed physiology and climatic conditions over fermentation, drying and roasting up to chocolate ingredients such as sugar (andersson et al., 2006; afoakwa et al., 2008; muñoz et al., 2019). the purpose of this review is to analyze the current knowledge about the impact of these factors and their interaction on the composition of taste and aroma-active substances as well as respective precursors and how specific flavor profiles can be explained through them. gaps in research as well as potential applications in cocoa processing and chocolate manufacturing are discussed. origin and distribution of the cacao tree and the genetic determination of the flavor potential the cacao trees origin are the rainforests of the eastern andean slopes of the amazonian basin. in these areas, the greatest genetic diversity has been observed. from the center of origin a natural dis tribution along the rivers of the amazonian basin took place (fig. 1a) (bartley, 2005; motamayor et al., 2008). at trade level, cacao trees from this huge area are grouped as 'forastero' (rohsius, 2007). scientifically, the group of forastero is divided into eight genetic clusters (motamayor et al., 2008). approximately 1,500 years bc cacao was distributed by olmec’s to central america where it gained increasing cultural and economic importance, especially in the civilizations of maya and aztecs. it was cultivated in plantations and apparently subjected to breeding (bartley, 2005; rohsius, 2007). this lead to a clear genetic distinction from cacao trees in the amazonian basin. the cacao from these areas in central america is named 'criollo' at both, trade and scientific level and forms an independent ninth genetic cluster (motamayor et al., 2008). the genetic diversity manifests also in the biochemical composition of the cacao seeds. whilst forastero seeds are mostly of dark violet color, criollo seeds are usually of lighter color up to white, due to a lack in anthocyanin (fig. 1a). sensory attributes also are affected. criollo is often described as mild and less astringent and bitter (elwers et al., 2009). recently, traces of an even earlier cultivation and use of cacao located in south america at the santa ana-la florida site in southeast ecuador, dating back about 5,300 years bc, have been reported by zarillo et al. (2018). a younger cultivation history might have the cacao nowadays grown in ecuador. first records about commercial plantings are from the europeans and date back 420 years (fig. 1a). according to historical sources, the first plantations were located at the guayas river shores and spread in its tributaries the rivers daule and babahoyo – upward rivers – probably the origin of the trade name 'arriba' of ecuadorean cocoa (loor et al., 2010). however, a previous distribution by the native population seems most likely (rohsius, 2007; zarillo et al., 2018). selections for distribution might have been made based on the aromatic, floral fruit pulp aroma (lieberei, personal communi cation). as traditional cultivar, this cacao also forms a distinct tenth genetic cluster named 'nacional' at scientific level (motamayor et al., 2008), a term sometimes also used by the trade, i.e. 'nacional' or 'arriba nacional'. nacional is known to develop a strong floral aroma (loor et al., 2010). criollo was the first cocoa encountered by europeans after the discovery of the american continent in 1492 a.d. when approximate ly 100 years later the cocoa consumption in europe increased strongly, the cultivation of criollo was extended to the caribbean islands, including trinidad (rohsius, 2007). in 1670 a.d. these plantations were largely destroyed by a disease and new cacao trees from the populations in south america, discovered in the meanwhile, were introduced. in the rehabilitated plantations, now containing criollo fig. 1: historical distribution of the cacao tree (theobroma cacao l.) in the americas (a) and worldwide (b) modified from rohsius (2007). red dot: center of origin. (map: http://www.reliefs.ch/blasse_weltkarte.jpg. iimages of cocoa taken from rohsius (2007)). the biochemistry of cocoa flavor 301 and cacao from the genetic clusters in the amazonian basin, crossings occurred. recalling their origin trinidad, these crossings are named trinitario at scientific and at trade level (fig. 1a) (bartley, 2005; rohsius, 2007). they can be assigned to different genetic clusters, depending on whether the criollo or the forastero background is dominant (johnson et al., 2009; gopaulchan et al., 2017). thus, whilst at scientific level ten genetic clusters are distinguished, at trade level there are four groups: forastero, criollo, nacional and trinitario. from criollo, nacional and trinitario trees so called fine or flavor cocoa is produced (international cocoa organization, 2019). it is characterized by the presence of ancillary flavor notes such as the floral aromas in case of nacional seeds (loor et al., 2010), fruity notes in case of trinitario cocoa and nutty flavors, characteristic for example for some criollo varieties (e.g. sukha and butler, 2005). such aromas do not occur in most forastero seeds. for them a strong basic chocolate aroma is typical (sukha et al., 2007). accordingly, the potential to develop fine flavor notes is genetically determined. therefore, the genetic structure of the plantations is one essential criterion to gain fine flavor status. two more criteria need to be ful filled, appropriate post-harvest processing, i.e. fermentation, and the exportation of cocoa. approximately 8% of the cocoa produced worldwide is fine or flavor cocoa, 92% are bulk cocoa (quarterly bulletin of cocoa statistics, 2019). from 1660 a.d. on the spanish distributed criollo trees to asia and east africa via the pacific route (fig. 1b) (bartley, 2005; rohsius, 2007). indeed, in several countries and regions, such as madagascar and java, criollo descendants are still planted and they have fine flavor status (international cocoa organization, 2019). in 1746 a.d., the first plantations of forastero, of genotypes belonging to the amelonado cluster, were established in bahia (bartley, 2005; rohsius, 2007; motamayor et al., 2008). indeed, brazil nowadays produces bulk cocoa (international cocoa organization, 2019). from bahia, starting in 1822 a.d., cocoa trees were distributed to west africa (fig. 1b). descendants of these trees were later referred to as 'west african amelonado' (rohsius, 2007). nowadays, more than 76% of the worldwide cocoa production is produced in west africa (quarterly bulletin of cocoa statistics, 2019). because of the genetic background of the trees, exclusively bulk coco is produced (international cocoa organization, 2019). 17.2% of the cocoa comes from south america, 6.3% from asia and oceania (quarterly bulletin of cocoa statistics, 2019). from 1880 a.d. on, trinitario varieties were distributed from trinidad to south america, asia and africa (fig. 1b) (bartley, 2005; rohsius, 2007). indeed, countries with trinitario plantations, such as trinidad itself or vietnam have fine flavor status. beside the genetic structure of the plantations, they also do fulfill the criteria of appropriate fermentation and exportation of cocoa, like all the countries mentioned. a full list of the countries producing and exporting fine or flavor cocoa can be found in the annex c of the international cocoa agreement (international cocoa organization, 2019). because of this historical distribution and the genetic structure of plantings it is connected with, as well as traditional processing protocols analyzed in the following paragraphs, countries are associated with specific flavor profiles. in the last decades, changes in the genetic composition of plantations have been observed worldwide (meinhardt, 2019). these changes are due to new selections and breeding approaches, the introduction of genetic material from other countries and farmer selections. consequently, typical traditional flavor profiles are changing. for studies about the development of specific flavor profiles, it is therefore important to analyze the genetic background of cocoa samples. meinhardt (2019) has recently proposed a methodology based on single nucleotide polymorphisms (snps), allowing the verification of the genetic background of single fermented and dried cocoa seeds. based on this method, one hundred seeds per cocoa sample can easily be analyzed, providing detailed insight into the genetic background. the great genetic diversity, described by motamayor et al. (2008), further underlines the necessity of a more detailed understanding of the impact of the genetic clusters and the genotypes on flavor and how specific flavor attributes are obtained. cocoa seed and fruit pulp morphology cocoa fruits are of various shape and color and contain up to 50 seeds. each seed is surrounded by an aqueous, sweet-sour tasting and sometimes aromatic fruit pulp, which derives from the fruits endocarp (fig. 2) (lieberei and reisdorff, 2007). the aroma of the fruit pulp that can be of floral or fruity character has been linked to the fine flavor attributes of the seeds. it is supposed that the respective aroma-active substances migrate into the seeds during fermentation (e.g. eskes et al., 2018). the fruit pulp stays attached to a hardened but flexible seed shell, protecting the embryo from mechanical damage (lieberei and reisdorff, 2007). it contributes to between 10 and 14% of the seeds dry weight. the embryo consists of two large storage cotyledons, comprising 86 90% of the dry weight (muñoz et al., 2019), a small hypocotyl and an even smaller radicle (fig. 2). cocoa seeds are recalcitrant and contain about 27 to 40% of water upon ripeness (stoll, 2010). fermentation, drying and their impact on the flavor profile fresh cocoa seeds have a very low storage stability, because of their high water content and the aqueous fruit pulp. they are characterized by an unpleasant bitter taste and a mouth-drying astringency, except for some criollo genotypes. fresh cocoa seeds do not develop any chocolate aroma and no typical bitterness during roasting, because they do not contain the necessary precursors (ziegleder and biehl, 1988; stark and hofmann, 2005). these attributes drastically change during primary post-harvest processing, consisting of fermentation and drying. for fermentation, the seeds with the attached fruit pulp are removed from the fruits and transferred into wooden boxes or heaped on banana leaves. sizes do vary and the boxes may contain from 200 kg of seeds up to several tons. during fruit opening, a spontaneous inoculation of the fruit pulp with microorganisms from the environment takes place. further inoculation may come from the boxes, the surface of the banana leaves or from fruit flies (schwan and wheals, 2004; weckx and de vuyst, 2016). formulations of starter cultures have been suggested (e.g. lefeber et al., 2012), but are not frequently applied so far. the microorganisms find in the fruit pulp an ideal growth media. their activity results in a degradation of the fruit pulp, heating of the fermentation mass and acidification of the seed tissue (schwan and wheals, 2004; weckx and de vuyst, 2016). fig. 2: cocoa seed morphology 302 d. kadow under these conditions, the chocolate aroma precursor formation from seed storage components initiates and the precursors of typical cocoa bitterness are produced. substances responsible for unpleasant bitterness and astringency are oxidized (ziegleder and biehl, 1988; stark et al., 2006; elwers et al., 2009; voigt et al., 2016). the use of starter cultures can reliably improve the fermentation, potentially leading to higher consistency within this important processing step and consequently also with regard to flavor quality (lefeber et al., 2012). the oxidation processes and the aroma precursor formation proceed during the drying process, following upon completion of fermentation (ziegleder, 2016). the residual moisture content is reduced to approximately 7%. the cocoa seeds become storage stable for several years, are low in unpleasant bitterness and astringency and develop chocolate aroma and typical cocoa bitterness during roasting. fermentation time may vary from as few as two days up to seven days. with increasing fermentation time unpleasant bitterness and astringency are more and more reduced. increasing amounts of chocolate flavor precursors are produced (rohsius, 2007; kumari et al., 2016), resulting in increased chocolate flavor intensity after roasting. however, fine flavor aroma, such as floral notes, seem to be reduced in their intensity with increasing fermentation time and can be completely lost (eskes et al., 2018). indeed, cocoa with fine flavor potential is usually fermented for less time than bulk cocoa, to obtain an optimal flavor profile (rohsius, 2007). such traditional local fermentation protocols thus are essential for the origin-specific flavor profiles. in other words, the flavor potential is genetically determined, but the flavor profile is defined during fermentation and drying. hereby, fermentation protocols include the opportunity to diversify flavor. cocoas with distinct flavor profiles, for example with light chocolate aroma and pronounced floral notes, or with intense chocolate aroma and light or no floral notes, can be produced from the same batch of fresh beans using adapted protocols. however, the potential to develop floral aroma or other fine flavor notes remains genetically determined. beside the duration, also the turning interval has great impact onto the flavor profile (caobisco/eca/fcc cocoa beans manual, 2015). the concept of terroir in cocoa terroir is a highly important concept in viticulture (van leeuwen and seguine, 2006). in cocoa, where terroir effects similar to wines are implied in many origin specific cocoas and chocolates, the impact has not yet been systematically tested (sukha et al., 2017). in general, terroir can be defined as an interactive ecosystem, in a given place, including climate, soil and the crop plant (seguin, 1988; van leeuwen and seguine, 2006). human factors, such as history, socioeconomics, as well as crop-cultivation and processing techniques, are also part of terroir. due to the many factors being involved and because these factors are interacting, terroir is difficult to study (seguin, 1986; van leeuwen and seguine, 2006). for cocoa, especially the importance of traditional origin-specific fermentation protocols for the flavor profile is well known and has been described in the above paragraph. in addition to this, sukha et al. (2017) report impact of the growing environment on the inten sity of the astringency, the bitter taste and the fruity aroma. the processing location may apparently affect the intensity of fruity, floral and nutty aromas and of the acidic taste. it remains however elusive, which components of the processing location and which en vironmental factors affect the flavor development. additional insides to this are provided by hegmann (2015), who shows that higher amounts of rainfall may potentially result in increased intensity of floral notes. findings of zug et al. (submitted) indicate that there might be an impact of the soil ph on components contributing to astringency and bitterness. however, as pointed out by sukha et al. (2017), systematical studies are needed to better understand terroir effects in cocoa. quality analysis by means of the cut test fermentation and subsequent drying go along with a change in color of the cotyledons. fresh seeds can be violet, slight-pink, rosé or white, depending on the genetic background (rohsius, 2007). when dried unfermented, violet seeds are of light-violet color and waxy consistence (fig. 3). they are called 'slaty'. upon acidifica tion, the color turns into intense dark violet (fig. 3). these seeds are still under-fermented and called 'violet'. with ongoing fermentation, the color starts turning brownish, often from the center of the seeds. such seeds are partially fermented. only upon completion of the fermentation process, their cotyledon color turns fully brownish (fig. 3). consequently, the respective seeds are called 'brown' (rohsius, 2007). thus, cotyledon color allows an assumption about the flavor attributes of a batch of cocoa. when a batch contains significant amounts of slaty seeds, it will usually be characterized by unpleasant bitterness and astringency and a lack of chocolate flavor after roasting. with increasing amounts of partially and fully fermented seeds and a residual quantity of violets, light chocolate aroma will become characteristic. fine aroma attributes are pre served. when most of the seeds are fully fermented, the chocolate flavor will be well pronounced, but fine aroma might be lost. an assumption about the genetic background also is possible, as slight-pink, rosé and white seeds maintain their lighter appearance during fermentation and drying, turning to light brown, beige or remain of almost white color (rohsius, 2007). forastero seeds are usually dark violet, turning dark brown during fermentation and drying. in contrast, pure criollo seeds stay almost white (fig. 1a). trinitario seeds can display a mixture of colors, from dark brown, over light brown to beige and almost white. the ratios may largely vary. however, exceptions do occur. catongo for example, a fora stero cacao, has white seeds too (rohsius, 2007). the evaluation of cotyledon color, the cut test, is a widely used industry standard (caobisco/eca/fcc cocoa beans manual, 2015). ' fresh cocoa bean ingredients and their impact on flavor cocoa beans are characterized by a unique composition of different nutrients. beside fat, which contributes to about 50% of the dry weight, the seeds contain 12% protein and 7% carbohydrates. more over, they are rich in phenolic substances, about 15% of the dry weight. alkaloids contribute to about 4% of the dry weight (e.g. kadow et al., 2015). both groups, phenolic substances and alkaloids, are of key importance for the flavor profile of fresh cocoa seeds. amongst the phenolic substances are epicatechin, the predominant one in quantity, and catechin. both are often linked to unpleasant bitterness and astringency (e.g. elwers et al., 2009), especially in unfermented and under-fermented seeds. the flavor attributes of epicatechin and catechin are described as both, bitter and puckering astringent (stark et al., 2006). elwers et al. (2009) observed no significant variation between seeds with different genetic background regarding the epicatechin concentration. however, the concentration varies from 14 21 g · kg-1 of cocoa nibs. broader variation was found fig. 3: color change of cocoa seeds during fermentation and drying. (images of cocoa taken from rohsius (2007)). the biochemistry of cocoa flavor 303 for catechin. whilst some genoytpes do not contain any catechin, in others up to 1.1 g · kg-1 were measured (elwers et al., 2009). to valuate the contribution of a substance to a sensory attribute, its threshold concentration (tc) for human perception needs to be considered (e.g. stark et al., 2006). only if the concentration is above threshold, a contribution to the taste profile is likely and the substance can be regarded as probably taste-active. the tc of the bitter taste of epicatechin and catechin in cocoa butter is 2.1 g · kg-1 and 0.9 g · kg-1, respectively. dividing the concentration measured in the seeds by the tc, the dose over threshold factor (dot) is calculated, i.e. the factor by that the concentration exceeds the specific tc. a dot factor greater than one makes it likely, that the respective substance contributes to the taste profile (stark et al., 2006). the dot factor for bitter taste of epicatechin varies from 6.5 to 10.3, whilst for catechin a maximum dot of 1.2 results, based on the concentrations reported by elwers et al. (2009). thus, in fresh beans epicatechin contributes most likely to the unpleasant bitter taste. catechin might contribute as well. the dot factors for the puckering astringency caused by epicatchin vary from 9.5 to 15.1 between samples. for catechin a dot of 0.9 is not exceeded. thus, epicatechin contributes most likely to the unpleasant astringency of unfermented and under-fermented seeds. catechin apparently does not. alkaloids, in particular theobromine, also contribute to the bitter taste of fresh cocoa seeds. the concentration in fermented and dried seeds varies from 9.1 to 17.6 g · kg-1 (rohsius, 2007), depending on the genetic background. the respective dot factor range is 15.3 to 29.5. during fermentation and drying the theobromine content slight ly decreases to approximately 81% of the initial amount (peláez et al., 2016). thus, it can be assumed that the dots in unfermented seeds are slightly higher, 19 to 37 approximately. caffeine occurs in lower concentrations, 0.5 to 4.2 g · kg-1 in fermented dried seeds (rohsius, 2007). dot factors are between 0.4 and 3.1. however, caffeine seems to decrease to approximately 50% of the initial amount during fermentation and drying (muñoz et al., 2019). ac cordingly, dot factors in fresh seeds might be as high as 0.8 to 6.2. nevertheless, it apparently depends on the genotype, if caffeine contributes to the bitter taste of fresh seeds or not. for puckering astringency, also conjugates of cinnamic acid and amino acids are of importance. caffeic acid aspartate for example occurs in concentrations of up to 3.3 g · kg-1 (elwers et al., 2009). having a low tc, the dot factor of caffeic acid aspartate can be as high as 59 in fresh seeds, indicating its importance for puckering astringency. however, some genotypes do only contain marginal amounts or even no caffeic acid aspartate (elwers et al., 2009). fresh fruit pulp ingredients and their impact on flavor solid in under-ripe fruits, the fruit pulp turns mucilageous in fully ripe fruits, because of the fruit own pectinolytic activity. the fruit pulp of ripe fruits is aqueous, containing about 83% of water (pettipher, 1986). it is also rich in carbohydrates. a sucrose concentration of 13 up to 43 g · kg-1 is typical (pettipher, 1986), explaining the sweet taste. the tc for sucrose is 4.3 g · kg-1, in water (stark et al., 2006). the dot range is 3 to 10, accordingly. the sweetness is contrasted by a pleasant acidity, caused by the approximately 2% of organic acids that the fruit pulp contains. the greatest part of it is ci tric acid. 3 to 13 g · kg-1 are typical. the tc of citric acid is 0.5 g · kg-1 in water (stark et al., 2006). the respective dot range is 6 to 26. amongst these major components, the fruit pulp contains variable amounts of aromatic substances: terpenes, alcohols and esters. fruit pulp of fine or flavor cocoas seems to contain much higher amounts than fruit pulp of bulk cocoas does (kadow et al., 2013). in line with this, the aroma intensity for example for floral notes of such genotypes, apparently caused by linalool and β-ocimene, is rated as high as nine and five on a scale from zero to ten in solid phase microextraction gc-olfactometry analysis. no floral aroma was observed in the bulk cocoa fruit pulp sample (kadow et al., unpublished data). kadow et al. based their study i.e. also the genotype selection on previous work by eskes et al. (2007). the authors observed as well floral and fruity aroma only in the fruit pulp of the fine flavor genotypes they analyzed. these aroma notes were absent in bulk cocoa fruit pulp (eskes et al., 2007). linalool has often been linked to fine flavor quality and the floral notes of ecuadorean arriba (ziegleder, 1990). besides linalool, also 2-phenylethanol may contribute to floral aroma of fresh fruit pulp. depending on the genotype, chetschik et al. (2018) report concentrations of 250 μg · kg-1 of 2-phenylethanol and 14 μg · kg-1 of linalool. like for taste-active substances, aromatic substances need to be above tc to contribute to the overall aroma impression. the tcs for 2-phenylethanol and linalool are 211 and 37 μg · kg-1, respectively. apparently, only 2-phenylethanol contri butes to the floral aroma of this specific fruit pulp, at least in its fresh status. 3-methylbutyl-acetate found in concentrations of 5 μg · kg-1 may provide a fruity character instead (chetschik et al., 2018). the tc for 3-methylbutyl-acetate can be as low as 0.75 μg · kg-1. beside the genetic impact, the linalool concentration in the fruit pulp can be influenced by the degree of fruit ripeness and by climatic conditions. hegmann (2015) report 10-fold increased linalool amounts during the rainy season. the findings add further information and evidence to the concept of terroir in cocoa. biochemical changes during fermentation and their impact on flavor changes of fruit pulp ingredients the anaerobic phase beside sucrose, the fruit pulp contains significant amounts of fructose and glucose (pettipher, 1986). together with the acidic ph of about 3.6 ideal growth conditions for yeasts do result. amongst other yeasts and in most of the fermentations worldwide, saccharomyces cerevisiae establishes in the fruit pulp at the beginning of the fermentation process (fig. 4). pichia species such as pichia kluyveri and candida species have also often been found (schwan and wheals, 2004; de vuyst and weckx, 2016). tight packaging of the cocoa seeds in the fermentation boxes and in the heaps together with the metabolic activity of the yeasts result in anaerobic conditions. consequent ly, the yeasts do degrade the pulp sugars to alcohol, mainly ethanol (fig. 4). in addition, they secret pectinase, resulting in further liquefaction of the fruit pulp (schwan and wheals, 2004; de vuyst and weckx, 2016). pichia and candida species do additionally metabolize the citric acid, causing an increase of the ph value of the fruit pulp (schwan and wheals, 2004). moreover, p. kluyveri produces elevated amounts of esters with fruity aroma (fig. 4). it is therefore used as starter culture (crafack et al., 2013). also saccharomyces cerevisiae, strain h5s5k23, has been suggested for starter culture formulations, as it contributes to enhanced and consistent ethanol production (lefeber et al., 2012). the increasing ph value, resulting from the citric acid degradation, allows the growth of bacteria. lactic acid bacteria (lab) do establish in the fermentation mass, shortly after the yeasts, amongst them many lactobacillus species such as l. plantarum and l. fermentum. the great majority of lab isolated from cocoa fermentations utilize glucose via the embden-meyerhof pathway yielding more than 85% lactic acid (fig. 4). however, some species utilize glucose via the hexose monophosphate pathway producing 50% lactic acid, and in addition ethanol, acetic acid, glycerol and mannitol (schwan and wheals, 2004). at which time point in the fermentation lab can establish could depend on the citric acid concentration. if the concentration is rather low, as observed for example in malaysian cocoa, lab could establish earlier, resulting in a lab dominated fermentation. this may have impact on the fla304 d. kadow vor profile, as lactic acid has physico-chemical properties that differ from those of acetic acid and is hardly removed from the seeds during further processing. in their study, ho et al. (2015) conclude that lab may not be required for successful fermentation of cocoa seeds. however, lefeber et al. (2012) report that a full starter culture, comprising beside s. cerevisiae h5s5k23 also l. fermentum 222 and a. pasteurianus 386b have been suggested for starter culture formulations as this allows a consistent production of high-quality fermented dry cocoa beans (lefeber et al., 2012) the aerobic phase with its increasing liquefaction, the fruit pulp starts draining from the fermentation mass. oxygen does re-enter. at this point in time, the fermentation mass is often turned, to allow equal oxygen distribution. with oxygen available, acetic acid bacteria (aab), such as acetobacter aceti and gluconobacter oxydans, start dominating in the fermentation mass. in their review, schwan and wheals (2004) provide lists of yeast, lab and aab species detected in cocoa fermentations. aab oxidize the ethanol, previously produced by yeast, to acetic acid (fig. 4). concentrations of a maximum of 6 g · l-1 of fruit pulp have been observed. the oxidation goes along with a heating of the fermentation mass and temperatures of 48 °c are usually reached (schwan and wheals, 2004). the acetic acid starts migrating into the seed tissue, where 1.5 to 12 g · kg-1 are found in fermented and dried seeds (rohsius et al., 2010). at the beginning, this migration seems to take place mainly via the micropyle (fig. 2; fig. 4). the seed shell at this point is impermeable for acetic acid, which is accumulating in a sclerenchymatic cell layer with thick cell walls (andersson et al., 2006). thus, the velocity of the seed tissue acidification seems to depend on how fast the micropyle opens, a seed physiological variable, and on how fast the sclerenchymatic cell layer is saturated with acetic acid. if genetic variability exists with regard to this cell layer, and thus regarding the velocity of the acetic acid migration, has not yet been investigated. fine flavor substances the aromatic alcohols, terpenes and esters, which the fruit pulp contains depending on the genotype, also undergo changes during the fermentation process. 2-phenylethanol increases 480fold, reaching a concentration of 120mg · kg-1 (chetschik et al., 2018). like for tastants the dot, an odor activity value (oav), i.e. the 2-phenylethanol concentration divided by its tc, can be calculated for aroma substances (frauendorfer and schieberle, 2008). the oav of 2-phenylethanol increases from 1.2 in the fresh fruit pulp to 569 during fermentation. accordingly, 2-phenylethanol now strongly contributes to the floral aroma of the fruit pulp. fruit pulp aroma attributes are often used as indicator for fine flavor quality (eskes et al., 2018). however, the findings of chetschik et al. (2018) indi cate that at least some floral aromas might be rather weak in ripe fruits and do strongly increase only during fermentation. in the seeds, the concentration of 2-phenylethanol increases from 42 μg · kg-1 to 16 mg · kg-1(chetschik et al., 2018). thus, the concentration in the fruit pulp is higher than in the seed tissue at any time during fermentation and might be the driving force of a migration into the cotyledons as suggested by eskes et al. (2007; 2012; 2018). the authors found that when floral aroma notes are present in the fresh fruit pulp, these notes can be detected by means of sensory evaluation in the seeds after fermentation and drying (eskes et al., 2007). when adding aromatic annona fruit pulp to bulk cocoa fermentations, the annona aroma traits were found in the dried seeds (eskes et al., fig. 4: physicochemical networks during standard cocoa fermentation. based on the discussions with reinhard lieberei, christina rohsius and silke elwers. the biochemistry of cocoa flavor 305 2012). indeed, when subjecting fresh cocoa seeds to an air stream containing linalool or β-ocimene, the substances could both be detected in the seed tissue afterwards (kadow et al., unpublished data). the oav for 2-phenylethanol in the seed tissue increases from 0.2 and thus below threshold to 76. accordingly, 2-phenylethanol confers most likely floral aroma to the seeds. a substantial decrease with increasing duration of the fermentation, especially in the seeds, has not been observed (chetschik et al., 2018). thus, a loss of fine flavor attributes during prolonged fermentation is not necessarily caused by a loss of the respective fine aroma substances. the linalool concentration increases 12-fold in the fruit pulp during fermentation, reaching a concentration of 170 μg · kg-1 (chetschik et al., 2018), corresponding to an increase of the oav from below threshold to 4.6. however, fresh cotyledons already contain 114 μg · kg-1. after five days of fermentation a concentration of 1130 μg · kg-1 is reached. thus, the concentration is lower in the fruit pulp than in the cotyledons at any time of the fermentation process. a gradient, explaining a migration into the seeds, is not present in the case of linalool. it seems to be produced in both tissues and to a greater extent in the cotyledons (chetschik et al. 2018), where with an oav of 31 linalool most likely contributes besides 2-phenylethanol to the floral aroma. accordingly, fine flavor components may be fruit pulp derived, as in the case of 2-phenylethanol, or seed tissue derived as for linalool (fig. 4). however, it needs to be considered that the fruit pulp largely consist of water. linalool has a low solubility in water. the cotyledons instead contain high amounts of fat, with presumably better linalool solubility. thus, a simple comparison of concentration gradients based on the weight of the tissues, may not adequately reflect the actual linalool migration gradient. 2-phenylethanol, which is clearly fruit pulp derived, can be synthesized through the ehrlich pathway. interestingly, this pathway depends on s. cerevisiae, present in cocoa fermentations worldwide. phenylalanine is metabolized first into phenylpyruvate, next into phenylacetaldehyde and finally into 2-phenylethanol (chetschik et al., 2018). however, the phenylalanine content of the fruit pulp seems to be rather low. 1.3 mg·kg-1 have been found by pettipher (1986). this quantity is not sufficient to explain the 120 mg·kg-1 of 2-phenylethanol, reported by chetschik et al. (2018). thus, a 2-phenylethanol release from glycosylated precursors seems more likely (fig. 4). however, it needs to be considered that pettipher (1986) did his measurements on bulk cocoa fruit pulp. it cannot be excluded, that fruit pulp of fine flavor cocoas has higher phenylalanine concentrations. another possibility is that phenylalanine is released from proteins, especially from storage proteins in the seeds, during fermentation. nevertheless, when the free amino acid release from storage proteins initiates, the yeast populations in the fruit pulp already decreased. in addition, if storage protein derived phenylalanine would be the source of 2-phenylethanol formation, high concentrations should be found in any cocoa, not only in fine flavor cocoa. changes of cotyledon ingredients astringent and bitter-tasting substances the acidification caused by the migration of acidic acid, together with the increasing temperature, result in cotyledon tissue death and thus in cell disintegration (schwan and wheals, 2004). an aqueous and a fatty reaction space do result (e.g. ziegleder, 2014). substrates and enzymes, previously stored in separate cell compartments get mixed. such as the phenolic substances and polyphenol oxidase (ppo) in the aqueous reaction space (fig. 4). upon oxygen availabili ty and a sufficiently high ph, the ph optimum of cocoa ppo is 6.4, ppo oxidizes catechin and epicatechin into the respective chinones. the chinones presumably react with proteins, carbohydrates, cell wall constituents and other cell substances, forming large insoluble polymers (afoakwa et al., 2008; elwers, 2008). however, the detailed reaction pathways that ppo activity leads to, still need further investigation. as consequence of the polymerization, the concentration of soluble catechin and epicatchin in the cotyledons decreases. elwers et al. (2009) report residual amounts of 8% for epicatechin and 1% for catechin. only soluble epicatechin and catechin seem to contribute to unpleasant bitterness and astringency. accordingly, the respective dot factors for bitterness and astringency decrease (fig. 4). caffeic acid aspartate seems to be less affected. 33% of the initial amount remain soluble (elwers et al., 2009). however, many analyses on this decrease during fermentation, such as the ones of elwers et al. (2009), have been carried out on dried seeds. thus, the impact of fermentation and the impact of drying cannot be fully distinguished. future studies should consider to shock-freeze sample aliquots immediately in liquid nitrogen, to compare their biochemical composition to the one after drying. flavor precursor formation moreover, during fermentation a seed storage protein degradation by seed own enzymes takes place (voigt and biehl, 1995). it already starts during the first 24 hours of fermentation (kumari et al., 2016). these early degradation processes might be due to initiating germination of the seed. cocoa seeds are recalcitrant and germination seems to be triggered by oxygen availability, thus quickly initiating after fruit opening (lieberei, personal communication). upon acidification and heating of the seeds, protein degradation greatly accele rates, as the involved proteases have a low ph and a high temperature optimum (hansen et al., 1998). the degradation consists in a twostep reaction. first, endoproteases cut the storage proteins into larger peptides of approximately 16 amino acids length on the first day of fermentation. secondly, exoproteases cleave single amino acids (aa) from theses peptides (fig. 4), resulting in increasing amounts of free aa and peptides of 13 to 14 aa length after 2 to 3 days of fermentation, 6 to 12 aa after 4 to 5 days of fermentation and finally 2 to 3 aa after 6 to 7 days of fermentation. in this way, from the vicilinlike globulin alone, contributing to 23% of the storage protein, about 560 different peptides can be formed (kumari et al., 2016). the carbohydrates as well do undergo modification processes. in particular, sucrose is cleaved into fructose and glucose by invertase (hansen et al., 1998). together with the free aa, these reducing sugars are precursors for the chocolate aroma formation during roasting (afoakwa et al., 2008). in addition, some of the peptides, such as arginine-asparagin-asparagin-proline-tyrosine-tyrosine-phenylalanine-proline-lysine, seem to contribute to chocolate aroma formation too (voigt et al., 2016). diand tri-peptides, such as valine-proline, are bitter-taste precursors (fig. 3). they yield bitter tastants during the roasting process (stark and hofmann, 2005). peptides are apparently also the precursors of nutty aroma notes (fig. 4) (voigt et al., 2018). the formation of the specific peptides is favored by ph values above five. when the ph value is lower, ideally at about 4.8, increased amounts of shorter peptides and free aa seem to be produced (voigt et al., 2018), i.e. bitter taste and chocolate aroma precursors. fine flavor substance biosynthesis with regard to fine flavor substances, de wever (2020) recently showed that in fine flavor cocoa the expression of genes from the secondary metabolites pathway including the terpenoid pathway is strongly upregulated during fermentation. this might explain the tenfold increase of the linalool concentration in the cotyledon tissue reported by chetschik et al. (2018). in bulk cocoa, no such upregulation of the respective genes was found (de wever, 2020). the findings are further evidence that fine flavor substances are not always fruit pulp derived, but can be produced also in the cotyledons, depending on the type of substance. 306 d. kadow biochemical changes of cotyledon ingredients during drying and their impact on flavor aroma precursor formation during the drying process, the free aa and the reducing sugars undergo further modifications. they react with each other, forming amadori compounds, such as fructosyl-leucine and fructosyl phenylalanine. amadori compounds also are important precursors of chocolate aroma development (fig. 4). ziegleder (2016) has studied their formation during the drying process in detail. recently, andruszkiewicz et al. (2020) found that also diand tri-peptides do react with the reducing sugars yielding the respective amadori and heyns compounds. amongst them also fructosyl-valin-prolin. presumably, these compounds do also function as aroma precursors (andruszkiewicz et al., 2020) suggesting a trade-off between dipeptide-based bitter taste and chocolate aroma. depending on the drying protocol, more or less di-peptides could react to amadori and heyns compounds, hereby changing the ratios of bitter taste and chocolate aroma precursors. however, it still needs to be investigated if the diand tri-peptide based amadori and heyns compounds are really formed during the drying process or already during fermentation. astringent and bitter-tasting substances the oxidation of phenolic substances apparently proceeds, as long as a sufficient amount of free water is available. consequently, unpleasant bitterness and astringency are further reduced. elwers et al. (2009) report epicatechin concentrations of 1.1 to 1.7 g · kg-1 in fermented dried seeds, corresponding to dot factor ranges of 0.5 to 0.8 for bitter taste and 0.8 to 1.2 for puckering astringency. catechin concentrations were as low as 11 mg · kg-1. the dot factor for bitterness as well as for puckering astringency is 0.01. thus, in the studied samples epicatechin and catechin do not contribute to the bitter taste of fermented dried seeds. only epicatechin contributes marginally to the puckering astringency. however, tran et al. (2015) report residual epicatechin and catechin concentrations of up to 4 g · kg-1 and 0.25 g · kg-1, respectively. in such samples, epicatechin may contribute to both, bitterness and puckering astringency. the respective dot factors are 1.9 and 2.8. the catechin concentration is still below threshold. the greater amounts found by tran et al. (2015) might be a result of the genetic background or, more likely, caused by a lower degree of fermentation. residual amounts of cinnamic acid aspartate can be as high as 1.1 g · kg-1 (elwers et al., 2009). the respective dot for puckering astringency is 19. thus, cinnamic acid aspartate remains an important contributor to puckering astringency after fermentation and drying. interestingly, it was found in higher concentrations in criollo seeds, described as low in astringency, than in other cocoas (elwers et al., 2009). theobromine and caffeine do not undergo modifications during fermentation and drying. however, leaching during fermentation results in a decrease especially of the caffeine concentration (muñoz et al., 2019). nevertheless, theobromine still significantly contributes to the bitter taste of fermented dried seeds (fig. 4). the contribution of caffeine is genotype dependent, as the concentration can be below tc. acidic tastants moreover, during drying, acetic acid evaporates, resulting in reduced intensity of the acidic taste. the concentrations found by rohsius et al., (2010) correspond to a dot factor range of 12 to 100. drying technicians often report that quick drying, e.g. in 3 to 4 days, results in higher residual acetic acid concentrations in the seeds. these observations are supported by the findings of jinap and thien (1994) and ziegleder (2016). higher acididty is thought to cause not only a more acidic taste, but also more intense unpleasant bitterness and astringency. the observations might be explained with the ph optimum of the ppo. only after evaporation of larger acetic acid quantities, the oxidation rate would be sufficiently high. however, it could well be that sufficient oxidation is mainly a question of time. fine flavor and ancillary aromas intermediate duration of drying, i.e. five days, is thought to favor the preservation of fruity aroma notes. extending it to about seven days, results in a dominant chocolate aroma instead. finally, with very slow drying regimes, i.e. about ten to fourteen days, tobaccoand leather-like aromas seem to occur (e.g. ingemann fine cocoa). more research is needed to elucidate the aroma-active substances and the biochemical processes occurring during drying. quality analysis through quantification of flavor-related ingredients the knowledge about the substances contributing directly, or indirectly as precursors, to cocoa flavor, can be used for quality analysis. within the cocoa atlas for example, 143 cocoa samples from 32 origins were analyzed with regard to their free aa content, the free sugar concentration, the polyphenol amount as a simple sum parameter and other valuable ingredients (rohsius et al., 2010). samp les from ecuador were found to contain between 5.7 and 12.6 mg · g-1 fat free dry matter (ffdm) of free aa. in the ghana samples 13.8 to 17.3 mg · g-1 ffdm were found. unfermented cocoa contains up to 4 mg · g-1 ffdm, well fermented seeds instead 8.9 to 14.9 mg · g-1 ffdm of free aa. thus, whilst the ghana samples are all well fermented, some of the ecuador samples are almost not fermented. the analytical picture becomes more detailed when looking additionally at the reducing sugars. the ghana samples contain between 7.4 and 11.1 mg · g-1 ffdm of glucose and fructose, the ecuador samples 3.3 to 7.9 mg · g-1 ffdm. the reason for the low amount in some of the ecuador samples is the incomplete cleavage of sucrose, resulting from a low degree of fermentation. whilst the residual sucrose amounts exceed 23 mg · g-1 ffdm in some of the ecuador samples, the highest amount found in the ghana samples was 1.1 mg · g-1 ffdm. in several of the ghana samples sucrose was not detectable. finally, the total phenolics amount can be considered. the ghana samples contained 80 to 96 mg · g-1 ffdm. with 65 mg · g-1 ffdm, in some of the ecuador samples the total phenolics concentration was lower. however, the ones with low free aa concentration contained as much as 140 mg · g-1 ffdm total phenolics (rohsius et al., 2010). such high concentrations are linked with an intensity of three to four, on a scale with a maximum of five, for unpleasant bitterness and astringency (kadow, unpublished data). accordingly, the mentioned cocoa seed ingredients can be used as biochemical quality parameters. however, the work-up procedures and measurements are rather time intensive. near infrared spectroscopy (nirs) has proofed to be a suitable tool to predict the concentration of many biochemical quality parameters within seconds and in a single measurement (kräehmer et al., 2015). the velocity of nirs allows its use in quality testing upon the arrival of batches of cocoa, as well as in-process applications. biochemical changes in the cotyledons during roasting and their impact on flavor aroma-active substances strecker aldehydes during roasting, numerous volatiles are formed from the previous obtained aroma precursors, amongst them strecker aldehydes. 3-methylbutanal for example, having malty aroma attributes, increases 64fold during roasting. phenylacetaldehyde, having honeylike aroma, does also increase 64-fold. the precursors are glucose the biochemistry of cocoa flavor 307 and fructose forming diketones in a first reaction, and leucine and phenylalanine, respectively. the strong increase of both substances during roasting is a valuable indicator for their importance to the aroma profile (frauendorfer and schieberle, 2008). however, more important is the oav, the factor that the substance concentrations are above the respective threshold for human perception. 3-methylbutanal has an oav of 2610, phenylacetaldehyde an oav of 250. both substances indeed are amongst the 25 substances indispensable for typical chocolate flavor (frauendorfer and schieberle, 2006). however, when combining glucose and leucine in roasting models, the formation of 3-methylbutanal is marginal. 25-times more of 3-methylbutanal are formed, when the respective amadaori compound fructosyl-leucine is used as precursor. this demonstrates, that amadori compounds, and not free aa and reducing sugars, are the key precursors of chocolate aroma formation (ziegleder, 2016). it might also explain the above-described observation that prolonged drying results in dominant chocolate aroma and diminished fruity notes. the formation of amadori compounds mainly takes place when the residual moisture is below 20% (ziegleder, 2016). prolonged drying might result in a prolongation of this phase and in increased amadori compound formation. consequently, the chocolate aroma intensity would increase, dominating the aroma profile. in addition to the aroma-active strecker aldehydes, granvogl et al., (2012) report the detection of oxazolines in dark chocolates, amongst them 2-isobutyl-5-methyl-3-oxazoline. the substance is rapidly hydrolyzed upon contact with water, yielding 3-methylbutanal. thus, an important part of the chocolate aroma substances might be released only during chocolate consumption i.e. upon the contact with saliva (granvogl et al., 2012). it remains to be determined, during which processing step the oxazolines are formed in cocoa. pyrazines pyrazines do also strongly increase during roasting. 2-ethyl-3,5-dimethylpyrazine for example does as well increase 64-fold, corresponding to an oav of 14. it has a potato chip-like aroma character (frauendorfer and schieberle, 2008). 2-ethyl-3,5-dimethyl pyrazine can apparently be released from peptide precursors such as arginine-asparagin-asparagin-proline-tyrosine-tyrosine-phenylalanine-proline-lysine, identified by voigt et al. (2016). however, further research is needed to fully understand the role of peptides in chocolate aroma formation. it might well be that the glucosyland fructosyl-diand tri-peptides first found in cocoa beans by andruszkiewicz et al., (2020) are the more important aroma precursors, as in the case of the free aa-derived amadori compounds. furans furans as well are of key importance for chocolate aroma. 4-hydroxy-2,5-dimethyl-3(2h)-furanon for example increases 16-fold during the roasting process and has an oav of 25. its aroma character is described as caramel-like (frauendorfer and schieberle, 2008). it can be formed from the precursor mixture glucose and phenylalanine. however, again the formation is more efficient, i.e. 21-fold, when the respective amadori compound fructosyl-phenylalanine is used, underlining once more the importance of amadori compounds as chocolate aroma precursors (ziegleder, 2016). their formation mainly takes place during the drying process, when the residual humidity is between 7 and 20%. the optimum temperature for increased amadori compound formation is about 60 °c (ziegleder, 2016). consequently and as mentioned previously, the drying process needs great attention to optimal develop the flavor profile of cocoa. the odor object chocolate aroma some substances do not show any increase during roasting, but maintain their high oav. this is the case for instance for 3-methyl-butanoic acid, having an oav of 390 before and after roasting. 3-methylbutanoic acid can be produced from free aa by yeast and bacteria (coban and demirci, 2017). it has, as a single substance, a rancid aroma character (frauendorfer and schieberle, 2008). thus, it might be regarded as an off-flavor, present in the samples analyzed, but this is apparently not the case. frauendorfer and schieberle (2006) demonstrate that 3-methyl-butanoic acid, together with the strecker aldehydes, pyrazines, furans and other aroma-active substances, forms a new odor object (hofmann et al., 2014), divers from the aroma attributes of the single substances: typical chocolate aroma. in total, 24 substances are needed to reproduce the typical aroma of roasted cocoa nibs and powder. fraundorfer and schieberle (2006) provide a list with all 24 substances in their publication. fine flavor substances for fine flavor cocoas, usually milder roasting conditions i.e. lower roasting temperatures are applied to preserve the fine aroma notes (caobisco/eca/fcc cocoa beans manual, 2015). as discussed, linalool and 2-phenylethanol are key substances for floral fine aroma. in unroasted criollo seeds frauendorfer and schieberle (2008) report oavs of 29 and 76 for linalool and 2-phenylethanol, respectively. upon roasting of the seeds at 95 °c and for 14 minutes, the linalool concentration remained stable. the 2-phenylethanol concentration increased from 16 mg·kg-1 to 34.3 mg·kg-1, corresponding to an oav of 163 in the roasted seeds (frauendorfer and schieberle, 2008). thus, fine aroma substance concentration does not necessarily decrease during roasting. that fine aroma attributes may nevertheless be reduced in their intensity or lost during roasting could be due to the formation of a new odor object (hofmann et al., 2014) with increasing concentration of strecker aldehydes, pyrazines and furans at higher roasting temperatures. accordingly, the odor object would change from mild chocolate aroma with clearly perceivable floral notes to intense chocolate aroma. additional studies with multiple roasting regimes would be of interest to clarify this point. for such studies, it is not only of great importance to consider exact quantities and thresholds of the aroma-active substances, but also the respective ratios (hofmann et al., 2014). which substances are responsible for fruity and nutty aroma notes still needs to be determined. roasted bulk cocoa seeds do as well contain linalool and 2-phenylethanol. however, the concentrations are marginal in contrast to fine flavor seeds. frauendorfer and schieberle (2006) report a linalool concentration of 70 μg · kg-1 and a 2-phenylethanol concentration of 0.6 mg · kg-1. the oavs are as low as 2 and 3 for linalool and 2-phenylethanol, respectively. thus, fine aroma substances do not exclusively occur in fine flavor cocoas, but in bulk cocoa the concentrations are too low for a significant contribution to the aroma profile. taste-active and astringent substances peptide-derived bitter tastants the taste-active substance profile also undergoes major changes during the roasting process. diand tri-peptides such as valine-pro line undergo cyclization, yielding bitter-tasting diketopiperazines, cyclo(l-proline-l-valine) when valine-proline is the precursor. the amount of cyclo(l-proline-l-valine) formed during roasting increases with increasing fermentation time, as more and more valine-proline is produced. how much of the peptide precursors react to diketopiperazines also depends on the roasting conditions. andruszkiewicz et al. (2019) report a two-fold increase of cyclo(lproline-l-valine) at elevated roasting temperatures in comparison to lower temperature roasting regimes. in fully fermented and roasted seeds a cyclo(l-proline-l-valine) concentration of 1.7 g · kg-1 was found, corresponding to a dot factor of 2 in cocoa butter. in total, up to 34 diketopiperazines have been detected in cocoa (andruszkiewicz et al., 2019). an additive effect is assumed regarding their bitter taste, making them key bitter tastants of roasted cocoa (stark 308 d. kadow and hofmann, 2005; stark et al., 2006). variation might also occur, depending on the storage protein quantity. kumari et al. (2018) report quantities ranging from 2 to 13% of protein, in cocoa seeds from different origin. for example about 3.5% in cocoa seeds from indonesia and up to 12% in samples from ivory coast. this should have impact on the flavor precursor quantity, which can be obtained during fermentation. indeed, rohsius (2007) finds free aa amounts of about 7 mg · g-1 ffdm in some indonesian samples, whilst those from ivory coast contain 12 to 18 mg · g-1 ffdm. however, the author also reports free aa amounts of up to 25 mg · g-1 ffdm for some of the samples from indonesia (rohsius, 2007). this underlines the importance of considering the multiple factors that influence the biochemical changes and thus the flavor development in cocoa along the complex processing chain. alkaloid bitter tastants theobromine is more or less process stable and still contributes to the bitter taste after fermentation, drying and roasting. a concentration corresponding to a dot factor of 19 in cocoa butter has been reported. in contrast, the caffeine concentration was below threshold (stark et al., 2006). however, as pointed out previously, depending on the genotype, the caffeine concentration can also be above threshold. in addition to this, zhang et al. (2014) report the formation of cate chin-c-spiro-glycosides during maillard reactions. one of these spirocyclic catechin maillard products significantly suppresses the bitterness of caffeine. the compound was identified in cocoa and increases 3-fold during roasting (zhang et al., 2014). thus, cocoa bitterness might be further modulated through partial suppression of the alkaloid bitter taste. bitter and astringent substances the epicatechin concentration further decreases during roasting, due to degradation and epimerization into catechin (urbańska et al., 2019). stark et al. (2006) find an epicatechin concentration of 2.5 g · kg-1 in roasted cocoa nibs. this corresponds to a dot factor of 1.2 for its bitter taste and 1.8 for astringency. with 0.69 g · kg-1 the catechin concentration is higher than in the fermented and dried bean samples analyzed by elwers et al. (2009) and tran et al. (2015). this might simply depend on a variation between samples. however, the catechin concentration might also increase during roasting, due to epimerization of epicatechin (urbańska et al., 2019). anyhow, the concentration is still below threshold of human perception for bitterness as well as astringency. in summary, the bitter taste changes completely during fermentation, drying and roasting, from a phenolic substances and alkaloid based one, to diketopiperazine and alkaloid dominated bitterness. thus, presumably diketopiperazines and alkaloids together are responsible for the typical bitter taste of cocoa and chocolate (stark et al., 2006). purely astringent substances caffeic acid aspartate also seems to decrease further during roasting. stark et al. (2006) report a concentration of 0.48 g · kg-1, corres ponding to dot factor of 8.5 for its puckering astringency. another n-phenylpropenoyl amino acid strongly contributing to puckering astringency is n-caffeoyl-l-dopa. a concentration of 0.31 mg · kg-1 was found in roasted seeds (stark et al., 2006). this corresponds to a dot factor of 15. another group of substances with astringent character is newly formed. during roasting, epicatechin and catechin react in nonenzymatic reactions with glucose, yielding phenol glycoconjugates, such as catechin-6-c,8-c-β-d-diglucopyranoside and epicatechin8-c-β-d-glucopyranoside. they are described as velvety, smoothly astringent (stark and hofmann, 2006) and may contribute to a pleasant mouth-filling sensation similar as in red wines. epicatechin8-c-β-d-glucopyranoside has a dot of 5.5, catechin-6-c,8-c-β-ddiglucopyranoside a dot of 9.5 in roasted cocoa (ep 1 856 988 a1). in addition, flavan-3-ol-c-glycosides seem to modulate bitterness. when added to cocoa beverages, the bitterness of the beverages is describe as milder, softer and more pleasant. when added to theobromine solutions, the bitterness of the solution strongly decreases (stark and hofmann, 2006), similar to the observations made by zhang et al. (2014) regarding catechin-c-spiro-glycosides. increased concentrations of flavan-3-ol-c-glycosides were found in alkalized samples, with the respective dots being 13.5 for catechin6-c,8-c-β-d-diglucopyranoside and 6.5 for epicatechin-8-c-β-dglucopyranoside (ep 1 856 988 a1). this might explain the taste attributes of alkalized cocoa, which are often described as milder and low in bitterness and astringency. acidic substances finally, the acidic taste strongly depends on the acetic acid formed during fermentation and on citric acid, with dot factors of 9 and 20, respectively. citric acid, already present in the fresh fruit pulp, might migrate into the seeds during early stages of fermentation. it could also be produced seed internally e.g. through the citric acid cycle (kumari, 2018). in addition, succinic acid and malic acid slightly contribute to the overall acidity. a dot of 1.9 is reported for succinic acid. the concentration of malic acid is at threshold and the dot is consequently as low as 1 (stark et al., 2006). both acids can be derived through the citric acid cycle as well (kumari, 2018). in the sample analyzed by stark et al. (2006), lactic acid did not contribute to the acidic taste. its concentration was below threshold. however, the lactic acid concentration in fermented dried seeds may strongly vary (rohsius, 2007; tran et al., 2015), presumably depending on the fermentation process. however, during the anaerobic phase of fermentation also seed internal lactic acid formation seems to occur (kadow et al., unpublished data). considering the maximum lactic acid amounts found by rohsius (2007) a dot of 10 could be reached for lactic acid. in total, 34 taste-active substances are needed to reproduce the typical taste-profile of roasted cocoa nibs. a list with all 34 substances can be found in the publication of stark et al. (2006). taste-saturation effects to fully understand the importance of each of the flavor-active substances, saturation effects in human perception need to be consi dered. saturation for n-caffeoyl-l-dopa is reached at a concentration of 0.6 g · kg-1, corresponding to a dot factor of 64 in aqueous solution. this means that even if the concentration further increases, the intensity of the astringency perceived does not. the reported ncaffeoyl-l-dopa amount of 0.3 g · kg-1 (stark et al., 2006) is below saturation and higher amounts would result in increased astringency. in contrast, for γ-aminobutyric acid, causing as well astringency, saturation is reached at a concentration of 8.2 mg · kg-1, corresponding to a dot factor of 4. the γ-aminobutyric acid amount detected in roasted cocoa was as high as 520 mg · kg-1. taking into account only the dot of 251 would result in an overestimation of the contribution of γ-aminobutyric acid to the astringency (stark et al., 2006). also variations in the γ-aminobutyric acid concentration, from 250 to 1500 mg · kg-1, as reported by stoll (2010), i.e. a dot factor range of 121 to 727, need to be considered under the aspect of saturation. the variations observed are not sensorial perceived. in addition, the intensity of the astringency perceived is 2 on scale with a maximum of 5 for γ-aminobutyric when saturation is reached. in contrast, the maximum intensity for n-caffeoyl-l-dopa is 5, pointing out again the importance of this substance for puckering astringency (stark et al., 2006). saturation effects do also occur regarding the bitter tastants. for cyclo(l-pro-l-val) saturation occurs at a dot of more than 64. at the biochemistry of cocoa flavor 309 such high concentrations, the bitter intensity of cyclo(l-pro-l-val) is 5 on a scale with a maximum of 5. this underlines the bitter potential and the importance of diketopiprazines as bitter tastants in cocoa (stark and hofmann, 2005). impact of the matrix on the perception of taste-active substances the impact of aqueous and fatty matrices when determining taste thresholds or training panelists, it is of vital importance to consider the impact of the matrix used to dissolve the taste-active substances. basic trainings on bitter taste for example are often carried out in aqueous solution (e.g. caobisco/eca/fcc cocoa beans manual, 2015). in water, caffeine for example has a bitter taste tc of 750 μmol· kg-1. in cocoa butter, the taste threshold increases nine-fold to a tc of 6.900 μmol· kg-1 (hofmann, 2015). the bitter taste tc of epicatechin as well increases nine-fold. similarly, the tc for its astringent character increases six-fold. amongst the astringent substances also quercetin-3-o-β-d-glucopyranoside has been detected in cocoa (stark et al., 2006). it has a very low tc in water, of only 0.65 μmol· kg-1. based on this tc, the quantities found in cocoa correspond to a dot of 156 (stark et al., 2006), suggesting a strong contribution of the substance to cocoa astringency. however, in cocoa butter the tc increases 369-fold (hofmann, 2015). consequently, the quantities found in cocoa only correspond to a dot 0.4. accordingly, quercetin-3-o-β-d-glucopyranoside most likely does not contribute to cocoa astringency. considering only the tc in water, the importance of quercetin-3-o-β-d-glucopyranoside for cocoa astringency would have been strongly overestimated. the example further underlines how essential the consideration of matrix effects is. the impact of sugar addition such above described matrix effects also occur in the presence of sugar. therefore, during chocolate manufacturing, the impact of the single substances on the flavor profile changes once again. the addition of sugar results in an increase of the tc for bitter taste of cate chin and epicatechin. consequently, the dot factors decrease from 0.7 to 0.3 for catechin and from 1.2 to 0.6 for epicatechin, based on the concentrations found by stark et al. (2006). thus, in chocolate both substances do not contribute to the bitter taste. in contrast, the tc for the bitter taste of cyclo(l-pro-l-val) decreases, resulting in an increment of the dot from 2 to 27. the concentration is still far below saturation of human perception. these findings further underline the importance of cyclo(l-pro-l-val) for the typical bitter taste of chocolates (stark et al., 2006). the tc of theobromine increases slightly. the dot factor for its bitter taste decreases from 19 to 17. nevertheless, also after sugar addition, theobromine remains an important contributor to the bitter taste. the tc of caffeine is as well affected. the dot factor range for caffeine after sugar addition is 0.3 to 2.5 based on the concentrations observed by rohsius (2010). astringency is as well affected by the addition of sugar. the tc for puckering astringency of catechin and epicatechin increases and the dots decrease from 0.5 to 0.3 for catechin and from 1.8 to 1.7 for epicatechin, according to the concentrations found by stark et al. (2006). thus, a marginal contribution of epicatechin to puckering astringency in the final chocolate product is possible. of greater importance remains n-caffeoyl-l-dopa. its tc increases as well, but the dot is still as high as 4.5 after sugar addition (stark et al., 2006). about the impact of sugar addition onto the tc for human perception of the flavan-3-ol-c-glycosides, no data have been published so far. changes of the aroma-active substance ratios during conching during the dry-phase of conching residual moisture and volatile acids, especially acetic acid, are effectively reduced in their concentration in the chocolate mass (e.g. beckett, 2008). beside acetic acid, other aroma-active substances are also affected and their concentration changes during conching. strecker aldehydes apparently show a general decline, with 3-methylbutanal decreasing by 28-36% (counet et al., 2002). in contrast to this, several pyrazines and furans apparently increase in their concentration. 2-ethyl-3,5-dimethylpyra zine only slightly, with a 1.4-fold increase. 4-hydroxy-2,5-dimethyl3(2h)-furanon more substantial. 1.7 to 3.4-fold higher concentrations were found after conching (counet et al., 2002). thus, not only the concentration of key aroma-active substances changes, but also the ratios between them. danzl and zardin (2014) describe a slight loss in chocolate aroma intensity during conching. however, the chocolate mass also becomes more harmonious in its flavor profile (danzl and zardin, 2014). quality analysis by means of aromaand taste-active substances the detailed knowledge about the substances responsible for a specific flavor attribute can be used in the training and formation of sensory panels. so far, in the first phase, trainings are often based on the recognition of the basic tastes, i.e. bitter, sweet, acidic, salty and umami, in aqueous solution. in a second phase, fine aroma attributes such as floral are trained, using for instance orange blossom water. in the third and final phase, panelists are exposed to a wide variety of origin chocolates to build a mental library of associations, linked to key chocolate flavor descriptors (caobisco/eca/fcc cocoa beans manual, 2015). making use of the reviewed knowledge, the cocoa specific tastants and aroma-active substances can be used to recognize in a first training phase single attributes of interest, for example puckering astringent, bitter or floral. the substances can be mixed into cocoa butter, instead of water, to best match the matrix of cocoa liquors and chocolates. in a second phase, different intensity levels can be trained, still on single attribute basis. this also allows the introduction and learning of optimum intensities for specific attributes. in a third phase, attributes can then be combined, until full taste or aroma profiles are used. in a fourth and final phase, the pane lists evaluate liquors and chocolates. these liquors and chocolates should be analyzed with regard to their flavor-active substance composition, to guarantee suitability as standardized training material. analysis of the tasteand aroma-active substances and their precursors can also be used for quality control purposes. it can be applied in the testing of the raw material, i.e. the fermented, dried seeds, and for the identification of new suitable origins, matching a required flavor profile. additional testing can be implemented along further critical control points of the processing chain, for example after roasting and grinding and after conching. in this way, a holistic monitoring of flavor quality can be established. however, such an approach requires high-throughput analytics. outlook on future research approaches most of the studies about flavor quality and its development have been carried out with a limited number of samples, given the complex analytical work behind them. often, only a small part of the processing chain is considered, e.g. fermentation or the roasting process. new studies should possibly be carried out with greater sample number and cover all influence factors i.e. critical control points (ccp) of the processing chain. key ccp are, based on the reviewed literature: · the genetic background of the seeds · fruit pulp aromaand taste-active substances as well as precursors · the microbiome during fermentation · fermentation parameters e.g. temperature, ph, duration and turning interval · seed aromaand taste-active substances as well as precursors during fermentation 310 d. kadow · the drying regime e.g. temperature, residual moisture and duration · seed aromaand taste-active substances as well as precursors during drying · aromaand taste-active substances after roasting · the impact of the other chocolate ingredients e.g. sugar in addition, factors such as the growing environment of the cacao e.g. climate and soil, fruit ripeness and characteristics of the processing location e.g. type of fermentation, box size and material should be monitored. high throughput analytics are needed to process the samples. for the genetic background of the cocoa beans and the taste-active substances, such tools are available (kauz et al., 2018; meinhardt, 2019). for aroma-active substances, hyper-fast-gc has the potential to become a key tool. a metagenomics-based approach to monitor cocoa fermentation microbial communities has recently been published (agyirifo et al., 2019). acknowledgements this review article is written in memory of and dedicated to prof. dr. reinhard lieberei. with him, research has lost a pioneer of interdisciplinary and applied work. there has been no topic, no question and no finding that he would not have had a valuable comment on. he never got tired of integrating new ideas and using new techniques to re-look at open research questions. this way of thinking is reflected in the article and shown here in detail for cocoa. due to his multi disciplinary thinking today physiology and biochemistry processes on development 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(eds.), modern methods of plants analysis, 321-390. springer-verlag, berlin, germany. ziegleder, g., 1990: linalool contents as characteristic of some flavor grade cocoas. z. lebensm. unters. forsch. 191, 306-309. ziegleder, g., 2014: flavour-release und aromaverteilung – kakao und schokolade. 50th ivlv symposium on chocolate. freising, germany. ziegleder, g., 2016: influence of cocoa drying on the flavour potential. 52. meeting ivlv chocolate technology. freising, germany. address of the corresponding author: daniel kadow, runder tisch kakao hamburg, chausseestraße 56, 10115 berlin, germany e-mail: daniel.kadow@rundertischkakao.de © the author(s) 2020. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1007/s00217-006-0551-2 http://dx.doi.org/10.1016/j.foodres.2015.09.019 http://dx.doi.org/10.1007/s00217-019-03333-w http://dx.doi.org/10.1080/09571260600633135 http://dx.doi.org/10.1111/j.1438-8677.1995.tb00496.x http://dx.doi.org/10.1016/j.foodchem.2015.07.068 http://dx.doi.org/10.1016/j.foodchem.2018.02.045 http://dx.doi.org/10.1038/s41559-018-0697-x http://dx.doi.org/10.1021/jf502040e journal of applied botany and food quality 92, 81 87 (2019), doi:10.5073/jabfq.2019.092.011 11szent istván university, department of mecicinal and aromatic plants, budapest, hungary 2institute of natural fibre and medicinal plants, poznań, poland effect of harvest date on yield and secondary compounds of lemon balm (melissa officinalis l.) éva németh-zámboriné1*, katarzyna seidler-łożykowska2, krisztina szabó1 (submitted: december 3, 2018; accepted: february 22, 2019) * corresponding author summary the quality of the drug of lemon balm (melissa officinalis l.) is influenced by several factors, among which the effect of ontogenesis has practically not been studied before. five varieties (‘lorelei’, ‘lemona’, ‘quedlinburger niederliegende,’, ‘gold leaf’, ‘soroksár’) were sampled at vegetative, budding, full flowering and after flowering phases at two locations (budapest and poznań) and their dried leaves analysed. the accumulation of volatile compounds showed maximum values (0.08-0.46 ml/100 g dry weight) in budding phase (budapest) or during flowering (poznań). the content of total phenolics was highest (226-431 mg gallic acid equivalent/g dry weight) in vegetative stage and in some cases similar values were measured until budding. after a sharp decrease at flowering time in several cases, a second peak was detected at the end of the vegetation period. similarly to the total phenolics, also the total flavonoid content reached the highest level (0.239-1.152% dry weight) at the first half of the vegetation period however, with characteristic differences between habitats. in cultivation, the highest essential oil content may be reached later than highest polyphenol content, however harvesting at budding time may assure a good quality from both aspects with advantageous fresh and drug yields. the described dynamics of the accumulation of the investigated secondary metabolites was depending more on the habitat and less from the cultivar. keywords: phenological phase, essential oil, flavonoids, polyphenols, antioxidant capacity, habitat introduction lemon balm (melissa officinalis l.) is a valuable perennial medicinal plant, which originates from the mediterranean area and western asia but it is already commonly cultivated under diverse ecological conditions in the temperate regions. the herb (melissae herba) and the leaves (melissae folium) are used as the components of digestive, antispasmodic and antiviral remedies (ema, 2014). based on up-to-date results, phenolics, flavonoids and hydroxy cinnamic acid derivatives are considered to be primarily responsible for most of the medicinal properties of the plant, most likely in connection with its considerable antioxidant activity (dastmalchi et al., 2008). the content of essential oil, which has a characteristic citrus like odor, varies in the herb of lemon balm from 0.01 to 0.3%. it consists mainly of monoterpene aldehydes (geranial, neral, citronellal) and sesquiterpenes (β-caryophyllene and β-caryophyllene oxide) (patora et al., 2003; seidler-lozykowska et al., 2017). it is already a well-known fact, that growth, development and biologically active material content of medicinal and aromatic plants may be significantly influenced by genetic (internal) and environmental (external) factors, the effects of which are however, in many cases characteristic for the target species. recent studies on the morphological and chemical intraspecific variability of lemon balm revealed considerable differences among accessions (talle et al., 2012; szabó et al., 2016). the environment may also influence the performance of the species, concerning both morphological traits and active compounds (sari and ceylan, 2002; manukyan, 2011; moqubeli et al., 2011; németh-zámbori et al., 2017). surprisingly, the least studied factors seems to be the ontogenesis. ayanoglu et al. (2005) provided data on the essential oil accumulation of lemon balm at two locations, depending on harvest time. however, a reproducible determination of sampling time and accurate information on the actual phenological phase of the plants is frequently missing in the references (patora et al., 2003; rusaczonek et al., 2007; dastmalchi et al., 2008; oniga et al., 2010; moqubeli et al. 2011; etc.). in other papers, a single sampling was carried out at diverse phases, e.g. before flowering (carnat et al., 1998), beginning of flowering (talle et al., 2012), when 50% of the plants had flowers (sari and ceylan, 2002), at full bloom stage (khalid et al., 2008) or at the end of flowering (farahani et al., 2009). reliable complex studies about the changes of biomass and active ingredients of lemon balm during the development are still missing. independent of the fact, that usually the leaves of lemon balm are used and processed, the optimal harvesting time may be of primarily importance concerning drug quality. the example of many species, − among others also numerous lamiaceae species − show that there might be significant changes during ontogenesis in the accumulation of secondary compounds (németh-zámbori, 2015). to clarify the above questions and optimize lemon balm production, we investigated the role of the developmental phase on the growth, development and drug quality. five cultivars were included into the experiment, which was conducted under two different environmental conditions. materials and methods plant material and experimental locations the experiments were conducted in open field plots of the szent istván university of budapest, hungary (47o54’n 19o14’e) and of the institute of natural fibers and medicinal plants of poznań, poland (52o25’n 16o58’e). the main soil parameters of the experimental plots are presented in tab. 1. the mean temperatures, marginal values of the temperatures and sum of precipitation before each harvest in both locations are summarized in tab. 2. irrigation was applied only in the very dry periods to avoid dying of the plants. five lemon balm (melissa officinalis l.) varieties were included into the trial: ‘lorelei’ (mediseeds, switzerland); ‘lemona’, ‘quedlinburger niederliegende’ (in the followings ‘quedlinburger’), ’gold leaf’’ (each from jelitto, germany) and ‘soroksár’ (enkraft bt., hungary). seed sowing and seedling raising was made in greenhouse. planting of 50 seedlings/genotype into open field plots was carried out at the beginning of june 2014, to a spacing of 45 × 45 cm. the harvesting treatments were carried out in the second vegetation year, in 2015. five healthy representative individuals from each plot were cut in three replicates in the following phenological phases: 1. vegetative 82 é. németh-zámboriné, k. seidler-łożykowska, k. szabó stage (no flowering stems appear still), 2. plant budding (flowering shoots with buds without white petals), 3. full flowering (50% of flowers are open); 4. after flowering (no flowers are visible and green seeds are developing). sample preparation the fresh mass of the plant samples was weighed individually, and then the shoots were dried in a shaded, well-ventilated place. the samples were separated into leaf and stem parts and only the leaves were used for chemical analysis. after drying, the individual leaf samples were mixed creating a representative bulk sample for each treatment and replicates. the phytochemical analyses were carried out on these homogeneous bulk samples in three further, technical replicates at the department of medicinal and aromatic plants, budapest. phytochemical analyses essential oil content (eo): 50 g of each sample was hydrodistilled for three hours in a clevenger-type apparatus recommended by the pharmacopoeia hungarica vii. the eo content was calculated as volume (ml) of oil per 100 g of dried weight (d.w.) determined in three hours, at 105 °c. total phenolic content (tpc): for the determination of the tpc 1 g powdered dried plant material was extracted by boiling in 100 ml distilled water and then was allowed to stand for 24 h. then the extracts were filtered and stored frozen until the measure ments took place. the total phenolic content was determined by the modified method of singleton and rossi (1965) and was expressed as mg of gallic acid equivalents (gae) per g of dry weight of extract. total flavonoid content (tfc): the flavonoid content was determined according to the method given in the pharmacopoeia hungarica viii for equiseti herba using half of the amounts of materials described there. shortly, 0.4 g dried and powdered plant material was extracted by 1 ml hexametilentetramine, 20 ml acetone and 2 ml hcl for 30 minutes, and then it was filtered. afterwards the extraction was repeated by 20 ml acetone twice and diluted by water and ethyl-acetate. the absorbance was measured at 760 nm after incubation for 30 min and expressed in mg isoquercitroside (qe) per g of dry weight of plant material. antioxidant capacity (ac): the frap assay was performed accor ding to the method of benzie and strain (1996), and the frap values of samples were calculated from a standard curve equation and expressed as mg ascorbic acid equivalent (aae) per g of dry weight of extract. statistical analysis the results were analysed with an ibm spss 22.0 statistical program. manova was used for evaluating the effects of variety and phenological phase for data obtained in each location. homogeneity of variances was tested by levene’s method. depending the homo geneity of variances measurement, a tukey hsd or games-howel test was used for the pairwise comparisons of the variances. confidence level was 5%. results and discussion yields highest fresh mass of the plants was produced in the flowering period (tab. 3). in budapest, highest yields were achieved in full flowering (in case of ‘lorelei’ and ‘soroksár’ not significantly different from the previous phase). unfortunately, in this growth area, the development of ‘gold leaf’ was severely disturbed, several individuals exhibited a decay and after budding phase the measurements could not be continued. in poznań, all of the five cultivars gave the highest yields at the beginning of flowering (budding phase), although in case of two cultivars the values did not differ significantly from the previous (‘gold leaf’) and the following (‘quedlinburger’) phases. in case of the fresh shoot mass the interaction between cultivar and phenophase was not significant in either location. the dry leaf mass – drug yield of melissae folium – followed similar tendencies (tab. 3). in budapest, highest drug yield were achieved at full flowering stage. in this aspect, the cultivar × phenophase interaction was significant only in poznań. in poland, the full flo wering stage proved to be optimal only for cultivar ‘quedlinburger’, while in case of the other varieties budding (‘lorelei’ and ‘soroksár’) or vegetative phase (‘gold leaf’ and ‘lemona’) assured the largest drug mass. the latter results might have been influenced also by the decreasing leaf proportion in the shoot mass, which was detected already from budding stage. leaf ratio in the total shoot mass was 66.6% in vegetative phase, 41.0% in budding, 38.4% in full flowering and 40.0% after flowering as a mean of the data of the examined cultivars. tab. 1: soil characteristics of the experimental locations ph humus % no3-n mg/kg p2o5 mg/kg k2o mg/kg ca % mg mg/kg budapest 6.91 0.87 7.73 583 189 0.51 32.1 poznań 5.18 2.58 8.46 395 131 1.36 27.4 vegetative buds flowering after flowering tab. 2: weather conditions during 30 days before lemon balm harvests in poznań and budapest phenological phase location harvest date average temperature range sum of precipitation day temperature (oc) (oc) (mm) budapest 25 may 15.6 10.7 – 20.4 82.8 poznań 08 june 13.4 1.6 – 24.9 39.6 budapest 17 june 20.0 11.8 – 24.1 59.8 poznań 28 june 15.3 4.6 – 32.5 77.8 budapest 25 july 21.5 15.0 – 21.8 32.8 poznań 03 august 17.0 6.3 – 34.1 82.2 budapest 31 august 22.3 7.6 – 39.1 84,2 poznań 06 september 20.7 7.2 – 37.1 68.8 effect of harvest date on lemon balm 83 in the practice, this crop is harvested before flower development and repeated cuttings can be carried out in the same year depending on the region and climate (hoppe, 2013). seidler-lozykowska et al. (2013) and szabó et al. (2016) described significant variation in yields of different lemon balm accessions. nevertheless, no detailed former study is known about the dynamics of biomass production of the species during the vegetation cycle, comparable with the recent findings. essential oil content the eo content of the harvested drug showed significant differences thorough the phenophases (tab. 4). budding phase was detected as the optimal time for all cultivars in budapest. in the following period, the essential oil content decreased sharply again except ‘lemona’. in poland, the maximum values of the volatile accumulation were reached at full flowering except ‘gold leaf’. this phenomenon and the later maximum accumulation compared to budapest may stay in connection with the very cold weather during spring and the fact that the plants could grow slowly. the cooler environment (tab. 1) might be an explanation also for the generally much lower essential oil values in poznań, where the highest mean value is only 42% of the maximum mean value in budapest. no variety × phenophase interaction could be determined in budapest, while it was a significant one (p<10%) in poznań. the range of the eo content is fully comparable with other references (bomme et al., 2002; patora et al., 2008; seidler-lozykowska et al., 2017). in own earlier investigations in a pot experiment on the same cultivars 0.07-0.29 ml/100 g eo content was determined in vegetative phase (szabó et al., 2016). based on investigation of 15 accessions, chizzola et al., (2018) mentioned that first cut leaves (in june) gave more oil, than those of the second cut (in august). ayanoglu et al. (2005) declared the before flowering phase as assuring highest oil contents but differences were significant only in one of the experimental locations. in related species frequently the early flowering or full flowering period has been detected as the optimal phase for volatile accumulation, both in our own works and in those of other authors: in hyssopus officinalis l. (németh et al., 2001), in mentha piperita l. (zámboriné and tétényi, 1988), in majorana hortensis mönch. (sellami et al., 2008), salvia officinalis l. (mirjalili et al., 2006). latter authors suggest that the ecological role of the higher volatile accumulation during flowering might be attracting of pollinators and defeating some diseases. total phenolic content in general, the tpc was higher at the beginning of plant development (tab. 5). in budapest, it showed maximum values already during the first sampling time at least for ‘lorelei’ (431.9 mg gae/g d.w.) and ‘soroksár’ (340.2 mg gae/g d.w.) cultivars. at flowering time phenolic content decreased in all of the samples while interestingly, at the end of the vegetation time it increased again to variable extent (except ‘lemona’). in poznań, all of the cultivars provided the highest phenolic content at the vegetative phase. compared to the later phases the advantage was significant in each case. similarly to the findings in budapest, in three cultivars also a second peak could be observed. however, this second peak was detected during flowering, not in the last phase, as in budapest. no variety × phenophase interaction has been established at either of the locations. rusaczonek et al. (2007) and chizzola et al. (2018) stated that tpc was higher in leaves compared to stem parts. considering the larger leaf proportion in the earlier phases, it could contribute to the peak values of phenolics in the beginning of vegetation. however, it cannot be an explanation for the second increase because leaf ratio has already dropped at this time (see above). it lets to assume, that changing metabolomic processes during ontogenesis could be responsible for changing values. in case of two cuts carried out at different times (in june and august) but in the same, vegetative phase after regrowth, as in the trial of chizzola et al. (2018), no significant change in the tpc was measured and this ascertains the role of the phenological phase in phenolic accumulation. at the same time, the effect of weather conditions cannot be excluded either (némethzámbori et al., 2017). tab. 3: fresh mass and drug yield of the tested cultivars in four phenological phases in two location (between subject effect variety × phenophase according to manova: fresh mass: pbudapest=0.167; ppoznan=0.455; dry mass: pbudapest=0.213; ppoznan=0.011) cultivar fresh mass of shoots (g/plant) dry mass of leaves (g/plant) phenological phase phenological phase vegetative budding flowering after flowering vegetative budding flowering after flowering budapest ‘gold leaf’ 36 bc 55 ad 6 bb 10 ac ‘lemona’ 140 aab 122 abc 150 ab 106 bc 24 ba 22 bcb 29 ab 20 cb ‘lorelei’ 158 ba 193 aa 197 aa 161 ba 21 ca 32 ba 44 aa 41 aa ‘quedlinburger’ 116 bb 107 bc 198 aa 105 bc 20 ca 24 bb 45 aa 27 bb ‘soroksár’ 143 ba 166 ab 153 ab 124 cb 26 aba 31 aa 30 ab 22 bb mean 119 129 175 124 19 24 37 28 poznań ‘gold leaf’ 78 ab 72 ac 42 bc 34 bc 50 ab 33 bc 12 cc 8 cd ‘lemona’ 94 ca 152 ab 124 bb 70 cb 63 aa 61 ab 47 bb 29 cc ‘lorelei’ 56 cc 180 aa 124 bb 110 ba 38 cc 70 aa 51 bb 51 ba ‘quedlinburger’ 86 bab 152 ab 154 aa 96 bab 57 aba 61 ab 66 aa 42 bb ‘soroksár’ 50 dc 172 aa 146 ba 106 ca 35 cc 69 aa 61 aa 46 bab mean 73 146 118 83 49 59 47 35 capital letters indicate sign. diff. (p<0.05) among phenophases in the same variety each location separately; lower case letters indicate sign. diff. (p<0.05) among varieties at the same phenophase in each location separately 84 é. németh-zámboriné, k. seidler-łożykowska, k. szabó two peaks for the accumulation of tpc were published also by kindlovits et al. (2016) in yarrow (achillea collina), the first in green bud early flowering phase (178.0-233.4 mg gae/g) and the next one in overblown phenological phase (170.9-258.5 mg gae/g). in marjoram, sellami et al. (2009) established that tpc of the marjoram herb was highest in vegetative phase before flowering. in parallel, they found that eo content showed a maximum at full flowering. although they did not investigate the after flowering phenological phase, the results show large similarity with our ones. the authors suggest, that phenolic accumulation in this early phenological phase might have a role in plant defence mechanism, which is taken during flowering by volatile secondary compounds. total flavonoid content the tfc of the tested cultivars exhibited a decreasing tendency during the phenological phases (tab. 6). in budapest, highest flavo noid contents were measured in the samples collected during vege tab. 4: essential oil content (ml/100g d.w.) in herb of lemon balm cultivars in four phenological phases in two location (between subject effect variety × phenophase according to manova: pbudapest=0.333; ppoznan=0.068) cultivar phenological phase vegetative budding flowering after flowering mean s.d. mean s.d. mean s.d. mean s.d. budapest ‘gold leaf’ 0.30 ba 0.001 0.46 aa 0.037 ‘lemona’ 0.13 cb 0.021 0.41 aa 0.042 0.28 ba 0.005 0.37 aba 0.021 ‘lorelei’ 0.10 bc 0.020 0.13 ac 0.020 0.05 cd 0.020 0.08 bb 0.001 ‘quedlinburger’ 0.06 cd 0.021 0.32 ab 0.035 0.10 bc 0.020 0.07 bb 0.001 ‘soroksár’ 0.07 bc 0.003 0.12 ac 0.021 0.12 ab 0.021 0.05 bc 0.021 mean 0.13 0.29 0.11 0.12 poznań ‘gold leaf’ 0.09 c 0.004 0.11 c 0.001 0.17 b 0.001 0.38 a 0.039 ‘lemona’ 0.09 b 0.004 0.10 b 0.015 0.14 a 0.001 0.13 a 0.021 ‘lorelei’ 0.01 c 0.001 0.06 b 0.015 0.09 a 0.001 0.07 a 0.001 ‘quedlinburger’ 0.02 c 0.004 0.05 b 0.001 0.08 a 0.004 0.02 c 0.016 ‘soroksár’ 0.05 b 0.004 0.07 a 0.016 0.00 c 0.00 c mean 0.05 0.08 0.11 0.12 capital letters indicate sign. diff. (p<0.05) among phenophases in the same variety each location separately; lower case letters indicate sign. diff. (p<0.05) among varieties at the same phenophase in each location separately tab. 5: total phenolic content (mg gae/g d.w.) of herb of lemon balm cultivars in four phenological phases in two location (between subject effect variety × phenophase according to manova: pbudapest=0.123; ppoznan=0.566) cultivar phenological phase vegetative budding flowering after flowering mean s.d. mean s.d. mean s.d. mean s.d. budapest ‘gold leaf’ 226.0 bd 30.5 249.1 ad 15.0 ‘lemona’ 303.3 bc 29.8 394.8 aa 33.9 222.1 ca 11.5 160.0 dd 12.8 ‘lorelei’ 431.9 aa 35.8 352.6 bb 18.5 163.7 cb 9.4 336.2 ba 17.0 ‘quedlinburger’ 334.0 ab 31.3 360.8 ab 38.2 165.4 bb 14.4 193.6 bc 19.7 ‘soroksár’ 340.2 ab 27.5 308.1a bc 27.3 167.9 cb 26.5 294.4 bb 40.6 mean 327.1 333.1 179.8 246.1 poznań ‘gold leaf’ 354.7 ab 21.2 244.1 cc 18.9 270.4 bc 44.4 222.4 ca 29.4 ‘lemona’ 410.8 aa 56.6 289.6 cb 36.4 331.9 bb 22.3 205.5 da 21.4 ‘lorelei’ 361.6 ab 22.4 316.3 ba 38.12 221.3 cd 23.7 163.5 db 19.9 ‘quedlinburger’ 369.0a bb 32.5 321.5 ba 40.14 410.2 aa 27.1 220.2 ca 12.7 ‘soroksár’ 364.4 ab 27.1 300.8 bab 37.91 249.5 ccd 51.2 146.9 db 19.2 mean 372.1 294.5 296.7 151.7 capital letters indicate sign. diff. (p<0.05) among phenophases in the same variety each location separatel; lower case letters indicate sign. diff. (p<0.05) among varieties at the same phenophase in each location separately effect of harvest date on lemon balm 85 tative phase in case of all cultivars. the values did not show large deviations among varieties (0.624-0.693 mg qe/g d.w.) except ‘gold leaf’ (0.239% d.w.). after the first sampling, a continuous decrease of the tfc could be measured in all cultivars. in ‘lemona’ however, the change was only a moderate one and this variety showed also a smart second peak at the end of the vegetation cycle. the mean values of the flavonoid content were by 18-44% higher in poznań, than in budapest. the maximum contents could be detected at budding phase and the decrease started after that. the only exception is ‘gold leaf’, which showed the highest contents in the earliest phase, similarly to the findings in budapest. after the first peak, a continuous decreasing tendency and significantly lower values of tfc were measured in each of the cultivars. the interaction between variety and phenophase was a significant one only in poznań (p<10%). there are practically no former data on the role of plant development in this aspect. the tfc detected by carnat et al. (1998) in lemon balm leaves is comparable with our data. however, only leaves har vested just before flowering of a single accession were investigated by those authors. the values of the tfc were characteristically different between the two growing areas, therefore investigating the spectrum of components depending on location might be of interest in the future. antioxidant capacity in budapest, the frap ac of the samples showed peak values in budding stage (tab. 7). after budding, a sharp decrease was measured in all cultivars and only in ‘lorelei’ a smart second peak detected at the end of the vegetation. similarly to this, in poznań maximum values for ac were measured in budding phase (with the exception of ‘gold leaf’). after that, a continuous decrease could be observed, however, the differences among phenological phases were less characteristic, than in budapest. ‘gold leaf’ exhibited a very different behavior than the other varieties, as it showed increasing ac during the developmental pha ses. in the first two phases, the ac data are comparable with the values in budapest, but in the last two phases the values in poznań are more than twofold compared with the hungarian data. the variety × phenophase interaction is not significant at either habitat. a lower variability of frap ac of lemon balm compared to the active ingredients (volatile, polyphenols) was described also in other studies (szabó et al., 2016; chizzola et al., 2018). drought stress may elevate it to some extent (németh-zámbori et al., 2017) however, no data were found on the changes of ac during ontogenesis of this species. in summary, we established that based on the investigation of five lemon balm cultivars at two different locations, the accumulation of volatile compounds showed a single peak with maximum values after the appearance of the generative organs. phenoloid type compounds showed highest concentrations in earlier phases, and in several samples, another peak appeared towards the end of the vegetation period. in practice, determination of optimal harvesting time may be oriented according to the goal of the production. highest essential oil yield may be reached later than highest polyphenol content. however, harvesting at budding time seems to assure the best quality from both aspects. this timing may also assure an advantageous fresh and drug yield. the study revealed, that the described dynamics of accumulation of the investigated secondary metabolites is depending more on the habitat and much less on the intraspecific cultivar. acknowledgements this research was supported by the higher education institutional excellence program (20430-3/2018/fekutstrat) awarded by the ministry of human capacities within the framework of plant breeding and plant protection researches of szent istván university and by the national research, development and innovation fund (otka -nn108633). references ayanoglu, f., arsan, m., hatay, a., 2005: effects of harvesting stages, harvesting hours and drying methods on essential oil content of lemon tab. 6: total flavonoid content (mg qe/g d.w.) in herb of lemon balm cultivars in four phenological phases in two location (between subject effect variety × phenophase according to manova: pbudapest=0.865; ppoznan=0.0622) cultivar phenological phase vegetative budding flowering after flowering mean s.d. mean s.d. mean s.d. mean s.d. budapest ‘gold leaf’ 0.239 aa 0.011 0.222 ab 0.027 ‘lemona’ 0.648 aa 0.002 0.547 ba 0.007 0.463 ca 0.010 0.523 ba 0.002 ‘lorelei’ 0.693 aa 0.087 0.560 ba 0.007 0.414 cb 0.016 0.299 db 0.014 ‘quedlinburger’ 0.624 aa 0.004 0.498 bb 0.010 0.389 cb 0.038 0.223 dc 0.022 ‘soroksár’ 0.641 aa 0.029 0.482 bb 0.003 0.407 cb 0.020 0.303 db 0.009 mean 0.569 0.462 0.418 0.337 poznań ‘gold leaf’ 0.520 ac 0.015 0.393 bc 0.022 0.354 cd 0.025 0.409 bc 0.026 ‘lemona’ 0.736 ba 0.009 0.852 ab 0.036 0.617 cc 0.033 0.432 dbc 0.003 ‘lorelei’ 0.642 cb 0.039 0.871 ab 0.031 0.723 ba 0.004 0.543 da 0.013 ‘quedlinburger’ 0.752 ba 0.001 0.854 ab 0.043 0.663 cb 0.011 0.573 da 0.002 ‘soroksár’ 0.667 bb 0.049 1.152 aa 0.026 0.644 bb 0.026 0.473 cb 0.015 mean 0.663 0.824 0.600 0.486 capital letters indicate sign. diff. (p<0.05) among phenophases in the same variety each location separately; lower case letters indicate sign. diff. (p<0.05) among varieties at the same phenophase in each location separately 86 é. németh-zámboriné, k. seidler-łożykowska, k. szabó balm grown in eastern mediterranean. intl. j. bot. 1, 138-142. doi: 10.3923/ijb.2005.138.142 benzie, i.f., strain, j.j., 1996: the ferric reducing ability of plasma (frap) as a measure of „antioxidant power”: the frap assay. anal. biochem. 239, 70-76. doi: 10.1006/abio.1996.0292 bomme, u., feicht, e., rinder, r., 2002: ergebnisse aus mehrjährigen leistungsprüfungen mit ausgewählten herkünften von zitronenmelisse (melissa officinalis l.). z. arznei-gewürzpfl. 7, 268-376. carnat, a.p., carnat, a., fraisse, d., lamaison, j.l., 1998: the aromatic and polyphenolic composition of lemon balm (melissa officinalis l. subsp. officinalis) tea. pharm. acta helvetiae 72, 301-305. doi: 10.1016/s0031-6865(97)00026-5 chizzola, r., lohwasser, u., franz, ch., 2018: biodiversity within melissa officinalis: variability of bioactive compounds in a cultivated collection. molecules, 23, 294: doi:10.3390/molecules23020294 dastmalchi, k., dorman, h.j.d., oinonen, p.p., darwis, y., laakso, i., hiltunen, r., 2008: chemical composition and in vitro antioxidative activity of lemon balm (melissa officinalis l.) extract. lwt food sci. technol. 41, 391-400. doi: 10.1016/j.lwt.2007.03.007 ema, 2014: community herbal monograph on melissae folium (2013). european medicines agency, committee on herbal medicinal products, no. ema/hmpc/196745/2012 retrieved 3rd december 2018 from http:// https://www.ema.europa.eu/medicines/herbal/melissae-folium farahany, h.a., valadabadi, s.a., daneshian, j., khalvati, m.a., 2009: evaluation changing of essential oil of balm (melissa officinalis l.) under water deficit stress conditions. j. med. plants res. 3, 329-333. hoppe, b. 2013: handbuch des arzneiund gewürzpflanzenbaus. vol. 5. bernburg, germany: saluplanta e.v. khalid, k.a., cai, w., 2011: the effects of mannitol and salinity stresses on growth and biochemical accumulations in lemon balm. acta ecol. sinica 31, 112-120. doi: 10.1016/j.chnaes.2011.01.001 kindlovits, s., cserháti, b., inotai., k, zámboriné-németh, é., 2016: ontogenetic variation of active agent content of yarrow (achillea collina becker). j. appl. res. med. aromat. plants. doi: 10.1016/j.jarmap.2016.01.003 manukyan, a., 2011: effect of growing factors on productivity and quality of lemon catmint, lemon balm and sage under soilless greenhouse production: i. drought stress. med. aromat plant sci. biotechnol. 5, 119125. mirjalili, h.m., salehi, p., sonboli, a., vala, m.m., 2006: essential oil variation of salvia officinalis aerial parts during its phenological cycle. chem. nat. compd. 42, 19-23. doi: 10.1007/s10600-006-0027-4 moqubeli, e., fathollahi, s., olfati, j.a., peyvast, g.a., hamidoqli, y., bakhshi, d., 2011: investigation of soil condition on yield and essential oil in lemon balm. south-west j. hortic. biol. environ. 2, 87-93. németh, é., varga, e., franke, r., 2001: variabilität des ätherischen ölgehaltes und deren ursachen in hyssopus officinalis l. z. arznei gewurzpflanzen 6, 29-34. németh-zámbori, é., 2015: natural variability of essential oil components. in: baser, k.h.c., buchbauer, g. (ed.), handbook of essential oils, science, technology, and applications, 87-126. 2nd ed. boca-raton, us, crc press taylor and francis group. németh-zámbori, é., szabó, k., rajhárt, p., inotai, k., seidlerlozykowska, k., radácsi, p., 2017: variability of phenolic compounds of four lamiaceae species in consequence of different water supply. acta sci. pol-hortoru. 16, 13-24. doi: 10.24326/asphc.2017.4.2 oniga, i., vlase, l., toiu, a., benedec, d., duda, m., 2010: evaluation of phenolic acid derivatives and essential oil content in some melissa officinalis l. varieties. farmacia 58, 764-769. patora, j., majda, t., gora, j., klimek, b., 2003: variability in the content and composition of essential oil from lemon balm (melissa officinalis l.) cultivated in poland. j. endocrinol. invest. 26, 950-955. pharmacopoea hungarica, 1986: vii. ed. tomus i. budapest, hungary: medicina könyvkiadó, 395-398. pharmacopoea hungarica, 2004: viii. ed. tomus ii. budapest, hungary: medicina könyvkiadó, 2208-2209. rusaczonek, a., zebrowska, m., waszkiewicz-robak, b., slusarczyk, e., 2007: evaluation of phenolic compounds content and antioxidant capacity of herbs. pol. j. food nutr. sci. 57, 483-488. sari, a.o., ceylan, a., 2002: yield characteristics and essential oil composition of lemon balm (melissa officinalis l.) grown in the aegean region of turkey. turk. j. agric. for. 26, 217-224. seidler-lozykowska, k., bocianowski, j., krol, d., 2013: the evaluation of the variability of morphological and chemical traits of the selected tab. 7: frap antioxidant activity (mg aae/g d.w.) of herb of lemon balm cultivars in four phenological phases in two location (between subject effect variety × phenophase according to manova: pbudapest=0.556; ppoznan=0.848) cultivar phenological phase vegetative budding flowering after flowering mean s.d. mean s.d. mean s.d. mean s.d. budapest ‘gold leaf’ 351.1 ab 29.72 357.4 ac 20.35 ‘lemona’ 431.6 ba 47.10 476.8 aa 69.16 186.8 ca 9.81 119.7 dc 3.52 ‘lorelei’ 432.0 aa 35.82 425.3 ab 29.88 131.5 cb 18.72 184.1 ba 8.29 ‘quedlinburger’ 430.0 aa 30.73 445.9 aab 53.51 126.0 bb 14.53 98.2 cc 14.13 ‘soroksár’ 378.9 bb 33.41 420.9 ab 29.55 142.5 cb 13.63 146.3 cb 15.82 mean 404.7 425.3 146.7 137.1 poznań ‘gold leaf’ 349.3a bb 28.00 301.3 bb 16.53 373.1 ad 10.52 399.5 ab 18.53 ‘lemona’ 404.2 ba 20.50 462.7 aa 57.82 450.1 aa 24.53 396.6 bb 19.48 ‘lorelei’ 361.7 cb 22.41 450.0 aa 29.57 401.2 bc 15.55 429.0a ba 26.83 ‘quedlinburger’ 346.4 db 16.22 460.4 aa 34.32 424.8 bb 26.56 383.7 cc 73.13 ‘soroksár’ 368.4 cb 41.26 447.4 aa 33.75 423.6 ab 17.18 410.5 bab 8.99 mean 366.0 424.4 329.8 321.8 capital letters indicate sign. diff. (p<0.05) among phenophases in the same variety each location separately; lower case letters indicate sign. diff. (p<0.05) among varieties at the same phenophase in each location separately https://www.sciencedirect.com/science/article/pii/s1872203211000059 https://www.sciencedirect.com/science/article/pii/s1872203211000059 f:\cikk\poznan_budapest_katarzyna fele\acta ecol sinica http://dx.doi.org/10.1016/j.jarmap.2016.01.003 effect of harvest date on lemon balm 87 lemon balm (melissa officinalis l.) genotypes. ind. crops prod. 49, 515520. sellami, i.h., maamouri, e., chahed, t., wannes, w.a., kchouk, m.e., marzouk, b., 2009: effect of growth stage on the content and composition of the essential oil and phenolic fraction of sweet marjoram (origanum majorana l.). ind. crops prod. 33, 395-402. doi: 10.1016/j.indcrop.2013.05.027 singleton, v.l., rossi, j.a., 1965: colometric of total phenolics with phosphomolybdic-phosphotungstic acid reagents. am. j. enol. vitic. 16, 144-158. szabó, k., malekzadeh, m., radácsi, p., ladányi, m., rajhárt, p., inotai, k., tavaszi-sárosi, sz., németh, é., 2016: could the variety influence the quantitative and qualitative outcome of lemon balm production? ind. crops prod. 83, 710-716. doi: 10.1016/j.indcrop.2015.12.027 talle, b., darvish, f., mohammadi, a., abbaszadeh, b., rohami, m., 2012: assessment of relationship between effective traits on yield and compounds of essential oil and morphological traits of lemon balm (melissa officinalis l.) accessions using path analysis and canonical correlation. j. basic appl. sci. res. 2, 3719-3723. zámboriné, n.é., tétényi, p., 1988: studies on the stimulation of the stolon development of peppermint. herba pol. 34, 129-135. address of the orresponding author: prof. dr. éva németh-zámbori, department of medicinal and aromatic plants, szent istván university, 1518 budapest, p.o. box 53, hungary e-mail: zamborine.nemeth.eva@kertk.szie.hu © the author(s) 2019. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). art18 journal of applied botany and food quality 82, 108 113 (2009) 1section urban horticulture, institute for horticultural sciences, humboldt university berlin, berlin, germany 2department of horticulture, virginia tech, blacksburg, va, usa impact of glucosinolate structure on the performance of the crucifer pest phaedon cochleariae (f.) m.m. uddin1, ch. ulrichs1, j.g.tokuhisa2, i. mewis1 (received august 28, 2008) summary glucosinolates (gs) are sulfur-rich secondary metabolites found in the brassicaceae and other related families of the order brassicales. gs consist of structurally-related compounds with different side chains. to explore the possibility that various side chain confer divergent biological activities to individual gs, we have investigated the performance of the specialist pest beetle, phaedon cochleariae (f.) on arabidopsis thaliana l. mutants and columbia wild-type (wt) which differ in the main group of gs. plant lines of a. thaliana altered for the expression of mam3, because of the introduction of an overexpression construct of mam3 (mam3+) or containing double knockouts of cyp79b2 and cyp79b3 (cyp79b2-/cyp79b3-) were used for the study in comparison to the wt. a. thaliana genotypes differed in their gs profiles. the highest gs content was present in the wt followed by mam3+ and cyp79b2-/ cyp79b3-. a modified aliphatic gs content was detected for the mam3+ as compared to the wt lines. furthermore, indolyl gs were completely absent in cyp79b2-/cyp79b3-. the percentage weight increase of larvae raised on each of the three plant genotypes was significant different. larval performance was poorest on plants of cyp79b2-/cyp79b3and best on wt, but there was no significant difference found in percentage weight increase on mam3+ and wt. there was no correlation between the weight increase of the larvae on genotypes and induced levels of aliphatic, indolyl, and total gs. however, the poor performance of beetle larvae on cyp79b2-/ cyp79b3-compared to wt and mam3+ might be explained by comparable high aliphatic gs levels of this mutant, a different induction of secondary metabolites, and the absence of indolyl gs. basic knowledge about the relationship of gs structures and their insect pests may help in further resistance breeding of crucifer crops. introduction glucosinolates (gs) are sulphur-rich β-thioglucosides present within the brassicaceae and other related families of the order brassicales (rodman et al., 1996). all gs contain a common core structure, but have variable side chains including aliphatic, aromatic, or indolic structures (wittstock and halkier, 2002; kliebenstein et al., 2005). to date, more than 120 gs have been identified in plants (fahey et al., 2001), including 34 compounds present in the model plant arabidopsis thaliana l. (kliebenstein et al., 2001). in plants gs and their hydrolyzing enzymes, β-thioglucosidases, which are also called myrosinases, are stored in different cell compartments or specialized cells (lüthy and matile, 1984; koroleva et al., 2000). but when the plant is damaged, hydrolysis occurs and biological active products such as isothiocyanates, thiocyanates, and nitriles are released. gs have dual roles in plant-insect interactions. although they function as defense compounds, several insects that are specialists have life cycles that are closely linked to the presence of such secondary compounds in their brassicaceous host plants (renwick, 2002). they are known to stimulate feeding in a number of insects that feed exclusively on crucifers (david and gardiner, 1966; nault and styer, 1972; tanton, 1977; larsen et al., 1985). brassicaceae species and individual plants vary within concentration and composition of gs (fahey et al., 2001; kliebenstein et al., 2001). additionally, many brassicaceous plants produce higher levels of indolyl gs after herbivory (kortisas et al., 1991; bodnaryk, 1992; griffiths et al., 1994; bauer et al., 1998; agrawal et al., 1999a). gs and/or their breakdown products have a variety of effects upon organisms that come in contact with them, but do not always control the interaction between plants and their potential herbivore or invading microorganisms (chew, 1988b). distinct gs can have different effect on the same insect herbivore. for example, p-hydroxybenzyl gs is involved in plant resistance (antixenosis) against feeding by the flea beetle, phyllotreta cruciferae (goeze), in seedlings of yellow mustard, sinapis alba l. (bodnaryk, 1991).whereas, allyl gs and indol-3ylmethyl, which are present in seedlings of indian mustard, brassica juncea (l.) and oilseed rape, brassica napus l., respectively, are not determinants of feeding behavior by p. cruciferae (bodnaryk and palaniswamy, 1990). the mustard leaf beetle, phaedon cochleariae (f.) (coleopterans: chrysomelidae), is an insect pest of crucifer crops that is common to europe (finch and jones, 1986). when this insect emerges in large numbers, it can quickly devastate crucifer fields with especially severe effects on young seedlings. hence, a study of the insect/hostplant interaction including the feeding behavior can lead to agriculturally relevant management strategies. tanton (1977) and nielsen (1978) found that p. cochleariae responded to a variety of gs in feeding bioassays, with distinct preferences for the aliphatic allyl gs (sinigrin) compared to the aromatic compounds: p-hydroxybenzyl gs (sinalbin) and benzyl gs (glucotropaeolin). however, these studies have limited insights into the impact of gs structure on the larvae and beetle performance of p. cochleariae. we have taken this opportunity to investigate the effect of different gs classes on the feeding and growth of the p. cochleariae larvae by using a. thaliana genotypes with desired gs profiles. materials and methods plant cultivation and experimental conditions seeds from single seed propagation were sown on petri plates containing solidified murashige-skoog media. after stratification for three days at 4°c, germinated seedlings were transferred to 7 x 6 cm pots filled with sterile potting mix (gramoflor). plants were kept in a growth chamber at 21 ± 1°c, 60 ± 5% relative humidity, at 200 µmol m-2 s-1 light intensity, and on a 10.5 : 13.5 (l : d) photoperiod. twenty-six days old plants were used for the force feeding experiments. insect rearing the start population of p. cochleariae was obtained from bayer crop science (mohnheim, germany). this beetle species was initially 0 50 100 150 200 250 300 350 400 cyp79b2-/b3mam3+ wt genotype a a a b 0 50 100 150 200 250 300 350 400 cyp79b2-/b3mam3+ wt genotype l a rv a lw e ig h t in c re a s e [% ] a ab b a reared on pak-choi plants (brassica chinensis l.) following by savoy cabbage (brassica sabauda l.) in the laboratory of the urban horticulture section at humboldt university berlin. pak-choi plants were provided as food in insect cages. for egg laying the beetles were provided the severed stem of savoy cabbage. after the beetles laid eggs on savoy cabbage, which was transferred to a new cage for larval hatching. larvae were fed with savoy cabbage leaves, which were replaced on alternate days until larval pupation. pupae were transferred to another cage for beetle hatching. this procedure was repeated to obtain a sufficient number of insects all at the 1st instar stage for the bioassays. force feeding experiments lines (three each) of two different a. thaliana mutants with modified gs profiles and the corresponding columbia wild-type (wt) were used for the bioassays. the first lines were the double knockout mutant of cyp79b2 and cyp79b3 (cyp79b2-/cyp79b3-, a gift from j. chory, the salk institute for biological studies, california). further information on the construction of the mutant can be reviewed in zhao et al. (2002). the second plant mutant lines are characterized by over expression of mam3 (mam3+) and were generated as described in textor et al. (2007). first instars of p. cochleariae were weighted before release on the plants, one larva per plant. the pots were covered with small cages made from transparent plastic cylinders and fine mesh gauze. after four days pre-conditioning to their respective plant lines, the larvae were weighted and transferred to a new plant of the same genotype. after three days the final larval weight was determined. ten replicates were made for each of the nine lines of mutants and wt. four plants of each genotype were harvested following larval feeding at 30 days after planting (first harvest) as well as 33 days after planting (second harvest), frozen in liquid nitrogen and stored at -80°c prior to gs analysis. data from chemical analysis and bioassays data were analyzed by performing variance analysis with following mean comparison test by using systat 11.0. glucosinolate analysis plant samples were freeze-dried and whole plants were grounded. samples of 20 mg were extracted in 70% methanol following the procedure described by mewis et al. (2005). extraction was done in five replications for each treatment and genotype. to quantify gs content, an internal standard p-hydroxybenzyl gs (purified from sinapis alba seeds as potassium salt) was added initially to the first methanol extract. the gs of extracts were analyzed as desulfo gs and for this purpose the extracts were desulfated on deae sephadex a-25 mini columns with aryl sulfatase solution (sigma-aldrich corp h 1 from helix pomatia); column wash steps are as described in mewis et al. (2005). a 40 µl aliquot of each gs extracts was run on a dionex summit p680a hplc system equipped with an asi-100 auto sampler and a pda-100 photodiode array detector. gs were separated on a narrow bore column (acclaim tm 120, 250-2.1, rp18, dionex) at a flow rate of 0.4 ml / min and a column temperature of 25°c. a 43 min gradient program with eluents: a) ultra pure h2o (milli q quality) and b) 40% acetonitrile (hplc grade) consisted of 0 1 min 99.5% a, 1 8 min 80%, 10 19 min 50%, 22 28 min 1.0% a, 33 36 min 99.5% a. the eluent was monitored for absorbance at 229 nm and gs were identified using purified standards, retention time, and uvvis spectra according to brown et al. (2003). the gs amount was calculated from hplc peak areas using response factors of desulfo gs at 229 nm. results insect performance on genotypes with different glucosinolate profiles percentage weight increase of larvae force-fed on each of the genotypes was significantly different in the first experiment (fig. 1a). larval performance was poorest on the cyp79b2-/cyp79b3mutants followed by mam3+, and wt. there was no significant difference in percentage weight increase determined after feeding on mam3+ and wt. although the same trend was observed in the second experiment (fig. 1b), the differences observed were not significant. to explain the impact of different types of gs on p. cochleariae larval performance, simple correlation of the larval weight increase to induced total gs contents were performed. we found that there was no correlation with r = 0.136 between the weight increase of the larvae and increasing levels of induced total gs among all genotypes. the performance of larvae was comparable poor on cyp79b2-/ cyp79b3mutants with lowest gs levels. although a different effect of gs classes was expected, no correlation of larvae performance to induced indolyl (r = 0.231) or aliphatic gs (r = 0.032) contents of genotypes was found (fig. 2 a and b). fig. 1: percentage weight increase of p. cochleariae larvae on different a. thaliana genotypes in the first (a) and second experiment (b) (different letters indicate significant differences among treatments, fisher’s lsd p < 0.05) performance of phaedon cochleariae 109 110 m.m. uddin, ch. ulrichs, j.g.tokuhisa, i. mewis r = 0.032 0 1 2 3 4 5 6 7 8 0 10 20 30 40 50 60 70 80 induced alipahtic glucosinolate content [µmol/g dry weight] cyp79b2-/b3mam3+ colwt b r = 0.213 0 1 2 3 4 5 6 7 8 0 5 10 15 20 25 30 induced indolyl glucosinolate content [µmol/g dry weight] w e ig h t in c re a se o f p . co c h le a ri a e la rv a e [m g ] a glucosinolate induction of arabidopsis thaliana genotypes by phaedon cochleariae the experimental a. thaliana mutant lines differed in their gs profile compared to the wt (tab. 1). in the wt the major aliphatic and indolyl gs were 4-methylsulfinylbutyl gs and indol-3-ylmethyl gs, respectively. in contrast to wt, 3-methylsulfinylpropyl gs was the dominant aliphatic gs in mam3+ and levels of the long chain aliphatic gs, c7 and c8, were increased. overall, levels of aliphatic gs were lower than in wt but with about similar levels of indolyl gs. the major gs compound detected in cyp79b2-/b3was 4-methylsulfinylbutyl gs followed by 3-methylsulfinylpropyl gs, as in wt, but indolyl gs were complete absent in this mutant. similar results for constitutive and induced levels were observed for the two experiments. the accumulation of gs following herbivory damage differed in the three genotypes (tab. 1). after feeding of p. cochleariae, levels of total gs increased in all genotype lines, but the accumulation was only significant with the cyp79b2-/b3mutant (tukey’s hsd test p = 0.004). in the wt, a marked increase of indol-3-ylmethyl and 1methoxyindol-3-ylmethyl gs levels was observed after larval feeding (tab. 1). also the major methylsulfinyl gs increased after herbivory of p. cochleariae. in the mam3+ mutant levels of nearly all aliphatic (except 4-methylthiobutyl gs and 8-methylsulfinyloctyl gs) and indolyl gs increased after feeding by larvae. the major aliphatic gs, 4-methylsulfinylbutyl gs increased about two/three-fold. as with the wt response, a strong increase of indol-3-ylmethyl and 1methoxyindol-3-ylmethyl gs was observed after beetle herbivory. only methylsulfinyl gs were induced by larvae feeding in cyp79b2/b3-, wherein this gs increased about two-fold in this mutant. discussion it has been shown that gs and their corresponding hydrolysis products determine plant resistance in crucifers such as a. thaliana and brassica sp., especially against generalist insects (lambrix et al., 2001, reviewed in kliebenstein et al., 2004). however, gs are known to influence the behavior of insects that specialize on crucifer plants (renwick, 2002; mewis et al., 2002). the effects of gs are assumed to vary depending on the attacking insect herbivore as well as the concentration and composition of gs present in the plants. many studies have demonstrated that gs and their hydrolysis products stimulate feeding in crucifer specialists (hicks, 1974; chew, 1988a; barlet and williams, 1991; li et al., 2000). that gs and their corresponding hydrolysis products can adversely affect the performance of specialists as well is reported by agrawal and kurashige (2003). they reported a negative effect of higher gs concentrations on the lepidopteron pieris rapae l. to study the effect of gs classes on the crucifer specialist p. cochleariae, we used different mutant lines of a. thaliana with modified gs profiles and their corresponding wt. larval performance in the experiments, measured as percentage weight increase, was best on columbia wt, followed by mam3+, and the double knock out mutant cyp79b2-/cyp79b3-. this was opposite to detected constitutive and induced gs contents in genotypes, whereby levels were highest in the wt and lowest in cyp79b2-/cyp79b3-. furthermore, we did not find a negative correlation of larval performance to increasing total gs contents of genotypes. this differs to other studies, where weight increase of crucifer specialist larvae was slower on plants with higher gs contents (nayar and thorsteinson, 1963; ahman, 1986; louda and collinge, 1992; wolfson, 1982; agrawal and kurashige, 2003; rohr et al., 2006). for example, li et al. (2000) reported that there was a negative correlation of larvae weight increase in plutella xyllostella (l.) and spodoptera eridania (stoll) to an increasing allyl gs concentration. also rohr et al. (2006) found that larval performance of pieris brassicae l. was negatively related to increasing gs contents in a. thaliana ecotypes. in contrast, kliebenstein et al. (2005) demonstrated that plant feeding damage was positively correlated to increasing levels of aliphatic gs in the crucifer specialist p. xylostella, whereas negative relationships of larval performance and increasing gs levels were detected for the generalist caterpillars trichoplusia ni (hübner) and spodoptera exigua (hübner). in our present study the insect-plant interaction seems to be complex, but it can be noted that the p. cochleariae performance was worse on the mutant which contain only aliphatic gs. furthermore, beetles feeding induced aliphatic gs to a greater extent in cyp79b2-/cyp79b3and mam3+ compared to the wt at the end of the experiment (second harvest, tab. 1). the study of siemens and mitchellolds (1996) with phyllotreat cruciferae (goeze) and p. xylostella showed that herbivory of these insects varied curvilinearly with gs levels in brassica campestris (l.), with maximum herbivory at intermediate gs levels. such relationship could be also true for p. cochleariae. a. thaliana ecotypes differ especially in their aliphatic but not their indolyl gs structures (kliebenstein et al., 2001; reichelt et al., 2002). from the high variability of aliphatic gs it is assumed that structurally different gs and their hydrolysis products have different biological effects. for example, higher oviposition preference by fig. 2: correlation between weight increase of p. cochleariae larvae and induced aliphatic (a) and indolyl glucosinolate (b) content of a. thaliana genotypes (1st experiment) performance of phaedon cochleariae 111 tab. 1: glucosinolate content of arabidopsis thaliana genotypes in the first experiments, control plants and plants after p. cochleariae feeding (induction of single compounds is in grey) genotype glucosinolate glucoinolate content (µmol/g dry weight) first harvest second harvest control phaedon control phaedon 3-methylsulfinylpropyl 4.34 5.04 4.77 5.76 4-methylsulfinylbutyl 26.07 27.47 24.72 36.96 5-methylsulfinylpentyl 0.71 0.86 0.72 1.08 6-methylsulfinylhexyl 0.13 0.21 0.17 0.26 4-hydroxyindol-3-ylmethyl 0.10 0.19 0.27 0.32 7-methylsulfinylheptyl 0.97 0.96 0.93 1.12 4-methylthiobutyl 1.30 1.29 1.18 1.10 indol-3-ylmethyl 0.86 6.51 5.24 10.14 8-methylsulfinyloctyl 0.00 0.00 0.00 0.64 4-methoxyindol-3-ylmethyl 1.28 1.24 0.32 0.90 1-methoxyindol-3-ylmethyl 0.57 1.13 0.00 1.20 7-methylthioheptyl 0.10 0.14 0.05 0.09 8-methylthiooctyl 0.19 0.22 0.13 0.20 total 36.63a 45.27a 38.48a 59.76a 3-methylsulfinylpropyl 11.53 12.78 11.46 15.31 4-methylsulfinylbutyl 4.22 7.07 3.61 10.13 5-methylsulfinylpentyl 0.24 0.42 0.25 0.49 6-methylsulfinylhexyl 0.38 0.66 0.58 1.14 4-hydroxyindol-3-ylmethyl 0.07 0.14 0.12 0.33 7-methylsulfinylheptyl 1.77 2.90 2.14 3.23 4-methylthiobutyl 0.35 0.39 0.16 0.34 indol-3-ylmethyl 5.59 7.59 6.60 12.79 8-methylsulfinyloctyl 1.22 0.29 0.00 0.04 4-methoxyindol-3-ylmethyl 1.34 1.42 1.10 1.83 1-methoxyindol-3-ylmethyl 0.47 1.94 0.08 0.49 7-methylthioheptyl 0.47 0.93 0.63 0.73 8-methylthiooctyl 0.75 1.29 0.81 1.54 total 28.38a 37.81a 27.53a 48.38b 3-methylsulfinylpropyl 1.68 2.34 2.19 3.68 4-methylsulfinylbutyl 9.36 14.91 10.79 21.73 5-methylsulfinylpentyl 0.28 0.49 0.32 0.75 6-methylsulfinylhexyl 0.07 0.13 0.06 0.19 4-hydroxyindol-3-ylmethyl 0.00 0.00 0.00 0.00 7-methylsulfinylheptyl 0.32 0.67 0.32 0.86 4-methylthiobutyl 1.10 1.03 0.96 0.85 indol-3-ylmethyl 0.00 0.00 0.00 0.00 8-methylsulfinyloctyl 1.22 2.73 1.68 3.65 4-methoxyindol-3-ylmethyl 0.00 0.00 0.00 0.00 1-methoxyindol-3-ylmethyl 0.00 0.00 0.00 0.00 7-methylthioheptyl 0.06 0.10 0.06 0.06 8-methylthiooctyl 0.14 0.22 0.17 0.15 total 14.21a* 22.60b 16.53a 31.93b *different letters indicate significant differences among treatments, tukey’s hsd test p < 0.05 w t m am 3+ cy p7 9b 2/b 3112 m.m. uddin, ch. ulrichs, j.g.tokuhisa, i. mewis hellula undalis (f.) was noted for aliphatic alkenyl gs than indolyl gs (mewis et al., 2002). also kliebenstein et al. (2002b) found evidence for diverse biological activities of gs by studying their variation and impact on plant resistance against the generalist t. ni. the authors suggested that gs with alkenyl side chains were more of a deterrent to the test insect than gs with non-alkenyl side chains. rohr et al. (2006) observed a correlation of side chain structure of gs in a. thaliana ecotypes and the performances of a generalist and specialist lepidopteron, s. exigua and p. brassicae, respectively. here larval weight gain was greater on the 3-hydroxypropyl gs-containing ecotypes than on methylsulphinyl gs-producing ecotypes. furthermore, giamoustaris and mithen (1995) reported an increasing damage of brassica napus l. plants by the cabbage stem flea beetle, psylliodes chrysocephala (l.), with plants containing gs with shorter side chains and increased levels of hydroxylation. our initial results with p. cochleariae do not support the notion of different responses to structurally distinct classes of gs by a specialist. however, in this study p. cochleariae fed on only a limited number of gs, which probably represents a subset of all the gs that the species may encounter. however, further studies with different specialist insects are needed to identify general effects of gs structures on feeding behavior that could be employed by plant breeders to develop insect resistance crop plants. acknowledgement we thank dr. joanne chory (the salk institute for biological studies, california, us) for providing the cyp79b2-/cyp79b3mutant lines. also thanks to dr. peter meisner and gerd trautmann (bayer crop science, mohnheim, germany) for sending the starting population of phaedon cochleariae beetles. the authors further thank dr. jonathan gershenzon (max-planck institute for chemical ecology jena, germany) for his collaborative help. references agrawal, a.a., 1999a: induced responses to herbivory in wild radish: effects on several herbivores and plant fitness. ecology 80, 1713-1723. agrawal, a.a., kurashige, n.s., 2003: a role for isothiocyanates in plant resistance against the specialist herbivore pieris rapae. j. chem. ecol. 29, 1403-1415. ahman, i., 1986: toxicities of host secondary compounds to eggs of the brassica specialist dasineura brassicae. j. chem. ecol. 12, 1481-1488. barlet, e., williams, i.e., 1991: factors restricting the feeding of the cabbage stem flea beetle ( psylliodes chrysocephala). entomol. exp. appl. 60, 233-238. bauer, r., staedler, e., monde, k., takasugi, m., 1998: phytoalexins from brassica (cruciferae) as oviposition stimulants the cabbage root fly, delia radicum. chemoecology 8, 163-168. bodnaryk, r.p., 1991: developmental profile of sinalbin (p-hydroxybenzyl glucosinolate) in mustard seedlings, sinapis alba l., and its relationship to insect resistance. j. chem. ecol. 17, 15431556. bodnaryk, r.p., 1992: effects of wounding on glucosinolates in the cotyledons of oilseed rape and mustard. phytochemistry 31, 2671-2677. bodnaryk, r.b., palaniswamy, p., 1990: glucosinolate levels in cotyledons of mustard brassica juncea (l.) and rape, b. napus (l.) do not determine feeding rates of flea beetle, phyllotreta cruciferae (goeze). j. chem. ecol. 6, 2735-2746. brown, p.d., tokuhisa, j.g., reichelt, m., gershenzon, j., 2003: variation of glucosinolate accumulation among different organs and developmental stages of arabidopsis thaliana. phytochemistry 62, 471-481. chew, f.s., 1988a: biological effects of glucosinolates. in: cutler, h.g. 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j.a.a., 2002: the chemical world of crucivores: lures, treats and traps. entomol. exp. appl. 104, 35-42. rodman, j.e., karol, k.g., price, r.a., sytema, k.j., 1996: molecules, morphology and dahlgren’s expanded order capparales. systematic botany 21, 289-307. rohr, f., ulrichs, c., mucha-pelzer, t., mewis, i., 2006: variability of aliphatic glucosinolates in arabidopsis and their influence on insect resistance. comm. appl. biol. sci. ghent university, 71, 507-515. siemens, d.h., mitchell-olds, t., 1996: glucosinolates and herbivory by specialists (coleoptera: chrysomelidae, lepidoptera: plutellidae): consequences of concentration and induced resistance. environ. entomol. 25, 1344-1353. tanton, m.t., 1977: response to food plant stimuli by larvae of the mustard beetle phaedon cochleariae. entomol. exp. appl. 22, 113-122. textor, s., kraker, de, j.-w., hause, b., gershenzon, j., tokuhisa, j.g., 2007: mam3 catalyzes the formation of all aliphatic glucosinolate chain lengths in arabidopsis. plant phys. 144, 60-71. wittstock, u., halkier, b.a., 2002: glucosinolate research in the arabidopsis era. trends in plant science 7, 263-270. wolfson, j.l., 1982: developmental responses of pieris rapae and spodoptera eridania to environmentally induced variation in brassica nigra. environ. entomol. 11, 207-213. zhao, y., hull, a.k., gupta, n.r., goss, k.a., alonso, j., ecker, j.r., normanly, j., chory, j., celenza j.l., 2002: trp-dependent auxin biosynthesis in arabidopsis: involvement of cytochrome p450s cyp79b2 and cyp79b3. genes developm. 16, 3100-3112. address of the author: dr. inga mewis, humboldt university berlin, institute for horticultural sciences, urban ecophysiology, lentzeallee 55, 14195 berlin, germany << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb 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/pagesize [907.087 680.315] >> setpagedevice 116 obituary obituary professor dr. reinhard lieberei on march 5th 2019 professor reinhard lieberei, one of the leading scientists in the field of applied botany, passed away in gorleben at the age of 70. he was born on july 4th 1948 in seefeld, a small city in the north of germany. from 1968 to 1974, he studied biology at the tu braunschweig. for his phd, which he achieved in 1976, he joined the group of prof. dr. böle biehl and studied the activation of chloroplastic polyphenol oxidases. thereafter, for his habilitation, he entered the field of phytopathology and chemical ecology. in this context, reinhard lieberei’s research was focussed on various diseases of the cyanogenic rubber tree, hevea brasiliensis. he demonstrated that the hcn, which is liberated from injured hevea leaves as potential defense mechanism against herbivores, strongly impairs the defense against pathogens by inhibiting the biosyn thesis of phytoalexins. related to this project, reinhard lieberei did his first research stay in the central amazon rain forest region in brazil. the experiences of this enthralling adventure led to his ongoing interest in the tropics. back in germany, in 1984, he obtained his venia legendi in botany from the faculty of natural science at the tu braunschweig. after his habilitation, reinhard lieberei held a position as assistant professor at the technical university braunschweig, until, in 1989, he accepted a professorship at the university of hamburg in the institute for applied botany. he held this position until his retire ment in 2013. in the university, he was strongly engaged within the academic self-administration, e.g., as head of the institute, head of the department or head of the faculty. in hamburg, reinhard lieberei’s research area was broadened by introduction of various physiological and biochemical aspects of plant tissue and organ culture, and − most importantly − the work on ecologically and economically sustainable agro-production in the tropics, in particular on value adding processing of cocoa beans, namely the control of fermentation. by further elucidating the physiological basis of cocoa fermentation and the intrinsic meta bolic processes, he successful carried on the seminal work of his mentor, prof. böle biehl. reinhard lieberei became a well known and renowned specialist for his studies of cocoa fermentation. this scientific work – apart from the relevance for basic science − had an impact on processes in cocoa plantations. in this way, reinhard lieberei represented, like no other, the ideal of an “applied plant biologist”. by his enthusiasm, he inspired and motivated many students to enter the field of plant biology. it was one of the major maxims of reinhard lieberei that he should pass on his knowledge and experiences to others. accordingly, he was a very successful academic teacher and mentor. this is evidenced by the large number of scientists that were educated, trained and shaped by reinhard lieberei and his scientific philosophy. in addition to prof. lieberei’s successful research, his tremendous achievements as an academic teacher and his extensive administrative work, reinhard lieberei always was also commited to support the scientific community. already in his early career, he was chairman of the association “host pathogen interactions” within the german phytomedicinal society (deutsche phytomedizinische gesellschaft). from 1996 to 2005, he was president of the german society of applied botany (vereinigung für angewandte botanik). in his incumbency, he has made many efforts to modernise and update the society. from 2008 to 2013, he acted as editor-in-chief for the journal of applied botany. due to his comprehensive knowledge of plant biology and his extensive experience in the tropics, he was appointed as scientific head of the shift programme (studies on human impact on forests and floodplains in the tropics), an important and extensive venture of the german federal ministry of education and research. for about six years (1996-2003), he was responsible for coordination and guidance of the various scientific endeavors involving sustainable management in brazil. because of his well known reputation, he was made a part of many other multilateral research projects, e.g., ecoman (ecosystem management in atlantic rain forest rural areas). after his retirement he continued being active in the field of applied botany, and in 2018, he realized his lifelong dream by establishing a corresponding museum: the museum für nutzpflanzen in gorleben, which was opened in october 2018. although the scientific work of reinhard lieberei was mainly established in field of applied botany, his research also had a strong impact in basic science, especially the interface between plant physiology, biochemistry and ecology. his contributions to this interdisciplinary area of research was based on his comprehensive knowledge and experience is each branch of plant biology. in this regard, reinhard was one of the last and rare polymaths in modern plant biology. dirk selmar institute for plant biology, tu braunschweig, braunschweig, germany prof. dr. reinhard lieberei (1948-2019) art29 journal of applied botany and food quality 82, 179 182 (2009) 1 vegetable research department, national research center, dokki, cairo, egypt 2 botany department, national research center, dokki, cairo, egypt 3 institute for horticultural sciences, humboldt university of berlin, germany hormonal changes, growth and yield of tomato plants in response to chemical and bio-fertilization application in sandy soils w.a. el-tohamy1, h.m. el-abagy1, n.h.m. el-greadly2, n. gruda3 (received may 4, 2008) summary the response of tomato plants to chemical and bio-fertilization under sandy soil conditions was investigated. the experiments were conducted in nubaria region, egypt. tomato plants were treated with microbein or a mixture of phosphorine and biogein as bio-fertilizers under different rates of the recommended nitrogen and phosphorus fertilization (100% of n and p, 75% of n and p and 50% of n and p). in addition, plants of three treatments received only the rates of chemical fertilizers and were not treated with the bio-fertilizers. vegetative growth measurements, yield, hormonal changes in leaves, and n, p and k contents of leaves were recorded to study the effects of these treatments. the results showed that bio-fertilization significantly increased the vegetative growth of tomato plants (including plant height, number of branches, number of leaves and the fresh weight of plants) and yield compared to non-treated plants. growth and yield of tomato plants was negatively affected by the low chemical fertilization treatments especially at 50% of n and p while biofertilization enhanced growth and productivity under such conditions. tomato plants which were treated with a mixture of phosphorine and biogein had higher growth and yield than plants treated with microbein. bio-fertilization resulted in higher n, p and k contents of leaves and higher indole acetic acid (iaa), gibberellins (ga3) and cytokinins. the possible effects of the treatments are discussed. introduction tomato is one of the most important vegetables grown in egypt. the production of vegetables with minimum chemical residues and avoiding environmental pollution requires minimizing the use of chemicals during the production process including minimizing the use of chemical fertilizers. however, new-reclaimed lands in the desert in egypt are characterized with poor soil fertility that requires the addition of high levels of chemical fertilizers. using bio-fertilizers may help reducing the amounts of chemical fertilizers added to the soil and improve tomato production under sandy soil conditions (amer et al., 2003). bio-fertilizers application resulted in improvement of growth and yield of different vegetable crops, as for instance, pepper (abdalla et al., 2001), garlic (ali et al., 2001) and cucumber (el-sanafawi, 2006). the positive effects of bio-fertilizers on growth and productivity of plants could be attributed to the effect of different strain groups of microorganisms such as nitrogen fixers, nutrients mobilizing group which improve the availability of metals and increase the levels of extractable n, p, k, fe, zn and mn as stated by el-karamany et al. (2000). this may help minimizing the amounts of chemical fertilizers and improve their application efficiency and subsequently avoiding environmental pollution by the access of these chemicals. the present study was designed to explore the various responses of tomato plants to different applications of bio-fertilizers under different levels of chemical fertilizers and to study these effects under sandy soil condition in the new-reclaimed lands. material and methods the experiments were carried out under sandy soil conditions at the experimental station of the national research center in nubaria region (egypt) during two successive seasons of 2006 and 2007. physical and chemical properties of the soil are presented in tab. 1. tomato (lycopersicon esculentum l.) seedlings ‘super strain b’ were transplanted on the 10th of april. seedlings were transplanted on ridges of 70 cm width with a spacing of 30 cm in the row. soil preparations before transplanting were followed according to the recommendations of the ministry of agriculture, egypt. the treatments included three levels of chemical fertilizers: 100, 75 and 50% of the recommended dose of both nitrogen and phosphorus fertilizers (100% of both n and p were calculated as 0.1 kg m-2 of ammonium sulfate and of superphosphate respectively). potassium was kept at 100% of the recommended dose for all treatments. the bio-fertilization treatments included: microbein at a rate of 1 g 2.5 g-1 seeds. microbein functions as biodenitrogen fixer, nutrient mobilizer and growth promoter and it is composed of a selected group of micro-organisms (yakout and greish, 2001), such as bacillus sp., azospirillum sp. and pseudomonas sp. a mixture of phosphorine (a set of p-dissolving bacteria including bacillus sp.) and biogein (contains n-fixing bacteria including azotobacter sp.) at a rate of 1g 2.5 g-1 seeds of each. all bio-fertilizers used in this study are produced under supervision of the ministry of agriculture in egypt. tab. 1: physical and chemical properties of the experimental soil. physical properties sand (%) clay (%) silt (%) texture field capacity % wilting point % 90.08 9.26 0.66 sandy 16.57 5.25 chemical analysis ph ca mg na k hco3 cl mequivalent/l 8.2 7.02 0.527 0.982 0.31 1.3 0.566 180 w.a. el-tohamy, h.m. el-abagy, n.h.m. el-greadly, n. gruda both chemical and bio-fertilization treatments were applied as follows. 1100% of n and p + microbein 275% of n and p + microbein 350% of n and p + microbein 4100% of n and p + phosphorine+biogein 575% of n and p + phosphorine+biogein 650% of n and p + phosphorine+biogein 7100% of n and p (without bio-fertilization). 875% of n and p (without bio-fertilization). 950% of n and p (without bio-fertilization). the following measurements were recorded: plant parameters and yield: (plant height, number of leaves, number of branches and plant fresh weight) were recorded 60 days after transplanting. the total number of fruits and the total yield were recorded at the end of the experiment. chemical measurements: nitrogen and potassium contents of plant leaves were recorded according to fao (1980), and phosphorus content according to troug and meyer (1939). endogenous phytohormones: samples for determination of endogenous hormones including indole acetic acid (iaa), gibberellins (ga3) and total cytokinins were taken in fresh shoots. identification and determination of acidic hormones (iaa and ga3) were carried out by gas liquid chromatography (glc). samples were extracted according to the method adopted by badr et al. (1971). cytokinins fractions were extracted as previously mentioned for the acidic hormones and were detected by hplc. statistical analysis nine treatments were arranged as a completely randomized block design of two factors (chemical fertilization and bio-fertilization) with four replicates. analysis of variance was calculated according to snedecor and cochran (1967). least significant difference (l.s.d.) at 5% was used to compare between means. results and discussion effects of chemical and bio-fertilization treatments on vegetative growth and yield of tomato plants the results showed that chemical and bio-fertilization treatments significantly increased vegetative growth including plant height, number of branches, number of leaves and fresh weight of plants in both seasons (tab. 2). however, the interaction between chemical tab. 2: effects of chemical and bio-fertilization treatments on vegetative growth and yield of tomato plants. analysis of variance refers to 1st or 2nd season. least significant difference (l.s.d.) at 5% was used to compare means within each column. treatments plant height number of number of number of plant fresh total yield (cm) branches leaves fruits/plant weight (g) (kg m-2) 1st season 100% np microbein 47.3 4.25 45.3 44.0 101.0 9.44 biogein+phosphorine 47.8 5.00 57.25 50.2 108.4 12.25 without biofertilizers 47.0 3.25 34.5 33.3 81.72 6.70 75% np microbein 42.3 4.00 35.25 37.8 79.8 8.31 biogein+phosphorine 42.8 4.3 44.75 43.5 91.9 8.66 without biofertilizers 38.7 2.25 31.5 36.0 68.7 6.01 50% np microbein 39.5 3.25 32.2 31.7 75.15 6.63 biogein+phosphorine 39.8 3.29 36.25 39.5 79.52 7.22 without biofertilizers 34.0 2.24 30.0 22.00 61.17 4.60 l.s.d. at 5% (biofertilization) 1.71 0.34 2.45 0.6 4.37 0.16 (chemical fertilization) 1.71 0.34 2.45 0.6 4.37 0.16 (interaction) n.s. n.s. 4.25 0.9 n.s. 0.28 2nd season 100% np microbein 45.2 4.6 43.4 40.2 92.3 8.68 biogein+phosphorine 45.6 4.08 52.2 48.2 104.7 11.76 without biofertilizers 44.2 3.08 32.7 31.2 77.9 6.41 75% np microbein 39.1 3.72 32.1 35.1 74.2 7.73 biogein+phosphorine 39.3 3.95 41.6 40.4 85.5 8.05 without biofertilizers 37.6 2.18 30.5 29.1 66.6 5.83 50% np microbein 37.5 3.05 31.1 29.8 70.6 6.23 biogein+phosphorine 38.4 3.15 33.8 38.3 77.1 7.01 without biofertilizers 32.8 2.16 28.8 21.1 58.7 4.41 l.s.d. at 5% (biofertilization) 1.63 0.32 2.32 0.53 4.11 0.16 (chemical fertilization) 1.63 0.32 2.32 0.53 4.11 0.16 (interaction) n.s. n.s. 4.01 0.91 n.s. 0.27 hormonal changes, growth and yield of tomato plants 181 and bio-fertilization was not significant for plant height, number of branches and fresh weight of plants. the combination of biogein and phosphorine had the best results on tomato growth and yield under all chemical fertilization levels. the combinations of chemical and bio-fertilization resulted in the highest plant growth and productivity in contrast to chemical fertilization alone. these results are in agreement with the results found by amer et al. (2003) who indicated that the bio-fertilization improved tomato productivity under sandy soil conditions. the mixture between biogein and phosphorine gave higher effects. this may be due to the fact that it contains different beneficial microbial strains, which can help fixing nitrogen and make the nutrients available to the plants. as indicated by el-karamany et al. (2000), the positive effects of bio-fertilizers on growth and productivity of plants could be attributed to the effect of different strain groups of microorganisms including nitrogen fixers, nutrients mobilizing group which improve the availability of metals and increase the levels of extractable n, p, k, fe, zn and mn. the results indicated that bio-fertilization can compensate some of the chemical fertilizers added to the soil which positively improved tomato growth and productivity. effects of chemical and bio-fertilization treatments on chemical composition of leaves nitrogen, phosphorus and potassium content of leaves were significantly increased by all chemical and bio-fertilization treatments. however, a combination of chemical and bio-fertilization treatments had higher results than the chemical fertilization alone (tab. 3). the interactions were also significant between chemical and biofertilization except for the phosphorus content in the first season. decreasing the level of chemical fertilization resulted in lower n, p and k contents of leaves while combining bio-fertilization enhanced n, p and k contents compared to plants received only chemical fertilizers without bio-fertilization. higher n and p fertilization combined with bio-fertilization of biogein and phosphorine mixture had the best effect on increasing n, p and k contents of leaves. bio-fertilization markedly increased the n, p and k contents of leaves which can be attributed to the fact that bio-fertilizers application such as microbein and the mixture of biogein and phosphorine resulted in nitrogen fixation and increased availability of other mineral nutrients. el-karamany et al. (2000) indicated that the positive effects of bio-fertilizers on growth and productivity of plants could be attributed to the effect of different strain groups of microorganisms such as nitrogen fixers, nutrients mobilizing group which improve the availability of metals and increase the levels of extractable n, p, k, fe, zn and mn. effects of chemical and bio-fertilization treatments on hormonal changes the high growth and yield of tomato plants in response to biofertilization application cannot be explained by only compensating some of the plant nutritional requirements. the measurements of hormonal contents of tomato plants in this study help exploring the possible roles of bio-fertilizations on promoting plant growth and productivity. the effect of chemical and bio-fertilization on hormonal changes of tomato plants is illustrated in tab. 4. bio-fertilization treatments had positive effects on the hormonal changes of tomato plants compared to non-inoculated plants. the contents of gibberellins (ga3), indole acetic acid (iaa) and cytokinins increased in response to bio-fertilization treatments especially at the treatment of biogein and phosphorine mixture, indicating that bio-fertilization had pronounced effects on the hormonal changes of tomato plants. the inoculation by some microorganisms encouraged the production of some activating hormones, which play an important role for plant growth and development. as shown in tab. 4, bio-fertilization increased contents of iaa, cytokinins and ga3. forlani et al. (1995) and el-khawas (1995) reported that several bacterial strains isolated from the rhizosphere of various crops were able to produce auxins. on the other hand, cacciari et al. (1989) indicated that phytohormones can be produced from different microorganisms such as azospirillum brailense and arthrobacter giacomelloi in single and mixed batch culture and resulted in higher productivity of gibberellins, cytokinins and auxins. on tomato plants, banerjee and chandra (1978) observed high amounts of iaa produced by nfixing bacteria. moreover, rodelas et al. (1997) stated that the main mechanism by which some bacteria such as azotobachter and azospirillium can benefit plant development and yield may not be fully understood unless bacterial production of biologically active substances such as phytohormones, amino acids and water soluble vitamins are related to growth promoting ability of bacterial strains. the present study demonstrated that bio-fertilization had a pronounced effect on increasing contents of stimulating hormones. in addition to the effects of bio-fertilizers on compensating some of the nutrients required by plants, these hormones promoted plant growth and productivity. tab. 3: effects of chemical and bio-fertilization treatments on chemical composition of leaves. analysis of variance refers to 1st or 2nd season. least significant difference (l.s.d.) at 5% was used to compare means within each column. treatments n% p% k% 1st season 100% np microbein 4.13 0.72 3.74 biogein+phosphorine 5.16 0.94 4.31 without biofertilizers 3.42 0.64 3.06 75% np microbein 4.25 0.68 3.69 biogein+phosphorine 4.98 0.92 4.25 without biofertilizers 3.32 0.62 3.04 50% np microbein 4.04 0.65 3.63 biogein+phosphorine 4.71 0.85 4.27 without biofertilizers 3.32 0.56 3.01 l.s.d. at 5% (biofertilization) 0.07 0.01 0.02 (chemical fertilization) 0.07 0.01 0.02 (interaction) 0.12 n.s. 0.04 2nd season 100% np microbein 3.68 0.62 3.31 biogein+phosphorine 4.88 0.9 4.08 without biofertilizers 3.25 0.57 2.77 75% np microbein 3.95 0.63 3.43 biogein+phosphorine 4.63 0.85 3.95 without biofertilizers 3.22 0.6 2.95 50% np microbein 3.8 0.61 3.41 biogein+phosphorine 4.57 0.82 4.41 without biofertilizers 3.19 0.54 2.89 l.s.d. at 5% (biofertilization) 0.06 0.01 0.02 (chemical fertilization) 0.06 0.01 0.02 (interaction) 0.10 0.02 0.04 182 w.a. el-tohamy, h.m. el-abagy, n.h.m. el-greadly, n. gruda tab. 4: effects of chemical and bio-fertilization treatments on gibberellins (ga3), indole acetic acid (iaa) and cytokinins contents of tomato leaves. least significant difference (l.s.d.) at 5% was used to compare means within each column. treatments ga3 iaa cytokinins ng/g fresh weight 100% np microbein 46.28 36.78 29.05 biogein+phosphorine 67.55 47.78 35.53 without biofertilizers 32.33 25.78 24.30 75% np microbein 42.55 34.03 27.05 biogein+phosphorine 63.80 42.55 38.29 without biofertilizers 27.28 25.05 22.83 50% np microbein 39.58 31.55 25.04 biogein+phosphorine 60.31 45.80 37.55 without biofertilizers 26.81 24.05 20.03 l.s.d. at 5% (biofertilization) 0.67 0.65 0.59 (chemical fertilization) 0.67 0.65 0.59 (interaction) 1.16 1.12 1.02 in conclusion, the present study showed that under sandy soil conditions, the application of bio-fertilizers had stimulating effects on the promoting hormones in plants, helped reducing the use of chemical and improved tomato growth and productivity. the use of mixture of some bio-fertilizers had the best effects in this respect. references abdalla, a.m., risk, f.a., adam, s.m., 2001: the productivity of pepper plants as influenced by some fertilizers under plastic house conditions. bull. fac. agric. 52, 625. ali, a.h., abdel-mouty, m.m., shaheen, a.m., 2001: effect of bio-nitrogen organic and in-organic fertilizer on the productivity of garlic (allium sativum) plants. egypt. j. appl. sci. 16, 173. amer, a.h., el-shimi, i.z., zayed, g.a., 2003. response of tomato plants grown in newly reclaimed sandy soils to bio and mineral fertilization. annals of agric. sc. moshtohor 41, 925-938. badr, s.a., martin, g.c., hartmann, h.t., 1971: a modified method for extraction and identification of abscisic acid and gibberellin-like substances from the olive (olea europaea). physiologia plantarum 24, 191-198. banerjee, m., chandra, a.k., 1978: auxin production potentiality of nitrogen fixers isolated from the phyllosphere of crop plants. current science 47, 962-963. cacciari, l., lippi, d., pietrosanli, w., 1989: phytohormone-like substances produced by single and mixed diazotrophic culture of azospirillum and arthrobacter. plant and soil 115, 151-153. el-karamany, m.f., ahmed, m.k.a., bahr, a.a., kabesh, m.o., 2000: uilization of bio-fertilizers in field crop production. egypt. j. appl. sci. 15, 137. el-khawas, h.m., 1995: idole acetic acid production by natural soil microresidents. egypt. j. appl. sci. 10, 575-582. el-sanafawi, e.m., 2006: effect of some bio-fertilizers on growth and productivity of cucumber plants grown under plastic house conditions. j. agric. sci. mansoura univ. 31, 393-400. fao, 1980: soils and plant analysis. soils bulletin 38, 250. forlani, g., pastorelli, r., favilli, f., 1995: root colonization efficiency, plant growth promoting activity and potentially related propertiesassociated bacteria. j. gene and breed. 49, 343-351. rodelas, b., gonzoalez-lopez, j., martinez, m.v., salmeron, v., revillas, j.j., sierra, s., 1997: production of vitamins by soil diazotrophic microorganisms in soil. biol. and biochem. 1, 39-45. snedecor, g.w., cochran, w.g., 1967: statistical methods (6th ed.) iowa state univ. press, ames, iowa, usa. troug, e., meyer, a.a., 1939: improvement in denigess, calorimetric method for phosphorus and arsenic. indian engineering annual., 136-139. yakout, g.m., greish, m.h., 2001: responce of faba bean crop to phosphatic, foliar and bio-fertilization under new reclaimed sandy soil conditions. in: horst, w.j., schenk, m.k., bürkert, a., claassen, n., flessa, h., frommer, w.b., goldbach, h., olfs, h.-w., römheld, v., sattelmacher, b., schmidhalter, u., schubert, s., v. wirén, n., wittenmayer, l. (eds.), plant nutrition – food security and sustainability of agro-ecosystems through basic and applied research, 850-851. kluwer academic publishers, dordrecht. address of the authors: el-tohamy, w.a. (ph.d.) and h.m. el-abagy (ph.d.) vegetable research department, national research center, dokki, cairo, egypt. n.h.m. elgreadly (ph.d.), botany department, national research center, dokki, cairo, egypt. e-mail: wael_eltohamy@hotmail.com dr. habil. n. gruda humboldt university of berlin, institute for horticultural sciences, lentzeallee 55/57 14195 berlin, germany << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedjoboptions true 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/pagesize [907.087 680.315] >> setpagedevice vorgabe neu journal of applied botany and food quality 80, 88 92 (2006) laboratory of plant ecology, ghent university, ghent, belgium effects of severe water stress on partitioning of 14c-assimilates in tomato plants h. zgallaï, k. steppe, r. lemeur (received march 27, 2006) summary tomato plants (lycopersicon esculentum mill. cv nikita) were grown hydroponically and subjected to severe water stress induced by addition of peg-6000 to the nutrient solution. the peg-treatment clearly impaired growth. leaf photosynthesis decreased during the experiment. moreover, the decrease in photosynthesis was associated with a decrease in dry weight of the shoot compared to the root. also leaf area expansion, stomatal conductance and transpiration decreased. water stress enhanced the transport of 14c-assimilates from the source leaf to the lower parts of the plant where the assimilates were incorporated in the lower stem, the leaves below the source leaf and the roots. it was observed that 14c was much more concentrated in the roots compared to the other plant parts. introduction water stress is one of the most important environmental constraints that limit plant productivity (bradford and hsiao, 1982). plant productivity is determined by a complex series of events leading from co 2 fixation in the chloroplasts, formation of phloem-mobile and storage metabolites, and delivery of these metabolites to sink tissue (madores and lucas, 1995). this partioning of carbon determines plant health and vigor during times of stress and subsequently leads to good or bad harvests (houpis et al., 1999). the relationship between carbon translocation and water stress is interesting to study as water is a major constituent of tissue, a reagent in chemical reactions, and a solvent for and mode of translocation for metabolites and minerals. with the occurrence of water stress many of the physiological processes associated with growth are affected. photosynthetic rate reductions due to increased water stress have been observed in a wide variety of plant species. stomatal effects are usually considered to be the first and the major limitation to co 2 fixation (cornic and massacci, 1996; zgallaï et al., 2005). however, leaf photosynthesis may also be affected by accumulation of photosynthates due to changes in translocation or utilization of the assimilates (shinohara et al., 1995). inhibition of assimilate translocation by water stress has been observed in a number of plant species (geiger and fondy, 1991). wardlaw (1990) has indicated that the major effect of water stress on translocation is the delay and the reduction of transfer rate of assimilates from the assimilating tissues to the sink tissues. munns (1988) reported that the reductions in translocation observed under water stress are probably the result from the reduction in growth and the slowing down in carbohydrate utilization prior to a reduction in photosynthesis. disagreement exists as to the relative sensitivity of photosynthesis and translocation to water stress (sung and krieg, 1979). determination of translocation or the carbon balance of the whole plant requires that the carbon balance of the source leaf is determined by the 14co 2 steady-state feeding method (shishido et al., 1999). this method allows calculation of the proportion of total carbon accumulated in the sink organ that was supplied by the source leaf, as well as the growth contribution of the source leaf to the main plant sinks. these data are vital to determine the productivity of the source leaf under severe water stress. the modified sink-source relationship caused by water stress has been largely neglected in the past, although the assessment is of high importance to gain fundamental insights into the carbon metabolism during stress conditions (shishido et al., 1999). in this paper, we examined the effects of severe water stress induced by polyethylene glycol-6000 (peg-6000) on the carbon metabolism in tomato plants (lycopersicon esculentum mill. cv. nikita). both photosynthesis and carbon translocation were measured in this study to elucidate the effects of severe water stress on the carbon metabolism. to investigate the relationship between translocation and water stress, the 14c incorporation of a leaf exposed to 14co 2 , and the subsequent distribution of the c-assimilates in different parts of the plants were analysed. materials and methods plant material and treatment tomato (lycopersicon esculentum mill. cv. nikita) seeds were germinated in pots containing sand and peat. seedlings were grown in a greenhouse at 20 / 32 °c, a relative humidity of 60 % / 70 %, and under normal day-light conditions. after 14 d, plants were transplanted to vermiculite and irrigated with a nutrient solution. the nutrient solution was aerated using an air compressor. the ph of the nutrient solution was monitored daily and kept at 6.0 with addition of hcl (0.1 mol/l) or naoh (0.1 mol/l). at this time, the plant material was subdivided into two sets: one control and one treatment group. each group consisted of 16 plants, with the plants being randomly arranged over two nutrient film drains. the osmotic potential of the nutrient solution (ψ s ) of the treatment group was changed gradually at a rate of -0.04 mpa/d by addition of polyethylene glycol-6000 (peg-6000). the addition of peg was suspended when ψ s reached -1.22 mpa (after 32 d; progressive severe stress) and a series of comparative investigations on treated and non-treated plants were performed. the concentration of peg-6000 (in g/g water) needed for this level of water stress was determined using the equation given in michel and kaufmann (1973). the water potential of the nutrient solution (ψ s ) was measured using filter paper discs (0.25 cm2). measurements were made with a thermocouple psychrometer (sample chambers type c52; wescor inc, logan, utah, usa) following the method described by spanner (1951). measurement of photosynthesis, transpiration and stomatal conductance photosynthesis (p n ), transpiration (e) and stomatal conductance (g s ) of four fully expanded mature leaves of different control (ψ s = -0.16 mpa) and treated plants (ψ s = -1.22 mpa) were determined using an infrared gas analysis system for co 2 and h 2 o respectively (walz compact co 2 /h 2 o porometer; heinz walz, efeltrich, germany). the analyzer was connected to a leaf chamber in which the leaf received a photosynthetic photon flux density of 900 µmol m-2 s-1. the flow of the air through the chamber was 600 ml min-1. after the measurement the leaf areas were measured using an automatic leaf area meter (li-cor, walz, germany) calibrated to ± 0.1 cm2. spatial distribution of photosynthetic assimilates translocation was measured using the 14co 2 steady-state feeding method (shishido et al., 1999). the radioactive pulse was given to the fourth leaf counted from the base of the shoot of tomato plants (fig. 1). the treated leaf consisted of only the three terminal leaflets (the other leaflets were removed just before treatment). the remaining leaflets were confined hermetically in a transparent cylindrical cuvette (crs087, pp systems, uk). 15 µl na 2 14co 3 (specific activity 53 mci mmol-1) (amersham, uk) was put in a reservoir inside the cuvette and 100 µl hcl (10-5 m) was added to the carbonate to form 14co 2 . the 14co 2 pulse was given during 15 min under natural light conditions. at the end of the translocation period (23 h), the plants were quickly divided into different parts (fig.1). for quantitative analysis of the amount of radioactivity in the different plant parts the sample was placed in a scintillation counter. the sample was combusted in oxygen (dry combustion) by a sample oxidizer (packard instrument co. model 306 tri-carb sample oxidizer) by which the carbon is evolved as 14co 2 . extracts were counted in a liquid scintillation counter (nuclear chicago isocap. 300) using a toluene-base scintillation liquid containing 0.015 % popop (w/v), 0.6 ppo (w/v) and triton x (2 parts of toluene / 1 part triton x). the radioactivity in the various parts of the plants was expressed in two ways as described by mor and halevy (1979): (a) as the percentage of radioactivity recovered in the plant, excluding the source leaf, and (b) as a relative specific activity (rsa), which is the ratio of the specific activity in a given part (soluble radioactivity/g dry weight) to specific activity of the whole plant. fresh and dry weight of different parts of the plant were determined with a digital balance (sartorious b310s, göttingen, germany), calibrated to ± 0.001 g. results effect of severe water stress on photosynthesis, transpiration and stomatal conductance the effect of severe water stress on the assimilation rate (p n ), transpiration rate (e) and stomatal conductance (g s ) are shown in tab. 1. after the severe water stress, the assimilation rate and the stomatal conductance decreased to about 71 % of the control, but the intercellular partial pressure of co 2 was identical (data not shown). the results indicate that the stomata of the leaves of the peg-treated plants were nearly completely closed, causing stomatal limitation of photosynthesis. the decrease in photosynthesis and stomatal conductance was associated with a reduction in transpiration rate (tab. 1). tab. 1: effects of severe water stress on the net photosynthesis rate (p n ), the stomatal conductance (g s ), the transpiration rate (e) and the shoot to root ratio of tomato plant grown in a nutrient solution. values indicate the mean (n = 4). different letters within the column show significantly differences using duncan’s test. treatment p n g s e shoot/root (µmol m-2 s-1) (mmol m-2 s-1) (mmol m-2 s-1) control 13.02±0.47a 131.72±12.65a 30.00±1.45b 1.70±0.01a stressed 3.87±0.02b 38.20±3.01b 17.19±4.14a 1.38±0.01b fig. 1: diagram showing the site of application of the solution containing the radiolabelled compound and the cut plant parts of tomato plant before counting. symbols used: r = roots, sb = stem below source leaf, lb = leaves below source leaf, sa = stem above source leaf, la = leaves above source leaf. cuvette site of application reservoir r la sa la sb lb lb lb the dry weight of the shoot, the root, the ratio shoot/root and the leaf area were significantly (p< 0.05) reduced by the imposed water stress (fig. 2, tab. 1). effects of water stress on translocation 89 effect of severe water stress on the distribution of photosynthetic assimilates the radioactive pulse was given to the fourth leaf counted from the base of the shoot (fig. 1). the relative distribution of total 14c radioactivity 23 h after 14co 2 exposure recovered in each plant part is shown in fig. 3. the pattern of 14c distribution exhibited some marked differences between control and peg-treated plants. for control plants, 14c was found mainly in the upper and lower stem and in the roots. the leaves contained less than 4 % of the recovered radioactivity (fig. 3) which was equally distributed between the leaves below and above the source leaf. the main difference in the radioactivity pattern found after application of severe water stress concerned the distribution of the 14c in the lower parts of the plants. the largest increase was observed in the roots (an increase of 10.2 % compared to control plant), but also in the stem and the leaves below the source leaf an increase was detected (6.2 and 2.7 % respectively). fig. 4 also shows the distribution of the radioactivity expressed in terms of relative specific activity (rsa) in the different parts of the tomato plants 23 h after application of the pulse to the source leaf. the rsa indicates that the stem and the roots are labelled most (the source leaf excluded). compared to the control plant, the distribution of radioactivity in the whole plant was significantly different for tomato plants exposed to severe water stress. except for the stem part above the source leaf, the rsa was higher in all distinguished parts of the peg-treated plants compared to the control plants. discussion in this study we investigated the response of hydroponically grown tomato plants to long-term severe water stress. addition of peg6000 to the nutrient solution was used to impose gradual water stress on the plants by exposing their root system to the solution. no toxic effects of peg were observed and, hence, this osmoticum was considered as suitable to induce water stress (kocheva et al., 2005; zgallaï et al., 2005). the water-stressed plants were clearly retarded in growth. both photosynthesis and transpiration decreased significantly (tab. 1) due to the rapid response of stomata to decreasing water availability (buckley, 2005). the closure of the stomata was observed by the sharp decrease in g s during severe water stress. this was also demonstrated by büssis et al. (1998) and zgallaï et al. (2005). hence, photosynthesis and transpiration are very sensitive to severe water stress. another well-observed adaptation response in relation with water stress is a reduction in shoot growth relative to root growth (turner and begg, 1981; bota et al., 2004). also our study indicates a decrease in shoot / root ratio for the water-stressed tomato plants (tab. 1). meyer and boyer (1981) and shinohara et al. (1995), fig. 2: changes in shoot and roots dry weight and leaf area expressed as a percentage of the same parameters in the control plants. 0 20 40 60 80 100 120 control stressed tr e atm e nt s p e rc e n ta g e to t h e c o n tr o l shoot root leaf area p er ce n ta ge o f co n tr ol ( % ) control stressed control plant stressed plant fig. 3: effect of severe water stress on the distribution pattern of (14c) carbon accumulated in each sink from a common source leaf. distribution values of the sinks are expressed as percentages of the total 14c accumulated in the source leaf. same symbols are used as in fig. 1. control plant stressed plant r = 11.4 % lb = 1.8 % sa = 48.8 % la = 1.3 % sb = 36.7 % lb = 4.5 % sa = 25 % la = 6 % r = 21.6 % sb = 42.9 % 90 h. zgallaï, k. steppe, r. lemeur reported that the decrease in shoot / root ratio mostly resulted from relative reductions in leaf growth. this significant shift in the shoot/ root ratio during water stress supports the idea of changes in biomass allocation (sharp and davies, 1985; shinohara et al., 1995; bota et al., 2004). changes in translocation were studied using the 14co 2 steady-state feeding method (shishido et al., 1999). the total 14co 2 fixation in control and treated source leaves was set equal to 100%. the proportion of carbon supplied by the source leaf to sink organs could be analysed. it was observed that water stress clearly affected the export of photosynthetic assimilates from the source leaf in the plant. after 23 h of translocation, the portion of 14c moved out of the source leaf in the control plant was equally distributed between the upper (50.1 %) and lower parts (49.9 %) (fig. 3). in contrast, in severe stressed plants, the percentages of radioactive distribution were 31 % and 69 % for the upper and lower plant parts, respectively. hence, the largest portion of 14c fixed by the source leaf was transferred to the lower parts of the plant (fig. 3) and there was much less movement of 14c towards the upper parts. this was confirmed by the relative specific activity measurements (fig. 4). hence, severe water stress had a significant effect on the movement of c-assimilates, whereby the roots received more photosynthetic assimilates compared to the control plants. these results suggest that tomato plants under severe water stress translocate more c-assimilates towards the roots in order to maintain root growth, which means survival (or uptake of water), even during conditions of severe water stress. these findings were supported by shinohara et al. (1995) and ohashi et al. (2000) who also observed changes in the distribution of assimilates, together with a decrease in shoot/root ratio and an increase of assimilates in the roots during water stress. in this study we used 14c to estimate the relative distribution of radiocarbon in tomato plants under severe water stress. the parameters we used were the percentage of radioactivity recovered in the plant and the relative specific activity (rsa), which emphasized the ability of various organs to draw metabolites (mor et al., 1981). this method enabled us to analyse the carbon balance of each individual sink, thereby obtaining information necessary to evaluate the productivity of plants under water stress condition. the higher translocation of photosynthetic assimilates towards the roots indicated that water-stressed tomato plants tried to maintain root growth and, hence, tried to survive even during the unfavourable growing conditions imposed. acknowledgements the authors wish to thank helga pien and philip deman for their technical assistance, their enthusiastic support and highly valuable contribution to the experimental set-up. references bota, j., stasyk, o., flexas, j., medrano, h., 2004: effect of water stress on partioning of 14c-labelled photosynthates in vitis vinifera. funct. plant biol. 31, 697-708. bradford, k.j., hsiao, t.c., 1982: physiological responses to moderate water stress. in: lange, o.l., nobel, p.s., osmond, c.b., ziegler, h. (eds.), encyclopedia of plant physiology, new series, 12b, 264-324. springer, berlin. buckley, t.n., 2005: the control of stomata by water balance. new phytol. 168, 275-292. büssis, d., kauder, f., heineke, d., 1998: acclimation of potato plants to polyethylene glycol-induced water deficit. i: photosynthesis and metabolism. j. exp. bot. 49, 1349-1360. cornic, g., massacci, a., 1996: leaf photosynthesis under drought stress. in: baker, n.r. (eds.), photosynthesis and the environment, 347-366. kluwer academic publishers, the netherlands. geiger, d.r., fondy, d.b.r., 1991: regulation of carbon allocation and partitioning: status and research agenda. in: bonnemain, j.l., delrot, s., lucas, w.j., dainty, j. (eds.), recent advances in phloem transport and assimilate compartimentation, 1-9. ouest editions, nantes. houpis, j.l.j., rogers, h.h., helms, j.a., costella, m.p., 1999: translocation of carbon-14 in pinus ponderosa seedlings subjected to longterm sulphur dioxide and water stress. trans illinois state academy of science 92, 191-202. kochev, k.v., busheva, m.c., georgrev, g.i., lambrev, p.h., goltsev, v.n., 2005: influence of short-term osmotic stress on the photosynthetic activity of barley seedlings. biol. plant 49, 145-148. madores, m.a., lucas, w.j., 1995: carbon partitioning and source-sink interactions in plants. current topics in plant physiology, vol. 13. aspp, rockville, md. meyer, r.f., boyer, j.s., 1981: osmoregulation, solute distribution, and growth in soybean and seedlings having low water potentials. planta 151, 482-489. michel, b.e., kaufmann, m.r., 1973: the osmotic potential of polyethylene glycol 6000. plant physiol. 51, 914-916. mor, y., halevy, a.h., 1979: translocation of 14c-assimilate in roses. i: the effect of the age of the shoot and location of the source leaf. physiol. plant 45, 117-182. mor, y., spiegelstein, h., halevy, a.h., 1981: translocation of 14cassimilate in roses. ii: the effect of shoot darkening and cytokinin application. physiol. plant 52, 197-200. munns, r., 1988: why measure osmotic adjustment? j. plant biol. 15, 717726. ohashi, y., saneoka, h., fujita, k., 2000: the effect of water stress on growth, photosynthesis, and photoassimilate translocation in soybean and tropical pasture legume siratro. soil sci. plant nutr. 46, 417-425. sharp, r.e., davies, w.j., 1985: root growth and water uptake by maize plants in drying soil. j. exp. bot. 36, 1441-1456. shinohara, y., akida, k., maruo, t., ito, t., 1995: effect of water stress on fig. 4: effect of severe water stress on the distribution of radioactivity (expressed in terms of relative specific activity) in tomato plants 23 h after exposure to 14co 2 . the radioactive pulse was given to the fourth leaf counted from the base of the plant. averages of three replications are presented. the different letters within the histogram indicate significantly differences at a 5 % level. same symbols are used as in fig. 1. 0 2 4 6 8 la lb sa sb r plants parts control stressed d c d b a b c c a r el at iv e sp ec if ic a ct iv it y (r s a ) plants parts b plant parts effects of water stress on translocation 91 the fruit yield, quality, and physiological condition of tomato plants using the gravel culture. acta hort. 396, 211-218. shishido, y., kumakura, h., nishizawa, t., 1999: carbon balance of a whole plant and the contribution of source leaves to sink growth using the 14co 2 steady-state feeding method. physiol. plant 106, 402-408. spanner, d.c., 1951: the peltier effect and its use in the measurement of suction pressure. j. exp. bot. 2, 145-168. sung, f.j.m., krieg, d., 1979: relative sensitivity of photosynthetic assimilation and translocation of 14carbon to water stress. plant physiol. 64, 852-856. turner, n.c., begg, j.e., 1981: plant-water relations and adaptation to stress. plant and soil 58, 97-131. wardlaw, i.f., 1990: the control of carbon partitioning in plants. new phytol. 116, 341-381. zgallaï, h., steppe, k., lemeur, r., 2005: photosynthetic, physiological and biochemical responses of tomato plants to polyethylene glycolinduced water deficit. j. integrat. plant biol. 47, 1470-1478. address of the authors: laboratory of plant ecology, ghent university, coupure links 653, b-9000 ghent, belgium e-mail: kathy.steppe@ugent.be 92 h. zgallaï, k. steppe, r. lemeur << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true 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<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> >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice art06 journal of applied botany and food quality 82, 30 34 (2008) mendel university of agriculture and forestry in brno, horticultural faculty lednice, czech republic formation of anaerobic products in sweet cherries and their partial release into ambient atmosphere under low oxygen storage jan goliáš, marek fruhwirt (received september 24, 2007) summary concentrations of acetaldehyde, ethanol and ethyl acetate in the flesh of ripe, sweet cherries were consistently enhanced by very low oxygen conditions in the ambient atmosphere. at the onset of storage the initial concentration of ethanol in the flesh was 6.0 and 6.4 mg l-1 for the cvs. 'kordia' and 'vanda' respectively. after 28 days under anaerobic conditions the concentration in the flesh of cv. 'kordia' was 5,502 ± 35 mg l-1, which during the next 10 days of storage in air was increased to 6,476 ± 771 mg l-1. the production of ethanol under anaerobic conditions increased during the storage period from 101 µg kg-1h-1 to 550 µg kg-1h-1 from the 16th to the 27th day. the ethanol released from fruit stored at ulo and ca atmospheres was accumulated in the ambient atmosphere at very low concentrations, two orders lower than in the anaerobic atmosphere, at concentrations from 1.2 to 1.6 µg kg-1h-1. the concentration of ethyl acetate during the first 10 days under very low oxygen atmosphere did not increase, because biosynthesis practically ceased. at the start it was 0.76 mg l-1 and after 10 days it had increased to only 0.88 mg l-1. until the fruit is exposed to a normal supply of oxygen from the air, there is no change in the esterification rate. some quantitative relations relate to the production and subsequent release of ethyl acetate from intact fruit, which under subsequent ambient atmosphere storage was detected at very low trace concentrations, of 20 to 80 nl l-1 overall, for all the different storage regimes. introduction the analysis of gas compounds emitted by stored fruit is an interesting research area for characterizing different varieties of the same fruit or for checking its condition by monitoring the volatiles in the ambient atmosphere. a knowledge of headspace modification can help to identify changes in the headspace profile induced by stress. modification of the gas mixture, a general stress indicator is a response which has already been intensively investigated (tiang et al., 2001; spotts et al., 2002; martines-romero et al., 2006). the emitted compounds, namely acetaldehyde, ethanol and ethyl acetate, are formed by the enzymatic conversion of glucose by anaerobic glycolysis. apart from these compounds, a wide range of chemically different compounds, some of them of approximately very high polarity, can be identified depending on the particular sampling technique employed. a commonly used method is the "dynamic headspace" method. the volatiles are concentrated onto an adsorbent, such as activated charcoal carbon molecular sieves or porous polymers, e.g. tenax (vercammen et al., 2000) followed by liquid or thermal desorption. presently, thermal desorption is the method of choice because it can be coupled on-line to the analytical system, which guarantees the highest sensitivity and reduces the risks of contamination. for thermal desorption, the adsorbents have a number of disadvantages, the most important of which are that high molecular weight and polar solutes can only be desorbed at relatively high temperatures. very low oxygen conditions are those where the oxygen concentration in the atmosphere surrounding tissues is insufficient to support aerobic metabolism. such conditions induce anaerobic respiration, when acetaldehyde and ethanol accumulate. brief periods of anaerobic conditions reduce post-harvest decay, help maintain general fruit quality, and are potential disinfestation treatments. low-o2 conditions are considered beneficial for apple storage, as they maintain colour and firmness longer than when stored in air (dixon and hewett, 2000; romanazzi et al., 2003). postharvest loss of quality of sweet cherries is essentially due to softening and loss of acidity, and microbiological decay also influences the storability of sweet cherries (petracek et al., 2002; esti et al., 2002; bernalte et al., 1999; lurie, 1998; venturi et al., 2002). the objective of this study was to monitor the production of the anaerobic metabolites ethanol, acetaldehyde and ethyl acetate under the experimental gas mixtures of ultra-low oxygen (ulo), controlled atmosphere (ca) and very low, fluctuating anaerobiosis (fan) with 0.2 0.9 % of oxygen, in sweet cherry fruits stored at a temperature of 3°3.5°c. after identifying the gaseous compounds released from intact fruit into the surrounding atmosphere, the concentrations were measured. material and methods plant material and preparation of atmosphere sweet cherry fruits were purchased from agro stošíkovice in south moravia. cv. 'vanda' and 'kordia' were harvested at the optimal stage of maturity (based on visual appearance) and after harvest were chilled to 2.5 3.0°c for 6 hours. the fruits were divided into four groups, treated as follows: fan fluctuating anaerobiosis 0.2 % o2 + 0.9 % co2, ca controlled atmosphere 2.0-2.5 % o2 + 6.5-7.0 % co2 ulo ultra-low oxygen 1.0-1.2 % o2 + 0.5 % co2 ra regular atmosphere 21 % o2 + 0,03 % co2 these gas mixtures were maintained for 28 31 days. the chamber with fan atmosphere was fitted with an additional circuit for sampling the ambient atmosphere, which allowed the sampling of fruit for measuring of metabolit volatiles at three terms as shown in fig. 1 3. the co2 and o2 levels in the chambers were monitored with a precision of 0.1 % (dual gas analyser o2/co2, model ts12e, fa arelco arc, fontanay, france) every few minutes initially and once a day thereafter. a concentration of <0.5 % o2 was reached within ten minutes of commencing purging and remained constant until the chambers were opened again. collection of samples and measurement of substances in the liquid phase at the beginning and at the end of the gas mixture treatments, a homogenous juice was separated from 12 fruits of each cultivar for each treatment and frozen to -18°c. chromatographic analysis followed. the defrosted samples were filtered with glass pre-filters, polypropylene housing, 0.2 µm pore size, (alltech, associates, inc., il) and 1 µl of the non-diluted filtrate was injected into the packing 0 20 40 60 0 10 20 30 40 5days mg l -1 0 0 day cv.kordia 10 days 21 days 28 days fig. 1: concentration of acetaldehyde in flesh of fruit stored in fan – fluctuating anaerobiosis – 0.2 % o2 + 0.9 % co2, after 10, 21 and 28 days exposed to air, (means and standard errors, n = 6). 0 2000 4000 6000 8000 0 10 20 30 40 5days mg l -1 0 0 day cv.kordia 10 days 21 days 28 days fig. 2: concentration of ethanol in flesh of fruit stored in fan – fluctuating anaerobiosis – 0.2 % o2 + 0.9 % co2, after 10, 21 and 28 days exposed to air (means and standard errors, n = 6). 0 5 10 15 20 25 0 10 20 30 40 5days mg l -1 0 0 day cv.kordia 10 days 21 days 28 days fig. 3: concentration of ethyl acetate in flesh of fruit stored in fan – fluctuating anaerobiosis – 0.2 % o2 + 0.9 % co2, after 10, 21 and 28 days fruit were exposed in air (means and standard errors, n = 6). column (length 1.2 m, inner diameter 3 mm) filled with porapak p (watters, ass., inc., framingham, mass, usa). crushed teflon was added periodically to the injection space of the chromatograph to adsorb the ballast substance contained in the liquid sample. gc conditions: oven temperature 92°c, detector temperature 150°c, injector temperature 120°c, carrier gas he (12 ml/min), fid, chrom 5, laboratory equipment, czech republic. the quantitative study of acetaldehyde, ethanol and ethyl acetate was carried out with absolute calibration. the calibration and the analyses were carried out under identical conditions. dynamic sampling the fruits was placed into spherical 500 ml jars. the jars were sealed with two gas ports. purified air flowed into the jars at 50 ml min-1 at 20°c prior to sampling. to the outlet port was sealed the enrichment column filled by tenax ta. after the 180 g of sweet cherry fruit had equilibrated in the sampling assembly for 1 h, dynamic sampling was performed by connecting a packed enrichment tube to the outlet of the sampling unit and applying a flow of air at 50 ml min-1 for 60 min. conditioning of the enrichment column packed with tenax ta was performed by heating at 300°c for 0.3 h under a flow of 100 ml min-1 of nitrogen. desorption of the trapped components was carried out using a td-2 thermal desorption unit mounted on top of the gc agilent 4890d injector. for all experiments desorption was in the splitless mode using helium at a flow rate of 150 ml min-1. the td-2 was programmed from 190°c to 200°c with a final time of 2 min. when desorption was completed, the split valve was closed after 0.7 min. the capillary gc-td system consisted of an hp 4890d gc. the column used during all experiments was a 30 m 0.25 mm i.d and 0.25 µm film. the oven was programmed from 35°c to 200°c at a rate of 3°c min-1. helium was the carrier gas at 1.2 ml min-1. the instrument was equipped with a thermal desorption (td-2) unit for dynamic sampling. statistical analysis comparisons were made of different storage conditions, cultivars by using unistat, versin 7 (by means of the standard error method) and quantification of content during the gas treatment and subsequent storage in a normal atmosphere at 3°c. differences of mean values were judged using a significance level of p=0.05. results and discussion content of metabolites under different storage conditions at the onset of storage the initial concentration of acetaldehyde in the flesh was 4.6 and 4.2 mg l-1 for the cvs. 'kordia' and 'vanda' respectively. at the same time onset of storage in healthy fruits there was ethanol content 15.1 and 6.4 mg l-1 respectively for these same cultivars (fig. 1 and 2). the concentration of ethyl acetate in the first 10 days under a very low oxygen atmosphere did not increase by much, because formation of this metabolites were stopped. at the start there was 0.76 mg l-1 and after 10 days were increased to only 0.88 mg l-1 (fig. 3). not until the fruit is exposed to a full supply of oxygen in air is there a change in the rate of ethyl acetate (fig. 3). after the end of the storage periods and the influence of the gas mixtures, significant differences in ethanol, acetaldehyde and ethyl acetate in the fruit flesh were seen (fig. 1 3). the highest ethanol concentration was observed after 30 35 days under fan conditions, being 7.0 g/l in the flesh of the fruit (fig. 2). for at least 10 days after being returned to aerobic conditions, the ethanol that was produced during the anaerobic phase had still not degraded. the gas mixtures ulo and ca did not promote the accumulation of anaerobic metabolites such as acetaldehyde and ethanol (fig. 4 and 5), and both the cultivars demonstrated tolerance of anaerobiosis. the higher concentration of these alcohols and esters with short c-chains suggests fermentative metabolism and increased esterase activity (mattheis et al., 1992, meheriuk et al., 1995). formation of anaerobic products 31 32 jan goliáš, marek fruhwirt the change in acetaldehyde, ethanol and ethyl acetate concentration after removal of fruit from low o2 atmosphere removal of fruit from a stress atmosphere of 0.2 0.9 % o2 fan and transfer to an atmosphere of 21 % o2 resulted in a moderately exponential decline in etoh (fig. 2). the time taken for acetaldehyde and etoh levels to return to those before the introduction of stress is dependent on the time duration of the stress. after the second removal from anaerobic conditions (after 20 days), the etoh levels did not significantly decline. however, after 30 days the ethanol production always increased, which confirmed the much reduced capacity of the fruit flesh to oxidize the accumulated compounds into their simple components. probably a period of around 30 days under very low o2 concentrations represents the maximum period for the fruit to be able to metabolize the ethanol before physiological failure state in (fig. 1, 2 and 3). formation of ethyl acetate a brief period of aerobic conditions enhances concentrations of ethyl acetate and anaerobic volatiles while suppressing acetate esters and acetaldehyde for a wide range of apple cultivars (dixon and hewett, 2000). oxygen is considered to be an essential co-factor for esterification of alcohols in fruit tissue by supplying nadh from aerobic respiration. in the absence of o2, esterification reactions stop and concentrations of free alcohol increase. on return to aerobic conditions, these alcohols are metabolised either to esters or to shorter-chain compounds before esterification or they diffuse out from the tissue. the rate of release of gases from the intact skin can be formulated as a permeability constant (k µg kg-1h-1) related to the concentration of compounds in the percolating gas flowing over the intact fruit to the concentration of these compounds occuring in the liquid phase in the fruit flesh. in the fruit from anaerobic conditions the permeability constant k value at 10 days was established as 1.28 10-4 µg kg-1h-1, at 20 days as 0.98 10-4 µg kg-1h-1, and at 30 days as 0.68 10-4 µg kg-1h-1. however, there are additional, undesirable chemicals produced which can also affect the quality of fruit. ethyl acetate production is linked to off-flavours that are persistent after ca storage for 7 days or more, for many kinds of fruit (larsen and watkins, 1995; paull, 1999). ethanol concentrations but not acetaldehyde concentrations affected the formation of off-flavours. the current sensory note were not detected at the end of fruit storage in the gas mixture labelled as fan. detection of traces of ethanol in ambient atmosphere this study has shown that, in all cases, exposure to very low oxygen has the potential to change the aroma compounds in the ambient atmosphere. however, the amount of ethanol in the flesh of the fruit clearly increased, and a portion of this is released in gaseous form through the skin into the surrounding atmosphere (fig. 6 and 7), and therefore this volatile was accumulated in the hermetically sealed chambers. apart from the possibility of absorption by the chamber walls and the small fluctuations in consequence by flushing with nitrogen, it could be that the control of oxygen levels in the storage atmosphere depends on the fermentation rate (fig. 8, 9, 10). therefore it is desirable to know the content of ethanol as well as of the other measured compounds. the production of ethanol in anaerobic conditions ( fan atmosphere) increases in stored fruit from 101 µg kg-1h-1 to 550 µg kg-1h-1 over 16 to 27 days (fig. 6), which practically constituted a straight-line increase. the ethanol released from fruit stored in ulo and ca atmospheres was accumulated in the ambient atmoshere at very low concentrations, two orders lower than in the fan atmosphere (fig. 7). the diffusion of gases into the ambient atmosphere depends on the actual rate of formation of anaerobic compounds in the fruit flesh and also the corresponding concentration in the surrounding atmosphere. 0 2 4 6 8 0 10 20 30 40 5days mg l -1 0 ca cv.kordia ra cv. kordia 0 5 10 15 20 0 10 20 30 40days mg l -1 50 cv.kordia fig. 4: effect of o2 level on the ethanol (etoh) in pulp of cvs. 'kordia' and 'vanda' in ulo – ultra-low oxygen – 1.0 -1.2 % o2 + 0.5 % co2 (means and standard errors, n = 6). fig. 5: effect of o2 level on the ethanol (etoh) in pulp of cvs. 'kordia' and 'vanda' in ca – controlled atmosphere – 2.0 2.5 % o2 + 6.5 7.0 % co2 (means and standard errors, n = 6). 0 200 400 600 16 21 27days µg kg-1 h-1 fan fig. 6: production of ethanol (etoh) (µg kg-1h-1) released from intact fruit continuously stored in fan – fluctuating anaerobiosis – 0.2 % o2 + 0.9 % co2, after 16, 21 and 27 days (means and standard errors, n = 3). formation of anaerobic products 33 traces of acetaldehyde and ethyl acetate in ambient atmosphere acetaldehyde, as a closely associated by-product of anaerobic glycolysis, was produced in the flesh at very much lower rates than ethanol, but the presence or absence of oxygen under the threshhold of aerobic metabolism had similar effects. the actual concentrations during the storage period essentially differentiated the treatments into two groups – very low oxygen atmosphere (fan atmosphere) and the second group – ulo and ca atmospheres (fig. 9). the same specificy the relationship applies to the production and subsequent release from intact fruit of ethyl acetate. this was detected at very small concentrations in the ambient atmosphere of the storage chamber, at traces from 20 to 80 nl l-1, for all storage regimes (fig. 10). acknowledgements we thank the ministry of agriculture of the czech republic, project no.1g58071 for financial support. µ g k g -1 h -1 0,8 1 1,2 1,4 1,6 16 21 27days g kg -1 h -1 ulo ca fig. 7: production of ethanol (etoh) (µg kg-1h-1) released from intact fruit continuously stored in ulo – ultra-low oxygen – 1.0 1.2 % o2 + 0.5 % co2 and ca – controlled atmosphere – 2.0 2.5 % o2 + 6.5 7.0 % co2 after 16, 21 and 27 days (means and standard errors, n = 3). 0,0 0,6 1,2 1,8 2,4 12 17 19 23 25 28days g l-1 fan ulo ca fig. 8: concentration of acetaldehyde (act) (µg/l) in ambient atmosphere of storage room in fan – fluctuating anaerobiosis – 0.2 % o2 + 0.9 % co2, ulo – ultra-low oxygen – 1.0 1.2 % o2 + 0.5 % co2, ca – controlled atmosphere – 2.0 2.5 % o2 + 6.5 7.0 % co2 (means and standard errors, n = 3). µg l-1 0 10 20 30 40 50 60 12 17 19 23 25 28days g l-1 fan ulo ca fig. 9: concentration of ethanol (etoh) (µg/l) in ambient atmosphere of storage room in fan – fluctuating anaerobiosis – 0.2 % o2 + 0.9 % co2, ulo – ultra-low oxygen – 1.0 1.2 % o2 + 0.5 % co2, ca – controlled atmosphere – 2.0 2.5 % o2 + 6.5 7.0 % co2 (means and standard errors, n = 3). fig. 10: concentration of ethyl acetate (etoac) (ng/l) in ambient atmosphere of storage room in fan – fluctuating anaerobiosis – 0.2 % o2 + 0.9 % co2, ulo – ultra-low oxygen – 1.0 1.2 % o2 + 0.5 % co2, ca – controlled atmosphere – 2.0 2.5 % o2 + 6.5 7.0 % co2 (means and standard errors, n = 3). 0 20 40 60 80 100 120 12 17 19 23 25 28days ng/l fan ulo ca references bernalte, m.j., hernandez, m.t., vidal-aragon, m.c., sabio, e.,1999: physical, chemical, flavor and sensory characteristics of two sweet cherry varieties grown in 'valle del jerte' (spain). j. food quality 22, 403-416. dixon, j., hewlett, e.w., 2000: exposure to hypoxia conditions alters volatile concentrations of apple cultivars. j. sci. food agric. 81, 22-29. esti, m., cinquanta, l., sinesio, f., moneta, e., di matteo, m., 2002: physicochemical and sensory fruit characteristics of two sweet cherry cultivars after cool storage. food chem. 76, 399-405. larsen, m., watkins, c.b., 1995: firmness and concentrations of acetaldehyde, ethyl acetate and ethanol in strawberries stored in controlled and modified atmospheres. postharvest biol. tec. 5, 39-50. lurie, s., 1998: postharvest heat treatments. postharvest biol. tec. 14, 257-269. martíinez-romero, d., alburquerque, n., valverde, j. m., guillen, f., castillo, s., valero, d., serrano, m., 2006: postharvest sweet cherry quality and safety maintenance by aloe vera treatment: a new edible coating. postharvest biol. tec. 39, 93-100. mattheis, j.p., buchanan, d.a., fellman, j.k., 1992: identification of headspace volatile compounds from 'bing' sweet cherry fruit. phytochem. 31, 775-779. meheriuk, m., girard, b., moyls, l., beveridge, h.j.t., mckenzie, d.-l. harrison, j., weintraub, s., hocking, v., 1995: modified atmosphere µ g k g -1 h -1 34 jan goliáš, marek fruhwirt packaging of 'lapins' sweet cherry. food res. int. 28, 239-244. paull, e., 1999: effect of temperature and relative humidity on fresh commodity quality. postharvest biol. tec. 15, 263-277. petracek, d., joles, d.w., shirazi, a., cameron, a.c., 2002: modified atmosphere packaging of sweet cherry (prunus avium l., cv. 'sams') fruit: metabolic responses to oxygen, carbon dioxide, and temperature. postharvest biol. tec. 24, 259-270. romanazzi, g., nigro, f., ippolito, i., 2003: short hypobaric treatments potentiate the effect of chitosan in reducing storage decay of sweet cherries. postharvest biol. tec. 29, 73-80. spotts, r.a., cervantes, l.a., facteau, t.j., 2002: integrated control of brown rot of sweet cherry fruit with a preharvest fungicide, a postharvest yeast, modified atmosphere packaging, and cold storage temperature. postharvest biol. tec. 24, 251-257. tian, s., fan, q., xu, y., wang, y., jiang, a., 2001: evaluation of the use of high co2 concentrations and cold storage to control monilinia fructicola on sweet cherries. postharvest biol. tec. 22, 53-60. venturini, m.e., oria, r., blanco, d., 2002: microflora of two varieties of sweet cherries: burlat and sweetheart. food microbiol. 19, 15-21. vercammen, j., sandra, p., baltussen, e., sandra, t., david, d., 2000: considerations on static and dynamic sorptive and adsorptive sampling to monitor volatiles emitted by living plants. j. hrc 23, 547-553. address of the authors: mendel university of agricultural and foresty in brno, institute of postharvest technology of horticultural products, valticka 337, cz 691 44 lednice, czech republic. e-mail: golias@mendelu.cz << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false 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/pagesize [907.087 680.315] >> setpagedevice journal of applied botany and food quality 92, 355 -359 (2019), doi:10.5073/jabfq.2019.092.047 1institute of genetics and physiology, hebei academy of agriculture and forestry sciences, shijiazhuang, hebei, pr china 2plant genetic engineering center of hebei province, shijiazhuang, pr china salicylic acid alleviates chilling injury in ‘huangguan’ pear yeqing guan1, 2, chuangqi wei1, 2, yudou cheng1, 2, junfeng guan1, 2 * (submitted: august 7, 2019; accepted: november 27, 2019) * corresponding author summary chilling injury (ci) often occurs in ‘huangguan’ pear (pyrus bret schneideri rehd) at low temperature storage, which is characterized by brown spot on the fruit surface. in this study, the ‘huangguan’ pear fruit was soaked either with salicylic acid (sa) or distilled water (control) and subsequently stored at 0 ℃. the results showed that 5 mm and 10 mm sa treatments significantly reduced the ci index of the fruit compared with the control, but had no significant effect on fruit firmness, soluble solids content (ssc) and titratable acid (ta) content. further study on the mechanism of ci showed that 5 mm sa treatment increased the content of sa in peel, enhanced the activities of ascorbic acid peroxidase (apx), glutathione reductase (gr) and superoxide dismutase (sod), reduced the accumulation of phenols in the later stage, decreased the activity of polyphenol oxidase (ppo) before the occurrence of ci, inhibited the expression of ppo1 and ppo5 genes in peel, and significantly down-regulated expression of lox1 and pld4, which code for lipoxygenase and phospholipase d, respectively. these results indicated that sa treatment increased the antioxidant capacity of the peel, inhibited the degradation of cell membrane lipids, reduced the appearance of brown spot on the fruit surface and alleviated ci during cold storage in ‘huangguan’ pear. keywords: salicylic acid; pear; antioxidant; phenol; chilling injury introduction ‘huangguan’ pear (pyrus bretschneideri rehd) has become one of the main cultivars in china with the characteristics of early maturity, beautiful appearance and good quality. in order to extend its shelf life, low-temperature storage is often adopted after harvest for ‘huangguan’ pear, however, it is sensitive to low temperature and easy to cause chilling injury (ci), mainly manifested by the brown spots on the fruit surface (li et al., 2017), with an incidence of about 30% and a serious incidence of over 90%, resulting in huge economic losses (li et al., 2011). salicylic acid (sa) is an endogenous small molecule phenolic com pound, which plays an important role in regulating stress resistance, growth and development in plants (han et al., 2017; supapvanich et al., 2017). studies have shown that sa enhanced ci in cucumber (cao et al., 2009), tomato (aghdam et al., 2014), pineapple (lu et al., 2010), li (luo et al., 2011), pomegranate (sayyari et al., 2011) and peach (cao et al., 2010; yang et al., 2012) fruits. excessive reactive oxygen species (ros) lead to the occurrence of ci caused by oxidative stress, and sa enhances the activities of superoxide dismutase (sod), ascorbic acid peroxidase (apx), and glutathione reductase (gr) and therefore sa is involved in chilling resistance (siboza and bertling, 2013; siboza et al., 2017). ci is also closely related to the damage of cell membranes and the oxidation of phenolic substances. lipoxygenase (lox) and phospholipase d (pld) are involved in cell membrane lipid degradation. sa treatment reduces the activities of pld and lox, maintaining cell membrane integrity and slowing down ci in tomato fruit (aghdam et al., 2014). phenolic metabolism is related to the occurrence of brown spot (li et al., 2011; li et al., 2017), and polyphenol oxidase (ppo) catalyzes the phenolic oxidation and plays an important role in the process of tissue browning (lu et al., 2011; tareen et al., 2012). so far, there is no relevant information on the effect of sa treatment on ci of pear fruit. therefore, in this study, ‘huangguan’ pear was treated with sa to study the changes of the activities of some enzymes and the expression of genes associated with ci, and to explore the mechanism of sa slowing down ci in ‘huangguan’ pear fruit. materials and methods materials ‘huangguan’ pear fruit were harvested from an orchard located in jinzhou city (38.03356° n, 115.0441° e), hebei province on august 14, 2016. fruit without pests, diseases and mechanical injury were selected. the fruit were soaked in 5 and 10 mm sa (sangon biotech co. ltd., shanghai, china) solution for 30 minutes, the control was distilled water, and then naturally dried at room temperature overnight, and subsequently stored at 0 ℃. after the determination of fruit quality, the peel tissue was sampled, immediately frozen in liquid nitrogen and then stored at -80 ℃ for use. triplicate per treatment, 10 fruit per replicate. determination of fruit quality and ci index firmness was determined by fruit hardness tester (gy-4, zhejiang, china), soluble solids content (ssc) was determined by portable sugar tester (pal-1, atago, japan), titratable acid (ta) content was determined by acid-base titration with reference to cao et al. (2007). according to the method of li et al. (2017), the classification of ci as brown spot area on fruit surface with 0, 0-25%, 25%-50% and > 50% was assigned as grade 0, 1, 2 and 3, respectively. the ci index = σ (each ci grade × the corresponding fruit quantity) / (3 × the total number of fruit). determination of sa content frozen peel was ground using liquid nitrogen as coolant and a ground sample of 50 mg was extracted over night at -20 °c in 0.5 ml 80% (v/v) methanol. the supernatant, obtained after centrifugation at 10000 g for 60 min at 4 ℃ was filtered with a c-18 solid phase extraction column and subsequently dried with nitrogen gas. the dried sample was dissolved in 0.5 ml 0.01 m pbs (ph=7.2-7.4), then the supernatant was obtained after centrifuged at 10000 g for 15 min at 4 ℃. the content of sa was determined by using plant sa elisa kit (ml036342; enzyme-linked biotechnology co., ltd., shanghai, china) with a microplate reader (rayto rt-6100, shenzhen, china). the content of sa was calculated based on a standard curve. the sa content was expressed as μg·g-1 fw. 356 y. guan, c. wei, y. cheng, j. guan determination of enzyme activity frozen peel was ground using liquid nitrogen as coolant and a ground sample of 50 mg was extracted in 0.5 ml 0.01 m pbs (ph=7.2-7.4). the supernatant was obtained after centrifuged at 5000 g for 15 min at 4 ℃ and used as crude enzyme extract. the activities of sod, apx, gr and ppo were determined by using plant sod, apx, gr and ppo elisa kits (ml397001, ml764550, ml764402 and ml763547; enzyme-linked biotechnology co., ltd., shanghai, china) with a microplate reader (rayto rt-6100, shenzhen, china), respectively. the unit of catalytic activity (1 u) of sod, apx, gr and ppo was defined as conversion of 1 μmol substrates into products per minute, i.e. 1 u = 1 μmol·min-1. the enzyme activity was expressed by the unit of enzyme activity in the crude enzyme extract per gram (u·g-1 fw). determination of phenolic content the determination of total phenolic content refers to singleton and rossi (1965), which is based on the folin-ciocalteau method. frozen peel samples where ground using liquid nitrogen and subsequently 0.5 g aliquots were combined with 5 ml 80% (v/v) ethanol and extracted by ultrasonic treatment. the supernatant was obtained after centrifuged at 10000 g for 15 min at 4 ℃. the absorbance was measured at 760 nm. the standard curve was carried out with gallic acid, and the phenol content was expressed as mg·g-1 fw. rna extraction and real-time fluorescence quantitative pcr (qrt-pcr) analysis rna extraction: the frozen peel was ground with liquid nitrogen, and the total rna was extracted from 100 mg ground sample according to the instruction of rna prep pure plant kit (tiangen biotechnology co., ltd., beijing, china). qrt-pcr analysis: 500 ng total rna was reverse transcribed into cdna by referring to the instructions of the primescripttm rt reagent kit and subsequently applying the gdna eraser kit (takara, dalian, china). the sybr® premix ex taqtm ii kit (tlirnaseh plus) (takara, dalian, china) was used according to manufacturer’s instructions to perform rt-pcr analysis with the 7500 qrt-pcr system (abi, applied biosystems inc., usa). with pear pbactin2 as internal reference gene (li et al., 2017), the gene expression was defined as 1.0 at initial value in the control, and the relative quantitative expression of gene was referred to the 2(-δδct) method (zhang et al., 2017). quantitative pcr primers were designed according to ‘dangshan suli’ pear genome (http://www. ncbi.nlm.nih.gov/genome/12793). the sequence of primers is shown in tab. 1. statistical analysis data were analyzed by spss software (version 18.0, spss inc., chicago, usa). results were expressed as the mean ± standard error (se) of triplicate value. the duncan’s multiple range test of single factor was used to analyze the significant difference (p < 0.05). results effect of sa on fruit ci and quality in ‘huangguan’ pear ci occurred on the fruit surface after 10 days of cold storage at 0 ℃ in ‘huangguan’ pear, and afterwards, the ci index increased slowly in sa treatment in contrast to that in control. this indicated that sa treatment significantly inhibited the occurrence of ci, but there was no significant difference between the two concentrations of sa treatment (fig. 1). it was found that firmness, ssc and ta content in sa treatment had no significant difference compared with the control at 0 ℃ during a short-term storage (30 days) in ‘huangguan’ pear fruit (tab. 2), which indicated that sa had no significant effect on fruit quality. in order to further reveal the mechanism of sa inhibiting ci, the fruit treated with 5 mm sa were selected for further study. tab. 1: the sequence of primers gene name genbank accession no. forward primer reverse primer pbppo1 hq729709 5´-tccctactcacaaagcccaag-3´ 5´-gacctccaagaccaagaagca-3´ pbppo5 gu906266 5´-accaaaacaaaaaccattccac-3´ 5´-cagccactccaccatacagg-3´ pblox1 xm_009378409 5´-cttccaagttttcgatggaatg-3´ 5´-tgacacgcttgaatcttcaacc-3´ pbpld4 xm_009369269.2 5´-tccccctcattctcctcatca-3´ 5´-tgataaggtatgcatcgtccact-3´ pbactin2 gu830959 5´-ggacattcaacccctcgtct-3´ 5´-atccttctgacccataccaacc-3´ tab. 2: effect of sa on fruit quality in ‘huangguan’ pear storage treatment firmness ssc ta time (d) (n) (%) (%) control 68.2 ± 3.50 a 12.2 ± 0.19 a 0.141 ± 0.011 b 5 mm sa 68.2 ± 3.50 a 12.2 ± 0.19 a 0.141 ± 0.011 b control 69.2 ± 3.41 a 12.2 ± 0.38 a 0.163 ± 0.004 a 5 mm sa 66.0 ± 3.00 a 12.4 ± 0.09 a 0.155 ± 0.008 ab values are mean ± se of three replicates. mean values within each column followed by the same letter are not significantly different at level p = 0.05. 0 30 effect of sa on sa content in peel the sa content in the peel showed a significant upward trend, and it was significantly higher in sa treatment than that in control (fig. 2). effect of sa on antioxidant enzymes activities in peel the sod activity decreased temporarily after 5 days of cold storage at 0 ℃, and afterwards, it was significantly higher in sa treatment fig. 1: changes of ci index in ‘huangguan’ pear by sa treatment during cold storage. values are mean ± standard error (se) of three replicates. different letters represent significant differences at p = 0.05. sa alleviates ci in pear 357 than that in control at day 10 and day 15, and significantly lower at day 20 (fig. 3a). this indicated that sa treatment increased the sod activity of peel at the early stage of cold storage. the activities of apx and gr of peel in fruit treated with sa increased continuously after cold storage at 0 ℃, and were significantly higher than in control samples during the whole period (fig. 3b, 3c). effect of sa on phenolic content, ppo activity and its gene expression in peel the phenolic content of peel increased slowly under cold storage in control, and it was significantly lower in sa treatment than that in control after day 20, but there was no significant difference from day 5 to day 15 (fig. 4a). the changing trend of ppo activity in sa treatment was basically the same as that in control. it increased significantly at day 5 and then decreased significantly in the control. the ppo activity was significantly lower in sa treatment at days 5 and 10 than that in control, and was significantly higher at days 15 and 20, but there was no significant difference at day 30 (fig. 4b). further analysis of ppo gene expression showed that ppo1 and ppo5 gene expression greatly increased in the control under cold storage (fig. 5). the relative expression of ppo5 increased at a lower level (fig. 5b), but a change of ppo1 was not obvious in sa treatment (fig. 5a). the expression level of ppo in sa treatment from day 10 to day 30 was significantly lower than that in control (fig. 5). effect of sa on the expression of lox1 and pld4 genes in peel the relative expression of lox1 was significantly up-regulated during cold storage. compared with the control, the expression of lox1 was significantly lower in sa treatments at days 10 and 15, and afterwards there was no significant difference between the two treatments (fig. 6a). the expression of pld4 gene showed an increasing trend in the control after day 5, and it was significantly lower in sa treatment compared with the control (fig. 6b). discussion this study showed that sa treatment significantly reduced the ci index and occurrence of brown spot in ‘huangguan’ pear during low temperature storage (fig. 1), which was consistent with previous studies on tomato, cucumber, pineapple, plum and peach fruits (aghdam et al., 2014; cao et al., 2009; lu et al., 2010; luo et al., 2011; wang et al., 2006). this was closely related to the increase of sa content after sa treatment (fig. 2). antioxidant enzymes can inhibit oxidative stress induced by excessive reactive oxygen species. aghdam and bodbodak (2013) stated that sa alleviated ci by improving antioxidant activity and reducing oxidative stress in fruit. the results showed that sa enhanced the activities of apx, gr and sod in the peel during cold storage period (fig. 3), it indicated that sa treatment increased the antioxidant level and thus helped to enhance the chilling resistance of the pear fruit. it fig. 3: changes of activities of sod (a), apx (b) and gr(c) of peel in ‘huangguan’ pear by sa treatment during cold storage. values are mean ± se of three replicates. different letters represent significant differences at p = 0.05. fig. 2: changes of sa content of peel in ‘huangguan’ pear by sa treatment during cold storage. values are mean ± se of three replicates. different letters represent significant differences at p = 0.05. 358 y. guan, c. wei, y. cheng, j. guan fig. 6: changes of relative expression level of lox1 (a) and pld4 (b) genes of peel in ‘huangguan’ pear by sa treatment during cold storage. values are mean ± se of three replicates. different letters represent significant differences at p = 0.05. fig. 4: changes of total phenolic content (a), ppo activity (b) of peel in ‘huangguan’ pear by sa treatment during cold storage. values are mean ± se of three replicates. different letters represent significant differences at p = 0.05. fig. 5: changes of relative expression level of ppo1 (a) and ppo5 (b) genes of peel in ‘huangguan’ pear by sa treatment during cold storage. values are mean ± se of three replicates. different letters represent significant differences at p = 0.05. is consistent with the results on pineapple, papaya, apricot and peach fruits (lu et al., 2010; promyou and supapvanich, 2016; wang et al., 2015; wang et al., 2006). the ci is manifested as brown spot on the fruit surface in ‘huangguan’ pear fruit. phenolic substances are oxidized to quinones by ppo to result in browning (lu et al., 2011). low temperature conditioning could reduce ci of ‘huangguan’ pear by decreasing ppo activity and inhibiting its gene expression (li et al., 2017). this study showed that sa had no significant effect on the phenolic content of peel in the early stage (5-15 days), but significantly decreased ppo activity (fig. 4) and significantly inhibited the expression of ppo1 and ppo5 genes (fig. 5). therefore, the lower ppo activity of peel reduced by sa contributed to slowing down the browning process, which was similar to the results on sponge gourd, pineapple and plum fruits (han et al., 2017; lu et al., 2010; luo et al., 2011). ci of fruit is closely related to pld, lox activity and their gene expression, which was associated with membrane lipid degradation (aghdam and bodbodak, 2013). the lower activities of pld and lox under sa treatment reduced ci (cao et al., 2010; rui et al., 2010). this study demonstrated that sa treatment significantly sa alleviates ci in pear 359 decreased the expression of lox1 and pld4 genes at cold storage (fig. 6). this indicated that sa helped to maintain the integrity of cell membranes and weakened the lipid peroxidation, thus alleviated the ci of ‘huangguan’ pear fruit. conclusion sa treatment mainly increased sa content and the activities of apx, gr, sod, decreased the ppo activity, and down-regulated gene expression of ppo1, ppo5, pld4 and lox1 of peel, accompanied by the inhibition of brown spot on fruit surface, thus alleviated ci in ‘huangguan’ pear. acknowledgements this research was supported by the hebei natural science fund (c2017301074), the system of industrial technology for modern agriculture of hebei province (hbct2018 100207), the project of modern agricultural innovation (2019-2-1-1) of hebei province, china. references aghdam, m.s., asghari, m., khorsandi, o., mohayeji, m., 2014: alleviation of postharvest chilling injury of tomato fruit by salicylic acid treatment. j. food sci. tech. 51, 2815-2820. doi: 10.1007/s13197-012-0757-1 aghdam, m.s., bodbodak, s., 2013: physiological and biochemical mecha nisms regulating chilling tolerance in fruits and vegetables under post harvest salicylates and jasmonates treatments. sci. hortic. 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https://www.sciencedirect.com/science/article/pii/s0308814610008769#! https://www.sciencedirect.com/science/article/pii/s0308814610008769#! https://www.sciencedirect.com/science/article/pii/s0308814610008769#! http://dx.doi.org/10.1016/j.foodchem.2010.07.036 http://dx.doi.org/10.1080/14620316.2013.11512965 http://dx.doi.org/10.1016/j.scienta.2017.07.023 http://dx.doi.org/10.1016/j.scienta.2016.11.046 http://dx.doi.org/10.1016/j.scienta.2012.04.027 http://dx.doi.org/10.1016/j.postharvbio.2006.04.010 http://dx.doi.org/10.1016/j.scienta.2014.10.055 http://dx.doi.org/10.1021/jf2041164 http://dx.doi.org/10.2298/abs160816002z vorgabe neu journal of applied botany and food quality 81, 86 94 (2007) 1 university of hohenheim, institute for landscape and plant ecology (320) 2 university of exeter, school of biosciences on the consistencies between csr plant strategies and ellenberg ecological indicator values jürgen franzaring1, andreas fangmeier1, roderick hunt2 (received april 30, 2007) summary one strand of british comparative plant ecology has used experimental measurements of innate traits under standardized conditions to confirm plant ‘strategies’ or ‘functional types’. the sheffield (grime) school has now established csr-signatures for 1010 species. in contrast, a central-european approach (göttingen or ellenberg school) has emphasized the unity of plants with their natural habitats by allocating ‘ecological indicator values’ (eiv´s; german: zeigerwerte) for over 2700 species, which describe the ecological behavior of each species in their plant associations. in this paper we assess the levels of compatibility and congruence between these two approaches using large datasets that include some previously unexamined traits. despite there being a wide gap between these plantand environment-based starting points, we discover that both approaches lead to similar conclusions regarding patterns of evolutionary tradeoffs and ecological processes. in particular, the comparisons support the major evolutionary generalization that plant life has, in effect, aligned itself along a continuum between one trait-group that confers rapid acquisition of resources and another that confers long-term resource conservation. introduction if it is true that in natural plant communities ‘there are many more actors on the stage than roles that can be played’ (colasanti et al., 2001) then the huge dimensionality of such communities can be reduced by viewing them not as collections of species (‘actors’), but of groups or types (‘roles’). the result of this simplification is that comparisons and functional analyses involving communities of widely differing species composition can be greatly facilitated (díaz and cabido, 2001; westoby et al., 2002; pausas et al., 2003; díaz et al., 2004). classical taxonomic knowledge has, therefore, been augmented in recent decades by integrated studies both of innate plant traits (‘plant functional groups’) and of coherent groups of traits (‘strategies’, or ‘plant functional types’). such approaches have been used to address many ecological topics, particularly in the fields of environmental change and biodiversity. some of the more recent studies were initiated by the igbp global change and terrestrial ecosystem (gcte) programme (smith et al., 1997) and protocols have now been established for the standardized determination of over twenty functional traits for vascular species (cornelissen et al., 2003). in addition, a catalogue of the life history traits of many bryophytes has been assembled by söderström and gunnarsson (2003). for the british flora, an extensive autecological database was presented by grime et al. (1988) and by hodgson et al. (1995), both since updated by grime et al. (2005). parallel work from central europe was presented by kleyer (1995) and in the biolflor database (klotz et al., 2002), while the leda database (knevel et al., 2003) represents species from nw europe. most of the ‘soft traits’ in the gcte ‘minimal list’ can easily be categorized or measured in the field in different parts of the world and will undoubtedly help to increase understanding of plant processes on many different scales. for example, using twelve of the gcte traits, díaz et al. (2004) assembled evidence for a common strand of evolutionary specialization in plants drawn from three different continents and recognized a general principle that apparently involved a trade-off between the rapid acquisition of plant resources and their protection within long-lived tissues. similar findings describing the leaf economics spectrum of plants using six traits in twelve biomes were reported by wright et al. (2004). besides having purely academic interest, life history traits and plant functional-type classifications have also found practical applications in biological databases designed for nature conservation purposes and in habitat models which address environmental planning issues. poschlod et al. (2000) proposed the development of biological flora databases in which vegetative and regenerative life history traits would be included so as to improve estimates of species extinction risk within the central european flora. also, information on plant traits, functional types and ecological behavior of plants may be useful in ecotoxicological risk assessment studies, as it has been shown that phytotoxicity is generally most acute in fastgrowing, productive species. examples of this phenomenon have been documented in relation to so 2 (ashenden et al., 1996), ozone (reich, 1987; sellden and pleijel, 1995; franzaring et al., 1999) and herbicide vapor (franzaring et al., 2001). moreover, the co 2 ‘fertilization effect’ may also be more pronounced in such species (hunt et al., 1993; poorter, 1998; poorter and nagel, 2000), with consequent implications for future shifts in community structures. the functional cataloguing of plants within european comparative plant ecology has been much influenced by the contrasting approaches of philip grime in the uk and heinz ellenberg and others in continental europe. the british approach has used experimentation to measure innate plant traits under standardized conditions and to derive plant functional types, while the continental approach has invoked more traditional geobotanical principles, explicitly emphasizing the unity between plants and their natural habitats. relatively few studies have been in a position to compare these two approaches in detail (fichtner and schulze, 1992; thompson et al., 1993; meerts, 1997), so here we assess the levels of compatibility and congruence between british and continental european plant types, and individual plant traits, using large datasets that include some previously unexamined variables. two ecological approaches: csr pfts and eiv´s the sheffield system of c-s-r plant functional types (grime 1974, 1977, 1979, 2001; grime et al., 1987, 2005) describes the ‘strategy’ of plant species in the established phase of growth and recognizes three primary functional types: ruderal, stress tolerator and competitor. many transitional types intervene along the three continuous primary axes (see hunt et al., 2004 for a fuller explanation of ‘c-s-r space’). also using the c-s-r approach, a 7-member classification of plant species from eastern germany (n > 2200) was put forward by frank and klotz (1990) and was subsequently used in the german biolflor database (klotz et al., 2002). the german classification is a simplification of the british original, which uses a 19-member system (hodgson et al., 1995; grime et al., 1997; hunt et al., 2004). practical tools have been developed to allocate a c-s-r type to an individual species (hodgson et al., 1999) and to derive a composite ‘c-s-r functional signature’ for entire vegetation samples (hunt et al., 2004). recent users of the method include moog et al. (2005). in continental europe, in contrast to this experimental approach, the göttingen school developed a system of ‘ecological indicator values’ (eiv´s; german: zeigerwerte) describing the ‘ecological behavior’ or ‘realized ecological niche’ of different plant species (n > 2700). these indicator values address species’ apparent preferences with regard to water availability (ellenberg-f), soil reaction (ellenberg-r), nutrient availability (ellenberg-n), and several other environmental factors (ellenberg et al., 2001). the validity of indicator values in other regions of europe has also been explored by diekmann (1995) for southern scandinavia, by hawkes et al. (1997), pyatt (1997), hill et al. (2000) and hill et al. (2004) for the british isles, by ertsen et al. (1998) and schaffers and sýkorá (2000) for the netherlands, and by schmidtlein (2005) within germany. hermy et al. (1999) used ivs in a study of ancient and recent forest plants of europe and drew implications for forest conservation. in these studies, regionally corrected numbers may be introduced for some species (e.g. grapow and pignatti, 1993). in the usa, some studies have recognized the potential usefulness of ivs (e.g. peet et al., 2003; niemi and mcdonald, 2004), but such values are not yet generally available for this region. ellenberg eivs have generally proved to be closely related to physicochemical field measurements, especially if phytosociological factors are taken into account (wamelink et al., 1998, 2002). the ivs can thus be of value, for example, in modeling succession in different regions so long as the mathematical problems associated with their ordinal scaling (moeller, 1992) are recognized. the -n and -r ivs, especially, have received widespread use in recent years in interpreting the effects of acidification, eutrophication and nitrogen deposition (ellenberg, 1985; bürger, 1988; ter braak and wiertz, 1994; wilson et al., 1995; diekmann et al., 1999; hill et al., 2000; preston et al., 2003; un-ece and ec, 2003). ellenberg ivs have also been used to study the relationships between species richness, nutrient supply and productivity (cornwell and grubb, 2003). a review by diekmann (2003) discusses the strengths and weaknesses of using weighted ellenberg values and suggests further ways to calibrate ivs with reference to new field measurements and other forms of ecological classification. for our comparisons, we gathered information on plant traits, and on british and continental european functional types, from a variety of sources. materials and methods data on specific leaf area (sla), relative growth rate (rgr) and leaf area ratio (lar) were extracted from geyger (1964), grime and hunt (1975), poorter et al. (1990), shipley and peters (1990), reiling et al. (1992), hunt and cornelissen (1997), pleijel and danielsson (1997), garnier et al. (1997), van der werf et al. (1998), wardle et al. (1998), ermolova et al. (1998), reich et al. (1998, 1999) and from our own measurements. data on rgr, sla and lar were available for 203, 171 and 123 species, respectively. most of the more recent publications made use of the standardized protocols prescribed by hendry and grime (1993). data on the c-s-r types of 1010 plant species were extracted from hunt et al. (2004) and ecological ivs were extracted from ellenberg et al. (2001), comprising about 2700 species. data on feed value, mowing tolerance and hemerobia (urbanity) of european plant species were extracted from the biolflor database (klotz et al., 2002). average mineral nutrient concentrations in plants were extracted from a sheffield database (thompson et al., 1997), and from cornelissen et al. (1997), balatova-tulackova (1993), körner (1989), reich (1999), and from our own measurements. data on nitrogen, phosphorus and potassium contents were available for 192, 127 and 126 plant species, respectively. data on thousand-seed weights were extracted from apel (2002) for 340 central european species. for comparison, we obtained field observations for ph and foliar nitrogen content in three local european floras: from sheffield (thompson et al., 1997), from an investigation by duvigneaud and denaeyer-de smet (1970) in hautes-vagnes (belgium), and from höhne (1962), who presented data from saxonia (eastern germany). all data were converted to rank form and spearman rank correlation coefficients were calculated using spss v.11.0. tab. 1 presents the complete correlation matrix; each entry consists of the coefficient (spearman’s rho), its level of significance and the number of cases (species) involved. results and discussion comparability of sources c-s-r types and frequency fig. 1 a gives an overview on how the central european flora behaves within c-s-r space, using the sheffield dataset (maximum 1100 species). the largest groups of species are those of intermediate type: 11% and 10% of the flora are csrand cr-strategists respectively, while the extreme types c, s and r account for only 3, 7 and 8% of the sample. klotz et al. (2002), who classified 2700 species but on the basis of seven c-s-r types only, found that 29 and 26% of the species could be regarded as cand csr-strategists, respectively. in both studies, the general picture of the contribution of different strategists to the central european vegetation is comparable, though the classification by hunt et al. (2004) gives the better resolution of types. fig. 1 b, using the sheffield c-s-r database, shows that the most common european species tend to be the more competitive ones, while the species with strong ruderal and stress-tolerant components tend to be rarer. these rare agriophytes, and species associated with extremely stressful (e.g. dry and rocky) habitats, commonly appear among listings of endangered species in the ‘red data books’ of many european countries. inter-flora comparisons of plant traits measurements of functional traits may reveal different values in the same species studied in different climatic regions and habitats. however, the ranking within the whole sample will more or less stay the same when sampling is made from plants in the same life stage under favorable conditions of measurement (hunt and cornelissen, 1997; cornelissen et al., 2003). fig. 2 a shows an example of this for foliar n measured in tharandt (germany) and in sheffield (england), and fig. 2 b shows that the fallen litter and soil-surface ph measurements for plants collections from these two sites are also closely comparable. plant traits and functional types plant traits interrelations between sla and rgr have been demonstrated elsewhere (e.g. grime et al., 1997, wright and westoby, 2003) and were also strongly present in our study (r = 0.47**). our calculations csr and ellenberg 87 t ab . 1 : s pe ar m an r an k co rr el at io n co ef fi ci en ts ( r ho ) be tw ee n sp ec ie s da ta f or p la nt t ra it s, f un ct io na l ty pe s, i nd ic at or v al ue s an d di st ri bu ti on al i nd ic es . * d en ot es s ig ni fi ca nc e at p < 0 .0 5, * * at p < 0 .0 1 an d ** * at p < 0 .0 01 . n um be rs o f ca se s ar e sh ow n be ne at h th e co rr el at io n co ef fi ci en ts . t he v ar ia bl es , a ll u se d in r an k fo rm , a re : c , t he s pe ci es ’s c -c oo rd in at e w it hi n th e c -s -r s ys te m o f cl as si fi ca ti on ; s , t he s -c oo rd in at e di tt o; r , t he r -c oo rd in at e di tt o; r g r , s ee dl in g re la ti ve g ro w th r at e; s l a , t yp ic al s pe ci fi c le af a re a; l a r , t yp ic al l ea f ar ea r at io ; s r r , t yp ic al s ho ot :r oo t ra ti o; t s w , m ea n th ou sa nd -s ee d w ei gh t; n % , ty pi ca l pe rc en t n it ro g en in d ry m at te r; p % , t yp ic al p er ce nt p ho sp ho ru s in d ry m at te r; k % , t yp ic al p er ce nt p ot as si um in d ry m at te r; n p, n it ro g en :p ho sp ho ru s ra ti o; n k , n it ro ge n: po ta ss iu m r at io ; c a% , p er ce nt c al ci um in d ry m at te r; e ll l , e ll en be rg l -v al ue ; e ll t , e ll en be rg t -v al ue ; e ll k , e ll en be rg k -v al ue ; e ll f , e ll en be rg f -v al ue ; e ll r , e ll en be rg r -v al ue ; e ll n , e ll en be rg n -v al ue ; f re q, f re qu en cy i n c en tr al e ur op e; m to l, t ol er an ce o f m ow in g; f va l, f oo d va lu e to l iv es to ck ; u rb , ur ba ni ty i nd ex . f or d ef in it io ns o f va ri ab le s an d da ta s ou rc es , se e te xt . s r e ll l e ll t e ll k e ll f e ll r e ll n r g r s l a l a r s r r t s w n % p k % n p n k c a % f re q m to l f v a l u rb -0 ,4 2 ** * -0 ,3 7 ** * -0 ,0 5 -0 ,0 6 0 ,1 8 0 ,3 3 ** * 0 ,1 8 0 ,3 2 ** * 0 ,0 6 -0 ,0 1 -0 ,0 1 -0 ,0 6 0 ,2 0 ** * 0 ,1 5 * 0 ,1 5 * -0 ,0 7 0 ,0 1 0 ,3 4 ** * 0 ,0 1 0 ,1 6 ** * -0 ,0 3 0 ,0 8 0 ,1 6 ** * c 1 0 1 0 1 0 1 0 7 9 0 5 9 4 6 9 1 6 5 7 6 1 4 7 2 0 1 9 4 1 6 9 1 2 5 9 5 3 9 7 2 5 1 1 9 9 1 9 9 1 9 2 1 3 5 1 3 1 7 5 8 3 6 5 3 6 5 7 2 6 -0 ,6 0 ** * -0 ,0 5 -0 ,2 0 ** * -0 ,1 5 ** * -0 ,2 3 ** * -0 ,2 1 ** * -0 ,6 0 ** * -0 ,5 9 ** * -0 ,3 4 -0 ,1 1 0 ,0 0 -0 ,0 3 -0 ,2 5 ** * -0 ,4 2 ** * -0 ,2 7 ** * 0 ,0 5 0 ,1 1 -0 ,1 8 * -0 ,1 7 -0 ,2 0 ** * -0 ,0 4 -0 ,4 5 ** * s 1 0 1 0 7 9 0 5 9 4 6 9 1 6 5 7 6 1 4 7 2 0 1 9 4 1 6 9 1 2 5 9 5 3 9 7 2 5 1 1 9 9 1 9 9 1 9 2 1 3 5 1 3 1 7 5 8 3 6 5 3 6 5 7 2 6 f v a l 0 ,1 5 ** 0 ,1 0 ** 0 ,2 9 ** * -0 ,0 2 -0 ,0 6 0 ,0 6 0 ,3 1 ** * 0 ,5 5 ** * 0 ,3 5 0 ,1 0 0 ,0 3 -0 ,1 1 * 0 ,1 5 * 0 ,3 6 ** * 0 ,4 2 ** * -0 ,0 9 -0 ,4 4 ** * 0 ,2 6 ** 0 ,0 2 0 ,3 1 ** * 0 ,0 2 0 ,3 0 ** * r 3 5 6 7 9 0 5 9 4 6 9 1 6 5 7 6 1 4 7 2 0 1 9 4 1 6 9 1 2 5 9 5 3 9 7 2 5 1 1 9 9 1 9 9 1 9 2 1 3 5 1 3 1 7 5 8 3 6 5 3 6 5 7 2 6 m to l 0 ,2 8 ** * 0 ,4 3 ** * 0 ,1 9 0 ,0 3 -0 ,1 1 ** 0 ,0 7 -0 ,2 2 ** * 0 ,1 1 -0 ,0 7 -0 ,0 8 -0 ,1 7 -0 ,1 5 ** -0 ,1 5 * -0 ,0 8 -0 ,2 6 ** * -0 ,0 6 0 ,1 3 -0 ,1 4 -0 ,2 2 ** * -0 ,0 5 -0 ,0 5 0 ,0 3 e ll l 3 5 6 3 6 5 5 9 4 6 9 0 6 5 6 6 1 3 7 2 0 1 9 3 1 6 8 1 2 4 9 4 3 9 5 2 4 9 1 9 7 1 9 7 1 9 0 1 3 3 1 2 9 7 5 6 3 6 3 3 6 3 7 2 4 f re q 0 ,3 0 ** * 0 ,1 7 ** 0 ,2 8 ** * 0 ,0 0 -0 ,2 5 ** * 0 ,2 6 ** * 0 ,1 0 * 0 ,1 3 -0 ,1 6 -0 ,1 1 -0 ,2 3 0 ,0 7 -0 ,1 6 * 0 ,0 2 -0 ,1 7 -0 ,1 5 0 ,2 7 * -0 ,0 3 -0 ,2 8 ** * 0 ,0 4 0 ,0 0 0 ,2 2 ** * e ll t 7 0 1 3 5 4 3 5 4 5 3 4 5 1 1 4 8 8 5 4 7 1 1 7 1 0 5 7 5 6 2 2 9 3 1 5 4 1 1 5 1 1 5 1 0 9 7 7 6 6 5 6 8 2 3 1 2 3 1 5 3 5 c a % 0 ,1 8 * -0 ,0 6 0 ,0 0 0 ,0 5 -0 ,0 9 * 0 ,2 9 ** * 0 ,1 6 ** * 0 ,1 8 * 0 ,1 8 * -0 ,1 5 0 ,0 1 0 ,0 1 -0 ,0 3 0 ,0 6 0 ,0 3 -0 ,1 1 0 ,1 0 0 ,0 5 0 ,1 6 ** * 0 ,0 7 0 ,1 8 ** 0 ,1 7 ** * e ll k 1 2 6 7 8 7 8 1 2 5 5 8 3 5 5 7 6 3 6 1 5 1 1 3 2 9 6 7 3 3 3 9 2 1 5 1 7 3 1 7 3 1 6 6 1 1 6 1 1 5 6 6 4 3 0 3 3 0 3 6 3 4 n k -0 ,0 5 -0 ,2 8 * -0 ,4 2 ** * -0 ,0 7 0 ,0 7 -0 ,0 9 * 0 ,2 7 ** * 0 ,2 1 * 0 ,0 9 -0 ,0 8 -0 ,2 3 * -0 ,0 1 0 ,1 0 0 ,2 6 ** * 0 ,0 4 0 ,0 7 -0 ,0 2 -0 ,1 1 -0 ,0 3 -0 ,0 6 -0 ,0 9 -0 ,2 6 ** * e ll f 1 3 0 7 8 7 8 1 2 8 9 2 5 2 4 6 1 0 1 4 6 1 2 3 9 4 7 1 3 3 3 1 9 4 1 5 5 1 5 5 1 4 8 9 9 1 0 3 6 2 8 2 7 2 2 7 2 6 0 0 n p -0 ,1 7 * 0 ,1 3 -0 ,0 2 -0 ,1 9 ** 0 ,1 4 -0 ,0 7 0 ,3 6 ** * 0 ,1 2 0 ,1 0 0 ,1 2 -0 ,0 3 0 ,2 6 0 ,0 1 0 ,0 4 0 ,1 3 0 ,1 0 0 ,0 9 0 ,4 8 -0 ,1 0 * 0 ,0 1 0 ,1 4 * 0 ,1 5 ** * e ll r 1 8 5 1 1 0 1 1 0 1 8 5 1 2 8 1 3 5 5 7 0 1 2 9 1 0 1 7 2 5 9 2 8 7 1 7 2 1 3 9 1 3 9 1 3 5 9 9 9 5 5 9 1 2 6 2 2 6 2 5 5 9 k % 0 ,2 3 ** 0 ,1 6 0 ,3 4 ** * 0 ,1 7 * 0 ,2 7 ** -0 ,6 7 ** * 0 ,1 3 0 ,3 8 ** * 0 ,3 1 ** * 0 ,1 9 -0 ,0 5 0 ,1 5 ** 0 ,3 8 ** * 0 ,5 3 ** * 0 ,4 8 ** * -0 ,0 5 -0 ,0 9 0 ,3 1 ** * 0 ,2 1 ** * 0 ,1 6 ** 0 ,0 8 0 ,4 4 ** * e ll n 1 8 7 1 1 0 1 1 0 1 9 1 1 3 1 1 3 5 1 9 1 1 7 1 1 4 6 1 0 8 8 3 3 5 7 2 1 6 1 7 5 1 7 5 1 6 8 1 1 4 1 2 0 6 9 3 3 3 0 3 3 0 6 6 1 p % 0 ,3 4 ** * 0 ,0 7 0 ,1 4 0 ,2 1 ** 0 ,1 6 0 ,0 0 0 ,5 2 ** * 0 ,3 8 ** * 0 ,4 7 ** * -0 ,0 1 0 ,0 8 -0 ,1 9 * 0 ,0 8 0 ,3 7 ** * 0 ,3 8 ** * -0 ,1 6 -0 ,3 9 ** * 0 ,0 7 0 ,2 4 ** 0 ,3 3 ** * 0 ,0 7 0 ,4 2 ** * r g r 1 8 7 1 1 1 1 1 1 1 9 1 1 3 0 1 3 5 1 9 2 1 9 8 1 3 6 1 1 9 8 7 1 4 4 1 1 3 9 2 9 2 9 0 8 0 7 0 1 8 0 1 3 7 1 3 7 1 8 6 n % 0 ,1 7 ** -0 ,0 6 -0 ,0 1 0 ,1 3 * 0 ,3 9 0 ,0 9 0 ,1 9 ** 0 ,5 7 ** * 0 ,5 9 ** * -0 ,1 1 0 ,3 2 ** -0 ,1 8 * 0 ,1 4 0 ,1 6 0 ,4 6 ** * 0 ,1 5 -0 ,3 4 ** 0 ,1 2 0 ,2 9 ** * 0 ,2 4 ** 0 ,2 2 * 0 ,2 8 ** * s l a 2 4 0 1 5 0 1 5 0 2 4 1 1 3 1 1 3 5 1 9 2 1 9 5 1 9 5 1 2 4 8 2 1 1 9 1 0 9 8 2 8 2 8 2 7 1 5 3 1 5 7 1 2 4 1 2 4 1 6 2 t s w 0 ,0 1 0 ,1 7 * 0 ,0 9 -0 ,1 5 ** 0 ,1 9 0 ,1 6 0 ,1 5 0 ,2 2 * 0 ,0 6 0 ,1 8 * -0 ,2 3 * 0 ,0 1 0 ,0 9 0 ,2 9 * 0 ,1 0 -0 ,2 8 * -0 ,1 1 0 ,1 7 0 ,0 6 -0 ,0 2 -0 ,0 1 0 ,2 1 * l a r 3 8 0 2 2 9 2 2 9 3 8 2 8 8 7 9 1 1 0 1 1 0 1 1 0 1 5 7 7 7 9 9 8 2 6 0 6 0 6 0 5 3 4 4 1 1 3 9 5 9 5 1 2 1 -0 ,1 1 0 ,1 4 0 ,1 3 0 ,1 1 0 ,0 3 -0 ,0 7 0 ,1 8 0 ,1 1 -0 ,0 9 -0 ,0 4 -0 ,0 4 s r r u rb m to l f va l f re q c a % n k n p k % p % n % 7 6 7 4 5 4 5 4 5 4 5 1 4 4 8 7 7 0 7 0 9 2 s ig n if ic a n c e o f c o e ff ic ie n ts : *, p < 0 .0 5 ; ** , p < 0 .0 1 ; ** *, p < 0 .0 0 1 . 88 jürgen franzaring, andreas fangmeier, roderick hunt 3 2 5 2 10 3 11 7 6 3 4 8 8 3 3 8 3 7 3 r s c percent of csr-signature 6 5 4 5 5 5 4 4 4 4 3 4 4 5 2 5 4 3 4 r s c frequency (medians) in central europe (a) percent of sample within each type (b) median frequency in c. europe 7 7 5 5 7 6 5 6 6 6 5 2 3 2 2 5 3 3 4 r s cmedians of nellenberg per csr-signature 8 8 5 8 7 5 6 5 5 5 5 5 5 5 4 5 6 4 6 r s cmedians of fellenberg per csr-signature 6 4 7 11 3 1 3 1 2 3 1 1 1 5 1 12 3 1 5 r s c mean 1000 seed weight (g) per csr-signature 0,3 0,4 0,4 0,2 0,4 0,4 0,3 0,5 0,4 0,4 0,3 0,2 0,2 0,2 0,1 0,3 0,2 0,3 0,3 r s c mean % dm of foliar p per csr-signature (f) median ellenbergf value for each type (e) median ellenberg n value for each type (c) mean foliar p concentration for each type (% dm) (d) mean 1000-seed weight for each type (g) fig. 1: distribution of plant species within ‘csr-space’: (a) percentages of central european sample of species occupying each type, n = 826; (b) median frequency of csr types within the central european sample, n = 784; (c) mean foliar p concentrations (% dry matter), n = 127; (d) mean thousand-seed weights (g), n = 340; (e) median ellenberg-n values, n = 750; (f) median ellenberg-f values, n = 682. data on frequency of species and on -n and -f indicator values from ellenberg et al., 2001, and on csr types from hunt et al., 2004; data on plant traits form various sources (see text). also show that both sla and rgr are positively correlated with foliar k, but less strongly so with por nconcentration. an association between leaf nitrogen content on a dry weight basis and mean rgr was previously confirmed by cornelissen et al. (1997), and fichtner and schulze (1992) found that the response of rgr to n-supply is strongly and positively correlated with nitrophily. such findings support the theory of poorter et al. (1990), who postulated that natural selection in a nutrient-rich environments favors species with a high specific leaf area, high leaf area ratio and rgr, whereas selection in nutrient-poor habitats leads to species with inherently low specific leaf area, leaf area ratio (lar) and rgr. these morphological and physiological differences are generally paralleled by the differences in chemical composition; slowly-growing species contain relatively less nutrient content but relatively more cell-wall material such as lignin, hemicellulose, cellulose, polysaccharide-bound ferulic acid and hydro-xyproline-rich protein (niemann et al., 1992). csr and ellenberg 89 ellenberg indicator values from tab. 1, an overview of the correlations between plant traits and ellenberg eivs is possible. the highest correlations were found between leaf macronutrients (in the order p > k > n) and the ellenbergn value (an index of affinity to high nutrient conditions). relationships between soil nutrients and ellenberg-n values, which describe the nitrophily of a plant species, have previously been confirmed in comparisons made by rastin (1992), meerts (1997) and schaffers and sýkorá (2000). however, the present correlation between foliar n and ellenberg-n has not previously been reported. the comparisons in fig. 2 c-e show that this relationship is stronger in the sheffield dataset than in those from hautes-vagnes (belgium) or tharandt (germany), perhaps indicating that differences in local land use and nitrogen availability have an impact on the realized niche of species. ellenberg-n also correlates here with rgr, confirming hill et al. (2000) and schaffers and sýkorá (2000), who suggested that ellenberg-n values represented ‘productivity values’. ellenberg-n values are also positively correlated with sla, indicating that plants from resource-rich environments tend to have thin, delicate leaves, while plants from unproductive habitats tend to have physically stronger leaves, also noted by grime et al. (1997). due to the generally more robust conditions, sla values recorded in the field are generally lower than in glasshouse-grown plants, but such comparisons generally show a consistency in ranking across a group of species (poorter and de jong, 1999). ellenberg-r values (indices of affinity to high ph) show highly significant correlations with leaf ca and mg concentrations and a low correlation to thousand-seed weight. schaffers and sýkorá (2000) suggested calling ellenberg-r values ‘calcium values’, an idea which is supported by present study. r-values also correlate with ph of the plant litter (fig. 2 f) and the c:n ratios of green and dead plant material (not shown). it has repeatedly been shown that weighted ellenberg-r values are well correlated with soil ph (diekmann, 2003). this is confirmed by the sheffield data, in which soil ph was measured in the rooting zone (fig. 2 g). however, the positive correlation between tsw and ellenberg-r values cannot be explained. ellenberg-f values (indices of affinity to environmental moisture) show a slight correlation with leaf p and k contents, as well as with rgr and sla, probably indicating a general association between moisture availability and productivity. they are positively associated with the species frequency, indicating correctly that many of the rarer species in central europe are associated with the drier habitats. the slight negative correlation between continentality (ellenberg-k) and nitrogen content is also supported by the gradient in nitrogen content of pine needles observed between central europe and the polar circle (bergmann, 1998). the positive relationship between n:k values and ellenberg-t (affinity to higher temperature) cannot be explained, but the negative correlation between ellenberg-l (affinity to full light conditions) and thousand-seed weight may indicate that under brighter conditions small seeds may have a better prospect of establishing successfully while, in shade, large-seeded species will have an advantage. c-s-r type while rgr and sla were among the plant traits used to originally confirm the c-s-r system of classification (grime et al., 1997), and they again exhibit clear relations to c-s-r position (tab. 1), the associations between c-s-r type and certain other traits have not much been demonstrated. fig. 1 c, d show the mean p concentrations and thousand seed weights for each of the 19 csr strategies represented in the sample. the p contents of plant leaves (fig. 1 c) show a clear trend along the s-axis of c-s-r space, i.e. the highest amounts occur on the ruderal and competitive side of the triangle, along with the highest rgrs and slas, though foliar n does not reproduce this trend so well (data not shown). lower nutrient contents in slow-growing species like the stress-tolerators, and their neighbors in c-s-r space, make it imperative for such types not to lose resources, while fastgrowing species on the c to r margin ruderals may safely rely upon a high nutrient turnover because, even when their short-lived leaves are lost, their nutrient content may generally be reclaimed from the environment. larger seeds are associated with the long-lived competitive species (fig. 1 d), while both ruderals and stress tolerators produce smaller seeds on average, a consequence of short life cycles in the former and low resource availability in the latter. it would have been advantageous to have had data on reproductive allocation to complete this part of the picture, but unfortunately this was not available on the scale required. interrelations between nutrient cycling, plant longevity, morphological and physiological traits have also been demonstrated in australian and north american floras by craine et al. (2002) and wright and westoby (2003), respectively. an evolutionary specialization in the form of a trade-off between gaining and conserving resources was confirmed by díaz et al. (2004) and wright et al. (2004), though reich et al. (2003) found a less clear interrelation between resource supply rate and ecophysiological performance. relations between functional types: csr, ellenberg and klotz indicator values fig. 1 e shows the median ellenberg-n and -f values for plants of each of 19 c-s-r types. the ellenberg-n dimension maps almost completely onto the s-axis of c-s-r space, with only a suggestion of a bias towards c rather than towards r. on the other hand, ellenbergf values very convincingly reflect the c-axis, confirming that while types s and r may often be common in drier habitats; competitive species are able to dominate only at moister sites. from tab. 1, a partial parallel with c-s-r position emerges for ellenberg values for soil reaction (-r); the many calcicole examples of the s-strategy are sufficient to establish a correlation here. another trend emerges within continentality (-k, which in europe is entrained with temperature -t), where s-types are favoured at high -k and -t values, and still another with light (-l), where ruderal types are excluded from closed, dense communities. at the same time, ruderal types emerge as being mowing-tolerant, stress tolerators as mowing intolerant and competitors as mowingindifferent. the klotz urbanity value maps onto all three c-s-r axes, but the way in which this affinity to urban habitats is expressed within c-s-r theory involves a delicate balance of stress, disturbance and eutrophication. surprisingly, no c-s-r correlations emerge for feed value, where the issue of chemical defenses may confound the otherwise simple relationship expected for the s-dimension. csr strategies and plant families six plant families within our central european sample contain 30 or more species which have been classified according to the csr system. the brassicaceae (41 represented) and caryophyllaceae (31 species) are both associated mainly with r-strategies, and the rosaceae (31 species) have a predominantly s-type strategy. however, the 77 species from the asteraceae have strong components in both cand r-types and the 65 poaceae and the 40 leguminosae each span the whole of the c-s-r spectrum. taxonomically-driven trait clusters like those for seed size (moles et al., 2005) are thus possible, but not inevitable. 90 jürgen franzaring, andreas fangmeier, roderick hunt r2 = 0,54 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 n % dm (tharandt) n % dm (sheffield) r2 = 0,69 2 3 4 5 6 7 8 2 3 4 5 6 7 8 ph (soil) sheffield ph (litter) tharandt r2 = 0,26 0 1 2 3 4 5 6 7 0 2 4 6 8 10 n % dm (tharandt) r2 = 0,60 0 1 2 3 4 5 6 7 0 2 4 6 8 10 n% dm (sheffield) r2 = 0,19 0 1 2 3 4 5 6 7 0 2 4 6 8 10 n% dm (haute belgique) ellenberg n r2 = 0,72 0 1 2 3 4 5 6 7 8 9 0 2 4 6 8 10 soil ph ellenberg r r2 = 0,17 0 1 2 3 4 5 6 7 8 9 0 2 4 6 8 10 ellenberg r ph (litter) (c) % n in dm (tharandt) (d) % n in dm (sheffield) (e) % n in dm (haute belgique) (f) litter ph (tharandt) (g) soil ph (sheffield) (a) % n in dm (tharandt) (b) litter ph (tharandt) % n in dm (sheffield) soil ph (sheffield) ellenberg-n ellenberg-r ellenberg-r fig. 2: (a) relationship between foliar n concentration in species collected from local floras at tharandt (se-germany) and sheffield (uk), n = 19; (b) relationship between ph of plant litter at tharandt and surface-soil ph at sheffield, n = 15 species; relationship between foliar n concentrations and ellenberg-n values in plant species collected in (c) tharandt (n = 44), (d) sheffield (n = 60), and (e) hautes vagnes, belgium (n = 64); (f) relationship between litter ph and ellenberg-r values for species collected at tharandt (n = 34) and (g) between soil ph and ellenberg-r values for species collected at sheffield (n = 43). tharandt data from höhne (1962) and sheffield data from unit of comparative plant ecology (ucpe, download under http:// www.shef.ac.uk/uni/academic/n-q/nuocpe/ucpe/); belgian data on nutrient contents from duvigneaud and denaeyer-de smet (1970). csr and ellenberg 91 conclusion our interpretation of existing plant ecological information confirms a high degree of congruence between many of these ecological indicator values and the established c-s-r strategies of european plant species. the c-s-r axis which has repeatedly emerged most strongly from objective syntheses of plant trait values is the s-dimension (grime et al., 1988; 1997; díaz et al., 2003; grime et al., 2005). this is the axis most strongly correlated here (in number and in significance of coefficients) with indices from the ellenberg and klotz classifications and with measured plant traits. the r-k classification of macarthur and wilson (1967) successfully recognized (in its r-strategy) what would later be postulated as the r-dimension within c-s-r, but did not further differentiate the cand svariants embedded within the k-strategy. in a similar manner, many of the continental european ecological indices, and individual trait axes, represent single c-s-r dimensions but fail to distinguish between the others. for example, ellenberg-n (nitrogen regime) and foliar-p both map clearly onto the s-dimension (fig. 1 c, f) but fail to distinguish rand c-types; and ellenberg-f (water regime) correlates well with the c-dimension (fig. 1 f) which, in a continent of predominantly mesic habitats, equates very successfully to median frequency (fig. 1 b). in general, the interrelations reported here were most prominent among measures of plant productivity and nutrient ecology: the s-axis of the c-s-r system is revealed in twelve alternative guises here. there is thus a clear congruence of classifications at work, with the same underlying phenomenon being described from a number of different directions. this phenomenon, recently discussed by díaz et al. (2004), again points towards a continuum of evolutionary specialization that ranges on the one hand between trait-groups that confer rapid acquisition of resources and, on the other hand, those that confer longterm resource conservation. the c-s-r classification of plant strategies, with its reliance upon two great drivers of success in the established phase (degree of resource-limitation and degree of biomass destruction), can thus be regarded as a parsimonious synthesis of plants’ evolutionary responses to the natural environment and hence, of their capacity to respond to biotic and abiotic pressures within ecological timescales. the centraleuropean ecological indicator approach is also capable of interpreting many aspects of environmental change and it also reinforces many of the most fundamental tenets from the csr approach. references apel, c., 2002: samen von wildpflanzen. conrad apel gmbh, darmstadt. ashenden, t.w., hunt, r., bell, s.a., williams, t.g., mann, a., booth, r.e., 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<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> /sve <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> >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice vorgabe neu journal of applied botany and food quality 81, 49 55 (2007) 1nees-institute for biodiversity of plants, university of bonn, germany 2institute for crop science and resource conservation (inres – phytomedicine), university of bonn, germany efficiency of self-cleaning properties in wheat (triticum aestivum l.) a.k. stosch1, a. solga1, u. steiner2, e.-c. oerke2, w. barthlott1, z. cerman1 (received february 26, 2007) summary an experimental study was carried out to assess the efficiency of self-cleaning properties of three wheat cultivars and their potential in the protection against blumeria graminis f. sp. tritici, a fungus that causes powdery mildew. leaf samples with intact epicuticular structure were compared to such with wiped wax crystals. contact angles were determined and the surfaces were subjected to a standardized contamination test with hydrophobic fluorescence powder. another set of samples was inoculated with conidia of b. graminis and, after various time intervals, exposed to artificial fog or rain. for the intact surfaces of all cultivars contact angles of about 165° were measured. it is therefore suggested that wheat should be termed superhydrophobic. the wiping of the wax crystals led to a significant decrease of contact angles. this fact underlines the importance of surface roughness for achieving extreme water-repellency. in the standardized contamination test significantly more particles remained on the wiped surfaces than on those who had been left intact. this result was ascribed to increased adhesion on the smoothed samples. the inoculation with subsequent precipitation revealed a significantly better removal effect of conidia from intact than from wiped surfaces. this was irrespective of the wheat cultivar. in general, conidia were more effectively removed by rain than by fog. this was probably due to the higher kinetic energy and the greater amount of water when using rain. if fog application was delayed by 3 hours a higher percentage of conidia remained on the surface. as possible causes are discussed increased adhesion by conidia secretions or the development of primary germ tubes. despite its highly efficient self-cleaning properties proved here, wheat is frequently infected by blumeria graminis. we conclude that the high water content of the mildew conidia, the ability of blumeria graminis to germinate at very low humidities and its rapid irreversible adhesion are effective adaptations in order to overcome the barrier of a superhydrophobic self-cleaning surface. introduction most higher plant species from major systematic groups do not have smooth but rough, microand sometimes also nanostructured surfaces. for the description of the surface characters four different categories are usually distinguished (cf. barthlott, 1981): 1. the cellular pattern, 2. the shape of the epidermal cells, 3. the relief of outer cell walls, 4. superimposed epicuticular secretions. the latter are termed epicuticular waxes and occur in a remarkable variety (e.g. jeffree, 1986; barthlott et al., 1998). microand nanostructures of plant surfaces have several functions which, so far, only partially have been proved while others are still being discussed. they affect resistance to uv radiation and probably have an impact on the air flow at the surface (barthlott and wollenweber, 1981; pfündel et al., 2006). furthermore, the structures change the degree of wettability and make a surface either more hydrophilic or more waterrepellent. the combination of a microstructure and superimposed hydrophobic epicuticular wax crystals leads to extreme waterrepellency with contact angles exceeding 150° (‘superhydrophobicity’, cf. marmur, 2004). examples of common plant species with such extremely water-repellent surfaces are nasturtium (tropaeolum majus), lily of the valley (convallaria majalis) and the sacred lotus (nelumbo nucifera). the superhydrophobicity is linked with an amazing phenomenon: the surfaces are self-cleaning, i.e. various contaminants as well as pathogenic microorganisms are washed away by water droplets rolling off (schwab et al., 1995; barthlott and neinhuis, 1997). regarding the colonization of pathogens the superhydrophobicity has an additional effect: as the availability of water on the surface is strongly reduced, the majority of spores is unable to germinate. however, this holds not for some specialists causing powdery mildew (juniper, 1991). wheat plays a vital role as food, feed and, in the future, probably also as an energy source. with an annual production of about 550 million tonnes the cereal is among the three most important crops worldwide (fao, 2004). moreover, it is the staple for nearly 40% of the world’s population (wiese, 1991). a large portion of the wheat harvest is destroyed by pests and diseases every year. for the period from 1988 to 1990 the damage caused by those factors has been estimated at 244 billion us-dollars (oerke et al., 1994; agrios, 1997). from the economical point of view, powdery mildew of wheat is one of the most important diseases (braun, 1995). powdery mildews are capable to infect wheat despite the strong waterrepellency of its surface. this water-repellency is caused by a dense cover of wax crystals which are predominantly flat (‘platelets’, barthlott et al., 1998) but which, depending on the part of the plant and the stage of growth, also occur as tubules and rodlets (troughton and hall, 1967). consequences of a mildew infection for the host are nutrient deficiency, a reduction of photosynthesis and an increase of respiration and transpiration. yield losses up to 40% have been recorded (wiese, 1991). in the majority of studies on the initial stages of mildew infection conducted yet the focus was on specific adaptations of the pathogen enabling it to stick rapidly to a surface and to colonize the host (kunoh, 1981; carver et al., 1995a; carver et al., 1995b; carver et al., 1996; carver et al., 1999). on the other hand, relatively few studies gave priority to the properties of the host surface preventing it from mildew formation and to environmental parameters influencing the colonization success of the pathogen (e.g. merchán and kranz, 1986b; merchán and kranz, 1986a; holthuis et al., 1990). the results of these studies indicate that precipitation is a key factor in removing conidia from the wheat surface and hence in reducing powdery mildew infection. however, no survey which has been carried out so far focused on the potential self-cleaning properties of wheat and the evaluation of their efficiency. the present experimental study attempts to fill this gap: a) for the first time, it investigates whether wheat can be considered as superhydrophobic and self-cleaning; b) it attempts to elucidate the relationship between the epicuticular structure of wheat as the outermost line of defence and the plant’s susceptibility to mildew infection; c) it examines the effect of time between deposition and precipitation on the success of a powdery mildew in colonizing the wheat surface. we confined our investigations to the physical aspects of superhydrophobicity and self-cleaning properties, physiological processes were not taken into consideration. in order to demonstrate independence of hydrophobicity phenomena on plant surface from resistance due to specific genes, wheat cultivars both with and without resistance genes were used in the tests. material and methods biological material the experiments were conducted with the three winter wheat cultivars kanzler, kris and ludwig. cultivar kris is known to have the resistance genes pm2 and pm6 against powdery mildew, whereas kanzler and ludwig lack such genes (bundessortenamt, 2002). the wheat seedlings were grown in 9x9 cm pots containing organic soil (klasmann-deilmann, geeste-groß hesepe, germany) in a greenhouse at 20-23°c and 70-80% relative humidity. a photoperiod of 16 h (>300 µe m-2 s-1) per day was ensured by supplementary lights (philips sgr 140, hamburg, germany). pots were watered daily with tap water avoiding water splashes to wet the plants. for all tests the second leaves of 14-days-old seedlings (growth stage gs 12) were used. the obligate, host-specific and ubiquitous parasite blumeria graminis (dc.) speer form species tritici em. marchal was chosen as the pathogen in all inoculation experiments. the fungus was maintained on wheat (triticum aestivum cv. kanzler). since only young and vigorous conidia should be used in the tests, host plants were shaken at the day before conidia harvest. in this way, old conidia were removed from the plants and a harvest of a homogeneous inoculum was ensured (cf. mount and slesinski, 1971). preparation and treatment of samples both for the standardized contamination test and for the inoculation with mildew conidia wheat leaves with intact wax crystals and such with destroyed epicuticular structure were required. the latter were obtained by wiping the leaf surfaces with cotton (cf. schwab et al., 1995). though the wax crystals were destroyed by this method the surface chemistry was not affected because a thin amorphous wax film remained on the leaves. the success of the procedure was controlled by scanning electron microscopy (sem, fig. 1). below, surfaces that have been wiped will be called ‘wiped surfaces’. as preparation for the processes of contamination, inoculation and microscopical investigation of wheat surfaces, pieces of 1x2 cm were cut off the leaves and were fixed on sem aluminium stubs with double-sided adhesive tape. a standardized contamination test was performed to assess the selfcleaning properties of the leaf surfaces (fürstner, 2002; fürstner et al., 2005). five samples with intact epicuticular structure and five with wiped crystals of each cultivar were put in a contamination chamber. furthermore, four sem-stubs with conducting tabs (w. plannet gmbh, wetzlar, germany) were added which served as reference and which were required for the determination of the density of deposited particles. in the contamination chamber 0.5 g of a hydrophobic fluorescence powder (osram leuchstoff f20, osram gmbh, schwabmünchen, germany) were dispersed with a ventilator. the particle size of the powder varied between 2 and 30 µm. after a sedimentation period of 60 minutes, the stubs were taken out and all except the reference stubs were moved to a fog chamber. in this chamber they were arranged on a plate inclined 45°. a fine mist of deionized water with droplet sizes between 8 and 20 µm, produced by a high pressure fog system (osberma, engelskirchen-osberghausen, germany), was injected into the chamber. the process took 5 minutes, corresponding to a precipitation amount of 1.5 mm. after the treatment the samples remained in the chamber for 60 minutes and were subsequently dried in an oven. the inoculation with conidia was carried out in a home-made inoculation chamber. four samples with intact epicuticular structure, four with wiped crystals and four sem-stubs with conducting tabs were randomly placed on a plate at the bottom of the chamber. the latter were required to monitor inoculum density. freshly harvested conidia were put in a dish close to the top of the apparatus and were dispersed by an air blast. afterwards the samples were left in the chamber until further processing. the inoculation was follwed by an exposition of the leaf samples either to a fine mist or to artificial rain. the fog treatment has already been described above. for simulation of rain the method described by fürstner (2002) was applied. in this procedure each sample was sprinkled with 50 ml deionized water which fell from a container furnished with 24 cannulas and installed 15 cm above the sample holder. the droplet size of the artificial rain was approximately 2 mm. in different experimental series the treatment with fog and that with artificial rain were carried out one, two and three hours after inoculation, respectively. sample analysis in order to determine wettability, static contact angles were measured. for this purpose, samples of 1x3 cm were cut off the leaves and were fixed on glass slides, using double-sided adhesive tape. the measurement was conducted with a contact angle meter (oca 30, dataphysics instruments gmbh, filderstadt, germany), using 15µl droplets and the young-laplace-fit. prior to further examination by scanning fig. 1: sem images of triticum aestivum cv. kanzler leaf surfaces. left: wax crystals intact, right: amorphous wax film after wiping. 50 a.k. stosch, a. solga, u. steiner, e.-c. oerke, w. barthlott, z. cerman kanzler kris ludwig n u m b e r o f co n id ia 0 200 400 600 800 1000 1200 1400 1600 1800 2000 b) electron microscopy, all samples were coated with gold in a sputter coater (balzer union scd 040, balzer-pfeifer gmbh, asslar, germany). the samples of the standardized contamination test were investigated in a cambridge stereoscan 200 (leica, bensheim, germany) coupled with a cathodoluminescence-detector. the fluorescence images taken by this method were analyzed with the program scion image (scion corporation, maryland, usa). then the coverage of the plant samples with particles relative to that of the reference stubs was calculated. the samples of the inoculation with conidia were examined in a leo 440i sem (carl zeiss smt, oberkochen, germany). ten sem micrographs were taken of each sample and the total number of conidia displayed in each micrograph was counted. data analysis differences between pairs of means were investigated by using student’s t-test. if normality and homogenity of variances were not given and could not be achieved by data transformation, the nonparametric mann-whitney u-test was applied. two-way analyses of variance were carried out to test the influence of the factors wheat cultivar and epicuticular structure on the variable number of conidia. in order to meet the assumptions of anova or at least to improve homocedasticity, three different transformations were utilized: log x, log x+1 and 1/x. the effect of time between conidia deposition and precipitation (fog, rain) on the penetration success with intact wheat surfaces was studied by calculating the ratio of remaining conidia to the amount of conidia on the reference stubs. afterwards multiple comparison tests (tukey hsd) were conducted to reveal differences between the time intervals (1, 2 and 3 hours). all statistical analysis was carried out with spss 12.0, graphs were created with sigmaplot 9.0 (both spss science, chicago, usa). results surface wettability contact angles around 165° were determined for the intact surfaces of all three cultivars (tab. 1). the contact angles of the samples with wiped epicuticular waxes ranged from 105° to 110°. variability was distinctly higher with the wiped surfaces. the difference between intact and disturbed structure was always highly significant (p < 0.001). tab. 1: surface wettability of the three wheat cultivars with intact and wiped wax crystals. means of 15 measurements with single standard deviation. contact angles [°] wheat cultivar waxes intact waxes wiped kanzler 165.8 ± 3.5 109.7 ± 6.5 kris 166.8 ± 2.8 105.6 ± 8.4 ludwig 163.0 ± 2.9 106.2 ± 6.2 standardized contamination test the analysis of the fluorescence images revealed relative coverages with particles below 1% for the surfaces with intact and around 10% for the samples with disturbed structure, respectively (tab. 2). in all three cultivars differences between the samples with intact and those with wiped waxes were highly significant (p < 0.001). on the other hand, differences in coverage between the cultivars were negligible. tab. 2: coverage of three wheat cultivars with fluorescence powder particles. comparison between surfaces with intact and wiped wax crystals. means of five replicates with single standard deviation. relative coverage [%] wheat cultivar waxes intact waxes wiped kanzler 0.7 ± 0.6 11.9 ± 3.6 kris 0.3 ± 0.2 10.4 ± 2.2 ludwig 0.9 ± 0.7 10.9 ± 1.9 effect of simulated fog after inoculation and fog treatment the number of conidia on the surfaces with wiped wax crystals was always many times higher than that on intact wax structure (fig. 2 a-c). the relationship between the surface structure and the number of conidia was significant irrespective of the interval between inoculation and artificial precipitation (tab. 3). no influence of the cultivar on the number of remaining conidia could be detected which reflects the similar coverage of the different cultivars. again, this held for all intervalls between inoculation and fog treatment. kanzler kris ludwig n u m b e r o f co n id ia 0 200 400 600 800 1000 1200 1400 1600 1800 2000 a) self-cleaning properties in wheat 51 kanzler kris ludwig n u m b e r o f co n id ia 0 20 40 60 80 100 a) kanzler kris ludwig n u m b e r o f co n id ia 0 200 400 600 800 1000 1200 1400 1600 1800 2000 c) fig. 2: number of blumeria graminis conidia remaining on the surfaces of three wheat cultivars with intact (open bars) and wiped (filled bars) wax crystals after inoculation and fog treatment. intervals between inoculation and fog treatment: a) 1 hour, b) 2 hours, c) 3 hours. means of four replicates with single standard deviation. tab. 3: results of the two-way anovas: effects on the variable number of conidia of the factors epicuticular structure and wheat cultivar and their interaction. three different time intervals. *: p < 0.05, ***: p < 0.001, n.s. = not significant. interval structure cultivar structure x cultivar 1 h *** n.s. * 2 h *** n.s. n.s. 3 h *** n.s. n.s. were detected when fog treatment was performed two or three hours after inoculation as compared to a treatment already after one hour. effect of simulated rain since no remarkable difference between the two and the three hours interval in the fog treatment was found, the two hours interval was omitted in subsequent experiments. thus, artificial rain was applied only one and three hours after inoculation. in general, artificial rain produced a much higher reduction of conidia on the surfaces than fog (fig. 4 a-b, notice the different scales of the y-axes). as with the fog treatment, significantly more conidia were found on wiped than on intact surfaces (tab. 4), and no relationship between the wheat cultivar and the number of remaining conidia was detectable. kanzler kris ludwig n u m b e r o f co n id ia 0 200 400 600 800 1000 1200 1400 1600 1800 2000 c) fig. 3: percentage of blumeria graminis conidia remaining on the intact surfaces of three wheat cultivars after inoculation and fog treatment. intervals between inoculation and fog treatment: 1 hour (open bars), two hours (filled bars), three hours (hatched bars). means of four replicates with single standard deviation. bars sharing the same letter are not significantly different (p < 0.05). fig. 3 compares the relative coverages of the intact surfaces that have been treated with fog one, two and three hours after inoculation, respectively. no significant differences were found for the cultivar kanzler. in contrast, on kris and ludwig significantly more conidia kanzler kris ludwig p e rc e n ta g e o f co n id ia 0 5 10 15 20 a a a m n n x y y fig. 4: number of conidia remaining on the surfaces of three wheat cultivars with intact (open bars) and wiped (filled bars) wax crystals after inoculation and rain treatment. intervals between inoculation and rain treatment: a) 1 hour, b) 3 hours. means of four replicates with single standard deviation. tab. 4: results of the two-way anovas: effects on the variable number of conidia of the factors epicuticular structure, wheat cultivar and their interaction. two different time intervals. ***: p < 0.001, n.s. = not significant. interval structure cultivar structure x cultivar 1 h *** n.s. n.s. 3 h *** n.s. n.s. kanzler kris ludwig n u m b e r o f co n id ia 0 20 40 60 80 100 b) 52 a.k. stosch, a. solga, u. steiner, e.-c. oerke, w. barthlott, z. cerman fig. 5: percentages of blumeria graminis conidia remaining on the intact surfaces of three wheat cultivars after inoculation and rain treatment. intervals between inoculation and rain treatment: 1 hour (open bars), three hours (hatched bars). means of four replicates with single standard deviation. bars sharing the same letter are not significantly different (p < 0.05). the percentages of conidia remaining on the intact surfaces after artificial rain were several times lower than after fog treatment (fig. 5). moreover, none of the three cultivars showed significant differences between the one and the three hours interval. discussion the formation of microand nanostructured superhydrophobic surfaces is only one among several options for plants to defend themselves against pathogens. eliminating spores before they stick and germinate has the advantage that no further physiological efforts to resist a pathogen attack have to be made. the first question was whether intact wheat surfaces can be termed superhydrophobic and self-cleaning. taking into account recent studies on the topic wetting and non-wetting (e.g. quéré, 2002; yoshimitsu et al., 2002; marmur, 2006), the contact angles of about 165° determined for all cultivars clearly match the criterium of superhydrophobicity. the values correspond to those reported in earlier studies on wheat and its surface properties (troughton and hall, 1967; holloway, 1969). the significant decrease of contact angles after wiping points to the indispensability of surface roughness and heterogenous wetting with air in the cavities under droplets for the purpose of achieving extraordinarily high contact angles (cf. dettre and johnson, 1964). the enhanced variation found for the wiped surfaces is probably due to surface heterogenity caused by the treatment. values around 110° were also found by holloway (1969) who determined contact angles on smooth films of isolated wheat waxes on glass. furthermore, it has been suggested that contact angles in this order of magnitude occur only if wax is involved at the cuticle surface (holloway, 1970). in addition to the visual documentation (fig. 1), this confirms that the wiping process in fact only destroyed the epicuticular structure and that it did not completely remove the wax layer. since particles were almost completely removed from the intact wheat surfaces in the standardized contamination test, it is justified to add triticum aestivum the the group of plants with self-cleaning leaf surfaces. the elimination of particles was even more complete than in lotus (nelumbo nucifera), the classic example for self-cleaning plants (cf. barthlott and neinhuis, 1997). this can be explained by considering the differences in surface sculpture between lotus and wheat: the leaves of lotus are papillose on the micrometer scale and the papillae are additionally covered with epicuticular wax crystals (barthlott and neinhuis, 1997; wagner et al., 2003). the wheat surface, on the other hand, consists of elongate, slightly convex cells with a dense layer of wax platelets (carver et al., 1995b; koch et al., 2006). as a consequence, if small particles on lotus leaves are not reached and picked up by droplets with high kinetic energy they remain between the epidermal papillae. in contrast to this, on wheat leaves even small particles lie always on top of the wax layer and can be removed even by very fine droplets with low kinetic energy. the 10-30 times higher coverage of the samples with wiped wax crystals after fog treatment is probably due to increased adhesive forces between smoothed wax films and deposited particles (burton and bhushan, 2006). furthermore, it is likely that lump-like wax accumulations produced by wiping prevented particles from getting removed. the results of the inoculation with blumeria graminis conidia and subsequent fog treatment agree well with the findings of the standardized contamination test. again, the high efficiency of the self-cleaning mechanism in case of an intact epicuticular structure was apparent. significantly less conidia on the wheat leaves with intact structure as compared to those with partially removed waxes have also been observed by merchán and kranz (1986b). as expected, artificial rain generally washed off much more conidia than fog. it is suggested that both the higher kinetic energy of the larger rain droplets and the amount of water which was about 150 times higher in the rain treatment led to this result. the stronger impact of rain in comparison to the fine mist probably also caused the less distinct differences between intact and disturbed surfaces (fig. 4 a-b). the fact that neither in the fog nor in the rain treatment wheat cultivars had an influence on the results indicates similarity of surface texture within the experimental sets. the third aspect examined in this study was whether the amount of time passing from deposition of conidia on intact wheat surfaces to the start of artificial precipitation has an influence on the colonization success of blumeria graminis. the significant relationship that was revealed in the fog treatment for the cultivars kris and ludwig and the tendency found for the cultivar kanzler can be explained by initial processes that start right after the first contact between conidium and surface. within the first few minutes the production of extracellular material (ecm) including proteins begins (kunoh et al., 1988; kunoh et al., 1990; nicholson and kunoh, 1995). 10 to 15 minutes after initial contact of the conidium with the surface a second phase of ecm release can be observed: cutinases are exuded which dissolve the wheat cuticle (pascholati et al., 1992; meguro et al., 2001). 45-50 minutes later the first primary germ tubes (pgts) are formed, followed by secondary germ tubes emerging after 2-3 hours (carver et al., 1995a; green et al., 2002). the development of appressoria starts not until 8-9 hours and is therefore of minor importance here (cf. carver et al., 1995b; nicholson, 1996). considering these first steps of the infection process it is suggested that sticking by ecm and/or the growth of pgts caused increased adhesion which resulted in significantly higher percentages of remaining conidia on kris and ludwig (see also nicholson and epstein, 1991). the development of pgts had also been observed in the sem while counting remaining conidia. in the rain treatment no significant differences could be established. this result is most likely due to the generally much more effective removal of conidia by rain than by fog (see above). referring to holthuis et al. (1990), a substantial washing-off with rain is to be expected until the full development of appressoria. in the present study no differences between the cultivar with resistance genes and the two cultivars without such genes could be observed. it is likely that the occurrence of resistance genes has an impact only kanzler kris ludwig p e rc e n ta g e o f co n id ia 0 5 10 15 20 a a m m x x self-cleaning properties in wheat 53 in later stages of the infection process and that the integrity and functional efficiency of the surface microstructure is the decisive factor within the first hours after conidia deposition. even though wheat can have different types of epicuticular waxes (troughton and hall, 1967), the shape of the latter should be of minor importance for the self-cleaning efficiency (cf. barthlott and neinhuis, 1997). it could be shown that the epicuticular waxes of wheat provide an efficient protection against the attachment of particles and conidia contamination. however, despite this effective self-cleaning mechanism proved here, fungi causing powdery mildew like blumeria graminis are pathogens with a worldwide distribution and a strong impact on the global wheat harvest. different reasons may account for this discrepancy. first of all, it is unlikely that the epicuticular waxes in a wheat canopy are completely intact. for example, rubbing of plants, mechanical impact by rain droplets and kinks of leaves and culms cause wax erosion and provide spots at which pathogens can start their attack (troughton and hall, 1967; wilson, 1984; baker and hunt, 1986; cleugh et al., 1998). it has to be mentioned that such surface defects are partly repaired by regeneration processes (cf. wolter et al., 1988; neinhuis et al., 2001; koch et al., 2004). secondly, there is evidence that surfactant-containing agrochemicals alterate the wax fine structure of wheat and other crops (wolter et al., 1988; noga et al., 1991). as shown in experiments with brassica oleracea (neinhuis et al., 1992), such an alteration of micromorphology reduces the self-cleaning ability of the surface and thus facilitates powdery mildews to adhere to the plant surface. in addition to agrochemicals also airborne pollutants can cause a degeneration of epicuticular waxes (turunen and huttunen, 1990). thirdly, if precipitation occurs mainly as fog or dew with low kinetic energy, the interval between the deposition of a conidium and the precipitation event must be very short. as already discussed above, conidia of blumeria graminis adhere rapidly after their first contact with the surface. in consequence, their removal is only possible when free water is available without delay. moreover, it has been demonstrated that blumeria graminis stores considerable quantities of water in its spores (yarwood, 1950). this property enables the mildew to germinate at low relative humidity (yarwood, 1978) without the presence of free water, i.e. under conditions that are unfavourable for most other fungal diseases (harrison et al., 1994). it is therefore suggested that pathogens causing powdery mildew have evolved a high degree of adaptation and that they have overcome the barrier of a superhydrophobic self-cleaning surface. a disturbance of the epicuticular structure does not inevitably increase mildew infestation. recent studies suggest that particularly the epicuticular waxes provide chemical (carver et al., 1996) and/or topographical (wynn, 1981) information which stimulates fungal development. acknowledgements we thank the university of bonn for funding this study as part of the interdisciplinary research projekt prior. mareike rüdinger kindly assisted with the experiments. günter rebing suggested stylistic improvements in the english version. references agrios, g.n., 1997: plant pathology. academic press, san diego. baker, e.a., hunt, g.m., 1986: erosion of waxes from leaf surfaces by simulated rain. new phytol. 102, 161-173. barthlott, w., 1981: epidermal and seed surface characters of plants: systematic applicability and some evolutionary aspects. nord. j. bot. 1, 345-355. barthlott, w., neinhuis, c., 1997: purity of the sacred lotus, or escape from contamination in biological surfaces. planta 202, 1-8. barthlott, w., neinhuis, c., cutler, d., ditsch, f., meusel, i., theisen, i., wilhelmi, h., 1998: classification and terminology of plant epicuticular waxes. bot. j. linn. soc. 126, 237-260. barthlott, w., wollenweber, e., 1981: zur feinstruktur, chemie und taxonomischen signifikanz epicuticularer wachse und ähnlicher sekrete. trop. subtrop. pflanzenwelt 32, 7-67. braun, u., 1995: the powdery mildews (erysiphales) of europe. gustav fischer verlag, jena. bundessortenamt, 2002: beschreibende sortenliste getreide, mais, ölfrüchte, leguminosen (großkörnig), hackfrüchte (außer kartoffeln). deutscher landwirtschaftsverlag, hannover. burton, z., bhushan, b., 2006: surface characterization and adhesion and friction properties of hydrophobic leaf surfaces. ultramicroscopy 106, 709-719. carver, t.l.w., ingerson, s.m., thomas, b.j., 1996: influences of host surface features on development of erysiphe graminis and erysiphe pisi. in: kerstiens, g. 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(eds.), biology of the plant cuticle, 216-249. blackwell publishing, oxford. quéré, d., 2002: fakir droplets. nat. mater. 1, 14-15. schwab, m., noga, g., barthlott, w., 1995: bedeutung der epicuticularwachse für die pathogenabwehr am beispiel von botrytis cinereainfektionen bei kohlrabi und erbse. gartenbauwissenschaft 60, 102-109. troughton, j.h., hall, d.m., 1967: extracuticular wax and contact angle measurements on wheat (triticum vulgare l.). aust. j. biol. sci. 20, 509525. turunen, m., huttunen, s., 1990: a review of the response of epicuticular wax of conifer needles to air pollution. j. env. qual. 19, 35-45. wagner, p., fürstner, r., barthlott, w., neinhuis, c., 2003: quantitative assessment to the structural basis of water repellency in natural and technical surfaces. j. exp. bot. 54, 1295-1303. wiese, m.v., 1991: compendium of wheat diseases. aps press, st. paul (minnesota). wilson, j., 1984: microscopic features of wind damage to leaves of acer pseudoplatanus l. ann. bot. 53, 73-82. wolter, m., barthlott, w., knoche, m., noga, g., 1988: concentration effects and regeneration of epicuticular waxes after treatment with triton x-100 surfactant. angew. bot. 62, 53-62. wynn, w.k., 1981: tropic and taxic responses of pathogens to plants. annu. rev. phytopathol. 19, 237-255. yarwood, c.e., 1950: water content of fungus spores. am. j. bot. 37, 636639. yarwood, c.e., 1978: water and the infection process. in: kozlowski, t.t. (ed.), water and plant disease, 141-173. academic press, new york. yoshimitsu, z., nakajima, a., watanabe, t., hashimoto, k., 2002: effects of surface structure on the hydrophobicity and sliding behavior of water droplets. langmuir 18, 5818-5822. address of the authors: anne kathrin stosch, dr. andreas solga, prof. dr. wilhelm barthlott and zdenek cerman (corresponding author), nees-institute for biodiversity of plants, university of bonn, meckenheimer allee 170, d-53115 bonn, germany. hd dr. ulrike steiner and pd dr. erich-christian oerke, institute for crop science and resource conservation (inres – phytomedicine), university of bonn, nussallee 9, d-53115 bonn, germany. self-cleaning properties in wheat 55 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) 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<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> >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice journal of applied botany and food quality 95, 121 128 (2022), doi:10.5073/jabfq.2022.095.016 1hungarian department of biology and ecology, babes-bolyai university, cluj-napoca, romania 2department of horticulture, sapientia hungarian university of transylvania, targu mures, romania enhancement of biomass production, salinity tolerance and nutraceutical content of spinach (spinacia oleracea l.) with the cuticular wax constituent triacontanol bernat tompa1, janos balint2, laszlo fodorpataki1,2* (submitted: october 10, 2021; accepted: july 19, 2022) * corresponding author summary the present study investigates the effect of foliar application of tri acontanol (tria) on various physiological parameters with regard to crop yield and quality attributes of spinach (spinacia oleracea l.) under normal growth conditions and salinity stress. plantlets were grown for 21 days in perlite-containing pots supplemented with hoagland’s nutrient solution, then they were subjected to 0 (control) or 150 mm nacl. two concentrations of tria (25 nm and 1 μm) were applied during seed germination and as foliar spray treatment, in itself or simultaneously with salt stress at 4-day intervals for three weeks. exogenous application of tria enhanced germination ener gy and capacity, as well as shoot and root biomass of young spinach plants. inhibition of net co2 assimilation (pn) and of potential quantum yield of photosystem ii (fv/fm), caused by salt stress, was significantly reduced by treatment with triacontanol. increment of carotenoid pigment and ascorbate (vitamin c) content, as well as reduction of membrane lipid peroxidation due to triacontanol treatment improves the health-promoting quality of spinach leaves developed under high salinity conditions. the presented results offer a novel solution for optimization of spinach cultivation on soils affected by high salinity, as well as for an increased content of health-promoting metabolites of spinach leaves upon human consumption. introduction plant growth regulators (pgrs) are widely used in modern agriculture and horticulture. approximately 40 active ingredients are in use and appropriate combination/concentration of these may significantly improve the overall plant growth, yield and quality (rademacher, 2015). pgrs include naturally occurring plant growth substances (translocatable phytohormones or local bioregulators) and synthetic compounds, which mostly are chemical analogs that alter hormone levels (hormone releasing agents or synthesis inhibitors) or hormone sensitivity (hajam et al., 2017). exogenous application of pgrs provides an alternative approach to counteract the adverse effects of abiotic stresses including high salinity (khanam and mohammad, 2018), heavy metal toxicity (asgher et al., 2015) or drought stress (ullah et al., 2012), and increase stress tolerance in plants by regulating gene expression and a number of physiological-biochemical processes (upreti and sharma, 2016). conventional plant breeding methods for improving plant tolerance to abiotic stress are time consuming, laborious, costly and dependent on existing genetic variability, while application of pgrs can be a wise strategy to maximize plant productivity under adverse growth conditions. the most suitable and efficacious technique is the foliar application of diluted aqueous solutions of pgrs for optimization of genetic potential of a crop. it is expected that the need to raise agricultural production will lead to the increased use of pgrs. out of various pgrs, triacontanol (tria) is a natural constituent of plant epicuticular waxes, which can exert its stimulatory effects at considerably low concentrations. due to its non-toxic and not-polluting properties, tria is known to be a potential plant growth stimulator of many agricultural and horticultural crops (naeem et al., 2012). the exogenously applied tria has been reported to enhance vege tative growth (sarwar, 2017), water and mineral nutrients uptake (asadi karam et al., 2017a), accumulation and synthesis of compatible organic compounds (shahbaz et al., 2013). tria modulates hormonal balance (pang et al., 2020), nitrogen assimilation (naeem et al., 2009) and activities of antioxidant enzymes (asadi karam et al., 2017b), leading to the enhancement of growth and quality characteristics of crops. owing to the high-efficiency and broadspectrum of tria, it plays an important role in plant growth and development, but mechanisms by which tria exerts its effects remain unelucidated. furthermore, tria may play a crucial role in improvement the plant tolerance against several abiotic stresses such as transplantation shock, heavy metal toxicity or drought, and it can help ameliorate the harmful effects of different adverse growth conditions (zaid et al., 2020). salt stress in one of the major abiotic factors limiting production and quality of food crops worldwide. approximately 20% of total cropland and 33% of irrigated agricultural land are affected by salinity. it is expected that by 2050, half of the croplands worldwide will become salinized and world population will surpass 9.5 billion people (uri, 2018). consequently, the world population will not have sufficient fertile soils to grow the food it requires, thus there is a strong need for more productive and stress-tolerant crops in the globally shrinking agricultural lands. soil salinization is accelerating as a result of in trusion of water contaminated with salt ions (poor drainage), excess irrigation and fertilization practices, soil erosion (deforestation) on large scale, and slow natural salt leaching processes (moreira barradas et al., 2015; singh, 2015; cuevas et al., 2019). salt stress manifests in physiological drought, which reduces turgor pressure leading to reduction in the cell expansion and growth. high salinity is known to inhibit plant growth mainly due to osmotic stress in a first phase (rasool et al., 2013), toxicity of sodium and chlorine ions on a longer time scale of exposure (hasanuzzaman et al., 2013), and hormonal imbalance (farooq et al., 2015). the combination of these factors is responsible for the oxidative stress caused by over-accumulation of reactive oxygen species (ros). excess production of the extremely unstable ros results in disruption of membrane lipids via peroxidation of unsaturated fatty acids, which causes production of highly reactive lipid peroxidation-derived mole cules such as 4-hydroxy-2-nonenal, 4-hydroxy-2-hexenal, malondialdehyde and acrolein. these unstable molecules are named reactive carbonyl species (rcs) and high concentrations of rcs can cause irreversible damage in plant cells, which ultimately leads to cell death (yalcinkaya et al., 2019). to scavenge ros, plants have evolved a highly integrated defense system consisting of several antioxidants, which provide protection from the oxidizing effects of ros and 122 b. tompa, j. balint, l. fodorpataki counteract their electron acquiring properties. lipid peroxidation is inhibited by naturally occurring carotenoids (β-carotene, zeaxanthin, lutein), because these photosynthetic pigments perform an essential photoprotective role by quenching triplet state chlorophyll molecules, scavenging singlet oxygen and dissipating of the harmful excess excitation energy under stress conditions. in chloroplasts, peroxisomes and cytosol, ascorbate (vitamin c) has a key role in hydrogen per oxide scavenging via ascorbate peroxidase, while in the apoplast and vacuoles, it is the main reductant of phenolic radicals generated under oxidative stress. being a major component of the foyer-halliwellasada cycle, ascorbate helps to modulate oxidative stress tolerance by controlling ros detoxification, both alone and in cooperation with glutathione (gsh). besides its antioxidative role, ascorbate has an important role in a complex and well-orchestrated plant response network to environmental stress, performing multiple tasks in redox signaling, regulation of enzyme activities, modulation of stress-related genes expression, biosynthesis of phytohormones and growth regulation (veljović-jovanović et al., 2017). like in case of most glycophytes, yield and quality of spinach is adversely affected by salt stress. for example, fresh weight, area and k+ content of leaves, photosynthetic rate (pn), ascorbate (asa) and glutathione (gsh) content have been found to be reduced in spinach under saline conditions (hoque et al., 2007; ors and suarez, 2017). spinach belongs to the amaranthaceae family, it is an annual (rarely biennial) plant regarded as the second most nutritious leafy green crop in the world. nutritionally, spinach is a rich source of vitamin a (9380 iu/100 g of edible portion), vitamin k1 (483 mg/100 g of edible portion), folate (194 mg/100 g of edible portion), which are essential for various metabolic activities in the human body (usda, 2019). spinach is a good source of antioxidants and has one of the highest orac (oxygen radical absorbance capacity) values of any vegetable (ou et al., 2002). the health benefits of spinach include anti-cancer and anti-obesity properties, prevention of macromolecular oxidative damage and age-related macular degeneration (roberts and moreau, 2016). the recent development of baby leaf spinach coupled with an upswing of nutrition concerns has been responsible for a considerably increased consumption of spinach in the last decade (morelock and correll, 2008). it is a moderately salt-tolerant plant, which allows its cultivation on croplands where salinity problem already exists or is likely to be salinized in the near future. thus, there is a strong need for further improvement of new technologies in order to increase salt tolerance of spinach, as well as to manage the present and future challenges of low productivity and increasing global demands. according to the publications available at present, there is no information pertaining to effects of triacontanol on physiological and biochemical parameters of spinach plants under salt stress conditions, except for one of our recent studies (tompa and fodorpataki, 2021). therein, we show that triacontanol increases the fresh shoot biomass of young spinach plants and neutralizes the deleterious effects of salt stress on the photosynthetic pigment content of leaves. our hypothesis is that tria acts as a chemical signal that triggers specific reactions in plants, leading to stimulation of physiological processes and enhancement of defense mechanisms under environmental stress conditions. the main objective of this work is to investigate the influence of salt stress, triacontanol and their combination on growth and metabolic processes of spinach, related to productivity, salinity tolerance and health-promoting quality. a step towards sustainable development goal 2 “zero hunger” our study can help improve the use of plant growth regulators and other natural bioactive compounds in cost-effective and environ mental-friendly horticultural and agricultural practices, thus enhancing yield and nutritional composition of crop plants under unfavorable conditions of cultivation, such as high soil salinity related to a dryer and warmer climate. all of these can represent a step toward reaching the sustainable development goal (sdg) #2 “zero hunger”. materials and methods plant material and growth conditions the experiments were carried out with spinach (spinacia oleracea l.) plants belonging to the ‘popey’ cultivar. this was selected from three frequently grown cultivars (‘popey’, ‘matador’ and ‘viroflay’, the seeds being purchased from zki and garafarm ltd.), largely used in nutrition as fresh leafy vegetables (mainly in salads). based on previous studies, the ‘popey’ variety was found to be moderately tolerant to high salinity and was selected for experiments concerning the alleviation of the negative effects of salt stress with triacon tanol. for investigation of germination dynamics, uniform sized healthy seeds, 100 in four replications for each treatment setup, were selected and sterilized in 0.1% (w/v) sodium hypochlorite solution for 10 minutes, then washed twice with distilled water. for the other experiments, sterilized seeds were pre-hydrated for 24 hours in aerated distilled water and germinated in polyethylene zipper bags, between filter paper sheets moistened with distilled water. spinach seeds require cold stratification in order to break the embryonic dormancy cycle and to germinate more uniformly. therefore, germination temperature in the growth chamber was set at 5.5 °c for a 12-hour dark period and 22 °c for a 12-hour light period, for four days. at the end of the fourth day, the seeds were kept at 22 °c for another three days until the sowing. after the germination period, seedlings with similar size were planted separately in pots filled with perlite, watered regularly with hoagland’s nutrient solution (ph 6.5) and grown for three weeks in a growth chamber under controlled environmental conditions. the temperature was kept at 22 °c and the relative air humi dity at 60%. the daily photoperiod was set to 14 hours of light and 10 hours of darkness, the photon flux density was 260 μm photons m-2 s-1 photosynthetically active radiation. to reveal the physiological effects of tria in spinach under normal growth conditions, three experimental variants were set up, each with five plantlets: the control provided with basic hoagland solution, a series exposed to 25 nm tria and another to 1 μm tria treatment (triacontanol being purchased from the nutri-tech solutions pty ltd). two concentrations of tria were applied as foliar spray in every fourth day for three weeks. control plants were sprayed with deionized water. to investigate the compensation capacity of tria under salt-stressed conditions, four experimental variants were set up, each with five plantlets: the control, a series exposed to 150 mm nacl, a series treated with 1 μm tria and exposed simultaneously to salt stress induced with 150 mm nacl, and a series treated with 1 μm tria, but not subjected to salt stress. nacl was dissolved in the nutrient solution used for watering the substrate, while tria solution was pulverized on the aboveground parts of the plants. growth conditions during the treatments were similar with those created for the development of seedlings after germination. germination dynamics surface sterilized seeds, four repetitions of 100 seeds each, were soaked for 24 hours in different aqueous solutions according to ex perimental variants: control lots were immersed in distilled water, while treated seeds were covered by aqueous solutions of 1 μm tri acontanol or 150 mm sodium chloride, and mixtures of these. after 24 hours, all seeds were put at equal distances from each other in polyethylene zipper bags, between double-layered filter papers tho roughly imbibed with distilled water for control lots and with the appropriate solutions, according to the three different treatments. the seeds were transferred in a growth chamber with 5.5 °c for a 12-hour improved spinach cultivation with triacontanol 123 dark period and 22 °c for a 12-hour light period, with 70% relative humidity, for 12 days. the filter papers were kept saturated with the treatment solutions. the germinated seeds (with the radicle emerged through the seed tegument) were recorded every third day during the morning hours. finally, for each treatment, the germination percentage (gp) and the germination energy (ge) were calculated as the average of the four replications. growth and biomass analyses at 42 days after sowing, plants from each experimental variant were uprooted carefully, were washed with tap water to remove all adhering perlite particles and were dried using blotting papers. shoot and root lengths were recorded using a measuring scale. the plants were cut to separate roots and shoots, to determine the fresh weights of underground and aboveground vegetative organs. the same plants were then dehydrated in a hot-air oven at 80 °c for three days to determine their dry weights. measurement of leaf gas exchange parameters gas exchange parameters were monitored in situ on the abaxial side of the fourth fully expanded leaf from the base of stem (developed during the triacontanol treatment), with a ciras-2 type leaf gas exchange meter (pp systems). in the measurement chamber the tempera ture was maintained at 22 °c, co2 concentration at 410 μmol mol-1, the relative air humidity at 60% and light intensity at 500 μmol m-2 s-1 photosynthetic photon flux density. stomatal conductance (gs), intensity of transpiration (e) and net photosynthetic carbon dioxide assimilation (pn) were the main physiological parameters registered in the middle of the daily photoperiod. determination of induced chlorophyll fluorescence parameters conventional parameters of induced chlorophyll fluorescence were determined in situ on the fourth emerging leaf, with an fms-2 type fluorometer (hansatech), in order to evaluate energetic efficiency of light use in photochemical reactions of photosystem ii (psii). on dark-adapted leaves, just before the onset of the light period, the ground fluorescence (fo) was measured by illuminating the leaves with a faint red light flash (0.1 μmol m-2 s-1), while maximum fluorescence (fm) was recorded during a subsequent saturating light pulse (10000 μmol m-2 s-1 for 0.5 s). the potential quantum yield of photo system ii (psii) was calculated as the fv/fm ratio, where fv is the difference between fm and fo (lichtenthaler et al., 2005). quantification of photosynthetic pigments photosynthetic pigments were extracted from the fourth fully expanded leaf, from the same leaf blade region on which gas exchange and chlorophyll fluorescence measurements were performed in vivo straight before pigment determination. extracts were obtained from 0.25 g of leaves (fresh weight) finely homogenized in 5 ml 80% (v/v) acetone, then the supernatant resulting from centrifugation for 10 min at 4000g and 4 °c was used for absorbance measurements, using a uv-vis spectrophotometer (jasco). concentrations of chlorophyll a, chlorophyll b and carotenoids were calculated according to well burn (1994), by measuring the absorbance of the solution at the wavelengths 663 nm, 646 nm and 470 nm. measurement of malondialdehyde and other tbars oxidative damage of membranes was evaluated on the basis of formation of malondialdehyde (mda) and other thiobarbituric acid reactive substances (tbars) due to peroxidation of unsaturated fatty acids in membrane lipids. 0.5 g fresh root samples were homogenized with 5 ml of 0.1% (w/v) trichloroacetic acid (tca) solution in a pre-chilled mortar, the homogenate was centrifuged at 15 000 g for 15 min, then 2 ml of the supernatant was transferred in a test tube and 4 ml of 10% (w/v) tca with 0.5% (w/v) 2-thiobarbituric acid (tba) were added. the mixture was heated at 95 °c for 30 min and then quickly cooled in an ice bath. the cooled solution was centrifuged at 10 000 g for 5 min, and the absorbance of the supernatant was measured at 532 nm and 600 nm. the absorbance at 600 nm (due mainly to interference of anthocyanin pigments) was deducted from the value obtained at 532 nm. mda concentration of samples was calculated using the extinction coefficient of 155 mm-1 cm-1 (heath and packer, 1968). determination of ascorbic acid content and of the reduced to oxidized vitamin c ratio ascorbic acid content was determined by homogenizing 0.5 g spinach leaf with 4 ml of 6% trichloroacetic acid in a prechilled mortar. the mixture was centrifuged for 13 min at 15 600 g and 4 °c, then 2 ml of supernatant was transferred to sodium phosphate buffer (ph 7.4) containing trichloroacetic acid, dithiothreitol, orthophosphoric acid, ethanolic solution of 2,2’-dipyridyl, n-ethylmaleimide and ferric chloride. after one hour of incubation of this extract mixture at 42 °c with permanent mixing, absorbance of the solution was measured at 525 nm with a spectrophotometer. the oxidized form of vitamin c present in leaf tissues (dehydroascorbate) was reduced to ascorbate by dithiothreitol. then the total ascorbic acid content was determined photometrically by the 2,2’-dipyridyl method, and concentration of dehydroascorbate was calculated as the difference between total and reduced ascorbate. a standard curve was obtained with known concentrations of pure ascorbic acid dissolved in 5% trichloroacetic acid, in the range of 25-100 nm (kampfenkel et al., 1995). statistical analysis all experimental variants were set in 5 replicates, except for germination, in which case experiments with 100 seeds for each treatment were conducted in four repetitions. measurements of physiological parameters were repeated three times. experimental data were statistically processed in r software environment (version 4.1.0.), using the shapiro-wilk test for normality and bartlett’s test for homogenei ty of variances. data were represented as the mean ± standard error (se). the significant differences were determined by the one-way anova test and the post-hoc tukey hsd test. differences were considered statistically significant at p < 0.05. results and discussion germination germination is an important developmental stage for seedling establishment and therefore it plays a key role in crop production. seed priming is a simple, low-cost, easily performable technique, which improves the seed performance and provides faster and synchronized germination. application of 1 μm triacontanol as seed priming substance did not cause spectacular changes in the dynamics of spinach seed germination, but it considerably alleviated the delaying and inhibitory effects of high salinity (fig. 1). in the case of only triatreated seed lots, the final germination percentage was higher by 8% after twelve days as compared to the control seeds, but this did not represent a major increment, even it was statistically significant. soil salinity is one of the major constraints of seed germination, because it creates a negative external osmotic potential to prevent water uptake and exerts a toxic effect on the germinating seeds through the excess amount of na+ and clions. ion toxicity and osmotic stress are re124 b. tompa, j. balint, l. fodorpataki sponsible for both inhibition and delay of seed germination and seedling establishment. under a salt stress induced by 150 mm nacl less than 19% of seeds germinated until the ninth day and around 34% germinated until the end of the 12 days period. these impairments caused by high salinity were significantly counteracted by priming of seeds with 1 μm of tria: the germination percentage of seeds exposed to 150 mm nacl was increased from 18% to about 28% on the ninth days, and the final germination percentage was increased from 34% to 52%. a similar conclusion was drawn by sarwar (2017), when priming with 50 μm triacontanol decreased time to start emergence and improved final germination capacity in case of cucumber seeds exposed to salt stress induced by 50 mm nacl. seed priming with aqueous solution of 1 μm triacontanol helped to maintain a significantly higher germination percentage and reduced the time needed for germination in salt-stressed spinach seeds, thus improving salinity tolerance at this early and sensitive developmental stage. on a longer time scale, better germination greatly contributes to the establishment of vigorous crop stands and an abundant final yield. vegetative growth the root system influences plant productivity via its phenotypic traits such as root length, biomass, branch density, volume, and contact area. foliar application of tria at 25 nm and 1 μm concentrations appreciably enhanced the root dry biomass (fig. 2a), but significantly reduced the length of spinach main root axis (fig. 2b). the highest root dry biomass values were recorded upon treatment with 1 μm tria. influence of triacontanol resulted in a shorter, but more densely branched root system, which makes efficient use of a smaller soil volume through the denser network of ramifications. allocation of assimilates is supposedly redirected to formation of more lateral roots, which increases overall absorption surface for water and mine ral nutrients. on the other hand, shorter roots could be detrimental in situations when the soil surface dries out regularly, while longer roots can reach deeper into the soil for moisture. benefits of a densely branched and shorter root system highly depend on irrigation regimes and cultivation practices. tantos et al. (2001) reported a significant increase in the number of roots per plant as result of tria application in the rooting media used for the micropropagation of apple and sour cherry. because spinach is a leafy vegetable, the fresh biomass of the vege tative shoot is particularly important for its production. treatment with 25 nm triacontanol of non-stressed plantlets resulted in a considerably increased shoot fresh biomass, while higher tria concentration did not lead to higher shoot weight than induced by 25 nm (fig. 3a). a similar positive influence of triacontanol was observed on total (root and shoot) dry biomass of six weeks old spinach plants. in this case, the stimulatory effect of tria principally refers to production of organic metabolites (photosynthates), as water content of plant organs is not modified significantly by triacontanol (fig. 3b). naeem et al. (2011) reported that foliar application of tria at 1 μm concentration significantly enhanced the length of shoots, the number and surface area of leaves, and the fresh and dry biomass of the wild mint. increment exerted by tria on the yield and content of active constituents (menthol, l-methone, isomenthone and menthyl acetate) of the essential oil augmented the medicinal value of this herb. triacontanol did not stimulate the growth of vegetative organs in proportion to its concentration, which may suggest that it has only a signaling, triggering role in this process and that its effect is amplified during signal transduction. cascading effect results in enhanced metabolism processes and the accumulation of various intermediate compounds, for example elevated intracellular ca2+ levels leading to changes in gene expression (islam et al., 2021). treatment with very small concentrations of tria improves the vegetative growth and biomass production of young spinach plants in a cost-effective way, which is not negligible given the growing demands for baby spinach. * ** 0 10 20 30 40 50 60 70 80 90 100 3 6 9 12 g er m in ati on p er ce nt ag e time (days) control 150 mm nacl 150 mm nacl + 1 µm tria 1 µm tria c b a 0 10 20 30 40 50 60 70 ø 25 nm tria 1 µm tria ro ot d ry b io m as s (m g) a. a b b 0 50 100 150 200 250 300 ø 25 nm tria 1 µm tria ro ot le ng th (m m ) b. b a a 0 20 40 60 80 100 120 140 160 ø 25 nm tria 1 µm tria to ta l d ry b io m as s (m g) b. b a a 0 200 400 600 800 1000 ø 25 nm tria 1 µm tria sh oo t f re sh b io m as s (m g) a. fig. 1: effects of nacl and/or triacontanol (tria) on germination of spinach seeds. significant differences between salt-stressed seeds with and without tria treatment are marked with * (p<0.05) and **( p<0.01). vertical bars represent ± standard error from means (n=4) fig. 2: effects of two concentrations (25 nm and 1 μm) of triacontanol (tria) on the root dry biomass (a.) and root length (b.) of spinach (ø stands for the control group). vertical bars represent ± standard error from means (n=5), different letters above the columns indicate significant differences at p < 0.05 (tukey’s hsd test) fig. 3: effects of two concentrations (25 nm and 1 μm) of triacontanol (tria) on fresh shoot weight (a.) and total dry biomass (b.) of young spinach plants (ø stands for the control group). vertical bars represent ± standard error from means (n=5), different letters above the columns indicate significant differences at p < 0.05 (tukey’s hsd test) photosynthetic performance photosynthesis is one of the most fundamental and intricate phy siological processes in all green plants, which is determining crop production, but it is severely affected by several unfavorable environmental conditions, including soil salinity. a simultaneous measurement of the responses of leaf gas exchange and induced chlorophyll fluorescence to salt stress provides an effective mean to understand the main limitations of photosynthesis in vivo. salt stress alters photosynthetic mechanisms by disrupting thylakoid membranes, improved spinach cultivation with triacontanol 125 modifying the electron transport chain, changing enzymatic activi ty during chlorophyll synthesis, and the coordinated functioning of the different steps of the calvin cycle. net photosynthetic rate (pn), measured through carbon dioxide uptake of spinach leaves, was drastically decreased (by an average of 65%) in plants exposed to 150 mm nacl (fig. 4a). this reduction was alleviated significantly by simultaneous application of 1 μm triacontanol, while in case of non-stressed plants tria caused a 28% increment of net carbon dioxide assimilation rate in comparison to control plants. tria stimulated the opening of stomata proportionately to its quantity used, thus enhancing their conductivity for carbon dioxide and for water vapor (fig. 4b). similar results were reported by shahbaz et al. (2013) for canola seedlings, as well as by naeem et al. (2011) for wild mint. net photosynthetic rate is limited by stomata closure, which is controlled by turgor pressure of the leaf cells. the leaf turgor pressure can be highly affected by abscisic acid (aba), which is responsible for the hydroactive stomatal closure under physiological drought. water deficit enhances aba generation in roots and xylem-mediated aba transport to shoots. an increase in ph of xylem sap up-regulates the transport of aba to the guard cells on leaf surfaces, where it regulates the stomatal closure through the involvement of ca2+ (shahid et al., photosynthesis may be associated with increased chlorophyll content of spinach leaves. plant pigments, particularly leaf chlorophyll contents have widely been appraised as indicators of photosynthesis and ultimately plant productivity. the fact that leaf spraying with tria causes a moderate, but statistically significant increase of the total chlorophylls in spinach leaves (fig. 5b.) may be related to the fact that triacontanol stimulates the biosynthesis of chlorophyll pigments. chen et al. (2003) obtained similar results, in which case tria increased the contents of total chlorophylls by 25.1%, and fv/fm and effective quantum yield of photosystem ii (φpsii) were higher in the treated rice plants. pang et al. (2020) demonstrated that exogenous application of tria stimulates expression of the genes encoding for protoporphyrinogen iii oxidase (ppox), chlorophyllide a oxygenase (cao) and divinyl chlorophyllide a 8-vinyl-reductase (dvr), thus it plays a substantial role in chlorophyll biosynthesis. another explanation for the better utilization of light energy in photochemical reactions under salt stress is due to the larger size and/or higher degree of organization of the light-harvesting antenna complexes (lhcii+lhci), as well as a higher efficiency of excitation trapping at the reaction centers of psii, caused by triacontanol. c b a 0 50 100 150 200 250 300 350 400 ø 25 nm tria 1 µm tria g s (m m ol h 2o m -2 s1 ) b. b d c a 0 1 2 3 4 5 6 7 8 ø 150 mm nacl 150 mm nacl + 1 µm tria 1 µm tria pn (µ m ol c o 2 m -2 s1 ) a. fig. 4: net co2 assimilation rate (pn) and stomatal conductivity (gs) in leaves of spinach plants exposed to high salinity, with or without triacontanol (tria) treatment. ø stands for the control group. vertical bars represent ± standard error from means (n=5), different letters above the columns indicate significant differences at p < 0.05 (tukey’s hsd test) 2020). pang et al. (2020) found that triacontanol inhibits aba signal transduction with down-regulation of genes involved in the process, thus it compensates for stomatal closure induced by physiological drought caused by high salinity. by increasing the amount of the available carbon dioxide due to regulation of stomatal movements, tria can prevent excessive yield loss caused by high salinity in the rhizosphere. the maximal photochemical efficiency (also known as potential quantum yield) of photosystem ii, indicated by the fv/fm chlorophyll fluorescence ratio, was not influenced by foliar spraying with 1 μm triacontanol in spinach plants which were not exposed to salt stress (fig. 5a). fv/fm of plants grown for twenty-one days in the presence of high salinity was markedly reduced. in plants exposed to 150 mm nacl, treatment with 1 μm tria totally counteracted the lowering effect of severe salt stress on this physiological marker of photosynthetic light use efficiency. decrease of the potential quantum yield is an early indicator of disturbance of the light reactions of photosynthesis when environmental stressors impair energy conservation in plants, and tria significantly compensated for the inhi bition effect of high salinity. borowski et al. (2000) observed a significant increase of the maximal quantum yield of psii (fv/fm) and of the efficiency of excitation capture by open psii reaction centers (fv’/fm’), as well as a reduction of non-photochemical quenching coefficient (qnp), when triacontanol was applied to tomato. plants treated with triacontanol at the doses of 0.3 and 3.0 μg had significantly higher yields of fruits than control. improvement in b a a 0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 ø 25 nm tria 1 µm tria to ta l c hl or op hy ll co nt en t ( m g g1 ) b.a b a a 0,5 0,55 0,6 0,65 0,7 0,75 0,8 0,85 ø 150 mm nacl 150 mm nacl + 1 µm tria 1 µm tria fv /f m a. fig. 5: potential quantum yield of photosystem ii (fv/fm) and total chlorophyll content in leaves of spinach plants exposed to high salinity, with or without triacontanol (tria) treatment. ø stands for the control group. vertical bars represent ± standard error from means (n=5), different letters above the columns indicate significant differences at p < 0.05 (tukey’s hsd test) degree of oxidative membrane damage the degree of oxidative membrane damage of salt-stressed spinach seedlings was shown by increased malondialdehyde (mda) and related thiobarbituric acid-reactive substances (tbars) content, these compounds being toxic products of fatty acid degradation. tbars increased by 28% in plants exposed to 150 mm nacl, compared to control seedlings. simultaneous application of 1 μm tria with salinity significantly reduced tbars formation in spinach leaves (fig. 6). thylakoid membranes contain a high percentage of polyunsaturated fatty acids (mainly in monogalactosyl-diacylglycerol), thus very susceptible to lipid peroxidation. a slight perturbation in the composition of thylakoid membranes can lead to impaired photochemical reaction and energy coupling between reaction centers and antenna complexes. in the present study, it was found that exo genous application of 1 μm tria mitigated the negative effects of salt stress on membrane lipid peroxidation and exhibited a protective capacity for biological membranes. similar results were reported in case of salt-stressed coriander, when treatment with 10 μm tria reduced h2o2 and malondialdehyde formation by modulating the cellular redox status and the activity of antioxidant enzymes, such as superoxide dismutase and catalase (asadi karam and keramat, 2017). inhibition of light-induced lipid peroxidation by tria was observed previously in isolated spinach chloroplasts (ramanarayan et al., 2000). under stressful conditions tria is able to increase the activities of antioxidant enzymes such as ascorbate peroxidase (apx), guaiacol peroxidase (gpx) and glutathione reductase (gr), 126 b. tompa, j. balint, l. fodorpataki so reducing the oxidative stress (asadi karam et al., 2017a). in the human organism, malondialdehyde-modified low-density lipoprotein (mda-ldl) acts as a marker of oxidative stress and is associated with atherosclerotic cardiovascular diseases, such as aortic stiffness (hou et al., 2020).the current results support the idea that treatment with micromolar amounts of triacontanol as foliar spray may efficiently alleviate oxidative membrane damage induced by salt stress in spinach plants, thus reducing the malondialdehyde content in the human diet which contains this leafy vegetable. carotenoid content of spinach leaves carotenoid content of spinach leaves is not influenced by the pre sence of triacontanol under stressless growth conditions. salt stress generated by 150 mm sodium chloride decreased total carotenoid content by 29% (fig. 7). simultaneous application of 1 μm tria completely counteracted the deleterious effect of salinity on the carotenoid pigment concentration, thus restoring the health-promoting quality of these spinach leaves due to the antioxidative protective role of these carotenoids for every living organism. in the absence of salt stress, naeem et al. (2011) reported that exogenous application of tria significantly improved the carotenoids content of a mint species, exceeding the control by 26%. carotenoids have two important roles in photosynthetic organisms. firstly, as light-harvesting pigments, they effectively extend the range of blue light absorbed by the photosynthetic apparatus, secondly, they play a crucial role in the dissipation of harmful excess excitation energy under stressful conditions which may impair the use of light energy in carbon assimilation. based on their capacity to neutralize singlet oxygen, hydroxyl and organic peroxyl radicals, carotenoids have a general protective role against oxidative damage. humans cannot synthesize carotenoids and must ingest them in food or via supplementation. they primari ly exert antioxidant effects, but carotenes also have a pro-vitamin a function, while lutein and zeaxanthin constitute macular pigments in the eye. the benefit of lutein in reducing progression of age-related macular eye disease and cataracts was proven experimentally. plantderived carotenoids also produce improvements in cognitive function and cardiovascular health, and may help to prevent some types of cancer (eggersdorfer and wyss, 2018), therefore an increased carotenoid content of spinach plantlets upon human consumption has a demonstrated health-promoting role. influence of foliar spraying with triacontanol on the vitamin c content ascorbic acid is one of the most abundant water-soluble antioxidants in plants. as plant-based foods constitute the principal source of vitamin c in the human diet, the possibility of increasing the ascorbate content of plants to improve their nutritional value has received considerable attention in recent years. this antioxidant molecule is considered as an essential component of cell signaling pathways triggering adaptive plant responses. triacontanol by itself did not exert any significant influence on the vitamin c content of spinach leaves, when it was used in the concentration of 1 μm as foliar spray on the ‘popey’ spinach cultivar. salt stress exerted by 150 mm sodium chloride, with no tria treatment, slightly but significantly increased the ascorbic acid content of leaves, as a defense reaction to the oxidative stress induced by a prolonged exposure to high salinity. when treatment with 1 μm triacontanol was applied simultaneously with the high salinity, the vitamin c content was approximately four times as much as in control leaves (fig. 8). foliar application of tria increased not only the overall vitamin c content of spinach leaves, but it also led to an elevated asa/dha ratio in comparison to triauntreated, nacl-exposed plants. this shows an enhanced capacity to cope with oxidative stress, as the reduced ascorbate has the capaci ty to quench hydrogen peroxide, and its recovery from the oxidized forms is crucial for a sustained protective capacity. a similar role of fig. 8: influence of tria on vitamin c content and on reduced ascorbic acid (asc) to oxidized dehydroascorbate (dha) ratio of spinach leaves exposed to salt stress. ø stands for the control group. vertical bars represent ± standard error from means (n=5), different letters above the columns indicate significant differences at p < 0.05 (tukey’s hsd test) b a c b 0 100 200 300 400 500 600 700 800 900 ø 150 mm nacl 150 mm nacl + 1 µm tria 1 µm tria tb a rs (n m g1 fr es h w ei gh t) a b a a 0 50 100 150 200 250 300 350 400 450 ø 150 mm nacl 150 mm nacl + 1 µm tria 1 µm tria ca ro te no id c on te nt (µ g g1 fr es h w ei gh t) d c d d b b a b 0 40 80 120 160 200 ø 150 mm nacl 150 mm nacl + 1 µm tria 1 µm tria a sc or bi c ac id c on te nt (µ g g1 fr es h w ei gh t) dha asa fig. 6: membrane lipid peroxidation assayed with the amount of thiobarbi turic acid-reactive substances (tbars) in roots of spinach plants exposed to salinity, with or without triacontanol (tria) treatment. ø stands for the control group. vertical bars represent ± standard error from means (n=5), different letters above the columns indicate significant differences at p< 0.05 (tukey’s hsd test) fig. 7: influence of triacontanol (tria) on carotenoid pigment content in leaves of spinach exposed to 150 mm nacl. ø stands for the control group. vertical bars represent ± standard error from means (n=5), different letters above the columns indicate significant differences at p < 0.05 (tukey’s hsd test) improved spinach cultivation with triacontanol 127 s-methylmethionine (vitamin u) was demonstrated in lettuce seedlings exposed to salt stress induced by 150 mm nacl (fodorpataki et al., 2016). increment of the ratio between the reduced and the oxidized form of vitamin c is associated with a stronger antioxidative defense system and increased activity of monodehydroascorbate reductase (mdhar) and dehydroascorbate reductase (dhar) enzymes. mdhar and dhar activity increased by 39.11% and 273%, respectively, when canola seedlings were pretreated with 10 μm tria before cd-induced oxidative stress (asadi karam et al., 2017b). these results clearly indicate the protective ability of tria to modulate the redox status of plant cells under unfavorable environmental conditions. however, the role of exogenously applied tra in the ascorbic acid biosynthesis needs to be elucidated. plants synthesize vitamin c is the highest amounts especially under environmental conditions which cause oxidative stress. vitamin c cannot be produced by the human organism, so a systematic supply with this vitamin through alimentation is required for normal cell functions, for immune stimulation and synthesis of collagen. severe deficiency leads to scurvy, whereas a limited vitamin c intake causes increased susceptibility to infections. therefore, new strategies aimed to increase vitamin c content in plants would be of interest to improve human health. conclusions treatments with very small concentrations of triacontanol is con sidered a profitable mean of augmenting quality and production of crop plants. in spinach, it improves vegetative growth and biomass production, and also stimulates seed germination. under high salinity conditions, tria improves the nutritional value of spinach leaves, by inducing a higher carotenoid and vitamin c content, and by reducing the generation of toxic products of oxidative membrane damage (such as malondialdehyde). adverse effects of high salinity of soil solution may be compensated for by very low amounts of tria pulverized on leaves. improvement of photosynthetic functions was indicated by a more efficient net photosynthetic carbon dioxide assimilation as well as by a more efficient use more of the incident light energy when photosynthesis was impaired by salt stress. development and production of crop plants may be improved by use of tria in an environmental-friendly approach. the present study can serve as a foundation for additional laboratory and field studies optimizing the application of triacontanol in crop production, as part of innovative cropping technologies. our results may contribute to enhance yield and nutritional composition of crop plants cultivated in areas affected by increasing soil salinity, in relation with global climate change and with the need to ensure higher quantities and better qualities of horticultural products for a continuously increasing human population, in a hastily changing environment. conflict of interest no potential conflict of interest was reported by the authors. references asadi karam, e., keramat, b., 2017: foliar spray of triacontanol improves growth by alleviating oxidative damage in coriander under salinity. ind. j. plant physiol. 22, 120-124. doi: 10.1007/s40502-017-0286-z asadi karam, e., keramat, b., sorbo, s., maresca, v., asrar, z., mozafari, h., basile, a., 2017a: interaction of triacontanol and arsenic on the ascorbate-glutathione cycle and their effects on the ultrastructure in coriandrum sativum l. environmental and experimental botany 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(eds.), protective chemical agents in the amelioration of plant abiotic stress, 491-509. wiley. doi: 10.1002/9781119552154.ch25 orcid: bernat tompa https://orcid.org/0000-0002-3474-3203 janos balint https://orcid.org/0000-0002-0351-888x laszlo fodorpataki https://orcid.org/0000-0003-2140-0137 address of the corresponding author: dr. laszlo fodorpataki, department of horticulture, sapientia hungarian university of transylvania, ro-540485 targu mures, romania e-mail: lfodorp@gmail.com, fodorpataki.laszlo@ms.sapientia.ro © the author(s) 2022. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1080/17429145.2011.619281 http://dx.doi.org/10.1007/s10725-011-9588-8 http://dx.doi.org/10.1016/j.scienta.2009.02.030 http://dx.doi.org/10.1016/j.agwat.2017.05.003 http://dx.doi.org/10.1021/jf0116606 http://dx.doi.org/10.3390/plants9040488 http://dx.doi.org/10.1007/s00344-015-9541-6 http://dx.doi.org/10.1016/s0031-9422(00)00201-6 http://dx.doi.org/10.1007/978-1-4614-4747-4_1 http://dx.doi.org/10.1039/c6fo00051g http://dx.doi.org/10.17957/ijab/15.0356 http://dx.doi.org/10.1080/17429145.2013.764469 http://dx.doi.org/10.3390/agronomy10070938 http://dx.doi.org/10.1016/j.ecolind.2015.04.027 http://dx.doi.org/10.1007/s002990000282 http://dx.doi.org/10.2478/ausae-2021-0006 http://dx.doi.org/10.1007/978-81-322-2725-0_2 http://dx.doi.org/10.1007/978-3-319-74057-7_3 http://dx.doi.org/10.1016/s0176-1617(11)81192-2 http://dx.doi.org/10.1016/j.envexpbot.2019.06.004 http://dx.doi.org/10.1002/9781119552154.ch25 https://orcid.org/0000-0002-3474-3203 https://orcid.org/0000-0002-0351-888x https://orcid.org/0000-0003-2140-0137 art06 journal of applied botany and food quality 81, 136 139 (2007) institute of vegetable and ornamental crops grossbeeren/erfurt e.v., grossbeeren, germany changes in quercetin and kaempferol concentrations during broccoli head ontogeny in three broccoli cultivars* angelika krumbein, heidi saeger-fink, ilona schonhof (received may 25, 2007) summary three broccoli cultivars – spear broccoli ‘emperor’, crown broccoli ‘marathon’ and violet broccoli ‘viola’ – were harvested during head ontogeny from start of head development until over maturity stage (five stages) in three different years. the aglycones quercetin and kaempferol were analysed at optimised conditions of acid hydrolysis by hplc. heads of over maturity stage had the highest contents of quercetin and kaempferol. however, the genotype fundamentally determined the quantity and course of the increase in flavonols. mini broccoli, as a new trend to market vegetables, has lower content of flavonols than the commercial stage, which indicates a reduction in health potentials. harvesting broccoli heads of over maturity stage should be used as raw material, e.g. for the design of new functional foods. introduction polyphenols are powerful antioxidants in vitro, scavenging a range of reactive species and binding transition metals in forms less active in catalysing free radical damage (halliwell, 2006). an epidemiological study in the netherlands (the zytphen study) suggests an inverse correlation between the incidence of coronary heart disease and stroke, and the dietary intake of flavonoids, especially quercetin (hertog et al., 1993). quercetin, the main representative of the flavonol class, found in high concentrations in onions, apples, red wine, broccoli, tea and ginkgo biloba, influences some carcinogenic markers and has minor effects on plasma antioxidant biomarkers in vivo, although some studies failed to find this effect (williamson and manach, 2005). flavonoids have in common a basic c6-c3-c6 skeleton structure, consisting of two aromatic rings and a heterocycle containing one oxygen atom. flavonoids usually occur in plants as glycosides substituted with various sugars – mostly glucose, but also galactose, rhamnose, and other sugars. in broccoli, five different glycosides of quercetin and kaempferol were identified, whereby quercetin 3-osophoroside and kaempferol 3-o-sophoroside were the main flavonoid glycosides (price et al., 1998). phenolic compounds may be affected by numerous preharvest factors. vallejo et al. (2003a) found that, besides genetic variation, extreme agronomic and environmental conditions (late seasons and rich sulphur fertilisation) induce different stress situations on broccoli, enhancing the phenolic content, such as flavonoids and hydroxycinnamoyl derivatives. in contrast, organic farming and water stress decreased the overall production of phenolics in broccoli, such as glycosylated flavonoids and hydrocycinnamic esters (robbins et al., 2005). the developmental stage at which a vegetable is harvested can also influence its phytochemicals. in fruit vegetables, such as pepper, it was found that immature green peppers had a very high flavonoid content, while green, immature red and ripe red peppers demonstrated a 4to 5-fold reduction (marin et al., 2004). otherwise, the flavonoid concentration of peppers decreased, increased or remained constant, while maturity was dependent on the cultivar (howard et al., 2000). in leafy vegetables such as baby spinach, it was found that younger baby spinach leaves had the highest concentration of total flavonoids (bergquist et al., 2005). furthermore, flavonol glycosides decreased with increasing age of pear leaves (andreotti et al., 2006). besides the consumption of fully developed broccoli heads by consumers, broccoli can also be used as a raw material and as ready-to-eat mini broccoli – a new market product (schreiner et al., 2006). vallejo et al. (2003b) found that the total flavonoid concentration and caffeoyl-quinic acid derivatives, sinapic and ferulic acid derivatives in broccoli increased from the first development stage until over maturity. they investigated broccoli grown in a relatively warm climate in spain, with relatively low head diameters at the commercial maturity stage of 13-14 cm, in comparison with 21-23 cm head diameters from northern europe. the aims of this study were (1) to determine the influence of head ontogeny on quercetin and kaempferol concentration in broccoli under the relatively cool climatic conditions of northern europe, and (2) to clarify whether similar trends can be observed between both flavonoids – quercetin and kaempferol – and between broccoli cultivars. samples were taken at five stages of head development for three broccoli cultivars in three different years. furthermore, based on the method of hertog et al. (1992), conditions of acid hydrolysis were optimised for the determination of the aglycones quercetin and kaempferol in broccoli. materials and methods plant material three broccoli cultivars were used – ‘emperor’ (spear type, heavy green), ‘marathon’ (crown type, grey-green) and ‘viola’ (violet type) – and experiments were performed in autumn in three different years: experiment i in 2002, planting date: july 25, harvest date: september 13 to november 4, with 17.4°c daily mean temperature and 246 µmol m-2 s-1 daily mean sum of photosynthetic photon flux density (ppfd); experiment ii in 2003, planting date: july 22, harvest date: september 9 to october 13, with 17.5°c daily mean temperature and 263 µmol m-2 s-1 daily mean sum of ppfd; and experiment iii in 2004, planting date: july 22, harvest date: september 10 to october 26, with 16.6°c daily mean temperature and 289 µmol m-2 s-1 daily mean sum of ppfd. the experiments were set up with 3-fold field repetition (sandy soil, ph 6.8) with a plot size of 20 m2, containing 5.5 plants m-2. cultivation (fertilisation, irrigation and plant protection) was performed according to ‘integrated production’ guidelines (winkhoff, 1992). protection nets, which reduced light by 20%, were used to minimise chemical plant applications. all cultivars were planted at the same time and harvested at different times once the respective head size had been reached. since the broccoli types differed with regard to their typical head size at harvest, measurement of head diameters was performed by an additional determination of the physiological stage at harvest. according to the bbch scale (uniform coding of phenologically similar growth stages * this paper was presented at the 42th meeting of the „deutsche gesellschaft für qualitätsforschung (pflanzliche nahrungsmittel) dgq e.v.“ of plant species) by meier (1997), we classified the harvested heads in five development stages: 1, start of head development; 2 and 3, mini broccoli; 4, fully developed heads (usual commercial stage); and 5, over maturity stage (first individual flowers visible). the head diameter was measured following the head surface curvature (tab. 1). tab. 1: head surface curvature diameter (cm) at harvest at five head development stages. values represent the mean of three experiments and tree replications. head development stages convar. 1 2 3 4 5 emperor 6.40±0.78 12.35±0.75 16.04±0.71 21.77±0.95 23.32±2.01 marathon 6.62±0.74 12.12±0.75 16.13±0.82 22.34±1.74 26.47±2.92 viola 6.10±0.58 11.49±0.96 15.95±0.96 21.90±1.61 23.33±3.08 flavonoid analysis a mixed sample of florets (bud and second-order branching) of each replication was taken, consisting of 10 heads at development stage 1 and 5 heads each at stages 2 to 5. only the florets were used for analyses, since these are the broccoli parts that are commonly consumed (schonhof and krumbein, 1996). the material was immediately deep-frozen (-40°c), then freeze-dried and finely ground. flavonols were determined as their aglycones after acid hydrolyses. unless otherwise stated, extracts of broccoli samples were prepared in the following way: 40 ml of 62.5% aqueous methanol was added to 0.5 g of the freeze-dried broccoli sample. 10 ml of 8 m hcl was added to this extract. thus, the extraction solution consisted of 1.6 m hcl in 50% aqueous methanol (v/v). after refluxing at 90°c for 2h, the extract was allowed to cool, was adjusted to 100 ml with 50% methanol, and sonicated for 5 min. the extract was then filtered through a 0.45 µm filter for hplc analyses. the flavonoid composition and concentration were determined using a series 1100 hplc (agilent, waldbronn, germany) equipped with a diode array detection system. a prodigy column ods(3) (250 x 4.6 mm, 5 µ, 100å) (phenomenex, aschaffenburg, germany) was used with a security guard c18 (4 x 3.0 mm) at a temperature of 25°c. solvent a was water + 0.1% tfa (trifluoroacetic acid) + 2% thf (tetrahydrofuran); solvent b was acetonitrile. the following gradient was used: 30 35% b (5 min), 35 39% b (12 min), 39 90% b (5 min), 90% b isocratic (2 min), 90 30% b (5 min), 30% b isocratic (5 min). the chromatogramme were monitored at 270 nm with a flow rate of 1 ml min-1. contents were quantitatively determined by calibration curves of the related pure standards (quercetin, fluka, buchs, swizerland; kaempferol, carl roth gmbh, karlsruhe, germany) . the results were converted to 100 g fresh matter (fm). quercetin and kaempferol were identified by hplc-esi-ms2, using agilent 1100 series (ion trap) in the negative ionisation mode. nitrogen was used as dry gas (12 l min-1, 350°c) and nebuliser gas (40 psi). to compare the results with the hplc-dad, the same column material with a shorter length (150 x 4,6 mm, 5 µ, 100 å) was used. tfa in solvent a was replaced by 0.5% (v/v) acetic acid, thf in solvent a was omitted and the same water/acetonitil gradient and a flow rate of 0.6 ml min-1 were used. quercetin and kaempferol were identified from the deprotonated molecular ions [m h]with m/z 301 and 285, respectively. furthermore, in ms2 querecetin and kaempferol showed a characteristic mass fragment of ring a at m/z 151, which is typical for several flavonoids (march et al., 2006). statistical analysis the results were evaluated using analysis of variance, and least significant differences were calculated using tukey’s honest-significantdifference (hsd) test (5% significance level). the data from different planting dates (three years) were pooled, resulting in nine replications per treatment. results and discussion optimised sample preparation and hplc method of flavonoids in broccoli the extraction efficiency of 0.5 g of a freeze-dried broccoli sample with 50% and 70% aqueous methanol was comparable, but higher than the use of 30% aqueous methanol (data not shown). fivty percent aqueous methanol was therefore used for all analyses. the hydrolysis of all glycosides to aglycons offers a practical method for the quantitative determination of flavonoids in foods (hertog et al., 1992). the authors found that the completeness of hydrolysis for the determination of quercetin, kaempferol, myricetin, luteolin and apigenin investigated in cranberry, onion, leek, lettuce, endive and celery depended on both the reaction period and acid concentration, and that the matrix composition was an important factor determining the rate of hydrolysis. therefore, the influence of hydrolysis times up to 6h (0.5, 1, 2, 3, 4, 6h) and three different hydrochlorid acid concentrations (1.2 m hcl, 1.6 m hcl, 2 m hcl) on a peak area of quercetin and kaempferol in the same freeze-dried broccoli material was investigated. figs. 1 and 2 show the results for quercetin and kampferol, respectively, in broccoli florets. for both flavonols, the highest concentration in broccoli was reached when 1.6 m hcl was used, with a hydrolysis time of 2h. a hydrolysis time of 4 h and more led to a loss of flavonols. this corresponds to studies performed by hertog et al. (1992) who proposed a hydrolysis with 1.2 m hcl in boiling 50% aqueous methanol with a reaction period not exceeding 2h and, eventually, a second analysis under identical conditions with a reaction period of 4h for the quantification of kaempferol glucosides. we found that 1.2 m hcl needs longer hydrolysis times of 3 h for the determination of quercetin and kaempferol in broccoli, and is therefore ineffective. 1.6 m hcl and a hydrolysis time of 2 h were therefore used for all analyses. using a prodigy ods column and optimised water (tfe, thf)/ acetonitrile gradient (see material and method), a good separation of quercetin and kaempferol from other unidentified compounds could be attained within 15 minutes. the recovery of quercetin and kaempferol in broccoli florets spiked with 50% of the original level was 93% for both flavonols. coefficients of variation ranged from 4.3% to 3.5% for quercetin and kaempferol, respectively. 0 20 40 60 80 100 120 0 1 2 3 4 5 6 7 time of hydrolysis % p e a k a re a 1.2 m hcl 1.6 m hcl 2.0 m hcl fig. 1: influence of hydrolysis time and acid concentration on quercetin. results are expressed as a percentage of the highest peak area found in broccoli florets. the values represent the mean of two replicates ± standard deviation. flavonoids in broccoli 137 138 angelika krumbein, heidi saeger-fink, ilona schonhof effect of head ontogeny the changes of quercetin and kaempferol with head ontogeny showed the same trend in the three experiments, enabling the values to be averaged over three years. heads of over maturity stage (stage 5, first individual flowers visible) had the highest contents of quercetin and kaempferol (figs. 3-5). valejo et al. (2003) also found that the total flavonoid concentration in broccoli increased from the first development stage until over maturity stage, although the head weights investigated by vallejo et al. (2003b) were far lower – with an average of 260 g for fully developed heads compared to 352 g for ‘emperor’ and ‘viola’, as well as 396 g for ‘marathon’ in our experiments. however, the genotype fundamentally determined the quantity and course of the increase in flavonols. the highest concentration of quercetin and kaempferol was found in the cultivar viola in the over maturity stage, with 6.8 and 9.9 mg (100 g fm)-1, respectively. the concentration of quercetin increased 3-fold in heads of over maturity stage compared to the beginning of the head formation (stage 1) in ‘emperor’, 5-fold in ‘marathon’ and 14-fold in ‘viola’. kaempferol correspondingly increased 5-fold in ‘emperor’, 8-fold in ‘marathon’ and 17-fold in ‘viola’. this means that kaempferol increased more intensively than quercetin during head development. while a continuous increase in quercetin and kaempferol took place in ‘viola’ in all examined head stages of development, both flavonols increased in ‘marathon’ in fully developed heads (stage 4), and once again in heads of over maturity stage (stage 5). similar results were obtained found for kaempferol in ‘emperor’. the concentration of quercetin remained unchanged in ‘emperor’, even up to the fully developed heads. changes in the ratios of quercetin to kaempferol were determined during head ontogeny. the proportional composition of about 45% quercetin and 55% kämpferol remained relatively constant from the head formation up to fully developed heads in ‘viola’; the proportion of kaempferol then increased up to 59% in heads of over maturity stage. the proportion of kaempferol increased during head ontogeny by 17% and 15% in ‘emperor’ and ‘marathon’, respectively. differences in the quantitative formation of quercetin and kaempferol during head formation may be caused by the various enzymes involved in each flavonol’s synthesis from the flavanone naringenin. the hydroxylation of naringenin to dihydrokaempferol is catalysed by the enzyme flavanone 3-hydroxylase (fht), and further conversion to kaempferol is catalysed by the enzyme flavonol synthase (fls) (forkmann and heller, 1989). in contrast, besides these enzymes the biosynthesis of quercetin also needs the enzyme flavonoid 3’hydroxylase (f3’h), which uses the flavanone naringenin and/or the 0 20 40 60 80 100 120 0 1 2 3 4 5 6 7 time of hydrolysis % p e a k a re a 1.2 m hcl 1.6 m hcl 2.0 m hcl fig. 2: influence of hydrolysis time and acid concentration on kaempferol. results are expressed as a percentage of the highest peak area found in broccoli florets. the values represent the mean of two replicates ± standard deviation. b aaaa c b ab a a 0 2 4 6 8 10 1 2 3 4 5 head development stage m g (1 00 g fm )1 quercetin kaempferol fig. 3: quercetin and kaempferol concentrations of broccoli florets with advanced head development during five development stages in cv emperor. values represent the mean of three experiments and three replications. different letters indicate significant differences during head development for each flavonol (p<0.05 by tukey’s hsd test). d c bc ab a d c bc ab a 0 2 4 6 8 10 1 2 3 4 5 head development stage m g (1 0 0 g fm )-1 quercetin kaempferol a a ab b c a a a b c 0 2 4 6 8 10 1 2 3 4 5 head development stage m g (1 00 g fm )1 quercetin kaempferol fig. 4: quercetin and kaempferol concentrations of broccoli florets with advanced head development during five development stages in cv marathon. values represent the mean of three experiments and three replications. different letters indicate significant differences during head development for each flavonol (p<0.05 by tukey’s hsd test). fig. 5: quercetin and kaempferol concentrations of broccoli florets with advanced head development during five development stages in cv viola. values represent the mean of three experiments and three replications. different letters indicate significant differences during head development for each flavonol (p<0.05 by tukey’s hsd test). flavonoids in broccoli 139 dihydroflavonol dihydrokaempferol as a substrate for quercetin biosynthesis and/or hydroxylates kaempferol directly to quercetin (forkmann and heller, 1989). the activity of f3’h was found in the microsomal fraction; the reaction required molecular oxygen and nadph (forkmann and heller, 1989). according to different applications, different head development stages of broccoli should be produced and offered to guarantee a high level of the desired flavonols quercetin and kaempferol. dependent on the genotype, fully developed broccoli heads eaten by the consumers already contain relatively high concentrations of flavonols. out of the investigated cultivars, ‘viola’ had the highest flavonol concentration. consumers should not only eat green broccoli, but could also benefit especially from consuming the violet variety, although, purple broccoli does not seem to correspond to consumers’ aesthetic expectations (schonhof et al., 2004). mini broccoli as a new trend to market vegetables has lower contents of flavonols than the commercial stage, which indicates a reduction in health potentials. harvesting broccoli heads of over maturity stage resulted in maximum flavonol levels. for this reason, these stages should be used as raw material, e.g. for the design of new functional foods. acknowledgements this research was supported by the german federation, and the federal states of brandenburg and thuringia. we would like to thank a. jankowsky for her flavonoid analyses and u. zentner, and e. buesch for their technical assistance. references andreotti, c., costa, c., treutter, d., 2006: composition of phenolic compounds in pear leaves as affected by genetics, ontogenesis and environment. scientia hort. 109,130-137. bergquist, s.m., gertsson, u.e., knuthsen, p., olsson, m.e., 2005: flavonoids in baby spinach (spinacia oleracea l.): changes during plant growth and storage. j. agric. food chem. 53, 9459-9464. halliwell b., 2006: polyphenols: antioxidant treats for healthy living or covert toxins. j. food agric. 86, 1992-1995. hertog, m.g.l., feskens, e.j.m., hollman, p.c.h., katan, m.b., kromhout, d., 1993: dietary antioxidant flavonoids and risk of coronary heart disease: the zutphen elderly study. lancet 349, 699-699. hertog, m.g.l., hollman, c.h., venema, d.p., 1992: optimization of a quantitative hplc determination of potentially anticarcinogenic flavonoids in vegetables and fruits. j. agric. food chem. 40, 1591-1598. howard, l.r., talcot, s.t., brenes, c.h., 2000: changes in phytochemical and antioxidant activity of selected pepper cultivars (capsicum species) as influenced by maturity. j. agric. food chem. 48, 1713-1720. forkmann, g., heller, w. 1998: biosynthesis of flavonoids. in: sankawa, u. 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2003b: changes in broccoli (brassica oleracea l. var. italica) health-promoting compounds with inflorescence development. j. agric. food chem. 51, 3776-3782. williamson, g., manach, c., 2005: bioavailability and bioefficacy of polyphenols in humans. ii. review of 93 intervention studies am. j. cin. nutr. 81 (suppl.), 243-255. winkhoff, j., 1992: integrierter anbau von gemüse in der bundesrepublik deutschland. zentralverband gartenbau, bundesfachgruppe gemüsebau, bonn. address of the authors: dr. angelika krumbein, heidi saeger-fink, ilona schonhof, institute of vegetable and ornamental crops großbeeren/erfurt e.v., department quality, theodor-echtermeyer-weg 1, d-14979 großbeeren, germany corresponding author: krumbein@igzev.de << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb 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/pagesize [907.087 680.315] >> setpagedevice journal of applied botany and food quality 86, 126 132 (2013), doi:10.5073/jabfq.2013.086.017 agricultural biotechnology and phytopathology laboratory, department of botany, university of karachi, pakistan impact of seaweeds on fluorescent pseudomonas and their role in suppressing the root diseases of soybean and pepper syed ehteshamul-haque, ghulam nabi baloch, viqar sultana, jehan ara, r.m. tariq, mohammad athar* (received october 4, 2012) summary incorporation of organic matter in soil before sowing of seeds or transplanting the seedlings has been reported to increase microbial activity in soil, especially fluorescent pseudomonas that play a vital role in suppressing the root rotting fungi and parasitic nematodes invading plant roots. in this study, dry powder of seaweeds sargassum binderi, s. tenerrimum, halimeda tuna, stoechospermum marginatum, padina tetrastromatica, stokeyia indica and solieria robusta were applied as soil amendment two weeks before sowing of soybean seeds or transplanting of pepper seedlings in screen house and in field experiments. application of some seaweeds and topsin-m (fungicide) and carbofuran (nematicide) showed more or less similar suppressive effects on root rotting fungi macrophomina phaseolina, rhizoctonia solani, fusarium solani and root knot nematode meloidogyne javanica on soybean and pepper plants. seaweed also showed a positive effect on plant growth by enhancing fresh shoot weight and plant height. incorporation of seaweeds in soil increased the population of fluorescent pseudomonas around the roots of soybean and pepper compared to a non-seaweed control. however, the increased population of fluorescent pseudomonas around the roots did not correlate with disease suppression. introduction management and manipulation of natural communities of antagonistic microorganisms through organic amendments have received less attention, in spite of the fact that these strategies have resulted in highly effective forms of biological control (hoitink and boehm, 1999). soil amendments have the potential to provide disease control through a variety of mechanisms, including chemicals which produce antimicrobial compounds during decomposition (brown and morra, 1997; tenuta and lazarovits, 2002), and biological (chen et al., 1988; mazzola, 2004). plants with therapeutic effects have received the attention of scientists as an alternate method of disease control, which would also protect our environment from the use of hazardous chemicals. the direct application of seaweeds in farming is a practice that extends over hundreds of years, and it is often more successful than using chemical fertilizers. seaweeds contain elaborated secondary metabolites that play a significant role in the defense of the host against predators and parasites (paracer et al., 1987; sultana et al., 2008; 2009). the use of brown algae, sargassum spp., showed significant (p<0.05) control of root infecting fungi on sunflower (ara et al., 1996) and root knot nematode in okra (ara et al., 1997). there are reports that the delivery of microbial antagonists with urban and agricultural wastes as mulches was found to be very effective in suppressing root pathogens of avocado and citrus (casale et al., 1995). similarly, addition of some compost to soil increased the population of plant growth promoting rhizobacteria (pgpr) in the tomato rhizosphere exhibiting antagonism towards fusarium oxysporum f. sp. radicis-lycopersici, pythium ultimum and rhizoctonia solani (alvarez et al., 1995). the present report describes the effect of soil amendment with seaweeds on root diseases of soybean in screen house experiments. the impact of soil amendments on the population of fluorescent pseudomonas and their role in protecting soybean and pepper from root rotting fungi and root knot nematode under field condition has also been reported. materials and methods seaweeds dictyota indica, padina tetrastromatica, stoechospermum marginatum, stokeyia indica, sargassum swartzii, solieria robusta and halimeda tuna collected from buleji beach, karachi, were used in these experiments, topsin-m (fungicide) and carbofuran (nematicide) were used to compare the efficacy of these seaweeds with common commercial pesticides. the experiments were conducted both under screen house and field conditions on soybean (glycine max (l.) merr.), and under field condition on pepper (capsicum annuum l.). screen house experiment dry powder of seaweeds was mixed with sandy loam soil (ph 8.1) to give a concentration of 1.0% w/w. the soil was naturally infested with 3-7 sclerotia of macrophomina phaseolina g-1 of soil as determined by wet sieving and dilution plating (sheikh and ghaffar, 1975), 2-6% surface colonization of rhizoctonia solani on sorghum seeds (10 sterilized seeds were spread on 100 g wet soil in a petri dish with 5 replicates of each sample) used as baits (wilhelm, 1955) and 3,000 cfu g-1 of soil of a mixed population of fusarium oxysporum and f. solani as determined by soil dilution (nash and snyder, 1962). one kg of amended soil was transferred to 12 cm diam. clay pots. the pots were watered daily to allow the decomposition of the organic substrate. after three weeks, 6 seeds of soybean were sown in each pot and pots were kept randomized on a screen house bench of department of botany at 50% whc (keen and raczkowski, 1921) with four replicates of each treatment. after germination, only four seedlings were kept and excess were removed. the seedlings were inoculated with meloidogyne javanica eggs/juveniles at 2000 eggs/j2 (2nd stage juveniles) per pot. topsin-m (20 ml/pot of 200 ppm) served as positive control against root infecting fungi, whereas carbofuran at 0.1 g per pot served as positive control against nematode. to determine the efficacy of seaweeds and pesticides on the root pathogens and plant growth, plants were uprooted after six weeks of nematode inoculation. observations were made on plant height, fresh shoot weight, root length and root weight. nematode infection was determined by counting the numbers of galls per root system (taylor and sasser, 1978). to determine nematode penetration and infection by root-infecting fungi, roots from each plant were cut into 1cm long pieces and five pieces of tap roots from each plant were used for assessment of fungal infection. the remaining roots were mixed thoroughly, and 1 g sub-sample was wrapped in muslin cloth and dipped in boiling 0.25% acid fuchsin stain for 3-5 minutes. * corresponding author impact of seaweeds on fluorescent pseudomonas 127 roots were left in the stain to cool, and then washed under tap water to remove excess stain. roots were then transferred to vials containing glycerol and water (1:1 v:v) with a few drops of lactic acid. roots were macerated in an electric blender for 45 seconds and the resulting slurry was suspended in 50 ml water. numbers of juveniles and females in 5 ml sub samples were counted with the aid of a dissecting microscope and numbers of nematode/g root were calculated (siddiqui and ehteshamul-haque, 2001). to determine the incidence of fungal infection, 1 cm long root pieces from tap roots (five pieces from each plant) were surface disinfested with 1% ca(ocl)2 and plated onto potato dextrose agar amended with penicillin (100,000 units/litre) and streptomycin (0.2 g/litre). colonies of macrophomina phaseolina, rhizoctonia solani and species of fusarium were recorded after incubation for 5 days at 28°c. the experiment was conducted twice. data were subjected to analysis of variance (anova) and means were separated using the least significant difference (lsd) according to gomez and gomez (1984). field experiments the efficacy of seaweeds were also examined in 2 x 2 m field plots at the crop diseases research institute, pakistan agricultural research council, karachi university campus, using soybean and pepper as test crops. dry powder of seaweeds: padina tetrastomatica, stoechospermum marginatum, stokeyia indica, sargassum swartzii, solieria robusta, halimeda tuna, and dictyota indica were mixed in sandy loam soil at 70 g per two meter rows and watered 2-3 days interval to allow the organic matters to decompose. the soil had a natural infestation of 6-18 sclerotia/g of soil of macrophomina phaseolina, 5-12% colonization of rhizoctonia solani on sorghum seeds used as baits and 2800 cfu/gm of soil of mixed population of fusarium oxysporum and f. solani. after two weeks, seeds of soybean (glycine max) were sown at 50 seeds per two meter row. after germination, each row was inoculated with aqueous suspension of meloidogyne javanica eggs/juveniles at 2000/two meter row. seeds treated with topsin-m served as positive control against root infecting fungi while carbofuran at 0.5 g per meter served as positive control against nematode. each treatment was randomized and replicated four times. plants were watered 2-3 days intervals depending upon requirement of plants. to assess the effect of seaweeds on infection of root infecting fungi and root knot nematode, plants were uprooted after 30 and 60 days (at 4 plants per replicate; a total of 16 plants per treatment) of nematode inoculation. roots were washed under running tap water and incidence of root infecting fungi and root knot nematode on roots were determined as described above. population of fluorescent pseudomonas around the roots was determined using s-1 selective medium for fluorescent pseudomonas (gould et al., 1985). data on plant growth were also recorded, where plant heights, root lengths fresh shoot weights and root weights were recorded for 4 plants per replicate and averaged. in case of pepper, three-week-old seedlings of equal size, raised in steam sterilized soil were transplanted in each row at 12 seedlings per row. after one week of seedling transplantation, each row was inoculated with aqueous suspension of meloidogyne javanica eggs/ juveniles at 2000/ two meter row. other details were similar except observations were recorded after 45 and 90 days of nematode inoculation. results screen house experiment application of seaweeds stokeyia indica, solieria robusta and halimeda tuna caused a complete suppression of macrophomina phaseolina infection on soybean roots (tab. 1), whereas 6.2% rhizoctonia solani infection was observed only in control plants. of the seaweeds used, s. robusta also caused complete suppression of fusarium solani infection. other seaweeds p. tetrastromatica, stoechospermum marginatum, sargassum swartzii, halimeda tuna and dictyota indica also significantly (p<0.05) reduced f. solani infection (tab. 1). seaweeds also caused significant suppressive effect on nematode infection by reducing the numbers of galls per root system and nematode’s penetration in roots. maximum reduction in gall formation was achieved by the application of s. swartzii, whereas h. tuna caused maximum reduction in nematode penetration in roots followed by solieria robusta and stoechospermum marginatum (tab. 1). seaweeds also showed significant beneficial effect on plant growth. maximum increase in root length was produced by solieria robusta followed by tab. 1: effect of seaweeds on the infection of root rotting fungi macrophomina phaseolina, rhizoctonia solani, fusarium solani, f. oxysporum on soybean roots after 45 days of nematode inoculation in screen house experiment. treatments m. phaseolina r. solani f. solani no. of knots females/juveniles infection % per gram roots control 18.7 6.2 25 52.6 337.5 topsin-m 18.7 0 12.5 50.9 420 carbofuran 0 0 6.2 45.2 510 stokeyia indica 0 0 18.7 28.2 147 halimeda tuna 0 0 12.5 26.5 40 dictyota indica 6.2 0 6.2 43.8 182 padina tetrastromatica 6.2 0 12.5 49.6 270 sargassum swartzii 12.5 0 6.2 19.0 112 stoechospermum marginatum 6.2 0 12.5 46.1 77 solieria robusta 0 0 0 28.9 76 lsd0.05 for fungal pathogens treatments= 10.61 pathogens= 5.82 37.61 1661 1mean values in column showing differences greater than lsd values are significantly different at p< 0.05. 2mean values in rows for fungi showing differences greater than lsd values are significantly different at p< 0.05. 128 s. ehteshamul-haque, g.n. baloch, v. sultana, j. ara, r.m. tariq, m. athar stokeyia indica. solieria robusta and stokeyia indica also caused maximum fresh root weight while maximum shoot length was achieved by the application of s. indica (tab. 2). field experiment soybean after 30 days, no infection of macrophomina phaseolina was found on any plant. all the test seaweeds caused a significant (p<0.05) reduction in rhizoctonia solani, fusarium solani and f. oxysporum infection (tab. 3). application of dictyota indica, padina tetrastromatica, sargassum swartzii, and stoechospermum marginatum resulted in complete suppression of f. solani. padina tetrastromatica, s. marginatum and solieria robusta also caused complete inhibition of f. oxysporum on soybean roots (tab. 3). effectivenes of s. marginatum and s. robusta were also found after 60 days. seaweeds caused a significant increase in pgpr population in the rhizosphere of soybean. dictyota indica caused maximum increase in pgpr population in soil around the roots (tab. 4). greater plant height was produced by s. robusta, whereas maximum fresh shoot weight was achieved by the application of s. marginatum (tab. 4). after 60 days, halimeda tuna and solieria robusta completely suppressed macrophomina phaseolina infection. while other seaweeds stokeyia indica, dictyota indica and stoechospermum marginatum also significantly (p<0.05) reduced m. phaseolina (tab. 3). all the test seaweeds also significantly (p<0.05) prevented rhizoctonia solani and fusarium solani infection. complete suppression of f. oxysporum was also achieved by the application of h. tuna, s. marginatum and solieria robusta (tab. 3). tab. 2: effect of seaweeds on the growth of soybean after 45 days of nematode inoculation in screen house experiment. treatments plant height (cm) fresh shoot weight (g) root length (cm) fresh root weight (g) control 32.8 4.0 32.6 3.05 topsin-m 35.6 3.5 35.6 3.1 carbofuran 37.6 3.5 27.4 3.8 stokeyia indica 46.3 4.5 41.2 4.6 halimeda tuna 42.8 4.1 35.5 3.6 dictyota indica 44.9 3.8 35.9 3.9 padina tetrastromatica 40.4 4.3 34.6 3.5 sargassum swartzii 41.7 4.3 33.2 4.3 stoechospermum marginatum 39.9 4.0 42.9 4.0 solieria robusta 43.4 4.4 33.1 4.6 lsd0.05 14.71 0.81 10.11 1.01 1mean values in column showing differences greater than lsd values are significantly different at p< 0.05. tab. 3: effect of seaweeds on the infection of root rotting fungi macrophomina phaseolina, rhizoctonia solani, fusarium solani f. oxysporum and meloidogyne javanica on soybean roots in field experiment. treatments m. phaseolina r. solani f. solani f. oxysporum no. of knots females/juveniles per gram roots 30 days 60 days 30 days 60 days 30 days 60 days 30 days 60 days 30 days 60 days 30 days 60 days control 0 25 62.5 87.5 25 75 43.7 18.7 0.81 18 6.8 165 topsin-m 0 0 12.5 62.5 0 25 0 6.2 1.1 11 0.5 77 carbofuran 0 0 0 18.7 0 6.2 0 0 1.8 17 1.1 87 stokeyia indica 0 12.5 31.2 56.2 12.5 25 25 6.2 0.62 12 6.7 30 halimeda tuna 0 0 18.7 50 18.7 12.5 6.2 0 0.43 13 4.7 70 dictyota indica 0 6.2 6.2 62.5 0 18.7 6.2 6.2 1.1 11 6.7 35 padina tetrastromatica 0 50 12.5 56.2 0 56.2 0 12.5 1.0 10 3.8 120 sargassum swartzii 0 31.2 25 50 0 43.7 18.7 12.5 0.43 7 1.3 37 stoechospermum marginatum 0 12.5 18.7 18.7 0 31.2 0 0 0.72 9 1.3 25 solieria robusta 0 0 18.7 25 6.2 31.2 0 0 0.48 8 0.5 42 treatments= 14.41 pathogens= 9.11 ns ns ns 931 1mean values in column showing differences greater than lsd values are significantly different at p< 0.05. 2mean values in rows for fungi showing differences greater than lsd values are significantly different at p< 0.05. impact of seaweeds on fluorescent pseudomonas 129 stoechospermum marginatum caused maximum reduction in nematode infection by reducing nematodes penetration in roots followed by s. indica (tab. 3). greater root weight was produced by s. indica. highest increase in pgpr population was achieved by the application of padina tetrastromatica (tab. 4). greater plant height and fresh shoot weight were produced by h. tuna, whereas maximum root length was resulted by the application of s. robusta followed by p. tetrastromatica (tab. 4). pepper after 45 days, application of seaweeds sargassum binderi, s. tenerrimum, halimeda tuna, stoechospermum marginatum, padina tetrastromatica and stoekyia indica caused complete suppression of root rotting fungi m. phaseolina and r. solani. highest reduction in f. solani infection was achieved by the application of stoekyia indica and stoechospermum marginatum (tab. 5). seaweeds also caused significant suppressive effect on root knot nematode. highest reduction in root galls was caused by solieria robusta followed by topsin-m (tab. 5). halimeda tuna also significantly reduced gall formation on roots as compared to control. incidence of roots associated fluorescent pseudomonads did not show any correlation with diseases suppression (tab. 6). in many cases, increased population of fluorescent pseudomonas in the rhizosphere did not cause greater reduction of root rotting fungi and root knot nematode as compared to plants having less population of fluorescent pseudomonas in the rhizosphere. greater plant height was achieved by the application of stoekyia indica, while s. robusta tab. 4: effect of seaweeds on the growth of soybean and population of rhizospheric fluorescent pseudomonas (pgpr) in field experiment. treatments plant height (cm) fresh shoot weight (g) root length (cm) fresh root weight (g) pgpr (x1000) 30 days 60 days 30 days 60 days 30 days 60 days 30 days 60 days 30 days 60 days control 12.6 26.4 2.0 11.1 6.8 6.7 0.39 1.06 41 497 topsin-m 13.9 34.4 2.4 14.7 7.3 12.9 0.44 1.2 50 256 carbofuran 14.4 38.0 3.0 18.0 6.5 13.0 0.40 1.6 93 111 stokeyia indica 15.4 30.1 3.1 14.4 6.3 14.3 0.38 3.5 49 627 malimeda tuna 12.0 49.7 2.9 23.4 7.5 12.9 0.57 1.3 250 667 dictyota indica 14.2 36.2 2.7 13.5 7.1 13.2 0.38 1.1 306 496 padina tetrastromatica 14.1 31.8 2.9 11.2 6.5 14.4 0.37 1.0 200 687 sargassum swartzii 16.0 31.6 2.5 11.0 6.7 10.6 0.39 0.9 187 455 stoechospermum marginatum 15.9 35.5 3.2 12.1 7.1 12.9 0.40 0.9 113 647 solieria robusta 17.6 35.9 2.9 13.9 6.8 15.8 0.8 1.2 82 377 lsd0.05 4.61 10.31 1.31 ns 1.41 3.91 0.71 2.61 1951 3651 1mean values in column showing differences greater than lsd values are significantly different at p< 0.05. tab. 5: effect of seaweeds on the infection of root rotting fungi macrophomina phaseolina, rhizoctonia solani, fusarium solani and root knot nematode meloidogyne javanica on pepper roots in field experiment. treatments m. phaseolina r. solani f. solani no. of knots females/juveniles per gram roots 45 days 90 days 45 days 90 days 45 days 90 days 45days 90 days 45 days 90 days control 45 25 31.2 25 62.5 31.2 38.5 18 23 77.5 topsin-m 0 0 6.2 0 37.5 0 9.0 11 8.5 52.5 carbofuran 0 6.2 0 0 12.5 6.2 17.3 17 15.2 24.5 sargassum binnderi 0 6.2 0 0 18.7 6.2 26.1 12 16.7 45.0 s.tenerrimum 0 6.2 0 6.2 18.7 6.2 29.4 13 13.7 49.2 halimeda tuna 0 12.5 0 6.2 25 0 13.0 11 15.7 32.0 stoechospermum marginatum 0 0 0 0 6.2 0 26.7 10 13.2 24.2 padina tetrastromaticai 0 0 0 0 25 0 13.6 7 17.5 39.5 stokeyia indica 0 6.2 0 0 6.2 6.2 18.4 9 11.2 73.2 solieria robusta 6.2 0 0 0 18.7 0 8.4 8 6.5 22.2 lsd0.05 treatments = 6.71 fungal pathogens = 3.72 23.11 14.31 11.31 41.81 1mean values in column showing differences greater than lsd values are significantly different at p< 0.05. 2mean values in rows for fungi showing differences greater than lsd values are significantly different at p< 0.05. 130 s. ehteshamul-haque, g.n. baloch, v. sultana, j. ara, r.m. tariq, m. athar produced maximum fresh shoot weight while stoechospermum marginatum produced maximum root length (tab. 6). seaweed amendment application was still found effective after 90 days in suppressing the macrophomina phaseolina, rhizoctonia solani and fusarium solani infection (tab. 5). solieria robusta, padina tetrastromatica and stoechospermum marginatum completely controlled the m. phaseolina, r. solani and f. solani infection on pepper roots (tab. 5). solieria robusta and s. marginatum significantly (p<0.05) reduced gall formation and penetration of nematodes in roots (tab. 5). population of fluorescent pseudomonads in the pepper rhizosphere did not correlate with disease suppression (tab. 6). greater plant height was observed for s. robusta followed by s. indica. the highest fresh shoot weight was produced by using stoekyia indica followed by solieria robusta. greater root length was found in plants grown in s. marginatum amended soil (tab. 6). discussion control of root-infecting fungi using antagonistic plants and phytochemicals offers an alternate strategy to the prevalent use of synthetic pesticides. a wide variety of plant materials have been found effective against plant parasitic fungi associated with crop plants (ehteshamul-haque et al., 1995; 1996; mansoor et al., 2007; mazzola, 2004). plant pathogenic fungi and plant parasitic nematodes are serious threat to modern agriculture and important limiting factor of low yield of most of the field crops. marine biosphere is an untapped reservoir of agrochemically potent compounds. in this study, seaweeds like sargassum swartzii, solieria robusta, stoechospermum marginatum, halimeda tuna, and stokeyia indica showed more or less similar suppressive effect on root rotting fungi and root knot nematode like chemical fungicides (topsin-m) and nematicide (carbofuran). considerable evidence has been accumulated in recent years to support and identify the benefits associated with the use of seaweed in crop production systems. seaweed extracts have been reported to increase plant resistant to pests and diseases, plant growth, yield and quality (yvin et al., 1989; jolvet et al., 1991; verkleij, 1992). application of seaweed to plants can result in decreased levels of nematode attack (ara et al., 1997; wu et al., 1997; 1998). seaweeds contain elaborate secondary metabolites that play a significant role in the defense of the host against predators and parasites which offer a potential novel approach to control population of plant parasitic nematodes (jacobs et al., 2003; paracer et al., 1987). in this study, application of some seaweed significantly increased rhizosphere population of fluorescent pseudomonas (pgpr) on soybean and pepper. pgpr have been reported to improve plant growth either through direct stimulation or by suppression of pathogens (raajmakers and weller, 1998; weller et al., 2002). of the various rhizosphere bacteria, fluorescent pseudomonads are aggressive colonizers of the rhizosphere of various crop plants and have broad spectrum antagonistic activity against plant pathogens (parveen et al., 1998; weller et al. 2002). species of pseudomonas are also reported to induce systemic resistance in plants against invading pathogens (de meyer et al., 1999; zhou and paulitz, 1994). these characteristics make these species good candidates to use for biocontrol against soil-borne plant pathogens (de meyer et al., 1999; parveen et al., 1998; weller et al., 2002). pseudomonas aeruginosa has been reported to suppress root knot infection on pepper, water melon, guar, pumpkin (parveen et al., 1998), okra (ara et al., 1997) and tomato (siddiqui and ehteshamulhaque, 2001). the increased population of pgpr in this study however did not correlate with disease suppression. the seaweed could affect cell metabolism through the induction of the synthesis of antioxidant molecules which could favor plant growth and plant resistant to stress (zhang and schmidt, 2000). anti-oxidants enzymes provide a degree of crop protection from free radical oxidants arising from normal metabolism and any number of biotic and abiotic stresses. wu et al., (1997) demonstrated that the betaines present in different extract decreased the nematode infestation. presumably, besides stimulating the population of pgpr in the rhizosphere, seaweeds suppressed the root rotting fungi and root knot nematode via producing antimicrobial compounds or synthesis of antioxidant molecules. seaweeds contain 1-aminocyclopropane-1-carboxylic acid (acc) which has antimicrobial activity (nelson and van standen, 1985). similarly polyphenols are well known for their antioxidant activity are widely distributed in seaweeds (tariq et al., 2011). tab. 6: effect of seaweeds on the growth of pepper and population of rhizospheric fluorescent pseudomonas (pgpr) in field experiment. treatments plant height (cm) fresh shoot weight (g) root length (cm) fresh root weight (g) pgpr (x1000) 45 days 90 days 45 days 90 days 45 days 90 days 45 days 90 days 45 days 90 days control 27.5 43.9 12.9 32.0 9.4 20.2 4.0 4.8 29.5 3.0 topsin-m 30.5 51.0 16.2 43.4 12.4 20.5 2.8 5.8 46.4 4.2 carbofuran 28.8 48.0 15.5 35.6 21.5 20.2 2.8 4.5 18.9 5.7 sargassum binnderi 27.5 53.1 14.6 36.5 10.9 19.2 2.2 5.1 44.6 53.6 s.tenerrimum 31.4 45.3 18.0 23.7 36.3 17.9 3.1 3.9 61.7 37.0 halimeda tuna 25.8 44.8 14.1 30.3 13.4 18.3 2.1 2.9 36.1 21.6 stoechospermum marginatum 29.4 39.2 11.6 21.6 33.9 35.9 2.5 2.7 12.9 3.0 padina tetrastromaticai 29.5 45.3 13.8 42.1 17.7 19.6 3.0 4.5 87.0 4.5 stokeyia indica 32.3 59.5 21.6 57.4 12.6 22.1 3.7 6.8 59.0 8.3 solieria robusta 29.6 61.6 25.5 52.2 14.2 19.5 2.7 4.3 95.2 15.4 lsd0.05 6.31 15.01 10.51 24.81 5.11 5.61 1.61 3.31 51.71 ns 1mean values in column showing differences greater than lsd values are significantly different at p< 0.05. impact of seaweeds on fluorescent pseudomonas 131 acknowledgments financial support (2005-2007) provided by the pakistan agricultural research council, islamabad under the agricultural linkage program (alp) with usa is sincerely acknowledged. references alvarez, m.a., gauge de, s., antoun, h., 1995: effect of compost on rhizosphere 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ehteshamul-haque, agricultural biotechnology and phytopathology laboratory, department of botany, university of karachi, karachi-75270, pakistan. dr. ghulam nabi baloch, department of botany, university of karachi, karachi-75270, pakistan. 132 s. ehteshamul-haque, g.n. baloch, v. sultana, j. ara, r.m. tariq, m. athar prof. dr. viqar sultana, biotechnology and drug development laboratory, department of biochemistry, university of karachi, karachi-75270, pakistan. prof. dr. jehan ara, post harvest technology and food biochemistry laboratory, department of food science and technology, university of karachi, karachi-75270, pakistan. dr. r.m. tariq, department of zoology, university of karachi, karachi75270, pakistan prof. dr. mohammad athar, pest detection and emergency projects, california department of food and agriculture, 3288 meadowview road, sacramento, ca 95832, usa. e-mail: atariq@cdfa.ca.gov) journal of applied botany and food quality 93, 197 203 (2020), doi:10.5073/jabfq.2020.093.024 1instituto de horticultura, departamento de fitotecnia. universidad autónoma chapingo, estado de mexico 2instituto de alimentos, departmento de ingenieria agroindustrial. universidad autónoma chapingo, estado de mexico fruits of the pitahaya hylocereus undatus and h. ocamponis: nutritional components and antioxidants lyzbeth hernández-ramos1, maría del rosario garcía-mateos1*, ana maría castillo-gonzález1, carmen ybarra-moncada2, raúl nieto-ángel1 (submitted: may 2, 2020; accepted: september 15, 2020) * corresponding author summary the pitahaya (hylocereus spp.) is a cactus native to america. despite the great diversity of species located in mexico, there are few studies on the nutritional and nutraceutical value of its exotic fruits, ancestrally consumed in the mayan culture. an evaluation was made regarding the physical-chemical characteristics, the nutritional components and the antioxidants of the fruits of h. ocamponis (mesocarp or red pulp) and h. undatus (white pulp), species of great commercial importance. the pulp of the fruits presented nutritional and nutraceutical differences between both species. the red pulp of h. ocamponis presented the highest content of betalains (15.94 mg 100 g-1), ascorbic acid (10.13 aae mg 100 g-1) and antioxidant activity (2009.58 μm te 100 g-1) compared to that of h. undatus. the seeds of both species had a higher content of linoleic acid (ω-6) compared to other fatty acids. the underused skin (epicarp) of the white pulp pitahaya presented a higher content of betalains (19.83 mg 100 g-1) than that found in the pulp and the red skin of the other species (13.21 mg 100 g-1). the red pitahaya that is for regional consumption presented a better functional quality. the skin of both species could be a source of pigments in the food industry. keywords: hylocereus ocamponis, hylocereus undatus, linolenic acid, phenolic compounds, betalains and antioxidant activity. introduction in mexico there are located approximately nine of the eighteen species identified so far of the genus hylocereus britton & rose (cálix de dios, 2004). it is one of the most wide y distributed genera of the family cactaceae. it is characterized by a crassulacean acid metabolism (cam) photosynthetic metabolism, which allows the production of biomass in arid and dry conditions according to its habitat (mercado-silva, 2018). however, these hemiepiphytic plants respond to stressful situations, grow in temperate, tropical and semi-arid areas of mexico, in wild or cultivated conditions, in varying annual precipitation (400 to 4000 mm), at altitudes from 0 to 2500 m above sea level (garcía-rubio et al., 2015) and survive at temperatures above 38-40 °c. mexico, central america and northern south america could be considered the center of origin of the genus hylocereus due to the number of species located in those regions. the great adaptability of these cacti has allowed their cultivation in various countries (india, thailand, china, australia, taiwan, malaysia, the philippines, vietnam, cambodia, indonesia, israel, the united states) among others (garcía-rubio et al., 2015; ibrahim et al., 2018). the non-climacteric fruit of the pitahaya is a globose or subglobose berry, with a sweet and juicy pulp (mesocarp) with small seeds, commonly known as dragon fruit because of the presence in the epicarp of bracts. in addition, it is important to point out that “pitaya” and “pitahaya” have been used incorrectly as synonyms (ibrahim et al., 2018; le bellec et al., 2006). “pitaya” corresponds to the genus stenocereus (quiroz-gonzález et al., 2018), while “pitahaya” corresponds to the genus hylocereus (wu et al., 2006). research on pitahaya was done in this study. the fruit of h. ocamponis (salm-dyck) britton & rose, known as pitahaya solferina (red epicarp, red, pink or purple mesocarp) is a native species of mexico (garcía-rubio et al., 2015), little documented, underused and of low commercial demand. it has recently caught the attention of consumers due to the pigmentation of the pulp because of its betalain content (ibrahim et al., 2018). the species h. undatus (haw.) britton & rose (red or pink epicarp, white mesocarp) is the most economically important, cultivated and exported one. finally, another underutilized species is h. megalanthus (k. schumann ex vaupel) ralf baue (yellow epicarp, white mesocarp) of regional consumption, which is cultivated in backyards in different regions of mexico; it is a native species of colombia, one of the important producing countries worldwide (mercado-silva, 2018). currently, the consumption of these exotic fruits is increasing, due to their nutritional, mineral, but mainly functional attributes. studies report that depending on the species (h. monacanthus, h. mega lanthus, h. polyrhizus and h. undatus), the fruit is a source of carbohydrates (pectin, simple sugars, hemicellulose), protein, high fiber content, minerals (mainly potassium, magnesium, calcium, phosphorus in a lower concentration, zinc, iron), antioxidant com pounds (phenolic acids, flavonoids), pigments (betalains), vitamins c and b, triterpenoids (cholesterol, campesterol, stigmasterol, and β-sitosterol) (ibrahim et al., 2018; lim et al., 2010; mercado-silva, 2018), some volatile components (flavor components) and a high content of functional lipids present in the seeds (palmitic, oleic and linoleic acids) (akram and mushtaq, 2019). however, the nutritional and nutraceutical properties of h. ocamponis are little known as well as the inhibitory effect of betanin against steatohepatitis in mice (lugo-radillo et al., 2020). few studies on this species were carried out in other countries (wybraniec et al., 2007), these ingredients provide health benefits, which include the pre vention and treatment of illnesses (wildman and kelley, 2007) and aid in preventing some health problems of this century such as obesity, cardiovascular disorders (badimon et al., 2010), cancer, osteoporosis, arthritis, diabetes and cholesterol among others (das et al., 2012). recent epidemiological studies have shown that the frequent consumption of fruits and vegetables is positively associated with a lower risk of chronic diseases including type 2 diabetes mellitus and obesity. a high consumption of fruits and vegetables ensures an intake of appropriate amounts of nutrients, dietary fibers, and nonnutrient phytochemicals. mercado-silva (2018) have pointed out the nutritional composition of h. undatus (dietary fiber, soluble fiber, insoluble fiber, and protein), the contents of calcium, phosphorus, iron and antioxidants (phenolic compounds and vitamin c) cultivated in mexico. however, the characteristics of h. ocamponis are unknown. 198 l. hernández-ramos, m. del rosario garcía-mateos, a.m. castillo-gonzález, c. ybarra-moncada, r. nieto-ángel in addition, wybraniec et al. (2007) showed the presence of beta cyanins in the peel and flesh of fruits of h. ocamponis and beta cyanins in the peel of h. undatus. these metabolites possess various biological activities from which their antioxidant activity, anticancer properties and antimicrobial activity stand out (wu et al., 2006; gengatharan et al., 2015). betalains are nutraceutical com ponents of restricted distribution, mainly in the family of the order caryophyllales. these pigments are biosynthetically derived from the bethalamic acid, and are grouped into betacyanins (red pigments) and betaxanthines (yellow pigments). they are responsible for the color of the pulp and the skin of cacti fruits such as the pitaya, the prickly pear, the xoconostle and the pitahaya (quiroz-gonzález et al., 2018; lópez et al., 2015). currently, the identification of these metabolites justifies some of the medicinal properties of the different parts of the plant, ancestrally used in the mayan culture (wybraniec and mizrahi, 2002). to the fruit, it is attributed anti-diabetic, anti-cancer, hepatoprotective, anti-inflammatory, prebiotic, hypolesterolemic properties and the reduction of blood pressure (ibrahim et al., 2018). flower infusions are consumed as a diuretic and as a hypoglycemic. regarding those with a skin, for the treatment of some chronic degenerative diseases, mainly due to the presence of some nutraceutical and antioxidant com ponents (betalains, phenolic compounds and flavonoids) (ibrahim et al., 2018). in recent decades, research on the antioxidant properties of cacti fruits have intensified due to the growing interest of the consumer in eating foods with nutraceutical quality that contribute to the improvement of health and the prevention of diseases (ibrahim et al., 2018). there are reports of the chemical components in the fruits of some species grown in other countries, but it must be considered that the different edaphoclimatic conditions can mainly modify their nutraceutical quality and consequently their medicinal properties (akram and mushtaq, 2019; ibrahim et al., 2018). in mexico, despite the great diversity and as a greater center of origin of cacti (novoa et al., 2015) there is little information on the nutritional and nutraceutical quality of most of them. the contribution of this paper focuses on providing information on the physical and chemical characterization as well as the nutritional and nutraceutical quality of the h. ocamponis fruits grown in mexico, information that is not reported. these properties of the h ocamponis fruits were compared with those of h. undatus. both species were cultivated in the same study area. the few studies that have been carried out on these species correspond to fruits grown in the american continent. therefore, the objective was to determine the nutritional characteristics and the content of antioxidant components of the fruits of the red pulp pitahaya (hylocereus ocamponis) and the white pulp pitahaya (h. undatus) cultivated in mexico, in order to contribute with information on its nutraceutical potential, mainly concerning the red pulp dragon fruit with a higher degree of pigmentation. materials and methods vegetal material the fruits of two species of pitahaya (hylocereus undatus and h. ocamponis) were obtained from a commercial produce farm in molcaxac, puebla, mexico (18º43'36'' n and 97º54'48'' w at an altitude of 1843 m above sea level), with a precipitation of 655 mm and a mean annual temperature of 19.2 °c. the fruits were harvested at commercial maturity during the second fruition of the plant, free of pests and physical damage. physical characterization the evaluated variables were the equatorial diameter (ed), length (l), skin thickness, width of bracts and length of the apical bracts using a digital vernier (inox ip54 caliper, grass valley, usa). the relationship between l/ed was considered to determine the roundness index. also, the number of bracts and seeds per fruit of each species was counted. the weight of the fruit, pulp, seeds, skin and the proportionality of pulp, seeds and skin were determined using an electronic scale (scout pro sp2001 ohaus®, usa). the skin firmness was evaluated with a penetrometer (ft 327, qa supplies®, usa) equipped with an 8 mm thick plunger tip. in addition, the color was evaluated with a color tec-pcm® portable colorimeter (cole palmer, illinois, usa) and was expressed in luminosity (l*), hue angle (hue) and color saturation index (chroma). chemical characterization the evaluated chemical parameters were the ph using a potentio meter (hi2221 hanna instruments, woonsocket, usa), the titratable acidity using the technique described by the association of official analytical chemists (aoac, 2005), the total soluble solids (ºbrix) using a digital refractometer (pal-1 atago®, japan), as well as the content of total soluble sugars by the anthrone method described by witham et al. (1971). proximate analysis the pulp with seeds was dried in an oven at 65 °c and ground in a thomas-wiley mill (model 4, thomas scientific, usa). percentages of moisture, lipids, crude fiber, ash and carbohydrates were deter mined using the methods described by the aoac (2005). mineral quantification the contents of p, k, ca, mg, s, na, fe, zn, mn, cu, b, mo and ni were quantified by an inductively coupled plasma optical emission spectrometry (icp-oes, model 725-es, agilent®), prior to a multielement acid digestion in microwaves. the total nitrogen content was determined by the dumas combustion method (aoac, 2005). fatty acid analysis three batches of about 4 kg (8-9 fruits) of fresh pitahaya per batch were processed in order to obtain 70 g seed/batch. the seeds were dried to constant weight in a circulation stove with forced air at 60 °c. the dried pitahaya seeds of both species were crushed sepa rately in a mortar. the extraction of the oil was carried out using the soxhlet method with hexane for 3 hours. the fatty acids were converted into their methyl esters using the method proposed by aoac (2012). the identification and quantification of the fatty acids were performed using a gas chromatograph (hp hewlett packard model 6890 gc system, usa) coupled to a flame ionization detector. the separation was carried out using a capillary column (60 m × 0.25 mm × 0.20 μm), helium as a carrier gas at a constant flow rate of 0.73 ml min-1. the temperature-time conditions of the injector and the detector were adjusted to 250 °c for 19.66 min. nutraceutical quantification preparation of the extract for the quantification of total phenolic compounds, flavonoids and antioxidant activity by abts and frap, an extract of acetone was prepared. the content of the nutraceutical components was determined separately in the skin and in the pulp with seeds according to what is described by wu et al. (2006), with some modifications. for the preparation of the extract, 1 g of the ground vegetable tissue was mixed with 10 ml of 80% (v/v) acetone. the mixture was homogenized by stirring in a vortex, sonicated for 10 min at room temperature and the extraction was carried out twice with the same residue. the extract was kept refrigerated until analysis. nutritional components and antioxidants of hylocereus fruits 199 quantification of total soluble phenolic compounds the quantification of these metabolites was carried out according to the method described by singleton and rossi (1965). it was mixed 0.1 ml of the previously prepared acetone extract, 0.1 ml of the folin-ciocalteu reagent (1 n), 4.5 ml of distilled water and 0.3 ml of 2% (w/v) na2co3. the mixture was incubated at room temperature and in the dark for 2 h. the absorbance of the mixture was read at 760 nm on a spectrophotometer (genesys 10s, thermoscientific, usa). the content of total phenolic compounds was determined using a standard gallic acid curve (y = 0.0011x 0.0084; r2 = 0.998). the results were expressed as mg gallic acid equivalent per 100 g of fresh sample (mg gae 100 g-1fresh weight). flavonoid quantification a mixture was prepared with 0.5 ml of previously prepared acetone extract, 1.5 ml of 95% (v/v) ethanol, 0.1 ml of 10% (w/v) alcl3 solution, 0.1 ml of ch3cook (1 m) solution, and 2.8 ml of distilled water, and then incubated for 30 min. subsequently, the absorbance was determined at 415 nm in a spectrophotometer. the flavonoid concentration was determined from a standard quercetin curve (y = 0.006x 0.0026; r2 = 0.996). results were expressed in mg quercetin equivalents per 100 g of fresh weight (mg qe 100 g-1 fresh weight). betalains quantification the extraction of betalains from the skin and from the pulp was performed separately with 80% (v/v) methanol. for the preparation of the extract, 1 g of the ground vegetable tissue was mixed with 15 ml of 80% (v/v) methanol. the mixture was homogenized by stirring in a vortex, sonicated for 10 min at room temperature, and the extraction was carried out twice with the same residue. subsequently, the absorbance of the extracts was read on a spectrophotometer (genesys 10 s). the content of total betalains, betacyanins and betaxanthines was determined according to the method described by wu et al. (2006) using the formula: b [mg g-1] = a·df·mw·v / ε·l·w; where b = concentration of betacyanins or betaxanthines, a = absorbance at 538 nm (betacyanins) or 483 nm (betaxanthines), df = dilution factor, mw = molecular weight (550 g mol-1 for betanin and 308 g mol-1 for indicaxanthin), v = gauging volume (ml), ε = molar extinction coefficient (betanin: 60,000 l mol-1 cm-1 and indicaxanthin: 48,000 l mol-1 cm-1), w = weight of the sample (g) and l = cell length (1 cm). the results were expressed as content of total betalains (betacyanins + betaxanthines) for every 100 g of fresh sample. ascorbic acid quantification the ascorbic acid (vitamin c) content was determined by iodometric titration using the method proposed by suntornsuk et al. (2002), with modifications. an extract was prepared with 20 g of ground pitahaya pulp and 25 ml of 2n sulfuric acid. the solution was filtered and mixed with 100 ml of distilled water and 3 ml of 1% (w/v) starch as an indicator. the solution was titrated with 0.1n iodine previously standardized with a 0.1n sodium thiosulfate solution. each ml of iodine (0.1 n) was equivalent to 8.806 mg of ascorbic acid. quantification of antioxidant activity abts method. a 7 mm solution of abts (2,2’-azino-bis(3-ethylbenzothiazolin-6-sulfonic acid), sigma-aldrich) in distilled water and another 2.45 mm potassium persulfate solution were prepared. both were combined at a 1:1 ratio. the mixture was left to stand for 16 h in the dark, to allow the generation of the free radical, and was diluted with anhydrous ethanol until obtaining an absorbance of 0.7 ± 0.01 at a wavelength of 734 nm (wu et al., 2006). to determine the antioxidant capacity, 30 μl of the previously prepared acetone extract per species and 3 ml of the abts·+ radical were placed. the mixture was incubated in the dark for 30 min. finally, the absorbance reading of the mixture at a wavelength of 734 nm was taken on a spectrophotometer. the antioxidant activity was quantified using a standard trolox curve (6-hydroxy-2,5,7,8-tetramethylchroman-2carboxylic acid, sigma-aldrich) (y = 29.599x + 9.238; r2 = 0.988). results were expressed in μm trolox equivalents per 100 g of fresh weight (mg te 100 g-1fresh weight). frap method the frap test was carried out according to the procedure described by benzie and strain (1996), with some modifications. it was mixed 100 μl of the previously prepared acetone extract, 3 ml of the frap reagent (2.5 ml of 300 mm acetate buffer at ph = 3.6, 0.25 ml of 10 mm solution of 2,4,6-tripyridyl-s-triazine (tptz; sigma-aldrich) in 40 mm hcl and 0.25 ml of 20 mm fecl3) and 300 μl of h2o. the mixture was incubated for 30 min at 37 °c. subsequently, its absorbance was read from the mixture at a wavelength of 593 nm on a spectrophotometer. the antioxidant activity was calculated using a trolox-based standard curve (y = 0.7419x + 0.0297; r2 = 0.990). results were expressed in μm trolox equivalents per 100 g of fresh weight (mg te 100 g-1 fresh weight). statistical analysis all data was expressed as the mean ± standard error of six repetitions, except for the physical characteristics of the fruit (twenty repetitions). the experimental unit was three pitahaya fruits. a student’s t-test was applied to differentiate the physical, chemical, nutritional cha racteristics and the fatty acid profile between the two pitahaya species. for the analysis of the data of the nutraceutical components and the antioxidant activity, it was performed an analysis of variance and a tukey comparison of means (p ≤ 0.05) using the sas software version 9.2. results and discussion physical characteristics of the fruits the two pitahaya species presented differences in their fruits’ morphological characteristics (shape, color of the epicarp or skin, and quantity and shape of their bracts) (fig. 1). the fruits of h. ocamponis (mesocarp or red pulp) were characterized by their elongated shape with an abundant number of thin bracts compared to the fruits of fig. 1: pitahaya: a) white pulp fruit (hylocereus undatus); b) red pulp fruit (hylocereus ocamponis). 200 l. hernández-ramos, m. del rosario garcía-mateos, a.m. castillo-gonzález, c. ybarra-moncada, r. nieto-ángel h. undatus (mesocarp or white pulp). it is a description similar to that reported by le bellec et al. (2006) for h. ocamponis, who pointed out that the fruits with red pulp, oblong shape and length between 10 and 15 cm were similar to those of h. purpusii. however, in thisresearch, the fruits of h. ocamponis presented thin acicular bracts in their skins, which differentiates them from h. purpusii. color was the most distinctive attribute of the mesocarp of the pitahaya fruit, ranging between red shades (hue) (lower values) for the red pulp of h. ocamponis than in those of h. undatus (tab. 1). however, the pulp of h. undatus presented a higher luminosity (l*) and a low color purity (low chroma values), which translates into a light gray pulp that is not very attractive to the consumer when compared to the red tone pulp of h. ocamponis. vaillant et al. (2005) reported a positive correlation between chroma and the concentration of betacyanins, which are pigments that provide red colorations. in this research, the pulp of h. undatus presented a very low concentration of betalains when compared to the pulp of h. ocamponis. the skin of h. undatus presented a lower hue value when compared to that of h. ocamponis, which means that the skin of the first species has a redder coloration. hue, chroma and luminosity (l*) values in the epicarp were close to those reported by ortiz and takahashi (2015). the red coloration in the pulp and in the skin of the pitahaya (hylocereus spp.) is the result of the synthesis and accumulation of betalain pigments. betalains assist in the prevention of cancer and prevent the oxidation of lipid membranes due to their antioxidant properties. they do not show toxic effects in humans when consumed in fruits in comparison with synthetic pigments and are used in the food, pharmaceutical and cosmetic industries (ibrahim et al., 2018; quiroz-gonzález et al., 2018). the fruits of both species presented an average weight of 478.9 g, with the pulp being the most abundant part of the fruit (62.5%), followed by the skin (34.2%). the two species presented high firmness values (55 to 70 n), higher than that reported by other authors on pitahaya fruits (h. undatus) cultivated in mexico (4.2 7.1 n) (osuna enciso et al., 2011) as well as in other cacti fruits like the pitaya (stenocereus pruinosus and s. stellatus) and the prickly pear (opuntia megacantha) (1.59-2.45 and 14.9 ± 5.2 n, respectively) (garcía-cruz et al., 2016; ríos-alegría et al., 2018), which is a property that provides a longer shelf life to the fruits of hylocereus spp. compared to those of stenocereus spp. it is important to note that the two species of pitahaya studied were found cultivated in a highly calcareous soil (40.02% caco3) which could explain the high firmness values. these results indicated that the pitahaya is a fruit with a hard consistency, making it less susceptible to mechanical damage during its post-harvest handling. awang et al. (2011) re ported that the calcium in the pitahaya fruits (h. polyrhizus) treated with cacl2 increased its firmness from 19.2 to 23.6 n because this element has an important role in the functionality and integrity of the cell membrane when binding to the polar headof phospholipids. abd el-rahman et al. (2019) pointed out that the ca+2 maintains the integrity of the middle lamella of the cell wall due to its union with the pectic acid and the formation of calcium pectates which generates a lower weight loss and a higher firmness. chemical characteristics of the fruits the white pitahaya presented lower values of ph, soluble solids and total soluble sugars compared to those of the red pitahaya (tab. 1). the observed differences are possibly due to the variability between species or due to its cam metabolism, where the co2 fixation is carried out by the enzyme phosphoenolpyruvate carboxylase (pep) and the accumulation in vacuoles of some organic acids (malic acid) in the cacti fruits. these values coincided with those reported by ortiz and takahashi (2015) in the fruits of h. undatus (ph = 4.6; 12.2 ºbx and 0.27% of titratable acidity). flavor is a parameter that determines the quality of a fruit, a result of the relationship between organic acids and sugars (tss/ta). in this research, the tss/ta value (55.5 tss/ta) indicated that the fruits of h. undatus had a more acidic flavor compared to those of h. ocamponis (tab. 1). van to et al. (2002) indicated that vietnamese consumers of white pitahaya (h. undatus) preferred less sweet fruits when the tss/ta ratio was 40. however, it is important to consider that the best evaluation of the taste of a fruit is obtained through a sensory evaluation, which allows to have a knowledge regarding consumer preferences. nutritional components of the fruits the proximate and mineral analysis did not show significant differences in the content of moisture, carbohydrates, crude fiber, na and s between the fruits of the two species (tab. 2). mercado silva (2018) reported values of moisture (83-89 %), ash (0.4-0.68%), p (16-19 mg 100 g-1), fe (0.30-0.65 mg 100 g-1) and ca+2 (6.0-10 mg 100 g-1), in the fruits of the h. megalanthus and the h. undatus species, which are similar to those found in this research. the fruits of the white pitahaya (h. undatus) presented a higher nutritional tab. 1: physical and chemical characterization of pitahaya fruits with white (hylocereus undatus) and red pulp (h. ocamponis). variable h. undatus h. ocamponis morphological characteristics equatorial diameter (cm) 8.60 ± 0.12 a 8.50 ± 0.15 a length (cm) 12.28 ± 0.21 a 13.56 ± 0.21 b shape index 1.43 ± 0.02 a 1.60 ± 0.02 b fruit weight (g) 466.94 ± 18.81 a 490.82 ± 22.21 a mesocarp weight (g fruit -1) 278.26 ± 16.65 a 325.51 ± 28.69 a epicarp weight (g fruit-1) 167.60 ± 7.86 a 153.87 ± 9.53 a seed weight (g fruit-1) 10.26 ± 0.36 a 7.95 ± 0.41 b number of seeds (seeds fruit -1) 6571 ± 652 a 5282 ± 232 a mesocarp (%) 60.84 ± 2.32 a 64.09 ± 2.79 a epicarp (%) 36.86 ± 1.04 a 31.57 ± 0.72 b seeds (%) 2.20 ± 0.08 a 1.62 ± 0.08 b number of bracts 18.00 ± 0.56 a 31.20 ± 1.02 b width of bracts (cm) 4.14 ± 0.15 a 2.94 ± 0.07 b apical bracts length (cm) 6.43 ± 0.83 a 6.08 ± 0.84 a epicarp physical attributes thickness (mm) 5.45 ± 0.18 a 4.44 ± 0.18 b firmness (n) 57.86 ± 2.97 a 65.13 ± 4.63 a lightness (l*) 36.08 ± 2.25 a 35.69 ± 1.34 a hue angle (hue) 19.08 ± 2.50 a 26.44 ± 1.91 b chroma 41.18 ± 2.14 a 42.94 ± 2.45 a pulp physical attributes lightness (l*) 55.71 ± 2.83 a 21.66 ± 2.71 b hue angle (hue) 48.37 ± 6.56 a 16.73 ± 2.11 b chroma 4.09 ± 0.70 a 36.70 ± 2.71 b chemical characterization ph 4.60 ± 0.11 a 5.00 ± 0.02 b titratable acidity 0.33 ± 0.02 a 0.15 ± 0.01 b (ta, % malic acid) total soluble solids (tss, ºbrix) 14.41 ± 0.26 a 15.63 ± 0.40 b tss / ta ratio 51.92 ± 3.09 a 106.36 ± 5.05 b total soluble sugars (%) 11.06 ± 0.63 a 13.14 ± 0.63 b data are expressed as mean ± standard error of six repetitions, except for the physical characteristics of the fruit (twenty repetitions). different letters in the same row indicate significant statistical differences by t-student test (p ≤ 0.05). tss: total soluble solids, ta: titratable acidity. nutritional components and antioxidants of hylocereus fruits 201 value compared to the fruits of h. ocamponis. an exception was the content of fe and cu (tab. 2). the differences in mineral levels between these species were probably due to the differential ex pression of the absorption capacity, transport and accumulation of minerals, because both were grown in a produce farm with the same chemical characteristics in the soil (gupta et al., 2016). on the other hand, in the fruits of both species the predominant mineral was potassium, which had the highest concentration in the white pitahaya, but no na was found. the intake of a white pitahaya fruit could contribute with 20% of the potassium daily requirements (4.5-4.7 g day-1) in persons between 9 and 70 years of age (brown, 2011), contributing to a decrease in blood pressure and in the risk of suffering a cerebrovascular accident by consumers (soto, 2018). in general, the fruits of the white and red pitahaya had a higher content of carbohydrates, lipids, p, k, ca and mg compared to that reported in other cactifruits such as the pitaya (s. pruinosus) (8.510.2%, 0.100.12%, 0.61-0.63%, 3.4-3.6 mg kg-1, 1.1-1.2 mg kg-1, 0.90-0.95 mg kg-1 and 2.15-2.35 mg kg-1, respectively) (garcía-cruz et al., 2013) and with the carbohydrate, protein and lipid content in the xoconostle fruits (opuntia spp.) (4.64-7.25%, 0.14-0.39% and 0.08-0.32%, respectively) (lópez et al. 2015). fatty acid profile of the pitahaya seed oil content such as that of polyunsaturated fatty acids in the seeds of h. undatus (25.49% dry weight) was higher than that found in h. ocamponis (18.11% dry weight), but similar to that reported in h. undatus (28.37% dry weight) by lim et al. (2010). in the seeds of both species, the linoleic acid (omega-6) was the one with the highest concentration (tab. 3). lim et al. (2010) reported higher palmitic, linolenic and linoleic acid values than those found in this research, possibly due to genetic factors, edaphoclimatic factors, sample storage times and oil extraction techniques (akram and mushtaq, 2019). the pitahaya fruit is consumed with seed, which leads to the consumption of fatty acids that are mainly unsaturated and which play an important role in human nutrition and which are associated with a reduced risk of pathologies such as coronary heart and cardiovascular disease (mensink and katan, 1992). nutraceutical content the pulp of the red pitahaya (h. ocamponis) presented the highest levels of betalains and ascorbic acid, as well as the highest antioxi dant activity (tab. 4). however, the skin of the white pitahaya had a higher content of betalains than that of the red pitahaya. furthermore, there were no differences in the antioxidant activity in this tissue between both species. the differences in the concentration between species and tissues are possibly due to genetic variability. when felker et al. (2008) were carrying out research on prickly pear fruits (opuntia ficus-indica) of different pigmentation, they did not find differences in the genomic dna, which is why they pointed out that the absence or presence of pigmentation in this cactus could be due to a defect in the transcription factor associated with the main enzymes involved in the biosynthesis of betalains. the concentrations of betacyanins found in this study were similar to those reported by wu et al. (2006) in the pulp of h. polyrhizus (10.3 ± 0.22 mg 100 g-1 fresh weight). wybraniec and mizrahi (2002) reported higher concentrations in eight species and hybrids of h. polyrhizus, h. purpusii, h. costaricensis and h. undatus culti vated in israel (23 to 39 mg betanin equivalent 100 g-1 of fresh pulp) perhaps due to either analysis methods or differences between species or edaphoclimatic conditions. it is important to note that there are no reports of the levels of betaxanthines (yellow color pigments) in the skin of the pitahaya. the consumption of fruits with these pigments could assist in the prevention of cancer. these pigments also prevent the oxidation of membrane lipids due to their antioxidant properties (livrea and tesoriere, 2006) and do not present toxic effects in humans as some synthetic pigments. these underused resources could be a source of dyes for use in the food and pharmaceutical industries, since these pigments are more stable to changes in ph than the anthocyanins found mainly in strawberries (tanaka et al., 2008). in the present study, the content of phenolic compounds in the tis sues of the two species (tab. 4) was similar and higher than that reported by wu et al. (2006) in the pulp and skin of the pitahaya h. polyrhizus (42.4 ± 0.04 and 39.7 ± 5.39 mg gae 100 g-1). in contrast, the highest flavonoid content was detected in the skin of both species when compared to the pulp. these metabolites have tab. 2: nutritional components in pitahaya fruits with white (hylocereus undatus) and red pulp (h. ocamponis). variable h. undatus h. ocamponis proximate analysis moisture (%) 86.12 ± 0.59 a 86.67 ± 0.53 a carbohydrates (%) 11.05 ± 0.37 a 11.47 ± 0.51 a protein (%) 0.93 ± 0.03 a 0.76 ± 0.02 b lipids (%) 0.67 ± 0.02 a 0.39 ± 0.01 b crude fiber (%) 0.27 ± 0.02 a 0.32 ± 0.01 a ash (%) 0.71 ± 0.03 a 0.43 ± 0.01 b energetic value (kcal 100 g-1) 53.89 ± 1.60 a 52.38 ± 2.03 a mineral content (mg 100 g-1 fw) n 140.18 ± 7.30 a 110.62 ± 5.43 b p 19.43 ± 1.01 a 14.66 ± 0.72 b k 294.23 ± 8.63 a 175.93 ± 8.63 b ca 8.33 ± 0.43 a 6.66 ± 0.33 b mg 33.31 ± 1.74 a 23.99 ± 1.18 b s 13.88 ± 0.72 a 14.66 ± 0.72 a na 0.00 ± 0.00 a 0.00 ± 0.00 a ni 0.00 ± 0.00 a 0.00 ± 0.00 b fe 0. 20 ± 0.01 a 0.36 ± 0.02 b zn 0.28 ± 0.02 a 0.16 ± 0.01 b mn 0.08 ± 0.00 a 0.05 ± 0.00 b cu 0.02 ± 0.00 a 0.02 ± 0.00 b b 0.15 ± 0.01 a 0.13 ± 0.01 b mo 0.00 ± 0.00 a 0.00 ± 0.00 b data are expressed as mean ± standard error of six repetitions. values reported in fresh weight (fw). different letters in the same row indicate significant statistical differences by t-student test (p ≤ 0.05). tab. 3: fatty acid compositions (%) of pitahaya seed oil white (hylocereus undatus) and red pulp (h. ocamponis). variable h. undatus h. ocamponis saturated fatty acid 18.68 ± 0.22 a 21.37 ± 0.27 b monounsaturated fatty acid 28.85 ± 0.35 a 34.07 ± 0.42 b polyunsaturated fatty acid 52.50 ± 0.63 a 44.94 ± 0.56 b palmitic acid (c16:0) 12.22 ± 0.15 a 13.90 ± 0.17 b stearic acid (c18:0) 7.17 ± 0.09 a 8.06 ± 0.10 b oleic acid (c18:1) 27.50 ± 0.33 a 32.68 ± 0.41 b linoleic acid (c18:2) 50.77 ± 0.65 a 44.88 ± 0.56 b linolenic acid (c18:3) 0.36 ± 0.00 a 0.43 ± 0.01 b data are expressed as mean ± standard error of three repetitions. different letters in the same row indicate significant statistical differences by t-student test (p ≤ 0.05). 202 l. hernández-ramos, m. del rosario garcía-mateos, a.m. castillo-gonzález, c. ybarra-moncada, r. nieto-ángel been reported mainly in higher concentrations in the flowers of the family cactaceae (iwashina, 2015). these are particular synthesis sites, as protective metabolites against biotic and abiotic stress and as part of the plant resistance mechanism against diseases (panche et al., 2016). phenolic compounds, due to their diversity and wide distribution, are the most important group of natural antioxidant metabolites. there are epidemiological evidences that show the health benefits and the contribution of these compounds in the pre vention of some degenerative diseases (soto-hernández et al., 2017). these metabolites are compounds with the ability to chelate heavy metals, modulate some enzymes and neutralize free radicals (reactive oxygen species produced by oxidative stress in the orga nism, affecting lipoproteins, lipids of blood plasma and other biomolecules) (soto-hernández et al., 2017). on the other hand, the ascorbic acid is another important constituent of food, which possesses a high antioxidant activity (naidu, 2003). in the present study, the content of ascorbic acid found in both species was lower than the concentration reported by vaillant et al. (2005) in three varieties of hylocereus sp. cultivated in nicaragua (12.3 to 17.1 mg aae 100 g-1 fresh weight), probably because the cam metabolism in the fruits of hylocereus favors the formation of malic acid compared to that of other organic acids. the pulp of both species showed a higher antioxidant activity than in the skin, but in the pulp of h. undatus, it could be associated with a synergistic effect of different metabolites. these results indicated that the pitahaya fruits had a higher antioxidant potential than the fruits of some cacti such as the prickly pear (opuntia ficus-indica) from yellow, red and white cultivars (531, 420 and 430 μm te 100 g-1 fresh weight, respectively) (butera et al., 2002) and that of the red pulp pitaya (stenocereus stellatus) (921 μm te 100 g-1 of pulp fresh weight) (garcía-cruz et al., 2016). pearson’s correlation coefficient showed a positive correlation between betalains (0.49) and the antioxidant activity in the pulp of the red pitahaya (h. ocamponis). in contrast, the correlation was positive between flavonoids (0.87) and phenolic compounds (0.45) with the antioxidant activity in the pulp of the white pitahaya (h. undatus). conclusions the white pulp pitahaya fruit of h. undatus presented a higher nutritional value than the red pulp fruit of h. ocamponis due to a higher content of protein, lipids, n, p, k, ca, mg, b in the mesocarp, and oleic acid along with linoleic acid in the seeds. in contrast, the red pulp of h. ocamponis presented the highest nutraceutical potential given the higher betalain content and antioxidant activity, despite being a fruit of lower commercial demand and consumption. on the other hand, the skin of h. undatus is an underused resource, which could be a source of antioxidant components associated to the presence of betalains, to be used agroindustrially to obtain natural pigments in the food industry. conflict of interest no potential conflict of interest was reported by the authors. references abd el-rahman, a., zagzog, o., el-naggar, n., gad, m. 2019: enhancing quality attributes of guava fruits during cold storage by using edible composite coating (chitosan) and calcium nitrate. j. product. dev. 24(4), 787-805. doi: 10.21608/jpd.2019.72491 akram, s., mushtaq, m. 2019: dragon (hylocereus megalanthus) seed oil. in: ramadan, m. 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pulp and epicarp of pitahaya fruits with white (hylocereus undatus) and red (h. ocamponis). variable hylocereus undatus hylocereus ocamponis pulp epicarp pulp epicarp total betalains (mg 100 g-1) 0.02 ± 0.00 c 19.83 ± 5.06 a 15.94 ± 2.89 ab 13.21 ± 2.19 b betacyanins (mg 100 g-1) 0.01 ± 0.00 c 14.66 ± 3.76 a 11.98 ± 2.03 ab 9.65 ± 1.67 b betaxanthins (mg 100 g-1) 0.01 ± 0.00 c 5.17 ± 1.40 a 3.97 ± 0.88 ab 3.55 ± 0.53 b total soluble phenolic (mg gae 100 g-1) 126.03 ± 29.71 a 139.39 ± 36.47 a 132.47 ± 21.58 a 127.88 ± 12.88 a flavonoids (mg qe 100 g-1) 4.82 ± 0.52 b 22.57 ± 2.91 a 5.32 ± 0.29 b 25.68 ± 2.08 a ascorbic acid (mg aae 100 g-1) 8.50 ± 0.23 b nd 10.13 ± 1.18 a nd aa by abts (mm te 100 g-1) 1118.20 ± 102.71 b 812.60 ± 80.32 b 2009.58 ± 159.66 a 817.35 ± 27.28 b aa by frap (mm te 100 g-1) 506.55 ± 38.48 b 500.24 ± 47.41 b 1164.47 ± 111.91 a 365.69 ± 41.48 b data are expressed as mean ± standard error of six repetitions. values reported in fresh weight. different letters in the same row indicate significant statistical differences by tukey’s test (p ≤ 0.05). gae: gallic acid equivalents, qe: quercetin equivalents, aae: ascorbic acid equivalents, te: trolox equivalents, nd: variable not detected http://dx.doi.org/10.21608/jpd.2019.72491 http://dx.doi.org/10.1007/978-3-030-12473-1_36 http://dx.doi.org/10.5897/ajmr10.541 http://dx.doi.org/10.1111/j.1755-5922.2010.00189.x http://dx.doi.org/10.1021/jf025696p http://dx.doi.org/10.1111/j.1744-7348.2008.00222.x http://dx.doi.org/10.1006/abio.1996.0292 nutritional components and antioxidants of hylocereus fruits 203 garcía-cruz, l., valle-guadarrama, s., salinas-moreno, y., joaquín-cruz, e., 2013: physical, chemical, and antioxidant activity characterization of pitaya (stenocereus pruinosus) fruits. plant food hum. nutr. 68(4), 403-410. doi: 10.1007/s11130-013-0391-8 garcía-cruz, l., valle-guadarrama, s., salinas-moreno, y., lunamorales, c., 2016: postharvest quality, soluble phenols, betalains content, and 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(ed.), handbook of nutraceuticals and functional foods, 1-21. crc press. boca raton, eua. doi: 10.1201/9781420006186.ch1 witham, f.h., blaydes, d.f., devlin, r.m., 1971: experiments in plant physiology. van nostrand reinhold company. new york, usa. 245 p. wybraniec, s., mizrahi, y., 2002: fruit flesh betacyanin pigments in hylocereus cacti. j. agric. food chem. 50(21), 6086-6089. doi: 10.1021/jf020145k wybraniec, s., nowak-wydra, b., mitka, k., kowalski, p., mizrahi, y., 2007: minor betalains in fruits of hylocereus species. phytochemistry. 68(2), 251-259. doi: 10.1016/j.phytochem.2006.10.002 wu, l., hsu, h.-w., chen, y.-c., chiu, c.-c., lin, y.-i., ho, j.a., 2006: antioxidant and antiproliferative activities of red pitaya. food chem. 95, 319-327. doi: 10.1016 / j.foodchem.2005.01.00 orcid lyzbeth hernández-ramos https://orcid.org/0000-0002-6621-2116 maría del rosario garcía-mateos https://orcid.org/0000-0003-2552-3951 ana maría castillo-gonzález https://orcid.org/0000-0002-8661-6433 maria carmen ybarra-moncada https://orcid.org/0000-0002-9634-008x raúl nieto-ángel https://orcid.org/0000-0003-4234-682x address of the corresponding author: maria del rosario garcía-mateos, instituto de horticultura. departamento de fitotecnia. universidad autónoma chapingo. carr. méxico-texcoco km 38.5. chapingo, estado de méxico. méxico. rosgar08@hotmail.com © the author(s) 2020. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1007/s11130-013-0391-8 http://dx.doi.org/10.1016/j.postharvbio.2015.07.004 http://dx.doi.org/10.17129/botsci.282 http://dx.doi.org/10.1016/j.lwt.2015.06.052 http://dx.doi.org/10.1007/s11157-016-9390-1 http://dx.doi.org/10.1111/jfbc.12491 http://dx.doi.org/10.1177/1934578x1501000675 http://dx.doi.org/10.1016/j.foodchem.2009.09.002 http://dx.doi.org/10.1051/fruits:2006021 http://dx.doi.org/10.1177/1934578x20932013 http://dx.doi.org/10.1161/01.atv.12.8.911 http://dx.doi.org/10.1016/b978-0-12-803138-4.00045-9 http://dx.doi.org/10.1186/1475-2891-2-7 http://dx.doi.org/10.1093/aobpla/plu078 http://dx.doi.org/10.4238/2015.november.18.5 http://dx.doi.org/10.1017/jns.2016.41 http://dx.doi.org/10.17660/actahortic.2018.1194.192 http://dx.doi.org/10.1016/j.rmclc.2018.01.001 http://dx.doi.org/10.1016 / s0731-7085 (01) 00661-6 http://dx.doi.org/10.1111/j.1365-313x.2008.03447.x http://dx.doi.org/10.1051/fruits:2005007 http://dx.doi.org/10.17660/actahortic.2002.575.72 http://dx.doi.org/10.1201/9781420006186.ch1 http://dx.doi.org/10.1021/jf020145k http://dx.doi.org/10.1016/j.phytochem.2006.10.002 http://dx.doi.org/10.1016/j.foodchem.2005.01.00 vorgabe neu journal of applied botany and food quality 80, 111 115 (2006) 1norwegian university of life sciences, department of plant and environmental sciences, ås, norway 2norwegian university of life sciences, department of chemistry, biotechnology and food science, ås, norway antioxidant activity in commonly grown and consumed vegetables: a screening survey anne-berit wold1, trude wicklund2, karin haffner1 (received april 20, 2006) summary a diet rich in fruits and vegetables is associated with a reduced risk of developing cardiovascular diseases and certain types of cancer. this positive effect is related to bioactive phytochemicals found in plants. the vegetables were grown in the field or in greenhouses at the norwegian university of life sciences (59º40’n) during the years 2000-2002. the vegetables were harvested at commercial maturity and analysed for dry matter and antioxidant activity assessed by the frap (ferric reducing ability of plasma) assay. there was a large variation in antioxidant activity both between and within different species. the highest antioxidant activity was observed in kale, red cultivars of cabbage and table beet. the lowest antioxidant activity was observed in lettuce, cucumber, carrots and tomato. the vegetables possessing a red colour showed higher antioxidant activity with the exception of carrots and tomatoes. introduction epidemiological studies have shown that a diet rich in fruits and vegetables is associated with reduced incidences of several chronic diseases, and a reduced risk of developing cardiovascular disease and certain types of cancer. a review of approximately 200 epidemiological studies examining the relationship between intake of fruits and vegetables and different types of cancer found that in 128 of 156 dietary studies the consumption of fruits and vegetables had a protective effect (block et al., 1992). this beneficial effect of fruits and vegetables is related to their content of bioactive compounds (phytochemicals), which may be carotenoids, phenolics, alkaloids, nitrogen containing compounds and organosulfur compounds. the most studied are the phenolics and carotenoids (liu, 2004). especially the phenolic compounds are effective antioxidants (rice-evans et al., 1997). the combination of phytochemicals from fruits and vegetables was proposed to be responsible for the potent antioxidant and anticarcinogenic activity of these foods. taken alone the individual antioxidants studied in clinical trials do not appear to have consistent positive/preventive effects. synergistic activity between antioxidants has been observed. therefore the consumer should obtain their phytochemicals from a wide variety of fruits and vegetables. the antioxidant activity in fruits and vegetables may be influenced by cultivar, geographic origin, growing season, and agricultural practices. a systematic screening of total antioxidant activity in dietary plants (halvorsen et al., 2002) revealed a large variation in the antioxidant content of vegetables. large variation was also observed within the species. this variation may have several causes, including genetic and environmental influences as well as differences in plant organ and tissue examined. brassica vegetables are important contributors of vitamins and antioxidants in human diets, the major antioxidants being vitamins c and e, carotenoids, and phenolic compounds (podsedek, in press). many previous studies have been limited to the use of samples without the knowledge of genotype, growing conditions or post-harvest conditions. the aim of this work was to assess the antioxidant activity in vegetables commonly grown and consumed in norway. the nordic growth season is fairly short with long daily light periods and relatively low temperatures. the special climatic conditions may influence the content of secondary plant metabolites including bioactive phytochemincals. material and methods plant material vegetables were grown in the field and in greenhouses at the norwegian university of life sciences (59º40’n) in the years 20002002. the vegetables included in the screening are listed in tab. 1. fertilisation, irrigation and plant protection corresponded to the standard cultivation procedures for each species. vegetables were harvested at commercial maturity. the vegetables were analysed for antioxidant activity by means of the frap (ferric reducing/antioxidant power) assay. products from at least two seasons were analysed. fifteen units of product (head, root, bulb or fruit) of each vegetable were harvested. the products were split into three samples each consisting of 5 units. approximately ¼ of each product unit was used for analyses. onions were analysed both freshly harvested and dried for 2 weeks at a temperature of 30 ºc the first year and for 4 weeks at 20 ºc the second year. the cured onions were stored at 0 ºc. tomatoes were harvested mature green and vine ripe and analysed as mature green, vine ripe and postharvest ripened fruits. cucumbers were harvested at two different maturities and both small (348 g) and large (576 g) cucumbers were analysed. samples were frozen at -20 ºc until further analyses. sample preparation for the frap assay samples were homogenized in a food processor, and 3 g of the homogenised material were dissolved in 30 ml of methanol. three replicates were prepared from each sample. the bottles were flushed with nitrogen before closing, the samples were then mixed and sonicated in a water-bath at 0 °c for 15 min. the extracts were stored at -20 °c until analysis. three samples of 1.5 ml of the extract were centrifuged at 12,402 x g for 2 min at 4 °c. the concentration of total antioxidants in the supernatant was measured in triplicate. frap assay the ferric reducing ability of plasma (frap) assay was used to measure the concentration of total antioxidants. frap was determined in extracts by the method of benzie and strain (1996), with the exception that the sample was not diluted with water (halvorsen et al., 2002). a technicon ra 1000 system (technicon instruments corporation, new york, usa) was used for the measurements of absorption changes that appear when the tptzfe3+ (2,4,6-tri-pyridyl-s-triazine) complex is reduced to the tptztab. 1: vegetables included in the screening survey. vegetable botanical name cultivar 2000 2001 2002 cucumber* cucumis sativus ventura x x early cabbage brassica oleracea var. capitata ladi x x white cabbage brassica oleracea var. capitata bartolo x x x red cabbage brassica oleracea var. capitata autoro x x x rovite x x primero x x savoy brassica oleracea var. sabauda taler x x x cauliflower (white) brassica oleracea var. botrytis fremont x x x cauliflower (green) alverda x x cauliflower (red) grafitti x brussels sprouts brassica oleracea var. gemmifera content x x x kale brassica oleracea var. sabellica ekstra moskruset x x x red kale brassica oleracea var. sabellica redbor x x iceberg salad lactua sativa var. capitata set x match x roxet x x brandon x x lollo rosso lactua sativa var. foliosum malibu x x anthony x x concorde x x radiccio rosso cihcorium intybus var. foliosum indigo x x spinach spinacea oleracea viking 290 x x broccoli brassica oleracea var. itallica maraton x x x montop x x lord x x red broccoli brassica oleracea var. itallica be 1891 x x swede brassica napus var. rapifera vige x x carrot daucus carota var. sativus nantes duke x x x yukon x x x onion allium cepa jumbo x x red onion allium cepa red baron x x rødbete beta vulgar var. conditiva boltardy x kosak x tomato* lycopersicon esculentum liberto x x favorita (cherry) x x durinta (truss) x x * vegetables grown in greenhouses fe2+ form in the presence of antioxidants. an intense blue colour with the absorption maximum at 593 nm develops. the measurements were performed at 600 nm. an aqueous solution of 500 µm feso4 x 7 h2o was used for calibration of the instrument. dry matter dry matter was determined by drying approximately 5 g of homogenized material for 24 h at 104 ºc, followed by stabilising at room temperature in a dessicator and weighing the final product. results and discussion kale was found to have the highest antioxidant activity followed by red cabbage, table beet, red broccoli, red cauliflower and lollo rosso lettuce (tab. 2). the lowest antioxidant activity was found in iceberg lettuce, carrots (tab. 2), cucumber and tomatoes (tab. 3) with the other vegetables at intermediate values. low antioxidant activity in tomato, carrot and lettuce was also observed by bahorun et al. (2004). the antioxidant activity in brassica vegetables examined ranged from 0,11 2,44 mmol/100g fresh weight. kale showed the highest values followed by cultivars of red cabbage, red broccoli, red cauliflower, brussels sprouts, broccoli cultivars, savoy cabbage, yellow cauliflower, early cabbage, cauliflower and white cabbage. examining carotene, tocopherol and ascorbate contents in subspecies of brassica oleracea, kurilich et al. (1999) found that kale had the highest level of vitamins, followed by broccoli and brussels sprouts with intermediate levels, and cabbage and cauliflower containing the lowest concentrations. 112 anne-berit wold, trude wicklund, karin haffner tab. 2: antioxidant activity (frap) in field grown vegetable species and cultivars. the letter n indicates number of replicates. n = 3 means three replicates for one growing season, n = 6 means three replicates for two growing seasons and n = 9 means three replicate for three growing seasons. vegetable cultivar sample dry matter % frap mmol/100g fw frap min-max mean red kale redbor n=3 14.5 2.44 2.30-2.55 2.44 kale ekstra moskruset n=9 15.9 2.38 1.95-2.94 2.38 red cabbage autoro n=9 8.7 1.90 1.52-2.15 1.60 rovite n=6 9.0 1.82 1.68-1.98 primero n=6 7.1 1.08 0.99-1.20 table beet boltardy n=3 14.6 1.56 1.52-1.61 1.46 kosak n=3 14.0 1.36 1.28-1.43 red broccoli be1891 n=3 15.5 1.34 1.29-1.41 1.34 broccoli lord n=6 9.5 0.41 0.37-0.46 0.36 montop n=6 9.6 0.34 0.32-0.39 marathon n=9 9.6 0.34 0.26-0.48 lollo rosso malibu n=6 6.1 1.16 0.37-2.34 0.95 anthony n=6 4.8 1.05 0.66-1.40 concorde n=6 4.1 0.65 0.42-1.11 radiccio rosso indigo n=6 6.3 0.47 0.16-0.85 0.47 red cauliflower grafitti n=3 7.2 1.11 1.05-1.23 1.11 red onion/cured red baron n=6 12.1 0.89 0.47-1.48 0.89 spinach viking 290 n=6 8.4 0.77 0.66-1.12 0.77 red onion/fresh red baron n=6 15.3 0.75 0.48-1.20 0.75 onion/cured jumbo n=6 12.1 0.71 0.33-1.48 0.71 brussels sprouts content n=9 18.0 0.65 0.54-0.72 0.65 onion/fresh jumbo n=6 11.7 0.60 0.33-1.12 0.60 savoy cabbage thaler n=9 11.0 0.26 0.19-0.40 0.26 swede vige n=6 9.1 0.26 0.18-0.38 0.26 yellow cauliflower alverda n=6 9.4 0.21 0.17-0.24 0.21 early cabbage ladi n=6 5.7 0.12 0.11-0.14 0.12 cauliflower fremont n=9 7.2 0.12 0.11-0.13 0.12 white cabbage bartolo n=9 10.2 0.11 0.07-0.17 0.11 carrot nantes duke n=9 10.4 0.04 0.02-0.05 0.04 yukon n=9 10.4 0.04 0.02-0.05 iceberg lettuce zet n=3 4.0 0.05 0.032-0.067 0.038 match n=3 3.3 0.03 0.028-0.031 brandon n=6 3.6 0.05 0.004-0.08 roxett n=6 3.3 0.02 0.009-0.032 the antioxidant activity in salads ranged from 0,02 1,16 mmol/ 100 g fresh weight. the lollo rosso cultivars had the highest frap values followed by radiccio rosso whereas the iceberg cultivars showed the lowest antioxidant activity. red-pigmented oak leaf lettuce has been found to have higher antioxidant activity than green cultivars of lettuce including butter and batavia salads (nicolle et al., 2004). red oak leaf lettuce had 3.5 times higher antioxidant capacity than green lettuce. analysing phenolic acids in lollo rosso, ferreres et al. (1997) found the red tissue to contain more than the green and white tissue. the red tissue also showed higher flavonoid content than green tissue while white tissue had the smallest amount. cured onion showed higher antioxidant activity than fresh onion, and the red cultivar had higher values than the white one (tab. 2). coloured vegetables were found to have a higher antioxidant activity than the paler varieties. anthocyanin is the abundant flavonoid that causes intense coloration. anthocyanins are found to have a protective effect against cancer (cooke et al., 2005). the red colour of red cabbage is caused by anthocyanins belonging to flaovonoids. red cabbage contains more than 15 different anthocyanins (dyrby et al., 2001; mazza and miniati, 1993). classifying vegetables according to their major phenols proteggente et al. (2002) found fruits and vegetables rich in anthocyanins to possess the highest antioxidant capacity, followed by those rich in flavanones and flavonoles (onions, leeks, lettuce, broccoli, spinach and cabbage), while the hydroxycinnamate-rich (tomato) showed lower antioxidant capacity. a strong correlation between antioxidant activity and total phenolics and total flavonoids has been found in vegetables (bahorun et al., 2004). in this study the tomatoes did not show a high antioxidant activity compared to the other colourful vegetables (tab. 3). this may be explained by the choice of extraction procedure. antioxidants in vegetables 113 the methanol extraction prior to the frap assay does not dissolve fat-soluble antioxidants such as lycopene. the flavonoids and the glucoside flavonoids are extracted by methanol and measured in the frap assay as part of the total antioxidant capacity. this would mean that the potential antioxidant activity in tomato cultivars is underestimated. this would also be the case for carrots, which contain considerable amounts of carotenoids. when estimating the requirement of antioxidants both the antioxidant activity in individual fruits and vegetable species but also the consumption frequency should be considered. chun et al. (2005) found asparagus and bell pepper to have high antioxidant activity whereas tomatoes and potatoes were lower. considering daily consumption potatoes showed the highest antioxidant intake followed by tomatoes. asparagus and bell peppers were shown to contribute relatively little to the total antioxidant intake due to their low daily consumption. the post-harvest ripened and vine ripe tomatoes had higher antioxidant activity than mature green fruits. there were no significant differences between vine ripe and post-harvest ripened tomatoes (wold et al., 2004), indicating that with respect to tomatoes agronomical practises such as post-harvest ripening did not influence the antioxidant activity. cucumbers are usually harvested early, as small, immature, dark green fruits. harvested at a later stage the cucumbers are larger with a lighter green colour. cucumbers were low in antioxidant activity and there were no differences observed in antioxidant activity between small immature and larger cucumbers. following studies should also include total phenolics and the content of individual flavonoids and vitamins to give a better understanding of the contribution of single components to antioxidant activity in fruits and vegetables. acknowledgement the authors wish to thank the norwegian research council for financing the project „bioactive compounds in fruit, berries and vegetables and their capabilities to regulate disease-associated genes“ (2000-2002), rune blomhoff at institute for nutrition research, uio for use of laboratory equipment, liv berge and karin svinnset at the department of plant and environmental sciences and tor brun at department of chemistry, biotechnology and food science for valuable technical assistance, and halvard baugerød for critical reading of the manuscript. references bahorun, t., luximon-ramma, a., crozier, a., aruoma, o.i., 2004: total phenol, flavonoid, proanthocyanidin and vitamin c levels and antioxidant activities of mauritian vegetables. j. sci. food agric. 84, 15531561. benzie, i.f.f., strain, j.j., 1996: the ferric reducing ability of plasma (frap) as a measure of „antioxidant power“: the frap assay. anal. biochem. 239, 70-76. block, g., patterson, b. subar, a., 1992: fruit, vegetable and cancer prevention: a review of the epidemiological evidence. nutr. cancer 18, 1-29. cooke, d., steward, w.p., gescher, a.j., marczylo, t., 2005: anthocyans from fruits and vegetables – does bright colour signal cancer chemopreventive activity? eur. j. cancer 41, 1931-1940. chun, o.k., kim, d.-o., smith, n., schroeder, d., han, j.t., lee, c.y., 2005: daily consumption of phenolics and total antioxidant capacity from fruits and vegetables in the american diet. j. sci. food agric. 85, 1715-1724. dyrby, m., westergaard, n., stapelfeldt, h., 2001: light and heat sensitivity of red cabbage extract in soft drink model systems. food chem. 72, 431-437. ferreres, f., gil, m.i., castaner, m., tomas-barberan, f.a., 1997: phenolic metabolites in red pigmented lettuce (lactuca sativa). changes with minimal processing and cold storage. j. agric. food chem. 45, 42494254. halvorsen, b.l., holte, k., myhrstad, m.c.w., barikmo, i., hvattum, e., remberg, s.f., wold, a.b., haffner, k., baugerød, h., andresen, l.f., moskhaug, j.ø., jacobs, d.r., blomhoff, r., 2002: a systematic screening of total antioxidants in dietary plants. j. nutr. 132, 461-471. kurilich, a.c., tsau, g.j., brown, a., howard, l., klein, b.p., jeffery, e.h., kushad, m., wallig, m.a., juvik, j.a., 1999: carotene, tocopherol, and ascorbate contents in subspecies of brassica oleracea. j. agric. food chem. 47, 1576-1581. liu, r.h., 2004: potential synergy of phytochemicals in cancer prevention: mechanism of action. j. nutr. 3479s-3485s. mazza, g., miniati, e., 1993: anthocyanins in fruits, vegetables, and grains. crc press, boca raton. nicolle, c., carnat, a., fraisse, d., lamaison, j.-l., rock, e., michel, h., amouroux, p., remesy, c., 2004: characterisation and variation of antioxidant micronutrients in lettuce (lactuca sativa folium). j. sci. food agric. 84, 2061-2069. podsedek, a., 2005: natural antioxidant capacity of brassica vegetables: a review. lebensmittel-wissenschaft u. -technologie (in press). tab. 3: antioxidant activity (frap) in tomato cultivars and cucumber grown in green houses and harvested at different maturity. the letter n indicates number of replicates. n = 6 means three replicate for two growing seasons. vegetable cultivar maturity sample dry matter % frap mmol/100g fw min-max mean tomato liberto green n=6 5.6 0.17-0.22 0.20 liberto post-harvest ripe n=6 4.9 0.26-0.29 0.28 liberto vine ripe n=6 6.0 0.24-0.30 0.30 favorita green n=6 7.4 0.25-0.42 0.32 favorita post-harvest ripe n=6 6.5 0.39-0.46 0.42 favorita vine ripe n=6 7.7 0.41-0.52 0.48 durinta green n=6 5.9 0.11-0.21 0.17 durinta post-harvest ripe n=6 4.7 0.22-0.28 0.24 durinta vine ripe n=6 5.5 0.20-0.26 0.26 cucumber small n=6 3.4 0.03-0.06 0.05 large n=6 3.2 0.02-0.05 0.04 114 anne-berit wold, trude wicklund, karin haffner proteggente, a.r., pannala, a.s., paganga, g., van buren, l., wagner, e., wisman, s., van de put, f., dacombe, c., rice-evans, c.a., 2002: the antioxidant activity of regular consumed fruit and vegetables reflects their phenolic and vitamin c composition. free radic. res. 36, 217233. rice-evans, c.a., miller, n.j., paganga, g., 1997: antioxidant properties of phenolic compounds. trends plant sci. 2, 152-159. wold, a.-b., rosenfeld, h.j., holte, k., baugerød, h., blomhoff, r., haffner, k., 2004: colour of post-harvest ripened and vine ripened tomatoes (lycopersicon esculentum mill.) as related to total antioxidant capacity and chemical composition. int. j. food. sci. technol. 39, 295302. address of the authors: anne-berit wold1*, trude wicklund2 and karin haffner1 1norwegian university of life sciences, department of plant and environmental sciences, p. o. box 5003, n-1432 ås, norway, 2norwegian university of life sciences, department of chemistry, biotechnology and food science, p. o. box 5003, n-1432 ås, norway *corresponding author: anne-berit.wold@umb.no antioxidants in vegetables 115 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false /maxsubsetpct 100 /optimize true /opm 1 /parsedsccomments true 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<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> >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice art24 journal of applied botany and food quality 82, 148 151 (2009) 1 jimma agricultural research center, jimma, ethiopia, 2 university of addis ababa, ethiopia, applied microbiology, 3 university of bonn, germany, inres-phytomedizin occurrence of fungal diseases of coffea arabica l. in montane rainforests of ethiopia a. zeru1, f. assefa2, g. adugna1, h. hindorf 3 (received august 20, 2008) summary coffee berry disease (cbd), colletotrichum kahawae, coffee wilt disease (cwd), gibberella xylarioides and coffee leaf rust (clr), hemileia vastatrix are the three major diseases reducing production and consumption of coffee in ethiopia. a survey was conducted from july to september 2005 for cbd and cwd and from 2003 until 2007 for clr in montane rainforest coffee areas of ethiopia to estimate the occurrence and distribution of these diseases. diseases were prevalent in all the surveyed forest coffee areas of ethiopia: harenna, bonga, berhane-kontir and yayu. depending on the forest coffee area the mean percent incidence of cbd ranged from 2 to 40 % in general and from 2 to 17.9 % at berhane-kontir and bonga, respectively. the mean incidence of cwd varied from 2.4 % at berhane-kontir to 16.9 % at yayu forest coffee areas. the mean incidence of clr also varied for instance in 2005 from 32.2 % at berhane-kontir to 96 % at harenna forest coffee areas. the detection of the diseases during our surveys requires an integrated management of major coffee diseases for a sustainable conservation and wise use of coffee in montane rainforests of ethiopia. introduction coffee is the most important commodity crop in ethiopia. current contributions of coffee provide more than 60 % of the country’s foreign exchange earning over 5 % of the gdp, 12 % of the agricultural output and 10 % of the government revenues (csa, 2002). coffee production systems in ethiopia are grouped into four broad categories, namely forest coffee, semi-forest coffee, garden coffee and coffee plantations with an average production of 10, 34, 35 and 21 % of the total production, respectively (mctd, 1992). many abiotic and biotic stresses are the major constraints of the coffee production in the country. the most important ones are fungal diseases that attack fruits, leaves, stems and roots and reduce the yield and marketability (derso, 1997). the major coffee diseases in ethiopia are coffee berry disease, cbd, coffee wilt disease, cwd and coffee leaf rust, clr (hindorf, 1998) . cbd is a disease caused by the fungal pathogen colletotrichum kahawae waller & bridge, inducing an anthracnose of green and ripe coffee cherries. the variation in frequency of cbd from one area to the other in garden coffee and coffee plantation systems was reported by many authors (iar, 1997; biratu, 1995; tesfaye and sokar, 2000; jirata and assefa, 2000; tesfaye and abate, 2000). derso (1997) estimated the overall national loss due to cbd on landraces between 24 and 30 %. also the presence of resistant cultivars was reported (van der graaff, 1981). cwd is a vascular wilt disease syndrome, commonly referred to as tracheomycosis and induced by gibberella xylarioides heim & saccas. cwd is known to attack all species of coffea including the wild indigenous lines in tropical africa (wrigley, 1988; coste, 1992). the disease was reported to be prevalent on coffea excelsa in the central african republic and cameroon, on robusta varieties in zaire and ivory coast (booth, 1971; coste, 1992) and found on arabica coffee in ethiopia, mainly in plantations near agaro, jimma and bonga in the early 1970’s (kranz and mogk, 1973). clr caused by hemileia vastatrix berkeley & broome, was firstly reported in ethiopia in 1934 (sylvain, 1955). according to wondimu (1991) the importance of clr is increasing with an estimated national percent tree attack of 12.9 % which raised to 36 % after ten years. an integrated disease management approach of forest coffee is required to conserve and use forest coffee sustainability in ethiopia. however, there exists very little background information on major coffee disease frequencies in the montane rainforest coffee of ethiopia. therefore, the main objective of the surveys was to estimate the frequency of cbd, cwd and clr across 4 montane rainforest coffee areas of ethiopia. these forest coffee areas contain a large genetic pool of arabica coffee representing a potential source for the benefit of present and future human generations. materials and methods descriptions of field sites disease surveys were conducted in 4 montane rainforest coffee areas in the southeast (harenna/bale mountains) and southwest (bonga/ kaffa, berhane-kontir/bench maji and yayu/illubabor) of ethiopia (tab. 1). assessment of coffee berry disease (cbd) in each forest coffee area, cbd assessments were taken in 5-7 plots across the forest coffee by considering the existing field variation, especially land gradients, presence and absence of forest coffee. in each 100 x 100 m plot assessments were conducted on the same trees diagonally following procedures used by tesfaye and sokar (2000). in visual assessments 10 trees/plot were randomly taken and diagnosed for presence and absence of the disease frequency on each tree. thereafter disease frequency was calculated as the number of diseased trees divided by total observed trees x 100. assessment of coffee wilt disease (cwd) in each forest coffee area cwd assessments were taken in 5-8 plots across the forest coffee area. in each 100 x 100 m plot, 30-50 trees were diagnosed diagonally and consecutively following the procedure tab. 1: description of the study sites in montane rainforests of southwestern ethiopia. forest area altitude (m) co-ordinates latitude(n) longitude (e) harenna 1400 1700 6° 30’ 39° 45’ berhane-kontir 1100 1200 7° 06’ 35° 26’ bonga 1600 1900 7° 19’ 35° 03’ yayu 1400 1800 8° 23’ 35° 47’ of adugna et al. (2001) and cabi (2003). healthy and diseased dying or dead trees, showing typical characteristic internal and external symptoms of the disease and/or sign of the pathogen, were visually observed and recorded. internal symptoms like black and/ or brown stripes were observed after scratching off the bark of diseased trees to expose the wood for proving the causal agent. then the numbers of healthy and diseased trees were counted and the incidence of cwd was computed as the number of diseased trees divided by the total number of observed coffee trees x 100. assessment of coffee leaf rust (clr) in each forest coffee area two representative sites were selected. the assessments for clr were carried out randomly within each 100 x 100 m forest coffee area on 100 trees counting the frequency of leaf attack (%) and scoring the intensity of pustules per leave in the following scale: 1 = no pustule, 2 = 1 pustule, 3 = 2 pustules and 4 = > 3 pustules. to obtain a longer termed overview on the development of the clr situation in montane rainforests we include data scored since 2003 in the same way. data analyses excel microcomputer statistical software was employed to design graphs. results and discussion the three major diseases of coffee, namely coffee berry disease (cbd), coffee wilt disease (cwd) and coffee leaf rust (clr) were found in association with forest coffee in harenna, bonga, berhanekontir and yayu montane rainforests of ethiopia (fig. 1). leaf blight, ascochyta tarda stewart and coffee bean darkening or bacterial blight, pseudomonas syringae pv. garcae (amaral, teixeira & pinheiro) young, dye & wilkie were also observed in the forest coffee, but not scored during our surveys. occurrence of cbd in the montane rainforest coffee of ethiopia the frequency and intensity of cbd varied among and within forest coffee areas depending on environmental conditions like altitude, rainfall, temperature etc. and genetic diversity of the forest coffee. survey results indicated that the disease frequency ranged from 0-50 %, 20-60 %, 0-20 % and 0-50 % and the intensity from 0-15 %, 12.5-22.5 %, 0-6.5 % and 0-7.8 % in forest coffee areas of harenna, bonga, berhane-kontir and yayu, respectively (fig. 1). the mean frequency ranged between 6 % at berhane-kontir and 40 % at bonga, respectively. high frequencies of cbd may be explained by the particularly high rainfall found in relatively high altitudes of bonga and to some extent in yayu. similarly, cook (1975) explained that high rainfall, high humidity or wetness and relatively low temperatures persisting for long periods and favouring the cbd development are existing at higher altitudes, where these conditions generally prevail. in general our results confirmed the observations of cook (1975) and are shown in fig. 2. in addition to expected results in yayu the disease was also found at low altitudes (<1500 m.a.s.l) ranging from a frequency of 0 to 40 %, but achieved a frequency upto 50 % in medium altitudes. this indicated that cbd can be very important in lower altitudes too. there occurred no cbd at low lands of harenna (around majete) and berhane-kontir (around gizmeret) forest coffee areas. these results are the first informations in both areas for the existence of cbd infestations in limited pocket parts of harenna (around mekabaldo) and berhane-kontir (around wesheka) forest and semi-forest coffee areas. occurrence of cwd in the montane rainforest coffee of ethiopia cwd was found in all assessed forest coffee areas suffering considerably in coffee tree losses. its frequency varied from 0-16 %, 0-10 %, 0-6 % and 0-30 % in forest coffee areas of harenna, bonga, berhane-kontir and yayu, respectively. the mean frequency varied between 2.4 % at berhane-kontir and 16.9 % at yayu (fig. 1). the disease seems to expand and damage coffee trees particularly in yayu and harenna. coffee farmers in yayu, bonga and harenna are accustomed to work in groups and use cutlasses (bushman knives) to slash weeds around coffee trees once per year from july to mid september and thereby occasionally coffee trees are wounded becoming then susceptible for an attack of the pathogen. the difference in genetic diversity of autochthonous arabica coffee as well as differences in cultural practices (exposure of coffee trees to wounding) from one forest population to the other and within the same forest coffee area could be the cause of variations in the extent of cwd incidence across forest coffee areas. adugna et al. (2001) also reported that cwd incidence ranged from 45 to 69 % on coffee plantation fields at gera and bebeka, respectively. reports on cwd incidences on arabica coffee in ethiopia 49 years ago resulted in very low mean percent frequencies across the forest coffee areas as compared to the national cwd survey results of ethiopia (cabi, 2003). our results also showed frequency variations from one forest coffee area to others, even from one particular plot to another plot within one forest coffee population. according to booth (1971) the fungus is known to penetrate coffee trees through wounds either above or below the ground. adugna et al. (2001) also reported that any parts of infected trees, except seeds, and possibly the adjacent fig. 1: occurrence of coffee berry disease (cbd), coffee wilt disease (cwd) and coffee leaf rust (clr) in montane rainforest coffee of ethiopia (error bars are standard errors). 0 10 20 30 40 50 60 70 80 90 100 1000 1200 1400 1600 1800 2000 altitude [m] f re q u e n cy [% ] i harenna ii bonga iii berhanekontir iv yayu fig. 2: frequency of coffee berry disease (cbd) in montane rainforest coffee of ethiopia depending on altitudes coffee diseases in ethiopia 149 150 a. zeru, f. assefa, g. adugna, h. hindorf asymptomatic coffee trees and soils serve as survival organ and could be potential sources of the pathogen for infections. in harenna forest coffee flocks of cattles have grazed and moved along the coffee trees while damaging coffee trees, seedlings and carrying the inoculum on their bodies from area to area. in berhane-kontir people intensively use drying and dead coffee trees as firewood and carry this across the forest coffee areas. this could easily be a methods for distributing ascospores of the perfect stage. all the above mechanisms most probably aggravated the spread and distribution of the disease in the forest coffee and increasing damaging effects of the disease in the areas. occurrence of clr in montane rainforest coffee of ethiopia in all investigated forest coffee areas coffee leaf rust (clr) was prevalent to a high extent (fig. 1). results of the assessments, counting exactly the number of infected trees for the frequency and scoring the intensity on single infected leaves, showed more or less high frequencies of clr in all forest coffee areas varying from 32.2 % at berhane-kontir to 96 % at harenna forest coffee (fig. 1). the highest clr frequency occurred in 2005 at harenna followed by yayu. young seedlings under the forest coffee at harenna were covered completely with rust sori influencing the survival and further growth. in addition to the annual disease occurrence in 2005 we present data on the intensity of clr during the complete observation period from 2003 2007. the tendency of an increase of the disease in all autochthonous coffee areas of ethiopia is shown in the positive slope of the regression curve (fig. 3). field plots in each forest coffee locality. differences in genetic diversity of autochthonous arabica coffee as well as the variations in the intensity of cultural practices from one forest population to the other and within the same forest coffee population could be the cause of the extent of cwd frequencies across forest coffee areas. even though cwd was reported in earlier times 49 years ago occurring on arabica coffee in ethiopia, our survey results showed very low mean percentage of disease frequencies across the forest coffee areas due to a possible high diversity among autochthonous forest coffee germplasms and low cultural practices in these forest coffee areas. in all assessed forest coffee localities clr occurred more or less frequently attacking up to 96 % of the trees with an increasing tendency during the last 5 years. the observations on the presence of all three major fungal diseases in the autochthonous coffee of montane rainforests in ethiopia need a careful integrated management of the diseases for sustainable conservation and wise use of the forest coffee gene-poole with its still high diversity (coce i, 2007). acknowledgements the surveys could be carried out as part of a joint project “conservation of wild coffee in montane rainforests of ethiopia (coce)” coordinated by the “center for development research (zef)” at bonn university and were financially supported by the german ministry of education and research (bmbf). we kindly acknowledge the fruitful cooperation and assistance of the staff of the “jimma agricultural research center” of eiar, addis ababa/ethiopia. references adugna, g., hulluka, m., hindorf, h., 2001: incidence of tracheomycosis, gibberella xylarioides (fusarium xylarioides), on arabica coffee in ethiopia. j. plant dis. prot. 108, 136-142. biratu, t., 1995: studies on colletotrichum population of coffea arabica l. in ethiopia and evaluation of the reactions of coffee germplasms. phd. thesis, bonn, 231. booth, c., 1971: the genus fusarium. commonwealth mycological institute: kew, surrey, england, 237. cabi, 2003: surveys to assess the extent of coffee wilt disease in east and central africa. final technical report. cabi regional center, nairobi/ kenya, 49. cook, r.t.a., 1975: screening coffee plants for resistance to cbd. annual report crf ruiru/kenya coste, r., 1992: coffee: the plant and the product. macmillan press ltd, london, 328. csa, 2002: annual statistical abstract. addis ababa/ethiopia. derso, e., 1997: coffee diseases and their significance in ethiopia. asic 17, 723-726. hindorf, h., 1998: current diseases of coffea arabica and c. canephora in east africa causing crop losses. med. fac. landbouww. univ. gent 63, 861-865. iar, 1997: jimma national coffee research center progress report for the period 1994, part 1: coffee. melko/ethiopia. jirata, m., assefa, s., 2000: status of cbd in the oromiya region. in: proc.worksh. control cbd in ethiopia, 13-15 august 1999, addis ababa/ ethiopia, 9-17. kranz, j., mogk, m., 1973: gibberella xylarioides heim et saccas on arabica coffee in ethiopia. phytopath. z. 78, 365-366. mctd, 1992: report on yield assessment survey: planning, monitoring and evaluation team, mctd, addis ababa/ethiopia, 5-60. sylvain, p.g., 1955: some observations on coffea arabica l. turrialba 5, 37. conclusion and recommendations major coffee diseases such as cbd, cwd and clr were assessed in the montane rainforest areas: harenna (bale mountains), berhanekontir, bonga and yayu. cbd frequencies varied at the areas in which forest coffee was grown depending on environmental conditions and genetic diversities of the forest coffee. the mean percentage of cbd varied from 6 to 40 % and 2 to 17.9 % at berhane-kontir and bonga, respectively. results of these surveys showed that cbd widely occurred in bonga forest coffee areas followed by the disease distribution in yayu. cwd was prevalent in all assessed forest coffee areas, where it has achieved already considerable coffee tree losses. its mean frequency ranged from 2.4 % at berhane-kontir forest area to 16.9 % at yayu forest coffee area with certain variations among sampled forest coffee fig. 3: intensity of coffee leaf rust (clr) from 2003 until 2007 in montane rainforest coffee of ethiopia. 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 sep 03 jan 04 may 04 sep 04 jan 05 may 05 sep 05 jan 06 may 06 sep 06 jan 07 period sep 2003 mar 2007 d is ea se in d e x d i harenna berhane kontir bonga yayu *di class 1: no disease; class 2: one rust pustule/leaf; class 3: two rust pustules/leaf and class 4: three and more rust pustules/leaf coffee diseases in ethiopia 151 tesfaye, a., sokar, i., 2000: the status of coffee berry disease in minor coffee growing regions. in: proc. worksh. control cbd in ethiopia, 13-15 august 1999, addis ababa/ethiopia, 29-34. tesfaye, n., abate, s., 2000: status of cbd in snnp. in: proc. worksh. control cbd in ethiopia, 13-15 august 1999, addis ababa/ethiopia, 18-28. van der graaff, n.a., 1981: selection for arabica coffee types resistant to cbd in ethiopia. mededel. landbouwh..wageningen/netherlands, 110. wondimu, m., 1991: epidemiology and resistance of coffee leaf rust in ethiopia. ministry of coffee and tea development. addis ababa/ ethiopia. wrigley, g., 1988: coffee. tropical agriculture series. longman sci. & techn. publ., new york/usa, 342-344. address of the author: 1jarc, pob. 192, jimma, ethiopia 3university, inres-phytomedizin, nussallee 9, 53115 bonn, germany << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) 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/pagesize [907.087 680.315] >> setpagedevice art01_oehl.indd journal of applied botany and food quality 85, 129 133 (2012) 1agroscope reckenholz-tänikon research station art, ecological farming systems, zürich, switzerland 2 universidad católica de temuco, facultad de recursos naturales, escuela de agronomía, temuco, chile 3 zurich-basel plant science center, institute of botany, university of basel, basel, switzerland 4 institute of plant production and agroecology in the tropics and subtropics, university of hohenheim, stuttgart, germany ambispora reticulata, a new species in the glomeromycota from mountainous areas in switzerland and chile 1* fritz oehl, 2 claudia castillo, 3 david schneider, 1verena säle, 4 ewald sieverding (received may 5, 2012) * corresponding author summary a new glomeromycotean fungus, ambispora reticulata, was found in the swiss alps and in the chilean andes. only acauloambisporoid spores were detected so far, 87-131 x 125-150 µm in diameter and having a three-layered, yellow-brown to brown outer wall, a bi-layered, hyaline middle wall and a generally three-layered, hyaline inner wall. the middle wall has a characteristic reticulate outer surface with irregular triagonal to octagonal (usually tetra to hexagonal) pits that are surrounded by ridges. as known for all ambispora species with acaulo-ambisporoid spore formation, the middle wall is a substantial part of the pedicel which connects the spore with the mycelium. the new species is a frequent member of arbuscular mycorrhizal fungal communities in mountainous and subalpine grasslands of the swiss alps at 1000-2100 m above sea level. it occurred less frequent in high alpine grasslands and at altitudes below 1000 m, where the fungus was found in a conservation tillage and a low-input tillage system. it was also detected in evergreen and in deciduous forests in the andes of southern chile at elevations of 550-1600 m. introduction in the last two decades, biodiversity studies have worldwide been intensifi ed. this is especially true for arbuscular mycorrhizal fungi (amf) of the glomeromycota (e.g. błaszkowski, 1993; vestberg, 1995; castillo et al., 2006, 2010; sánchez-castro et al., 2011; bezerra et al., 2011; souza et al., 2012). while until 1990, only ca. 130 glomeromycotean species were described (schenck and pérez, 1990), today we count approximately 250 species. also remarkably, the more recently published new amf species (e.g. gamper et al., 2009; furrazola et al., 2011; rodriguez et al., 2011) have been described by an steadily increasing number of research groups (stürmer, 2012). high amf species and genus diversities were for instance revealed in central europe (jansa et al., 2002, 2003; gamper et al., 2004; oehl et al., 2005a, 2010) even up to the highest altitudes and harshest environments in the swiss alps where higher plants live (oehl et al., 2011a; körner, 2011). while several species were found in all habitats investigated, others were characteristic for specifi c soils (oehl et al., 2003, 2005b), soil depths (oehl et al., 2005a), land use practices (oehl et al., 2009) or altitudes (oehl et al., 2006, 2011e, 2012). one unknown amf species was mainly found in mountainous to subalpine grasslands of the swiss alps at 1000-2100 m above sea level (a.s.l.). it was simultaneously found also in mountainous forests in the andes of southern chile (castillo et al., 2006). it is here published under the epithet ambispora reticulata. the new species did not reproduce in single spore bait cultures. it is described from spore morphological characters as these are clearly indicating that the species belong to ambispora in the archaeosporales, archaeosporomycetes (spain et al., 2006; walker et al., 2008; oehl et al., 2011c). materials and methods study sites and soil sampling soil samples were taken between march 2000 and april 2009 all over switzerland from about 100 tillage and conservation tillage farming sites in the lowlands, and from in total > 400 grassland sites in the lowlands, mountainous and alpine areas from altitudes between 300 and 3000 m a.s.l. the soils have developed on different geological bedrocks from nutrient poor jurassic sandstones over granite and gneiss rocks to carbonatic loess sediments, limestones and ultrabasic serpentinites (e.g. oehl et al., 2005b, 2010, 2011a). undisturbed soil cores from 0-10 cm depth were collected at study sites. spores of glomeromycota were separated from the soil samples by the wet sieving, decanting and subsequent sugar gradient centrifugation technique (sieverding, 1991). in chile, soil samples were taken from replicated plots of a nutrient recycling experiment in an evergreen natural rainforest and a deciduous secondary forest in the experimental station san pablo de tregua of the university austral de chile (valdivia, chile, 39°30-39°38’s and 72°0272°09’w) at an elevation between 550 and 1600 m a.s.l. (castillo et al., 2006). spores were separated as given above. am fungal bait cultures bait cultures were established in switzerland directly after soil sampling as described in oehl et al. (2005b, 2011a) using a sterilised substrate (terragreen (american aluminium oxide, oil dry us special, type iii r, > 0.125 mm; lobbe umwelttechnik iserlohn, germany) -loess mixture 3:1; ph-kcl 6.2; organic carbon 0.3 %; available p (na-acetate) 2.6 mg kg-1; available k (na-acetate) 350 mg kg-1) in pots of different sizes (1 l in 2003-2005; oehl et al., 2011a, 3.5 l in 2009-2010, and 9 l in 2000-2002; oehl et al., 2005b). sub-samples of the fi eld samples represented natural fi eld inocula and were placed as a thin sandwich layer between two substrate layers, about 5 cm below the surface. above the soil inocula, about 5-7 seeds of each of the four bait plants, plantago lanceolata l., lolium perenne l., trifolium pratense l., and in some experiments also hieracium pilosella l. were sown. two weeks-old trifolium pratense plants received 1 ml of a 1:5 with water diluted 12 h old culture of rhizobium trifolii (dsm 30138, from dsmzdeutsche sammlung von mikroorganismen und zellkulturen gmbh, braunschweig, germany), grown in liquid dsmz 98 medium at 27 c. an automated watering system (tropf-blumat, weninger gmbh, a-6410 telfs) was installed and the cultures were kept in the greenhouse under ambient light and temperature conditions for 16-32 months. the spore formation was monitored in the bait cultures in bior four-monthly intervals as described in oehl et al. (2009, 2011a). the new fungus reproduced spores only in two of approximately 650 bait cultures established. all trials to 130 f. oehl, c. castillo, d. schneider, v. säle, e. sieverding reproduce the new fungus in so-called amf mono-species cultures so far failed. morphological analyses the described morphological characteristics of spores and their subcellular structures are based on observations of specimens mounted in polyvinyl alcohol-lactic acid-glycerol (pvlg; koske and tessier, 1983), in a mixture of pvlg and melzer’s reagent (brundrett et al., 1994), a mixture of lactic acid to water at 1:1, melzer’s reagent, and in water (spain, 1990). the terminology of the spore structure basically is that suggested by spain et al. (2006) for ambispora species, and by oehl et al. (2011b) for all glomeromycotean taxa. photographs in fig. 1-9 were taken with a digital camera (olympus model dp70-cu) on a compound microscope (zeiss axioplan). specimens mounted in pvlg and the mixture of pvlg and melzer’s reagent were deposited at the mycological herbarium of the eth zürich (z+zt, zurich, switzerland). latin diagnosis ambispora reticulata oehl & sieverd. sp. nov. (fig. 1-9) mycobank mb 800269 sporae acaulo-ambisporoideae fl avo-fuscae vel fuscae, 87-131 x 125-150 µm, formatae appendice, tunicis tribus; tunica media duobus stratis hyalinis reticulum tetragonale at hexagonale formans, depressionibus 3-7.5(-10) µm in diametro et 0.5-2.5 µm profundis. typus hic designatus # 57-5701 (zt myc 24171). etymology. latin, reticulata referring to the reticulate ornamentation of the middle wall. holotype holotype (here designated: slide nr. 57-5701; accession number zt myc 24171) and isotypes (zt myc 24172) were isolated from soil samples taken from the rhizosphere of an high mountainous pasture characterized by nardus stricta, at cuolms dil run alp (46°42’57’’n; 8°57’54’’e), surrein-sumvitg, surselva, kanton graubünden, switzerland, at about 1600 m a.s.l. paratypes isolated from several locations in the swiss alps and central lowlands of switzerland (see below) were also deposited at z+zt (zt myc 24173-24175). description acaulo-ambisporoid spores are formed by the fungus, but so far sporiferous saccules and glomo-ambisporoid spores were not detected. the acaulo-ambisporoid spores are yellow brown to brown, globose to oval to fi g-like, 87-131 x125-150 µm in diameter (fig. 12) consisting of three walls: outer wall (ow), middle wall (mw) and inner wall (iw; fig. 3). outer spore wall consists of three layers (owl1-owl3) and is in total 4-9 µm thick (fig. 2-3). owl1 is hyaline, unit, 0.5-1.0 µm thick (fig. 1), evanescent (fig. 2) and thus, usually diffi cult or not to observe in mature spores. second layer (owl2) is yellow-brown to brown, laminated, 3-8 µm thick. it sometimes swells up to 10-20 µm in pvlg (plus melzer reagent) under pressure applied on the cover slide (fig. 4). owl3 is hyaline, 0.5-1.5 µm thick, often tightly adherent to owl2, but can be separated under pressure. swelling laminae of owl2 stain pinkish, and owl3 stains pinkish purple to purple in melzer’s reagent (fig. 4). with age, ow shows many fi ssures, a feature that becomes more obvious when pressure is applied on the cover slide (fig. 3). middle wall is hyaline, bi-layered (mwl1-mwl2) and 1.53.5 µm thick (fig. 5-6). both layers are tightly adherent to each other. mwl1 is semi-fl exible, 0.8-2.5 µm thick and has a reticulate surface ornamentation (fig. 6-7). the pits are irregular triagonal to octagonal (usually tetrato hexagonal) and are 3-7.5(-10) µm wide and 0.5-2.5 µm deep (fig. 6, 7). pits are surrounded by ridges that are 0.5-1.2 µm wide. inner mwl2 is unit, smooth and 0.8-2.1 µm thick. none of the layers reacts to melzer’s reagent. inner wall is hyaline, with (two to) generally three layers (iwl1iwl3) that are 1.2-3.0 µm thick in total (fig. 8). iwl1 is < 0.5 µm thick, and often appears to be missing (fig. 5-6). iwl2 is 1.2-2.5 µm thick and rigid, and iwl3 is very thin and usually very diffi cult to detect since tightly adherent to iwl2. none of the layers reacts to melzer’s reagent, but in old spores iwl3 sometimes becomes yellow (fig. 8). pedicel at spore base is formed by the outer wall and the outer layer of the middle wall (fig. 2, 9). it is 7-14 µm broad and 4-16 µm long, respectively, at the spore base. the pedicel ow layers are 3-6 µm thick at spore base and taper to 1.2-2.2 µm within a few µm distances from the base. on the spore surface, they may form a wide pore (= collar), which is 5-13 µm in diameter. the continuation of mwl1 is reticulate to undulate at the pedicel (fig. 9). mwl1 wall layer regularly is only 0.5-1.5 µm thick at pedicel. distribution. in all regions of the swiss alps and in southern chile, where the fungus was found, spores of am. reticulata were common, but they were rarely found in numbers > 1 g-1 soil. in switzerland, they were frequently isolated from the rhizosphere of mountainous to subalpine grasslands at 1000-2100 m a.s.l. up to the tree line with vegetation characterized by the dominance of nardus stricta or trisetum fl avescens, in soils of ph 3.6-5.9. spores were rarely found in alpine nardus stricta grasslands and generally absent in (high) alpine grasslands characterized by carex ferruginea or sesleria caerulea. they were also less frequently found in lower mountainous and lowland grasslands. in detail, am. reticulata was found in eastern switzerland at piz nadels (trun and surrein-sumvitg, surselva, chantun grischun) between 15002000 m a.s.l.in nardus stricta grasslands, in southeastern switzerland at spadla alp (sent, engiadina, chantun grischun) at 2300 m a.s.l. in a nardus stricta grassland, in central switzerland in the gotthardfurka region between 1500-1900 m a.s.l. (hospental and realp, kanton uri; airollo, cantone ticino) in nardus stricta and trisetum fl avescens grasslands, at axalp (brienzer see) between 10001800 m a.s.l., and at grindelwald (grosse scheidegg) between 12002000 m a.s.l. (berner oberland, kanton bern) in nardus stricta and trisetum fl avescens grasslands, and in southwestern switzerland at 1000-1800 m a.s.l. (la valette, champex d’en bas, bourg-st.-pierre, all canton valais) in trisetum fl avescens grasslands. recently, they were also detected in the canton berne in an extensively managed conventional crop rotation system in niederösch (three years of temporary grassland and one year of potatoes production) and in a conservation tillage system in rubigen (7 year crop rotation with winter wheat, sugar beet, winter wheat, maize, winter barley and one year of grass/clover). the soil types were a broad range of eutric to dystric cambisols in the swiss alps, and a luvisol and calcaric cambisol in the lowlands of berne, that had developed on a broad range of bedrocks: acidic sandstones, siliceous gneiss and granite ambispora reticulata sp. nov. 131 fig. 1-9: ambispora reticulata – all acaulo-ambisporoid morphs. 1-2. uncrushed spores isolated from fi eld soils with a pedicel (pcl) formed by the outer and the middle wall. 3. crushed spores with outer, middle and inner wall: ow, mw and iw; ow showing multiple fi ssures which are especially seen in aged spores. 4. crushed spore in pvlg & melzer’s reagent, with slight pinkish staining reaction on layer owl2 and purple reaction of layer owl3. 5-8. bi-layered mw (mwl1-mwl2) and triple layered iw (iwl1-iwl3). middle wall has a diagnostic tetragonal to hexagonal reticulate ornamentation on the outer surface. rarely depressions have three or more than six sides. 9. wall of the pedicel (pcl) is a continuation of mwl1 having fl exible to semi-fl exible appearance. rocks, calcareous ‘bündner schiefer’ schists, moraine sediments and carbonatic limestones. the soil ph was always < 6.0 in the mountainous areas, while it was 6.0 and 7.1 in the two lowland sites. available p (na-acetate, see oehl et al., 2011a) was between 3.215.2 mg kg-1 in the mountainous to low alpine areas and 17.2 and 42.8 mg kg-1 in the two lowland soils. in chile, am. reticulata was found in an evergreen forest dominated by nothofagus dombeyi and laureliopsis philippiana tree species and a deciduous forest dominated by nothofagus alpina at mountainous altitudes between 550-1600 m a.s.l. (castillo et al., 2006). soil ph was 4.6 and 5.4, and available p was 6.1 and 3.6 mg kg-1, respectively. discussion ambispora reticulata can be easily distinguished from all other glomeromycotean fungi by its spore wall structure since the reticulate ornamentation on the middle spore wall is hitherto unique 132 f. oehl, c. castillo, d. schneider, v. säle, e. sieverding and thus, diagnostic. in the genus ambispora there are only two other fungi with ornamentation on the middle wall. these are am. appendicula and am. jimgerdemannii that both have an alveolate middle wall, in which both middle wall layers are ornamented on their interface (rose et al., 1979; schenck et al., 1984; spain et al., 2006), while in am. reticulata the reticulum is only on the outer surface of the middle wall. the outer wall (ow) of aging spores of am. reticulata regularly shows many fi ssures. this is a feature that becomes more obvious when pressure is applied to the cover slide of permanent specimens in pvlg, and is also known for the acaulo-ambisporoid morph of most of the other ambispora species. these are am. appendicula, am. gerdemannii, am. fennica, am. nicolsonii, and am. brasiliensis (spain et al., 2006; goto et al., 2008; oehl et al., 2011d), while this is not observed with am. granatensis which has a shorter-lived, since substantially thinner ow (palenzuela et al., 2011). remarkably, spores of am. reticulata were never found with sporiferous saccules attached, although in addition of fi eld sampled spores, also bait cultured spores were analyzed. the reason of not detecting sporiferous saccules in these bait cultures might be that the intervals between single sampling periods were four months in these pots instead of two months. in the shorter intervals we usually found sporiferous saccules attached with e.g. acaulospora nivalis, ac. alpina, am. fennica, am. appendicula, otospora bareae (e.g. oehl et al., 2006, 2009, 2012; palenzuela et al., 2008). hence, the mode of spore formation of am. reticulata still has to be discovered. also the glomoid morph of am. reticulata, if existent, still has to be identifi ed. in the genus ambispora, glomoid morphs are known only for some species but not for all (spain et al., 2006; walker et al., 2007). in switzerland, the new species was frequently found in mountainous to subalpine regions between 1000 and 2100 m a.s.l., whereas it was regularly absent in higher alpine areas and only sporadically found in lowland areas. in the chilean andes, am. reticulata was hitherto revealed solely from mountainous areas between 550 and 1600 m a.s.l. indicating that the preferential occurrence of this fungus may be in medium altitudes within such colder climates. distinct altitude respective climatic preferences in the swiss alps and in sierra nevada (spain) were recently described also for other ambispora species, as well as for several other glomeromycotean fungi like acaulospora alpina, ac. nivalis, ac. punctata, glomus badium, pacispora robigina and tricispora nevadensis (oehl and sieverding, 2004; spain et al., 2006; oehl et al., 2005a, 2006, 2011e, f, 2012; palenzuela et al., 2010). by studying different ecosystems on global scales, we will gradually get a clearer picture about the biogeography of the glomeromycota, that are counted among the most important soil micro-organisms, since they deliver a series of ecosystem services like soil aggregation, soil erosion protection, plant growth promotion, plant health and pathogen suppression, and seedling survival at sites as for example stressed by rillig and mummey (2006), smith and read (2008) and van der heijden and horton (2009). acknowledgements this study has been supported by the swiss national science foundation within the programme nfp48 ‘landscapes and habitats of the alps’ and snsf project 315230_130764/1. financial support was also received from fondecyt chile which made visits of southern chile possible for the last author, and recently also for f. oehl (fondecyt project 11090014). the fi rst author also thanks the family giuanna albin-pelican (hostel in surrein, surselva) for great hospitality during several collections at the type location between 2003 and 2007. we are also grateful to urs zihlmann (agroscope art) and our collaborators claudia maurer, andres chervet and wolfgang sturny at the bodenschutzfachstelle of the kanton bern for the excellent support at the lowland sampling sites. references becerra, a.g., cabello, m.n., bartoloni, n.j., 2011: native arbuscular mycorrhizal fungi in the yungas forests, argentina. mycologia 103, 280-290. błaszkowski, j., 1993: comparative studies on the occurrence of arbuscular fungi and mycorrhizae (glomales) in cultivated and uncultivated soils of poland. acta mycol. 28, 93-140. brundrett, m., melville, l., peterson, l., 1994: practical methods in mycorrhizal research. mycologue publications, university of guelph, guelph, ontario, canada. castillo, c.c.g., borie, f., godoy, r., rubio, 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arguments for diagnoses based on unaltered wall structures. mycotaxon 38, 71-76. spain, j.l., sieverding, e., oehl, f., 2006: appendicispora: a new genus in the arbuscular mycorrhiza-forming glomeromycetes, with discussion of archaeospora. mycotaxon 97, 163-182. stürmer, s., 2012: 163-182 a history of the taxonomy and systematics of arbuscular mycorrhizal fungi belonging to the phylum glomeromycota. mycorrhiza 22, 247-258. van der heijden, m.g.a., horton, t.r., 2009: socialism in soil? the importance of mycorrhizal fungal networks for facilitation in natural ecosystems. j. ecol. 97, 1139-1150. vestberg, m., 1995: occurrence of some glomales in finland. mycorrhiza 5, 329-336. walker, c., vestberg, m., demircik, f., stockinger, h., saito, m., sawaki, h., nishmura, i., schüssler, a., 2007: molecular phylogeny and new taxa in the archaeosporales (glomeromycota): ambispora fennica gen. sp. nov., ambisporaceae fam. nov., and emendation of archaeospora and archaeosporaceae. mycol res. 111, 137-153. walker, c., 2008: ambispora and ambisporaceae resurrected. mycol res. 112, 297-298. address of the authors: fritz oehl, agroscope reckenholz-tänikon research station art, ecological farming systems, reckenholzstrasse 191, ch-8046 zürich, switzerland, e-mail: fritz.oehl@art.admin.ch. claudia castillo, universidad católica de temuco, facultad de recursos naturales, escuela de agronomía, casilla 15-d, temuco, chile. david schneider, zurich-basel plant science center, institute of botany, university of basel, hebelstrasse 1, ch-4056 basel, switzerland. verena säle, agroscope reckenholz-tänikon research station art, ecological farming systems, reckenholzstrasse 191, ch-8046 zürich, switzerland. ewald sieverding, institute of plant production and agroecology in the tropics and subtropics, university of hohenheim, garbenstrasse 13, d70593 stuttgart, germany. art020a journal of applied botany and food quality 80, 14 18 (2006) dr. khanaqa research institute, riyadh, kingdom of saudi arabia truffle production in the kingdom of saudi arabia – potential and limitation azad khanaqa (received august 17, 2005) summary in saudi arabia desert truffles are much appreciated for culinary despite high prices. this is attributed to high demand throughout the year and a limited offer. so far, only naturally occurring truffles have been traded on local markets. the aim of the present investigation was to find out whether it is possible to establish and propagate truffles under semi arid conditions in saudi arabia using olive plants as host. the field experiment was installed at al-khalidiah farm-tebrak situated in the vicinity of riyadh and comprised an area of 150 ha. previously raised olive cuttings were inoculated with termania nivea and tirmania pinoyi and kept under controlled greenhouse conditions prior to be transplanted into the field. although olive plants developed well, yields of truffles was very weak after four years. the incorporation of a suitable soil on separated areas modified the situation and truffles started to develop well, and up to 14 kg/ha were yielded. unusual heavy rainfalls, however, inhibited the development of the fruit bodies of truffles resulting in a sharp decrease of the yields. based on the promising results obtained during a four-year period, it was decided to continue to produce desert truffles taking into account the experiences gained in the past years. 1. introduction truffles are rich-flavoured fruit bodies of a subterranean mushroom. they develop only in symbiosis with the root system of certain hardwood trees. under ideal soil conditions, the roots of these trees are infected with fungal spores of spreading mycelium and the truffles start to grow. since time immemorial, this most precious of all edible mushrooms has exhibited a mystical aura. the mystery of its formation seemed impossible to solve and this unique treasure of nature could be reaped only after laborious searching and with great fortune. although highly valued by gourmets, truffles remained a well-protected secret of nature. the southern french truffle, a jewel of gastronomy since roman times, is now almost extinct. this aromatic fungus has become so rare and so expensive that it is barely worth looking for. this lack has prompted both a minor economic eruption and a real social crisis in some parts of europe where truffle hunting was once an important source of income and a popular pastime. many rural people who used to collect truffles to supplement agricultural incomes are now facing a significant challenge due to the gradual extinction of wild truffles. at the turn of the century the southern plateau in france, known as „le causse“ was producing 500 tons of the black truffle which played a key role in the development of french cuisine and, even in 1960, eighty (80) tons were still being exported annually. during 1998-1999 the harvest for truffles had dropped to (6) tons and the truffle season produced a minuscule haul. in contrast to the european forest truffles, the so-called desert truffles are found in arid and semi arid zones of the mediterranean basin, in iraq and kuwait, the sahara, saudi arabia and part of the maghreb. for centuries, people from the middle east have also appreciated the highly prized desert truffle. it is being recognized as beneficial for many medical problems including the treatment of gout, muscular and arthritic pains, and high cholesterol levels. with regard to their nutritional value literature reveals the following information: of approximately 20% dry matter, 20-27% consists of proteins (of which about 85% is digestible by humans, 3-7.5% of fat (including unsaturated as well as saturated fatty acids), 7-13% of crude fibre and close to 60% of carbohydrates (ackerman et al., 1975; aldelaimy, 1977; al-shabibi et al., 1982; sawaya et al., 1985; bokhari et al., 1989). in arab countries, desert truffles have long been prized for culinary. during their relative short fruiting season in years of abundance, they are marketed at prices comparable to those of meat, and indeed, served also instead of meat. in years of scarcity, they command even very high prices. according to investigations of the author at various markets in saudi arabia the demand for truffles exceeds by far the offer. only in riyadh the annual offer varies between 1-1.2 t whereas the demand is much higher. for the whole country it is estimated to be more than 50 t per year. consequently, the price for one kilogram truffle remains high and may attain 200 us$ per kg. to collect desert truffles, one needs not resort to trained dogs or pigs, as is the case with european forest truffles. the fruit bodies form close to the soil surface, and as they swell, they lift up the soil to form little cracked mounds recognizable to trained eye (awameh and alsheihk, 1979a). in some areas of the arabian gulf region, royal families claim the truffle crop during seasons of abundance and have truffle lands patrolled until most of the crop is harvested. the objective of the present research was to investigate the possibility to produce naturally occurring truffles and to determine whether agricultural practices exert an impact on the development of desert truffles under semi-arid conditions in saudi arabia using olea europea as host plant. in the following the term truffle is used for termania spp. which is found in association with helianthemum spp. in desert habitats. 2. materials and methods 2.1. location and conditions the present research activities were carried out at al-khalidiah farmtebrak situated in the vicinity of riyadh. the soil in this farm is a sandy soil (tab. 3) and was not considered by the author to be very suitable for truffle production. however, the owner of the farm insisted to carry out the investigations on his farm. the climate in this area is semi-arid with a distinct moderate rainy season from january to march showing a very irregular distribution. temperatures are highest in the dry season (june-october) and relative humidity is very low (tab. 1). according to taxonomic identification carried out by dr. giovanni pacioni in 2004 at the dipartimento di scienze ambientali, università l´aquila, italy, it was confirmed that the truffles species used in the experiment were: tirmania nivea (fig. 1 and 3) and tirmania pinoyi (fig. 2 and 4) often used under the same name of „zubaidi“ (that is buttery) in arab countries. the identification was carried out on the basis of morphological characteristics (see tab. 2) and by allozyme analysis (pacioni et al., 1997). according to pacioni (personal communication), tirmania nivea genes seem to be fixed in homozygosis, whereas tirmania pinoyi shows a remarkable level of heterozygosis. 2.2. ecology of tirmania tirmania nivea and tirmania pinoyi have been demonstrated to form a unique type of mycorrhizae with desert annuals belonging to the family cistaceae. helianthemum ledifolium (l.) mill and h. salicifolium (l.) mill (awameh and alsheik, 1979a; awameh and alsheik, 1979b). the habitat and ecology of termania spp. have been studied in kuwait (awameh and alsheikh, 1979a). terminia spp. develops well in high ph calcareous soils (fortas and chevalier, 1992; giovanntti et al., 1994). they occur in gravely gypsiferous, gypsiferous-saline, or saline deserts as described by halwagy and halwagy (1974a). sporocarp production is correlated with amount and seasonal distribution of rainfall, an important factor for the development of the host plant. according to alsheikh and trappe (1983) under semiarid conditions in kuwait, a minimum of 180 mm precipitation welldistributed from october through march, produces a rich development of annuals, including helianthemum spp. sporocarps of tirmania form in the soil at depths of 1-6 cm among roots of annuals. as the sporocarps enlarge, they raise a mound of soil that cracks as it dries. tab. 2: characteristics of tirmania nivea and tirmania pinoyi morphological tirmania nivea tirmania pinoyi characteristics (natural zubaidi truffel) (khanaqa's zubaidi truffel) asci spontaneously blushing blushing after treatment with iodine reactive with alcohol spores smooth broadly wrinkled and round ellipsoids peridium white almost without whitish with sticking sticking sand sand fig. 3: characteristic spore of tirmania nivea, (pacioni and el-kholy, 1994), bar = 10µm tab. 1: weather statistics from al-khalidiah farm-tebrak temperature (ºc) months mean daily mean daily precipitation humidity minimum maximum (mm) (%) june 22.8 42.1 0.0 14 july 23.4 43.0 0.0 15 august 23.3 42.2 0.0 14 september 20.2 40.5 0.0 18 october 15.3 34.8 2.5 24 november 11.0 28.4 2.9 37 december 7.7 22.5 8.0 46 january 6.5 21.5 18.0 56 february 8.5 24.4 27.0 50 march 12.5 27.7 24.4 35 april 17.1 33.3 16.0 33 may 21.0 38.7 7.3 22 desert truffle production 15 fig. 1: naturally occuring „zubaidi“ truffel; tirmania nivea fig. 2: khanaqa's „zubaidi“ truffel; tirmania pinoyi 2.3. soil analysis chemical and grain size analysis of the soils (tab. 3) were carried by the inspection diagnostics analysis consultation laboratories (idac) in riyadh. original soil (s1) designates the soil at alkhalidiah farm whereas introduced soil (s2) refers to soil from an area about 30 km north of riyadh and which was later incorporated at the experimental site. this area is known for its desert truffles which can be found in association with helianthemum spp. powder (naphthalene acetic acid 0.80 gm + vitamin b1 – hydrochloride 0.30gm) to activate root growth prior to be transplanted into the nursery of a greenhouse with automatic temperature regulation. mean temperature was kept at 25ºc and cuttings were sprayed with water 5 times a day. at a root length of 5-7 cm cuttings were transplanted into plastic bags (3 litres) containing a pear light/peat moss mixture (50/50, v/v). 2.5. inoculation with truffle spores 4-7 weeks after transplanting, cuttings were ready (root length > 10 cms) for inoculation. locally occurring truffle species (tirmania nivea and tirmania pinoyi, see above taxonomic identification) were selected for the preparation of the inoculation media which consisted of truffle spores and hypheae and a specific truffle media previously produced and successfully tested in the laboratory of dr. khanaqa's institute at hannover/germany (khanaqa, 1994) . about 10 ml of truffle media were added to each plant in the plastic bag and kept in nursery under controlled greenhouse conditions. after six weeks 100 randomly selected samples of the inoculated plants were checked under the microscope for root infection. as soon as infection rate was higher than 80% olive plants were ready for transplantation on the land. 2.6. plantation establishment, planting density and maintenance the layout of the olive plantation (150 ha) was arranged on a grid where each plant is equidistant from its four neighbours. distance between plants and between rows was 3 metres giving a total number of 1111 trees per hectare. in order to overcome insufficient rainfall during crucial periods of the year, a droplet irrigation system was installed and each olive plant was daily provided with 20 litres of water. no fertilizers or chemicals were applied. however, at the beginning of transplantation each plant received 3 kg of compost and again 3 months after transplantation with the following chemical composition (ph 7.7; organic matter, 54.5%; total nitrogen, 2.1%; calcium, 3.9%; phosphorous, 0.9%; potassium 2.3%; magnesium, 0.5%). 2.7. statistical analyses statistical analyses were carried out using systat. analysis of variance, was used to test the differences among the four harvests. 3. results and discussion the olive plantation was installed in 1997 and all plants began to take root and developed well (fig. 5). the following description of the results and the observations made are based on a 4-year lasting development process and each year an additional treatment was evaluated. in february 2001, four years after implementation of the olive plantation, 250 plants randomly selected per hectare were checked for truffle infection. development of truffles was very poor resulting in very modest yield. on the average less than 3 kg per ha were found (tab. 4). these findings were very unsatisfying and we began to search for the reasons. we checked the soil (s1) where the olive plantation had been installed. the results of the soil analysis revealed a high percentage of sand and gravel (98%) and a very low percentage of silt (1%) indicating that this soil has an unfavourable granulation for truffle production. in addition the electrical conductivity (ec) was also high (4.5 ms/cm) which indicated the presence of salts in the soil. further chemical analysis (tab. 3) confirmed this assumption and chloride and sodium were found in 2.4. preparation of cuttings for truffle inoculation in saudi arabia end of december is the most suitable time to take cuttings from a variety of trees (e.g. olive trees) for truffle inoculation. selected healthy mother trees were used for the production of cuttings. each cutting had about 3-4 buds and was dipped into a hormone 16 azad khanaqa fig. 4: characteristic spores of tirminia pinoyi tab. 3: chemical and grain size analysis of the soils description and units original soil (s1) introduced soil (s2) (sand) (loamy sand) sand + gravel (%) 98.0 77.1 silt (%) 1.0 14.0 clay (%) 1.0 8.9 water content (%) 2.2 9.1 ph 7.69 8.1 ec (ms/cm) 4.5 1.05 total chloride, (mg/kg) 629 270 sulphate, (mg/kg) 3095 850 bicarbonate, (mg/kg) 267 150 calcium, (mg/kg) 671 957 magnesium, (mg/kg) 103 70 sodium, (mg/kg) 380 130 phosphorus, (mg/kg) 14.53 17.1 potassium, (mg/kg) 121 104 copper, (mg/kg) 0.07 0.08 iron, (mg/kg) 0.56 0.41 manganese, (mg/kg) 0.11 0.36 zinc, (mg/kg) 0.08 0.1 calcium carbonate, (%) 27.08 38.22 high concentration – 629 mg/kg and 380 mg/kg respectively. these findings clearly explained the weak development of truffles resulting in very low yields. in order to overcome this problem, we decided to incorporate a soil with better characteristics for truffle production. after intensive search a suitable soil (s2) was found which originated from a region about 30 km north of riyadh. as we discovered later, this area is known for its desert truffles which can be found in association with helianthemum spp. from the original olive plantation 4 ha were separated and each of the olive plants received 25 kg of soil (s2) which was thoroughly mixed and incorporated. in february 2002, again 250 plants per ha were randomly selected from the original plantation as well as from the previously separated area in order to examine the development of truffles. the results clearly showed the positive effect of the incorporated soil. in the original soil of the plantation truffle development was again very weak, however, in the separated area yield of truffle reached 10 kg/ha (tab. 5, treatment 2 and 2a respectively). in order to confirm the results, an additional area (4 ha) was separated from the original plantation in 2002. this new area was also treated with (s2) as previously described. in the year 2003, plants were checked in the 3 treatments (3-3b). again we obtained a very poor development of truffles in the area of the original plantation. in the two separated areas, however, the results were even significantly higher when compared to the yields in 2a of the previous year. between the treatments 3a and 3b and between 2a and 3b significant differences were obtained. this finding clearly indicates a time course improvement of the development of truffles in the presence of (s2). the results in the year 2003 led to a yield of 75 kg. however, in order to follow this positive development we installed an additional area and repeated the same procedure as described above. in february 2004, the development of truffles was checked in the treatments 4-4c. surprisingly, the development of truffles had dramatically decreased. in the original treatment (4) no truffles at all were found and even in the other treatments (4a-4c) only a reduced development was observed. in general, we found 50-60% less truffles when comparing the results of 3a/3b with 4a/4b. we assume that these poor yields are attributed to unexpected heavy rains which occurred during the months december 2003 and january 2004. between the 10th and 16th of january 2004 about 200 mm rain were recorded and in some areas even temporary floods occurred. these heavy rain falls probably swept away many truffle spores and therefore the development of fruit bodies did not take place. in mediterranean countries (france and italy), this phenomenon occurs quite often after heavy rainfalls during the winter season (personal communication to the author). considering the positive results of the second and third year, it was decided to continue with the experiment. means were investigated on how (s2) could be transported in large quantities to al-khaladiah farm to be incorporated into the local soil. we look forward to repeat these promising results which clearly revealed that the production of desert truffles is feasible. fig. 5: olive plantation, three years old tab. 4: results of yields (kg/ha) in the years 2001 2004. average of treatments (1-4c, n = 250), s.e. in parentheses year of harvest and extrapolated yield sequential treatments kg/ha 1) 2001 (original plantation) < 3 2) 2002 (original plantation) < 2 2a) 2002 (4 ha separated in 2001 from original plantation 10.00 (1.4) + incorporated s2) 3) 2003 (original plantation) < 2 3a) 2003 (4 ha separated in 2001 from original plantation 14.2 (2.2) + incorporated s2) 3b) 2003 (additional 4 ha, separated in 2002 from original plantation 11.1 (1.6) + incorporated s2) 4) 2004 (original plantation) nil 4a) 2004 (4 ha separated in 2001 from original plantation 4.7 (1.2) + incorporated s2) 4b) 2004 (additional 4 ha, separated in 2002 from original plantation 5.8 (1.1) + incorporated s2) 4c) 2004 (additional 4 ha, separated in 2003 from original plantation 4.9 (1.4) + incorporated s2) 4. conclusions desert truffles are so far known not cultivated anywhere in the world. in developed countries, where fertile soils can readily be worked by modern agriculture methods, cultivation of desert truffles based on perennial host plants transposed from their natural habitat is not likely to be a profitable venture at present. introduction may be costly, and desert truffles fetch far lower prices than truffles from temperate regions. in desert regions, however, where the truffles are indigenous like in many arabian countries, domestication of the truffle symbiosis may reasonably be expected to be successful. it would not involve new introductions and since an appreciative local market already exists, considerable returns could be expected. tirmania nivea and t. pinoyi are known for their economic value in the countries where they occur and provide a much-desired source of food. however, crops vary markedly from year to year because of variations in weather. therefore, development of cultivation methods, e.g. through irrigation with drip irrigation in droughty years, could provide an important step in productive use of arid lands. as a result, cultivated imitation of nature would represent an advance over collection from the wild. moreover, by using an appropriate perennial crop as host plant, such as olea europea, a dual benefit from this association could be achieved i.e. the production of truffles and the desert truffle production 17 production of fruits from the trees. in addition, due to the presence of trees a microclimate will be established giving evidence to further benefits: 1) soil formation will be promoted because the trees will prevent soil erosion 2) water holding capacity will be improved in the planted area, 3) decreased surface run-off due to an improved water retention by the trees resulting in enhancing the volume of underground water and, 4) trees will serve as windbreaks and sandstorm breakers. besides the annual costs for maintenance, the only investment to make is to purchase the truffle-infected seedlings and plant them in the designated areas. the experience gained with above described project are quite promising and will certainly have a great impact on the local and external market, where the cultivation of truffles on a large scale would allow more people to enjoy this exotic delicacy. acknowledgements the author wishes to thank prof. giovanni pacioni (dipartimento di scienze ambientale, università degle studi dell´aquila, italy) for the taxonomic identification of the tirmania spp. collected in the area of research and for permission to use the fig. 3 and 4 for demonstration purposes. references ackerman, l.g.f., van wyk, p.j., du plessis, l.m., 1975: some aspects of the composition of the kalahari truffle or n´abba. south african food rev. 4, 145-146. al-delaimy, k.s., 1977: protein and amino acid composition of truffle. canadian institute food sci. technol. j. 10, 221-222. al-shabibi, m.m.a., toma, s.j., hadad, b.a., 1982: studies on iraqi truffles: proximate analysis and characterization of lipids. canadian institute food sci. technol. j. 15, 200-202. alsheikh, a.m., trappe, j.m., 1983: desert truffles: the genus tirmania. trans. br. mycol. soc. 81, 83-90. awameh, m., alsheikh, a.m., 1979a: laboratory and field study of four kinds of truffle (kameh), terfezia and tirmania species for cultivation. mushroom sci. 10, 507-517. awameh, m., alsheikh, a.m., 1979b: characteristics and ascospore germination of white kameh (tirmania nivea and t. pinoyi). annales de phytopathologie 11, 223-29. bokhari, y.h.a., suleiman, a.a., basalah, m.o., 1989: the fatty acid components of the desert truffle „al kamah“ of saudi arabia. j. food protection 52, 668-669. fortas, z., chevalier, g., 1992: effet des conditions de culture sur la mycorhization de l' helianthemum guttatum par trois especes des genres terfezia et tirmania d'algerie. can. j. bot. 70, 2453-2460. giovannetti, g., roth-bejerano, n., zanini, e., kagan-zur, v., 1994: truffles and their cultivation. horticultural rev. 17, 71-107. halwagy, r., halwagy, m., 1974a: ecological studies on the desert of kuwait i. the physical environment. journal university kuwait 1, 75-86. khanaqa, a., 1994 : trüffelkultur. internal publication, universität hannover, germany. pacioni, g., el-kholy, h., 1994: tartufi del deserto egiziano: micologia e vegetazione mediterranea 9, 69-84. pacioni, g., frizzi, g., mirando, m., el-kholy, h., 1997: allozyme characterization of some terfeziaceous fungi (pezizales, ascomyctina). mycotaxon 61, 427-432. sawaya, w.n., al-shalhat, a., al-sogor, a., al-mohamed, m., 1985: chemical composition and nutritional value of truffles of saudi arabia. j. food sci. 50, 450-453. present address of the author: dr. azad khanaqa, riyadh 11547, p.o. box 69428, kingdom of saudi arabia corresponding address: wülferoderstraße 32, d-30880 laatzen, germany 18 azad khanaqa << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false /maxsubsetpct 100 /optimize true /opm 1 /parsedsccomments true /parsedsccommentsfordocinfo true /preservecopypage true /preserveepsinfo true /preservehalftoneinfo false 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<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> >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice art12_saha.indd journal of applied botany and food quality 85, 207 211 (2012) 1indian agricultural research institute, new delhi, india 2india institute of sugarcane research, lucknow, u.p., india antifungal acetylinic thiophenes from tagetes minuta: potential biopesticide supradip saha1*, suresh walia1, a. kundu1, b. kumar1, deeksha joshi2 (received august 11, 2012) * corresponding author summary apart from thiophenes, which possess wide range of biocidal activity, aerial parts of tagetes sp contain essential oil. oil components were reported to have antifungal activity, thus making whole plant of tagetes very useful for exploiting as natural fungistatic agent. in the present study, tagetes minuta grown in north western himalayan condition were evaluated for its potential for use as antifungal agent. flower essential oil showed minimal antifungal activity. whereas, leaf essential oil was found signifi cant antifungal activity against three phytopathogenic fungi out of eight tested fungi. ed50 values were 165, 175 and 110 µg ml-1 against rhizoctonia solani, sclerotinia sclerotiorum and sclerotium rolfsii, respectively. thiophene rich extract of tagetes minuta was found comparatively lesser active (ed50: 233-484 µg ml-1) than leaf essential oil against the same fungi. the present study shows that essential oil from leaves and thiophene rich extracts from marigold roots have signifi cantly good antifungal activity against a number of soil borne and foliar plant pathogens. the easy availability of these plants makes it an attractive potential candidate for development of natural fungicide. introduction plant secondary metabolites and their derivatives have been evaluated as viable alternatives to the persistent and less environmentally friendly synthetic fungicides (dixon, 2001). although a large number of phytochemicals are known for their insect control properties, only a few of them are known to impart antifungal activity. the use of pesticides of natural origin is becoming appealing because of the problem of environmental pollution arising from the use of persistent pesticides. to move forward in the discovery of natural plant metabolites, plant extracts were screened for potential candidates with antifungal activity (crouse, 1998). some of the most promising botanicals for use as pesticides have been extracted from species of plants in the families meliaceae, rutaceae, asteraceae, annonaceae, labiatae, and canellaceae (jacobson, 1989). although much of the literature on natural products in agricultural fi eld concerns insect control, a smaller but emerging body of papers has reported the effectiveness of plant extracts and essential oils for controlling microbial pathogens of plants. marigold (tagetes sp.), belonging to the asteraceae family, is a commonly occurring plant all over the world and is well known for a wide range of biological properties. the plant has been credited with having anticancerous and antiageing effects (block et al., 1992). it contains carotenoids which is used as food colorants and feed additives (timberlake and henry, 1986). besides this the plant also has pharmacological properties as it contains fl avonoids (terschuk et al., 1997). the foliar parts of this plant possess essential oils, known for antibacterial and insecticidal properties (piccaglia et al., 1997); and thiophenes which have a marked biocidal activity (hulst et al., 1989). these polyacetylene derivatives, the activity of which depends on photodynamic activation (hudson and towers, 1991), act as antibiotics, insecticides, nematicides and fungicides (gommers, 1981; mares et al., 1990; hudson, et al., 1983; romagnoli et al., 1994, 1998). further, constituents of essential oil, mainly (z)and (e)-ocimenones, along with piperitone, piperitenone, limonene, tagetone and caryophyllene, are the major terpenes present in leaves (vasudevan et al., 1997). in addition, hethelyi et al. (1986) assumed that the presence of linalool and linalyl acetate characterizes indian tagetes patula oil. due to the high degree of chemodiversity observed within essential oils of tagetes sp., biological activities also subjected to vary. however, even though the various properties of the tagetes plant are well known, less attention has been focused on the studies of biological activity of essential oils from tagetes species. therefore, the present work was aimed at developing an antifungal formulation from the total plant extract. interestingly, synergestic effect on egg hatchability of globodera rostochinensis was observed when treated with a combination of t. erecta leaf and root extracts (sasanelli and vito, 1991). furthermore, juvenile of m. incognita populations were more suppressed by stem and whole plant rather than root portions (siddiqui and alam, 1988). present study was aimed to evaluate the antifungal activity of the whole plant parts. thus in vitro antifungal activity was evaluated against eight important plant pathogenic fungi. antifungal formulation may be developed using essential oil from leaves and thiopene rich extract from roots and shoots. materials and methods general experimental procedure all chemicals and reagents were procured from merck® india ltd. double-distilled water was used throughout the analysis. gcms analysis was carried out on a trace-gc (thermo finnigan) coupled with fi son md-800 quadrupole mass detector and db-17 capillary column (30m × 0.32 mm i.d.; fi lm thickness 0.25 mm). temperature programming was done from 75-250 °c at 5 °c min-1. helium was used as the carrier gas at 1 ml min-1 fl ow rate. mass spectra was recorded over 40-400 amu range at 1 scan s-1 with ionization energy of 70ev and ion source temperature was 250 °c. the split ratio was 1:20. plant material the plant material (leaves, fl ower and roots) was collected during the month of september from experimental farm of vivekananda institute of hill agriculture located at hawalbagh (altitude: -1220 m a.s.l., latitude: -29°38’4.8”n, longitude: -79°37’48.6”e) in north western himalayan region. the identifi cation of the plants was confi rmed from department of botany, almora inter college, kumaon university, almora, india. mancozeb and β-pinene, a constituent of essential oil was used as positive control (procured from merck®). 208 supradip saha, suresh walia, a. kundu, b. kumar, deeksha joshi test fungi the essential oils / extracts were tested for their antifungal properties against eight plant pathogenic fungi. pyricularia grisea was isolated from blast infected fi nger millet leaves; rhizoctonia solani and fusarium solani were isolated from infected roots of french bean, sclerotium rolfsii from infected collar portion of french bean, fusarium oxysporum f.sp. pisi from wilted pea plants, sclerotinia sclerotiorum from white rot infected pea plants, fusarium oxysporum f.sp. lentis from wilted lentil plants and alternaria solani from tomato leaves. extraction of essential oil leaves (200 g) and fl ower (250 g) of tagetes sp. were subjected to hydro-distillation separately for 3 h using a clavenger apparatus to obtain essential oil. yield of essential oil was 0.3-0.4% and 0.20.3% from leaves and fl ower respectively. the oil was dried with anhydrous sodium sulfate and preserved in a sealed vial at 4 °c until the moment of analysis. extraction and purifi cation of thiophene extraction of thiophene from shoots and roots was done by following the modifi ed method of margl et al., 2002. finely chopped air dried shoot and roots (600 g) from tagetes sp., were extracted with methanol: water (3:1 v/v, 2500 ml) twice at room temperature with the help of mechanical stirrer and fi ltered. volume of combined extract was reduced in vacuo at 45 °c. the concentrated extract was sequentially partitioned into hexane: chloroform (2:1 v/v). the partitioned organic solvent fractions were concentrated to dryness by rotary evaporation at 35 °c to get dark green coloured thiopene rich concentrate (625 mg). dark green thiophene rich extract was dissolved in minimum quantity of hexane, fi ltered and concentrated to dryness. then the residue was dissolved in et2o and passed through column, preconditioned with et2o and packed with 60120 mesh silica gel. purifi ed extract was then subjected to gc-ms analysis. characterization tic of root and shoot extract was presented in fig. 1. extract contains essential oil component as well as acetylenic thiophenes. first compound in the tic eluted as dihydrotagetone and it was confi rmed by its mass fragmentation pattern. the compound was eluted at 2.267 minute. molecular ion peak was detected as m/z 154 [m]+ with the mass fragmentation of m/z 139, 125, 108, 93, 81, 71, 58 (tab. 1). second compound eluted in the tic was confi rmed by its molecular ion peak of m/z 152 and fragments of 137, 109, 95, 81, fig. 1: tic of thiophene rich extract. antifungal thiophenes from tagetes minuta 209 69, 58 as piperitone. α-terpineol (m/z 154, 136, 121, 111, 93, 83, 71, 58) was eluted at 5.801 minute. compound at rt of 6.534 min was remain unidentifi ed. three thiophenes were identifi ed as 5-(3-buten1-ynyl)-2,2’-bithienyl (bbt) (37.57 min), α-terthienyl (47.354 min) and 5-(4-acetoxy-1-butynyl)-2,2’-bithienyl (bbtoac). molecular ion peak of these three compounds were m/z 218, 248 and 216 respectively with characteristic fragmentation pattern. antifungal bioassay the cultures were maintained on pda slants at 25 °c and were subcultured in petri dishes prior to testing. weighed quantities of essential oil and extract (extracted under diffused sunlight) to be tested were dissolved in acetone (1 ml) and incorporated into the molten medium (at 45 °c) to reach the desired concentration. pda containing only acetone served as control while β-pinene at 250 µg ml-1, 500 µg ml-1 and 1000 µg ml-1 served as a positive control. medium was poured into each sterile petri dish under aseptic conditions and left to settle. circular discs (5 mm diameter) of the test mycelia mats (punched-in mycelia mats grown in sterile petri dishes) were inoculated centrally and left to grow. in all cases, triplicates were maintained. radial growth inhibition of test fungi was evaluated. the diameter of fungal growth (mm) was noted at 72 h after inoculation and subsequently every 24 h for a total period of 240 h. these data were subjected to analysis of variance using completely randomised blocks. the percentage inhibition relative to the control, i, was converted to corrected percentage inhibition, ic, using the following formula: ic = [(i − cf)/(100 − cf)] × 100 cf = [(90 − c)/c] × 100 where cf is the correction factor, 90 is the diameter of the petri dish (mm) and c is the diameter of the fungus in the control (mm). statistical analysis four replicates were used for each experimental treatment. the effective concentration for 50% inhibition of mycelial growth, ed50 (µg ml-1), was calculated from the ic data using the basic ld50 program v.1.1 as described by trevors, 1986. percentages were angular transformed (arc sine) for analysis and analysed by complete randomised design. results three monoterpenes and three thiophenes were identifi ed from the shoot and root extract of tagetes minuta. terpenoids were identifi ed as dihydrotagetone, piperitone and α-terpineol by their mass fragmentation pattern. out of these three monoterpenes, one is acyclic i.e. dihydrotagetone and other two are cyclic monoterpenes. romagnoli et al., 2005 reported these compounds to be present in fl ower essential oil along with other monoterpenes. the antifungal activity of fl ower essential oil is not so encouraging (tab. 2). percent inhibition of fl ower essential oil at 1000 µg ml-1 was ranged between 8.9-35.1%. highest activity was found against fusarium oxysporum pisi and least activity was recorded against pyricularia grisea. antifungal activity of leaf essential oil against eight phytopathogenic fungi is presented in tab. 3. antifungal activity was comparatively superior in leaf essential oil than fl ower. ed50 value ranged between 110-1016 µg ml-1. leaf essential oil was most active against sclerotium rolfsii and least active against fusarium oxysporum lentis. the in vitro results were classifi ed according to aligiannis et al., 2001 and duarte et al., 2005 who proposed that, when the minimum inhibitory concentration (mic) is below 500 µg ml-1, the antifungal activity is considered strong, and, if the extracts/compounds display an mic between 600 and 1500 µg ml-1, the antifungal activity is considered moderate. compounds with an mic above 1600 µg ml-1 are considered weak. as evident from the data, leaf essential oil exhibited strong activity against rhizoctonia solani, sclerotinia sclerotiorum and sclerotium rolfsii. medium antifungal activity was recorded against other fi ve phytopathogenic fungi. the commercial reference fungicide, mancozeb, was highly effective against all four test fungi. whereas, essential constituent β-pinene showed ed50 value less than 180 µg ml-1. antifungal activity of thiophene rich extract from shoot and root is presented in tab. 4. ed50 value against eight phytopathogenic fungi ranged between 233 to 2227 µg ml-1. the extract was most active against r. solani and least active against p. grisea. hyphal growth inhibition was maximum in r. solani, s. sclerotiorum and s. rolfsii and the activity was found to be strong. while, moderate antifungal activity was recorded in f. solani, a. solani, f. oxysporum pisi and f. oxysporum lentis. weak antifungal activity was evidenced against p. grisea. tab. 1: mass spectra of compounds present in the purifi ed root extract. compound rt m/z mass spectra (m/z, relative intensity) [min] dihydrotagetone 2.267 154 58(100), 71(41), 81(54), 93(37), 108(54), 125(9), 139(32), 154(31) piperitone 3.784 152 58 (25), 69(49), 81(100), 95(5), 109(10), 137(6), 152(12) α-terpineol 5.801 154 58(92), 71(100), 83(27), 93(78), 111(78), 121(15), 136(19), 154(18) bbt 37.570 216 69(8), 95(29), 171(29), 216(100), 217(15), 218(12) α-terthienyl 47.354 248 58(5), 69(10), 127(14), 171(9), 203(15), 248(100) bbtoac 51.054 276 95(6), 171(13), 203(16), 216(100) tab. 2: hyphal inhibition of eight major plant pathogens upon application of fl ower essential oil at 1000 µg ml-1 after 3 days. pathogen inhibition (%) rhizoctonia solani 5.7 fusarium solani 13.8 sclerotinia sclerotiorum 8.9 fusarium oxysporum pisi 35.1 scleroium rolfsii 19.5 pyricularia grisea 26.4 fusarium oxysporum lentis 22.9 alternaria solani 21.9 210 supradip saha, suresh walia, a. kundu, b. kumar, deeksha joshi discussion the marigold plant has been credited with a number of properties including antimicrobial / insecticidal / nematicidal activity (natarajan et al., 2006; piccaglia et al., 1997; hulst et al., 1989; romagnoli et al., 1994). in the present work, a comparative study of the antifungal activity of essential oils / extracts from different parts of the tagetes plant against eight important plant pathogenic fungi was done. out of six identifi ed compounds, three were monoterpenic derivative and are component of essential oil. three compounds namely, dihydrotagetone, piperitone and α-terpineol mostly came from shoot portion of the tagetes minuta plant. other three identifi ed compounds were thiophenes and root portion contributed its lion share (downum, 1983). antifungal activity of tagetes sp. published so far is related both to the presence of thiophenic compounds alone and with uv-a irradiation (mares et al., 1990; romagnoli et al., 1994). number of species also reported for different activity including antifungal activity. reports on exploration of total plant extract of t. minuta for fungistatic activity is not so well documented. though uhlenbroek and bijloo (1958) found that nematicidal or nematostatic effects of tagetes were due to α-terthienyl, sasanelli and vito (1991) suggested that there was a synergistic effect of leaf and root extract on egg hatchability of g. rostochinensis. siilar effect was observed by siddiqui and alam, 1988, where it was concluded that stem and whole plants was more effective in suppressing juveniles of nematode than root extract. all the different compounds (essential oils and extract) from tagetes plant as well β-pinene exhibited antifungal activity against the eight pathogens. however, there were signifi cant differences in the antifungal activity of the compounds from different parts of the plant. it was observed that leaf essential oil and root extract at 1000 µg ml-1 showed the highest inhibitory activity against all the eight pathogens which persisted for more than 6 days. leaf essential oil showed hyphal growth inhibition of 34.1 to 85.6% while root extract treatment showed inhibition of 21.6 to 77.8% after 6 day against various pathogens. even at lower concentrations of 500 µg ml-1 and 250 µg ml-1 leaf essential oil resulted in signifi cant reduction in hyphal growth of fi ve of the pathogens both 3 and 6 days after treatment. however, the essential oil from fl owers was not highly potent and exhibited minimal antifungal activity (< 35%) at all three concentrations against the 8 pathogens. its antifungal activity was particularly low against the fast growing pathogens r. solani and s. sclerotiorum. marigold (tagetes sp.) plant is a commonly available plant in all regions of the world. the present study shows that essential oil from leaves and thiophene rich extracts from marigold roots have high antifungal activity against a number of soil borne and foliar plant pathogens. the easy availability of these plants makes them an attractive potential candidate for development of plant based biopesticide. tab. 4: antifungal activity thiophene rich extract against eight phytopathogenic fungi. ed50 χ2exp fiducial limit regression equation [µg ml−1] (3df, 95%) rhizoctonia solani 233 3.17 191-284 y = 1.77+1.37x sclerotinia sclerotiorum 484 4.37 352-667 y = 2.36+0.98x fusarium solani 3656 5.85 1195-11187 y = 2.42+0.72x alternaria solani 1045 6.64 634-1722 y = 2.18+0.93x pyricularia grisea 2227 2.00 819-6063 y = 2.82+0.65x fusarium oxysporum pisi 1131 6.69 611-2094 y = 2.63+0.78x fusarium oxysporum lentis 1244 5.56 680-2275 y = 2.42+0.83x sclerotium rolfsii 285 4.31 232-350 y = 1.78+1.31x mancozeb 32 β-pinene <180 tab. 3: antifungal activity of leaf essential oil against eight phytopathogenic fungi. ed50 χ2exp fiducial limit regression equation [µg ml−1] (3df, 95%) rhizoctonia solani 165 2.20 128-213 y = 2.50+1.13x sclerotinia sclerotiorum 175 4.32 135-225 y = 2.59+1.08x fusarium solani 1776 0.86 642-4915 y = 3.14+0.57x alternaria solani 751 4.74 393-1436 y = 3.29+0.60x pyricularia grisea 708 5.33 360-1393 y = 3.10+0.68x fusarium oxysporum pisi 3716 1.23 890-15520 y = 3.04+0.55x fusarium oxysporum lentis 6975 0.46 843-57700 y = 3.24+0.46x sclerotium rolfsii 110 2.43 84-144 y = 2.49+1.22x mancozeb 32 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antifungal properties of the leaf oils of tagetes minuta l. and t. fi lifolia lag. j. essent. oil res. 6, 617-621. address of the corresponding author: e-mail: s_supradip@yahoo.com journal of applied botany and food quality 86, 24 32 (2013), doi:10.5073/jabfq.2013.086.004 school of life science, shanxi university, taiyuan, people’s republic of china antimicrobial activities of nitellopsis obtusa (desvaux) groves and chara vulgaris l. j. cai, s. xie1*, j. feng (received february 26, 2013) * corresponding author summary the extracts of nitellopsis obtusa (desvaux) groves and chara vulgaris l. were investigated for antimicrobial activities against the following common microorganisms using an agar diffusion method: staphylococcus aureus, bacillus subtilis, escherichia coli, proteus vulgaris, saccharomyces cerevisiae, and candida albicans. the extracts strongly inhibited the growth of gram-positive bacteria, but did not show inhibitory activity against gram-negative bacteria and yeast. in this study, novel methods for extraction of antimicrobial substances from n. obtusa and c. vulgaris were reported. the optimum extraction conditions were investigated using single factor experimental design and l273(13) orthogonal experimental design. results showed that the optimum extraction conditions for n. obtusa are: solid to liquid ratio 1:20, temperature 85°c, ethanol concentration 50%, extraction time 6h; and the optimum extraction conditions for c. vulgaris are: solid to liquid ratio 1:15, temperature 85°c, ethanol concentration 70%, extraction time 10h. stability study demonstrated neuter and alkaline conditions enhanced the antimicrobial activities from n. obtusa and c. vulgaris, and both extracts were stable under ultraviolet (uv). these data suggest that extracts from both n. obtusa and c. vulgaris might be of potential use as bactericidal agents. introduction for many years, antibiotics have been used to treat bacterial diseases. however, microorganism resistance to antibiotics has increased in parallel (bronzwaer et al., 2002). several studies have been conducted using synthetic antimicrobials, which often cause undesirable side effects (levy and marshall, 2004). in order to prolong the storage stability of foods, synthetic antimicrobials are widely used in industrial processing. however, according to toxicologists and nutritionists, there are side effects associated with the use of some synthetic antimicrobials in food processing (kajiwara et al., 2006; nguyen et al., 2008). plant diseases caused by microorganisms are the major problem in world agriculture because they cause tremendous losses in crop yield (fletcher et al., 2006). synthetic antimicrobials are also widely used in the control of plant diseases. however, the use of synthetic antimicrobials cause hazards to human health and may directly increase environmental pollution (fletcher et al., 2006). because of these associated problems, the need for new antimicrobial agents has lead to the search of new sources of potential antimicrobials (carson and riley, 2003). in the last several decades, various plant extracts have been the focus of great interest from researchers all over the world because they represent natural resources (sivakumar et al., 2008; cai et al., 2009; dan et al., 2010; hashem et al., 2010). plants produce a wide variety of physiologically active substances. these secondary metabolites have various functions, including antimicrobial activity (yazaki et al., 2008). charophytes are one of the most structurally complex green algae with a worldwide distribution (zaneveld, 1940; wood and imahori, 1965). china has a wealth of charophyte resources in both fresh-waters and estuarine systems (han and li, 1994; ling et al., 2000). they are commonly used as fertilizers of farmland and diets of aquatic animals (zaneveld, 1940). in recent years, research of charophytes has focused on their physiological activities, ecological characters, and taxonomy (lan et al., 2003; kotta et al., 2004; vouilloud et al., 2005). however, there are fewer reports in the area of antimicrobial activity, and only chara zeylanica, chara conirari, and nitella hyaline were reported to have antimicrobial activities (ghazala and shameel, 2005; khalid et al., 2010). therefore, the objectives of the present study are (1) to perform assessment of the antimicrobial activities in eight solvent extracts from nitellopsis obtusa (desvaux) groves and chara vulgaris l., for the first time, against six microorganisms; (2) to test and optimize extraction that can give maximal antibacterial activities; (3) to evaluate the stability of the extracts under different ph and uv. materials and methods plant materials and microorganisms nitellopsis obtusa (desvaux) groves and chara vulgaris l. were collected from jinci park, taiyuan, shanxi province, china, in july 2010. taxonomic identification was performed by prof. xie shulian, shanxi university. microorganisms: staphylococcus aureus (atcc 25923), bacillus subtilis (atcc 6633), escherichia coli (atcc 25922), proteus vulgaris (atcc13315), saccharomyces cerevisiae (atcc 2601), and candida albicans (atcc 10231). all microorganisms were from the american type culture collection (atcc). preparation of plant extracts extraction was performed as previously described (khalid et al., 2010) using eight solvents with different polarities. n. obtusa and c. vulgaris were cut into small pieces (2-4cm) respectively. each were washed several times with running tap water, then with sterile water and dried at 40°c (abdel-monaim et al., 2011). dry materials were grounded to fine powders in a grinder, then 10g of each powders blended in 50ml of 80% methanol, 80% ethanol, 80% acetone, 80% chloroform, 80% ethyl acetate, 80% butanol, 80% benzene, 80% petroleum ether solutions at room for 48h (zhang et al., 2008). after filtration, the extracts were concentrated to 1 mg/ml by using rotary evaporation. the 1 mg/ml of extracts were evaluated for their antimicrobial activities as described below. antimicrobial assays determination of antimicrobial activity was accomplished by agar diffusion method (adm; michielin et al., 2009). the agar surface was perforated with 6 mm diameter holes, aseptically cut and filled with 50μl of extracts. 80% methanol, 80% ethanol, 80% acetone, 80% chloroform, 80% ethyl acetate, 80% butanol, 80% benzene and 80% petroleum ether solutions were used as negative controls. after diffusion of the solution in each hole, the plates were inverted and incubated at 37°c for 24 hours for bacteria and 28°c for 48 hours for yeast. antimicrobial activities were determined by measuring the radius of the zone of inhibition around the hole. each treatment was replicated nine times. antimicrobial activity of two charophytes 25 single factor experiment the four factors including solid to liquid ratio (g:ml), extraction temperature (°c), ethanol concentration (%), extraction time (h) could affect extraction efficiency. single factor experiments were applied to decide appropriate levels. for each single factor, five different levels were designed, with other factors being kept constant. for each experiment, a total of 12g sample was added to corresponding volume of ethanol and extracted as described in tab. 1. four replicates were used for each extraction. after filtration, the extracts were concentrated to 0.5 mg/ml by using rotary evaporation. s. aureus was used as a test organism by inoculating it into separate mixtures at a concentration of 107 cfu/ml. then, antimicrobial activity of extracts from each sample was analyzed by adm to choose three reasonable levels of four factors for orthogonal experimental design. each treatment was replicated nine times. orthogonal experimental design on the basis of single factor experiments, three levels of four factors were selected as described in tab. 2. then orthogonal array l27(313) matrix was used to determine the optimum extraction conditions of antimicrobial substances from n. obtusa and c. vulgaris, with the consideration of the interactions between the parameters (jia et al., 2010; li et al., 2010; zhou et al., 2010). for each experiment, a total of 12g sample was added to corresponding volume of ethanol and extracted as described in (tab. 3). four replicates were used for each extraction. the extracts were concentrated to 0.5 mg/ml by using rotary evaporation. s. aureus was used as a test organism by inoculating it into separate mixtures at a concentration of 107 cfu/ ml. then, antimicrobial activity of extracts from each sample was analyzed by adm. each treatment was replicated nine times. stability assays the effect of ph on antimicrobial activity in extracts was examined by ph stability assays (cheikhyoussef et al., 2009; wu et al., 2008). tests were conducted in two sets: test sets of extracts from n. obtusa and c. vulgaris were adjusted with 5m naoh or 5m hcl to different ph values ranging from 5 to 9. control sets were prepared using the same method with 80% ethanol except that no extracts was added. to test the impact of uv, each extract was incubated under uv (256rim, 6w, 5cm) for a period ranging from 1h to 5h (jia et al., 2010; chen and dai, 2012). then, antimicrobial activity was analyzed by adm. each treatment was replicated nine times. statistical analysis each data was presented as mean ± standard error (n = 9). anova (one-way analysis of variance) and duncan’s multiple range test were carried out to determine significant (p < 0.05) differences between the means. the analyses were carried out using spss package software (version 17.0). tab. 1: single factor experimental design factors conditions levels 1 2 3 4 5 solid to liquid ratio (g:ml) temperature 50°c 1:5 1:10 1:15 1:20 1:25 ethanol concentration 80% extraction time 10h temperature (°c) solid to liquid ratio 1:5 40 55 70 85 97 ethanol concentration 80% extraction time 10h ethanol concentration (%) solid to liquid ratio 1:5 40 50 60 70 80 temperature 50°c extraction time 10h extraction time (h) solid to liquid ratio 1:5 4 6 8 10 12 temperature 50°c ethanol concentration 80% tab. 2: factors and levels of orthogonal experiment of n. obtusa extraction factors levels solid to liquid ratio temperature ethanol concentration extraction time (a) (b) (c) (d) 1 1:5 40°c 40% 4h 2 1:10 70°c 50% 6h 3 1:20 85°c 60% 8h 26 j. cai, s. xie, j. feng tab. 3: factors and levels of orthogonal experiments for c. vulgaris extraction factors levels solid to liquid ratio temperature ethanol concentration extraction time (a) (b) (c) (d) 1 1:5 70°c 40% 4h 2 1:15 85°c 50% 6h 3 1:20 97°c 70% 10h results antimicrobial activity initial antimicrobial screening tests using extracts obtained by eight solvents from n. obtusa and c. vulgaris indicated that there was almost no antimicrobial activity against gram-negative bacteria (e. coli and p. vulgaris) and yeast (s. cerevisiae and c. albicans). extracts obtained by methanol, ethanol, and acetone had greater activity against gram-positive bacteria (s. aureus and b. subtilis) than other solvents, with an inhibition zone ranging from 7 mm and 15 mm. the ethanol extracts had the highest inhibition zone values and were significantly (p < 0.05) different from other solvents (fig. 1). single factor experiments as can be seen from fig. 2 (a), antimicrobial activities of n. obtusa and c. vulgaris increased with an increasing solid to liquid ratio. maximum extraction yields of antimicrobial substances were achieved at 1:10 ratio for n. obtusa and 1:20 ratio for c. vulgaris, then antimicrobial activities decreased with increasing ratio. for n. obtuse extracts, anova shows that the best solid to liquid ratios with significant (p<0.05) difference (selected one level which was more resource saving, when there was no significant difference between two or more levels), were level 1 (1:5), level 2 (1:10), level 4 (1:20), and selected them for orthogonal experimental design in tab. 2. for c. vulgaris extracts, best solid to liquid ratios with significant (p<0.05) difference (selected one level which was more resource saving, when there was no significant difference between two or more levels) were level 1 (1:5), level 3 (1:15), level 4 (1:20), and selected them for orthogonal experimental design in tab. 3. increase in temperature led to greater antimicrobial activities in extracts (fig. 2 (b)), and the highest antimicrobial activities with significant (p<0.05) difference were observed at 85°c. however, increasing temperature did not improve the antibacterial activity at 97°c. for n. obtuse extracts, anova shows that the best extraction temperatures with significant (p<0.05) difference (selected one level which was more resource saving, when there was no significant difference between two or more levels), were level 1 (40°c), level 3 (70°c), level 4 (85°c), and selected them for orthogonal experimental design in tab. 2. for c. vulgaris extracts, best extraction temperatures with significant (p<0.05) difference (selected one level which was more resource saving, when there was no significant difference between two or more levels), were level 3 (70°c), level 4 (85°c), level 5 (97°c), and selected them for orthogonal experimental design in tab. 3. fig. 2 (c) showed the effect of ethanol concentration on the antimicrobial activities in extracts from n. obtusa and c. vulgaris. antimicrobial activities of n. obtusa and c. vulgaris increased significantly (p<0.05) with increasing concentration until the equilibriums (60% for n. obtuse and 70% for c. vulgaris) were reached. for n. obtuse extracts, anova shows that the best extraction concentrations with significant (p<0.05) difference (selected one level which was more resource saving, when there was no significant difference between two or more levels), were level 1 (40%), level 2 (50%), level 3 (60%), and selected them for orthogonal experimental design in tab. 2. for c. vulgaris extracts, best extraction concentrations with significant (p<0.05) difference were level 1 (40%), level 2 (50%), level 4 (70%), and selected them for orthogonal experimental design in tab. 3. fig. 2 (d) depicted the effect of different extraction time on the antimicrobial activities in extracts from n. obtusa and c. vulgaris. for n. obtuse, the antimicrobial activity increased significantly (p<0.05) with extraction time extended, and equilibrium reached at 8h. anova shows that the best extraction times with significant (p<0.05) difference (selected one level which was more resource saving, when there was no significant difference between two or more levels), were level 1 (4h), level 2 (6h), level 3 (8h), and selected them for orthogonal experimental design in tab. 2. for c. vulgaris, the highest inhibition zone value was observed at 10h, thereafter, inhibitory activity decreased gradually. anova shows that the best extraction times with significant (p<0.05) difference (selected one level which was more resource saving, when there was no significant difference between two or more levels), were level 1 (4h), level 2 (6h), level 4 (10h), and selected them for orthogonal experimental design in tab. 3. optimization of extraction condition orthogonal experimental design, the main method of fractional factorial design, can effectively screen out key variables by several representative experiments (antony, 2006; kilickap, 2010). from experimental results, it was inferred that antimicrobial activities of n. obtusa and c. vulgaris were influenced by both different factors at different levels and their interactions. the term l27(313) of an orthogonal array implies 27 groups of experiments (tab. 4). this array handles up to four factors at three levels each. the subscripts 1, 2, and 3 represent the value of a designed factor at levels 1, 2, and 3 respectively. in other words, these subscripts designate each special trial run of the experiment. for example, in the first row of tab. 4 (following the indicated subscripts), the factor level of factor a (which is assigned to the first column of the array) is 1, and the level of factors b, c and d are 1 as well. the first trial run of this experiment will be designed as a level set {1, 1, 1,1} for factors a, b, c and d according to tab. 2 and tab. 3. therefore, the first experiment, for n. obtuse extracts, was carried under solid to liquid ratio 1:5 (g:ml), temperature 40°c, extraction concentration 40%, extraction time 4h conditions. for c. vulgaris extracts, the first experiment was carried under solid to liquid ratio 1:5 (g:ml), temperature 70°c, extraction concentration 40%, extraction time 4h conditions. the other experiments will perform in the same way, and the experimental results of the orthogonal design were shown in tab. 4. factors that influence antimicrobial activity of n. obtusa were listed in a decreasing order as follow: b > a > c > d. the individual levels within each factor were ranked as in fig. 3: a: 3 > 2 > 1; b: 3 > 2 > 1; c: 2 > 3 > 1; d: 2 > 3> 1. factors that influence antimicrobial activity of c.vulgaris were listed in a decreasing order as follow: antimicrobial activity of two charophytes 27 fig. 1: antimicrobial activities in extracts from n. obtuse (a) and c. vulgaris (b) against s.aureus and b.subtilis. a negative result was defined as an inhibition zone of 6mm. greater than 6mm indicated positive result of the presence of antibacterial substance. inhibition zones of controls were all 6mm. different letters indicated significant differences (p<0.05; one-way analysis of variance (anova) and duncan’s multiple range test). bars represent the means ± standard deviation. each was replicated nine times. c > a > d > b. the individual levels within each factor were ranked as in fig. 3: a: 2 > 3 > 1; b: 2 > 1 > 3; c: 3 > 2 > 1; d: 3 > 2 > 1. because interactions between factors are complex, only low-order interactions were analyzed while high-order (three-, four-, and fiveorder) interactions were neglected. tab. 5 and tab. 6 summarize the analysis of variance (anova) of factors and their second-order interactions that affect antimicrobial activity. the term ‘‘interaction’’, indicated by inserting the ‘‘×’’ symbol between the two interacting factors, is used to describe the condition in which the effect of one factor’s influence upon the result is dependent on the condition of the other factor. in tab. 5 and tab. 6, f-ratio is defined as f = msf/mse, where msf and mse represent respectively mean square of factors or interactions, mean square of errors. df, ss and ms respectively represent degree of freedom, sum of squares and mean square. if the calculated value f is greater than critical value fα [e.g. f0.05(2,6) = 5.14], then that factor or interaction is statistically significant. in tab. 5, if significant level α = 0.01, then a (solid to liquid ratio) and b (temperature) were statistically significant factors that affect antimicrobial activity of n. obtusa. when significant level α = 0.05, interaction a × b was statistically significant. therefore, factors a, b and interaction a × b were regarded as dependent factors and interaction in extraction of antimicrobial substances. c (ethanol concentration), d (extraction time) and interactions a × c, b × c were regarded as independent factors and interactions. optimum values of these factors for extraction of antimicrobial substances from n. obtusa were a3b3c2d2, solid to liquid ratio 1:20, temperature 85°c, ethanol concentration 50%, extraction time 6h (tab. 4). in tab. 6, if significant level α = 0.05, then c (ethanol concentration) was the statistically significant factor that affect antimicrobial activity of c. vulgaris. therefore, factor c was regarded as dependent factor in antimicrobial activity. a (solid to liquid ratio), b (temperature), d (extraction time) and interactions a × b, a × c, b × c were regarded as independent factors and interactions. optimum values of these factors for extraction of antimicrobial substances from c. vulgaris were a2b2c3d3, solid to liquid ratio 1:15, temperature 85°c, ethanol concentration 70%, extraction time 10h (tab. 4). 28 j. cai, s. xie, j. feng effect of ph and uv on the antimicrobial activity effect of ph was tested over a wide range (ph 5 to ph 9). as shown in fig. 4 (a), maximum efficiency of antibacterial activity from n. obtuse extracts was observed when ph was 8 and 9, whereas the extract from c. vulgaris exhibited ph stability in the range from 6 to 9. data from ph stability suggest that antimicrobial agents in extracts from n. obtusa and c. vulgaris are different. to test the uv stability of extracts, we investigated the antimicrobial activities of different treatments. there are no statistically significant (p<0.05) differences between samples that were exposed to uv light for different times (fig. 4 (b)), implying that these extracts are not impacted by exposure to uv. discussion in the present study, we evaluated the antimicrobial activities in extracts from n. obtusa and c. vulgaris against 6 microorganisms. like most of antimicrobial activity results from algae (reichelt and borowitzka, 1984), extracts from n. obtusa and c. vulgaris were highly effective in inhibiting the growth of s. aureus and b. subtilis (gram-positive bacteria). however, they showed no antimicrobial activities against e. coli, p. vulgaris (gram-negative bacteria), and s. cerevisiae, c. albicans (yeast). these results might be attributed to the differences in the outer membrane structure and permeability between gram positive bacteria, gram negative bacteria and yeast. bacterial membrane has peptidoglycan layer, while yeast membrane consists of dextran and mannan. gram positive and gram negative bacteria are reported to have further differences in cell wall composition (ventosa et al., 1998). gram positive cells contained more teichoic acid in cell walls that might be more sensitive to the extracts from n. obtusea and c. vulgaris. the extracts might inhibit synthesis of teichoic acid, and thus the biosynthesis of cell walls thereby. to our knowledge, s. aureus and b. subtilis are resistant to various antibiotics (adedapo et al., 2008). our results indicate that there is a possible alternative therapy for these antibiotics resistant strains. this therapy is very important because these types of microorganisms are difficult to treat and often require alternative therapy (vila et al., 2010). solvent played a key role in extraction of antimicrobial substances from n. obtusa and c. vulgaris. several extraction methods have been reported using solvents with different polarities, such as methanol, ethanol, chloroform, ethyl acetate, acetone, and petroleum ether (cheung et al., 2003). in this study, the ethanol extracts were found to be highly effective against microorganisms (fig. 1). ethanol has a high dielectric constant and cohesive energy, as compared with other solvents, which provides strong bounding between solvent molecules and polar compounds from the solutes, causing their dissolution (xie and lu, 2004). moreover, the results revealed that ethanol caused damages to cell walls and the cell membranes of n. obtusa and c. vulgaris, and thus enabled the solvent penetrate more effectively into cellular tissue and antimicrobial substances to be released rapidly. in addition, ethanol has several advantages such as low toxicity, economical, and lower boiling point (xie and lu, 2004). thus, ethanol was considered and also demonstrated as an ideal extraction solvent in our studies. there are many factors affecting the extraction. among them, the solid to liquid ratio, extraction temperature, the concentration and extraction time are key factors (xiong et al., 2007). single factor experiment was performed by one factor varied with different levels while other factors being fixed. when the solid to liquid ratio was too low, the contact between antimicrobial substances and solvents was not sufficient enough, and it was not conducive to extract maximal amount of antimicrobial substances. when the solid to liquid ratio was too high, concentration time would be long and antimicrobial components might be decomposed (fig. 2 (a); yang et al., 2010). increasing temperature enhanced diffusivity and thus the yields of antimicrobial activities in extracts were increased with higher temperature (liu et al., 2002). when temperature was too high, ethanol fig. 2: effect of solid to liquid ratio (a), extraction temperature (b), ethanol concentration (c), and extraction time (d) on antimicrobial activities in extracts from n. obtusa and c. vulgaris against s.aureus. different letters indicated significant differences (p<0.05; one-way analysis of variance (anova) and duncan’s multiple range test). bars represent the means ± standard deviation. each was replicated nine times. antimicrobial activity of two charophytes 29 tab. 4: result analysis of orthogonal experiments l27 (313) factors inhibition zone (mm) experiment solid extraction extraction extraction n. obtusa a c. vulgaris a no. to liquid ratio temperature concentration time (a) (b) (c) (d) 1 1 1 1 1 8.4 7.75 2 1 1 2 2 9.09 7.9 3 1 1 3 3 9.40 12.33 4 1 2 1 2 10.77 7.38 5 1 2 2 3 9.43 10.89 6 1 2 3 1 9.58 10.28 7 1 3 1 3 11.19 10.04 8 1 3 2 1 11.94 7.67 9 1 3 3 2 12.16 12.3 10 2 1 1 2 9.65 11.08 11 2 1 2 3 10.02 9.87 12 2 1 3 1 9.95 11.03 13 2 2 1 3 11.54 10.37 14 2 2 2 1 11.04 11.25 15 2 2 3 2 11.79 13.23 16 2 3 1 1 13.95 8.75 17 2 3 2 2 14.27 10.44 18 2 3 3 3 14.64 11.7 19 3 1 1 3 9.06 9.22 20 3 1 2 1 11.01 11.58 21 3 1 3 2 10.77 8.77 22 3 2 1 1 12.04 10.46 23 3 2 2 2 13.02 10.4 24 3 2 3 3 12.98 8.97 25 3 3 1 2 14.77 8.91 26 3 3 2 3 16.05 9.17 27 3 3 3 1 14.35 10.26 k1jb 10.22 9.71 11.26 11.36 k2j 11.87 11.35 11.76 11.81 k3j 12.67 13.70 11.74 11.59 k1jc 9.62 9.95 9.33 9.89 k2j 10.86 10.36 9.91 10.05 k3j 9.75 9.92 10.99 10.28 rkd 2.45 3.99 0.5 0.45 rke 1.24 0.41 1.66 0.52 okf a3 b3 c2 d2 okg a2 b2 c3 d3 a80% ethanol was used as control. a negative result was defined as an inhibition zone of 6mm. greater than 6mm indicated positive result of the presence of antibacterial substance. each value was means of nine determinations. b kij = (1 / 9) ∑ mean inhibition zone of n. obtusa at factor j (j = a, b, c, d). c kij = (1 / 9) ∑ mean inhibition zone of c. vulgaris at factor j (j = a, b, c, d). d rij = max {kij} − min {kij}, j and i mean factor and setting level here, respectively. e rij = max {kij} − min {kij}, j and i mean factor and setting level here, respectively. f o means the optimum combination of conditions for n. obtusa, is a3b3c2d2. g o means the optimum combination of conditions for c. vulgaris, is a2b2c3d3. 30 j. cai, s. xie, j. feng volatilization was accelerated and the solid to liquid ratio was lowed, and thus the yields of antimicrobial activities were decreased (fig. 2 (b)). shorter extraction times and lower ethanol concentration would result in incomplete extraction, longer extraction times and higher concentration would lead to waste of time and energy (fig. 2 (c and d); bernardo-gil et al., 2009). the orthogonal experimental design was used to study optimization of parameters for efficient extraction of antimicrobial substances from n. obtusa and c. vulgaris. the advantage of orthogonal experimental design is that it is economical for characterizing a complicated process in fewer experiments. however, it requires a specialized experimental design to properly set up the test and specialized statistics to analyze data (díez et al., 2008). the results (tab. 4, 5 and 6) revealed that factor a (solid to liquid ratio), factor b (temperature), and interaction a × b had significant effects on the antimicrobial activity of n. obtusa. for c. vulgaris, ethanol concentration had significant effect on the antimicrobial activity, while the other factors and interactions were identified as insignificant factors and interactions under the selected conditions based on anova. we concluded that solid to liquid ratio and temperature were the two major factors affecting extraction of n. obtusa, and ethanol concentration was the major factor affecting extraction of c. vulgaris. thus, we should pay more attention to these factors in extraction. in summary, the optimum extraction condition for n. obtuse was defined as below: solid to liquid ratio: 1:20, temperature: 85°c, ethanol concentration: 50%, extraction time: 6h, and the best combination of extraction parameters for c. vulgaris was: solid to liquid ratio: 1:15, temperature: 85°c, ethanol concentration: 70%, extraction time: 10h. compared with conventional extraction conditab. 5: results of variance (anova) analysis for n. obtuse source ss df ms f a significance b a 28.2044 2 14.1022 69.54 ** b 72.6127 2 36.3064 179.03 ** c 1.4213 2 0.7107 3.50 d 0.9023 2 0.4512 2.22 a×b 3.747 4 0.9368 4.62 * a×c 1.9536 4 0.4884 2.41 b×c 1.6777 4 0.4194 2.07 error 1.2167 6 0.2028 total 111.7357 26 a significant parameter, f0.05(2, 6) = 5.14, f0.01(2, 6) = 10.92, f0.05(4, 6) = 4.534, f0.01(4, 6) = 9.148. b *, ** and blank indicate significant different, more significant different and no significant different, respectively. tab. 6: results of variance (anova) analysis for c. vulgaris source ss df ms f a significance b a 8.3716 2 4.1858 2.9805 c 12.7238 2 6.3619 4.53 * a×c 13.848 4 3.462 2.4651 error 25.2785 18 1.4044 total 60.2219 26 a significant parameter, f0.05(2,18) = 3.55, f0.01(2,18) = 6.01, f0.05(4,18) = 2.93, f0.01(4,18) = 4.58. b *, ** and blank indicate significant different, more significant different and no significant different, respectively. fig. 3: effect of each parameter on antimicrobial activities of n. obtusa and c. vulgaris. parameters of n. obtusa: a, solid to liquid ratio (g:ml): a1: 1:5, a2: 1:10, a3: 1:20; b, temperature: b1: 40°c, b2: 70°c, b3: 85°c; c, ethanol concentration: c1: 40%, c2: 50%, c3: 60%; d, extraction time: d1: 4h, d2: 6h, d3: 8h. parameters of c. vulgaris: a, solid to liquid ratio (g:ml): a1: 1:5, a2: 1:15, a3: 1:20; b, temperature: b1: 70°c, b2: 85°c, b3: 97°c; c, ethanol concentration: c1: 40%, c2: 50%, c3: 70%; d, extraction time: d1: 4h, d2: 6h, d3: 10h. fig. 4: effect of ph (a) and uv (b) on antimicrobial activities in extracts from n. obtusa and c. vulgaris against s.aureus. for ph effect, the control sets were prepared using 80% ethanol that no extract was added. different letters indicated significant differences (p<0.05; one-way analysis of variance (anova) and duncan’s multiple range test). bars represent the means ± standard deviation. each was replicated nine times. antimicrobial activity of two charophytes 31 tions, our optimum extraction conditions in this study are economic, convenient and efficient (li et al., 2010). further, this extraction method meets the actual needs and is also compliant with environmental regulations. environmental factors often influence the efficacy of bactericides (qasem and abu-blam, 1995). in this study, we tested whether ph and uv could influence the antimicrobial activity. the ph of extraction is a significant factor, which may affect the extraction procedure. maximum efficiency of antibacterial activity from n. obtuse extracts was observed when ph was 8 and 9, whereas the extract from c. vulgaris exhibited ph stability in the range from 6 to 9 (fig. 4 (a)). it was observed that greater antimicrobial activities were obtained under neuter and alkaline conditions. as exposure time changed, no statistically significant (p<0.05) differences were observed between different uv treatments. the result (fig. 4 (b)) showed that extracts were stable following exposure to uv light. these results indicate that n. obtusa and c. vulgaris have a great potential for the extraction of antibacterial substances. the antibacterial activities against gram-positive bacteria should be applied to plants in the field, food process, and medicine. acknowledgments we thank dr. j. a. jiao 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pseudoacacia linn1 extracts against cucumber powdery mildew fungus, sphaerotheca fuliginea. crop prot. 27, 920-925. zhou, w., zhang, x.y., xie, m.f., chen, y.l., li, y., 2010: infraredassisted extraction of adenosine from radix isatidis using orthogonal experimental design and lc.chromatographia 72, 719-724. address of the corresponding author: shulian xie, school of life science, shanxi university, taiyuan 030006, china. e-mail: xiesl@sxu.edu.cn vorgabe neu journal of applied botany and food quality 80, 145 149 (2006) 1 department of biology and biochemistry, birzeit university, birzeit, palestine 2 federal center of technological education, sao vicente do sul, brazil 3 kompetenzzentrum für obstbau, ravensburg, germany quality and biochemical changes of sweet cherries cv. regina stored in modified atmosphere packaging j. harb1, a.a. saquet2, r. bisharat1, j. streif 3 (received june 26, 2006) summary biochemical and quality changes of sweet cherries cv. regina were assessed over three consecutive years after storage in different modified atmosphere packaging (map) liners, with or without hydrocooling, compared to regular atmosphere (ra) storage. all plastic liners used in the experiment resulted in co 2 -enrichment and o 2 -reduction inside packages, with the following impact on fruit quality after five weeks of storage: improved retention of fruit firmness and red color of skin, no significant effect on acidity and total soluble solids, and minimal loss of fruit weight. fruit decay was absent under both storage conditions (ra and map), probably due to rain-protected cultivation of cherry trees. stalks of map-fruits remained fresher than control fruits, obviously due to higher relative humidity condition inside packages. ma-packaged cherries were preferred by the taste panel, while cold-stored fruits were criticized due to flat and slightly bitter taste. the atp concentration in air stored fruits was higher than in ma-packaged fruits, while adp level was higher in ma-packaged fruits. further, ma-packed sweet cherries exhibited higher antioxidant potential and ascorbic acid content than air-stored fruits. moreover, hydrocooling did not cause any significant effect compared to nontreated fruits. introduction map refers to the technique of sealing actively respiring produce like fruits in polymeric film packages to modify o 2 and co 2 partial pressures within the packages (kader and watkins, 2000). in their study on table grapes, artés-hernández et al. (2004) used 35 mm thick microperforated polypropylene (pp) to generate a modified atmosphere packaging of 15 kpa o 2 and 10 kpa co 2 . the changes in o 2 and co 2 concentrations within a package depend on the interaction between respiration of the commodity and the permeability properties of the packaging film and/or microperforations (beaudry et al., 1992). consequently, researchers stated that it is crucial to use films with suitable gas permeability, and to determine the time needed for the development of the concentrations of the respiratory gases inside the package, to achieve any success with map (evelo, 1993). it was found that map can double the shelflife of tomatoes, citrus, cucumber and apples compared to the nonpackaged fruits, when the permeability characteristics of the package matched the respiration rate of the produce (cameron et al., 1989; ait-oubahou et al., 1994). usually it is desirable to generate an atmosphere low in o 2 and/or high in co 2 to influence the metabolism of the produce being packaged, and/or to retard the activity of decaycausing organisms, which resulted in most cases in better storability of fruits and an extended shelf life. skog et al., (2003) reported that map reduced decay by 50% and significantly maintained firmness with ‘hedelfinger’ cherries. in addition to atmosphere modification, map improves moisture retention, which had greater influence on preserving stalks freshness than o 2 and co 2 levels (jobling, 2001). however, the atmosphere modification inside packages may induce various undesirable effects. fermentation and off-flavors may develop if decreased o 2 levels cannot sustain aerobic respiration (kays, 1997). similarly, injury will occur if co 2 exceeds tolerable levels. production of compounds that contribute to characteristic taste of many fruit, including apple, banana, pear, peaches, strawberries and others, can be adversely affected by low o 2 and elevated co 2 (mattheis and fellman, 2000). synthesis of aroma compounds are generally suppressed by high co 2 and low o 2 concentrations (harb et al., 2000), in part by their action on ethylene perception, but also via oxidative processes, including respiration, required for substrates production (saquet et al., 2003). further findings by crisosto et al. (2002) indicate that rachis browning was accelerated and a trained panel perceived ‘off-flavor’ in grapes (cv. ‘redglobe’) exposed to > 10 kpa co 2 partial pressure in the packages. the aim of this work was to study the influence of plastic liners on the fruit quality and storability of sweet cherries cv. ‘regina’ cultivated under rain covers. rain-protection of sweet cherries during fruit ripening, in particular during harvest period, is essential in various regions in western-europe to avoid fruit cracking. materials and methods over three consecutive years various plastic liners in several maptrials were used. in all experiments, cherry fruits were obtained from a rain-protected orchard at the kompetenzzentrum obstbau-bodensee (kob) located in south-west germany, in lake of constance area. fruits were picked and selected for uniformity in size and color and absence of decay and external injuries, and packed at the same day. experiments in 2001: fruits were divided into 16 samples, each sample amounts about 2000 g. eight samples were enclosed in pvc plastic bags that were kept open (control treatment). a second set of eight samples were enclosed in 30 µm life-plus® gusseted polyethylene bulk liners supplied from danisco flexible, bristol-uk (ma-packed treatment). analyses of quality parameters were conducted as described for experiment 2003. experiments in 2002: repetition of the 2001 experiments. experiments in 2003: the following treatments were conducted: control treatment: fruits were stored in cold storage without precooling. the cherries were enclosed in macro-perforated ldpe-liners (50 µm) for the purpose of increasing the relative humidity without altering gas composition around fruits. map experiment: fruits were enclosed in two different plastic films (sj 304 and sj 604) delivered from a chilenian manufacturer (san jorge, santiago, chile) and designed specifically for sweet cherries. furthermore, the 30 µm life-plus® gusseted polyethylene bulk liner supplied from danisco flexible was used also. hydrocooling experiment: fruits were cooled in ice-water for 10 minutes before enclosure in various liners (304, lifespan, or ldpe). the aim of hydrocooling trial was to investigate the impact of hydrocooling on various quality aspects, in particular on the freshness of stem condition. all samples were stored at 0 °c for the entire storage period. from each treatment two samples were taken every two weeks, and gas composition inside packages was determined using a micro-gc (model cp 2002p, chrompack,); detector: thermal conductivity detector (tdc), column for o 2 -determination: molsieb at 40 °c, column for co 2 -determination: hayesep at 45 °c. fruits were analyzed for the following quality parameters: weight loss: upon storage the initial weights of all samples were recorded, and at each sampling date, the weights of two samples for each treatment were recorded directly after removal from room, to avoid any condensation of water on the fruits. fruit firmness: firmness was measured using a nondestructive instrument (firmtech2; up gmbh, ibbenbüren, germany) designed for soft fruits, which measured the maximum force needed to compress the fruit tissues. results are given in g mm-1. total soluble solids (tss): the tss in the fruit juice was determined with a digital refractometer (atago, japan). titratable acidity: 10 ml of fruit juice were diluted with 100 ml distilled water and titrated with 0.1 n naoh solution to ph 8.1. skin color: fruit color was assessed using a chromameter (minolta, japan). ten fruits per replicate were measured. the color assessment with this instrument is specified by the coordinates l*, a*, b* in a three-dimensional color space. l* represent the brightness, whereas a* is the green (with negative values) and the red component (with positive values), and b* is the blue (with negative values) and the yellow component (with positive values). ascorbic acid (aa) concentration: all steps of this determination procedure were carried out under cold and dark condition to minimize the degradation of vitamin c. at each sampling date fruit sections were obtained from at least 15 fruits per replicate, two replicate for each treatment, shock frozen in liquid n 2 , and ground to fine powder. six grams of fruit powder was added to 15 ml of 3 % hpo 3 solution and homogenized for 30 seconds. after centrifugation at 14 000 g for 20 minutes, the supernatant was filtered and 20 µl were used for hplc determination under the following conditions: column: prontosil 60-5-c18-h; size: 4.0 x 125 mm; particle size: 5.0 µl; eluent: tetra-n-butylammoniumhydrogensulfate (2.5 g) + methanol (55 ml) in 1 l h 2 o; flow: 800 µl·min-1; temperature: 25.0 °c; pressure: 16.0 mpa. an ascorbic acid standard solution (20 mg 100 ml-1) was used to calculate the vitamin c concentration of the fruits. determination of antioxidants capacity of the water soluble compounds (acw): determinations were carried out by photochem system (analytik jena ag) with photo-chemo-luminescence method. standard kits from analytik jena, germany, were used to measure water soluble antioxidants. acws were measured using the same extract used for vitamin c determination after dilution with water at a ratio of 1:10 according to the protocol provided with the acw determination kit (analytik jena, germany). atp/adp extraction and assay: the extraction and assay of atp and adp were carried out according to saquet et al. (2003). the concentrations of atp and adp were determined by bioluminescence technique using a kit from bio-orbit oy (finland). 1 g of the lyophilized fruit powder was homogenized in a 5%-tca solution plus edta (2 mm) and incubated in ice for 30 min. samples were then centrifuged at 18000 g at 4°c for 15 min. an aliquot of 10 µl of the supernatant was diluted with tris-edta-buffer (ph 7.75) and assayed with a luminometer (model 1250, lkb-wallac, finland) at 25 °c. for adp-determination, samples were incubated with pyruvate kinase at 25 °c for 30 min., during which adp was converted into atp. an internal atp-standard was used for calculating atp and adp concentrations. sensory test: a taste panel, with a minimum of four people, evaluated the sensory quality of the cherry fruits from different storage conditions after four and seven weeks. the panelists looked for both visual quality criteria, such as stalks freshness (green color and dryness) and fruit color, and taste criteria, such as sweetness, acidity, crispness, and off-flavour. all tests were performed after a shelf-life period of 24 hours at 20°c in air. the visual properties (appearance, color, stem condition, injuries) and organoleptical impression were judged by numerical scores between 1 and 5 as follows: for decay: 1 = no decay, and 5 = strong infection; for color: 1 = fresh as at harvest time, and 5 = very dark; for stalks condition: 1 = fresh as at harvest time and 5 = dry and brown color; for taste: 1 = very good, and 5 = very bad. furthermore, a detailed discussion was conducted at each session to describe the quality of sweet cherries. therefore, our results will be shown in both numerical as well as a descriptive manner. statistical analysis: all results were subjected to analysis of variance (anova) using the costat-software (cohort software, monterey, ca, 1998), and mean separations were calculated by duncan’s multiple range test at p < 0.05. results and discussion partial pressures of gases inside the ma-packages: fig. 1 shows changes in gas composition occurred over a storage period of five weeks after using various map-liners. the lifespan liner led to the strongest reduction in co 2 partial pressure, although it was not low enough to slow the ripening process according to wills et al. (1998). co 2 partial pressure increased up to 5 kpa in all liner treatments and this influenced some aspects of fruit ripening and quality as shown in tab. 1. none of the tested liners was clearly superior over the others and all liners affected fruit quality and freshness mainly by increasing the relative humidity around the fruit more than by changing the gas composition inside because stem freshness was clearly better than control. quality parameters: tab. 1 shows the influence of map-treatments and hydrocooling on various quality parameters after five weeks of storage in 2003. fruit firmness decreased significantly upon cold storage (control), but preserved upon storage of fruits in plastic liners, and hydrocooling did not contribute with most treatments to the preservation of firmness. changes in the total soluble solids, which reflect the amount of sugars in fruits, were minimal and did not differ significantly between control and all map treatments. concerning the titratable acidity, fruits in all conditions suffered great losses, which possibly reflect that the gas composition developed inside plastic liners was not effective in reducing the respiration rate of fruit, and no significant differences were registered between all treatments. however, significant differences were recorded in color attributes (a* and b* values). the a*-value, which gives the red 146 j. harb, a.a. saquet, r. bisharat, j. streif component of color decreased with both treatments. however, a significant lower decrease was registered with map-fruits, which indicates that increased co 2 -level inside the liner may have preserved the red pigments in map stored fruits more than in control fruits. concerning the b*-values, which reflect the blue/yellow components of color, a significant lower decrease was registered also in mapfruits compared to control fruits. the l*-values did not differ significantly between both treatments. hydrocooling of cherries for two minutes in ice water soon after harvest did not affect fruit quality, when fruits were analysed after five weeks storage period. our results are in agreement, although partially, with that of meheriuk et al. (1995), who succeeded in maintaining the quality of sweet cherries cv. ‘lapins’ through storing in ldpe-bags (30 µm) with an immediate flushing of bags with a gas mixture composed of 5% o 2 + 5% co 2 . arjona et al. (1994) also found that yellow passion fruit wrapped with vf-60 plastic film and stored for 15 and 30 days show less weight loss, while maintaining external appearance. moreover, kupferman and sanderson (2005) mentioned also good results, but concluding that controlling fruit temperature is more important than using map, particularly if sweet cherries are to be stored for less than 10 days. in another experiment, map using 80 µm ldpe film retarded fruit softening and inhibited development of peel and flesh disorders of japanese ‘fuyu’ persimmon (ben-arie and zutkhi 1992). however, fruit quality deteriorated more rapidly in a 60 µm package, which was attributed to specific physiological effects of the different atmospheric equilibria established due to film thickness. weight loss: results show that cold-stored fruits without packaging lost more weight than ma-packaged fruits (data not shown). this was attributed to the buildup of a high relative humidity inside mapackages. othieno and thompson (1993) reported that sweet corn packed in polypropylene film with no perforations showed a reduced rate of weight loss. arjona et al. (1994) found also that yellow passion fruit wrapped with vf-60 plastic film and stored for 15 and 30 days showed less weight loss. it is not expected that any differences in respiration rates between treatments could cause such a difference in weight loss. visual inspection and sensory test: panelists were not able to detect differences between control and map stored fruits, in respect to external fruit quality (appearance and color) (tab. 2). however, significant differences were detected in stalks condition and taste. it 0 5 10 15 20 25 0 1 2 3 4 5 weeks of storage c o 2 a n d o 2 p a rt ia l p re ss u re ( kp a ) sj 3 304 (co2) sj 6 604 (co2) lifespan (co2) sj 304 (o2) sj 6 604 (o2) lifespan (o2) a a b a a a fig. 1: changes in partial pressures of co 2 and o 2 inside various mapliners filled with 500 g sweet cherries cv. regina, and stored at 0 °c, over a storage period of five weeks in 2003. different letters indicate significant differences between treatments at p < 0.05, duncan’s multiple range test. tab. 1: influence of various map-treatments with or without hydrocooling (+hc) on various quality parameters of sweet cherries cv. regina. measurements were taken at harvest time and after storage period of five weeks at 0 °c in 2003. assessment control sj 604sj 304sj 304lifespanlifespanuntight untight time liner liner liner liner liner enclosure1 enclosure +hc +hc +hc firmness at harvest ………………………………….......……........ 277 …………………………………………………………….…….…............ (g mm-1) after 5 weeks 237 d* 343 a 317 abc 311 bc 288 c 327 ab 312 bc 322 ab tss at harvest ………………………………….......……….... 17.7 …………………………………………………………………….............. (%) after 5 weeks 18.0 a 17.2 a 17.6 a 17.1 a 17.6 a 17.6 a 17.7 a 17.3 a acidity at harvest ………………………………….......……..….. 11.4 ……………………………………………………………….…....…......... (g l-1) after 5 weeks 9.0 a 8.9 a 8.5 a 8.2 a 8.9 a 8.5 a 8.5 a 7.9 a color at harvest …………………………………….........…….. 27.2 ……………………………………………………………….......…........... (l*) after 5 weeks 24.9 a 25.2 a 25.0 a 26.3 a 25.1 a 24.7 a 25.0 a 25.5 a color at harvest ………………………………….......……..….. 13.6 …………………………………………………………………....….......... (a*) after 5 weeks 10.3 a 10.6 a 9.1 bc 9.7 ab 9.7 ab 8.9 bc 8.3 c 8.2 c color at harvest ………………………………….......……….... 1.7 ………………………………………………………………..…....…........ (b*) after 5 weeks 1.6 a 1.4 ab 1.2 cd 1.3 bc 1.3 bc 1.3 bc 1.2 cd 1.0 d 1 fruits were enclosed inside a pvc plastic film without tight enclosure with the aim of increasing relative humidity around fruits without changing the gas composition around fruits. * means within lines followed by different letters indicate significant differences between treatments at p < 0.05, duncan’s multiple range test. modified atmosphere packaging of sweet cherries 147 is well known that stalks condition plays crucial role in the consumer acceptance, and it is obvious from tab. 2 that stalks of ma-packed fruits remained fresher than control fruits. the high relative humidity inside packages may contribute highly to this positive stalks appearance. reduced dehydration and better condition of stalks were reported also by horvitz et al. (2004) with cherries stored in ma up to 42 days. tab. 4: changes in atp, adp concentrations and atp/adp ratio of sweet cherries cv. regina stored for five weeks (year 2003). atp adp atp:adp (nmol g-1 dw) (nmol g-1 dw) ratio at harvest time 55.3 c* 16.9 c 3.3 a control fruit (after 5 weeks) 194.7 a 46.4 b 4.2 b map fruit (after 5 weeks) 168.8 b 51.4 a 3.3 a * means within each column followed by different letters indicate significant differences between treatments at p < 0.05, duncan’s multiple range test. tab. 3: ascorbic acid content and potential of water soluble antioxidants (acw) of mapacked sweet cherries cv. regina at harvest time and after five weeks in storage at 0 ºc. year 2003. ascorbic acid (mg 100g-1 fw) acw. (µg g-1 fw) control map control map harvest time ……… 2.8 ……… ......… 1.65 ……. after 5 week 1.9 b* 2.3 a 1.26 b 1.36 a * means within rows -for each parameterfollowed by different letters indicate significant differences between treatments at p < 0.05, duncan’s multiple range test. tab. 2: percentages of weight losses and scores of consumer perception of sweet cherries cv. regina after six weeks storage period in 2001. before tasting, fruits were conditioned for 24 hours at room temperature. scores for fruit color and appearance: 1 = fresh as at harvest time, 5 = very dark; scores for stem condition: 1 = fresh as at harvest time, 5 = dry and brown colored; scores for fruit taste: 1 = very good, 5 = very bad. weight stem fruit fruit fruit loss (%) freshness color appearance taste (%) (1-5) (1-5) (1-5) (1-5) map (life-plus 30µm) 3.1 b* 1.7 b 2.8 a 5.0 a 2.7 b control 7.4 a 3.5 a 2.5 a 5.0 a 4.3 a *means within each column followed by different letters indicate significant differences between treatments at p < 0.05, duncan’s multiple range test. atpand adp-levels: tab. 4 shows the changes in atp and adp levels in sweet cherries at harvest time, and after five weeks in store. at harvest time, fruits contained the minimal levels compared with fruits stored for five weeks. however, significant differences occurred between control and ma-packaged fruits, by which air stored sweet cherries contained significantly higher atp-concentrations than mapackaged fruits, whereas adp levels show an inverse situation. significant differences were registered also in atp-adp ratios, by which control fruits had significantly the highest ratio. co 2 -enriched atmosphere inside ma-packages may possibly reduced the respiration rate, and consequently the turn over of energy carriers may be slow enough to cause the accumulation of adp in map-fruits; lower respiration rate was registered also with ca-storage of sweet cherries cv. regina (harb et al., 2003). modified atmosphere packaging lengthened the postharvest life of cherry fruit by retaining firmness, reducing the rate of acidity loss, and by retaining stalks freshness. in conclusion, map seems to exert a positive impact on both external and internal quality of fruits, which means better storability of sweet cherries. however, map should be seen as a complementary measure, and not as a substitute for cold temperature; cherries held at lower temperature are generally superior to those held at slightly warmer temperatures (kupferman and sanderson, 2005). references ait-oubahou, a., el-otmani, m., pech, j., chiragui, t., 1994: improving postharvest storage and handling of horticultural commodities in morocco. final research report for eu, eu grant ts2a-0176. artés-hernández, f., aguayo, e., artés, f., 2004: alternative atmosphere treatments for keeping quality of ‘autumn seedless’ table grapes during long-term cold storage. postharvest biol. technol. 31, 59-67. arjona, h., matta, f., garner, j. jr., 1994: wrapping in polyvinyl chloride film slows quality loss of yellow passion fruit. hortscience 29, 295-296. beaudry, r., cameron, a., shirazi, a., dostal-lange, d., 1992: modifiedatmosphere packaging of blueberry fruit: effect of temperature on package o 2 and co 2 . j. amer. soc. hort. sci. 117, 436-441. ben-arie, r., zutkhi, y., 1992: extending the storage life of ‘fuyu’ persimmon by modified-atmosphere packaging. hortscience 27, 811813. cameron, a., boylan-pett, w., lee, j., 1989: design of modified atmosphere packaging systems: modeling oxygen concentration within sealed packages of tomato fruits. j. food sci. 54, 1414-1416. crisosto, c., garner, d., crisosto, g., 2002: carbon dioxide-enriched atmospheres during cold storage limit losses from botrytis but accelerate rachis browning of ‘redglobe’ table grapes. postharvest biol. technol. 26, 181-189. evelo, r., 1993: modelling modified atmosphere systems, 147-154. in: „cost 94; post-harvest treatment of fruit and vegetables: systems and operations for post-harvest quality“. proceeding of workshop, september moreover, our results with odor volatiles (data are not shown), allow us to believe that co 2 -level created inside packages did not reach the injurious level. larsen (1993) found that strawberries packed in ldpe-films developed very strong off-flavor, which is reflected through an increase in the content of acetaldehyde, ethanol, and ethyl acetate, and a decrease in the content of methyl ester, hexanal, and trans-2-hexenol. the current results revealed the preference of taste panel to ma-packed sweet cherries, in particular due to more juicy and crunchy taste. furthermore, these fruits were judged to be more acidic, although panelists criticized the weak odor of fruits – no offflavor-, while with control fruits flesh dryness and slight bitter and a non-homogenous taste were peceived. dryness is partly a result of higher dehydration of cold-stored fruits. hydrocooling of sweet cherries before map (year 2003) did not caused any significant improvement in the consumer acceptance and stalks condition after five weeks of storage (data not shown). vitamin c and antioxidants: tab. 3 shows further aspects related to quality that is highly important for human health. both ascorbic acid content and the potential of water soluble antioxidants of fruits decreased in sweet cherries stored either in air or inside the mapliners, compared to those analyzed at harvest time. this reflects a gradual oxidation of constituents with time, and it is obvious that map slows down this degradation. 148 j. harb, a.a. saquet, r. bisharat, j. streif 14-15 leuven, belgium. harb, j., streif, j., bangerth, f., 2000: response of controlled atmosphere (ca) stored ‘golden delicious‘ apples to the treatments with alcohols and aldehydes as aroma precursors. gartenbauwissenschaft 65, 154-161. harb, j., streif, j., saquet, a., 2003: impact of controlled atmosphere storage conditions on storability and consumer acceptability of sweet cherries ‘regina’. j. hort. sci. biotechnol. 78, 574-579. horvitz, s., yommi, a., lopez-camelo, a., godoy, c., 2004: effects of maturity stage and use of modified atmospheres on quality of sweet cherries cv. sweetheart. revista-de-la-facultad-de-ciencias-agrarias (universidad-nacional-de-cuyo) 36, 39-48. jobling, j., 2001. modified atmosphere packaging: not as simple as it seems. good fruit and vegetables 11, 1-3. kader, a., watkins, c., 2000: modified atmosphere packaging – towards 2000 and beyond. horttechnology 10, 483-486. kays, s.j., 1997: postharvest physiology of perishable plant products. van nostrand reinhold, ny. kupferman, e., sanderson, p., 2005: temperature management and modified atmosphere packaging to preserve sweet cherry quality. acta horticulturae 667, 523-528. larsen, m. 1993. flavour changes in strawberries packed in modified atmospheres. acta horticulturae 368, 78-82. mattheis, j., fellman, j., 2000: impact of modified atmosphere packaging and controlled atmosphere on aroma, flavor and quality of horticultural produce. horttechnology 10, 507-510. meheriuk, m., girard, b., moyls, l., beveridge, h., mckenzie, d., harrison, j., weintraub, s., hocking, r., 1995: modified atmosphere packaging of ‘lapins’ sweet cherry. food research international 28, 239-244. othieno, j., thompson, a., 1993: modified atmosphere packaging of sweetcorn, 247-254. in: „cost 94; post-harvest treatment of fruit and vegetables: systems and operations for post-harvest quality“. proceeding of workshop, september 14-15 leuven, belgium. saquet, a., streif, j., bangerth, f., 2003: impaired aroma production of long-term ca-stored ‘jonagold’ apples as affected by atp and adp levels, pyridine nucleotides and fatty acid metabolism. j. hort. sci. biotechnol. 78, 695-705. skog, l., schaefer, b., smith, p., 2003: on-farm modified atmosphere packaging of sweet cherries. acta horticulturae 628, 415-422. wills, r., mcglasson, b., graham, d., joyce, d., 1998: postharvest. cab international, wallingford oxon. addresses of the authors: dr. j. harb, department of biology and biochemistry, birzeit university, p. o. box 14, birzeit, west bank, palestine (via israel) r. bisharat, department of biology and biochemistry, birzeit university, p. o. box 14, birzeit, west bank, palestine (via israel) dr. a. saquet, federal center of technological education, sao vicente do sul, brazil dr. j. streif, kob, schuhmacherhof 6, d-88213 ravensburg modified atmosphere packaging of sweet cherries 149 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false 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<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> >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice journal of applied botany and food quality 92, 343 354 (2019), doi:10.5073/jabfq.2019.092.046 1department of beverage research, geisenheim university, geisenheim, germany 2department of vegetable crops, geisenheim university, geisenheim, germany 3department of biometry and population genetics, justus liebig university, giessen, germany 4leibniz institute of vegetable and ornamental crops, großbeeren, germany chemical composition of field grown radish (raphanus sativus l. var. sativus) as influenced by season and moderately reduced water supply christine schlering1, 2*, helmut dietrich1, matthias frisch3, monika schreiner4, ralf schweiggert1, frank will1, jana zinkernagel2 (submitted: june 25, 2019; accepted: october 25, 2019) * corresponding author summary seasonal variations in water availability as increasingly provoked by climate change pose severe challenges for vegetable production, particularly for crops requiring reliable and high water supply for achieving satisfactory quality. in contrast to most previous studies applying severe water deficits, we examined the effects of moderate water deficits on the chemical composition of red radish tubers during three consecutive years with variable climatic conditions. radish were cultivated in open field, applying two different water supply treatments and following a randomized block design comprising four sets of six plots each. the resulting water reductions of 3-20% led to a significant increase of dry matter-based myo-inositol levels, whereas those of selected minerals and trace elements, phenolics and glucosinolates decreased. anthocyanin levels remained unchanged. freshmatter related levels of most analytes increased upon reduced water treatments due to higher dry matter contents. while pigment levels in radish remained unchanged, mild water deficit affected other qualityrelated parameters such as pungency-related glucosinolates. keywords: vegetables, polyols, glucosinolates, anthocyanins, drought, water deficit introduction plant responses to changing environmental conditions are complex and vary between cultivation season and year. abiotic stress factors such as water deficiency induce several changes in various physiological plant processes (prasad et al. 2008), generally related to negative effects on plant growth, yield and quality. future scenarios suggest that increasing global surface temperatures will very likely lead to higher incidences of precipitation-free periods such as for example in germany (schmidt and zinkernagel, 2017) or south and west european countries (nikulin et al., 2011), particularly because rainfall patterns are shifting upon temperature changes. for example, 2018 proved to be the warmest and one of the driest years in germany since weather records began. as a result of climate change, growing conditions undergo changes and inadequate water supply might become an increasingly important agricultural challenge for fruit and vegetable production, and drought-stress tolerant cultivars might be desired. climate change conditions including uncommon temporal limitations or excess in water supply, increased regional mean temperatures, and enhanced radiation intensity induces numerous physiological reactions in plants that could change their chemical composition of valuable phytochemicals, clearly influencing the quality of their harvest products (wang and frei, 2011). while the effects of considerable drought stress events on plant physiology have been investigated in detail (taiz and zeiger, 2010; akinci and dorothy, 2012), the impact of a mild reduced water supply on the quality of vegetables has only been insufficiently studied so far. moreover, even less is known about the effects of moderate water shortage on the plant’s chemical composition in open field cultivation, caused by precipitation shifts from summer to winter as expected for many parts of europe in the context of climate change (bindi and olesen, 2011). depending on its duration and intensity, reduced water supply will affect vegetable crop production in different ways and levels from physiology to crop quality. while perennial crops such as grapevine and fruit trees seem to be well adapted for mild water deficits, vegetable crops – particularly freshly marketed products such as spinach as well as tomato and cucumber – appear to be more susceptible to water shortage (costa et al., 2007; nishihara et al., 2001). the drought-induced responses modulate large parts of the plant metabolism and the resulting effects are highly cropspecific (wang and frei, 2011) and often hardly predictable. root vegetables might be affected in a particular way, especially if water deficits occurring during tuber or root initiation (daryanto et al., 2016), since drought increases the rigidity of the soil and, thus, might affect growth of the produce. for instance, red radish (raphanus sativus l. var. sativus) might be particularly affected due to their short tuber growth periods. red radish belongs to the botanical family brassicaceae and occurs in many different color variations and shapes (papetti et al., 2014). economically important cultivars often include those with red round hypocotyl tubers, which are eaten predominantly as raw food and are being harvested at a very early stage of development long before flowering. they provide a range of nutrients as well as quality-related and bioactive compounds, which also have been associated with physiological benefits for human health (manchali et al., 2012). for instance, they were reported to contain considerable amounts of vitamin c (28 mg/100 g fresh weight), glucosinolates, and phenolic compounds like anthocyanins (schreiner et al., 2002; souci et al., 2008). glucosinolates are important for the often desired mild, moderate or strong pungency of radish, addi tionally their breakdown products being associated with health promoting such as anti-carcerogenic effects (hirata et al., 2018). besides their importance for the color and consumer acceptance of radish, anthocyanins have been related to health-promoting properties due to their high antioxidant capacity (yao et al., 2004). furthermore, radish is rich in dry matter, soluble sugars, proteins, and minerals (souci et al., 2008). moderate water reduction may support or affect the accumulation of quality-related and health promoting compounds in red radish. earlier studies have shown that inadequate water supply can lead to higher concentrations of primary and secondary metabolites in vegetables other than red radish (koyama et al., 2012; schreiner et al., 2009; zhang et al., 2008). moreover, varying climate conditions also contribute to alterations in the chemical composition of the product, as shown for bioactive compounds 344 c. schlering, h. dietrich, m. frisch, m. schreiner, r. schweiggert, f. will, j. zinkernagel in different brassica vegetables (aires et al., 2011). therefore, we sought to study the effect of a mild reduced water supply on quality relevant morphological and physio-chemical traits of the red radish tubers in open field cultivation during three successive years including four cultivation sets, thus resembling more practically relevant conditions as compared to experiments in controlled environments like greenhouses or growth chambers. material and methods chemicals all reagents and solvents used were at least of analytical or hplc quality unless specified differently. folin-ciocalteu’s phenol reagent and sodium carbonate were purchased from merck (darmstadt, germany); l-(+)-ascorbic acid from carl roth (karlsruhe, ger many); d-(+)-catechin-hydrate, myo-inositol (> 99.5%, hplc) and anhydrous glycerol from sigma aldrich (steinheim, germany). pelargonidin-3-o-glucoside obtained from extrasynthese (genay cedex, france). l-aspartic acid (ph. eur., usp) was received from applichem (darmstadt, germany). plant material and experimental setup red radish cv. celesta f1 (enza zaden, dannstadt-schauernheim, germany) were grown from seeds sown in the field. cultivation experiments were conducted under open field conditions on a sandy loam in a plot installation at geisenheim university, germany (49°59´ n, 7°58´ e). a total of four cultivation sets were grown during the years 2015 (1 set), 2016 (2 sets) and 2017 (1 set) as shown in tab. 1. each set consisted of six circular plots (marked by a, c, f, h, m and p) with a diameter of 11.9 m. each plot was subdivided into four quarters (subplots) for the simultaneous cultivation of three different vegetable crops with an annual crop rotation. each subplot quarter was again divided into three further segments for conducting the different irrigation treatments following a randomized block design, while the outer segment of each subplot was excluded from harvest (fig. 1). the irrigation levels remained assigned to the subplots during the full duration of the experiments, irrespective of the annual crop rotation. the size of each segment was at least 6.5 m², respectively. cultivation and water supply the red radish seeds were sown in rows with a spacing of ca. 0.125 m between the rows and a sowing distance of ca. 0.033 m in the rows. fertilization was carried out uniformly with calcium ammonium nitrate according to commercial standard specifications for radish cultivation (109 kg/n ha-1) based on mineralized n (no3--n) in 0-15 cm soil depth. crop protection was applied equally to all plots, whereby application depended on the growing set. the insecticide karate zeon (syngenta, maintal, germany) was used against flea beetles (psylliodes spp.) and goldor bait (basf, ludwigshafen, germany) against wireworms (elateridae) once per cultivation set. the removal of weeds was conducted manually. water supply was carried out as follows: drip irrigation was initiated when the soil moisture tension fell below -20 kpa in 10 cm depth and the wellirrigated segments (ctr) were provided with 100% water supply (6.2 l/m2 per irrigation), as controlled by a tensiometer with electronic pressure sensor (tensio-technik, bambach gbr, geisenheim, deutschland). segments assigned to reduced water supply (rws) were provided with 63% (2015, 2016-2), 68% (2016-1) and 51% (2017), respectively, of the aforementioned water amounts by irrigation. since natural precipitation dominated the moisture tension, the total amount of irrigation events and, consequently, the total amount of supplied water was highly variable from set to set. as a result, the extent to which water supply had been reduced was variable, amounting to a reduction of 10% (2015), 15% (2016-1), 20% (2016-2) and 3% (2017), respectively, when considering both irrigation and precipitation (tab. 1). leaf water potential was measured two days before harvest (2016-1 and 2016-2) or just before harvest (2017) by using a scholander pwsc 3000 (soil moisture equipment corp., usa). to ensure uniform germination and seedling growth, the soil of both the full and reduced water supply treatments was kept moderately moist by receiving the same, sufficiently high water amounts during initial stage. depending on the growing period, this initial stage lasted for 9 days (2015), 7 days (2016-1, 2016-2) and 12 days (2017). tab. 1 summarizes further detailed information on cultivation data and water supply. harvest a total of 120 plants per segment were harvested randomly when tuber diameter reached 25-30 mm, representing the commercially targeted maturation stage for harvest. to avoid border effects, outer rows were omitted from harvest. specifically, harvest dates were 26, 23, 23 and 33 days after sowing for the cultivation sets 2015, 20161, 2016-2 and 2017, respectively (tab. 1). after harvest and shoot removal with a knife, tubers were cleaned twice manually with fresh tap water to remove adhering soil particles. then, radish tubers were frozen at -80 °c until analyses. climatic data local climate data were supplied by the local weather station, which was located at 100 meters distance to the experimental field site in geisenheim. climatic parameters for each cultivation period are summarized in tab. 1. based on detailed weather situations of daily temperature and air humidity profile (supporting information, fig. a.1 and a.4), the different growing periods were characterized fig. 1: circular structure of the open field experimental setup. each quarter of the circle represents a plot divided into three subplots. red radish was grown in one of the four plots. subplots differed in the irrigation regimes. the outer subplot (marked by dot pattern) was excluded from the harvest. chemical composition of field grown radish 345 and assigned as follow: 2015 (warm & moist), 2016-1 and 2016-2 (warm & dry) and 2017 (cold & moist). strictly speaking, the cultivation periods 2015 and 2017 were characterized by strong variations of global radiation and air humidity (supporting information, fig. a.2 and a.3), whereas the conditions in 2016 were much more constant. sample preparation for all analyses, except for the determination of ascorbic acid and anthocyanins, 25 radish tubers of each sample were lyophilized (beta 2-8 ldplus, martin christ gefriertrocknungsanlagen gmbh, osterode am harz, germany) prior to grinding with a laboratory mill (ika m 20; ika-werke, staufen, germany). for the analyses of anthocyanins, the colored external tissues of 15 radish tubers were carefully separated with a vegetable peeler, frozen immediately at -80 °c, and ground into a fine powder under liquid nitrogen with the aforementioned laboratory mill. dry matter content was determined from freeze-dried material. d-glucose, d-fructose, titratable acidity, l-malic acid and fumaric acid as well as inorganic anions like nitrate, phosphate, sulfate and chloride were determined from aqueous extracts prepared from the lyophilized powder. for this purpose, an aliquot of 8-10 g of lyophilized radish powder was thoroughly homogenized with 500 ml ultrapure water at room temperature for approx. 10 s at high level (setting 2) using a stainless steel food blender (waring blender, waring commercial, torrington, ct 06790, usa). after transferring the extract into an 800 ml beaker using 150 ml ultrapure water, extraction was continued for 10 min under continuous magnetic stirring, followed by a single ultrasoundassisted extraction step in an ultrasound water bath for another 5 min. after centrifuging for 5 min at 4596 × g to separate liquid and solid phases, the supernatants were collected, filtered, and stored at -25 °c until analyses. extraction procedures for all other target analytes are given below. chemical analysis unless otherwise noted, ifu-methods (international fruit juice union, paris) were used for determination of routine parameters in aqueous extracts, such as sugars, total acidity, organic acids and inorganic anions. sugars and polyols d-glucose and d-fructose were determined with enzymatic kits (r-biopharm, darmstadt, germany) using a konelab 20 xti analyzer (thermofisher, dreieich, germany). determination of polyols was carried out in duplicates as follows. an aliquot of 400 mg of lyophilized powdered radish was extracted twice with 12 ml ultrapure water in a shaking water bath for 1 h at 45 °c. after centrifuging for 10 min at 12.857 × g, supernatants were collected, made up to a volume of 25 ml and filtered through a 0.2 μm membrane (pes filter; vwr, darmstadt, germany). extracts were injected (10 μl) into a hpaecpad system (dionex/thermofisher biolc system, ics 5000+, ics 3000 sp, thermo fisher scientific, dreieich, germany) equipped with an anion-exchange carbopac ma 1 column (250 × 4 mm, thermo fisher scientific, dreieich, germany) and a guard column of the same material, both operated at 15 °c. separation was achieved by isocratic elution with 500 mmol/l naoh at a flow rate of 0.5 ml/ min. quantitation was conducted with linear external calibrations of myo-inositol and glycerol. organic acids titratable acidity, calculated as citric acid, was measured potentiometrically after titration to ph 8.1 with 0.3 m naoh (titroline alpha, schott, mainz, germany). l-malic acid was determined using enzymatic kits (r-biopharm, darmstadt, germany). fumaric acid was determined on a summit hplc-uv system (dionex, idstein, germany) using a serial column connection of a luna c18 250 × 4.6 mm and a rezex fast fruit 100 × 7.8 mm (phenomenex, aschaffenburg, germany). an isocratic elution profile with diluted aqueous phosphoric acid (1 ml h3po4 (0.6%)/100 ml) and a flow rate of 0.7 ml/min was used. quantitation was carried out by uv detection at 230 nm using an external calibration. determination of ascorbic acid was conducted by iodometric titration. for this, an aliquot of 100 g of fresh frozen radish were homogenized in 200 g of oxalic acid (1%, w/v) using a waring blender. after centrifuging at 4596 × g for 10 min at 4 °c, the supernatant was acidified with aqueous sulfuric acid (10%) prior to ascorbic acid determination by automatic potentiometric titration (schott-titrator, titrinoline alpha plus; software, titrisoft) with 1/128 mol l-1 iodide-iodate solution. results were expressed as ascorbic acid in mg g-1 fresh weight. inorganic anions nitrate, sulfate and phosphate were analyzed by ion chromato graphy on an ics 2100-system on an ionpac as 17 column using a koh-gradient (4-45 mmol/l in 20 min, flow rate 0.5 ml/min) and suppressed conductivity detection (dionex, idstein, germany). tab. 1: cultivation and climate data during the different experimental periods. das: days after sowing. irrigation (mm): total irrigation amount including irrigation during initial stage. total water amount (%) rws: water amount (%) of the reduced variants including watering after sowing. growing season 2015 2016-1 2016-2 2017 sowing date (year-month-day) 2015-08-06 2016-06-29 2016-08-29 2017-04-19 beginning of water supply differentiation (das) 15 7 7 28 harvest date (das) 26 23 23 33 temperature sum (°c) 565.1 488.1 483.6 408.9 daily mean air temperature (°c) 21.7 20.3 20.2 12.0 mean relative air humidity (%) 67.1 83.8 84.2 68.4 global radiation sum (mj/m²) 444.1 480.5 372.8 590.9 daily mean global radiation (mj/m²) 16.4 20.0 15.5 17.4 wind speed sum (m/s) at height of 2 m 38.4 32.1 27.4 43.8 evaporation sum (mm) 92.4 86.6 61.1 94.0 precipitation sum (mm) 21.2 9.2 12.8 62.9 number of differentiated irrigation events 2 6 5 1 irrigation (mm) 93 56 43 48 total water amount (mm) incl. precipitation 125 65 56 111 total water amount (%) rws 90% 85% 80% 97% 346 c. schlering, h. dietrich, m. frisch, m. schreiner, r. schweiggert, f. will, j. zinkernagel quantitation was based on linear calibrations with a multi-ion re ference solution (bernd kraft, duisburg, germany). chloride was analyzed by potentiometric titration with an agcl-electrode using a schott titrator titroline alpha plus combined with a sample changer tw alpha plus and a piston burette titronic universal (schott, mainz, germany). total carbon and nitrogen elemental analyses of radish samples for carbon and nitrogen were carried out in duplicate by the dumas combustion method (vario max cns, elementar analysensysteme gmbh, langenselbold, germany), combusting 300 mg of freeze-dried radish powder at 950 °c. aspartic acid was used as reference substance. minerals and trace elements for minerals and trace elements analyses, the principle of the kjeldahl digestion with the gerhardt turbotherm rapid digestion unit (c. gerhardt gmbh & co. kg, königswinter, germany) was applied as follows: an aliquot of 250 mg of freeze-dried powdered plant material was mixed with 10 ml of a specifically designed digestion solution (420 ml 18 m h2so4, 330 ml aqueous 30% h2o2 p.a., 0.48 g se (black powdered), 14.0 g lithium sulfate (suprapur)). after digestion at 100 °c for 100 min, the extract was made up to 50 ml with ultrapure water. elemental analyses for p, k, ca, mg, mn, zn and cu were executed with an icp-oes (spectro arcos, spectro analytical instruments, kleve, germany). total phenols an aliquot of 500 mg of freeze-dried powdered radish was extracted twice with 12 ml of 80% aqueous methanol under ultrasonication for 30 min and consecutive centrifugation for 10 min at 12.857 × g. the combined supernatants were made up to 25 ml with extraction solvent and stored at -28 °c until spectrophotometric analyses with the folin-ciocalteu reagent and a linear (+) catechin calibration as described previously (singleton and rossi, 1965). anthocyanins separation, identification and quantitation of single anthocyanins in the radish skin was carried out by hplc-pda-ms using the procedure described by will and dietrich (2006). in brief, an aliquot of 5 g of powdered fresh radish skin was extracted twice with 12 ml of aqueous 85% methanol acidified with 1% formic acid (v/v), using a cooled ultrasonic bath for 30 min in the dark. after centrifugation, supernatants were collected and combined, made up to a volume of 25 ml with extraction solvent and filtered through a 0.2 μm membrane (chromafil o-20/25 ptfe syringe filter; machereynagel, düren, germany) prior to injection. chromatographic and mass spectrometric parameters, settings and equipment was used as reported earlier (will and dietrich, 2006). injection volume was 4 μl and flow rate 250 μl/min at 40 °c. anthocyanins were identified by comparison of retention times, uv/vis absorption maxima, and mass spectra to those of authentic reference substances or literature data (jing et al., 2012; wu and prior, 2005). quantitation was carried out at 520 nm using an external calibration with pelargonidin3-o-glucoside. glucosinolates identification and quantitation of glucosinolates were determined according to wiesner et al. (wiesner et al., 2013). statistical analysis compositional data was analyzed with regard to both dry and fresh matter. dry matter-related data should allow evaluations irrespective of different water contents in the plant material due to reduced irrigation treatments, while fresh matter-related data should represent the nutritional composition of the harvested edible plant material. data were analyzed by fitting a linear mixed-effect model using the lmer-function within the lme4-package (bates et al., 2015) of the statistical software r (r core team, 2016). the evaluation of the single cultivation sets based on a model with respect to the random plot-effects (eq. 1), while the total dataset was evaluated with respect to the interaction of plot and year (eq. 2): y = water supply + plot (eq. 1) y = water supply + plot:year (eq. 2), where y represents the analyzed parameter, water supply (control, reduced) was a fixed factor and plot as well as the interaction plot:year were random factors. comparisons of means derived from different treatments were considered significantly different if p-values < 0.10. pairwise comparison of least-squares means was carried out with lsmeans-package (lenth, 2016) to estimate fixed-effects of the treatment as well as random effects for the plot and season. significances of random effects were calculated by the lmertestpackage (kuznetsova et al., 2016). the results for the treatment (control, reduced) were evaluated on basis of adjusted data generated from the model mentioned above. principal component analysis (pca) was carried out by using the r-packages factominer (le et al., 2008) and factoextra (kassambara and mundt, 2017). to avoid an overweight of single anthocyanins and glucosinolates, only total amounts were considered for principal component analysis (pca). three missing values were calculated by the missmdapackage (josse and husson, 2016). results and discussion evaluation of cultivation sets and season the entire data set was visualized by pca after correcting for random plot-effects (cf. eq.1) by the least-square means method. the corresponding loading plot revealed a strong differentiation of the cultivation sets by their chemical composition and points out the seasonal effects on the quality of the produce. the first two principal components (pcs) accounted for 66.4% of the total variance in case of dry matter evaluation (fig. 1) and 62.1% in relation to fresh matter evaluation (data not shown). consequently, the chemical composition was strongly dependent on the season with all its individual climatic conditions, which was already shown to affect selected substances in brassica varieties (aires et al., 2011; schreiner, 2005). cultivation sets from 2015 and 2017 partly showed an overlap, while both sets from 2016 were clearly separated within the pca loading plot (fig. 1). the total water amounts were higher in 2015 (125 mm) and 2017 (111 mm) compared to 2016-1 (65 mm) and 2016-2 (56 mm), respectively. these years clusters also differed in mean relative air humidity, which were 67.1% (2015) and 68.4% (2017) as compared to the datasets 2016-1 (83.3%) and 2016-2 (84.2%) (tab. 1). the resulting higher leaf-air vapor pressure difference in 2015 and 2017, respectively, might have altered parameters such as transpiration and tissue temperature, which in turn may affect ion uptake, carbon assimilation and water transport (grantz, 1990). nevertheless, both sets from 2016 were clearly separated within the pca loading plot (fig. 1). effects of reduced water supply on the chemical composition of radish biomass when comparing chemical parameters across the full dataset by chemical composition of field grown radish 347 pca, the data points of the well-irrigated radish samples were mainly clustered in sectors with positive pc2, while those of the less irrigated radish samples were mainly clustered in sectors with negative pc2 (fig. 2a). most of the individuals were separated by the second principal component pc2 (dim2), which accounted for 11.4% of the total variance. accordingly, the first two principal components (dim1+dim2) accounted for 38.2% of the total variance in the data set. the corresponding variables are represented in the loading plot (fig. 2b). the most important variables with high contribution to dim2 were contents of anions like phosphate and chloride as well as content of phosphorous (p), carbon (c), malic acid and total phenols. dim1 was mainly influenced by contents of minerals like ca, mg, n, k, p and zn as well as nitrate and anthocyanins. apart from these multivariate results, univariate statistical evaluation shown in tab. 2 was carried out to support the multivariate approach. while the content of malic acid, phosphate, p and mn as well as total phenols and glucosinolates decreased in samples derived from reduced water supply, the content of polyols like inositol and glycerol as well as cu increased in the same group. in brief, except for polyols and cu, all other targeted constituents remained constant or were diminished under reduced water supply (tab. 2). when subjecting the fresh matter-related data to pca, the distinction of both treatments was substantially clearer (fig. 3a) than when using dry matter-related data (fig. 2a). the second principal component (dim2) explain ing 17% of the total variance. similarly to the dry matter-related evaluation, the first two principal components accounted for 47.3% of the total variance. however, discrimination of both treatments was mainly based on primary metabolites like sugars and polyols, total carbon, total phenols, ascorbic acid as well as sulfate and copper (fig. 2a-b). univariate evaluation (tab. 2) confirmed these results. total glucosinolates decreased under reduced water supply. in contrast, contents of many other constituents such as polyols showed a significant increase. this is possibly being related to a concomitantly significantly increase in dry matter content (tab. 2), which was also shown for carrot tap roots subjected to drought stress just prior to harvest (sørensen et al., 1997). sugars and polyols the dry matter-related content of sugars such as glucose and fructose was similar irrespective of the water supply applied (0.1072 < p < 0.2932 and 0.1992 < p < 0.6536, resp., tab. 2). similarly, differences in fresh matter-related contents of glucose and fructose were insignificant, except for the set 2016-1, where glucose and fructose were significantly higher under reduced water supply cultivation conditions (p = 0.0129 and p = 0.058, respectively). in this latter set (2016-1), the overall glucose and fructose levels (1.40-1.48 and 0.991.03 g/100g fw) were higher than in all other sets (1.11-1.34 and 0.74-0.89 g/100 g fw). as shown in tab. 1, the set of 2016-1 was grown under comparably warm and high radiation conditions, thus possibly evoking highest sugar accumulation as a result of enhanced photosynthetic rates. in agreement with our results, schreiner et al., (2002) found that contents of glucose and, in particular, fructose as metabolites of photosynthesis were highest in radish grown at high mean light intensities and high mean temperatures. by analogy to glucose and fructose, highest levels of polyols were also found in the warm and well-irradiated growing period 2016-1 (tab. 2). however, in contrast to glucose and fructose, both dry and fresh matter-related concentrations of polyols like glycerol and myoinositol were significantly increased in 2016 and 2015 upon reduced water supply (tab. 2 and 3), except for the set 2017 grown under cold fig. 2: principal component analysis of the original, non-adjusted dataset including all radish cultivation sets (2015 (red circles), 2016-1 (green triangles), 2016-2 (blue squares) and 2017 (violet crosses)). score plot represents the individual samples of each cultivation set (separated by color) and plot (marked by the letters a, c, f, h, m and p) based on dry matter-related chemical analyses. fig. 3: multivariate evaluation by pca (pc1+pc2) of the total radish dataset including all cultivation sets (2015, 2016-1, 2016-2 and 2017) based on dry matter-analyses. score plot (a) represents all individual plot samples (marked by the letters a, c, f, h, m and p) classified according control ctr (blue circles) and reduced water supply rws (red triangles). the corresponding loading plot (b) shows the related variables determined by the chemical analyses. 348 c. schlering, h. dietrich, m. frisch, m. schreiner, r. schweiggert, f. will, j. zinkernagel (daily mean air temp.: 12 °c) and comparably well-watered (97% relative to control, tab. 1) conditions. supporting the hypothesis that water supply might have played a role, the reduced water supply in the set of 2015 was at 90% relative to full supply, already reaching a marginal statistical significance (p = 0.1303). those of 2016-1 and 2016-2 were at a more intensely reduced water supply, i.e. at 85% and 80% rel. to full water supply, consequently yielding statistically significant differences (tab. 2 and 3). our data also shows that the climatic conditions in the set of 2016-1 resulted in an increase of total polyols, mainly that of inositol (tab. 2). polyols have been reported to act as compatible solutes in the osmotic adjustment and as osmoprotectants in plants, being accumulated to osmotically significant levels without adverse effects on the plant’s metabolism (nuccio et al., 1999). in this context, dietrich et al. (2007) found strong increases of sorbitol in pear and apple juice exposed to drought stress. in agreement with our findings, strong increases of myo-inositol were observed in drought-stressed pea (pisum sativum l.) in both the field experiment and under greenhouse conditions (charlton et al., 2008). in our study, we show for the first time that the accumulation of myo-inositol and glycerol in radish might be elicited at already comparably small reductions in water supply, i.e., at ≤ 10% of the full water supply as indicated in our study. organic acids the organic acids studied in our experiment were hardly affected by moderate water reduction, except for malic acid, which was negatively affected in 2015, but not in 2016-1, 2016-2 and 2017. the reason for these observations remains unclear. similarly, ascorbic acid levels remained unchanged by mildly reducing water supply, suggesting that the nutritional value with regard to vitamin c re mained unaffected irrespective of the water supply. however, by analogy to glucose, ascorbic acid levels were highest in the sets 2016-1 and 2016-2 grown in the warm and well-irradiated conditions, possibly being associated as glucose represents the biosynthetic precursor of ascorbic acid. in contrast to mildly reducing water supply, stronger drought stress was earlier shown to lead to an increase of ascorbic acid in fresh matter of hydroponically grown leafy vegetables like lettuce, spinach and turnip leaf (koyama et al., 2012). in agreement with these results, we also found higher ascorbic acid levels in radish under mildly reduced water supply with statistical significance (p = 0.0891 and p = 0.0517, respectively) for the set 2016-1 as well as the total dataset. anions nitrate (no3-) contents were only insignificantly influenced by reduced water supply (tab. 2 and 3, fig. 2a-b). however, no3contents were substantially lower in 2016-1 (11.1-13.8 mg/g dm) and 2017 (17.95-18.96 mg/g dm) in contrast to other cultivation sets (26.5-32.8 mg/g dm) as shown in tab. 1. high radiation and evapotranspiration rates might have led to balanced conditions between absorption and assimilation of no3-, resulting in lower no3--levels. however, frequent stress-related conditions for the respective radish plants just before harvest might have affected no3--levels in the set of 2016-1 (tab. 1). in agreement with this hypothesis, sørensen et al. (1997) found lowered nitrate levels in carrots (daucus carota l.) even when drought stress occurred just prior to harvest. in contrast to 2016-1, where irrigation was finished two days prior to harvest, irrigation in 2016-2 was finished 6 days before harvest due to adequate soil humidity, probably having led to more continuous no3uptakes in contrast to 2016-1. further study is needed though. although the dry matter-related content of phosphate (po43-) was significantly decreased by water supply reduction when considering the overall dataset, the significant decline of po43was only observed in 2016-2, when water reduction was highest, i.e., to 80% of full water supply. since phosphate mobility in the soil depends on moisture (taiz and zeiger, 2010), reduced soil moisture might have diminished the uptake into the tubers (da silva et al., 2010). similarly to the other anions, sulfate (so42-) and chloride (cl-) levels were hardly affected by reduced water supply, except for the set 2017, where a significant increase in clfrom 0.89 to 0.96 mg/100 g dm (p = 0.0375) was observed (tab. 2). while the levels of so42and cl were highest in 2016-1 and 2016-2, when irrigation events were more frequent compared to the other sets, the mild water deficit evoked in our study appeared to be insufficient to significantly affect sulfate and chloride uptake. carbon water supply reductions did not influence dry matter-related total carbon (c) as shown by multivariate and univariate analyses. fresh matter-related total carbon showed to be significantly higher in samples grown under reduced water supply (1.75-1.95 g/100 g fm) than in control samples (1.67-1.91 g/100 g fm). these findings correlate well to significantly higher contents of dry matter in radish grown under reduced water supply. in agreement, multivariate evaluation confirmed a considerable contribution of c to the separation of both variants on fresh matter basis (fig. 3a-b). fig. 4: multivariate evaluation by pca (pc1+pc2) of the total radish dataset including all cultivation sets (2015, 2016-1, 2016-2 and 2017) based on fresh matter-analyses. score plot (a) represents all individual plot samples (marked by the letters a, c, f, h, m and p) classified according control ctr (blue circles) and reduced water supply rws. the corresponding loading plot (b) shows the related variables determined by the chemical analyses. chemical composition of field grown radish 349 nitrogen mild reductions in water supply had no significant impact on dry matter-related total nitrogen (n) in radish (tab. 2, fig. 2a-b), while it was associated with an apparently significant increase of fresh matter-related nitrogen contents in the warm and moist year 2015 and the cold and moist year 2017 (tab. 3). in these years, the total nitrogen levels were substantially higher (94.5-104.0 mg/100 g fw) than in 2016 (72.1-81.6 mg/100 g fw). because soil n availability is negatively affected by drought events and this strongly influences nutrient absorption and uptake by plants, it might be assumed that n mobility had been temporarily restricted due to insufficient total water amounts in 2016 (56-65 mm) in contrast to 2015 (125 mm) and 2017 (111 mm). in addition, lower air humidity in 2015 (67.1%) and 2017 (68.4%), in contrast to 2016 (83.3 to 84.2%), might have led to higher n levels. gisleröd et al. (1987) found decreased levels of selected macronutrients in greenhouse plants when exposed to higher air humidity. further studies showed that negative effects on the plants’ nitrogen level are alleviated with drying-rewetting cycles (he and dijkstra, 2014), possibly explaining the absent treatment effect in 2016-1 and 2016-2, although effects were expected due to several short-time drought events. potassium by analogy to our observations on nitrogen levels, water supply limitations exerted only insignificant effects on dry matter-related contents of potassium according to both pca analysis and univariate statistical evaluation (fig. 2a-b, tab. 2). in contrast, significantly increased fresh matter-related contents were observed in the sets of 2015 and 2017 with reduced water supply (tab. 3), possibly being a result of generally increased dry matter, which in turn resulted from reduced moisture content. since potassium plays multiple important functional roles in plant cells (taiz and zeiger, 2010), its level should be strictly regulated by plant metabolism. in agreement, afzal et al. (2014) also found only insignificant effects of drought stress on dry matter-related k+ concentrations in leaves and roots of mungbean (vigna radiata (l.) wilczek). in our study, radish showed highest absolute k+ levels in 2017 (232.6-255.6 mg/100 g fw; 51.553.2 mg/g dw) when precipitation was most frequent and abundant, as compared to the sets of other years (198.6-222.3 mg/100 g fw; 39.0-47.4 mg/100 g dw). under these conditions in 2017 (cold and moist), however, k+ levels notably increased upon water supply limitation (p = 0.0405, fresh matter; p = 0.0711, dry matter). phosphorous the content of phosphorous (p) in radish dry matter was significantly decreased by moderately reduced water supply. by analogy to po43-, the decline of p was highly significant for the total dataset as well as for the single cultivation sets 2015 and 2016-2 (tab. 2). multivariate analyses by pca support this result (fig. 2a-b). greenway et al. (1969) found that even a small decrease in water potential reduces p uptake by tomato plants (lycopersicon esculentum mill.), while water uptake and transport were generally lower in gradually treated plants in contrast to plants suddenly exposed to low water potential. however, p uptake was much less for a considerable time even if plants were exposed to stress for only one hour. further studies in agreement with these and our findings have been reported (sardans and peñuelas, 2004; sánchez-rodríguez et al., 2010). calcium reduced water supply led to lower calcium (ca2+) contents in radish dry matter, being significant in 2016-1 (p = 0.0266) and 2016-2 (p = 0.0907), but not in the cultivation sets of 2015 and 2017. contrary to k+ or n, absolute ca2+ content was higher in reducibly watered samples in 2017, but water reduction was very low (tab. 1). in gene ral, ca2+ acts as important constituent of the middle lamella of the cell wall and it is required in numerous metabolic processes (taiz and zeiger, 2010) including responses to abiotic stress like drought (hu and schmidhalter, 2005; knight et al., 1997). however, sánchez-rodríguez et al. (2010) found no differences in the ca2+ accumulation of tomato leaves when plants were treated by moderate drought stress, although its uptake was significantly affected. hu and schmidhalter (2005) concluded that even if its uptake is reduced, the accumulation of ca2+ declines very slightly. in low-transpiring organs like tubers or fruits, which are supplied predominantly via the phloem, its concentration is considerably lower than in leaves (marschner, 1986). in agreement, we did not observe any consistent effect of reduced waters supply on the ca2+ content in red radish. magnesium similarly to other minerals, effects on magnesium (mg2+) with regard to reduced waters supply on radish were apparently insignificant as shown by multivariate analyses (fig. 2a-b). however, univariate evaluation demonstrated an increase in the mg2+ content due to reduced water supply in 2016-1 (p = 0.0946) (tab. 2). in agreement, pulupol et al. (1996) found only insignificant effects of deficit irrigation on the dry weight-based levels of magnesium in tomato fruits grown in a glasshouse. however, fresh weight-based mg2+ contents have been found to be higher in fruits of deficit irrigated plants than in fruits of well-watered plants (pulupol et al., 1996), which also applies to radish fresh matter grown under moist conditions in 2015 (p = 0.0642) and 2017 (p = 0.0387) (tab. 3). micronutrients (fe, zn, mn, cu) dry matter-related iron (fe) contents were reduced in 2015 (p = 0.0846), whereas zinc (zn) and copper (cu) were increased in 2017 (p = 0.0297 and p = 0.0523, respectively) by slight water reduction (tab. 2). these findings might be related to the involvement of these micronutrients in photosynthesis and their catalytic role in the antioxidant systems of plants (hänsch and mendel, 2009). evaluation of the total dataset indicated a significant dry matter-related decrease of manganese (mn) and a significant increase in cu upon water supply limitation. similar observations have been reported earlier (sánchez-rodríguez et al., 2010). pca analyses supported the contribution of these micronutrients to the variants’ differentiation (fig. 2b). because micronutrient transport through the soil and towards the root is governed by diffusion, diminished levels were expected when limiting water supply. considering the total fresh matter-related dataset (tab. 3), significantly higher contents of zn and cu were found for samples with limited water supply in 2017. our results showed generally reduced contents of fe in 2016-1 and 2017 (tab. 2), when global radiation was highest. the reduced contents of fe in the set of 2016-1 are in agreement with the trend found for most macronutrients. however, this results are contradictory to zancan et al. (2006), who found iron accumulation in roots and leaves of maize provoked by high uv-b radiation. phenolic compounds reduced water supply led to significant reductions (p = 0.0523) in the dry matter-related content of total phenols (tab. 2), while fresh matter-related contents remained unchanged (tab. 3). previous studies reported variable effects of drought stress on levels of polyphenols including anthocyanins in the respective plants (robbins et al., 2005). in agreement with literature, the quantitatively most abundant antho cyanins in radish skin were acylated derivatives, namely pelargo 350 c. schlering, h. dietrich, m. frisch, m. schreiner, r. schweiggert, f. will, j. zinkernagel ta b. 2 : in flu en ce o f r ed uc ed w at er s up pl y on th e dr y m at te r ( d m )re la te d le ve ls o f c on st itu en ts o f r ad is h tu be rs fr om d iff er en t c ul tiv at io n se ts (2 01 5, 2 01 61, 2 01 62 an d 20 17 ). l in ea r m ix ed m od el , t -t es t, pva lu es : < 0. 1, < 0 .0 5 (* ), < 0. 01 (* *) a nd < 0 .0 01 (* ** ), nd = n ot d et er m in ed . c t r : c on tr ol tr ea tm en t w ith fu ll w at er s up pl y. r w s: r ed uc ed w at er s up pl y tr ea tm en t. r ad is h 20 15 r ad is h 20 16 -1 r ad is h 20 16 -2 r ad is h 20 17 r ad is h to ta l c t r r w s pva lu e c t r r w s pva lu e c t r r w s pva lu e c t r r w s pva lu e c t r r w s pva lu e l ea f w at er p ot en tia l ψ [m pa ] nd nd n d 0. 15 0. 21 0. 01 23 * * 0. 16 0. 24 0. 00 02 * ** 0. 07 0. 09 0. 00 06 * ** nd nd n d d ry m at te r [ % ] 4. 79 4. 97 0. 03 37 * 5. 00 5. 17 0. 08 86 4. 64 4. 71 0. 00 35 * * 4. 61 4. 75 0. 17 36 4. 76 4. 90 0. 00 01 * ** su ga rs a nd p ol yo ls g lu co se [m g/ g] 25 4. 5 24 4. 6 0. 29 32 28 1. 1 28 6. 5 0. 15 77 27 2. 9 28 3. 7 0. 10 72 24 7. 06 23 9. 92 0. 18 46 26 9. 5 27 1. 6 0. 95 50 fr uc to se [m g/ g] 18 4. 3 17 8. 9 0. 34 52 19 7. 3 19 9. 2 0. 63 87 16 0. 2 16 2. 3 0. 65 36 16 8. 39 16 2. 01 0. 19 92 18 0. 6 18 0. 1 0. 39 63 po ly ol s to ta l [ m g/ g] 2. 07 2. 18 0. 13 03 3. 66 4. 40 0. 00 09 * ** 1. 71 1. 85 0. 00 72 * * 1. 97 2. 01 0. 50 62 2. 48 2. 81 0. 00 09 * ** in os ito l [ m g/ g] 1. 75 1. 85 0. 14 67 3. 15 3. 81 0. 00 19 * * 1. 37 1. 51 0. 00 71 * * 1. 41 1. 43 0. 60 12 2. 09 2. 39 0. 00 10 * * g ly ce ro l [ m g/ g] 0. 32 0. 34 0. 27 22 0. 51 0. 60 0. 00 21 * * 0. 35 0. 34 0. 45 49 0. 56 0. 58 0. 55 21 0. 39 0. 42 0. 01 97 * o rg an ic a ci ds to ta l a ci di ty [m g/ g] 15 .0 14 .6 0. 31 11 11 .2 10 .1 0. 37 01 12 .3 11 .8 0. 67 68 10 .6 0 10 .0 9 0. 08 16 12 .8 12 .2 0. 11 09 m al ic a ci d [m g/ g] 36 .1 34 .7 0. 04 54 * 30 .5 29 .7 0. 14 87 34 .9 34 .3 0. 18 65 35 .6 6 35 .7 0 0. 96 80 33 .8 32 .9 0. 02 35 * fu m ar ic a ci d [m g/ g] 3. 39 3. 49 0. 49 69 4. 86 4. 63 0. 29 60 4. 81 6. 39 0. 31 58 2. 52 2. 70 0. 81 72 4. 35 4. 34 0. 87 18 a sc or bi c ac id [m g/ g] 5. 64 5. 47 0. 29 69 5. 80 6. 03 0. 23 39 6. 45 6. 45 0. 98 72 5. 21 5. 30 0. 30 32 5. 96 5. 98 0. 63 62 a ni on s n itr at e [m g/ g] 32 .8 32 .3 0. 82 00 13 .8 11 .1 0. 10 77 27 .8 26 .5 0. 54 18 17 .9 5 18 .9 6 0. 45 86 24 .7 7 23 .2 9 0. 35 40 ph os ph at e [m g/ g] 11 .4 8 11 .1 6 0. 49 93 12 .9 0 12 .1 2 0. 16 68 11 .1 4 10 .1 7 0. 02 11 * 10 .0 4 9. 89 0. 66 74 11 .8 4 11 .1 5 0. 00 92 * * su lf at e [m g/ g] 2. 52 2. 75 0. 52 39 3. 99 4. 13 0. 23 28 4. 00 4. 05 0. 79 45 3. 47 3. 32 0. 58 19 3. 51 3. 64 0. 56 80 c hl or id e [m g/ g] 0. 88 0. 98 0. 53 87 1. 90 1. 92 0. 88 35 1. 79 1. 27 0. 14 02 0. 89 0. 96 0. 03 75 * 1. 52 1. 38 0. 39 48 e le m en ts c ar bo n [m g/ g] 37 0. 7 36 9. 9 0. 54 56 38 2. 9 38 2. 6 0. 78 38 37 2. 8 37 1. 7 0. 65 34 36 8. 53 36 7. 43 0. 49 87 37 5. 5 37 4. 8 0. 29 69 m ac ro nu tr ie nt s po ta ss iu m [m g/ g] 44 .9 5 44 .7 6 0. 79 44 40 .0 2 38 .9 5 0. 27 18 47 .3 5 46 .0 5 0. 41 33 51 .5 4 53 .1 8 0. 07 11 44 .1 0 43 .2 6 0. 66 44 n itr og en [m g/ g] 20 .2 4 20 .3 6 0. 64 06 14 .6 9 14 .1 4 0. 14 97 17 .6 2 16 .7 2 0. 10 60 20 .9 3 21 .6 0 0. 18 40 17 .5 2 17 .0 7 0. 48 93 ph os ph or ou s [m g/ g] 4. 15 4. 03 0. 02 41 * 4. 29 4. 17 0. 32 20 4. 67 4. 35 0. 00 81 * * 4. 51 4. 48 0. 65 85 4. 37 4. 18 0. 00 56 * * c al ci um [m g/ g] 3. 57 3. 59 0. 72 81 3. 01 2. 86 0. 02 66 * 3. 54 3. 22 0. 09 07 2. 91 3. 07 0. 08 24 3. 37 3. 22 0. 22 59 m ag ne si um [m g/ g] 1. 30 1. 33 0. 35 91 0. 97 1. 03 0. 09 46 1. 26 1. 19 0. 21 25 1. 29 1. 35 0. 17 56 1. 20 1. 16 0. 58 56 m ic ro nu tr ie nt s ir on [μ g/ g] 90 .2 7 76 .8 5 0. 08 46 68 .5 7 70 .2 9 0. 70 39 94 .7 0 97 .0 0 0. 79 62 84 .0 2 70 .7 6 0. 28 25 84 .8 4 81 .3 8 0. 18 73 z in c [μ g/ g] 22 .6 5 22 .3 2 0. 43 37 20 .2 7 20 .1 4 0. 85 11 23 .4 3 23 .2 5 0. 67 63 22 .1 1 23 .1 1 0. 02 97 * 22 .1 2 21 .9 0 0. 71 66 m an ga ne se [μ g/ g] 11 .1 9 10 .8 7 0. 21 30 9. 45 9. 47 0. 93 89 10 .8 5 10 .5 1 0. 29 25 9. 91 8. 98 0. 13 12 10 .5 2 10 .2 8 0. 03 55 * c op pe r [ μg /g ] 4. 29 5. 39 0. 19 07 5. 83 6. 11 0. 64 88 5. 68 5. 77 0. 62 23 5. 21 5. 86 0. 05 23 5. 27 5. 76 0. 03 97 * p he no lic c om po un ds to ta l p he no ls [m g/ g] 11 .0 10 .8 0. 43 05 11 .2 11 .0 0. 08 49 11 .5 9 11 .1 6 0. 14 87 10 .1 7 10 .1 7 0. 97 50 11 .2 5 10 .9 9 0. 05 23 to ta l a nt ho cy an in s [m g/ g pe el ] 20 .3 6 21 .3 2 0. 15 70 26 .3 2 23 .9 6 0. 09 09 33 .9 9 31 .5 1 0. 10 40 29 .1 9 29 .5 3 0. 38 14 26 .8 9 25 .6 0 0. 12 17 g lu co si no la te s to ta l g lu co si no la te s [μ m ol /g ] 11 .9 7 12 .3 0 0. 79 59 28 .5 2 21 .4 9 0. 01 59 * * 26 .3 2 23 .6 7 0. 43 96 16 .0 6 16 .6 0 0. 05 28 22 .2 7 19 .1 6 0. 06 70 a lip ha tic g lu co si no la te s [ μm ol /g ] 11 .2 2 11 .5 3 0. 80 26 27 .8 8 21 .0 0 0. 01 53 * * 25 .5 5 23 .0 9 0. 46 27 15 .5 1 16 .0 1 0. 05 23 21 .5 5 18 .5 4 0. 06 91 in do lic g lu co si no la te s [μ m ol /g ] 0. 75 0. 79 0. 70 86 0. 64 0. 49 0. 20 62 0. 76 0. 58 0. 02 60 * 0. 55 0. 59 0. 24 19 0. 72 0. 62 0. 08 87 chemical composition of field grown radish 351 ta b. 3 : in flu en ce o f r ed uc ed w at er s up pl y on th e fr es h m at te r ( fm )re la te d le ve ls o f c on st itu en ts o f r ad is h tu be rs fr om d iff er en t c ul tiv at io n se ts (2 01 5, 2 01 61, 2 01 62 an d 20 17 ). l in ea r m ix ed m od el , t -t es t, pva lu es : < 0. 1, < 0 .0 5 (* ), < 0. 01 (* *) a nd < 0 .0 01 (* ** ), nd = n ot d et er m in ed . c t r : c on tr ol tr ea tm en t w ith fu ll w at er s up pl y. r w s: r ed uc ed w at er s up pl y tr ea tm en t. r ad is h 20 15 r ad is h 20 16 -1 r ad is h 20 16 -2 r ad is h 20 17 r ad is h to ta l c t r r w s pva lu e c t r r w s pva lu e c t r r w s pva lu e c t r r w s pva lu e c t r r w s pva lu e l ea f w at er p ot en tia l ψ [m pa ] nd nd n d 0. 15 0. 21 0. 01 23 * 0. 16 0. 24 0. 00 02 * ** 0. 10 0. 15 0. 00 06 * ** nd nd n d d ry m at te r [ % ] 4. 79 4. 97 0. 03 37 * 4. 64 4. 71 0. 08 86 4. 78 4. 98 0. 00 35 * * 4. 61 4. 75 0. 17 36 4. 76 4. 90 0. 00 01 * ** su ga rs a nd p ol yo ls g lu co se [g /1 00 g ] 1. 22 1. 22 0. 97 32 1. 40 1. 48 0. 01 29 * 1. 27 1. 34 0. 07 34 1. 11 1. 15 0. 43 01 1. 25 1. 30 0. 03 15 * fr uc to se [g /1 00 g ] 0. 88 0. 89 0. 86 12 0. 99 1. 03 0. 05 80 0. 74 0. 76 0. 36 32 0. 76 0. 78 0. 61 91 0. 84 0. 87 0. 10 60 po ly ol s to ta l [ m g/ 10 0 g] 9. 93 10 .8 4 0. 06 40 18 .2 6 22 .4 7 0. 00 12 * * 36 .9 3 39 .2 3 0. 04 94 * 42 .2 4 42 .7 9 0. 72 67 26 .8 4 28 .8 3 0. 00 11 * * in os ito l [ m g/ 10 0 g] 8. 39 9. 18 0. 06 81 15 .7 0 19 .4 4 0. 00 19 * * 29 .4 6 32 .0 2 0. 02 99 * 30 .1 9 30 .4 5 0. 82 05 20 .9 3 22 .7 7 0. 00 06 * ** g ly ce ro l [ m g/ 10 0 g] 1. 55 1. 65 0. 10 56 2. 56 3. 03 0. 00 17 * * 7. 47 7. 21 0. 26 60 12 .0 5 12 .3 4 0. 63 87 5. 91 6. 06 0. 34 52 o rg an ic a ci ds to ta l a ci di ty [m g/ 10 0 g] 71 .6 7 73 .3 3 0. 56 23 55 .0 0 56 .6 7 0. 79 26 56 .6 7 53 .3 3 0. 36 32 48 .3 3 48 .3 3 1. 00 00 58 .3 3 57 .5 0 0. 66 43 m al ic a ci d [m g/ 10 0 g] 17 1. 66 17 5. 00 0. 36 32 15 3. 33 15 3. 33 1. 00 00 16 3. 33 16 0. 00 0. 17 47 16 0. 00 17 0. 00 0. 20 31 16 2. 09 16 4. 58 0. 26 56 fu m ar ic a ci d [m g/ 10 0 g] 16 .2 0 16 .8 3 0. 36 15 24 .7 3 23 .8 9 0. 33 58 23 .5 9 23 .6 3 0. 94 88 10 .7 3 12 .3 0 0. 59 45 18 .8 1 19 .1 6 0. 64 67 a sc or bi c ac id [m g/ 10 0 g] 26 .9 8 27 .1 7 0. 77 96 28 .9 5 30 .9 8 0. 08 91 29 .9 4 30 .3 5 0. 60 99 24 .4 3 24 .8 6 0. 38 60 27 .5 7 28 .3 4 0. 05 17 a ni on s n itr at e [m g/ 10 0 g] 15 6. 61 16 0. 59 0. 66 72 68 .8 5 57 .1 7 0. 15 67 12 9. 17 12 4. 49 0. 62 69 80 .4 6 90 .9 4 0. 08 69 10 8. 77 10 8. 30 0. 91 65 ph os ph at e [m g/ 10 0 g] 54 .9 2 55 .2 8 0. 89 17 64 .3 4 62 .5 6 0. 53 15 51 .6 9 47 .9 2 0. 06 03 45 .1 7 47 .5 8 0. 12 62 54 .0 3 53 .3 4 0. 53 12 su lf at e [m g/ 10 0 g] 12 .0 4 13 .5 8 0. 39 44 19 .9 7 21 .3 4 0. 06 93 18 .5 5 19 .0 7 0. 56 22 15 .5 7 15 .7 3 0. 89 95 16 .5 3 17 .4 3 0. 11 53 c hl or id e [m g/ 10 0 g] 4. 23 4. 84 0. 39 83 9. 52 9. 63 0. 86 13 8. 32 5. 88 0. 14 62 4. 03 4. 53 0. 06 75 6. 53 6. 22 0. 52 44 e le m en ts c ar bo n [g /1 00 g ] 1. 77 1. 84 0. 03 92 * 1. 91 1. 95 0. 02 07 * 1. 73 1. 75 0. 19 62 1. 67 1. 77 0. 17 70 1. 77 1. 83 0. 00 46 * * m ac ro nu tr ie nt s po ta ss iu m [m g/ 10 0 g] 21 5. 16 22 2. 30 0. 01 55 * 19 9. 39 19 8. 61 0. 86 30 21 9. 88 21 6. 72 0. 67 84 23 2. 64 25 5. 56 0. 04 05 * 21 6. 77 22 3. 30 0. 07 59 n itr og en [m g/ 10 0 g] 96 .7 7 10 1. 06 0. 00 88 * * 73 .1 8 72 .1 3 0. 52 82 81 .8 5 78 .6 9 0. 25 46 94 .5 2 10 4. 01 0. 04 69 * 86 .5 8 88 .9 7 0. 13 11 ph os ph or ou s [m g/ 10 0 g] 19 .8 8 20 .0 3 0. 37 80 21 .3 7 21 .2 4 0. 80 66 21 .6 9 20 .5 1 0. 05 34 20 .2 1 21 .6 9 0. 13 23 20 .7 9 20 .8 7 0. 80 06 c al ci um [m g/ 10 0 g] 17 .0 4 17 .8 1 0. 11 88 15 .0 1 14 .5 7 0. 12 92 16 .4 2 15 .1 5 0. 16 31 13 .1 5 14 .8 0 0. 01 69 * 15 .4 1 15 .5 8 0. 59 58 m ag ne si um [m g/ 10 0 g] 6. 20 6. 59 0. 06 42 5. 14 4. 96 0. 19 69 5. 85 5. 58 0. 32 90 5. 84 6. 50 0. 03 87 * 5. 76 5. 91 0. 24 40 m ic ro nu tr ie nt s ir on [μ g/ 10 0 g] 43 3. 98 38 2. 79 0. 17 48 34 2. 23 35 8. 24 0. 45 20 45 1. 42 45 6. 74 0. 88 85 37 7. 74 34 1. 80 0. 50 64 38 4. 89 40 2. 16 0. 39 76 z in c [μ g/ 10 0 g] 10 8. 22 11 0. 80 0. 27 98 10 1. 03 10 2. 71 0. 61 31 10 8. 78 10 9. 39 0. 78 59 99 .9 9 11 1. 04 0. 01 99 * 10 4. 50 10 8. 49 0. 01 62 * m an ga ne se [μ g/ 10 0 g] 53 .5 6 54 .0 2 0. 71 45 47 .0 9 48 .2 8 0. 36 52 50 .3 7 49 .4 2 0. 43 95 44 .7 5 43 .2 6 0. 64 70 48 .9 6 48 .7 4 0. 82 79 c op pe r [ μg /1 00 g ] 20 .5 0 26 .7 3 0. 14 36 29 .0 9 31 .1 1 0. 49 15 26 .3 5 27 .1 9 0. 32 22 23 .5 4 28 .1 4 0. 01 52 * 24 .8 7 28 .2 9 0. 00 94 * * p he no lic c om po un ds to ta l p he no ls [m g/ g] 53 .3 4 54 .4 1 0. 42 28 55 .8 7 56 .6 6 0. 42 63 57 .9 3 57 .6 3 0. 82 91 46 .0 1 49 .0 8 0. 20 09 53 .2 9 54 .4 5 0. 13 64 to ta l a nt ho cy an in s [m g/ g pe el ] 12 8. 35 13 9. 14 0. 03 49 * 13 1. 17 12 2. 13 0. 15 53 15 8. 04 14 8. 32 0. 19 93 13 6. 99 13 8. 30 0. 35 81 13 8. 64 13 6. 97 0. 57 03 g lu co si no la te s to ta l g lu co si no la te s [μ m ol /g ] 57 .0 7 61 .0 4 0. 54 04 14 2. 23 10 9. 68 0. 02 53 * 12 2. 07 11 1. 04 0. 47 24 74 .1 9 76 .6 3 0. 06 35 98 .8 9 89 .6 0 0. 10 67 a lip ha tic g lu co sin ol at es [μ m ol /g ] 53 .4 8 57 .1 8 0. 55 45 13 9. 05 10 7. 19 0. 02 45 * 11 8. 53 10 8. 31 0. 49 68 72 .7 5 74 .9 6 0. 08 01 95 .9 5 86 .9 1 0. 10 76 in do lic g lu co si no la te s [μ m ol /g ] 3. 59 3. 86 0. 39 01 3. 19 2. 50 0. 24 58 3. 54 2. 72 0. 02 99 * 1. 44 1. 67 0. 06 28 2. 94 2. 69 0. 20 19 352 c. schlering, h. dietrich, m. frisch, m. schreiner, r. schweiggert, f. will, j. zinkernagel nidin-3-o-( p-coumaroyl-)-diglucoside-5-o-(malonyl-)-glucoside and pelargonidin-3-o-(feruloyl)-diglucoside-5-o-(malonyl)-gluco side, accounting for approx. 80% of the total anthocyanins (wu and prior, 2005) (data not shown). limited water supply led to only insignificant effects on total anthocyanins in radish. univariate analysis indicated a dry matter-related decline of total anthocyanins in radish skin upon limited water supply in 2016 (tab. 2), whereas a fresh matter-related increase was found in 2015. in agreement, multivariate analyses did not indicate a strong contribution of anthocyanins to the differentiation of samples grown under full or reduced water supply (fig. 2a-b). interestingly, both fresh and dry matterrelated contents of anthocyanins were highest in 2016-2 followed by 2017 (tab. 2 and 3). in the context of seasonal effects on radish tuber color, schreiner et al. (2002) found most intense red coloration at high photosynthetic photon flux density, hypothesizing its production to be dependent on light intensity, quality and duration (mazza and miniati, 1993). in addition, an earlier study revealed that radish gained more distinctive red shades at lower mean temperatures (schreiner et al., 2002). in our study, lowest mean temperature and highest global radiation occurred in 2017, which also showed high amounts of anthocyanins apart from the set 2016-2 (tab. 2 and 3). in brief, mild water supply reduction did not significantly affect anthocyanin contents, representing a most important commercial quality trait due to its direct correlation with the tubers’ color. glucosinolates reduced water supply led to decreased dry matter-related contents of total, aliphatic and indolyl glucosinolates. most abundant glucosinolates in radish were aliphatic derivatives such as glucoraphasatin (4-methylthio-3-butenyl glucosinolate) and glucoraphenin (4-methylsulfinyl-3-butenyl glucosinolate). in earlier studies, drought stress has been shown to more likely increase glucosinolate concentration in brassica species such as brassica carinata (schreiner et al., 2009), b. rapa ssp. rapifera l. (zhang et al., 2008) and b. napus l. (jensen et al., 1996). however, the intensity of drought appears to be a decisive impact factor in glucosinolate increase, because, under minor drought, glucosinolate concentrations did not increase in brassica napus plants (jensen et al., 1996) and decreased in brassica oleracea var. italica. (robbins et al., 2005) thus, robbins et al. (2005) also observed decreasing concentrations of mainly indolyl-glucosinolates in broccoli (b. oleracea var. italica) induced by a slightly reduced water supply. our results showed that total glucosinolates concentration was highest in 2016 (21.49-28.52 μmol/g dm) as compared to the other years (11.97-16.60 μmol/g dm). in 2016, precipitation was lowest and, thus, irrigation events were frequently released, but total water amounts were considerately lower compared to 2015 and 2017. zhang et al. (2008) also proved significant interactions of growing season and water supply for turnip, which underlines the seasonal-dependent effects on glucosinolates. conclusion in conclusion, slightly reduced water supply situations, as expected in the future due to climate change, were shown to alter the chemical composition of red radish grown in open field cultivation. the effects expectedly appeared to vary in dependence of the respective environmental conditions. dry matter contents were mainly enhanced by moderate water reduction and the same was found for polyols, which seem to be highly sensitive to slight water lowering. being important for color and pungency of the produce, anthocyanin and glucosinolate levels were shown to be only marginally affected. in brief, our results indicate that red radish plants are able to cope with moderately reduced water supply, only marginally affecting the overall quality of its commercialized tubers. however, limitations of water supply in areas of vegetable production may increase by the on-going climate change and thus exceed those exposed in our simulation, ultimately aggravating the effects observed in our study and thus still impacting crop quality. funding sources the loewe excellence cluster face2face from the hessian state ministry of higher education, research and the arts (wiesbaden, germany) funded this study. supplementary data information on the daily mean weather conditions during the different cultivation periods 2015, 2016-1, 2016-2 and 2017 is supplied in fig a.1-4 as supporting information. references afzal, a., gulzar, i., shahbaz, m., ashraf, m., 2014: water deficit induced regulation of growth, gas exchange, chlorophyll fluorescence, inorganic nutrient accumulation and antioxidative defense mechanism in mungbean (vigna radiata (l.) wilczek). j. appl. bot. food qual. 87, 147156. doi: 10.5073/jabfq.2014.087.022 aires, a., fernandes, c., carvalho, r., bennett, r.n., saavedra, m.j., rosa, e.a.s., 2011: seasonal effects on bioactive compounds and anti oxidant capacity of six economically important brassica vegetables. molecules 16, 6816-6832. doi: 10.3390/molecules16086816 akinci, s., dorothy, l.m., 2012: plant water-stress response mechanisms. in: rahman, i.m.m., hasegawa, h. 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will https://orcid.org/0000-0003-0369-3087 jana zinkernagel https://orcid.org/0000-0001-6817-091x address of the corresponding author: christine schlering, department of soil science and plant nutrition, depart ment of microbiology and biochemistry, geisenheim university, germany e-mail: christine.schlering@hs-gm.de © the author(s) 2019. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1016/j.envexpbot.2004.11.004 http://dx.doi.org/10.1002/jpln.200700079 supplementary material i supplementary material fig. a.1-4: daily mean weather conditions during the different cultivation periods 2015, 2016-1, 2016-2 and 2017: (1) mean air temperature [°c], (2) global radiation sum [w/m²], (3) daily mean relative humidity [%] and (4) daily mean evapotranspiration [mm]; das = days after sowing. 1 art06 journal of applied botany and food quality 80, 40 47 (2006) 1departamento de ciencias químicas, universidad de la frontera, temuco, chile 2instituto de botánica, universidad austral de chile, valdivia, chile 3institute of crop production and agroecology in the tropics and subtropics, university of hohenheim, stuttgart, germany diversity of mycorrhizal plant species and arbuscular mycorrhizal fungi in evergreen forest, deciduous forest and grassland ecosystems of southern chile c.g. castillo1, f. borie1, r. godoy2, r. rubio1, e. sieverding3 (received january 9, 2006) summary in the valdivian rainforest region of the southern chilean andes three main ecosystems are found: primary evergreen forests, secondary deciduous forests, and grassland areas. the secondary forest and the grasslands are habitually the result of the clearance of the primary forest some 60 years ago. the secondary forest consists mainly of the deciduous tree species nothofagus alpina; forest management practices such as crown thinning and clearance of the understorey are applied to the secondary forest to improve its economic value. the grasslands are used by extensive cattle grazing. soils in this region are acid andosols with high organic matter content, high exchangeable aluminum and low levels of available phosphorus. the main objective of this study was to investigate the diversity of arbuscular mycorrhizal (am) plant species and of arbuscular mycorrhizal fungi (amf) in these three ecosystems. the highest diversity with 53 plant species was found in the evergreen forest with 77.4% of them am, while in the grassland 91% of the 22 plant species were am. the deciduous forest had 11 plant species only and the lowest proportion of am plant species (55%). thirty-nine am fungal species were found in total, of which most are being reported for the first time from southern chile. thirteen fungal species were of the acaulospora genus, 10 of glomus, 4 species each of scutellospora and archaeospora, 3 species each of pacispora and entrophospora, and one species each of paraglomus and diversispora. amf species were more abundant in the grassland (29 spp.) than in the evergreen forest (20 spp.) which is likely related to a higher relative proportion of am plant species in the grassland. four amf species were present in all the ecosystems, and 15 species were apparently quite specific as they were only found in one of the ecosystems. noteworthy was the lack of paraglomus and scutellospora spp. in any of the forest ecosystems, and the relatively higher presence of entrophospora spores in those ecosystems. it was concluded that the diversity of the amf species in the ecosystems is strongly influenced by the proportion of am plant species in each ecosystem and that their diversity is not related to soil chemical properties. introduction in the area between 35ºs and 55ºs latitude in southern chile about 4,1 million ha are covered with a primary rain forest which is evergreen, and 1,5 million ha with a secondary forest which consist mainly of the deciduous tree nothofagus alpina. this secondary forest and large grassland areas are the result of clearance and burning of the primary forest, some 60 years ago (conaf et al., 1997). the secondary forest has multiple uses being the most important firewood production; it represents an important source of income for forest owners. the grassland areas are more or less intensively used for cattle production. due to the economic, social, cultural and environmental interest in the evergreen forest, appropriate and sustainable forest management systems are now being developed which try to avoid the destruction or damage of its ecological components and which aim at conserving a biological diversity as high as possible (lara et al., 2003). the soil on which the evergreen forest grows has low ph with the associated, often high and toxic soluble aluminum concentrations and the very low phosphate availability (álvarez et al., 2005). in this environment, mycorrhizal fungi are of primary importance for many if not the majority of plant species, and for the functioning of ecosystems (barea et al., 1997; smith and read, 1997; van der heijden, 2002). there are two main types of mycorrhizas: ectomycorrhiza (ec) which is formed by many forest tree species with a number of fungal species of the basidiomycetes and ascomycetes classes of fungi, and endomycorrhizas. ec is frequently found in forest plant species of the temperate zones, and pinaceae and fagaceae form this kind of symbiosis also in southern chile (garrido, 1988; palfner, 2001). of the endomycorrhizas, the arbuscular mycorrhiza (am) type is by far the most important worldwide because it occurs with more than 60% of the species of the plant kingdom (trappe, 1987). am is commonly found in many, but not all forest species and in most herbaceous species, shrubs, ferns and grasses. the fungal species involved in the formation of am belong to the glomeromycota (schüssler et al., 2001). some plant families, like ericaceae or orchideaceae form very specific mycorrhizas (smith and read, 1997) without which the plants cannot survive under natural conditions. the main purpose of this study was to investigate the occurrence of am plant species and arbuscular mycorrhizal fungal (amf) species in the evergreen forest ecosystem of southern chile and the two ecosystems which developed from it when it was destroyed. this should give information in which way the diversity of mycorrhizal plants and amf species is affected in a long term after slashing and burning the primary forest. such information may have value for the future in conserving the diversity of a native soil microbiological resource like amf species. materials and methods study area the study was carried out at the experimental station san pablo de tregua of the university austral de chile, valdivia. it is located in the panguipulli region, province of valdivia in the andean mountains (39º30’-39º38’s, 72º02’-72º09’w) at an altitude between 550 and 1600 m above sea level (a.s.l.). the locality has an approximate surface of 2180 ha. almost 90 % of the site is covered by undisturbed evergreen primary forest with an excellent state of conservation. the primary forest is representative for southern chile; it belongs to the evergreen nothofagus dombeyi – laureliopsis philippiana subtype and is in an advanced state of maturity. shadow tolerant tree species such as saxegothaea conspicua and dasyphyllum diacanthoides have begun to displace those plant species which cannot regenerate under shade, such as n. alpina. the soil at the site is an andosol. it derives from recent volcanic ashes and belongs to the liquiñe soil series. the soil is a loamy sand, acidic to moderately acidic, deeply to moderately deeply developed (0.7 -1.5 m) with good water infiltration capacity, good drainage, high water holding capacity and with high contents of organic matter (15 -25%) in the upper part (lara et al., 2002). the land is mountainous, with a complex topography and few plane land surfaces. fifty % of the forestland is made up of hills with a relatively low inclination (0 -10º); more than 10% of the forestland is situated on hills with an inclination of more than 25º. the climate at the location is temperate with a mean annual temperature of 11º c, with a minimum of 5º c in august and a maximum of 20º c in february. the number of days with frost during the year ranges from 30 to 50. the annual rainfalls range between 4000 to 5000 mm, with 50% of the rain between may and august while the summers are short and dry with up to 200 mm per month. most of the precipitation falls as snow, above 1000 m a.s.l. ecosystems three ecosystems dominate the area and were selected for the study: evergreen forest ecosystem: this is the natural evergreen forest with the dominant trees having an estimated age of more than 200 years. the basal trunk area of the mature tree vegetation was 119 m2 per ha in 2002, distributed over about 40-100 trunks per ha. the forest is dominated by n. dombeyi, l. philippiana and s. conspicua trees. the canopy is not completely closed. the degree of disturbance is low. the understorey has a rich community of epiphytes and climber plants. deciduous forest ecosystem: a small sector of the area, about 10% of the experimental location, has a secondary forest dominated by the deciduous tree species n. alpina which represents more than 90% of the tree trunks in number per ha. there are a few companion plant species such as n. obliqua, n. dombeyi, weinmannia trichosperma, l. philippiana, s. conspicua and eucryphia cordifolia. the estimated age of the trees is 50 years, and the basal trunk area was 30 m2 per ha distributed over about 500 to 1000 trunks per ha. this basal area was obtained after a silvicultural management practice in 2002, which consisted of crown thinning and the reduction of the original basal trunk area per ha by 40%. grassland ecosystem: the same location also contains small patches with natural grasslands between the above-mentioned forest ecosystems. the grassland ecosystem has a high diversity of grasses and herbs. it is extensively used by cattle grazing. root and soil sampling root and soil samples were collected at the end of autumn, in may 2003. plant species were identified at the same time. the nomenclature of the plant species follows that of marticorena and quezada (1985). to establish the presence of the mycorrhiza type of the represented species in the vascular flora, root material from 3 individual specimen of each species was collected at each study site. root samples were obtained at a soil depth down to 15 to 30 cm for the forest and bushy species. fine roots were excavated starting from the trunk and working out towards the fine roots. roots were collected from young and mature trees. roots of the climbers, ferns, grasses and herbaceous species were collected at a soil depth from 0 to 20 cm. soil sampling for soil analyses and the first set of amf spore separation and species identification was done in 5 plots of each ecosystem, in may 2003. plots had been randomly established within each ecosystem area. the plots had been selected for nutrient recycling and for hydrological studies of the institute of botany of the universidad austral, valdivia. within each plot of 100 m2 surface area, 15 samples were taken using a 20-cm-long tube sampler, and crossing the plot in a diagonal way. at the forest sites, the litter layer was cleared prior to soil sampling. samples from each plot were bulked to give five replicates per ecosystem. soil samples were brought to the lab and stored in a freezer until analyses. a second set of soil samples for the separation of spores and the identification of amf species was taken in the ecosystems, in november 2004. soil analysis methods the key parameters of the chemical soil fertility in each ecosystem were determined: soil ph was measured by glass electrode in a 1:2.5 soil:water suspension. exchangeable aluminum (al) and iron (fe) were analysed by aas after previous extraction with dtpa at ph 7.3 (sadzawka et al., 2000). available phosphorus (p) was determined after extraction with a solution of 0.5 m nahco3 at ph 8.5 (olsen and sommers, 1982). total p was determined according to dick and tabatabai (1977) and soil organic matter (om) was analyzed using the dichromate oxidation method (walkley and black, 1934). analyses of mycorrhizas roots were cleared and fungal structures were stained following the methods described by brundrett et al. (1996). mycorrhizal structures were observed in a microscope at 50x magnification. spores of amf were extracted and separated from the soil using the wet-sieving and decanting procedure described by sieverding (1991). spores were transferred to petri-dishes and those of the first sampling date (may 2003) were counted. spores were isolated under a stereomicroscope and were fixed in polyvinyl alcohol-lactic acidglycerol (pvlg) (koske and tessier, 1983), and a mixture of pvlg and melzer’s reagent (brundrett et al., 1996) to obtain permanent specimen. for the taxonomic classification main morphological spore characteristics such as color, diameter, type and number of spore walls, and the morphology of the subtending hypha at the point of spore attachment were observed under a high-power light microscope at 100x and 400x magnification. for the species identification the instructions given by schenck and perez (1990) tab. 1: characteristics of soils in the evergreen forest (ef), deciduous forest (df) and grassland (gr) ecosystemsa ecosystem ph om (%) al (mg kg-1) fe (mg kg-1) p (mg kg-1) available total ef 4.56 b 21.85 ab 321 a 66.4 a 6.11 a 1763 b df 5.40 a 24.92 a 376 a 35.8 b 3.64 b 1487 c gr 5.37 a 15.31 b 341 a 24.1 b 2.93 b 2095 a a values are averages of five replicates per ecosystem. treatment means followed by the same letter are not significantly different (p<0.05). mycorrhiza diversity in chilean forest ecosystems 41 and invam (international culture collection of arbuscular and vesicular-arbuscular endomycorrhizal fungi, see internet homepage: www.invam.caf.wvu.edu) or in species descriptions were followed. diversispora (walker and schüssler, 2004) and pacispora (oehl and sieverding, 2004) were identified using descriptions of spores of the species. all isolated specimen were deposited at the laboratory of plant nutrition of the universidad la frontera, temuco, chile. results soil fertility the ph was not different in soils under grassland and deciduous forest, whereas the soil under evergreen forest was significantly more acidic compared to the grassland (tab. 1). with regard to the organic matter content, the soil under grassland had the lowest content. the grassland also showed the lowest available p content whereas the evergreen forest soil had the highest. in contrast, the total p content was highest in soil under grassland as compared to both forest systems. exchangeable aluminum was not different in all the sites studied but fe was much higher in the evergreen forest than in the other two ecosystems. distribution of plant species and mycorrhizas the evergreen forest had the most diverse botanical composition with 53 plant species (tab. 2) of which 77.4% formed am, 17% were non-mycorrhizal and only 5.6% formed ec including one species which was associated with ericoid mycorrhiza. of the 18 tree species 12 were associated with am, the 5 species of the proteaceae were non-mycorrhizal. nothofagus dombeyi of which some individuals had developed a large canopy forms ec. the understorey was not dense but the stability of the ecosystem permitted the presence of a rich community of 14 shrub species of which 13 were am. the shrub of the ericaceae family formed an ericoid mycorrhiza. the 6 climbers were all am and only the epiphytic fascicularia bicolor was non-mycorrhizal. of the 8 ferns the asplenium sp. and the hymenophyllaceae were non-mycorrhizal, the others formed am. of the 6 herbaceous species the loasa sp. was non-mycorrhizal. the deciduous forest was dominated by n. alpina which is ec as was the other member of the fagaceae. with the exception of the proteaceae tree species, the other trees were am. an understorey was almost absent. two fern species were present, one of them am, and the bromeliaceous species greigea landbecki formed am. in the grassland 22 herbaceous and grass species were found, 20 of them were am. the species of the juncaceae and polygonaceae were non-mycorrhizal. tab. 2: list of vascular plants and their mycorrhizal status in chilean evergreen forest (ef), deciduous forest (df) and grassland (gr) ecosystems. plant type scientific name family ecosystema ef df gr trees aextoxicom punctatum r. et p. aextoxicaceae am amomyrtus luma (mol.) legr. et kause myrtaceae am amomyrtus meli (phil.) legr. et kausel myrtaceae am dasyphyllum diacanthoides (less.) cabr. asteraceae am drimys winteri j.r. et g. forster winteraceae am embothrium coccineum j.r. et g. forster proteaceae n eucryphia cordifolia cav. eucryphiaceae am am gevuina avellana mol. proteaceae n laureliopsis philippiana (looser) schodde monimiaceae am am lomatia dentata (r. et p.) r. br. proteaceae n lomatia ferruginea (cav.) r. br. proteaceae n n lomatia hirsuta (lam.) diels ex macbr. proteaceae n luma apiculata (dc.) burret myrtaceae am maytenus magellanica (lam.) hook. f. celastraceae am myrceugenia planipes (h. et a.) berg myrtaceae am nothofagus alpina (p. et e.) oerst fagaceae ec ec nothofagus dombeyi (mirb.) oerst fagaceae ec ec nothofagus obliqua (mirb.) oerst fagaceae ec saxegothaea conspicua lindl. podocarpaceae am am weinmannia trichosperma cav. cunoniaceae am am shrubs aristotelia chilensis (mol.) stuntz elaeocarpaceae am azara lanceolata hook. f. flacourtiaceae am berberis buxifolia lam. berberidaceae am berberis linearifolia phil. berberidaceae am chusquea culeou desv. gramineae am desfontainia spinosa r. et p. desfontainiaceae am drimys andina (reiche) r.a. rodr. et quez. winteraceae am fuchsia magellanica lam. onagraceae am gaultheria phyllireifolia (pers.) sleumer ericaceae er griselinia ruscifolia (clos.) taub cornaceae am myoschilos oblonga r. et p. santalaceae am myrceugenia parvifolia (dc.) kausel myrtaceae am ovidia pillo-pillo (gay) meisn. thymeleaceae am ribes magellanicum poir. saxifragaceae am 42 c.g. castillo, f. borie, r. godoy, r. rubio, e. sieverding amf species and distribution in ecosystems in total, 39 amf species could be distinguished on the basis of morphological criteria (tab. 3) from the sampling in autumn; the additional sampling in late spring did not result in more amf species but some of the species could only be identified after having obtained additional specimen from the second sampling. thirty four species were identified unequivocally according to descriptions in the literature. short descriptions of the 5 unknown amf species are given in tab. 4. the major number of species of amf was observed in the grassland (29), followed by the evergreen forest (20), and the deciduous forest with only 14 species. in the three ecosystems studied, 13 acaulospora spp., 4 archaeospora spp., one diversispora sp., 3 entrophospora spp., 10 glomus spp., 3 pacispora spp., one paraglomus sp., and 4 scutellospora spp. were isolated. no gigaspora sp. was observed in any sampling. in the total study area a third of the amf species belonged to the genus acaulospora, and a quarter to the genus glomus, the rest being of other genera. while this relative distribution in number of species in these two genera was more or less the same in the evergreen forest tab. 2 (continued) plant type scientific name family ecosystema ef df gr climbers asteranthera ovata (cav.) hanst. gesneriaceae am and campsidium valdivianum (phil.) skottsb. bignoniaceae am epiphytics fascicularia bicolor (n. et z.) bromeliaceae n hydrangea integerrima engl. hydrangeaceae am hydrangea serratifolia (h. et a.) f. phil. hydrangeaceae am luzuriaga radicans (r. et p.) philesiaceae am mitraria coccinea cav. gesneriaceae am ferns asplenium dareoides a.n. desv. aspleniaceae n n blechnum hastatum kaulf. blechnaceae am blechnum blechnoides (keyserl.) blechnaceae am am blechnum chilense (kaulf.) mett. blechnaceae am hymenophyllum pectinatum cav. hymenophyllaceae n hymenophyllum sp. hymenophyllaceae n hypolepis poeppigii (kunze) r.a. rodr. dennstaedtiaceae am lophosoria quadripinnata (j.f. gmel.) c. chr. lophosoriaceae am herbs acaena ovalifolia r. et p. rosaceae am achillea millefolium l. asteraceae am agrostis capillaris l. poaceae am dichondra sericea sw. convolvulaceae am dysopsis glechomoides (rich.) muell. arg euphorbiaceae am fragaria chiloensis (l.) duch. rosaceae am greigea landbeckii (lechlerex phil.) phil. ex f. phil. bromeliaceae am am holcus lanatus l. poaceae am hypericum perforatum l. clusiaceae am hypochaeris radicata l. asteraceae am juncus procerus e. meyer juncaceae n loasa sclareifolia juss. loasaceae n lotus uliginosus schkuhr. fabaceae am medicago sp. fabaceae am mentha piperita l. labiatae am mentha spicata l. labiatae am nertera granadensis (mutis ex r.f) druce rubiaceae am osmorrhisa chilensis h. et a. apiaceae am plantago lanceolata l. plantaginaceae am poa annua l. poaceae am prunella vulgaris l. lamiaceae am ranunculus minutiflorus bert. ex phil. ranunculaceae am rumex acetosella l. polygonaceae n senecio vulgaris l. asteraceae am solanum sp. solanaceae am taraxacum officinale weber asteraceae am trifolium pratense l. fabaceae am trifolium repens l. fabaceae am nr. total 53 11 22 nr. species with arbuscular mycorrhiza (am) 41 6 20 nr. species without mycorrhizas (n) 9 2 2 nr. species with ec and er mycorrhizas 3 3 0 aam: arbuscular mycorrhizal; n: non-mycorrhizal; ec: ecto-mycorrhizal; er: ericoid mycorrhizal; (-) species absent mycorrhiza diversity in chilean forest ecosystems 43 and the deciduous forest, there was a strong deviation in the grassland ecosystem where more than 40% of the species belonged to acaulospora and only 21% to glomus. species of paraglomus and scutellospora were not present in any of the forest ecosystems. four amf species were found in all the three ecosystems: acaulospora alpina, a. mellea, g. etunicatum and g. macrocarpum. many of the other species appeared to be highly specialized, hence occurring in only one of the three ecosystems. the total number of amf spores was highest in the evergreen forest with 3164 spores per 100 g dry soil, significantly lower in the grassland with 1001 spores and significantly lower still in the deciduous forest with 456 spores. counting the spores of each individual amf species was laborous because some species have very similar spore morphology, making difficult their differentiation under a stereo-microscope at low magnification. therefore, for comparing the relative occurrence of spores of the amf genera in the three ecosystems we pooled all spore counts of the species of each genus (fig. 1). while in the evergreen forest the number of acaulospora and glomus spores were each about one-third of the total spore number, the relative number of acaulospora spores decreased in the deciduous forest and increased in the grassland ecosystem. by contrast, the relative numbers of glomus spores increased in the deciduous forest and decreased in the grassland. scutellospora spores were only present in the grassland. noteworthy was also the relatively higher spore number of entrophospora in the two forest ecosystems than in the grassland. tab. 3: genera and species of the glomeromycota found in the evergreen forest (ef), deciduous forest (df) and grassland (gr) ecosystems in southern chile. genus species name ecosystema ef df gr acaulospora acaulospora alpina oehl, sykorova & sieverd. + + + acaulospora cavernata blaszk. + acaulospora colossica p.a. schultz, bever & j.b. morton + + acaulospora dilatata j.b. morton + + acaulospora koskei blaszk. + + acaulospora laevis gerd.&trappe + + acaulospora longula spain & n.c.schenck + + acaulospora mellea spain & n.c. schenck + + + acaulospora paulinae blaszk. + acaulospora scrobiculata trappe + acaulospora spinosa c. walker & trappe + acaulospora thomii blaszk. + acaulospora sp. a + + archaeospora archaeospora leptoticha (n.c. schenck & g.s. sm.) j.b. morton & d. redecker + archaeospora trappei (r.n. ames&linderman) j.b. morton & d. redecker emend spain + + archaeospora sp. a + + archaeospora sp. b + + diversispora diversispora spurca (c.m. pfeiff., c. walker & bloss) c. walker & a. schüssler + + entrophospora entrophospora baltica blasz. + entrophospora infrequens (i.r. hall) r.n. ames & r.w. schneid. + + entrophospora schenckii sieverd. & s. toro + + glomus glomus brohultii r.a. herrera, ferrer & sieverd. + glomus claroideum n.c. schenck & g.s. sm. emend c. walker & vestberg + + glomus diaphanum j. b. morton & c. walker + glomus etunicatum w.n becker & gerd. + + + glomus fasciculatum (thaxt.) gerd. & trappe emend. c. walker & koske + glomus geosporum (t.h. nicolson & gerd.) c. walker + + glomus invermaium i.r. hall + glomus laccatum blaszk. + glomus macrocarpum tul.& c. tul. + + + glomus rubiforme (gerd. & trappe) r.t. almeida & n.c. schenck + + pacispora pacispora dominikii (blaszk.) oehl & sieverd. + pacispora sp. a + pacispora sp. b + + paraglomus paraglomus occultum (c. walker) j.b. morton & d. redecker + scutellospora scutellospora auriglobosa (i.r. hall) c. walker & f.e. sanders emend. c. walker & i.r. hall + scutellospora calospora (t.h. nicolson & gerd.) c. walker & f.e. sanders + scutellospora dipurpurascens j.b. morton & koske + scutellospora weresubiae koske & c. walker + nr. of species 20 14 29 a (-): absence or (+): presence of arbuscular mycorrhizal fungal species. 44 c.g. castillo, f. borie, r. godoy, r. rubio, e. sieverding discussion it was not surprising that the evergreen forest had a higher plant species diversity than the grassland vegetation and the secondary deciduous forest (tab. 2). the main timber producing trees of both the forest ecosystems formed ectomycorrhiza. trees of the fagaceae are known ec species also in the temperate zones (smith and read, 1997). myrtaceae can form dual associations of ec and am (http:/ /www.ffp.csiro.au/research/mycorrhiza/ozplants.html, 2005); the two myrtaceae amomythus and myrceugenia were am in our study. in both the forest ecosystems the relative number of non-mycorrhizal tree species was about the same. the non-mycorrhizal proteaceae were only found in the evergreen forest and not in the deciduous forest, however. the understorey vegetation in the evergreen forest was almost completely am with the exception of an epiphyte and 3 ferns which were non-mycorrhizal. the understorey vegetation was poor in the deciduous forest. the natural grassland had a diverse plant species community which was somewhat lower in number of plant species than that of temperate natural grasslands in regions of europe and north america where similar studies on the mycorrhizal status were performed earlier (e.g. read and haselwandter, 1981; miller, 1987). of the two nonmycorrhizal plant species found was rumex acetocella (polygonaceae) reported earlier to be non-mycorrhizal (harley and harley, 1987) although other rumex spp. can be am. juncus procerus was non-mycorrhizal; other juncaceae can form am (silva et al., 2001). even though ectomycorrhizal tree species represented the main trunk area per ha in both the forest ecosystems, a surprisingly high number of am plant species could survive even in view that there must have been competition between ec and am in the soil. strong dominance of ectomycorrhizal hyphal networks in the soil can be detrimental for the establishment of am understorey species (booth, 2004) which may be applicable also for the chilean deciduous forest with a higher number of trunks of ec trees per ha. in the chilean evergreen forest, however, many of the am understorey species grow directly in the crown area of the dominant ectomycorrhizal n. dombeyi trees. this may indicate that in the evergreen forest with a relative lower number of stems of ec trees per ha, the competition between the two mycorrhizal types was low. the deciduous forest had a significantly lower amf spore concentration and lower diversity of amf species (tab. 3) than the evergreen forest or the grassland. it is not likely that the slight differences in soil chemical properties between the two forest systems, such as increased organic matter in the deciduous forest as compared to the evergreen forest, have much to do with the ecosystem differences in the amf communities. this is because the soil characteristics (tab. 1) of the deciduous forest were very similar to those of the grassland ecosystem which had a high amf diversity. hence, the reason for the differences in the amf populations must be related to the am plant species community which was proportionally highest in the grassland followed by the evergreen forest and finally the deciduous forest, respectively. we relate the low number of am plant species and amf species in the deciduous forest to the dominating presence (in terms of stem number per ha) and competition of ec tree species. thirty-nine species of the glomeromycota were found at the research location, 13% of them had not been previously described to our knowledge. this is not a surprisingly high number of unknown species, given that this was the first survey of amf species in southern tab. 4: morphological characteristics of spores of non-identified arbuscular mycorrhizal fungal species in southern chilean forest ecosystems species characteristics of spores acaulospora sp. a yellow to light brown, globose spores, 100-125 µm in diam., with 2-2.5 µm thick yellow outer wall which is ornamented with pits, 1-2 µm deep and 2-5 µm wide. archaeospora sp. a subhyaline to dull cream yellow, globose to subglobose spores, 100-120 x 150-160 µm in diam., with a 1-3 µm thick outer wall which is ornamented with a pentagonal to hexagonal reticulum, openings 5-10 x 10-20 to 27-37 x 25-50 µm wide, and ridges about 2-3 µm wide and 2-3 µm high; germinal wall 3-layered and 1-2.5 µm thick. archaeospora sp. b dull white globose spores, 130-180 µm in diam., with a up to 5 µm thick outer wall which is ornamented with tiny thin spines. the next (germinal) wall is ornamented with a crenulate (pitted wave-like) structure and is 3-6 µm thick. pacispora sp. a light yellow to orange yellow, globose to subglobose spores, 100-125 µm in diam. with a 3-layered outer thin wall and a 3-layered inner wall. outer layer ornamented with edged warts 5-10 µm broad at the bases, and up to 7.5 µm high and formed in a distance of 5-7 µm to each other. pacispora sp. b yellow to yellow orange subglobose tear like spores, 87-105 x 125-150 µm in diam. with a 3-layered outer wall and a 3-layered inner wall. the outer wall is hyaline to white and 4-5 µm thick in total. the inner wall is light yellow and has a reticulate ornamentation on the outer layer; the openings of the reticulum are 5-7.5 µm wide and 2.5 µm deep. fig. 1: relative number of spores belonging to the different arbuscular mycorrhizal fungal (amf) genera, in the evergreen forest ecosystem (ef), deciduous forest ecosystem (df) and grassland ecosystem (gr). total spore number in each ecosystem = 100%. fungal genera are: sc: scutellospora; pr: paraglomus; pa: pacispora; gl: glomus; en: entrophospora; ar: archaeospora; ac: acaulospora; di: diversispora. mycorrhiza diversity in chilean forest ecosystems 45 chile and given that so far only about 170 amf species have been described worldwide in the phylum glomeromycota (invam; see: www.invam.caf.wvu.edu). oehl et al. (2003) detected a total of 45 amf species in 8 field sites in agro-ecosystems in central europe, of which about 20% were not previously described. the relative number of tentatively new species in our study was thus about the same. we identified the amf species on the basis of their spore morphology and did not apply molecular biological tools to detect them in the roots. the identification method on the basis of spore morphology is appropriate for a survey of amf in a region, as there is no reason to believe that molecular biological tools are more efficient in detecting amf species in field soils. both the methods have disadvantages as explained by sanders (2004). mycorrhizal species may seasonally sporulate (pringle and bever, 2002). we believe that we discovered most of the amf species present in the ecosystems at the study location because the times of sampling corresponded to the end of the growing season (autumn) and to a warm season at the end of the spring. in both the forest ecosystems acaulospora spp., glomus spp. and the rest represented each about one third of the relative species number. acaulospora spp. dominated in the grassland ecosystem in southern chile which is in contrast to european grasslands where glomus spp. clearly represent the majority of the amf community (blaszkowski, 1993; oehl et al., 2005). the relative proportion of spores of entrophospora spp. was higher in the forest ecosystems than in the grassland, and there is another study (johnson and wedin, 1997) reporting that entrophospora spp. were only formed in a native tropical forest ecosystem and not in grasslands. to our knowledge no reliable information is available about the occurrence of amf species in deciduous forests, anyway. only 4 of the 39 amf species were found in all the three ecosystems, and thus can be considered able and fit to survive and form spores under very diverse conditions. these 4 fungal species must be considered ecological generalists. glomus etunicatum is known as such a generalist from european studies (blaszkowski, 1993; oehl et al., 2004) and g. macrocarpum is frequent in european grasslands and elsewhere in the world. the other two generalists were acaulospora spp. which is not surprising as species of this genus occur frequently in acidic tropical soils (sieverding, 1989). acaulospora alpina is known to occur at high mountainous regions of the swiss alps (oehl et al., 2006). the number of species found in only one of the three ecosystems investigated was relatively high with 19 amf species. also other studies from european grasslands and agroecosystems (oehl et al., 2004; 2005) and from tropical ecosystems (johnson and wedin, 1997) report that some fungal species occur only under specific ecological conditions. as yet it is not possible to relate their occurrence to specific ecological factors. however, it is an interesting finding of our study that paraglomus and all scutellospora spp. were only isolated from the grasslands. these grassland plots were in the neighborhood of the forests so that a wider distribution could have been expected. paraglomus occultum and scutellospora spp. were often reported from grassland savanna areas with acidic soils from colombia and venezuela (sieverding and toro, 1986; herrerapedraza et al., 2001). we assume that paraglomus and scutellospora spp. prefer open, un-shaded areas like grasslands. it is as yet unknown what function the different amf species play in each of the investigated ecosystems. it is assumed that a high diversity of amf species is likely to be more beneficial for a plant community than a low number (van der heijden et al., 1998). whether or not the low plant species diversity and low number of am plant species in the deciduous forest ecosystem was related to the relatively low fungal species diversity remains unknown. it is known, however, that a diverse fungal population is very helpful in the establishment of seedlings of am forest species (landim, 2003). we can conclude from this study that the only way to maintain a high plant species diversity with its associated am plant species and amf community is by conserving the native rainforest ecosystem. grasslands with a broad plant species community appear to be good alternatives to the native evergreen rainforests in conserving a high biodiversity of amf but it is clear that this is at the expense of the forest vegetation diversity. the deciduous secondary forest with an almost mono-specific n. alpina cover appears to be economically attractive in the first view but results in an ecological degraded ecosystem with low biodiversity, including low diversity of amf species. forest management practices should aim to increase the diversity of tree species of the secondary forest with the aim also to increase the soil microbiological resources like amf communities. acknowledgements this research was supported by grants c-13755-28 and fondecyt 1020306, provided by the fundación andes and the comisión nacional científica y tecnológica, chile, respectively. this paper is a contribution to forecos po4-065-f. we are grateful for help in field sampling by cefor-uach. references álvarez, e., fernández-marcos, m.l., monterroso, c., fernándezsanjurjo, m.j., 2005: application of aluminium toxicity indices to soils under various forest species. forest ecol. manage. 211, 227-239. barea, j.m., azcón-aguilar, c., azcón, r., 1997: interactions between mycorrhizal fungi and rhizosphere microorganisms within the context of sustainable soil-plant systems. in: gange, a.c., brown, v.k. 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(eds.), mycorrhizal ecology, 243-265. ecological studies, vol. 157, springer, berlin. walkley, a., black, i.a., 1934: an examination of the degtjareff method for determining soil organic matter and the proposed modification of the chromic acid titration method. soil sci. 37, 29-38. walker, c., schüssler, a., 2004: nomenclature clarifications and new taxa in the glomeromycota. mycol. res. 108, 1105-1106. address of authors: dr. c. g. castillo, r. rubio, prof. dr. f. borie, departamento ciencias químicas, facultad de ingeniería y ciencias, universidad de la frontera, temuco, chile. prof. dr. r. godoy , instituto de botánica, universidad austral, valdivia, chile. priv. doz. dr. e. sieverding (corresponding author: sieverdinge@aol.com), institute of plant production and agroecology in the tropics and subtropics, university of hohenheim (380), 70593 stuttgart, germany. mycorrhiza diversity in chilean forest ecosystems 47 << /ascii85encodepages false /allowtransparency false 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181 (2012) 1leibniz-institute for agricultural engineering potsdam-bornim e.v., potsdam, germany 2humboldt-universität zu berlin, division urban plant ecophysiology, section quality dynamics/postharvest physiology, berlin, germany impact of postharvest uv-c and ozone treatments on microbiological properties of white asparagus (asparagus offi cinalisof white asparagus (asparagus offi cinalisof white asparagus ( l.) k. hassenberg1, s. huyskens-keil2, w.b. herppich1* (received august 14, 2012) summary to meet the increasing demand for safe and high quality fresh white asparagus and the recent food safety regulations, optimization of postharvest handling, processing and storage is essential. modern sanitation techniques relying on physical methods and/or generally recognized as safe (gras) compounds are desired for reducing microbiological spoilage. to evaluate the effects of aqueous ozone and uv-c on the microbial load of spears, samples were uv-c irradiated (254 nm, 1 kj m-2) and/or washed with ozonated water (approx. 3 ppm or 4.5 ppm at 10 °c), and analyzed at three times during a four day storage. also, the potential effects of initial natural microbial loads, and precondition of the spears in terms of water and sugar contents on the responsiveness of asparagus to these treatments were determined in detail over four growing seasons. the initial microbial loads (mould and yeasts, and aerobic mesophilic total bacterial counts) of white asparagus spears varied considerably during the different harvest seasons of this four-year study. this variability could not be explained by the variance of climatic conditions nor by the respective water and sugar content. furthermore, there was never a clear cut relation of the initial microbial load and the growth of pathogens during four-day storage at 20 °c in nearly water vapour saturated atmosphere. neither washing the spears with ozonated water (3 or 4.5 ppm) nor treating them with uv-c radiation (1 kj m-2) systematically and signifi cantly affected their microbial loads during storage. in addition, the assumption that a combination of both treatments could synergistically improve the effect of each treatment could not be verifi ed during this long-term study. in conclusion, microbial load and pathogen development in asparagus spears are highly persistent and, thus, to meet hygienic requirements further investigations will be necessary. introduction white asparagus (asparagus offi cinaliswhite asparagus (asparagus offi cinaliswhite asparagus ( l.) is a very important and popular crop in germany. in 2010, it was cultivated on an area of 22.900 ha, representing 20 % of the total area used for vegetable production (statistisches bundesamt, 2011). asparagus is considered as a nutritionally highly valuable vegetable (makus, 1994; siomos, 2003), containing important antioxidant and anticancerogenic compounds (eichholz et al., 2012). at present, asparagus is mainly purchased as fresh commodity; the demand for minimally processed fresh-cut convenience product, however, largely increased during recent years. as a consequence, quality assurance has to now focus both on the retardation of metabolic processes, e.g. reduction of undesired spear toughening or losses of value adding components (huyskens-keil and herppich, 2012; huyskens-keil et al., 2011), and on meeting the recently intensifi ed hygienic requirements. total economic losses within the entire postharvest chain have been estimated as high as approx. 45 % for fresh fruits and vegetables in europe (gustavsson et al., 2011). for asparagus, supermarket losses alone may range between 8 and 11 % in the usa (buzby et al., 2009); this is, to a large part, due to microbiological spoilage (barth et al., 2009; kadau, 2005). consequently, for asparagus, optimization of postharvest handling and processing as well as storage is essential to reduce economic losses and improve food supply chain management. former standard chemical sanitation treatments applying chlorine or methylbromide include the risk of hazardous by-products formation, such as carcinogenic trihalomethane (thm) (brungs, 1973; wei et al., 1985). as a conseuqence, these applications are meanwhile forbidden by german law (lfbg, 2011). more recently, newly developed hygienisation techniques progressively include physical treatments (e.g. uv-irradiation, gamma-irradiation) or fumigation with generally recognized as safe (gras) compounds such as ozone (jamieson et al., 2009). indeed, uv-irradiation and ozone fumigation or washing with ozonated water gain more and more importance in this context. numerous studies have already been focused on the bactericidal and fungicidal capacity of ozone and uv-c treatments (karaca and velioglu, 2007; hassenberg et al., 2007 and 2008; escalona et al., 2010; gonzález-aguilar et al., 2010; horvitz and cantalejo, 2012). as a strong oxidiser, ozone exhibits effi cient bactericidal and fungicidal properties and very effectively destroys various microorganisms by oxidative breakdown of carbon residues in the washing water or adhering to the surface of the produces. ozone is effi cent at low concentrations, it requires only a short contact times, does not generate hazardous residues in or on the treated product and rapidly auto-decomposes to oxygen (jamieson et al., 2009). applied as gas or dissolved in water, ozone has been used to sanitizing produces, thus, extending storage time of perishable food (hassenberg et al., 2008). in addition, as a component of the water desinfection system, ozonation can also be implemented into excisting washing processes (e.g. artés et al., 2009). hence, gaseous and solved ozone has been successfully applied to several produces such as tomatoes, strawberries, grapes and plums (tzortzakis et al., 2007; rodoni et al., 2010), carrots (hassenberg et al., 2008), apples (achen and yousef, 2001), lettuce (hassenberg et al., 2007) and cantaloupe (rodgers et al., 2004). on the other hand, uv irradiation is predominantly applied for effective surface decontamination. uv-c (wavelength range 190 nm to 280 nm) directly damages microbial dna (artés et al., 2009) or induces the biosynthesis of plant secondary metabolites (schreiner et al., 2012) which, in turn, may inhibit or retard germination of bacterial or fungal spores (perkins-veazie et al., 2008). as ozone, uv-c irradiation does not produce harmfuls chemical residues but is lethal to most types of microorganisms. in addition, it does not require extensive safety equipment during utilization (artés et al., 2009). uv-c irradiation effectively retarded microbial growth on sliced zucchini squashes even at moderately cold temperatures of 5 °c and 10 °c during 12 days of storage (erkan et al., 2001). uv-c irradiation also effectively reduced salmonella spp. and escherichia coli o157:h7 on ‘red delicious’ apples, leaf lettuce and tomatoes (yaun et al., 2004). further, uv-c treatment controlled botrytis cinerea and penicillium expansum on ‘empire’ apples (wilson* corresponding author uv-c and ozonated water treatment of white asparagus 175 et al., 1997), showed germicidal effect on total viable count on fresh-cut apples (manzocco et al., 2011) and signifi cantly inhibited monilinia fructicola on ‘yali’ pears (li et al., 2010). for green asparagus, the effect of uv-c irradiation on the quality were tested (poubol et al., 2010); information about its infl uence on the natural microfl ora of white asparagus spears, however, are not available. hence, one aim of the presented investigation was to evaluate the effect of ozonated water and uv-c irradiation, or a combination of both, on the microbial load of white asparagus spears during shelf-life. for this purpose, spears were treated with uv-c and/or ozonated washing water and were analyzed at three times during four day storage. untreated spears were used as controls. in addition, it has not yet been determined in detail whether the initial natural microbial load may potentially infl uence the responsiveness of white asparagus spears to uv-c and/or ozone treatments. this also applies to important spear quality parameter such as water and sugar contents. these attributes indicate the physiological status of the produce, which is of importance for the growth of bacteria and for the germination of fungal spores (coates and johnson, 1997; sholberg and conway, 2000). consequently, all the above criteria were tested over the range of four growing seasons and analysed in relation to the effectivness of uv-c and/or ozone treatments. material and methods plant material and experimental setup in four independent experiments (2006, 2008, 2009 and 2011), white asparagus spears (‘gijnlim’), freshly harvested from commercial fi elds near berlin (2006 and 2008: spargelgut diedersdorf, diedersdorf, germany; 2009 and 2011: spargelhof nottebohm gbr, kartzow, germany), were immediately transported to the laboratory, washed, sorted (according ec quality standard class i), cut to a length of 22 cm (mean spear diameter: 1.8 ± 0.2 cm) and randomly separated into 12 batches of approximately 500 g. thereafter, three batches of spears each were either kept a) untreated (controls), b) submerged in ozonated water (approx. 3 ppm (2006, 2008 and 2011) or 4.5 ppm (2009) at 10 °c (thermostat 45; haake, karlsruhe, germay) for 30 s, c) irradiated with uv-c (254 nm; vl-6c, 6 w-254 nm tube, power: 12 w, vilber lourmat, marne-la-vallee, france) at 1 kj m-2, or d) both washed with ozonated water and uv-c-irradiated. ozone was generated at rates of 0.02 g min-1 by a ‘bewazon 1’ ozone generator (bwt water technology ltd., schriesheim, germany); ozone concentrations were measured with a dr 2800 photometer (hach lange gmbh, berlin, germany) applying a complementary chlorine/ozone cuvette test (dr. bruno lange gmbh & co., düsseldorf, germany). in addition, to evaluate the effect of tap water washing on microbial fl ora (e), spears were submerged in tap water at 10 °c for 30 s in 2011. after treatments, spears were stored at 20 °c in water vapour saturated atmosphere for up to 4 d. all climatic data given were continuously recorded (automatic weather station, toss gmbh, potsdam, germany) at the leibnizinstitute for agricultural engineering potsdam-bornim, potsdam, germany. microbiological analysis for the determination of the aerobic mesophilic total bacterial counts (2009 2011), yeast and mould counts (2006 2011), 3 asparagus spears each were used for the control and each treatment. therefore, 5 g from the apical, the middle and the base section of each spear was placed in a stomacher bag under sterile conditions. after adding 45 ml of ringer solution, the resulting mixture was homogenized in a stomacher (bagmixer 400, interscience, st. nom, france) for 2 min and aliquot diluted. for the analysis of the aerobic mesophilic total bacterial count, 100 µl of it was spread plated on plate count agar (pca, plates, merck, darmstadt, germany) and incubated at 30 °c for 2 d. in the case of yeast and mould counts, 100 µl was plated on rose-bengal chloramphenicol agar (rbc plates, merck, darmstadt, germany) and incubated at 25 °c for 7 d. determination were performed twice for each spear. analysis of quality parameter on days 0 (n = 12), 2 and 4 (n = 6) of the experiment, spears of each treatment were randomly taken out of storage. for each spear, fresh mass (fm, electronic balance bp 210 s, sartorius ag, göttingen, germany) and total length were determined. at positions 2.5 cm, 7.5 cm, 12.5 cm and 18 cm from the spear base, the diameters of the spears were determined by means of an electronic calliper. at these positions, the spears were cut, and from each section, tissue sap was extracted by freezing/thawing procedure. this sap was analysed for tss (total soluble solid content; pr 1 digital refractometer, leo kuebler gmbh, karlsruhe, germany), osmotic content (vapro 5520, wescor inc., logan, ut, usa) and sugar content (hplc). all sections were dried in an oven (85 °c) to constant mass for determination of the spear dry mass (dm). from fresh and dry mass, spear water content was calculated as wcdm = (fm-dm)/dm and values were given in g gdm-1. the osmotic content was related to spear dry mass and given in mol kgdm-1. for hplc analysis of free sugar content, 0.15 ml of tissue sap were diluted with distilled water to a volume of 1.5 ml and cleared by micromembrane fi ltration (0.45 µm pvdf-spritzenfi lter, rotilabo®, carl roth gmbh + co. kg, karlsruhe, germany). after clearing, the extracts were stored on ice until hplc-analysis with a dionex hplc (dionex, sunnyvale, usa) using a eurokat h column (300 mm x 8 mm, 10 µm) and 0.01 n h2so4 as the mobile phase (rate: 0.8 ml min-1 at 63 pa) and a refractive index detector (ri 71, shodex, techlab, erkerode, germany). statistical analysis all data were statistically analysed (anova) with winstat (r. fitch software, staufen, germany). treatments means were statistically compared using the duncan’s multiple range test (p < 0.05). results initial natural microbial loads of freshly harvested asparagus spears and climate data in general, the loads of freshly harvested spear with yeasts and moulds were low, often below detection limits, and ranged up to 1.1·105 ± 2.9 ·104 cfu g-1 and 4.6 ·103 ± 2.6 ·103 cfu g-1, respectively (tab. 1). no signifi cant effects of harvest season or year on these tab. 1: initial natural microbial loads (yeasts, moulds and total microbial counts) of freshly harvested ‘gijnlim’ asparagus spears, as determined during four harvest seasons. sample yeasts moulds total bacterial counts year (cfu gfm-1) (cfu gfm-1) (cfu gfm-1) 2006 1.1 ·105 ± 2.9 ·104 2.6 ·103 ± 1.1 ·103 nm 2008 1.7 ·104 ± 2.6 ·104 4.0 ·103 ± 2.6 ·103 nm 2009 < 103 ± 0 2.5 ·103 ± 2.6 ·103 1.7 ·105 ± 5.8 ·104 2011 < 103 ± 0 < 103 ± 0 6.0 ·105 ± 9.6 ·105 nm – not measured 176 k. hassenberg, s. huyskens-keil, w.b. herppich uv-c and ozonated water treatment of white asparagus tab. 2: daily means (± sd) of air temperature, relative air humidity, global radiation and total monthly precipitation during time of asparagus production in all years of investigation. sample month mean daily mean daily mean daytime total monthly year year year air temperature rel. humidity global radiation precipitation (°c) (%) (w m-2) (ml month-1) april 8.9 ± 3.4 78.9 ± 11.0 142 ± 58 50.4 2006 may 14.8 ± 2.6 66.8 ± 14.4 212 ± 63 36.4 june 19.0 ± 4.5 67.5 ± 10.3 258 ± 64 13.3 april 8.6 ± 3.5 79.4 ± 12.8 148 ± 83 64.2 2008 may 12.2 ± 4.5 63.4 ± 9.6 251 ± 54 6.3 june 11.0 ± 3.7 60.4 ± 11.8 267 ± 68 36.3 april 13.2 ± 2.8 61.4 ± 10.3 267 ± 68 2.1 2009 may 15.3 ± 3.0 69.5 ± 11.1 213 ± 78 73.9 june 16.4 ± 3.1 74.8 ± 7.7 201 ± 71 62.8 april 13.4 ± 3.3 70.1 ± 11.9 170 ± 65 29.0 2011 may 16.2 ± 4.4 64.7 ± 10.0 237 ± 61 20.6 june 19.4 ± 3.1 70.6 ± 9.8 244 ± 79 25.0 tab. 3: frequency of detected mould species on asparagus spears independent of treatment and harvest year. species frequency (%) acremonium sp. 36.8 alternaria sp. 5.3 aspergillus sp. 15.8 botrytis cinerea 5.3 cladosporium sp. 36.8 fusarium sp. 68.4 paecilomyces sp. 5.3 penicillium sp. 84.2 parameters could be detected. this was valid for both fi eld locations. it also applied for the total microbial counts, which were found to be 4.1·105 ± 2.2 ·105 cfu g-1 on average. there was a broad range of weather conditions during the three months of asparagus harvest seasons among all years investigated (tab. 2). in 2011, climate was warm and sunny, with a constant but moderate precipitation. in contrast, in 2008, it was rather cold during harvest season with few rain events in may. on the other hand, 2009 was warm and wet compared to the other years. in nearly 85 % of all samples, irrespective of any treatment or harvest year, penicillium spec. moulds could be detected (tab. 3). in addition, fusarium spp., cladosporium spec. and acremonium spec. showed a relatively high frequency. on the other hand, moulds of alternaria spec., botrytis cinerea, paecilomyces spec. were only rarely isolated. ozone and uv-c treatment effects on microbial loads of stored asparagus spears analysed directly after application, no effect of any postharvest treatment on yeast and mould counts, and aerobic mesophilic total bacterial counts could be detected; hence, data were not shown. during storage, both counts of yeasts and moulds tended to increase by more than one log in all experiments, irrespective of the treatments (fig. 1). in most cases, however, changes were statistically not signifi cant. nevertheless, after four d of storage, a slight inhibition of yeast growth was always observed in treated spears compared to controls, except in 2011 (fig. 1 a, c, e, g). in addition, no clear cut systematic effects of any treatment were monitored although the combined application of both o3 and uv tended to yield the lowest yeast loads of spears at the end of storage. results of mould counts were more inhomogeneous (fig.1). only in the fi rst experiment, all treatments signifi cantly inhibited mould growth after four day storage compared to controls (fig. 1 b). in contrast, treatments even yielded partially signifi cantly higher mould counts in the other experiments (fig. 1 d, f, h). also, none of the treatments tested could signifi cantly inhibit the increase of the aerobic mesophilic total bacterial counts during four days of storage (fig. 2). in addition, washing the spears with ozonated water at a higher concentration of 4.5 ppm (fig. 2 a) compared to that of 3 ppm (fig. 2 b) did not result in lower aerobic mesophilic total bacterial counts. furthermore, combination of ozonated washing water and uv-c irradiation could not signifi cantly reduce to increase of total bacterial counts (fig. 2). analysis of quality parameters initial produce quality largely and signifi cantly varied between the different harvest seasons as indicated by spear water content, osmotic content, tss and total sugar contents (fig. 3). spear quality was, in general, low in 2006 (lowest water content, osmotic and total sugar content, and low soluble solid); while it was highest in 2010 (moderate water content, high osmotic, soluble solid and total sugar content). in 2009, spears had the highest mean water content but, on the other hand, only moderate osmotic, soluble solid and total sugar contents. in all years, mean water content of controls did not change during four days of storage (fig. 3; cont-4). in contrast, all other parameters (osmotic content, tss, sugar content) declined in untreated spears during storage. these differences were statistically signifi cant in 2006 and 2008, but not in 2009 and 2011. compared to the controls, quality parameters were not systematically affected by either treatment. nevertheless, spears exposed to the combined treatment uv-c and ozonated water treatment of white asparagus 177 fig. 1: means (± sd; n = 3) of yeast counts (a, c, e, g) and mould counts (b, d, f, h) of fresh and stored (at 20 °c in water vapour saturated air for up to 4 days) asparagus spears in 2006, 2008, 2009 and 2011. before storage, spears were either untreated ( open bars), washed with ozonated water (90 s, 3 or 4.5 ppm; hatched bars), irradiated with uv-c (1 kj m-2; crossed bars) or both washed with ozonated water and irradiated ( fi lled bars). different letters indicate signifi cant differences between means (duncan’s multiple range test, p < 0.05). ; crossed bars) or both washed with ozonated water and irradiated ( fi lled ; crossed bars) or both washed with ozonated water and irradiated ( fi lled showed signifi cantly higher retention of total sugars and osmotic content in 2011 (fig. 3). discussion coates and johnson (1997) stated that a wide range of preharvest factors may affect the postharvest susceptibility of produces to phytopathogens. among others, weather is assumed to have a prevailing effect (webb and mundt, 1978; sholberg and conway, 2000). although climatic conditions during the different harvest seasons were highly variable, ranging from dry, sunny and hot in 2011 and wet, cloudy and cool in 2010 to, wet, sunny and warm in 2009 neither yeast or mould counts nor total bacterial counts of asparagus spears refl ected this variability in the present study. from the climatic conditions, highest microbial load could be expected in 2009 (sholberg and conway, 2000); however, in this year all counts were relatively low. on the other hand, initial counts were highest in 2006 with its moderate climate with relatively low precipitation. in this context, goßmann et al. (2008) could not verify a clear infl uence of temperature and, especially, precipitation on the microbial loads of asparagus spears from different locations with fusarium species. the fact that overall climatic conditions seem to have a low effect on the initial microbial counts may at least partially depend on the sandy soils in which the analyzed asparagus were cultivated in brandenburg region. though the area is less suitably for the cultivation of asparagus (martinez et al., 2007), these soils are mostly well-drained, thus preventing water logging, which would facilitate the growth of fungal pathogens (brückner et al., 2008). hence, only low loads of soilborne moulds were found in this region (brückner et al., 2008). on the other hand, the generally low microbial loads found during all years of investigation agreed well with early fi nding of web and mundt (1978). these authors reported negligible contaminations on to 4 days) asparagus spears in 2006, 2008, 2009 and 2011. before storage, spears were either untreated ( open bars), washed with ozonated water 178 k. hassenberg, s. huyskens-keil, w.b. herppich uv-c and ozonated water treatment of white asparagus fig. 2: means (± sd; n = 3) of total bacterial counts of fresh and stored (at 20 °c in water vapour saturated air for up to 4 days) asparagus spears in 2009 and 2011. before storage, spears were either untreated ( open bars), washed with ozonated water (90 s, 3 ppm (2009) or 4.5 ppm (2011); hatched and 2011. before storage, spears were either untreated ( open bars), washed with ozonated water (90 s, 3 ppm (2009) or 4.5 ppm (2011); hatched and 2011. before storage, spears were either untreated ( open bars), washed with ozonated water (90 s, 3 ppm (2009) or 4.5 ppm (2011); hatched and 2011. before storage, spears were either untreated ( open bars), washed with ozonated water (90 s, 3 ppm (2009) or 4.5 ppm (2011); hatched bars), irradiated with uv-c (1 kj m-2; crossed bars) or both washed with ozonated water and irradiated ( fi lled bars). different letters indicate ; crossed bars) or both washed with ozonated water and irradiated ( fi lled bars). different letters indicate ; crossed bars) or both washed with ozonated water and irradiated ( fi lled bars). different letters indicate ; crossed bars) or both washed with ozonated water and irradiated ( fi lled bars). different letters indicate ; crossed bars) or both washed with ozonated water and irradiated ( fi lled bars). different letters indicate ; crossed bars) or both washed with ozonated water and irradiated ( fi lled bars). different letters indicate ; crossed bars) or both washed with ozonated water and irradiated ( fi lled bars). different letters indicate signifi cant differences between means (duncan’s multiple range test, p < 0.05). green asparagus spears compared to various other vegetables such as beans or cucumbers. in the present study, this fi nding applies both for aerobic mesophilic total bacterial as well as mould and yeast counts. the initial loads of the former closely refl ect those reported by berrang et al. (1990; 104 105 cfu g-1) and lescano et al. (1993; 5.0 ·104 cfu g-1). in contrast, starting counts of fungal pathogens were more than one log lower than reported earlier for white ‘argenteuil’ spears from argentina (lescano et al. 1993; 7.2 ·104 cfu g-1). the fi nding that most of the fungal pathogens identifi ed in the present study, belong to penicillium spec., fusarium spec., and cladosporium spec., while moulds of aspergillus spec., alternaria spec. and botrytis cinerea contributed to only a minor and highly variable degree to the overall counts, closely refl ects earlier reports on the endophytic fungal loads of asparagus spears (web and mundt, 1978; kadau, 2005). on the other hand, moulds of acremonium spec. and paecilomyces spec. have not been described for asparagus before. especially the former moulds, however, seem to be a regular microbial load in brandenburg asparagus spears. in contrast to goßmann et al. (2008), the occurrence and prevalence of fusarium spec. did obviously not depend on the sampling location or sampling date. although the initial microbial load varied considerably during the different harvest seasons, there was never a clear cut relation to the growth of pathogens during storage. although spears stored under conditions favourable for microbial growth (tamm and flückiger, 1993; samson et al., 2010), i.e. 20 °c and water vapour saturated atmosphere, their growth was generally rather limited and changes highly variable, not at least in the 2011 season. huyskens-keil et al. (2012) reported a significant effect of postharvest applied aqueous ozone and uv-c on textural properties of white asparagus spears. in the present study, it has been hypothesized that the initial natural microbial load might potentially infl uence the responsiveness of white asparagus spears to uv-c and/ or ozone treatments. the higher the initial load the more effective might the phytopathgens spread (sholberg and conway, 2000) and, as a consequence, the less effective could the treatment be. in addition, high produce water content and, on the other hand, sugar content might provide an optimal growing medium for pathogenes. up to now, this has not yet been studied in detail. however, from the presented results it seems clear now that these factors did not affect the microbial growth in control samples nor did they infl uence the effects of any of the chosen treatments. in fact, none of the investigated treatments yielded a satisfying reduction or control of the microbial load on fresh, unprocessed white asparagus spears during storage for up to four days. this is surprising because both ozone (achen and yousef, 2001; tzortzakis et al., 2007; hassenberg et al., 2007; 2008; horvitz and cantalejo, 2012) and uv-c radiation (wilson et al., 1997; marquenie et al., 2002; allende et al., 2006; li et al., 2010; escalona et al., 2010) have been proven to be effective for this purpose in many fresh produces (karaca and velioglu, 2007). on the other hand, poubol et al. (2010) also reported that even uv-c irradiation of 3.6 kj m-2 did not show any inhibitory effect on the growth of the natural microfl ora including total microbial counts, coliforms and fungi in green asparagus. maybe the effect of radiation may increase with radiative doses as shown for gamma irradiation (lescano et al., 1993). however, further enhancement of uv doses may be limited by the potential sensitivity of produces to this radiation. very similar unsatisfying ambiguous results were obtained for washing with ozonated water. this is in contrast to the fi ndings of sothornvit and kiatchanapaibul (2009), who reported a reduction of microorganisms by nearly 1 log after washing with ozonated water (0.1 mg l-1; 10 °c, 15 min) compared to tap water-washed spears. in addition, horvitz and cantalejo (2012) obtained a successful washing time dependent decline in microbial populations when washing bell pepper with ozonated water (1 ppm). in this experiment, however, gaseous ozone (0.7 ppm) proved to be more effective. in addition, chlorine solution seemed to be advantageous over ozone (sothornvit and kiatchanapaibul, 2009; horvitz and cantalejo, 2012). on the other hand, the use of latter solution is limited by new legislations (hassenberg et al., 2008; lfbg, 2011). the application of the higher ozone concentration of 4.5 ppm did not result in a better inhibition of microorganisms compared to the application of 3 ppm. further increase in ozone concentration is limited by the solubility of this gas, which strongly depends on the temperature and decreased with increasing water temperature (langlais et al., 1991). in this context, achen and yousef (2001) proposed the use of continuous bubbling of ozone gas in the washing water to guarantee a high ozone concentration. nevertheless, it has been shown that high concentration ozone treatment may potentially result in produce quality losses (forney, 2003; liew and prange, 1994). in addition, in several studies, treated spears showed signifi cantly higher counts than untreated but washed controls. hence, careful washing with clean fresh tap water may be very effective in reducing the microbial load of asparagus spears. indeed, this has been indicated in earlier studies (osuna et al., 1995; simón et al., 2004; sothornvit and kiatchanapaibul, 2009). on the other hand, water quality cannot always be guaranteed in practise due to the and 2011. before storage, spears were either untreated ( open bars), washed with ozonated water (90 s, 3 ppm (2009) or 4.5 ppm (2011); hatched uv-c and ozonated water treatment of white asparagus 179 fig. 3: means (± sd; n ≤ 6) of water content (a), osmotic content (b), total soluble solids content (c) and the sum of the contents of all soluble sugars (d) of fresh and stored (at 20 °c in water vapour saturated air for up to four days) asparagus spears in 2006 ( open bars) 2008 ( crossed bars), 2009 ( hatched bars) and 2011 ( fi lled 2008 ( crossed bars), 2009 ( hatched bars) and 2011 ( fi lled 2008 ( crossed bars), 2009 ( hatched bars) and 2011 ( fi lled 2008 ( crossed bars), 2009 ( hatched bars) and 2011 ( fi lled 2008 ( crossed bars), 2009 ( hatched bars) and 2011 ( fi lled 2008 ( crossed bars), 2009 ( hatched bars) and 2011 ( fi lled 2008 ( crossed bars), 2009 ( hatched bars) and 2011 ( fi lled 2008 ( crossed bars), 2009 ( hatched bars) and 2011 ( fi lled 2008 ( crossed bars), 2009 ( hatched bars) and 2011 ( fi lled 2008 ( crossed bars), 2009 ( hatched bars) and 2011 ( fi lled bars). before storage spears were either untreated (cont), washed with ozonated water (90 s, 3 or 4.5 ppm; o3), irradiated with uv-c (1 kj m-2; uv) or both washed with ozonated water and irradiated (o3+uv). different letters indicate signifi cant differences between means (duncan’s multiple range test, p < 0.05). air for up to four days) asparagus spears in 2006 ( open bars) to be very promising according to the hurdles concept (scott, 1989). a combination of different treatments may induce synergistic effects, as has been reported by various authors (garzia et al., 2003; pan et al., 2004; sothornvit and kiatchanapaibul, 2009; xu and du, 2012; song et al., 2012). so, xu and du (2012) showed that combined treatment of pears with yeast antagonist and uv-c irradiation was more effective than the single treatments alone. furthermore, washing celery and cherries with chlorine dioxide solution in combination with uv-c irradiation resulted in a better produce decontamination then single applications of each treatment (song et al., 2012). however, these results could not be verifi ed during the presented long-term study on white asparagus spears. conclusions the initial microbial loads (mould and yeasts, and aerobic mesophilic total bacterial counts) of white asparagus spears varied considerably during the different harvest seasons of this fi ve-year study. this variability could not be explained by the variance of climatic conditions or the place of harvest. it was also not related to the respective water or sugar contents of spears. furthermore, there was never a clear cut relation between the initial microbial load and the growth of pathogens during four-day storage at 20 °c in nearly water vapour saturated atmosphere. neither washing the spears with ozonated water (3 or 4.5 ppm) nor treating them with uv-c radiation (1 kj m-2) systematically and signifi cantly affected their microbial loads during storage. in addition, the assumption that a combination of both could synergistically improve the effect of each treatment could not be verifi ed during the presented long-term study. microbial load and pathogen development in asparagus spears seem to be highly persistent and thus, other application methods of ozone or other postharvest treatments (e.g. ethanol, heat impulse techniques) meeting hygienic requirements have to be considered in further investigations. acknowledgements the authors thank veronika egert, corinna rolleczek, janett schiffmann 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energy. int. j. food microbiol. 90, 1-8. addresses of the authors: dr. karin hassenberg, dr. werner b. herppich, leibniz-institute for agricultural engineering potsdam-bornim, department for horticultural engineering, max-eyth-allee 100, d-14469 potsdam, germany corresponding author’s e-mail: wherppich@atb-potsdam.de dr. susanne huyskens-keil, humboldt-universität zu berlin, division urban plant ecophysiology, section quality dynamics/postharvest physiology, lentzeallee 55/57, d-14195 berlin, germany journal of applied botany and food quality 86, 221 (2013) buchbesprechung the alternatives growth and defense: resource allocation at multiple scales in plants rainer matyssek, ulrich lüttge und heinz rennenberg das buch umfasst alle wissenschaftlichen beiträge, die anlässlich des internationalen leopoldina-symposiums 2011 mit dem titel „the alternatives growth and defense: resource allocation at multiple scales in plants“ präsentiert wurden. im mittelpunkt der tagung stand die konfliktsituation, die unterschiedlichen ökophysiologischen anforderungen von kulturpflanzen einerseits und die jeweilige ressourcen-bereitstellung andererseits miteinander in balance zu bringen. dabei bestand der anspruch der organisatoren insbesondere darin, ein umfassenderes, prozessbasiertes verständnis von „systembiologie“ zu etablieren, das es gestattet, eine integrierte betrachtungsweise des intensiven ressourcenaustausches einer pflanze mit ihren abiotischen und biotischen umwelteinflüssen erzielen zu können. die insgesamt 28 beiträge sind den folgenden vier themenbereichen zugeordnet: 1. den objekten: wirtspflanze, pathogen, symbiont 2. den prozessen: wettbewerb versus erleichterung 3. den skalen: räumliche und zeitliche musterbildung 4. den systemen: holobionten und hierarchische theorie es wird ein mechanistisches modell des pflanzlichen gedächtnisses vorgestellt, das u.a. dazu genutzt werden kann, die wichtigsten bedürfnisse einer pflanze wie wachstum und verteidigung optimieren zu können. anhand ausgewählter beispiele (z.b. rinde von piceaarten) wird darüber hinaus auf induzierte abwehrmechanismen, die nach befall durch pflanzenschädlinge auftreten, näher bezug genommen. es wird in diesem zusammenhang auch darauf hingewiesen, dass durch den einsatz von „metabolomics“und „genomics“-werkzeugen in den letzten jahren das verständnis für zahlreiche pflanzliche abwehrstrategien deutlich gewachsen ist. außerdem wird am beispiel der parasitären pflanze cuscuta reflexa auch auf pflanzen-pflanzen-interaktionen bezug genommen und die in diesem zusammenhang beobachteten molekularen vorgänge, die zu einer resistenz gegenüber dem parasiten führen, demonstriert. weitere beiträge beziehen sich auf die oberirdische und unterirdische konkurrenzsituation zwischen unterschiedlichen pflanzenarten um licht und nährstoffe. am beispiel von fichte und rotbuche wird gezeigt, in welcher weise erhöhte kohlendioxid und ozongehalte in der atmosphäre einfluss auf wachstum und ressourcenallokation nehmen. schließlich werden auch einige beispiele für koexistenzfördernde interaktionen bei pflanzen angeführt. es wird postuliert, dass insbesondere diese prozesse dafür zu verantwortlich zu machen sind, dass über geologische zeiträume hinweg pflanzliche biodiversität bis heute erhalten geblieben ist. zum abschluss des buches werden die globalen veränderungen dargestellt, die sich bis zum jahr 2050 bei einem prognostizierten bevölkerungswachstum auf mindestens 9 milliarden menschen ergeben werden. es werden in diesem kontext möglichkeiten zur erntesteigerung diskutiert (biotechnologische verfahren, vermehrter anbau von c4-pflanzen, optimierung der photosyntheseleistung). der leser erhält durch die einzelnen buchbeiträge aktuelle informationen hinsichtlich der existierenden konflikte bei wachstum und stressabwehr zusammen mit den jeweils daraus resultierenden kosten-/nutzen-bewertungen. darüber hinaus besteht die möglichkeit, sich anhand der umfangreichen literaturhinweise am ende jedes beitrags, noch detaillierter mit den einzelnen fachthemen auseinanderzusetzen. der text wird durch insgesamt 92 schwarz/ weiß und farbfotos illustriert und liefert insbesondere für wissenschaftler (sowie fortgeschrittene studenten), die sich speziell für die bereiche pflanzenzüchtung, pflanzenbau und pflanzenernährung interessieren, zahlreiche neue denkanstöße. dr. h. schulz e-mail: hartwig.schulz@jki.bund.de bibliografie: rainer matyssek, ulrich lüttge und heinz rennenberg, the alternatives growth and defense: resource allocation at multiple scales in plants, nova acta leopoldina bd. 114, nummer 391, wissenschaftliche verlagsgesellschaft mbh, stuttgart, 1. auflage 2013, 369 seiten mit 39 farbigen und 53 schwarz/weiß abbildungen sowie 19 tabellen, preis: 28,95 €, isbn: 978-3-8047-3057-1 art12 journal of applied botany and food quality 82, 69 75 (2008) institute of human nutrition and food science, university of kiel, germany apolipoprotein e genotype, vitamin e, and alzheimer’s disease prevention p. huebbe, g. rimbach (received june 9, 2008) summary alzheimer’s disease (ad) is a multi-causal neurodegenerative disorder and the most common form of dementia in the elderly. although extensively investigated, the exact underlying molecular and cellular mechanisms of ad remain to be fully elucidated. amongst other factors, ad may be associated with increased oxidative stress and chronic inflammation. although dietary antioxidants, in particular vitamin e, have been related to a reduction of ad risk, data from clinical studies are still contradictory. aside from increasing age, one key risk factor for sporadic ad is the apolipoprotein e4 genotype. as major component of lipoproteins the apolipoprotein e (apoe) is of crucial importance in the distribution of cholesterol and lipids within the brain and thus, involved in neuronal membrane repair mechanisms. however, apoe4 has been associated with several altered cellular features including an impaired neuronal repair function and a higher neuronal vulnerability towards oxidative insults leading to an increased ad risk. in this context, the role of antioxidant supplementation as a primary prevention strategy for subjects at high risk including carriers of the apoε4 allele, is discussed. alzheimer’s disease alzheimer’s disease (ad) is the most common neurodegenerative disorder of the elderly and the most frequent cause of dementia during aging in western countries. every year 250,000 new ad patients are confirmed in germany. prevalence of ad increases with age from 0.4% at <65 years of age to 10% at >65 years of age. only a small percentage of the ad cases exhibits a familial trait with an early disease onset (<65 years of age), more than 90% of the patients develop a sporadic form (laws et al., 2003). the progressive disorder is characterised by massive neuronal loss in specific brain regions important for cognition and memory such as the hippocampus and parts of the cortex. histopathological hallmarks of the ad are extracellular depositions containing amyloid β (aβ) peptides and intracellular neurofibrillary tangles (nft) of the hyperphosphorylated tau protein. a profile of cellular abnormalities occurs in ad including oxidative stress, mitochondrial dysfunction and impaired regulation of cellular calcium homeostasis (mattson, 2000). furthermore, ad brains exhibit profound loss of synapses, microglial activation and inflammatory processes (pratico and trojanowski, 2000; selkoe, 2002). the amyloid precursor protein (app) is considered to play a central role in the ad pathology corroborated by the fact that autosomal dominant inherited forms of the alzheimer’s dementia are mainly caused by mutations of three genes including app and presenilines-1/2 (ps-1 and ps-2), which are involved in app processing. the transmembrane glycoprotein app comprises a large extracellular c-terminal domain, a membrane-spanning domain and an intracellular n-terminus (gralle and ferreira, 2007). major consequences for ad pathology arise from the amyloid β sequence, which is located partly in the extracellular and the trans-membrane domain of app. proteolytic activity of enzymes called βand γ-secretase (see fig. 1) contributes to the release of the toxic aβ fragment whereas α-secretase cleavage hinders aβ production (haass et al., 1992). the secreted aβ peptides are hydrophobic and form insoluble fibrils that aggregate and accumulate in the extracellular matrix as amyloid plaques. the main aβ species consist of 40 to 42 amino acids with the 42 amino acid form being deposited first (iwatsubo et al., 1994). amyloid plaques are supposed to produce reactive oxygen species and thereby injuring surrounding neuronal membranes and initiating neurite degeneration (hensley et al., 1995). the association of amyloid plaques with astrocytes and activated microglia induce cytokine production and inflammatory processes (selkoe, 1999). thus, occurrence of amyloid plaques is accompanied by chronic inflammatory burden which may in turn contribute to neuronal degeneration. apolipoprotein e apolipoprotein e (apoe) is one of the different classes of apolipoproteins in the body and is a main regulator of cholesterol transport. fig. 1: scheme of the alternative cleavage pathways of the transmembrane protein amyloid precursor protein (app) by either α-secretase (non-amyloidogenic) or β-secretase (amyloidogenic). 70 p. huebbe, g. rimbach neuronal body aβ aβ plaques density apoe3 < apoe4 cytoskeletal destabilisation preference for hdl-like particles apoe3 > apoe4 intracellular cholesterol transport apoe3 > apoe4 repair of synaptic ends apoe3 > apoe4 neurite outgrowth apoe3 > apoe4 aβ clearance apoe3 > apoe4 mitochondrial dysfunction apoe3 < apoe4 formation of neurofibrillary tangles apoe3 < apoe4 neuronal body aβ aβ plaques density apoe3 < apoe4 cytoskeletal destabilisation preference for hdl-like particles apoe3 > apoe4 intracellular cholesterol transport apoe3 > apoe4 repair of synaptic ends apoe3 > apoe4 neurite outgrowth apoe3 > apoe4 aβ clearance apoe3 > apoe4 mitochondrial dysfunction apoe3 < apoe4 formation of neurofibrillary tangles apoe3 < apoe4 several tissues synthesize apoe and of these, the liver and the brain are major sites of apoe expression. moreover, besides apoj, apoe is the major apolipoprotein in the brain suggesting its primary role in the central nervous system. apoe is considered to distribute cholesterol from astrocytes to neurons based on the fact that astrocytes are the main source of apoe and cholesterol in the brain whereas neurons express receptors to endocytose apoe and cholesterol from lipoproteins (laws et al., 2003). nevertheless, a part of activated microglia and many neurons have been shown to express apoe in response to injury (xu et al., 2006) suggesting that apoe may play a pivotal role in inflammatory and repair processes in the brain. the apoe gene is subjected to numerous single nucleotide polymorphisms and the common polymorphism gives rise to three apoe genotypes, apoe2, apoe3 and apoe4. from these arise three homozygous (e2/2, e3/3, e4/4) and three heterozygous (e2/3, e3/4, e2/4) phenotypes with different distribution throughout population and different susceptibility towards ad progression. apoe allelic distribution varies worldwide, but the apoε3 allele is always the most abundant. in a selected german population allelic distribution was reported to be 8.1% for ε2, 78.2% for ε3 and 13.6% for ε4 (singh et al., 2006). tab. 1: apoe genotype frequency in the general population (usa) and in ad-patients (according to raber et al., 2004). apoe genotype population ad ε2/ε2 1 % 0.1 % ε2/ε3 12 % 4 % ε3/ε3 60 % 35 % ε3/ε4 21 % 42 % ε4/ε4 2 % 16 % the frequency of individual phenotypes is shifted in ad patients compared to general population, whereby frequency of the e3/4 phenotype increases to 42% (from 21% in general) and of the e4/4 genotype to 16% (from 2% in general) (raber et al., 2004). thus, apoε4 allelic frequency has been reported to increase from approximately 14% in the control population to around 40% in the ad population (poirier et al., 1993) and age of disease onset is decreased in association with apoe4 (locke et al., 1995). in addition, there is also an apparent gene dosage effect according to the number of the apoε4 alleles. thus, the risk for ad rises from 20% when no apoε4 allele is present to 90% when two copies of the apoε4 allele are present in 42 families with late onset ad (corder et al., 1993). on the other side, it has been shown that the apoε2 allele is associated with a delayed age of onset, and in addition, the ε2 allele is under-represented in ad compared to control population (poirier et al., 1993). from these findings it was proposed that the ε4 allele increases ad risk whereas the ε2 allele of the apoe gene has a protective effect against the development of ad. the apoe protein contains 299 amino acids and has a mass of approximately 34 kda. the primary structure of the three common isoforms varies at two sites of the protein: 112 and 158. apoe2 has two cystein and apoe4 two arginine residues at these positions whereas apoe3 has a cystein at position 112 and an arginine residue at position 158. in apoe2, cystein at position 158 results in less effective binding to the ldl receptor family and type iii hyperlipoproteinaemia, a lipid disorder characterised by increased plasma level of triglycerides and cholesterol (mahley et al., 2006). in apoe4, the arginine residue at position 112 causes a domain interaction which is associated with reduced protein stability and different lipoprotein preferences compared to apoe3 and e2. due to the conformational difference, apoe4 favours to bind to large lower-density lipoproteins, vldl and ldl, whereas apoe3 and e2 prefer smaller, cholesterol-rich particles, hdl (hatters et al., 2006). this contributes to differences in lipoprotein metabolism and thus, total cholesterol and ldl cholesterol levels are elevated in apoε4 carriers. due to higher remnant catabolism, the ldl receptors were supposed to be down regulated thereby increasing ldl level in apoε4 carriers (mahley and rall, 2000). on that account, apoe4 was associated with an increased risk for cardiovascular disease but more evident seems to be the involvement of apoe4 in neurodegenerative processes. apoe and neurodegeneration several cellular features in the brain seem to be altered in the apoe4 as compared to the apoe3 genotype (see fig. 2). due to the limited ability of neurons to regenerate, the maintenance of synaptic function is of crucial importance. specific brain areas such as the hippocampus are able to induce formation of presynaptic extensions, and this process of compensatory synaptogenesis is accompanied by increased apoe expression. thus, synaptic repair is a key function of apoe and it has been reported to be impaired in apoε4 carriers (poirier, 1994). fig. 2: role of apoe3 and apoe4 in neuronal functions. synaptic end apoe, vitamin e, and alzheimer’s disease 71 in the cerebrospinal fluid cholesterol and lipids are transported in small high-density lipoprotein-like particles (ladu et al., 2000) and thereby can be delivered to sites of injury for repair of cells and synaptic ends. as the preference of apoe4 is lower for high-density than for lowdensity lipoproteins and apoe4 containing lipoprotein remnant catabolism is increased in relation to apoe3, insufficient neuronal uptake and intracellular cholesterol transfer may be likely to account for impaired neuronal membrane repair in the apoe4 genotype. in this regard apoe4 compared to apoe3 has been shown to inhibit neurite outgrowth (bellosta et al., 1995; holtzman et al., 1995). further information on the impact of the apoe genotype are gained from transgenic mouse models, where apoe4 has been reported to be associated with a smaller number of presynaptic ends, a higher plaque density and increased degree of tau phosphorylation. moreover, memory, cognitive performance and synaptic plasticity are impaired in the apoe4 genotype (raber et al., 2000; mahley et al., 2006). as mentioned above, the production and accumulation of amyloid β are major cornerstones in the pathogenesis of ad. in this context, decreased clearance and increased deposition of aβ in the apoe4 genotype contribute to the detrimental impact of apoe4 compared to apoe3 in ad progression. furthermore, the apoe4 isoform has been shown to enhance aβ production in vitro and this effect has been attributed to mediation through the domain interaction caused by the arginine residue at position 112 (ye et al., 2005). in transgenic mice with human apoe3 or apoe4 replacing murine apoe in the mouse genome it has been demonstrated that alpha-secretase expression is decreased in apoe4 compared to apoe3 mice (fig. 3) suggesting a shift from the non-amyloidogenic to the amyloidogenic cleavage pathway in relation to the apoe genotype (huebbe et al., 2007b). are assumed to be key mechanisms for the aβ-independent neuropathology involving apoe4 (mahley et al., 2006). while many aspects of the apoe4-ad association have been discussed lately, one of the first putative mechanisms was supposed to be the apoe allele-specific antioxidant activity. the apoe4 isoform has been shown to protect neuronal cells in vitro against oxidative insults to a lesser extent than apoe3 (fig. 4), whereas apoe2 exhibited most protection (miyata and smith, 1996; huebbe et al., 2007a). moreover, in stably transfected macrophages the apoe4 relative to the apoe3 genotype increases the production of superoxides, a prominent member of reactive oxygen species (jofre-monseny et al., 2007a). 0 0.2 0.4 0.6 0.8 1.0 1.2 mrna activity re la ti ve le ve l apoe3 apoe4 * * 0 0.2 0.4 0.6 0.8 1.0 1.2 mrna activity re la ti ve le ve l apoe3 apoe4 * * fig. 3: relative mrna and activity level of non-amyloidogenic α-secretase in the brain of apoe3 and apoe4 transgenic mice. * indicates a significant (p<0.05) apoe4 genotype dependent effect compared to apoe3. (according to huebbe et al., 2007b). besides all, cognitive impairment and functional brain abnormalities related to apoe4 have been found also in non-demented subjects (baxter et al., 2003; caselli et al., 2004; reiman et al., 2004) suggesting a global effect of the apoe genotype on brain function prior to the onset of ad symptoms. thus, mitochondrial dysfunction associated with ad has been observed to be more pronounced in apoe4 subjects (gibson et al., 2000) and disruption of mitochondrial regulation of energy and glucose metabolism in neurons has been shown in both healthy and demented apoε4 carriers (small et al., 1995). due to the lower apoe4 protein stability, increased formation of apoe4 fragments has been supposed to interact with the cytoskeletal components and to induce formation of neurofibrillary tangles (huang et al., 2001). mitochondrial and cytoskeletal alterations caused by apoe4 fragments ce ll v ia b il it y [% u nt re at ed ce ll s] apoe3 apoe4 h2o2 0 20 40 60 80 100 120 * control ce ll v ia b il it y [% u nt re at ed ce ll s] apoe3 apoe4 h2o2 0 20 40 60 80 100 120 * control fig. 4: effect of apoe3 and apoe4 conditioned media on neuronal cell viability following hydrogen peroxide challenge. * indicates a significant difference between apoe3and apoe4-cultured cells. (according to huebbe et al., 2007a). oxidative stress is linked to inflammatory processes and increased innate immune activity in the brain. importantly, activation of immune response including production of pro-inflammatory cytokines is higher in apoe4-macrophages (jofre-monseny et al., 2007b) and apoe4microglia (maezawa et al., 2006a) compared to the apoe3 genotype. neurotoxicity mediated by activated microglia, cells mediating the innate immune response in the brain similar to macrophages, is more pronounced in apoe4 than apoe3 transgenic mice (maezawa et al., 2006b). moreover, production of anti-inflammatory mediators such as interleukin-10 is lower in the apoe4 compared to the apoe3 genotype (jofre-monseny et al., 2007b). vitamin e as alterations in the oxidant/antioxidant balance are considered a primary feature of ad the lower antioxidant activity of apoe4 may be associated with a higher requirement of dietary antioxidants in apoε4 carriers (dreon and peroutkal, 2001). in this regard, vitamin e is of particular interest since it is the major lipid soluble antioxidant in the human body. vitamin e is the most effective chain-breaking antioxidant in biological membranes, where it contributes to membrane stabilisation and protection of functional molecules and cellular structures against damage from free radicals (meydani, 1995). furthermore, vitamin e is especially suitable to improve antioxidant status in the brain by easily crossing the blood-brain-barrier. vitamin e is a generic term used to describe at least four forms of (α-, γ-, β-, δ-) tocopherols and tocotrienols (fig. 5), which exhibit the biological functions in varying activity (rimbach et al., 2002). compared to the other tocopherols α-tocopherol is preferentially incorporated into lipoproteins and secreted into the plasma facilitated by the α-tocopherol transfer protein (α-ttp) (brigelius-flohe and traber, 1999). furthermore, hepatic degradation of vitamin e to the corresponding 72 p. huebbe, g. rimbach evidence of a role for vitamin e in protecting against the onset of ad and its progression in humans is based largely on epidemiological data, where lower plasma, brain and cerebrospinal fluid (csf) vitamin e levels were detected in ad patients relative to matched control groups (tohgi et al., 1994; grundman and delaney, 2002; rinaldi et al., 2003). morris and co-workers observed a lower rate of development of ad in individuals who regularly consumed vitamin e supplements relative to the non-supplement users in a 4 year prospective study of non-ad elderly participants at baseline (morris et al., 2002). additionally, in vitro studies support a potential role for vitamin e in ad prevention (halks-miller et al., 1986; behl et al., 1992; luchsinger and mayeux, 2004; huebbe et al., 2007a). however, only one clinical trial in subjects with established ad suggested that a high dose vitamin e supplementation (2000 iu/d) delayed disease progression (sano et al., 1997). on the other hand, there have been several contradictory clinical studies showing no benefit from vitamin e supplementation (1000-2000 iu/d) in ad patients (klatte et al., 2003; fillenbaum et al., 2005; petersen et al., 2005). albeit the discussion whether high dietary vitamin e intake is beneficial or not, the molecular mechanisms of potential protective effects remain poorly understood. whereas beneficial effects of vitamin e may be partly attributable to its antioxidant activity, thereby reducing oxidative modification within the neuronal cell, it is likely that vitamin e also mediates its biological role through non-antioxidant molecular targets in the brain tissue. the first observations of a cell signalling role for vitamin e were made in 1991 by azzi and co-workers (boscoboinik et al., 1991) who demonstrated that vitamin e inhibited protein kinase c activity and cell proliferation in cultured cells. advances in microarray technology have allowed us to investigate genes differentially expressed in various tissues including liver (barella et al., 2004; rimbach et al., 2004), testes (rota et al., 2004), prostate (siler et al., 2004), cortex (gohil et al., 2003), hippocampus (rota et al., 2005) and total brain homogenates (roy et al., 2002) in response to vitamin e deficiency thereby offering the possibility of more insight into the molecular functions of vitamin e. in this context, several ad related genes have been identified to be regulated by dietary alpha-tocopherol. in particular, vitamin e strongly affected a number of genes that were associated with hormone activities and hormone metabolism, nerve growth factor, apoptosis, dopaminergic neurotransmission, and clearance of amyloid beta (aβ) and advanced glycated end products (age) (rota et al., 2005). these data demonstrate that vitamin e in vivo may affect the expression of an array of genes encoding for proteins directly or indirectly involved in the prevention of ad pathogenesis. water-soluble hydroxychromans is shifted to favour of non-α-vitamin e forms (sontag and parker, 2002). however, in vitro, in contrast to α-tocopherol, γ-tocopherol is suggested to act as a trap for membranesoluble electrophilic reactive nitrogen species (christen et al., 1997). furthermore, tocotrienols are supposed to posses neuroprotective properties at a much lower concentration than α-tocopherol and may exhibit cholesterol lowering properties in vitro and in vivo (packer et al., 2001). a number of cell culture and animal studies using models of neurodegeneration have suggested a protective role for vitamin e in the reduction of oxidative damage, glycation and amyloid beta toxicity (usuki et al., 2001). the symptoms of vitamin e deficiency in alphatocopherol transfer protein (α-ttp) knock-out mice include neurological impairment, ataxia and dysfunctional reflexes accompanied by increased oxidative damage in the brain tissue. vitamin e supplementation has been shown to decrease oxidative damage and prevent neurodegeneration in ttp knock out mice (yokota et al., 2001). in our own studies pre-incubation of neuronal cells with different vitamin e forms exhibited significant protection against peroxide-induced cell death (fig. 6). fig. 5: molecular structure of vitamin e stereoisomers. r 2 4´ 8´ 3 2 or r 1 oh ch3 ch3 ch3 ch3h h ch3 r 2 4´ 8´ 3 2 or r 1 oh ch 3 ch3 ch3 ch3h h ch3 tocopherols tocotrienols 0 50 100 ce ll v ia bi li ty [% o fu nt re at ed ce ll s] * * * * tbhp α-tocopherol γ-tocopherol α-tocotrienol γ-tocotrienol + + + + + + + + + 7 5 25 0 50 100 ce ll v ia bi li ty [% o fu nt re at ed ce ll s] * * * * tbhp α-tocopherol γ-tocopherol α-tocotrienol γ-tocotrienol + + + + + + + + + 0 50 100 ce ll v ia bi li ty [% o fu nt re at ed ce ll s] * * * * tbhp α-tocopherol γ-tocopherol α-tocotrienol γ-tocotrienol + + + + + + + + + 7 5 25 fig. 6: protective effects of a pre-incubation of neuronal cells with different vitamin e forms against peroxide induced cell death (tert butyl hydroperoxide, tbhp). *indicates significant (p<0.001) effects of a vitamin e pre-treatment on neuronal viability (according to huebbe et al., 2007a). r1 r2 r3 αααααch3 ch3 ch3 βββββch3 h ch3 γγγγγh ch3 ch3 δδδδδh h ch3 apoe, vitamin e, and alzheimer’s disease 73 apoe genotype and dietary vitamin e as the complexity of pathological cellular features contributes to the lack of therapeutic success and the prevention of ad is being more and more of primary importance the identification of subjects at high ad risk is required most. accordingly, the apoe genotype as well as lifestyle and dietary factors have been discussed as determinants of ad disease risk. it is of considerable interest if one’s risk for ad may be reduced by preventive strategies similar to those that reduce the risk of cardiovascular disease (mattson, 2004). interestingly, it has been shown that the apoe4 genotype enhances adverse effects of cigarette smoking on cardiovascular disease risk (humphries et al., 2001) and that apoε4 carriers benefit to a certain extent from dietary antioxidant supplementation as far as inflammatory gene expression is concerned (majewicz et al., 2005). recent studies suggest that vitamin e metabolism is different in apoe4 to non-apoe4 subjects (proteggente et al., 2006) indicating a lower peripheral α-tocopherol concentration in apoε4 carriers due to increased retention of vitamin e in plasma (lodge et al., 2004). furthermore, in our own studies lower α-tocopherol concentrations were evident in tissues of apoe4 compared to apoe3 mice with the highest difference in the lung, whereas plasma α-tocopherol tended to be higher in the apoe4 as compared to the apoe3 genotype. we hypothesize that the vitamin e uptake from circulating low-density and high-density lipoproteins via lipoprotein receptors is affected by the apoe genotype (jofre-monseny, 2007). mas and co-workers (2006) proposed that the apoe4 genotype is associated with a functional vitamin e deficiency in the brain due to an impaired delivery of this antioxidant to the neuronal tissue (mas et al., 2006). in consequence, the benefit of a dietary vitamin e supplementation may partly depend on the apoe genotype. in a prospective study, morris and co-workers observed a positive association of vitamin e and the lower risk of ad only among individuals who were non apoε4 carriers (morris et al., 2002). on that account, supplementation of apoε4 carriers with vitamin e as suggested by dreon and peroutkal (2001) to reduce the risk for ad is still an open issue. furthermore, systematic investigations on the peripheral vitamin e metabolism in relation to the apoe genotype are needed. acknowledgments g.r. is supported by grants of the german ministry of education and science (bmbf 0313856a) and the international foundation for nutrition research and education (isfe). references barella, l., muller, p.y., schlachter, m., hunziker, w., stocklin, e., spitzer, v., meier, n., de pascual-teresa, s., minihane, a.m., 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potential therapeutic target. proc. natl. acad. sci. usa 102, 18700-18705. yokota, t., igarashi, k., uchihara, t., jishage, k., tomita, h., inaba, a., li, y., arita, m., suzuki, h., mizusawa, h., arai, h., 2001: delayedonset ataxia in mice lacking alpha-tocopherol transfer protein: model for neuronal degeneration caused by chronic oxidative stress. proc. natl. acad. sci. usa 98, 15185-15190. address of the authors: p. hübbe, g. rimbach, institute of human nutrition and food science, christian albrechts university, d-24098 kiel, germany. email: huebbe@foodsci.unikiel.de << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true 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/pagesize [907.087 680.315] >> setpagedevice journal of applied botany and food quality 93, 149 158 (2020), doi:10.5073/jabfq.2020.093.019 1leibniz institute for agricultural engineering and bioeconomy (atb), potsdam, germany 2agromechatronics – sensor-based process management in agriculture, technische universität berlin, germany 3institute of horticulture in erfurt; department of fruit growing, education and research, germany estimation of daily carbon demand in sweet cherry (prunus avium l.) production martin penzel1,2, monika möhler3, cornelia weltzien1,2, werner b. herppich1, manuela zude-sasse1* (submitted: january 27, 2020; accepted: may 22, 2020) * corresponding author summary in cherry production, the assimilate supply to the fruit is a crucial factor for growth and formation of quality parameters. the assimilate supply per fruit is limited by the relative growth capacity of trees, represented by the leaf area to fruit ratio (la:f). in the present study, the required leaf area per fruit (lademand [cm² fruit-1]) of two sweet cherry cultivars, 'bellise' and 'regina', was estimated in 2018 and 2019, based on measured and interpolated values of fruit growth and fruit respiration rates. lademand changed daily with an overall increase during fruit development, showing average values in stage iii in 2018 and 2019 of 139 cm² and 175 cm² in 'bellise', while 199 cm² and 212 cm² were found in 'regina', respectively. estimated lademand for both cultivars was compared with measurements in cherries grown on girdled branches. in both years, estimated values exceeded measured values. in both years, positive correlation between la:f and fresh mass, soluble solids content, and coloration was observed. the data obtained can be applied to evaluate the tree’s crop load for precise management. keywords: carbon balance, dry mass, fruit growth rate, fruit quality, fruit respiration, precision horticulture introduction fruit quality of sweet cherry (prunus avium l.) is frequently de scribed by fruit size, coloration, soluble solids content (ssc), and texture (kappel et al., 1996). the consumer’s acceptance and corresponding market value highly depend on these quality attributes primarily determined by scion-rootstock combination (gonçalves et al., 2006), management, crop load (whiting and lang, 2004), and environmental factors (blanco et al., 2019). however, the underlying determinant is the fruit growth that enables development of fruit quality parameters, whereas the main variable of quality, the fruit size, even directly depends on the growth of the fruit. major driver of fruit growth and accordingly of yield, is the leaf area per fruit ratio (la:f) (whiting and lang, 2004). in sweet cherry, the annual shoot growth is crucial for the development of fruiting branches, because branches usually start to bear fruit from the 3rd year. heterogeneity in soil properties within an orchard, frequently occurring in soils formed by glacial and post glacial deposits, influences the generative and the vegetative growth of stone fruit trees (käthner and zude-sasse, 2015; molina et al., 2016), especially the growth of annual shoots. therefore, tree size, total leaf area and potential yield per tree vary within orchards heterogeneous in soil properties. considering that leaves provide the major source of carbohydrate necessary for fruit growth (kappes and flore, 1986), the la:f of trees determines the magnitude of the potential fruit growth and the formation of quality parameters, which is limited by the seasonal environmental conditions at the growing location (cittadini et al., 2008, roper and loescher, 1987; whiting and lang, 2004). for cherry grown on p. avium seedling rootstocks, 16.8% of the seasonal above-ground dry mass of the whole tree was found in the fruit at harvest (kappel, 1991). however, recent research pointed out that trees grown on dwarfing rootstocks provided larger percentages of carbohydrates to fruit than trees grown on seedling rootstocks (ayala and lang, 2018). furthermore, different leaf populations supply varying percentage of assimilated carbohydrates to the fruit. the contribution of leaves of current season’s extension, non-fruiting spurs, and fruiting spurs of cherry trees grown on a dwarfing rootstock ranged from 17.5% to 79% at the different stages of fruit development (ayala and lang, 2018). the quantity of spurs usually depends on the number of buds per tree, which is set during pruning. leaves can start to export assimilates 3 d 4 d after unfolding and current seasons extension shoots from 17 d after bud break (kappes and flore, 1986). though, it can be assumed that the percentage of carbohydrates supplied by the actual leaf populations on the different branches of the trees varies considerably during the season. consequently, the leaf number per spur can be described as a function of growing degree day accumulation, but to a different extent for each type of leaf (eisensmith et al., 1980). for fruiting trees grown on p. avium rootstock at harvest, 45% of the la was found on current season’s shoots, 34% on one-year-old shoots, and the rest on older wood. on non-fruiting trees, 54% of la grew on current season’s shoots, 28% of the la on one-year-old shoots and the rest on older wood (kappel, 1991). the influence of crop load and, particularly, the la:f on fruit quality parameters was frequently investigated (overholser and claypool, 1934; facteau et al., 1983; roper and loescher, 1987; whiting and lang, 2004; cittadini et al., 2008; usenik et al., 2010) showing positive correlations between la:f and fresh mass, ssc and coloration of sweet cherry fruit. however, the specific leaf area necessary for high fruit growth and for high fruit quality varies considerably. in the trials of overholser and claypool (1934), the highest fruit mass of 7.4 g was achieved at 203 cm² la:f, whereas usenik et al. (2010) reported a fruit mass of 8.4 g at 99 cm² la:f. these differences may result from different cultivar, rootstock and tree size in the experiments as these factors affect yield efficiency, carbon partitioning to the fruit and light interception (gonçalves et al., 2006). furthermore, the la:f in the above studies were all measured at harvest when fruit reached their final size. however, daily fruit growth and respiration rates, and the resulting daily fruit carbon demand of stone fruits underlie seasonal changes (dejong and walton, 1989), as does the percentage of carbon partitioned to the fruit (ayala and lang, 2018). consequently, it can be hypo thesized that the leaf area necessary to supply the carbon demand of growing fruit varies during their development. it, additionally, depends on the ambient incident radiation intercepted by the leaves. information on the la:f, necessary to supply the carbon demand of developing fruit, is crucial for precise crop load management. particularly, the actual status of crop load at different timing during the season needs to be assessed. in some years, excessive crop load can occur, leading to unfavorable la:f. this, in turn, results in high yield of small fruit (whiting and lang, 2004), not reaching the 150 m. penzel, m. möhler, c. weltzien, w.b. herppich, m. zude-sasse commercial quality requirements. the required crop load management should adjust a balanced la:f. this enables fruit growing to a marketable size, homogeneous fruit calibers and high yield. in commercial sweet cherry production, crop load management can be performed via pruning (lauri and claverie, 2005), mechanical flower thinning (spornberger et al., 2014) or chemical thinning (stern et al., 2009). for precise management, information on the leaf area demand of individual trees is needed to assess the tree’s actual crop load. in order to develop a method to estimate the seasonal leaf area demand for sweet cherry, the objectives of the present research were (i) to quantify the daily fruit carbon demand during the period of fruit development, (ii) to model the corresponding leaf area demand per fruit with values from the literature, and (iii) to compare the results with fruit grown in the field. material and methods orchard layout and treatment trials were carried out in 2018 and 2019 on sweet cherry (prunus avium l.) trees of the cultivars 'bellise' and 'regina', planted in 2009 in the fruit production area of werder, germany (52.379971 n, 12.866811 e). trees were trained in a central leader system on gisela 5 rootstock with planting distance of 2 m × 4 m. the area covered by the tree canopy, gcovered [m²], was 3.2 m². the management of the orchards was carried out according to the federal regulations of good horticultural practice. trees (n = 30) per cultivar were labelled. full bloom was observed on the 17.04. in 'bellise' in both years, while in 'regina' full bloom varied (2018: 25.04., 2019: 23.04.). gas exchange, dry mass, elemental carbon, and growth of fruit were analyzed weekly for estimating carbon balance and leaf area demand, lademand. fruit quality was measured at harvest. to compare the lademand with la:f and their effect on fruit quality, an additional trial with adjusted leaf:fruit ratio (l:f) was performed in the same orchard in 2018 and 2019. in both years at 13 d after full bloom (dafb) three year-old fruiting branches (n=18) per cultivar, well exposed at the outside of the canopy, in 1.20 m height, forming approximately rectangular angles with the trunk, were girdled and three l:f (1:1, 2:1, 4:1) were adjusted. initially, tips of the growing shoots and new leaf growth during the season were removed. analyses of fruit co2 gas exchange and fruit growth in order to analyze the seasonal course of fruit’s dark respiration rate, rdt [mg kg-1 h-1], fresh mass, fm [g], diameter, d [mm], and elemental carbon content, c [g], was measured in weekly intervals over the entire fruit developmental period in 2018 (n=30) and 2019 (n=40) for trees of both cultivars. d and fm were measured immediately after harvesting. afterwards, respiration of 3 samples á 5 fruit in 2018 and 4 samples á 10 fruit in 2019 were measured in a closed cuvette system at 20 °c (±1 °c) in 2018 and at 10 °c (±1 °c) and 20 °c (±1 °c) in 2019. in 2018, gas samples were taken before closing the cuvettes and after 16 h using a glass syringe. the concentration of co2 in the internal air [μmol · mol–1] was analyzed with a gas chromatograph (gc 17a, shimadzu europa gmbh, duisburg, germany) at operating temperature of 60 °c. in 2019, fruit were placed in acrylic glass cuvettes equipped with continuously logging nir-co2 sensors (fya600co2, ahlborn, germany). both systems were calibrated with 0 and 1000 ± 20 ppm co2 (linde, germany). rdt was calculated (equ. 1) considering the fruit volume, cuvette volume and actual atmospheric pressure in the system described by brandes and zude-sasse (2019). (1) rdt [mg kg-1 h-1] = δco2 · fm-1 · δt-1 subsequently, at each measuring date, 15 fruit per cultivar were dried at 80 °c to constant mass. the dry sample was homogenized with a mixer mill (mm400, retsch technology, haan, germany) at a frequency of 30 hz for 1 min. c content, crel [%], of the homogenized dry mass (dm) was analyzed with an elemental analyzer (vario el iii, elementar analysensysteme gmbh, hanau, germany) at 1150 °c. in 2018, the measured values of crel were interpolated to obtain daily data over the growing season by means of curve fitting, while reducing the square error (table curve 2d version 5.01, systat software inc., usa) and expressed as function of dafb (equ. 2, 3). similarly, equ. 2, 3 were applied in 2019 to estimate the absolute c content of fruit from the dm. (2) crel(dafb)bellise = 49.9 + 10.02 · (exp (-0.5 · ((dafb 61.2) · 12.6-1)2)); r² = 0.85 (3) crel(dafb)regina = (49.84 1.947 · dafb + 0.02686 · dafb2) · (1 0.0379 · dafb + 0.00049 · dafb2)-1; r² = 0.87 modelling of fruit growth, daily carbon demand and corresponding leaf area demand the daily elemental carbon demand per fruit, cdaily [g d-1], results from fruit growth, changes in storage carbohydrates, and respired carbon. in the present study, fruit growth and changes in storage carbohydrates of the fruit were considered as sum referred to as absolute growth rate, agrc [g d-1], defined as daily change in carbon content per fruit. absolute growth rates for d, fm and c were calculated for each period (equ. 4-6). (4) agrd [mm d-1] = δd · δt-1 (5) agrfm [g d-1] = δfm · δt-1 (6) agrc [g d-1] = δc · δt-1 in 2018, measurements started 10 dafb and 8 dafb for 'bellise' and 'regina', respectively. the daily respired carbon per fruit, rdaily [g], was calculated from the measured respiration rate of fruit in the field, rfield [mg kg-1 h-1], and daily values of fm (equ. 7), assuming that no daily course in rdt occurred. additionally, co2 was converted into c by multiplying with the factor 0.27 representing the fraction of atomic mass of c (12.01 g mol-1) of the molecular mass of co2 (44.01 g mol-1). (7) rdaily [g d-1] = rfield · fm · 24 · 0.27 · 10-3 rfield was calculated from equ. 8, where tmean is the average daily temperature [°c] in 2 m height recorded with temperature sensor (pt100 1/3 class b, imetos® 3.3, pessl instruments gmbh, austria) in the neighboring orchard (52.453684 n, 12.824633 e). (8) rfield [mg kg-1 h-1] = rd10°c + (tmean 10) · (rd20°c rd10°c) · 10-1 if tmean < 20 °c rd20°c + (tmean 20) · (rd20°c rd10°c) · 10-1 if tmean > 20 °c q10-20 values were calculated at each measurement date (equ. 9). the q10-20 values were applied in 2019 for temperature correction con sidering the same cultivar. additionally, to estimate rd10°c in 2018, q10-20 of 2019 at the different measurement dates were applied in 2018 considering cherries at the same fruit size. (9) q10-20 = rd20°c · rd10°c-1 cdaily was quantified by the sum of agrc and rdaily (equ. 10). (10) cdaily [g d-1] = agrc + rdaily daily carbon demand in sweet cherry 151 to estimate the leaf area demand, lademand [cm²], necessary to assimilate cdaily, the cdaily was related to the daily assimilated c per tree, pdaily [g d-1], gcovered and the leaf area index of the tree, lai [m² m-²], assuming that the fraction of the assimilated carbohydrates partitioned to fruit, cpart [0-1], was 0.5 (equ. 11). pdaily was calculated by use of equ. 12, modified from the equation of the canopy daily net photosynthesis integral of charles-edwards (1982), analogue to the canopy photosynthesis model for apple developed by lakso and johnson (1990). (11) lademand [cm²] = cdaily · (pdaily · gcovered -1 · lai-1 · 10-4 · cpart) -1 (12) pdaily [g d-1] = (α · s · h · maxjco2 · (1 exp · (-k · lai · fmax-1))) · (α · k · s + h · maxjco2)-1 · pt · gcovered · 0.27 the leaf photochemical efficiency, α [μg j-1], and rate of light saturated photosynthesis, maxjco2 [g m-2 s-1], were set 5.027 and 0.000788, transformed from flore (1994) under the assumption that the fraction of photosynthetic active radiation (400-700 nm), par, from the solar radiation is 0.5 and the conversion factor from μmol s-1 m-1 (par) to w m2 (par) is 0.2188 (mccree, 1972). the daily integral of solar radiation, s [mj m-2 d-1], was recorded by a pyranometer (cmp 3, kipp & zonen, the netherlands) in the spectral range of 300-2800 nm. the day length, h [s], resulted from the daily hours where solar radiation (> 0 w m-2 h-1) was abundant multiplied with 3600 s h-1. the canopy light extinction coefficient, k, was set 0.6 (cittadini et al., 2008), the maximum daily light inter ception, fmax, 0.7. the lai was assumed to increase with time from 0.5 at full bloom to a final value of 3.2 at 98 dafb, when extension shoot growth was completed, in a sigmoid course (equ. 13). the curve was fitted with table curve 2d, using the data of steiner et al. (2015), hrotko (personal communication) and final lai of 3.2, measured on neighboring trees (n=6; data not shown) when the canopy was fully developed. (13) lai(dafb) = 0.5 + 2.8010247 · (1 exp (-0.033901796 · dafb)) the temperature dependence of pdaily was considered by applying the normalized equation, pt [0-1], from higgins et al., 1992 (equ. 14) for the average temperature in the hours when solar radiation was abundant, tday [°c]. (14) pt [0-1] = (11.985 + 3.846 · tday 0.20867 · tday² + 0.0053294 · tday³ 0.0000535728 · tday4) · 16.1694-1 fruit quality and leaf area analyses d of all fruit was measured weekly. at harvest, fm, ssc [%brix], hue angle, and apparent modulus of elasticity, e [kpa], were quantified for all fruit using a caliper, a refractometer (dr-301-95, krüss, germany), a texture analyzer (ta.xt, stable micro systems ltd., uk) with a spherical steel body (diameter d = 6.35 mm) and a colori meter (cm-2600d, minolta co. ltd., japan), respectively. e was calculated from the deformation, l [mm] at a force, f, of 0.5 n (equ. 15; mohsenin, 1986), where μ is the poisson’s ratio, which was assumed 0.5. r1 and r2 are the radii of the individual cherry measured crosswise. (15) e [mpa] = (0.531 · f · (1-μ²)) · l1.5 · (1· r1-1 + 1· r2-1 + 4· d-1)-0.5 additionally, all branches were defoliated after harvest and individual leaf area were measured with a leaf area meter (ci-203, cid bio-science, camas, wa usa). in 2019, strong winds caused branch breakage on 'regina' trees one week prior to harvest. therefore, data of 9 branches could not be recorded on the last date. analysis of variance of d considering the different la:f treatments on each measurement date at 5% confidence level was carried out with software r (version 3.4.1, r core team, 2018) using the package ‘userfriendlyscience’ (peters, 2018). regression equation of la:f and quality attributes were calculated with the program table curve 2d (version 5.01, aisn, systat software, ca, usa). results fruit development in 2018 and 2019, trees of the early cultivar 'bellise' flowered 8 d and 6 d earlier than those of 'regina'. fruit of 'bellise' trees achieved the final size in a shorter period of time than those of 'regina' in both years (2018: 10 d, 2019: 7 d). in 2018, fm of 'regina' fruit exceeded that of 'bellise', but fruit of both cultivars were similar in d (fig. 1). in 2019, 'bellise' fruit exceeded d and fm of 'regina' fruit. fruit of 'bellise' grew to a final fm of 9.1 ± 1.4 g and 8.7 ± 1 g in 2018 and 2019, respectively, while those of 'regina' reached 10.0 ± 0.5 g and 8.4 ± 1.2 g, respectively. the dry mass content, dmrel [% fm], of 'bellise' fruit varied between 11% 26% and 13% 21%, while those of 'regina' showed a variation of 10% 17% and 13% 22% during fruit development in 2018 and 2019, respectively. flowers had an average dmrel of 14%, mature fruit of 17% and 20% for 'bellise' and 'regina', respectively. crel of 'bellise' and 'regina' fruit was 49% and 50%, respectively, at the beginning of the 2018 season. values of fruit of both cultivars were similar until 39 dafb and 28 dafb for 'bellise' and 'regina', respectively, when dmrel increased to 61% and 62%, respectively, at harvest. seasonal course of crel of fruit of both cultivars was inter polated using smoothing average and described as function of dafb (equ. 2, 3). the seasonal course of fruit diameter, d, followed a double sigmoid curve in both years (fig. 1), indicating the stone hardening stage with reduced fruit growth from 20 27 dafb in 'bellise' and 19 27 dafb in 'regina' in 2018, while in 2019 the stone hardening periods were extended between 19 33 dafb and 20 41 dafb, respectively. in comparison, the c contents steadily increased in both years and in fruit of both cultivars until 2 and 3 weeks prior to harvest. during the last weeks of fruit development, the increase in c content of all fruit was pronouncedly accelerated. the derived growth rates fig. 1: time course fresh mass, diameter and carbon content of developing 'bellise' (closed symbol, solid line) and 'regina' (open symbol, dotted line) sweet cherry fruit during two growing seasons. error bars show the standard deviation. 152 m. penzel, m. möhler, c. weltzien, w.b. herppich, m. zude-sasse (fig. 2) show similar patterns for both cultivars. in 2018, all growth rates peaked in 'bellise' fruit at 20 dafb, where agrd achieved the highest values during fruit development. the first growth peak before stone hardening was less pronounced in 'regina'. growth rates showed unexpected minima in 2018, which were not found in 2019. the typical minimum during the stone hardening stages were measured for fruit of both cultivars in 2019. the decrease of fruit growth rates indicated the optimum harvest date. only 'regina' fruit in 2018 didn’t reach the optimum harvest date, since the experiment was ended at the commercial harvest appearing earlier. the integral of growth rates of 'bellise' fruit were enhanced in comparison to that of 'regina'. during fruit development, rd20 decreased from full bloom until harvest in a course typical for non-climacteric fruit (fig. 3). seasonal changes in rd20 in the two years were similar for fruit of both cultivars, though the average values during the last two weeks of fruit development were enhanced in 2019 compared to 2018. maximum rd20 of 908 mg kg-1 h-1, 702 mg kg-1 h-1 in fruit of 'bellise' and of 'regina', respectively, were measured at the beginning of fruit development, while rd20 were minimal (45 mg kg-1 h-1, 38 mg kg-1 h-1) at harvest and the week before in 2018. in 2019, rd20 were higher dur ing this time (76 mg kg-1 h-1, 82 mg kg-1 h1). in 2019, values of rd20 were 1.7 3.5 and 2.2 3.4 fold higher than rd10 for all fruit (fig. 3, 4). q10-20 varied during the season (fig. 4). carbon demand in fruit of both cultivars, the temperature-corrected fruit respiration rate, rfield, was highest after full bloom and declined afterwards (fig. 5). during the initial 10 d of fruit development, average air temperature was, below 21 °c. in 2018, rfield was lowest during the last days of fruit development in fruit of both cultivars, while in 2019 the lowest rfield were measured on may 15 due to prevailing low temperature of 7.6 °c for samples of both cultivars. at 25 dafb and 43 dafb, elevated rfield were recorded for 'regina' cherries as a response to elevated temperature of 17.2 °c and 24.1 °c, respectively. at these days, 'bellise' fruit was less responsive to the high temperature. the amount of carbon respired per day per fruit, rdaily, increased during fruit development from bloom until the final fruit size was achieved (fig. 5). highest annual value of rdaily of 'bellise' fruit was 0.0040 g d-1 (42 dafb, 2018) and 0.0059 g d-1 (49 dafb, 2019); that of 'regina' cherries were 0.0040 g d-1 (36 dafb, 2018) and 0.0069 g d-1 (53 dafb, 2019). integration of rdaily over the entire fruit development yielded 0.12 g c respired per fruit (56 d, 2019) in 'bellise' whereas 'regina' totally respired 0.17 g c (63 d, 2019). dark respiration accounted for 10% and 13% of the total c demand of developing 'bellise' fruit in 2018 and 2019, respectively, with daily variation of 2% 44% and 5% 37%. for fruit of 'regina', the average percentage of dark respiration of the total c demand was 9% and 15%, with daily variation of 3% 27% and 3% 41% in 2018 and 2019, respectively. the daily carbon consumption per fruit, cdaily considering fruit respiration and fruit growth were lowest at full bloom in 2019 ('bellise': 0.004 g d-1; 'regina': 0.002 g d-1) and highest at harvest in 2018 ('bellise': 0.09 g d-1, 'regina': 0.06 g d-1) (fig. 6). starting at 40 dafb and 47 dafb (2018) as well as at 39 dafb and 40 dafb fig. 2: time course of absolute growth rates in fresh mass (agrfm), dia meter (agrd), and elemental carbon (agrc) of developing 'bellise' (closed symbol, solid line) and 'regina' (open symbol, dotted line) sweet cherry fruit in two years. fig. 3: time course of fruit dark respiration rate at 10 °c (rd10; dotted and dash-dotted line) and 20 °c (rd20; solid and dashed line) of deve loping 'bellise' (closed symbol) and 'regina' (open symbol) sweet cherry fruit in two years. error bars show the standard deviation. fig. 4: q10-20 values for fruit respiration between 10 °c and 20 °c of 'bellise' (closed symbol) and 'regina' (open symbol) cherries during fruit development in 2019. error bars show the standard deviation. fig. 5: time course of average daily air temperature, measured at 2 m height, tmean, fruit respiration rate calculated for tmean, rfield, and daily respired carbon per fruit, rdaily, of developing 'bellise' (closed symbol, solid line) and 'regina' (open symbol, dashed line) sweet cherries during the growing seasons 2018 and 2019. daily carbon demand in sweet cherry 153 (2019) in 'bellise' and 'regina' fruit, respectively, cdaily increased until harvest with reduced rate around harvest. from 16 dafb till harvest 'bellise' cherries consumed 0.93 g c and 0.92 g c in 2018 and 2019, respectively, whereas 'regina' cherries consumed 1.18 g c and 1.09 g c in 2018 and 2019, respectively. values of both years point out an enhanced total c demand of 'regina' fruit compared to 'bellise' fruit. the daily assimilated c per tree, pdaily, was lowest at full bloom in 2018 ('bellise': 10 g d-1; 'regina': 7 g d-1) and early in the season in 2019 ('bellise': 17 dafb; 'regina': 11 dafb) and increased over the season with fluctuations (fig. 6). highest values (37 g d-1, 2018) were observed at harvest for 'bellise' cherries at the same time when lai reached the maximum of 2.8 considering the period of fruit development. maximum value of pdaily (37 g d-1) of 'regina' fruit was observed at 43 dafb, when solar radiation, s, achieved its seasonal maximum (28 mj). in 2019, pdaily of 'bellise' cherries was maximal (35 g d-1) at 49 dafb, when s peaked. 'regina' trees showed maximum pdaily (36 g d-1) at harvest when lai was 3.0. the magnitude of pdaily coincided with that of s, a major determinant of pdaily. the integral of pdaily of 'bellise' was 1347 g c and 1205 g c in the 2018 (52 d) and 2019 (56 d), respectively. during fruit development, total pdaily of 'regina' trees was 1664 g c in 2018 (62 d) and 1484 g c in 2019 (63 d). the seasonal course of lademand derived from cdaily varied considerably over both seasons (fig. 6). resulting, the seasonal course of lademand was inverse to that of s. minima in s coincided with peaks in lademand, e.g. at 29 dafb, 35 dafb, 41 dafb, and 50 dafb for 'bellise' cherries in 2019 (fig. 6). a maximum of 643 cm² was estimated for the lademand at 60 dafb in 2018 of 'regina' cherries when tfield was 13 °c and s was minimal (8 mj). in 'bellise' cherries, lademand was highest (504 cm²) at 49 dafb in 2018 as a result of cdaily being maximal despite relatively high s and tfield. during fruit development lademand increased. however, temporary reduction in lademand appeared corresponding to the stages of fruit growth (fig. 6, tab. 1). in the last days of fruit development after stone hardening, lademand varied between cultivars and years due to differences in cdaily and pdaily. 'regina' fruit had a total higher lademand in 2018 and 'bellise' appeared more demanding in 2019. results considering adjusted l:f the average individual leaf area from the trial was 23 cm², 26 cm² for 'bellise' and 28 cm², 28 cm² for 'regina' in 2018 and 2019, respectively. results from the trial on untreated trees and adjusted l:f on girdled branches were compared considering the la and number of fruits measured in the laboratory (tab. 2). in 2018, 'bellise' fruit grown with 115 cm² la:f reached enhanced size from 20 dafb until harvest compared to fruit d of treatments with la:f of 19 cm² and 50 cm², while treatments with 19 cm² and 50 cm² showed no difference (fig. 7). in 2018, in the real world experiment with no thinning treatment (control) and resulting unknown la:f of 'bellise' fruit, lademand was higher than the actual la:f tab. 1: leaf area demand [cm²] of sweet cherry fruit considering the 8 day interval in two years. values in parenthesis are mean values with local maxima excluded, compare fig. 6. cultivar/year average lademand fruit-1 [cm²] 1-8 dafb 9-16 dafb 17-24 dafb 25-32 dafb 33-40 dafb 41-48 dafb 49 dafb-harvest 'bellise' 2018 101 112 (101) 70 164 (124) 464 2019 16 48 89 106 (84) 90 (56) 209 (188) 328 'regina' 2018 27 47 75 152 98 330 (307) 2019 10 30 99 (75) 56 89 157 249 fig. 6: time course of changes in total carbon demand per fruit, cdaily, solar radiation, s, estimated carbon assimilation, pdaily, and daily leaf area demand per fruit, lademand, of developing 'bellise' (solid line) and 'regina' (dashed line) sweet cherries in the growing season 2018 and 2019 under the assumption that 50 % of the assimilated carbon is partitioned to fruit. 154 m. penzel, m. möhler, c. weltzien, w.b. herppich, m. zude-sasse between 41 dafb and harvest. d of fruit grown at 115 cm² la:f was 26.5 mm, which is 0.7 mm less than d of the untreated fruit from the untreated trees. in 2019 at 20 dafb, d of 'bellise' fruit grown on low (25 cm²) la:f was significantly (p<0.005) lower than that of fruit grown on medium (59 cm²) or high (110 cm²) la:f (tab. 2). from 26 dafb until harvest, differences between d of fruit of all treatments were significant (26 dafb: p<0.001, 33 dafb harvest: p<0.0001). fruit grown at 110 cm² la:f achieved higher d than that of untreated fruit with an average estimated lademand of 260 cm². in 2018, d of 'regina' fruit grown on low la:f was lower than that of fruit grown at 63 cm², 132 cm² from 27 dafb to harvest (fig. 7). fruit grown at high la:f had lower d then untreated fruit from the control tress, and la:f was also lower lademand of untreated fruit. in 2019, 'regina' fruit grown at 23 cm² la:f and 68 cm² la:f showed no differences in d until 56 dafb when d of fruit grown at 68 cm² la:f exceeded d of fruit grown at 23 cm² la:f to harvest. fruit grown at la:f of 132 cm² exceeded d of fruit from the other treatments from 34 dafb to harvest. at harvest, d of fruit grown at 132 cm² la was higher than d of untreated fruit with lademand of 208 cm² (when peaks were excluded). in addition, fruit maturity was delayed in every trial on trees of the lowest la:f treatment in comparison to those of untreated trees. the leaf area to fruit ratio (la:f) affected fm, coloration and ssc, while no effect on e was found considering fruit of both cultivars in two years (fig. 8, tab. 3). in 2018, fresh mass of 'bellise' fruit pro nouncedly increased with la:f in the range of 11 cm² to 126 cm². in 2019 lowest fm of 'bellise' fruit of 3.7 g was observed when grown at 14 cm² la:f and highest fm of 10.9 g at 136 cm² la:f. fm of tab. 2: diameter of cherries (d) grown at adjusted leaf to fruit ratios (l:f) on girdled branches (n=18) and corresponding leaf area to fruit ratio (la:f) measured in the laboratory; and leaf area demand per fruit (lademand), estimated for crop found in untreated trees (control, n=30), see fig. 6. cultivar year l:f la:f lademand d [cm²] [cm²]* [mm] 'bellise' 2018 1 19 22.9 ± 1.6a 2 50 23.4 ± 1.5a 4 115 26.5 ± 1.4b control 226 (202) 27.2 ± 1.2b 2019 1 25 21.0 ± 1.8 a 2 59 25.0 ± 2.0b 4 110 27.1 ± 1.7c control 260 26.7 ± 1.2c 'regina' 2018 1 32 22.4 ± 1.2a 2 63 25.0 ± 1.6b 4 132 25.4 ± 1.6b control 237 (208) 27.0 ± 0.8c 2019 1 23 20.9 ± 1.5a 2 68 23.4 ± 2.4b 4 132 25.6 ± 1.6c control 214 25.2 ± 1.5c *from 41 dafb – harvest, values in parenthesis are mean values with local maxima excluded fig. 7: time course of changes in fruit diameter of sweet cherry cultivars 'bellise' (a: 2018, c: 2019) and 'regina' (b: 2018, d: 2019) developing at different leaf:fruit ratio (star: 1:1, open triangle: 2:1, closed triangle: 4:1). error bars show the standard deviation. fig. 8: means of fresh mass (fm), hue angle (), ssc () and modulus of elasticity (e;) of sweet cherries of 'bellise' (closed symbol, solid line) and of 'regina' (open symbol, dotted line) grown with different leaf area to fruit ratio (la:f) in 2018 and 2019. error bars show the standard deviation. daily carbon demand in sweet cherry 155 'regina' cherries increased similarly with la:f (tab. 2). hue angle of 'bellise' fruit decreased with increasing la:f in both years, while results of 'regina' fruit showed no clear trend. ssc was in expected ranges, showing an influence of la:f on ssc. in 'regina' fruit, ssc increased with la:f from 10 %brix to 21 %brix in 2018 and 16% brix to 22 %brix in 2019. in contrast, la:f and e showed no correlation. discussion fruit development fruit of both cultivars showed typical growth patterns for sweet cherry (lilleland and newsome, 1934) reaching similar fruit sizes. in the present study, time course of the development of fresh mass and diameter did not differ between fruit of both cultivars in 2018. in 2019, however, fm and d development of 'regina' fruit was slower than those of 'bellise'. furthermore, in 'regina' cherries, overall fruit development from full bloom to harvest was generally retarded in both years. the generally higher standard deviation of data observed in 2018 but not in 2019 may be due to the dry and hot weather in former year. the findings that absolute growth rates in both fresh mass (agrfm) and diameter (agrd), but not in total carbon content (agrc), decreased during mid growing period (35 dafb – 41 dafb) irrespective of year and cultivar was also described earlier (lilleland and newsome, 1934). this effect was explained by independent growth of flesh, endocarp and kernel in stone fruit as well as by changes in fruit composition during fruit development. the assumed enhanced growth rate of early mature 'bellise' in comparison to 'regina' fruit was confirmed for agrfm and agrd. in sweet cherry (brüggenwirth et al., 2016) and peach (dejong et al., 1987), dry mass-based absolute growth rates peaked shortly before final fruit size was reached. in contrast, the dry mass-based carbon content, agrc, increased further. consequently, considering fruit quality, harvest should be as late as possible because agrc and ssc continue to increase even after the end of fruit size growth (proebsting and mills, 1981). as present dry mass-based data clearly indicate, the increase in ssc did not only result from a concentrating effect due to the reduction of fruit water content. the seasonal changes in fruit dmrel observed for both cultivars and seasons are common in cherries and occur due to water potential gradient-induced vascular water flow (brüggenwirth et al., 2016). generally, dmrel of mature fruit at harvest are expected to increase with leaf area per fruit (overholser and claypool, 1934). consistently, ssc and la:f were positively correlated in the pre sent study. crel, measured during early season well reflected former results, though values gathered at the end of the season exceeded data reported for sweet cherry pits (53.9%, petrov et al., 1999), whole peaches (47.5%, dejong and walton, 1989) and mature apples (44.5%, lakso and denning, 1996). the seasonal courses of rdt and rdaily were found to be similar for fruit of both cultivars in the present study. in addition, very similar rdt was measured earlier in sweet cherries at the end of the fruit development (sekse, 1988). in the present study, higher q10-20 values were measured than q10 values published for sour cherry ranging from 1.5 2 (flore and layne, 1999) and fruit development averaged q20-30 values of 1.9 for peach (dejong et al., 1987). how ever, q12-20 reported for pear in shelf life was higher (brandes and zude-sasse, 2019), while q20-30 of peach (dejong et al., 1987) fluctuated during the season with no visible trend. in the literature, average percentages of dark respiration on total carbon demand were estimated as 13% 23% for sweet cherry (loescher et al., 1986), 30.9% for sour cherry (flore and layne, 1999), and 16.3%, 20.5% for peach considering two cultivars (dejong and walton, 1989). the latter results were in the daily range of the values found in the present study. in contrast, the total percentage of dark respiration was lower than values reported earlier. although fruit photosynthesis reduces net co2 losses by refixation of respirational co2 (pavel and dejong, 1993), fruit photosynthesis was not considered in the presented estimations, nor in the previous analyses. in sour cherry, fruit photosynthesis contributed 11.2% of the fruit’s carbon balance and this rose up to 29.7% in stage ii, when expressed on a daily basis, especially for fruit grown in exposed positions (kappes and flore, 1986). in the present investigation, the difference in cdaily for both cultivars originated mostly from agrc, because respiration had a much lower contribution to cdaily than agrc. leaf area to fruit ratio daily carbon demand per fruit, cdemand, increased during fruit development, resulting in an increased lademand. furthermore, peaks of cdemand and lademand appeared, potentially exceeding the available leaf area, which highly depends on solar radiation. additionally, when lademand was elevated on days with low photosynthesis, fruit growth could have been maintained by consumption of malate, which is available in fruit, and of other stored reserves previously partitioned to the fruit. previous research on apple demonstrated shaded fruit had reduced growth rates after 30 hours of 90% shade (zibordi et al., 2009). in the present study, on none of the days solar radiation was below 10 % of the average, and periods of low light lasted no longer than one day. therefore peaks of lademand were extab. 3: equations relating fruit quality attributes, y, and leaf area to fruit ratio (la:f), x, and corresponding coefficient of determination, r², of 'bellise' and 'regina' sweet cherry fruits in two years. cultivar attribute year regression equation r² 'bellise' fm [g] 2018 y = 132.041 · exp(-0.0002536 · x) + 136.7 0.58 2019 y = 9.146 · exp(-0.017219 · x) + 11.05 0.65 hue angle [°] 2018 y = 1.15 + 24.824 · (1 + 0.009796 · x)-0.5 0.78 2019 y = 14.87 + 11.274 · exp(-0.010588 · x) 0.61 ssc [%brix] 2018 y = 247.343 · exp(-0.00028148 · x) + 256.02 0.47 2019 y = 338.642 · exp(-0.00026261· x) + 347.13 0.55 'regina' fm [g] 2018 y = 8.5609 · exp(-0.023849 · x) + 10.02 0.40 2019 y = -10.2947 · exp(-0.01468 · x) + 11.2 0.28 hue angle [°] 2018 y = 9.32 + 27.877 · (1 + 0.305193 · x)-0.5 0.56 2019 y = 15.69 + 10.82519 · exp(-0.020641686 · x) 0.69 ssc [%brix] 2018 y = 17.7442 · exp(-0.00987962 · x) + 24.25 0.52 2019 y = 11.118 · exp(-0.0067048 · x) + 24.01 0.41 156 m. penzel, m. möhler, c. weltzien, w.b. herppich, m. zude-sasse cluded, when they appeared for less than 2 d. carbon partitioning to the fruit is influenced by various factors such as weather, source-sink relations, developmental stage, sink-strength or position of the fruit. in the present study, the estimation was obviously simplified. ayala and lang (2018) reported that the percentage of assimilated carbon available to fruit varied between 17.5% and 79% depending on leaf population and time. leaves on fruiting spurs provided the highest percentage of assimilates to the fruit, while leaves on current seasons extension shoots contributed lowest to the fruit carbon yield. moreover, in the late season, when daily cdemand of developing fruit was highest, cpart of every leaf population increased. speculating that cdemand in the present study was reduced by 10% due to fruit photosynthesis and cpart during the final 10 days before harvest was 65% (average cpart at 56 dafb from ayala and lang, 2018), the lademand in the period 41 dafb till harvest was 135 cm² and 166 cm² for 'bellise' fruit, 149 cm² and 147 cm² for 'regina' fruit in 2018 and 2019, respectively. such weighing of the type of leaves resulted in estimations closer to the actual values of the la:f confirmed in the trial with girdled branches. though, comparison between the estimated lademand and measured la:f in the experiment with girdled branches and adjusted l:f has limited validity as the leaf population of the girdled branches consisted of fruiting spur leaves close to the fruit with high cpart. whereas, observations on random fruiting branches of 'regina' showed that the percentage of fruiting spur leaves per total leaf numbers varied from 17% 58% (data not shown). this indicates a generally higher leaf area demand of fruit on whole trees in comparison to the adjusted branches, due to the abundance of leaves from non-fruiting shoots and current season’s extension shoots with lower cpart. furthermore, girdling interrupted vascular connections of the branches to other sink organs within the tree and removal of the shoots tips reduced the sink organs of the branches to mainly fruit. as a result of the absence of other strong sink organs and its competition for assimilates, cpart of the girdled branches can be assumed to be elevated. in general, there is lack of data (not necessarily practical knowledge) on the temporal composition of the different leaf populations in the varying training systems. flore and layne (1999) reported that leaf emergence on sour cherries of spurs and long shoots occurred at 30 d and 60 d after bud break, respectively, demonstrating seasonal variation. loescher et al. (1986) found that the growth of current season’s long shoots was completed at 114 d after bud break in ‘bing’ sweet cherry. the seasonal course of the percentage of different leaf populations on the total leaf area per tree, the exposure of leaf to solar radiation (eisensmith et al., 1980), and carbon partitioning to the fruit need to be further investigated in sweet cherry to increase the accuracy of carbon balance estimation. also, estimation models for the final leaf area of sweet cherry, early in the season need to be further developed, to enable prediction of each tree’s potential maximum yield and, together with the carbon demand of targeted fruit size, the fruit bearing capacity (fbc) of the tree. fbc is defined as the crop load of a specific tree, which enables fruit growth to a certain size. knowledge of the tree-specific fbc would improve current methods to evaluate the tree’s current crop load status at any time during the season for precision management. nevertheless, the strong daily variation in lademand was figured out for fruit of both cultivars in all fruit growth stages. also, it was demonstrated that differences in lademand between two cultivars yielding early and later mature fruit occurred, but with varying results, not clearly stating if one cultivar has a different lademand when comparing both years. such varying cultivar-dependent responses to the seasonal occurring growth factors are well known in practice. the estimated leaf area demand in the experiment on girdled bran ches provide the explanation for lower size fruit grown at a la of 19 cm² 32 cm² and 50 cm² 68 cm² compared to fruit grown at 110 cm² 132 cm² la:f. the growth in every treatment was source limited by carbon availability. in the two treatments with the lowest l:f, the leaf area was sufficient to fulfil lademand of the fruit in the first stages of fruit development, while lademand exceeded the adjusted la:f in the period from 20 dafb 40 dafb and afterwards. the treatment with 110 cm² 132 cm² la:f allowed similar fruit size as the untreated real world fruit. lademand of the untreated fruit was lower than the 110 cm² 132 cm² la:f until 20 dafb, whereas it was similar until 40 dafb. in 2019, lademand of untreated fruit exceeded la:f of 110 cm² 132 cm² la. fruit quality minimum quality requirement for sweet cherry on the fresh market is in general d ≥ 26 mm, although individual retailers also accept d ≥ 24 mm. however, the enhanced fruit size is strongly desired. 'bellise' cherries with d of 26 mm showed a fm of 8 g, 'regina' fruit of 26 mm had a fm of 8.8 g. on the basis of the regression equations as shown in tab. 2, 'bellise' required 101 cm² la:f and 64 cm² la:f to achieve fm of 8 g in 2018 and 2019, respectively. 'regina' required 83 cm² la:f and 99 cm² la:f in 2018 and 2019, respectively, to achieve fm of 8.8 g. the corresponding ssc at this la:f was 16 %brix for fruit of both cultivars in 2018 and 14 %brix and 18 %brix in 2019 for 'bellise' and 'regina', respectively. in terms of taste, fruit should have 18 %brix (proebsting and mills, 1981). considering the relationship of fruit quality attributes and la:f (tab. 2), e.g. la:f of 137 cm², 109 cm² for 'bellise' cherries in 2018, 2019, respectively, were necessary to produce a cherry of 18 %brix. data demonstrate that the leaf area demand to produce a high-quality fruit based on fm was higher in 'bellise' cherry in comparison to 'regina' in 2018, but lower than 'regina' in 2019. when the optimum la:f was based on ssc, 'bellise' fruit production required a higher la to produce fruit of 18 %brix in both years compared to 'regina' fruit production. therefore, no conclusive statement can be made, which cultivar has a higher leaf area demand to produce fruit of a marketable quality. additionally, the high temperature in both years had strong influence on the results. june 2019 was one of the hottest months recorded in germany within the last 70 years. in 2019 during fruit growth 105 h with average temperature above 30 °c were measured, spread over 11 d with maximum temperature of 37 °c. in 2018 during fruit development 52 h with average temperature > 30 °c were measured, spread over 11 d. the maximum air temperature in 2018 from full bloom to harvest was 32 °c. in general the effect of extreme weather events need to be further investigated on fruit growth and fbc of cherries. however, the obtained results confirm findings of former studies showing that d, fm, ssc and coloration can be expressed as function of la:f, while no relation between texture and la:f existed (cittadini et al., 2008). the positive correlation between la:f and d, fm, ssc, and coloration originates from an increasing carbohydrate supply when la:f is increasing during source limited fruit growth. when the carbon supply from the tree exceeds the carbo hydrate demand of the growing fruit, the fruit growth will be sink limited (warren wilson, 1967). therefore, the functions to describe the relation between la:f and fm or ssc should be a saturation curve, in which the asymptote indicates sink limited growth. a linear relationship between la:f and fm (facteau et al., 1983; roper and loescher, 1987; cittadini et al., 2008) may be due to measuring in the range of la:f where growth is still source limited. fruit firmness and e are supposed to be influenced by the water relations within the tree. positive correlations between la:f and to ssc, d, fm, and coloration (tab. 4) was expected and reported earlier. usenik et al. (2010) reported for 'lapins'/gisela 5 that fm of 8.4 g was achieved at 93 cm² la:f, which confirmed facteau et al. (1983) reporting l:f of 3.4 to achieve fm of 8.4 g of 'bing' and 'lambert' cherries, which equals 99 cm² la:f, when average la is assumed daily carbon demand in sweet cherry 157 30 cm². in contrast for 'bing'/gisela 5, regression between la:f and fm showed that la:f of 175 cm² were necessary to produce a fruit of 8.4 g (whiting and lang, 2004), whereas 'bing'/p. mahaleb required la:f of 216 cm² to grow to a final mass of 8.4 g (cittadini et al., 2008). in other studies (overholser and claypool, 1934; roper and loescher, 1987) fruit did not achieve a final fruit mass of 8.4 g at the highest la:f. additionally, cittadini et al., 2008 found that there is no correlation between la:f and firmness. despite the positive correlations between la:f and main quality attributes, the actual leaf area demand to produce a certain fruit quality varies in the literature, as the trials were conducted with different scion-rootstock combinations in different growing regions. two studies (facteau et al., 1983; usenik et al., 2010) describe la:f around 100 cm² as sufficient to achieve a fm of 8.4 g, which is considered marketable. other studies reported la > 170 cm² to produce a cherry of 26 mm, representing a fm of 8.4 g. in the present study, lademand was lower in early fruit developmental stage, but > 200 cm² in the last weeks of fruit development, where cdemand is highest considering the entire fruit development. results from the girdled branches trial with adjusted la:f demonstrated that the production of fruit of marketable size and taste requires 99 cm² 137 cm² la:f capturing leaves from fruiting spurs. for a concluding result more trials are necessary, but as a recommendation the la:f in the first 20 dafb may be below 100 cm². afterwards it should be maintained > 100 cm² and in stage iii exceed 170 cm² or 200 cm² depending on cultivar and target fruit size. conclusion to produce a high fruit quality in terms of size, coloration and ssc, a high la:f over 170 cm² la:f should be maintained during the whole season, but are of enhanced importance in the third developmental stage of the fruit as the carbon demand in this period is the highest of the whole season. in the first stage the la:f can be lower, which is usually the case due to maturation of leaves during the same period. estimations of the leaf area demand based on the tree carbon balance enables to calculate an individual tree’s fruit bearing capa city (fbc) from the total la of the tree in different growing systems, however, the leaf area demand was maybe slightly overestimated in the present study. the requested daily carbon demand and derived leaf area demand per fruit determines the optimum crop load and can be used to evaluate tree’s current crop load at any time during the season. acknowledgements we wish to thank stefan lindicke for technical support in his orchard, corinna rolleczek and gabriele wegner for sampling and laboratory analysis over the two years of experiments. for financial support we acknowledge the ministry of agriculture, environment and climate protection of the federal state of brandenburg and the agricultural european innovation partnership (eip-agri) for the research grant. conflict of interest statement the authors declare no conflict of interest. references ayala, m., lang, g., 2018: current season photoassimilate distribution in sweet cherry. j. am. soc. hortic. sci.143 (2), 110-117. doi: 10.21273/jashs04200-17 blanco, v., zoffoli, j.p., ayala, m., 2019: high tunnel cultivation of sweet cherry (prunus avium l.): physiological and production variables. sci. hortic.-amsterdam 251, 108-117. doi: 10.1016/j.scienta.2019.02.023 brandes, n., zude-sasse, m., 2019: respiratory patterns of european pear (pyrus communis l. ‘conference’) throughout preand postharvest fruit development. heliyon 5, e01160. doi: 10.1016/j.heliyon.2019.e01160 brüggenwirth, m., winkler, a., knoche, m., 2016: xylem, phloem, and transpiration flows in developing sweet cherry fruit. trees 30, 1821-1830. doi: 10.1007/s00468-016-1415-4 charles-edwards, d.a., 1982: physiological determinants of crop growth. academic press australia, north ryde. cittadini, e.d., van keulen, h., de ridder, n., valles, m., rodriguez, m., peri, p., 2008: effect of fruit-to-leaf area ratio on fruit quality and vegetative growth of 'bing' sweet cherry trees at optimal leaf area index. acta hortic. 795, 677-680. doi: 10.17660/actahortic.2008.795.107 dejong, t.m., doyle, j.f., day, k.r., 1987: seasonal patterns of reproductive and vegetative sink activity in early and late maturing peach (prunus persica) cultivars. physiol. plantarum 71, 83-88. doi: 10.1111/j.1399-3054.1987.tb04621.x dejong, t.m., walton, e.f., 1989: carbohydrate requirement of peach fruit growth and respiration. tree physiol. 5, 329-335. doi: 10.1093/treephys/5.3.329 eisensmith, s.p., jones, a.l., flore, j.a., 1980: predicting leaf emergence of 'montmorency' sour cherry from degree-day accumulation. j. am. soc. hortic. sci.105, 75-78. facteau, t.j., chestnut, n.e., rowe, k.e., 1983: relationship between fruit weight, firmness, and leaf/fruit ratio in lambert and bing sweet cherries. can. j. plant sci. 63, 763-765. doi: 10.4141/cjps83-096 flore, j.a., 1994: stone fruit. in: schaffer, b., anderson, p.c. 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(eds.), the collection and processing of field data, 77-123. john wiley & sons, new york, usa. whiting, m.d., lang, g.a., 2004: 'bing' sweet cherry on the dwarfing rootstock 'gisela 5': thinning affects fruit quality and vegetative growth but not net co2 exchange. j. amer. soc. hort. sci. 129, 407-415. doi: 10.21273/jashs.129.3.0407 zibordi, m., domingos, s., corelli grappadelli, l., 2009: thinning apples via shading: an appraisal under field conditions. j. hortic. sci. biotechnol. 84 (6), 138-144. doi: 10.1080/14620316.2009.11512611 orcid martin penzel https://orcid.org/0000-0001-9617-2082 cornelia weltzien https://orcid.org/0000-0002-2951-3614 werner herppich https://orcid.org/0000-0002-6274-4596 manuela zude-sasse https://orcid.org/0000-0002-8930-7855 address of the corresponding author: manuela zude-sasse, leibniz institute for agricultural engineering and bioeconomy (atb), max-eyth-allee 100, 14469 potsdam, germany e-mail: mzude@atb-potsdam.de © the author(s) 2020. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1093/treephys/26.1.93 http://dx.doi.org/10.1016/0304-4238(92)90032-8 http://dx.doi.org/10.17660/actahortic.1986.184.15 http://dx.doi.org/10.21273/jashs.116.2.201 http://dx.doi.org/10.21273/hortsci.31.3.443 http://dx.doi.org/10.17660/ejhs.2015/80.5.5 http://dx.doi.org/10.17660/actahortic.1996.416.19 http://dx.doi.org/10.17660/actahortic.1990.276.15 http://dx.doi.org/10.17660/actahortic.2005.667.51 http://dx.doi.org/10.1016/0002-1571(72)90045-3 http://dx.doi.org/10.1016/j.eja.2015.09.010 http://dx.doi.org/10.1111/j.1399-3054.1993.tb05506.x http://dx.doi.org/10.17605/osf.io/txequ http://dx.doi.org/10.1080/00015128809437993 http://dx.doi.org/10.1080/01448765.2013.844079 http://dx.doi.org/10.1016/j.scienta.2009.06.015 http://dx.doi.org/10.1016/j.scienta.2010.06.008 http://dx.doi.org/10.21273/jashs.129.3.0407 http://dx.doi.org/10.1080/14620316.2009.11512611 art11 journal of applied botany and food quality 81, 165 171 (2007) 1institute of forest genetics and forest tree breeding, georg-august university göttingen, germany 2landesbetrieb wald und holz nordrhein-westfalen, außenstelle arnsberg forstgenbank nrw chloroplast dna analysis in oak stands (quercus robur l.) in north rhine-westphalia with presumably slavonian origin: is there an association between geographic origin and bud phenology? o. gailing1, h. wachter2, j. heyder2, h.-p. schmitt2, r. finkeldey1 (received august 6, 2007) summary slavonian oaks (quercus robur subsp. slavonica) have been introduced into germany in the second half of the 19th century from the lowlands of the rivers save and drava in today’s croatia. if compared to indigenous oak stands, they are characterized by good growth, comparatively low seed production and a late bud burst. based on the information of european-wide variation patterns at chloroplast dna markers in oaks we adapted chloroplast microsatellites for the analysis of all oak stands of presumably slavonian origin in the münsterland and lower rhine regions. we were able to distinguish between slavonian haplotypes with no natural occurrence in the study area and indigenous types that do not occur in the balkan region. a generally high differentiation among stands was observed at chloroplast markers (gst = 0.674). based on the haplotype information and historic records we found that stands with slavonian material have been established between the years 1878 and 1903. in a total of 910 analysed trees the slavonian haplotypes 5, 2 or 17 were the most frequent ones but a considerable amount of samples with indigenous haplotype 1 or haplotype10 with presumed origin in southwestern europe was also present. a clear association between haplotype 2 and late bud burst was detected in adult stands and in a field trial established with seeds from slavonian and indigenous oak stands. the information about the haplotype composition in all slavonian stands can be used as reference for the certification of reproductive material. the analysis of cpdna haploytpes in old oak stands that had been established before the introduction of foreign seed material can give valuable information for the identification of indigenous oak stands. introduction slavonian pedunculate oaks had been introduced into germany (münsterland, north rhine-westphalia) in the second half of the 19th century when extensive seed trade by railway started. historical documents and genetic marker analyses using chloroplast dna indicated that slavonian oaks originated from the lowlands of the rivers save and drava in today’s croatia (gailing et al., 2007; gehle, 1999; hesmer, 1955; wachter, 2001). slavonian oaks in germany are characterized by late bud burst, fast growth and long clear boles when compared to indigenous oaks that had been planted at the same time in the same stand (gailing et al., 2003; wachter, 2001). earlier studies indicated that stands established with slavonian oaks flushed two or three weeks later than neighboring indigenous oak stands, possibly resulting in better resistance to late frosts and lower susceptibility to pests (e.g. feeding damage by the european oak leaf roller tortrix viridana) (gailing et al., 2003; wachter, 2001). however, the number of presumably slavonian stands that have been characterized at chloroplast dna markers and for bud phenology was rather limited. chloroplast dna (cpdna) markers can give valuable information about the geographic origin of oaks and other angiosperms. the chloroplast genome is uniparentally inherited in maternal lineages; usually, recombination does not occur. thus, patterns of seed dispersal can be recognized at cpdna markers (dumolin-lapègue et al., 1998). an european-wide inventory of more than 2600 populations of white oaks using pcr-rflps of four non-coding chloroplast regions (petit et al., 2002b) revealed a total of 45 haplotypes with a characteristic distribution in europe (petit et al., 2002b). in a former study we adapted chloroplast microsatellites (deguilloux et al., 2003; weising and gardner, 1999) for the analysis of oak stands in the münsterland region. by applying these markers we were able to distinguish most chloroplast haplotypes and found complete congruence with the pcr-rflp procedure (gailing et al., 2007). these and an additional new marker were used in the present study in order to determine the haplotype composition of all putative slavonian oak stands in north rhine-westphalia (germany). three control stands were included in the analysis that have been identified to be indigenous according to their growth habit, bud phenology and /or due to the early stand establishment before the introduction of slavonian oaks into germany. here, we present the results of the cpdna analysis of 33 newly investigated stands. haplotype frequency and diversity within and among stands were analysed by including data from 17 formerly analysed stands (gailing et al., 2003; gailing et al., 2007). the aims of the present study are to give an overview of the haplotype composition of all pedunculate oak stands with presumably slavonian origin in north rhine-westphalia and to determine the time frame in which stands with different origins (haplotypes) have been established. more specific aims are the development of reliable and easyto-use genetic markers as a basis for the certification of reproductive material of slavonian oaks in germany and to lay a basis to distinguish between indigenous and introduced plant material. finally, we want to test for an association between geographic origin of oaks (identified by the chloroplast haplotype) and the putatively adaptive character bud burst. materials and methods plant material a total of 50 stands (mean of 18.2 samples per population) of quercus robur in the münsterland and from the lower rhine region have been studied including all putative stands of slavonian oak (quercus robur ssp. slavonica) in north rhine-westphalia (tab. 4). most of them have been characterized as slavonian oaks according to their growth habit, bud phenology (late flushing) and according to historical documents. samples from one stand (croatia) were collected directly in croatia (vinkovsi). three of the stands were characterized to be indigenous q. robur according to growth habit and historic documents and were used as references. seventeen of the stands were included in earlier studies (gailing et al., 2003; gailing et al., 2007). additionally, four provenances of presumably slavonian origin grown in a field trial in dormagen (abt. 33c/f, frh. v. nagel-doornick; abt. 35c1; abt. 161 a1, frh. v. boeselager; abt. 343a3, fa peine) and a reference stand (frh. v. vittinghoff-schell) were characterized at chloroplast dna markers and for bud phenology (20 trees per 166 o. gailing, h. wachter, j. heyder, h.-p. schmitt, r. finkeldey provenance). the field trial was established in 1993 with seedlings grown from seed material that was collected in stands in 1988. dna isolation total genomic dna was extracted from fresh leaves (a small slice of about 1 cm2) or from buds with the dneasy plant kit for 96 probes from qiagen (hilden, germany). the amount of dna was checked on 1% agarose gels after staining with ethidium bromide. pcr-rflp technique the following non-coding regions of the chloroplast genome trndtrnt with taqi (dt), trnc-trnd with taqi (cd), psa-trns with hinf i (as) were studied with the pcr-rflp technique (demesure et al., 1995). the pcr profile was as described by demesure et al. (1995). pcr amplification was performed in a 15 µl volume containing about 10 ng template dna, 1x qiagen pcr buffer; 1x q-solution (qiagen), 2 mm of mgcl2, 0.20 mm each dntp (fermentas); 0.5 µm of each primer and 1u taq dna polymerase (qiagen). the restriction reactions were performed by adding 5 µl of pcr product to a mix containing 3.5 µl h2o, 1.0 µl 10x enzyme buffer (roche) and 5 units of the enzyme. the reactions were incubated for 5h at 37°c (hinfi) and at 65°c (taqi). the restriction fragments were separated on a 8% polyacrylamid (paa) gel, stained with sybr gold (molecular probes) and visualized under uv light. the interpretation of the restriction pattern followed petit et al. (2002b). control probes were used to assign the resulting patterns to haplotypes described in the european-wide inventory of oak species at cpdna markers. chloroplast microsatellites thirteen chloroplast microsatellites developed for dicotyledonous angiosperms (weising and gardner, 1999) or specifically for oak (deguilloux et al., 2003) were tested for polymorphisms (gailing et al., 2007). additionally one cpssr marker (odt) has been developed from sequencing the trnd-trnt region of the chloroplast genome. the sequencing reactions were carried out with the big dye terminator v. 1.1 cycle sequencing kit (applied biosystems). primers were designed using the software primer 3 (rozen and skaletsky, 2000). in total, six cpssrs were polymorphic in our sample (tab. 1). two of these markers (ucd4, udt4) were sufficient to distinguish between all main haplotypes detected by the pcr-rflp method (gailing et al., 2007). only h5 and h7-26 could not be distinguished by cpssrs. however, they were easily distinguishable by amplification of the trnd-trnt region and restriction with taqi. differences in the restriction pattern were clearly recognized on a 2% agarose gel. data analysis as in most angiosperms the chloroplast genome in oaks is maternally inherited reflecting the dispersal by seeds (dumolin-lapègue et al., 1998). since it is equivalent to a single haploid locus the following analyses are based on the haplotype. haplotypes were determined on the basis of the combination of different chloroplast microsatellite markers (see tab. 2). absolute haplotype frequencies were determined in each stand. haplotype frequencies were used as input to calculate within-population diversity (hs) and total diversity of haplotypes (ht) in the program rarefac (petit et al., 1998). likewise, allelic richness as a measure adjusting for different sample sizes was calculated after rarefaction in the program rarefac (petit et al., 1998) with a rarefraction size equalling the smallest sample size. gst ((ht-hs)/ ht) was calculated for the partitioning of diversity among stands. rst was also calculated as a similar measure of population differentiation that is derived from estimates of allelic richness (petit et al., 1998). assessment of bud burst bud burst was scored on a scale ranging form 0 (buds closed) to 5 (leaves fully expanded). when samples were collected in the mid of may 2006, stands with extreme phenotypic values for bud burst were assessed (all trees of a given stand without leaves, or all leaves fully expanded). the experimental plot in dormagen has been established with seedlings from four different stands of presumably slavonian origin and one reference stand (vittinghoff-schell) in year 1993. pronounced differences in the dating of bud burst have been detected with late and early flushing trees growing side by side. early and late flushing trees were randomly selected on april 30th in 2007. bud phenology was assessed for individual trees using a scale from 0 (buds closed) to 5 (leaves fully expanded). the haplotype of each tree was determined as described above. results pcr rflps combining the information from chloroplast regions cd, dt and as a total of 7 haplotypes could be distinguished that corresponded tab. 1: characterization of chloroplast microsatellites locus name primer sequences (5’-3’) repeat size (bp) na ta (°c) location author udt4 fam-gataatataaagagtcaaat (a)9 144-145 2 touchdown intergenic trne-trnt deguilloux et al. 2003 ccgaaaggtcctatacctcg ucd4 fam-ttatttgtttttggtttcacc (t)12 93-96 4 touchdown intergenic ycf6-psbm deguilloux et al. 2003 tttcccatagagagtctgtat ukk4 hex-ttgtttacctataattggagc (t)9 109-110 2 53 intergenic matk-trnk deguilloux et al. 2003 tagcggatcggttcaaaactt ccmp2 fam-gatcccggacgtaatcctg (a)11 233-234 2 50 5‘to trns weising and gardner atcgtaccgaggggttcgaat 1999 ccmp10 hex-tttttttttagtgaacgtgtca (t)14 111-112 2 52.5 intergenic rp12-rps19 weising and gardner ttcgtcgdcgtagtaaatag 1999 odt fam-gagcgtctcgcaaattgtta (a11) 228-229 2 51 intergenic trnd-trnt this study ttaccgctgttcatttgctc chloroplast dna analysis of slavonian oak stands 167 to haplotypes h1, h2, h4, h5, h7-26, h10* and h17 of the european-wide inventory (petit et al., 2002b). haplotypes 7 and 26 (h726) are closely related most likely due to a post-colonisation mutation event (petit et al., 2002a) and and are summarized as h7-26 in the following. the comparison with the distribution of haplotypes in europe showed that h1, h4 and h10* are described for the study area in germany (münsterland and lower rhine region) but are missing in the balkan region (bordács et al., 2002; könig and stauber, 2004; könig et al., 2002; petit et al., 2002b). h1 is the most frequent type in the western part of germany and dominant in central europe. it had its glacial refugia most likely in southern italy (petit et al., 2002a). h10* reveals a center of distribution in the southwest and west of europe with presumed refugia on the iberian peninsula. h4 rarely occurs, mainly in germany, hungary, poland and romania (petit et al., 2002b). h2 and h17 are frequent in croatia but do not occur naturally in germany. h5 has a center of distribution in the balkan region but is comparatively rare in germany. in western germany natural populations with this haplotype are apparently missing (könig and stauber, 2004). h5 was also found in all samples of the reference stand from croatia (tab. 4) h2, 5, 7-26, 17 have a center of distribution in the balkan region (bordács et al., 2002; petit et al., 2002b) but none of the haplotypes is restricted to this region. while haplotype h7-26 is mainly concentrated in low mountain ranges in the western part of croatia, haplotypes 2, 5, and 17 occur in the lowlands of the river save between zagreb and the serbian border. h2 was also found in the dinarides in croatia. only haplotype 7-26 occurs both in natural stands in the study area (könig and stauber, 2004), and in populations from the balkan region, but it is comparatively rare (n= 20) in the studied stands. identification of haplotypes with cpssrs all but haplotypes h5 and h7-26 could be distinguished with the combination of chloroplast microsatellites ucd4 and udt4 (tab. 2). these two variants can be separated by pcr-rflps of trnd-trnt with taqi. the results of the cpssr method were fully congruent with the results obtained from the pcr-rflp procedure (see also gailing et al., 2007). additionally, two variants of what was hitherto haplotype 5 were identified at chloroplast microsatellite odt (tab. 2). both types showed about the same frequency in the analysed stands. most of the variation of those two types is distributed among populations (gst =0.72, calculated for populations where h5 is dominating). a higher frequency of one or the other type in stands with predominant haplotype 5 is indicated by asterisks in tab. 4. variation within and among stands at cpdna markers complete haplotype information has been obtained for a total of 910 adults trees in 50 stands. the most frequent haplotype was h5 (n = 405) followed by h2 (n = 169), h1 (n = 154), h10* (n = 93), h17 (n = 54), h7-26 (n = 21) and h4 (n = 14). most stands either showed only one haplotype (15 stands) or they are characterized by one predominant haplotype, a result that is reflected by high differentiation values among stands (gst = 0.674, rst = 0.734). the mean haplotype diversity and allelic richness within stands was 0.243 and 1.85, respectively. thirteen stands had a haplotype diversity higher than 0.5 (probability to encounter two different haplotypes in a stand) due to a mixture of two or more (up to four) haplotypes per stand. stands with predominant haplotypes 2 or 5 show the lowest haplotype diversity and allelic richness when compared to stands with other dominating haplotypes. haplotypes 2 and 5 were the predominant types in eight and 20 stands, respectively. the indigenous haplotype 1 was the most frequent variant in six stands, and three additional stands (controls) were fixed on this type. this indigenous haplotype was detected in several stands in low frequency (tab. 4). the oldest stand analysed established between 1826 and 1864 when seed transfer was less extensive revealed the indigenous haplotype 1. the slavonian haplotypes 2 and 5 occurred in stands established between 1880 and 1895 as predominant types (tab. 3). another slavonian type (h17) that is not indigenous to germany occurred as the dominant type in six stands established between 1886 and 1912, the most recent one (kottenforst 154b, 1912) was established from nursery-produced seedlings. a long period of time during which a stand had been established was not always associated with high haplotype diversity. for example, stand 76a (cappenberg) was fixed on the indigenous type 1 and had been established between 1826 and 1864. however, while nine out of 20 stands established by direct sowing were fixed on only one haplotype, the five stands established from young nursery-produced seedlings showed a mixture of haplotypes reflected in higher haplotype diversity (hs = 0.468 (0.279-0.611)) as compared to stands established from seeds (hs = 0.232 (0 0.659)). association of cpdna haplotypes with bud burst late bud burst was mainly observed in stands with predominant haplotype 2 (tab. 4). in stand „steprath“ trees with haplotypes 1, 10 tab. 2: characterization of chloroplast haplotypes with microsatellites. the size of the fragments is given in base pairs (bp) haplotype ucd4 udt4 ukk4 odt ccmp2 ccmp10 1 95 145 110 228 233 112 2 93 145 110 228 233 111 4 95 144 109 229 233 112 5a 94 144 109 229 233 112 5b 94 144 109 228 233 112 7-26 94 144 109 229 233 112 10-11-12 95 143 109 228 234 111 17 96 145 109 228 234 111 ccmp2, ccmp10 (weising and gardner, 1999), ucd4, udt4, ukk4 (deguilloux et al., 2003), odt. haplotype 5 und haplotype 7-26 can be distinguished by the primer-enzyme combination trnd-trnt taqi. no distinction was made between southwest european types h10, h11 and h12 (designated as h10* in the text). haplotypes 7 and 26 (h7-26) are closely related most likely due to a post-colonisation mutation event (petit et al., 2002a) and can not be distinguished with chloroplast microsatellites. 168 o. gailing, h. wachter, j. heyder, h.-p. schmitt, r. finkeldey and 7-26 (n = 4) showed completely unfolded leaves in early may 2006, while buds of trees with haploytpe 2 were still mostly closed (n = 16). the same observation had been made in stands with mixed haplotype composition of individual trees in the münsterland region (gailing et al., 2003). also in the experimental plot of dormagen where slavonian and indigenous oaks grow in the same environment, a strong association of slavonian haplotype 2 with late bud burst was detected in early may 2007 (bud stage 3.56 ± 1.64, n = 32). plants with haplotype 2 originating from stand 161a1 (freiherr von boeselager) revealed an even later bud burst (bud stage 2.43 ± 1.87, n = 12) than plants from stand 343a3 (forstamt peine, bud stage = 4.35 ± 0.81, n = 20). a few plants with slavonian haplotype 5 show a slightly earlier bud stage (bud stage 4.70 ± 0.47, n = 27) than plants with indigenous haplotype 1 (n = 22) or southwest european haplotype 10* (n = 9, all bud stage 5, leaves completely unfolded). discussion certification of reproductive material of slavonian oaks stands that had been established in the late 19th century with plant material from croatia (slavonian stands) show several favourable characteristics as fast growth, a long clear bole, and late bud burst that may result in a lower susceptibility to late frost and pest damage (wachter, 2001). on the other hand, seed set and natural regeneration in slavonian oaks in germany is lower than in indigenous pedunculate oaks possibly due to the colder climate in the study area (lower annual mean temperatures) and/or restricted gene flow between indigenous and slavonian oaks. for example, it was shown that flushing dates of indigenous oaks and slavonian oaks grown side by side are nearly non-overlapping (gailing et al., 2003). however, gene flow between slavonian oaks and indigenous oaks and the effect on potentially adaptive characters as growth characteristics and seed production has not yet been studied. the data on the haplotype composition available for all oak stands of slavonian origin lays the basis for the certification of reproductive material from these stands. since cpdna markers are maternally inherited in angiosperms with all seeds and natural regeneration showing the same chloroplast information as their mother tree, they show high uniformity within but a clear differentiation between stands. this is especially true for barochorous, wind-pollinated tree species such as oaks where seed dispersal is quite restricted if compared to dispersal by pollen. thus, chloroplast markers are specifically suited to test and falsify the origin of reproductive material of slavonian and indigenous oaks. since many stands of slavonian oaks with a specific haplotype (h2) are uniform for favourable growth and stem characteristics and bud phenology (wachter, 2001), the analysis of seeds and young plants may allow to conclude on the phenotype of adult trees (see below experimental plot in dormagen). however, gene flow by pollen from neighboring stands may affect potentially adaptive traits as bud burst (see below) or growth rate and other yield characteristics. the analysis of the highly informative cpssrs markers allows the identification of the chloroplast haplotype even in older material or wood samples with highly degraded dna (see below). an accurate distinction of small informative size differences can be performed in high-resolution capillary electrophoresis. by using internal standards an unambiguous distinction between chloroplast haplotypes is easily achievable (gailing et al., 2007). identification of slavonian and indigenous oak stands evidence for the establishment of stands with non-indigenous material is unambiguous for stands showing predominant haplotypes 2 or 17 that do not occur naturally in germany. for stands showing predominant haplotypes with a wider distribution range in europe a clear distinction between indigenous and introduced material based on cpdna alone is not possible. especially in germany the diversity of different haplotypes is comparatively high due to a mixing of cpdna lineages from different glacial refugia (petit et al., 2003). for example, h10* is most abundant in southwestern and western europe but is a rarer occurrence in the study area. however, for at least one stand with h10* the establishment with nonautochthonous material is well-documented (159a in westtünnen). this stand is characterized by late bud burst and late leave fall, a combination of characters that is not found in indigenous and slavonian stands in the same area and that may be interpreted as an adaptation to different climatic conditions in southwestern europe (gailing et al., 2003). also h5 that was identified as the most common haplotype in the slavonian stands of the study area does also naturally occur in germany but according to historical documents and cpdna analyses is most likely absent in natural stands of the study area (könig and stauber, 2004; könig et al., 2002). since extensive seed transfer started with the expansion of railway connections in the second half of the 19th century, allochthonous oak populations are unlikely to be found among those established before 1860. the cpdna analysis confirmed that seed material from slavonian oaks was introduced only during the second half of the 19th century with the earliest occurrence of the slavonian haplotypes h2 and h5 in 1880 and 1878, respectively. according to historical documents and cpdna haplotype information slavonian stands were established (from seeds) in germany in the münsterland and lower rhine region between 1878 and 1903. the further analysis of the haplotype composition of stands established before that time might help to identify cpdna haplotypes that are characteristic for stands indigenous to the study area in the münsterland and lower rhine regions. since cpssr primers amplify short informative regions of the tab. 3: estimate of haplotype diversity for stands with different dominating haplotypes dominant year year s n hs average hs range na narange haplotype dominant type h1 1826-1905 1826-1907 7 154 0.237 0 0.659 0.91 0 2.43 h2 1880-1894 1880-1894 8 166 0.175 0 0.363 0.78 0 1.76 h5 1880-1895 1878-1912 20 406 0.142 0 0.611 0.58 0 2.48 h7-26 1894 1881-1894 1 20 0.503 1.55 h10* 1886-1907 1880-1907 6 93 0.321 0 0.753 1.03 0 2.74 h17 1886-1912 1880-1912 6 54 0.541 0.485 0.602 1.42 1 1.90 hs: genetic diversity of haplotypes; na: allelic richness, s: number of stands where the haplotype is dominating, n: total number of samples with the respective haplotype. h4 occurs in 14 samples and dominates in none of the stands. h4 occurred at low frequency in stands established between 1886 and 1912. chloroplast dna analysis of slavonian oak stands 169 tab. 4: haplotype frequencies in quercus robur stands forest district stand year area planting/ collection chloroplast haplotypes (ha) sowing h1 h2 h4 h5 h7 h10* h17 fa bergisch gladbach, königsforst 76b 1891 5.0 seeds buds closed may-06 0 19 0 0 0 0 0 h2 fa bergisch gladbach, königsforst 127c 1887 3.4 seeds bud stage 5 may-06 0 0 0 20* 0 0 0 h5 fa bonn, kottenforst 10b2 1893 1.5 plants may-06 2 0 0 16 1 1 0 h5 fa bonn, kottenforst 37c 1907 2.2 seeds may-06 8 0 0 1 0 9 0 h10* fa bonn, kottenforst 40b 1907 2.2 seeds low growth may-06 0 0 0 0 0 19 0 h10* fa bonn, kottenforst 70d 1903 2.5 seeds may-06 0 0 0 9* 0 0 10 h17 fa bonn, kottenforst 85a 1912 3.7 plants bud stage 5 may-06 0 0 0 4 0 0 8 h17 fa bonn, kottenforst 85b 1905 4.1 seeds may-06 10 0 4 0 0 2 0 h1 fa bonn, kottenforst 85d 1904 8.6 seeds may-06 0 0 0 0 0 19 0 h10* fa bonn, kottenforst 89aa 1904 8.4 seeds may-06 7 0 0 1 0 1 5 h1 fa bonn, kottenforst 95b 1903 2.8 seeds may-06 0 0 0 4 0 12 4 h10* fa bonn, kottenforst 134a/c 1902/1900 6.1 plants/seeds low growth may-06 5 0 6 2 0 7 0 h10* fa bonn, kottenforst 154b 1912 3.2 plants may-06 0 0 3 5 0 0 11 h17 freiherr von der leyen 17c 1885 7.1 ? bud stage 5 may-06 1 0 0 15* 0 3 0 h5 fürst zu salm-salm, rhede 105/4a1/a7 ? ? may-06 17 0 0 0 0 3 0 h1 gut ulenburg 4c ? 1.6 ? may-06 17 0 0 0 1 1 0 h1 stadt viersen 36b/38 1886 16.7 seeds may-06 0 0 1 18 0 0 0 h5 steprath 3h 1881 0.5 ? buds may-06 1 16 0 0 1 2 0 h2 mostly closed letmathe, h. blix, cappenberg flur 2/155 1888 1.0 ? may-06 0 0 0 20** 0 0 0 h5 letmathe, estermann 116a1 1894 2.2 ? may-06 1 17 0 0 1 1 0 h2 letmathe, graf v. kanitz 77c 1883 4.4 seeds may-06 2 0 0 18* 0 0 0 h5 letmathe, graf v. kanitz 76a 1826-1864 9.5 ? may-06 20 0 0 0 0 0 0 h1 letmathe, graf v. kanitz 19a 1883 1.6 seeds may-06 0 0 0 16* 0 0 0 h5 letmathe, graf v. kanitz 32h/39a ? 2.0 ? may-06 0 0 0 20** 0 0 0 h5 letmathe, frhr. v. boeselager 159a 1886 2.2 late bud burst, apr-02 0 0 0 0 0 10 0 h10* late leave fall letmathe, frhr. v. boeselager 159b 1887 3.3 early bud burst apr-02 9 1 0 0 0 0 0 h1 letmathe, frhr. v. boeselager 160b 1887 2.0 early bud burst apr-02 10 0 0 0 0 0 0 h1 letmathe, frhr. v. boeselager 161a1 1887 1.2 seeds late bud burst apr-02 0 8 0 2 0 0 0 h2 letmathe, frhr. v. boeselager 161a2 1879 1.0 seeds intermediate apr-02 0 4 0 4 0 2 0 h2/h5 bud burst letmathe, schulze-becking 54b1/b2 1880 1.5 may-06 0 6 0 13** 0 0 1 h5 münsterland, br deutschland 1b1a ~1894 0.4 plants late bud burst jul-05 0 17 0 1 2 0 0 h2 münsterland, br deutschland 1b1b ~1890 0.5 plants intermediate jul-05 2 4 0 12 0 0 2 h5 bud burst münster, ziebell, lüdinghausen flur 1/299 1880 4.0 ? may-07 0 19 0 0 0 1 0 h2 obereimer, gr. von plettenberg 14d1 1890? 1.0 ? may-07 10 0 0 9 1 0 0 h1 obereimer, gr. von plettenberg 104a 1895 1.8 ? may-06 1 0 0 19** 0 0 0 h5 obereimer, gr. von plettenberg 106g 1888 2.9 ? may-06 0 0 0 19** 0 0 0 h5 obereimer, veba 43 ~1894 0.7 ? jul-05 0 18 0 1 1 0 0 h2 warendorf, frh. v. nagel-doornick 13a 1886 1.5 ? jul-05 2 0 0 5 0 0 12 h17 warendorf, frh. v. nagel-doornick 24b ~1887 1.8 seeds jul-05 0 0 0 17 1 0 0 h5 warendorf, frh. v. nagel-doornick 33c 1878 1.3 seeds jul-05 0 0 0 20 0 0 0 h5 warendorf, frh. v. nagel-doornick 33d 1879 1.9 seeds jul-05 20 0 0 0 0 0 0 h1 warendorf, frh. v. nagel-doornick 33f 1880 0.8 seeds jul-05 0 0 0 20 0 0 0 h5 warendorf, frh. v. nagel-doornick 36e ~1882 1.5 seeds jul-05 0 20 0 0 0 0 0 h2 warendorf, frh. v. nagel-doornick 50b ~1883 1.6 seeds jul-05 2 0 0 17 0 0 0 h5 warendorf, schulze-pellengahr 4h 1893 1.2 ? may-06 0 0 0 20** 0 0 0 h5 warendorf, schulze-sutthoff flur 2/77 ~1890 0.6 ? jul-05 0 0 0 19 0 0 0 h5 warendorf, suttorp, everswinkel flur 11/129 ? 2.0 ? may-07 0 20 0 0 0 0 0 h2 warstein-rüthen, kirche anröchte 32c 1894 4.2 ? may-06 5 0 0 1 12 0 0 h7 westkirchen, quante flur 2/90-92 ~1889-1890 2.2 seeds jul-05 2 0 0 17 0 0 1 h5 croatia, vinkovsi 2007 may-06 0 0 0 20** 0 0 0 h5 sum haplotypes 154 169 14 405 21 93 54 a: samples were collected in the southwestern part of the stand. h7 is a shortcut for h7-26. stand were either established by direct sowing (seeds) or from nursery-produced seedlings (plants). *: higher frequency of h5a **: higher frequency of h5b 170 o. gailing, h. wachter, j. heyder, h.-p. schmitt, r. finkeldey chloroplast genome that are present in high copy numbers per cell in comparison with nuclear dna markers, those markers can also be analysed in highly degraded samples (for example old wood samples) (deguilloux et al., 2004; rachmayanti et al., 2006). the analysis of old and / or prehistorical wood samples at these markers might give additional information on the haplotype composition in oak stands before extensive long-distance seed transfer started. more recent studies indicate that authentic dna can be retrieved from wood samples up to 1,000 years of age (liepelt et al., 2006). association between haplotype and phenotype slavonian provenances with haplotypes 2 and 5 of pedunculate oak showed an up to three week later bud burst than neighboring indigenous oak stands in the münsterland region (gailing et al., 2003). these differences were found in four consecutive years where trees with h2 showed an even later bud burst than those with h5 (gailing et al., 2003). in the present study bud burst has been assessed on a fixed date (april 30th) in a field trial in dormagen containing plants with slavonian, indigenous and southwest european haplotypes. while a strong association of haplotype 2 with later bud burst was detected, only minor differences in flushing date were recorded between individuals with slavonian haplotype 5 and non-slavonian haplotypes, possibly due to the assessment of the trait relatively late in spring. progenies from stand 161a1 (freiherr von boeselager) with uniform late bud burst (gailing et al., 2003) also showed the latest bud burst in the field trial reflecting a putatively strong genetic component (heritability) of the trait. since also other stands with slavonian haplotypes show a uniformly later bud burst than neighboring indigenous oak stands (gailing et al., 2003; gailing et al., 2007; wachter, 2001), underlying genetic differences have possibly evolved in response to different environmental (climatic) conditions in the regions of origin. while chloroplast markers can indicate geographic origin, they are obviously not directly involved in the control of bud burst. thus, due to the wide geographic distribution of most haplotypes, a prediction of the phenotype only from the haplotype information is in most cases not possible. accordingly, kremer et al. (2002) found in general no significant impact of chloroplast haplotype (maternal origin) on phenotypic traits such as bud burst in a test with 16 provenances of q. petraea. the association between haplotype and late bud burst found for slavonian stands in this study is most likely due to the fact that slavonian oaks had been introduced into germany from a restricted area in the lowlands of the rivers save and drava with similar environmental conditions. in order to resolve the genetic basis of adaptive differences in bud burst, an intraspecific crosses between adults trees of slavonian and german origin with pronounced differences in flushing date have been produced. qtls (quantitative trait loci) will be located on genetic linkage maps calculated in a progeny of 192 full-sibs (gailing, submitted). acknowledgements we wish to thank mrs. olga artes and mrs. o. dolynska for the technical help in the laboratory. for the help in collecting the plant material we thank mrs. l. schulze, mr. rogina. references bordács, s., popescu, f., slade, d., csaikl, u., lesur, i., borovics, a., kézdy, p. et al., 2002: chloroplast dna variation of white oaks in the northern balkans and in the carpathian basin. forest ecol. manage. 156, 197-209. deguilloux, m.-f., dumolin-lapegue, s., gielly, l., grivet, d., petit, r.j., 2003: a set of primers for the amplification of chloroplast microsatellites in quercus. mol. ecol. notes 3, 24-27. deguilloux, m.-f., pemonge, m.-h., petit, r.j., 2004: dna-based control of oak wood geographic origin in the context of the cooperage industry. ann. forest sci. 61, 97-104. demesure, b., sodzi, n., petit, r., 1995: a set of universal primers for amplification of polymorphic non-coding regions of mitochondrial and chloroplast dna in plants. mol. ecol. 4, 129-131. dumolin-lapègue, s., pemonge, m.-h., petit, r., 1998: association between chloroplast and mitochondrial lineages in oaks. mol. biol. evol. 15, 13211331. gailing, o., 2007: qtl analysis of leaf morphological characters in a quercus robur full-sib family (q. robur x q. robur subsp. slavonica). plant biology (submitted). gailing, o., wachter, h., leinemann, l., hosius, b., finkeldey, r., schmitt, h.-p., heyder, j., 2003: characterisation of different provenances of late flushing pedunculate oak (quercus robur l.) with chloroplast markers. allg. forstund jagdzeitung 174, 227-231. gailing, o., wachter, h., schmitt, h.-p., curtu, a.-l., finkeldey, r., 2007: characterization of different provenances of slavonian pedunculate oaks (quercus robur l.) in münsterland (germany) with chloroplast dna markers: pcr-rflps and chloroplast microsatellites. allg. forstund jagdzeitung 178, 85-90. gehle, t., 1999: genetische differenzierung der eiche (quercus robur) in nordrhein-westfalen. allg. forstund jagdzeitung 170, 183-187. hesmer, h., 1955: die späteiche in westfalen und im rheinland. forstarchiv 26, 197-203. könig, a.o., stauber, t., 2004: haplotypenbestimmung als hilfsmittel. in: löbf/nrw (ed.), vitalität und genetische variabilität der eiche in nordrhein-westfalen. löbf/nrw, recklinghausen. könig, a.o., ziegenhagen, b., van dam, b., csaikl, u., coart, e., degen, b., burg, k. et al., 2002: chloroplast dna variation of oaks in western central europe and genetic consequences of human influences. forest ecol. and manage. 156, 147-166. kremer, a., kleinschmit, j., cottrell, j., cundall, e.p., deans, j.d., ducousso, a., könig, a.o. et al., 2002: is there a correlation between chloroplastic and nuclear divergence, or what are the roles of history and selection on genetic diversity in european oaks? forest ecol. manage. 156, 75-87. liepelt, s., sperisen, c., deguilloux, m.-f., petit, r., kissling, r., spencer, m., de beauliue, j.-l. et al., 2006: authenticated dna from ancient wood remains. ann. bot. 98, 1107-1111. petit, r., aguinagalde, i., beaulieu, j., bittkau, c., brewer, s., cheddadi, r., ennos, r. et al., 2003: glacial refugia: hotspots but not melting pots of genetic diversity. science 300, 1563-1565. petit, r., brewer, s., bordács, s., burg, k., cheddadi, r., coart, e., cottrell, j. et al., 2002a: identification of refugia and post-glacial colonisation routes of european white oaks based on chloroplast dna and fossil pollen evidence. forest ecol. manage. 156, 49-74. petit, r., csaikl, u., bordács, s., burg, k., coart, e., cottrell, j., van dam, b. et al., 2002b: chloroplast dna variation in european white oaks. phylogeography and patterns of diversity based on data from over 2600 populations. forest ecol. manage. 156, 5-26. petit, r., el mousadik, a., pons, o., 1998: identifying populations for conservation on the basis of genetic markers. conservation biol. 12, 844855. rachmayanti, y., leinemann, l., gailing, o., finkeldey, r., 2006: extraction, amplification and characterization of wood dna from dipterocarpaceae. plant mol. biol. reporter 24, 45-55. rozen, s., skaletsky, h.j., 2000: primer3 on the www for general users and for biologist programmers, in: krawertz, s., misener, s. (eds.), bioinformatic methods and protocols: methods in molecular biology, 365386, humana press, totowa, nj, united states. wachter, h., 2001: untersuchungen zum eichensterben in nrw. schriftenreihe der landesforstverwaltung nrw 13, 1-112. weising, k., gardner, r.c., 1999: a set of conserved pcr primers for the chloroplast dna analysis of slavonian oak stands 171 analysis of simple sequence repeat polymorphisms in chloroplast genomes of dicotyledonous angiosperms. genome 42, 9-19. address of the authors: oliver gailing and reiner finkeldey, institute of forest genetics and forest tree breeding, georg-august university göttingen, germany, büsgenweg 2, d-37077 göttingen, email: ogailin@gwdg.de h. wachter, j. heyder and h.-p. schmitt, landesbetrieb wald und holz nordrhein-westfalen, außenstelle arnsberg forstgenbank nrw, obereimer 2a, d-59821 arnsberg << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot 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/pagesize [907.087 680.315] >> setpagedevice vorgabe neu journal of applied botany and food quality 81, 29 35 (2007) 1agronomy faculty, national university of colombia, bogotá, colombia 2 department of fruit science, humboldt-university berlin, germany production, seeds and carbohydrate contents of cape gooseberry (physalis peruviana l.) fruits grown at two contrasting colombian altitudes g. fischer 1, g. ebert 2, p. lüdders 2† (received february 6, 2007) summary in the boyacá region (5ºn 73ºw) of colombia, cape gooseberry ecotypes ‘kenya’, ‘south africa’ and ‘colombia’ were grown at 2,300 and 2,690 m altitude above sea level during 10 months. with increasing altitude a reduction in fruit production was observed, principally through the smaller fruit number, whereas fruit weight units were not affected. the two african ecotypes developed heavier fruits but with a smaller fruit number per plant compared to those of the colombian origin. the greatest harvest peak at 2,300 m was obtained five months after planting (128 fruits/plant) and decreased continuously during the following pickings. at 2,690 m highest harvest peak was obtained 10 months after planting (78 fruits/plant). elevation also influenced fruit development, which lasted 75 days at 2,690 m and 66 days at 2,300 m. percentage of dry matter and sucrose accumulation in fruits increased with decreasing altitude. fruit glucose and fructose contents remained unaffected by the altitudinal factor. at the high location, fruits produced a smaller number and weight of seeds. introduction cape gooseberry (physalis peruviana l.) originates from the andean highlands of south america. it belongs to the family solanaceae, in which the genus physalis includes about 100 species, which form their fruits in an inflated calyx (legge, 1974). in many parts of the andes, cape gooseberries grow wild up to 3,000 m above sea level (fao, 1982). the plant productivity in poor soils, easiness of cultivation, and low requirement for water and fertilizer has made it an attractive potential crop (mccain, 1993). in colombia, it is not only an important source of vitamins (a and c) for the highland farmers, but also has become the second important export fruit (fischer et al., 2005). the tropics are known as the zone of thermal uniformity without marked seasons in all altitudes. lauer (1986) states clearly that temperature has the main effect on altitude classification of tropical mountains. thus, rundel et al. (1994) denominate the climate in tropical alpine environments as having summer every day and winter every night. barcelo et al. (2001) characterized high elevations as regions with high light intensity, low temperature, great diurnal temperature fluctuations, frequent situations of drought, and low co2 and o2 partial pressures. among these, high ultraviolet radiation has been demonstrated to have profound effects on field crops (kakani et al., 2003). growth, production, and quality of fruits are strongly influenced by tropical altitude (fischer, 2000). climatic factors, especially temperature and light intensity, have a strong influence on the nutritional quality of fruits and vegetables, but the effect of site elevation on fruit quality has been studied only in few cases (schaffer and andersen, 1994). lulo (solanum quitoense) develops fruits in andean regions of 15-16°c mean temperature in 140160 days, but with increased temperatures of 17-20°c the duration of fruit development may be only 120-130 days (eraso, 1991). peach varieties, grown at higher elevations (2,500 m) in mexico, delayed their harvest season from 20 to 40 days (perez, 2001). fischer et al. (2000) found that two altitude conditions in colombia, 2,300 and 2,690 m, had no effect on organic acid (citric, malic, tartaric, and ascorbic acid) accumulation in cape gooseberry fruits, but, in contrast, β-carotene and total provitamin a carotenoids were significantly higher at the lower altitude. temperature during fruit growth and maturation influenced quality by either hastening or delaying horticultural maturity (arpaia, 1994). final fruit size is influenced by various factors and is determined by the genetically fixed cell number and also genetically contributed capacity of cell elongation dependent on the environmental conditions (friedrich and fischer, 2000). developing seeds play an important role in fruit development (crane, 1969). seeds are centers of metabolic activity (dennis, 1984), and once have initiated seeds development these seem to exert a predominant effect on fruit growth in many species. although fruit growth and ingredient accumulation are affected by tropical altitude markedly, there is no information about the influence of the tropical altitudinal microsite conditions on growth, seeds, and non-structural carbohydrate contents of cape gooseberry fruits growing on the slopes of the colombian east cordilleras. results of the present ecophysiological study help to make this crop better known and ready for genetic and cultural-technical improvements. the colombian highland farmers will be able to select appropriate locations for cropping physalis in order to enhance fruit production and improve its nutritional quality. material and methods the ecotypes ‘colombia’, ‘kenya’ and ‘south africa’ were propagated by seeds and transplanted after 10 weeks into experimental fields in villa de leyva at 2,300 m and tunja at 2,690 m, both locations in the boyacá department (5ºn 73ºw) of colombia. the climate conditions at 2,300 m were: 17ºc mean air temperature, 19.5ºc mean soil temperature, 66,6% relative humidity (rh), 837 mm annual precipitation, 5.3 daily sunshine hours, and 148 mw uv-b radiation. at 2,690 m, the climate conditions were: 12,5ºc mean air temperature, 16,8ºc mean soil temperature, 79% rh, 302 mm annual precipitation, 5.3 daily sunshine hours, and 160 mw uv-b radiation. because of the close distance to the equator, the monthly mean air temperature oscillated only between 16.6 and 17.9ºc at 2,300 m and 11.0 and 15.1ºc at 2,690 m during the 44 weeks of the growing period. during a period of 24 hours, the leaf temperatures in the surrounding air, inside the calyx and the soil (10 cm depth) were measured. the cape gooseberries were planted at a distance of 1 x 1 m and supported with a v-frame system. the soil at both sites was sandy loam with a corrected ph value of 5.7 and an organic matter content of 4.5%. fertilization, irrigation, plant protection, and pruning practices were applied according to local procedures. a polyfactorial block design was used with three replicates on two locations. date was analyzed using the sas program to determine least significant differences (lsd). fruits were harvested when skin color turned from light-orange to orange, indicating the physiological maturity (fischer et al., 1997). seeds were taken from mature fruits, counted and weighed to evaluate the site and ecotype effect. fruits were picked between 7.00 to 8.00 a.m., and these used for sugar analysis immediately frozen. sucrose, glucose, and fructose contents were determined by high-performance liquid chromatography (hplc) using waters associates 6000a pump, u6k universal injector, r-401 differential refractometer, and waters uv/vis photo diode array detector. for separation, bondapak c18 column (10 µm, 30 x 0.39 cm) was used. isocratic separation of sugars was achieved at 20°c using acetonitrile:water ratio 83:17. injecting 30 ml, the flow rate was adjusted to 1.2 ml·min-1. sample preparation was made following the method of ganzedo and luh (1986). each sample consisted of six replicates representing 20-25 g fruit fw. total carbohydrates were defined as the sum of sucrose, glucose, and fructose. results fruit development fruit development appeared to be affected slightly by the altitude (fig. 1) and followed a sigmoid curve with a marked increase in diameter during the first 20 days reaching nearly 65% of their final size at this early stage. up to 30 days of development, there was no observed effect of the altitudes on fruit diameter, after which fruits at the low elevation grew faster and presented significantly greater diameters than those at the high site, especially between 50 and 60 days of development. at harvest, after 70 days, fruits at the 2,690 m site had reached nearly the same size as those at 2,300 m, because of their over-proportional growth during the last stage (fig. 1). after 70 days, fruit diameter at the low site was still higher than at the 2,690 m location as a main effect (fig. 1). fruits of south african plants were 2.5 cm, significantly the largest, and those from colombia with 1.9 cm were the smallest among the three origins (tab. 1). fig. 1: influence of the altitude (2,300 and 2,690 m) on the development of fruit diameter of three cape gooseberry ecotypes during the experiment in colombia; vertical bars indicate significant differences (lsd) at p < 0.05. days past fruit set 0 10 20 30 40 50 60 70 f ru it d ia m e te r (c m ) 0,0 0,5 1,0 1,5 2,0 2,5 2,300 m 2,690 m the altitude exerted significant effects on the duration of fruit development (observed past fruit set), however this parameter appeared to be unaffected by the plant origin. at the 2,690 m site, fruit development lasted about 75 days, but only 66 days at the low site (tab. 1). the african types needed significantly more time to complete fruit development than the colombian ecotype. as in all other studied yield components, the colombian type did not show any altitude dependence, not even in the period of fruit development (tab. 1). 30 g. fischer , g. ebert , p. lüdders tab. 1: effect of the altitude (2,300 and 2,690 m) on the fruit dry matter accumulation, fruit development period, fruit number and fruit yield of three cape gooseberry ecotypes in colombia; different letters indicate significant differences (lsd) at p < 0.05. parameter altitude mean altitude (m) kenya s. africa colombia fruit development (days) 2,300 63 d 66 d 70 c 66 b 2,690 75 ab 77 a 72 bc 75 a fruit diameter (cm) 2,300 2.31 b 2.56 a 1.86 c 2.24 a 2,690 2.21 b 2.45 a 1.84 c 2.17 b fruit dm accumulation (%) 2,300 17.5 bc 21.7 a 19.6 ab 19.6 a 2,690 16.3 c 16.5 c 18.5 bc 17.1 b unit fruit weight (g) 2,300 6.5 a 6.8 a 4.1 c 5.8 a 2,690 5.9 b 6.7 a 4.3 c 5.6 a fruit number/plant 2,300 226 cd 292 b 383 a 300 a 2,690 156 e 190 de 348 a 231 b fruit yield (kg fw/plant) 2,300 1.468 b 1.970 a 1.564 b 1.667 a 2,690 916 c 1.230 b 1.494 b 1.213 b ecotype fruit dry matter accumulation in general, more dry mass was accumulated in fruits at the low altitude, than in those at the 2,690 m site (tab. 1). kenyan plants not only had the least fruits, but also the lowest dry matter accumulation comparing the three ecotypes. on an average, the kenyan and the colombian ecotypes appeared to have exactly the same fruit dry mass accumulation. the south african ecotype accumulated a considerably higher dry mass in fruits at the 2,300 m location (tab. 1) causing the altitude mean difference. the other two ecotypes only showed tendencies towards this behavior. fruit production the effect of altitude on fruit weight unit was less than on yield, but ecotype differences were more noticeable with this parameter. fruits produced by the colombian type had a markedly lower (35%) unit weight than the others (tab. 1). the south african origin developed significantly highest fruit weight unit. comparing interaction means, a pronounced altitude effect was only produced by the kenyan ecotype (tab. 1), which had significantly higher fruit weight unit at the lower location (2,300 m). conversely, the colombia ecotype tended to have heavier fruits at the high altitude. the accumulated fruit number, harvested until the end of the experiment in may, differed significantly with the altitude, similar to the yield, but also with the plant origin. as a main effect, with lowering the altitude from 2,690 to 2,300 m, significantly more fruits were picked (tab. 1). at its native site, the colombian ecotype produced a noticeably higher fruit number than the two african types, 48% more fruits than the kenyan and 34% more than the south african. nevertheless, from latter origin more fruits were picked than from kenyan plants. with increasing altitude, harvested fruit fresh weight decreased markedly as a main effect (tab. 1). comparing the three ecotypes, main effect means showed uniformly higher fruit fresh weight of the south african and colombian plants than of the kenyan type. as an interaction, only harvested fruit weight of the african origins was significantly higher at the 2,300 m site compared to that of the 2,690 m location (tab. 1). the fruit production of the colombian ecotype remained unaffected by the factor altitude. at the 2,690 m site, south african cape gooseberries produced a clearly heavier fruit yield than the kenyans, however, at this site, colombian plants had highest fruit production of the three ecotypes used. fruit harvest course with reference to the fruit harvest course, realized between november and august, this parameter was significantly affected by the altitude. at the 2,300 m site, the first fruit harvest in december was the highest one (128 fruits/plant), then showed a progressively decreased production in two further harvest peaks, which followed at two-month intervals (fig. 2). after the last peak in april (50 fruits/ plant), production declined and finally increased once more, but slightly, from june to august (12 ➝ 30). conversely, at 2,690 m, fruit numbers picked in december and january were very low (11 and 12 fruits/plant, respectively). in may, highest fruit numbers (78 fruits/plant) were collected at this high site, but afterwards producion decreased dramatically in july (6 fruits) and then increased up to 21 fruits in august. seed content seed number and seed weight differed greatly with altitude and the ecotypes used. in general, number of seeds per fruit and g fruit fw, as well as seed weight per fruit and per unit, increased significantly with lowering the altitude from 2,690 to 2,300 m (tab. 2). in regard of the total seed weight per fruit, differences between the locations were most distinct. fruits at the low site had a 28% higher seed weight than those at the high altitude. however, the parameter seed weight unit was only slightly affected by the site factor. south african fruits not only developed remarkably higher total seed weight, but also much heavier unit seeds than the two other ecotypes. unit seeds from south african fruits were more than twice as heavy as these of colombian fruits. in respect of the seed number per fruit, the kenyan type had significantly higher values than the others. however, colombian fruits contained markedly most seeds per gram fruit fw. although the low altitude increased all recorded seed components, such as seed number and weight per fruit, as well as unit seed weight and number per g fruit, only the kenyan ecotype reacted with a significant increase in fruit weight. the only small differences related to the unit seed weight and seed number per gram fruit between the two locations make apparent that altitude did not exert a sufficient influence on the seeds to induce different fruit weights (tab. 1, 2). 0 20 40 60 80 100 120 140 160 180 h a rv e st e d f ru its ( n /p la n t) 0 20 40 60 80 100 120 140 160 180 kenya south africa colombia 2,300 m 2,690 m nov dec jan feb mar abr may jun jul aug 0 20 40 60 80 100 120 140 160 180 2,690 m 2,300 m fig. 2: effect of the altitude (2,300 and 2,690 m) on the course of harvested fruits during the experiment in colombia (means of three cape gooseberry ecotypes); vertical bars indicate significant differences (lsd) at p < 0.05. cape gooseberry fruits at two colombian altitudes 31 carbohydrate composition glucose content was modified by the ecotypes and was affected by the altitude only in interaction with the plant origin. the colombian ecotype accumulated significantly more glucose in the fruits than the african types did; both the latter had similar glucose values. as an interaction, with increasing altitude kenyan fruits contained markedly more glucose, although south african and colombian ecotypes were not affected by the elevation (fig. 3). similar to glucose the fructose content was also not clearly dependent on the altitude, but noticeably on the ecotype and strong interaction effects were observed. as with the glucose, colombian fruits also contained most fructose compared to the african ecotypes. as an interaction, kenyan fruits accumulated significantly more fructose at the 2,690 m altitude than at the low site, whereas for colombian fruits it was exactly the opposite (fig. 3). sucrose accumulation in fruits was significantly related to the altitude and the ecotype. with decreasing altitude, sucrose content increased, as a main effect. fruits from the kenyan ecotype concentrated remarkably more sucrose than those from south africa. colombian fruits had intermediate sucrose values. further examination of the interaction effect shows that with increasing altitude sucrose content of the two african ecotypes was significantly reduced; the native type colombia only showed a tendency towards this reaction (fig. 3). fruit sucrose content increased with decreasing altitude, whereas analyzed monosaccharides, glucose, and fructose remained unaffected. the parallel increase of fruit dry matter accumulation (tab. 1) indicates that sucrose is the principal component of fruit dry matter. total carbohydrate content remained unaffected by the altitude; however, it was significantly related to the ecotype. in general, the colombian and kenyan ecotypes accumulated more carbohydrates in fruits than the south african. at both locations, the kenyan ecotype concentrated remarkably more total carbohydrates in fruits than the south african origin (fig. 3). kenya s.africa colombia altitude kenya s.africa colombia altitude 0 200 400 600 800 1000 1200 1400 2,300 m 2,690 m d b c c a b a a fructose c a rb o h yd ra te s (m g . 1 0 0 g -1 f w ) 0 200 400 600 800 1000 1200 1400 c ab bc a ab a a glucose 0 1000 2000 3000 4000 5000 b a b c ab bc a b sucrose 0 1000 2000 3000 4000 5000 6000 7000 bc c a ab a a total carbohydrates c a a fig. 3: effect of the altitude (2,300 and 2,690 m) on the glucose, fructose, sucrose, and total carbohydrate content of fruits of three cape gooseberry ecotypes in colombia; different letters indicate significant differences (lsd) at p < 0.05. 32 g. fischer , g. ebert , p. lüdders tab. 2: effect of the altitude (2,300 and 2,690 m) on the fruit seed content of three cape gooseberry ecotypes in colombia; different letters within the same column indicate significant differences (lsd) at p < 0.05. altitude ecotype fruit s e e d (m) (origin) g fw n fruit-1 mg fruit-1 mg unit-1 n g-1 fruit 2,300 kenya 6.0 b 348 a 320 c 0.92 c 58 b south africa 7.7 a 277 c 459 a 1.65 a 36 d colombia 4.9 c 312 b 234 de 0.73 e 66 a mean ecotypes 6.2 ns 312 a 388 a 1.10 a 54 a 2,690 kenya 5.7 b 292 bc 269 d 0.92 c 51 c south africa 7.1 a 249 cd 380 b 1.52 b 35 d colombia 4.6 c 245 d 197 e 0.80 d 53 c mean ecotypes 5.8 ns 262 b 282 b 1.08 b 47 b discussion fruit development and production in regard of fruit diameter growth, the high yielding african ecotypes utilized the site advantage more efficiently and began 30 days before harvest to increase fruit size faster than at the high altitude. however, fruit diameter and fruit weight unit was only slightly affected by the altitude. probably, the mean air temperature of 12.5ºc at the high elevation site was not sufficient enough to affect these parameters more, which is in agreement with agusti (2004), who indicated that, generally, temperatures below 10ºc decrease fruit diameter. fruit development period of the two african ecotypes was prolonged by nearly 3 days for each 100 m of altitude increase. similarly, but more drastically each 100 m of altitude increased growth cycles in banana by one month (samson, 1986). peach varieties grown at higher elevations (2,500 m) in mexico showed harvest season delays from 20 to 40 days (perez, 2001). also ‘valencia’ oranges, grown in the trujillo state of venezuela, showed accelerated fruit growth rates at lower altitudes as compared to the higher located zones (zambrano and quintero, 2001). dry matter accumulation in fruits was increased at 2,300 m, which may indicate higher sink strength or a more efficient translocation of photosynthates as compared with the high altitude field. according to brown (1984), the yield potential of a crop is viewed as a product of the photosynthate produced and the fraction of photosynthate diverted to the fruit. factors known to reduce plant growth, such as low temperatures, water stress, and mineral nutrient deficiencies, also reduce translocation (friedrich and fischer, 2000). the low temperatures at the high location, especially night air temperatures and soil temperatures may explain the declined dry matter allocation to fruits and, on the other hand, the longer fruit development period. regarding fruit set and, therefore, fruit number produced, a critical night temperature has to be taking into account. in tomatoes, the optimal range for fruit set lies between 15 and 20°c (varga and bruinsma, 1986), so that night temperatures recorded at the high altitude of between 5.7 and 13.0°c can have limited fruit set of the cape gooseberries. although the critical night temperature for fruit set in cape gooseberries is still unknown, we observed (data yet not published) a normal fruit set in these species at air temperatures of 12°c, but growth rate and thereby fruit setting were obviously slowed as compared to the 19°c regime. it appears that the higher air temperature regime at the low altitude (2,300 m) with a mean temperature of 17.4°c and a mean maximum of 20.5°c hastened vegetative growth and reproductive maturity (kinet et al., 1985) as compared to the high elevation with 12.5 and 15.1°c, respectively. fischer et al. (2005) established a mean growth temperature between 13 and 18ºc as optimum for cape gooseberries. with reference to evans (1975) it appears evident that the duration of photosynthetic optimal temperatures during the day is more important than the daily mean temperature. thus, cape gooseberry plants at the low altitude received supposed optimal temperatures >20°c for 9 hours per day as compared to only 5 hours at the high elevation. in the altitude experiment, an effect of the root zone temperature cannot be excluded. although soil temperature at the 2,690 m site had a mean value of 16.8°c, the recorded minimum of 13.0°c was four degrees lower than that at 2,300 m. similar to own findings under glasshouse conditions, in where 15°c root zone temperature reduced fruit number markedly as compared to 22ºc (fischer and lüdders, 1992), the low soil temperatures <15°c during 15 hours per day at 2,690 m could have contributed to a shorter shoot growth (lower node number) and, thereby, a reduced fruit set. a close relationship between yield and vegetative plant parts in cape gooseberry was also found by asna et al. (1988), who recorded a high correlation between fruit yield and leaf width as well as plant height. these findings correspond well with our results, demonstrating that the higher yielding plants at the 2,300 m site had larger leaves recorded during all readings and plants were higher, especially during first 24 weeks of experiment (fischer and lüdders, 2002). in tomatoes, fruit weight and volume were also positively correlated with the leaf area per fruit (stenvers and staden, 1976), however total yield depended mainly on the number of fruits. these findings agree completely with the present results. with regard to the two monthly production peaks at the low altitude, this seems to indicate that marked new fruit setting only took place when fruits were fully developed or already harvested. this corresponds with the findings of brown (1984), who indicated that when a plant develops a heavy fruit load, the fruits seem to have priority for the photosynthates over vegetative plant parts. thus, when fruit terminates its intensive sink activity growth, shoot elongation and fruit set increases (friedrich and fischer, 2000). in the present study, after harvesting the fruits (removal of the sinks) again an elongation growth of shoots could have taken place and new fruits were induced in the developing nodes. marcelis (1993) found an increased leaf production in cucumber when number of fruits decreased. the higher number of fruit bearing nodes at the lower elevation affected yield significantly, taking into account that, for the december harvest, the shoot length, which was built in october when fruits were set, was more than 3.5 times higher at 2,300 m than at the 2,690 m location (fischer and lüdders, 2002). the recorded yields of 1.7 kg/plant until may and 2.1 kg/plant (correspond to 21 t·ha-1) during one year at the 2,300 m site are in the reported international range, although increased harvests are possible through the application of trellis systems (fischer et al., 2005). plant yield has been considered a result of photosynthetic (source) capacities on the one hand and capacity to utilize photosynthate (sink activity) on the other (brown, 1984). in plants with indeterminate growth habit such as cape gooseberry, in which reproductive and vegetative growth occurs simultaneously, the two components may compete for photosynthetic products. although higher nar were recorded at the 2,690 m site (fischer and lüdders, 2002), fruit yields expressed as fruit fresh weight per plant were higher at the 2,300 m location, in particular for the african ecotypes. yield potential at the lower site were, however, less higher because of higher unit fruit weights, but rather through an increased fruit number. the higher fruit number for plants at 2,300 m was particularly due to the elongated shoot development with increased numbers of fruitbearing nodes, at least during first 32 weeks of the experiment. seed content many studies have shown a link between seed number, berry development, and sugar and acid content (coombe, 1989; boselli et al., 1995). there exist a marked correlation between the number of seeds and the final fruit size (crane, 1969). the development of seed and fruit are strongly coordinated by hormonal signals, which are originated in the seeds (schopfer and brennicke, 2006). cape gooseberry fruits at two colombian altitudes 33 in two grapevine cultivars, the final size of the berry was related to its seed number: one seeded berries were smaller than 2-34 seeded berries (boselli et al., 1995). these data can be confirmed in our study comparing the colombian and kenyan ecotype, but not with the south african that had lower seed content, but a much higher fruit weight than the colombian origin. probably, in the south african ecotype, hormone production of the heavier seed attracted a higher amount of carbohydrates than in the other types. friedrich and fischer (2000) stated that a strong basipetal flow of auxins results in that fruit develops a high capacity of attraction for fotoassimilates and mineral elements and so can grow more than fruits containing only few seeds. thus, ho (1992) observed that seeded tomato fruits accumulate more dry matter and compete better for assimilate than fruit with fewer of no seeds. in tomato, varga and bruinsma (1986) concluded that the growth of tomato fruit is not directly caused by cell elongation in the pericarp due to seed-produced indol acetic acid, but rather by indirectly profiting from the sink activity exerted by the developing seeds. in agreement with results of varga and bruinsma (1986), who observed that tomato fruits with a lower seed number took more time in developing, we found the same tendency in the cape gooseberries growing at the 2,690 m altitude (tab. 1, 2), but there, certainly, the lower temperature exerted its effect too on amplifying fruit development period. studying the same three cape gooseberry ecotypes grown in the 2,464 m high nuevo colón municipality in the colombian boyacá department peña and ayala (2006) found that ‘colombia’ ecotype presented the highest seed index (number of seeds /100 g fruit weight) and had the most important relationship between the fruit fresh weight and the weight of seeds per fruit (r2 = 0.87). carbohydrate composition like the majority of edible fruits, cape gooseberry contains three major sugars, i.e. sucrose, glucose, and fructose (sugiyama et al., 1991). the increased sucrose amounts at the warmer site (2,300 m) point out that there is a positive relation between the temperature and translocation rates of this sugar (brown, 1994). guardiola and garcia-luis (1993) stated that a lower organ temperature can increase the viscosity of the phloem solution and, thereby, reduce transport rates. in the present experiment, sucrose content was especially high in kenyan fruits as the only ecotype, which produced significantly greater fruits and highest seed number at the 2,300 m site as compared to 2,690 m. thus, it seems that temperature affected the translocation of assimilates into the fruit largely through its effect on the organ with the greater demand for assimilates (wardlaw, 1980). kozlowski (1992) points out that the capacity of reproductive structures to import carbohydrates varies with the growth characteristics of the fruit, besides plant age and vigor as well as time of the experiment. with the higher seed content at the lower altitude there could be an increased sink capacity of these fruits, which in turn allows a higher inflow of sucrose (friedrich and fischer, 2000). ripening berries are a strong sink for solutes from photosynthesis and reserve organs (coombe, 1989). this relationship has been attributed to hormones such as auxins, gibberellins and cytokinins formed in the seeds and spread into the pulp, thus promoting cell division and extension and modifying pulp composition (scienza et al., 1978). references agusti, m., 2004: fruticultura. ediciones mundi-prensa, madrid. arpaia, m.l., 1994: preharvest factors influencing postharvest quality of tropical and subtropical fruit. hortscience 29, 982-985. asna, a.b., yaday, s.p., srisvastava, s.r., pandey, p.k., jaiswal b.k., 1988: correlation and path coefficient analysis in cape gooseberry (physalis peruviana l.). south ind. hort. 36, 5-7. barcelo, j., nicolas, g., sabater, b., sanchez, r., 2001: fisiología vegetal. ed. pirámide, madrid. boselli, m., volpe, b., di vaio, c., 1995: effect of seed number per berry on mineral composition of grapevine (vitis vinifera l.) berries. j. hort. sci. 70, 509-515. brown, r.h., 1984: growth of the green plant. in: tesar, m.b. (ed.), physiological basis of crop growth and development, 153-174. amer. soc. agron., madison, wisconsin. coombe, b.g., 1989: the grape berry as a sink. acta hort. 239, 149-158. crane, j.c., 1969: the role of hormones in fruit set and development. hortscience 4, 1969-1970. dennis, f.g. jr., 1984: fruit development. in: tesar, m.b. (ed.), physiological basis of crop growth and development, 265-289. amer. soc. agron., madison, wisconsin. eraso, b., 1991: ecological effects on the physiology of the lulo, solanum quitoense. in: hawkes, j.g., lester, r.n., nee, m., estrada, n. (eds.), solanaceae iii: taxonomy, chemistry, evolution, 451453. royal botanic gardens kew and linnean society of london. evans, l.t. (ed.), 1975: crop physiology. cambridge university press, london. fao, 1982: fruit bearing forest trees: technical notes. fao forestry paper 34, 140-143. fischer, g., 2000: ecophysiological aspects of fruit growing in tropical highlands. acta hort. 531, 91-98. fischer, g., lüdders, p., 1992: effect of root-zone temperature and tropical altitude on growth and development of cape gooseberry (physalis peruviana l.). acta hort. 310, 189-198. fischer, g., ebert, g., lüdders, p., 2000: provitamin a carotenoids, organic acids and ascorbic acid content of cape gooseberry (physalis peruviana l.) ecotypes grown at two tropical altitudes. acta hort. 531, 263-267. fischer, g., lüdders, p., 2002: efecto de la altitud sobre el crecimiento y desarrollo vegetativo de la uchuva (physalis peruviana l.). revista comalfi (bogotá) 29, 1-10. fischer, g., lüdders, p., gallo, f., 1997: qualitätsveränderungen der kapstachelbeere während der fruchtreifung. erwerbsobstbau 39, 153-156. fischer, g., miranda, d., piedrahíta, w., romero, j. 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(ed.), grundlagen des pflanzenbaues in den tropen und subtropen, 26-45. verl. ulmer, stuttgart. legge, a.p., 1974: notes on the history, cultivation and uses of physalis peruviana l. j. royal hort. soc. 99, 310-314. marcelis, l.f.m., 1993: leaf formation in cucumber (cucumis sativus l.) as influenced by fruit load, light and temperature. gartenbauwissenschaft 58, 124-129. mccain, r., 1993: goldenberry, passionfruit & white sapote: potencial for cool subtrpical areas. in: janick, j., simon, j.e. (eds.), new crops, 479486. wiley, new york. peña, j.f., ayala, j.d., 2006: relación semilla/fruto en tres ecotipos de uchuva (physalis peruviana l.). thesis, agronomy faculty, national university of colombia, bogotá. perez, s., 2001: factors associated with peach yield, quality and postharvest behavior. in: dris, r., niskanen, r., jain, s.m. (eds.), crop management and postharvest handling of horticultural products. vol. quality management, 157-167. science publishers, enfield, new hampshire. rundel, p.w., smith, a.p., meinzer, f.c. (eds.), 1994: tropical alpine environments – plant form and function. cambridge univ. press, cambridge. samson, j.a., 1986: tropical fruits. 2nd edition. tropical agriculture series, longman, new york. schaffer, b., andersen, p.c. (eds.), 1994: handbook of environmental physiology of fruit crops. vol ii: subtropical and tropical fruits. crc press, boca raton, florida. schopfer, p., brennicke, a., 2006: pflanzenphysiologie. elsevier, münchen. scienza, a., miravalle, r., visai, c., fregoni, m., 1978: relationships between seed number, gibberellin and abscisic acid levels and ripening in cabernet sauvignon grape berries. vitis 17, 361-368. stenvers, n., staden, o.l., 1976: growth, ripening and storage of tomato fruits (lycopersicon esculentum mill.). iii. influence of vegetative plant parts and effects of fruit competition and seed number on growth and ripening of tomato fruits. gartenbauwissenschaft 41, 253-259. sugiyma, n., roemer, k., bünemann, g., 1991: sugar pattern of exotic fruits from the hannover market, germany. gartenbauwiss. 56, 126-129. varga, a., bruinsma, j., 1986: tomato. in: monselise, s.p. (ed.), handbook of fruit set and development, 461-481. crc press, boca ratón, florida. wardlaw, i.f., 1980: translocation and source-sink relationships. in: carlson, p.s. (ed.), the biology of crop productivity, 297-339. academic press, new york. zambrano, j., quintero, i., 2001: quality evaluation of ‘valencia’ orange fruits (citrus sinenesis) from three altitudinal levels of trujillo state, venezuela. hortscience (abstract) 36, 595. address of the authors: prof. dr. gerhard fischer1, facultad de agronomía, universidad nacional de colombia, a.a. 14490, bogotá, colombia. dr. georg ebert2, prof. dr. peter lüdders†, department of fruit science, humboldt-university berlin, albrecht-thaer weg 3, d-14195 berlin, germany. 1 corresponding author: gerfischer@gmail.com 2 now: agricultural advisory department, k+s kali gmbh, p.o.box 102029, d-34111 kassel, germany cape gooseberry fruits at two colombian altitudes 35 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile 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<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> >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice journal of applied botany and food quality 86, 71 78 (2013), doi:10.5073/jabfq.2013.086.011 department of horticulture, faculty of agriculture, shiraz university, shiraz, iran growth and physiological responses of grafted and non-grafted cultivars of ziziphus spina-christi to salinity akhtar shekafandeh*, shohreh takhti (received may 27, 2013) * corresponding author summary two grafted cultivars of z. spina-christi (‘danehgird’ and ‘danehboland’ were grafted on wild seedlings) and non-grafted seedlings were grown in soil and perlite mixture (1:1 v/v) and treated with 0 (0 ds m-1), 3.2 (5 ds m-1), 6.4 (10 ds m-1) and 12.8 (20 ds m-1) g l-1 nacl. after 16 weeks, salt stress resulted in a substantial decrease in root length, stem, leaf surface, lateral root number, root, stem and leaf fresh and dry weight in both grafted cultivars and seedling rootstock. these reductions were predominance in seedling rootstock than in grafted cultivars. in all organs (leaf, stem and root) of grafted ‘danehboland’ cultivar, the k+/na+ ratio was significantly higher than non-grafted wild seedling in saline and non-saline conditions. the proline and soluble sugar also was significantly higher in the leaves of ‘danehboland’ cultivar than non-grafted control. the results imply the predominance of the scion genotype in determining salt tolerance in comparison with rootstock seedling. introduction widespread in tropical and subtropical regions, ziziphus spinachristi (l.) desf. belonging to the rhamnaceae family is a spiny shrub or small tree (johnston, 1963) that are naturally distributed in southern parts of iran and locally named ‘konar’. this species is a multipurpose plant. in addition to high value of non wood products, particularly leaves and shoots saponin, tannin substrates and fruit nutrition, this species is ecologically and economically important for its tolerance to drought and salinity (sudhersan and hussain, 2003; sohail, 2009). the fruit is a drupe (about 1 to 1.5 cm in diameter) and is consumed either fresh or dried by the rural population (saied et al., 2008). in the south of iran, a wide variability in yield, fruit size, peel colour was observed among the genotypes (torahi, 2011). at present, the seedlings are used as rootstock for cultivars with high fruit quality (takhti and shekafandeh, 2012). salinity is a main problem in arid and semi-arid regions of the world (gebauer et al., 2004). global warming and low rainfall intensify this difficulty. the saline growth condition causes many unfavorable effects on plant growth and development at physiological and biochemical levels, which are due to low osmotic potential of soil solution (osmotic stress), specific ion effects, nutritional imbalance or combination of these factors (parida and das, 2005; arzani, 2008). due to water scarcity and exacerbated soil salinity in some part of iran, cultivation of other fruit trees such as citrus is limited, so, orchardists established spina-christi grafting orchards in which selected genotypes with superior edible fruit are grafted on spinachristi seedling. despite the fact that the most documents on salt tolerance of plants reported the important role of rootstocks, for they represent the first part to control the uptake and translocation of nutrients and salts throughout the plant (munns, 2002; zrig et al., 2011), there are many reports that clarify the predominant role of scion genotype in plant growth and improvement of salt effect (moya et al., 2002; santa-cruz et al., 2002; chen et al., 2003). colla et al. (2010) expressed that grafting is an integrative mutual process and both scion and rootstock can influence growth and greater root to shoot ratio and lower accumulation of na+ and/or cl in shoots than ungrafted or self-grafted. the proline accumulation has often been proposed as a valuable marker for the selection of salt tolerance genotypes (zid and grignon, 1991). in relation to spina-christi ‘konar’, despite its highly appreciated fruits in local markets and increasing the area under cultivations, there is limited information on physiological responses and the extent of salt tolerance of these grafted plants. the objective of this research was to study the effect of different levels of nacl salinity on growth parameters and physiological responses of three selected local genotypes non-grafted wild type, ‘danehgird’ and ‘danehboland’ grafted on wild spina-christi seedlings. material and methods one year-old grafted ‘konar’ plants (‘danehgird’ and ‘danehboland’ cultivars were grafted on wild seedlings) and non-grafted seedlings were used in this experiment. the plants were transferred into 20 liter plastic pots without drainage and filled with a mixture of soil (laom soil) and perlite (1:1, v:v). field capacity of soil mixture was measured using cell pressure device (santa barbara calif soil equipment co.) and accordingly, plants were irrigated once every three days. commercial fertilizer npk (20:20:20) including; iron (1 g l-1), manganese (0.5 g l-1), zinc (0.5 g l-1), copper (0.04 g l-1), boron (0.2 g l-1), molybdenum (0.04 g l-1) and calcium nitrate (0.5 g l-1) was applied to pots with irrigation water each week. the pots were placed into a plastic greenhouse with natural sunlight and temperature range 32± °c and 20± °c in the day and at night respectively. after 10 weeks when plants were well established, salinity treatments were applied. for preventing salinity shock, the salts were applied in three times with irrigation water. treatments were 0 (0 ds m-1), 3.2 (5 ds m-1), 6.4(10 ds m-1) and 12.8 (20 ds m-1) g l-1 nacl. at the ends of experiment (after 16 weeks), the leaf number of all treatments were counted. leaf area was measured by leaf area meter (am 200, adc bio scientific ltd. taiwan). all 8 plants in each replicate were individually harvested, the lateral roots were counted, stem and root length were measured with a ruler, then they were divided into stems, leaves and roots after recording their fresh weight, all plant organs were oven-dried at 80 °c for 48 h then weighed again. ion content dry-ashing of plant material was obtained at 500 °c for 6 hr. sodium (na+) and potassium (k+) contents of samples were measured after digestion process in 1 n nitric acid solution (hno3-), using the flame emission photometry. chloride (cl−) content was analyzed by titration with 0.1n silver nitrate (agno3) in the presence of potassium bichromate according to a modified colorimetric method of mohr (mathieu and pieltain, 2003). 72 a. shekafandeh, s. takhti chlorophyll content fresh leaf samples were washed with deionized water prior to extraction to remove any surface contamination. one g leaf sample was squashed in 80% acetone using a pestle and mortar. the mixture was centrifuged at 4800 rpm for 20 min. the optical density of the supernant was measured at 663 and 645 nm wavelengths and chlorophyll content was calculated using the following equations. mg chl g f.w. = [20.2 (od645nm) + 8.02 (od663nm)] × v / f.w. × 1000 where v is final volume of solution (ml) and f.w. leaf fresh weight (mg). proline contents to determine proline content of the leaves, 0.5 g plant material was homogenized in 10 ml of 3% aqueous solution of sulphosalicylic acid and the homogenate filtered through whatman # 2 filter paper. two ml of filterate was reacted with 2 ml acid ninhydrin and 2 ml of glacial acetic acid in a test tube for 1 h at 100 °c and the reaction terminated in an ice bath. the reaction mixture was extracted with 4 ml toluene, mixed for 15 ~ 20 seconds. chromophore containing toluene was aspirated from the aqueous phase and the absorbance was read at 520 nm in a spectrophotometer. the proline concentration (μ mole g-1 f.w.) was calculated based on a standard curve. soluble sugars content soluble sugars were extracted from 0.1 g fresh leaves in 5 ml of 80% ethanol placed in a water bath at 70 °c for 30 min. the insoluble remains were removed by centrifuging at 5000 g for 10 min. one ml of the resulted extract was mixed with 5ml of sulfuric acid and 1 ml of 5% phenol solution. the mixed solution was vortexed after cool down to room temperature. the absorbance was recorded at 490 nm using a spectrophotometer. the soluble sugar content of each sample was determined using a standard curve for glucose and expressed as mg glucose g-1 f.w. (mccready et al., 1950; dubois et al., 1956). starch content from previous section, the solid debris remaining in the centrifuge tube was washed, re-extracted and re-centrifuged four-times using 80% (v/v) ethanol. the starch content in the samples was determined colorimetrically using anthrone method (mccready et al., 1950). the absorbance was read at 630 nm in a digital spectrophotometer as described by lópez et al. (2002). statistical analysis a factorial experiment 3 (two grafted and one non-grafted ‘konars’) × 4 (4 levels of salinity) was arranged in complete randomized design (crd) with 4 replicates and 2 plants in each replication. data were subjected to analysis of variance using the spss software (ver. 13.0) spss inc. mean differences were determined by tukeys tests at p≤0.05. results the results showed that leaf area, stem and root length were significantly reduced by increasing salinity. however, the reduction in growth parameters varied according to cultivar and measured factor so that, in 20 ds m-1 nacl, the highest stem reduction (58.1%) was observed in ‘danehboland’ and lowest reduction (50.5%) in wild cultivar. in contrast, the highest root length reduction (78.6%) was observed in wild types and the lowest root reduction was observed in grafted ‘danehboland’ by 44% (tab. 1). the effect of salinity on lateral root number, root fresh and dry weight showed a significant genotypic variation (tab. 2). in nonsaline condition, scions ‘danehboland’ and ‘danehgird’ promoted significantly lateral root number in comparison with control. the highest reduction in lateral root number was found in wild type rootstock (without grafting), which reached by 62.5% at 20 ds m-1 nacl, whereas the lowest one was obtained in ‘danehboland’ with 50% decreased when compared to control plants. regardless of salt treatments, scion ‘danehboland’ also produced more root fresh (15.4 g) tab. 1: sodium chloride effect on leaf surface, stem and root length of non-grafted wild, grated ‘danehgird’ and ‘danehboland’ cultivars of z. spina chiristi. cultivar nacl leaf surface mean stem length mean root length mean ds m-1 (mm2) (cm) (cm) non-grafted 0 14.0a-d 80.6b 19.8bc wild 5 13.2c-f 60.1de 11.4f 10 12.6def 50.0ef 7.0g 20 11.9f 39.9fg 4.2g 12.9b 57.6b 10.6c grafted 0 14.9ab 83.0ab 21.7ab danehgird 5 14.2abc 64.3cd 17.6cd 10 13.3c-f 50.1ef 16.1de 20 12.3ef 35.8g 13.2ef 13.7a 58.3b 17.1b grafted 0 15.3a 94.1a 24.4a danehboland 5 14.2abc 74.6bc 19.8bc 10 13.4b-e 53.1de 18.1cd 20 12.4ef 39.4fg 13.9ef 13.8a 65.3a 19.1a in each column, means followed by the same letter are not significantly different at p≤0.05, using tukey’s test. (n=8). responses of grafted and non-grafted z. spina-christi to salinity 73 and dry weight (8.45 g) than scion ‘danehgird’ (13.8 and 7.55 g respectively) and wild type (4.6 and 2.6 g respectively) (tab. 2). leaf and stem fresh and dry weight were also significantly reduced by salinity treatments. however, this reduction is varied according to cultivar (tab. 3). for example, in 20 ds m-1 nacl wild type, ‘danehboland’ and ‘danehgird’ showed 77.5, 68 and 79% reduction in leaf dry weight respectively in comparison with control plants. regardless of salinity levels, scion ‘danehboland’ showed higher stem fresh (12.4 g) and dry weight (7.7 g) than scion ‘danehgird’ (11 and 6.7 g respectively) and wild type (4.6 and 2.67 g). data showed that grafted cultivars had different behavior in na+ and k+ uptake in comparison to non-grafted wild type. in all grafted and non-grafted plants, with increasing salinity in culture media the na+ concentrations in different organs (root, stem and leaf) increased, however, regardless of different level of salinity, the accumulation of na+ was significantly lower in all organs of ‘danehboland’ and tab. 2: sodium chloride effect on lateral root number, root fresh and dry weight of non-grafted wild, grafted ‘danehgird’ and ‘danehboland’ cultivars of z. spina-christi. cultivar nacl lateral mean root f.w. mean root d.w. mean ds m-1 root number (g) (g) non-grafted 0 19.8d 8.4d 5.3de wild 5 16.2de 5.4e 3.1fg 10 12.3e 3.0f 1.6gh 20 7.4f 1.5f 0.67h 13.9c 4.6c 2.7b grafted 0 31.7b 20.7a 11.8ab danehgird 5 25.8c 15.9b 8.6c 10 19.9d 11.7c 6.1d 20 13.2e 6.9de 3.8ef 22.6b 13.8b 7.6a grafted 0 38.9a 21.8a 13.1a danehboland 5 32.4b 17.3b 10.7b 10 26.2c 12.1c 6.2d 20 18.9d 7.9d 3.8ef 29.1a 15.4a 8.5a in each column, means followed by the same letter are not significantly different at p≤0.05, using tukey’s test. (n=8). tab. 3: sodium chloride effect on leaf, stem fresh and dry weight of non-grafted wild, grafted ‘danehgird’ and ‘danehboland’ cultivars of z. spina-christi. cultivar nacl leaf mean leaf mean stem f.w. mean stem d.w. mean ds m-1 f.w. (g) d.w. (g) (g) (g) non-grafted 0 6.2a 3.4ab 7.8ef 4.7efg wild 5 3.3bcd 1.9cd 5.1gh 2.9ghi 10 1.8def 1.2c-f 3.5hi 2.0hi 20 1.2f 0.7ef 2.2i 1.1i 3.1ab 1.8a 4.6c 2.7c grafted 0 4.0b 2.3bc 16.5b 10.4b danehgird 5 3.0b-e 1.5c-f 11.2d 7.4cd 10 2.3c-f 1.1def 9.5de 5.3ef 20 1.5ef 0.7ef 6.8fg 3.6fgh 2.7b 1.4b 11.0b 6.7b grafted 0 6.8a 4.1a 19.1a 13.3a danehboland 5 3.7bc 1.9cde 13.8c 8.4c 10 2.3c-f 0.8def 10.3d 5.7de 20 1.8def 0.6f 6.4fg 3.5fgh 3.6a 1.8a 12.4a 7.7a in each column, means followed by the same letter are not significantly different at p≤0.05, using tukey’s test. (n=8). 74 a. shekafandeh, s. takhti ‘danehgird’ cultivars in comparison to wild type (tab. 4, tab. 5 and tab. 6). for example, leaf na+ concentration in ‘danehboland’ and ‘danehgird’ was 11.4 and 12.4 mg g-1 d.w. respectively which was significantly lower than wild type (17.5 mg g-1 d.w.). in 5, 10 and 20 ds m-1 nacl, leaves of all genotypes showed a significant decrease in k+ concentration (tab. 6). the same trend observed for accumulation of k+ in stem, and root. regardless of salt concentration, all organs (leaf, stem and root) of grafted cultivars maintained a higher ratio of k+/na+ than wild type. (tab. 4, tab. 5 and tab. 6). with increasing salinity, the clcontent in all plant parts (leaf, stem and root) of grafted and non-grafted cultivars increased. but this accumulation is more pronounced in roots than stems and leaves tab. 4: sodium chloride effect on root na+, k+ (mg g-1 d.w.) and k+/na+ ratio of non-grafted wild, grafted ‘danehgird’ and ‘danehboland’ cultivars of z. spina-christi. cultivar nacl root na+ mg mean root k+ mg mean root mean ds m-1 g-1 d.w. g-1 d.w. k+/ na+ non-grafted 0 24.2de 20.3cd 0.8bcd wild 5 28.8c 17.2ef 0.6d 10 34.3b 16.1fg 0.5d 20 37.9a 14.7g 0.4d 31.3a 17.1c 0.6c grafted 0 14.3g 21.7bc 1.5bc danehgird 5 18.1f 18.7de 1.0bcd 10 22.4e 17.3ef 0.8cd 20 25.4d 15.3fg 0.6d 20.1b 18.2b 1.0b grafted 0 9.0h 30.1a 3.5a danehboland 5 14.4g 23.1b 1.6b 10 18.8f 20.3cd 1.1bcd 20 22.9de 16.4efg 0.7cd 16.3c 22.5a 1.7a in each column, means followed by the same letter are not significantly different at p≤0.05, using tukey’s test. (n=8). tab. 5: sodium chloride effect on stem na+, k+ (mg g-1 d.w.) and k+/na+ ratio of non-grafted wild, grafted ‘danehgird’ and ‘danehboland’ cultivars of z. spina-christi. cultivar nacl stem na+ mean stem k+ mean stem k+/na+ mean ds m-1 mg g-1 d.w. mg g-1 d.w. non-grafted 0 15.2e 28.8c 1.9cd wild 5 21.7cd 23.2d 1.2def 10 26.4b 18.3e 0.7ef 20 31.0a 15.6f 0.5f 23.6a 21.5c 1.1c grafted 0 10.0fg 31.6ab 3.2b danehgird 5 15.4e 27.7c 1.8cde 10 19.8d 22.3d 1.1def 20 23.2c 17.0ef 0.7ef 17.1b 24.6b 1.7b grafted 0 7.7g 34.0 4.5a danehboland 5 11.1f 29.3bc 2.7bc 10 16.4e 23.0d 1.4def 20 21.8d 17.8ef 0.8def 14.2c 26.0a 2.4a in each column, means followed by the same letter are not significantly different at p≤0.05, using tukey’s test. (n=8). responses of grafted and non-grafted z. spina-christi to salinity 75 (tab. 7), which means the plants limited the translocation of clto aerial parts. in all cultivars, with increasing nacl the amount of sugar increased and the amount of starch decreased and in all levels of salinity, ‘danehboland’ leaves showed significantly higher sugar and starch contents in comparison with non-grafted wild type and grafted ‘danehgird’ (fig. 1). the leaf proline content increased with increasing the salinity in all cultivars and in contrast the amount of chlorophyll decreased. in all levels of salinity, scion-stock relationship had no effect on chlorophyll content in comparison with nongrafted wild type, but in 3.2 and 6.4 g l-1 nacl scion ‘danehboland’ cultivar showed higher proline concentration in comparison with others (fig. 2). tab. 6: sodium chloride effect on leaf na+, k+ (mg g-1 d.w.) and k+/na+ ratio of non-grafted wild, grafted ‘danehgird’ and ‘danehboland’ cultivars of z. spina-christi. cultivar nacl leaf na+ mean leaf k+ mean leaf k+/na+ mean ds m-1 mg g-1 d.w. mg g-1 d.w. non-grafted 0 12.3ef 30.7b 2.5cde wild 5 15.7cd 25.7c 1.7ef 10 18.6b 22.2de 1.2ef 20 23.6a 18.1g 0.8f 17.5a 24.2c 1.5b grafted 0 8.3gh 35.6a 4.3ab danehgird 5 10.1fg 30.3b 3.0bcd 10 13.4de 24.2cd 1.8def 20 17.7bc 18.4fg 1.1f 12.4b 27.1b 2.5a grafted 0 7.4h 36.8a 5.0a danehboland 5 9.1gh 32.4b 3.6abc 10 12.4ef 26.2c 2.1def 20 16.7bc 21.2ef 1.3ef 11.4b 29.2a 3.0a in each column, means followed by the same letter are not significantly different at p≤0.05, using tukey’s test. (n=8). tab. 7: sodium chloride effect on leaf, stem and root cl− (%) of non-grafted wild, grafted ‘danehgird’ and ‘danehboland’ cultivars of z. spina-christi. cultivar nacl root cl− mean stem cl− mean leaf cl− mean ds m-1 % % % non-grafted 0 5.1h 2.9h 3.0g wild 5 7.6fg 3.9gh 4.4fg 10 8.9ef 6.7cde 6.6de 20 11.6cd 8.1bc 6.8de 8.3b 5.4c 5.2c grafted 0 5.5gh 3.6h 4.5fg danehgird 5 7.9f 5.4efg 5.5ef 10 9.1ef 7.2bc 7.2cd 20 13.1c 8.8b 7.3bcd 8.9b 6.2b 6.1b grafted 0 10.2de 4.4fgh 7.1cd danehboland 5 12.9c 5.8def 8.5abc 10 19.5b 7.8bc 8.9ab 20 22.7a 9.9a 9.2a 16.3a 6.9a 8.4a in each column, means followed by the same letter are not significantly different at p≤0.05, using tukey’s test. (n=8). 76 a. shekafandeh, s. takhti discussion salt stress resulted in a considerable decrease in leaf surface, shoot and root length, lateral root number, root, stem an leaf fresh and dry weight in both grafted cultivars and seedling rootstock of spinachristi ‘konar’. these reductions were predominant in seedling rootstock than in grafted cultivars (tab. 1, tab. 2 and tab. 3), which means that different cultivars grafted on the same seedling rootstock improved the growth behavior of the root system. in respect to some traits such as lateral root number, shoot and root length, grafted ‘danehboland’ cultivar even achieved better than grafted ‘danehgird’. hence, the scion genotype played a major role in establishing the growth rate of grafted ‘konars’, which is in accordance with the results obtained in salt stress citrus (banuls and primomillo, 1995; chen et al., 2003) and tomato (santa-cru, 2002). in grape vines, sivritepe et al. (2010) explained the preference of the scion genotype by determining variation in leaf-levels physiological characters of grafted vines. the most important parts of the plant exposed with soil-related stress factors such as salinity are root systems. the enhanced salt tolerance of grafted ‘konar’ cultivars may be associated with the root system. in fact, in non-saline condition, grafting significantly increased lateral root and root dry weight in comparison with nongrafted seedling rootstock. in salt stress condition, two of the above factors decreased, but the reduction in grafted plants was significantly smaller than in non-grafted seedlings. in tomatoes, he et al. (2009) also observed that root dry mass declined at high nacl in comparison with non-saline conditions, but the decrease was smaller in rootstock-grafted plants. in non-saline condition, the na+ ion content in root of the grafted ‘konar’ cultivars were significantly lower than na+ ion content in root of seedling rootstock, that means, scion genotypes limited the na+ uptake from root zone. in the same trend, in salt stress condition (5, 10, 20 ds m-1 nacl) the accumulation on na+ in root of grafted plants were significantly less than non-grafted controls. in addition, na+ content in stems and leaves of rootstock seedlings and in grafted ‘konar’ cultivars were less than their roots in all levels of salt treatments conditions. the preservation of na+ in the roots of two grafted ‘konars’ aid to decline its concentration in the leaves that may help to avoid affecting plant metabolism processes. in this respect, tabatabaie (2010) previously reported similar results with olive tree cultivars subjected to high salt stress, where salt tolerance fig. 2: sodium chloride effect on leaf total chlorophyll (mg g-1 f.w.) and leaf proline content (µmole g-1 f.w.) of non-grafted wild, grafted ‘danehgird’ and ‘danehboland’ cultivars of z. spina-christi. in each measured factor (chlorophyll, proline), means followed by the same letter are not significantly different at p≤0.05, using tukey’s test. the data are the means±se (n=8). fig. 1: sodium chloride effect on leaf sugar, starch content mg g-1 d.w. of non-grafted wild, grafted ‘danehgird’ and ‘danehboland’ cultivars of z. spina christi. in each measured factor (sugar, starch), columns followed by the same letter are not significantly different at p≤0.05, using tukey’s test. the data are the means±se ( n=8). responses of grafted and non-grafted z. spina-christi to salinity 77 in olive trees was hypothesized to be associated with the ability to reduce the uptake and/or transport of saline ions. in both grafted cultivars and seedling rootstock, with increasing salinity, the leaf k+/na+ ratio decreased, which can be attributed to a failure in the k+/na+ selectivity mechanisms. in the leaves of two grafted cultivars the k+/na+ ratio was recorded above 2 in all salt concentrations, except in 20 ds m-1 nacl for grafted ‘danehboland’ and in 10 ds m-1 and 20 ds m-1 in grafted ‘danehgird’ (tab. 6). a high k+/na+ ratio in the leaves is often considered as a salt-tolerance marker (chartzoulakis et al., 2002; dasgan et al., 2002; meena et al., 2003). so, scion genotypes had an ameliorating effect on k+/ na+ ratio which helps the grafted plants do better their metabolic functions. the finding from chloride analysis showed that both grafted ‘konars’ and seedling rootstock exhibited a significant variation at all salinity levels and in all plant parts (leaf, stem and root). grafted and ungrafted ‘konar’ plants limited the translocation of clto the aerial parts; which is an important mechanism to avoid the deleterious effects of salinity in plants (chartzoulakis, 2005), however, grafted ‘danehboland’ cultivar accumulated significantly more clin their different parts than non-grafted seedlings. it is interesting to note that this grafted cultivar had higher root, stem and leaf fresh and dry weight and also accumulated more k+ than non-grafted rootstock. it seems that this cultivar uses clto balance electrical charge and ph of their cells (roussos et al., 2006). k+ is the major cation within plants which counter balance negative charge of anions and stabilizes the ph, osmotic potential and turgor pressure within cells. it also plays a crucial role in the activation of the enzyme involved in the metabolism (chelli-chaabouni et al., 2010). in the present study, a positive correlation was recorded between proline and nacl concentration in the leaves of non-grafted wild seedlings and two grafted cultivars. the same trend was observed between soluble sugar and salt concentration. it has been shown that these compatible solutes play a major role in osmoregulation (hare et al., 1998; parida and das, 2005; rejskovà et al., 2007). in this regard, grafted ‘danehboland’ achieved better than grafted ‘danehgird’ and non-grafted wild type. the results are in agreement with those previously reported by hokmabadi et al. 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(ishs) 864, 45-48. http://www.actahort.org/books/864/864_4.htm. zid, e., grignon, c., 1991: les tests de sélection précoce pour la résistance des plantes aux stress. cas des stress salin et hydrique. in: aupelf-uref (ed.), l’amélioration des plantes pour l’adaptation aux milieux arides, 91-108. john libbey eurotext, paris. zrig, a., tounekti, t., mohamed vadel, a., ben mohamed, h., valero, d., serrano, m., chatara, c., khemira, h., 2011: possible involvement of polyphenols and polyamines in salt tolerance of almond rootstocks. plant physiol. biochem. 49, 1313-1322. address of the authors: dr. akhtar shekafandeh and eng shohreh takhti, department of horticulture, faculty of agriculture, shiraz university, shiraz, iran, p.o. box 7144465186. e-mail; shekafan@shirazu.ac.ir e-mail; shoreh_g_t@yahoo.com journal of applied botany and food quality 86, 167 171 (2013), doi:10.5073/jabfq.2013.086.022 institute of animal nutrition and functional plant compounds, university of veterinary medicines, vienna, austria self-incompatibility and male sterility in six matricaria recutita varieties b. faehnrich*, p. nemaz, ch. franz (received july 25, 2013) * corresponding author summary on purpose to develop suitable mother lines for a large scale hybrid production of the medicinal crop species matricaria recutita [l.] rauschert investigations to – determine the proportions of self and cross fertilization, – identify self-incompatible genotypes and – observe spontaneous or induced male sterility were undertaken. the seed production per flower head under isolated and under untreated control-conditions was evaluated in six german chamomile varieties. under assumption of equal behaviour of plants concerning their reproduction rate results showed opposed reactions of the auto-tetraploid ‘manzana’ and the diploid ‘degumille’ to isolation. moreover, slightly significant interactions between cultivars and treatment concerning seed production per flower head indicate the influence of isolation being cultivar specific. an average of 22 % of self-fertilization under open pollination conditions is displayed over all cultivars. highly significant differences between cultivars reveal cultivar specific self-incompatibility, at which the diploid varieties ‘bona’ and ‘degumille’ exhibit the highest levels of 25 % and 28 %, respectively. 9 of 1105 plants showed spontaneous single flower head aberrations indicating possible tendencies to male sterility. spontaneous pollen sterility, estimated after aceto-carmine staining, reached mean levels between 0.5 % (‘bona’) and 2.4 % (‘lutea’). the progeny after crossings across all six varieties showed an average of 10.20 % pollen sterility indicating a tendency of increasing pollen sterility by intra-specific crossings. nevertheless, as the best way to raise suitable mother lines the development of vegetatively propagated self-incompatible diploid genotypes can be recommended. introduction german chamomile (matricaria recutita) is known to be a mainly out-crossing plant (massoud, 1988; cortnummé, 1980; letchamo, 1992), but exact values of proportions of crossand self-fertilization are not available and also might differ by variety and/or ploidy level (grube, 2009). the incidence of self-incompatibility and the maintenance and propagation of self-incompatible genotypes is an appreciated basis for successful artificial crossings for hybrid breeding. prior investigations on seed development after crossand selffertilization in allopolyploid achillea-species (asteraceae) showed a high rate of self-incompatibility (vetter et al., 1998; jezek, 1989). for matricaria no reliable data are available. under the aspect of raising suitable mother lines for hybrid breeding naturally occurring and induced male sterility is sought (faehnrich et al., 2012). as the chamomile’s flower head is build up by hermaphroditic disc flowers and female ray flowers (massoud, 1988; cortnummé, 1980), naturally occurring aberrations with a higher ray flower/ disc flower ratio indicate tendencies towards male sterility. the same does increased pollen sterility. artificial inter-cultivar crossings could, by genetic alteration, change this reproductive trait (becker, 1993). material and methods six matricaria recutita [l.] rauschert varieties were included in these recent trials, comprising three diploid ones (‘bona’, ‘degumille’ and ‘hungarian 2’) and three colchicine induced autotetraploid ones (‘manzana’, ‘lutea’ and ‘hungarian 1’). while the accessions ‘hungarian 1’ and ‘hungarian 2’ were supplied by the university of budapest, faculty of horticultural sciences, all of the other varieties have been maintained in own experimental stations of the university of veterinary medicines, vienna, austria for about 20 years. ‘manzana’ is a protected variety since 1986, ‘degumille’, ‘bona’ and ‘lutea’ are described, discrete varieties since 1977, 1984 and 1995, respectively (heine et al., 2009). purity of defined ploidy level in distinct varieties was assessed by flow cytometry of 1800 individuals (300 per variety) and ranged from 81 % (‘hungarian 1’) to 100 % (‘bona’). only ‘lutea’ exhibited lower ploidy purity with 61 % of 4x-plants (detailed data not shown). to prove self-incompatibility and the rate of self-/cross pollination 300 plants were included in a trial with all six above mentioned varieties and two treatments. ploidy levels of these plants were assumed to be according to their origin and the performed ploidy level testings. all of the tested plants were raised from the same batch of seeds as the above mentioned plants. treatments included isolation with micro-perforated crispac bags covering whole single plants and untreated, open pollination control conditions. both versions took place in the same green house, under the same environmental growing situation. 25 plants were kept per factor combination (variety and treatment). the number of seeds per flower head was assessed by counting visually as fertile determinable fruits (achenes, slightly curved, with a length of 1-2 mm and a width of app. 0.3 mm, striped) under light microscope. in case of the control version one flower head per plant, in case of isolation up to three flower heads per plant after frequent hand self-pollination were assessed. if all three flower heads did not produce any seeds, the plant was defined as a self-incompatible plant. to determine self pollination against cross pollination always the first examined flower head was taken into account. to assess potentially male sterile plants on the one hand 1105 plants deriving from the above mentioned varieties (100 300 plants per variety) were observed regarding the occurrence of flower head aberrations like missing hermaphroditic disc flowers or additional rings of female ray flowers. both effects increase the female/hermaphroditic floret rate per flower head. from all suspicious flower heads seeds were sown to raise and examine the f1-generation. on the other hand pollen fertility was estimated under light microscope after aceto-carmine staining of patted-off mature pollen of one flower head per plant, using again a trial set of 25 plants per accession under open pollination conditions in the green house. additionally 150 diallele reciprocal artificial crossings between all varieties were undertaken to compare the rate of pollen sterility/fertility after intercultivar crossing. statistical analysis included analysis of variance with one factor (cultivar) for both treatments (isolated and control) in terms of produced seeds per flower head as well as analysis of variance with two factors (cultivar and treatment) over all data to reveal different behaviour of chamomile cultivars under isolation. to compare 168 b. faehnrich, p. nemaz, ch. franz ratios of self-/cross pollination among cultivars, means of produced seeds per flower head under isolation were calculated as percentages of mean amounts under control conditions and tested against average equal values for all cultivars/varieties per chi-square-test after weighting the percentages. a similar procedure was performed to gain results concerning self-incompatibility (comparing percentages of self-incompatible plants per accession) and potential ms-(male sterile) plants (comparing percentages of relevant flower head aberrations per cultivar). to assess occurring pollen sterility 150 data of plants grown under normal green house conditions of six varieties (25 replications per variety) were compared with data from the progeny (35 plants) after intra-specific crossings using univariat analyses of variance and t-test. results comparing the means of seeds per flower head between cultivars/ varieties within different treatments results show no significant influence of cultivars (p-value for control = 0.108, for isolation = 0.119), but post-hoc-tests (duncan) show ‘manzana’ (4x) and ‘degumille’ (2x) belonging to different homogenous groups. under open pollination (control) conditions ‘manzana’ exhibits the lowest amount of produced seeds (mean of 20.40 seeds), while ‘degumille’ shows the highest amount (mean of 58.27 seeds). under isolated conditions the proportion is exactly reversed, i.e. ‘degumille’ shows the lowest (mean of 3.46 seeds), ‘manzana’ the highest amount of seeds per flower head (mean of 13.94 seeds). the diploid, and therefore rather original or natural cultivars ‘bona’ and ‘degumille’ (the tetraploid cultivars ‘manzana’ and ‘lutea’ are colchicine-induced polyploids) exhibit two very differing columns of means of seeds per flower head (43.05 vs. 8.50 for control and isolation for ‘bona’ and 58.27 vs. 3.46 for ‘degumille’, resp.), i.e. under isolation the seed production drops very severely in diploids. ‘manzana’ (4x) does not produce so many seeds under normal (control) conditions but is rather insensible to conditions of isolation (20.40 vs. 13.94) (fig. 1). conducting analysis of variance with two factors (cultivar and treatment) over all data a significant influence of treatment (p = 0.000) and a slight significance of interactions between cultivar and treatment (p = 0.025) comes out. both calculations indicate the influence of isolation being cultivar specific. at this stage we cannot reveal whether this influence derives from different sensitivities to stress situations like isolation bags or from genetically caused self sterility effects. open pollinated control plants perform selfand cross-pollination; isolated plants can only perform self-pollination. just based on these considerations, percentages of selfand cross-pollination relying on the means of number of seeds per flower head were calculated. the same reproductive behaviour of plants of the same variety and ploidy level under isolation and under ‘normal’ conditions was assumed. results show an average of 22% self-pollination under open pollination conditions with a significant influence of cultivar (p = 0.000, fig. 2). " " " " " " " " " " " " " " fig. 1: comparison of seed production between cultivars/varieties and treatments, different letters indicate statistical difference at the p < 0.05 level fig. 2: calculated percentages of selfand cross-fertilization under open pollination conditions, based on the means of produced seeds per flower head under isolation and under open pollination conditions self-incompatibility was defined as 3 examined flower heads in succession per isolated plant without any seed production despite of frequent hand pollination. tab. 1 shows the number and percentages of measured and as self-incompatible determined plants in six varieties. tested by chi-square-tests a statistically highly significant difference between cultivars can be detected (p = 0.000). remarkable is the high self-incompatibility of the diploid cultivars ‘bona’ and ‘degumille’ with almost 28 % and 25 %, respectively. to reveal tendencies to develop male sterility (ms), relevant flower head aberrations (9 of 1105 plants) were labelled and counted. no significant difference between cultivars (p = 0.564) did appear, although ‘bona’ and ‘degumille’ (both diploid) seem not to tend to flower head alterations (tab. 2). the f1-generation of the selected flower heads was raised, but no renewal of relevant flower head alteration did occur. pollen sterility data of the parental generation showed no significant influence of cultivar (p = 0.518) and no completely pollen sterile plant appeared in the parental generation. mean percentages of pollen sterility ranged from 0.55 % (‘bona’) to 2.20 % (‘lutea’) (fig. 3). compared with the f1-generation after crossings across dif self-incompatibility and male sterility in matricaria 169 ferent varieties significant differences in pollen sterility (p = 0.000) could be detected, although also in the f1-generation no completely sterile plant occurred. the parental vs. the f1-generation displayed mean pollen sterility values of 1.29 % and 10.20 %, respectively (fig. 4 and 5). details of mean pollen sterility of progeny in comparison with their respective parental cultivars are shown in tab. 3. the data indicate the increase of pollen sterility by inter-cultivar crossings. discussion looking at figure 1 not only the cultivar-specific reaction to isolation can be discovered but also – knowing that ‘degumille’ and ‘bona’ are diploid and ‘manzana’ and ‘lutea’ auto-tetraploid – a ploidy level dependant behaviour concerning reproductive traits under isolation can be assumed. obviously diploids suffer more from isolation while tetraploids in general produce fewer seeds but their reaction to forced self-fertilization is not dramatic. whether this phenomenon derives from a general lower ability of diploids to cope with stress situations or from a genetic disposition of diploids to – as far as possible – avoid selfing cannot be proven in these investigations, because no other trials on stress-situations were performed. however, in various publications decreasing seed production and lower selfincompatibility is stated for polyploid individuals (grube, 2009; becker, 1993). the increased adaptability of polyploids to extreme environmental conditions is explained as a result of higher genetic variability and therefore a higher probability to produce suitable genotypes (kuckuck et al., 1985; singh, 1992). genomic rearrangements and instability in plant polyploids play a role to develop this high variability (chen et al., 2006; wang et al., 2010). as one of the most important causes for decreased fertility in polyploids altered physiologic features due to the increase of cell size is postulated (kuckuck et al., 1985). provided that the reproductive behaviour of chamomile plants under open pollination condition resembles to isolated, but apart from that identical conditions, proportions of selfand of cross pollination in different varieties can be assumed (fig. 2). on the other hand one could also assume a mainly out-crossing plant being forced to do selfing will display a higher fertilization rate and seed production by self pollination than under open pollination conditions where cross-fertilization is possible. no other stress-tests to discover stress " " " " " " " " " " " " " " " " tab. 1: detected percentages of self-incompatibility in six chamomile varieties cultivar 3x0 seeds-plants of measured % of self incompatible plants manzana 1 16 6.3 bona 5 18 27.8 degumille 6 24 25.0 hungarian 1 0 17 0.0 hungarian 2 1 15 6.7 lutea 4 23 17.4 total 17 113 15.0 tab. 2: detected percentages of plants with flower head aberrations in dicating tendencies to male sterility in the parental generation of six chamomile varieties cultivar plants of measured % of potential with potential ms-plants ms-formations manzana 3 300 1.0 bona 0 100 0.0 degumille 0 100 0.0 hungarian 1 0 100 0.0 hungarian 2 1 205 0.5 lutea 5 300 1.7 total 9 1105 0.8 fig. 3: mean percentages of pollen sterility in six varieties of the parental generation fig. 4: mean percentages of pollen sterility in parental vs. f1-generation after crossings across different varieties 170 b. faehnrich, p. nemaz, ch. franz susceptibility of different varieties were performed. therefore, to be sure about the proportions of selfand cross pollination the performance of trials with phytochemical or molecular markers to assess the parents and their progeny without creating isolation stress would be necessary. similar studies over five generations with phytochemical markers in chamomile, but with different investigation targets were conducted by mader (1998). easily recognizable phenotypic markers are not available in chamomile. in these present trials the whole referring plant (as one genotype) was put under an isolation bag to assess self compatibility or not, in other trials only single flower heads were isolated (vetter et al., 1998; cortnummé, 1980). nevertheless, the proportions of selfand cross fertilization show a significant influence of cultivar and a tendency of diploids (e.g. ‘bona’ and ‘degumille’) to perform low rates of self fertilization. ploidy level purities within the single cultivars were very high. ‘degumille’ (2x) exhibited 98 %, ‘bona’ (2x) 100 % according individuals. in continuation of these thoughts it is not surprising that self-incompatibility also presents a significant cultivar-specification, and shows to be high in diploids (tab. 1). this message could be of high value for breeders as the chance to develop suitable mother lines for artificial crossings from selected, self incompatible and vegetatively propagated diploid plants seems to be high. in order to test different options to create suitable mother lines for cross breeding purposes, naturally occurring flower head aberrations indicating the development of male sterility (ms) by performing more ray flowers and/or fewer disc flowers were assessed. after finding 9 of 1105 potential ms-plants (tab. 2) no renewal of such aberrations occurred, neither in the f1-generation raised from the seeds of the selected and suspicious flower heads nor among the seedlings of the base plants. this observation suggests these appearances to be only somatic and not genetically determined. an f2-generation could not be established. the fact that these aberrations only occurred at single flower heads and never concerned the whole plant underlines a similar approach. therefore the establishment of mother lines via spontaneously occurring ms-plants cannot be recommended without conducting further research. possibly there could be a chance to generate ms-aberrations by induced mutations (matsumara et al., 2010). based on investigations and publications concerning changes of pollen sterility/fertility after interand intra-specific crossings and/ or in different ploidy levels (lambrou et al., 2001, pers. comm.; lössl, 1999; köhler et al., 1991; kempe and gils, 2011; kaul, 1988) diallele reciprocal crossings between all varieties were conducted and pollen fertility was tested before and after the crossings. controlled crossings could enhance the chance to develop decreased fertility in general and male sterility in particular (lössl, 1999). this effect could have its origin in a disposition of cms (cytoplasmatic male sterility) or could be determined solely genetically (gms, genetic male sterility). both cases would have to be proven in numerous progeny tests (kuckuck et al., 1985; odenbach, 1997). as our trials discovered significant statistical differences in pollen sterility between the two tested generations (parental and f1-generation), pollen fertility/sterility of cuttings of the f1-generation will be continued to be tested and female fertility/sterility of the f1-generation will be assessed by observing seed production and germination rates. certainly, more investigations have to be undertaken to ensure and specify the effect of intra-specific crossings on male or female fertility. according to the outcomes of self-incompatibility tests and different focused kinds of male sterility the most appropriate approach to create suitable mother lines for hybrid production seems to be the development of diploid (esp. ‘bona’ and ‘degumille’) lines from vegetatively propagated self-incompatible plants. also an approach via pollinating maintainers to establish self-incompatible mother lines – similar to cms-maintainer lines, according to pank et al. (2001) – is worth consideration. further investigations to detect self-incompatibility in an early stage of plant development, e.g. by watching the growth of the pollen tube by light microscope, are recommended. acknowledgements the authors wish to thank prof. dr. johannes novak for his helpful suggestions and comments concerning experimental design and statistical analyses. this work was part of a funded project (nr. 22038911), sponsored by the fnr (fachagentur nachwachsender rohstoffe) of the bmelv (german federal ministry of food, agriculture and consumer protection). references becker, h., 1993: pflanzenzüchtung. ulmer, stuttgart. chen, zj., ni, z., 2006: mechanisms of genomic rearrangements and gene fig. 5: infertile pollen (yellow, in the lower left corner) vs. fertile pollen (red, the other three) in the f1-generation tab. 3: detailed percentages of pollen sterility of parental cultivars and their progeny, respectively cultivar pollen sterility cultivar pollen sterility pollen sterility of in %, of father in %, in %, mother mean of mean of mean of cultivar cultivar progeny manzana 1.20 lutea 2.20 9.93 manzana 1.20 hung. 1 0.84 n/a manzana 1.20 hung. 2 1.27 n/a lutea 2.20 hung. 1 0.84 8.82 lutea 2.20 manzana 1.20 4.76 hung. 2 1.27 bona 0.55 8.20 hung. 2 1.27 degumille 1.45 7.58 hung. 1 0.84 lutea 2.20 8.41 degumille 1.45 bona 0.55 4.21 bona 0.55 degumille 1.45 15.53 bona 0.55 hung. 2 1.27 20.33 self-incompatibility and male sterility in matricaria 171 expression changes in plant polyploids. bioassays 28, 240-252. cortnummé, j., 1980: beiträge zur züchtung der kamille (matricaria chamomilla l.). diplomarbeit, technische universität münchenweihenstephan. faehnrich, b., franz, c., 2012: effects of gibberellic acid as a gametocide on different genotypes of german chamomile (matricaria recutita [l.] rauschert). j. appl. bot. food qual. 85, 73-74. grube, m., 2009: populationen – biologie, genetik, ökologie. vorlesungsunterlagen ss 2009, uni graz. heine, h., eger, h., franz, c., blüthner, w-d., hoppe, b., 2009: kap. 2.3 sortenwesen. in: saluplanta (hrsg.), handbuch des arzneiund gewürzpflanzenbaus, band 1, grundlagen des arzneiund gewürzpflanzenbaus i, 609-640. verein für arzneiund gewürzpflanzen saluplanta e.v., bernburg. jezek, b., 1989: kreuzungsversuche an europäischen vertretern des achillea millefolium-komplexes. diplomarbeit, universität für bodenkultur und veterinärmedizinische universität, wien. kaul, m.l., 1988: male sterility in higher plants. springer, berlin. kempe, k., gils, m., 2011: pollination control technologies for hybrid breeding. mol. breed. 27, 417-437. köhler, r., horn, r., lössl, a., zetsche, k., 1991: cytoplasmic male sterility in sunflower is correlated with the co-transcription of a new open reading frame with the atpa gene. mol. gen. genet. 227, 369-376. kuckuck, h., kobabe, g., wenzel, g., 1985: grundzüge der pflanzenzüchtung, 5., neubearbeitete und erweiterte auflage. walter de gruyter, berlin, new york. lambrou, m., bein-lobmaier, b., franz, ch., 2001: cytological analysis of diand tetraploid plants of matricaria recutita (asteraceae) and their hybrid progeny. personal communication. letchamo, w., 1992: ökologische, genetische und ontogenetische einflüsse auf wachstum, ertrag und wirkstoffgehalt von diploiden und tetraploiden kamillen, chamomilla recutita (l.), rauschert. dissertation, justusliebig-universität gießen. lössl, a., 1999: die cytoplasmatisch männliche sterilität und cms aus helianthus petiolaris bei der sonnenblume. cytoplasm genome research, http://www.lossl.de/index.htm. mader, e., 1998: experimentelle überprüfung des hardy-weinberg gleichgewichts von bisabolol-chemotypen bei tetraploiden kamillen, diplomarbeit, veterinärmedizinische universität wien. massoud, h., 1988: quantitative vererbung einiger ertragsmerkmale und der hauptkomponenten des ätherischen öls von kamille, matricaria chamomilla l. (syn. chamomilla recutita l.). dissertation, technische universität münchen. matsumara, a., nomizu, t., futurani, n., hayashi, k., minamiyama, y., hase, y., 2010: ray florets color and shape mutants induced by 12 c 5+ ion beam irradiation in chrysanthemum. sci. hortic. 123, 558-561. odenbach, w., 1997: biologische grundlagen der pflanzenzüchtung. parey buchverlag, berlin. pank, f., vender, c., van niekerk, l., junghanns, w., langbehn, j., blüthner w.-d., novak, j., franz, c., 2001: combining ability of origanum majorana l. strains – agronomical traits and essential oil content: results of the field experiment series in 1999. journal of herbs, spices and medicinal plants 9, 31-37. singh, r., 1992: chromosomal abnormalities and fertility in induced autotetraploid helianthus annuus in c1 and c2 generation. cytologia 57, 277281. vetter, s., franz, c., 1998: samenbildung bei kreuzungen und selbstungen mit polyploiden achillea-arten (asteraceae). z. arzn. gew.pfl., 11-14. wang, y., jha, a.k., chen, r., doonan, j.h., yang, m., 2010: polyploidy-associated genomic instability in arabidopsis thaliana. genesis 48, 254-263. address of the authors: dipl. ing. dr. bettina faehnrich, pietro nemaz and em. o. univ. prof. dr. chlodwig franz, institute of animal nutrition and functional plant compounds, university of veterinary medicines, veterinaerplatz 1, a-1210 vienna, austria. e-mail: bettina.faehnrich@vetmeduni.ac.at vorgabe neu journal of applied botany and food quality 80, 171 178 (2006) institute of plant molecular physiology and biotechnology, university of bonn, germany particle bombardment as a strategy for the production of transgenic high oleic sunflower (helianthus annuus l.) sherin mohamed, robert boehm, heide schnabl* (received august 2, 2006) summary in order to develop an efficient and reproducible protocol for genetic engineering of high oleic helianthus annuus l. genotypes (cv. capella and swsr2 inbred line) important parameters of a particle bombardment strategy have been optimized, such as gold particle size, particle acceleration pressure, distance between macrocarrier assembly and target plate, pre-culture period of the explant and number of bombardments per explant. these parameters were evaluated on the basis of resulting gus activity coupled with regeneration frequency and efficiency as well as plant cell vitality. split shoot apices were used as explants. the maximum gus activity was observed at 1550 psi acceleration pressure combined with 6 cm target distance and 1.6 µm gold particle size. a pre-culture of one day prior to bombardment gave the best results. in addition, two subsequent bombardments increased the gus activity of cv.capella and swsr2 inbred line 1.6 and 2.1 fold, respectively, compared to explants bombarded once. the optimized bombardment conditions were applied for estimating the transformation frequency which reached 3.1 and 4.5 % for high oleic cv.capella and swsr2 inbred line, respectively. this frequency was calculated on the basis of positive pcr results of putative transgenic plants and in relation to the total number of bombarded explants. abbreviations: mug – 4-methylumbelliferyl-ß-glucuronide; 4-mu – 4-methylumbelliferone; bap – 6-benzylaminopurine; gus – βglucuronidase; ms – murashige and skoog; nos – nopaline synthase gene; nptii – neomycin phosphotransferase gene; psi – pounds per square inch; se – standard error; sim – shoot induction medium introduction sunflower (helianthus annuus l.) is one of the three most important annual oil-bearing crops world-wide following soybean (glycine max l.) and rapeseed (brassica napus l.) (weber et al., 2003). high oleic sunflower oil can also be used as food oil or deep-frying fat (fick and miler, 1997; dorrel and vick, 1997). its high oxidative performance of oleic acid and its very low content of polyunsaturated fatty acids combined with low content of stearic acid make them suitable for industrial applications like cosmetics, pharmaceuticals, detergents, lubricants, metal working fluids, surfactants or for chemical synthesis. sunflower is known as one of the most recalcitrant species for tissue culture and genetic transformation. therefore, progress in sunflower transformation has been restricted for many years by the limitations of an available regeneration system and problems in combining regeneration and transformation within the same cells (potrykus, 1990). particle bombardment is a popular method of direct gene delivery into cells, tissues and organs. this technique uses pressurized helium to accelerate sub-cellular size microprojectiles of tungsten or gold coated with dna (or other biological material) into cells over range of velocities necessary to optimally transform many different cell types (bathnagar et al., 2002). in the first report on the introduction of a foreign gene by particle bombardment of sunflower meristem explants, the regenerated plants showed gus expression sectors indicating that chimeric plants had been produced (bidney, 1990). transient expression of the gus gene has been induced in sunflower cotyledonary explants and immature zygotic embryos at different developmental stages after microprojectile bombardment (hunold et al., 1995). small embryos of approximately 1.5-2.0 mm in diameter were the most suitable for efficient transient gus expression (laparra et al., 1995; hunold et al., 1995) and multiple shoot formation, but the conversion rate of transient to stable transformation was shown to be very low (hunold et al., 1995). the limited success of dna transfer into sunflower cotyledons by microprojectile bombardment is likely due to the strong cuticle (hunold et al., 1995). several factors have been described to influence the applicability and efficiency of biolistic gene transfer such as genotype (koprek et al., 1996), particle size (bhat et al., 2001), pre-culture period prior to gene transfer (rasco-gaunt et al., 1999), acceleration pressure (koprek et al., 1996; bhatnagar et al., 2002; tadesse et al., 2003), the adjustable distances between rupture disc and target plate (bhat et al., 2001; rasco-gaunt et al., 1999) and number of bombardments (lonsdale et al., 1990). the aim of this study was to optimize the physical and biological parameters in order to work out the highest transformation efficiency without compromising the plant cell vitality and regeneration frequency and to deliver a strategy for obtaining stable transformants of high oleic sunflower, a hybrid (cv. capella) and an inbred line (swsr2). material and methods plant material and explant preparation seeds of high oleic sunflower (helianthus annuus l.), cv. capella and inbred line swsr2, kindly provided by südwestsaat (rastatt, germany), were surface sterilized for one min with 70 % (v/v) ethanol, rinsed in a 6 % (v/v) sodium hypochlorite solution plus one drop tween-20 for one hour and washed three times with sterile water. seeds germinated on a ms-based medium (murashige and skoog, 1962) containing ms salts 2.3 g l-1, sucrose 2 % (w/v), 2-(n-morpholino) ethanesulfonic acid (mes) 3.2 mm, phytoagar 7.5 g l-1 and ph adjusted to 5.7 with naoh (1 m). seedlings grew for 10 days in growth chamber at 25 ± 1°c and a light period of 12 h (115 µ e m-2 s-1). after 10 days, aseptic shoot apices (4-5 mm length) were bisected longitudinally according to malone-schoneburg et al. (1994) and used as explants in this study. plasmid isolation preparation of plasmid dna from e. coli dh5α carrying the plasmid pbi121 was performed as described by sambrook and russel (2001) using the alkaline lysis method. this plasmid containes the gus gene under the transcriptional control of the camv35s promoter* corresponding author. and the selectable nptii marker gene under the control of nos promoter. preparation of the gold particles and coating with dna preparation of the gold particles was performed according to the method of sanford et al. (1993) and stored at -20°c at a final concentration of 60 mg ml-1. the particle suspension was thawed when required and vortexed vigorously to resuspend the particles. 50 µl of microcarriers were taken in an eppendorf tube and while vortexing continuously (for uniform dna precipitation onto microcarriers) the following was added sequentially: 5 µl dna (1 µg µl-1), 50 µl 2.5 m cacl 2 and 20 µl 0.1 m spermidine. contents were vortexed for 5-6 min, microcarriers were allowed to settle for 1 min and pelleted by spinning for 2 sec at 14,000 rpm. the liquid was removed and replaced by 140 µl of 70 % (v/v) ethanol for washing. the first washing was followed by washing with 100 % (v/v) ethanol and finally particles were resuspended in 48 µl of 100 % ethanol. these coated particles were kept at 4 ºc, used within 1 h of preparation and 6 µl of the coated particle suspension were loaded onto the macrocarrier membrane which was allowed to dry for 10 min prior to use. particle bombardment and plant regeneration various physical and biological parameters were applied in a single or multifactorial way using pbi121 coated gold particles for cv. capella hybrid and swsr2 inbred line. these tested parameters included different gold particle size (1 and 1.6 µm), particle acceleration pressures (0, 450, 900, 1550 and 1800 psi), distance between macrocarrier assembly and target plate (6 and 9 cm), preculture of the explant (0, 1 and 2 days) and number of bombardments per explant (1 and 2 shots). split shoot apices were grouped in the center of 4 cm petri dishes on 2 % (w/v) autoclaved agarose gel (the cut surface facing up). the bombardment was performed according to sanford et al. (1993) using a biolistic® pds-1000/he particle delivery system (biorad, germany). bombarded explants were cultured on shoot induction medium (sim2, mohamed et al., 2003) containing ms salts 4.3 g l-1, myo-inositol 0.6 mm, thiamine-hcl 0.3 µm, glycine 26.6 µm, nicotinic acid 4.1 µm, pyridoxine-hcl 2.4 µm, sucrose 3 % (w/v), bap 0.4 µm, ph 5.7 and plant-agar 6 g l-1 without any selecting agent. culture conditions were 25 ± 1°c and a light period of 12 h (115 µ e m-2 s-1). after three weeks, explants were sub-cultured on fresh sim2 and incubated under the same conditions. under these conditions, multiple-shoot regeneration could be observed and the biggest regenerant of each explant was used for further molecular analysis. measurement of plant cell vitality regenerated shoots from the bombardment were chosen randomly to measure the vitality by using a pulse-amplitude modulated fluorescence measurement system (pam 2000 fluorometer, waltz, germany) as described by schreiber and bilger (1993). vitality was measured as a yield, which represents the essence of fluorescence quenching analysis by the saturation pulse method and calculated according to the equation fm' – ft yield = fm' ft = the parameter which represents the basic fluorescence yield at any given time. fm' = the parameter defined as the maximal fluorescence yield reached in a pulse of saturating light with an illuminated sample. histochemical gus activity assay ß-glucuronidase (gus) activity was assayed according to jefferson et al. (1987). regenerated shoots (five weeks after bombardment in the transformation experiments testing different parameters and 8 weeks after bombardment in the final representative transformation experiment) were immersed in gus staining solution (0.1m na 2 hpo 4 ; ph 7.0, 10 mm naedta, 0.5 mm k-ferricyanide, 0.5 mm k-ferrocyanide, 0.1 % (v/v) triton-x-100, 1 mm x-gluc (5 bromo-4-chloro-3-indolyl glucuronide) and 20 % (v/v) methanol) (kusogi et al., 1990) and vacuum infiltration was applied at 200 mbar for 10 min, then incubated overnight in the dark at 37°c. before microscopic analysis, chlorophyll was removed by extraction in an ethanol series (70 %, 96 %) for 24 h. untreated explants were cultured under identical conditions and served as negative control. by this assay qualitative data concerning the specificity of the gus gene expression in the tissue were obtained. after the histochemical assay, positive plants were used for pcr analysis. fluorometric gus activity assay the fluorometric gus activity assay was performed according to jefferson et al. (1987). for testing different parameters, the assay was performed four weeks after bombardment. in the final representative transformation experiment it was performed 8 weeks after bombardment. leaf tissue from transformed and non-transformed regenerants was ground to homogeneity with a pestle and mortar in the presence of liquid nitrogen. tissue was extracted in microcentrifuge tubes with extraction buffer (2 ml g-1 ground tissue) containing 50 mm nah 2 po 4 , ph 7.0, 10 mm edta, 0.2 % (v/v) triton x-100 and 10 mm ß-mercaptoethanol, centrifuged to pellet the debris and the supernatant was collected. protein content of this crude extract was quantified according to bradford (1976), mixed with 500 µl mug solution (1 mm 4-methylumbelliferyl-ß-glucuronide in 20 % (v/v) methanol). the reaction was carried out in the dark at 37°c for 1 h. and stopped with 400 µl 0.2 m na 2 co 3 . the assays were analyzed in a spectro fluorometer (fluoro-max, spex, germany) and the fluorescence was recorded at an excitation wavelength of 365 nm and an emission wavelength of 455 nm. the readings were compared to readings of 4-mu standards of varying concentrations and were plotted against time to determine the amount of 4-mu produced. gus activity was calculated as micromoles of 4-mu formed per mg protein and min. plant dna extraction and polymerase chain reaction analysis dna from transformed (positive gus activity assay), putatively transformed and non transformed plants was extracted according to the ctab method (doyle and doyle, 1987; cullings, 1992) and digested with ecori. detection of gus and nptii in these samples was conducted using pcr with the following primers: gus primers 5´-atg tta cgt cct gta gaa ac-3´ and 5´-ctt cac tgc cac tga ccg ga-3´, which were designed to amplify an approximately 830 bp dna fragment of the gus gene and nptii primers 5´-aca aga tgg att gca agg-3´and 5´-aac tcg tca aga cga tag-3´ which amplify an approximately 800 bp dna fragment of the nptii gene. pcr reaction was performed in 50 µl reaction mix containing 50-100 ng ecori digested genomic dna, 5 µl 1.5 mm mgcl 2, 5 µl 10 x taq dna polymerase buffer, 0.75 µl 0.2 mm dntp, 12.5 µl 0.25 µm of each primer and 2 u taq dna polymerase. as a positive control, the corresponding plasmid was used as a template. dna samples were denatured for 5 min at 95°c and amplified during 32 cycles, denaturation for 1 min at 95°c, annealing for 1 min at 64°c for gus and at 58°c for nptii, extension for 1 min at 72°c. cycling was closed with a final extension step for 172 sherin mohamed, robert boehm, heide schnabl 10 min at 72°c. amplified products were separated on a 0.8 % (w/v) agarose gel and the dna was visualized with ethidium-bromide on a uv transilluminator (sambrook and russel, 2001) at 305 nm. results evaluation of different physical and biological parameters of bombardment two microcarrier gold particle sizes (1 and 1.6 µm) were used at four particle acceleration pressures (0, 450, 900, 1550 and 1800 psi) in combination with 6 and 9 cm target distance. moreover, pre-culture durations of the explant (0, 1 and 2 days) and number of bombardments per explant (1 and 2 shots) were studied to support the introduction of dna with minimal tissue damage or interference with the regeneration potential. evaluation of these parameters was based on histochemical and fluorometric gus activity assays coupled with shoot induction frequency (%), which was calculated as number of regenerated explants per total number of explants, and plant cell vitality. these experiments were laid out as a complete randomized design and each treatment was based on four replicates of forty explants. effect of gold particle size, particle acceleration pressure and target distance on gus activity fig. 1 and 2 show that the general pattern of response was similar in both tested genotypes (cv. capella and swsr2) with respect to gus activity. the highest fluorometric activity value resulted by using 1.6 µm particle size regardless of helium pressure and target distance used. moreover, there was a direct correlation between the fluorometric gus activity and the helium pressure up to 1550 psi. when the helium pressure exceeded this value, the fluorometric gus activity dramatically decreased. increasing the target distance to 9 cm induced a reduction in the fluorometric gus activity with respect to the particle size and the helium pressure used. in comparison, the fluorometric gus activity of cv. capella and swsr2 inbred line plants which were bombarded with 1.6 µm particle size and 1550 psi at 9 cm was 3.7 and 2.9 times lower than those bombarded with the same particle size and helium pressure at 6 cm, respectively (fig. 1a and 2a). increasing the target distance to 9 cm could not be compensated by the elevation of acceleration pressure with the use of any particle size. a helium pressure of 1550 psi in combination with a target distance of 6 cm and 1.6 µm particle size resulted in the highest gus expression frequency which amounted to 33.3 and 30.8 % for cv. capella and swsr2 inbred line, respectively (fig. 1b and 2b). there was no gus expression observed in the plants which were bombarded at 9 cm and 450-900 psi using either particle size of both genotypes. effect of gold particle size, particle acceleration pressure and target distance on plant regeneration and cell vitality the cell vitality of the bombarded tissues appeared to be inversely related to the helium pressure at either of the two target distances (fig. 1c and 2c). at the highest helium pressure (1800 psi) the bombarded tissues with 1.6 µm particle size at 6 cm distance were extensively damaged in both genotypes. shoot induction frequency was also affected by varying helium pressure. this effect was more pronounced at 6 cm target distance with 1.6 µm particle size. using a combination of 1.6 µm particle size, 6 cm target distance and 1800 psi the shoot induction frequency was decreased to 12.5 and 7.5 % of cv. capella and swsr2 inbred line, respectively (fig. 1d and 2d). considering the overall effects of acceleration pressure on the gus activity as well as the cell vitality and the tissue culture response, a helium pressure of 1550 psi, combined with 6 cm target distance and 1.6 µm particle size was found to be acceptable for bombardment of sunflower split shoot apices. effect of pre-culture duration and number of shots per explant on gus activity all the hitherto optimized parameters (shown above) were applied in subsequent transformation experiments. these experiments were designed to find out the effect of pre-culture duration and number of shots per explant on the transformation events as well as cell vitality and tissue culture response. as presented in fig. 1e and 2e, the bombardment of the explants for two times resulted in the highest levels of gus activity in the fluorometric assay. in comparison, when the explants were bombarded twice after one day of pre-culture, an increase in gus activity of 1.6 and 2.1 fold was achieved in cv. capella and swsr2 inbred line, respectively, compared to explants bombarded one time. moreover, pre-culture duration also affected gus activity. when explants were cultured for one day prior to bombardment, higher values in the fluorometric assay were obtained compared to 0 and 2 days of culture. in terms of the histochemical gus activity assay, the gus expression decreased by19.6 and 53 % with increasing the pre-culture duration to 2 days in cv. capella and swsr2 inbred line, respectively, when explants were bombarded one time. meanwhile, there was a variation in the general pattern of gus expression between the two genotypes when the explants were bombarded twice. the lowest gus expression frequency was 12.5 % in cv. capella and resulted from pre-culture of the explants for one day prior to the double bombardment. conversely, the highest gus expression frequency was 25 % in swsr2 inbred line and resulted from the same condition (fig. 1e and 2e). effect of pre-culture duration and number of shots per explant on plant regeneration and cell vitality influence of pre-culture duration and number of shots per explant on the shoot induction and the cell vitality is illustrated in fig. 1f and 2f. shoot induction frequency and the cell vitality increased in dependency of pre-culture duration. number of bombardment per explant affected shoot induction frequency as well as the cell vitality. cv. capella was highly affected by the number of shots per explant compared to swsr2 inbred line. in conclusion, on the basis of the results obtained during the optimization experiments, the optimized bombardment conditions are: 1550 psi of acceleration pressure in combination with 6 cm target distance, 1.6 µm gold particle size and pre-culture the explants for one day prior to a double bombardment. estimation of transformation frequency all the resulting optimal parameters were applied in final representative transformation experiments using 95 split shoot apices of cv. capella and 110 of swsr2 inbred line. regenerated plantlets 8 weeks after bombardment were subjected to histochemical and fluorometrical gus activity assay as well as to molecular analysis. the transformation frequency was calculated on the basis of pcr analysis and recorded as a percentage of the total number of cocultivated explants. as shown in tab. 1 the recorded number of gus expressing plants was 1 and 4 in cv.capella and swsr2 inbred line, respectively. gus sunflower transformation by particle bombardment 173 0 1 2 0 1 2 0 1000 2000 fluorometric assay histochemical assay 0 10 20 30 40 1 shot 2 shots pre-culture duration (day) µ m o l m u /m g p ro te in /m in g u s ex p re s s in g s h o o ts (% ) un tre at ed 0 1 2 0 1 2 0 25 50 75 shoot induction (%) cell vitality 0.00 0.25 0.50 0.751 shot 2 shots pre-culture duration (day) s h o o t in d u ct io n ( % ) y ie ld e f 0 1 2 0 1 2 0 1000 2000 fluorometric assay histochemical assay 0 10 20 30 40 1 shot 2 shots pre-culture duration (day) µ m o l m u /m g p ro te in /m in g u s ex p re s s in g s h o o ts (% ) un tre at ed 0 1 2 0 1 2 0 25 50 75 shoot induction (%) cell vitality 0.00 0.25 0.50 0.751 shot 2 shots pre-culture duration (day) s h o o t in d u ct io n ( % ) y ie ld e f 45 0 90 0 15 50 18 00 45 0 90 0 15 50 18 00 0 500 1000 1500 6 cm 9 cm target distance (cm) and helium pressure (psi) µ m o l m u /m g p ro te in /m in . u nt re at ed 45 0 90 0 15 50 18 00 45 0 90 0 15 50 18 00 0.00 0.25 0.50 0.75 6 cm 9 cm tar get distance (cm) and helium pressure (psi) y ie ld 45 0 90 0 15 50 18 00 45 0 90 0 15 50 18 00 0 10 20 30 40 1.0 µm 1.6 µm t arg et distance (cm) and helium pressur e (psi) g u s e x p re s s in g s h o o ts ( % ) 6 cm 9 cm un tre at ed 45 0 90 0 15 50 18 00 45 0 90 0 15 50 18 00 0 25 50 75 1.0 µm 1.6 µm 6 cm 9 cm target distance (cm) and helium pressure (psi) s h o o t in d u ct io n ( % ) a b c d 45 0 90 0 15 50 18 00 45 0 90 0 15 50 18 00 0 500 1000 1500 6 cm 9 cm target distance (cm) and helium pressure (psi) µ m o l m u /m g p ro te in /m in . u nt re at ed 45 0 90 0 15 50 18 00 45 0 90 0 15 50 18 00 0.00 0.25 0.50 0.75 6 cm 9 cm tar get distance (cm) and helium pressure (psi) y ie ld 45 0 90 0 15 50 18 00 45 0 90 0 15 50 18 00 0 10 20 30 40 1.0 µm 1.6 µm t arg et distance (cm) and helium pressur e (psi) g u s e x p re s s in g s h o o ts ( % ) 6 cm 9 cm un tre at ed 45 0 90 0 15 50 18 00 45 0 90 0 15 50 18 00 0 25 50 75 1.0 µm 1.6 µm 6 cm 9 cm target distance (cm) and helium pressure (psi) s h o o t in d u ct io n ( % ) a b c d fig. 1: assessment of different particle bombardment parameters affecting the transformation efficiency of split shoot apices for high oleic h. annuus l. cv. capella. a d: different target distances, different helium pressures and different gold particle sizes and its effect on (a) gus expression level, (b) gus activity in the histochemical assay, (c) vitality and (d) shoot induction frequency. the bombardment were applied without pre-culture of the explants and a single shot per plate. e f: different pre-culture durations and numbers of shots per plate and its effect on (e) gus activity and (f) shoot induction frequency and vitality. here, bombardments were applied under 6 cm distance between macrocarrier assembly and target plate using 1.6 µm gold particles and 1550 psi helium pressure. results are data of at least three replicates and error bars represent the se. for protocol details see materials and methods. expression was complete and uniform in the transformed plants, whereas no expression was detected in the untreated plants (control) (see fig. 3). the fluorometric gus activity assay results reflect that swsr2 inbred line had a slightly higher average gus expression than the cv. capella. the fluorometric value of swsr2 was 1723.2 µmol mu mg protein-1 min-1. whereas the corresponding value recorded from cv. capella was 1509.6 µmol mu mg protein-1 min-1 (tab. 1). the specific amplified fragments of 830 and 800 bp for gus and nptii, respectively, were amplified in the transformed regenerated plantlets of cv. capella and swsr2 inbred line (fig. 4), whereas no amplified band was detected in the non-transformed plants. from 30 and 40 tested pcr plants 3 and 5 plants were positive with either of the two primers of cv. capella and swsr2 inbred line, respectively (tab. 1). this indicates that some of the pcr positive plants did not express the gus gene. the transformation frequency of cv. capella was 3.1 %, while the corresponding frequency of swsr2 was 4.5 % in relation to the total number of explants used in the experiment. 174 sherin mohamed, robert boehm, heide schnabl 0 1 2 0 1 2 0 1000 2000 fluorometric assay histichemical assay 0 10 20 30 40 1 shot 2 shots pre-culture duration (day) µ m o l m u /m g p ro te in /m in g u s exp re ssin g s h o o ts (% ) u nt re at ed 0 1 2 0 1 2 0 25 50 75 100 shoot induction (%) cell vitality 0.00 0.25 0.50 0.751 shot 2 shots pre-culture duration (day) s h o o t in d u c ti o n ( % ) y ie ld e f 0 1 2 0 1 2 0 1000 2000 fluorometric assay histichemical assay 0 10 20 30 40 1 shot 2 shots pre-culture duration (day) µ m o l m u /m g p ro te in /m in g u s exp re ssin g s h o o ts (% ) u nt re at ed 0 1 2 0 1 2 0 25 50 75 100 shoot induction (%) cell vitality 0.00 0.25 0.50 0.751 shot 2 shots pre-culture duration (day) s h o o t in d u c ti o n ( % ) y ie ld e f un tre at ed 45 0 90 0 15 50 18 00 45 0 90 0 15 50 18 00 0 25 50 75 100 6 cm 9 cm 1.0 µm 1.6 µm target distance (cm) and helium pressure (psi) s h o o t in d u c ti o n ( % ) 45 0 90 0 15 50 18 00 45 0 90 0 15 50 18 00 0 10 20 30 40 1.0 µm 1.6 µm 6 cm 9 cm target dist ance (cm) and helium pressure (psi) g u s e xp re s s in g s h o o ts ( % ) un tre at ed 45 0 90 0 15 50 18 00 45 0 90 0 15 50 18 00 0.00 0.25 0.50 0.75 6 cm 9 cm target distance (cm) and helium pressure (psi) y ie ld a b c d 45 0 90 0 15 50 18 00 45 0 90 0 15 50 18 00 0 250 500 750 1000 6 cm 9 cm target distance (cm) and helium pressure (psi) µ m o l m u /m g p ro te in /m in . fig. 2: assessment of different particle bombardment parameters affecting the transformation efficiency of split shoot apices for high oleic h. annuus l. swsr2 inbed line. a d: different target distances, different helium pressures and different gold particle sizes and its effect on (a) gus expression level, (b) gus activity in the histochemical assay, (c) vitality and (d) shoot induction frequency. the bombardments were applied without preculture of the explants and a single shot per plate. e f: different pre-culture durations and numbers of shots per plate and its effect on (e) gus activity and (f) shoot induction frequency and vitality. here, bombardments were applied under 6 cm distance between macrocarrier assembly and target plate using 1.6 µm gold particles and 1550 psi helium pressure. results are data of at least three replicates and error bars represent the se. for protocol details see materials and methods. discussion the ability to accelerate dna-coated particles directly into intact tissue by particle bombardment technique has expanded the range of organisms that can be genetically transformed. however, the efficiency of microparticle bombardment depends on a large number of physical, biological and environmental factors (christou, 1992; sanford et al., 1993; koprek et al., 1996; rasco-gaunt et al., 1999; bhat et al., 2001; bhatnagar et al., 2002; tadesse et al., 2003), which have to be optimized for every plant tissue. in the present investigation different physical and biological factors were optimized to obtain an efficient biolistic gene transfer protocol for high oleic sunflower genotypes. the results showed that the highest gus expression values resulted from using 1.6 µm gold particle size in both tested genotypes. the same size of gold particle size was successfully employed in the transformation of sunflower cotyledons (vischi et al., 1999), buffel grass calli (bhat et al., 2001) and sorghum shoot tip and immature embryo (tadesse et al., 2003). changes in helium pressure were found to affect the level of the gene expression. there was a direct proportion between the gus expression and the helium pressure up to 1550 psi. this could be related to high penetration rate of the gold particles. on other hand, sunflower transformation by particle bombardment 175 a higher pressure (1800 psi) caused a dramatically reduction in the gus expression due to the decreased vitality of the explants. eventually, the cell vitality was affected by varying helium pressure and was inversely related to it. these results were related to the penetration of those heavy metal particles into intact cells or tissues which may provoke various levels of tissue wounding that can have various effects on subsequent plant regeneration (tadesse et al., 2003). the data explain that the enhanced cell damage with increasing the helium pressure resulted in decrease of the gus expression. our results and explanation were in accordance with gaunt et al. (1999), bhat et al. (2001), bhatnagar et al. (2002) and tadesse et al. (2003). in general, increase of the target distance to 9 cm induced a reduction in the gus activity regardless to the particle size and the helium pressure used. similarly, tadesse et al. (2003) bombarded different types of sorghum explants at 6 cm target distance and reported that increasing the target distance could not be compensated by the elevation of acceleration pressure in any of the explants. thus, a helium pressure of 1550 psi in combination with a target distance 6 cm and 1.6 µm particle size resulted in the highest gus transformation frequency. tab. 1: summary of transformation results of split shoot apices from high oleic h. annuus l. genotypes cv. capella and swsr2 using biolistic gene transfer method. genotype cv. capella swsr2 total number of used explants 95 110 number of positive plants 1 4 in fluorometric gus activity assay gus frequency* (%) 1,1 3.6 activity in the fluorometric assay (µmol mu mg protein-1 min-1). 1509.6±130.8 1723.2±101.3 mean ± se number of plants tested with pcr 30 40 number of pcr-positive plants 3 5 transformation frequency* (%) 3.1 4.5 (*) the percentage was calculated in relation to the total number of bombarded explants and pcr was performed with gus and nptii primers 14-16 weeks after bombardment. transformation frequency was calculated on the basis of positive pcr plants. fig. 3: histochemical gus assay of two high oleic sunflower genotypes, cv. capella and swsr2 inbred line. (a) multiple shoots from direct shoot apices regeneration on sim2 medium, (b) untreated shoot and (c) gus expressing shoot after gus staining assay. fig. 4: pcr analysis of transformed plants of high oleic h. annuus l. genotypes, cv. capella and swsr2 inbred line, 14-16 weeks after co-cultivation. 100 ng of ecori digested genomic dna was amplified with specific primers to gus and nptii genes. (a) with gus primers and (b) with nptii primers, lane (1) molecular marker dna, lane (2-6 and 12-16) transformed swsr2 inbred line plants, lane (7-9 and 19-21) transformed cv. capella plants, lane (10, 17) positive control (plasmid dna) and lane (11, 18) negative control (untreated plants). 176 sherin mohamed, robert boehm, heide schnabl a b c in an evaluation of the number of shots per explant, the results show that bombarding the explants two times resulted in the highest levels of gus activity. these results were in agreement with pereira and erickson (1995), schöpke et al. (1997) and bhatnagar et al. (2002) who revealed that double bombardment of the same tissue increased the number of transformed explants. moreover, our results clearly demonstrated the importance of preculture phase which seems to be a general feature with the current technology (harwood et al., 1995; hunold et al., 1995; pereira and erickson, 1995; nehlin et al., 2000). when explants were cultured for one day prior to bombardment, the highest gus expression frequency was obtained compared to 0 and 2 days of culture. we hyopothesize that the increase of the gus expression after 1 day of pre-culture is due to the reduction of bombardment shock and, consequently, to tissue injury. folling and olesen (2002) suggested that the positive effect of pre-culture on transformation efficiency of wheat could be related to the easiness by which plasmid dna reaches the chromosomes as well as to the damaging effect of the bombardment. therefore, a helium pressure of 1550 psi in combination with a target distance of 6 cm, 1.6 µm gold particle size, bombardment the explants twice and pre-culture the explants for one day prior to bombardment were found to be a compromise between cell vitality and gus expression frequency. for an estimation of the resulting transformation frequency the optimized parameters were applied on split shoot apices of cv. capella and swsr2 inbred line and the plants were subjected to histochemical, fluorometric and molecular analysis. with regard to histochemical analysis, two plants of cv. capella and one plant of swsr2 inbred line did not express the gus gene despite of their positive pcr results. we suggest that gene silencing took place which possibly resulted from the interaction among the multiple integrated copies of transgene (sanford, 1990; kumpatla et al., 1997) or when additional copies of an endogenous gene are expressed ectopically involves homology dependent gene silencing (hdgs) (reddy et al., 2003) or dna methylation (al-kaff et al., 2000; reddy et al., 2003). similar results were previously observed in potato (ottaviani et al., 1993), pearl millet (lambé et al., 1995) and soybean (reddy et al., 2003). the fluorometric gus activity and the transformation frequency results reflected that the swsr2 inbred line showed a higher response than cv. capella for the present transformation, since the transformation frequency amounted to 3.1 and 4.5 % for cv. capella and swsr2 inbred line, respectively. in accordance, stable transformation was achieved via particle bombardment for different plant species such as alfalfa (pereira and erickson, 1995), cassava (zhang et al., 2000), potato (romano et al., 2001), barley (manoharan and dahleen, 2002), wheat (chugh and khurana, 2003), orchid (men et al., 2003), soybean (reddy et al., 2003), sorghum (tadesse et al., 2003) and rice (cho et al., 2004). additionally, particle bombardment appears to be the best technique for gene transfer into conifers (humara et al., 1999). finally, the results showed for the first time that the biolistic method can be successfully combined with direct regeneration system (avoiding dedifferentiated cell stages) in producing high oleic h. annuus l. plants. acknowledgements the authors want to thank mrs. gröhne (südwestsaat) for the provision of seeds of cv. capella and swsr2 inbred line. this project was supported by the egyptian gouvernment (grants to sh. m.) which is greatfully acknowledged. references al-kaff, n.s., kreike, m.m., covey, s.n., pitcher, r., page, a.m., dale, p.j., 2000: plants rendered herbicide-susceptible by cauliflower mosic virus-elicited suspension of a 35s promoter-regulated transgene. nat. biotechnol. 18, 995-999. bhat, v., dalton, s.j., kumar, s., bhat, b.v., gupta, m.g., morris, p., 2001: particle-inflow-gun-mediated genetic transformation of buffel grass (cenchrus ciliaris l.): optimizing biological and physiological parameters. j. appl. genet. 42, 405-412. bhatnagar, s., kapur, a., khurana, p., 2002: evaluation of parameters for high efficiency gene transfer via particle bombardment in indian mulberry. indian j. exp. biol. 40, 1387-1392. bidney, d., 1990: expression of ß-glucuronidase in sunflower apical meristems following microprojectile bombardment. in: abstracts 7th international congress on plant tissue and cell culture, amsterdam. bradford, m.m., 1976: a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. anal. biochem. 72, 248-254. cho, m.j., yano, h., okamoto, d., kim, h.k., jung, h.r., newcomb, k., le, v.k., yoo, h.s., langham, r., buchanan, b.b., lemaux, p.g., 2004: stable transformation of rice (oryza sativum l.) via microprojectile bombardment of highly regenerative green tissues derived from mature seed. plant cell rep. 22, 483-489. christou, p., ford, t.l., kofron, m., 1992: the development of a varietyindependent gene-transfer method for rice. trends biotech. 10, 239-246. chugh, a., khurana, p., 2003: regeneration via somatic embryogenesis from leaf basal segments and genetic transformation of bread and emmer wheat by particle bombardment. plant cell tiss. organ cult. 74, 151161. cullings, k.w., 1992: design and testing of a plant-specific pcr primer for ecological and evolutionary studies. mol. ecol. 1, 233-240. dorrell, g., vick, a., 1997: properties and processing of oilseed sunflower. in: schneiter, a.a. 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<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> >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice journal of applied botany and food quality 86, 217 220 (2013), doi:10.5073/jabfq.2013.086.030 departamento de química, universidad de las palmas de gran canaria, unidad asociada al csic, campus de tafira, las palmas de gran canaria, canary islands, spain screening of the antioxidant properties of crude extracts of six selected plant species from the canary islands (spain) milagros rico*, idayra sánchez, cristina trujillo, norma pérez (received september 24, 2013) * corresponding author summary the extracts of six common plants from the canary islands were screened for their antioxidant activities and compared with several phenolic compounds of natural origin (quercetin, catechin, rutin and gentisic acid) and synthetic (butylated hydroxyanisole (bha) and butylated hydroxytoluene (bht)). the in vitro antioxidant activity determined by using the 1,1-diphenyl-2-picrylhydrazyl (dpph) method revealed that plantago major l., artemisia canariensis (bess.) lessing and bidens aurea (dryand.) sherff exerted greater activity than the other plants (90.9%, 89.0% and 88.2% inhibition rate, respectively). the most active plants were bidens aurea (dryand.) sherff and plantago major l. (9.5 and 7.2 trolox μmol equivalents), when the cupric ion reducing antioxidant capacity assay (cuprac) was used. all the plants species exhibited higher antioxidant capacities than the synthetic antioxidants bha and bht. among the natural phenolic compounds, gentisic acid was the most active. however, two of the plant extracts showed higher antioxidant activity than any other of the pure compounds studied, even than that of gentisic acid. the use of reversed phase high performance liquid chromatography (rp-hplc) allowed the identification of the natural phenolic constituents listed above in bidens aurea (dryand.) sherff and plantago major l. extracts. catechin and quercetin were the most prominent phenolic compounds. the presence of phenolic compounds in the plant extracts and their high antioxidant activities underline their phytomedicinal potentials. these plants may be exploited in the production of health foods and as an antioxidant carrier in the food and pharmaceutical industries. introduction the canary islands are an exceptional enclave in the world on account of their privileged climate. the temperature in the islands ranges from 17 º to 24 ºc all year round. the islands are volcanic and mountainous, and the varying altitudes create diverse habitats and ecosystems, making them very interesting from a botanical point of view. the high level of solar radiation forces plants to develop defence mechanisms against ultraviolet radiation through the accumulation of antioxidant substances. the following plant species grow anywhere and everywhere naturally: withania aristata (aiton) pauquy (solanaceae) (w. aristata p.), locally called orobal, are native of the canary islands, the mediterranean and the arabian region; plantago major l., popularly called llantén, is a perennial medicinal herb that belongs to the highly diverse genus plantago of the plantaginaceae family; chenopodium ambrosioides l. (ch. ambrosioides l.), locally called pasote, is an herb of the chenopodiaceae family, indigenous to south america, though it has been distributed to much of the world; bidens aurea (dryand.) sherff (asteraceae), called canary tea, is an european herb widely distributed in the mediterranean areas and commonly used as digestive and sedative; forsskahlea angustifolia retz. (urticaceae) (f. angustifolia r.), popularly known as ratonera, it is present in the seven canary islands; artemisia thuscula cav. (asteraceae), a synonym of artemisia canariensis (bess.) lessing, is an endemic canary islands species, widespread in all the islands in which different species of nitrophilous vegetation are dominant. it is most often found in coastal and mid zones of western canary islands (100 -350 m). these species were selected because they have been used by the canary folklore medicine as panacea for a great diversity of health problems and have traditionally been consumed as infusion or herbal tea (ortega et al., 2000; samuelsen, 2000; darias et al., 2001; martín-herrera et al., 2008). these plant species are estimated to be botanical medications in widespread domestic use among the population. plant polyphenols have been implicated in diverse functional roles, including plant resistance against microbial pathogens and animal herbivores such as insects (antibiotic and antifeeding actions), protection against solar radiation, besides reproduction, nutrition, and growth (harborne and williams, 2000). phenolic compounds have also been reported to prevent diseases resulting from oxidative stress. they have the ability to counteract the damaging effects of free radicals in tissues and thus, they are believed to protect against cancer, atherosclerosis, heart diseases and several diseases (bors et al., 1990; dillard and german, 2000; yao et al., 2004; dai and mumper, 2010; ferrazano et al., 2011; quideau et al., 2011). by other hand, the most widely food synthetic preservatives: butylated hydroxytoluene (bht) and butylated hydroxyanisole (bha) have recently been restricted because of serious concerns about their carcinogenic potential (ito et al., 1986; kahl and kappus, 1993). toxic effects of bha and bht often occur only after high dosage and long-term treatment. however, bha induces in animals tumors of the forestomach, which are dose dependent, whereas bht induces liver tumors in long-term experiments. because there is no indication of genotoxicity of bha and bht, all published findings agree with the fact that bha and bht are tumor promoters. therefore, in recent decades, there has been a great interest in finding new and safe antioxidants from natural sources to replace the synthetics used in food preservation. the chemical diversity of antioxidants makes it difficult to separate and quantify individual antioxidants from the vegetable matrix and the total antioxidant power is often more meaningful to evaluate health-beneficial effects because of the cooperative action of antioxidants (gao et al., 2011). recent studies revealed beneficial effects of plant extracts in ghee (butter oil) during accelerated oxidation in order to understand its potential use as an antioxidant in the food industries (ghandi et al., 2013). the addition of mango seed kernel crude extracts (with high contents of phenolic compounds) at a level of 5% or above was more effective in prolonging the stability of buffalo ghee than the addition of bha at the permitted level in ghee (0.02%) (puravankara et al., 2000). by other hand, macaroni products incorporated with vegetal preparations exhibited improved antioxidant properties: the content of polyphenols increased from 0.46 to 1.80 mg per g of macaroni and the carotenoid content increased from 5 to 84 μg per g of macaroni, without affecting its cooking, textural and sensory properties (ajila et al., 2010). the main objectives of this work consisted in the following, therefore: (1) to determine the antioxidant activities of 218 milagros rico, idayra sánchez, cristina trujillo, norma pérez extracts derived from the plant species listed above with regard to their potential uses; (2) to compare the antioxidant activity of several pure phenolic compounds namely quercetin, catechin, rutin and gentisic acid (widely distributed in vegetables, fruits, tea, coffee, wine and other products (rababah et al., 2011; lópez et al., 2013; rico et al., 2013) with those of the synthetic antioxidants bht and bha. mataterials and methods chemicals methanol (of hplc grade) was obtained from panreac (barcelona, spain) with formic acid, ammonium acetate, copper(ii) chloride and 96% ethanol provided by merck (hohenbrunn, germany) of analytical quality. the 1,1-diphenyl-2-picrylhydrazyl (dpph), 6hidroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid (trolox) and 2,9-dimethyl-1,10-phenanthroline (neocuproine) were from sigmaaldrich chemie (steinheim, germany). polyphenols quercetin and (+) catechin, were purchased from sigma-aldrich chemie; rutin and gentisic acid were supplied by merck (hohenbrunn, germany). plant material w. aristata p., p. major l., ch. ambroioides l., b. aurea s. and f. angustifolia r. were colleted at rincon de tenteniguada (977 m altitude, valsequillo, gran canaria) in october, 2012. a. canariensis l. was collected in tafira (320 m altitude, gran canaria) in october, 2012. soon after collection, aerial parts of the plants were separated, shaken, weighed and frozen. the frozen samples were then freeze-dried and pulverized into powder using a blender (moulinex, 600 w, ecully cedex, france) and were subsequently kept in the dark at -20 °c under nitrogen. the dried residues were weighed and the yield for water was calculated. a voucher specimen has been deposited at the herbarium of the viera y clavijo botanical garden in las palmas de gran canaria: w. aristata p. (lpa: 30929); plantago major l. (lpa: 30930); ch. ambrosioides l., (lpa: 30931); bidens aurea (dryand.) sherff (asteraceae) (lpa: 30932); f. angustifolia r. (lpa: 30933-30935), artemisia canariensis (bess.) lessing (lpa: 30936). preparation of the samples for dpph and cuprac assays plant extracts: freeze-dried plant material (1.0 g) was extracted with methanol (18 ml) for 1 h at room temperature by mixing using a multipoint magnetic stirrer (anm-10006, paris, france). each extract was filtered for removal of plant particles. after centrifugation at 3000 rpm for 10 min, the supernatant was collected and filtered through 0.45 mm filter paper and stored at 4 ºc. solutions of pure phenolic compounds: all the solutions (of the natural and synthetic pure phenolic compounds) were prepared at the concentration of 200 μg ml-1. free radical scavenging activity on dpph the reducing ability of the antioxidants on the dpph radical was evaluated by measuring the loss of 1,1-diphenyl-2-picrylhydrazyl (dpph) color at 515 nm after reaction with test extracts (bondet et al., 1997). the sample solution (15 ml) was rapidly mixed with one ml of a solution of 0.1 mm dpph. after 20 min incubation in darkness at ambient temperature (23 °c), the reduction of the dpph radical was measured by monitoring the decline in absorbance (abs) against a methanol blank at 515 nm using a shimadzu 1700 uvvis spectrophotometer. the percentage inhibition was calculated by application of the equation: rsa = 100 (1 − abs in the presence of sample/abs in the absence of sample). the estimation of the rsa was carried out in triplicate, and the results were averaged. values mean ± standard deviation of the three measurements. cupric ion reducing antioxidant capacity (cuprac) then cupric ion reducing capacities were determined according to a previously reported method (apak et al., 2004). a solution of cucl2 (125 μl 1.0x10-2 m), 125 μl of neocuproine ethanolic solution (7.5x10-3 m) and 125 μl of nh4ac buffer solution were added to a test tube, followed by mixing; 20 μl of sample followed by 605 μl of water were then added (total volume, 1 ml) and mixed well. absorbance against a reagent blank was measured at 450 nm after 30 min. the estimation of the rsa was carried out in triplicate, and the results were averaged. the same procedure was repeated for all standard trolox solutions (0.2-0.6 mm) and a standard curve was obtained with the following equation: absorbance = 0.2859 x + 0.0762; r2 = 0.9998. the results were expressed as trolox μmol equivalents (tr). determination of the phenolic profile by rp-hplc the phenolic compounds catechin, gentisic acid, rutin and quercetin were quantified in line with a previously reported method (lópez et al., 2011). in brief, the elution conditions applied were 0 -5 min, 20% b isocratic; 5 -30 min, linear gradient from 20% to 60%b; 30 35 min, 60%b isocratic; 35 -40 min, linear gradient from 60% to 20% b; and, finally, washing and reconditioning of the column. each standard was individually tested to determine its retention times (rt) as follows: (+) catechin (rt: 12.7 min), gentisic acid (rt: 17.1 min), rutin (rt: 28.1 min) and quercetin (rt: 34.6 min) were well resolved. simultaneous monitoring was set at 270 nm ((+)-catechin), 324 nm (gentisic acid) and 373 nm (rutin and quercetin) for quantification. reproducibility, expressed as relative standard deviation (rsd), ranged from 1.91% to 5.81%. the accuracy was expressed as the recovery of standard compounds added to the pre-analyzed sample. the recovery was found to be in the range of 87.97%-115.79%. the limits of detection (lods) were found to be in the range of 0.00030.1230 mg ml-1 and the limits of quantification (loqs) were observed in the range of 0.0008-0.4100 mg ml-1. results evaluation of the water content the content of water was as follows (tab. 1): b. aurea s. > p. major l. > ch. ambroioides l. > w. aristata p. > a. canariensis l. > f. angustifolia r. tab. 1: content of water of the plant species (%). plant species water content b. aurea s. 89.6 p. major l. 88.6 c. ambroioides l. 80.5 w. aristata p. 68.4 a. canariensis l. 62.7 f. angustifolia r. 43.2 free radical scavenging activity on dpph in tab. 2 is shown the relative antioxidant efficiency of six plant extracts by quenching dpph radical. among the plants p. major antioxidant properties of canary plants 219 l., a. canariensis l. and b. aurea s. exerted more potent radical scavenging activity than any of the other samples (90.9%, 89.0% and 88.2% inhibition rate, respectively). the plant extract with the weakest scavenging potency was ch. ambrosoides l. (29.1%), which had significantly lower activity than the other plant extracts. however, ch. ambrosoides l. gave higher activity than bha. among the pure phenolic compounds of natural origin tested, gentisic acid showed the highest activity (67.8%), even much higher than those of the synthetic antioxidants bha and bht (22.7% and 5.0%, respectively) (tab. 2). discussion according to the existing food additive regulations, bha and bht are lawful for use individually or in combination at a maximum level of 0.02%, or 200 ppm, based on the lipid content of food products (pratt, 1996; reische et al., 1998). therefore, we used the concentration 0.2 g l-1 to test the antioxidant activity of the pure compounds. because the extracts are complex mixtures that include active components at lower levels, they were prepared by solving 55.6 mg of freeze-dried plant material in 1 ml of methanol. methanol was selected as extracting solvent because several studies have reported that high levels of phenolic compounds are associated with the use of polar solvents in the extraction (hayouni et al., 2007; lópez et al., 2011). the in vitro antioxidant activities determined by using the dpph and cuprac assays revealed that all the plant extracts exhibited much higher antioxidant activities than the synthetic phenolic compounds bha and bht, which are used as preservatives in many foods, cosmetic products and drugs (tab. 2 and tab. 3). by other hand, the most active phenolic compound of natural origin was gentisic acid (rsa: 67.8%; rc: 3.17 tr), which gave lower activity than those of the b. aurea s. and p. major l. extracts. among the samples, the plant extracts exhibited the highest antioxidant activity compared to the individual standards probably due to a synergistic effect of combining the antioxidants (jacobovelazquez and cisneros-zevallos, 2009). correlation was found between the activities of the pure compounds determined by both methods. however, there is no simple correlation between the radical scavenging activities and the copper reducing capacities of the plant extracts. this may be due to the fact that the cuprac method offers distinct advantages over other electron transfer based assays: applicability to both hydrophilic and lipophilic antioxidants (unlike dpph); the redox reaction giving rise to a colored chelate of cu(i)-neocuproine is relatively insensitive to a number of parameters adversely affecting dpph, i.e., air, sunlight, humidity, and ph, to a certain extent. moreover, the extracts are complex mixtures with active components that may vary the antioxidant potential or the responses to different kinds of assays. the lack of correlation is in agreement with other literature (apak et al., 2007; badarinath, 2010). quercetin, catechin, rutin and gentisic acid were identified in the extracts of p. major l. and b. aurea s., confirming their phytomedicinal potentials as natural sources of well-known antioxidant compounds (silva et al. 2002). conclusions the results of this study confirmed that the selected plant materials, especially p. major l. and b. aurea s. gave higher antioxidant activities than those of the synthetic compounds bha and bht, commonly used as food preservatives. in addition, the presence cupric ion reducing antioxidant capacity (cuprac) the cuprac assay was used to study the ability of the antioxidants in the extracts to reduce cupric copper to the cuprous form. when the reducing capacities (rc) of the plant extracts were compared, b. aurea s. and p. major l. showed the highest capacities (9.5 and 7.17 tr, respectively) (tab. 3). the extract of f. angustifolia r. gave the weakest activity (1.49 tr). however, all the plant extracts exhibited higher antioxidant activities than those of the synthetic antioxidants tba and tbh (1.26 and 0.09 tr, respectively) and natural quercetin, catechin and rutin (2.17, 0.96 and 0.62 tr, respectively). gentisic acid showed the highest activity among the natural phenolic compounds (3.17 tr). tab. 2: radical scavenging activities of the samples expressed as inhibition percentage. phenolic compound rsaa plant species rsaa bht 5.0 ± 0.1 p. major l. 90.9 ± 0,5 bha 22.7 ± 0.4 a. canariensis l. 89.0 ± 0,4 quercetin 28 ± 2 b. aurea s. 88.2 ± 0.3 catequin 21.3 ± 0.5 w. aristata p. 47 ± 3 rutin 12 ± 1 f. angustifolia r. 45 ± 2 gentisic acid 67.8 ± 0.9 ch. ambrosoides l. 29.1 ± 0.4 a values represented mean ± standard deviation of three measurements. tab. 3: cupric ion reducing capability expressed as trolox μmol equivalents (tr). phenolic compound rca plant species rca tbh 0.09 ± 0.06 b. aurea s. 9.5 ± 0.1 tba 1.26 ± 0.02 p. major l. 7.17 ± 0.01 quercetin 2.17 ± 0.00 a. canariensis l. 2.4 ± 0.1 catequin 0.96 ± 0.00 ch. ambrosoides l. 2.2 ± 0.2 rutin 0.62 ± 0.00 w. aristata p. 1.88 ± 0.00 gentisic acid 3.17 ± 0.02 f. angustifolia r. 1.49 ± 0.00 avalues represented mean ± standard deviation of three measurements. determination of the phenolic profile by hplc the proposed polyphenols quercetin, catechin, rutin and gentisic acid were identified in the extracts of the most active plants, plantago major l. and bidens aurea (perkins) sherff and the results are summarized in tab. 4. catechin and quercetin were the most abundant compounds of those under study. tab. 4: polyphenol contents in plant extracts presented as average values ± standard deviation of two measurements in mg per gram of freezedried plant material. plantago major l. bidens aurea (perkins) sherff catechin 1637 ± 79 1142 ± 24 gentisic acid 97 ± 1 59 ± 3 rutin 345 ± 30 287 ± 11 quercetin 592 ± 32 473 ± 13 sum 2671 1961 220 milagros rico, idayra sánchez, cristina trujillo, norma pérez of well-known antioxidant compounds afford more than sufficient arguments for researching the viability of the use of the selected plants in the health and food industries in general, as well as in the pharmaceutical industry. acknowledgments the authors would like to express their gratitude to maría 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accepted: november 27, 2020) * corresponding author summary more than 1000 different cacao varieties are described. only few are considered as fine or flavour cocoas, meaning they have the potential to develop special flavour characteristics after appropriate fermentation and drying. it is assumed that aroma compounds located in the fruit pulp migrate into the seed during fermentation. we studied the fine aroma potential of five cacao varieties selected at catie, costa rica, by analysing aroma compounds in their fresh fruit pulps using headspace spme-gcms. pulps of unripe, ripe and overripe fruits harvested in the dry and rainy season, respectively, were compared to the control genotypes eet 62 and sca-6, both known for high amounts of fine aromas described as e.g. “fruity”, “floral” or “spicy”. all genotypes contained a basic content of the two dominating esters 2-pentanol acetate and 2-heptanol acetate, combined with a mixture of aroma-active compounds with small peak areas that form the variety-specific aroma character. total aroma diversity and intensity increased during ripening. aroma profiles were more diverse when fruits ripened during the dry season, whereas aroma intensity was higher in the rainy season. thus, the fruit and environmental conditions prior to harvest can already play a decisive role for the aroma potential of the cacao pulp. due to their aroma profiles, the varieties from catie can be classified as fine or flavour cocoas. keywords: cocoa; flavour; fine or flavour cocoa; secondary plant metabolites introduction the seeds of the cacao tree (theobroma cacao l.) are the key raw material for chocolate products. they are the source of income for millions of small-scale farmers in tropical and subtropical countries. industry and trade distinguish between bulk cocoa and fine or flavour cocoa. whereas bulk cocoas are characterised by a broad chocolate flavour that builds up during the fermentation and drying process and usually lack any special flavour attributes, fine cacao varieties additionally develop special aromas described for example as fruity, floral or spicy (sukha, 2008) that originate from plant secondary metabolites. the most important volatile and non-volatile fine aroma compounds in cocoa belong to the groups of terpenes, alcohols, aldehydes, carboxylic acids, methyl ketones and esters (aprotosoaie et al., 2016; ziegleder, 1990; eskes et al., 2007; kadow et al., 2013). they derive from different synthesis pathways, differ in their chemical properties and undergo transformation during cocoa post-harvest processes (belitz, grosch and schieberle, 2009). in addition to these compounds, pyrazines and furan derivatives play a key role in the flavour of chocolate liquors as they display e.g. nutty notes and enhance the overall flavour (afoakwa et al., 2008; aprotosoaie et al., 2016; belitz, grosch and schieberle, 2009; rodriguezcampos et al., 2011; schieberle and pfnuer, 1999). fine or flavour aroma compounds can derive from different tissues within the seeds or are a result of yeast and bacterial activity during fermentation (schwan and wheals, 2004), but most of them are formed or stored in the fruit pulp (eskes et al., 2007; pino et al., 2010). aldehydes for example can be found in the fresh fruit pulp, but are also built within the seeds during the fermentation process in the course of strecker degradation from amino acids (belitz, grosch and schieberle, 2009; afoakwa et al., 2008). also, they derive from primary alcohols via autoxidation or enzymatic lipid peroxidation of saturated and unsaturated fatty acids. however, due to the fatty acid pattern of the cacao seeds (with a ratio of palmitic, stearic, oleic and linoleic acid of 25:37:34:3, respectively) the autoxidation process can be largely excluded (belitz, grosch and schieberle, 2009). secondary alcohols like 2-nonanol can be oxidized to ketones and reduced to esters under the influence of carboxylic acids. with some rare exceptions (e.g. ancient criollos) most fresh cacao seeds are inedible, they must first be fermented and dried to reduce bitterness and astringency. cacao seeds are usually fermented spontaneously in their own mucilaginous fruit pulp. yeasts and bacteria utilize sugars from the pulp for their metabolism and enable mole cules from the pulp to enter the cacao cotyledons during the fermentation process (schwan and wheals, 2004). chetschik et al. (2018) found the same aroma compounds in the cacao fruit pulp and in the cocoa beans: since many aroma compounds are lipophilic, it can be assumed that these substances are transferred from the cacao pulp to the cotyledons and accumulated in storage fats (kadow et al., 2013). depending on variety, season and region, cacao seeds contain between 45% and 60% fat (pires et al., 1998; lockwood and eskes, 1996; rohsius, 2010), so that large quantities of the less polar aromatic substances from the pulps could be stored in the lipid vacuoles of the mesophyll cells after fermentation (rohsius, 2007). cacao fruit pulp may therefore play a crucial role as a precursor in the aroma development, composition and intensity of the cocoa beans and ultimately has the potential to contribute to the organoleptic character of the chocolate liquor. fruit pulp is a combination of modified mesocarp and endocarp and develops during the ripening process of the cacao fruit (lieberei and reisdorff, 2012; andersson et al., 2006). during the last weeks of fruit ripening, the pulp begins to accumulate sugars and acids while the content of pectin decreases (rohsius, 2007). due to biochemical changes within the fruit the pulp becomes more viscous and softer towards the end of fruit ripening. even when ripe, cacao fruits remain on the tree and do not fall off (rohsius, 2007). since ripe fruits can only be distinguished from unripe fruits by their colour and the cacao pulp aroma diversity 267 cacao tree produces fruits all year around (see phillips-mora et al., 2013, fig. 13), knowledge and proper harvest management are indispensable. nonetheless, cacao farmers often harvest unripe or already overripe fruits and combine and process all beans together. this may influence the overall aroma of the fruit pulp in the fermented cocoa mass and has an effect on the final cocoa bean aroma. drought increases the aroma concentration in many fresh fruits like wine grapes (deluc et al., 2009; savoi et al., 2016) and its processed products like wine (savoi et al., 2020). an effect of the seasons on the aroma of cocoa beans or cacao fruit pulp has not yet been studied, though several studies show an effect of environmental conditions and season on the molecular composition of cocoa beans (niether et al., 2017; de araujo et al., 2018; sukha, 2008). here, we studied the aroma potential of five cacao varieties (catie r1, catie-r4, catie-r6, pmct-58 and ics-95 (t1)) from catie, costa rica (phillips-mora et al., 2013) by analysing the aroma characteristics of the fresh fruit pulps. these clones are the first materials accessible to farmers which possess, to a greater or lesser degree, a combination of desirable characteristics, particularly resistance to frosty pod (moniliophthora roreri), yield potential and flavour quality (phillips et al., 2009; phillips-mora et al., 2013). they are now widely distributed in central america and mexico and are being evaluated for cultivation in brazil (phillips-mora et al., 2017). unripe, ripe and overripe fruits were harvested in the dry and in the rainy season to analyse the influence of the ripening stages and the effect of climatic conditions during fruit ripening on aroma volatiles in the pulp. the intensity and diversity of aroma compounds were compared to those detected in the two control genotypes eet 62 (“nacional” cacao, according to delgado et al., 2003) and sca-6 (“contamana” cacao, according to motamayor et al., 2008) that are known for their fine aroma notes (kadow et al., 2013). we hypothesize that (1) the cacao varieties from catie have the potential to develop a diversity of aroma compounds due to their genetic background, (2) intensity and composition of different aroma substances increase during fruit ripening and (3) pulp aroma intensity is enhanced in fruits of the dry season as shown for other fruit crops. material and methods study location and fruit sampling fruit sampling of the potential fine or flavour varieties (catie-r1, catie-r4, catie-r6, ics-95 (t1), pmct-58) and the varieties used as control genotypes (eet 62, sca-6) was conducted at the international cacao collection (ic3) of the tropical agricultural research and higher education center (catie). the institute is located in turrialba, in the central highland of costa rica (602 m a.s.l.) with a mean annual precipitation of 2645 mm and a mean temperature of 22.5 °c (arciniegas, 2005). from all varieties, up to three unripe, ripe and overripe fruits, respectively, were harvested each in november 2012 (ripening during the rainy season) and in april 2013 (ripening during the dry season). fruit ripening stages were determined visually by the typical fruit colour characteristics of each variety and their changes during ripening (phillips-mora et al., 2013). due to production limitations, the number of three fruits could not be reached for all varieties, seasons and ripening stages resulting in a total of 51 samples from the rainy and 67 samples from the dry season. the correct number of fruits used for analysis is shown in the annex table 1a. the fruits were harvested in the morning hours, disinfected, individually wrapped in 4 cm thick foam, stored in a well isolated shipping box and immediately sent by express (3-4 days) to the institute of food chemistry at the university of hamburg, germany, for analysis. analysis of volatile aroma compounds in fresh cacao fruit pulps cacao fruits were opened and the ripening stage of the pulps determined: pulps of unripe fruits were firmly connected with cotyledons and mesocarp of the fruit, starch degradation had not yet occurred; ripe fruit pulps had slightly dissolved from the mesocarp already and the pulps were aqueous; overripe fruits presented pulps completely separated from the mesocarp and, depending on the variety, they were of slightly brown colour. from each fruit, two fresh pulp samples of each 5 g including the testa were removed from the cotyledons using a scalpel and directly weighed into a 20 ml headspace crimp neck vial n 20 (macherey nagel). vials were closed gastight using an aluminium crimp cap type n20 (8 mm, septum: blue silicone/ ptfe colourless, 3 mm, macherey nagel). samples were stored at -80 °c. for the analysis, each sample vial was heated in a water bath at 30 °c for 15 min. volatile aroma compounds accumulated in the headspace were obtained by solid-phase micro-extraction (spme) during 15 min at 30 °c using a pdms / dvb fibre (65 μm, needle size 24ga, spme fiber assembly, stableflex, supelco, sigmaaldrich group). analytes were then manually injected into the gas chromatograph (6890 series gc-system, agilent technologies) and separated into the individual substances for subsequent identification and quantification in the mass spectrometer (s 5973 network mass selective detector, agilent technologies) (tab. 1). chromatograms were evaluated using openchrom 9.0. for aroma identification, mass spectra of the volatiles were compared with the reference spectra of the nist library (national institute of standards and technology). fruit pulp samples were analysed in duplicate. one sample of the control genotype eet-62 indicated with 5.29e + 09 the highest mean total peak area of volatile aromas in its pulp of ripe fruits. this was used as reference area for calculating the proportional peak area of all samples. aroma compounds detected in the fresh fruit pulps were assigned to the aroma groups “green”, “herbal”, “fruity”, “floral”, “spicy”, “woody” and “earthy” (tab. 2, according to mosciano, 1989; 1990a, b; 1991a, b, c, d; 1992a, b; 1993a, b; 1995a, b, c; 1996a, b; 1997a, b; 1998; 2000; 2001a, b; 2009 via the good scents company, surburg and panten (2006), nozaki et al. (1997), hui (2010), lan-phi et al. (2009)). the difference between the sum of peaks and the total peak area was grouped as “others” together with four aroma compounds that were either not clear in their aroma characteristics or unassignable to one of the other aroma groups. statistical analysis a multivariate pca (principal component analysis) was performed on 67 pulp samples from the dry season only, using all the detected aroma compounds to give a descriptive overview over the global dataset and the relation of the levels of the factors “variety” (catie r1, catie-r4, catie-r6, ics-95, pmct-58, eet 62, sca-6) and “ripening” (unripe, ripe, overripe). a second pca was performed from 95 pulp samples from ripe and overripe fruits (pooled) for the factors “variety” (catie-r1, catie-r4, catie-r6, ics-95, pmct-58, eet 62, sca-6) and “season” (dry, rainy). pca plots were visualized with the ggfortify r package (horikoshi and tang, 2016) within the statistical programming environment r (r core team, 2020). spearman’s rank correlation (ρ) for non-normally distributed data was used to evaluate bivariate relationships between the aroma groups. linear mixed-effects models (lmertest, kuznetsova et al., 2017) were used to study the factors influencing the aroma groups (response variables) of the fruit pulp. in the first model with samples from the dry season only, the fixed factors “variety” (catie-r1, catie-r4, catie-r6, ics-95, pmct-58) and “ripening” (unripe, ripe, overripe) and their interaction were analysed. in a second model using only ripe and overripe (pooled) fruits from both seasons, the effects of the fixed factors “variety” (catie-r1, catie-r4, catie-r6, 268 e. hegmann, w. niether, w. phillips, c. rohsius, r. lieberei ics-95, pmct-58) and “season” (dry and rainy) and their interaction on the aroma groups as response variables were analysed. the number of pulp analyses per treatment combination (replication, four levels) was added as a random factor in both models and “ripening” (ripe and overripe) was added as a random factor in the second mo del. when statistical differences were found, a post-hoc pair comparison of least significant means (lsmeans r package, lenth, 2016) was applied. when necessary, data were box cox-transformed to meet the normality and homoscedasticity requirements. data are shown as mean with the standard error of means. data frames were managed with the plyr r package (wickham, 2011). graphs were designed with the ggplot2 r package (wickham, 2016). results in total, we detected 56 aroma compounds in the fresh fruit pulps out of eight aroma groups (tab. 2). not all volatiles were found in all varieties. we detected 22 and 26 aroma compounds in eet 62 and sca-6, respectively. in the catie varieties, many of these aroma compounds were detected in addition to others summing up to a total of 35 aroma compounds in catie-r1, 44 in catie-r4, 37 in catie-r6, 33 in pmct-58 and 41 in ics-95. the evaluation of the gcms chromatograms of both seasons showed that eet 62 had the largest mean total peak area (5.29 e+09), followed by ics-95 (3.89 e+09) and sca-6 (3.73 e+09). the varie ties catie-r4 (2.91 e+09), catie-r1 (2.99 e+09) and catie-r6 (2.99 e+09) showed similar mean total peak areas. the genotype pmct-58 presented the smallest mean total peak area with 2.46 e+09, meaning less than half of eet 62. across all varieties, we observed an effect of the season on the expression of aroma compounds: aroma profiles were more diverse in fruit pulps from the dry season whereas aroma intensity (greater mean peak areas) was higher in samples from the rainy season (tab. 2). all varieties showed many different “fruity” compounds. in comparison, the amount of detected compounds associated with the groups “woody”, “earthy” and “spicy” was rather low. in ics-95 and eet 62 the aroma group “green” dominated with 2-heptanol acetate as principal compound. the cacao varieties catie-r4 and catie-r6 showed very similar aroma compositions with dominating peaks in the aroma groups “floral”, “fruity” and “spicy”, such as the acyclic monoterpenes α-ocimene and α-myrcene. in addition, we detected traces of the sesquiterpene α-bergamotene (“woody”) as well as the ester linalyl acetate (“herbal”) in these varieties, similar to the control sca-6. the pulp aroma of catie-r1 consisted primarily of alcohols, esters and ketones. the high proportion of “fruity” aromas based on 2-pentanol acetate and 2-pentanone. in pmct-58 we found aromas that were either not present in the other varieties or only to a smaller extent, such as the “floral” aromas styrallyl alcohol, styrallyl acetate and benzacetaldeyde or the “woody” α-gurjunene (tab. 2). pmct58 did not show any “earthy” compounds. the first two components of the pca explain 38.65% of the variance between the samples (fig. 1). the aroma compounds were distributed in the pca according to their aroma groups (fig. 1a). the first axis of the pca (pc1) was mainly loaded with compounds of the “green” (e.g. pentyl-furan and 2-octenal) and “herbal” (e.g. 3-octanone and 1-hexanol) on the positive side, on compounds of the “floral” (e.g. trans-linalooloxide and styrallyl-alcohol) and “fruity” groups (e.g. pentanol-acetate and trans-β-farnesene) on the negative side. the second component of the pca (pc2) was loaded with compounds of the “fruity” group (e.g. 2-heptanol and 2-nonanone) on the positive side and “woody” groups (e.g. α-bergamotene and α-copaene) on the negative side. the variety sca-6 was separated from eet 62, pmct 58 and ics95 (pc1) and could be separated from catie-r4 (pc2). ics-95 and eet 62 were clustered to the positive side of pc2 (fig. 1b), where “fruity” aroma compounds were found, only the unripe fruits of ics-95 spread to the positive side of pc1 and the negative side of pc2, like the unripe fruits of catie-r4 (fig. 1b and 1c). the different ripening stages of pmct-58 were clustered all together. catie-r1 and catie-r6 as well as sca-6 spread along the negative sides of pc1 and pc2. the clusters of the ripe and overripe fruits were similar, overlapping with the areas where also “floral” and “fruity” aroma compounds were computed, while the cluster of the unripe fruits mainly overlapped with the “green”, and partly with “herbal” aroma compounds (fig. 1c). pulp aroma intensity and composition change throughout the fruit ripening process (fig. 2, tab. 3). in the pulp of unripe cacao fruits, we found less aroma diversity than in ripe and overripe fruits. all varieties except catie-r1 displayed very low concentrations of “floral” and “spicy” notes in unripe fruits and “woody” aroma substances were not detected in any of the cacao varieties at this ripentab. 1: gcms structure and conditions gas chromatograph 6890plus mass-spectrometer s 5973 injector kas 4 transferline 300 °c program start-temp.: 200 °c, hold for 30 s; 12 °c min-1 at 240 °c, hold for 10 min ms source 230 °c front inlet mode: pulsed splitless ms quad 150 °c pressure: 48,5 kpa mass scan 40-400 pulse pressure: 250 kpa solvent delay 0.5 min pulse time: 30 s purge flow: 40.1 ml min-1 purge time: 28,8 s column db-wax (agilent j&w, 30 m, 0,25 mm inner diameter, 0,25 μm, catalogue-no.122-7032) carrier gas helium 4,6 flow 1 ml min-1; constant oven temperature program: rate temperature hold [°c min-1] [°c] [min] initial 40 3 ramp 1 3 100 0 ramp 2 10 150 0 ramp 3 15 240 5 cacao pulp aroma diversity 269 9 associated with the groups “woody”, “earthy” and “spicy” was rather low. in ics-95 and eet 62 the 196 aroma group “green” dominated with 2-heptanol acetate as principal compound. the cacao varieties 197 catie-r4 and catie-r6 showed very similar aroma compositions with dominating peaks in the aroma 198 groups “floral”, “fruity” and “spicy”, such as the acyclic monoterpenes -ocimene and myrcene. in 199 addition, we detected traces of the sesquiterpene -bergamotene (“woody”) as well as the ester linalyl 200 acetate (“herbal”) in these varieties, similar to the control sca-6. the pulp aroma of catie-r1 consisted 201 primarily of alcohols, esters and ketones. the high proportion of “fruity” aromas based on 2-pentanol 202 acetate and 2-pentanone. in pmct-58 we found aromas that were either not present in the other 203 varieties or only to a smaller extent, such as the “floral” aromas styrallyl alcohol, styrallyl acetate and 204 benzacetaldeyde or the “woody” -gurjunene (table 2). pmct-58 did not show any “earthy” 205 compounds. 206 207 table 2 aroma compounds detected in fresh cacao fruit pulps of seven cacao varieties and their 208 assignment to aroma groups. studied ripening stages: 1= unripe; 2= ripe; 3= overripe. not all ripening 209 stages for all clones were available at both seasons. further details in annex table 1 210 mean peak area < 5.0e+06 mean peak area < 1.0e+08 > 1.0e+09 not detected mean peak area < 5.0e+07 mean peak area > 1.0e+09 211 ar om a gr ou p clone catie-r1 catie-r4 catie-r6 pmct-58 ics-95 eet 62 sca-6 season dry rainy dry rainy dry rainy dry rainy dry rainy dry rainy dry rainy ripening stage 1,2,3 3 1,2,3 3 1,2,3 3 1,2,3 2 1,2,3 1,2 2 2 2 2 aroma compound gr ee n trans-2-nonenal cis-6-nonenal trans-2-cis-6-nonadienal trans-2-octenal trans-2-hexenal cis-3-hexenyl acetate 2-pentylfuran 2-heptanol acetate hexanal cis-3-hexenol heptanal he rb al 5-methyl-3-heptanone 2-methyl-3-buten-2-ol 1-octen-3-yl actetate 3-octanone 1-hexanol -cadinene trans-ocimene linalyl acetate fr ui ty -ocimene benzaldehyde -cubebene 2-nonanone 10 2-undecanone 2-pentanone 2-octanol acetate 2-pentanol acetate 2-undecanol 2-heptanol 2-nonanol octanal trans--farnesene flo ra l linalool trans-linalooloxide epoxylinalool benzyl acetate nonanal 2,3-butanediol diacetate benzacetaldehyde styrallyl acetate styrallyl alcohol acetophenone sp ic y -myrcene trans-caryophyllene 2-octanol w oo dy -gurjunene -bergamotene -copaene ea rt hy cis-linalool oxide 2-octanone 3-octanol 1-octen-3-ol ot he rs 2-pentanol 1-pentanol 3-methyl-1-butanol 2-heptanone tab. 2: aroma compounds detected in fresh cacao fruit pulps of seven cacao varieties and their assignment to aroma groups. studied ripening stages: 1= unripe; 2= ripe; 3= overripe. not all ripening stages for all clones were available at both seasons. further details in annex tab. 1a. 270 e. hegmann, w. niether, w. phillips, c. rohsius, r. lieberei ing stage. volatiles with “fruity” attributes increased in all varieties with increasing ripening stage and were highly concentrated in pulps of overripe fruits. at this stage, ics-95, catie-r4 and catie-r6 displayed an enhanced share of “herbal”, “floral” and “spicy” aromas as well. ics-95 stood out for its relatively high concentration of “green” components, in addition to “earthy” volatiles. tab. 4 shows a significant positive correlation between the aroma groups “spicy” with “floral” and “herbal”. thus, when “spicy” aromas increased, the aroma groups “herbal” and “floral” increased as well. on the contrary, the aroma groups “green” and “earthy” decreased with an increase of “fruity” volatiles. aroma components combined in the group of “others” increased with the aroma group “green”, but were negatively correlated with the aroma groups “floral” and “spicy”. due to the overlapping of the samples from ripe and overripe fruits and the distinct pattern of the unripe samples (fig. 1), we deleted the samples from unripe fruits and pooled the ripe and overripe samples to check the influence of the dry and the rainy season, respectively, on the aroma profile. the first two components of the pca explain 37.66% of the variance of the dataset (fig. 3). the positive side of pc1 is loaded with aroma compounds of the “fruity” aroma group (e.g. 2-octanol-acetate and nonanol), and the negative side is loaded with compounds of the “floral” (e.g. linalool and trans-linalooloxide) and “woody” aroma groups (e.g. α-bergamotene and α-copaene) (fig. 3a). pc2 is loaded with compounds of the “fruity” (e.g. 2-nonanone and 2-heptanol) and “earthy” aroma groups (e.g. 2-octanone) on the positive side, and “floral” (e.g. benzyl-acetate and nonanal) and “woody” aroma groups (e.g. α-gurjunene) on the negative side. we observed a strong separation of the varieties ics-95 and eet 62 from the other genotypes towards the positive side of pc1 (“fruity” aroma compounds) (fig. 3b). pmct-58 was clustered in the centre, as well as catie-r1. catie-r4 and catie-r6 stretched along pc1 directing to the “floral” and “woody” aroma groups. the samples from the dry season were separated from the rainy season (fig. 3c). the dry season samples were mostly located at the positive side of pc1 and the negative side of pc2 (“fruity” to “floral” and “woody” aroma groups). the samples of the rainy season spread along the negative side of pc1 for the varieties catie-r1, catie-r4 and catie-r6 (“floral” and “woody”) and to the “fruity” and “earthy” side for eet 62 and ics-95. all studied varieties showed higher aroma concentrations in pulps of fruits that ripened during the rainy season (fig. 4, tab. 5). especially the concentration of “fruity” and “floral” aroma increased and to a lesser extend the remaining aroma groups. the difference between the season was highest in the genotypes ics-95 and catie-r6. the varieties catie-r4 and catie-r6 displayed a pronounced increase in the total concentration of “spicy” and “floral” aroma towards the rainy season. discussion fine aroma potential of the cacao varieties from catie the chromatograms of all studied cacao fruit pulps showed largest peak areas for the esters 2-pentanol acetate (“fruity”) and 2-heptanol acetate (“green”) (tab. 2). these two esters also accounted for the largest share of total pulp aroma in comparable studies using cacao pulps from colombian varieties (pino et al., 2010) and eet 62, sca-6, ccn 51 (kadow et al., 2013), respectively. a determination or differentiation of fine or flavour cocoa from bulk cocoa by these compounds is not sufficient. the formation of variety-specific fine aromas is rather caused by a combination of compounds with smaller peak areas. the concentrations of some minor compounds might have been below the odour threshold (kadow et al., 2013), but in combination with other volatiles they could provide a new aroma quality through so-called “additive effects”, which can be decisive for the overall aroma (belitz, grosch and schieberle, 2009). the pulp aroma of sca-6 showed high proportions of “spicy” and “floral” terpenes and alcohols and was similar to the varieties catie-r4 and catie-r6. the latter are siblings from the cross “uf 273 (t1) × pa-169” (phillips-mora et al., 2013) and in comparison with the other selections of catie, both showed highest peak areas of the monoterpenes trans-ocimene, α-ocimene and myrcene as well as the floral terpenealcohol linalool. catie-r1 showed traces of the complex sesqui terpenes α-copaene (“woody”) and trans-β-farnesene (“fruity”), as do catie-r4 and catie-r6. these similarities can be related to the fig. 1: aroma compounds in the fruit pulps of seven cacao varieties for three ripening stages (during dry season); a) loading of the principle component pc1 and pc2 with the aroma compounds sorted by aroma groups; b) distribution of the fruit pulp samples indicated by the factor variety c) distribution of the fruit pulp samples indicated and clustered by the factor ripening stage cacao pulp aroma diversity 271 fig. 2: share of aroma groups detected in the cacao fruit pulps of the five varieties catie-r1, catie-r4, catie-r6, ics-95, pmct-58 in three different fruit ripening stages. shown are mean values and standard error. maximum total peak area was detected in one sample of eet 62 and used as reference area. for comparison, we show the data of the varieties eet 62 and sca-6 for the ripening stage “ripe”. 272 e. hegmann, w. niether, w. phillips, c. rohsius, r. lieberei fact that all three varieties have the same genetic mother uf-273 (t1) (phillips-mora et al., 2013). uf-273 (t1) is a hybrid between the “nacional” variety from ecuador (63%) and the “matina” forastero variety from costa rica (32%) (phillips-mora et al., 2017; mataquirós et al., 2017). since floral aroma is a distinctive feature of the “nacional” variety, this is possibly the origin of this trait in the three catie-r clones. according to andersson et al. (2006), properties of the pulp are formed from the tissue of the mother plant (mesocarp and endocarp). it can therefore be assumed that the aroma profile of the fruit pulp is principally determined by the maternal genes and the characteristics encoded by them. the potential of the varieties from catie to build up these aroma compounds in the fruit pulp allows a classification as fine or flavour cocoas. ripe cacao fruits can develop a broader aroma spectrum we showed for the first time analytically, that aroma active-compounds with small peak areas vary greatly depending on the ripening stage of the fruit. during the fruit ripening process, the complexity of aroma compounds increases significantly. the proportion of “her bal” and “green” substances decreases in favour of “spicy”, “fruity” and “floral” compounds. it must be assumed that the initial amount of pulp derived aromatic compounds is crucial for the fermentation outcome. findings from amores (2006, cited in voigt and lieberei, 2014) who observed that the floral terpene alcohol linalool appears in the cotyledons on the 3rd day of fermentation suggest that migration of these substances occur from the pulp into the cotyledon. also eskes et al. (2009) showed that aromas migrate from the outer part into the seed by adding aromatic fruit pulp of theobroma grandiflorum and annona muricata to the fermentation of cacao seeds. as a result, these aromatic compounds were perceived sensorially in the chocolate made from it. thus, cacao fruits should be fully ripe when harvested in order to exploit the entire potential of volatile fine aroma tab. 4: aroma groups. correlation matrix of the aroma groups with the correlation coefficient ρ and the level of significance (* p-value > 0.05; ** p-value > 0.01; *** p-value > 0.001; n.s. not significant) green herbal fruity floral spicy woody earthy herbal -0.21 n.s. fruity -0.70 * -0.38 n.s. floral -0.35 n.s. 0.38 n.s. 0.00 n.s. spicy -0.40 n.s. 0.59 * -0.07 n.s. 0.79 *** woody 0.03 n.s. 0.25 n.s. -0.03 n.s. 0.50 n.s. 0.46 n.s. earthy 0.26 n.s. 0.42 n.s. -0.60 ** -0.15 n.s. -0.02 n.s. 0.03 n.s. others 0.75 *** -0.17 n.s. -0.42 n.s. -0.51 ** -0.51 ** -0.04 n.s. 0.23 n.s. fig. 3: principal component analysis (pca) over the aroma compounds detected in all the cacao fruit pulp samples (dry season). a) loading of the principle component pc1 and pc2 with the aroma compounds sorted by aroma groups; b) distribution of the fruit pulp samples indicated by the factor variety: c) distribution of the fruit pulp samples indicated and clustered by the factor season. tab. 3: results from linear mixed-effects models for the aroma groups and the factors variety (including the five varieties catie-r1, catie-r4, catie-r6, ics-95, pmct-58), ripening stage and their interaction; f-value and level of significance: * p-value > 0.05; ** p-value > 0.01; *** p-value > 0.001; n.s. not significant variety ripening variety:ripening f-value f-value f-value green 47.9 *** 55.8 *** 7.6 *** herbal 1.7 n.s. 5.8 ** 2.0 n.s. fruity 43.5 *** 121.2 *** 12.5 *** floral 6.7 *** 19.6 *** 5.7 *** spicy 4.6 ** 17.2 *** 5.5 *** woody 4.6 ** 3.2 n.s. 4.7 *** earthy 31.1 *** 9.3 *** 6.2 *** others 25.1 *** 33.2 *** 1.8 n.s. cacao pulp aroma diversity 273 the aroma potential of the resulting bean composition. otherwise, selecting or blending fruits based on their ripening stage may open up new avenues to obtain chocolate products with differentiated aromas and flavours. the change in aroma properties of the fruit pulp during ripening implies a biological function. the predominantly lipophilic and volatile fine aroma compounds diffuse with increasing vapour pressure through the pericarp (personal communication lieberei, 2015). this explains the observations and assumptions of warren and emamdie (1992) that squirrels select fruits by odour and fruit colour and prefer certain cacao genotypes, such as sca-6. in addition, they prefer ripe fruits over unripe fruits (warren and emamdie, 1992). by selecting especially mature seeds from ripe fruits, rodents play a key role in the natural seed dispersal of the cacao tree. aroma intensification during the rainy season the prevailing climate during fruit ripening strongly influences the aroma of the cacao pulp. in our study, the highest intensity of fine aromas was found in the samples of fruits harvested during the rainy season while highest aroma diversity was detected in fruits harvested during the dry season. it is well known that spicy and medicinal plants exposed to drought stress react with an increase in aromatic compounds deriving from their secondary metabolism (al-gabbiesh et al., 2014; nowak et al., 2010). under water deficit, the stomatal conductance of the cacao leaves decreases (lahive et al., 2018), limiting photosynthesis and the protein metabolism (larcher, 1994). when carbon assimilation is reduced, the plant uses the available energy primarily to form lipids and structural elements (phospholipids in membranes) (taiz and zeiger, 2007). the down-regulation of the protein metabolism may reduce the synthesis of volatile fine aroma compounds from amino acids, which could explain the lower intensity of aroma components in the cacao fruit pulp in the dry season. in addition, when leaves heat up due to high ambient temperatures and radiation, the transpiration rate increases (larcher, 1994) and water may be removed from developed fruits. the increasing water withdrawal from the cacao fruits may have contributed to the observed changes in the synthesis of aroma compounds, as it is already described for other aromatic plants (al-gabbiesh et al., 2014; nowak et al., 2010). in the rainy season, however, clouds and precipitation provide optimal growing conditions for the cacao tree, meaning sufficient energy is available for the synthesis of aroma-relevant pulp compounds and in high amounts. for several cocoa producing countries, changes in mean ambient temperature and precipitation patterns are expected or already observed (läderach et al., 2013, niether et al., 2018,). the effect of seasons on the cacao fruit pulp aroma might be enhanced by a climate change induced extension of dry seasons, reducing the aroma intensity of the whole year harvest when blended up. that makes it even more important for the fine or flavour cocoa industry to recommend and establish cocoa production systems that are resilient to climate change like agroforestry systems (niether et al., 2018) or support breeding for drought tolerant varie ties (lahive et al., 2018). conclusion previous findings that aroma intensity and aroma composition of fine or flavour cocoa varieties vary between genotypes were confirmed for five cacao selections from costa rica (catie-r1, catie-r4, catie-r6, ics-95, pmct-58). in addition, we showed for the first time analytically that aroma intensity and composition of the fruit pulp vary between different harvest seasons and change with the ripening stage of the fruit. the results of the present study confirm that the fine aroma potential of the cacao pulp results from a complex combination of genetic pretab. 5: results from linear mixed-effects models for the aroma groups and the factors variety (including the five varieties catie-r1, catie r4, catie-r6, ics-95, pmct-58), season (dry and rainy) and their interaction. f-value and level of significance: * p-value > 0.05; ** p-value > 0.01; *** p-value > 0.001; n.s. not significant. variety season variety:season f-value f-value f-value green 221.3 *** 111.9 *** 32.1 *** herbal 25.1 *** 170.9 *** 12.8 *** fruity 6.3 *** 636.3 *** 1.8 n.s. floral 33.9 *** 268.3 *** 28.5 *** spicy 25.6 *** 155.8 *** 16.5 *** woody 15.4 *** 0.2 n.s. 16.5 *** earthy 129.9 *** 34.2 *** 29.1 *** others 19.5 *** 151.0 *** 13.0 *** fig. 4: share of the aroma groups per varieties (pooled data from ripe and overripe fruits) displayed for two seasons (mean). maximum total peak area was detected in one sample of the control genotype eet 62 and used as reference area (100 %). notes. however, cocoa producers often harvest fruits of different ripening stages, especially when harvest is not done regularly: some fruits are still unripe while others are almost overripe, which affects 274 e. hegmann, w. niether, w. phillips, c. rohsius, r. lieberei disposition, as shown by the different varieties, environmental influences such as climatic conditions and the ripening stage of the fruit. however, the extent to which aroma compounds appear in the beans highly depends on the degree to which they can migrate from the pulp into the seeds during fermentation. thus, the implementation of best practices during post-harvest management is another crucial step to maintain and increase aroma properties. combining different cacao varieties may increase the aroma character of the chocolate, but only if ripe fruits are harvested. industry and consumers should be aware that cocoa is a natural product and that the aroma composition of e.g. single origin chocolates made out of certain cacao varie ties can vary throughout the year. further research on the biochemical background is needed to understand the detailed mechanisms and transfer processes in the formation of fine aromas in theobroma cacao l. this includes the role of bacteria and yeasts (naturally occurring or added starter cultures) in the migration of aroma compounds from the fruit pulp into the cotyledons during fermentation. along with that, the variability and quantity of aroma relevant compounds within the existing varieties need to be examined, especially those compounds discriminating between fine or flavour cocoas and bulk cocoas. acknowledgements this publication is based on a data set raised in a phd thesis conducted under the guidance of professor reinhard lieberei. it was supported by his comprehensive expertise on plant physiology and ability to capture interrelations between the cacao plant and postharvest processing of cacao, leading our discussions beyond a typical biologist’s horizon. we thank adriana arciniegas and the field workers of the cacao breeding program at catie for providing excellent cacao fruit material from ic3, costa rica. we also thank sascha rohn and katrin ulbrich (food chemistry, university of hamburg, germany) for providing the headspace-gcms-spme equipment and for great assistance in fruit pulp analytics. this research was partly supported by the chocolate manufacturer rausch gmbh, germany. conflict of interest no potential conflict of interest was reported by the authors. references al-gabbiesh, a., kleinwächter, m., selmar, d., 2014: influencing the contents of secondary metabolites in spice and medicinal plants by deliberately applying drought stress during their cultivation. jordan j. biol. sci. 8(1), 1-10. andersson, m., koch, g., lieberei, r., 2006: structure and function of the seed coat of theobroma cacao l. and its possible impact on flavour precursor development during fermentation. j. appl. bot. food qual. 80, 48-62. aprotosoaie, a.c., vlad luca, s., miron, a., 2016: flavor chemistry of cocoa and cocoa products − an overview. compr. rev. food sci. f., vol. 15. doi: 10.1111/1541-4337.12180 belitz, 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(wood strawberry) is an old food, ornamental, and medicinal plant. already the roman poet ovid mentioned fragaria twice and very likely meant fragaria vesca (darrow, 1966). in the middle ages, the wood strawberry became a christian symbol and is to find in many paintings from that time. fragaria vesca is the most widely distributed species of the genus fragaria and appears circumpolar throughout europe, northern america, and northern asia. since the middle ages, the so-called ‘semperflorens’ or ‘alpine’ forms of f. vesca ssp. vesca have been cultivated in european gardens (shulaev, 2010) together with f. moschata l. and f. viridis duch., two other wild species occurring in european forests. after the 1750’s the cultivated wild-types were replaced by the garden strawberry (f. ×ananassa duch.) with its bigger fruits and a unique pleasant flavor. f. vesca (2n = 2x = 14) has a very small genome of 240 mb. as a diploid herbaceous plant with remontant behavior in case of the forma semperflorens, it became the model plant for molecular studies in the rose family (rosaceae) (hollender et al., 2012). in 2010, genome sequencing was completed for a hawaiian accession of the wood strawberry (shulaev et al., 2010). the octoploid f. ×ananassa duch. (2n = 8x = 56), which derives from a spontaneous hybridization of the two octoploids f. chiloensis (l.) miller and f. virginiana miller, is not a direct descendant of f. vesca. however, genome research revealed that the two parental species of the garden strawberry derived from different diploid ancestors including f. vesca. (rousseau-gueutin et al., 2009; shulaev et al., 2010). since the middle ages, wild-type strawberries were praised for their intense flavor (darrow, 2005). the aroma of wild strawberry is perceived as very aromatic and more herbaceous than those of f. ×ananassa cultivars (hirvi and honkanen, 1982). therefore, f. vesca serves as a comparison with regard to flavor. in direct comparison of cultivars and wild-types, the sensory impression of the latter was described as much more sweetish-aromatic than those of the f. ×ananassa cultivars but with some astringent and bitter impressions (ulrich et al., 2007). f. vesca is characterized by outstanding flowery notes like violet and acacia. but especially in the white mutant f. vesca f. alba (ehrh.) staudt, these impressions sometimes were described by the testers with negative statements like over-aromatic and perfume-like. by gas chromatographyolfactometry (gco) experiments, the flowery impressions were assigned to the content of the aromatic ester methyl anthranilate whereas the herbaceous impressions are caused by a high content of terpenoids. fragaria vesca always was involved in breeding experiments. since the typical high aromatic flavor was missed in the f. ×ananassa cultivars, cross-breeding started already in 1918 by elisabeth schiemann (kuckuck, 1980). successful ways of species introgression with the cultivated strawberry using cross-breeding and chromosome doubling were first described by scott (1951). hereby, decaploid fertile clones could be obtained. the first decaploid cultivars were introduced under the denomination fragaria × vescana by bauer and bauer (1979), combining characteristics of f. vesca and f. ×ananassa. synthetic octoploids were obtained from the inclusion of fragaria vesca in the breeding pedigree by evans (1977) and bors and sullivan (2005). nowadays, breeding programmes with f. vesca focus on the inheritance of methyl anthranilate and several resistances (pinker et al., 2012). the aroma of the wood strawberry was investigated in detail for the first time by drawert et al. (1973). the content of 40 identified main peaks in f. vesca exceeds those of the f. ×ananassa cultivar cv. 'revata' by the factor of 4.7, which is in agreement with later findings of ulrich et al. (1997) (factor 3.2 in comparison to cv. 'elsanta'). additionally, aharoni et al. (2004) discussed the differences in the patterns of volatile organic compounds (vocs) between f. vesca and f. ×ananassa. in this paper the differences are interpreted as a gain and loss of metabolic diversity caused by domestication and breeding. in plant breeding this process also is known as funnel effect (ulrich et al., 1997; ulrich and olbricht, 2011) or bottlenecking (doebley et al., 2006). a general shortcome of several published investigations of voc profiles is the neglect of the environmentally caused dynamic of the volatile patterns. it is unquestioned that beside genetic prerequisites outer factors like maturity at harvest, location, climate, irrigation, harvest time as well as the attack by pathogens have an eminent influence on biosynthetic network and hence on the development of the whole volatile patterns (quantitative and qualitative!) (forney et al., 2000; azodanlou et al., 2004; olbricht et al., 2011). to quote an example, jouquand et al. (2008) observed changes in ester contents of fruit from cultivated strawberries between two harvest dates of the same season up to 350 %. this volatile dynamic was in the same order like those caused by the cultivars. consequently, the use of samples from only few or even one harvest date gives results which represent only a ‘snapshot’ of the possible dynamic of metabolite patterns. therefore, the topic of this investigation is to study the diversity of volatile patterns in a collection of 16 f. vesca accessions in comparison to selected f. ×ananassa cultivars in two harvest years 38 d. ulrich, k. olbricht by using a sampling strategy which prevents the influence of maturity and harvest date during one season. materials and methods plant material and cultivation fragaria vesca is the most distributed wild species of the genus and exists with four subspecies, six formae and several cultivars (staudt, 1962; staudt, 1989). due to this biogeography, a high diversity is to expect. therefore, 16 accessions from different locations from the most eastern habitat at lake baikal in siberia, from middle and southern europe and northern europe with scandinavia and iceland were investigated as well as two of the three described north american subspecies and three cultivars (fig. 1). the used accessions are listed in tab. 1. five very distinct european f. ×ananassa cultivars were chosen to serve as a comparison. they were selected with regard to their breeding history and demonstrate with different aroma patterns the process of domestication over a period of nearly 80 years. all fragaria vesca accessions were cultivated in the field at dresden-weixdorf, germany, longitude 13.8°, latitude 51.145°, 200 m above sea level, on sandy loam on gravel ground (german classification: 'ackerzahl' 37). each accession was growing in a quadrate of 1 sqm containing plants of different ages according to their natural habitats. neither chemical treatment nor extra fertilizer were applied, except for two applications of insecticide during the time of flowering in order to prevent damage by the strawberry weevil (anthonomus signatus say) in both years. irrigation only was applied to avoid damage by drought. the cultivars (f. ×ananassa) were planted on the same location in rows on a field under the same conditions and with two additional treatments of fungicide during the time of flowering in order to prevent infection by grey mold (botrytis cinerea pers.). in 2011 and 2012, fruits of one year old plants were harvested. harvest and storage all available, fully ripe, typical and healthy fruits of all fragaria vesca accessions and all available fruits from six clone plants of each f. ×ananassa cultivar were harvested (fig. 2). the weight per f. vesca accession varied depending on the fruit size (for example between 115 g with 198 fruits for 'multiplex' and 1054 g with 902 fruits for 'red wonder' in 2012). all fruits were immediately frozen at minus 20 °c after the sepals were removed. extraction of fruit volatiles by immersion stir bar sorptive extraction (imm-sbse) to prepare an enzyme inhibited strawberry juice, all frozen fruits of one accession or cultivar of the whole season were pooled. one mass part of fruits without sepals were homogenized in one volume part of a solution of 18.6 % (m/v) nacl by a household mixer for 2 min. the homogenate was centrifuged 4000 rpm for 30 min. one hundred milliliter of the supernatant were mixed with 10 μl internal standard (0.1 % (v/v) 2,6-dimethyl-5-hepten-2-ol dissolved in ethanol). for each sample, three head-space vials containing 3 g nacl each for saturation were filled with 10 ml of the supernatant, sealed with magnetic crimp caps including septum, and stored at 4 °c for up to three weeks. an aliquot of 8 ml of the saturated homogenate but without the solid nacl deposit was transferred in an empty glass vial for volatile isolation by sbse. a stir bar with 0.5 mm film thickness and 10 mm length coated with polydimethysiloxan (pdms) was placed in the liquid (gerstel, mülheim an der ruhr, germany). the stir bar was moved at 350 rpm at room temperature for 45 min. after removal from the strawberry juice, the stir bar was rinsed with purified water, gently dried with a lint-free tissue and then transferred into a glass tube for thermal desorption and subsequent gc analysis. gas chromatography – mass spectrometry the parameters for the thermal desorption unit (tdu, gerstel) and the cold injection system (cis4, gerstel) were the following: thermal desorption at 250 °c, cryo trapping at -150 °c. the tdu-cis4 unit was used in gerstel-modus 3: tdu splitless and cis4 with 15 ml/ min split flow. the analyses were performed with an agilent technologies 6890n fig. 1: map of origins for f. vesca accessions. the denotation is adequate to tab. 1. fragaria vesca diversity 39 tab. 1: list of used accessions of fragaria vesca and their origin and of five fragaria ×ananassa cultivars. number accession / cultivar abbreviation origin / year of introduction 1 f. vesca f. alba st. 08/101 foralb gatersleben, via g. staudt 2 f. vesca f. alba 'south queen ferry' sferry scotland, south queen ferry, (near edinburgh), united kingdom, leg. hohlfeld 3 f. vesca ssp. vesca st. 94, 13 'baikal' baikal lake baikal, siberia, russia, leg. popova 4 f. vesca ssp. bracteata st. 98, 04-4 bracte nass river, british columbia, canada 5 f. vesca ssp. americana st. 14324 americ saint-armand, missisquoi river, near montreal, canada, via botanical garden montreal 6 f. vesca f. semperflorens 'red wonder' redwon cultivar 7 f. vesca f. semperflorens 'yellow wonder' yelwon cultivar 8 f. vesca ssp. vesca 'island' island botanical garden rejkavik, iceland 9 f. vesca ssp. vesca 'kaiserpfalz tilleda' tilled tilleda, saxony-anhalt, germany, leg. roentzsch 10 f. vesca ssp. vesca 'korsika' korsik st. bonifatius, corsica, france, leg. olbricht 11 f. vesca ssp. vesca 'multiplex' multip cultivar 12 f. vesca ssp. vesca 'weimar' weimar valley of the river ilm, garden of j. w. v. goethe, weimar, thuringia, germany, leg. gerischer 13 f. vesca ssp. vesca 'böhmen' boehme bohemian sandstone mountains, near stimmersdorf, bohemia, czech republic, leg. olbricht 14 f. vesca ssp. vesca 'tüchersfeld' tuefel tüchersfeld, franconia, germany, leg . olbricht 15 f. vesca ssp. vesca 'süd-öland 1' oeland oeland, sweden, leg. drewes-alvarez 16 f. vesca ssp. vesca 'großolbersdorf' olbers großolbersdorf, ore mountains, saxony, germany, leg. olbricht 17 f. ×ananassa cv. 'alba' alba italy, 2003 18 f. ×ananassa cv. 'mara de bois' mdb france, 1991 19 f. ×ananassa cv. 'mieze schindler' ms germany, 1933 20 f. ×ananassa cv. 'polka' polka netherlands, 1980 21 f. ×ananassa cv. 'elegance' eleg england, 2009 gas chromatograph equipped with an agilent 5975b quadrupol ms detector. compounds were separated on a polar column zb-wax plus 30 m length x 0.25 mm id x 0.5 μm film thickness. helium was used as a carrier gas with a column flow rate of 1.1 ml/min. temperature programme: 45 ºc (3 min), temperature gradient 3 k/min to 210 ºc (30 min). the mass spectrometer was used with electron ionization at 70 kev in the full scan mode. all samples were run with two analytical repetitions from an identical part of the supernatant. data processing the resulting total ion chromatograms (tic) were integrated using the agilent chemstation™ routine with an initial threshold of 15 for all samples. the value of the initial threshold was chosen in such a manner that the most intense chromatograms contain about 100 integrated peaks. for a non-targeted data processing, the integration results were imported subsequently as raw data (txt-formatted) into the chemometrical software chromstattm 2.6 from analyt-mtc (müllheim, germany). the semi-quantitative results were expressed as non-dimensional values (counts or relative concentrations). statistical analyses were performed using the software statistica 7.1 from statsoft (tulsa, usa) and multi experiment viewer from tm4 software development team (www.tm4.org). brix and acid measurements brix values in °brix as a correlated value for sugar content were measured for the strawberry juice of all frozen fruits of one accession / cultivar using the portable 'quick brix' (mettler toledo, schwerzenbach, germany). from the same juice, an acid equivalent on the base of citric acid was determined by a semi-automatic titrator (716 dms titrino 06, metrohm, filderstadt, germany) with given results in % acid equivalent. results volatiles the semi-quantitative data of volatile organic compounds of 21 genotypes analyzed during two harvest seasons are displayed as so-called heat maps in fig. 3. the concentration level is converted in a color code from deep blue (c = 0) to deep red (c ≤ 15). the heat maps represent the raw data in an identical order for both harvest years. the group of f. vesca accessions is located above the five cultivars. the heat map patterns are similar for both years but not identical. because of the specific entire sampling strategy of fruits described above, the concentration levels of volatiles represent an average concentration pattern of all fruits from the whole harvest season independent of sampling frequency. since identical plant material was used in both harvest seasons, the resulting differences 40 d. ulrich, k. olbricht must be caused by the climatic conditions only. within the f. vesca group, high diversity in vocs is evident. aroma relevant substances like ethyl hexanoate (lox, ester biosynthesis), mesifuran (furanone), myrtenal (terpenoid) and g-decalactone (lox pathway) vary in a wide range. between the f. vesca and f. ×ananassa, group characteristic differences occur. small esters like methyl butanoate, ethyl butanoate, methyl hexanoate and ethyl hexanoate as well as hexanoic acid occurs in higher amounts in the cultivars. in opposite, ketones (2-pentanone, 2-heptanone, 2-nonanone), c6compounds like hexanol and hexenols are clearly more abundant in f. vesca. another difference occurs in the group of terpenoids (e.g. terpinen-4-ol, myrtenal, myrtenyl acetate, a-terpineol and myrtenol) which were also found in higher concentrations in the f. vesca group. one exception is the monoterpene linalool with higher amounts in f. ×ananassa than in f. vesca. the key compound methyl anthranilate is evident in all f. vesca accessions. the concentrations vary in a wide range between values of 9.19 (accession no. 8, 'island') and 980 (accession no. 16, 'olbers') in 2011 and 6.75 (accession no. 8, 'island') and 359.81 (accession no. 16, 'olbers') in the season 2012. in accordance with former findings (ulrich et al., 2009; ulrich and olbricht, 2011), methyl anthranilate was found in two of the five f. ×ananassa cultivars in medium concentration between values of 6.13 to 27.17. in fig 4a the diversity of some exemplary volatiles discussed above is shown as box-whisker-plots using the entire data from both years (2011 and 2012). the plots represent the medium values both for the f. vesca (left, v) and f. ×ananassa (right, c) group. the magnitude of metabolic diversity within these groups is characterized by the standard deviation and the minimum/maximum values. 2-heptanone, 1-hexanol, myrtenyl acetate and methyl anthranilate represent the metabolites with generally higher amounts in wild types whereas ethyl hexanoate and linalool belong to volatiles with higher concentrations in the f. ×ananassa cultivars. as an exception, the ethyl butanoate content of the cultivar 'elegance' is marked with an asterisk. the ester content of this cultivar is extremely depleted and therefore marked as outlier (see section discussion). several volatiles show a typical skewed distribution of the data with a mean value shifted to lower values. in general, the range between minimum and maximum values is much higher in f. vesca than in f. ×ananassa for most of the volatiles although the chosen five cultivars represent the most diverse breeding results from ca. 80 years of european breeding history. myrtenyl acetate is not detectable in the evaluated cultivars but in the majority of investigated f. vesca. generally, all median values of the demonstrated volatiles are significantly different between both groups. for comparison of the two harvest years, a data reduction by pca and a rank correlation analysis (spearman’s rank) was performed. the results of pca are shown in fig. 5 as score and parameter plots for each harvest season separately. in analogy to the heat map in fig. 3, the results for 2011 and 2012 are similar with some differences in details. in the score plots (left), the groups of f. vesca and f. ×ananassa cultivars are clearly separated. the parameter plot of 71 parameters (67 volatiles, sum of volatile, brix value, acid content, ratio of brix and acid value) has generally a similar structure fig. 2: typical fruits of sixteen f. vesca accessions and one f. ×ananassa cultivar. the denotation of f. vesca accessions is adequate to tab. 1. fragaria vesca diversity 41 for both years. the calculation of rank coefficients (tab. 1) gives a quantitative measure of the similarity of volatile patters between the two seasons. the coefficient values are distributed between 0.67 (f. vesca, accession no. 8, 'island') and 0.92 (f. ×ananassa cv. 'mara de bois') with means of 0.77 for f. vesca and 0.88 for the cultivars, respectively. non-volatiles the distribution of brix values (brix), titratable acid (acid) as well as the ratio are shown in fig. 4b using the entire data from both years (2011 and 2012). in contrast to the volatile data, brix and acid values are characterized by a nearly ideal gaussian distribution. the mean values of brix are not significantly different between the wild and cultivated strawberries whereas acid values and accordingly the ratio carry significant differences. interestingly, the range of acid values in f. ×ananassa cultivars is very small in comparison to f. vesca. discussion berry sampling method the laborious sampling strategy for ripe and healthy berries was used to prevent temporary influences which occur between different harvest dates within the whole season. taking into account that changes of metabolite contents between different harvest dates within one season may arise in the same order like genotype differences (jouquand et al., 2008), the high similarity between the heat map patterns (fig. 3) and the pca plots (fig. 5) is an argument for the benefits of this kind of sampling. additionally, this fact is supported by the rank correlation results for all of 67 vocs as well as brix, acid and the ratio brix/acid in two years (tab. 2). the spearman’s rank show high values between 0.77 for the f. vesca accession and 0.88 for f. ×ananassa cultivars. strawberry aroma types the diploid wood strawberry f. vesca is not a direct ancestor of our cultivated f. ×ananassa which is a hybrid of the two octoploid species f. chiloensis (l.) miller and f. virginiana miller. nevertheless, from the view of sensory quality, the aroma of cultivars often has been judged in comparison to those of wild species, especially to those of the wood strawberry (ulrich et al., 1997; hoberg et al., 2000). improving the aroma of new f. ×ananassa cultivars is a declared aim of all modern breeding programmes whereas the aroma of f. vesca often is described as the most powerful strawberry character. therefore, the volatile patterns of sixteen f. vesca accession 20 11   20 12   fig. 3: heat map of the metabolite patterns including 67 volatiles from 16 f. vesca accessions and four cultivars (f. ×ananassa). 45 volatile compounds are fully identified by co-elution of authentic substances, the remaining by ms library search. the relative concentrations of metabolites (counts) are color coded from deep blue (c = 0) to deep red (c = 15). concentration values above c = 15 (around 10 % of all values) are also coded in deep red. 42 d. ulrich, k. olbricht f. ×ananassa types represent five cultivars with very diverse aroma types regarding the classification by ulrich et al. (1997 and 2009). this classification divides strawberries in terms of their content of methyl anthranilate (ma) and short chain esters of butanoic, hexanoic or 2-methyl butanoic acids (esters) in three main aroma types: ‘ma high’(1) and ‘ma low (2)’ with the ‘ma low’ types subdivided into an ‘ester high’ (2a) and an ‘ester low’ (2b) type . 'mieze schindler' (type 1) is an old german cultivar (introduction 1933) with intensive aroma which often is characterized with the notation ‘vesca-like’. 'mara de bois' (also type 1) is a modern highly aromatic french cultivar (introduction 1991) and an exception in the range of modern cultivars. its rich aroma pattern is explained by the pedigree which includes the old european germplasm. this cultivar is mainly used for house gardens and as gourmet fruit. the cultivar 'polka' (introduction 1980) represents the fresh, fruity aroma type 2a whereas the cultivar 'alba' (type 2b) belongs to the modern high yielding cultivars with medium to low aroma strength (introduction 2003). 'elegance' (introduction 2009) represents a high performance cultivar with firm and best looking fruit but with extremely low volatile values. one of the most abundant volatiles in ripe f. vesca berries is methyl anthranilate (ma), which is a product of the shikimate pathway synthesized via chorismate (ulrich et al., 1997; bohlmann et al., 1996). this compound varies within the sixteen f. vesca accessions by a magnitude of three orders (fig. 4a). ma is characterized by a typical sweetish-flowery smell like acacia. in combination with others, this compound causes the unique flavor of f. vesca and in addition the preferred sensory quality of few f. ×ananassa cultivars like 'mieze schindler' and 'mara de bois'. because of several bioactivities, ma became an important industrial chemical, and synthetic forms are widely used as odorant, artificial flavor, bird repellent and oxidation inhibitor (wang and de luca, 2005). in higher concentrations ma gives an unpleasant note like soapy (komes et al., 2006) in strawberries and foxy in grapes (wang and de luca, 2005). fig. 4: elementary statistics for the data set including the values of the harvest years 2011 and 2012. a) box-whisker-plots of six representative volatiles for the group of f. vesca accessions and f. ×ananassa cultivars. b) box-whisker-plots of value for brix, acid content and the relation brix/acid. rectangle – mean; box – standard deviation; whisker – minimum/maximum. nomenclature of genotypes: v – f. vesca, c – f. ×ananassa cultivars. box labels with different characters indicate a significant statistical difference of the mean values. asterisk – cultivar ‘elegance’ is characterized by untypical low ester contents (outlier). b   a   c   d   e   f   g   h   i   k   l   m   *   a)   aa   aa   ba   bb   cb   ca   b)   were compared with five f. ×ananassa cultivars. while the sixteen f. vesca accessions represent important habitats of their geographic distribution around the northern hemisphere (staudt, 2009) the fragaria vesca diversity 43 odor was found in higher amounts in cultivars which is, on the one hand, in agreement with the findings of chambers et al. (2012) who postulated a mutation in a nerolidol synthase (fanes1) in octoploids (f. ×ananassa) in comparison to the diploids (f. vesca). on the other hand, the linalool concentrations are generally lower in f. vesca but not absent like postulated by chambers et al. (2012). this result supports the hypothesis that linalool synthesis may arise also from an alternative biosynthesis than fanes1. in contrast to most of the terpenoids, the group of small fruit esters (methyl butanoate, ethyl butanoate, methyl hexanoate and ethyl hexanoate) gives a positive, fruity impact to aroma and is found in higher amounts in f. ×ananassa cultivars. influence of domestication and breeding on metabolite patterns and organoleptic properties the comparison of metabolic patterns of f. vesca and f. ×ananassa gives an interesting insight in mechanisms of domestication and breeding even if the cultivated forms do not descend directly from the diploids. on the other hand, phylogenetic research reveals that f. vesca shares important sequences with the genome of f. ×ananassa (aharoni et al., 2004) and is considered as one of the close 20 11 20 12 fig. 5: principal component analysis using semi-quantitative data of 67 volatiles, the sum of volatiles as well as brix value, acid content and the ratio of brix to acid value. left – score plot of 16 f. vesca accession and five f. ×ananassa cultivars. right – parameter plot. comparison of terpenoid and ester patterns in comparison with f. ×ananassa cultivars, f. vesca accessions produce higher amounts and a higher diversity of terpenoid derived compounds (fig. 3 and fig. 4a). it can be interpreted as a result of the large biogeographical distribution of the wild species. the compounds mentioned above (terpinen-4-ol, myrtenal, a-terpineol, myrtenol and linalool) carry important bioactivities like attractants, pheromones, kairomones, fungizides etc. (n.n. 2013). the loss of terpenoids in cultivated strawberries may be accompanied with a loss of resistance features against different diseases and herbivores. until now this topic is rarely investigated and also not in the focus of this research. furthermore, this compound group has an impact on sensory characteristics due to low odor thresholds (pet’ka et al., 2012; larsen and poll, 1992). the generally higher amounts of terpenoids in wild accession may have a negative influence on sensory quality induced by a turpentine-like, herbaceous, woody odor (aharoni et al., 2004) and harsh flavor (simon, 1985). this is in agreement with the astringent characters found by descriptive sensory tests in comparison of wild strawberries from europe and north america with the standard cultivar f. ×ananassa cv. 'elsanta' in an earlier study (ulrich et al., 2007). as an exception, the terpenoid linalool with a fresh-flowery, citrus-like and pleasant 44 d. ulrich, k. olbricht tab. 2: rank correlation analysis between two harvest years based on semi quantitative data of 67 volatiles, the sum of volatiles as well as brix, acid-value and the quotient of brix and acid-value. accession spearments rank 2011-2012 1 foralb 0.81 2 sferry 0.80 3 baikal 0.74 4 bracte 0.80 5 americ 0.86 6 redwon 0.83 7 yelwon 0.80 8 island 0.67 9 tilled 0.80 10 korsik 0.75 11 multip 0.69 12 weimar 0.78 13 boehme 0.72 14 tuefel 0.76 15 oeland 0.76 16 olbers 0.71 mean wildtypes 0.77 17 alba 0.88 18 mdb 0.92 19 ms 0.86 20 polka 0.85 21 eleg 0.83 mean cultivars 0.88 relatives to the octoploid species f. virginiana and f. chiloensis (rousseau-gueutin and olbricht, 2009). the tremendous diversity of aroma patterns within the f. vesca accessions is believed to be an adaptation to the different requirements of the ecosystems which were occupied by this strawberry species. today’s strawberry cultivars are characterized by rather different patterns of secondary metabolites. as described above for vocs, early domestication steps and in particular the breeding seem to give advantages to strawberry types with pleasant flavor perceptions. in consequence, metabolites with bitter and astringent (sesquiterpenes), herbaceous, harsh and unpleasant odors (monoterpenes) were downgraded. in contrast, the content of sensorial pleasant small esters is high in cultivars. sugar and acid contents follow the same direction. cultivars carry high or medium sugar but low acid contents and lead to high sugar-acid ratios. the f. vesca accessions in this study are characterized by high acid values and very diverse brix-acid ratios. additionally, high acid contents involve also astringency especially in combination with low sugar values. evidence exists that organoleptic properties of plants played an important role during domestication and even in early breeding activities. diamond (2002) discussed this topic regarding the bitterness (or non-bitterness) of almond and acorns. early selection and breeding work can be characterized as a trend toward desirable sensory sensations such as sweet, non-bitter, moderate sour and low astringent. this is because humans have an inborn preference for sweet sensations and an aversion against bitter and astringent food (shepherd, 2006). in the data set presented here, this trend can be seen clearly in the comparison of the patterns of esters and the acid content (or brix-acid ratio). excellently tasting cultivars possess high concentrations of short chain esters (butanoates, hexanoates, hexenoates and 2-methylbutanoates) in combination with high values of brix-acid ratio and a moderate firmness. in strawberries, like in other fruits, the flavors appreciated by consumers are best represented in old cultivars and so-called land races or heirloom varieties (alston, 1992). breeding activities of the past decades on f. ×ananassa cultivars for high-yielding and firm strawberries were accompanied with a dramatic loss of genetic diversity in modern cultivars. between old and modern cultivars, a loss of one third of observed alleles (30 % 35 %) was determined by molecular analysis using ssr markers (gil-ariza et al., 2009, horvath et al., 2011). additionally, an acceleration of this process was found because further 18 % of alleles disappeared in only one decade in the 1990s. as discussed above, this so-called funnel effect influences also qualitative and quantitative traits of volatile patterns. the most impressive example for the funnel effect in flavor caused by breeding investigated in the present research is the cultivar 'elegance'. the ester contents of this cultivar depleted of high degree (fig. 4a), which resulted in poor and consequently untypical f. ×ananassa flavor. methodical principals in volatile analysis aharoni et al. (2004) postulated a strong selective advantage of strawberry types containing the sensorial pleasant compounds linalool (sweetish, floral, citrus like note) and nerolidol (rose, apple, green note) passed on to preferred domesticated strawberries. also in the present analyses, linalool content is significantly higher in cultivars whereas nerolidol was not detectable in all of the f. vesca accessions and was found in only two f. ×ananassa cultivars ('polka' and 'mara de bois') in the first harvest year. but some important aspects are still unconsidered in this discussion. firstly, if correlation between metabolite profiles of the fruit and sensory perceptions are in the focus of discussion, an important prerequisite of the instrumental analysis is that the sample preparation methodology must approximately mimic the eating process. the used methodology in the present research provides for this aspect using homogenization (lox activity of disrupted tissue) with following enzyme inhibition of the homogenate using high salt concentrations. that is that measurements using whole, undamaged fruit do not represent the metabolite profiles which are active in the human mouth. therefore, statements about individual aroma compounds based on whole fruit (aharoni et al., 2004; azodanlou et al., 2003) represent more relations to signaling processes (herbivores) than those of human sensory. secondly, a selective advantage based on human sensory impressions cannot directly be judged by simple instrumental analysis results (concentration values). the sensory impact of vocs follows the socalled aroma value concept (rothe and thomas, 1963). the aroma impact in the human nose corresponds to the aroma value (av) (or in synonym the odor activity value (oav)) which is calculated by the ratio of the voc concentration (c) and the odor threshold (ot) in the same matrix. strawberry character impact compounds in cultivars differ in their concentration in a range of about two orders resulting in corresponding avs in a range of six orders (larsen and poll, 1992). the highest avs in cultivars were found for the three esters ethyl butanoate (453,000), ethyl hexanoate (4,700) and methyl butanoate (614) as well as the furanone 2,5-dimethyl-4-hydroxy-3(2h)-furanone (1,170). in contrast, the avs of terpenoids are 100-fold lower (larsen and poll, 1992). based on earlier results of ulrich fragaria vesca diversity 45 et al. (1997), the avs for the key compound methyl anthranilate can be estimated as about 100 for the f. ×ananassa cultivar 'mieze schindler' and up to 5,000 for the f. vesca accession no. 16, 'olbers'. these calculations confirm the findings of the present research that determining compounds for preferred strawberry aroma types are esters of butanoic and hexanoic acids in combination with methyl anthranilate and furaneol. additionally, a high acceptance demands high sugar contents (brix value), moderate acid content (jouquand et al., 2008) and hence a high sugar/acid ratio value. the metabolic differences and sensory relationships discussed in the present paper are in total analogy to those of other fruits and vegetables like apple and carrot (ulrich and olbricht, 2011). aroma patterns of successful cultivars are stable an additional aspect is the voc dynamic regarding the harvest years which is illustrated by the metabolite comparison of wild (f. vesca) and cultivated (f. ×ananassa) types in two years. the influence of the harvest year to the profiles of 20 character impact compounds in a whole f1 population of cultivated strawberries was published by olbricht et al. (2008 and 2011). the degree of variation triggered by harvest year is very different for every single aroma compound. this effect causes a high dynamic of volatile patterns. generally, groups of stable and unstable metabolites were found in strawberries. in the present paper, the stability of the metabolite patterns including 67 compounds as well as brix value, acid content and the ratio brix to acid value regarding the two harvest seasons can be judged using the rank correlation coefficients in tab. 2. obviously, the stability is significantly lower in the wild types f. vesca (0.77) than in f. ×ananassa cultivars (0.88). a conclusion for breeding is that successful cultivars like those used in this research are characterized not only by sensory suitable concentration patterns of vocs but also by robust patterns with regard to yeardependent, environmentally caused influences. additionally, this is supported by the two f. vesca cultivars (‘redwon’ and ‘yelwon’), which are a product of plant breeding too and show next to f. vesca ssp. americana comparable high values of spearmans rank. acknowledgements the authors thank anne doedtmann, ursula gerischer, angela ludwig, henning wagner, veronika waurich, kirsten weiß and lisette wutzky for technical realisation as well as petra scheewe for helpful discussion. references aharoni, a., giri, a.p., verstappen, f.w.a., bertea, c.m., sevenier, r., sun, z.k., jongsma, m.a., schwab, w., bouwmeester, w.h., 2004: gain and loss of fruit flavor compounds produced by wild and cultivated strawberry species. plant cell 16, 3110-3131. alston, f.h., 1992: flavour improvement in apples and pears through plant breeding. in: patterson, r.l.s., charlwood, b.v., macleod, g., williams, a.a (eds.), bioformation of flavours, royal society of chemistry, cambridge, uk. azodanlou, r., darbellay, c., luisier, j.l, villettaz, j.c., amado, r., 2003: quality assessment of strawberries (fragaria species). j. agr. food chem. 51, 715-721. azodanlou, r., darbellay, c., luisier, j.l, villettaz, j.c., amado, r., 2004: changes in flavour and texture during the ripening of strawberries. eur. food res. technol. 218, 167-172. bauer, r., bauer, a., 1979: hybrid breeding in the genus fragaria: spadeka − a new variety with the aroma of wild strawberry. erwerbs-obstbau 21, 151-158. bohlmann, j., lins, t., martin, w., eilert, u., 1996: anthranilate synthase from ruta graveolens − duplicated as alpha genes encode tryptophansensitive and tryptophan-insensitive isoenzymes specific to amino acid and alkaloid biosynthesis. plant physiol. 111, 507-514. bors, r.h., sullivan, j.a., 2005: interspecific hybridization of fragaria vesca subspecies with f. nilgerrensis, f. nubicola, f. pentaphylla, and f. viridis. j. am. soc. hortic. sci. 130, 418-423. chambers, a., whitaker, v.m., gibbs, b., plotto, a., folta, k.m., 2012: detection of the linalool-producing nes1 variant across diverse strawberry (fragaria spp.) accessions. plant breeding 131, 437-443. darrow, g.m., 1966: the strawberry: history, breeding, and physiology. holt, rinehart and winston, new york, chicago and san francisco. 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(ed.), evaluation of quality of fruits and vegetables, 315-328. avi publishing co., inc.: westport, conn., usa., kansas city, mo., usa. staudt, g., 1962: taxonomic studies in the genus fragaria. typification of fragaria species known at the time of linnaeus. can. j. botany 40, 869-886. staudt, g., 1989: the species fragaria, the taxonomy and geographical distribution. acta hortic. 265, 23-33. staudt, g., 2009: strawberry biogeography, genetics and systematics. acta hortic. 842, 71-84. ulrich, d., hoberg, e., olbricht, k., 2009: strawberry aroma analysis as selection tool in breeding. acta hortic. 842, 899-902. ulrich, d., hoberg, e., rapp, a., kecke, s., 1997: analysis of strawberry flavour − discrimination of aroma types by quantification of volatile compounds. z. lebensm. unters. for. 205, 218-223. ulrich, d., komes, d., olbricht, k., hoberg, e., 2007: diversity of aroma patterns in wild and cultivated fragaria accessions. genet. resour. crop ev. 54, 1185-1196. ulrich, d., olbricht, k., 2011: fruit organoleptic properties and potential for their genetic improvement. in: jenks, m.a. bebeli, p.j. (eds.), breeding for fruit quality. john wiley & sons, inc., hoboken, nj, usa. wang, j., deluca, v., 2005: the biosynthesis and regulation of biosynthesis of concord grape fruit esters, including ‘foxy’ methylanthranilate. plant j. 44, 606-619. address of the corresponding author: detlef ulrich, julius kühn-institute (jki), federal research centre for cultivated plants, institute for ecological chemistry, plant analysis and stored product protection, erwin-baur-straße 27, d-06484 quedlinburg, germany. e-mail: detlef.ulrich@jki.bund.de art07 journal of applied botany and food quality 81, 140 144 (2007) 1, 2 humboldt-universität zu berlin, institute for horticultural sciences, section quality dynamics/postharvest physiology and section fruit science, berlin, germany 3 technical university of berlin, institute for food technology and food chemistry, berlin, germany uv-b induced changes of phenol composition and antioxidant activity in black currant fruit (ribes nigrum l.)* s. huyskens-keil1, i. eichholz2, l.w. kroh3, s. rohn3 (received july 6, 2007) summary information on uv-b elicitor mediated changes on phenolic composition and antioxidant activity of black currant (ribes nigrum l.) are scanty. in the present study physiological ripe black currant fruits were harvested and exposed to uv-b radiation with different exposure and adaptation times. the influence of uv-b on phenolic profile and quantitative composition as well as on the corresponding antioxidant activity was investigated. antioxidant activity was screened with electron spin resonance spectrometry (esr), while phenolic compound composition was conducted by hplc analysis. total phenol content and phenolic composition (flavonols, anthocyanins, hydroxycinnamic and hydroxybenzoic acids) increased to a large extent during uv-b treatment, irrespective of the adaptation time. anthocyanins are concluded to absorb uv radiation within a short time, meanwhile flavonols and phenolic acids are assumed to have an impact on antioxidant protection of uv-b mediated tissue damage. moreover, antioxidant activity significantly correlated with different phenolic compounds and increased to a similar extent by uv-b exposure. introduction polyphenols belong to the secondary plant metabolites and are present in a wide range of fruits and vegetables. based on the chemical structure phenolic compounds are marked by a broad spectrum of health-promoting functions such as antioxidant, antimutagenic and anticancerogenic properties (halliwell, 1996). there is clear evidence for an association between the free-radical binding character of phenols and the prevention of degenerative diseases, e.g. cancer and cardiovascular diseases (world cancer research fund, 1997; bazzano et al., 2002). berry fruits such as black currant (ribes nigrum l.) are a rich source of phenolic compounds (kähkönen et al., 2001; benvenuti et al., 2004). the antioxidant activity of black currant fruits is based on the high content of flavonols (myricetin and quercetin derivatives) (anttonnen and karjalainen, 2006) and anthocyanins (delphindin and cyanidin derivatives) (slimestad and solheim, 2002). moreover, hydroxycinnamic acids (caffeic acid and coumaric acid) and hydroxybenzoic acid (gallic acid and hydrobenzoic acid) also contribute to the antioxidant activity, but to a smaller extent (häkkinen et al, 1999; kähkönen et al., 2001; mättää et al., 2003). up to now, research on antioxidant phenolic plant compounds in black currant was mainly focused on the effect of cultivars, seasonal timing and cultivation practices (kähkönen et al., 2001; mikkonen et al., 2001; moyer et al., 2002; anttonnen and karjalainen, 2006; tabart et al., 2006). however, information on changes in phenolic composition and antioxidant activity in black currant as affected by ultraviolet-b (uv-b) radiation is scanty. in recent years, increased uv-b radiation (280-315 nm) by air pollution-induced ozone depletion has raised awareness of the effects of uv-b on the ecosystem. though only a small portion of the total solar spectrum, uv-b has a large photobiological effect (kerr et al., 2003). there are many reports on potential consequences of an increase in uv-b radiation on plants, however, a rather limited understanding the effect on secondary plant metabolites is present. the increasing exposure of plants to uv-b causes protective mechanisms in plant response to this anthropogenic source of stress. additionally to morphological changes the protective stress response triggers distinct changes in the plant’s secondary metabolism resulting in an accumulation of various phytochemicals (e.g. phenolic compounds) and their antioxidative activity (schreiner and huyskens-keil, 2003; eichholz et al., 2005; huyskens-keil et al., 2006; schreiner and huyskens-keil, 2006). ). anthocyanins and flavonoids have various physiological functions (i.e. antioxidative and antitumor activity) and reveal uv protective effects (higashio et al., 2001; indorf, 2001; kolb et al, 2001; higashio, 2005). in general, the synthesis of phenolic compounds can also be stimulated by uv exposure in postharvest as it was reported for example for quercetin in onion and strawberry, flavonoids in brokkoli and black currant (higashio et al., 2005; costa et al., 2006; huyskens-keil et al., 2006). however, uv stress mediated changes in phenol profile and antioxidant activity are dependent on the duration of stress, adaptation time of plants to uv stress and physiological status of the plant at the time of uv exposure. this study contributes to an assessment of a short-term uv-b stress induced impact on the dynamics of secondary plant metabolites in postharvest, exemplarily demonstrated on phenolic composition and antioxidant activity of black currant. furthermore, the effect of targeted uv application in postharvest for increasing health-promoting phytochemical compounds and thus, nutritional value of fruits and vegetable products is focused. material and methods plant material and experimental setup three years old plants of ribes nigrum l. cv ‘titania’ (n=120) were cultivated in 10 l containers with compo sana® rose substrate, (compo gmbh & co. kg, münster) and manna lin m fertilizer (wilhelm haug gmbh & co. kg, ammerbuch, germany). fruits were harvested at a fully developed ripening stage (according to the maturity index as described below) in july 2006. special attention was paid on picking berries of the appropriate maturity stage according to fruit firmness and maturity index (sugar-acid-ratio) considering sample collection from all sections of the bush. after picking, one part of fruit samples (250 g pe-trays) was subjected to uv-b radiation for 60, 90 or 120 min using an uv-b fluorescence light source (fl 20se, 305-310 nm) with an average fluency rate of 8.2 ws m-2 at a distance of 30 cm to fruits. after an adaptation time of 2 or 22 h fruits were shock-frozen in liquid nitrogen and kept at -25 °c before freeze drying (christ alpha 1-4, christ; osterode, germany). dried samples were grinded for subsequent extraction and analysis of total phenolic content and phenolic composition, i.e. flavonoid subclasses (flavonols, anthocyanins, hydroxycinnamic and hydroxybenzoic acids). the experiment was conducted with three * this paper was presented at the 42th meeting of the „deutsche gesellschaft für qualitätsforschung (pflanzliche nahrungsmittel)“ dgq e.v. replicates per uv-b treatment and adaptation time. non-treated fruits (250 g with three repetitions) were used as control. maturity index soluble solids of 150 g fruit per treatment with three replications were determined using a hand refractometer (leo kübler gmbh, germany). for the determination of titratable acidity fruits were homogenized (ultra-turrax t25, ika-labortechnik, staufen, germany). thereafter, 2.5 grams of the homogenate were dissolved with 2.5 ml of distilled water. titratable acidity was determined by titration with 0.1 n naoh until ph 8.1 and expressed as percent total organic acid on basis of citric acid being the main acid component in black currant fruits. phenolic compounds analysis of phenolic compounds for the analysis of phenolic compounds, extraction was conducted following the method described by kähkönen et al. (2001) and connor et al. (2002) using acidified methanol (0.1% hydrochloric acid). an aliquot of 0.5 g grinded sample was mixed vigorously with 3 ml of acidified methanol (0.1% hydrochloric acid) and centrifuged for 15 minutes at 3000 rpm. the supernatant was collected in a 10 ml volumetric flask. the residue was treated again twice with 3 ml acidified methanol and 15 minutes centrifugation. supernatants were collected and standardised to a final volume of 10 ml. the extracts obtained were used for the determination of the phenol content, composition, and antioxidant activity. determination of the total phenol content total phenol content in the fruit extracts were determined using the folin-ciocalteu method with results expressed as milligrams gallic acid equivalents (gae) per gram of dry matter. absorbance was measured at 765 nm (lkb-novaspek ii, pharmacia, freiburg, germany). appropriate dilutions of extracts were prepared by duplicate with distilled water (1+29; v,v). the mixture develops a dark blue color and was left to react for one hour. duplicate lecture of samples were measured spectrophotometrically at 765 nm. analysis of the phenolic compounds using hplc-dad in the present study, the concentrations of two major flavonoid subclasses, flavonols and anthocyanins, and two phenolic acid groups, hydroxycinnamic and hydroxbenzoic acids, were determined by hplc-dad. an analytical hewlett packard 1100 series hplc instrument equipped with an autosampler, quaternary hplc pump and diode array detector was used. analytical separation of the phenolic compounds was carried out on a 250 mm x 4.6 mm, 5 µm, fluofix 120e column (wako pure chemical industries, osaka, japan) with a two solvent mobile phase (eluent a = water/acetic acid/acetonitrile (94.5 + 0.5 + 5; v,v,v); eluent b = acetic acid/acetonitrile (5 + 95; v,v). the eluent gradient used for all extracts is described in tab. 1. the identification of phenolic compounds on the basis of the compound group’s characteristic absorption wavelength was conducted according to kähkönen et al. (2001). two milliliter aliquots of the extract (50 mg dm ml-1) were concentrated by drying with nitrogen and then re-dissolved to a volume of 500 µl with hplc grade methanol. the injection volume was 20 µl, and the flow rate 1 ml min-1. contents of anthocyanin (detection wavelength 520 nm), flavonol (365 nm), and hydroxycinnamic acid (320 nm) were quantified as cyanidin-3glucoside, rutin, and chlorogenic acid equivalents, respectively. total concentration of each representative subclass was calculated from the concentration obtained from a 1 mm standard solution of the compounds mentioned above. results were expressed as milligrams per gram dry matter (dm). determination of the antioxidant activity by electron spin resonance spectrometry (esr) the degradation of a stable synthetic radical, fremy’s salt (potassium nitrosodisulfonate), in presence of antioxidants in the black currant extracts was monitored with esr as applied by rösch et al. (2003). appropriate extract dilutions (1+29; v,v) were prepared and 100 µl aliquots were allowed to react for 20 min with an equal volume of a solution of fremy’s salt (1 mm in phosphate buffer, ph 7.4). esr spectra of fremy’s radical were obtained with a miniscope ms100 spectrometer (magnettech gmbh, berlin, germany). the antioxidant activity expressed as mm fremy’s salt reduced by 1 g dried sample (mm per g dm), was calculated by comparison with a control reaction with 100 µl fremy’s salt 1 mm and 100 µl phosphate buffer. statistical analysis all data were statistically analysed (anova) with spss 13.0 (spss inc., usa). significant differences were determined by tuckey’s test (p < 0.05). the mean variability was indicated by the standard deviation. results and discussion total phenol content in the ripe black currants studied were found to be slightly lower in comparison to the contents reported in the literature, revealing 14-18 mg/g dw and 22-27 mg/g dw, respectively (kähkönen et al., 2001). this might be due to genetic differences of the plant material, differences in the physiological ripening stage of the fruit at the time of harvest and/or environmental conditions during plant growth. the biosysnthesis of phenolic compounds was affected by uv-b radiation in postharvest (fig. 1). uv-b treatments resulted in a continuous increase of phenols showing a peak at 90 minutes. however, adaptation times did not significantly affect total phenol content. the rapid physiological response of phenols to uv radiation in postharvest is also reported for other horticultural products, e.g. brassica spp., vaccinium spp. (huyskens-keil et al., 2006). it is assumed that an uv stress mediated increase of phenylalanin ammonia-lyase activity occurrs leading to an acceleration of the biosynthesis of phenolic compounds (pluskota et al., 2005). anthocyanin synthesis in black currant was promoted by increasing exposure time of uv-b (fig. 2). the highest increase of anthocyanin (60%) was found at 120 min uv treatment, irrespectively of the adaptab. 1: analysis of phenolic compounds using hplc-dad gradient time (min) eluent b (%) 0-4 0-4 4-5 2-4 5-10 2-4 10-25 4-8 25-40 8-22 40-45 22-28 45-50 28 50-55 28-45 55-60 45 60-61 45 phenol composition and antioxidant activity in black currant fruit 141 142 s. huyskens-keil, i. eichholz, l.w. kroh, s. rohn tation time. similar results were reported for strawberries and peaches, where uv mediated increase of anthocyanins was associated with an increase in phenylalanin ammonia-lyase activity (kataoka and beppu, 2004; higashio et al., 2005). in addition to phenylalanin ammonia-lyase induction, uv radiation increases also the activity of other enzymes involved in flavonoid synthesis: chalcon synthase, chalcon isomerase, and dihydroflavonol-4reductase (tomas-barberan and espin, 2001). in the present study also flavonols were affected by the uv-b treatment (fig. 3). with increasing uv-b exposure periods flavonol synthesis was activated revealing a drastic increase of 76% after 120 minutes. the results demonstrate that flavonols showed a similar response to uv in comparison to anthocyanins. hydroxycinnamic and hydroxybenzoic acids are biochemical precursors of flavonols and anthocyanins in the shikimate pathway. therefore, both precursors showed an early reaction to uv exposure: hydroxycinnamic acid increased by 98% after 60 min uv-b exposure, whereas the dominating hydroxybenzoic acid only increased by 35% (fig. 4). correlation between total phenol content and phenolic compounds revealed a strong correlation with hydroxybenzoic acids (r=0.71, p<0.01), anthocyanins (r=0.75, p<0.01) and flavonols (r=0.66, p<0.01). no correlation was found between total phenol content and concentration of hydroxycinnamic acid. antioxidant activity in harvested black currant fruits was significantly induced (21%) by uv-b radiation, however only after 120 min uv exposure with an adaptation time of 22 h (fig. 5). tendentiously, antioxidant activity accelerated with increasing adaptation times revealing a longer biochemical reaction time to uv mediated stress. fig. 2: uv-b induced changes of anthocyanin content in black currant fruits. vertical bars indicate significant differences p < 0.05 (tukey test). 0 , 0 1 , 0 2 , 0 3 , 0 4 , 0 5 , 0 6 , 0 7 , 0 co nt ro l uv 60 _2 h uv 60 _2 2h uv 90 _2 h uv 90 _2 2h uv 12 0_ 2h uv 12 0_ 22 h u v b r a d i a t i o n ( m i n ) / a d a p t a t i o n t i m e ( h ) a n th o c y a n in c o n te n t [m m o l/ g d w ] d c c c b a a 0,0 5,0 10,0 15,0 20,0 25,0 co nt ro l u v6 0_ 2h u v6 0_ 22 h u v9 0_ 2h u v9 0_ 22 h u v1 20 _2 h uv 12 0_ 22 h uv-b radiation (min)/adaptation time (h) t o ta l p h e n o l c o n te n t [m g /g d w ] b abab ab a a a fig. 1: uv-b induced changes of total phenol content in black currant fruits. vertical bars indicate significant differences p < 0.05 (tukey test). 0 , 0 0 , 1 0 , 2 0 , 3 0 , 4 0 , 5 0 , 6 0 , 7 0 , 8 0 , 9 c on tro l u v 60 _2 h u v 60 _2 2h u v 90 _2 h u v 90 _2 2h u v 12 0_ 2h u v 12 0_ 22 h u v b r a d i a t i o n ( m i n ) / a d a p t a t i o n t i m e ( h ) f la v o n o le c o n te n t [m m o l/ g d w ] d c bc bc ab aa fig. 3: uv-b induced changes of flavonol content in black currant fruits. vertical bars indicate significant differences p < 0.05 (tukey test). 0 ,0 0 ,5 1 ,0 1 ,5 2 ,0 2 ,5 c o n tro l u v 6 0 u v 9 0 u v 1 2 0 u v -b ra d ia tio n [m in ] p h e n o li c c o m p o u n d s [m m o l/ g d w ] h yd ro xyc in n a m ic a cid h yd ro xyb e n zo ic a cid ab b b b c b a fig. 4: uv-b induced changes of hydroxycinnamic and hydroxybenzoic acids in black currant fruit. vertical bars indicate significant differences p < 0.05 (tukey test). a strong correlation between antioxidant activity (esr) and the different phenolic compounds were found: total phenols (r=0.63, p<0.01), anthocyanins (r=0.76, p<0.01); hydroxybenzoic acids (r=0.71, p<0.01) and flavonols (r=0.70, p<0.01). no correlation was found between esr and hydroxycinnamic acid, presumably due to their low contribution and thus low antioxidant activity. rice-evans et al. (1995) also 0,00 0,05 0,10 0,15 0,20 0,25 0,30 0,35 0,40 co nt ro l uv 90 _2 h uv 90 _2 2h uv 60 _2 h uv 60 _2 2h uv 12 0_ 2h uv 12 0_ 22 h uv-b radiation (min)/adaptation time (h) m m o l f re m y s s a lt / g d w b ab ab ab ab ab a fig. 5: uv-b induced changes of antioxidant activity in black currant fruit. vertical bars indicate significant differences p < 0.05 (tukey test). phenol composition and antioxidant activity in black currant fruit 143 reported a low antioxidant activity of caffeic acid in comparison to the high antioxidant activity of anthocyanins (delphinidin and cyanidin), flavonols (quercetin and myricetin) and hydroxybenzoic acids (gallic acid) in black currant. in conclusion, the results demonstrate that the synthesis of phenolic compounds and associated antioxidant activity of black currant fruits is stimulated by a short term uv-b radiation in postharvest. anthocyanins are concluded to be able to absorb uv radiation within a short time and thus have a uv-b protective effect in black currant as it is also known for other fruits (jaakola et al., 2004; yang et al., 2005; hagen et al., 2007). flavonoids have various physiological functions such as antioxidant or antitumor activity as well as uv protective effects (higashio et al., 2001; kolb et al, 2001; higashio, 2005). in the present study, flavonols and phenolic acids are also assumed to have an impact on the antioxidant protection of uv-b mediated tissue damage in black currant. further studies on the dynamic of uv mediated changes in secondary plant compounds and antioxidant activity of black currant will be conducted. acknowledgements the authors gratefully acknowledge the laboratory staff inge dressel of the section quality dynamics/postharvest physiology. references anttonen, m., karjalainen, r.o., 2006: high-performance liquid chromatography analysis of black currant (ribes nigrum l.) fruit phenolics grown either conventionally or organcally. j. agric. food chem. 54, 7530-7538. bazzano, l., he, j., ogden, g., vupputuri, s., loria, c., meyers, l., whelton, p., 2002: fruit and vegetable intake and risk of cardiovascular disease in us adults: the first national health and nutrition examination survey epidemiologic follow-up study. am. j. clin. nutr. 76, 93-99. benvenuti, s., pellati, f., melegari, m., bertelli, d., 2004: polyphenols, anthocyanins, ascorbic acid, and radical scavenging activity of rubus, ribes, and aronia. j. food sci. 69, 164-169. connor, a.m., luby, j.j., hancock, j.f., berkheimer, s., hanson, e.j., 2002: changes in fruit antioxidant activity among blueberry cultivars during cold-temperature storage. j. agric. food chem. 50, 893-898. costa, l., vicente, a.r., civello, p.m., chaves, a.r., martinez, g.a., 2006: uv-c treatment delays postharvest senescence in broccoli florets. postharvest biol. technol. 39, 204-210. eichholz, i., rohn, s., kroh, l.w., huyskens-keil, s., 2005: influence of location and fertilization on the antioxidative activity of highbush blueberries (vaccinium corymbosum l.). in: eklund, t., schwarz, m., steinhart, h., their, h.p., winterhalter, p. (eds.), proceedings of the euro food chem xiii: macromolecules and their degradation products in food – physiological, analytical and technological aspects, vol. 2, 388-391. hamburg. isbn 3–936028–31–1. häkkinen, s., heinonen, m., kärenlampi, s., mykkänen, h., ruuskanen, j., törroönen, r., 1999: screening of selected flavonoids and phenolic acids in 19 berries. food res. intern. 32, 345-353. hagen, s.f., borge, g.i.a., bengtsson, g.b., bilger, w., berge, a., haffner, k., solhaug, k.a., 2007: phenolic contents and other health and sensory related properties of apple fruit (malus domestica borkh., cv. aroma)effect of postharvest uv-b irradiation. postharvest biol. technol. 45, 1-10. halliwell, b., 1996: oxidative stress, nutrition and health. experimental strategies for optimization of nutritional antioxidant intake in humans. free radic. res. 25, 57-74. higashio, h., ippoushi, k., ito, h., azuma, k., 2001: effect of uv irradiation on content of flavonoids in spinaci leaves. acta hort. 553, 567-568. higashio, h., hirokane, h., sato, f., tokuda, s., uragami, a., 2005: effect of uv irradiation after harvest on the content of flavonoid in vegetables. acta hort. 682, 1007-1012. huyskens-keil, s., ulrichs, ch., mewis, i., schreiner, m., 2006: ultraviolet irradiation: elicitor to promote the synthesis of phytochemicals in vegetables in preand postharvest. acta hort. (in press). indorf, a., 2001: licht und uv-b-wirkung auf die bildung von lignin, flavonoiden und tanninen in keimlingen der waldkiefer. phd thesis, albrecht-ludwigs-universität freiburg jaakola, l., määttä-riihinen, k., kärenlampi, s., hohtola, a., 2004: activation of flavonoid biosynthesis by solar radiation in bilberry (vaccinium myrtillus l.) leaves. planta 218, 721-728. kähkönen, m.p., hopia, a.i., heinonen, m., 2001: berry phenolics and their antioxidative activity. j. agric. food chem. 49, 4076-4082. kataoka, i., beppu, k., 2004: uv irradiance increases development of red skin color and anthocyanins in ‘hakuho’ peach. hort. sci. 39, 1234-1237. kerr, j.b., seckmeyer, g., 2002: surface ultraviolet radiation: past and future. in: scientific assessment of ozone depletion – global ozone research and monitoring project – report no. 47. world meteorological organization, genf. kolb, c.a., käser, m.a., kopecky, j., zotz, g., riederer, m., pfündel, e.e., 2001: effects of natural intensities of visible and ultraviolett radiation on epidermal ultraviolett screening and photosynthesis in grapes leaves. plant physiol. 127, 863-875. mättää, k.r., kamal-eldin, a., törrönen, a.r., 2003: high-performance liquid chromatography (hplc) analysis of phenolic compounds in berries with diode array and electrospray ionization mass spectrometric (ms) detection: ribes species. j. agric. food chem. 51, 6736-6744. mikkonen, t.p., määttä, k.r., hukkanen, a.t., kokko, h.i., törrönen, a.r., kärenlampi, s.o., karjalaien, r.o., 2001: flavonol content varies among black currant cultivars. j. agric. food chem. 49, 3274-3277. moyer, r., hummer, k., wrolstad, r.e., finn, c., 2002: antioxidant compounds in diverse ribes and rubus germplasm. acta hort. 585, 501505. pluskota, w.e., michalczyk, d.j., gorecki, j., 2005: control of phenylalanine ammonia-lyase gene promoters from pea by uv radiation. acta physiol. plant. 27, 229-236. rice-evans, c.a., miller, n.j., bolwell, p.g., bramley, p.m., pridham, j.b., 1995: the relative antioxidant acitivies of plant-derived polyphenolic flavonoids. free radical res. 22, 375-383. rösch, d., bergmann, m., knorr, d., kroh, l.w., 2003: structure-antioxidant efficiency relationships of phenolic compounds and their contribution to the antioxidant activity of sea buckthorn juice. j. agric. food chem. 51, 4233-4239. schreiner, m., huyskens-keil, s., 2003: effect of film packaging and surface coating on primary and secondary plant compounds in fruit and vegetable products. j. food engineer. 56, 237-240. schreiner, m., huyskens-keil, s., 2006: phytochemicals in fruit and vegetables: health promotion and postharvest elicitors. crit. rev. plant sci. 25, 267-278. slimstad, r., solheim, h., 2002: anthocyanins from black currants (ribes nigrum l.). j. agric. food chem. 50, 3228-3231. tabart, j., kevers, c., pincemail, j., defraigne, j.o., dommes, j., 2006: antioxidant capacity of black currant varies with organ, season, and cultivar. j. agric. food chem. 54, 6271-6276. tomas-barberan, f., espin, j., 2001: phenolic compounds and related enzymes as determinants of quality in fruit and vegetables. j. sci. food. agric. 81, 853-876. world cancer research fund, 1997: american institute of cancer research, food, nutrition and the preservation of cancer: a global perspective. washington, usa yang, y., yao, y., xu, g., li, c., 2005: growth and physiological responses to drought and elevated ultraviolet-b in two contrasting populations of hippophae rhamnoides. physiol. plant. 124, 431-440. 144 s. huyskens-keil, i. eichholz, l.w. kroh, s. rohn addresses of the authors: dr. susanne huyskens-keil1, ines eichholz2, humboldt-universität zu berlin, institute for horticultural sciences, 1section quality dynamics/postharvest physiology and 2section fruit science, lentzeallee 75, d-14195 berlin, germany prof. dr. l.w. kroh, dr. sascha rohn, technical university of berlin, institute for food technology and food chemistry, tib 4/3-1, gustav-meyer-allee 25, d-13355 berlin, germany corresponding author: dr. susanne huyskens-keil, email: susanne.huyskens@agrar.hu-berlin.de << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false /maxsubsetpct 100 /optimize true /opm 1 /parsedsccomments true /parsedsccommentsfordocinfo true /preservecopypage true 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/pagesize [907.087 680.315] >> setpagedevice journal of applied botany and food quality 86, 222 (2013) vereinigung für angewandte botanik e.v. nachruf auf prof. dr. dr. h.c. hans-jürgen jäger völlig unerwartet verstarb am 18. august 2013 herr prof. dr. dr. h.c. hans-jürgen jäger, ehemaliger direktor des instituts für pflanzenökologie der justus-liebig universität gießen und langjähriger chef-herausgeber der zeitschrift „journal of applied botany and food quality – angewandte botanik“ im alter von 71 jahren. hans-jürgen jäger wurde im august 1942 in bad hersfeld geboren und studierte von 1964 -1969 an der justus liebig universität (jlu) in gießen biologie und chemie. 1971 promovierte er am damaligen institut für pflanzenökologie der jlu mit einem biochemisch ausgerichteten thema zur immissionsökologischen wirkung von schwefeldioxid auf kulturpflanzen. es folgten wissenschaftliche tätigkeiten am gleichen institut mit zeitweise größeren arbeitsgruppen als wissenschaftlicher assistent und dozent auf widerruf bzw. dozent auf zeit. 1979 wurde er nach erfolgreicher habilitation zum professor auf zeit ernannt. im jahre 1983 erhielt hans-jürgen jäger einen ruf auf die stellung als direktor und professor an das institut für produktionsund ökotoxikologie der ehemaligen bundesforschungsanstalt für landwirtschaft (fal) in braunschweig. dieses institut leitete er bis ende 1989 vor ort. daneben war er als honorarprofessor an der jlu tätig. anfang 1990 verließ er braunschweig und kehrte an die jlu in gießen zurück, nachdem er dort den ruf auf den lehrstuhl für pflanzenökologie angenommen hatte. in personalunion neben seiner tätigkeit als lehrstuhlinhaber in gießen fungierte er bis mitte 1991 als leiter des braunschweiger institutes. im gleichen jahr wurde ihm von der pannon universität keszthely, ungarn der ehrendoktortitel dr. h.c. für seine unterstützung und kooperationsarbeiten zu problemen des „european crop loss by air pollutants“ verliehen. seine professur und seine funktion als institutsdirektor in gießen hatte er bis zu seiner pensionierung im august 2007 inne. hans-jürgen jäger konzentrierte sich im laufe seiner gesamten wissenschaftlichen arbeit auf stoffbezogene umweltproblematiken in terrestrischen ökosystemen, die aus eingriffen des menschen in die biogeochemischen elementkreisläufe der erde resultieren. hieraus entstand schon früh die spezielle ausrichtung seiner forschung auf die immissionsökologie bzw. die ökotoxikologie von luftverunreinigungen im pflanzenökologischen kontext, die im laufe seiner weiteren tätigkeit und bis zum ende seiner laufbahn auf fragen im zusammenhang mit dem anthropogenen klimawandel ausgedehnt wurden. zur etablierung dieser wissenschaftlichen arbeitsgebiete hat hans-jürgen jäger wesentliche grundlagen beigetragen. seine arbeiten an diesen problemkreisen wurden sowohl unter stark grundlagenbezogenen aspekten als auch immer unter hohem anwendungsbezug durchgeführt. mit dieser ausrichtung verbunden war nicht nur eine hohe wissenschaftliche reputation sondern auch die berufung in zahlreiche kommissionen, gremien und gutachterkreise, die mit umweltproblemen befasst waren (u.a. enquete-kommission „schutz der erdatmosphäre“ des 12. deutschen bundestages; senatskommission der dfg; beirat für naturschutz des bmu; forschungsbeirat „waldsterben“ der bundesregierung; vdi-arbeitsgruppen der kommission reinhaltung der luft). seine forschungsergebnisse sind in 270 wissenschaftlichen originalarbeiten, zwei büchern und zahlreichen sonstigen publikationen niedergelegt. als hochschullehrer hat er sowohl die klassischen ökologischen bzw. pflanzenökologischen fächer vertreten als auch immer wieder neu auftauchende aktuelle themen der angewandten ökologie und angewandten botanik in seinen vorlesungen und übungen thematisiert. die vereinigung für angewandte botanik ist hans-jürgen jäger zu besonderem dank verpflichtet. über 20 jahre lang, von 1983 bis 2007 fungierte er als chef-herausgeber der zeitschrift „angewandte botanik“, die später in „journal of applied botany – angewandte botanik“ bzw. „journal of applied botany and food quality“ umbenannt wurde. im laufe dieses zeitraums hat er die zeitschrift mit hohem engagement und mit einem klaren anspruch auf inhaltliche qualität sorgfältig betreut. durch den tod von hans-jürgen jäger verlieren wir eine persönlichkeit, die mit wegweisend war für die experimentelle ökologische und ökotoxikologische forschung in deutschland. die vereinigung für angewandte botanik wird hans-jürgen jäger ein ehrendes andenken bewahren. braunschweig, im oktober 2013 prof. dr.hans-joachim weigel deceased member prof. dr. dr. h.c. hans-jürgen jäger, former director of the institute of plant ecology at the justus-liebig-universität in gießen, germany, passed away at the age of 71 on 18th of august 2013. hans-jürgen jäger received his phd in plant ecology at the justus liebig university gießen in 1971. he did research as a post-doc and assistant professor (dozent) at the institute of plant ecology in gießen and finished his habilitation in 1979. in 1983 he became director of the institute of productionand ecotoxicology at the former federal research centre of agriculture (fal) in braunschweig, germany. in 1989 he went back to the university of gießen and took over the position of the chair of plant ecology at the institute of plant ecology. he was director of this institute until his retirement in 2007. throughout his career in gießen and braunschweig, hans-jürgen jäger’s research interests were in experimental plant ecology related to environmental problems of biogeochemistry, the impact of pollutants, ecotoxicology and global climate change issues. for more than 20 years from 1983 – 2007, he was editor in chief of the scientific journal “journal of applied botany and food – angewandte botanik” (formerly “angewandte botanik”) published by the german society of applied botany (gesellschaft für angewandte botanik e.v.). braunschweig, october 2013 prof. dr. hans-joachim weigel journal of applied botany and food quality 86, 138 142 (2013), doi:10.5073/jabfq.2013.086.019 agricultural biotechnology and phytopathology laboratory, department of botany, university of karachi, karachi, pakistan management of root diseases of eggplant and watermelon with the application of asafoetida and seaweeds ghulam nabi baloch, samrah tariq, syed ehteshamul-haque, mohammad athar, viqar sultana, jehan ara (received october 10, 2012) summary eggplant (solanum melongena l.) and watermelon (citrullus lanatus (thunb.) matsum. & nakai) are highly susceptible to root rotting fungi fusarium solani, f. oxysporum, macrophomina phaseolina and root knot nematode (meloidogyne spp.) causing huge losses each year in pakistan. in field experiments, application of asafoetida, a medicinal gum from ferula assafoetida and seaweeds spatoglossum variabile, stokeyia indica and melanothamnus afaqhusainii showed significant suppressive effect on root rotting fungi fusarium solani, macrophomina phaseolina and root knot nematode meloidogyne incognita) attacking watermelon and eggplant and improved plant growth in soil naturally infested with root rotting fungi and artificially infested with root knot nematode. length of vine of watermelon, shoot length of eggplant and fresh shoot weight were higher in seaweed and asafoetida treated plants as compared to control or topsin-m, a fungicide, treated plants. seaweed and asafoetida treated plants also showed earlier fruiting than control or fungicide treated plants. at farmer’s field seaweed showed similar suppressive effect on f. solani and m. phaseolina and root knot nematode on watermelon in soil naturally infested by these pathogens. application of seaweed produced healthy plants and number of fruits and weight were significantly higher in seaweed and asafoetida treated plants. asafoetida and seaweeds offer a nonchemical means of disease management. introduction cucurbits and vegetable crops are highly susceptible to a number of root and soilborne diseases causing great losses in yield and quality (sharma et al., 2004; chehri et al., 2010). watermelon (citrullus lanatus (thunb.) matsum & nakai) is a cucurbit fruit, grown throughout the world and consumed as fresh fruit. fusarium wilt of watermelon caused by fusarium oxysporum f. sp. niveum is among the most important diseases of watermelon (zhou and everts, 2004). the disease is well known as mature watermelon vine decline (sudden wilt of watermelon). infected plants also showed the presence of fusarium solani, macrophomina phaseolina, rhizoctonia solani, monosporascus cannonballus and pythium spp. besides f. oxysporum (boughalleb and el mahjoub, 2006). the disease is best controlled through the use of wilt resistant cultivars and crop rotations for a minimum of 5 to 7 years (martyn, 1996). however, resistance in commercial cultivars often no longer is effective due to the presence of the highly aggressive race of f. oxysporum (martyn, 1987). similarly, among the various vegetables, eggplant (solanum melongena) is one of the most common and extensively grown all over the world. eggplant wilt complex caused by a number of fungal genera such as fusarium, verticilium, rhizoctonia, sclerotium and phytophthora take a considerable portion of produce annually (najar et al., 2011). moreover, eggplant is highly susceptible to toot knot nematodes, species of meloidogyne (zarina and shahina, 2010). several nonchemical methods including addition of organic amendments are an effective method for controlling soilborne pathogens and diseases in various field crops (chellemi, 2002; huber, 1980; zhou and everts, 2004). marine bioactive substances extracted from seaweeds have been used for several decades to enhance plant growth and productivity. (rathore et al., 2009; stirk and van staden, 1997). the application of seaweeds as organic soil amendment, has been increased in the recent years due to raising awareness about the adverse effect of chemical pesticides (sultana et al., 2007; 2008; 2009; 2011). similarly, asafoetida or hing, a dry latex or resinous gum from ferula assafoetida has been widely used in various indigenous systems of medicine in india and pakistan. it is regarded as an effective remedy for worms and other intestinal parasites. from old ages, farmers in malir, karachi area of sindh, are applying asafoetida for preventing the plants from root diseases particularly nematode attacks and increasing yield. however, scientific reasons are still unclear. in this study, efficacy of some seaweeds and asafoetida were evaluated in field plots on watermelon and eggplants in suppressing the soilborne diseases and their effect on plant growth. the experiment was also conducted at farmer’s field on watermelon. materials and methods asafoetitda (ferula assafoetida) was purchased from local market whereas seaweeds melanothamnus afaqhusainii shameel, spatoglossum variabile fig. et de notar [= s. lubricum fig. et de notar] and stokeyia indica thivy et doshi [= cystoseira indica (thivy et doshi) mairh] were collected at the coastal area of karachi at low tide. the seaweeds were washed under tap water, dried under shade, ground to powder and stored in polyethylene bags at room temperature until used. field plot experiments the experiments were conducted at the crop diseases research institute, pakistan agricultural research council, karachi university campus, karachi. dry powder of seaweeds melanothamnus afaqhusainii, spatoglossum variabile and stokeyia indica were mixed in sandy loam soil at 70 g per two meter rows and watered 2-3 days interval to allow the organic matters to decompose, whereas aqueous suspension of asafoetida (200 pp) was applied at 400 ml per 2 meter row. the soil had a natural infestation of 5-16 sclerotia/g of soil of macrophomina phaseolina (sheikh and ghaffar, 1975), 5-12% colonization of rhizoctonia solani on sorghum seeds used as baits (wilhelm, 1955) and 2500cfu/gm of soil of mixed population of fusarium oxysporum and f. solani as determined by soil dilution (nash and snyder, 1962). after two weeks of seaweed decomposition, 12 seeds of watermelon were sown in each row. whereas in the case of eggplant, three-week-old seedlings of equal size, raised in steam sterilized soil, were transplanted in each row at 12 seedlings per row. after one week of seed germination or seedling transplantation, each row was inoculated with aqueous suspension of meloidogyne incognita eggs/juveniles at 2000 / two meter row. topsin-m (200 ppm) at 400 ml / 2 meter row and carbofuran (1 g / management of root diseases of eggplant and watermelon 139 2 m row) served as positive control against fungi and nematode respectively. each treatment was replicated four times and plants were watered 2-3 days intervals depending upon requirement of plants. to determine the efficacy of seaweeds and asafoetida on the root pathogens and plant growth, plants were uprooted after six weeks of nematode inoculation. observations were made on yield, plant height, fresh shoot weight, root length and root weight. nematode infection was determined by counting the numbers of galls per root system (tayler and sasser, 1978). to determine nematode penetration and infection by root-infecting fungi, roots from each plant were cut into 1 cm long pieces and five pieces of tap roots from each plant were used for assessment of fungal infection. the remaining roots were mixed thoroughly, and 1 gram sub-sample was wrapped in muslin cloth and dipped in boiling 0.25% acid fuchsin stain for 3-5 minutes. roots were left in the stain to cool, and then washed under tap water to remove excess stain. roots were transferred to vials containing glycerol and water (1:1 v:v) with a few drops of lactic acid. roots were macerated in an electric blender for 45 seconds and the resulting suspension was suspended in 50 ml water. numbers of juveniles and females in five 5 ml sub samples were counted with the aid of dissecting microscope and numbers of nematode/g root were calculated (siddiqui and ehteshamulhaque, 2001). to determine the incidence of fungal infection, 1 cm long root pieces from tap roots (five pieces from each plant) were surface disinfested with 1% ca (ocl)2 solution and plated onto potato dextrose agar amended with penicillin (100,000 units/l) and streptomycin (0.2 g/l). after incubation for 5 days at 28°c, colonies of macrophomina phaseolina, rhizoctonia solani and species of fusarium were recorded. the experiment was repeated twice. data were subjected to analysis of variance (anova) and means were separated using the least significant difference (lsd) according to gomez and gomez (1984). farmer’s field experiment efficacy of asafoetida and seaweeds in protecting the watermelon from root rotting fungi and root knot nematode were also evaluated at farmer’s field in malir, karachi. watermelon field about 6 acres was selected before the sowing of seeds. fifteen meter rows were selected for each treatment at different locations in the field and dry powder of seaweeds melanothamnus afaqhusainii, spatoglossum variabile and stokeyia indica were mixed in soil at 35 g per meter and watered 2-3 days interval to allow the organic matters to decompose, whereas aqueous suspension of asafoetida (200 pp) was applied at 200 ml per meter. the soil had variable natural infestation of root rotting fungi and root knot nematode. after two weeks of amendment, seeds of watermelon were sown in each row. observations were recorded after 80 days of seed germination. results field plot experiment eggplant fusarium solani and macrophomina phaseolina were found in higher frequencies in control plants than r. solani or f. oxysporum. plants grown in seaweed or asafoetida amended soil showed less infection of m. phaseolina than control plants. spatoglossum variabile was also found effective against f. solani (tab. 1). seaweed and asafoetida also showed significant suppressive effect on root knot nematode by reducing gall formation on roots and nematode’s penetration in roots as compared to untreated control (tab. 2). efficacy of asafoetida and seaweed against root knot nematode is comparable with carbofuran, a nematicide. taller plants and greater fresh shoot weight was produced by s. indica (tab. 2). watermelon application of seaweeds spatoglossum variabile, melanothamnus afaqhusainii and asafoetida caused a suppressive effect on root rotting fungi by reducing the infection of m. phaseolina, r. solani and f. oxysporum on watermelon roots. stokeyia indica was effective against f. solani (tab. 3). seaweeds and asafoetida also showed suppressive effect on root knot nematode by reducing the nematode’s penetration in roots (tab. 4). vine length and fresh weight were significantly higher in watermelon grown in spatoglossum variabile amended soil than other treatments (tab. 4). plants grown in seaweed or asafoetida amended soils also showed earlier emergence of fruits than control of pesticides treatments. farmer’s field experiment watermelon infection of fusarium solani was found in higher frequencies in control or treated watermelon plants. however, plants grown in s. variabile amended soil showed less infection of f. solani than other treatments, whereas topsin-m treated plants showed no infection of f. solani (tab. 3). macrophomina phaseolina infection was found in control plants only. nematode penetration in watermelon roots were found less in s. indica s. variablie, asafoetida amended soil or in topsin-m treated plants (tab. 4). number of fruits and fruit weight were higher in plants grown in asafoetida or seaweed tab. 1: effect of asafoetida and seaweed on the infection of root infecting fungi on eggplant in field plot experiment. no. treatments m. phaseolina r. solani f. solani f. oxysporum infection % 1. control 68.7 6.2 31.2 6.2 2. topsin-m 12.5 0 12.5 0 3. carbofuran 18.7 31.2 43.7 6.2 4. asafoetida 6.2 50 43.7 0.0 5. stokeyia indica 0.0 0.0 50 0.0 6. spatoglossum variabile 0.0 6.2 18.7 0.0 7. melanothamnus afaqhusainii 12.5 12.5 25 0.0 lsd0.05 treatments= 10.31 pathogens= 7.82 1mean values in column showing differences greater than lsd values are significantly different at p< 0.05. 2mean values in rows showing differences greater than lsd values are significantly different at p< 0.05. 140 g.n. baloch, s. tariq, s. ehteshamul-haque, m. athar, v. sultana, j. ara tab. 2: effect of asafoetida and seaweed on the infection of root knot nematode and growth of eggplant in field plot experiment. no. treatments no. of knots j2/females plant fresh root fresh /g roots height shoot length root (cm) wt. (g) (cm) wt. (g) 1. control 92.5 192 14.2 20.2 7.7 11.7 2. topsin-m 23.2 124 21.2 29.7 5.2 4.1 3. carbofuran 16 80 26.5 33.7 11 10.2 4. asafoetida 16.5 80 18.7 32.3 9.7 12.7 5. stokeyia indica 7 72.7 38.5 40.1 10.5 11.7 6. spatoglossum variabile 12 79.2 24.5 29.5 8.2 4.5 7. melanothamnus afaqhusainii 24.2 102.5 17 27.6 6.7 2.6 lsd0.05 35.11 15.31 9.61 14.01 3.11 3.61 1mean values in column showing differences greater than lsd values are significantly different at p< 0.05. tab. 3: effect of asafoetida and seaweeds on the infection of root infecting fungi on watermelon in field plot experiment (after 45 days of nematode inocu lation) and at farmer’s field (after 80 days of sowing). no. treatments m. phaseolina r. solani f. solani f. oxysporum infection % field plot experiment 1. control 31.2 25 37.5 18.7 2. topsin-m 43.7 25 50 12.5 3. carbofuran 12.5 6.2 43.7 18.7 4. asafoetida 6.2 0 18.7 0 5. stokeyia indica 50 12.5 18.7 0 6. spatoglossum variabile 6.2 6.2 31.2 6.2 7. melanothamnus afaqhusainii 0 0 50 0 farmer’s field experiment 1. control 18.7 6.2 37.5 0 2. topsin-m 0 0 0 0 3. asafoetida 0 0 12.5 0 4. stokeyia indica 0 6.2 37.5 0 5. spatoglossum variabile 0 0 18.7 0 6. melanothamnus afaqhusainii 0 0 31.2 0 lsd0.05 field plot experiment treatments= 9.071 pathogens= 6.862 experiment at farmer’s field treatments= 7.31 pathogens= 5.22 1mean values in column showing differences greater than lsd values are significantly different at p< 0.05. 2mean values in rows showing differences greater than lsd values are significantly different at p< 0.05. amended soil than control plants. vine length and fresh weight was found significantly higher in plants grown in m. afaqhusainii amended soil (tab. 4). discussion protecting the plant roots from soilborne plant diseases with organic amendments and improving plant growth and yield is practiced since long by the farmers throughout the world. the suppressive effect of organic amendments on diseases is primarily associated with a reduction in pathogen inoculums density in amended soil (chun and lockwood, 1985; sun and huang, 1985; subbarao et al., 1999). in this study, soil amendment with seaweed or asafoetida in general showed suppressive effect on soilborne pathogens and have a positive effect on growth of watermelon both in field plots and at farmer’s field. plant grown in seaweed or asafoetida amended soil showed earlier fruiting, greater number of fruits and weight than control plants. it was reported that seaweed extracts increased plant resistant to pests and diseases, improve plant growth, yield and quality (rathore et al., 2009; stirk and van staden, 1997; verkleij, 1992). application of seaweed to plants can result in decreased levels of nematode attack (ara et al., 1997; wu et al., 1997; 1998), due to presence of betaines (wu et al., 1997) and root rotting fungi (sultana et al., 2007; 2008; 2009; 2011) due to 1-aminocyclopropane-1-carboxylic acid (acc), which has antimicrobial activity (nelson and van standen, 1985) or due to volatile compounds and essential oils (kajiwara et al., 2006). seaweed could also affect cell metabolism through the induction of the synthesis of antioxidant molecules which could favor plant growth and plant resistance to stress (zhang and schmidt, 2000). tariq et al. (2011) has already reported the presence of polyphenols and antioxidant activity of seaweeds from the karachi management of root diseases of eggplant and watermelon 141 coast. in this study, the potential of asafoetida in suppressing the soilborne diseases has been confirmed. asafoetida or hing, is regarded as an effective remedy for worms and other intestinal parasites and posseses antimicrobial and antioxidant activities (iranshahy and iranshahi, 2011). biological activity of sesquiterpene coumarins from ferula species has also been reported (nazari and iranshahi, 2011). similarly lee et al. (2009) reported antiviral and cytotoxic agents from ferula assafoetida. growth inhibition of plant pathogenic fungus fusarium by the essential oils extracted from asafetida has also been reported (sitara et al., 2008). due to the damaging influence on crop yields, the root knot nematodes meloidogyne incognita, m. javanica, m. arenaria and m. hapla are considered economically important pests worldwide (robertson et al., 2006). the damages are much higher in tropical and subtropical countries where environmental factors favor their survival and dispersal (sikora and fernandez, 2005). in this study, the application of asafoetida and seaweeds besides, suppressing the same root rotting fungi also showed significant reduction in gall formation on roots and nematode’s penetration into roots of eggplant and improve plant growth. eggplant is susceptible to several insects and pest, particularly fusarium wilts (f. oxysporum f. sp. melongenae) and root knot nematodes (meloidogyne spp.) which reduced the yield (phap et al., 2010). due to high susceptibility, eggplant is widely used for maintaining the pure culture of root knot nematode (siddiqui et al., 2001; sikander et al., 2009). in this study, efficacy of asafoetida and seaweed is comparable to carbofuran, a commercial nematicide. nonchemical management of soilborne pests has been practiced for centuries. only in the last 40 years agricultural producers have come to rely on synthetic chemicals for the control of soilborne pest and diseases (chellemi, 2002). however, due to raising awareness about the adverse effect of chemicals, organic forming is gaining popularity (sultana et al., 2011). the ability of seaweeds and asafoetida in reducing the root knot nematode on a highly susceptible crop showed its great potential in future nematode pest management. acknowledgements financial support provided the dean, faculty of science, university of karachi is sincerely acknowledged. we are also thankful to dr. aly khan, director, crop diseases research institute, pakistan agricultural research council, karachi university campus, karachi and mr. feroz baloch, a progressive farmer for providing the space and other facilities for conducting the experiments. references ara, j., ehteshamul-haque, s., sultana, v., ghaffar, a., qasim, r., 1997: use of sargassum species for the control of meloidogyne javanica in okra. nematol. medit. 25, 125-128. boughalleb, n., el mahjoub, m., 2006: watermelon sudden decay in tunisia: identification of pathogenic fungi and determination of primary agents. pak. j. biol. sci. 9, 1095-1103. chehri, k., abbasi, s., redd, k.r.n. salleh, b., 2010: occurrence and pathogenicity of various pathogenic fungi on cucurbits from kermanshah province, iran. afr. j. microbiol. res. 4, 1215-1223. chellemi, d.o., 2002: nonchemical management of soilborne pests in fresh market vegetable production systems. phytopath. 92, 1367-1372. chun, d., lockwood, j.l., 1985: reductions of pyhthium ultimum, thielaviopsis basicola and macrophomina phaseolina populations in soil associated with ammonia generated from urea. plant disease 69, 154158. gomez, k.a., gomez, a.a., 1984: statistical procedures for agri cultural research. 2nd ed. wiley, new york. huber, d.m., 1980: the use of fertilizers and organic amendments in the control of plant disease. in: pimental, d. (ed.), handbook series in agriculture. sect. d. crc press, florida. tab. 4: effect of asafoetida and seaweeds on the infection of root knot nematode and growth of watermelon in field plot (after 45 days of nematode inocu lation) and at farmer’s field (after 80 days of sowing). no. treatments no. of j2/females no. of fruit vine fresh root fresh knots /g roots fruits/ wt. length shoot length root plant (g) (cm) wt. (g) (cm) wt. (g) field plot experiment 1. control 1.25 5.75 0 -58.7 16.2 14.2 0.85 2. topsin-m 0.25 1.5 0 -63.5 39.0 22 2.41 3. carbofuran 0.25 0.75 0 -66 23.5 15 0.87 4. asafoetida 0 0.75 0.75 -70 20.9 12.2 1.13 5. stokeyia indica 0 1 0.5 -67.5 32.2 16.7 0.99 6. spatoglossum variabile 0 0.75 1.25 -92 38.7 15.3 1.87 7. melanothamnus afaqhusainii 0 0.75 1.0 -81.5 36.4 14 1.92 lsd0.05 0.641 1.391 0.881 -27.01 18.61 7.81 0.671 farmer’s field experiment 1. control 4.5 6.5 09 356 124 86 13.1 3.9 2. topsin-m 3.5 1.8 14 1264 164 164 12.6 6.2 3. asafoetida 2.8 2.5 18 3847 188 156 12.5 3.1 4. stokeyia indica 5.5 0.75 17.5 1871 129.7 120 12.7 2.2 5. spatoglossum variabile 2.8 0.75 16 2585 204 154 12.7 2.5 6. melanothamnus afaqhusainii 3 5.75 19.5 4265 227.5 185 12.2 4.2 lsd0.05 1.71 1.121 2.11 46.81 6.81 5.01 ns 0.51 1mean values in column showing differences greater than lsd values are significantly different at p< 0.05. 142 g.n. baloch, s. tariq, s. ehteshamul-haque, m. athar, v. sultana, j. ara iranshahy, m., iranshahi, m., 2011: traditional uses, phytochemistry and pharmacology of asafetida (ferula assafoetida oleo-gum-resin) − a review. j. ethnopharmacol. 134, 1-10. kajiwara, t., matsui, k., aleakabe, y., murakawa, t., arai, c., 2006: antimicrobial browning inhibitory effect of flavor compounds in seaweeds. j. appl. phycol. 18, 413-422. lee, c.l., chiang, l.c., cheng, l.h., liaw, c.c., abd el-razek, m.h., chang, f.r., wu, y.c., 2009: influenza a (h1n1) antiviral and cytotoxic agents from ferula assafoetida. j. nat. prod. 72, 1568-1572. martyn, r.d., 1987: fusraium oxysporum f. sp. niveum race 2. a highly aggressive race new to the united states. plant disease, 71, 233-236. martyn, r.d., 1996: fusarium wilt of watermelon. in: zitter, t.a., hopkinsand, d.l., thomas, c.e. 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of fusarium wilt of watermelon by soil amendment with hairy vetch. plant disease 88, 13571365. address of the author: mr. ghulam nabi baloch, agricultural biotechnology and phytopathology laboratory, department of botany, university of karachi, karachi-75270, pakistan. miss samrah tariq, agricultural biotechnology and phytopathology laboratory, department of botany, university of karachi, karachi-75270, pakistan. prof. dr. syed ehteshamul-haque, agricultural biotechnology and phytopathology laboratory, department of botany, university of karachi, karachi-75270, pakistan. dr. mohammad athar, pest detection and emergency projects, california department of food and agriculture, 3288 meadowview road, sacramento, ca 95832, usa. e-mail: atariq@cdfa.ca.gov prof. dr. viqar sultana, biotechnology and drug development laboratory, department of biochemistry, university of karachi, karachi-75270, pakistan. prof. dr. jehan ara, post harvest technology and food biochemistry laboratory, department of food science and technology, university of karachi, karachi-75270, pakistan. art01 summary nitrogen-fixing and phosphate-mobilizing bateria, as well as mycorrhizal fungi, can influence plant nutrition beneficially and thus be used as biofertilizers in agriculture. this paper briefly reviews the role of wheat genotypes in the interaction of wheat with soil microorganisms like phosphate solubilizing and nitrogen fixing bacteria, specifically azotobacter sp., and with mycorrhizal fungi for the development of sustainable wheat crop production. the role of rhizosphere microorganisms and the mechanisms, factors affecting response of bioinoculants and the possibilities of breeding wheat genotypes responsive to these bioinoculants for sustainable wheat production in semi-arid tropics are discussed. introduction wheat (triticum aestivum l.) is one of the major sources of energy, protein and fiber in human diet, staple food for nearly 35% of the world population and hence the most important cereal crop globally. semi dwarf, high yielding wheat varieties developed in the mid sixties led to an impressive increase in wheat yields in several conventional wheat belts (rajaram, 1999). however, the gain in yield based on breeding (or genetic traits) has been rather low since the mid nineties. the expected global demand for wheat in the year 2020 will vary between 840 to 1,050 million tons (rosegrant et al., 1995). to meet this growing demand, the global average grain yield must increase from the current 2.5 t/ha to 3.8 t/ha. india is the second largest producer of wheat in the world after china. in india, wheat production has increased 11 times from 6.46 million tons in 1950 to 72 million tons in 2003, while the average wheat yield has increased nearly four times from 0.66 tons/ha in 1950 to 2.7 tons/ ha in 2003. however, to meet the food security requirement 1.5 million tons of additional wheat per year would be required. it will require production targets of wheat to be enhanced from 95 million tons at present to 109 million tons by 2020. such a daunting task may not be achieved easily considering the various natural and technological constraints. the conventional breeding methods provide at best only one per cent yield gain per year against the much-needed 2 per cent yield gain consistently necessary over the years. biofertilizers biofertilizers are formulated products of living cells of different types of microorganisms that have the ability to mobilize nutrients form non-usable to usable form through biological processes. these microorganisms come from the soil biosphere and hence are parts of the plant’s natural environment. the microorganisms that are in use as biofertilizers in wheat broadly include the free living and associative nitrogen fixing and phosphate solubilizing rhizobacteria and the mycorrhizal fungi that are capable of mobilizing non-available nutrients from soil and transporting them to and across plant roots, e.g. phosphorus (hayman and mosse, 1971) and zinc (swaminathan and verma, 1979). to these has been recently added the plant growth promoting rhizobacteria (pgpr) which stimulate plant growth and repress root diseases by a variety of mechanisms. the contribution of vesicular arbuscular mycorrhiza (vam) and azotobacter to growth and development of many plant families has long been recognized (wani et al., 1988). earlier studies of such types of symbioses in wheat have described considerable variability for the colonization by vam and response to azotobacter. even when wheat is colonized, biomass and yield improvement due to symbiosis depends on the nutrient absorbing efficiency of the fungal symbiont (hetrick et al., 1991; veirheilig and ocampo, 1991), fertility of the soil (young et al., 1985) and genotype of the cultivar (azcon and ocampo, 1981; hetrick et al., 1991). a wide variation in plant dependency and degree of vam infection was found in different cultivars of wheat (triticum vulgare) in response to vam fungus glomus mosse inoculation (azcon and ocampo, 1981). comprehensive reviews examined the benefits that biofertilizers including am association can have to increase soil fertility and crop production in sustainable farming and for agroecosystems including organic farming (wu et al., 2005; gosling et al., 2006). rationale wheat yield per se is closely associated with input responsiveness. the use of higher doses of chemical fertilizers than ever before, are needed to maintain the yield levels of wheat world over. besides, there are large areas in asia and africa where wheat is grown under rainfed and/or limited water and nutrient supply situations and consequently with poor yield. in these areas, soils are generally poor in mineral nutrients, organic carbon and rhizospheric activity and crops encounter intermittent moisture stress. also in vast areas soils are either calcareous (high ph) or acidic (low ph) and hence phosphorus (p) and zinc (zn) fixation is witnessed. survey of literature reveals that irrespective of the levels of nitrogen fertilizer applied to wheat crop following rice, treatment with azotobacter used as biofertilizer singly or in combination with vam, leads to yield improvement of wheat. therefore, nowadays there is a greater awareness to use microbial bioinoculants (biofertilizers) such as azotobacter and mycorrhizal fungi as a component of integrated nutrient management strategies to attain higher input use efficiency (nutrients and water), to cope with increasing fertilizer costs, and their long term adverse effects on agricultural ecosystems like increased nutrient imbalances and declining productivity, etc. the beneficial effects of the use of bioinoculants include increase in plant growth and yield, better quality, improved crop uniformity and reduction in p and micronutrient fertilizer requirement, and reduced losses due to environmental stresses and diseases (allen and boosalis, 1983; behl et al., 2006). all these benefits translate into increased profits for farmers through improved production efficiencies and increased crop values. therefore, selection of efficient wheat varieties responsive to bioinoculants and applied inorganic nutrients for high and low input conditions in different agroecological zones is very important to harness synergy between crop genotypes, microbial inoculants and inputs like nutrient and water. journal of applied botany and food quality 81, 95 109 (2007) 1 department of plant breeding, ccs haryana agricultural university, hisar, india 2 institute of vegetable and ornamental crops, igz, grossbeeren, germany 3 institute of microbiology, friedrich schiller university, jena, germany 4 department of microbiology, ccs haryana agricultural university, hisar, india wheat x azotobacter x va mycorrhiza interactions towards plant nutrition and growth – a review rishi k. behl1, sabine ruppel2, erika kothe3, neeru narula4 (received july 23, 2007) 96 rishi k. behl, sabine ruppel, erika kothe, neeru narula vesicular-arbuscular mycorrhizae (vam) the vesicular arbuscular mycorrhiza (vam) is a mutually beneficial symbiosis of fungi which are found associated with the majority of agricultural plants. they are ubiquitous in geographic distribution occurring with plants grown in arctic, temperate and tropical regions alike. vam occur over a broad ecological range from aquatic to desert environments. the fungi belong to the glomeromycota with the genera endogone, glomus, entrophosphora, gigaspora, acaulospora, scutellospora. they are obligate symbionts which have not been cultured on nutrient media yet because of their obligately biotrophic life style which makes it essential for vam fungi to infect and spread inside a living root. the symbiotic association between plant roots and mycorrhizal fungal mycelia has been termed mycorrhizae, where endomycorrhizae (here: vesicular-arbuscular mycorrhiza) colonize the root cortical cells intracellularly (smith, 1980; harley and smith, 1983). vam colonization originates from hyphae arising from soil borne propagules. root exudates induce chemotactic growth and branching. upon contact with the root appresoria-like structures are formed to penetrate the root surface. intercellular growth proceeds from which hyphae invade cortical cells. hyphae grow into cells and differentiate into tree-like dichotomously branching arbuscules. these arbuscules do not invade the cortex cell’s cytoplasma membrane but rather are surrounded by the intact plant cell membrane. between both fungal and plant plasma membrane, an extramatrical compartment allows nutrient transfer from plant to fungus (for photosynthesis products) and vice versa (for nutrient and water). when colonization is well established, oval structures, called vesicles, may form for which mainly storage functions for p as phospholipids have been described (barea, 1991). to assess the role of vam as modifiers of soil fertility, the main quantitative estimate to be considered is the extent of external hyphae in soil associated with mycorrhizal roots (abbott and robon, 1985). vam have been associated with increased plant growth and with enhanced accumulation of plant nutrients, mainly p and zn. cu and s are mainly increased through greater soil exploration by mycorrhizal hyphae. it has also been suggested that vam stimulate plant growth by physiological effects other than by enhancement of nutrient uptake or by reducing the severity of diseases caused by soil pathogens. the survival and performance of vam fungi is affected by the host plant, soil fertility, cropping practices, soil management practices, as well as biological and environmental factors. the results of field trials conducted in india reviewed (wani and lee, 1995) indicated that vam inoculations increased yields significantly in around 50% of the trials and the response varied with soil type, soil fertility, and vam cultures. maximum root colonization and sporulation occurs in low fertility soils. however, yield increases in barley grown in soil with 40 ppm available p (na 2 hco 3 -extractable) was observed by vam inoculation. internal p concentration of roots rather than external p concentration in soil controls root colonization by vam function (menge et al., 1978). application of farm yard manure stimulated vam, while in bare soil between harvesting of one crop and sowing of the next crop during the rainy season mycorrhizal spores in soil were reduced significantly. longer periods free of cultivation reduced mycorrhizal colonization of crops, and cereals in rotation with legumes showed higher root colonization and higher numbers of vam propagules than cereals grown in monocropping (harinikumar and bagyaraj, 1988). higher temperatures generally result in better root colonization in the temperate zones. however, the reverse may be true in the tropics. application of fungicides (like benomyl), soil solarization and prolonged water logging can significantly reduce the number of vam propagules in soil (wani and lee, 1995). luttger (1996) found no infection with vam even though about 170 spores existed on one experimental field where sewage water was used for irrigation. reasons for that might be either a high c and n content of the field, or the sensibility of vam-spores to the pollutants in sewage water. a key determinant for the ability of a root systems to acquire nutrients from the soil is the extent to which it is colonized by appropriate mycorrhizal fungi. the mycorrhizal condition is actually the normal status of most terrestrial plants and it greatly enhances the possibilities of nutrient uptake. the formation and activity of this symbiosis, in turn, is affected by soil fertility, as the extramatrical mycelium helps the plant to acquire nutrients from the soil (barea, 1991). the ecological and economic interest in vam can simply be deduced from the fact that about four-fifths of all land plants including agronomically important crops form this type of mycorrhizae which suggests coevolution of plants and vam. inoculums of the obligate symbiotic vam fungi have to be produced in co-culture with roots of host plants. the major constraint with vam application in annual crops has been the production of high quality, ‘clean pure’ inoculum of prepared fungal species and possibly even ecotypes not contaminated with other microorganisms. since the vam fungi are heterokaryons with up to 5000 different genotypes within the mycelium propagated from a single spore, such ecotypes might be established only by selection during growth under the specific conditions of the plant cultivation. however, companies producing vam in certified quality are now on the rise throughout europe. the other interaction partner, the plant, is more easily accessible to genetic experiments and cultivar selection. one biological approach to manage vam symbiosis is therefore to select plant genotypes for their ability to form an efficient symbiosis with the indigenous vam population of the field soil. it is also known that crop management, i.e, crop rotation and fertilizer management, has an impact on the amount and composition of the vam fungal population and may, therefore, have a considerable effect on its efficiency to promote crop productivity (johnson et al., 1992). vam infection and wheat plant growth there is little host specificity for vam fungi, but the competitive ability of a given species with native strains may influence the dominance of a certain endomycorrhizal fungus in a root system. cultivars of wheat have been shown to vary in the level of colonization by vam fungi (hetrick et al., 1992; johnson et al., 1992). furthermore, the degree to which wheat cultivars are colonized by and benefit from vam is a plant heritable trait which can be improved through plant breeding (johnson et al., 1992). landraces rely more on this symbiosis than modern wheat cultivars. the yield improving potential of vam should be taken into account in the breeding of nutrient efficient wheat varieties (kapulnik and kushnir, 1991). vam can increase wheat plant growth and yield, improve crop uniformity and reduce fertilizer and pesticide/fungicide requirements. veirheilig and ocampo (1991) reported that colonization of all cultivars was independent of the amount of inoculum and the time interval of inoculation. however, it has been reported that an increase in inoculum potentially enhances effectiveness (haas and krikun, 1985), and that the time interval of inoculation may play an important role in effectiveness of vam infection (hepper, 1985). after inoculation, the dry weight of some cultivars increased, which may indicate that the increased amount of vam inoculum increased the effectiveness of colonization. however, this effect varied depending on the cultivar and the time of reinoculation. no relationship was found between the colonization level and the effectiveness of the symbiosis (hepper, 1985). the growth temperature affects the colonization level of the wheat cultivars. colonization was enhanced at 35 over 24°c, but temperature changes did not reduce the susceptibility of the plants even when grown at low temperature (ishac et al., 1986). although a similar level of vam colonization was observed in some cultivars grown at different temperatures, vam increased only shoot dry weight. veirheilig and ocampo (1991) found no significant differences in shoot dry weight and root : shoot (r:s) ratio between inoculated and un-inoculated wheat triple interactions towards plant nutrition and growth 97 cultivars in earlier sampling, but after 6 weeks of growth, the inoculated cultivars showed higher shoot dry weight and lower r:s ratio than the un-inoculated cultivars. several other authors have found no relationship between the percentage of infection and the dry weight of infected plant. hence, measurement of succinate dehydrogenase activity has been proposed as a parameter of fungal affectivity (ocampo and barea, 1985). hetrick et al. (1991) demonstrated that plants that rely heavily on mycorrhizal symbiosis (mycotrophic versus low mycotrophic plants) have a more plastic root morphology, i.e., they maintain a less fibrous root system in response to colonization by the mycorrhizal symbiont. in facultative mycotrophs such as wheat, however, root morphology appears to be a fixed characteristic, unchanged by mycorrhizal colonization of roots. therefore, the observed variation in root morphologies among wheat cultivars and ancestors, and similarty of morphologies within cultivars or ancestors depending on whether or not they are mycorrhizal, suggests that the morphological differences observed are genetic characteristic of the plant themselves rather than changes induced by symbiosis. luttger et al. (1993) demonstrated that genotypically determined levels of infection by the indigenous vam fungi population did have an impact on grain yield. cultivars with high grain yield under reduced mineral fertilization exhibited two-fold more vam infections at the stage of tillering. the higher grain yield obtained in mycorrhizal inoculated plants is due rather to an increase in grain weight. it is suggested that the mechanism by which vam inoculation affects grain yield is established at an early stage of plant growth rather than at the later stage of maturation. luttger (1996) reported results of extensive studies on wheat-vam interactions in low versus high input genotypes under different agronomic management. dependent upon the location, irrigation, fertilizer level and previous crop, the infection of the wheat roots varied between 0-70%. the roots of the wheat genotypes fertilized with rock phosphate showed significantly higher infection compared to the fertilizer treatment with easy soluble single super phosphate. in general, an early (crown root initiation stage) and intensive colonization of the wheat with vam was beneficial for the grain yield in two subsequent years, particularly with reduced fertilizer application. at anthesis, the vam infection was either positively or negatively correlated with above ground biomass or grain yield, dependent on the availability of water. in general, the influence of mycorrhizal infection increased with reduced ground water table. an impact of amf to improved nutrient uptake was only evident at the lowest fertilizer level with rock phosphate. the increased infection rates led to significantly higher p concentrations in the above-ground biomass and an increase in zn uptake. the symbiosis of a wheat genotype with amf seems to be under genotypic control. independent of the location and fertilizer level, the wheat cultivars c 306 and pbw 226 had high infection rates in both years. phosphate acquisition and uptake vam enhances plant growth by improved mineral nutrition (barea and azcon-aguilar, 1983; harley and smith, 1983; sreenivasa and bagyaraj, 1988) which includes increased p uptake by the plant occurring in three steps: 1) mobilization and absorption of phosphate from soil by vam hyphae, 2) translocation of phosphate along hyphae from external to internal cortical mycelia, and 3) the transfer of phosphate to cortical root cells, ready to be used by the plant (barea,1991). since under normal conditions where no fertilizers are applied, the concentration of available phosphate in soil is very low (10-6 m), the hyphal network of vam make closer contacts than roots with the surface of sparingly available phosphate particles in soil and absorb it more rapidly than the roots (hetrick et al., 1996). also, fungal hyphae may produce organic acids like citrate and affect phosphate solubilization thus improving p nutrition of the host (clarke and mosse, 1981; sreenivasa and bagyaraj, 1988; mcarthus and knowles, 1993; weber et al., 1993). although genotypic and environmental factors influence symbiosis, a prolonged, stable and compatible relation between plants and vam is based primarily upon the transfer of p from symbiont to host that has a net p deficit (barea, 1991). vam thus is important for crop production on soils with high p fixation such as calcareous soils of the semiarid regions of the tropics. the fungus obtains photosynthates and other growth factors from the host, and in turn increases the functional root surface area through hyphal extension improving absorption of nutrients and water from soil (edriss et al., 1984). mycorrhizal plants frequently show resistance to drought (nelson and safir, 1982) and environmental stresses (dehne, 1986). several studies have shown that biofertilization helps expansion of the root systems, leads to better seed germination and better plant growth (barea et al., 2005). tarafdar and marschner (1994) studied phosphatase activity of vam fungi in rhizosphere and phyllosphere with mycorrhizal and non-mycorrhizal wheat (triticum aestivum). they found that in the root compartments the activity of acid phosphatase was higher than for alkaline phosphatase, with both enzymes enhanced with vam supplied with organic p. p application also increased the percentage of infected root length and phosphatase activity was correlated with hyphal length which was greatest within 10 mm of the root surface. mycorrhizal inoculation increased plant dry weight, p content and total p uptake irrespective of p source and soil type. mycorrhizal infection accounts for 24-34% of the total plant p when supplied as organic p which also stimulated mycorrhizal infection and hyphal growth. they also demonstrated the efficient use of phytate p by phosphatase of mycorrhizal hyphae. vam infection improves p uptake per unit root length (p influx). manske et al. (1995) observed that in an field trial in northern india, the roots of all 20 wheat lines screened were infected by the native vam fungi in the soil. increased fertilizer doses reduced the infection by native vam. with reduced fertilizer application amf infection at the stage of tillering resulted in higher grain yield. manske et al. (2000) concluded that plant factors influencing p uptake efficiency are mainly associated with root characteristics. they evaluated 42 spring wheat genotypes, grown on an acid andisol in mexico, with and without p fertilization to identify traits associated with improved p uptake efficiency. amf infection rate was positively correlated with p uptake in to wheat shoots (r=0.47). however, this explains only 25% of the variance of p uptake. other traits like rld (root length density) and phosphatatses excretion by the roots were also important. vam-colonization and acid phosphatase activity were to a lesser degree correlated with plant p uprake efficiency. yao et al. (2001) evaluated three wheat (t. aestivum) genotypes with high (81(85)), intermediate (fengxiao) and low (nc37) p efficiency in a pot experiment with low or adequate p supply either inoculated with the arbuscular mycorrhizal fungus glomus versiforme or uninoculated. the mycorrhizal dependency of the genotypes with relatively high p efficiency was lower than that of the genotypes with lower p efficiencies. linear correlation analysis revealed that mycorrhizal dependency was primarily controlled by p uptake efficiency. in the second pot experiment the same three genotypes were grown in low p soils. higher hyphal length density arising from carbohydrate translocation led to more p uptake and this may account for higher mycorrhizal dependency. manske and vlek (2002) emphasized the importance of wheat nutrient uptake efficiency and suggested that genetic variation can be exploited to manipulate root characteristics such as rld to improve nutrient use efficiency in different agroecological niches. plant growth response to vam infection also depends on the balance between the costs and benefits of the symbiosis. the energy required 98 rishi k. behl, sabine ruppel, erika kothe, neeru narula to build and maintain an extensive amf-infected root system needs to be offset by the improved nutrient uptake of the plant, a condition that may apply in marginal soils. from the plant’s side, vam fungi are strong competitors for assimilates which is especially relevant if c availability for growth and grain filling is limited. depending on the host plant genotypes or on p supply, 4-14% of the photosynthates are allocated to amf-infected roots (harris and paul, 1987). response of plant growth to amf usually declines as stress conditions are relaxed, e.g. sufficient nutrients are supplied. the enhanced p uptake is gained at the cost of host photosynthates utilized by amf. however, if p becomes non-limiting and photosynthetic utilization by amf is not curtailed, the amf plant may become carbon limited, a situation that leads to parasitic effects of amf (vlek et al., 1996). neumann and george (2004) conducted studies to quantify the contribution of amf to p nutrition of the host plant when the p availability of the soil was limited by drought. to investigate the potential of amf hyphae in taking up p from dry soil, mycorrhizal (+m) and non-mycorrhizal (-m) sorghum bicolor l. plants were grown in a vertical split root system that consisted of two compartments placed one upon the other. the upper compartment was filled with well fertilized soil and the plant roots were allowed to grow into the lower compartment through a perforated bottom. the lower compartment was filled with an expanded clay substrate and nutrient solution, to supply the plants with water and all nutrients except p. the soil in upper compartments was either dried or kept moist during a period of four weeks before harvest. the total plant p content did not differ significantly between the mycorrhized and non-mycorrhized plants within the treatment with sufficient water. in contrast, the p content of mycorrhizal plants was almost doubled when the soil in the upper compartment was dried. the concentration of all elements except p in plant shoot tissue was sufficient for adequate plant growth. p concentrations in the shoots of non-mycorrhizal, water stressed plants indicated p deficiency, and these plants also had significantly lower dry matter and transpiration compared to the plants in all other treatments. the authors concluded that plant mycorrhizal colonization seems to be particularly beneficial to p uptake from dry soil. wheat genome and vam infection variation in response to vam is partly governed by the difference in the genetic background of the host plant and only partly by differences in the vam genotype. bread wheat is a hexaploid species with three different genomes (a, b, d), which can be used to study genomic effects and/or interaction in response to vam inoculation through analytical breeding. furthermore, modification of the vam response during wheat evolution or domestication can be detected. the active role of the host plant and the importance of the host genotype in supporting the symbiosis and the benefits from it might be of considerable practical importance. such information is most valuable in breeding programs, especially for developing wheat cultivars adapted to marginal soils where mycorrhiza is most effective. kapuluik and kushnir (1991) reported that mycorrhizal dependency was higher in representatives of d genome donors. the nature of the response to vam in hexaploid wheat was controlled by the factors of the a and b genomes which are epistatic over those located in the d genomes. the high mycorrhizal colonization and vam dependency which was found in t. timophivee var. araraticum may indicate special genomic affinity possessed by the g genome of wheat in vam interact ion. hetrick et al. (1991) observed that there was no mycorrhizal dependence in triticum monococcum (a genome), aegiops speltoids (s (b) genome) or in ab genome ancestors triticum carthlicum, t. orientale, t. paleocoichicum and t. persicum. mycorrhizal dependency in d genome ancestors was more variable, as seen with t. tauschi var. typica which lack dependency whereas, t. tauschi var. meyeri and var. strangulata were dependent on mycorrhizal symbiosis. the spring wheat cultivars chinese spring 3008, spelta 2603, pavon 762980 and norm 293025 and the winter wheat cultivars tam 200, wrangler, saluda and karl were not dependent on mycorrhizae, whereas winter wheat cultivars tam 107 and century were dependent. therefore, it has been concluded that dependency in diploid ancestors with the a, s (b) and d genomes existed, but dependency was lost in tetraploid ancestors. hetrick et al. (1991) suggested that presence of dependency in modern wheat cultivars of abd genome is probably derived from the d genome. it is thus evident that response to mycorrhizae is considerably influenced by the host genotype especially in a facultative mycotrophic plant such as wheat. assuming that genetic control for mycorrhizae is oligogenic or polygenic, it is expected that genes governing host response are scattered over the a, b and d genomes of hexaploid wheat. disomic substitution lines of chromosomes of mycorrhiza responsive c591, an old indian wheat variety bred into the background of less responsive genotypes of chinese spring will be useful genetic material to elucidate chromosome specific response to mycorrhiza. laxminarayan et al (1993) found that there are significant differences both between a, b and d genome and between seven disomic chromosomes of each genome. they found that 3a and 3d were in general responsive to azotobacter while 4a and 4d showed responsiveness to mycorrhiza, and 7b to azospirillum in comparative evaluation of c591 disomic substitution lines. azotobacter azotobacter chroococcum, a gram negative bacterium belonging to the family azotobacteraceae of the proteobacteria is a coherent group of aerobic, free living diazotrophs able to fix atmospheric nitrogen in nitrogen free or nitrogen poor medium with organic carbon compounds as energy source. several properties of azotobacter are considered to be responsible for their beneficial effects on associated plants. these include: • the ability to produce ammonia, vitamins and growth substances which enhance seed germination (brown,1975; narula et al., 1981; narula and tauro, 1986). merbach and ruppel (1992) found that in pot experiments with soil as substrate, microbial colonization of wheat (triticum aestivum l.), resulted in higher rates of 14co 2 uptake, of 14c release by roots and of 15n uptake compared with plants in sterile cultures. • the production of iaa and other auxins, gibberellins and cytokinins (martinez-toledo et al., 1988) which help enhancing root growth and nutrient adsorption. scholz-seidel and ruppel (1992) investigated the superanatant of d5/23, a free living beneficial strain isolated from the phyllosphere of winter wheat. nonhydroxylated cytokinins (i6a.i6ade) were detected by elisa and two auxin compounds, 3indoleacetic acid and 3-indole-lactic acid were detected by hplc. • the inhibition of phytopathogenic fungi through production of antifungal substances (sharma and chahal, 1988; verma et al., 2001). • the production of siderophores (neiland, 1981; page, 1987; suneja and laxminarayana, 1993; suneja et al., 1996) which solubilizes fe+3 and suppress plant pathogens through iron deprivation. azotobacter vinelandii is also reported to have the ability to synthesize siderophores under fe deficient conditions (tindale et al., 2000). the numbers of azotobacter in soils in various parts of the world are usually below 104 cells g-1. azotobacter develops more intensively in the root zone of plants than free in soils (dey, 1973). these findings suggest that the root secretion of the plants may be providing the means of existence to azotobacter. recently, behl et al. (2006) have reviewed role of azotobacter in sustainable wheat production in semi arid tropics. therefore, only our recent work on various aspects of azotobacter and methods used by some german researchers in enterobacter radicincitans, which can be used in future research of azotobacter are briefly described. triple interactions towards plant nutrition and growth 99 manske et al. (2000) found that wheat varieties showed distinct differences in the response of grain yield to azotobacter inoculation. when grain yield was improved, p and n utilization efficiency were also enhanced, especially in cultivar wh542. in contrast, some cultivars improved their p and n uptake efficiency without transferring this advantage into higher grain yield (cultivars wh157 and cpn3004). in wh147 and hd2329, azotobacter usually led to greater effect on improved p versus n uptake efficiency. increase in uptake of plant nutrients (n,p,k) in wheat upon inoculation with azotobacter chroococcum, both under green house and field conditions, has also been reported (kumar et al., 1999; narula et al., 2000; kumar et al., 2001a,b). mrkovacki and milic (2001) elaborated that bacteria of the genus azotobacter synthesizes auxins, cytokinins and ga-like substances and these growth promoters are primary substances controlling the enhanced growth. these hormonal substances, which originate from the rhizosphere or root surface, affect the growth of the closely associated higher plants. in order to guarantee the high effectiveness of the inoculants and microbial fertilizers it is necessary to find the compatible partners, i.e. a particular plant genotype and a particular azotobacter strain that will form a good association. vasudeva et al. 2002 reported response of disomic chromosome substitution lines of wheat variety c591 to azotobacter chroococcum strains mac27 and mac37 in a pot experiment using different soil types. they observed significant differences in viable count, plant growth parameters and nutrient uptake of different disomic lines. in general, allelels for response to azotobacter chroococcum inoculation were found to be spread over all the three genomes (a, b, d). however, significant differences among different disomic lines both within a genome and over genomes were evident. due to energetic limitations the microbiological associative nitrogen fixation from the air alone is not able to cover the nitrogen demand of the plant. it is therefore necessary to find diazotrophic strains which are able to fix atmospheric nitrogen in the presence of additional sources and to excrete nitrogen into the surrounding medium. bela et al. (1996) developed deprepressed strains of azotobacter (mac, ala, msx, mal) which can fix nitrogen in the presence of nitrogen in the soil. ruppel and merbach (1995) found that the diazotrophic strains azospirillum sp and pantoea agglomerans could fix atmospheric n 2 in the presence of ammonium nitrate in nutrient broth as sole nitrogen source. further, ruppel and merbach (1997) investigated the dinitrogen fixing ability of two diazotrophic bacterial strains, pantoea agglomerans and azospirillum spp., in hydroponic experiments with wheat plants using 15n 2 incubation. enrichment of 15n was detected in plant growth media, and in roots and shoots of wheat plants grown 26 days in 15n 2 enriched atmosphere. highest 15n amounts were found in wheat shoots. thus, the form of nitrogen applied and the bacterial strain inoculated affected plant growth, by nitrogen uptake and the amount of biologically fixed dinitrogen. ammonia or nitrate supply to plants did not repress 15n 2 fixation. the agronomic and soil health benefits are higher when the inoculated bacteria are able to survive, multiply and colonize in the rhizosphere of crop species. large numbers of reports available in literature are based on viable counts in soil suspension samples from rhizospheric soils (behl et al., 2006). however, these do not furnish settlement and colonization behaviour of inoculated bacteria. this necessitate to develop methods to detect inoculated bacteria in situ and its settlement behaviour, on, into, or along plant roots and/or shoots, if bioinoculation technology is to be developed into a viable technology for sustainable agriculture. ruppel et al. (1992) investigated the ability of pantoea agglomerans to colonize various regions and tissues of the wheat plant (triticum aestivum l.) upon inoculation by using different inoculation methods and inoculum concentrations. in addition, elisa and transmission electron microscopy were used to determine the ability of bacterial cells to grow and survive both on the surface and within internal tissue of the plant and the response of the plant to bacterial infection. p. agglomerans was found to be located in roots, stems and leaves. colony developments of bacterial cells were observed in leaves and stems on the surface of epidermis, in the vicinity to stomata cells, within intercellular spaces of the mesophyll and within xylem vessels. inoculated bacterial cells were found to be able to enter host tissues, to multiply in the plant and to maintain a delicate relation between endophyte and host. recently, narula et al. (2005) and vasudeva et al. (2007) have shown formation of paranodules (endophytic) in wheat upon inoculation with phytohormone producing azotobacter chroococcum strains as well as phytohormones. kumar et al. (2007) studied establishment of thirteen strains of a. chroococcum on plant roots in relation to root exudates and development of chemotactic response in an indian wheat variety, wh711, and two varieties of cotton (diploid and tetraploid). analysis of root exudates revealed the presence of sugars and simple carbohydrates (glucose), amino acids (glutamate, lysine) and organic acids (citric acid, maleic acid, malonic acid). the bacterial strains showed preference for one root exudate over the others. two strains, ht54 and is-16, preferred wheat exudates at least two fold over cotton exudates. the other strains preferred cotton and differentiated between diploid and tetraploid species. this study revealed a high degree of adaptation between the host plant and the most abundant diazotrophs. remus et al. (2000) investigated pantoea agglomerans d5/23 for its colonization sites using an immunological detection method (double antibody sandwich enzyme linked immunosorbent assay, daselisa), migration within individuals of different plant species and root and shoot samples using electron microscopy. inoculation with p. agglomerans led to increase in grain yield of different wheat (triticum aestivum) cultivars. the same strain was also able to colonize the rhizosphere and phyllosphere of different cereals due to its ability to migrate within the plant. the authors observed that p. agglomerans colonized the root and plant-growth medium of wheat to a greater extent than those of rye (secale cereale) and barley (hordeum vulgare), whereas the colonization of shoot was higher in rye and barley compared to wheat. thus, the host plant has also a role in determining bacterial colonization behaviour. narula et al. (2007) studied colonization of a. chroococcum strain mac27 on wheat roots in terms of colonization sites, migration and survival of the bacteria. also, the affectivity of inoculation of a. chroococcum and p. agglomerans d5/ 23 strain on wheat plant parameters under green house condition was investigated. root tips had significant titres of inoculants as compared to the basal root parts. a. chroococcum colonized roots as well as soil and also migrated along roots. both a. chroococcum and p. agglomerans were found to increase plant growth, plant dry matter, as well as n and p uptake. depending upon two elements of diversity, i.e. abundance and adaptability, soil microorganisms impact many soil processes and productivity. knowledge of their interactions, roles and functions is therefore, vital to our understanding of soils and their sustainability. with the advent of recent techniques like real-time pcr it is now possible to quantify and localize specific bacterial target genes in plant tissue. a significant colonization of brassica oleracea leaves and roots with enterobacter radicincitans cells was measured (ruppel et al., 2006). the introduced bacteria proliferated on and inside the root and that they colonized the intercellular spaces of the root cortex layer. it would interesting to know more about interaction between n availability and prokaryotic diversity particularly in a well-characterized system for long-term field experiment if biologically oriented sustainable wheat production strategies are to be developed and validated. ruppel et al. (2007) measured the highest prokaryotic potential functional diversity and community composition on a loamy sandy soil without n fertilization, indicating an efficient as well as versatile utilization of the substrates in this soil. both substrate utilization richness and substrate utilization evenness, the two constituents of 100 rishi k. behl, sabine ruppel, erika kothe, neeru narula the functional diversity, decreased with increasing n supply. these differences suggest a dominance of population adapted to utilizing readily available substrates. also, prokaryotic diversity and n availability were interrelated in this sand soil system. azotobacter is the favoured bioinoculants promoted for cotton-wheat cropping systems in india. bhatia (2006) investigated 76 free-living diazotrophs isolated from cotton crop soils. all these isolates were found to be gram-negative, and morphological and further biochemical characterization also showed close resemblance of these isolates to azotobacter spp. divided the isolates into two different clusters and four sub-clusters. thus, regional specificity was observed in 16s rrna analyses, which could be due to domestication of these isolates in different agro-ecological niches. different methods based on serology, homology of amplified dna, protein and plasmid profiles have been used for strain identification. all these methods are laborious and time consuming. none of these techniques provides in situ detection in soil. antibiotic or ammonium analogue resistance (lakshminarayana et al., 2000; wilson et al., 1994) or introduced genes that can be easily monitored on a chromogenic substrate (wilson et al., 1994) have been proposed as a better way to identify strains. kumar et al. (2006) attempted to genetically tag azotobacter chroococcum strains with lacz and gfp to study the colonization behaviour on wheat (t. aestivum) and cotton (gossypium sp.) in soil under controlled conditions. 103-104 cfu g-1 soil of strain ht 57 lac z were found to colonize roots of both cotton and wheat crops whereas 1.7 x 104 7.2 x 104 cfu g-1 soil of strain e12 gfp was colonizing wheat roots and 1.6 x 104 9.3 x 104 cfu g-1 soil of e12 gfp colonized cotton roots. tagged strains were found to colonize mostly root tips in both crops. fate of inoculated strains in the rhizosphere 1. crop productivity, in general, is determined by plant genotype, agronomic inputs favourable soil/rhizosphere conditions including plant microbe interactions and is positively correlated with survival, multiplication and colonization of favourable micro-organisms in the rhizosphere. inoculation of plants with an efficient strain of a microbial inoculant might lead to fast proliferation due to root exudates or other carbon source(s) upon which bioinoculants might adapt, increase in viable counts/numbers through mutualism among the rhizosphere microorganism community, inhibition of the inoculated strain due to other microorganisms/ strains or mineralization, or through mutation generating altered efficiency or altered host preference of the inoculated strain. in order to guarantee the high effectiveness of microbial inoculants it is necessary to find the compatible partners, i.e. a particular wheat genotype and a particular azotobacter strain that will form a good association. triple interactions seed inoculation with rhizobacteria may also stimulate the infection of roots by the indigenous vam community. synergistic effects between azotobacter and glomus were reported (bagyaraj and menge, 1978; ishac et al., 1986). bagyaraj and menge (1978) recovered larger populations of bacteria (including actinomycetes) from the rhizospheres of tomato plants inoculated with the mycorrhizal fungus glomus fasciculatus and azotobacter chroococcum either individually or together. plants inoculated with both g. fasciculatus and a. chroococcum had greater numbers of bacteria and actinomycetes in the rhizosphere than plants inoculated with either g. fasciculatus and a. chroococcum alone. inoculation of tomato with g. fasciculatus increased a. chroococcum population in the rhizosphere which was maintained at a high level for a longer time. inoculation of tomato roots with a. chroococcum enhanced infection and spore production by g. fasciculatus. the dry weights of tomato plants inoculated with both g. fasciculatus and a. chroococcum were significantly (62%) greater than non-inoculated plants. behl et al. (2003) observed similar effects in wheat, and brown and carr (1984) found that dual inoculation of roots of lettuce seedlings with amf and a. chroococcum produced larger plants than either inoculum alone in the partially sterilized, p deficient soil. singh (1992) found that inoculation of n 2 fixing (azospirillum brasilense, a. lipoferum and azotobacter chroococcum) and p solubilizing (bacillus polymyxa and pseudomonas striata) bacteria enhanced root volume and percent vam root colonization of pennisetum padicillatum in the presence of glomus macrocarpum. the number of vam spores, after the inoculation, was also increased after inoculation with these two groups of bacteria. singh and kapoor (1998) found that root colonization by vam fungi in the sandy soil of low fertility was low. inoculation with amf (glomus sp. 88) improved root colonization of wheat. at maturity, grain and straw yields as well as n and p uptake improved significantly following inoculation with po 4 3--solubilizing microorganisms bacillus circulans and cladosporium herbarum or the vam fungus. these increases were higher on combined inoculation of p solubilizing microbes and the vam fungus with mussoorie rock phosphate amendment. in general, a larger population of p solubilizing microbes was maintained in the rhizosphere of wheat in treatments with vam fungal inoculation and rock phosphate amendment. diederichs and manske (1991) observed that azotobacter stimulated the mycorrhization rate in wheat roots, which, in combination with the improved total root length, resulted in an even greater enhancement of the vam infected root length. both effects, particularly the stimulated mycorrhization of the roots, may result in an improved p uptake, especially when plant available p in the soil is low. the benefits of the azotobactervam association were plant genotype dependent. plant growth promoting rhizobacteria (pgpr) in the rhizo-mycosphere may influence mycorrhizal function and biomass of the mycosymbiont. however, definite research in this area is still scare. all rhizobacterial associations improved mycorrhization of wheat roots. however, the degree of root colonization varied not only with different pgpr species but also with different isolates in some iaa producing pgprs which enhanced sporulation of vam fungi by 45%. g. fasciculatum and azotobacter chroococcum inoculation enhanced the p concentration in shoots at tillering (manske et al., 2000), although there was no effect on the shoot growth at this early stage. at the same time, mycorrhization rate in the wheat roots was higher with the dual inoculation than in the non-inoculated control. this effect was not significant with a. chroococcum alone. however, with time (at the stage of anthesis), the treatments with a. chroococcum alone or in combination with g. fasciculatum, improved the vam infected root length. this effect was not only caused by an improved total root length, but also by a significantly higher vam infection. grain yield was improved by about 5% in the ten wheat cultivars tested. zaidi and khan (2005) studied the interactive effect of rhizotrophic microorganisms on growth, yield, and nutrient uptake of wheat (triticum aestivum l.) in a pot experiment using sterilized soil deficient in available p. positive effects on plant vigor, nutrient uptake, and yield of wheat plants was recorded after a. chroococcum, phosphate solubilizing pseudomonas striata g. fasciculatum inoculation. the available p status of the soil improved significantly following the triple inoculation. wu et al. (2005) evaluated the effects of four biofertilizers containing an arbuscular mycorrhizal fungus (g. mosseae or g. intraradices) with or without n 2 -fixer (a. chroococcum), p-solubilizer (bacillus megaterium) and k-solubilizer (b. mucilaginous) on soil properties and the growth of zea mays. the application of biofertilizer containing amf and three species of bacteria significantly increased the growth of zea mays, nutritient assimilation of the plant (total n, p and k), and soil properties. the arbuscular mycorrhizal fungi had a higher root infection triple interactions towards plant nutrition and growth 101 rate in the presence of bacterial inoculation. by contrast, the amf seemed to have an inhibiting effect on the p-solubilizing bacteria. the nutrient deficiency in soil resulted in a larger population of nitrogenfixing bacteria and higher colonization of amf (wu et al., 2005). thus, the findings (bagyaraj and menge, 1978; singh, 1992; zaidi and khan, 2005; wu et al., 2005) suggest a synergistic or additive interaction between g. fasiculatus and a. chroococcum on plants, the combined inoculation with p-solubilizers and an amf along with rock phosphate amendment can improve crop yields in nutrient-deficient soils. rhizotrophic microorganisms can interact positively in promoting plant growth, as well as n and p uptake of crop plants, leading to improved yields. root exudates the associated heterotrophic rhizobacteria of the rhizoplane and rhizosphere depend on the carbohydrates exuded by the plant roots. barber and martin (1976) determined that under sterile soil conditions between 5 to 10% of the net photosynthates of wheat may be released by the roots, whereas 12 to 18% is released in the non-sterile system. the latter amount of carbon release would approximate 10 to 25% of the dry matter increments of the plants. the fact, that the presence of microorganisms can cause a plant to release up to 25% (merbach and ruppel, 1992) of its photosynthate through the root indicate that we may be able to modify or manipulate associative microflora by artificial inoculation of efficient strains, thus increas-ing the carbohydrate availability to vam since there is a close relationship between vam and rhizosphere microflora. in order to meet each others needs, host plant and amf adjust through metabolic changes. consequently, amf induced root exudation will boost microbial communities as a whole to grow in rhizosphere with beneficial effects on plant growth. thus, dual inoculation of efficient strains of azotobacter chroococcum and glomus fasiculatum in responsive wheat varieties adapted to low input stress conditions could be profitably used to maximize wheat production by harnessing favourable plant-microbe interaction. the chemotactic responses of the plant-growth-promoting rhizobacteria azotobacter chroococcum and pseudomonas fluorescens to roots of vesicular-arbuscular mycorrhizal infected (glomus fasciculatum) tomato plants were determined (sood, 2003). a significantly greater number of bacterial cells of wild strains were attracted towards vesicular-arbuscular mycorrhizal infected tomato roots compared to non-vesicular-arbuscular mycorrhizal tomato roots. substances exuded by roots served as chemo-attractants for these bacteria and also for the vam. p. fluorescens was strongly attracted towards citric and malic acids, which were predominant constituents in root exudates of tomato plants. a. chroococcum showed a stronger response towards sugars than amino acids, but the response was weakest towards organic acids. the effects of temperature, ph, and soil water matric potential on bacterial chemotaxis towards roots were also investigated. in general, significantly greater chemotactic responses of bacteria were observed at higher water matric potentials (0, -1, and -5 kpa), slightly acidic to neutral ph (6, 6.5 to 7), and at 20-30oc (depending on the bacterium) than in other environmental conditions. it is suggested that chemotaxis of p. fluorescens and a. chroococcum towards roots and their exudates is one of the several steps in the interaction process between bacteria and vesicular-arbuscular mycorrhizal roots. possible mechansims mediating wheat-vam-azotobacter interactions the synergistic or additive interactions between vam and azotobacter could be attributed to several mechanisms which are summarized below. 1. hormonal effects a. plant growth azotobacter is one of the microorganisms which are able to synthesize large amounts of biologically active substances. it has been reported that azotobacter chroococcum produces plant growth regulators in n-free media. it is known that the beneficial effects on plant growth that result from inoculation with azotobacter are caused by these growth regulators, besides its main function of n 2 -fixation (brown, 1974). several metabolites are contained in cell free supernatants of azotobacter cultures including auxins, gibberellins and cytokinins (azcon and barea, 1975; martinez-toledo et al., 1988). auxins among several other activities, control root formation and relax the cell wall, gibberellins increase leaf and root growth (paleg and west, 1972) and cytokinins are involved in many basic processes of plant growth. all these activities could additionally be expected to influence the formation and function of mycorrhizae. two possible effects were suggested by carr (1981) for the stimulating effect of azotobacter on mycorrhiza: if absorbed into the roots, it can directly enhance of the metabolic activity of the mycorrhiza, or the increasing leaf size could enhance the potential for photosynthesizing nutrient supplies for the endophytes within the plants. tsavkelova et al. (2006) reviewed the microbial producers of plant growth regulators and their practical use. phytohormones are viewed as specific mediators in interaction between various organisms inhabiting the same ecological niche, the biological role of which is not limited to processes taking place in plants. however, specific compounds leading to such changes are yet to be identified. b. enhanced nutrients uptake this effect may be related to hormone production by azotobacter. these substances are known to increase the number of root hairs, lateral roots and root mass. hence, the absorptive capacity of the root system for nutrients is expanded directly due to enhanced root growth or indirectly due to the presence of more sites created for vam development. these effects may explain the significant increase in nutrient uptake by plants dually inoculated with azotobacter and vam compared with those singly inoculated with vam. 2. n 2 -fixation n 2 -fixation would be expected to be the principal mechanism by which azotobacter interacts with amf. the possible affinity of azotobacter for vam may be mainly based on phosphate availability. vam enhances phosphate availability (gerdman, 1968) and p deficiency is known to limit n 2 -fixation. amf may supply azotobacter with an adequate phosphate source in exchange for nitrogen. however, results of n 2 -flxation in plants dually inoculated with azotobacter and vam did not show conclusive information. the functional relationship between plant-vam-rhizobacteria is complex and many questions are still open (bonfante, 2003). the effects of triple interactions vary due to genetic variability in each partner and their interaction for nutrient and water use and induced disease resistance, signal molecules produced by and receptors in each partner are not well understood to characterize triple interactions. understanding of these signaling pathways and symbioses will lead to the development of mixed inocula for a given genotype of wheat and thus open new avenues for the practical use to harness the potential of bioinoculants for sustainable crop production genotype dependent response of bioinoculants genetic variability exists among wheat genotypes for acquisition and assimilation of mineral nutrients (dambroth and el-bassam, 1983; 102 rishi k. behl, sabine ruppel, erika kothe, neeru narula manske et al., 2000). egle et al. (1999) compared the p efficiency of three newly developed genotypes of wheat chilwuh, baukauz and pgoseri with an old variety curinda in the field at two p levels. all four genotypes responded to p application, however, the new varieties were classified as p efficient due to their significantly higher yields at both levels of p as compared to the old variety. this high level of p efficiency is mainly due to more effective p uptake. manske et al. (2001) concluded, from a set of semi dwarf spring wheat (t. aestivum) genotypes evaluated in acid and calcareous soils without and with p fertilization, that for combining improved p uptake and p utilization efficiency in the same genotypes, simultaneous selection under both high and low p conditions is required. manske et al. (2002) studied impact of rht dwarfing genes on utilization efficiency, total p uptake and related traits in the varietal backgrounds of two tall wheat cultivars, maringa and nainari 60 and sets of near-isogenic lines carrying different combinations of alleles rht-b1b, rht-d1b rht-b1c under conditions of adequate nutrient supply and irrigation. dwarfing genes rht-d1b and rht-b1b led to improved p uptake in maringa and p transfer in maringa and nainari60. the rht-b1c genotypes showed low p uptake, thick roots and high p concentration in vegetative biomass. gill et al. (2004) classified 30 wheat varieties by metroglyph analysis into eight different groups on the basis of their grain yield performance and p uptake which proved useful in identifying varieties suitable for cultivation in different soil p regimes and selection of parents for recombination breeding to develop p efficient cultivars. according to this study, varieties wh711 and pbw343 with a combination of high grain yield and high p uptake will suit the high p fertility soils, whereas, raj3765 and wh283 with high grain yield (5348 kg/ha) despite low p uptake would prove better for low p regimes as both of them recorded high index score. inter-mating between varieties belonging to hgy-hp (pbw343 and wh711) and hgy-lp (raj3765 and wh283) would further expand genotypic variability and thus frequency of recombinants exhibiting different grain yield and p uptake levels. this would be quite helpful to develop p efficient cultivars. however, the effect of host genotypes on amf infection and root characters and its interaction with host and co-inoculation with azotobacter have not been adequately investigated. although genotypic differences for amf infection in indian wheat genotypes under different growing conditions have been reported (manske et al., 2000), information on whether such differences are heritable, as reflected in cross combinations is not available. can this approach be effective in breeding wheat genotypes with high amf infection and better root systems? in order to address this question, two sets of field experiments were conducted to determine the effects of plant genotype and azotobacter survival on amf infection in some wheat crosses involving ecogeographically and genetically divergent parents grown under low mineral nutrient input conditions (for first set: sharma et al., 2001; behl et al., 2002; behl et al., 2003; sharma et al., 2006; for second experimental setup: behl et al., 1999; singh et al., 2004; singh et al. 2007a; singh et al. 2007b). viable counts of inoculated azotobacter were determined from rhizospheric soil (after bela et al., 1986) and amf infection in roots (after phillips and hayman, 1970; jalali and domsch, 1975), in addition to measuring total root length (tennant, 1975). root and plant traits inoculation of amf and amf + azc led to increase in peduncle length, flag leaf area, number of grains spike-1, grain weight, biological yield and grain yield per plant (fig. 1). various wheat varieties and crosses showed different responses to inoculation with amf and amf + azotobacter. among the parents, maximum response to inoculation with amf + azotobacter was evident flag leaf area, number of grains spike-1, grain yield and biological yield in wh147. in crosses, this parent contributed towards higher magnitude for these traits. varietal response was found to be heritable. co-inoculation of amf with azotobacter led to an increase in the viable count of azotobacter in the wheat rhizosphere. amf and azotobacter complement each other and result in improved plant growth. maximum azotobacter counts were found 80 days after sowing (5.4 x 106) in amf + azotobacter treatments of cross wh147 x pbw175 (335 x 104), which may have been due to more root exudates containing amino acids, carbohydrates, organic acids and growth promoting substances (vancura and harizlikuva, 1972; leinhos and vacek, 1994), supporting better plant-amf-microbe interaction and nutrient mobilization efficiency of fungal (hetrick et al., 1992) and bacterial symbionts. the bacterial population increased at faster rates in the rhizosphere of each cross when amf was co-inoculated. it is well known that wheat roots secrete carbonaceous exudates, which could help in proliferation of amf and azotobacter (manske et al., 2000). varietal differences for root exudates thus, may be responsible for differences in viable counts of azotobacter as observed. behl et al. (2003) observed that wheat genotypes differed from each other for root biomass, total root length and amf infection of roots. pbw175 followed by wh542 had maximum root biomass, root length and amf infection in roots on the basis of two years average over all the treatments (tab. 1). inoculation of azotobacter chroococcum along with amf had complementary effects on amf infection particularly in pbw 175. a comparison of the average over two years for parents and hybrids indicated that wh147 had the lowest magnitude and pbw175, on the contrary, the highest magnitude for all root traits (tab. 1). in cross combination, pbw175 showed overdominance for root traits. thus, the potential for higher root biomass, total root length and amf infection of roots for pbw 175 appeared to be heritable. although amf infection of roots in amf treatments was more or less similar for all crosses, inoculation of amf + azotobacter had better effects on amf in wh147 x pbw175 and wh147 x wh157.wheat crosses responded differently to amf treatment as maximum increases were noted for root biomass in cross wh147 x wh157, total root length in cross wh147 x pbw175, while f 1 crosses were similar to each other for amf infection of roots. in dual inoculation, total root length increased in each of the three crosses with a maximum being noted for wh147 x pbw175. micro and macro nutrient uptake singh et al. (2004) showed best effects of dual inoculation with amf (g. manihotis) and rhizobacteria (a. chroococcum) on biomass, vam fig. 1: effect of bioinoculants on grain yield (g) in wheat crosses 0 5 10 15 20 25 wh 147 x wh 157 wh 147 x pbw 175 wh 147 x wh 542 wheat crosses control amf amf + azc y ie ld ( g ) triple interactions towards plant nutrition and growth 103 infection in roots and nutrient content in three spring wheat varieties and their f 1 hybrids under low ph soil (ph 3.8). hybrid wh533 x raj3077 exhibited higher vam infection in roots (35.14%), p content (670ppm), fe, cu, mn and zn while wh147 x r3077 produced higher plant biomass (3.00 g plant-1) and had higher n content and uptake. regarding micronutrients, dual inoculation of vam and azotobacter increased the cu and zn contents in shoots, while vam inoculation produced better results for fe and mn content in the shoots (singh et al., 2007a). the cross wh533 x raj3077 recorded high values for the micronutrients fe (287 ppm) and mn (55.0 ppm) under vam and zn (29.43 ppm) under dual inoculation. for cu content, wh533 (15.6 ppm) among parents and wh147 x raj3077 (14.80 ppm) among hybrids were best. cross wh533 x raj3077 exhibited heterotic effects (increased performance of f 1 ) for fe contents under vam and dual inoculation and for cu and mn contents under vam influence. vam infection in roots showed significant positive associations with nitrogen and zn content under azotobacter inoculation and with p, fe, cu and mn contents were the best under vam inoculation. under dual inoculation, vam infection in roots exhibited significant positive correlation with p content (r=0.65), p uptake (r=0.53), fe (r=0.85), cu (r=0.36) and zn (r=0.64) content. gene effects experiments involving diverse wheat genotypes differing for their adaptation to various agronomic and ecological conditions revealed genotype dependent responses to the inoculation of azotobacter and amf individually, as well as in combination. therefore, it was imperative to work out gene effects controlling response of inoculants for different morpho-physiological plant and root traits involved in yield building and nutrient uptake. sharma et al. (2007) found that bio-inoculants enhanced the mean performance of most characters. crop season also showed considerable effect on impact of bio-inoculants. the joint scaling test (cavalli, 1952) revealed adequacy of additive-dominance model of gene effects (jinks and jones, 1958) for root biomass, root length density, flag leaf area, tillers/plant, grain weight and grain yield in all the crosses and bio-inoculants treatments in two years. the amf treatment brought about changes in the magnitude and significance of additive component for root biomass, plant height, flag leaf area in all the three crosses. both additive (d) and dominance (h) components were affected with respect to grain yield in wh147xwh157 and wh147xwh542. the dominant component was important for tillers/plant, grain yield, root length in control as well as bio-inoculant treated populations of wh147xpbw175, but treatment of amf and amf + azotobacter reduced the magnitude of h and increased the magnitude of d. digenic interactions were prominent for grains/spike in wh147xwh157. magnitude of digenic interactions was higher under bio-inoculation. simple pedigree and bulk pedigree methods are suggested to capitalize on adequate additive gene effects for developing bio-inoculants responsive wheat genotypes. dual inoculation of efficient strains of a. chroococcum and g. fasiculatum in responsive wheat genotypes adapted to low input stress conditions might be profitably used to maximize wheat production. however, intensely amf infected roots even at moderate nutrient deficiency are important during early plant growth when roots are too small to provide high demand for minerals for shoot growth. selection of rapid amf colonization in wheat may lead to improved total root length and root biomass. wh147 x pbw175 can be used for breeding pure lines in wheat for sustainable agriculture (low input genotypes responsive to biofertilizers like amf and azotobacter). likewise, selection in cross wh147 x wh157 is expected to be fruitful to develop bio-inoculant responsive genotypes for moderate soil salinity conditions. on the other hand, cross wh147 x wh542 would yield potent recombinants for high input conditions. in any eventuality, such genotypes will be efficient to make better use of applied nutrients and thus will be suitable for sustainable wheat production under diverse agro-ecological conditions. also, such genotypes would hold promise for organic regimes as well. singh et al. (2004) evaluated the impact of bio-inoculants in low input field conditions on magnitude and direction of gene effects and mean performance of some morphological and productivity traits. the application of bioinoculants influenced genetic effects like for days to flowering, days to maturity, flag leaf area, spike length, grains/ spike, 1000 grain weight and harvest index where complex genetic interactions were changed to simple additive-dominance gene effects in the cross wh147 x raj3077. considerable magnitude of additive and additive x additive gene effects for plant height, days to flowering, grains per spike and grain weight suggested that simple pedigree selection would be effective to develop wheat genotypes which are responsive to dual inoculation of amf and azotobacter. in this context, selection of potent recombinants in crosses involving wheat variety wh147 suitable for medium fertility and water deficient conditions holds great promise to develop bio-inoculants responsive high yielding genotypes for stress prone, low input conditions. the impact of bio-inoculants on the magnitude and direction of gene effects was assessed for and mean performance for root length density, root biomass per plant, amf colonization in roots and micronutrients uptake (cu, fe, mn, zn) in wheat under low input field conditions (singh et al., 2007a). root length density, root biomass per plant, tab. 1: effect of inoculation on root biomass, root length density and amf infection in wheat genotype root biomass (g) root length density (m) am infection in roots (%) c amf azc + mean c amf azc + mean c amf azc+ mean amf amf amf parents wh147 0.74 1.09 1.20 1.00 3.60 4.07 4.52 4.06 33.1 39.3 47.5 40.0 wh157 0.63 0.95 1.15 0.90 2.24 4.12 3.75 3.37 36.3 42.5 50.4 43.0 pbw175 1.23 1.55 1.61 1.48 3.70 4.91 6.58 5.06 50.6 60.6 63.1 58.1 wh542 1.01 1.32 1.83 1.38 3.6 4.79 6.19 4.85 37.6 47.3 56.5 47.1 crosses wh147xwh157 0.77 1.30 1.26 1.11 2.97 3.93 5.69 4.19 30.2 44.0 47.1 40.4 wh147x pbw175 1.05 1.08 1.37 1.16 3.66 5.04 5.66 4.78 36.6 45.2 53.3 45.0 wh147x wh542 0.83 1.02 1.02 0.95 2.95 3.56 4.63 3.71 33.4 45.7 52.8 43.9 sd 0.21 0.21 0.28 0.22 0.59 0.56 1.01 0.58 6.6 6.8 5.6 6.1 data given represent mean values of two years. c= control, amf = arbuscular mycorrhizal fungi, azc= azotobacter 104 rishi k. behl, sabine ruppel, erika kothe, neeru narula amf colonization in roots and zn and mn content were found maximum under dual inoculation of amf + azotobacter in all the three crosses. the joint scaling tests revealed that additive-dominance gene effects were mainly operative in governing expression of root biomass, cu and zn content in all the three crosses under the three treatments (i.e. control, amf and amf + azotobacter). singh et al. (2007b) also analyzed the data from second set of our experiments to know the impact of bio-inoculants in low input field conditions on the magnitude and direction of gene effects and mean performance of n and p use in wheat. bio-inoculation of amf and amf + azotobacter exhibited positive impact on mean performance of all the wheat crosses. the mean performance of amf was maximum in the cross wh147 x wh533 for n and p response (%), n and p use index (%) and p content (ppm), whereas, for n and p uptake it was maximum in the cross wh147 x raj3077. the n and p response and their use index were better when combined treatment of amf + azotobacter was given in all the three crosses. adequacy of additive-dominance model for p uptake (mg/plant) in all the three crosses under all the three treatments (i.e. control, amf, amf + azotobacter) suggested that additive (d) and dominance (h) gene effects mainly governed inheritance of this trait. in all the cases, digenic interactions were present where duplicate type of epistasis prevailed except in p content for the control in the cross wh147 x wh533, where complementary type of interaction was present. from both analyses, it appears that pedigree selection in crosses wh147 x wh533 and wh147 x raj3077 can be effective for breeding pure lines in wheat combining high yield and nutrient use efficiency (low input genotypes responsive to biofertilizers like amf and azotobacter)for sustainable agriculture). candidate genotypes for breeding of improved associative cultivars tall wheat varieties carry the rht gene. such varieties produce low endogenous hormone. supply of hormone in any form eventually leads to increase in root and shoot lengths and consequently root and shoot biomasses (gale and marshall, 1975). pbw 175 is a tall type genotype having an efficient root system (sangwan et al., 1999) tailored to extract more water from deeper soil profiles under stress conditions. in one of our earlier studies, amf infection was observed to be higher under dryness and nutrient stress conditions (manske et al., 1995) in wheat genotypes tolerant to abiotic stresses (manske et al., 1999). in general, the capability of rhizobacteria to produce plant growth regulating substances of the auxin type may contribute to their stimulating effects on plant growth (sattar and gaur, 1987; leinhos and vacek, 1994). the phytohormone indole acetic acid (iaa) affects root elongation and lateral root formation (hirsch et al., 1997). plant root systems are complex and are comprised of several types of structural and functional characteristic (renegal, 1997). efficient water and mineral uptake is closely related to roots and their growth (kapulnik et al., 1985). the stimulation of root biomass and total root length for plants grown in dual inoculation, as observed in our studies, may be the result of cumulative effects of favorable plantamf-microbe interactions. total root length (m) was maximum in wh147 x pbw175 control treatments, and increased in each cross with amf and amf + azotobacter treatments, being lowest in wh147 x wh542. this could be possible as plants may have had fibrous and plastic roots (hetrick et al., 1991). the bacterial population increased at faster rates in the rhizosphere of each cross when amf was co-inoculated. it is well known fact that wheat roots secrete carbonaceous exudates, which could help in proliferation of amf and azotobacter (manske et al., 1999). azotobacter excretes phytohormones, which improves growth of plant roots, and amf may solubilize p from surrounding areas and make it available to roots. dual inoculation of efficient strains of azotobacter chroococcum and glomus fasiculatum in responsive wheat genotypes adapted to low input stress conditions would be profitably used to maximize wheat production. however, intensely amf infected roots even at moderate nutrient deficiency are important during early plant growth when roots are too small to provide the high demand for minerals for shoot growth. selection of rapid amf colonization in wheat may lead to improved total root length and root biomass. wh147 x pbw175 can be used for breeding pure lines in wheat for sustainable agriculture (low input genotypes responsive to biofertilizers like amf and azotobacter). breeding strategies mycorrhizal dependence is a genetic trait that is strong in some wheat genotypes and absent in other (hetrick et al., 1992). mycorrhizal dependence is an index of plant response at particular p level (plenchette et al., 1983). only relative relationship between cultivars can be inferred from this index. the p level in the soil was relatively low; therefore, both the degree of growth and the number of cultivars responding to mycorrhizae would decline with increasing p level in the soil. the significant correlation between root fibrousness and mycorrhizal dependence suggests that the use of a morphological trait such as root architecture is probably a more reliable predictor of plant benefit from the symbiosis than the colonization by the fungal symbiont. using low fertility soil and fungal symbiont infection of wheat, hetric et al. (1992) suggested that old hexaploid wheat, landraces and older cultivars are more consistent and highly responsive to the symbiosis than modern wheat cultivars. the germplasm selection under fertilized condition could have reduced the frequency of genes for mycorrhizal dependence in wheat as under high fertility conditions vam infection is less and wheat genes responsive to vam may not be expressed. symbiosis creates unique problems because the phenotype is the product of an intimate association between different organisms. as a result, the development of the symbiotic phenotype can be influenced by genes which affect each organism’s independent existence as well as those which determine their joint association. such factors have the potential to produce levels of complexity between genotypes and environments that are reflected in the phenotype which are not easily unraveled and thus not easily stabilized. it is, perhaps, important to emphasize at this point that the agronomic objective is the phenotype rather than the genotype. inferences about genotype are made from phenotype and assessment of the strength and stability of this relationship is of central importance to the speed and precision with which genetic patterns can be altered in populations. thus, the first tasks in any breeding program are to identity and quantify phenotypic variations for relevant traits. often environment and genotype x environment interaction influence phenotypic expression and create bias to unknown extent. under such situation selection is jeopardized. such a situation warrants use of dna markers which are ubiquitous and independent of environment. however, such dna markers with universal application are yet to be identified. if the gene effects for vam-wheat symbiosis are additive or additive x additive simple selection of wheat lines will be effective. however, non-additive gene effects are the basis for poor heritability, intense selection and specific combining ability in such cases are important to breed for vam-azotobacter responsive wheat genotypes. however, the available literature points to the possibilities that wheat response to bio-inoculants being heritable and genotype dependent, targeted and efficient breeding of wheat genotypes for biologically oriented land use system is possible using a combination of conventional and molecular approaches. outlook soil is a habitat for a vast, complex and interactive community of soil organisms whose activities largely determine the chemical and physical triple interactions towards plant nutrition and growth 105 properties of the soil. soil micro flora plays an important role in the maintenance of soil fertility because of their ability to carry out biochemical transformations and also due to their importance as a source and sink for mineral nutrients. trophic interactions between plants, soil microflora control patterns and rates of organic matter decomposition, nutrient cycling, mobilization, and nutrient uptake by plants. soil structure and porosity are much influenced by the activities of soil organisms, especially moisture, temperature, and aeration. in turn, soil structure and porosity determine community structure and regulate soil fertility. microorganisms also influence the crop productivity through control of insect pests and diseases in sustainable development of agriculture (lynch and bragg, 1985; barea et al., 2005). under the present day energy crisis there is an urgent need to improve the potential benefits of bioinoculants like azotobacter and vam for sustainable and enhanced wheat production for food security. pgpr have the potential to improve plant health and productivity and they are particularly suitable for low input sustainable agriculture applications, such as biocontrol. inoculants bacteria are often applied in seed coatings. after sowing, the inoculants bacteria must be able to establish themselves in the rhizosphere at population densities sufficient to produce a beneficial effect. therefore, efficient inoculants bacteria should survive in the rhizosphere, make use of nutrients exuded by the plant root, proliferate, be able to efficiently colonize the entire root system and be able to compete with indigenous microorganisms. inadequate bio-control in the field experiments has often been correlated to poor root colonization. identification of the genes and traits involved in the processes of inoculation and root colonization will give a more detailed insight into plant-bacterial interactions and lead to more efficient application of inoculant strains. the survival and competitive ability and performance of the improved bioinoculant strains must be improved for different agro-ecological niches. plant species can define the bacterial communities present in the rhizosphere via the selection of genetically diverse populations or by favouring specific dominant groups. however, despite the acceptance that plant extracellular signals influence microbial populations in the rhizosphere, the most microorganisms residing there, little is known about the genes involved and roles they may play in plant-microbe interactions. traditional molecular techniques, such as transposon mutagenesis, provide researchers with important information about the production and regulation of biocontrol determinants. modern techniques now allow molecular studies of pgpr not just in isolation but as a part of the complex interacting communities that occur in rhizosphere (franks et al., 2006). use of pcr based molecular markers, as in case of enterobacter radicincitans by some german researchers, should be made to unravel facts involved in genotypemicrobe interaction in specific agro-ecological conditions to harness favourable interactions for better understanding and application. yet our understanding of the links between microbial diversity and soil/ rhizosphere functions remains poor, we have still to discover an easy and comprehensive methods for measuring functional microbial diversity among individual community members. in addition, current measurements of microbial function determine only the overall rate of an entire metabolic process and are still unable to identify directly the microbial species involved. the characterization of novel genes involved in plant-microbe interaction is vital for understanding the response of rhizospheric bacterial populations to plant signals at a molecular level. by incorporating techniques such as in vivo expression technology with recent advances in transcriptome profiling, researchers can search for genes induced or repressed by plant signals. psuedomonas fluorescens genes that are specifically expressed in the rhizosphere (rhi genes) have been identified using ivet (rainy, 1999). more than 20 genes have been identified, of which 14 showed significant homology to genes that are involved in nutrient acquisition, stress response or secretion (bloemberg and lugtenberg, 2001). our understanding of the rhizospheric function of these genes can be furthered by application of proteomics, metabolomics and metagenomics, while marker gene technology can provide spatial and temporal information on specific gene expression. to elucidate the role and function of pgpr in the rhizosphere, a number of complementary techniques may therefore, be used to generate information and further overall bacteria-plant interactions. appropriate management practices and compatible wheat genotypes need to be provided to ensure maximum contribution from bioinoculants. host controlled factors play an important role in regulating responses of bio-inoculants but not have received due share of attention by researchers. in coming years, transgenics will occupy larger areas under agriculture world over. targeted genetic traits for improved plant nutrition include greater plant tolerance to low fe available in alkaline soils, enhanced acquisition of soil inorganic and organic p and increased assimilation of soil n (motavalli et al., 2004). possible changes in soil microbial activity due to differences in the amount and composition of root exudates, changes in microbial functions and alterations in microbial populations resulting from gene transfer from the transgenic crops and the effects management practices for transgenic crops, respectively would warrant evaluations of the impact of the diverse transgenic crops to an improved understanding of soil biological functions and processes. genotypic variation exists for vam infection and azotobacter response and the expression of variability is better under low input condition. hence, it is possible to select and breed wheat genotypes under low input conditions. many plant genes might be involved (quantitative trait loci, qtls) and can be identified for their functions in host response to bio-inoculants. pyramiding of such qtls, mostly available in traditional varieties, local land races, should be done in the background of agronomically superior genotypes for high and low input systems. therefore, more efforts should be done in this direction which will certainly prove effective in the advancement of wheat improvement programme for biologically oriented land use system. transcriptomic studies related to plant-fungus combinations in different natural field conditions make important contributions to understand the ecological context of the symbiosis (franken, 2006). besides roots, it would be imperative to study the photosynthetic part of the plant as well as the developing fruits in any transcriptome analysis as has been performed in one case for leaves of tomato plants (taylor and harrier, 2003). barea et al. (2005) described microbial cooperation in the rhizosphere. they highlighted that soil microbial populations are immersed in a frame-work of interactions known to affect plant fitness and soil quality. they are involved in fundamental activities that ensure the stability and productivity of both agricultural systems and natural ecosystems. strategic and applied research has demonstrated that certain co-operative microbial activities can be exploited, as a low-input biotechnology to help sustainable, environmentally-friendly, agro-technological practices. plant growth stimulating rhizobacteria can be propagated in vitro in huge quantities as pure cultures in biofermenters at low cost. diazotrophs, particularly azotobacter chroococcum are increasingly being used as bioinoculants for wheat and other crops to increase crop productivity (narula et al., 2005) in india and elsewhere. in india, azotobacter is being marketed with success over past two decades and intensive research and development efforts are being made in public and private sector institutions. however, there is still need to optimise the efficiency of these products. the discovery of many traits and genes that are involved in the beneficial effects of pgprs has resulted in a better understanding of the performance bio-inoculants in the field, and provides the opportunity to enhance the beneficial effects of pgpr strains by genetic modification (bloemberg and lugtenberg, 2001). some strong correlations between the presence 106 rishi k. behl, sabine ruppel, erika kothe, neeru narula of an organism with microbial processes or soil structure and function would be helpful in making wise choices prior to making large investments in such sequencing projects (kent and triplett, 2002). as a result, linkage of specific organisms to ecosystem function over time and space is fundamental work that must go forward. genetic tagging of azotobacter chroococcum with lac z and gfp markers as reported by kumar et al. (2007) might be useful in to study colonization behaviour and ecological studies besides other molecular methods. mutualism among azotobacter strains and vam species and wheat host genotype must be studied both in vitro and in vivo so as to find best combination for different environmental conditions. research should be intensified to establish diversity in azotobacter spp. and vam from different wheat growing localities, an effective associative/ endophyte system in wheat by identifying effective bio-inoculants strains and plant genotypes so as to identify ideal plant genotypemicrobial inoculants partners. in order to harness synergy among wheat, va mycorrhiza and azotobacter, following aspects deserve attention for future work: (i) identify biological markers for interaction between azotobacter and am fungi and /or psb to select the most compatible combinations for wheat plant inoculation, (ii) develop azotobacter strains that can provide more nitrogen to plants/vam through nitrogen fixation or excretion of ammonia without compromising survival and colonization in rhizosphere under diverse ecological conditions, (iii) identify physiological features of bacterium that have role in microbe-microbe-plant association, (iv) study role and regulation(gene expression) of phytohormones like gibberellins, kinetins and cytokinins and particularly iaa pathway in wheat-vam-azotobacter interaction, (v) study role of root exudation and exudates in triggering and silencing bacterial genes in relation to their impact on processes like chemotaxis, survival and colonization determining plant bacterial-fungal interactions, and (vi) use of microarrays and proteomics to elucidate cross communication among wheat roots-vam and azotobacter in the rhizosphere. likewise, the identification of genes involved in the yield and grain quality in response to inoculation with mycorrhiza and and azotobacter as well as use of such gene as functional markers in wheat breeding or as elements of constructs for transgenic plants with improved traits may be quite useful. references abbott, l.k., robon, a.d., 1985: formation of external hyphae in soil by four species of vesicular-arbuscular mycorrhizal fungi. new phytol. 99, 245-255. allen, m.f., boosalis, m.g., 1983: effects of two species of va mycorrhizal fungi on drought tolerance of winter wheat. new phytol. 93, 67-76. azcon, r., barea, j.m., 1975: synthesis of auxins, gibberellins and cytokinins by azotobacter vinelandii and a. beyerinckii related effects produced on tomato plants. plant and soil 43, 609-619. azcon, r., ocampo, j.a., 1981: factors affecting the vesicular-arbuscular infection and mycorrhizal dependence of thirteen wheat cultivars. new phytol. 87, 677-685. bagyaraj, d.j., menge, j.a., 1978: interaction between va mycorrhiza and azotobacter and their effects on the rhizosphere microflora and plant growth. new phytol. 80, 567-573. barber, d.a., martin, j.k., 1976: the release of organic substances by cereal roots into soil new phytol. 76, 69-80. barea, j.m., 1991: vesicular-arbuscular mycorrhiza as modifiers of soil fertility. adv. soil sci. 15, 1-40. barea, j.m., azcon-aguilar, c., 1983: mycorrhizas and their significance in nodulating nitrogen fixing plants. adv. agron. 3, 1-54. barea, j.m., pozo, m.j., azcon, r., azcon-aguilar, c., 2005: microbial cooperation in the rhizosphere. j. exp. bot. 56, 1761-1778. behl, r.k., sharma, h., kumar, v., narula, n., 2003: interaction between mycorrhiza, azotobacter chroococcum and root characteristics of wheat varieties. j. agron. crop sci. 89, 151-155. behl, r.k., narula, n., vasudeva, m., sato, a., shinano, t., osaki, m., 2006: harnessing wheat genotype x azotobacter strain interactions for sustainable wheat production in semi arid tropics. tropics 15, 121-133. behl, r.k., sharma, h., kumar, v., singh, k.p., 2002: effect of dual inoculation of va mycorrhizae and azotobacter on flag leaf characters in wheat. arch. acker. pfl. boden 48, 1-7. behl, r.k., singh, r., moawd, a., vlek, p.l.g., 1999: response of wheat varieties and their crosses to dual inoculation of va mycorrhiza and azotobacter. ufz bericht, int. symp. june 3-5, 1999, 273-275. bela, s., dahiya, p., lakshminaryana, k., 1986: isolation and characterization of mutants of azotobacter chroococcum derepressed for nitrogenase. in: singh, r., nainawatee, h.s., sawhney, s.k. 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(eds.), plant microbe interactions in sustainable agriculture, 62-88. proceedings of the seminar on natural resource management, ccs haryana agricultural university, max mueller bhawan, new delhi. wani, s.p., chandrapalaih, s., zambre, m.a., lee, k.k., 1988: association between n 2 fixing bacteria and pearl millet plants: responses, mechanisms and persistence. plant and soil 110, 289-302. weber, e., saxena, m.c., george, e., marschner, h., 1993: effect of vesicular-arbuscular mycorrhiza on vegetative growth and harvest index of chickpea grown in northern syria. field crop res. 32, 115-128. wilson, k.j., sessitsch, a., akkermans, a.d.l., 1994: molecular markers as tools to study the ecology of microorganisms. in: ritz, k., dighton, j., giller, ke. (eds.), beyond the biomass, compositional and functional analysis of soil microbial communities, 149-196. john wiley, chichester. wu, s.c., cao, z.h., li, z.g., cheung, k.c., wong, m.h., 2005: effects of biofertilizer containing n-fixer, p and k solubilizers and am fungi on maize growth: a greenhouse trial. geoderma125, 155-166. yao, q., li, x., christie, p., 2001: factors affecting arbuscular mycorrhizal dependency of wheat genotypes with different phosphorus efficiencies. j. plant nutr. 24, 1409-1419. young, j.l., davis, e.a., rose, s.l., 1985: endomycorrhizal fungi in breeder wheat and triticale cultivars field-grown on fertile soil. agron. j. 77, 219224. zaidi, a., khan, s., 2005: interactive effect of rhizotrophic microorganisms on growth, yield, and nutrient uptake of wheat. j. plant nutr. 28, 20792092. address of the corresponding author: r. k. behl, department of plant breeding, ccs haryana agricultural university, hisar-125004, india. email: rkbehl@hau.ernet.in << 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/pagesize [907.087 680.315] >> setpagedevice art07 journal of applied botany and food quality 82, 35 40 (2008) institute of agricultural and nutritional sciences, professorship plant nutrition, martin-luther university halle-wittenberg, halle (saale), germany phytohormonal effects on rhizosphere processes of maize (zea mays l.) under phosphorus deficiency l. wittenmayer, a. deubel, w. merbach (received november 13, 2007) summary effects of the hormones indole-3-acetic acid (iaa), gibberellic acid (ga3), and trans-zeatin (t-z) on growth, p status and rhizosphere processes of maize (zea mays l., cv. 'bezemara') were investigated in a pot experiment at two levels of phosphorus availability (+p: water soluble phosphate and -p: sparingly soluble tricalcium phosphate). six weeks after seed germination, plants were harvested and analysed for dry weight, shoot length, root surface, p concentration, acid phosphatases activity (acid pase) in shoot and rhizosphere and the content of carboxylic acids and sugars in the rhizosphere. anova was used to estimate the effects of treatments on measured parameters. hormone application via rhizosphere had a highly significant effect on the growth of whole plants, their p status and rhizosphere processes. ga3 and t-z promoted quantitatively shoot and root growth and morphological changes, whereas iaa affected the chemical composition of the rhizosphere. in several parameters, the effects of hormone treatment depended on the p status of plants indicating different sensitivity of +p and -p plants to plant growth regulator (pgr) application (significant interaction of hormone application × p availability). the findings help to improve our knowledge, why pgr treatments and plant growth promoting rhizo-microorganisms have varying effects on plants depending on growth conditions. abbreviations acid pase: acid phosphatases activity, iaa: indole-3-acetic acid, ga3: gibberellic acid, pgr: plant growth regulator, t-z: trans-zeatin introduction the plant growth promoting effects of rhizo-microorganisms include the production of a vast range of metabolites (biologically active substances) that may affect plant growth directly after being taken up by the plant, or indirectly by modifying the soil environment (walker et al., 2003). phytohormones like auxins (indole-3-acetic acid, iaa), gibberellins (gibberellic acid, ga3) or cytokinins (transzeatin, t-z) belong to this group of substances (frankenberger and arshad, 1995). it is well documented that cultures of several plant growth promoting rhizo-microorganisms (pgpr) possess a great potential to produce these phytohormones in considerable amounts (martinez-toledo et al., 1988; scholz-seidel and ruppel, 1992; gyaneshwar et al., 2002) and, thus, it is assumed that they might support plants in responding to environmental stress (itai, 1999). unfortunately, a direct link between microbial capacity for hormone formation and the plant growth promoting effects of hormones in the rhizosphere is still missing. it is well known that hormonal effects depend on time of application (developmental stage of plant) and its duration (arteca, 1996). sensitivity of plant tissue to pgr also plays an important role (trevawas, 1981). there are very few experiments with applications of plant growth regulators to the rhizosphere of intact plants. leinhos and bergmann (1995) and lippmann et al. (1995) conducted experiments with a single application of iaa to maize plants. due to its fast metabolisation in the presence of maize roots within a few hours (crozier et al., 1988), the hormonal effect of a single application in the rhizosphere is temporarily limited and hardly comparable with microbial, continuous hormone formation at the root surface. in order to understand the microbial effect of hormone formation in soil on plant growth, observations on a long-term scale are necessary, i.e. the pgr effect should exceed its half-life time substantially and correspond with the timescale of plant development. thus, the hormonal effects have to be investigated within days or weeks rather than within a few hours. stress conditions are a good background for elucidating plant growth promoting effects and their mechanisms. nutrient deficiency is a common and very important abiotic stress factor. particularly, mechanisms of plant response to p starvation are being intensively investigated (schilling et al., 1998; neumann and römheld, 2001; randall et al., 2001; yun and kaeppler, 2001; abel et al., 2002; lynch and ho, 2005). they include morphological, physiological and biochemical processes (jones, 1998; ahmad et al., 2001; pinton et al., 2001). there is data available about the effects of hormones on root architecture (leinhos and bergmann, 1995; lippmann et al., 1995; lópez-bucio et al., 2002). their effect on rhizosphere physiology and chemistry is still poorly understood. the aim of this experiment was to investigate the long-term effects of the three plant growth promoting phytohormones indole-3-acetic acid (iaa), gibberellic acid (ga3), and trans-zeatin on plant growth and rhizosphere processes depending on phosphorus availability. a continuous microbial biosynthesis of phytohormones in the rhizosphere was simulated by controlled delivery of plant growth regulators by hormone-containing nutrient solutions. thus, this experiment is an approach to elucidate long-term effects of phytohormone-producing plant growth promoting rhizobacteria on plants coping with nutrient deficiency. material and methods plant cultivation the maize cultivar 'bezemara' was used in a pot experiment. seeds were germinated in quartz sand (1 to 2 mm particle size) wetted with saturated caso4 solution. after seedlings developed a root length of about 1 cm, six germinated maize seeds were transferred to cultivation pots containing 1.5 kg of quartz sand. the pots were covered inside by a proliferated plastic bag. for harvest, this bag was pulled out of the pot carrying the intact root system. after four days, the seedling number per pot was adjusted to four. the ruakura nutrient solution (smith et al., 1983) was used in this experiment with the following modifications: iron citrate was replaced by an equimolar amount of fe(iii) chloride. in treatment with low phosphorus availability (-p), the sole p source (65 mg p per pot) was sparingly soluble ca3(po4)2 mixed with quartz sand before planting. the quantities of other nutrient elements involved in this replacement (potassium, sulphur, calcium) were adjusted to the concentration of the full nutrient solution (+p). their values are given in tab. 1. 36 l. wittenmayer, a. deubel, w. merbach plants were cultivated in a vegetation station under ambient conditions from 15 may to 28 june and harvested six weeks after germination. hormone application for hormone treatment, stock solutions of indole-3-acetic acid (iaa), gibberellic acid (ga3) and trans-zeatin (t-z) were prepared using pure ethanol as a solvent. 400 µl of the appropriate stock solution were added to one litre of +p or -p nutrient solution resulting in 0.1 mm (iaa or ga3) or 0.01 mm (t-z) hormone-containing application solution. in the control treatment ('0'), 400 µl of pure ethanol were used. hormone-containing nutrient solutions were prepared every day immediately before application to plants to ensure precise original doses of growth regulators avoiding their chemical or microbial destruction. during the first two weeks, the daily hormone dose per plant was 0.6 µmol (iaa or ga3) and 0.06 µmol (t-z). considering plant biomass, these amounts have been increased every two weeks by 100% of the initial values. during the experiment, the total volume of nutrient solution used for one pot was 1650 ml. this amount is equivalent to 65 mg of p – the quantity used in -p treatment. thus, total p amounts in +p and -p treatments were the same. analysis of rhizosphere solution in order to collect the rhizosphere solution, plants were transferred into two-litre beakers by lifting the root system with the attending substrate by the plastic bags. then the plastic bags were removed carefully with scissors and one litre of water was added. gently dipping of the maize roots in the solution removed the sand from the root surface. after two minutes, the roots were pulled out of the solution. then the dipping solution was filtered through glass wool, frozen in liquid nitrogen and lyophilized (saarnio et al., 2004). water-soluble neutral (carbohydrates) and acidic (carboxylic acids) compounds in the maize rhizosphere were collected and determined by hplc as described elsewhere (gransee and wittenmayer, 2000). in order to separate acidic and neutral compounds, the solid phase extraction technique was applied (johnson et al., 1996) before chromatographic quantification. water soluble phosphorus in the rhizosphere solution was measured according to murphy and riley (1962). the sigma diagnostics procedure number 104 (sigma diagnostics inc, st. louis, mo, usa) was used to measure acid phosphatases activities (acid pase) in the rhizosphere using p-nitrophenyl phosphate as a substrate (tabatabai and bremner, 1969). for the determination of rhizosphere acid pase, aliquots of root dipping solutions were frozen in liquid nitrogen and lyophilized at -30°c. the dry residue was dissolved in an appropriate volume of a tris buffer. protein quantification was conducted according to bradford (1972). determination of growth characteristics the root surface was estimated using the methylene blue method according to sattelmacher et al. (1983). in order to characterize the p status of plants, p concentration in shoots and roots was determined using ammonium vanadate molybdate (gericke and kurmies, 1952) and the acid phosphatase activity was measured in the third and fourth fully developed leaves under the same conditions as for rhizosphere solution. statistical analysis a two-factorial experimental design was used in anova: factor a – hormone application (0, iaa, ga3 and t-z), factor b – phosphorus availability (+p water soluble phosphate; -p sparingly soluble tricalcium phosphate). each treatment included four replicates. the total number of pots was a × b × r = 4 × 2 × 4 = 32. for statistical analysis, sas software 9.1.2 (sas institute cary, nc usa) was used. the t test (p < 0.05) was used for comparison of ±p means for the same hormone treatment. significant differences are indicated by boldface font in tab. 2-4. for comparisons of mean values between different hormone treatments of similar p availability, tukey test (p < 0.05) was used (dörfel and bätz, 1980; warnstorff, 2000). results plant growth phosphorus nutrition of maize plants exclusively from sparingly soluble tricalcium phosphate resulted in a substantial increase in root surface (+17% in control treatment, tab. 2). the application of hormones via nutrient solution had a similar effect. in +p treatment, the iaa, ga3 and t-z applications induced an 8%, 30% and 36% increase in root surface, respectively. in -p plants, root surface was increased by 11%, 23%, and 26% by the respective hormone treatments. the hormones affected not only the root system, but also the shoot. ga3 and t-z significantly increased the shoot length but decreased its dry weight indicating remarkable morphological changes. phosphorus nutrition from sparingly soluble tricalcium phosphate resulted in a decrease of plant dry weight in all treatments. the differences between +p and -p plants were relatively small compared to experiments using p free substrates for p-deficiency induction (e. g. gaume et al., 2001). in control treatment, the -p induced decrease in plant dry weight was only 11%. nutrition with ca3(po4)2 affected mainly the shoot. in all treatments, the -p plants had a significant lower shoot dry weight than the +p plants (0: -19%, iaa: -18%, ga3: -14%, and t-z: -10%), whereas the roots tend to increase their dry weight in p limited conditions. different responses of shoot and root to p availability resulted in different root : shoot ratios of +p and -p plants. in control treatment, this value was increased by 37% due to p deficiency. iaa application of +p tab. 1: element concentration (mg l-1) in applied +p and -p nutrient solutions. nutrient element +p -p n 261.6 261.6 p 40.5 0 k 241 241 s 60.9 88.6 ca 128.2 128.2 mg 21.1 21.1 cu 0.0406 0.0406 zn 0.253 0.253 mn 0.507 0.507 fe 10.1 10.1 b 0.507 0.507 mo 0.0102 0.0102 cl 29.3 29.3 na 14.6 14.6 phytohormones in rhizosphere processes 37 plants also increased this parameter slightly by 10% resembling this p deficiency response of maize plants. the highest root : shoot ratio was observed in iaa treatment of p deficient plants (0.43). rhizosphere solution within eight hours, the original p concentration of 1.31 mm in the +p nutrient solution was lowered in the rhizosphere of maize plants to 0.27 mm (tab. 3). in the rhizosphere of -p plants, the concentration of water-soluble p was half as high. iaa and t-z applications had no effect on p concentration in the rhizosphere neither in +p nor in -p maize plants. in contrast, ga3 tended to increase the concentration of water-soluble p in the -p rhizosphere, indicating an inhibited p uptake by roots rather than an improved solubilization of the sparingly soluble tricalcium phosphate. in this treatment, p concentration and p uptake by organs and by total plant were the lowest as well. because of the lower shoot dry matter production combined with lower p concentrations, also iaa and t-z reduced total p uptake of plants in +p treatments, whereas p uptake in -p treatment was not affected. generally, plants supplied with water-soluble phosphate had a significant higher p concentration in shoot and in root. acid phosphatases activity (acid pase) in the shoot is considered to be an indicator for p stress. the maize cultivar 'bezemara' did not show significant differences in enzyme activity between +p and -p plants in control and iaa treatments, but in ga3 and t-z (tab. 3). higher activity of acid pase in the rhizosphere is assumed to be an adaptation process to low available p in soil. highest rhizosphereenzyme activity was observed in iaa plants independent of p availability, the lowest in ga3 treatment (tab. 4). significant differences depending on available p were measured in control and t-z treatments, surprisingly with higher activities in the +p rhizosphere. the ph of the diluted rhizosphere solution varied depending on p availability and hormone treatment. though the original ph value of both nutrient solutions was identical (6.0), the ph of the rhizosphere solution was different. nutrition with water-soluble phosphate increased the ph in comparison to nutrition with tricalcium phosphate. furthermore, the rhizospheres of control plants at both p levels were more acidic than those of corresponding hormone treatments. no substantial differences between hormone treatments could be observed. among water soluble carbohydrates, sucrose, glucose and fructose were the quantitatively most important compounds. p deficiency tab. 3: effect of hormone application and phosphorus availability on p status of maize cultivar 'bezemara'. parameter hormone application and p availability* 0 iaa ga3 t-z +p -p +p -p +p -p +p -p 0.27 0.11 0.26 0.13 0.28 0.15 0.27 0.13 0.05 shoot 2.66 1.46 2.34 1.52 1.98 1.30 2.27 1.51 0.24 root 1.49 0.79 1.32 0.99 1.09 0.76 1.13 0.88 0.25 shoot 28.5 12.7 22.0 11.7 16.7 9.5 19.5 11.6 1.9 root 4.8 2.8 4.1 3.3 2.6 2.0 2.9 2.6 0.9 plant 33.3 15.6 26.2 15.0 19.3 11.5 22.4 14.1 2.2 acid pase in shoot in 5.7 6.9 6.6 6.6 6.1 11.3 4.6 8.4 3.2 nmol s-1 mg-1 protein for explanation of * and ** see tab. 2. lsd** (tukey, p < 0.05) p concentration in mg g-1 dw p uptake inmg per pot water-soluble p in rhizosphere in mm tab. 2: effect of hormone application, phosphorus availability (+p = high, -p = low) on growth of maize cultivar 'bezemara'. parameter hormone application and p availability* 0 iaa ga3 t-z +p -p +p -p +p -p +p -p dry weight shoot 10.7 8.7 9.4 7.7 8.5 7.3 8.6 7.7 0.8 in g root 3.2 3.6 3.1 3.3 2.3 2.6 2.6 2.9 0.6 plant 13.8 12.3 12.5 11.0 10.8 9.9 11.2 10.6 1.3 root : shoot ratio 0.30 0.41 0.33 0.43 0.28 0.36 0.30 0.38 0.06 shoot length in cm 100 92 98 91 130 121 120 109 8 root surface in dm² 84 98 91 110 109 121 114 125 2 *) significant differences between +p and -p treatment at the same hormone application are indicated by boldface (t test, p<0.05) **) for comparison of +p or -p means of the different hormone applications. lsd** (tukey, p < 0.05) 38 l. wittenmayer, a. deubel, w. merbach reduced monosaccharide exudation partially significant in all hormone treatments. lowest glucose and fructose contents were observed in ga3 and t-z treatments at both p levels. the most common carboxylic anions were trans-aconitate, citrate, succinate, malate and oxalate. due to their marginal role in psolubilization, the identified monocarboxylic acids lactate and acetate are not included in this table. generally, +p plants contained in their rhizosphere more acid anions than -p plants. furthermore, ga3 induced a general reduction in acid anions in the rhizosphere. discussion by using the method of continuous application of hormone containing nutrient solution it is possible to investigate hormonal long-term effects on plant growth, p status and rhizosphere processes of the maize cultivar 'bezemara'. the factorial analysis of variance in the experiment showed that hormone treatment had a significant effect on the investigated parameters. both phosphorus deficiency and hormone application resulted in an increase of root surface. thus, phytohormones resemble p deficiency response in roots. considering the data of other maize cultivars (deubel et al., 2007), the effect of hormone treatment on root surface was significantly modified by genotype. thus, there was a genotype-induced variation in response-sensitivity to hormone treatment. root morphology and architecture play an important role in nutrient acquisition in marginal soils (schubert and mengel, 1989; liao et al., 2001). particularly, the formation of root hairs is significant in phosphorus uptake by the plant (jungk, 2001). föhse et al. (1991) observed that up to 90% of total p uptake is contributed to root hairs. in our experiment, an increase in the root surface area did not result in an improved p acquisition. in soil, root hairs can penetrate small soil pores, as if 'mining' nutrients therein (michael, 2001). in coarse quartz sand used in this experiment, this adaptation response would not result in an increased availability of phosphorus because p was not caught in small soil compartments accessible only to root hairs. in this context, it should also be mentioned that another phytohormone not considered in this experiment has a strong influence on root morphology: ethylene (schmidt, 2001; arshad and frankenberger, 2002). the most frequent interaction occurred between hormone application and p status of plants indicating that the same phytohormone treatment of +p and -p plants may result in a different response to the plant growth regulator applied. lópez-bucio et al. (2002) observed a different response of arabidopsis to plant growth regulator treatment depending on p status of plants. the authors assume that p status of plants influences sensitivity to exogenously applied hormones. varying growth response due to changing sensitivity of plant roots to auxin production by microorganisms is also discussed by björkman (2004). inactivation of hormones by the formation of conjugates (ljung et al., 2001, 2002; leclere et al., 2002), increased catabolism (kerk et al., 2000) or reduced transport to the sensitive tissue (ljung et al., 2001) may affect sensitivity of plant tissue to pgr. crozier et al. (1988) observed that maize roots were making the predominant contribution to catabolism of rhizosphere iaa in colonized roots. the activity of acid phosphatases is considered to be a good indicator for p deficiency in plants (barrett-lennard and greenway, 1982; mclachlan and de marco, 1982) particularly in the rhizosphere (tarafdar and jungk, 1987; ascencio, 1997). comparing the acid pase of 23 barley cultivars, römer et al. (1995) found that not all cultivars respond to p deficiency by increasing acid pase activity in the shoots. in several cases the authors observed higher enzyme activities in +p plant than in -p plants in comparison with different cultivars. thus, the acid pase was more strongly affected by genotype than by p availability. in our experiment with the cultivar 'bezemara', the enzyme activity in shoots was influenced by p only in ga3 and t-z treatments. thus, a general influence of a singly investigated growth parameter, e.g. p availability, on the enzyme activity, cannot be concluded. it is well documented that in root exudates carboxylic acids may dissolve ca3(po4)2 replacing phosphate by carboxylic anions (jones et al., 2003, jones, 1998). in order to solubilise p amounts covering the p content of maize plants in -p treatments (tab. 3), equivalent quantities of calcium ions had to be neutralised by carboxylic anions tab. 4: effect of hormone application, phosphorus availability on rhizosphere characteristics of maize cultivar 'bezemara'. parameter hormone application and p availability* 0 iaa ga3 t-z +p -p +p -p +p -p +p -p 14.0 3.2 21.2 23.6 4.5 4.2 10.6 3.3 8.4 6.95 5.90 7.26 6.32 7.22 6.27 7.19 6.26 0.31 0 0.754 0 0.726 0 0.556 0 0.686 0.046 sucrose 0.04 0.06 0.09 0.04 0.06 0.02 0.04 0.05 0.05 glucose 1.13 0.62 1.35 0.92 0.48 0.26 0.50 0.33 0.73 fructose 0.51 0.20 0.61 0.32 0.12 0.08 0.14 0.07 0.25 citrate 0.74 0.10 0.66 0.41 0.19 0.08 0.40 0.06 0.16 trans-aconitate 0.41 0.05 0.32 0.08 0.13 0.03 0.26 0.03 0.15 oxalate 0.12 0.06 0.08 0.09 0.03 0.05 0.10 0.03 0.07 succinate 1.47 0.67 1.04 0.73 0.22 0.33 0.56 0.25 0.62 malate 0.80 0.08 0.45 0.32 0.15 0.06 0.47 0.05 0.24 for explanation of * and ** see tab. 2. concentration in rhizosphere in mm lsd** (tukey, p < 0.05) acid pase in rhizosphere in nmol s-1 mg-1 protein ph of rhizosphere solution. (1 : 10 v/v) diluted neutralized ca derived from ca3(po4)2 solubilisation in mmol phytohormones in rhizosphere processes 39 (tab. 4). thus, from the measured concentration of carboxylic anions in the rhizosphere solution conclusions about their exudation rates cannot be drawn because a manifold portion of carboxylic acids have been bound in insoluble calcium compounds. due to the low solubility of calcium salts (calculation according to seidell and linke, 1952) with citrate and oxalate of 1.5 and 0.5 mmol l–1, respectively, a precipitation of the exuded organic acid anions in the vicinity of root surface might have been occurred as observed for lupine plants in a calcareous soil by dinkelaker et al. (1989). this would explain the lower concentration of organic acids in -p treatments. fore example, citrate concentration in the control -p treatment was 0.1 mm. considering the volume of the rhizosphere solution (0.11 l), 0.011 mmol of dissolved citrate were found in this treatment. to neutralise the released ca amount of 0.754 mmol, a 46 times higher citrate amount is necessary. even the sum of all identified dissolved organic acids of the rhizosphere solution is only a small fraction of the quantity necessary for neutralisation of the calcium. the assumption of a higher carboxylic acid exudation rate of -p plants is supported by the substantial lower ph values in their rhizosphere indicating additional proton release by co-exudation with carboxylic anions. the content of sugars and carboxylic anions in the maize rhizosphere was promoted by the applied iaa doses. from our data, it cannot be concluded that other hormones do not affect rhizosphere chemistry. the observation of negligible effects of ga3 and t-z is valid only for the applied doses of these phytohormones. for iaa is known that depending on its concentration, this hormone can stimulate or inhibit root growth (pilet, 1996, 1998a,b; björkman, 2004). werner et al. (2001) found that in cytokinin-deficient plants, root growth was promoted. in our pot experiment, the applied t-z amount stimulated root growth. obviously, not only the strength but also the direction of hormone effects (stimulation/inhibition), depends on their applied doses (ludwig-müller, 2004). since hormonal effects depend on pgr doses, for elucidation of the optimum effect of a specific pgr on plant growth and particular rhizosphere parameters, various concentrations should be tested. furthermore, only single hormonal factors were tested here. in a plant-rhizosphere continuum, multiple hormonal interactions occur. there are indications that only the combination of various hormones (e.g. iaa, gibberellic acid, and kinetin) can induce rhizosphere effects similar to those produced by plant growth promoting microorganisms (tien et al., 1979). thus, in future experiments, applications of various doses of pgr and their combinations should be tested for their long-term rhizosphere processes. acknowledgments the authors want to thank mrs. chr. birke and mrs. e. pfeil for their technical assistance. the english text was revised by g.c. calderone, trenton nj, usa. references abel, s., ticconi, c.a., delatorre, c.a., 2002: phosphate sensing in higher plants. physiol. plant. 115, 1-8. ahmad, z., gill, m.a., qureshi, r.h., 2001: genotypic variation of phosphorus utilization efficiency of crops. j. plant nutr. 24, 1149-1171. arshad, m., frankenberger, w.t., jr., 2002: ethylene. agricultural sources and applications. kluwer academic/plenum publishers, new york, boston, dordrecht, london and moscow. arteca, r.n., 1996: plant growth substances: principles and applications. chapman and hall, new york, albany, bonn, boston, cincinnati, detroit, london, madrid, melbourne, mexico city, pacific grove, paris, san francisco, singapore, tokyo, toronto, washington. ascencio, j., 1997: root secreted acid phosphatase kinetics as a physiological marker for phosphorus deficiency. j. plant nutr. 20, 9-26. barrett-lennard, e.g., greenway, h., 1982: partial separation and characterization of soluble phosphatases from leaves of wheat grown under phosphorus deficiency and water deficit. j. exp. bot. 33, 694-704. björkman, t., 2004: effect of trichoderma colonization on auxin-mediated regulation of root elongation. plant growth regul. 43, 89-92. bradford, m.m., 1972: a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. anal. biochem. 72, 248-254. crozier, a., arruda, p., jasmim, j.m., monteiro, a.m., sandberg, g., 1988: analysis of indole-3-acetic acid and related indoles in culture medium from azospirillum lipoferum and azospirillum brasilense. appl. environ. microbiol. 54, 2833-2837. deubel, a., wittenmayer, l., merbach, w., 2008: einfluss von phytohormonen und p-versorgung auf wachstum und wurzelabscheidungen von mais. in: merbach, w., deubel, a., augustin, j. 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ramos. agron. j. 87, 902-908. agron. j. 92, 1042-1045. werner, t., motyka, v., strnad, m., schmülling, t., 2001: regulation of plant growth by cytokinin. proc. nat. acad. sci. usa 98, 10487-10492. yun, s.j., kaeppler, s.m., 2001: induction of maize acid phosphatase activities under phosphorus starvation. plant soil 237, 109-115. address of authors: dr. lutz wittenmayer, dr. annette deubel, prof. dr. wolfgang merbach (corresponding author), institute of agricultural and nutritional sciences, pofessorship plant nutrition, martin-luther university halle-wittenberg, adam-kuckhoff-straße 17b, d-06108 halle, germany. e-mail: wolfgang.merbach@landw.uni-halle.de lutz.wittenmayer@landw.uni-halle.de annette.deubel@landw.uni-halle.de << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) 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/pagesize [907.087 680.315] >> setpagedevice art30 journal of applied botany and food quality 82, 183 192 (2009) university of natural resources and applied life sciences, vienna, department of applied plant sciences and plant biotechnology, institute of agronomy and plant breeding calibration and validation of the crop growth model lintul for grain amaranth (amaranthus sp.) d.m. gimplinger, h.-p. kaul (received september 18, 2008) summary grain amaranth, a c4 plant, is a promising pseudocereal due to its valuable grain components. knowledge of crop growth parameters is crucial for the introduction of a new crop, and the use of a crop model can help to understand yield formation and yield limiting processes. the aim of the study was to parameterise and validate the model lintul for grain amaranth. basically, lintul estimates dry matter production from daily intercepted radiation and light use efficiency under potential growth conditions, i. e. without occurrence of any other limiting factors. a field experiment with the a. hypochondriacus genotypes „neuer typ“ and „anderer typ“ was carried out under semiarid conditions in 2004 and 2005. field data of individual years were used for parameterisation while independent observations of the other year allowed for cross-validation, respectively. the estimated light use efficiency ranged between 2.5 and 2.8 g mj-1 (total biomass per accumulated par). specific leaf area estimates were lower in observations of 2004 (0.014 m2 g-1) than in observations of 2005 (0.018 m2 g-1). the light extinction coefficient of both genotypes measured before heading was 1.1. the effective sum of temperature (above a given threshold of 8°c) to anthesis was 554°c d for the genotype „neuer typ“ and 640°c d for „anderer typ“ in both years. the effective sum of temperature to maturity was 1127°c d in 2004, and 1249°c d in 2005 independent of the genotype. model predictions of total biomass agreed well (rmse: 92 to 136 g m-2) with the observed biomass throughout the growing cycle including final harvest (between 749 and 1172 g m-2). the estimated grain yield over time (rmse: 47 to 112 g m-2) matched the field observations including final grain yield (between 220 and 367 g m-2) with less accuracy. the leaf area index was overestimated throughout the growing cycle from heading onwards to seed filling. the sharp initial increase in grain yield was underestimated suggesting that currently produced assimilates could not meet the growth capacity of the young seeds but might be complemented by internal re-translocation of biomass. introduction grain amaranth is a promising crop due to its valuable grain components. however, the low grain yield level in comparison to common cereals seems to constrict the future market opportunities of the crop. the generally low yield potential of grain amaranth is due to various reasons. firstly, breeding work on amaranth has been marginal compared to common crops. secondly, physiological properties of the plant might restrict the yield level. so far, little is known quantitatively about processes that limit the formation of grain yield in amaranth. the detailed analysis of crop growth parameters, the analysis of plant growth and development over time and in dependency on weather conditions may help to better understand yield formation and to reveal physiological limitations of growth and grain production. lintul (light interception and utilisation) is a generic crop growth model that follows a mechanistic approach. it was developed by simplifying the model sucros and was originally applied to the growth of potato (spitters, 1990; spitters and schapendonk, 1990). later it was also used for different growth analysis purposes in maize (farré et al., 2000), rapeseed (habekotté, 1997), crambe (mathijssen and meijer, 1995) and grassland (schapendonk et al., 1998; rodriguez et al., 1999). lintul type models have the advantage that only low data input is required and model parameterisation is facilitated (bouman et al., 1996). thus, the lintul approach was also used for agro-ecological characterisation of potato production (van keulen and stol, 1995), for the prediction of potential and water-limited global food production (penning de penning de vries et al., 1995) and for the simulation of wheat production under climatic change (wolf et al., 2002). the objective of the present study was to parameterise and validate the model lintul for grain amaranth under potential growth conditions. the obtained parameter values shall deliver insight into limitations of growth and yield formation of grain amaranth compared to other crops. this knowledge can help to improve crop management decisions as well as breeding efforts. furthermore, the parameterisation shall enable to include the niche crop in agro-ecological modelling approaches. the following questions were addressed: – which is the range of the required physiological crop parameters for grain amaranth? are there differences between the tested amaranth genotypes? how do parameter values differ from those of other grain crops or c4 plants? – is the parameterised model able to predict leaf area, total crop biomass and biomass of individual plant compartments, especially grain yield accurately over time when compared to independent field data? materials and methods field experiments field experiments were conducted on the experimental farm großenzersdorf (48°15´n, 16°37´e; 153 m a. s. l.) in eastern austria during the growing seasons of 2004 and 2005. mean annual precipitation at the location is 546 mm, mean annual temperature is 9.8°c. tab. 1 shows weather data of the two vegetation periods. the soil type at the location was classified as a chernozem of alluvial origin which is rich in calcareous sediments. the texture ranges between silty loam and loamy silt. content of humus is between 2.2 and 2.3%. tab. 2 gives information on the field experiments. due to the high level of soil mineral nitrogen and the high supply of p and k at the site, no fertiliser was added. plots were irrigated twice, at the beginning of flowering and at the start of grain filling. the experimental design was a randomised block in a split plot arrangement with two replications. two grain amaranth genotypes were established on main plots, two crop densities on subplots. the desired crop densities of 8 and 70 plants m-2 were established by hand-thinning. the breeder g. dobos, zeno projekte, vienna, austria, provided the seeds of the genotypes which result from progenies of crossbreds: „neuer typ“ (a. hypochondriacus) is an 184 d.m. gimplinger, h.-p. kaul tab. 1: mean monthly temperature, total monthly precipitation and total incident monthly photosynthetic active radiation (par) during the 2004 and 2005 growing seasons year may june july aug. sep. oct. temperature 2004 13.8 17.4 19.9 20.7 15.4 11.6 (°c) 2005 15.7 18.6 20.5 18.7 16.2 10.4 precipitation 2004 51.0 51.0 24.8 21.8 32.4 53.6 (mm) 2005 43.4 43.4 94.6 65.4 34.8 4.0 incident par 2004 232.0 280.9 299.0 220.6 154.7 67.4 (mj m-2) 2005 302.3 222.2 213.5 189.0 134.2 83.8 tab. 2: details of the field experiments during the 2004 and 2005 growing seasons 2004 2005 soil mineral n, 0–90cm 5 may, 163 kg n ha-1 12 may, 166 kg n ha-1 p (cal), 0–30cm / 30–60cm 88 / 74 mg kg-1 92 / <20 mg kg-1 k (cal), 0–30cm / 30–60cm 255 / 222 mg kg-1 264 / 80 mg kg-1 soil ph 7.7 7.5 sowing date 27 may 11 may fertilisation none none 21 july, 20 mm 6 july, 20 mm 3 august, 20 mm 18 july, 20 mm insecticide 15 june, deltamethrin 30 may, deltamethrin weed control mechanical and hand-weeding mechanical and hand-weeding row spacing 37.5 cm 37.5 cm neuer typ sparse crop 9 pl. m-2 8 pl. m-2 crop density neuer typ dense crop 49 pl. m-2 49 pl. m-2 (across sampling dates) anderer typ sparse crop 9 pl. m-2 8 pl. m-2 anderer typ dense crop 51 pl. m-2 53 pl. m-2 sum of temp eff (°c d) neuer typ 554 554 from emergence to anthesis anderer typ 641 640 date 1 = emergence 7 june 0.0 25 may 0.0 date 2 28 june 198.1 16 june 202.4 date 3 8 july 305.4 30 june 392.1 sum of temp eff (°c d) date 4 26 july 532.1 13 july 517.2 at sampling dates date 5 22 july 640.9 date 6 16 aug. 808.6 2 aug. 809.7 date 7 9 aug. 876.8 date 8 = maturity 16 sept. 1126.5 12 sept. 1248.9 irrigation treatments early-maturing, strongly branching, semi-dwarf type reaching a plant height between 85 and 100 cm at the experimental site. „anderer typ“ (a. hypochondriacus) is similar to „neuer typ“ with respect to grain maturity, but about one week later in flowering. it is hardly branching, shows a rather compact inflorescence and reached plant heights between 115 and 130 cm under given conditions. plants were sampled between emergence and maturity at six dates in 2004 and at eight dates in 2005. at each sampling date, total aboveground biomass was determined by harvesting 1.125 m2 per plot. ten randomly chosen plants per plot were used for the determination of the dry matter weights (48h, 100°c) of green leaves, senescent leaves on the plant, stems (including chaff), seeds and roots. roots were sampled by digging out cuboids (40 x 40 x 28 cm3) of the soil round the harvested plant using a spade. subsequently, roots were separated from earth, cleaned and dried. total crop biomass was computed as the sum of harvested above ground biomass per area and root biomass. root biomass resulted from aboveground biomass multiplied by the percentage of root weight of ten plants. leaves with more than 50% yellow-coloured blade area were defined as being senescent. the green leaf area of ten plants was determined using a li-3100 area meter (licor). model description and implementation the model lintul was used for simulating crop growth of grain amaranth due to its comparatively low data requirements which allow for a wide-spread application. lintul is based on the linear relationship between produced biomass and the amount of radiation intercepted by the crop canopy which is a reasonable assumption under favourable growth conditions (monteith, 1977). the applied version lintul 1 simulates potential crop growth under wellwatered conditions, ample nutrient supply and the absence of pests, diseases and weeds (van ittersum et al., 2003). biomass production is based on intercepted radiation according to lambert-beer’s law and an experimentally derived value of light use efficiency. the produced biomass is partitioned among various crop organs (leaves, stems, storage organs and roots) according to partitioning coefficients (kg organ biomass kg-1 total biomass) defined as a function of the development stage of the crop. the software modelmaker (version 4, cherwell scientific) was used to implement the model equations and to run simulations. model equations were obtained from the version lintul 1 as published by the de wit graduate school for production ecology (1997). lintul for amaranth 185 initial values average dry weights of plant organs (stems, leaves, roots) and leaf area at emergence were determined by sampling 75 seedlings per genotype grown under green house conditions to enable quick and homogeneous germination (tab. 3). sampling was carried out when more than 90% of the plants had emerged. a windias colour image analysis system was used for measuring initial leaf area. weather data daily global incoming radiation and average daily temperature were obtained from an automatic weather station close to the experimental plots. photosynthetic active radiation (par) was calculated as 50% of global radiation. the sum of temperature was calculated by accumulating the daily values of effective temperature after emergence above a base temperature of 8°c. this threshold temperature was chosen in accordance with the base temperature for leaf appearance in amaranthus retroflexus which was found to be about 8.5°c (gramig and stoltenberg, 2007). estimation of model parameters light use efficiency was estimated by a linear regression of total biomass per area (including roots and senescent leaves on the plant) on cumulative intercepted radiation. the last sampling date was excluded because of unmeasured weight losses due to scattered leaves and negligible increase in biomass. this approach was already applied by farré et al. (2000) who estimated the light use efficiency of maize on the basis of measurements before anthesis only. cumulative intercepted radiation was calculated by summarising daily intercepted radiation according to the function of lambert-beer. the essential daily leaf area index was derived by linear interpolation between leaf area indices of green leaves at sampling dates. a crop-specific light extinction coefficient of 1.1 (standard error: 0.27) was determined by using a sunscan canopy analysis system (setting: beer’s law) at ear emergence in 2005. the instrument was not available in 2004, but a substantial year effect on this parameter is not expected. the sunscan device uses a probe of 1 m length for measurements of incident and transmitted photosynthetic active radiation. four measurements per plot were carried out, two measurements along the rows and two measurements rectangular to the rows. one measurement consisted of one reading above the canopy followed by four readings measured in trans-sections below the canopy. no effects of genotype or crop density on the light extinction coefficient were observed. the initial relative growth rate of leaf area was assumed to increase exponentially over thermal time as long as the temperature sum is below 330°c d and the leaf area index is below 0.75 according to spitters et al. (1989). a linear regression was calculated of the natural logarithm of the increase in leaf area index, i. e. ln(lai/ laiinitial), on the effective sum of temperature. during later development stages, it was assumed that leaf area expands in proportion to the increase in leaf dry weight, and it is calculated by multiplying the increase in leaf dry weight by the specific leaf area. specific leaf area was determined by a linear regression of leaf area on leaf weight across all sampling dates. leaf senescence was assumed to be driven by ageing which starts at anthesis and by self shading which starts beyond a critical leaf area index of 4 according to spitters et al. (1989). it was assumed that the death rate due to ageing depends on average daily temperature using a tabled function with linear interpolation (<10°c, 0.03; 15°c, 0.04; >30°c, 0.09). the death rate due to shading was supposed to increase linearly till a maximum of 0.03 at a leaf area index of 8. anthesis was defined as the development stage when about 50% of the inflorescence showed anthers. daily produced biomass was allocated to the organs roots, green leaves, stems (including chaff) and seeds by distribution factors obtained from the plant samplings. the relative fraction shares were tabulated and linearly interpolated as a function of thermal time. the portion of biomass allocated to each plant organ was derived by dividing the increase in biomass of a plant organ by the increase in total biomass between subsequent harvest dates. the slight decrease in weight of leaves or roots at late growth stages was assumed to show that no more biomass was allocated to these plant organs. a slight increase in stems (including chaff) between the last two harvest dates was ignored, and it was assumed that the produced biomass was totally allocated to the seeds. model testing and statistical analysis calibration and validation of the model were done separately for each genotype (across both crop densities). cross validation was carried out by using independent field data of two years (2004 and 2005). parameter estimates based on the field experiment in 2004 were used for the simulation of growth and development under environmental conditions of 2005 and observed field data of 2005 were used for the validation of the model, and vice versa. the parameter estimates based on regression equations were statistically evaluated by calculating the significance of the regression line and the standard error of the parameters. comparisons were made between simulated and observed values. root mean squared errors and mean absolute percent errors were calculated using the software irene (integrated resources for evaluating numerical estimates), version 1, developed by the research institute for industrial crops, bologna, italy (fila et al., 2001). furthermore, regression analyses between simulated and observed values were carried out. results model parameterisation model parameters based on the data of different years and genotypes allow the characterisation of environmental and genotypic effects on crop growth. estimated model parameters and regression statistics are given in tab. 4. all regressions were highly significant. the estimated light use efficiency ranged between 2.48 and 2.84. obviously, both genotypes were more efficient in producing biomass in 2005 than in 2004. the 2005 environment was characterised by higher precipitation in july and august, but by lower radiation throughout the growing season compared to the 2004 environment. environmental differences also affected specific leaf area and initial growth rate of leaf area. the higher specific leaf area in 2005, i.e. thinner leaves, is presumably due to higher rainfalls. however, the initial growth rate of leaf area was higher in the 2004 environment than in the 2005 environment. genotypic differences were obvious with regard to crop development and also with respect to the allocation of biomass to plant organs. thermal time from emergence to full flowering was shorter with tab. 3: initial values: dry matter weight of plant organs and leaf area per seedling (means across genotypes) start value (per seedling) standard error of mean stem (g pl-1 ) 0.211 x 10-3 9.55 x 10-6 root (g pl-1) 0.219 x 10-3 30.4 x 10-6 leaf (g pl-1) 0.380 x 10-3 17.9 x 10-6 leaf area (m2 pl-1) 0.0147 x 10-3 0.586 x 10-6 186 d.m. gimplinger, h.-p. kaul tab. 4: estimated parameters based on field experiments in 2004 and 2005 (lue = light use efficiency, sla = specific leaf area, igrl = initial relative growth rate of leaf area, sum of temp eff = temperature sum above a base temp. of 8°c) year genotype lue standard sign. (p) sla standard sign. (p) igrl standard sign. (p) sum of (g mj-1) error (m2 g-1) error (°c-1 d-1) error temp eff (°c d) to anthesis 2004 neuer typ 2.58 0.103 <0.0001 0.0144 0.578 x 10-3 <0.0001 0.0290 1.620 x 10-3 0.0031 554 anderer typ 2.48 0.056 <0.0001 0.0136 0.775 x 10-3 <0.0001 0.0298 0.707 x 10-3 0.0151 641 2005 neuer typ 2.72 0.053 <0.0001 0.0177 0.632 x 10-3 <0.0001 0.0272 0.304 x 10-3 0.0071 554 anderer typ 2.84 0.085 <0.0001 0.0178 0.383 x 10-3 <0.0001 0.0268 0.272 x 10-3 0.0065 640 „neuer typ“ than with „anderer typ“, and consequently grain yield formation started earlier with „neuer typ“. the portion of biomass allocated to leaves of both genotypes was highest during early plant development when plant height was about 10 cm and the first leaves were formed. from the phase of stem elongation onwards (305390°c d), a higher rate of biomass was distributed to leaves in „anderer typ“ than in „neuer typ“ tab. 5. tab. 5: estimated dry matter partitioning coefficients based on field experiments in 2004 and 2005 year genotype sampling sum of root stem + chaff leaf seed date temp eff (°c d) 2004 neuer typ 1 0 0.270 0.261 0.469 0.000 2 198 0.145 0.188 0.668 0.000 3 305 0.161 0.451 0.388 0.000 4 532 0.082 0.816 0.102 0.000 5 no sampling 6 809 0.000 0.278 0.000 0.722 7 no sampling 8 1127 0.000 0.000 0.000 1.000 2004 anderer typ 1 0 0.270 0.261 0.469 0.000 2 198 0.170 0.188 0.642 0.000 3 305 0.185 0.387 0.429 0.000 4 532 0.098 0.673 0.228 0.000 5 no sampling 6 809 0.007 0.636 0.000 0.357 7 no sampling 8 1127 0.000 0.000 0.000 1.000 2005 neuer typ 1 0 0.270 0.261 0.469 0.000 2 202 0.107 0.180 0.713 0.000 3 392 0.152 0.484 0.365 0.000 4 517 0.080 0.796 0.124 0.000 5 641 0.037 0.779 0.067 0.117 6 810 0.012 0.249 0.000 0.740 7 877 0.044 0.030 0.000 0.926 8 1249 0.000 0.000 0.000 1.000 2005 anderer typ 1 0 0.270 0.261 0.469 0.000 2 202 0.104 0.167 0.729 0.000 3 392 0.176 0.380 0.445 0.000 4 517 0.101 0.687 0.212 0.000 5 641 0.105 0.809 0.086 0.000 6 810 0.023 0.672 0.000 0.305 7 877 0.037 0.324 0.000 0.640 8 1249 0.000 0.000 0.000 1.000 model testing the time-courses of simulated and observed total biomass (including senescent leaves and root biomass), leaf area index of green leaves and grain yield of the two environments (2004 and 2005) are given in fig. 1, 2 and 3, respectively. fig. 4 shows regressions of simulated versus observed values throughout the growing season. total biomass was simulated well by the model in both environments. lintul for amaranth 187 with respect to crop density, the model calculates necessarily higher total biomass for the dense stands due to higher initial values. root mean squared errors of predicted biomass figures for the 2004 and the 2005 environment were similar and amounted to values between 92 and 136 g m-2 (tab. 6). relative root mean squared errors were between 18 and 28% of the mean observed biomass. the leaf area index (fig. 2) and the weight of green leaves (data not shown) were highly over-predicted by the model, especially throughout the period from heading to seed filling (sampling dates 3-6). this overestimation was most evident when simulating crop growth under environmental conditions of 2004 with parameters based on observations of 2005. while the observed leaf area index was between 1.2 and 2.3 at the most, the simulated values mounted up to values between 2.1 and 3.4 under growing conditions of 2005 and even to values between 3.9 and 5.9 under growing conditions of 2004. as a consequence, root mean squared errors were high, being 71-219% of the mean observed leaf area index. regression of simulated versus observed leaf area index showed that the overestimation was highest at sampling date 4 when the crops started flowering. day of year l a i (m 2 m -2 ) neuer typ 2004 0 2 4 6 150 175 200 225 250 275 neuer typ 2005 0 2 4 6 150 175 200 225 250 275 anderer typ 2005 0 2 4 6 150 175 200 225 250 275 anderer typ 2004 0 2 4 6 150 175 200 225 250 275 fig. 2: simulated and observed leaf area index of green leaves of two genotypes at two plant densities during crop development in 2004 and 2005 ❍ observed (sparse crop) ● observed (dense crop) simulated (sparse crop) simulated (dense crop) t o ta lb io m a ss (g m -2 ) day of year neuer typ 2004 0 500 1000 1500 150 175 200 225 250 275 c anderer typ 2004 0 500 1000 1500 150 175 200 225 250 275 neuer typ 2005 0 500 1000 1500 150 175 200 225 250 275 anderer typ 2005 0 500 1000 1500 150 175 200 225 250 275 fig. 1: simulated and observed total biomass of two genotypes at two plant densities during crop development in 2004 and 2005 ❍ observed (sparse crop) ● observed (dense crop) simulated (sparse crop) simulated (dense crop) 188 d.m. gimplinger, h.-p. kaul ∑ = . − . n i i ii nm me 1 1 100 ( ) n me n i ii∑ = − 1 2 g ra in yi e ld (g m -2 ) day of year neuer typ 2004 0 100 200 300 400 150 175 200 225 250 275 anderer typ 2004 0 100 200 300 400 150 175 200 225 250 275 neuer typ 2005 0 100 200 300 400 150 175 200 225 250 275 anderer typ 2005 0 100 200 300 400 150 175 200 225 250 275 fig. 3: simulated and observed grain yield of two genotypes at two plant densities during crop development in 2004 and 2005 ❍ observed (sparse crop) ● observed (dense crop) simulated (sparse crop) simulated (dense crop) the time-course of grain yield formation (fig. 3) was simulated with less accuracy than the production of total biomass. especially the sharp rise in grain weight at the beginning of grain formation was underestimated by the model. root mean squared errors were between 47 and 112 g m-2, i. e. 31-37% of the mean observed grain yield. grain yield levels at maturity were predicted quite satisfactorily, but some deviations from observed yield levels had to be noticed. discussion experimental conditions the experimental site provided appropriate conditions for potential growth. the high level of soil mineral nitrogen and the high level of available phosphor and potassium ensured ample supply of nutrients. the typical growth limiting factor at the site is the low precipitation. therefore, plots were irrigated twice during the growing cycle. grain yield in irrigated plots was higher than in neighbouring not irrigated plots reported in a different field study (gimplinger et al., 2008). this effect was especially obvious in the 2004 environment with lower precipitation. higher yield in irrigated plots suggests that the irrigation treatments met the water demands of the crop. diseases did not occur. flea beetles (phyllotreta spp.) and the sugar-beet weevil (bothynoderes sp.) which infested plants at emergence were controlled by an insecticide. mechanical and handweeding provided a weed-free environment. the attained level of grain yield was similar to the level of yield found in field experiments under favourable conditions elsewhere (jamriška, 1996; aufhammer and kübler, 1998). mean observed grain yields at harvest of „neuer typ“ were 338 and 309 g m-2, observed grain yields of „anderer typ“ were 331 and 232 g m 2 in the 2004 and 2005 environment, respectively. due to the small data base, crop density effects were not taken into account. yet, it is known that rising plant density affects the allocation patterns of biomass, e. g. slightly decreases the harvest index (gimplinger et al., 2008). as parameterisation was based on genotypes (averaged across densities), crop density differences calculated by the model merely reflect the differences in start values depending on the defined number of seedlings per area. model parameterisation model predictions are highly dependent on model parameters. therefore, parameters should be defined precisely and deserve closer attention. farré et al. (2000) analysed the sensitivity of the model lintul to changes in input parameters in growth simulations of tab. 6: statistical measures for observed versus simulated values (all sampling dates) rmsea rmse ma%eb absolute relative (g m-2) (% of mean (m2 m-2) observed) (%) total biomass neuer typ 2004 135.7 28.4 30.7 neuer typ 2005 91.8 20.7 60.0 anderer typ 2004 97.0 17.8 19.6 anderer typ 2005 96.3 21.0 75.0 leaf area index neuer typ 2004 1.56 219.0 161.0 of green leaves neuer typ 2005 0.76 90.7 113.5 anderer typ 2004 1.62 155.1 82.8 anderer typ 2005 0.79 71.1 125.0 grain yield neuer typ 2004 112.0 37.1 33.7 neuer typ 2005 57.9 31.2 49.5 anderer typ 2004 84.9 35.7 41.4 anderer typ 2005 47.0 31.1 26.9 armse: root mean squared error calculated as where e i is the i th estimated value, m i is the ith measured value, and i…n is the number of values. bma%e: mean absolute percent error calculated as lintul for amaranth 189 (a) (b) (c) y = 66.17 + 0.98x r2 = 0.94 y = 0.08 + 1.67x r2 = 0.62 y = -22.06 + 0.92x r2 = 0.70 0 500 1000 1500 0 500 1000 1500 observed total biomass (g m-2) s im u la te d to ta lb io m a s s (g m -2 ) date 2 date 3 date 4 date 5 date 6 date 7 date 8 0 1 2 3 4 5 6 0 1 2 3 4 5 6 observed lai (m2 m-2) s im u la te d l a i (m 2 m -2 ) date 2 date 3 date 4 date 5 date 6 date 7 date 8 0 100 200 300 400 0 100 200 300 400 observed grain yield (g m-2) s im u la te d g ra in y ie ld (g m -2 ) date 5 date 6 date 7 date 8 fig. 4: simulated versus observed values of total biomass (a), leaf area index (lai) of green leaves (b) and grain yield (c) maize. they found that the simulated grain yield was particularly sensitive to changes in light use efficiency, but also to changes in the light extinction coefficient and in the rate of leaf senescence whereas changes in specific leaf area or in the partitioning coefficients to root and shoot hardly affected crop yield. in lintul, the effective sum of temperature controls plant development. however, no data were available to examine the base temperature of the used grain amaranth species carefully. therefore the estimated base temperature might also be a potential source of inaccurate model predictions. the estimated model parameters of the tested amaranth genotypes resulted in reasonable values compared to earlier findings in amaranth and other crops. hardly any differences between genotypes could be noted except for the dry matter portions partitioned to each plant organ. yet, differences between years were obvious for several parameter estimates. light use efficiency several studies revealed that light use efficiency is not constant throughout the development of a crop, e.g. throughout the growth of maize (sinclair and muchow, 1999), wheat (calderini et al., 1997) and rapeseed (habekotté, 1997). in the presented study, the linear regression of biomass of amaranth against the accumulated par (based on a constant light extinction coefficient) did not suggest any variability in light use efficiency throughout the growing period. only shortly before grain maturation light use efficiency decreased 190 d.m. gimplinger, h.-p. kaul probably due to senescent leaves scattered to the ground. however, the data of this period were excluded from the calculation of light use efficiency. light use efficiency estimates might have been more accurate if the periodic measurements of biomass and leaf area were accompanied by periodic measurements of radiation intercepted by the canopy. light use efficiency also includes root growth, but it is often expressed with reference to shoot biomass per accumulated par only. for comparisons with other findings, the recalculated light use efficiency for our data based on shoot biomass ranges between 2.26 and 2.57 g mj-1. these values found under semiarid conditions are slightly lower than the light use efficiency between 2.44 and 2.83 g mj-1 reported earlier by kaul et al. (2000) and values from 2.54 to 3.02 g mj-1 found by kruse (1996) under moister conditions in southwest germany. higher light use efficiency values in the 2005 environment might also be due to the higher precipitation in this year, indicating slight drought stress despite irrigation in 2004. the calculated light use efficiency of amaranth is similar to values of sorghum, but it is lower than most findings from other c4 plants and lower than that of c3 cereals (sinclair and muchow, 1999). the low light use efficiency of amaranth compared to maize and sugarcane can be attributed to the comparably high protein and fat content of amaranth grains. it is well known that differences in the energy content of the biochemical constituents strongly contribute to variations in light use efficiency (sinclair and muchow, 1999). the lower light use efficiency might also be due to the subtype of c4 photosynthesis. leakage of co2 out of the bundle sheath and energy costs for refixing co2 are greater in plants following the nad-me subpathway, as found in amaranthus, than in plants using the nadpme subpathway, and greater in c4 dicotyledons than in monocotyledons (pearcy and ehleringer, 1984; kubasek et al., 2007) because c4 dicotyledones lack the suberised lamella in the walls separating the mesophyll and the bundle sheath cells in the leaves (kigel, 1994). light extinction coefficient lintul is based on a constant light extinction coefficient. yet, it is known to be variable throughout the life cycle of most crops (van heemst, 1998) and it can be reduced by drought (farré et al., 2000). kruse (1996) also reported that the extinction coefficient of amaranth is variable during the season amounting to 1 during heading and decreasing to 0.8 towards harvest. in our experiment, the single value was based on measurements taken before heading in 2005 only and amounted to 1.1 for both genotypes. yet, own measurements in a field study without irrigation treatments in 2006 led to the same value (data not shown) and suggest little variability of this parameter at the site. in comparison to other crops, the light extinction of amaranth canopies is high which is typical for a dicotyledonous species showing horizontally oriented leaves. specific leaf area it is known that specific leaf area is not constant throughout the growing season and can be affected by environmental conditions (van delden et al., 2000). for example, water stress during ageing of a crop may reduce specific leaf area (ludlow, 1975). apart from the growth phase after emergence, our linear regression plots used for parameter estimation did not suggest reductions of the specific leaf area (of green leaves) during ageing. however, differences of specific leaf area between years were noted, and the lower specific leaf area found in 2004 might be explained again by some water stress occurring although plots were irrigated. this is in accordance with liu and stützel (2004) who found that water stress significantly reduced specific leaf area of young vegetable amaranth plants (amaranthus spp.) grown in pots. estimates of specific leaf area between 0.014 and 0.018 m2 g-1 are low compared to the range found in c3 cereals like wheat, barley and rice (0.020) or c3 dicots like oilseed rape (0.019-0.022) or sunflower (0.025-0.035) (boons-prins et al., 1993). however, the specific leaf area of amaranth is similar to values of c4 monocots like maize (0.016, boons-prins et al., 1993) and sorghum (0.019, van heemst, 1988), and it is higher than the specific leaf area of sugarcane (0.08-0.012, van heemst, 1988). measurements of the thin and tiny cotyledons at emergence resulted in high specific leaf area which was similar to cotyledons of maize and sorghum (boonsprins et al., 1993; van heemst, 1988). growth rate of leaf area the original version of lintul calculates leaf area expansion as an exponential function of temperature from emergence onwards till a leaf area index of 0.75 or a temperature sum of 330°c d is reached. thereafter, leaf area is assumed to expand in proportion to the increase in leaf dry weight. the analysis of leaf area expansion versus thermal time up to a leaf area index of 1.0 showed that the relative leaf area growth rates of potato, winter and spring wheat were 0.018, 0.007 and 0.011°c-1 d-1, respectively (van delden et al., 2000). compared to these crops, the initial relative growth rate of the tiny amaranth leaves between 0.027 and 0.030°c-1 d-1, as estimated from our data, is remarkably high which could be connected with the comparatively high base temperature of 8°c for amaranth. it is critical to define the right switching point from temperatureto radiation-driven leaf expansion, and differences between crops arise. van delden et al. (2001) found that model performances were best when using a threshold leaf area index of 1.0 in potato and of 1.5 in wheat. presumably a crop-specific switching point determined for grain amaranth might improve model performance. changing model parameters towards a later switching point resulted in a reduced initial growth rate and subsequently more accurate model predictions especially with respect to leaf area (data not shown). biomass allocation patterns when comparing allocation patterns of grain amaranth with those determined for maize (farré et al., 2000), clear differences become obvious. the dry matter fraction allocated to roots in amaranth was only half of the fraction found in maize. to some extent, our comparatively rough method of root sampling might account for this effect. outstanding in grain amaranth is the high fraction of biomass partitioned to leaves at emergence and the even higher fraction allocated to leaves up to the sampling date where plants have up to 15 leaves and reach a plant height of about 10 cm, i. e. at about 200 °c d. model testing the time course of total biomass production of grain amaranth was simulated quite well by lintul for both genotypes in both tested environments. total biomass at harvest also was predicted quite satisfactorily. the determined maximum leaf area index of the tested amaranth genotypes between 1.2 and 2.3 is rather low compared to values reported by others. kruse (1996) for example recorded a leaf area index of more than 4 in amaranth stands sown at a row distance of 30 cm. outstanding is the severe overestimation of leaf area by the model, especially in the simulations of crop growth in 2004 due to the parameter estimates based on field data of 2005. the high specific leaf area and the high light use efficiency found under field conditions in 2005 promoted these overestimates of the leaf area as well as the lintul for amaranth 191 high allocation of biomass to leaves in early growth stages. when analysing the growth of maize by using the model lintul 2 (including water limitations), farré et al. (2000) also found that the leaf area index was overestimated. yet, this was only obvious in suboptimal irrigation treatments due to underestimated leaf senescence. when calibrating the closely related model wofost for several crops in different environments, boons-prins et al. (1993) stressed that it was often necessary to adopt dry matter partitioning especially when too much leaf area was calculated while at the same time the produced storage organs were underestimated. however, in our study the properly simulated grain yields and the highly overestimated leaf weights conflict with a simple shift of dry matter from leaves to storage organs. the agreement between observed and simulated grain yield was not as accurate as the prediction of total biomass. the model underestimated the steep dry matter increase at the beginning of grain formation. this fact might indicate that not only current assimilates are used for grain filling, but also mobilised assimilates from other plant organs. in lintul it is assumed that grain filling is exclusively based on current photosynthesis. as already stressed by spitters (1990), this assumption does not hold for a number of crops, e.g. cereals. it is well known that reserve assimilates stored in other plant organs (e.g. in stems) may contribute to the growth of storage organs. to include these translocation processes it would be necessary to add an algorithm to the original lintul model. habekotté (1997) included an additional state variable for reserve assimilates in the model lintul-brasnap for rape. when current assimilation cannot not meet the growth capacity of the seeds, additional assimilates are supplied from this reserve pool. it may be assumed that similar adaptations of lintul for amaranth would result in more accurate predictions of grain yield formation. conclusions the parameterisation of lintul for amaranth yielded reasonable and significant values. parameters were affected by environmental conditions. differences between years could be noted especially for light use efficiency and specific leaf area. obviously, accurate model predictions require model parameterisation under adequate environmental conditions. the estimated light use efficiency was slightly lower than found earlier in amaranth and low compared to other c4 crops like sugarcane or maize, but similar to sorghum. it was also lower than the light use efficiency of cereals. the determined light extinction coefficient was rather high compared to other dicotyledonous plants. specific leaf area was slightly lower than values found in cereals or oilseed rape. lintul properly simulated the production of total biomass of grain amaranth under potential growth conditions. total biomass at harvest and also the time course of biomass formation were predicted accurately. grain yield levels at harvest were simulated less well, yet satisfactorily. model simulations revealed that leaf weight and leaf area throughout the period from heading to seed setting were overestimated while the sharp rise in grain yield at the beginning of yield formation was underestimated. presumably reserve assimilates from leaves or other plant organs contribute to the production of grain yield. in summary, the simple model lintul can simulate the progress of growth and yield formation of amaranth rather exactly. however, model predictions of grain yield are hardly precise enough for using the model as a crop management tool on individual fields. acknowledgements acknowledgements are due to the staff of the institute of agronomy and plant breeding, especially to m. schütze, h. heschel and j. schodl, and to the staff of the experimental farm groß-enzersdorf, especially to k. ofner and h. ulrich. references aufhammer, w., kübler, e., 1998: vergleichende untersuchungen zur anbauwürdigkeit der getreidearten rispenhirse (panicum milleaceum) und kanariensaat (phalaris canariensis) sowie der pseudogetreidearten buchweizen (fagopyrum esculentum), reismelde (chenopodium quinoa) und amarant (amaranthus sp.). bodenkultur 49, 3, 159-169. boons-prins, e.r., de koning, g.h.j., van diepen, c.a., penning de vries, f.w.t., 1993: crop specific simulation parameters for yield forecasting across the european community. simulation 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integrated resources for evaluating numerical estimates. research institute for industrial crops, via di corticella 133, bologna, italy. http:/ /www.isci.it\tools (03.09.2007). gimplinger, d.m., schulte aufì m erley, g., dobos, g., kaul, h.-p., 2008: optimum crop densities for potential yield and harvestable yield of grain amaranth are conflicting. eur. j. agron. 28, 119-125. gramig, g.g., stoltenberg, d.e., 2007: leaf appearance base temperature and phyllochron for common grass and broadleaf weed species. weed technol. 21, 1, 249-254. habekotté, b., 1997: evaluation of seed yield determining factors of winter oilseed rape (brassica napus l.) by means of crop growth modelling. field crop res. 54, 137-151. jamriška, p., 1996: vplyv odrôd na úrodu semena láskavca (amaranthus sp.). rostlinná výroba 42, 3, 109-114. kaul, h.-p., kruse, m., aufhammer, w., 2000: yield and radiation use efficiency of pseudocereals compared with oats. pflanzenbauwissenschaften 4,1, 9-14. kigel, j., 1994: development and ecophysiology of amaranths. in: paredeslópez, o. (ed.), amaranth: biology, chemistry, and technology, 39-73. crc press, boca raton. kruse, m., 1996: vergleichende untersuchungen zur lichtund stickstoffnutzung von amarant-, reismeldeund buchweizenbeständen. phdthesis, university of hohenheim. cuvillier, göttingen. kubasek, j., setlik, j., dwyer, s., 2007: light and growth temperature alter carbon isotope discrimination and estimated bundle sheath leakiness in c4 grasses and dicots. photosyn. res. 91, 47-58. liu, f., stützel, h., 2004: biomass partitioning, specific leaf area, and water use efficiency of vegetable amaranth (amaranthus spp.) in response to drought stress. sci. hort. 102, 15-27. ludlow, m. m., 1975: effect of water stress on the decline of leaf net photosynthesis with age. in: marcelle, r. 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(eds.), simulation and systems management in crop protection. pudoc, wageningen, 147-181. van delden, a., pecio, a., haverkort, a. j., 2000: temperature response of early foliar expansion of potato and wheat. ann. bot. 86, 355-369. van delden, a., kropff, m. j., haverkort, a. j., 2001: modelling temperatureand radiation-driven leaf area expansion in the contrasting crops potato and wheat. field crop res. 72, 199-142. van heemst, h.d.j., 1988: plant data values required for simple crop growth simulation models: review and bibliography. simulation reports cabott no. 17, wageningen. van ittersum, m.k., leffelaar, p.a., van keulen, h., kropff, m.j., bastiaans, l., goudriaan, j., 2003: on approaches and applications of the wageningen crop models. eur. j. agron. 18, 201-234. van keulen, h., stol, w., 1995. agro-ecological zonation for potato production. in: haverkort, a.j., mackerron, d.h.l. (eds.), potato ecology and modelling of crops under conditions limiting growth, 357-371. kluwer academic publishers, dordrecht. wolf, j., van oijen, m., kempenaar, c., 2002: analysis of the experimental variability in wheat responses to elevated co2 and temperature. agric. eco-syst. environm. 93, 227–247. address of the authors: dr. daniela m. gimplinger and prof. dr. hans-peter kaul, institute of agronomy and plant breeding, department of applied plant sciences and plant biotechnology, university of natural resources and applied life sciences, gregor-mendel-straße 33, a-1180 vienna, austria. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true 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/pagesize [907.087 680.315] >> setpagedevice art13_saha.indd short-title: igr activity of thevetia nerifolia journal of applied botany and food quality 85, 212 215 (2012) 1national institute of research on jute and allied fiber technology, kolkata, west bengal 2assitant director of agriculture, department of agriculture, govt. of west bengal 3indian institute of natural resins and gums, ranchi, jharkhand 4division of agricultural chemicals, indian agricultural research institute, new delhi, india insect growth regulatory activity of thevetia nerifolia juss. against spodoptera litura (fab.) deb prasad ray1*, debashis dutta2, s. srivastava3, b. kumar4, supradip saha4 (received august 11, 2012) * corresponding author summary screening for insect growth regulatory activity (igr) of thevetia nerifolia leaf extracts were evaluated against spodoptera litura (fab.). methanol extract of leaves provided 53.8 % larval mortality, 29.6 % pupation and 22.3 % adult emergence at 2.5 % concentration level. the extract was further subfractioned with solvents of different polarity in search of better igr activity and chloroform extract was found to be most active in terms of larval mortality (27.5-61.5 %), pupation (28.4-60.2 %) and adult emergence (19.8-52.8 %). gi50 of the extract was recorded to be 3.02 %. activity was attributed to the glycosides present in the extract. introduction out of several agriculturally important insects, spodoptera litura is an economically important polyphagous pest in india, china, and japan. it is causing considerable economic loss to many vegetable and fi eld crops. it is a polyphagous insect present in high numbers in tropical countries even during rainy seasons with at least 48 families (ulrichs and mewis, 2004). it is the fi rst lepidopteron pest to develop resistance in india (kumar and reghupathy, 2001; srivastava and joshi, 1965). therefore, an alternative to chemical pesticide is imminent to control the pest. botanical pesticides are projected as the key plant protection in the new millennium. they are relatively safe and viable alternative to synthetic pesticides as they reduce or prevent insect feeding. numerous plant species have been reported to possess pest control properties but only a few seem to be ideally suited for practical utilization. yellow oleander (thevetia sp.) is a small tree belongs to apocynaceae family. it is one of the unexplored plant species for insecticidal or insect growth regulatory activity, though other biological activities were studied (ray et al., 2009; oji and okafor, 2000; ambang et al., 2007; gata gongalves et al., 2003). satpathi and ghatak (1990) found that methanolic extracts of seeds of thevetia nerifolia merr, at 1.0 per cent resulted in 100 per cent mortality of fourth instar larvae of p. xylostella, 12-24 h after treatment when applied topically. pesticidal property of thevetia sp. was reported against diamond back moth and other agriculturally important insect pests (lingappa et al., 2004; freedman et al., 1979; mclaughlin et al., 1980; reed et al., 1981 and 1982). igr and larvicidal activity of thevetia sp. was evaluated against two species of mosquito and found good larvicidal but very little igr activity (lapcharoen et al., 2005. thevetia neriifolia leaf extract was evaluated against tribolium confusum adults and it was found that acetone extract was found to be the most effective toxicant followed by ethyl acetate, petroleum ether and methanol extracts (khanam et al., 1995). antifertility potential of thevetia peruviana in male albino rats was also reported (gupta et al., 2011). the present study was therefore undertaken to evaluate the insect growth regulatory and larvicidal action of the thevetia nerifolia leaf extracts against spodoptera litura (fab.). materials and methods plant material fresh leaves of thevetia nerifolia were collected from paschim medinipur district of west bengal. the botanical identifi cation of the plant was done at department of botany, vidyasagar university, paschim medinipur, west bengal, india. insect rearing insect cultures of spodoptera litura (f.) (noctuidae: lepidoptera) were maintained in the laboratory on castor leaves (ricinus communis l.). rearing conditions were a 12 h photo regime at 28±1 °c and 60 % relative humidity. insect cultures were continuously refreshed with new one, found in the vicinity of the research farm of the indian agricultural research institute, new delhi, india. preparation of extracts leaves (2 kg) were chopped and extracted by soaking with hexane (3 × 3 lit) for overnight at room temperature in order to remove fatty materials. hexane extract was collected and evaporated under vacuum at < 40 °c using rotary evaporator (heidolph, germany). hexane extracted plant materials were then extracted with methanol at 50 °c. extract was fi ltered and concentrated under vacuum to obtain a viscous residue. further fractionation of methanol extract was done using hexane, chloroform and ethyl acetate. each extract was collected and concentrated separately under vacuum at <45 °c using rotary evaporator. contact toxicity dry fi lm method was adopted for bioassay of extracts. one milliliter of extract and fraction solution was poured in each rimless glass test tube (25 x 200 mm). the tubes were rotated gently at 100 rpm using hand rotating wheel. angle of the tubes was adjusted in such a way that the solution covered three-fourth of the inner surface of the tube. the process was continued till the solution dried up leaving behind a uniform fi lm of extract on the inner glass surface. dry fi lm with acetone was prepared in a similar manner as a control. first instar larvae (12 h old) of spodoptera litura were released using a hair brush, the tubes were plugged with cotton and wrapped in muslin cloth. the larvae were allowed to remain in contact with dry fi lms or 4 h and mortality was recorded thereafter. three replications per treatment were used for the experiment. moribund larvae were also counted as dead one. growth regulatory activity the test compounds (500 mg) were weighed accurately in a 5 ml volumetric fl ask and dissolved into 0.5 ml of suitable solvent. the volume was then made to 5 ml to obtain 10 % stock solution. from these stock solutions, different concentrations (0.5, 1.0, 2.0, 3.0 and 5.0 %) were prepared separately by serial dilution with 0.5 % emulsifi ed water, which in turn was prepared by dissolving 5ml of tween 80 emulsifi er in 1 l of distilled water. short-title: igr activity of thevetia nerifolia 213 for insect growth regulatory activity, third-instar larvae of spodoptera litura weighing between 30 and 60 mg were treated with various concentrations of the test emulsions under potter’s direct spray tower at a pressure of 340 g cm-2. the sprayed dishes were dried for fi ve min under a fan after which the larvae were transferred to separate rearing bottles. the larvae, similarly sprayed with emulsifi ed water, served as a control. larval weight was taken at 3 and 7 days after treatment. ten replications were used per dose for the test. the raw data on different parameters were subjected to angular transformation (arc sine percentage)1/2 and analyzed statistically by complete randomized design. analysis of variance was done, and means were separated by square difference, i.e., critical difference. inhibition concentration (ic50) was determined based on probit analysis. insect growth regulatory activity (igr) was calculated from percent reduction in larval weight gain over control and calculated as igr (%) = [(weight gain in control-weight gain in treatment)/ weight gain in control] × 100 observations on the biological parameters viz, larval survival, weight of full grown larvae, larval duration, pupation and adult emergence were also recorded. data analysis growth inhibition (gi50) values were calculated by using a basic ld50 program version 1.1 as described by trevors, 1986. the raw data on different parameters were subjected to angular transformation (arc sine percentage) and analyzed by complete randomized design. analysis of variance was done, and means were separated by critical difference (cd) using the statistical package for social sciences (spss, version 10). results larval mortality by contact toxicity was presented in tab. 1. as extraction concentration increased, toxicity indices also increased, in a dose-dependent manner. perusal of the data revealed that with increase in concentrations, there was increased larval mortality. lc50 of methanol extract was recorded as 2.54 %, wheras hexane extract did not show any contact toxicity. among subfractions of methanol extracts, lc50 ranged between 1.14 to 2.54 %. among the sub fractions, chloroform extract showed maximum activity and water extract provided maximum survival of the larvae. thevetia leaf extracts lowered food consumption and reduced larval growth rate, which was measured in larval weight reduction and calculated as growth inhibition percentage (tab. 2). hexane extract provided no growth regulatory activity upto 1.5 % concentration, wheras methanol extract showed promising igr activity against spodoptera litra. igr activity ranged between 9.9 to 31.4 % in 0.5-2.5 % concentration. among the four sub fractions of methanol extract, chloroform provided best and hexane showed least igr activity. igr activity ranged between 9.4 to 46.8 % in the concentration range between 0.5-2.5 %. gi50 of methanol extract was 7.20 % and among its subfractions, chloroform extract recorded gi50 of 3.02 % (tab. 3). perusal of the data revealed that the methanloic extract showed the mean larval mortality varied from 19.8 to 53.8 % (tab. 4). among subfractions, all the extracts showed similar range of bioactivity. larval mortality ranged between 24.5 to 71.2 % across the solvent polarity range. water extract comparatively provided least survival of the fi rst instar larvae. the extracts were also infl uenced the pupation process also (tab. 4). pupation varied between 29.6-78.2 % in methanol extract and hexane extract provided no effect on pupation. pupation percentage ranged between 20.4 to 60.2 % across subfractions. in terms of adult emergence, ethyl acetate subfraction of methanol provided least adult emergence. it ranged between 14.846.5 % upon application of 0.5-2.5 % of the extract. hexane and chloroform fractions of methanol extract recorded comparatively lesser impact on adult emergence of spodoptera litura. discussion contact toxicity of the leaf extracts were found not so promising. chloroform and hexane subfractions of methanol extract showed comparatively better activity than other extracts. compounds responsible for the activity seem to be of medium polarity range. el-shazly, 2000 also used fractionation of ethanolic extract of nerium oleander for screening of insecticidal activity. neriifolin was also isolated from the fractionation of the extract. thevetia sp. also reported to have the same cardiotonic glycoside, neriifolin (mclaughlin et al., 1980). ethanol extract of thevetia peruviana provided moderately good mosquito larvicidal activity and the lc50 ranged between 211.4-346.4 mg l-1 (lapcharoen et al., 2005). these fi ndings are supported by evans and kaleysa raj (1988). though the investigation studied the effect using aqueous extract of the plant. these studies except lapcharoen et al. (2005) did not mention any effects on molting and metamorphosis of the test insect. t. tab. 1: contact toxicity of different extracts of thevetia nerifolia leaf against fi rst instar larvae of spodoptera litura. extracts lc50 χ2exp fiducial limit regression equation (%) (3df, 95%) methanol 2.54 7.09 2.11-3.05 y = 4.36+1.58x methanol-hexane 1.16 5.15 1.04-1.31 y = 4.84+2.45x methanol-chloroform 1.14 0.69 0.99-1.31 y = 4.16+1.75x methanol-ethylacetate 1.27 7.22 1.15-1.40 y = 3.85+1.68x methanol-water 1.79 1.92 1.53-2.08 y = 4.51+1.93x tab. 2: insect growth regulatory (%) activity of thevetia nerifolia against s. litura. conc [%] hexane methanol methanol fractions hexane chloroethylwater form acetate 0.5 9.9 4.2 9.4 5.8 9.8 1.0 13.4 5.8 20.3 13.4 12.7 1.5 18.2 15.9 26.1 17.7 22.3 2.0 6.5 26.1 24.3 39.0 29.4 35.2 2.5 10.6 31.4 27.4 46.8 31.6 38.9 214 d.p. ray, d. dutta, s. srivastava, b. kumar, s. saha short-title: igr activity of thevetia nerifolia peruviana did not show any igr properties against two mosquito species (lapcharoen et al., 2005). our fi ndings are in contrast with the earlier study. igr activity was recorded in fi rst instar larvae of s. litura. feeding detergency was also observed in the fi rst instar larvae of s. litura and the igr activity might be attributed to the antifeedancy of the extracts. probable mode of action is likely to be juvenile hormone mimics like juvocimenes of sweet basil, miroesterol derivatives of pueraria mirifi ca etc.. though we could not present the antifeedant data but feeding detergency was observed, which is in agreement with reed et al., 1982. glycoside from t. nerifolia like neriifolin, thevetin a & b, peruvoside was found to be major ingredient for acting as feeding deterrent to the striped cucumber beetle, acalymma vittatum. glycosides like triterpenic saponins were found to have antifeedant and igr activity against spodoptera litura (saha et al., 2010). larval, pupal and adult survival was infl uenced by the persistence of the active molecules in the insect hemolymph (tab. 4). igr activity was characterized by initial larval mortality followed by mortality of pupa and adult. elongation of pupal period was not clearly observed, though minor changes in the pupation were recorded as compared to control insects. larval and pupal mortality during the respective stages, and molting could either be due to the presence of toxic ingredients in the extract or the imbalances between growth stimulating and growth-inhibiting hormones. further studies are needed to establish a more exact explanation for the cause of death. it is not clear whether this is due to mimicking of the juvenile hormone, to blocking hormone degradation, or to some other physiological interference. various functions occurring during molting and metamorphosis may be affected, such as cuticle tanning and tab. 3: gi50 of different solvent extracts against spodoptera litura. extracts gi50 χ2exp fiducial limit regression equation (%) (3df, 95%) methanol 7.20 1.78 3.28-15.8 y = 3.96+1.22x methanol-hexane 5.67 3.37 3.39-9.46 y = 3.64+1.80x methanol-chloroform 3.02 1.12 2.29-3.97 y = 4.16+1.75x methanol-ethylacetate 4.82 1.31 3.01-7.71 y = 3.85+1.68x methanol-water 4.10 3.26 2.74-6.11 y = 4.02+1.60x tab. 4: igr activity of thevetia nerifolia leaf extracts against spodoptera litura. treatments concentration larval mortality pupation adult emergence (%) (%) (%) (%) methanol extract 0.5 19.8 78.2a (62.2) 61.3a (51.3) 1.0 22.6 72.2a (58.2) 50.1b (45.1) 1.5 27.5 58.3b (49.8) 49.2b (44.5) 2.0 36.7 42.5b (40.7) 38.1c (38.1) 2.5 53.8 29.6c (32.9) 22.3d (22.2) methanol-hexane fr. 0.5 24.5 56.5a (48.7) 45.3a (42.3) 1.0 39.5 53.3a (46.8) 42.3a (40.7) 1.5 48.4 42.7b (40.8) 35.3b (36.5) 2.0 55.8 39.2b (38.7) 28.2c (32.1) 2.5 59.8 33.4b (35.3) 21.4d (27.6) methanol-chloroform fr. 0.5 27.5 60.2a (50.9) 52.8a (46.6) 1.0 30.4 48.8b (44.3) 40.6b (39.5) 1.5 41.8 39.1b (38.7) 30.2c (33.3) 2.0 57.1 30.6c (33.5) 22.6d (28.4) 2.5 61.5 28.4c (32.2) 19.8d (26.4) methanol-ethylacetate fr. 0.5 27.0 57.5a (49.3) 46.5a (43.0) 1.0 38.4 48.5b (44.1) 39.5b (38.9) 1.5 47.7 36.2c (36.7) 30.2c (33.3) 2.0 61.8 26.5d (30.9) 18.5d (25.4) 2.5 66.4 20.4d (26.9) 14.8d (22.6) methanol-water fr. 0.5 29.5 56.8a (48.9) 46.8a (43.2) 1.0 39.8 52.3a (46.8) 40.2b (39.3) 1.5 51.9 41.8b (40.3) 32.1c (34.5) 2.0 67.5 29.8c (33.1) 22.5d (28.3) 2.5 71.2 23.4c (28.9) 19.6d (26.3) figure in parentheses are arc sin transformation values. subgroup columns sharing the same letter are not signifi cantly different (p < 0.05) d.p. ray, d. dutta, s. srivastava, b. kumar, s. saha short-title: igr activity of thevetia nerifolia 215 hardening, but actual target of action needs more study to confi rm. with the present state of knowledge, it can be concluded that igr actions of the methanol extract was clear and might be attributed to the glycosides present in the extract. acknowledgement the authors are thankful to dr. s. saha for helping in preparation of manuscript and head, division of agricultural chemicals, indian agricultural research institute, new delhi for laboratory facilities to pursue the research work. references ambang, z., bekolo, n., petga, e., ngohdooh, j.p., asnga, a., 2007: effect of crude extracts of thevetia peruviana seeds on development of leaf spot disease of groundnut caused by cercospora sp. african crop sci. conf. proc. 8, 797-800. el-shazly, m.m., el-zayat, e.m., hermersdorfer, h., 2000: insecticidal activity, mammalian cytotoxicity and mutagenicity of an ethanolic extract from nerium oleander (apocynaceae). annals appl. biol. 136, 153-157. evans, d.a., kaleysa, r.r., 1988: extracts of indian plants as mosquito larvicides. ind. j. med. res. 88, 38-41. freedman, b., nowak, l.j., kwolek, w.f., berry, e.c., guthrie, w.d., 1979: a bioassay for plant-derived pest control agents using the european com borer. j. econ. entomol. 72, 541-545. gata gongalves, l., nogueira, j.m.f., matos, o., sausa, r., 2003: photoactive 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(lepidoptera: noctuidae). ind. j. ent. 27, 102-104. trevors, j.t., 1986: a basic programme for estimating ld50 value using the ibm-pc. bull. environ. contam. toxicol. 37. ulrichs, c.h., mewis, i., 2004: seasonal abundance of two armyworm species, spodoptera exigua (h€ubner) and spodoptera litura (f.) in the philippines. commun. agric. appl. biol. sci. 69, 323-328. address of the authors: deb_iari@rediffmail.com journal of applied botany and food quality 87, 175 181 (2014), doi:10.5073/jabfq.2014.087.025 1department of pharmacy, faculty of medicine, university of niš, serbia 2institute for biology and human genetics, faculty of medicine, university of niš, serbia 3department of ecology and biology, faculty of sciences, university of niš, serbia assessment of polyphenol content, in vitro antioxidant, antimicrobial and toxic potentials of wild growing and cultured rue dragana r. pavlović1*, marija vukelić2, stevo najman2, milica kostić1, bojan zlatković3, tanja mihajilov-krstev3, dušanka kitić1 (received march 12, 2014) * corresponding author summary ruta graveolens l. (rue) is an edible medicinal plant that is traditionally used in various countries. this study aimed to investigate and compare the phenolic content, antioxidant capacity, antibacterial and cytotoxic activities of the methanolic and ethanolic extracts of wild growing and cultured rue. the total phenolic content of the tested extracts varied from 57.90 to 166.91 mg of catechin equivalent (ce)/g of extract and the total flavonoid content from 4.18 to 26.87 mg of rutin equivalent (ru)/g of extract. all the tested samples exhibited significant antioxidant potential in dpph radicals scavenging and lipid peroxidation inhibition assays (comparable with activity of rutin in the same test systems), antimicrobial activity determined by microdilution method (particularly against gram (+) bacteria strains) and ability to induce inhibition of hela cells growth and proliferation (up to 71.81 %). in addition, rue-treated hela cells showed various morphological changes after 72 h of incubation with rue extracts. extracts from wild growing rue with the highest polyphenol, tannin and flavonoid contents demonstrated the strongest activities in all tested systems. the present study also emphasized the fact that the rue leaves and herb should be harvested at the beginning of blossoming stage in order to achieve the maximal level of secondary metabolites and optimal pharmacological effects. introduction ruta graveolens l. (rutaceae), rue, is a shrub-like herbaceous perennial aromatic plant that is native to mediterranean region (townsend, 1968). it is cultivated in the gardens all over the world, though preferably grows in mediterranean climates. rue is highly used in the traditional medicine in various countries to treat a variety of ailments, ranging from absence of menstruation to rheumatism and various mental conditions (pdr, 2000; tucakov, 1997). due to the lack of sufficient evidence of the efficacy of the drug in the proposed indications and the unfavorable ratio of benefit to risk, the commission e gave the drug, rue leaves and herb, a negative rating. rue can cause contact dermatitis, phototoxic reactions, severe liver and kidney damage, while serious undesirable effects can occur even after administration of therapeutic doses (blumenthal, 1998; fritz weiss and fintelmann, 2000). this edible plant has been part of east asian diets for many years and has dual function as food and medicine (yang et al., 2006). in the european union, r. graveolens herb may be used as spice and flavoring agent in certain food products as for instance baked goods, frozen dairy products, soft candy or non-alcoholic beverages. r. graveolens and its essential oil have been approved for generally recognized as safe status by the united states food and drug administration and may be added to human food as flavoring agents (up to 2000 and 10 000 μg/kg, respectively). rue herb and oil are also allowed in the united states in animal feedstuffs at the same levels (ema, 1999). the plant contains active compounds like flavonoids, alkaloids, coumarin derivatives, lignans and essential oils (pdr, 2000). the drug (rue herb and/or leaves) is antimicrobial, abortifacient, and photosensitizing. as the current information shows, it expresses pharmacological functions including anti-inflammatory, analgesic, antiandrogenic, antihyperlipidemic, antihyperglycemic, xantine oxidase inhibition and anticancer activities, among others (asgarpanah and khoshkam, 2012; yang et al., 2006). vitkova and philipov (1999) conducted a comparative phytochemical study of rue with material from natural bulgarian populations of the species and from cultivated specimens. their conclusion was that cultivation of rue revealed changes in the second metabolite composition. however, as far as our literature survey could ascertain, our study is the first comparative study of effects of wild growing and cultured ruta graveolens. the focus of our research was to explore and compare antimicrobial and antioxidant activity alongside phenolic contents of ethanolic and methanolic extracts of plants collected from two different localities (rocky slopes and private garden). additionally, the antiproliferative/cytotoxic activity of these extracts on the human cervix adenocarcinoma hela s3 cell line was included in this study, due to the fact that rue is well-known traditional uterine remedy (pdr, 2000; tasić et al., 2004; tucakov, 1997). therefore the study sought to elucidate the impact of plant growth conditions and geographical location on these properties. material and methods general plant materials were collected from ruta graveolens l. (rutaceae) in 2007 at two different locations: 1) sićevačka gorge, sićevo (e serbia), cca. 590 m a.s.l., rocky and bushy slopes at the limestone ground; gps coordinates: 43°20’34.60”n; 22°6’30.79”e; 2) niš (novo selo), (se serbia), cca. 180 m a.s.l., cultivated in the garden; gps coordinates: 43°19’3.39”n; 21°48’44.41”e. the aerial parts of the wild growing and cultivated plants were collected at the beginning of the flowering season and the aerial parts of the wild growing plants were also collected at the end of the flowering season. the plant was identified and authenticated by the taxonomist dr. bojan zlatković. the voucher herbarium specimens (accession number 3106 hff) have been deposited in the herbarium collection of the faculty of pharmacy, university of belgrade. the plant material was reduced to a fine powder and extracted with ethanol (70 %, v/v) or methanol (80 %, v/v) by percolation, as described in european pharmacopeia 6.0.10 (ph. eur. 6.0, 2007). ethanolic and methanolic extracts of wild growing plants collected at the beginning (re and rm) and at the end of the flowering season (r2e and r2m), and of cultured plants (rde and rdm) were obtained after evaporation to the dryness under reduced pressure below 40 ºc. the reference chemicals such as rutin and catechin used for calibration curves were purchased from sigma-aldrich (st. louis, usa), 176 d.r. pavlović, m. vukelić, s. najman, m. kostić, b. zlatković, t. mihajilov-krstev, d. kitić and cisplatin as commercial cytostatic from pfizer (australia). all other chemicals, reagents and solvents used were of analytical grade. spectrophotometric measurements were performed using evolution 60 thermo scientific spectrophotometer (fisher scientific, uk) and multiskan ascent no354 (thermolabsystems, finland) elisa microplate reader. incuterm raypa®trade (catalonia, spain) and jouan, eg 110ir (saint herblain, france) were used for incubation and vibramix 30 (tehtnica, slovenia) for shaking of microplates. phenotype changes of hela cells were evaluated by invert microscope (observer z1, carl zeiss, germany). all results are presented as mean ± standard deviation. data obtained by mtt assay after hela cells treatment with rue extracts were compared to control (untreated cells) using paired sample ttest. statistical analysis was performed with commercial statistical software package. values of p<0.01 and p<0.001 were accepted for statistical significance and marked on figures as * and **, respectively. determination of constituents determination of total flavonoids the content of total flavonoids was determined spectrophotometrically using aluminium chloride (lamaison and carnat, 1990). quantification was done on the basis of a standard curve with rutin and results were expressed as mg rutin (ru)/g of dry extract. determination of total polyphenols the total phenolic content of extracts was estimated using the folin-ciocalteu colorimetric procedure (makkar et al., 2000). quantification was performed using a standard curve with (+)-catechin (concentration span 0.1-1 mg/ml). the results were expressed as catechin equivalents (mg ce/g extract) per g of sample. determination of total tannins total tannins content was determined by the same folin-ciocalteu procedure after removal of tannins by their adsorption on insoluble matrix – polyvinylpolypyrrolidone (makkar et al., 2000). the assay was carried out with clear supernatant and results were expressed as mg ce/g of sample. determination of antioxidant capacity dpph assay the free radical scavenging activities were determined using a stable 1,1-diphenyl-2-picrylhydrazyl (dpph) free radical (cuendet et al., 1997). various concentrations of samples were mixed with methanol or ethanol solution of dpph˙ (0.05mm), vigorously shaken and left in the dark at room temperature for 30 min. inhibition of dpph free radical in percent was calculated according to: % dpph= (ab – as /ab) x 100, where ab is the absorbance of the control reaction (containing ethanol or methanol instead of test solution), and as is the absorbance of the sample. dose response curves were constructed and dpph results calculated as the concentration of sample required for scavenging 50 % of the free radical (ic50). concentrations are expressed in mg/ml. rutin was used as reference compound. inhibition of lipid peroxidation inhibition of lipid peroxidation in β-carotene-linoleic acid assay was determined according to the slightly modified method described by koleva et al. (2002). in brief, 200 μl of freshly prepared β-carotenelinoleic acid emulsion were added to 20 μl of sample (different concentrations of ethanol extracts in ethanol, 70 % v/v, or methanol extracts in methanol, 80 %, v/v) in each well of 96-well microtitration plate. samples were prepared in triplicate for each concentration used. the plate was shaken on a microplate shaker and read in microplate reader using a 450 nm filter immediately (t = 0 min) and after 120 min (t = 120 min) of incubation at 55 °c. the percentage inhibition of β-carotene bleaching by the samples was calculated according to formula (baros et al, 2007): % inhibition = (a120 / a0) x 100 where a120 is the absorbance of the sample at t = 120 min and a0 is the absorbance of the sample at t = 0 min. ic50 value, which denotes the concentration of sample that is required for decreasing the β-carotene bleaching at 50 %, was calculated and expressed in mg/ml. rutin was used as reference compound. assessment of antimicrobial activity for the antibacterial bioassays six bacterial strains were used, three gram-positive: bacillus cereus (atcc 10876), listeria monocytogenes (atcc 15313) and staphylococcus aureus (atcc 6538), and three gram-negative: escherichia coli (atcc 25922), pseudomonas aeruginosa (atcc 27853) and salmonella enteritidis (atcc 13076). candida albicans (atcc 10231) and aspergilus niger (atcc 16404) were used for antifungal estimation. all microorganisms were from the american type culture collection (atcc). the bacterial and fungal inoculates were made up from overnight broth cultures. suspensions with microorganisms were adjusted to 0.5 mcfarland standard turbidity according to consensus standard of the national committee for clinical laboratory standards (nccls, 2003). determination of mic (minimum inhibitory concentration) and mbc/mfc (minimum bactericidal/fungicidal concentration) was carried out by micro-well dilution method according to the nccls (2003). serial doubling dilutions of the tested extracts were prepared in the 96-well microtiter plates in inoculated nutrient broth. the final volume was 100 μl and the final bacterial concentration was 2x106 cfu/ml in each well and 2x105of spores for fungal strains. the plates were incubated for 24 h at 37 °c for bacteria and 48 h at 25 °c for fungi. microbial growth was determined by adding 20 μl of 0.5 % triphenyl tetrazolium chloride (ttc) aqueous solution (sartoratto et al., 2004). the minimal concentration where there was no visible growth was defined as minimal inhibitory concentration (mic). for mbc/mfc determination broth was taken from each well and inoculated into mueller hinton agar at 37 °c for 24 h for bacteria or in malt extract agar at 25 °c for 48 h for fungal strains. mbc/mfc was defined as the lowest concentration of extract which had killed 99.9 % of microorganism cells (nccls, 2003). chloramphenicol, streptomycin, and nystatin were used as positive control for gram-positive bacteria, gram-negative bacteria and fungal strains, respectively. a 10 % solution of dimethyl sulfoxide (dmso) in water, used for extracts dissolving, was used as a negative control. determination of cell viability and proliferation viability and proliferation of hela s3 cells (human cervical adenocarcinoma cell line) after treatment with ethanolic and methanolic extracts of cultured and wild growing rue (both collected on the beginning of flowering period) were determined by mtt test (iso, 1998; kostić et al., 2008). dry extracts were dissolved in dmem (dulbecco’s modified eagle’s minimal essential medium, paa laboratories gmbh) enriched with l-glutamine, 100 iu/ml penicillin, 100 gu/ml streptomycin and 10 % comparative study of effects of wild growing and cultured ruta graveolens 177 fetal calf serum (fetal calf serum, gibco, united kingdom) to the concentration of 1 mg/ml (stock solutions), followed by filtration through syringe filters (pore diameter 0.2 μm, millipore, bedford, ma, usa). further, stock solutions were diluted in previously described culture media. final concentrations of the extracts were 0 (drug-free control), 0.1, 1, 10, 100 and 1000 μg/ml. for viability testing 1x105 cells in 0.1 ml of extracts were seeded in sterile 96well plate and incubated in a humidified incubator at 37 °c in an atmosphere of 5 % co2 for 24 hours. for proliferation testing 1x104 cells in 0.1 ml of extracts were seeded in sterile 96-well plate and incubated in a humidified incubator at 37 °c in an atmosphere of 5 % co2 for 72 hours. the medium in which cells were incubated was drawn at the end of the incubation and the cells were washed with 0.1 ml of pbs (phosphate buffered saline) and 0.02 ml of mtt was added. after 4 hour incubation at 37 ºc, the formazan crystals were dissolved with 0.1 ml of isopropanol. spectrophotometric measurement of the intensity of mtt reduction was carried out at optical density of 540 nm on the multi-channel photometer. to get inhibition of cell viability (%), which was the parameter of cytotoxic effects, and inhibition of cell proliferation (%), which was the parameter of cytostatic effects, a (absorbance at 570 nm) of a sample with cells grown in the presence of various concentrations of the extracts was subtracted and divided with control optical density (the a of control cells grown only in culture medium), and multiplied by 100. cisplatin was used as a positive control. mtt test was performed through three independent experiments in triplicates. hela cells morphological characteristics were observed after 72 h incubation with extracts under the invert microscope. results and discussion phenolic content since food phenolics from spices may augment the body’s source of natural antioxidants, their consumption could offer protection against chronic diseases caused by free radicals and could also augment cellular defenses against oxidative damage (otunola et al., 2013). also, the putative therapeutic effects of many traditional medicines have been ascribed to phenolics in particular due to their antioxidant activity. in order to compare wild growing and cultivated rue, the first approach was to quantify total phenolic contents in methanolic and ethanolic extracts alongside with their antioxidant activity. considerable amounts of phenolic compounds were found in rue extracts (tab. 1). the total phenolic content varied from 57.90 to 166.91 mg of catechin equivalent (ce)/g of extract and the total flavonoid content from 4.18 to 27.72 mg of rutin equivalent (ru)/g of extract. the tannin fraction represents more than half of the total polyphenolic compounds (from 50.45 % in rde to 61.71 % in rm). it was established that rutin content was influenced by the climatic condition. according to vitkova and philipov (1999), the aboveground parts of r. graveolens from experimental plots in bulgaria manifested a slightly enhanced quantity of rutin as compared to those from the natural populations. our results indicated three to four fold lower content of rutin in extracts obtained from cultivated compared to wild growing plants. also, we agreed with colleagues in conclusions that the above-ground parts of the plants had the highest rutin content at the beginning of blossoming and that quantity of tannins manifested a decrease in cultivated plants. we emphasize that extraction yields were notably higher for extracts obtained from wild growing rue. namely, extraction yields for ethanolic and methanolic extracts of wild growing plants collected at the beginning (re and rm) and at the end of the flowering season (r2e and r2m), and of cultured plants (rde and rdm) were 27.3, 23.3, 23.95, 19.45, 13.2 and 14.2 % (w/w), respectively. antioxidant activity antioxidant activity was determined through two complementary test systems: dpph free radical scavenging and inhibition of the lipid peroxidation in β-carotene-linoleic acid system. results are presented in tab. 1. according to dpph assay, our samples possess notable and quite similar anti-radical activity with ic50 range from 36.36±1.20 μg/ml (r2m) to 59.49±4.31 μg/ml (rde). all extracts also showed considerable, although less pronounced activity against lipid peroxidation. ic50 values in β-carotene-linoleic acid test system varied in wider extent: from 31.06±2.30 μg/ml (r2m) to 185.55±1.86 μg/ml (rde). methanolic extract of wild rue collected at the end of flowering season (r2m) exhibited the strongest antioxidant activity. its antioxidant effects are comparable and in the β-carotene-linoleic acid test system even stronger than antioxidant activity of rutin. ethanolic extract of wild rue collected at the beginning of flowering season (re) also exhibited significant activities in dpph and β-carotene-linoleic acid tests with ic50 values 36.41±1.37 μg/ml and 70.81±2.40 μg/ml, respectively. tab. 1: phenolic contents of rue extracts and ic50 values of ruta graveolens extracts and rutin in antioxidant assays. extract total polyphenols tannins flavonoids radical scavenging inhibition of lipid (mg ce/g) (mg ce/g) (mg ru/g) activity ic50 peroxidation ic50 (μg/ml) (μg/ml) rm 154.04±3.53 95.06±1.05 26.87±0.36 40.65±2.53 93.95±3.83 r2m 87.55±1.84 49.89±3.16 23.58±0.16 36.36±1.20 31.06±2.30 rdm 57.90±1.24 29.36±0.12 4.18±0.09 45.08±1.40 105.62±3.47 re 166.91±2.62 102.69±1.64 27.72±0.15 36.41±1.37 70.81±2.40 r2e 95.87±2.34 53.55±1.96 26.77±0.27 36.43±2.01 91.63±6.91 rde 71.72±2.35 36.18±2.62 7.10±0.07 59.49±4.31 185.55±1.86 rutin 2.68±0.25 33.29±1.49 rm – methanolic extract of wild growing plants collected at the beginning of the flowering season r2m – methanolic extract of wild growing plants collected at the end of the flowering season rdm – methanolic extract of cultured plants collected at the beginning of the flowering season re – ethanolic extract of wild growing plants collected at the beginning of the flowering season r2e – ethanolic extract of wild growing plants collected at the end of the flowering season rde – ethanolic extract of cultured plants collected at the beginning of the flowering season 178 d.r. pavlović, m. vukelić, s. najman, m. kostić, b. zlatković, t. mihajilov-krstev, d. kitić among the tested samples, extracts obtained from cultivated plants (rdm and rde) have the strikingly lower total polyphenols, tannins and flavonoids values. thus, cultivation of the rue revealed changes in the second metabolite composition. accordingly, as we expected, these extracts showed lower antioxidant activity in both applied tests. these differences were determined by the ecological factors of the environmental characteristic of the respective locations and the plants capability for adaptation to the new conditions. the climatic and the soil conditions in the area of sićevo george rise to considerable spring and summer water shortages. it is known that drought is a stress factor for plants and entails intensification of phenolic biosynthesis which apparently is equally valid for r. graveolens (kitić, 2006; tavarini and angelini, 2013). significant influence of environmental conditions on the phenolic compounds and consequently to antioxidant capacity of various fruits and medicinal plants have been well described, indicating the important role played by location, uv radiation, temperature, water stress and mineral nutrient availability in determining antioxidant capacity (tavarini and angelini, 2013). the lower flavonoid, total polyphenolic and tannin content in cultivated plants from novo selo could be explained with the greater precipitation in the region during the period of investigation, type of soils and the high level of underground water and water supply in garden conditions. antimicrobial activity the antimicrobial activity of methanolic and ethanolic extracts of r. graveolens collected at the beginning of flowering season was tested against selected microorganisms. the samples exhibited moderate antimicrobial activity against most of the bacterial and fungal strains tested. inhibitory effects on the growth of gram-positive bacteria were more potent (tab. 2.). methanolic rue extracts were active in inhibiting of the growth of all the gram-positive bacteria and p. aeruginosa (mic 25 or lower than 25 mg/ml) except low activity of rdm against strains of s. aureus (mic 100 mg/ml). the anti-microbial activity of rue methanolic extract against p. aeruginosa has already been reported and attributed to presence of alkaloids, flavonoids, terpenoids, steroids and tannins (benazir et al., 2011). according to ivanova et al. (2005) methanolic extract of cultivated rue and its fractions showed no activity against the gram-negative strain e. coli and the fungus c. albicans in disk diffusion method. same samples demonstrated a good antibacterial activity against staphylococcus aureus, streptococcus pyogenes, listeria monocytogenes and bacillus subtilis and no inhibition of corynebacterium diphtheriae. our results for rm and rdm antimicrobial activity string along these findings. for ethanolic extract of wild rue (re), the most prominent effect was achieved on listeria monocytogenes with equal concentrations of mic and mbc, 6.25 mg/ml. the lowest inhibitory activity was noticed for s. enteritidis (50 mg/ml), while mbc for all tested gramnegative bacterial species was not reached at the highest concentration applied (100 mg/ml). re exhibited the same antifungal activity on c. albicans and a. niger (mic=mbc 25 mg/ml). the major classes of secondary metabolites identified in rue herb were alkaloids, flavonoids, coumarin derivatives, lignans, terpenoids, primary and secondary alcohols, quinines, fatty acids, steroids and other minor components (benazir et al., 2011; pdr, 2000). besides methoxypsoralens, that are generally cytotoxic and have been widely reported to be fungitoxic, quinoline and quinolone alkaloids are important to the rue plant in defense against plant pathogens (oliva et al., 2003). as referent antimicrobial drugs, streptomycin, chloramphenicol and nystatin exhibited obviously higher antimicrobial activity than rue extracts which is additionally substantiated with obtained mic/ mbc values. however, extracts of wild rue showed a better antimicrobial activity than cultivated rue extracts and the most active of all tested samples was ethanolic extract of wild rue. influence on hela cell viability and proliferation the mtt test has been widely used as rapid and sensitive method for screening anticancer drugs as well as for the assessment of cytotoxic tab. 2: minimum inhibitory concentration (mic) and minimum microbicidal concentration (mmc) values for ruta graveolens extracts and positive controls. rm rdm re rde positive control* microorganism mic/mbc mic/mbc mic/ mbc mic/mbc mic/ mbc mg/ml/mg/ml mg/ml/mg/ml mg/ml/mg/ml mg/ml/mg/ml μg/ml/μg/ml gram-positive bacteria staphylococcus aureus 25 / >100 100 / >100 12.5 / 25 50 / 100 1/8 bacillus cereus 12.5 /100 25 / >100 6.25 / 25 25 / 50 4/16 listeria monocytogenes 12.5 /100 25 / >100 6.25 / 6.25 50 / >100 8/16 gram-negative bacteria pseudomonas aeruginosa 12.5 / 25 12.5 / 12.5 6.25 / >100 50 / 100 8/8 salmonella enteritidis >100 />100 100 / >100 50 / >100 12.5 / >100 4/4 escherichia coli 100 / >100 >100 />100 12.5 / >100 100 / 100 16/16 fungal strains candida albicans 100 / 100 25 / >100 25 / 25 50 /100 8/16 aspergillus niger 100 / 100 100 / >100 25 / 25 50 /100 16/16 *positive control: streptomycin for gram-positive bacteria, chloramphenicol for gram-negative bacteria and nystatin for fungal strains rm – methanolic extract of wild growing plants collected at the beginning of the flowering season r2m – methanolic extract of wild growing plants collected at the end of the flowering season rdm – methanolic extract of cultured plants collected at the beginning of the flowering season re – ethanolic extract of wild growing plants collected at the beginning of the flowering season r2e – ethanolic extract of wild growing plants collected at the end of the flowering season rde – ethanolic extract of cultured plants collected at the beginning of the flowering season comparative study of effects of wild growing and cultured ruta graveolens 179 and cytostatic effect of different samples (kostić et al., 2008). this colorimetric assay is based on the uptake and the reduction by mitochondrial succinate dehydrogenase of the yellow water soluble substrate mtt (3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide) to an insoluble purple formazan product. process depends both on the number of cells present and on mitochondrial activity per cell (chiba et al. 1998). cells that were used are adherent epithelial cells, and the possible toxic effect of the rue extracts was evaluated through the possible change of their characteristics. reduction of adherent phenotype was sign of the toxic effect. the round forms of living cells was considered as non-adherent cells. impact of different concentration of rue extracts on hela cells morphology was shown on fig. 1. in control culture, hela cells (fig. 1d) expressed typical morphology of adherent cells, and no apoptotic changes were observed. in contrast, treatment with rue extracts for 72 h had dramatic effects on cell proliferation as well as cell morphology. upon rue extracts exposure, the proliferation of hela cells decreased remarkably also, most of the cells were rounded, reduced in size and detached from the substratum of the culture dish. in addition, cells exposed to the extract revealed typical apoptotic morphology (fig. 1c). decreased cell proliferation and reduction of adherent cells in presence of tested extracts were observed. morphological changes in higher level were observed in cells that were incubated in a medium containing higher concentrations of all types of extracts. in the lowest applied concentration phenotypic changes were also present as phyllopodic and thread-like extensions and changes of cell shape. the hela cell viability and proliferation were significantly reduced by rue extract treatment. the impacts of different concentrations of methanolic and ethanolic extracts of r. graveolens collected at the beginning of flowering season on hela cells after 24 h of incubation and after 72 h of incubation (as % of inhibition) were presented on fig. 2 and 3, respectively. since longer exposure to rue extracts leads to stronger inhibition of hela cells growth and proliferation, these effects are time-dependent. viability of hela cells in presence of extracts (except lowest concentrations of ethanolic extract of cultured rue) was lower than viability of untreated control. re showed maximal cytotoxic effect in this assay (inhibition of cell viability was 38.98 %). all investigated samples (except lowest concentrations of ethanolic extracts of wild growing plants and of cultured plants collected both at the beginning of the flowering season) showed statistically significant (p<0.001) inhibition of hela cells proliferation after 72 h of incubation. maximal inhibition (71.81±3.81 %) was measured in culture with greatest concentration of wild rue ethanolic extract. there are no statistically significant differences between effects of 4 investigated extracts. our results indicate notable in vitro antiproliferative activity of r. graveolens in hela cells (fig. 3). cells viability and proliferation were not inhibited in a strictly dosedependent manner (fig. 2, fig. 3), although strongest inhibition was found at the highest rue concentrations applied. hela cell line proved to be more sensitive to rue extracts obtained from wild growing plants. preethi et al. (2006) stated that prooxidant activity of high concentrations of r. graveolens extract may be responsible for cytocidal action and its ability to produce tumor reduction. our study confirmed only a strong antioxidant activity in tested concentration span of rue extracts. bearing in mind that oxidative stress is commonly connected to cell death (fulda et al., 2010), antioxidant activity of rue extracts anyway contributes to their overall effect on hela cell growth and viability. the present study, on one hand, brings forward the new data regardfig. 1: phenotype changes of hela cells in a) 0.1 μg/ml re – ethanolic extract of wild growing plants, b) 10 μg/ml rde – ethanolic extract of cultured plants, c) 1000 μg/ml re – ethanolic extract of wild growing plants, and d) untreated cells (control). 180 d.r. pavlović, m. vukelić, s. najman, m. kostić, b. zlatković, t. mihajilov-krstev, d. kitić ing the phenolic content, antioxidant, antimicrobial and cytotoxic activities of ruta gravoelens considering the fact that rue is still frequently used in many parts of the world either as a spice or as a medicinal plant. on the other hand, the comparative study of wild growing and cultivated plants extracts confirmed the effects of different soil and climatic conditions on their final pharmacological effect. our observation that ethanolic extract of wild rue collected at the beginning of flowering season, containing the highest polyphenol, tannin and flavonoid contents, demonstrated the marked activities in all tested systems, emphasizing the fact that the rue leaves and herb should be harvested at the beginning of blossoming stage and that wild growing plants present rich source of secondary metabolites. acknowledgements this research was supported by the ministry of education and science of the republic of serbia (grant no. iii 41018 and iii 46013). authors would like to thank to prof. branislava lakušić for kindly providing accession number for ruta graveolens in the herbarium collection of the faculty of pharmacy, university of belgrade. references asgarpanah, j., khoshkam, r., 2012: phytochemical and pharmacological properties of ruta graveolens l. j. med. plants res. 6 (23), 3942-3949. barros, l., ferreira, m.-j., queirós, b., ferreira, i.c.f.r., baptista, p., 2007: total phenols, ascorbic acid, β-carotene and lycopene in portuguese wild edible mushrooms and their antioxidant activities. food chem. 103, 413-419. benazir, j.f., suganthi, r., renjini devi, m.r., suganya, k., monisha, k., nizar ahamed, k.p., santhi, r., 2011: phytochemical profiling, antimicrobial and cytotoxicity studies of methanolic extracts from ruta graveolens. journal pharmacy research 4 (5), 1407-1409. blumenthal, m., 1998: the complete german commission e monographs – therapeutic guide to herbal medicines. american botanical council, austin, texas. chiba, k., kawakami, k., tohayama, k., 1998: simultaneous evaluation of cell viability by neutral red, mtt and cristal violet staining assays of the same cells. toxicol. in vitro 12, 251-258. cuendet, m., hostettmann, k., potterat, o., 1997: iridoid glucosides with free radical scavenging properties from fagraea blumei. helv. chim. acta 80, 1144-1152. european pharmacopeia 6.0., 2007: 6th edition council of europe, strasbourg. fritz weiss, r., fintelmann, v., 2000: herbal medicine. thieme medical publishers, incorporated. fulda, s., gorman, a.m., hori, o., samali, a., 2010: cellular stress responses: cell survival and cell death. int. j. cell biol. 2010, 1-23. iso 10993: 1998. ivanova, a., mikhova, b., najdenski, h., tsvetkova, i., kostova, i., 2005: antimicrobial and cytotoxic activity of ruta graveolens. fitoterapia 76, 344-347. kitić, d., 2006: divlji bosiljak hemijsko i mikrobiološko ispitivanja, zadužbina andrejević, beograd. koleva, i.i., van beek, t.a., linssen, j.p.h., de groot, a., evstatieva, l.n., 2002: screening of plant extracts for antioxidant activity: a comparative study on three testing methods. 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including a new quinolone alkaloid. j. agri. food fig. 3: cytostatic activity of different ruta graveolens samples (rm – methanolic extract of wild growing plants, rdm – methanolic extract of cultured plants, re – ethanolic extract of wild growing plants, rde – ethanolic extract of cultured plants) on hela cells (% of inhibition) after 72 h of incubation. differences of the effects of different rue extracts in different concentrations to untreated control were checked using t-test, significant differences p<0.001 being marked with (**). fig. 2: the influence of different rue samples on hela cell viability (% of inhibition of cell viability) after 24 h of incubation. differences of the effects of different ruta graveolens extracts (rm – methanolic extract of wild growing plants, rdm – methanolic extract of cultured plants, re – ethanolic extract of wild growing plants, rde – ethanolic extract of cultured plants) in different concentrations to untreated control were checked using t-test, 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bertoni as a source of bioactive compounds: the effect of harvest time, experimental site and crop age on steviol glycoside content and antioxidant properties. j. sci. food agric. 93, 2121-2129. the european agency for the evaluation of medicinal products, 1999: committee for veterinary medicinal products: ruta graveolens. summary report. emea/mrl/542/98-final. townsend, c.c., 1968: ruta l. in: tutin, t.g., heywood, v.h., burges, n.a., moore, d.a., valentine, d.h., walters, s.m., webb, d.a. (eds.), flora europaea 2, 227. university press, cambridge. tucakov, j., 1997: lečenje biljem. vii edition, rad, beograd. vitkova, a., philipov, s., 1999: phytochemical study of ruta graveolens l. phytologia balcanica 5/1, 53-67. yang, r.-y., tsou, s.c.s., lee, t.-c., wu, w.-j., hanson, p.m., kuo, g., engle, l.m., lai, p.-y., 2006: distribution of 127 edible plant species for antioxidant activities by two assays. j. sci. food agr. 86, 23952403. address of the corresponding author: department of pharmacy, faculty of medicine, university of niš, bulevar dr zorana djindjića 81, 18000 niš, serbia e-mail: anagard@medfak.ni.ac.rs journal of applied botany and food quality 86, 190 197 (2013), doi:10.5073/jabfq.2013.086.026 1humboldt-universität zu berlin, division urban plant ecophysiology, section quality dynamics/postharvest physiology, berlin, germany 2tarbiat modares university, department of biological science, tehran, iran 3technische universität berlin, institute of food technology and chemistry, berlin, germany 4university of hamburg, hamburg school of food science, hamburg, germany interaction of drought stress and uv-b radiation – impact on biomass production and flavonoid metabolism in lettuce (lactuca sativa l.) elham rajabbeigi 1, 2, ines eichholz1, nina beesk3, christian ulrichs1, lothar w. kroh3, sascha rohn4, susanne huyskens-keil1* (received may 30, 2013) * corresponding author / author has equally contributed to the present manuscript summary the response of plants to stress such as uv-radiation or drought highly depends on the species, cultivar, plant organ, developmental stage, and furthermore, is influenced by ecophysiological interactions. drought stress as well as uv irradiation are the most adverse factors for plant growth and productivity. in the present study, the interactive effect of uv-b and drought stress on biomass, primary and secondary metabolites, and mediated enzyme activity of phenylalanine ammonia-lyase (pal, ec 4.3.1.5) was investigated in lettuce (lactuca sativa l.). it was found that biomass production decreased in response to both stressors, while dry matter, total phenolic content and the flavonol quercetin were not significantly affected by uv-b and drought stress, neither solely nor in combination. in contrast, anthocyanins and luteolin accumulated only in response to drought stress. however, the precursor amino acid proline as well as the activity of pal increased under conditions of increased uv-b and water deficit. thus, the present results deduce that both stressors acted either synergistically or to some extent antagonistically in terms of inducing plant protective mechanisms. introduction plant growth and quality are affected by various environmental impacts. therefore, acclimatization of plants to changing environmental conditions is essential for their growth and survival. drought stress is one of the most important environmental stressors, adversely affecting crop productivity and quality. in many habitats, water shortage is the main limiting factor of plant productivity being regarded as a consequence of global climate change (tesar et al., 2007). drought stress induces various physical and/or chemical modifications in plants. liu et al. (2011) reported that the photosynthetic electron chain in plants is affected and thus, photosynthetic pigments decreased in response to water deficit (ben ahmed et al., 2009). drought stress may also alter the synthesis of secondary plant compounds. for example, the content of phenolic compounds such as caffeic acid was found to be reduced in ipomoea batatas roots (mao et al., 2004), whereas total soluble phenols, quercetin and betulinic acid were increased in hypericum brasiliense (shoots) (abreu and mazzafera, 2005) under drought stress conditions. further, selected amino acids are also involved in drought stress responses: proline is widely distributed in plants and is accumulated in larger amounts than other amino acids in drought stressed plants (irigoyen et al., 1992). for various crops, it is reported that the proline content significantly increased when water is limited, e.g. for maize and bean plants (shoots and roots) (mohammadkhani and heidari, 2008), and for alfalfa plants (leaves) (irigoyen et al., 1992). thus, drought stress mediated changes in primary and secondary plant compounds differ depending on the plant and morphological plant part used for consumption. in recent years, increasing uv-b radiation resulting from air pollution-induced ozone depletion has raised awareness of the effects of uv-b on the ecosystem. though, only a small portion of the total solar spectrum, uv-b in the range of 280-315 nm has a large effect, as it may induce photobiological stress and activates the plant defence system, leading to an accumulation of secondary plant metabolites and corresponding enzymes in plant tissues (teramura, 2006). many studies report potential consequences of uv-b radiation on plants (e.g. jansen, 2010), but there is still a rather limited understanding of the effect on secondary plant metabolites. secondary plant metabolites such as phenolic compounds (flavonoids, hydroxycinnamic acids) act often as uv-b absorbing components or as natural antioxidants in plants (treutter, 2010). the protective health effects of flavonoids are attributed to their radical scavenging and metal-chelating abilities and can be ascribed to their capacity to transfer electrons as free radicals, chelate heavy metals, activate antioxidant enzymes, reduce α-tocopherol radicals and/or inhibit oxidases (heimler et al., 2007). thus, uv light may act as a natural elicitor of secondary metabolite responses in higher plants. recently, the targeted application of uv irradiation has gained importance among food scientists. main targets are sanitation purposes and prevention of postharvest diseases (terry and joyce, 2004) as well as improving the functionality of fruits and vegetables, i.e. health-promoting properties of plant food (schreiner and huyskens-keil, 2006; schreiner et al., 2012). the general emerging elicitor effects of low uv-b radiation, triggering distinct changes, e.g. in the accumulation of phenolic compounds were reported by huyskens-keil et al. (2007), treutter (2010) and schreiner et al. (2012). this was specifically documented for quercetin in onion bulbs and anthocyanins in strawberry fruits (higashio et al., 2005) as well as for diverse flavonoids in brassica sprouts (huyskens-keil et al., 2008) and black currant fruits (huyskens-keil et al., 2007). uv-b as well as drought are known to promote the activity of enzymes playing an important role in phenol metabolism, such as phenylalanine ammonia-lyase (pal, ec 4.3.1.5) (dixon and paiva, 1995; treutter, 2010). stress mediated pal synthesis induced by uv-b has been documented for lettuce (caldwell and britz, 2006) and white asparagus spears (eichholz et al., 2012). the promotion of pal activity through water deficit conditions was reported for pepper plants (sung et al., 2005). further, plant responses to uv-b are known to interact with the water availability of plants, i.e. in general, water limitation is reported to lower the uv-b sensitivity of plants (gwynn-jones et al., 1999). however, the sensitivity of plants to uv-b or drought differs among species, populations and varieties, and depends upon physiological stage of the plant and duration of stress impact (abreu and mazzafera, 2005; schreiner et al., 2009; liu et al., 2011). information on changes of the secondary plant compounds as affected by drought stress in combination with uv-b radiation is contradictive to a certain extent and still limited (hofmann et al., 2003). for the present study, lettuce (lactuca sativa l.) was used as a model uv-b, drought stress and lactuca sativa 191 plant, as it is one of the most important dietary leafy vegetables that is primarily consumed fresh or consumed as fresh-cut convenience product (putnam et al., 2000). lettuce reveals high contents of health-promoting compounds such as phenolic components (gallic acid, caffeic acid), carotenoids (lutein), and dietary fiber (chu et al., 2002; caldwell, 2003; nicolle et al., 2004). moreover, it is reported that lettuce indicates plant protective mechanisms towards stress impacts, e.g. drought stress or uv-b irradiation, by activation of genes being responsible for pal biosynthesis (oh et al., 2010) or by triggering the synthesis of anthocyanins and other flavonoids, respectively (tsormpatsidis et al., 2010). consequently, the aim of the present study was to investigate the interactive effect of drought stress and uv-b radiation on the plant defense reaction of lettuce analyzing the amino acid proline as a stress indicator as well as the profile of flavonoids with the corresponding pal activity. this study might contribute to an assessment of multiple moderate stress factors on the dynamics of health promoting plant metabolites in vegetables as demonstrated for lettuce. material and methods plant material, water regime and uv-b treatment seeds of lactuca sativa var. capitata cv. teodore rz® were obtained from rijk zwaan ltd., netherlands. all plants were grown for two months (april-may 2011) in a greenhouse located at the experimental station of the humboldt-universität zu berlin in berlindahlem, germany. a completely randomized block design was used for experimental purposes. two weeks before harvest, plants were divided into two groups. the first group (well-watered: ww) and the second group (drought stress: ds) were grown in a peat substrate with 45% and 25% water capacity, respectively. the different water capacities were determined daily and obtained gravimetrically. one week before harvest, each group (ww and ds treatment, each n=30 plants) was again divided into sub-groups, i.e. well-watered without uv-b (control), well-watered with uv-b (uvb), water-deficit without uv-b (ds), water-deficit with uv-b (ds+uvb). the two sub-groups uvb and ds+uvb (each n=15 plants) were subjected to uv-b radiation at 0.11 kj m-2 for 5 days using an uv-b fluorescence light source (fl-20se, 305-310 nm, philips gmbh, germany) with an average fluency rate of 8.2 ws m-2 at a mean distance of 20 cm to plants. the other two sub-groups were used as control. selected water conditions and uv-b treatments have been evaluated in preliminary experiments. after harvest, all plants were weighted in order to determine the total above-ground biomass. afterwards, all infected or damaged leaves were removed and plants were weighted again to obtain the marketable yield that is presented as biomass in this study. during the experiment, the following parameters were determined: dry matter, proline content, total phenolic content, flavonoid profile, and pal activity. chemical analysis for the chemical analysis, harvested plants (n=15 per treatment) were shock-frozen in liquid nitrogen and stored at -25 °c. for phenolic compound analysis (total phenolic content, flavonoids) and the determination of proline, samples were lyophilized (alpha 1-4. christ, osterode am harz, germany), ground and stored in desiccators until further analysis. additionally, for determination of chlorophyll and carotenoid contents as well as pal activity, frozen samples were stored at -80 °c until further analysis. analysis of chlorophyll and carotenoids frozen samples (500 mg fresh weight) were homogenized in 15 ml aceton: hexan (4/5, v/v). the homogenate was centrifuged at 4000 rpm for 20 min (labofuge, heraeus, usa). in the hexan layer of the supernatant the contents of total carotenoids, β-carotene, lutein, lycopene, chlorophyll a and chlorophyll b were assayed spectrophotochemically as described by goodwin (1980). the absorbance of the extract was measured at 445 nm, 450 nm, 453 nm, 505 nm, 645 nm and 663 nm, respectively. pigment contents were expressed as micrograms or milligrams per gram dry matter (μg g-1 or mg g-1 dm). extraction of phenolic compounds and analysis of total phenolic content for the analysis of total phenolic content, extraction was conducted according to connor et al. (2002) using acidified methanol (0.1% hydrochloric acid). an aliquot of 0.5 g freeze-dried sample was mixed with 3 ml of acidified methanol and centrifuged for 15 min at 4000 rpm and repeated three times. supernatants were collected and standardized to a final volume of 10 ml. total phenolic content of the leave’s extract was determined using the folin-ciocalteu methodology (slinkard and singleton, 1977). absorbance was measured photometrically after one hour incubation time at a wavelength of 765 nm (lkb-novaspek ii, pharmacia, freiburg, germany). results were expressed as milligrams gallic acid equivalents (gae) per gram dry matter (mg gae g-1 dm). hplc analysis of selected flavonoids lyophilized samples (0.2 g) were extracted with 10 ml of acidified methanol (0.1% hcl) and centrifuged at 1210 × g for 10 min. the supernatants were evaporated in a water bath at 50 °c and 200300 mbar in a rotator evaporator. then, dried samples were resolved with 2 ml of 2 n hcl and 2 ml of methanol (hplc grade) and heated in water bath at 80 °c for 2 hours. the volume was set to 5 ml with methanol (hplc grade). these extracts were kept at -20 °c for hplc analysis. an analytical hewlett packard 1100 series hplc instrument equipped with an autosampler, quarternary hplc pump and diode array detector was used. analytical separation of the flavonoids was carried out on a 150 × 3 mm, prodigy od 53 column (phenomenex aschaffenburg, germany) with a two solvent mobile phase (eluent a = water/acetic acid/acetonitrile (98.5/0.5/1; v/v/v); eluent b = acetonitrile). the eluent gradient used for all extracts was described as follows: 0% b (5 min); 0-4% b (4 min); 4% b (6 min); 4-8% b (15 min); 8-22% b (15 min); 22-28% b (5 min); 28% b (5 min); 2845% b (10 min), and 45-0% (1 min). the detection was performed at 280 nm, 325 nm and 365 nm, simultaneously. the injection volume was 20 μl, the flow rate was 0.5 ml min-1 and the column temperature was 30 °c. total concentration of quercetin and luteolin was obtained from a 1 mm standard solution. results were expressed as milligrams per gram dry matter (mg g-1 dm). anthocyanin analysis for anthocyanin analysis, the absorbance of a methanolic extract, similarly prepared for the hplc analyses, was measured spectrophotometrically (heraeus, usa) at a wavelength of 510 nm. the anthocyanin content was calculated by using the molar extinction coefficient of 29,600 l cm-1 mol-1 and the molecular weight of 449.2 g mol-1 of cyanidin-3-glucoside. the anthocyanin content was calculated on the base of dry matter as milligrams cyanidin-3glucoside per gram dry matter (mg cy g-1 dm). pal activity assay phenylalanine ammonia-lyase (pal, ec 4.3.1.5) activity was determined as described by zucker (1968) with slight modifications. 192 e. rajabbeigi, i. eichholz, n. beesk, c. ulrichs, l.w. kroh, s. rohn, s. huyskens-keil an aliquot of 6 g of fresh lettuce tissue was homogenized with 50 ml 0.025m borate buffer (ph 8.8) containing 5 mm of 2-mercaptoethanol. the homogenate was filtrated and centrifuged at 11000 rpm at 4 °c for 15 min. the supernatant obtained was used as a crude extract for pal analysis. the reaction solution consisted of 1.5 ml of a 0.025 m borate buffer (ph 8.8), 0.5 ml of 0.1 m l-phenylalanine and 0.3 ml of the crude enzyme solution. the pal activity was measured after an incubation period of 60 min at 37 °c photometrically (heraeus, usa) at 290 nm to determine the increase in cinnamic acid as a product. protein concentration of the extract was determined according to bradford (1976) method using bsa as standard. the enzyme activity was expressed as picokatal per milligram protein (pkat mg-1 protein). proline assay proline content was determined according to bates et al. (1973) with slight modifications. a freeze-dried sample (40 mg) was mixed with 1.5 ml of 3% aqueous sulfosalicylic acid in reaction tubes. samples were centrifuged at 0 °c and 11000 rpm for 30 min. to 300 μl of the supernatant 300 μl of ninhydrin and 300 μl of glacial acetic acid were added and mixed in a reaction tube for 1 h at 90 °c. reaction was stopped in an ice bath for 5 min. the mixture was extracted with 900 μl toluene and centrifuged 4000 rpm for 10 min. the absorbance of the toluene layer was determined photometrically at 520 nm. pure proline (l-proline, 99%, roth, germany) was used as a standard. results were expressed as milligrams per gram dry matter (mg g-1 dm). statistic calculations the statistical evaluation was performed using spss 13.0 (spss inc., usa). significance of differences was conducted using anova with a tukey test (p ≤ 0.05). the mean variability was indicated by the standard deviation. results and discussion effect of drought stress and uv-b radiation on biomass production and dry matter content in the present study, biomass production, i.e. above-ground fresh weight of plants of all treatments revealed a significant loss compared to the control plants (fig. 1). uv-b treated plants and plants grown under both stress conditions (ds+uv-b) exhibited significantly lower biomass production than plants grown only under drought stress conditions. the worldwide increase of drought stress conditions is hypothesized to lead to a reduction of biomass production, and thus a reduction of total plant yield (farooq et al., 2009). during water deficiency periods, plants close their stomata to prevent water loss by transpiration which correspondingly permits less co2 intake into plants. this is associated with a decline in photosynthetic activity leading to a reduced biomass production (farooq et al., 2009). however, in the present study, photosynthetic pigments were not affected by drought stress conditions (data not shown) indicating a limited effect of drought stress on photosynthesis in lettuce. this is underlined by the results obtained on the dry matter content of lettuce studied. the dry matter content of lettuce grown under water-deficit conditions (ds) was significantly higher compared to uv-b treated plants, but not significantly different to control plants (fig. 2). in general, it is reported that dry matter content of plants grown under limited water availability increases due to the higher accumulation of assimilates (marschner, 1995) that are necessary for maintenance of plant metabolism or activation of stress responses (roitsch, 1999). carbohydrates might accumulate under drought stress conditions, e.g. sucrose and sugar alcohols (sircelj et al., 2005), being osmoprotectants protecting plants from extreme oxidative stress (rontein et al., 2002). while crop biomass production was highly sensitive to uv-b radiation (fig. 1), dry matter content remained constant in response to uv-b (fig. 2). this data is supported by findings of cechin et al. (2007), who reported that inhibition of photosynthesis is a key reaction leading to a decline in biomass production of crops. plants exposed to uv-b might allocate more energy for other physiological activities than biomass production, especially defense mechanisms fig. 1: biomass production (g plant-1) of lettuce plants at harvest in response to drought stress, uv-b radiation and the combination of both stress factors. vertical bars represent standard deviation, and different letters indicate differences at the significance level p ≤ 0.05 (tukey test). (control = well-watered; ds = drought stress, i.e. water-deficit; uv-b = well-watered with uv-b radiation of 0.11 kj m-2; ds+uv-b = drought stress i.e. water-deficit in combination with uv-b). fig. 2: dry matter content (%) of lettuce at harvest in response to drought stress, uv-b radiation and the combination of drought stress and uv-b radiation. vertical bars represent standard deviation, and different letters indicate differences at the significance level p ≤ 0.05 (tukey test). (control = well-watered; ds = drought stress, i.e. water-deficit; uv-b = well-watered with uv-b radiation of 0.11 kj m-2; ds+uv-b = drought stress i.e. water-deficit in combination with uv-b). uv-b, drought stress and lactuca sativa 193 resulting in an uv-b mediated synthesis of protective pigments, such as carotenoids, anthocyanins or phenolic acids (gao et al., 2004). plant response to uv-b is also dependent on other ecophysiological factors (caldwell, 2003), some of them even in an antagonistic manner (alexieva et al., 2001). in the present study, uv-b treatment of drought stressed plants amplified the effect of both stress factors on biomass production compared to drought stress alone. however, additional drought events in uv-b treated plants did not lower dry matter production of lettuce. thus, uv-b might have a stronger impact on biomass production in lettuce than drought stress. effect of drought stress and uv-b radiation on proline content the amino acid proline is a pre-requisite marker of drought stress (alexieva et al., 2001) and may also act as a protective factor against uv stress (he et al., 2011). in the present study, proline increased only tendentiously in response to drought stress compared to control plants, while uv-b treated plants showed no significant effect on the proline content (fig. 3). previous studies documented an accumulation of intracellular proline concentration in response to osmotic stresses, such as drought (singh et al., 1973). this amino acid is generally assumed to serve as a physiologically compatible solute that maintains a favorable osmotic potential between cells (pollard and wyn, 1979), and is also involved in alleviating cytosolic acidosis in plants under stress conditions (kurkdjian and guern, 1989). also, enhanced uv-b radiation has been reported to increase concentrations of free proline (hofmann et al., 2003). however, results of the present study did not confirm this finding. this might be due to the uv-b radiation dosage applied, which did not influence proline synthesis at this stage. from the present findings it is assumed that drought conditions revealed a tendentiously higher influence on proline content in lettuce than uv-b radiation. the combination of uv radiation and drought stress applied to the lettuce plants of the present study revealed a pronounced increase in comparison to the single stress factor: proline concentrations rose up to 1.5 times higher than in control plants and plants subjected to single uv-b treatment (fig. 3). when uv-b irradiation and drought stress is applied simultaneously or successively, the specific impact of uv-b on drought is reported to be reduced compared to the single stress factor (oh et al., 2010). authors assumed that plant response to drought stress might be masked or compensated by other stress factors. the latter supports the present findings, however, the compensated effect of combined stress on proline content was found to occur only tendentiously compared to single drought stress conditions. effect of drought stress and uv-b radiation on phenolic compounds plant tissue protection against stress is a concerted action of diverse enzymatic and non-enzymatic antioxidant mechanisms. enzymatic defence includes e.g. peroxidases (pod), glutathione reductase (gr) and phenoloxidases (ppo), while non-enzymatic antioxidant network includes e.g. the synthesis of phenolic compounds (especially flavonoids), carotenoids and ascorbic acid (mandal et al., 2009). thus, phenolic compounds provide important physiological and ecological duties, being mainly involved in protection against different types of stress (ayaz and lu, 2000). total phenolic content in the present study, single drought or uv-b treatment as well as the combination of both stressors did not lead to changes in total phenolic content of lettuce plants compared to control plants (fig. 4). this is in contrast to various studies, where one of the main actions of plants in stress situations such as drought or uv-b exposure is the synthesis and accumulation of phenolic compounds. an increase in total phenolic content as a response to drought stress and uv radiation was reported in numerous reports (abreu and mazzafera, 2005; huyskens-keil et al., 2008; treutter, 2010). polyphenols act against stress in plants, however, dependent on the stress factor different phenol groups or single compounds are synthesized and accumulated (dixon and paivia, 1995). thus, changes within the fig. 3: proline content (mg g-1 dm) of lettuce at harvest in response to drought stress, uv-b radiation and the combination of drought stress and uv-b radiation. vertical bars represent standard deviation, and different letters indicate differences at the significance level p ≤ 0.05 (tukey test). (control = well-watered; ds = drought stress, i.e. water-deficit; uv-b = well-watered with uv-b radiation of 0.11 kj m-2; ds+uv-b = drought stress i.e. water-deficit in combination with uv-b). fig. 4: total phenolic content (mg gae g-1 dm) of lettuce at harvest in response to drought stress, uv-b radiation and in combination of drought stress and uv-b radiation. vertical bars represent standard deviation, and different letters indicate differences at the significance level p ≤ 0.05 (tukey test). (control = well-watered; ds = drought stress, i.e. water-deficit; uv-b = well-watered with uv-b radiation of 0.11 kj m-2; ds+uv-b = drought stress i.e. water-deficit in combination with uv-b). 194 e. rajabbeigi, i. eichholz, n. beesk, c. ulrichs, l.w. kroh, s. rohn, s. huyskens-keil phenolic profile could occur without influence on the total content of phenolic compounds. moreover, the assay used for the analysis of the total phenolic content determines the changes of all compounds with reducing abilities as sum parameter (al-duais et al., 2009). consequently, when considering the results of the flavonoid profile, the effect of the different treatments on phenolic compounds of lettuce demonstrated a more specific picture. flavonoid profile in this study, the main flavonoids of lettuce detected were quercetin, luteolin and anthocyanin. for quercetin, stress application did not lead to significant changes. however, results revealed a tendentious increase in uv-b treated plants and tendentiously decline of quercetin in lettuce grown under combined stress conditions (ds+uv-b) (fig. 5). in contrast, analysis of luteolin showed different results (fig. 6). here, drought exhibited a significantly promoted synthesis of luteolin compared to control plants. uv-b treated plants and plants grown under both stress factors (ds+uv-b) experienced no significant changes in luteolin concentration. here, only a tendentious accumulation of luteolin was found. drought stress activates genes associated with the protection through polyphenol synthesis, leading to an accumulation of different phenolic compounds, such as chlorogenic acid, chicoric acid, caffeic acid, quercetin, luteolin, and others (mao et al., 2004; abreu and mazzafera, 2005). drought not only influences the water status of plants, but it also leads to oxidative stress (zhu, 2002). antioxidants such as phenolic compounds are able to prevent oxidative burst of plant cells and thus, protect plants from damage of proteins, lipids, dna as well as rna (apel and hirt, 2004). however, this study suggests that in drought stressed plants, luteolin might play a more pronounced role in the protective mechanisms in lettuce than quercetin. flavonoids strongly absorb uv and their biosynthesis is known to be accelerated by uv irradiation (schreiner et al., 2012). it seems that uv-b stimulates selectively those flavonoids with antioxidant properties. uv-b shielding by flavonols, i.e. increase of quercetin in response to uv irradiation was also reported for birch seedlings (betula verrucosa ehrh.) (lavola et al., 1997). also, chappell and hahlbrock (1984) found that uv radiation lead to the accumulation of pigments and phenolic compounds as a plant defense mechanism. it is also documented that uv-b radiation activates genes for enzymes of the phenolic compound synthesis, such as pal and chalcone synthase (caldwell and britz, 2006). thus from the present results, it is concluded that quercetin and only to a certain extent luteolin might be specific uv-b protectants in lettuce. a study by nogues et al. (1998) showed that uv-b radiation delayed and reduced the harmful effects of drought stress in pea (pisum sativum l.) plants by reducing transpirational loss via influencing stomatal conductance and leaf area development. furthermore, in two mediterranean pine species (pinus spp.) elevated uv-b radiation in field experiments alleviated drought symptoms similar to drought stress, such as needle loss (petropoulou et al., 1995), assumed to be caused by inducing stomata closure for optimizing plant water economy. in the present study, it seems that additional uv-b mediated the effect of drought stress, thus, less luteolin was tendentiously required to be synthesized, while additional drought events on uv-b treated plants significantly inhibited quercetin accumulation. thus, the combined treatment of drought stress and uv-b might not lead to an accumulated response on flavonoids. anthocyanin content in the present study a strong increase in the total anthocyanin content in response to drought stress occurred (fig. 7). when plants were exposed to uv-b radiation, the amount of anthocyanin were not significantly affected and increased only tendentiously. furthermore, combined stress impact (drought + uv-b) only led to a tendentiously higher total anthocyanin content compared to the control. chalker-scott (2002) concluded that anthocyanins may also prevent desiccation through osmotic effects, although it was not clear, how this mechanism could explain anthocyanin accumulation. further, drought inhibits nutrient uptake of macroand micronutrients due to low soil water availability. especially nitrogen deficit fig. 5: quercetin content (mg g-1 dm) of lettuce at harvest in response to drought stress, uv-b radiation and in combination of drought stress and uv-b radiation. vertical bars represent standard deviation, and different letters indicate differences at the significance level p ≤ 0.05 (tukey test). (control = well-watered; ds = drought stress, i.e. water-deficit; uv-b = well-watered with uv-b radiation of 0.11 kj m-2; ds+uv-b = drought stress i.e. water-deficit in combination with uv-b). fig. 6: luteolin content (mg g-1 dm) of lettuce at harvest in response to drought stress, uv-b radiation and in combination of drought stress and uv-b radiation. vertical bars represent standard deviation, and different letters indicate differences at the significance level p ≤ 0.05 (tukey test). (control = well-watered; ds = drought stress, i.e. water-deficit; uv-b = well-watered with uv-b radiation of 0.11 kj m-2; ds+uv-b = drought stress i.e. water-deficit in combination with uv-b). uv-b, drought stress and lactuca sativa 195 detrimentally affect photosynthetic function and efficiency and lower the levels of calvin cycle enzymes (sugiharto et al., 1990). this enhances the accumulation of foliar anthocyanins in leaves of many plant species (kumar and sharma, 1999). the latter finding might explain the enhanced total anthocyanin content of lettuce under drought stress conditions in this study. among the several flavonoid subclasses, anthocyanins are exceptional uv-b protectants. plants enhance their total anthocyanin content as a response towards stress for photoprotecting their photosynthetic systems. several studies demonstrated an increased anthocyanin concentration induced by uv-b treatment (huyskens-keil et al., 2007; treutter, 2010). the results of this study contradict these findings. additionally, also sullivan et al. (1996) found no change in the concentrations of uv-absorbing compounds in response to uv-b radiation. they suggested that these qualitative differences in uv-b absorbing compounds among species are due to the different adaptation mechanisms in terms of their uv-screening capability and disparity in uv-b responses. when both stress treatments were combined, the uv-b effect on total anthocyanin content reduced only tendentiously the effect of drought stress (fig. 7). thus, uv-b radiation and drought might have acted synergistically and thus, increasing uv-b radiation might alleviate the effect of drought stress in lettuce plants. effect of drought stress and uv-b radiation on pal activity flavonoids are synthesized through the phenylpropanoid pathway (dixon and paiva 1995) in which three important enzymes regulate their biosynthesis: (1) phenylalanine ammonialyase transforms phenylalanine into cinnamic acid; (2) chalcone-flavanone isomerase being responsible for an early step of flavonoid biosynthesis; and (3) peroxidase(s) degrading phenolic compounds in the cell vacuole or activate precursor molecules (liu et al., 1995). in the present study, application of uv-b radiation and drought stress alone could not stimulate pal activity (fig. 8). when drought stress and uv-b were applied in combination, pal activity was promoted in comparison to control plants. it was shown in various studies that pal activity increased in response to stress impacts like uv and drought stress (oh et al., 2010; shehab et al., 2010). however, the present results demonstrated that also enzymes other than pal might be involved in the defense mechanism. previous studies indicated that uv radiation can affect chalcone synthase activity or other enzymes being involved in flavonoid biosynthesis like further synthases or isomerases (christie and jenkins, 1996). conclusion plant adaptation to stress factors is associated with increased levels of antioxidant constituents that may prevent stress damage. only to a certain extent, plants and other organisms in nature are affected by only a single stress factor. instead, they typically respond to a combination of several factors such as uv radiation, drought stress, increased atmospheric co2, mineral nutrient availability, tropospheric air pollutants, and temperature. in summary, the present study revealed that lettuce plants appeared to be less sensitive to drought stress compared to uv-b in terms of biomass production and dry matter in contrast to the strong reaction regarding secondary plant metabolites. thus, the effect of drought stress on secondary metabolites of lettuce were more pronounced than those of uv-b radiation, leading the assumption that the latter mediating drought stress effect which needs to be included in further investigations. furthermore, the role of different flavonoids in stress adaptation of lettuce needs to be studied more in detail. however, the direction and extent of an interaction between uv-b and drought stress also depends on the physiological stage of the plant as well as on the time and duration of stress exposure which has to be considered in the future. acknowledgments we would like to thank mrs. susanne meier and mrs. sigrun witt of the division urban plant ecophysiology for their excellent analytical support. fig. 8: phenylalanine ammonialyase (pal) activity (nkat mg-1 protein) of lettuce at harvest in response to drought stress, uv-b radiation and in combination of drought stress and uv-b radiation. vertical bars represent standard deviation, and different letters indicate differences at the significance level p ≤ 0.05 (tukey test). (control = well-watered; ds = drought stress, i.e. water-deficit; uv-b = wellwatered with uv-b radiation of 0.11 kj m-2; ds+uv-b = drought stress i.e. water-deficit in combination with uv-b). fig. 7: total anthocyanin content (mg cy g-1 dm) of lettuce at harvest in response to drought stress, uv-b radiation and in combination of drought stress and uv-b radiation. vertical bars represent standard deviation, and different letters indicate differences at the significance level p ≤ 0.05 (tukey test). 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division urban plant ecophysiology, section quality dynamics/postharvest physiology, humboldt-universität zu berlin, lentzeallee 55/57, 14195 berlin, germany nina beesk, department of food analysis, institute of food technology and food chemistry, technical university of berlin, gustav-meyer-allee 25, 13355 berlin, germany prof. dr. dr. christian ulrichs, division urban plant ecophysiology, humboldt-universität zu berlin, lentzeallee 55/57, 14195 berlin, germany prof. dr. lothar w. kroh, department of food analysis, institute of food technology and food chemistry, technical university of berlin, gustavmeyer-allee 25, 13355 berlin, germany prof. dr. sascha rohn, university of hamburg, hamburg school of food science, grindelallee 117, 20146 hamburg, germany dr. susanne huyskens-keil (corresponding author), section quality dynamics/postharvest physiology, division urban plant ecophysiology, humboldt-universität zu berlin, lentzeallee 55/57, d-14195 berlin, germany art03 journal of applied botany and food quality 80, 19 24 (2006) 1department of wood science, hamburg university, germany 2centro agronômico tropical de investigaciôn y enseñanza, turrialba, costa rica 3museum of natural science, karlsruhe, germany study on the wood anatomy, annual wood increment and intra-annual growth dynamics of podocarpus oleifolius var. macrostachyus from costa rica josef bauch 1, ligia quiros ², guna noldt 1, petra schmidt ³ (received october 10, 2005) summary tropical countries in the future will have an increasing demand for softwoods which favours mixed plantations possibly with minor portions of native conifer species. in this context numerous tropical species of the podocarpaceae can be of ecological and economical interest. in costa rica the native species podocarpus oleifolius var. macrostachyus (parl.) buchholz & gray could be attractive for the establishment of manmade forests. however, profound knowledge on growth characteristics and wood properties is missing. in particular, information on the annual wood increment and intra-annual growth dynamics under natural site conditions at higher altitudes where p. oleifolius var. macrostachyus competes with hardwood species is not available and therefore the objective of this study. at the cordillera de talamanca (approx. 2,700 m a.s.l.), costa rica, an old-growth stand was chosen from which in total 5 trees (40 to 80 cm diameter at dbh) were sampled by taking stem sections and discs (2 trees)or increment cores (3 trees). during the period from october 1998 to december 2000, two trees of the site were pinned monthly for exact determination of the annual wood increment and the intra-annual growth dynamics in relationship to climate. the results of one of these pinned trees are demonstrated. it turned out that at high altitude the annual wood increment of old growth trees amounts to 1-2 mm (diameter) only. the tracheids show a rather constant cell wall thickness (2.5-4.5 µm) throughout the year, but a very variable radial cell diameter from 29 to 61 µm. the exact age of the tree cannot be determined anatomically, as there are no distinct tree-ring boundaries, but only very moderately developed terminal bands of flattened tracheids which do not circle the entire circumference of the stem. the monthly pin-labelling documents that during the dry season from about january until march in 1999 and 2000, virtually no cells were formed. however, with the beginning of the rainy season, about 59 % of the wood increment resulted from the months april to june. this increment rate already decreased from july to september to 32 %. due to a distinct decrease in precipitation towards the end of the year, only 9 % of the wood increment were formed in the last quarter of the year, mainly consisting of the hardly visible terminal and flattened tracheids. the wood of this species is of very homogeneous structure and certainly attractive for highquality wood production. introduction in the tropical country costa rica the demand for commercially important softwoods will increase in the future, whereas in former decades the limited portion of native conifer species was considerably exploited already (arias and dohrenbusch, 2001). in particular, species of the podocarpaceae family would be of interest for their cultivation, for which they are also estimated in other tropical countries (e.g. günter et al., 2004). however, preliminary results indicate that natural regeneration does not guarantee fertilized female cones due to increased distance between the individual trees. the establishment of mixed plantations enables the cultivation of minor portions of the native species podocarpus oleifolius var. macrostachyus (parl.) buchholz & gray, which reaches a height of up to 30 m and a diameter of more than 1 m in old-growth sites (buchholz and gray, 1948; laubenfels, 1990) and with good wood properties (malavassi, 1995; bauch et al., 2006). unfortunately, there is hardly any documentation (blaser and camacho, 1991; orozco, 1991) on the annual wood increment for this native species in costa rica and in particular no data exist on the intra-annual growth dynamics in relation to exogenous influences such as precipitation and day/ night temperature gradients at high altitudes. in the subsequent study an old-growth site of podocarpus oleifolius var. macrostachyus of the cordillera de talamanca was chosen for a preliminary investigation on wood characteristics, annual wood increment and intra-annual growth dynamics. this qualitative and quantitative wood characterization will further allow us to evaluate the potential of this species for mixed plantations at high altitudes. material and methods site and tree selection of a natural forest in order to obtain information on wood structure and growth characteristics of the native species podocarpus oleifolius var. macrostachyus (parl.) buchholz & gray in a natural forest (fig. 1a) at high altitude stem sections, discs and increment cores of in total 5 old growth trees were selected at the cordillera de talamanca, approx. 2,700 m a.s.l. (9°33´ north, 83°40´ west), costa rica. in former years, this species was studied taxonomically (e.g. laubenfels, 1990; hammel et al., 2003) and finally determined as a var. of podocarpus oleifolius and thus no longer as a separate species (fig. 1b). podocarpus oleifolius var. macrostachyus (parl.) buchholz & gray only contributes a few per cent portion (~7%, quiros unpubl.) to the mixed hardwood species (e.g. predominantly quercus costaricencis, quercus copeyensis, drimys winteri, weinmannia pinnata), attainable heights of about 30 m and a diameter of more than 1 m. in october 1997 discs of one tree (dbh approx. 60 cm) were collected for wood structural studies. at october 16, 1998 we started with the cambial analysis by 26 monthly executed pin-markers (december 1999 left out) ending in december 2000 on two trees of the same site (dbh approx. 40 cm). due to a complete prohibition for tree felling at that site only one tree of both could be felled on april 26, 2001 and the pinned stem sections collected (1m 4m tree height). for additional microscopic observations and confirmations of the anatomical results, six increment cores from three other trees at the same site were collected in july 2003. the annual precipitation at that site amounts to about 2,500 mm, exhibiting high variation during the rainy season from april to about november/december and the dry months from january to march (blaser, 1987; anonymus, 2001, fig. 2, tab. 1). these intra-annual dynamics of the water supply with distinct dry months leads to a drastical water deficit in the soil (comp. blaser, 1987). the average monthly temperatures are fairly constant ranging between 10.0 and 11.8° c (fig. 2), however, the day/night temperature gradient is fig. 1: experimental site. a. old growth trees of podocarpus oleifolius var. macrostachyus (parl.) buchholz & gray. b. leaves and female cones of an investigated tree. fig. 3: wood structure of podocarpus oleifolius var. macrostachyus (parl.) buchholz & gray. a. segment of a disc with very weak growth increment zones. s sapwood, h heartwood. b. cross section containing a moderately developed band of radially flattened tracheids formed in november/december (→→→→→ border of an increment zone). all tracheids exhibit more or less constant wall thickness. diffuse distributed axial parenchyma (pa). 20 josef bauch, ligia quiros, guna noldt, petra schmidt considerable and moreover, also varies significantly from day to day with measured minima of almost 0°c during the night (blaser, 1987). the duration of daily sunshine differs remarkably from 7.5 hours (monthly average) in february to 3.0 hours in september (segura and venegas, 1999). the soil consists of a moderately fertile humus layer with a deficit in nitrogen and phosphorus (grubb, 1976). according to nuhn (1978) this soil type is classified as litosole. gitudinal and ray parenchyma. the individual tracheid characteristics were determined histometrically such as radial tracheid diameter, cell wall thickness and tracheid length on sections and on macerated tissue by a digitized image analysis system (analysis®, olympus hamburg), adjusted on a ax 70 microscope. determination of annual growth increment and intra-annual growth dynamics referring to former pinning experiences (kuroda and kiyono, 1997; bauch and dünisch, 2000) small pieces of razor blades were put into the cambial tissue of the selected trees monthly to verify the start of the wound response exactly to the date of treatment even after more than two years. the consistent monthly treatment allowed to determine the intra-annual growth dynamics triggered at the cambial zone. despite of the low annual increment, the wound response caused by the pin-marks immediately led to an abrupt and drastical reduction of tracheid length and tracheid cross area as well as to an accumulation of longitudinal parenchyma at the wound spot. the structurally recognizable increment zones, the intra-annual growth dynamics and the variation of the radial cell diameter of the tracheids were also recorded with the digitized image analysis system. results anatomical structure of the woody tissue the structure of wood with special reference to growth increment zones (fig. 3a) was studied on cross, radial and tangential wood sections from sapwood and heartwood from discs and cores of the five selected trees. in dry condition the brownish wood colour does not remarkably differ between sapwood and heartwood. it is striking that only weakly formed bands of radially flattened tracheids indicate wood increment zones (fig. 3b). however, they are not continuously formed circularly within the stem which means that they do not automatically indicate the exact age of the tree. due to relatively equal cell wall thickness (2.5-4.5 µm) of the tracheids (fig. 3b) across the woody tissue including thin walled longitudinal and ray parenchyma, the wood reveals a homogeneous appearance (comp. tab. 2). the tracheid length ranges from 3.1 to 4.9 mm. however, the radial diameters of the tracheids vary considerably from cell to cell with a maximum of 61 µm and a minimum of 29 µm (unpinned sapwood), measured on macerated tracheids and confirmed by means of cross sections. but the flattened tracheids, which occur at the border of an annual increment show radial cell diameters of less than 20 µm (comp. fig. 3b). the anatomical and histometrical results do not allow any precise conclusion with reference to annual wood increment and intraannual growth dynamics. consequently, pinning studies were chosen for a profound determination of wood formation. annual wood increment by means of 26 monthly executed pins from october 16, 1998 to december 18, 2000, it could be proven that radially flattened tracheids – often only a few tangentially oriented series – are formed during tab. 1: mean of precipitation [mm] per month available from the neighbourhood of the selected forest site at the cordillera de talamanca for the years 1999 and 2000 (anonymus, 2001). month 1999 2000 january 9.4 26.7 february 129.8 25.6 march 25.0 14.7 april 175.8 50.6 may 450.2 368.1 june 437.4 395.4 july 94.8 186.3 august 448.8 110.8 september 377.7 572.6 october 529.5 423.8 november 264.9 162.4 december 207.1 46.0 0 100 200 300 400 500 600 j f m a m j j a s o n d months m e a n o f p re c ip it a ti o n p e r m o n th (m m ) 0 2 4 6 8 10 12 m e a n o f te m p e ra tu re p e r m o n th ( ° c ) fig. 2: mean of precipitation and air temperature per month for the neighbourhood of the selected forest site at the cordillera de talamanca for the years 1942-1985 (blaser, 1987). microscopical observations of the structural characteristics of the wood cross-sectioned slides from the sapwood and heartwood of the discs and cores of the five selected trees, treated with safranin-astrablue, were used to determine variation in percentage of tracheids and lontab. 2: histometrical results for the adult wood of podocarpus oleifolius var. macrostachyus tracheids proportion [%] axial parenchyma proportion [%] rays proportion [%] tracheid length [mm] wall thickness tang [µm] 91.8 92.9 94.2 1.7 2.2 2.8 4.1 4.9 5.5 3.1 4.0 4.9 2.5 4.5 (→6.5) wood structure of podocarpus oleifolius var. macrostachyus 21 november and december (figs. 3b, 4). this is the period when the monthly amount of precipitation usually decreases. fig. 4 illustrates that the cells 6 and 7 were formed in november december 2000, and the cells 28 and 29 were identified as being formed in november december 1999. for the years 1999 and 2000 from january to march no cells were formed. the annual growth rate can be determined as 0.86 mm for both years. this means an average of only about 1.7 mm increase in wood diameter (dbh) per year based on nine months cambial cell formation. not differ in cell wall thickness from others, but some show reduced radial cell diameters (comp. fig. 3b). under temperate climate this special „latewood“-phenomenon is visible in species of juniperus and pinus strobus which obviously is strongly genetically controlled (larson, 1995). 50 49 pa 1 3 2 4 pa 5 6 7 8 9 10 11 12 13 14 16 17 15 pa 18 19 20 21 22 pa 23 24 25 26 27 28 29 31 30 32 33 pa 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 0 10 20 30 40 50 60 70 0 10 20 30 40 50 cell number r a d ia l c e ll d ia m e te r [µ m ] 1999 2000 2001 fig. 4: variation of radial cell diameter of an individual continuous series of radially oriented cells from april 26, 2001 (felling date) to april 1999 (pa axial parenchyma cell). intra-annual growth dynamics in addition to the determination of the annual wood increment, the study of the intra-annual growth dynamics might indicate to which extent environmental parameters such as monthly amount of precipitation, average temperature, sharp gradients of day and night temperature and hours of sunshine will influence the quality and quantity of wood formation. the cross sections with the 26 pin-marks in total give precise information on the number and radial diameter of the monthly formed tracheids and longitudinal parenchyma cells as shown in the selected example (fig. 4). the measurements for the years 1999 and 2000 were averaged and the data grouped in 3 months (fig. 5). it could be demonstrated that during january to march the cambium apparently does not produce any cells. this is in accordance with the dry months with 4.2 % mm precipitation refered to the amount of the whole year (fig. 5). the cambial activity begins at the period with a high amount of precipitation starting in april (april june 33.9 % mm). the corresponding increment from april to june shows the highest values with 58.4 % of the radial increment of the entire year. in july and august, the amount of precipitation (tab. 1) is retrogressive (in several years even significantly lower, comp. fig. 2), but increases again in september and october (tab. 1). the individual meteorological measurements for these months in 1999 and 2000 (anonymus, 2001) were in accordance with the average values of 1942-1985 (blaser, 1987). the wood increment from july to september equals 32.4 % of the entire year. apparently from july onwards also other parameters such as limited duration of sunshine (segura and venegas, 1999) may contribute to a retardant cambial cell division. from october to december, the amount of precipitation (29.5 %) significantly decreases (tab. 1). the few cell series formed from october to december correspond to an increment rate of only 9.2 % of the entire annual increment (fig. 5), which is ecophysiologically an interesting fact. moreover, these tracheids do discussion this preliminary study of the wood anatomical characteristics, the annual wood increment and the intra-annual growth dynamics of podocarpus oleifolius var. macrostachyus offers hints on wood characteristics and growth potential at high altitude conditions. the investigated trees show only faint growth ring boundaries with mostly incomplete series of radially flattened tracheids, which do not allow to determine the exact age of the tree. this finding corresponds to the growth characteristics of the long-lived podocarpus neriifolius and other tropical asian species of this genus (buckley et al., 1995) which also do not permit a dendrochronological dating. however, other species of the genus podocarpus from outside of the tropics develop distinct annual growth boundaries, such as podocarpus nubigens in chilean patagonia (swed and inoue, 1987). the histometrical data reveal that podocarpus oleifolius var. macrostachyus is superior in qualitative aspects (such as fiber characteristics)to the wood of the commercially important species podocarpus salignus from chile and argentina (bauch et al., 2006). the pinning experiments allowed investigations of the annual growth increment and the intra-annual growth dynamics triggered in the cambium as documented in detail by fritts (1976) and larson (1995) for trees grown under temperate climate. for tropical softwood species, hardly any data for annual wood increment and intra-annual growth dynamics are available (jacoby, 1989), whereas for tropical hardwoods numerous case studies were published in recent years (e.g. breitsprecher and bethel, 1990; worbes, 1999; worbes and junk, 1999; dünisch et al., 2003). the monthly and annually determined growth rates of a dominant tree of podocarpus oleifolius var. macrostachyus exhibit full accordance throughout the years 1999 and 2000. the total annual increase fig. 5: intra-annual growth dynamics, monthly determined by pinning examplified for one tree. wood increment (i) illustrated as relative average values every three months for the years 1999 and 2000 in comparison to the distribution of precipation (p). 0 58.4 9.2 4.2 32.4 29.5 32.4 33.9 0 10 20 30 40 50 60 70 jan.-march april-june july-sept. oct.-dec. r e la ti v e w o o d i n c re m e n t i [% ] 0 10 20 30 40 50 60 70 r e la ti v e a m o u n t o f p re c ip it a ti o n p [ % ] i p 22 josef bauch, ligia quiros, guna noldt, petra schmidt in wood diameter is about 1.7 mm, which corresponds to some individual dendrometer measurements of the same site with about 1.9 mm total increase in diameter (dbh) per year (quiros, unpubl.). comparative studies on p. oleifolius in montane forests (2,000 m a.s.l.) in ecuador reveal an even lower annual increment (homeier, 2004). individual dendrometer measurements in a secondary managed forest of costa rica show that under climatically more moderate conditions this softwood species can compete with commercially important native hardwoods reaching an increase of up to about 4-5 mm diameter increase per year (quiros, unpubl.). nevertheless, it should be also evaluated in how far this conifer species can cope sociologically with hardwoods under mixed plantation condition. there are studies for the species podocarpus nubigena as a shade-tolerant species (lusk, 1996; gutierrez et al., 2004). for podocarpus oleifolius var. macrostachyus corresponding experiences do not exist. the monthly determined growth rates reveal some relationship to exogenous influences at the experimental site. the wood formation is triggered by the begin of the rainy season in april and it ends in december with the onset of dry season. in contrast to the genetically controlled relatively uniform cell wall thickness of the tracheids (comp. larson, 1995) throughout the entire year, the considerable variability of the lumen area from tracheid to tracheid depends obviously on short term influences, which have an direct impact on the primary cell wall phase. with adequate water supply this period of primary cell wall formation with radial tracheid enlargement lasts for picea abies only 7-14 hours and is mainly induced during night time (dünisch and bauch, 1994). during the night the water content in the sapwood tracheids can be increased via the fine roots and supply subsequently the cambium. ongoing experiments under controlled conditions with temperature variation also indicate an influence of the existing air temperature during night time (dünisch unpubl.). at the cordillera de talamanca at about 2,700 m a.s.l., the annual growth increment and the intra-annual growth dynamics will primarily depend on the distribution of the annual precipitation, but also the variation of day/night temperature has to be considered. conclusion the preliminary investigation on wood anatomical characteristics of podocarpus oleifolius var. macrostachyus (parl.) buchholz & gray demonstrates a tropical conifer with considerable intra-annual dynamics in wood production, obviously controlled mainly by climatic conditions. the data of our study suggest that high altitudes are most likely not suitable for plantations due to the limited annual wood increment of the trees. however, with regard to the ecological amplitude and the potential for sociological competition this species can be recommended for regeneration concepts in mountainous humid regions of more moderate altitude levels (e.g. räber, 1991; quiros unpubl.). this conclusion is underlined by some investigations of the wood properties (comp. malavassi, 1995; bauch et al., 2006). without doubt, this wood species can be recommended for its high-quality homogeneous raw material with reference to lumber with decorative value for furniture. acknowledgements we are indebted to the institute costarricense de electricidad, san josé, costa rica supplying us with the meteorological data of the selected experimental site. thanks are also directed to dr. h. j. saa, costa rica and to dr. d. kelch, california for taxonomic information. dr. j. homeier and dr. s. günter deserve our gratitude for supplying us with comparative samples of p. oleifolius d. don ex lamb, from ecuador. for cooperation in the experimental work, we thank pd dr. g. koch, c. lehringer, t. schwarz, s. voiß, and c. waitkus. references anonymus, 2001: meteorological data of the site villa mills 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rafael and soler areas, patagonia. bulletin glacier res. 4, 125-132. worbes, m., 1999: annual growth rings, rainfall-dependent growth and longterm growth patterns of tropical trees from the caparo forest reserve in venezuela. j. ecology 87, 391-403. worbes, m., junk, w.j., 1999: how old are tropical trees? the persistence of a myth. iawa journal 20, 255-260. address of the authors: prof. dr. josef bauch, dr. guna noldt, department of wood science, hamburg university, leuschnerstr. 91, d-21031 hamburg. ligia quiros, centro agronômico tropical de investigación y enseñanza, 7170 turrialba, costa rica. dr. petra schmidt, museum of natural science, erbprinzenstr. 13, d-76133 karlsruhe. e-mail: jbauch@holz.uni-hamburg.de (corresponding author). 24 josef bauch, ligia quiros, guna noldt, petra schmidt << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) 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<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> /sve <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> >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice journal of applied botany and food quality 86, 16 23 (2013), doi:10.5073/jabfq.2013.086.003 1 unit fruit science, center of life and food sciences weihenstephan, technische universität münchen, freising, germany 2 fraunhofer institute for process engineering and packaging ivv, freising, germany 3 department for physical biochemistry, center of life and food sciences weihenstephan, technische universität münchen, freising, germany flavanols in the nuclei of the tea bush (camellia sinensis) – broadening the perspectives to human health w. feucht1, m. schmid2, j. polster3, h. dithmar3, d. treutter1 (received january 16, 2013) summary the nuclei of the tea shrub (camellia sinensis l.) contain flavanols. based on the dynamic, fine-tuned subnuclear changes of the flavanol pattern during the cell cycle, it can be postulated that these blue staining phenols play a role in organizing basic mechanisms of chromatin remodelling. silenced interphase nuclei of mature parenchyma cells indicate a pronounced diffuse distribution of blue stained flavanols using the selective reagent p-dimethylamino cinnamaldehyde (dmaca). by contrast, in activated nuclei the flavanols reflect a variable, mosaic-like blue patterning enclosed by white interchromatin spaces. subnuclear expression of euchromatin displays relative tiny blue dots of flavanols as compared with the largersized blobs of heterochromatin. from metaphase to telophase, the chromosomes stain a fairly dark blue on flavanols with a more or less diffuse appearance. those nuclei running through mitotic interphases from g 1 to g 2 have well-defined flavanol-free nucleoli. the flavanol pattern of meristematic chromosomes found in the tea plant is basically also valid for herbaceous plants, such as hyacinthus romanus l., tulipa gesneriana l. and allium cepa l., which genuinely do not contain nuclear flavanols. this was verified by incubation of their rootlets in solutions of green tea and epigallocatechin gallate (egcg). also human nuclei demonstrate an easy import of added flavanols and the resulting blue stained chromatin reflects in much the same way the structural modifications as already described for the plant nuclei. flavanols have the potential to associate to the histones of chromatin which inhibits a possible oxidation. even small fragments of histones can aggregate to catechin as shown on the basis of kinetic measurements for the h4-core peptide hakrkt and its acetylated product hak(ac)rk(ac)t. introduction in many tree species the flavanols, generally known as catechins, are deposited preferentially in vacuoles of enlarging cells. histological studies, uv-vis spectroscopic titrations and reaction kinetic investigations as well laser microsurgery indicated that also nuclei of certain tree species are targets for flavanols (feucht and polster, 2001; polster et al., 2003; polster et al., 2006). recently, these results were analytically confirmed by high resolution two photon excitation techniques applied to nuclei of taxus baccata (muellerharvey et al., 2012). many coniferous species show a significant nuclear affinity for flavanols in contrast to woody angiosperms as dealt in a recent review (feucht et al., 2012 b). a prominent example of the angiosperms displaying a high affinity of nuclei for flavanols is the tea plant (feucht et al., 2004). hplc investigations showed the presence of catechin (c), epicatechin (ec), epigallocatechin (egc), epigallocatechin gallate (egcg), procyanidin b2 and epicatechin gallate (ecg) in leaves and anthers of the tea plant (feucht et al., 2005). most of these flavanols were examined on their association behaviour towards histone proteins (feucht et al., 2004, 2005, 2007). newest results indicate that the aggregation of flavanols to histones seems to be dependent on the epigenetic histone code (feucht et al., 2012 a). these findings appear to be of particular importance because histones are known to be linked with epigenetic and transcriptional activities (jenuwein and allis, 2001). acetylation and methylation imposing relaxation or compaction of nucleosomes play also a major role in histone modifications (gasser, 2002). biological activities of phenolics located in nuclei have been exhaustively discussed by bidel et al. (2010). it is interesting that the subnuclear flavanol distribution of the tea bush is basically similar to that found in the distantly related hemlock as a coniferous species (feucht et al., 2011). the present publication pays particular attention to the fine-mapping of the nuclear flavanols during cell cycling of camellia sinensis. to carry on a further step, green tea flavanols were added to three herbaceous species and human nuclei lacking flavanols in the native state. material and methods biological material five tea bushes (camellia sinensis l.), 8 years old, were grown in the greenhouse. during the early phase of growth young anthers of sprouting flowers were sampled. the anthers between 100-150 µm in length were most active in cell cycling. over a period of three years a total of about 3.500 to 4.000 nuclei of the tea bush were studied. young rootlets of allium cepa l., hyacinthus romanus l. and tulipa gesneriana l. were used to study the affinity of their nuclei for flavanols. the plants were cultivated in a greenhouse and the rootlets were sampled at a length of about 3 to 7 mm. then, they were incubated for 3 h in green tea. a total of about 500 nuclei per plant species were examined by microscopy. in the case of human cells a total of about 200 nuclei from nasal mu-about 200 nuclei from nasal mucosa, saliva secretion and skin (forehead) epidermis were sampled. then, they were incubated as outlined above in green tea and egcg solutions. in this context, dna patches (calf thymus, sigma ) were likewise incubated for 3-4 h in egcg (concentrations as described above). histology to study the dynamics of flavanol patterning nuclei from very young and older anthers are directly stained and studied by microscopy (zeiss axiom apparatus). direct staining of fresh cells with the dmaca reagent is necessary because any embedding procedures would result in some loss of the soluble flavanols. the staining reagent (0.5% p-dimethylamino cinnamaldehyde dissolved in 1.5 m sulphuric acid and methanol 1:4, v/v) is very specific for flavanols. the cells are stained on a microscope slide for 15-25 min. then, most of the reagent is withdrawn with a strip of filter paper, and after adding few drops of water the flavanols switched rapidly to bright blue. the stained tissues are gently squashed with a cover-glass, so that the cells of the tissues separated easily from one another. for flavanols in nuclei 17 17 dna detection dapi staining (4`, 6-diamidino-2-phenylindole dihydrochloride, serva) the zeiss fluorescence apparatus (iii rs) was used (fig. 2b). histological absorption measurements in the visible range light microscopic images of the anther nuclei were obtained with a zeiss axioscope, then scanned with a nicon coolscan iv ed. thereafter, a digieye 2.3 apparatus from verivide ltd. leicester, united kingdom, as described by (lau et al 2011), is used to determine the relative absorption in % of the scanned blue coloured nuclei at 640 nm. this measurement method is based on a noncontact imaging system. the image is captured by a high resolution digitalcamara in a unique controlled lighting environment, the digieye cube. the measurements device was calibrated according to the digieye calibration routine. this experimental set up leads to most reliable absorption measurements. the captured image is then displayed on a calibrated monitor. digieye is well suited to measure very small and irregular shaped samples by selecting and retrieving colour data from any pixel in the high-resolution image. thus far, the method permits a more refined analysis of the nuclei from the tea plant. as stated by lau et al. (2011) the values measured by the digieye have very strong positive correlations with those measured by classical spectrophotometry. chemicals for kinetic measurements using the uv-vis spectroscopy the influence of histone peptides on the oxidation degradation behaviour of catechin was studied in dependence on time (fig. 4). the peptides hakrkt and hak(ac)rk(ac)t were prepared from peptides & elephants gmbh (nuthetal, germany; article no: ep-01641 and ep01716); (+)-catechin (puriss.; article no: 6200) and tris = tris(hydroxymethyl)amino methane (pufferan®, ≥ 99.9%, p.a.) were obtained from carl roth (karlsruhe, germany), ethanol (for spectroscopy; “uvasol”) from merck (darmstadt, germany), oxygen 4.5 (content 12 l; „minican“; 12 bar) from linde (unterschleißheim, germany), hydrochloric acid (min. 25%; puriss. p.a.) from riedelde haën (sigma aldrich, seelze, germany) and magnesiumchloride hexahydrate (analar normapur, 99.0-102.0%) from vwr (leuven, belgium). the preparation of the buffers and catechin-peptidesolutions as well the procedure of the uv-vis spectroscopic kinetic measurements are described in feucht et al. (2012 a). results differential flavanol patterning of activated and silenced nuclei of tea cells the cytological approach using directly the fresh nuclei provides a global view on the entire nuclei. the most striking feature of the silenced anther nuclei is pronounced diffuseness of the slightly undulated blue stained nucleoplasm (fig. 1 a, b). other silenced, inactive tissues from the tea bush, such as petioles and sepals from fully developed flowers indicate a similar diffuse appearance (not shown). on the contrary, each interphase nucleus from the meristematic cluster (fig. 1 c) shows its own chromatin landscape. by now, transcriptional mrna synthesis is high because prominent nucleoli are evident. these nuclei, measuring between 7 to 9 µm in diameter, are more or less loosened and display a fine-structured network of blue chromatin mottling on a mosaic-like white background (fig. 1 c). stronger magnification of those activated interphase nuclei (fig. 1 d-g) reveals fairly blue heterochromatin blobs as well as smaller pale blue dots of euchromatin. the mosaic structures of the four nuclei show a wide variability since the white flavanol-free areas are considerably different in both, size and subnuclear distribution. the most opened and active configuration is reflected by fig. 1 g, the chromatin-free nucleolus of which is, as usual, without flavanols. sometimes larger blobs of heterochromatin or nuclear organizer regions (nors) come to lie close to the nucleolar periphery. fig. 1 g shows clearly half the flavanol absorption of fig. 1 f . during s phase a doubling of dna takes place, and then the histones and also the flavanols should attain a two-fold increase which is really exemplified by an increase of absorption from 24 to 48 units (fig. 1 g and f). by anaphase, the separating sister chromatides are pushed to the opposite poles and then contracted to form two synchronously structured telophase nuclei (fig. 1 h). the two daughter nuclei exhibit rather small colourless interchromatin spaces being interspersed throughout the closely located, dark blue mottled chromatin blobs. the absorption data from the activated nuclei (fig. 1 c) range from 44 to 66 and indicate a higher variation than those of the silenced ones. a dense and diffuse blue cluster of silenced cells (fig. 1, a) ranges from 64 to 70 absorption units and shows a low variation. application of flavanols to nuclei of herbs and humans it is clearly of evident importance to know whether in herbs or humans which are genuinely without nuclear flavanols take them up in vitro. there is no doubt that a general affinity of chromosomes for flavanols is of immense significance for the plant kingdom and in particular for human health. three plant species which genuinely do not contain nuclear flavanols were investigated (fig. 1 i-t). egcg (10 µm) in watery solution was externally supplied. it is striking that supplied egcg (fig. 1 i, k, m) and flavanols of the green tea (fig. 1 j, l, n) are capable to bind to nuclei. the rootlets of the three herbaceous species were investigated in the following order (fig. 1): hyacinthus (i, j), tulipa (k, l) and allium (1 m, n). after an incubation period of 3 hours each nucleus reflects a certain individuality of the flavanol landscape. the characteristic symptoms found in the nuclei of hyacinthus have more or less sharply mottled mosaic structures and show a change from fairly blue (i) to a rather faint blue (j). two large nucleoli are seen in each nucleus. in tulipa, treatment with egcg (fig.1 k) resulted in a rather diffuse, only lightly mottled flavanol pattern throughout the nucleus. in such a diffuse blue environment even the two nucleoli display a tinge of blue. a very contrasting crisp structure is shown when using the treatment with green tea extract (fig. 1 l). in allium, the nucleoplasm is strongly granulated by both euchromatin and heterochromatin (fig. 1 m, incubated in egcg) whereas the following nucleus displays a fine mottled lighter blue and a dispersal of dense heterochromatin blocks (fig. 1 n, green tea). additionally, the very large nucleolus is nearly completely encircled by a visibly dense blue border line. finally, human nuclei were sampled from epithelial cells of nasal mucosa (o, p), saliva secretion (q, r), and from cells of the forehead surface (s, t). it is to assume that some nuclei are affected by certain degrees of ageing which might affect structural aspects and also the flavanol pattern. each of the three groups was treated for 2 to 4 h with egcg (fig. 1 o, q, s) or green tea extract (fig. 1 p, r, t). nasal mucosa imbibed in egcg shows a rather deep flavanol staining which is characterized by a conspicuous dot-like mottling (fig. 1 o), especially near the nuclear periphery. the second nucleus from nasal mucosa treated with green tea extract is rather low in amount of flavanols, except the darker blue envelope area (fig. 1 p). the low affinity for flavanols, except the envelope region, might be due to an advanced stage of degeneration. both nuclei from saliva secretions (fig. 1 q, r) indicate a marked flavanol reaction. the first one (fig. 1 q), treated with egcg, reveals an apparent dark blue, diffuse flavanol reaction with condensed dark 18 w. feucht, m. schmid, j. polster, h. dithmar, d. treutter fig. 1: endogenous nuclear flavanols from camellia sinensis and externally added flavanols (egcg and green tea) to nuclei of herbaceous plants and humans. absorbance units (au) measured at 640 nm are given in [brackets] as single values or as the mean ± standard deviation for the nuclei visible in the figures. diameters of the nuclei from tea bush range from 7 to 9 µm including those of fig. 1a and 1c. a. silenced nuclei from the mature anther epidermis (7 µm in diameter) [au 66.6 ± 2.1]. b. magnification of one diffuse nucleus from fig. a [au 68.4]. c. activated nuclei of a young anther during maximal cell cycling representing a meristematic cluster [60.1 ± 7.8]. d-g. magnification of activated nuclei showing the typical mosaic pattern of flavanols. [au: d, 57.2; e, 59.0; f, 48.1; g, 24.4] h. developing daughter cells of telophase with euchromatin interspersed with sharply mottled, darkly stained blobs of heterochromatin [au 57.4; 63.5]. herbaceous nuclei from hyacynthus (i, j,tulipa (k, l) and allium (m, n). egcg (i, k, m) [au: i, 66.0; k, 71.9; m, 74.1] and green tea (j, l, n). [au: j, 44.0; l, 63.9; n, 60.0], diameters of the nuclei range from 8-9 µm. human nuclei from nasal mucosa (o, p), saliva secretion (q, r) skin (forehead) epidermis (s, t). egcg (p, r, t) [au: p, 51.1; r, 78.0; t, 49.2] and green tea (o, q, s) [au: o, 73.4; q, 79.; s, 65.3] diameters of the nuclei range from 5-6 µm. blotches, and a large part of the envelope line is exceptionally dark blue. the second nucleus (fig. 1 r), imbibed in green tea, shows curious morphological details. the envelope is without flavanol reaction, probably a sign of degeneration. also the organization of the nucleolar region appears to be anomal, as evidenced by two heavily stained dots and a partially darkly blue border line around the nucleolus. an epidermal skin cell (forehead) treated with egcg developed throughout the nuclear area a number of tiny but deep blue spots which are clearly interrupted by fairly pale blue areas (fig. 1 s). in flavanols in nuclei 19 19 addition, this nucleus is characterized by a sharply delimited, dark blue envelope. the second nucleus from skin, treated with green tea extract (fig. 1 t), shows a moderate blue chromatin without pronounced blobs of heterochromatin. the two rather enlarged nucleoli are characteristic of a stronger transcriptional activity. summing up, the nuclei of the herbaceous plants and human cells range in flavanol absorption values from 44 to 79 which are roughly comparable with those of the tea nuclei. flavanols during mitosis of tea cells the morphological change from g2 to the mitotic structures is dramatic because the nuclear envelope is removed and elongated chromosome structures become visible (fig. 2 and 3). it is commonly accepted that metaphase chromosomes are very compacted compared with the interphase (arrow, fig. 2 a). pronounced dna density during metaphase is affirmed by the bright uv-fluorescence when stained with dapi (fig. 2 b) whereas the interphase (arrow) appears lighter blue. fig. 2 c shows the transit stage from metaphase to anaphase. at the spindle axis there is a fairly blue staining deposition of chromatin and some pale chromosomes begin to move toward the poles. a certain degree of diffusion is recognizable. fig. 2 d shows an example in which a more concrete, long armed and zigzagging chromosome is visible. hook shaped chromosome segments showing blue streaks can be seen in fig. 2 e. other chromosome segments (fig. 2 f) stain a darker blue, appear denser crowded and curved to semi-circles. the next fig. 2 g shows one of the two daughter nuclei during early telophase configuration, with rather uniform organization of chromosomes which are more or less bended. finally, both daughter nuclei of a progressed telophase (fig. 2 h) appear in a more diffuse state, in contrast to the pronounced fine-grained blue mosaic as seen in fig. 1 h. the frequently dark blue areas of nuclei support the idea that flavanols, aside from their affinity to histones, probably also bind to dna. fig. 2 i shows a sample of dna (calf thymus) incubated for 3 h in a watery solution of egcg (50 µm) with dark blue staining. stability of catechin in presence of modified histone peptides in a recent paper, it was shown that not only histone proteins but also fragments of them synthetically produced have the potential to reduce more or less strongly the oxidative degradation of catechin (feucht et al., 2009 and 2012 a). thus, the fragment 71-85 of the h4-core protein (tytehakrktvtamd) leads to a significant decrease of the rate of the oxidative degradation. even distinctly smaller peptides of the h4-core fragment can exert an influence on the protection of catechin degradation. the time dependent degradation of catechin in the presence of oxygen is shown for the wavelength 434 nm in fig. 4 (two curves). in the presence of the peptide hakrkt being a segment of tytehakrktvtamd the oxidative degradation is significantly retarded (see fig. 4). when the two lysine amino acids are acetylated according to the epigenetic histone code, the peptide hak(ac)rk(ac)t reduces also the degradation of catechin, however to a less extend than the unmodified peptide. it is well documented that both lysine positions of the peptide can be acetylated in the h4core protein (zhang et al., 2003; hyland et al., 2005). discussion flavanols involved in fine-mapping of the chromatin landscape in the literature, a large body of data is offered indicating that nuclear chromatin arrangement is precisely regulated by both genetic and epigenetic information (exner and henning, 2008). the findfig. 2: curved and compacted chromosomes during mitosis. a. compacted metaphase compared with a loosened interphase nucleus (arrow). b. uv micrograph to visualize dna of both metaphase and interphase (arrow). c. transit from metaphase to anaphase with longer and shorter arms of diffuse chromosomes. d. stretched chromosome with a zigzag configuration and condensed flavanols confined to distinct sectors. e. curved chromosomes during anaphase with pale blue and moderate blue sectors. f. semicircles of chromosomes during anaphase. g. one daughter cell at early telophase shows bending and intermingling chromosomes with variable density of flavanols. h. diffuse early telophase configuration. i. dna (calf thymus) incubated in watery egcg solution (20 µm). 20 w. feucht, m. schmid, j. polster, h. dithmar, d. treutter fig. 3: four proposed properties of flavanols to counteract harmful effects during mitotic destabilization of the chromosomes. 1. affinity of flavanols to dna and histones might provide more stability against too strong bending, compaction, stretching and breakage of chromosomes, especially with regards to exposed tails and loops. 2. flavanols are involved in optimal balancing of enzymatic activities, for example of separases, rnases and polymerases. 3. flavanols act in oxygen radical scavenging to allow a reductive nuclear milieu being favourable for mitosis and growth promotion. 4. flavanols bind to specific histone proteins in dependence on the epigenetic histone code, and thus influence directly the gene activity. fig. 4: absorbance-time (a434 vs. t) curves of catechin degradation (0.2 mg catechin/ml, 0.69 mm) in the presence of peptides hakrkt and hak(ac)rk(ac) each about 1 mg/ml. further conditions: 20 °c, 0.1 m tris buffer ph 8 with 3.3% ethanol, 10 mm mgcl2 (see feucht et al., 2012 a), and gassed with o2; aa means amino acid, 6 refers to hexapeptide, ac represents acetylated. ing that the blue staining flavanols associate with histones (polster et al., 2006) prompted us to study more thoroughly possible links between the chromatin activity and patterning of flavanols. recent studies with hemlock (tsuga canadensis) indicated that the blue coloured flavanol landscape from activated chromatin is organized as a mosaic-like structure being quite different from the evenly diffuse blue appearance of silent chromatin (feucht et al., 2011). evidently, the flavanol characteristics of the tea nuclei (fig. 1 d-g) share a strong similarity with those of the very distantly related hemlock fir. other plant species which genuinely are without nuclear flavanols, as shown in the rootlets of allium cepa l., hyacinthus romanus l. and tulipa gesneriana l. obviously respond with blue colouration when flavanols are applied externally. moreover, also human nuclei follow this pattern. notably, all these flavanol-treated nuclei (fig. 1 i-t) indicate a similar spatial mosaic arrangement as shown by those of the tea plant (fig. 1 d-g). in humans, it would be of particular medical concern to apply new methods which allow an import of externally supplied flavanols into the nuclei to yield protection against oxidative damage. new analytic methods being much more sensitive in detecting lowest amounts of nuclear flavanols would be very promising (mueller-harvey et al., 2012). destruction of hamster nuclei by aflatoxins could be significantly reduced by adding catechin in vitro (bauer et al., 2009). various tissues of the cow (brain, kidney, liver and spleen) were shown to be a strong sink for added flavanols (polster et al., 2002). when young piglets were fed with appleor red-grape pomace various flavanols could be detected in ileum, liver and kidney and white blood cells responded with changes in mrna expression (sehm et al., 2011). intranuclear flavanol distribution and flavanol-free nuclear spacing regarding the blue-white mosaic pattern of active nuclei (fig. 1 cg) there is evidently a large-scale change in both size and staining intensity of the chromatin blobs. consequently, the variation of the nuclear flavanols (fig. 1, a1-a6 and c1-c11) is significantly higher in meristematic clusters. the distinct values for blue colour absorption are 7.4 % as compared with 1.4 % of the silenced ones. obviously, flavanols respond to changes in activity of local intranuclear micro flavanols in nuclei 21 21 domains as already shown for hemlock (feucht et al., 2011). in fig. 1 f twice the absorption value of flavanols is found compared with fig. 1 g. it is to note here that during s phase a doubling of dna from 2 c to 4 c takes place. this is accompanied by a doubling of histones which, in turn, would imply that also the flavanols increase on parallel lines. in either case, the s phase period might span over a time space of about 5 to 8 hours. so, by this time there might be single nuclei which just have reached only amounts of 30% or 60% and so on regarding the final values of dna, histones plus flavanols. in essence, variability in flavanol aggregation within a cluster of nuclei should be expected. two cell clusters of the same anther can be similar or different in density of flavanols (tab. 1). such a feature is due to meristematic cell lineages starting from one initial cell and finally consisting of 4 or seldom 8 daughter cells (feucht et al., 2011). lateron, the lineage is not prolonged, however, by longitudinal divisions of 2 or 4 lineage cells, about 8 to 12 daughter cells are established which are visibly synchronized in size, shape and density of nuclear flavanols. a cluster rather low in flavanols may relate to different aspects. for example, the import of histones into the nuclei can be restricted by a limited availability of sufficient cellular energy and atp (breewer and goldfarb, 1990). a most extreme flavanol loading of nuclei from yellow flecked upper epidermal cells might be due to special stress conditions imposed by fungal infection (tab. 1). irradiation stress is less likely since the tea shrubs are grown in the greenhouse. are flavanols involved in modulating the histone-dna contacts and gene transcription? transcription activities are particularly focussed to distinct regions of chromosomes as for instance to lysine residues of the core histones h3 or h4 (mersfelder and parthun, 2006). so far, if flavanols interact with acetylated or methylated histone domains, then they inevitably should modulate distinct patterns of gene expression. roughly, light or dark blue flavanol patterning should result in expression and repression of the tea nuclei. the flavanol-free interchromatin ‘channels’ (fig. 1 d-g) can be considered as transport highways to achieve high transcription activity. they facilitate movement of many compounds, for example non-histone proteins and small rnas which are necessary for any transcriptional activity of a nucleus (gasser, 2002; misteli, 2007). non-histone proteins coordinate the multiple activities of transcription (gorski et al., 2006) or special protein kinases together with rnas spread out to distinct target sites within nuclei and chromosomes (li and reinberg, 2011). despite the relative low amount of dna in heterochromatin according to richards and elgin (2002), nevertheless a deep blue staining was found for flavanols (compare fig. 1 g). these roughly spherical bodies of highly folded heterochromatins are considered to be engaged primarily in gene silencing. however, fransz et al. (2003) argue that methylation combined with silencing, for example of histone lysines, is not generally sufficient to form heterochromatin. perhaps, in chromatin blobs the dense flavanols may additionally be involved in impeding faulty intermingling of genes. as discussed by dorier and stasiak (2010) such an intermingling is a high-risk incident. cross-linking of proteins with polymeric phenolic compounds up to tannins is, roughly speaking, widely accepted. however, monomeric catechins, as located in nuclei (feucht et al., 2008) by far have not gained a proper scientific interest regarding the affinity of phenols to proteins. the variable mode and strength of flavanol-binding to specific histone sites can be manipulated by a number of facilities, such as van der waals interactions, hydrogen bonds, hydrophobic effects, electrostatic forces and molecular size of the interacting protein and phenol structures (hagerman, 2012). there is surely a potential link between different binding modes and biological activity during gene transcription. yet, reversible binding should be of crucial importance for chromatin remodelling. do nuclear flavanols operate as antioxidants? principally, dna wrapped round the histones is continuously exposed to oxidative stress (apel and hirt, 2004) and mitosis, depleted from the nuclear membrane, surely suffers from more oxidative damage. fragmentation of dna can be markedly reduced by monomeric catechin at 0.5 mm (huang et al., 2006). flavanols (catechins), donating electrons or hydrogens, are well-known to operate in an antioxidative fashion (stangl et al., 2006). transcriptionally important basic amino acids of histones, such as lysine, arginine and histidine, are protected against oxidative stress by proanthocyanidins, mostly dimeric catechins, of pomegranate juice (guo et al., 2008). collectively, transcription is prone to increased rates of oxidative dna damage. this point needs more attention. histone buffer solutions, even if gassed with oxygen, nevertheless are capable to inhibit oxidation of catechin (feucht et al., 2009). there is another crucial point. during mitosis, the nuclear membrane becomes disassembled so that the chromatides are additionally exposed to a novel source of cytoplasmic oxygen radicals. even a low-level oxidative stress can be responsible for cell cycle arrest (clopton and saltman, 1995). under such conditions also the auxin molecules (iaa) involved in mitosis might suffer from rapid oxidative degradation. auxin nestled in a reductive cell environment enforces the transcriptional processes during cell cycling (exner and henning, 2008; spoel et al., 2010). in this connection, it should be emphasized that catechin together with auxin is capable to double the fresh weight of callus tissues as compared with auxin applied alone (feucht and nachit, 1977). using callus cultures (feucht and treutter, 1995), after a 4 weeks growth period the content of residual iaa was roughly 8-fold in the culture media containing iaa plus catechin compared with iaa alone. most exciting, however, in these experiments was a highly significant increase of proteins in the catechin-treated fast dividing callus tissues compared with the controls. yet, it is not correct to delimit the role of ros (reactive oxygen species) molecules solely as being hazardous by-products of metabolic activities (noctor et al., 2007). at subtoxic levels, they also operate as dynamic signals which activate transcription factors (van breusegem et al., 2001). both, ros-mediated signaling of histidine kinase and gene expression are tightly integrated into the cell cycling processes (apel and hirt, 2004). support flavanols the physical stability of overstretched, sharply bent or over-compacted chromatin? from prophase to telophase the nucleosome territories are exposed to extreme physical stress (fig. 2 c-h, fig. 3). at transit from prophase to metaphase kinesin-like motor proteins manipulate a tight congression of chromosomes along the metaphase plate. then, at transit from metaphase to anaphase the chromatides suffer from strong physical tension by too much pulling forces from microtubule motors. intrachromosomal cohesion is known to be achieved by adherence-type proteins. cohesins keep sister chromosomes together and readily also small chromatin granules (peric-hupkes and van steensel, 2008). is there a functional similarity or even cooperation of cohesines and flavanols in stabilizing supercoiled dna as well as in the case of extended dna stretches looping out from chromosomes? 22 w. feucht, m. schmid, j. polster, h. dithmar, d. treutter during the cell cycle, special conditions account for a more fluid appearance of blue-coloured chromatin, at least temporally (fig. 2 c, h). indeed, diffusion-driven mechanisms are in play during microtubule flux of the poleward sliding nucleosomes (berg et al., 1981). hereby, proteins or histones might be covered by a fluid hydration shell allowing a lower friction during anaphase movement. in this context, of utmost importance is the finding that flavanols also bind to dna as shown for egcg by chemo-physical methods (kuzuhara et al., 2006). a similar result, based on histochemical blue staining reaction, is obtained if dna (from calf thymus) is incubated in egcg or green tea flavanols (fig. 2 i). dna and rna are faced by nucleases and these enzymes are repressed by egcg (tichopad et al., 2005). active euchromatin was found to be very sensitive to nucleases (paul and ferl, 1998). complexation of various polyphenols with dna results in a reduction of strand breaks (bidel et al., 2010). apart from the pulling stress as discussed above, a basic problem of the nucleus is that the thousand-fold compaction of the genome, as produced by curled and folded structures up to 700 nm, provide an enormous intranuclear pressure. here again, the pressure stress might be damped down by flavanols attached to histones and supercoiled dna. phenolic hydroxyl groups build hydrogen clamps, for instance with proline-rich protein complexes which, as explicitly emphasized by haslam (1998), are exposed to conditions of overstretching, bending and distortion. if a single hydrogen bond (o-h---o) is broken, then the whole group of a hydrophilic, hydrogen-bonded network will dissipate (haslam, 1998). all the features discussed here advocate for an intricate involvement of the nuclear flavanols in regulating nucleosome conformation. however, no individual factor such as the flavanol molecule is capable of playing an isolated physiological role. as most of these complex interactions as yet are barely understood, the flavanols located in nuclei need much more detailed basic research. references apel, k., hirt, h., 2004: reactive oxygen species: metabolism, oxidative and signal transduction. ann. rev. plant biol. 55, 373-399. bauer, j., neubauer, k., dithmar, h., polster, j., feucht, w., 2009: flavanols in nuclei and cytoplasm: reduction of the micronuclei inducing effect of aflatoxin b1 in v79 cells through catechin. adv. food sci. 31, 82-87. berg, o.g., winter, r.b., von hippel, p.m., 1981: diffusion-driven mechanisms of protein translocation on nucleic acids. models and theory. biochemistry 20, 6929-6948. bidel, l.p.r., coumans, m., baissac, y., doumas, p., jay-allemand, c., 2010: biological activity of phenolics in plant cells. in: santos-buelga, c., escribano-bailon, m.t., lattanzio, v. 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m.a., 2003: identi fication of novel histone post-translational modifications by peptide mass fingerprinting. chromosoma 112, 77-86. address of the corresponding authors: w. feucht, d. treutter, fg obstbau, dürnast 2, 85354 freising, germany e-mail: dieter.treutter@wzw.tum.de art19 journal of applied botany and food quality 82, 114 121 (2009) 1institute for landscape and plant ecology (320), universität hohenheim, stuttgart, germany 2german research centre for food chemistry and hans-dieter-belitz-institute for cereal research, garching, germany 3landesanstalt für landwirtschaftliche chemie, universität hohenheim, stuttgart, germany 4johann heinrich von thunen-institute, federal research institute for rural areas, forestry and fisheries, institute for biodiversity, braunschweig, germany does elevated atmospheric co2 allow for sufficient wheat grain quality in the future? p. högy1, h. wieser2, p. köhler2, k. schwadorf3, j. breuer3, m. erbs4, s. weber1, a. fangmeier1 (received september 22, 2008) summary to identify future impacts on biomass production and yield quality of important c 3 crops, spring wheat was grown in association with 13 weed species in a mini-face (free-air carbon dioxide (co 2 ) enrichment) system under ambient (375 µl l-1) and elevated (526 µl l-1) co 2 concentrations. wheat productivity was assessed at maturity and grain yield was subjected to various chemical analyses and baking quality tests. co 2 enrichment acted as carbon ‘fertiliser’ and increased the aboveground biomass production of wheat by 18.8% as there was a trend towards higher stem biomass. although not statistically significant, wheat grain yield was increased by 13.4% due to a significant establishment of more grains per unit ground area. at the same time, thousand grain weight was non-significantly shifted towards smaller grain size classes, which may result in negative consequences for the crop market value. as a result of the co 2 induced physiological and biochemical modifications, concentration of total grain protein was significantly decreased by 3.5%, reducing the wheat grain quality with potentially far-reaching impacts on the nutritional value and use for processing industry. although often not significant, the concentrations of amino acids per unit of flour were decreased by 0.2 to 8.3% due to elevated co 2 thereby affecting the composition of proteinogenic amino acids. furthermore, gluten proteins tended to decline. within the significant decreased gliadins, αand ω5-gliadins were significantly reduced under co 2 enrichment; there was also a negative trend for ω1,2and γ-gliadins. changes in certain essential minerals were found as well, although not statistically significant. concentrations of sodium, calcium, phosphorus and sulphur were slightly lowered and those of potassium and magnesium were slightly increased due to co 2 enrichment. the micro-element molybdenum was increased, while concentrations of iron, zinc, copper, manganese and aluminium were decreased. with regard to rheological and baking parameters defining the cereal quality for industrial processing, the resistance of the dough was significantly reduced by about 30%, while the extensibility was non-significantly increased by 17.1% under co 2 enrichment. moreover, the bread volume was decreased non-significantly by about 9%. elevated co 2 is obviously affecting grain characteristics important for consumer nutrition and health, industrial processing and marketing. experimental evidence for these changes is still poor but deserves further attention. introduction before the industrial revolution, the global atmospheric carbon dioxide (co 2 ) concentration was around 280 µl l-1 and has increased since then considerably to approximately 380 µl l-1 today. a further increase to about 550 µl l-1 by the year 2050 has been predicted (ipcc, 2007). beside indirect impacts via climate changes such as temperature increase and alterations in precipitation pattern, co 2 enrichment is acting directly on c 3 crops such as wheat in agroecosystems (ipcc, 2007; ziska and runion, 2007). the co 2 -induced impacts may be determined by interactions with other components such as the presence of biotic (pests, diseases, weeds) or abiotic (temperature, light, soil water, nutrients) stresses (fangmeier and jäger, 2001; ainsworth and long, 2005). wheat (triticum aestivum l.) is one of the most important c 3 crops worldwide. co 2 -induced consequences include increased photosynthesis as well as improved waterand nitrogen(n) use efficiencies. biomass and grain yield production of wheat in terms of tonnage is positively affected by co 2 enrichment (e.g. amthor 2001; ziska and bunce, 2007). in earlier chamber-based experiments, the gain in wheat grain yield was shown to occur by means of increases in thousand grain weight (tgw; mckee et al., 1997), grain number per ear (mckee et al., 1997; mulholland et al., 1997) or the number of flowering tillers per unit ground area (havelka et al., 1984; fangmeier et al., 1996). a recently published data evaluation of free-air co 2 enrichment (face) experiments by högy and fangmeier (2008) showed that tgw was significantly increased by 3.8% under elevated co 2 , indicating a potential positive effect on wheat grain quality in terms of milling quality and hence economic value. since co 2 enrichment alters the carbon(c) and n-metabolism in vegetative plant parts (cotrufo et al., 1998; loladze, 2002), the availability of metabolites for the grain during filling may be changed, thus affecting the chemical composition of grains with regard to different market and utilisation requirements. although grain quality is as important as yield, much less attention has been paid to possible effects of elevated co 2 concentrations on grain quality traits. potential co 2 effects on grain quality aspects other than concentration of total grain protein are scarcely published (kimball et al., 2002; loladze, 2002; högy and fangmeier, 2008). the total concentration of grain protein is a major determinant of grain price and is strongly and positively correlated with breadmaking quality in particular due to its impact on dough strength (macritchie, 1978; lawlor and mitchell, 2001). it has been shown that 80% of the n present in the vegetative plant parts of wheat will be distributed to the grain after anthesis (osaki et al., 1991; conroy and hocking, 1993; fangmeier et al., 1997). as a consequence of the co 2 -induced modifications in plant metabolism, grain protein concentration is generally decreased in face experiments (högy and fangmeier, 2008; taub et al., 2008). overall, the grain protein concentration is negatively related to grain yield (evans, 1993; pleijel et al., 1999), but this is not simply caused by dilution due to increased concentrations of non-protein components (gifford et al., 2000). currently, the mechanisms by which co 2 enrichment decreases proteins are not well understood. especially the unique chemical and physical properties of wheat grain protein fractions are the main factors explaining the widespread use of wheat (finney, 1985; pommeranz, 1988). accumulation of structural and metabolic proteins (albumins and globulins) appears to be sink-regulated and starts earlier (triboï et al., 2003), whereas storage protein accumulation is constrained by n sources to the developing grain (martre et al., 2003). while albumins and globulins have only a minor impact on dough strength and associated characteristics such as loaf volume, the storage proteins (monomeric gliadins and large disulfide linked polymeric glutenins) are primarily responsible for many aspects of grain quality (macritchie et al., 1990; weegels et al., 1996). especially the ratio between gliadins and glutenins affects plasticity, strength and elasticity of dough, which are traits fundamental to processing high-quality bread. wieser et al. (2008) recently reported that the amount of albumins and globulins was unaffected by co 2 enrichment, while the gluten proteins, especially the n-rich ω5-, ω1,2and α-gliadins and high molecular weight glutenin subunits, were decreased in a face experiment using winter wheat. moreover, metabolic proteins are rich in sulphur (s) containing amino acids such as cysteine (cys) and methionine (met) as well as essential amino acids, especially lysine (lys), resulting in a high nutritive value, while gluten proteins are rich in glutamine (gln) and proline (pro). in previous open-top chamber (otc) experiments, relatively more essential amino acids were found under co 2 enrichment (manderscheid et al., 1995; högy et al., 1998), indicating positive effects on the nutritive value. the composition of grain proteins and the balance of strength and elasticity are fundamental in determining the physical properties of dough formation and product quality (weegels et al., 1996; cornish et al., 2006). blumenthal et al. (1996) reported that co 2 enrichment did not cause significant or consistent impacts on dough properties except of the lowered peak resistance by 15.9%. dough extensibility and farinograph dough development time were also affected by co 2 enrichment in the same chamber-based experiment (blumenthal et al., 1996), which was largely associated with the decline in total protein concentration. in a prior face experiment, kimball et al. (2001) found a 6% increase in optimum mixing time for bread dough due to elevated levels of co 2 . currently, data of dough rheological properties from face experiments are still missing. moreover, significant decreases in volume of bread loaves were observed in chamber-based (blumenthal et al., 1996; fangmeier et al., 1999) and in face experiments (kimball et al., 2001). with regard to minerals, co 2 -induced impacts have been reported in otcs by manderscheid et al. (1995) for magnesium (mg), calcium (ca), s, iron (fe) and zinc (zn), by fangmeier et al. (1999) for ca, fe and s, by de la puente et al. (2000) for mg, potassium (k) and ca and by wu et al. (2004) for p, k and zn. data from face field studies are not available. the impacts of co 2 enrichment on the quality traits of wheat are currently not well understood and the experimental outcomes are often contradictory, suggesting both negative and positive implications for the nutritional value and industrial processing of grains in the future. therefore, in our field study we tested whether the co 2 -induced impacts on photosynthesis and nutrient allocation of wheat plants have consequences on grain yield and grain quality with regard to food safety. wheat grains were subjected to analyses of general quality aspects (number of grains per unit area, grain yield, tgw, grain size pattern), several chemical analyses (concentrations of macroand micro-elements, amino acids, total proteins and protein composition) as well as mixing, rheological and baking quality tests to comprise whether these parameters were affected by realistic future concentrations (ambient + 150 µl l-1 co 2 ) in a face system. materials and methods experimental co2 treatments, environmental monitoring and plant cultivation the experiments were performed in 2005 at the heidfeldhof farm managed by the plant breeding station of the universität hohenheim (9°11´e, 48°42´n, 395 m above sea level) south of stuttgart, germany. spring wheat (triticum aestivum l. cv. triso) as main crop and 13 associated weed species were grown on circular field plots (2 m in diameter). experimental treatments were (i) ambient co 2 concentrations without exposure system (control), (ii) ambient co 2 concentrations with exposure system (ambient) and (iii) elevated co 2 concentrations with exposure system (face) with five replicates each. the principle of operation and the performance of the face system have been described by erbs and fangmeier (2006) and erbs et al. (2008). the co 2 exposure was started before emergence of the plants (04. april 2005). to achieve the target gas concentrations of 150 µl l-1 above ambient during daylight hours (average from 7.00 a.m. to 8.00 p.m.), pure co 2 (westfalen gas, germany) was added and concentrations achieved at canopy height were monitored at the centre of each field plot. wind speed and direction (siggelkow, germany; reference height 1 m) as well as air temperature, relative humidity (rotronic, switzerland) and solar radiation (kipp & zonen, the netherlands; reference height 2 m, respectively) were measured at the centre of the field site. precipitation was recorded at the nearby weather station stuttgart airport from the "deutscher wetterdienst", which is about 2 km from the field site. the amount of rain is similar at both sites (j. franzaring, personal communication). local soil (clayey to loamy) was used for field-grown plants in the 15 plots. wheat (13 lines with 15 cm row spacing; 200 plants m-2) and weed species were sown on 24. march 2005. based on agronomic practice, plots were fertilised with 140 kg n ha-1, 30 kg phosphorous (p) ha-1 and 60 kg k ha-1, which was supplied in 50/25/25% portions at different development stages of the spring wheat (ec 13-29, ec 30-39 and ec 40-59, respectively; tottman and broad, 1987). additionally, a trace element fertiliser (k&s kali, germany) was used at ec 30-39 containing 0.03 kg boron (b) ha-1, 0.45 kg mg ha-1, 0.36 kg s ha-1 and 0.03 kg manganese (mn) ha-1. time domain reflectometry (tdr) sensors for soil moisture (imko, germany) were horizontally installed in a depth of 15 cm. plants were manually irrigated when the volumetric soil moisture declined to 25% with the same amount of water applied to all treatments. irrigation volumes were recorded and included into the calculation of total water supply. wheat plants of the central square metre of each plot were harvested at maturity (01. august 2005) and separated into leaves, stems and ears. the biomass of the different wheat fractions was determined after drying until constant weight at 60°c, except for the ears (30°c). grains were removed from ears by threshing, ground to wholemeal flour using a laboratory mixer mill (mm 301, retsch, germany) and stored for quality analyses such as concentrations of macroand micro-elements and amino acids. in a second step, the grains were milled to refined flour (type 550) for analyses of protein types, mixing and rheological properties and baking tests using a brabender quadrumat junior mill. determination of grain quality parameters the tgw was determined (condator "e", pfeuffer, germany) and grains were separated into different size classes (> 2.8 mm; 2.82.5 mm; 2.5-2.2 mm; < 2.2 mm) using a sortimat (type k, pfeuffer, germany). the s and n concentrations of wholemeal flour were determined using an elemental analyser (vario el, elementar analysensysteme, germany) according to iso 10694 (1995), while the n concentration of refined flour was obtained by the dumas combustion method using a leco fp-328 (leco, usa). the total protein concentration was calculated by multiplying the n concentration by a conversion factor of 5.7 for wheat grain. the individual macroand microelements were determined after acid digestion using (i) inductivelycoupled plasma optical emission spectrometry (icp-oes, vista pro radial, varian, usa) for aluminium (al), calcium (ca), fe, k, mg, sodium (na), p and (ii) inductively-coupled plasma mass-spectrometry (icp-ms, elan 6000, perkin elmer sciex, usa) for b, copper co 2 enrichment and wheat grain quality 115 116 p. högy, h. wieser, p. köhler, k. schwadorf, j. breuer, m. erbs, s. weber, a. fangmeier (cu), mn, molybdenum (mo), zn according to the method of vdlufa vii 2.2.2.5. (2003) and vdlufa vii 2.2.2.6. (2003). prior to analysis the samples were digested with highly pure nitric acid (baker instra-analysed, usa) at 220°c and approximately 10 mpa in quartz vessels using a high pressure digestion system (ultraclave iii, mls, germany). the amino acid concentrations were analysed according to the european commission directive 98/64/ec (1998). the protein types in wheat flour were quantified by a modified osborne fractionation followed by reversed-phase high-performance liquid chromatography (rp-hplc) using the protocol of wieser et al. (1998). the mixing and rheological properties and the baking quality tests were analysed according to micro-scale methods described previously (kieffer et al., 1998). data, replication, statistics the statistical significances of the co 2 effects on wheat biomass fractions and grain quality aspects were tested by an analysis of variance (anova) using spss pc (version 15 for windows). data from ambient and face treatments were used for the analyses, while data from the control treatment was excluded from the evaluation since differences to ambient treatments were not significant. results were expressed as probability (p) levels (ns, p > 0.1; numbers in parenthesis indicate trends with 0.1 > p > 0.05; numbers without parenthesis indicate p < 0.05). data from face treatments (four replicates) were given as co 2 response in relation to the ambient treatments (five replicates) of the experiment (ambient = 100%). results climatic conditions and experimental co2 concentrations in 2005, the cultivation period (130 days) was rather moist with frequent thunderstorms and high precipitation in single rain events. from sowing to maturity, the plants received 425 mm of water. the averages ± standard deviations of daily minimum, mean and maximum temperatures averaged over the cropping season were 9.2 ± 4.8°c, 14.8 ± 5.3°c and 20.1 ± 6.4°c, respectively, and the growing degree days (baseline > 5°c) were 1120°c. the mean seasonal daylight co 2 concentrations achieved were 375 ± 11 µl l-1 (ambient) and 526 ± 5 µl l-1 (face), demonstrating that the exposure system worked satisfactorily. biomass production, grain yield and grain quality traits co 2 enrichment increased the wheat aboveground biomass at final harvest by 18.8% (p = ns) with a trend towards increased stem weight due to co 2 enrichment (tab. 1). leaf and ear biomass were increased by 13.5 and 13.3%, respectively; however, these increases were not statistically significant. grain yield increased non-significantly by 13.5%, resulting from a co 2 -induced increase in the number of ears (+22.4%, p = 0.031) and grains (+14.8%, p = ns) produced per unit of area. concomitantly, the grain number per ear and the harvest index was not significantly affected under elevated co 2 . also the tgw remained unaffected under co 2 enrichment, although there were slight but non-significant shifts towards smaller grains according to the results obtained for different grain size classes. the concentrations of macroand micro-elements were not significantly affected by co 2 enrichment (tab. 2). however, the macroelements k and mg were increased, whereas na, ca, p and s were decreased. the concentrations of micro-elements such as fe, zn, cu, mn and al were also decreased, while the only co 2 -induced increase was observed for mo. the total protein concentration in wheat grains was significantly decreased in the high-co 2 treatment, whereas the protein yield (i.e. protein harvested per ground area) was not changed. the co 2 -induced impact on protein concentrations was slightly larger (-4.2%) when protein was measured in refined rather than whole-grain flour (data not shown). tab. 1: aboveground biomass production, grain yield traits and grain size classes of wheat grown in the field at either ambient (ambient) or elevated (face) co 2 concentrations. values represent means and standard deviations of the replicates per treatment, the calculated co 2 effect (ambient = 100%) and level of statistical significance of the one-way analysis of variance (anova). wheat traits ambient face co2 effect p-level wheat production [g dw m-2] aboveground 1041 ± 191 1236 ± 169 +18.8% ns leaves 106.7 ± 26.9 121.1 ± 17.2 +13.5% ns stems 450.4 ± 75.0 568.8 ± 78.1 +26.3% (0.054) ears 479.4 ± 102.7 543.1 ± 139.2 +13.3% ns yield parameters ear number [no. m-2] 435.2 ± 50.6 532.6 ± 58.4 +22.4% 0.031 grain number [no. m-2] 8747 ± 1892 10044 ± 2729 +14.8% ns grain number [no. ear-1] 19.99 ± 2.76 19.02 ± 5.46 -4.9% ns grain yield [g dw m-2] 350.9 ± 84.0 398.2 ± 129.8 +13.5% ns harvest index 0.34 ± 0.04 0.32 ± 0.08 -5.3% ns tgw [g 1000 grains-1] 39.95 ± 1.00 39.19 ± 2.30 -1.9% ns grain size classes [% grains] > 2.8 mm 55.6 ± 3.3 52.6 ± 7.7 -5.4% ns 2.8 – 2.5 mm 32.9 ± 2.4 31.7 ± 2.7 -3.8% ns 2.5 – 2.2 mm 7.6 ± 1.1 10.2 ± 3.1 +35.2% ns < 2.2 mm 3.9 ± 1.3 5.5 ± 2.1 +40.1% ns ns = no significance at p < 0.05; number in parenthesis indicates trend (0.1 > p > 0.05). co 2 enrichment and wheat grain quality 117 tab. 2: concentrations of macroand micro-elements of wheat grains grown in the field at either ambient (ambient) or elevated (face) co 2 concentrations. values represent means and standard deviations of the replicates per treatment, the calculated co 2 effect (ambient = 100%) and level of statistical significance of the one-way analysis of variance (anova). nutrient elements ambient face co2 effect p-level macro-elements [mg kg-1 dw] na 10.2 ± 2.2 9.8 ± 1.6 -4.1% ns k 892 ± 202 4943 ± 144 +1.0% ns ca 439 ± 13 434 ± 36 -1.1% ns mg 1812 ± 54 1835 ± 44 +1.3% ns p 5070 ± 105 5060 ± 67 -0.2% ns s 1864 ± 47 1835 ± 47 -1.6% ns micro-elements [mg kg-1 dw] fe 63.4 ± 3.1 1.3 ± 2.2 -3.4% ns zn 36.2 ± 1.6 36.0 ± 2.2 -0.6% ns cu 6.85 ± 0.42 6.66 ± 0.24 -2.8% ns mn 26.4 ± 3.0 25.5 ± 3.7 -3.4% ns mo 0.75 ± 0.14 0.76 ± 0.15 +0.9% ns al 260 ± 154 247 ± 118 -5.1% ns ns = no significance at p < 0.05. in agreement with the responses in protein concentration, the concentrations of all amino acids per unit of flour weight were decreased by 0.2 to 8.3% in the face treatment (fig. 1). of the proteinogenic amino acids, some are called essential amino acids because they cannot be synthesised de novo by humans and must be obtained from food. the semi-essential amino acids, however, are not normally required in the diet, but must be supplied exogenously to specific populations because the metabolic pathways that synthesise these amino acids in adequate amounts are not fully developed. in the present study, the co 2 effects were only significant for non-essential glycine (gly) and essential valine (val), while a trend was observed for non-essential gln. there was no clear pattern whether different amino acid types were more or less reduced in the high-co 2 treatment. correspondingly, if amino acid concentrations were calculated on a per protein basis, there were decreases in the concentrations of gly, valine (val), methionine (met), gln, leucine (leu), pro, isoleucine (ile) and aspartic acid (asp) by 1.1 to 5.5% in the face treatment (fig. 2). the only significant co 2 effect was observed for gly, while for val only such a trend was found. concomitantly, nonsignificant co 2 -induced increases of tryptophan (trp), arginine (arg), lys, tyrosine (tyr), cys, phenylalanine (phe), serine (ser), alanine (ala), histidine (his) and threonine (thr) were observed by 0.5 to 3.3%. the protein fractions of albumins/globulins were not affected by co 2 enrichment (fig. 3). glutens tended to be decreased by co 2 enrichment. the gluten-related gliadin fraction was significantly decreased under elevated co 2 , while the glutenins remained unaffected. as a consequence, the glutenin-gliadin ratio was increased, although this effect was not statistically significant. within the gliadin proteins, ω5-gliadins and α-gliadins were significantly decreased in the high 0 -10 -9 -8 -7 -6 -5 -4 -3 -2 -1 ala tyr met gln phe gly pro asp arg ile val thr lys leu cys his ser trp (0.082) 0.016 0.018 essential semi-essential essential for children non-essential r e la ti v e c h a n g e i n c o n c e n tr a ti o n [ % d w ] 0 -10 -9 -8 -7 -6 -5 -4 -3 -2 -1 ala tyr met gln phe gly pro asp arg ile val thr lys leu cys his ser trp (0.082) 0.016 0.018 essential semi-essential essential for children non-essential r e la ti v e c h a n g e i n c o n c e n tr a ti o n [ % d w ] fig. 1: relative responses of amino acids [% dw] in wheat grains due to co 2 enrichment. the results of the statistical analyses are denoted as levels of probability (p) of the one-way analysis of variance (anova) at p < 0.05, numbers in parenthesis indicate a trend (0.1 > p > 0.05). -6 -5 -4 -3 -2 -1 0 1 2 3 4 ala tyr met gln phe gly pro asp arg ile val thr lys leu cys his ser trp 0.015 (0.095)r e la ti v e c h a n g e i n c o n c e n tr a ti o n [ % p ro te in ] essential semi-essential essential for children non-essential -6 -5 -4 -3 -2 -1 0 1 2 3 4 ala tyr met gln phe gly pro asp arg ile val thr lys leu cys his ser trp 0.015 (0.095)r e la ti v e c h a n g e i n c o n c e n tr a ti o n [ % p ro te in ] essential semi-essential essential for children non-essential fig. 2: relative responses of amino acids [% protein] in wheat grains due to co 2 enrichment. the results of the statistical analyses are denoted as levels of probability (p) of the one-way analysis of variance (anova) at p < 0.05, numbers in parenthesis indicate a trend (0.1 > p > 0.05). 118 p. högy, h. wieser, p. köhler, k. schwadorf, j. breuer, m. erbs, s. weber, a. fangmeier co 2 treatment (fig. 4). moreover, ω1,2-gliadins and γ-gliadins tended also to be decreased under elevated co 2 . the mixing properties such as water absorption, dough development time and degree of dough softening showed no significant responses to elevated co 2 concentrations (tab. 3). with regard to the rheological properties, the dough resistance was significantly decreased in the high-co 2 treatment. in contrast, the dough extensibility was increased by 17.1%, although this co 2 effect was not statistically significant. the area of the extensogram was not affected. furthermore, elevated co 2 exposure non-significantly decreased the loaf volume by 8.9%. discussion co2-induced impacts on biomass allocation, grain yield and yield components of wheat in the present study, no significant co 2 effect on aboveground biomass production of wheat was observed, although wheat production was increased by 18.8%. the biomass allocation towards stems tended to increase due to co 2 enrichment, while that allocated to leaves and ears was not affected. nevertheless, ear number per area was shown to significantly increase in response to co 2 exposure, whereas no significant co 2 effect was found for grain yield, although the latter was increased by 13.5%. the present grain yield gain is in accordance with increases by 10 to 16% found in previous face studies when co 2 levels were raised from 380 to 550 µmol l-1 at ample supplies of n and water (kimball, 2004, 2006; kimball et al., 2001, 2002; long et al., 2006; schimel, 2006). however, the mean total grain yields were lower than in regional cultivation trials and reached a mean of 35 dt ha-1 (ambient), which may have resulted from the lower plant density of wheat plants and the competitive pressure from weeds. in our study, other grain yield components such as grain number per unit area and grain number per ear as well as the harvest index remained unaffected in the highco 2 exposure. the tgw was also unaffected in the face treatment. this is in agreement with results from previous face studies (kimball et al., 2001; manderscheid et al., 2004), while li et al. (2001) observed an increase in tgw by 7% under high-co 2 conditions. in summary, tgw may be affected due to elevated co 2 , but in an inconsistent fashion, depending on the experimental conditions. in the present study, a non-significant shift to smaller grain sizes was observed, resulting in a lower market value of the grains. no results on this topic are available from other face experiments, while significant shifts to smaller grain sizes were also found in otcs (högy, 2002). tab. 3: co 2 -induced impacts on wheat grains and consequences for mixing and rheological properties of flour and loaf volume. values represent means and standard deviations of the replicates per treatment, the calculated co 2 effect (ambient = 100%) and level of statistical significance of the oneway analysis of variance (anova). be, brabender units; n, newton. grain quality traits ambient face co2 effect p-level mixing properties water absorption [ml] 4.66 ± 0.08 4.67 ± 0.03 +0.1% ns dough development time [min] 12.6 ± 1.8 12.5 ± 0.6 -0.8% ns degree of dough softening [be] 45.0 ± 6.1 43.8 ± 11.1 -2.8% ns rheological properties dough resistance [mn] 298 ± 68 198 ± 48 -33.8% 0.041 dough extensibility [mm] 78.9 ± 22.8 92.4 ± 9.9 +17.1% ns area of extensogram [n x mm] 11.6 ± 4.0 11.2 ± 2.5 -2.9% ns loaf volume [ml] 32.4 ± 9.6 29.5 ± 2.0 -8.9% ns ns = no significance at p < 0.05. ambient face 0 500 1000 1500 2000 2500 r e la ti v e a m o u n t [a u m g -1 d w ] albumin/ globulin gliadin glutenin gluten ns ns (0.078) 0.017 ambient face 0 500 1000 1500 2000 2500 r e la ti v e a m o u n t [a u m g -1 d w ] albumin/ globulin gliadin glutenin gluten ns ns (0.078) 0.017 fig. 3: grain protein fractions of wheat grown in the field at either ambient (ambient) or elevated (face) co 2 concentrations. values are means and standard deviations of all replicates per treatment. the results of the statistical analyses were expressed as levels of probability (p) of the one-way analysis of variance (anova) at p < 0.05, ns = no significance at p > 0.05, numbers in parenthesis indicate a trend (0.1 > p > 0.05). fig. 4: relative responses of gliadin fractions [% dw] in wheat grains due to co 2 enrichment. the results of the statistical analyses are denoted as levels of probability (p) of the one-way analysis of variance (anova) at p < 0.05, numbers in parenthesis indicate a trend (0.1 > p > 0.05). 0.022 0 200 400 600 800 ω5-gliadin ω1,2-gliadin α-gliadin γ-gliadin (0.064) 0.018 (0.072) ambient face r e la ti ve a m o u n t [a u m g -1 d w ] 0.022 0 200 400 600 800 ω5-gliadin ω1,2-gliadin α-gliadin γ-gliadin (0.064) 0.018 (0.072) ambient face r e la ti ve a m o u n t [a u m g -1 d w ] co 2 enrichment and wheat grain quality 119 alterations of mineral composition in wheat grains due to co2 enrichment since most of the minerals in wheat grains originate from the redistribution from vegetative pools during grain filling, co 2 enrichment may cause serious alterations in the concentrations of macroand micro-elements, probably diminishing the nutritional value. in our study, reductions due to elevated co 2 were not statistically significant, although all concentrations except for k, mg and mo were decreased between 0.2 to 5.1%. until today, knowledge with regard to co 2 -induced impacts on the elemental composition of wheat grains from face experiments is scarce. effects of co2 enrichment on concentration and composition of wheat grain proteins in the present study, the total protein concentration per dry weight was 15.7% (ambient) and 15.2% (face), which is quite high for summer wheat produced under n supply conventional for regional agronomic practice. the grain protein concentration was significantly decreased by 3.5% under co 2 enrichment, which is in accordance with earlier findings from face experiments (kimball et al., 2001, 2002; kimball, 2004; högy and fangmeier, 2008; taub et al., 2008). in contrast, wieser et al. (2008) reported that the protein concentration of wheat grains was lowered by about 14% at comparable co 2 enrichment (ambient + 150 µl l-1). since total protein concentration greater than 11.5% is required for adequate breadmaking quality, crop fertilisation practices may need to be altered in a future high-co 2 environment in order to achieve the quality requirements. unfortunately, the co 2 -induced reductions in grain quality may not easily be overcome by increases in n fertilisation since this may translate into higher biomass and yield production rather than into enhanced redistribution of n to the grains (fangmeier et al. 1999; weigel and manderscheid, 2005). results from previous, european-wide studies in otcs suggest that the loss in grain protein under co 2 enrichment is similar irrespective of the amount of n supply to the wheat crop (fangmeier et al., 1999). also taub et al. (2008) stated that even under high n treatments, wheat still experiences a reduction in protein concentration by 10%. therefore, co 2 enrichment is likely to make the wheat grain quality poorer in the future with regard to nutritional value and use for processing industry. it remains open whether more n fertilisation will compensate the co 2 -induced loss in grain protein. moreover, in our face study the grain protein yield was unaffected, while piikki et al. (2008) reported a significant increase of protein yield in chamber-based experiments, resulting from a higher grain yield caused by elevated co 2 concentrations. taub et al. (2008) suggested that the co 2 effect on human nutrition may differ between consumers of refined or whole-grain flour since co 2 may affect the protein concentration in endosperm less than in other grain parts. here we directly examined this possibility and found no clear evidence for this suggestion. in the present study, the composition of grain protein fractions was affected by co 2 enrichment, resulting in a serious impairment of the bread-making quality. the albumins and globulins were unaffected, whereas the gluten proteins tended to be decreased in the high-co 2 treatment. also wieser et al. (2008) reported that both gluten proteins and their fractions were decreased in a comparable face experiment. our results demonstrate that among the gluten proteins, the nand gln-rich gliadins showed a more pronounced co 2 -induced decrease, while the glutenins remained unaffected. although not significant in the present study, comparable increases of the glutenin-gliadin ratio were reported in chamber-based (blumenthal et al., 1996) and face (wieser et al., 2008) experiments. within the gliadin fraction, the ω5and ω1,2-gliadins with a high n requirement showed a more pronounced decrease due to co 2 enrichment than did the αand γgliadins, which is in accordance with wieser et al. (2008). currently, no further information on this topic is available in the literature. co2-induced impacts on amino acid concentration and composition of wheat grains along with the reduced protein concentration and the observed changes in protein composition in wheat grains, the concentrations of amino acids were affected, too. all concentrations of amino acids were reduced between 0.2 and 8.3% in the present face study, although effects were only significant for gly and val, while a trend was observed for gln. no clear co 2 -induced impacts on the pattern of different types of amino acids were observed in our face study. in contrast, manderscheid et al. (1995) and högy et al. (1998) reported increases in the concentrations of essential amino acids in wheat grains under elevated co 2 concentrations in chamber-based experiments. moreover, in the present study changes in the composition of amino acids were found under co 2 enrichment. no comparable results have been reported from other co 2 experiments using face techniques. based on otc experiments, högy et al. (1998) reported that the amino acid composition of wheat grains was affected by elevated co 2 , which likely indicated a decline in grain storage proteins, while metabolic proteins were unchanged or increased due to co 2 enrichment. currently, this view is supported by decreased gluten proteins found in high-co 2 conditions (wieser et al., 2008). since amino acids such as asn, and marginally gln as well as asp, are involved in the formation of acryl amide during industrial processes like baking, the possible concentration of acryl amide may decrease too in flour-derived products in a high-co 2 world. however, there is no existing experimental database related to this topic to draw final conclusions. effects of co2 enrichment on mixing and rheological properties and loaf volume of wheat flour in our study, the peak resistance was significantly decreased by 33.8%, which is in accordance with the results reported by blumenthal et al. (1996) from chamber-based experiments, although the present co 2 -induced impact was nearly twice as large. no significant co 2 effects on dough extensibility and farinograph dough development time were found in the present face study, which is in contrast with the study by blumenthal et al. (1996). while in the present face experiment the volume of bread loaves was unaffected under co 2 enrichment, significant decreases were reported in a previous experiment (kimball et al., 2001). however, the change in grain protein is often more pronounced than co 2 -induced impacts on the functional properties for wheat processing (rudorff et al., 1996; lawlor and mitchell, 2001). conclusions the present results demonstrate that especially the n-rich protein fractions, which are important for the visco-elastic properties of wheat grains, were negatively affected under co 2 enrichment. although it seems that co 2 -induced impacts on wheat grain quality may be relatively small, we should be concerned about it. moreover, as the magnitude of co 2 -induced effects on grain quality aspects is often variable because of the sensitivity of the results to experimental conditions such as cultivar, location, climate, agronomic practice and more, it is important to realise multiple-year face field experiments at different sites. despite the importance in terms of grain quality, the database concerning possible co 2 effects is currently small. further research is needed, especially with regard to potential 120 p. högy, h. wieser, p. köhler, k. schwadorf, j. breuer, m. erbs, s. weber, a. fangmeier technological adjustments by breeding, by agronomic management practices and by processing to maintain the quality requirements for all consumers of wheat grains in a future co 2 -enriched world. acknowledgements the authors express thanks to mrs. g. gensheimer and the work of all assistants for largely contributing to the hohenheim mini-face experiment. we also thank dr. j.-j. kim and mrs. a. axthelm for excellent technical support. the authors would like to thank the reviewers for their careful reading and very helpful and constructive comments. references ainsworth, e.a., long, s.p., 2005: what have we learned from 15 years of free-air co2 enrichment (face)? meta-analytic review of the responses of photosynthesis, canopy properties and plant production to rising co2. new phytol. 165, 351-371. amthor, j.s., 2001: effects of atmospheric co2 concentration on wheat yield: review of results from experiments using various approaches to control co2 concentration. field crop. res. 73, 1-34. blumenthal, c., rawson, h.m., mckenzie, e., gras, p.w., barlow, e.w.r., wrigley, c.w., 1996: changes in wheat grain quality due to doubling the level of atmospheric co2. cereal chem. 73, 762-766. conroy, j.p., hocking, p., 1993: nitrogen nutrition of c3 plants at elevated atmospheric co2 concentrations. physiol. plant. 89, 570-576. cornish, g.b., bekes, f., eagles, h.a., payne, p.i., 2006: prediction of dough properties for bread wheat. in: wrigley, c., bekes, f., bushuk, w. 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/pagesize [907.087 680.315] >> setpagedevice vorgabe neu journal of applied botany and food quality 81, 56 61 (2007) institute of plant molecular physiology and biotechnology, university of bonn protease activity in the medium of larch (larix spec.) embryogenic suspension cultures and medium-protein stabilization by compatible solutes philipp westhofen, ghada ismail, kurt zoglauer, robert boehm* (received march 5, 2007) summary the stability of recombinant, secreted protein in the medium of transgenic plant cell cultures heavily determines the resulting protein yield, which is a crucial factor for every production system. in order to gain more knowledge about the feasability of rapidly growing larch (larix sp.) embrogenic cell cultures as a possible expression system for recombinant proteins, spent cell culture medium was characterized in this study. an accumulation of endogenous proteins could be observed in the medium of larch embrogenic suspension cultures which reached up to 1750 µg per g fresh weight. in contrast, low protease activity accumulated within a typical 14-day culture period in the medium. this activity was up to 20 times lower than the protease activity in two callus-derived suspension cultures of tobacco (genotype sr1 and by-2) which were measured in parallel. to asses the stability of foreign proteins, medium aliquots were spiked with immunoglobulin g (igg) and the amount of protein degradation was determined after 23 h of incubation by sds-page. the loss of igg was comparable in three different larch genotypes, resulting in a mean loss of 18 % during the incubation time. this loss could remarkably be diminished by the addition of ectoin derivatives, known to be protein-protective „compatible solutes“ of bacterial origin. the most effective one was hydroxyectoin which resulted in a 76 % reduction of the observed igg degradation. the stabilization of proteins in plant cell culture medium by compatible solutes is shown here for the first time. the possible mechanism of the stabilizing effect is discussed. abbreviations: 2.4-d, 2.4-dichlorophenoxy acetic acid, cim, callus induction medium, ms, murashige and skoog, pbs, phosphate buffered saline introduction plant cell suspension cultures may offer a viable alternative to established bacterial and animal cell culture systems for the production of recombinant therapeutic proteins since they are able to generate complex oligomeric proteins in a biologically active manner without the risc of human pathogenic contamination (doran, 2000). moreover, they can be grown on simple, cost-effective media under controlled conditions without the release of transgenic plants into the environment. the secretion of the expressed proteins into the medium is an important feature for it enables a simplified purification strategy (fischer et al., 1999a) without the need to harvest and extract the cell material. this also is the basis for sophisticated bioreactor cultures using perfusion-, semi-continuous or continuous fermentation strategies (mcdonald et al., 2005; lee et al., 2004). plant cell culture media generally consist of inorganic compounds (nutrients and trace elements) and some organics like vitamins or sugar. this aqueous, low salt-environment can be, however, a critical factor for the stability of secreted proteins, resulting in inactivation or degradation after secretion (tsoi and doran; 2002, sharp and doran, 2001). additionally, co-secreted proteases were detected in tobacco suspension cultures, causing loss of recombinant target protein (schiermeyer et al., 2005; kwon et al., 2003). the recovery of protein stability is therefor one crucial parameter that heavily determines the overall yield of recombinant protein from plant in vitro-cultures. for this, several protein-protective agents have been added like gelatin (kwon et al., 2003), polyvinylpyrrolidone (lacount et al., 1997) or protease inhibitors (bateman et al., 1996). baur et al. (2005) have co-expressed human serum albumin to stabilize secreted recombinant protein. a new class of protein-protective substances are the compatible solutes (kempf and bremer, 1998; galinski, 1993). these low-molecular-weight molecules are taken up or produced by halophilic bacteria to protect themselves against water stress in extreme environments. the protein-protective properties have been applied to plant cells by transformation with the relevant biosynthesis enzymes (bohnert and shen, 1999), but they never have been tested as a medium additive for plant cell cultures yet. embryogenic cultures of larch (larix spec.) originated by direct somatic embryogenesis from zygotic embryos (klimaszewska, 1989) are propagating rapidely in suspension culture (jain, 1998; silveira et al., 2004). moreover, the genetic transformation could be established (ismail et al., 2004; taryono, 2000). this makes this type of plant material a promising tool for biotechnological application like mass propagation (silveira et al., 2004; archambault et al., 1994) or production of foreign proteins under controlled conditions (doran, 2000). to further test the suitablility of these cultures for protein production, the possible secretion of proteases into the medium over the culture period was tested in comparison with tobacco suspension cultures. moreover, the stability of exogeneously added immunoglobuline g (igg) was tested in spent culture medium aliquots. finally, the influence of the addition of compatible solutes (ectoin, hydroxyection, homoectoin) to the medium on protein (igg) stability was tested here for the first time. material and methods chemicals azocasein, imunnoglobulin g (technical grade, i 8640) and polyvinylpyrrolidone were purchased from sigma (st. louis, usa). ectoin and homoectoin were from bitop gmbh (witten, germany). hydroxyectoine was synthesized at the institute of arteriosclerosis research, university of münster, germany (schnoor et al., 2004). all other chemicals were of the highest purity available. plant cell culture embryogenic cultures of larix decidua mill., genotypes 4/93 and s90, and larix x eurolepis genotype 58 were maintained on msg medium (becwar et al., 1990) which is a modified murashige and skoog´s (ms) medium with vitamins but with decreased nitrate and sup-* corresponding author plemented with 9 µm 2.4-dichlorophenoxyacetic acid (2.4-d), 2.25 µm 6-benzylaminopurine (bap), 10 mm l-glutamine and solidified with 0.3 % (w/v) gelrite. suspension cultures were initiated by adding embryonal masses (3 g fw) to 50 ml liquid msg-medium in a 300 ml erlenmeyer-flask and maintaining on a rotary shaker at 80 rpm and 21°c ± 2°c in the dark. they were subcultured every 14 days. additionally, callus-derived suspension cultures of tobacco (nicotiana tabacum l.) cv petite havana „sr1“ and cv. bright yellow 2 (by-2) have been used for control experiments. sr1 cells were cultivated in liquid callus induction medium (cim), which consists of basal msmedium (4.6 g/l ms-salts, 30 g/l sucrose, ph 6.3) supplemented with 0.5 mg/l 2.4-d. the genotype by-2 was cultivated in liquid by2 medium, which consists of a modified ms medium (4.3 g/l mssalts, 30 g/l sucrose, ph 5.8) supplemented with 0.6 mg/l thiamine/ hcl, 200 mg/l kh 2 po 4 and 0.5 mg/l 2.4-d. cultures were cultivated on a rotary shaker under identical conditions as decribed above. for fresh weight determination, suspensions were passed over a sterilized nylon sieve (50 µm mesh width) to separate the cells from the medium. the plant cells were weighted after removel of residual liquid using sterilized filter paper protein preparation from medium medium of suspension cultures of l. decidua genotypes 4/93 and s90 was harvested 3, 7, 10 and 14 days after inoculation and the fresh weight was determined as decribed above. protein quantification was performed by bradford method (bradford, 1976). 1 ml-aliquots of the media were concentrated by filtering over regenerated cellulose (amicon ultracel ym-10 columns, millipore, billerica, usa). the concentrats were adjusted to 21 µl with water and used for sds-page. soluble cellular protein extraction for soluble cellular protein control, 0.8-1 g fresh weight of cell material of each genotype was ground in liquid nitrogen to a fine powder with mortar and pestle. total soluble protein was extracted using 2 ml extraction buffer (100 mm 2-(n-morpholino) ethane sulfonic acid, ph 6.0, 1 mm dithiotheritol, 10 % (w/v) glycerol,) per g of tissue. cell debris was removed by centrifugation at 14,000 rpm and 4°c for 15 min and the supernatant was the crude extract of soluble proteins. protein quantification was done by bradford method (bradford, 1976). protease activity assay protease activity in the medium was determined by azocasein degradation after sharp and doran (2001). briefly, 650 µl medium aliquots were incubated with 350 µl azocasein solution (1 % (w/v) azocasein in pbs) for 16 h at 25°c. the azocasein was precepitated by adding 500 µl of icecold 20 % (w/v) trichloro acetic acid. after centrifugation at 14,000 rpm at 4°c for 10 min, the supernatant was used for subsequent photometrical analysis at 340 nm. for background correction, a blank assay using fresh instead of spent medium was used and the value was substracted from spent medium measurements. igg stability assay medium aliquots of 500 µl each were taken from 7 days-old suspension cultures of all three genotypes. in experiments where somatic embryo material was included in the aliquots, a total of 650 µl was taken. after spiking each aliquot with 5 µg igg, they were incubated for 6 or 23 h at 21°c on a rotary shaker at 80 rpm. for protein precipitation, two volumes of icecold ethanol were added and the samples were stored at -20°c overnight. after harvesting the protein by centrifugation at 14,000 rpm and 4°c for 30 min, the resulting pellet was washed with 70 % (v/v) ethanol, air-dried and resolved in 21 µl of water for subsequent analysis using sds-page. for testing the influence of different additives on the igg stability, medium aliquots were processed as described above, but samples were adjusted to 0.1 m ectoine, 0.1 m hydroxyectoine, 0.1 m homoectoine or 0.75 g/l polyvinylpyrrolidone before spiking the samples with 5 µg igg. sds-page and protein quantitation 6 µl of 5 x laemli buffer (62.5 mm tris/hcl ph 6.6, 2 % (w/v) sds, 10 % (v/v) glycerol, 0.8 % (w/v) bromphenol blue) and 3 µl 0.5 m dithiothreitol were added to each sample which was denatured afterwards at 100°c for 5 min. proteins were spearated on a 12.5 % sds-page with 25 ma per gel. after staining of the gels using coomassie brilliant blue (for spiking experiments) or silver staining protocol (for naturally secreted proteins), they were photographed using a hv c20a video camera (hitachi, japan). for quantitation, band intensity was analyzed using quantity onetm software version 4.2.1.(biorad, hercules, usa). after background substraction, the intensity of the igg heavy chainbands of each sample was determined. the intensity of a prominent protein band of a medium protein of ca. 50 kda that occurred in all media aliquots was used as an internal standard. two igg samples (2,5 and 5 µg) were also included in each gel and the resulting intensity of the heavy chain-bands was used to quantify the amount of igg in the medium samples. results protein accumulation in the medium of embryogenic larch cultures to preliminary clarify whether embryogenic cultures of larch at all secrete any natural proteins into the medium, samples of 1 ml were taken from genotypes 4/93, s90 and 58 3, 7, 10 and 14 days after inoculation and the protein content was determined. all three genotypes showed a time-dependend accumulation of proteins. the amounts, however, were highly genotype-dependent. at the end of the 14 dayculture, medium proteins had amounted to 110 µg/g fw (genotype 4/93), 320 µg/g fw (genotype s90) and 507 µg/g fw (genotype 58) (see fig. 1 a). the protein content remained quite low during the first 10 days of culture in genotypes 4/93 and s90, indicating only slight protein secretion. the protein accumulation in the medium of the tobacco genotypes were comparable, resulting in 487 µg/g fw and 250 µg/g fw for sr1 and by-2, respectively. on day 14, another 1 ml aliquot was concentrated and the total medium protein was subjected to sds-page. fig. 1 b illustrates an example gel picture of larch genotypes 4/93 and s90 and the tobacco genotypes. especially genotype 4/93 shows a wast variety of secreted proteins and its size partially exceeds 100 kda. when compared with the protein pattern of equivalent amounts of soluble cellular proteins of cultured embryos, some clear differences could be detected. however, some of the bands indeed seem to have an equivalent in the soluble cellular protein fraction. protease activity in spent medium in a first set of experiments, protease activity in the medium of the cultures was monitored over a standard culture time period of 14 days (fig. 2). for this, a protease activity assay based on the digestion of azocasein was applied (sharp and doran, 2001). medium aliquots were incubated with azocasein solution. after precipitaion, the protease activity in the medium of larch embryogenic cultures 57 fig. 2: (a) growth rate and (b) protease activity in the medium of l. decidua genotype 4/93(▲) and s90 (■), l. x eurolepis 58 (▼), n. tabacum genotype sr1 (♦) and by-2 (✳) over a culture period of 14 days. protease activity was determined by azocasein assay as described in materials and methods. the data are mean of 2-3 independent replicates. error bars represent standard error. 0.0 2.5 5.0 7.5 10.0 12.5 15.0 0 200 400 600 800 culture time [d] b io m a ss [% o f in o cu lu m ] 0.0 2.5 5.0 7.5 10.0 12.5 15.0 0.0 0.1 0.2 0.3 0.5 1.0 culture time [d] a b s 3 6 0 a b supernatant contained the non-precipitable peptide fragments resulting from protease activity which can be quantified photometrically. the media of all three larch genotypes showed only minute amounts of protease activity which were slightly increasing over the culture time (fig. 2 b). for comparison, the protease activity was also determined in tobacco suspension cultures of the genotypes sr1 and by-2. here, protease acitivity was reasonably higher. the genotype sr1 by far showed highest protease activity accumulation over time (fig. 2 b). here, protease activity was approx. 20 times higher at day 14 than the larch genotype 4/93, although the biomass was comparable. the medium of genotype by-2 also showed a fast increase of protease activity during the exponential growth phase but then is decreasing during the stationary phase. when compared with the larch culture medium, by-2 medium exhibits an approx. 6 times higher protease activity at the beginning of the stationary phase (day 7) compared to the mean of the genotypes 4/93 and s90 at the comparable phase (day11). normalized to the same amount of biomass, the by-2 medium still shows approx. 3 times higher protease activity per g fresh weight (data not shown). stability of igg in spent culture medium to evaluate the stability of secreted recombinant proteins in the medium of transgenic cell lines in future applications, immunoglobulin g (igg) was added to spent medium aliquots from the exponential growth phase of the different cultures (7 days after inoculation). spiked aliquots were incubated for 23 h and the protein was recovered afterwards by ethanol precipitation. a precipitation using methanol, desalting of the samples using sephadex-g-25 prior to precipitation or freeze-drying the samples instead of precipitation did not result in higher protein recovery from the samples (data not shown). to quantify and compare the recovered igg, samples were subjected to sds-page. the resulting heavy chain-bands of the igg were quantified according to their intensity in the coomassie-stained gels and compared to spiked samples without incubation time. results are shown in fig. 3. they clearly show a loss of exogeneously added igg over the incubation time which was approx. 16 % (genotype s90), 18 % (genotype 58) and 20 % (genotype 4/93), respectively. the controls (no incubation time) differ from the originally added 5 µg igg due to a non-quantitative ethanol-precipitation, which, however, is comparable in all controls. the control experiment using fresh instead of spent medium resulted in approx. 95 % recovery of the added igg (data not shown), indicating that the effect measured in the spent medium cannot be due to unspecific sticking of the proteins to the tube walls during the experiment. effect of protein-protective medium additives in order to find conditions of enhanced protein stability in spent medium of larch embryogenic cultures, polyvinylpyrollidone, ectoin fig. 1: naturally secreted proteins by larch embryogenic culture. (a) accumulation of total protein in the medium of l. decidua genotype 4/93(▲) and s90 (■), l. x eurolepis 58 (▼), n. tabacum genotype sr1 (♦) and by-2 (✳) over a culture period of 14 days. the data are mean of 2 independent replicates. error bars represent maximal divergence of values. (b) representative silver-stained sds-page of 1 ml-medium aliquots of culture medium of genotypes s90, 4/93, sr1 and by-2 (m) and the same protein amount of total soluble cellular proteins (s) 14 days after inoculation. 0 5 10 15 0 500 1000 1500 2000 2500 culture time [d] t o ta l m e di um p ro te in [µ g *g f w -1 ] a 58 philipp westhofen, ghada ismail, kurt zoglauer, robert boehm fig. 4: influence of different protein-protective additives to the stability of igg in spent medium of larch embryogenic cultures of the genotypes (a) l. decidua 4/94, (b) l. decidua s90 and (c) l. x eurolepis 58. 5 µg igg were exogeneously added to medium aliquots without additives or in the the presence of different ectoins (100 mm each final concentration) or 0.075 % (w/v) polyvinylpyrrolidone (pvp) and recovered after 23 h of incubation. control : igg addition and recovery without incubation time. the data are mean of 5-7 independent replicates. error bars represent standard error. c on tro l no a dd itiv e ec to in hy dr ox ye ct oi ne ho m oe ct oi ne pv p 0 1 2 3 4 5 -16% -6% -0% -0% -9% a µ g ig g a ft er 2 3 h c on tro l no a dd itiv e ec to in hy dr ox ye ct oi ne ho m oe ct oi ne pv p 0 1 2 3 4 5 -20% -11% -7% -9% -12% b µ g ig g a ft er 2 3 h co nt ro l no a dd itiv e ec to in hy dr ox ye ct oi ne ho m oe ct oi ne pv p 0 1 2 3 4 5 -30% -12% -12% -18% -24% c µ g ig g a ft er 2 3 h fig. 3: loss of exogenuously added immunoglobulin g (igg) in spent larch embryo culture medium over 23 h. 7 day-old medium aliquots (0.5 ml each) were spiked with 5 µg igg and incubated for 23 h. the proteins were ethanol-precipitated and the heavy chain was used for densitometric quantitation on sds-page. the data are mean of 5-11 independent replicates. error bars represent standard error. 4/93 s90 58 0 1 2 3 4 5 0 h 23 h larch genotype a m ou nt o f ig g [ µ g] and two ectoin derivatives, hydroxyectoine and homoectoine, have been tested as medium additives. basically, the same protein stability assay using igg-spiked medium aliquots were deployed but with the addition of the mentioned substances. the results are summarized in fig. 4, demonstrating that the stability of igg could significantly be enhanced in media aliquots of all larch genotyopes to a similar extent. whereas the loss of igg was 16-30 % without additives, the addition of 0.1 m ectoin could reduce this loss to a mean of 10 %. similarly, the igg loss was reduced to 9 % in the case of 0.1 m homoectoin. the addition of 0.1 m hydroxyectoin led to the best results, reducing the loss of igg to a mean of 6 % per 23 h of incubation. this is in the range of the control using fresh medium (see above) and clearly demonstrates the usefullness of the addition of 100 mm hydroxyectoin to the culture medium of embryogenic larch cultures. the addition of 0.75 g/l pvp did not draw a homogeneous picture and resulted in an average reduction of igg loss of only 15 % discussion the overall protein yield is of paramount importance for an economic feasibility of cell culture-based expression systems. this yield is not only a function of gene expression level, but also of mrna stability, efficiency of translation and post-translational modifications. the stability of the protein once secreted into the medium also greatly attributes to the protein yield. however, in the case of plant cell cultures, secreted proteins has been shown to be subject to degradation by cosecreted proteases (schiermeyer et al., 2005; kwon et al., 2003), adsorption, aggregation or inactivation by interaction with anorganic media components (sharp and doran, 2001) or adsorption to glass walls (tsoi and doran, 2002). we therefore attempted to characterize the protein stability in the medium of larch embryogenic cultures, which may are an interesting production platform for recombinant proteins. this was done by means of protease activity determination and degradation assay of spiked igg in spent cell culture medium. igg was chosen as an example for a future target protein for genetically modified larch embryogenic strains. in first preleminary experiments, we tested the medium for the occurrence of naturally secreted proteins and compared this pattern with the soluble cellular protein pattern. whereas several bands have a size equivalent in the soluble cellular protein extract, some appear exclusively in the medium, thus are putative secreted proteins. the occurrence of natural protein secretion is widely recognized in callusbased suspension cultures, e.g. in sunflower (mita et al., 1997) or lupin (wojtaszek et al., 1998; wink, 1994), as well as in somatic embryo-based suspension cultures, e.g. carrot (van hengel et al., 2001). it is regarded as a common phenomenon for plant cell cultures protease activity in the medium of larch embryogenic cultures 59 besides the secretion of low-molecular weight compounds like amino acids and organic acids (wink, 1994). the secreted proteins have function in cell wall biosynthesis, cell signalling (van hengel et al., 2001; loopstra, 2004), pathogen defence or are stress-related (mita et al., 1997; wojtaszek et al., 1998). to further characterize secreted proteins in embryogenic larch cell cultures, a protease activity assay was performed which showed significant, but low amounts of protease activity in the medium of all three genotypes which slightly increases over the culture period. however, it remains questionable whether the detected protease activity results from the release of cytosolic or vacuolar proteases from destroyed cells or whether they are actively secreted. although the medium was taken from cell in the exponential growth phase, there is always a little amount of dead cells within the population, as revealed by fluorescein-staining (unpublished observations). comparative analysis of protease activity in tobacco suspension cultures (genotypes sr1 and by-2) revealed a considerable higher amount and pronounced accumulation of protease activity which more probably results from secretion. the existence of proteases in these genotypes have also been observed by kwon et al. (2003) for sr1 and by schiermeyer et al. (2005) for by-2. in the latter, the oberserved decrease of protease activity in the stationary phase probably is due to protein degradation as was also noticed in lupin cell cultures (wink, 1985). in order to assess the stability of foreign protein in spent larch cell culture medium, we examined the fate of exogenuously added igg in 7 days-old medium. after 23 h of incubation, the loss of igg was at an average of 18 %. a decrease in secreted recombinant protein has also been reported in tobacco (sharp and doran, 2001; bodeutsch et al., 2001) and tomato suspension culture (kwon et al., 2003b). this loss can be attributed to degradation by proteases as observed in tobacco cell cultures (kwon et al., 2003a; sharp and doran, 2001b). a loss of protein can also be due to media component interaction (tsoi and doran, 2002). no degradation or aggregation products, however, could be observed. the loss of the added igg cannot be attributed clearly to one of these possibilities since we were not able to recover the test protein from fresh-medium control samples. when plant cell cultures are to be used as production systems for recombinant proteins, secretion of these proteins into the medium is of great importance to facilitate the dowenstream processing (fischer et al., 1999). thus, several medium additives have been tried for enhancement of protein stability. one strategy is to add protease inhibitors (bateman et al., 1997), but more common is the addition of substances which unspecifically stabilize the recombinant protein, using dimethyl sulfoxide (wahl et al., 1995), gelatin or poylvinylpyrrolidone (wongsamuth and doran, 1996; lacount et al., 1997) or bovine serum albumin and salt (james et al., 2000). recently, protein stabilization was achieved by co-expression of human serum albumin in the moss physcomitrella patens (baur et al., 2005). to find out conditions where recombinant proteins secreted into the larch embryo medium are stabilized, we applied the protein stability assay to spent medium samples with addition of protein-protective substances. besides pvp, a polymer known to stabilize proteins in aqueous solutions (lacount et al., 1997), we tested members of the compatible solutes, a new class of protein-protective substances (galinski, 1993; kempf and bremer, 1998). ectoins belong to compatible solutes of bacterial origin, which recently has been identified to occur in extremophilic bacteria like halmononas elongata (sauer and galinski, 1998). ectoin has been shown to support periplasmic protein expression in bacteria (barth et al., 2000) and to confer hyperosmotic tolerance in transgenic tobacco by-2 cells that produce this compatible solute (nakayama et al., 2000). ectoines never have been tested as medium additive in plant cell cultures, although the general protein-protective properties have been shown (galinski et al., 2000). here, we firstly report that ectoins successfully can recover protein stability when added to the medium in concentrations of 0.1 m. the addition of hydroxyectoin was most successful, resulting in 73 % reduction of the igg loss over 23 h. these results indicate the usefulness of ectoin and ectoine derivatives as protein-protective agent in plant cell cultures. however, it could not be clearified whether this protein-protective property is due to a specific protection against proteases or an unspecific osmotic stabillization effect making the aquous medium environment more comfortable for proteins – or both. acknowledgements the authors want to thank e.a. galinski (institute of micobiology and biotechnology, university of bonn) for the generous gift of ectoin and ectoin derivatives and s.schillberg (fraunhofer institute of molecular biology and applied ecology, aachen) for the provision of tobacco by-2 cells. this project was done in the framework of coordinated research of the center of molecular biotechnology (cembio) of the bonn university. references archambault, j,, williams, r.d., lavoie, l., pépin, m-f., chavarie, c., 1994: production of somatic embryos in a helical ribbon impeller bioreactor. biotechnol. bioeng. 44, 930-943. barth, s., huhn, m., matthey, b., klimka, a., galinski, e.a., engert, a., 2000: compatible-solute-supported periplasmic expression of functional recombinant proteins under stress conditions. appl. environ. microb. 66, 1572-1579. bateman, k.s., congiu, m., tregear, g.w., clarke, a.e., anderson, m.a., 1997: bacitracin significantly reduces degradation of peptides in plant cell cultures. biotechnol. bioeng. 53, 226-231. baur, a., reski, r., gorr, g., 2005: enhanced recovery of a secreted recombinant human growth factor using stabilizing additives and by coexpression of human serum albumin in the moss physcomitrella patens. plant biotech. j. 3, 331-340. becwar, m.r., mnoland, t.l., wyckoff, j.l., 1989: maturiation, germination and conversion of norway spruce (picea abies l.) somatic embryos to plants. in vitro cell dev. biol. plant 25, 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(eds.), plant tissue culture 100 years since gottlieb haberlandt, 175-202. springer press, vienna, new york, 2003. address of the authors: philipp westhofen, ghada ismail and dr. robert boehm (corresponding author), institute of plant molecular physiology and biotechnology, university of bonn, karlrobert-kreiten-straße 13, d-53115 bonn, germany. e-mail:r.boehm@uni-bonn.de kurt zoglauer, institute of biology, humboldt-university berlin, invalidenstraße 42, d-10115 berlin, germany. protease activity in the medium of larch embryogenic cultures 61 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false 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<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> >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice vorgabe neu journal of applied botany and food quality 80, 150 154 (2006) 1federal research centre for nutrition and food, institute for biochemistry of cereals and potatoes, germany 2research institute of organic agriculture, switzerland nutritional quality of organic and conventional wheat* g. langenkämper1, c. zörb1, m. seifert1, p. mäder2, b. fretzdorff1, t. betsche1 (received june 26, 2006) summary the popularity of organic food and the farming area managed according to organic agriculture practices have been increasing during the last years. it is not clear, whether foods from organic and conventional agriculture are equal with respect to nutritional quality. we chose wheat (triticum aestivum l., cv. titlis) as one of the most important crop plants to determine a range of substances relevant for human nutrition in crops from organic and conventional agriculture systems. wheat grains of 2003 originating from a long term field experiment, the swiss dok trial, consisting of bio-dynamic, bio-organic and conventional farming systems were used. thousand seed weight, protein content, phosphate levels, antioxidative capacity, levels of phenols, fibre, fructan, oxalate and phytic acid were determined in whole wheat meal from the various organic and conventional growing systems of the dok trial. levels of these substances fell into a range that is known to occur in other wheat crops, indicating that wheat from the dok trial was not special. clearcut differences were observed for none-fertilised wheat, which was significantly lowest in thousand seed weight, protein and significantly highest in total oxalate. for the majority of the nutritionally important substances analysed, there were no significant differences between bio-dynamic, bio-organic, and conventional growing systems. only protein content and levels of fibres were statistically different. taken together, the magnitude of observed variations was very small. the results of our investigations do not provide evidence that wheat of one or the other agriculture system would be better or worse. introduction organic food is perceived by many consumers as safer and healthier than conventionally produced food (e. g. bourn and prescott, 2002; kuhnert et al., 2003). the question arises, whether this perception can be confirmed on scientific grounds. in order to establish a basis to draw sound conclusions with respect to nutritional quality, it is necessary to analytically determine a range of substances and physiological parameters with relevance for human nutrition in both, organic and conventional food. this approach of comparing key nutrients of food, including wheat, from both farming systems has been used in a number of studies. however, it is difficult to draw firm conclusions from most of these studies for several reasons: i) the analysed food was purchased from retailers, ii) samples from different farm locations, with varying soil types and micro climate were used, iii) statistical analyses of the data were insufficient, iv) the analytical methods employed are now subject to criticism (worthington, 1998; bourn and prescott, 2002; williams, 2002). the dok field trial has been conducted continuously since 1978 (mäder et al., 2002). the experiment uses a field plot design where different organic and conventional farming systems with identical crop varieties are run side by side, thus eliminating variation in climatic conditions and soil types. exclusively, the mode of fertilisation and crop protection practises are different in the various systems. the levels of applied plant nutrients are distinctly lower in the organic systems (see materials and methods for details). wheat grains from the dok experiment provide an excellent basis for any comparative study addressing different farming systems. using these materials, we have set out to clarify by biochemical analyses, whether there are any differences in relative concentrations of nutritionally relevant substances and parameters in wheat from bio-dynamic, bioorganic and conventional farming systems. materials and methods wheat production wheat grains (triticum aestivum l., cv. titlis) were harvested in 2003 from the dok field trial conducted near basel, switzerland. the dok field trial was started in 1978 by the research institute of organic agriculture (fibl, frick, ch) and the agroscope reckenholz-tänikon research station art (zürich, ch). refer to mäder et al. (2002) for a detailed description of the field trial. in our study, wheat material was used from two organic farming systems defined as bio-dynamic (dyn) and bio-organic (org) and two conventional systems, one with mineral fertiliser plus farmyard manure (conv) and the other with mineral fertiliser only (mineral). in the organic systems fertiliser levels corresponded to 1.4 livestock units per hectare and in the conventional systems fertiliser was supplied at the level of the plant-specific swiss standard recommendation [fertiliser high]. for each of the four systems there was a second fertilisation level applying half of the above described standard levels [fertiliser low]. nutrient supply with respect to n, p and k was between 34 and 51% lower in the organic systems (mäder et al., 2002). additionally, wheat grains from plots without fertilization (none) were included. these plots received nutrients from two cultivations of green manure in a seven year crop rotation and n-entries by air (ca. 40 kg/ha). crop rotation, varieties, and tillage were identical in all systems. analyses thousand seed weight was determined by recording the mass of three times 100 grains. whole wheat meal was obtained by grinding wheat grains (100 g from each field plot) in a teflon laminated mill using a sieve with 0.5 mm pore size (retsch, germany). air dried whole wheat meal was used in all analyses. nitrogen levels were determined using the procedure of kjeldahl (en iso 3188, 1994). factor 5.7 was applied to calculate protein content. phosphate content was determined according to gericke and kurmies (1952). antioxidant capacity of whole wheat meal was determined using dpph (2,2-diphenyl-1-picrylhydrazyl) as described (miller et al., * the paper was presented at the 41th meeting of the „deutsche gesellschaft für qualitätsforschung (pflanzliche nahrungsmittel) dgq e. v.“ 2000). standard curves were obtained for the reaction of dpph with trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), a water soluble analogue of vitamin e. data were expressed as µmol trolox equivalents (te) per g whole wheat meal. phenols were measured with the folin-ciocalteu reagent (merck) as described (peterson et al., 2002). for soluble phenols 2 g of whole wheat meal were stirred in 20 ml methanol for 30 min at 25°c. extracts were brought to a volume of 25 ml with methanol and were filtered (fretzdorff, 2005). total phenols were extracted using alkaline conditions as described by piber and köhler (2004) with minor modifications: whole wheat meal was 100 mg and the volumes were adapted. after drying of the organic phase in a rotating evaporator, the residues were resuspended in 5 ml methanol. the folinciocalteu reaction was performed with 0.25 ml suspension. ferulic acid was used as a standard for calibration. soluble and total oxalic acid were determined enzymatically as described by fretzdorff (2005). phytic acid levels were determined according to wheeler and ferrel (1971). fructan was determined using the aoac method 999.03 (megazyme, ireland) as described by fretzdorff and welge (2003). soluble and insoluble dietary fibre was determined with an enzymatic-gravimetric method (anon., 1997), based on the method of lee et al. (1992). statistical evaluation was carried out using the tukey-test algorithm of the students range using sas software (institute inc., cary, nc usa). significance was tested at the 5% level. for each of the various wheat farming systems, samples of at least three individual field plots were analysed. results and discussion thousand seed weights were not significantly different between wheat samples from the various farming systems, regardless of the fertilisation level. only wheat that was grown without any fertilisation had a significantly lower thousand seed weight (fig. 1a). these results with wheat from the 2003 harvest confirm results obtained with wheat grown in three crop rotations between 1978 and 1998 in the dok experiment (mäder et al., unpublished). the independence of the thousand seed weight from the farming system indicates that development of endosperm, germ and bran was similar in all systems, if there was fertilisation. thus, possible differences that might be found in levels of nutritional parameters in wheat from organic and conventional systems cannot be attributed to variations in proportions of endosperm, germ and bran. however, since total wheat yields in the dok trial were decreased in the organic systems between 10 and 14% (mäder et al., 2002, mäder et al., unpublished), it is most probable that the number of tillers was reduced in the organic systems. by doing so, the plant is capable of achieving fully developed seeds also at the lower level of available nutrients. variation in crude protein content was observed (fig. 1b). as expected, protein content increased with the amount of fertiliser applied. protein content was significantly highest in wheat that received exclusively mineral fertiliser. the trend of increased protein content in mineral fertilised wheat was observed in other studies comparing organic and conventional systems (steineck and liebhard, 1984; bolling et al., 1986; eltun, 1996). wheat protein levels were relatively high, i. e. between 13 and 15%, in all systems of the dok trial that used fertiliser (fig 1b). as for phosphate, there were no significant differences between the farming systems, nonefertilisation included (fig. 1c). this finding is in full agreement with the results of other experiments showing that phosphorus was sufficiently available for plants in the various dok field plots (oehl et al., 2002). antioxidants such as vitamins c and e, carotenoids and polyphenols are of particular interest in animal and human nutrition. it has been reported that these antioxidants can lower the risk of developing cardiovascular disease, certain types of cancer and age-related degenerative processes due to their radical scavenging capacity (stanner et al., 2004). in order to assess the antioxidative capacity of the various wheat samples, the free radical scavenging capacity was determined and samples were analysed for soluble and total phenolic compounds. for none of these parameters any significant difference was found between wheat samples from the farming systems using fertiliser (fig. 2a-c). none-fertilised wheat, however, had increased levels of soluble phenolic compounds (fig. 2c). fig. 1: basic properties of wheat grains grown at different farming conditions in 2003. thousand seed weight (a), protein content (b) (nitrogen content x 5.7), phosphate levels (c). values are means of replicates from at least three independent field trials. standard errors of the mean are shown. statistical significance was tested using the tukeytest algorithm of the students range (α = 0.05). differing small print letters above histograms indicate significant differences. (org = bioorganic, dyn = bio-dynamic, conv = conventional). thousand seed weight 0 10 20 30 40 50 g aaa aaa a b protein 4 8 12 16 % cd de bc bcd bcd a e ab phosphate 0 50 100 150 200 250 300 µ g / g aa a aa a a a or g dy n co nv m in er al fertiliser low level none or g dy n co nv fertiliser high level a b c thousand seed weight 0 10 20 30 40 50 g aaa aaa a b protein 4 8 12 16 % cd de bc bcd bcd a e ab phosphate 0 50 100 150 200 250 300 µ g / g aa a aa a a a or g dy n co nv m in er al fertiliser low level none or g dy n co nv fertiliser high level a b c nutritional quality of organic and conventional wheat 151 antioxidative capacity and phenol levels are considered as indicators for „stress conditions“. the lack of differences in these parameters is considered as evidence that organic agriculture would not induce „stress conditions“ for plants because of possible mal-nutrition. dietary fibres comprise mainly carbohydrates that are resistant to human digestion (asp, 2004). there is increasing evidence from epidemiological studies that dietary fibre intake is health-promotive, in particular with regard to obesity, diabetes, coronary heart disease and certain types of cancer (miller jones, 2004). because of these favourable features, the levels of different components of dietary fibre were determined. it was observed that levels of soluble fibre were elevated in the bio-dynamic system [fertiliser high] and were significantly decreased in the conventional system [fertiliser high] (fig. 3a). the levels of soluble fibre in the other systems were intermediate (fig. 3a). levels of insoluble fibre did not vary to a large extent, but were significantly increased in mineral fertilised wheat versus conventional wheat [fertiliser high] (fig. 3b). taken together, no relationship was found between dietary fibre level and farming system, organic versus conventional. fructan-levels were equal in wheat from organic and conventional farming systems (fig. 3c). however, fructan was moderately increased in nonefertilised wheat (fig. 3c), probably indicating a stress condition due to lack of nutrients. in human and animal nutrition oxalic acid is considered an „undesired compound“, occurring frequently in plant tissues, including cereal grains (fretzdorff and betsche, 1998). health related problems associated with oxalic acid are reaching from irritation of intestinal mucosa caused by oxalate crystals, reduction of the bioavailability of mg, ca and fe, to increasing the risk in development of renal stones for humans who are genetically predisposed (franceschi fig. 2: antioxidative capacity (a), expressed as µmol trolox equivalents (te) per g whole wheat meal, levels of total phenols (b) and soluble phenols (c) in wheat grains grown at different farming conditions in 2003. for details see legend to fig 1. antioxidati ve capacity 2 4 6 8 10 12 14 16 t e / g aa a aaa a a total phenols 0 0.5 1.0 1.5 2.0 m g / g a a a aaa a a soluble phenols 0 0.1 0.2 0.3 0.4 0.5 0.6 m g / g a ab ab bb bb b or g dy n co nv m in er al fertiliser low level none or g dy n co nv fertiliser high level a b c antioxidati ve capacity 2 4 6 8 10 12 14 16 t e / g aa a aaa a a total phenols 0 0.5 1.0 1.5 2.0 m g / g a a a aaa a a soluble phenols 0 0.1 0.2 0.3 0.4 0.5 0.6 m g / g a ab ab bb bb b or g dy n co nv m in er al fertiliser low level none or g dy n co nv fertiliser high level a b c fig. 3: levels of soluble fibre (a), insoluble fibre (b) and fructan (c) in wheat grains grown at different farming conditions in 2003. for details see legend to fig 1. soluble fibre 0.5 1.0 1.5 2.0 2.5 3.0 % bc ab bc bc a ab ab c insoluble fibre 0 2 4 6 8 10 % b ab ab ab ab a ab ab fructan 0 0,2 0,4 0,6 0,8 1,0 % ab ab ab ab ab ab a b or g dy n co nv m in er al fertiliser low level none or g dy n co nv fertiliser high level a b c soluble fibre 0.5 1.0 1.5 2.0 2.5 3.0 % bc ab bc bc a ab ab c insoluble fibre 0 2 4 6 8 10 % b ab ab ab ab a ab ab fructan 0 0,2 0,4 0,6 0,8 1,0 % ab ab ab ab ab ab a b or g dy n co nv m in er al fertiliser low level none or g dy n co nv fertiliser high level a b c 152 g. langenkämper, c. zörb, m. seifert, p. mäder, b. fretzdorff, t. betsche fig. 4: levels of total oxalate (a), soluble oxalate (b) and phytic acid (c) in wheat grains grown at different farming conditions in 2003. for details see legend to fig 1. total oxalate 10 20 30 40 50 60 m g / 1 0 0 g a bb b b bb b 0 soluble oxalate 5 10 15 20 m g / 1 0 0 g a a a a aa a a or g dy n co nv m in er al fertiliser low level none or g dy n co nv fertiliser high level phytic acid 0 2 4 6 8 10 12 m g / g ab b abab ab ab a ab a b c total oxalate 10 20 30 40 50 60 m g / 1 0 0 g a bb b b bb b 0 soluble oxalate 5 10 15 20 m g / 1 0 0 g a a a a aa a a or g dy n co nv m in er al fertiliser low level none or g dy n co nv fertiliser high level phytic acid 0 2 4 6 8 10 12 m g / g ab b abab ab ab a ab a b c and libert, 1987; dunwell et al., 2000). total and soluble oxalate were not significantly different between any of the wheat samples originating from the farming systems using fertiliser (fig. 4ab). it has been observed that levels of oxalate can vary, in vegetable plants (e. g. franceschi and libert, 1987) and also in wheat grain (betsche and fretzdorff, 1997), with the amount and type of fertiliser applied. our results show that oxalate is equal in wheat grain from organic and conventional farming systems. this finding further supports the proposal that organic agriculture is not a „stress condition“ for wheat in the dok trial. the result that a significantly increased level of total oxalate was detected in none-fertilised wheat (fig. 4a), is corroborative. oxalate oxidase activity was determined, but no significant differences in activity were detected between any of the wheat samples (data not shown). oxalate oxidase activity is correlated with germination and sprouting (e. g. betsche and fretzdorff, 2005). phytic acid has long been regarded as an anti-nutrient, because it can form complexes with mineral ions rendering them unavailable for intestinal uptake (lopez et al., 2002). however, more recently, also positive effects of phytic acid have been proposed, e. g. a role as an antioxidant through its iron chelating properties (minihane and rimbach, 2002) and a function as an anti-cancer substance (shamsuddin, 2002). in the dok wheat samples, levels of phytic acid did not vary to a large extent between the different farming systems (fig. 4c). conclusion thousand seed weight, protein content, phosphate levels, antioxidative capacity, levels of phenols, fibre, fructan, oxalate and phytic acid were determined in whole wheat meal from the various organic and conventional growing systems of the dok trial (2003 harvest). levels of these substances fell into a range that is known to occur in other wheat crops, indicating that wheat from the dok trial was not special. none-fertilised wheat was different from wheat stemming from either organic or conventional farming systems. there were no significant differences between bio-dynamic, bio-organic, and conventional growing systems for the majority of the nutritionally important substances analysed. all in all, if differences were observed, the magnitude of variations was very small. taken together, the results do not provide evidence that wheat of one agriculture system or the other would be better or worse. acknowledgements we like to thank the agroscope reckenholz-tänikon research station art (zürich, ch) for providing the wheat material. we thank c. heistermann, m. krome, a. meyer-wieneke, b. nierle, and e. schieseck for skilful technical assistance and u. leckeband for help with preparing the figures. the financial support from the „bundesprogamm ökologischer landbau“, project no. 02oe069, is gratefully acknowledged. references anon., 1997: bestimmung der ballaststoffe in lebensmitteln. amtliche sammlung von untersuchungsverfahren nach § 35 lmbg l 00.00-18. beuth verlag, berlin. asp, n.-g., 2004: definition and analysis of dietary fibre in the context of food carbohydrates. in: van der kamp, j.w., asp, n.-g., miller jones, j., schaafsma, g. 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(nz) georg langenkämper, corresponding author: georg.langenkaemper @bfel.de:, dr. christian zörb, dr. mathias seifert and dr. thomas betsche, federal research centre for nutrition and food, institute for biochemistry of cereals and potatoes, schützenberg 12, d-32756 detmold, germany dr. paul mäder, research institute of organic agriculture, ackerstrasse, ch5070 frick, switzerland 154 g. langenkämper, c. zörb, m. seifert, p. mäder, b. fretzdorff, t. betsche << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true 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<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> >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice art12 journal of applied botany and food quality 81, 172 174 (2007) 1federal research centre for nutrition and food, institute for biochemistry of cereals and potatoes, detmold, germany 2university of applied sciences, lippe and höxter, department of life sciences technologies, lemgo, germany free sugars in spelt wholemeal and flour christian zörb1*, thomas betsche1, georg langenkämper1, jürgen zapp2, mathias seifert1 (received august 7, 2007) summary spelt (triticum aestivum l. ssp. spelta) is experiencing a renaissance in europe and north america, where it is used for baking, brewing, production of pasta, and self-supplied animal feed. one of the characteristics of spelt is that in comparison to modern wheat it is more resistant to harsh climatic and poor soil conditions. in contrast to wheat the hulls remain on the grain after threshing. drawbacks are that spelt yields are quite low compared to modern wheat. the subject of the current study was to gain information about the composition of soluble sugars and their concentrations in spelt wholemeal and flour. high performance liquid chromatography (hplc) was used for analysis. concentrations of nine free sugars in spelt wholemeal and flour are reported. flour cumulative free sugar concentrations were 63% lower than in wholemeal. for comparisons, we also analyzed wholemeal of wheat. the cumulative concentration of free sugars was 27% lower than in spelt wholemeal. however, when published data for sugar concentration ranges of wheat are taken into account, the total concentration of free sugar was not different between spelt and modern wheats. low concentrations of xylose and stachyose were detected in spelt. higher concentrations of fructans such as 1-kestose and kestotetraose were detected in spelt when compared with wheat. generally, concentrations of free sugars in spelt were in the range of free sugar levels published for wheat, except for maltose which was higher in spelt. introduction spelt wheat (triticum aestivum l. ssp. spelta) is one of the earliest wheat species cultivated and has been used as bread cereal since ancient times (abdel aal et al., 1998; rösch, 1998). later, it has been replaced by high-yielding cultivars of common bread wheat (triticum aestivum l. ssp. vulgare). drawbacks of spelt are that the hulls are not removed during threshing and that yield is quite low compared to modern wheat varieties. modern wheat varieties intended for human consumption have compact ears, lose their hulls during threshing, and produce high yields. spelt is more resistant to harsh climatic and poor soil conditions (rösch et al., 1998), and better resists to pests and plant diseases. these features render spelt highly suitable for organic agriculture (skrabanja et al., 2001). moreover, spelt can be grown in sub-alpine regions as well as in low-input farming (nesbitt and samuel, 1996). for this reasons, spelt is experiencing a renaissance in europe and north america (mielke and rodemann, 2007), where it is increasingly used for baking, brewing, and the production of pasta in a number of regional specialties such as the not fully matured grain ‘grünkern’, i.e. not fully matured grain torrefied after harvest, of southern germany. another field of application is self-supplied animal feed. today, a variety of spelt-based products are available including flour, bread, breakfast cereals, pasta and crackers. it has been proposed that this cereal has valuable nutritional and/or physiological properties, an argument which has been used to help to promote the consumption of these products (marques et al., 2007). it is a matter of fact that interest in alternative cereals and the demand for organically produced foods, creating a niche market for spelt, is growing. as a consequence, prices for spelt flour are up to 50% above the price of respective wheat flour in european countries (von büren et al., 2001). carbohydrates such as starch, the cell wall compounds cellulose, hemicelluloses, and pentosans and soluble sugars are the most abundant constituents of the cereal grain, making up 80% of the total dry matter (täufel et al., 1959). because there is a lack of sound scientific data on the composition and concentration of nine soluble sugars in spelt, we determined these sugars by means of high performance liquid chromatography (hplc). taken together, we present results of spelt wholemeal and flour. we also compare the spelt data with published results of soluble sugar concentration ranges of modern wheats. materials and methods spelt wholemeals and flours the spelt samples were purchased in grocery stores or from mill operating companies from the harvest of 2006. four samples of each, wholemeal and flour (type 630), were collected from the marketplace. spelt wholemeals were obtained from adler-mühle, bahlingen; bronnmühle, rottenburg; aurora hamburg; diamant, hamburg. spelt flours (type 630) were obtained from pfälzische mühlenwerke, mannheim aurora hamburg; rosenmühle landshut; and adlermühle, bahlingen. four wholemeal samples (bread wheat) were collected and analyzed. analytical procedures soluble sugars from 4 g of each flour and wholemeal sample were extracted with 50 ml of 80% ethanol at 60° c for 30 min. the extraction was repeated once (munzquiz et al., 1992). the two extracts were combined and centrifuged for 10 min at 1500 g at room temperature. the supernatant was concentrated by rotary evaporation from 100 mbar to 30 mbar and the dry residue was resuspended in 8 ml water (double destilled) and sonicated for 3 min in an ultra sonic bath. the suspension was centrifuged for 10 min at 20° c and 1500 g. the supernatant was filtrated through a strata™ x 60 mg cartridge (phenomenex, germany) which was previously equilibrated with 2 ml methanol and 2 ml water, subsequently. to 4 ml of the filtrate 6 ml acetonitrile was added. after centrifugation (see above), 50 µl of the supernatant was applied to the high performance liquid chromatography system (hplc) for sugar separation. analysis was done by hplc according to munzquiz et al. (1992) with a modified mobile phase (see below). the chromatographic equipment consisted of a kontron hplc system, a geminyx data module (spectro bionova, germany), and an ri detector 7515a (erc, germany). a stainless-steel analytical column cc 250/4.6 nucleosil 100-5 nh2 (macherey & nagel, germany) was used together with a precolumn cc 8/4 nucleosil 100-5 nh2 (macherey *corresponding author & nagel, germany). the mobile phase, was degassed (acetonitrilewater 6:4, w/w), and the flow-rate was set to 0.5 ml/min. the column temperature was maintained at 30° c. quantification was done on the basis of peak heights. external standards were used for calibration. a linear response was obtained in the range of 0.0 4.0 mg ml-1 with a correlation coefficient of 0.99. statistical analyses and accuracy results were expressed as means obtained for four samples from wholemeal or flour of spelt, respectively. each sample was extracted and analyzed at least two times. standard errors of the mean (se) were calculated. the reproducibility of the extraction of soluble sugars was calculated from three independent extractions and the standard error of the mean ranged between 0.1% and 5.6%. the recovery of sugars tested were 70% for fructose, 95% for glucose, 90% for sucrose, 70% for maltose, 81% for 1-kestose, 85% for raffinose, 98% for kestotetraose and 78% for stachyose. results the overlay of two chromatograms derived from two separate hplc runs from flour and from wholemeal of spelt is shown in fig. 1. in total, nine free sugars could be detected and quantified. the nonidentified peaks were relatively small. differences in sugar concentrations between flour and wholemeal from spelt were quantitative. no qualitative difference was found (fig. 1). mean values and standard errors of the mean from all four samples of wholemeal and flour (type 630) from spelt are shown in fig. 2. in spelt wholemeal the concentrations of sucrose, 1-kestose, raffinose, and maltose were between 2 and 7.5 mg g-1 dw and were threeto two-fold higher than in spelt flour. concentrations of kestotetraose, fructose, glucose, xylose, and stachyose were below 0.6 mg g-1 dw in wholemeal. in flour, the concentrations of these sugars were approximately half of those in wholemeal (tab. 1). the concentrations of free sugars in spelt were in the following order: sucrose > 1-kestose > raffinose > maltose >> kestotetraose > fructose = glucose = xylose > stachyose (fig. 2). four wheat wholemeal samples were analyzed for soluble sugars. the comparison of free sugar concentrations in wholemeal of spelt and wheat revealed higher sugar concentrations in spelt except for raffinose, which was higher in wheat. neither xylose nor stachyose were detected in wholemeal from wheat. in total 17.1 mg and 6.3 mg of free sugar g-1 dw were detected in wholemeal and flour of spelt, respectively. in wheat wholemeal 12.5 mg free sugar g-1 dw was found in total (tab. 1). these results nicely match published data of sugar concentrations in wheat (summarized by pomeranz, 1988) shown in tab. 1. 0 5 10 15 20 25 200 100 150 50 r i in te ns it y [m v ] time [min] sucrose raffinose kestotetraose glucose 1-kestose stachyose xylose start fructose maltose 0 5 10 15 20 25 200 100 150 50 r i in te ns it y [m v ] 200 100 150 50 r i in te ns it y [m v ] 150 50 r i in te ns it y [m v ] time [min] sucrose raffinose kestotetraose glucose 1-kestose stachyose xylose start fructose maltose fig. 1: overlay of hplc chromatograms of free sugars from spelt wholemeal and spelt flour (type 630). the upper chromatogram represents wholemeal, the lower chromatogram flour. 0 1 2 3 4 5 6 7 8 9 su cr os e 1ke st os e ra ffi no se m al to se ke st ot et ra os e fru cto se gl uc os e xy lo se st ac hy os e c o n c e n tr a ti o n [m g /g ] 0 1 1 2 2 3 su cr os e 1ke st os e ra ffi no se m al to se ke st ot et ra os e fru cto se gl uc os e xy lo se st ac hy os e c o n c e n tr a ti o n [m g /g ] spelt flour fig. 2: concentrations of free sugars in spelt wholemeal (left) and flour (right). were from four different companies each. sugar concentrations were determined by hplc and are given as mg g-1 dry weight. standard errors of the mean (± se) were calculated from four independent samples. note the different scales! spelt flourspelt wholemeal free sugars in spelt wholemeal and flour 173 discussion because there is a lack of a sound scientific basis of spelt based data were determined. concentrations of nine free sugars in wholemeal and flour of spelt were determined by hplc (tab. 1). the main advantage of hplc by comparison to enzymatic methods lies in the reliable simultaneous detection of a set of sugars from one extract with a relatively low detection limit. the 63% lower cumulative free sugar concentrations flour by comparison with wholemeal from spelt, which reflects the removal of free sugars during the milling process. fructose amounts to only 1.5% of total free sugars in spelt. sucrose, 1-kestose, raffinose, and maltose show the largest reduction by milling, with kestotetraose, fructose, glucose, xylose and stachyose being less affected (fig. 2). fretzdorff and welge (2003) reported higher concentrations of kestotrioses compared to larger fructooligosaccharides. the second issue of our investigation was to compare the concentrations of free sugars from spelt and wheat. the concentration of cumulative free sugars from spelt was 27% higher than that of wholemeal of wheat analyzed in this work (tab. 1). however, when many published data are considered (summarized by pomeranz, 1988), sugar concentrations in spelt are within the range observed for wheats (tab. 1). xylose and stachyose could not be detected at all in wheat in our work while other workers found low concentration (tab. 1), indicating a slight difference in sugar composition in wheat and spelt. sucrose was found the sugar most abundant in both, spelt and wheat, the sucrose concentration in spelt being significantly higher (tab. 1). additionally, higher concentrations of fructans such as 1-kestose and kestotetraose were detected in spelt compared to wheat (tab. 1). in our work using hplc, arabinose was not detected in spelt and wheat. in contrast, barron et al. (2007) reported arabinose in wheat, although little. tab. 1: comparison of mean values and ± se of free sugar concentrations from wholemeal and flour (type 630) from four different spelt wholemeal and flour (triticum aestivum l. ssp. spelta) samples and from four different samples of wholemeal wheat (triticum aestivum l. ssp. vulgare). sugar concentrations are given in mg g-1 dry weight. (± se), standard error of the mean. spelt wheat wholemeal flour wholemeal range* sucrose 7.47 ± 0.20 2.30 ± 0.05 5.91 ± 0.13 5.4 15.5a 1-kestose 3.08 ± 0.05 1.28 ± 0.02 2.00 ± 0.04 2.6 4.1a raffinose 2.46 ± 0.03 0.79 ± 0.01 2.61 ± 0.04 1.9 6.8a maltose 2.35 ± 0.12 0.99 ± 0.08 1.37 ± 0.1 0.0 1.8a kestotetraose 0.58 ± 0.01 0.27 ± 0.01 0.33 ± 0.01 n.a. fructose 0.36 ± 0.12 0.25 ± 0.01 0.17 ± 0.01 0.6 0.8a glucose 0.36 ± 0.05 0.21 ± 0.01 0.15 ± 0.01 0.3 0.9a xylose 0.32 ± 0.01 0.17 ± 0.01 n.d. n.a. stachyose 0.16 ± 0.12 0.08 ± 0.05 n.d. 0.06b σ sugars 17.14 6.34 12.54 n.d., concentration not detectable; n.a., data not available; *concentration ranges according to [a] pomeranz (1988); [b] henry and saini (1989) generally, concentrations of free sugars in spelt were in the range of free sugar levels published for wheat, except for maltose which was higher in spelt (tab. 1). acknowledgements the excellent assistance of annette meyer-wieneke during the sample preparation and analysis is gratefully acknowledged. references abdel-aal, e.-d., sosulski, f.w., hucl, p., 1998: origins, characteristics, and potentials of ancient wheats. cereal foods world 43, 708-715. barron, c., surget, a., rouaux, x., 2007: relative amounts of tissues in mature wheat (triticum aestivum l.) grain and their carbohydrate and phenolic acid composition. j. cereal sci. 45, 88-96. büren von, m., stadler, m., lüthy, j., 2001: detection of wheat adulteration of spelt flour and products by pcr. eur. food res. technol. 212, 234-239. fretzdorff, b., welge, n., 2003: fructanund raffinosegehalte im vollkorn einiger getreidearten und pseudo-cerealien. getreide mehl brot 57, 38. henry, r.j., saini, h.s., 1989: characterization of cereal sugars and oliogosaccharides. cereal chem. 66, 362-365. marques, c., d’auria, l., cani, p.d., baccelli, c., rozenberg, r., ruibal-mendieta, n.l., petitjean, g., delacroix, d.l., quentinleclercq, j., habib-jiwan, j.l., meurens, m., delzenne, n.m., 2007: comparison of glycemic index of spelt and wheat bread in human volunteers. food chem. 100, 1265-1271. mielke, h., rodemann, b., 2007: der dinkel, eine besondere weizenart – anbau, pflanzenschutz, ernte und verarbeitung. nachrichtenbl. deut. pflanzenschutzd. 59, 40-45. munzquiz, m., rey, c., cuadrago, c., 1992: effect of germination on the oligosaccharide content of lupine species. j. chromatogr. 607, 349-352. nesbitt, m., samuel, d., 1996: from staple crop to extinction? the archeology and history of hulled wheats. in: padulosi, s., hammer, k., heller, j. (eds.), hulled wheats, 41-100. proceedings of the first international workshop on hulled wheats. international plant genetic resources institute, rome. pomeranz, y., 1988: wheat chemistry and technology. american association of cereal chemists, minnesota, usa. rösch, m., 1998: the history of crops and crop weeds in south-western germany from the neolithic period to modern times, as shown by archaeobotanical evidence. veget. hist. archaeobot. 7, 109-125. skrabanja, v., kovac, b., golob, t., liljeberg elmståhl, h.g.m., björck, i.m.e., kreft, i., 2001: effect of spelt wheat flour and kernel on bread composition and nutritional characteristics. j. agric. food chem. 49, 497-500. täufel, k., romminger, k., hirschfeld, w., 1959: oligosaccharide von getreide und mehl. z. lebensm. unters. forsch. 109, 1-12. address of the authors: dr. chr. zörb, bundesforschungsanstalt für ernährung und lebensmittel, institut für biochemie von getreide und kartoffeln, schützenberg 12, d32756 detmold 174 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/pagesize [907.087 680.315] >> setpagedevice art25 journal of applied botany and food quality 82, 152 157 (2009) institute of crop science and resource conservation, horticultural science, university of bonn polyphenol content and antioxidant capacity of apple fruit: effect of cultivar and storage conditions anne matthes, michaela schmitz-eiberger (received october 2, 2008) summary the benefits of fruits and vegetables are often attributed to their high antioxidant content. research supports a role of secondary plant metabolites particulary polyphenols in the prevention of degenerative diseases e.g. cardiovascular diseases and cancer. apple fruit are an important source of secondary plant metabolites and one of the major phenol sources being consumed during the whole year. the present investigation was undertaken to determine antioxidant capacity in selected apple cultivars depending on cultivar and different modes of postharvest storage. additional storage at 20 °c was tested to simulate the conditions at the consumers’ home (shelf life). antioxidant capacity differed between the cultivars. cold storage (1 °c) for 4.5 months increased the antioxidative capacity and polyphenol content in most of the cultivars. shelf life led to a decrease in polyphenol content and in antioxidant capacity. storage under controlled atmosphere led to low increases of both antioxidant capacity and polyphenol content. in some cultivars polyphenol content remained stable. after the shelf life period lower values for antioxidant capacity were determined, in combination with no changes in phenol content. correlation analysis showed a positive correlation between total phenols and antioxidant capacity (teacvalue). lipophilic antioxidants decreased during storage. storage experiments indicated that a high content of polyphenols and antioxidants can be sustained by optimal storage conditions, these fruit may contribute to an antioxidant rich diet and may impart health benefits. introduction in the mid-1990s the first epidemiological studies were published in which the intake of fruits and vegetables was inversely correlated to the mortality of cardiovascular diseases and other degenerative diseases (hertog et al., 1993a; scalbert et al., 2005b). current evidence strongly supports the role of polyphenols in prevention of cardiovascular diseases, cancers and osteoporosis and assumes a contribution of polyphenols in preventing neurodegenarative diseases and diabetes mellitus (scalbert et al., 2005b). increased formation of oxygen radicals, which may result in attack of different biomolecules such as lipids of biomembranes, dna and other proteins, may be related to this age-related diseases. the protective effects of fruit and vegetable consumption may be attributed to the antioxidant properties of secondary plant metabolites such as carotenes, ascorbic acid and polyphenols, which are due to their ability to act as metal chelators, reductants, hydrogen donators and singulett oxygen quenchers (rice-evans, 1995). some animal and in-vitro studies support this relationship (eberhard et al., 2000; halliwell et al., 2005). especially polyphenols belong to the most important bioactive components; a direct positive correlation between phenol content and antioxidative capacity was reported several times (kalt et al., 1999; schmitz-eiberger et al., 2003). recent studies reported that cells respond to polyphenols by a direct interaction with receptors and enzymes that modify the redox status of the cell (scalbert et al., 2005a). halliwell et al. (2005) reported that phenols might exert positive effects directly in the gastrointestinal tract, including binding of prooxidant iron, scavening of reactive nitrogen, chlorine and oxygen species. therefore, phenolic compounds commonly found in fruits and vegetables improve the quality and nutrional value for the consumer, and moreover the significance of the content of secondary plant metabolites will rise for the food producers in the future. total polyphenol intake is estimated to about one gram per day for people who eat several servings of fruit and vegetables during the day. that means the intake is ten times higher than that of ascorbic acid (scalbert and williamson, 2000; manach et al., 2004). yet not only the total intake should be high but also the bioavailability and metabolism of various substances should be taken into consideration in order to evaluate their biological activity in the body. in central europe apples are an important product in the plant food market as they are available at markets the whole year, and for that reason they are one of the most important sources for secondary metabolites besides coffee, tea and onion in the dutch diet (hertog et al., 1993b). polyphenols are not homogenously distributed in apple fruit, higher phenol content was found in the peel than in the flesh (lattanzio et al., 2001; solovchenko and schmitz-eiberger, 2003; wolfe et al., 2003). the main phenols in the flesh consisted of catechins, procyanidines, phloretin glycosides and chlorogenic acid, the peel contains higher amounts of these components and quercetin glycosides (burda et al., 1990; van der sluis et al., 2001). during storage of apples changes of the content of bioactive components may occur as a result of metabolic degradation, respiration and synthesis processes. especially during storage at ambient temperature in supermarkets or at consumers’ homes there might be changes affecting the quality of the fruits. changes might be attributed to diseases and physiological disorders. fruit phenolics have also been implicated in pathogen resistance (mayr et al., 1997) and some physiological disorders occuring during storage (lattanzio et al., 2001), so that a high phenol content can enhance postharvest disease resistance. several studies on the impact of cold storage or controlled atmosphere on polyphenol composition of apple fruits have been carried out (awad and de jager, 2000; van der sluis et al., 2001; napolitano et al., 2004). it can be concluded from these studies, that polyphenols are stable during storage, however other studies observed an increase. up to now it has not often been investigated in how far shelf life influences the content of secondary plant metabolites and antioxidant capacity. the aim of this study was to determine polyphenol content and the antioxidative capacity of selected apple cultivars and to evaluate whether the phenol content correlates with antioxidant capacity. furthermore we wanted to evaluate the effects of different storage conditions and the influence of shelf life on antioxidant content. materials and methods plant material and storage conditions fruit of 19 apple cultivars (malus domestica, borkh.) were cultivated at the centre of competence klein-altendorf, germany. the ‘aw 106’ cultivar is a new selection developed by the centre of competence. streif-index was used to determine maturity and as a consequence the optimal harvest date for every cultivar. the index was calculated as fruit firmness/ [soluble solid content x starch degradation value] (höhn et al., 1990). the harvest lasted from the beginning of september (‘elstar’, ‘gala’, ‘honeycrisp’) to the end of october (‘fuji kiku’). subsequent to harvest the fruit were washed and the antioxidants extracted. to minimize variation ten fruits were randomly selected for analysis. for storage experiment fruit were stored in controlled atmosphere conditions (ca) (1.5 °c, 1.2% o2, 2.5% co2) or in a cold chamber (1 °c) for 4.5 months. subsequent to this, some fruits were stored at shelf life conditions (20 °c) for two weeks to simulate the conditions at the consumers’ home or during the handling in the production chain. storage experiments were conducted with six cultivars: ‘wellant’, ‘golden delicious’, ‘honeycrisp’, ‘mairac’, ‘pinova’ and ‘topaz’. preparation of extracts extraction of hydrophilic antioxidants was carried out with a potassium phosphate buffer (10 mm di-potassium hydrogen phosphate (k2hpo4), 10mm potassium dihydrogen phosphate (kh2po4), ph 7.1) containing 10 mm sodium dietyldithiocarbamate trihydrate (dieca) and 2mm ethylenediaminetetraacetic acid (edta). peel and pulp of a composite sampling of ten fruit was homogenized with buffer in a relation 1:1.5 w/v with a retsch (retsch, haan, germany) grinder. the extracts were prepared from the same ratio of peel and pulp for each cultivar. the core was removed before extraction. after incubation for four hours at room temperature, the homogenates were centrifuged at 4 °c for 15 min at 3500 rpm. the supernatant was collected and stored at -80 °c until analysis. the preparation of the lipophilic extract was carried out as described by schmitz and noga (2000). two gram of chilled pulp were homogenized with 3 ml hexane using a retsch homogenizer. samples were centrifuged at 4 °c for 10 min at 4000 rpm, the supernatants were collected, blown up with nitrogen and filled up to 2 ml with hexane. until analysis the extracts were stored at -80 °c. analysis of antioxidant capacity and total phenol content the determination of the antioxidant capacity of the water-extract was performed as reported by miller et al. (1993) with some modifications. the method is based on the oxidation of 2,2’-azinobis(3-ethylbenzthiazoline-6-sulfonic acid (abts) to form a radical cation abts . + in the presence of hydrogen peroxide and metmyoglobin. the radical has a maximum of absorbance at 734 nm and a direct scavening of the formed radical by hydrogen-donating antioxidants resulted in a decrease of absorbance at 734 nm. the absorbance was measured after incubating for 6 min from start of the reaction in a lambda 5/15 spectrophotometer (perkin elmer). 6hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (trolox), a water-soluble vitamin e analogon, was used as a standard and results were expressed as mg trolox g-1 fw. total phenols were measured using folin-ciocalteau reagent (singleton and rossi, 1965). an aliquot of 0.5 ml of the sample was mixed with 0.5 ml of destilled water and 0.5 ml of the folinciocalteau reagent was added. after 30 sec 5 ml of aqueous sodium carbonate were added, the mixture was incubated for 30 min at room temperature in the dark. extinction was measured at 720 nm. total phenol content was calculated using gallic acid as standard. results were expressed as mg gallic acid units (gau) 10-2 g-1 fw. the determination of the antioxidant capacity of lipophilic extracts was determined according to chevolleau et al. (1992). this method is based on the decoloration of β-carotene by radicals that are generated during the oxidation of linoleic acid at 50 °c. one milligram β-carotene was solved in 10 ml chloroform. to 5 ml of the carotine-chloroform-solution 20 mg linolic acid and 200 mg tween 40 were added. after vaporization of chloroform with nitrogen the substances were filled up with distilled water to 100 ml. an aliquot of 200 µl of every sample were vaporized with nitrogen, then mixed with 5 ml of the carotene-chloroform solution. the solutions were incubated in a 50 °c water bath and the extinction was measured every 30 min at 470 nm for a period of two hours. antioxidant capacity was calculated as me g-1 fw. statistical analysis analyses were performed at least in duplicate. the experimental data were analysed using the statistic program spss 12.0 for windows (munich, germany). the statistical analysis was performed by one way analysis of variance (anova), with a significance level of α = 0.05. comparisons of mean values were performed by the tukeytest. a normal distribution and variance homogeneity was implied. any correlation was calculated as pearson’s coefficient of correlation. results total phenol content of apple cultivars ranged between 25 44.3 mg gau 10-2 g-1 fw and a cultivar variation was noticed (tab. 1). at harvest total phenol content was highest in the cultivars ‘cox orange’, ‘fuji kiku’, ‘rubinette’ followed by ‘delia’, ‘pinova’ and ‘jonagold’. lowest contents were measured in the cultivars ‘aw 106’, ‘braeburn’ and ‘rubens’ (tab. 1). ‘pinova’ and ‘topaz’ fruit stored under ca conditions for 4.5 months showed a slight increase in total phenol content (fig. 1). this was not the case for the cultivars ‘wellant’, ‘honeycrisp’ and ‘golden tab. 1: polyphenol content (mg gau 10-2 g-1 fw), antioxidant capacity of hydrophilic extracts (mg trolox g-1 fw; teac-values) and lipophilic extracts (me g-1 fw) in selected apple cultivars at harvest (mean ± se). cultivar total phenols teac-value lipophilic antioxidant capacity cox orange 44.3 ± 2.1 11.1 ± 0.0 4.1 fuji kiku 37.1 ± 0.0 13.8 ± 0.4 29.1 rubinette 35.8 ± 1.5 11.1 ± 0.1 31.2 delia 35.3 ± 0.6 8.3 ± 0.6 60.7 pinova 35.1 ± 1.3 11.7 ± 0.5 27.4 jonagold 34.3 ± 0.8 10.2 ± 0.1 17.2 diwa 34.1 ± 0.4 11.3 ± 0.1 31.1 topaz 33.1 ± 0.6 9.5 ± 0.3 15.9 wellant 32.7 ± 2.0 9.7 ± 0.2 200.2 cameo 31.4 ± 0.4 10.0 ± 0.1 18.2 mairac 31.3 ± 0.8 7.5 ± 0.1 16.3 grenstar 29.3 ± 0.0 6.9 ± 0.4 19.8 elstar 29.2 ± 1.1 8.3 ± 0.2 23.1 honeycrisp 28.6 ± 0.2 9.6 ± 0.2 2.7 golden delicious 27.3 ± 0.2 9.0 ± 0.5 50.5 kanzi 26.0 ± 0.6 8.3 ± 0.2 33.8 aw 106 25.7 ± 0.1 6.7 ± 0.1 56.8 braeburn 25.1 ± 0.5 2.5 ± 0.2 34.9 rubens 25.0 ± 0.3 7.2 ± 0.2 37.5 polyphenols in apple fruit as affected by storage 153 154 anne matthes, michaela schmitz-eiberger delicious’, in which phenol content remained stable and for the cultivar ‘mairac’ in which phenol content declined. during an additionally shelf life period in most cultivars no differences in polyphenol content were observed. just in ‘wellant’ and ‘pinova’ fruit higher concentrations were found after the shelf life period in comparison to that determined after ca-storage (fig. 1). phenol concentration was affected by cold storage, which resulted in an increase in ‘pinova’ and ‘topaz’ fruit and a significant increase in the cultivar ‘golden delicious’. in the other cultivars phenol concentration was constant during storage. subsequent shelf life at ambient temperature led to different reactions in the fruit. in four cultivars (‘golden delicious’, ‘pinova’, ‘mairac’ and ‘honeycrisp’) phenol content did not change, an increase was observed for ‘wellant’ and a significant decrease was determined in ‘topaz’, phenol content was only 50% in comparison to that measured at harvest. total phenol content was more dependent on the cultivar but on storage conditions (fig. 1). antioxidant capacity of the hydrophilic extract was measured using the teac-method. highest teac-values at harvest were measured in ‘fuji kiku’, ‘pinova’ and ‘cox orange’ followed by ‘rubinette’ and ‘diwa’. the lowest antioxidant capacity was measured in ‘braeburn’ (tab. 1). stored cultivars showed an increase in teac-values if stored for 4.5 months irrespective of the storage condition (fig. 2). this could not be observed in cold stored ‘pinova’ fruit and in ca-stored ‘golden fig. 1: phenolic compounds in different apple cultivars at harvest, after cold and ca storage and after an additional shelf life (sl) period (mean ± se). values followed by different letters within a cultivar are significantly different at 5% significance level. wellant golden delicious honeycrisp to ta l p h e n o ls [m g g a u /1 0 0 g f w ] 0 20 40 60 80 100 harvest ca ca + sl cold storage cold storage + sl mairac pinova topaz to ta l p h e n o ls [m g g a u /1 0 0 g f w ] 0 20 40 60 80 100 harvest ca ca + sl cold storage cold storage + sl ab b ab a ab b ab ab a ab ab b a b ab bc c bc ab a ab b ab a ab a ab b ab ab wellant golden delicious honeycrisp to ta l p h e n o ls [m g g a u /1 0 0 g f w ] 0 20 40 60 80 100 harvest ca ca + sl cold storage cold storage + sl mairac pinova topaz to ta l p h e n o ls [m g g a u /1 0 0 g f w ] 0 20 40 60 80 100 harvest ca ca + sl cold storage cold storage + sl wellant golden delicious honeycrisp to ta l p h e n o ls [m g g a u /1 0 0 g f w ] 0 20 40 60 80 100 harvest ca ca + sl cold storage cold storage + sl mairac pinova topaz to ta l p h e n o ls [m g g a u /1 0 0 g f w ] 0 20 40 60 80 100 harvest ca ca + sl cold storage cold storage + sl ab b ab a ab b ab ab a ab ab b a b ab bc c bc ab a ab b ab a ab a ab b ab ab mairac pinova topaz t e a c [m g t ro lo x /g f w ] 0 2 4 6 8 10 12 14 16 harvest ca ca + sl cold storage cold storage + sl wellant golden delicious honeycrisp t e a c [m g t ro lo x /g f w ] 0 2 4 6 8 10 12 14 16 harvest ca ca + sl cold storage cold storage + sl b a a a a b a b c d a ab a a c bc c a a b b a b b b cd b c ab a mairac pinova topaz t e a c [m g t ro lo x /g f w ] 0 2 4 6 8 10 12 14 16 harvest ca ca + sl cold storage cold storage + sl wellant golden delicious honeycrisp t e a c [m g t ro lo x /g f w ] 0 2 4 6 8 10 12 14 16 harvest ca ca + sl cold storage cold storage + sl mairac pinova topaz t e a c [m g t ro lo x /g f w ] 0 2 4 6 8 10 12 14 16 harvest ca ca + sl cold storage cold storage + sl wellant golden delicious honeycrisp t e a c [m g t ro lo x /g f w ] 0 2 4 6 8 10 12 14 16 harvest ca ca + sl cold storage cold storage + sl b a a a a b a b c d a ab a a c bc c a a b b a b b b cd b c ab a fig. 2: teac-values in different apple cultivars at harvest, after cold and ca storage and after an additional shelf life (sl) period (mean ± se). values followed by different letters within a cultivar are significantly different at 5% significance level. polyphenols in apple fruit as affected by storage 155 delicious’ where antioxidant capacity remained unchanged. storage at 20 °c for two weeks led to a significant decrease of antioxidant activity in most cultivars. for the cultivars ‘mairac’, ‘golden delicious’ (ca-stored) and ‘topaz’ (ca-stored) no changes in teacvalues could be observed (fig. 2). correlation analysis showed that total phenol content was positively correlated to the antioxidant activity (tab. 2). a strong correlation between teac-values and the total phenol content was found after harvest, cold and ca-storage with additional shelf life. a moderate relationship was calculated for the fruit after ca-storage and no correlation was found after cold storage (tab. 2). antioxidant capacity of the lipophilic extract was highest in the cultivar ‘wellant’, followed by ‘delia’, ‘aw 106’ and ‘golden delicious’. lowest antioxidant capacity was found in the cultivars ‘cameo’, ‘jonagold’, ‘mairac’ and ‘topaz’ (tab. 1). lipophilic antioxidants decreased during storage in the cultivars ‘wellant’, ‘pinova’ and ‘topaz’. in fruit of ‘mairac’, ‘honeycrisp’ and ‘golden delicious’ highest antioxidant capacity was determined after cold storage in comparison to the values at harvest and after ca-storage. an additional shelf life period led to a further decrease in antioxidant capacity. tendentiously higher values were found in cold stored fruit in comparison to fruit stored under ca conditions (tab. 3). discussion phenolic compounds are important secondary plant metabolites and contribute to any health promoting effects of fruits and vegetables. food producers and consumers spend increasing interest in the amount of certain health promoting substances. therefore a close investigation is necessary to find out whether this metabolites can be enhanced by breeding and the selection of special cultivars. as apples are distributed the whole year it is important to investigate if this metabolites remain stable during storage and in how far any degradation processes can be decelerated by improving the storage conditions and in how far degradation processes are influenced by shelf life. several studies reported on the content of different bioactive components in apple fruit. these studies were mainly focused on the peel of the fruit, because higher concentrations of polyphenols were found there (lattanzio et al., 2001; solovchenko and schmitzeiberger, 2003; wolfe et al., 2003). we used whole apples, because usually apple fruit are consumed as whole fruits and water-soluble antioxidants are distributed in the pulp respectively. the variation of single components of polyphenols between individual apples was 10-30% in a study conducted by van der sluis et al. (2001), so we used composite samples. polyphenol content was shown to be cultivar dependent. schmitzeiberger et al. (2003) investigated bioactive components in 31 apple cultivars. they found a large cultivar variation with highest contents in cultivars used for must and juice production. some of the results of the current study are consistent with that of 2003 (e.g. low teacand folin-values in the cultivar braeburn), contradictionary results were e.g. obtained for the cultivar ‘pinova’ and ‘aw 106’. this showed that the antioxidant content of apple fruit is affected by environmental conditions which support the biosynthesis of polyphenols, so that content may vary at different harvest years. in conflict to this van der sluis et al. (2001) observed no seasonal effect on antioxidant capacity and flavonoid concentration in four apple cultivars when they compared the results of three different harvest years. polyphenols seem to be stable during storage as shown here and in several other studies. awad and de jager (2000) observed no significant changes in flavonoid content upon storage for 30 weeks under ca and cold conditions in the skin of the apple cultivars ‘jonagold’ and ‘elstar’. there were no significant differences in flavonoid concentration between fruit stored under ca and regular conditions (0 °c). except for catechin the data of awad and de jager (2000) showed that flavonoid and chlorogenic acid concentration remained constant during two weeks shelf life. lattanzio et al. (2001) determined a decrease in phenolics, particulary phloridzin after ten days of shelf life in ‘golden delicious’ fruit. van der sluis et al. (2001) reported that ca and storage in a cold chamber did not influence flavonoid concentration and antioxidant activity of whole apple fruit of various cultivars. this is consistent with the findings of golding et al. (2001) who found no great changes in the concentration of the major phenolics during long-term storage. napolitano et al. (2004) observed an increase in the flesh of ‘annurca’ apple fruit in antioxidant activity during cold storage, which was correlated with the concentrations of catechin and phloridzin. in disagreement to this burda et al. (1990) found a decrease of catechin, but total phenol concentration stayed at a constant level. another study detected a decrease in anthocyanins with simultaneously increasing phenol content (leja et al., 2001). leja et al. (2001) reported an increase in total phenol content and a doubling of antioxidant activity after four months of storage in a cold chamber and under ca conditions. an additional storage at 16 °c led to a further increase in total phenol content and to maintainance of antioxidant activity. the present results are different from tab. 2: pearson’s correlation coefficients (r) between phenolic compounds and antioxidant capacity (teac-values) of selected apple cultivars. phenolic compounds harvest ca ca + sl cold cold storage storage + sl antioxidant 0.761 ** 0.5 0.82 ** 0.072 0.876 ** capacity ** significant at p < 0.01 tab. 3: lipophilic antioxidant capacity (me g-1 fw) of selected apple cultivars at harvest, after cold and ca-storage and after additional shelf life (sl) period (mean). cultivar harvest ca ca + sl cold storage cold storage + sl wellant 200.2 a* 10.2 bc 13.4 b 12.7 b 6.6 c gd 50.5 b 6.2 b 14.5 b 340.2 a 4.8 b honeycrisp 2.7 d 5.7 cd 15.3 b 21.7 a 6.8 c mairac 16.3 a 6.5 a 0 a 117.2 a 3.3 a pinova 27.4 a 12.7 bc 2.5 c 22.1 ab 2.2 c topaz 15.8 a 5.7 bc 0.3 c 12.7 ab 3.9 c * means with same letters within a cultivar are not significantly different, tukey-test (p < 0.05). 156 anne matthes, michaela schmitz-eiberger those reported by tarrozi et al. (2004) who found lower phenol values in the peel of apple fruit after cold storage for three months, no further effect was seen after six month. no effect of cold storage was shown for gallic acid concentration in the pulp of the fruit. napolitano et al. (2004) reported a decrease of antioxidant capacity in a water extract of apples during storage of the fruit, which was related to the ascorbic acid degradation occuring during storage or any polymerization of free phenols into less water-soluble polymers. this effect was also shown here, even though the polyphenol content was constant during shelf life the teac-values decreased. this could be explained by lower amounts of synergistic antioxidants e.g. ascorbic acid (data not shown) and other lipid-soluble antioxidants, which could also be seen by lower antioxidant activity of lipophilic extracts after storage. an increase of polyphenol content could be due to ethylene action. this phytohormone stimulates the activity of the key enzyme in polyphenol biosynthesis, the phenylalanine ammonium lyase (pal), which leads to formation of polyphenols (blankenship and unrath, 1988; leja et al., 2001; napolitano et al., 2004). pal activity has been shown to have a direct influence on total flavonoids in apples (lister et al., 1996). leja et al. (2001) found a marked ethylene evolution in stored apple fruit, especially when stored in air, and reported on increased polyphenol contents during storage. tomasbarberan and espin (2001) reported that pal activity is higher at lower temperatures. at the same time activity of enzymes responsible for polyphenol degradation, polyphenoloxidase, are inhibited by lower temperatures. leja et al. (2001) stated that an increase in phenol content could be due to lower activity of polyphenoloxidase at low temperatures, so that oxidation processes were minimalized. increases in polyphenol content during shelf life can be explained by a higher ethylene production which results in a stimulation of pal (napolitano et al., 2004). however, reyes (2003) indicated that ethylene and temperature did not significantly affect accumulation of polyphenols. but in his study phenol amount in purple flesh potatoes increased through mechanical damage, that led to activation of polyphenol biosynthesis probably via signalling pathways such as salicylic and jasmonic acids. a high number of correlation studies can be found in literature. in consistence with our results a good correlation between antioxidant capacity and total phenol content was found in several studies (kalt et al., 1999; arnous et al., 2001; schmitz-eiberger et al., 2003). in contrast to this no correlation between the antioxidant capacity and any single phenolic compounds was found (arnous et al., 2001; van der sluis et al., 2001; schmitz-eiberger et al., 2003). the lacking correlation after cold storage might be explained by synthesis of any other antioxidants during cold storage, so that antioxidant capacity was maintained. antioxidant activity is a result of several phytochemicals present in the fruit and their synergistic effects. the benefits of fruit and vegetable consumption can not be attributed to a single compound but to synergistic and additive effects between different phytochemicals (liu, 2003). the contribution of ascorbic acid to the total antioxidant activity of fruits was determined to be generally lower than 15% (wang et al., 1996), other authors calculated a contribution of ascorbic acid to antioxidant capacity of only 0.4% (kalt et al., 1999; eberhard et al., 2000; schmitz-eiberger et al., 2003; wolfe et al., 2003). however, synergistic effects between polyphenols and other antioxidants were described in earlier studies (fryer, 1992; saucier and waterhouse, 1999; eberhard et al., 2000). previous studies showed that the antioxidant and antiproliferative activities of apples are the consequence of synergistic activities of phenolics rather than ascorbic acid (eberhard et al., 2000; arnous et al., 2001). much attention has recently been paid to the possible health benefits of dietary phenolics which have stronger antioxidant activities than ascorbic acid. there is increasing evidence that phenolics can be absorbed into the human body in amounts that are sufficient to exert antioxidant properties in vivo. first studies showed a bioavailability of 52% of quercetin glycosides present in onions (hollmann et al., 1997) and of 33% of chlorogenic acid present in a supplement (olthof et al., 2001). the relative bioavalibility of quercetin from apples was only one-third of that from onions (hollmann et al., 1997). that shows that bioavailability depends on the amount and kind of metabolite present in the food source and the food itself. nevertheless further investigations have to be executed to investigate the absorption of different secondary metabolites, their tissue uptake, plasma concentrations and transport as well as metabolism and elimination, so that the metabolites can be identified that exert potential health benefits. conclusions we conclude that polyphenolics vary among cultivars, they remain relatively stable during cold storage and storage at controlled atmosphere. shelf life led to lower concentrations of antioxidants, which indicates that metabolism and turnover is higher at room temperature. the results obtained in the present study showed that bioactive components can be preserved by storage and can therefore obtain the high nutrional quality of apple fruits for the consumer. consuming cultivars with high content of antioxidants even after storage may contribute to an antioxidant rich diet and may impart health benefits. references arnous, 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(eds.), free radicals and oxidative stress: environment, drugs and food additives, 103-116. portland press, london. reyes, f., 2003: wounding stress increases the phenolic content and antioxidant capacity of purple flesh potatoes. j. agric. food chem. 51, 5296-5300. saucier, c.t., waterhouse, a.l., 1999: synergistic activity of catechin and other antioxidants. j. agric. food chem. 47, 4491-4494. scalbert, a., williamson, g., 2000: dietary intake and bioavailability of polyphenols. j. nutr. 130, 2073-2085. scalbert, a., johnson, i.t., saltmarsh, m., 2005a: polyphenols: antioxidants and beyond. am. j. clin. nutr. 81 (suppl.), 215-217. scalbert, a., manach, c., morand, c., remesy, c., jimenez, l., 2005b: dietary polyphenols and the prevention of diseases. crit. rev. food sci. 45, 287-306. schmitz, m., noga, g., 2000: selected plant components and their antioxidative capacity in hydrophilic and lipophilic extracts of phaeseolus vulgaris, malus domestica and vitis vinifera leaves. gartenbauwiss. 65, 65-73. schmitz-eiberger, m., weber, v., treutter, d., baab, g., lorenz, j., 2003: bioactive components in fruits from different apple varieties. j. appl. bot. 77, 167-171. singleton, v.l., rossi, j.a., 1965: colorimetry of total phenols with phosphomolybdic-phosphotungstic acid reagents. am. j. enol. vitic. 16, 144-158. solovchenko, a., schmitz-eiberger, m., 2003: significance of skin flavonoids for uv-b-protection in apple fruits. j. exp. bot. 54, 19771984. tarozzi, a., marchesi, a., cantelli-forti, g., hrelia, p., 2004: cold storage affects antioxidant properties of apples in caco-2 cells. j. nutr. 134, 1105-1109. tomas-barberan, f.a., espin, j.c., 2001: phenolic compounds and related enzymes a determinants of quality in fruits and vegetables. j. sci. food agric. 81, 853-876. van der sluis, a.a., dekker, m., de jager, a., jongen, w.m.f., 2001: activity and concentration of polyphenolic antioxidants in apple: effect of cultivar, harvest year and storage conditions. j. agric. food chem. 49, 3606-3613. wang, h., cao, g., prior, r.l., 1996: total antioxidant capacity of fruits. j. agric. food chem. 44, 701-705. wolfe, k., xianzhong, w., rui hai, l., 2003: antioxidant activity of apple peels. j. agric. food chem. 51, 609-614. address of the authors: dipl. oec. troph. anne matthes and priv.-doz. dr. michaela schmitz-eiberger, university of bonn, institute of crop science and resource conservation, horticultural science, auf dem huegel 6, d-53121 bonn, germany e-mail: amatthes@uni-bonn.de << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false 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/pagesize [907.087 680.315] >> setpagedevice art08 journal of applied botany and food quality 80, 63 68 (2006) university of hamburg, biocenter klein flottbek, institute of applied botany, hamburg, germany physiological characteristics of microcyclus ulei (p. henn.) v.arx. – a fungal pathogen of the cyanogenic host hevea brasiliensis r. lieberei (received january 17, 2006) summary the ascomycete microcyclus ulei causes premature leaf shedding of the rubber tree, hevea brasiliensis. during the development of this biotrophic fungus in the tissues of infected leaves hcn is liberated from the lesions. despite high hcn concentrations in the leaf tissue the pathogenic fungus develops infection hyphae. the reaction of m. ulei to hydrogen cyanide and the biochemical properties of a fungal β-glucosidase which is active on the cyanogenic precursors of the host plant are reported. introduction microcyclus ulei is the causative agent of south american leaf blight (salb), the most serious disease of rubber tree hevea brasiliensis in south america. the biotrophic pathogenic ascomycete is characterized by its narrow host range, attacking genotypes of four (h. brasiliensis, h. benthamiana, h. spruceana, h. guianensis (chee, 1976) out of eleven species of the genus hevea (schultes, 1970). within the species hevea brasiliensis and h. benthamiana a distinct race-cultivar relationship was described (junqueira et al., 1986), additionally first reports on the attack of genotypes of h. pauciflora were reported. the host-pathogen relations varied strongly with the fungal isolates, underlining the fact that m. ulei reveals a highly variable pathogen race structure with a range of obviously freely combining virulence factors. using molecular approaches, recently new contributions to the genetic polymorphism of the pathogen were given by le guen et al. (2004). in an enlarged field study mattos et al. (2003) analysed the variability of m. ulei isolates from southeast bahia. fifty isolates taken from 29 rubber tree clones were tested with respect to their qualitative and quantitative development on young leaf stages of twelve hevea sp. clones. these clones were either pure h. brasiliensis, h. pauciflora, f1 hybrids of various crossings of the species h. brasiliensis and h. benthamiana or backcrossings of these f1 hybrids with h. brasiliensis. the authors evaluated virulence profiles of the isolates tested and concluded, that at least 36 isolates turned out to be new races of the pathogen, besides the description of 11 other races by rivano (1997). only two isolates of these 36 new races revealed similar properties to the isolate profiles described earlier by junqueira et al. (1986). the importance of this high variability and the high potential to adapt to genotype properties of the host species is discussed by mattos et al. (2003) with special view on the difficulties in resistance breeding. under this aspect of virulence variability it is interesting to point out that the pathogenic range of m. ulei still is restricted to hevea spp. first steps of host range amplification by this fungus have recently been reported by hagen et al. (2003) who reported the physiological interaction of m. ulei with manihot esculenta crantz leaves. it is assumed that the narrow host range of this pathogen might be due to typical physiological properties of hevea species which facilitate the attack by m. ulei. the ability to accumulate cyanogenic compounds and to store them in considerable amounts is a characteristic biochemical feature of both, the genus hevea and the genus manihot. in both genus the cyanogenic storage compounds are the β-glucosides linamarin and lotaustralin. the compounds are accumulated in vacuoles and after cell destruction these glucosides are cleaved and hcn is liberated. the β-glucosidases cleaving these compounds (linamarase) are of apoplastic origin (selmar et al., 1987). cultivars of h. brasiliensis with a high capacity to liberate hcn are readily susceptible to m. ulei (lieberei, 1988, 1989), whereas low cyanogenic genotypes of h. brasiliensis and genotypes of h. viridis and h. pauciflora, characterized by a reduced ability to liberate hcn (lieberei, 1988), reveal the typical host-pathogen cultivar specific resistance pattern (e.g. pa31, mattos et al., 2003; chee and wastie, 1980). hcn is liberated from hevea leaves infected by m. ulei throughout the entire process of pathogenesis from penetration of host tissues to conidium formation. the time course of hcn liberation has been quantified, and these results also allow to calculate the cyanide concentration inside the leaf tissue (lieberei, 1986). this volatile and readily soluble compound diffuses directly after liberation from precursors through the tissues surrounding the infected area, thus both the plants and the pathogen are in contact with the hcn formed. a more detailed description on the physicochemical properties of hcn in leaf tissue is provided by lieberei et al. (1996). studies of the influence of hcn on m. ulei isolates in pure culture revealed that the pathogens are tolerant to hcn. vegetative mycelial development is enhanced in pure culture, but spore formation is inhibited by the presence of hcn (lieberei et al., 1983). this tolerance to hcn and the stage specific promotion of mycelial growth by hcn suggest a strong physiological adaptation of the pathogen to the cyanogenic properties of the host plant. this adaptation is of considerable interest for the understanding of biochemical coevolution between host and pathogen. the knowledge of these biochemical interactions will contribute important aspects to the practical development of resistance screening steps and for planning of molecular biological approaches to modify host preconditions against the pathogens. molecular approaches to change the biochemical qualities of the host plants with respect to the cyanogenesis is underway in the important crop plant cassava (m. esculenta) and it must be taken into consideration to transfer this work to rubber tree germplasm. the focus of this paper is to analyse the metabolic adaptation of m. ulei, the specialized biotrophic fungal plant pathogen to its cyanogenic host plant, hevea species. the analysis of cyanide resistant respiration of the fungus, the substrate specificity of the fungal-β-glycosidase to the cyanogenic precursor of the host and the reaction of the pathogen to β-cyanoalanine, a detoxification product of hcn, formed by the plant, is presented and the consequences of these findings for breeding and selection are discussed. abbreviations cr = cyanide resistant respiration, β-ca = β-cyanoalanine, psa = potato sucrose agar. materials and methods fungus culture m. ulei was cultivated on potato sucrose agar (psa 0.5 %) ph 5.0 at 23°c and 90 min of light (osram w/30, 40 cm distance) according to chee (1978). it was subcultured every 14 days using spores and mycelium. for spore mass production cultures were kept dark for 15 days, followed by light induction for three days. mass cultures were run in 300 ml erlenmeyer flasks with 80 ml psa 0.5 % (lieberei et al., 1983). spore isolation spore forming cultures after light induction were thoroughly shaken with 6 ml sterile distilled water (2 x). the spores were liberated easily and normally contained about 90 % of two celled spores. suspension density was adjusted using an „improved double neubauer-chamber“. cultivation of mycelia in liquid culture 5 ml of a freshly isolated spore suspension (1.2 x 105 spores l-1) were mixed with 25 ml of potato-sucrose-medium (0.5 %). the resulting spore density was 2.0 x 104 spores l-1. the suspensions were cultured at 23° c in the dark in a 3 mm liquid layer. after 5 days the mycelia were harvested by filtration on schleicher and schuell filters (0.2 µm), suspended in phosphate buffer 0.067 mmol l-1 ph 5.0 amended with 0.5 % sucrose. these samples were taken to respiration measurements in a ysi yellow springs instruments model 53 biological oxygen monitor equipped with clark electrode ysi 5331. spore exsudates freshly isolated conidia were adjusted to 8 x 104 spores ml-1 and left in sterile petri dishes in sterile tap water at 23°c for 24 hrs. the liquid layer did not exceed 3 mm, the germination rate was counted at onset of the experiment and after 24 hrs. the exsudates were isolated by filtration using filters of schleicher and schuell 0.2 µm pore size. concentration was done by freeze drying. βββββ-glucosidase enzyme activity was estimated according to selmar et al. (1987). results cyanide resistant respiration of the pathogen the mitochondrial electron transport chain is very sensitive to hcn because that substance strongly inhibits the cytochrome a/a3 dependent oxidase, however, in all cases so far studied, residual, cyanide-resistant o2 consumption in the presence of hcn is seen. the pathway can be determined using specific inhibitors. hcn inhibits the main respiratory pathway, whereas salicylhydroxamic acid inhibits the alternative respiratory path. the time course of inhibition of respiration and the concentration of inhibitors vary and must be identified experimentally for the organisms under study. six minutes after addition of hcn to mycelia of m. ulei liquid cultures, maximal inhibition was reached (tab. 1). at a hcn concentration of 1 mmol l-1, the o2 consumption of the samples was inhibited by about 67.4 %. enhancement of the hcn concentration up to 5 mmol l-1 did not lead to higher rate of inhibition. the alternative path of respiration was inhibited by salicylhydroxamic acid (sham) (schönbaum et al., 1971). incubation with 2.5 mmol l-1 tab. 1: inhibition pattern of respiration and cyanide resistant respiration of m. ulei. inhibitory compound final concentration of inhibition of respiration inhibitor [mol/l] in [% ] of control 5.0 c 10 –4 41.3 kcn 7.5 c 10 –4 59.6 1.0 c 10 –3 67.4 5.0 c 10 –3 67.4 1.0 c 10 –3 44.4 sham 2.5 c 10 –3 52.4 5.0 c 10 –3 52.5 the kcn solution for inhibitory action was prepared in the measuring buffer and was adjusted to the physiological ph of the cultivation media directly in the moment of addition in order to avoid interferences by other factors like ph-changes. sham, salicyl hydroxamic acid was solubilized in dmso solution in such concentrations, that 1% (vol/vol) of dmso was never exceeded. cn-resist. o2-consumption relative amount total o2-consumption [nmol o2 x mg dw-1 x min] [mg] dry weight [growth] respiration cn-resist. respiration [%] cn-resist. o2-consumption fig. 1: oxygen consumption and growth pattern of m. ulei with respect to sensitivity to hcn for 30 minutes was sufficient to reach maximum inhibitory action (tab. 1). the total amount of inhibition of mycelial respiration observed depended on the developmental status of the fungal samples used (fig. 1): oxygen consumption of fresh spores at the onset of germination is inhibited by hcn to less than 50 % of the control, whereas that of 5 days old cultures was inhibited up to almost 80 %. the conidia of m. ulei show large constitutive amount of cyanide resistant o2 consumption. the absolute amount of cyanide resistant respiration rises during mycelial development, but due to the enhance64 r. lieberei ment of total o2 consumption of the culture, the relative amount of cyanide resistant respiration is diminished. the cyanide resistant o2 consumption of m. ulei is inhibited to a great extent by sham, but residual o2 consumption persists. this low residual level of oxygen consumption is inhibited neither by hcn, nor by sham. this type of non-inhibited o2 consumption is also known for other fungi (rissler and miller, 1977). in general this finding supports the view that m. ulei has a high biochemical potential to grow in the presence of hcn. induction of cyanide resistant respiration in mycelia by hcn when mycelia of m. ulei growing in liquid culture media are treated with small concentration of kcn, the inhibition pattern of o2 consumption is considerably changed. overall o2 consumption in cyanide treated cultures is significantly higher than in control cultures without addition of kcn (1 mmol l-1). the additional o2 consumption found in cyanide-treated cultures is completely due to enhanced activities in the cyanide resistant pathway (tab. 2). βββββ-glucosidase production as is known from many other fungi, m. ulei produces soluble exoenzymes during germination and early fungal growth. the first colonization phase of the host tissue by this biotrophic pathogen is leading to hcn liberation due to breakdown of linamarin from plant origin. hcn liberated is able to promote fungal development. in order to know if the fungus is able to interact with hcn liberation which promotes fungal development and impairs plant resistance reactions, it was studied if the fungus itself produces enzymes able to catalyse the cleavage of cyanogenic glucosides present in the host plant tissue. ungerminated spores of m. ulei contain an active β-glucosidase (tab. 3). about 30 % of this activity can be washed off from the spores with distilled water, about 70 % are attached to the walls. both, spores and supernatants (30,000 g) from spore homogenates or from spores ultrasonicates contain the same β-glucosidase activity, thus the β-glucosidase present in spores is wall bound and is located in the periphery, easily accessible to aqueous treatment (tab. 3). in the course of germination, the extracellular level of β-glucosidase activity is strongly enhanced and is partially soluble. however, there is also a variable amount of the β-glucosidase activity still bound to fungal cell walls. the amount of β-glucosidase produced during germination is mostly soluble. the higher the amount of germinated spores the higher is the amount of β-glucosidase in the germination medium (tab. 3). βββββ-glucosidase characteristics the β-glucosidase found in the germination medium reveals a broad ph optimum from ph 5.0 to 5.6 with half maximal rates at ph 4.0 and 6.5 (fig. 2). the soluble enzyme exhibits β-glucosidase activity, but also catalyses the cleavage of α-glucosides, β-galactosides and cellobiosides (cellobiose, amygdalin) to a very limited extent (tab. 4). of the natural substrates, the cyanogenic glucoside linamarin, the main cyanogenic compound in tissues of the rubber tree, is readily cleaved. the km of m. ulei β-glucosidase for linamarin is 4.2 mmol l-1 for linamarin in contrast to 0.82 for the artificial model substrate p-nitrophenol-β-d-glucopyranoside (p-npg). the vmax for linamarin is very high and about twice that of p-npg, i.e. the turn over is high. the fungal enzyme reveals a higher affinity for the cyanogenic substrate of the host plant than the enzyme of host plant itself. the linamarin cleaving β-glucosidase of hevea brasiliensis has a km for tab. 2: influence of culture treatment with hcn on the oxygen consumption hcn preculture (15 hours) o2-consumption o2-consumption under 1 x 10 –3 mol hcn/l nmol o2 nmol o2 % of o2-consumption min x mg dw min x mg dw without cyanide no cyanide 49.2 23.4 47.6 cyanide (2 x 10 –4 mol/l ) 112.9 95.1 84.9 tab. 3: β-glucosidase activity in spores and germination medium sampleno. germination time of germination β-glucosidase activity β-glucosidase activity relation of in germination medium attached to free to bound (free β-glucosidase) germinated spores β-glucosidase [%] [hours] [nmol x s-1l-1] [nmol x s-1l-1] [%] 1 15 24 0.3 6.3 4.8 2 16 24 0.3 5.5 5.5 3 18 24 0.2 3.3 6.1 2 0 2.6 6.2 41.9 4 91 24 93.4 6.6 1415.1 5 74 24 73.2 5.2 1407.7 spore isolates 1, 2, 3 were isolated from undefined h. brasiliensis genotypes, mato grosso spore isolates 4 and 5 were isolated from h. brasiliensis ian 717, amazonas, manaus germination was run at 25 °c in the dark in sterile tap water at 3mm liquid layer physiological characteristics of microcyclus ulei 65 linamarin of 7.6 mmol l-1 (selmar et al., 1982). the relative cleavage rates (vmax linamarin: vmax pnpg) are 1.56 for m. ulei β-glucosidase and 2.8 for hevea β-glucosidase. thus, the soluble extracellular β-glucosidase of the pathogen produced during early germination stages can contribute to linamarin cleavage and hcn liberation. in other words, the hcn-liberation capacity, as defined in lieberei (1988) is influenced by the pathogen. influence of βββββ-cyanoalanine on germination and mycelial development of m. ulei β-cyanoalanine (β-ca) is a detoxification product of cyanide in plants. the enzyme β-cyanoalanine synthase, which is present in cyanogenic as well as in non-cyanogenic plants (e.g. miller and conn, 1980), produces β-cyanoalanine. it catalyzes the reaction of cysteine with hcn to form β-cyanoalanine and h2s. β-ca is neurotoxic to mammals. it has been suggested that this compound causes disturbances in biomembranes. the presence of β-cyanoalanine in tissues of cyanogenic plants during hcn liberation has been established (castric et al., 1972). βcyanoalanine synthase activity of hevea brasiliensis is high (körner, 1994) and during cyanogenesis caused by fungal attack, β-cyanoalanine is assumed to be present in the tissues. in order to evaluate the influence of β-ca on m. ulei, in vitro germination tests with βca were carried out. spore germination and mycelial development of m. ulei are significantly inhibited by β-ca (fig. 3.). at 90 µmol l-1 β-ca in the germination medium, the ungerminated spores develop very fragile germ tubes and do not grow out to form normal hyphae. less than 1 % of spores form new germination buds. when β-ca is added to germinating spores after a 20 hours period of dark incubation, a distinct inhibition of spore formation takes place. at 90 µmol β-ca less than 2 % of dark incubated spores react with new spore formation after light induction. in fig. 4 the general spore formation pattern is shown which can be reached after light induction in standard assays. fig. 2: activity of m. ulei β-glucosidase with respect to ph substrate: p-np-glucoside fig. 4: spore formation in germination tests after artificial induction of sporulation by light tab. 4: kinetic properties of β-glucosidase from m. ulei substrate km mmol/l vmax mol p-np min –1 activity per hour µmol/h nkat p-nitrophenyl-β-d-glucopyranoside 0.82 1.4 p-nitrophenyl-β-d-galactopyranoside minimal activity minimal activity < 0.006 p-nitrophenyl-α-d-glucopyranoside minimal activity minimal activity < 0.39 p-methylumbelliferyl-β-d-glucopyranoside 0.81 0.89 cellobiose minimal activity minimal activity < 0.0005 salicin minimal activity minimal activity < 0.006 linamarin 4.2 2.27 prunasin minimal activity minimal activity < 0.34 amygdalin minimal activity minimal activity < 0.02 fig. 3: influence of β-cyanoalaine on spore germination and mycelium development spores with germtubes and germination buds germinated spores spores produced after light induction mmol -ca l[ ] 100 80 60 40 20 [%] 66 r. lieberei discussion young mycelia of m. ulei reveal a high potential of cyanide resistant respiration. addition of small amounts of cyanide to mycelial suspension in culture media, enhances respiratory o2 consumption. the additional o2 consumption appears to be completely due to enhanced cyanide resistant respiration. as shown previously (lieberei et al., 1983), the growth of young mycelia of m. ulei in pure culture in the presence of hcn is higher than in cultures without added hcn. the changes in energy metabolism that are expressed by enhanced cyanide resistant respiration obviously do not impair the vegetative development of the fungus. the flexibility of fungi to use alternative pathways of energy metabolism when the cytochrome dependent respiration pathway is inhibited also is known from neurospora crassa (sturani et al., 1977) and other fungi. n. crassa develops enhanced fermentative metabolism and enhanced cyanide resistant respiration. the cyanide tolerant fungal pathogen stemphylium loti of the cyanogenic host plant lotus corniculatus also responds with high cyanide resistant respiration when the cytochrome dependent respiration is blocked by hcn (rissler and miller, 1977). the role of cyanide resistant respiration in higher plants is still under discussion and its primary role to produce atp is not proven (e.g., lambers, 1982). for microorganisms, the cyanide resistant respiratory path is assumed to be a metabolic way to form atp (palmer, 1981). the amount of atp produced per glucose in the cyanide resistant pathway is far less than in the cytochrome dependent pathway, but it is more than in fermentative processes (day et al., 1980). m. ulei is a very slow growing fungus in culture (blasquez and owen, 1957; chee, 1978) and it does not undergo sexual stages in pure culture. in the vegetative growth phase, it is cyanide insensitive, but morphogenetic changes such as sporophore formation and production of conidia are inhibited by cyanide (lieberei et al., 1983). spore formation of m. ulei is correlated with a significant rise in cyanide sensitive respiration (fig. 1), after more than 70 hrs. of incubation and with a considerable rise in dry weight. this metabolic pattern is in accordance with the general theory of turian (1977), that for the onset of morphogenetic processes in fungi, the fermentative pathway has to be diminished and cytochrome dependent energy metabolism is needed. according to this idea, the growth of m. ulei can be described in the following way: energy metabolism of germinating conidia and young mycelia is realized predominantly via fermentation and/or cyanide resistant respiration. the hyphae are produced by spending the storage compounds of the conidia. due to this fact there is no essential gain in dry weight in this developmental phase. the cyanide sensitive respiratory path is correlated with the ability of the fungus to form spores. the amount of atp produced per reduced carbon substrate rises. the amount of dry matter produced also rises. when these aspects are transferred to the infection process, the following scheme arises: during penetration and early colonization of rubber tree leaf tissue by m. ulei there is liberation of hcn: m. ulei can adapt its energy metabolism to the presence of hcn. in contrast, the early active resistance responses of the host plant are impaired by hcn (lieberei et al., 1989). after four days of colonization the fungus induces conidiophore production, again there is a hcn burst. one day later the conidiophores break through the epidermis, the amount of hcn outside the leaf tissue is low, the morphogenetic process of spore formation takes place. another very distinct biochemical adaptation of m. ulei to its cyanogenic host plant is seen in the expressed specificity of the fungal βglucosidase to the cyanogenic substrate of the host tissue. specificity of enzymes to their substrates is always tested in artificial situations under optimized assay conditions in the laboratory and, therefore, they will not always give good arguments for the discussion of functionality, but specifically this β-glucosidase has greater affinity to the host plant’s natural substrate than the host enzyme itself. furthermore, a series of tests of β-glucosidases of cyanogenic and non-cyanogenic plants to various cyanogenic and non-cyanogenic substrates showed that normally especially high substrate specificity of the plants β-glucosidase co-occurs to the special cyanogenic glucosides present in the plants under study (hösel and nahrstedt, 1975; hösel, 1981). the sensivity of m. ulei to β-ca is striking and shows how a biochemical factor such as hcn which gives rise to sensitivity of a plant to a biochemical adapted fungal pathogen, could be turned biochemically into a potential resistance factor by the plant, but for hevea no free β-ca has been reported in healthy or in infected leaf tissue (e.g. mevenkamp, 1986, unpublished). according to further plant selection on biochemical level it would be very interesting to look for hevea plants revealing low levels of cyanogenic capacity (lieberei, 1988) and to look for plants with a potential for developing transient pools of β-ca in the course of pathogen attack. biochemical features like these may help to design directed molecular markers and may allow the design of biological approaches to develop rubber tree material tolerant to the most devastating rubber tree disease of the new world. new selection approaches on molecular level should use the knowledge of resistance physiology in that way, that markers for cyanogenic features and the detoxification processes should be included in the modern dna-chip technologies for breeding and selection. acknowledgements this project was financially supported by the deutsche forschungsgemeinschaft (grant li 238/2-1 to 2-4) in combination with the gtz. substantial support of the federal brazilian agricultural research institution embrapa was also given. the author wishes to express the appreciation to dr. luadir gasparotto and dr. vincente h.f. moraes for fruitful scientific discussions and to karin puttfarken, volker nölting and ricardo pessoa rebello for technical help. references blasquez, c.h., owen, j.h., 1957: physiological studies of dothidella ulei. phytopathology 47, 727-732. castric, p.a., farnden, k.j.f., conn, e.e., 1972: the formation of asparagine from β-cyanoalanine. arch. biochem. biophys. 152, 62-69. chee, k.h., 1976: assessing susceptibility of hevea clones to microcyclus ulei. ann. appl. biol. 84, 135-145. chee, k.h., 1978: south american leaf blight of hevea brasiliensis: culture of microyclus ulei. trans. br.mycol. soc. 70, 341-344. chee, k.h., wastie, r.l., 1980: the status and future prospects of rubber diseases in tropical america. rev. plant pathol. 59, 541-548. day, d.a., arron, g.p., laties, g.g., 1980: nature and control of respiratory pathway in plants: the interaction of cyanide-resistant with the cyanide sensitive pathway. in: stumpf, p.k., conn, e.e. (eds.), the biochemistry of plants – a comprehensive treatise, vol. 2, 197-241. acad. press new york, london. hagen, j., gasparotto, l., moraes, v.h.f., lieberei, r., 2003: reaction of cassava leaves to microcyclus ulei, causal agent of south american leaf blight of rubber tree. fitopatol. bras. 28, 477-480. hösel, w., 1981: glycosylation and glycosidases. in: stumpf, p.k., conn, e.e. (eds.), the biochemistry of plants a comprehensive treatise, physiological characteristics of microcyclus ulei 67 vol. 7, 725-753. acad. press new york, london. hösel, w., nahrstedt, a., 1975: spezifische glucosidase für das cyanoglucosid triglochinin. hoppe seyler’s z. physiol. chemie 356, 12651275. junqueira, n.t.v., chaves, g.m., zambolim, l., gasparotto, l., alfenas, a.c., 1986: variabilidade fisiologica de microcyclus ulei. fitopatol. bras. 11, 823-833. junqueira, n.t.v., chaves, g.m., zambolim, l., alfenas, a.c., gasparotto, l., 1988: reação de clones de seringuera a vários isolados de m. ulei. pesquisa agropecuaria brasileira 23, 877-893. körner, r., 1994: resistenzkomponenten von kautschukbäumen (hevea spp.) gegen blattschadpilze. ph.d. thesis, fachbereich biologie, university of hamburg. lambers, h., 1982: cyanide resistant respiration: a non phosphorylating electron transport pathway acting as an energy overflow. physiol. plant. 55, 478-485. le guen, v., rodier-goud, m., troispoux, v., xiong, t.-c., brottier, p., billot, c., sequin, m., 2004: characterization of polymorphic microsatellite markers for microcyclus ulei, causal agent of south american leaf blight of rubber trees. molecular ecology notes 4, 122-124. lieberei, r., 1984: cyanogenese und resistenz – physiologische studien zur wirt-pathogen beziehung bei cyanogenen pflanzen, durchgeführt am wirt-pathogen system hevea brasiliensis – microcyclus ulei. habilitationsschrift an der technischen universität braunschweig. lieberei, r., 1986: cyanogenesis during infection of hevea brasiliensis with microcyclus ulei. j. phytopathology 115, 134-146. lieberei, r., 1988: relationship of cyanogenenic capacity (hcn-c) of the rubber tree hevea brasiliensis to susceptibility to microcyclus ulei, the agent causing south american leaf blight. j. phytopathology 122, 5467. lieberei, r., schrader, a., biehl, b., chee, k.h., 1983: effect of cyanide on microcyclus ulei cultures. j. rubber res. inst. malaysia 31, 227-235. lieberei, r., biehl, b., giesemann, a., junqueira, n.t.v., 1989: cyanogenesis inhibits active defense reactions in plants. plant physiology 90, 33-36. lieberei, r., fock, h.p., biehl, b., 1996: cyanogenesis inhibits active pathogen defence in plants: inhibition by gaseous hcn of photosynthethic co2 fixation and respiration in intact leaves. j. appl. bot. 70, 230-238. mattos, c.r.r., garcia, d., pinard, f., le guen, v., 2003: variabilidade de isolados de microcyclus ulei no sudeste da bahia. fitopatol. bras. 28, 502-507. mevenkamp, g., 1986: charakterisierung und intrazelluläre lokalisierung der β-cyanoalaninsythase in blättern von hevea brasiliensis (1985/1986). diploma thesis, faculty of natural sciences, tech. university braunschweig. mc mahon, j.m., arteca, r.n., 2000: molecular control of ethylene production by cyanide in arabidopsis thaliana. physiologia plantarum 109, 180-187. miller, j., conn, e.e., 1980: metabolism of hydrogen cyanide by higher plants. plant physiol. 65, 1199-1202. palmer, j.m., 1981: cyanide-resistant respiration of eukaryotic cells. in: vennesland, b., conn, e.e., knowles, c.j., westkey, h., wissing, f. (eds.), cyanide in biology, 437-449. acad. press london. rissler, j.f., millar, r.l., 1977: contribution of a cyanide insensitive alternative respiratory system to increases in formamide hydrolase activity and to growth in stemphylium loti. plant physiol. 60, 857-861. rivano, f., 1977: la maladie sud-américaine des feuilles de l’hévéa. i. variabilité du pouvoir pathogène de microcyclus ulei. plantations recherche développement 4, 104-110. schönbaum, g.r., bonner, w.d. jr., storey, b.t., bahr, j.t., 1971: specific inhibition of the cyanide insensitive pathway in plant mitiochondria by hydroxamic acids. plant physiol. 47, 124-128. schultes, r.e., 1970: the history of taxonomic studies in hevea. bot. rev. 36, 197-276. selmar, d., lieberei, r., biehl, b., voigt, j., 1987: hevea linamarase – a non specific β-glycosidase. plant physiol. 83, 557-563. sturani, e., chimenti, r., trezzi, f., martegan, e., 1977: effects of chloramphenicol on some aspects of growth in neurospora crassa. j. submicrosc. cytol. 9, 83-95. turian, g., 1977: fungal differentiation. in: meyrath, j. bu’lock, j.d. (eds.), biotechnology and fungal differentiation, fems-symposium 4. acad. press, london, new york, toronto, sydney, san francisco. address of the author: r. lieberei, university of hamburg, biocenter klein flottbek, institute for applied botany, ohnhorststrasse 18, d-22609 hamburg 68 r. lieberei << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 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<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> >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice journal of applied botany and food quality 86, 47 54 (2013), doi:10.5073/jabfq.2013.086.007 institute of botany, gottfried wilhelm leibniz university hannover, germany comparative analysis of methods analyzing effects of drought on the herbaceous plant lablab purpureus s. guretzki, j. papenbrock (received march 1, 2013) summary due to the changing climatic conditions, there is an enlargement of land areas with insufficient rainfall and therefore a reduction in the cultivated area for common crops. hence, it is now important to find plants that are adapted to these drought conditions. the focus of our research was to apply and compare different methods to quantify the impact of drought stress on plants. lablab purpureus is considered to be drought tolerant. therefore, we used l. purpureus genotypes from three continents cpi 36903 (europe), cpi 52508 (africa) and ha-4 (asia) as examples for our study. all genotypes were screened for their tolerance to drought stress by various methods to obtain quantitative data on the drought stress tolerance of individual genotypes and to find out which methods are especially suitable for the measurement of drought tolerance. classical methods such as leaf size, plant height, biomass, and plant water content were investigated. in addition, by chlorophyll fluorescence measurement effects of drought on the photosynthetic system were examined. infrared thermography was used in order to make the changes in leaf temperature in plants stressed by drought compared to unstressed plants visible. the methods were complemented by the measurement of leaf conductivity. results indicate a difference in the usability of the methods for the determination of drought stress. finally, a set of methods is assembled based on suitability for drought tolerance analysis in plants. the methods include classical growth parameters, including dry weight biomass, plant water content (pwc) and leaf size determination, as well as height measurements of the plants. the stomata behavior is analyzed by leaf conductivity and infrared thermography, both methods complete the set for drought tolerance identification. based on the results of these methods a ranking of the examined genotypes with respect to their drought tolerance is created. introduction because of global warming the climate conditions in e.g. africa, south asia and east asia will become more arid (dai, 2011). however, the precipitation amount is a key factor for the productivity of crops (rosenzweig et al., 2001). common crops have to deal with these new environmental conditions. the detection of new crop plants or genotypes of crop plants that are particularly adapted to more arid conditions is therefore a major objective to preserve the livelihood of the local populations. lablab purpureus (l.) sweet (synonyms: dolichos purpureus, dolichos lablab (ncbi-taxonomy)), the experimental plant in this study, is referred to as drought tolerant in maass et al. (2010). l. purpureus belongs to the eudicots, core eudicots, fabids order fabales, family fabaceae (the angiosperm phylogeny group (apgiii), 2009). the herbaceous plant is perennial but is often grown as an annual plant. l. purpureus occur as bushy, semi-erect and prostate growth habit types. the stem is twining, the leaves are alternate and trifoliate. flowers exist in different colors (white, pink, red, purple). pods and seeds vary in color and size. various parts of the plant are used as food (flowers, leaves, pods, root tubers, seeds) or fodder (www.lablab.org, university of agricultural sciences, bangalore). l. purpureus is cultivated as a component of mixed cropping schemes or home gardens, wherein the plant is known especially in africa, south asia and south east asia (maass et al., 2010). studies showed that this species is adapted to drought, but there are differences in terms of drought tolerance within the species as summarized in maass et al. (2010). however, the differences in drought tolerance have not been quantified for this species so far. also for other herbaceous species studies on the quantitative analysis to measure the impact of drought stress on plants are rare. there are the more traditional parameters measured, including the determination of the fresh weight and dry weight biomass, plant water content, as well as development of plant height and leaf size to investigate the effects of stress on plants. in addition, there are new non-destructive methods which allow a rapid screening of plants. this includes the determination of leaf chlorophyll fluorescence by pam-imaging in order to get information about the state of the photosynthetic system under stress conditions (woo et al., 2008; sperdouli and moustakas, 2012). the behavior of stomatal conductivity can be observed over a large area by infrared thermography, a further rapid screening method (grant et al., 2006). this makes the method interesting for phenotype screening and breeding programs (chaves et al., 2003). another option for stomatal conductivity measurements is the use of a porometer (jones et al., 2002; grant et al., 2006). the combination of infrared thermography and chlorophyll fluorescence for the observation of changes in transpiration rate and photosynthesis can be used for detection of early plant stress and stress tolerance screening (chaerle et al., 2007, 2009). munns et al. (2010) concluded that stomatal conductance is a growth rate indicator for plants under water stress and thermography is a good screening method. together it is possible to find the best genotypes for different growth conditions. berger et al. (2010) points out that chlorophyll fluorescence is helpful for the detection of servere drought stress. for early drought stress detection chlorophyll fluorescence seems only useful in combination with other methods to gain a more comprehensive picture of the plant stress response. the objective of this study is to find methods that are suitable for recognizing the impact of drought stress on plants and methods that are appropriate in a combined way for a reliable detection of drought-tolerant genotypes in the case of l. purpureus as an example for an herbaceous species. more precisely, to compile methods those allow the drought stress detection before the leaves wilt. the drought tolerance results correlated with the results of genetic analyses can be used to find the best genotypes for field experiments within future breeding programs. material and methods plant material, growth conditions and drought treatment seeds of lablab purpureus (l.) sweet were originally obtained from dr. b.l. maass, international centre for tropical agriculture (ciat), nairobi, kenya (cpi 36903, cpi 52508) and from dr. m.b. gowda, 48 s. guretzki, j. papenbrock university of agricultural sciences, bangalore, india (ha-4). cpi 36903 (southern ukraine, europe) and cpi 52508 (mozambique, africa) are semi-domesticated genotypes (maass and usongo, 2007). the origin of ha-4 is karnataka, southern india, asia. for germination, seeds were soaked in water at room temperature overnight and transferred on type cl t soil (einheitserde, sinntalaltengronau, germany) in pots of 12 cm diameter the following day. the soil contains 30% clay. plants were grown in the greenhouse in a 12 h light/dark rhythm at a temperature of 22°c/22°c. when the outdoor light conditions did not ensure sufficient light intensity inside the greenhouse, additional light was switched on to obtain a constant quantum fluence rate of approx. 350 µmol m-2 s-1 (sodium vapor lamps, son-t agro 400, philips, amsterdam, netherlands). plants were grown in the greenhouse for five weeks under wellwatered conditions. the drought experiments started in week six under the same greenhouse conditions. on day one, the plants were watered for the last time before the plants were set to the experimental conditions. experimental groups differ in soil volumetric water content (vwc); the control group (35% vwc) and the drought group (20% vwc) were used to reach a moisture content level near field capacity and near the permanent wilting point of the soil, respectively, in accordance to the manual of the used device. for the measurement of vwc time domain reflectometry (tdr) (fieldscout, spectrum technologies, plainfield, usa) was used. for daily irrigation, water was added based on water deficit calculation (d) of the fieldscout, 1 mm = approx. 7.8 ml (experiment (exp.) 1) and approx. 8.0 ml (exp. 2) for a pot with approx. 710 cm3 (exp. 1) and approx. 750 cm3 (exp. 2) volume according to the formula of truncated cones. the water contained 0.25% wuxal top n fertilizer (aglukon, düsseldorf, germany). five plants per treatment were grown for every genotype with a distance of approx. 7.5 cm (exp. 1) and approx. 35 cm (exp. 2) between the pots. growth parameters the growth parameters plant height, leaf size, biomass and plant water content (pwc) were determined. the plant height was measured with a folding yardstick; thereby the length of the longest shoot was used. the leaf of l. purpureus is divided into three leaflets. instead of the leaf area, the approximate leaf size was calculated measuring the length of the paired leaflets and the terminal leaflet plus petiole (between paired leaflets and terminal leaflet). afterwards, both values were multiplied to obtain the leaf size. the fresh and dry weight for biomass and pwc were determined by harvesting above-ground plant material, which was then dried in an incubator at approx. 110°c for 48 h. plant material was weighed before drying for fresh weight (fw) and after drying for dry weight (dw). for biomass data dw was used. pwc was calculated using the formula pwc = (fw dw)/ fw * 100. measurements for quantification of the drought stress effects the effect of drought stress was examined by porometry, thermal imaging measurements and pam-imaging. porometry and thermal imaging measurements were done on attached leaves as nondestructive methods. stomatal conductance (mmol m-2 s-1) of leaves was determined by the use of the porometer ap4 (delta-t devices, burwell, uk) in the morning. the measurements were performed on either of the two youngest fully expanded leaves, wherein only the higher value was used for further calculations (exp. 2) or one leaf was measured over the entire experimental period (exp. 1). thermal imaging investigation was carried out with the camera t360 (flir systems, wilsonville, usa) in accordance with grant et al. (2006) in the afternoon. to ensure consistent measurements, the camera was turned on at least 30 min before taking the first picture. the youngest fully expanded leaves were examined. one leaflet of the paired leaflets of a leaf was used as dry (tdry) the other one as wet (twet) reference. dry reference leaflets were covered with petroleum jelly and wet reference leaflets were wetted with water on both sides. the terminal leaflet of the same leaf was used as sample (tleaf). tdry was measured at least five min after the application of petroleum jelly, twet immediately after using the water. in addition to tleaf a stomatal conductance measurement was performed on the terminal leaflet. the thermal imaging pictures were analyzed using the software flir quickreport 1.2 sp2 (flir systems, wilsonville, usa). the object parameters were set for each image to emissivity 0.95, reflected apparent temperature 23°c, atmospheric temperature 23°c, relative humidity 45%/50% and distance 0.2 m. based on the results the index ig was calculated using the formula ig = (tdry tleaf)/ (tleaf twet). additionally the crop water stress index (cwsi) was calculated from cswi = (tdry tleaf)/ (tdry twet). whether there is an influence of drought stress on photosynthesis was investigated by chlorophyll fluorescence using an imaging pam m series device and imagingwin v2.32 software (heinz walz, effeltrich, germany). the measurements were performed either on a young fully expanded leaf (using cut off leaves; exp. 2) or one leaf was measured over the entire experimental period (using attached leaves; exp. 1) in the morning. for analysis of the photosynthetic system light curves were analyzed as presented by the manufacturer. through the use of the filter plate imag-max/f the effective par values are about 15% lower. before the measurement, the plants were dark adapted for 20 min. the parameters fv/fm (maximal ps ii quantum yield), y(ii) (effective ps ii quantum yield), y(npq) (quantum yield of regulated energy dissipation), y(no) (quantum yield of non-regulated energy dissipation), npq/4 (nonphotochemical quenching/4) and etr (electron transport rate) were analyzed (for background information: baker, 2008; sperdouli and moustakas, 2012). fv/fm values were obtained from the falsecolor images created by imagingwin software. etr values were determined using a mean value of par 396-801 μmol quanta m-2 s-1. the other parameters were analyzed based on the par 396 (approx. growth light intensity) and 801 (approx. twice the growth light intensity) results. statistical analysis all statistical analyzes were conducted with r 2.15.2 (www.rproject.org) in combination with r studio v0.97.248 (rstudio, boston, usa). box plots were drawn using ggplot2 version 0.9.3 (wickham, 2009). significant differences (p <0.05) were determined by the welch t-test analysis. results development of classical growth parameters under drought stress the classic growth parameters, gain in biomass dw, plant water content, leaf size (exp. 1) and plant height growth (exp. 2) were analyzed (fig. 1, 2). drought stress results in a decrease of biomass dw by slower growth in stressed plants (fig. 1a, 2a). in experiment set up 1 ha-4 was the least affected genotype among the groups. the strongest decrease in exp. 2 was found for genotype cpi 52508 (cg: 5.9 g; dg: 4.7 g), ha-4 showed no impact of drought stress (cg: 4.3 g; dg: 4.3 g). significant differences (p<0.05) between stressed and unstressed plants for biomass dry weight measurements were found for cpi 36903 and cpi 52508. there is a reduction in plant water content under drought stress conditions, too (fig. 1b, 2b). significant differences (p<0.05) among the groups occurred only in genotype cpi 52508 exp. 2 (cg: 79.7%; dg: 78.3%). generally, there drought tolerance screening 49 were only small changes in the drought stress plants in comparison to unstressed plants. there was also a considerable decrease in leaf size and plant growth, in the case of drought stressed plants. the strongest difference in leaf sizes between both groups (fig. 1d) was found in cpi 52508 (cg: 253 cm2; dg: 118 cm2), ha-4 showed a smaller difference (cg: 267 cm2; dg: 157 cm2). the reduction of leaf size under drought stress is stronger in younger leaves (fig. 1d) compared to older leaves (fig. 1c). the decrease was in younger leaves between 21% (ha-4) and 39% (cpi 52508) and in older leaves between 41% (ha-4) and 53% (cpi 52508). the height growth of the plant was affected particularly in cpi 36903 (cg: 47%; dg: 31%). the slightest effect was found in ha-4 (cg: 31%; dg: 23%). however, it must be considered that l. purpureus is a twining plant and thereby the measurement of plant height to obtain the growth rate was impaired. in general, a large mean variation especially of the controls can be observed although the seed material was homogenous and also the plantlets had a homogenous phenotype. the conditions in the greenhouse might have local maxima and minima resulting in a large mean deviation, based on single measuring points collected from five different plants. determination of leaf conductance the leaf conductivity was measured in the morning by porometry (fig. 3). drought stress led to a reduction in leaf conductance. in summary, the lowest difference between control and drought group showed cpi 52508 (exp. 2 cg: 220 mmol m-2 s-1; dg: 141 mmol m-2 s-1 (fig. 3b)) in both experiments. ha-4 was most affected by drought stress (exp. 2 cg: 212 mmol m-2 s-1; dg: 84 mmol m-2 s-1 (fig. 3b)). cpi 36903 behaved uneven; in exp. 1 the differences among the groups are similar to ha-4 and with larger space available per plant similar to cpi 52508 in exp. 2. significant differences (p<0.05) among drought stressed and unstressed plants were found for all genotypes. analysis of the impact of drought stress on l. purpureus by infrared thermography the influence of drought stress on surface temperature of plant leaves was analyzed by using an infrared thermography camera (fig. 4). drought stress leads to an increase in leaf temperature by closed stomata (fig. 4a). only ha-4 (cg: 30°c; dg: 28.8°c) showed a significant difference (p<0.05) between drought stressed and unstressed plants. cpi 36903 was stronger affected by drought stress (cg: 26.2°c; dg: 24.6°c). cwsi and ig values drop under drought stress, wherein the results of both indices were homologous (fig. 4b and 4d). the biggest differences between the two groups were measured in ha-4, followed by genotypes cpi 36903 and cpi 52508. the cwsi decrease for ha-4 (cg: 0.39; dg: 0.05) was 88% and in comparison only 28% for cpi 52508 (cg: 0.28; dg: 0.20). there were no significant differences (p<0.05) between the groups. leaf conductance measured during the same time to substantiate the results of the infrared thermography camera showed lower values under drought stress (fig. 4c). drought stress led to a large decrease for cpi 36903 (cg: 158 mmol m-2 s-1; dg: 44.5 mmol m-2 s-1), the decreases for ha-4 and cpi 52508 were also above 50%. the leaf conductance confirms again that cpi 52508 was least affected by drought stress. measurement of various chlorophyll fluorescence factors during drought stress chlorophyll fluorescence was examined by factors etr, fv/fm, npq/4, y(ii), y(npq) and y(no) through a light curve (fig. 5, 6). fig. 1: effects of water limitation after 15 days on (a) biomass dry weight (g), (b) plant water content (%) and after eight days on (c) size older leaf (cm2) and (d) size younger leaf (cm2); n=10, genotypes with * = p<0.05, exp. 1. 50 s. guretzki, j. papenbrock fig. 2: effects of water limitation after nine days on (a) biomass dry weight (g) (n=5), (b) plant water content (%) (n=5) and (c) plant height growth (%) (n=4-5); genotypes with * = p<0.05, exp. 2. fig. 3: effects of water limitation after eight days on stomatal conduc tance through porometry in mmol m-2 s-1 (a) exp. 1 (n=10) and (b) exp. 2 (n=5); genotypes with * = p<0.05. fv/fm showed no notable differences between control and drought groups for all genotypes (fig. 5b, 6b), except a drought group increase for cpi 36903 in exp. 2 (cg: 0739; dg: 0788). stress leads to fv/fm reduction. drought stress caused also a decrease in rate of electron transport (fig. 5a, 6a). etr decreased under drought stress especially in ha-4 (exp. 1 cg: 60.28; dg: 44.37; exp. 2 cg: 63.75; dg 61.82), an increase under drought stress was found for cpi 36903 (exp. 1 cg: 49.03; dg: 57.44)17%; exp. 2 cg: 53.18; dg: 54.88). factor npq/4 increased under stress conditions (fig. 5c, 6c). also that was particularly apparent for ha-4 (exp. 1 cg: 0.345; dg: 0.453; exp. 2 cg: 0.341; dg 0.384). drought stress indicates a decrease in y(ii) and thereby a change in y(npq) and y(no) (fig. 5d, 6d). ha-4 was again most negatively affected by drought stress. generally, there were only significant differences in cpi 36903. based on the results cpi 36903 tends to be classified between the other two genotypes with respect to drought tolerance. ha-4 has a lower tolerance to drought. overall, the results of chlorophyll fluorescence were very inconsistent and provide only evidences for drought tolerant genotypes. result summary of the different drought stress measurement techniques tab. 1 summarizes the results of the stress methods used in this study. a strong negative influence of drought stress on the genotype of a species is represented by a low score. a high score indicates that the influence of drought stress on the genotype is low. genotypes with high scores are therefore more drought tolerant in comparison to the others. ha-4 (values 6 in the scale) appears to be the least drought tolerant genotype, cpi 36903 (8) is slightly more drought tolerant. cpi 52508 (10) is the most drought tolerant genotype of the three genotypes used in this study. it is noticeable that ha-4 is the most tolerant genotype, if the classical growth parameters are used. in the other methods ha-4 is the least adapted genotype. the results for cpi 52508 are exactly the opposite. discussion the effectiveness of the determination of traditional growth parameters relating to drought tolerance investigations the determination of classical growth parameters, e.g. leaf area measurements, is helpful in the screening of drought tolerance (jones, 2007). the impact of drought leads to a reduced growth of plants. the limited availability of water leads to reduced turgor and restrictions in mitosis. this results in a lower rate of cell division and elongation, and therefore in reduced growth (farooq et al., drought tolerance screening 51 fig. 4: effects of water limitation after eight days on (a) leaf temperature (°c), (b) index ig, (c) stomatal conductance (mmol m-2 s-1) and (d) crop water stress index (cwsi) based on infrared thermography analysis; n=5, genotypes with * = p<0.05, exp. 2. fig. 5: effects of water limitation after eight days on (a) etr, (b) fv/ fm and under ll conditions through chlorophyll fluorescence measurements (c) npq/4, (d) y(ii) y(no) y(npq); n=6, genotypes with * = p<0.05, exp. 1. 52 s. guretzki, j. papenbrock 2009). biomass, pwc, leaf size and plant height decreased under drought conditions in l. purpureus. especially genotypes cpi 36903 and cpi 52508 indicated significant differences between the control groups and drought treatment groups. leaf size and plant height measurements combine the advantages of being non-destructive methods; biomass and pwc determination belong to the destructive methods. reduction in leaf dry weight and stem dry weight, plant height and leaf area was already observed in faba bean (vicia faba l.) under drought stress conditions (zabawi and dennett, 2010). a study on the effects of drought stress at different growth stages of the mung bean (vigna radiata l.) also showed a reduction in plant height. drought stress affected the measured plant height in different magnitude depending on the growth stage (ranawake et al., 2011). a comparison of two common bean (phaseolus vulgaris l.) varieties, one heavily influenced and the other one less affected by drought stress in yield, was done. a stronger decrease in relative water content and relative growth rate was shown in the variety that was more influenced by drought stress (lizana et al., 2006). in conclusion, the results suggest that traditional growth factors can help to find drought tolerant genotypes in l. purpureus. but some of the techniques belong to the destructive methods. furthermore results of the classical growth parameters reflect the impact of drought stress on the plant as a whole. it is therefore useful to study further non-destructive and non-invasive methods according to their suitability to detect drought tolerance and indicate specifically the impact of drought on the plants. for this we tested the turgor pressure probes for non-invasive online-monitoring of the water relations of intact leaves zimmermann et al. (2008). however, problems arose in the operation of this system in terms of l. purpureus: the leaves were injured because they are too thin and grow very fast. with respect to genotype screening a lot of probes are required or the probes need to be repositioned frequently. this is in contrast to the aim that methods should allow a fast high-throughput screening of genotypes. infrared thermography as a valuable addition to the measurement of stomatal conductance stomatal conductance and infrared thermography are techniques that can be useful in an analysis of drought tolerances (jones, 2007). fig. 6: effects of water limitation after nine days on (a) etr, (b) fv/ fm and under ll conditions through chlorophyll fluorescence analysis (c) npq/4, (d) y(ii) y(no) y(npq); n=3, genotypes with * = p<0.05, exp. 2. tab. 1: result summary of used stress methods. in this case each genotype is assigned to a value of one to three. three corresponds to a low negative influence and one represents a strong negative influence of drought for the pants. genotypes with similar values get the same rating. genotype growth parameters leaf conductance infrared chlorophyll sum thermography fluorescence ha-4 3 1 1 1 6 cpi 36903 2 2 1 3 8 cpi 52508 1 3 3 3 10 drought tolerance screening 53 under drought stress conditions stomata closure leads to a reduction of water loss for the plants. conductivity and leaf temperature measurements allow the observation of stomata behavior. both are non-destructive methods. stomata conductivity measurements with a porometer have the disadvantage that the measurement is possible only punctually on a leaf. this results in a large mean variance of the data (fig. 3). in contrast, it is possible to investigate the stomata behavior and thereby temperature changes of a leaf or even of complete plants with infrared thermography. the differences in stomatal conductance between drought and control groups in l. purpureus are significant. the differences in tleaf measurements were significant, the thermal indices ig and cwsi showed nonsignificant differences between stressed and unstressed plants in l. pupureus. a study by grant et al. (2006) proves that the results of stomatal conductance measurements using a porometer correlate with the results of infrared thermography indices. for this grapevines (vitis vinifera l.), french beans (p. vulgaris) and lupins (lupinus albus l.) were examined. for p. vulgaris differences between well watered plants and drought stress plants were found for tleaf and the thermal indices ig and cwsi. it is suggested that measurements of tleaf are probably sufficient for the comparison of different genotypes. for the calculations of the thermal indices ig and cwsi measurements of the minimum temperature for a leaf (in this study twet), and the maximum achievable leaf temperature (in this study tdry) is required at the same time as tleaf measurements. the consequence is that the thermal indices are less susceptible to fluctuations in ambient conditions over a specific time period (idso et al., 1981; jones, 1999). otherwise, any changes in surface temperature may originate from changing environmental conditions. thus, the thermal indices ig and cwsi should be used to observe the stomatal behavior in long time experiments (grant et al., 2006). in conclusion, measurements of stomatal conductance in combination with tleaf are the best for the identification of drought tolerant genotypes. the suitability of chlorophyll fluorescence in drought tolerance screenings under mild to moderate drought stress, the closing of the stomata is the main reason for changes in photosynthesis as summarized by medrano et al. (2002). the analysis of chlorophyll fluorescence in both experiments led to no meaningful results. the measurements of fv/fm, etr, npq/4, y(ii), y(no) and y(npq) showed almost no significant differences. some of the drought stressed groups showed even better values in comparison to the control groups of the same genotypes. for example, fv/fm is considered as fast-measuring factor in the case of stress for plants. for non-stressed c3 plants values of about 0.83 (björkman and demmig, 1987) are expected. these approx. values were obtained with one exception in the analysis of the two experiments done here by both control groups and drought groups of the genotypes. chlorophyll fluorescence does not appear to be sensitive enough to detect early symptoms of drought stress, at least in l. purpureus. this assumption is supported by a study with arabidopsis thaliana (l.) heynh. plants. here, drought stress was initiated by complete withheld of water. a change in the measured values of fv/fm, npq and y(ii) occurred only after long-term (more than ten days) drought stress. etr and y(no) measurements behaved similarly, at first there was no impact of stress and then both factors reflected strong signs of stress (woo et al., 2008). only a slightly decrease of fv/fm was found for p. vulgaris seven days after stopping irrigation (miyashita et al., 2005). another study with a. thaliana compared the behavior of the photosynthetic system under mild, moderate and severe drought stress. in this experiment, water was withheld until the soil water content reached 66-68% for mild drought stress, 50-52% for moderate drought stress and 43-45% for severe drought stress in comparison to the soil water content of the control group. severe drought stress caused the strongest changes in comparison to the control group. but mild drought stress led in comparison to moderate drought stress to larger photosynthesis modifications. it was concluded that the response of the plant matches with the “threshold for tolerance model” (sperdouli and moustakas, 2012). according to this model, tolerance mechanisms are started with lag time or induced by threshold concentrations (barceló and poschenrieder, 2002). moderate stress caused less damage to the plant, because stress adaptation processes and repair mechanisms started in the plant whereas during mild drought conditions the stress threshold was not reached. therefore, the plants were more affected under mild drought stress reflected by stronger altered chlorophyll fluorescence values in comparison to the moderate group (lichtenthaler, 1998; sperdouli and moustakas, 2012). the results of our study showed only small differences in etr, fv/fm, npq/4, y(ii), y(no) and y(npq) between the control and drought stress groups indicating a moderate stress level of the plant. important for the characterization of drought tolerance in different genotypes is therefore the correct strength of drought stress, then chlorophyll fluorescence measurements might be used efficiently. new measurement protocols could provide more reliable data in early symptoms of drought stress on the photosynthetic system. burke et al. (2010) measured fv/fm at two time points by harvesting leaf punches. the chlorophyll fluorescence measurement thereby loses the advantage of being a non-destructive method. in summary, measurements of chlorophyll fluorescence in the case of l. purpureus are not advised without reservation. this is demonstrated by the measurement results: the differences between the unstressed and stressed plants are usually too low in order to make statements about the drought tolerance of the tested genotypes. conclusion for the screening of drought-tolerant genotypes under moderate drought stress traditional methods such as leaf size measurements and biomass determination as well as new techniques like infrared thermography are suitable. chlorophyll fluorescence is only appropriate to examine the impact of severe drought stress conditions or in recovery experiments. because l. purpureus is a twining plant, the measurement of the more traditional growth parameter plant height is difficult. overall, the combination of several methods is recommended. based on the results a combination of infrared thermography and porometer measurements in conjunction with traditional growth parameter like biomass investigations is advisable for l. purpureus under greenhouse conditions. these methods are also suitable for other species because measurements are easy and quick to handle. thereby the different effects of drought stress on the plant can be analyzed in order to filter out drought tolerant genotypes from a selection of genotypes. it must be considered that the selected genotypes of these greenhouse experiments have to be tested under field conditions. finally, the yield of the required plant product of the selected genotypes in field conditions is most important for the growers. acknowledgements l. purpureus seeds were kindly provided by dr. b.l. maass, international centre for tropical agriculture (ciat) nairobi, kenya, and by dr. m.b. gowda, university of agricultural sciences, 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procedure for quantitative assessment of drought survival using chlorophyll fluorescence. plant meth. 4, 27. zabawi, a.g.m., dennett, m.d.d., 2010: responses of faba bean (vicia faba) to different levels of plant available water: i. phenology, growth and biomass partitioning. j. trop. agr. food sci. 38, 11-19. zimmermann, d., reuss, r., westhoff, m., gessner, p., bauer, w., bamberg, e., bentrup, f-w., zimmermann, u., 2008: a novel, noninvasive, online-monitoring, versatile and easy plant-based probe for measuring leaf water status. j. exp. bot. 59, 3157-3167. address of the authors: sebastian guretzki, jutta papenbrock (corresponding author), institute of botany, leibniz university hannover, herrenhäuserstr. 2, 30419 hannover, germany. e-mail: jutta.papenbrock@botanik.uni-hannover.de vorgabe neu journal of applied botany and food quality 81, 36 40 (2007) 1) norwegian university of life sciences, department of plant and environmental sciences, ås 2) norwegian institute for agricultural and environmental research, arable crops division, section kise, nes fruit quality in strawberry (fragaria x ananassa duch. cv. korona) at three times during the season and with two fertilizer strategies anne-berit wold1), nina opstad2) (received february 12, 2007) summary effects on fruit quality of preplant fertilization and fertigation in strawberry were studied in a field experiment established august 2003 in south east norway. half the field was fertilized at planting and fertigated with a low nutrient rate from spring 2004 (t1), while the second half was unfertilized at planting but fertigated with a relatively high nutrient rate from spring 2004 (t2). berries were harvested three times per week during the first harvesting year. samples of berries from early season, mid season and late season were analysed for soluble solids, ph, titratable acidity, colour, vitamin c, antioxidant activity (frap) and mineral concentration. the results were correlated to leaf mineral concentration in dry matter. t1 gave larger and darker berries compared to t2. the other quality parameters and mineral content in strawberry fruits were not significantly influenced by the two fertilizer strategies. soluble solids, ph, titratable acidity and colour varied significantly through the season, while vitamin c and antioxidant activity (frap) was unaffected by fertilizer treatment, berry size and time of season. the results gave a general view of the quality parameters in the strawberry cultivar ‘korona’. introduction a number of preharvest factors have been found to influence on postharvest quality of berry crops, including genetics, climate, water management and other cultural practices (prange and deell, 1997). genotype present the main variation in fruit composition, and the content of flavonoids and phenolic compounds are found to vary significantly between different berry species, with certain similarities within family and genera (häkkinen et al., 1999). strawberries contain high levels of antioxidant compounds (halvorsen et al., 2002), with a considerable variation between different cultivars (davik et al., 2006). both kallio et al. (2000) and davik et al. (2006) found differences between cultivar in the sugar and acid composition in strawberry. variation in climate at different geographical sites and between years was also found to influence on the quality factors in these experiments. cultural practices such as ground cover and mulching affect fruit quality as well (neuweiler et al., 2003; moor et al., 2005; anttonen et al., 2006), and mulch type was suggested to affect fruit quality through changes in the soil moisture (neuweiler et al., 2003). the results from hansen (1995) and kirnak et al. (2003) also indicated that water stress reduced fruit size and increased soluble dry matter. application of fertilizer has a great impact on various yield and fruit quality parameters in strawberry, and the major point of interest has been berry size, saleable yields and the content of soluble solids. as it has become well known that a diet rich in fruits and vegetables reduce the risk of developing cardiovascular diseases, cancer and a number of chronic diseases, fertilizer effects on the antioxidant content in assorted horticultural crops has also been reported (jeppson, 2000; lee and kader, 2000; anttonen et al., 2006). strawberries contain high levels of antioxidant compounds (halvorsen et al., 2002), and although genotype has the greatest impact on antioxidant content in berries, cultivation methods that may improve the quality and health benefits of strawberries for fresh consumption is of current interest. the aim of the present study was to establish possible effects from time of fertilizer application on selected fruit quality parameters in the strawberry cultivar ‘korona’. in addition to the fertilizer effects, the experiment gave a good opportunity to show quality parameters in fruits of the strawberry cultivar ‘korona’ at three harvesting times. absolute and relative fruit growth rate varies according to rank order in the cyme (forney and breen, 1985), and effects on fruit quality of harvest dates are documented (sjulin and robbins, 1987; anttonen et al., 2006). ripe berries from early season, mid season and late season were therefore analysed in the present trial. material and methods the trial was conducted from 2003 to 2004 at apelsvoll research center division kise, norway (60o40’n, 10o11’e), on a morainic loam soil. the field was planted on august 22nd 2003 using certified rooted plug plants. plants of the cultivar ‘korona’ were planted in double rows, on low ridges covered with black polyethylene mulch. plant spacing was 25 cm within the row, 40 cm between the double rows and 160 cm between ridges. the whole field was equipped with a pressure compensated drip irrigation system connected to a fertilizer injector. the tubes were placed under the polyethylene mulch in the centre of the ridges. before planting, a compound fertilizer equivalent to 500 kg ha-1 of 6-5-20 npk with micronutrients (fullgjødsel® npk 6-5-20 mikro) was applied to half of the rows (treatment 1; t1) (tab. 1). the rows in the latter half were not fertilized prior to planting (treatment 2; t2). fertigation was carried out in both treatments twice a week from may to august in 2004 and 2005, using calcinit™(15.5% n, 19.0% ca) and super-ba™rød (7-4-22 npk + micronutrients). all fertilizers were from yara. in the first cropping year, t1 and t2 were fertigated with different amounts of nutrients in the same volume of water. in t2 the ec was kept constant at 2.4 ms cm-1 and the ratio between calcinit™and superba™was 5:7, to get a total nutrient composition similar to t1. t1 was fertigated concurrently, but ec was kept on 1.6 ms cm-1. at the end of the first cropping season, the total amount of water and nutrients that had been applied was similar for both treatments for the first two years. the amount of n added during 2003 and 2004 was 80 kg ha-1 in total, which is the recommended fertilizer amount per year for ‘korona’ in norway. the experimental design was a tab. 1: summary of the fertilizer treatments in the trial presented as kg n ha-1 year-1. treatment 2003 2004 (2005) t1 301) 502) 802) t2 0 802) 802) 1) broadcasted compound fertilizer 2) fertigation randomized complete block with three replicates, each replicate counting 200 plants. first fully developed leaves for dry matter analyses were sampled at the same times as the berry samples were harvested, dried at 70oc and ground. berries were harvested three times per week on 50 plants per plot during the harvesting period in 2004 from june 21st to july 19th. samples of berries from early season, mid season and late season were registered and frozen at -18oc for quality analyses, while subsamples were dried for dry matter analyses. dry matter analyses leaf macronutrient concentration in dry matter were analysed after sulphuric/hypochloric acid digestion (allen et al., 1974), using a skalar autoanalyser for n and p, flame photometry for k and atomic absorption spectrophotometry for ca and mg. the same method was used when analysing mineral concentration of the berries. fruit quality analyses colour for colour analyses the juice was diluted to a 5 % solution by adding distilled water. the absorbance was measured at 515 nm using a spectrophotometer (shimadzu uv mini 1240, shimadzu corporation, kyoto, japan). frap assay fruits were homogenised in a food processor, and 3 g of the homogenised material was dissolved in 30 ml of methanol. three replicates were prepared from each sample. the bottles were flushed with nitrogen before closing, the samples were then mixed and sonicated on a water-bath at 0 °c for 15 min. the extracts were stored at -20 °c until analysis. three samples were centrifuged at 3250 g for 4 min at 4 °c. the concentration of total antioxidants was measured in triplicates of the supernatant. the ferric reducing ability of plasma (frap) assay of benzie and strain (1996) was used with minor modifications (halvorsen et al., 2002). a konelab 30i (termo, finland) was used to measure the antioxidant activity. the measurements were performed at 600 nm. an aqueous solution of 500 µm feso 4 x 7 h 2 o was used for calibration of the instrument. vitamin c 50 g of frozen material was made up to 150 g using 1 % oxalic acid. the material was homogenized for 1 minute and filtered through a schleicher and schuell filter (520 b1/2, folded). the samples were then filtered through an activated sep-pack c18 filter (waters) and a millipore filter (0,45 µm) before injection. prior to use the sep-pack filter was activated using 5 ml of methanol and 5 ml of ultra pure water. the first 2 ml of the analytic sample was discarded. the hplc analysis was performed according to williams et al. (1973) using an agilent system comprising hp1100 liquid chromatograph, auto sampler and uv detector (agilent technologies, oslo, norway). monitoring of the chromatography and data processing were performed by means of chemstation software. the separation was conducted on a 250 x 4.6mm zorbax sb-c18 5 µm column (agilent technologies, oslo, norway). the mobile-phase was 0.05m kh 2 po 4 for isocratic elution at 25 ºc. the flow was 1 ml min-1. the injection volume was 5 µl and the time was set to 5 min. l-ascorbic acid was measured at 254 nm. sugars, acids and total dry matter samples of juice were prepared for analyses of soluble solids and tritratable acidity. soluble solid concentration was determined by a digital refractometer (atago refractometer model pr-100, atago co, ltd. tokyo, japan), the titratable acidity by a radiometer end-point titrator (radiometer end point titration system, ets 822, copenhagen, denmark) and calculated as citric acid. dry matter was determined by drying approximately 5 g of homogenized material for 24 h at 104 ºc and weighing after stabilizing at room temperature in a dry container. statistics all data were subjected to analysis of variance (anova) using minitab® statistical software (release 14). significant differences between treatments were identified at the 5% probability level. for correlations, the pearson correlation coefficient (r) was calculated. results preplant fertilization in august 2003 (t1) was positive for yield and berry size (opstad et al., unpublished), but the fertilizer treatments in this trial had only minor effects on the quality parameters recorded. only fruit colour was significantly darker in t1 (p=0.002), tab. 2: quality parameters in strawberry fruits with two fertilizer strategies at three harvesting times during 2004. vitamin c frap berry size colour (mg 100 g-1 (µmol g (gram berry-1) ref ph % acid (5%) fresh weight) fresh weight-1) time date t1 t2 t1 t2 t1 t2 t1 t2 t1 t2 t1 t2 t1 t2 early season 25.06.04 11.37 11.80 3.60 3.61 0.84 0.84 0.07 0.06 57.16 54.36 17.53 18.09 36.43 31.90 mid season 05.07.04 10.20 10.63 3.41 3.44 0.90 0.90 0.12 0.09 55.39 57.26 18.05 17.84 18.13 13.30 late season 15.07.04 12.00 12.47 3.48 3.48 0.93 0.98 0.09 0.07 55.99 57.39 18.57 19.12 9.00 9.00 analysis of variance (p-value) source treatment 0.058 0.604 0.527 0.002 0.872 0.723 0.001 time <0.001 <0.001 0.006 <0.001 0.728 0.566 <0.001 treatment x time 0.997 0.727 0.604 0.264 0.137 0.909 0.020 fruit quality in the strawberry cultivar korona 37 while t2 tended to have a higher concentration of soluble solids (p=0.058) (tab. 2). soluble solids, ph, acidity and colour all varied with time of season. ph was highest in early season and lowest in mid season, while acidity was increasing through the season. antioxidant content was similar in both treatment and at all sampling times, while berry size was affected by treatments until the last sampling date as well as with sampling time (tab. 2). this indicates that vitamin c and frap was unaffected of both treatment, sampling time and berry size. neither the concentration of n, p, k, ca or mg in dry matter of strawberry fruits showed any variation with time or treatment (tab. 3). n, k and ca concentration in leaf dry matter varied significantly between the three sampling times, while concentration of ca and mg was affected by treatments (tab. 4). there were no correlations between fruit quality parameters, mineral concentration in berries or mineral concentration in leaves or sap (data not presented). discussion the lack of fertilizer response on fruit quality in strawberry in the present trial is probably due to the small difference in fertility level in t1 and t2 in the cropping year. fruit quality may not be easily affected by moderate fertilizer rates, and alleyne and clark (1997) and miner et al. (1997) found no fertilizer effects on various fruit quality components. the fertilizer application timing in the present trial significantly affected yield parameters, probably by augmenting flower differentiation in autumn (opstad and sønsteby, unpublished). the only statistical significant fertilizer effects on fruit quality was, however, concerning fruit colour, and the preplant fertilized plots receiving the lowest fertilizer rate during the harvesting season had the darkest fruit colour. application of 50 g n per grape vine increased the level of anthocyanins and the density of colour of grape must, compared to no fertilizer n application (delgado et al., 2004). application of 200 g per vine resulted in a colour density in-between the control and 50 g treatment. the fertilizer rate applied in our trial in t1 in 2004 may therefore have been optimal for colour development. alternatively, the positive effect on fruit colour in t1 may have been due to translocation of n from the autumn fertilization (archbold and mackown, 1995) as the fertilizer applications in t2 was quite moderate. application of k had no effect on colour density in grape (delgado et al., 2004). in our trial, plants fertigated with the highest amount of fertilizer during the harvesting season (t2) tended to yield fruits with a higher content of soluble solids, corresponding to the results of wang and lin (2002). also nestby (1998) registered higher sugar content when applying 124 kg n ha-1 than at lower n levels. the opposite was found by voth et al. (1967) and hansen (1995), were the lowest fertilizer rate resulted in the highest soluble solids content. other trials have, however, found no fertilizer effects on content of soluble solids (alleyne and clark, 1997; miner et al., 1997; jeppson, 2000). all of the measured quality parameters except content of antioxidants and vitamin c varied between sampling times. the content of soluble solids decreased from early season to mid season, and maximized at the end of season. also miner et al. (1997) and hansen (1995) found a higher concentration of total and soluble solids in berries at the late picking dates, and hansen (1995) suggested this to be an effect of increased access to assimilates due to removal of the tab. 3: mineral concentration in strawberry fruits with two fertilizer strategies at three harvesting times during 2004. n p k ca mg time date t1 t2 t1 t2 t1 t2 t1 t2 t1 t2 early season 25.06.04 0.933 1.103 0.197 0.237 1.260 1.400 0.127 0.123 0.097 0.107 mid season 05.07.04 1.017 1.070 0.207 0.217 1.260 1.363 0.107 0.117 0.097 0.103 late season 15.07.04 1.017 0.983 0.213 0.213 1.337 1.280 0.113 0.103 0.097 0.097 analysis of variance (p-value) source treatment 0.312 0.185 0.293 0.949 0.231 time 0.839 0.939 0.944 0.703 0.647 treatment x time 0.409 0.384 0.353 0.887 0.647 tab. 4: mineral concentration in strawberry leaves with two fertilizer strategies at three sampling times during 2004. n p k ca mg time date t1 t2 t1 t2 t1 t2 t1 t2 t1 t2 early season 25.06.04 2.463 2.510 0.290 0.327 1.367 1.410 0.677 0.653 0.237 0.233 mid season 05.07.04 1.977 2.047 0.277 0.280 1.300 1.227 0.647 0.537 0.230 0.210 late season 15.07.04 1.673 1.913 0.280 0.313 1.437 1.503 0.610 0.460 0.240 0.213 analysis of variance (p-value) source treatment 0.151 0.456 0.837 0.023 0.010 time <0.001 0.324 0.045 0.040 0.116 treatment x time 0.550 0.441 0.589 0.365 0.227 38 anne-berit wold, nina opstad strongest sinks by picking of the primary berries. this could also explain the decrease in soluble solids in berries harvested mid season in the present trial, when a large number of berries were competing for assimilates. these differences between sampling time of ripe berries suggest that time of season must be considered when comparing fruit quality in relation to varieties or cultivation conditions. a decrease in shelf life from early to late season has also been found (opstad et al., unpublished), as well as differences in fruit maturation related to fertilizer treatments (opstad and sønsteby, unpublished). ideally, fruit samples should therefore be defined in relation to fruit rank order before comparing fertilizer and harvest time effects on fruit quality. a significant negative relationship between fruit size and total antioxidant capacity has been observed for strawberry cultivars (davik et al., 2006) and high-bush blueberry (vaccinium corymbosum) cultivars (remberg et al., 2006). this was explained by the surface to volume ratio, as the peel fraction of fruits have a significantly higher antioxidant capacity compared to pulp (guo et al., 2003). in this study, berry size decreased during the season without any significant influence on antioxidant activity. wang and lin (2000) found a linear relationship between the content of anthocyanins and antioxidant activity measured by orac assay (oxygen radical absorbance capacity), but in our trial the significantly darker fruit colour in the t1 treatment was not associated with antioxidant activity measured by frap assay. thus, other components than anthocyanins, like ellagic acid (anttonen et al., 2006), contribute significantly to the antioxidant capacity in strawberries. the vitamin c content has also been found to correlate negatively with fruit size where smaller fruits contained more vitamin c compared to larger fruits (moor et al., 2004), but in this study the vitamin c content was not influenced by the berry size. moor et al. (2005) found that vitamin c content in strawberry is not easily influenced by cultural practises, and in tomato fruits, fertilization using electrical conductivity of 2 and 5 ms cm-1 had no significant effect on antioxidant activity (wold et al., 2004). in contrast, the same trial demonstrated a significant increase in dry matter, soluble solids and titratable acids with higher electrical conductivity. the lack of correlations with berry size in our trial, could possibly be due to unknown effects of sampling time that is not correlated to berry size. the concentration of macro nutrients in berries analysed in this trial was not affected by neither fertilizer treatments nor sampling time, while a statistical significant effect was found of sampling time in respect to mineral elements in leaf dry matter. only concentration of ca and mg in leaf dry matter was affected by the fertilizer treatments. surprisingly, the fertilizer treatment where the highest fertilizer amount was applied during the harvesting season (t2), showed a decrease in concentration of all mineral elements in fruits from early to late season, while in the preplant fertilized treatment (t1) there was an increase in npk concentration from early to late season. these changes were, however, not significant. archbold and mackown (1995) found that fruit under development was a greater sink for stored fertilizer n acquired in the months before cropping than for newly absorbed n, and may explain these tendencies in our results. the concentration of all macronutrients in the berries in our trial was lower than the concentration found in dry matter of three strawberry cultivars and clones in florida (albregts and howard, 1980). this is probably due to differences between cultivars or climatic factors, as the fertilizer treatments used in our trial are according to recommended fertilizer rates. n concentration in fruit dry mass in blackberry (rubus l., subgenus eubatus) was unaffected by fertilizer timing, and only the control was significantly lower than other fertilizer treatments (alleyne and clark, 1997). in conclusion, moderate fertilizer rates applied in late autumn or during the harvesting season resulted in only marginally different effects on fruit quality parameters, and only fruit colour was significantly affected by the fertilizer treatments. although significant differences between harvest date was found for berry size, soluble solids, ph, titratable acids and colour in fruits, and n, k and ca concentration in leaves, no effects on antioxidant activity or fruit mineral concentration was found. other factors probably affected fruit composition more than application of fertilizer. acknowledgement the technical assistance of signe hansen, kari grønnerød and karin svinnset at norwegian university of life sciences, department of plant and environmental sciences are greatly appreciated. we would also like to thank professor finn måge for revising the manuscript. references 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sci. biotechn. 73, 563-568. neuweiler, r., bertschinger, l., stamp, p., feil, b., 2003: the impact of ground cover management on soil nitrogen levels, parameters of vegetative crop development, yield and fruit quality of strawberries. eur. j. hort. sci. 68, 183-191. prange, r.k., deell, j.r., 1997: preharvest factors affecting postharvest quality of berry crops. hortsci. 32, 824-830. remberg, s.f., wold, a.-b., kvaal, k., appelgren, m., haffner, k., 2006: an approach towards rapid optical measurements of antioxidant activity in blueberry cultivars. j. appl. bot. food qual. 80, 36-39. sjulin, t.m., robbins, j.a., 1987: effects of maturity, harvest date, and storage time on postharvest quality of red raspberry fruit. j. amer. soc. hort. sci. 112, 481-487. voth, v., uriu, k., bringhurst, r.s., 1967: effect of high nitrogen applications on yield, earliness, fruit quality, and leaf composition of california strawberries. proc. amer. soc. hort. sci. 84, 249-256. wang, s.y., lin, h.s., 2000: antioxidant activity in fruits and leaves of blackberry, raspberry, and strawberry varies with cultivar and developmental stage. j. agric. food chem. 48, 140-146. wang, s.y., lin, s.-s., 2002: composts as soil supplement enhanced plant growth and fruit quality of strawberries. j. plant nutr. 25, 2243-2259. williams, r.c., baker, d.r., schmidt, j.a., 1973: analyses of watersoluble vitamins by high-speed ion exchange chromatography. j. chromatographic sci. 618-624. wold, a.-b., rosenfeld, h.j., baugerød, h., blomhoff, r., 2004: the effect of fertilization on antioxidant activity and chemical composition of tomato cultivars (lycopersicon esculentum mill.). eur. j. hort. sci. 69, 167-174. address of the authors: anne-berit wold1 and nina opstad 2 * 1 norwegian univ. life sci., dept. of plant and environm. sci. pb. 5003, n-1432 ås, norway. 2 norwegian inst. agr. environm. res. (bioforsk) arable crops div., sect. kise, n-2350 nes h. norway. * corresponding author: nina.opstad@bioforsk.no 40 anne-berit wold, nina opstad << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false /maxsubsetpct 100 /optimize true /opm 1 /parsedsccomments true /parsedsccommentsfordocinfo true /preservecopypage true 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<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> >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice journal of applied botany and food quality 87, 97 103 (2014), doi:10.5073/jabfq.2014.087.015 1department of horticulture and food science, university of craiova, craiova, romania 2department of chemistry, university of craiova, craiova, romania evolution of antioxidant activity and bioactive compounds in tomato (lycopersicon esculentum mill.) fruits during growth and ripening violeta nour1, ion trandafir2, mira e. ionica1 (received november 30, 2013) summary the interest in the consumption of tomato (lycopersicon esculentum mill.) is, to a large extent, due to its content of bioactive compounds and their importance as dietary antioxidants. during the growth and ripening process, there are quantitative and qualitative changes in the fruit composition which determine the nutritional quality and antioxidant potential at each stage. two half determinate early hybrids cultivars (prekos and balkan) and one indeterminate mid-early hybrid cultivar (reyana) were considered for this study. fruits from plants grown on sandy soil in an unheated greenhouse were collected at three growth and six maturity stages. antioxidant activity, dry matter, soluble solids, titratable acidity, ascorbic acid, lycopene, b-carotene, chlorophylls and total phenolic contents were monitored. during fruit growth, dry matter, soluble solids and titratable acidity recorded a slight decrease, polyphenols and b-carotene contents remained almost the same while ascorbic acid content and antioxidant activity increased continuously. the stage of ripening significantly influenced the content of all bioactive compounds as well as the antioxidant activity of tomato fruits. the first stages of ripening were characterized by a slight decrease of the dry matter content and by an increase of the titratable acidity, while in the last two stages of ripening these variations reversed. ascorbic acid and total phenolics content increased as maturity progressed from mature green to pink or light red stage and decreased afterward. lycopene started to accumulate since turning and sharply increased in the last three stages, on average 36 % of the lycopene content being accumulated in the last stage of ripening. in terms of hydrophilic antioxidant activity, depending on the cultivar, the pink or light red stages were the ones with the greatest potential. although there were significant differences among the contents of bioactive compounds and antioxidant activity of the three cultivars studied, their patterns of variation during the nine stages were quite similar. introduction tomatoes are widely known for their outstanding antioxidant content, including their high concentration of lycopene and excellent amounts of other conventional antioxidants like vitamin c and tocopherols, additional carotenoids (b-carotene, lutein, and zeaxanthin), trace minerals (selenium, copper, manganese and zinc) and phytonutrients including flavonoids (naringenin, rutin, kaempferol, and quercetin) and hydroxycinnamic acids (caffeic, ferulic, and coumaric acid) (capanoglu et al., 2010; fernandez-ruiz et al., 2011). recently, researchers started to identify new phytonutrients in tomatoes that help provide us with health benefits including the glycoside esculeoside a (fujiwara et al., 2007), the flavonoid chalconaringenin (yamamoto et al., 2004; slimestad and verheul, 2005) and 9-oxo-octadecadienoic acid, a fatty-acid derivative (kim et al., 2011). tomato has been identified as a functional and nutraceutical food because intake of tomatoes has long been linked to health benefits (canene-adams et al., 2005). fresh tomatoes and tomato products have been shown to diminish the prevalence of certain types of cancer (campbell et al., 2004) and to support the heart health by lowering total cholesterol, ldl cholesterol, and triglycerides, and the risk of atherosclerosis by preventing unwanted aggregation of platelet cells in the blood (willcox et al., 2003). furthermore, many studies have been conducted involving tomato and specific antioxidant protection of the bones, liver, kidneys, and bloodstream. in the last years the antioxidant composition as well as antioxidant capacity has been extensively studied while development of tomato cultivars having high antioxidants content is a significant trend in plant research (frusciante et al., 2007). aside from the genetic potential of the cultivar, agronomic and environmental conditions, ripening stage of fruit and post-harvest storage are known to affect the chemical composition of tomatoes (dumas et al., 2003; hernández et al., 2007). tomato fruit ripening is a complex process characterized by various morphological, physiological, biochemical and molecular transformations. these include degradation of chlorophylls, synthesis and storage of carotenoids (mainly lycopene and b-carotene) and aromatic compounds, alterations in organic acid metabolism, and a softening of the fruit tissue which occur in conjunction with an increase in co2 production (the respiratory climacteric) and an increase in ethylene production by the fruit. the amount of other important antioxidants, such as ascorbic acid and phenolics, is also variable during tomato ripening, in physiological response to various abiotic and biotic stresses (ilahy et al., 2011; domínguez et al., 2012). all these events determine major changes in texture, color, flavor, and aroma but also greatly affect the content of antioxidant compounds and the antioxidant activity of tomato fruits (dellapenna et al., 1986; pék et al., 2010). since tomato fruits are harvested at different ripening stages (from breaker to deep-red) depending on the consumer and market preference, the quantification of different antioxidants, as well as their variation during ripening, is of great relevance both to human health and to commercial purposes (ilahy et al., 2011). the aim of this study was to investigate the patterns of variation of dry matter, soluble solids, titratable acidity, and of the major antioxidant compounds (lycopene, b-carotene, phenolics, ascorbic acid) during growth and ripening of tomato fruits. as hydrophilic antioxidant acitivity depends upon synergistic effects among all hydrophilic antioxidants and their interaction with other constituents, its evolution was also monitored. fruits samples from three tomato cultivars grown simultaneously on a sandy soil representative of southern oltenia (romania) were analysed at nine different stages of the growth and ripening process. materials and methods plant material three different round-type tomato (lycopersicon esculentum mill.) cultivars were used in these experiments: prekos and balkan, two half determinate, early hybrids cultivars, and reyana, an indeterminate mid-early hybrid cultivar. plants were grown in an unheated greenhouse covered with polymeric film located at dabuleni, oltenia region, in southwestern romania (43°80' n, 24°08' e) during the spring growing season (march-july). 98 v. nour, i. trandafir, m.e. ionica seeds were sown on 20 january in alveolus plates filled with peat and transplanted into pots (12 cm diameter) after six weeks. plants for each genotype were transplanted to the greenhouse at the end of march in a randomised complete block design with two replicates. planting distances were 30 cm on the row and 100 cm between rows. each tomato cultivar was arranged in two replicated plots containing 40 plants. the soil was representative of southern oltenia and classified as sandy soil (psamosol) with neutral to weak basic reaction and low natural fertility. mean daily temperatures averaged between 6 °c in march and 22.7 °c in july. the three cultivars were grown simultaneously in the same greenhouse following traditional agronomic techniques for plant nutrition and prevention of pathogens and subjected to identical cultural practices of drip irrigation and fertilization doses. the plants were fertilized with nutrient solution of monoammonium phosphate (map) (12-61-0) through irrigation water until blossom, water-soluble npk fertilizer (polyfeed) 19-19-19 (0.5 %), three applications up to fruit formation and multi-k potassium nitrate fertiliser from first ripening until end of the crop cycle. the conventional methods also included weed control with synthetic chemical herbicides (fusilade s 2 l/ha and sencor 70 wp 0.4 kg/ha) and plant pathogen control with synthetic chemical pesticides (dithane m 45 0.2 % and topsin m 0.1 %). sampling fruits were collected by hand at three growth and six maturity stages. the growth stages were established at 7, 17 and 27 days after pollination. according to yamaguchi (1983), the stages of maturity were classified as follows: mature green (mature fruits with surface completely green, varying from light to dark green), breaker (first appearance of yellow or pink color but not more than 10 %), turning (yellow or pink color between 10 to 30 %), pink (pink or red color between 30 to 60 %), red (red color more than 60 % but less than 90 %), and deep-red (over 90 % red surface; desirable table ripeness). fruit samples from each plot and stage were chosen randomly in four repetitions, four fruits in each repetition, for each stage selecting those samples which showed uniformity in external color, size and shape. immediately after collection, fruits from each repetition were washed in tap water, blotted with a paper towel and cut into halves, the seeds were removed and the pericarp and mesocarp were ground to a homogeneous puree in a waring blender for about 2 min. part of the sample was immediately used for some analyses (dry matter content, soluble solids content and titratable acidity). the other part was frozen at -18 °c and used to determine the lycopene, b-carotene, chlorophylls, phenolics and ascorbic acid contents as well as the hydrophilic antioxidant activity. experiments were executed in three repetitions, and the results were expressed as mean ± standard deviation of values from four experiments performed in triplicate. determination of physico-chemical characteristics dry matter content (%) was determined gravimetrical ly by drying 5 g tomato homogenate in a laboratory oven (memmert, germany) set at 70 °c until constant weight was reached. soluble solids content (%) was measured with a digital refractometer (euromex, arnhem, the netherlands) after tomato homogenate clarifica tion by centrifugation (5000 × g, 10 min). titratable acidity (% as citric acid) was measured by titrating the water extract of tomato homo genate with a sodium hydroxide solution (0.1 n) using phenolphthalein as indicator. determination of antioxidant compounds extraction and analysis of ascorbic acid the ascorbic acid content was determined by reversed-phase highperformance liquid chromatography (hplc) method as described by nour et al. (2010). samples of 5 g of tomato homogenate were mixed and diluted to 100 ml with 0.1 n hcl. after 30 minutes the extraction solution was centrifuged at 5000 × g for 10 minutes. the supernatant was passed through a 0.45 μm pore size filter prior to hplc analysis. the liquid chromatograph was a finningan surveyor plus system (thermo electron corporation, san jose, ca, usa) equipped with a diode array detector (dad) set at 245 nm. the separation was performed on a hypersil gold aq (25 cm × 4.6 mm id, 5 μm) column using a 50 mm water solution of kh2po4 buffer adjusted to ph 2.8 with ortho-phosphor ic acid as mobile phase. the temperature of the column was set at 10 °c and the flow rate at 0.7 ml/min. results are expressed in mg/100 g fresh weight (fw). potassium di hydrogen orthophosphate and phosphoric acid were of analytical purity (sigma-aldrich, germany). ultrapure water was obtained from a milli-q water purification sys tem (tgi pure water systems, usa). extraction and analysis of chlorophyll and carotenoids determination was based on a spectrophotometric analysis following the method developed by nagata and yamashita (1992) for the simultaneous determination of chlorophyll and carotenoids in tomato fruit. the samples were thawed in the dark in a refrigerator at 4 °c to avoid carotenoid oxidation. samples of 1.0 g of tomato puree were extracted with 16 ml of acetone-hexane (4:6) solvent. after 15 min of shaking in a test tube and phase separation, the absorbance of the hexane layer was measured in a 1 cm path length quartz cuvette at 663, 645, 505, and 453 nm using a uv-vis spectrophotometer (varian cary 50 uv-vis, varian co., usa). carotenoid and chlorophyll contents were calculated according to the equations: lycopene (mg/100 ml) = 0.0458 × a663 + 0.204 × a645 + 0.372 × a505 0.0806 × a453; β-carotene (mg/100 ml) = 0.216 × a663 1.22 × a645 0.304 × a505 + 0.452 × a453; chlorophyll a (mg/100 ml) = 0.999 × a663 0.0989 × a645; chlorophyll b (mg/100 ml) = 0.328 × a663 + 1.77 × a645. the results were finally expressed in mg/100 g fw. determination of total phenolic content total phenolic content was measured spectrophotometrically using the folin-ciocalteau colorimetric method (singleton and rossi, 1965) with gallic acid (99 % purity, sigma) as a calibration standard. folin-ciocalteu reagent (2n, merk) and anhydrous sodium carbonate (99 % purity, sigma) were also used. samples (3 g of tomato homogenate) were extracted with 5 ml methanol in an ultrasonic bath for 45 min at ambient temperature. after extraction, samples were centrifuged at 5000 × g for 5 min and supernatants were filtered through 0.45 μm polyamide membranes. 100 μl of each tomato methanolic extract were mixed with 5 ml of distilled water and 500 μl of folin-ciocalteau reagent. after 30 sec to 8 min, 1.5 ml of sodium carbonate (20 % w/v) was added. the reaction mixture was diluted with distilled water to a final volume of 10 ml. the same pro cedure was also applied to the standard solutions of gallic acid. the absorbance at 765 nm of each mixture was measured on a varian cary 50 uv spectrophotometer (varian co., usa) after incubation for 30 min at 40 °c. results were expressed as mg of gallic acid equivalents (gae)/100 g fw. determination of hydrophylic antioxidant activity the hydrophilic antioxidant activity was measured using the dpph (2,2-diphenyl-1-picryl hydrazyl) assay as described by oliveira et al. (2008), with some modifications. the extraction of samples was made according to the same pro tocol as described for total phenolic content. each methanol tomato extract (50 μl) was mixed with 3 ml of methanolic solution containing 0.004 % (v/v) dpph. the mixture evolution of bioactive compounds in tomato fruits during growth and ripening 99 was shaken vigorously and allowed to stand at room temperature in the dark for 30 min, at which time the decrease in absorbance at 517 nm was measured using a varian cary 50 uv-vis spectrophotometer. the dpph free radical scav enging ability was subsequently calculated with respect to the trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), which was used as a standard reference. methanol (merck, germany), 2,2-diphe nyl-1-picrylhydrazyl (dpph) (sigma-aldrich, germany), and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox) (merck, germany) were used. the radical was freshly prepared and protected from the light. a blank control of methanol/water mixture was run in each assay. all assays were conducted in triplicate. results were expressed in mmol trolox/100 g fw. statistical analysis the results were statistically analyzed using statgraphics centurion xvi software (statpoint technologies, warrenton, va, usa). differences among means were determined using one-way analysis of variance (anova), followed by lsd multiple range test for multiple comparisons at p < 0.05. results and discussion in the first stage of growth the average dry matter content of the three cultivars was 8 %. during fruit growth the dry matter content registered a continuous decrease (fig. 1). this trend can be explained by the fact that dry matter content in tomatoes is mainly determined by dietary fibre and organic acid content (frusciante et al., 2007). as regards the evolution of dry matter content during ripening, there were no significant differences among the first three stages (from mature green to turning), in the pink stage a significant decrease registered while in the red and deep red stages the dry matter content significantly increased. the final average dry matter content of the three cultivars was 6.24 %. the highest content was observed for balkan cultivar (6.78 %) while prekos showed the lowest value (5.84 %). the variation of the soluble solids content of tomato fruits can be seen in fig. 2. there were no significant differences among the soluble solids contents of the three cultivars in the first stage of fruit growth. a slight decrease of soluble solid content was observed during fruit growth and a significant increase occurred during the last two stages of fruit ripening as reported in previous studies (helyes and lugasi, 2006; opara et al., 2012). this was due to partial breakdown of nonreducing sugars and other polysaccharides and their subsequent inversion to reducing sugars in the course of fruit ripening. similarly to dry matter, in the deep red stage balkan cultivar registered the highest soluble solids content (5.8 %), significanly higher than reyana and prekos between which no significant differences (p > 0.05) were found (5.1 % and 4.9 % respectively). fig. 1: variation of dry matter content of tomato fruits during growth and ripening (mean±sd). different letters within the same stage indicate significant differences (p < 0.05) among cultivars. fig. 2: variation of soluble solids content of tomato fruits during growth and ripening (mean±sd). different letters within the same stage indicate significant differences (p < 0.05) among cultivars. fig. 3: variation of titratable acidity of tomato fruits during growth and ripening (mean±sd). different letters within the same stage indicate significant differences (p < 0.05) among cultivars. the level of acidity in tomato fruits is an important parameter associated with sensory attributes like flavor and astringency. according to helyes and lugasi (2006), general impression of flavors of tomato fruit is basically determined by the ratio of sugars and acids. the evolution of titratable acidity of tomato fruits is presented in fig. 3. titratable acidity recorded a decrease during fruit growth and was the lowest in the mature green stage (average 0.335 %), results in good agreement with those reported by helyes et al. (2006). our results however differ from previous studies concerning the evolution of the titratable acidity during ripening. in the following stages titratable acidity increased, reached a maximum value in the pink or red stage depending on cultivar and slightly decreased in the final stage of maturity, when it has reached a mean value of 0.357 %. malate is the predominant acid in tomato and the metabolism of malate has been a strong focus of research on tomato fruits because the acid plays an important metabolically active role. some climacteric fruits such us tomato appear to utilize malate during the respira100 v. nour, i. trandafir, m.e. ionica tory burst (goodenough et al., 1985; kortstee et al., 2007), which explains the decrease in titratable acidity recorded in the early stages of ripening. the decline of the titratable acidity in the final stages of ripening is atributed to their use as substrate for respiration. helyes et al. (2006) reported that acid content remained almost the same during the ripening process (averagely 0.47 %). no significant differences were found between the final values of titratable acidity for the three cultivars. the evolution of the ascorbic acid content of the investigated tomato cultivars at the nine different stages are shown in fig. 4. the results showed that ascorbic acid levels were significantly different between the studied growth and ripening stages (p < 0.05). during fruit growth ascorbic acid content increased continuously while during ripening ascorbic acid content continues to increase as maturity progressed from mature green to pink or light red stage and decreased afterward. the decrease in ascorbic acid could be attributed to its susceptibility to oxidative destruction as impacted by the ripening environments. different patterns of variation were evidenced for the studied tomato cultivars during ripening. the highest amount of ascorbic acid was recorded at the pink stage in the cultivars balkan (32.74 mg/100 g fw) and prekos (23.2 mg/100 g fw), but at the red-ripe stage for the cultivar reyana (23.64 mg/100 g fw). the results are in good agreement with those of other authors (giovanelli et al., 2009; ilahy et al., 2011) who reported similar variations of ascorbic acid content during ripening (increase during the early stages of ripening, followed by a decline). other studies in tomatoes have revealed, however, that the ascorbic acid content remained practically constant in the first stages of ripening (until the pink stage), and increased slightly in the last, when the fruits were fully ripe (cano et al., 2003). in addition to ripening stage, it is known that environmental and genotypic factors contribute to the nutritional qualities of tomato (dumas et al., 2003; lenucci et al., 2006). in our study we found also that all along the ripening process, balkan cultivar exhibited significant higher amounts of ascorbic acid than prekos and reyana cultivars. reached a maximum at the pink stage and then declined significantly during the last two stages of ripening (red and deep red stages). helyes et al. (2006) found also the lowest polyphenols content in tomato fruits from mature green stage while the highest content at the pink stage while lugasi (2006) reported that the phenolic content of the tomato fruit does not change during ripening. ilahy et al. (2011) observed different patterns of variation in phenolic content between the studied cultivars, with peaks in the amount of phenols reached either at the orange-red ripening stage or at the green and green-orange stages of ripening. raffo et al. (2002) found also that total phenolics content showed slight, but significant, decreases at later stages of ripeness. they considered that the decrease of phenolic compounds in the fruit may be associated with the involvement of these compounds in the defense mechanisms against reactive oxygen species, which are produced in great quantities during the climacteric crisis as a consequence of the increase of the respiratory rate of the fruit. at the deep red ripe stage the polyphenol content of the fruits ranged from 23.26 to 26.15 mg gae/100 g while at the pink stage the total phenolic content varied between 29.39 and 38.56 mg gae/100 g. our data are in agreement with the results reported by oliveira et al. (2013) for immature, mature and ripe tomato fruits cultivated conventionally. fig. 5 shows the evolution of total phenolics content at different growth and maturity stages. the pattern of changes of the phenolic compounds during fruit growth and ripening can vary and it depends on the species or even the variety (egea et al., 2009). our results showed that although there were significant differences between total phenolics content of the three cultivars studied, the patterns of variation were quite similar both during fruit growth and ripening. total phenolics content remained almost constant during fruit growth, continuously increased during the first stages of ripening, fig. 4: variation of ascorbic acid content of tomato fruits during growth and ripening (mean±sd). different letters within the same stage indicate significant differences (p < 0.05) among cultivars. fig. 5: variation of total phenolics content of tomato fruits during growth and ripening (mean±sd). different letters within the same stage indicate significant differences (p < 0.05) among cultivars. fig. 6: variation of lycopene content of tomato fruits during growth and ripening (mean±sd). different letters within the same stage indicate significant differences (p < 0.05) among cultivars. evolution of bioactive compounds in tomato fruits during growth and ripening 101 carotenoids are known to be unique constituents of a healthy diet and have been associated with reducing the risk of several degenerative disorders, including various types of cancer, cardiovascular or ophthalmological diseases (stahl and sies, 2003). in most fruits with carotenoids, like apricot, mango, orange, papaya, pepper, persimmon, tomato, etc., the ripening is accompanied by an increase of carotenoids biosynthesis. therefore, in these fruits, the ripening degree determines the fruit carotenoid contents (egea et al., 2009). in tomato fruit this increase is due mainly to the accumulation of lycopene and β-carotene which is influenced by the genetic potential of the variety and environmental factors, especially temperature and light (helyes and lugasi, 2006). lycopene is the most important antioxidant compound of the mature tomato fruit and the one which determines its red color. change in the concentration of lycopene during growth and ripening of the investigated tomato cultivars is shown in fig. 6. up to the breaker stage the lycopene content of the fruits was nearly nil while from turning stage it increased sharply. on average 36 % of the lycopene content was synthesized and accumulated in the sixth maturity stage while helyes and lugasi (2006) found that almost half (46 %) of the lycopene content was synthesized and accumulated in this last stage. at the deep-red stage, reyana cultivar showed the highest content of lycopene (3.38 mg/100 g fw) followed by balkan (2.91 mg/100 g fw) and prekos (2.8 mg/100 g fw). these concentrations fell well inside the interval described by other authors (hernández et al., 2007; nour et al., 2013). the concentration of β-carotene showed a gradual and linear increase during fruit ripening (fig. 7). this constant increase of the β-carotene concentration was reported also by giovanelli et al. (1999) while cano et al. (2003) reported that β-carotene content increased until breaker and pink stages and decreased afterwards. egea et al. (2009) asserted also that there is an old controversy regarding the accumulation of β-carotene during fruit ripening. some studies have found a constant increase in its concentration during ripening, while others reported that β-carotene reaches its maximum concentration before the fruit reaches total ripeness, the differences being attributed to the different growing conditions and cultivars. in the red-ripe stage the β-carotene content of reyana and balkan cultivars was within the literature concentration range (0.56 and 0.86 mg/100 g fw respectively) as reported by frusciante et al. (2007) while prekos cultivar registered a higher content (1.35 mg/ 100 g fw). as previous reported (ilahy et al., 2011) chlorophylls decrease during tomato ripening, being substituted by carotenoids, mainly lycopene (fig. 8 and 9). when studying the antioxidant evolution of tomato fruit during ripening, the separate study of the hydrophilic and lipophilic fractions is asserted since, as they are determined by different antioxidant compounds, they will have different evolutions. cano et al. fig. 7: variation of b-carotene content of tomato fruits during growth and ripening (mean±sd). different letters within the same stage indicate significant differences (p < 0.05) among cultivars. fig. 8: variation of chlorophyll a content of tomato fruits during growth and ripening (mean±sd). different letters within the same stage indicate significant differences (p < 0.05) among cultivars. fig. 9: variation of chlorophyll b content of tomato fruits during growth and ripening (mean±sd). different letters within the same stage indicate significant differences (p < 0.05) among cultivars. fig. 10: variation of hydrophilic antioxidant activity of tomato fruits during growth and ripening (mean±sd). different letters within the same stage indicate significant differences (p < 0.05) among cultivars. 102 v. nour, i. trandafir, m.e. ionica (2003) established that hydrophilic antioxidant activity represented 71-85 % of the total antioxidant activity of the tomato fruit, the lowest contribution being observed in the last stage of tomato ripening, when the lipophilic antioxidant activity increased as the result of lycopene accumulation in the fruit. in our study only the hydrophilic antioxidant activity was monitored during fruit growth and ripening (fig. 10). a continuous increase could be observed during development of tomato fruit until the pink or light red stages where, depending on the cultivar, hydrophilic antioxidant activity reached maximum values and then declined significantly. different results were reported by cano et al. (2003) who found that the hydrophilic antioxidant activity and ascorbic acid content remained practically unchanged even though the phenol content increased during ripening, but also by ilahy et al. (2011) who recorded the highest hydrophilic antioxidant activity in tomato fruits at the green stage of ripening and the lowest value at the red-ripe stage. hydrophilic antioxidant activity correlated well with the total phenolics content (r = 0.77; p > 0.05) and ascorbic acid content (r = 0.78; p > 0.05), which proves once more that these bioactive components contribute significantly to the hydrophilic antioxidant capacity of tomato fruits. cano et al. (2003) found also that the levels of ascorbic acid and hydrophilic antioxidant activity were strongly correlated. conclusions the experiments conducted have shown that the composition of valuable nutrients including total soluble solids, organic acids, ascorbic acid, carotenoids as well as phenolics and antioxidant activity of the tomato fruit is strongly influenced during growth and ripening stages. total phenolics showed their highest levels at the pink stage while lycopene and b-carotene reached their highest levels in deepred tomato fruits. although the pink or red tomato had the greatest amount of vitamin c, the losses of ascorbic acid at the end of tomato fruit maturation were not great and the beneficial effect of ripening on the biologically-active carotenoids showed the deep-red stage to be valuable from the nutritional point of view. the hydrophilic antioxidant capacity of all investigated tomato cultivars clearly decreased during the last two stages of ripening. high 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(ed.), world vegetables, 291-312. avi book, new york, ny. yamamoto, t., yoshimura, m., yamaguchi, f., kouchi, t., tsuji, r., saito, m., obata, a., kikuchi, m., 2004: anti-allergic activity of naringenin chalcone from a tomato skin extract. biosci. biotech. bioch. 68, 1706-1711. address of the authors: violeta nour, mira e. ionica, department of horticulture and food science, university of craiova, a.i. cuza street 13, 200585 craiova, romania ion trandafir, department of chemistry, university of craiova, calea bucuresti street 107, 200529 craiova, romania journal of applied botany and food quality 87, 286 290 (2014), doi:10.5073/jabfq.2014.087.040 1institute for food technology, university of novi sad, serbia 2institute of general and physical chemistry, university of beograd, serbia comparative study of white cabbage, traditional variety and hybrid intended for biological fermentation biljana r. cvetković1*, lato l. pezo2, mladenka pestorić1, bojana filipčev1, žarko kevrešan1, jasna mastilović1 (received march 13, 2014) * corresponding author summary traditional serbian variety of white cabbage, cultivar “futoški ” (33 samples) and hybrid “bravo” (10 samples) were investigated in this study for their applicability to biological fermentation. different chemical, physical, texture and sensory characteristics of raw cabbage heads were investigated. obtained experimental results were analyzed using descriptive statistics, while correlation analysis showed the relations between different assays. also, principal component analysis (pca) has been applied to classify and discriminate between different cultivars cabbage heads. furthermore, standard score has been introduced to enable more comprehensive comparison between the investigated samples, in order to find the optimum sample, regarding observed chemical, physical, texture and sensory properties. pca analysis showed that the best sample for cabbage cultivar “futoški ” was sample 9, while sample 34 was the best for hybrid “bravo”, regarding their chemical, morphological and sensory characteristics. introduction cabbage (brassica oleracea var capitata) is a widely used vegetable in serbia. farmers cultivate hybrids because of their higher yield, compacted head, uniform quality and resistance to diseases, but traditional varieties are still highly prized because of their taste, tradition and suitability for fermentation. traditional varieties are characterized by loose heads suitable for spontaneous fermentation, since brine diffuses easier into the loose heads (cvetković et al., 2008). white cabbage “futoški ” is a traditionally grown and fermented variety in serbia, but it is also recognized in the eu. one of the most important commercial products obtained from brassica vegetables is sauerkraut, which results from the lactic acid fermentation of shredded and salted white cabbage. proper cabbage fermentation depends on cabbage variety (dobričević et al., 2004; stamer et al., 1969). fermented cabbage is traditionally produced in the balkans using whole heads of cabbage instead of shredded cabbage (johanningsmeier et al., 2007; wiander and palva, 2008; lu et al., 2003). fermentation time lasts 1-1.5 months (niksic et al., 2005). for proper whole cabbage head fermentation important quality requirements are minimum 3 % sugar content (malinowska-pan’czyk, 2012; niketić, 1988) and gentle cabbage leaves with less expressed venation. moreover, compact head is not a desirable attribute because it retards the progress of fermentation process by lowering the rate of the salt diffusion into the cabbage tissue (cvetković et al., 2012). in this paper, cultivar “futoški ” and hybrid “bravo” were analyzed as a raw vegetable, with their morphological, chemical and sensory characteristics in the purpose of their suitability for whole cabbage head fermentation. experimental results have been subjected to analysis of variance (anova) to show relations between applied assays (physical, chemical and sensory). in order to enable more comprehensive comparison between the investigated samples, standard score (ss) has been introduced. principal component analysis (pca) has been applied to classify and discriminate between the analysed samples and different cultivars. materials and methods plant material cabbage heads, cultivar “futoški ”, were harvested from 33 parcels while cabbage heads, hybrid “bravo”, were collected from 10 parcels in futog district, northern serbia (province of vojvodina). they are late fall varieties. cabbages were harvested in fall 2012. both types of cabbage were treated with appropriate agro-ecological measures. soil preparation included deep plowing at 30 cm depth performed in autumn 2011. also, npk formulation 8:16:24 in the amount of 800 kg ha-1 and manure was added in amount of 30-40 t ha-1. seedlings were transplanted in mid-july 2012. cabbage crops were irrigated in 8-12 day intervals with 30-40 mm of water. young plants were treated with actarom 25 wg. further treatment was with dihtane-m24 and rodomil gold. against weeds, herbicide treflan was used. harvesting of cabbage heads started when heads were hard to the touch or when the first outer leaf was slightly cracked (not the whole head). cabbage heads were analyzed after removing the outer leaves. chemical analysis sugar content was analyzed according to luffschoorl titrimetric method (egan et al., 1981). dry matter content was determined by drying sample to constant weight, in an oven at 105 oc. soluble solid content was determined using digital an atr st plus, schmidthaensch refract meter, germany. crude protein content was analyzed by kjeldhal method with a conversion factor of 6.25. ash content was determined after incineration of samples in a muffle furnace at 550 oc for 6 h. the content of cellulose was determined according to the standard procedure of kürschner-hanak (kurschner and hanak, 1930). physical analysis raw cabbage head weight was measured by scale balance (tovarna tehtnic, celje, slovenia), and the width of raw cabbage was measured with nonius at the site of widest cabbage cross-section. the height of cabbage head was measured in the middle of the cross section from the stem base to the top of the cabbage head. stem lenght of cabbage heads were also measured. all measurements were done on ten cabbage heads and descriptive statistics are presented in following tables. sensory analysis of raw cabbage raw cabbage heads were evaluated sensorially, by seven attributes in order to determine differences between the observed two cabbage varieties. six trained panelists evaluated four cabbage heads from one cabbage sample. a scale from 0 = “not dectetable” to 15 = “very strong” was used for attributes: color of outer leaves, leaf venation, biological fermentation of white cabbage 287 leaf thickness, leaf elasticity cabbage heads compaction. flavor was described as wetness and sweetness. the intesity of each attribute was rated by panelists by marking a 0-100 unstructured scale with the description of extreme points as previous reported (johanningsmeier et al., 2007). panelist also evaluated color of outer leaves from 0 (“very light green”) to 15 (“very dark green”), compared to the adopted color standards. instrumental texture analysis cabbage leaf mechanical properties were analyzed by punch test on a texturometer ta-xt2 (stable micro systems, england). test settings were: test speed 1.0 mm/s, distance 10 mm, trigger force 5 g. for the measurements, the first two or three outer leaf layers were removed from the cabbage head. the next four leaves were then placed on the holed platform and punched with a spheric probe. the tissue between the main veins (midrib and 2° veins) was analysed. the exact punch position was on the left-hand side of the midway between leaf tip and base, between the midrib and the leaf margin, avoiding main veins. measurings were repeated on four cabbage heads collected from one parcel. mean burst strenght and mean distance to burst were determined as indicators of leaves hardness and elasticity. statistical analyses descriptive statistical analyses for all the obtained results have been expressed by means, standard deviation (sd), variance, minimums and maximums, for each cabbage cultivar. collected data have been subjected to one-way analysis of variance (anova) for the comparison of means. furthermore, principal component analysis (pca) has been applied successfully to classify and discriminate between different cabbage cultivars. the evaluation of one-way anova and pca analyses of the obtained results has been performed using statsoft statistica 10.0® software (statsoft, tulsa, ok). determination of normalized standard scores (ss) min-max normalization is one of the most widely used technique to compare various characteristics of complex samples determined using multiple assays, where samples are ranked based on the ratio of raw data and extreme values of the measurement used. since the units and the scale of the data from various parameters are different, the data in each data set should be transformed into normalized scores, according to following equations: , in case of “the higher, the better” criteria, or in case of “the lower, the better” criteria, where: xi represents the raw data. normalized scores of the most of physical, chemical and sensory properties were evaluated using the above written equations, except for a few parameters, which are evaluated according to optimal values, using trapezoidal function, as follows: in case of “optimal range” criteria, where: m and n are minimum and maximum of optimal range values (written in tab. 1, 2, and 3). the sum of normalized scores of a sample for different measurements, when averaged give a single unit less value termed as ss, which is a specific combination of data from different measuring methods with no unit limitation. this approach also enables the ease of employing some other set of cabbage cultivars to this elaboration in the future comparisons. ss for physical, chemical and sensory quality for cabbage are calculated and the results are presented in tab. 1, 2, 3 and 4. results and discussion chemical properties of raw cabbage, cultivar “futoški ” and hybrid “bravo” are shown in tab. 1. the obtained results for the content of sugar, soluble solids, ash and proteins are in line with other authors (holzapfel et al., 2003; martínez et al., 2010). standard scores for chemical properties have been evaluated (tab. 1), showing that cultivar “futoški ” achieved slightly higher result (especially for lower cellulose and protein content). chemical parameters were coded as c1-c6, as designated in tab. 1. according to the experimental results, regarding physical characteristics of the cabbage head, there were statistically significant differences in all parameters, except for the stem lenght. tab. 2 shows that mean value of the height/width ratio, for hybrid “bravo”, is close to 1 (0.97). this means that the hybrid had almost regular round shape, whereas cultivar “futoški ” had distinctively oval-shaped heads (0.85). this is a good feature of “futoški ”cabbage head, because oval heads allow more dense packing of cabbage heads in fermentation barrels, in comparison with round-headed cabbage cultivars and hybrids. densely packed heads contribute to the achievement of more favorable conditions for anaerobic fermentation. evaluated standard scores for physical attributes are presented in tab. 2. hybrid “bravo” was scored much higher regarding physical properties. physical properties were coded as p1-p5 (tab. 2). sensory characteristics are shown in tab. 3, and they were evaluated by their intensity in order to characterize and compare between cabbage cultivar “futoški ” and hybrid “bravo”. the results showed that cabbage cultivar “futoški ” had a lighter colored outer leaves. cultivar “futoški ” leaves had less pronounced venation, a desirable characteristic in culinary practice which is in accordance with other authors (balkaya et al., 2005). heads of hybrid “bravo” are very compact, while cultivar “futoški ” heads are loose. calculated standard scores for sensory evaluation (tab. 3) showed that cultivar “futoški ” performed better regarding its geometrical and color attributes, and also organoleptic features. sensory attributes were coded as s1-s13, (3). textural properties of cabbage leaves for the analyzed cabbage cultivar and hybrid are presented in tab. 4. skin strength represented hardness of cabbage leaves and bravo had higher value (1064.39) for hardness than cultivar “futoški ” (727.83) which has a gentler structure. that can be explained by higher content of cellulose in hybrid “bravo” (tab. 1). distance indicates elasticity of cabbage leaves; raw hybrid “bravo” showed higher elasticity (79.34) than cultivar “futoški ” (78.86). the pca allows a considerable reduction in a number of variables and the detection of structure in the relationship between measuring parameters, different cabbage cultivars that give complimentary information. for visualizing the data trends and the discriminating efficiency of the used descriptors, a scatter plot of samples using the first two principal components (pcs) issued from pca of the data matrix is obtained (fig. 1). as can be seen, there is a neat separation of the two varieties of cabbage, according to the used assays for all chemical, physical and sensory characteristics. quality results showed that the first two principal components, accounting for 50.52 % of the total variability can be considered sufficient for data representation and the first two principal components for integrated chemical, physical , , 288 b.r. cvetković, l.l. pezo, m. pestorić, b. filipčev, ž. kevrešan, j. mastilović and sensory properties. pca graph showed good discrimination characteristics between cultivar “futoški ” and hybrid “bravo”, which were found different mostly due to variables located on the right side of factor plane, c5 (cellulose content), p5 (height/width ratio of cabbage head) and a few sensory parameters (s4 -, color of outer leaves, s5 leaf nervature, s6 leaf thickness, s10 head density and s13 acerbity). all of these characteristics are considered inappropriate for cabbage head used for fermentation, and samples having the lowest c5, p5, t1, s4, s5, s6, s10 and s13, located at the left side of pca biplot graphic seems to be the better than others. samples located at the left side of graph also showed better ss results, 0.67-0.76. best sample for cabbage cultivar “futoški ” was sample 9, while sample 34 was the best for hybrid “bravo”. the chemical characteristics for optimal sample 9, were: c1=7.20; c2=9.09; c3=7.50; c4=0.64; c5=0.17 and c6=1.19, while physical properties were: p1=2.0·103; p2=19.75; p3=16.25; p4=6.50; p5=0.82. textural characteristics were: t1=505.96; t2=79.08, while sensory attributes gained for optimal sample 9 were: s1=1.00; s2=0.50; s3=1.00; s4=1.87; s5=2.33; s6=1.97; s7=11.98; s8=1.63; s9=1.60; s10=2.57; s11=5.77; s12=7.68 and s13=4.97. sensory attributes gained for optimal sample 34, for hybrid “bravo” were: c1=7.20; c2=8.17; c3=7.83; c4=0.51; c5=0.30; c6=1.30; p1=1.5·103; p2=16.00; p3=13.63; p4=6.00; p5=0.85; t1=1007.13; t2=78.51; s1=0.25; s2=1.00; s3=0.75; s4=8.90; s5=9.05; s6=10.33; s7=4.30; tab. 1: chemical characteristics of raw cabbage, cultivar “futoški“ and hybrid “bravo” (g/100g). total sugar total dry soluble solids ash cellulose proteins content matter content content content content codes c1 c2 c3 c4 c5 c6 cultivar “futoški”, ss=0.82, n=33 samples average 5.94 9.34 7.20 0.63 0.58 1.50 st. dev 1.09 0.99 0.72 0.07 0.41 0.34 min. 4.16 7.00 5.33 0.51 0.07 0.96 max. 8.06 12.05 8.30 0.77 1.85 2.28 variance 1.18 0.99 0.52 0.00 0.17 0.11 hybrid “bravo”, ss=0.73, n=10 samples average 6.14 9.46 7.39 0.64 1.09 1.35 st. dev 1.29 0.76 0.91 0.09 0.58 0.19 min. 4.13 8.17 5.50 0.51 0.30 1.14 max. 7.56 10.48 8.13 0.84 1.78 1.79 variance 1.66 0.58 0.82 0.01 0.34 0.04 polarity + 7-10 6-8 + polarity: ‘+’ = the higher the better criteria, ‘−’ = the lower the better criteria. tab. 2: physical characteristics of raw cabbage heads, cultivar “futoški” and hybrid “bravo” weight width height stem length height/width relation (g) (cm) (cm) (cm) codes p1 p2 p3 p4 p5 cultivar “futoški”, ss=0.47, n=33 samples average 1797.95 17.67 14.98 6.89 0.85 st. dev 434.80 1.47 1.35 1.08 0.04 min. 1015.50 14.75 12.25 4.50 0.76 max. 2651.25 20.00 17.50 9.25 0.91 variance 189047 2.18 1.83 1.17 0.00 hybrid “bravo” , ss=0.77, n=10 samples average 1383.75 14.55 14.02 6.78 0.97 st. dev 458.78 1.51 1.55 1.06 0.07 min. 620.00 11.88 10.63 4.75 0.85 max. 2244.50 16.88 16.38 8.25 1.08 variance 210478 2.27 2.40 1.11 0.00 polarity 1500-2000 15-19 12-15 polarity: ‘+’ = the higher, the better criteria, ‘−’ = the lower, the better criteria. biological fermentation of white cabbage 289 s8=2.93; s9=3.50; s10=12.57; s11=3.60; s12=5.37 and s13=3.67. the main differences between these two samples were due to before mentioned characteristics c5, p5, s4, s5, s6, s10 and s13, especially in sensory evaluation. conclusions a comparative analysis of raw cabbage properties for domestic variety “futoški ” and hybrid “bravo” showed that raw cabbage cultivar “futoški ” has good physical, chemical, texture and sensory characteristics desirable for fermentation process. the optimal characteristics for both cabbage cultivars were determined. standard score analysis showed that “futoški ” cultivar is dominant within almost all characteristics of major importance for fermentation process. this is especially evident after pca analysis, which showed very neat discrimination between these two cultivars. analysis showed that both cabbages have adequate sugar content needed for fermentation process. acknowledgement these results are part of projects supported by the ministry of education, science and technological development of the republic of serbia, iii 46001 and tr-31055. 2011-2014 references balkaya, a., yanmaz, r., apaydin, a., kar, h., 2005: morphological characterisation of white head cabbage (brassica oleracea var. capitata subvar. alba) genotypes in turkey. new zeal. j. crop hort. 33(4), 333341. tab. 3: sensory evaluation of raw cabbage, cultivar “futoški“ and hybrid “bravo“ shape violet coverage color of leaf leaf leaf head root head wetness sweetness acerbity color with outer venation thickness elasticity section color density presence leaves leaves color codes s1 s2 s3 s4 s5 s6 s7 s8 s9 s10 s11 s12 s13 cultivar “futoški”, ss=0.85, n=33 samples average 0.84 0.93 0.94 4.46 4.50 5.28 9.37 3.15 2.77 5.89 6.05 7.97 3.09 st. dev 0.15 0.17 0.13 1.53 1.26 2.00 1.95 1.09 1.27 2.46 1.76 1.72 1.95 min. 0.50 0.25 0.50 1.87 2.33 1.97 4.70 1.45 1.23 2.13 3.13 4.03 0.35 max. 1.00 1.00 1.00 8.77 7.63 9.30 12.50 6.13 6.87 10.43 9.35 11.40 8.37 variance 0.02 0.03 0.02 2.35 1.59 4.00 3.80 1.20 1.60 6.05 3.10 2.96 3.80 hybrid “bravo”, ss=0.36, n=10 samples average 0.13 1.00 0.45 10.41 11.36 10.65 3.50 4.16 4.03 12.62 4.99 6.24 6.19 st. dev 0.18 0.00 0.39 1.14 1.23 2.59 1.89 2.01 1.43 0.87 2.80 2.04 2.93 min. 0.00 1.00 0.00 8.13 9.05 4.77 1.83 1.80 2.20 10.93 2.20 3.20 2.33 max. 0.50 1.00 1.00 11.65 13.00 12.85 7.45 7.73 6.30 13.70 10.38 9.47 11.03 variance 0.03 0.00 0.15 1.31 1.51 6.71 3.56 4.05 2.06 0.75 7.86 4.17 8.58 polarity + + + + + polarity: ‘+’ = the higher, the better criteria, ‘−’ = the lower, the better criteria tab. 4: texture analysis of cabbage leaves, cultivar “futoški” and hybrid “bravo”. skin strength (g) elasticity (mm) cultivar “futoški”, ss=0.95, n=33 samples average 727.83 78.86 st. dev 232.20 0.63 min. 396.08 77.55 max. 1351.17 79.97 variance 53914.61 0.40 hybrid “bravo”, ss=0.83, n=10 samples average 1064.09 79.34 st. dev 237.66 0.53 min. 654.54 78.51 max. 1421.58 80.25 variance 56484.28 0.28 fig. 1: biplot for characteristics of cabbage heads. 290 b.r. cvetković, l.l. pezo, m. pestorić, b. filipčev, ž. kevrešan, j. mastilović cvetković, b., bardić, ž., jokanović, m., mastilović, j., 2008: technological quality of biofermented white cabbage, cultivar futoški. food processing, quality and safety 35(2), 93-97. cvetković, b.r., pestorić, m.v., gubić, j.m., novaković, a.r., mastilović, j.s., kevrešan, ž.s., červenski, j.f., 2012: the dynamics of the fermentation process and sensorial evaluation of sauerkraut, cultivar futoški and hybrid bravo-comparative study. proceedings of 6th central european congress on food-cefood congress. institute of food technology, novi sad (serbia). dobričević, n., borosić, j., zutić, i., novak, b., toth, n., 2004: quality of different cabbage cultivars intended for biological fermentation. iii balkan symposium on vegetables and potatoes 729, 423-427. holzapfel, w., schillinger, u., buckenhüskes, h., farnworth, e., 2003: sauerkraut. handbook of fermented functional foods, 343-360. johanningsmeier, s., mcfeeters, r.f., fleming, h.p., thompson, r.l. 2007: effects of leuconostoc mesenteroides starter culture on fermentation of cabbage with reduced salt concentrations. j. food sci. 72(5), 166-172. kurschner, k., hanak, a., 1930: determination of cellulose. z. untersuch. lebensm. 59, 484. malinowska-pan’czyk, e., 2012: 10 fermented vegetables products. fermentation: effects on food properties, 231. martínez, s., olmos, i., carballo, j., franco, i., 2010: quality parameters of brassica spp. grown in northwest spain. int. j. food sci. tech. 45(4), 776-783. niketić, a.g., 1988: tehnologija voća i povrća. naučna knjiga, poljoprivredni fakultet, beograd, 139-158. niksic, m., niebuhr, s.e., dickson, j.s., mendonca, a.f., koziczkowski, j.j., ellingson, j.l.e., 2005: survival of listeria monocytogenes and escherichia coli o157:h7 during sauerkraut fermentation. j. food protect. 68(7), 1367-1374. stamer, j., dickson, m., bourke, j., stoyla, b., 1969: fermentation patterns of poorly fermenting cabbage hybrids. appl. microbiol. 18(3), 323327. wiander, b., palva, a., 2008: sauerkraut and sauerkraut juice fermented spontaneously using mineral salt, garlic and algae. agr. food sci. 20(2), 169-174. address of the corresponding author: e-mail: biljana.cvetkovic@fins.uns.ac.rs art08_raeini.indd water stress and transgenic strawberry response journal of applied botany and food quality 85, 182 187 (2012) 1department of horticulture, 2department of agricultural engineering, sari agricultural sciences and natural resources university, sari, iran the response of transgenic strawberry plants overexpressing a drought induced gene to water stress vida chalavi 1, mahmoud raeini-sarjaz 2* (received may 24, 2012) * corresponding author summary transgenic strawberry plants expressing a chitinase gene were evaluated for their performance during water stress. transgenic and non-transgenic plants were assigned to three different soil water contents (swc). they were kept under well-watered, moderately watered or stressed water conditions. at fi nal stage of experiment, dry matter components, leaf area, photosynthesis rate, water-use effi ciency (wue) and water use per leaf area (wula) were measured. transgenic lines showed vigorous growth as compared with non-transgenic plants. leaf area (la), leaf dry matter (ldm), root dry matter (rdm) and total dry matter (tdm) of well-watered and water-stressed plants of transgenic lines were signifi cantly higher than those of non-transgenic plants. the wue increased signifi cantly in transgenic lines, while water use (wu) per leaf area reduced in transgenic plants relative to control plants. photosynthetic rates were not different between transgenic and non-transgenic plants. soil water contents signifi cantly affected dry matter production, and photosynthetic rates. transgenic plants also showed vigorous growth in comparison to non-transgenic plants when grown in vitro. shoot, root and total fresh and dry weight of in vitro transgenic lines were signifi cantly higher than those of nontransgenic plants. introduction cell dehydration is a common factor in major environmental stresses such as drought, high salinity and cold temperature. although water is essential for growth and development of all plants, a high diversity in adaptation to water availability exist among different plant species. in one extreme, resurrection plants become desiccated and completely air-dry during drought spans and upon rehydration they revive and resume growth. in another extreme, some higher plant species are adapted for life in the water. in response to environmental stresses, many plant species express a similar set of genes and produce common proteins. the precise functions of most of these proteins are not well known. many stress-induced genes have been identifi ed and isolated from wild species, which demonstrate excellent tolerance to biotic and abiotic stresses (leckband and lorz, 1998; oldach et al., 2001). some of these genes have been introduced into cultivated plants in order to improve their performance under stress conditions or to study the gene function (chalavi et al., 2003). chitinases are proteins of 25-35 kds molecular weight and their expression is induced by biotic and abiotic stresses in higher plants (hong and hwang, 2006). these proteins are widely distributed in bacteria, fungi, animals, and plants (park et al., 2000; merecedes dana et al., 2006; punja and zhang, 1993). plant chitinases are endo-chitinases and are able to hydrolyze chitin of hypha walls, it is speculated that these enzymes are involved in plant defense against fungal attack and resistance to abiotic stresses (chalavi et al., 2003; schickler and chet, 1997; mercedes dana et al., 2006). moreover, the induction of chitinase gene by osmotic stress (chen et al., 1994) implies that in addition to the known function in plant defense against fungal diseases, this protein might play other roles in plant metabolism (kasprzewska, 2003). in recent years, there are some reports indicating the involvement of chitinases in fl ower formation (takakura et al., 2000; neale et al., 1990; lotan et al., 1989), fruit ripening (peumans et al., 2002; robinson et al., 1997), embryogenesis (de jong et al., 1992), seed germination (caruso et al., 1999), and insect resistance (van der westhuizen et al., 1998). chitinases are also assumed to be involved in freezing tolerance (yeh et al., 2000; hiilovaara et al., 1999). other reports indicated the role of chitinases in drought tolerance (yu et al., 1997) and enhancement plant fungal defence mechanisms (karasuda et al., 2003). clendennon et al. (1998) hypothesized that a class iii banana pulp-specifi c chitinase is a storage protein. due to its expression pattern and deduced amino acid sequences the authors dismissed the possibility of its involvement in plant defensive mechanism. on the other hand, the principal role of a lupinus albus l. class iii chitinase, constitutively expressed in vegetative organs and developing seeds, assumed to be in antifungal activity (regalado et al., 2000). water use effi ciency (wue), dry matter production per unit of consumed water, is one of the factors which evaluate plant physiological behavior under favorable or stressful environmental conditions. it has been shown that wue increases during water stress for plant species that could cope with other environmental conditions (raeini-sarjaz and barthakur, 1997). in the present study, we hypothesized that transgenic strawberry plants constitutively expressing a drought induced chitinase gene may have improved tolerance to water stress. we therefore produced transgenic strawberry lines overproducing chitinase protein. these strawberry transgenic lines were phenotypically normal. the growth parameters of strawberry transgenic lines, japarameters of strawberry transgenic lines, japarameters of strawberry transgenic lines, j and jm, with nontransformed one, j, were compared. materials and methods plant material: production of transgenic plants: the production of transgenic strawberry cv. joliette plants have been described eleswhere (chalavi et al., 2003). briefl y, the coding region of a chitinase gene from lycopersicon chilense (a wild tomato), induced by drought and abscisic acid (chen et al., 1994), was placed under the transcriptional control of caulifl ower mosaic virus (camv) 35s of binary pbin19 vector (bevan, 1984). the t-dna of the pbin19 vector contains a kanamycin (kan) selection marker gene. the resulting vector was introduced into agrobacterium tumefaciens strain lba4404 (hoekma et al., 1983) in a mix electroporation. in vitro shoots of strawberry (cv. joliette), were transferred by stipule transformation method (chalavi et al., 2003). trandgenic shoots were recovered and rooted on ms (murashige and skoog, 1962) medium containing 100 mgl-1 kanamycin for selection and 500 g ml-1 carbenicillin for killing agrobacterium. positively trandgenic water stress and transgenic strawberry response 183 plantlets, identifi ed by pcr (polymerase chain reaction), northen and southern blot analysis (chalavi et al., 2003), were propagated and used in this experiment. in vitro grown plants to evaluate the effect of chitinase gene on growth enhancement of strawberry (cv. joliette), two transgenic lines along with nontransformed shoots, as control, were grown on half-strength murashige and skoog (1962) salts and b5 vitamins (gamborg et al., 1968), solidifi ed with 0.6% of agar. the culture was kept at 26 oc under 16 h photoperiod and 50-70 µmol m-2 s-1 light intensity. after 2 months, plants were removed from culture medium, washed with deionized water, blotted with paper towels and the fresh weight of shoots and roots were recorded. to measure the dry weight, shoots and roots were dried in an oven at 60 oc for 72 h. soil grown plants strawberry transgenic and control (non-transformed) in vitro rooted plantlets were transferred to sterile soil and acclimated for 2 weeks under plastic cover. after 6 weeks, plants were transplanted in 2-l pots containing 1.6 l of composite soil made up of soil, sand and vermiculite (75, 12.5 and 12.5%, respectively). potted plants were randomly assigned to different soil water contents (swc) as described elsewhere (raeini-sarjaz and cahalavi, 2011); well-watered (w0), moderately-watered (w1), and waterstressed (w2). two transgenic lines, ja). two transgenic lines, ja). two transgenic lines, j and jm, and control strawberry plants were grown in pots under a completely randomized design. five pots (replications) were used for each treatment. well-watered plants were initially watered to 100% soil water content (swc) and re-watered when soil water dropped to 80%. soil water content of w1 and w2 plants initially were kept to 80% and 50% swc and water was supplied when swcs reached 60% and 30%, respectively. the total amount of the supplied water to each plant was calculated at the end of the experiment. plants were fertilized uniformly with 20-20-20 npk fertilizer every week during experiment. gas exchange measurements: after three weeks, when plants had 4 extended leaves, all plants were watered to 100% swc. one hour later, when plants were acclimatized to new condition, instantaneous leaf photosynthesis rates (pi) were measured on newly developed leaves of all plants of different long-term soil water contents and transgenic lines using a photosynthesis measurement system, li-6400 (licor inc., lincoln, nebraska, usa). the photon fl ux density was kept constant at 800 µmol m-2 s-1 and mean co2 concentration was closed to 400 ppm. after one day, when soil water content dropped to 60%, photosynthesis rates were measured again on the same leaves. plant shoot and root measurements: after four weeks, the plants’ shoot and root components were harvested and weighed. leaf area of each plant was measured using a leaf area meter, li-3100 (licor inc., lincoln, nebraska, usa). plants growth components were dried in an oven at 60 oc for 72 h to determine leaf dry matter (ldm), petiole dry matter (pdm), root dry matter (rdm) and total dry matter (tdm) with an electronic balance of ± 0.001 g precision. water use effi ciency (wue) of each treatment was calculated as total dry matter production (tdm) to total used water (wu) as transpiration, by plant, and evaporation from pot surface. statistical methods data presented as mean ± std. fresh and dry weight data were evaluated using a two factor analysis of variance (anova) procedures for soil grown plants and one-way anova for in vitro plants. when the main treatment effects showed signifi cant differences, the tukey’s post hoc tests were used to determine if signifi cant differences exist between transgenic lines and water treatments. associations between different growth components were determined using pearson product-moment correlation coeffi cients. the accepted level of signifi cance was p<0.05. results in vitro experiment shoot, root and total fresh weight (sfw, rfw and tfw, respectively) of in vitro plants were signifi cantly higher (p<0.001) in transgenic plants relative to nontransgenic ones (tab. 1). sfw of jajaj and jm, were signifi cantly higher by 59.6 and 55.3% compared with control plants. rfw of the jawith control plants. rfw of the jawith control plants. rfw of the j and jm was also higher (136.4 and 128.2% respectively) compared to non-transgenic plants. the same trend was observed for tfw, which increased by 74.1 and 86.2% for jajaj and jm, respectively, compared to control plants (tab. 1). shoot dry weight (sdw) of jashoot dry weight (sdw) of jashoot dry weight (sdw) of j and jm were higher by 50 and 62.5%, respectively, compared to control plants. root dry weight (rdw) of jajaj and jm also increased by 200 and 400%, respectively, compared to non-transgenic control plants. total dry weight of transgenic, jato non-transgenic control plants. total dry weight of transgenic, jato non-transgenic control plants. total dry weight of transgenic, j and jm, plants increased by 68 and 100%, respectively, compared to control plants (tab. 1). tab. 1: mean (± std) in vitro shoot (sfw), root (rfw) and total fresh weight (tfw), and shoot (sdw), root (rdw) and total (tdw) dry weight data of transgenic and non-transgenic plants (g/plant). fresh weight (g/plant) treatments shoot root total transgenic jatransgenic jatransgenic j 0.75±0.19a 0.26±0.12 a 1.01±0.29 a transgenic jm 0.73±0.12 a 0.35±0.13 a 1.08± 0.20 a non-transgenic 0.47±0.12 b 0.11±0.05 b 0.58±0.15 b dry weight (g/plant) transgenic jatransgenic jatransgenic j 0.12±0.03 a 0.03±0.01 a 0.15±0.04 a transgenic jm 0.13±0.02 a 0.05±0.02 a 0.18±0.04 a non-transgenic 0.08±0.02 b 0.01±0.01 b 0.09±0.02 b means in each column that carry the same letter are not signifi cantly different (p<0.05). pot experiment the effects of strawberry transgenic lines and soil water content on overall performance of examined plants were highly signifi cant (p<0.001). leaf and root dry matters (ldm and rdm, respectively) of transgenic plants were signifi cantly higher than those of control plants (p<0.0001). in well-watered condition, japlants (p<0.0001). in well-watered condition, japlants (p<0.0001). in well-watered condition, j and jm transgenic plants produced almost 40% more leaf dry matter compared with control plants. when ldm of moderate watered plants were compared, transgenic plants (jacompared, transgenic plants (jacompared, transgenic plants (j and jm) performance were similar to those of control plants (tab. 2). while in water stressed plants leaf dry matter of transgenic plants jaleaf dry matter of transgenic plants jaleaf dry matter of transgenic plants j and jm was higher by 51.0 and 41.8%, respectively, compared to that of control plants (tab. 2). mean rdm of transgenic plants was signifi cantly higher (41.6%) than that of non-transgenic plants. total dry matter (tdm) of 184 v. chalavi, m. raeini-sarjaz water stress and transgenic strawberry response transgenic plants also was signifi cantly (p<0.001) higher than that of control plants. well-watered transgenic jaof control plants. well-watered transgenic jaof control plants. well-watered transgenic j and jm plants overgrew non-transgenic control plants by 40 and 44.8%, respectively. under moderate water condition tdm of transgenic and control plants were not statistically different, while transgenic plants (jawere not statistically different, while transgenic plants (jawere not statistically different, while transgenic plants (j and jm) for stressed-water condition outperformed those of control plants by 53.4 and 43.8%, respectively (tab. 2). plant leaf area (la) had the same trend as dry matter components; there was an overall signifi cant difference (p<0.0001) between la of transgenic plants and that of control plants. for well-watered transgenic jatransgenic jatransgenic j and jm plants leaf area increased by 27.5 and 29.3%, respectively, in comparison to non-transgenic control plants. again, when moderate watered plants were considered no differences were found between transgenic and non-transgenic plants, while severely stressed transgenic plants (jastressed transgenic plants (jastressed transgenic plants (j and jm) leaf area increased by 46.4 and 32.4%, respectively, compared with control plants (tab. 2 and fig. 1). soil water contents also signifi cantly (p<0.001) affected la (fig. 1). the expression of chitinase gene in strawberry had no effect on specifi c leaf area (sla) or root to shoot ratio (tab. 3). water use (wu) during plant growth and development was highly affected by transgenic strawberry and soil water content (p<0.001). mean water consumption of transgenic plants increased by 10% relative to control plants. well watered and water-stressed transgenic plants on average consumed 16% and 21% more water, respectively, relative to those of control plants, while moderate watered transgenic plants and corresponding control plants were not different (tab. 3). when water use per leaf area (wu la-1) was considered, non-transgenic plants on average consumed more water (14%) compared to transgenic plants (tab. 3). in all three lines both wu and wu la-1 increased as soil water content increased (tab. 3 and fig. 1b). transgenic lines and soil water content had signifi cant (p<0.001) effects on water use effi ciency (wue). mean wue of transgenic ja and jm plants was signifi cantly higher (24% and 15%, respectively) than that of control plants (tab. 3). transgenic well, moderate and stress watered plants wues increased (23%, 16% and 19%, respectively) relative to those of control plants (tab. 3 and fig. 1c). the effect of soil water content on wue was also highly signifi cant and stressed plants on average produced higher dry matter for unit of consumed water (5.32 g/kg) relative to moderate and well watered plants (4.71 and 3.59 g/kg , respectively) (fig. 1c). mean instantaneous photosynthetic rates of different lines under different soil water conditions were not statistically different when watered to 100% or 60% of swc (tab. 4). while soil water content signifi cantly (p<0.001) affected mean photosynthetic rates, when soil water content was kept at 100% or 60% (tab. 4). mean photosynthetic rates across different lines under 60% swc signifi cantly dropped for both well and moderate soil contents, while it did not changed under longterm stressed plants (tab. 4). discussion the main fi nding of this study is that the constitutive and high level expression of chitinase gene in strawberry (chalavi et al., 2003) enhanced growth of transgenic plants. to our knowledge, this study is the fi rst study to evaluate the effects of a chitinase gene expression and soil water content on growth of strawberry plants. chitinase enzymes have been suggested to be implicated in a number of plant normal metabolic functions such as hypocotyle, leaf, root and seed developments (robinson et al., 1997). although chitinase activity has been correlated with growth, the mechanism of such involvement is not well known. stress factors, mainly pathogen infections and abiotic stresses are responsible for chitinases induction (kasprzewska, 2003). the role of these enzymes is usully considered as a defence mechanism against pathogens (chalavi et al., 2003; regalado et al., 2000). besides defence mechanism of chitinases against pathogen attack (patil et al., 2000), it is speculated that, they may get involved in stress response and also in growth and development processes (regalado et al., 2000) by tab. 2: the effect of soil water content and strawberry lines on mean leaf area (la, cm2), leaf (ldm), petiole (pdm), root (rdm) and total (tdm) dry matter (g/plant) of transgenic and non-transgenic strawberry plants (mean ± sd). treatments la ldm pdm rdm tdm transgenic jatransgenic jatransgenic j well-water 1759.9±106.2 9.69±0.77 2.99±0.14 2.31±0.42 14.96±1.08 moderate 1419.4±79.6 7.24±0.45 2.37±0.30 1.74±0.27 11.34±0.97 stress 1086.7±77.6 4.96±0.42 1.77±0.47 1.30±0.22 8.04±0.67 maen* 1422.0±298.9a 7.29±2.07a 2.37±0.56a 1.78±0.54a 11.45±3.08a transgenic jm well-water 1783.9±108.6 9.80±0.98 3.14±0.28 2.55±0.82 15.48±10.08 moderate 1335.1±216.8 6.41±1.38 2.21±0.41 1.40±0.43 10.02±2.20 stress 982.5±102.2 4.65±0.71 1.62±0.13 1.26±0.15 7.54±0.93 mean* 1367.2±374.1a 6.95±2.45a 2.32±0.71a 1.73±0.82a 11.01±3.90a non-transgenic well-water 1379.8±73.8 6.98±0.74 2.27±0.22 1.44±0.28 10.69±1.22 moderate 1227.4±93.5 6.41±0.92 1.97±0.25 1.46±0.20 9.84±1.27 stress 742.2±95.6 3.28±0.48 1.08±0.19 0.88±0.21 5.24±0.78 mean* 1116.5±295.8b 5.56±1.84b 1.77±0.57b 1.26±0.37 b 8.59±2.73b *means across different soil water contents for each strawberry line. means in each column that carry the same letters are not signifi cantly different (p<0.05). water stress and transgenic strawberry response 185 generating or degrading signal molecules (van-der holst et al., 2001). it is assumed that overproduction of ethylene, a plant growth regulator, by enhanced chitinase activity might be involved in enhanced plant growth and development (zhong et al., 2002; kasprzewska, 2003). patil et al. (1997) have suggested a role for chitinase in cell wall disruption, presumably in the cell division cycle. they found some correlation between growth and chitinase activity in the seedlings and suspension cultures of transgenic and wild type tobacco plants. in our study in both in vitro and pot experiments, strawberry plants overexpressing chitinase gene showed signifi cant growth enhancement compared to non-transgenic plants. the jathe jathe j line had a high level of chitinase gene expression showed superiority to jm plants (chalavi et al., 2003), which had a lower expression of the gene, and to non-transformed control plants. soil grown transgenic plants produced 25 and 30% more dry weight and leaf area compared to control plants. likewise, the growth enhancement of the in vitro transgenic plants was much higher and transgenic plants dry weight increased more than 83% compared to non-transgenic plants. when plants were exposed to different soil water contents, the growth of both well and stress watered transgenic plants enhanced signifi cantly as compared with non-transformed plants. interestingly, moderate watered transgenic plants showed no advantage to corresponding control plants. these results could happen if strawberry endogenous chitinase genes were induced only at optimal and stress water conditions, but not at moderate soil water condition. the same circumstance was reported by alvin et al., (2001). when they subjected their transgenic and control tobacco plants to progressive water stress, the endogenous binding protein (bip) was induced in tobacco control plants after withholding water for 1 week, and it was declined after 3 weeks of water defi cit the binding protein levels in transgenic plants was steady and high before and during period of progressive water stress. contrary to our data and patil et al. (1997) fi ndings, bolar et al. (2001) have reported a negative correlation between chitinase activity and growth in transgenic apple plants. they produced transgenic apple lines expressing endochitinase gene from trichoderma harzianum. expression of endochitinase had a negative effect on apple growth; those plants with highest level of endochitinase activity did not grow when transferred to soil. the same authors (bolar et al., 2001) also produced transgenic apple plants simultaneously expressing two chitinase genes, an endochitinase and an exochitinase. growth of these transgenic plants was negatively affected by the level of endochitinase activity and irrespective to the level of exochitinase. tab. 3: the effect of soil water contents and strawberry lines on mean specifi c leaf area (sla, cm2 g-1), shoot to root ratio, water use (wu, kg/plant), water-use effi ciency (wue, g kg-1) and water use per leaf area (wu la-1, g cm-2) of transgenic and non-transgenic strawberry plants (mean ± sd). treatments sla shoot/ root water use wue wu la-1 transgenic jatransgenic jatransgenic j well-water 5.49±0.14 0.181±0.013 4.07±0.07 3.62±0.11 2.32±0.03 moderate 5.09±0.05 0.180±0.010 2.15±0.05 5.28±0.11 1.51±0.01 stress 4.56±0.09 0.194±0.017 1.36±0.08 6.00±0.43 1.25±0.07 mean* 5.05±0.19 a 0.185±0.008a 2.525±0.53a 4.98±0.51a 1.67±0.29 b transgenic jm well-water 5.48±0.14 0.193±0.012 3.92±0.12 3.97±0.31 2.21±0.11 moderate 4.76±0.17 0.158±0.012 2.15±0.15 4.61±0.25 1.62±0.04 stress 4.72±0.17 0.202±0.008 1.44±0.08 5.26±0.25 1.47±0.07 mean* 4.99± 0.22 a 0.184±0.016a 2.504±0.49a 4.61±0.34a 1.77±0.26 b non-transgenic well-water 5.05±0.19 0.155±0.007 3.43±0.08 3.15±0.15 2.49±0.05 moderate 5.20±0.19 0.175±0.008 2.32±0.07 4.24±0.20 1.90±0.06 stress 4.42±0.11 0.202±0.020 1.12±0.08 4.68±0.24 1.52±0.10 mean* 4.89± 0.21a 0.177±0.015a 2.290±0.44b 4.02±0.36 b 1.96±0.20a *means (± std) across different soil water contents for each strawberry lines. means in each column that carry the same letters are not signifi cantly different across strawberry lines (p< 0.05). tab. 4: mean (± std) photosynthetic rates (µmol m-2 s-1) of transgenic and non-transgenic strawberry plants across different soil water contents. water mean1¥ treatments well 10.84±0.39 10.18±1.44 10.45±0.55 10.49±0.96 b moderate 10.87±0.98 10.95±1.51 10.58±1.40 10.80±1.33 b stress 11.46±0.69 12.12±0.68 11.61±0.47 11.72±0.69 a mean*2 11.05±0.81 11.08±1.52 10.86±1.06 well 3.78±1.29 4.80±2.50 4.11±1.17 4.44±1.90b moderate 5.06±1.37 6.39±3.32 7.5.0±2.28 5.90±2.62b stress 9.38±3.16 10.07±2.47 11.07±1.59 10.04±2.72a mean**2 6.19±3.25 7.08±3.82 7.33±3.19 * during photosynthesis measurements swc kept at 100% fc. ** during measurements swc kept at 60%. 1 and 2, means across different soil water contents and different strawberry lines, repectively, when watered to 100 and 60% swc. ¥means in the last column for each separate row that carry the same letter are not signifi cantly different (p<0.05). water water strawberry lines line j line j aline jaline j line jm control 186 v. chalavi, m. raeini-sarjaz water stress and transgenic strawberry response therefore, they concluded that only the level of endochitinase activity had the negative effect on growth of apple plants. of transgenic lines, which could be an indirect marker for longterm higher stomatal conductance (raeini-sarjaz and chalavi, 2011). it has been shown that carbon isotope discrimination against 13co2 in leaf tissues is correlated to dry matter production (zacharisen et al., 1999) and leaf stomatal conductance (osório et al., 1998; farquhar et al., 1989). carbon isotope discrimination due to genotype or growth environment is considered as an indicator of higher long-term stomatal conductance (farquhar et al., 1989). therefore, higher dry matter production in transgenic plants could be due to larger leaf area and higher long-term stomatal conductance. the 13c enrichment of leaf tissues signifi cantly reduced in transgenic plants compared to control plants (raeini-sarjaz and chalavi, 2011), while dry matter increased. this could indicate a longterm higher stomatal conductance to ambient co2 relative to nontransgenic plants. the vigorous growth enhancement of transgenic plants for both well and stress watered plants could be related to this long-term stomatal conductance increase. therefore, the expression of chitinase in these lines kept the stomata open for longer time relative to non-transgenic plants, perhaps via direct or indirect effects on mechanisms which control stomatal conductance. however, this higher performance was not seen for moderate watered transgenic plants compared to corresponding treatment of control plants. the same trend was reported for 13c enrichment of moderate watered transgenic plants (raeini-sarjaz and chalavi, 2011). it seems under this experimental condition and probably due to the induction of strawberry endogenous chitinase gene, the moderate watered control plants were able to compete with their transgenic counterpart plants. the high level of expression of chitinase (chalavi et al., 2003) in transgenic lines not only enhanced plant growth, it also increased plants’ tolerance to a fungal disease (chalavi et al., 2003) and abiotic stresses such as water stress. water consumption of transgenic plants reduced per leaf area compared to non-transgenic ones. transgenic lines also demonstrated enhanced wue and produced more dry matter per unit of consumed water in comparison to non-transgenic ones, which is in agreement with dry matter production and water used per leaf area of these lines. water stress also enhanced wue in both transgenic and control plants, in agreement with fi ndings of grant et al. (2010) and liu et al. (2007). in conclusion although our results demonstrates that the chitinase gene is possibly involved in the growth enhancement of transgenic lines relative to non-transgenic ones under well water and water stressed conditions, further investigation at the protein level would provide more information. such study might help to defi ne the correlation between the transgene transcript levels, the corresponding protein content and the growth of transgenic plants. references alvin, f.c., carolino, s.m.b., cascardo, j.c.m., nunes, c.c., martinez, c.a., otoni, w.c., fontes, e.p.b., 2001: enhanced accumulation of bip in transgenic plants confers tolerance to water stress. plant physiol. 126, 1042-1054. bevan, m., 1984: binary agrobacterium vector for plant transformation. nucl. acids res. 12, 8711-8721. bolar, j.p., norelli, j.l., harman, g.e., brown, s.k., aldwinckle, h., 2001: synergistic activity of endochitinase and exochitinase from trichoderma atroviride (t. harzianum) against the pathogenic fungus (venturia inaequalis) in transgenic apple plants. transgenic res. 10, 533-543. caruso, c., chilosi, g., caporale, c., leonardi, l., bertini, l., magro, p., buonocore, v., 1999: induction of pathogenesis-related proteins in germinating wheat seeds infected with fusarium culmorum. plant sci. 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g., 2000: chitinase genes responsive to cold encode antifreeze proteins in winter cereals. plant physiol. 124, 1251-1263. yu, l.x., djebrouni, m., chamberland, h., lapontaine, j.g., tabaeizadeh, z., 1997: chitinase: differential induction of gene expression and enzyme activity by drought stress in the wild (lycopersicon chilense dun.) and cultivated (l. esculentum mill.) tomatoes. j. plant physiol. 155, 745-753. zacharisen, m.h., brick, m.a., fisher, a.g., ogg, j.b., ehleringer, j.r., 1999: relationships between productivity and carbon isotope discrimination among dry bean lines and f2 progeny. euphytica 105, 239-250. zhong, r., kays, s.j., schroeder, b.p., ye, z.-h., 2002: mutation of a chitinaselike gene causes ectopic deposition of lignin aberrant cell shapes and overproduction of ethylene. plant cell 14, 165-179. address of the author: mahmoud raeini-sarjaz, department of agricultural engineering, sari agricultural sciences and natural resources university, po-box 578, sari, iran. e-mail: m.raeini@sanru.ac.ir; mraeini@gmail.com journal of applied botany and food quality 87, 243 248 (2014), doi:10.5073/jabfq.2014.087.034 1departamento de biología, facultad de ciencias, universidad de la amazonía, florencia, caquetá, colombia 2institute of plant production and agroecology in the tropics and subtropics, university of hohenheim, stuttgart-hohenheim, germany arbuscular mycorrhiza in colombian coffee plantations fertilized with coffee pulps as organic manure raúl hernando posada1, ewald sieverding2* (received december 23, 2013) * corresponding author summary the distribution of arbuscular mycorrhizal (am) fungal structures in roots, in soil surrounding roots and in amended coffee pulps (cp) was investigated in 12 coffee plantations in colombia. fresh cp had been added to plants 6 -10 months before sampling. the questions were whether soil chemical and physical parameter and soil depth had an effect on mycorrhiza. root colonization rates with am in-oot colonization rates with am increased in cp amended-plants (f = 7.75, p< 0.001) as compared to a non-amended control. significantly more roots, cp and am root colonization were found in the upper soil layer (f = 41.24, 9.54, 6.60 respectively, p< 0.001), while root external mycelium and cp colonization with am were not affected by soil depth (f = 14.82, p> 0.05). external mycelium length differed between locations (f = 5.89, p< 0.001) and was inversely correlated with soil water content (r = -0.655, p = 0.02). external mycelium length per am colonized root was higher in the lower soil layer (f = 14.82, p< 0.05). soil aeration seemed to be an important physical characteristic for mycorrhiza development in and around coffee roots. higher mycorrhiza colonization in cp amended-plants might be an adaptive strategy for nutrients acquisition, and am external mycelium that colonizes cp might take up nutrients directly during cp decomposition. introduction coffee (coffea arabica l.) is one of the most important cash crops in tropical countries of central and south america, as well as of tropical africa and asia (iica and promecafe, 1997). coffee is often grown in nutrient-deficient soils, where its establishment, development and production are limited by low levels of available phosphate (p) (gaur and adholeya, 2002). the response curves to p fertilization indicate that coffee is an obligate mycotrophic plant depending on am for its growth (osorio et al., 2002; de almeida et al., 2003) and field trials have shown that arbuscular mycorrhizal fungi (amf) can increase coffee growth and productivity up to 62 % (siqueira et al., 1998) via improved uptake of potassium and p (siqueira et al., 1994). in coffee seedlings propagated in vitro, vaast et al. (1996) found that am enhanced root and shoot growth and plant p status, resulting in a lower root-to-shoot ratio compared to non-mycorrhizal plants. in adult coffee plants, mycorrhizal root colonization rates varied from 4 to 80 %, indicating the existence of environmental factors that influence am under field conditions (saggin-junior and siqueira, 1996). the consistent effects of am on plant development and productivity are higher in young seedlings and are diminished with crop age. in order to conserve soil fertility, it is recommended to add organic amendments to coffee plantations, including fresh organic manure, compost and coffee pulp (fedecafe, 2003; vaidya et al., 2008). the coffee pulp (cp) is the main byproduct in the wet separation of coffee beans from fresh berries. the pulp consists of both the exocarp and mesocarp of berries. for every ton of coffee beans, about two tons of cp are produced (hofmann and baier, 2003). organic matter content of dry cp is > 90 %; cp is rich in nitrogen (n, about 1-2 %) and low in p (< 0.1 %), while the c:n ratio is about 30 (blandón et al., 1998). fresh cp is superficially incorporated into the upper soil layer around growing coffee plants. the slow decomposition of cp releases nutrients, which in turn are taken up by microorganisms or plants. organic amendments, like cp, can increase water-holding capacity and reduce leaching of mineralized nutrients from soils. latter is especially important in hilly landscapes with coffee plants (arellano et al., 2000). positive effects of such amendments are also increased soil aggregation, decreased soil compaction, and increased microbial activity (celik et al., 2004; zeleke et al., 2004; gryndler et al., 2006). the general consensus is that organic amendments have beneficial effects on development of am (gryndler et al., 2006; vaidya et al., 2008). it was reported that the mycelium of amf could colonize rotten residues and organic materials in diverse ecosystems (aristizabal et al., 2004, posada et al., 2012), but its distribution in decomposing cp has not been evaluated. superficial cp incorporation in coffee plantations implies more abundant nutritional supply to the topsoil. although soil depth effects on amf communities and root colonization were investigated with many plants in the past (sieverding, 1991; oehl et al., 2005; smith and read, 2008), root external mycelium was seldom investigated at different soil depths (steinaker and wilson, 2008). the root external mycelium may not only provide an increased surface area for interactions between plants and soil particles and between organic matter and their decomposing microorganisms, but also provide an important pathway for the translocation of energy-rich plant assimilates (products of photosynthesis) to microorganisms in deeper soil zones (finlay, 2008). on the other hand, soil parameter such as water content (stevens and peterson, 1996; schack-kirchner et al., 2000), ph (heijne et al., 1996; álvarez-sánchez et al., 2011), organic carbon (c) content (zhu and miller, 2003), and interactions between soil parameter (posada et al., 2008) may affect am mycelium development, spore production and root colonization. this study aimed to answer the following questions: 1) do cp amendments influence the occurrence and distribution of am in roots, rhizosphere (soil around roots) and cp in coffee plantations grown under different soil chemical and physical conditions? 2) are mycorrhizal parameters in roots, rhizosphere, and cp influenced by soil depth? materials and methods soil sampling was conducted in a coffee production area of caquetá, colombia. the area is at an altitude between 882 and 1450 m.a.s.l., with a mean annual temperature of 17 oc, annual rainfall of 3800 mm and air humidity of 80 %. the soil type in the area is oxisol. the sampling was carried out in a total of 12 coffee farms, in two farms each located in doncello (do) (1º41’n, 75º20’w), florencia (fl) (1º38’n, 76º01’w), puerto rico (pr) (1º51’n, 75º16’w), paujil (pa) (1º35’n, 75º22’w), montañita (mo) (1º46’n, 75º24’w) and san vicente (sv) (2º16’n, 74º58’w), in 2013. coffee varieties were “caturra” and “arabica”. 244 r.h. posada, e. sieverding six samples were taken in the two farms in florencia before cp application; these were used as controls. all farms had applied and incorporated one kg of fresh cp in a one m radius around the stem of the trees in coffee plantations, 6 10 months before sampling. four soil samples were collected at a distance of 50 cm from the trunk of a plant, with three plants per farm. samples were collected with a soil corer of 3.8 cm diameter to 10-cm depth. each sample was divided for the upper soil layer (0-5 cm, top) and the lower (5-10 cm, bottom), placed in plastic bags, and brought to the laboratory. samples were thoroughly homogenized and separated into decomposing cp, roots and soil. the proportion of cp, roots and soils were estimated as their fresh weight. to characterize the cp at each depth, the areas of 15 randomly selected fragments were measured using sigma scan pro 5.0 (spss inc.); the area was used as an indicative for decomposition stage of cp – smaller size indicating more advanced decomposition. soil water content was measured using subsamples by drying at 80 oc for 72 h. sub-samples of soils from the same farm were mixed and homogenized. this pooled sample (400 g) was sent to the instituto geográfico agustín codazzi (igac), bogota, for physical and chemical characterization: texture was determined by bouyoucos method (bouyoucos, 1962), ph in h2o (1:1) by potentiometer, cation exchange capacity (cec) in kcl, saturation of bases in %, organic c according to walkley-black (nelson and sommers, 1982), and plant available p according to bray ii (fixen and grove, 1990). a dried pooled cp sample (2 g) of each farm was sent to igac for the determination of c and n content, by dry combustion and thermal conductivity using an elemental analyzer (leco 1000). external mycelia of am fungi were measured according to herrera et al. (1986) as follows: a 2 % h2o2 dilution was added to a defined air-dried sample, blended for 30 s, rinsed on a 45 μm-mesh-opening sieve, air dried for 48 h and weighed. a sub-sample of 0.02 g of this was mixed with 2 drops of glycerin (100 %) on a microscope slide. the number of coenocytic hyphae that intersected four squared transects (two horizontal and two vertical) on each slide were counted at 100 times magnification under the microscope. the mycelium length per gram of dry sample was calculated as: average number of intersections/transect x 1.57 mm/intersection x 20 transects/22 mm square cover-slip x weight of material from the 45 μm sieve/0.02 g sub-sample (modified from herrera et al. (1986)). the results were expressed in meters of root external am mycelium per gram of dry sample. sub-samples of cp fragments were cleared by the koske and gemma (1989) technique, as modification of phillips and hayman (1970). decomposing cp was carefully cleaned and submerged in koh, extremely lignified tissues were exposed to a mix (1:1 v/v) of 2 % h2o2-nh4oh (schenck, 1982). cleared samples were acidified with 1 % hcl during 60-600 s and amf mycelium was stained with acid trypan blue (0.5 g l-1 trypan blue) at 85 oc for 1 h. to assess the amf colonization of cp, small randomly selected cp fragments were placed adjacent on microscope slides to form lines 2 mm broad and 18 mm long, and mounted in polyvinyl-lacto glycerol under a cover slip. on each slide nine transects of each line were observed for amf colonization. cp fragments were considered to be colonized if they contained either coenocytic mycelium with unilateral angular projections (nicholson, 1959) or coenocytic hyphae with terminal vesicles or spores. cp colonization rate was estimated as the number of colonized intersections, divided by the total number of observed intersections multiplied by 100. roots from each sample were cleared according to the koske and gemma (1989) method, as a modification of phillips and hayman (1970). the cleared roots were acidified with 1 % hcl during 3001200 s and the am fungal structures were stained with acid trypan blue (0.5 g l-1 trypan blue) at 85 oc for 1 h. the stained roots were mounted on microscope slides for assessment of percent am colonization by the magnified intercept method (mcgonigle et al., 1990). for statistical analyses, am root colonization and soil water content did not require normalization. colonization of cp by am mycelium was normalized by x-1/2 transformation; the external mycelium was normalized by sen x transformation and the remained variables were normalized by – log 10(x+1) transformation (zar, 1999). in each location, no large numerical differences were observed in soil characteristics between the two farms, except for el vergel in puerto rico, where p content was much higher. thus, we decided to process the data for the am parameters by location. two-way anova and the least significant difference (lsd) post-hoc test (statistica ver. 6.0 programme of statsoft, 2001) were used to evaluate statistical differences between locations and soil depths for cp and root colonization by amf, soil water content, am mycelium length, root and cp fractions. the kruskal-wallis test was employed to compare the area of cp fragments between locations and soil depths, separately. spearman’s rank correlation coefficients were computed for all pairwise combinations of measured variables (zar, 1999). results soils in the study area were clay-sandy-loams with a ph between 3.8 and 4.7. the contents of organic c and for plant available p were low, except for el vergel with an intermediate level of p (tab. 1). cec was very low in all locations. highest c contents were found in doncello and florencia with 2.6 3.6 %; it varied in the other locations from 1.9 % to 2.7 %. content of n in soils was between 0.08 0.22 %. significant differences were found among locations in soil water contents, cp fragment areas, am root colonization and root external mycelium length (tab. 2). root colonization was lower in control plants than in cp-amended plants. significantly more roots and cp fractions, as well as higher am root colonization were observed in the top soil layer than in the bottom layer (tab. 2). however, there were no significant interactions between locations and soil depths. calculating the root external mycelium length in relation to percentage of root or cp fraction in soil, more mycelium per unit root was found in deeper soil layers, while there was no such clear indication of mycelium length with the cp fraction (tab. 3). on the other side, soil depth did not influence the mycelium length per am colonized-root, but mycelium length per am colonized-cp was consistently lower in the bottom soil layer (tab. 3). the ph, cec, organic c, base saturation, available p, and water contents did not indicate any influence on am root colonization or on am colonization of cp. c:n ratios in cp, organic c and soil water content were negatively correlated with am mycelium in soils (r = -0.8738, p = 0.023; r = -0.7096, p = 0.010; r = -0.6551, p = 0.02, respectively). more cp fragments, more roots, and higher am root colonization were found in the top soil layer than in the bottom layer. although there were no significant differences between soil depths in cp colonization with am or external mycelium length (tab. 2), in the majorities of the locations, more mycelium per am colonized-cp was found in the upper soil layer (tab. 3). discussion the present study showed that cp amendments resulted in higher am root colonization of field grown coffee (66 ± 19 % vs. 35 ± 6 % without amendment). osorio et al. (2002) reported increases in height, leaf number, root, shoot and total dry weight but not in root colonization of coffee seedlings amended with cp under greenhouse conditions. our results indicate and confirm that amendments arbuscular mycorrhiza in coffee 245 tab. 1: chemical properties of soils and decomposing coffee pulp (cp) in six locations of caquetá, colombia. locationa farm soil cp ph cation base organic available % c % n c:n exchange saturation carbon % phosphorus capacity % mg / kgb meq/100 g do los naranjos. 4.1 2.9 81.9 2.4 nd 3.5 0.13 26.9 la argelia. 3.9 3.3 82.8 1.8 nd 2.6 0.08 32.5 fl la primavera. 3.9 4.7 91.4 2.8 0.6 3.6 0.18 20.0 las brisas. 3.9 3 89.6 1.9 nd 3.2 0.21 15.2 mo peñas altas. 4.5 1 26.8 1.7 3 2.7 0.18 15.0 la esperanza. 4.3 1.6 42.7 1.4 nd 2.4 0.15 16.0 pa la florida. 3.8 3.2 89.6 1.7 0.6 2.9 0.22 13.2 sombra palestina. 3.9 2.3 83.7 1.9 2.6 2.9 0.11 26.4 pr la floresta. 4.6 1.4 32.6 0.5 4.3 2.1 0.22 9.6 el vergel. 4.7 1.1 24.9 1.8 24.5 2.6 0.21 12.4 sv la lindosa. 4.5 0.7 32.7 1.2 nd 1.9 0.15 12.7 el cedral. 4.4 1.4 63.1 1.3 nd 2.2 0.21 10.5 a do: doncello; fl: florencia; mo: montañita; pa: paujil; pr: puerto rico; sv: san vicente. b nd: not detected. tab. 2: soil water content, fraction of roots and coffee pulps (cp) in soils, area of cp fragments, and mycorrhizal parameters investigated in top layer (0-5 cm) and bottom layer (5-10 cm) at different locations of caquetá, colombia. locationa soil depth water content % roots cp fraction cp fragments mycorrhizal root am external cp fraction % % mm2 colonization % mycelium mycorrhizal (m g-1 soil) colonization % do top 44±4 d 0.9±0.7 3.5±2.6 19.0±16.7 a 81.6±17.3 c 1.9±1.7 a 17.1±20.4 bottom 41±3 0.2±0.1 0.9±0.8 11.9±11.9 74.7±12.9 1.3±0.9 25.4±30.0 fl top 47±3 e 0.7±0.3 2.0±0.3 16.4±11.2 a 69.8±16.4 b 4.4±2.0 a 13.3±9.2 bottom 43±3 0.1±0.1 1.2±1.3 17.7±16.1 53.2±17.7 2.0±1.3 13.4±6.9 mo top 32±3 ab 1.5±1.1 2.7±2.8 9.4±8.7 a 62.2±17.4 b 4.2±2.6 ab 7.4±7.0 bottom 33±3 0.2±0.1 0.6±0.4 9.5±7.2 59.2±24.7 3.7±0.5 8.5±9.9 pa top 35±3 bcd 0.6±0.4 1.9±1.9 238.7±275.1 b 78.3±10.4 bc 6.2±4.7 abc 16.6±13.7 bottom 39±9 0.1±0.0 1.0±1.2 289.5±225.6 63.6±6.9 2.8±2.1 16.5±18.6 pr top 35±2 bc 0.6±0.3 0.5±0.3 9.5±12.0 a 62.7±24.6 bc 5.2±3.7 bcd 9.6±9.4 bottom 34±6 0.2±0.1 1.0±1.2 7.3±5.9 68.0±19.0 5.5±1.5 14.4±15.6 sv top 27±6 a 0.8±0.6 1.1±0.3 297.8±274.2 b 71.2±14.2 b 6.8±4.0 bcd 13.9±16.2 bottom 28±6 0.2±0.2 0.8±0.6 283.4±263.6 44.8±23.5 6.7±1.1 17.2±20.5 cb top 43±2 d 37.2±5.0 a 2.5±0.9 a bottom 39±3 32.5±6.6 2.1±0.8 anovas location (l) 22.39** 1.75 1.61 5.94** 7.75** 6.74** 0.83 depth (d) 0.87 41.24** 9.54** 1.36 6.60** 3.81 0.54 l x d 1.36 1.24 2 1.14 0.96 0.11 * significant at p < 0.025; ** significant at p < 0.001 a: do: doncello; fl: florencia; mo: montañita; pa: paujil; pr: puerto rico; sv: san vicente. b: control sample. -: not evaluated. means of column that share the same letter are localities that do not differ significantly. 246 r.h. posada, e. sieverding with organic manure have positive effects on am root colonization of adult plants under field conditions (muthukumar and udaiyan, 2000; gosling et al., 2010; montalba et al., 2010). amendments of cp consistently resulted in more cp fragments in the upper soil layer, which is not surprising as cp was only superficially incorporated. root distribution of coffee plants was highest in this layer, too, as well as am root colonization (tab. 2). we had expected more roots in deeper soil, as the upper layer was more affected by seasonal and daily changes in water contents and temperatures. perhaps the superficial proliferation of nutrimental sources led to more roots and higher am rates in the upper layer as was earlier reported by cuenca et al. (1983) for coffee grown under shade trees. in this study there was no relationship between c:n ratio of cp and the cp fragment areas, indicating that cp decomposition processes were similar at all locations. it can generally be assumed that the microbial activity and organic matter decomposition is high at soil temperatures of 15-25 ºc in the tropics, when water is not limiting the microbial activity. we found that the absolute am mycelium length in soils was inversely related to c:n ratios of cp, together with soil water content and organic c. the decreased absolute presence of root external am mycelium with higher water contents must be related to the likely higher release and availability of mineralized nutrients from cp, and potentially to poor soil aeration in water saturated soils. both factors, lack of soil aeration (muthukumar et al., 1997; bramley et al., 2007) and higher nutrient availability (smith and read, 2008; talbot et al., 2008; fitter, 2011) were reported to have negative impacts on am. colonization of cp by mycelium of am fungi was so far unknown. it is of interest to note that the colonization of cp was not significantly influenced by different soil physical and chemical characteristics at the different locations, nor by soil depth. also, the colonization rate of cp itself was totally independent on cp fragment size, cp nutrient content or c:n relation in cp, indicating that am mycelium can explore this source of organic manure at all stages of decomposition for nutrient extraction. these nutrients are theoretically then moved to the plant by the root external mycelium. although the total external mycelium was decreased by more nutrient availability, the relative root external mycelium length was not impacted by cp fractions size and by the am colonized cp fractions, at the two soil depths. this may indicate that the root external mycelium was not sensitive to nutrients potentially released by cp. further on, relating root external mycelium length to root growth, it is clear that relatively more mycelium per unit root was produced in the lower soil layer. this may lead to the conclusion that root external mycelium is much more important for nutrient acquisition from the lower soil layer, since there the distance between roots and cp fractions was bigger than in the upper soil layer. this may be of great biological importance because the nutrient uptake by the external mycelium may compensate for the patchiness of cp and heterogeneity in nutrient availability at a micro-site level (degens et al., 1996), and thus may lead to a more uniform nutrient uptake by plants (pedersen and sylvia, 1996). thus, in general, amf mycelium may result in a greater nutrient acquisition form nutrient-rich patches like cp (vaidya et al., 2008). we conclude that the application of cp, and the direct uptake of nutrients from organic sources like cp via am has great biological importance for a sustainable nutrition of coffee in soils with low nutrient availability of the tropics. acknowledgements the authors are thankful to the amazonia university, caquetá, colombia, and the federación nacional de cafeteros de colombia (fedecafe), for the economic support. the logistic support of fedecafe regional caquetá is highly acknowledged. thanks are given to the grupo de investigaciones en microorganismos tab. 3: relations of root external mycelium length (m g-1) with root fraction (% w/w), cp fraction (%) in soil, am colonized-root (%), and am colonized-cp fragments (%). locationa soil depth mycelium per root fraction mycelium per cp fraction mycelium per ammycelium per am (m g-1 / %) (m g-1 / %) colonized-root (m g-1 / %) colonized-cp fragment (m g-1 / %) do top 2.1 0.5 0.02 0.11 bottom 6.5 1.4 0.02 0.05 fl top 6.3 2.2 0.06 0.33 bottom 20 1.7 0.04 0.15 mo top 2.8 1.6 0.07 0.57 bottom 18.5 6.2 0.06 0.44 pa top 10.3 3.2 0.08 0.37 bottom 28 2.8 0.04 0.17 pr top 8.7 10.4 0.08 0.54 bottom 27.5 5.5 0.08 0.38 sv top 8.5 6.2 0.10 0.48 bottom 33.5 8.3 0.15 0.39 cb top 0.07 bottom 0.06 anova depth (f) 14.82* 0.02 0.02 2.05 * significant at p < 0.05 a for abbreviations see tab. 1 b control sample. -: not evaluated. arbuscular mycorrhiza in coffee 247 simbiontes (gims), specially to luis antonio franco, faver alvarez carrillo and juan carlos suarez for facilitating the sampling and laboratory activities. references álvarez-sánchez, j., johnson, n.c., antoninka, a., chaudhary, v.b., lau, m.k., owen, s.m., sánchez-gallen, i., guadarrama, p., castillo, s., 2011: large-scale diversity patterns in spore communities of arbuscular mycorrhizal fungi. in: pagano, m.c. 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(purple loosestrife). mycorrhiza 6, 99-104. vaast, p., zasoski, r.j., bledsoe, c.s., 1996: effects of vesicular-arbuscular mycorrhizal inoculation at different soil p availabilities on growth and nutrient uptake of in vitro propagated coffee (coffea arabica l. ) plants. mycorrhiza 6, 493-497. vaidya, g.s., keshab, s., khadge, b.r., johnson, n.c., wallander, h., 2008: organic matter stimulates bacteria and arbuscular mycorrhizal fungi in bauhinia purpurea and leucaena diversifolia plantations on eroded slopes in nepal. rest. ecol. 16, 79-87. zar, j.h., 1999: biostatistical analysis. prentice hall, new jersey. zeleke, t., grevers, m.c.j., si, b.c., mermut, a.r., beyene, s., 2004: effect of residue incorporation on physical properties of the surface soil in the south central rift valley of ethiopia. soil tillage res. 77, 35-46. zhu, y.g., miller, r.m., 2003: carbon cycling by arbuscular mycorrhizal fungi in soil-plant systems. trends plant sci. 8, 407-409. address of the authors: dr. raul posada, diagonal 151b no. 136a-75 cs 93. villas de hato chico ii, bogotá, colombia e-mail: raulposada@hotmail.com priv. doz. dr. habil. ewald sieverding, institute of plant production and agroecology in the tropics and subtropics, university of hohenheim, garbenstr. 13, d-70593 stuttgart-hohenheim, germany e-mail: sieverdinge@aol.com art14 journal of applied botany and food quality 82, 76 82 (2008) 1 institute of vegetable and ornamental crops großbeeren/erfurt e.v., kühnhausen, germany 2 institute of systematic botany, friedrich-schiller-university jena, jena, germany the correlation between ovule quality parameters and the seed yield at cyclamen persicum mill. s. reinhardt1, a. ewald1, f. hellwig2 (received february 15, 2008) summary are there indicators that the seed yield at cyclamen persicum is predetermined by the quality of ovules? this was the main question of these investigations. the aim of our study was to investigate why only some of the available ovules develop into mature seeds. we surmised that the quality of the ovules played an important role in this. in order to corroborate this theory, we examined specific ovule parameters and their correlation with seed yield. the parameters included the levels of callose in the nucellus, the heterogeneity of embryo sacs, the deviants in callose inclusion and the number of ovules examined by light and fluorescence microscopy. there is still considerable disagreement on the biological significance of the inclusion of callose in ovules. in our study, we were able to show that the inclusion of callose is essential for fertilization in the case of c. persicum. this appears to contradict the findings reported for other plant species, where the inclusion of callose has been evaluated as a sign of ovule degeneration. however, the results of our study clearly demonstrate that seed yield is already determined by the maternal plant during the ovule development phase, i. e. shortly before and at the beginning of anthesis. some ovule parameters allow predictions to be made about the expected seed yield for the studied genotypes of c. persicum. introduction cyclamens are reproduced exclusively using seeds. also, cyclamens still have a high economic value, with approximately 25 to 30 million plants sold in germany alone. in germany, they represent about 10% of the total annual production of greenhouse flowering potted plants (anonymous, 2000). the number of cultivators and growers must steadily grow to meet the demand for potted plants. since the trade in cyclamens is such big business, growers always strive to generate high quantities of seeds, independent of the genotype applied. however, differences appear in practice between individual genotypes and growing seasons. the causes for this are not fully known. in addition, a certain quantity of seed is commercially expected to produce a certain (stable) number of descendants. this requires a high quality of the seeds in respect of their germinating capacity and motivation force. we know from other plant families and types that not all ovules develop into ripe seeds (rodrigo and herrero, 1997; akhalkatsi et al., 1999). this phenomenon is thought to be an expression of an evolutionary, essentially sexual selection. it appears self-evident that, if the mother plant is deprived of nutrient resources, only a part of the ovules can thrive. therefore, a competitive situation develops among the ovules. the ovules experience this competition repeatedly in a similar manner. above all, before fertilization, the biochemical composition of the cytoplasm must be in the ovule, as well as in the embryo sac, for an optimal penetration of the pistil. this is obtained by a complex interplay of proteins, carbohydrates and inorganic substances (raghavan, 2000). following fertilization, the ovules depend on nutrition from the mother plant. it is often reported that the competitive pressures occur at this stage. in quercus suber l., only one of the six ovules (on rare occasions two) will generally fertilize (boavida et al., 1998) and develop into a ripe seed, blocking the development of the others. even if two ovules are fertilized, only one continues to develop, while the other degenerates. furthermore, it is reported that with cerasus vulgaris mill., a low yield of seeds correlates, among other things, with a high number of degenerated ovules, or anomalously developed embryo sac tissue (dys, 1984). experiments with buckwheat (fagopyrum sp.), have shown that fertilization does not take place with abnormal embryo sac tissue (obendorf et al., 1992). with hormathophylla spinosa (cruciferae), only 50% of the existing ovules usually develop into ripe seeds (gomez and zamora, 2003). in this context, the phenomenon of callose inclusion in the ovules is highly interesting. it was not known whether the inclusion of callose in the nucellus of the ovules had a verifiably negative effect on pollen tube growth or pollination. to determine this, the studies for callose inclusion in the ovule were repeated for some flowers in which pollination was induced a few days before the start of anthesis. examinations of the ovules of prunus armeniaca showed that there was a great variance regarding development conditions (alburquerque et al., 2002). here, a genotype-dependent development of the embryo sac tissue was found at the time of the anthesis. also with butomus umbellatus, fertilization failure was linked to deformed or incompletely developed ovules (fernando and cass, 1997). examinations at the leibniz institute of vegetable & ornamental crops in erfurt (germany) have shown that higher yields were achieved with cyclamen, if repeated dustings took place at the plants (ewald and schwenkel, 1997). this is conceivably to be seen as an indication of different development steps of the ovules of a placenta at the beginning of anthesis. also, with pistacia vera, it is reported that the ovules are still unripe at the time of the anthesis, and only if the pistil reaches the ovule, a ripe embryo sac becomes available (martinez-palle and herrero, 1998). in order to recognize causes for maturity, yield, and quality differences in the seeds, it is necessary to study the reproduction structures and processes prior to fertilization and in the early phases of the seed development. materials and methods plant material the plants were taken from the c. persicum cultivar ‘sylvia’ (2n = 2x = 48). ten plants had been cloned from a single plant (sk), while 20 more were obtained by sowing self-generated descendants of the same plant (ss). furthermore, 20 plants from a population (sp), and ten plants of a autotetraploid clone (tk) with 96 chromosomes were used. the clones of the diploid kind ‘sylvia’, as well as the autotetraploid clone, had been generated with a somatic embryogenesis procedure (schwenkel and winkelmann, 1998). the process of cultivating these plants took place under relative constant temperature and humidity conditions in the greenhouse. light microscopic preparation the ovaries were prepared from flowers at the beginning of anthesis and boiled in an aniline blue solution in a water bath at 100°c for 5 min. then the ovaries were carefully crushed in propantriol on a slide. this method of preparation was carried out using dark field microscopy (leitz dmrb, germany) for the subsequent callose rating under uv light stimulation as well as for examining embryo sac heterogeneity. four different examination periods were selected for the callose rating (three days before the start of anthesis, the anthesis itself, three to five days after the start of anthesis, and ten days after the start of anthesis). to identify the deviants from the callose synthesis, all specimens that had been rated according to the extent of the callose inclusion were examined. for the embryo sac heterogeneity studies, ten plants were used from each genotype. five flowers were used from each plant so that a total of 50 flowers were included in the study for each genotype. at least 100 ovules from each flower or placenta were analysed for their embryo sac surface areas, which gave a total volume of 5000 ovules per genotype for this study. in total, approximately 20.000 ovules were analysed. the surface area measurement of the embryo sac was carried out according to the planimetric method using the bioscitec cellexplorer2001. statistical analyses of embryo sac heterogeneity for the purposes of analyzing the parameter „embryo sac heterogeneity“, the coefficients of variation (as percentages) for all flowers and ovaries were calculated from the quotient of standard deviation and mean. the mean values for the resultant coefficients of variation were then determined, and a box & whisker plot was prepared for each plant of each genotype. in order to allow a comparison between the individual genotypes, the overall means of the coefficients of variation for each genotype were calculated. statistical analyses of callose inclusion four different procedures were employed for the evaluation of callose inclusion in the ovules. a score of 0 means that there was no callose inclusion in the ovules, a score of 1 that there was the beginning of callose inclusion with some callose in the ovules, a score of 2 that two thirds of the ovule area were filled with callose and a score of 3 that the ovule was completely filled with callose. the plant appraisal scores (rating method) were ranked and then subjected to a mann-whitney rank sum test. in order to evaluate the extent of callose inclusion in ovules during the various phases of ovule development, the quotient of the rank sums and number of investigated ovules per genotype was calculated and plotted in a line graph. statistical analyses of seed yield for the statistical analysis, we used statistica 6.1 (statsoft gmbh germany) and microsoft office excel 2003. to analyse the seed yield, the number of seeds per seed vessel was recorded. this gave the total seed yield. this includes the seed yield for each plant, taking into account biological selection and the competition between the ovules as well as the vitality (and therefore the quality) of the mother plant. the total seed yield is composed of the net yield, the gross yield and the seed vessel yield. the net yield indicates the number of yielded seeds per seed vessel. the gross yield describes the number of seeds in an ovarium that have been developed from the available ovules per placenta. finally, the seed vessel yield describes the proportion of yielded vessels in relation to the number of pollinated flowers. to allow a comparison between the different genotypes, the variation co-efficient of all flowers were calculated, following which we determined the mean values of the variation co-efficients of all genotypes. correlation analyses regression analyses were used in order to determine the correlation between embryo sac heterogeneity and the extent of callose inclusionas well as the deviations from standard (rating appraisal) callose inclusion in the ovules. anova (analysis of variance) and manova, with a subsequent newman-keuls test, were used for the statistical analysis of the surface area of embryo sacs. in order to test for correlations between ovule quality and subsequent seed yield parameters, regression analyses and spearman’s rank correlation tests were used. results callose inclusion a phenomenon that was observed in the not pollinated flowers of c. persicum was the temporal occurrence of callose inclusions in the cell walls of the nucellus. a particularly large amount of callose was included at the start of anthesis. it was found that the intensity of the inclusion increased or decreased at different points during the flower development (fig. 1). the callose inclusion began three days before the start of anthesis, during which a high concentration of callose was seen in the area of the chalaza (fig. 2a). however, in the remaining areas of the nucellus, only a small concentration of callose was observed. this means that, although it was very easy to differentiate the nucellus from the remaining ovule after uv stimulation, there were no large deposits of callose in the cell wall area. at the beginning of anthesis, the concentration of callose greatly increased in the area of the nucellus, and all cell walls of the the embryo sacs were affected by callose inclusions from this point on (fig. 2b). the ss was the one genotype whose values were lower than the others , three days before and 3-5 days after the start of anthesis (fig. 1). this variation however fig. 1: the levels of callose inclusion intensity (all genotypes) at different times of flower development, multifactoral analyses of variance α = 0.05. 3dba = three days before anthesis. ab = at the beginning of anthesis. 3-5 daa = three to five days after anthesis. 10 daa = ten days after anthesis. n sk, ss, sp = 120 flowers = 12000 ovules, n tk = 86 flowers = 8600 ovules. (0 no callose inclusion in the ovules. score 1 beginning of callose inclusion. score 2 ovule was filled with callose to 2/3 of the ovule area. score 3 ovule was completely filled with callose). sk ss sp t k 3 dba ab 3-5 daa 10 daa period of investigat ion 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 1,8 2,0 2,2 2,4 2,6 2,8 3,0 3,2 c al lo se in cl u si o n in te n si ty cylamen persicum, ovule quality parameters 77 78 s. reinhardt, a. ewald, f. hellwig was not significant. during the examination of callose inclusion three to five days after the start of anthesis, the inclusion was observed to decrease gradually (fig. 2c). the intensity of fluorescence decreased and it was now possible to identify areas in the nucellus that were free from callose inclusions. this was even more evident ten days after the start of anthesis, at which point the callose inclusion had almost completely disappeared in the area of the nucellus. only a weak fluorescence could still be detected, which meant that it was no longer possible to differentiate clearly between the nucellus and the rest of the ovule. the growth of the pollen tubes in ovules with callose inclusions in the area of the nucellus and the micropyle was not affected or inhibited in any way. the callose included in the area of the obturator or the micropyle clearly served as a type of guide for the orientation of the pollen tubes (fig. 3). it was observed several times that the pollen tube grew in this precise area of the ovule. in each case, only one pollen tube grew in an ovule. furthermore, seed development was established in all genotypes after pollination. callose inclusion deviants it was observed in all genotypes that some ovules from the same ovary had homogeneous levels with regard to the extent of the callose inclusion, while others did not. this had different characteristics and will be termed deviants (tab. 1). the deviants were characterised by the fact that the intensity of the fluorescence was weaker or noticeable stronger. in a period between three days before and three to five days after the start of anthesis, the genotype sk had the most deviants with 15.0%, while genotypes sp (2.2%) and tk (1.9%) had the fewest. this showed that in addition to the previously recorded structural irregularities, there were also physiological irregularities in unpollinated ovules. embryo sac heterogeneity at the start of anthesis, there was a high heterogeneity regarding size and form of the embryo sac within the ovules of an ovary (fig. 4). these differences, however, were also observed between different flowers on a plant, as well as between individual plants of a genotype. the comparative studies of the individual embryo sacs were based on the different embryo sac surface areas. in order to compare the genotypes with each other, the coefficients of variation (as percentages) fig. 2: callose inclusion in the ovules at different times of flower development of genotype sp. a. three days before the beginning of anthesis. b. during the beginning of anthesis. c. five days after anthesis. the picture shows the area of the embryo sac inside the ovules with callose inclusion. a. b. c. tab. 1: callose deviants at different times. 3 dba = three days before the beginning of anthesis. ab = at the beginning of anthesis. 3-5 daa = three to five days after beginning of anthesis. 10 daa = ten days after the beginning of anthesis. period for examination 3 dba ab 3-5 daa 10 daa total total total total invest. invest. invest. invest. genotype ovules deviants % ovules deviants % ovules deviants % ovules deviants % sk 1501 102 6.8 1929 57 2.9 1399 210 15.0 1953 52 2.7 ss 2340 22 0.9 2246 40 1.8 2135 141 6.6 1915 155 8.1 sp 2778 26 0.9 4453 58 1.3 3348 75 2.2 2955 43 1.5 tk 2496 18 0.7 2496 10 0.4 1548 29 1.9 373 10 2.7 fig. 3: pollen tube penetration in the ovule through the obturator. callose inclusion in the nucellus and the obturator (o = obturator, pt = pollen tube, c = callose inclusion in area of embryo sac). c o ➞➞➞➞➞ pt ➞➞➞➞➞ cylamen persicum, ovule quality parameters 79 of all flowers were calculated. it was observed that the genotype sk (cv% = 18.0) had the largest variability of all individual plants (tab. 2). there were significant differences between the genotypes. the total values of the individual plants fell into a wide range, similar to the values previously established within a placenta. with the ss genotype, there was also a wide range of values, although the range was narrower than with sk. sp, on the other hand, displayed the least variation (tab. 3). the values were basically characterised by their relative homogeneity. tk was also found to be relatively homogeneous with regard to these values. as a result, it was possible to identify differences regarding the embryo sac surface area on a placenta, both between different ovaries of an individual plant and between different individual plants of a genotype. in addition, the examined genotypes varied considerably in terms of the range of values. relationship between the ovule parameters it was possible to determine a relationship between the values of the embryo sac heterogeneity and the total mean values of the deviants from callose rating. both factors correlated positively with each other. this correlation is highly significant for p<0.01 (tab. 5). these differences between plants could also be observed in the direct comparison of the individual genotypes. there was also a correlation between the heterogeneity of the embryo sacs and the extent of the callose inclusion. the two factors correlated negatively with each other. it was evident that a high embryo sac heterogeneity correlated with a weak callose inclusion in the embryo sacs. the homogeneity of the embryo sac surface area, however, correlated with a high callose inclusion. the same relationship between individual plants could also be observed between the genotypes under examination. the genotypes sk and ss had higher variation co-efficients with regard to the embryo sac heterogeneity than the genotypes sp and tk (tab. 2). it was also evident that the genotypes sk and ss had the lowest levels of callose inclusion at the start of anthesis. the genotypes sp and tk, on the other hand, had significantly higher levels of callose (fig. 1). there were also highly significant correlations between the mean number of ovules per ovary, the callose inclusion and the embryo sac heterogeneity (tab. 5). seed yield only about 20% of all the ovules in the diploid genotypes developed into visually ripe seeds (tab. 4). there were no significant differences within the diploid genotypes in terms of the gross yield (tab. 4). clear and sometimes significant differences within the examined genotypes were observed in the net yield as well as the seed vessel yield. the sp genotype returned the highest values for both parameters under examination, and sk had the lowest values within the diploid genotypes. these differences were also observed in the total seed yield. the sp genotype had the highest total seed yield. in contrast, the total seed yield for the sk genotype was the lowest, falling short – by a significant margin – of the total seed yield for the sp and ss genotypes. relationship between ovule parameters and seed yield in this context, it was possible to establish a correlation between the previously determined and examined ovule quality parameters and the parameters of the seed yield (tab. 5). the strongest correlations were found between ovule and seed parameters at the diploid genotypes. it was evident that there was a strong correlation between the extent of the callose inclusion, the net yield and the seed vessel yield, furthermore between embryo sac heterogeneity, callose deviants and all parameters of seed yield. the strongest correlation was observed between the net yield (seeds per vessel) and the extent of the callose inclusion and the embryo sac heterogeneity. discussion the levels of callose inclusion were similar for all genotypes under examination. an increase in the callose inclusion was observed in all genotypes three days before and during the start of anthesis. three to five days after start of anthesis, there was a clear reduction in the callose inclusion. as before, there is still disagreement over the precise development of this inclusion, the biochemical details of the callose synthesis and the biological nature or consequences of this phenomenon. callose has a comparable biochemical structure to cellulose. fig. 4: heterogeneity in the size of ovules and of embryo sacs in ovules at the beginning of anthesis (ov = ovules; es = embryo sacs) ➞➞ ➞➞➞ es es ov ➞➞➞➞➞ tab. 2: embryo sac area heterogeneity (see also picture 4) of all genotypes at the beginning of anthesis. coefficient of variation cv in %, mean values with the same letter within the parameter don’t vary significantly (newman-keuls test, α = 0.05). genotype cv% sk 18.0 (a) ss 16.8 (a) sp 13.1 (b) tk 14.2 (c) tab. 3: embryo sac surface area of all genotypes and normal range of the ovules concerning embryo sac surface area at the beginning of anthesis (scale 1:64 means 1 µm2 = 64 µm2 = area x 64). genotype normal range total number ovules at ovules out of (area µm², 1:64) of ovules normal range normal range (%) (%) sk 361.9 461.9 6559 46 54 ss 287.0 387.0 5126 45 55 sp 246.2 346.2 8280 68 32 tk 376.2 476.2 5116 47 53 80 s. reinhardt, a. ewald, f. hellwig it is a polyglucan of β 1➞3 glycosidic bonded glucose elements that have predominantly linear bonds. alongside this, 1➞4 or 1➞6 bonds can occur between neighbouring molecule chains caused by the buildup of hydrogen bonds. the polymer has a helical structure to which the aniline blue bonds. under uv stimulation, this leads to a greenyellow fluorescence. the biosynthesis of the callose occurs on rosetteshaped protein complexes of the plasma membrane. it is assumed that the callose-synthase-complex and the cellulose-synthase-complex are identical. the enzyme responsible for this reaction is the celluloseglycosyltransferase and is part of a membrane complex in the plasmalemma (strasburger, 1998; richter, 1988). in a normal situation, the cellulose synthesis as a main element of the cell wall is catalysed on this enzyme complex. sometimes this reaction fails. instead of β1➞4 glucan bonds, β-1➞3 bonds are now linked. for this reaction, calcium is essential as a co-factor (gayle and franceschi, 2000). both the cellulose-synthase-complex and the callose-synthase-complex can specifically or randomly react to biotic and abiotic stress (verma and hong, 2001). it was demonstrated on gossypium hirsutum that carbon can be included both in cellulose and in callose, benefiting from the presence of calcium (amor et al., 1995). although callose inclusion has been observed in many studies about different plant families and representatives of plant families, it is still unclear whether this is a defensive mechanism on the part of the ovule against selfpollination (le roux et al., 1996) or an early indication of the ovule’s increasing degeneration (stösser and anvari, 1982; dittmann, 1998; detzel, 1998), or even an indication of fungal infection of the ovule (cohan et al., 1990; jacobsen and berry, 2002). in this context, however, the discussion is about whether callose inclusion leads to an intermittent isolation of the embryo sac from the surrounding tissues, as a result of the associated restricted permeability between the cell walls and the impaired metabolite transport between the cells or the tissues. also under discussion is whether this causes an unimpaired, autonomous development of the embryo sac, and whether it protects it against damaging exogenic influences (kapil and tiwari, 1978; tiwari, 1982; kapil and bhatnagar, 1981; bouman, 1984; colombo and angenent, 1999). during the development of the megaspore mother cell, or more precisely, during mitotic cell division, the occurrence of callose around the dyads, triads, and tetrads was a frequently observed phenomenon (porceddu et al., 1999; tucker, 2001). this callose inclusion around these cells probably causes the degeneration of the three megaspores (rodkiewicz, 1970; huang and russel, 1992). fundamental studies into callose inclusion in the context of embryo genesis were carried out on arabidopsis thaliana (webb and gunning, 1994). callose inclusion has often been observed in connection with a sterility of this ovule. this was also the case with medicago sativa (rosellini et al., 1998 and 2003; kolyasnikova, 1985), and other medicago types (oryol et al., 1986). ovules with callose inclusion are therefore deemed sterile, not functionable (knox et al., 1986) and non-viable (dumas and knox, 1983). however, indications of degeneration are often associated with callose inclusion, as shown in studies on prunus dulcis (pimienta and polito, 1982), prunus armeniaca (rodrigo and herrero, 1997) and other prunus types (stösser and anvari, 1982). in carica papaya, callose was found in 90% of the aborted ovules, in these cases in the region of the funiculus (bautista-calles et al., 1999). this indicates the selective permeability of the callose-affected cell walls, and the consequently impaired metabolite transport into the ovule. in contrast to the possible functions of the callose inclusion mentioned above, however, the question whether the callose inclusion may be linked to the pollen tube growth, or more precisely, with the pollen tube direction, is still in the focus of discussion. an example for this dicussion is the case of pistacia vera, in which callose appeared primarily in the region of the ponticulus, where the cells of the ponticulus form and discharge callose. after pollen tube penetration, the ovule separates from the remaining tissue because of the callose (martinez-palle and herrero, 1995). in ipomoea purpurea, it was observed that the transmission tissue in the stigma, through which the pollen tube has to grow, had a higher concentration of callose (diaz-pontones, 2000). even in galanthus nivalis, in addition to polysaccharides, proteins, lipids, and calcium, callose was found in the micropylar channel and these substances had a direct influence on pollen tube growth. part of the reason for this was that the proportion of the substances greatly decreased after pollination, and these substances did not occur in sterile ovules (chudzik and sniezko, tab. 5: correlations between callose inclusion intensity, callose deviants, embryo sac area heterogeneity, mean number of ovules per ovary and yield parameters of diploid genotypes (sk, ss, sp). regression analyses, with * marked values are significant p<0.05 and with ** marked values are significant p<0.01. callose inclusion intensity callose deviants embyo sac area heterogeneity callose inclusion intensity 1 callose deviants 0.45* 1 embryo sac area heterogeneity 0.56* 0.76** 1 ovules per ovary 0.75** 0.35 0.60** gross yield (seeds per ovary) 0.34 0.55* 0.54* net yield (seeds per vessel) 0.72** 0.57* 0.74** seed vessel yield 0.63** 0.49* 0.51* tab. 4: key data from all genotypes concerning the mean number of ovules per ovary and yield parameters gross yield (seeds per ovary), net yield (mean number of seeds per vessel), seed vessel yield (developed vessels after pollination), mean values with the same letter within the parameter don’t vary significantly (t-test, α = 0.05). genotype mean number gross yield % net yield seed vessel yield total seed yield of ovules per ovary n % % sk 145 (a) 21.0 (a) 30.5 (b) 23 (b) 4.6 (b) ss 190 (b) 21.2 (a) 40.3 (b) 54 (bc) 12.5 (a) sp 237 (c) 23.6 (a) 56.0 (a) 75 (a) 17.9 (a) tk 211 (d) 13.1 (b) 27.8 (b) 40 (bc) 5.4 (b) cylamen persicum, ovule quality parameters 81 2003). the same phenomenon was observed in a study about paspalum in which the callose served as a type of guide, used by the pollen tube to enter the inside of the ovule via the micropylar channel (chao, 1971). the successful development of the pollen tube in a calloseaffected ovule was also observed in arabidopsis thaliana (raghaven, 2000). this theory is further supported by the fact that in fundamental studies on callose inclusion in petunia hybrida, a periodicity of the inclusion was seen, showing that the inclusion begins during pollination, and ends after pollination (esser, 1963). our own studies on cyclamen persicum confirm this theory. on the one hand, the same degree of periodicity was observed, and on the other hand, we found on several occasions that pollen tubes were developing into calloseaffected ovules. the callose concentration was especially high in the area of the obturator and the micropylar channel at the moment of pollen tube penetration, which probably means that the callose should be viewed here as a functional pollen tube attractant. therefore, the callose inclusion can be defined as a phenomenon in functional ovules. this also corresponds to the fact that there is a negative correlation between the parameters ‘embryo sac heterogeneity’ and ‘extent of the callose inclusion’. the callose inclusion and the associated biological efficacy is therefore higher in ovules with rather homogeneously developed embryo sacs. the deviants identified in the callose rating are therefore regarded as non-functional or partially functional ovules. this view is also supported by the close correlation between the embryo sac heterogeneity and the deviants from the callose rating. in addition to the different basic forms of the ovules on a placenta, it was also possible to identify heterogeneous shapes and surface qualities of the embryo sacs. the phenomenon of disparity between embryo sacs has been reported in many studies. in medicago sativa, the embryo sac heterogeneity is regarded as the cause for poor seed yield (oryol and kazachkovskaya, 1991). embryo sac heterogeneity was even reported for prunus sp. as an indication for the lack of a functional ovule, linked with a poor yield (eaton and jamont, 1964). with ipomoaea nil, there was a connection between aborted ovules and simultaneous embryo sac degeneration (miyajima et al., 2003). this was also observed with olives (rapoport and rallo, 1990). with petunia inflata, a correlation was reported between embryo sac heterogeneity and failed pollination, which also caused a reduced seed yield (lee et al., 1997). basic studies on arabidopsis thaliana showed conclusively that a normally developed gametophyte was necessary for direct pollen tube growth, whereas defective ovules with abnormally developed embryo sacs were not pollinated (hülskamp et al., 1995). this demonstrates that pollination is possible only when a completely formed embryo sac, including egg apparatus, is present (shimizu and okada, 2000). the shape and size of the cyclamen embryo sac is determined by the shape and size of the ovule. a small embryo sac indicates that the ovule has formed abnormally. therefore, it can be interpreted as a failed formation with limited functionality. on the basis of the fore-going studies, the ovule characteristics described earlier – i.e. ‘embryo sac heterogeneity’, ‘callose inclusion’, and ‘callose inclusion deviants’ – were classified as ovule quality parameters. it appeared that only a fraction of the large number of ovules available on a placenta developed into ripe seeds. this meant that in the diploid genotypes, only approx. 20% of all ovules developed into ripe seeds (only 13% of the tk genotype). the abort rate was therefore between 80% and 87%. this phenomenon is well documented: with melilotus officinalis, only one ovule out of eight developed into a ripe seed (akhalkatsi et al., 1999), with prunus armeniaca, only one in two ovules succeeded (rodrigo and herrero, 1997), the same success rate observed for quercus suber (boavida et al., 1999), while, in hormatophylla spinosa, the proportion of ripe seeds from the existing ovules usually falls short of the 50% mark (gomez and zamora, 2003). it is therefore assumed that the failure on the maternal side is determined (akhalkatsi et al., 1999; karkkainen et al., 1999), among other causes, by a lack of available nutrients (stephenson, 1981). in addition, ovule sterility is also a major reason for poor seed yield, as was demonstrated in the case of trifolium repens (thomas, 1996). however, a relationship has also been frequently established between abnormal ovule development, and therefore abnormally formed embryo sacs, and a poor seed yield (oryol and kazachkovskaya, 1991; eaton and jamont, 1964). this kind of relationship was also found in the present studies. with an increasing homogeneity of the embryo sacs of the ovules on a placenta, the seed yield was also high, whereas a lower seed yield correlated with a high heterogeneity of the embryo sacs. this is possibly an indication of the vitality and therefore the quality of the mother plant. there was also a correlation between the extent of the callose inclusion and the seed yield. this could also be seen as an indication for the quality of the mother plant, much in the same way as the embryo sac heterogeneity. from this, it can be concluded that a high quality of the mother plant also means an increased availability of nutrients for the developing seeds and ovules. this also benefits the seed yield. the correlation of the deviants from the callose rating and the seed yield also supports this theory. consequently, it may be concluded, on the one hand, that the ability of the mother plant to create a large number of ovaries is an indication for its vitality, and, on the other hand, that the quality of this mother plant allows a high seed yield to be achieved. the presence of any irregularities in the ovule or seed development can be measured and predicted reliably on the basis of the parameters ‘embryo sac heterogeneity’, ‘callose inclusion in the ovules’, and ‘callose inclusion deviants’. as is shown in the present studies, however, the parameters ‘number 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e.v., kühnhäuser straße 101, 99189 kühnhausen, germany prof. dr. frank hellwig institute of systematic botany, friedrich-schilleruniversity jena, philosophenweg 16, 07743 jena, germany << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false /maxsubsetpct 100 /optimize true /opm 1 /parsedsccomments true 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/pagesize [907.087 680.315] >> setpagedevice journal of applied botany and food quality 86, 55 58 (2013), doi:10.5073/jabfq.2013.086.008 1 ministry of food, agriculture and livestock, alata horticultural research station directorate, erdemli-mersin, turkey 2 faculty of agriculture, ataturk university, erzurum, turkey 3 faculty of agriculture, university of erciyes, kayseri, turkey 4 faculty of science, ataturk university, erzurum, turkey determination of genetic diversity among wild grown apricots from sakit valley in turkey using srap markers h. pinar 1, m. unlu 1, s. ercisli 2*, a. uzun 3, m. bircan 1, k.u. yilmaz 3, g. agar 4 (received december 19, 2012) * corresponding author summary sequence-related amplified polymorphism (srap) marker was employed for the first time to analyze genetic diversity of 57 seed propagated early-maturated wild grown apricot genotypes sampled from different parts of sakit valley in mediterranean region of turkey. from total 19 primer combinations investigated, 16 could amplify clearly and consistently. they produced a total of 87 fragments, of which 56 (64.3%) were polymorphic bands. all bands obtained from me3-em2, me2-em10 and me2-em6 primers were polymorphic. the cluster analysis revealed that the 57 genotypes were grouped into three major clusters. the similarity ratio among genotypes was between 0.73 and 0.94. there were no identical genotypes. the study revealed that the srap marker system is useful for identification and genetic diversity analysis of wild grown apricots. introduction turkey is one of the richest countries in terms of biological diversity, due to its specific geographic position between asia and europe and characteristic ecological, climatic and geomorphologic conditions (ercisli, 2004). apricot trees can be grown both temperate and subtropical zones in the world. china, the irano-caucasian region (turkey and iran), central asia, europe and north america are the main apricot producer regions in the world (halasz et al., 2010). turkey has been dominating world apricot production for a long time and the country is one of the main diversity centres of this unique fruit (ercisli, 2009). the central asia is the oldest and the primary genetic source of apricot groups and central asian accessions are self-incompatible; the irano-caucasian apricots which are mostly the cultivated ones are mostly self-incompatible, with large fruits and low chilling requirements (mehlenbacher et al., 1991; romero et al., 2003; halasz et al., 2005). one of the most widely spread wild edible fruits in turkey are apricots. they are called ‘zerdali’ in turkish. continuous seed propagation of wild apricots hundreds of years in different agro climatic conditions in turkey is responsible for high phenotypic variability. owing to the long-lasting process of natural selection, wild grown apricots have adapted to ecological conditions of habitats and developed natural resistance mechanisms to biotic and abiotic environmental factors (ercisli, 2009). wild grown apricot (prunus armeniaca l.) is an economically important fruit crop in particular for local people living in rural areas in turkey. it is a multi-purpose fruit tree and besides its fresh edible fruits, it is used in diverse ways because the fruits have distinct taste and aroma. edible fruits of wild apricots have been used from the past till now as dry fruit, processed into jam, marmalade, fruit juice etc. in turkey. traditional uses and drying of apricot fruits have been found to be of great significance in the socio-economy of people in these areas. the bitter seeds of wild grown apricots are valuable material for pharmacology to treat cancer. in turkey, all apricot cultivars are grafted on seedlings obtained from wild apricot seeds. more recently, wild grown apricot fruits have been gaining more importance particularly in fruit juice industry in turkey and there is growing interest in wild grown apricot juice because of its better sugar/acidity balance compared to juice from cultivated apricots. it is also believed that most of turkish apricot cultivars were directly selected among wild grown apricots populations within several regions of turkey (altindag et al., 2006). the wild grown apricots display great phenotypic diversity in turkey and they have potential to be used practically in apricot breeding programmes. natural populations of wild grown apricots in turkey are mainly found on field borders, some forest edges, and wide areas of lowland and mountains. besides this, they also can be found in extensive fruit orchards (ercisli, 2009). in turkey, there are several valleys such as sakit valley, aras valley and coruh valley which have important wild grown apricot genetic resources (durgac, 2001; ercisli, 2009). the diversity in apricots has been studied with pomological, morphological and phenological characteristics for a long time (guerriero and watkins, 1984). in recent years, dna-based markers are widely used to clarify the genetic relationship among the apricot accessions (romero et al., 2003; yilmaz et al., 2012). for germplasm characterization and breeding and commercialization of promising apricot cultivars, precise characterization and discrimination of the genotypes are prerequisite. different types of marker such as morphological, molecular, biochemical systems have been used for identification in horticultural plants (ercisli et al., 2007; kafkas et al., 2008; pedryc et al., 2009). however, due to the effects of environmental factors, assessment of morphological and pomological traits may be ambiguous. therefore, markers independent from the environment are necessary for reliable identification and discrimination of genotypes and cultivars. dna markers are independent from environmental interactions and they show high level of polymorphism. they are considered invaluable tools for determining genetic relationships/diversity. various types of dna markers are now available. among them, rapd developed by williams et al. (1990) has been commonly used in apricot to assess genetic variability and relationships among cultivars (marinello et al., 2002; ercisli et al., 2009). more recently, issr (chenjing et al., 2005; yilmaz et al., 2012), aflp (krichen et al., 2006; yuan et al. 2007), ssr (maghuly et al., 2005; pedryc et al., 2009) and srap (uzun et al., 2010) techniques have frequently been used in apricot to characterize different genotypes belonging to diverse ecogeographical groups. sequence related amplified polymorphism (srap) is a pcr based marker system as described by li and oiros, 2001. the sraps is a simple and efficient marker system that can be adapted for a variety of purposes in different crops. it is simple, has reasonable throughput rate, discloses numerous co-dominant markers, targets open reading frames (orfs), and allows easy isolation of bands for sequencing (li and quiros, 2001). this marker system has been used to determine genetic diversity in several horticulture plants such as 56 h. pinar, m. unlu, s. ercisli, a. uzun, m. bircan, k.u. yilmaz, g. agar peach [prunus persica (l.) batsch.] and nectarines (ahmad et al., 2004), persimmon (diospyros kaki l.f.) (guo and luo, 2006), apricots (uzun et al., 2010) and citrus (uzun et al., 2009). to date, there is no report on determining the genetic diversity and characterization of wild grown apricots by srap markers in turkey and in the world. this study aimed to determine the genetic diversity among seed propagated apricots sampled from sakit valley in mediterranean region of turkey. material and methods study area and plant material the study area, sakit valley, one of the famous wild apricot growing areas, is located in the east mediterranean region of turkey. the altitude of this valley is ranging from 120 to 1150 meters above sea level (m a.s.l.). a total of 57 sakit apricot genotypes were collected from different part of the sakit valley. all 57 genotypes are preselected according to their high yield capacity. they were free from pests and diseases. dna extraction and srap analysis genomic dna was extracted from young leaves of 57 wild apricot genotypes by the ctab method as described by uzun et al. (2009). dna concentration was measured with a microplate spectrophotometer (biotek instruments, inc. vinooski, usa), and dna was diluted to 10 ng/ml using te (10 mm tris-hcl, 0.1 mm edta, ph 8.0). a total of 19 srap primer combinations were used in this study (tab. 1). pcr reaction components and pcr cycling parameters for srap analysis was performed as described by uzun et al. (2009). each of the 15 µl reactions consisted of 1.33 mm of primers, 200 µm of each dntp, 1.5 µl of 10x pcr buffer (biorun, nantes, france), 2 mm of mgcl2, 0.8 µg/µl bovine serum albumine, 5.8 µl ddh2o, 1 unit of taq polymerase (biorun, nantes, france) and 20 ng of template. dna thermal cycler (sensoquest progen scientific ltd. mexborough, south yorkshire, uk) was used and cycling parameters included 2 min of denaturing at 94 ºc, five cycles of three steps: 1 min of denaturing at 94 ºc, 1 min of annealing at 35 ºc and 1 min of elongation at 72 ºc. in the following 35 cycles, the annealing temperature was increased to 50 ºc, and the extension per cycle was set to 5 min at 72 ºc. pcr products were separated on a 2% agarose gel in 1x tbe buffer (89 mm tris, 89 mm boric acid, 2 mm edta) at 115 volt for 2.5-3 h. the fragment patterns were photographed under uv light for further analysis. a 100 bp standard dna ladder was used as the molecular standard to confirm the appropriate markers for srap analysis. data analysis each band was scored as present (1) or absent (0) and data were analyzed with the numerical taxonomy multivariate analysis system (ntsys-pc program ver. 2.11) software package (rohlf, 2000). a similarity matrix was constructed by using srap data based on (dice, 1945) coefficient. then, the similarity matrix was used to construct a dendrogram using the upgma (unweighted-pair group method arithmetic average) to determine genetic relationships among the studied genotypes. results and discussion genetic diversity the srap primers, total fragments, polymorphic fragments and polymorphism percentage are given in tab. 1. in the present study, a total of 87 bands were presented from the 19 selected srap primers among 59 individual wild grown apricot genotypes. of these, 56 fragments (64.3%) were polymorphic (tab. 1). the primers me3-em2, me2-em10 and me2-em6 gave the highest polymorphism ratio (100%) while me1-em6, me6-em2 and me7-em3 did not give polymorphic bands (0%). the number of bands per primer ranged from 1 to 9 with mean value of 4.6 (tab. 1). the results obtained in this study show that there are high levels of polymorphism in wild grown apricots in sakit valley of turkey and all of them were distinguished with the srap markers. previously, the number of fragments per pcr reaction for apricot cultivars was reported as 3.5 (ruthner et al., 2006) and 4.1 (sanchez-perez et al., 2005) for ssr markers, as 6.5 (uzun et al., 2007) and 9.8 (ercisli et al., 2009) for rapd markers and as 5.4 for srap markers (uzun et al., 2010). turkish germplasm was studied by akpinar et al. (2010), uzun et al. (2010) and yilmaz et al. (2012) and genetic diversity and relationships among the accessions were determined using rapd, issr, srap and ssr markers. they found high genetic diversity among turkish apricot cultivars. yuan et al. (2007) reported a 72% polymorphism in apricot based on aflp data. this is comparable to the srap based analysis performed in this study. in apricot, the high level of genetic differentiation could be explained by the mating system and by low migration rates. most fruit trees under cultivation are derived from allogamic wild progenitors in which cross-pollination was maintained by self-incompatibility. genetically, domestication of fruit trees means changing the reproductive biology by shifting from sexual reproduction (in the wild) to vegetative propagation (maghuly et al., 2005). genetic relationships among the accessions the data obtained from srap analyses were used to perform genetic similarity analysis among the 57 wild grown apricot genotypes. tab. 1: list of srap primers used in this study, their numbers of total and polymorphic fragments and percentage of polymorphism srap primers total polymorphic polymorphism fragments fragments (%) me8-em6 5 3 60 me3-em2 4 4 100 me7-em10 4 2 50 me2-em10 4 4 100 me2-em6 5 5 100 me1-em6 2 0 0 me3-em12 4 3 75 me2-em12 6 5 83 me5-em8 4 2 50 me6-em5 6 1 17 me2-em2 6 2 33 me7-em6 8 6 75 me7-em3 4 2 50 me4-em12 3 2 67 me5-em2 3 2 67 me6-em2 2 0 0 me7-em3 1 0 0 me9-em10 7 5 71 me13-em15 9 8 89 mean 4,6 2,9 64.3 total 87 56 molecular characterization of wild apricots by srap markers 57 the similarity matrix was calculated using 87 srap fragments according to dice’s coefficient method (dice, 1945). then, the similarity matrix was used to perform upgma cluster analysis. all genotypes used in this study were distinguished. the analyzed wild grown apricot genotypes had similarity levels ranging from 0.73 to 0.94 (fig. 1). in the dice’s coefficient based upgma dendrogram, wild grown apricot genotypes clustered into three main groups (i, ii and iii) (fig. 1). group i, ii and iii consisted of five, fourty-seven and five genotypes, respectively. group i, ii and iii also further divided into 2 subgroups. there were no identical genotypes on the dendrogram (fig. 1). the most closely related genotypes in this study were ‘34’ and ‘36’; ‘27, 31 and 38’; ‘17 and 21’; ‘14 and 26’; ‘13 and 29’ and ‘44 and 45’ with 98% similarity ratios. uzun et al. (2010) reported genetic similarity between 0.77 and 0.97 among 28 turkish apricot cultivars. maghuly et al. (2005) reported that the east european, west european and irano-caucasian groups were very similar compared to central asian cultivars and other apricot species. the european genetic base (mediterranean basin and continental europe) was reported as much narrower and european cultivars share a common genetic base and are interrelated, which makes classification based on genetic distances difficult (hagen et al., 2002). in previous studies, it was observed that heterozygosity among apricot cultivars decreased from china to middle europe and the middle european and chinese apricots were clarified to be distantly related (pedryc et al., 2009). it can be assumed that cultivars from east of turkey are closer to irano-caucasian group than to european group and the ones from west of turkey including sakit valley are closer to european group (halasz et al., 2010). conclusions the srap marker system is becoming the marker of choice for characterization and genetic diversity studies in a wide range of plants. the study described in this paper shows that srap analyfig. 1: dendrogram of the 57 wild grown apricot genotypes using upgma method obtained from srap marker 58 h. pinar, m. unlu, s. ercisli, a. uzun, m. bircan, k.u. yilmaz, g. agar sis is a powerful tool for the characterization of wild grown apricot genotypes. in our study, the srap markers were used for the first time in wild grown apricot and distinguished genotypes efficiently with high level of polymorphism. genetic variation among the wild grown apricots may have potential for breeding new cultivars by selection and hybridization for high yield, quality and resistance to biotic and abiotic stress conditions. the diversity determined between wild grown apricot genotypes was probably due to seed propagation. it can be concluded that investigated wild growing fruit species have a great future potential in genetic research, as well as in biodiversity research. it is necessary to carry out further inventorisation and evaluation of investigated wild growing apricots to utilize them in the most appropriate way, as well as conservation of interesting accessions in the gene banks. the most valuable collected specimens should be involved in plant breeding programmes that can create new cultivars. references ahmad, r., potter, d., southwick, m., 2004: genotyping of peach and nectarine cultivars with ssr and srap molecular markers. j. am. soc. hortic. sci. 129, 204-210. akpinar, a.e., kocal, h., ergul, a., kazan, k., selli, m.e., bakir, m., aslantas, s., kaymak, s., saribas, r., 2010: ssr-based mole cular analysis of economically important turkish apricot cultivars. genet. mol. res. 9, 324-332. altindag, m., sahin, m., esitken, a., ercisli, s., guleryuz, m., 2006: biological control of brown rot (moniliana laxa ehr.) on apricot (prunus armeniaca l. cv. hacihaliloglu) by bacillus, burkholdria, and pseudomonas application under in vitro and in vivo conditions. biol. control 38, 369-372. chenjing, f., yuanhui, z., xiuying, x., 2005: genetic diversity revealed by issr marker in apricot. j. agric. univ. hebei 28, 52-55. dice, l.r., 1945: measures of the amount of ecologic association between species. ecology 26, 297-302. durgac, c., 2001: selection and fruit development of sakit apricots and determination of chilling period of sakit valley. phd thesis. cukurova university institute of natural and applied sciences. ercisli, s., 2004: a short review of the fruit germplasm resources of turkey. genet. res. crop evol. 51, 419-435. ercisli, s., agar, g., orhan, e., yildirim, n., hizarci, y., 2007: interspecific variability of rapd and fatty acid composition of some pomegranate cultivars (punica granatum l.) growing in southern anatolia region in turkey. biochem. syst. ecol. 35, 764-769. ercisli, s., 2009. apricot culture in turkey. sci. res. essays 4, 715-719. ercisli, s., agar, g., yildirim, n., esitken, a., orhan, e., 2009: identification of apricot cultivars in turkey (prunus armeniaca l.) using rapd markers. rom. biotech. lett. 14, 4582-4588. guerriero, r., watkins, r., 1984: revised descriptor list for apricot (prunus armeniaca). ibpgr secretariat, rome, cec secretariat, brussels. guo, d.l., luo, z.r., 2006: genetic relationships of some pcna persimmons (diospyros kaki thunb.) from china and japan revealed by srap analysis. genet. res. crop evol. 53, 1603-1797. hagen, l., khadari, b., lambert, p., audergon, j.m., 2002: genetic diversity in apricot revealed by aflp markers: species and cultivar comparisons. theor. appl. genet. 105, 298-305. halasz, j., hegedus, a., herman, r., stefanovits-banyai, e., pedryc, a., 2005: new selfincompatibility alleles in apricot (prunus armeniaca l.) revealed by stylar ribonuclease assay and s-pcr analysis. euphytica 145, 57-66. halasz, j., pedryc, a., ercisli, s., yilmaz, k.u., hegedus, a., 2010: s-genotyping supports the genetic relationships between turkish and hungarian apricot germplasm. j. am. soc. hortic. sci. 135, 410-417. kafkas, s., ozgen, m., dogan, y., ozcan, b., ercisli, s., serce, s., 2008: molecular characterization of mulberry accessions in turkey by aflp markers. j. am. soc. hortic. sci. 133, 593-597. krichen, l., trifi-farah, n., marrakchi, m., 2006: variability, organisation and identification of tunisian apricot (prunus armeniaca l.) cultivars using aflp markers. acta hortic. 717, 251-254. li, g., quiros, c.f., 2001: sequence-related amplified polymorphism (srap) a new marker system based on a simple pcr reaction, its application to mapping and gene tagging in brassica. theor. appl. genet. 103, 455-461. maghuly, f., borroto fernandez, e., ruthner, s., pedryc, a., laimer, m., 2005: microsatellite variability in apricot (prunus arme niaca) reflects their geographical origin and breeding history. tree genet. genomes 1, 151-165. marinello, l., sommella, m.g., sorrentina, a., 2002: identification of prunus armeniaca cultivars by rapd and scar markers. biotechnol. lett. 24, 749-755. mehlenbacher, s.a., cociu, v., hough, l.f., 1991: apricots (prunus). in: moore, j.n., ballington, j.r. (eds.), genetic resources of temperate fruit and nut crops, 63-109. acta hortic. 290. pedryc, a., ruthner, s., herman, r., krska, b., hegedus, a., halasz, j., 2009: genetic diversity of apricot revealed by a set of ssr a markers from linkage group g1. sci. hortic. 121, 19-26. romero, c., pedryc, a., munoz, v., llacer, g., badenes, m.l., 2003: genetic diversity of different apricot geographical groups determined by ssr markers. genome 46, 244-252. rohlf, f.j., 2000: ntsys-pc, numerical taxonomy and multivarite analysis system, version 2.11. new york, exeter, setauket. ruthner, s., pedryc, a., krska, b., romero, c., badenes, m.l., 2006: molecular characterization of apricot (prunus armeniaca l.) cultivars using cross species ssr amplification with peach primers. int. j. hortic. sci. 12, 53-57. sanchez-perez, r., ruiz, d., dicenta, f., egea, j., martinez-gomez, p., 2005: application of simple sequence repeat (ssr) markers in apricot breeding: molecular characterization, protection, and genetic relationships. sci. hortic. 103, 305-315. uzun, a., gulsen, o., aka-kacar, y., aras, v., demirel, a., bircan, m., paydas, s., yildiz, a., 2007: characterization of new apricot cultivars by rapd markers. j. appl. hortic. 9, 132-135. uzun, a., yesiloglu, t., aka-kacar, y., tuzcu, o., gulsen, o., 2009: genetic diversity and relationships within citrus and related genera based on sequence related amplified polymorphism markers (sraps). sci. hortic. 121, 306-312. uzun, a., gulsen, o., seday, u., bircan, m., yilmaz, k.u., 2010: srap based genetic analysis of some apricot cultivars. rom. biotech. lett. 15, 5396-5404. williams, j.g.k., kubelik, a.r., livak, k.j., rafalski, j.a., tingey, s.v., 1990: dna polymorphism amplified by arbitrary primers are useful as genetic markers. nucleic acids research 18, 6531-6535. yilmaz, k.u., paydas-kargi, s., dogan, y., kafkas, s., 2012: genetic diversity analysis based on issr, rapd and ssr among turkish apricot germplasms in iran caucasian eco-geographical group. sci. hortic. 138, 138-143. yuan, z., chen, x., he, t., feng, j., feng, t., zhang, c., 2007: population genetic structure in apricot (prunus armeniaca l.) cultivars revealed by fluorescen-aflp markers in southern xinjiang, china. j. genet. genom 37, 1037-1047. address of the corresponding author: e-mail: sercisli@gmail.com art17_kramer.indd journal of applied botany and food quality 85, 235 247 (2012) 1institute of food science and biotechnology, chair plant foodstuff technology, hohenheim university, stuttgart, germany 2 department of genetics, plant breeding and seed science, university of agriculture in krakow, poland 3 institute for breeding research on horticultural and fruit crops, federal research centre for cultivated plants, julius kühn-institute, quedlinburg, germany effects of cultivation year and growing location on the phenolic profi le of differently coloured carrot cultivars maike kramer 1, anna maksylewicz-kaul 2, rafal baranski 2, thomas nothnagel 3, reinhold carle1, dietmar r. kammerer1* (received june 20, 2012) summary carrots (daucus carota l.) are economically and nutritionally important crops that, apart from carotenoids, contain numerous phenolic compounds which are assumed to exert health benefi cial effects. the total phenolic contents of fruits and vegetables are known to depend on cultivar and growing conditions; however, studies examining the variability of a collection of carrots comprising differently coloured cultivars are rare. therefore, the objective of the present study was to investigate the phenolic compounds of ten differently coloured carrot cultivars considering the effects of three cultivation years at two growing locations. although total phenolic contents varied in a wide range, both purple cultivars ‘anthonina’ and ‘deep purple’ signifi cantly exceeded those of yellow, orange, red, and uncoloured cultivars (p ≤ 0.05) with amounts from 4,113 to 11,737 mg [kg dry matter (dm)]-1. in contrast to the purple roots, the other generally were characterised by far lower polyphenol contents ranging from 33 to 1,369 mg (kg dm)-1. interestingly, the values did not considerably vary within these cultivars. in the present study, contrary to cultivar specifi c effects, the infl uence of growing location was found to be rather weak, supposedly due to similar climatic conditions at both locations. similarly, variation of phenolic contents from year-to-year was less pronounced. in conclusion, the selection of breeding material was found to be of utmost importance regarding the expression of polyphenols in differently coloured carrots. introduction benefi cial effects of phytochemicals on human health have been demonstrated in numerous studies. phytochemicals are plant secondary metabolites, with phenolic compounds forming one of the most important groups. more than 10,000 phenolic compounds have so far been identifi ed in a wide range of different plant materials. they can be classifi ed into various sub-groups according to their number and arrangement of carbon atoms, comprising phenolic acids, fl avonoids, stilbenes, and lignans (carle, 2007; 2010). phenolic acids can be further sub-divided into two groups based on their basic carbon skeleton, namely cinnamic acid derivatives and benzoic acid derivatives, while hydroxycinnamic acids are more common (manach et al., 2004). due to the fact that phenolic compounds are widely distributed within fruits and vegetables, they constitute an important factor in plant-based human diets (bravo, 1998; kammerer et al., 2005). their benefi cial health effects are associated with the prevention of the risk of cardiovascular diseases, cancer, and cataract formation, as well as a number of other degenerative diseases (shahidi and naczk, 2004). such health benefits are ascribed to their antioxidant activity, due to their chemical nature predestined for free radical scavenging (leopoldini et al., 2011; rice-evans et al., 1997), as well as antimicrobial, antiviral, and further pharmacological actions (shahidi and naczk, 2004). carrots (daucus carota l.) are a popular vegetable cultivated worldwide. traditionally, in the northern hemisphere, it may be considered as the most important carotene source for human nutrition. however, as can be deduced from their browning potential, carrots also contain a variety of phenolic compounds, mainly phenolic acids that may be easily oxidised in the presence of oxygen. the most abundant compounds in carrot roots are 5-o-caffeoylquinic acid (chlorogenic acid), 3-o-caffeoylquinic acid (neochlorogenic acid), 3,4and 3,5-di-o-caffeoylquinic acids, 3-, 4-, and 5-o-feruloylquinic acid, or 3and 5-pacid, or 3and 5-pacid, or 3and 5-coumaroylquinic acids (alasalvar et al., 2001; klaiber et al., 2005). consequently, phenolic acids in carrots have been associated with a protective effect against bacterial infection (babic et al., 1993), interacting with proteins in their oxidised form (friedman, 1997), and also infl uencing sensory properties (phan, 1974). according to previous reports, bitter and sour taste perceptions were intensifi ed with increasing total soluble phenolics (talcott and howard, 1999a), and a di-caffeic acid derivative showed a clear-cut correlation with bitterness in raw carrots (kreutzmann et al., 2008). furthermore, phenolic acids were shown to be directly involved in colour degradation (talcott and howard, 1999b) and enzymatic browning (chubey and nylund, 1969) of carrots. the total phenolic contents of differently coloured carrot roots ranged from 7.7 to 74.6 mg (100 g fresh weight)-1 (alasalvar et al., 2001), and the wide range of values was shown to be cultivar dependent (bozalan and karadeniz, 2011; metzger and barnes, 2009; nicolle et al., 2004; sun et al., 2009; talcott and howard, 1999a). in addition, polyphenol levels may be affected by numerous other factors, including growth conditions, such as climate (manach et al., 2004), location (talcott and howard, 1999a), and fertilisation (smolén and sady, 2009), as well as post-harvest conditions including storage (heredia and cisneros-zevallos, 2009; klaiber et al., 2005; kramer et al., 2012; sarkar and phan, 1979) and processing (gonçalves et al., 2010; howard et al., 1994). theoretically, differing growing locations and cultivation years entail different environmental infl uences, which may lead to varying phenolic profi les and levels in various coloured carrot cultivars. therefore, the present study was conducted to provide evidence about the effect of cultivar and cultivation on the content and profi le of phenolic compounds in methanolic extracts of ten differently coloured carrot cultivars (white, yellow, orange, red, and purple roots) as investigated by hplc analyses. the effects of two different european locations (poland and germany) over three consecutive growing seasons in 2008, 2009, and 2010 were also considered. materials and methods plant material the roots of ten carrot cultivars (daucus carota l.) of different origins and colours from fi eld trial studies undertaken in 2008, 2009, and 2010 were included in the analysis. ‘white satin’ 236 m. kramer, a. maksylewicz-kaul, r. baranski, t. nothnagel, r. carle, d.r. kammerer (ws), ‘yellowstone’ (ys), ‘nerac’ (nf), and ‘deep purple’ (dp) were obtained from bejo samen (sonsbeck, germany), and ‘line 710015’ (li), ‘nutrired’ (nr), ‘santa cruz’ (sc), and ‘anthonina’ (an) from seminis vegetable seeds (neustadt, germany). ‘blanche 1⁄2 longue des vosges’ (bv) was a julius kühn-institute selection from gene bank accession 126 of the institut national d’horticulture (angers, france), and ‘pusa kesar’ (pk) was from warwick genetic resources unit, warwick university (wellesbourne, uk). the cultivars were grown in a collaborative research project at two different geographical locations near kraków, poland (malopolska region; 50° 07' n, 19° 59' e) [pl] on loess-brown soil and in quedlinburg, germany (north harz foreland region; 51° 47′ n, 11° 8′ e) [d] on loess-black soil in 2008, 2009, and 2010 with four replications in each case. field trial characteristics at the two locations are specifi ed in tab. 1. after harvest aliquots of approx. 1 kg of carrot roots (marketable quality) of each cultivar and each replication were collected. the samples were washed with tap water, drained, sliced into cubes (1x1x1 cm), and frozen at -80 °c. in 2008, the samples were lyophilised at hohenheim university. in 2009 and 2010, lyophilisation was carried out by gft sitte (oppin, germany). finally, the carrot samples were ground in a centrifugal mill zm1 (retsch gmbh, haan, germany) in 2008 and with a ball mill mm 301 (retsch gmbh, haan, germany) in 2009 and 2010. subsequently, the powder was stored at -20 °c until analysis. phenolic compound analysis extraction and fractionation of phenolic compounds phenolic compounds were isolated according to the procedure described by kammerer et al. (2004) with minor modifi cations. in brief, aliquots of 1 g carrot powder were extracted by stirring with 20 ml of methanol/water (60/40, v/v, acidifi ed with 0.01% hcl) for 1 h after fl ushing the fl ask with nitrogen. the extracts were centrifuged (10 min, 3480g), and the material was re-extracted with 20 ml of the same solvent (30 min). after fi ltration the combined supernatants were evaporated to dryness in vacuo at 30 °c, and the residue was dissolved in 3 ml of deionised water (ph 3.5). the resulting extract was used for further purifi cation. fractionation of phenolic compounds was performed using end-capped c18sep-pak cartridges (chromabond 1000, macherey-nagel, düren, germany) which were activated with 3 ml of methanol and rinsed with 10 ml of deionised water. an aliquot of 2.5 ml of the extract was made up to 5 ml with deionised water. after adjusting the ph to 7.0, the solutions were applied to the cartridge. non-anthocyanin phenolics were subsequently eluted with 10 ml of deionised water and 10 ml of 0.01% hcl (fraction i) and 20 ml of ethyl acetate (fraction ii). fraction iii containing anthocyanins, which was eluted with methanol/0.01% hcl, was discarded. the eluates were concentrated in vacuo, and the residues obtained were dissolved in 50% aqueous methanol (fraction i) and in pure methanol (fraction ii), respectively, membrane-fi ltered (0.45 µm), and used for hplc-dad-msn analyses. all analyses were performed in duplicate. chromatographic separation of phenolic compounds coupled with diode array and ms detection chromatographic separation was performed on a phenomenex (torrance, ca, usa) c18 hydro-synergi column (150 x 3.0 mm i.d., 4 µm particle size) equipped with a c18 ods guard column (4.0 x 2.0 mm i.d.). column temperature was 25 °c. the mobile phase consisted of 2% (v/v) acetic acid in water (eluent a) and 0.5% acetic acid in water and methanol (10/90, v/v; eluent b). the gradient used for the separation of phenolics in fraction i was 0% b to 35% b (35 min), 35% b to 75% b (20 min), 75% b to 100% b (2 min), tab. 1: field trial characteristics of the two growing locations in the years 2008, 2009, and 2010 location kraków [pl] quedlinburg [d] 2008 vegetation period 16.05.2008-23.09.2008 14.05.2008-17.09.2008 temperature [°c] 16.9 17.0 soil temperature [°c] 18.2 19.0 rel. humidity [%] n/a 79.3 rainfall [mm] 317.9 226.4 irrigation [mm] +15 (2 applications) +21 (3 applications) total water [mm] 332.9 or 2.6/day 247.4 or 2.0/day 2009 vegetation period 02.07.2009-08.10.2009 13.05.2009-08.09.2009 temperature [°c] 17.4 16.9 soil temperature [°c] 17.3 17.5 rel. humidity [%] n/a 80.3 rainfall [mm] 178.3 284.8 irrigation [mm] +0 +20 (2 applications) total water [mm] 178.3 or 1.8/day 304.8 or 2.6/day 2010 vegetation period 08.06.2010-28.09.2010 18.05.2010-13.09.2010 temperature [°c] 17.6 16.9 soil temperature [°c] 19.2 18.0 rel. humidity [%] n/a 85.0 rainfall [mm] 451.2 274.2 irrigation [mm] +0 +20 (2 applications) total water [mm] 451.2 or 4.0/day 294.2 or 2.5/day n/a not available cultivation effects on polyphenolic compounds in carrots 237 100% b isocratic (5 min), 100% b to 0% b (2 min). for fraction ii a gradient programme was used as follows: 0% b to 100% b (83 min), 100% b isocratic (5 min), 100% b to 0% b (2 min). total run time was 69 min and 95 min, respectively. the injection volume for all samples ranged from 5-40 µl. polyphenol separation was monitored separately at 280 nm (hydroxybenzoic acids) and 320 nm (hydroxycinnamic acids) at a fl ow rate of 0.4 ml/min. additionally, uv/vis spectra were recorded in the range of 200-600 nm at a spectral acquisition range of 1.25 scans/s (peak width 0.2 min). analyses were performed using a series 1100 hplc system (agilent, waldbronn, germany), equipped with a degasser, a binary gradient pump, a thermoautosampler, a column oven, and a diode array detection (dad) system controlled by agilent chemstation software (ver. a.09.03). the system was coupled on-line with a bruker (bremen, germany) esquire 3000+ ion trap mass spectrometer fi tted with an esi source. data acquisition and processing were performed using esquire control software (ver. 3.1). negative ion mass spectra of the column effl uent were recorded in the range m/z 50-1000 at a scan speed of 13,000 th/s (peak width 0.6 th, fwhm). nitrogen was used both as drying gas at a fl ow rate of 9.0 l/min and as nebulising gas at a pressure of 40.0 psi. the nebuliser temperature was set at 365 °c. helium was used as collision gas for collisioninduced dissociation (cid). the fragmentation amplitude was 1.5 v. characterisation and quantitation of phenolic compounds phenolic compounds and one amino acid were quantitated using calibration curves of the respective standards or related reference compounds [caffeic acid, quercetin dihydrate, p-coumaric acid, p-hydroxybenzoic acid, and ferulic acid from roth (karlsruhe, germany); chlorogenic acid, vanillic acid, and tryptophan from sigma (st. louis, mi, usa)] and molecular weight correction factors according to chandra et al. (2001). the results were expressed in mg per kg dry matter (dm), as means of four replications. characterisation of individual compounds was based on uv spectra, mass spectra, and retention times. statistical analysis data were subjected to analysis of variance (anova). as a fi rst step, means were separated by using tukey’s multiple comparison test (p ≤ 0.05). further, multivariate data analysis was performed, adopting a stepwise approach as described by kienzle et al. (2011) for harvest maturity specifi cation of mango fruit with some modifi cations. the detected compounds of each variety (combination of cultivar from each growing location and cultivation year) were processed by means of principal component analysis (pca) to fi nd out similarities and differences between the varieties. for a closer distinction of the varieties, agglomerative hierarchical cluster analysis (ca) was further applied to the data matrices outlined above. the complete-linkage clustering was based on maximum euclidian distances, considering all pcs. dendrograms showed maximum distances as heights of the clusters. data evaluation was performed with the sas software package (sas institute, cary, north carolina; software version 9.1). results characterisation of phenolic compounds and tryptophan tentative assignment of phenolic compounds and tryptophan in root extracts of differently coloured carrot cultivars was based on their chromatographic behaviour. characteristic uv spectra, hplc retention times, and hplc-msn data matched those of authentic reference compounds and were in accordance with literature data (clifford, 2003; fang et al., 2002; hossain et al., 2010; kammerer et al., 2004; klaiber et al., 2005). whenever uv and lc-ms data of the compounds did not allow their unambiguous identifi cation, the substances were designated as derivatives. basically, these investigations provided evidence of similar phenolic profi les of individual cultivars independent of different cultivation years and growing locations. hence, fig. 1 displays typical chromatographic profi les of fraction i of fi ve carrot cultivars of different root colours (white, yellow, orange, red, and purple) cultivated in pl in 2008. phenolic acids comprising a benzoic acid basic structure were detected at 280 nm because of their maximum absorption in a wavelength range of 200 290 nm (robbins, 2003). due to the additional conjugation, cinnamate derivatives showing a broad secondary absorbance band from 270 360 nm (robbins, 2003) were monitored at 320 nm. hplc and ms data of individual compounds are summarised in tab. 2. for the majority of compounds, base line separation was achieved. in general, 20 phenolic substances were identifi ed, mainly hydroxycinnamic acids and hydroxybenzoic acids, one fl avonoid, and one aromatic amino acid. the hydroxycinnamic acids comprised derivatives of caffeic acid, such as 3-o-caffeoylquinic acid, 5o-trans-caffeoylquinic acid, 5-o-cis-caffeoylquinic acid, further monocaffeoylquinic acids, 4,5-di-o-caffeoylquinic acid, additional caffeic acid and di-caffeic acid derivatives, as well as ferulic acid derivatives (1-o-feruloylquinic acid, 4-o-feruloylquinic acid, 5o-feruloylquinic acid, other feruloylquinic acids, and ferulic acid derivatives). furthermore, a range of p-coumaric acid derivatives (5-p(5-p(5-coumaroylquinic acid) and of mixed conjugates of ferulic and caffeic acids (4-o-feruloyl-5-o-caffeoylquinic acid, caffeic / ferulic acid derivatives) were detected. usually such compounds form simple esters with quinic acids or sugars. in contrast, ferulic acid was found in un-esterifi ed form. the hydroxybenzoic acids were deduced from vanillic acid and p-hydroxybenzoic acid. additionally, quercetin-3-o-galactoside and the aromatic amino acid tryptophan were detected. infl uence of cultivar on the contents of phenolic compounds content levels of the phenolic compounds and tryptophan were determined in ten carrot cultivars harvested in 2008, 2009, and 2010 on two growing locations (d and pl). in tab. 3 mean contents of different carrot cultivars are presented, listed by carrot root colour and also including statistical signifi cances (p ≤ 0.05). the highest amounts of total phenolics have been determined in purple carrots with values ranging from 4,113 to 11,737 mg (kg dm)-1. in contrast, all other cultivars being devoid of anthocyanins showed considerably lower contents of colourless phenolics [33 to 1,369 mg (kg dm)-1] (tab. 3). interestingly, despite this broad range, the total phenolic contents did not differ signifi cantly within white, yellow, orange, and red carrot cultivars. furthermore, mainly hydroxycinnamic acids were detected among phenolic acids in carrot roots (tab. 2). considering the major phenolic compounds, a vanillic acid derivative dominated in ‘white satin’, whereas ferulic acid derivatives prevailed in the yellow cultivar ‘yellowstone’. in cultivars ‘blanche 1⁄2 longue des vosges‘ (white) and ‘line 710015’ (yellow) the major compounds varied depending on cultivation year and growing location. in orange, red, and purple roots, predominantly 5-o-transcaffeoylquinic acid (chlorogenic acid) was observed, yet the highest contents were found in purple cultivars ‘anthonina’ and ‘deep purple’ where it constantly represented more than 50% of the total phenolic content. although there was a great range of chlorogenic acid contents in white, yellow, orange, and red roots (0 64% of total phenolic contents), these amounts did not signifi cantly differ. moreover, 5-o-cis-caffeoylquinic acid was detected in all cultivars, showing highest quantities in purple roots. in contrast to chloro238 m. kramer, a. maksylewicz-kaul, r. baranski, t. nothnagel, r. carle, d.r. kammerer fig. 1: chromatographic profi les at a wavelength of (a) 280 nm and (b) 320 nm of fraction i of fi ve differently coloured carrot cultivars (samples from 2008, pl). for peak assignment see tab. 2. genic acid, trace amounts of 3-o-caffeoylquinic acid (neochlorogenic acid) were determined only in ‘pusa kesar’ (red), while higher amounts were found in both purple cultivars ‘anthonina’ and ‘deep purple’. further caffeic acid derivatives were only present in minor amounts in white and yellow cultivars. however, in orange, red, and purple roots these compounds have been found in increased amounts. while ferulic acid derivatives dominated with 28%, 49%, 55%, and 48% in the roots of ‘white satin’, ‘blanche 1⁄2 longue des vosges’, ‘yellowstone’, and ‘nutrired’, respectively, they occurred in far lower contents in all other cultivars. free ferulic acid was detected in six (‘white satin’, ‘blanche 1⁄2 longue des vosges’, ‘yellowstone’, ‘santa cruz’, ‘anthonina’ and ‘deep purple’) of ten cultivars. derivatives of caffeic and ferulic acids were primarily detected in purple cultivars. all other cultivars showed far lower amounts, except for ‘line 710015’. the phenolic acid 5-pamounts, except for ‘line 710015’. the phenolic acid 5-pamounts, except for ‘line 710015’. the phenolic acid 5coumaroylquinic acid constituted a minor constituent of all cultivars under investigation. among hydroxybenzoic acids, vanillic acid derivatives, and a phydroxybenzoic acid hexoside were quantitated. interestingly, phydroxybenzoic acid hexoside was not found in white and yellow cultivars. remarkably, purple carrots were devoid of hydroxybenzoic acids. instead, the fl avonoid quercetin-3-o-galactoside was detected, which did not occur in the remaining cultivars. finally, tryptophan was determined in all carrot roots, most frequently in red cultivars. infl uence of cultivation year on phenolic contents due to the relatively large number of cultivars and compounds detected, multivariate data analyses were performed. hence, the mean values of the data set were fi rst sorted by growing location. generally, on both locations (d and pl) purple cultivars could be clearly discriminated from the differently coloured carrot roots (fig. 2). among purple carrots ‘anthonina’ and ‘deep purple’, the results of year 2008 were outstanding, whereas the values of 2009 and 2010 did not differ considerably. surprisingly, also the score for ‘pusa kesar’ of year 2008 took a separate position (fig. 2). while in d this variety was likely to be an outlier, the content of roots grown in pl was merged in one cluster comprising non-purple coloured cultivars. the values of all other roots were grouped in approximate positions. considering their total phenolic contents, individual cultivars behaved differently depending on cultivation years. no clear trend could be detected. throughout the years the most striking differences in contents were found for roots of ‘pusa kesar’ the amounts of which dropped from 2008 to 2010 by a factor of 8.5 and 4.6-fold in pl and d, respectively. infl uence of growing location on phenolic contents regarding the effect of location, pca and ca were performed with means sorted by cultivation year. in agreement with the observations described above, purple cultivars ‘anthonina’ and ‘deep purple’ stood out compared to the cultivars being devoid of anthocyanins (fig. 3). however, only in 2010 purple roots of both growing locations were found in separate positions. in 2008 and 2009, the scores for purple cultivar ‘anthonina’ were clearly separated from cultivation effects on polyphenolic compounds in carrots 239 tab. 2: characteristic data of phenolic compounds from different carrot cultivars. peak numbers and retention times refer to hplc traces in fig. 1. peak no. retention time hplc/dad [m-h]hplc/esi(-)-msn experiment tentative identifi cation [min] uv spectrum m/z m/z (% base peak) λmax [nm] fraction i i-1 14.9 238, 308sh, 327 365 ms2[365]: 203(100) caffeic acid derivative ms3[365→203]: 97(100), 129(52) i-2 16.0 227, 254, 292 659 ms2[659]: 329(100) vanillic acid derivative ms3[659→329]: 167(100) i-3 16.9 235, 308sh, 325 353 ms2[353]: 191(100), 179(86) 3-o-caffeoylquinic acida i-4 19.1 229, 278 203 ms2[203]: 116(100) tryptophana i-5 20.1 255 299 ms2[299]: 137(100) p-hydroxybenzoic acid hexoside ms3[299→137]: 93(100) i-6 20.6 283 472 ms2[472]: 404(100), 300(66) not identifi ed ms3[472→404]: 275(100), 386(67) i-7 23.7 258 329 ms2[329]: 125(100) not identifi ed ms3[329→125]: 97(100) i-8 23.8 268 443 ms2[443]: 161(100), 237(79), 437(71), 281(58) not identifi ed ms3[443→161]: 101(100) i-9 25.2 298 508 ms2[508]: 405(100) not identifi ed ms3[508→405]: 276(100) i-10 26.8 234, 291, 314 355 ms2[355]: 193(100) ferulic acid derivative ms3[355→193]: 149(100), 134(73) i-11 27.3 240, 307sh, 326 353 ms2[353]: 191(100) 5-o-trans-caffeoylquinic acida (chlorogenic acid) i-12 28.2 252, 327 658 ms2[658]: 385(100) not identifi ed ms3[658→385]: 191(100), 193(58) i-13 28.5 258 431 ms2[431]: 329(100) not identifi ed ms3[431→329]: 125(100), 203(58) i-14 29.5 294, 318 355 ms2[355]: 193(100) ferulic acid derivative ms3[355→193]: 134(100), 149(87) i-15 30.3 233, 316 353 ms2[353]: 191(100) 5-o-cis-caffeoylquinic acid i-16 31.0 250, 327 365 ms2[365]: 185(100), 203(94) caffeic acid derivative ms3[365→185]: 141(100) i-17 32.5 269 281 ms2[281]: 237(100) not identifi ed ms3[281→237]: 171(100), 123(89), 207(74) i-18 32.8 312 658 ms2[658]: 385(100) not identifi ed ms3[658→385]: 191(100), 193(86) i-19 33.9 232, 312 337 ms2[337]: 191(100) 5-p5-p5-coumaroylquinic acida i-20 34.3 239, 308 367 ms2[367]: 173(100) 4-o-feruloylquinic acid i-21 36.5 237, 305sh, 326 367 ms2[367]: 191(100) 5-o-feruloylquinic acid i-22 39.1 239, 308sh, 328 367 ms2[367]: 191(100) 1-o-feruloylquinic acid i-23 39.8 241, 308sh, 329 365 ms2[365]: 203(100) caffeic acid derivative ms3[365→203]: 115(100), 97(90), 85(76), 69(50) i-24 39.9 238, 303sh, 323 193 ms2[193]: 134(100) ferulic acida i-25 41.6 235, 324 379 ms2[379]: 341(100), 185(69), 203(64) caffeic acid derivative ms3[379→341]: 179(100) i-26 44.6 237, 308sh, 328 379 ms2[379]: 185(100) ferulic acid derivative ms3[379→185]: 141(100) i-27 44.9 243, 307sh, 326 193 ms2[193]: 131(100), 134(79) ferulic acid derivative, e.g. isoferulic acid i-28 45.6 238, 308sh, 328 527 ms2[527]: 365(100) di-caffeic acid derivative ms3[527→365]: 203(100) i-29 47.7 238, 308sh, 326 541 ms2[541]: 379(100) caffeic / ferulic acid derivative ms3[541→379]: 185(100) i-30 48.9 234, 308sh, 327 541 ms2[541]: 379(100) caffeic / ferulic acid derivative ms3[541→379]: 185(100) i-31 54.2 229, 304sh, 324 577 ms2[577]: 355(100), 193(61), 505(53) ferulic acid derivative ms3[577→355]: 355(100), 163(70) fraction ii ii-1 17.2 267, 296 329 ms2[329]: 167(100), 209(72) vanillic acid hexoside ii-2 17.7 230, 279 203 ms2[203]: 159(100) tryptophana ii-3 17.9 230, 279 431 ms2[431]: 329(100), 125(88) not identifi ed ms3[431→329]: 203(100), 125(56) 240 m. kramer, a. maksylewicz-kaul, r. baranski, t. nothnagel, r. carle, d.r. kammerer ii-4 19.3 239, 306sh, 326 353 ms2[353]: 173(100), 179(56) caffeoylquinic acid ii-5 20.1 239, 306sh, 326 341 ms2[341]: 281(100), 179(94), 251(53) caffeic acid derivative ms3[341→281]: 179(100) ii-6 20.2 232, 304sh, 325 355 ms2[355]: 217(100) not identifi ed ms3[355→217]: 202(100) ii-7 21.0 240, 307sh, 326 353 ms2[353]: 173(100), 191(81) caffeoylquinic acid ii-8 22.3 235, 304sh, 327 341 ms2[341]: 281(100), 179(65) caffeic acid derivative ms3[341→281]: 179(100) ii-9 23.1 235, 304sh, 325 355 ms2[355]: 217(100) not identifi ed ms3[355→217]: 202(100) ii-10 23.8 238, 306sh, 327 353 ms2[353]: 173(100), 191(86) caffeoylquinic acid ii-11 24.7 242, 307sh, 327 353 ms2[353]: 191(100) 5-o-trans-caffeoylquinic acida (chlorogenic acid) ii-12 25.5 242, 304sh, 330 355 ms2[355]: 193(100), 217(58) ferulic acid derivative ms3[355→193]: 134(100) ii-13 25.8 258 431 ms2[431]: 329(100) not identifi ed ms3[431→329]: 125(100), 203(75), 179(57) ii-14 26.4 237, 328 355 ms2[355]: 193(100), 217(64) ferulic acid derivative ms3[355→193]: 134(100), 149(49) ii-15 27.8 240, 305sh, 324 355 ms2[355]: 265(100), 235(97), 295(78), 193(65) ferulic acid derivative ms3[355→265]: 193(100) ii-16 27.1 240, 316 353 ms2[353]: 191(100) 5-o-cis-caffeoylquinic acid ii-17 30.4 236, 306sh, 326 355 ms2[355]: 265(100), 295(90), 235(77) ferulic acid derivative ms3[355→265]: 193(100) ii-18 31.0 306sh, 328 367 ms2[367]: 191(100) feruloylquinic acid ii-19 32.2 208, 306sh, 326 367 ms2[367]: 191(100) feruloylquinic acid ii-20 34.1 238, 305sh, 326 367 ms2[367]: 191(100) feruloylquinic acid ii-21 35.6 237, 303sh, 323 193 ms2[193]: 134(100) ferulic acida ii-22 37.4 236, 307sh, 326 365 ms2[365]: 203(100) caffeic acid derivative ms3[365→203]: 97(100) ii-23 38.7 230, 268, 294sh 373 ms2[373]: 193(100), 343(76), 219(59) not identifi ed ms3[373→193]: 178(100) ii-24 39.1 231, 278 565 ms2[565]: 361(100) not identifi ed ms3[565→361]: 165(100), 361(91), 346(69), 179(58) ii-25 41.4 239, 308sh, 327 527 ms2[527]: 365(100) di-caffeic acid derivative ms3[527→365]: 203(100) ii-26 42.8 256, 356 463 ms2[463]: 301(100) quercetin-3-o-galactoside ms3[463→301]: 301(100) ii-27 44.0 243, 306sh, 329 515 ms2[515]: 353(100) 4,5-di-o-caffeoylquinic acid ms3[515→353]: 173(100) ii-28 46.4 237, 306sh, 323 541 ms2[541]: 379(100) caffeic / ferulic acid derivative ms3[541→379]: 185(100) ii-29 47.9 236, 308sh, 328 529 ms2[529]: 367(100) 4-o-feruloyl-5-o-caffeoylquinic ms3[529→367]: 173(100), 193(53) acid ii-30 50.9 240, 308sh, 328 531 ms2[531]: 337(100) ferulic acid derivative ms3[531→337]: 217(100), 193(98), 337(92), 175(91) ii-31 52.3 235, 323 503 ms2[503]: 267(100), 311(73) not identifi ed ms3[503→267]: 267(100), 252(71) ii-32 54.9 235, 303sh, 325 577 ms2[577]: 355(100), 193(69) ferulic acid derivative ms3[577→355]: 163(100), 355(64) ii-33 58.9 265 327 ms2[327]: 229(100), 211(70) not identifi ed ms3[327→229]: 211(100), 229(51) ii-34 62.8 256 329 ms2[329]: 229(100), 211(65) not identifi ed ms3[329→229]: 229(100), 230(97), 127(58), 211(58) ii-35 64.5 235, 307 385 ms2[385]: 279(100), 383(95), 367(93), 337(66), 342(59) not identifi ed ms3[385→279]: 279(100) ii-36 65.2 261 615 ms2[615]: 495(100) not identifi ed ms3[615→495]: 137(100) ii-37 71.2 228, 269 597 ms2[597]: 459(100) not identifi ed ms3[597→459]: 415(100) a identifi cation with standard peak no. retention time hplc/dad [m-h]hplc/esi(-)-msn experiment tentative identifi cation [min] uv spectrum m/z m/z (% base peak) λmax [nm] cultivation effects on polyphenolic compounds in carrots 241 t ab . 3 : m ea n co nt en ts ( n= 4) o f in di vi du al p he no li c co m po un ds i n di ff er en tl y co lo ur ed c ar ro t ro ot s. p he no li c co nt en t [m g (k g d m )1 ] d p l d p l 20 08 20 09 20 10 20 08 20 09 20 10 20 08 20 09 20 10 20 08 20 09 20 10 w hi te c ul ti va rs ‘w hi te s at in ’ ‘b la nc he 1⁄2 l on gu e de s v os ge s’ v an il li c ac id d er iv at iv e 11 8. 3 b b 74 .8 a a 87 .2 a b 97 .1 b a 92 .2 b a 64 .3 a a 33 .9 a a 38 .3 a b 33 .5 a b 32 .6 b a 32 .6 b a 20 .6 a a v an il li c ac id h ex os id e nd nd nd nd nd nd nd nd nd nd nd nd t ry pt op ha n 41 .4 a a nd a nd a 43 .7 b a nd a nd a 11 .4 a a nd a nd a 19 .7 b a nd a nd a p -h yd ro xy be nz oi c ac id h ex os id e nd nd nd nd nd nd nd nd nd nd nd nd 3o -c af fe oy lq ui ni c ac id nd nd nd nd nd nd nd nd nd nd nd nd 5o -t ra n sc af fe oy lq ui ni c ac id 1. 9 a a 0. 8 a a 0. 4 a a 21 .9 a b 29 .3 a b 37 .7 a b 2. 8 a a nd a a 10 .2 a a 70 .3 b b 4. 0 a b 37 .6 a b b 5o -c is -c af fe oy lq ui ni c ac id nd a nd a a 0. 1 b a nd a 8. 3 c b 2. 9 b b nd a a nd a a 0. 8 a a 2. 4 ab b 0. 2 a a 4. 1 b b c af fe oy lq ui ni c ac id 9. 4 c a 4. 7 b a 0. 0 a a 35 .1 c b 20 .3 b b 5. 3 a b nd nd nd nd nd nd 4, 5di -o -c af fe oy lq ui ni c ac id nd nd nd nd nd nd nd nd nd nd nd nd c af fe ic a ci d de ri va ti ve 3. 3 b a nd a nd a 1. 8 b a nd a nd a 0. 8 b a nd a a nd a 32 .3 b b 2. 8 a a nd a di -c af fe ic a ci d de ri va ti ve 1. 1 a a nd a nd a 15 .3 b a nd a nd a 0. 2 a a nd a nd a 34 .9 b b nd a nd a 5p 5p 5-c ou m ar oy lq ui ni c ac id 1. 3 a a nd a nd a a 1. 7 b a nd a 0. 6 a b 2. 7 a a nd a 1. 4 a a 4. 1 a a nd a 0. 7 a b f er ul ic a ci d 2. 2 b a nd a nd a 1. 4 b a nd a nd a 1. 2 b a nd a nd a 2. 6 b b nd a nd a 1o -f er ul oy lq ui ni c ac id 4. 4 b a 1. 0 a b nd a a 13 .0 b b nd a a 1. 0 a b 2. 2 b a nd a a 0. 9 ab a 23 .3 b b 1. 4 a a 1. 3 a a 4o -f er ul oy lq ui ni c ac id nd a nd nd 1. 4 b b nd a nd a 0. 4 a a nd a nd a 2. 7 b b nd a nd a 5o -f er ul oy lq ui ni c ac id 10 .0 b a 1. 1 a a 0. 4 a a 14 .4 b a nd a a 2. 0 a b 7. 2 b a nd a a 2. 4 ab a 26 .3 b b 3. 7 a a 2. 6 a a f er ul oy lq ui ni c ac id nd a a 19 .8 b a 3. 2 a a 5. 8 a b 62 .6 b b 9. 3 a b 11 .4 a a 62 .9 b a 33 .4 a a 19 .0 a b 97 .5 b b 20 .8 a a f er ul ic a ci d de ri va ti ve 54 .8 b a 23 .0 a a 13 .0 a a 40 .2 c a 24 .6 b a 10 .8 a a 17 .6 b a 18 .3 b a 10 .1 a b 38 .2 c b 13 .5 b a 5. 2 a a 4o -f er ul oy l5o -c af fe oy lq ui ni c ac id -c af fe oy lq ui ni c ac id -c af fe oy lq ui ni c ac id nd nd nd nd nd nd nd nd nd nd nd nd c af fe ic / f er ul ic a ci d de ri va ti ve nd a nd nd 1. 6 b b nd a nd a nd a nd nd 5. 5 b b nd a nd a q ue rc et in -3 -o -g al ac to si de nd nd nd nd nd nd nd nd nd nd nd nd t ot al p h en ol ic s 24 8. 1 b a 12 5. 2 a a 10 4. 3 a a 29 4. 4 b a 23 7. 4 b b 13 3. 9 a b 91 .7 a a 11 9. 4 a a 92 .7 a a 31 3. 8 b b 15 5. 7 a a 92 .9 a a y el lo w c ul ti va rs ‘y el lo w st on e’ ‘l in e 71 00 15 ’ v an il li c ac id d er iv at iv e 32 .8 b a 24 .8 a b a 20 .4 a b 23 .1 a b a 27 .4 b a 15 .2 a a 16 .7 a a 11 .5 a a 52 .8 b b 11 .7 a a 10 .2 a a 9. 8 a a v an il li c ac id h ex os id e nd nd nd nd nd nd nd nd nd nd nd nd t ry pt op ha n 20 .7 b a nd a nd a 25 .8 b a nd a nd a 14 .4 b a nd a a nd a 16 .0 b a 4. 6 a b nd a p -h yd ro xy be nz oi c ac id h ex os id e nd nd nd nd nd nd nd nd nd nd nd nd 3o -c af fe oy lq ui ni c ac id nd nd nd nd nd nd nd nd nd nd nd nd 5o -t ra n sc af fe oy lq ui ni c ac id nd a a 0. 3 a a 15 .8 b a 19 .7 a b 5. 6 a b 21 .1 a a 0. 4 a a 4. 5 a a 9. 8 a a 7. 7 a b 4. 9 a a 85 .0 b b 5o -c is -c af fe oy lq ui ni c ac id nd a a nd a a 2. 9 b a 1. 5 a b 0. 8 a b 3. 5 b a nd a a 0. 8 a a 0. 6 a a 0. 6 a b nd a a 7. 6 b b c af fe oy lq ui ni c ac id nd nd nd nd nd nd nd nd nd nd nd nd 4, 5di -o -c af fe oy lq ui ni c ac id nd nd nd nd nd nd nd nd nd nd nd nd c af fe ic a ci d de ri va ti ve nd a a 1. 4 b b nd a a 2. 7 b b nd a a 0. 6 a a 2. 4 b a nd a 2. 2 b a 5. 5 a a nd a 19 .1 b b di -c af fe ic a ci d de ri va ti ve nd a nd nd 17 .1 b b nd a nd a nd a nd nd 4. 6 b b nd a nd a 5p 5p 5-c ou m ar oy lq ui ni c ac id 0. 4 b a 0. 3 a a nd a a 1. 5 c b nd a a 0. 7 b b 2. 0 b a 0. 1 a a 0. 2 ab a 1. 3 ab a 0. 1 a a 3. 4 b b f er ul ic a ci d nd a nd nd 2. 0 b b nd a nd a nd nd nd nd nd nd 1o -f er ul oy lq ui ni c ac id 1. 7 b a nd a a 0. 5 ab b 10 .6 b b 0. 9 a b nd a a 2. 6 b a 0. 2 a a 1. 9 ab a 2. 0 a a nd a a 1. 4 a a 4o -f er ul oy lq ui ni c ac id nd a nd nd 2. 4 b b nd a nd a nd a nd nd 1. 9 b b nd a nd a 5o -f er ul oy lq ui ni c ac id 3. 9 b a 1. 3 ab b nd a a 14 .7 b b nd a a 1. 6 a a 6. 9 b a 0. 6 a a 0. 5 a a 10 .2 b a nd a a nd a a f er ul oy lq ui ni c ac id 5. 7 a a 13 .0 a a 7. 0 a a 7. 7 a a 42 .3 b b 26 .6 a b b 2. 0 a a 7. 8 a a 1. 4 a a 11 .2 a a 15 .3 a a 5. 0 a a f er ul ic a ci d de ri va ti ve 42 .5 a a 36 .5 a a 35 .4 a a 56 .0 b b 20 .6 a a 21 .0 a a 18 .0 b a 6. 9 a a 14 .8 a b b 31 .4 b a 6. 1 a a 3. 5 a a 242 m. kramer, a. maksylewicz-kaul, r. baranski, t. nothnagel, r. carle, d.r. kammerer p he no li c co nt en t [m g (k g d m )1 ] d p l d p l 20 08 20 09 20 10 20 08 20 09 20 10 20 08 20 09 20 10 20 08 20 09 20 10 4o -f er ul oy l5o -c af fe oy lq ui ni c ac id -c af fe oy lq ui ni c ac id -c af fe oy lq ui ni c ac id nd nd nd nd nd nd nd nd nd nd nd nd c af fe ic / f er ul ic a ci d de ri va ti ve nd a nd nd 4. 9 b b nd a nd a nd nd nd nd nd nd q ue rc et in -3 -o -g al ac to si de nd nd nd nd nd nd nd nd nd nd nd nd t ot al p h en ol ic s 10 7. 7 a a 77 .6 a a 82 .0 a a 18 9. 6 b b 97 .7 a a 90 .3 a a 65 .4 a b a 32 .6 a a 84 .3 b a 10 4. 1 ab a 41 .3 a a 13 4. 7 b a o ra ng e cu lt iv ar s ‘s an ta c ru z’ ‘n er ac ’ v an il li c ac id d er iv at iv e 13 .9 b b 5. 4 a a 7. 2 a b 8. 5 a a 7. 8 a a 5. 8 a a 44 .9 b b 18 .5 a a 20 .8 a b 21 .2 b a 24 .4 b a 11 .7 a a v an il li c ac id h ex os id e nd nd nd nd nd nd nd nd nd nd nd nd t ry pt op ha n 20 .3 b a nd a a nd a 38 .7 c a 16 .0 b b nd a 12 .5 b a nd a nd a 16 .0 a a nd a nd a p -h yd ro xy be nz oi c ac id h ex os id e nd a nd a 3. 8 b b nd nd nd a nd a nd a 2. 6 b a nd a nd a 7. 0 b b 3o -c af fe oy lq ui ni c ac id nd nd nd nd nd nd nd nd nd nd nd nd 5o -t ra n sc af fe oy lq ui ni c ac id 72 .4 a a 19 .6 a a 22 .5 a a 17 4. 3 a b 16 8. 4 a b 31 0. 1 b b 86 .4 a a 35 .6 a a 8. 1 a a 11 0. 9 a a 15 6. 2 a b 18 3. 9 a b 5o -c is -c af fe oy lq ui ni c ac id 3. 5 a a 3. 2 a a 4. 1 a a 6. 8 a b 17 9. 9 b b 19 .5 a b 2. 9 a a 4. 9 a a 1. 5 a a 3. 8 a a 9. 2 b b 18 .8 c b c af fe oy lq ui ni c ac id nd nd nd nd nd nd nd nd nd nd nd nd 4, 5di -o -c af fe oy lq ui ni c ac id nd nd nd nd nd nd nd nd nd nd nd nd c af fe ic a ci d de ri va ti ve 2. 9 b a nd a nd a a 3. 9 a a nd a 60 .9 b b 25 .1 b a nd a nd a a 53 .2 b a nd a 58 .1 b b di -c af fe ic a ci d de ri va ti ve 32 .8 a a nd a nd a 22 7. 7 b b nd a nd a 31 .6 a a nd a nd a 90 .7 b a nd a nd a 5p 5p 5-c ou m ar oy lq ui ni c ac id 5. 2 a a 1. 3 a a 2. 7 a a 5. 0 b a nd a a 11 .0 c b 0. 8 a a nd a a 0. 6 a a 1. 0 a a 1. 2 ab b 2. 0 b b f er ul ic a ci d 1. 5 b a nd a nd a 3. 0 b b nd a nd a nd nd nd nd nd nd 1o -f er ul oy lq ui ni c ac id 17 .8 b a 1. 1 a b 1. 7 a a 58 .5 b b nd a a 1. 4 a a nd a 0. 9 a a 2. 6 a a nd a 3. 0 b b 2. 6 b a 4o -f er ul oy lq ui ni c ac id tr ac es nd nd tr ac es nd nd 2. 1 b a nd a a nd a 2. 9 b a 0. 8 a b nd a 5o -f er ul oy lq ui ni c ac id 20 .3 b a 2. 4 a b 3. 4 a a 25 .4 b a nd a a 3. 5 a a 26 .9 b a 1. 8 a a 3. 6 a a 21 .6 b a 5. 6 a a 2. 5 a a f er ul oy lq ui ni c ac id 1. 6 a a 16 .8 b a 8. 9 a a 16 .9 a b 13 5. 3 b b 44 .3 a b 7. 2 a a 41 .7 b a 13 .2 a a 10 .7 a a 57 .8 b a 63 .0 b b f er ul ic a ci d de ri va ti ve 41 .7 b a 25 .7 a b 18 .4 a a 59 .0 c b 10 .6 a a 27 .1 b a 39 .8 b a 24 .2 a b b 19 .9 a a 61 .0 b a 13 .6 a a 16 .3 a a 4o -f er ul oy l5o -c af fe oy lq ui ni c ac id -c af fe oy lq ui ni c ac id -c af fe oy lq ui ni c ac id nd nd nd nd nd nd nd nd nd nd nd nd c af fe ic / f er ul ic a ci d de ri va ti ve 3. 4 b a nd a nd a 22 .4 b b nd a nd a 5. 7 a a nd a nd a 17 .5 b a nd a nd a q ue rc et in -3 -o -g al ac to si de nd nd nd nd nd nd nd nd nd nd nd nd t ot al p h en ol ic s 23 7. 2 b a 75 .6 a a 72 .6 a a 65 0. 0 a b 51 7. 9 a b 48 3. 6 a b 28 5. 8 a a 12 7. 5 a a 72 .9 a a 41 0. 4 a a 27 1. 8 a b 36 5. 9 a b r ed c ul ti va rs ‘n ut ri re d’ ‘p us a k es ar ’ v an il li c ac id d er iv at iv e 12 .5 a a 9. 4 a a 11 .6 a b 13 .1 b a 6. 8 a a 8. 3 a a 34 .3 b b 11 .8 a a 9. 8 a b 10 .3 b a 10 .4 b a 5. 9 a a v an il li c ac id h ex os id e nd a 7. 9 b a 0. 8 a a nd a 15 .1 b b 1. 0 a a nd nd nd nd nd nd t ry pt op ha n 23 .7 b a 8. 8 a a nd a 10 6. 5 b b nd a a nd a 12 4. 9 a a 25 .0 a a 0. 7 a a 67 .2 a a 44 .1 a a 1. 9 a b p -h yd ro xy be nz oi c ac id h ex os id e 8. 5 b a nd a 5. 3 b a 16 .5 b b nd a 7. 8 b b 39 .9 b a 9. 9 a b 7. 6 a a 39 .2 c a 4. 8 a a 9. 7 b a 3o -c af fe oy lq ui ni c ac id nd nd nd nd nd nd 3. 4 b b nd a nd a 1. 0 b a nd a nd a 5o -t ra n sc af fe oy lq ui ni c ac id 29 .3 b a 6. 2 a a 64 .7 c a 20 3. 5 b b 66 .9 a b 17 9. 6 b b 58 4. 0 b b 46 .6 a a 55 .2 a a 34 0. 8 b a 41 7. 7 b b 10 5. 2 a b 5o -c is -c af fe oy lq ui ni c ac id 5. 7 b a 0. 5 a a 8. 2 b a 12 .0 b b 2. 9 a b 14 .1 b a 23 .8 b b 5. 1 a a 6. 0 a a 13 .0 a a 30 .4 b b 8. 4 a b c af fe oy lq ui ni c ac id nd nd nd nd nd nd nd nd nd nd nd nd 4, 5di -o -c af fe oy lq ui ni c ac id nd a nd a 3. 2 b a nd a nd a 14 .5 b a nd nd a nd nd a 71 .0 a a nd a c af fe ic a ci d de ri va ti ve nd nd nd a nd a nd a 6. 3 b b 13 5. 9 b b nd a nd a 92 .9 b a nd a nd a di -c af fe ic a ci d de ri va ti ve nd a nd nd 33 .2 b b nd a nd a 22 4. 9 b a nd a nd a 25 9. 7 b a nd a nd a 5p 5p 5-c ou m ar oy lq ui ni c ac id 2. 0 a a 0. 2 a a 1. 0 a a 0. 7 a a nd a a 2. 6 a a 23 .3 b b 1. 3 a a 1. 4 a a 7. 5 b a 1. 1 a a 4. 2 ab b f er ul ic a ci d nd nd nd nd nd nd nd nd nd nd nd nd 1o -f er ul oy lq ui ni c ac id 8. 9 b a 0. 8 a a 2. 8 a b 28 .1 b b 1. 4 a a 1. 7 a a nd a 4. 7 b a 1. 9 ab a nd a nd a a 2. 2 b a 4o -f er ul oy lq ui ni c ac id 1. 8 b a nd a nd a 2. 9 b a nd a nd a 4. 5 a a 0. 5 a a nd a 1. 0 b a nd a a nd a cultivation effects on polyphenolic compounds in carrots 243 p he no li c co nt en t [m g (k g d m )1 ] d p l d p l 20 08 20 09 20 10 20 08 20 09 20 10 20 08 20 09 20 10 20 08 20 09 20 10 5o -f er ul oy lq ui ni c ac id 29 .4 b a 1. 5 a a 5. 2 a a 35 .2 b a 1. 2 a a 3. 3 a a 69 .2 b b 13 .2 a a 8. 7 a a 13 .9 b a nd a a 6. 0 ab a f er ul oy lq ui ni c ac id 5. 7 a a 50 .8 a a 26 .7 a a 10 .0 a b 28 6. 8 c b 46 .8 b a 24 .5 a a 57 .7 a a 63 .2 a a 51 .2 a a 16 9. 4 b b 51 .0 a a f er ul ic a ci d de ri va ti ve 34 .4 a a 45 .1 a a 37 .0 a b 45 .3 a a 12 1. 0 b b 12 .2 a a 49 .0 b b 12 .0 a b 6. 3 a a 23 .1 b a 5. 4 a a 5. 1 b a 4o -f er ul oy l5o -c af fe oy lq ui ni c ac id -c af fe oy lq ui ni c ac id -c af fe oy lq ui ni c ac id nd nd nd nd nd nd nd nd nd nd nd nd c af fe ic / f er ul ic a ci d de ri va ti ve nd a nd nd 6. 1 b b nd a nd a 27 .6 a a nd a nd a nd a nd nd q ue rc et in -3 -o -g al ac to si de nd nd nd nd nd nd nd nd nd nd nd nd t ot al p h en ol ic s 16 1. 9 a a 13 1. 2 a a 16 6. 4 a a 51 3. 3 b b 50 2. 1 b b 29 8. 3 a b 13 69 .3 b b 18 7. 6 a a 16 0. 9 a a 92 0. 7 b a 75 4. 4 b b 19 9. 5 a a p ur pl e cu lt iv ar s ‘a nt ho ni na ’ ‘d ee p p ur pl e’ v an il li c ac id d er iv at iv e nd nd nd nd nd nd nd nd nd nd nd nd v an il li c ac id h ex os id e nd nd nd nd nd nd nd nd nd nd nd nd t ry pt op ha n 40 .8 b a nd a nd a 96 .0 b a nd a nd a 35 .6 b a nd a nd a 62 .4 b a nd a nd a p -h yd ro xy be nz oi c ac id h ex os id e nd nd nd nd nd nd nd nd nd nd nd nd 3o -c af fe oy lq ui ni c ac id 96 .3 b a 40 .8 a a 50 .1 a b 25 6. 7 b b 36 .0 a a 27 .8 a a 65 .6 a a 43 .0 a a 38 .9 a a 12 2. 9 b a 28 .9 a a 75 .7 a b a 5o -t ra n sc af fe oy lq ui ni c ac id 52 68 .4 a a 84 98 .2 b a 32 79 .3 a a 69 69 .6 b a 92 12 .4 b a 30 73 .6 a a 35 65 .7 a a 31 67 .4 a a 34 33 .3 a a 51 46 .9 a b a 65 30 .6 b b 26 51 .3 a a 5o -c is -c af fe oy lq ui ni c ac id 31 .3 a a 10 5. 0 b a 13 1. 2 c a 28 .8 a a 14 0. 6 b a 16 8. 5 b b 12 4. 7 a a 10 6. 8 a a 14 0. 9 a a 14 1. 0 a a 22 2. 4 a b 12 2. 3 a a c af fe oy lq ui ni c ac id nd a nd a 24 3. 6 b b nd a nd a 16 5. 3 b a nd a nd a 11 9. 2 b a nd a nd a 10 3. 1 b a 4, 5di -o -c af fe oy lq ui ni c ac id 65 .8 a a 31 .7 a a 36 .4 a b 65 .1 b a 34 .4 a b a nd a a 14 .6 a a 17 .1 a a 29 .7 a a 49 .2 b a 27 .8 a b a nd a a c af fe ic a ci d de ri va ti ve 50 0. 9 c a 31 1. 6 b a 94 .3 a a 84 8. 1 b b 23 6. 4 a a 17 8. 8 a a 21 1. 2 a a 14 3. 1 a a 22 5. 8 a a 44 7. 2 a a 25 3. 0 a a 41 8. 3 a a di -c af fe ic a ci d de ri va ti ve 20 9. 5 b a nd a nd a 47 5. 6 b b nd a nd a 20 0. 3 b a nd a a nd a 40 5. 7 b a 24 .0 a b b nd a 5p 5p 5-c ou m ar oy lq ui ni c ac id 7. 1 a a 27 .3 b b 12 .9 a a 7. 4 a a 8. 9 a a 9. 4 a a 11 .4 a b 30 .9 b b 11 .9 a b 6. 2 a a 9. 3 a a 6. 9 a a f er ul ic a ci d 96 .6 b a 1. 8 a a 15 .8 a b 11 4. 4 b a 1. 7 a a nd a a 21 .2 a b a 34 .9 b b 16 .5 a b 98 .1 b b 4. 8 a a nd a a 1o -f er ul oy lq ui ni c ac id nd nd nd nd nd nd nd nd nd nd nd nd 4o -f er ul oy lq ui ni c ac id nd nd nd nd nd nd nd nd nd nd nd nd 5o -f er ul oy lq ui ni c ac id 24 .9 b a 10 .6 a a 7. 5 a a 57 .3 b b 8. 8 a a 7. 8 a a 0. 6a a 0. 4 a b 0. 1 a a 2. 1 a a 0. 1 a a 0. 2 a a f er ul oy lq ui ni c ac id nd a 39 .7 b a 85 .9 c a nd a 28 .0 a a 24 2. 6 b b nd a 12 .0 a a 18 3. 9 b a nd a 57 .0 a b 25 5. 0 b a f er ul ic a ci d de ri va ti ve 21 18 .1 b a 19 80 .4 b a 12 92 .4 a a 20 79 .9 b a 19 11 .9 b a 88 0. 8 a a 23 87 .9 a a 18 50 .8 a a 12 07 .8 a b 19 85 .5 b a 24 04 .1 b a 48 0. 3 a a 4o -f er ul oy l5o -c af fe oy lq ui ni c ac id -c af fe oy lq ui ni c ac id -c af fe oy lq ui ni c ac id 49 .1 a a 83 .8 a a 82 .6 a b 42 .1 a b a 65 .3 b a nd a a 8. 0 a a 35 .0 b a nd a 14 3. 9 c b 73 .0 b b nd a c af fe ic / f er ul ic a ci d de ri va ti ve 11 1. 3 b a nd a nd a 25 6. 2 b b nd a nd a 13 5. 8 b a nd a nd a 41 3. 1 b a nd a nd a q ue rc et in -3 -o -g al ac to si de 58 .3 a a 52 .6 a a 52 .0 a b 67 .2 c a 53 .2 b a nd a a 43 .8 a a 52 .2 a a 47 .6 a b 57 .9 b a 70 .3 b b nd a a t ot al p h en ol ic s 86 78 .2 b a 11 18 3. 4 c a 53 83 .9 a a 11 36 4. 5 b b 11 73 7. 4 b a 47 54 .7 a a 68 26 .4 a a 54 93 .7 a a 54 55 .6 a a 90 82 .1 b a 97 05 .3 b b 41 13 .0 a a nd n ot d et ec te d ab c v al ue s de ri ve d fr om s am pl es w it hi n on e lo ca ti on a nd d if fe re nt c ul ti va ti on y ea rs w it h di ff er en t lo w er c as e le tt er s ar e si gn ifi c an tl y di ff er en t a b c v al ue s de ri ve d fr om s am pl es o f di ff er en t lo ca ti on s w it hi n on e cu lt iv at io n ye ar w it h di ff er en t up pe r ca se l et te rs a re s ig ni fi ca nt ly d if fe re nt 244 m. kramer, a. maksylewicz-kaul, r. baranski, t. nothnagel, r. carle, d.r. kammerer fig. 2: infl uence of cultivation year on phenolic compounds of different carrot cultivars grown at location d (a+b) and pl (c+d): (a/c) pca scatter plots of the fi rst two principal components (pc 1/pc 2) and (b/d) dendrograms of complete-linkage cluster analysis. main clusters and their secondary and tertiary subgroups shown by cluster analysis are indicated in the pca plots (a/c) by light-grey areas, black-rimmed and black-dotted ellipses, respectively. those of ‘deep purple’. moreover, for 2009 the growing locations of the latter were grouped into different clusters (fig. 3). considering the other cultivars, for 2008 the values of ‘pusa kesar’ of both growing locations could be distinguished from other white, yellow, orange, and red cultivars. interestingly, in 2010 the means of both red cultivars ‘pusa kesar’ and ‘nutrired’ scored in a tertiary subgroup. however, the location did not have any infl uence on this subgroup. total phenolic contents of roots grown in pl and d varied in a wide range. remarkably, in pl higher total phenolic contents were found for most carrot roots, except for ‘pusa kesar’ grown in 2008 as well as ‘anthonina’ and ‘deep purple’ cultivated in 2010. although there was a great variability, the variation among cultivars did not differ notably between both growing locations within the years (tab. 3). discussion the aim of this study was to clarify the role of genotype and environment (growing location and cultivation year) on the phenolic contents of carrot cultivars exposed to european growing conditions. in summary, 20 phenolic compounds have been identifi ed in carrot roots. proof has been furnished that there was a broad variation throughout differently coloured cultivars, while purple carrots behaved differently. in accordance with the fi ndings of alasalvar et al. (2001) purple roots accumulated highest amounts of total phenolics, only comprising hydroxycinnamic acids, with 5-o-trans-caffeoylquinic acid being the predominant compound (kreutzmann et al., 2008; sun et al., 2009). assuming a dry matter content of approx. 10%, the contents in purple carrot roots were on the same level as reported in previous studies (alasalvar et al., 2001; kammerer et al., 2004; kreutzmann et al., 2008). results of pca and ca support the outstanding position of purple cultivars and hence the strong infl uence of genotype (fig. 2 and 3). also the cultivation year showed an effect on purple roots, since the values of year 2008 could clearly be separated from those of 2009 and 2010 (fig. 2). however, this may also partly be due to different sample preparations after harvest (see material and methods). in contrast, growing location did not exert such a clear-cut infl uence on polyphenol contents. in 2010, the differences between both locations (fig. 2) may be explained by differing water supply (tab. 1). in carrot cultivars being devoid of anthocyanins (white, yellow, orange, and red roots), considerably lower total phenolic contents, comprising hydroxycinnamic and hydroxybenzoic acids, were observed, which was also refl ected by chlorogenic acid amounts. despite the great variations of total phenols between the cultivars, differences were insignifi cant as also previously reported by sun et al. (2009), supposedly due to the heterogeneity of sample material within the cultivars (mattila and hellström, 2007). however, based on pca and ca results, carrot ‘pusa kesar’ (red) was grouped in a separated position in 2008, whereas in 2010 the values of both cultivation effects on polyphenolic compounds in carrots 245 red cultivars ‘pusa kesar’ and ‘nutrired’ differed from the other roots. again, infl uence of cultivation year and growing location was of minor relevance. consequently, the results of the present study support the effects of cultivar to be predominant, although solely purple cultivars were found to signifi cantly differ when compared with further differently coloured genotypes. in agreement with previously published data, considerable variability was found among carrot cultivars regarding their phenolic contents, in particular in purple carrots (alasalvar et al., 2001; kreutzmann et al., 2008; metzger and barnes, 2009; nicolle et al., 2004; sun et al., 2009). consistently, also for different orange carrot genotypes it has been shown that much of the variation in phenolic acids can be attributed to the genetic diversity among cultivars (bozalan and karadeniz, 2011; talcott and howard, 1999a). moreover, simon et al. (1982) reported that beside phenolics various other attributes such as fl avour attributes, total sugars, carotenoids, or total terpenoids are known to be rather infl uenced by carrot genotype than by climate or soil. talcott and howard (1999a) described a considerable influence of different growing areas on phenolic compounds in carrots, assuming adverse growing conditions or improper post-harvest handling to be responsible for this effect. in contrast, the observed location fig. 3: infl uence of location on phenolic compounds of different carrot cultivars grown in 2008 (a+b), 2009 (c+d), and 2010 (e/f): (a/c/e) pca scatter plots of the fi rst two principal components (pc 1/pc 2) and (b/d/f) dendrograms of complete-linkage cluster analysis. main clusters and their secondary and tertiary subgroups shown by cluster analysis are indicated in the pca plots (a/c) by light-grey areas, black-rimmed and black-dotted ellipses, respectively. 246 m. kramer, a. maksylewicz-kaul, r. baranski, t. nothnagel, r. carle, d.r. kammerer infl uence in the present study was found to be minor, since phenolic variation among cultivars in different years was comparable at both locations. this may be due to similar climatic conditions (tab. 1), as kraków (pl) and quedlinburg (d) are situated on adjoining latitudes, and also identical harvesting practices were used. the year-to-year variations between 2008 and 2009 / 2010, especially observed in purple roots, may have resulted from different sample processing after harvest as mentioned above. therefore, we suggest the infl uence of cultivation year to be of minor relevance, because the climatic conditions, which were considered to be of major infl uence on polyphenol content (manach et al., 2004) did not vary signifi cantly between the years of observation. also søltoft et al. (2010) did not fi nd signifi cant year-to-year variation of phenolics in carrots grown at one location in different cultivation systems. in conclusion, the results demonstrate a variation in phenolic compounds between differently coloured carrot cultivars. in particular, purple cultivars could be clearly distinguished from cultivars being devoid of anthocyanins without using their anthocyanin contents for analysis. when grown on different locations having similar climatic conditions, variations of phenolic patterns are marginal. therefore, selection of cultivar for a given commodity is an important factor in providing raw material with specifi ed phenolic patterns. thus, these results provide an indication for breeders and the processing industry. however, further investigations are required to compile a more comprehensive database considering different environmental conditions such as temperature, water, and light. furthermore, due to their outstanding phenolic contents, our fi ndings suggest the inclusion of purple carrot into human diets to exploit their putative health benefi cial effects. acknowledgements this research project was supported by the deutsche forschungsgemeinschaft (dfg) in bonn, germany (grant number: ca 225/4-1) and by the polish ministry of science and higher education (grant number: 97/n-dfg/2008/0). the authors also thank mrs. erika müssig and mr. jaroslav kasperzec for their excellent technical assistance. references alasalvar, c., grigor, j.m., zhang, d., quantick, p.c., shahidi, f., 2001: comparison of volatiles, phenolics, sugars, antioxidant vitamins, and sensory quality of different colored carrot varieties. j. agric. food chem. 49, 1410-1416. babic, i., amiot, m.j., nguyen-the, c., aubert, s., 1993: changes in phenolic content in fresh ready-to-use shredded carrots during storage. j. food sci. 58, 351-356. bozalan, n.k., karadeniz, f., 2011: carotenoid profile, total phenolic content, and antioxidant activity of carrots. int. j. food prop. 14, 10601068. bravo, l., 1998: polyphenols: chemistry, dietary sources, metabolism, and nutritional signifi cance. nutr. rev. 56, 317-333. carle, r., 2010: sekundäre pflanzenstoffe. in: biesalski, h.k., bischoff, s.c., puchstein, c. 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(eds.), methods in polyphenol analysis, 315-336. the royal society of chemistry, cambridge. fang, n., yu, s., prior, r.l., 2002: lc/ms/ms characterization of phenolic constituents in dried plums. j. agric. food chem. 50, 3579-3585. friedman, m., 1997: chemistry, biochemistry, and dietary role of potato polyphenols. a review. j. agric. food chem. 45, 1523-1540. gonçalves, e.m., pinheiro, j., abreu, m., brandão, t.r.s., silva, c.l.m., 2010: carrot (daucus carota l.) peroxidase inactivation, phenolic content, and physical changes kinetics due to blanching. j. food eng. 97, 574-581. heredia, j.b., cisneros-zevallos, l., 2009: the effect of exogenous ethylene and methyl jasmonate on pal activity, phenolic profi les and antioxidant capacity of carrots (daucus carota) under different wounding intensities. postharvest biol. technol. 51, 242-249. hossain, m.b., rai, d.k., brunton, n.p., martin-diana, a.b., barryryan, c., 2010: characterization of phenolic composition in lamiaceae spices by 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ammonia-lyase activity and phenolic acid content of minimally processed carrot sticks. j. agric. food chem. 53, 1065-1072. kramer, m., bufler, g., ulrich, d., leitenberger, m., conrad, j., carle, r., kammerer, d.r., 2012: effect of ethylene and 1methylcyclopropene on bitter compounds in carrots (daucus carota l.). postharvest biol. technol. 73, 28-36. kreutzmann, s., christensen, l.p., edelenbos, m., 2008: investigation of bitterness in carrots (daucus carota l.) based on quantitative chemical and sensory analyses. lwt-food sci. technol. 41, 193-205. leopoldini, m., russo, n., toscano, m., 2011: the molecular basis of working mechanism of natural polyphenolic antioxidants. food chem. 125, 288-306. manach, c., scalbert, a., morand, c., rémésy, c., jiménez, l., 2004: polyphenols: food sources and bioavailability. am. j. clin. nutr. 79, 727-747. mattila, p., hellström, j., 2007: phenolic acids in potatoes, vegetables, and some of their products. j. food compos. anal. 20, 152-160. metzger, b.t., barnes, d.m., 2009: polyacetylene diversity and bioactivity in orange market and locally grown colored carrots (daucus carota l.). j. agric. food chem. 57, 11134-11139. nicolle, c., simon, g., rock, e., amouroux, p., rémésy, c., 2004: genetic variability infl uences carotenoid, vitamin, phenolic, and mineral content in white, yellow, purple, orange, and dark-orange carrot cultivars. j. am. soc. hortic. sci. 129, 523-529. phan, c.t., 1974: biochemical studies on the development of bitterness in stored carrots. acta hortic. 38, 309-319. rice-evans, c.a., miller, n.j., paganga, g., 1997: antioxidant properties of phenolic compounds. trends plant sci. 2, 152-159. robbins, r.j., 2003: phenolic acids in foods: an overview of analytical methodology. j. agric. food chem. 51, 2866-2887. cultivation effects on polyphenolic compounds in carrots 247 sarkar, s.k., phan, c.t., 1979: naturally-occurring and ethylene-induced phenolic compounds in the carrot root. j. food prot. 42, 526-534. shahidi, f., naczk, m., 2004: phenolics in food and nutraceuticals. crc press, boca raton, florida. simon, p.w., peterson, c.e., lindsay, r.c., 1982: genotype, soil, and climate effects on sensory and objective components of carrot fl avour. j. am. soc. hortic. sci. 107, 644-648. smolén, s., sady, w., 2009: the effect of various nitrogen fertilization and foliar nutrition regimes on the concentration of sugars, carotenoids and phenolic compounds in carrot (daucus carota l.). sci. hortic. 120, 315-324. søltoft, m., nielsen, j., laursen, k.h., husted, s., halekoh, u., knuthsen, p., 2010: effects of organic and conventional growth systems on the content of fl avonoids in onions and phenolic acids in carrots and potatoes. j. agric. food chem. 58, 10323-10329. sun, t., simon, p.w., tanumihardjo, s.a., 2009: antioxidant phytochemicals and antioxidant capacity of biofortifi ed carrots (daucus carota l.) of various colors. j. agric. food chem. 57, 4142-4147. talcott, s.t., howard, l.r., 1999a: chemical and sensory quality of processed carrot puree as infl uenced by stress-induced phenolic compounds. j. agric. food chem. 47, 1362-1366. talcott, s.t., howard, l.r., 1999b: phenolic autoxidation is responsible for color degradation in processed carrot puree. j. agric. food chem. 47, 2109-2115. address of the authors: hohenheim university, institute of food science and biotechnology, chair plant foodstuff technology, garbenstraße 25, 70599 stuttgart, germany journal of applied botany and food quality 86, 205 211 (2013), doi:10.5073/jabfq.2013.086.028 1centro de investigación en alimentación y desarrollo, a.c. (ciad, ac), hermosillo, sonora, mexico 2instituto tecnológico de sonora (itson), obregon, sonora, mexico 3department of cell biology and molecular genetics, the university of maryland, md, usa 4department of food science and technology, bihar agricultural university, sabour, bhagalpur, bihar, india antimicrobial and antioxidant properties of byproduct extracts of mango fruit v. vega-vega1, b.a. silva-espinoza1, m.r. cruz-valenzuela1, a.t. bernal-mercado1, g.a. gonzález-aguilar1, s. ruíz-cruz2, e. moctezuma3, md. w. siddiqui4, j.f. ayala-zavala1* (received may 28, 2013) * corresponding author summary byproducts of fruit processing could have higher content of phenolic compounds that can act as antimicrobial and antioxidant agents. in this context, the main objective of this study was to obtain extracts from peel, seed, and unused flesh of haden, ataulfo and tommy atkins mango varieties, in order to measure their antioxidant and antimicrobial properties. the extraction was performed using different methods, such as methanolic-polar, methanolic-non-polar, ethanolic-polar, ethanolic-non-polar and water infusion. the total phenolic content of the ethanolic-non-polar extract from seed of mango haden showed 875.06 mg/g, dpph ec50: 0.04 mg/ml, causing a 100 % inhibition of bacteria pathogens applying 25 mg/ml and inhibition of 89.78 % against alternaria applying 6.25 mg/ml. the flesh always showed the lowest content and bioactivity of the tested parameters. these results demonstrate the antimicrobial and antioxidant potential uses of fruit byproducts as sources of bioactive compounds. introduction mango products are well demanded by consumers for their flavor, convenience and bioactive compounds; however, during processing considerable generation of byproducts can be an issue (kim et al., 2007). depending on the mango variety, the peel and seed contribute about 15-20 % and 10-25 % of the whole fruit weight, respectively (berardini et al., 2005; abdalla et al., 2007). after industrial processing of mango, considerable amounts of peel and seeds are discarded, resulting in high economic loss to the manufacturer, as well as an impact to the environment (puravankara et al., 2000). for example, in india the mango-processing industry annually generates 3 x 105 tons of seed, approximately (soong and barlow, 2004). therefore, it is necessary to consider alternative uses for these mango byproducts, in order to avoid environmental problems and to create new revenue sources, providing greater economic returns to the agribusiness (ayala-zavala et al., 2011). several studies have shown that the content of phytochemical compounds is higher in peel and seeds with respect to the edible tissue of several fruits. the total phenolic contents in the peels of lemons, oranges, and grapefruits were 15 % higher than that of the pulp of these fruits (gorinstein et al., 2001). eight selected clingstone peach cultivars were studied and it was reported that the peels contained 2-2.5 times the amount of total phenolic compounds as contained in the edible product (chang et al., 2000). peels from apples, peaches, pears as well as yellow and white flesh nectarines were found to contain twice the amount of total phenolic compounds as that contained in fruit pulp (gorinstein et al., 2001). several studies show that peel and seed byproducts of fresh-cut fruits have high antioxidant capacity, and in many cases, even higher than the pulp (soong and barlow, 2004; ayala-zavala et al., 2010). phenolic compounds are some of the most important bioactive compounds in the mango fruit, mainly found in peels and seeds (ribeiro et al., 2008). the antioxidant capacity is attributed mainly to some of these phenolic compounds, which also have high antimicrobial capacity (kabuki et al., 2000). ayala-zavala et al. (2010) reported that the seed of mango var. ‘kent’ presents 3,727.52 mg of gallic acid equivalent (gae) per 100 g of fresh weight (f.w), which is higher than that reported in the pulp (48.84 mg gae/100 g f.w). this ‘kent’ mango phenolic compound seed value is higher than other fruit byproducts, such as pineapple, papaya and mandarin peel (113.24, 108.88 and 352.05 mg gae/100 g of fresh weight, respectively). moreover, catechin, chlorogenic acid, and phloridzin, three phenolic compounds that are abundant in apple processing byproducts, exhibited varying degree of inhibitory action toward the growth of tested food pathogenic and spoilage bacteria, fungi, and yeasts (muthuswamy and rupasinghe, 2007). phenolic compounds can be found in different tissues of fruits, e.g. i) peels are rich in phenolic compounds with antioxidant, antimicrobial and colorant properties, some of these are natural defenses against pathogens attack and environmental conditions; ii) the pulp posses a lower content of phenolic compounds, however, it is the main source of dietary antioxidants for humans, and finally iii) the seed can be also rich in phenolics, mainly tannins with antioxidant an antimicrobial properties that protect this tissue to perpetuate the regeneration of the species (ayala-zavala et al., 2011). the interest of some research groups has focused on the search for natural sources of antioxidant and antimicrobials, as well as an evaluation of their properties, to preserve food quality. this interest in natural antioxidants and antimicrobial, is due to the increased potential health risks associated with consumption of synthetic antioxidants and antimicrobial compounds (seneviratne and kotuwegedara, 2009). in this context, the analysis of natural compounds with antioxidant and antimicrobial potential is getting attention. therefore, the objective of this study was to obtain, using several methods, extracts from the peel and seed of different varieties of fresh-cut mango (‘haden’, ‘ataulfo’ and ‘tommy atkins’) in order to assess their antioxidant and antimicrobial properties. materials and methods sample preparation and characterization mangoes varieties ‘haden’, ‘ataulfo’ and ‘tommy atkins’ were obtained from a local store in the city of hermosillo, sonora, mexico. mangoes were selected according to color and free of physical defects in a commercial maturity stage (tab. 1). all fruit was washed with chlorinated water (250 ppm) for 3 min and air dried at 25 °c. fruits were weighted and the peel and seed were removed, the pulp was cut into cubes of 2 cm per side. yield was calculated weighting the amount of peel and seed byproducts, as well as finished products and compared with the initial weight of raw material. byproducts (peel, seed and unused pulp) were used for the extraction of phenolic compounds. 206 v. vega-vega, b.a. silva-espinoza, m.r. cruz-valenzuela, a.t. bernal-mercado, g.a. gonzález-aguilar, s. ruíz-cruz, e. moctezuma, md. w. siddiqui, j.f. ayala-zavala preparation of phenolic extracts methanol and ethanol were used in the extraction process that has been proved to generate extracts with high antioxidant capacity (oboh and rocha, 2007; muthuswamy et al., 2008; ayalazavala et al., 2012a). samples (10 g) were left to macerate in 100 ml of each alcohol: water (7:3) in darkness for 10 days at 25 °c. after that time, the extracts were filtered and the solvent removed using a rotary evaporator. the aqueous fraction was lyophilized and hydrolyzed (10 ml of naoh 4m) for 4 h in the absence of light, subsequently, an acid hydrolysis was performed with hcl 4m taking every sample to ph 2. the hydrolyzed extract was subject to liquid-liquid separation by two washes with 20 ml of ethyl acetate, yielding two fractions: polar (aqueous phase) and non polar (ethyl acetate phase) (oboh and rocha, 2007). solvent was evaporated from the non polar extract and it was re-suspended in de-ionized water. overall, methanolic polar (mpe), methanolic non polar (mnpe), ethanolic polar (epe) and ethanolic non polar extracts (enpe) with a concentration of 25 mg of extract of tissue per ml of water were obtained. water infusion was used as an alternative extraction process. lyophilized byproducts (1 g) were mixed with 40 ml of distilled water; the mixture was boiled (100 °c) for 30 min. the crude extract was centrifuged at 2,348 x g, 4 °c for 8 min. subsequently filtered through layers of organza, the filtrate obtained (concentration 25 mg/ml) was frozen at -35 °c until further use (muthuswamy et al., 2008). all the extractions were performed by triplicate and the extract yield, total phenolic, flavonoid contents, antioxidant capacity and antimicrobial activity were measured. total phenolic and flavonoid contents total phenolic content was measured by the methods described by singleton and rossi (1965), with some modifications. extracts (50 μl) were mixed with 3 ml of h2o and 250 μl of folinciocalteu’s phenol reagent 1n. after 8 min of equilibration time, 750 μl of na2co3 (20 %) and 950 μl of h2o were added to the mixture, and incubated for 30min at room temperature. after incubation, the absorbance was read at 765 nm with an uv-vis spectrophotometer (cary, model 50 bio, varian, italy). concentration of total phenolic compounds was calculated using a standard curve of gae and expressed as milligrams per gram of dry weight. flavonoid content was determined based on the method described by zhishen et al. (1999). one ml of each extract was mixed with 5 % nano2, 10 % alcl3 and naoh 1m and measured spectrophotometrically at 415 nm using quercetin as standard. the results were expressed as mg of quercetin equivalents (qe) per g of dry weight. dpph radical scavenging activity• this assay is based on the measurement of the scavenging ability of antioxidants towards the stable radical dpph• (gonzález-aguilar et al., 2007). a 3.9 ml aliquot of a 0.0634 mm of dpph• solution, in methanol was added to 0.1 ml of each extract. the mixture was shaken in a vortex and kept 30 min in the dark. absorbance was then read in an uv-vis (cary, model 50 bio, varian, italy) spectrophotometer, at a wavelength of 515 nm. results were expressed as ec50 (concentration of the antioxidant extract required to reduce the absorbance of the radical by 50 %) in mg/ml. analyses were performed in triplicate per each sample. trolox equivalent antioxidant capacity (teac) this assay is based on the ability of the antioxidants to scavenge the blue-green abts•+ radical compared to the scavenging ability of the water-soluble vitamin e analogue, trolox (re et al., 1999). the abts•+ radical cation was generated by the interaction of 5 ml of 7 mm abts solution and 88 μl of 0.139 mm k2s2o8 solution. after the addition of 2970 μl of abts solution to 30 μl of extracts (0.2 g/ml) or trolox standards (0 to 20 μm range), the absorbance was monitored exactly 1 and 6 min after the initial mixing. the percentage of absorbance inhibition at 734 nm was calculated and plotted as a function of that obtained for the extracts and the trolox standard. the final teac value was calculated by using a regression equation between the standard concentration and the inhibition percentage and expressed as trolox equivalents (μmol te) per g of dry weight. antifungal assay the antifungal potential of byproducts extracts was tested against alternaria alternata using the central inoculation method, the extracts were diluted in the agar (ayala-zavala et al., 2012b). for this 6.25 mg/ml of each extract were added to petri dishes containing potato dextrose agar, and then inoculated in the center with the fungi. the efficiency of the treatments was evaluated at 5 days of incubation at 25 °c. controls were pure agar inoculated with the fungus and without exposure to any extract, no effect of the solvent on the fungal growth was observed. mycelial area was recorded (cm2) in triplicate by the digital analysis of the image of every plate using the uthscsa imagetool version 3.0 software. results were expressed as inhibition percentage of mycelial growth of a. alternata at day 5. antibacterial assay the antibacterial ability of every extract against salmonella enterica subsp. enterica serovar. choleraesuis atcc 14028, listeria monocytogenes atcc 7644, escherichia coli o157:h7 atcc 43890 and staphylococcus aureus atcc 65384, was assessed. a loopful (~20 μl) of the bacterium was transferred to a tube containing 10 ml of tryptic soy broth and incubated overnight at 37 °c. initial inoculums of 1 x 108 cfu/ml were placed in tubes with 1 ml of mueller hinton broth and then 1ml of extract was added (25 mg/ ml), including a control tube without the extract. the tubes were incubated at 37 °c by 24 h and the cfu of each bacteria in presence and absence of extracts were determined on mueller hinton agar. results were expressed as a percentage of growth inhibition compared with controls. tab. 1: physicochemical characterization of studied mango varieties ‘haden’, ‘ataulfo’ and ‘tommy atkins’. parameters evaluated haden ataulfo tommy atkins sst (%) 12.37 ± 0.23* 14.40 ± 0.13 14.13 ± 0.16 firmness (n) 97.66 ± 0.29 105.77 ± 0.39 109.10 ± 0.18 ph 3.42 ± 0.02 2.87 ± 0.02 3.16 ± 0.01 acidity (% citric acid) 0.77 ± 0.03 2.07 ± 0.03 0.97 ± 0.07 peel color l 52.20 ± 2.27 64.90 ± 1.72 59.48 ± 2.76 °hue 110.74 ± 5.52 102.10 ± 1.69 101.86 ± 4.97 chroma 44.08 ± 3.38 55.72 ± 1.44 43.49 ± 1.73 pulp color l 72.42 ± 1.36 76.64 ± 1.44 75.68 ± 1.12 °hue 94.52 ± 1.39 93.14 ± 1.02 97.52 ± 1.24 chroma 72.78 ± 2.79 67.52 ± 2.54 57.21 ± 3.58 *average of three determinations ± standard error antimicrobial and antioxidant properties of mango byproducts 207 statistical analysis the experimental design consisted of a 3x3x5 factorial arrangement: 3 = variety (‘haden’, ‘ataulfo’, ‘tommy atkins’), 3 = tissue (peel, seed, pulp) and 5 = extracts (mpe, mnpe, epe, enpe, water infusion) for the evaluation of the total phenolics, flavonoids, and antioxidant capacity. for the evaluation of the antimicrobial activity a factorial arrangements of 3 x 2 x 5: 3 = variety (‘haden’, ‘ataulfo’, ‘tommy atkins’), 2 = tissue (peel, seed), and 5 = extracts (mpe, mnpe, epe, enpe, water infusion) were used, respectively. anova was performed (p ≤ 0.05) to estimate significant differences between treatments and tukey-kramer was the mean test used for comparison (p ≤ 0.05) using the ncss software (2007). results and discussion yield of fresh-cut mango byproducts fresh-cut mango byproducts, composed of peel, seed and useless pulp, represented 40 % of the total weight of the fruit, while freshcut produce in the form of cubes yielded 60 % of the total weight. amongst byproduct, seed yields from 11-12.33 %, peel 8.90-13 % and unused pulp 15.3-59 % (tab. 2). authors like berardini et al. (2005) reported that industrial processing of mango yields of 3385 % of pulp, 7-24 % of peel and 9-40 % of seed, which depends on the variety of mango and the type of product to be manufactured with the pulp. although the range is wide, this is indicative of the large percentage of byproducts that minimum processing of mango generates, which usually have no further use. previous studies demonstrated that the minimal processing of several kinds of fruits produced high amounts of byproducts. the processed fruits were apples (malus domestica var. golden delicious), mandarins (citrus reticulata), papayas (carica papaya var. maradol), pineapples (ananas comosus var. premium cayenne) and mangos (mangifera indica var. kent). processing of apples produced 10.91 % of pulp and seed (core) byproducts and 89.09 % of apple slices. mandarins processing produced 16.05 % of peels and 83.95 % of wedges. processing of papayas produced 6.51 % of seeds, 8.47 % of peels, 32.06 % of unusable pulp (due to the lack of shape uniformity in a cube) and 52.96 % of cubes. pineapples processing produced 9.12 % of core, 13.48 % of peels, 14.49 % of pulp, 14.87 % of top and 48.04 % of slices. mangos ‘kent’ produced 13.5 % of seeds, 11 % of peels, 17.94 % unusable pulp and 57.56 % of cubes (ayala-zavala et al., 2010). it has to be highlighted that regarding the considerable amounts of fruit byproducts of the minimal processing, it can be considered the possibility of creating alternative uses to give added value to this wasted material. antioxidant capacity of the fresh-cut mango byproducts extracts fig. 1 shows the content of phenolic compounds (total phenolics and flavonoids) from fresh-cut mango byproducts extracts and fig. 2 shows the antioxidant capacity (dpph and teac) of the extracts obtained from such tissues. a significant effect of the three evaluated mango varieties was found (p ≤ 0.05). regarding the global effect of the variety (including all the tissues and the extractions): haden and ataulfo had the highest total phenolics (168 and 145 mg gae/g, respectively), flavonoids (58 and 91 mg qe/g), antioxidant capacity expressed by dpph ec50 (0.8456 and 0.5406 mg/ml), and teac values (45.93 and 45.88 mmol te/g), followed by tommy atkins (phenolics = 128 mg gae/g, flavonoids = 56 mg qe/g, dpph ec50 = 1.23 mg/ml, and teac = 37.5 mmol te/g). no significant differences between haden and ataulfo were found (p ≥ 0.05), but between ataulfo and haden with tommy atkins, the antioxidant capacity was significantly different (p ≤ 0.05). a significant effect (p ≤ 0.05) of the evaluated tissues on the antioxidant properties of the extracts was observed, disregarding the effect of the variety and extraction. the seed had the highest values of the evaluated parameters (phenolics = 346 mg gae/g, flavonoids = 126.9 mg qe/g, dpph ec50 = 0.307 mg/ml, and teac = 112 mmol te/g), followed by the peel (phenolics = 91 mg gae/g, flavonoids = 78 mg qe/g, dpph ec50 = 1.438 mg/ml, and teac = 16 mmol te/g), and finally the pulp (phenolics = 4.42 mg gae/g, flavonoids = 1.9 mg qe/g, dpph < 50 % inhibition at mg/ml, and teac = 0.25 mmol te/g). also, a significant effect of the different extraction solvents on the antioxidant status of the extracts was observed (p ≤ 0.05), overall enpe showed higher values (phenolics = 230 mg gae/g, flavonoids = 46 mg qe/g, dpph ec50 = 0.716 mg/ml, and teac = 77 mmol te/g), and mnpe (phenolics = 186 mg gae/g, flavonoids = 107 mg qe/g, dpph ec50 = 0.51 mg/ml, and teac = 64 mmol te/g), followed by mpe (phenolics = 143 mg gae/g, flavonoids = 93 mg qe/g, dpph ec50 = 1 mg/ml, and teac = 22 mmol te/g), epe (phenolics = 109 mg gae/g, flavonoids = 50 mg qe/g, dpph ec50 = 0.72 mg/ml, and teac = 37 mmol te/g), and water infusion (phenolics = 67.8 mg gae/g, flavonoids = 47 mg qe/g, dpph ec50 = 1.41 mg/ml, and teac = 13 mmol te/g). in the triple interaction among factors (variety-tissue-solvent of extraction), the enpe of mango seed variety haden showed the highest values (phenolics = 875 mg gae/g, flavonoids = 164 mg qe/g, dpph ec50 = 0.04 mg/ml, and teac = 272 mmol te/g), followed by the same extract of the variety tommy atkins (phenolics = 746 mg gae/g, flavonoids = 112 mg qe/g, dpph ec50 = 0.095 mg/ml, and teac = 232 mmol te/g), and the mnpe of the same tissue of the variety ataulfo (phenolics = 424 mg gae/g, flavonoids = 261 mg qe/g, dpph ec50 = 0.133 mg/ml, and teac = 168 mmol te/g). in general, there was a positive relation between phenolic content and antioxidant capacity. comparing the results of this study with previous knowledge the antioxidant content and activity of extracts from mango byproducts are high. matsusaka and kawabata (2010) reported the total phenolic content and the antioxidant capacity of the non-edible parts of some tropical fruits, resulting that the mango seed and mango peel possess the major content of phenolics (153 and 123.80 mg/g dw respectively) compared to avocado, starfruit, canistel, passion fruit, tab. 2: fresh-cut mango and byproducts yield. variety and part of the fruit yield (%) haden peel 13.0 ± 0.58* seed 11.0 ± 1.15 unused pulp 15.33 ± 0.58 final product (cubes) 60.67 ± 1.15 ataulfo peel 8.90 ± 0.09 seed 11.83 ± 0.08 unused pulp 22.27 ± 1.13 final product (cubes) 57.00 ± 1.15 tommy atkins peel 11.67 ± 0.58 seed 12.33 ± 0.29 unused pulp 17.00 ± 1.50 final product (cubes) 59.00 ± 2.29 *average of three determinations ± standard error 208 v. vega-vega, b.a. silva-espinoza, m.r. cruz-valenzuela, a.t. bernal-mercado, g.a. gonzález-aguilar, s. ruíz-cruz, e. moctezuma, md. w. siddiqui, j.f. ayala-zavala fig. 1: total phenolics and flavonoids from fresh-cut mango byproducts extracts. *average of three determinations ± standard error, pme: polar methanolic extract; npme: non polar methanolic extract; pee: polar ethanolic extract; npee: non polar ethanolic extract. fig. 2: antioxidant capacity from fresh-cut mango byproducts extracts. *average of three determinations ± standard error, **reported as inhibition % at the concentration 25 mg/ml, because the ec50 was not found antimicrobial and antioxidant properties of mango byproducts 209 kiwifruit, papaya and kiwano. also the mango byproducts, seed (4188 teac μmol/g dw) and peel (1846 teac μmol/g dw) showed higher antioxidant potencial by the dpph and abts radical scavenging assays than the avocado seed (462 teac μmol/g dw), passionfruit peel (75.4 teac μmol/g dw), papaya peel (58.4 teac μmol/g de) and kiwano peel (14.2 teac μmol/g dw). in the other hand, the mango seed possess major phenolic content than apple peel (1144 mg gae/100g dw), kiwifruit peel (820 mg gae/100g dw) and pink grapefruit peel (2335 mg gae/100g dw) (wijngaard et al., 2009). the phenolics content found in these studies for mango byproducts were similar to that obtained during the infusion in our study, nevertheless the enpe yield major phenolic contents than the byproducts of some tropical fruits. these results confirm the findings from previous studies that showed that the extraction of phenolics compounds with hydro-alcoholic mixtures yield higher values (bucic-kojic et al., 2009). the differences may be attributed to the process and the solvent used in the extraction, the variety of the fruits and the antioxidant compounds characteristics of each byproduct extract. with this in mind, the mango byproducts have shown to be a potent source of phenolic compounds with high antioxidant capacity. antimicrobial activity of the fresh-cut mango byproducts extracts fig. 3 shows the antifungal activity of the extracts from fresh-cut mango byproducts varieties haden, ataulfo and tommy atkins against a. alternata at a concentration of 6.25 mg/ml. a significant global effect (p ≤ 0.05) of the evaluated variety (including all the tissues and the extractions) on the antifungal activity of the obtained extracts was found. ataulfo and haden varieties had the highest antifungal activity (37.5 and 34 %, respectively), showing no significant differences between them (p ≥ 0.05), followed by tommy atkins (33 %), which showed no significant difference as between tissues was observed, disregarding the effect of the variety and extraction compared to haden (p ≥ 0.05). a significant difference (p ≤ 0.05), with the highest antifungal activity values found in the peel (33 % inhibition), followed by the seed (37 %). on the other hand, there was a significant difference (p ≤ 0.05) in antifungal activity depending on the type of extraction solvent used for this study. the mpe showed the highest antifungal capacity (42 %), followed by mnpe (36 %), enpe (36 %), epe (31 %), and finally water infusion (30 %). all the interactions were significant (p ≤ 0.05), with the enpe obtained from the seed of the variety haden showing the highest inhibition (89.78 %) of the mycelial growth of a. alternata. the water infusion and the mpe obtained from the seed of the variety tommy atkins had antifungal values of 89.69 and 87.34 %, respectively, and no difference (p ≥ 0.05) was found between them. fig. 4 and 5 show the inhibition of bacterial strains by the presence of the bioactive extracts. a significant effect (p ≤ 0.05) of the evaluated varieties (including all the tissues and the extractions) was observed against food pathogens survival. the extracts from the haden variety showed higher values of inhibition percent of bacterial strains (e. coli 0157:h7 = 48 %, s. choleraesuis = 47.6 %, l. monocytogenes = 48.5 %, and s. aureus = 48 %), followed by ataulfo (e. coli 0157:h7 = 49 %, s. choleraesuis = 47 %, l. monocytogenes = 47.8 %, and s. aureus = 47.8 %), and tommy atkins (e. coli 0157:h7 = 47.5 %, s. choleraesuis = 46 %, l. monocytogenes = 45 %, and s. aureus = 46 %), all presenting significant differences (p ≤ 0.05) amongst them. similarly, a significant effect (p ≤ 0.05) of the extracted tissue and extraction solvent was found. amongst the different tissues, disregarding the effect of the variety and extraction, the extracted peel showed higher values of inhibition (e. coli 0157:h7 = 47.7 %, s. choleraesuis = 48 %, l. monocytogenes = 47 %, and s. aureus = 47.6 %), followed by the seed (e. coli 0157:h7 = 48.6 %, s. choleraesuis = 46.6 %, l. monocytogenes = 46.9 %, and s. aureus = 47 %). similarly, a significant effect (p ≤ 0.05) of the extraction solvent was observed (disregarding the effect of the variety and tissues), no differences were found (p ≥ 0.05) in methanolic (e. coli 0157:h7 = 50 %, s. choleraesuis = 49 %, l. monocytogenes = 50 %, and s. aureus = 50 %) and ethanolic (e. coli 0157:h7 = 49.9 %, s. choleraesuis = 50 %, l. monocytogenes = 50 %, and s. aureus = 50 %) extracts, but significant differences were found (p ≤ 0.05) with the water infusion extraction method (e. coli 0157:h7 = 42 %, s. choleraesuis = 34 %, l. monocytogenes = 37 %, and s. aureus = 37 %). considering the factors interactions, all were significant (p ≤ 0.05). the extracts that showed 100 % inhibition of all food pathogens were: haden (seed mnpe and epe and peel mpe); ataulfo and tommy atkins (seed and peel mnpe, mpe, enpe, epe), while the water infusions of all tissues exhibited the lowest inhibition. in general, the most sensitive bacterial strains to the presence of bioactive extracts were the gram positives: l. monocytogenes and s. aureus, with 100 % inhibition values obtained with the epe, enpe, mpe, and mnpe as compared against gram negative strains of e. coli o157:h7 and s. choleraesuis. similar results have been reported by kabuki et al. (2000), showing that extracts from mango seed are more effective against gram-positive bacteria than gram-negative bacteria. gallotannins (tannins, penta-, hexa-, and hepta-o-galloylglucose) extracted from mango kernels inhibited the proliferation of grampositive food spoilage bacteria and reduced the growth of gramnegative e. coli, although the growth of lactic acid bacteria was not inhibited (engels et al., 2009). this effect could be attributed to the differences in structure of cell envelope, including cytoplasmic membrane and cell wall components of gram-positive bacteria, as compared to gram-negative species (hugo and russell, 1987). fig. 3: inhibition percentage of mycelial growth of alternaria alternata at day 5. *concentration 6.25 mg/ml. **average of three determinations ± standard error. 210 v. vega-vega, b.a. silva-espinoza, m.r. cruz-valenzuela, a.t. bernal-mercado, g.a. gonzález-aguilar, s. ruíz-cruz, e. moctezuma, md. w. siddiqui, j.f. ayala-zavala fig. 4: percentage of inhibition of gram negative bacterial strains exposed to bioactive extracts from fresh-cut mango byproducts. *concentration 25 mg/ml. **average of three determinations ± standard error. fig. 5: percentage of inhibition of gram positive bacterial strains exposed to bioactive extracts from fresh-cut mango byproducts. *concentration 25 mg/ml. **average of three determinations ± standard error. antimicrobial and antioxidant properties of mango byproducts 211 considering the total phenolic content of the obtained extracts, it can be related that these compounds could be responsible of their antimicrobial activity. the antimicrobial mechanisms of these compounds are not well understood; however, it has been suggested that phenolic compounds will cause changes in the membrane through its interaction with the carboxyl groups of the hydrophilic amino acids of the protein in the cell membrane, thus altering its permeability (raybaudi-massilia et al., 2009). phenolic compounds will cause an alteration of ph and electrical potential, causing the release of protons to the outside. thus, there will be a coagulation of cytoplasmic content in the bacteria, accompanied by a loss of normal cell metabolism, leading to cell death (raybaudi-massilia et al., 2009). although, the antimicrobial activity of the extracts is attributed to phenolic compounds it is recommended to identify the profile of compounds in the different tissues, consider that other natural compounds with antimicrobial activity could be found in mango tissues, e.g. terpenoids (negi et al., 2002). conclusions the present study demonstrates a significantly higher total antioxidant capacity, phenolic content and antimicrobial activity of mango byproducts than of the edible portions. the npee obtained from the seed and peel, of mango var. ‘ataulfo’ and ‘haden’ showed the highest antioxidant and antimicrobial activity. this study opens the possibilities of the potential applications of the obtained extracts as antioxidants and antimicrobial agents. acknowledgements authors thank the financial support of the mexican council for science and technology conacyt and the national program for the improvement of the professorate promep. references abdalla, a.e.m., darwish, s.m., ayad, e.h.e., el-hamahmy, r.m., 2007: egyptian mango byproduct 1. compositional quality of mango seed kernel. food chem. 103, 1134-1140. ayala-zavala, j.f., rosas-domínguez, c., vega-vega, v., gonzález aguilar, g.a., 2010: antioxidant enrichment and antimicrobial protection of fresh cut fruits using their own byproducts: looking for integral exploitation. j. food sci. 75, 175-181. ayala-zavala, j.f., vega-vega, v., rosas-domínguez, c., palafoxcarlos, h., villa-rodriguez, j.a., siddiqui, m.w., dávila-aviña, j.e. gonzález-aguilar, g.a., 2011: agro-industrial potential of exotic fruit byproducts as a source of food additives. food res. int. 44, 18661874. ayala-zavala, j.f., perez-carlon, j.j., esqueda, m., gonzalezaguilar, g.a., leyva, j.m., cruz-valenzuela, m.r., moctezuma, e., 2012a: polar fractionation affects the antioxidant properties of methanolic extracts from species of genus phellinus quel. (higher basidiomycetes). int. j. med. mushrooms. 14, 563-573. ayala-zavala, j.f., silva-espinoza, b.a., cruz-valenzuela, m.r., villegas-ochoa, m.a., esqueda, m., gonzalez-aguilar, g.a., calderon-lopez, y., 2012b: antioxidant and antifungal potential of methanol extracts of phellinus spp. from sonora, mexico. rev. iberoam. micol. 29, 132-138. berardini, n., knödler, m., schieber, a. carle, r., 2005: utilization of mango peels as a source of pectin and polyphenolics. innov. food sci. emerg. technol. 6, 442-452. bucic-kojic, a., planinic, m., tomas, s., jakobek, l., seruga, m., 2009: influence of solvent and temperature on extraction of phenolic compounds from grape seed, antioxidant activity and colour of extract. int. j. food sci. tech. 44, 2394-2401. chang, s., tan, c., frankel, e.n., barrett, d.m., 2000: low-density lipoprotein antioxidant activity of phenolic compounds and polyphenol oxidase activity in selected clingstone peach cultivars. j. agr. food chem. 48, 147-151. engels, c., knodler, m., zhao, y.y., carle, r., ganzle, m.g., schieber, a., 2009: antimicrobial activity of gallotannins isolated from mango (mangifera indica l.) kernels. j. agr. food chem. 57, 7712-7718. gonzález-aguilar, g.a., villegas-ochoa, m.a., martínez-téllez, m.a., gardea, a.a., ayala-zavala, j.f., 2007: improving antioxidant capacity of fresh-cut mangoes treated with uv-c. j. food sci. 72, 197-202. gorinstein, s., martin-belloso, o., park, y.s., haruenkit, r., lojek, a., ciz, m., caspi, a., libman, i., trakhtenberg, s., 2001: comparison of some biochemical characteristics of different citrus fruits. food chem. 74, 309-316. hugo, w.b., russell, a.d., 1987: in pharmaceutical microbiology. 4th ed. oxford, blackwell scientific publications. kabuki, t., nakajima, h., arai, m., ueda, s., kuwabara, y., dosako, s., 2000: characterization of novel antimicrobial compounds from mango (mangifera indica l.) kernel seeds. food chem. 71, 61-66. kim, y., brecht, j.k., talcott, s.t., 2007: antioxidant 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r.s., 2000: effect of antioxidant principles isolated from mango (mangifera indica l) seed kernels on oxidative stability of buffalo ghee (butter fat). j. sci. food agric. 80, 522-526. raybaudi-massilia, r.m., mosqueda-melgar, j., soliva-fortuny, r., martin-belloso, o., 2009: control of pathogenic and spoilage microorganisms in fresh-cut fruits and fruit juices by traditional and alternative natural antimicrobials. compr. rev. food sci. f. 8, 157-180. re, r., pellegrini, n., proteggente, a., pannala, a., yang, m., riceevans, c., 1999: antioxidant activity applying an improved abts radical cation decolorization assay. free rad. biol. med. 26, 1231-1237. ribeiro, s.m.r., barbosa, l.c.a., queiroz, j.h., knödler, m., schieber, a., 2008: phenolic compounds and antioxidant capacity of brazilian mango (mangifera indica l.) varieties. food chem. 110, 620-626. seneviratne, k.n., kotuwegedara, r.t., 2009: antioxidant activities of the phenolic extracts of seed oils and seed hulls of five plant species. food sci. technol. int., 15, 419-425. singleton, v.l., rossi, j.a., 1965: colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. am. j. enol. viticult. 16, 144-158. soong, y.y., barlow, p.j., 2004: antioxidant activity and phenolic content of selected fruit seeds. food chem. 88, 411-417. zhishen, j., mengcheng, t., jianming, w., 1999: the determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. food chem. 64, 555-559. address of the corresponding author: e-mail: jayala@ciad.mx journal of applied botany and food quality 87, 190 195 (2014), doi:10.5073/jabfq.2014.087.027 1department of horticulture, faculty of agriculture, ondokuz mayis university, samsun, turkey 2department of histology and embryology, faculty of medicine, ordu university, ordu, turkey 3department of histology and embryology, faculty of medicine, ondokuz mayis university, samsun, turkey the effect of different nursery conditions on some of the leaf and stomata characteristics in chestnuts ozturk ahmet1*, serdar umit1, gürgör pinar naile2, korkmaz adnan3 (received october 23, 2013) * corresponding author summary the aim of this study was to determine the effect of different nursery conditions on some leaf dimensions and stomatal characteristics in the chestnut. the study was carried out in the open field and under shaded (50 %) and unshaded greenhouse conditions. in this study, scions of the marigoule and unal cultivars were grafted on seedlings of the 554-14 genotype with the inverted radicle grafting method. grafting was performed during the second week of may in the first year and during the last week of april in the second year. significant differences in leaf dimensions and stomata density were found in different nursery conditions. the width and length of leaf lamina were higher in the shaded greenhouse (p<0.01). the ratio of the leaf lamina width/length (p<0.01) and stomata density (p<0.05) were higher in the open field condition. the highest correlation (r = 0.847) was determined between stomata length and stomata width/length ratio (p<0.01). these results indicate that the leaf lamina width and length increased in the shaded greenhouse unlike the stomata density which decreased slightly. however, stomata dimensions remained the same in all three nursery conditions. abbreviations: sem: standard error mean, ns: non significant introduction plants are able to modify their growth, development and physiology according to their environment. this ability of plants plays a key role in determining their tolerance to stresses and maintains efficient growth (murchie and horton, 1997; walters et al., 2003). stomatas are important organelles that affect the adaptation skills of plants (salisbury and ross, 1992; brownlee, 2001) by directing transpiration and photosynthesis and by giving clues about suitable plant cultivation. they are epidermal valves that are essential for plant survival because they control the entry of carbon dioxide assimilated in photosynthesis and optimize water use efficiency (schroeder et al., 2001; bergmann and sack, 2008). therefore, stomata play a vital role in the ability of land plants to balance water loss with photosynthetic performance. in leaves, the pattern of stomatal distribution is highly variable between species but is regulated by a mechanism that maintains a minimum of one cell spacing between stomata (casson and gray, 2008). many factors influence stomatal behavior in the plants (salisbury and ross, 1992). stomatal number, size and density show a variation that depends on the plant’s species, cultivar, clones and cultivation conditions (kliewer et al., 1985; caglar and tekin, 1999; caglar et al., 2004). the number, index, and size of stomata are, also, affected by light intensity (pazourek, 1970; thomas et al., 2003) light color (kim et al., 2004; lee et al., 2007; kilic et al., 2010). indeed, an increase in light intensity increased the stoma number, index and size. while blue and red light increases the stomata size and decreased the stomata number, respectively, white light increased the stomata number and size (lee et al., 2007). in addition the stomata density changes with characteristics such as dependence on drought, net photosynthesis production (bierhuizen et al., 1984), vegetative development phases (caglar and tekin, 1999) and altitude (caglar et al., 2004). leaves are the most important organs for photosynthesis and transpiration in plants, and the arrangement, size, shape and anatomy of them differ greatly in different environments (neophytou et al., 2007). leaf parameters such as size and shape are frequently recorded variables in plant research (serdar and kurt, 2011). the shape and structure of leaves varies considerably depending on climate, primarily due to the availability of light and the potential for water loss due to temperature and humidity. temperature, light intensity and relative humidity are the fundamental environmental factors in plant growing. these factors should be considered especially in micropropagation and the acclimatation of new cultivars to different ecologies. the morphological, physiological and anatomical characteristics of plants such as chestnut trees are largely influenced by developmental, environmental and cultivational factors (pereira-lorenzo et al., 1996; ozturk and serdar, 2011). the environmental conditions during and following grafting are one of the factors influencing the healing of the graft union and plant growth. the stomata density of fruit tree leaves can be related to the adaptation processes of any fruit tree. thus, it is worthwhile to investigate the suitability of open, shaded and unshaded greenhouse conditions for grafted seedling production systems. different studies have been carried out to investigate the effects of different nursery conditions on graft success and plant development in the chestnut (anagnostakis, 2007; ozturk et al., 2009; zarafshar et al., 2010; ozturk and serdar, 2011). but, there have been no studies concerned with the effects of different nursery conditions on leaf dimensions and stomata characteristics. the objective of this study was to determine the effects of open field, shaded and unshaded greenhouse conditions on the leaf and stomata characteristics of marigoule and unal chestnut cultivars. materials and methods plant materials this study was carried out in samsun, turkey. the unal and marigoule chestnut cultivars were used in the study. the scions of them were taken from an orchard in the province of samsun, turkey in february and were stored at 4 ± 0.5 °c until they were used for grafting. two or three bud scions were used for inverted radicle grafting. newly germinated seeds of chestnut genotype 554-14 (soylu and serdar, 2000) were used as rootstock. the inverted radicle grafts were carried out on 10th may in the first year and 29th april in the second year (ozturk et al., 2009). grafted seeds were planted in pots (30 x 40 cm) filled with a medium containing 3/4 soil + 1/4 ground pine needles and manure. the growing mediums were clay loam with 3.03 % and 1.62 % organic matter and ph 7.30 and ph 7.82 in the 2 experimental years, respectively. a. ozturk, u. serdar, p.n. gürgör, a. korkmaz 191 study design the study was carried out in the open field and under shaded and unshaded greenhouse conditions. shading was carried out with netting having a light transmission of 50 % and the samples were immediately covered with this netting after grafting. the experimental design was a randomized plot. three replications and 20 grafts per replication were used. leaf samples were taken from 10 plants per replication. the temperature, relative humidity and light intensity of the nursery conditions were measured using a datalogger (kimo kt100, france). data of nursery conditions measured during the experimental period are presented in fig. 1-3. leaf and stomata measurement in order to determine the stomata characteristics of the leaves, samples were taken from the middle part of shoots at the closing time of stomata, 09.00-11.00 a.m. on 25 and 26 september (sahin and soylu, 1991; serdar and kurt, 2011). two leaves per plant and 20 leaves per replication were sampled. after leaf sampling, 6 leaf pieces containing the membrane of the stomatal surface were taken from each leaf by the systematic random sampling method (korkmaz et al., 2000). in this preparation, firstly nail polish was applied to the areas in between the lateral veins of the lower surface containing stomata (sahin and soylu, 1991). after drying out for approximately five minutes, leaf pieces (2.0-2.5 cm2) containing the membrane of the stomatal surface was taken out with an adherent acetate sheet (bozoglu and karayel, 2006). after taking the stomatal surface, the length and width of the leaf lamina were measured immediately. the stomata count and measurements of dimensions including the width and length were done in three regions having an average area of 180 mm2 per sampling through 40 x 100 or 20 x 100 magnifying lenses. the length and width of leaf lamina were measured in five plants (approximately 50 leaves) and the width/length ratio of leaf lamina was calculated. all measurements were repeated in both years (2006 and 2007) in order to determine the effects of nursery conditions on the studied parameters. data analyses some leaf and stomata characteristics of the cultivars, including the effects of nursery conditions, cultivars and years and their interactions, were analysed by a three factored anova test. therefore the data was analyzed in a randomized block design as a factorial arrangement (3 x 2 x 2) of treatments. for data on leaf characteristics individual leaves were considered to be the experimental unit (n = 60 per treatment). when the f test was significant, differences were determined by duncan’s multiple range tests. all analyses were performed using the spss statistical package. results are presented as means and a pooled sem (standard error mean). correlation analysis was applied to assess whether the characteristics of the leaves depend on the stomatal behavior in chestnuts as influenced by the nursery conditions. fig. 2: the light intensity data of nursery conditions with respect to years fig. 1: the temperature data of nursery conditions with respect to years te m pe ra tu re (° c ) 192 some leaf and stomata characteristics in chestnut results and discussion leaf characteristics leaf dimensions and ratio of lamina width/lenght of chestnuts grown in the shaded and unshaded greenhouse and open field conditions are presented in tab. 1. nursery conditions affected the length, width, and width/length ratio of leaf lamina (p<0.01). except for the width/ length ratio of leaf lamina, the length and width was higher (p<0.01) in the shaded greenhouse. however, the width/length ratio of leaf lamina was higher (p<0.05) in the open field (tab. 2). lamina width and length were higher in the shaded greenhouse (4.6 and 17.8 cm respectively) (p<0.01) than both those grown in the unshaded greenhouse (3.4 and 13.6 cm respectively) and in the open field (3.5 and 12.9 cm respectively) (tab. 2). the plants grown in the shaded greenhouse were nearly 75 % wider and had longer leaves than those grown in the other conditions. in this study, the shaded and unshaded greenhouse and open field conditions had a mean temperature of 21.1, 21.4 and 18.9 °c, a mean relative humidity of 79.2, 75.7 and 76.5 %, and a mean light intensity of 1392, 3007 and 4130 lux, respectively (fig. 1-3). these results indicate that leaf dimensions may be affected by higher temperatures (fig. 1), a lower light intensity (fig. 2) and a higher relative humidity (fig. 3). these results confirm the findings of ozturk and serdar (2011). as compared to shaded leaves, those growing in the sun usually have smaller leaf dimensions (leaf width, length and area) and a higher dry mass per leaf area unit (lichtenthaler et al., 1981). leaf dimensions in the strawberry were higher in plants grown in the shade than those grown in unshaded and open field conditions (ozturk and demirsoy, 2004). increases in the area and number of leaves are a common response of leaves that are adapted to shadier conditions and of species that are shade tolerant or intermediate with respect to shade tolerance (lambers et al., 1998). the cultivar had a significant effect on the leaf dimensions (p<0.01) and also the leaf lamina width and length changed considerably between the two years (p< 0.01). the leaf lamina width and leaf lamina width/length ratio of the marigoule cultivar were wider (p<0.01) and higher (p<0.01) than that of the unal. the lamina length of the unal cv. was longer (p<0.01) than that of the marigoule cv. (tab. 1). it was observed that year had a significant effect on the dimensions of the leaf, except for the lamina width/length ratio. the lamina width and length in the second year was higher than that in the first year (p<0.01). on the contrary, the highest lamina width/length ratio was obtained from the plants grown in the open field in the first year (0.29). the interaction between nursery condition and cultivar had a significant effect on the lamina width and length (p<0.05). significant effects of nursery condition and year interactions on the leaf width (p<0.05) and leaf length (p<0.01) and leaf width/length ratio (p< 0.01) were observed (tab. 1). the highest lamina width and length was obtained from the plants grown in the shaded greenhouse in the second year (4.8 and 18.2 cm, respectively). the interaction between year and cultivar had no significant effect on the leaf dimensions and also there were significant effects of nursery condition x cultivar x year interaction on the leaf lamina width (p<0.05) (tab. 1). although the ratios obtained from leaf dimensions may be more stable, leaf dimensions can be a bit changeable according to environmental conditions and cultural practices (serdar and kurt, 2011). the same researchers reported that leaf dimensions (width and length of the leaf lamina, leaf length and leaf area) depend on genotypes rather than year and year x genotype in the chestnut. stomata characteristics stomata are necessary for gas exchange, and have an important role in environmental stress, ploidy levels and resistance to drought, disease and insects (mehri et al., 2009). the leaf stomata frequency of the genotypes can be related to the process of adaptation of the trees. the nursery conditions used in the present study affected the stomata density (p<0.05), but didn’t affect the width and length and width/length ratio of stomata (tab. 1). stomata density of chestnuts grown in the open and unshaded greenhouse conditions was higher (p<0.05) than that of those grown in shaded greenhouse conditions, 312.7, 292.6 and 273.4 per mm2 respectively (tab. 2). ryugo (1988) reported that stomatal frequency varied from 125 to over 1000 per mm2. moreover, light intensity has a strong effect on stomata density and dimensions (thomas et al., 2003). as compared to leaves in the shaded regions, leaves exposed to the sun usually have a greater stomata density (lichtenthaler et al., 1981). and also, stomata density reduced with different shading levels in banana leaves (israeli et al., 1995). in the present study, except for the stomata density, the width and length and width/length ratio of stomata were not affected by the nursery conditions. an increase in light intensity results in an increase in the stomata density, index and size (thomas et al., 2003). also, different colors of light affect the stomata number and size (kim et al., 2004; kilic et al., 2010). lee et al. (2007) reported that white light increased the stomata number and size while blue and red light increased the stomata size and decreased the stomata number. in our study, shading decreased the stomata number, but didn’t affect the stomata dimensions. thus, stomatal differentiation is seen to be very sensitive to a change in the supply of light. this result may be due to the dark green shading cover that was used in the study and may fig. 3: the relative humidity data of nursery conditions with respect to years a. ozturk, u. serdar, p.n. gürgör, a. korkmaz 193 tab. 1: the width and length and width/length of leaf lamina and the number, width and length and width/length of stomata in chestnuts grown in the shaded and unshaded greenhouse and open field condition with respect to years and cultivars nursery conditions cultivars leaf lamina stomata width length width/length number width length width/length first year (2006) shaded mariquel 5.44 16.31 0.33 417.96 17.00 21.67 0.78 unal 3.39 18.59 0.18 272.37 19.17 24.17 0.79 unshaded mariquel 3.73 11.88 0.31 390.01 17.17 22.67 0.76 unal 2.34 12.57 0.18 326.70 18.33 23.92 0.77 open mariquel 3.71 10.70 0.34 419.10 17.17 22.33 0.77 unal 2.48 10.74 0.23 313.15 18.83 23.67 0.80 second year (2007) shaded mariquel 5.61 16.68 0.33 222.60 18.17 23.33 0.78 unal 4.03 19.44 0.20 181.39 18.00 25.00 0.72 unshaded mariquel 4.73 14.63 0.32 263.38 18.67 24.67 0.76 unal 2.96 15.26 0.19 190.94 18.67 24.67 0.76 open mariquel 4.71 14.31 0.33 294.18 17.83 23.50 0.76 unal 3.26 15.73 0.20 225.17 19.22 25.44 0.76 pooled sem 0.032 0.152 0.002 1.366 0.12 0.16 0.04 main effect of nursery conditions ** ** ** * ns ns ns cultivar ** ** ** ** ** ** ns year ** ** n.s ** ns ** * nursery conditions x cultivar * * n.s n.s. ns ns ns nursery conditions x year * ** ** n.s. ns ns ns cultivar x year n.s n.s n.s n.s. ns ns ns nursery conditions x cultivar x year * n.s n.s n.s. * ns ns pooled sem. pooled standard error of the mean. ns. p > 0.05; *. p < 0.05; **. p < 0.01. also be a consequence of less light intensity in shaded conditions than in the other nursery conditions (fig 2). different light conditions decreased the stomata number in the lower surface of leaves of tomato seedlings, compared to the control and transparent cover (kilic et al., 2010). thomas et al. (2003) noted that in tobacco, the shading of mature leaves while exposing developing leaves to high light intensity resulted in a 12.7 % decrease in the stomatal index of the developing leaves compared with controls exposed only to high light intensity. pazourek (1970) stated that the stomatal density decreases with lower light intensities and the largest reaction to the light intensity appeared in leaves with a higher stomata density. also, ilgin and caglar, (2009) stated that plants grow in environments with a higher temperature may adapt to these conditions by reducing the number of stomata, or stomatal density on each leaf surface. in the present study, we tried to determine by how much the nursery conditions affect the leaf dimensions and stomata characteristics in the chestnut (tab. 1) and presented the mean data of temperature, light intensity and relative humidity of these nursery conditions (fig. 1-3). but, we could not determine how responsible temperature, light intensity or relative humidity is for these effects. tab. 2: the width and length and width/length ratio of leaf lamina and the number and width and length and width/length ratio of stomata in chestnuts grown in the shaded and unshaded greenhouse and open field condition nursery conditions leaf lamina stomata width (cm) length (cm) width / length number width (μ) length (μ) width / length shaded 4.62±0.99 a 17.80±1.60 a 0.26±0.07 b 273.36±2.24 b 18.08±0.99 23.54±1.6 0.76±0.02 unshaded 3.44±0.95 b 13.58±1.74 b 0.25±0.06 b 292.61±1.89 ab 18.21±0.98 23.98±1.1 0.75±0.02 open 3.54±0.85 b 12.87±2.38 b 0.27±0.06 a 312.72±1.92 a 18.26±1.02 23.74±1.4 0.77±0.02 significance ** ** ** * ns ns ns a.b means with different letters in the same column were significantly different (**p < 0.01. *p<0.05). 194 some leaf and stomata characteristics in chestnut the cultivar had a significant effect on the number and width and length of the stomata (p<0.01) but not on the stomata width/length ratio, and also there were significant effects of the year upon the stomata density and length (p<0.01) and stomata width/length ratio (p<0.05). the stomata density of the marigoule cv. was higher (p< 0.01) than that of the unal cv., 334.51 and 251.61, respectively. the stomata width and stomata width/length ratio of the unal cultivar was wider (p<0.01) and higher (p<0.01) respectively than that of the marigoule (tab. 1). stomata size and density show variation according to the plant’s species, cultivar, clones and cultivation conditions (caglar and tekin, 1999; caglar et al., 2004). the stomata density and stomata width/length in the first year was higher than that in the second year, (p<0.05; p<0.01) respectively. serdar and kurt (2011) reported that stomata width and length and the stomata width/length ratio change with respect to year and genotypes. stomata density changed in relation to the year and cultivars in the grape (kara and ozseker, 1999). in this study, except for the interaction of nursery condition x cultivar x year, there were no significant effects of the interaction of nursery condition x cultivar, nursery condition x year, cultivar x year on stomata characteristics in the chestnut. also there were significant effects of nursery condition x cultivar x year interaction on stomata width (p<0.05) in the chestnut. the unal cultivar grown in open conditions in the second year had a higher stomata width (p<0.05) than other nursery conditions. a linear correlation has been found amongst the lamina width and lamina width/length ratio (r=0.738) and lamina width/length and stomata width/length (r=0.717) in the present study. the highest correlation (r=0.847, p<0.01) was found between mean stomata length and stomata width/length ratio (tab. 3) respectively. sahin and soylu, (1991) cited that a significant linear correlation (r=0.758**) was found between the mean length and mean width of stomata of the cultivars in the chestnut. the mean length and width of lamina in chestnut genotypes varied from 17.7 to 21.6 cm and from 4.10 to 6.77 cm, respectively (serdar and kurt, 2011). these researchers reported that the mean length and width of stomata in the studied chestnut genotypes varied from 23.3 to 25.9 μm and 17.1 to 18.1 μm, respectively. in the present study, corresponding values for the grafted headings of unal and marigoule cultivars grown under different nursery conditions were 24.5-18.7 μm and 23.017.7 μm, respectively. these values indicate that the chestnut cultivars used in the current study acclimated to different nursery conditions well. conclusion in the present study, the results indicate that nursery conditions did affect the some of the leaf dimensions and stomata characteristics in the chestnut. shaded greenhouse conditions resulted in a higher lamina width and length but it did not affect either the ratio of leaf lamina width/length or the stomata dimensions. in this event, the role of temperature, light intensity and relative humidity was probably significant. however, we did not determine which factors are more influential which could be the focus of a future study. acknowledgements we thank dr. nuh ocak for critical review of the manuscript and thomas william harvey for english revision. references anagnostakis, s.l., 2007: effect of shade on growth of seedling american chestnut trees. northern j. apply forestry 24(4), 317-318. bergmann, d.c., sack, f.d., 2008: stomatal development. annual rev. plant biol. 58, 163-81. bierhuizen, j.f., bierhuizen, j.m., martakis, g.f.p., 1984: the effect of light and co2 on photosynthesis of various pot plants. gartenbauswissenschaft 49, 215-257. bozoglu, h., karayel, r., 2006: investigation of stomata densities in pea (pisum sativum l.) lines/cultivars. online journal biological science 6, 45-50. brownlee, c., 2001: the long and short of stomatal density signal. trend in plant sci. 6, 441-442. caglar, s., sutyemez, m., beyazit, s., 2004: stomatal density in some selected walnut 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proc. intl. symp. cool climate viticulture and enology, 198-216. korkmaz, a., ciftci, n., bosnak, m., agar, e., 2000: a simplified application of systematic field sampling and low-cost video recording setup for viewing disector pairs exemplified in the rat cochlear nucleus. j. micros. 200(3), 269-276. lambers, h., chapin, f.s., pons, t.l., 1998: physiological, biochemical, and anatomical differences between sun and shade leaves. in: plant physiological ecology, 26-36. springer-verlag, new york. lee, s.h., tewari, r.k., hahn, e.j., paek, k.y., 2007: photon flux density and light quality induce changes in growth, stomatal development, photosynthesis and transpiration of withania somnifera (l.) dunal. plantlets. plant cell tiss. org. cult. 90, 141-151. lichtenthaler, h.k., buschmann, c., doll, m., fietz, h.j., bach, t., kozel, u., meier, d., rahmsdorf, u., 1981: photosynthetic activity, chloroplast ultra-structure, and leaf characteristics of high-light and lowlight plants and of sun and 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195 µg m-3 nh3). reference plants (amb) were raised outdoors, where temperatures were 1.4°c lower than in the greenhouses. after the treatments plants were all overwintered outside to study whether the pre-winter growth advancement was still discernible in the following spring. shed leaves were collected weekly to follow how much shoot biomass was lost during and after the winter. cultivars responded differently to warming and exposure to ammonia. up to the winter shoot biomass was strongly increased by the advancing treatments. however, fi nal shoot mass in the following summer did not differ between cultivars and was unaffected by the higher temperatures in the preceding autumn. nevertheless, signifi cantly more biomass was observed in ammonia fumigated plants. higher autumn temperatures increased leaf shedding and advanced fl owering and senescence in the next spring so that plants showed a signifi cantly reduced seed mass, harvest index and oil yield at the fi nal harvest. obviously, the growth advancement in the preceding autumn by elevated temperatures negatively affected the availability of resources in the following spring. in contrast, plants that were grown at both elevated ammonia and temperature in the autumn showed a delayed fl owering, higher shoot and seed mass, increased harvest index and oil yield. we conclude that growth advancement by elevated autumn temperatures without the re-supply of nutrients increases leaf shedding during winter. nevertheless, the loss of resources in winter for re-growth in spring will certainly be of minor importance for yield formation as compared to the frost damage resulting from late spring frosts. introduction oilseed rape (osr) is currently the most important bioenergy crop in the temperate regions and much effort has been made to improve yields and the oil quality of this species in recent decades. climatic changes may shift the current osr crop belt in the near future and in its current cultivation range increasing temperatures may affect the phenology, speed up the crop development and eventually reduce the seed output due to the shorter crop growth cycle. at the same time, seed maturation, yield and oil quality may directly be affected by planting dates and high temperatures (morrison and stewart 2002; aksouh-harradj et al., 2006; pellet et al., 2008; baux et al., 2008; faraji et al., 2009). it may also be hypothesised that with the predicted climatic changes, new pests and diseases will occur in the future, while the rising winter temperatures may not be suited to the vernalization requirements of some winter crop cultivars any more. fig. 1 shows time series for osr yield increments since 1961 and changes in the average duration of the culture period between the years 1951 and 2008 for germany. because sowing and harvest dates have been moved to earlier and later dates, respectively, average winter osr culture periods increased from the 1950s to the 1980s but then decreased since the 1990s. at the same time osr fl owering occurred 15 days earlier in the decade 1999 to 2008 as compared to the climate normal period (fig. 2). it is unclear, however, whether and how strong the introduction of new cultivars has had an effect on the observed phenological changes and the development of yields. there is also no information available whether yield increments over time and changes in crop duration are interrelated. fig. 1: changes in the average duration of the winter osr cultivation period (○, y-axis to the left) and yields (■, y-axis to the right) in germany. phenological records (1951-2008) stem from the database of deutscher wetterdienst (dwd) and data on oilseed rape yields in germany (1961-2008) from faostat. effects arising from changes in the fl owering window due to deviations from the normal climatic program can be manifold. earlier onset of fl owering may be especially critical if pollinators are still absent and if late frosts occur. the fl owering window of osr is about one month in total and adverse climatic conditions may well occur during this rather long time period affecting at least part of the 250 275 300 325 350 1950 1970 1990 2010 d ay s fr o m so w in g to h a rv es t 0 20 40 60 80 d t h a -1 effects of autumn growth advancement in winter oilseed rape cultivars 135 fl owers. because the climate in central europe may become more variable in the future, the uniform phenological development may be impaired if the heat accumulation over time and the photoperiodic response of crops and cultivars do not match any more. besides changes in the beginning of fl owering, the alteration of the plant phenological program by climatic changes may also affect the timing of root and leaf development, which drive the resource acquisition and nutrient use effi ciency (nue) of plants (nord and lynch, 2009). while the timing of fertilizer application may be adapted to these new conditions, the availability of water in the future will depend on the timing and intensity of rainfall. however, variations in precipitation patterns under climatic change are currently much more diffi cult to forecast than changes in temperatures. responses of oilseed rape to higher spring temperatures peltonen-sainio et al. (2009 a, b) addressed the current and future potential of osr farming in northern europe suggesting that cropping of winter instead of spring varieties may be more favourable in a warmer climate. information is lacking, however, on problems with osr cropping in the southern european regions that might arise from climatic changes. the suitability of irrigated oilseed rape cultivation as a winter rotational crop in semiarid regions of the south-western us was addressed by adamsen and coffelt (2005). planting the same spring varieties as used in the great plains in november at maricopa agricultural center (arizona) lead to acceptable yields but plants produced too much vegetative biomass (straw) at the expense of seed mass. a reason for the lower harvest index may be the high temperatures in the study region that affected fl owering and seed fi lling. morrison and stewart (2002) found that fl oral fertility and seed fi lling were signifi cantly reduced at high air temperatures above 29.5°c. it may thus be suggested that the increasing frequency of hot spells even in the cool and temperate regions may reduce the seed set of osr. this could be the case in both winter and spring cultivars and, indeed, peltonen-sainio et al. (2007) have attributed the yield losses observed in finnish osr over the past 15 years to the increased occurrence of summer heat. in contrast to this study, the halt in osr farm yields since 1985 in the uk has been attributed to crop management factors (shorter rotations, direct drilling, lower application rates of nutrients) rather than to changing climatic factors and failures in germplasm improvement (berry and spink, 2006). still, the authors suggest that an increase in temperatures by 3°c will reduce the seed fi lling period by 13 days. using historical crop yield and climate data from canada, kutcher et al. (2010) found that the beginning of july, i.e. the beginning of the fl owering period, was the critical period in the determination of osr yields. low yields were strongly associated with high july temperatures whereas high yields were associated with greater-than-average precipitation, and to a lesser extent, cooler-than-average nocturnal temperatures. in contrast to the negative effects of high temperatures during fl owering and the seed determination period 19-25 days after mid-fl owering the earlier fl owering caused by warmer springs may also be regarded as a positive response. this is due to the fact that light refl ection by the fl owers will be reduced earlier so that leaf area and the assimilation of carbohydrates may overall profi t. one would then expect higher yields provided the absence of thermal stress later on. berry and spink (2006) state that bringing forward fl owering by one week could improve yields but it is unclear whether the earlier fl owering and increased yields in germany (fig. 1) are interrelated. still, the strong yield depressions by 20% in the hot summer 2003 and the warm spring in 2007 indicate adverse climatic infl uences on osr and give some idea on the effects of thermal stress in the future. fig. 2: beginning of fl owering of winter osr in germany during the wmo climate normal period 1961-1990 (left) as compared to the warmer decade 19992008 (right). phenological data for over 3500 stations (black dots) from deutscher wetterdienst (dwd). maps were produced on the basis of a digital elevation model (1 km x 1 km) with local trend surface prediction (bivand et al., 2008) using the statistical programming environment r. numbers in scale represent days of the year (doy, with doy 1 = 1 january). in most regions fl owering occurs 10 to 15 days earlier nowadays than during the climate normal period. 136 j. franzaring, i. holz, a. fangmeier responses of oilseed rape to elevated autumn and winter temperatures despite heat stress and heat spells probably occurring more frequently in the future, frost will still play a signifi cant or even increasing role in a warmer world (gu et al., 2008). this is because more severe damage or post-freeze setback may turn up in plants that experienced a strong pre-freeze phenological advancement due to warmer conditions. in winter crops, suffi cient biomass and cryoprotective compounds must be accumulated between the sowing and the onset of the winter to increase their frost hardiness. on the other hand, too much shoot biomass and premature shooting will increase the danger of frost damage due to the exposure of aerial plant parts and the occurrence of frost drought. in order to prevent premature shooting under climate change, farmers may apply plant growth regulators to keep plants in the rosette stage but this investment may narrow the benefi ts. to respond to the increased likelihood of shooting before the onset of winter, sowing of winter osr cultivars could eventually be moved to later dates in the near future. in spring cultivars, however, sowing dates should be as early as possible because late planting will result in lower yields, a reduced harvest index and lower oil contents (gross, 1964; kondra, 1977; johnson et al., 1995). while the concept of accumulated thermal time (growing degree days, gdd) is successfully applied to predict the development of spring and winter osr (morrison et al., 1989) in spring, not much information is available on the relationship between heat accumulation and pre winter growth of winter osr. according to diepenbrock (2006a) winter osr should be sown in germany between 15 august and 15 september at a density of 60 to 90 seeds per square metre. until winter plantlets should be in the 6to 8-leaf stage and have an optimum shoot dry weight of approximately 1 g per plant. this will give plants a high chance to survive the winter. growth completely ceases below 2°c and low temperatures will induce the accumulation of phospholipids in plant leaves, which renders them more resistant to frost dehydration (rapacz, 1998, 1999, 2002; christen and friedt, 2007). another prerequisite to withstand frost is the existence of a thick root collar containing large amounts of soluble carbohydrates, which will also benefi t a rapid regrowth in spring (lääniste et al., 2007). during the winter a mean of 50% of the dry shoot mass will normally be lost, but higher losses may occur due to frost damage (diepenbrock, 2006b). lääniste et al. (2007) have addressed the importance of sowing dates on winter hardiness of osr in estonia, which is currently the northern border for the winter cultivars of this crop. based on three years trials they found that winter survival was strongest in plants that had received 416 growing degree days (gdd above a base temperature of 5°c). while plants from late sowing treatments did not survive the winter also the plants from the early sowing variants were severely affected by frost damage. the authors found that at 500 gdd the percentage of overwintering plants was below 50%. effects of n-availability on the frost hardiness of plants another important non-climatic factor, which may strongly advance the development of winter crops, is of course the availability of fertilisers. while p and k fertilisers may be applied to the seedbed to increase yields, nitrogen availability in autumn does not have pronounced effects on the fi nal yields as long as the fertilisation is adequate in the following spring. according to colnenne et al. (2002) even severe autumn n defi ciencies will allow enough shoot mass accumulation to ensure suffi cient re-growth in spring. in fact, excess fertilisation in autumn may increase the danger of frost damage in crops due to the stronger built up of shoot biomass and the exposure of aerial plant parts to frost desiccation. apart from soil-n, elevated concentrations of ammonia near livestock farms may signifi cantly contribute to the n-supply of plants (sommer, 1988, sommer et al., 1991, 2009). the uptake rates of gaseous ammonia have been shown to be elevated at higher soil fertilisation rates, which may be explained by the higher leaf area index at higher availability of soil n that will lead to higher n deposition to the crop. also böhme et al. (2003) found that crop biomass and canopy area determine how much nitrogen will be scavenged from the atmosphere and made available for additional plant growth. while n utilisation from airborne ammonia will be largest in growing stands, plants will switch from sinks of nh3 to active sources during the senescence due to shifts in compensation points. bi-directional fl uxes of ammonia between the atmosphere and osr leaves have been studied by husted et al. (1995, 1996) and massad et al. (2009) under controlled conditions and by nemitz et al. (2000) and schjoerring et al. (2000) in the field. whereas it has been suggested that even low environmental concentrations of ammonia may reduce the frost hardiness of plants from acidic heathlands (e.g. calluna, sheppard and leith, 2002; sheppard et al., 2008), exposure to extremely high concentrations (1000 nl l-1) of ammonia did not affect low-temperature hardening of conifers and wheat (clement et al., 1995, 1999). physiological mechanisms potentially involved in the direct effects of ammonia on the frost hardiness of plants relate to the reduced accumulation of osmotic compounds (polyamines and carbohydrates) and phospholipids allowing plant tissues to adapt to and withstand low temperatures. while these relationships have not been well established, indirect effects of ammonia on the frost sensitivity of plants may occur due to the increase in shoot biomass. scope of the study we assumed that higher autumn temperatures and sub-acute concentrations of ammonia as an additional n-source would fi rst stimulate pre-winter plant growth and later increase the risk of leaf loss in winter oilseed rape. we further postulated different responses in different cultivars and that carry-over effects from the autumn treatments would be recognisable in the formation of yields and the quality of seeds in the following spring. to test these hypotheses, we grew different cultivars of osr in two consecutive experiments with an early and a late sowing date under ambient and elevated temperatures to quantify the additional biomass production of selected cultivars under warmer autumn conditions. we used a third treatment, in which plants were exposed to both elevated temperatures and subchronic concentrations of ammonia to study the additional stimulation of shoot growth. plants from the late sowing were over-wintered to observe how the treatments affected the shedding of leaves during winter and how much biomass and seeds were produced in the following spring. performing such pot-based studies under controlled climatic conditions and collecting the shed leaves at fi xed intervals may serve to address the resource use effi ciency as well as the progress of senescence in higher temporal resolution as compared to fi eld trials, in which shed biomass cannot be accounted for. materials and methods plant cultivation two individual experiments with different sowing dates were performed to investigate the effects of elevated autumn temperatures and sub-acute levels of ammonia on the growth of winter oilseed rape (osr). while plants from the fi rst experiment (early sowing date) were harvested before the winter to address the plant responses in late autumn, only the plants of the second experiment (late sowing date) were overwintered to study the effects of elevated autumn effects of autumn growth advancement in winter oilseed rape cultivars 137 temperatures and ammonia exposures on yield formation and seed quality in the following spring. in the fi rst experiment, osr was sown on 15 august 2008 (doy 228), which is three days earlier than the sowing date in hohenheim in recent years. in the second experiment the crop was sown fi ve weeks later on 22 september 2008 (doy 266). after sowing, each of the 48 pots (4 per cultivar * 4 cultivars * 3 treatments) was fi xed in packs of 6 pots on polystyrene racks placed above individual plastic water containers to be able to follow water consumption over time. water consumption was registered for each of the pots and water was refi lled on demand to the plastic containers at known amounts. the four cultivars used in the experiments ‘asgard’ (a), ‘express’ (e), ‘lorenz’ (l) and ‘viking’ (v) were provided by norddeutsche pfl anzenzucht hans-georg lembke kg (npz holtsee). seed lots of cultivars a, e, l and v had thousand seed weights (tsw) of 4.9, 4.5, 4.7 and 4.3, respectively. five seeds per pot were sown at a depth of 1.5 cm in plastic containers (truncated cone, l=13 cm, b=13 cm, h=13 cm, a=169 cm2, v=1.7 l) and reduced to one plant per pot representing a fi nal density of 60 plants m-2. pots were fi lled with 0.65 kg standard soil (fruhstorfer ld 80, archut-hawita gruppe, vechta) and equipped with glass fi bre wicks (type 6000, hecker werke gmbh, weil im schönbuch) for automatic watering. at water saturation the substrate has a maximum water content of 73.6 wt. % and 27.7 vol. %. the soil substrate is approved after en 12580 and contains bark humus, volcanic clay and peat (35% organic matter in dry mass and ph 6.1). it is enriched with radigen® slow release micronutrients and aquafl ow® slow release npk fertilisers and has a macronutrient content of 150 mg l-1 n and p2o5 each as well as 250 mg l-1 k2o. accounting for the pot volume and its area the initial nitrogen availability represented a value of 150.9 kg ha-1. until winter no further nutrients were supplied, because plants were harvested in the rosette stadium. in the late sowing experiment, which lasted until seed maturation, plants received 100 ml of a nutrient solution (vdi, 2008) on 23rd of march 2009, representing a second gift of 0.5 g n per pot. the vdi standard solution is used for curly kale (brassica oleracea l.) cultures and for its suffi cient sulphur concentrations was believed to be well suited for osr pot cultures. the total n availability (slow release fertiliser plus the nutrient solution) amounted to 0.755 g pot-1 and this quantity was taken into account to derive the n economy parameters nitrogen use effi ciency (nue) and nitrogen recovery in the seeds. treatments and monitoring of ammonia and climate three treatments were applied in the experiments. one set of plants was kept under ambient conditions (amb) in a fenced premise which was covered by a transparent roof to exclude rainfall. the two other treatments were grown in greenhouses (floratherm® alltop ka 200 krieger gmbh herdecke, l 200, b: 185 cm, h: 210 cm, v: 7 m3) to raise the air temperatures by ca. 2°c above the ambient conditions (elvt). in addition to the elevated temperatures one of these warm treatments was subjected to elevated ammonia concentrations (elvt+a). both greenhouses, i.e. treatments elvt and elvt+a, were equipped with a charcoal fi lter unit, a ventilator and fl exible tubes (ø15 cm), through which air was lead into the greenhouse. air was exchanged 54 times per hour in both of the greenhouses. to enrich the elvt+a treatment with ammonia, pure nh3 (quality 3.8, 40 l fl ask, swf friedrichshafen) was lead via a tefl on tube into the air stream. outlet pressure of the fl ask was reduced from 8 to 1 bar resulting in an average fl ow of 1.5 ml min-1 of the gas. this supply was calculated to yield a set ammonia concentration of about 200 µg m-3 (285 ppb) in the elvt+a treatment. in order to avoid placement effects the treatments were switched between the two greenhouses bi-weekly. at these dates also the pots were randomly re-arranged. ammonia concentrations were monitored daily in the elvt+a and weekly in the other treatments using the radiello® passive sampler method (sigma aldrich, fsm, 2006). the adsorbing cartridge is coated with phosphoric acid, which adsorbs ammonium at a sampling rate of 235 ml min-1. the quantity of ammonium was determined against standards at 635 nm by visible spectrometry (beckman coulter du 640b, fullerton) in the form of indophenol which develops from the reaction between phenol and sodium hypochlorite in the presence of cyanoferrate. in order to calculate ammonia concentrations, the nh4+ mass per sampler was divided by the product of the duration (minutes) of the exposure and the sampling rate. in order to obtain information on the temperatures and relative air humidity in the three treatments calibrated tinytag temperature loggers (gemini data loggers, uk) were operated on a 10 min routine inside and outside the greenhouses. measurements on oilseed rape in the early sowing experiment, germination and numbers of germinated seedlings were recorded. on day 7 after germination (7 dag) one seedling per pot was harvested at random and the number of seedlings per pot was reduced to 3 individuals to reduce plant competition. two more harvests took place 14 and 21 days after germination (14 dag and 21 dag) leaving just one plant per pot. leaf area and fresh weight of plant fractions leaf and stem were determined. harvested plants were put into labelled paper bags and were dried at 80°c for 24h in a drying cabinet. after drying, dry matter (dm) of the seedlings was determined and absolute (agr) and relative growth rates (rgr) were calculated accordingly. calculations of growth rates were based on average plant weights (n=4 per treatment and cultivar). leaf greenness was determined weekly with a spadmeter (minolta spad 502) to follow differences in chlorophyll contents in the different treatments. five measurements were taken on the top of the youngest completely unfolded leaf and averages of these measurements were recorded. in the late sowing experiment the same growth related parameters were studied. like in the early sowing experiment root biomass was not analysed. however, much effort was put on the harvest of dead leaf material, which was present after the winter and which accumulated during the senescence of the plants. shed leaves were collected every week and shoot length and fl owering phenology were recorded thoroughly. at the onset of fl owering, spad values were determined on a weekly basis, with measurements being performed on marked leaves at a height of 80 cm. we chose a leaf from a high position instead from the rapidly senescing rosette to be able to get extended information on the whole process of senescence. at the fi nal harvest shoot biomass was separated into stem, leaves, pod walls and seeds. while the fractions stem, leaves and pod walls were dried at 80°c, seeds were air dried to constant weight. finally, the dry weight of all fractions was determined. seeds of each plant were counted with a contador seed counting machine (pfeuffer gmbh kitzinigen, germany) to derive thousand seed mass. seed oil contents were analysed using the near infrared spectroscopy (nirs) standard procedure outlined in vdlufa (2002). n concentrations of leaves that had developed before the winter and seeds harvested the following spring were determined by elemental analyses and seed pkconcentrations were determined by icp-oes following acid digestion. data evaluation and statistics from the original biomass data determined at several harvests, specifi c leaf area (sla), relative growth rates (rgr) and absolute growth rates (agr) were calculated after hendry and grime 138 j. franzaring, i. holz, a. fangmeier (1997). all the biological data were subjected to descriptive statistical analyses and one or two-factorial anova to identify signifi cant treatment effects as well as treatment* cultivar interactions. posthoc tukey honest signifi cant differences (hsd) tests were used for multiple comparisons to study whether there were signifi cant differences in the response of the chosen cultivars. all statistical analyses and most of the graphical representations were performed using the open source programme r and different libraries (r development core team, 2009). results and discussion climate and ammonia concentrations the daily mean temperatures recorded inside the greenhouses (treatments elvt and elvt+a) and outside (amb) are presented in fig. 3. the temperature curve from 15 august 2008 (early sowing of osr) to 24 june 2009 (harvest of osr from the late sowing experiment) is also compared to the long term daily temperature means at stuttgart, germany. as can be seen in tab. 1, ambient temperatures in the autumn 2008 were slightly higher than the long term average. however, in december 2008 and january 2009 mean temperatures remained below the long term average for those months. the extended frost spell early in the year therefore proved to be well suited to study the effect of advanced autumn growth on the winter hardiness of osr. during the warm autumn treatment (elvt) in the early sowing experiment (15.08.-13.10.2008) temperatures were 2.4°c higher than in the amb treatment. in the late sowing experiment, in which the treatments lasted from 22.09. to 19.12.2008 (88 d) the realised temperature increase was only 1.4°c. mean temperatures did not differ between the two greenhouses, which served to increase the temperatures (elvt and elvt+a). mean daily ammonia concentrations during the early sowing experiment were 2.5 (± 0.7) µg m-3 outside (amb), 7.3 (± 6.4) µg m-3 inside the untreated greenhouse (elvt) and 275 (± 99) µg m-3 inside the ammonia treated greenhouse (elvt+a). in the late sowing experiment the mean daily nh3-concentrations ranged from 6.8 (± 6.4) µg m-3 outside (amb) to 6.4 (± 3.2) µg m-3 inside the untreated greenhouse (elvt) and 195 (± 93) µg m-3 inside the ammonia fig. 3: mean daily temperatures during the growth experiments as compared to the long term daily averages and changes in day length. warm autumn treatments lasted from 15 august to 19 december 2008. grey bars indicate the duration of the experiments i (early sowing) and ii (late sowing). long term temperatures are based on the 1991-2005 time series from stuttgart-airport (data available from deutscher wetterdienst, dwd). treated greenhouse (elvt+a). the ammonia treatments resulted in sub-acute ammonia concentrations comparable to those measured after slurry applications. responses during the autumn treatments seeds germinated at a rate of almost 100% with no signifi cant differences between the cultivars and the three treatments in both the early and late sowing experiments. however, there were slight cultivar differences in the speed of germination in the follow order a>v>l>e, but the different speed of germination was unrelated to growth differences subsequently evolving in the three treatments. nevertheless, the availability of seed resources had slight effects in the seedling establishment phase. in the amb treatments of the early sowing experiment, thousand seed masses (tsm) of the cultivars were positively associated with their absolute growth rates (agr) between 7 and 21 days after germination, while the opposite relationship was observed in the plants that were exposed to higher temperatures. at the same time, the osr cultivar ‘viking’ with low availability of seed resources (low tsm) responded more strongly to the atmospheric nitrogen fertilisation in the elvt+a treatment than the other lines (data on growth performance in intermediate harvests not shown). osr plants from all cultivars and from both the early and late sowing experiments were able to absorb ammonia via their shoots and achieved a signifi cantly higher shoot mass than in the other treatments. fig. 4 indicates that shoot mass was highest in the elvt+a treatments and reached an average of 20 g dm in the early and 3.5 g dm in the late sowing experiment. the results confi rm that advancing the plant development by higher temperatures and elevated ammonia concentrations may strongly increase the shoot biomass produced before the onset of the winter. in both of the experiments shoot biomass of the elvt and elvt+a treatments was well above a shoot dry weight of 1 g, which has been considered the minimum plant biomass for the winter survival by diepenbrock (2006a). even the plants from the late sowing experiment in the amb treatment had approached this value indicating that osr sowing could be moved to the beginning of september. effects of autumn growth advancement in winter oilseed rape cultivars 139 tab. 1: mean temperatures during the experiments, temperature sums accumulated until the individual harvests and mean shoot biomasses recorded across the four different osr cultivars. temperature sums (growing degree days, gdd) refer to daily temperature means above a threshold of 5°c. dag represents days after germination. long-term data (1991-2005) are based on daily records from stuttgart-airport (data from dwd). day mean temperatures (°c) heat units (gdd>5°c) shoot biomass (g dm) length longoutside inside diffeoutside inside diffeoutside inside increment (hrs) term amb elvt rence amb elvt rence amb elvt (%) experiment 1 sowing date 15.08.08 dag 0 harvest 1 01.09.08 dag 7 13.9 18.1 19.8 22.9 3.1 266 330 64 0.055 0.10 96.5 harvest 2 08.09.08 dag 14 13.7 17.4 19.5 22.6 3.1 363 447 84 0.35 0.54 54.5 harvest 3 15.09.08 dag 21 13.5 16.8 18.9 21.7 2.8 442 538 96 1.46 2.44 67.4 harvest 4 13.10.08 dag 49 12.7 14.7 15.9 18.3 2.4 652 795 143 15.1 16.5 9.3 experiment 2 sowing date 22.09.08 dag 0 harvest 1 06.10.08 dag 7 11.8 12.5 11.2 13.1 1.9 83 109 26 0.01 0.01 0.0 harvest 2 13.10.08 dag 14 11.6 11.9 12.6 14.6 2.0 156 193 37 0.03 0.05 111 harvest 3 20.10.08 dag 21 11.4 11.1 12.9 15.0 2.1 207 265 58 0.08 0.26 201 harvest 4 13.11.08 dag 45 10.7 9.3 11.0 12.9 1.9 261 339 78 0.89 2.49 180 harvest 5 24.06.09 dag 268 11.5 7.1 8.8 9.2 0.4 1263 1341 78 45.7 43.2 -5.3 mean temperature (22.09. to 19.12.) 6.5 7.7 9.1 1.4 fig. 4: box plots showing the mean shoot biomass and harvest index of four osr cultivars grown in early and late sowing experiments. autumn treatments refer to ambient temperatures (amb: 1), elevated temperatures (elvt: 2) and elevated temperatures plus ammonia (elvt+a: 3). winter shoot biomass from the early sowing experiment (a) was harvested on 13 october 2008 (49 dag) and that of the late sowing experiment (b) one month later (45 dag). final shoot biomass of the late sowing experiment (c) was harvested on 24 june 2009 (268 dag) and harvest index (d) relates to the contribution of fi nal seed to shoot mass. 140 j. franzaring, i. holz, a. fangmeier the strong increase in shoot mass due to warmer autumns and a luxurious supply of fertilisers (here airborne ammonia) may induce premature shooting and increase the frost sensitivity of plants. the maximum leaf number prior to the onset of the winter period should not exceed 8 leaves according to diepenbrock (2006a), but in the early sowing experiments plants had 11.4 in the amb and 13.3 leaves on average in both the elvt and elvt+a treatments. it may thus be expected that plants would have not survived the following cold winter. however, plants were harvested prior to the winter. large differences in leaf numbers were found between the cultivars. while v had only 8.2 leaves in the amb treatment, the other cultivars produced 11.7 (e), 12.2 (a) and 13.2 (l) leaves on average in the early sowing experiment. while a, e and v did not signifi cantly increase leaf numbers due to warming in the elvt treatment, cultivar l signifi cantly increased leaf numbers to 18. it may thus be followed that l should not be sown too early, whereas v is much less temperature sensitive. the plants from the late sowing experiment produced less than 8 leaves before the winter but all plants from the late sowing experiment survived the cold winter of 2008/2009. leaf numbers were 5.1, 7.4 and 7.7 in the amb, elvt and elvt+a treatments, respectively and like in the early sowing experiment cultivar l responded strongly to elevated temperatures with 8.25 leaves on average (data not shown). the accumulated heat sum (gdd above a threshold of 5°c) until the fi nal harvest of the early sowing experiments was 652 and 795 in the amb and the elvt treatments, respectively. according to lääniste et al. (2007) such thermal sums would be much too high for the survival of osr under cool winter conditions. however, these fi ndings were made in estonia and might not be representative for milder oceanic climates. winter hardiness of osr in estonia was strongest in plants that had received 416 gdd between sowing and the onset of the winter. the plants from present late sowing experiments received only 261 and 339 gdd in the amb and elvt treatments between sowing (21.09.2008) and 31 december but all of the plants survived the following cold winter. responses during the following spring the development of plants subjected to the three autumn treatments was followed thoroughly in the following spring including the regular collection of shed leaves, the determination of shoot length and growth stages and weekly measurements of leaf greenness (spad). at the fi nal harvest (24 june 2009), the biomass allocated to leaves, stems and the reproductive organs (pod walls and seeds) was determined and oil contents were analysed in the dry seeds. total dry shoot mass averaged to 45 g plant-1 dm. this is comparable to the average shoot weight of 17 34 g dm per plant, which results from the shoot mass production of 100 to 200 dt ha-1 (=1 to 2 kg m-2) at a density of 60 plants m-2. average seed mass averaged to 12 g plant-1, which is also comparable to the seed yield in the fi eld. at a fi nal plant density of 40 plants per square metre, seed production per plant ranges between 8 and 14 g dm per plant yielding 320 to 560 g m-2 (32 to 56 dt ha-1). fig. 5 shows a comparison of the leaf shedding and the leaf greenness in the four cultivars over time. the cultivars a and v had somewhat higher initial spad values than the other two cultivars, indicating that there were slight differences in the chlorophyll concentrations between different cultivars. results of two-way anovas indicating the existence of signifi cant differences between the cultivars and cultivar * treatment interactions are shown in tab. 2. chlorophyll degradation and probably the onset of senescence in the spring occurred earlier in a than in the other cultivars despite its higher initial spad values. at the same time, cultivars v and e had somewhat higher initial spad values in the plants that were treated with ammonia in the previous autumn. however, signifi cant cultivar * treatment interactions were only observed on 28 april. a highly signifi cant effect of the warming treatments was found for the leaf mass that was shed during winter and the fi nal cumulative leaf mass (see fi rst and last observation 61 and 143 doy in fig. 5). in the warm treatment cultivars a and l shed more biomass during the winter than in the other treatments, but only in cultivar a the strong leaf loss during the winter could not be compensated in the following spring (see fig. 5). furthermore, highly signifi cant cultivar differences and a strong cultivar * treatment interaction were observed in fi nal shoot length. while cultivar e reduced shoot length upon advanced growth by elevated autumn temperatures and ammonia concentrations, cultivar l produced taller plants in both of the advancing treatments and cultivar a in the elvt+a treatment. signifi cant cultivar differences and effects of the elevated temperatures and ammonia concentrations were also observed in the onset and duration of fl owering, while a signifi cant cultivar * treatment interaction across the treatments was only found in the onset of fl owering. plants of cultivar v grown at elevated autumn temperatures fl owered three days earlier than those grown at ambient temperatures. nevertheless, differences in the onset of fl owering and changed fl owering windows did not explain differences in fi nal seed output. while signifi cant cultivar differences were found for seed mass, with cultivar l being the most productive, signifi cant treatment effects were found for elevated temperatures and elevated ammonia. elevated autumn temperatures reduced seed mass, but elevated ammonia compensated this negative effect. also the growth traits stem mass, harvest index and oil yield showed opposite responses, with higher values when plants were subjected to a combination of elevated temperatures and elevated concentrations of ammonia. interestingly, thousand seed mass was unaffected by the autumn treatments, while anova across all treatments revealed signifi cant cultivar differences with cultivar a having the smallest seeds. pod numbers and weights of pod walls (siliques) were unaffected by the treatments and did not signifi cantly differ between cultivars, suggesting that these traits had not been changed much (or selected for) during the breeding history. nevertheless, the harvest index (fig. 4) showed highly signifi cant cultivar differences with the cultivars e and l having the highest and cultivar v the lowest values. adverse effects of the autumn treatments on the harvest index are primarily due to the growth enhancement of the vegetative plant parts at the expense of the seeds. finally, the seed oil contents appeared to be unaffected by the elevated temperatures and ammonia concentrations supplied in the previous autumn. however, signifi cant cultivar differences were observed, with cultivar l having the highest oil contents and the tallest cultivar v the lowest. when addressing relationships between the oil contents and other traits across treatments and cultivars a slight negative relationship (r2 = 0.49) was identifi ed between the mean oil contents and the seed n concentrations. the existence of a negative relationship between these variables and a trade-off between the synthesis of oils and proteins has also been found in other studies (refer to the review of rathke et al., 2006). also the spad values recorded on 16 april 2009 showed a slightly negative relationship (r2 = 0.15) to the oil contents that were determined at the end of the season. it therefore seems that plants with a high spring chlorophyll and presumably n status produce seeds with lower oil content. conclusion the pot-grown plants from present experiments produced comparable fi nal shoot and seed mass as plants grown in the fi eld. also effects of autumn growth advancement in winter oilseed rape cultivars 141 schulte auf’m erley et al. (2007) found that pots-in-greenhouse experiments are a simple but suited tool to study the performance of different osr cultivars under a variable n supply. when sown late, plants of the tested four osr cultivars strongly increased pre-winter shoot mass under elevated temperature (+180%) and even more so under elevated ammonia concentrations (+270%). relative shoot mass stimulations were lower when plants were sown earlier. the results indicate that osr was able to utilise gaseous ammonia to produce signifi cantly more biomass than the untreated plants. still we are not sure how much of the ammonia-n that was absorbed in the autumn was lost later on due to leaf shedding or due to outgassing. although the different treatments were ceased before the winter, spring growth of the plants remained different in the three treatments. final shoot mass was still 13% (pments. final shoot mass was still 13% (pments. final shoot mass was still 13% ( <0.05) and seed mass 16% (p(p( <0.05) higher in the plants that were fumigated with ammonia in the previous autumn. in contrast, the plants that were subjected to higher autumn temperatures had a 5% (ns) lower shoot mass and a 13% (p13% (p13% ( <0.05) lower seed mass than those grown under ambient temperatures. the fi ndings show that pre-winter growth enhancement has discernible effects on the performance of plants in the following spring. reasons are the loss of resources due to the shedding of leaf mass in the winter. nevertheless, the effects of autumn growth advancement by elevated temperatures and the leaf loss due to winter frost will be of minor importance as compared to late frost events eventually occurring in spring. furthermore, we suggest that growth advancement due to atmospheric ammonia will probably not play a signifi cant role at ambient concentrations, which are normally lower than 50 µg m-3. acknowledgements student assistants zorica kauf, felix frey and sebastian weller are thanked for taking care of the plants and performing part of the analyses. time series of the phenological data were obtained from deutscher wetterdienst (dwd), offenbach. references adamsen, f.j., coffelt, t.a., 2005: planting date effects on flowering, seed yield, and oil content of rape and crambe cultivars. ind. crops prod. 21, 293-307. aksouh-harradj, n.m., campbell, l.c., mailer, r.j., 2006: canola response to high and moderately high temperature stresses during seed maturation. can. j. plant sci. 86, 967-980. baux, a., hebeisen, t., pellet, d., 2008: effects of minimal temperatures on low-linoleic rapeseed oil fatty-acid composition. eur. j. agron. 29, 102-107. berry, p.m., spink, j.h., 2006: a physiological analysis of oilseed rape yields: past and future. j. agr. sci. 144, 381-392. bivand, r.s., pebesma, e.j., gomez-rubio, v., 2008: applied spatial data fig. 5: development of leaf loss during winter and spring (above), cumulative leaf loss (centre) and leaf greenness (below) in four osr cultivars subjected to development of leaf loss during winter and spring (above), cumulative leaf loss (centre) and leaf greenness (below) in four osr cultivars subjected to warming treatments in the previous autumn. x-axis represents time in days of the year (doy). treatments refer to amb (○), elvt (●) and elvt+a (●). for the existence of signifi cant treatment effects refer to tab. 2. note that the fi nal cumulative leaf loss represents the dry mass of all leaves that were produced by a plant from sowing to harvest. 142 j. franzaring, i. holz, a. fangmeier t ab . 2 : e ff ec ts o f g ro w th e n h an ce m en t b y i n cr ea se d a u tu m n t em p er at u re s an d e le va te d c o n ce n tr at io n s o f th e ga s am m o n ia i n f o u r cu lt iv ar s o f w in te r o il se ed r ap e. d at a re fe r to t h e p la n t p er fo rm an ce ( p h en o lo g y, s pa d a n d b io m as s) i n t h e fo ll ow in g s p ri n g w it h m ea n s ac ro ss f o u r re p li ca te s p er c u lt iv ar i n e ac h o f th e th re e tr ea tm en ts . c u lt iv ar s: ‘ a ’ a sg ar d , ‘e ’ e x p re ss , ‘l ’ l o re n z, ‘ v ’ v ik in g . r es u lt s o f th e a n o v a i n d ic at e si g n ifi c an t d if fe re n ce s b et w ee n c u lt iv ar s (c ), t re at m en ts ( t ) an d i n te ra ct io n s b et w ee n t ) an d i n te ra ct io n s b et w ee n t c * t . s ig n ifi c an t te m p er at u re e ff ec ts w er e te st ed u si n g t h e co m p ar is o n b et w ee n a m b a n d e l v t , c * t . s ig n ifi c an t te m p er at u re e ff ec ts w er e te st ed u si n g t h e co m p ar is o n b et w ee n a m b a n d e l v t , c * t w h il e te st s fo r am m o n ia e ff ec ts r es te d o n t h e co m p ar is o n b et w ee n t h e e l v t a n d e l v t + a t re at m en ts . s ig n ifi c an ce l ev el s: * p < 0 .0 5 , * * p < 0 .0 1 a n d * * * p < 0 .0 0 1 . a re fe rs t o l ea f m as s th at h ad b ee n s h ed d u ri n g t h e w in te r; l ea ve s w er e co ll ec te d o n 1 m ar ch 2 0 0 9 . b r el at es t o a ir d ri ed s ee d s. t re at m en ts a m b e l v t e l v t + a t em p . n h 3 c om b in ed a m b ie n t te m p er at u re e le va te d t em p er at u re e le va te d t em p er at u re e ff ec t e ff ec t e ff ec t p lu s am m on ia c u lt iv ar s a e l v a e l v a e l v l ea f lo ss d ur in g w in te r (g d m )a 1. 21 1. 58 1. 27 1. 27 4. 21 3. 30 3. 67 3. 56 3. 42 3. 18 2. 50 4. 53 ** * ** * o ns et o f f lo w er in g (d o y ) 10 2 10 2 10 2 10 5 10 0 10 1 10 2 10 2 10 2 10 2 10 3 10 2 ** ** * ** * * f lo w er in g du ra ti on ( da ys ) 26 26 25 27 26 25 25 25 25 24 25 27 ** ** * f in al s ho ot l en gt h (c m ) 14 3 14 8 13 9 15 5 14 0 14 0 15 0 15 4 15 3 13 0 15 4 15 5 * ** * * ** * ** * s pa d ( 16 a pr il 2 00 9) 52 .8 46 .9 46 .7 49 .1 50 .6 46 .0 49 .2 50 .0 52 .3 48 .9 46 .5 53 .5 * * ** s pa d ( 21 a pr il 2 00 9) 49 .1 46 .3 45 .3 47 .5 50 .8 46 .2 47 .7 50 .5 48 .2 48 .6 46 .4 51 .1 * * ** s pa d ( 28 a pr il 2 00 9) 39 .1 44 .3 43 .8 45 .7 48 .5 42 .0 45 .2 48 .1 44 .4 44 .7 44 .9 53 .0 * * ** * * ** * s pa d ( 5 m ay 2 00 9) 40 .2 41 .8 42 .0 43 .9 42 .0 41 .1 42 .3 41 .3 38 .6 42 .2 43 .3 50 .0 * * * s pa d ( 13 m ay 2 00 9) 21 .6 35 .5 34 .2 40 .3 1. 0 35 .7 28 .2 30 .6 11 .2 35 .4 36 .1 36 .6 ** * * ** * ** * l ea f m as s to ta l (g d m ) 8. 1 7. 2 7. 8 8. 0 9. 2 10 .0 10 .4 9. 8 10 .8 10 .6 10 .2 11 .6 ** * ** * s te m m as s (g d m ) 15 .8 14 .1 17 .0 16 .8 12 .3 12 .9 15 .8 14 .4 15 .9 14 .1 17 .8 14 .7 * ** * * ** p od n um be r 18 8 18 4 18 8 15 5 15 1 16 9 16 5 15 3 18 5 19 5 16 3 16 3 p od w al ls ( g d m ) 10 .0 9. 7 9. 8 8. 8 8. 4 8. 9 9. 5 8. 3 10 .2 10 .8 9. 7 8. 5 s ee d m as s (g d m )b 12 .7 12 .1 13 .2 11 .7 10 .3 11 .2 12 .0 9. 7 12 .4 13 .2 13 .6 10 .8 * * * * s ho ot m as s (g d m ) 46 .6 43 .1 47 .8 45 .3 40 .2 43 .0 47 .7 42 .2 49 .3 48 .7 51 .3 45 .6 * h ar ve st i nd ex 0. 27 0. 28 0. 28 0. 26 0. 26 0. 26 0. 25 0. 23 0. 25 0. 27 0. 27 0. 24 ** * ** * ** * * ** * ** t ho us an d se ed m as s (g ) 2. 9 2. 8 3. 2 3. 0 3. 1 3. 3 3. 1 2. 9 2. 8 3. 2 3. 5 3. 0 * s ee d oi l co nt en ts ( % ) 50 .2 53 .3 53 .5 50 .5 52 .6 52 .7 53 .6 51 .6 50 .2 53 .0 54 .3 50 .4 ** ** * ** * o il y ie ld s (g p la nt -1 ) 6. 4 6. 5 7. 1 5. 9 5. 4 5. 9 6. 4 5. 0 6. 2 7. 0 7. 1 5. 4 * * * ** s ee d n c on ce nt ra ti on s (% ) 2. 3 2. 2 2. 0 2. 4 2. 1 2. 2 2. 0 2. 3 2. 2 2. 2 1. 9 2. 3 ** * ** * ** * n u e s ( g se ed g -1 a va il ab le n ) 16 .8 16 .0 17 .5 15 .4 13 .6 14 .8 15 .9 12 .9 16 .4 17 .5 18 .1 14 .3 * * * * n u e o ( g oi l g1 av ai la bl e n ) 8. 4 8. 6 9. 4 7. 8 7. 2 7. 8 8. 5 6. 6 8. 3 9. 3 9. 4 7. 18 * * * ** cultivar treat. c*t cultivar treat. c*t cultivar treat. c*t effects of autumn growth advancement in winter oilseed rape cultivars 143 analysis with r. springer verlag, new york. böhme, f., merbach, i., weigel, a., russow, r., 2003: effect of crop type and crop growth on atmospheric nitrogen deposition. j. plant nutr. soil sci. 166, 601-605. christen, o., friedt, w., 2007: winterraps. das handbuch für profis. dlg verlag, frankfurt. clement, j.m., venema, j.h., van hasselt, p.r., 1995: short-term exposure to atmospheric ammonia does not affect low-temperature hardening of winter wheat. new phytol. 131, 345-351. clement, j.m.a.m., van hasselt, p.r., van der eerden, l.j.m., dueck, t.a., 1999: short-term exposure to atmospheric ammonia does not affect frost hardening of needles from threeand fi ve-year-old scots pine trees. j. plant physiol. 154, 775-780. colnenne, c., meynard, j.m., roche, r., reau, r., 2002: effects of nitrogen defi ciencies on autumnal growth of oilseed rape. eur. j. agron. 17, 11-28. diepenbrock, w., 2006a: raps. saat und bestandsbildung. in: heyland, k.v., hanus, h., keller, e.r. 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arbeitsanleitung für nirs-untersuchungen an körnerraps im rahmen des qualitätssicherungssystem nirs/nit. verband deutscher landwirtschaftlicher untersuchungsund forschungsanstalten, http://www.vdlufa.de/nirs. address of the authors dr. jürgen franzaring, dr. ingo holz, prof. andreas fangmeier, institute for landscape and plant ecology (320), university of hohenheim, ökologiezentrum 2, august-von-hartmann-str. 3, d-70599 stuttgart, germany. art08 journal of applied botany and food quality 81, 145 150 (2007) 1department of biology, university of isfahan, isfahan, iran 2school of botany, the university of melbourne, parkville, australia accumulation of lead and zinc by plants colonizing a metal mining area in central iran s.m. ghaderian1, g.r. hemmat1, r.d. reeves2, a.j.m. baker2 (received february 1, 2007) summary the irankouh area, located in central iran, is a vast mountainous region with mineralized soils and several active zinc and lead mining and smelting sites. in this study plants and soils from 5 different sites in this area were collected and analyzed for zn and pb. analysis of soils from different sites showed the expected high concentrations of zn and pb up to 23,000 and 18,000 µg g-1 for total, 30 and 20 µg g-1 for exchangeable, 1 and 0.6 µg g-1 for water-soluble fractions, respectively. plants collected from these sites total 67 species from 66 genera and 29 families. most of these are annual herbs found also on non-metalliferous soils in this region. the concentrations of zn and pb in the leaf dry matter of plants were variable, with up to 4800 µg g-1 for zn and 740 µg g-1 pb in matthiola chenopodiifolia and pinus elderica, respectively. a significant positive correlation was detected between the concentrations of zn and pb in plant dry matter and those in soils. the concentrations of zn and pb in the leaves of most species collected were significantly higher than for other plants from non-metalliferous soils. some accumulator plants found in this area could have potential for soil clean-up by phytoextraction. introduction heavy metal contamination is a most serious form of environmental pollution and has received widespread attention owing to toxicity to a wide variety of organisms. high concentrations of heavy metals in the soils are due to both natural processes as a result of weathering of rock minerals, and human industrial activities such as mining and smelting. on metal contaminated soils, a range of plants which have evolved physiological mechanisms to tolerate heavy metal toxicity can survive and reproduce (baker, 1987). some tolerant plants are restricted to these metalliferous soils, either absolutely or regionally, and are recognized as absolute or local metallophytes. others, occurring on both contaminated and uncontaminated soils as tolerant and non-tolerant races in the same region, are recognized as pseudometallophytes (baker, 1987). the latter group can be divided into selective, indifferent and accidentals (baker and proctor, 1990). some endemic forms of tolerant plants have been described on metalliferous soils, as with the zinc floras in western europe (brooks, 1998). the tolerance of plants to heavy metals is achieved either by metal exclusion or metal accumulation. some plants on heavy metal-contaminated soils accumulate extraordinary concentrations of heavy metals into their above ground parts. according to baker and brooks (1989) ‘hyperaccumulators’ of co, cu, cr, ni or pb are those containing more than 1000 µg g-1 metal on a dry weight basis; for mn and zn the critical concentration was set at 10000 µg g-1. to date more than 450 metal hyperaccumulator species belonging to 45 families have been identified, of which about 18 and 5 species are zinc and lead hyperaccumulators, respectively, mostly from contaminated sites of europe (shimwell and laurie, 1972; reeves and brooks, 1983; reeves and baker, 2000). many hyperaccumulators are endemic to metalliferous or metal contaminated soils and thus are absolute metallophytes. while zn is an essential element for plants and is normally present at concentrations of 10-200 µg g-1, pb is neither essential nor beneficial in plant nutrition and is generally present at about 1-10 µg g-1 in plant tissues. hyperaccumulation of pb by plants is exceedingly rare, owing to the readiness with which it can be precipitated as the insoluble sulfate in the rhizosphere, hence minimizing potential uptake and transport to the aerial parts of the plants (baker and brooks, 1989). there are about 8000 plant species in iran, belonging to 150 families; about 1727 of these species are endemic to the country (jalili and jamzad, 1999). due to the shortage of rainfall in central iran, most plant species in these areas are herbaceous. while there are numerous natural metalliferous and metal-contaminated soils in different parts of iran, little information is available regarding their flora, concentration of metals in plants and any relationship between the concentrations of metals in plants and soils. the aims of this study were to identify the plant species growing on mineralized and contaminated soils in the irankouh zn and pb mining area and to determine the concentrations of zn and pb in its soils and plants. materials and methods site description the irankouh mining area is located 20 km southwest of isfahan in central iran in a mountain area at 1670-2750 m above sea level, between 32° 28´ and 32° 37´ n, and 51° 31´ and 51° 37´ e, in an area about 25 km by 3 km. the climate is semi-dry and the average annual rainfall is about 130 mm, mostly in winter and to some extent in autumn and spring. the maximum and minimum temperatures in summer and winter in this area are about 45°c and -10°c, respectively. there are several zn and pb mining sites, mostly exploited as open mines; some have been active during the last 60 years. in some parts the surface soils naturally contain high concentrations of zn and pb. owing to the mining activity, and the distribution of dust and spoil, surrounding soils in a vast area have been affected by zn and pb. ore concentration using flotation methods, and smelting, are carried out close to the mining sites, where dust and contaminated water cover the surroundings. in the present study, for collection of soils and plants, three different sites near three open-air mines (site1, colahdarvazeh; site 2, gooshfile; and site 3, tapehsorkh), one small spoil heap (site 4) and an area around the smelting operation (site 5) were selected. soil and plant sampling and analysis during june 2003-july 2004, plants growing on the above sites were collected for identification, analysis and preservation of reference specimens. soil samples were taken from 0-15 cm depth from the same areas as the plants. the soils were air-dried and sieved to < 2 mm. for the analysis of total zn and pb, subsamples of 4 g were ground to pass a -80 mesh sieve (< 190 µm) and oven-dried at 70°c to constant 146 s.m. ghaderian, g.r. hemmat, r.d. reeves, a.j.m. baker weight. a further subsample of 0.5 g was transferred to a kjeldahl digestion tube for extraction with 10 ml of a 3:1 hcl/hno3 mixture. tubes were left at room temperature overnight and were then transferred to a heating block. each was covered with an air condenser and the heating control adjusted so that the mixture refluxed gently at 80°c for 2 hours. after cooling, the digests were filtered through a moistened filter paper into 50 ml volumetric flasks. flasks were made up to volume with distilled water. analysis for zn and pb was performed by atomic absorption spectrophotometry (aas, phillips model pu 9100). for the analysis of exchangeable elements, 20 g of air-dry soil which had been sieved to < 2 mm was placed in a 100 ml screw-cap polythene bottle, 50 ml of 1m nh4no3 solution added, and the suspension was shaken for 2 hours at 20°c in an end-over-end shaker. after shaking, the soil suspension was left to stand for 5 min, and then filtered into a clean bottle. the filtrate was then acidified to 0.2% hno3 for analysis of the above elements by aas. for the analysis of water-soluble elements, a 3-inch pot was filled with soil which had been sieved to < 2 mm and watered with distilled water via its saucer. the saucer was refilled as required over 4 days to allow the sample to approach filled capacity. a conical flask with side-arm extension and a büchner funnel with a 9 cm filter paper were placed on a top pan balance. the flask was connected to a vacuum line and wet soil from the pot was added gradually until the funnel was full. after 10 min, sufficient h2o-extractable had been drawn into the flask and the apparatus was disconnected. the apparatus including the collected solution and the büchner funnel was reweighed. the h2o-extractable was decanted into an aas tube and acidified with concentrated hcl to produce a final concentration of 4% hcl. the above elements were measured by aas. the soil in the büchner funnel was spread out and air-dried for a week and then reweighed. the concentration of zn and pb in h2o-extractable could then be related to the soil in terms of w/w (dry weight) by calculating and compensating for water content. the ph of the soil was determined using a glass electrode after 10 g of soil had been stirred well in 30 ml distilled water in a beaker and allowed to stand for about 30 min. for the analysis of plants for zn and pb, leaf materials were washed well with double distilled water and dried at 70°c for 48 h. then about 0.5 g dry leaf sample was weighed into 25 ml beakers and ashed in a muffle furnace for 14 h at 480°c. the ash was taken up in 5 ml 10% hno3 and the digest finally made up to 10 ml in 10% hno3. the solutions were analyzed for zn and pb by aas. results the soils of the irankouh mining area are of near neutral ph, except site 5 (ph 6), and are highly enriched with zn and pb (tab. 1). the total concentrations are as high as 23500 µg g-1 zn in site 1 and 18000 µg g-1 pb in site 5 (tab. 1). for exchangeable metals, the highest concentrations of zn are 25-30 µg g-1 (sites 5 and 1), and the highest pb concentrations are 17-21 µg g-1 (sites 1 and 5). for h2o-extractable metals, the concentrations are low, only up to 1 µg g-1 zn and 0.6 µg g-1 pb (both at site 5) (tab. 1). in our study, 67 species of vascular plants were collected from different sites of the irankouh mining areas (tab. 2). they belong to 66 genera and 29 families. major families represented are: asteraceae (10), brassicaceae (9), poaceae (6), apiaceae (5), lamiaceae (5) and chenopodiaceae (4). most plants are herbaceous annuals, biennials or perennials. there are only 6 shrubs and 3 man-planted tree species: pinus eldarica, platanus orientalis and elaeagnus angustifolia. these trees have been planted around sites 5 and 2. the concentrations of zn and pb in the leaves of collected plants are shown in tab. 2. the zn concentrations ranged from 35 µg g-1 in peganum harmala (site 2) to 4800 µg g-1 in matthiola chenopodiifolia (site 1). in addition ebenus stellata (site 1), heliotropium lasiocarpum (site 5), diptychocarpus strictus (site 1), phragmites australis (site 5), kochis sp. (site 4), descurainia sophia (site 5), scorzonera tortuosissima (1), tamarix ramosissima (site 5) and capparis spinosa (site 5) contained zn at 2000-3000 µg g-1. concentrations of zn in 23 species were more than 1000 µg g-1, belonging to site 1 (10 species), site 5 (9 species), site 3 (2), site 2 (1 species) and site 4 (1 species). the range of pb concentrations was between 8 µg g-1 in acantholimon aspadanum (site 2) and 740 in pinus eldarica (site 5). elaeagnus angustifolia (site 5), scorzonera tortuosissima (site 5) and stachys inflata (site 3) contained up to 600-700 µg g-1 pb. concentrations of pb in 15 species were more than 300 µg g-1, belonging to sites 1, 5 and 3 with 9, 5 and 1 species, respectively. there were 9 species with both >1000 µg g-1 zn and 300 µg g-1 pb in their leaves; among these, scorzonera tortuosissima and ebenus stellata contained 2400 and 2900 µg g-1 zn and 650 and 500 µg g-1 pb, respectively. there was no significant positive correlation between the concentrations of zn and pb in these 9 species (r = 0.05, p>0.05). there was a significant positive correlation between the concentrations of zn in 20 species with more than 1000 µg g-1 zn and the tab. 1: means (n=5) and ranges (below) of total (t), exchangeable (e) and h2o-extractable (h2o-e) zn and pb concentrations, and soil ph for the study sites. site no. zn (µg g-1) pb (µg g-1) ph t e h2o-e t e h2o-e 1 16350 18.3 0.32 3700 13.5 0.35 7.0 10000-23500 10-30 0.30-0.35 1200-6400 10-17 0.30-0.40 6.9-7.1 2 5850 2.0 0.29 2150 1.35 0.25 6.9 4400-7700 1.5-2.5 0.27-0.31 1900-2400 1.2-1.5 0.20-0.30 6.8-7.0 3 5150 11.0 0.35 4200 2.25 0.36 7.2 3000-8500 10-12 0.30-0.40 3000-5700 2.0-2.35 0.30-0.40 7.1-7.3 4 11000 19.5 0.41 2900 3.4 0.31 7.4 6500-18500 18-21 0.40-0.42 1400-4400 3.0-4.0 0.25-0.35 7.35-7.46 5 5700 23.5 0.90 14800 20 0.57 6.0 5200-6250 22-25 0.80-1.00 12000-18000 18-21 0.55-0.60 5.9-6.2 accumulation of lead and zinc by plants colonizing a metal mining area in central iran 147 tab. 2: concentrations of zn and pb in leaf dry matter (µg g-1) of plants from sites in the irankouh mining area. key: ah: annual herb; bh: biennial herb; ph: perennial herb; s: shrub; t: tree †single specimens, or range of values where 2-4 specimens were analyzed. species plant form site zn† pb† apiaceae eryngium billardieri f. delaroche ah 2 80 150 ferula sp. ah 1 135 45 psammogeton canescens (dc.) vatke ah 1 420 65 pycnocycla spinosa decne. ex boiss. ah 1,2 330-515 175-205 zosima radians boiss. & hohen. ah 1 800 115 asclepiadaceae cynanchum acutum l. ph 1 1135 70 asteraceae acroptilon repens (l.) dc. ah 5 920 240 anthemis gayana boiss. ah 3 365 80 artemisia sieberi besser ah 3 220 60 centaurea gaubae (bornm.) wagenitz ah 3 180 58 centaurea ispahanica boiss. ah 1 730 380 koelpinia tenuissima pavl. & lipsch. ah 1 1360 360 pulicaria gnaphaloides (vent.) boiss. ph 2 260-305 40-105 scorzonera tortuosissima boiss. ah 1,5 750-2400 270-650 senecio glaucus l. ah 1 1030 370 zoegea purpurea fresen. ah 2 120 45 boraginaceae heliotropium lasiocarpum fisch. & c. a. mey. ah 5 1120-2800 220-250 lappula sp. ah 3 355 70 brassicaceae cardaria draba (l.) desv. ph 1,4 600-675 50-90 descurainia sophia (l.) webb ex prantl ah 5 2125 450 diptychocarpus strictus (fisch.) trautv. ah 1 455-2670 185-335 erysimum crassicaule (boiss.) boiss. bh 5 1800 150 isatis cappadocica desv. ph 1 465 65 malcolmia africana (l.) r. br. ph 1,4 490-1050 20-115 matthiola chenopodiifolia fisch. & c.a. mey. ph 1 500-4800 160-340 pseudocamelina sp. bh 1 520-1280 100-180 sisymbrium septulatum dc. ah 5 1725 310 capparaceae capparis spinosa l. ph 1,5 860-2460 45-230 cleome foliolosa dc. ah 2,3 340-535 50 chenopodiaceae anabasis setifera moq. ph 4 570 160 girgensohnia oppositiflora (pall.) fenzl ah 1 665 25 kochis sp. ah 4 2780 225 salsola kali l. ph 3,4 430-1390 25-86 dipsacaceae scabiosa olivieri coult. ah 1 580 350 elaeagnaceae elaeagnus angustifolia l. t 5 520 660 ephedraceae ephedra strobilacea bunge ex a. lehm. s 1,2 45-85 10-30 euphorbiaceae euphorbia striatella l. ph 1 800 100 fabaceae astragalus sp. ph 1 690 315 alhagi persarum boiss. & buhse ph 1 1800 180 ebenus stellata boiss. s 1 2910 500 geraniaceae erodium oxyrrhynchum m. bieb. ah 1 400 300 148 s.m. ghaderian, g.r. hemmat, r.d. reeves, a.j.m. baker lamiaceae hymenocrater incanus bunge ph 2 350 52 lallemantia royleana fisch. & c.a. mey. ah 3 430 60 salvia sp. ah 3 140 40 stachys inflata benth. ah 1,2,3 295-1635 154-640 ziziphora tenuior l. ah 3,4 265-430 115-155 malvaceae alcea aucheri (boiss.) alef. ph 2 160 30 moraceae ficus johannis boiss. s 1 1175 190 papaveraceae roemeria hybrida (l.) dc. ah 3 430 40 pinaceae pinus eldarica medw. t 5 600 740 platanaceae platanus orientalis l. t 2 125 50 plumbaginaceae acantholimon aspadanum bunge ah 2 50 8 poaceae bromus tectorum l. ah 3 430 60 eremopyrum orientale (l.) jaub. & spach ah 3 1000 25 pennisetum orientalis l. c. rich. ph 1 435 85 phragmites australis (cav.) trin. ex steud. ph 1,5 950-2120 70-120 poa sinaica steud. ph 1,3 105-350 30-50 stipa barbata desf. ph 1 210 50 polygonaceae pteropyrum aucheri jaub. & spach s 1 235 130 resedaceae reseda lutea l. ah 4,5 725-1050 60-215 rosaceae amygdalus lycioides spach s 3 100 20 scrophulariaceae linaria michauxii chav. ah 1 400 50 scrophularia striata boiss. ph 3 285 40 solanaceae solanum nigrum l. ah 2 160 50 tamaricaceae tamarix ramosissima ledeb. s 5 2600 97 urticaceae parietaria judaica l. ph 3 100 50 zygophyllaceae peganum harmala l. ph 2 35 20 concentrations of total (r = 0.45, p<0.05), exchangeable (r = 0.48, p<0.05) and h2o-extractable (r = 0.45, p<0.05) zn in the soil. also, a significant positive correlation exists between the concentrations of pb in 14 species with more than 300 µg g-1 pb and the concentrations of total (r = 0.72, p<0.05), exchangeable (r = 0.58, p<0.02) and h2o-extractable (r = 0.56, p<0.05) pb in the soil. discussion the soils of all sites studied contain predictably high concentrations of pb and zn. this is due to natural mineralization in the parent rocks and to contamination by mining and smelting during the last 60 years. the concentrations of zn and pb in sites 1-3, shown in tab. 1, are generally typical of mineralized soils in this area, although contaminated dusts arising from mining activity to some extent may have added to these elevated concentrations. high concentrations of these metals in site 4 are due to the spoil heap which has been leveled and left unchanged for nearly 20 years, whereas the high concentrations in site 5 are mainly from the smelting operation. there is considerable variation in concentration both within and between sites pointing to the heterogeneous nature of mineral and mine wastes (ghaderian, 1998; wenzel and jockwer, 1999; dahmani-müller accumulation of lead and zinc by plants colonizing a metal mining area in central iran 149 et al., 2000). concentrations of zn in sites 1-4 are higher than those of pb, mainly because the irankouh mining area is more enriched with zn minerals, and pb is the secondary metal. much of the metal is insoluble and not immediately available for plants and is therefore not directly toxic. however, heavy metals in the h2o-extractable and exchangeable forms may be directly accessible to organisms in the soil (lorenz et al., 1997; pollard et al., 2002). thus, the high concentrations of zn and pb in the h2o-extractable and exchangeable fractions at some sites suggest extreme toxicity of the soil. according to prüess (1994) the limits of nh4no3-exchangeable zn and pb in soil are 5 and 0.3 µg g-1, respectively; above these concentrations, the assessment of risk and remediation needs is required. these concentrations are exceeded in all the soils in this study, except for exchangeable zn in site 2. the highest concentrations of mean zn and pb (exchangeable and h2o-extractable) were in site 5, where minerals were processed and smelted. the ph here is 6 and due to the operations here the soils have more moisture. generally, solubility and availability of metals in soils depend on many factors, including total metal content, ph and moisture regime (alloway, 1995). most plants collected in this area were annual or perennial herbs; this dominant vegetation form is due to the climate of the area, with low annual rainfall typical of central iran. among the plants collected, 15 species are endemic to iran (rechinger, 1963-1997), but not exclusively endemic to these metalliferous areas. as these plants naturally grow on these metal substrata, they can be categorized as pseudometallophytes (baker, 1987; pollard et al., 2002). whether these plants are physiological races of metal tolerant species should be clear in further investigations. according to the definition of baker and brooks (1989), the criteria for hyperaccumulation for zn and pb are >10000 and 1000 µg g-1, respectively. on this basis, none of the collected species in the irankouh mining area was a hyperaccumulator of either metal. the highest concentration of zn (4800 µg g-1) was recorded in leaves of matthiola chenopodiifolia (brassicaceae), collected from different parts of site 1. the highest pb concentrations (above 500 µg g-1) were found in planted trees of pinus eldarica (740 µg g-1) and elaeagnus angustifolia (660 µg g-1), as well as in naturally growing plants of scorzonera tortuosissima (650 µg g-1), stachys inflata (640 µg g-1) and ebenus stellata (500 µg g-1). to date 18 and 5 hyperaccumulators of zn and pb, respectively, have been reported (reeves and baker, 2000), mostly from europe, but zn and pb hyperaccumulation does not occur in most plants from pb and zn contaminated soils (shimwell and laurie, 1972; reeves, 1988; wenzel and jockwer, 1999; reeves et al., 2001). in contrast, ni hyperaccumulators are rather more abundant in temperate and tropical ultramafic areas (reeves, 1992; reeves et al., 1999). yang et al. (2002) believed that the criterion of 10000 µg g-1 zn is too high for defining a zn hyperaccumulator plant, and suggested 3000 µg g-1 leaf dry wt instead. thus, the zn accumulation in m. chenopodiifolia is notable and this plant seems to have potential for phytoremediation in some metal contaminated areas. although there was no pb hyperaccumulator at irankouh, the existence here of 3 naturally occurring plants with > 500 µg g-1 pb is notable. the significant positive correlations between the mean concentrations of zn and pb in the soil (as total, exchangeable or h2o-extractable) and the mean concentrations of these metals in dry leaves of plants which accumulate >1000 and 300 µg g-1, respectively, could indicate a cause-and-effect relationship. such correlations can indicate a metal-accumulation tolerance strategy. baker et al. (1994), however, did not find such a correlation between indices of metal tolerance in different populations of thlaspi caerulescens and concentrations of the metals in the soils. as some soils of the irankouh area naturally contain high concentrations of zn and pb, most plants growing on these substrata should be adapted to these metal stress conditions. more work should be done to determine the correlations of the zn and pb concentrations of each accumulator plant with its own substratum. however, it is possible in areas adjacent to mines and smelters, in particular, that part of the measured heavy metals may be from external deposition not removed in sample washing. it is almost impossible to know how much might have been externally deposited on leaf surfaces. one consequence, unfortunately, is that because the contaminant is essentially soil material, it will generate strong positive correlation between soil metal and that apparently present in the plant. this is not a great problem when plant analysis is used as a biogeochemical prospecting method, but can introduce uncertainties into discussions of the physiology of heavy metal uptake into plants from the soil via the soil solution and the root system. conclusion plants on zn and pb enriched soils of the irankouh area were collected and identified, and the concentrations of these metals in the soils and plants were determined. none of the 67 collected species were recognized as true endemics on these substrata: all were pseudometallophytes. total zn and pb concentrations in the soils were high, in the range 3000-23500 µg g-1 zn and 1200-18000 µg g-1 pb. the bioavailable concentrations of these metals as nh4no3-exchangeable and h2o-extractable were high enough to produce toxicity to nontolerant plants. the concentrations of zn and pb in the plant leaves indicated that most contain elevated amounts of these metals. the concentrations of zn were up to 4800 and 2800 µg g-1 in matthiola chenopodiifolia and heliotropium lasiocarpum, respectively. the highest pb concentrations were in planted trees of pinus eldarica (740 µg g-1) and elaeagnus angustifolia (660 µg g-1) and in the naturally growing species scorzonera tortuosissima (650 µg g-1) and stachys inflata (640 µg g-1). despite high concentrations of bioavailable metals in the rhizosphere, no species showed hyperaccumulation of zn or pb. there were significant positive correlations between the concentrations of zn or pb in the soils and in the leaves of accumulator plants. this suggests the adaptation of some metal tolerant plants to the toxic concentrations of heavy metals in the soils by the uptake mechanism. these accumulator plants may be valuable in phytoextraction of metal-contaminated soils in some parts of central iran. acknowledgements we thank m. r. rahiminejad for his help in identification of plants, m. nogherian for his geological advice and bama co. for their help in analysis of the samples. this research was carried out using an msc grant to grh, offered by the graduate school, university of isfahan. references alloway, b.j. (ed.), 1995: heavy metals in soils. 2nd edn. blackie academic and professional, london. baker, a.j.m., 1987: metal tolerance. new phytol. 106, 93-111. baker, a.j.m., brooks, r.r., 1989: terrestrial higher plants which hyperaccumulate metallic elements – a review of their distribution, ecology and phytochemistry. biorecovery 1, 81-126. baker, a.j.m., proctor, j., 1990: the influence of cadmium, copper, lead, and zinc on the distribution and evolution of metallophytes in the british isles. pl. syst. evol. 173, 91-108. 150 s.m. ghaderian, g.r. hemmat, r.d. reeves, a.j.m. baker baker, a.j.m., reeves, r.d., hajar, a.s.m., 1994: heavy metal accumulation and tolerance in british populations of the metallophyte thlaspi caerulescens j. & c. presl (brassicaceae). new phytol. 127, 6168. brooks, r.r., 1998: plants that hyperaccumulate heavy metals. cab international, cambridge. dahmani-müller, h., van oort, f., gélie, b., balabane, m., 2000: strategies of heavy metal uptake by three plant species growing near a metal smelter. environ. pollut. 109, 231-238. ghaderian, s.m., 1998: the effect of toxic heavy metals upon fungi of the genus pythium isolated from soil. ph.d thesis, university of sheffield, uk. jalili, a., jamzad, z., 1999: red data book of iran. rifr, tehran. lorenz, s.e., hamon, r.e., holm, p.e., domingues, h.c., sequeira, e.m., christensen, t.h., mcgrath, s.p., 1997: cadmium and zinc in plants and soil solutions from contaminated soils. plant soil 189, 21-31. pollard, a.j., powell, k.d., harper, f.a., smith, j.a.c., 2002: the genetic basis of metal hyperaccumulation in plants. crit. rev. plant sci. 21, 539-566. prüess, a., 1994: einstufung mobiler spurenelemente in böden. in: rosenkranz, d., einsele, g., harress, m. (eds.), ergänzbares handbuch der maßnahmen und empfehlungen für schutz, pflege und sanierung von böden, landschaft und grundwasser, 15. lieferung, i/94. erich schmidt verlag, berlin. rechinger, k.h. (ed.), 1963-1997: flora iranica, 1-172. akademische drucku. verlagsanstalt, graz, austria. reeves, r.d., 1988: nickel and zinc accumulation by species of thlaspi l., cochlearia l., and other genera of the brassicaceae. taxon 32, 309-318. reeves, r.d., 1992: the hyperaccumulation of nickel by serpentine plants. in: baker, a.j.m, proctor, j., reeves, r.d. (eds.), the vegetation of ultramafic (serpentine) soils, 253-278. intercept ltd, andover, u.k. reeves, r.d., baker, a.j.m., 2000: metal accumulating plants. in: raskin, i., ensley, b.d. (eds.), phytoremediation of toxic metals: using plants to clean up the environment, 193-229. john wiley & sons inc., ny, usa. reeves, r.d., baker, a.j.m, borhidi, a., berazaín, r., 1999: nickel hyperaccumulation in the serpentine flora of cuba. ann. bot. 83, 29-38. reeves, r.d., brooks, r.r., 1983: hyperaccumulation of lead and zinc by two metallophytes from mining areas of central europe. environ. pollut. 31, 277-285. reeves, r.d., kruckeberg, a.r., adigüzel, n., krämer, u., 2001: studies on the flora of serpentine and other metalliferous areas of western turkey. s. afr. j. sci. 97, 513-517. shimwell, d.w., laurie, a.e., 1972: lead and zinc concentration of vegetation in the southern pennines. environ. pollut. 3, 291-301. wenzel, w.w., jockwer, f., 1999: accumulation of heavy metals in plants grown on mineralized soils of the austrian alps. environ. pollut. 104, 145-155. yang, x., xinxian, l., wuzhong, n., chenxin, f.u., 2002: sedum alfredii h: a new zn hyperaccumulating plant first found in china. chinese sci. bull. 19, 1634-1637. address of the authors: dr. s.m. ghaderian (corresponding author) (email: ghaderian@sci.ui.ac.ir) and g.r. hemmat, department of biology, faculty of sciences, university of isfahan, isfahan, 81746-73441, iran. professor a.j.m. baker and professor r.d. reeves, school of botany, the university of melbourne, parkville, vic 3010, australia. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true 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/pagesize [907.087 680.315] >> setpagedevice journal of applied botany and food quality 86, 59 65 (2013), doi:10.5073/jabfq.2013.086.009 1 department of horticulture, faculty of agriculture, mustafa kemal university, antakya, hatay, turkey comparison postharvest quality of conventionally and organically grown ‘washington navel’ oranges elif çandır1, müge kamiloğlu1, durmuş üstün1, gülcan tuğçe kendir1 (received june 4, 2013) summary this study aimed to compare postharvest quality of conventionally and organically grown ‘washington navel’ oranges. oranges from the conventional and certified organic citrus orchards were harvested at commercial maturity and kept at 4°c for 5 months. changes in weight loss, juice percentage, titratable acidity (ta), total soluble solid (tss), sugars (fructose, glucose and sucrose), organic acids (citric, malic and ascorbic acid) content and incidence of fungal decay and chilling injury were determined at a month interval during storage. conventionally grown oranges had lower weight loss and higher juice percentage than organically grown oranges during storage. rind color (l*, c*, hº), tss, sugar (fructose, glucose and sucrose) and malic acid content were not affected by the production systems at harvest and during storage. in both conventionally and organically grown oranges, rind color become darker (lower l*), more intense (higher c*) and deeper orange color (lower hº) while malic acid content remained constant during 5 months of storage. as storage time extended, a significant increase in tss and sugar content and a decrease ta and citric acid content occurred in fruits from both production system. compared to conventionally grown oranges, organically grown oranges had lower ta and citric acid, but better taste scores since they attained higher tss/ta ratio at harvest and during storage. the taste of conventionally and organically grown oranges was rated as an acceptable throughout the storage period. although there was no significant difference in ascorbic acid content of fruits between two production systems at harvest, lower ascorbic acid content was found in organically grown oranges, compared to conventionally grown oranges during storage. incidence of fungal decay was low in conventionally and organically grown oranges after 5 months of storage and the production system did not affect the sensitivity to fungal decay. chilling injury was not observed any of fruits from both production systems throughout storage period. introduction the global organic food and drink sales reached 54.9 billion us dollars in 2009 with a three-fold increase from 18 billion us dollars in 2000 (willer and kilcher, 2011). the consumer demand for organic foods continues to expand rapidly due to the perception that organic products are safe, clean, more nutritious, healthy, bettertasting and environmentally friendlier than conventionally grown foods (bourn and prescott, 2002; lester, 2006). the production of citrus fruits in turkey has been increasing steadily in the past 20 years, reached about 3.5 million tons in 2009 (faostat, 2009). turkey is among the top four citrus producers in the mediterranean basin and ranks tenth in the world. oranges are the main citrus fruit grown in turkey, accounting for about 48% of total citrus production. the increase in citrus production has resulted in some potential marketing problems, especially for commonly grown citrus cultivars including ‘washington navel’ oranges (demirkeser et al., 2009). thus, the citrus industry in turkey has been tending to organic production expanding due to export market opportunities for organic citrus fruits (demirkeser et al., 2009). the 68.210 hectares of citrus fruit are grown worldwide (willer and kilcher, 2011). turkey was accounted for 1.2% of the world’s area of organic citrus fruits. the effect of the production system on citrus fruit quality has already studied. citrus fruits produced in organic farms had greater juice percentage and sugar content (lester et al., 2007); more soluble solids, a lower maturation index (duarte et al., 2010), higher malic, citric and ascorbic acid concentration (duarte et al., 2012), higher contents of minerals and carotenoids and better sensory quality (beltran-gonzalez et al., 2008) than those produced by conventional farming systems but the responses depended on citrus species and cultivar (duarte et al., 2010; 2012) and harvest season (lester et al., 2007). some of the authors did not find significant differences in some quality parameters between conventional and organic citrus fruits (rapisarda et al., 2005; perez-lopez et al., 2007; lester et al., 2007; beltran-gonzalez et al., 2008; esch et al., 2010; camin et al., 2011). few studies monitored the postharvest life and quality of organically versus conventionally grown fresh fruits such as kiwi (amodio et al., 2007), apple (deell and prange, 1992) and grapefruit (chebrolu et al., 2012a). the aim of this study was compare postharvest quality of conventionally and organically grown ‘washington navel’ oranges. materials and methods plant material ‘washington navel’ oranges were obtained from the commercial citrus orchards, one organic and one conventional in seyhan, adana during 2010 and 2012 growing seasons. the orchards were located close to each other (1 km apart), with trees grafted on the same rootstock and of about the same age to allow a valid comparison of organic versus conventional fruits. production systems were defined in tab. 1. fruits were harvested at commercial maturity from 7 year-old trees which were grafted on sour orange and planted 6 m x 6 m. after harvest, fruits were transported to the postharvest laboratory at the horticultural department of mustafa kemal university (antakya-hatay), where they were sorted for size, color uniformity, and absence of surface defects. three replicates per treatment were then kept at 4°c and 85-90% relative humidity for 5 months. each replicates contained 10 fruits. evaluation of postharvest quality postharvest quality was assessed monthly intervals during storage. fruits were numbered and individually weighted to determine weight loss. weight loss was calculated as percentage loss of initial weight. rind color was determined with a minolta chroma meter cr-300 (osaka, japan). color measurements were recorded using the cie l*a*b* color space. from these values, hue angle (h°) and chroma (c*) values were calculated as h°=tan−1 (b*/a*) and c*=(a*2+b*2)1/2, color values for each fruit were computed as means of two measurements taken from opposite sides at the equatorial region of the 60 e. çandır, m. kamiloğlu, d. üstün, g.t. kendir fruit. juice was extracted by using an electric rotary juicer. juice percentage was calculated by weighing the juice for each sample and dividing by the original fruit weight. total soluble solids (tss) content and titratable acidity (ta) were assessed in juice obtained from ten fruits per replicates. tss content was determined with a refractometer (atago model atc-1e) and ta by titration of 5 ml of fruit juice with 0.1 n naoh to ph 8.1 and expressed as g citric acid 100 ml-1 juice. the incidence of fungal decay was assessed and expressed as percentage of fruit infected by fungal pathogens. fruits were examined visually for chilling injury (ci) symptoms such as peel pitting or brown staining. incidence of ci was expressed as the percentage of fruits with ci. a trained panel consisting of 10 people evaluated the sensory quality (taste) of the fruits based on a hedonic scale of 1 (disliked extremely) to 9 (liked extremely) at the beginning of the experiment and monthly throughout the storage period. extraction and hplc analysis of sugar and organic acids sugars were extracted from the oranges following a modified method of lee and coates (2000). exactly 5 ml of sample was diluted with deionized distilled water to total volume of 10 ml. after vortexing for a minute, the sample was centrifuged (rotina 380r hettich, tuttlingen, germany) for 5 min at 9,418 × g (6500 rpm) and 5°c. twenty microliters of sample was injected directly into the hplc after filtration through a millex-hv 0.45 μm filter (millipore, bedford, ma). organic acids (citric and malic) ascorbic acid were extracted according to a modified version of the method described previously (lee, 1993; lee and coates, 1999). a sample of juice (5 ml) was pipetted into a 50 ml centrifuge tube containing 5 ml of 2.5% metaphosphoric acid. after centrifugation at 9,418 × g (6500 rpm) for 5 min at 5°c, the supernatant was recovered. twenty microliters of sample was filtered using a millex-hv 0.45 μm filter and injected directly into a shimadzu hplc. hplc analyses of sugars and organic acids were performed on lc10a equipment consisting of lc-10ad pumps, an in-line degasser, a cto-10a column oven, an scl-10a system controller, an spd 10avp, a photo diode array detector, a refractive index detector and operated by lc solution software (shimadzu, japan). sugars were separated using an ec nucleosil carbohydrate 250 mm × 4 mm i.d. column (macherey-nagel, düren, germany) at 25°c. the mobile phase included acetonitrile: deionized distilled water (80:20, v/v) at a flow rate of 2 ml min-1. organic acids were separated on a transgenomictm icsep ion300 300 mm × 7.8 mm i.d. column (transgenomic, san jose, ca, usa) at 65°c. the mobile phase used was 0.0085 n h2so4 at a flow rate of 0.4 ml min-1. sugars and organic acids were detected using a refractive index and photo diode array detector (210 nm for citric and malic acid and 244 nm for ascorbic acid), respectively. quantification was performed according to external standard method by the comparison of retention times to those of authentic standards, purchased from merck kgaa (darmstadt, germany) and chem service (west chester, usa). statistical analysis the data were analyzed as a factorial experiment in a completely randomized block design by analysis of variance (anova) using sas software of sas institute, cary, n.c. (sas, 1999). statistical means were across the two growing seasons. mean separation was performed by fisher’s least significance test at p<0.05 level using sas’s proc glm procedure. results and discussion weight loss increased as storage time extended and reached to 10.11% and 11.94% after 5 months of storage in conventionally and tab. 1: fertilizer, weed and insect control inputs of conventionally and organically grown washington navel orange orchards production system input description and rate application conventional fertilizer n 280 kg ha-1 1 p 100 kg ha-1 1 k 280 kg ha-1 1 attack df (foliar nutrient spray): 1 zn 0.168 l ha-1; fe 0.140 l ha-1; mn 0.112 l ha-1; mg 0.084 l ha-1; b 0.042 l ha-1; cu 0.028 l ha-1; mo 0.001 l ha-1; s 0.322 l ha-1 insect control applaud 1.82 l ha-1 1 neoran 1.4 l ha-1 1 malathion 14 l ha-1 1 citrus oil 35 l ha-1 1 weed control cultivation 2 irrigation flood irrigation organic fertilizer compost: 1 n115 kg ha-1; p 57.5 kg ha-1; k 115 kg ha-1 pattrone (foliar nutrient spray) : 2 free amino acid 630 g ha-1; inorganic n 142 g ha-1; organic n 142 g ha-1; organic acid 578.62 g ha-1 insect control citrus oil 35 l ha-1 1 flowable sulphur 16.8 l ha-1 1 2-3 predatory insects (cryptolaemus montrouzieri) per tree 1 10 predatory insects (leptemastix dactylopii) per tree 1 weed control cultivation 2 irrigation drip irrigation comparison of conventionally and organically grown oranges 61 organically grown oranges, respectively (fig. 1a). conventionally grown oranges had lower weight loss than organically grown oranges during storage. consistent with our results, conventional grapefruits showed lower water loss than organic grapefruits during 4 weeks of storage at 9°c (chebrolu et al., 2012a). fruit size of organically grown oranges (263.31 g to 318.42 g) was higher than that of conventionally grown oranges (223.65 g to 247.40 g) as result of lower number of fruits per tree in organically grown trees (data not shown). the farming system had a significant influence on the mean fruit weight and equatorial diameter which could be assigned to the lower yield per tree resulting in larger orange fruits due to lower assimilate competition under organic farming (roussos, 2011). according to ketsa (1990), the largest tangerines lost 1.661.69 times as much weight as did the smallest fruits during shelf life. in agreement with the findings of ketsa (1990), larger fruit size of organically produced fruits compared to conventionally produced fruits caused higher weight loss. juice percentage decreased from 52.91% to 45.24% in conventionally grown oranges and from 49.06% to 41.53% in organically grown oranges during storage (fig. 1b). these results are in agreement with the findings of ozdemir and dundar (2006) who found that juice percentage of orange fruits significantly decreased with the increasing of storage periods. juice percentage was higher in conventionally grown oranges than organically grown oranges at harvest and during storage. similar finding was reported for conventional versus organic acid limes (rangel et al., 2011). in contrast to our results, the previous study showed that grapefruits produced in organic farms had greater juice percentage than those produced by conventional farming systems (lester et al., 2007). in other studies, no significant differences in juice percentage were found for citrus fruits between two production systems (rapisarda et al., 2005; duarte et al., 2010; nunes et al., 2010; camin et al., 2011). in our study, compare to conventionally grown oranges, lower juice percentage in organically grown oranges might be as a result of being larger fruit which often have proportionately lower juice content than smaller fruit in oranges (davies and zalman, 2004). rind color parameters of l* (lightness) and hue angle (hº) values decreased, but chroma (c*) value increased during storage (fig. 2). these changes indicate that rind color become darker (lower l* value), more intense (higher c* value), deeper orange color (lower hº). previous studies demonstrated changes in peel color from green yellow to yellow-orange and a decrease in l* value in ‘washington navel’ oranges with the increasing of storage period (özdemir et al., 2008). conventionally and organic grown oranges had similar rind color l*, c* and hº values at harvest and during storage (fig. 2). duarte et al. (2010) found higher l* value in organically grown citrus fruits, but more intense color in conventionally grown citrus fruits. lester et al. (2007) reported that early season conventionally grown citrus fruits had a darker, more intense, reddishyellow color. as the production season advanced, peel lightness, and color intensity differences from the two systems became nonsignificant. fig. 1: changes in weight loss and juice percentage of conventionally and organically grown ‘washington navel’ oranges during storage at 4°c. fig. 2: changes in rind color l*, c* and h° values of conventionally and organically grown ‘washington navel’ oranges during storage at 4°c. conventionally and organically grown oranges contained about 10% of total soluble solid (tss) content at harvest (fig. 3a). an increase in the tss content of conventionally and organically grown oranges occurred during 5 months of storage. increases in tss content have been previously reported in stored ‘pineapple’ oranges for 6 weeks at 4°c, ‘valencia’ oranges for 12 weeks at 1°c (davis et al., 1973), ‘hamlin’ oranges for 9 weeks at 15°c (echeverria and ismail, 1987), ‘valencia’ oranges for 24 weeks at 4°c and 6°c (ozdemir and dundar, 2006; özdemir et al., 2008) and navel oranges for 6 weeks at 5°c (obenland et al., 2008). our findings were in agreement with previous reports on orange fruits. in our study, there was no significant difference in tss content be62 e. çandır, m. kamiloğlu, d. üstün, g.t. kendir tween conventionally and organically grown oranges at harvest and during storage. compared with conventional farming, higher tss content was reported in organically grown grapefruits (lester et al., 2007) and oranges, mandarins and lemons (duarte et al., 2010). in agreement with our results, a similar tss content at harvest under organic and conventional orchard management were reported for ‘navelina’ and ‘tarocco’ (rapisarda et al., 2005; camin et al., 2011), ‘salustiana’ (roussos, 2011) and ‘valencia’ oranges (nunes et al., 2010), ‘clemenules’ (perez-lopez et al., 2007) and ‘hernandina’ mandarins (beltran-gonzalez et al., 2008). chebrolu et al. (2012a) found no significant difference in tss content of grapefruits at harvest and throughout a 5-week storage period at 9c°. conventionally and organically grown oranges contained 1.982.05 g 100 ml-1 of fructose, 1.82-1.83 g 100 ml-1 of glucose and 3.27-3.30 g 100 ml-1 of sucrose at harvest (fig. 5). sugars content of conventionally and organically grown oranges were similar at harvest and during storage. lester et al. (2007) found lower total sugars in early and late season ‘rio red’ grapefruits from organic production systems, compare to conventional production systems, but midseason grape fruits from both production systems had similar sugar content. consistent with our results, sugar content and sugar composition were not affected by the production system in oranges (roussos, 2011) and kiwifruits (amodio et al., 2007) at harvest or during storage. in both conventionally and organic grown oranges, changes in fructose, glucose and sucrose content were not statistically significant for 2 months and then a significant increases in individual sugars occurred in the last 3 months of storage (fig. 5). in ‘hamlin’ oranges, a concurrent increase in sucrose and decrease in fructose were measured during the first 5-6 weeks of storage, and then fructose increased in the last 4 weeks of storage while glucose remaining unchanged during the 10 weeks of storage (echeverria and ismail, 1987). ahmad et al. (1989) reported increases in reducing and non-reducing sugars in ‘blood red’ sweet oranges during 5 weeks of storage at 8-19°c. the increases in individual sugars (fig. 5) were consistent with increase in tss content (fig. 3a) during storage in our study. according to echeverria and ismail fig. 3: changes in total soluble solid (tss) and titratable acidity (ta) of conventionally and organically grown ‘washington navel’ oranges during storage at 4°c. fig. 4: changes in sugar/acid ratios (tss/ta) and taste scores of conventionally and organically grown ‘washington navel’ oranges during storage at 4°c. fig. 5: changes in sugars content of conventionally and organically grown ‘washington navel’ oranges during storage at 4°c. (1990), the increase in sugar concentration (due mainly to sucrose) accounted for the measured rise in tss content for ‘hamlin’ oranges. comparison of conventionally and organically grown oranges 63 an acid loss was observed in fruits from both production systems during storage (fig. 3b) as reported previously for citrus fruits (erkan and pekmezci, 2000; ozdemir and dundar, 2006; obenland et al., 2008). at harvest, conventionally and organically grown oranges had titratable acidity (ta) 1.53% and 1.22% and reached 1.11% and 0.87%, respectively. this is in contrast to what was observed by duarte et al. (2010) who found that compared with conventional farming, fruits from organic farming were more acid and had a greater °brix and lower a lower maturation index (tss/ta ratio). lester et al. (2007) reported that organically grown grapefruits had higher ta than conventionally grown grapefruits during early and midseason, but differences in ta from the two systems became non-significant at late season. no differences were observed for ta between conventional and organic oranges (rapisarda et al., 2005; camin et al., 2011), limes (rangel et al., 2011), mandarins (beltran-gonzalez et al., 2008), grapefruits (chebrolu et al., 2012a), kiwifruits (amodio et al., 2007) and apples (deell and prange, 1992) at harvest or during storage. the minimum acceptable maturity level for navel oranges is a 6.5 to 1 ratio of sugar to acid (oecd, 2010). in conventionally and organically grown oranges, the initial tss/ta ratio was about 6.6 to 1 and 8.2 to 1 and increased to reached 9 to1 and 12 to 1, respectively, during storage (fig. 4a). the increase in tss content and decrease in ta lead to a progressive increase in the tss/ta ratio as storage time advanced. likeability (hedonic score) was unchanged for 3 weeks of storage in conventionally grown oranges and for 4 weeks of storage in organically grown oranges and then the scores had declined from 7 (like moderately) to 6 (like slightly) (fig. 4b). the taste of fruits was still rated as acceptable (≥6) throughout the storage period. obenland et al. (2009) reported that sensory panelists increasingly liked navel oranges as ta declined and ssc/ ta rose during navel orange maturation and that the increase in likability did not plateau until ssc/ta values of 18 or more were reached. in our study, it is possible that the loss of acidity had some role in the decline in flavor quality in this cultivar as suggested by obenland et al. (2011). in citrus fruits, a higher ssc/ta ratio is generally desired because the fruit are perceived to be sweeter, although too high of a ratio can cause the fruit to be bland (obenland et al., 2009). compared to conventionally grown oranges, organically grown oranges had lower ta (fig. 3b), but better taste scores (fig. 4b), since they attained higher tss/ta ratio (fig. 4a). the difference experienced in our study from previous studies could be the result of different degree of maturity of conventionally and organically grown oranges at harvest. organic versus conventional production system inputs can change ripening patterns (perkinsveazie and lester, 2008). at harvest, conventionally and organically grown oranges had citric and malic acid content ranging from 1.20 g 100 ml-1 to 1.42 g 100 ml-1 and 0.17 g 100 ml-1 to 019 g 100 ml-1, respectively (fig. 6). citric acid content decreased during storage period (fig. 6a). citric acid has been reported to decrease in stored ‘pineapple’ and ‘valencia’ (davis et al., 1973) and ‘hamlin’ oranges (echeverria and ismail, 1987). malic acid content remained constant in both conventionally and organically grown oranges during storage (fig. 6b), as reported by davis et al. (1973) in ‘valencia’ and by echeverria and ismail (1987) in ‘hamlin’ oranges. reduction in ta was mainly to the result of a decrease in citric acid because of steady levels of malic acid during storage. conventionally grown oranges had higher citric acid content, compared to organically grown oranges. however, malic acid content was similar fruits from both production systems. the farming systems did not result in any significant difference in the individual organic acids of ‘salustiana’ oranges (roussos, 2011) and kiwifruits (amodio et al., 2007) at harvest or during storage. duarte et al (2012) reported that the concentration of citric acid and malic was generally higher in citrus fruits from organic production, in comparison to conventional production, but similar or higher concentration of citric and malic was found in some of conventionally grown mandarin, lemon and orange cultivars. conventionally and organically grown oranges contained 35.77 mg 100 ml-1 % and 35.97 mg 100 ml-1 of ascorbic acid, respectively at harvest. a range of about 29 to 74 mg of ascorbic acid 100 ml-1 of juice was reported for orange varieties (nagy, 1980; rapisarda et al., 2005; duarte et al., 2012; roussos, 2011; chebrolu et al., 2012b). ascorbic acid content of conventionally and organically grown oranges was within range of previous studies. a greater vitamin c content in citrus fruits from organic compared to conventional farming systems was reported for ‘navelina’, ‘tarocco’ (rapisarda et al., 2005), ‘valencia late’ and ‘baia’ oranges (duarte et al., 2010) and ‘rio red’ grapefruits (lester et al., 2007; chebrolu et al., 2012ab), however, we found that conventionally and organically grown oranges had similar ascorbic acid content at harvest. in agreement with our results, no differences in ascorbic acid content were detected between the fruits from organic or conventional farming in ‘clemenules’ mandarins (perez-lopez et al., 2007), ‘dalmau’, ‘newhall’, ‘lanelate’, ‘rohde’ (duarte et al., 2010; 2012), ‘salustiana’ (roussos, 2011), ‘valencia’ and navel oranges (esch et al., 2010), acid limes (rangel et al., 2011). based on previous reports, nitrogen fertilizers, especially at high rates, seem to decrease the concentration of vitamin c in citrus and other fruits and vegetables (nagy, 1980; lee and kader, 2000; rapisarda et al., 2005). duarte et al. (2012) reported that ascorbic acid concentration in the juice was generally higher in citrus fruits produced in the organic orchards, but the response depended on species and cultivar. of the six types of fruits (lemons, oranges apples green fig. 6: changes in organic acids content of conventionally and organically grown ‘washington navel’ oranges during storage at 4°c. 64 e. çandır, m. kamiloğlu, d. üstün, g.t. kendir kiwi, and mangoes) analyzed by esch et al. (2010), only one, lemon, demonstrated a significantly higher vitamin c concentration for organically grown versus conventionally grown fruit. lester et al. (2007) found higher ascorbic acid content early and midseason ‘rio red’ grapefruits from organic production system to compare conventional production system but differences in ascorbic acid from the two systems became non-significant at late season. chebrolu et al., (2012a) compared vitamin c levels of organic and conventional ‘rio red’ grapefruits for two growing season. in the first year, organic grapefruits showed significantly higher levels of vitamin c over conventional grapefruits while no significant difference was found in organic and conventionally grown grapefruits for vitamin c levels in the second year. previous reports suggested that other factors such as growing season, storage, and shipping conditions, time of harvest, species and cultivars may have also important influence on vitamin c content as well as the growing methods employed by organic and conventional farmers (lee and kader, 2000; lester et al., 2007; esch et al., 2010; duarte et al., 2010; 2011; chebrolu et al., 2012a). ascorbic acid content showed a significant decreased during storage and this decrease was higher in organically grown oranges than conventionally grown oranges (fig. 6c). a loss of vitamin c was reported previously for citrus fruits under refrigerated conditions (nagy, 1980; lee and coates, 1999; erkan and pekmezci, 2000; chebrolu et al., 2012a). higher water loss after harvest result in a more rapid loss of vitamin c (lee and kader, 2000; nunes et al., 1998). the decreases in total acids in the juice were also closely correlated with the loss of vitamin c (nagy, 1980). comparing to conventionally grown oranges, a lower ascorbic acid level in organically grown oranges could be as a result of a higher weight loss and a lower titratable acidity during storage period, as suggested previously. incidence of fungal decay was low at about 7% in conventionally and organically grown oranges after 5 months of storage (tab. 2). we observed infections mainly caused by penicillium digitatum and p. italicum on fruits. consistent with our result, conventionally and organically grown grapefruits did not show significant difference in the incidence of fungal decay during storage at 9°c (chebrolu et al., 2012a). nunes et al. (2010) concluded that the sensitivity to green or blue mold is determined better by the origin of orange fruits than by the system of production and the reduction or elimination of chemical synthesis compounds in the citrus production does not cause a significant increase in sensitivity of fruits to penicillium spp. chilling injury was not observed any of fruits from both production systems throughout storage period. our previous study showed that no or slight incidence of chilling injury occurred in ‘washington navel’ oranges after 5 months at 4°c (özdemi̇r et al., 2008). in conclusion, conventionally grown oranges had higher juice percentage, citric acid, ta and lower weight loss than organically grown oranges at harvest and during storage. rind color (l*c* hº), tss, fructose, glucose and sucrose and malic acid content were not affected by the production systems. compared to conventionally grown oranges, organically grown oranges had lower ta, but better taste scores since they attained higher tss/ta ratio. there was no significant difference in the ascorbic acid content of fruits between two production systems at harvest, but lower ascorbic acid content was found in organically grown oranges, compared to conventionally grown oranges during storage. acknowledgements the authors wish to thank the taner bugay from tareks tarımsal ürünler ltd. şti. 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ifoam, bonn and fibl, frick, switzerland. address of the authors: assoc. prof. dr. elif çandır (corresponding author), assist. prof. dr. müge kamiloğlu, durmuş üstün, and gülcan tuğçe kendir, mustafa kemal university, faculty of agriculture, department of horticulture, 31034 antakya, hatay, turkey. e-mail: eerturk@mku.edu.tr art21 journal of applied botany and food quality 82, 131 135 (2009) 1institute for horticulture science, urban horticulture, humboldt university berlin, germany variability of aliphatic glucosinolates in arabidopsis thaliana (l.) – impact on glucosinolate profile and insect resistance f. rohr1, ch. ulrichs1, i. mewis1 (received november 11, 2008) summary the glucosinolate(gs)-myrosinase system of brassicaceae, including the model plant arabidopsis thaliana (l.), comprises a defence which is effective especially against generalist herbivores. based on their side chain structure gs are grouped into aliphatic, aromatic, and indolyl gs. indolyl gs are widely distributed among a. thaliana ecotypes and the brassicaceae family, but the presence of aliphatic gs is variable and under strong genetic control. we investigated the effect of aop gene expression on the side chain modifications of gs and the impact on insect resistance. aop2 and aop3 genes from mr-0 and sap-0 ecotypes, respectively, were crossbred into the methylsulfinyl gs producing gie-0. successful crosses were heterozygote plants which produced allyl (aop2) or 3-hydroxypropyl gs (aop3). after self-pollination, the chemical profile of the f3 generation of plants was screened to identify homozygote lines. homozygote lines producing 3-hydroxypropyl gs were compared to methylsulfinyl gs, which were used to study the impact of gs structure on insect performance in first experiments. our experiments revealed that methylsulfinyl gs containing ecotype lines were more resistant to the generalist caterpillar spodoptera exigua (hübner) and to the specialist caterpillar pieris brassicae (l.) than the lines containing hydroxypropyl gs as main compounds. introduction plants live in a dangerous world and are constantly being attacked by various enemies, such as fungi, bacteria, and insects. they have developed effective strategies in order to overcome their enemies. the defense strategy of plants in families in the brassicales order and of the model plant arabidopsis thaliana (l.) comprises glucosinolates (gs) as their secondary plant metabolites. gs are stable, hydrophilic compounds localized in the plant’s cell vacuole or in specialized cells, separated from their hydrolyzing enzymes, the myrosinases (β-thioglucoside glycohydrolases) (koroleva et al., 2003). the principal biologically active compounds, such as isothiocyanates and nitriles, are released after tissue damage, when gs and myrosinase come into contact. the formation of the hydrolysis products depends on the chemical structure of the aglycone side chain, the presence of protein factors like the epithiospecifer protein (esp), and other reaction conditions (lambrix et al., 2001). to date more than 120 gs varying in their aglycone side chain are described (fahey et al., 2000). studies with a. thaliana and different brassica species revealed that they are synthesized via different biochemical pathways (kliebenstein et al., 2001a; raybouldt and moyes, 2001). in a. thaliana, three major classes of gs are distinguished: aliphatic gs, which derive principally from methionine precursors, aromatic gs from phenylalanine, and indolyl gs from tryptophan precursors. indolyl gs are uniformly distributed in a. thaliana ecotypes and members of the brassicaceae family (kliebenstein et al., 2001a; fahey et al., 2001). in contrast, the presence of aliphatic gs is highly variable in the brassicaceae and under strong genetic control. the variability of aliphatic gs is determined by polymorphism at only a few different loci. studies on aop genes and gs contents in ecotypes showed that they naturally produce either hydroxyalkyl (e.g 3-hydroxypropyl) or alkenyl (e.g. allyl) gs with production of either aop2 or aop3 (kliebenstein et al., 2005). aop genes are a result of gene duplication and encode for 2-oxoglutarate-dependent dioxygenases (kliebenstein et al., 2001c). the gene product of aop2 causes the production of alkenyl gs of a methylsulfinyl precursor, whereas aop3 leads to the formation of hydroxy-alkyl gs in presence of c3 precursors are present. a. thaliana ecotypes that accumulate methylsulfinyl gs, like columbia, possess a non-functional aop2 and/or aop3, or the expression of these genes is blocked. there is a lack of studies that investigate the importance of aliphatic gs variability in regard to plant defense against herbivorous insects. preliminary examinations indicated that the variability of the aop genes in a. thaliana ecotypes influences the host plant’s resistance. further investigations shall reveal the influence of aop gene expression on the side chain modifications of gs and the impact on insect resistance in more detail. the first approach was to crossbreed aop2 and aop3 genes into a methylsulfinyl gs producing c3 ecotype to obtain allyl and 3-hydroxypropyl gs containing plants in the filial generation. to evaluate the host-plant resistance of homozygote lines with a chemically different phenotype, we tested the feeding performance of two lepidopteron pest species on these lines. we used the specialist pieris brassicae (l.) and the generalist spodoptera exigua (hübner) for the bioassays. in the present study, the first results for methylsulfinyl gs producing lines versus hydroxypropyl gs containing lines are presented. material and methods crossbreeding of aop2 and aop3 into a methylsulfinyl gs containing ecotype the 3-methylsulfinylpropyl gs producing ecotype gie-0 (n1193) was selected for the experiments, because preliminary bioassays revealed that this ecotype is highly resistant to insects. its crossing partners were the hydroxypropyl gs accumulating ecotype sap-0 (n1506) and mr-0 (n1373), whereby sap-0 was most suitable to insects in pilot studies. female-sterile crossing lines of gie-0 were produced by making the stamen unripe. then the pistil was spread with pollen of sap-0. to guarantee that at least one cross contained the desired gs profile (hydroxypropyl or allyl gs) in the f1, 10 parallel crossings were carried out each time. the seeds of successful crosses were used to obtain at least five plants. subsequently the gs profiles of filial generations (self-pollination) were examined. plants containing 3-hydroxypropyl, allyl, and 3-methylsulfinyl gs were used for seed collection. once can only tell which plant is homozygous for aop3, aop2 or aop0 (non-functional aop2/3) in the f3 generation, because of the constant gs profile. only homozygote lines which are stable for aop and esp were used for insect bioassays. plants used for the bioassays were cultivated in a climate chamber at 21 ± 1 °c, 60 ± 5% relative humidity, at 200 µmol m-1s-1 light intensity and a 10 : 14 (l : d) photoperiod. 132 f. rohr, ch. ulrichs, i. mewis glucosinolate analysis for gs analysis, 4 to 5 leaves of 2-week old plants were frozen in liquid nitrogen, freeze-dried, and grinned with a pestle directly in the tube. gs were extracted as described in detail in mewis et al. (2005). 4-hydroxybenzyl gs (sinalbin, purified from sinapsis alba seeds as potassium salt) was used as internal standard for quantification of gs in extracts. gs in extracts were desulfated with 75 µl aryl sulfatase solution (h-1 from helix pomatia, sigma-aldrich) on deae sephadex a-25 mini columns. 40 µl of desulpho gs extracts were run on a dionex p680a hplc system equipped with a narrow bore column (acclaimtm 120, 250 2.1, 5 µm, rp18, dionex). for analysis of gs a 43 min gradient program was used consisting of the following eluents: a) millipure water and b) 40% acetonitrile (hplc grade). the run was composed of 0.5% b (1 min), 0.5 20% b (7 min), 20% b (2 min), 20 50% b (9 min), 50% b (3 min), 50 99% b (6 min), a 5 min hold at 99% b, 99 0.5% b (3 min), and a 7 min final hold at 0.5% b. gs were monitored at 229 nm. for gs identification, internal standards, retention time, and uv spectra were used. insect bioassay pieris brassicae (l.) larvae were obtained from an established rearing in the urban horticultural department at humboldt university. eggs were originally ordered from insect service gmbh (berlin). for the rearing, the adult laid its eggs on kohlrabi (brassica oleraceae var. gongylodes) and were reared until the 2nd instar on these plants. then they were transferred to savoy cabbage (brassica oleraceae var. sabauda) and maintained there until pupation. spodoptera exigua (hübner) eggs and larvae were kindly given by bayer crop science (monheim, germany). rearing is described in rohr et al. (2006). for the bioassays, 10 lines of methylsulfinyl (msop) gs producing plants and 10 lines of hydroxypropyl (ohp) gs producing plants were used. the test insects were 2nd instars of s. exigua and p. brassicae. the experiments were conducted in plastic cages covered with fine mesh gauze containing one larva per plant. the initial and final (after 72 h) larval weights were determined. in addition, the plant damage was estimated, according to stotz et al. (2000). analysis of glucosinolate hydrolysis product leaf samples (200 mg) of ecotypes were ground by using a pestle with 1.0 ml of milliq water in 4 ml glass vials. phenyl cyanide (50 µl; diluted 1 : 5000 in 1% meoh/h2o) was added as internal standard. the tubes were quickly sealed with a septum cap and left standing for 10 min at room temperature. after the addition of 2 ml of dichloromethane through the septum, the tube was vortexed for 10 s and centrifuged at 5000 g. the dichloromethane layer was then removed, dried, and filtered by passing through a short column of anhydrous sodium sulfate to remove water residues. the aqueous layer of the first extraction was re-extracted with 2 ml dichloromethane. extracts were combined and concentrated under nitrogen to 200 µl and analyzed by gc-ms using an agilent 6890 series gas chromatograph (agilent technologies, waldbronn, germany) with a db5 column (j & w scientific, 30 m, 0.25 mm i.d., and 0.25 µm film). a 1 µl sample was split-less injected at 200 °c. a temperature program of 35 °c for 3 min, a 10 °c/min ramp to 230 °c and a cool down with a total analysis time of 37 min was used, and helium was used as carrier gas. for product identification by using standards and ms libraries, the column was coupled to an agilent 5973n quadrupole mass detector. rt-pcr analysis total rna of mr-0, gie-0, sap-0, and col-0 (reference ecotype) was isolated from frozen rosettes with trizol® reagent (invitrogen, karlsruhe) following the standard protocol and including the high salt precipitation step. rna was converted to cdna by reverse transcription according to the promega (madison, wi) protocol. a primer (0.5 µg), oligodt12-18 (invitrogen, carlsbad, ca) was added to 2 µg of total rna with a total of 8 µl volume and was heated to 65 °c for 5 min. cdna was synthesized by adding 0.5 mm dntps, 200 units of moloney murine leukemia virus reverse transcriptase (promega) and the buffer supplied for this enzyme in a total volume of 20 µl. the mixture was incubated at 37 °c for 1 h. the volume was adjusted to 50 µl followed by heating for 10 min at 70 °c. the pcr reaction was optimized and 2 µl of rt reaction was used as a template for the tests. the following reaction condition were used for the pcr in a total volume of 20 µl, 1 x pcr buffer (promega), 0.2 mm dntp’s, 2.1 mm mgcl2, 0.5 µmol of the forward and reverse primer, 1 unit of taq dna polymerase (promega). the following pcr program was performed using a biometra gradient cycler: 2 min 96 °c, 30 cycles of 15 s at 94 °c, 30 s 54 °c, and 20 s at 72 °c, followed by a 5 min final at 72 °c. actin8 (ac8, at1g49240) was used as reference gene and was designed to be intron spanning for possible detection of dna contamination. the ac8 forward (f) primer was 5’-atgaagattaaggtcgtggcac and the reverse (r), 5’gtttttatccgagtttgaagaggc. following primers were designed to amplify aop genes: aop2 (at4g03060) f: 5’cacgtgtccaaaaccggac r: 5’attgtcgaaactcggaatcaag aop3 (at4g03050) f: 5’gaaagaagacgagatacgcag r: 5’cttgaaaccacgtccaaaca the intensities of bands were visualized by gel electrophoresis on a 1.5% agarose gel containing ethidium bromide on a kodak image station by using kodak mitm software. all primers successfully amplified a band of correct size. pcr products were cloned into the t-overhang vector pcr2.1 with the topo ta cloning kit (invitrogen). pcr products were fully sequenced to confirm their fidelity. results characteristics of parent ecotypes in order to investigate the influence of aop genes on side chain modifications of gs and their impact on insect resistance, ecotypes with different c3 main gs were crossed to obtain ecotype lines containing methylsulfinyl, hydroxypropyl or allyl gs. the parent ecotypes had the following characteristics. gie-0 is accumulating mainly 3-methylsulfinylpropyl gs and does not show aop3 expression (fig. 1). low expression of aop2 was detected, indicating a non-functional enzyme like in col-0. aop2 in the allyl gs containing ecotype mr-0 was greatly expressed, whereas expression of aop3 was not detected. the predominantly 3-hydroxypropyl gs accumulating ecotypes sap 0 was the only ecotype with aop3 expression. the gc-ms analysis revealed that gie-0 produces nitriles as hydrolysis products, whereas the dominating hydrolysis product of mr-0 and sap-0 were isothiocyanates. gs profil of gie-0 x sap-0 crosses although we were able to successfully cross the methylsulfinyl gs producing ecotype gie-0 with sap-0 and mr-0 to achieve the desired gs profiles (either 3-hydroxypropyl or allyl gs, in the f1 generation) detailed data will be presented only for gie-0 x sap-0. the seeds of successful crosses were used, and gs profiles of filial generations after self-pollination were examined. 3-hydroxypropyl, allyl, and 3-methylsulfinylpropyl gs containing plants were used for seed glucosinolates and insect resistance 133 collection. in fig. 2 (a, b) hplc chromatogram examples for ecotype lines producing 3-hydroxypropyl as well as 3-methylsulfinylpropyl gs used for screening, are presented. to obtain homozygous plant lines for aop3, gs profiles of five plants per line were analyzed by hplc in the f3 generation. for methylsulfinyl gs producing plants (genotype aop0, nonfunctional aop2/aop3), only one plant was analyzed each time. according to their stable gs profiles, 10 homozygote lines containing aliphatic 3-methylsulfinyl (3msop) gs, as well as 10 lines producing aliphatic 3-hydroxypropyl (3ohp) gs as main compounds, were identified and selected for the bioassays with insects (tab. 1). the main indolyl gs of lines were 3-indolylmethyl gs followed by 4-methoxy-3-indolylmethyl gs and 1-methoxy-3indolylmethyl gs. the total gs content in ohp producing lines was up to twofold higher than in msop producing lines (tab. 1). plants obtained from seeds of these lines were used for the bioassays with different insect herbivores. tab. 1: aliphatic, indolyl, and total glucosinolate content in leaves of a. thaliana ecotype lines producing 3-hydroxypropyl gs (ohp) or 3methylsulfinylpropyl gs (msop). glucosinolate content [µmol/g dry weight] line aliphatic indolyl total ohp1 55.87 5.15 61.02 ohp2 72.54 10.42 82.97 ohp3 40.52 6.32 46.84 ohp4 60.73 9.39 70.12 ohp5 52.23 7.53 59.77 ohp6 61.56 9.46 71.03 ohp7 57.04 7.90 64.95 ohp8 42.20 6.32 48.53 ohp9 27.28 5.54 32.83 ohp10 35.06 7.18 42.25 msop1 12.94 4.72 17.67 msop2 9.45 3.72 13.17 msop3 8.10 5.40 13.51 msop4 19.07 6.06 25.14 msop5 43.57 11.53 55.10 msop6 26.80 5.65 32.45 msop7 7.52 4.30 11.82 msop8 12.04 5.70 17.74 msop9 23.64 4.94 28.59 msop10 26.58 5.55 32.13 host plant suitability of ecotype lines for spodoptera exigua and pieris brassicae larvae insect performance data, measured as percentage weight gain of larvae within three days, on msop or ohp producing lines are presented in fig. 3. the percentage larval weight gain on chemically fig. 1: expression of aop genes in a. thaliana ecotypes compared to the reference gene actin 8. fig. 2: hplc chromatogram examples of lines producing 3-hydroxypropyl gs (a) or 3-methylsulfinylpropyl gs. aop 2 aop 3 actin 8 g ie -0 m r0 s a p -0 c o l0 a b int. standard int. standard min mau mau min 134 f. rohr, ch. ulrichs, i. mewis different ecotype crosses was significant different for both lepidopteron species tested (fig. 3). when the larval weight increase on the msop and ohp phenotypes is compared, the generalist s. exigua and the specialist p. brassicae performed better on 3-hydroxypropyl gs producing lines than on lines containing 3-methylsulfinylpropyl gs. fig. 3: average percentage weight gain of second instars of caterpillars on ecotype lines (anova, ** p <0.05. * p <0.1). discussion the gs-myrosinase defence system has been an object of extensive study in the past several years. however, many aspects are far from being clear, such as the function of variability of aliphatic gs in a. thaliana and other brassicaceae. in our study we examined the impact of aop gene expression on the side chain modification of aliphatic gs and the impact on host-plant resistance. our study supports the finding of kliebenstein et al. (2001b), which state that the two genes, aop2 and aop3, which encode for 2oxoglutarate-dependent dioxygenases, are responsible for side chain hydroxylation of aliphatic gs. aop2 from mr-0 crossed into the 3methylsulfinylpropyl gs producing ecotype gie-0 resulted in an alkenyl-producing phenotype. aop3 introduced into gie-0 leads to the conversion of methylsulfinylalkyl to the hydroxyalkyl form, whereby aop3 expression was detected only for sap-0. the methylsulfinyl gs accumulating ecotype gie-0 is caused by the absence of aop3 and a non-functional aop2. ecotypes vary not only in their main gs, but also other factors like plant morphology and other secondary metabolites than gs can influence host-plant resistance (larkin et al., 1996; harrewijn et al., 2001). therefore, we created ecotype lines which posses a chemically distinct gs profile with a preferably similar genetic background to run feeding studies with insects of different specialization. weight gain of lepidopteron larvae, one generalist: s. exigua and one specialist: p. brassicae, was significantly higher on 3-hydroxypropyl gs (ohp) containing ecotype lines than on 3-methylsulfinyl gs (msop) producing ecotype lines. the better performance of ohp lines corresponds to a higher mean gs content compared to gs contents in msop lines. this suggests a strong effect of side chain hydroxylation on insect performance. our results are consistent with our previous study with common ecotypes (rohr et al., 2006). it is known that gs and different types of hydrolysis-product produced influence the plant resistance especially against generalist insects (kliebenstein et al., 2002b; kroymann et al., 2003). but different compounds within a chemical group, like the gs, can have effects on specialized herbivorous insects as well (bartlett et al., 1994). the reason for ohp lines being more suitable for generalist and specialist lepidopteron species than msop lines might be due to the short chemical structure of 3-hydroxypropyl gs and/or different reactivity of this compound compared to 3-methylsulfinylpropyl gs. further investigations regarding the reaction of this compound within the insect gut could confirm this assumption. the preference of generalist and specialist insect species for short chain aliphatic gs like 3-hydroxypropyl gs has been reported also in other studies (giamoustris and mithen, 1995). also lambrix et al. (2001) showed a feeding preference of the generalist trichoplusia ni (hübner) for f2 lines of ecotype crosses da(1)-12 x ei-2, containing 3-hydroxypropyl gs. furthermore, they determined that t. ni preferred feeding on lines producing nitriles as hydrolysis products. in our study with ecotype lines, we did not find any dependence of hydrolysis products, isothiocyanates or nitriles, on host plant suitability (data not shown). although our study indicates that gs side chain influences plant resistance against insect, further studies are needed to reveal the biochemical basics, which lead to the distinct effects of chemically different compounds to insects. acknowledgement we thank dr. peter meisner and gerd trautmann (bayer crop science, mohnheim, germany) for sending the start population of s. exigua. the authors further thank dr. michael reichelt and dr. jonathan gershenzon (max-planck institute for chemical ecology jena, germany) for their collaborative help in analyzing the glucosinolate hydrolysis products. the project was funded by dfg (deutsche forschungsgesellschaft, gz me 2095/4-1). references bartlet, e., kiddle, g., williams, i., wallsgrove, r.m., 1999: woundinduced increases in the glucosinolate content of oilseed rape and their effect on subsequent herbivory by a crucifer specialist. entomol. exp. appl. 91, 163-167. fahey, j.w., zalcmann, a.t., talalay, p., 2001: the chemical diversity and distribution of glucosinolates and isothiocyanates among plants. phytochemistry 56, 5-51. harrewijn, p., oosten, van a.m., piron, p.g.m., 2001: natural terpenoids as messengers: a multidisciplinary study of their production, biological functions, and practical applications. dordrecht, boston: kluwer academic 2001, 440. kliebenstein, d.j., gershenzon, j., mitchell-olds, t., 2001a: comparative quantitative trait loci mapping of aliphatic, indolic and benzylic glucosinolate production in arabidopsis thaliana leaves and seeds. genetics 159, 359-370. kliebenstein, d.j., lambrix, v.m., reichelt, m., gershenzon, j., mitchell-olds, t., 2001b: gene duplication in the diversification of secondary metabolism: tandem 2-oxogluterate-dependent dioxygenases control glucosinolate biosynthesis in arabidopsis. plant cell 13, 681693. koroleva, o.a., davies, a., deeken, r., thorpe, m.r., tomos, a.d., hedrich, r., 2000: different myrosinase and ideoblast distribution in arabidopsis and brassica napus. plant physiol. 127, 1750-1763. lambrix, v., reichelt, m., mitchell-olds, t., kliebenstein, d.j., gershenzon, j., 2001: the arabidopsis epithiospecifer protein promotes the hydrolysis of glucosinolates to nitriles and influences trichoplusia ni herbivory. plant cell 13, 2793-2807. larkin, j.c., young, n., prigge, m., marks, m.d., 1996: the control of trichome spacing and number in arabidopsis. development 122, 9971105. mewis, i., appel, h.m., hom, a., raina, r., schultz, j.c., 2005: major signaling pathways modulate arabidopsis thaliana (l.) glucosinolate accumulation and response to both phloem feeding and chewing insects. plant physiol. 138, 1149-1162. raybould, a.f., moyes, c.l., 2001: the ecological genetics of aliphatic glucosinolates and insect resistance 135 glucosinolates. heredity 87, 383-391. rohr, f., ulrichs, c., mucha-pelzer, t., mewis, i., 2006: variability of aliphatic glucosinolates in arabidopsis and their influence on insect resistanc. comm. appl. biol. sci, ghent university, 71/ 2b, 507-515. stotz, h.u., pittendrigh, b.r., kroymann, j., weniger, k., fritsche, j., bauke, a., mitchell-olds, t., 2000: induced plant defense responses against chewing insects ethylene signalling reduces resistance of arabidopsis against egyptian cotton worm but not diamondback moth. plant physiol. 124, 1007-1017. address of the authors: franziska rohr, institute for horticulture science, urban horticulture, humboldt university berlin, lentzeallee 55-57, 14195 berlin-dahlem, germany. << /ascii85encodepages false /allowtransparency false 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/pagesize [907.087 680.315] >> setpagedevice journal of applied botany and food quality 87, 62 66 (2014), doi:10.5073/jabfq.2014.087.009 1 meisterschule ebern für das schreinerhandwerk, ebern, germany 1 universidade federal do parana, dept. de solos e engenharia agricola, curitiba-pr, brazil significance of xylem water conductance for the compatibility of maté phenotypes (ilex paraguarensis) for grafting oliver dünisch1*, bruno reissmann2 (received july 2, 2013) * corresponding author summary in this study the compatibility for grafting of root stocks and buds of three recognized leaf phenotypes of ilex paraguarensis st. (maté) hil. from south brazil was investigated. special regard was given to the anatomical structure and the sap flow in the secondary xylem of the grafted plants. significant differences of the wood structure expressed in terms of vessel portion, vessel member length, and intervessel perforations were found in low compatible phenotypes, while highly compatible phenotypes had a very similar wood structure. in grafts of highly compatible phenotypes six months after grafting the water conducting system of root stocks and buds was strongly linked to each other, while in grafts of low compatible phenotypes the formation of connecting vessel elements was rare. in the first month after grafting the xylem sap flow in the buds of all grafts was significantly higher compared to their root stocks, while in the second month in grafts of highly compatible phenotypes, the water transport in the root stocks and in the buds was balanced. in contrast, in grafts of low compatible phenotypes even in the second month the water transport in the root stocks was lower compared to the buds causing the dieback of a high portion of these plants. introduction maté-tea produced from leaves and young branches of ilex paraguariensis st. hil. (aquifoliaceae) is an important non-wood forest product in south brazil as well as in parts of argentina, paraguay, and uruguay. due to its content of caffeine and theobromine, in this region the consumption of maté tea is similar to the consumption of coffee or black tea in other regions of the world. for the production of health and wellness teas, considerable amounts of maté are also exported to asia, europe, and north america (ohem and hölzl, 1990). depending on the origin of the seeds, the age of the plants, and the sampling time the extractive content and the flavour of the leaves strongly vary (kawakani and kobayashi, 1991; martinez et al., 1997; valduga et al., 1997; scherer et al., 2002). in spite of the high variability within the species breeding programs for the improvement of the quality and the quantity of maté production are rare. so far, in practice localy known varieties of leaf phenotypes (shape and colour of the leaves) are used for seed propagation, while vegetative propagation is only used in smaller scales (rosse and fernandes, 2002). ecological studies and investigations on the chemical composition of the leaves showed significant differences between leaf phenotypes with regard to their drought tolerance and the flavour of the final product (baas, 1973; 1975; reissmann et al. 1999). consequently, with regard to the ecological stability of maté plants under different site conditions and high quality maté production appropriate combinations of root stocks and buds are desired. in former studies reissmann et al. (2003) and dünisch et al. (2004) found a significant correlation between the wood structure in the secondary xylem, the morphology of the leaves, and the adaptation to drier sites in different leaf phenotypes from south brazil. it turned out that ecologically best adapted phenotypes did not produce the best quality of maté, while the phenotypes, which produced the best quality of maté were more sensitive to drought conditions. therefore in this study the compatibility of root stocks and buds of three recognized leaf phenotypes of ilex paraguarensis from south brazil for grafting was investigated. special regard was given to the anatomical structure and the sap flow in the xylem of the grafted plants. material and methods plant material and grafting procedure one hundred and fifty plants each of the maté phenotypes “amarelinha”, “cinza”, and “sassafras” of ilex paraguariensis st. hil. (detailed description of the phenotypes by reissmann et al., 2003) were obtained by vegetative propagation from five 15-yearold trees each grown at the fazenda bitumirim in the state of paraná, brazil (25°15‘s, 50°45‘w, 870 m above sea level; annual precipitation 1,570 mm, mean air temperature 19.1 °c). for root formation the cuttings were cultivated in a “knop” nutrient solution for two months. after that the plants were transferred into pots with a standard soil substrate (dünisch et al., 2006) and cultivated in the greenhouse of the fazenda bitumirim under subtropical climate conditions. after two years of growth grafts with all nine possible combinations of root stocks and buds of the three phenotypes were produced (50 plants per treatment). for copulation grafting cuttings for roots stocks and buds were produced with sterile cutter knifes at approximately 100 mm plant height. before grafting the cut fases of the two internodes were disinfected with 0.5 % chinosol. the connecting zone was covered with grafting wax (lauril, germany) and sealed with parafilm. during a six month period the survival rate of the grafts was monitored weekly. microscopical characteristics of the xylem six months after grafting samples from the root stock, the grafting zone, and the bud of three plants of each treatment were collected. after harvest the samples were fixed in a formalin/acetic acid/alcohol (faa) solution. the relevant shoot portions were embedded in polyethylene glycol (peg 1500) with increasing concentration (peg 1500:h2o, 1:2, 1:1, 2:1, 1:0, 1:0). transverse, radial, and tangential sections were prepared with a sliding microtome (reichert, austria; section thickness: 15 μm). the sections were mounted on glass slides in drops of a 25 % solution of nh4oh (watanabe et al., 2004). the sections were covered with coverslips and were analysed using a light microscope (axioskop mc 80 zeiss, germany). microphotos were taken with a digital camera adjusted on the microscope. xylem water conductance of maté phenotypes 63 the cross sections of the root stocks and the buds were used to determine the percentage of cell type and the radial and tangential diameter of the vessels. the percentage of cell type was obtained by means of an integration ocular lens (leucodiff; höster and spring, 1971). for the quantification of the percentage of cell type 400 spots each per increment zone were analysed. the diameter of the vessels was measured directly in the microscope with an eye piece micrometer (50 measurements per treatment). the number of bars per vessel perforation plate was counted in the radial sections. the length of the vessel members was determined after maceration in a jeffrey´s solution. the length of 60 vessel members was measured in each fraction by means of an image analyzer (sigmascanpro). the mean value and the standard deviation of each parameter were calculated. the significance of differences between phenotypes was assessed by anova at p ≤ 0.05 by fisher’s f-test. staining of the water conducting xylem and calculation of hydraulic conductivity the water conducting system of the secondary xylem was marked using 1% methylenblue as a tracer (fig. 1). shoot samples (100 mm length) of five plants each of the phenotypes “amarelinha”, “cinza”, and “sassafras” were cut under water to avoid air embolism. dye was introduced into the samples via the transversal cut with a pressure of 10 cm water column for 5 minutes (dünisch and morais, 2002). staining of the samples was quantified microscopically with regard to stained and unstained cell types and the maximum distance of stained cells from the transversal cut, where dye was applied. the hydraulic conductivity of different cell types was calculated as the portion of the cross section areas of stained cells in different distances from the transversal cut (2 mm steps). fig. 1: staining of the water conducting xylem in the stem of a three-yearold maté plant (phenotype “amarelinha”) with methylene blue. scale bar = 2 mm. xylem sap flow measurements xylem sap flow measurements were carried out according to granier (1985; 1987) with a constant heating method using heated and unheated thermocouples (up gmbh, osnabrück, germany). probes were installed 30 mm below (root stock) and 30 mm above (bud) the grafting zone. the upper sensor was continuously heated with a constant power supply (156 mw). data were recorded continuously in 10-minutes averages on skye datahog dataloggers (skye instruments, wales, u.k.). in order to calculate the xylem sap flow from the temperature gradients measured with the thermocouples, the measuring system was calibrated by correlating temperature gradients with the water flux (sap volume) through 100 mm shoot pieces of the three phenotypes (erbreich, 1997). for all three phenotypes a similar relationship was found, close to the relationship described by granier (1985; 1987). results survival rate of grafted maté plants six months after grafting the survival rate of the grafts varied between 6 % and 94 % (tab. 1). the survival rate of grafts produced from root stocks and buds of the same morphotype was between 82 % and 94 %. except in plants with root stocks of the morphotype “sassafras”, the survival rate of grafts produced from root stocks and buds of different leaf phenotypes was strongly reduced. in particular the survival rate of grafts with root stocks of the morphotype “cinza” and buds of the morphotype “amarelinha” was extremely low (6 % corresponding to 3 plants). in these grafts 39 out of the 50 scions died in the first three months of the experiment. anatomical characteristics of the secondary xylem of the grafted maté plants in the secondary xylem of the grafts vessels, fibre tracheids, axial and ray parenchyma were present. in the xylem formed before grafting the distribution of the xylem elements was diffuse. no significant differences of the vessel diameter and the percentile portion of fibre tracheids were found between the three phenotypes (tab. 2). in contrast the portion of vessels was significantly higher in the xylem of the phenotypes “amarelinha” and “sassafras” compared to the phenotype “cinza”. the overlap in class size distribution of measured vessel portions between the phenotypes “amarelinha” and “cinza” was 0 %, between “amarelinha” and “sassafras” 87 %, and between “cinza” and “sassafras” 1 % (fig. 2). significant differences in the length of the vessel members and the number of bars in their scalariform perforation plates were found between the three phenotypes. the longest vessel members and the highest number of vessel bars were found in the xylem of the morphotype “amarelinha”, while the shortest vessel members and the lowest number of vessel bars were found in the xylem of the phenotype “cinza”. the overlap in class size distribution of the length of vessel members between the phenotypes “amarelinha” and “cinza” was 29 %, between “amarelinha” and “sassafras” 61 %, and between “cinza” and “sassafras” 63 % (fig. 3). the overlap in class size distribution of counted vessel bars between the phenotypes “amarelinha” and “cinza” was 0 %, while the overlap between “sassafras” and “cinza” and “”sassafras” and “amarelinha” was 14 % and 5 %, respectively (fig. 4). six months after grafting in the connecting zone of best compatible phenotypes (high survival rate, table 1) the xylem tissue of root stocks and buds was strongly linked to each other in particular by the formation of connecting vessel elements and fibre tracheids tab. 1: survival rate (%) of grafted maté plants produced from different combinations of root stocks and buds of the phenotypes “amarelinha”, “cinza”, and “sassafras” six months after grafting. 50 plants per treatment. morphotype buds amarelinha cinza sassafras amarelinha 82 38 34 root stocks cinza 6 94 62 sassafras 80 84 90 64 o. dünisch, b. reissmann (fig. 5b/c). the lowest formation of conneting vessel elements and fibre tracheids and the strongest formation of a protective layer were found in root stocks of the phenotype “cinza” connected to buds of the phenotype “amarelinha” (fig. 5c). water transport in the secondary xylem of the grafted maté plants the transport of water in the secondary xylem of the two-years-old plants was equally distributed over the shoot cross section (fig. 1). water transport was restricted to vessels and fibre tracheids. due to the very low hydraulic conductivity of fibre tracheids compared to vessels (tab. 2), in the shoot of all phenotypes the predominant portion of water was transported in the vessels. however, the hydraulic conductivity of the vessels in the xylem of the morphotype “cinza” was strongly reduced compared to the phenotypes “amarelinha” and “sassafras” (reduction 66 % and 39 %, respectively). during the first month after grafting significant differences in xylem sap flow between the root stocks and the buds of the grafts were found (tab. 3a). in all grafts xylem sap flow was significantly higher in the buds compared to the root stocks. highest differences in xylem sap flow between root stocks and buds were found in grafts with root stocks of the phenotype “cinza” and buds of the phenotype “amarelinha” (0.004 l cm-2 day-1 and 0.039 l cm-2 day-1) corresponding to a water loss of the buds of approximately 0.53 l during the first month. except in grafts of combinations of root stocks and buds of the phenotypes “cinza” and “amarelinha”, “amarelinha” and “cinza”, and “amarelinha” and “sassafras” in the second month after grafting the xylem sap flow of root stocks and buds of all grafts was balanced (tab. 3b). the strong differences of xylem sap flow between the root stocks and buds in “cinza-amarelinha”, “amarelinha-cinza”, and “amarelinha-sassafras” – grafts even in the second month after grafting corresponded to the low survival rate of these combinations of root stocks and scions (tab. 1). discussion in terms of leaf morphology, shoot anatomy, and chemical composition, the variability within the species ilex paraguariensis st. hil. (maté plant) is very high (baas, 1973; 1975; reissmann et al., 1999). therefore over decads best cultivars were selected with regard to their ecological stability and the quality of the final product. in total, six to nine distinct varieties of ilex paraguariensis are tab. 2: anatomical characteristics of the water conducting system and relative hydraulic conductivity in the xylem of the phenotypes “amarelinha”, “cinza”, and “sassafras”. mean value ± standard deviation. values followed by different letters differ significantly at p<0.05 (fisher’s f-test). characteristics amarelinha cinza sassafras vessel portion (%) 28 ± 7a 16 ± 4b 26 ± 2a vessel diameter (µm) 78 ± 15a 79 ± 17a 74 ± 18a vessel member length (µm) 832 ± 132a 499 ± 98b 698 ± 112c number of vessel bars 23 ± 4a 15 ± 3b 20 ± 3c per perforation plate fibre tracheids portion (%) 43 ± 5a 47 ± 5a 42 ± 2a relative hydraulic conductivity 100 34 73 vessels (%) relative hydraulic conductivity 2 8 3 fibre tracheids (%) fig. 4: class size distribution (%) of the number of vessel bars per perforation plate in the xylem of the phenotypes “amarelinha” (rough dotted line), “cinza” (fine dotted line), and “sassafras” (full line). class size: 1. 500 measurements per phenotype. fig. 2: class size distribution (%) of vessel portions (%) in the xylem of the phenotypes “amarelinha” (rough dotted line), “cinza” (fine dotted line), and “sassafras” (full line). class size: 1 %. measuring area: 1 mm². fig. 3: class size distribution (%) of vessel member length (µm) in the xylem of the phenotypes “amarelinha” (rough dotted line), “cinza” (fine dotted line), and “sassafras” (full line). class size: 50 μm. 1500 measurements per phenotype. (fig. 5a). in grafts of less compatible combinations of root stocks and buds the formation of connecting vessels and fibre tracheids was strongly reduced. in these cases the formation of a protective layer of parenchyma cells with dark staining compounds within was observed xylem water conductance of maté phenotypes 65 described in the literature (coelho et al., 2002). inheritance studies indicate a high genetic variability between, but a smaller genetic variability within the varieties (scherer et al., 2002). consequently, the genetic variation within the phenotypes selected for our experiment is supposed to be low, although the grafts were produced from five – genetically not identical plants of each phenotype. the high survival rate of grafts produced from root stocks and buds of identical phenotypes (blanc samples) proves that an appropriate grafting procedure for the production of the experimental plants was chosen. studies of moore (1983), wang and kollmann (1996), and espen et al. (2005) underline the significance of the restoration of the continuity of the vascular tissue for the compatibility of root stocks and buds in grafts. our tracer experiments showed that in the secondary xylem of ilex paraguariensis, vessels as well as fibre tracheids make part of the water conducting vascular tissue. however, in the three phenotypes the hydraulic conductivity of the vessel system was 4 to 50 times higher than in the tissue of fibre tracheids. in agreement with investigations of becker et al. (1999) this result confirms the extraordinary importance of the vessel system for the water transport in the shoot. in terms of vessel portion, vessel member length and perforations, significant differences between the three phenotypes were found, while the anatomical structure and the portion of fibre tracheids of the three phenotypes were very similar. a low portion of short vessels with a reduced number of perforations reduce significantly the maximum capacity of water transport in the xylem (tyree and zimmermann, 2002). in contrast, bigger and longer vessels are more susceptible to air embolism under drought conditions (sperry and ikeda, 1997). consequently, the water conducting system of the phenotype “amarelinha” could be considered to be the most effective, but most vulnerable, while the water conducting system of the phenotype “cinza” is supposed to be the least effective, but the most resistant to drier periods of the 3 morphotyps. the distribution of the anatomical characteristcs of the vessel system in the phenotype “sassafras” had a strong overlap to the vessel system of the phenotype “amarelinha” as well as to the vessel system of the phenotype “cinza”. this explains the high survival rate of grafts with fig. 5a-c: wood structure of the connecting zone between the root stock of the phenotype “cinza” and the bud of (a) the phenotype “cinza”, (b) the pheno type “sassafras”, and (c) the phenotype “amarelinha” six months after grafting. ft = fibre tracheids, pl = protective layer, v = vessel. scale bar = 200 μm. tab. 3: xylem sap flow (l cm-2 day-1) in the root stocks and in the buds of grafted maté plants produced from different combinations of root stocks and buds of the phenotypes “amarelinha”, “cinza”, and “sassafras” in the (a) first and in the (b) second month after grafting. five plants per treatment. (a) buds phenotype amarelinha cinza sassafras root stock bud root stock bud root stock bud root stocks amarelinha 0.012±0.004 0.043±0.009 0.007±0.003 0.023±0.009 0.009±0.006 0.029±0.008 cinza 0.004±0.001 0.039±0.010 0.002±0.001 0.025±0.007 0.005±0.002 0.024±0.011 sassafras 0.007±0.003 0.032±0.011 0.003±0.001 0.030±0.011 0.008±0.002 0.031±0.010 (b) buds phenotype amarelinha cinza sassafras root stock bud root stock bud root stock bud root stocks amarelinha 0.060±0.003 0.062±0.005 0.011±0.004 0.017±0.006 0.017±0.005 0.028±0.006 cinza 0.009±0.002 0.022±0.003 0.036±0.006 0.037±0.007 0.022±0.005 0.029±0.010 sassafras 0.061±0.009 0.057±0.004 0.032±0.003 0.029±0.003 0.049±0.008 0.048±0.007 66 o. dünisch, b. reissmann root stocks or buds of the phenotype “sassafras”. due to the lower quality of the leaves for maté production and the low vulnerability of the water conducting system under drought conditions, the phenotype “sassafrass” seems to be most suitable for the production of universal root stocks in grafts. in addition, the results also show that vessel member length is the best wood anatomical indicator for the prediction of the compatibility of cuttings from different maté varieties in grafts. in highly compatible grafts the water cohesion between root stocks and buds was restored by the formation of a high number of connecting vessel elements. this requires almost identical vessel characteristics in the root stock and the bud. in low compatible grafts (e.g. phenotypes “amarelinha”/”cinza”) only fibre tracheids (no significant differences between phenotypes) are suitable connecting elements for the restoration of the water transport between the root stocks and the scions. however, due to the lower water conductivity of fibre tracheids compared to vessels the buds of these grafts suffer from a restricted water supply (tyree and ewers, 1991; tyree and zimmermann, 2002). this was confirmed by the xylem sap flow measurements below and above the grafting zone, indicating a high transpiration of the buds and a very low water flux from the root stock to the scion. according to shigo (1984) the observed formation of a “protective layer” in the root stock of incompatible grafts is considered to compartimentalize the root stock from the incompatible bud favoring the chance for the formation of coppice shoots in the root stocks. the balanced water flux in the root stocks and the buds of compatible grafts in the second month of growth indicates that in these grafts the restoration of the water conducting system of the secondary xylem is very fast and lasts between a couple of days and 5 weeks as a maximum. acknowledgements we thank the cnpq/capes, brasilia, and the german academic exchange service (daad), bonn for financial support. furthermore we express our deep gratitude to afonso oliszewski and dalnei neiverth, ervateira bitumirim for providing plant material and for sample collection. the improvement of the manuscript by the unknown reviewers is especially appreciated. references baas, p., 1973: the wood anatomical range in ilex (aquifoliaceae) and its ecological and phylogenetic significance. blumea 21, 193-259. baas, p., 1975: vegetative anatomy and the affinities of aquifoliaceae, sphenostemon, phelline, and oncotheca. blumea 22, 312-411. becker, p., tyree, m.t., tsuda, m., 1999: hydraulic conductances of angiosperms versus conifers: similar transport sufficiency at the wholeplant level. tree physiol. 19, 445-452. coelho, g.c., mariath, j.e.a., schenkel, e.p., 2002: populational di versity on leaf morphology of mate (ilex paraguariensis st. hil., aquifoliaceae). braz. arch. biol. technol. 45, 47-51. dünisch, o., morais, r., 2002: regulation of xylem sap flow in a deciduous, a semi-deciduous, and an evergreen meliceae species of the amazon. trees 16, 404-416. dünisch, o., reissmann, c.b., oliszeski, a., 2004: variability of vessel characteristics in the xylem of ilex paraguariensis st. hil. (mate-tree) from south brazil. iawa j. 25, 449-458. dünisch, o., fladung, m., nakaba, s., watanabe, y., funada, r., 2006: influence of overexpression of a gibberelin 20-oxidase gene on the kinetics of xylem cell development in hybrid poplar (populus tremula l. x p. tremuloides michx.). holzforschung 60, 608-617. erbreich, m., 1997: untersuchungen zur xylemwasserleitung ausgewählter tropischer wirtschaftsbaumarten in zentralamazonien. master thesis, university of hamburg. espen, l., cocucci, m., sacchi, g.a., 2005: differentiation and functional connection of vascular elements in compatible and incompatible pear/ quince internode micrografts. tree physiol. 25, 1419-1425. granier, a., 1985: une nouvelle methode pour la mesure de flux de seve brute dans le tronc des arbres. annales des sciences forestières 42, 193200. granier, a., 1987: mesure du flux de seve brute dans le tronc du douglas par une nouvelle methode thermique. ann. sci. for. 44, 1-14. höster, h.r., spring, c., 1971: zur bestimmung der zellartenanteile im holzgewebe. mikroskopie 27, 220-225. kawakami, m., kobayashi, a., 1991: volatile constituents of green mate and roasted mate. j. agric. food chem. 39, 1275-1278. martinez, m.a.p., peletto, j.p., basualdo, n., 1997: distribution of flavonoid aglycones in ilex species (aquifoliaceae). biochem. syst. ecol. 25, 619-622. moore, r., 1983: vegetative compatibility responses in plants. baylor university press, 89-105. ohem, n., hölzl, j., 1990: der mate – eine genußund heilpflanze aus dem mittleren südamerika. pharm. ztg. 41, 2737-2746. reissmann, c.b., radomski, m.i., quadras, r.m.b, 1999: chemical composition of ilex paraguariensis st. hil. under different management conditions in seven localities of paraná state. braz. arch. biol. technol. 42, 187-194. reissmann, c.b., dünisch, o., boeger, m.r., 2003: beziehung zwischen ernährungsbiologischen (fe, mn, ca) und strukturellen merkmalen ausgewählter morphotypen der mate-pflanze (ilex paraguariensis st. hil.). in: hüttl, r.f. (ed.), boden, wald und wasser, 146-171. shaker verlag, aachen. rosse, l.n., fernandes, j.s.c., 2002: choice of traits for genetic improvement in erva-mate by means of multivariate techniques. ciênc. florestal 12, 21-27. scherer, r., urfer, p., mayol, m.r., belingheri, m.l., marx, f., janssens, m.j.j., 2002: inheritance studies of caffeine and theobromine content of mate (ilex paraguariensis) in misiones, argentina. euphytica 126, 203-210. shigo, a.l., 1984: compartmentalization: conceptual framework for understanding how trees grow and defend themselves. phytopathol. 22, 189-214. sperry, j.s., ikeda, t., 1997: xylem cavitation in roots and stems of douglas-fir and white fir. tree physiol. 17, 275-280. tyree, m.t., ewers, f.w., 1991: the hydraulic architecture of trees and shrubs and other woody plants. new phytol. 119, 345-360. tyree, m.t., zimmermann, m.h., 2002: xylem structure and the ascent of sap. springer verlag berlin. valduga, e., freitas, r.s., reissmann, c.b., nakashima, t., 1997: caracterização química da folha de ilex paraguariensis st. hill. (ervamate) e de outras espécies utilizadas na adulteração do mate. ceppa 15, 25-36. wang, y., kollmann, r., 1996: vascular differentiation in the graft union of in vitro grafts with different compatibility. structural and functional aspects. j. plant physiol. 147, 521-533. watanabe, y., kojima, y., ona, t., asada, t., sano, y., fukazawa, k., funada, r., 2004: histochemical study on heterogeneity of lignin in eucalyptus species ii. the distribution of lignins and polyphenols in the walls of various cell types. iawa j. 25, 283-295. addresses of the authors: oliver dünisch, meisterschule ebern für das schreinerhandwerk, gleusdorfer straße 14, 96106 ebern, germany e-mail: duenisch@meisterschule-ebern.de bruno reissmann, universidade federal do parana, setor de ciencias agrarias, dept. de solos e engenharia agricola, rua dos funcionarios 1540, 80035-050 curitiba-pr, brazil art13 journal of applied botany and food quality 81, 175 177 (2007) 1 institut für allgemeine botanik und pflanzenphysiologie, ag spezielle botanik, justus-liebig-universität gießen 2 institut für biochemie und biologie, ag biozönoseforschung/spezielle botanik, universität potsdam 3 institut für spezielle botanik, friedrich-schiller-universität jena 4 institut für allgemeine botanik und pflanzenphysiologie, friedrich-schiller-universität jena small scale analysis of population structure in the woody cornelian cherry cornus mas l. (cornaceae) by aflp accentuates the need for a population based conservation strategy v. wissemann*1, h. baumbach2, s. müller3, y. venus4, f.h. hellwig3 (received august 29, 2007) summary we investigated population differentiation among and within three populations (two natural, one artificial) of the cornelian cherry (cornus mas l., cornaceae) to examine the extent of gene flow from planted cornelian cherries commonly used in planting vegetations of public parks or streets into natural stands. additionall we assessed if natural populations show any intrapopulational and/or interpopulational differentiation pointing towards restricted gene flow with possible necessity for a population based conservation strategy rather than a taxon based strategy. results clearly indicated within and between population structure a radius of isolation by distance for pollen and seed dispersal of about 5.0 km. interestingly genetic distance did not support coherence of the two natural populations but mirrored the historical origin of the innertown population from diverse natural sources reflecting the traditional use and selection of edible varieties from nature. the nem value of 1.25 implicates the prevention of population differentation. however the low level of genetic diversity and distance at all might mislead the interpretation and the degree of distance reflects more ancient similarities than actual geneflow. given this observable isolation by distance, conservation biology of cornus mas requires a population based strategy rather than a broad taxon based strategy. introduction introgression of non autochthonous genes from introduced plants of different origin into local genepools is one of the major issues in conservation biology. here we assess the population structure of the cornelian cherry cornus mas l. (cornaceae) at two natural stands in thuringia and compare the genetic diversity with an artifical population based on samples of garden and town origin from jena. the primary question to which no information for cornus exists is if there is gene flow between populations and to what extent introgression from planted cornelian cherries, commonly used in planting vegetations of public parks or streets, can be detected. there is no information available if natural populations show any intrapopulational and/or interpopulational differentiation pointing towards restricted gene flow with possible necessity for a population based conservation strategy rather than a taxon based strategy. natural populations of the mainly south-east european and asia minor distributed species (hegi, 1927) cornus mas are rare in germany, the thuringian populations are of special interest because they might be outposts at the warm and dry limestone slopes of the saale valleys and have been explicetly mentioned in the post-renaissance herbals e.g. by zwinger (1744) and since then (hegi, 1927). as an early pasture for bees and a traditional used edible fruit cornus mas is an important element of the native flora (bartels, 1993; friedrich and petzold, 1993). around jena, cultural history reflects heavily on cornus mas, for a long time hiking poles and batons for student leagues, the so called „ziegenhainer“ were made in the small village ziegenhain next to jena and distributed through germany (traeger, 1993). the populations we studied were an artificial one from plants grown in the town jena, a population from wöllnitz thought to be used for the production of the ziegenhainer poles and samples from a large natural population from the conservation area of kunitz, 5 km north of jena. we assessed within and between population diversity and tied the results to the questions of distance of geneflow among populations, introgression of non-autochthonous gene pools into natural stands either by pollination or planting and dispersal abilities of pollen and fruits/seed. the guiding hypothesis assumes, that the two natural populations are more similar to each other than the artificial population and that the genetic diversity of the artificial town population is higher due to its diverse origin than the natural populations, reflecting the traditional use and selection of edible varieties. material and methods sampling and dna isolation in 2004 ninety plants were sampled from the three thuringian populations around jena: kunitz, wöllnitz, and jena town (30 plants per population). geographic distance of kunitz is 5.0 km north of jena, wöllnitz is about 2.5 km west of jena. plant material was dried in silica gel, ground with sterile sea sand and stored at -20 °c until dna extraction. total genomic dna was isolated using the qiagen (hilden, germany) plasmid mini kit following the protocol of hellwig et al. (1999). extracted dna was quantified photometric at 260 nm. aflp analysis the complete aflp procedure can be taken from vos et al. (1995) but we used irdlabelled primers for the selective pcr amplification and an automated dna sequencer as described in baumbach (2005). 35 primer combinations were screened and checked for band polymorphism, band brightness, band/background contrast, number of amplification products and reproducibility of band patterns. four primer combinations have been selected for aflp analysis: i) ecoriaac/msei-cac (44 loci, 37-482 bp), ii) ecori-aac/msei-cct (37 loci, 36-471 bp), iii) ecori-aag/msei-cgt (38 loci, 48-449 bp), iv) ecori-aag/msei-ctt (53 loci, 34-474 bp). they yielded a total of 172 well-amplified and highly reproducible polymorphic bands. it was possible to distinguish all 90 sampled plants as separate aflp phenotypes. all selective pcr amplifications were performed twice. only bands which could be detected both times unequivocally were used for further analyses. data analyses aflp patterns were compiled into a 0/1-matrix (state „0“ for absence, „1“ for presence of a band) using the „saga generation 2“ software (li-cor biosciences, bad homburg, germany). bands of 176 v. wissemann, h. baumbach, s. müller, y. venus, f.h. hellwig equal fragment sizes were interpreted as homologous. different staining intensities of homologous bands were not interpreted as different bands. the partitioning of genetic variance within and among populations was assessed by an analysis of molecular variance (amova, implemented in arlequin ver. 2.000, schneider et al. 2000). the fixation index φst (in analogy to fst) was calculated with the arlequin software to estimate gene flow [(nem=¼(1/φst-1)] between populations. principal coordinates analysis was calculated with ntsyspc ver. 2.11a (exeter software, setauket, usa) using a distance matrix based on the band similarity coefficient (lynch 1990). percentage of average heterozygosity, and percentage of polymorphic loci (calculated with tfpga; miller, 1997) were used as parameters for description of genetic variability. heterozygosity was calculated under the assumption of hardy-weinberg equilibrium (band state „0“ is counted as homozygous recessive genotype). results analysis of molecular variance (amova) genetic variation within populations exceeds variation among populations clearly (17 % vs. 83 %). the corresponding φst-value is 0.167. under the assumption of wright’s island model gene flow between populations is nem=¼(1/φst-1)=1.25 (tab. 1). tab. 1: results of amova computation: source of variation (sv), degrees of freedom (df), sum of squares (sq), variance components (vc), and fixation index (φst). source of variation: among populations (ap), within populations (wp). level of significance (based on 1023 iteration steps): ***p < 0.001. sv df sq vc variation φ nem ap 2 329.58 4.71 va 16.7 % wp 87 2043.47 23.49 vb 83.3 % φst=0.167*** 1.25 total 89 2373.05 28.20 parameters of genetic variability analysis of genetic variability revealed the highest level of variability in the artificial jena town population, supporting the view of multiple and diverse origin. wöllnitz again exhibits significant genetic diversity contributing to our assumption of wöllnitz as a population with human impact by economic use and potential introduction and cultivation of useful genotypes. kunitz shows the lowest average heterozygosity indicating a viable but isolated natural population. tab. 2: genetic variability parameters of the examined cornus mas l. populations. population sample size average polymorphic heterozygosity loci 1 (kunitz) 30 25.2 % 79.7 % 2 (wöllnitz) 30 29.0 % 89.0 % 3 (jena) 30 33.0 % 93.6 % total 90 33.7 % 100 % the pco-analysis reveals a highly scattered artificial population due to the multiple and diverse origin of the samples. a similar degree is found in the wöllnitz population with a slight substructering maybe due to planting. kunitz is represented as an entire population (with a few exception). due to the low level of genetic differentiation at all, we interpret the coherence of the kunitz population as a sign of isolation by distance from the remainder populations. genetic distances in general genetic distances (nei, 1972) among all three populations are low. wöllnitz (2) and jena (3) are more similar to each other than to kunitz (1). kunitz (1) and wöllnitz (2) are less similar to each other indicating isolation between the two assumed natural populations wöllnitz and kunitz. genetic distances between wöllnitz-jena and kunitz-jena support the view of the town population being originated from collected samples from natural stands (more from wöllnitz, less from kunitz) tab. 3: nei’s original (1972) distance (1) kunitz, (2) wöllnitz, (3) jena population 1 2 3 1 ***** 2 0.106 ***** 3 0.088 0.057 ***** discussion our guiding hypothesis assumed, that the two natural populations are more similar to each other than the artificial population and that the genetic diversity of the artificial town population is higher due to its diverse origin than the natural populations, reflecting the traditional use and selection of edible varieties. the data corrects this hypothesis partially. indeed, the genetic diversity of the artificial town population is higher than the within population differentiation of the naturals stands. this diversity mirrors the historical development of the town population from multiple introductions, either from commercially grown cornelian cherries or from transplantations from natural sources into the homes garden. pco analysis indicates, that some of the genotypes can not be distinguished from the wöllnitz population, pointing towards transplantation of tasty edible varieties from nature principal coordinates analysis fig. 1: principal coordinates plot of 90 cornus mas individuals for the first two principal coordinates estimated with 172 aflp markers. variation explained: 73.8 % (pco i: 49.7 %, pco ii: 24.1 %). population structure in cornus mas l. (cornaceae) 177 into the town (preferred) or vice versa to produce useful wood for the production of the ziegenhainer (less likely). what is not supported is our expectation, that the two natural stands are more similar to each other than to the town population. wöllnitz (2) and jena (3) are more similar to each other than to kunitz (1). kunitz (1) and wöllnitz (2) are less similar to each other indicating isolation between the two assumed natural populations wöllnitz and kunitz, but gene flow between town and wöllnitz. the unity of the population in kunitz reflects its isolated position. obviously the distance of 5 km from jena and wöllnitz prevents more or less the pollen transfer by bees or seed/fruit transport although bartels (1993) reports endozooic seed dispersal by birds. still, analysis of pollen fertility as a dimension of fitness does not indicate any fitness decrease within and between populations (müller, 2004; nohlen, 2007). the nem value of 1.25 implicates the prevention of population differentiation. however the low level of genetic diversity and distance at all might mislead the interpretation and the degree of distance reflects more ancient similarities than actual geneflow. given this observable isolation by distance, conservation biology of cornus mas requires a population based strategy rather than a broad taxon based strategy. acknowledgements we like to thank the thuringian landesverwaltungsamt, weimar, t. rohde for the allowance to collect plant material from the conservation area „nsg 149 grosser gleisberg“ letter 7.7.2003, az: 601.12-8512.05-149.shk-03.001601.1-ro. references bartels, h., 1993: gehölzkunde. einführung in die dendrologie. e. ulmer, stuttgart. baumbach, h., 2005: genetische differenzierung mitteleuropäischer schwermetallsippen von silene vulgaris, minuartia verna und armeria maritima unter berücksichtigung biogeographischer, montanhistorischer und physiologischer aspekte. dissertationes botanicae 398. borntraeger, stuttgart. friedrich, g., petzold, h., 1993: obstsorten. 300 obstsorten in wort und bild. neumann, radebeul. hellwig, f., nolte, m., ochsmann, j., wissemann, v., 1999: rapid isolation of total cell dna from milligram plant tissue. haussknechtia 7, 29-34. lynch, m., 1990: the similarity index and dna fingerprinting. mol. biol. evol. 7, 478-484. miller, m., 1997: tools for population genetic analysis (tfpga) 1.3: a windows program for the analysis of allozyme and molecular population genetic data. department of biological sciences – northern arizona university. müller, s., 2004: die variabilität von cornus mas l. im mittleren saaletal. magisterarbeit, institut für spezielle botanik, fsu jena. nei, m., 1972: genetic distance between populations. amer. naturalist 106 (949), 283-292. nohlen, f., 2007: untersuchungen zur reproduktionsbiologie von cornus mas l. und cornus sanguinea l. staatsexamensarbeit, institut für spezielle botanik, fsu jena. traeger, i., 1993: von lichtenhainer kännchen und ziegenhainer stöcken. burschenschaftliche blätter 3, 136-137. vos, p., hogers, r., bleeker, m., reijans, m., van de lee, t., hornes, m., friters, a., pot, j., paleman, j., kuiper, m., zabeau, m., 1995: aflp: a new technique for dna fingerprinting. nucleic acids res. 23, 4407-4414. schneider, s., roessli, d., excoffier, l., 2000: arlequin ver. 2.000. university of geneva. zwinger, t., 1744: theatrum botanicum. j. bischoffs, basel. address of the authors: volker wissemann (corresponding author), institut für allgemeine botanik und pflanzenphysiologie, ag spezielle botanik, justus-liebig-universität gießen, senckenbergstr. 17, d-35390 giessen (volker.wissemann@bot1.bio.uni-giessen.de) henryk baumbach, institut für biochemie und biologie, – biozönoseforschung / spezielle botanik –, universität potsdam, maulbeerallee 1, d14469 potsdam sylvia müller, frank h. hellwig, institut für spezielle botanik, friedrichschiller-universität jena, philosophenweg 16, d-07743 jena y. venus institut für allgemeine botanik und pflanzenphysiologie, friedrichschiller-universität jena, dornburger straße 159, 07743 jena << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false /maxsubsetpct 100 /optimize true /opm 1 /parsedsccomments true /parsedsccommentsfordocinfo true /preservecopypage true /preserveepsinfo true 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/pagesize [907.087 680.315] >> setpagedevice vorgabe neu journal of applied botany and food quality 80, 116 128 (2006) institut für pflanzenökologie, justus-liebig-universität giessen plant functional types and elevated co2: a method of scanning for causes of community alteration u. grüters*, s. janze, c. kammann, h.-j. jäger (received april 28, 2006) summary in this paper, a general method for an a posteriori plant functional type (pft) analysis of global change effects on community composition is developed. we apply the method to a case study, specifically the giessen-face experiment. this experiment involves a central european meadow that has been exposed to moderate co2-enrichment since may 1998. the method for an a posteriori pft-analysis: the method consists of four working steps and uses a combination of standard gradient analysis and random forests (rf). (1) the trait composition of the species is studied using principal components analysis. species trait information is gathered from databases. natural pft, i.e. groups of species with similar trait-sets, are identified specifically for the community under study. (2) a ranking of the species according to standardized/absolute co2 abundance response is obtained from redundancy analysis. initially, species with a response above or below the median are grouped into three response groups (rg) each having similar behaviour, i.e. positive/negative or no-response. (3) the outlyingness measure of rf is used to shift rg boundaries until satisfactory rg homogeneity is achieved. rf is utilized to find the best traits for the rg classification. the behaviour of species representative of the rg is derived from rf class centers. (4) from knowledge gained in steps 1-3, hypotheses about the causes underlying the community alteration are built. strengths/weaknesses of the method are discussed. application of the method to the case study: the community consists of three natural pft. five species are summer-green forbs of varying competitiveness. four species are evergreen ruderal forbs characterized as (semi-) basal rosette plants. the third natural pft contains evergreen, more or less competitive species, mostly grasses, but also a few forbs. negative standardized co2-response was practically restricted to two natural pft, i.e. the summer-greens, irrespective of their competitiveness, and the evergreen ruderals. standard positive response covered part of the evergreen competitive natural pft. among them was glechoma hederacea, one of the forbs with the greatest similarity to grasses. two hypotheses were formulated to explain the response pattern: (1) summer-greens lost in competition with evergreens, because the annual time-integral they can use for enhanced growth was more limited with year-round co2-enrichment. (2) as rosette plants, ruderal evergreens lagged behind evergreen competitors because periods with full sunlight, which enabled them to gain additional carbon, were shorter for them. absolute responses were additionally dependent on dominance patterns. the most striking difference to standard responses was the restriction of positive response to (sub-)dominant grasses. introduction a wide range of species-abundance responses to elevated co2 has been reported for free air co2-enrichment (face) experiments conducted on grassland ecosystems (campbell et al., 2000; körner, 2000; nowak et al., 2004). often, pre-defined or so-called a priori plant functional types (pft) have been used to summarize results of biomassor abundance-shifts at the species level. the most frequently deployed classification for temperate grassland communities is based on tradition or on taxonomy (grasses and forbs, lüscher et al., 1996; vasseur and potvin, 1998; edwards et al., 2001; marissink and hansson, 2002). this classification encompasses two problems. firstly, in many experiments no definitive response was detected for these groups, e.g. grasses in canadian pasture (vasseur and potvin, 1998), swedish grassland (marissink and hansson, 2002), and c3 grasses in the short-grass steppe (morgan et al., 2004). secondly, the membership in one of the groups does not comprise per se an explanation for contrasting behaviour. as a consequence, the usefulness of this classification has been questioned (teyssonneyre et al., 2002). however, the concept of pft has recently undergone major advancements. in the novel conceptual framework, the first step of pft analysis is experimental manipulation of a global change factor and the a posteriori identification of response groups (rg), each consisting of species with similar behaviour (chapin et al., 2000; chapin et al., 2002; díaz et al., 2002; lavorel and garnier, 2002; chapin, 2003). in the classification of response groups, species traits play a pivotal role. distinct species trait-sets are assumed to cause the species to respond differently (lavorel and garnier, 2002; chapin et al., 2002). in our opinion, a posteriori pft analysis has the potential to overcome the predicament of elevated co2 research summarized by körner (2000) as „it is possibly one of the greatest frustrations of researchers in recent years that no reliable functional groups with respect to co2responsiveness could be identified“. the aim of this paper is to develop a general method for an a posteriori pft analysis of global-change effects on plant community composition. the method characterizes the trait composition of the initial plant community, provides a ranking of species responses to the global change factor, identifies response groups and scans for species traits which caused the detected community alteration. it combines standard gradient-analysis techniques and random forests (rf) (breiman, 2001), the 21st century advancement to classification and regression trees (cart). the method is applied to an elevatedco2 case study, namely the giessen-face experiment – a long-term ecosystem study, in which a central-european old meadow has been exposed year-round to moderate co2-enrichment (20% above ambient level). 1. description of the giessen-face case study 1.1 experimental site the grassland research area is situated on the floodplain of a river near giessen, germany (at 50°32’n; 8°41.3’e, 172 m above sealevel). due to the floodplain location the soil is fluvic gleysol with a texture of sandy clay loam over a clay layer (fao classification). between 1996 and 2001 the site received a mean annual precipitation of 573 mm and the average annual air temperature amounted to 9.2 °c. for at least 50 years the grassland on this area has been managed as a meadow, mown twice a year. over the whole time the area was fertilized with nitrogen at a rate of 50 80 kg n ha-1 a-1. this management regime was maintained after the establishment of the research station in 1993/1994. however, the nitrogen supply in the years 1993-1995 (80 kg n ha-1 a-1) was reduced to 40 kg n ha-1 a-1 in all years following. each spring, 600 kg ha-1 a-1 of 10% p2o5 + 15% k2o + 3% mgo; 33% cao + mgo were supplied to the site. throughout a pre-experimental phase (1993-1996), soil moisture, composition of the vegetation and above-ground biomass at harvest were monitored. the monitoring revealed that the area was under the influence of a soil moisture gradient. these results enabled to choose locations with equal baseline conditions for the replicate rings. 1.2 experimental design in 1997 three face ring pairs, resembling mid-face size, were installed along the detected soil moisture gradient (ambient co2 (a) and elevated co2 (e) combined with „dry“ (a1, e1), „intermediate“ (a3, e3) and „wet“ soil conditions (a2, e2)). the co2-enrichment commenced in april 1998. since then, the enrichment has been in daily operation throughout the entire year from 2 h after sunrise to 2 h before sunset. the exposure system allows for relative increment of ambient co2 levels with resembling the diurnal cycle in the ambient. the co2 concentration was set at 20% above ambient levels to simulate concentrations expected to be reached within the next 25 40 years (houghton et al., 2001). the soil moisture was measured once daily with four permanentlyinstalled tdr sensors in each co2 ring (imko, type p2g; vertically inserted in 0 15 cm depth). the four annual means of soil moisture were pooled to give an average for the ring. further experimental details can be found elsewhere (jäger et al., 2003; kammann et al., 2005). 1.3 sampling the plant community the vegetation on the research site is an arrhenatheretum elatioris (br.-bl.) filipendula ulmaria sub-community (rodwell et al., 1992). species richness is moderate on the site and amounts to 16 species on 4.5 m² plots and 35 species on 100 m² plots, respectively. the species-area relation can be described best by the exponential model: number of species = 18.462 log10 (area) – 2.3742 (r² = 0.9062) where area denotes the plot size in m². the dominant species are the grasses arrhenatherum elatius (l.) and holcus lanatus (l.) along with the forb galium mollugo (l.). the abundance of the species in the community was estimated with twice-annual vegetation surveys, preceding each summer (sum: may 28 until june 16) and autumn harvest (aut: august 25 until september 11) by one week. the surveys took place on two 4.5 m² permanent plots located in the northern and southern half of each ring. %coverages for the species were estimated with the relevé method. the values were pooled to give ring averages (3 replicates of co2treatment). species having a constancy of less than 10% among plots were excluded from further analysis. since the original vegetation data held units of %-coverage it had to be logarithmically transformed. by this means a model based on the ecologically more meaningful multiplicative scale could be constructed (lepš and šmilauer, 2003). multivariate analysis of pre-experimental data revealed no significant differences in species composition between plots selected later for a and e treatments, respectively. fig. 1: an outline of the method for an a posteriori pft analysis: for further explanation see text. abbreviations: pca = principal components analysis, rda = redundancy analysis, rf = random forests plant functional types and elevated co2 117 2. the method for an a posteriori pft analysis an outline of the method for the a posteriori pft analysis of global change effects is given in fig. 1. basically, the method consists of four working steps. step 1 uses information from species traitbases and a combination of principal components analysis (pca, ter braak and šmilauer, 1998) and random forests (rf, breiman, 2001) to characterize the trait composition of the species and to identify groups of species with similar trait-sets, hereafter called natural pft. step 2 provides a ranking of species responses to the global change factor from partial redundancy analysis (rda, ter braak and šmilauer, 1998). we distinguish two types of ranking. the first is based on standardized species responses to remove effects of varying initial abundance, while the second is obtained from absolute shifts in species abundance. in steps 3 and 4 the random forests algorithm is used to identify homogeneous response groups and to scan for possible causes of community change by an examination of the most important traits for the classification of response groups. the entire gradient analysis (pca and rda) was conducted with canoco 4.5 (ter braak and šmilauer, 1998). random forests were calculated with the similarly-named r-package (liaw and wiener, 2006), which is a translation of breiman’s and cutler’s original fortran code (breiman and cutler, 2006) to r. additionally, in an explorative phase, the partition platform of jmp 5.0.1 (sas institute inc., nc, usa) was used to calculate cart. the following sections 2.1 2.4 describe the working steps in more detail, while the respective sections 3.1 3.4 apply the working steps to the case study. in principle, the discussion resembles this scheme by first dealing with the method and then with the application. 2.1 characterizing the trait composition of the species a pca was conducted to examine the trait composition of the species present in the community. the necessary species trait information was gathered from databases (fitter and peat, 1994; hodgson et al., 1995; kleyer, 1995). the life history traits included are shown in tab. 1. in a few instances missing values were supplemented from schubert and vent (1990) and oberdorfer (1990). leaf nitrogen concentrations were taken from a field survey made in the uk (thompson et al., 1997) and from the glopnet database (wright et al., 2004). with a total of 24 traits, in part categorical and in part numerical variables, a relatively large trait number was incorporated. a core set of six traits was chosen based on the emphasis these traits were given in the co2 literature (seedling growth rate, competitiveness, ruderality, stress tolerance, leaf n-concentration, root type), but many more traits previously not known to be influential were also included. traits that had missing values for more than 8 of the 27 species (<33%) were excluded from the analysis. for the remaining traits, missing values were imputed iteratively from species proximities using random forests (see section 2.3). this procedure was not suitable for traits with more than 33% missing values, since the imputation integrates the effects of all other traits and, hence, imputed traits with large numbers of missing values tended to become „super“-predictors in steps 3 and 4. the linear pca was chosen, because detrended correspondence analysis (dca) revealed that trait composition varied only moderately among species. before conducting the pca, categorical variables had to be replaced by a set of dummy variables for each category (with 0 = category absent, 1 = category present). pca results were centered and standardized by traits to remove effects resulting from the different units of the traits. the resulting correlation biplot (fig. 2) provides a visualization of the relationships of interest. as usual, correlations can be assessed from the ordination diagram using the biplot rule (lepš and šmilauer, 2003). in principle, the ordination diagram elucidates similarities among the trait composition of the various species and sheds light on relationships among the traits. by this means, it is possible to identify groups of species with similar trait-sets, i.e. natural pft. 2.2 species ranking according to standardized/absolute co2-response we distinguish between a species ranking according to standardized co2-response and one that is founded on absolute abundanceshifts due to the co2-treatment. the working „sub-“steps for obtaining these two types of species ranking are detailed in the following: a) dca: with a gradient length of 1.264 (= average standard deviation of species turnover, ter braak and šmilauer, 1998) the species composition varied only moderately among plots and, hence, the linear method rda was chosen for direct gradient analysis of the ln-transformed %-coverage data. b) redundancy analysis (rda) and empirical modeling: all explanatory variables considered to affect the community were included in an empirical model (soil moisture, year and season of vegetation survey, co2, co2 x soil moisture, co2 x year, co2 x season). the model structure took into account only co2-effects beyond existing plot differences. the co2-treatment of the year prior to the onset of enrichment (yr0 = 1997) was coded as 0 for all plots, though (before-after-control-impact = baci-design, ter braak and šmilauer, 1998). this concept particularly allowed for separate statistical testing of co2-effects and co2 x time interactions. assuming cyclical behaviour of the species data, experimental time was treated by separating year (yr0..yr4) and season of the vegetation survey (sum, aut), which represents the most general treatment of time (ter braak and šmilauer, 1998). annual averages of soil moisture were assigned to the data of the corresponding year irrespective of the season in which the vegetation survey was made. treatment of soil moisture as a variable that changes with the year has important implications: time effects did not include those of fluctuating soil moisture and co2 x soil moisture interactions did not contain effects of co2-induced water savings. this was not a problem, since tdr sensor data did indicate that elevated co2 induced neither prolonged water savings nor a general shift in soil moisture (kammann et al., 2005). c) statistical testing: the statistical analysis was conducted in the usual way (full model, main effects, interactions, lepš and šmilauer, 2003). statistical testing was performed by distribution-free monte-carlo permutations that were restricted according to the experimental design (only whole-plots freely exchanged). to additionally increase the power of the test, permutations were extended to the split-plot level (alleviated test: cyclic shifts, dependent across whole-plots). this sort of statistical test has also been used by verdonschot and ter braak (1994) and by ter braak and šmilauer (1998). d) partial rda and ranking of standardized species responses: in the partial rda the effects of all significant explanatory variables were partialled out as covariables, leaving co2 as the only explanatory variable. the species scores along the first ordination axis resulting from the analysis of absolute species composition (samples not standardized by norm, species centered and standardized) reflect standardized co2-responses, that are neither dependent on the initial species abundance in the community (lepš and šmilauer, 2003) nor on the transformation of the original data. 118 u. grüters, s. janze, c. kammann, h.-j. jäger tab. 1: list of generative and vegetative traits used in the a posteriori pft analysis. trait abbr src categories scale, var type 1. generative traits a. traits concerning individual reproduction time of 1st reproduction trep 2 6 (of 10) classes, mths, yrs (03,1,2,3,6,nd) ord, num flowering start flows 1 5 (of 12) classes, month (3,4,5,6,7) ord, num flowering period flowp 1 7 (of 12) classes, months (1,2,3,4,5,8,12) ord, num no. of diaspores/shoot num 2 3 (of 4) classes, counts (1, 2, nd) ord, num b. traits concerning mating system/pollination pollination type pol 2 4 (of 6) classes (ins = insects, wind = wind, apo = apomixis, nd) nom, cat c. traits concerning dispersal in space and time dispersule weight dispw 1 4 (of 7) classes, mg, (2,3,4,5) ord, num dispersule form dispf 1 3 (of 7) classes (fr =fruit, sd = seed, nd) nom, cat agency of dispersal ad 2 4 (of 4) classes(anim = animals ,wind = wind, nom, cat aqu = water,unsp = unspecific) seed bank longevity seedb 1 4 (of 4) classes (1,2,3,nd) ord, num d. traits concerning germination and establishment seasonal time of germination gs 2 7 (of 10) classes (early = early spring, sprin = spring, nom, cat easum = early summer, sum = summer, aut = autumn, shed, nd) seedling growth rate sgr 3 species-specific maximum values, g g -1 d -1 ord, num 2. vegetative traits a. traits concerning aboveground vertical expansion shoot height canh 1 5 (of 10) classes , mm, (1,2,3,4,5) ord, num canopy structure cans 1 3 (of 5) classes (b = basal rosette, s = semi-basal rosette, l = leafy) nom, cat leaf size size 2 5 (of 8) classes, cm2, (1 = lep = leptophyllous, 2 = nan = nanophyllous, ord, num 3 = mic = microphyllous, 4 = mes = mesophyllous, nd) leaf phenology leafp 1 4 (of 5) classes (sa = seasonal aestival, sh = seasonal hibernal, nom, cat ep = evergreen partially, ea = evergreen always) leaf n concentration leafn 5, 6 species-specific values , mg g -1 ord, num b. traits concerning belowground expansion root type rt 2 7 (of 10) classes (1tube = tuber root, tuss = tussock roots, nom, cat rhiz = rhizomes, stol = stolons, elon = elongated, deep = deep rhizomes, nd) rooting depth deep 2 3 (of 4) classes, m, (0.5, 1, 1.5, nd) ord, num mycorrhiza type myc 1 4 (of 7) classes (va = vssicular-abuscular, pos = positive, nom, cat neg = negative, nd) c. traits concerning life history, life form, lateral expansion, csr-status life history lifeh 1 3 (of 6) classes (as = summer annual, aw = winter annual, p = perennial) nom, cat life form lifef 1 3 (of 8) classes (th = therophytes, ch = chamaephytes, nom, cat h = hemikryptophytes) lateral spread latsp 1 5 (of 6) classes, mm (1,2,3,4,5) ord, num branching bra 2 4 (of 5) classes (no = without branching, akro = akrotony, nom, cat bas = basitony, nd) competitiveness c-radius 2, 4 4 (of 5) classes (-2, -1, 0, 1) ord, num stress-tolerance s-radius 2, 4 4 (of 5) classes (-2, -1, 0, 1) ord, num ruderality r-radius 2, 4 5 (of 5) classes (-2, -1, 0, 1, 2) ord, num src = source: 1 = hodgson et al. (1995), 2 = kleyer (1995), 3 = fitter and peat (1994), 4 = hodgson et al. (1999), 5 = thompson et al. (1997), 6 = wright et al. (2004) plant functional types and elevated co2 119 e) ranking of absolute species responses: to obtain absolute abundance shifts due to co2 the following calculations were performed (ter braak and šmilauer, 1998): spec_abundance = mean_coverage + mean_coverage * (exp(b_spec_ax1 * c_co2_ax1 * co2_std + b_spec_ax2 * c_co2_ax2 * co2_std)-1)/10 where spec_abundance denotes the %-coverage of either ambient or elevated co 2 -treatment, shifted from the overall mean of the experimental period by only co 2 . the expression exp(..-1)/10 (= e (..-1)/10) ) accomplishes back-transformation of the natural logarithm ln (10 * y+1) of the original vegetation data (y). b_spec_ax1, b_spec_ax2 are the non-centered, not standardized scores of one of the species on the first or second ordination axis, respectively. likewise, c_co2_ax1, c_co2_ax2 denote the scores of the standardized explanatory variable co 2 (co2_std) on these axes. the absolute shift in %-coverage due to the co2-treatment was calculated by subtracting results for ambient from those for elevated co2. species were ranked accordingly. 2.3 identification of standardized/absolute co2-response groups in the a posteriori pft analysis proposed here, identification of rg involves finding traits that are shared by group members and that contrast them to species outside the rg. in principle, this represents a classical classification/clustering problem. nonetheless, traditional unsupervised learning, such as k-means clustering, is not well suited for the problem at hand, because it has difficulties with mixed variable types (duda et al., 2001). in contrast, the supervised-learning method cart can easily handle large numbers of both numerical and categorical variables, but requires setting-up rg beforehand from the species ranking. in an explorative phase, classification trees provided detailed insight into the relationships among species and species traits. basically, interpretation of the rule sets for rg, which were based on visual inspection and other criteria, was straightforward (duda et al., 2001). however, experimentally shifting the class boundaries changed results dramatically. this is a known problem of classification trees and has been referred to as instability (breiman and cutler, 2006). furthermore, cart (in jmp) does not provide a measure for assessing rg homogeneity. in conclusion, the classifying capabilities of cart appeared to be limited (compare breiman and cutler, 2006). the basic problem outlined at the beginning of this section was not solved. random forests (rf) were invented by breiman (2001) to overcome problems known from cart (breiman and cutler, 2006). rf builds a population of decision trees for the classification. in the construction of these trees two randomization schemes are used: a boot-strapped sample of cases (i.e. the species) is chosen from the classes (i.e. the rg) using stratified sampling with replacement. these cases are utilized to train the decision tree, whereas the rest of the cases, the „out-of-bag“ cases, form a test dataset and provide a means of calculating an unbiased error-estimate for the tree. at each node the best splitter is determined from 5 randomly-chosen traits. unlike cart, splitting of a tree in rf is not proceeded until terminal nodes are found for all cases and the tree is eventually subsequently pruned. instead, splitting is stopped when a terminal node is reached for each class. thus, rf does not overfit (breiman and cutler, 2006) and seems to be even better in this respect than supported vector machines (truong et al., 2004). the rf classifier works by summing up the votes of the trees for which a case is a test case and dividing by the number of trees. when the rf vote is above a class-specific cutoff-threshold the case is given that class. the overall rf error-rate is obtained from the proportion of times a case is misclassified by the rf averaged over all cases. the resulting rf classifier is considered to be unexcelled among current classifying techniques (breiman and cutler 2006). the importance of variables (i.e. the traits) is found as the mean decrease in classification accuracy, when the variable in the original dataset is replaced by a variable randomly permuted from the original variable distribution. additionally, variable importance can be calculated separately for each class. in principle, variable importances focus on the general pattern in the data and represent a sort of change in perspective when compared with the detailed rule sets of cart. from the importance of a variable for the classification one cannot read out the class-specific values of each variable. towards this end, class centers are calculated from the class-specific mode for categorical variables or from the class median for numerical variables (k-nearest neighbours = class members – 1). in essence, class centers give the value of the variable for a typical class member. however, the most versatile measures in rf are the species proximities, which resemble euclidean distances among the species. they are calculated from the number of times two cases end up in the same terminal node of a tree, summed over all trees and normalized by the number of trees in the forest. species proximities can be used for replacement of missing values (compare section 2.1). they are also useful for detecting outliers, defined as cases having low proximity to the other members of their class. they provide a means of solving the problem outlined in the beginning of this section: when a mislabelled case at the boundary of a class is detected by its outlyingness the class boundary is shifted. the process is repeated, until rg-homogeneity is satisfactory. following truong et al. (2004), we considered this to be the case when maximum outlyingness fell below 3. in the alternative case a non-boundary species is an outlier, it might be possible to augment the class size, thereby increasing the „common denominator“ among the species. before applying this refinement of class boundaries, however, it is necessary to have an initial set-up of the classes. one might suppose that, using statistical criteria for the separation of species into nonresponsive and significantly responding groups is straightforward. however, this is not the case as can be illustrated with the results of the alleviated statistical testing (compare section 3.2): since the whole set of species was significantly affected by co2, any subset consisting of the most responsive species was also significant, but to a greater degree. however, due to the low co2-significance of the whole suite of species, removal of only the most responsive species reduced the set to non-significance. statistical testing of a single species was not possible for most of the species (in canoco), possibly because sufficient constancy is required for doing this. thus, because of the wide range of uncertainty with respect to species significance, almost any grouping could be justified. due to these difficulties we instead used the simpler approach of lososová et al. (2004) and divided the set into species responses below (non-responsive) and above the median (positive/negative response). special provisions had to be made because rg were imbalanced, i.e. they held different numbers of species. chen et al. (2004) advise using the balanced rf scheme of r in the imbalanced case. this is done by down-weighting the bootstrapping sample size(s) of the majority class(es). however, the authors also state that this procedure results in a loss of information, since a large part of the majority class(es) is not used for the construction of the classifier. due to the low number of species this was not acceptable in our case. thus, 120 u. grüters, s. janze, c. kammann, h.-j. jäger adjustments were only made to the cutoff-thresholds for the classes to achieve balancing of the classifier. 2.4 inventory of causes responsible for co2-induced community alteration step 4 of the method scans for species traits which caused the detected community alteration. to accomplish this task, results from the class centers are interpreted based on knowledge of trait relationships gained in section 2.1. the inventory of causes is solely based on verbal inference, though. 3. application to the case study 3.1 characterizing the trait composition of the species a visualization of the species locations and trait composition among the species is presented in fig. 2. with 15.4%, the first ordination axis from pca, i.e. the horizontal axis, explains most of the variation in the trait composition of the species. it covers an evergreen – summer-green life strategy gradient. along this axis we find the evergreen strategy on the left and the summer-green strategy on the right. on the vegetative side, summergreenness is associated with a tuber root, larger leaves, a larger shoot height and a tendency to exhibit higher leaf-nitrogen concentrations. we may ask whether the storage capacity of the tuber root is a prerequisite for high n-concentration and for a summer-green life strategy in general. among the generative traits, pollination by insects is most prominent. a late time of first generative reproduction might be causally related to summer-greenness, since summer-greens use a shorter time period of the year for active growth. moreover, summergreens tend to have heavier dispersules. evergreens, on the other hand, are pollinated by wind, but their seeds/ fruits are dispersed by animals. their dispersules appear to stay in the seed bank longer. one of the most prominent vegetative traits is possession of tussock roots. besides, the evergreens are characterized by a higher seedling growth rate. there is a slight tendency, that these species possess the vesicular-arbuscular mycorrhiza type more often. the second ordination axis, i.e. the vertical axis, explains slightly less variation in the trait composition than the first. here, the explained variation amounts to 13.2%. this axis covers a gradient of competitiveness at the bottom towards ruderality at the top. the competitors are mostly defined by a number of vegetative traits: a leafy canopy structure with leaves all along the stem and taller shoots appear to lead to a fast lateral spread. additionally, they tend to possess mycorrhiza. however, among generative traits, only a delayed flowering start is a characteristic of competitive life strategy. ruderals, on the other hand, are herbaceous basal or semi-basal rosette plants and consequentially have an evergreen life strategy (to partially avoid competition later in the year). as a consequence, they start flowering early in the year, leading to a long flowering period. eventually, they can escape the mowing in a flowering state as well, due to the rosette canopy structure. the late time of first generative reproduction might also be causally related to the (semi-)basal canopy structure, although these species, when grown in isolation, tend to have a high seedling growth-rate (sgr). plantago and rumex, in fact, have the secondand third-highest sgr (sgr > 0.243 g g -1 d -1) within the community. as ruderals they produce large numbers of fruits/seeds, which are dispersed by wind. as fig. 2 shows, ruderals are defined primarily by generative traits. looking at the species’ location in the ordination diagram, it is most apparent that the grasses cover only a very small region at the fig. 2: ordination diagram of the trait composition for the species obtained from principal components analysis (pca): a) species location with envelopes around putative natural pft b) generative traits c) vegetative traits. for abbreviations used see tab. 1. plant functional types and elevated co2 121 tab. 2: statistical analysis using monte-carlo permutation tests: co2 = co2-treatment, mois = soil moisture, yr0 = 1997, year before co2-enrichment started, yr1= 1998, 1st year of co2-enrichment,..., sum = summer records, aut = autumn records, %-var. expl. all canonical axes = %-variance explained by all canonical axes, p-value i = p-value of design-based test, p-value ii = p-value of alleviated test. null hypothesis tested explanatory variables covariables % var. expl. p-value i all canonical axes p-value ii co2, mois, yr0...yr4, h01 co2 x mois, 0.2860 full model co2 x yr0 …co2 x yr4, 0.1340 co2 x sum, co2 x aut h02 0.0550 main effects 0.0105 0.1550 0.0455 0.1370 0.1155 0.1040 0.0315 h03 co2 x mois, interactions co2 x yr0 ...co2 x yr4, co2, mois, yr0...yr4, sum, aut 6.5 co2 x sum, co2 x aut co2, mois, yr0...yr4, sum, aut, h03a co2 x mois co2 x yr0 ...co2 x yr4, 3.8 co2 x sum...co2 x aut co2, mois, yr0...yr4, sum, aut, h03b co2 x yr0 ...co2 x yr4 co2 x mois, 2.7 co2 x sum...co2 x aut co2, mois, yr0...yr4, sum, aut, 0.9035 co2 x mois, co2 x yr0...co2 x yr4 0.7555 evergreen end of the 1st axis and intermediate along the 2nd, the competitor-ruderal axis. merely arrhenatherum elatius seems to be slightly more competitive than the other grasses. the trait-sets of grass species are thus very similar to each other. in contrast, herbaceous species possess a much wider spectrum of traits. most interestingly, some of them even resemble the traitsets of the grass species. with respect to species co2-responses, this implies the following: when the trait-set enables grasses to respond to elevated co2 in a certain way, it would not be surprising if the most similar herbaceous species (glechoma hederacea, ranunculus acris, galium verum) responded in the same way. therefore, we drew an envelope around the grasses and the three forbs and put them in the same natural pft. a second natural pft consists of 4 herbaceous species at the ruderal end of the vertical axis. the third natural pft contains 5 forbs at the summer-green end of the horizontal axis. their dissimilarity to the first natural pft is most pronounced. among them is likely the most competitive species within this community: the forb filipendula ulmaria. some of the trait vectors in fig. 2b and 2c point towards particular specialists among the species: bromus hordeaceus is a grass that is unique because it is an annual with hibernal leaf phenology. taraxacum officinale is the only species which forms seeds asexually without a requirement for pollination (apomixis). besides, lathyrus pratensis is the sole legume, but with 48.6 mg g -1 its leaf n concentration is still exceeded by that of glechoma hederacea (52.4 mg g -1). in conclusion, due to the wide spectrum of traits possessed by herbaceous species in the community under study it would be completely misleading to put them into one natural pft, named „the forbs“. 3.2 species-ranking according to their standardized/absolute co2-response results obtained from statistical testing of the empirical model are shown in tab. 2. the design-based test did not show any significant effects of the environmental variables. however, when monte-carlo permutations were extended to the split-plot level (alleviated test), the main effects and the effect of moderate co2-enrichment were significant. since none of the interactions was significant, co2 appeared to alter the community in a constant manner over the investigation period (1998-2001). standardized responses provide the only way to get a true unbiased picture of co2-effects on species abundances. as shown in fig. 3a, the standardized response of the dominant grass arrhenatherum elatius, but also that of the forb glechoma hederacea, was positive and above the median threshold. hence, both were put in a positive initial response group (rg_std +). however, the fact that 5 more grasses follow glechoma in the ranking, makes glechoma an outstanding forb. this favourite position represents yet another case in which the a priori pft concept of grasses/forbs would have failed. it is inconsistent with a sole stimulation of grasses, but is consistent with its similarity to grasses. while this supports the validity of the sum, aut 37.7 co2, mois, yr0...yr4 sum, aut 31.2 h02a co2 mois, yr0...yr4, sum, aut 10.0 h02b mois co2, yr0...yr4, sum, aut 8.8 h02c yr0...yr4 co2, mois, sum, aut 17.2 h03c co2 x sum, co2 x aut 1.0 0.6100 0.6470 0.7160 0.7255 0.3870 0.4390 122 u. grüters, s. janze, c. kammann, h.-j. jäger natural pft concept, the negative response of galium verum weakens that view. overall, nine forbs and only two grasses responded negatively (above the median threshold) and were grouped into an initial negative rg (rg_std -). the rest of the species, a heterogeneous mixture of grasses (mostly) and forbs, were considered non-responsive by definition (rg_std 0). fig. 3b, at first allows us to assess the absolute response of the whole community. in fact, the reaction of the community as a whole was intrinsically low: all species that responded positively to the co2treatment amount to a total of +1.26 %-coverage, whereas all negative responses summed up to -1.64 %-coverage. this clearly confirms results of the statistical test based on the experimental design. however, unlike standardized responses, absolute species responses are superimposed by the dominance patterns in the community. consequently, the two dominant grasses arrhenatherum elatius (µ = 39.9 %-coverage) and holcus lanatus (µ = 17.8 %-coverage) took over the leading position of the positive absolute response. due to its low abundance glechoma hederacea responded less to elevated co2 in absolute terms. from its absolute response glechoma would not have been identified as the outstanding forb. nonetheless, together with four grasses, this species was still put into rg_abs +, since its co2-induced abundance-shift was larger than the median. on the negative side, in contrast, geranium pratense and filipendula ulmaria did hold the leading position despite their low abundance, whereas the dominant forb galium mollugo (µ = 34.0 %-coverage) was only third. basically, with the exception of poa trivialis, the rg_abs – did consist only of forbs. once again, in absolute terms a heterogeneous mixture of grasses and forbs was non-responsive (rg_abs 0). 3.3 identification of standardized/absolute co2-response groups the adjustment of the class boundaries is carried out in this section. at the end of this process we obtain homogeneous standardized/ absolute co2-response groups. processing the initial standardized rg revealed that the outlyingness of galium mollugo exceeded 5. therefore, galium mollugo was transferred from rg_std 0 into rg_std –. with a total of two species the initial rg_std + was likely too small for the rf classifier to work under the given constraints: it was not possible to balance the classifier by merely reducing the cutoff-threshold for rg_std +. moreover, the outlyingness of arrhenatherum was beyond 30. following suggestions made in section 2.3, the size of rg_std + was augmented to reach satisfactory rg homogeneity. the final rg_std + contained the four most responsive species (see fig. 3a). tab. 3 shows the importance of the traits for the classification and presents the class centers for the final rg, i.e. the trait-sets of a representative rg member. besides that, tab. 3 contains the class centers for the natural pft of evergreen competitors in which rg_std + was nested. overall, the random forest misclassified 42.4% of the out-of-bag test cases (error rg_std = 43%, error rg_std 0 = 33%, error rg_std + = 51.0%). in part, this large error rate was due to the number of traits that did not contribute to the classification and solely represented noise. additionally, this implies that the species number was at its lower limit for sound rf classification. nevertheless, when only the five most important traits were incorporated, the error rate improved markedly amounting to 23.8% (error rg_std = 18%, error rg_std 0 = 28%, error rg_std + = 25%). arr_ ela alo_ pra hol_ lan tri_ fla gle_ hed poa_ pra ant_ odo agr_ ten car_ pra ran_ acr tar_ off fes_ pra fes_ rub gal_ ver lol_ per bro_ hor agr_ rep sax_ gra dac_ glo poa_ tri lat_ pra pla_ lan rum_ ace san_ off gal_ mol fili _ulm ger_ pra -0,8 -0,3 0,2 0,7 1,2 shift in % coverage due to co2 enrichment dominant grass, µ = 17.8 % dominant grass, µ = 39.9 % rg_abs + median threshld arr_ ela gle_ hed alo_ pra tri_ fla ant_ odo poa_ pra agr_ ten car_ pra hol_ lan fes_ rub dac_ glo ran_ acr fes_ pra poa_ tri bro_ hor gal_ mol agr_ rep lol_ per sax_ gra lat_ pra tar_ off san_ off gal_ ver pla_ lan ger_ pra rum_ ace fili _ulm -0,8 -0,6 -0,4 -0,2 0 0,2 0,4 species scores a b median median median median fig. 3: ranking of species-specific a) standardized co2-responses, b) absolute co2-responses. horizontal lines mark the median threshold. abbreviations: ger_pra = geranium pratense, fil_ulm = filipendula ulmaria, rum_ace = rumex acetosa, pla_lan = plantago lanceolata, san_off = sanguisorba officinalis, lat_pra = lathyrus pratensis, sax_gra = saxifraga granulata, gal_ver = galium verum, lol_per = lolium perenne, agr_rep = agropyron repens, bro_hor = bromus hordeaceus, gal_mol = gallium mollugo, poa_tri = poa trivialis, tar_off = taraxacum officinale, fes_pra = festuca pratensis, dac_glo = dactylis glomerata, ran_acr = ranunculus acris, fes_rub = festuca rubra, car_pra = cardamine pratensis, agr_ten = agrostis tenuis, hol_lan = holcus lanatus, pea_pra = poa pratensis, ant_odo = anthoxanthum odoratum, tri_fla = trisetum flavescens, arr_ela = arrhenatherum elatius, alo_pra = alopecurus pratensis, gle_hed = glechoma hederacea plant functional types and elevated co2 123 in contrast to the standardized rg, the initial absolute rg were kept unchanged. the largest outlyingness among the species amounted to less than 3 (poa trivialis). tab. 4 shows results concerning variable importance for the classification and class centers for the absolute rg. here, the random forest misclassified 50.0% of the test cases, when they were out-of-bag (error rg_abs = 47%, error rg_abs 0 = 54%, error rg_abs + = 49%). limiting the incorporated traits to the five best classifying ones reduced the overall error rate to acceptable 24.6% (error rg_abs = 13%, error rg_abs 0 = 22%, error rg_abs + = 39%). 3.4 inventory of causes responsible for co2-induced community alteration the final step of the a posteriori pft analysis is the inventory of causes responsible for differing rg behaviour. rf identified the flowering period, the time of first generative reproduction and the root type as the best classifying traits for the standardized rg (tab. 3). species belonging to rg_std have the longest flowering period and reproduce themselves late. while the long flowering period is an indicator for summer-greenness and ruderality (see fig. 2c), the late first generative reproduction and the pollination by insects designate the species as summer-greens. although at least one clear indicator of summer-greenness (tuber root) was lacking, half of the species were summer-greens and two of them (filipendula ulmaria, geranium pratense) have even decreased in abundance most strongly with co2-enrichment (see fig. 3a). the strong negative response of the most competitive species (filipendula) suggests that ruderality did not play a major role at the summer-green end. this might explain, at least in part, that clear indicators of ruderality were lacking in the class centers of rg_std -: the number of dispersules was intermediate and the same was true for the r-radius itself. nonetheless, three of the four ruderals were members of this group. species in rg_std –, thus, largely belonged to two natural pft: summer-greens irrespective of their competitiveness and ruderal evergreens. species in rg_std + are primarily flagged as evergreens: early time of first reproduction was the best classifying trait. the majority of the species is always evergreen and pollinated by wind. furthermore, the large lateral spread representing the second best classifying trait for the group denotes the species as competitors. this is supported by the leafy canopy structure, the large shoot height and the slightly later flowering start. in line with this is the short flowering period, as it is an indicator for both, degree of evergreen character and competitiveness. however, rg_std + was nested within the evergreen competitive natural pft. comparison of their class centers indicates that rather more competitive species within this natural pft qualified for positive response. species in rg_std + did exhibit larger shoot height, later time of first flowering, shorter flowering period, later flowering start and faster lateral spread than those in the respective natural pft. in conclusion, evergreen competitors were the winners in the elevated co2-treatment, while summer-greens, irrespective of their competitiveness, and evergreen ruderals lost in abundance. given that ruderals are (semi-) basal rosette plants, this suggests that overshadowing by neighbours for part of the year caused them to fall behind. but why should the summer-greens lag behind as a group? it has often been reported that arrhenathereta remain green and are tab. 3: variable importance for the classification of standardized rg (decreasing order, in mean decrease of accuracy) and respective class centers including that for the natural pft evergreen competitors (= natural pft ec). encircled numbers (➊➋➌➍➎) denote the importance of a variable for the classification of the specific rg. for trait abbreviations used see tab. 1. trait mean decrease accuracy rg_std rg_std 0 rg_std + natural pft ec flowp 3.27 4 ➊ 3 ➊ 2 ➌ 3 trep 2.51 2.5 ➋ 1.8 ➋ 1 ➊ 1.74 rt 1.98 rt_elon ➌ rt_tuss rt_stol, tuss ➍ rt_tuss num 1.59 1.26 ➍ 1 ➎ 1.5 1 seedb 1.26 1 2 1 ➎ 1 size 1.24 4 4 ➍ 4 4 leafp 0.74 leafp_ea, sa leafp_ea leafp_ea leafp_ea r-radius 0.59 1.67 2 1.5 2 sgr 0.54 0.171 ➎ 0.187 0.191 0.186 leafn 0.53 29.44 24.3 ➌ 26.95 25.49 myc 0.46 myc_va myc_va myc_pos, va myc_va latsp 0.46 3 3 3.5 ➋ 3 pol 0.30 pol_ins pol_wind pol_wind pol_wind flows 0.27 5 5 5.5 5 c-radius 0.12 2 2 2.5 2 cans 0.12 cans_s, l cans_s cans_l cans_l s-radius 0.11 1.42 2 1.5 2 lifeh 0.00 lifeh_p lifeh_p lifeh_p lifeh_p dispf -0.04 fruit fruit fruit fruit canh -0.07 3 2 3.5 2 ad -0.27 ad_unsp ad_anim, unsp ad_anim, unsp ad_unsp deep -0.50 1 1 1 1 dispw -0.52 4 3 3 3 124 u. grüters, s. janze, c. kammann, h.-j. jäger able to maintain a positive carbon balance in winter under the mild sub-atlantic climate that prevails in central-europe (ellenberg, 1996 and citations therein). thus, it is hypothesized that the more limited annual time-integral they can use for enhanced vegetative growth with year-round co2-enrichment caused the competitive loss of summer-greens. absolute species responses reflect the dominance patterns within the community as well as the differences in standardized responses. the five best traits for the classification of absolute rg are leaf phenology, time of first flowering, shoot height, root type and seedling growth rate. species of rg_abs – are primarily defined by summer-greenness and related traits (low seedling growth rate, large shoot height, pollination by insects). unlike rg_std -, traits associated with ruderality receded in importance for the classification of rg_abs -. the last remnant of this association is likely the long flowering period exhibited by species of rg_abs -. the overall low abundance of ruderals in this community is likely to explain this finding. early time of first flowering is the best predictor of positive absolute response. as we know from pca, this trait is primarily associated with the evergreen life strategy. the leaf phenology class center of rg_abs + confirms this. two other important classifying traits, namely leafy canopy structure and fast lateral spread, define the respective species as competitors. in principle, this reflects the observation that competitive species tend to be the dominant species as well. 4. discussion 4.1 the method to use a metaphor: when compared with the common practice of measuring a few eco-physiological traits on the dominants and relating their response to those traits, knowing the „curriculum vitae“ of all the individual species from a database makes a difference. as visualized in fig. 2, this knowledge allows to identify natural pft specific to the community under study. furthermore, this knowledge makes aware of the (causal) relationships among the traits and provides a background for evaluating the trait-sets of the response groups. unlike the a priori pft grasses/forbs, natural pft were able to give an explanation for the positive response of the forb glechoma among otherwise only grasses, but, unfortunately, could not elucidate why the strikingly similar galium verum responded negatively. even at closer inspection more indicators for the opposite ranking of the two species were present in the incorporated traits (e.g. galium leafy vs. glehoma semi-basal rosette). thus, we believe that in the long run only incorporation of more traits could solve this issue. in working step 1 and throughout the method missing values were a critical issue that could not be solved entirely. missing values are present in all current databases. however, this situation will presumably change with the leda traitbase, since the aim of this eu project was to fill in knowledge gaps and make standardized species traits for the northwest-european flora available online (knevel et al., 2003). tab. 4: variable importance for the classification of absolute rg (decreasing order, in mean decrease of accuracy) and respective class centers. encircled numbers (➊➋➌➍➎) denote the importance of a variable for the classification of the specific rg. for trait abbreviations used see tab. 1. trait mean decrease acuracy rg_abs rg_abs 0 rg_abs + leafp 2.27 leafp_sa ➊ leafp_ea ➌ leafp_ea ➌ trep 2.25 2.22 1.97 ➋ 1.75 ➊ canh 1.68 4 ➌ 2 ➊ 3.5 rt 1.58 rt_elon ➎ rt_elon, tuss ➎ rt_tuss ➍ sgr 1.37 0.166 ➋ 0.184 0.182 leafn 0.99 29.44 25 ➍ 40.55 cans 0.84 cans_s cans_s cans_l ➋ flowp 0.56 4 ➍ 3 2.5 latsp 0.55 3 2.5 4.5 ➎ pol 0.50 pol_ins pol_wind pol_ins, wind dispf 0.38 fruit fruit fruit myc 0.33 myc_pos myc_va myc_va size 0.28 4.27 4 4 c-radius 0.17 2.11 2 2.5 num 0.11 1 1 1.5 lifeh -0.32 lifeh_p lifeh_p lifeh_p deep -0.34 1 1 1 r-radius -0.38 1.69 2 1.5 seedb -0.58 1 2 1.5 dispw -0.69 5 3 4 flows -0.70 5 5 4.5 ad -0.73 ad_unsp ad_unsp ad_unsp s-radius -0.75 1.43 1.79 1.5 plant functional types and elevated co2 125 the empirical model of working step 2 suffered primarily from the low magnitude of the community response. this likely originated in the applied moderate co2-enrichment, but also in the much lower effect size concerning leaf area when compared with aboveground biomass (see kammann et al., 2005). besides, the power of the statistical tests was low due to statistical noise present in vegetation survey data. nonetheless, since the ranking of absolute species responses was in good agreement with results of measured biomass for a priori pft (kammann et al., 2005), we proceeded with the a posteriori pft analysis as described. the ranking of species according to their standardized response was independent of the initial abundances and thus provides an accurate means for comparing responses. the ranking based on absolute abundance shifts, on the other hand, enabled us to assess the magnitude of the community response and to make comparisons with biomass results. the low species number caused classification problems in working step 3. thus, the method would be more suitable for experimental scales providing species numbers above the current 27. when species numbers are much larger, as for instance in observational studies, a regression tree approach – as used by de bello et al. (2005) – might be the better choice. however, due to its unexcelled classifying capabilities doing this with random forests might still be advantageous. overall, the initial set-up of rg and the adjustment of the rg boundaries using the outlyingness measure accomplished rg homogeneity. results of the rf-classifier were satisfactory when only the best classifying traits were incorporated. however, according to the rf classifier negative standardized co2-response was not entirely restricted to the summer-green and the ruderal evergreen natural pft. in principle, a better congruence with the natural pft could be obtained by decreasing the outlyingness threshold beyond the value reported in the literature (which was not done here). in a posteriori pft analysis the trait-set of a response group is assumed to cause the response behaviour. working step 4 followed this scheme. however, as the cautionary subtitle „a method of scanning for causes…“ suggests, the interpretation of the rg class centers as cause is tentative. we believe that these inferences have more the status of „hypotheses“ and need to be verified either by species-specific eco-physiological measurements or by an organismbased plant community model. in the future, a posteriori pft analysis could benefit a lot from the integration of quantitative methods for causal inference as described by shipley (2000) and pugesek (2002). causal reasoning could start from basic vegetative traits (e.g. summergreen leaf phenology, basal canopy structure) via a causal chain present in the database, leading to the best classifying traits of rf (flowering period, time of first flowering), and end with the species scores of standardized co2-response. in any case, applying bayesian networks or structural equation models (sem) to our case study was not feasible because of the extensive data requirements and because important assumptions of sem were violated in the data (petraitis et al., 1996). the method proposed here relies primarily on standard multivariate data-analysis techniques, well-established in ecology. the additional key component of random forests is readily available and can be learned with ease. these might represent advantages over the more laborious canonical correlation analysis utilized by lavorel et al. (1998). a large number of species can be incorporated into the pft analysis as outlined and numerous traits beyond a core set with known significance to the problem can be checked for relevance. this might be beneficial in comparison with the three matrix approach of pillar and sosinski (2003) in which an optimal classification is iteratively sought. in principle, this method may well be applied to investigate the impact of global change factors beyond elevated co2 on plant community composition. 4.2 application to the case study similar trait-sets cause species to react in the same way to a global change factor, and natural pft track these similarities. they do not need to be homogeneous with respect to a priori pft grasses/forbs. presumably this is one of the most important explanations for the frequent inconsistencies which arose when classification into grasses/ forbs was applied. in essence, negative standardized response to co2-enrichment was limited to species belonging to two of the natural pft, i.e. summergreens and ruderal evergreens, while the positive standard response covered competitive evergreen species. poorter (1993) and poorter et al. (1996) found fast-growing species (cand r-type) more responsive to elevated co2 than slow-growing ones (s-type). hunt et al. (1993) screened a total number of 36 individual species for their co2-responsiveness and, on the contrary, found competitiveness alone to be the best predictor of the speciesspecific co2-stimulation. later, poorter et al. (1996) claimed that hunt’s results also point towards stimulation of fast-growing species (cand r-type). in principle, it makes sense that an attribute defining the space acquisition capabilities of a species is an excellent predictor of the co2-responsiveness of isolated plants (grime, 2001). however, a fundamental difference may exist among factors governing species’ response in an established ecosystem and those effective on isolated plants as in hunt’s experiments or in non-natural conditions towards which poorter’s meta-analyses were biased. in established ecosystems the response pattern is likely to be modified by biotic factors. in the meadow under study, the management regime repeatedly frees rosette plants from the competition for light immediately following the first cut in early summer and after the second cut during autumn and early spring of the next year. these time periods appear to enable such ruderals to stay at low abundance in the community under study. it is during these time periods that they receive full sunlight and presumably achieve the largest additional carbon gain with co2enrichment. this is quite distinct from the situation at the forest floor or with artificial, permanent low light, where the co2-induced stimulation of growth is most pronounced (hättenschwiler, 2001). when light levels are close to the light compensation-point, not enabling positive carbon balance under ambient conditions, co2enrichment will even lead to an infinite relative increment of carbon gain due to the enhanced quantum yield (ehleringer and björkman, 1977). essentially, in this study evergreen ruderals seem to have lagged behind evergreen competitors because of the more limited time-integral with full sunlight available to them. this parallels our hypothesis that summer-green species lost in competition because of the more limited annual time-integral they can use for enhanced vegetative growth with year-round co2-enrichment. this argumentation is further supported by the fact that even the most competitive summer-greens decreased in abundance. however, the possession of a tuber root as part of the inferior „trait syndrome“ of summer-greens was a surprise. it contradicts studies in which storage organs were found to prevent species from establishing a source-sink-imbalance and enable them to retain the initial stimulation of photosynthesis and growth due to co2 (poorter, 1993; poorter et al., 1996; lüscher et al., 1998; poorter and navas, 2003). however, we remind the reader that moderate co2-enrichment 126 u. grüters, s. janze, c. kammann, h.-j. jäger was applied here and that this exposure regime is less likely to induce a severe source-sink-imbalance than the co2-doubling applied in other experiments. notably the summer-green lathyrus pratensis, representing the sole legume within the community, also decreased in abundance. in many experimental studies the co2-response of leguminous species was more pronounced than that of species without the ability to fix atmospheric nitrogen (poorter, 1993; lüscher et al., 1996; lüscher et al., 1998; teyssonneyre et al., 2002). when this was not the case, either an inferior competitiveness for light (teyssonneyre et al., 2002) or a phosphorous limitation (stöcklin et al., 1998) was held responsible for the finding. since lathyrus pratensis has considerable canopy height, the first explanation is rather unlikely here. a rough estimation of the annual p-budget of the ecosystem suggests that phosphorus was also not limiting here (see parameters below): p-supply in fertilizer = 26.2 kg ha-1 a-1 p-withdrawal by harvests = 13.3 kg ha -1 a -1 (assuming average n-withdrawal with harvests = 100 kg n ha-1 a-1, p: n ratio in harvested plant tissue = 0.133 and low phosphorus leaching/mobility in the soil) the sole remaining explanation, however, is that the summer-green trait-set was of higher relevance and that the hypothesized mechanism, however, caused the response of the legume. in the giessen-face study – as in many other experiments – a priori pft have been studied based on above-ground biomass, which was separated into grasses, forbs and legumes, respectively. a significant increase in above-ground biomass of up to 11% was largely attributable to a stimulation of the a priori pft „grasses“ (kammann et al., 2005). in accordance with this is the ranking of absolute species responses and the finding that positive absolute response was almost restricted to dominant and subdominant grasses. the absence of a total coverage stimulation found here is in line with the unchanged lai development reported by kammann et al. (2005) for the elevated co2-treatment. both findings indicate that the „taller“ plants had lower leaf area ratio with co2-enrichment. the findings further confirm results of the meta-analysis of ainsworth and long (2005), in which c3 grasses did show significant biomass increment under face conditions, but non-significant lai-stimulation. identification of pft in a natural ecosystem exposed to elevated co2 is a major challenge. founded on knowledge of the „curriculum vitae“ for all individual species and based on the method for a posteriori pft analysis, it was possible to build several novel hypotheses regarding the causes underlying co2-induced community alteration. acknowledgements we gratefully acknowledge financial support from the hessian agency for environment and geology (hessisches landesamt für umwelt und geologie, hlug) and the german science foundation (deutsche forschungsgemeinschaft, dfg). petr šmilauer and jan lepš provided valuable advice concerning canoco during their course on multivariate analysis of ecological data. furthermore, we appreciate the assistance of a. streitfert and c. roth, proofreading by g. richters, and maintenance work of s. martis, j. senkbeil, w. stein, g. meyer, j. franz and b. lenz on the face site. references ainsworth, a.e., long, s.p., 2005: what have we learned from 15 years of free-air co2 enrichment (face)? a meta-analytic review of the responses of photosynthesis, canopy properties and plant production to rising co2. new phytol. 165, 351-372. bassirirad, h., constable, j.v.h., lussenhop, j., kimball, b.a., norby, r.j., oechel, w.c., reich, p.b., schlesinger, w.h., zitzer, s., sehtiya, h.l., lim, s.i.s., 2003: widespread foliage δ15n depletion under elevated co2: inferences for the nitrogen cycle. glob. change biol. 9, 15821590. breiman, l., friedman, j., stone, c.j., olshen, r.a., 1984: classification and regression trees. wadsworth, redmont. breiman, l., 2001: random forests. mach. learn., 45, 5-32. breiman, l., cutler, a., 2006: random forests. http://www.stat.berkeley. edu/~breiman/randomforests/ caldwell, b.d., hart, a.l., 1996: competition between grasses and trifolium repens with elevated atmospheric co2. in: bazzaz, f.a., körner, c. 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nature 428, 821-827. address of the authors: institut für pflanzenökologie, justus-liebig-universität giessen, heinrichbuff-ring 26-32, d-35392 giessen, germany * corresponding author: dr. u. grüters e-mail: uwe.grueters@bot2.bio.uni-giessen.de 128 u. grüters, s. janze, c. kammann, h.-j. jäger << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 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<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> >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice journal of applied botany and food quality 87, 108 116 (2014), doi:10.5073/jabfq.2014.087.017 1department of life sciences and biotechnology (sveb), university of ferrara, ferrara, italy 2promociòn de la investigatiòn, universidad estatal amazònica, puyo, pastaza, ecuador 3centro de investigatiòn y valoración de la biodiversidad “civabi”, universidad politecnica salesiana, wilson, quito, ecuador 4department of life sciences, university of modena and reggio emilia, modena, italy biological and chemo-diverse characterization of amazonian (ecuador) citrus petitgrains a. guerrini1*, d. rossi1, a. grandini1, l. scalvenzi2, p.f. noriega rivera3, e. andreotti4, m. tacchini1, a. spagnoletti1, i. poppi1, s. maietti1, g. sacchetti1 (received november 20, 2013) * corresponding author summary six amazonian petitgrain samples from c. nobilis lour., c. aurantium l., c. limon l. and mixture of citrus spp. (rutaceae), named cn, ca, cl1, cl2, c1 and c2, were chemically characterized by gc-ms and 13c nmr and evaluated for antioxidant acitivity (dpph and b-carotene bleaching tests), for antimicrobial properties (disk diffusion method) and for antifungal capacity (agar vapour assay). cn, c1, c2 samples evidenced the most interesting results: cn (gterpinene/linalool chemotype: 14.3 % / 41.6 %, with a considerable amount of thymol: 9.0 %), and c1 (linalool, 18.3 %; sabinene, 11.6 %; thymol, 5.5 %), showed relevant antioxidant activity with both dpph (ic50 = 3.52 and 5.48 mg/ml, respectively) and b-carotene (ic50 = 0.387 and 0.491 mg/ml, respectively). antibacterial properties of cn and c1 against p. mirabilis (mic = 0.61 mg/ml for both) and b. subtilis (mic = 0.61 and 0.44 mg/ml, respectively) were most probably due to thymol. c2 (geranial: 34.7 %, neral: 33.1 %) evidenced a valuable bioactivity against c. albicans (mic = 0.44 mg/ml). the 50 % growth inhibition (ic50) of the dermatophytes t. mentagrophytes and n. cajetani was reached with amounts of c1, c2 and cn less than 4 ml/plate. bioactivity of amazonian citrus spp. cn, c1 and c2 essential oils suggests their potential use as food preservatives or additives in cosmeceuticals as preventive against dermatophytic fungal infections. introduction herbs and spices have been employed since ancient times as flavouring and preservatives agents for food, but only in the last decade scientific research has focused its interest on essential oils and extracts as natural sources of antimicrobial and antioxidant compounds, as safer alternative additives for food preservations (burt, 2004; shaaban et al., 2012). while citrus essential oils are usually cold-extracted with mechanical systems (as stated in european pharmacopoeia, vii ed.), the socalled petitgrain oils are obtained by distillation of citrus leaves, buds and small branches from citrus spp. adult plants (dugo et al., 2010). the composition of citrus leaves essential oils are not as well defined as the correspondent peel oils (dugo et al., 2010) and studies concerning these aspects have been often reviewed (lota et al., 2001a; lota et al., 2001b; lota et al., 2002; dugo et al., 2010). due to its pleasant and characteristic fragrance, bitter orange (sour orange, bigarade) petitgrain is the most important and appreciated among leaf essential oils: it is widely used in perfumery for preparation of eau de colognes, lotions and soaps because of its good resistance to alkaline medium. sour orange plants are cultivated mainly in the mediterranean countries (france, italy, spain) and in paraguay (lota et al., 2001a; dugo et al., 2010). the best known and most employed species is citrus aurantium l. other citrus spp. petitgrains (lemon, mandarin, etc.) are afforded at small quantities. lemons (citrus limon in particular) are cultivated in mediterranean countries, southern california and argentina while mandarins (other several citrus spp. and hybrids) are cultivated in mediterranean countries, japan, brazil, argentina, united states (known as tangerines) and australia (lota et al., 2000; dugo et al., 2010). although there is extensive literature on citrus spp. petitgrain composition, very few papers concern biological properties or report correlations among phytochemical data, chemodiversity and biological activities (dugo et al., 2010; shaaban et al., 2012). in fact it is known that the wide chemodiversity which characterizes this kind of phytocomplexes could be due to several variables, affecting the chemical profile, as geographical origin, time of collection and cultivars (guerrini et al., 2011). these variables can determine chemical diversities in essential oils obtained from the same species which necessarily could reflect different bioactivities and possible functional uses. in light of these premises, in ecuadorian traditional ethnomedicine citrus spp. leaves are used to treat stomachaches (rios et al., 2007). the present paper represents the first report about citrus spp. petitgrain essential oils from plants grown at the margin of the amazonian forest (ecuador). the aim is to evaluate possible, distinctive bioactivity properties of chemotypes driven due to this peculiar geographical origin. in fact, it is known that the amazonian biodiverse hot-spot is characterized by an biotic and abiotic set of conditions which can force the secondary plant metabolism to peculiar and unique profiles with corresponding interesting bioactivities (ryder wilke et al., 2010; rossi et al., 2013). moreover, the ethnobotanical use of citrus spp. leaves is sometimes performed without particular attention to the single species since the shape is similar and they are collected without distinction. this fact contributes to have preparations with mixed-species leaves (mainly as flavouring and anxiolytic agents) (hanazaki et al., 2000). in the present work, the study of essential oils obtained from mixture of citrus leaves would mime this ethnobotanical evidence. therefore, different amazonian citrus spp. petitgrains from single and mixed species leaves were evaluated for their chemical composition through gc, gc-ms and 13c nmr in order to point out the possible chemodiversity aspects related to their geographical origin and to check for functional properties (antimicrobial, antifungal, antioxidant) to valorize their possible applicative uses in food and/or health fields. material and methods chemicals all the solvents employed for chemical analyses and bioassays were chromatographic grade. solvents and pure compounds were all purchased from sigma-aldrich italy (milano, italy). thymus vulgaris essential oil, thymol-chemotype employed as reference phytocomplex (sacchetti et al., 2005), was purchased from extrasynthese (genay, france). microbial culture media were obtained from oxoid italia (garbagnate, italy). amazonian citrus petitgrains 109 plant material c. nobilis (named cn), c. aurantium (named ca), c. limon (named cl1 and cl2) fresh leaves and fresh leaves mixture of genus citrus spp. (named c1 and c2) were purchased by fundacion chankuap (quito, ecuador), non-governmentive organization which has as main target the valorization of amazonian sources recovering plant material to directly obtain commercial products from natives, with the cooperation of our research about ecuadorian amazonian bio-about ecuadorian amazonian biodiversity. for what concerns the essential oil mixtures, no information have been given by fundacion chankuap regarding the different citrus species employed and their quantitative ratio. leaves were collected in september 2010 from wild adult plants growing in three different locations on the outskirts of the wasak’entsa reserve in eastern ecuador (77°.15” w/2°.35” s) and positively identified by fundacion chankuap (quito, ecuador). dried specimens were deposited at the department of biology and evolution, university of ferrara, code c1, c2, ca1, cn1, cl1, cl2. essential oils isolation essential oils were in situ extracted for 8 hours through steam distil-8 hours through steam distillation of c. limon, c. aurantium, c. nobilis, mixture of citrus spp. fresh leaves (approximately 10 kg) using a mobile essential oil dis-(approximately 10 kg) using a mobile essential oil dis-10 kg) using a mobile essential oil distiller (essential oil company, portland, or, usa) set up follow-essential oil company, portland, or, usa) set up following the parameters reported in literature (horwitz, 2003). essential oil yields have been achieved through three different distillations of fresh plant material belonging to citrus spp. the petitgrains were dried over anhydrous sodium sulfate and stored in airtight glass vials with teflon-sealed caps at -18.0±0.5 °c in the absence of light until analysis. gas chromatography essential oil samples were analyzed and the relative peak areas for individual constituents averaged. the relative percentages were determined using a thermoquest gc-trace gas-chromatograph equipped with a fid detector and a varian factorfour vf-5ms poly-5 % phenyl-95 %-dimethyl-siloxane bonded phase column (i.d., 0.25 mm; length, 30 m; film thickness, 0.15 μm). operating conditions were as follows: injector temperature 300 °c; fid temperature 300 °c, carrier (helium) flow rate 1 ml/min and split ratio 1:50. oven temperature was initially 55 °c and then raised to 100 °c at a rate of 1 °c/min, then raised to 250 °c at a rate of 5 °c/min and finally held at that temperature for 15 min. one μl of each sample dissolved in ch2cl2 was injected. the percentage composition of the oils was computed by the normalization method from the gc peak areas, without using correction factors. gas chromatography-mass spectrometry essential oil constituents were then analyzed by a varian gc-3800 gas chromatograph equipped with a varian ms-4000 mass spectrometer using electron impact and hooked to nist library. the conditions were the same reported for gc analysis and the same column was used. the ms conditions were as follows: ionization voltage, 70 ev; emission current, 10 μamp; scan rate, 1 scan/s; mass range, 29-400 da; trap temperature, 150 °c, transfer line temperature, 300 °c. the constituents of the volatile oils were identified by comparing their relative retention time, ki and the ms fragmentation pattern with those of other essential oils of known composition, with pure compounds and by matching the ms fragmentation patterns and retention indices with the above mentioned mass spectra libraries and with those in the literature (adams, 2007). in order to determine the kovats index of the components, a c8-c22 n-alkanes (sigma-aldrich) was added to the essential oil before injecting in the gc–ms equipment and analyzed under the same conditions as above. nmr spectroscopy 13c nmr spectra were recorded at 100.58 mhz and at temperature of 303 k with a varian gemini-400 spectrometer. the essential oils were dissolved in cdcl3 (70 mg/0.8 ml) into a 5 mm nmr and solvent signal was used for spectral calibration (central line of triplet at 77.0 ppm). chemical shifts (ppm) and peak attribution were based on comparisons of the resonances in 13c nmr spectrum of the essential oil with those of pure standards and mixture of these (a-pinene, sabinene, b-pinene, d-limonene, g-terpinene, linalool, citronellal, 4-terpinenol, citral, thymol) present in our spectral library (guerrini et al., 2006) or according with those of literature (kubeczka, 2002), sdbs (saito et al., 2009). biological activities antioxidant, antifungal and antimicrobial activities were performed comparing all the data with those obtained with appropriate pure synthetic compounds and/or commercial thymus vulgaris essential oil, in order to have positive control references with single compounds or comparable phytocomplexes reputed for their functional bioactivities. the use of a phytocomplex known for its chemical and biological properties (e.g. thyme essential oil) as a positive reference results particularly indicative of the real functional efficacy of a tested extract (maietti et al., 2013). data reported for each assay are the average of three determinations of three independent experiments. antifungal and antimicrobial strains according to previously described methodology (guerrini et al., 2006; maietti et al., 2013), citrus petitgrain antifungal and antimicrobial activities were performed with agar vapor method and standard disk diffusion technique respectively. for antibacterial assays, gram-positive (enterococcus foecalis atcc 29212, staphylococcus aureus atcc 29213 and bacillus subtilis atcc 7003) and gram-negative (escherichia coli atcc 4350, proteus mirabilis atcc 29852 and klebsiella oxytoca atcc 29516) bacterial strains were employed. antifungal activity was assessed on yeast candida albicans atcc 48274, on phytopathogen strains (botrytis cinerea micheli atcc 48339, pythium ultimum trow, kindly supplied by prof. g. d’ercole (institute of vegetal pathology, university of bologna, italy), magnaporthe grisea atcc 64413) and dermatophyte strains (trichophyton mentagrophytes var. mentagrophytes (robin) blanchard cbs (centraal bureau voor schimmelcultures, baarn, the netherlands) 160.66, nannizzia cajetani ajello ihme (institute of hygiene and epidemiology mycology (ihme) brussels, belgium) 3441 and trichophyton rubrum (castellani) sabouraud ihme 4321). antimicrobial activity: disks diffusion method mother cultures of each bacteria were set up 24 h before the assays in order to reach the stationary phase of growth. the tests were assessed by inoculating from the mother cultures petri dishes with proper sterile media with the aim of obtaining the microorganisms concentration 106 cfu/ml. for bacteria, aliquots of dimethyl sulfoxide (dmso) were added to the essential oils in order to obtain a 0.01-50.0 mg/ml concentration range and then deposited on sterile paper disk (6 mm diameter, difco). bioactivity against the yeast candida albicans was also processed. mother cultures were set up inoculating 100 ml yepd liquid medium (yeast extract and potato dextrose) in 250 sterile flasks and 110 a. guerrini, d. rossi, a. grandini, l. scalvenzi, p.f. noriega rivera, e. andreotti, m. tacchini, a. spagnoletti, i. poppi, s. maietti, g. sacchetti incubated in the dark at 30 °c in order to assess growth curves. from each mother cultures at the stationary phase of growth, broth dilutions were made to obtain the strain concentration of 105 cfu/ml to inoculate petri dishes with agarized yepd for bioassays. then, 10 μl of dmso-essential oil sample solutions were prepared in order to have an assay range 0.01-50.0 mg/ml, and then deposited on sterile paper disk (6 mm diameter, difco). the petri dishes were successively incubated at 30 °c in the dark and checked for evaluating the growth inhibition after 48 h. both for bacteria and candida streams, the lowest concentration of each essential oil showing a clear zone of inhibition was taken as the mic (minimum inhibitory concentration). negative controls were set up with 10 μl of dmso in the test solution, while positive ones were assessed with t. vulgaris essential oil. antifungal activity: agar vapour assay biological activity of citrus petitgrains against three phytopathogenic and three dermatophytic fungi was performed by using the agar vapour method (maietti et al., 2013). they were grown in petri plates (90 mm) supplemented with 15 ml/plate of potato dextrose agar, inoculated with 6 mm plugs from stationary-phase cultures. the plates were then incubated for 24 h at 26 ± 1 °c. successively, sterilized filter paper discs (diameter 9.0 mm) were absorbed with different volumes of citrus petitgrains samples ranging from 0.20 to 25.00 ml, and placed inside the upper lid of each plate. plates were kept in an inverted position, tightly sealed with parafilm, and incubated for 7 days at 26 ± 1 °c. blanks served as a negative control. commercial t. vulgaris essential oil was prepared as described above for petigrain samples, with volumes ranging from 0.20 to 25.00 ml, and considered as a positive control reference phytocomplex. three replicates were made for each treatment. after 7 days the results were determined as the inhibition of radial growth and expressed as the amount of essential oil that led to 50 % inhibition of growth in each fungal strain (ic50). antioxidant activities radical scavenging and antioxidant properties of essential oils were performed through different assays, namely the dpph assay and the b-carotene bleaching test according to previously described methods. this approach permits the antioxidant effectiveness of an essential oil to be more carefully defined, as it is almost impossible to express the antioxidant activity as an absolute value that is universally recognizable, besides being expressed by only one type of assay (maietti et al., 2013). t. vulgaris essential oil was used as positive controls. essential oils antioxidant activity was considered as the ic50, calculated from inhibition curves obtained by plotting the % inhibition against oil concentration. all the data collected for each assay are the average of three determinations for three independent experiments. statistical analysis relative standard deviations and statistical significance (student’s t test; p < 0.05), one-way anova and lsd post hoc fisher’s honest significant difference test, were given, where appropriate, for all data collected. all computations were made using the statistical software statistica 6.0 (statsoft italia srl). results chemical fingerprinting steam distillation of citrus spp. leaves provided petitgrains with yield from 0.20 ± 0.02 g/100g for cn to 0.29 ± 0.03 g/100g for cl1 and density that covered a range of 0.82-0.92 g/ml (tab. 1). the ecuadorian ca petitgrain exhibited as major components sabinene (38.3 %), trans-e-ocimene (6.7 %), linalool (8.8 %): other minor components were 3-carene (8.9 %), d-limonene (7.9 %), bmyrcene (3.4 %), 4-terpinenol (2.5 %), a-pinene (1.9 %), geranial (1.9 %), b-pinene (1.8 %). cl1 essential oil evidenced an high abuncl1 essential oil evidenced an high abundance of limonene (52.7 %) and linalool (15.1 %) and as minor compounds citronellal (3.1 %), sabinene (2.7 %) and carvone (2.6 %), instead in cl2 petitgrain predominated sabinene (36.1 %) followed by limonene (24.1 %), linalool (4.7 %), 4-terpinenol (3.9 %), g-terpinene (3.9 %), citronellal (3.6 %), trans-b-ocimene (3.2 %), aterpinene (2.8 %), b-myrcene (2.6 %). citrus nobilis petitgrain evi-evidenced high abundance of linalool (41.6 %) and appreciable contents of γ-terpinene (14.3 %) and thymol (9.0 %), followed by trans-eocimene (10.9 %), p-cymene (4.1 %), a-pinene (3.6 %), b-pinene (3.1 %) limonene (2.8 %) as minor compounds. finally, linalool (18.3 %), sabinene (11.6 %), limonene (11.1 %), γ-terpinene (10.6 %), thymol (5.5 %), b-pinene (4.9 %), trans-e-ocimene (4.8 %) and p-cymene (3.4 %) were the most characteristic compounds for c1 petitgrain samples; c2 petitgrain, instead, evidenced geranial (34.7 %) and neral (33.1 %) followed by b-myrcene (5.4 %), linalool (4.7 %), and geraniol (3.1 %) as the most abudant chemicals (tab. 1). to contribute to define a metabolomic fingerprinting of citrus spp. essential oils, 13c-nuclear magnetic resonance (nmr) of the most abundant chemical standard compared with whole essential oil spectrum was performed confirming the evidences emerged by gc-ms (supplementary materials, tab. 5 and fig. 1). mono-dimensional 13c spectrum revealed typical and numerous diagnostic signals for characterizing the chemical makeup of carbons and, therefore, of the functional groups typical of the examined molecules. antioxidant activities the essential oils examined evidenced interesting antioxidant properties with sligthly different among the samples (tab. 2). in particular, cn petitgrain exhibited a good radical antioxidant activity both with dpph test (ic50 = 3.52 ± 0.25 mg/ml) and b-carotene bleaching assay (ic50 = 0.387 ± 0.021 mg/ml). these results are particularly relevant if compared to that obtained with commercial thymus vulgaris essential oil (ic50 = 1.24 ± 0.10 mg/ml), taken as reference phytocomplex (sacchetti et al., 2005). c1 petitgrain, that showed a relative abundance of g-terpinene (10.6 %) and thymol (5.5 %), probably to relate to the interesting dpph activity (ic50 = 5.48 ± 0.45 mg/ ml), displayed instead a lower activity in b-carotene bleaching test (ic50 = 0.491 ± 0.041 mg/ml). thymol has been tested as pure compound in dpph and b-carotene bleaching tests suggesting that the antioxidant capacity displayed by essential oils could be mainly due to the presence and the abundance of this substance. antimicrobial activity evaluation of antibacterial activity (tab. 3), expressed as mic (minimun inhibitory concentration), revealed that cn petitgrain was the most active among the citrus spp. phytocomplexes against gram-negative as well as gram-positive bacteria. the most interesting results were against p. mirabilis for cn, c1 and ca, against b. subtilis for cn and c1 and finally against e. coli for cn and ca since mics were comparable. the antibacterial properties of cn petitgrain were relevant also against s. aureus and e. foecalis (0.78 ± 0.08 and 0.95 ± 0.09 mg/ml) if compared to the positive control t. vulgaris. no remarkable inhibition activity was observed against k. oxytoca. c2 petitgrain was instead particularly active against the yeast c. albicans, with a mic of 0.44 ± 0.05 mg/ml. amazonian citrus petitgrains 111 tab. 1: chemical composition of citrus petitgrains compound kia rab cn ca cl1 cl2 c1 c2 α-thujene 930 1.8 0.5 0.2 1.0 1.1 0.1 α-pinene 939 3.6 1.9 0.7 2.7 2.7 0.2 sabinene 977 0.4 38.3 2.7 36.1 11.6 0.6 β-pinene 979 3.1 1.8 0.9 3.4 4.9 0.2 6-methyl-5-hepten-2-one 986 0.3 0.1 1.9 β-myrcene 991 0.7 3.4 0.3 2.6 1.1 5.4 α-phellandrene 1003 0.1 0.6 0.2 p-mentha-1(7),8-diene 1004 0.6 3-carene 1009 8.9 α -terpinene 1017 0.4 1.0 0.3 2.8 0.4 p-cymene 1025 4.1 0.5 1.9 0.3 3.4 0.5 d-limonene 1029 2.8 7.9 52.7 24.1 11.1 0.7 1,8-cineole 1031 0.2 0.6 cis-z-ocimene 1031 0.8 0.2 0.8 0.4 0.4 trans-e-ocimene 1037 10.9 6.7 3.2 4.8 0.6 γ-terpinene 1051 14.3 1.6 0.3 3.9 10.6 0.6 cis-sabinene hydrate 1060 0.1 0.3 0.4 0.8 0.1 trans-linalool oxide 1073 0.1 1.5 cis-linalool oxide 1087 1.6 isoterpinolene 1088 0.3 terpinolene 1089 1.6 1.7 0.5 0.8 0.9 0.2 p-cymenene 1091 0.9 0.4 0.2 linalool 1097 41.6 8.8 15.1 4.7 18.3 4.7 1,3,8-p-menthatriene 1110 0.3 0.3 cis-p-ment-2-en-1-ol 1122 0.1 0.7 cis-limonene oxide 1138 0.5 trans-p-ment-2-en-1-ol 1141 0.8 0.3 trans-limonene oxide 1142 0.1 0.5 isopulegol 1150 0.2 0.1 0.8 citronellal 1153 1.4 3.1 3.6 0.8 0.3 cis-linalyl oxide 1174 0.2 trans-linalyl oxide 1176 0.1 4-terpinenol 1177 0.2 2.5 0.7 3.9 0.6 0.2 α-terpineol 1189 0.2 0.6 0.5 0.3 0.4 0.7 cis-dihydrocarvone 1193 0.6 transdihydrocarvone 1201 0.3 trans-carveol 1217 1.4 citronellol 1226 0.3 0.5 0.7 0.3 cis-carveol 1229 0.6 nerol 1230 0.3 0.7 neral 1238 1.6 0.1 0.2 33.1 carvone 1243 2.6 geraniol 1253 0.1 3.1 geranial 1267 1.9 0.1 0.3 34.7 perillaldehyde 1272 0.4 1.0 112 a. guerrini, d. rossi, a. grandini, l. scalvenzi, p.f. noriega rivera, e. andreotti, m. tacchini, a. spagnoletti, i. poppi, s. maietti, g. sacchetti compound kia rab cn ca cl1 cl2 c1 c2 citronellyl formate 1274 0.9 2-undecanone 1294 1.3 geranyl formate 1298 0.5 0.4 carvacrol 1299 δ-elemene 1338 0.1 citronellyl acetate 1353 0.3 0.9 0.2 0.1 neryl acetate 1362 0.5 0.8 0.2 0.1 geranyl acetate 1381 0.4 0.4 β-elemene 1391 1.4 0.1 methyl methylantranilate 1406 0.3 0.2 13.1 3.1 trans-β-caryophyllene 1419 0.9 0.8 0.6 1.3 0.2 cis-carvyl propanoate 1422 0.6 γ-elemene 1433 trans-α-bergamotene 1435 0.2 α-humulene 1455 0.1 0.4 0.2 β-(e)-farnesene 1457 0.2 2-tridecanone 1470 0.5 bicyclogermacrene 1500 0.2 0.2 0.3 0.4 germacrene a 1509 0.7 0.2 germacrene b 1561 0.1 spathulenol 1578 0.1 caryophyllene oxide 1583 0.5 1-methoxy-9(e)-octadecen 1651 0.7 β-sinensal 1700 0.6 α-sinensal 1757 0.3 0.1 0.2 total identified 99.8 99.0 96.7 98.7 98.8 98.9 extraction yield (g/100g) 0.20±0.02 0.23±0.01 0.29±0.03 0.29±0.01 0.23±0.01 0.24±0.03 a arithmetic indices calculated on a varian vf-5ms column b relative peak area calculated by gc-fid. the major components (bold letters) of samples were identified by 13c nmr tab. 2: antioxidant activity of citrus petitgrains performed by dpph, β-carotene bleaching assays and compared to commercial thymus vulgaris essential oil and pure compound thymol. ic50± sd (mg/ml) essential oils dpph β-carotene bleaching cn 3.52 ± 0.25 0.387 ± 0.021 ca 7.12 ± 0.50 0.432 ± 0.037 cl1 9.90 ± 0.71 0.986 ± 0.088 cl2 7.45 ± 0.61 0.521 ± 0.038 c1 5.48 ± 0.45 0.491 ± 0.041 c2 8.41 ± 0.71 0.788 ± 0.066 thymol 0.60 ± 0.05 0.09 ± 0.011 thymus vulgaris 1.24 ± 0.10 0.164 ± 0.013 antifungal activity the most interesting results concerning antifungal activities (tab. 4) were exhibited by c2 petitgrain, particularly against dermatophytes species (t. mentagrophytes, n. cajetani), that showed ic50 less than 0.20 ml/plate, comparable to positive control t. vulgaris essential oil: however, the concentrations corresponding to the 100 % growth inhibition was better for c2. this essential oil was the most active also against phytopathogens, but less active than the reference standard t. vulgaris. c1 and cn petitgrains also showed good activity against all tested fungi reaching values of 50 % inhibition at concentration comprised from 2 to 8 ml/plate. the most sensitive fungal strain, however, appears to be t. rubrum. the study of activity of citral (mixture of neral/geranial) standard, the most abundant component in c2, against phytopathogens and dermatophytes, confirmed the best activities against t. mentagrophytes, n. cajetani, t. rubrum: in particular, t. mentagrophytes was the most sensitive fungus since it evidenced 50 % inhibition at concentration less than 0.20 ml/plate. amazonian citrus petitgrains 113 tab. 3: antimicrobial activity of citrus petitgrains compared to commercial thymus vulgaris essential oil, thymol and citral. mic (mg/ml) ± sd essential oils gram-positive bacteria gram-negative bacteria yeast s. aureus b. subtilis e. foecalis k. oxytoca e. coli p. mirabilis c. albicans atcc29213 atcc7003 atcc29212 atcc29516 atcc4350 atcc29852 atcc48274 cn 0.78 ± 0.08 0.61 ± 0.05 0.95 ± 0.09 2.17 ± 0.22 0.61 ± 0.06 0.61 ± 0.05 1.74 ± 0.19 ca 8.46 ± 0.64 1.52 ± 0.16 1.06 ± 0.11 3.38 ± 0.35 0.68 ± 0.07 0.68 ± 0.07 3.38 ± 0.34 cl1 4.06 ± 0.41 3.79 ± 0.37 1.99 ± 0.19 8.12 ± 0.61 6.32 ± 0.62 4.06 ± 0.41 0.88 ± 0.09 cl2 3.50 ± 0.31 1.73 ± 0.17 3.89 ± 0.38 6.05 ± 0.55 3.89 ± 0.38 3.89 ± 0.37 3.46 ± 0.33 c1 1.10 ± 0.12 0.44 ± 0.04 1.93 ± 0.18 4.38 ± 0.42 2.19 ± 0.22 0.61 ± 0.06 1.75 ± 0.18 c2 7.94 ± 0.59 1.94 ± 0.18 3.97 ± 0.37 6.17 ± 0.41 1.76 ± 0.17 3.97 ± 0.39 0.44 ± 0.08 thymol 0.31 ± 0.06 0.28 ± 0.03 0.52 ± 0.05 1.10 ± 0.21 0.29 ± 0.03 0.31 ± 0.02 0.90 ± 0.07 citral 0.50 ± 0.09 0.30 ± 0.07 0.58 ± 0.06 0.20 ± 0.03 0.25 ± 0.05 0.70 ± 0.07 0.42 ± 0.04 t. vulgaris 0.11 ± 0.01 0.11 ± 0.01 0.11 ± 0.01 0.40 ± 0.04 0.06 ± 0.01 0.12 ± 0.01 0.06 ± 0.01 tab. 4: antifungal activity of citrus spp. petitgrains compared to commercial t. vulgaris essential oil, citral and thymol. samples phytopathogens dermatophytes p. ultimum m. grisea b. cinerea n. cajetani t. mentagrophytes t. rubrum var. mentagrophytes cn 4.0 ± 0.2 6.3 ± 0.4 3.4 ± 0.3 3.7 ± 0.2 3.6 ± 0.2 2.7 ± 0.2 ca 12.7 ± 0.9 16.1 ± 1.1 15.3 ± 1.2 9.3 ± 0.9 >25 14.2 ± 1.2 cl1 8.3 ± 0.7 >25 20.7 ± 1.5 >25 19.9 ± 1.8 12.0 ± 1.1 cl2 10.0 ± 1.0 >25 18.6 ± 1.5 17.6 ± 1.9 18.3 ± 1.7 16.7 ± 1.4 c1 3.2 ± 0.4 7.9 ± 0.5 6.2 ± 0.4 6.7 ± 0.7 4.8 ± 0.3 2.0 ± 0.2 c2 2.2 ± 0.2 2.4 ± 0.3 1.9 ± 0.2 <0.20a <0.20b 1.9 ± 0.1 t. vulgaris 0.40 ± 0.10 0.38 ± 0.08 0.23 ± 0.04 <0.20c <0.20a <0.20c citral 1.2 ± 0.2 1.3 ± 0.1 1.4 ± 0.1 0.44 ± 0.02 <0.20b 0.88 ± 0.05 thymol 1.1 ± 0.1 2.2 ± 0.3 1.6 ± 0.2 1.1 ± 0.2 1.2 ± 0.4 0.8 ± 0.2 all the values are expressed as ic50 (ml/plate) ± standard deviation a100% growth inhibition at concentration of 2.0 ml/plate b100% growth inhibition at concentration of 1.0 ml/plate c100% growth inhibition at concentration of 5.0 ml/plate discussion the purpose of the current study was to compare the chemical composition of amazonian citrus spp. leaves essential oils with those reported in literature to determine possible different chemotypes and biological activities with the final aim to valorize their commercial use. the distillaton yields were average values among those reported for ca (blanco tirado et al., 1995; lota et al., 2001a) and cn (lota et al., 2001b); instead for cl petitgrains were lower than those re-, 2001b); instead for cl petitgrains were lower than those reported for other lemon species (vekiari et al., 2002). the ecuadorian ca petitgrain was comparable to an atypical saatypical sabinene/trans-e-ocimene chemotype, as previously reported (lota et al., 2001b). cl1 petitgrain exhibited an atypical composition with high abundance of limonene (52.7 %) and linalool (15.1 %), as reported for the meyer cultivar (c. meyeri) (lota et al., 2002), cl2 petitgrain could be defined as limonene (24.1 %)/sabinene (36.1 %)/ linalool (4.7%) chemotype, standing out the most common lemon chemotype characterized by limonene (17.8-33.5 %), a-pinene (10.5-25.1 %), geranial (8.6-22.6 %), neral (5.9-16.1 %) (lota et al., 2002). c. nobilis petitgrains (cn) evidenced a γ-terpinene/linalool chemotype, because of high abundance of linalool (41.6 %) and appreciable contents of γ-terpinene (14.3 %) and thymol (9.0 %). a similar chemotype was pointed out in a systematic research on petitgrains derived from 58 corsical mandarin cultivars from different species and 41 cultivars belonging to c. reticulata blanco (lota et al., 2000; lota et al., 2001b). mandarin leaves essential oil composition from plants of different geographical origins, floridian c. tangerine hort. ex tan and israelian c. reticulata, evidenced high content of linalool, thymol and γ-terpinene (attaway et al., 1967; fleisher and fleisher, 1990; fleisher and fleisher, 1991), while colombian c. reticulata petitgrain was characterized by an high abundance of linalool (52.66 %), but less content of γ-terpinene (1.95 %) (blanco tirado et al., 1995). the chemical composition of citrus spp. leaves essential oils (c1 and c2) does not allow to deduce any consideration about the species employed and their abundance, but it is an important starting point in making suggestions about the comparison between the bio114 a. guerrini, d. rossi, a. grandini, l. scalvenzi, p.f. noriega rivera, e. andreotti, m. tacchini, a. spagnoletti, i. poppi, s. maietti, g. sacchetti activities of the mixtures and the other essential oils. however, the studies concerning plants growing in amazonia are particularly interesting since the amazonian basin is one of the most important biodiversity hotspots where the ecological conditions and high density and diversity of species per unit area drive the plant secondary metabolism to biosynthetic pathways which are particularly rich in different chemical structures (ryder wilkie et al., 2010; rossi et al., 2013). this aspect could explain the slight differences in chemical composition detected for the essential oils, with particular reference to those belonging to c. limon (cl) samples. the confirmation of the gas chromatographic results by nmr experiments suggests this spectroscopic technique as suitable for the identification, quality control, or fraud detection of essential oils providing their good and fast discrimination. moreover, these kinds of evidences reinforce the role of non-chromatographic approach as potential tool to discriminate chemotypes, cultivar and hybrids as already suggested elsewhere (lota et al., 2001b; guerrini et al., 2006; guerrini et al., 2011). all these chemical profiles obtained through gc-ms and confirmed by nmr spectroscopy, evidence that amazonian biodiversity does not induce strong chemodiversity among citrus spp. petitgrains examined, if compared to what related literature reports, even if interesting differences regarding minor compounds were found. the examined essential oils evidenced that cn petigrain revealed the highest antioxidant activity, if compared to results obtained with commercial t. vulgaris essential oil, taken as reference phytocomplex (sacchetti et al., 2005), c1 petitgrain, with relative abundance of g-terpinene (10.6 %) and thymol (5.5 %), showed also interesting data. the antioxidant capacity displayed by essential oils could be mainly due to the presence and the abundance of thymol, as experimental results evidenced. however, with particular reference to cn sample, the relevant abundance of g-terpinene (14.3 %) could be also suggested as responsible of this biological property (choi et al., 2001), together with the presence of thymol (9.0%) (ruberto and baratta, 2000), as well as methyl-n-methylanthranilate (13.1 %) (el-ghorab et al., 2003). tab. 5: chemical shifts (13c) of compounds in citrus spp. petitgrains compound chemical shift 13c α-pinene 144.5/116.0/47.0/40.7/38.0/31.4/31.2/26.3/23.0/20.9 sabinene 154.5/101.5/37.6/32.6/30.1/29.0/27.9/19.8/19.7/16.0 β-pinene 152.2/105.9/51.6/40.7/40.5/27.0/26.1/23.5/21.8 β-myrcene 146.2/139.0/131.9/124.3/115.7/113.1/31.6/26.7/25.6/17.6 3-carene 131.4/119.4/28.3/23.7/20.8/18.4/16.8/16.6/13.2 p-cymene 145.9/135.1/129.0/126.2/33.7/24.1/20.9 d-limonene 150.3/133.8/120.6/108.3/41.0/30.8/30.6/27.9/235/20.8 trans-e-ocimene 1415/133.7/122.1/110.6/ 27.3/25.7/17.7/11.6 γ-terpinene 140.6/131.2/118.9/116.0/34.5/31.6/27.5/23.0/21.3 linalool 145.0/132.0/124.3/111.7/73.5/42.0/27.9/25.7/22.9/17.7 citronellal 202.7/131.8/124.0/51.0/36.9/27.8/25.7/25.4/19.9 4-terpinenol 133.8/118.4/71.7/36.8/34.6/30.8/27.0/23.3/16.8 neral 190.9/163.9/133.7/128.6/122.2/32.6/27.0/25.6/25.1/17.7 geraniol 139.5/131.4/124.1/123.6/59.4/39.4/26.3/25.7/17.6/16.2 geranial 191.4/163.9/132.9/127.4/122.5/40.6/25.7/25.6/17.7/17.6 thymol 152.5/136.6/131.8/126.2/121.5/116.0/26.7/22.7/20.9 methyl 169.0/151.2/134.7/131.8/115.4/110.6/51.5/29.1 methylantranilate fig. 1: 13c spectra of citrus spp. petitgrains cn petitgrain was the most effective against all the bacteria strains: mic values of ca and c1 samples were instead lower and comparable. the amounts of thymol in cn (9.0 %) and c1 (5.5 %) petitgrains could be one of the possible reasons for the antibacterial activity (burt, 2004). c2 petitgrain was instead particularly active against the yeast c. albicans, with a mic of 0.44 ± 0.05 mg/ml probably due to the high abundance of neral (33.1 %) and geranial (34.7 %), previously described as anti-candida spp. agents (silva et al., 2008) and confirmed by our results. trying to relate antimicrobial activity with chemical data, thymol has been assayed as pure compound, but no remarkable results were obtained. however, it should be stressed that higher antibacterial capacity of thyme essential oil than that of thymol could be due to a sinergic interaction involving more chemicals, thymol included. this suggestion plays certainly a role in the activities displayed by petitgrains. the most interesting results concerning antifungal activities (tab. 4) were exhibited by c2 petitgrain due to the high abundance of citral, as confirmed by experimental data. the good activities of c1 and cn petitgrains could be explained with the relative abundance of thymol, tested by us as pure compound and previously described as antifungal agent in vitro and in vivo against dermatomycoses (sokovic et al., 2008). finally, the particular interesting bioactivity of the essential oil mixtures confirmed the amazonian ethnobotany which amazonian citrus petitgrains 115 often does not discriminate citrus species in using leaves for traditional preparations, emphasizing synergic expression of different extracts/chemical compounds to have better biological performances. conclusions this first report about amazonian citrus spp. petitgrains evidenced their chemical characterization by gc and gc-ms and remarked the use of nmr as useful tool to characterize and discriminate chemotype for identification, quality control and fraud detection of essential oils (lota et al., 2001b; guerrini et al., 2006; guerrini et al., 2011). however, no remarkable difference emerged with other citrus spp. petitgrains from other geographical regions, even if interesting differences regarding minor compounds were found. in particular amazonian cn petitgrain, γ-terpinene/linalool chemotype on the basis of chemical composition defined by gc/ms and nmr, and c1 petitgrain revealed both interesting in vitro antibacterial and radical scavenging activities. result highlights that these two essential oils could be potentially employed as food preservatives or functional constituents in food supplements and/or health herbal products. moreover the antidermatophytic properties of c1, cn and the most interesting c2 leaf essential oils suggest their possible application in cosmeceuticals as antidermatophytic additives, not only as single essential oil but also as mixtures. acknowledgements the authors wish to thank dott.ssa immacolata maresca for skil-immacolata maresca for skilful technical assistance. a special thanks is also due to fundación chankuap (quito, ecuador) for the invaluable cooperation in the promotion of ecuadorian amazonic biodiversity and in the practical realization of this work. the research has been financially supported by a grant of the university of ferrara (far 2010). references adams, r.p., 2007: identification of essential oil components by gas chromatography/mass spectrometry. 4th ed. allured publishing co.: carol stream, illinois, usa. attaway, j.a., pieringer, a.p., barabas, l.j., 1967: the origin of citrus flavor components-iii. a study of the percentage variations in peel and leaf oil terpenes during one season. phytochemistry 6, 25-32. blanco tirado, c., stashenko, e.e., combarisa, m.y., martinez, j.r., 1995: comparative study of colombian citrus oils by 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(euphorbiaceae) stem bark essential oil as possible mutagen-protective food ingredient against heterocyclic amines from cooked food. food chem. 139, 439-447. ruberto, g., baratta, m.t., 2000: antioxidant activity of selected essential oil components in two lipid model systems. food chem. 69, 167-174. ryder wilkie, k.t., mertl, a.l., traniello, j.f.a., 2010: species diversity and distribution patterns of the ants of amazonian ecuador. plos one 5(10), e13146. sachetti, g., maietti, s., muzzoli, m., scaglianti, m., manfredini, s., radice, m., bruni, r., 2005: comparative evaluation of 11 essential oils from different origin as functional ingredients for foods as antioxidants, antiradicals and antimicrobials. food chem. 91, 621-632. saito, t., hayamizu. k., yanagisawa, m., yamamoto, o., wasada, n., 2013: spectral database for organic compounds, sdbs (free site organized by national institute of advanced industrial science and technology (aist), japan): http://sdbs.ds.aist.go.jp/sdbs/cgi-bin/cre_index.cgi. shaaban, h.a.e., el-ghorab, a.h., shibamoto, t., 2012: bioactivity of essential oils and their volatile aroma components: review. j. essent. oil res. 24, 203-212. silva, c.b., guterres, s.s., weisheimer, v., schapoval, e.e., 2008: antifungal activity of the lemongrass oil and citral against candida spp.. braz. j. infect. dis. 12, 63-6. sokovic, m., glamoclija, j., ciric, a., kataranovski, d., marin, p.d., vukojevic, j., brkic, d., 2008: antifungal activity of the essential oil of thymus vulgaris l. and thymol on experimentally induced dermato116 a. guerrini, d. rossi, a. grandini, l. scalvenzi, p.f. noriega rivera, e. andreotti, m. tacchini, a. spagnoletti, i. poppi, s. maietti, g. sacchetti mycoses. drug dev. ind. pharm. 34, 1388-93. vekiari, s.a., protopapadakis, e.e., papadopoulou, p., papanicolaou, d., panou, c., vamvakias, m., 2002: composition and seasonal variation of the essential oil from leaves and peel of a cretan lemon variety. j. agric. food chem. 50, 147-153. address of the corresponding author: department of life sciences and biotechnology (sveb), university of ferrara, c.so ercole i d’este 32, i-44121 ferrara, italy. e-mail: grrlsn@unife.it art31 journal of applied botany and food quality 82, 193 198 (2009) 1institute of agricultural and nutritional sciences, martin luther university halle wittenberg, halle (saale), germany 2department of geography, university of zurich, zürich, switzerland 3institute of food technology, hohenheim university, stuttgart, germany root exudation of phloridzin by apple seedlings (malus x domestica borkh.) with symptoms of apple replant disease a. hofmann1, 2, l. wittenmayer1, g. arnold3, a. schieber3, w. merbach1 (received january 29, 2009) summary this study investigates the occurrence of the flavonoid phloridzin (phloretin-2'-o-β-d-glucoside) in root exudates of apple seedlings showing growth reduction related to apple replant disease (ard). the disease is most likely caused by a complex of soil-borne fungi and bacteria, but the etiology remains to be elucidated. information on specific exudation processes in the rhizosphere of apple seedlings could contribute to our understanding of the conditions triggering ard development. to procure ard symptoms, apple seedlings (malus x domestica borkh.) were grown in ard-conductive soil. root exudates were collected by submerging the roots in a solution of 0.05 mm cacl2 for a period of 4 h. the fraction of phenolic root exudates was analyzed using hplc/dad (high performance liquid chromatography/diode array detector). results suggest that (i) phloridzin is a constant root exudate of apple seedlings. it was the most abundant phenol in the collected exudates from replant-diseased as well as healthy seedlings. (ii) phloridzin exudation, related to root dry matter, was the most intensive at the onset of ard symptom development and lower during the period when symptoms were most severe or outgrown. (iii) in comparison to healthy seedlings, the phloridzin exudation of apple replantdiseased seedlings was significantly higher only at the onset of ard symptom development, suggesting a response of the plants to infection. the finding of phloridzin in the root exudates of malus x domestica borkh. might have consequences for research on the etiology of ard. specialized pathogenic microorganisms could be attracted by this distinct compound. since it is very characteristic of apple plants, phloridzin might be the compound that ard-causing microorganisms utilize to recognize their host. for practical applications, phloridzin root exudation could therefore be a parameter in evaluating ardsusceptibility of different rootstocks. introduction apple replant disease is a soil-borne disease, which has been reported to occur in apple-growing areas throughout the world, in europe (savory, 1966; hoestra, 1968; manici et al., 2003), north america (willet et al., 1994), and australia (dullahide et al., 1994). symptoms include general growth reduction, browning of roots, and stunting of the shoot (savory, 1966; hoestra, 1968; caruso et al., 1988). these symptoms have been described for malus as well as other species of the rosaceae family, subfamily maloideae (otto and winkler, 1995). the fact that sterilization of soil leads to healthy plants supports the hypothesis that ard is caused by soil-borne microorganisms. pathogens suggested to be involved are actinomycetes (otto and winkler, 1977; westcott et al., 1986), bacteria such as bacillus subtilis (utkede and li, 1988), and fungi, e.g., pythium intermedium (sewell, 1980; manici et al., 2003). mazzola (1998) suggests that rather than by a single organism, ard is caused by a complex of several microbial species. to reduce effects of apple replant disease, orchard management relies on land shifting or sterilization of affected soil by fumigation, e.g. with methyl bromide. because of its general toxicity to soil organisms, several studies of the past years have focused on finding alternative techniques to fumigation, like organic soil amendments or alternative rootstock genotypes to combat apple replant disease (mazzola et al., 2001; rumberger et al., 2004; wilson, 2004; yao et al., 2005). in order to find effective techniques, research on the underlying mechanisms of the disease is still necessary. information on rhizosphere processes during the disease might be pivotal in explaining the involvement of microorganisms in ard and might provide new strategies against ard. we hypothesize that specific biochemical conditions in the rhizosphere of apple roots, like the root exudation of certain compounds, trigger plant-microbe interactions that lead to the disease. it has been suggested that particular or rare compounds from root exudates of a given plant species might have specific functions in the rhizosphere of that species (schroth and hildebrand, 1964; rovira, 1969; werner, 2001). for flavonoids, belaños-vasquez and werner (1997) and jain and nainawatee (2002) suggested a role of signal molecules for microorganisms in the rhizosphere. signal molecules in root exudates might attract specialized microorganisms to their host plants. this could help explain the specificity of certain plantmicrobe interactions. o o oh oh oh o oh oh oh ch 2 oh fig. 1: phloridzin (phloretin-2'-o-β-d-glucoside) the flavonoid phloridzin (phloretin-2'-o-β-d-glucoside, fig. 1) could be such a root exudate with specific functions in the rhizosphere of apple seedlings. it is a compound characteristic to the genus malus, yet not exclusive to that genus, since it has been detected also in fragaria (hilt et al., 2003). in apple plants, phloridzin has been detected in roots, leaves, buds and fruit (börner, 1959; raa, 1968; antolovich et al., 2000; hrazdina, 2003), but not yet reliably identified as a part of the root exudates (szabó and wittenmayer, 2000). börner (1959) was the first to examine the role of phloridzin in relation to the symptoms of apple replant disease (ard). he determined phloridzin concentrations in apple plant residues from soil of former apple orchards to assess possible toxic effects on newly planted apple trees. the concentrations and persistency of phloridzin were too low to explain the occurring symptoms. börner later suggested that, instead of leaching from apple plant residues, 194 a. hofmann, l. wittenmayer, g. arnold, a. schieber, w. merbach phloridzin in soil could also originate from active root exudation of apple plants and could thus be directly released into the rhizosphere (börner, 1960). the objectives of our study were to (i) prove the presence of phloridzin in the fraction of phenolic root exudates of malus x domestica borkh., (ii) quantify changes in phloridzin exudation in relation to development of replant disease symptoms, and (iii) investigate differences in phloridzin exudation between healthy and ard-infected plants. materials and methods preparation of cultivation substrates the original soil was collected from the topsoil (approx. 0-30 cm) of a previously cleared apple orchard site suspected to be affected by ard. this soil was sieved (5 mm mesh size) for homogenization and in order to remove large plant residues. part of the soil was sterilized by gamma radiation (60co, radiation dose 35 kgy, gamma service produktbestrahlung, radeberg, germany). chemical soil parameters of the original and sterilized soil were compared using standard methods of soil analysis. no differences in ph or plantavailable macroand micronutrients were observed. the cultivation substrates, hereafter referred to as "ard-conductive soil" and "sterilized ard soil", were prepared by mixing the original or gamma-radiated soil with an equal amount of coarse quartz sand (1-2 mm). the sand was added to improve aeration in the pots and facilitate root growth. apple seedling cultivation in order to investigate the possibility of phloridzin root exudates being involved in the development of ard, we carried out a three-month in-vitro experiment with apple seedlings grown in ard-conductive soil and sterilized soil for control. apple seedlings were grown from seeds (g. j. steingaesser, miltenberg, germany) of malus x domestica borkh., a variety used for tall tree rootstocks. when three pairs of primary leaves had developed, the seedlings were transplanted into the prepared substrates. the pots had volumes of 0.2 to 0.9 l, in accordance with expected plant size at future dates of the root exudates collection. they were lined with thin plastic bags (perforated at the bottom) to prevent root system damage at the time of removing the plants from containers for measurements. seedlings were watered daily to maintain the water-holding capacity of the substrate at 80%. plants were sprayed with dimethoate (against aphids) and also with sulfur and fenarimol (as alternating applications, against mildew). fertilization was not included in the experiment. seedlings did not show any symptoms of nutrient deficiency. collection of root exudates we collected root exudates of the grown seedlings by submersion of the roots in a solution of 0.05 mm cacl2 (egle et al., 2003). this approach is similar to the dipping method described and evaluated by gransee and wittenmayer (2000), who concluded that the method is suitable for nearly complete sampling of root exudates. collection of root exudates was started 30 days after the transfer of the seedlings into the substrates and repeated on every ninth day for a total of ten collection dates, corresponding to 30, 39, 48, 57, 66, 75, 84, 93, 102 and 111 days after the transfer into the substrates. the seedlings were carefully removed from the pots and substrate. the roots were gently rinsed with water to prevent damage of the root system. subsequently, the entire root systems of three intact seedlings were submerged in a solution of 0.05 mm cacl2 in a glass beaker wrapped with aluminum foil to prevent the roots from light exposure. the plants were set up in a climate chamber (22°c, 70 % relative humidity, light intensity 500 µm m-2 s-1). after one hour of equilibration, the solution was discarded and replaced with fresh solution of 0.05 mm cacl2. the plants were then allowed to continue exudation into the solution for 4 h. the total amount of root exudates is attributed to this time period. the solution containing the root exudates was shock-frozen in liquid nitrogen, lyophilized at -25°c, and stored in the dry state at -70°c. separation and quantification of the phenolic component for analysis of the phloridzin contents in the acquired root exudates, we chose an established hplc-method originally applied in food technology for the detection of phenolic compounds in fruit juices (schieber et al., 2001). the phenolic compounds of the root exudates were separated by solid phase extraction using reversephase cartridges (dsc18, 3 ml, 500 mg, particle size 56 µm, supelco, bellefonte, pa, usa). about 25 mg of the lyophilized sample was dissolved in 2 ml of 0.5% acetic acid. prior to extraction, the columns were activated with 2 ml of methanol and conditioned with 2 ml of 0.5% acetic acid. the phenolics were eluted from the column with 2 ml of methanol. the solvent was evaporated at 25 °c in vacuo and the phenolics were redissolved in 0.2 ml acetonitrile and 0.8 ml 2 % acetic acid for hplc analysis. the c18 columns were weighed before and after extraction to determine the amount of insoluble residues. hplc analysis of the phenols was performed as described by schieber et al. (2001) using a reversed-phase stationary phase (125 aqua c18 column, 250 x 4.6 mm i.d., particle size 5 µm, phenomenex, torrance, ca, usa). the hplc system (merck/vwr international, darmstadt, germany) consisted of a degasser l-7614, hplc-pump l-7100, column thermostat l-7350 and a diode-arraydetector l-7455. phloridzin was identified by comparison of the retention time with that of the authentic reference substance (fluka, seelze, germany) and by lc-ms/ms (liquid chromatography/ tandem mass spectrometry). determination of plant parameters upon collection of root exudates, plant parameters were determined in order to relate them to phloridzin exudation and quantitatively describe the growth reduction of replant-diseased apple seedlings. the following root and shoot parameters were measured: root surface (sattelmacher et al., 1983), root and shoot dry weight (gravimetrically), and leaf surface by digital image analysis (software analysis 3.0, soft imaging system gmbh, münster, germany). statistics for measurements, nine apple seedlings per cultivation substrate were harvested. the nine seedlings were pooled in groups of three for collection of exudates, allowing three replicates in hplc analysis (n=3). plant parameters were determined individually for the selected nine plants (n=9). for data analyses, spss 11 software was used (chicago, il, usa). normal distribution was confirmed with the kolmogorov-smirnovtest. significant differences were detected using one factorial analysis of variance and duncan-test. results apple replant disease symptoms apple seedlings grown in soil conductive to ard displayed significant growth reductions of shoots and roots and were considered phloridzin exudation by apple seedlings 195 "apple replant-diseased plants", whereas seedlings grown in the same but sterilized soil did not show any symptoms of ard and were thus considered „healthy plants“. after the cultivation period of 111 days, seedlings grown in ard soil had built up only 34% of the shoot dry matter of healthy plants. leaf area was 40% and root dry matter was 30% compared to healthy plants (data not shown). growth reduction is visualized in fig. 2 a c. the prerequisite for the comparison of the phloridzin root exudation of diseased and healthy plants was thus achieved. phloridzin in root exudates phloridzin was found to be the most abundant compound in the phenolic component of the root exudates of both replant-diseased and healthy apple seedlings throughout the measurement period (81 d). beside phloridzin (phloretin-2'-o-β-d-glucoside), one more compound was identified in the phenolic root exudates, namely phloretin (4‚2'‚4'‚6' tetrahydroxydihydrochalcon), the aglycone of phloridzin. authentication was achieved by lc-ms/ms (quasi molecular ion peak [m-h]at m/z 435 for phloridzin, and a fragment of m/z 273 for the phloretin aglycon, hilt et al., 2003). other compounds were present unregularily or in small quantities and could not be identified. fig. 3 shows a typical chromatogram indicating the spectrum of various substances of the phenolic fraction of the root exudates with phloridzin being the dominant compound. total root exudates, defined as the soluble component of the lyophilized exudation solution, increased with plant age (tab. 1). healthy plants did not consistently exude significantly higher amounts of total root exudates, yet there was a tendency toward this for the last four measurements (tab. 1). concentrations of phloridzin in the root exudates were significantly higher for diseased seedlings 30 days after transfer into ardconductive soil (tab. 1), indicating quantitative and qualitative changes in root exudation as a first response to infection. subsequent measurements of phloridzin concentrations did not show differences between the treatments. when phloridzin exudation was considered in relation to synthesizing capacity of seedlings, such as root dry weight and leaf area, the same pattern was noticed: phloridzin exudation was significantly higher for the replant-diseased seedlings only at the first measurement (30 days after transplanting into cultivation substrates, tab. 2). for subsequent analyses of exudates, a general trend of higher phloridzin exudation by replant-diseased seedlings could be observed (tab. 2). discussion amounts of exuded phloridzin in comparison to carbohydrates or organic acids (szabó and wittenmayer, 2000), phenolic compounds occur at a lower concentration in apple seedlings and in their root exudates. the phenolic compound phloridzin was exuded by apple seedlings in the range of only a few micrograms per plant during a four-hour time period. this is two magnitudes lower than the amount of, for example, citric acid, as found by egle et al. (2003) using the same method for quantification of root exudates in lupin. in the study by hrazdina (2003), values for phloridzin contents in roots of (micropropagated) apple seedlings are in a range of 400 to 1200 µg g-1 root fresh weight. our results for exudation of phloridzin per g root dry weight were in the range of 2 to 30 µg. assuming a root dry matter content of 20%, we can state that less than one-hundredth of the phloridzin amount accumulated in the root was released into the rhizosphere. phloridzin should be apparently considered a trace substance and not a significant carbon source for potential root-pathogenic organisms. for replant-infected seedlings, leakage from cells might occur, since fig. 2: a c significant growth reduction of apple replant-diseased (left) compared to healthy (right) apple seedlings, in parentheses time after transfer of seedlings to cultivation substrate. a) shoots (111 d); b) root system (93 d); c) leaves (111 d). a) b) c) 196 a. hofmann, l. wittenmayer, g. arnold, a. schieber, w. merbach 1 2 3 fig. 3: hplc-chromatogram of phenolic root exudates of replant-diseased apple seedlings (30 days after transplanting in ard-conductive soil). 1 – injection peak, 2 – phloridzin, 3 – phloretin. tab. 2: phloridzin exudation (µg) per g root dry weight and per dm2 leaf area during 4 h. comparison between apple seedlings cultivated in apple replant disease conductive soil or in the same but sterilized soil. time after exuded phloridzin per root dry weight (µg g-1)a exuded phloridzin per leaf area (µg dm-2)a transfer to soil (d) ard-conductive soil sterilized ard soil pd ard-conductive soil sterilized ard soil p 30 30.2b (2.4)c 6.8 (2.5) *** 5.2 (0.5) 1.5 (0.3) *** 39 15.6 (9.4) 8.1 (0.3) n.s. 4.1 (2.4) 2.6 (0.2) n.s. 48 9.4 (2.7) 5.7 (3.2) n.s. 4.0 (1.1) 2.2 (1.2) n.s. 57 5.8 (3.7) 1.7 (0.1) n.s. 3.8 (2.5) 0.7 (0.1) n.s. 66 6.4 (5.1) 5.0 (2.2) n.s. 4.2 (3.7) 2.2 (0.6) n.s. 75 7.5 (5.5) 2.7 (0.9) n.s. 2.6 (1.8) 0.6 (0.3) n.s. 84 6.6 (2.2) 6.8 (1.5) n.s. 2.1 (0.5) 1.3 (0.2) n.s. 93 5.8 (2.5) 5.4 (1.5) n.s. 2.3 (0.8) 1.8 (0.6) n.s. 102 11.8 (8.5) 2.8 (1.1) n.s. 5.2 (3.0) 1.2 (0.6) n.s. 111 4.5 (2.3) 10.6 (11.7) n.s. 1.9 (0.6) 5.3 (4.7) n.s. a n=3 b mean c standard deviation of the mean d degree of significance of p: p <0.05*, p <0.01**, p <0.001***, n.s. not significant tab. 1: root exudates (mg) released per seedling and concentration of phloridzin (%) in exudates during 4 h. comparison between apple seedlings cultivated in apple replant disease conductive soil or in the same but sterilized soil. time after root exudates per plant (mg)a phloridzin concentration in root exudates (% )a transfer to soil (d) ard-conductive soil sterilized ard soil pd ard-conductive soil sterilized ard soil p 30 0.77b (0.18)c 1.17 (0.09) * 0.14 (0.04) 0.03 (0.00) ** 39 1.08 (0.13) 0.78 (0.08) * 0.11 (0.08) 0.14 (0.01) n.s. 48 0.66 (0.14) 0.70 (0.14) n.s. 0.28 (0.16) 0.25 (0.19) n.s. 57 0.68 (0.47) 1.00 (0.71) n.s. 0.31 (0.20) 0.10 (0.05) n.s. 66 0.63 (0.27) 1.11 (0.29) * 0.34 (0.22) 0.24 (0.14) n.s. 75 1.78 (0.33) 0.68 (0.57) * 0.20 (0.16) 0.42 (0.19) n.s. 84 2.03 (0.11) 2.79 (2.50) n.s. 0.19 (0.06) 0.29 (0.14) n.s. 93 2.70 (0.33) 4.94 (0.69) n.s. 0.13 (0.05) 0.19 (0.06) n.s. 102 3.34 (1.27) 5.30 (1.69) n.s. 0.32 (0.22) 0.14 (0.03) n.s. 111 3.22 (0.95) 12.41 (7.81) n.s. 0.16 (0.05) 0.30 (0.13) n.s. a n=3 b mean c standard deviation of the mean d degree of significance of p: p <0.05*, p <0.01**, p <0.001***, n.s. not significant phloridzin exudation by apple seedlings 197 the root cortex of infected plants becomes heavily damaged, as described by caruso et al.(1989) in a histological study. to prevent contamination of the exudates with leaked compounds during the collection, the equilibration step was applied. the raised phloridzin values for the first measurement (30 d upon the transfer into cultivation substrate) in our study might point to leakage as a result of cell damage inflicted by root pathogens. however, the results indicate that the total root exudation was not consistently higher for the diseased plants. the trend was even the opposite towards the end of the observation period, when the healthy plants had developed root systems three times larger than the diseased plants, with an adequately higher capacity to exude compounds into the rhizosphere. thus the raised phloridzin content at the early-measurement point was genuine. furthermore, at this early measurement point, the phloridzin concentration in the exudates was increased, suggesting a qualitative plant response to the contact with the ard-conductive soil. phloridzin – a constant component in root exudate of apple seedlings in previous approaches (szabó and wittenmayer, 2000; wittenmayer und szabó, 2000), a specific substance was searched for within the root exudates of malus species that could attribute the specificity of ard to a rhizosphere interaction, involving a distinct root exudate and an attracted specific pathogen. the results of this study show that the suspected phloridzin indeed occurs in root exudates of the examined apple seedlings. it was exuded in detectable amounts throughout the entire observation period. otto et al. (1993), who investigated the infestation of apple seedling roots with actinomycetes in relation to ard, observed that apple seedlings were susceptible to infection while the shoot was growing, whereas seedlings with a dormant terminal bud did not show newly infected root tissue. this observation gave rise to the hypothesis that a malus-specific compound may be exuded during a certain time period, and during that period the compound serves as an attractant to pathogens. this hypothesis was disproved by our finding that phloridzin was exuded throughout the measurement period, and not at specific periods only. however, the fact that a higher exudation of phloridzin was found at the very beginning of the measurements, points to a quantitative and qualitative response of the plants just shortly upon transfer to the ard-conductive soil, i.e., during the period when most likely the infection occurs. in a study on the influence of polyphenols on the resistance of apple leaves to the fungal pathogen venturia inaequalis, raa (1968) found an antimicrobial effect produced by phloridzin. taking this finding into account, the higher root exudation of phloridzin could be interpreted as a defense reaction of apple seedlings, though not an effective one, against microorganisms involved in ard. in another study of the role of phloridzin in plant disease (dreyer and jones, 1981), two opposite effects were suggested: repellence as well as attraction. these reversal effects have been explained by harborne (1994). secondary plant substances, whose primary function is in defense, might become attractant substances if a microorganism would have overcome the repellency in order to avoid competitors. since phloridzin occurs only in small amounts, the original function in defense might be ineffective, as we found. meanwhile its second possible function as attractant or signal molecule should be considered. conclusions with this study we were able to (i) confirm the occurrence of phloridzin in root exudates of malus x domestica borkh. seedlings. phloridzin was the dominant compound in the fraction of detected phenolic root exudates. this substance was present in all samples throughout the observation period in replant-diseased as well as healthy plants. therefore, we conclude that phloridzin is a constant component of root exudates in malus. (ii) in apple replant-diseased plants, phloridzin exudation was the most intensive at the onset of ard symptom development and lower during the period when symptoms were most severe or outgrown. we therefore deduce that the symptom expression itself did not stimulate phloridzin exudation. we suggest that the higher phloridzin exudation at the onset of the disease can be considered as a response of the plants to infection. this is also supported by (iii) a significant difference in the amount and concentration of exuded phloridzin between the diseased and healthy apple seedlings 30 days after the transplanting. seedlings cultivated in ard-conductive soil exuded significantly more phloridzin compared to healthy seedlings. considering our results, we assume two functions for phloridzin exudation: the primary one, as a part of the defense mechanism, but failing to suppress disease development, supported by results (ii) and (iii); and the secondary, as a host-signaling compound utilized by microorganisms, which is supported by result (i). as a constant component of the root exudates of apple plants, phloridzin might have become an attractant for microorganisms that have developed resistance to this plant defense mechanism. this possible secondary function of phloridzin should be taken into account in future investigations on possible ard pathogens. for future testing of apple rootstocks we propose to screen seedlings with different phloridzin exudation patterns for their susceptibility to ard. references antolovich, m., prenzler, p., robards, k., ryan, d., 2000: sample preparation in the determination of phenolic compounds in fruits. analyst 125, 989-1009. belaños-vasquez, m.c., werner, d., 1997: effects of rhizobium tropici, r. etli, and r. leguminosarum by phaseoli on nod gene-inducing flavonoids in root exudates of phaseolus vulgaris. molecular plantmicrobe interactions 10, 339-346. börner, h., 1959: the apple replant problem. i. the excretion of phlorizin from apple root residues. contributions of the boyce thompson institute of plant research 20, 39-56. börner, h., 1960: liberation of organic substances from higher plants and their role in the soil sickness problem. botanical rev. 26, 393-424. caruso, f.l., neubauer, b.f., begin, m.d., 1989: a histological study of apple roots affected by replant disease. canad. j. botany 67, 742-749. dreyer, d.l., jones, k.c., 1981: feeding deterrency of flavonoids and related phenolics towards schizaphis graminum and myzus persicae: aphid feeding deterrents in wheat. phytochemistry 20, 2489-2493. dullahide, s.r., stirling, g.r., nikulin, a., stirling, a.m., 1994: the role of nematodes, fungi, bacteria, and abiotic factors in the etiology of apple replant problems in the granite belt of queensland. aust. j. exp. agric. 34, 1177-1182. egle, k., römer, w., keller, h., 2003: exudation of low molecular weight organic acids by lupinus albus l., lupinus angustifolius l. and lupinus luteus l. as affected by phosphorous supply. agronomie 23, 511-518. gransee, a., wittenmayer, l., 2000: qualitative and quantitative analysis of water-soluble root exudates in relation to plant species and development. j. plant nutr. soil sci. 163, 381-385. harborne, j.b., 1994: the flavonoids: advances in research since 1986. chapman & hall/crc, 601. hilt, p., schieber, a., yildirim, c., arnold, g., klaiber, i., conrad, j., beifuss, u., carle, r., 2003: detection of phloridzin in strawberries (fragaria x ananassa duch.) by hplc-pda-ms/ms and nmr spectroscopy. j. agric. food chem. 51, 2896-2899. hoestra, h., 1968: replant diseases of apple in the netherlands. meded. 198 a. hofmann, l. wittenmayer, g. arnold, a. schieber, w. merbach landbouwhogeschool wageningen nederland 68-13. hrazdina, g., 2003: response of scab-susceptible (mcintosh) and scabresistant (liberty) apple tissues to treatment with yeast extract and venturia inaequalis. phytochemistry 64, 485-492. jain, v., nainawatee, h.s., 2002: plant flavonoids: signals 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247-260. sattelmacher, b., klotz, f., marschner, h., 1983: vergleich von zwei nichtdestruktiven methoden zur bestimmung der wurzeloberflächen. z. pflanzenern. bodenk. 146, 449-459 (in german with english summary). savory, b.m., 1966: specific replant diseases. commonwealth agricultural bureaux, farnham royal, bucks, england. szabó, k., wittenmayer, l., 2000: pflanzenspezifische wurzelabscheidungen als mögliche ursache der spezifität der bodenmüdigkeit bei rosaceen. angew. bot. 74, 191-197 (in german with english summary). schieber, a., keller, p., carle, r., 2001: determination of phenolic acids and flavonoids of apple and pear by high-performance liquid chromatography. j. chromatography a 910, 265-273. schroth, m.n., hildebrand, d.c., 1964: influence of plant exudates on root infecting fungi. ann. rev. phytopath. 2, 101-132. sewell, g.w.f., 1980: effects of pythium species on the growth of apple and their possible causal role in apple replant disease. ann. appl. biol. 97, 31-42. utkhede, r.s., li, t.s.c., 1988: the role of fungi, bacteria, and their interactions in apple replant disease complex in soils of british columbia. acta horticult. 233, 75-80. werner, d., 2001: organic signals between plants and microorganisms. in: pinton, r., varanini, z., nannipieri, p. (eds.), the rhizosphere, 197-222. marcel dekker inc., new york. westcott, s.w. iii, beer, s.v., israel, h.w., 1986: infection of apple roots by actinomycetes associated with soils conductive to apple replant disease. plant disease 70, 1125-1128. willett, m., smith, t.j., peterson, a.b., hinman, h., stevens, r.g., ley, t., tvergyak, p., williams, k.m., maib, k.m., watson, j.w., 1994: growing profitable apple orchards in replant sites: an interdisciplinary team approach in washington state. horttechnology 4, 175-181. wilson, s., andrews, p., nair, t.s., 2004: non-fumigant management of apple replant disease. sci. horticult. 102, 221-231. wittenmayer, l., szabó, k., 2000: the role of root exudates in specific apple (malus x domestica borkh.) replant disease (sard). j. plant nutr. soil sci. 163, 399-404. yao, s., merwin, i.a., abawi, g.s., thies, j.e., 2005: soil fumigation and compost amendment alter soil microbial community composition but do not improve tree growth or yield in an apple replant site. soil biol. biochem. 38, 587-599. address of authors: anett hofmann, university of zurich, department of geography, winterthurer strasse 190, ch-8057 zürich, switzerland dr. lutz wittenmayer, prof. dr. wolfgang merbach (corresponding author), martin-luther university, institute of agricultural and nutritional sciences, julius-kühn-straße 25, d-06112 halle (saale), germany e-mail: wolfgang.merbach@landw.uni-halle.de gabi arnold, dr. andreas schieber, hohenheim university, institute of food technology, garbenstraße 25, d-70599 stuttgart, germany << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile 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/pagesize [907.087 680.315] >> setpagedevice art08 journal of applied botany and food quality 80, 155 159 (2006) institute of vegetable and ornamental crops großbeeren / erfurt e.v., department quality, germany distribution of nutritive compounds and sensory quality in the leafs of chives (allium schoenoprasum l.)* bernhard brueckner, henrike perner (received june 26, 2006) summary the distribution of sulphur, thio-sulphinates (pyruvic acid method), ssc, colour, freshand dry matter was analysed in three sections, base, centre, and tip, along the leaf tube of chives. also the intensity of 28 sensory attributes was determined in these sections by a trained quantitative, descriptive panel. the sections differed in their mouthfeel attributes – centre and basis were juicier and crisper than the tips, which were strawy/fibrous and drier. at higher pyruvic acid and ssc concentrations in the tips, more punceny and sweetness was expected, but no increased values were found. very low juiciness to convey pungent and sweet compounds and very low fresh matter related to leaf length were identified as possible reasons for this inconsistency. introduction chive leaves, grown under greenhouse forcing conditions show a gradual change of green colour along the leaf tube. the earlier, prior plant parts toward the leaf tip have more time to synthesise chlorophyll, leading to more intensive, dark green colour. basal leaf parts are often much brighter. according to observations in the production chain the brighter, basal parts offer an increased culinary quality, particularly because of more juiciness. dissolved compounds in the augmented amount of juice may be the reason for increased flavour intensity. preliminary measurements confirmed the assumed higher water content in these plant parts. imported chives from kenya or israel, present on the market, show much less inhomogeneity with regard to colour, but compound and sensory quality distribution is not known. if improved sensory properties of brighter leaf areas can be demonstrated scientifically, a balanced ratio of brighter and darker leaf sections should be recommended from a consumer perspective. this study was proposed to determine the distribution of nutritive and sensory properties along the leaf tubes of chives. material and methods plant material german chive leaves (cv. divonne, rijk zwaan, welver) were harvested on june 5th , 2005 and delivered to the gartenbauzentrale papenburg after 3 to 5 days of forcing in the greenhouse according to usual professional methods. the israeli chives were imported from sharon binyamin, carmel, israel and were obtained on the same day as the harvested material from the gartenbauzentrale papenburg. the israeli and german samples were cooled during the ½ day road transport to grossbeeren, germany on june, 6th. here the containers were opened and stored overnight at 4 ± 2 °c and 80 % relative humidity in a cold storage room. one half of the german samples was analysed together with the israeli samples the next day. the other half of the german samples was stored for one week under the described cool storage conditions. the israeli samples were not stored. instead, fresh israeli samples were obtained in the same manner one week later. because the previous history of the israeli chives was not known in detail, the samples were regarded as random samples. definition of leaf sections the chive leaves were divided into three longitudinal sections. the basal part was cut at a length of 8 cm, the central part at 12 cm, and the tip end had a length of 8-12 cm according to the actual growth length. the analyses were conducted for all sections separately. colour measurement leaf colour was measured with a minolta lr 321 colour-meter (minolta camera co., osaka, japan) using a white standard and standardized light type d65. colour measurements were expressed in the l*a*b* scale, where l* denotes luminescence on a 0 to 100 scale from black to white, a* (+) red or (-) green and b* (+) yellow or (-) blue light. an average of twenty single measurements on two bundles of chives was recorded for each sample. dry matter content, soluble solid compounds and weight loss the dry matter content was determined after drying four replicates of 50 g each for three days at 80 °c. soluble solids compounds (ssc) were determined by extracting the juice from at least 15 g fresh sample in a household centrifuge juicer and measured with a handheld refractometer (digital refractometer pr-1, atago, tokyo, japan). the measurement is based on the capacity of sugar in a juice to deviate light and gives the proximate sugar content in °brix (ahlers, 2001). weight losses of four replicates with 50 g chive leaves in each sample were determined after a 24 hour period of uncovered storage in the cold storage room. pyruvic acid and sulphur total pyruvic acid (pa) was analysed in 20 g ruptured shoot tissue using the method described by schwimmer et al. (1961) and randle et al. (1993). the fresh shoot tissue was homogenised with 30 ml distilled water, after 20 min mixed with 5 % trichloroacetic acid solution (1:1) and left standing for 1h. the filtrate was mixed with the indicator 0.0125 % 2,4-dinitrophenylhydrazine in 2n hcl, then alkalined with 0.6 n naoh, and the measured transmission at 420 nm. background levels of pa of intact onion tissues were assumed to be negligible and constant (yoo and pike, 2001), so that background pyruvic acid was not measured. total sulphur was analysed in an elementary analyser (high temperature oxidation) and detected with non-dispersive infrared technique (ndir) (multi ea 2000, analytik jena ag, germany). * the paper was presented at the 41th meeting of the „deutsche gesellschaft für qualitätsforschung (pflanzliche nahrungsmittel) dgq e.v.“ sensory analysis the descriptive sensory analysis was conducted to characterise the attributes of the different sections of the chive leaf material using a trained panel similar to the method of (stone et al., 1993). eight judges were selected who had successfully passed standardised tests for olfactory, taste and colour sensibility as well as for commemoration, verbal abilities and creativity. approximately 20 h training over a 10-week period for establishing the methodological foundations was followed by another 20 h training with allium species. leaf samples were presented individually and in random order on a cutting board. leaves were cut and squeezed according to a specified scheme. a four digit code was assigned to the samples. 28 odour-, flavour-, mouthfeeland aftertaste attributes were distinguished and assessed using unstructured line scales with the anchor points 0 – not perceptible and 100 – strongly perceptible. among the flavour attributes were: green/grassy, leek-like, garliclike, onion-like, cabbage-like, sweet, bitter, fruity, earthy. mouthfeel attributes were: burning/pungent, adstringent, dry/juicy, crisp, strawy/ fibrous. statistical analyses the data were analysed using the software packages statistica v. 4.1 (statsoft), and spss v. 7.5 (spss inc.). analytical data were subject to anova and duncan-test at the significance level of p=0.05. results instrumental colour values the colour values differed between the three sections of both geographic origins in a similar way. the basal section was brighter, green and yellow colour notes were somewhat more intensive than the central and apical section. the section differences were minor in the israeli samples. between the samples large luminescence (l*) differences were found (tab. 1). tips and central parts of israeli chives produced values of 34 to 38 units, thus characterising dark material. the second sample was darker than the first. with l* values of 40 to 42 the basal sections were significantly brighter than the other sections. the german greenhouse samples were brighter than the israeli samples. the luminescence values of the fresh german samples decreased significantly from the basis to the tip (tab. 1). the one week storage period of the german chives had no influence on their luminescence (l*) values. the intensity of green colour is indicated by negative a* values in the cie-lab system (tab. 1). the tip and central sections of the israeli samples were not always significantly different in their a* values. but the basal parts had significantly lower (greener) values at both sampling dates compared to the tip and central section. the german samples had lower a* values than the israeli samples. the german samples were less green at the basal and central sections than the tip sections. there was no a* change during storage. the figure b* indicates the intensity of yellow colour notes (tab. 1). similar to a* values, the israeli b* values show a significant higher yellow colour intensity in the central and top sections compared to the basal section. the german samples had significantly increasing yellow colour intensities towards the tip of the leaf. storage did not affect b* values. dry matter content dry matter content was significantly different between the sections (tab. 2). the basal sections contained the least, the tip sections the highest percentage of dry matter per total mass. in all sections the israeli samples had significantly higher dry matter ratio than the german samples. after storage the dry matter ratio of german samples was significantly higher in the central section only. soluble solid compounds the concentration of soluble solid compounds (ssc) increased from the basis towards to the tips (tab. 2). very high values were detected in one sample from israel; but at both dates, the israeli samples had higher concentrations in all sections compared to the papenburg material. storage led to a slight, not significant increase of ssc in each of the three sections. weight loss no differences were found in the weight loss of all samples and between the sections, because of large variation within the material. there was a tendency for higher losses in the tip section of the leaves, especially in stored german sample (tab. 2). pyruvic acid concentration the indirect pyruvic acid method allows to determine the amount of thio-sulphinates, organo-sulphur compounds, which are responsible for the allium flavours and pungency (randle, 1992; randle et al., 1993). the pyruvic acid concentration was highest in the tip sections, and slightly lower in the central and basal sections (tab. 3). only in the israeli sample this difference was significant. the pyruvic acid concentration in the chive leaves from germany was in all sections significantly higher than those in the israeli samples. the one week storage of the german sample did not affect pyruvic acid contents. tab. 1: results of the instrumental colour measurement (cielab-system). luminescence green a* yellow b* l* germany 07.06.05 basis 53.5 g -16.7 a 33.5 f centre 46.0 f -16.8 a 29.9 e tip 38.9 d -15.3 b 21.4 c germany 14.06.05 basis 53.4 g 16.4 a 34.7 f center 44.7 f -16.9 a 29.4 e tip 38.6 cd 14.9 bc 21.2 c israel 07.06.05 basis 42.0 e 15.0 bc 24.1 d center 38.0 bcd 13.7 de 19.1b tip 36.5 abc 13.5 e 19.0 b israel 14.06.05 basis 39.9 d 14.4 cd 21.5 c center 35.9 ab 12.6 f 15.5 a tip 34.3 a -11.8 g 14.2 a different subscript letters in a column indicate significant differences at the p=0.05 level. 156 bernhard brueckner, henrike perner sulphur concentration many compounds of allium species, responsible for aroma and human health related effects, contain sulphur as unique organosulphur compounds (goldman et al., 1999). a high concentration of total sulphur in the chive leaves may lead to a high concentration of organosulphur compounds, and thus increased health relevance. the concentration of total sulphur in the fresh matter of the different leaf sections corresponded with the concentration of pyruvic acid. the highest concentrations of sulphur in all samples were found at the tip, the lowest values at the leaf basis (tab. 4). during the one week storage of the german samples, significant losses of sulphur were recorded. the total amount of sulphur was reduced by 25 %, probably because of gaseous losses. beside these losses, the remaining concentration was higher than in the israeli material. sulphur content in plants can not only be influenced by processes after harvest, but also by sulphur fertilisation. distribution of fresh matter fresh matter was not evenly distributed along the length of the leaves. in the basal section with its bulkier and more succulent tubes 43.7 % of the total fresh matter were allocated, 39.4 % in the centre and 16.9 % in the tip sections of german samples. in israeli chives 41.3 % were found in the basal section, 41.6 % in the centre and in the tips only 17.1 % of the total fresh matter was allocated. the order of increasing contents was reverted, when absolute contents of sulphur and pyruvic acid in the different sections were calculated (tab. 5). sensory attributes the perceivable sensory attributes are crucial for the consumer appreciation of a food product, but sensory determination of product attributes, especially of fresh fruit and vegetables are performed not on a regular basis. often the concentration of compounds is used to estimate sensory properties, but interaction of attributes can lead to modification of the correlation of compounds and sensory attributes (lavin et al., 1998; lawless et al., 1998; brückner et al., 2000; bruckner et al., 2005). tab. 4: total sulphur concentration related to fresh matter (mg/100g fm) sulphur concentration german sample german sample israeli sample israeli sample (mg/100 g fm) fresh stored first date second date basis 77.6 e 53.2 cd 33.0 a 29.9 a centre 80.1 ef 59.0 d 38.6 ab 31.0 a tip 88.4 f 62.1 d 55.2 cd 46.0 bc different subscript letters in a row or column indicate significant differences at the p=0.05 level. tab. 5: absolute amount of sulphur and pyruvic acid contained in the different sections of the chive leaves. total sulphur (mg) pyruvic acid (µmol) in 100 g chives, compounds german sample israeli sample german sample israeli sample are contained in the fresh first date fresh first date basis 33.9 13.6 559 267 centre 31.6 16.1 529 255 tip 14.9 9.4 238 143 sum 80.4 39.1 1326 665 tab. 2: dry matter ratio, soluble solid compounds and weight loss dry matter soluble solid weight loss (%) compounds (%) (°brix) germany 07.60.05 basis 6.31 a 3.90 a 15.10 ab centre 6.86 a 4.20 ab 18.55 ab tip 8.52 de 5.43 cd 18.75 ab germany 14.06.05 basis 6.42 a 4.69 abc 17.95 ab centre 7.42 b 4.90 bcd 17.80 ab tip 9.03 ef 5.75 de 22.25 b israel 07.06.05 basis 8.08 cd 4.31 ab 13.65 a centre 9.19 f 5.05 bcd 13.75 a tip 11.37 h 6.46 ef 17.35 ab israel 14.06.05 basis 7.80 bc 5.40 cd 16.65 ab centre 8.94 ef 7.03 fg 14.55 a tip 10.73 g 7.74 g 18.65 ab different subscript letters in a column indicate significant differences at the p=0.05 level. tab. 3: pyruvic acid concentration related to fresh matter. pyruvic acid german german israeli sample (µmol/gfm) sample fresh sample stored first date basis 12.813 c 14.589 cd 6.459 a centre 13.404 c 13.934 cd 6.128 a tip 14.060 cd 15.474 d 8.387 b different subscript letters in a row or column indicate significant differences at the p=0.05 level. nutritive compounds and sensory quality of chives 157 onion flavour 38 40 42 44 46 48 50 52 54 basis centre tip fresh stored green/grassy flavour 48 50 52 54 56 58 60 62 64 basis centre tip fresh stored mouthfeel straw y/fibrous 0 10 20 30 40 50 60 basis centre tip leaf section in te n s it y ( 0 -1 0 0 ) . german fresh german stored israel 1 israel 2 mouthfeel juicy/not dry 0 5 10 15 20 25 30 35 40 45 50 basis centre tip leaf section in te n s it y ( 0 -1 0 0 ) . german fresh german stored israel 1 israel 2 fig 1: intensities of the sensory flavour attributes onion (left) and green/grassy (right). fig 2: intensities of the sensory mouthfeel attributes strawy/fibrous (left) and juicy, not dry (right). sensory differences between the sections several sensory attributes of the sections differed significantly. the most pronounced differences were identified in the mouthfeel modality. tips were far less juicy and crisp, but more strawy/fibrous and drier (fig. 2). the flavour attributes differed little between the sections. despite the increased concentration of pyruvic acid (related to fresh matter), which should indicate higher pungency, the perceived pungency of the tips was not above that of the central and the basal section (fig. 3). the concentration of soluble solid compounds (ssc) was higher in the leaf tips compared to the central and basal sections (fig. 4). the difference and the absolute values were even higher in the israeli samples, than in the german samples. ssc concentration usually indicates sugar concentration, but sweetness was even lower in the tips in israeli material. sensory changes after one week storage of german chive sensory changes during the one week storage of the german chives were small but in some cases significant. strawy/fibrous mouthfeel increased (fig. 2), onion flavour and green/grassy notes decreased (fig 1). comparison of the sensory attributes of german and israeli chives the israeli samples differed in their attributes juicy, not dry, crispy, strawy/fibrous. but israeli samples of both dates were much less juicy and crispy and more strawy/fibrous and drier than the german sample. the israeli sample of the first date differed even more from the german sample than the israeli sample of the second date, in which the sensory intensities of the basal section coincided with those of the tip section of the german sample. pungent notes of german samples were significantly more intensive than of both israeli samples, which were well correlated with the pyruvic acid concentration (fig. 3). sweetness of the german sample was intermediate, above the first, but below the second israeli sample (fig. 4). the concentration of ssc was higher in both israeli samples but very low juiciness may have modified the perception of sweetness. chiveand leek-like notes were also more intensive in the german sample. conclusion the properties of chives are not distributed evenly along the leaf tube. younger tissue, especially when forced in the greenhouse contains more water, which leads to more juiciness and less strawy or fibrous impressions. though the older tissue towards the tips contains higher organosulphur compound concentrations on a fresh weight basis, flavour was not perceived more intensive. the tips of the german sample were in its colour and mouthfeel properties partly comparable to those of the central and basal part of the israeli samples. but towards its basal parts the german chives were juicier, whereas the tips of the israeli material were even less juicy and more strawy and fibrous. the reason for the large differences in ssc and organosulphur compounds between the german and israeli material is not known. therefore the different growing conditions and cultivar differences should be studied in this respect. another important question is, how the significant sensory differences in appearance, colour, mouthfeel and flavour and their combinations can influence consumer appreciation and preference. 158 bernhard brueckner, henrike perner pyruvic acid 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 1,8 basis centre tip leaf section c o n c . (g /1 0 0 g f m ) . german fresh german stored israel 1 mouthfeel pungent 0 10 20 30 40 50 60 70 80 basis centre tip leaf section in te n s it y ( 0 -1 0 0 ) . german fresh german stored israel 1 israel 2 fig. 3: results of the instrumental determination of the pyruvic acid concentration (left) and the intensity of the sensory mouthfeel attribute pungent (right). references ahlers, w., 2001: brix values. http://www.kennzeichnungsrecht.de, ref type: internet communication brückner, b., auerswald, h., 2000: instrumental data – consumer acceptance. in: shewfelt, r.l., brückner, b. (eds.), fruit and vegetable quality: an integrated view, 178-198. technomic publishing, lancaster. pa, usa. bruckner, b., schonhof, i., kornelson, c., schrodter, r., 2005: multivariate sensory profile of broccoli and cauliflower and consumer preference. italian j. food sci. 17, 17-32. goldman, i.l., kader, a.a., heintz, c., 1999: influence of production, handling, and storage on phytonutrient content of foods. nutrition reviews 57, 46-52. lavin, j.g., lawless, h.t., 1998: effects of color and odor on judgments of sweetness among children and adults. food qual. pref. 9, 283-289. lawless, h.t., heymann, h., 1998: psychological and psychophysical foundations of sensory function. in: lawless, h.t., heymann, h., (eds.), sensory evaluation of food – principles and practices, 28-82. fig. 4: concentration of soluble solid compounds (ssc), (left) and intensity of the sensory taste attribute sweet (right). soluble solid compounds (ssc) 0 1 2 3 4 5 6 7 8 9 basis centre tip leaf section c o n c e n tr a ti o n ( °b ri x ) . german fresh german stored israel 1 israel 2 sw eet taste 0 5 10 15 20 25 30 basis centre tip leaf section in te n s it y ( 0 -1 0 0 ) . german fresh german stored israel 1 israel 2 randle, w.m., 1992: onion germplasm interacts with sulfur fertility for plant sulfur utilization and bulb pungency. euphytica 59, 151-156. randle, w.m., bussard, m.l., 1993: pungency and sugars of short-day onions as affected by sulfur nutrition. j. amer. soc. hort. sci. 118, 766770. schwimmer, s., weston, w.j., 1961: onion flavor and odor – enzymatic development of pyruvic acid in onion as a measure of pungency. j. agric. food chem. 9, 301. stone, h., sidel, j.l., 1993: sensory evaluation practices. academic press, san diego. yoo, k.s., pike, l.m., 2001: determination of background pyruvic acid concentration in onions, allium species, and other vegetables. scientia horticulturae 89, 249-256. address of the authors: institut für gemüseund zierpflanzenbau, theodor-echtermeyer-weg 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greifswald productivity of reed (phragmites australis trin. ex steud.) in continental-arid nw china in relation to soil, groundwater, and land-use n. thevs*, s. zerbe, f. gahlert, m. mijit, m. succow (received march 16, 2007) summary reed (phragmites australis trin. ex steud.) is a cosmopolitan plant species which can build up large stands in wetlands, floodplains, and on sites where groundwater is available. phragmites australis provides many ecosystem services, such as the production of raw material (e.g. house construction or organic fuel). in the desert regions of central asia, reed occurs along river, e.g. the tarim, syr darya, amu darya, and serves as fodder plant and raw material for construction and paper production. in those arid regions, reed occurs on submerged sites as well as non-flooded sites in a wide variety of phenotypes, ranging from so-called „giant reed“ (2-4 m high) to dwarf-like thorny reed not exceeding 40 cm stem length. we investigated the net primary production of the different phenotypes and their distribution with regard to soil and groundwater salt content and regarding grazing. the phenotypes were characterized through stem length, stem diameter, number of leaves per stem length, leaf weight ratio, leaf length, and leaf width. the net primary production reached 6,004 kg/ha · a on a non-grazed site, which is submerged for one month in late summer. the depth of the closed capillary fringe before onset of the flood was 2.2 m. the electric conductivity at the closed capillary fringe (determined from a water saturated soil extract) was 2 ms/cm. stem length and stem diameter did not decrease with increasing soil and groundwater salt content, as expected. conversely, stem length and stem diameter decreased and leaf weight ratio increased with increasing grazing intensity. thus, grazing turned reed into dwarf-like thorny phenotypes. non-grazed reed stands are the most productive ecosystems of the riparian vegetation at the tarim and have a high potential to be used as raw material plant. we conclude that biomass harvesting could be an alternative to grazing with regard to sustainable land use. introduction reed (phragmites australis trin. ex steud.) as a cosmopolitan plant species occurs all over the world and can build up large stands in wetlands, floodplains, and on sites where groundwater is available (björk, 1967; björndahl, 1983; hanganu et al., 1999; ostendorp, 1993; rodewald-rudescu, 1974; schieferstein, 1997). natural reeds range from mono-specific stands to diverse vegetation types (dierschke, 1994; ellenberg, 1996). additionally to its role in ecosystem functioning (e.g. self-purification of water, habitat for a broad range of animals; ostendorp, 1993), phragmites australis provides many ecosystem services (costanza et al., 1997), such as the production of raw material for different purposes (e.g. organic fuel). reed also is a key species in the floodplains of the streams in central asia, e.g. the tarim river (thevs, 2005; 2006a; 2006b), syr darya, and amu darya (ogar, 2003). in these continental-arid regions, where large deserts with sparse or without any vegetation occur, it serves, for example, as fodder plant and is intensively grazed by sheep and goat (thevs, 2006b), as shown in fig. 1. additionally, it is used as raw material for paper production and mats (mijit and thevs, pers. observation). in the tarim river floodplain, reed stands naturally occur in a mosaic together with forests mainly built-up by the tree species populus euphratica. *corresponding author fig. 1: reed (phragmites australis) grazed by sheep and goat, site 1. the trees are populus euphratica. reed has a phenotypic plasticity, as reported by vretare et al. (2001) with pot experiments with submerged reed. it is well adapted to highly fluctuating water levels (pagter et al., 2005). in continental-arid regions, reed occurs on submerged sites as well as non-flooded sites in a wide variety of phenotypes, ranging from so-called „giant reed“ over „medium-sized reed“ to „small reed“ or „claw reed“, respectively (gao and xu, 1995; liu et al., 2005; xu et al., 1995). giant reed has stem heights of 200-400 cm and 15-30 nodes and is found on submerged or periodically flooded sites. these stands are partly used to harvest reed as raw material as mentioned above. medium-sized reed has stems 40-200 cm high with 10-20 nodes. it is found on nonflooded sites. small reed has stems with an average growth height of 10-20 cm, not exceeding 40 cm, and 8-12 nodes and occurs on sites with salt accumulation, i.e. with a salt content of 37.1 g/kg in the upper 30 cm of the soil (gao and xu, 1995). the latter reed appears sturdy with contracted internodes. leafs are hard and thorny and thus resemble claws. consequently, the phenotypes of phragmites reeds seem to correspond with the flood regime and topsoil salinization (gao and xu, 1995; xu et al., 1995). according to haslam (1969; 1970), contracted internodes are due to nutrient deficiency and salt. hellings and gallagher (1992) showed in a pot experiment that reed growth decreases with increasing salt content. reed has to take up water from the groundwater and the saturated soil horizon above the groundwater, when not flooded. karunaratne et al. (2004) showed that harvest of reed in spring and particularly in summer removes nutrients from the plant and consequently the nutrient content of the rhizomes decreases. however after harvesting reed, leaf production increases while stem height and diameter decrease. in our study, we hypothesize that under the continental-arid climate in southern xinjiang (1) productivity of phragmites australis is reflected by different phenotypes and corresponds to the salt content in the saturated soil horizon above the groundwater. therefore, we investigate the variety of different phenotypes in the tarim river floodplain in relation to the salt content of the saturated soil horizon above the groundwater and the groundwater depth. (2) we consider a relationship between stand biomass and net primary production, respectively, and different phenotypes. the aspect of productivity has to be seen against the background that reed could be used as raw material and thus is a major source of income for the local people. it serves also as building material for house construction, e.g. roof thatching and insolation, and as energy source (wichtmann, 1999; wichtmann et al., 2000). low quality reed can be used as packing material (wichtmann, 1999). (3) we also hypothesize that grazing affects the phenotype of reed. material and methods study area and investigation sites our investigation area is the tarim huyanglin nature reserve at the middle reaches of the tarim river in xinjiang, nw china (fig. 2). the tarim river is 1,321 km long and flows along the northern edge of the tarim basin. the tarim basin is bordered by the mountain ranges of the tian shan in the north and northeast, the pamir in the west, and the kunlun mountains and tibetan plateau, respectively, in the south and southeast. the surrounding mountain ranges exceed up to more than 7,000 m above sea level and cut off all humid air currents. consequently, the climate of the study area is extremely continentalarid with a mean annual precipitation of less than 50 mm and a mean annual potential evapotranspiration of more than 2,500 mm. the mean annual air temperature is 10.6 °c. the average air temperature in january is -9 °c and in july 25 °c, thus reflecting the strong continental character of the climate (weili xian difangzhe bianzuan weiyuanhui, 1993; xinjiang weiwuer zizhiqu shuili ting and xinjiang shuili xuehui, 1999; yuan and li, 1998). fig. 2: location of the study area, the tarim huyanglin nature reserve. the tarim river is fed by melting water and precipitation in the surrounding mountain ranges. about 75 % of the annual run-off of the tarim river is concentrated in the months july, august, and september (fig. 3) resulting in annual summer flood periods (song et al., 2000; giese et al., 2005). during the annual floods, the water level rises 3-4 m above base flow. fig. 3: monthly runoff of the tarim river at yengi bazar, western boundary of the tarim huyanglin nature reserve. in contrast to the lower reaches of the tarim river which has been strongly degraded due to excessive use of the water resources in the past decades, the tarim huyanglin nature reserve still has a natural to near-natural floodplain character (hai et al, 2006; hou et al., 2007; 2007a; song et al., 2000). however, most of the reed stands are more or less heavily grazed by goat and sheep. the grazing system is transhumant. the herds are kept at the families’ homes during the winter. in spring, they are brought to pastures close to the river and graze there until the summer. in summer, just before the onset of the summer flood, the herds are moved to pastures at the edge of the floodplain out of the reach of the flood. in autumn, the herds are brought back to the herders’ homes (thevs, 2006b). in the tarim huyanglin nature reserve, four investigation sites were established near the settlement of iminqäk (fig. 2) where different phenotypes from so-called „small reed“ to „giant reed“ were observed. site 1 is grazed for 3-4 months per year with 32 sheep and 128 goat in 2002 and 37 sheep and 300 goats in 2003 on an area of 93 ha (data gathered by thevs and mijit, 2003). the herd was brought to site 1 every day during the grazing period. site 2 is only occasionally grazed, because it does not belong to any of the herders in the vicinity of iminqäk. site 3 and 4 is not grazed. determination of groundwater depth and soil sampling on the four investigation sites, soil profiles were drilled with a machine driven soil borer into the closed capillary fringe above the groundwater layer. the depth of the closed capillary fringe served as proxy for groundwater depth. the profiles were drilled in july 2004, i.e. during base flow and before onset of the flood season, in order to record the deepest water level that the plants have to endure. in the field, the depth of the closed capillary fringe was recorded according to ag boden (1994) as proxy for the groundwater level. the depth of the closed capillary fringe is determined by knocking onto the soil borer. if water comes out of the soil sample, the closed capillary fringe is hit. furthermore, the depth of oxidized (iron mottles) and reduced horizons was recorded. from each investigated plot, soil samples were taken from the closed capillary fringe and from the top 100 cm of the soil profile in order to determine salinization. the electric conductivity of the saturation extract was measured after schlichting et al. (1995) and served as a proxy for salt content of the groundwater. phragmites australis in the tarim river floodplain 63 š reed sampling the net primary production (npp) was determined on the basis of the stand biomass at the end of the vegetation season (sept. 2004 and oct. 2006, respectively) using a correction of 10 % added to the stand biomass. this correction applies for non-grazed reed and takes losses due to herbivores and dead plant parts into account (kvet, 1971; westlake, 1965). as the summer flood was very low in 2004 and site 1 therefore was grazed till autumn, we there analysed the stand biomass in 2006, when grazing was stopped due to the summer flood. in order to determine the biomass, 8 sub-plots were randomly placed on each site in july 2004, september 2004, and october 2006, respectively. each sub-plot had an area of 1 m x 0.5 m. all living and dead stems were counted. on the basis of the living stems, the stem density was calculated. in each sub-plot, the 10 stems closest to the centre line were cut and leafs were separated from the stem. leafs and stems were oven-dried (24 h at 105 °c) and weighted to determine the total plant and leaf weight (björndahl, 1983). derived from the average total plant weight of the 10 sampled plants and the stem density, the above ground stand biomass was calculated and values given for the area of one hectare. furthermore from each harvested plant, the stem length and basal stem diameter were measured and number of leafs was counted as morphological data (björk, 1967; björndal, 1983; dykyjova et al., 1973). these data were also retrieved from 30 plants of site 1 in july 2004. from randomly chosen plants from each site, the length and width of leafs were measured. data analysis from the data obtained by the above described procedure, the following indices were calculated: leaf weight ratio, leafs per stem length, and leaf area quotient (björndahl, 1983). leaf weight ratio = leaf weight [g] / total plant weight [g] leafs per stem length [m-1] = number of leafs / stem length [m] leaf area quotient = leaf length [cm] / leaf width [cm] the data of each of the four sites were compared with each other regarding significant differences with a one-way analysis of variance (anova) with the duncan post-hoc test. the data of the two sampling dates of each site were tested for significant differences using the ttest. the statistical analysis was done with the spss program. results groundwater depth and salinization the characterization of sites and soils are given in tab. 1. site 1 on average is flooded for two months per year, which is the longest flood period of all sites. this site also shows the highest capillary fringe (1.3 m) and the lowest electric conductivity at the closed capillary fringe (1.8 ms/cm) as well as in the top 100 cm of the soil profile (2.0 ms/cm). in contrast, site 2, which is not flooded at all, shows by far the highest electric conductivity in the top 100 cm (52.6 ms/cm) compared to all other sites. however, the electric conductivity at the closed capillary fringe of site 2 (2.9 ms/cm) does not exceed that of site 3 (3.3 ms/cm). while the electric conductivity in the top 100 cm differs between the sites, the electric conductivity at the closed capillary fringes of the four sites does not differ very much. the soil profiles of sites 1, 2, and 4 have a greyish, reduced horizon within 3 m below surface, indicating that the closed capillary fringe does not sink below 3 m for longer time periods. reed biomass, production, and morphology sites 3 and 4 both harbour the reed stands with the highest biomass (fig. 4) as well as the tallest reed plants (fig. 5) with the highest diameter (fig. 6). the reed stands sampled in summer on site 1 and 2 are more or less sturdy with contracted internodes, i.e. the number of leafs per stem length is relatively high (fig. 7). as indicated by the comparatively high leaf weight ratio of the reed of the sites 1 and 2 (fig. 8), in those two reed stands more biomass of the plants is concentrated in the leaf than in the reed stands of sites 3 and 4. fig. 4: biomass in summer (left square shaped dots and bars of each pair) and autumn (right diamond shaped dots and bars of each pair). dots represent means, bars represent standard deviation. * indicates significant differences at α < 0.05 between summer and autumn for one site. letters indicate significant differences at α < 0.05 after the duncan post-hoc test between sites. the npp of sites 3 and 4, both not grazed, is 5,129 kg/ha · a and 6,004 kg/ha · a, respectively. the biomass at the end of the vegetation season on sites 3 and 4 is 4,663 kg/ha and 5,458 kg/ha, respectively. in contrast, on site 1, which is the most intensively grazed site, a biomass at the end of the vegetation season of only 159 kg/ha was recorded (fig. 4). the number of leafs per stem length and the leaf weight ratio of the reed of site 1 drops from 119 leafs per meter and 0.59, respectively, in summer to 42 leafs per meter and 0.24, respectively, in autumn (fig. 7 64 n. thevs, s. zerbe, f. gahlert, m. mijit, m. succow tab. 1: flood duration, soil characterization, and grazing intensity of the four reed stands investigated in iminqäk at the tarim river. site parameter site 1 2 3 4 flood duration per year [month] 2 0 1 1 depth of closed capillary fringe [m] 1.3 2.5 2.9 2.2 depth of iron mottles [m] 0 1.0 . 0 depth of greyish reduced soil horizon [m] . 2.7 2.9 2.2 electric conductivity at closed capillary fringe 1.8 2.9 3.3 2.0 [ms/cm] electric conductivity in the top 1 m 2.0 52.6 8.5 2.7 of the soil profile {ms/cm] grazing intensity* 2 1 0 0 * grazing intensities: 2 intensively grazed, 1 occasionally grazed, and 0 not grazed. fig. 7: number of leafs per stem length in summer (left square shaped dots and bars of each pair) and autumn (right diamond shaped dots and bars of each pair). dots represent means, bars represent standard deviation. * indicates significant differences at α < 0.05 between summer and autumn for one site. letters indicate significant differences at α < 0.05 after the duncan post-hoc test between sites. fig. 5: stem length in summer (left square shaped dots and bars of each pair) and autumn (right diamond shaped dots and bars of each pair). dots represent means, bars represent standard deviation. * indicates significant differences at α < 0.05 between summer and autumn for one site. letters indicate significant differences at α < 0.05 after the duncan post-hoc test between sites. fig. 6: diameter in summer (left square shaped dots and bars of each pair) and autumn (right diamond shaped dots and bars of each pair). dots represent means, bars represent standard deviation. * indicates significant differences at α < 0.05 between summer and autumn for one site. letters indicate significant differences at α < 0.05 after the duncan post-hoc test between sites. fig. 8: leaf weight ratio in summer (left square shaped dots and bars of each pair) and autumn (right diamond shaped dots and bars of each pair). dots represent means, bars represent standard deviation. * indicates significant differences at α < 0.05 between summer and autumn for one site. letters indicate significant differences at α < 0.05 after the duncan post-hoc test between sites. and 8). the reed of site 1, sampled in summer, has the highest leaf weight ratio, while sampled in autumn, it has the lowest leaf weight ratio of all reed stands investigated (fig. 8). the leaf width ranges from 1.0 cm (site 1) to 1.9 cm (site 3), as shown in fig. 9. the means of the leaf area quotients on sites 1, 2, 3, and 4 are 2.6, 6.2, 13.7, and 13.6, respectively, with sites 1, 2, and 3 and 4 being significantly different from each other (duncan post-hoc test, α < 0.05). discussion the reed plants sampled in this investigation are similar to those reed plants sampled by gao and xu (1995) and xu et al. (1995) in northern xinjiang with regard to stem length, number of leaves per stem length, leaf length, and leaf width. the reed of sites 3 and 4 correspond to the so-called „giant reed“, of site 2 to „middle reed“, and the reed of site 1 is equivalent to „small reed“ (gao and xu, 1995; liu et al., 2005; xu et al., 1995). the reed plants of site 3 and 4 appear in the same phenotype as it is prevalent on sites in scandinavia (björk, 1967). phragmites australis in the tarim river floodplain 65 we attribute the differences in the phenotypes of reed plants and reed stands, respectively, mostly to grazing. consequently, this is in accordance with findings of haslam (1969). the four sites all have a closed capillary fringe not deeper than 3 m. the presence of a greyish reduced horizon not deeper than 3 m in the soil profiles of sites 1, 2, and 4 indicates that the closed capillary fringe does not fall below 3 m for prolonged time periods. thus, groundwater supply for reed within 3 m soil depth is ensured on all four sites. considering the results with regard to electric conductivity at the closed capillary fringe, the four sites are rather similar with respect to water supply and salt concentration of the groundwater. furthermore, electric conductivity is well below the limiting value for reed of 12 ms/cm, stated by serag (1996). the salt contents in soils under different reed phenotypes shown by gao and xu (1995) differ widely in the topsoil (0-30 cm), but become more and more similar towards the sub-soil. this corresponds to our results. the salt contents in the topsoil differ between sites, while the salt contents in the closed capillary fringe hardly differ between sites. furthermore, we found reed stems in summer sheltered from grazing within groups of trees and shrubs on site 1 (fig. 1, left side), which were as high as the reed stems on sites 3 and 4. therefore we conclude that the soil and water conditions on site 1 would fit tall non-sturdy reed stands, but grazing turned the reed into the short and sturdy reed which we sampled for our analysis. fig. 9: leaf length (left square shaped dots and bars of each pair) and leaf width (right diamond shaped dots and bars of each pair), both in cm. dots represent means, bars represent standard deviation. letters indicate significant differences at α < 0.05 after the duncan post-hoc test between sites. tab. 2: net primary production (npp, dry matter) of reed stands in different regions of the world, sorted from highest to lowest production. site npp [kg/ha*a] reference lake manzala, nile delta, egypt (submerged reed) 46,800 khedr (1989), quoted from serag (1996) hutubi, zhonggar basin, xinjiang, china (submerged reed) 46,200 gao and xu (1995) nile delta, egypt (submerged reed) 44,000 serag (1996) tingitan peninsula, nw-marokko 22,960 ennabili et al. (1998) krankesjön, sweden (submerged reed) 23,810 björk (1967) ringsjön, sweden (groundwater level 0-30 cm) 22,100 björk (1967) akigase park in saitama prefecture, japan 19,800 karunaratne et al. (2003) nile delta, egypt (saltmarsh) 17,000 serag (1991), quoted from serag (1996) danube delta, rumania (fresh water) 9,810-16,320 hanganu et al. (1999) danube delta, rumania (salt water) 6,190-10,740 hanganu et al. (1999) sweden 9,900 graneli (1984) japan, arakawa flood plain 6,900 asaeda et al. (2006) tarim, xinjiang, china (non-grazed reed, 1-2 month flooded per year) 5,129-6,004 thevs et al. (this study) seddinsee (near berlin), germany 1,520-4,200 rolletschek et al. (1999) tarim, xinjiang, china (occasionally grazed reed, salinized topsoil) 1,773 thevs et al. (this study) hutubi, zhonggar basin, xinjiang, china (non-salinized topsoil, not flooded) 1,372 1 gao and xu (1995) tarim, xinjiang, china (groundwater level 1.6-2 m) 930-1,590 zhang et al. (2002) tarim, xinjiang, china (groundwater level 2.3 m) 510 zhang et al. (2002) hutubi, zhonggar basin, xinjiang, china (salinized topsoil) 277 1 gao and xu (1995) fiolen, sweden (submerged reed) 230 björk (1967) tarim, xinjiang, china (grazed reed) 159 1 thevs et al. (this study) 1 stand biomass was determined 66 n. thevs, s. zerbe, f. gahlert, m. mijit, m. succow š grazing, particularly in spring, poses stress to the reed plants. the young shoots are browsed by the animals and nutrients are removed from the reed (ausden et al., 2005; esselink et al., 2000). according to karunaratne et al. (2004), the nutrient resources of the rhizome are reduced by harvest in spring and summer compared to nonharvested reed. reed plants harvested, increase leaf growth and decrease stem growth, as found on site 1. consequently, through grazing the nutrient balance is strongly negatively affected. on site 1, after the herd was removed before autumn 2006, the reed increased in growth height again and became less sturdy, as it was not grazed. as shown in tab. 2, the reed stands investigated at the tarim river have a lower productivity compared to submerged reed. compared to other non-submerged sites in xinjiang, the sites 3 and 4 have a high productivity. the lower productivity of the other sites from xinjiang, shown in tab. 2, might be due to grazing. the annual increment of wood of populus euphratica, the main forest building tree and natural wood source at the tarim river, ranges from 1,795 to 3,459 kg/ha · a (xinjiang linkeyuan zaolin zhisha yanjiusuo, 1989) and 736 to 1,472 kg/ha · a (wang et al., 1996) on sites in the tarim huyanglin nature reserve. thus, with regard to the dominating vegetation and land-use types, non-grazed reed stands are the most productive ecosystems of the riparian vegetation at the tarim and have a high potential to be used as raw material plant. stands with sturdy reed yielding low year-end biomass can be turned into reed which yields much higher biomass. we conclude that biomass harvesting could be an alternative to grazing with regard to sustainable land use. acknowledgements we are indebted to the volkswagen foundation, the heinrich-böll foundation (both germany), and the german academic exchange service (daad), which financially supported our research work in china. references ag boden, 1994: bodenkundliche kartieranleitung. schweizerbart’sche verlagsbuchhandlung, stuttgart. asaeda, t., rajapakse, l., manatunge, j., sahara, n., 2006: the effect of summer harvesting of phragmites australis on growth characteristics and rhizome resource storage. hydrobiologia 553, 327-335. ausden, m., hall, m., pearson, p., strudwick, t., 2005: the effects of cattle grazing on tall-herb fen vegetation and molluscs. biol. conserv. 122, 317-326. björk, s., 1967: ecologic investigations of phragmites communis. gleerup, lund. björndahl, g., 1983: structure and biomass of phragmites stands. ph.d. thesis, university gothenburg, gothenburg. costanza, r., d’arge, r., de groot, r., farber, s., grasso, m., hannon, b., limburg, k., naeem, s., o’neill, r.v., paruelo, j., raskin, r.g., sutton, p., van den belt, m., 1997: the value of the world’s ecosystem services and natural capital. nature 387, 253-260. dierschke, h., 1994: pflanzensoziologie. ulmer, stuttgart. dykyjová, d., hejný, s., kvet, j., 1973: proposal for international comparative investigations of production by stands of reed (phragmites communis). folia geobot. phytotax. 8, 435-442. ellenberg, h., 1996: vegetation mitteleuropas mit den alpen: in ökologischer, dynamischer und historischer sicht. ulmer, stuttgart. ennabili, a., ater, m., radoux, m., 1998: biomass production and npk retention in macrophytes from wetlands of the tingitan peninsula. aquat. bot. 62, 45-56. esselink, p., zijlstra, w., dijkema, k.s., van diggelen, r., 2000: the effects of decreased management on plant-species distribution patterns in a salt marsh nature reserve in the wadden sea. biol. conserv. 93, 61-76. gao, h., xu, j., 1995: correlation of growth forms of phragmites australis with soil water and salt. grassland in china 6, 20-23 (chinese). giese, e., mamatkanov, d.m., wang, r., 2005: wasserressourcen und deren nutzung im flussbecken des tarim (autonome region xinjiang / vr china). zentrum für internationale entwicklungsund umweltforschung, discussion papers 25. justus-liebig-university giessen, giessen. graneli, w., 1989: influence of standing litter on shoot production in reed, phragmites australis (cav.) trin. ex steudel. aquat. bot. 35, 99-109. hai, y., wai, l., hoppe, t., thevs, n. 2006: half a century of environmental change in the tarim river valley. an outline of the causes and remedies. in: hoppe, t., kleinschmit, b., roberts, b., thevs, n., halik, ü. 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(eds.), watershed and floodplain management along the tarim river in china’s arid northwest, 373-393. shaker, aachen. vretare, v., weisner, s.e.b., strand, j.a., graneli, w., 2001: phenotypic plasticity in phragmites australis as a functional response to water depth. aquat. bot. 69, 127-145. wang, s., chen, b., li, h., 1996: euphrates poplar forest. china environmental science press, beijing. weili xian difangzhe bianzuan weiyuanhui (compiling commission of the encyclopaedia of weili county), 1993: encyclopaedia of weili county. xinjiang university press, urumqi (chinese). westlake, d.f., 1965: some basic data for investigations of the productivity of aquatic macrophytes. in: proceedings of the ibp symposium on primary productivity in aquatic environments, 229-248. italian hydrobiologial institute, pallanza. wichtmann, w., 1999: utilization of reed (phragmites australis). arch. nat. conserv. landscape res. 38, 217-231. wichtmann, w., knapp, m., joosten, h., 2000: utilisation of biomass from fen peatlands. z. kulturtech. landentw. 41, 32-36. xinjiang linkeyuan zaolin zhisha yanjiusuo (reforestation and combating desertification research centre of xinjiang), 1989: research on techniques for the revitalization of populus euphratica forests). xinjiang linkeyuan zaolin zhisha yanjiusuo, urumqi (chinese). xinjiang weiwuer zizhiqu shuili ting (water resource adminitration of xinjiang uyghur autonomous region), xinjiang shuili xuehui (water resource science association of xinjiang), 1999: hydrology and water resources of xinjiang’s rivers, xinjiang hygiene and technology press, urumqi (chinese). xu, j., li, w., gao, h., an, s., 1995: study on morpha and structure of three distinct growth forms of phragmites australis. grassland china 5, 49-52. address of the authors: niels thevs, institute of botany and landscape ecology, university greifswald, grimmer strasse 88, d-17487 greifswald, germany niels.thevs@uni-greifswald.de muzappar mijit, institute of resource and environmental sciences, xinjiang university, shengli lu 14, 830046 urumqi, china 68 n. thevs, s. zerbe, f. gahlert, m. mijit, m. succow << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false /maxsubsetpct 100 /optimize true /opm 1 /parsedsccomments true /parsedsccommentsfordocinfo true /preservecopypage true 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<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> >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice art18_buchbesprechung.indd journal of applied botany and food quality 85, 248 (2012) buchbesprechung dc-atlas − dünnschichtchromatographie in der apotheke peter pachaly und angelika koch auch heute kommt der identifi zierung von arzneistoffen, drogen und zubereitungen in der apotheke mittels dünnschichtchromatographie (dc) noch eine bedeutende rolle zu. daher stellt die 7. aktualisierungslieferung zum dc-atlas eine wichtige grundlage für eine zuverlässige inhaltsstoffl iche charakterisierung der in der ph. eur. aufgenommenen monographien dar. in der aktualisierten fassung wurden insgesamt 24 monographien zusätzlich aufgenommen, davon vier aus dem bereich der phytopharmazie (artesunat, citronellöl, lavendelöl, gynostemmablätter). außerdem sind einige, als lifestyle-präparate eingestufte stoffe, wie z.b. dehydroepiandrosteron (dhea) und pregnenolon neu aufgenommen worden, während nicht mehr im arzneibuch aufgeführte stoffe wie melatonin, citrus aurantium und synephrin aus der sammlung entfernt wurden. der aktualisierte dc-atlas enthält somit insgesamt 288 drogenund arzneistoff-monographien. es ist zu begrüßen, dass erstmalig zur charakterisierung chemisch einheitlicher substanzen auch deren atr-ir-spektren präsentiert werden. es wäre jedoch bestimmt nützlich, wenn in einer nachfolgenden lieferung auch für die anderen monographien entsprechende ir-spektren als identifi zierungshilfe mitgeliefert werden könnten (auch wenn es sich hier oftmals um komplexe stoffgemische handelt). außerdem sollte jeweils die physikalische maßeinheit auf der abzisse (cm-1) angegeben werden. teilweise sind detaillierte wellenzahlangaben in den spektren aufgeführt; hier sollte bei der nächsten aktualisierung auf mehr einheitlichkeit geachtet werden. pyridin als fließmittelkomponente wurde bereits in den vorangegangenen lieferungen vollständig eliminiert. im hinblick auf den arbeitsschutz und die ökologische unbedenklichkeit wäre es nunmehr wünschenswert, wenn bei weiteren aktualisierungen auch für dichlormethan und toluol geeignete alternativen in den aufgeführten fließmittelsystemen gefunden werden könnten. in den vorschriften wurde die verwendung von 5 x 5 cm dc-platten weitgehend beibehalten, wobei auch häufi g hptlc-platten zum einsatz kommen. den einzelnen arbeitsvorschriften vorangestellt sind drei kapitel, die auf die theoretischen grundlagen, die laborausrüstung und die arbeitstechnik der dünnschichtchromatographie näher bezug nehmen. abbildungen, die noch die verwendung von benzol als fließmittel zeigen (abb. 10), sollten entsprechend aktualisiert werden. da in den dc-anwendungen auch vermehrt hptlc-platten zum einsatz kommen, sollte auch die densitometrie als alternative detektionsmethode der hptlc in kapitel 3 zumindest kurz vorgestellt werden. insgesamt stellt die aktualisierte lieferung zum dc-atlas eine wertvolle basis zur identifi zierung von arzneistoffen, drogen und zubereitungen dar und sollte daher in keinem apothekenlabor fehlen. dr. h. schulz e-mail: hartwig.schulz@jki.bund.de bibliografi e: peter pachaly und angelika koch, dc-atlas − dünnschichtchromatographie in der apotheke, deutscher apotheker verlag, stuttgart, 1. aufl age inkl. 7. aktualisierungslieferung, mit 288 drogen und arzneistoff-monographien, loseblatt-ausgabe, 870 seiten, zahlreiche farbabbildungen, isbn: 978-3-8047-3014-4 (7. aktualisierungslieferung), isbn: 978-3-8047-3013-7 (gesamtwerk einschließlich 7. aktualisierungslieferung) obwohl zahlreiche ernährungsratgeber auf dem buchmarkt angeboten werden, stehen bisher nur vergleichsweise wenige fachbücher, die sich mit den heute relevanten nahrungsmittelunverträglichkeiten (nmu) näher auseinander setzen, zur verfügung. das buch beleuchtet dabei nicht nur die theoretischen hintergründe und pathophysiologischen zusammenhänge der behandelten unverträglichkeiten, sondern es liefert den betroffenen auch eine reihe nützlicher hinweise, wie man im alltäglichen leben mit den gesundheitlichen einschränkungen besser zurechtkommen kann. der autor stellt die heute bekannten diagnostischen und therapeutischen möglichkeiten vor und versucht dabei, die oftmals komplexen ursachen bei nahrungsmittelunverträglichkeiten mit dem notwendigen tiefgang zu erklären. in einem einführenden kapitel werden zunächst einige fachtermini erläutert und auf die systematik der einzelnen nmu eingegangen. die sich anschließenden kapitel gehen dann auf die wichtigsten nmu näher ein. hierbei erfährt der leser details über prävalenz, ätiologie, klinik, diagnostik und therapie der vier bedeutendsten nahrungsmittel-unverträglichkeiten laktose-, fruktose-, histamin-intoleranz und zöliakie. der fokus liegt dabei allerdings eher auf den beiden in der öffentlichkeit weniger bekannten nmu, der histamin-intoleranz und der zöliakie, während der laktose-intoleranz und fruktosemalabsorption etwas weniger platz eingeräumt wird. besonders erwähnenswert ist der anamnese-bogen „bei verdacht auf nmu“, der inklusive einer auswertungsschablone im internet zum kostenlosen download angeboten wird. ein umfassendes schlagwortregister zeichnet das fachbuch zusätzlich als geeignetes nachschlagewerk aus. bei einer neuaufl age würde man sich allerdings noch mehr therapeutische ansätze wünschen, die hier insbesondere mit blick auf den leserjournal of applied botany and food quality 85, 249 (2012) buchbesprechung nahrungsmittelunverträglichkeiten axel vogelreuter kreis der ernährungsund gesundheitsberater etwas zu kurz kommen. anhand des umfangreichen literaturverzeichnisses (20 seiten) besteht jedoch für interessierte die möglichkeit, sich zu den einzelnen themen weitere detailinformationen zu beschaffen. das buch liefert vor allem den in gesundheitsberufen arbeitenden bei der beratung, begleitung und therapie ihrer patienten eine wertvolle unterstützung. die kapitel sind klar gegliedert und der text ist trotz der umfangreichen fachlichen details gut verständlich verfasst. farblich abgesetzte textblocks fassen die wesentlichen inhalte der einzelnen abschnitte in form kurzer merksätze zusammen, sodass die wesentlichen kernaussagen der einzelnen kapitel als „infothek“ für den leser schnell abrufbar sind. anhand der zahlreichen abbildungen und tabellen gelingt es, die verständlichkeit der textinhalte noch weiter zu verbessern. der autor erreicht mit seinem buch nicht nur die fachlich geschulte leserschaft, sondern liefert vor allem den von nahrungsmittelunverträglichkeiten betroffenen personen wichtige hintergrundinformationen – nicht nur hinsichtlich der jeweiligen krankheitsbilder selbst, sondern darüber hinaus auch zu deren möglichen ursachen sowie ersten therapeutischen ansätzen. dr. h. schulz email: hartwig.schulz@jki.bund.de bibliografi e: axel vogelreuter, lebensmittel-unverträglichkeiten, wissenschaftliche verlagsgesellschaft mbh, stuttgart, 1. aufl age 2012, 230 seiten mit 41 farbigen abbildungen sowie 34 farbigen tabellen, preis: 34,80 €, isbn: 978-3-8047-2938-4. art04 journal of applied botany and food quality 80, 25 30 (2006) institute of freshwater ecology and inland fisheries, berlin effects and metabolism of the phenylurea herbicide isoproturon in the submerged macrophyte ceratophyllum demersum l. constanze pietsch, eberhard krause, b. kent burnison, christian e.w. steinberg, stephan pflugmacher*) (received november 24, 2005) summary phenylurea herbicides such as isoproturon (ipu) restrain photosynthesis by connection to the d1 protein in the photosynthetic apparatus in target plants such as weeds in crop fields. direct effects of herbicides on organisms, which are not a target of the pesticide, have been examined seldom. since a many of agriculturally used pesticides are found in surface waters in agricultural areas, we determined the effects on the photosynthetic oxygen production of the submerged macrophyte ceratophyllum demersum using concentrations of ipu ranging from 0.2 µg/l to 200 µg/l ipu. at environmental relevant concentrations of ipu, the photosynthetic oxygen release was impaired. a reduction of the photosynthetic oxygen release showed a time dependency with the assigned herbicide concentrations. furthermore, this study presents the first indications for metabolism of ipu in the aquatic plant c. demersum. introduction the environment encounters an increasing number of chemicals of anthropogenic origin. similar to animals, plants are also able to metabolize and biotransform a great variety of xenobiotics. research on such mechanisms has mainly focused on terrestrial plants, but those plants seem not to be as sensitive as aquatic plants (garten and frank, 1984). the submerged freshwater macrophyte ceratophyllum demersum l., which is also called coontail, is one of the most widespread species in european lakes and rivers and can thus represent a high macrophyte biomass. due to the abundance of this higher aquatic plant, it can play a major role in detoxifying agricultural xenobiotics but detoxification increases energy demands and consequently plant reproduction may be impaired. one of the most extensively used pesticides in conventional european agriculture is the phenylurea herbicide isoproturon [3-(4-isopropylphenyl)-1,1-dimethylurea (ipu)] (federal environmental protection agency, 2000). ipu is used for preand postemergence control of annual grasses as well as broad leave weeds in wheat, rye and barley crops. each year approximately 30 t of pesticides reach the surface water in germany with ipu contributing 2 t annually. as a result of its extensive use and its properties of moderate persistence and relatively low adsorption, ipu is detected in groundand surface waters in europe in levels which exceed the european commission drinking water limit of 0.1 µg l-1 (spliid and køppen, 1998). although information about effects of herbicides on terrestrial and aquatic organisms is increasing, there is little known about effects of ipu on freshwater macrophytes. few studies focused on rooted macrophytes like elodea densa and ludwigia natans (grouselle et al., 1995; feurtet-mazel et al, 1996). rana and kumar (1995) showed that elodea densa is far more sensitive to high doses of ipu than ipomoea aquatica and the composition of phytoplankton was changed by exposure to this herbicide. from these studies it is apparent that aquatic plants show different sensitivity to herbicides like ipu due to their differences in morphology and metabolism. ipu is known to be a selective systemic herbicide, absorbed by the roots, and rapidly transported through the xylem to the leaves. the mode of action is the inhibition of the photosynthetic electron transport (berger and heitefuss, 1991). in the macrophyte elodea densa grouselle et al. (1995) showed, that the main binding site for ipu is represented by the d1 protein of the photosynthetic apparatus. the bioconcentration of ipu in aquatic plants exposed to this herbicide ranges from 100 to 1,200 depending on the initial ipu concentration and is higher than usually calculated by numerical models (crum et al., 1999; merlin et al., 2001). biotransformation of ipu in aquatic organisms is possible as shown for some bacteria (sørensen and aamand, 2001). additionally, ecotoxicological data showed that ipu and some of its metabolites could be harmful to aquatic organisms like amphibians (greulich et al., 2002). the first phase of metabolism of ipu involves hydroxylation of carbon atoms to create reactive functional groups and is catalyzed by cytochrome p450-dependent monooxygenases (glässgen et al., 1999). in a second step, these activated electrophilic derivatives can be conjugated to glutathione, catalyzed by glutathione-s-transferase (gst) in phase ii (george, 1994). the hydrophilic products of conjugation can be transported into cell compartments or cell walls in the third phase of biotransformation. gst is widely distributed in organisms and may be implicated in cell-line resistance to pesticides (hayes and wolf, 1988; glässgen et al., 1999). biotransformation of aquatic contaminants by conjugation to glutathione is well documented and especially the biotransformation of herbicides is often followed by conjugation to this molecule (lamoureux and rusness, 1989). in the present study, we describe effects of ipu on the photosynthetic oxygen production and the metabolism of this herbicide in ceratophyllum demersum to evaluate the fate of ipu which represents the first step for environmental risk assessment. material and methods plant material ceratophyllum demersum was collected during the summer season from different sites around berlin. identification of the plant species was done according to casper and krausch (1980). plants were cultivated continuously non axenically prior to the experiment for three years in distilled water containing 0.09 g/l cacl2, 0.03 g/l nahco3 (merck; darmstadt, germany) and 0.03 g/l of commercial seasalt (sera gmbh; heinsberg, germany), and in 100 l tanks. supplementary light was provided by daylight lamps at a light / dark cycle of 12 : 12 h. temperature was maintained at 22 24 °c. only young shoots were used for the experiments. measurement of photosynthetic oxygen production one gram fresh weight (fw) of the coontail was exposed to 1000 ml medium containing different concentrations (0.2, 2, 20 and 200 µg/ l) of isoproturon (riedel-de haen; seelze, germany). each exposure was performed in four replicates. in control experiments no isoproturon was applied to the medium. the measurements of photosynthetic oxygen production of the plant were performed using a phosy-mess 4000 (inno concept; straussberg, germany), 100 % light intensity (si unit: 2000 lx) and a dark/light/dark cycle of 10/ 12/10 min under constant temperature of 20 °c. measurements were taken with a clark electrode (wtw eo 196-1,5; sensortechnik meinsberg gmbh, meinsberg, germany). the rates were calculated in mg o2 * h -1* g fw-1. extraction of isoproturon metabolites and analysis by hplc one gram of coontail was exposed to 2000 ml medium containing 25 µg/l ipu. after 24 hours the plant was rinsed thoroughly and frozen in liquid nitrogen and stored under -80 °c until extraction of metabolites. in order to follow the fate of ipu in the surrounding medium 250 ml of the exposure solution were applied onto solidphase cartridges (sep-pak plus tc18 environ. cartridges, waters, milford, massachusetts, us) equilibrated with 5 ml methanol followed by 5 ml sodium phosphate buffer (0.1 m, ph = 6.5) to enrich the target compounds. the sorbent was eluted with 1 ml of methanol and the ipu metabolites analysed by hplc. the frozen plant material was grounded using a mortar and liquid nitrogen. the frozen plant powder was added slowly to 10 ml of 70 % methanol and stirred for 30 minutes on ice. the solid cell material was separated by centrifugation (12,100 x g, 5 minutes). the supernatant was analysed by hplc directly to reduce the possibility of produced oxidation artifacts. analyses of ipu and its metabolites were performed using a liquid chromatograph (waters, milford, massachusetts, us) with an injection valve with a 100-µl loop. ipu and its metabolites were seperated using a lichrospher 100 rp-18 column (250 x 4 mm, 5 µm; merck, darmstadt, germany) and a guard column of the same material (4 x 4 mm). the separation took place at 40 °c at a flow rate of 1 ml/min. the 996 diode array uv detector was set at 240 nm. the separation was performed with a liquid gradient consisting of water and acetonitrile in a composition as described by haas (1997). the run of one sample took 65 minutes. hplc and electron spray injection – mass spectrometry reverse-phase hplc separations were carried out on the plant extract according to kertscher et al. (1998) on a vydac c18 column (150 x 1 mm i.d., 5 µm, type 218tp5115), using a dual syringe pump as solvent delivery system (applied biosystems, 140b, mixing chamber 60 µl). the injection volume was 100 µl. mobile phases a and b consisted of water, 0.06% tfa, and acetonitrile-water (8:2, v/v), 0.05% tfa, respectively. runs were performed at a linear gradient of 8 to 60% b in 40 min and an eluent flow rate of 30 µl/min. the total flow was divided at the column outlet resulting in the introduction of 4 µl/min in a uv detector (785a, applied biosystems) with micro cell type zu (lc packings), λ = 220 nm, and 26 µl/min via a fused-silica capillary directly to the electron spray interface. mass spectrometry was performed on a triple quadrupole instrument (tsq 700, finnigan mat, bremen, germany) equipped with an electron spray ion source (api-esi) operating in the positive mode and with a capillary temperature of 200 °c and a high voltage of 4.5 kv. nitrogen was introduced as sheath gas (3.4 bar) and auxiliary gas (960 ml/min). isopropanol-propionic acid was applied as sheath liquid (25:75, v/v; 4 µl/min). statistics analysis of significance of the photosynthetic oxygen production between control and exposures was performed using one-way analysis of variance (anova) followed by newman-keuls test (spss 9.0 for windows). results photosynthetic oxygen production as shown in fig. 1, there is a time-dependent reduction of the photosynthetic oxygen production in c. demersum after exposure to all four concentrations of ipu compared to control measurements. photosynthetic oxygen production was significantly (p = 0.05) inhibited after 48 hours exposure to 0.2 µg/l ipu compared to control. although the exposure to 2 µg/l ipu for 48 hours caused a reduction of the photosynthetic oxygen release of 54 % compared to control, but this decrease was not significant. a 66 % reduction of the photosynthetic oxygen production compared to control was observed after 48 h-exposure to 20 µg/l ipu. measurements of photosynthetic oxygen production in c. demersum after treatment with the highest ipu concentration (200 µg/l) for 48 hours showed a significant decrease of 70 % when compared to control. 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 co nt ro l 1 m in 30 m in 1 h 2 h 4 h 6 h 24 h 48 h co nt ro l 1 m in 30 m in 1 h 2 h 4 h 6 h 24 h 48 h 0.2 µg/l ipu 2 µg/l ipu 200 µg/l ipu20 µg/l ipu * * * * * ** * fig. 1: time-dependent reduction of photosynthetic oxygen release of c. demersum after exposure to the four concentrations of ipu (0.2; 2; 20 and 200 µg/l). m g o 2 · h -1 · g f w -1 m g o 2 · h -1 · g f w -1 hplc analysis hplc analysis of the medium after exposure to 25 µg/l isoproturon (fig. 2b) showed that c. demersum is able to metabolize ipu to quite a large amount of metabolites indicated by distinct peaks with lower retention times than ipu. in the plant extract no peak was detectable at the retention time of pure ipu (26 min, fig. 2a). mass spectrometry analysis of the in-vivo extract from the coontail by mass spectrometry showed that ceratophyllum demersum is able to transform the herbicide ipu (m/z = 207.0) to a variety of hydroxylated, demethylated and conjugated metabolites. our measurements showed the metabolites didesmethyl-ipu and monodesmethyl-ipu identified as signals at masses of 176.0 and 193.0 (m/z), respectively (fig. 3a). the metabolite isopropenyl-ipu occurred at a mass of 205.1 (m/z). metabolite oh-isopropyl-ipu exhibited a protonated ion at m/z 223.0 (tab. 1). a glutathione metabolite of oh-isopropyl-ipu was detec26 constanze pietsch, eberhard krause, b. kent burnison, christian e.w. steinberg, stephan pflugmacher ted at a mass of 527.1 (m/z) (fig. 3b). as a degraded glutathione conjugate of the same metabolite occurred cysteine-oh-isopropylipu at m/z of 341.0 (tab. 1). different derivates of monodesmethyl-ipu were detected. a very distinct peak showed the conjugate of cysteine and monodesmethylipu at m/z 543.1 (fig. 3b). the metabolite hydroxy-monodesmethylipu showed a protonated peak at 208.9 mass units (fig. 3a). the degraded glutathione conjugates of the same molecule were found as a defined peak at a mass of 327.1 (m/z), which was identified as the conjugate of cysteine and oh-monodesmethyl-ipu (tab.1). the peaks at the masses of 404.8 and 294.0 (m/z) corresponded to the degraded glutathione conjugates of didesmethyl-ipu (γ-glutamylcysteine-didesmethyl-ipu and cysteine-didesmethyl-ipu, respectively). based on the mass spectrometry of the ipu metabolites, fig. 4 represents a possible scheme for the metabolization of ipu in the freshwater macrophyte c. demersum. discussion photosynthetic oxygen production herbicides control a broad spectrum of weeds without affecting the crops. but action of pesticides in agriculture is not confined to the target organisms alone and it is necessary to evaluate their effects on aquatic ecosystem. because of its common use ipu is widely distributed in the environment. concentrations in a french river surface water reached up to 2.6 µg/l (irace-guigand et al., 2004). kirby and sheahan (1994) reported short-lived values in agricultural run-off waters occurred concentrations up to 17 µg/l ipu and a maximum concentration of 500 µg/l of this herbicide was noted for drain water from a crop field after rainfall event by johnson et al. (1996). consequently, the concentrations used in this study are environmentally relevant. for photosynthesis in plants is considered as a process sensitive to environmentally changes and ipu is acting as an inhibitor of the photo-dependent electron transport at the photosystem ii level, effects on metabolism and growth of plants are the consequence (berger and heitefuss, 1991). despite the non-significant reduction of the photosynthetic oxygen release after 48 h exposure of coontail to 2 µg/l ipu, the lower concentrations of isoproturon (0.2 and 2 µg/l) already had a pronounced effect on this important metabolic funcfig. 2: hplc elution profiles of isoproturon (a) and its metabolites in the in-vivo extract (b) from ceratophyllum demersum detected at 240 nm with reference to the elution time. a b b tab. 1: apparent metabolites in the in-vivo extract from c. demersum after exposure to 25 µg/l ipu analysed by esi-tof. mass peak metabolite 176.0 didesmethyl-ipu 193.0 monodesmethyl-ipu 195.0 oh-didesmethyl-ipu 205.1 isopropenyl-ipu 207.0 ipu 208.9 oh-monodesmethyl-ipu 223.0 oh-isopropyl-ipu 294.0 cysteine-didesmethyl-ipu 327.1 cysteine-oh-monodesmethyl-ipu 341.0 cysteine-isopropyl-ipu 404.8 γ-glutamyl-cysteine-didesmethyl-ipu 527.1 oh-isopropyl-ipu 543.1 cysteine-monodesmethyl-ipu fig. 3: chosen esi spectra (150-220 and 400-600 m/z) of in-vivo extract from c. demersum after exposure to 25 µg/l ipu for 24 h. m/z : a: 1 = didesmethyl-ipu(176.0); 2 = monodesmethyl-ipu (193.0); 3 = oh-didesmethyl-ipu (195.0); 4 = isopropenyl-ipu (205.1); 5 = ipu (207.0); 6 = oh-monodesmethyl-ipu (208.9); b: 1 = γglutamyl-cysteine-didesmethyl-ipu (404.8); 2 = oh-isopropyl-ipu (527.1); 3 = cysteine-monodesmethyl-ipu (543.1). 149.0 163.2 177.4 191.6 205.8 220.0 mass (m/z) 0 154.9 0 10 20 30 40 50 60 70 80 90 100 % in te n s ity 1 4 5 6 3 2 400 440 480 520 560 600 mass (m/z) 0 42.3 0 10 20 30 40 50 60 70 80 90 100 % in te n s ity 1 2 3 a b % in te n sity metabolism of isoproturon in ceratophyllum demersum 27 tion in c. demersum in this study. after exposure to 20 µg/l ipu the negative effect on photosynthesis was even stronger and the photosynthestic oxygen release was inhibited significantly beyond an exposure time of 2 hours to 200 µg/l ipu. this is in agreement with the observations of feurtet-mazel et al. (1996), showing that the dissolved oxygen concentration in the surrounding medium decreased due to inhibition of photosynthesis in elodea densa. damage to aquatic organisms by pesticides is known to be related to their potential toxicity and their duration of exposure. additionally, age of plant shoots seems to be of importance as shown by weinberger and greenhalgh (1985) for absorption of the pesticide aminocarb to ceratophyllum demersum. as a consequence, we used young shoots of coontail only. grouselle et al. (1995) showed that the photosynthetic inhibition in plants by ipu is mainly due to specific binding to the d1 protein, a 32-kda protein of the photosystem ii within the thylakoid membranes. ipu causes a rapid bioaccumulation in aquatic macrophytes (crum et al., 1999). high bioconcentration factors for aquatic macrophytes indicate that low ipu concentrations show a clear saturation tendency in these plants (feurtet-mazel et al., 1996). this saturation effect is caused by the specific binding of ipu to the d1 protein, while exposure to higher ipu concentrations results in lower bioconcentration factors in elodea densa due to non-specific binding of ipu (grouselle et al., 1995). this may be caused by the lipophilic properties of ipu resulting in an higher toxicity to macrophytes than to algae (kirby and sheahan, 1994). a concentration of 10 µg/l ipu caused significant negative effects on the growth of elodea densa, and concentrations higher than 200 µg/l ipu were followed by a total growth inhibition (feurtet-mazel et al., 1996). furthermore binding of ipu to d1 protein in algae and higher plants results in a breakdown of the photosynthetic synthesis of adenosine triphosphate and nicotinamide adenine dinucleotide phosphate and thus further impact on metabolism can be expected. hplc analysis after an exposure of 24 hours to ipu (25 µg/l), the parent compound is no longer detected in the medium, but quite a variety of herbicide metabolites were observed by hplc. this indicates that metabolization of ipu by this macrophyte is rather fast as already shown by crum et al. (1999). depending on weather conditions ipu might be more persistent in soil than indicated by its half-life (johnson et al., 1996), and even if farmers follow good agricultural practice, herbicides applied to winter cereals might reach nearby ditches and streams and thus contaminate surface waters. the average half-life of ipu in the water column ranged from approximately 50 to 60 days (peres et al., 1996). cooper et al. (2004) showed that vegetated agricultural ditches affect the mitigation of pesticides. vegetation in ditches plays an important role in the transformation of contaminants and can thus be an environmental benefit. macrophytes accelerate the ipu removal from the water column while its adsorption to sediment and photochemical degradation play a negligible role (merlin et al., 2001; amine-khodja et al., 2004). additionally, buffer zones between field crops and ditch banks can reduce pesticide deposition on ditches. though the presence of macrophytes may result in lower environmental pesticide concentrations the accumulated contaminants can also be a long-term source for aquatic contaminants and thus further threaten organisms living in or off aquatic plants. according to merlin et al. (2001) aquatic macrophytes are able to accumulate approximately 45 % of the ipu applied. average bioconcentration factors for ipu, calculated by grollier et al. (1996), were close to 10 in elodea densa and ludwigia natans. the rapid metabolization of ipu in our experiments also revealed the shift to derivatives with lower retention time indicating the production of more hydrophilic metabolites. because reference standards for ipu metabolites are rare, further characterization of metabolites by mass spectrometry was applied. fig. 4: metabolic scheme for isoproturon in ceratophyllum demersum based on the esi-tof elution profiles at 240 nm. the protonized masses (m/z) and abbreviated trivial names as used in the present article are indicated together with the metabolic structures. metabolites in brackets were not detectable in c. demersum but are possible short-lived intermediates. h3c h3c n h o nho h h h3c ch3 ch3 n h o n h2c n o h n ch3 ch3 h3c h3c h3c h3c n h o nho ch3 ch3 h3c h3c ch3 n h o n h h3c h3c n h o nho h ch3 h3c h3c n h o n h h 28 constanze pietsch, eberhard krause, b. kent burnison, christian e.w. steinberg, stephan pflugmacher mass spectrometry methods like time-of-flight secondary ion mass spectrometry (tof-sims) are not being employed very often in plant metabolic studies, but our results are in agreement to those by glässgen et al. (1999), who showed the metabolization of ipu in wheat and soybean cell cultures was mainly by hydroxylation and dealkylation. these reactions are catalysed by cytochrome p450 enzymes. a ring-methyl hydroxylation of ipu was shown in wheat cell cultures (mougin et al., 1991). in c. demersum we also found the hydroxylated derivative isopropyl-ipu. robineau et al. (1998) reported that the monooxygenase cyp76b1 in helianthus tuberosus actively metabolizes phenylureas via a double n-dealkylation. in this plant the herbicide ipu is mainly metabolized to monoand didealkylated derivatives with the second demethylation being the rate limiting step of detoxification. the dealkylated derivatives of ipu identified in this study were monodesmethyl-ipu and didesmethyl-ipu which are both further processed to hydroxylated metabolites. the generation of oh-didesmethyl-ipu might be possible via demethylation of the monodealkylated ipu derivative and followed by hydroxylation. on the other hand the detoxication of monodesmethyl-ipu via hydroxylation to oh-monodesmethyl-ipu and further processing to oh-didesmethyl-ipu might be more important in c. demersum. the existence of the desaturated metabolite isopropenyl-ipu is in agreement to the observations of glässgen et al. (1999) though its importance remains unknown. in summary the tolerance and selectivity of phenylureas in plants seems to rely on the presence and relative expression of at least two p450 enzymes. further research is needed to determine the importance of the different possible detoxication pathways. the second step of biotransformation consists of the conjugation of xenobiotics to glutathione (gsh) or other cell-internal molecules. in our study the only glutathione conjugate detectable in coontail is glutathione-hydroxy-isopropyl-ipu (gs-oh-isopropyl-ipu), but the amount of cysteine conjugates and γ-glutamyl derivatives indicates that the majority of the produced glutathione conjugates is degraded rapidly. the conjugation of monodesmethyl-ipu to glutathione seems to be of importance as indicated by the pronounced peak of the cysteine derivative of this metabolite on the esi spectrum. it is also known that herbicides can be activated by biotransformation. though trixier et al. (2001) showed that hydroxylation of phenylureas leads to non-toxic derivatives, demethylation can generate products of high toxicity. according to müller (1986), the metabolite monodesmethyl-ipu can be still phytotoxic. glässgen et al. (1999) showed that the metabolism of ipu in monocotyledon plants is different from those in dicotyledonous plants. the same author showed that cell culture suspensions of the monocotyledonous wheat produced not as much of the metabolite monodesmethyl-ipu as did cells of the dicotyledonous soybean. meanwhile the wheat cells seemed not to be affected by ipu as much as soybean cells. thus the different metabolism of ipu in monocotyledonous and dicotyledonous plants may be the reason for differences in sensitivity to this herbicide. for ipu seems to be reacting selectively on dicotyledonous plants, this might explain the pronounced impact on the cormophyte c. demersum. in conclusion, this study shows a great impact of ipu on photosynthetic oxygen production of the aquatic macrophyte c. demersum. furthermore the metabolization of ipu by c. demersum showed that this plant behaves like a typical dicotyledonous plant and this can partially explain its sensitivity to ipu. acknowledgement the authors would like to thank dr. kerstin greulich for advising in the ipu related work and for helpful discussions. references amine-khodja, a., boulkamh, a., boule, p., 2004: photochemical behaviour of phenylurea herbicides. photochem. photobiol. sci. 3, 145156. berger, b., heitefuss, r., 1981: use of isoproturon, alone and in combination with other compounds, on winter wheat and winter barley. weed res. 31, 9-18. casper, j.s., krausch, h.-d., 1981: pteridophyta und anthophyta, in: ettl, h., gerloff, j., heynig, h. (eds.), süßwasserflora von mitteleuropa. band 24, 2.teil, 477-482. fischer verlag, jena. cooper, c.m., moore, m.t., bennett, e.r., smith, s. jr., farris, j.l., milam, c.d., shields, f.d. jr., 2004: innovative uses of vegetated drainage ditches for reducing agriculturall runoff. water sci. technol. 49, 117-123. crum, s.j.h., van kammen-polman, a.m.m., leistra, m., 1999: sorption of nine pesticides to three aquatic macrophytes. arch. environ. con. tox. 37, 310-316. federal environmental protection agency, 2000: estimation of the pesticide input in surface waters of germany. federal environmental protection agency report 3/2000, berlin, germany. feurtet-mazel, a., grollier, t., grouselle, m., ribeyre, f., boudou, a., 1996: experimental study of bioaccumulation and effects of the herbicide isoproturon on freshwater rooted macrophytes (elodea densa and ludwigia natans). chemosphere 32, 1499-1512. garten, c.t., frank, m.l., 1984: comparison of toxicity to terrestrial plants with algal growth inhibition by herbicides. environ. sci. division publication no. 2361, ornl/tm-9177, oak ridge nat. lab., oak ridge, tennesee. george, s.g., 1994: enzymology and molecular biology of phase ii xenobiotic-conjugation enzymes in fish. in: malins, d.c., ostrander g.k. 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104-110. robineau, t., batard, y., nedelkina, s., cabello-hurtado, f., leret, m., sorokine, o., didierjean, l., werck-reichhart, d., 1998: the chemically inducible plant cytochrome p450 cyp76b1 actively metabolizes phenylureas and other xenobiotics. plant. physiol. 118, 10491056. sørensen, s.r., aamand, j., 2001: biodegradation of the phenylurea herbicide isoproturon and its metabolites in agricultural soils. biodegradation 12, 69-77. spliid, n.h., køppen, b., 1998: occurrence of pesticides in danish shallow ground water. chemosphere 37, 1307-1316. trixier, c., sancelme, m., bonnemoy, f., cuer, a., veschambre, h., 2001: degradation products of a phenylurea herbicide, diuron: synthesis, ecotoxicity, and biotransformation. environ. toxicol. chem. 20, 13811389. weinberger, p., greenhalgh, r., 1985: the sorptive capacity of an aquatic macrophyte for the pesticide aminocarb. j. environ. sci. health b20, 263-273. addresses of the authors: constanze pietsch, pd dr. stephan pflugmacher, leibniz institute of freshwater ecology and inland fisheries, müggelseedamm 301/310, d-12587 berlin prof. c.e.w. steinberg, humboldt university at berlin, institute of biology, arboretum, späthstraße 80/81, d-12437 berlin dr. eberhard krause, research institute for molecular pharmacology, robert rössle straße 10, d-13125 berlin dr. b. kent burnison, national water research institute, canadian center for inland waters, 867 lakeshore road, l7r 4a6 burlington, ontario, canada 30 constanze pietsch, eberhard krause, b. kent burnison, christian e.w. steinberg, stephan pflugmacher << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages 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<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> >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice vorgabe neu journal of applied botany and food quality 80, 179 186 (2006) (1)institute of human nutrition and food science, christian-albrechts-university kiel (2)institute of animal nutrition and physiology, christian-albrechts-university kiel ochratoxin a-induced cytotoxicity in liver (hepg2) cells: impact of serum concentration, dietary antioxidants and glutathione-modulating compounds* christine bösch-saadatmandi(1), christoph hundhausen(1), laia jofre-monseny(1), ralf blank(2), siegfried wolffram(2), gerald rimbach(1) (received august 15, 2006) * the paper was presented at the 41th meeting of the „deutsche gesellschaft für qualitätsforschung (pflanzliche nahrungsmittel) dgq e. v.“ abbreviations bso, buthionine sulfoximine; cat, catechin; dmso, dimethyl sulfoxide; dtnb, dithio-bis-nitrobenzoic acid; egcg, epigallocatechin gallate; fcs, foetal calf serum; gsh, glutathione; iarc, international agency for research on cancer; nac, n-acetylcysteine; no, nitric oxide; nr, neutral red; oatp, organic anion-transporting polypeptide; ota, ochratoxin a; pbs, phosphate buffered saline; que, quercetin; ros, reactive oxygen species; rosac, rosmarinic acid; rpmi, roswell park memorial institute; α-toc, α-tocopherol; α-toc-p, α-tocopherol phosphate summary ochratoxin a (ota) is a nephroand hepatotoxic mycotoxin produced by various species of the genera aspergillus and penicillium. ota is known to bind with high affinity to plasma proteins which may have a substantial impact on its bioavailability and, thus, on its toxicity. however, the underlying mechanisms of ota-induced cellular toxicity have not yet been fully elucidated. it has been suggested that oxidative damage contributes to its cytotoxic effects. dietary antioxidants such as vitamin e and polyphenols may therefore counteract ota-induced cell death. furthermore, compounds influencing the intracellular level of glutathione (l-gamma-glutamyll-cysteinylglycine, gsh), the most abundant thiol antioxidant in mammalian cells, may have an impact on ota-induced cytotoxicity. in this study we investigated the effects of serum concentrations as well as different dietary antioxidants on the viability of ota-exposed liver (hepg2) cells. additionally, we determined the intracellular gsh-levels after incubation with ota and n-acetylcysteine (nac, a precursor of gsh) or buthionine sulfoximine (bso, an inhibitor of gamma-glutamylcysteinyl synthetase). incubation of human hepatoma cells (hepg2) for 24 h with increasing concentrations of ota (0.25 50 µmol/l) in the presence of 0, 2.5, 5, or 10% foetal calf serum (fcs) resulted in a dose-dependent decrease in cell viability. decreasing the serum concentrations in the cell culture medium led to increased cell mortality. pre-treatment for 24 h with rrr-α-tocopherol (α-toc), rrr-α-tocopherolphosphat (α-tocp), epigallocatechin gallate (egcg), quercetin (que), catechin (cat), and rosmarinic acid (rosac) at concentrations of 25, 50 and 100 µmol/l did not prevent ota-induced toxicity. α-toc-p, egcg, and que even amplified the cytototoxic effects of ota in hepg2. supplementation with nac and bso in the presence of ota did not substantially change cell viability, although bso treatment resulted in depletion of cellular gsh. ota treatment increased gsh levels at concentrations of 50 and 100 nmol/l, but decreased cellular gsh at the higher concentrations (250, 500, and 1000 nmol/l). the decrease in cellular gsh concentrations was less pronounced than for bso. our results indicate that the cytotoxicity of ota in hepg2 cells, which is strongly dependent on the protein concentration in the cell culture medium, can not be prevented by pre-incubation with dietary antioxidants. although cellular gsh levels are influenced by ota incubation, mechanisms other than oxidative stress are likely to be involved in ota-induced cell death in hepg2 cells. introduction the mycotoxin ochratoxin a (ota), mainly produced by aspergillus ochraceus and penicillium verrucosum, is the most toxic of the three known members of the ochratoxin family (ochratoxin a, b, and c). ota was discovered and its chemical structure described in 1965 (van der merwe et al., 1965). chemically, ota consists of a dihydroisocumarin moiety joined by a peptide bond to 1-phenylalanine (fig. 1). ota can be found in many foods and beverages, e.g. cereal grains, coffee, and wine (fazekas et al., 2002; studerrohr et al., 1995; van egmont et al., 1994). due to its ubiquitous occurrence in foods, it is virtually impossible to completely avoid ingestion of ota. both in animals and humans, a wide range of toxic effects of ota has been reported, including renal and hepatic toxicity (bendele et al., 1985), genotoxicity (creppy et al., 1985; bose and sinha, 1994), neurotoxicity (miki et al., 1994; bruinink et al., 1998), and immunotoxicity (harvey et al., 1992; petzinger and ziegler, 2000). severe human renal diseases, such as endemic nephropathy (ben), chronic interstitial nephritis and karyomegalic interstitial nephritis, may be a result of continued exposure to ota (simon et al., 1996). moreover, the international agency for research on cancer has classified ota as a class 2b carcinogen (possible human carcinogen) (iarc, 1991). fig. 1: chemical structure of ochratoxin a the toxicity of ota is largely determined by its protein binding properties. in blood, 99% of ota is bound to serum proteins (mainly albumin), which prolongs its systemic half-life, delays its elimination and may, therefore, amplify its toxicity (chu, 1971; chu, 1974). the exact mode of action of ota toxicity is as yet unclear. the toxicity of ota has been explained by three major mechanisms: 1. inhibition of protein synthesis via inhibition of phenylalanine-trna synthetases (creppy et al., 1979; creppy et al., 1998), 2. inhibition of mitochondrial function (moore and truelove, 1970; meisner, and chan, 1974; wei et al., 1985), and 3. generation of reactive oxygen species (ros) which may oxidise dna, proteins, lipids, and other macromolecules (rahimtula et al., 1988; omar et al., 1990; schaaf et al., 2002; domijan et al., 2005; kamp et al., 2005). therefore, antioxidants may counteract ota-induced cytotoxicity. recent studies have shown that supplementation with vitamin e or several polyphenols partly diminished the toxic effects of ota (schaaf et al., 2002; baldi et al., 2004; renzulli et al., 2004; guerra et al., 2005). furthermore, an involvement of the glutathione (gsh) system in ota cytotoxicity, has been suggested. gsh (γglutamylcysteinylglycine) functions as a free radical scavenger, and serves as a nucleophilic co-substrate for glutathione transferases in the detoxification of xenobiotics. it has been shown that preincubation with n-acetylcysteine (nac), a precursor of gsh, completely prevented ota-induced cell death in kidney (llc-pk1) cells (schaaf et al., 2002). the objective of the present study was to assess the impact of serum protein concentration in the cell culture medium and the effect of dietary antioxidants on ota-induced cytotoxicity in hepg2 cells. furthermore, we aimed at investigating whether modulation of cellular gsh levels affects ota-induced cytotoxicity in human liver cells in culture. materials and methods cell culture and determination of ota-cytotoxicity the human hepatoblastoma derived cell line hepg2 was cultured in rpmi medium supplemented with 10% fcs, 2 mmol/l glutamine, 100 iu/ml penicillin and 100 µg/ml streptomycin under standard conditions (37°c, 5% co 2 ). confluent cells were harvested by trypsination and seeded at 1 x 105 cells per well in 24-well plates. the medium was changed every 48 h. in dose-response experiments, hepg2 cells were exposed to increasing concentrations of ota ranging from 0.25 50 µmol/l for 24 h. the fcs concentrations in the medium were 0, 2.5, 5 or 10%. in a separate experiment, hepg2 cells were incubated with ota at concentrations of 10 1000 nmol/l to find out the highest non-toxic ota concentration under serum-free conditions. the cytotoxicity of ota was determined employing the neutral red (nr) assay (valacchi et al., 2001). this test is based on the ability of viable cells to incorporate nr in lysosomes which is photometrically measured at 540 nm. cell viability was expressed as percentage of solvent-exposed control (0.1% methanol) survival. for each incubation period, two independent experiments were performed in triplicate (n = 6). pre-incubation with test substances to study the potential protective effects of vitamin e and polyphenols on ota-induced cytotoxicity, cells were pre-treated with rrrα-tocopherol (α-toc), rrr-α-tocopherolphosphate (α-toc-p), epigallocatechin gallate (egcg), quercetin (que), catechin (cat), and rosmarinic acid (rosac) (fig. 2) for 24 h at concentrations of 25, 50, and 100 µmol/l (non-toxic concentrations for hepg2 cells, data not shown). 100 mmol/l stock solutions were prepared in dimethyl sulfoxide (dmso), ethanol or an acetic acid:ethanol mixture (1:3) for the polyphenols, α-toc and α-toc-p, respectively. stock solutions were further diluted in culture medium containing 10% fcs with a maximum solvent concentration of 0.1%. cells were washed twice with pbs and incubated for another 24 h with 1, 5 or 15 µmol/l ota in serum-free rpmi. additionally, hepg2 were pre-incubated for 24 h with 0, 0.2, 0.5, 1, 2, or 4 mmol/l nac or 0, 5, 25, 50, 100, or 500 µmol/l bso. bso and nac were prepared as 100 mmol/l stock solutions in pbs. the ph of the nac stock solution was adjusted to ph 7 with naoh. subsequently, cells were washed twice with pbs and incubated for 24 h with 1, 5 or 15 µmol/l ota in fcs-free medium. two or three independent replicates were performed in triplicate. determination of cellular glutathione levels total cellular gsh content was determined by the method of griffith with minor modifications (griffith, 1980). hepg2 cells were grown at a density of 9 x 105 cells per well in 6-well plates for 48 hours and incubated with ota, bso, or nac at increasing concentrations. after 24 h, the incubation medium was aspirated; cells were washed with pbs and harvested by trypsination. due to the limited cell material, cells of three wells were pooled together. cell pellets were collected after centrifugation and stored immediately at -80°c. to measure glutathione concentrations, cell pellets were resuspended in lysis buffer (pbs, 0.05% triton-x (w/v), 0.05 mmol/l edta) and homogenized. the cell suspension was treated with an equal volume of 10% sulfosalicylic acid (w/v) and separated by centrifugation (12,000 x g, 5 min, 4°c). 270 µl of the supernatant was mixed with 20 µl of a 150 mmol/l phosphate buffer (including 1% bovine serum albumin, ph 7.5) and 30 µl triethanolamin (50%). sample (75 µl) was added to the reaction mixture containing 350 µl of 0.28 mmol/l nadph and 50 µl of 6 mmol/l dtnb (5-5'dithio-bis2-nitro-benzoic acid). the reaction was started by addition of 50 µl glutathione-reductase (10 u/ml) and the increase in absorption at 412 nm was followed for 180 min in 20 sec intervals. each sample was measured in duplicate. results were calculated by comparison of the difference in absorbance per minute with data from a standard curve generated with oxidised glutathione. the protein content of the cell suspension was determined with the commercially available bca protein kit from pierce. three independent replicates were performed in duplicate. statistical analysis data are presented as mean with standard deviation. the statistical program spss (version 13.0) was used to assess the effects of various treatments by analysis of variance (anova) followed by the post hoc test dunnett. in the case of inhomogeneous variances multiple t-tests were applied. differences were considered significant if p < 0.05. results cytotoxicity of ota incubation of hepg2 cells with concentrations of ota ranging from 0.25 to 50 µmol/l for 24 h resulted in a dose-dependent decline in cell viability (fig. 3). decreasing fcs concentrations were paralleled by a marked increase in ota-induced cytotoxicity, with the strongest effects at ota concentrations between 0.25 and 15 µmol/l. in cells cultured in serum-free medium the strongest cytotoxicity was observed at ota concentrations between 10 (100% viability) and 250 nmol/l (53% viability). however, in medium containing 5 or 10% serum, 24 h exposure to 1 µmol/l ota did not lead to cell death at all, while 0 or 2.5% serum decreased cell viability to 52 and 87%, respectively. the minimum level of cell viability (approximately 40%) was reached upon incubation with 15 µmol/l ota and did not 180 christine bösch-saadatmandi, christoph hundhausen, laia jofre-monseny, ralf blank, siegfried wolffram, gerald rimbach decrease further even upon incubation with as much as 50 µmol/l ota in fcs-free medium (fig. 3).the highest non-toxic ota concentration was 10 nmol/l (fig. 4). due to the higher bioavailability of ota under these conditions, serum-free medium was chosen for further experiments. pre-incubation experiments with antioxidants and glutathione-modulators a 24 h pre-incubation of hepg2 cells with α-toc, α-toc-p, egcg, que, cat, and rosac at concentrations of 25, 50, or 100 µmol/l and a subsequent incubation with 1, 5, or 15 µmol/l ota for 24 h did not prevent the cytotoxic effects of ota (fig. 5). treatment with αtoc-p, egcg, and que even enhanced ota cytotoxicity dosedependently. no changes in cell viability were observed upon prefig. 2: chemical structures of the antioxidants α-tocopherol catechin α-tocopherol phosphate (disodium salt) epigallocatechin gallate rosmarinic acidquercetin ochratoxin a (µmol/lochratoxin a (µmol/lochratoxin a (µmol/lochratoxin a (µmol/l)))) fig. 3: influence of foetal calf serum (fcs) concentration on the viability of hepg2 cells after 24 h incubation with ochratoxin a (0 50 µmol/l). data are mean ± sd from two experiments in triplicate (n = 6). vi a b ili ty vi a b ili ty vi a b ili ty vi a b ili ty (% ) (% ) (% ) (% ) ochratoxin a (nmol/lochratoxin a (nmol/lochratoxin a (nmol/lochratoxin a (nmol/l)))) fig. 4: viability of hepg2 cells after 24 h incubation with ochratoxin a (0 1000 nmol/l). data are mean ± sd from two experiments in triplicate (n = 6). effect of antioxidants on ota-induced cytotoxicity in hepatocytes 181 incubation with α-toc, cat and rosac. cellular α-toc levels remained unchanged in response to ota treatment (data not shown), cellular concentrations of egcg, que, cat, and rosac were not determined. to further investigate the involvement of cellular glutathione levels in ota-induced cytotoxicity, additional pre-incubations with the glutathione modulators bso and nac were carried out. incubation with nac did not protect ota-treated cells from cytotoxicity (fig. 6a). pre-incubation with 500 µmol/l bso decreased cell viability by 10%, while lower concentrations of bso were without effect (fig. 6b). effect on cellular glutathione levels as shown in fig. 7a, incubation with bso for 24 h resulted in a substantial and dose-dependent depletion of cellular glutathione levels in hepg2 cells. concentrations as low as 5 µm decreased cellular gsh by about 40% compared to untreated controls. a complete gshdepletion was observed at 100 µmol/l bso. supplementation with nac did not increase cellular gsh (data not shown). incubation with ota decreased gsh levels at concentrations of > 0.25 µmol/l (fig. 7b). 1 µmol/l ota led to an approximately 30% decrease in cellular gsh as compared to controls. in contrast, lower concentrations (0.05 or 0.1 µmol/l) of ota resulted in a moderate increase in gsh levels in hepg2 cells. discussion in line with previous studies our data indicate that ota induces cytotoxicity in hepg2 cells in a dose-dependent manner (baldi et al., 2004; renzulli et al., 2004; hundhausen et al., 2005). more0000 10101010 20202020 30303030 40404040 50505050 60606060 70707070 80808080 100100100100 90909090 0000 20202020 10101010 30303030 40404040 50505050 60606060 70707070 80808080 100100100100 90909090 v ia b il it y v ia b il it y v ia b il it y v ia b il it y (% ) (% ) (% ) (% ) v ia b il it y v ia b il it y v ia b il it y v ia b il it y (% ) (% ) (% ) (% ) 1111 5555 15151515 1111 5555 15151515 ochratoxinochratoxinochratoxinochratoxin a (a (a (a (µµµµmol/l)mol/l)mol/l)mol/l) ochratoxinochratoxinochratoxinochratoxin a (a (a (a (µµµµmol/l)mol/l)mol/l)mol/l) ----toctoctoctoc ----toctoctoctoc----pppp 0000 10101010 20202020 30303030 50505050 40404040 60606060 70707070 80808080 100100100100 90909090 0000 10101010 20202020 40404040 30303030 50505050 60606060 70707070 80808080 100100100100 90909090 v ia b il it y v ia b il it y v ia b il it y v ia b il it y (% ) (% ) (% ) (% ) v ia b il it y v ia b il it y v ia b il it y v ia b il it y (% ) (% ) (% ) (% ) 1111 5555 15151515 1111 5555 15151515 ochratoxinochratoxinochratoxinochratoxin a (a (a (a (µµµµmol/l)mol/l)mol/l)mol/l) ochratoxinochratoxinochratoxinochratoxin a (a (a (a (µµµµmol/l)mol/l)mol/l)mol/l) egcgegcgegcgegcg quequequeque 0000 10101010 30303030 20202020 40404040 50505050 60606060 70707070 80808080 100100100100 90909090 0000 10101010 20202020 30303030 50505050 40404040 60606060 70707070 80808080 100100100100 90909090 v ia b il it y v ia b il it y v ia b il it y v ia b il it y (% ) (% ) (% ) (% ) v ia b il it y v ia b il it y v ia b il it y v ia b il it y (% ) (% ) (% ) (% ) 1111 5555 15151515 1111 5555 15151515 ochratoxinochratoxinochratoxinochratoxin a (a (a (a (µµµµmol/l)mol/l)mol/l)mol/l) ochratoxinochratoxinochratoxinochratoxin a (a (a (a (µµµµmol/l)mol/l)mol/l)mol/l) catcatcatcat r osacrosacrosacrosac 0000 10101010 20202020 30303030 40404040 50505050 60606060 70707070 80808080 100100100100 90909090 0000 20202020 10101010 30303030 40404040 50505050 60606060 70707070 80808080 100100100100 90909090 v ia b il it y v ia b il it y v ia b il it y v ia b il it y (% ) (% ) (% ) (% ) v ia b il it y v ia b il it y v ia b il it y v ia b il it y (% ) (% ) (% ) (% ) 1111 5555 15151515 1111 5555 15151515 ochratoxinochratoxinochratoxinochratoxin a (a (a (a (µµµµmol/l)mol/l)mol/l)mol/l) ochratoxinochratoxinochratoxinochratoxin a (a (a (a (µµµµmol/l)mol/l)mol/l)mol/l) ----toctoctoctoc ----toctoctoctoc----pppp 0000 10101010 20202020 30303030 40404040 50505050 60606060 70707070 80808080 100100100100 90909090 0000 10101010 20202020 30303030 40404040 50505050 60606060 70707070 80808080 100100100100 90909090 10101010 20202020 30303030 40404040 50505050 60606060 70707070 80808080 100100100100 90909090 0000 20202020 10101010 30303030 40404040 50505050 60606060 70707070 80808080 100100100100 90909090 v ia b il it y v ia b il it y v ia b il it y v ia b il it y (% ) (% ) (% ) (% ) v ia b il it y v ia b il it y v ia b il it y v ia b il it y (% ) (% ) (% ) (% ) 1111 5555 151515151111 5555 15151515 1111 5555 151515151111 5555 15151515 ochratoxinochratoxinochratoxinochratoxin a (a (a (a (µµµµmol/l)mol/l)mol/l)mol/l) ochratoxinochratoxinochratoxinochratoxin a (a (a (a (µµµµmol/l)mol/l)mol/l)mol/l) ----toctoctoctoc ----toctoctoctoc----pppp 0000 10101010 20202020 30303030 50505050 40404040 60606060 70707070 80808080 100100100100 90909090 0000 10101010 20202020 40404040 30303030 50505050 60606060 70707070 80808080 100100100100 90909090 v ia b il it y v ia b il it y v ia b il it y v ia b il it y (% ) (% ) (% ) (% ) v ia b il it y v ia b il it y v ia b il it y v ia b il it y (% ) (% ) (% ) (% ) 1111 5555 15151515 1111 5555 15151515 ochratoxinochratoxinochratoxinochratoxin a (a (a (a (µµµµmol/l)mol/l)mol/l)mol/l) ochratoxinochratoxinochratoxinochratoxin a (a (a (a (µµµµmol/l)mol/l)mol/l)mol/l) egcgegcgegcgegcg quequequeque 0000 10101010 20202020 30303030 50505050 40404040 60606060 70707070 80808080 100100100100 90909090 0000 10101010 20202020 40404040 30303030 50505050 60606060 70707070 80808080 100100100100 90909090 v ia b il it y v ia b il it y v ia b il it y v ia b il it y (% ) (% ) (% ) (% ) v ia b il it y v ia b il it y v ia b il it y v ia b il it y (% ) (% ) (% ) (% ) 1111 5555 151515151111 5555 15151515 1111 5555 151515151111 5555 15151515 ochratoxinochratoxinochratoxinochratoxin a (a (a (a (µµµµmol/l)mol/l)mol/l)mol/l) ochratoxinochratoxinochratoxinochratoxin a (a (a (a (µµµµmol/l)mol/l)mol/l)mol/l) egcgegcgegcgegcg quequequeque 0000 10101010 30303030 20202020 40404040 50505050 60606060 70707070 80808080 100100100100 90909090 0000 10101010 20202020 30303030 50505050 40404040 60606060 70707070 80808080 100100100100 90909090 v ia b il it y v ia b il it y v ia b il it y v ia b il it y (% ) (% ) (% ) (% ) v ia b il it y v ia b il it y v ia b il it y v ia b il it y (% ) (% ) (% ) (% ) 1111 5555 15151515 1111 5555 15151515 ochratoxinochratoxinochratoxinochratoxin a (a (a (a (µµµµmol/l)mol/l)mol/l)mol/l) ochratoxinochratoxinochratoxinochratoxin a (a (a (a (µµµµmol/l)mol/l)mol/l)mol/l) catcatcatcat r osacrosacrosacrosac 0000 10101010 30303030 20202020 40404040 50505050 60606060 70707070 80808080 100100100100 90909090 0000 10101010 20202020 30303030 50505050 40404040 60606060 70707070 80808080 100100100100 90909090 v ia b il it y v ia b il it y v ia b il it y v ia b il it y (% ) (% ) (% ) (% ) v ia b il it y v ia b il it y v ia b il it y v ia b il it y (% ) (% ) (% ) (% ) 1111 5555 151515151111 5555 15151515 1111 5555 151515151111 5555 15151515 ochratoxinochratoxinochratoxinochratoxin a (a (a (a (µµµµmol/l)mol/l)mol/l)mol/l) ochratoxinochratoxinochratoxinochratoxin a (a (a (a (µµµµmol/l)mol/l)mol/l)mol/l) catcatcatcat r osacrosacrosacrosac * * vi a b ili ty (% ) vi a b ili ty (% ) vi a b ili ty (% ) vi a b ili ty (% ) vi a b ili ty (% ) vi a b ili ty (% ) vi a b ili ty (% ) vi a b ili ty (% ) vi a b ili ty (% ) vi a b ili ty (% ) vi a b ili ty (% ) vi a b ili ty (% ) ochratoxin a (µmol/lochratoxin a (µmol/lochratoxin a (µmol/lochratoxin a (µmol/l)))) ochratoxin a (µmol/lochratoxin a (µmol/lochratoxin a (µmol/lochratoxin a (µmol/l)))) ochratoxin a (µmol)/lochratoxin a (µmol)/lochratoxin a (µmol)/lochratoxin a (µmol)/l)))) ochratoxin a (µmol/lochratoxin a (µmol/lochratoxin a (µmol/lochratoxin a (µmol/l)))) ochratoxin a (µmol/lochratoxin a (µmol/lochratoxin a (µmol/lochratoxin a (µmol/l)))) ochratoxin a (µmol/lochratoxin a (µmol/lochratoxin a (µmol/lochratoxin a (µmol/l)))) * ** * * * * * * * * * * * * * * α α fig. 5: cytotoxicity of ochratoxin a (24 h, 1 15 µmol/l) in hepg2 cells after pre-incubation with rrr-α-tocopherol (α-toc), rrr-α-tocopherolphosphate, epigallocatechin gallate (egcg), quercetin (que), catechin (cat), and rosmarinic acid (rosac) (24 h, 0 , 25 , 50 , and 100 µmol/l). data are mean ± sd from two experiments in triplicate (n = 6). *indicates significant differences when compared to control (p < 0.05). 182 christine bösch-saadatmandi, christoph hundhausen, laia jofre-monseny, ralf blank, siegfried wolffram, gerald rimbach fig. 7: total glutathione (gsh, in % of control) after 24 h incubation with buthionine sulfoximine (bso, a) or ochratoxin a (ota, b) at the given concentrations. data are mean ± sd from two experiments in duplicate (n = 4, a) or triplicate (n = 6, b). *indicates significant differences when compared to control (p < 0.05). 0 10 20 30 40 50 60 70 80 90 100 0 5 10 25 50 100 bso (µm) to ta l g s h ( % o f c o n tr o l) 0 10 20 30 40 50 60 70 80 90 100 0 5 10 25 50 100 bso (µm) to ta l g s h ( % o f c o n tr o l) a 0 20 40 60 80 100 120 140 0 0.05 0.1 0.25 0.5 1 ota (µm) to ta l g s h ( % o f c o n tr o l) 0 20 40 60 80 100 120 140 0 0.05 0.1 0.25 0.5 1 ota (µm) to ta l g s h ( % o f c o n tr o l) b bso (µmol/l) ota (µmol/l) * * * over, we demonstrated a strong impact of serum protein concentration in the cell culture medium on the viability of ota-exposed hepg2 cells. a decrease in serum concentration led to a dramatic increase in ota-induced cell death in hepg2 cells. importantly, under serumfree conditions even nanomolar ota-concentrations resulted in a considerable loss of cell viability. concerning the protein binding properties of ota, a high affinity of ota for plasma proteins, both in animals and in humans, has been reported (ringot et al., 2006). in a wide range of species, including quail, mouse, rat and monkey, the fraction of free ota in plasma was found to be less than 0.2% (hagelberg et al., 1989). furthermore, it is well-known that ota toxicity depends on its half-life in the body (o’brien and dietrich, 2005), which in turn has been suggested to be influenced by binding of ota to proteins (chu, 1971; chu, 1974). this is supported by findings that albumindeficient rats had a 20to 70-times faster ota clearance from the systemic circulation than non-deficient rats (kumagai, 1985). however, studies investigating the dose-response relationship between the protein concentration in the cell culture medium and ota toxicity are lacking. for this reason, a major focus of the present study was to determine the impact of different fcs levels in the culture medium on ota-induced cell death in hepatocytes. we observed that a decrease of serum concentration in the medium was paralleled by a dramatic increase in ota toxicity, which may indicate strong protein binding of ota, possibly linked with inhibited cellular uptake and, thus, lower toxicity in hepg2 cells. consistently, a previous study revealed decreased neurotoxic effects of ota by protein binding to bovine serum albumin in embryonic chick brain and neural retina cell cultures (bruinink and sidler, 1997). on the other hand, protein binding alone does not always decrease ota fig. 6 cytotoxicity of ochratoxin a (24 h, 1 15 µmol/l) on hepg2 cells after pre-incubation with n-acetylcysteine (nac, a) (24 h, 0 4 mmol/l) or buthionine sulfoximine (bso, b) (24 h, 0 – 500 µmol/l). data are mean ± sd from two experiments in triplicate (n = 6).*indicates significant differences when compared to control (dunnett; p < 0.05). 0 10 20 30 40 50 60 70 80 90 100 ochratoxin a (µmol/l) v ia b il it y % nac 0 mmol/l nac 0.2 mmol/l nac 0.5 mmol/l nac 1 mmol/l nac 2 mmol/l nac 4 mmol/l 5 1 15 0 10 20 30 40 50 60 70 80 90 100 ochr atoxin a (µmol/l) v ia b il it y % bso 0 µmol/l bso 5 µmol/l bso 25 µmol/l bso 50 µmol/l bso 100 µmol/l bso 500 µmol/l 5 1 15 0 10 20 30 40 50 60 70 80 90 100 ochratoxin a (µmol/l) v ia b il it y % nac 0 mmol/l nac 0.2 mmol/l nac 0.5 mmol/l nac 1 mmol/l nac 2 mmol/l nac 4 mmol/l 5 1 15 0 10 20 30 40 50 60 70 80 90 100 ochr atoxin a (µmol/l) v ia b il it y % bso 0 µmol/l bso 5 µmol/l bso 25 µmol/l bso 50 µmol/l bso 100 µmol/l bso 500 µmol/l 5 1 15 * * * fig. 6: cytotoxicity of ochratoxin a (24 h, 1 15 µmol/l) on hepg2 cells after pre-incubation with n-acetylcysteine (nac, a) (24 h, 0 4 mmol/l) or buthionine sulfoximine (bso, b) (24 h, 0 500 µmol/l). data are mean ± sd from two experiments in triplicate (n = 6). *indicates significant differences when compared to control (dunnett; p < 0.05). a b effect of antioxidants on ota-induced cytotoxicity in hepatocytes 183 toxicity (stojkovic et al., 1984). it has been suggested that binding of ota to two small serum proteins (molecular mass 20.000 da), being able to pass through the glomerular membrane, might explain the ota-mediated nephrotoxic effect in mammals (stojkovic et al., 1984). taken together, variations in the degree of protein binding as well as selective binding of ota to distinct proteins may contribute to the often reported species-specific differences in ota toxicity (dietrich et al., 2001; o’brien et al., 2001; heussner et al., 2002). in the present study, we investigated the potential cytotoxic effects of ota at nanomolar concentrations. previous studies have revealed that nanomolar ota concentrations induced damage to renal and brain cells, due to an induction of apoptosis (gekle et al., 2000) and a loss of neuronal enzyme activity (monnet-tschudi et al., 1997). our study, however, is the first to demonstrate that ota causes cell death in hepatocytes at nanomolar concentrations, which is probably due to the absence of foetal calf serum (fcs) in the culture medium. under serum-free conditions, incubation with 50, 100, or 250 nmol/l ota for 24 hours resulted in an approximately 10, 40, or 60% decrease in viability of hepg2 cells, respectively. we observed toxicity with concentrations as low as 50 nmol/l ota, which is within the range of ota detected in human blood and serum. between 6 and 26% of human blood and serum samples from the balkan area contained ota in the range of 1 35 ng/ml (2.5 88 nmol/l) and 1 40 ng/ml (2.5 100 nmol/l) (petkova-bocharova et al., 1988), respectively, paralleled by an increased occurrence of the balkan endemic nephropathie (ben) (petkova-bocharova et al., 1988). considering the elimination half-life of ota in humans of approximately 36 days (studer-rohr et al., 2000), which is substantially longer than that reported for other species – mice 40 h; rats 55-120 h (galtier et al., 1979; hagelberg et al., 1989); pigs 72-120 h (galtier et al., 1981); monkeys 820 h (kuiper-goodman and scott, 1989) – it is apparent that ota constitutes a higher risk in humans. our study also demonstrated that at least 45% of the cells were still viable after incubation with 50 µmol/l ota for 24 h. in serum-free medium this maximum toxicity was reached at ota concentrations as low as 0.25 µmol/l. incubation with 1 50 µmol/l ota did not further increase ota-induced cell death in hepg2 cells. in accordance with our results it has been shown in llc-pk1 cells that higher ota-concentrations (high nmol/l to low µmol/l) or longer times of exposure (96 h) did not further decrease cell viability (dreger et al., 2000). one hypothesis, which might explain the survival of a considerable number of hepg2 cells, suggests the existence of ota carrier systems being saturated at low otaconcentrations and, thus, not being able to increase intracellular ota-transport at higher ota-concentrations. such ota carriers, belonging to the family of human organic anion transporters, are well-known in proximal tubule cells from mice (jung et al., 2001; babu et al., 2002). however, studies investigating the transport of ota in liver cells are rare. it has been proposed, however, that the organic anion-transporting polypeptide (oatp) is involved in otauptake in hepatocytes (kontaxi et al., 1996). neither the toxicokinetics of ota, nor its precise toxicity mechanisms have been established to date. nevertheless, the involvement of reactive oxygen species (ros) in ota toxicity seems to be likely (rahimtula et al., 1988; gautier et al., 2001; schaaf et al., 2002). therefore, one objective of our study was to counteract ota toxicity by pre-incubation of hepg2 cells with various antioxidant test substances. we pre-incubated liver cells with 25, 50, or 100 µmol/l of the respective antioxidants. concerning α-toc these concentrations are similar to physiological plasma concentrations, which have been reported in the range of 25 40 µmol/l (hensley et al., 2004), referring to flavonoid concentrations used in the cell culture experiments however exceeded physiological plasma concentrations (0.1 10 µmol/l) (manach et al., 2004). to avoid interactions between ota and the test compounds in the culture medium, cells were washed twice with pbs before incubation with ota. none of the test compounds prevented hepg2 cells from ota-induced cell death. this is in contrast to studies reporting protective effects of antioxidants towards ota-induced cell damage. for example, 24 h of pre-treatment with 50 µmol/l cyanidin-3-o-beta-glucopyranoside or 50 µmol/l rosmarinic acid inhibited the cytotoxicity of 10 µmol/l ota in hepg2 cells by 25 and 35%, respectively (renzulli et al., 2004; guerra et al., 2005). another experiment, also using ota in serum-free medium, showed that three hours of pre-incubation with 1 nm α-toc decreased ota-induced cytotoxicity in bovine mammary epithelium cells by 10% (baldi et al., 2004). the reasons for these conflicting results remain unclear, although cell-specific differences might be one plausible explanation. we also found that egcg, que, and α-toc-p enhanced the toxic effects of ota. α-toc-p inhibits cell proliferation (ogru et al., 2004) and modulates membrane fluidity (rezk et al., 2004). the latter might be the reason for a facilitated uptake of ota into hepg2 cells, and thus, for the observed synergistic toxic effects of α-toc-p and ota. moreover, it has been demonstrated that both egcg and que are able to exert not only antioxidant, but also pro-oxidant activity, partly due to the formation of hydrogen peroxide (metodiewa et al., 1999; elbling et al., 2005; ferraresi et al., 2005), which might explain the increased cytotoxicity of ota in hepg2 cells. we further investigated the importance of the intracellular antioxidant glutathione for the prevention of ota toxicity. several studies report a depletion of glutathione by buthionine sulfoximine (bso), often paralleled by either loss of cell viability or increased susceptibility to compounds exerting oxidative stress (wright et al., 1998; anderson et al., 1999; long et al., 2000; anderson and reynolds, 2002; honda et al., 2004). however, it has been shown that hepg2 cells are able to survive bso treatment, which may be explained by an up-regulation of bcl-2, a protein preventing apoptosis (d’alessio, 2004). in the present study we confirmed the resistance of hepg2 cells to bso treatment and showed, additionally, that there were no synergistic effects of ota and bso on cytotoxicity in hepg2 cells. although gsh was dose-dependently depleted by both bso and ota (fig. 7), pre-treatment with bso did not enhance cell death (fig. 6). furthermore, nac did not increase cellular gsh levels (data not shown), and was not able to prevent hepg2 cells against the toxic effects of ota (fig. 6). consistent with these findings are the results of a study with cultured rat embryonic cells demonstrating a decrease of gsh induced by ota, but no protection due to exogenous gsh supplementation (hong et al., 2000). on the other hand, it has been shown that nac completely protected llc-pk1 from otainduced cell death (schaaf et al., 2002). interestingly, we observed a biphasic effect of ota on intracellular gsh levels in hepg2 cells. 50 and 100 nmol/l ota increased cellular gsh to 116 and 121% compared to controls, respectively, while ota at concentrations between 0.1 and 1 µmol/l dose-dependently decreased cellular gsh levels to approximately 70%. this effect may indicate that gsh is involved in the detoxification of ota, possibly by directly binding to ota as proposed by dai and co-workers (2002). on the other hand, it has been shown for brain cells that ota-exposure leads to an increased generation of nitric oxide (no) (zurich et al., 2005), which in turn is detoxified through conjugation with gsh. in conclusion, the presented data indicate a dose-dependent effect of foetal calf serum concentrations in the cell culture medium on ota-induced cytotoxicity in hepg2 cells. supplementation with dietary antioxidants did not counteract cytotoxic effects of ota. although cellular gsh levels are modulated by ota, mechanisms other than oxidative stress are likely to be involved in ota-induced cell death in hepg2 cells. acknowledgements the authors gratefully thank susan pollard from the school of food biosciences, university of reading, uk and dr. jan frank from this department for their critical reading of the manuscript and helpful comments. c. bösch-saadatmandi and c. hundhausen contributed equally to this manuscript. references anderson, c.p., reynolds, c.p., 2002: synergistic cytotoxicity of buthionine sulfoximine (bso) and intensive melphalan (l-pam) for neuroblastoma cell lines established at relapse after myeloablative therapy. bone marrow transplant. 30, 135-140. anderson, c.p., tsai, j.m., meek, w.e., liu, r.m., tang, y., forman, h.j., reynolds, c.p., 1999: depletion of glutathione by buthionine sulfoxine is cytotoxic for human neuroblastoma cell lines via apoptosis. exp. cell 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with glutathioneinduced inhibition of ap24 activation of nuclear dna fragmentation. cancer res. 58, 5570-5576. zurich, m.g., lengacher, s., braissant, o., monnet-tschudi, f., pellerin, l., honegger, p., 2005: unusual astrocyte reactivity caused by the food mycotoxin ochratoxin a in aggregating rat brain cell cultures. neuroscience 134, 771-782. address of the authors: christine bösch-saadatmandi, christoph hundhausen, laia jofre monseny, gerald rimbach1, institute of human nutrition and food science, christianalbrechts-university, hermann-rodewald-strasse 6, d-24118 kiel, germany. ralf blank, siegfried wolffram, institute of animal nutrition and physiology, christian-albrechts-university, hermann-rodewald-strasse 9, d-24118 kiel, germany. 1corresponding author: rimbach@foodsci.uni-kiel.de 186 christine bösch-saadatmandi, christoph hundhausen, laia jofre-monseny, ralf blank, siegfried wolffram, gerald rimbach << /ascii85encodepages false /allowtransparency false /autopositionepsfiles 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<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> >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice journal of applied botany and food quality 87, 117 123 (2014), doi:10.5073/jabfq.2014.087.018 1nucleo de investigación en producción alimentaria 2 escuela de agronomía, facultad de recursos naturales, universidad católica de temuco, temuco, chile 3 university hohenheim, institute of plant production and agroecology in the tropics and subtropics, stuttgart, germany effect of seed treatment with natural products on early arbuscular mycorrhizal colonization of wheat by claroideoglomus claroideum c.g. castillo1, 2, c. fredericksen2, r. koch2, e. sieverding3* (received august 25, 2013) * corresponding author summary commercially available natural products (np) were applied to seed of winter wheat which was sown in a sandy soil infected with the arbuscular mycorrhizal fungus claroideoglomus claroideum. the aim was to investigate whether an isoflavonoid (formononetin), different humates/algae extracts and inorganics (dolomitic lime, silicates) improved the early mycorrhization process. experiments were carried out under controlled conditions in small pots in growth chambers. plants were harvested between 14 and 29 days after treatment. the results showed that the isoflavonoid accelerated the mycorrhiza formation by increasing the number of mycorrhizal infection points with consequently higher infection frequency, intensity, and mycorrhized root biomass. a dolomitic limestone also improved the mycorrhizal infection process. no effects were found by the humates, extracts of algae and a silicate product. while in general the mycorrhization was most influenced by lower dose rates of np (0.1 and 1 mg seed-1), a higher rate of 10 mg seed-1 had lower and sometimes negative effects on the mycorrhization. on the other side, highest np doses had positive effects on some plant growth parameter, which may have been related to the potassium content of e.g. the humate products, or because these products had plant growth promoting effects. it can not be excluded that some products, like the dolomitic limestone had an indirect effect on the mycorrhiza development via influencing other micro-organisms in the wheat rhizosphere. introduction arbuscular mycorrhizal fungi (amf) of the glomeromycota play important roles for plant nutrition and resilience of plants after abiotic stress situations (smith and read, 2008), in particular under acid soil conditions. improved root health and higher tolerance to root pathogens and nematodes were reported, too (whipps, 2004; kaewchai et al., 2009). research in various agronomic plants have shown that early, quick and extensive mycorrhizal root colonization is one of the most important pre-requisites to make use of the symbiosis for crop production (garcía-garrido et al., 2009). this applies in particular for short season crops, or annual crops like cereals. accordingly, technologies were developed to inoculate crops with arbuscular mycorrhizal fungi at time of planting. inoculation is nothing else then placing a substrate with a high and concentrated mycorrhizal infection potential under the germinating seed of a plant. this is making sure that the first roots get immediately infected, and more and better than with the native amf population which concentration is often low in natural soils (sieverding, 1991; salvioli et al., 2012). it is well known that cereals like wheat, barley and oats are mycorrhizal, and under acidic soil conditions, like in volcanic derived andosols of southern chile, amf are important for phosphate (p) uptake of the crop and for tolerating aluminum (al) stress conditions of these soils (mora et al., 2006). problematic low p soils are also found in other parts of the world and in south america, like in argentina, bolivia, paraguay and uruguay where wheat production is of primary socio-economic importance. when the mycorrhizal symbiosis is to be managed, the inoculation technology (see above: artificially increased inoculum potential under the seed) is likely not practical and not economic, considering that likely the equivalent value of 1 t grain (e.g. 150-200 usd) or more may be necessary for application of inocula per hectare, as found by own internet searches. also, as far as we know, in the above mentioned countries no suitable commercial amf inoculum is available. the alternative to applying amf inocula is the management of the native amf population by agronomic practices in such a way that the early mycorrhizal development is enhanced. this strategy was proposed longer time ago (sieverding, 1991) and some agronomic practices, like incorporation of p fertilizers enhanced amf in wheat (covacevich et al., 2007). also, selection of wheat varieties with strong early root growth appears to be a potential measure to obtain improved early mycorrhizal infection rates (castillo et al., 2012), while some fungicide seed treatments may or may not decrease the root colonization (burrows and ahmed, 2007; murillo-williams and pedersen, 2008). seed treatment with chemical or biological products which improve early mycorrhizal infection would indeed be the easiest practical way to influence amf in extensive large acreage cereal production, as farmers are adapted to treat wheat seed with pesticides anyway. it was thus the objective of the here presented research to investigate whether some selected inert natural products (np) affect the early mycorrhizal colonization in wheat. one np was found some 20 years ago to improve the early am infection of plants: formononetin, an isoflavon which is extracted from clover roots (nair et al., 1991; tsao et al., 2006). this still patented active showed a quite consistent improvement in mycorrhization of roots and of yields (davis et al., 2005; westphal et al., 2008). it is used as a reference np in our studies. other np are often advertised that they improve the native amf action but research results of their effects are not available. these other natural products are either humic and or fulvic acids, extracts of algae, dolomite limestone and sodium silicates, and are all commercially available not only in chile but also elsewhere. we investigated the effects in growth chambers at optimum temperatures using one amf fungal species, claroideoglomus claroideum (n.c. schenck & g.s. sm.) c. walker & a. schüssler. this fungal species was isolated from cultivated soils of southern chile and had proven infectivity in different soils and different agronomic crops (castillo et al., 2006; castillo et al., 2010; castillo et al., 2013). the results presented here are part of a series of growth chamber and field experiments with the aim to evaluate the effect of different np, as well as micro-organisms on early amf root colonization in different cereals species and different soils. the current paper reports the results of three sequential and related experiments to determine the effect of np on germination of wheat seeds and early amf root colonization parameter and growth parameter of the plant. 118 c.g. castillo, c. fredericksen, r. koch, e. sieverding materials and methods soil substrate for bio-assays sandy soil was used in the experiments which had been sterilized in an autoclave for one hour each on two consecutive days. the sand had ph 6.7 and was low in available nutrients. the sand was mixed with 2.5% (v/v) of a soil substrate containing 57 spores per ml of the mycorrhizal fungus cl. claroideum, so that the final mixture contained about 1-2 spores per ml. this is a relative low number of spores in field soils but was chosen on purpose as sub-optimal so that the np could promote the fungus for infection. this amf species had been multiplied on maize as host plant in a soil-perlite mixture for 6 months. the fungal species originated from a soil of the araucanía region in southern chile where it is frequently found. for the experiments the soil was added to 250 ml plastic pots. wheat variety the triticum aestivum l. variety “kumpa-inia” was selected for the experiments because this had shown low root growth and low infection rates in earlier experiments (castillo et al., 2012). we thus expected good responses in growth parameter to the application of np if there were any. for the experiments in soil, the seeds physiological dormancy was broken through pre-drying by heating in a furnace at 30 ºc for 7 days (ista, 2008). seed had not been treated with fungicides or insecticides before use. seed was surface sterilized with sodium-hypochlorite, washed and placed on wet filter paper to germinate. when the first signs of germination were visible, equal progressed seed was place in planting holes one in each pot. one ml of either water (control) or water with np at specific concentrations was added onto each seed so that the seed and the soil below the seed were wetted with the np. the seed was then tapped with the sand. natural products (np) the following np were used: (b) borregro ha-2 powder, company borregaard ligno tech bridgewater, nj, usa: it is a complete water soluble potassium (k) humate product, made by extraction of high quality leonardite ore. it is 50 % humic acid and 50 % fulvic acid. (d) disper alghum gs, company disper, alicante, spain (in chile: am ecological, santiago de chile): complete water soluble product containing humic extracts from american leonardite and seaweed extract from ascophyllum nodosum. (m) myconate®, company plant health care usa agriculture, pittsburgh, usa: powder product of a potassium salt water-soluble form of formononetin (7-hidroxy, 4’-metoxy isoflavone). (n) natural green®, company natural green gmbh, cologne, germany: finely milled energized product derived from natural deposits consisting of dolomite limestone, mainly caco3 (79.19 %) and mgco3 (4.62 %). (p) powhumus wsg-85®, company humintech gmbh, düsseldorf, germany: contains water soluble potassium salt of humic and fulvic acids (80-85 % w/w) from leonardite. (q) quick sol®, company beyond international inc., miami, usa (in chile: am ecological, santiago de chile): water soluble sodium silicate, contains silicon (36 %), sodium (6 %), humic and fulvic acids (each 1 %). all products were dissolved or dispersed in water and 1 ml solution was applied per seed containing 0.1 mg (low concentration), 1 mg (normal concentration) and 10 mg (high concentration) of each product. the concentrations were derived from the use recommendation of myconate® for optimum mycorrhizal colonization (koide, 1999). considering a weight of about 40 g per 1000 wheat seeds, and 100 kg of wheat seed ha-1, the 2.5 x 106 ha-1 seeds will have received 0.25, 2.5 or 25 kg np product. seed germination tests seed germination percentage were evaluated according to the rules of the international seed testing association (ista, 2004) using four replicates of 25 seeds per treatment sowing previously above pleated sterile moistened paper; 1 ml of differently concentrated np was added to each seed as well as control to which sterile water was added. after applying the np on the seeds, the plates were tightly sealed with parafilm. the experiment was conducted in a semi-controlled growth chamber adjusted to 25 °c ± 2 °c day/night temperatures, and a 16 h photoperiod at 310 μmol m-2 s-1 of photosynthetic photon flux density. for all np treatments, including the control, evaluations took place in accordance with ista (2008) at eight days after treatment. in some cases, also plumule elongation and radicle length were measured. mycorrhization bioassays the experiments were conducted in a growth chamber with permanent 25 °c ± 2 °c temperature, and a 16 h photoperiod at 310 μmol m-2 s-1 of photosynthetic photon flux density. the experimental set up was a complete randomized design, with five replicates per treatment and harvest. plants were watered daily, no fertilizer were applied. five pots of each treatment were harvested at 14 or 15 days after planting, dap (h1); 17 dap (h2); 20 dap (h3); 24 dap (h4) and 29 dap (h5). these harvests corresponded to plant growth stages 1.12 and 1.13 (two and three leaves unfolded) (zadock et al., 1974). for each harvest, plant shoots were harvested and the dry weight was determined; before, the length of the longest leave was measured from the old germinated seed up to the tip. roots were carefully washed to remove any sand adhered to the surface, length of the longest root (from germinated seed to tip) was measured and fresh weight and dry weight were determined. a subsample of fresh root material was taken randomly after the whole root sample had been cut into about 1 cm long segments. this subsample was used for determination of am colonization. am fungal structures in roots were stained with 0.05 % trypan blue at 60 ºc for 5 min in a water bath (koske and tessier, 1983) after heating in 2.5 % koh at 60 ºc for 12 min, rinsing then in a few changes of water, and acidification in 1 % hydrochloric acid at room temperature for one hour. fungal colonization was evaluated according to trouvelot et al. (1986) and expressed as frequency percentage (f%), mycorrhization intensity percentage (m%) and mycorrhizal infection entry points (ep) in root cortex. twenty randomly chosen root fragments of 1 cm in length of each treatment were mounted on slides and examined microscopically at 100-400x magnification under an olympus ys100 light microscopy. frequency percentage was assessed by relating the number of infected 1 cm root segments over number of total root segments. intensity of am infection in the root cortex was determined by the five-class classification system following the method described by trouvelot et al. (1986) where the numbers indicate the proportion (%) of root cortex colonized by the fungus (alarcón and cuenca, 2005; castillo et al., 2012), whilst ep are the number per 20 root segments. finally, the root biomass infected with mycorrhiza was calculated by multiplying the root fresh weight with the mean intensity of am infection divided by 100 (castillo et al., 2012) where the infection intensity classes were transformed to percentage scales. statistical analysis the data obtained were submitted to the shapiro-wilk normality test and then an anova of one factor was carried out using the duncan a posteriori multiple range means separation test (p ≤ 0.05). all analyses were conducted with the computer program spss version 12.0 (spss, 2003). natural products 119 results effect of different np the seed germination test showed that the 0.1 and 1 mg seed-1 rate had no negative effect on seed germination (fig. 1). at 10 mg seed-1, products d, m, q decreased the germination to rates between 90 and 95 %, although not significantly. mycorrhizal entry points, frequency and intensity of root infection, and infected root biomass in general appeared to be highest at the lowest concentration of the np and lowest at the highest concentrations (fig. 2). myconate (m) increased the mycorrhizal parameter at the lowest concentration, significantly. frequency of root infection was also increased by d and n, at the lowest concentration. infected root biomass was significantly increased over the control with b, d, m, n at the lowest dose rate. at the medium dose rate only m increased mycorrhizal entry points and frequency of infection, while at the high dose m and p and q decreased the infection parameter. growth parameters were influenced by the np and the dose rate used (tab. 1). while plant length was often higher than c when d, m, n and q where applied at the medium and high rate, b and p increased height at medium rate, only. root length had significantly increased often with the highest dose of the np, except with b. shoot dry weight increased in general with higher dose of the np. root dry matter was also generally highest at the highest np dose. effect of myconate (m) and natural green (n) on mycorrhizal and plant development over time while in the first experiment harvest of the plants was very early, the second experiment aimed to define the development of the mycorrhization over time. two np were selected, m as reference product and n. natural green had shown some intermediate effects on the mycorrhization and on the plant growth parameter in the first experiment at 15 dap. in this experiment only one dose was tested: 1 mg np plant-1. all mycorrhizal parameter increased over time and both np had positive effects on the development of the mycorrhiza in roots, as shown by numbers of entry points, frequency and intensity of infection and infected root biomass (fig. 3). both, m and n increased the mycorrhization often significantly over the control plants, and more with more advancement over the time. however, it should be noted that the mycorrhizal infection intensity was very low in general and less than 1% of the root (fig. 3, frequency) was infected up to 24 days after planting. plant growth parameter increased over time (tab. 2). while plant height was not influenced by np, root length, shoot dry weight and root dry weight increased through np already from the second harvest on. 7   the seed germination test showed that the 0.1 and 1 mg seed-1 rate had no negative effect on seed germination (fig. 1). at 10 mg seed-1, products d, m, q decreased the germination to rates between 90 and 95%, although not significantly. fig. 1: seed germination percentage of t. aestivum var. “ kumpa-inia” with application of borregro (b), dispher alghum (d), myconate (m), natural green (n), pow humus (p), quick sol (q) and control without addition np (c) at three concentrations: 0.1 mg ml-1 seed-1 (d-1); 1 mg ml-1 seed-1 (d-2), and 10 mg ml-1 seed-1 (d-3). no significant differences were found between values at each concentration using duncan (p�0.05). mycorrhizal entry points, frequency and intensity of root infection, and infected root biomass in general appeared to be highest at the lowest concentration of the np and lowest at the highest concentrations (fig. 2). myconate (m) increased the mycorrhizal parameter at the lowest concentration, significantly. frequency of root infection was also increased by d and n, at the lowest concentration. infected root biomass was significantly increased over the control with b, d, m, n at the lowest dose rate. at the medium dose rate only m increased mycorrhizal entry points and frequency of infection, while at the high dose m and p and q decreased the infection parameter. d-1 d-2 d-3       fig. 1: seed germination percentage of t. aestivum var. “kumpa-inia” with application of borregro (b), dispher alghum (d), myconate (m), natural green (n), pow humus (p), quick sol (q) and control without addition np (c) at three concentrations: 0.1 mg ml-1 seed-1 (d-1); 1 mg ml-1 seed-1 (d-2), and 10 mg ml-1 seed-1 (d-3). no significant differences were found between values at each concentration using duncan (p≤0.05). tab. 1: plant parameters of t. aestivum var. “kumpa-inia” with application of borregro (b), dispher alghum (d), myconate (m), natural green (n), pow humus (p), quick sol (q), and a control (c) without addition np, at three concentrations: 0.1 mg ml-1 seed-1 (d-1); 1 mg ml-1 seed-1 (d-2), and 10 mg ml-1 seed-1 (d-3). natural products plant parameters doses c* b d m n p q d-1 13.5 b 18.4 ab 15.5 b 23.7 a 13.1 b 18.3 ab 17.4 b height (cm) d-2 13.5 b 20.8 a 20.2 a 20.8 a 19.5 a 20.1 a 20.8 a d-3 13.5 b 18.1 ab 23.1 a 23.3 a 20.8 a 18.4 ab 19.8 a d-1 16.2 b 24.2 ab 24.8 ab 31.7 a 20.8 ab 30.2 a 23.7 ab root lenght (cm) d-2 16.2 b 20.3 b 39.7 a 21.8 b 27.6 ab 24.6 ab 25.6 ab d-3 16.2 b 15.9 b 29.1 a 27.6 a 25.5 a 27.2 a 26.6 a shoot dry weight d-1 15.3 bcd 17.7 abc 20.0 a 20.3 a 10.7 d 15.0 cd 17.3 abc (mg plant-1) d-2 15.3 b 20.0 ab 27.7 a 22.7 ab 19.7 ab 25.0 ab 24.7 ab d-3 15.3 b 30.3 a 28.3 ab 28.0 ab 23.0 ab 21.3 ab 23.0 ab root dry weigth d-1 12.0 b 15.0 ab 19.0 a 11.7 b 16.7 ab 11.3 b 18.3 a (mg plant-1) d-2 12.0 c 17.7 bc 18.0 b 16.0 bc 24.7 a 18.3 b 20.7 ab d-3 12.0 c 29.0 ab 33.7 a 30.3 ab 21.3 bc 33.3 a 27.0 ab in each row, for each dose, values not sharing a letter in common differ significantly according to duncan test (p≤0.05). *values for c were repeated in each concentration as a reference value for the nps. 120 c.g. castillo, c. fredericksen, r. koch, e. sieverding d-1 d-2 d-3 fig. 2: mycorrhizal colonization parameters: entry points, frequency of infection, infection intensity, and infected root biomass (irb) in t. aestivum var. “kumpa-inia” with application of borregro (b), dispher alghum (d), myconate (m), natural green (n), pow humus (p), quick sol (q) and control without addition np (c) at three concentrations: 0.1 mg ml-1 seed-1 (d-1); 1 mg ml-1 seed-1 (d-2), and 10 mg ml-1 seed-1 (d-3). columns for each concentration sharing a letter in common do not differ significantly according to duncan test (p≤0.05). discussion for this study, we took mycorrhizal infection entry points in roots as one of the main parameter because its number may be the direct result of the np treatments on germination of fungal propagules and branching of mycorrhizal germination structures in soil. the infection intensity was quantified by conventional methods as this was a method applicable under the conditions in chile, and because real time pcr was considered, at the time of the start of the experiments, a poor method for quantifying mycorrhizal colonization in roots (könig et al., 2010). the results of this investigation showed that np, when used as seed treatments, can differ strongly in their effects on early mycorrhiza formation in wheat. the investigated products can be characterized into three groups: 1) potassium salts of plant extracts (m), 2) potassium salts of lignates, humates and sea weed extracts, and mixtures thereof (b, d, p), and 3) inorganics (n, q). only m has been more intensively investigated and tested in different agronomic crops for mycorrhiza effects, in the past (nair et al., 1991; davies et al., 2005; novais and siqueira, 2009). the isoflavonoid formononetin stimulated amf spore germination, hyphal branching in the soil and root colonization by am fungi (nair et al., 1991; siqueira et al., 1991) and increased the number of appressoria and/or entry points to the roots of soybeans (silva-junior and siqueira, 1997). in our study, entry point number of the am fungus cl. claroideum was also most increased by m in wheat roots, and was higher than with any other np, with some exception for n. as a result of this, higher infection frequency and intensity were found. because root dry matter production also developed better, higher root biomass was occupied with mycorrhizal structures when the wheat seed was treated. myconate led to quicker and stronger mycorrhization of wheat as compared to no seed treatment. assuming natural products 121 that myconate has little or no potassium fertilizing effect at the low concentration used, we conclude that the early and quicker mycorrhization process was responsible for the mostly significant increase in particular of root length and root and shoot dry matter formation of the plant. the shoot:root ratio was also wider (tab. 2) which is often a clear indicator for the activity of mycorrhiza (smith et al., 2011). the results also indicate that the stimulation of mycorrhiza by formononetin with the consequent growth enhancement can take place even when the root infection intensity is low and below or near 1 % only. it was clear from the results that the lignates/humates (b, p) and mixtures of humates with algae extracts (d) had little stimulating effects on the mycorrhizal development. it may be that the one single measurement of mycorrhiza parameter at 15 dat was too early in the experiment, or that the dose rate of the products was not adequate and too high. obviously was the highest dose of p inhibiting the entry point formation and doubtless that in particular the root dry matter production was increased over the control at the highest dose of these np. it may be, but needs confirmation, that this effect was caused by the potassium which was applied with the humates, as well as with the myconate. dispher algum contains some extracts of algae, and such extracts are known to have plant growth stimulating hormonal effects (ördög et al., 2004; norrie and keathley, 2006; khan et al., 2009). it was striking that dry matter production at the lower doses of this product was significantly better than in the control and some of the other products. we assume that this increase was due to other effects than to mycorrhiza. of the two inorganics the dolomitic limestone (n) stimulated the mycorrhization of the plants, and significantly over time. shoot and root dry weight was also higher which may have been an effect of the better and quicker mycorrhization process. the effect of n on the mycorrhiza formation cannot easily be explained. it is unlikely that direct nutritional aspects (ca, mg) have played any role in the growth improvement as the rates were far too low (2.5 kg ha-1 in the second experiment). neutralization effects of toxic elements by the limestone can also be excluded as the soil used in the experiment was fig. 3: mycorrhizal colonization parameters: entry points, frequency of infection, infection intensity, and infected root biomass (irb) in t. aestivum var. “kumpa-inia” with application of myconate (m), natural green (n), and a control (c) without addition np at 14 (h1), 17 (h2), 20 (h3), 24 (h4), and 29 (h5) days after treatment. data of each harvest not sharing a letter in common differ significantly according to the duncan test (p≤0.05). 10   significantly over the control plants, and more with more advancement over the time. however, it should be noted that the mycorrhizal infection intensity was very low in general and less than 1% of the root (fig. 3, frequency) was infected up to 24 days after planting.   fig. 3: mycorrhizal colonization parameters: entry point, frequency infection, infection intensity, and infected root biomass (irb) in t. aestivum var. “ kumpa-inia” with application of myconate (m), natural green (n), and a control (c) without addition np at 14 (h1), 17 (h2), 20 (h3), 24 (h4), and 29 (h5) days after treatment. data of each harvest not sharing a letter in common differ significantly according to the duncan test (p�0.05). plant growth parameter increased over time (tab. 2). while plant height was not influenced by np, root length, shoot dry weight and root dry weight increased through np already from the second harvest on. tab. 2: plant growth parameters of t. aestivum var. “kumpa-inia” with application of myconate (m), natural green (n), and a control without addition of np (c) at 14 (h1), 17 (h2), 20 (h3), 24 (h4), and 29 (h5) days after treatment. cl. claroideum plant parameters harvest c m n height (cm) h1 20.2 a 21.2 a 22.7 a h2 21.8 a 23.3 a 22.5 a h3 24.0 a 27.2 a 27.3 a h4 30.5 a 31.5 a 31.1 a h5 32.0 a 30.2a 32.7 a root lenght (cm) h1 16.8 b 19.8 b 26.0 a h2 13.0 b 20.2 a 23.3 a h3 18.4 b 26.8 a 23.3 ab h4 15.5 b 24.7 a 19.3 ab h5 19.0 b 35.0 a 35.7 a shoot dry weight h1 17.8 c 23.2 b 27.6 a (mg plant-1) h2 23.1 b 28.9 a 30.8 a h3 26.5 c 38.9 b 42.2 a h4 41.0 b 44.1 a 46.5 a h5 55.6 b 81.8 a 83.7 a root dry weigth h1 5.7 b 5.7 b 8.3 a (mg plant-1) h2 6.6 b 9.6 ab 11.9 a h3 10.4 b 10.8 b 14.5 a h4 13.2 a 15.6 a 16.0 a h5 15.4 b 23.3 a 24.5 a values in each row not sharing a letter in common differ significantly according to the duncan test (p≤0.05). 122 c.g. castillo, c. fredericksen, r. koch, e. sieverding not very acidic. it is possible that this product had an indirect effect on mycorrhiza formation by influencing positively other soil microorganisms in the rhizosphere of wheat seedlings as the experiment was not under strict sterile conditions. it was frequently shown that helper bacteria can stimulate the mycorrhizal infection processes (johansson et al., 2004; miransari, 2011), and it may be that such micro-organisms stimulated mycorrhizal propagule germination and hyphal branching in soil as the number of mycorrhizal infection entry points was almost as high as with myconate. it is known since longer time that rhizobacteria can produce plant hormones and hormone type products like salicylic acids (meyer and höfte, 1997). latter not only can induce resistance against pathogens in plants but also can influence positively the plant physiology and plant growth (hayat et al., 2007). more research is required on this subject as such effects must be powerful as they resulted in significant better plant establishment during the first 29 days after seed treatment. the silicate based product (q) had no effect on the mycorrhization process but surprisingly the root dry matter production was significantly better that in none treated wheat. silicates are reported to strengthen the plant tissue against attacks by pathogens or to form physical barriers against soil fungi on roots (shen et al., 2010), but it appears here that it did not inhibit mycorrhiza infection ratings more than other products, even at high dose rates. natural products, when applied to seed of plants, may have a number of different effects on the early development of mycorrhiza and plants. some natural products like formononetin and substances in sea weed extracts may mimic flavonoids which are exudated by plant roots (steinkellner et al., 2007). such substances can be important signaling compounds for the establishment of microb-plant interactions, like arbuscular mycorrhiza (catford et al., 2006). indirect effects of np on other soil micro-organisms are also possible, and these rhizosphere micro-organisms can produce phytohormone like substances (e.g. oligosaccharides, salicylic acids) which influence plant growth (tsavkelova et al., 2006) without stimulating mycorrhiza. the concentration of np used for seed treatment appears to play a role for promoting the mycorrhizal infection process. in general, lower rates are more effective than higher rates. it is unclear whether this has something to do with phytotoxic effects; the seed germination (fig. 1) was numerically slightly inhibited by some np (d, m, q) but not to levels which were seen critical for wheat establishment. whether or not the initial positive effect of e.g. myconate and natural green, on mycorrhiza will translate to more wheat yield, can only be found in field experiments. acknowledgements this study was financed by fondecyt project 11090014, from comisión nacional de investigación científica y tecnológica, conicyt, chile. references alarcón, c., cuenca, g., 2005: arbuscular mycorrhizal in coastal sand dunes of the paraguaná, península venezuela. mycorrhiza 16, 1-9. burrows, r., ahmed, i., 2007: fungicide seed treatments minimally affect arbuscular-mycorrhizal fungal (amf) colonization of selected vegetable crops. j. biol. sci. 7, 417-420. castillo, c.g., borie, f., godoy, r., rubio, r., sieverding, e., 2006: diversity of mycorrhizal plant species and arbuscular mycorrhizal fungi in evergreen forest, deciduous forest and grassland ecosystems of southern chile. j. appl. bot. food qual. 80, 40-47. castillo, c.g., rubio, r., borie, f., sieverding, e., 2010: diversity of arbuscular mycorrhizal fungi in horticultural production systems of southern chile. j. soil sci. plant nutr. 10, 407-413. castillo, c.g., puccio, f., morales, d., borie, f., sieverding, e., 2012: early arbuscular mycorrhiza colonization of wheat, barley and oats in andosols of southern chile. j. soil sci. plant nutr. 12, 511-524. castillo, c.g., morales, a., rubio, r., barea, j.m., borie, f., 2013: interactions between native arbuscular mycorrhizal fungi and phosphate solubilizing fungi and their effect to improve plant development and fruit production by capsicum annuum l. afr. j. microbiol. res. 7, 33313340. catford, j.g., staehelin, c., larose, g., piche, y., vierheilig, h., 2006: systemically suppressed isoflavonoids and their stimulating effects on nodulation and mycorrhization in alfalfa split-root systems. plant soil 285, 257-266. covacevich, f., echeverría, h.e., aguirrezabal, l.a.n., 2007: soil available phosphorus status determines indigenous mycorrhizal colonization of field and glashouse-grown spring wheat from argentina. applied soil ecology 35, 1-9. davies, f., calderon, c., huaman, z., gómez, r., 2005: influence of a flavonoid (formononetin) on mycorrhizal activity and potato crop productivity in the highlands of peru. sci. hortic.-amsterdam 106, 318329. garcía-garrido, j.m., lendzemo, v., castellanos-morales, v., steinkellner, s., vierheilig, h., 2009: strigolactones, signals for parasitic plants and arbuscular mycorrhizal fungi. mycorrhiza 19, 449459. hayat, s., ali, b., ahmad, a., 2007: salicylic acid: biosynthesis, metabolism and physiological role in plants. in: hayat, s., ahmad, a. 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(eds.), physiological and genetical aspects of mycorrhizae, 217-221. inra press, paris. tsao, r., papadopoulos, y., yang, r., young, j.c., mcrae, k., 2006: isoflavone profiles of red clovers and their distribution in different parts harvested at different growing stages. j. agr. food chem. 54, 57975805. tsavkelova, e.a., klimova, s.y., cherdyntseva, t.a., netrusov, a.i., 2006: hormones and hormone-like substances of microorganisms: a review. appl. biochem. micro. 42, 229-235. westphal, a., snyder, n., xing, l., 2008: effects of inoculations with mycorrhizal fungi of soilless potting mixes during transplant production on watermelon growth and early fruit yield. hortscience 43, 354-360. whipps, j.m., 2004: prospects and limitations for mycorrhizas in biocontrol of root pathogens. can. j. bot. 82, 1198-1227. zadock, j.c., chang, t.t., konzak, c.f., 1974: a decimal code for growth stages of cereals. weed res. 14, 415-421. address of the corresponding author: priv. doz. dr. ewald sieverding, institute of plant production and agroecology in the tropics and subtropics, university stuttgart-hohenheim, garbenstraße 13, 70599 stuttgart, germany. e-mail: sieverdinge@aol.com journal of applied botany and food quality 87, 16 23 (2014), doi:10.5073/jabfq.2014.087.003 university of bonn, institute of nutrition and food sciences (iel) – food technology, germany effect of selected plant extracts on the inhibition of enzymatic browning in fresh-cut apple b. wessels1, s. damm, b. kunz, n. schulze-kaysers*1 (received september 11, 2013) * corresponding author 1 these authors contributed equally and are considered co-first authors summary this study describes the evaluation of the anti-browning effect of 36 plant extracts, divided into three groups: (1) fruits, vegetables, and oil seeds, (2) herbs and tea plants, and (3) medicinal plants, on minimally processed fresh apples. the extracts were applied to freshcut apple slices as dipping solutions. development of browning was analyzed by measuring l*, a*, and b* values. the greatest inhibition of browning was caused by extracts from pumpkin seed (group 1), hibiscus flower (group 2), and pelargonium root (group 3). however, the latter caused intense passive staining. the inhibitory potential might be attributable to the antioxidative activity of secondary plant metabolites, especially phenolic compounds. furthermore, these bioactive substances might influence enzyme activity directly by acting as competitive or non-competitive inhibitors. introduction fresh-cut fruits and vegetables have become a popular snack in recent years because of an increased awareness by consumers of the health benefits of fresh fruits and vegetables, as well as an increased demand for convenience food (toivonen, 2006). a major concern is enzymatic browning, which can lead to changes in colour, taste and texture of fresh-cut products and is thus associated with quality deterioration. enzymatic browning is induced by the action of polyphenoloxidase (ppo) on its phenolic substrates in the presence of oxygen. the oxidation products that are formed can undergo further reactions resulting in complex polymers, which are responsible for browning (mcevily et al., 1992). especially potatoes, mushrooms, bananas, peaches, and apples are prone to oxidative browning (mcevily et al., 1992). for browning control of fresh cut fruits and vegetables ascorbic acid and its derivatives can be used due to their reducing properties; however these effects are temporary. long-term antibrowning effects can be achieved by sulphur dioxide and sulphites. thus, these food additives are industrially used to inhibit ppo-induced browning reactions for a wide range of products (mcevily et al., 1992), including fresh cut apples sold as intermediate products to gastronomic business areas. however, sulphiting can reduce the uptake of thiamine by degradation of the vitamin and lead to asthmatic reactions in sensitive individuals (mcevily et al., 1992; rangan and barceloux, 2009). hence, the replacement of such compounds is an important issue for the food industry. several approaches, such as the use of dipping treatments with reducing or complexing agents, edible coatings and modified atmosphere packaging have been explored to control the browning of fresh-cut apples (omsoliu et al., 2010; rojas-graü et al., 2009). however, owing to concerns about food safety, off-flavours and off-odours, economic feasibility, and the effectiveness of inhibition, only a few substances and methods have shown potential for use in the food industry (mcevily et al., 1992; park, 1999). thus, further investigation of alternative methods is required. one approach is the application of plant extracts to inhibit browning in apple products. such extracts could serve as reducing agents because of the antioxidant capacity of the secondary plant metabolites present. furthermore, phenolic compounds might influence ppo activity directly by acting as, for example, competitive or non-competitive inhibitors. promising results have been obtained with rhubarb juice, pineapple juice, and green tea extract, which were all able to prevent discolouration of cut surfaces of apples (soysal, 2009; son et al., 2000; lozanode-gonzalez et al., 1993). at the same time several phenolic compounds can be ppo substrates depending on the presence and position of substituents (chang, 2009). to compare the anti-browning effects of different plant-derived ppo inhibitors on fresh-cut apples it is essential to conduct assays under identical conditions. in the study described herein, 36 plant extracts were screened and their polyphenol content and antioxidant capacity determined to identify promising anti-browning agents. material and methods plant material and chemicals apple cv. ‘jonagold’ was chosen for all investigations in order to obtain comparable results for all extracts. the fruits were delivered by meyer gemüseverarbeitung gmbh (twistringen, germany). following harvest at commercial maturity, the fruits were stored at 10 12 °c until processed. folin-ciocalteu reagent, 6-hydroxy-2,5,7,8-tetramethylchromane-2carboxylic acid (trolox), and 2,2’-azino-bis(3-ethylbenzothiazoline6-sulphonic acid) diammonium salt (abts) were obtained from fluka (neu-ulm, germany). caffeic acid was purchased from sigma aldrich (steinheim, germany). all other chemicals were obtained from merck (darmstadt, germany). dipping solutions of plant extracts thirty-six different plant extracts that were divided into three major groups (tab. 1) were tested. except for the grapefruit extract, which was obtained from eurochem feinchemie gmbh (gröbenzell, germany), all extracts were provided by frutarom switzerland ltd (wädenswil, switzerland). the extracts were used as aqueous dipping solutions at a concentration of 1 % (w/v). after dilution, the extract solutions were autoclaved (121 °c, 15 min) and centrifuged (7025 g, 10 min). as a reference, 0.14 % sodium bisulphite solution was prepared by the same method except for the centrifugation step. characterization of plant extracts the plant extracts were characterized by determining the ph value, the total polyphenol content (tpp), and the antioxidative capacity of the 1 % aqueous extract solutions. the tpp of the extracts was determined by the folin-ciocalteu method, modified after tanner and brunner (1987) and ritter (1994). folin-ciocalteu reagent (75 μl) was mixed with 15 μl of extract and 1260 μl of distilled water. after 3 min, 150 μl of a saturated na2co3 solution were added. after 1 h, the absorbance of the sample was measured at 720 nm. the tpp was calculated from a calibration curve obtained using caffeic acid and thus was expressed as caffeic acid equivalents. the antioxidant capacity of the extract was determined using the plant extracts as inhibitors of enzymatic browning 17 tab. 1: plant extracts investigated for their anti-browning properties (extract sources, botanical names, and commercial names according to supplier: www.frutarom.com, www.eurochem-feinchemie.com). group i: fruits, vegetables & oil fruits/ seeds extract source botanical name plant part extraction solvent commercial name acai euterpe oleracea mart. fruit water acai 4:1 grapefruit citrus paradisi macf. fruit water grapefruitextrakt ws “superberry“ vitis vinifera l., fruit water superberry 4000 punica granatum l., vaccinium macrocarpon ait., vaccinium corymbosum l., vaccinium myrtillus l., fragaria vesca l., rubus idaeus l. grape vitis vinifera l. seed water opc grape seed baobab adansonia digitata l. fruit ethanol u. d. broccoli brassica oleracea l. var. italica seed co2 (supercritical) broccoraphanin horseradish armoracia rusticana root ethanol u. d. artichoke cynara scolymus l. leaf water artichoke leaf powder extract garlic allium sativum l. bulb ethanol white garlic powder extract pumpkin curcubita pepo l. seed ethanol pumpkin seed powder extract soy glycine max. l. hypocotyl ethanol soylife 40% flax linum usitatissimum l. hull ethanol linumlife extra cranberry vaccinium macrocarpon ait. fruit water cranberry high pac purslane portulaca oleracea l. herb ethanol purslane herb powder extract group ii: herbs & tea plants extract source botanical name plant part extraction solvent commercial name oregano origanum vulgare l. herb water origanox ws rosemary rosmarinus officinalis l. leaf water rosemary leaf powder extract lemon balm melissa officinalis l. herb water balm leaf powder extract green tea camellia sinensis l. leaf ethanol green tea leaf powder extract chamomile chamomilla recutita l. flower ethanol chamomile flower powder extract hibiscus hibiscus l. flower water u. d. pink rockrose cistus incanus l. herb ethanol pink rockrose herb powder extract green mate ilex paraguariensis a. st.-hil. leaf ethanol finomate nettle urtica dioica l. leaf water nettle leaf powder extract sage salvia officinalis l. leaf water sage leaf powder extract olive olea europaea l. leaf ethanol benolea group iii: medicinal plants extract source botanical name plant part extraction solvent commercial name red wine vitis vinifera l. leaf water red vine leaf powder extract pelargonium pelargonium sidoides dc. root ethanol pelargonium root powder extract goldenrod solidago sp. herb ethanol goldenrod herb powder extract eyebright euphrasia sp. herb water eyebright herb powder extract java tea orthosiphon stamineus benth. leaf ethanol java tea powder extract bearberry arctostaphylos uva-ursi (l.) spreng. leaf water bearberry leaf powder extract chasteberry vitex agnus-castus l. fruit ethanol chaste berry powder extract oat avena sativa l. herb water oats herb powder extract willow salix sp. bark water willow bark powder extract passion flower passiflora incarnata l. herb ethanol passion flower herb powder extract ivy hedera helix l. leaf ethanol ivy leaf powder extract u. d. = under development 18 b. wessels, s. damm, b. kunz, n. schulze-kaysers method of kao and chen (2006) with modifications. an aqueous solution of 7 mm abts was prepared, to which 2.45 mm potassium persulphate were added to generate the abts radical (abts•+) by incubation in the dark at ambient temperature for 12 h. the abts•+solution obtained was diluted with 1 mm pbs buffer (ph 7.4) to an absorbance at 734 nm of 1.00 (± 0.02). trolox dissolved in 1 mm pbs buffer was used as a reference. ten microliters of the standard or sample were mixed with 1 ml of abts•+; as a control, 10 μl of pbs buffer were mixed with 1 ml of abts•+. after 60 min, absorbance at 734 nm was recorded in triplicate against pbs as a blank. the degree of quenching of the abts•+ radical was calculated as follows (fogliano et al., 1999): inhibition (%) = 1 – as/ac × 100 where ac was the absorbance of the control and as the absorbance of the sample at 734 nm. the antioxidant property of the extracts was calculated from a calibration curve of abts•+ inhibition plotted against trolox concentration and was expressed as trolox equivalent antioxidant capacity (teac). slicing and dipping selected apples of uniform size and colour were manually washed, cored, and sliced longitudinally using a manual corer-slicer (tchibo gmbh hamburg, germany). for each extract, three apple slices (thickness of inner cutting edge 9 mm, width 25 – 35 mm) were soaked in dipping solution at ambient temperature for 2 min. the excess liquid was removed and the samples were stored at room temperature for 4 h. colour measurement the development of browning in the treated apple slices and the reference slices was measured with a colorimeter (cr 400, konica minolta, tokyo, japan) with the standard illuminant d65. the instrument was calibrated with a white tile before the first measurement. colour (l*, a* and b* values) was measured in untreated apple slices (initial colour directly after cutting; lc, ac, bc), at zero time (directly after dipping; l0h, a0h, b0h), and after 4 h (l4h, a4h, b4h). the results are presented as the means of three replicates of three measured samples. the difference in lightness (∆l4h), which is associated with the degree of browning (maskan, 2006), was calculated as: l0h – l4h. the passive staining by the plant extracts was determined separately as the total colour change given by the parameter ∆e, which was calculated using the following equation: ∆e = √∆l2 + ∆a2 + ∆b2 with ∆l = l0h – lc, ∆a = a0h – ac, and ∆b = b0h – bc. statistical analysis to determine significant differences between treatments, the experimental data for ∆l4h were analyzed with duncan’s multiple range tests (p = 0.05) as implemented in spss 18.0 (ibm deutschland gmbh, ehningen, germany). prior to this post-hoc test, normal distribution and homogeneity of the variance were tested by kolmogorov-smirnov test and levene test. results many secondary plant compounds, such as polyphenols and carotenoids, are known to have antioxidative properties (moure et al., 2001). thus, they might serve as reducing agents to prevent food products from enzymatic browning by, for example, reducing pigment intermediates such as quinones. some polyphenols are also known to inhibit ppo competitively, non-competitively, or by acting as a chelating agent (chang, 2009). fruits, vegetables, and oil fruits/ seeds are rich in polyphenols, carotenoids, and other antioxidative secondary plant compounds. fourteen extracts from such sources were tested as anti-browning agents (group i). also many herbs and tea plants contain significant amounts of secondary plant metabolites, such as flavonoids, which possess strong antioxidant properties (ivanova et al., 2005). hence, 11 extracts from herbs and tea plants were tested (group ii). medicinal plants are known to contain considerable amounts of phenolic acids and flavonoids, especially hydrolyzed and condensed tannins, respectively. as described above, tannins are able to inactivate ppo by binding copper or proteins. in the present study, 11 extracts from medicinal plants were tested for their inhibitory effect on browning in apple slices (group iii). fig. 1 shows the ∆l4h values of the extract-treated apple slices, which represent the difference in browning. the total change in colour of the apple slices that had occurred after dipping is an indicator of the passive staining by the extracts and data are shown in tab. 2. as ph influences ppo activity, the ph values of the used extract solutions were measured. the results are shown in tab. 3. fig. 1: browning of fresh-cut apples treated with 1 % plant extract solutions 4 h after treatment compared to the sodium bisulfite solution (reference). apple samples were treated with extracts from (a) fruits, vegetables, and oil fruits/seeds, (b) herbs and tea plants, and (c) medicinal plants. vertical bars represent the standard deviation of the mean (n = 3). reference reference reference plant extracts as inhibitors of enzymatic browning 19 tab. 2: passive staining of apples by the 36 chosen plant extracts (expressed as ∆e); for comparison: the sodium bisulfite solution (reference) had a ∆e of 0.76. fruits, vegetables passive staining herbs and tea plants passive staining medicinal plants passive staining and oil fruits / seeds (∆e) (∆e) (∆e) pumpkin seed 2.04 hibiscus flower 3.94 pelargonium root 26.25 garlic bulb 2.54 green tea leaf 6.94 oats herb 1.35 soy hypocotyl 2.49 pink rockrose herb 4.85 chasteberry fruit 3.60 cranberry fruit 2.01 nettle leaf 7.01 bearberry leaf 2.53 broccoli seed 1.88 sage leaf 3.51 eyebright herb 3.02 grapefruit fruit 2.83 chamomile flower 0.99 red wine leaf 6.49 baobab fruit 0.43 green mate leaf 1.40 passion flower herb 4.13 “superberry“ fruit 14.11 oregano herb 2.60 willow bark 6.60 flaxseed hull 2.85 rosemary leaf 4.51 goldenrod herb 1.27 acai fruit 1.85 lemon balm herb 3.55 java tea leaf 6.37 horseradish root 1.72 olive leaf 2.04 ivy leaf 1.54 grape seed 23.03 artichoke leaf 0.96 purslane herb 7.53 tab. 3: ph values, tpp (mg/100 ml) and teac (mm) of plant extract solutions (1 %) at room temperature (18 – 22 °c); for comparison: the sodium bisulfite solution (reference) had a ph of 3.7. group i group ii group iii fruits, vegetables ph tpp teac herbs and ph tpp teac medicinal plants ph tpp teac and oil fruits/seeds (mg/ (mm) tea plants (mg/ (mm) (mg/ (mm) 100 ml) 100 ml) 100 ml) pumpkin seed 5.7 12.38 2.43 hibiscus flower 2.8 18.79 2.83 pelargonium root 5.0 484.00 131.80 garlic bulb 6.0 16.46 4.25 green tea leaf 4.4 445.52 162.96 oats herb 5.6 26.93 3.81 soy hypocotyl 5.3 41.82 22.96 pink rockrose herb 4.9 196.14 36.83 chasteberry fruit 4.7 41.21 7.10 cranberry fruit 3.6 2.73 17.02 nettle leaf 7.0 52.29 7.99 bearberry leaf 4.4 408.36 83.84 broccoli seed 6.0 13.25 15.34 sage leaf 5.3 174.78 31.01 eyebright herb 5.6 55.64 8.90 grapefruit fruit 4.4 141.62 45.28 chamomile flower 4.8 61.65 13.54 red wine leaf 4.7 131.96 34.48 baobab fruit 3.2 14.31 2.87 green mate leaf 5.0 309.97 40.70 passion flower herb 5.1 61.82 14.65 superberry fruit 3.8 311.43 49.89 oregano herb 4.5 275.43 62.95 willow bark 5.4 214.54 42.43 flaxseed hull 6.4 88.58 22.98 rosemary leaf 5.3 204.02 34.44 goldenrod herb 5.1 145.86 36.03 acai fruit 3.6 4.92 7.44 lemon balm herb 5.6 249.66 33.09 java tea leaf 5.4 187.78 32.59 horseradish root 5.2 8.62 1.66 olive leaf 4.4 132.83 22.33 ivy leaf 4.6 61.00 10.98 grape seed 4.0 623.78 106.13 artichoke leaf 5.4 37.10 2.50 purslane herb 5.3 74.65 15.23 discussion extracts of group i the greatest inhibition of browning was shown by pumpkin seed extract; apple slices treated with this extract showed only a slight darkening over the 4 h period of measurement. analysis with duncan’s multiple range test (p = 0.05) showed that pumpkin seed, garlic bulb, and soy hypocotyl extracts formed a homogeneous subgroup of inhibitors (subgroup 1). in this subgroup, ph did not influence the inhibitory effect of the extracts, which had ph values between 5.3 and 6.0. these values were similar to the optimal ph (5.5) for ppo from the apple cv. ‘golden delicious’ that was reported by soysal (2009). approximately half of the other extracts had ph values lower than 5 but were identified to be less potent inhibitors of browning. one class of secondary plant compounds that is characteristic of the three extracts in subgroup 1 and present in considerable amounts are phytoestrogens. the major class that is present in pumpkin seed and garlic are lignans, such as secoisolariciresinol, whereas in soy the main phytoestrogens are isoflavones, such as genistein, daidzein, glycitein, and their glycosides, as well as lignans (murphy and 20 b. wessels, s. damm, b. kunz, n. schulze-kaysers hendrich, 2002). although phytoestrogens are known to possess antioxidant activity (moure et al., 2001) in subgroup 1 only soy extract showed a strong antioxidant activity, with a teac value of 22.96 mm (tab. 3), which was correlated with its polyphenol content (fig. 2a). not cover all the extracts from plants that contain high amounts of lignans. the highest concentrations of lignans are found in flaxseed (3710 μg/g dry weight) followed by pumpkin seeds (214 μg/g dry weight) (adlercreutz and mazur, 1997). however, in the present study, pumpkin seed extract was a more potent inhibitor of browning than flaxseed hull extract. a reason for flaxseed lignans being weak ppo inhibitors might be their complex structure. lignans are present mainly in bound form and usually occur as a lignan complex of esterified secoisolariciresinol diglucosides (struijs et al., 2009). karioti et al. (2007) investigated the inhibition of tyrosinase by lariciresinol glycosides from species of lamiaceae and found that diglycosides are less potent inhibitors than glycosides, probably because of a lack of free hydroxyl groups. given that the secoisolariciresinol content of broccoli (4.14 μg/g dry weight) is much lower than that of flaxseed (adlercreutz and mazur, 1997), a different class of substances must be responsible for the higher anti-browning effects of broccoli seed extract. these compounds might be glucosinolates, which are sulphurand nitrogencontaining substances derived from amino acids and glucose and are found in considerable amounts in brassica vegetables (verkerk and dekker, 2008). recently, narváez-cuenca et al. (2011) found out that sodium hydrogen sulphite reacts with o-quinones formed during ppo reactions, particularly with caffeic acid-containing compounds. following a nucleophilic attack sulpho-adducts are formed, which are not involved in further browning reactions. it needs to be investigated, whether chlorogenoquinones formed of chlorogenic acid, which is a main phenolic compound of apples, is attacked by sulphur groups of glucosinolates in a similar way. the secoisolariciresinol content in broccoli is similar to that in garlic (3.79 μg/g) (adlercreutz and mazur, 1997), but the degree of browning indicated by ∆l4h is higher for apples treated with broccoli seed extract than those treated with garlic bulb extract. the inhibition of apple ppo by the latter might be the result of sulphuric compounds that are found in allium species. garlic is rich in alliin, which is transformed enzymatically into water-soluble, lipidsoluble, and volatile compounds by alliinase following mechanical rupture of cells (crozier et al., 2006). given that aqueous extract solutions were used in the study, the main active agents in the garlic bulb dipping solution would be (partially) water-soluble substances such as n-acetylcysteine. it is known to inhibit apple ppo (son et al., 2001) and loquat ppo (ding et al., 2002), although the exact inhibitory mechanism is unclear. comparably, kim, kim and park (2005) have shown that onion extract can inhibit browning of pears and attributed it to the thiol-containing compounds. given that soy hypocotyl extract belonged to subgroup 1, the results suggest that isoflavones also act as an inhibitor of apple ppo. tyrosinase inhibition by isoflavones that are contained in soy was demonstrated by chang et al. (2007). these authors found that daidzein, glycitein, daidzin, and genistin act as competitive inhibitors that reversibly inhibit the monophenolase activity of mushroom tyrosinase. in addition, there is evidence that the position and number of the hydroxyl group in the a-ring of the isoflavone structure could affect both the strength and mode of inhibition of isoflavones on mushroom tyrosinase (chang et al., 2007). however, details on the mechanism of inhibition remain unclear. within the extracts of fruits, vegetables, and oil fruits/seeds the correlation coefficients between tpp and ∆l4h (0.07) and teac and ∆l4h (0.03) respectively underline the lack of correlation between antibrowning and reducing properties. grape seed and “superberry” fruit extract showed significantly higher values for teac and tpp but did not have strong ppo inhibitory potential. the high teac values of grape seed extract can mainly be attributed to the procyanidins. the antioxidant potential of proanthocyanidins is 20 times higher than that of vitamin e and 50 times higher than that of vitamin c (shi et al., 2003). hence, the inhibition of browning by fig. 2: linear correlation of total polyphenol content (mg/100 ml) and antioxidant capacity (mm/100 ml). fresh-cut apple samples were treated with 1 % solutions of extract from (a) fruits, vegetables, and oil fruits/seeds (ac: acai fruit, ar: artichoke leaf, bb: baobab fruit, br: broccoli seed, cr: cranberry fruit, fs: flaxseed hull, ga: garlic bulb, gr: grapefruit fruit, grs: grape seed, hr: horseradish root, pk: pumpkin seed, pl: purslane herb , sb: superberry fruit, sh: soy hypocotyl), (b) herbs and tea plants (ch: chamomile flower, gm: green mate leaf, gt: green tea leaf, hb: hibiscsus flower, lb: lemon balm herb, ne: nettle leaf, ol: olive leaf, or: oregano, pr: pink rockrose herb, rm: rosemary leaf, sa: sage leaf), and (c) medicinal plants (be: bearberry leaf, cb: chasteberry fruit, ey: eyebright herb, go: goldenrod herb, iv: ivy leaf, jt: java tea leaf, oa: oats herb, pe: pelargonium root, pf: passion flower herb, rw: red wine leaf, wi: willow bark). hence, this extract might act as reducing agent to retard enzymatic browning. in addition to their reducing properties, the phytoestrogens that are present in the extracts could influence ppo activity directly as enzyme inhibitors. in the present study, subgroup 1 did plant extracts as inhibitors of enzymatic browning 21 procyanidins acting as reducing agents may be expected. the low level of inhibition of ppo by grape seed extract suggests that an additional factor in the extract counteracts the positive effects of the procyanidins with respect to browning. this factor might be the high content of (+)-catechin and gallic acid, which act as ppo substrates (selinheimo et al., 2007; kubo et al., 2000). “superberry” fruit extract, which is obtained from seven different berries [grape and pomegranate (main ingredients), cranberry, blueberry, bilberry, strawberry, and raspberry], also showed high values for teac and tpp. the predominant polyphenols in dark blue-, red-, and purple-colored fruits, such as blueberries, cranberries, and strawberries, are anthocyanins. almost all of these fruits also contain hydroxycinnamic acids, hydroxybenzoic acid derivatives, flavonols, and flavanols, as well as proanthocyanidins (naczk and shahidi, 2006). the antioxidant properties of these classes of polyphenols indicate that they act mainly as reducing agents, and thus may inhibit browning temporarily. however, given that compounds from grape and pomegranate are the main ingredients, the extract will also contain considerable amounts of ppo substrates, such as (+)-catechin, gallic acid, chlorogenic acid, and ellagic acid. extracts of herbs and tea plants among the extracts tested, only hibiscus flower extract showed strong potential as an inhibitor of browning and represented a separate homogeneous subgroup, as indicated by duncan’s multiple range test. the extract even seemed to bleach the apple slices, as indicated by the negative value for ∆l4h. the antioxidative properties of hibiscus (hibiscus sabdariffa) flavonoids (e. g. hibiscitrin, gossytrin, quercetrin, sabdaretin, and gossypetin-8-, -7and -3-glucosides (hegnauer, 1990)) and anthocyanins (e. g. cyanidin-3-diglycosides and cyanidin-3,5 bismonoglucoside (hegnauer, 1990)) might explain the ppo inhibition. however, given that the teac and tpp values of hibiscus flower extract were the lowest among the extracts from herbs and tea plants, the anti-browning effect cannot be attributed solely to polyphenols. the ph of the extract seems to play an important role because it was lower than 3, and thus is within the range of potential to influence ppo activity. mcevily et al. (1992) mentioned that ppo is inactivated completely at ph levels below 3. the low ph of the extract derived from flowers of hibiscus sabdariffa might reflect the high content of a number of organic acids, such as citric acid, malic acid, tartaric acid, oxalic acid, and hibiscus acid (hiller and loew, 2009). besides lowering the ph organic acids are able to form complexes with copper, the cofactor of ppo and thus to influence the enzyme activity. for instance oxalic acid was determined to be a noncompetitive inhibitor due to the binding with copper to form an inactive complex (yoruk and marshall, 2003). among the tested extracts derived from herbs and tea plants, tpp and teac did not correlate with the anti-browning properties of the extracts, approved by the correlation coefficients 0.02 and 0.04. as already mentioned, hibiscus flower extract showed the lowest antioxidant capacity and polyphenol content. the highest values were observed for green tea leaf extract. this finding is consistent with previous studies which indicated that green tea flavonoids possess strong antioxidative potential that is correlated with the total phenolic content of tea (soysal, 2009). green tea leaf extract ranked second with respect to the inhibition of browning. this finding is consistent with the results reported by soysal (2009), who found that flavonoid-rich green tea extract has an inhibitory effect on apple ppo activity. it is reasonable to assume that the inhibitory effect of green tea leaf extract on browning depends on flavonoids, in particular catechins, which can be divided into free catechins and esterified catechins (hara, 2001). besides acting as reducing agents, flavonoids with a free 3-hydroxy group inhibit tyrosinase by chelating copper (parvez et al., 2007). due to their structure, free catechins in green tea extract belong to the group of copper chelating agents. but, as mentioned above, (+)-catechin can also act as ppo substrate. the esterified catechins (–)-epicatechin gallate (ecg) and (–)-epigallocatechin gallate (egcg) have been identified as competitive tyrosinase and melanogenesis inhibitors, respectively (lin et al., 2008; selinheimo et al., 2007; parvez et al., 2007). it is assumed that gallate moieties bound to the 3-hydroxyl position of flavon-3-ols are responsible for the strong inhibitory effect of ecg and egcg (parvez et al., 2007). furthermore, ppo might be deactivated by reaction with tannins, such as esterified green tea catechins and condensed tannins based on green tea catechins. tannins are known to form complexes with heavy metal ions such as copper (kraal et al., 2006). given that ppo contains copper in its active site (mcevily, 1992), the enzyme might be complexed by the tannins in the extract. furthermore, tannins can bind proteins resulting in protein precipitation (crozier et al., 2006). relating to enzymes it was shown that ß-glucosidase and esterases can be precipitated by tannins from the bark of betula, salix, and pinus species (juntheikki and julkunen-tiitto, 2000). however, the exact mechanisms of ppo inhibition by green tea flavonoids remain to be determined. duncan’s multiple range test indicated that pink rockrose herb extract and green tea leaf extract formed a homogeneous subgroup. their comparable anti-browning effects might be attributable to the identical profile of catechins in green tea and pink rockrose (pomponio et al., 2003). other extracts within the group of herbs and tea plants also showed high ttp contents that were correlated with teac (fig. 2b). however, given that these extracts did not show promising antibrowning effects, they are not discussed further. extracts of medicinal plants among the group of medicinal plants, ph had no influence on the inhibitory effect of the extracts. the ph values of the extracts ranged from 4.4 to 5.6, which was close to the ph optimum of 5.5 for apple ppo (soysal, 2009). thus, the effects on browning can be attributed to the effects of substances present in the extracts. the greatest inhibition of browning was achieved with pelargonium root extract, for which only a slight colour change was observed over the period of measurement. duncan’s multiple range test (p = 0.05) indicated that the extracts from pelargonium root, oats herb, and chasteberry fruits formed a homogeneous subgroup of inhibitors. pelargonium root extract showed the strongest inhibition of browning and the highest levels of teac and tpp. the polyphenols in pelargonium root extract are mainly flavonoids, tannins, and coumarins, especially umckalin (wiesenauer, 2011). the antibrowning potential might be attributed to the reducing properties of the polyphenols, but this effect is only temporary. on the other hand, particular flavonoids and tannins can inactivate ppo by complexing copper or reacting with proteins. similar mechanisms might occur in apples treated with chasteberry fruit extract which also contains tannins. in addition, chasteberry fruits contain neolignans, such as ficusal, vladirol, and balanophonin (chen et al., 2011), which might be responsible for the anti-browning potential of chasteberry extract. tian, kang, kim and kim (2005) found out that honokiol, a neolignan found in the cortex of magnolia species, inhibits mushroom tyrosinase significantly. in contrast to pelargonium root and chasteberry fruit extracts, oats herb extract does not contain tannins. oats herb contains mainly flavonoids; nevertheless, the overall content of phenolic compounds in oats herb extract was low, as indicated by the results of the folin and teac assays. hence, the main anti-browning activity cannot be attributed to reducing compounds. additional secondary plant compounds that are contained in oats herb are the phytoalexins 22 b. wessels, s. damm, b. kunz, n. schulze-kaysers avenalinum i – iii. no information is available on the potential effects of these substances on apple ppo. oats herb also contains minerals such as calcium. the extract was recovered with water (tab. 1), so it is expected to be rich in minerals compared with extracts obtained by ethanolic extraction. thus, minerals might contribute to the inhibitory effect of oats herb extract. ayaz et al. (2008) reported that several minerals, including calcium, reduced the activity of ppo extracted from medlar (mespilus germanica l.) fruit. the results of the teac and folin assays were not correlated with the anti-browning activity (∆l4h) of the extracts of medicinal plants, underlined by the correlation coefficients 0.001 and 0.01. the extracts that showed the highest antioxidant activity and polyphenol content were scattered among all the homogeneous subgroups resulting from duncan’s multiple range test, namely pelargonium root extract (subgroup 1), bearberry leaf extract (subgroup 2), willow bark extract (subgroup 3), and java tea leaf extract (subgroup 4). in bearberry leaf extract, arbutin, which is a hydroquinone glycoside, might inhibit ppo competitively because of its structural similarities with the substrate tyrosine. this knowledge is useful owing to the use of arbutin in depigmentation products in the cosmetic industry in recent years (zhu and gao, 2008). willow bark and java tea leaf extracts did not show promising anti-browning effects and are not discussed further. comparison of anti-browning activity among the three extract groups according to tab. 1 showed that extracts from groups 1 and 3 were stronger inhibitors than extracts from group 2, as indicated by the maximum ∆l4h values. browning and total colour change of extract-treated apple slices in comparison with the reference the passive staining was negligible for most of the extracts that were identified as potential inhibitors of browning (tab. 2). only treatment with pelargonium root extract, which was the strongest inhibitor within the extract group of the medicinal plants, resulted in orange staining. the ∆e value for pelargonium root extract was at least six times higher than ∆e for apples treated with hibiscus flower or pumpkin seed extract (fig. 3). the latter two extracts showed ∆e values that were similar to those of the reference samples, which were treated with sodium bisulphite. regarding the development of browning, only treatment with hibiscus flower extract resulted in a negative ∆l4h value which is comparable to the reference and represents bleaching of the apples. the ∆l4h of apples treated with pumpkin seed extract was close to zero and little passive staining of the apple slices was observed. overall, hibiscus flower and pumpkin seed extracts showed the highest potential among the extracts investigated to replace sodium bisulphite as an anti-browning agent. conclusion the aim of this study was to identify the most effective antibrowning agent(s) for fresh-cut apple from among different plantderived extracts. thirty-six extracts that were categorized into three main groups were tested for their inhibitory effects on enzymatic browning. the extracts that showed the highest potential in each group were: pumpkin seed extract (group 1), hibiscus flower extract (group 2), and pelargonium root extract (group 3). pumpkin seed and hibiscus flower extract can be considered as substitutes for sulfites, whereas pelargonium root extract is not suitable because of the strong passive staining. in general, it was shown that the content of reducing compounds, determined as tpp and teac, does not correlate with the anti-browning activity of the investigated plant extracts. thus, browning control by plant extracts is not only based on reduction of o-quinones, but might also be attributed to competitive and noncompetitive ppo inhibition. further investigations are needed to clarify, whether the anti-browning effects might be attributed to particular substances or in case of hibiscus flower extract resulted from the low ph. likewise, the effectiveness, the economic feasibility, the effects on sensorial quality parameters, and the nutritional hazards of the practical application of extracts like pumpkin seed and hibiscus flower extract have to be investigated. in addition, research on the microbiological potential of these extracts should be conducted because sulfurization is also used to preserve food. acknowledgements the study was carried out with financial support from the commission of the european communities within the seventh framework programme for research and technological development (fp7), grant agreement no. 226930, title ‘replacement of sulphur dioxide (so2) in food keeping the same quality and shelf-life of the products’, acronym so2say coordinated by ttz bremerhaven. the study does not necessarily reflect its views or the view of the project partners and in no way anticipates the commission’s future policy in this area. the authors thank frutarom switzerland ltd (rütiwisstraße 7, ch-8820 wädenswil, switzerland) and eurochem feinchemie gmbh (industriestraße 29, d-82194 gröbenzell, germany) for kindly providing the plant extracts as well as meyer gemüsebearbeitung gmbh (hinterm holze 10, 27239 twistringen) for providing the apples. references adlercreutz, h., mazur, w., 1997: phyto-oestrogens and western diseases. 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(eds.), bioactive compounds in foods, 31-51. blackwell publishing, oxford. wiesenauer, m., 2011: erkrankungen der ableitenden harnwege. in: wiesenauer, m. (ed.), phytopraxis, 241-261. springer medizin verlag, berlin, heidelberg. wolfe, k.l., kang, x.m., he, x.j., dong, m., zhang, q.y., liu, r.h., 2008: cellular antioxidant activity of common fruits. j. agric. food chem. 56, 8418-8426. yoruk, r., marshall, m.r., 2003: a survey on the potential mode of inhibition for oxalic acid on polyphenol oxidase. j. food sci. 68, 24792485. zhu, w.y., gao, j., 2008: the use of botanical extracts as topical skinlightening agents for the improvement of skin pigmentation disorders. j. invest. derm. symp. p. 13, 20-24. address of the authors: dipl. oec. troph. birgit wessels, institute of nutrition and food sciences (iel) – food technology, university of bonn, römerstr. 164, 53117 bonn, germany sandra damm, institute of nutrition and food sciences (iel) – food technology, university of bonn, römerstr. 164, 53117 bonn, germany prof. dr.-ing. habil. benno kunz, institute of nutrition and food sciences (iel) – food technology, university of bonn, römerstr. 164, 53117 bonn, germany dr.-ing. nadine schulze-kaysers (*corresponding author), institute of nutrition and food sciences (iel) – food technology, university of bonn, römerstr. 164, 53117 bonn, germany. e-mail: nadine.schulze@uni-bonn.de vorgabe neu journal of applied botany and food quality 81, 41 44 (2007) 1humboldt university berlin, institute for horticultural sciences, section fruit science / 3section quality dynamics and postharvest physiology 2 technical university berlin, institute of food technology and food chemistry, department of food analysis influence of location and fertilization on antioxidant acitivity in highbush blueberries (vaccinium corymbosum l.) i. eichholz1, s. rohn2, l.w. kroh2, s. huyskens-keil3 (received february 23, 2007) summary highbush blueberry cultivars ‘bluecrop’ and ‘reka’ were growing in two variants of mulching and fertilizing systems on formerly used farmland. fruits were harvested at two picking dates and analyzed for their content of phenolic compounds and antioxidant activity. these data were compared with samples of two forest soil locations from the brandenburg region (beelitz and klaistow). the results showed significant differences between cultivars, both harvest times and different locations. the variations in fertilization and ground cover (with or without mulch) showed significant differences. moreover, it is demonstrated that without ground cover and commercial fertilization higher contents of total phenolic compounds and an increase in antioxidant activity tendentiously occurred. this result paralleled the decline in vegetative growth and was associated with drought stress. introduction secondary plant metabolites, especially phenolic compounds, which are responsible for flavour and colour, are being produced by all plant material for the protection of biotic and abiotic influences. moreover it is known that phenolic compounds block free radicals, which are activated due to oxidative stress or environmental pollution (riceevans et al., 1995). therefore, phenols have been suggested to reduce cellular damage and play a role in preventing harmful diseases such as cancer and coronary heart disease (kalt and dufour, 1997). berry fruits are one of the richest sources of antioxidants in our diets (kähkönen et al., 1999). especially blueberries have received much attention due to high levels of plant phenolic compounds and thus high antioxidant activity (prior et al., 1998). blueberry bushes originally grow on heathland or forest soil. however, in germany there are attempts to cultivate blueberries on formerly used farmland. on such location soil condition are not favourable due to high soil ph, low organic matter content and low water storability. moreover, there is a lack of symbiosis of mycorrhiza organism, which is essential for nutrition especially for nitrogen supply (yang et al., 2002). therefore, soil preparation (mulching) and fertilization are important features for cultivation on farmland soil. furthermore, the foliar applications of boron support growing and fruiting of plants, as boron is known for enhancing fruit set and yield of many crops, e.g. for apple (wójcik, 2006) and black currant (wójcik, 2005) as well as fruit berry number of blueberries (blevins et al., 1996). boron also plays an important role as a drought stress inhibitor. werminghausen (1957) reported that plants with higher boron level showed a descreased transpiration rate under drought conditions. therefore, the aim of the present study was to investigate the effect of different nitrogen and boron supply as well as ground cover systems of formerly used farmland on changes in bioactive secondary plant metabolites of highbush blueberries. materials and methods plant material plant material was obtained from the highbush blueberry cultivars ‘bluecrop’ and ‘reka’. bushes were planted in 1997 in peat filled holes on formerly used farmland in berlin-dahlem. in 2004 soil minerals data comprised of 31 mg p 2 o 5 , 62 mg k 2 o, 33 mg mgo per 100 g soil, soil acidity was 5.58 (cacl 2 ) with an organic matter content of 2.97%. the plants were cultivated in two ground cover (with and without pine bark mulch) and fertilization variants. fertilization was conducted from may to mid july with a nutrient supply of 10 g n29 g p 2 o 5 83 g k 2 o13 g mgo66 mg b per plant in first fertigation variant (f1). plants of second nutrient supply system (f2) received an increased nitrogen fertilization (14 g/ plant) and additionally boron foliar applications (400 mg/ plant). ripe berries were picked at two harvest dates providing 70-80% of total crop (‘reka’: 19/07/04 and 29/07/04; ‘bluecrop’: 28/07/04 and 04/08/04). samples of each variant and cultivar were frozen immediately and stored at -20°c until further analysis. these samples were compared with blueberries of two forest soil locations from the brandenburg region, klaistow and beelitz. chemical analysis fifteen berries of each variant were carefully crushed and 1 ml of juice was filtered through filtrations tubes (20 µm pore size, supelco, deisenhofen, berlin) and frozen immediately at -30°c until analysis. antioxidant activity was determined by electron spin resonance (esr) spectroscopy and trolox equivalent antioxidant capacity (teac) assay. esr was performed as described by rösch et al. (2003), using a table spectrometer miniscope ms 100 (magnettech, berlin, germany). the signal intensity of a stabilized synthetic radical (potassium nitrosodisulfonate (fremy´s salt), sigmaaldrich, steinheim, germany) was obtained after 20 min, by which the time of reacting with blueberry antioxidants was completed and expressed as moles of fremy´s salt reduced by one mole antioxidant/l. teac assay was carried out on a specord 40 (analytik jena, jena, germany) spectrophotometer as described by re et al. (1999) with abts (2,2´-azinobis (3-ethylbenzothiazonline-6-sulfonic acid, sigmaaldrich, steinheim, germany) as free radical and trolox (6hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, sigmaaldrich, steinheim, germany) as a standard at a wavelength of 732 nm. results were expressed as mmol trolox/100 ml. total soluble phenols were analyzed photometrically with folinciocalteu reagent (sigma-aldrich, steinheim, germany) by the method of jennings (1981), using gallic acid (serva, heidelberg, germany) as a standard at a wavelength of 765 nm. results were expressed as mg gallic acid /100 ml. statistic calculations the statistic calculations were performed with spss 11.0 by spss inc., chicago, usa (2001). significances of differences were conducted with a tukey test (p< 0.05). results and discussion influence of location on total phenol content and antioxidant activity there were significant differences between the forest soil locations beelitz (22.0 mmol fremy´s salt/ l) and klaistow (27.7 mmol fremy´s salt/ l) when using esr method (fig. 1). teac assay determined differences between the locations beelitz (14.4 mmol trolox/ 100 ml) and klaistow (24.8 mmol trolox/ 100 ml), as well as beelitz and berlin-dahlem (27.0 mmol trolox/ 100 ml). significant differences in total phenol analysis were observed between the locations beelitz (69.8 mg gallic acid/ 100 ml) and berlin-dahlem (125.9 mg gallic acid/ 100 ml). plants of berlin-dahlem are more stressed by wind and unfavourable soil conditions. furthermore open farmland berries were influenced by a high solar radiation in comparision to forest locations. stress as well as increased light conditions increase phenolic metabolism (feucht and treutter, 1989; häkkinen, 2000) and might explain the higher levels of bioactive compounds in berlin-dahlem. the forest soil location beelitz showed the lowest content of total phenols as well as of antioxidants in all analysis, probably due to a higher berry weight and higher soil nitrogen content. high nitrogen levels decrease the content of phenolic compounds (feucht and treutter, 1989; häkkinen, 2000). berry weight again is negative correlated with the content of phenolic compounds (connor et al., 2002b; moyer et al., 2002). however, variation between locations is depending on local micro climate (temperature, irradition, moisture) and soil conditions in blueberry cultivation (jones, 1999 in howard, 2003; häkkinen, 2000; moyer et al., 2002). in contrast, prior et al. (1998) reported no differences between the blueberry growing locations orgeon, new jersey and michigan. influence of cultivation on total phenol content and antioxidant activity esr data ranged from 23.6 to 28.9 mmol fremy´s salt/l, for teac method 24.5 to 27.5 mmol trolox/ 100 ml and for total phenol content from 120.1 to 134.4 mg gallic acid/ 100 ml (fig. 2). the present results demonstrate that blueberries without ground cover, lower nitrogen and boron fertilization (f1om) had higher contents of total phenolic compounds and an increase in antioxidative activity. this result is assumed to be related to a stress mediated process, i.e. the non covered soil variant resulted in a low organic matter content, high soil ph and thus insufficient waterstorage. this might also explain the decline in vegetative growth. in contrast the f2om variant revealed a lower content of bioactive compounds due to additional boron application. boron could have restricted the influx of substrate into the pentose-phosphate pathway and the synthesis of phenols (lee and aronoff, 1967 in mondy and munshi, 1993). mondy and munshi (1993) reported a decreased phenol concentration in potato tubers after boron treatment. also al-yousif et al. (1994) determined a reduced phenol content in date palm and sorghum leaves due to an increased boron level. fig. 2: influence of cultivation on total phenol content and antioxidant activity; f1m= commercial fertilization with mulch, f1om= commercial fertilization without mulch, f2m= increased nitrogen and boron supply with mulch; f2om= increased nitrogen and boron supply without mulch: tukey test (p < 0.05) f1m f1om f2m f2om cultivation systems 5 10 15 20 25 30 35 m m ol f re m ys ' sa lt /l antioxidant activity (esr) b a b b 24.5 23.928.9 23.6 f1m f1om f2m f2om cultivation system 5 10 15 20 25 30 35 m m ol t r o l o x / 10 0 m l antioxidant activity (teac) a a a a 25.6 25.627.5 24.5 f1m f1om f2m f2om cultivation system 20 40 60 80 100 120 140 160 180 m g ga ll ic a ci d / 10 0 m l phenol content a a a a 125.7 134.4 123.3120.1 fig. 1: influence of location on total phenol content and antioxidant activity, tukey test (p < 0.05) berlin-dahlem beelitz klaistow location 5 10 15 20 25 30 35 m m ol f re m ys ' sa lt /l antioxidant activity (esr) ab b a 25.4 27.722.0 berlin-dahlem beelitz klaistow location 5 10 15 20 25 30 35 m m ol t r o l o x / 10 0 m l antioxidant activity (teac) b a a 27.0 24.814.4 berlin-dahlem beelitz klaistow location 20 40 60 80 100 120 140 160 180 m g ga ll ic a ci d / 10 0 m l phenol content b a ab 125.9 94.869.8 42 i. eichholz, s. rohn, l.w. kroh, s. huyskens-keil influence of cultivar and harvest time on total phenol content and antioxidant activity comparision of cultivars showed statistic differences in antioxidant activity at the first harvest date between ‘bluecrop’ (22.3 mmol fremy´s salt/l) and ‘reka’ (28.5 mmol fremy´s salt/l) by esr method (fig. 3). in teac assay differences were found between cultivars of the second harvest date (‘bluecrop’: 30.9 mmol trolox/ 100 ml; ‘reka’: 22.3 mmol trolox/ 100 ml). in respect to total phenols significant differences were observed for the first harvest date between ‘bluecrop’ (105.2 mg gallic acid/ 100ml) and ‘reka’ (132.7 mg gallic acid/ 100ml) as well as for the second date (‘bluecrop’: 164.9 mg gallic acid/ 100ml; ‘reka’: 95.8 mg gallic acid/ 100ml). variation in bioactive compounds of different cultivars are affected genetically, also described by prior et al. (1998), connor et al. (2002a; 2002b), moyer et al. (2002) and howard et al. (2003). comparision of harvest times showed differences in the cultivar ‘reka’ (first date: 28.5 mmol fremy´s salt/l; second date: 23.2 mmol fremy´s salt/l) using esr method. significances in teac assay of harvest times occurred at both harvest dates for ‘bluecrop’ (first date: 23.1 mmol trolox/ 100ml; second date: 30.9 mmol trolox/ 100 ml) and ‘reka’ (first date: 25.9 mmol trolox/ 100 ml; second date: 22.3 mmol trolox/ 100ml). total phenol content differed significantly between harvest time and cultivar, i.e. ‘bluecrop’ (first date: 105.2 mg gallic acid/ 100 ml; second date: 164.9 mg gallic acid/ 100 ml) as well as of ‘reka’ (first date: 132.7 mg gallic acid/ 100 ml; second date: 95.8 mg gallic acid/ 100 ml). however, antioxidant activity (teac) and total phenol analysis showed that bioactive components of the medium-ripened cultivar ‘bluecrop’ were significantly higher in the second harvest in comparision to the first harvest, whereas in contrast the first harvest of the early-ripened cultivar ‘reka’ had higher levels of total phenols and antioxidants. the results of the esr method showed a similar trend. total phenolic content and antioxidant activity of blueberries decrease during fruit ripening (kalt, 2003). although all berries were harvested at a fully ripe stage, fruits of ‘bluecrop’ second crop showed a lower sugar/acid ratio, i.e. an earlier ripening stage (10.3) in comparison to the first harvest date (16.0). moreover, in the cultivar ‘bluecrop’ increased level of phenolic compounds and antioxidants of the second harvest date may also be caused by the decline in fruit weight from 1.9 g to 1.4 g, respectively. this is also consistent with findings by howard et al. 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w.q., goulart, b.l., demchak, k., li, y., 2002: interactive effects of mycorrhizal inoculation and organic soil adjustments on nitrogen acquisition and growth of highbush blueberry. j. amer. soc. hort. sci. 127, 742-748. addresses of the authors: 1 humboldt universität zu berlin, institut für gartenbauwissenschaften, fachgebiet obstbau, albrecht-thaer-weg 3, d-14195 berlin 2 technische universität berlin, institut für lebensmitteltechnologie und lebensmittelchemie, fachgebiet lebensmittelanalytik, tib 4/3-1, gustavmeyer-allee 25, d-13355 berlin 3 humboldt universität zu berlin, institut für gartenbauwissenschaften, lehrund forschungsgebiet produktqualität/qualitätssicherung, lentzeallee 75, d14195 berlin 44 i. eichholz, s. rohn, l.w. kroh, s. huyskens-keil << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) 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<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> >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice journal of applied botany and food quality 87, 30 35 (2014), doi:10.5073/jabfq.2014.087.005 1department of pharmacy, bahauddin zakariya university, multan, pakistan 2 the patent office, karachi, pakistan 3 department of food science, human nutrition unit, university of parma, parma, italy 4 siteia.parma interdepartmental centre, university of parma, parma, italy phenolic profile and antioxidant potential of selected plants of pakistan imran imran1, muhammad zia-ul-haq2*, luca calani 3, teresa mazzeo4, nicoletta pellegrini 3 (received january 12, 2013) * corresponding author summary antioxidants play an important role in inhibiting and scavenging radicals, thus providing protection to humans against infectious and degenerative diseases. literature shows that the antioxidant activity is high in medicinal plants. realizing the fact that, this investigation was carried out to evaluate the in vitro antioxidant capacity of methanolic extracts of acacia leucophloea (bark), albizia lebbeck (bark, flower, seed), capparis decidua (root), cicer arietinum (seeds) and grewia asiatica (leaves). barks showed the highest phenolic content as compared to seeds, leaves and roots and the order observed was a. lebbeck bark> a. leucophloea bark> g. asiatica leaves> c. decidua root >a. lebbeck flowers> a. lebbeck seeds> c. arietinum seeds. phenolic compounds were identified based on their mass spectral characteristics in each extract. antioxidant capacity measured by three commonly-benched methods, teac, frap and trap assays indicated that all extracts are a good source of natural antioxidants. investigated extracts appeared to have potential as a health supplement rich in natural antioxidants and merits further intensive study. the results of this study will promote the reasonable usage of these plants in food and pharmacy industries as well as in alternative medicine and natural therapy. introduction it is well known that reactive oxygen species (ros) formed in vivo, such as superoxide anion, hydroxyl radical and hydrogen peroxide, are highly reactive and potentially damaging transient chemical species. the oxidative damages caused by ros on lipids, proteins and nucleic acids may trigger various chronic diseases, such as coronary heart disease, atherosclerosis, cancer and aging (uttara et al., 2009; barnham et al., 2004). the antioxidants can delay or inhibit the oxidation of lipids and other molecules by inhibiting the initiation or propagation of oxidative chain reactions and they can thus prevent or repair the damage done to the body’s cells by ros (tachakittirungrod et al., 2003). an imbalance between antioxidants and ros results in oxidative stress, leading to cellular damage. it is possible to reduce the risk of chronic diseases and prevent disease progression with dietary antioxidants (stanner et al., 2000). among these, natural antioxidants are regarded as safer than synthetic antioxidants. therefore, there is a considerable interest in finding new and safe antioxidants from natural sources to replace these synthetic antioxidants (rahimi et al., 2005; chanwitheesuk et al., 2005). plants have many phytochemicals which are a potential source of natural antioxidant, e.g. phenolic diterpenes, flavonoids, alkaloids, tannins and phenolic acids (amro et al., 2002; cai et al., 2004; moure et al., 2001). in recent years, considerable attention has been directed towards the identification of plants with antioxidant ability that may be used for human consumption. pakistan is considered as a paragon of valuable food, medicinal and aromatic plants thanks to its comprehensive latitudinal spread and immense altitudinal range. magnificent mountain tops in northern areas to fertile plain areas irrigated by rivers in punjab and sindh, arid regions of thal, thar and baluchistan to the coastal mangrove forests of the arabian sea supports an extensive array of exotic plant species (zia-ul-haq et al., 2013 a, b). however, few studies have explored the antioxidant capacity of these plants. in this investigation, the total antioxidant capacity (tac) and the characterization of phenolic compounds of several pakistan plants, i.e. acacia leucophloea (bark), albizia lebbeck (bark, flower, seed), capparis decidua (root), cicer arietinum (seeds) and grewia asiatica (leaves) were explored. as there is no single and widely acceptable assay method for evaluating antioxidant capacity, a battery of assays was used for measuring total antioxidant capacity. materials and methods plant material acacia leucophloea (bark), albizia lebbeck (bark, flower, seed), capparis decidua (root), cicer arietinum (seeds) and grewia asiatica (leaves) were procured from department of agronomy, bahauddin zakariya university, multan and voucher specimen number (no.1804-2008) was deposited in herbarium of department of botany of same university. the plant material (1 kg for each) was crushed separately to coarse powder separately with help of pestle and mortar and macerated with aqueous methanolic mixture (5 l; 80:20; v/v) at room temperature for fifteen days with occasional shaking. the extracts obtained were filtered through filter paper under vacuum and concentrated under reduced pressure in a rotary evaporator (model q-344b – quimis, brazil) using a warm water bath (model q-214m2 – quimis, brazil) to obtain a thick gummy mass, which was further dried in a desiccator and stored in air-tight vial till further use. chemicals the 6-hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic acid (trolox), 2,2-azinobis (3 ethylben zothiazoline-6-sulfonic acid) diammonium salt (abts), and 2,4,6-tripyridyl-s-triazine (tptz) were purchased from sigma-aldrich (st. louis, mo, usa). rphycoerythrin (r-pe) was purchased from prozyme (san leandro, ca, usa); 2,2-azobis (2-amidinopropane) dihydrochloride (abap) was purchased from waco chemicals (richmond, va, usa). all chemicals and solvents used were hplc-grade and purchased from carlo erba (milan, italy). ultrapure water from a milliq system (millipore, marlborough, ma, usa) was used throughout the experiments. determination of total phenolic content (tpc) and tac for the determination of tpc and tac a weighed amount of methanolic extract sample (between 50 and 130 mg depending on the sample) was dissolved in 10 ml of 1 % formic acid methanol solution. the extracts were kept at 4 °c at dark prior to the analysis. antioxidant potential of selected plant extracts 31 tpc analysis the total phenolic content of each extract was determined using the method previously described (adom and liu, 2002 ). briefly, the extracts were oxidized with folin-ciocalteu reagent and the reaction was neutralized with sodium carbonate. the absorbance of the resulting blue color was measured at 760 nm. data are expressed as mg catechin equivalents per g plant extract. tac analyses plant extracts were analyzed for their antioxidant capacity by three different tac assays: trolox equivalent antioxidant capacity (teac) assay (pellegrini et al., 2003), ferric reducing antioxidant power (frap) assay (benzie and strain, 1999) and total radical-trapping antioxidant parameter (trap) assay (ghiselli et al., 1995). the teac and trap values were expressed as micromoles of trolox per g plant extract, frap values were expressed as micromoles of fe2+ equivalents per g plant extract. hplc-esi-ms/ms analysis of phenolic compounds phenolic compounds were analysed using a water 2695 alliance separation module equipped with a micromass quattro micro api mass spectrometer fitted with an electrospray interface (esi) (waters, milford, ma, usa). a preliminary investigation on phenolic profiles of selected plants was carried out by means of ms scan analysis, operating in negative ion mode from 100 to 1000 mass-tocharge ratio (m/z). then, different multiple reaction monitoring (mrm) methods were developed for all sample types, based on the obtained ms scan data. separations were performed using a waters atlantis dc18 3 μm (2.1 x 150 mm) reverse phase column (waters), with the mobile phase, pumped at a flow rate of 0.17 ml/min. the mobile phase was a 30-min linear gradient of 5 to 30 % acetonitrile in 1 % aqueous formic acid. the esi source worked in negative ionisation mode. source temperature was 120 ºc, desolvation temperature was 350 °c, capillary voltage was 2.8 kv, cone voltage was 35 v, desolvation gas (n2) 750 l/h, cone gas (n2) 50 l/h. the collision energy for ms/ms identifications was set at 30 ev, and the collision gas used was argon. statistical analysis to verify the association among the total antioxidant capacity and total phenols method, pearson correlation analysis was performed using the spss statistical software (version 19.0, spss inc., chicago, il); p-values £ 0.01 were considered significant. results and discussion data on tpc (tab. 1) indicated that barks were the botanical part of plants with the highest amount of phenolic compounds, with a. lebbeck containing higher amount than a. leucophloea. this is in agreement with previous findings that reported that phenolics are present more in leaves, flowering tissues and woody parts, such as stems and barks, and in less amount in seeds (larson, 1988; gallo et al., 2010). between seeds analysed, a. lebbeck contained greater amount of phenolics than c. arietinum. a. lebbeck flowers had intermediate phenolic content between bark and seed. the order of tpc was the following: a. lebbeck bark > a. leucophloea bark > g. asiatica leaves > c. decidua root > a. lebbeck flowers > a. lebbeck seeds > c. arietinum seeds. the mass spectral characteristics of phenolic compounds identified in the samples as phenolic acids, flavonoids and condensed tannins were reported in tab. 2. as example, fig. 1 and 2 report the chromatographic profile of some polyphenols identified in a. lebbeck flowers. among phenolic acids the hydroxycinnamate derivates were present mostly than hydroxybenzoate derivates. about flavonoids, the flavonols kaempferol and quercetin derivates were the most representative compounds detected in extracts. the condensed tannins (procyanidins) were identified only in a. lebbeck bark (fig. 3). among these flavonoids, at least three b-type dimers of procyanidins were identified, presenting a [m -h]at m/z 577 and typical fragment ions at m/z 125, 287, 289, 407, 425, formed by a ring cleavage (m/z 125), interflavanic bond cleavage through the quinone methide mechanism (m/z 289 and 287), retro-diels alder tab. 1: total phenol content (mg catechin equivalents/g). data are expressed as the mean ± standard deviation (n = 3). plant total phenol content (mg/g) acacia leucophloea bark 218.50 ± 2.95 albizia lebbeck bark 388.51 ± 5.83 albizia lebbeck flowers 8.21 ± 0.05 albizia lebbeck seeds 6.86 ± 0.07 capparis decidua root 36.26 ± 0.22 cicer arietinum seeds 1.47 ± 0.03 grewia asiatica leaves 118.52 ± 0.90 tab. 2: tentative identification of phenolic compounds based on their mass spectral characteristics n° compound [m-h](m/z) qualifier ions (m/z) 1 gallic acid 169 125 2 galloyl-hexoside 331 169 3 coumaric acid 163 119 4 caffeic acid 179 135 5 coumaric acid-hexosides 325 163, 119 6 caffeic acid-hexosides 341 179, 135 7 ferulic acid-hexosides 355 193, 134 8 syringic acid-hexosides 359 197 9 vanillic acid-hexosides 329 167 10 sinapic acid-hexosides 385 223,149 11 caffeoylquinic acids 353 191, 179, 173 12 feruloylquinic acids 367 191 13 apigenin-hexosides 431 269 14 kaempferol-rhamnosides 431 285 15 kaempferol-hexosides 447 285 16 quercetin-hexosides 463 301 17 quercetin-glucuronide 477 301 18 kaempferol-glucuronide 461 285 19 quercetin-rutinoside 609 301, 447 20 kaempferol-rutinoside 593 285, 447 21 quercetin-dirhamnoside755 609, 463, 301 hexoside 22 kaempferol-dihexoside 609 447, 285 23 epicatechin 289 137, 245 24 epicatechin derivative 289, 245, 137 25 procyanidin dimers b-type 577 125, 287, 289, 407, 425 26 procyanidin trimers b-type 865 577, 575, 289 27 procyanidin tetramers 1153 575, 577 b-type 32 i. imran, m. zia-ul-haq, l. calani, t. mazzeo, n. pellegrini fig. 1: mrm chromatograms of coumaric acid-hexosides. it is possible to note at least two isomers (14.85 and 17.34), identified through the loss of hexose moiety 325>163 and further fragmentation of coumaric acid 163>119. fig. 2: mrm chromatogram of quercetin-rutinoside (609>301), identified through the loss of rutinosyl moiety (308 amu) and subsequent ionization of formed quercetin. (rda) cleavage and loss of a water molecule (m/z 425 and 407). instead, five procyanidin trimers were identified, characterized by a [m-h]at m/z 865 besides typical fragment ions at m/z 577, 575, 289 formed through the same fragmentation pattern observed for procyanidin dimers. moreover, the bark of a. lebbeck contained several tetramers, identified through their [m-h]at m/z 1153, besides fragmentation forming ions at m/z 577 (dimers) and its quinone counterpart at m/z 575 (li et al., 2007; gu et al., 2003). g. asiatica leaves had the highest number of phenolic compounds (tab. 3), associated to a high tpc, followed by c. decidua root, while few phenolic compounds were identified in c. arietinum seed and a. leucophloea bark. as the lc-ms/ms instrument allows to identify up to fourth degree of polymerization, the high tpc found in the two barks analysed was probably owed to procyanidins with a higher molecular weight than tetramers, which also justified the high values of tac measured by teac and frap assays (tab. 4). the presence of tannins in methanolic extracts of a. leucophloea bark has been previously demonstrated (anjaneyulu et al., 2010). several phenolic compounds were identified by hplc-esi-ms/ms analysis in other botanic parts of a. lebbeck (i.e., flowers and seed), even though the tpc values were lower than those in bark as well as their tac values (tab. 4). the five plant species considered in this study were subjected to antioxidant capacity screening using different testing methods, i.e. teac, frap and trap assays, and the results were present in tab. 4. teac and frap values were in agreement, with a. lebbeck bark and a. leucophloea bark having the highest values followed by c. decidua root and g. asiatica leaves. conversely, these latter plants exhibited the highest trap values followed by a. leucophloea bark and a. lebbeck seeds. this discrepancy could be related to the different sensitivity of tac assays toward different phenolic compounds. in the case of trap assay, its low sensitivity of high polymerised polyphenol rich matrices could be owed to the putative interaction of phycoerythrin (used as a target molecule in the trap assay) with high molecular weight polyphenols, such as proanthocyanidins and gallotannins (hagerman and butler, 1981). conversely, high polymerized phenolic compounds exhibit higher teac values than simple ones (hagerman et al., 1998). finally, due to the low tpc and a low number of phenolic compounds identified by hplc-esi-ms/ms analysis, c. arietinum seeds showed the lowest tac value regardless of the assay applied. similar results have been obtained analysing the tac of chickpea by teac assay (han and baik, 2008; pellegrini et al., 2006). pearson correlation analysis was performed to corroborate relationships between teac, frap, trap values and tpc of plants. strong positive and significant correlations between total antioxidant capacity measured by frap and teac assays and total phenols were observed. in particular, the best correlation was observed between frap and tpc (r = 0.964, p value £ 0.01), whereas the teac and tpc values were less correlated (r = 0.833, p value £ 0.01). conversely, no correlation was found between trap and total phenolic content. these positive correlations observed between frap and teac assays and tpc are consistent with previous findings (srivastava et al., 2012) and support the hypothesis that phenolic antioxidant potential of selected plant extracts 33 fig. 3: mrm chromatograms of b-type procyanidins. in detail dimers identified through the interflavanic bond cleavage (577>289), as well as for trimers (865>577) and tetramers (1153>575). in the tetramers it is possible to note the quinone dimers formation (m/z 575). compounds contribute significantly to the total antioxidant capacity of medicinal plants. the lack of correlation between trap assay and tpc is probably due to the before mentioned low sensitivity of this assay to high polymerised phenolic compounds. conclusions investigated extracts appeared to have potential as a health supplement rich in natural antioxidants and merits further intensive study. the results of this study will promote the reasonable usage of these plants in food and pharmacy industries as well as in alternative medicine and natural therapy. references adom, k.k., liu, r.h., 2002: antioxidant activity of grains. j. agric. food chem. 50, 6182-6187. amro, b., aburjai, t., al-khalil, s., 2002: antioxidative and radical scavenging effects of olive cake extract. fitoterapia. 73, 456-461. anjaneyulu, e., ramgopal, m., hemalatha, s., balajj, m., 2010: phytochemical analysis, antimicrobial and antioxidant activity of the bark extract of acacia leucophloea l. global j. biotech. biochem. 54, 231-236. barnham, k., masters, c.l., bush, a.i., 2004: neurodegenerative diseases and oxidative stress. nat. rev. drug dis. 3, 205-214. benzie, i.f.f., strain, j.j., 1999: ferric reducing/antioxidant power assay: direct measure 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fluorescence-based method for measuring total plasma antioxidant capability. free radic. biol. med. 18, 29-36. gu, l., kelm, m.a., hammerstone, j.f., zhang, z., beecher, g., holden, j., haytowitz, d., prior, r.l., 2003: liquid chromatographic/electrospray ionization mass spectrometric studies of proanthocyanidins in foods. j. mass spectrom. 38, 1272-1280. hagerman, a.e., butler, l.g., 1981: the specificity of proanthocyanidinprotein interactions. j. bio. chem. 256, 4494-4497. hagerman, a.e., riedl, k.m., jones, g.a., sovik, k.n., ritchard, n.t., hartzfeld, p.w., riechel, t.l., 1998: high molecular weight plant polyphenolics (tannins) as biological antioxidants. j. agric. food chem. 46, 1887-1892. han, h., baik, b.k., 2008: antioxidant activity and phenolic content of lentils (lens culinaris), chickpeas (cicer arietinum l.), peas (pisum sativum l.) and soybeans (glycine max), and their quantitative changes during processing. int. j. food sci. tech. 43, 1971-1978. katalinic, v., milos, m., kulisic, t., jukic, m., 2006: screening of 70 34 i. imran, m. zia-ul-haq, l. calani, t. mazzeo, n. pellegrini tab. 3: phenolic profile of plant extracts phenolic a. lebbeck a. lebbeck a. lebbeck a. leucophloea c. arietinum c. decidua g. asiatica compound bark flowers seeds bark seeds root leaves 1 + +* +* 2 + 3 + 4 + 5 + +* + + 6 + + + 7 + +* + + + 8 + 9 +* + + 10 + + + 11 +* +* + + 12 + 13 + + + 14 + 15 + 16 + +* + 17 + 18 + 19 + + + 20 + + 21 + 22 + 23 +* 24 + 25 + 26 + 27 + +: present; *: trace (present at limit of detection) tab. 4: teac, frap and trap values of plant extract analysed. data are expressed as the mean ± standard deviation (n =3). plant teac μmol/g frap μmol/g trap μmol/g albizia lebbeck flowers 33.11 ± 0.99 176.55 ± 8.15 53.65 ± 2.90 albizia lebbeck bark 502.56 ± 2.37 2561.50 ± 46.74 43.66 ± 1.13 albizia lebbeck seeds 41.38 ± 1.56 48.20 ± 2.29 62.08 ± 3.07 acacia leucophloea bark 682.95 ± 3.06 2234.43 ± 83.55 90.91 ± 3.84 cicer arietinum seeds 3.34 ± 0.01 23.71 ± 1.06 3.72 ± 0.00 capparis decidua root 384.91 ± 3.07 338.68 ± 12.57 202.21 ± 0.00 grewia asiatica leaves 72.47 ± 1.62 939.89 ± 46.96 353.63 ± 10.72 medicinal plant extracts for antioxidant capacity and total phenols. food chem. 94, 550-557. larson, r.a., 1988: the antioxidants of higher plants. phytochem. 27, 969-978. li, h.b., cheng, k.w., wong, c.c., fan, k.w., chen, f., jiang, y., 2007: evaluation of antioxidant capacity and total phenolic content of different fractions of selected microalgae. food chem. 102, 771-776. moure, a., cruz, j.m., franco, d., dominguez, j.m., sineiro, j., dominguez, h., nunez, m.j., parajo, j.c., 2001: natural antioxidants from residual sources. food chem. 72, 145-171. pellegrini, n., serafini, m., salvatore, s., delrio, d., bianchi, m., brighenti, f., 2006: total antioxidant capacity of spices, dried fruits, nuts, pulses, cereals and sweets consumed in italy assessed by three different in vitro assays. mol. nutr. food res. 50, 1030-1038. pellegrini, n., 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chem. 102, 938-953. tachakittirungrod, s., okonogi, s., chowwanapoonpohn, s., 2007: study on antioxidant activity of certain plants in thailand: mechanism of antioxidant action of guava leaf extract. food chem. 103, 381-388. uttara, b., singh, a.a., zamboni, p., mahajan, r.t., 2009: oxidative stress and neurodegenerative diseases: a review of upstream and downstream antioxidant therapeutic options. curr. neuropharma. 7, 65-74. zia-ul-haq, m., shahid, s.a., ahmed, s., ahmad, s., qayum, m., khan, i., 2012: anti-platelet activity of methanolic extract of grewia asiatica l. leaves and terminalla chebula retz. fruits. j. med. plant res. 6, 2029-2032. zia-ul-haq, m., shahid, s.a., ahmed, s., ahmad, s., qayum, m., khan, i., 2012: antioxidant potential of various parts of ferula assafoetida l. j. med. plant res. 6, 3254-3258. zia-ul-haq, m., ćavar, s., qayum, m., imran, i., defeo, v., 2011: compositional studies: antioxidant and antidiabetic activities of capparis decidua (forsk.) edgew. int. j. mol. sci. 12, 8846-8861. address of the corresponding author: e-mail: ahirzia@gmail.com art26.indd journal of applied botany and food quality 82, 158 162 (2009) 1 department of plant physiology, faculty of agriculture and economics, agricultural university of cracow, kraków, poland 2 f. górski’ institute of plant physiology, polish academy of sciences, kraków, poland cell membrane permeability and antioxidant activities in the rootstocks of miscanthus x giganteus as an effect of cold and frost treatment a. płażek1, f. dubert2, k. marzec1 (received november 26, 2007) summary the aim of the study was to estimate the ability of miscanthus x giganteus to acquire frost tolerance. field grown rootstocks were transferred into pots and cultivated in a glasshouse at 20°c. after 5 weeks plants were pre-hardened at 12°c for a further 2 weeks and then hardened at 5°c for another 3 weeks. after this time, plants were frozen at -8°c or -15°c for 1, 3 or 5 days, after which their regrowth at 20°c was investigated. the membrane permeability (electrolyte leakage), activity of the catalase (cat), non-specific peroxidase (px), and protein content in stolons were measured, before and after pre-hardening, as well as after hardening and freezing. both pre-hardening and hardening decreased membrane permeability of the rootstock cells, and this effect was observed further, after 5week of regrowth at 20°c. freezing at both temperatures increased ion leakage gradually over the period of treatment. on the basis of total ion content, damage to the cell membranes of frozen stolons after recovery was stated. prehardening increased cat activity, while hardening did not alter it. however, after 5-week de-hardening, cat activity decreased significantly. freezing at -8°c for 5 days increased significantly the activity of this enzyme. at -15°c cat activity was lower than in the control after only one day of freezing. px activity decreased both in the rootstocks of cold (12°c and 5°c) and frost treated plants. protein content increased significantly in the stolons of both pre-hardened and hardened plants, although not immediately after cold treatment, but only after a 5-week re-growth period in a glasshouse at 20°c. this phenomenon was observed also in the stolons of plants frozen at -15°c for 5 days. from frozen rootstocks no new stems in regrowth conditions were obtained. the results obtained indicated, that although frozen stolons cannot produce new shoots, they do demonstrate some metabolic vitality. so, it could be supposed that the frost susceptibility of studied plants resulted from the strong sensitivity of shoot apical meristems to the cold. further studies will analyse the survival of miscanthus in milder frost temperatures. abbrevations: cat – catalase, edta – ethylenediaminetetraacetic acid, fw – fresh weight, ppfd – photosynthetic photon flux density [µmol m-2 s-1], px – non-specific peroxidase, ros – reactive oxygen species, introduction miscanthus x giganteus is a sterile triploid (hybrid of the diploid miscanthus sinensis and tetraploid m. sacchariflorus), which reproduces only vegetatively from rootstocks. recently, miscanthus has received the interest of producers because of the possibility of its use as a biofuel or cellulose source. this is a perennial plant of a c4-photosynthesis type, characterized by high co2 absorption, high yield potential and low water consumption. it does not demand high mineral nutrition and can be cultivated on poor soils. moreover, because of many tangled stolons it prevents soil erosion. these properties are important for contemporary, sustainable agriculture designed to keep wastelands in a good condition (lewandowski, 2006). however, miscanthus plants are sensitive to frost, especially during the first winter. the loss of many plants results in the necessity of spring replanting, so increasing production costs. the consequence of vegetative reproduction is a small range of genetic variability within this species, so it is very difficult to select plants with higher frost resistance. the mechanisms responsible for freezing tolerance are not very well understood, and have a very wide background. herbaceous plants from temperate regions survive temperatures ranging from -5°c to -30°c, while plants from tropical regions generally have little or no capacity to survive even the slightest frost. many important crops, such as rice, maize, soybean, cotton and tomato are chill sensitive and incapable of cold acclimation. moreover, they cannot tolerate ice formation in their tissues. nevertheless, the temperature threshold for chilling damage is lowered even in chill-sensitive crops by prior exposures to sub-optimal low temperatures (chinnusamy et al., 2007). freezing tolerance is induced during cold hardening at non-freezing temperatures. cold acclimation involves the stabilization of plasma membranes against freeze-induced damages, synthesis of proteins preventing molecules from precipitation and direct physical damages caused by the accumulation of intercellular ice. cold acclimation includes an increase in fatty acid desaturation in membrane phospholipids and changes the levels and types of membrane sterols and cerebrosides (thomashow, 1998). murata and los (1997) reported that changes in membrane fluidity under cold treatment regulate the activity of receptor-mediated phospholipase c and protein kinase c, key enzymes, in signal transduction during thermoacclimation. low temperature evokes the accumulation of proteins, especially those of small molecular weight with antifreeze properties (thomashow, 1998; karimzadeh et al., 2000; chinnusamy et al., 2007). these proteins protect membranes and other cell structures from mechanical injuries, regulate cell metabolism, and control their osmotic potential (antikainen and griffith, 1997; yu and griffith, 1999; thomashow, 1998; chinnusamy et al., 2007). oxidative stress is the secondary effect of numerous abiotic and biotic stresses. exposure to low temperatures causes changes in the activity of various antioxidant enzymes, which is connected with the higher production of reactive oxygen species (ros). in maize, many studies have linked chilling tolerance to antioxidant capacity (pastori et al., 2000). hydrogen peroxide is recognized as a signal molecule initiating the defence responses of plants to many stressors. this compound mediates the cross-talk between signalling pathways, and is a key signalling molecule contributing to the phenomenon of cross-tolerance. hence, the mediating role of h2o2 takes into account any programme aimed at improving crop tolerance to environmental stresses (neill et al., 2002). among genes up-regulated by hydrogen peroxide are those, which code the proteins required for peroxisome biogenesis, senescence-related proteins, antioxidant enzymes, protein kinases and phosphatases, and dna damage repair proteins (desikan et al., 2000). hydrogen peroxide also regulates stomata closure in response to abscisic acid (allan and fluhr, 1997). the accumulation of hydrogen peroxide under cold treatment in many plant species was observed (scebba et al., 1998; seppänen and fagerstedt, 2000; pastori et al., 2000). the plant defence antioxidant mechanism includes enzymatic and non-enzymatic scavengers of ros, so their amount in cells may be estimated on the basis of antioxidant activities. the main antioxidant enzymes that scavenge hydrogen peroxide are catalases and peroxidases. they are activated by different h2o2 concentrations (mittler, 2000), and catalase (cat) as chilling-sensitive, while peroxidase (px) as chilling-tolerant are recognized (scebba et al., 1999). the aim of the study was to estimate, the processes taking place in the rootstocks of miscanthus x giganteus undergoing pre-hardening or hardening temperatures, and if the changes induced by low temperature are stable during regrowth in a warm glasshouse. the frost tolerance was estimated on the basis of changes in membrane permeability, as well as the activities of enzymes scavenging hydrogen peroxide in the rootstocks of plants undergoing pre-hardening, hardening and frost treatment. material and methods plant growth conditions in august, the rhizomes of micanthus were transferred from the field to pots and grown in a glasshouse in natural light at 20°c. the pots contained a mixture of soil : peat : sand (2 : 2 : 1 v/v/v) at ph 5.8. sixweek-old plants were pre-hardened for 2 weeks in a growth chamber (12°c, 10 h-photoperiod, 250 µmol m-2 s-1 of ppfd) and than hardened for a further 3 weeks (5°c, 8 h-photoperiod, light intensity as above). after this time the plants were frozen at -8oc or -15oc for 1, 3, or 5 days in the dark. some pre-hardened, hardened and frozen plants were taken for analyses, while others were transferred to a glasshouse (conditions as described above) for 5 weeks after which their regrowth ability was examined. pre-hardened plants transferred to 20°c for 5 weeks were marked as rp (regrowth after prehardening), hardened ones as rh (regrowth after hardening, also known as dehardened plants), and frozen plants for 1, 3 or 5 days, as r1, r3 and r5 respectively, for each frost temperature. control plants were grown in a glasshouse and were divided into three groups: c1, c2 and c3 – plants growing in the glasshouse for 5, 7 and 10 weeks respectively. their age responded to age of prehardened (c2) and hardened as well as frozen plants (c3). c1 was the initial control. the estimation of membrane permeability and total ion content per g of fw was performed on stolons from prehardened, hardened and frozen plants, as well as after 5-week regrowth at 20°c, while in the case of other measurements, frozen plants after the regrowth period were not analyzed. analyses ion leakage the conductivity was expressed as the ion leakage via cell membranes. stolon disks (approximately 0.3 g of fw) were placed into vials containing 13 cm3 of ultrapure water, and were shaken (100 rpm) at 20°c. after 2 h, the electrical conductivity (e1) was measured using a conductometer (ci 317, elmetron, poland). then, the vials with the samples were boiled for 5 min and shaken again. the conductivity was re-measured and this value was taken to represent the total ion content (e2) in the tissue. membrane permeability was expressed as a percentage of total electrolyte leakage (e1x100/e2). moreover, total ion content per g of fw in the stolons was calculated. the measurements were done in 50 replicates (10 discs from any of 5 stolons for each treatment). catalase (ec 1.11.1.6) (cat) activity frozen samples of stolons (each weighing 0.2 g of fw) were ground to a fine powder with liquid nitrogen and extracted with 50 mm of phosphate buffer (ph 7.5). the extracts were centrifuged at 4°c for 10 min at 14 000 × g and the supernatants were used as the crude extracts. cat activity was assayed in a reaction mixture (3 cm3 final volume) composed of 50 mm of phosphate buffer ph 7.5, to which 30% (w/v) h2o2 was added to reach an absorbance value in the range of 0.5200.550 a at 240 nm. the reaction was started by adding 200 µl of crude extracts. cat activity was measured by monitoring the decrease in absorbance at 240 nm (using spectrophotometer lkb ultrospec ii, umea, sweden) as a consequence of h2o2 consumption. the decrease in absorbance of 0.0145 a corresponded to 1 µmol h2o2 decomposed by cat (aebi, 1984). the activity of the enzyme was expressed as µmol h2o2 decomposed per minute per g of fw of stolons. catalase assays were done in 15 replicates (3 samples were collected from any of 5 stolons for each studied object). total peroxidase (px) activity peroxidase activity was measured according to the method described by bergermeyer (1965). stolon discs were ground to a fine powder with liquid nitrogen and extracted with 50 mm of phosphate buffer (ph 7.0) and 1 mm edta (sigma-aldrich). the extract was centrifuged (14 000 rpm) at 4°c for 10 min and the supernatant was used as the crude extract. two cm3 of 50 mm phosphate buffer (ph 7.0) was mixed with 12 µl of 0.5% p-phenylenediamine and 12 µl of crude extract. the oxidation of p-phenylenediamine was initiated by the addition of 12 µl of buffered h2o2 (0.15 cm3 of 30% h2o2 (w/v) mixed with 50 cm3 of extract buffer) to a prepared mixture. the absorbance was measured at 460 nm. the total peroxidase activity was expressed as an increase in absorbance of the sample after 1 min and expressed as activity per 1 mg of fw. the determination of px activity was completed in 15 replicates. protein determination protein concentration was determined according to bradford (1976) using the bio-rad (munich, germany) protein assay reagent. bovine serum albumin (sigma-aldrich) was used as a calibration standard. the measurements were taken in 15 replicates. statistical analysis all the effects of various temperatures on studied parameters in stolons were tested with the f-test (analysis of variance anova, statistica 5.0). results statistical analysis showed the significant effect of applied temperatures on all studied parameters. although the pre-hardening temperature did not influence the membrane permeability to a significant degree, some tendency to improve (the decrease) cell membranes permeability under a 12°c-treatment could be seen (fig. 1). after 5-week-growth after pre-hardening at 20°c, a considerable decrease in ion leakage from the stolon cells (rp) was observed. a temperature of 5°c caused a significant decrease in ion leakage immediately after hardening. less membrane permeability remained on the same level after 5-weekgrowth (rh) in glasshouse conditions. in the case of freezing at -8oc and -15oc, the membrane damages increased throughout the duration of the treatment. electrolyte leakage from cells of stolon frozen at -8°c and -15°c for 1 day (r1 and r2) in regrowth conditions, did not change from those tested just after freezing, while the membrane permeability of rootstocks frozen at both frost temperatures for 3 and 5 days (r3 and r5) decreased at the control temperature. total ion content in the stolons of pre-hardened plants did not differ significantly from the control (c2), although an increase in this para frost hardening of miscanthus x giganteus 159 meter could be seen especially after the recovery period (fig. 2). hardening caused a considerable increase in ion content in tissue compared with c3 plants and after 5-weeks of de-hardening this process was even stronger. frozen stolons demonstrated no significant difference in ion content compared to the rootstocks of hardened plants. however, some influence of prolonged time and a stronger frost on a parameter decrease was observed. after 5-week regrowth at 20°c independent of the time and temperature of frost treatment, an almost total ion efflux from the stolons was observed. cat activity during prehardening at 12°c increased significantly and after 5-week-recovery at 20°c, it did not change (fig. 3). the rootstocks of hardened plants demonstrated few changes in cat activity, while during de-hardening (rh) at 20°c they showed a decrease. generally, both applied frost temperatures did not cause significant changes in cat activity, and only freezing at -8°c for 5 days increased the activity of this enzyme. px activity decreased along with plant age (c1, c2, c3) (fig. 4). prehardening resulted in a decrease in the activity of this enzyme, and the decrease was also observed later in the stolons of plants transferred back to a glasshouse at 20°c (rp). neither hardening nor -5°c exerted changes in px activity. plant freezing at -15°c for 5 days increased px activity in comparison with frozen plants at -8°c and at -15°c for 1 and 3 days. however, the level of px activity in the stolons of frozen plants at -15°c for 5 days, returned to the level of the c3 plant rootstocks. protein content per fw unit strongly increased in both pre-hardened and hardened plants, not immediately after cold treatment, but only after 5-week regrowth in a glasshouse at 20°c (fig. 5). this phenomenon was observed also in the stolons of plants frozen at -15°c for 5 days. analysis after freezing showed that many stolons demonstrated no visible damage. however, in the case of both frost treatments (temperature x time) no new shoots from rootstocks kept in regrowth conditions were obtained. this result suggests that temperatures applied independently of time treatment were lethal for miscanthus apical meristems. 0 10 20 30 40 50 60 70 80 90 100 treatments c1 c2 p rp c3 h rh 1 r1 3 r3 5 r5 1 r1 3 r3 5 r5 -8oc -15oc pe rc en ta ge io n le ak ag e fig. 1: electrolyte leakage from the rootstocks of prehardened (p) at 12°c, hardened (h) at 5°c and frozen at -8°c and -15°c for 1, 3 or 5 days miscanthus plants (-8/1, -8/3, -8/5; -15/1, -15/3 -15/5). c1 – initial control; c2 – control for prehardened plants; c3 – control for hardened and frozen plants. rp – pre-hardened plants regrowing at 20°c for 5 weeks; rh – hardened plants re-growing at 20°c for 5 weeks; r1, r2 and r3 – frozen plants at both frost temperatures for 1, 3 or 5 days respectively and regrowing at 20°c. mean of 50 replicates ± se. 0 200 400 600 800 1000 1200 1400 1600 1800 2000 treatments c1 c2 p rp c3 h rh 1 r1 3 r3 5 r5 1 r1 3 r3 5 r5 -8oc -15oc µs � g -1 f w fig. 2: ion content [µs g-1 fw] in the rootstocks of prehardened, hardened and frozen miscanthus plants. treatment labels the same as on fig. 1. mean of 50 replicates ± se. fig. 3: catalase activity in the rootstocks of prehardened, hardened and frozen miscanthus plants. treatment labels the same as on fig. 1. mean of 15 replicates ± se. discussion the main problem for plants originating from other climatic zones acclimatizing to polish environmental conditions, is their weak resistance to the cold and freezing temperatures in winter. for thermophilic plants even temperatures of 10°c during spring or autumn can evoke cold damage. in the case of miscanthus, the first winter in the field determines its further cultivation. changes in the structure of cell membranes are often observed as the 0 0,2 0,4 0,6 0,8 1 1,2 1,4 treatments c1 c2 p rp c3 h rh 1 3 5 1 3 5 -8oc -15oc m g p ro te in � g -1 f w fig. 5: protein content in the rootstocks of prehardened, hardened and frozen miscanthus plants. treatment labels the same as on fig. 1. mean of 15 replicates ± se. fig. 4: non-specific peroxidase activity in the rootstocks of prehardened, hardened and frozen miscanthus plants. treatment labels the same as on fig. 1. mean of 15 replicates ± se. 160 a. płażek, f. dubert, k. marzec effects of cold temperatures (yoshida and umera, 1990; murata and los, 1997; thomashow, 1998). these changes relate especially to the proportion between the amounts of saturated and non-saturated fatty acids, the polar phospholipids content, and h+-atpase activity, which is closely connected with the selective characteristic of membranes. according to carruthers and melchior (1986), selective characteristics of cell membranes significantly influence the activity of several membrane-enzymes. this fact points to the positive role of cold in the activation of membrane proteins, functioning as receptors in the signal transduction initiated during the defence responses to any stress. in the presented experiment, ion leakage from cells was measured in the rootstocks of miscanthus. after the first cold days in autumn the leaves of this species dry very quickly, and as such the metabolic changes effected by prehardening, hardening and frost were studied in the stolons, because their physiological condition before and during winter determines whether miscanthus survives the winter. quamme and brownlee (1997) used ion leakage from apple-tree root pieces for estimation of resistance to winter injury of rootstocks of various cultivars. the laboratory tests of frost resistance agreed with reports of field survival. the prehardening of miscanthus plants did not change significantly the membrane permeability of stolon cells. however later, during deprehardening, the positive effect of this treatment on the selectivity of membranes was observed. hardening caused a considerable decrease in ion leakage from cells and this effect remained during de-hardening. cold-improving membrane permeability could be concluded on the basis of total ion content in the tissue. in the earlier study, płażek and żur [2003] stated that in winter rape, spring barley and meadow fescue, cold hardening at 5°c decreased the membrane permeability of leaf cells, while de-hardening increased electrolyte leakage to the control level. in the frozen stolons, ion leakage increased gradually throughout the time treatment, possibly resulting from frost damaged tissue. also, in this case after 5-weeks re-growth at 20°c, the electrolyte leakage decreased similarly to the effect observed by de-prehardened and de-hardened rootstocks. this result could be explained by partial ion efflux from damaged tissue to the soil, although many stolons seemed to be uninjured. this is why no other measurements in stolons re-grown after frost-treatment were performed. arora and palta (1991) stated in freeze-thawed onion bulbs a higher ion leakage rate, which decreased during recovery. the authors explained this was the result of frost reduction of plasma membrane atpase activity, which then returned to the control level. in wheat under water stress, pereyra et al. (2006) observed no changes in membrane structure and permeability in root cells contrary to those of the leaves. kang and saltveit (2002) stated that chilling maize, cucumber and rice seedlings at 15°c, affected the increase in electrolyte leakage from leaves but did not influence this parameter in radicals. the change pattern of cat and px activity in miscanthus rootstocks under cold treatment, differ from the effects noted in leaves by other authors (scebba et al., 1999; back and skinner, 2003; ella et al., 2003). these results could indicate that roots differ in reaction to the stresses from leaves. oxidative stress has been proposed as one of the reasons for coldtemperature damage (scebba et al., 1999). reactive oxygen species are supposed to be responsible for frost-induced injuries because they are produced at higher concentrations during freezing stress. the higher tolerance to oxidative stress evoked by freezing is induced by cold-acclimation temperatures (scebba et. al., 1998). ros generation is different in various plant parts. scebba et al. (1999) showed, that the roots of cold-treated wheat demonstrated higher an activation of sod (superoxide dismutase) and cat, while in cold-hardened leaves a decrease in activity of both enzymes compared to the control was observed. the main source of ros in leaves are electron transport via thylacoids and mitochondrial membranes, and the reactions taking place in peroxisomes during photooxidation, while in rhizomes ros are produced mainly during the electron transportation chain in mitochondria. hydrogen peroxide could moreover be generated in apoplast in both organs – leaves and rhizomes. in miscanthus stolons, prehardening temperature of 12°c increased cat activity, while a hardening temperature of 5°c did not change it. the cold effect (the decrease in cat activity) was observed much later in de-hardened plants, although interpretating this result is not easy. a significant increase in activity of this enzyme in the stolons of frozen plants was observed at -8°c, but only after 5 day-treatment. the activity of antioxidants in frozen rootstocks during recovery was not measured, because no plants from these rootstocks were obtained, although it could indicate that both applied freezing temperatures were lethal for this plant species. cat is recognized as a cold-sensitive enzyme, and usually its lower activation level in cold is observed (leipner et al., 1999; płażek and żur, 2003), although during recovery its activity increases to the control level (ella et al., 2003; płażek and żur, 2003]. thus, the decrease in cat activity in stolons, 5 weeks after hardening, may be difficult to explain. it’s very possible, that despite no obvious visible damage, in the cold aftermath some metabolic processes in miscanthus rootstocks were altered. although no correlation between cell membrane permeability and cat activity was found, it could be supposed that in stolons of prehardened and hardened plants, this enzyme protected cell structures against the cold. px was drasticly inhibited in the miscanthus stolons during prehardening, while lower temperatures during hardening and freezing did not influence px activity, except at -15°c lasting for 5 days, when px activity increased to the control level. peroxidase is recognized as cold-tolerant, so the obtained result relating to the cat increase and px decrease in frost indicates, that rhizomes react differently from leaves and the interpretation of these results could not be compared to processes occurring in the leaves. moreover, the increase in cat activation could be a response to the dramatic decline in px activation, and it was caused by an increase in hydrogen peroxide accumulation. the different response of miscanthus stolons at 12°c and the much lower temperatures could suggest, that this plant species may also be classified as cold-sensitive equally with maize, sorghum and cotton, for which even 15°c is a stress-temperature. low temperature evokes the accumulation of various proteins, especially those of small molecular weight (thomashow, 1998; karimzadeh et al., 2000; chinnusamy et al., 2007). these proteins with antifreeze properties protect membranes and other cell structures from mechanical injuries, regulate metabolic processes, and control the osmotic potential of cells (antikainen and griffith, 1997; yu and griffith 1999; thomashow, 1998; chinnusamy et al., 2007). cold hardening resulted in protein accumulation especially in winter cereals (antikainen and grffith 1997; yu and griffith, 1999). these proteins demonstrated mainly antifreeze properties. karimzadeh et al. (2000) confirmed higher protein accumulation in the roots of hardened wheat compared to the plants growing at 20°c. according to some authors (see karimzadeh et al., 2000), the cold treatment in various plant species had no effect on protein synthesis, for example in lolium temulentum. in contrast, the tropical cereal sorghum bicolor grown initially at 35°c, responded markedly to low temperatures. in the studied miscanthus rootstocks, a drastic increase in protein accumulation after prehardening at 12°c and hardening at 5°c was observed, but only after 5-week regrowth at 20°c. freezing of plants at -8°c for 5 days caused some increase in the protein content in stolons, although this change was not statistically significant. in contrary, the influence of -15°c for 5 days, increased (by 3.5 times) the accumulation of proteins in miscanthus stolons, than in the case after freezing for 1 or 3 days. from the frozen stolons no new stems in re-growth conditions were obtained. this result could be explained by the strong sensitivity of the meristematic tissues of miscanthus to the cold. according to field observations, after the first autumn chilling, the leaves of this plant frost hardening of miscanthus x giganteus 161 species remain green, while the apical part of stems inside the leaf sheaths become brown and atrophy. the results obtained indicated, that although frozen stolons can not produce new shoots they do demonstrate some metabolic vitality. further studies will analyze the survival possibility of miscanthus in milder frosts than applied in the presented experiment. conclusions 1. prehardening at 12°c and hardening at 5°c decrease cell membrane permeability, while freezing temperatures increase ion leakage from the cells of miscanthus rootstocks. less membrane permeability remains during further plant cultivation, which could be recognized as some symptoms of the cold acclimation of this plant species. 2. a temperature of 12°c significantly increases catalase activity, and decreases non-specific peroxidase activity in miscanthus stolons. 3. prehardening, hardening and frost temperatures do not influence protein accumulation in miscanthus stolons. however, a significant increase in protein content does occur in the aftermath of chilling. 4. the frost sensitivity of miscanthus is determined by a high susceptibility of apical meristems to frost. references aebi, h., 1984: catalase in vitro. methods of enzymol. 105, 121-126. allan, a.c., fluhr, r., 1997: two distinct sources of elicited reactive oxygen species in tobacco epidermal cells. plant cell. 9, 1559-1572. antikainen, m., griffith, m., 1997: antifreeze protein accumulation in freezing-tolerant cereals. physiol. plant. 99, 423-432. arora, r., palta, j.p., 1991: a loss in the plasma membrane atpase activity and its recovery coincides with incipient freeze-thaw injury and postthaw recovery in onion bulb scale tissue. plant physiol. 95, 846-852. baek, k., skinner, d.z., 2003: alteration of antioxidant enzyme gene expression during cold acclimation of near-isogenic wheat lines. plant sci. 165, 1221-1227. bergermeyer, h.u., 1965: methods of enzymatic analysis. verlag chemie gmbh weinheim/bergstr., academic press, new york and london. bradford, m.m., 1976: a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. anal. biochem. 72, 248-254. chinnusamy, v., zhu, j., zhu, j.k., 2007: cold stress regulation of gene expression in plants. trends plant sci. 12, 444-451. carruthers, a., melchior, d.l., 1986: how bilayer lipids affects membrane protein activity. trends biochem. sci. 11, 331-335. desikan, r., neil, s.j., hancock, j.t., 2000: hydrogen peroxide-induced gene expression in arabidopsis thaliana. free radical biology and. medicine. 28, 773-778. ella, e.s., kawano, n., ito, o., 2003: importance of active oxygen-scavenging system in the recovery of rice seedlings after submergence. plant sci. 165, 85-93. kang, h., saltveit, m.e., 2002: chilling tolerance of maize, cucumber and rice seedling leaves and roots are differentially affected by salicylic acid. physiol. plant. 115, 571-576. karimzadeh, g., francis, d., davies, m.s., 2000: low temperature-induced accumulation of proteins is sustained both in root meristems and in callus in winter wheat but not in spring wheat. ann. bot. 85, 769-777. leipner, j., fracheboud, y., stamp, p., 1999: effect of growing season on the photosynthetic apparatus and leaf antioxidative defenses in two maize genotypes of different chilling tolerance. environ. exp. bot. 42, 129-139. lewandowski, i., 2006: miscanthus – a multifunctional biomass crop for the future. in: jeżowski, s., wojciechowicz, m.k., zenkteler, e. (eds.), alternative plants for sustainable agriculture, vol. 5, 83-90. pagen, centre of excellence in plant agrobiology and molecular genetics. institute of plant genetics, polish academy of sciences, poznań. mittler, r., 2002: oxidative stress, antioxidants and stress tolerance. trends plant sci. 7, 405-410. murata, n., los, d.a., 1997: membrane fluidity and temperature perception. plant physiol. 115, 875-879. neil, s.j., desikan, r., clarke, a., hurst, r.d., hancock, j.t., 2002: hydrogen peroxide and nitric oxide as signalling molecules in plants. j. exp. bot. 53, 1237-1247. pastori, g., foyer, c.h., mullineaux, p., 2000: low temperature-induced changes in the distribution of h2o2 and antioxidants between the bundle sheath and mesophyll cells of maize leaves. j. exp. bot. 51, 107-113. pereyra, m.a., zalazar, c.a., barassi, c.a., 2006: root phospholipids in azospirillum-inoculated wheat seedlings exposed to water stress. plant physiol. biochem. 44, 873-879. płażek, a., żur, i., 2003: cold-induced plant resistance to necrotrophic pathogens and antioxidant enzyme activities and cell membrane permeability. plant sci. 164, 1019-1028 quamme, h.a., brownlee, r.t., 1997: cold hardiness evaluation of apple rootstocks. vi international symposium on integrated canopy, rootstock, environmental physiology in orchard systems. ishs acta hort. 451, scebba, f., sebastiani, l., vitagliano, c., 1998: changes in activity of antioxidative enzymes in wheat (triticum aestivum) seedlings under cold acclimation, physiol. plant. 104, 747-752. scebba, f., sebastiani, l., vitagliano, c., 1999: protective enzymes against activated oxygen species in wheat (triticum aestivum l.) seedlings: responses to cold acclimation. j. plant. physiol. 155, 762-768. seppänen, m.m., fagerstedt, k., 2000: the role of superoxide dismutase activity in response to cold acclimation in potato. physiol. plant. 108, 279-285. tomashow, m.f., 1998: role of cold-responsive genes in plant freezing tolerance. plant physiol. 118, 1-7. yoshida, s., uemura, m., 1990: responses of the plasma membrane to cold acclimation and freezing stress. in: larsson, c., mřller, i.m. (eds.), the plasma membrane: structure, function and molecular biology, 293-319. springer-verlag, berlin, heidelberg, yu, x., griffith, m., 1999: antifreeze proteins in winter rye leaves form oligomeric complexes. plant physiol. 119, 1361-1369. address of the authors: a. płażek, k. marzec, department of plant physiology, faculty of agriculture and economics, agricultural university of cracow, podłużna 3, 30-239 kraków, poland (the experiment was conducted in this institution) f. dubert, f. górski’ institute of plant physiology, polish academy of sciences, niezapominajek 21, 30-239 kraków, poland corresponding author: rrplazek@cyf-kr.edu.pl 162 a. płażek, f. dubert, k. marzec art09 journal of applied botany and food quality 82, 47 54 (2008) 1leibniz-institute for agricultural engineering potsdam-bornim, dept. horticultural engineering 2humboldt university berlin, section quality dynamics / postharvest physiology 3institute of vegetable and ornamental crops großbeeren / erfurt, dept. quality effects of saline irrigation on growth, physiology and quality of mesembryanthemum crystallinum l., a rare vegetable crop. w.b. herppich1, s. huyskens-keil2, m. schreiner3 (received december 11, 2007) summary world wide increased desertification due to recent global changes enhances the need of irrigation, which, in turn, provokes the risk of soil salinization. furthermore, limited fresh water resources may increasingly constrain the use of low quality irrigation water. hence, intensified use of halotolerant crop plants will be necessary, even in europe. commercial use of halophytes as fresh food is limited. several facultative halophytic members of aizoaceae are nowadays used as special crop plants. a rare leafy vegetable species is the common ice plant mesembryanthemum crystallinum, a crassulacean acid metabolism (cam) species, which is mostly cultivated in india, california, australia, and new zealand. it is also known in europe as a quickly cooked tender vegetable. with their succulent, mellow, slightly salty tasting leaves and young shoots, m. crystallinum is getting interesting as delicious cool flavored salad greens during recent years. however, it is a perishable product and thus, shelf live is short. on the other hand, cam capacity of m. crystallinum can be largely enhanced by saline irrigation. increased cam potentially reduces water and carbon losses. in this project we studied whether moderate salt treatment affects physiology, growth and yield of this rare crop plant. furthermore, we investigated whether such treatment that enhances the irreversible c3 to cam shift in young leaves of this cam species, potentially prolongs shelf live. results showed that moderate salt treatment did not negatively influence growth, yield and sensory quality. when in cam, leaves showed reduced transpiration water losses and cam also reduced carbon losses during storage. introduction recent models of global changes prognosticate a world wide dramatically increase in desertification (schneider et al., 2007). this is not only true for the arid and semi arid regions of the world. desertification may also spread throughout low land regions of europe that are now still well supplied with precipitation. even if the velocity of changes might by lower than predicted an enhanced need of irrigation seems to be unavoidable in many cases. this may further limit fresh water resources and increasingly constrains the use of low quality irrigation water (kundzewicz et al., 2007). combined with a higher evaporation demand this, in turn, may largely provoke the risk of soil salinization. hence, intensified use of halotolerant crop plants will be necessary, even in europe. however, many important food crop species are more or less sensitive to salinity stress (shannon and grieve, 1999). hence, there will be an increased demand for new crop species or improved varieties. conventional but also modern molecular based (e.g. marker assisted selection) plant breeding programs are normally slow and it is not clear whether the can reach the goals at all. the same applies to genetic engineering approaches because of the very complex nature of halotolerance (yamaguchi and blumwald, 2005). one relative rapid approach might be the development of naturally halotolerant plants into valuable crop species (glenn et al., 1999) or the increased cultivation of halotolerant crop species. in this context, commercial use of halophytes as fresh food is nowadays very limited. few exceptions are e. g. aster tripolium, batis maritime, crambe maritime, portulaca oleraceae, or salicornia bigelovvii (national research council, 1990; shannon and grieve, 1999; el-haddad and noaman, 2001). several facultative halophytic members of aizoaceae are nowadays used as special crop plants since many years. since approximately two hundred years tetragonia tetragonioides (pall.) o. kunze has been grown as a spinach-like vegetable in new zealand, australia, japan, india, california and europe (vogel, 1996). young leaves and tender shoots of this species can also be eaten as a fresh and tasty salad. however, in europe new zealand spinach lost most of its economic relevance to spinach during the last decades. as a convenience product deep frozen spinach (spinaca oleracea) is much easier to handle than new zealand spinach. although it contains some oxalate (ahmed and johnson, 2000) tetragonia has a low nitrate and a high vitamin c content. in contrast to new zealand spinach, aptenia cordifolia (l.f.) n.e.br., commonly called baby sun rose, is getting increasingly interesting as a delicious salad during recent years, even in germany (herppich et al., 2004). leaves of a. cordifolia contain small amounts of some soothing and bloodstream stimulating alkaloids such as mesembrin, mesembrinin und tortuosamin (smith et al., 1996). an old, but nowadays rare salad green is the common ice plant mesembryanthemum crystallinum (vogel, 1996). this species is an ephemeral or pseudo-biennial prostrate white flowering herbaceous weedy plant with simple, ovate, succulent leaves and stems which are densely covered with bladder cells. these cells give the plant a glistening appearance, and hence the species name „ice-plant“ (adams et al., 1998). it has been shown that plants of this species grew best under moderately saline conditions (winter, 1973). according to vogel (1996), m. crystallinum is mostly cultivated in india, california, australia, and new zealand. with their succulent, mellow, slightly salty tasting leaves and young shoots, this species is a delicious cool flavored salad green. in europe it is also known as a quickly cooked tender vegetable. sold in the delicatessen shops m. crystallinum can reach reasonable price in germany (e.g. 25 € per kg in a well-known berlin department store). however, this product is highly perishable and should be stored in a cool and humid atmosphere or sold packed in plastic bags. nevertheless, their shelf live is very short and may not extend 2 to 3 days (vogel, 1996). m. crystallinum is a cam plant (winter and von willert, 1972), i.e. it possesses the ability to fix internal or external co2 at night into organic acids, which are stored in the vacuoles. during the daytime organic acids are set free into the cytoplasm and decarboxylated to give free co2 which is than finally fixed in the normal c3 photosynthesis. this resulted in the well known diurnal pattern of co2 uptake which is mostly governed by the related changes in internal co2 concentration (ci). ci also controls stomatal opening. as a consequence, stomata are at least partially closed during daytime, largely reducing transpiration and, hence, water losses in cam plants. it has been shown that even mild salt treatment largely enhances the cam capacity of the ice plant (winter, 1973; herppich 48 w.b. herppich, s. huyskens-keil, m. schreiner et al., 1992; 1995; 1996; 1997). from the above it may be concluded that irrigation of m. crystallinum plants with saline water may stimulate growth and enhance cam capacity, and may, thus, increase the keeping quality of the harvested product. hence, in the presented project we studied whether moderate salt treatment affects physiology, growth and yield. furthermore, we investigated whether such treatment that enhances the irreversible c3 to cam shift in young leaves of this cam species (cushman et al., 1990; herppich et al., 1992), potentially prolongs shelf live. materials and methods plant material plants of mesembryanthemum crystallinum were grown from seeds on soil (fruhstorfer erde typ-p, industrie-erdenwerk-archut gmbh co. kg, lauterbach, germany) in a glasshouse under semi-controlled conditions (td/n = 18/14°c; rfd/n = 40/55%) and natural light (mean photosynthetic photon fluence rate (pfr) approximately 400 µmol m-2 s-1; max pfr < 1300 µmol m-2 s-1). nine days after germination seedlings were pricked out into plastic pots (ø 9 cm). at day 24 after germination plants were finally transplanted in large plastic pots („bc 29“; ø 28 cm, v = 10 l). plants were watered daily with tap water and regularly fertilized by top dressing (0.88 g ca(no3)2 and 0.25 g mgso4 per plant). ninety days after germination each plant was approximately 69 cm wide and 16 cm high. it developed 12 shoots with approximately 50 shoot tips. experimental design and methods on days 64 and 85 after germination 12 plants each were randomly selected for the presented experiments. six plants (+ nacl) were watered with nacl-solution (150 mmol l-1) every 2nd. the remaining six plants (nacl) were further irrigated with tap water. these 12 pots were placed in 4 rows, alternating salt-treated and untreated plant. on day 69 after germination the measurements were performed with the first set of plants. on days 90, 104 and 133 after germination plants of the second batch were used for the investigations. this means that in this second set leaves and shoot tips were repeatedly harvested as recommended for the practical use (vogel, 1996). free acidity for the analysis of the diurnal changes in the free acid content the largest fully developed leaves of each shoot of each plant (3 plants per treatment) were harvested early in the morning (approx. 7 am) and in the afternoon (approx. 3 pm). the leaves of each harvesting time were combined to give two mixed-samples per plant (approximately 15 g), weighed (fresh mass, fm), roughly chopped, mixed with 50 ml distilled water and finally homogenised for 2 min (20000 rpm, ultra-turrax, ika® werke gmbh & co. kg, staufen, germany). in a graduated flask the volume of the homogenate was replenished to 250 ml with distilled water, the mixture was shaken and filtered. finally, 50 ml of the filtrate was titrated with naoh solution (1 mol l-1) to a ph of 8.1. the free acid concentration (mmol l-1) of the samples (n = 6 per treatment) were calculated from the added volume of the naoh solution. leaf sodium content approximately 6 g of leaf material per plant was harvested in the morning and in the afternoon, weighed, and dried at 60°c in the oven for 48 h and re-weighed (dry mass, dm). afterwards the dry material was ground, dried at 85° for additional 4 h and then ashed at 500°c in a muffle kiln for 4 h. afterwards, the ash was dissolved in 5 ml water, mixed with 5 ml 6 mol l-1 hcl-solution and stirred for 30 min. the solution was brought to 100 ml with distilled water and filtered. the sodium concentration of the solution was determined with an atomic absorption spectrometer (vario 6, analytik jena, jena, germany). transpiration measurements approximately 100 g leaves and shoot tips per plant and treatment were harvested in the morning (7 am), weighed, and placed loosely into pre-weighed open containers. these containers were stored at 8°c and 70% relative air humidity (corresponding to a water vapour pressure deficit, ∆w, of 0.32 kpa mpa-1) in a climate chamber (type vb1014, vötsch industrietechnik gmbh, balingen-frommern, germany) for 3 days. every day, the containers with leaves were weighed at 08:45. from the mass losses over time and the respective initial fresh mass mean transpiration rates (jh2o) were calculated as mmol h -1 gfm -1. average leaf conductance (gh2o; mmol h-1 gfm-1) for water vapour was calculated as the ratio of jh2o and ∆w (von willert et al., 1995). co2 and o2 gas exchange for the determination of leaf gas exchange approx. 40 g of freshly harvested pre-weighed plant material per plant and treatment was sealed in an air-tight plastic container (18.5 cm x 15.5 cm x 9 cm). these containers were stored at 8°c in a climate chamber (type vb1014, vötsch industrietechnik gmbh, balingen-frommern, germany) for 3 days. each container was fitted with a standard septum through which gas samples (5 ml) were taken with a syringe every day at the same time (8 am). the co2 and o2 concentration of these samples were measured with a checkmate 900 head space gas analyser (pbi dansensor, ringsted, denmark). from the variation in the co2 and o2 concentration of the samples and the respective fresh mass the actual respiration rates jco2 and jo2 were calculated (mmol h-1 kg-1). furthermore, the respiratory quotient (rq) was determined as the ratio of jco2 and jo2. chlorophyll fluorescence analysis on days 88 to 94 d and 104 to 118 after germination photosynthetic activity of salt-treated and untreated plants was investigated by means of chlorophyll fluorescence analysis. using a minipam fluorometer (heinz walz gmbh, effeltrich; germany) the steady state fluorescence signals ft and f0, respectively, and the maximum fluorescence signals fm’ and fm were automatically (rec-modus) recorded under natural light conditions, i.e. without additional actinic irradiation, in 30-min intervals over several 24 h-periods. the 2030-b leaf-clip holder kept attached to the respective leaf for the whole measurement time without any visible damage to the leaf tissue. from the recorded fluorescence signal measured on illuminated leaves the actual photochemical efficiency of photosynthesis (φpsii) was calculated as φpsii = ∆f/fm’ = (fm’ ft) / fm’. the photosynthetic electron transport rate through photosystem ii (etr) was determined as etr = φpsii * ppfd * a * f (e.g. herppich et al., 1998). the absorptivity a of the succulent leaves in the visible range was taken as 0.68 (eller et al., 1983) and the light distribution factor between photosystem i and ii, f, was set to 0.5 (krall and edwards, 1992). in addition, non-photochemical quenching (npq), an indicator of total non-radiative dissipation of absorbed light energy, was determined as npq = (fm -fm’) / fm’ (bilger and björkman, 1990). the fluorescence signals recorded on dark adapted leaves (fm and f0) cam and postharvest quality of mesembryanthemum crystallinum 49 during the night were used to calculate the maximum photochemical efficiency of photosystem ii, fv/fm (= (fm f0)/ fm), which is an indicator of the potential maximum performance of photosynthesis. this parameter is also a valuable measure of the intactness of the photosynthetic apparatus (von willert et al., 1995). statistical analysis all data were statistically analysed (anova) with winstat (r. fitch software, staufen, germany). significant differences were determined by the duncan’s multiple range test (p < 0.05). the mean variability was indicated by the standard deviation. results biomass, nacl content and cam capacity saline irrigation of m. crystallinum affected growth only in plants older than 105 days (fig. 1a). up to this date salt-treated and wellirrigated plants showed exact the same increase in fresh mass. at the last harvest date however, fresh mass of salt-treated plants was significantly higher than that of well-watered. as expected, repeated moderate salt treatment resulted in significantly enhanced leaf na+ content (fig. 1b). the initial mean na+ content of leaves of well-watered plants (0.44 ± 0.03 mmol gdm-1) increased by only about 50% at the end of the experiment. intermediate watering with 150 mmol l-1 nacl-solution led to approximately 60% higher leaf na+ contents. in contrast, salt treatment did not significantly affect mean leaf water content (fig. 1c) or leaf dry matter content (fig. 1d). nevertheless, salinity-treated plants tended to have slightly higher water content at a lower dry mass content at the end of the experiment. irrespective of the treatment all plants showed a clear nocturnal accumulation of free acidity (∆h+) indicating that mature plants utilize cam under all conditions (fig. 2). intermediate saline irrigation only very slightly affected the capacity of the plants to perform cam. however, development of plants led to a significant increase in the maximum morning content of free acidity (fig. 2b). nevertheless, mean diurnal acid difference, i.e. cam capacity, as well as mean maximum morning content declined again with further aging in salt-treated plants of m. crystallinum. leaf conductance and respiration salt treatment significantly reduced the average leaf conductance for water vapour (fig. 3a) compared to well-irrigated plants. leaf conductance generally declined with plant development. however, this effect was more pronounced in well-watered plants finally leading to lower leaf conductance in these plants at the end of the harvest season. after 3 days of storage leaf conductance was low in all plants irrespective of the treatment. in this case the effect of plant development was no longer significant. the results of mean leaf conductance clearly reflected the fact that d 60 80 100 120 140 0 20 40 60 80 100 120 d ry m a ss c o n te n t (g g f m -1 )c 60 80 100 120 140 time after sowing (d) 0 5 10 15 20 w a te r c o n te n t (g g d m -1 ) b 0.0 0.2 0.4 0.6 0.8 1.0 1.2 n ac lc o n te n t (m m o l g d m -1 ) 0 50 100 150 200 250 300 f re s h m as s p e r p la n t (g ) a fig. 1: average (mean ± sd; n = 6) fresh mass (a), nacl content (b), water and dry mass content (c) of salt-treated (squares) and well-irrigated (triangles) plants of m. crystallinum during the entire growth period. 60 80 100 120 140 time after sowing (d) 0 20 40 60 80 100 120 h + c o n c e n tr a ti o n (m m o l l-1 ) fig. 2: free acid concentration of salt-treated (squares) and well-irrigated (triangles) plants of m. crystallinum (mean ± sd; n = 6) harvested in the morning (closed symbols) and in the afternoon (open symbols) during the entire growth period. 50 w.b. herppich, s. huyskens-keil, m. schreiner moderately salt treated plants lost significantly less water (approximately 20%) than well-watered plants, both during the initial and the late storage, except again at the last harvest date (fig. 3b). nevertheless, water losses generally declined by approximately 70% with plant development. both co2 release and o2 uptake of harvested leaves of m. crystallinum were always approximately 10 to 15% higher in well-watered than in salt-treated plants (fig. 4 a, b). gas exchange in general significantly declined during the 3 days of storage. nevertheless, this decline did not affect the effect of the treatment. the respiratory quotient (rq), a helpful indicator of the average respiratory source, of freshly harvested leaves was always significantly higher than 1 (1.10 ± 0.05; fig. 4c). during storage the rq declined significantly at the early harvest or in younger plants, respectively, when the quotient was slightly lower than 1 (0.97 ± 0.02). this effect was largely reduced and the rq of stored leaves increased during further plant development. no clear effect of the treatment could be detected. a comparison of co2 release and leaf conductance to water vapour revealed that the observed seemingly decline in leaf respiration during storage was nearly exclusively due to a reduced leaf conductance for gas transfer. both co2 release and leaf conductance were highly linearly related (fig. 5) in well-watered (r2 = 0.87) and salt-treated (r2 = 0.70) plants. this evaluation further illustrates that the reduction of co2 release with plant development also mainly resulted from a decline in leaf conductance with plant maturation. again, the initial 60 80 100 120 140 time after sowing (d) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 t o ta l w a te r lo s s (g k g f m -1 ) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 l e a f c o n d u c ta n c e (m m o l h -1 g f m -1 ) a b fig. 3: (a) average (mean ± sd; n = 3) leaf conductance for water vapour transfer and (b) total water loss of freshly harvested (open and closed bars) and 3 d-stored (dark grey and light grey bars) well-irrigated (closed and dark grey bars) and salt-treated (open and light grey bars) plants of m. crystallinum (i.e. closed bar = freshly harvested + well-irrigated; open bar = freshly harvested + salt-treated; light grey bar = 3 d-stored + salt-treated; dark grey bar = 3 d-stored + wellirrigated). 0.0 0.5 1.0 1.5 2.0 2.5 3.0 o 2 u p ta k e (m m o l h -1 k g f m -1 ) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 c o 2 re le a s e (m m o l h -1 k g f m -1 ) 60 80 100 120 140 time after sowing (d) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 r es p ir a to ry q u o ti e n t r q a b c fig. 4: average rates (mean ± sd; n = 3) of co2 release (a) and o2 uptake of freshly harvested (open and closed bars) and 3 d-stored (dark grey and light grey bars) well-irrigated (closed and dark grey bars) and salt-treated (open and light grey bars) plants of m. crystallinum (i.e. closed bar = freshly harvested + well-irrigated; open bar = freshly harvested + salt-treated; light grey bar = 3 d-stored + salt-treated; dark grey bar = 3 d-stored + well-irrigated). (c) means of the respiratory quotient as calculated from the rates of co2 release and o2 uptake. cam and postharvest quality of mesembryanthemum crystallinum 51 co2 release and leaf conductance for water vapour of samples harvested at different times after sowing were significantly linearly related (r2 = 0.87). nevertheless, at a given leaf conductance wellirrigated plants always exhibit a significantly higher co2 release and hence a higher respiration activity. furthermore, after 3 days of storage rates of co2 release and o2 uptake (data not shown) tended to decline without concomitant corresponding reduction in leaf conductance indicating a direct effect of storage on respiration. capacity in leaves of m. crystallinum without negatively affecting productivity. the presented results clearly indicated that repetitive irrigation with saline water did not negatively affect plant growth in m. crystallinum. in fact, in this facultative halophytic species the salt treatment applied even acts as a nacl-nutrition. our results are 0.0 0.5 1.0 1.5 2.0 leaf conductance (mmol h-1 gfm -1) 1.0 1.5 2.0 2.5 3.0 c o 2 re le a se (m m o l h -1 kg f m -1 ) du rat ion of sto rag e / 69 d / 90 d / 104 d / 133 d well-irrigated r2 = 0.87 open symbols salt-treated r2 = 0.70 closed symbols fig. 5: respiratory co2 release rates as a function of the respective leaf conductance for water vapour transfer of salt-treated (closed symbols) and well-irrigated (open symbols) plants of m. crystallinum as measured 69 (circles), 90 (squares), 104 (triangles) and 133 days after sowing. fig. 6: dependence of (a) photosynthetic electron transport rates, etr, and (b) non-photochemical quenching, npq, on incident photosynthetic active photon fluence rates of approximately 90 days old salt-treated (squares) and well-irrigated (triangles) plants of m. crystallinum. 0 200 400 600 800 1000 1200 photon fluence rate (µmol m-2 s-1) 0 1 2 3 4 n p q (r e l. u n it s ) 0 40 80 120 160 e t r (µ m o lm -2 s -1 ) a b photosynthesis determined 88 to 94 d after germination, salt-treated plants of m. crystallinum had significantly higher maximal mean fv/fm (0.809 ± 0.011 versus 0.786 ± 0.002) (data not shown). also, saturated photosynthetic electron transport rates (etr, measured at t = 26 ± 8 °c) of salt-treated plants were higher (107 ± 27 µmol m-2 s-1) than in well-watered (68 ± 24 µmol m-2 s-1). this was valid irrespective of the photosynthetic fluence rates during measurement (fig. 6a). the lower gross photosynthesis in the well-watered plants mainly resulted from a higher activity of the non-photochemical fluorescence quenching mechanisms (fig. 6b). these results fit very well with the higher cam capacity of salt-treated plants at this age. in contrast, investigated 104 to 118 d after germination, photosynthetic electron transport at a constant, and, according to the results given in fig. 6a, nearly saturating pfr of 368 ± 57 µmol m-2 s-1 was increased in well-watered plants (fig. 7) if compared with salt-treated plants at the same temperature range (115 ± 29 µmol m-2 s-1 versus 84 ± 14 µmol m-2 s-1). in the latter, etr started to decline above a threshold of 25°c while in well-watered plants gross photosynthesis nearly linearly increased with temperature. again these results reflect the analysis of cam capacity. during the later harvest period the salt-treated plants showed a lower nocturnal acid accumulation than well-watered plants and hence a smaller internal photosynthetic co2 source. discussion effects of intermittent low-salt treatment on cam capacity one major goal of this study was to show that even a practically meaningful intermittent low-salt treatment could enhance the cam 10 15 20 25 30 leaf temperature (˚c) 0 20 40 60 80 100 120 140 160 e t r (µ m o l m -2 s -1 ) r2 = 0.841 r2 = 0.801 fig. 7: photosynthetic electron transport rates, etr, as a function of leaf temperature of approximately 116 days salt-treated (squares) and well-irrigated (triangles) plants of m. crystallinum. 52 w.b. herppich, s. huyskens-keil, m. schreiner in accordance with those published by winter (1973). this author found that only nacl-concentrations above 300 mmol l-1 lead to a reduction of growth in hydroponically cultured plants of m. crystallinum, while maximum growth was obtained with a 100 mmol l-1 nacl-solution. furthermore, neither water content nor dry matter content seemed to be affected by the irrigation regime used in our experiments. hence, moderately saline or low quality irrigation water certainly does not exert any stress on but even increase the physiological activity and yield of this rare crop. this conclusion is further substantiated by the study of the photosynthesis using chlorophyll fluorescence analysis. photosynthetic performance, a sensitive indicator of general metabolic activity, was significantly higher in salt-treated plants when investigated about 90 d after sowing. this applied both for the maximum efficiency of photosystem ii, i.e. the potential capacity under optimal conditions (von willert et al., 1995; maxwell and johnson, 2000), and for the actual photosynthetic electron transport rates, i.e. that part of the maximum efficiency actually used by plants (herppich, 2001). this higher photosynthetic activity of salt-treated plants might be explained by a general physiological stimulation in response to salinity. it may also reflect increased chloroplastic carbon dioxide availability during times of higher irradiance due to the higher cam capacity and thus increased nocturnal acid accumulation of those plants. the presented results seemingly contrast those reported earlier (e.g. keiller et al., 1994; baker et al., 2004). those authors found a clear reduction of actual photosynthetic activity if plants of m. crystallinum were salt-treated. however, in these investigations nacl concentrations of 400 mmol l-1 were used which represent true stress conditions even for a halophytic plant (winter, 1973). on the other hand, maximum photochemical efficiency was not affected by the salt treatment, as reported by baker et al. (2004). nevertheless, baker et al. (2004) also reported that older leaves were more affected by the salt treatment than younger and denoted it to a probable acceleration of senescence. this interpretation would also nicely explain the finding in the presented study that in older plants salt-treated leaves showed a lower photosynthetic activity than un-treated. the applied intermittent salt treatment resulted in the expected increased nocturnal accumulation of free acid (∆-h+) and, hence, enhanced cam capacity. however, the obtained differences between the ∆-h+ of well-watered and salt-treated plants were not very pronounced. instead, well-watered plants also showed clear and distinct night time acid accumulation. as all these plants were always abundantly watered to yield high productivity this result unequivocally indicates that no environmental stress is necessary to induce the c3 to cam shift in fully developed plants of m. crystallinum. hence, our study confirmed earlier investigations (von willert et al., 1977; cushman et al., 1990; chu et al., 1991; herppich et al., 1992) postulating plant development being the crucial parameter controlling the ability of plants to perform cam. in contrast, winter and holtum (2007) recently concluded from „continuously monitoring net co2 exchange of whole shoots from the seedling stage until seed set“ that „the induction of the cam pathway for carbon acquisition in m. crystallinum is under environmental control“. this conclusion was based on whole-plant gas exchange measurements only. however, it is well established that the cam capacity in plants of m. crystallinum strictly depends on leaf age. hence, the large young still developing non-cam as well as senescent, out-of-cam leaves might have „diluted“ the weak nocturnal co2 uptake of mature leaves. furthermore, nocturnal accumulation of organic acid is the decisive feature making up cam at all while night-time co2 uptake is an important but secondary pattern. it is well known that cam gas exchange can be easily shifted to what is called a typical c3 pattern without clearly affecting night time acid accumulation in flexible cam plants (herppich, 1997; de mattos and lüttge, 2001). the relatively small partial increase in cam capacity due to the salt treatment may also contrast the popular hypothesis that it is the indirect drought stress resulting from a nacl mediated lower water availability which intensifies cam in m. crystallinum (piepenbrock and schmitt, 1991; winter and gademann, 1991). the mean water content of salt-treated plants tended to be higher than in well-irrigated plants in nearly all cases. furthermore, early morning leaf water content was slightly higher than that of the afternoon samples in most plants irrespective of the treatment (data not shown). this may reflect some transpiration water loss or the enhance ability to take up water as a result of the cam induced increase in osmotic content (herppich, 2004). hence, the presented results support the thesis that cam induction in well-irrigated and cam enhancement in salttreated plants was not due to any physiological drought stress (herppich and herppich, 1997b). interestingly, salt treatment enhances cam only in plants younger than 133 d. in fact, nocturnal acid accumulation, and, hence, cam capacity was significantly reduced in salt-treated m. crystallinum plants at the end of the harvest season. an acceleration of plant development by the salt-treatment untimely leading to senescence would be a plausible explanation. nevertheless, herppich et al. (1992) found that in young, still developing plants salt stress (300 mmolnacl l -1) reversibly retarded development in this species. this was evident when salt stress was relieved from those plants at different stages of life cycle. hence, low-salt treatment of mature plants as used in our study might show different effects than in young still developing plants. effects of intermittent low-salt treatment on keeping quality the second aim of the presented investigation was to proof that a higher cam capacity can improve the keeping quality of harvested leaves of the salad green m. crystallinum under practical low temperature (8°c) conditions thereby simulating sale conditions in cooled, mist-equipped display cases or short-term storage in cold rooms. the results of our postharvest gas exchange measurements conclusively show that this was indeed the fact. plants with a higher ∆-h+ had always lower values of leaf conductance. hence, these plants suffered lower water losses, both at the beginning and at the end of a three-day storage period. as this was also valid when wellirrigated plants showed the higher cam capacity at the last harvest date, the reduced stomatal conductance was not an effect of salt treatment as might be concluded from earlier studies (winter and gademann, 1991; dai et al., 1994). in contrast, it was directly linked to cam capacity. however, as was the magnitude of ∆h+, leaf conductance and water losses were strongly dependent on plant age largely declining with progressing development. during storage, the differences in leaf conductance between treatments declined. at the third day after harvest stomata seemed to be more or less completely closed. hence, the remaining mean value of leaf conductance of 0.22 ± 0.06 mmol kg-1 h-1 nearly represented the water vapour conductance of the cuticle. assuming a degree of succulence (von willert et al., 1995) of 4 gh2o dm-2 (herppich et al., 1992) this value equals a leaf conductance of approximately 0.05 mmol m-2 s-1. although this value is very low (approx. 10 times lower) if it is compared with mesophytic c3 plants (von willert et al., 1995), it lies well in the range of cuticular conductance reported for other succulents (herppich, 1997). the observed reduction of leaf conductance during postharvest storage and during plant development largely affected co2 and o2 gas exchange. in fact, the close linear relationship between these parameters clearly indicated that most of the variation in co2 loss and o2 uptake was not due to a direct reduction of mitochondrial activity by water loss but simply resulted from the increase in diffusion resistance. this effect was obviously independent of the cam and postharvest quality of mesembryanthemum crystallinum 53 treatment. nevertheless, the evaluation of the data indicated that wellwatered plants showed higher co2 release rates at a given level of leaf conductance. thus, this may be due to their lower cam performance resulting in a lower capacity to retain respiratory co2 by a re-fixation into malic and/or citric acid (herppich, 2004). since o2 uptake is also higher it is more probably that well-watered plants exhibit higher respiratory activity. however, this seems to contrast the finding that the oxidative capacity of isolated mitochondria showed the tendency to increase with the shift from c3 to cam in m. crystallinum (peckmann et al., 2004). on the other hand, wellwatered plants had already developed a significant cam capacity very similar to that of salt-treated plants in our study. furthermore, although respiration activity of seedlings of this species was reported to increase at higher nutrient solution salinity (von willert, 1974), low salt concentrations (50 mmolnacl l -1) resulted in an intermitted reduction of respiration. also, koyro (2006) found that dark respiration declined with increasing nacl-salinity in the halophytic cash crop plantago coronopus. hence, it is not clear whether the reduction of dark respiration with the salt-treatment as observed in our study is due to cam induction or is a nacl-effect. nevertheless, our data show that intermittent salt-treatment can indeed significantly reduce the postharvest carbon loss and thus, increase keeping quality of this highly perishable produce. furthermore, we can speculate that a generally increased cam with its higher ability to refix mitochondria derived co2 in the detached harvested leaves could be the reason for the reduced carbon loss at the end of the storage period. the unavoidable water losses after detaching lead to drought stress, which, in turn, has been shown to amplify cam capacity (piepenbrock and schmitt, 1991), sometimes interpreted as drought-induction of cam (piepenbrock et al., 1994). the given assumption may be further substantiated by the fact that the reduction of carbon loss is not fully reflected by the same variation in o2 uptake rates leading to a significantly reduced rq with means less than 1, at least in younger plants. although we can not exclude that the change in the rq were due to a reduced availability of organic acids in storage, this hypothesis seems to be not highly probable in m. crystallinum (piepenbrock and schmitt, 1991; piepenbrock et al., 1994; adams et al., 1998). nevertheless, a rq above 1 observed under most conditions may indicate that malic and citric acid are preferred substrates of dark respiration. an important quality aspect that might be influenced by the cultivation method is taste, especially affected by the salt content. as a facultative halophytic species plants of m. crystallinum might accumulate large amounts of nacl in their leaves and shoot if they are salt-treated (herppich et al., 1992; adams et al., 1998). high salt content is highly undesired and might render the plants useless for human consumption. however, a mean na+-content of 0.81 mmol gdm-1 in salt-treated and 0.50 mmol gdm-1 in well-watered plants was very low and a daily uptake up to 150 mmol nacl is quite safe within the human diet (elmadfa and leitzmann, 1998). these values lie in the range reported for soil grown plants of m. crystallinum watered with tap water (herppich et al., 1992). the sodium content found in leaves of salt-treated plants in this investigation is, thus, approximately 10 times less than found earlier if plants were continuously irrigated with a 300 mmol l-1 nacl-solution (herppich et al., 1995). furthermore, the effect of salt-treatment on sensorial quality of m. crystallinum plants used in these experiments was also evaluated by a small, untrained consumer panel. the panelists indicated no negative impact such as an unacceptable salty or stringent taste (data not shown). conclusion in general, the knowledge of preharvest treatments affecting postharvest physiology and quality dynamics is an essential tool for a comprehensive product physiological oriented quality management. the results of this study provide clear evidence that intermittent lowsalt treatment was effective to prevent quality loss and to optimize quality assurance of m. crystallinum leaves due to the enhancement of cam capacity. furthermore, the presented results clearly show that fully-developed well-watered, soil-grown plants of m. crystallinum do not need any stress to induce cam. references adams, p., nelson, d., yamada, s., chmara, w., jensen, r.g., bohnert, h.j., griffiths, h., 1998: transley review no. 97. growth and development of mesembryanthemum crystallinum (aizoaceae). new phytol. 138, 171-190. ahmed, a.k., johnson, k.a., 2000: the effect of the ammonium: nitrate nitrogen ratio, total nitrogen, salinity (nacl) and calcium on the oxalate levels of tetragonia tetragonioides pallas. kunz. j. hortic. sci. biotechnol. 75, 533-538. barker, d.h., marszalek, j., zimpfer, j.f., adams, w.w. iii, 2004: changes in photosynthetic pigment composition and absorbed energy allocation during salt stress and cam induction in mesembryanthemum crystallinum. funct. plant biol. 31, 781-787. bilger, w., björkman, o., 1990: role of the xanthophyll cycle in photoprotection elucidated by measurements of light-induced absorbance changes, fluorescence and photosynthesis in leaves of hedera canariensis. photosynth. res. 25, 173-185. chu, c., dai, z., ku, m.s.b., edwards, g.e., 1990: induction of crassulacean acid metabolism in the facultative halophyte mesembryanthemum crystallinum. plant physiol. 93, 1253-1260. cushman, j.c., michalowski, c.b., bohnert, h.j., 1990: developmental control of crassulacean acid metabolism inducibility by salt stress in the common ice plant. plant physiol. 94, 1137-1142. dai, z., ku, m.s.b., zhang, d., edwards, g.e., 1994: effects of growth regulators on the induction of crassulacean acid metabolism in the facultative halophyte mesembryanthemum crystallinum l. planta 192, 287-294. de mattos, e.a., lüttge, u., 2001: chlorophyll fluorescence and organic acid oscillations during transition from cam to c3-photosynthesis in clusia minor l. 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d-14469 potsdam, germany, wherppich@atb-potsdam.de s. huyskens-keil, humboldt-universität zu berlin, section quality dynamics/postharvest physiology, lentzeallee 75, d-14195 berlin, germany m. schreiner, institute of vegetable and ornamental crops großbeeren/erfurt, dept. quality, theodor-echtermeyer-weg 1, d-14979 großbeeren, germany << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedjoboptions true /dscreportinglevel 0 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/pagesize [907.087 680.315] >> setpagedevice art14_golier.indd journal of applied botany and food quality 85, 216 -223 (2012) department of agriculture, forest and food sciences, university of torino, grugliasco (to), italy changes in pasture and cow milk compositions during a summer transhumance in the western italian alps a. gorlier, m. lonati, m. renna, c. lussiana, g. lombardi, l.m. battaglini (received july 22, 2012) summary the changes occurring in pasture and milk compositions during summer grazing were studied following a transhumance of a dairy cattle herd in the western italian alps. during three consecutive grazing periods (p1, p2, and p3) the cows exploited, in sequence, mountain pastures located at 1200-1260 m a.s.l. (a1), alpine pastures at 2000-2200 m a.s.l. (a2), and then returned to a1 pastures. the botanical and nutritional compositions of pastures, as well as cow milk yield, gross composition and fatty acid (fa) profi le were assessed during the transhumance. within the pastures, a cluster analysis allowed the recognition of fi ve vegetation types and seven vegetation sub-types; their allocation and plant species composition differed among the exploited grazing areas. the average pastoral values were signifi cantly higher in the mountain (a1p1, a1p3) than in the alpine pastures (a2p2) due to the abundance of highand medium-quality forage species such as dactylis glomerata l., polygonum bistorta l., and festuca rubra s.l.. nevertheless, the nutritional quality of the herbage offered to the animals did not differ between a1p1 and a2p2, while it was signifi cantly higher in a1p3 due to a younger vegetation phenological stage. the nutritional parameters were found to be correlated to the pasture botanical composition and phenology: organic matter digestibility and net energy for lactation were correlated negatively to the phenological stage and the specifi c contribution (sc) of poaceae and positively to the sc of fabaceae and asteraceae. milk yield signifi cantly declined while milk protein increased during the grazing season, following the advance of cows’ stage of lactation. milk fat and lactose percentages did not vary signifi cantly among the grazing periods. the same was also observed for milk fa, with the exception of palmitic acid, whose level was lower in a2p2 if compared to the other two periods. signifi cant correlations were found between the percentages of some fa in milk and the sc of the main botanical families of the grazed pastures. in particular, linoleic acid was negatively correlated with the sc of poaceae and positively correlated with the sc of fabaceae. results showed that the changes in the nutritional composition of pastures depended on variations in pasture botanical composition and phenology at the time of grazing, and that such factors concurred with animal-related factors in affecting milk quality during the grazing season. introduction in the alps, semi-natural pastures are the main feed sources for ruminants during summer. they are traditionally exploited according to a vertical transhumance, following their gradual availability at growing altitudes (dodgshon and olsson, 2007). in recent years, semi-natural pastures have also become a key factor for the traceability and the quality of dairy products (martin et al., 2005). this added value arises from the consideration of the multiple effects of grazing on animal performance. milk yield and composition are affected by a combination of factors including diet (sutton, 1989) and animal-related aspects (coulon et al., 1991). nevertheless, when animals graze, additional variables infl uence milk characteristics. botanically diverse pastures are known to confer peculiar organoleptic and nutritional qualities to milk compared with either conserved forages or cereal-based concentrate feeds. in particular, dairy products obtained from grass-fed ruminants are very rich in benefi cial fatty acids (fa), such as polyunsaturated fatty acids (pufa) which have some interest for their positive effects on human health (van dorland et al., 2006). furthermore, there is evidence that grazing in alpine areas per se can affect milk fat composition as well. severe environmental conditions due to altitude, topography, and climate, along with the frequently unstable nutritional quality of herbage, lead to animals’ increased energy requirements and physiological changes (i.e., increased body fat mobilisation, ruminal ecosystem alterations) which have some bearing on milk production (zemp et al., 1989; leiber et al., 2004). alpine pasture types (jouglet et al., 1992; cavallero et al., 2007) and the factors determining changes in their nutritional and chemical compositions (jeangros et al., 1999; bovolenta et al., 2008) have been described in literature. similarly, the nutritional aspects and the chemical composition of milk produced in alpine areas have been well deepened (collomb et al., 2001; collomb et al., 2002a). several studies also aimed at understanding the relationships existing between alpine pastures and milk quality during grazing (jeangros et al., 1997; buchin et al., 1999; collomb et al., 2002b; de noni and battelli, 2008). however, to date, only few of these studies investigated the compositional changes in both pastures and milk following a dairy herd under the usual farming conditions of a transhumance and providing at the same time a full description of pastures’ botanical composition (buchin et al., 1999; de noni and battelli, 2008). by contrast, given that such movements of herds are widespread in all european mountain areas and considered the increasing consumers’ interest in traditional dairy products, it should be important to determine which factors usually affect pasture and milk quality in natura. in particular, understanding the effects of changes in pasture botanical composition and timing of grazing on pasture nutritional composition could help in improving grazing management during summer and, consequently, animal performance and the quality of milk and derived products (elgersma et al., 2006). therefore, the goals of this study were: i) to assess the botanical and nutritional compositions of semi-natural pastures grazed during a transhumance in the western italian alps, ii) to assess yield, gross composition, and fa profi le of the milk produced by the herd, and iii) to determine which factors led to changes in pasture and milk compositions during the transhumance. materials and methods study sites and experimental design this trial was carried out in the mont avic natural park (aosta valley, northwest italy) during an entire summer grazing season. a dairy farm breeding a herd of thirty-six lactating aosta red pied cows was selected as representative of the traditional pasture-based pasture and cow milk quality changes during a summer transhumance 217 farming system typical of this alpine area. all cows were multiparous, in mid lactation (176 ± 30 days in milk at the beginning of the trial), and adapted to the alpine environment. their feed source was limited to fresh grass from pastures. from the beginning of june to the end of july (p1), the herd exploited mountain pastures located at 1200-1260 m a.s.l. (a1). in august (p2), the cows moved to alpine pastures located at 2000-2200 m a.s.l. (a2), while in september (p3) they returned to the mountain pastures already exploited at the beginning of the trial (a1). thus, during the grazing season three experimental periods were identifi ed (a1p1, a2p2, and a1p3) (tab. 1). the cows were managed according to a rotational grazing system, with paddocks grazed in sequence both in the mountain and alpine areas. the surface of paddocks and the stocking periods ranged respectively from 0.4 to 1.6 hectares and from 3 to 8 days depending on vegetation composition, herbage biomass, and site conditions (e.g., topography, stone cover). vegetation surveys pasture botanical composition was surveyed using a vertical pointquadrat method (daget and poissonet, 1969) along 25 m transects laid out on representative and homogeneous areas. twenty-two surveys were performed before exploitation (a1p1 and a2p2). five surveys were repeated in a1 during the regrowth period (p3). all plant species were identifi ed (pignatti, 1982) and their relative cover was computed as specifi c contribution (sc) percentage (daget and poissonet, 1969). vegetation pastoral values (pv) were determined according to daget and poissonet (1972). vegetation sampling and analysis representative mixed grass samples (500 g) were collected at each grazing area to determine the herbage nutritional composition (tab. 1). sampling was carried out two days before exploitation following herd movements within the grazing areas. the phenological stage of the most abundant species was recorded using the lambertin’s schedule (lambertin, 1990) and 1×1 m2 area of each pasture was harvested to assess the herbage biomass (t dm ha-1). after collection, the samples were immediately oven-dried at 60°c for 48 hours and ground to pass a 1-mm screen using a cyclotech mill (tecator, höganäs, sweden). the samples were analyzed for dry matter (dm) (930.15; aoac, 2000), crude protein (cp) (978.04; aoac, 2000), neutral detergent fi bre (ndf), acid detergent fi bre (adf), acid detergent lignin (adl) (van soest et al., 1991), organic matter digestibility (omd) (aufrère, 1982), and ash (930.05; aoac, 2000). the net energy for lactation (nel) was calculated on the basis of the inra energy system of feedstuffs evaluation (jarrige, 1988). milk sampling and analysis milk yield recording and milk sampling started in a1p1 after a threeweek period provided to allow cow rumen adaptation to summer grazing. in a2p2 and a1p3 milk yield recording and milk sampling started fi ve days after the entrance in each grazing area to support rumen adaptation to vegetation types (tab. 1). milk samples to be analyzed for their gross composition were immediately transported to the laboratory in a portable refrigerator at 4°c, while the samples destined to the assessment of the fa composition were frozen until analyzed. milk fat, protein, and lactose percentages were assessed by infrared spectroscopy (milkoscan 4000, foss electric, hillerød, denmark). for fa analysis, milk fat extraction was obtained by centrifugation at 8000 g for fi ve minutes at -4°c. the fatty acid methyl esters (fame) were prepared by esterifi cation (aoac, 2000) and determined by using a gaschromatograph (perkin-elmer p-e 8700, perkin-elmer co., norwalk, ct, usa) equipped with a db-wax (j&w scientifi c inc., folsom, ca, usa) capillary column (60 m × 0.53 mm id, 1.0 µm tab. 1: features of the grazing season and experimental design. dairy herd 36 aosta red pied cows grazing periods june 1st july 27th july 28th august 31st september 1st october 6th codes p1 p2 p3 grazing areas mountain pastures alpine pastures mountain pastures codes a1 a2 a1 coordinates 45°41’n, 7°36’e 45°39’n, 7°35’e 45°41’n, 7°36’e altitude (m a.s.l.) 1200-1260 2000-2200 1200-1260 surface (ha) 9.0 9.2 6.9 notes fi rst grazing event fi rst grazing event second grazing event grazing management system rotational grazing (paddocks) fodder fresh grass from pasture milking system by hand (shed) surveys and samplings a1p1 a2p2 a1p3 botanical surveys on the fi rst grass growth, before the fi rst exploitation on the regrowth, before the second exploitation number 6 16 5 vegetation sampling 2 days before grazing number 7 5 4 milk sampling 3 weeks after moving 5 days after moving into the grazing area into the grazing area number 7 7 7 218 a. gorlier, m. lonati, m. renna, c. lussiana, g. lombardi, l.m. battaglini fi lm thickness). the column temperature was held at 180°c for one minute and then raised 4°c min-1 to a fi nal temperature of 225°c, where it remained for 45 minutes. the injector was maintained at 250°c and the fl ame ionization detector (fid) at 270°c. peaks were identifi ed by comparing their retention times with pure fame standards (restek corporation, bellefonte, pa, usa; matreya inc., pleasant gap, pa, usa). milk fa composition was expressed as a percentage of each individual fa per total fa detected. statistical analysis a two-level classifi cation system, based on pasture types and subtypes, was used to describe pasture vegetation (cavallero et al., 2007). to identify homogeneous vegetation groups – in terms of species composition – botanical data from the fi rst grass growth (a1p1 and a2p2) were classifi ed by cluster analysis performed using the sc (©clustan graphics 5.27 software; wishart, 1987). the similarity matrix was calculated using pearson’s correlation, while between-groups linkage was selected as agglomeration method. one-way anova was used to compare the characteristics of pastures (i.e., sc of the main botanical families, phenological stage, pv, herbage biomass), the nutritional composition of herbage, yield, gross composition, and fa profi le of milk among the three experimental periods. the assumption of equal variances was assessed by levene’s homogeneity of variance test. the bonferroni post-hoc test was performed to test the difference between each pair of means. correlations between the herbage nutritional parameters, milk quality, and vegetation attributes (i.e., sc of the main botanical families, phenological stage) were calculated using the pearson’s correlation test. statistical analyses were performed using ©spss (2007). signifi cance was declared at p<0.05. results botanical composition and attributes of pastures a total of 145 plant species belonging to 38 botanical families were identifi ed in the study area. five vegetation types and seven vegetation sub-types were recognized (fig. 1). the botanical composition of types and sub-types is shown in tab. 2. the average herbage biomass, pv, and sc of the main botanical families in the three experimental periods are given in tab. 3. poaceae, fabaceae, asteraceae, polygonaceae, and cyperaceae were the most widespread families in the pastures (78% of sc). the abundance of poaceae and cyperaceae did not differ signifi cantly among the grazing areas. fabaceae and polygonaceae species were signifi cantly more abundant in the mountain (a1p1 and a1p3) than in the alpine pastures (a2p2), while asteraceae species were signifi cantly more abundant in a1p3 with respect to both a1p1 and a2p2 pastures (tab. 3). overall, dicotyledonous species sc were signifi cantly higher (p<0.05) in the mountain regrowth (60.9% in a1p3) than in the alpine pastures (37.0% in a2p2). the mountain pastures had signifi cantly higher pv than the alpine pastures. in fact, in the a1p1 area, vegetation sub-types with higher pv ascribable to type polygonum bistorta and type festuca rubra s.l. were dominant (about 77% of total a1 surface), while sub-types with lower pv ascribable to type bromus erectus were confi ned to steep heat slopes covering few areas (about 23% of surface). by contrast, in the a2p2 pastures, sub-types with lower pv (type nardus stricta and type carex sempervirens) were more widespread (about 80% of total a2 surface) than those with higher pv (type festuca rubra s.l.; about 20% of surface). by comparing a1p1 and a1p3, the mountain pastures were shown not to have undergone signifi cant changes in their pv and their botanical composition did not differ with the exception of asteraceae abundance. however, the amount of herbage biomass was signifi cantly lower at the regrowth stage than at the beginning of the season. nutritional values of pastures the phenological stage and nutritional values of pastures during the three experimental periods are shown in tab. 3. the growth stage of pastures differed signifi cantly among the three periods. on average, pastures were grazed by the cows at the full fl owering stage in a1p1, at the end of the fl owering stage in a2p2, and at the beginning of the heading stage in a1p3. dm percentages did not differ among the experimental periods; fig. 1: dendrogram of the vegetation data obtained through cluster analysis. for the botanical composition of vegetation types and sub-types refer to tab. 2. the survey codes refer to areas (a1 and a2) and to periods (p1 and p2) of the study. pasture and cow milk quality changes during a summer transhumance 219 t ab . 2 : b ot an ic al c om po si ti on o f th e ve ge ta ti on t yp es a nd s ub -t yp es i n th e m ou nt ai n (a 1p 1) a nd a lp in e (a 2p 2) p as tu re s, a nd b ot an ic al c om po si ti on o f a 1p 3 m ou nt ai n pa st ur es a ft er t he fi r st e xp lo it at io n. t he m ea n (x ) an d s ta nd ar d d ev ia ti on ( s d ) of s pe ci fi c c on tr ib ut io ns a re g iv en f or t he t en m os t ab un da nt s pe ci es o f su bty pe s (t he s pe ci es h av in g cu m ul at iv e s c > 25 % a re i n bo ld f on t) . f lo ri st ic n om en cl at ur e fo ll ow s p ig na tt i (1 98 2) . a re a /p er io d m ou n ta in p as tu re s – a 1p 1 (1 20 012 60 m a .s .l .) m ou n ta in p as tu re s – a 1p 3 (1 20 012 60 m a .s .l .) of g ra zi n g ty p e 1 p ol yg on u m b is to rt a 2 b ro m u s er ec tu s 3 f es tu ca r u br a s. l. s u b -t yp e co d e 1. 1 2. 1 3. 1 x s d x s d x s d † x s d x s d p ol yg on u m b is to rt a 12 .4 1. 4 b ro m u s er ec tu s 26 .7 9. 0 t ri se tu m fl a ve sc en s 13 .8 t ar ax ac u m o ffi c in al e 12 .9 3. 4 t ar ax ac u m o ffi c in al e 23 .9 6. 4 a gr os ti s te n u is 11 .3 7. 3 f es tu ca r u b ra s .l . 9. 7 3. 6 f es tu ca r u br a s. l. 13 .4 t ri se tu m fl a ve sc en s 12 .0 5. 3 f es tu ca r u br a s. l. 9. 5 7. 2 t ar ax ac u m o ffi c in al e 9. 6 4. 1 c a re x p a ll es ce n s 5. 9 8. 4 c a re x ca ry o p h yl le a 7. 7 p ol yg on u m b is to rt a 11 .0 2. 3 d a ct yl is g lo m er a ta 6. 7 5. 1 d a ct yl is g lo m er a ta 7. 2 3. 3 l o tu s co rn ic u la tu s 4. 5 0. 8 b ro m u s er ec tu s 6. 9 t ri fo li u m r ep en s 8. 4 1. 9 p o ly g o n u m b is to rt a 6. 7 7. 6 t ri fo li u m p ra te n se 7. 0 9. 7 t ri se tu m fl a ve sc en s 3. 8 5. 4 l u zu la c a m p es tr is s .l . 6. 1 d a ct yl is g lo m er a ta 7. 9 4. 6 h o lc u s la n a tu s 6. 4 4. 5 r u m ex a ce to sa 5. 1 3. 9 a g ro p yr o n r ep en s 3. 1 4. 5 a ch il le a m il le fo li u m 5. 7 a g ro st is t en u is 7. 0 4. 9 a ch il le a m il le fo li u m 6. 3 2. 5 t ri se tu m fl a ve sc en s 5. 0 2. 3 a ch il le a m il le fo li u m 3. 1 4. 3 l a th yr u s p ra te n si s 3. 7 r a n u n cu lu s a cr is 6. 7 1. 1 t ri fo li u m p ra te n se 6. 2 6. 6 a n th o xa n th u m o d o ra tu m 4. 9 7. 8 o n o n is s p in o sa 2. 8 3. 9 r a n u n cu lu s bu lb o su s 3. 7 a ch il le a m il le fo li u m 5. 6 3. 7 l o li u m p er en n e 5. 2 3. 8 a ch il le a m il le fo li u m 4. 8 3. 6 h ie ra ci u m p il o se ll a 2. 8 3. 9 l o tu s co rn ic u la tu s 3. 3 g a li u m m o ll u g o 4. 3 3. 9 p la n ta g o l a n ce o la ta 3. 7 1. 5 r a n u n cu lu s a cr is 3. 7 1. 3 b ra ch yp o d iu m r u p es tr e 2. 7 1. 7 p la n ta g o l a n ce o la ta 2. 4 l a th yr u s p ra te n si s 3. 9 4. 5 b ra ch yp o d iu m r u p es tr e 3. 5 3. 0 a re a / p er io d of g ra zi n g a lp in e p as tu re s – a 2p 2 (2 00 022 00 m a .s .l .) ty p e 3 f es tu ca r u br a s. l. 4 n ar du s st ri ct a 5 c ar ex s em pe rv ir en s s u b -t yp e co d e 3. 1 3. 2 4. 1 4. 2 5. 1 x s d † x s d x s d x s d x s d f es tu ca r u br a s. l. 22 .0 f es tu ca r u br a s. l. 18 .2 4. 5 n ar du s st ri ct a 22 .4 7. 8 a n th ox an th u m a lp in u m 11 .8 3. 6 c ar ex s em pe rv ir en s 16 .1 6. 2 t ri se tu m fl a ve sc en s 13 .0 n ar du s st ri ct a 15 .3 6. 1 c ar ex f u sc a 22 .0 3. 9 n ar du s st ri ct a 11 .6 5. 9 p la n ta go s er pe n ti n a 15 .0 4. 8 p o a a lp in a 12 .4 p h le u m a lp in u m 14 .3 9. 8 a n th o xa n th u m a lp in u m 11 .4 7. 1 f es tu ca o vi n a s. l. 10 .3 2. 0 l eo n to d o n h el ve ti cu s 14 .1 3. 8 t ri fo li u m p ra te n se 10 .7 p o a a lp in a 10 .6 9. 3 l eo n to d o n h el ve ti cu s 6. 3 5. 9 g eu m m o n ta n u m 8. 1 2. 2 n a rd u s st ri ct a 6. 5 5. 8 a g ro p yr o n r ep en s 10 .2 c a re x fu sc a 3. 8 6. 0 c a re x st el lu la ta 5. 8 4. 7 c a re x se m p er vi re n s 5. 9 6. 6 f es tu ca o vi n a s .l . 5. 3 2. 2 p o ly g o n u m b is to rt a 6. 8 g eu m m o n ta n u m 3. 6 3. 9 f es tu ca o vi n a s .l . 4. 4 6. 7 p o a a lp in a 5. 8 2. 7 a g ro st is a lp in a 4. 9 5. 7 l eo n to d o n h el ve ti cu s 4. 0 a n th o xa n th u m a lp in u m 3. 5 4. 5 v io la b ifl o ra 3. 9 2. 6 p o te n ti ll a g ra n d ifl o ra 4. 7 5. 6 p o a a lp in a 3. 2 2. 5 a n th o xa n th u m a lp in u m 3. 4 t ri fo li u m p ra te n se 2. 7 3. 4 p o a a lp in a 3. 1 4. 0 p la n ta g o s er p en ti n a 4. 6 3. 3 s o ld a n el la a lp in a 3. 0 3. 0 p o te n ti ll a g ra n d ifl o ra 3. 4 s o ld a n el la a lp in a 2. 7 3. 2 s o ld a n el la a lp in a 2. 6 3. 5 l eo n to d o n h el ve ti cu s 4. 0 4. 2 c a m p a n u la s ch eu ch ze ri 2. 7 2. 4 p h le u m a lp in u m 2. 3 p o te n ti ll a g ra n d ifl o ra 2. 5 2. 4 t ri ch o p h o ru m c a es p it o su m 2 .4 3. 0 a g ro st is t en u is 2. 7 3. 3 a n th o xa n th u m a lp in u m 2. 5 1. 4 † s ub -t yp es i nc lu di ng o nl y on e su rv ey . 220 a. gorlier, m. lonati, m. renna, c. lussiana, g. lombardi, l.m. battaglini however, all other nutritional parameters varied signifi cantly during the season. most of them did not differ between a1p1 and a2p2, but the pasture quality was signifi cantly higher in a1p3, as demonstrated by the higher cp, omd, and nel and the lower ndf. the phenological phase of plants was signifi cantly correlated with all considered nutritional parameters, with the exception of the adf and adl percentages: it was positively correlated to dm and ndf percentages and negatively to cp, nel, and omd (tab. 4). many nutritional parameters were also correlated to the sc of poaceae, fabaceae, and asteraceae. in particular, nel and omd were found to be correlated negatively to poaceae sc and positively to asteraceae and fabaceae sc (tab. 4). milk yield, gross composition and fatty acid profi le average bulk milk yield, gross composition and fa profi le in the three grazing periods are shown in tab. 5. during the transhumance, a decrease in milk yield was observed, specifi cally from 380 kg herd-1 day-1 at the fi rst sampling in june to 60 kg herd-1 day-1 at the last sampling in october. average milk yield signifi cantly differed among the three experimental periods, being about 50% lower both in a2p2 compared to a1p1 and in a1p3 compared to a2p2. the protein percentage was signifi cantly lower in a1p1 with respect to a2p2 and a1p3. the percentage of fat showed higher values in a2p2 and a1p3 if compared to a1p1 but such variations were not signifi cant. the lactose percentage did not vary signifi cantly tab. 3: attributes and nutritional values of the pastures grazed by the herd during the three experimental periods. area / period of grazing a1p1 a2p2 a1p3 sem inter-area / period bonferroni post-hoc signifi cant comparisons differences† at p < 0.05‡ pasture attributes herbage biomass (t dm ha-1) 3.3 3.0 1.8 0.167 f = 27.605*** a1p1 vs a1p3 a2p2 vs a1p3 phenological stage§ 400.7 497.2 212.5 29.713 f = 42.310*** a1p1 vs a2p2 a1p1 vs a1p3 a2p2 vs a1p3 pastoral value 33.3 20.4 45.4 2.614 f = 15.708*** a1p1 vs a2p2 a2p2 vs a1p3 poaceae (%) 43.2 47.2 38.3 2.593 f = 0.905 fabaceae (%) 11.1 3.0 11.9 1.376 f = 6.892** a1p1 vs a2p2 a2p2 vs a1p3 asteraceae (%) 11.3 7.8 25.6 1.862 f = 12.944*** a1p1 vs a1p3 a2p2 vs a1p3 polygonaceae (%) 9.7 1.7 9.2 1.254 f = 7.066** a1p1 vs a2p2 a2p2 vs a1p3 cyperaceae (%) 5.4 15.6 0.0 2.106 f = 7.108 nutritional values dm (%) 25.0 29.4 21.8 1.417 f = 2.464 cp (%dm) 10.1 10.9 13.3 0.416 f = 12.129** a1p1 vs a1p3 a2p2 vs a1p3 ndf (%dm) 58.1 59.4 41.6 2.115 f = 29.233*** a1p1 vs a1p3 a2p2 vs a1p3 adf (%dm) 37.2 33.2 29.3 1.026 f = 11.991** a1p1 vs a1p3 adl (%dm) 6.5 4.4 6.5 0.374 f = 5.613* a1p1 vs a2p2 ash (%dm) 5.9 4.8 9.1 0.466 f = 35.942*** a1p1 vs a1p3 a2p2 vs a1p3 omd (%dm) 51.1 50.5 68.0 2.184 f = 21.870*** a1p1 vs a1p3 a2p2 vs a1p3 nel (mj kg dm-1) 3.9 3.8 5.2 0.165 f = 19.822*** a1p1 vs a1p3 a2p2 vs a1p3 abbreviations: dm, dry matter; cp, crude protein; ndf, neutral detergent fi bre; adf, acid detergent fi bre; adl, acid detergent lignin; omd, organic matter digestibility; nel, net energy for lactation. †signifi cance: *p<0.05; **p<0.01; ***p<0.001. ‡vs = versus. §lambertin phenology scale (p = poaceae and cyperaceae; o = other families): 100 = p/o: vegetative stage; 200 = p: 70% of spikes in stems, o: 70% of fl owering bottoms; 300 = p: 70% of spikes out of stems (spikes close to stems), o: 70% of fl owering bottoms opened; 400 = p/o: full fl owering stage; 500 = p: lactic corns, o: fl owers withered; 600 = p: doughy corns, o: starting fructifi cation (fruits formed); 700 = p: hard corns, o: fruits fully ripened; 800 = p/o: end of vegetation. tab. 4: pearson’s correlation coeffi cients† between the nutritional values of the grazed herbage, the phenological stages, and the specifi c contributions of the main botanical families. dm ndf adf adl cp nel omd phenological stage 0.625** 0.784** 0.353 −0.474 −0.548* −0.752** −0.747** poaceae 0.555* 0.460 −0.139 −0.857** −0.391 −0.504* −0.503* fabaceae −0.408 −0.531* 0.033 0.580* 0.367 0.547* 0.534* asteraceae −0.540* −0.549* −0.111 0.699** 0.454 0.642** 0.630** polygonaceae −0.356 −0.410 0.100 0.470 0.225 0.433 0.410 cyperaceae 0.440 0.391 0.103 −0.434 −0.160 −0.448 −0.418 abbreviations: dm, dry matter; ndf, neutral detergent fi bre; adf, acid detergent fi bre; adl, acid detergent lignin; cp, crude protein; nel, net energy for lactation; omd, organic matter digestibility. †signifi cance: *p<0.05; **p<0.01. pasture and cow milk quality changes during a summer transhumance 221 throughout the grazing season. the fa percentages of milk did not show signifi cant differences among periods, except in the case of palmitic acid (c16:0), whose level was signifi cantly lower in alpine (a2p2) than in mountain milk (a1p1 and a1p3). signifi cant correlations were found between the percentages of some fa in milk and the sc of the main botanical families of the grazed pastures (tab. 6). palmitic acid was positively correlated with the sc of polygonaceae. oleic acid (c18:1 c9) was negatively correlated with the sc of polygonaceae and positively correlated with the sc of cyperaceae. linoleic acid (c18:2 c9c12) was negatively correlated with the sc of poaceae and positively correlated with the sc of fabaceae. the total monounsaturated fatty acid (mufa) percentage was negatively correlated with the sc of polygonaceae and positively correlated with the sc of cyperaceae. the total pufa percentage was instead positively correlated with the sc of fabaceae. no signifi cant correlations were observed between milk fa and the sc of asteraceae. discussion in the alpine farming systems, dominant plant species and available herbage biomass at the time of grazing, along with the topographic features of pastures (e.g., altitude, slope), are usually the main factors taken into account to set grazing management strategies (jouglet et al., 1992). pv, as deduced from vegetation composition, is particularly considered by pastoralists the most effective tool to assess pasture carrying capacity (daget and poissonet, 1972). instead, other relevant factors such as plant phenology hardly support management decisions because of logistic constraints, with consequences on the quality of herbage actually offered to ruminants during the grazing seasons. in the study area, mountain and alpine pastures, whose vegetation types and sub-types can be considered representative of those usually grazed in the western alps (cavallero et al., 2007), showed signifi cant differences in their botanical composition and pv. in particular, the average pv were higher in the mountain than in the alpine pastures due to the abundance of highand medium-quality forage species such as dactylis glomerata, polygonum bistorta, and festuca rubra s.l.. nevertheless, mountain and alpine pasture fi rst growth did not display, as expected, signifi cant diftab. 6: signifi cant pearson’s correlation coeffi cients between milk fatty acids and the specifi c contributions of the main botanical families in pastures. milk fatty acids botanical families (pearson’s correlation coeffi cient†) c16:0 polygonaceae (r = 0.614*) c18:1 c9 polygonaceae (r = −0.587*); cyperaceae (r = 0.643*) c18:2 c9c12 poaceae (r = −0.610*); fabaceae (r = 0.697*) σmufa polygonaceae (r = −0.589*); cyperaceae (r = 0.603*) σpufa fabaceae (r = 0.653*) abbreviations: mufa, monounsaturated fatty acids; pufa, polyunsaturated fatty acids. †signifi cance: *p<0.05. tab. 5: bulk milk yield, gross composition and fatty acid (fa) profi le during the three experimental periods. area / period of grazing a1p1 a2p2 a1p3 sem inter-area / period bonferroni post-hoc signifi cant comparisons differences† at p < 0.05‡ milk yield (kg herd-1 day-1) 325.8 160.0 86.0 27.546 f = 77.209*** a1p1 vs a2p2 a1p1 vs a1p3 a2p2 vs a1p3 milk gross composition (%) fat 3.87 4.23 4.14 0.077 f = 1.952 protein 3.21 3.63 3.75 0.060 f = 26.255*** a1p1 vs a2p2 a1p1 vs a1p3 lactose 4.82 4.72 4.76 0.019 f = 3.020 fatty acids (% of total fa) c10:0 1.97 2.38 2.26 0.110 f = 1.164 c12:0 2.26 2.98 2.75 0.161 f = 1.796 c14:0 9.70 8.67 9.77 0.283 f = 1.948 c14:1 c9 1.27 1.23 1.44 0.057 f = 1.297 c15 aiso 0.92 0.97 0.90 0.028 f = 0.514 c15:0 1.62 1.74 1.97 0.065 f = 3.088 c16:0 25.40 23.62 25.55 0.357 f = 5.305* a1p1 vs a2p2 a2p2 vs a1p3 c16:1 c9 1.35 1.47 1.37 0.105 f = 0.121 c18:0 17.63 17.27 16.15 0.397 f = 1.213 c18:1 c9 34.59 36.98 34.45 0.567 f = 2.880 c18:2 c9c12 1.79 1.48 1.88 0.146 f = 0.723 c18:3 c9c12c15 1.52 1.23 1.52 0.123 f = 0.649 σsfa 59.49 57.61 59.35 0.630 f = 0.981 σmufa 37.21 39.68 37.26 0.557 f = 2.955 σpufa 3.30 2.71 3.40 0.263 f = 0.716 σufa 40.52 42.39 40.66 0.629 f = 0.974 abbreviations: sfa, saturated fatty acids; mufa, monounsaturated fatty acids; pufa, polyunsaturated fatty acids; ufa, unsaturated fatty acids. †signifi cance: *p<0.05; ***p<0.001. ‡vs = versus. 222 a. gorlier, m. lonati, m. renna, c. lussiana, g. lombardi, l.m. battaglini ferences in herbage allowance and quality, as they were both grazed at advanced phenological stages with digestibility levels extremely low for dairy cows (jarrige, 1988). on the contrary, the regrowth of mountain pastures in september, which did not differ from the fi rst growth for its pv, was found to have the highest nutritional quality. during the entire grazing season plant phenology concurred strongly with plant species composition in determining such changes in pasture nutritional profi les. plant growth normally results in increased fi bre content and decreased energy values (schubiger et al., 2001), but it is known that the extent to which nutritional values shift and their rapidity depend on the species, being generally greater in grasslands rich in poaceae than in those rich in dicotyledonous (jarrige, 1988; farruggia et al., 2008). moreover, regrowths are known to have a higher leaf:stem ratio and anticipated development stages, which usually determine higher digestibility values and lower productivity relative to the fi rst growth (schubiger et al., 2001). the differences among pastures and the correlations between nutritional values, phenological stages and the sc of poaceae, asteraceae, and fabaceae confi rmed such tendencies. although plant senescence effects are usually well known by farmers, in alpine environments short vegetative season normally determines rapid plant development and decline in herbage quality (van dorland et al., 2006), while differences in site conditions and plant species composition lead to a high variability of growth rates among pastures (farruggia et al., 2008), which altogether do not allow to easily manage the timing of grazing during the transhumances. in the western alps, pasture quality changes such as those observed in this study are commonly considered to affect milk yield and composition during the summer grazing season (battaglini et al., 2003). given that in the study area calving traditionally occurs in december (barmaz, 1992), a drop in milk yield and some changes in milk nutritional composition were expected due to the advance in the cows’ stage of lactation. however, many other factors affected milk yield and composition throughout the season, especially as a consequence of herd transfers between mountain and alpine areas. the general grazing conditions in alpine pastures (i.e., walking, altitude) coupled with low herbage quality, likely concurred in reducing milk yield by 50% in a short period as a2p2 (about one month). in fact, high altitude grazing adaptation can be generally achieved by the animals within a few weeks, but it is associated with a concomitant decline in herbage intake and milk yield due to animal stress (van dorland et al., 2006). instead, further decrease in milk yield after return to a1p3 occurred mainly as cows were at the end of the lactation. since herbage allowance was lower compared to the previous considered periods, regrowth quality probably allowed supporting lower animal requirements at the end of the grazing season (bovolenta et al., 2008). concerning milk gross composition, fat was expected to increase signifi cantly during lactation as it was observed for protein. nevertheless, fat is known as the most sensitive parameter in milk to dietary infl uences (sutton, 1989) and percentages varied notably during the entire season probably because of variations in herbage quality. in particular, although changes were not signifi cant, milk fat values increased in a2p2 compared to a1p1 in correspondence to the drop in milk yield and the supposed increased body fat mobilisation due to energy defi cits (leiber et al., 2006). no signifi cant variations were also observed for lactose, but such result was expected as this parameter is known to be approximately constant in milk (sutton, 1989). in this study, the fa levels in milk were generally consistent with those of previous reports for cows grazing natural pastures (collomb et al., 1999, 2002a; de noni and battelli, 2008). however, fa percentages did not vary as expected in correspondence of variations in pasture botanical composition and quality. in fact, when cows graze grasslands rich in dicotyledonous plant species or at young growth stages, as in a1p3, higher concentrations of long-chain mufa and pufa (i.e. oleic, linoleic, and α-linolenic acids), along with lower concentrations of saturated fatty acids (sfa) (i.e., myristic and palmitic acids) are expected in milk fat because of the higher availability of long-chain unsaturated fatty acid (ufa) precursors in the diet (collomb et al., 2001). instead, only palmitic acid percentage varied signifi cantly during the season, being lower in the alpine area in correspondence of lower abundances of dicotyledonous such as fabaceae and polygonaceae species. since it is well known that feeding has a dominant role on milk fa composition if compared to animal-related factors (palmquist et al., 1993) and that other factors (e.g., altitude, botanical diversity) can affect milk fa as well (leiber et al., 2005), it is hypothesised that the overall conditions of the grazing areas coupled with their botanical and nutritional composition concurred to the values observed in milk fa. in particular, the synthesis of shortand medium-chain fa by the mammary gland usually declines notably when cows are in negative energy balance and when a proportionately high dietary fi bre causes digestive modifi cations in the animals, as during alpine grazing, confi rming that the a2p2 grass nel was likely insuffi cient to cover animal energy needs (palmquist et al., 1993; khanal et al., 2008). although animal intake was not taken into account in the present study, it is also reasonable to hypothesise that herbage intake varied during the grazing season, being lower as expected in a2p2 because of the effect of alpine grazing conditions (leiber et al., 2006). finally, the correlations found between milk fa composition and the sc of some botanical families support the role played by these families in the synthesis of some fa pools (collomb et al., 2002b). in particular, fabaceae and cyperaceae abundances confi rmed to be positively correlated, while poaceae and polygonaceae were negatively correlated, with some representative individual fa as well as mufa and pufa in milk. by contrast, no signifi cant correlations involved other dicotyledonous families (e.g., asteraceae, apiaceae, and rosaceae) whose presence has been reported to increase the level of pufa in other studies (collomb et al., 2002b; de noni and battelli, 2008). in conclusion, considering pasture and milk compositions together allowed understanding which factors normally affect animal performance during traditional transhumances of dairy cow herds. in mountain and alpine areas with high-diverse pasture types, botanical and pv evaluations could be not suffi cient, per se, to assure a constant herbage quality to dairy cows. managing the botanical composition along with the timing of grazing can be an effective strategy for optimizing the quality of the herbage offered to ruminants throughout a grazing season, in particular for vegetation types dominated by poaceae species. early exploitation in both mountain and alpine areas would generally lead to higher herbage energy values, improving animal performances and the quality of milk and derived dairy products. acknowledgements the authors gratefully acknowledge prof. andrea cavallero for supervision of the project. the authors wish also to thank massimo bocca and the mont avic natural park for support and technical assistance in the fi eldwork, sergio valsoaney and jean paul allasinaz for fi eldwork, claudio and roberto bosonin for allowing the study to be performed in their farm. the research was supported by a grant from the région autonome vallée d’aoste and the mont avic natural park. references aoac, 2000: offi cial methods of analysis of aoac international. 17th edition. association of offi cial analytical chemists, gaithersburg, md, pasture and 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(eds.), seasonal landscapes, 85-101. springer, dordrecht, nederland. elgersma, a., tamminga, s., ellen, g., 2006: modifying milk composition through forage. anim. feed sci. tech. 131, 207-225. farruggia, a., martin, b., baumont, r., prache, s., doreau, m., hoste, h., durand, d., 2008: quels intérêts de la diversité fl oristique des prairies permanentes pour les ruminants et les produits animaux? inra prod. anim. 21, 181-200. jarrige, r., 1988: alimentation des bovins, ovins & caprins. inra publications, paris, france. jeangros, b., scehovic, j., troxler, j., bachmann, h.j., bosset, j.o., 1999: comparaison de caractéristiques botaniques et chimiques d`herbages pâturés en plaine et en montagne. fourrages 159, 277-292. jeangros, b., troxler, j., conod, d., scehovic, j., bosset, j.o., bütikofer, u., gauch, r., mariaca, r., pauchard, j.p., sieber, r., 1997: etude des relations entre les caractéristiques des herbage et celles du lait, de la crème et du fromage de type l’etivaz ou gruyère: i. présentation du projet. rev. suisse agric. 29, 23-34. jouglet, j.-p., bornard, a., dubost, m., 1992: eléments de pastoralisme montagnard. tome 1: végétation. equipements. etudes du cemagref, série montagne n° 3. cemagref, gap, france. khanal, r.c., dhiman, t.r., boman, r.l., 2008: changes in fatty acid composition of milk from lactating dairy cows during transition to and from pasture. livest. sci. 114, 164-175. lambertin, m., 1990: calcul de la charge d’un alpage par une approche phyto-ecologique. institut agricole régional aoste publ., aosta, italy. leiber, f., kreuzer, m., jörg, b., leuenberger, h., wettstein, h.r., 2004: contribution of altitude and alpine origin of forage to the infl uence of alpine sojourn of cows on intake, nitrogen conversion, metabolic stress and milk synthesis. anim. sci. 78, 451-466. leiber, f., kreuzer, m., leuenberger, h., wettstein, h.r., 2006: contribution of diet type and pasture conditions to the infl uence of high altitude grazing on intake, performance and composition and renneting properties of the milk of cows. anim. res. 55, 37-53. leiber, f., kreuzer, m., nigg, d., wettstein, h.r., scheeder, m.r.l., 2005: a study on the causes for the elevated n-3 fatty acids in cows’ milk of alpine origin. lipids 40, 191-201. martin, b., verdier-metz, i., buchin, s., hurtaud, c., coulon, j.b., 2005: how do the nature of forages and pasture diversity infl uence the sensory quality of dairy livestock products? anim. sci. 81, 205-212. palmquist, d.l., beaulieu, a.d., barbano, d.m., 1993: feed and animal factors infl uencing milk fat composition. j. dairy sci. 76, 1753-1771. pignatti, s., 1982: flora d’italia. edagricole, bologna, italy. schubiger, f.x., lehmann, j., daccord, r., arrigo, y., jeangros, b., scehovic, j., 2001: valeur nutritive des plantes de prairies. 5. digestibilité de la matière organique. rev. suisse agric. 33, 275-279. spss, 2007: user’s guide, base 16.0 for windows. statistical package for social sciences inc., chicago, il, usa. sutton, j.d., 1989: altering milk composition by feeding. j. dairy sci. 72, 2801-2814. van dorland, h.a., wettstein, h.-r., kreuzer, m., 2006: species-rich swards of the alps: constraints and opportunities for dairy production. in: elgersma, a., dijkstra, j., tamminga, s. (eds.), fresh herbage for dairy cattle, 27-43. springer, dordrecht, nederland. van soest, p.j., robertson, j.b., lewis, b.a., 1991: methods for dietary fi ber, neutral detergent fi ber, and nonstarch polysaccharides in relation to animal nutrition. j. dairy sci. 74, 3583-3597. wishart, d., 1987: clustan user manual. university of edinburgh, edinburgh, uk. zemp, m., leuenberger, h., künzi, n., blum, j.w., 1989: infl uence of high altitude grazing on productive and physiological traits of dairy cows. i. infl uence on milk production and body weight. j. anim. breed. genet. 106, 278-288. address of the authors: alessandra gorlier (corresponding author: alessandra.gorlier@unito.it), michele lonati, manuela renna, carola lussiana, giampiero lombardi, and luca maria battaglini, department of agriculture, forest and food sciences, university of torino, via. l. da vinci 44, 10095 grugliasco (to), italy. journal of applied botany and food quality 87, 93 96 (2014), doi:10.5073/jabfq.2014.087.014 1department of horticulture, faculty of agriculture, university of süleyman demirel, isparta, turkey 2 fruit research station, eğirdir, isparta, turkey 3 department of soil, faculty of agriculture, university of süleyman demirel, isparta, turkey nutritional constituents of wild-grown black mulberry (morus nigra l.) f. koyuncu1, m. çetinbaş2*, e. ibrahim3 (received february 4, 2014) * corresponding author summary some chemical properties were determined in completely ripe fruits and fully developed leaves of black mulberry (morus nigra l.) genotypes, grown in mahmatlar, turkey. crude protein was found as the most abundant component in both fruits (10.25 %) and leaves (25.72 %). native black mulberry fruits had higher content of total sugar (6.25 %), crude fat (5.75 %) and crude protein than those of the other berries. analyses of mineral composition (k, na, p, ca, mg, zn, fe, mn and cu) results indicated that k was the main mineral of fruit followed by na and p. furthermore, black mulberry leaves were rich in sodium, calcium and potassium. black mulberry should be more widely used because of its potential nutrient contribution for human and also for feeding animals. abbreviations k: potassium, na: sodium, p: phosphorus, mg: magnesium, ca: calcium, fe: iron, mn: manganese, zn: zinc, cu: copper introduction morus nigra is a member of morus genus in the moraceae family. black mulberry grows wildly in various parts of the world especially in the temperate regions or mediterranean climates (verheij and coronel, 1991; tutin, 1996). in turkey, black mulberry is cultivated for fruit production and its dark shade in the summer. the perennial woody plant has a height of about 10 m (yaltirik, 1982). it flowers from april to may, and fruit ripens from july to september (koyuncu et al., 2004; koyuncu, 2004). black mulberry is considerably valued for its delicious fruits which are 2-3 cm long; they have a weight of approximately 5-6 g and black-purple color (koyuncu et al., 2004). the fruits fall from the tree when they are fully ripe. therefore, fruit harvesting is similarly difficult since fruit detachment is not easy to achieve unless they are fully ripe (gerasopoulos and stavroulakis, 1997). nevertheless, by the use of fruit collection nets in combination with tree shaking, followed by hand sorting (holland et al., 1992) or by growing short grass under the tree, harvesting is managed relatively easily. fruits are consumed fresh, dried, cooked or are used in preserves and tea. fruits of black mulberry have aromatic, laxative and anti-pyretic actions (chopra et al., 1988). the syrup is obtained from black mulberry fruits is believed to have medicinal purpose (baytop, 1984). in addition it is used in jam, marmalade, paste, pulp, jelly and juices. also, it has an exceptional coloring effect, especially for ice-cream. furthermore, fruits and leaves are used for pharmacogical actions all over the world. in our country, from its fruits we have some products such as pekmez. pekmez is defined as the prufication of the fresh and dried black mulberry from the external matters, and defined as a product whose unfermented juice’s concentration at the sun or in a vacuum till certain extend (anonim, 1996). since black mulberry fruits decompose very quickly, by making pekmez it is possible to have long lasting usage of the black mulberry (erdoğan and çakmakçi, 2005). the researches have shown that the leaves are antibacterial, astringent, diaphoretic, hypoglycemic, odontalgic and ophthalmic (duke and ayensu, 1985). the leaves are collected in the late autumn and can be used fresh but generally they were dried. they should be used internally in the treatment of colds, influenza, eye infections and nosebleeds (bown, 1995). as interest in fruit plants, especially berries, is now growing, it is important to investigate the composition of the berries commonly grown in each country. mineral compounds are essential protective nutrients for the maintenance of nutritional and healthy body. the human body cannot synthesize minerals therefore they must be obtained through the diet. adequate amounts of minerals should be included in the daily diet to supply of minerals for body. while it is clear that fruits have superior source of minerals in human nutrition. it is possible that many fruit varieties may have a better effect on human metabolism because of their different nutritional composition. therefore, some native fruits such as black mulberry are still important due to their nutrients, texture and medicinal properties. except for the limited study of koyuncu et al. (2004), koyuncu (2004a), koyuncu (2004b) on black mulberry, there is no detailed information on the nutritional composition of fruits and leaves of black mulberry. that information is essential to inquire about nutritive constituents of black mulberry. the research aimed to determine the nutritional value of 8 selected black mulberry genotypes. materials and methods sampling the fruits and leaves analyzed in this experiment were collected from native black mulberry genotypes, which were evaluated as promising by koyuncu et al. (2004) in mahmatlar, turkey. the fruits were harvested in july and transported to the laboratory in iceboxes immediately. the fruit samples were stored in a freezer at -80 oc until usage. the fully developed young leaf samples were collected from current year’s shoots in july representing whole tree from four sides to determine mineral composition of trees. proximate analysis basic proximate composition analyses were conducted on black mulberry fruits. for this purpose, fifty grams of each fruit sample were used after dried at 65 oc to constant weight. these samples were ground with a stainless-steel mill for analytic procedures. in accordance with aoac methods (1995), moisture was determined by weight loss after heating in an oven at 105 oc, ash in a muffle furnace at 550 oc for 5 h. to determine total sugars, a modified anthrone method (sanz et al., 1987) was used. crude fat as estimated by exhaustive extraction with petroleum ether using soxhlet apparatus according to the aoac (1984). crude protein was calculated from nitrogen, determined by kjeldahl method, multiplying the value by 6.25 as recommended by bremler (1965). 94 f. koyuncu, m. çetinbaş, e. ibrahim mineral elements analysis the mineral contents were analyzed on both leaf and fruit samples. samples were washed thoroughly with fountain water, dilute acid (0.2 n_hcl) and distilled water to remove surface residues, and dried at 70±5 oc. dried samples were ground with a stainless-steel mill for analytic procedures. nitrogen concentration in samples was determined according to kjeldahl method (bremler, 1965) in which 1g sample digested in concentrated h2so4 and distilled with naoh (40 %). the ammonium n was fixed in h3bo3 (2 %) and titrated with 0.1n h2so4. to determine k, na, p, ca, mg, zn, fe, mn and cu concentration of plant (leaf and fruit), dried samples were grinded then 1 g of samples were wet-digested in hno3+hclo4 acid mixture. digested material was dissolved and filled up to 100 ml with distilled water. phosphorus concentration was determined with molibdo-vanado phosphoric acid method using a shimadzu uv-1208 spectrophotometer at 430 nm. na, k and ca contents were evaluated using a flame photometer (jenway pfp 7). the other nutrients (mg, zn, fe, mn, cu) concentrations were measured using an atomic absorption spectrophotometer (varian aa240fs) (kaçar, 1972). statistical analysis all analyses were carried out in triplicate and the mean calculated. the data were analyzed using analysis of variance (anova). duncan’s multiple range test was used to compare mean values. significance was accepted at p<0.01 level. variety was the only independent variable. results and discussion data of biochemical analysis of fruits and leaves of 8 mulberry genotypes are summarized in tab. 1, 2, and 3. quantitative results indicate the difference among mineral contents of different genotypes. all samples present a similar profile composed of minerals. proximate composition of black mulberry fruit and leaf the moisture content of fresh fruits in the eight genotypes (m-5, m-8, m-11, m-14, m-17, m-18, m-22, m-28) were found to be between 77.30 % (m-17) 84.27 % (m-28). the average moisture content of black mulberries (morus nigra l.) is 81.6 % in antalya southern of turkey region which was found by ozdemir and topuz (1998). ercişli and orhan (2007) also found that the moisture content of fresh fruits 72.6 %. these differences can be attributed to ecological factors and also harvesting time. the average crude fat compounds were 5.75 %, and total ash compounds were determined between 0.36 % 0.12 % (tab. 1). crude fat compounds of genotypes were significantly different whereas ash compounds were not. the crude fat contents were lower than those reported by ercişli and orhan (2007). the fruits average total sugar was 6.25 %. in black mulberries sucrose concentration is low and total sugar mainly consists of reducing sugars (özdemir, 1998). in that sense, the sugars in black mulberry fruits are important for human. significant differences were found sugar concentration of fruits with the genotype m-11 showing the highest concentration (7.26 %) (tab. 1). our results coincide with the other studies (holland et al., 1992; anonymous, 2001) showing sugar concentrations in black mulberry fruits is between 6-9 %. the protein concentrations in black mulberry fruits ranged between 7.66 % 12.93 % according to other authors (bamikole et al., 2005; glosh et al., 2003; erdoğan and çakmakçi, 2005; kitahara et al., 2002; martin et al., 2002; machii et al., 2002; srivastava et al., 2006). in leaves, m-17 genotype (29.15 %) had the highest crude protein, m-14 had the lowest with 20.94 % (tab. 1). significant differences on crude protein concentration was found among genotypes in fruits and leaves (tab. 1). mineral composition of black mulberry fruit and leaf mineral concentrations of fruit and leaf from black mulberries were given at tab. 2 and 3. as seen in tab. 2, the main mineral of black mulberry is k, followed by na, p and mg. mineral concentrations of fruits showed differences among genotypes (except for mg and fe). the mean values of k, na, p, mg, ca, fe, mn, zn and cu were 1041 mg/100g, 277 mg/100g, 170 mg/100g, 128 mg/100g, 32 mg/100g, 7.07 mg/100g, 5.63 mg/100g, 3.02 mg/100g, 0.34 mg/ 100g, respectively. these findings agree with previous studies (cemeroğlu and acar, 1986; ercişli and orhan, 2007; erdoğan and çakmakçi, 2005; holland et al., 1992). in a research conducted by ozdemir and topuz (1998), black mulberry fruits in antalya had higher mineral contents than our results, but it was stated that the main minerals of black mulberry fruits were k, ca, p and mg. ercisli and orhan (2007) found higher p, k, ca and mn contents. looking at the contents of cu we saw similarity with the study but they also found lower zn. these differences may be due to ecological factors, growing conditions and genetic factors. mineral nutrition of plant is controlled by tab. 1: proximate composition of black mulberry fruits and leaves (%). fruit leaf crude protein genotype moisture total sugar crude fat ash crude protein m-5 81.97 a 7.05 a 5.65 b 0.17 10.04 c 29.07 a m-8 81.49 a 7.08 a 5.92 ab 0.24 9.76 c 25.34 b m-11 82.86 a 7.26 a 5.52 b 0.21 9.56 c 24.06 b m-14 82.35 a 6.44 b 6.71 a 0.36 10.36 bc 20.94 c m-17 77.30 b 5.45 d 6.79 a 0.23 10.31 bc 29.15 a m-18 84.20 a 5.09 d 6.06 ab 0.28 11.39 b 25.00 b m-22 84.03 a 6.11 bc 6.22 ab 0.13 12.93 a 28.59 a m-28 84.27 a 5.53 cd 3.15 c 0.12 7.66 d 23.61 b mean 82.31 6.25 5.75 0.21 10.25 25.72 max 84.27 7.26 6.79 0.36 12.93 29.15 min 77.30 5.09 3.15 0.12 7.66 20.94 *) different letters in the same column indicate significant differences, according to duncan’s multiple range test (p<0.01). nutritional status of black mulberry 95 environment, soil and plant factors (marschner, 1995). since the uptake of nutrients from the soil is genetically controlled, plant species and varieties show different response to nutrients even when they are grown in the same conditions (tsipoutidis et al., 1990; ershadi and talaie, 2001; erdal and baydar, 2005; kuçukyumuk, 2007). as shown in tab. 3, na, ca, k, mg and p values of black mulberry leaves varied from 649 mg/100g (m-5) to 3402 mg/100g (m-28). differences among the black mulberry genotypes were observed based on the mineral concentrations. it can be stated that most of the minerals constituting the leaves of black mulberry trees from isparta district were just constituted by na, ca, k and mg. in the study conducted by kitahara et al. (2002), it was pointed out that the mineral patters of leaf samples mostly consist of ca, k, and mg. the mineral composition of leaves depended not only on the types, but also on the growing conditions, such as soil and geographical conditions. in this study, na was predominant, followed by ca, k, mg, p, mn, fe, zn and cu. black mulberry fruits which are cultivated in isparta than other regions have rich nutrition elements. for this reason they are important raw material for food technology production (jam, marmalade, paste, pulp, jelly, juice, pekmez, etc.). their leaves have also rich nutrition elements like their fruits. leaves can be used for animal nutrition, tea production and they can be used for various purposes. karadut leaves which are cultivated in isparta region are very important for the reasons above. however, in order to produce certain crops from black mulberry industrially, relevant researches on this purpose should be conducted and orchards with appropriate varieties should be set up. references anonim, 1996: dut pekmezi. turk standardı ts12001, ankara, turkey. anonymous, 2001: mulbery, http://crtg.org/pubs/tt/mulberry.html. aoac, 1984: official methods of analysis, 14th edn. washington, dc: association of official analytical chemists. aoac, 1995: official methods of analysis, 16th edn. washington, dc: association of official analytical chemists. bamikole, m.a., ikhatua, m.i., ikhatua, u.j., ezenwa, i.v., 2005: nutritive value of mulberry (morus spp.) leaves in the growing rabbits in nigeria. pak. j. nutr. 4, 231-236. baytop, t., 1984: türkiye’ de bitkilerle tedavi. i̇stanbul universitesi yayınları, istanbul, turkey. bown, d., 1995: encyclopadia of herbs and their uses, dorling kindersley, londan, uk. bremler, j.m., 1965: total nitrogen (methods of soil analysis). am. soc. agron. inc. medison, usa. cemeroglu, b., acar, j., 1986: meyve sebze isleme teknolojisi, sanem matbaacılık, ankara, turkey. chopra, r.n., chopra, i.c., honda, k.l., kapur, l.d., 1988: chopra’s tab. 2: mineral composition of black mulberry fruits (mg/100 g). genotype k na p mg ca fe mn zn cu m-5 999 b* 205 e 150 c 148 33 bc 6.77 4.20 b 3.17 ab 0.58 a m-8 1254 a 247 cd 168 bc 105 42 ab 5.50 3.83 b 2.21 b 0.28 b m-11 818 c 309 ab 154 c 136 31 bcd 4.47 3.93 b 3.09 ab 0.34 b m-14 999 b 291 ab 157 bc 140 30 cd 6.62 4.93 b 3.39 ab 0.31 b m-17 1165 a 329 a 182 b 152 29 cd 6.65 4.40 b 2.07 b 0.22 b m-18 1228 a 279 bc 160 c 150 27 cd 5.61 5.93 b 2.77 b 0.36 b m-22 1264 a 229 de 164 bc 92 21 d 10.34 5.17 b 3.09 ab 0.36 b m-28 599 d 329 a 228 a 99 45 a 10.59 12.67 a 4.36 a 0.31 b mean 1041 277 170 128 32 7.07 5.63 3.02 0.34 max 1264 329 228 152 45 10.59 12.67 4.36 0.58 min 599 205 150 92 21 4.47 3.83 2.07 0.22 *) different letters in the same column indicate significant differences, according to duncan’s multiple range test (p<0.01). tab. 3: mineral composition of black mulberry leaves (mg/100 g). genotype na ca k mg p mn fe zn cu m-5 649 h* 1226 c 812 d 327 b 117 d 22.20 bc 12.50 b 4.84 0.48 abc m-8 1008 g 1759 a 1217 ab 431 ab 127 d 21.93 bc 16.60 ab 6.62 0.51 abc m-11 1368 f 1847 a 1103 bc 431 ab 153 bc 25.10 ab 13.97 b 6.31 0.44 bc m-14 1727 e 1788 a 978 c 387 ab 171 ab 27.23 ab 13.58 b 6.45 0.41 c m-17 2086 d 1660 a 1223 ab 372 b 162 ab 27.30 ab 20.70 a 5.27 0.53 ab m-18 2205 c 1542 ab 994 c 359 b 150 bc 26.20 ab 11.79 b 5.99 0.44 bc m-22 2684 b 1552 ab 1306 a 387 ab 180 a 30.00 a 15.38 b 7.49 0.56 a m-28 3402 a 1325 bc 818 d 521 a 137 cd 18.85 c 14.80 b 5.99 0.53 ab mean 1891 1587 1056 402 149 24.85 14.91 6.12 4.90 max 3402 1847 1306 521 180 30.00 20.70 7.49 5.60 min 649 1226 812 327 117 18.85 11.79 4.84 4.13 *) different letters in the same column indicate significant differences, according to duncan’s multiple range test (p<0.01). 96 f. koyuncu, m. çetinbaş, e. ibrahim indigenous drugs of india 2nd. dhar & sons pvt. ltd. duke, j.a., ayensu, e.s., 1985: medicinal plants of china. reference publications inc., isbn 0-917256-20-4. ercişli, s., 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(eds.), in flora europe psilotaceae to platanaceae second edition, 100-106. cambridge university press, australia. verheij, e.w.m., coronel, r.e., 1991: fruits and nuts. plant resources of south-east asia 2. edible, netherlands. yaltirik, f., 1982: morus. in: davis, p.h. (ed.), flora of turkey, 641-642. edinburgh university press, edinburgh. address of the corresponding author: m. çetinbaş, fruit research station, eğirdir, 32500, isparta, turkey. e-mail: melikecetinbas@gmail.com journal of applied botany and food quality 87, 157 167 (2014), doi:10.5073/jabfq.2014.087.023 department of fruits and orchard management, faculty of horticulture, bidhan chandra krishi viswavidyalaya, mohanpur, india understanding the pollen and ovule characters and fruit set of fruit crops in relation to temperature and genotype – a review k.s. thingreingam irenaeus*, sisir k. mitra (received august 7, 2013) * corresponding author summary plant reproduction is indispensable for maintaining crop productivity and species sustainability which basically involves pollen and ovule. productivity is influenced by temperature stress in various fruit crops worldwide and important factor for unfruitfulness is failure in fertilization. understanding of the plant reproductive characters in relation to genotype differentiated response across different temperatures will help to develop strategies in overcoming temperature stress. available literature suggests that variation in pollen viability, germination and tube growth and ovule normality exists among different fruit species, cultivars and flower types (even in the same plant as in litchi) and are strongly influenced by temperature stress and suitable temperature for proper pollen and ovule performance has been identified for various fruit crops. possible reasons to explain the mechanism for impairment of proper functioning of pollens and ovule are discussed. further, low and erratic fruit set, improper fruit development and stenocarpic fruits as a result of temperature stress leads to reduced yield. we suggest considering in vitro performance for establishing in vivo nutritional requirements and standardizing critical leaf nutrients by way of correlating with pollen germination, tube growth and ovule normality per cent. male and female parents can be identified for breeding purpose and region specific adoption based on temperature prevalence in the area. introduction climate change is the defining challenge for human development and ecological well being in the 21st century and increase in average global temperature of between 1.1 and 6.0 °c by the end of this century is predicted by the intergovernmental panel on climate change (ipcc). the consequence of 2 oc temperature rise is grave for potentially millions of people through death, injury and dislocation from flooding, fire and diseases, adverse affects on water quality, species extinction and reduced agricultural yields. to which extent temperature stress will influence plant survival and crop yield is critical in evolutionary, environmental, economic and social terms. global warming may represent another challenge to plant productivity and geographic distribution (hedhly et al., 2009) and tolerance to temperature stress is becoming a desirable trait. understanding of temperature stress physiology during plant reproductive process is still incomplete. a number of reproductive processes must occur in a highly concerted fashion for fertilization to occur and pollen development and function may be the most thermosensitive reproductive processes to high temperature (hedhly et al., 2009). the yield of plant species with reproductive structures of agricultural importance is exceptionally sensitive to temperature stress during flowering (zinn et al., 2010). heat stress can limit fertilization by inhibiting male and female gametophyte development, pollen germination and pollen tube growth (hedhly, 2011). however, moderately low temperature also reduces pollen performance and could result in dysfunctions of the fertilization processes given the short stigma receptivity intervals. male gametophyte development is especially sensitive to temperature both at preand post-pollination levels. pollination of flower is necessary to achieve a satisfactory fruit yield and improper pollination factors can drastically influence productivity and have serious economic impacts. since, successful pollination will be affected by pollen viability, germination and tube growth, impairment of these functions and qualities could threaten species reproduction and survival. also, the maternal tissues of the pistil and the female gametophyte, traditionally considered more tolerant, have also been reported to be sensitive to temperature stress. above these individual responses, temperature stress might lead to developmental asynchrony in pollen-pistil-ovule functioning leading to reduced fertilization levels (hedhly, 2011). thus, studies on the effect of temperature stress on reproductive process may be of great importance for choosing the appropriate variety for a certain area according to the microclimate and to elucidate the correlation between low fruit set and incidents of extreme temperature preceding and during flowering period. strong genotypedifferentiated response in low, high and moderate high temperature stress could be exploited by plant breeders towards producing new temperature tolerant varieties. effect of temperature on pollen characters of different fruit species air temperature is considered as the main environmental factor affecting the pollination and fertilization progress and was evident in sweet cherry (zhao et al., 2008). studies on the influence of genotype, temperature, nutrient media (types and sources, concentration, ph) on pollen germination and tube growth has been established in many crops both in vivo and in vitro though open field conditions are more susceptible to abiotic stress since they are directly exposed at environmental extreme. the effects of temperature on pollen germination and pollen tube growth have earlier been reported by several workers in various fruit species (mellenthin et al., 1972; luza et al., 1987; cerovic and ruzic, 1992a; egea et al., 1992). pollen viability and germination temperatures above the optimum can exert negative effects on plant reproductive development. a pronounced detrimental effect of high day/night (d/n) temperature regimes 27/22 °c and particularly 32/27 °c during floral development on pollen viability of litchi has been reported by (stern et al., 1996). the litchi originated in southern china and its center of cultivation is the ghuangzhou region, where temperatures during floral development (february and march) are ≈ 20/10 °c (groff, 1921). optimum temperature for some reproductive processes might reflect, indeed, species origin or adaptation. for example, while for many species native to or cultivated in temperate regions optimum temperatures in the range 1525 °c are usually recorded [examples include sweet cherry at 25 °c (weinbaun et al., 1984), or almond (prunus dulcis mill.) at 16 °c and peach at 23 °c (lewis, 1942). these adaptations can be best seen in natural populations that flower during extremely high or low temperatures. 158 k.s.t. irenaeus, s.k. mitra the rate of pollen viability in walnut was high (> 75 %) for all the cultivars studied (mert, 2009). similarly pollen viability rates of 32 different walnut cultivars were reported to vary between 81 % and 94 % (sutyemez, 2007). in another study pollen viability ratio of selected 19 walnut cultivars varied between 77-92 % (sutyemez and eti, 2006). in some previous studies, similar results for pollen viability were reported in hazelnut (49-97 %) (beyhan and odabas, 1995), chestnut (8.8-35.8 %) (beyhan and serdar, 2009), apricot (76-86 %), sweet cherry (67-81 %), and sour cherry (71 %) (bolat and pirlak, 1999). it was observed that no germination of pollen from any of the juglans regia (persian walnut) or juglans nigra (black walnut) cultivars occurred below 14 °c and maximum germination was obtained at 28 °c in j. regia and 32 °c in j. nigra (luza et al., 1987). temperature had significant effect on the germination percentage of different walnut cutivars (‘şebin’, ‘kaplan 86’, ‘yalova 3’, ‘pedro’, ‘hartley’ and ‘franquette’) (mert, 2009) and pollen germination rates increased significantly with increasing temperatures with highest germination rates at 27±1 °c temperature in both years (26.94-73.98 % and 22.78-70.86 %, respectively) of study while, in cv. ‘yunxin’ the optimum temperature for pollen germination was recorded at 25 °c (wu et al., 2008). usually, pollen germination is negatively affected by high or low ambient temperatures (westwood, 1978). in almond, pollen germination was observed at temperatures between 4.4 and 10 °c (griggs, 1975). optimum temperature required for pollen germination in apricot was about 15 °c and 20 °c (pirlak, 2002). pollen germination (egea et al., 1992; pirlak, 2002) and tube growth speed was low at 5 °c for apricot varieties and the germination rate increased with increasing temperature but temperatures above 25 °c caused a decrease in pollen germination rates (egea et al., 1992). similarly, investigation of pollen germination and pollen tube growth rate of different sweet cherry varieties at different temperatures (5, 10, 15 and 20 °c) showed that pollen germination was low at 5 °c and was optimum at 15 and 20 °c (pirlak, 2002). in cherimoya, 2025 °c was found optimum for pollen germination, while germination decreased at 30 and 35 °c; and at 10 °c, only 1.8 % of the pollen grains germinated (rosell et al., 1999). in vitro pollen germination of olive cultivars picudo, picual, manzanila, hojiblanca and gordal decreased at 35 °c (fernandezescobar, 1983) and reduced germination at 30 °c has also been reported for ‘manzanillo’ olive (cuevas et al., 1994) and was concluded that optimum temperature for pollen germination was 2025 °c while pollen of ‘amigdalolia’ olive germinated similarly at 25 and 30 °c (fernandez-escobar, 1983; cuevas et al., 1994). even a short-term exposure at high temperature extremes (40 °c) combined with low rh (20 %) prior in vitro culture had detrimental effect on pollen performance of four olive cultivars viz., koroneiki, mastoidis, amigdalolia and kalamata. pollen culture at 15 °c resulted in the lowest germination and tube growth rates and low temperature pre-incubation treatment (10 °c) showed no constant effect on pollen germination (koubouras et al., 2009). pear (vasilakakis and porlingis, 1985) and avocado (loupassaki et al., 1997) pollens germinated best at 25 °c and reduction in pollen germination occurred due to heat stress. pollen germination of different pistacia genotypes ranged from 83 % to 97 % (kamiab et al., 2007). in strawberry cv. ‘tochiotome’ both viability and germination were not significantly different between two day/night temperature (32/27 °c and 27/22 °c) regimes. approximately 90-93 % of ovules were fertilized at 27/22 °c however; the fertilized percentage of ovules at 32/27 °c was 52 % to 85 % depending on the inflorescence type. embryo development in both inflorescences was accelerated at 32/27 oc (pipattanawong et al., 2009). in mango, optimum temperature was found to be 15 and 33 °c (issarakraisila and considine, 1994) and 15-25 °c (sukhbivul et al., 2000) and at 10 and 30 °c pollen germination was reduced and that 25 °c was optimum for in vivo incubation (sukhbivul et al., 2000). the optimum temperature for pollen germination of citrus in vitro was of 25 °c, while pollen germination under different in vitro incubation temperatures, showed a progressive increase in germination rate from 15 °c to 25 °c, with a sharp decrease when temperature reached 30 °c (distefano et al., 2012). furthermore, temperature appears to have an effect on self-incompatibility reaction by affecting the place where pollen tubes are arrested. absence of germination at 10 °c, and the optimum germination temperature of 25 °c reflect the subtropical origins of all citrus species examined. the critical low temperatures for the induction of pollen sterility in lemon is 18 °c (soost et al., 1975), respectively and low temperature in the early spring may be an obstacle for the pollen germination and pollination. hence, temperature extremes either low or high can be detrimental or may lead to impairment of pollens. pollen tube growth pollen tube growth rate was evaluated under increasing temperatures, in datura stramonium (buchholz and blakeslee, 1927) where pollen tube growth rate increased linearly by a factor of 4.5 from 11 oc to 33 oc (from 1.28 mm/h to 5.86 mm/h). since then information on temperature affects to pollen tube growth rate, built up confirming this fact in different fruit species (mellenthin et al., 1972; lombard et al., 1927; socias i company, 1976). this response pattern of pollen tube growth with temperature seems to be a general phenomenon since it has been confirmed in many seed plants, and mathematical models for pollen tube growth in relation to temperature have been developed (jeffries et al., 1982). this variation in the rate of pollen tube growth was suggested to be the result of increased metabolism at higher temperatures, typical for most biological growth rates (weinbaum et al., 1987). it is now conclusively proved that temperature at bloom is one of the main factors affecting fruit set (mellenthin et al., 1972; vasilakakis and porlingis, 1972). since high temperatures accelerate and low temperatures retard pollen tube growth rate, it would be expected that fertilisation and, hence, fruit set would be enhanced by moderately high temperatures and reduced by low temperatures during bloom. however, this is not always the case, due to the fact that temperature also does have an effect on pistil development, accelerating development at high temperatures and slowing it down at low temperatures. consequently, high temperatures during flowering accelerate pollen tube growth, but also maturation and hence early degeneration of the stigma (burgos et al., 1991) and ovule development (postweiler et al., 1985; hedhly et al., 2004). temperature around 25 oc was found most favourable to accelerate in vivo pollen tube growth, to advance fertilization and to obtain a good initial fruit set in olive (cuevas et al., 1994). in avocado cultivars, pollen tube length further increased though pollen germination decreased after in vitro culture at 30 oc. an increase in temperature also reduced pollen germination but accelerated pollen tube growth in sweet cherry (hedhly et al., 2004). pollen tube growth rate of ‘manchurian’ crabapples and ‘golden delicious’ apple in the style increased quadratically with increasing temperature from 13 to 29 °c. pollen tube growth rate in the style increased with increasing day/night temperature from 7/0 to 24/7 °c. the time required for pollen tubes to grow to the base of styles decreased with increasing day/night temperature from 13/2 to 24/7 °c (yoder et al., 2009). in in vivo studies with litchi (stern and gazit, 1998), it was observed that high temperature regime (32/27 °c) had a pronounced detrimental effect on further pollen tube growth after reaching the base of the style. pollen tubes reached the ovary in ≈20 % of the flowers and in no flower did they reach the ovule. variation in the pollen tube growth was observed at different temperature according to cultivars with decline in tube growth at temperatures in excess of the optimum (irenaeus, 2012). pollen and ovule of fruit crops in response to temperature and genotype 159 the pollen tube growth of olive cultivars picudo, picual, manzanila, hojiblanca and gordal were found to decrease at 35°c (fernandezescobar et al., 1983). high temperatures in excess of the optimum (30 °c) resulted in an increase in the rate of pollen tube growth through the style for prunus avium, but decreased the number of pollen tubes to reach the base of the style (hedhly et al., 2004). pollen tube length increased with increasing incubation temperature and the longest pollen tube were measured at 25 °c in sweet cherry (koyuncu, 2006) while, in citrus, the most favourable temperature to accelerate in vivo pollen tube growth depended on the particular male female interaction and ranged between 15 and 25 °c (distefano et al., 2012). on the other hand, low temperatures have also been reported to slow pollen tube growth in pear (mellinthin et al., 1972; lombard et al., 1972), almond (socias i company et al., 1976), plum (jeffries et al., 1982), sweet cherry (guerrero-prieto et al., 1985) and apple (williams et al., 1970). as a general rule, low temperature during flowering slow pollen tube growth rate but extend the effective pollination period (epp) through an ovule life-extension effect (vasilakakis et al., 1985; tromp and borsboom, 1994). however, extreme low temperatures can shorten the epp if longevity of the ovule does not outweigh the slower growth rate of pollen tubes (lombard et al., 1972). high temperatures, while increasing pollen tube growth rate, shorten the epp by shortening both stigma and ovule receptivity. under the cold regime (17/22 °c), pollen tubes of litchi reached the ovary in all the flowers but did not proceed any further and no pollen tube reached the ovule reflecting a slow growth rate at this cold regime. under the cool (22/17 °c) and warm (27/22 °c) regimes, tubes reached the ovule in ≈35 % of the flowers (stern and gazit, 1998). the virtual absence of sexual reproduction of ‘tainong 1’ mango at low temperatures appears to be largely due to slow growth of pollen tube in vivo and to a low rate of successful fertilization (huang et al., 2010). however in olive, low temperature pre-incubation treatment (10 °c) had positive influence on tube length in four cultivars (koubouris et al., 2009). mechanisms leading to pollen impairment due to temperature stress report explaining the possible mechanism of heat stress leading to poor quality and performance of reproductive organs on specific fruit crop is scarce but can be explained from the evidences available in other plants. heat stress adversely affects pollen meiosis and germination, ovule development and viability and development of the embryo (peet et al., 1988). at high temperatures decreased pollen production may be related to anther indehiscence (porch and jahn, 2001) and lower pollen viability could be related to decreased carbohydrate metabolism (datta et al., 2001; karni and aloni, 2002), all of which could significantly influence nourishment of pollen mother cells which could lead to infertile pollen. the effect of heat stress on pollen viability was associated with carbohydrate metabolism during anther development (pressman et al., 2002). under optimal temperature soluble sugar concentration gradually increased in pollen. continuous high temperature prevented the increase in starch concentration and led to decrease soluble sugar in mature pollen. these possibly cause to decrease pollen viability. carbohydrates are the major nutrients which support pollen development (pacini, 1996), whereas for germination of pollen grains, simple sugars are the principal metabolic substances (stanley, 1971). anthers have the highest sink strength in the flower, and the large amounts of sugars are mobilized to the anthers to support early development (castro and clement, 2007). it was reported that male sterility could be induced in tobacco by metabolic engineering of the carbohydrate supply to the developing pollen (goetz et al., 2001). major alterations in gene expression under high temperature stress have been shown, paralleling tapetum degeneration and pollen sterility in rice (endo et al., 2009). among these genes, enzymes involved in carbohydrate metabolism (e.g. cell wall and vacuolar invertase, sucrose synthase) and transport, are gaining higher research interest as indicators of losses in pollen viability due to temperature fluctuations. both cold (oliver et al., 2005) and heat stress (pressman et al., 2005) have been shown to down regulate gene expression of several invertase and sucrose synthase isomorphs, and this inhibition was accompanied by a disruption of sucrose and starch turnover in developing pollen grains, and, hence, lower accumulation of soluble carbohydrates (oliver et al., 2005; jain et al., 2007). a complete understanding of temperature stress on pollen development must await further understanding of carbohydrate turnover during this phase. interestingly, in a recent study in barley and arabidopsis, it was observed that high temperature stress reduced endogenous synthesis of auxin in developing anthers, an effect that was suggested to be related to pollen sterility since exogenously applied auxin restored fertility (sakata et al., 2010). it may be concluded that continuous high temperature reduced the total number of pollens, viability and germination. in a study in mango, it was observed that microsporogenesis is sensitive to low and high temperature stress and the formation of fertile pollen require temperature within the range of 10-35 °c. the developmental phase from meiosis to the pre-vacuolate microspore was found to be the most temperature sensitive phase of pollen development, though sensitivity continued through the end of vacuolation and began in the late microsporocyte phase. the critical temperature for the formation of fertile pollen in the mango cv. ‘kensington’ seems to be about 10 °c. a night temperature below 10 °c (12 h regime) during meiosis resulted in pollen grains with low viability (<50 %). at the vacuolate stage, a night temperature of 9 °c (8 h) did not lower the percentage of pollen viability, but the viability of mature pollen was markedly reduced if the exposure to 9 °c lasts for 12 hours (issarakraisila and considine, 1994). low temperature during flower development has been found responsible for the occurrence of defective reproductive organs in different fruit crops like almond (egea and burgos, 1995), grape (ebadi et al., 1995) and persimmon (fukui et al., 1995). chilling injury has been implicated in damage to mango pollen (issarakraisila and considine, 1994) and probably also to mango female organs (issarakraisila et al., 1992) precise aspect of pollen development which fails under low temperature stress is unclear. meiosis is reported to be the stage of greatest sensitivity to low temperature in sorghum (brooking et al., 1976). previous analyses have associated a depression of respiratory activity in anthers during meiosis with loss of viability (toriyana and hinata, 1984). early abnormalities in rice anthers as a result of cooling at the young microspore stage included increased content of non-reducing sugars and starch, decreased inorganic phosphates and acid phosphatase activity and dilation of the tapetum. these responses were followed by degeneration of the microspores (nishiyama, 1984). natural low temperatures significantly affected pistil and male gametophyte development, resulting in pollen grains with low viability of mango. meiotic chromosomal irregularities, including univalents, multivalents, laggards, bridges and micronuclei were detected at higher incidences and significantly greater proportions of nucleolus fragmentation and dissolution were detected when temperatures were low. pollen tube growth was retarded under low temperature stress either in vivo or in vitro (huang et al., 2010). pollen numbers and germinability were less in low night temperature (lnt) of 10±2 °c and day temperature which did not exceed 24 °c and suggested that low temperatures hinder pollen functioning in pepper, by interfering with starch accumulation at four days before anthesis, thereby decreasing the concentrations of soluble sugars in the mature pollen grains (shaked et al., 2004). 160 k.s.t. irenaeus, s.k. mitra on the other hand, high temperatures impairment of mature pollen grains functioning was associated with a significantly higher sucrose and starch concentrations at four days before anthesis (as observed in pepper) (aloni et al., 2001) and that sucrose accumulated due to a reduction in its utilization and that starch degradation rather than biosynthesis was suppressed under high temperatures. these contradicting results may suggest a different mode of carbohydrate metabolism disturbances by different stresses or cultivar specifity. based on the demonstration (sheoran and saini, 1996) where levels of both reducing and non-reducing sugars in whole rice anthers increased substantially during the period of water stress, a suggestion (saini, 1997) that accumulation of sugars could be the result of disturbances in carbohydrate metabolism or of an inhibition of sugar utilization was made. inhibition of sugar utilization could be relevant to the higher concentration of either sucrose or reducing sugars at four days before anthesis of the pollen developed under low night temperature (shaked et al., 2004). at that stage, starch concentration was lower than that in the pollen grains at higher temperature (night temperature of 20±2 °c and similar day temperatures) and all other processes were probably slower. actin cytoskeleton alteration is also an early signal component of pollen in response to low temperature (lt) stress and actin cytoskeleton alteration may be one of the reasons for inhibition of pollen germination and pollen tube growth of pear (wu et al., 2012). depolymerization of the actin cytoskeleton in pollen will induce activation of pollen plasma membrane ca2+ channels (wang et al., 2004) and pollen tube growth prevention similar to self incompatibility as seen in pear (liu et al., 2007). identification of similar characteristics of low temperature induced depolymerization of the pear pollen actin cytoskleton [ca2+]cyt increase, and activation of pollen plasma membrane inward ca2+ and outward k+ channels was reported (wu et al., 2012). the activation of ca2+ channels was mediated by the actin cytoskeleton structure. low temperatures also induced intracellular ca2+ influx. moreover, the low temperatureelicited outward k+ current was mediated by increased [ca2+]cyt. when extracellular ca2+ influx was chelated by egta, the ltinduced outward k+ conductance decreased, indicating mediation by [ca2+]cyt these results were consistent with the outward k+ channel being activated by [ca2+]cyt suggesting that activation of outward k+ channels were the downstream targets of [ca2+ ]cyt increase in lt signal transduction. outward k+ conductance has been identified in pollen and pollen tubes for several years (fan et al., 2003). ca2+-activated outward k+ channel in pear pollen tubes could be stimulated by depolarization voltage and reciprocally regulated by heme and its catabolism product, carbon monoxide. the carbon monoxide-induced k+ outward flux largely inhibited pear pollen tube growth (wu et al., 2007). thus, excess k+ efflux can also hamper pollen tube growth. the cytoskeleton is an important factor regulating numerous cellular processes in plants (higaki et al., 2007) such as a track for cytoplasmic streaming and organelle movement (tominaga et al., 2000), positioning of organelles such as nuclei (ketelaar et al., 2002), and maintenance of cellular architecture (tominaga et al., 2000). role of polyamines among common polyamines (pas), some studies have shown that cellular free putrescine (put) was the most abundant polyamine followed by spermidine (spd) and spermine (spm), which were less abundant as seen in vitis vinifera l. (faure et al., 1991) and carrot (feirer et al., 1994). however, this difference in polyamines predominance cannot be generalized, but might depend on species, the developmental stage, the duration of treatments, or the physiological status of cell lines as well as the media examined (el meskaoui and trembaly, 2009). several lines of evidence have suggested that pas play a role in pollen germination and tube growth. in apple, pas are synthesized during pollen germination (bagni et al., 1981). both stimulation and inhibition of pollen germination and pollen tube growth in vitro by the addition of various concentrations of pas in various apple cultivars have been demonstrated (xu et al., 1999). however, the lower and higher concentrations of the pas were less efficient than an optimum concentration. few reports (xu et al., 1999; prakash et al., 1988) are available in the literature concerning pa synthesis inhibitor methyoxal-bis(guanylhydrazone) (mgbg) and pollen germination and pollen tube growth. pollen germination to different degrees in two apple cultivars was inhibited by mgbg and at higher concentrations of mgbg, germination was completely inhibited in both cultivars (xu et al., 1999). increasing the temperature, particularly to 35 oc, showed inhibitory effects on pollen germination of almond cv. ‘mamaei’. at a concentration of 0.05 mm putrescine and spermidine and 0.005 and 0.025 mm spermine caused longer pollen tube growth than that of the control at 10 oc, while higher concentrations tended to inhibit pollen tube growth. at 25 oc, most of the treatments had an inhibitory effect on pollen tube growth except for 0.25 mm putrescine and 0.005 mm spermine, which slightly stimulated pollen tube growth (sorkheh et al., 2011). addition of the polyamines biosynthesis inhibitors mgbg reduced polyamines biosynthesis, which indirectly reflected that polyamines are important to pollen germination and pollen tube growth. conversely, treatments of the pollen of almond with 2.5 mm spermidine and 0.25 mm spermine during the germination and pollen tube growth increased intracellular spermidine and spermine concentrations. it is similar to the studies showing a strong positive correlation between conifer embryo development or its morphological quality and a high spermidine concentration (minocha and minocha, 1995). exogenous polyamines serve merely as a nitrogen source for the plants (bagani and serafini-fracassini, 1985). also, polyamines can act as free radical scavengers and protect senescing membranes against lipid peroxidation. another possible mechanism is that polyamines might be active, not by themselves, but through their catabolic pathways or through their interaction with ethylene biosynthesis (kumar et al., 1997). ovule normality in response to temperature stress factors like ovule normality and longevity in fruit crops plays a decisive role in the effective pollination period, fertilization as well as determining fruit set. in higher plants fertilisation takes place after a pollen tube reaches a receptive ovule. however, various abnormalities during ovule development have been described in fruit species and these can often limit the epp, pollination and fruit set. these abnormalities include incomplete development of ovule structures in sour cherry (furukawa and bukovac, 1989), and abnormalities in ovule or embryo sac development during flowering in olive (rallo et al., 1981), almond (pimienta and polito, 1982) and litchi (stern et al., 1996; irenaeus, 2012). moreover, in normally developed ovules, a short ovule life span has been reported to be an important factor limiting the epp (cerovic and ruzic, 1992b). in ‘granada’ peach high temperature conditions delayed the female gametophytes (embryo sac) and promoted anomalies in the formation of male gametophytes. these promoted low pollen viability and a lack of synchrony in fertilization, thereby generating low fruit set percentages and yield (nava et al., 2009). detrimental effect was observed in ovule normality of litchi at high temperatures during floral development (stern et al., 1996). at extreme higher temperature (30/22 oc), ovule normality was affected in all the cultivars and flowers with normal ovule was recorded better at 20/15 oc (irenaeus, 2012). it was observed that litchi ovules lack embryo sac and egg cells (stern et al., 1996). while some of these abnormalities appear to have a genetic basis, early degeneration of the ovule pollen and ovule of fruit crops in response to temperature and genotype 161 seems to be environmentally affected and, thus, variable results can be obtained depending on location and year. the ovule degeneration is encompassed by callose layering at the chalazal end of the nucellus (stosser and ansari, 1982). later it was shown that as callose accumulates at the chalaza, translocation to the ovule is interrupted (pimienta and polito, 1982) and that this interruption is preceded by starch reduction in the ovule (rorigo an herrero, 1998). a shortening of style length, and abnormalities in ovary development have been reported under mild increases in temperature (an average of 3 oc higher than control) during the last week of flower development in apricot (rorigo an herrero, 2002), as well as under lower than optimal temperatures in mango (20/10 oc, day/night) (sukhbivul et al., 1999) resulting in lower fruit set. in ‘tainong 1’ mango, proportion of deformed ovaries with a short style for samples collected when temperatures are low during floral development is greater than that for samples collected at “normal” temperatures, however there is no significant difference occurred (huang et al., 2010). the length of stigmatic receptivity is shortened at high temperatures and enlarged at low temperatures in sweet cherry and peach regardless of the effect on the male side (hedhly et al., 2003; hedhly et al., 2004). temperature stress might affect stigma function by affecting the amount of exudates and their temporal availability to pollen grains (srinivasan et al., 1999). likewise, the analysis of carbohydrate content in chickpea flowers subjected to cold stress revealed a reduction in carbohydrate levels in aborted styles and ovaries compared to those retained in the plant (nayyar et al., 2005). a disruption in cell wall invertase activity was found under low temperature stress not only during the commonly reported pollen developmental phase but also in the stigma and the style (jain et al., 2007). taken together, these findings point out to a plausible effect of temperature stress on the sporophytic structures of the pistil and warrant further research. increasing the temperature from 5 oc to 25 oc in sweet and sour cherry (prunus cerasus l.) reduced ovule longevity from 5 days to 1-2 days (postweiler et al., 1985). similarly, a constant temperature of 20 oc in controlled growth chamber reduced ovule longevity in plum (prunus domestica l.) when compared to both field conditions and a lower constant temperature (cerovic et al., 2000). a temperature above 25 oc induced degeneration and/or suppression of embryo sac development in peach (kozai et al., 2004). callose deposition at the ovule chalazal end has been traditionally used as a microscopic feature to assess early ovule degeneration (vishnyakova, 1991). genotypic variation in pollen characters and ovule normality in response to temperature stress temperature effects on pollen grain germination seem to be cultivar or species dependent and optimum temperature for pollen germination and tube growth depends on species and varies between cultivars (mert, 2009; loupassaki et al., 1997). for example, in litchi, ‘floridian’ is much more susceptible to high temperature than ‘mauritius’, particularly during floral development but also with respect to male1 (m1) pollen germination in vitro at 35 oc. ‘floridian’ m1 pollen germinated at 15 oc, whereas, ‘mauritius’ m1 pollen did not (stern and gazit, 1998). high temperature regime also had a more severe detrimental effect on gynocium development in ‘floridian’ than ‘mauritius’ (stern et al., 1996). the difference between the two cultivars in their response to temperature may reflect the climate in their place of origin. ‘floridian’ is related to ‘brewster’ (degani et al., 1995), which probably is identical to the known ‘fujian’ cultivar ‘chenzee’ (cobin, 1954; groff, 1948). ‘chenzee’ is cultivated mainly in putian (25on latitude), whereas, ‘mauritius’, which is apparently identical to the chinese cultivar ‘tai so’, is cultivated mainly in the southern parts of fujian and guangdong (22-23on latitude) (gazit and goren, 1997). similarly, pollen performance in terms of viability, germination and tube growth were better from pollens of male2 (m2) than that of the respective male1 (m1) flowers in all the cultivars studied across different temperatures. in m1 pollens, lowest pollen viability was observed at 30/22 oc in all the cultivars and highest at 20/15 oc while in m2 flowers, the pollen viability was better at 20/15 oc for cultivars china, elaichi, early muzaffarpur and rose scented (93.89, 95.80, 96.41 and 92.60 %, respectively) and the viability decreased with increasing temperatures except for cultivar nafarpal whose pollen viability was found the same at both 30/22 oc and 26/20 °c (irenaeus, 2012). significant differences were observed in the pollen quality (viability, longevity, morphological homogeneity, pollen germination and pollen tube growth rate) among six species and cultivars of each prunus species including p. dulcis, p. armeniaca, p. domestica, p. ceracus, p. salicina and p. avium (sharafi, 2011). in sweet cherries, 20 °c was the optimum temperature for the in vitro germination of ‘bigarreu gaucher’ and ‘noble’ while 25 °c was optimum for ‘starks’ ‘bing’ ‘gold’ ‘vista’, ‘van’, ‘0900 ziraat’ and ‘stella’ (koyuncu and guclu, 2009). preincubation at 30 oc decreased pollen germination for olive cutivars ‘koroneki’, ‘kalamata’ and ‘amigdalolia’ but not for ‘mastoidis’, confirming the strong genotype-treatment interaction observed in olive (fernandezescobar et al., 1983). pollen tube growth on the pistil and stigma receptivity suggested that different peach genotypes responded differently to different temperature treatment and selections conserva 1566 and conserva 693 and cv. ‘maciel’ were tolerant to temperatures around 29 °c at beginning of blooming. such differences in pollen germination and tube growth within the same species were also found in pear (chagas et al., 2009; chagas et al., 2010). in juglans species, at 14 to 15 °c only pollen from early blooming varieties (serr, manregian, and early ehrhardt) germinated with 16.4 %, 2.2 % and 2.1 %, respectively. pollens of the later-blooming j. regia cultivars, ‘idaho’, ‘chico’, ‘sharkey’, ‘amigo’, ‘s. franquetta’, and ‘meylan’ did not germinate at temperatures below 16 to 18 °c and germination percentages ranging from 1.4 to 6.2 % in that temperature range (luza et al., 1987). best pollen germination for cultivars ‘şebin’, ‘kaplan 86’, ‘yalova 3’, ‘pedro’, ‘hartley’ and ‘franquette’ was noted at 27±1 °c (mert, 2009) while for cv. ‘yunxin’ germination occurred best at 25 °c (wu et al., 2008). percent germination of pollen of ‘manchurian’ crabapples and ‘golden delicious’ apple flowers on the stigmatic surface of ‘golden delicious’ pistils increased with increasing temperature from 13 to 29 °c in the first 24 and 48 h after pollination, respectively, but not thereafter. ‘manchurian’ was a more effective pollinizer of ‘golden delicious’ than was ‘golden delicious’ pollen. for example, at 24 or 29 °c, some ‘manchurian’ pollen tubes grew to the base of ‘golden delicious’ styles by 24 h after pollination. on the other hand, no ‘golden delicious’ pollen tube grew to the base of a ‘golden delicious’ style regardless of temperature and time (yoder et al., 2009). difference in pollen viability among the cultivars of hazelnut (hoseinava et al., 2010) and germination of pistachio vera l. (kamiab et al., 2007) was also obtained and pollen tube length ranged from 697 to 1270 μm (acar and kakani, 2010). in citrus, pollen performance is not only an inherent characteristic of the pollen genotype, but is largely dependent on the particular male-female combination and on genotype-temperature interactions (distefano et al., 2012). temperature requirement for the pollen germination and pollen tube growth in fruit species and cultivars may differ in early or late flowering period. for example, the desired temperature for optimum pollen germination of walnut was found comparatively low in early flowering cultivars than late flowering walnut counterparts (luza et al., 1987). a somewhat similar pattern of temperature responses by pollen was noted in a comparison of almond (prunus dulcis) and peach (p. persica), two closely related species that differ in bloom 162 k.s.t. irenaeus, s.k. mitra time (weinbaun et al., 1984). pollens from the earlier blooming almond had higher germination percentages at low temperatures and a lower temperature for maximum germination was needed. in vitro pollen germination and pollen tube growth of different pistacia genotypes varied with temperature. the maximum percentage of pollen germination and pollen tube length of genotypes, and tmin and tmax were the most important parameters describing genotypic tolerance to low and high temperatures (acar and kakani, 2010). a heat shock of 40 °c is sufficient to induce mrnas for heat shock proteins (hsp) in pollen of maize (hopf et al., 1982) revealing the induced resistence of plant in order to protect (defence mechanism) the cells against damage and/ or death. high temperature proved to be lethal for the pollen of the cultivars studied and the determination of hsp mrnas presence in the pollen might help explain why differential thermotolerance was observed among the cultivars studied. variations due to flower type in response to temperature stress in crop like litchi, there are two type of male flowers; male (m1) flower and the pseudohermaphrodite (m2) flower (functionally male) and differences in the pollen quality of this flower types has been reported. a consistent and usually significant advantage of m2 over m1 pollen was found when pollen from five cultivars (mauritius, floridian, no mai chee, wai chee and early large red) was germinated in vitro under five different (15, 20, 25, 30 and 35 °c) temperature regimes (stern and gazit, 1998). the germination rate of m2 pollen was consistently greater (p=0.01) than that of m1 pollen in ‘wai chee’, ‘no mai chee’, and ‘early large red’. the maximal germination reached 55 % to 59 % for m2 pollen, but only 8 % to 19 % for m1 pollen. the optimal temperature for m2 pollen germination was 30 °c in all cases. the same temperature was also optimal for the germination of ‘no mai chee’ m1 pollen, whereas, germination of ‘wai chee’ and ‘early large red’ m1 pollen was greatest at 25 °c. similarly, germination was better for m1 pollens at 24 oc in most of the cultivars and germination percentage decreased with increasing temperatures though for cultivar bombai it was higher at 28 oc. the pollens of m2 flower in most of the cultivars showed better germination at 28 oc though cultivars like bedana, elaichi and nafarpal germinated better at 30 oc. however, decrease in pollen germination was observed at lower and higher temperature extremes of 24 oc and 32 oc in all the cultivars (irenaeus, 2012). earlier, higher germination rates for m2 pollen were reported (mustard et al., 1953; costes, 1988) and such similarities in six cultivars (fivaz et al., 1994) though no such advantage of m2 over m1 was recorded in cv. bengal. pollen tube growth rate was also reported to be faster for m2 than m1 pollen (stern and gazit, 1998; irenaeus, 2012) with the maximum growth in cv. rose scented (irenaeus, 2012). higher fruit set was also obtained from the flowers pollinated by m2 pollens. they opined that the difference in viability of the two pollens must be phenotypic as they were identical genetically. no difference in the size and shape of the pollens were found by (stern and gazit, 1998). it was thus, assumed that m2 flowers secrete more nectar and sugar than m1 flowers (stern and gazit, 1996) which indicates that the m2 flower is a much stronger sink, and this may also manifest itself in a better supply of nutrients to its developing pollen. other factors affecting in vitro pollen germination and tube growth nutrient media validity of the in vitro evaluation of pollen germination is a predictor of in vivo behaviour (hormoza and herrero, 1999; tunistra and wedel, 2000) and therefore it is important to standardize the nutrient media to get the best pollen germination and tube growth. and since the fruit set and yield largely depends on the pollen quality, nutritional requirements for successful pollen germination and pollen tube growth in vitro will help growers and researchers to supply the plant with different type of nutrients at proportionate amount. correlation of pollen germination and ovule normality with nutritional requirements in vitro and in vivo leaf nutrient content at flowering may be considered for standardization of critical nutrient content. also correlation studies of these parameters at different temperature regimes is important so as to categorize the genotypes for region specific recommendation and differential response of the genotypes to temperature may be due to variation in its metabolic process at different temperature. this response to nutrient media was found to vary from species to species and cultivar to cultivar. suitable culture medium for pollen germination in vitro of wild apricot was reported as no. d contained 1 % agar, 10 % sucrose, and 0.01 % boric acid, where pollen germination rates was 75.4 % and suitable concentration of sucrose and boric acid had certain promotion function for germination (qiang et al., 2012). in avocado, maximum pollen germination could be achieved by using a medium containing 10 % sucrose and 23 % polyethylene glycol (peg) (alcaraz et al., 2011). the addition of peg to the pollen germination medium was recommended in anacardium (subbaiah, 1984). sucrose, boric acid, and calcium nitrate have been shown to be the key substrates for pollen germination in other species (tunistra and wedel, 2000). similarly use of sucrose and agar supplemented with boron as culture medium for pollen germination and tube growth has been done in surinam cherry (eugenia uniflora l.) (franzon et al., 2007). when the concentration of sucrose was 30 g l-1 and boric acid was 3 g l-1, the internal and external osmotic pressure of cherry cv. ‘duan bing’ could keep balance and the ratio of pollen germination was the highest (qiang et al., 2009). 15 % sucrose concentration gave the highest germination rates for walnut cultivars (sutyamez, 2007). other researchers also noticed that, 15 % and 20 % sucrose concentration gave the highest germination rates in various fruit species (sutyamez and eti, 2006; asma, 2008). generally, the best pollen germination rates in most of the walnut cultivars studied were obtained from 15 % and 20 % sucrose concentrations while in cv. ‘pedro’ best germination rates were obtained with 10 % sucrose concentration (mert, 2009). however, 100 g sucrose+10 mg boric acid+40 mg cacl2 l-1 was found best for germination of cv. ‘yunxin’ (wu et al., 2008). in pear, use of 10 g l-1 of agar combined with 90 g l-1 sucrose for the rootstock ‘taiwan mamenashi’ and 47.78 g l-1 for the ‘taiwan nashi-c’, with 795 to 838 mg l-1 of boric acid, under absence of calcium nitrate and ph between 5.2 and 5.8 and temperature of 28 °c, provided the best conditions to the germination of the pollen grains (chagas et al., 2010). similarly, for the aurora 1 cultivar, higher germination of pollen grains was obtained with the use of 48.29 g l-1 of sucrose, 10 g l-1 of agar, 400 mg l-1 of boric acid and ph 5.5. for the douradao cultivar, higher germination was obtained on medium containing 90 g l-1 of sucrose, 10 g l-1 of agar, 400 mg l-1 of boric acid, 369 mg l-1 of calcium nitrate and ph 6.5 (chagas et al., 2009). however, optimum temperature for germination was recorded to be 25 °c in both the cultivars. sucrose plays a nutritive role for pollen germination and no germination occurred in avocado at a concentration less than 5 % sucrose. variations in the effect of different sucrose concentrations are associated to different osmotic potentials (alcaraz et al., 2011). sucrose acted in the regulation of osmotic optimal conditions and nutritional source for pollen germination and pollen tube development (taylor and heper, 1997). peg is an osmotic regulator not metabolized in pollen that is thought to regulate the permeability of the plasma membrane (shivanna and sahwney, 1995) and can be highly effective to promote pollen germination and reduce bursting (vasil, 1987), although its mechanism of action is not well understood. pollen and ovule of fruit crops in response to temperature and genotype 163 duration of incubation for germination and growth pollen grains showed different rates of viability and germinability in relation to storage length, storage temperature, genotype and their interactions in four olive cultivars viz., ‘carolea’, ‘frantoio’, ‘leccino’ and ‘moraiolo’ though no linear correlation has been found between viability and germination rates (ferri et al., 2008). the maximum percentage of germinated pollen is obtained with eight hours after inoculation for ‘taiwan nashi-c’ and twelve hours for ‘taiwan mamenashi’ pear (chagas et al., 2010). poor fruit set due to temperature stress many cultivated and wild plant species within different climatic regions worldwide perform poorly and display erratic fruit and seed set when subjected during their flowering phase to high and low temperatures beyond their optimum needs. for example, in temperate fruit trees, a substantial reduction in fruit set has been shown in apricot (prunus armeniaca l.) after an increase in the average daily temperature as low as 3 °c during the week preceding anthesis (rodrigo and herrero, 2002), or in sweet cherry (prunus avium l.) during the first two weeks following anthesis (hedhly et al., 2007). similarly, results in peach (prunus persica l. batsch), show that a two month exposure to different constant temperatures in growth chambers, starting one month before anthesis, resulted in very low fruit set at above 25 °c (kozai et al., 2004). cherimoya (annona cherimola mill.), native to the tropical highlands of the andes, usually displays lower fruit set in tropical lowlands and in temperate regions. an increase in temperature from 20 °c to 30 °c during two consecutive years substantially reduced fruit set and increased the number of malformed fruits (higuchi et al., 136). mango has been shown to be prone to recurrently erratic fruit set with an increased proportion of stenocarpic fruits when temperature falls below 10 °c during flowering (sukhvibul, 2005). low temperatures adversely affected inflorescence and floral development (issarakraisila et al., 1992), causing morphological changes in styles, stigmas, ovaries and anther size in ‘nam dok mai’, ‘kensington’, ‘irwin’ and ‘sensation’ cultivars (sukhvibul et al., 1999). sternospermocarpic mangoes were reported to be caused by low temperatures (ram et al., 1976). low temperature during flowering might also interfere fertilization and/or ovule development in early stage of fruit development and hence resulted in sternospermocarpic fruit set (lim et al., 1996). a tendency towards ovule degeneration, both in pre-fertilised and post-fertilised stages may seriously impair fruit set. this appears to be a specific genetic character in ‘doyenne du comice’ pear that is responsible for the poor fruit set of this cultivar (jaumien, 1968). in the same way, the high proportion of both degenerated embryo sacs and embryo sacs that lack a functional egg cell is responsible for the poor fruit set of ‘constant’ apricot, regardless of weather conditions (eaton and jamont, 1965). conclusion a wide variation exists not only among the species in terms of pollen viability, germination, pollen tube growth, ovule normality and the specific requirements for the reproductive process to occur but also among the cultivars within the same species. also variation in these characters are not only genotypic but is also influenced greatly by temperature. optimum temperature at which such reproductive process can best occur has been assessed and established in many crop species and cultivars. nutritional requirements for in vitro germination and growth of pollens has also been standardized for many crop species and this can be considered for establishing in vivo nutritional requirements and standardizing critical leaf nutrient standards. accordingly, cultivars with higher pollen viability and germination and or higher temperature stress tolerance can be identified and selected for use as pollinizers, breeding purpose and region specific adoption based on temperature prevalence in the area. pollen release and germination ability might be suggested as a good criterion for determining plant response to high temperature and this can be used as selection criteria in breeding programmes for selecting heat tolerant genotypes. poor fruit set and yield may be largely attributed to impairment of pollen and ovule quality under high temperature is the most important factors to determine the fruit set ability. pollen performance is also dependent on the particular male-female combination and on genotype-temperature interactions as in citrus (distefano et al., 2012). decreased pollen number and viability at high temperature may be due to anther indehiscence, polyamine synthesis inhibition, degeneration of tapetum layer and/or decreased carbohydrate metabolism, while, there are contrary reports that at higher temperature, impairment of pollens is due to higher sucrose and starch accumulation as a result of reduced utilization and starch degradation rather than biosynthesis prior to anthesis. though the mechanisms in the reduction of pollen quality under low temperature has also been attributed to alterations in starch accumulations and decreased respiratory activity, the precise nature leading to poor pollen development is still not clear. depolymerization of actin cytoskeleton in low temperature stress is also reported which induce activation of 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effects of temperature and the combination of liquid lime sulfur and fish oil on pollen germination, pollen tube growth, and fruit set in apples. hortscience 44, 1277-1283. zhao, d.y., cheng, l.g., guo, l.d., jun, q.s., yu, m.h., 2008: influence of environmental factors on pollen performance of sweet cherry in solar greenhouse during pollination and fertilization period. j. fruit sci. 25, 506-509. zinn, k.e., tunc-ozdemir, m., harper, j.f., 2010: temperature stress and plant sexual reproduction: uncovering the weakest links. j. exp. bot. 61, 1959-1968. pollen and ovule of fruit crops in response to temperature and genotype 167 address of the authors: dr. k.s. thingreingam irenaeus1 (corresponding author) and prof. dr. s.k. mitra2, department of fruits and orchard management, faculty of horticulture, bidhan chandra krishi viswavidyalaya, mohanpur, po – krishi visavidyalaya, nadia 741252, wb, india. e-mail: 1angamire@yahoo.co.in; 2sisirm@vsnl.net journal of applied botany and food quality 88, 164 169 (2015), doi:10.5073/jabfq.2015.088.023 dr. y.s. parmar university of horticulture and forestry, nauni – solan, (h.p.) india exploitation of malus ests for development of ssr markers after in silico analysis era vaidya, rajinder kaur*, krishan kumar, neha sharma (received march 29, 2014) * corresponding author summary the domestic apple (malus × domestica) has been extensively characterized for est sequences and currently 330181 ests are available in ncbi. although this is a valuable resource for marker development, the redundancy in sequences makes the mining out of unique candidates for designing markers cumbersome. keeping this in view, the present study was undertaken with the aim to remove the data redundancy in apple ests and then to develop ssr markers. the est sequences were assembled into a non-redundant set of 5619 contigs and 59343 singletons (total sequences 64962), indicating 80 % reduction in data redundancy. these 64962 sequences were then used to mine out ssr motifs. 4575 est-ssrs were detected, with dinucleotide repeats being predominant (72 %), followed by trinucleotide repeats (25 %). these markers were validated by designing primer pairs. 25 of these designed primers were tested on a group of 48 apple accessions. all the markers gave robust amplification, generating 93 % polymorphism. these findings indicate the usefulness of est-ssrs in genome analysis. this study further emphasizes the importance of assembly of the vast amounts of data submitted in public databases. our results have generated a set of non redundant apple ests which is of prime importance for development of marker systems without any repetition or overlapping. introduction although a vast array of genetic marker systems are available in literature for crop genome analysis, their efficiency and density across the genomes varies considerably. markers based on ssrs are the markers of choice in genetics and breeding studies due to their multi-allelic nature, transferability over genotypes and extensive genome coverage. in apple a number of microsatellite markers have been developed and deployed for the study of genetic polymorphism (potts et al., 2012; zhang et al., 2012; kitahara et al., 2005; liebhard et al., 2002). however, most of these are genomic ssrs whose development is highly laborious, cumbersome and cost-intensive. advances in genomic technologies have generated a large number of ests, which are available in public databases, thereby offering an opportunity to develop est derived ssr markers by data mining. est-ssr markers are expected to possess high transferability across boundaries as they belong to relatively conserved genic regions of the genome. the large apple (malus × domestica) est database (comprising over 300 000 sequences) provides a valuable resource for developing well characterized molecular markers (newcomb et al., 2006; gasic et al., 2008; igarashi et al., 2008). however, it has been observed that large datasets are highly redundant and contain a large number of repetitions in sequences. little attempt has been made in reducing the redundancy present in apple est dataset, so as to develop a set of non-repetitive est sequences. keeping the above point of view, the present study was taken up with the aim to remove the data redundancy in apple ests by assembly into singletons and contigs. this non-redundant set was used to mine out ssrs, which were further analyzed for abundance and distribution of different types of repeats. these ssrs were then used to develop novel ssr markers for apple, which were tested on a set of 48 apple accessions to check for their usefulness in genetic analysis and related applications in genetics and plant breeding. material and methods retrieval of est sequences est sequences belonging to apple (malus × domestica) were ob tained in their fasta format from the est database on the ncbi website. a single text file containing all the sequences was compiled. est sequence assembly a non-redundant data set was derived from the redundant set of 330181 apple ests for clustering and assembly analysis. an automated trimming and screening for various contaminants and low quality and complexity sequences was done through egassembler web server (masoudi-nejad et al., 2006). the server clustered and assembled the sequences into contigs and singletons using cap3 (huang and madan, 1999) with the criterion of 80 % overlap identity between one end of a read to another end. the results were obtained in two different output files, one comprising of contigs and the other of singletons. for ssr identification, these two files were combined to form non-redundant sequence data set. mining of ssrs for detection of ssrs present in the est sequences, a modified version of a perl script ssr identification tool (ssrit) (temnykh et al., 2001; http://www.gramene.org/db/markers/ssrtool) was used. the ssrs were screened out by setting the search parameters to identify at least five repeats of ssr motifs with a maximum of ten base pairs. this software program takes a fasta formatted sequence file as an input and produces an output file with sequence name, number of ssrs in the sequence, ssr type, ssr motif, repeat number, sequence coordinates for ssr and the length of the sequence. primer designing and marker validation the ssrs detected were used for primer design. primer pairs were designed to flank the target ssr repeats using primer3 software (rozen and skaletsky, 2000) (www.frodo.wi/mit.edu/primer3/). the parameters for designing primers were as follows: primer length between 18 – 27 bp, gc content between 20 – 60 %, and optimum primer tm between 57 oc – 63 oc. the designed primer pairs were synthesised by eurofins mwg/operon (eurofins genomics, bangalore, india). 25 primer pairs were custom synthesized and were validated for their ability for amplification on a set of 48 apple accessions. est-ssr markers in apple 165 ssr-est similarity searches the sequences used for primer designing were also analyzed for putative functions by pairwise comparison against the swissprot/ uniprot protein database using blastx program at the uniprot server (http://www.uniprot.org/), assuming an e value of < 1e-5 as a significant criterion of homology. the most significant matches for each sequence were recorded. gene ontology descriptions were assigned to ssr-ests on the basis of swiss-prot protein sequence matches. ssr protein domain analysis the sequences containing ssrs were also searched for functional protein domains using prosite (http://www. http://prosite.expasy. org/prosite.html). it has been observed that some regions, important for the function of a protein and/or for the maintenance of its three dimensional structure have been better conserved than others during evolution. the properties of such groups of similar sequences are analyzed to derive a signature for a protein family or domain, which distinguishes its members from all other unrelated proteins. marker validation by pcr amplification and gel electrophoresis a pcr protocol was standardized to amplify est-ssr markers. the target sequences were amplified in 10 μl pcr reactions each containing 1×pcr buffer (genie, bangalore, india), 2.2 mm mgcl2, 0.3 mm dntps, 20 μm of each primer, 1 unit taq dna polymerase and 30 ng leaf dna. ssr markers were amplified in a thermal cycler (applied biosystems), with the following programme: 5 min initial denaturation at 95 oc, followed by 40 cycles each of 1 min denaturation at 94 oc, 1 min annealing at the tm for each primer and 2 min extension at 72 oc, with a final extension step of 5 min at 72 oc. the pcr products were then analysed on 2.5 % (w/v) agarose gels. electrophoresis was performed at 80v in 1×tae buffer (40 mm trisacetate, 1 mm edta; ph 8.0). the gels were stained with ethidium bromide (0.5 μg μl-1) and visualized under uv light. data analysis genetic diversity, defined as polymorphism information content (pic) (anderson et al., 1993), was used to measure allelic diversity at each ssr locus. pic values were calculated as follows: pic = 1−σpi2 where pi was the frequency of the ith allele in the set of genotypes analysed. the presence or absence of bands were encoded in binary mode by 0 and 1, respectively and used to derive a similarity matrix based on jaccard’s coefficients. a dendrogram was generated based on this matrix based on upgma clustering using ntsyspc ver. 2.02h software. results est sequence retrieval and assembly 330181 est sequences belonging to apple were retrieved from the est database. the obtained sequences were found to be related to different plant tissues such as leaves, stem and root. after sequence assembly, 5619 sequences were successfully assembled into contigs. the remaining 59343 showed no overlap with any ests, called as ‘singletons’. the whole dataset was reduced to 64962 sequences, prior to the ssr analysis. reduction in data redundancy in the assembled ests set of contigs and singletons was to be found 80.33 % (tab. 1). frequency of ssrs ssrs in the ests were detected using the online interface of ssrit. 849 (15 %) contigs and 3724 (6 %) singletons were found to contain ssrs. analysis of ssr-ests revealed that dinucleotide ssrs were the most common ssrs at 72 %. these were followed by trinucleotide ssrs at 25 %. tetranucleotide, pentanucleotide and hexanucleotide ssrs were less frequent, i.e. 1.24 % ssrs were tetranucleotide, 0.37 % were pentanucleotide and 0.61 % were hexanucleotide repeats, respectively. frequency of occurrence of different ssr motifs was also calculated. among the dinucleotide repeats, tc/ct motif was found to be the most frequent (37.76 %) and cg/gc (0.09 %) was least frequent (tab. 2). among trinucleotide repeats, the most frequent motif was ctc/tct/ctt/ttc/cct/tcc at 20.49 % and cgg/ccg/cgc/ gcg/gcc and tca/cat/atc/act were the least frequent motifs at 4.46 % (tab. 2). tab. 1: results of est sequence assembly total number number number number of % reduction of est sequences of singletons of contigs assembled sequences in redundancy 330181 59343 5619 64962 80.33 % tab. 2: distribution of repeat motifs s. no. repeat motif number frequency dinucleotide repeats 1. ag/ga 1159 34.84 % 2. ac/ca 185 5.56 % 3. tc/ct 1256 37.76 % 4. gt/tg 148 4.45 % 5. at/ta 575 17.28 % 6. gc/cg 3 0.09 % total 3326 trinucleotide repeats 7. ctc/tct/ctt/ttc/cct/tcc 234 20.49% 8. aac/cac/acc/cca/caa/aca 169 14.79% 9. cag/cga/agc/acg/gca/gac 143 12.52% 10. tgt/gtg/ttg/tgg/gtt/ggt 63 5.51% 11. cgg/ccg/cgc/gcg/gcc/gc 51 4.46% 12. tgc/gct/cgt/gtc/ctg/tcg 109 9.54% 13. tca/cat/atc/act 51 4.46% 14. gga/gaa/aag/aga/gag 215 18.82% 15. atg/tga/gat/gta/agt/tga 55 4.81% 16. taa/tta/aat/att/tat/ata 52 4.55% total 1142 166 e. vaidya, r. kaur, k. kumar, n. sharma tab. 3: list of est-ssr markers designed sr.no. sequence id sequence base pairs 1. gi226857985 f: cggtgcatgtacaacacgta 20 r: tccagcagaagaggtgatga 20 2. gi226857903 f: gtattggctccaccacaacc 20 r: gaaacccggtcgtgtaaaaa 20 3. gi226857895 f: aaagtggcgtaaggttgtgg 20 r: cacgctggacacatacatcc 20 4. gi226857649 f: tttcttctcttctccaatcctctg 24 r: ttgcagagaaagatcaaggtga 22 5. gi226857752 f: ggcccaaatgtcatcgaata 20 r: tattccaaattcccggactg 20 6. gi226814813 f: aacatatggggcagcgtatt 20 r: ttgtgatgcttgtggggata 20 7. gi226811802 f: tcctctggagtagcgagcac 20 r: accgtcaacgaggctcag 18 8. gi226805503 f: caacaaagccttttcccagt 20 r: ttattcggcctttgttttgg 20 9. gi226798745 f: cacgtttccaagagccttaca 21 r: tttgcccttaaaagttgggtta 22 10. gi226796317 f: tcatcgccaccttgatgata 20 r: agctcaaagaggccgtcata 20 11. gi226794476 f: cacttgggccaatttctgac 20 r: ccaaggagtgagtggaggag 20 12. gi|226814826 f: ttccaacgacaccaaccttt 20 r: gcgatgatgtatggcacaga 20 13. gi|226812665| f: tccgacaaatcaacattgga 20 r: ctgacggacgctcataacaa 20 14. gi|226810461 f: tgatgatgacgatgacgatg 20 r: gagcaaaagttgaaaccctca 20 15. gi|226810487| f: gggctgcttgatttgaactt 20 r: gaagctaaaacttcacccttga 22 16. gi|226810743| f: tgttcatgtcggtgctcaat 20 r: gacgatgatcaggccattct 20 17. gi|226811551| f: actgagggcttgtgttggtc 20 r: gcttctgaacgaggaagacg 20 18. contig 48-1 f: tggtgtgccctgaagtgtta 20 r: tatgggagcagcaaataccg 20 19. contig 51-1 f: aacaaggaacctattttccgaac 23 r: cagggagaaacgagaggtca 20 20. contig 175-1 f: ccagtagagggccaaaaaca 20 r: tctgtcaccggatagtgctg 20 21. contig 208-1 f: agcccaaaaggcagtctaca 20 r: acccccattttcagcctatc 20 22. contig 139-2 f: cgctccagaagtcaacatca 20 r: ggagctccaattccatttca 20 23. contig 10-1 f: tgttagatccggacccactc 20 r: agatggaaggggagaagagc 20 24. contig 1-2 f: tgaggtgaccacaagcaaag 20 r: caattcttttgtgcgcgtat 20 25. contig 27-1 f: ttttccccctcaagaacaaa 20 r: tgaagccgaaggttcatcat 20 est-ssr markers in apple 167 primer designing ssr primers were successfully designed for 25 sequences using primer 3, for the purpose of polymorphism study. 17 singletons and 8 contigs were selected from the assembled data set and used for primer designing (tab. 3). blastx analysis for sequence identification blastx analysis was carried out for the est sequences selected for primer designing. based on this analysis, a putative function could be assigned to 22 of the potential est-ssr markers (15 singletons and 7 contigs) assuming a threshold value of <1e-5. the annotation results indicated that 13 est-ssr sequences showed high homology with prunus persica proteins, while four were observed to reveal high similarity with well characterized malus proteins. analysis of ssr-protein domains the 25 ssr containing sequences used for primer deigning were also analyzed for protein families and domains through prosite. a functional protein domain could be assigned to 23 sequences. the sequences were recorded to possess functional domains responsible for protein signatures like 2fe-2s ferredoxin binding sites, anaphylatoxin domain signature, vwfc domain and thiolases active site. the ssr-fdms provide information regarding transcribed genetic markers having putative functions. ssr-ests of apple were gene ontologically assigned with their major molecular function, biological process and cellular component categories. gene ontology for the corresponding ssrs was determined on the basis of sequence, domain and motif similarity. this indicates an indirect role of above ssr-ests in development of genetic markers for the protein and enzymes involved in various cellular processes. marker validation by pcr amplification the 25 primer pairs were used to study genetic polymorphism in a set of 48 apple accessions (including cultivated varieties, rootstocks and wild relatives). all the primers amplified discrete dna fragments, 24 being polymorphic (96 %) and detected 94 alleles. the total level of polymorphism among the studied accessions of apple found to be 93 %. pic values were calculated for all the polymorphic primers. the pic was found to range from 0.07 to 0.81. fig. 1 shows amplification pattern obtained with primer e19. jaccard’s similarity matrix coefficient was obtained through ntsyspc. the similarity coefficient values ranged from 0.303 to 0.903. the generated dendrogram revealed a wide genetic base of the apple germplasm collection studied. the dendrogram generated for estssr markers divided the genotypes into three main clusters, a, b and c (fig. 2). group a could be further classified into subgroup a1 and a2. subgroup a1 comprised of ‘early red one’, ‘red fuji’, ‘compact winter banana’, ‘black ben davis’, ‘granny smith’, ‘spartan’, ‘top red’, ‘early mcintosh’, ‘well spur’, ‘anna’, ‘malus orientalis’, ‘chaubattia ambroise’, ‘ambred’, ‘malus baccata kashmir’, ‘malus sargentii’, ‘m7’, ‘red chief’, ‘hardeman’ and ‘vance delicious’. subgroup a2 contained ‘malus simcoe’, ‘malus baccata kinnaur’, ‘b9’, ‘gale gala’, ‘scarlet spur’, ‘m9’ and ‘m793’. group b formed the other major cluster. it could be subgrouped into b1 and b2. b2 was found to contain only two accessions, i.e. ‘gloster’ and ‘red spur’. ‘ b1 comprised of accessions, ‘ambroyal’, ‘bright-n-early’, ‘royal’, ‘malus baccata shillong’, ‘golden spur’, ‘m26’, ‘mm106’, ‘oregon spur’, ‘mutsu’, ‘malus eseltine’, ‘skyline supreme’, ‘silver spur’, ‘hardispur’, ‘golden delicious’, ‘scarlet gala’, ‘mm111’, ‘miller sturdy spur’ and ‘criterion’. group c contained ‘red delicious’ and ‘tydeman’s early worcester’. discussion est databases represent a potentially valuable resource for developing molecular markers for evolutionary studies. since est derived markers come from transcribed regions of the genome they are likely to be conserved across a range of genomes than other types of markers (pashley et al., 2006). this is promising, considering the increasing number of est-derived ssr markers in various crops. fig. 1: banding pattern with est-ssr primer e19; 1-48: apple accessions; mmolecular weight ladder 168 e. vaidya, r. kaur, k. kumar, n. sharma theoretically, the frequency of a repeat should decrease as its length increases. accordingly, in the present study dinucleotide repeats are reported to be more abundant among the ssr types (72 %). similar reports have been obtained in other plant species (li et al., 2002). the most dominant dinucleotide repeat motif was ct/tc followed by ga/ag (37 % and 34 %, respectively). a high abundance of (ga)n microsatellites has also been reported in some other plant genomes (guyomarc et al., 2002; gupta and varshney, 2000; suwabe et al., 2002). preference was given to trinucleotide repeats in ssrs, based on the hypothesis that di-, tetra-, or pentanucleotide variation would produce shifts in the reading frame that would result in negative selection and a lesser degree of polymorphism (varshney et al., 2005) marker validation and genetic diversity ests have been effectively utilized for the development of ssr markers which have been subsequently used in the genetic diversity analysis (kumar et al., 2011; vaidya et al., 2012). est-derived markers have been well documented in marker assisted selection that involves mainly the identification of qtl for important traits, genetic mapping, genetic diversity, and comparative genomics (cloutier et al., 2009; durand et al., 2010; kumar et al., 2011; vaidya et al., 2012). est-ssrs also have the potential for direct linkage association to functional genes encoding important horticultural traits. the level of polymorphism in the apple accessions revealed using the developed ssr markers, was relatively high, though a limited number of primer pairs had been used. a total of 25 est-ssrs were used to amplify dna from 48 apple accessions. all the primers yielded scorable bands, out of which 24 were found to be polymorphic. it was not possible to reach 100 % success rate to design and amplify ssr primers, as was also reported by szewc-mcfadden et al. (1996). the pic values, which provide an estimate of the discriminatory power of a locus or loci, recorded for all the informative primers were found to range between 0.07 to 0.81. the success rate for est-ssr primers (percentage of ssr primers producing discrete amplification products) in various reports was between 60 and 90 % (varshney et al., 2005). in this study, the relatively high level of polymorphism observed with apple est-ssrs at the cultivar level might suggest that the apple cultivars used in this study were derived from cross-pollination and propagated vegetatively, and thus they might have a high level of heterozygosity, which can increase the polymorphism level. conclusion the present study attempts to ascertain the frequency and distribution of ssrs in the apple est database and develop those est-ssrs for use in genetic studies. we demonstrated the utility of computational approaches for mining ssrs from ever increasing repertoire of publicly available plant est sequences present in different databases. the resulting est-ssr set is a valuable tool for further genetic and genomic applications. the developed 25 est-ssr markers have a high rate of pcr amplification and can be used in apple breeding and genetic studies. the use of these markers would reduce the cost and therefore facilitate cultivar identification, genetic distance assessments, gene mapping and possible marker-assisted selection (mas). the functional categorization of these markers corresponded to many genes with biological, cellular and molecular functions, thus providing an opportunity to investigate the consequences of ssr polymorphisms on gene function. references anderson, j.a., churchill, g.a., autrique, j.e., tanksley, s.d., sorrells, m.e., 1993: optimizing parental selection for genetic linkage maps. genome 36, 181-186. fig. 2: dendrogram showing genetic relationship among 48 apple accessions est-ssr markers in apple 169 cloutier, s., niu, z.x., datla, r., duguid, s., 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(eds.), bioinformatics methods and protocols: methods in molecular biology, humana press, totowa nj. suwabe, k., iketani, h., numone, t., kage, t., hirai, m., 2002: isolation and characterization of microsatellites in bassica rapa l. theor. appl. genet. 104, 1092-1098. szewc-mcfadden, a.k., kresovich, s., bliek, s.m., mitchell, s.e., mcferson, j.r., 1996: identification of polymorphic, conserved simple sequence repeats (ssrs) in cultivated brassica species. theor. appl. genet. 93, 534-538. temnykh, s., declerk, g., lukashova, a., lipovich, l., cartinhour, s., mccouch, s.r., 2001: computational and experimental analysis of microsatellites in rice (oryza sativa l.): frequency, length variation, transposon associations, and genetic marker potential. genome res. 11, 1441-1452. vaidya, e., kaur, r., bhardwaj, s.v., 2012: data mining of ests to develop dbest-ssrs for use in a polymorphism study of cauliflower (brassica oleracea var. botrytis). j. hortic. sci. biotech. 87, 57-63. varshney, r.k., graner, a., sorrels, m.e., 2005: genic microsatellite markers in plants: features and applications. trend biotechnol. 23, 4855. zhang, q., li, l., zhao, y., korban, s., han, y., 2012: evaluation of genetic diversity in chinese wild apple species along with apple cultivars using ssr markers. plant mol. biol. rep. 30(3), 1-8. address of the authors: department of biotechnology, dr. y.s. parmar university of horticulture and forestry, nauni – solan, himachal pradesh, india © the author(s) 2015. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 86, 212 216 (2013), doi:10.5073/jabfq.2013.086.029 1vegetable research department, national research center, dokki, cairo, egypt 2plant nutrition department, national research centre, dokki, cairo, egypt 3institute of plant sciences and resource conservation, division of horticultural sciences, university of bonn, bonn, germany drought tolerance and water status of bean plants (phaseolus vulgaris l.) as affected by citric acid application w.a. el-tohamy1, h.m. el-abagy1, m.a. badr2, n. gruda3 (received june 05, 2013) summary enhancement of drought tolerance of plants is a crucial concern in arid and semi-arid regions. using safe and environmentally-friendly tools and treatments for this purpose is needed to overcome the problems of water shortage with particular emphasis on sustainable resource management and environmental protection. this study investigated the water status and drought tolerance of beans. bean plants (phaseoulus vulgaris l.) were treated with citric acid (0.5, 1, 1.5 and 2 g/l) as a foliar application prior the exposition to drought stress conditions. physiological changes, such as leaf temperature, relative water content (rwc) and chlorophyll content of leaves, were recorded in response to citric acid application. the results revealed that the water status of bean plants under drought stress conditions was improved by citric acid application, indicated by higher rwc of leaves compared to control plants. the most effective level in this respect was 1.5 g/l. a similar trend was observed with total chlorophyll content of leaves. in addition, plant growth, productivity and quality parameters were significantly improved by application of citric acid compared to control plants. the possible roles of citric acid on water status and drought tolerance of bean plants are discussed. introduction bean plants (phaseolus vulgaris l.) are important protein source world-wide. however, they are sensitive to drought conditions. growth and productivity of bean plants are extremely affected by water stress (millar and gardner, 1972). moreover, according to el-tohamy et al. (1999) water stress resulted in a decline of leaf water potential, stomatal conductance, photosynthesis rate and all growth, productivity and quality parameters of bean plants. finding relatively safe tools and treatments to overcome the negative effects of drought stress or improve drought tolerance of sensitive plants could be of great value especially under arid and semi-arid conditions where shortage of water becomes a limiting factor for plant growth and productivity. recently, there is a tendency to explore the relationships between citric acid accumulation in plants and improvement of drought tolerance. for example, levi et al. (2011) found that the accumulation of some organic acids including citric acid could contribute to superior capacity of some lines of cotton to cope with drought. merewitz et al. (2012) indicated that the enhanced drought tolerance of some drought tolerant lines of creeping bentgrass was associated with the maintenance of accumulation of several metabolites including the organic acids that are mainly involved in the citric acid cycle. they concluded that the accumulation of these metabolites could contribute to improved drought tolerance. other authors, emphasized the importance of citric acid for improving quality of fruits (such as tomato, pear and citrus fruits) and its accumulation is evident under mild water stress (morinage and sykes, 2001; nahar and gretzmacher, 2002; papadopoulus et al., 2004; kang et al., 2009; and iwasaki et al., 2011). moreover, sun and hong (2011) reported that exogenous citric acid significantly increased internal citric acid concentration in plant tissues and improved plant growth and stress tolerance during exposure to saline stress conditions. on the other hand, darandeh and hadavi (2011) indicated that foliar spray of citric acid during growth stage significantly increased post-harvest vase life of the cut lilium flowers. it seems that this compound is able to be taken up by plant cells, although the leaf surface is covered with cutin. recently, burkhardt et al. (2012) studied the stomatal penetration by aqueous solutions and their results clearly confirmed the stomatal uptake of aqueous solutions. however, investigations concerning the effect of citric acid on water status of other sensitive plants such as bean plants are scarce in the literature. the present study aimed to explore the effects of citric acid on drought tolerance and water status of bean plants subjected to drought conditions. materials and methods the experiments were carried out at the national research center during two successive seasons 2011 and 2012. seeds of snap bean (phaseolus vulgaris l.) cv. ‘bronco’ were sown in 5 liter pots (filled with peat moss) in the second week of february for both seasons. at the 3rd leaf stage, bean plants were sprayed with citric acid at four levels (0.5, 1, 1.5 and 2 g/l). one day after treatments, plants were subjected to drought stress by withholding water for 7 days. control plants were also subjected to drought stress for the same period of time and not sprayed with citric acid (control 1), while an additional treatment of non-stressed plants were installed (control 2). the plant in control 2 were not treated by citric acid and kept at field capacity during the entire experiments. after stress period, all stressed-plants were re-watered and kept at field capacity for post stress observations on vegetative growth, generative development and yield as well as some quality parameters of pods. all plants were fertilized by using a nutrient solution (15-5-25, npk) during the entire experiments. the following parameters were recorded: physiological parameters: the second fully expanded leaves were used for the following measurements at the end of drought stress period: total chlorophyll content of leaves was measured using tys-a chlorophyll meter (zhe jang top instrument co. ltd., china), rwc of leaves according to turner (1981), and leaf temperature measurements by infrared thermometer (dt 8500-cheerman, china). post stress growth, quality and productivity observations: (plant fresh weight and branches number per plant) were recorded 60 days after sowing for both seasons. productivity measurements (weight of pods and pods number per plant) were recorded as well as pod characteristics (length and diameter). statistical analysis: the experiments were established as complete randomized block design with 4 replicates and analysis of variance was calculated according to snedecor and cochran (1967). least drought tolerance and water status of bean by citric acid 213 significant difference (l.s.d.) at 5% was used to compare the means for different treatments. results and discussion effects of citric acid on leaf temperature of bean plants subjected to drought stress previous investigations indicated that leaf or canopy temperature can be used as a cheap, fast and not-destructive screening tool for estimating drought tolerance among different plants such as maize (liu et al., 2011) and rice (hirayama et al., 2006). talebi (2011) studied the effect of drought stress on canopy temperature and total chlorophyll content on durum wheat and found that the genotypes with high yield in well-watered condition had also low canopy temperature and high chlorophyll content. he concluded that wheat genotypes with a low canopy temperature can maintain high transpiration and photosynthetic rate as well as produce a high yield under moisture-stressed conditions. leaf temperature of bean plants under drought stress as well as nonstressed plants for both seasons is illustrated in fig. 1. generally, although plants treated by citric acid had lower leaf temperature than control 1 plants, these differences were not significant between treatments. only non-stressed plants (control 2) showed significantly lower leaf temperature compared to other treatments. this was probably because, plants of this treatment were able to maintain higher transpiration rate, by which the leaf temperature was reduced. generally, the leaf temperatures of the second season were higher than the first season for all treatments, and this was related to the climatic variations between both seasons. fig. 1: effect of different levels of citric acid on leaf temperature of bean plants subjected to drought stress conditions .control 1: plants not treated by citric acid, but stressed. control 2: plants not treated by citric acid and kept at field capacity during the entire experiments. vertical bars present lsd value at 5%. effects of citric acid on chlorophyll content of leaves from bean plants subjected to drought stress chlorophyll content as an indicator of leaf photosynthesis is one of the criteria to access drought tolerance of plants. khayatnezhad and gholamin (2012) evaluated the effect of drought stress on this parameter and stress tolerance of different maize cultivars (zea mays l.) and found that different genotypes had different chlorophyll content and finally different resistance to drought stress. also, arjenak et al. (2012) indicated that chlorophyll content (and rwc) made difference between resistance and susceptible genotypes and thus, this attributes can be used as screening tool for drought tolerance in wheat. according to sun and hong (2011), when citric acid was exogenously applied significantly improved the plant growth under stress conditions and also induced defense mechanisms by increasing the activities of antioxidant enzymes. darandeh and hadavi (2011) found that foliar application of citric acid during growth stage significantly increased post-harvest vase life of the cut lilium flowers. in addition, the authors indicated that although any direct effect of citric acid on chlorophyll content was missing, the interaction effect between citric acid and malic acid on chlorophyll content was significant. in here presented study, plants sprayed with citric acid showed a higher total chlorophyll content compared to control for both seasons (fig. 2). the most effective concentration was 1.5 g/l of citric acid. citric acid seems to be a substance contributing to osmotic adjustment during drought stress and helps minimizing the injury caused by dehydration to plant tissues, which is indicated by higher chlorophyll content in leaves. although the mechanism behind this effect is not clearly explained, probably plays here the stomatal penetration by aqueous solutions a role. the results of burkhardt et al. (2012) supported the relevance of stomatal uptake of substances, as indicated by the different behaviors of adaxial/abaxial leaf sides. moreover, a ph decrease in the apoplastic continuum with related effects on fe and chlorophyll contents or distributions has been reported by kosegarten et al. (2001) and darandeh and hadavi (2011). fig. 2: effect of different levels of citric acid on total chlorophyll content of leaves of bean plants subjected to drought stress conditions. control 1: plants not treated by citric acid, but stressed. control 2: plants not treated by citric acid and kept at field capacity during the entire experiments. vertical bars present lsd value at 5%. effects of citric acid on water status of bean plants subjected to drought stress relative water content (rwc) in leaves is an indicator of plant water status and is markedly affected by drought stress. the rwc of non-stressed plants (watered at field capacity for the entire experiments, control 2) is indicated in fig. 3. the plants of this treatment represented the normal water status of plants and showed the highest rwc (see the comparison between the control 1 and control 2, for both seasons). interestingly, higher rwc were maintained also by citric acid applications at the end of drought stress period, especially at the level of 1.5 g/l, while control plants showed the lowest rwc in leaves of bean plants. this is in agreement with the statement of kramer (1983), who showed that the solutes involved in osmotic adjustment in response to drought stress vary, but consist of inorganic ions, carbohydrates and organic acids. merewitz et al. (2012) observed an accumulation of several metabolites during drought conditions, including organic acids that were mainly involved in the citric acid cycle. the authors concluded that the accumulation of these metabolites could contribute to improved plant drought tolerance (in this case of creeping bentgrass) due to their roles in the stress response pathways such as stress signaling, osmotic adjustment, and respiration for energy production. levi et al. (2011) also indicated that the improvement of drought tolerance of some cot214 w.a. el-tohamy, h.m. el-abagy, m.a. badr, n. gruda ton lines could be related to organic acid accumulation including citric acid. in addition, peckmann and herppich (1998) indicated that increased net citric acid accumulation in response to drought of aptenia cordifolia may result from the larger inhibition of the citrate degrading system relative to citrate synthesis. moreover, herppich and peckmann (1997) studied water relations and malic and citric acid accumulation in response to a short-term drought (10 days) in aptenia cordifolia and found that while water potential was reduced with decreasing water content, bulk leaf pressure potential remained constant due to active osmotic adjustment. the authors reported that the changes in tissue osmotic content, which were fully reversible upon rewatering, resulted largely from variations in citrate content. on the other hand, the investigation of sun and hong (2011) indicated that exogenous citric acid improved internal citric acid and helped improving stress tolerance of l. chinensis plants during saline stress conditions. the results from our study indicate that citric acid improved plant water status (as indicated by higher rwc) during drought stress and thus improved drought tolerance of bean plants. in accordance with here cited literature, the contribution of citric acid in improving water status maybe due to its role in osmotic adjustment during drought conditions. williamson and milburn (1995) found also that citric acid treatment resulted in the highest rwc in stems of acacia amoena in relation to water stress and indicated the involvement of citric acid in increasing hydraulic conductance. fig. 3: effect of different levels of citric acid on relative water content of leaves of bean plants subjected to drought stress conditions. control 1: plants not treated by citric acid, but stressed. control 2: plants not treated by citric acid and kept at field capacity during the entire experiments. vertical bars present lsd value at 5%. effects of citric acid on post stress growth, quality and productivity of bean plants subjected to drought stress in order to regulate water loss and prevent further dehydration, plants respond to drought stress by changes in their morphology, e.g. by decreasing leaf area, plant height and plant weight, and other plant parameters (gruda and schnitzler, 2000). this is in agreement with the results of here presented study. plant fresh weight and the number of branches per plants were reduced when the stress situation was induced, which is well documented by comparison of control 1 with control 2 for both seasons (tab. 1 and 2). not only the vegetative growth, but also the productivity and the quality of bean plants subjected to drought stress were negatively affected (tab. 1 and 2). however, the data clearly revealed that foliar application of citric acid ameliorated the negative effects of drought compared to control. vegetative growth parameters, expressed as fresh weight of plants and branches number per plant, generative development, expressed as number and weight of pods, as well as the quality of pods, expressed as length and pod diameter were improved by using of acid citric applications. these effects were significant especially at the level of 1.5 g/l of citric acid, being in accordance with results of physiological parameters. increasing the citric acid concentration to more than 1.5 g/l led not to better effects for all parameters measured. moreover, both seasons had the same trend concerning the effects of the treatments. the relatively better growth and productivity in the second (tab. 2) than the first season (tab. 1) could be related to climatic variations of the seasons. as it was mentioned before, leaf temperatures of the second season were higher than the first season (fig. 1), due to relatively warmer climate conditions during the growth period that in turn was much favorable for the enhancement of bean growth and productivity. the improvement of growth, productivity and quality of droughtstressed bean plants in response to citric acid application can be explained by the enhancement of water status during drought stress due to osmotic adjustment. as it was shown before, the physiological responses indicated a better water status especially at the level of 1.5 g/l of citric acid (fig. 3). changes in metabolites accumulation during drought and their relation to osmotic adjustment were discussed by silvente et al. (2012), who indicated that metabolic changes in response to drought conditions play a significant role in the adjustment of metabolism and physiology of the soybean varieties. sun and hong (2011) showed that plant growth was significantly improved by citric acid application during saline stress conditions. according to authors, citric acid seem to be an important component of the stress response in l. chinensis. tab. 1: effect of different levels of citric acid on vegetative growth, generative productivity and pod characteristics of bean plants subjected to drought stress conditions (first season). control 1: plants not treated by citric acid, but stressed. control 2: plants not treated by citric acid and kept at field capacity during the entire experiments. treatments plant fresh branches number of pod length pod diameter fresh weight weight (g/plant) number/plant pods/plant (cm) (cm) of pods (g/plant) citric acid (0.5 g/l) 165.53 4.67 13.67 7.50 0.67 57.60 citric acid (1 g/l) 178.77 6.67 14.67 8.17 0.70 58.93 citric acid (1.5 g/l) 243.12 7.67 16.33 9.81 0.77 64.13 citric acid (2 g/l) 207.47 6.33 15.33 9.17 0.70 61.00 control 1 162.00 4.00 13.33 7.33 0.63 54.50 control 2 314.50 10.00 33.67 12.17 1.00 116.67 l.s.d. at 5 % 15.30 1.82 2.18 2.12 0.20 9.70 drought tolerance and water status of bean by citric acid 215 it can be concluded that citric acid application helped improving water status of bean plants exposed to drought stress conditions and subsequently improved drought tolerance. citric acid can be applied during or before an expected drought period for improving drought tolerance of bean plants. using citric acid in combination with other technological tools, such as e.g., the application of drip irrigation, can minimize the problems of water shortage with particular emphasis on sustainable resource management and environmental protection. under the conditions of egypt spraying at a level of 1.5 g/l of citric acid is recommended. references burkhardt, j., basi, s., pariyar, s., hunsche, m., 2012: stomatal penetration by aqueous solutions – an update involving leaf surface particles. new phytologist 196, 774-787. darandeh, n., hadavi, e., 2011: effect of pre-harvest foliar application of citric acid and malic acid on chlorophyll content and post-harvest vase life of lilium cv. brunello. front plant sci. 2, 106. el-tohamy, w.a., schnitzler, w.h., el-behairy, u., singer, s.m., 1999: effect of long-term drought stress on growth and yield of bean plants (phaseolus vulgaris l.). j. appl. bot. – angewandte botanik 73, 173-177. arjenaki, f.g., jabbari, r., morshedi, a., 2012: evaluation of drought stress on relative water content, chlorophyll content and mineral elements of wheat (triticum aestivum l.) varieties. int. j. agri. crop sci. 4, 726-729. gruda, n., schnitzler, w.h., 2000: the effect of water supply on biomorphological and plant-physiological parameters of tomato transplants cultivated in wood fiber substrate (in german). j. appl. bot. 74, 233-239. herppich, w.b., peckmann, k., 1997: responses of gas exchange, photosynthesis, nocturnal acid accumulation and water relations of aptenia cordifolia to short-term drought and rewatering. j. plant physiol. 150, 467-474. hirayama, m., wada, y., nemoto, h., 2006: estimation of drought tolerance based on leaf temperature in upland rice breeding. breed. sci. 56, 47-54. iwasaki, m., fukamachi, h., imai, a., nonaka, k., 2011: effects of summer and autumn water stress on fruit quality of medium-late maturing citrus ‘harehime’. hort. res. (japan) 10, 191-196. kang, n.j., cho, m.w., kang, k.h., 2009: accumulation of soluble solids and activation of antioxidant enzymes by deficit irrigation in fresh tomato fruits. korean j. hort. sci. tech. 27, 343-352. khayatnezhad, m., gholamin, r., 2012: the effect of drought stress on leaf chlorophyll content and stress resistance in maize cultivars (zea mays). african j. microbiol. res. 6, 2844-2848. kosegarten, h., hoffmann, b., mengel, k., 2001: the paramount influence of nitrate in increasing apoplastic ph of young sunflower leaves to induce fe deficiency chlorosis, and the re-greening effect brought about by acidic foliar sprays. j. plant nutr. soil sci. 164, 155-163. kramer, p.j., 1983: water relations of plants. academic press, inc. san diego, california. levi, a., paterson, a.h., cakmak, i., saranga, y., 2011: metabolite and mineral analyses of cotton near-isogenic lines introgressed with qtls for productivity and drought-related traits. physiol. plantarum 141, 265275. liu, y., subhasha, c., yan, j., song, c., zhao, j., li, j., 2011: maize leaf temperature responses to drought: thermal imaging and quantitative trait loci (qtl) mapping. env. exp. bot. 71, 158-165. merewitz, e.b., du, h.m., yu, w.j., liu, y.m., gianfagna, t., huang, b.r., 2012: elevated cytokinin content in ipt transgenic creeping bentgrass promotes drought tolerance through regulating metabolite accumulation. j. exp. bot. 63, 1315-1328. millar, a.a., gardner, w.r., 1972: effect of soil and plant water stress potential on the dry matter production of snap bean. agron. j. 64, 559-562. morinaga, k., sykes, s.r., 2001: effect of salt and water stress on fruit quality, physiological responses, macroand micro-element contents in leaves of satsuma mandarin trees under greenhouse conditions. japan agri. res. quarterly 35, 53-58. nahar, k., gretzmacher, r., 2002: effect of water stress on nutrient uptake, yield and quality of tomato (lycopersicon esculentum mill.) under subtropical conditions. bodenkultur 53, 45-51. papadopoulos, i., metochis, c., seraphides, n., 2004: irrigation – fertigation of greenhouse tomato under saline conditions. technical bulletin – cyprus agri. res. inst. 220. peckmann, k., herppich, w.b., 1998: effects of short-term drought and rewatering on the activity of mitochondrial enzymes and the oxidative capacity of leaf mitochondria from a cam plant, aptenia cordifolia. j. plant phys. 152, 518-524. silvente, s., sobolev, a.p., lara, m., 2012: metabolite adjustments in drought tolerant and sensitive soybean genotypes in response to water stress. plos one 7(6): e38554. doi:10.1371/journal.pone.0038554 snedecor, g.w., cochran, w.g., 1967: statistical methods, 6nd ed. iowa state univ. press, ames, iowa, usa. sun, y.l., hong, s.k., 2011: effects of citric acid as an important component of the responses to saline and alkaline stress in the halophyte leymus chinensis (trin.). plant growth reg. 64, 129-139. tab. 2: effect of different levels of citric acid on vegetative growth, generative productivity and pod characteristics of bean plants subjected to drought stress conditions (second season). control 1: plants not treated by citric acid, but stressed. control 2: plants not treated by citric acid and kept at field capacity during the entire experiments. treatments plant fresh branches number of pod length pod diameter fresh weight weight (g/plant) number/plant pods/plant (cm) (cm) of pods (g/plant) citric acid (0.5 g/l) 170.70 6.4 16.0 7.9 0.75 58.80 citric acid (1 g/l) 180.82 7.3 16.4 9.7 0.78 62.93 citric acid (1.5 g/l) 245.61 9.7 18.8 11.6 0.90 69.93 citric acid (2 g/l) 210.02 7.9 16.7 10.5 0.80 64.20 control 1 167.07 5.1 14.6 7.6 0.69 56.83 control 2 321.04 13.0 37.0 12.3 0.97 120.67 l.s.d. at 5 % 15.71 1.33 2.37 1.88 0.14 7.52 216 w.a. el-tohamy, h.m. el-abagy, m.a. badr, n. gruda talebi, r., 2011: evaluation of chlorophyll content and canopy temperature as indicators for drought tolerance in durum wheat (triticum durum desf.). austral. j. basic appl. sci. 5, 1457-1462. turner, n.c., 1981: techniques and experimental approaches for the measurement of plant water status. plant soil 58, 339-366. williamson, v.g., milburn, j.a., 1995: cavitation events in cut stems kept in water: implications for cut flower senescence. sci. horticul. 64, 219232. address of the authors: prof. dr. w.a. el-tohamy and prof. dr. h.m. el-abagy, vegetable research department, national research center, dokki, cairo, egypt e-mail: wael_eltohamy@hotmail.com prof. dr. m.a. badr, plant nutrition department, national research centre, dokki, cairo, egypt pd dr. n. gruda, institute of plant sciences and resource conservation, division of horticultural sciences, university of bonn, bonn, germany art02 journal of applied botany and food quality 81, 110 120 (2007) department of plant ecology, justus liebig-university, giessen new insights into the dynamics of the glutathione-ascorbate redox system of plants edwin pahlich, christoph müller, hans-jürgen jäger (received april 18, 2007) summary the hallilwell-asada-foyer redox cascade (haf) is viewed as a h2o2 detoxifying system with a great variety of responses against environmental changes. the functional consequences of these responses are interpreted intuitively because a systemic analysis of the inherent dynamic potential of the haf is lacking. with the help of numerical modelling we show that in wheat roots parameter patterns are established which result in homeostatic states of haf over a vast range of environmental changes. the reduced fractions glutathione (gsh) and ascorbate (asc) remain on high levels even during dramatic changes in the enzyme activity ratios of glutathione reductase, dehydroascorbate reductase and ascorbat peroxidase. necessarily their oxidised counterparts dithioglutathione (gssg) and dehydroascorbate (dha) stay in these buffered regions on very low concentration levels. our modelling shows that redox ratios gsh/gssg and asc/dha can be modified additionally via changes in nadph/h2o2 ratios. thus, the redox states of gsh and asc can not simply be regarded as indicators for oxidative stress with respect to h2o2 levels. the involvement of the redox variables in other redox processes than the haf reaction (redox proteome) and / or their utilisation in metabolism (protein modification, detoxification of xenobiotics) are viewed to cause system relaxations of the redox variables. the re-establishment of their homeostatic ratios follow time courses which are redox moiety specific and are balanced according to the existing parameter patterns. despite of its detoxification function the haf balances the glutathione / ascorbate redox state in cells according to the prevailing physiological conditions. introduction the halliwell-asada-foyer cascade (haf) is a redox cascade which was originally viewed as a h2o2 detoxifying system (halliwell, 1978; halliwell, 1987; ishikawa and sies, 1989). detoxification is reached by transfering electrons from nicotineamide adenine nucleoside phosphate (nadph) to hydrogen peroxide (h2o2) via the cycle intermediates glutathione and ascorbate. the reactions are catalyzed by the enzymes glutathione reductase (gr), dehyroascorbate reductase (dhr) and the ascorbate peroxidase (apx). the enzyme activity patterns as well as the redox properties of the intermediates are highly responsive to changes of plant growth conditions such as salinity, temperature, ozone, heavy metals, oxygen, ph, and soil fertility and light stress (arrigoni, 1995; noctor and foyer, 1998; de marco and roubelakis-angelakis, 1999; pearce, 1999; mittler, 2002; blokhina et al., 2003; hernandez et al., 2004; foyer and noctor, 2005; foyer et al., 2005; smirnoff, 2005). this responsiveness is viewed as counteraction for fighting oxidative stress, caused by changes of the above mentioned environments. however, it is widely neglected whether other causes than oxidative stress, resulting from metabolic pertubations, could also lead to similar responses of the haf cascade. exploring this problem will be a focus of this paper. the functioning of any reaction system is primarily dependent on its parameter status (kacser and burns, 1973; reich and sel’kov, 1981) which is given by the values of rate constants of the individual enzymes and their numerical ratios. according to kacser and burns (1973) also co-enzymes and other (signaling) molecules and input to autput ratios in reaction cascades can reach parameter status under certain conditions. in the case of the haf this notion can be applied to stationary concentrations of nadph and h2o2 which are the input and output ports of the haf system. both, the rate parameters and the input (nadph) to output (h2o2) ratios are regarded as „minimal sets“ of determinants of the dynamic patterns of the haf system. a re-evaluation of oxidative stress phenomena in plants which is also based on genetic evidence opens new insights into the potentially damaging effects of reactive oxidative species (ros) but also directs the attention to their „oxidative signaling“ assignment (foyer et al., 1997; dat et al., 2000; foyer and noctor, 2005; foyer et al., 2005; halliwell, 2006). according to this extended point of view h2o2 is expected to act as signal molecule, as environmental messenger or enhancer for the regulation of developmental processes (lagrimini et al., 1997; lander, 1997; rao and davis, 2001; piquery et al., 2002; foreman et al., 2003; overmyer et al., 2003; foyer, 2005; foyer and noctor, 2005). it remains open how the effective h2o2 detoxifying function of haf can be correlated with the need of residual portions of h2o2 for signaling purposes. furthermore haf provides reducing power for the redox proteome and is also supporting glutathione for protein modification reactions as indicated in fig. 1 (scheibe, 1991; dietz, 2005; foyer and noctor, 2005; ströher and dietz, 2006). in addition glutathione and ascorbate serve particular regulatory and developmental purposes (ishikawa and sies, 1989; arrigoni, 1995; smirnoff, 1996; foyer et al., 1997; jimenez et al., 2002; foyer et al., 2005; foyer and noctor, 2005; smirnoff, 2005). the haf must be regarded as a multifunctional core unit of redox metabolism in cells which serves not only as detoxificant of h2o2, but also provides appropriate reduction power for cellular redox proteome, supports protein modification and balances the redox molecules in accordance with their physiological needs and is involved in the regulation of growth and development of plants via its components h2o2, glutathione and ascorbate. numerical modelling is ideal to unravel the multifaceted functions of the haf core process. a recent model by polle (2001) focused on detoxifying mechanism of the haf and simulated the functioning of the sod-asc-gsh cycle in cells. here we extend the approach by considering variable system parameter portraits according to reich and sel’kov (1981). these portraits identify the steadystate levels of variables as functions of their parameter sets. they quantify the redox ratios of the variables and allow flux calculations that are characteristic for these steadystates. the focus of this model is addressed to the question how the redox couples gsh / 2 gssg and asc / dha are shifted under changing enzyme activities and how they depend on the support (nadph) to demand (h2o2) ratio. it will be shown how transitory dynamics of the variables can cause „pseudo steady-states“ (heinrich and rapoport, 1977) which can give rise to misinterpretations of experimental data. material and methods in order to deal with a coherent set of data the model calculations were based on a systematic survey of varying growth conditions on the parameters and variables of haf in wheat roots (maldonado, 1998). only three experiments (4 and 9 days old seedlings grown in the dark, 7 days old seedlings with 3 days under a 14 h photoperiod) from this study are considered in this modelling which show the most significant differences in the responses of the enzyme activities of glutathione reductase (gr), dehydroascorbate reductase (dhr) and ascorbate peroxidase (apx) and of the concomitant redox changes of the gsh / 2gssg and asc / dha moieity ratios. it is intended to elucidate via modelling which effect on the system properties of the haf arise from these parameter changes. plant material: surface sterilized kernels of wheat seedlings (triticum aestivum l. cv. bezostaya) were grown in a growth chamber at 20° c in the dark on a 1cm layer of vermiculite which was placed on stainless steel sieves. the vermiculite was watered by wicks which were connected to an aerated water reservoir. the roots were allowed to grow through the sieve into the water. the analytical data considered here stem from roots of dark grown seedlings (4 and 9 days old, respectively) and from roots of seven days old seedlings which had been grown under a 14 h photoperiod for three days before harvest. the harvested root samples were frozen in liquid nitrogen and used for subsequent enzyme activity determinations. the moieties of nicotinamide adenine dinucleoside phosphate (nadp), glutathione (gsh) and ascorbate (asc) were analyzed from freshly prepared plant extracts. roots of seedlings were analyzed because they are lacking the chloroplast antioxidative system. according to the applied method (10 000 * g supernatant) the extracts did not contain mitochondria and peroxisomes. enzyme activities and metabolites: the enzyme activities of the glutathione reductase (e.c. 1.6.4.2) (gr), the dehydro-ascorbate reductase (e.c. 1.8.5.1) (dhr) and the ascorbate peroxidase (e.c. 1.11.1.11) (apx) were determined from 10 000 g supernatants according to procedures by smith et al. (1988), gogorcena et al. (1995) and kato et al. (1997), respectively. from these determinations the rate parameters vmax / km were calculated and used for modelling. the concentrations of variables glutathione gsh and of dithioglutathione (gssg) were determined according to griffith (1980), ascorbate (asc) and dehydroascorbate (dha) were determined according to okamura (1980) and the reduced and oxidized coenzymes nadph and nadp were measured as described by matsumura and miyachi (1980). these primary data of parameters and variables (nadph, glutathione and ascorbate) provide unique starting sets of data from one plant species and from directly comparable experiments for modelling. modelling of the antioxidative system: the rate equations for modelling the haf (fig. 1, solid lines) were formulated according to their scaled expressions (fig. 2). this formalism depicts the core processes of the haf (fig. 2) in form of its dimensionless variables and its scaled enzyme parameters (reich and sel’kov, 1981). scaled data facilitate comparibility of the system properties of the haf under changed sets of parameters at varying growth conditions. scaling converts experimentally measured results of highly differing magnitudes to comparable ranges between 0 and unity. scaled data of reactions of different orders are dimensionless which is a necessity for explicit mathematical modelling. interferences of the haf (fig. 1) with components of additional cellular redox processes like the reactions of the thiol redox proteins (dotted lines) are considered but are viewed as kinetically subordinate in comparison to the core process reactions (see later). in our model calculations was used a coherent set of experimental data from one plant organ. this is different to the modelling approach by polle (2001) who used a mix of data from different sources. the formalism for modelling haf is given by the following approach. the rate of reactions can be related to the concentrations of reactants by an equation of the type: eqn. 1: v = k · [a] . [b] . [c] where v is the reaction rate; k is the rate constant with a complex overall order and [a], [b] and [c] are substrate concentrations. the order of a reaction is a purely experimental quantity (laidler, 1978) and is from no concern in calculations with scaled and dimensionless entities. in our example each of the three reactions (fig. 1, solid lines) represents combinations of high and low substrate concentrations. kinetically we are dealing with pseudo monomolecular pseudo first order reactions (lehninger, 1977). model calculations occurred with dimensionless contents of the variables glutathione, ascorbate and nicotineamide adenine dinucleoside phosphate (fig. 2). scaling of the variables is justified by assuming „moiety conservation“ of the redox couples which can be expected for „snap shot conditions“ of a current physiological state in the moment of harvest. the scaling formalism is given in equations 2a c, including moiety conservation expressions of the variables (eqns. 3a c). fig. 1: the redox balancing potential of the antioxidative glutathione-ascorbat cascade (solid lines). the nadph pool provides reducing power to the system. electrons are transferred by the glutathione reductase (gr)(v1) and the dehydroascorbate reductase (dhr)(v2) to the ascorbate moiety (dha : asc). hydrogen peroxide (h 2 o 2 ) finally is reduced via the ascorbate peroxidase reaction (apx)(v3). included into the reaction cascade are interacting reactions (dotted lines). vp 1 , vp 3 and vp 4 symbolize consuming reactions of the reductants, vp 2 and vp 5 are consuming oxidants. frx ox /frx red = oxidized and reduced forms of thioredoxins; prot sh /prot ssg = thiol protein and glutathionylated protein; grx ox /grx red = oxidized and reducued forms of glutaredoxins; fe3+/fe2+ = oxidized and reduced 2-oxoglutarat (feii) dioxygenase; prx 1 /prx oxidized and reduced forms of peroxiredoxins. nadph gssg 2gsh asc dha h2o2 v1 v2 v3gr dhr apx 2h2o nadp frxox frxred vp1 grxsred grxsox vp3 fe3+ fe2+ vp4 prx prx1 vp5vp2 protsh protssg nadph gssg 2gsh asc dha asc dha h2o2 v1 v2 v3gr dhr apx 2h2o nadp frxox frxred vp1 grxsred grxsox vp3 fe3+ fe2+ vp4 prx prx1 vp5vp2 protsh protssg modelling the glutathione-ascorbate redox system of plants 111 112 edwin pahlich, christoph müller, hans-jürgen jäger β a b a c α .k2 β 2 .k2 β 2 .k3 r ( ) )(1 2 1 11 grkv ηβ ⋅−⋅⋅= ( ) )(1 222 dhrkv βα ⋅−⋅= )(33 apxrkv ⋅⋅= α b .. 1 3 k3 r k2 .1 9 ( ).k2 ka ...2 k3 r k2 2 .k2 2 ka 2 c . 1 3 ( ).k2 ka ...2 k3 r k2 ( ).k2 ka a + ...... 1 6 k3 r k2 2 ( ).k2 ka ...2 k3 r k2 ka .. 1 2 k3 r k2 . 1 27 ( ).k2 ka ...2 k3 r k2 3 .k2 3 ka 3 .. 1 9 k3 + ... + ... + ......k3 3 r 4 ka k2 ...8 k3 3 k2 2 r 4 ....12 k3 2 k2 2 r 3 ka ....10 k3 2 r 3 ka 2 k2 ..k3 2 r 3 ka 3 ....6 k3 k2 2 ka 2 r 2 ....2 k3 r 2 ka 3 k2 ...r k2 2 ka 3 3 .k2 3 2 ka 3 2 1 3 eqn. 2a: eqn. 2b: eqn 2c: with, eqn. 3a: eqn. 3b: eqn. 3c: the dimensionless concentration of h2o2 is defined as eqn. 4: r = h2o2 / km h2o2 with km h2o2 , the half saturation constant of ascorbate peroxidase (takeda et al., 1998). in anology to equation 1 the scaled and dimensionless forms of the rate equations derived from fig. 2 are: eqn. 5: eqn. 6: eqn. 7: where, gr, dhr and apx represent the scaled reactions of the gluthatione reductase (eqn 5), the dehydroascorbate reductase (eqn 6) and the ascorbate peroxidase (eqn 7) respectively. scaling of the rate parameters is achieved as depicted in tab. 2. assuming that the antioxidative system is in steady state, rate equations can be equated (v1 = v2 = v3) and solved symbolically for the variables α and β: with eqn. 8: eqn. 9: with a, b and c as written in equation 10. eqn. 10: the rate constants k1, k2 and k3 in equations 8 and 10 represent the scaled rate parameters. ka is the product of the scaled values of k1 and η. combining both constants saves computer capacity for calculations. additional redox rates (vps) in fig. 1 represent low leveled time hierachies as compared to the fast reaction rates v1, v2 and v3. they are omitted from the model calculations. the latter reactions are considered to adjust the redox ratios of the glutathione and ascorbate moieties. the assumptions for the model calculations are: 1) the redox system establishes steady-states. 2) the enzymes and their substrates / products are freely diffusible and not separated by compartmentalization. 3) the fraction of nadph of the total coenzyme moiety (nadph + nadp) was taken to be 60% in accordance with the experimental results. 4) the electron transfer through the core system is unidirectional because of the irreversibility of the reaction step of apx. the mono-dehydro ascorbate reductase (mdhr) has not been included into the model because it was practically not measurable in the root supernatant extracts. according to the experimental procedure we deal most likely with the cytosolic fraction of roots (isocitrate dehydrogenase was not measurable). secondly, mono-dihydroascorbate (mdha) disproportionates with a rate constant of 2 x 109 m-1min-1 (polle, 2001; blokhina et al., 2003) which assigns the reaction to a time hierarchy which is more efficient than the enzyme catalyzed reactions of the haf. only reactions with comparable time scales can form steady-state aggregates as defined by heinrich and rapoport (1977). finally, mdha most likely fulfils particular physiological functions in the context of trans-membrane redox catalysis (mittler, 2002). this view is underlined by the fact that the cytosolic isoform of this catalyst is located in the plasma membrane (asada, 1999). transmembrane separation and transport of dha and asc as well as disproportionation of mdha may influence the respective pools of our model but will be equilibrated by relaxations which are processes considered in our approach. omitting mdhr does not violate the general concept of our model. model calculations have been performed with mathcad 6 (mathsoft inc. cambridge, massachsetts, usa) according to the reaction mechanism depicted in fig. 2. the time courses of relaxation events were calculated with modelmaker (version 3, cherwell scientific, oxford, uk) which integrates numerically the differential equations 5, 6 and 7 using the runge-kutta algorithm. the figs. have been created in sigmaplot (version 8.02, spss, inc. usa). 0a asc=α ; 0 1 a dha=− α 0g gsh=β ; ( ) 0 1 2 1 g gssg=−⋅ β 0n nadph=η ; 0 1 n nadp=−η dhaasca +=0 gssggshg 20 += ++= nadpnadphn 0 modelling the glutathione-ascorbate redox system of plants 113 results and discussion steady state solutions of haf with scaled variables and parameters: the goal of this investigation was to elucidate systemic properties of the halliwell-asada-foyer detoxification system by means of numerical modelling. it was analyzed which steady-state redox ratios of glutathione and ascorbate can be established under varying enzyme parameter conditions, how the steady fluxes of electrons (the detoxifying potential) change during parameter alterations and how shifted input to output ratios (nadph / h2o2) effect the redox patterns of the variables. finally it was studied how relaxation processes equilibrate the variables after system perturbations. tab. 1 summarizes the scaled values of the oxidized fractions of the variables nadp, gssg and dha as well as their total moieties n0 (nadp++nadph), g0 (gsh+ 2 gssg) and asc0 (asc+dha) in root extracts of wheat seedlings. the differences of the dimensionless values (tab. 1) of the variables reflect the effect of the three growth conditions considered. nadp changed between 0.42 and 0.64. the fraction of 2gssg in the seedlings grown in the dark ranged between 0.1 and 0.15 but reached a value of 0.48 under a 14 h photoperiod. dha varied between 0.36 and 0.49. the fractions of the reduced forms of the variables are the differences to unity of the presented values. it can be seen (tab. 1) that the haf under the considered growth conditions is kept on a highly reduced state with respect to glutathione while ascorbate tends to less reduced fractions. the reduced nictotinadenine dinucleoside phosphate fraction shifts to approximately 0.5. in the latter case it should be considered that major portions of the reduced co-enzyme are assumed to be firmly bound to redox enzymes. nadph is therefore most likely represented by a higher fraction in roots as detected in measured soluble extracts of this organ. the total moieties of the co-enzyme (n0), glutathione (g0) and ascorbate (asc0) in wheat root extracts follow the ranking n0 < g0 << asc0 (tab. 1). the absolute values of the total moieties under the chosen experimental conditions of n0, g0 and asc0 varied between 0.12 to 0.39 µmol g-1 dry weight (dw), 1 to 1.5 µmol g-1 dw and 30 to 100 (or more) µmole g-1 dw respectively. this ranking is most likely also valid for respective extracts of other plants and might even be generalized (foyer and noctor, 2005). from the ranking of the pools of variables it is concluded that only the asc pool (and to a lesser extend the gsh pool) has the potential to detoxify bursts of h2o2 which are caused by environmental cues (okuda et al., 1991). this effect will hardly be reached with the cosubstrate nadph which typically appears in low free concentrations in cells. its low rate of generation in compartments like the cytoplasm by metabolic reactions like the pentose phosphate shunt (fukuoka and enomoto, 2001) or the isocitrate dehydrogenase contradicts the view of its function as a primary reductant to detoxify oxidants like h2o2. with nadph as primary donor of reducing power it must be considered that the reservoirs of gsh and asc can only be filled up under low oxidative demand. this point of view is supported by the fact that the glucose-6-phosphate dehydrogenase is regulated by a nadph-dependent negative feedback loop. increased nadph pools would inhibit its regeneration by a highly redox sensitive regulatory mechanism of the pentose phosphate shunt (maldonado and pahlich, 1997). most likely the antioxidative cascade functions as a pullregulated system (oliver, 2002) the nadph support of which could become the limiting process for fueling glutathione and ascorbate pools under severe oxidative stress in non-chloroplast compartments. an advantage of a high asc pool for detoxification arisis from its participation in two-substrate mechanisms of catalysis as in the ascorbate peroxidase (apx) reaction. based on ordinary enzyme kinetics it can be shown that a high substrate pool of asc in combination with a low pool of h2o2 results in very effective and complete destruction of the oxidant. this is in contrast to the reactions of catalase or the gsh or nadph dependent h2o2 reductases. the latter enzymes catalyse also two substrate reactions but with low substrate concentrations of the reductants and the oxidant. the consequence is that residual fractions of h2o2 survive on low levels if the reaction fades out at its final course of h2o2 destruction. it remains open wether these basic differences in the detoxification rates of h2o2 are involved in the regulation of h2o2 concentrations which are needed for signaling. fig. 2: symbolism for modelling the antioxidative cascade. the antioxidative cascade as depicted in fig. 1 is presented with the symbols used for model simulations. k1, k2 and k3 are the rate parameters of the reactions of the glutathione reductase (gr), the dehydroascorbate reductase (dhr) and the ascorbate peroxidase (apx) respectively (see methods for details). the greek letters symbolize the scaled fractions of the variables: η is nadph/n 0 , (1-η) is nadp / n 0 ; α is asc / a 0 , (1-α) is dha / a 0 , β is gsh / g 0 and ½ · (1-β) is gssg / g 0 . r is the scaled concentration of h 2 o 2 and is defined as [h 2 o 2 ] / km h2o2 , where km h2o2 is the half-saturation concentration of h 2 o 2 of the apx. ( )1 12 β⋅ − α+ 2 β⋅ ( )1 α−+ η r k1 gr k3 apx dhr k2 1 η− h2o ( )1 12 β⋅ − α+ 2 β⋅ ( )1 α−+ η r k1 gr k3 apx dhr k2 1 η− h2o tab. 1: the scaled oxidized fractions of the variables nicotinamide adenine dinucleoside phosphate (nadp), dithioglutathione (gssg) and dehydro ascorbate (dha) (lanes 1, 2 and 3) and their total moieties: n o is nadp + nadph, g o is 2 gssg + gsh and asc o is dha + asc (lanes 4, 5 and 6) from root extracts of three different growth experiments with wheat seedlings. the wheat seedlings were grown according to the conditions highlighted in the headlines. 4d dark (reference) scaled values total concentrations (per g dw) nadp 2gssg dha n o (µm) g o (µm) asc o (µm) 0.42 0.12 0.49 0.382 1.51 33.8 9d dark scaled values total concentrations (per g dw) nadp 2gssg dha n o (µm) g o (µm) asc o (µm) 0.64 0.15 0.36 0.118 1.0 31.1 4d dark + 3d 14 / 10 photoperiod scaled values total concentrations (per g dw) nadp 2gssg dha n o (µm) g o (µm) asc o (µm) 0.43 0.48 0.49 0.388 0.92 100.0 114 edwin pahlich, christoph müller, hans-jürgen jäger parameter patterns determine the redox ratios of the variables: the scaled rate parameters of gr, dhr, and apx i.e. k1, k2 and k3 are presented in tab. 2. the rate parameters are formulated as vmax / km ratios according to the generally accepted view that enzymes in cells are reacting at substrate concentrations in the range or even the lower range of their km values (which changes the simple michaelis and menten equation from v = vmax * s / [km + s] to the linear form v = [vmax / km] * s). the measured vmax values in tab. 2 support the impression that the respective antioxidative cascades of plants grown under the three conditions have developed fairly different „antioxidative properties“. however, the scaled values elucidate that despite of this impression the parametric system design is very similar in the two dark grown batches but seems to be different in the light / dark grown seedlings (tab. 2). the agreement between the measured and modelled redox moieties of glutathione and ascorbate is shown in fig. 3. the dimensionless glutathione and ascorbate values are found at the y-axis when k2 is at unity. in 4d and in the 9d old seedlings (fig. 3 a and b) the modelled dimensionless gsh values were 0.86 and 0.88 respectively, while in the dark-light treatment a value of 0.52 was found (fig. 3c). the redox states of glutathione predicted by the model are in good agreement with the measured values at the given set of parameters and at the h2o2 values indicated. in contrast, the simulated and observed asc values under the same parametric conditions are in disagreement. asc was predicted to be close to unity in all three cases. in reality the asc was present in far more oxidized states in all three batches (tab. 1). most likely the high fraction of dha is a preparation artefact as far as it results from mixing highly oxidised apoplastic and vascular portions (burkey et al., 1995; foyer et al., 2001) during homogenization with the intracellular fluid. the model predicts also, that asc under steady conditions is practically insensitive to changes of h2o2 under the measured parameter patterns. the robustness of the antioxidative system as judged from the parameter portraits: the parameter portrait of a system describes the „qualitative behaviour in the space of permissible parameter values“ (reich and sel’kov, 1981). with respect to haf this means that systematic changes of the set of the system parameters in a parameter portrait can visualize and predict the possible stationary values of glutathione and ascorbate. under the growth conditions tested (tab. 2) the dhr activity shows the most variable responses. therefore parameter k2 (x-axis in fig. 3) was allowed to change between unity and zero in the parameter portrait while parameters k1 and k3 were kept constant. the calculated steady values of the moieties of glutathione and ascorbate are found on the y-axis of fig. 3. following the x-axis downward to k2 = 0.1 continuously changes the experimentally determined parameter ratio k1 : k2 : k3 = 0.036 : 1 : 0.01 to k1 : k2 : k3 = 0.036 : 0.1 : 0.01. this is a dramatic parameter perturbation which might be caused in situ by environmental cues or developmental traits. surprisingly this dramatic parameter change is rather inefficient with respect to changes of the redox moieties of glutathione and ascorbate which stay at their initial redox patterns irrespective of the assumed enzyme parameter changes. the enzyme parameter patterns as measured in wheat roots reflect obviously a construct which has the potential to buffer the redox ratios of the variables. despite of major parameter shifts this behaviour – which provides stability to the redox ratios of the variables – visualizes homeostasis and is a system immanent property. this property primarily arises from the ratio of the rate parameters k1 : k2 : k3 under a given ratio of nadph : h2o2. stable pools of the reductants are the result despite of dramatic changes of the enzyme activity patterns. this is to our knowledge the first report of this kind which explains homeostasis of redox components of haf on a mechanistic background and not on intuitive reasons. however, the homeostatic property of the system vanishes if the k2values are approaching zero. the differences of the parameter ratios k1 : k2 : k3 are approaching the approximate range of 1 : 1 : 1. under such a regime of parameters the ratios of gsh : 2gssg and asc : dha are highly responsive to further parametric changes. a new reaction (and model) situation would arise with dhr approaching very low values. thus, the parameter portrait of the antioxidative system uncovers a bi-phasic character. there exsists a robust and stable branch (k1 = 1 ➝ 0.1) with highly unequal parameter values and a sensitive branch (k1 < 0.1) which arises from a set of parameter ratios which approach equal values. this structural difference within the reaction cascade elucidates that experimentally observed enzyme activity changes do not necessarily indicate functional changes with respect to the redox ratios of the variables glutathione and ascorbate. however, it is important for functioning whether an environmental cue hits the system at its homeostatic or its sensitive branch. the responses must be expected to be quite different. therefore the parameter portrait visualizes the „functional phenotype“ of the system in cells / tissues. the portrait also reflects system changes which might occur during plant development or under the influence of varying growth conditions. in order to better understand „stress induced“ metabolic responses it is necessary to consider the actual state of the haf system at the moment of perturbation. observations of pastori et al., (2000) in maize leaves resulted in a parameter pattern of the haf of gr : dhr : apx of 0.9 : 1 : 10 tab. 2: vmax values of the glutathione reductase (gr), the dehydroascorbate reductase (dhr) and the ascorbate peroxidase (apx) of dark grown seedlings (4 d; 9 d) and of seedlings with a 14/10 photoperiod during the last 3 d (lanes 2, 3 and 4) and the km values of their oxidized substrates (lane 5) respectively. on the right hand side of the table are arranged the rate parameters of the three enzymes (vmax / km, upper row in lanes 5, 6 and 7) and their scaled values k1, k2 and k3 (lower row in lanes 5, 6 and 7). scaling was achieved by dividing k1 and k3 by k2. growth conditions 4 d dark 4 d dark 9 d dark 4 d dark 4 d dark 9 d dark (reference) plus 3 d 14/10 (reference) plus 3 d 14/10 photoperiod photoperiod vmax [µm * min-1*g-1dw] km [µm] vmax / km [min-1*g-1dw] (upper row) k = scaled rate constants (lower rows) glutathione gssg 2.86 5.0 1.43 reductase 4 7 2 1.4 k1 = 0.036 k1 = 0.015 k1 = 0.036 dehydroascorbate dha 80 328 40 reductase 20 82 10 0.25 k2 = 1 k2 = 1 k2 = 1 ascorbate h 2 o 2 0.8 2.4 0.6 peroxidase 2 6 1.5 2.5 k3 = 0.01 k3 = 0.007 k3 = 0.015 modelling the glutathione-ascorbate redox system of plants 115 (20°c) and 2.7 : 1 : 0.18 (15°c). these findings identify a completely different situation in maize leaves as is described for wheat roots. the data point to a sensitive pattern of the haf rather than a homeostatic situation. the concomitant redox ratios gsh : gssg that are close to unity in leave extracts and about two in mesophyll extracts support this point of view. the ascorbate redox ratios (asc : dha) are about 1.3 and 0.14 and mirror an entirely different situation than observed in wheat roots. comparison of the model predictions with literature data is hard to achieve mainly because of incomplete sets of comparable system parameters and variables. input to output parameters: kacser and burns (1973) have shown that under certain steady conditions co-enzymes and other (signaling) molecules can receive the state of parameters. according to the formulation of our reaction model (fig. 2), which symbolized a snap shot condition at the moment of harvest of the plants, the reductant nadph and the oxidant h2o2 are treated as parameters. it will be shown that in combination with the enzyme parameters they must also be viewed as determinants of the redox state of glutathione and ascorbate. under physiological conditions it can be assumed that the rates of generation of nadph and of h2o2 and hence their levels are not necessarily interdependent. therefore the concentration ratios of nadph and of h2o2 will vary individually according to the physiological condition of cells. the effect of this variation has been modelled for two scenarios. in these calculations the enzyme rate parameters have been kept constant: 1) nadph to h2o2 ratio is 4 to 1 (fig. 3a: η = 0.58, r = 0.15) 2) nadph to h2o2 ratio is 2 to 1 (fig. 3d: η = 0.3, r = 0.15) the homeostatic level of gsh at high values of nadph relative to h2o2 is 0.86 (fig. 3a) while it decreases to about 0.73 at drastically decreased values of nadph relative to h2o2 (fig. 3 d). at the same time the homeostatic fractions of asc remains nearly unaffected in both cases. if h2o2 is increased relative to a fixed nadph value a similar effect on gsh (and asc) is obtained (not shown). the effect on the gsh level from decreasing nadph levels relative to h2o2 or from increasing h2o2 levels relative to nadph is vice versa. as has been shown for the enzyme rate parameters it is the ratio of these additional sets of parameters and not their absolute values which intab. 3: the parameter portrait of the antioxidative cascade. the normalized redox values of gsh, gssg, asc and dha (y-axis) are presented as functions of varying scaled values of k2 (x-axis) at constant values of k1 and k3 respectively. relative levels of gsh and gssg are depicted as solid and dotted lines and those of asc and dha are presented as dashed and dashdotted lines. the calculations were performed with the following parameters (taken from tab. 2); a: 4 days old dark grown seedlings, k1 = 0.036, k3 = 0.01, nadph = 0.6, η = 0.58, r = 0.15. the calculated values of gsh and asc (at x = unity) are 0.86 and 0.997 respectively. b: 9 days old dark grown seedlings, k1 = 0.036, k3 = 0.015, h = 0.36, r = 0.05. the calculated values of gsh and asc (at x = unity) are 0.88 and 0.998 respectively. c: 7 days old seedlings which are grown in a 14 / 12 h photoperiod during the last 3 days. k1 = 0.015, k3 = 0.007, η = 0.57, r = 0.3. gsh and asc (at x = unity) are calculated to be 0.52 and 0.99 respectively. d: gsh and asc under decreased nadph (η = 0.3), the rest of parameters and variables as in fig. 3a. the calculated values of gsh and asc (at x = unity) are 0.73 and 0.99 respectively. r e la ti ve c o n c e n tr a ti o n s 0.0 0.2 0.4 0.6 0.8 1.0 gsh gssg asc dha a b k2 0.00 0.05 0.10 0.15 0.95 1.00 r e la ti v e co n c en tr a ti o n s 0.0 0.2 0.4 0.6 0.8 1.0 k2 0.00 0.05 0.10 0.15 0.95 1.00 c d 116 edwin pahlich, christoph müller, hans-jürgen jäger fluence the redox ratios of the glutathione and ascorbate moieties. it is evident from these calculations that decreased / increased stationary concentrations of gsh can be caused by changing the enzyme rate parameter patterns, or the ratios of nadph levels relative to h2o2. under the parameter patterns considered the level of asc remains always on a highly reduced state. the redox ratios of the variables glutathione and ascorbate and their changes can not simply be interpreted as indicators of increased or decreased oxidative stress, i.e. changed levels of h2o2. electron fluxes and detoxifying efficiencies: the detoxifying efficience of the haf is best characterized by the electron fluxes through the system under the prevalent parametric conditions. therefore fluxes have been calculated with the measured parameters (tab. 1) and the redox levels of glutathione and ascorbate at k2 values of 1, 0.1, 0.05, 0.005 and 0.001 respectively (fig. 4). the relative fluxes derived from scaled parameters and variables show also biphasic patterns of different intensities and are bending downward at very low k2 values (fig. 4). this effect is most pronounced in plants that are grown under light. their parameter conditions promote highest electron fluxes despite that gr and apx and also gsh obtain lowest values. in comparison to this the fluxes in 4d and 9d old dark grown seedlings range on lower rates. all three fluxes approach similar values when k2 is becoming a limiting reaction step. under these conditions the regeneration of asc is fading out and the system does not agree any more to the concept formulated in fig. 2. it is surprising how far the rate parameter ratios can be changed without being mirrored by respective flux changes in the plateau ranges of the profiles. the example illustrates that electron flux (as metabolic fluxes in general) reflect sytem properties and can not be deduced intuitively from pool sizes of the redox variables or from enzyme activity changes of single reaction steps. it can be concluded that moderate enzyme activity alterations of the haf, which are reported in the literature, are only from minor consequences for changes of its detoxifying potential under respective parameter patterns. changing the input to output ratios nadph / h2o2 from 4:1 to 2:1 result in high fluxes if k2 is unity (fig. 6, in-out 2:1). but the entire system undergoes a shift from homeostatic to sensitive patterns in respect to decreasing k2. it should be noted that a very similar flux profile is reached under 10 fold increased k3. flux responses at decreased nadph input or at a 10-fold increase of k3 (fig. 6, in-out 2:1 and k3) are nearly indentical. thus, two entirely different parameter changes result in nearly identical system responses which again elucidates that system responses are hardly to foresee intuitively. modelling assumed genetic transformations of the haf: tailoring of plants for improving their (oxidative) stress performance via transformation of particular reaction steps is a widely followed concept (broadbent et al., 1995; noctor et al., 1997; xiang et al., 2001; strohm et al., 2002; rizhsky et al., 2002b; murgia et al., 2004; mittler and poulos, 2004; yamamoto et al., 2005). from the discussion in the previous section it can be seen which results might be received by over-expressing enzymes of the haf. if in 4 d dark grown seedlings the k1 would be over-expressed 10-fold then the measured parameter ratio k1 : k2 : k3 would change from the observed ratio 0.036 : 1 : 0.01 to the assumed ratio 0.36 : 1 : 0.01. the model predicts for this tailored parameter constellation that concentrations of asc and gsh approach their maximal levels, which is nearly unity for fig. 5: the effect on glutathione and ascorbate moieties of increased k1 and k3. the effect of a 10 fold increase of k1 (fig. 5a) or of k3 (fig. 5b) on the amount of gsh and asc. other conditions as described in fig. 3a. gsh and asc remain reduced under increased k1 (fig. 5a) but drop dramatically under increased k3 (fig. 5b). k2 0.0 0.2 0.4 0.6 0.8 1.0 s c a le d c o n c e n tr a ti o n s 0.0 0.2 0.4 0.6 0.8 1.0 gsh gssg asc dha a k2 0.0 0.2 0.4 0.6 0.8 1.0 b fig. 4: electron fluxes calculated with the parameters and variables of figs. 3 a to c at decreasing values of k2. the fluxes exert well established homeostatic ranges down to a nearly 100 fold decrease of k2 relative to constant values of k1 and k3. k2 (log-scale) 1 0.1 0.05 0.005 0.001 s c a le d f lu x e s 2 4 6 8 10 12 14 16 18 20 22 9d dark 4d dark 4d dark, 3d 14/10 photoperiod modelling the glutathione-ascorbate redox system of plants 117 asc (fig. 5a). due to the equilibrium of the gr reaction gsh can not exceed 0.9 on the relative scale. in contrast to expectations (increasing support of the cascade with gsh), the electron flux at increased k1 remains low and moves on a plateau over a wide range of k2 changes (fig. 6). this is easy to understand since the substrate dha is on its lowest level under these conditions (fig. 3) and necessarily opposes the parameter increase. checking these predictions experimentally is difficult. overexpression of a particular enzyme like gr might enhance also other enzymes like apx and dhar (strohm et al., 2002) and therefore change the entire rate parameter patterns. furthermore the effects arising from overexpression of a particular gene might be limited to a given developmental state and is different under other developmental conditions (strohm et al., 2002). another question is whether changes of single gene expressions can be reached in vivo as practiced by modelling. increased apx (k3) activities are expected to increase the rate of detoxification of h2o2. assuming a 10-fold increase of k3 in 4 d old seedlings changes the ratio of k1 : k2 : k3 to 0.036 : 1 : 0.1. the stationary gsh pool under these conditions is predicted to drop to a level of < 0.2 and the asc pool decreases to 0.6 (fig. 5 b). the predicted steady flux profile over changing k2 is very different (fig. 6, k3) to the control (fig. 4, 4d). the system has lost its homeostatic plateau and is changed into a k2-sensitive slope (fig. 6, k3). this parametric alteration causes a system change and switches it from a stable to a sensitive functional form. if electron flux is taken as a measure for antioxidative capacity then the system should become less effective with respect to h2o2 detoxification under this parameter constellation. overexpression of particular enzymes might show the expected response under certain growth conditions. but even slightly different growth conditions might perturb the system and evoke „flexible“ and unpredictable responses. it has been shown for instance that overexpression of tapx increases the resistance against o2 under given growth conditions but does not reinforce defense under changed light or heavy metal environments (murgia et al., 2004). even so we are convinced that modelling provides useful insights into the responsiveness and functioning of complex metabolic units like haf which can not be gained by guessings. but modelling „physiological reality“ (polle, 2001) is a much more complex task than modelling a defined „core unit“ of metabolism. glutathione and ascorbate consumption cause relaxations: glutathione and / or ascorbate can be withdrawn from the haf system for instance by intercellular transport (foyer et al., 2001) or by asc dependent hydroxylation of extracellular proteins (de tullio et al., 1999). export and re-import of gsh and / or gssg and asc and / or dha among cellular compartments such as the apoplast or the vascular system are also reported (smirnoff et al., 1996; horemans et al., 2000; heber et al., 2003; yamamoto, 2005). in any of these cases the redox balance within the haf will be disturbed and a re-adjustment according to the given parameter patterns by a relaxation process will be initiated. the time scales of the relaxations are different for any reaction because of individual relaxation times τ which are approximated by km / vmax values in enzyme catalyzed reactions (bücher and rüssmann, 1963; higgins, 1963). the smaller the relaxation time the more effective is the transition process. in 4 d old seedlings (tab. 1) are observed asc levels (fig. 3) which are out of redox balance and far too low according to model calculations. under the given set of parameters a relaxation would be unavoidable under these conditions (fig. 7). the pool of asc which is about 0.5 at time zero increases instantaneously and approaches its stationary value close to unity in an extremely short period of time. this reduction occurs on the cost of the gsh pool which at the same time drops from 0.86 to a value of less than 0.5. this drop is caused by the high k2 value or its respective relaxation time τ2 which is 0.013. in contrast to asc the regeneration of the stationary gsh level is slow because of the lower relaxation time τ1 of the gr which is 0.34. the consequence of these differences in the relaxation times of the two enzymes is that the gsh level reaches its original stationary value of 0.86 long after the adjustment of the asc pool. these remarkable differences in the time courses of the adjustments of the stationary values of perturbed systems can also be observed in physiological responses of even higher complexity as the haf like the the prolin acclimation in water stressed plants (pahlich, 1996). as long as it is open whether variables have reached their stationary fig. 6: electron fluxes under increased rate parameters k1 or k3 (other conditions as defined in fig. 5a) and under decreased amounts of nadph relative to h 2 o 2 (η : r is 0.3 : 0.15). increasing the rate parameter k1 is ineffective in respect to the homoeostatic patterns of the system. increasing amounts of k3 or decreasing levels of nadph relative to h 2 o 2 make the system sensitive to changes of the parameter k2. k2 (log-scale) 1 0.1 0.05 0.005 0.001 s c a le d f lu x e s 0 2 4 6 8 10 k1 10fold k3 10fold in-out 2:1 fig. 7: adjustment of the unbalanced dha : asc moiety over time according to the parameter patterns of 4d old seedlings. according to the system modelling (fig. 3a) the measured asc fraction (tab. 1) of seedlings is too low. the balance of the variables in dependence of the measured rate parameters is achieved in a two step relaxation process. dha is reduced immediately after the onset of the relaxation process on the cost of gsh which drops from 0.86 to about 0.45. in a second step gsh is approaching its steady value at a much lower rate than asc. the gsh : gssg values up to about 250 time units might be regarded as „pseudo steady-state“ values. time (min) 0 50 100 850 900 r e la ti v e c o n c e n tr a ti o n s 0.0 0.2 0.4 0.6 0.8 1.0 asc dha gsh gssg 118 edwin pahlich, christoph müller, hans-jürgen jäger values after experimental perturbations it must be assumed that they are part of a so called „pseudo steady-state“ (heinrich and rapoport, 1977). the variables are not yet balanced according to the parameter patterns and are still moving on a transitory trajectory. the level of the variables can be far away from their final and balanced states while accompanying metabolites have reached their balanced state already. in the presented case the decreasing levels of gsh at high asc contents in the initial phase of the relaxation will hardly provide a safe base for intuitive interpretations of the physiological significance of the state of haf. thiol-disulfide proteome, a subsystem of haf?: it is assumed that nadph, gsh and also asc are electron supporters of the thioldisulfide proteome which therefore are competing reactions to the detoxification of h2o2. the genetic multiplicity of the glutathione ascorbate cycle genes (mittler and poulos, 2005) and those of the thiol-disulfide proteome (dietz, 2005) in plants can cause highly diverse and tissue specific combinations of the proteom’s components with the antioxidative cascade. this multiplicity can not be treated with a simplifying dynamic model. but some aspects of the supposed interactions can be deduced from our findings. it can be assumed that the redox cycles of the thiol-disulfide proteins operate at lower concentration and rate scales than those of the antioxidative cascade. consumption of gsh or asc or direct changes of their redox ratios will necessarily cause relaxations of the haf. in any case the redox variables glutathione and ascorbate will be balanced according to the parameter structure of the antioxidative cascade. these suggestions might be taken as evidence that the antioxidative cascade functions as a superior balancing system which mirrors the cellular redox state according to the parameter ratios of the haf. does the antioxidative system operate at a steady-state?: the described properties of the antioxidative system are strictly valid only if the antioxidative system operates at steady-state. this assumption includes steady values of nadph and h2o2. a steady-state of the haf can most likely be established only under conditions when a moderate and continuous electron demand is needed for the detoxification of continuously produced metabolic h2o2 while the support with nadph remains undisturbed. however, increased burdens of h2o2 (e.g. caused by ozone spikes), changed availabilities of nadph, environmentally induced sudden enzyme activity changes will perturb the system due to drastically changed input to output ratios and enzyme rate parameter patterns. thus, the system is forced into relaxations with fluctuating concentrations of the variables according to individual relaxation times of the particular reactions (fig. 7). the system is removed from its initial steady state and will stay in motion until its self-organized search for a new possible steady-states is reached (von bertalanffy et al., 1977; peacock, 1983). we emphazise the core process of the antioxidative system as a nondecomposable set of enzymes (papin et al., 2003) which looses its particular system properties if parametric components are included or deleted. this undissectable reaction unit might be part of a broader metabolic network with superimposed regulatory properties. advanced analytical techniques (schwender et al., 2004) and mathematical network approaches, which are based on whole genome responses (papin et al., 2003), might help to uncover missing components of an extended antioxidative network. however the basic functioning of a minimal and unique pathway needs still be analyzed and characterized by a classical kinetic approach. based on these results it is doubtful whether system properties like homeostasis or efficiency of functioning of thiol linked redox processes can be deduced from genome expression data (foyer and noctor, 2005) or from proteome based insights (dietz, 2005). these techniques are best suited to identify system components. insights in functionality however, have to be based on kinetic data which have to be analysed via system modelling. system characteristics like homeostasis, emerge from the „supramolecular organization of the enzymes“ (srere, 1993; srere, 1994). this is a functional level beyond gene expression or proteom patterns. conclusions the modelling of the antioxidative system has uncovered properties which hitherto have been overlooked. the particular enzyme activity ratios of the antioxidative halliwell-asada-foyer (haf) cascade of wheat roots give rise to the formation of homeostasis. in the homeostatic range the redox ratios of the glutathione and ascorbate moieties remaine on a stable level as opposed to rather dramatic enzyme activity changes. the ratios of the homeostatic redox moieties 2gssg : gsh and dha : asc depend on the ratios of the respective enzyme parameter patterns of the system when the pools of nadph and of h2o2 are in steady-state. under the parameter conditions considered the redox moieties of glutathione and ascorbate were kept on highly reduced states. the measured level of dha most likely represents a fraction which is located in a non-regenerating compartment, probably the apoplast or the vascular system. major changes of the redox ratios of the glutathione and ascorbate moieties can also result from changes of the nadph to h2o2 ratio. decreasing concentrations of nadph relative to h2o2 cause identical redox properties of the redox variables with increasing h2o2 burdens relative to an unchanged nadph pool. the redox state of the glutathione and ascorbate moieties can not be regarded as indicators for oxidative stress in terms of increased h2o2 levels. the electron fluxes through the system exert also homeostatic branches. neither increased enzyme activities nor increased levels of the reduced states of the variables are indicators for increased fluxes or increased antioxidative capacities. high electron fluxes can occur under low fractions of gsh and asc and the system might be highly efficient with respect to h2o2 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vulgaris. biochimie 80, 295-301. xiang, c., werner, b.l., christensen, e.l.m., oliver, d.j., 2001: the biological functions of glutathione revisited in arabidopsis transgenic plants with altered glutathione levels. plant physiol. 126, 564-574. yamamoto, a., bhuiyan, n.h., waditee, r., tanak, y., esaka, m., oba, k., jagendorf, a.t., takabe, t., 2005: suppressed expression of the apoplastic ascorbate oxidase gene increases salt tolerance in tobacco and arabidopsis plants. j. exp. bot. 56, 1785-1794. address of the authors: department of plant ecology, justus liebig-university, heinrich-buff-ring 26 32, d-35392 giessen e-mail: edwin.pahlich@bot3.bio.uni-giessen.de christoph.mueller@uce.ie hans-juergen.jaeger@bot2.bio.uni-giessen.de << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb 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/pagesize [907.087 680.315] >> setpagedevice journal of applied botany and food quality 87, 67 73 (2014), doi:10.5073/jabfq.2014.087.010 1department of agronomy, faculty of agricultural sciences, universidad nacional de colombia, bogota, colombia 2 division urban plant ecophysiology, faculty of agriculture and horticulture, humboldt-universität zu berlin, berlin, germany 3institute of food chemistry, universität hamburg, hamburg, germany salinity effects on proline accumulation and total antioxidant activity in leaves of the cape gooseberry (physalis peruviana l.) diego miranda1*, gerhard fischer1, inga mewis2, sascha rohn3, christian ulrichs2 (received march 4, 2013) * corresponding author summary in colombia, the cape gooseberry is grown in salt-affected areas. the effect of increasing sodium chloride (0, 60 and 120 mm nacl) stress was investigated on the growth, proline content and total antioxidant activity (taa) in leaves of cape gooseberry plants kept in 2 l pots and grown under greenhouse conditions. plant leaves were analyzed 45, 55, 65 and 75 days after transplanting (dat). the vegetative growth (measured as total plant and organ dry weight [dw], leaf number and leaf area, as well as plant height) was significantly lower at 120 mm nacl, as compared to the control and 60 mm treated plants. at 60 mm nacl, all determined leaf parameters and total plant dw were markedly reduced, as compared to non-salinized plants. the leaf proline content increased significantly during the evaluation period and tended to be higher with increasing nacl concentrations, but without statistical differences. the taa, measured as μm fremy’s salt per g-1 fw, increased constantly during the evaluation period and, from day 55, was significantly higher than in leaves of non-salinized plants. after 75 dat of salt stress, the taa and proline content did not differ between the 60 and 120 mm nacl treatments. for all sampling dates, the 120 mm salt concentration significantly enhanced the free radical scavenging activity, as compared to the control and the 60 mm nacl treatment. all treatments showed a nearly 12% increase in the radical scavenging activity during the experiment’s duration. in conclusion, cape gooseberry plants protect themselves from salinity (120 mm nacl) stress by increasing leaf antioxidant activity, as confirmed by the higher radical scavenging activity, but not significantly with proline synthesis. introduction salt stress poses a major environmental threat to agriculture, limiting the productivity of crop plants (türkan and demiral, 2009). this is due to the fact that salinity affects most aspects of plant physiology, growth and development (borsani et al., 2003). most plants, especially glycophytes, are very sensitive to high na+ concentrations (kaya et al., 2007). in addition, salinity has a stronger effect on fruit crops than on field, forage or vegetable crops (mengel et al., 2001). high concentrations of na+ can lead to cell death by disturbing the intracellular homeostasis, which leads to membrane dysfunction, attenuation of metabolic activity, and secondary effects that cause growth inhibition (ashraf and mcneilly, 2004). however, some plants are able to tolerate saline and drought stress by reducing the cellular osmotic potential with a net increase in solute accumulation in a process called osmotic adjustment (munns, 2002). among the metabolic responses to salt stress, the synthesis of compatible osmolytes, as well as non-toxic organic compounds, is responsible for the osmotic equilibrium between the cytoplasm and different cell compartments. thus, many plants synthesize amino acids and amides (proline, alanine, glutamine, asparagine), quarternary ammonium bases (betaines), as well as various sugars, polyols (e.g., mannitol, sorbitol), and cyclites (pinitol) as a non-specific reaction to stress from excess salt, drought or frost (larcher 2003). these organic compounds are thought to mediate osmotic adjustment, protecting the subcellular structures and preventing oxidative damage with their free radical scavenging capacity (smirnoff, 1993). it is generally accepted that, under conditions of extreme salinity, proline accumulation serves as a defense against osmotic challenge by acting as a compatible solute and appears to be the preferred organic osmoticum in many plants (manchanda and garg, 2008). a characteristic pattern of stress metabolites is associated with different plant groups. for instance, soluble carbohydrates are found in poaceae and proline in brassicaceae (larcher, 2003). ionic and osmotic effects (tarakcioglu and inal, 2002) and oxidative stress (borsani et al., 2003) induced by salinity and sodicity can reduce plant growth and alter ionic ratios. oxidative stress is characterized by the overproduction of active oxygen species, predominantly represented by superoxide radicals (o2–), singlet oxygen (1o2), hydrogen peroxide (h2o2) and hydroxyl radical (oh·), which cause tissue injury (foyer et al., 1994). the degree of oxidative cellular damage in plants subjected to abiotic stress is controlled by the capacity of the antioxidative systems (acar et al., 2001) to overcome salt-mediated oxidative stress. plants detoxify reactive oxygen species (ros) by up-regulating antioxidative enzymes, such as superoxide-dismutase, ascorbate peroxidase, catalase, glutathione reductase and glutathione peroxidase (türkan and demiral, 2009). pre-harvest factors may affect the content and stability of phytochemicals in plants and influence their antioxidant capacity (wang, 2006). de pascale et al. (2003) reported that it is possible to improve the contents of carotenoids and ascorbic acid and, thereby, the antioxidant activity in tomatoes by irrigating with saline water. in colombia, the cape gooseberry (physalis peruviana l., solanaceae) was grown on 743 ha in 2011 (agronet, 2013) at sites ranging from 1800 to 2800 m above sea level. the cape gooseberry is not only an important source of vitamins (a and c) for colombian highland farmers but also has become the second most important export fruit (fischer et al., 2007). the cape gooseberry, like most horticultural crops, is glycophyte; generally believed to have little or no tolerance to salinity (grattan and grieve, 1999). if agricultural crops are to be used in potentially arable soils, a moderate degree of salt resistance may be useful to avoid yield reduction, especially in more arid regions (larcher, 2003). in that vein, the mechanisms involved in the responses to salt stress must be understood in order to improve productivity under salinity stress conditions (manchanda and garg, 2008). nearly 600,000 ha of the agriculturally cultivated area in colombia are affected by salinity (casierra et al., 2000). furthermore, cape gooseberry orchards on the bogota plateau are impaired by salinization. however, no attempts have been made to study proline accumulation and total antioxidant activity in cape gooseberry leaves 68 diego miranda, gerhard fischer, inga mewis, sascha rohn, christian ulrichs under these conditions. in order to verify the possibility of stressinduced proline synthesis and total antioxidant activity in cape gooseberry leaves, the effect of two increasing nacl concentrations was studied under greenhouse conditions. materials and methods plant material and management this experiment was carried out in the laboratories and glass greenhouse of the urban plant ecophysiology division at humboldt university, berlin, germany, using the p. peruviana ‘colombia’ ecotype. seeds were germinated in blond peat substrate. the seedlings were transplanted in the greenhouse 20 days after sowing. forty-five days after seeding, 340 seedlings were transplanted to black plastic pots (2 l) containing perlite as a substrate, where they grew over a period of 75 days. the pots were placed in double rows on elevated greenhouse tables, with a plant spacing of 0.5 m between rows and 0.35 m between plants within a row, resulting in a density of 5.7 plants per m2. the climatic conditions in the greenhouse during the experiment were 24.9/20.4 °c mean air day/night temperature, 60.3% mean relative humidity and 484.1 μm m-2 s-1 mean photosynthetic active radiation, as indicated in tab. 1. the day-length at the experiment’s start (7 of april) was 13.3 light hours, which increased to 17.0 light hours when the greenhouse experiment ended (30 of june). the nutrient solution for the adult cape gooseberry plants was prepared by dissolving 0.2 g kristalontm (19-6-20-3 + micronutrients) per liter of water. the electrical conductivity (ec) of the nutrient solution was 1.4 ds m-1. the application was made with a dosatron, which applied a 0.5% concentration with two applications per day, each 3 min in length. mine dry weight (dw). the leaf area was measured with a portable leaf area meter (model li-3000 a, li-cor, lincoln, ne). proline and total antioxidant activity determination samples of fully expanded leaves, obtained from the medium third of the plants and the medium third of the reproductive shoots, located after the first natural branch ramification, were collected at 45, 55, 65 and 75 dat, between 9.30 and 10.30 am, and, then, shock frozen in liquid nitrogen. the proline content was determined for the 0, 60 and 120 mm nacl treatments according to bates et al. (1973). purified proline (sigma-aldrich, steinheim, germany) was used as the standard. approximately, 0.5 g of plant material was homogenized in 10 ml of 3% aqueous sulfosalicylic acid and the homogenate was filtered through a paper filter (whatman # 2). two ml of filtrate were reacted with 2 ml ninhydrin and 2 ml of glacial acetic acid in a test tube for 1 hour at 100 °c and the reaction was terminated in an ice bath. the reaction mixture was extracted with 4 ml toluene and mixed vigorously with a test tube stirrer for 15-20 sec. the chromophore containing toluene was aspirated from the aqueous phase, warmed to room temperature and the absorbance read at 520 nm, using toluene for a blank. the proline concentration was determined from a standard curve and calculated on a fresh weight basis as follows: [(μg proline/ml × ml toluene) / 115.5 μg / μmoles]/[(g sample)/5] = μmoles proline/g of fresh weight material. the total antioxidant activity (taa) was determined using electron spin resonance spectroscopy (esr) (rohn and kroh, 2005), in accordance with the following procedure: 1-2 leaves of the middle part of the plant (using three plants per repetition) that were frozen in liquid nitrogen were ground with a mortar and pestle. 500 mg of sample and 5 ml of methanol (70%) were weighed in centrifugation tubes and vigorously shaken for 2 min. following centrifugation at 2000 rpm for 10 min (eppendorf centrifuge hermle z320, hamburg, germany), the undiluted supernatant was used for esr spectroscopy with fremy’s salt (potassium nitrodisulphonate) as a stabilized radical. the measurements were taken according to roesch et al. (2003). 100 μl of fremy’s salt (1 mm) + 100 μl of sample were measured for 20 min against a blind (100 μl buffer) and fremy’s radical esr spectrum was obtained after 20 min, when the reaction was complete. signal intensity was obtained by integration (phenolic compounds or further aromatic compounds) and antioxidant capacity (expressed as moles of fremy’s salt reduced by one mole antioxidant) was calculated by comparison with a control reaction using methanol. the results are expressed as units of μm fremy’s salt per g fresh weight (fw). free radical scavenging activity the quantitative measurement of the radical scavenging properties of the sample extracts of cape gooseberry leaves was carried out in a universal bottle. the reaction mixture contained 50 μl of test samples (or 80% meoh as a blank) and 5 ml of a 0.04% (w/v) solution of fremy’s salt in methanol. different known antioxidants, vitamin e (alpha tocopherol) and butylatedhydroxytoluene (bht, sigma) were used for comparison or as a positive control. the discoloration was measured at 517 nm after incubation for 20 min. the measurements were carried out at least in triplicate. fremy’s salt radical’s concentration was calculated using the following equation: fremy’s salt scavenging effect (%) = ao – a1 / ao x 100, where ao was the absorbance of the control and a1 was the absorbance in the presence of the sample extract of cape gooseberry leaves according to oktay et al. (2003). day night tab. 1: ranges of mean day and night temperature, relative humidity and photosynthetic active radiation (par) (±sd) in the greenhouse at the four sampling periods of the experiment. days after air temperature (ºc) relative par transplanting humidity (dat) (%) (μm m-2 s-1) 36-45 26.0 ± 4.5 20.9 ± 2.4 66 ± 3.6 535.8 ± 34.6 46-55 23.9 ± 4.8 19.5 ± 2.5 60 ± 5.7 502.7 ± 67.6 56-65 27.5 ± 5.2 22.3 ± 2.1 55 ± 2.0 500.4 ± 49.1 66-75 22.3 ± 4.4 19.1 ± 1.8 60 ± 3.9 397.6 ± 56.9 sodium chloride treatments three treatments of 0, 60 and 120 mm nacl were studied over 12 weeks. each plot consisted of 68 plants, 340 plants in total. nacl applications began 2 days after transplanting (dat). the electrical conductivity (ec) of the irrigation water was measured twice a week using a conductivity meter (hanna instruments hi 9811, ann arbor, mi) and averaged at: 0.70 (non-saline, control), 6.36 (60 mm nacl), and 12.53 (120 mm nacl) ds m-1 over the course of the experiment. the ph was recorded using a portable ph meter (testo 206, kkinstruments, berlin) and averaged at: 7.9, 8.06, and 8.12, respectively. samples of fully expanded leaves (six leaves per plant from three plants per repetition) were collected and shock frozen in liquid nitrogen before analysis. growth measurements the plants were harvested at 75 dat and separated into roots, stems and leaves. the plant material was dried at 70 ºc for 48 h to deter salinity in proline accumulation and antioxidant activity 69 statistical analysis the experiment involved a completely randomized design with three replicates. the data were statistically analyzed by anova using the sas software (sas 9.1); significant differences between treatments were calculated by tukey’s test (p≤0.05). results plant growth salinization of the irrigation water significantly affected all the measured vegetative growth parameters of the cape gooseberry (fig. 1 and 2). at 60 mm nacl, all the leaf parameters (dw, total area and number per plant) were significantly (p≤0.05) reduced. the considerably lowered leaf number (p≤0.05) with increasing salinity induced a significantly greater unit leaf area (p≤0.05), 40.8 cm2 at 60 mm nacl. at the intermediate salinity, only root and stem dw and plant height remained unchanged (p>0.05) by the salinity. the higher growth reductions at 120 mm nacl were found in root dw (46.8%), plant dw (34.3%) and leaf dw (33.9%), while plant height (99.0 cm) was only slightly, but significantly (p≤0.05), affected, i.e. 8.3% lower than the control plants. proline content the leaf proline content showed an overall increase during the time of salinity exposition, with no clear tendency from 45 to 65 dat, but, at 75 dat, the content was significantly higher (p≤0.05) than at 65 dat (tab. 2). the salt treated plants tended to have higher proline concentrations than the control plants, but without statistical differences (p>0.05). after 75 dat, both salinity levels exhibited the highest proline contents, which were nearly identical (about 0.8 μm g-1 fw). fig. 1: effect of nacl on root, stem, leaf and plant dry weight of cape gooseberry at 75 dat. bars indicate ±se. fig. 2: effect of nacl on plant height, leaf number, leaf area and unit leaf area of cape gooseberry at 75 dat. bars indicate ±se. tab. 2: effect of nacl salinity on the proline content (μm g-1 fw) in leaf tissue of cape gooseberry plants between 45 and 75 dat. nacl (mm) days after transplanting (dat) 45 55 65 75 0 0.51 aa 0.18 ac 0.23 ab 0.46 aa 60 0.64 ab 0.36 ac 0.56 ab 0.80 aa 120 0.54 ac 0.47 ac 0.68 ab 0.82 aa means in columns followed by the same letters are not significantly different according to tukey’s test (p≤0.05). lowercase letters are for comparing between nacl concentrations and uppercase letters between sampling days. total antioxidant activity in this methodology, the scavenging of the synthetic stable radical fremy’s salt was followed. the present study revealed that 3.11, 3.25 and 3.86 μm fremy’s salt were scavenged on average by 1 μm antioxidant present in the foliar tissue of plants subjected to stress with 0, 60 and 120 mm nacl, respectively. the total antioxidant activity (taa) increased constantly during the period of exposure to saline stress. the maximum taa was 4.18 μm fremy’s salt per g fw for plants exposed to 120 mm nacl at 75 dat, as compared to the minimum of 2.72 μm in the control plants on the first sampling date (tab. 3). the 120 mm nacl treatment resulted in a significantly (p≤0.05) higher taa than the lower salt concentrations at days 45, 55 and 65. tab. 3: effect of nacl salinity on the total antioxidant activity (μm fremy’s salt/g fw) in leaf tissue of cape gooseberry plants between 45 and 75 dat. nacl (mm) days after transplanting (dat) 45 55 65 75 0 2.72 bc 3.16 bb 3.15 bb 3.46 ba 60 2.74 bc 3.17 bb 3.29 bb 3.78 aba 120 3.28 ac 3.84 ab 4.18 aa 4.14 aa means in columns followed by the same letters are not significantly different according to tukey’s test (p≤0.05). lowercase letters are for comparing between nacl concentrations and uppercase letters between sampling days. free radical scavenging activity in all treatments, the free radical scavenging activities of the leaf plant extract increased until 55 dat (tab. 4), but, after that date, no clear tendency was noticeable. it was obvious that, at all sampling dates, the 120 mm salt concentration significantly enhanced (p≤0.05) the free radical scavenging activity, as compared to the control and 60 mm treated plants. in general, all the treatments showed a nearly 12% increase in scavenging activity over the course of the experiment. 70 diego miranda, gerhard fischer, inga mewis, sascha rohn, christian ulrichs discussion plant growth a plant’s metabolism is affected in several ways by salt stress and, therefore, growth is reduced (manchanda and garg, 2008). a lower biomass production induced by salinity is common in most plants (larcher, 2003) and total plant and organ dw and leaf number have been reported to be the most commonly used criteria in order to characterize salinity stress and tolerance (levitt, 1980). if the rooting medium has an excess of salts, the osmotic pressure will be increased, thereby decreasing the plant’s ability to take up water and slowing growth (manchanda and garg, 2008). a common result of a decrease in the osmotic potential of a plant’s cells under salt stress is a malfunction in the enzymatic system of the plant, which reduces co2 fixation and n assimilation and, consequently, causes a shortage in plant structural material production (aslam et al., 1984). also, the impaired photosynthesis under severe salinity stress, as a result of smaller leaves and a higher stored carbohydrate usage as compared to plants in a nonsalt stressed environment, reduces biomass production (mengel et al., 2001). furthermore, carbon dioxide uptake can be limited by the inadequate photosynthesis caused by stomatal closure, which further reduces the growth rate (zhu, 2001). root growth was extremely affected, expressed in the root biomass reduction (fig. 1) and low number of secondary roots (data not shown), at 120 mm nacl, in agreement with neumann (1995), who found that high salt concentrations inhibit the initiation of new roots and radical growth in the strawberry. azooz et al. (2004) described the reduced hormone delivery from root to leaves as the principal effect of salinity on the inhibition of crop growth. growing leaves are particularly sensitive to water stress (induced by salinity) because foliar tissue has very high transpiration rates (jones, 1992). furthermore, leaf expansion responds rapidly to changes in the water potential gradient between the substrate solution and the roots (cramer and bowman, 1991), which is markedly influenced under salinity conditions. in our study, the salinity conditions reduced the number of leaves (fig. 2) due to the lower plant growth rates (miranda et al., 2010a; munns and termaat, 2008) and shorter leaf duration (manchanda and garg, 2008) resulting from the premature leaf fall of adult and necrotic leaves, as compared to non-salinized plants. leaf growth variations under saline stress have been found to be correlated with foliar na+ content in triticum (schachtmann and munns, 1992) and the soybean (doğan, 2011). similarly, in the cape gooseberry, leaf na+ concentrations increased with greater nacl salinity (miranda et al., 2010b). abscission of old leaves is common under salt stress (munns and termaat, 1986) because the principal site of na+ toxicity, in a great number of plant species, is the leaf blade, where na+ accumulates after being placed in the transpiration stream (munns and termaat, 2008). our results indicated that stem elongation was less affected than total leaf area (fig. 2) by salinity stress, which coincides with the findings of chartzoulakis and klapaki (2000) in pepper plants. the impact of increasing salt concentrations on cape gooseberry growth shows that this plant begins to manifest a reduction in leaf dw starting at just 60 mm, where plant height, stem and root dm are not yet affected, which indicates a resistance to relatively high salt concentrations. miranda et al. (2010a) classified the cape gooseberry as a moderate salt tolerant crop, wherein 30 mm nacl stimulated growth indices such as crop growth rate, relative growth rate, unit leaf rate and leaf area, as compared to non-salinized plants. proline content the accumulation of compatible solutes, such as proline, a preferred organic osmoticum in many plant species, is one of the key mechanisms used by higher plants to respond to salt-stress conditions (larcher, 2003). compatible solutes are used for osmotic adjustment and for maintaining the functional state of macromolecules, probably by scavenging ros (xiong and zhu, 2002). proline accumulates more in the leaves of plants that are more tolerant to salinity stress than in salt sensitive plants (türkan and demiral, 2009). considering that the cape gooseberry is classified as a moderately salt tolerant plant (miranda et al., 2010a), this behavior could not be attributed to the accumulation of proline in the present study because the proline content of the two nacl treatments only tended to be higher than that of the control plants starting at 55 dat, because there were no statistical differences. the increase in proline content during the vegetative growth phase in the nacl treatments suggests that the plants tried to stabilize their protection mechanism (szabados and savouré, 2009) with increased salt accumulation in the leaves. also, in wheat plants, a salt stress provoked an increase in leaf proline content, but higher accumulation was recorded at the vegetative and boot stages than at the reproductive stage (ashram et al., 2007). an increase in leaf proline content also depends on environmental stress factors, such as high light intensity, extreme temperatures and low relative humidity, as claussen (2007) reported in tomatoes, but this behavior could not be confirmed in our experiment. furthermore, at the lowest medium temperature (22.3 °c) and par, the highest proline concentration was measured (tab. 1). total antioxidant activity pastori et al. (2000) stated that many stress situations can generate increased foliar antioxidant activity. higher levels of antioxidants, whether constitutive or induced, have been reported to induce a greater resistance to different types of environmental stress conditions (young and jung, 1999). in the present study, the taa increase with increasing nacl concentrations agrees with the results of de pascale et al. (2003), who observed that salinity increased lipophilic and hydrophilic antioxidant activities in tomato fruits. a correlation between antioxidant capacity and salinity tolerance was reported by türkam and demiral (2009) and other authors cited therein, for several plant species (cotton, rice, wheat, pea, sesame, among others) and fruit species, such as citrus fruits by guetadahan et al. (1997). according to wang et al. (2009), who found that tolerant species generally withstand saltor drought-induced oxidative stress with improved antioxidant enzyme activities, the cape gooseberry can be classified as a moderate salt tolerant species due to its increasing taas with increasing nacl concentrations. the moderate salt tolerance of this species has been previously reported in terms of germination (miranda et al., 2010c), growth indices tab. 4: effect of nacl salinity on the free radical scavenging activity (%) by antioxidants in the extract of foliar tissue of cape gooseberry plants between 45 and 75 dat. nacl (mm) days after transplanting (dat) mean 45 55 65 75 0 50.7 b 62.3 b 58.7 b 61.6 b 58.3 b 60 46.8 b 55.6 c 55.9 b 60.0 b 54.6 b 120 57.6 a 73.8 a 60.0 a 68.4 a 65.0 a means in the same column followed by the same letters are not significantly different according to tukey’s test (p≤0.05). salinity in proline accumulation and antioxidant activity 71 (miranda et al., 2010a) and leaf ca2+, k+ and na+ accumulation (miranda et al., 2010b). ramachandra et al. (2004) demonstrated that the antioxidant response level depends on stress duration and intensity, plant species, development, and metabolic state. results for the soybean have shown that the activity of antioxidant enzymes under salinity depends on kind, age and type of plant organs, as well as on the salinity level (doğan, 2011). adult physalis plants produced more taa than young plants (tab. 4), corresponding to the report of buchanan et al. (2002) that states that resistance or sensitivity to the stress depends on the developmental age of the plant. consequently, reactive oxygen species (ros) are continuously produced in plants. fruit crop maturity significantly influences antioxidant capacity (wang, 2006). however, whereas the taa increases with fruit maturity; in leaves of the red raspberry, blackberry and strawberry, the opposite was seen, i.e. the taa decreased with leaf age (wang and lin, 2000). given the high content of antioxidants in cape gooseberry fruits (ramadan, 2011), its cultivation in moderately saline soils or with saline water irrigation may also increase antioxidant activity in fruits, as found in tomatoes by de pascale et al. (2003), in the cois hc01 hybrid, when watering with saline water of up to 4.4 ds m-1 ec, and in the leovovil and cervil tomato varieties by gautier et al. (2010), using 7.6 ds m-1 water, and, in turn, also increase the content of total soluble solids (de pascale et al. (2003) in fruit organs. similar to the taa, the free radical scavenging activity also increased with increasing nacl concentrations. antioxidants use scavenging free radicals as a mechanism for inhibiting lipid peroxidation. the fact that ros-induced lipid peroxidation of membranes reflects stress-induced damage at the cellular level is well accepted (jain et al., 2001). the lack of difference found between the non-salinized plants and the plants treated with 60 mm nacl for free radical scavenging activity confirmed that this salt concentration was probably not high enough to produce oxidative stress conditions in the plants. on the other hand, in agreement with duh and yen (1997), the appropriate concentration of crude extracts of non-stressed cape gooseberry leaves may act as a free radical scavenger and may react with free radicals to convert them into more stable products and terminate radical chain reactions. wu et al. (2006) found, in p. peruviana leaf extracts, a strong superoxide anion scavenging, antioxidant, and anti-inflammatory activity, underlining the role of this fruit species in folk medicine. also, miranda et al. (2010b) found no change in leaf ca2+ concentrations in cape gooseberry plants with increasing salinity levels, which could indicate the effect of ca2+ in alleviating salinity stress symptoms due to its important role in stability and integrity of biological membranes and tissues (mengel et al., 2001). in addition, it could be possible that the irrigation and nutrition management in the present study did provide normal conditions, where the ros production produced by the low saline stress was efficiently scavenged by the plants’ antioxidant systems. wang (2006) reported that management practices such as fertilization and organic farming, as well as climatic and soil conditions (light, temperature, co2) influence a crop’s antioxidant content and activity. thus, in the present study, from days 56 to 65, the mean temperature increase was high, from 19.6 to 31.2 ºc, and significantly correlated with the taa increase (r=0.68; p<0.05). also, wang and zheng (2001) recorded a higher scavenging capacity of strawberry plants against active oxygen species when day/night temperatures were high (30/22 ºc and 25/22 ºc), as compared to low temperatures (18/12 ºc). ulrichs et al. (2008) found a higher antioxidant activity, expressed as lycopene, β-carotene and total phenolic contents, in tomatoes growing in soils inoculated with vesicular arbuscular mycorrhizal fungi, as compared to conventionally grown tomatoes. free radical scavenging activity only the plants that received the highest sodium chloride concentration considerably increased their free radical scavenging activity, as compared to the other treatments. as discussed above, a number of adverse abiotic factors can disturb equilibrium between ros production and scavenging activity; among these factors, prasad et al. (1994) and tsugane et al. (1999) mentioned high luminosity, temperature and salinity. according to wang et al. (2009), the elevated scavenging capacity of cape gooseberries treated with 120 mm nacl may reflect an increased ros scavenging capacity, in order to provide protection from oxidative damage to lipids of the plasma membrane due to salt and drought stresses. the percentage increase of free radical scavenging activity was almost the same between the nonand salinized plants, i.e. an increase of c. 12% in the leaves of all treatments. this indicates that the increased scavenging activity over the course of the experiment was determined only by the plant’s age, independent of the salt concentrations applied. the degrees of scavenging capacity for different active oxygen species can vary with plant species or cultivar. in fruits of blackberries, blueberries, cranberries, raspberries and strawberries, the scavenging capacity for superoxide radicals ranged from 40.8% to 72.0%, for hydrogen peroxide from 50.7% to 73.9%, for hydroxyl radicals from 52.4% to 77.3%, and for singlet oxygen from 6.3% to 17.4% (wang, 2006). these values are in accordance with the determined scavenging capacity of non-salinized cape gooseberry leaves (58.3%). the results of the present study suggest, in agreement with duh and yen (1997), that the appropriate concentration of crude extracts of cape gooseberry leaves may act as a free radical scavenger and may react with free radicals to convert them to more stable products and terminate radical chain reactions. conclusions the increasing nacl concentrations, from 0 to 120 mm nacl, affected leaf growth the most and, consequently, the biomass production of the plants. a nacl concentration of 60 mm did not affect the root and stem growth, which indicates a resistance to relatively high salt concentrations, which are too high for many other plants; so, this crop can be grown in many areas with ecs as high as 3 to 6 ds m-1 in the soil solution. the enhanced total antioxidant activity with the higher sodium chloride concentrations confirms that p. peruviana uses antioxidant production in order to alleviate salt stress. the plants exposed to the highest sodium chloride concentration considerably increased the free radical scavenging activity. for further studies on the cape gooseberry under salt conditions, it is recommended that different types of antioxidant enzymes and low-molecular antioxidants be determined in order to recognize the stress defense mechanism for use in plant selection and breeding purposes. beyond this preliminary experiment on proline and taa activity in cape gooseberry leaves in relation to salt stress, their contents in fruits should also be determined because other species have shown increased antioxidant activities in fruits grown under these stress conditions. acknowledgements the authors wish to thank professor lothar w. kroh for the use of the laboratory at the department of food chemistry and analysis, institute of food technology and food chemistry, technische universität berlin, germany. 72 diego miranda, 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water-induced oxidative stress and antioxidant defenses in rice plants. j. plant physiol. 155, 255-261. zhu, j.k., 2001: plant salt tolerance. trends plant sci. 6, 66-71. address of the authors: prof. dr. diego miranda, department of agronomy, faculty of agricultural sciences, universidad nacional de colombia, bogota, colombia. e-mail: dmirandal@unal.edu.co prof. dr. gerhard fischer, department of agronomy, faculty of agricultural sciences, universidad nacional de colombia, bogota, colombia sascha rohn, institute of food chemistry, universität hamburg, hamburg, germany prof. dr. dr. christian ulrichs and dr. inga mewis, division urban plant ecophysiology, faculty of agriculture and horticulture, humboldtuniversität zu berlin, berlin, germany art15.indd journal of applied botany and food quality 82, 83 89 (2008) palacký university in olomouc, faculty of science, department of botany, olomouc, czech republic embryo rescue of cucumber (cucumis sativus), muskmelon (c. melo) and some wild cucumis species (c. anguria, c. zeyheri, and c. metuliferus) skálová d.*, navrátilová b., lebeda a. (received september 19, 2007) summary cucumis sativus is one of the most economically important crops of the cucurbitaceae. recent cucumber cultivars are susceptible to some serious diseases and pests, including downy mildew, powdery mildew, nematodes, and spider mites. sources of resistance to these pathogens and pests were identified in some accessions of wild cucumis species. one possible way of introducing these resistances into cucumber germplasm is interspecific hybridization. however, c. sativus is sexually incompatible with nearly all other cucumis species, because of substantially different chromosome numbers, n = 7 in c. sativus versus n = 12 in c. melo and most wild cucumis species. overcoming this obstacle can be accomplished through the use of embryo rescue and/or ovule culture. results of experiments using these methods, especially of embryo rescue of cucumber and selected wild cucumis species after intraand interspecific hybridization, are summarized in this paper. various culture media and selected genotypes were tested in our experiments. successful regeneration of mature embryos of some cucumis spp. was observed on all types of media, and callus or sporadic plant formation from immature embryos and seeds occurred on media with coconut water and gibberellic acid. abbreviations ba – benzyladenin; iba – indole-3-butyric acid; ms – murashige and skoog medium (1962). introduction the cucurbitaceae family consist of approximately 118 genera and 825 species almost equally divided between the new and old world, with an emphasis on tropical regions. one of the most important genera is cucumis (jeffrey, 2001), which has two subgenera. subgenus cucumis considered to be of asiatic origin (n = 7), and subgenus melo miller of african origin (n = 12). subgenus cucumis includes cucumis sativus l. and c. hystrix chakrav. although cucumis hystrix resembles c. sativus morphologically and biochemically, its base chromosome number (n = 12) is the same as c. melo, which is included with wild cucumis species in subgenus melo (lebeda et al., 2007). cucumber cultivars are susceptible to some serious diseases and pests. genes for resistance to several pathogens and pests that are not known to occur in cucumber have been found in accessions of wild cucumis species (leppik, 1966; chen and adelberg, 2000; chen et al., 2003; lebeda et al., 2007). accessions of cucumis anguria l., c. metuliferus e. mey ex naud. and c. zeyheri sond. have high levels of resistance to root-knot nematode (den nijs and custers, 1990; wehner et al., 1990). very high levels of resistance to tetranychus urticae were found in some accessions of c. zeyheri (lebeda, 1996). cucumis metuliferus also displays resistance to squash mosaic virus (sqmv) and watermelon mosaic virus (wmv) (mccarthy et al., 2001). cucumis melo line mr-1 and some other accessions have some resistance to cucumber downy mildew (pseudoperonospora cubensis) (lebeda et al., 1996, 1999, 2007). one possible method of introducing these resistance genes to cucumber cultivars is via interspecific hybridization (lebeda et al., 2007). to this end, it is necessary to use unconventional techniques, including various methods of in vitro culture, due to hybridization barriers and embryo abortion in the early globular stage of embryo development (ondřej et al., 2001). one approach to overcome this crossability barrier, which is primarily caused by different chromosome numbers, is to use embryo culture, a method of embryo rescue (chen and adelberg, 2000; lebeda et al., 1996; skálová et al., 2004). the composition of the culture media has the major influence on the successful regeneration of isolated and cultivated embryos (skálová et al., 2004). successful embryo rescue techniques for the genus cucumis is considered important for future efforts toward interspecific hybridization (lebeda et al., 2007; skálová et al., 2004). the main purpose of this paper is to report our recent results in the research of cucumis spp. embryo rescue. selected wild species and new types of media were tested for supporting regeneration, especially of immature embryos. materials and methods plant material selected cucumis species were used for embryo rescue experiments (tab. 1). the plant material originated from the vegetable germplasm collection of the research institute of crop production (prague), gene bank department, olomouc workplace, czech republic (web site: <www.vurv.cz/>, evigez) and the usda-ars north central regional plant introduction station, iowa state university, ames, ia. the plants were cultivated in a glasshouse at the department of botany, palacký university in olomouc, czech republic (25°c / 15°c day/night; with daily watering and weekly fertilization by kristalon start (hydro agri, rotterdam, netherlands), 10 ml of fertilizer (19:6:20 n:p:k) /10 l of h 2 o), in organic substrate (florcom sp, bbcom s.r.o, letohrad, czech republic), and without pest control. embryo culture fruits of selected cucumis species (tab. 1) were harvested after three days or one, two and three weeks, and in the case of cucumis sativus and c. anguria, also after six weeks post hand self-pollination. for cross-pollination, c. sativus was used as a maternal parent and other cucumis species (tab. 1) as paternal parents, and the fruits were harvested two weeks after hand cross-pollination. they were surface sterilized with 70% ethanol (fruits were rinsed and then flamed) and seeds (s) were dissected. immature seeds (originating from the 3 day fruits after self-pollination and from 2 week fruits after cross-pollination) were cultured intact. zygotic embryos (e) were excised from the seeds of 1, 2, 3 and 6 week fruits while viewed using a stereoscopic binocular microscope under sterile conditions. embryos or seeds were cultivated on various media (tab. 2) in test tubes for six weeks at 25oc in the dark (5 ml of medium per tube). one of these was ok-medium, which acted as a control, and the others, on-, cw-, and ga-medium, were supplemented with components which are thought to have a positive influence on embryogenesis: casein hydrolysate, coconut water, and gibberellic acid. ms-medium (murashige and skoog, 1962) was used as a basis for macroelements, 84 skálová d., navrátilová b., lebeda a. microelements and vitamins. other components were supplemented depending on the type of medium (ascorbic acid, casein hydrolysate, αglutamin, iba, ba, sucrose, and agar), and the ph was adjusted to 5.8 before autoclaving. solutions with coconut water and gibberellic acid were filter-sterilized (steritope, millipore, billerica, ma) and added to the autoclaved part of the medium. after germination, the embryos or seeds were transferred to a culture room (22±2oc and 16h-day/8h-night photoperiod, 32 36 µmol · m-2 · s-1) and then the type and frequency (%) of regeneration of embryos of various ages and seeds on the media were evaluated and compared. we describe three types of regeneration in our experiments – 1. callus formation (c; no organogenesis was observed from isolated embryos and seeds; only callus without stems, roots or leaves); 2. abnormal plant formation (ap; indirect organogenesis from isolated embryos and seeds was observed, such as the formation of stems, roots or leaves on callus; vitrificated plants also occurred – callus formation on developed stems, roots or leaves) and 3. normal plant formation (p; direct organogenesis from isolated embryos and seeds was observed, with the development of entire plants) (fig. 1). the experiments were repeated. the frequency of regeneration was expressed in percent (average percentages were presented). in graphs, y-error bars were used to represent the standard deviation of means. results and discussion embryo rescue after hand self-pollination in cucumis species the most important aspects to consider regarding embryo development in cultures are the age of embryos (seeds), the genotype, a suitable fig. 1: various types of regeneration from isolated embryos or seeds of cucumis species (a – normal plant formation; b – abnormal plant formation; c – callus formation). tab. 1: cucumis species used for embryo rescue. cucumis species abbreviation accession number c. sativus (line sm-6514) cs cz 09h3900768 c. anguria var. longipes ca pi 249896 c. zeyheri cz pi 364473 c. melo (line mr-1) cm1 pi 124111 c. melo var. charentais cm2 pi 261778 c. metuliferus cme pi 292190 tab. 2: composition of culture media. culture basic other components medium medium ok ms 20mg/l ascorbic acid, 0.01mg/l iba, 0.01mg/l ba, 20mg/l sucrose, 8g/l agar (control medium) on ms 1g/l casein hydrolysate, 0.01mg/l iba, 0.01mg/l ba, 20g/l sucrose, 6g/l agar cw ms 5% coconut water, 200mg/l α-glutamin, 0.01mg/l iba, 0.01mg/l ba, 60g/l sucrose, 6g/l agar ga ms 0.3mg/l gibberellic acid, 0.01mg/l iba, 0.01mg/l ba, 20g/l sucrose, 8g/l agar explanatory notes: ms = murashige and skoog (1962); ok, on, cw, ga = abbreviations for ms-media supplemented with different type of additions supporting embryogenesis; iba = indole-3-butyric acid; ba = benzyladenin. a c b embryo rescue of cucumber 85 tab. 3: frequency (%) of successful regeneration of cucumis genotypes on various culture media. cucumis spp. cs ca cz cm1 cm2 cme age medium 3 days ok 22 35 6 6 0 20 on 61 7 8 11 5 30 cw 72 27 12 6 10 30 ga 72 – 4 17 0 23 1 week ok 56 10 10 60 40 10 on 50 20 10 25 0 10 cw 50 20 20 60 20 25 ga 60 60 80 40 28 2 weeks ok 20 50 55 89 30 0 on 78 20 40 89 20 5 cw 78 50 70 100 23 20 ga 40 40 40 100 30 15 3 weeks ok 100 90 80 50 43 100 on 86 80 90 45 100 100 cw 86 80 75 55 100 50 ga – – 60 40 83 75 6 weeks ok 100 100 – – – – on 100 100 – – – – cw 100 100 – – – – ga – – – – – – explanatory notes: cs, ca, cz, cm1, cm2, cme – selected genotypes of cucumis (tab. 1); ok, on, cw, ga – various types of media (tab. 2); – variant was not tested. culture medium, the concentration of sucrose and growth regulators and the influence of light (ondřej and navrátilová, 1999). our recent experiments focused on the selection of the most suitable genotypes and media for embryo rescue in the genus cucumis. embryos and seeds isolated from fruits in different time periods after hand self-pollination were cultivated on various types of media, and the type and frequency (%) of regeneration was compared (tabs. 3 and 4; fig. 4). for cucumber (c. sativus), the in vitro response of one genotype, sm 6514, was studied. we observed normal development of mature embryos (6 week) to entire plants on the okand on-media (100% regeneration; fig. 1). on the other hand, coconut water in cw-medium often supported indirect regeneration with callus development on mature isolated embryos (fig. 2). a similar effect of coconut water was noted by ondřej et al. (2000). callus formation and abnormal plant formation were noted during the development of the 1, 2 and 3 week embryos (especially on cw-medium; fig. 1). the 3 day seeds developed to plants on on-medium (61%), and callus development and abnormal plant development were recorded on ga-medium (72%). regeneration of 3 and 6 week embryos of c. anguria resembled results from c. sativus. the most suitable media for normal plant development were ok (90%; 100%) and on (80%; 100%), and abnormal plant formation was again noted on the cw-medium. immature embryos and seeds were regenerated to entire plants on ok-medium (fig. 2). for 3 week embryos of c. zeyheri, okand on-media (80%; 90%) were suitable, but for the 2 week embryos it was cw-medium (70%). ondřej et al. (2000) also found these media to be suitable for c. zeyheri. nuñez-palenius et al. (2006) reported a beneficial effect of coconut water on embryo resuce of cucumis melo, and goralski and przywara (1998) observed its positive effects on regeneration of immature embryos generally. regeneration of immature 1 week embryos and the 3 day seeds was the most efficient on ga-medium (60%). the positive influence of ga 3 (but at a different concentration) was recorded in previous experiments (ondřej et al., 2002). the most important species for interspecific hybridization of cucumber is considered to be muskmelon (c. melo (line mr-1)). we observed 100% regeneration of its 2 week embryos on cwand ga-media; however, the type of regeneration was only callus or abnormal plants (fig. 1). normal plants were obtained from embryos cultivated on okand on-media (89%). ondřej et al. (2000) reported that the most suitable medium for c. melo (line mr-1) was generally the okmedium. gibberellic acid (ga-medium) was suitable for the 3 day seeds and 1 week embryos (60%; 80%). embryos and seeds of a second genotype of muskmelon, c. melo var. charentais, regenerated less frequently than did line mr-1. the most suitable media for the 3 week embroys were onand cw(only callus formation; 100%). gibberellic acid and coconut water were suitable for all immature embryos (2 and 1 week) and seeds (3 day) (callus formation predominated) (fig. 2). the most suitable medium for c. metuliferus embryos was okmedium, but lower reduced concentrations of sucrose and growth regulators (ondřej et al., 2000). the 3 week embryos of c. metuliferus expressed the best results on okand on-media (100%). no regeneration of plants was observed on media with coconut water and gibberellic acid. nevertheless, these media were the best for the cultivation of the 2 week, 1 week and the 3 day seeds (mostly callus formation, rarely abnormal plant production was observed). it is clear from these experiments that each species responds differently to these in vitro culture conditions. our results confirm the fact that the successful cultivation (= obtaining the entire plants through direct organogenesis) of immature embryos and seeds is more complicated than is regeneration from mature embryos and seeds (preťová, 1995). 86 skálová d., navrátilová b., lebeda a. fig. 2: regeneration of embryos isolated at different time after a hand self-pollination from various cucumis genotypes (a – 1 week c. sativus embryo on cw medium; b – 2 week c. anguria embryo on ok-medium; c – 2 week c. melo (line mr-1) embryo on cw-medium; d – 2 week c. melo (var. charentais) embryo on ga-medium). tab. 4: types of regeneration of cucumis genotypes on various culture media. cucumis species cs ca cz cm1 cm2 cme age medium 3 days ok p, ap p p, ap c 0 c on p ap ap c c c cw ap ap p c c c ga c, ap – p c 0 c 1 week ok p p p, ap p c c on p ap ap ap c, ap c cw ap ap p c, ap c, ap c ga ap – p ap c c 2 weeks ok p p p p c c on p p p p c c cw ap ap p, ap ap ap ap, c ga ap – p, ap ap ap ap, c 3 weeks ok p p p p c p on p p p p ap p cw p, ap p, ap p, ap ap ap c ga – – – ap c c 6 weeks ok p p – – – – on p p – – – – cw p, ap p, ap – – – – ga – – – – – – explanatory notes: cs, ca, cz, cm1, cm2, cme – selected genotypes of cucumis (tab. 1); ok, on, cw, ga – various types of media (tab. 2); c – callus formation, ap – abnormal plant formation, p – normal plant formation; – variant was not tested. a c b d embryo rescue of cucumber 87 small roots or a shoot meristem, however callus formation predominated. two embryos developed into entire flowering plants and their hybrid origin was confirmed using isozyme analyses and flow cytometry. only callus formation was observed in our experiments with classic hybridization of cucumber with other cucumis species. with respect to the evaluation of the success of cross-pollination, the highest frequency of fruits was obtained with c. metuliferus as an paternal parent (74%; fig. 5). on the other hand, the worst partner for cucumber was c. anguria (19 % successful pollination; fig. 5). only callus formation was observed after hybridization with c. melo (line mr-1) or c. metuliferus. the frequency of regeneration was very low (1.5% for c. melo and 1.7% for c. metuliferus). ondřej et al. (2001) had similar results with interspecific hybridization in the genus cucumis. as suitable media for obtained hybrid embryos were embryo rescue after hand cross-pollination in cucumis species cucumis sativus as a maternal parent and c. melo and selected wild cucumis species (tab. 1) as paternal parents were used in interspecific hybridization. embryos and seeds isolated from fruits after two weeks of hand cross-pollination were cultivated on several types of media. because of the different embryos responses on various media that we observed from previous hand self-pollination, we focused on frequency of successful pollination (= formation of fruits; fig. 5) and on the type and frequency of regeneration after hand cross-pollination with various interspecific partners (tab. 5). the regeneration of embryos after interspecific hybridization of cucumis has been infrequently accomplished. in total, the regeneration of seven embryos from interspecific crosses of c. sativus × c. melo was observed (lebeda et al., 1996, 1999). five embryos developed fig. 3: regeneration of embryos isolated two weeks after a hand cross-pollination of c. sativus with other cucumis species (a – fruit developing from c. sativus × c. melo; b – fruit developing from c. sativus × c. zeyheri; c, d – callus formation on embryos from c. sativus × c. melo on ga-medium). tab. 5: results of interspecific hybridization of c. sativus with cucumis species. partner no. of no. of no. of no. of no. and type of successful pollinations obtained fruits isolated seeds isolated embryos regener. media cm1 27 13 260 260 8 (c) ok (3c), cw (2c), ga (3c) cm2 18 8 120 160 0 ca 11 2 80 0 0 cz 16 7 280 0 0 cme 8 6 240 0 4 (c) cw (2c), ga (2c) explanatory notes: partner for ih – partner for interspecific hybridization with c. sativus; cm1, cm2, ca, cz, cme – selected genotypes of cucumis (tab. 1); ok, on, cw, ga – various types of media (tab. 2); c – callus formation. a c b d 88 skálová d., navrátilová b., lebeda a. 15 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 ok on cw ga m e d iu m rege neration (%) cme cm2 cm1 cz ca cs fig. 4: frequency (%) of regeneration 3 day seeds of tested cucumis species (cs, ca, cz, cm1, cm2, cme – selected genotypes of cucumis species (tab. 1); ok, on, cw, ga – various types of media (tab. 2); y-error bars represent standard deviations). 0 10 20 30 40 50 60 70 80 90 100 cm1 cm2 ca cz cme partner for ih r eg en er a ti o n (% ) fig. 5: frequency (%) of successful hand cross-pollination (formation of fruits) c. sativus with cucumis species (cs, ca, cz, cm1, cm2, cme – selected genotypes of cucumis species (tab. 1); ih partner – partner for interspecific hybridization with c. sativus; y-error bars represent standard deviations). appeared cwand ga-. there was observed a positive influence of coconut water and gibberellic acid again on immature embryos (goralski and przywara, 1998; ondřej et al., 2002; nuñezpalenius et al., 2006). however, no direct or indirect organogenesis was noted from these hybridizations. it is necessary to use other strategies to obtain hybrids from crossing c. sativus with other cucumis species. for example the polyploidization of cucumber maternal plant was used and entire plants were developed after cross c. sativus × c. melo (skálová et al., 2006). conclusions from this study, it is evident that methods of embryo rescue in the genus cucumis were successful. direct organogenesis was observed in selected cucumis species cultivated on media supporting embryogenesis. an important hybridization partner for cucumber, c. melo line mr-1, regenerated with 10 % frequency but only formed callus. among wild cucumis species, immature c. metuliferus embryos (3 day), showed the highest frequency of regeneration (26%). these two genotypes were characterized by callus formation from interspecific hybridization with cucumber. the addition of coconut water (cw-medium) and gibberellic acid (ga-medium) to culture media had positive effects on the regeneration of immature embryos (27%; 29%). these same media supported callus formation, the only degree of regeneration, in the case of interspecific hybrids between c. sativus and c. melo or c. metuliferus. future work will concentrate on optimizing methods to increase the frequency of regeneration and make it possible to recover entire plants from the interspecific hybridization of cucumber with muskmelon. acknowledgements authors thanks to dr. m.p. widrlechner (usda-ars ncrpis, iowa state university, ames iowa, usa) and dr. h.s. paris (aro, newe yaar research center, ramat yishay, israel) for reading and comments to the first draft of the manuscript. this research was supported by the following grants: (1) msm 6198959215 (ministry of education, youth and sports, czech republic); and (2) qf 4108 (nazv, ministry of agriculture, czech republic). references chen, j.f., adelberg, j., 2000: interspecific hybridization in cucumis – pro gress, problem, and perspectives. hortsci. 35, 11-15. chen, j.f., staub, j., qian, ch., jiang, j., luo, x., zhuang, f., 2003: reproduction and cytogenetic characterization of interspecific hybrids derived from cucumis hystrix chakr. cucumis sativus l. theor. appl. genet. 106, 688-695. den nijs, a.p.m., custers, j.b.m., 1990: introducing resistances into cucumbers by interspecific hybridization. in: bates, d. et al. 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(ed.), mansfeld’s encyclopedia of agricultural and horticultural crops, vol. 3. springer-verlag, berlin, fig. 4: frequency (%) of regeneration 3 day seeds of tested cucumis species (cs, ca, cz, cm1, cm2, cme – selected genotypes of cucumis species (tab. 1); ok, on, cw, ga – various types of media (tab. 2); y-error bars represent standard deviations). 0 10 20 30 40 50 60 70 80 90 100 cm1 cm2 ca cz cme partner for ih r eg en er a ti o n (% ) fig. 5: frequency (%) of successful hand cross-pollination (formation of fruits) c. sativus with cucumis species (cs, ca, cz, cm1, cm2, cme – selected genotypes of cucumis species (tab. 1); ih partner – partner for interspecific hybridization with c. sativus; y-error bars represent standard deviations). embryo rescue of cucumber 89 heidelberg, new york, 1510-1557. lebeda, a., 1996: resistance in wild cucumis species to twospotted spider mite (tetranychus urticae). acta phytopathol. entom. hung. 31, 247-252. lebeda, a., křístková, e., kubaláková, m., 1996: interspecific hybridization of cucumis sativus x cucumis melo as a potential way to transfer resistance to pseudoperonospora cubensis. in: gómez-guillamón, m.l. et al. (eds.), cucurbits towards 2000. proceedings of the vith eucarpia meeting on cucurbit genetics and breeding, malaga (spain), 28-30 may, 31-37. lebeda, a., kubaláková, m., křístková, e., navrátilová, b., doležal, k., doležel, j., lysák, m., 1999: morphological and physiological characteristics of plant issued from interspecific hybridization of cucumis sativus x cucumis melo. acta hort. 492, 149-155. lebeda, a., widrlechner, m.p., staub, j., ezura, h., zalapa, j., křístková, e., 2007: cucurbits (cucurbitaceae; cucumis spp., cucurbita spp., citrullus spp.), chapter 8, 271-376. in: singh, r. 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(eds.), progress in cucurbit genetics and breeding research. palacký university in olomouc, olomouc, czech republic, 415-430. skálová, d., navrátilová, b., lebeda, a., gasmanová, n. 2006: embryo culture as a tool of interspecific hybridization of cucumis sativus and wild cucumis spp. in: holmes, g. (ed.), cucurbitaceae 2006. north carolina state university, raleigh (nc, usa), 2006, 51-59. wehner, t.c., cade, r.m., locy, r.d., 1990: cell, tissue and organ culture techniques for genetic improvement of cucurbits. in: bates, d. et al. (eds.), biology and utilization of the cucurbitaceae, cornell university press, ithaca, new york, 367-381. address of the authors: rndr. dagmar skálová, ph.d.*, rndr. božena navrátilová, ph.d. and prof. ing. aleš lebeda, drsc.; department of botany, faculty of science, palacký university in olomouc, šlechtitelu 11, 783 71 olomouc – holice, czech republic. *corresponding author (dagmar.skalova@upol.cz) art03_ercisli.indd journal of applied botany and food quality 85, 144 149 (2012) 1department of horticulture, faculty of agriculture, erzurum, turkey 2department of pomology, faculty of agriculture, zagreb, croatia 3department of horticulture, faculty of agriculture, gorukle, bursa, turkey 4department of botany, division of biology, faculty of science, zagreb, croatia genetic relationships among olive (olea europaea l.) cultivars native to croatia and turkey s. ercisli1*, d. bencic2, a. ipek3, e. barut3, z. liber4 (received february 9, 2012) * corresponding author summary the aim of the study is to determine genetic diversity and relationships among olive cultivars native to croatia and turkey. a total of twenty olive (olea europaea l.) cultivars including fourteen from croatia and six common cultivars from turkey were analyzed for genetic diversity and relationships by using six microsatellite markers (dca05, dca09, dca18, gapu71b, gapu101, udo43). the number of polymorphic alleles ranged from 2 (udo43) to 5 (dca09), with an average of 3.6 fragments per marker. upgma cluster analysis based on simple matching similarity matrix grouped cultivars into three main clusters. two pairs of cultivars from croatia ("buža muška" and "levantinka"; "vlmd6" and "drobnica") were thought to be different, although they produced identical ssr profi les. cluster analysis points to some genetic relationships between croatian and turkish olive cultivars. the results also indicate effi ciency of ssr markers to evaluate genetic diversity in olive and identify misnamed or synonym individuals. introduction the olive (olea europaea) is native to the coastal areas of the eastern mediterranean basin and it is estimated that the cultivation of olive trees began more than 7000 years ago (green, 2002). in the last few years, olive cultivation is steadily expanding to more geographical zones, in response to increased demand for olive oil consumption owing to its nutritional value and recognized health benefi ts. olives are now cultivated in many regions of the world with mediterranean climates, such as south africa, chile, peru, australia, argentina and usa (california) and in areas with temperate climates such as new zealand. however these countries shared 15-16 percent of world’s olive production (fao, 2009). the olive is of major agricultural importance in the mediterranean region and olive oil is a basic constituent of the mediterranean diet. present production of olives (olea europaea) is about 18.0 million tons green and black table olives and 3.3 million tons olive oil from 10.8 million ha (fao, 2009). of the total production, 86% is produced in the mediterranean region with spain, italy, greece and turkey as the main producing countries. mediterranean countries constituted a wide germplasm with a large number of cultivars. it is expected that there were more than 1200 olive cultivars in the world (bartolini et al., 2005) olive has a great commercial importance in both turkey and croatia and both countries have long tradition in olive growing (poljuha et al., 2008; ercisli et al., 2011). it is used for local consumption mainly as olive oil in both countries. in turkey, table olive consumption is also highly preferred. olive growing in turkey and croatia are well established mainly around aegean and mediterranean regions of turkey and dalmatia region and istria peninsula in croatia (ercisli, 2004; poljuha et al., 2008). turkey has continued olive production with its very old olive cultivars such as "domat", "uslu", "ayvalik", "gemlik" etc. for a long time. in contrast, new olive plantations has been established in croatia predominantly with cultivars introduced from italy including "leccino", "pendolino", "frantoio", (pribetic, 2006). however, there is increased interest to old autochthonous croatian olive cultivars such as "istarska bjelica" in new plantations in croatia as well because of its adaptation to local conditions and high oil quality (poljuha et al., 2008). turkey (the ottoman empire) had an intense and long-lasting infl uence on the entire balkan peninsula until to the nearer past. archeological and historical research in anatolia, turkey proves that this region was very important for the history of olive (durgac et al., 2010). most contemporary olive cultivars are fairly old and of unknown genetic background. olives have been propagated by vegetative means in croatia for centuries and the introduction and spread of cultivars in croatia may have come from countries that have in the past settled or conquered the area of the present croatia. until now, it has not been clarifi ed all around the mediterranean basin whether their olive cultivars have been introduced or are derived from local oleaster (hannachi et al., 2008). to evaluate genetic relationships, trace phylogenetic origin and extent of ecologically differentiation in the same species, plant breeders need to have a defi nitive identifi cation both of cultivars and selections of crop plants. reliable and rapid methods of identifi cation are also required for the establishment of plant variety rights (kjeldgaad and marsh, 1994). classifi cations and evaluations of the olive cultivars based on phenotypic expressions such as growth form, leaf morphology and fruit properties and information from these environmentally infl uenced morphological characteristics is not suffi cient to identify olive cultivars. it is a predominantly allogamous species showing a high degree of outcrossing which leads to considerable levels of heterozygosity and dna polymorphism among individuals. the wide genetic patrimony and the large number of synonyms and homonyms in olive require precise methods of discrimination for cultivar identifi cation and classifi cation. (angiolillo et al., 1999; rallo et al., 2000). olive cultivars have been the subject of multiple studies given the large number of synonyms and homonyms generated during many centuries of vegetative multiplication and exchange. nowadays several molecular markers (rflps, aflps, rapds, issrs, ssrs) are available and these markers provide an accurate and unambiguous tool for precise identifi cation of cultivated olive germplasm (carriero et al., 2002). similarly to other crops, simple sequence repeat (ssr) markers have been preferred until now because of their high level of polymorphism, co-dominant nature and reliable repeatability (szikriszt et al., 2011). ssr markers have been developed for olive by several groups (sefc et al., 2000; carriero et al., 2002; cipriani et al., 2002), and this marker system was found to be the most reliable, ef fective and easy-to-use for identifi cation of olive cultivars (sarri et al., 2006). simple sequence repeats (ssrs) are now widely used in olive (breton et al., 2008; poljuha et al., 2008; roubos et al., 2010; ercisli et al., 2011). in this study, six well-known cultivars from the western part of turkey and fourteen cultivars from dalmatia and istria regions of genetic relationships among olive cultivars 145 croatia were analyzed by simple sequence repeats (ssr) markers in order to determine the potential synonyms and genetic relatedness among them. materials and methods plant material in the present study, a total of 20 olive cultivars including six well-known turkish olive cultivars ("domat", "uslu", "ayvalik", "gemlik", "tavsan yuregi" and "memecik"), which dominate commercial olive production in turkey and 14 croatian olive cultivars ("istarska bjelica", "buža muška", "buža", "piculja", "mezanica", "bjelica dubrovnik", "murgulja", "uljarica", "levantinka", "oblica", "grčka", "drobnica", "lastovka" and "vlmd6") were used for ssr analysis. turkish and croatian cultivars were selected according to dominancy in both countries’ plantations. six olive cultivars from turkey share over 80% olive trees in turkey. the cultivar "buža " dominate old olive plantations in croatia over 50 percents (milotic and setic, 2005) and the autochthonous cultivar "oblica", makes up 75% of the total number of olive trees in croatia (strikic et al., 2007). the turkish cultivars were found together in the atatürk central horticultural research institute, yalova in turkey. "istarska bjelica", "buža muška" and "buža" were grown in istria region of croatia. "levantinka" and "vlmd6" were sampled from middle dalmatia and rest of the croatian cultivars were sampled from south dalmatia of croatia. dna isolation young leaves of olive trees were sampled for dna extraction. lyophilized leaf sam ples were ground to a fi ne powder using a mortar and pestle. dna samples were extracted from 150 mg powdered leaf samples using a modifi ed ctab method described by futterer et al. (1995). the concentrations of each dna sample were measured using a qubit fluorometer (invitrogen, carlsbad, ca, usa) and adjusted to 50 ng/ml for analysis. amplifi cation, scoring and analysis of ssr’s six previously developed and widely used ssr primers in molecular characterization of olive cultivars (dca-05, dca-09, dca-18, gapu-71b, gapu-101, udo-43) were used for amplifi cation of ssr analysis in this study. each 20-µl polymerase chain reaction (pcr) mixture for amplifi cation of ssr markers consisted of 1.0 u dna polymerase (fermentas, hanover, md, usa) with the reaction buffer supplied at 1x concentration, 0.4 µm of each primer, dntps at 0.25 mm each, and 50 ng template dna. thermal cycling conditions were: 2 min at 94°c; 40 cycles of 40 s at 94°c, 45 s at annealing temperature of each primer pair, and 1 min and 30 s at 72°c, and a fi nal extension step of 5 min at 72°c. an applied biosystems thermal cycler was used for these reactions. pcr products were separated on a 4% agarose sfr™ gel (amresco inc., solon, oh, usa) in 1x tbe (89 mm tris base, 89 mm boric acid, 2 mm edta). gels were stained with ethidium bromide (0.5 mg/ ml; sigma, st louis, mo, usa) and photographed. ssr markers were scored as present (1) or absent (0) and simple matching similarity coef fi cients (sneath and sokal, 1973) were calculated for all pair-wise comparisons among 20 olive cultivars. expected heterozygosity (he) and observed heterozygosity (ho) were calculated according to the method of nei (1973) using popgen32 software v.1.31 (yeh et al., 1997). a dendrogram demonstrating the relative genetic relationship was generated using ntsyspc version 2.11v (exeter software, setauket, ny) (rohlf, 2004) based on the unweighted pair-group method of arithmetic mean cluster analysis (upgma). tab. 1: selected characteristics of olive cultivars from croatia and turkey used in this study. variety fruit mass stone mass fruit shape oil content use of fruit origin (g) (g) (%) domat 5.30 0.86 oval 20.57 table turkey uslu 3.53 0.52 oval 21.50 table turkey ayvalik 3.65 0.54 cylindrical 24.72 oil turkey gemlik 3.73 0.53 oval 29.98 table turkey tavsan yuregi 6.08 0.83 heart 20.20 table turkey memecik 4.78 0.56 oval 24.50 table-oil turkey istarska bjelica 3.09 0.40 oval 23.80 oil croatia buža muška 2.90 0.63 ovoid 23.00 oil croatia buža 4.38 0.53 ovoid 20.05 table-oil croatia piculja 1.27 0.32 ovoid 18.77 oil croatia mezanica 3.00 0.59 spherical 25,60 table-oil croatia bjelica dubrovnik 2.97 0.50 ovoid 24.57 oil croatia murgulja 5.64 0.97 spherical 18,02 table croatia uljarica 2.73 0.65 spherical 25.57 oil croatia levantinka 4.50 0.52 ovoid 20.00 oil croatia oblica 5.05 0.8 ovoid 22.10 table-oil croatia grčka oil croatia drobnica 2.18 0,49 spherical 23.10 oil croatia lastovka 2.80 0.48 ovoid 24.00 oil croatia unnamed (vlmd6) oil croatia *croatian cultivars were reported by bakaric, p. (1995); bakaric, p. (2002); bencic, ð. (2000); bencic et al. (2009); bencic et al. (2010) and turkish cultivars were reported by canozer (1991). 146 s. ercisli, d. bencic, a. ipek, e. barut, z. liber tab. 2: simple sequence repeats (ssrs), no. of detected alleles, observed heterozygosity (ho) and expected heterozygosity (he) of 6 ssr markers on 20 olive cultivars investigated. ssr primers number expected observed of alleles heterozygosity heterozygosity (he) (ho) dca-05* 3 0.18 0.10 dca-09 5 0.63 0.40 dca-18 4 0.14 0.10 gapu-71b** 4 0.68 0.35 gapu-101 4 0.74 0.55 udo-43*** 2 0.45 0.50 total 22 average 3.6 0.47 0.33 * developed by sefc et al. (2000) **developed by carriero et al. (2002) ***developed by cipriani et al. (2002) results and discussion tab. 1 summarizes the data generated by the six ssr primer pairs in 20 olive cultivars from croatia and turkey. a total of 22 polymorphic alleles were identifi ed. the number of polymorphic alleles ranged from 2 (udo-43) to 5 (dca-09), with an average of 3.6 fragments per primer. on the other hand, ssr locus dca0-3 was excluded from our study because it was not polymorphic among the plant materials studied. expected heterozygosity (he) was the lowest as 0.14 at two loci (dca-18) whereas it was the highest as 0.74 in gapu-101 loci. observed heterozygosity (ho) was the highest in gapu-101 (0.55). except udo-43, expected heterozygosity (he) was higher than the observed values (ho) at all loci (tab. 2). according to ssr profi les of 20 autochthonous olive cultivars from turkey and croatia, two pairs of cultivars from croatia ("buža muška" and "levantinka"; "vlmd6" and "drobnica") were found to be synonyms. no synonyms were found among turkish cultivars. in addition, synonyms were not observed between turkish and croatian cultivars. the dendrogram derived from an upgma cluster analysis of the total 22 ssr markers is shown in fig. 1. three main distinct groups were observed in the dendrogram. group i consisted of "ayvalik" from turkey and "uljarica" from croatia with 77% similarity ratio and both cultivars have been utilized for olive oil production. group ii included 3 turkish and 6 croatian autochthonous cultivars ("murgulja", "tavsan yuregi", "domat", "grčka", "uslu", "lastovka", "levantinka", "buža muška" and "oblica"). in this fig. 1: the upgma dendrogram based on simple matching similarity matrix obtained using 22 ssr markers, illustrating the relative similarity among 20 olive cultivars from croatia and turkey. genetic relationships among olive cultivars 147 group, "murgulja" formed a subgroup and the rest of cultivars formed another subgroup within group ii. croatian and turkish cultivars distributed within group ii without any geographical isolation. croatian cultivars, "levantinka" and "buža muška" showed 100% identical ssr banding profi les. group iii included "gemlik", "memecik", "istarska bjelica", "buža", "piculja", "mezanica", "bjelica dubrovnik", "drobnica" and "vlmd6" cultivars. there were 3 subgroups within this cluster and turkish cultivars "gemlik" and "memecik" clustered together but related to croatian cultivars within group iii. there were 100% similarities between the last two croatian cultivars within group iii. similarity matrix indicated that the "ayvalik" and "tavsan yuregi" were the most distant cultivars among turkish samples with 0.45 similarity ratio. the highest similarity (0.86) was observed between "gemlik" and "memecik" olive cultivars among turkish samples. considering croatian samples, cultivars "murgulja" and "mezanica" was found the most distant from each other with a similarity ratio of 0.39. "levantinka" "buža muška" and "drobnica" "vlmd6" had identical allelic ssr profi le (tab. 3). we obtained a high level of polymorphism among olive cultivars in the present study. a high level of polymorphism in olive cultivars by using ssr markers was also reported previously (carriero et al., 2002; sarri et al., 2006; muzzalupo et al., 2006; gomes et al., 2009; alba et al., 2009). in our study, the number of average polymorphic alleles per primers (3.6) was higher than obtained by cipriani et al. (2002) and comparable to carriero et al. (2002), sarri et al. (2006) and muzzalupo et al. (2006) and lower than those reported by belaj et al. (2003), poljuha et al. (2008), alba et al. (2009) and roubos et al. (2010). in this study, we used relatively few ssr loci; nevertheless, the most were highly polymorphic and therefore allowed unequivocal identifi cation of all the plant material. previously gomes et al. (2009) and roubos et al. (2010) also discriminated olive cultivars by using only six ssr loci. the ssr locus dca-03 was excluded from our study because it was not polymorphic among the cultivars used. the marker udo-43 showed the lowest and dca09 showed the highest polymorphism in this study. in contrast, belaj et al. (2003) and alba et al. (2009) reported a high discrimination capacity of the udo-43 marker. however, alba et al. (2009) and noormohammadi et al. (2009) also found high polymorphism with dca-09 marker. poljuha et al. (2008) found that the markers dca-03, dca-10 and dca-16 were of high discrimination capacity among istrian olive trees. variations reported in the number of alleles in olive cultivars by different scientists may be related to variation in the loci studied as well as the number of genotypes and their localities (lopes et al., 2004). as indicated before some of croatian cultivars had identical allelic ssr profi le. previously poljuha et al. (2008) reported three synonym cultivars "crna", "karbonera" and "karbuna" from croatia by ssr analysis. ercisli et al. (2011) reported synonymous names of "ziraat" and "gemlik"; "isrange" and "tuz" and "patos" and "yag" olive cultivars from the black sea region in turkey. these results also indicat that synonym olive cultivars from croatia might be renamed or misnamed in a new locality where those were cultivated. our results also highlight the genetic diversity of olive cultivars grown in turkey and croatia. it seems that the six ssr markers used in this study had high discriminating capacity for 20 olive cultivars. ssr markers have been previously used in genetic diversity and relationships studies in olive cultivars and most scientists conclude that ssr markers are a powerful tool for cultivar identifi cation and analysis of genetic structure (khadari et al., 2003; belaj et al., 2003; gomes et al., 2009). as expected, the most closely related cultivars were within each gene pool ("levantinka" and "buža muška", "drobnica" and "vlmd6" from croatia with 100% identical ssr banding profi les and "gemlik" and "memecik" from turkey with 85% similarity ratio (fig. 1). the use of synonym or mislabeling is one of the most important problems in olive germplasm from different mediterranean countries. discrimination of synonymous cases in olive germplasm has also been reported by using ssr and other molecular markers (muzzalupo et al., 2006; gomes et al., 2009). no particular clustering was observed among cultivars from two countries and turkish olive cultivars distributed in all three main clusters and were clustered with croatian olive cultivars suggesting that turkish and croatian olive cultivars continue to be related. this result may be due to the germplasm exchange and selection for similar climatic tab. 3: similarity matrix among 14 croatian and 6 turkish olive cultivars based on "simple matching" coeffi cient. istarska buža buža mezamuruljarica bjelica oblica levangrčka vlmd6 droblasayvalik domat gemlik memetavsan istarska bjelica muška istria nica gulja piculja dubrovnik tinka nica tovka cik yuregi bjelica buža muška 0.77 buža istria 0.64 0.68 mezanica 0.68 0.64 0.68 murgulja 0.50 0.67 0.67 0.39 uljarica 0.59 0.45 0.68 0.55 0.39 piculja 0.91 0.68 0.64 0.77 0.44 0.68 bjelica dubrovnik 0.77 0.73 0.86 0.64 0.61 0.73 0.77 oblica 0.77 0.91 0.68 0.55 0.78 0.45 0.68 0.73 levantinka 0.77 1.00 0.68 0.64 0.67 0.45 0.68 0.73 0.91 grčka 0.59 0.73 0.68 0.64 0.72 0.45 0.59 0.73 0.82 0.73 vlmd6 0.83 0.78 0.89 0.61 0.67 0.72 0.78 0.94 0.78 0.78 0.72 drobnica 0.73 0.68 0.91 0.59 0.67 0.77 0.73 0.95 0.68 0.68 0.68 1.00 lastovka 0.68 0.91 0.77 0.64 0.72 0.55 0.68 0.73 0.82 0.91 0.73 0.83 0.77 ayvalik 0.64 0.59 0.64 0.50 0.50 0.77 0.64 0.68 0.59 0.59 0.59 0.83 0.73 0.68 domat 0.82 0.77 0.64 0.59 0.72 0.50 0.73 0.59 0.86 0.77 0.68 0.72 0.64 0.77 0.55 gemlik 0.73 0.77 0.73 0.68 0.61 0.59 0.64 0.77 0.68 0.77 0.68 0.94 0.82 0.77 0.73 0.64 memecik 0.77 0.64 0.59 0.73 0.44 0.55 0.68 0.64 0.55 0.64 0.55 0.78 0.68 0.64 0.59 0.68 0.86 tavsanyuregi 0.64 0.68 0.73 0.50 0.72 0.59 0.55 0.68 0.77 0.68 0.68 0.72 0.73 0.68 0.45 0.82 0.64 0.59 uslu 0.55 0.77 0.64 0.59 0.72 0.41 0.55 0.59 0.77 0.77 0.77 0.72 0.64 0.86 0.64 0.73 0.73 0.68 0.64 148 s. ercisli, d. bencic, a. ipek, e. barut, z. liber environments (e.g. selecting for similarly important adaptation genes). however, the divergence among croatian cultivars was most likely caused by breeding for adaptation and cultivar improvement after the introduction of olive cultivars from turkey and other related ancestral cultivars from mediterranean countries. for croatian olive cultivar improvement, it is possible to use those turkish olive cultivars that are related to croatian olive cultivars as parents to possibly add new alleles without introducing too much new genetic diversity, or the genetic diversity that is most closely related to previous/historical parents. these results also indicate that grouping genotypes based on the geographic origin is not useful in olive. besnard et al. (2001) found that olive genotypes from different countries clustered together within a group and they did not fi nd any grouping based on their geographical origins. the result was similar to poljuha et al. 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a. ipek, e. barut, department of horticulture, faculty of agriculture, 16059 gorukle, bursa, turkey z. liber, department of botany, division of biology, faculty of science, 10000 zagreb, croatia journal of applied botany and food quality 87, 227 233 (2014), doi:10.5073/jabfq.2014.087.032 1centro de investigación en alimentación y desarrollo, ac (ciad, ac), hermosillo, sonora, méxico 2instituto tecnológico de tepic, tepic, nayarit, méxico added dietary fiber affects antioxidant capacity and phenolic compounds content extracted from tropical fruit a.e. quirós-sauceda1, j.f. ayala-zavala1, s.g. sáyago-ayerdi2, r. vélez-de la rocha1, j.a. sañudo-barajas1, g.a. gonzález-aguilar1* (received january 15, 2014) * corresponding author summary the effect of fruit dietary fiber (fdf) on the antioxidant capacity and phenolic compounds (pc) content from the same fruits was investigated. fdf was incubated (ph 2.5, room temperature, 2 h) with methanolic extracts (me) containing pc. the phenolic compounds (total phenols and flavonoids) content and antioxidant capacity (dpph and teac) were analyzed in the resulting supernatants. results showed that the addition of fdf decreased the pc content (5-25 %) and their action as antioxidants (4-22 %), being mango fiber which affects in most proportion. wheat dietary fiber (wdf) was used as control, and its addition to me reduced in greatest proportion the phenolic content (16-38 %) and the antioxidant capacity (30-48 %) than the fdf. this suggests that apparently depending on the nature and type of dietary fiber present in the food, some physicochemical interactions between the polysaccharides that conforms the fiber and pc could occur and subsequently affect their quantification and mode of action at the level of antioxidant capacity. introduction several epidemiological studies have linked the consumption of tropical fruits to the prevention of chronic disease and different types of cancer (hooper and cassidy, 2006). the proposed beneficial effects of these foods have been attributed to their important source of numerous phytochemicals with potential biological activity, such as phenolic compounds (pc) (kris-etherton et al., 2002; gonzalezaguilar et al., 2010). the mechanisms by which pc confers health effects are mainly associated to the antioxidant protection, by scavenging free radicals, inducing antioxidant enzymes or inhibiting prooxidant enzymes. mango (mangifera indica l.), pineapple (ananas comosus l.), papaya (carica papaya l.) and guava (psidium guajava l.) are popular tropical fruits that presented high antioxidant potential, due to the high concentration of pc presented in their pulp (sancho et al., 2010; fu et al., 2011; palafox-carlos et al., 2012). however, to achieve any health beneficial effect, the pc must be first released from the food matrix in which is embedded (bioaccessibility) and consequently be bioavailable for absorption (manach et al., 2005; palafox-carlos et al., 2011). in plant cell, most pc are compartmentalized primarily within the vacuole, enclosed by tonoplast and cytoplasmic lipid membranes which are in turn compressed against the plant cell wall (padayachee et al., 2012b). mechanical and biochemical break down of food matrix results in cell rupture allowing the pc release and therefore favoring the bioavailability during digestion (padayachee et al., 2012b). however, the exposition of pc with the cell wall polysaccharideprotein matrix, produces a potential physical and chemical binding that promotes interactions in one or other way. there is evidence that pc interact physicochemically with polysaccharides during fruit synthesis as part of cell growth and development (palafox-carlos et al., 2011; padayachee et al., 2012b). these interactions may be beneficial, non-beneficial, or even detrimental for the bioactive effects attributed to pc. in previous reports, some authors have studied the interactions between different groups of pc and the primary components of the cell wall matrix, particularly polysaccharides of dietary fiber. renard et al. (2001) studied the interactions between apple cell walls and native polyphenols. they reported that hydroxycinnamic acids and epicatechin did not bind to cell walls. however, binding of procyanidins reached up to 0.6 g per g cell walls. motomura and yoshida, (2002) reported that cell walls from a range of fruits have been found to protect ascorbic acid from oxidation. subsequently, apple cell walls and their polysaccharides were shown to affect the antioxidant activity of quercetin and l-ascorbic acid (sunwaterhouse et al., 2007). sun-waterhouse et al. (2008) reported that fiber from onions have some type of favorable interaction with l-ascorbic acid, but not with quercetin. more recently, padayachee et al. (2012b; 2012a) studied the extent of anthocyanins and phenolic acids cell wall interaction, using cellulose and pectin as cell wall models. it was found that cellulose and pectin are each able to bind both compounds. consequently the interactions between different pc and dietary fiber components may prevent the bioaccessibility or release of the pc, and thereby affect its antioxidant effect as well as could potentially impact the nutritional content and functional potential of diets (sun-waterhouse et al., 2008). however, a beneficial interaction could occurs when a pc bound to fiber is not bioavailable for absorption in the small intestine; so it is transported to the large intestine where fermentation of fibrous material takes place and helps maintain good gut health (saura-calixto, 2011; padayachee et al., 2012b). the purpose of this study was to investigate the influence of the source of dietary fiber on the interaction with supplemented pc (total phenols and flavonoids), as well as the effect of the antioxidant capacity. materials and methods materials tropical fruits of mango cv. “haden”, pineapple cv. “esmeralda”, papaya cv. “maradol” and guava cv. “hawaiian white” were purchased from a local market in hermosillo, sonora, mexico, and used in this study. fruits free of defects and mechanical damage were selected and distributed into 2 lots, to obtain fiber and pc. fruit used for pc extraction was freeze-dried and stored until use. in addition, wheat dietary fiber (wdf) was obtained from a local store and used as fiber control. isolation of dietary fiber dietary fiber of tropical fruits were obtained as alcohol insoluble solids according to the methodology reported by sañudo-barajas et al. (2009). a sample of 100 g of cubed flesh from the pooled tissue was homogenized (ika® works, model t25, willmington, nc) with 300 ml of 95 % ethanol to dissolve low molecular weight sugars and 228 a.e. quirós-sauceda, j.f. ayala-zavala, s.g. sáyago-ayerdi, r. vélez-de la rocha, j.a. sañudo-barajas, g.a. gonzález-aguilar organic acids and the slurry was boiled with continuous stirring for 45 min to ensure enzyme inactivation and prevention of autocatalytic breakdown of polysaccharides (rose et al., 1998). the insoluble material was filtered through glass fiber filter, recovered and sequentially washed with 250 ml of ethanol, 250 ml of chloroform:methanol (1:1, v/v), and 250 ml of acetone. insoluble, acetone-washed material was oven-dried at 37 °c, weighed and stored in a desiccator until use. this crude extract was named fruit dietary fiber (fdf). dietary fiber characterization fdf and wdf were analyzed for total, soluble and insoluble dietary fiber contents by using the megazyme total dietary fiber analysis kit (megazyme international ireland ltd wicklow, ireland). in addition, the uronic acids, total sugars and neutral sugar and starch content were determined. the uronic acids was assayed in two mg of sample hydrolyzed in h2so4 and calculated from a standard curve prepared with galacturonic acid (gala) (sigma) as described by ahmed and labavitch (1977). for total sugars, 3 mg of sample were stirred with 3 ml of 67 % h2so4 for 4 h and aliquots were assayed by the anthrone reaction according to yemm and willis (1954), using glucose (sigma) for the standard curve. the neutral sugars composition was obtained from two mg of sample residue hydrolyzed with one mol l-1 trifluoroacetic acid at 121 °c for 1 h. after hydrolysis the samples were centrifuged and the residue was digested with 67 % h2so4 and used for cellulose assay using the anthrone reagent (crystalline cellulose sigma was used for a standard curve). the trifluoroacetic acid-soluble supernatants were dried and converted to alditol acetates (blakeney et al., 1983). for analysis, they were injected into a gas chromatograph (model 3800, varian inc.) with a 30 m x 0.25 mm i.d. capillary column (model db-23, j & w scientific, folsom, ca) as described in carrington et al., (1993). results were calculated using standards of rhamnose, fucose, arabinose, xylose, mannose, galactose, glucose and myo-inositol as internal standard (sigma). starch was determined in 250 mg of sample adding 300 u thermostable α-amylase and 3 ml mops buffer (50 mm, ph 7.0) and hydrolyzed during 45 min in a boiling water bath. thereafter, 100 u amyloglucosidase and 4 ml sodium acetate (200 mm, ph 4.5) were added; mixture was incubated for 45 min at 50 °c, then cooled and centrifuged until the sedimentation of insoluble solids. aliquots (0.1 ml) in triplicate were taken from the supernatant and mixed with the glucose oxidase/peroxidase reagent; the mixture was incubated for 10 min at 50 °c; absorbance was recorded at 510 nm using a cary 1e-uv spectrophotometer. by means of the glucose standard the concentration was obtained. phenolic compounds extraction freeze-dried samples (1 g) of tropical fruits were homogenized in 10 ml solution of 80 % methanol (ika® works, model t25, willmington, nc) at room temperature. the homogenate was sonicated for 30 min (bransonic ultrasonic co., model 2210, danbury, ct, usa) and then centrifuged at 14000 rpm for 15 min at 4 °c. the supernatants were collected and the pellet was resuspended again with 10 ml of 80 % methanol, under the conditions previously described. the two supernatants were mixed, filtered (whatman no. 1, springfield mill, maidstone kent, uk) and made up to a final volume of 30 ml. the final concentration of the methanolic extracts (me) was 0.033 g ml-1 which was stored at -25 °c to be used for later evaluations. phenolic compounds content and antioxidant capacity total phenols total phenols were determined according to singleton and rossi (1965), with some modifications. briefly, 30 μl of fruit me were added to 150 μl folin-ciocalteu reagent (dilution 1:10), after reacting for 5 min, 120 μl of 7.5 % na2co3 solution were added. the phenols were incubated for 2 h in the dark, and absorbance at 765 nm was measured using a microplate reader (bmg labtech inc., model omega, usa). the results were reported as mg of gallic acid equivalents (gae)/100 g of fresh weight. total flavonoids the total flavonoids content was measured using a colorimetric assay in accordance with the method of (zhishen et al., 1999). flavonoids were extracted 5 % nano2 10 % alcl3 and 1 mol l-1 naoh and measured spectrophotometrically at 510 nm using quercetin as standard. the results were expressed as mg of quercetin equivalents (qe)/100 g of fresh weight. dpph antioxidant capacity was evaluated by radical-scavenging activity using the dpph (2, 2-diphenyl-1-picryl-hydrazyl-hydrate) as reported by palafox-carlos et al. (2012). the stock solution was prepared by mixing 2.5 mg of dpph radical with 100 ml of pure methanol. the solution was adjusted at an absorbance of 1.0 ± 0.02 at 515 nm. samples of 20 μl of the me were placed in a microplate and 280 μl of dpph radical were added. the mixture was kept in the dark for 30 min. the absorbance was read using a microplate (bmg labtech inc., model omega, usa) reader device, at a wavelength of 515 nm. the capacity of the antioxidants to reduce the absorbance of the radical after incubation time was calculated and the results were expressed as μmol of trolox equivalents (te)/100 g of fresh weight teac abts•+ cation was generated through the interaction of 19.2 mg of abts (2’2-azino-bis(3-ethylbenzotriazoline-6-sulfonic acid)), dissolved in 5 ml of hplc-grade water and 88 μl of potassium persulfate (k2s2o8) (0.0378 g ml-1). it was incubated in the dark at room temperature for 16 h; then 1 ml of abts activated radical was taken and 88 ml of pure ethanol was added. the radical was adjusted at an absorbance of 0.7 ± 0.02 at 734 nm. the reaction was initiated adding 245 μl of abts•+ and 5 μl of me of tropical fruits. absorbance was monitored at 734 nm at 1 and 6 min. the percentage of inhibition was calculated and the results were expressed as μmol of te/100g of fresh weight. incubation system the incubation system was carried out to enhance interaction between the addition of fibers and pc contained in the me of tropical fruits. 300 mg of fdf were added in 6 ml of me ph 2.5 for 2 h. samples were mixed and shaken constantly in the dark at room temperature. in order to compare the different type of fibers, a wdf was used as a positive control in the same study. control solutions of the me of tropical fruits free of fiber were also incubated under the same conditions. after 2 h, supernatant was collected and evaluated for phenolic content and antioxidant capacity. moreover, a dynamic interaction was conducted to study the effect of incubation of both molecules with respect to the time. aliquots were taken at 0, 10, 30, 60, 90 and 120 min and antioxidant capacity was evaluated. statistical analysis results were expressed as means ± sd. data were statistically analyzed by one-way anova procedure, and the tukey-kramer multiple comparison tests were used at 95 confidence level. number cruncher statistical system version 6.0 software (ncss, llc) was used. four replicates were used for each experiment. dietary fiber affects phenolic compounds properties 229 results and discussion dietary fiber characterization the dietary fiber total composition of samples is summarized in tab. 1. among the fdf, the lowest content of total fiber was observed in mango and the highest in guava. insoluble fiber content ranged from 10.1 to 72.1 g/100 g in mango and guava, respectively. the soluble fiber content ranged from 10.7 in pineapple to 36.1 g/ 100 g in papaya. interestingly, mango and papaya fibers had the highest proportion of soluble fiber; whereas pineapple and guava fibers showed highest proportion of insoluble fiber. this relationship agrees with that reported by ramulu et al. (2003), indicating that mango and papaya were rich in soluble fiber whereas pineapple and guava contained mainly insoluble fiber. the total sugars range in samples from 47.5 to 91.4 g/100g in papaya and mango, respectively. papaya fiber had the highest content of uronic acids represented by pectin and soluble dietary fiber; whereas the cellulose concentration (representing insoluble dietary fiber) was higher in guava and pineapple fiber. in addition, starch was detected only in mango fiber. while, by definition, the starch content is not necessarily part of the fiber; however, it was determined and included in the fdf due to it has been demonstrated that starch molecules (amylose and amylopectin) interacts effectively with phenolic (barros et al., 2012). as for the wdf (used as control), this presented a high content of total dietary fiber compared to the fdf, being insoluble fiber the highest proportion. besides, wdf showed presence of starch. in terms of health benefits, both type of fibers are complementary and derived from individual polymeric composition, each fraction could play different physiological role depending on their fermentability and the reactivity of the sugars found in fiber. insoluble fiber relates to both water and other compounds absorption and intestinal regulation, whereas soluble fiber associates with cholesterol in blood and it has been observed that it influenced the intestinal absorption (anderson et al. 2009). in general, both types of dietary fibers are associated with the entrapment and interaction with different molecules with biological activity presented in foods. tab. 2 shows the neutral sugars composition of the fiber samples. glucose and galactose were the main neutral sugars in mango and papaya fibers, while xylose and arabinose were the major monosaccharides in pineapple and guava fibers. this contrasts the high proportion of soluble dietary fiber in mango and papaya, and insoluble dietary fiber in pineapple and guava; as these sugars are major for those types of fibers respectively. the rest of monosaccharides were maintained at relatively low concentrations. this is in line with results reported by others authors (medlicott and thompson, 1985; larrauri et al., 1997; gomez et al., 2006) for neutral sugars in same fruits. indeed, the high concentration of specific neutral sugars could be associated to the result of the transformation of some other sugars, by hydrolysis or bond. xylose and arabinose indicate the presence of arabinoxylans. galactose can be converted to galactan by hydrolysis. binding of arabinose and galactose generates arabinogalactans and xyloglucans have a backbone composed by xylose, galactose and fucose. although, these molecules may also bind to other compounds such as phenolic. in addition, the highest neutral sugars present in the control fiber (wdf) were glucose, arabinose and xylose. thus agrees with that reported in the literature, indicating that arabinoxylans are the major constituents in wheat bran (bataillon et al., 1998). phenolic compounds content and antioxidant activity determination of total phenols and flavonoids content as well as antioxidant activity of crude me are shown in tab. 3. total phenols varied in all fruits, papaya had lowest content but not different for mango, followed by pineapple and guava. moreover, the flavonoids content measured was lower than the content of total phenols; however, it showed the same trend. contrasting these results with others reported in the literature, we observed comparable values for the same fruits (rocha ribeiro et al., 2007; corral-aguayo et al., 2008; fu et al., 2011; rosas-dominguez, 2011). the variation found in fruits and previous studies, may be attributed to differences in varieties, climate and ripeness, among others. the antioxidant capacity evaluated by the dpph assay showed a range of radical scavenging capacity, 7.19 to 14.30 μmol te/100 g of fresh weigh. the highest activity was found in guava and the lowest in pineapple. with the teac assay the extracts showed higher activity than dpph assay but the same trend, showing the pineapple tab. 2: sum of the neutral sugars composition of fibers (g/100 g). fiber rha fuc ara xyl man gal glc total pineapple 0.79b 0.38b 9.63c 19.5c 1.23c 5.77c 2.91a 40.2b mango 0.64b 0.39b 5.09b 2.01a 0.45a 5.12c 75.9c 89.6d papaya 1.03c 0.35b 0.90a 2.74a 0.98b 2.82b 3.05a 11.8a guava 0.89bc 0.31b 5.39b 21.6c 0.43a 2.69b 2.45a 34.1b wheat 0.13a 0.04a 10.8c 12.7b 0.40a 1.18a 24.7b 49.9c (control) rha: rhamnose. fuc: fucose. ara: arabinose. xyl: xylose. man: mannose. gal: galactose. glu: glucose. means values in each column followed by a different letter are significantly different (p<0.05). tab. 3: phenolic compounds content and antioxidant activity of methanolic extracts of tropical fruits. fruit phenolic compounds antioxidant capacity total total phenols flavonoids dpph teac (mg gae/100g fw) (mg qe/100g fw) (μmol te/100 g fw) pineapple 77.59b 63.30c 7.196a 189.5a mango 55.85a 49.39b 10.80c 224.4c papaya 51.01a 34.71a 9.679b 325.7b guava 221.8c 108.2d 14.30d 959.1d fw: fresh weight. gae: gallic acid equivalents. qe: quercetin equivalents. te: trolox equivalents. means values in each column followed by a different letter are significantly different (p<0.05). tab. 1: total, soluble and insoluble dietary fiber, uronic acids, total sugars, starch and cellulose content of fibers (g/100 g). fiber tdf idf sdf ts ua cellustarch lose pineapple 76.1b 65.3c 10.7b 50.5ab 21.6b 43.3d nd mango 28.3a 10.1a 18.1c 91.4d 19.4b 10.6a 48.6b papaya 68.9b 32.8b 36.1d 47.5a 46.1c 30.1b nd guava 85.2c 72.1d 13.1b 56.4b 17.9b 43.6d nd wheat 93.5 d 89.0 e 4.50 a 72.5c 5.20a 35.1c 15.9a (control) nd: not detected. tdf: total dietary fiber. idf: insoluble dietary fiber. sdf: soluble dietary fiber. ts: total sugars. ua: uronic acids. means values in each column followed by a different letter are significantly different (p<0.05). 230 a.e. quirós-sauceda, j.f. ayala-zavala, s.g. sáyago-ayerdi, r. vélez-de la rocha, j.a. sañudo-barajas, g.a. gonzález-aguilar the lower activity, followed by mango, papaya and guava. the fact that both reactions have same mechanism explains the behavior between their results. moreover, the antioxidant activity did not follow the same tendency that pc present in the me; as reported by tabart et al. (2009). this can be explained largely because the antioxidant activity of pc depends on the structure, in particular the number and positions of the hydroxyl groups and the nature of substitutions on the aromatic rings (balasundram et al., 2006). in this sense, it appears that the differences in the antioxidant capacity of me are attributed to the specific structure and concentration of each pc present in them. effect of dietary fiber on phenolic compounds content and antioxidant capacity fig. 1 shows the total phenols content of me after 2 h of contact with fibers. total phenols decreased significantly (p<0.05) in all me of tropical fruits when both types of fibers were added. mango fdf mostly decreased total phenols content (23.72 %), followed by papaya (13.06 %), pineapple (10.82 %) and guava (5.86 %). nevertheless, addition of wdf increased 15 % the depletion of the phenols content, following the same trend than fdf, mango (38 %), papaya (31.30 %), pineapple (28.77 %) and guava (16.17 %). interestingly, the addition of fdf and wdf decreased at a higher ratio the phenols content in mango. by contrast, flavonoids content was affected in lowest proportion; however, similarly, the compounds were mostly decreased when wdf and mango fiber were present (fig. 2). the effect on the antioxidant capacity of me with the addition of fdf and wdf evaluated by dpph assay are shown in fig. 3. when fdf was added, me of tropical fruits shows a significantly decrease (p<0.05) in the dpph scavenging capacity. mango fiber decreased the antioxidant capacity in greater extent (22.13 %), followed by papaya (8.83 %), pineapple (5.76 %), and guava (4.69 %). moreover, addition of wdf decreased the antioxidant capacity of me in greater proportion, being the pineapple the highest reduction (48.27 %), followed by mango (41.58 %), papaya (33.20 %) and guava (30.35 %). similar results and the same tendency was showed in the effect on the antioxidant capacity assessed by teac (fig. 4). it was observed that the decrease of pc content as well as antioxidant capacity is not directly related to the concentration of fdf, it could be more related to the type of fiber present as well as by the type of sugars that conform it. for example, guava showed a higher content of fiber but less decreased of pc content and antioxidant capacity. however, this behavior could be attributed to previous reports that indicated that fig. 1: total phenols content of methanolic extracts of tropical fruits and reduction by adding fruit dietary fiber (fdf) and wheat dietary fiber (wdf). different letter in bars indicates significant difference (p<0.05) among fiber types by fruit. fig. 2: total flavonoids content of methanolic extracts of tropical fruits and reduction by adding fruit dietary fiber (fdf) and wheat dietary fiber (wdf). different letter in bars indicates significant difference (p<0.05) among fiber types by fruit. fig. 3: radical scavenging activity assessed by the dpph assay, of methanolic extracts (me) of tropical fruits by adding fruit dietary fiber (fdf) and wheat dietary fiber (wdf). different letter in the same fruit indicates significant difference (p<0.05) between fiber types. fig. 4: radical scavenging activity assessed by the teac assay, of methanolic extracts (me) of tropical fruits by adding fruit dietary fiber (fdf) and wheat dietary fiber (wdf). different letter in the same fruit indicates significant difference (p<0.05) between fiber types. dietary fiber affects phenolic compounds properties 231 guava is an excellent source of antioxidant dietary fiber; this indicates that fiber has associated phenols, which confer antioxidant activity to it (jimenez-escrig et al., 2001). in this sense, due to that guava fiber has in its structure associated pc, there could be no space to interact with other phenols. furthermore, it was observed that in all assays, the highest depletion of pc and antioxidant capacity occurred with the addition of wdf and mango fiber, which can be associated to the presence of starch; these fibers being the only ones that had starch in their structure. it is evident that the addition of both types of fibers affects adversely the pc content and consequently the antioxidant capacity of me. this could be attributed to physicochemical interactions that may occur between pc and the components of dietary fiber, causing pc physical trapping, consequently affecting their quantification and their action as antioxidants (palafox-carlos et al., 2011). next, will be described some possible explanations that could be causing the observed results. the group of complex polysaccharides that conform dietary fiber has the ability to act forming physicochemical interactions with the pc, thereby preventing its action as antioxidants (serrano et al., 2009). these interactions may occur by hydrogen and ester bonds (with ferulic and cinnamic acids), hydrophobic interactions, and covalent bonds or simply by a physical entrapment (palafox-carlos et al., 2011). however, the composition, functional group substitution and physical properties of the fibers are key factors for this interaction. in addition, another key factor is the type of pc present in the extract. pc that have more hydroxyl groups and also contain more hydrophobic domains provide hydrogen bonding and would promote stronger interactions. comparing between fiber types, wdf declined proportionally the phenolic compounds (total phenols and flavonoids) and antioxidant capacity of me of tropical fruits. wdf is an insoluble fiber type, consisting mainly of cellulose. according to its architecture, the cellulose has the ability to trap and bind phenolic. pc may be interacting through non-covalent interactions with neutral sugars (xyloglucans) present in cellulose chain (padayachee et al., 2012a). furthermore, when the cellulose are in contact with water or aqueous solutions (as study was conducted) can form multiple compact fibers by hydrogen bonds between the hydroxyl groups on different chains of glucose (le-bourvellec et al., 2004). thus, some pc can be caught in its structure, thereby reducing its bioaccessibility or release, in consequence affecting their bioactive action. also, molecular size and structure of pc are key factors that could influence the possible interactions between both molecules. therefore, architecture of the cellulose may be more conducive to pc penetration through and potentially binding to the surface than pectin composites (le-bourvellec et al., 2004). another possible mechanism of interaction is due to the presence of starch. it was reported that presence of starch can significantly decrease the phenols content, due to strong interactions with their constituents, particularly with the amylose. the amylose possess a linear nature that affords a more optimum configuration for stronger bond formation between starch and pc in solution, particularly hydrophobic interactions. however, also the amylopectin double helix structure might provide a physically trapped of pc within the bulky amylopectin matrix, without necessarily chemically interacting with the starch (barros et al., 2012). as for fdf, mango and papaya fibers were those that decreased in largest proportion the pc and antioxidant capacity of me. these fruits fibers are characterized by showing highest uronic acid composition that is associated with the content of pectin, the fibers also showed presence of cellulose. padayachee et al. (2012a) reported that pc presented in various fruits and vegetables had the ability to interact with both cellulose and pectin. as mentioned before, the architecture of the cellulose may be more conducive to pc penetrating through and potentially binding to the surface of the cellulose, than pectin composites. however, pectin components also play a role in binding pc, especially on prolonged exposure. pectin contains hydrophobic cavities that could potentially encapsulate pc, as le bourvellec et al. (2004) concluded. also, the portion of pectin polygalacturonic acid may be complex with some types of antioxidants through ca+ ions, which are the main cations present in the cell walls. furthermore, in less proportion to the cellulose, the porosity of the pectin composites might also be a contributing factor to pc entrapment. nevertheless, the high presence of starch in mango fiber could be associated with the greatest depletion of pc, as mentioned before. moreover, the major neutral sugars identified in the insoluble fibers (xylose and arabinose) and soluble fibers (glucose and galactose) may be directly involved in the interaction with the pc, facilitating their physics interactions (sun-waterhouse et al., 2008; padayachee et al., 2012a; sivam et al., 2013). dynamic interaction fig. 5 shows a dynamic interaction of the addition of fdf and wdf on the antioxidant capacity of the me of tropical fruits with respect to time. the addition of fdf shows a slow steady decline in the antioxidant capacity of me of tropical fruits; however, the addition of wdf shows a faster decrease. with the addition of both types of fibers, mango fruit was decreased antioxidant capacity in less time. this indicates that the physical interaction or entrapment between these compounds was carried out more easily and more quickly, in comparison with the rest of the tropical fruits. that could be attributed to the type of pc presents in mango me. fig. 5: antioxidant capacity depletion of methanolic extract (me) by adding fruit dietary fiber (fdf) and wheat dietary fiber (wdf) respect to time. conclusion this study has shown that addition of fdf to me containing pc decreased their phenolic content, as well as their antioxidant capacity. this can be attributed to the result of different physicochemical interactions between the two molecules, which affects the determination and antioxidant action of pc, as reported by other authors. however, it is shown that the type of fiber (soluble or insoluble) and the specific components, such as starch, are a key factors in this 232 a.e. quirós-sauceda, j.f. ayala-zavala, s.g. sáyago-ayerdi, r. vélez-de la rocha, j.a. sañudo-barajas, g.a. gonzález-aguilar process. however, is necessary further work to evaluate the type of the physicochemical interactions that can be carried out between fiber and phenols. spectroscopy studies (ir, nmr, uv/vis) are tools that can provide more information to understand this phenomenon. furthermore, it is important to know if this interaction affects the absorption and bioavailability of pc during digestion process. acknowledgements we thank ciad and conacyt-méxico for financial support. this work is part of the project “nutrigenómica e interacciones moleculares de fenoles y fibra dietaria del mango “ataulfo” (mangifera indica, l.) en un sistema murino” project 179574cb-2012-01. references ahmed, a.e.l.r., labavitch, j.m., 1977: a simplified method for accurate determination of cell wall uronide 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111(3), 580-585. tabart, j., kevers, c., pincemail, j., defraigne, j.-o., dommes, j., 2009: comparative antioxidant capacities of phenolic compounds measured by various tests. food chem. 113(4), 1226-1233. yemm, e., willis, a., 1954: the estimation of carbohydrates in plant extracts by anthrone. biochem. j. 57(3), 508. zhishen, j., mengcheng, t., jianming, w., 1999: the determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. food chem. 64(4), 555-559. address of the authors: a.e. quirós-sauceda, j.f. ayala-zavala, , r. vélez-de la rocha, j.a. sañudobarajas, g.a. gonzález-aguilar*, centro de investigación en alimentación y desarrollo, ac (ciad, ac), carretera a la victoria km 0.6, la victoria, hermosillo, sonora 83000, méxico e-mail corresponding author: gustavo@ciad.mx s.g. sáyago-ayerdi, laboratorio integral de investigación en alimentos, división de estudios de posgrado, instituto tecnológico de tepic, av. tecnológico 2595, cp 63175, tepic, nayarit, méxico art09 journal of applied botany and food quality 81, 151 157 (2007) department grassland management and forage growing, justus liebig-university, giessen, germany impact of long-term nutrient supply on plant species diversity in grassland: an experimental approach on conventionally used pastures j. oerlemans, w. opitz von boberfeld, d.wolf (received may 24, 2007) summary the research was initiated to determine the impact of long-term (16 years) differentiated n, p, k supply on the floristic diversity of conventionally used pastures classified as lolio-cynosuretum. at four different sites the factors n supply (0, 160 and 320 kg n ha-1 a-1), p supply (0.26 and 52 kg p ha-1 a-1) and k supply (0.66 and 133 kg k ha-1 a-1) were tested in 27 different supply combinations in a factorial design with three replicates. dry matter (dm) yields of the 1st cut, soil chemical values (ph, p and k concentrations) and the number of species were determined. site-independent n fertilisation had the largest influence on the diversity, reducing the number of species as a consequence of light competition due to an increase in biomass productivity as well as a decrease in soil ph-levels. mostly, also the factor k had a significant effect. recorded species numbers ranged from 7 up to 35 species 25 m-2. on most sites a ‘humpback relationship’ (unimodal relationship) could be observed between productivity and the species number, with maximum species numbers reached with first cut yield levels between 2.5 and 3.5 t dm ha-1. the humpback relationship between productivity and species richness however was not a curve, but an envelope filled with points, indicating that besides productivity also other factors influence the attained species number. in this view the highest number of species were recorded in case of a co-limitation of n and k, indicated by a n:k ratio in the above ground biomass between 1 and 1.5, as well as soil ph-levels between 4.5 and 5.5. introduction the increased use of mineral fertilisers and frequency of utilisation clearly benefited the agricultural productivity of grasslands however at the same time seriously degraded the botanical diversity of these grassland communities (chapman, 2001). recent results suggest however that preserving and restoring grassland diversity may be beneficial to maintaining desirable levels of several ecosystem processes such as biological control of potential pest organisms, ecological resilience, recycling or retention of nutrients, control of local microclimate and regulation of local hydrological processes and may therefore have applications in land management and agriculture. other important motivations to enhance floristic diversity include species conservation and aesthetic value of the landscape (altieri 1999; duelli and obrist, 2003; minns et al., 2001; sanderson et al., 2004). as a consequence grassland management is now confronted with the task of developing systems which regenerate or conserve a stable floristic composition of meadows and pastures so that biodiversity is encouraged or maintained. for such systems to result in improved biodiversity, detailed knowledge of the effects of soil nutrient supply on floristic succession is required. in various studies the relationship between ecosystem functioning and plant diversity has been approached differently, leading to diverging views on the role of nutrient supply/availability in controlling plant diversity. in large-scale, observational studies al-mufti et al. (1977) and grime (1979) find a hump-shaped relationship between biomass and plant diversity. connecting productivity with soil nutrient availability janssens et al. (1998) and mccrea et al. (2001) indicate that there also appears to be an unimodal relationship between soil extractable phosphorus (p) and potassium (k) and plant diversity. more recently, small-scale experimental approaches examining the effect of simulated random species extinctions on productivity, reveal a positive asymptotic relationship between species richness and productivity (bullock et al., 2001; hector et al., 1999; tilman et al., 1996; tilman et al., 2001). other studies focus on the hypothesis that plant species diversity is related to the number of limiting resources, in which plant species may co-exist that are differentially limited by resources (braakhekke and hooftman, 1999). in this view several studies have related the species diversity with ratios of n:p and n:k in the aboveground biomass (aerts et al., 2003; koerselman and meuleman, 1996; roem and berendse, 2000; verhoeven et al., 1996). considering how these different experimental and observational studies and approaches can be reconciled in setting targets applicable in practice a multiple-site n-, p-, k-fertiliser experiment was conducted on conventionally used pastures located in central germany. the effect of application of n-, pand k-fertilisers on floristic change in grassland is only obvious in most communities after many years. therefore the experiment started in 1986 after which vegetation surveys took place first in 2002. moreover, in order to reveal more direct causal relationships between influencing factors and floristic diversity the experiment was established as a factorial design. material and methods at four different sites in central germany the factors n supply (0, 160 and 320 kg n ha-1 a-1), p supply (0.26 and 52 kg p ha-1 a-1) and k supply (0.66 and 133 kg k ha-1 a-1) were tested in 27 different supply combinations during 16 consecutive years (1989 2002). the experiment was arranged on each site in a latin rectangle design with three replicates. the plot size amounted 25 m2. the pastures at all four sites were conventionally used, situated on mineral soils and classified as lolio-cynosuretum. the altitude as well as soil chemical values in unfertilised condition are for the different sites presented in tab. 1. in november 2001 the soil of each plot was sampled (15 core samples) between 0 and 10 cm depth and analysed for: ph (0.01 m cacl2) and exchangeable p and k (calcium acetate lactate extraction). before the first cut in may 2002 a complete list of the plant species present tab. 1: altitude and soil chemical properties (unfertilised condition) of experimental sites. altitude phosphorus potassium ph m above sea-level mg p 100 g-1 soil mg k 100 g-1 soil site 1 210 3.1 10.5 5.3 site 2 260 1.3 13.0 5.5 site 3 360 3.0 9.4 5.6 site 4 620 2.2 11.8 4.8 152 j. oerlemans, w. opitz von boberfeld, d.wolf in the treatment plots was recorded. subsequently the plots were mown and dry matter (dm) yields of this first cut determined by drying subsamples at 103 ºc. for selected supply combinations another subsample of the herbage from the first cut was analysed for n (kjeldahl) and k content (absorption spectrometry). the data were analysed by means of a factorial analysis of variance. to ensure normal distribution the species numbers were subjected to a square-root transformation before statistical analysis. results the soil ph is most influenced by n fertilisation which lowers the soil ph (tab. 2). on site 2 the highest soil ph values are found, here n supply lowers the soil ph least, with ph values ranging from 5.9 to 5.3. the lowest soil ph occurs on site 4 with values between 4.9 and 4.0. largest sources of variance for the soil p and k concentrations are, except for the k concentration on site 1, respectively the factors p and k. also n supply exerts an influence on the soil p and k content. on site 1, 2 and 3 the p concentration is reduced with n supply in combination with p fertilisation, here the interaction n x p is significant. the same applies for the soil k content with a combination of n and k fertilisation, the interaction n x k is significant on all sites. the highest soil-exchangeable p level is reached on site 1 with 14.4 mg p 100 g-1 soil, the lowest on site 2 with 1.2 mg p 100 g-1 soil. the soil k concentration ranges between 5.5 mg k 100 g-1 soil (site 1) and 53.4 mg k 100 g-1 soil (site 4). the productivity is on all sites significantly increased with n and k fertilisation, whereas on most sites a combination of both n and k increases the production more compared to solely applicating n or k; the interaction n x k shows a significance (tab. 3). on site 2 and 4 also p fertilisation raises the first cut yield. both the highest and the lowest production is registered on site 2 with first cut yields ranging between 1.8 and 6.1 ton dm ha-1. on the transformed species numbers the factor n exhibits the largest source of variance at all sites, with a reduction of the species number with increasing n supply (fig. 1). next to n-, k supply also lowers the number of species on site 1.3 and 4. on site 2 and 4 also p fertilisation results in occasional significant species number reductions. tab. 2: anova of soil ph, soil extractable p and k. mean squares soil ph soil extractable p soil extractable k d.f. site 1 site 2 site 3 site 4 site 1 site 2 site 3 site 4 site 1 site 2 site 3 site 4 row 2 0.068 * 0.031 0.054 0.240 ** 5.76 * 9.13 ** 9.21 ** 6.89 * 23.51 169.14 ** 4.50 106.72 column 2 0.144 ** 0.221 ** 0.259 ** 1.153 ** 0.31 8.38 ** 13.31 ** 38.14 ** 40.53 24.24 72.10 47.92 n 2 4.570 ** 0.729 ** 2.592 ** 2.003 ** 37.24 ** 18.08 ** 19.84 ** 7.64 * 1271.95 ** 434.08 ** 600.59 ** 778.39 ** p 2 0.253 ** 0.225 ** 0.023 0.223 ** 334.32 ** 285.29 ** 414.74 ** 179.70 ** 13.43 60.82 43.16 127.96 * k 2 0.012 0.053 0.097 * 0.024 0.64 0.85 2.80 1.20 1058.67 **1283.88 ** 3916.51 ** 6221.03 ** n x p 4 0.041 0.019 0.076 * 0.107 ** 4.00 * 4.80 ** 3.45 * 3.03 16.42 33.02 35.87 33.68 n x k 4 0.081 ** 0.011 0.110 ** 0.051 3.81 * 0.24 2.36 0.15 256.18 ** 61.57 * 307.88 ** 147.47 ** p x k 4 0.010 0.018 0.020 0.024 4.38 * 1.06 0.65 0.92 13.59 9.65 36.97 26.10 n x p x k 8 0.007 0.007 0.018 0.013 0.92 0.40 1.82 1.02 17.81 8.85 36.90 23.75 error 50 0.016 0.039 0.029 0.020 1.40 0.86 1.34 1.75 14.55 19.51 29.00 36.06 total 80 **significance at p < 0.01, *significance at p < 0.05 source of variation tab. 3: anova of dm yield 1st cut and number of species. mean squares dm yield 1st cut number of species1 d.f. site 1 site 2 site 3 site 4 site 1 site 2 site 3 site 4 row 2 0.691 1.080 0.558 0.401 0.157 0.411 * 0.559 0.009 column 2 0.035 0.505 1.044 0.631 0.936 ** 0.071 0.594 0.162 n 2 23.890 ** 23.333 ** 4.820 ** 1.817 ** 16.855 ** 2.735 ** 21.330 ** 11.054 ** p 2 0.922 15.452 ** 0.146 3.958 ** 0.227 0.448 * 0.073 0.915 ** k 2 3.102 ** 12.667 ** 2.488 ** 2.845 ** 1.289 ** 0.019 4.701 ** 1.427 ** n x p 4 0.877 0.670 0.244 0.688 0.239 0.133 0.116 0.027 n x k 4 1.549 * 1.454 * 0.455 1.038 * 0.076 0.040 0.248 0.176 p x k 4 0.967 1.034 0.025 0.164 0.118 0.058 0.209 0.054 n x p x k 8 0.438 0.488 0.131 0.223 0.069 0.078 0.170 0.039 error 50 0.471 0.565 0.328 0.358 0.105 0.121 0.190 0.106 total 80 **significance at p < 0.01, *significance at p < 0.05 1square root transformed source of variation nutrient and plant species diversity in pastures 153 fig. 1: number of species 25 m-2 depending on long-term n, p and k supply and site. discussion nutrient supply might influence the floristic diversity directly through soil nutrient availability (janssens et al., 1998) or indirectly through productivity, i.e. light competition (al-mufti et al., 1977; grime, 1979) and soil ph (marrs, 1993). tab. 4 shows the results of linear correlation analysis between the species number, soil ph and 1st cut yield. although not highly correlated, the species number and soil ph are, except for site 2, related positively. this agrees with observations from ejrnaes and bruun (1995), marrs (1993), mccrea et al. (2004) and roem and berendse (2000) who find higher floristic diversity on neutral or basic soils compared with diversity on acidic soils. n fertilisation reduces the soil ph on site 1, 3 and 4 probably because long-term n supply results in an accumulation of soil organic matter (bakker and berendse, 1999) which induces increased nitrification rates with a decrease of soil ph (mengel, 1991; opitz v. boberfeld, 1994). contrary on site 2 the reduction of the soil ph by n supply is less pronounced. possibly on this site the soil extractable p in unfertilised condition is that low that nitrification activity of soil organisms is depressed (alexander, 1965; janssens et al., 1998). bohner (2005) finds a relatively strong relationship between soil p content and the n mineralisation potential of grassland. the low soil p level-induced moderate reduction of the soil ph by n supply could explain the small decrease of the species number by n fertilisation as well as the deviating correlation coefficient between soil ph and the number of species for site 2, see fig. 1 and tab. 3. the exclusion of species caused by light competition with increasing productivity (grime, 1979) is reflected in negative correlation coefficients between 1st cut yields and the species number on all sites, see tab. 3. the large number of factors influencing floristic diversity prohibit however that coefficients of determination r2 > 0.5 are reached for the relation between the number of species and productivity as well as soil ph. the 1st cut yield and soil ph are not correlated; the differentiated n-, pand k application affect both diversity-influencing factors in a different way. the soil ph level is obviously reduced with long-term n fertilisation, p supply has however a buffering effect (mengel, 1991) and brings about a small rise in soil ph levels. productivity on the other hand is influenced by n and k supply; the n induced yield increase is further strengthened with k supply (see factor n and k as well as n x k interaction for 1st cut yield in tab. 2). the same n x k interaction on productivity is also described by voigtländer (1987). tab. 4: linear correlation coefficients between species number, soil ph and dm yield. soil ph dm yield 1st cut number of species site 1 0.59** 0.56** site 2 0.21 0.46** site 3 0.40** 0.58** site 4 0.46** 0.35** soil ph site 1 0.29* site 2 0.04 site 3 0.10 site 4 0.22* the significance of correlation is indicated as **p < 0.01 and *p < 0.05 154 j. oerlemans, w. opitz von boberfeld, d.wolf the graphic presentation of the relationship between productivity and the species number in fig. 2 reveals, except for site 2, an unimodal relation (humpback) according to al-mufti et al. (1977) and grime (1979). the hump-shaped relationships between diversity and productivity are not curves, but clouds filled with points. this variance can be related to the point of criticism from marrs (1993) that the humpback-model has been produced empirically from comparisons across a range of communities, and it is not clear if the relationship holds when the fertility, i.e. productivity, of a given site is manipulated. also moore and keddy (1989) warned that, although the model held for comparisons between vegetation types in their study of wetlands, the relationship was less useful for comparisons within vegetation types. moreover it indicates the problems of extrapolating from a model derived from a few points (14 in al-mufti and grime’s study) to a large area with many sample points (marrs, 1993). another approach towards explaining this variance comes from the ‘resource balance hypothesis’ from braakhekke and hooftman (1999). from the idea that species richness is related to the number of limiting resources, braakhekke and hooftman (1999) hypothesize that plant species diversity is favoured when actual resource supply ratios are balanced according to the optimum resource supply ratios for the vegetation as a whole. in this view the envelope filled with points occurs because high species diversity is not consequently reached when the productivity level is between 2.5 and 3.5 ton dm ha-1, however when also other diversity influencing factors, such as soil chemical values, are in an optimal range. further fig. 2 reveals that for the none of the sites an asymptotic relation between the species number and productivity (see also negative correlation between species number and 1st cut yield, tab. 3) can be observed. this in contradiction to the results of experimental research by bullock et al. (2001), hector et al. (1999), tilman et al. (1996) and tilman et al. (2001) that reveal a positive asymptotic relation between the number of species and productivity. loreau et al. (2001) and schmid (2002) state that the large-scale, observational approach as performed by al-mufti et al. (1977) and grime (1979) and the small-scale experimental approach conducted by bullock et al. (2001), hector et al. (1999), tilman et al. (1996) and tilman et al. (2001) examine different causal relationships under different sets of conditions. although this study is experimentally designed on similar plant communities – contrary to the observational research from al-mufti et al. (1977) and grime (1979) referring to different plant communities – the productivity is nevertheless not only influenced by plant diversity however also by the here differentiated soil nutrient availability. this differs from above-mentioned experimental researches where the effects of diversity on productivity are detected after the effects of other environmental factors have been removed. another difference between this fertiliser experiment and abovementioned experimental studies is the duration of the experiment. the obtained positive relationships between diversity and productivity refer to rather short-term experimental manipulations of diversity. one of the proposed mechanisms behind the positive relation between diversity and productivity, the ‘selection probability effect’ (huston, 1997) in which more diverse plant communities have a higher probability of containing, and becoming dominated by, a highly productive species, reflects only an initial condition (loreau, 2000). through a selective process towards dominance of highly productive species the number of species should be reduced again. sampling a large number of old permanent grasslands in west and central europe and in the uk janssens et al. (1998) and mccrea et al. (2001) resp. find also an unimodal relation between soil extractable p and k and the diversity of species. relating the soil k content with the number of species following the ceteris-paribus principle, i.e. regarding only the nand p-unfertilised variants, here the species richest communities are found for soil k concentrations between 10 and 30 mg k 100 g-1 soil (fig. 3). this is in accordance with the findings of janssens et al. (1998) and mccrea et al. (2001). fig. 2: relation between dm yield and the number of species 25 m-2 nutrient and plant species diversity in pastures 155 fig. 3: relation between soil extractable k and the number of species 25 m-2 fig. 4: relation between soil extractable p and the number of species 25 m-2 156 j. oerlemans, w. opitz von boberfeld, d.wolf tab. 5: 95%-confidence intervals for the means of soil ph, dm yield and n:k ratio in aboveground biomass for diversity level > 25 species 25 m-2. site 1 site 2 site 3 site 4 n = 6 n = 11 n = 25 n = 22 soil ph 5.2 – 5.5 5.4 – 5.6 5.4 – 5.6 4.5 – 4.8 dm yield (ton ha-1) 1st cut 2.7 – 3.8 2.2 – 3.6 2.5 – 2.9 2.7 – 3.2 n = 3 n = 5 n = 11 n = 11 n:k ratio in aboveground biomass 0.3 – 1.4 0.5 – 1.4 0.8 – 1.4 0.9 – 1.4 however, the relation between the soil p content and the species number for the nand k-unfertilised variants, contrary to the results of janssens et al. (1998) and mccrea et al. (2001), does not reveal an upper boundary of 20 species 100 m-2 when the soil p content exceeds 5 mg 100 g-1 soil (fig. 4). on site 2 and 4 the species diversity is reduced with p supply, which is probably an indirect p effect on the n availability through an increased legume proportion (data published in oerlemans, 2006) or an increased mineralisation regarding the yield increase on these sites with p supply, see tab. 2 and fig 1. nevertheless for soil p concentrations higher than 5 mg 100 g-1 soil on all sites still species numbers exceeding 20 species 25 m-2 are found. there is a probability that inter-correlations with other soil chemical characteristics occur for the relation between soil p content and diversity, and to a less extent also for the relation between soil k content and diversity, as found in the not-experimental designed empirical sampling research from janssens et al. (1998) and mccrea et al. (2001). as all soils are inherently low in p, the effects of agricultural improvement through fertiliser addition with accompanying influence on the floristic diversity, are most easily recognised in terms of the soil p level (critchley et al., 2002). moreover after cessation of fertiliser application, n availability may be reduced in 5 to 10 yr by mowing, whereas reducing k availability may take 15 to 20 yr and reducing p availability more than 50 yrs (olff and pegtel, 1994). the fact that in this factorial designed fertiliser experiment no upper boundary of 20 species 100 m-2 with soil p content exceeding 5 mg 100 g-1 soil occurs could be explained by that the soil p content in the empirical studies from janssens et al. (1998) and mccrea et al. (2001) could be seen as a good indicator of the long-term level of fertiliser application and utilisation intensity in present and past with accompanying floristic diversity (bohner, 2005). from the view that a co-limitation of nutrients through a differential resource limitation of species leads to increased diversity, the significant n x k interaction found for the 1st cut yield could be interpreted as a co-limitation of n and k on most sites. fig. 5 shows for the p-unfertilised variants the relation between the n:k ratio in the aboveground biomass and the species number. in accordance with results from roem and berendse (2000) an unimodal relationship with an optimum n:k ratio in aboveground biomass between 1 and 1.5 can be found. as for the 1st cut yield no interactions with p occur, a co-limitation of n and p can for this experiment not be derived. regarding the large number of factors influencing the floristic difig. 5: relation between n:k ratio in aboveground biomass and the number of species 25 m-2 nutrient and plant species diversity in pastures 157 versity on pastures as well as different approaches towards examining/ explaining the relations found between these influencing factors and diversity tab. 5 summarises some results: the 95%-confidence intervals for the means of soil ph, 1st cut yield and n:k ratio in aboveground biomass for the plots with a species number > 25 species 25 m-2 are shown. these confidence intervals could be indicative for setting targets applicable in the conservation or restoration of floristic diversity on pastures (lolio-cynosuretum) in central europe. references aerts, r., de caluwe, h., beltman, b., 2003: is the relation between nutrient supply and biodiversity co-determined by the type of nutrient limitation? oikos 101, 489-498. alexander, m., 1965: nitrification. in: bartholomew, w.v., clark, f.e. 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(eds.), competition and succession in pastures, 261-282. cab international, wallingford. critchley, c.n.r., chamber, b.j., fowbert, j.a., sanderson, r.a., bhogal, a., rose, s.c., 2002: association between lowland grassland plant communities and soil properties. biol. cons. 105, 199-215. duelli, p., obrist, m.k., 2003: biodiversity indicators: the choice of values and measures. agric. ecosyst. environ. 98, 87-98. ejrnaes, r., bruun, h.h., 1995: prediction of grassland quality for environmental management. j. environ. manage. 43, 171-183. grime, j.p., 1979: plant strategies and vegetation processes. john wiley & sons, london. hector, a., schmid, b., beierkuhnlein, c., caldeira, m.c., and 30 further co-authors, 1999: plant diversity and productivity experiments in european grasslands. science 286, 1123-1126. huston, m.a., 1997: hidden treatments in ecological experiments: reevaluating the ecosystem function of biodiversity. oecologia 110, 449460. janssens, f., peeters, a., tallowin, j.r.b., bakker, j.p., bekker, r.m., fillat, f., oomes, m.j.m., 1998: relationship between soil chemical factors and grassland diversity. plant and soil 202, 69-78. koerselman, w., meuleman, a.f.m., 1996: the vegetation n:p ratio: a new tool to detect the nature of nutrient limitation. j. appl. ecol. 33, 1441-1450. loreau, m., 2000: biodiversity and ecosystem functioning: recent theoretical advances. oikos 91, 3-17. loreau, m., naeem, s., inchausti, p., bengtsson, j., grime, j.p., hector, a., hooper, d.u., huston, m.a., rafaelli, d., schmid, b., tilman, d., wardle, d. a., 2001 biodiversity and ecosystem functioning: current knowledge and future challenges. science 294, 804-808. marrs, r.h., 1993: soil fertility and nature conservation in europe: theoretical considerations and practical management solutions. adv. ecol. res. 24, 241-300. mccrea, a.r., trueman, i.c., fullen, m.a., 2004: factors relating to soil fertility and species diversity in both semi-natural and created meadows in the west midlands of england. eur. j. soil sci. 55, 335-348. mccrea, a.r., trueman, i.c., fullen, m.a., atkinson, m.d., besenyei, l., 2001: relationships between soil characteristics and species richness in two botanically heterogeneous created meadows in the urban english west midlands. biol. cons. 97, 171-180. mengel, k., 1991: ernährung und stoffwechsel der pflanze. verlag gustav fischer, jena. minns, a., finn, j., hector, a., caldeira, m., joshi, j., palmborg, c., schmid, b., scherer-lorenzen, m., spehn, e., troumbis, a., 2001: the functioning of european grassland ecosystems: potential benefits of biodiversity to agriculture. outlook agr. 30, 179-185. moore, d.r.j., keddy, p. a., 1989: the relationship between species-richness and standing crop in wetlands: the importance of scale. vegetatio 79, 99106. oerlemans, j., 2006: langfristige effekte abgestufter n-, p-, k-gaben bei mähweiden verschiedener standorte auf die zusammensetzung der pflanzenbestände unter den aspekten floristischer diversität und agronomie. cuvillier verlag, göttingen. olff, h., pegtel, d.m., 1994: characterisation of the type and extent of nutrient limitation in grassland vegetation using a bioassay with intact sods. plant and soil 163, 217-224. opitz von boberfeld, w., 1994: grünlandlehre. biologische und ökologische grundlagen. verlag eugen ulmer, stuttgart. roem, w.j., berendse, f., 2000: soil acidity and nutrient supply ratio as possible factors determining changes in plant species diversity in grassland and heathland communities. biol. cons. 92, 151-161. sanderson, m.a., skinner, r.h., barker, d.j., edwards, g.r., tracy, b.f., wedin, d.a., 2004: plant species diversity and management of temperate forage and grazing land ecosystems. crop sci. 44, 1132-1144. schmid, b., 2002: the species richness-productivity controversy. trends ecol. evol. 17, 113-114. tilman, d., reich, p.b., knops, j., wedin, d., mielke, t., lehman, c., 2001: diversity and productivity in a long-term grassland experiment. science 294, 843-845. tilman, d., wedin, d., knops, j., 1996: productivity and sustainability influenced by biodiversity in grassland ecosystems. nature 379, 718720. verhoeven, j.t.a., koerselman, w., meuleman, a.f.m., 1996: nitrogenor phosphorus-limited growth in herbaceous, wet vegetation: relations with atmospheric inputs and management regimes. trends ecol. evol. 11, 494-497. voigtländer, g., 1987: düngung des grünlandes. in: voigtländer, g., jacob, h. (eds.), grünlandwirtschaft und futterbau, 123-182. verlag eugen ulmer, stuttgart. address of the authors: prof. dr. dr. h.c. wilhelm opitz v. boberfeld (corresponding author), dr. judith oerlemans and dr. daniel wolf, dept. grassland management and forrage growing, justus liebig-university, ludwigstr. 23, d-35390 giessen. e-mail: wilhelm.opitz-von-boberfeld@agrar.uni-giessen.de << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true 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/pagesize [907.087 680.315] >> setpagedevice art09_stoklosa.indd morphology and productivity of semileafl ess peas (pisum sativum l.) journal of applied botany and food quality 85, 188 197 (2012) 1department of crop production, university of agriculture, krakow, poland 2 department of agrotechnology and agricultural ecology, university of agriculture, krakow, poland morphological-developmental reaction and productivity of plants and canopy of semileafl ess pea (pisum sativum l.) after seed vaccination with rhizobium and foliar micronutrient fertilization t. zając1, a. klimek-kopyra1, a. oleksy1, a. stokłosa2, b. kulig1 (received april 13, 2012) summary the determinants of semileafl ess peas (pisum sativum l., cv. tarchalska) crop productivity were studied during two vegetative seasons: cool 2010 and warm 2011 in south part of poland (modzurów 50°09’n 18°07’e; 274 m. a.s.l.. peas was treated either with seed vaccine (nitraginatm) containing rhizobium bacteria or foliar micronutrient fertilizer (photreltm) or both of them. the range of peas response to treatments included biometrical measurements and also the measurements of vegetation indices namely, green area index (gai), normalized difference vegetation index (ndvi) and relative chlorophyll content (spad), carried out in the specifi c stages of development, which for the compared objects were generally insignifi cant. in the warmer growing season, pea plants grew better, what resulted in a very high yield of seeds per plant, determined by a greater number of large seeds. it was shown that the length and weight of pea pod and the number of seeds formed in the pod depends on its position on the particular node. the longest pods, characterized by the greatest weight and number of seeds, developed on the lower nodes: 1st and 2nd one. the pea pods forming on higher nodes, from the 3rd, had reduced number of fruits and the weight of a single seed. the shortest pods were growing out of the 5th and 6th nodes, at the top of the stem. analysis of the single pea seed mass shows a highly signifi cant effect of its position in the fruit on pod productivity. seeds located in the central part of the pod had the greatest mass, and this accuracy, as highly signifi cant, was found for the pods containing from 3 to 8 seeds. the tested agrochemical treatments did not differentiate the chemical composition of seeds. introduction many legume species are believed to be invaluable to organic or sustainable farming in temperate region (corre-hellou and crozat, 2005). cousin (1997) states that because pisum is of great importance as edible leguminous plants and therefore of economic importance, its planted area should expand. the intensive breeding of peas, carried out in the second part of the twentieth century, improved the genus genetically. increased sources of variation allowed breeders to create cultivars with unique botanical and agricultural characteristics, such as shorter growing period, short stem, and semileafl ess (type afi la) instead of traditional foliage. increased breeding of fi eld pea includes both, edible and fodder cultivars grown for seeds, which increased the attractiveness of this species for agriculture. the main purpose of such modifi cations was to reduce pea lodging (stelling, 1989) and to facilitate process of combined harvesting. semileafl ess morphotypes, as compared to conventional foliage types, use expanded tendrils in reducing lodging. however, despite the enhanced morphological features of the new morphotypes, lodging was not completely eliminated in peas. usually, lodging of semileafl ess pea plants and canopy occurs in the phase of maturation (the fi nal stage of development). it is recognized, that semileafl ess pea cultivars are less competitive than the cereal species in a cereallegume mixture (semere and froud-williams, 2001). currently available non-destructive measurements allow to measure vegetation indices such as the green area index (gai), the normalized difference vegetation index (ndvi) and the relative chlorophyll content (spad), in a canopy of plants linked by tendrils. measurements of vegetation indices can give a picture of ecophysiological state of semileafl ess pea plants and other crops in their characteristic phases of development. although prusiński (2007) stated that those indices did not specify productivity of semileafl ess pea precisely, which is determined by the unique agrobiological properties. it was empirically demonstrated that longer growing season and optimal weather conditions led to a higher yield of pea seeds of the semileafl ess cultivars that is more resistant to lodging (boros and sawicki, 1997; jeuffroy and sebillote, 1997; dore et al., 1998). it is emphasized that water availability and nitrogen content during the growing season determined pea production (jeuffroy and ney, 1997). jensen (1996) showed that peas utilize a small amount of nitrogen from the soil, for this reason placing the strains of rhizobium bacteria along with the pea seeds to the soil is compulsory. sadowsky and graham (2001) point out that most rhizobium strains, in a symbiosis relationship with the peas, require warm soil of a ph of 5.0 or higher. soil bacteria, rhizobium, bradyrhizobium, sinorhizobium and azorhizobium provide 80% of the total nitrogen required by pulses, a group of plants that provide 25-35% of useful proteins to the world (vance, 2001). it has been shown that vaccination of pea seeds with bacteria rhizobium leguminosarum (carranca et al., 1999; sadowsky and graham, 2001), including strains of rh. leguminosarum bv. viceae r 21 (huang and erickson, 2007) leads to several changes in morphological features of plants and improves seed yield. optimization of supplying essential micronutrients to pea plants, in order to stimulate growth, development and to increase yield of plants and canopy, can be obtained by foliar application to the plants in the green bud stage (szwejkowska, 2004). application of micronutrients in the form of foliar spray leads to a higher yield of seeds from a single shoot and plant (palcu et al., 2008) and increases the contents of nitrate reductase and glutamine synthetase in the shoots when supplied with molybdenum (hristozkova et al., 2006). however, the combined effect from bacterial vaccines application and the effect of leaf micronutrient fertilizer on the productivity of semileafl ess peas has not yet been conducted. in the previous studies, the seed yield and its components for the peas – edible or fodder cultivars, were usually analyzed with high degree of generality (duthion and pigeaire, 1991; ney and turc, 1993; dumoulin et al., 1994; uzun and acikgoz, 1998; uzun et al., 2005), regardless of differences in individual seed weight per pod. the aim of our study was to comparatively analyse the vegetation indices, productivity, and seed yield components of a single plant and the canopy of semileafl ess peas, as a function of the seed vaccination with rhizobium bacteria and foliar application of micronutrients. the studies included morphological features of plants and pods and the individual seed weight of peas, and their basic chemical composition. comparisons for semileafl ess morphotype were made morphology and productivity of semileafl ess peas (pisum sativum l.) 189 between plants and pods to estimate the correlations for the traits pairs. material, methods and research area the research was based on the fi eld experiment, conducted in four replicates and carried out in the experimental fi eld of bayer® company located in modzurów (50°09’n 18°07’e), silesian voivodeship. the experimental fi eld soil was umbrisol – slightly degraded chernozem, formed from loess, therefore the soil conditions were suffi cient for the pea needs. the soil ph was neutral (ph in 1mol/dm3 kcl 6.28) and the richness of topsoil layer was high: 19.1 mg/ 100g p2o5 , 21.7 mg/100g k2o, and 10.1 mg/100g mg. a randomized block design was adopted in the conducted fi eld experiment and it consisted of 4 replicates, and the size of each fi eld was 8.4 m2. the following pre-sowing doses were applied: phosphorus 60 kg . ha-1(p2o5) and potassium 60 kg . ha-1(k2o). ammonium nitrate was applied as a „starting dose” 20 kg . ha-1 n. one hundred and twenty germinating seeds of ‘afi la’ pea cv. ‘tarchalska’ were sown in 1 m2 with a span of 15 cm. the plant seeds were sown in the second week of april 2010 and in the fi rst week of april 2011. commercial vaccine nitraginetm, produced by the biofood company (poland), containing rhizobium leguminosarum bv. viceae was applied during sowing. the foliar fertilizer photrel was applied in the beginning of the pea budding. photrel contains 5% b, 7% mn, 0.4% mo, 13.3% mgo and 36.3% so3. fertilizer applied in 3 l ha-1 contained: 150 g b, 210 g mn, 12 g mo, 400 g mgoi , and 1081 g so3. at the end of the fl owering stage the presence of pee weevil (bruchus pisorum) and pea moth (laspeyresia nigricana) was controlled nurelletm d 550 ec (a.i. cypermethrin and chloropyrifos, producer: dowagro science). the collection of plants was carried out with a plot harvester. after collection the seeds were cleaned and the moisture content was determined. the fi nal seed yield from the plot was calculated for the water content of 14.5%. chemical composition of seeds was determined according to aoac (2005) methods. green area index (gai) of the canopy (m2 . m-2), normalized difference vegetation index (ndvi) and relative chlorophyll content (spad) were determined in characteristic development phases: fl ower buds, beginning of fl owering, fl owering declining and end of fl owering and development of fruits. before collection – during the ripening phase – 15 pea plants were sampled from the fi elds in order to perform the biometric analyses. morphological and productive features of pea were determined i.e. height of 1st reproductive pod in a shoot, length of reproductive shoot, plant weight, total shoot weight, stem weight, number of seeds per pod, weight of seeds. pod characteristics were determined i.e. length of pedicle, weight of pedicle, length of pedicle, weight of pod. the obtained results were processed by analysis of variance (anova) for a 1-factorial experiment (block design) or by linear or non-linear regression analyse, using statistica 9.1 (statsoft) software. weather course monthly precipitation and average temperature for 2010-2011 growing seasons in modzurow are presented in tab. 1. in 2010 intensive precipitation was observed in the seedling phase (may) and during the fl owering phase, which contributed to the growth and development retardation of vegetative and generative stages. this reduced plant density, number of seeds and pods, consequently resulted in a low yield. in 2011 the precipitation and average temperature were optimal for the plant growth. results after a warm march in the year 2010, the following months of vegetation were cooler, due to the higher cloudiness and heavy precipitations (tab. 1). excessive rainfalls in the early may, lasting for two weeks, resulted in the hinder germination of pea seeds, due to the excessive moisture of soil, which was also cold. moreover, after the heavy rainfall part of the fi eld was under the water. these stress factors resulted in lower density of semileafl ess pea canopy and lower values of the yield components. in 2011 the weather course, up to june, was favorable for peas. in both years the excessive rainfalls in the month of july caused a severe crop lodging (as shown in the last part of the paper), which hindered the maturation of plants, and later their combine harvesting. green area index (gai) of peas canopy throughout the examined stages of development, was in the range 1.35 5.61 m2 m-2 (tab. 2). in the fi rst decade of june value of this ratio was > 3, which allowed the utilization of photosynthetically active radiation at a level close to optimal. the maximum leaf surface peas developed in the fl owering stage, around 15 june 2010 and 8 june 2011 r. the objects tested did not differ statistically for this trait within the terms (except july 2011). additionally, during the phases of elongation and fl owering the object where both, nitragina(tm) + photrel(tm) was applied, was characterized by a slightly larger gai, in comparison with the other objects (tab. 2). there were no signifi cant differences of values of vegetation index (ndvi) within the examined objects at any time of measurement (tab. 2). the highest ndvi values were found at peas fl owering stage on 15th june 2010 and 8th june 2011. ndvi values during this period ranged from 0.545 to 0.615. the largest differences between ndvi values between years were recorded in the last phases of measurement (seed maturity), when ndvi values in 2010 were more than two times lower as compared to 2011 (tab. 2). those differences resulted from accelerated maturation of peas in 2010, because of high temperatures in july (tab. 1). leaf greenness index (spad), which expresses the relative content of chlorophyll and, indirectly, the degree of nitrogen nutrition of plants, ranged between 24.1 48.5 (tab. 2). the highest value of spad was noted on 15 june 2011 (fl owering stage). the lower values of spad in june 2010 (fl owering stage), might result from the dilution of nitrogen in the higher biomass of rapidly growing pea plants because of the abundance of water, which is also indirectly indicated by the value of the gai index in this term (tab. 2). tab. 1: weather course at research station at modzurów during 2010-2011 variable year march april may june july august temperature (oc) 2010 4.0 7.5 11.7 16.7 20.4 18.5 2011 2.0 9.7 13.2 17.4 17.3 18.9 precipitation (mm) 2010 17.0 66.5 193.2 103.5 208.5 95.1 2011 31.1 29.2 71.5 99.5 167.5 73.2 190 t. zając, a. klimek-kopyra, a. oleksy, a. stokłosa, b. kuligąc, a. klimek-kopyra, a. oleksy, a. stokłosa, b. kuligą morphology and productivity of semileafl ess peas (pisum sativum l.) in both years, growth of semileafl ess pea cv. ‚tarchalska’, vaccinated with rhizobium resulted in a reduced height of deposition of the pods on the fi rst reproductive node of the plants (tab. 3). the opposite trend was showed after foliar fertilizer application, which has increased the elongational growth of stems, increasing the height of pea plants and also the height of pods deposition, which was especially evident in 2011. at the same time the application of trace elements in a form of photreltm fertilizer, in the stage of buds formation, did not cause a signifi cant increase in production potential and yield. higher plants of pea undoubtedly increase the tendency for lodging during adolescence. foliar applied fertilizer did not change signifi cantly the generative growth. at the same time, the length of pea shoots treated both, with seed vaccine nitraginatm and foliar fertilizer photreltm has increased, but differences were not signifi cant. pea seeds vaccination in combination with foliar application of fertilizer also increased the number of nodes with pods, which was particularly evident in 2010 year, which was climatically less favorable for growth of plants. the same combination of seed vaccine and foliar fertilizer applied in 2010 increased the total mass of pea shoots, assessed at the end of the growing season. as a result, the highest seed yield per single plant of peas was obtained from this combination as well. pre-vaccination of pea with nitraginatm resulted also in the increase of the whole plant and the stem biomass. this was particularly pronounced in 2010 year, climatically unfavorable for peas, as a result of excessive rainfall during the growing season, which washed out mineral nitrogen compounds. from the humus layer of soil. more effi cient rhizobium, delivered to pea plants as a commercial product nitraginatm, provided nitrogen, which promoted the acceleration of plant growth to some extent, and increase in biomass as a result. harvest index of pea plants varied between the growing seasons. higher values of this index were reported in the year 2011, comparing to the year 2010 (tab. 3). density of pea plants before harvest was signifi cantly differentiated between the objects studied, as a cause of the lower plant density in objects of pea vaccinated with rhizobium bacteria and also with both, rhizobium and micronutriend fertilizer (tab. 3). however, the small values of coeffi cients of variation (cv%) for the pea crop productivity, are evidence of the similar plant density, which can be considered as a stability of fi eld replications per each object. the seed yield of pea varied between the growing seasons 2010 and 2011 by about 33%, due to agroclimatic conditions, which shows the sensitivity of semileafl ess morphotype of pea to the set of conditions that determine its growth and development, and as a consequence shape the crop productivity. fig. 1 shows the relations that occur between seed yield (y) from a single shoot, and the four independent variables. the dependent variable was poorly determined by the length of the fruiting part tab. 2: changes in the growth indices (gai, ndvi, spad) of semileafl ess peas canopy vaccinated with rhizobium bacteria (nitraginatm) or/and leaffertilized (photreltm) treatment vegetation years growth signifi cance cv % indices stages* control nitraginatm photreltm nitraginatm lsd level + photreltm 1 2 3.41 3.61 3.33 3.64 ns 0.857 23.5 3 4.98 5.48 5.00 5.61 ns 0.564 20.7 4 1 1.54 1.35 1.40 1.68 ns 0.254 16.6 2 3.89 3.95 3.83 4.14 ns 0.689 9.2 3 3.63 3.58 3.66 4.04 ns 0.238 9.4 4 3.09 2.72 2.93 2.98 0.241 0.036 6.7 1 2 0.611 0.612 0.606 0.591 ns 0.562 3.77 3 0.589 0.615 0.602 0.610 ns 0.127 2.77 4 0.184 0.153 0.180 0.193 ns 0.583 22.68 1 0.351 0.346 0.352 0.346 ns 0.993 9.07 2 0.554 0.545 0.558 0.557 ns 0.098 1.50 3 0.577 0.569 0.583 0.586 ns 0.060 1.79 4 0.418 0.407 0.424 0.437 ns 0.701 8.06 1 2 37.8 36.5 36.1 36.7 ns 0.337 3.7 3 44.7 42.7 44.5 43.9 ns 0.456 4.1 4 1 35.4 37.4 39.0 38.1 ns 0.299 14.1 2 43.5 44.2 46.0 45.0 ns 0.773 12.2 3 48.1 48.2 47.2 48.5 ns 0.989 17.9 4 22.6 28.2 25.2 24.1 ns 0.527 35.8 * growth stages of pea: 1 – flower buds phase (bbch 51-57), 2 – beginning of fl owering phase (bbch 62-64), 3 – flowering declining and end of fl owering phases (bbch 67-69), 4 – development of fruits (bbch 77-79) – (meier 2001) gai green area index [m2 m-2]; ndvi normalized difference vegetation index; spad relative chlorophyll content. gai** gai** (m2 m-2) ndvi spad 2010 2011 2011 2010 2010 2011 2011 2010 2011 2011 morphology and productivity of semileafl ess peas (pisum sativum l.) 191 tab. 3: comparison of morphologic and productive traits of semileafl ess pea, vaccinated with rhizobium bacteria (nitraginatm) or/and leaf-fertilized (photreltm) treatment traits unit year control nitraginatm photreltm nitraginatm lsd signifi cance cv % + photreltm level 2010 80.7 75.0 85.5 87.5 9.36 0.045 16.3 2011 78.5 74.7 81.5 79.1 ns 0.075 9.2 2010 61.1 54.3 61.1 60.5 ns 0.110 15.3 2011 58.8 57.5 64.2 57.9 4.77 0.024 11.6 2010 19.7 20.7 24.4 27.0 ns 0.099 39.8 2011 19.7 17.2 17.3 21.1 ns 0.091 27.2 2010 3.3 3.8 3.3 4.3 0.81 0.049 31.2 2011 3.7 3.5 3.4 4.0 ns 0.301 24.6 2010 5.7 6.3 5.1 7.4 1.61 0.036 37.7 2011 6.7 6.7 6.3 7.2 ns 0.451 23.4 2010 19.9 23.4 19.0 27.6 ns 0.087 45.7 2011 30.7 30.2 27.7 30.1 ns 0.685 25.0 2010 4.94 6.43 4.93 7.13 ns 0.054 46.2 2011 9.17 8.17 7.71 8.42 ns 0.251 24.0 2010 3.60 3.87 3.73 4.36 ns 0.545 38.9 2011 4.51 4.42 4.15 4.09 ns 0.689 25.9 2010 8.53 12.59 8.66 11.49 ns 0.111 39.9 2011 13.68 10.30 11.86 12.51 ns 0.385 22.6 2010 0.564 0.624 0.532 0.620 0.075 0.045 18.5 2011 0.670 0.650 0.647 0.673 ns 0.360 7.5 2010 69.5 56.9 72.7 52.6 6.67 0.000 16.2 2011 62.8 67.3 70.1 67.0 ns 0.189 11.9 2010 3.2 3.4 2.9 3.1 ns 0.076 8.3 2011 2.5 2.6 2.5 2.6 ns 0.092 6.8 2010 3.59 3.63 3.55 3.71 ns 0.833 6.7 2011 5.32 5.45 5.35 5.58 ns 0.919 9.8 * lodged scale – 1: total lodging; 9: vertical plants plant height plant height (cm) (cm) height to 1 height to 1 st st pod pod st podst st pod st (cm) (cm) length of length of (cm) fruiting stem fruiting stem fruiting stem reproductive reproductive (pcs) (pcs) node per stem node per stem node per stem pods per stem pods per stem (pcs) (pcs) seeds per plant seeds per plant (pcs) (pcs) seed mass seed mass (g) (g) stem mass stem mass (g) (g) plant mass plant mass (g) (g) harvest index harvest index (g g (g g -1) plant density plant density (pcs. m (pcs. m -2) lodging lodging scale* at harvest at harvest (1-9) (1-9) seed yield seed yield t ha-1 of stem (1a), as for this pair of features the correlation coeffi cient r2 = 0.181. another independent variables (1b, 1c) improved the prediction of seed yield per peas plant. moderate correlation was obtained for the number of nodes per shoot (r2 = 0.472) and for the number of pods per shoot (r2 = 0.621). a highly signifi cant linear relationship occurred between yield of pea seeds and the number of seeds per shoot (1d), as for this pair r2 = 0.9396. a further analysis of a yield of semileafl ess pea plant, including relations between reproductive nodes and pod characteristics, allowed the accurate estimation of their contribution to the creation of individual plant productivity (fig. 2). fig. 2a shows the changes in the number of pods per subsequent nodes of pea stem. it has been proven that the nodes of peas formed a similar number of pods, and their number was decreasing with increasing reproductive node layer. the number of seeds was highest at the fi rst node, and then visibly decreased at subsequent nodes (fig. 2b). the weight of a single pea seed was similar, if the seeds developed on the nodes labeled from 1 to 4 (fig. 2c). the seed weight of two top nodes, the 5th and 6th, was lower, but the differences between the objects signifi cantly increased. the seed yield per plant of pea (fig. 2d), was arranged in a similar way as the number of pods and seeds, but differences between the objects were not signifi cant. the relative contribution of seeds sub-yield per each reproductive node in a total yield of seeds per plant, did not differ signifi cantly between the objects (fig. 3a). as expected, the share of additional nodes in seed yield per plant decreased steadily. predictors of this trend were previously discussed, using data presented in fig. 2a-d. the input of reproductive nodes in the total number of seeds per pea plant was arranged similarly (fig. 3b). the individual contribution of each node, including the different pea treatments, in a total seed yield per plant was highly signifi cant (fig. 3c). supremacy of the 1st reproductive node is visible, as the contribution of the 2nd node is smaller, although the statistical difference between both nodes is negligible. the contribution of the other nodes in the pea seed yield per plant, was signifi cantly smaller. it should be emphasized that the overall contribution of reproductive nodes 1 to 4, in the overall yield per plant, was ca 97%. the other two upper nodes, the 5th and 6th had a little or no contribution to the total plant seed yield. these regularities, in a general way, have already been revealed, as fig. 1 shows the poor correlation coeffi cient between seed yield of pea 192 t. zając, a. klimek-kopyra, a. oleksy, a. stokłosa, b. kuligąc, a. klimek-kopyra, a. oleksy, a. stokłosa, b. kuligą morphology and productivity of semileafl ess peas (pisum sativum l.) ���������������������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������������� fig. 1: correlation indices for peas seed yield and length of fruiting part of stem (a); number of reproductive nodes (b); number of pods per stem (c) and number of seeds per stem (d) (n=120) fig. 2: relation of pod number of compared treatments (a); seed number (b); single seed weight (c); total seeds weight (d) per each of semileafl ess peas nodes (average from vegetative seasons); s.l. signifi cance level morphology and productivity of semileafl ess peas (pisum sativum l.) 193 plants, and the number of reproductive nodes on the stem. tab. 4 presents the data related to different morphological features of pods, which may contribute to of their individual productivity. as a consequence of the increase of reproductive nodes number per stem an increase in the number of pods was noted, which was recorded in 2010 for an object with a combined use of see vaccination (nitraginatm) and foliar fertilizer application (photreltm). this pattern of results largely determined the number of pods per shoot of peas. the objects were not signifi cantly differentiated in terms of number of seeds per pod. the different conditions during both vegetative seasons infl uenced the number of seeds per pod, which was visible especially in the year 2011, when a higher air temperature in the growing season occurred, and the fruits of pea contained more seeds as compared to the previous year. the features of pods as well as seed yield were characterized by a great diversity, which was discussed previously, and as evidenced by high variation coeffi cients. comparisons relating to pods were extended with information relating to the length and weight of pedicel. in 2011, peas’ pedicels were signifi cantly longer, but their weight was only slightly higher. the pod length was similar in the both years of study, and demonstrated signifi cant differences between objects for vegetative seasons were characterized by alternating arrangement. a higher proportion of seeds per pod was noted for the vegetative season 2011, seeds also had a higher weight. the share of seeds in the pod of semileafl ess peas was high and fl uctuated in a range between 81-86%. pod weight was higher in 2011, as a consequence of the presence of higher number of mature seeds. there was a moderate and positive correlation between the weight of seeds from a single pod of semileafl ess peas and the length of the fruit in both vegetative seasons (fig. 4a). for this pair of traits correlation coeffi cient value oscillated around r2 = 0.6, which demonstrates that the prediction of the productivity of pea fruit based on this trait is not very precise due to the varying number of seeds per pod, ranging from 1 to 8 pieces. the stronger correlation was observed for the mass of seeds per pod and the number of seeds per pod (fig. 4b). during the both vegetation periods, so climatically different, the correlation characteristics for this pair of traits was relatively constant and of linear relationship, as evidenced by the high value of the correlation coeffi cient r2. the number of pea seeds, developing in the pod or on the shoot, is strongly determined by seeds mass, and both these features are easy to determine. the other correlations, shown in fig. 4c and 4d were poor. correlation between the mass of seeds and their share in the pod weight, turned out to be poor, although the empirically stated share of seeds in the pod weight was high, in the range of 75-90% and 80-91%, for vegetative seasons 2010 and 2011, respectively. a comparison of the mass of a single pea seed, depending on their number and their individual position in the pod is presented in tab. 5; this summary has an acognitive context for semileafl ess pea morphotype, represented by cv. ‘tarchalska’, providing seeds for edible purpose. eight categories of pods, because of the number of educated seeds, were distinguished. a tendency to increase the weight of a single pea seed within a pod, with the increase of seeds number per pod was clearly indicated. this arrangement of fig. 3: evaluation of contribution of peas seeds sub-yields per each reproductiye node into total plant yield (a); individual contribution of reproductive nodes in total plant yield (means from all objects and two vegetative seasons) (b); contribution of number of seeds per each node into a total number of seeds per plant (c); comparison of relative mass of single seed per each node (d); s.l. signifi cance level 194 t. zając, a. klimek-kopyra, a. oleksy, a. stokłosa, b. kuligąc, a. klimek-kopyra, a. oleksy, a. stokłosa, b. kuligą morphology and productivity of semileafl ess peas (pisum sativum l.) tab. 4: traits and productivity of pods of semileafl ess peas in each vegetative season depending on seed vaccination with rhizobium bacteria (nitraginatm) or/and leaf-fertilization (photreltm) treatment traits unit year control nitraginatm photreltm nitraginatm lsd signifi cance cv % + photreltm level 2010 3.42 3.23 2.98 3.14 ns 0.296 49.3 2011 5.03 4.92 5.79 6.19 0.886 0.012 44.2 2010 0.085 0.089 0.084 0.120 0.027 0.028 40.8 2011 0.103 0.093 0.095 0.108 ns 0.471 29.3 2010 5.64 6.08 5.58 5.68 0.295 0.003 17.9 2011 5.88 5.30 5.29 5.30 0.296 <0.001 20.1 2010 1.045 1.231 1.163 1.166 ns 0.086 43.0 2011 1.587 1.438 1.442 1.363 0.1381 0.013 34.5 2010 0.861 1.016 0.975 0.981 ns 0.076 43.5 2011 1.344 1.236 1.235 1.175 ns 0.050 35.7 2010 0.183 0.215 0.200 0.202 ns 0.098 42.7 2011 0.243 0.204 0.207 0.189 0.025 <0.001 43.4 2010 81.8 82.3 81.1 81.2 ns 0.676 9.2 2011 83.8 85.6 85.5 86.0 1.33 0.005 5.7 2010 3.45 3.69 3.78 3.73 ns 0.356 35.2 2011 4.54 4.41 4.41 4.18 ns 0.231 29.7 2010 245 278 260 259 ns 0.057 13.0 2011 298 275 278 284 ns 0.194 11.2 pedicel length pedicel length (cm) (cm) pedicel mass pedicel mass (g) (g) pod length (cm) pod dry matter pod dry matter (g) (g) mass of seeds mass of seeds (g) (g) per pod per pod per pod stripped stripped (g) (g) pods mass pods mass pods mass share of seeds share of seeds (%) (%) per pod mass per pod mass per pod mass number of number of (pieces) (pieces) seeds per pod seeds per pod seeds per pod single seed single seed (mg)weight weight weight data refers directly to the location of the pod on the stem of peas. pods developing at the lowest nodes contained the highest number of seeds, which is showed in the fig. 2a and 2b. the second observed relation is that seeds located in the pod on the fi rst and the last position was characterized by a lower biomass. seeds located in the central part of the pod had the highest biomass, and the highly signifi cant tendency was found for the pods containing from 3 to 8 seeds. the compared pea objects did not differ signifi cantly in terms of chemical composition (tab. 6). total protein content in pea seeds was higher in 2010, as compared to 2011. the lower protein content in semileafl ess pea in 2011 was probably due to the higher weight of a single seed. however, in the both vegetative seasons slightly higher protein and fat content was noted for seeds of peas treated both, with nitraginatm vaccine and foliar fertilizer photreltm. fiber content in pea seeds was slightly higher in 2010, due to their smaller biomass, because larger seeds, collected in the following year were characterized by a lower content of this component. similar relations were noted for the fat and ash content, which proves that climatic conditions in a subsequent vegetation seasons change the content of the most important elements of the peas seeds to a small extent. discussion it was found that the seed yield and aboveground biomass of edible and fodder peas cultivars, are characterized by high variability, caused by habitat conditions and the weather course during the growth (jeuffroy and sebillote, 1997; baigorri et al., 1999; carranca et al., 1999; poggio et al., 2005; annicchiarico and iannucci, 2008). according to olszewski (2004) peas variable yielding, in a climatically diverse vegetative seasons is the result of interaction of different kind of stress factors that may cause the reversible changes in the plants resulting in the slowed down growth, but can also induce irreversible changes resulting in death of plants, which in consequence leads to a decrease in the canopy production potential. this phenomenon became apparent in our studies in the growing season 2010, when unfavorable agroclimatic conditions for peas, led to a decrease in plant and canopy yield. our study has shown that the productive potential of edible pea cv. ‘tarchalska’, representing semileafl ess morphotype, was differentiated stronger by climate variables during vegetative seasons, as compared to treatments applied. most of the analyzed morphological differences, as a quantitative picture of peas productivity in the full maturity stage, were generally insignifi cant. this pattern of results indicates the high stability of species traits on the one hand, on the other the effects of commercial preparations photreltm and nitraginatm, turned out to be weaker, as the creators of plant growth and development and, consequently, a crop yield. the main reason to apply foliar fertilizer to the peas was an expectation for a seed yield increase. czyż (1993) emphasizes that foliar application of three micronutrients solution (b+ mn + mo) increased the peas seed yield and protein content. szwejkowska (2004) found that an increase in the outlay for the cultivation of peas signifi cantly affect peas seed yield and partially compensate for the adverse weather conditions for this species during the growing season. in our studies semileafl es pea plants were developing in the contrasting weather conditions during two growing seasons. a variable course of precipitation and average air temperature during plant growth and development, lead to different quantitative and qualitative characteristics of individual plants and canopy. in the colder and more humid season 2010, semileafl ess pea canopy has developed greater assimilation area (gai) than during the next, drier season 2011. abundance of water boosted the development of plants, gai in the phase of full fl owering was of 5.48 m2 m-2, but this did not translate into a positive effect on seed yield from a single shoot, or canopy. obtained by prusiński (2007) gai of pea cultivars in the morphology and productivity of semileafl ess peas (pisum sativum l.) 195 ������������������������������������������������������������������������������������������������������� ����������������������������������������������������������� fig. 4: correlation coeffi cients for mass of peas seeds per pod and: pod length (a); seeds number (b); share of seeds in the pod mass (c); length of pedicels (d) full fl owering phase was in a range of 4.23-5.28 m2 m-2, and even though the semileafl ess peas cv. ‘venus’ was characterized by a signifi cantly lower gai rate as compared to cultivars of normal foliage, still it was characterized by the highest biomass yield and, consequently, the highest seed yield. in the semileafl ess type of peas cultivars a signifi cant role in photosynthesis play bracts and tendrils, and later also the green pods (kof et al., 2004). in our studies value of relative chlorophyll content (spad) was not differentiated by any treatment. the value of spad ratio depends on the color of leaves, which informs not only of the nutritional status, but also is an inherited trait, as reported by ambrose (2010). according to this author, genotypes of pea with dark-green bracts are characterized by higher spad values, > 60, and those of the bright green bracts have spad values < 30. in the present study we used the same genotype, so in both vegetative seasons the differences were only due to the habitat conditions and objects tested. ridao et al. (1996) under conditions of unlimited water availability(irrigation) obtained gai, of semileafl ess pea cultivars in the phase of the foliage development, ranging from 4 to 5 m2 m-2 depending on the growing season, with much smaller values for the non-irrigated sites. in this regard, the use of par gai (rpi = apar / par) was greater than 0.8. this study provides an objectively shown diversifi cation of 196 t. zając, a. klimek-kopyra, a. oleksy, a. stokłosa, b. kuligąc, a. klimek-kopyra, a. oleksy, a. stokłosa, b. kuligą morphology and productivity of semileafl ess peas (pisum sativum l.) tab. 5: comparison of mass (mg) of single seed of peas (n= 3109) in relation to the number of seeds per pod, including the position of seed in the pod position of seed in pod number of seeds lsd signifi cance per pod level 1st 2nd 3rd 4th 5th 6th 7th 8th n=769 n=749 n=670 n= 507 n=292 n= 99 n= 19 n= 4 1 259.5±72.7 2 243.3±52.5 243.2±46.9 ns 0.987 3 253.5±46.1 267.0±44.4 255.6±47.6 9.70 0.009 4 258.2±46.6 278.4±46.1 275.8±47.6 259.0±47.4 8.87 <0.001 5 271.9±44.7 289.6±45.4 290.4±47.1 289.5±43.7 266.1±48.9 9.18 <0.001 6 270.9±40.7 296.9±37.3 300.0±35.3 297.3±35.4 292.6±32.5 263.0±41.1 11.50 <0.001 7 278.0±43.3 311.3±30.0 324.0±29.7 326.7±27.4 324.7±28.3 308.7±33.4 290.7±32.6 23.53 < 0.001 8 292.5±20.6 355.0±23.8 367.5±20.6 352.5±26.3 355.0±28.9 350.0±23.1 327.5±37.7 317.5±9.6 36.36 0.005 lsd 29.29 27.86 30.41 31.57 35.23 36.83 ns signifi cance level <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.067 mean ± standard. deviation tab. 6: the chemical composition of peas seeds (g kg-1) and protein and starch yield (kg ha-1) in different vegetation seasons for plants vaccinated with rhizobium bacteria (nitraginatm) or/and leaf-fertilized (photreltm) treatment item years control nitraginatm photreltm nitraginatm lsd signifi cance cv % + photreltm level protein 2010 236.9 238.4 242.4 242.9 ns 0.509 2.68 2011 206.6 206.1 206.8 209.4 ns 0.892 2.88 starch 2010 396.7 400.6 394.8 400.8 ns 0.843 2.63 2011 396.5 405.8 396.4 404.8 ns 0.160 1.96 fibre 2010 57.3 54.8 56.4 60.2 ns 0.765 12.03 2011 49.5 48.5 50.7 47.3 ns 0.205 4.73 fat 2010 16.5 18.9 19.3 21.2 ns 0.706 28.08 2011 13.1 15.8 12.3 14.1 ns 0.479 22.81 ash 2010 28.3 30.0 29.5 29.6 ns 0.363 4.65 2011 23.3 22.9 24.1 23.6 ns 0.561 4.96 protein yield 2010 999.9 1016.8 1014.4 1062.0 ns 0.764 7.91 2011 1294.0 1322.1 1300.3 1376.8 ns 0.863 10.57 starch yield 2010 1675.2 1711.9 1647.0 1752.7 ns 0.735 7.76 2011 2483.2 2601.2 2493.7 2658.9 ns 0.762 9.99 productive potential of each of reproductive nodes of peas and it enabled to show the accurate range of components infl uencing the structure of seed yield at the level of a single plant. the presented data fi ll the gap relating to studies on productivity of semileafl ess pea plant that dominates these days in cultivation, as productively effi cient and characterized by high seeds yield. cv. ’tarchalska’ is a high-yielding cultivar in the conditions of southern poland (silesia province), because its seed yield (t ha-1) in the years 2008, 2009 and 2010 was 7.01, 5.02 and 6.10, respectively, exceeding the yielding of the other 11 cultivars (ryszka 2011). in the previous work zającącą et al. (2006) showed that the position of seed in a faba bean pod determines its individual weight: seeds located in the middle part of the pod reached the greatest mass, while the smallest seeds were formed in pods located in the upper part of the shoot. similar trend was also observed in our study, for a semileafl ess edible peas cv. ‘tarchalska’. we have found that the length and weight of pea pod and the number of seeds formed in the pod depends on its location on the frutinig part of a stem. the longest pods with the highest weight and the number of seeds have been found in the lowest nodes – from the 1st to the 2nd. slightly shorter pods were found in the 3rd and 4th nodes, located in the middle part of the stem. the shortest pods were found in the 5th and the 6th nodes, on top of the stem. morphology and productivity of semileafl ess peas (pisum sativum l.) 197 conclusions 1. agroclimate conditions highly differentiated growth of plants and canopy of semileafl ess pea in the south-western region of poland (modzurów). in the warmer growing season 2011, generative growth of plants was better, resulting in very high yield of seeds per plant, determined by higher number of large seeds. also the seed yield of peas from a unit area in that season was high. 2. length, weight and number of pod of pea depends on the pod location along the fruiting part of stem. the longest pods with the highest weight and the number of seeds develop in the lowest nodes – from the 1st to the 2nd. shorter pods grow on the 3rd and 4th nodes located in the middle part of stem. the shortest pods are obtained from the 5th and 6th nodes, on top of the stem. 3. seeds located in the central part of the peas’ pod have the greatest mass, as found with highly signifi cant correlation for the pods, containing from 3 to 8 seeds. the analysis of a single seed, demonstrates position as a major determinant of seed mass. acknowledgements this work was fi nancially supported from national research grant n n310 151837. references ambrose, m.j., 2010: field quantification of foliar chlorophyll content in pisum germplasm. pisum genetics 42, 7-10. annicchiarico, p., iannucci, a., 2008: adaptation strategy, germplasm type and adaptive traits for fi eld pea improvement in italy based on variety response across climatically contrasting enviroments. field crop res. 108, 133-142. aoac, 2005: offi cial methods of analysis. 18th ed. association of offi cial analytical chemists, washington, dc. baigorri, h., antolin, m.c., sanchez-diaz, m., 1999: reproductive response of two morphologically different pea cultivars to drought. eur. j. agron. 10, 119-128. boros, l., sawicki, j., 1997: evaluation of selected pea (pisum sativum l.) cultivars and forms. ii. stability of yielding parameters and trait interrelationships. zesz. probl. post. nauk rol. z. 446, 107-112 (in polish). carranca, c., de varennes, a., rolston, d., 1999: biological nitrogen fi xation by fi xation by fababean, pea and 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intensity of cultivation technology. acta sci. pol., agricult. 6, 43-51 (in polish). ridao, e., oliveira, c.f., conde, j.r., mínguez, m.i., 1996: radiation interception and use, and spectral refl ectante of contrasting canopies of autumn sown faba beans and semi-leafl ess peas. agr. forest meteorol. 79, 183-203. ryszka, k., 2011: groch siewny. in: wyniki 2010. porejestrowe doświadczalnictwo odmianowe 12/2011, 84-87 (in polish). sadowsky, m.j., graham, p.h., 2001: soil biology of the rhizobiaceae. the rhizobiaceae molecular biology of model plant-associated bacteria. in: spaink, h.p., kondorosi, a., hooykaas, p.j.j. (eds.), 155-172. kluwer academic publishers, dordrecht / boston / london. semere, t., froud-williams, r.j., 2001: the effect of pea cultivar and water stress on root and shoot competition between vegetative plants of maize and pea. j. appl. ecol. 38, 137-145. stelling, d., 1989: problems of breeding for improved standing ability of dried pea (pisum sativum l.) j. agron. crop sci. 163, 21-32. szwejkowska, b., 2004. the effects of methods of cultivation on pea seed production. fragm. agron. 3, 120-126 (in polish). uzun, a., acikgoz, e., 1998: effect of sowing season and seeding rate on the morphological traits and yields in pea cultivars of differing leaf types. j. agron. crop sci. 181, 215-222. uzun, a., bilgili, u., sincik, m., filya, i., acikgoz, e., 2005. yield and quality of forage type pea lines of contrasting leaf types. eur. j. agron. 22, 85-94. vance, c.p., 2001: legume symbiotic nitrogen fixation: agronomic aspects: the rhizobiaceae molecular biology of model plant-associated bacteria. in: spaink, h.p., kondorosi, a., hooykaas, p.j.j. (eds.), 509-529. kluwer academic publishers, dordrecht / boston / london. zając, t., kulig, b., strojny, j.,ąc, t., kulig, b., strojny, j.,ą 2006: analysis of empirical and indirect traits of traditional strain of fi eld bean pods (vicia faba l.). part 1. comparison of pods traits placed on three segments of shoot fruiting part. acta agr. silv. ser. agr. 48, 3-17 (in polish). address of the author: dr. agnieszka stokłosa, department of agrotechnology and agricultural ecology, university of agriculture, al. mickiewicza 21, 31-120 krakow, poland journal of applied botany and food quality 87, 182 189 (2014), doi:10.5073/jabfq.2014.087.026 department of horticulture, faculty of agriculture, shiraz university, shiraz, iran effect of 24-epibrassinolide on salinity-induced changes in loquat (eriobotrya japonica lindl) fatemeh sadeghi, akhtar shekafandeh* (received october 30, 2013) * corresponding author summery this study was carried out to investigate the role of 24-epibrassinolide (24-ebl) in inducing loquat plant salt tolerance. plants were treated with five levels of salt, so that the electrical conductivity (ec) of 0.5 (control), 2, 4, 6 and 8 ds m-1 was established in pots. then, the plants were sprayed with different concentrations of 24-ebl (0, 0.25, 0.5 and 0.75 mg l-1). under salt stress, the plant growth parameters and chlorophyll content decreased, while the total soluble sugars and proline contents considerably increased. however, the application of 24-ebl significantly ameliorated the plant growth by reducing the adverse effects of salinity on the examined parameters. the ion concentrations showed an increase in accumulation of na+ and cl coupled with a decrease in k+ with increasing salinity in medium. exogenous application of 24-ebl had a significant effect on leaf na+, k+, clcontents. out of different 24-ebl concentrations, the effects of 0.5 mg l-1 proved the best under stress conditions. introduction loquat (eriobotrya japonica lindl.) is a subtropical evergreen fruit tree of rosaceae family, (lin et al., 2007; zheng, 2007) which is grown commercially in subtropical to mild temperate climate. loquat is commonly propagated by seed; as a result, the plants possess variable performance and different fruit characteristics owing to heterozygosis and cross-pollination. therefore, in some parts of the world, the seedlings employ as a rootstock for cultivars with high fruit quality (lin, 2007). salinity is one of the major abiotic stresses that affects plant production and growth in many arid and semi-arid areas throughout the world (chelli-chaabouni et al., 2010). iran is now faced with salinity problems in about 34 % of its area in addition to harsh conditions of climate in about half of the country. this also signals that global climate change and consecutive years of drought is likely to increase salinity of the main agricultural lands (rahimian et al. 2013). the deleterious effects of salinity on plant growth are associated with low osmotic potential (water stress), nutritional imbalance, specific ion effect (salt stress), or a combination of these factors. all these factors cause adverse effects on plant growth and development at physiological and biochemical and molecular level (ashraf and harris, 2004). among the most common effects of salinity is growth inhibition by nacl. in many plants, compatible osmoprotectant metabolites, such as proline, glycine-betaine, and soluble sugars are produced to protect the cells against the damaging effects from salt stress (chelli-chaabouni et al., 2010). although, soil salinity directly affects root system, hence breeding and selection of salt tolerant rootstock for sustainable fruit production is inevitable, but this is a time consuming. for reducing adverse effects of salinity different strategies have also been developed. one of these approaches is; employing different types of phytohormones (houimli et al., 2008). among them, brassinosteroids, a recent class of plant hormone not only play prominent role in various physiological and biochemical processes in plants, like stem elongation, vascular differentiation, leaf bending and epinasty, induction of ethylene biosynthesis, regulation of gene expression, nucleic acid and protein synthesis and photosynthesis (hayat et al., 2010; krishna, 2003; yu et al., 2004; cao et al., 2005), but has also attracted increasing attention in studies addressing to adaptive response to environmental stresses, such as heavy metal (ali et al., 2008; hayat et al., 2007), salt (ali et al., 2007), temperature (wilen et al., 1995), drought (zhang et al., 2008). loquat is considered as having a moderate tolerance to drought but there are some conflicting evidences on the loquat salt tolerance. some sources have mentioned its moderate tolerance to nacl (gilman and watson 1993) and others its salt-sensitive (http:// aciar.gov.au/files/node/2275/mn050_part_5_pdf_19541.pdf). knowing about salt tolerance limit of loquat helps us in extending its cultivation in area with certain levels of salinity which are not suitable for citrus. so, the present investigation was conducted to discover the salt tolerance of seedlings, effect of 24-ebl on its salt tolerance, and to determine the interactive effects of salinity and 24ebl on plants morphophysiological characteristics. materials and methods plant material and treatments the loquat uniform seeds were extracted from mature fruits and immediately washed with tap water and placed at 4 ˚c for 2 weeks. germinated seedlings were allowed to grow in perforated polyethylene pots contained a mixture of peat-moss, sand and clay (1:1:1, v/v/v). then, when the height of the seedlings in pots was about 30 to 40 cm, the same vigour seedlings were transferred to 7 litre plastic pots filled with 6 kg soil mixture as mentioned above. the field capacity of the soil used for potting was determined according to the protocol described by richards (1949). potted seedlings were irrigated for 8 months to field capacity level. in order to achieve optimum seedling vegetative growth, fasemko complete fertilizer (ph 6.7) was applied to each pot with irrigation water each fortnight. the seedlings were grown in the greenhouse at day/night temperature: 30/25±4 °c and relative humidity of 40-45 % under natural sun light. after this period, when the seedlings were well established, a factorial experiment was conducted in completely randomized design with 4 replications. treatments were 5 levels of salt (nacl) × 4 levels of 24-epibrassinolide (24-ebl). the salts were applied to pots by irrigation water step-wise until an electrical conductivities (ec) of 0.5(control), 2, 4, 6 and 8 ds m-1 were attained. after that, plant were treated exogenously with 0.0, 0.25, 0.5 and 0.75 mg l-1 of 24-epibrassinolide (24-ebl) at run-off. this action was repeated 3 times with one week interval, and then six weeks after spraying, data were recorded. growth measurements at the end of experiment, plants were harvested and divided into shoots and roots, after measuring of shoot and root fresh weight epibrassinolid and salinity-induced changes 183 (fw), they were dried in the oven at 75 °c for three days to determine their dry weights (dw). leaf area was measured by delta-tdevices (england). chlorophyll content leaf chlorophyll content was measured with a chlorophyll-meter (spad-502, minolta co. japan) using 3 full expanded leaves. subsequently, 14 leaves with their chlorophyll content already measured by spad-502 were randomly detached from the plants to extract chlorophyll using the chemical method. extraction was performed with 80 % ethanol and the yielded solution was centrifuged at 8000 rpm for 10 min. chlorophyll content was colorimetrically determined using the following formula (a.o.a.c 1975): chlorophyll (mg g-1 fresh weight) = [20.2 (a 645) + 8.02 (a 663) x v] / (1000 w) where a is absorption value, v is ultimate volume of extract and w is leaf fresh weight. a regression line and equation were obtained using excel software, considering the spad-502 readings for the mentioned 14 leaves and colorimetrically determined chlorophyll contents. this equation was later used to estimate other chlorophyll readings from spad-502. total soluble sugar to measuring soluble sugars, 150 mg of dried leaf samples was extracted two times with ethanol. sample were centrifuged at 3500 rpm for 10 min, the volume of upper phase was reached to 25 ml. soluble sugars was measured according to method of dobios et al. (1956), the absorption was recorded using spectrophotometer at 490 nm (model uv-120-20.japan). estimation of free proline content proline content was measured using the method of bates et al. (1973). free proline was extracted from 0.5 g of leaf samples with 5ml 3 % sulpho salicylic acid and centrifuged at 5000 rpm for 10 min. supernatant was treated with acid-ninhydrin and acetic acid, tab. 1: effect of 24-epibrassinolide on loquat plant growth parameters under salt stress conditions 24-ebl nacl shoot mean shoot mean root mean root mean mg l-1 ec ds m-1 fw (g) dw (g) fw (g) dw (g) 0 cont(0.5) 297.4abc 105.1cd 80.2abc 30.8ab 2 298.7abc 129.26b 83.3ab 31.6ab 4 242.2f 92.2def 73.3bc 22.4de 6 161.8ghi 72.6ghi 58.1def 15.3fg 8 125.7j 224.1b 58.1i 91.5c 45fg 68b 13.1g 22.6b 0.25 cont(0.5) 298abc 128.5b 83.6ab 31.2ab 2 290bcd 118.7bc 84.8ab 30.8ab 4 276.6cde 121bc 81.1abc 25cd 6 166.9gh 82.4efg 56.5efg 16.2fg 8 141.5ij 234.8ab 68.6ghi 103.8b 46.3g 70.5ab 15.3fg 23.7ab 0.5 cont(0.5) 319.3a 148.2a 89.6a 32.2ab 2 304.9ab 155.2a 88.3a 35.1a 4 272.2de 104.1cd 89.6a 22.2de 6 163.5ghi 81.9efg 58.3def 20.5def 8 144.7hij 240.9a 71.2ghi 112.1a 51.2efg 75.4a 16.3fg 25.3a 0.75 cont(0.5) 298.7abc 115.2bc 81.2abc 32.3ab 2 299.8abc 116.8bc 84.3ab 32.3ab 4 254.3ef 96.5de 70.3cd 20.5def 6 172.3g 76.7fgh 60.2de 17.9efg 8 129.4j 230.9ab 63.7hi 93.8c 55.1efg 70.2ab 17.3efg 23.3ab analysis of variance (f values) salinity 333.03*** 78.7*** 50.73*** 65.27*** 24-ebl 3.04* 11.3*** 2.39ns 1.73ns salinity × 24-ebl 1.0 ns 2.62** 0.88ns 0.80ns in each column, means with the same letters are not significantly different (p<0.05) *, **, * * *; significant at 0.05, 0.01, and 0.001 levels, respectively. ns; non-significant 184 f. sadeghi, a. shekafandeh boiled for 1 h at 100 °c. the reaction was then terminated in an ice bath. reaction mixture was extracted with 2 ml toluene. absorbance of chromophore containing toluene was determined at 520 nm. mineral nutrient analysis the mineral composition of each plant organ (roots and leaves) was determined at the end of the experiment. leaves and roots were harvested and analysed for sodium, chloride and potassium. roots were intensively washed with de-ionized water. one gram of leaf samples was ashed in a muffle a furnace at 550 °c for 5 h. then the ash was dissolved in 10 ml 2 n hcl and reached volume to 100 ml with distilled water. potassium and sodium were determined using flame photometer (model pfp7, jenway, england). the chloride content of leaves and root were determined following the method of chapman and pratt (1961). data analysis statistical analyses were performed by proc glm of sas (sas.9.1) and means were compared by lsd test at p≤0.05. result shoot fresh and dry weight the results showed that salt stress caused a significant reduction in shoot fresh and dry weight. the main effect of exogenous application (foliar spray) of 24-ebl showed an increase in shoot fresh and dry weight and the highest values (240.9 and 112.1 g for fresh and dry weight respectively) were recorded in plants sprayed with 0.5 mg l-1 24-ebl (tab. 1). in the absence of 24-ebl, low concentration of salt (2 ds m-1) increased significantly shoot dry weight in comparison with control. there was a significant interaction between salinity and 24-ebl on shoot dry weight and the highest amount (155.2 g) was achieved in ec 2 ds m-1 when the plants were sprayed with 0.5 mg l-1 24-ebl which was significantly higher than control and other salt and 24ebl combinations. in ec 4 ds m-1 when plants were treated with 0.25 mg l-1 24-ebl, shoot dry weight increased significantly by 28.8 % compared with non-treated plants in the same salt condition (tab. 1). root fresh and dry weight root fresh and dry weight decreased significantly due to imposition of salt stress. electro conductivity (ec) of 2 ds m-1 had not destructive effect on development (tab. 1). the highest root fresh weight (89.6 g) achieved when plants were treated with 0.5 mg l-1 24-ebl in ec of 4 ds m-1 which was significantly higher than the root fresh weight in the same condition without 24-ebl. leaf area salinity and 24-ebl, and their interaction significantly influenced average leaf area in loquat seedlings. leaf area per plant decreased significantly in salt stress conditions (fig. 1). in control (ec 0.5 ds m-1), plant treated with 0.25-0.5 mg l-1 of 24-ebl led to maximum enhancement in leaf area (about 140.3 cm2). in ec of 2 ds m-1, 0.25 mg l-1 24-ebl increased nearly 38 % in average leaf area per plant. in ec of 4 ds m-1 or higher, application of 24-ebl was not able to decreased harmful effect of salt. chlorophyll chlorophyll was also significantly influenced by salinity and 24-ebl application. with increasing salt concentrations in the medium, leaf chlorophyll content decreased. there was a high interaction between salinity and 24-ebl on leaf chlorophyll content (fig. 2). in ec of 2 and 4 ds m-1, the highest leaf chlorophyll content was observed when plants were sprayed with 0.5 mg l-1 24-ebl which was the same as control. total soluble sugars (tss) the results showed that with increasing salt concentrations, leaf total soluble sugar as an osmoprotectant increased (fig. 3). in 6 ds m-1 nacl, plants treated with 0.5-0.75 mg l-1 of 24-ebl accumulated significantly higher tss than other combinations of salt and 24-ebl treatments. 60 fig. 1: effect of 24-ebl on loquat leaf area under salt stress. values are mean ± se of 12 leaves. different letters indicate significant differences between the treatments at 0.05 % level. fig. 2: effects of 24-ebl on loquat leaf chlorophyll under salt stress. values are mean ± se of 12 leaves. different letters indicate significant differences between the treatments at 0.05 % level. fig. 3: changes in total soluble sugars of loquat leaves under salt stress condition sprayed with different concentrations of 24-ebl. values are mean ± se of 6 plants. different letters indicate significant differences between the treatments at 0.05 % level. epibrassinolid and salinity-induced changes 185 proline in the absence of 24-ebl, with increasing salinity in pots, leaf proline content increased. application of 24-ebl enhanced the proline accumulation. for example, in ec 4 ds m-1, 0.5-0.75 mg l-1 of 24-ebl increased significantly leaf proline content in comparison to non-treated plants in the same salt concentration (fig. 4). mineral nutrients both sodium and chloride contents in the different plant organs (leaves and roots) increased with increasing salt concentration in the medium. the leaf sodium content was higher than that of root (tab. 2, 3). exogenous application of 24-ebl significantly changed the leaf na+ and clcontents under saline conditions (tab. 2). for example, in salinity of 6 ds m-1, when plants were treated with 0.5 mg l-1 24-ebl leaf na+ and clcontents decreased by 8.7 and 35.5 % respectively with comparison to non-treated plant under the same salt concentration. tab. 2: effect of 24-epibrassinolide on leaf na+, k+ and clions of loquat plants under nacl stress. 24-ebr nacl leaf na+ mean leaf k+ mean leaf clmean (mg l-1) ec ds m-1 (mg g-1 dw) (mg g-1 dw) (mg g-1 dw) 0 cont(0.5) 1.3g 13.7bc 1.3gh 2 1.5g 13.2cde 1.6f 4 8.5f 12.5fg 2.4de 6 14.9c 11.7hi 3.1c 8 16.3ab 8.5a 11.2i 12.5c 3.5ab 2.4a 0.25 cont(0.5) 1.4g 13.6bc 1.2h 2 1.3g 13.4bcd 1.6fg 4 8.4f 12.5fg 2.3e 6 14de 13.7bc 2.2e 8 16.1b 8.2bc 11.7hi 13b 3.7a 2.2b 0.5 cont(0.5) 1.3g 14ab 1.1h 2 1.1g 13.5bc 1.1h 4 8.1f 12.9def 1.5f 6 13.6e 13.3cde 2d 8 15.9b 8c 11.7hi 13.1ab 3c 1.9c 0.75 cont(0.5) 1.2g 14.4a 1.1h 2 1.3g 13.3cde 1.1h 4 8.4f 12.7efg 1.6fg 6 14.2d 13.8abc 3.1c 8 14.2d 8.3ab 12.2gh 13.3a 3.3bc 2c analysis of variance (f value) salinity 5093.7*** 55.82*** 226.3*** 24-ebl 6.38*** 11.99*** 13.46*** salinity × 24-ebl 1.81* 3.37*** 4.85*** in each column, means with the same letters are not significantly different (p<0.05) *, **, * * *; significant at 0.05, 0.01, and 0.001 levels, respectively. fig. 4: changes in proline of loquat leaves under salt stress condition sprayed with different concentrations of 24-ebl. values are mean ± se of 6 samples. different letters indicate significant differences between the treatments at 0.05 % level. 186 f. sadeghi, a. shekafandeh although significant reduction in leaf and root k+ occurred due to the addition of nacl in the growth medium (tab. 2, 3), exogenous application of 24-ebl altered the root and leaf k+ of the salt stressed and non-stressed plants. the highest leaf k+ (14.4 mg g-1 dw) and root k+ (8.5 mg g-1 dw) contents were observed in control and ec 2 ds m-1 respectively, when plants were treated with 0.75 mg l-1 of 24-ebl. in ec 6 ds m-1, exogenous application of 24-ebl (in all concentrations) increased significantly leaf k+ content compared with non-treated plant in the same salt condition. discussion after 10 days of salt treatment, leaves of plants grown in ec 8 ds m-1 showed the signs of toxicity. these symptoms were started from leaf margins and then extended on leaf surfaces. injury symptoms may be related to accumulation of cland/or na+ in the leaves. it has been also reported that chloride injury develop as marginal chlorosis of leaves of broad leaved trees followed by extensive scorching of leaf blades (kozolowski, 1997) which was in accordance with tab. 3: effect of 24-epibrassinolide on root na+, k+ and clions of loquat plants under nacl stress. 24-ebr nacl root na+ mean root k+ mean root cl_ mean mg l-1 ec ds m-1 (mg g-1 dw) (mg g-1 dw) (mg g-1 dw) 0 cont(0.5) 0.9h 7.8b 3.7f 2 1.7g 7.8b 4.7e 4 2.3cde 6.6cde 7d 6 2.6b 5.8fg 8.7c 8 2.9a 2.1a 5.7g 6.7b 10a 6.8a 0.25 cont(0.5) 0.9h 8.1ab 3.5f 2 1.6g 7.9b 4.6e 4 2.3cd 6.6cde 7.1d 6 1.9f 6.1fg 8.7c 8 2.3cd 1.7c 6fg 6.9ab 10a 6.7ab 0.5 cont(0.5) 0.9h 8.2ab 3.5f 2 1.7g 8.2ab 4.5e 4 2ef 6.9cd 6.8d 6 2.2def 5.7fg 8.4c 8 2.5bc 1.8bc 6.24ef 7.1a 9.5ab 6.5b 0.75 cont(0.5) 0.9h 7.7b 3.7f 2 1.5g 8.5a 4.2e 4 2.23cde 7c 7d 6 2.46bcd 5.9fg 8.8c 8 2.7ab 1.9ab 6.3def 7.1a 9.3b 6.6ab analysis of variance (f value) salinity 189.08*** 106.84*** 934.01*** 24-ebl 10.31*** 3.11* 2.70 ns salinity × 24-ebl 2.44* 0.96ns 1.00 ns in each column, means with the same letters are not significantly different (p<0.05) *, **, * * *; significant at 0.05, 0.01, and 0.001 levels, respectively. ns; non-significant our observation on loquat plants. in ec 68 ds m-1, 24-ebl improved the growth of plants thus decreased the symptoms of toxicity (fig. 5). the increasing of salt concentration in growth media, shoot and root fresh and dry weight significantly decreased. the effect of salt stress on growth reduction has been reported on different plants by others (ashraf and harris, 2004; anjum, 2008). application of brassinosteroids (brs) considerably reduced the growth inhibitory effect of salt stress as reflected in the growth of the plants. the organ growth-promotive effects of brs have been mainly related to cell expansion and division (mayumi and shibaoka, 1995) via enhancing microtubules and cellulose biosynthesis and thus changing mechanical characteristics of the cell wall (fujioka and sakurai, 1997). the role of homobrassinolide application in growth promotion under normal or stress conditions has also been reported in banana growth under stress conditions (nassar, 2004). houimli et al. (2008) also reported that 24-epibrassinolide increased salt tolerance in pepper plants which was in consistent with our result in loquat plants. it has been suggested that brassinosteroids could epibrassinolid and salinity-induced changes 187 fig. 5: in high salt condition (ec 8 ds m-1), loquat plants were treated with different concentration of 24-ebr; 0.0, 0.25, 0.5 and 0.75 mg l-1 from left to right respectively. application of 0.5 and 0.75 mg l-1 24ebl ameliorates loquat plant growth and decreased nacl toxicity symptoms, two plants on the right. have a positive role on root growth, if its concentration be greater than its threshold value and this is genotype dependent (mussig et al., 2003). in present study, salt stress reduced severely leaf surface area of loquat leaves. it is known that reduction in leaf area in salt-stressed plants can be explained by a decrease in leaf turgor, changes in cell wall properties and diminish in photosynthetic rate (rodriguez et al., 2005). brs determine leaf size by controlling the cell proliferation gradient at the transition zone between cell division and expansion through positive regulation of either the rate or duration of the cell proliferation (gudesblat and russinova, 2011). elkhallal et al. (2009) indicated that increase in the leaf area induced by brassinolide was further translated into improved growth of the plants as reflected in the enhancement in fresh and dry weights of the shoot system. under saline conditions, reduction of leaf chlorophyll content may be attributed to the increase in the activity of chlorophyll degrading enzyme chlorophylase, (reddy and vora, 1986). it was observed that brassinosteroids reduced the inhibitory effects of salt stress on leaf pigment levels and this could be one of the reasons for induced growth stimulation by brassinosteroids under saline conditions (anuradha and rao, 2003). castle et al. (2003) also reported that epibrassinolide improved the tolerance of barley to salt stress, by reducing damage via protecting cell ultra-structure and chloroplast membrane system (castle et al., 2003). in present study, until certain level of salt stress (ec 6 ds m-1) with increasing salinity total soluble sugar (tss) increased and then decreased. it is known that sugars act as osmoprotectant in stress condition. the main functions of them are osmotic regulation, carbohydrate storage and also sugars can cause cell membrane and protein stability. these can be done through the formation of hydrogen bonds between the carboxyl groups of proteins and polar chain of sugar (koster and leopold, 1988). reduction of tss in ec 8 ds m-1, may be due to induction of high osmotic pressure which affects hydrocarbon enzyme synthesis and consequently sugar content declined (doring and ludders, 1986). the results showed that in ec 4-6 ds m-1, exogenous application of 24-ebl enhanced leaf total soluble sugar content. some investigations found increases in the activity of enzymes of carbohydrate metabolism such as sucrose synthase, sucrose phosphate synthase and acid invertase, in the presence of brassinosteroids, suggesting a regulatory role for this hormone in carbohydrate metabolism (yu et al., 2004; lisso et al., 2006). leaf proline accumulation is a common metabolic response of higher plants to salinity stress. in addition to its role in osmotic adjustment as an osmolyte, it also plays an important role in a number of physiological and biochemical phenomena such as stabilizing sub-cellular structures, scavenging free radicals, and buffering cellular redox potential under stress conditions (srinivas and balasubramanian, 1995; hare and cress, 1997). shahid et al., (2011) reported that accumulation of proline in plant tissue due to ebl under stress may be associated with reduction in proline utilization due to the minimum protein formation, proline degradation and enhancement in proline formation due to the hydrolysis of proteins. the results of present study are also consistent with findings of wang and zhang (1993) on rice and vardhini and rao (2003) on sorghum. significant correlation was also observed between soluble sugar and proline (r2 = 0.68) (fig. 6). stewart et al. (1966) showed that proline accumulation was greater and most prolonged in leaves with higher sugar and starch contents. based on these results, the interpretation of the role of sugar in proline accumulation is that the oxidation of sugars furnishes α-ketoglutarate and nad(p)h for proline synthesis in leaves. fig. 6: relationship between proline and total soluble sugars in leaves of loquat in response to salinity. regression equation: r2 = 0.68; p < 0.001. it has been observed that plants exposed to nacl take up high amounts of na+, whereas the uptake of k+ is significantly reduced (asharaf and akhtar, 2004). similarly, the competition between k+ and na+ in the roots of plants is well known (garcia-sanchez et al., 2002; garcia-legaz, 2008), suggesting that the uptake mechanism of the two ions (na+ and k+) is similar (schroeder et al., 1994). potassium ion is the major cation within plants which counterbalances the negative charge of anions. the k+ ion stabilizes the ph, osmotic potential, and turgor pressure within cells. it also plays a crucial role in the activation of the enzymes involved in the metabolism and synthesis of proteins and carbohydrates (page and cera, 2006). moreover, k+ ions contribute to the osmotic adjustment of cells in salt stressed plants. in present study, under salt conditions, the na+ content in the shoots was higher than it in the roots. the transport and accumulation of na+ in the leaves often characterize tolerant includer species that compartmentalize toxic ions in the vacuoles (zid and grignon, 1991; niknam and mccomb, 2000; ashraf, 2004). however, if na+ sequestered in the vacuole of a cell, organic solutes should 188 f. sadeghi, a. shekafandeh accumulate in the cytoplasm to balance the osmotic pressure of the ions in the vacuole, the compounds that accumulate most commonly are proline (munns, 2000). as mentioned before, in our experiment the leaf proline contents increased. our results showed that in salt stress condition, the uptake of na+ reduced and that of k+ increased by exogenous application of 24ebl from root zone (tab. 3). the beneficial effect of 24-ebl may be related to the improvement of the ion balance in salt treated cells due to its cationic nature as reported by pirogovskya et al. (1996). ronsch et al. (1993) also suggested that brs can be employed to plants for effective absorption of minerals from the soil. it is worth noting that, where the amount of na+ decreased and the amount of k+ increased, plant growth ameliorated via increasing in shoots and root fresh weight (tab. 1). the low concentration of cl− in the leaves indicates the ability of the different plant parts, especially the roots, to limit cl− transport to the leaves, where stronger damage could be produced. leaf injury and defoliation are closely correlated with leaf cl− levels, and hence application of 24-ebl had an obvious influence on improving the damage. also, the reduced accumulation of cl− in leaves of 24ebl treated plants may be accentuated by the dilution effect of cl− because of the higher growth of 24-ebltreated plants under saline conditions. in conclusion, the detrimental effects of salt on growth and biochemical parameters were alleviated by 24-ebl application. out of the four 24-ebl concentrations, the effects of 0.5 mg l-1 proved the best under stress conditions. the application of 24-ebl improved the performance of plants under low level of salt and also decreased salt harmful effect under medium-salt concentrations (ec of 4-6 ds/m) especially on leaf chlorophyll content and increased in leaf proline content as osmoprotectant. it seems that loquat is a moderate salt tolerance and application of this hormone may be useful for declining salt effect in area with ec less than 4 ds/m. however, field trail must be performed. reference a. o. a. c., 1975: official methods of analysis of the association of official analytical chemist. 10th ed. washington, d. c.. ali, b., hasan, s.a., hayat, s., hayat, q., yadav, s., fariduddin, q., ahmad, a., 2008: a role for brassinosteroids in the amelioration of aluminium stress through antioxidant system in mung bean (vigna radiata l. wilczek). env. exp. bot. 62, 153-159. ali, b., hayat, s., ahmad, a., 2007: 28-homobrassinolide ameliorates the saline stress in chickpea (cicer arietinum l.). env. exp. bot. 59, 217223. anjum, m.a., 2008: effect of nacl concentrations in irrigation water on growth and polyamine metabolism in two citrus rootstocks with different levels of salinity tolerance. acta physiol. plant. 30, 43-52. anuradha, s., rao, s.s.r., 2003: application of brassinosteroids to rice seeds (oryza sativa l.) reduced the impact of salt stress on growth, prevented photosynthetic pigments loss and increased nitrate reductase activity. plant growth regu. 40, 29-32. ashraf, m., 2004: some important physiological selection criteria for salt tolerance in plants. flora 199, 361-376. ashraf, m., harris, p.j.c., 2004: potential biochemical indicators of salinity tolerance in plants. plant sci. 166, 3-16. asharaf, m., akhtar, n., 2004: influence of salt stress on growth, ion accumulation and seed oil content in sweet fennel. biol. plant. 48, 461464. bates, l.s., waldren, r.p., teare, i.d., 1973: rapid determination of free proline water stress studies. plant soil. 39, 205-207. cao, s., xu, q., cao,y., qian, k., an, k., zhu, y., binzeng, h., zhao, h., kuai, b., 2005: loss-of-function mutations in det2 gene lead to an enhanced resistance to oxidative stress in arabidopsis. physiol. plant. 123, 57-66. castle, j.t., montoyam, t., bishop, g.j., 2003: selected physiological responses of brassinosteroids: a historical approach. in: hayat, s., ahmad, a. 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alleviated the adverse effect of water deficits on photosynthesis and the antioxidant of soybean (glycine max l.). plant growth regu. 56, 257264. zheng, s.q., 2007: achievement and prospect of loquat breeding in china. acta hort. 750, 85-91. zid, e., grignon, c., 1991: tests early selection for plant resistance to stress. case of drought and salt stresses. in: plant breeding for adaptation to arid environments. ed aupelf-uref, john libbey eurotext, paris. 91-108. address of the author: dr. akhtar shekafandeh and eng fatemeh sadeghi, department of horticulture, faculty of agriculture, shiraz university, p.o. box 7144465186, shiraz, iran e-mail: shekafan@shirazu.ac.ir e-mail: fateme.sadeghi2775@yahoo.com vorgabe neu journal of applied botany and food quality 80, 160 164 (2006) institute of vegetable and ornamental crops grossbeeren/erfurt e.v., grossbeeren, germany effects of environmental factors on carotenoid content in tomato (lycopersicon esculentum (l.) mill.) grown in a greenhouse* angelika krumbein, dietmar schwarz, hans-peter kläring (received june 26, 2006) summary tomatoes are an important source of lycopene in the human diet. the effect of temperature (15°c 24°c), co 2 supply (380 1000 ppm) and nutrient concentration measured as electrical conductivity of the nutrient solution (ec, 2 9 ds m-1) on the content of carotenoids (lycopene, ß-carotene) were investigated in two tomato cultivars grown in a greenhouse. the cherry tomato cultivar supersweet was characterised by higher lycopene contents than the conventional round tomato ‘counter’. the results indicated that temperature has a significant influence on the biosynthesis of lycopene and ß-carotene during ripening. a temperature above 20°c seems to be optimal for lycopene production in the investigated cultivars, whereas a decrease to 15°c diminished the lycopene content. neither co 2 supply nor ec increase affected the carotenoid content under the conditions investigated. introduction tomatoes are low in fat and calories but rich in minerals, vitamins, and health-promoting secondary plant compounds. due to their high frequency in the diet, tomatoes are an important source of carotenoids. the carotenoid content increases with fruit ripening. lycopene is the most abundant carotenoid in ripe tomatoes with about 90% of total carotenoids, followed by ß-carotene. epidemiological studies, in vitro, and animal studies provide evidence that carotenoids may protect humans from several kinds of cancer (stahl and sies, 2005). a decreased risk of contracting prostate cancer was associated with high lycopene intake through consumption of tomatoes and tomato products (giovannucci, 2002). the protective effects of lycopene include antioxidant activity, such as the quenching of singlet oxygen and the scavenging of peroxyl radicals, induction of cell – cell communication, and growth control (sies and stahl, 1998). otherwise, with respect to the prevention of cardiovascular diseases and cancer, data for ß-carotene are conflicting (clarke and armitage, 2002; stahl and sies, 2005). the growth, yield and fruit quality of tomatoes can be influenced by their genetic potential and environmental factors like temperature, radiation intensity, co 2 concentration, and nutrient availability. greenhouse vegetable plants show positive effects from co 2 supply by increasing photosynthesis and yield (nederhoff, 1994). furthermore, it can also directly improve fruit quality. thus, li et al. (1999) showed that an increase in the co 2 concentration (from 380 to 1200 ppm) may compensate the negative effect of high salinity (5.2 and 7.0 ds m-1) on yield and slightly improve the tomato’s quality in terms of total soluble solids, glucose, and acidity. to our knowledge, no information has yet been published on the effect of co 2 supply on carotenoid content in tomatoes. it is well known that increasing the nutrient solution concentration in a soilless culture increases taste components like sugar and acids in tomatoes (dorais et al., 2001; auerswald et al., 1999), but the effects on carotenoids are still unclear. it was reported that increasing ec did not affect the content of lycopene and ß-carotene in tomatoes, but it was also found that an increase of these secondary metabolites may occur (dorais et al., 2001; krauss et al., 2006). temperature has a significant influence on the growth and development of tomato fruits. because of the shorter fruit growth period and faster initiation of new trusses at higher temperatures, early yields are higher at higher temperatures (van der plog and heuvelink, 2005). regarding carotenoids, temperatures below 12°c strongly inhibit lycopene biosynthesis, whereas temperatures above 32°c stop this process altogether (dumas et al., 2003). it was argued that high temperatures (35°c) specifically inhibit the accumulation of lycopene because they stimulate the conversion of lycopene into ß-carotene (hamauzu et al., 1998). the aim of the present study was to determine the effect of environmental conditions such as temperature, co 2 concentration, and nutrient solution concentration measured as electrical conductivity (ec), on the content of carotenoids (lycopene, ß-carotene) in tomatoes grown in a greenhouse. two tomato cultivars, representing different types, were investigated: a conventional round tomato (cv. counter) and a cherry tomato (cv. supersweet). materials and methods plant material the effects of the environmental conditions (temperature, co 2 concentration, ec of the nutrient solution) on the carotenoid content of the tomato fruits were investigated in a hydroponically grown crop in five greenhouse experiments at grossbeeren (52°n, 13°e) using the cultivars counter and supersweet. the combination investigated was replicated in two greenhouse compartments. plants were grown using the nutrient film technique. a nutrient solution was prepared by mixing rainwater and stock solutions according to the recipe of de kreij et al. (1997). in all experiments, variations in the environmental conditions were started when the second truss was flowering. carbon dioxide was varied in two experiments (spring and autumn). here, the set point for the co 2 supply was adjusted at 1000 ppm in half of the greenhouse compartments while the other compartments were not supplied with co 2 . each co 2 concentration was treated with two concentrations of the nutrient solution. to this end, the ec was adjusted at 2 and 9 ds m-1, where the higher concentration was achieved by a proportional increase of all nutrients. the realised ec was close to the set points, while the realised co 2 concentration depended on the set points but also on the outside temperature and global radiation. the mean co 2 concentrations during the daytime were 348 and 806 ppm in spring, and 393 and 997 ppm in autumn, respectively. the temperature was varied at three levels in three experiments (spring, summer and autumn). the realised mean daily temperature during the sensitive phase of the fruit ripening (the last 10 days before harvest) were 18.0, 19.9 and 22.0°c in spring, 19.9, 21.5 and 23.7°c in summer and 15.0, 17.6 and 20.3°c in autumn. fruits were harvested when they were red-ripe, and sorted manually * the paper was presented at the 41th meeting of the „deutsche gesellschaft für qualitätsforschung (pflanzliche nahrungsmittel) dgq e.v. into the colour stage 9-10 of the colour screening scale for the tomato (cbt-scale, anonymous, 1992). carotenoid analysis carotenoids (lycopene and ß-carotene) were determined by hplc (krumbein, 1996). the analyses were carried out as double estimations with 12 fruits of the cultivar counter and 30 fruits of the cultivar supersweet per replicate. after homogenisation, 1 g calcium carbonate, 30 g sodium sulphate and 30 ml acetone were added to15 g homogenised tomato, and the samples were homogenised for 2 minutes. the extract was then filtered under suction, and the solid materials were extracted repeatedly with acetone until the resulting filtrate was colourless. the extract was then filtered through a 0.45 mm filter for hplc analyses. the carotenoid composition and content were determined by hplc using a c-18 reversed-phase column lichrosphere 100 (5 µm, 250 x 4 mm; merck) with an isocratic eluent of 75% acetonitrile, 15% methanol and 10% methylene chloride. the analysis was carried out at a flow rate of 1 ml min-1. wavelengths of 470 nm and 455 nm were used for the determination of lycopene and ß-carotene, respectively. contents were quantitatively determined by calibration curves of the related pure standards. the results were converted to 100 g fresh matter (fm). statistical analysis the data were analysed using multifactorial analysis of variance (anova) and fisher’s f-procedure. this was followed by tukey’s hsd procedure, which was carried out separately for each cultivar and each experiment. the significance level in both tests was 0.05. results and discussion effect of co 2 supply and electrical conductivity of the nutrient solution the predominant carotenoid detected in tomato was lycopene followed by ß-carotene. the cultivars varied significantly in their content of lycopene in the two experiments in spring and autumn (tab. 1). the ‘supersweet’ cherry tomato had higher lycopene contents (between 5.5 -8.3 mg (100 g fm)-1) than the conventional round ‘counter’ tomato, which had contents in the range of 4.9 5.8 mg (100 g fm)-1 in the two experiments (figs. 1-2). the contents of ß-carotene varied between 0.54 0.79 mg (100 g fm)-1 and 0.59 0.68 mg (100 g fm)-1 in ‘supersweet’ and ‘counter’, respectively. they were low in comparison with the detected lycopene contents and with ß-carotene contents determined in other vegetables such as spinach (approximately 2 mg (100 g fm)-1) and leafy asian vegetables (2 4 mg (100 g fm)-1) (krumbein et al., 2005). they differed only in autumn with higher contents in the ‘supersweet’ variant. the concentration of lycopene in the tomato skin is approximately three to five times higher than in the whole tomato pulp (shi and le maguar, 2000). the greater skin to volume ratio of the cherry tomato could enhance their lycopene content. tab. 1: analysis of variance for lycopene and ß-carotene in two tomato cultivars grown at two co 2 concentrations and two ec values in spring and autumn. the probability levels of fischer’s f-procedure are given. significant effects are marked by bold numbers. factor lycopene ß-carotene p (spring) p (autumn) p (spring) p (autumn) cultivar 0.035 0.0096 0.39 0.0001 co 2 0.67 0.39 1.00 0.74 ec 0.27 0.72 0.23 0.74 co 2 supply increased and ec significantly diminished the yield of tomato in the spring and autumn experiment (data not shown). figs. 1-2 show the content of lycopene as the main carotenoid in tomato. co 2 supply did not affect the carotenoid content either at ec 2 or ec 9 ds m-1 in both cultivars (tab. 1, figs. 1-2). environmental stress generates oxidative stress in plants, producing free radicals and other derivatives of oxygen. the results of idso and idso (2001) indicated that co 2 supply appears to reduce oxidative stress in plants. for this reason, they concluded that plants growing in co 2 -enriched air generally experience less oxidative stress, so that they need less antioxidant protection, which thus allows them to produce smaller quantities of antioxidants. our results indicate that, under the investigated conditions, no oxidative stress was present. this fact is also supported by the investigations of different ec levels. the high level of 9 ds m-1 had no effect on the lycopene and ßcarotene concentration in comparison to ec 2 (tab. 1, figs. 1-2). contrary to these findings, de pascale et al. (2001) found in salt fig. 1: effect of co 2 supply on lycopene content in cv. counter grown in spring and autumn at ec 2 and ec 9. the values represent the mean of two replicates ± standard deviation. spring autumn ec 2 ec 9 ec 2 ec 9 348 806 348 806 393 997 393 997 0,0 2,0 4,0 6,0 co2 concentration (ppm) m g (1 00 g f m ) -1 carotenoids in tomato 161 grown tomatoes an increase in lycopene with increasing ec level from 0.5 4.4 ds m-1 and a decrease in lycopene with increasing ec level from 4.4 15.7 ds m-1. they explained the increase by stressinduced upregulation of the genes’ encoding for the enzymes involved in the key steps for lycopene biosynthesis. otherwise, rising salinity levels (3, 6.5 and 10 ds m-1) increased lycopene and ß-carotene content in tomato, relating to the fm basis, based on the concentration effect since plants under salinity accumulate less water and have a reduced water uptake (krauss et al., 2006). whether or not ec affects the content of lycopene and ß-carotene depends on the ec levels studied, the cultivars and the growing conditions (dorais, 2006). effect of temperature in the three experiments with different temperature levels the tomato cultivars can be differentiated by their lycopene content (tab. 2). here again, the ‘supersweet’ cherry tomato was characterised by higher lycopene contents than the conventional round ‘counter’ tomato (tab. 2, figs. 3 and 4). ‘supersweet’ also had slightly higher ß-carotene contents than ‘counter’, with values of between 0.31 0.45 mg (100 g fm)-1 and 0.26 0.38 mg (100 g fm) 1 in summer as well as 0.42 0.45 mg (100 g fm)-1 and 0.29 0.33 mg (100 g fm)-1 in autumn, respectively (tab. 2). the content of lycopene was influenced by temperature in spring and autumn (tab. 2). lycopene in the cultivar counter increased significantly from 5.1 mg (100 g fm)-1 at 18°c to 6.5 mg (100 g fm)-1 at 22°c in spring as well as from 2.4 mg (100 g fm)-1 at 15°c and 3.5 mg (100 g fm)-1 at 17.6°c, up to 6.3 mg (100 g fm)-1) at 20.3°c in autumn (fig. 3). in the cultivar supersweet the lycopene content increased significantly from 5.7 mg (100 g fm)-1 at 15°c to 10 mg (100 g fm)-1 at 20.3°c in autumn (fig. 4). the increase of lycopene with temperature in spring was only a tendency. increasing the temperature from 15.0°c to 20.3°c during the fruit ripening stage in autumn stimulated lycopene accumulation 2.6-fold and 1.8-fold in the cultivars counter and supersweet, respectively. increasing the temperature from 18°c to 22°c in spring increased the lycopene content 1.3-fold in ‘counter’. a temperature between 20 and 24°c seems to be optimal for the biosynthesis of lycopene. this is in agreement with robertson et al. (1995), who found that the regulation of lycopene formation in a cell suspension culture of the cherry type variety vfnt showed that lycopene exhibited a broader plateau between 18 26°c for maximum concentration, after which the lycopene-forming activity was sharply reduced at 14°c and completely inhibited at 32 °c. the colour of the tomato fruit develops best when the ambient temperature inside the greenhouse is kept at between 12°c and 21 °c; very low (<10 °c) or very high air temperatures (>30 °c) inhibit the normal fruit ripening and the development of lycopene (dorais et al., 2001). the effect of low temperatures on biosynthesis has also been discussed by dumas et al. (2003). lower temperature regimes (different day/night temperatures) at the ripening stage (variation of day temperature of between 25.6°c and 13.9°c as well as night temperatures of between 17.8°c and 2.8°c) decreased the content of lycopene but not that of ß-carotene. the content of ß-carotene in experiments presented here was only influenced in summer (tab. 2). there was a slight decrease from 0.38 mg (100 g fm)-1 at 19.9°c to 0.26 mg (100 g fm)-1 at 23.7°c and 0.45 mg (100 g fm)-1 at 19.9°c to 0.31 mg (100 g fm)-1 at 23.7°c in the ‘counter’ and ‘supersweet’ variants, respectively. while lycopene biosynthesis is maximal above 20°c, ß-carotene biosynthesis seems to be maximal below 20°c. spring autumn ec 2 ec 9 ec2 ec9 348 806 348 806 393 997 393 997 0,0 2,0 4,0 6,0 8,0 10,0 co2 concentration (ppm) m g (1 00 g f m ) -1 fig. 2: effect of co 2 supply on lycopene content in cv. supersweet grown in spring and autumn at ec 2 and ec 9. the values represent the mean of two replicates ± standard deviation. tab. 2: analysis of variance for lycopene and ß-carotene in two tomato cultivars grown at different temperatures in spring, summer and autumn. the probability levels of fischer’s f-procedure are given. significant effects are marked by bold numbers. factor lycopene ß-carotene p (spring) p (summer) p (autumn) p (spring) p (summer) p (autumn) cultivar 0.00011 0.00000 0.000004 0.14 0.004 0.0004 temperature 0.0027 0.54 0.00002 0.10 0.00077 0.24 162 angelika krumbein, dietmar schwarz, hans-peter kläring spring summer autumn 18.0 19.8 22.0 19.9 21.5 23.7 15.0 17.6 20.3 0,0 2,0 4,0 6,0 8,0 10,0 temperature (°c) m g ( 1 0 0 g f m ) -1 a aa a aa spring summer autumn 18.0 19.8 22.0 19.9 21.5 23.7 15.0 17.6 20.3 0,0 2,0 4,0 6,0 8,0 10,0 temperature (°c) m g ( 1 0 0 g f m ) -1 a ab b a aa acknowledgements this research was supported by the german federation, the federal states of brandenburg and thuringia. we would like to thank a. jankowsky for her carotenoid analyses and u. zentner, a. maikath, a. platalla, and j. lenk for their technical assistance. references anonymous, 1992: kleur-stadia tomaten. central bureau van de tuibouwveilingen in nederland (ed.), 2803 pe gouda. auerswald, h., schwarz, d., kornelson, c., krumbein, a., brückner, b., 1999: sensory analysis, sugar and acid content of tomato at different ec values of the nutrient solution. scientia horticulturae 82, 227-242. clarke, r., armitage, j., 2002: antioxidant vitamins and risk of cardiovascular disease. review of large.scale randomised trials. cardiovasc. drugs ther. 16, 411-415. de kreij, c., voogt, w., van den bos, a.l., baas, r., 1997: voedingsoplossingen voor de teelt van tomaat in gesloten teeltsystemen. proefstation voor bloemisterij en glasgroente, naaldwijk, the netherlands vg2. de pascale, st., maggio, a., fogliano, v., ambrosino, p., ritieni, a., 2001: irrigation with saline water improves carotenoids content and antioxidant activity of tomato. j. hort. sci. biotechnol. 76, 447-453. dorais, m., papadopolus, a.p., gosselin, a., 2001: greenhouse tomato fruit quality: the influence of environmental and cultural factors. horticultural reviews 26, 239-319. dorais, m., 2006: effect of cultural management on tomato fruit health qualities. acta hortic. (in press). dumas, y., dadomo, m., di lucca, g., grolier, p., 2003: effects of environmental factors and agricultural techniques on antioxidant content of tomatoes. j sci. food agric. 83, 369-382. giovannucci, e., 2002: a review of the epidemiologic studies of tomatoes, lycopene, and prostate cancer. exp. biol. med. 227, 852-859. hamauzu, y., chacin, k., ueda, y., 1998: effect of postharvest temperature on the conversion of 14c-mevalonic acid to carotenes in tomato fruit. j. fig. 3: effect of the temperature during ripening (the last 10 days before harvest) on the lycopene content in cv. counter grown in spring, summer and autumn. the values represent the mean of two replicates ± standard deviation. different letters show significant differences within each experiment (level of significance 5%). fig. 4: effect of the temperature during ripening (the last 10 days before harvest) on the lycopene content in cv. supersweet grown in spring, summer and autumn. the values represent the mean of two replicates ± standard deviation. different letters show significant differences within each experiment (level of significance 5%). carotenoids in tomato 163 jpn. soc. hortic. sc. 67, 549-555. idso, s.b., idso, k.e., 2001: effects of atmospheric co 2 supply on plant constituents related to animal and human health. environ. exp. bot. 45, 179-199. krauss, s., schnitzler, w.h., grassmann, j., woitke, m., 2006: the influence of different electrical conductivity values in a simplified recirculating soilles system on inner and outer fruit quality characteristics of tomato. j. agric. food chem. 54, 441-448. krumbein, a., 1996: schnelle hplc-methode zur bestimmung der carotinoide und chlorophylle in brokkoli. deutsche gesellschaft für qualitätsforschung xxxi, 41-44. krumbein, a., schonhof, i., schreiner, m., 2005: composition and contents of phytochemicals (glucosinolates, carotenoids and chlorophylls) and ascorbic acid in selected brassica species (b. juncea, b. rapa subsp. nipposinica var. chinoleifera, b. rapa subsp. chinensis and b. rapa subsp. rapa). j. appl. bot. food quality 79, 168-174. li, y., safi, m., gale, j., volokita, m., novoplansky, a.,1999: response of tomato plants to saline water as affected by carbon dioxide supplementation. i. growth, yield and fruit quality. j. hort. sci. biotechnol. 74, 132-237. nederhoff, e.m., 1994: effects of co 2 concentration on photosynthesis, transpiration and production of greenhouse fruit vegetable crops. phd thesis, wageningen, the netherlands. robertson, g.h., mahoney, n.e., goodman, n., pavlath, a.e., 1995: regulation of lycopene formation in cell suspension culture of vfnt tomato (lycopersicon esculentum) by cpta, growth regulators, sucrose, and temperature. j. exp. bot. 46, 667-673. shi, j., le maguer, m. 2000: lycopene in tomatoes: chemical and physical properties affected by food processing. crit. rev. biotechnol. 20, 293334. sies, h., stahl, w., 1998: lycopene: antioxidant and biological effects and its bioavailability in the human. proc. soc. exp. biol. med. 218, 121124. stahl, w., sies, h., 2005: bioactivity and protective effects of natural carotenoids. rev. biochim. biophys. acta 1740, 101-107. van der plog, by a., heuvelink, e. 2005: influence of sub-optimal temperature on tomato growth and yield: a review. j. hort. sci. biotechnol. 89, 652-659. address of the authors: dr. angelika krumbein, dr. dietmar schwarz, dr. hans-peter kläring institute of vegetable and ornamental crops großbeeren/erfurt e.v., theodorechtermeyer-weg 1, d-14979 großbeeren, germany, corresponding author: krumbein@igzev.de 164 angelika krumbein, dietmar schwarz, hans-peter kläring << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 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<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> >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice vorgabe neu summary influence of uniconazole and different salt concentrations on growth and development of broad bean (vicia faba l.) were investigated. addition of salt to the irrigation water caused significant reduction of plant growth but uniconazole treated plants revealed better biomass. the number of phenolic compounds in v. faba leaf extracts changed with the age of the leaves, and the antioxidative potentials of uniconazole treated plants were slightly lower. the activity of polyphenoloxidase and its activation after plastid membrane desintegration by sds were lower in uniconazole treated plants. further studies with varying sds-concentrations revealed a remarkable difference in membrane disintegration by sds in uniconazole pretreated plants. this finding supports the hypothesis that uniconazole enhanced stress tolerance after uniconazole treatment is based on physiological change in thylakoid membrane stability. introduction uniconazole ((e)(p-chlorophenyl(-4.4-dimethyl-2-(1,2,4-triazol-1yl)-1penten-3yl is a fungitoxic triazole which reveals plant growth regulator properties (mackay et al., 1990; fletcher and hofstra, 1988; cavins et al., 2003). furthermore, uniconazole treated plants develop a broad tolerance against a number of environmental stresses (fletcher and hofstra, 1988) including salt stress (bekheta, 2000) and cold stress (zhou and leul, 1998). the influence of uniconazole on plant metabolism is obviously due to interactions of this triazole compound with various endogenous growth regulators. triazoles in general, but uniconazole in particular, are known to block the biosynthetic way to gibberellin in several plant species at the step from ent-kaurene to ent-kaurenoic acid. this action interferes with the activity of endogenous giberellins. in fact, application of triazoles to rhododendron catawbiense and kalmia latifolia markedly reduced stem elongation (gert, 1995, 1997). the complex nature of plant responses to uniconazole comprise growth responses and morphological modifications as well as biochemical characteristics of the plants, i.e. enhanced chlorophyll content, accumulation of stress related amino acids, and variations in phospholipid composition of biomembranes (zhou and ye, 1996). the general biochemical stress responses of uniconazole treated plants against environmental factors has been compared to the reaction pattern of plants treated with abscisic acid (aba). exogenous treatment of plants with aba rises plant resistance against salinity, ozone, heat, chilling and freezing (e.g. mackay et al., 1990). aba is known to induce a fast stomatal closure in response to water deficit (zeevaat and creelmann, 1988). concomittant with water deficit induced stress the formation of reactive oxygen species (smirnoff, 1993, 1998) may occur, which cause membrane damages and lipid peroxidation. after treatment of plants with 2-aminoethanol or with uniconazole, membranes are transferred into a state which provides better resistance to stress factors (aziz et al., 1998; mascher et al., 2005; zhou and leul, 1998), and even the structural stability of organelles may be enhanced as a consequence of stress related biochemical changes. mascher et al. (2005) described the protective action of choline and other amines against oxidative membrane deterioration in pretreated plants. from the complex protective pattern of uniconazole arises the assumption that triazoles may cause changes of membrane properties. in order to develop a test system for membrane stability of salt stressed plants under uniconazole influence we chose the phenolase activation test with vicia faba. the broad bean is known to contain a tightly membrane bound, latent phenolase in its plastids (swain et al., 1966; angelton and flurkey, 1984). the enzyme is integrated in the thylakoids in a latent state and becomes active after following liberation from the membrane. this liberation from the membrane is slow in physiological buffers but can be enhanced by detergent-driven membrane disintegration (hutcheson et al., 1980). in some plants, especially in mosses, ppo latency is strongly expressed and has been correlated to strong membrane association by lipophilic domains (richter et al., 2005). this publication describes the ppo test system used in v. faba and presents some arguments for the assumption that enhancement of stress tolerance of v. faba caused by uniconazole is due to changes in membrane properties. material and methods seeds broad bean (vicia faba l.) seeds (variety: dicke bohne (goldblume o’lacys’ gmbh, düsseldorf z 020021/05) were soaked in water or uniconazole solution, respectively, at 20 ppm uniconazole for 8 hours at environmental temperature (ca. 18°c). after soaking, the seeds were air-dried on filter paper and were placed in floraton-3® garden soil: sand mixture of 3:1. the seeds were planted in 200 ml pots (2 seeds/ pot) from which they were repotted after 3 weeks into 1l pots. the seeds were covered by 1 cm of soil. irrigation started directly after seedling emergence with sodium chloride containing water with 2000, 4000 ppm of salt and salt free control solution. cultivation was carried out under greenhouse conditions with l:d of 14:10 at an overall range of about 400 µmol photons / m2 x s. the average temperature was 20 to 25°c at day time and 18°c at night period. root, shoot and leaves were harvested and quantified after given growth times, root length was estimated according to tennant (1975), root to shoot relation was used as relative growth parameter. the plants from soaking experiments were cultivated under the same greenhouse conditions as the plants cultivated for spraying treatments. after 28 days of cultivation uniconazole treated plants (seed soaking in 20 ppm aqueous solution) revealed better shoot and root biomass than those of the respective control plants. journal of applied botany and food quality 80, 129 134 (2006) national research centre, plant physiology department, dokki, cairo, egypt1 university of hamburg, ecology and biology of useful plants, biocenter klein flottbek, germany2 uniconazole-induced changes of stress responses of vicia faba: polyphenole oxidase activation pattern serves as an indicator for membrane stability mohamed a.g.a. bekheta1, rasim sahbaz2, reinhard lieberei2 (received may 2, 2006) , plant treatment plants in four developmental leaf stages were sprayed with a 20 ppm uniconazole-solution in water. after regrowing under greenhouse conditions, the plants were taken for chloroplast isolation, for estimation of total phenolics and for the analysis of the composition of phenolic compounds by hplc-standard separation procedure. the morphological features of sprayed plants showed the well-known and described morphological changes after uniconazole treatment: internodial stunting, even in the inflorescence region, and formation of smaller leaves in size. chloroplast isolation the plants can be divided into two groups: a) 1st group: plants produced from soaked seeds in 20 ppm uniconazol and irrigated with different concentrations of salt (nacl) (0, 2000 and 4000 ppm) b) 2nd group: plants sprayed with uniconazol at 20 ppm and irrigated with tap water. chloroplasts were isolated from freshly harvested young leaves of v. faba, ground in icecold posphate buffer 10 mmol/l, ph 6.4, which contained 0.6 mol/l sucrose. x g leaves were ground in 2 x ml of buffer volume. the homogenate was filtered through 4 layers of cheesecloth, and centrifuged at 900 x g for 8 min. the resulting sediment was the fraction called „unwashed chloroplasts“. the sediment was taken up in the original buffer volume, thoroughly resuspended and centrifuged again at 900 x g. the sediment was again taken up in the original buffer volume. the fraction was called „washed chloroplasts“ or c1. polyphenol analyses the quantitative analyses of polyphenolic substances were carried out in a rp-hplc method with detection by a photodiode array detector (pad), 225 to 540 nm, according to tikkanen and julkunen-tiitto (2003). preparation of samples, modified: 0.200 g fresh leaf material was weighed into a 10 ml-centrifugation tube. each sample was extracted three times for 30 s with 5 ml ice-cold methanol using an ultra-thurrax. after each extraction, the mixture was centrifuged for 10 min at 5,000 rpm. after centrifugation the supernatant was collected in a 50 ml round flask. the methanol was completely evaporated in a rotation evaporator at 40°c and 100 mbar. the residues were solubilized in 1.5 ml methanol (lichrosolv) for analysis. an acetonitrile gradient was used, which was composed of elution solutions: a: acetic acid solution, w = 2 % and b: acetonitile/h 2 o lichrosolv/glacial acetic acid, 400+90+10 (v/v/v) gradient parameter: time flow rate percentage of elution solution min ml/min % a % b 0 1.2 90 10 8 1.2 90 10 38 1.1 77 23 50 1.0 60 40 70 1.0 10 90 73 1.0 10 90 78 1.2 90 10 93 1.2 90 10 hplc-conditions: separating column: merck rp 18 (250 mm, ø 4 mm, 5 µm, endcapped) injection volume: 20 µl temperature: column temperature: 26°c detection: pda-detector (waters millipore 996; spectrum 225 nm -540 nm, polyphenols quantified at 280 nm) pump: 2 x knauer model 64 gradient-former knauer programmer model 50 autosampler: merck-hitachi as-2000 quantification: area integration sample estimation of antioxidative potential all samples were tested for antioxidative activity by means of teac method (=trolox® equivalent antioxidative capacity). it refers to a „in vitro“-test based on the abts substance (2.2-azino-di-[3-ethylbenzothiazolin-6-sulfonate]), which was developed by miller et al. (1993). by adding hydrogen peroxide, under catalysis of metmyoglobin, a colour develops by the formation of abts radicals in a time-limited increase at an extinction maximum at 734 nm. antioxidative substances, such as polyphenols, delay the beginning of the staining reaction, i.e. the onset of radical formation. the standard substance trolox®, a water soluble tocopherol derivative, causes also delays in radical formation. the delay produced by the polyphenolic substances from leaf discs of vicia faba material was correlated to the concentration of trolox® which causes the same delay during equivalent test-conditions. the antioxidative capacity of the samples is expressed as mmol/l trolox ® equivalents/cm². for preparation of samples 3 leaf discs (1 disc = 0.785 cm²) were put into a 2 ml flask containing a mixture of acetone/methanol/phosphate buffer (c = 5 mmol, ph 7.4) 3+1+1 (v/v/v), suspended and shaken for 15 min. at 1,400 rpm. the mixture was finally centrifuged at 13,000 rpm for 10 min. 20 µl of the supernatant was used for the test and buffer was added to 1 ml. 600 µl 0.5 mm/l abts was pipetted in a macro cuvette, 70 µl metmyoglobin-solution was added, and 20 µl sample solution followed. after adding 300 µl of a 2 % solution of h 2 o 2 in distilled water, the reaction started and was measured at 734 nm. total phenolic content the determination of the total phenolic content was carried out using folin-ciocalteu-phenolic reagent. for this purpose, 1 ml raw polyphenolic extract was resolved in 2 ml 2.5 % acetic acid. 0.2 ml of this diluted sample was given into a 10 ml graduated flask, 0.8 ml 2.5 % acetic acid, 2.0 ml 20 % na 2 co 3 solution and 0.5 ml folinciocalteu-phenolic reagent were added. the solution was shaken after the adding of each reagent. the flask was filled with distilled water. after mixing, the samples were heated in a water bath of 70°c for 10 min. after cooling down to room temperature, the blue colour of the polyphenols is stable for some hours. the polyphenolic content was measured at 730 nm; epicatechin was used as a reference. chlorophyll estimation chlorophyll a and chlorophyll b content were determined in an 80% acetone extract of the leaves according to lichtenthaler (1987). ppo estimation ppo activity was determined polarographically at 25°c with a ysi oxygen electrode 5331. the air saturated reaction mixture (3 ml) 130 mohamed a.g.a. bekheta, rasim sahbaz, reinhard lieberei, contained 7.5 mmol/l 4-methylcatechol in 67 mmol/l phosphate buffer (lieberei et al.,1981). the sds-activation of broad bean ppo was carried out according to swain et al. (1966). results plants grown from seeds soaked in 20 ppm uniconazole solution revealed a higher biomass production in roots, shoots and leaves than controls (tab. 1). under 2000 ppm salt, control plants were slightly higher in their fresh weight, under higher salt conditions (4000 ppm) growth was impaired. plants grown from uniconazole treated seeds were similar in qualitative stress response, but were higher in root growth compared with the corresponding salt treated plants without uniconazole. the root to shoot ratios under control conditions and in plants under low salt were higher than the in non-treated plants. thus, there is long lasting interference of uniconazole with growth pattern, which lasts at least up to 28 days after soaking. obviously the short uniconazole treatment of seeds causes changes in the plants which are leading to the long lasting differences in their reaction to environmental stress. the spectrum of phenolic compounds in v. faba methanolic leaf extracts changes with the age of the leaves. young leaves contain five groups of peaks, which form a typical qualitative pattern (fig. 1a). during leaf maturation, the number of groups is reduced and the number of peaks in the groups is also lower. uniconazole spraying of leaves leads to less extractable phenolics per leaf weight and to a peak composition in the young leaves (1b) which resembles the peak pattern of mature leaves in untreated plants (1c). a considerable part of the antioxidative potential of leaves is based on the extractable phenolics. in all cases studied, the uniconazole treated plants were lower in their antioxidative compounds, but the differences were not significant. the long lasting unionazole effect seems not to be due to an interference of the triazole treatment with the phenolics metabolism (fig. 2). chloroplast bound (or thylakoid bound) phenolase of v. faba is latent in vivo and thus cannot be detected by oxygen consumption in intact leaves. under standard conditions (7.5 mmol/l 4-methylcathechol) for phenolase quantification, a low activity of about 36 µm o 2 / h x g fw was detectable in control plants and in uniconazole treated plants. addition of sds in a final concentration of 0.1 % w/v gave rise to an immediate strong time dependent acceleration of phenolase activity and after 10 min of incubation, already 95 µmol o 2 / h x g fw were consumed. the activity in the sample stored at 4 °c rose over the whole storage time of more than 24 hrs, after which the activity of phenolase was about 20 times higher than the initial value (fig. 3). higher concentration of sds normally degrade the active phenolases of many plants, and the treatment results in irreversible loss of phenolase enzyme (lieberei et al., 1981; moor and flurkey 1990; kanade et al., 2006). the phenolase of v. faba, in contrast, is remarkably stable in the presence of sds. concentrations of 1.5 % sds lead to a very high activity, which is reached after about 2 hrs of incubation. no losses of activity, which might be the result of enzyme deterioration, are detectable within this time span (fig. 4). tab. 1: influence of salt stress on uniconazole treated vicia faba plants. morphological characteristics of non flowering plants after 4 weeks of culture. samples number leaf-fw stem-fw root-fw nodulation stem-length length of of leaves primary root [g] [g] [g] [cm] [cm] 0-0 7 3,9 4,24 6,12 28 16 0,75 0-2000 8 5,19 5,03 7,6 22 27 0,74 0-4000 7 3,7 3,41 4,84 + 19 18 0,68 20-0 8 5,4 5,26 9,1 + 27 35 0,85 20-2000 8 5,97 6,21 11,33 + 20 27 0,93 20-4000 7 4,57 4,64 6,18 17 20 0,67 r = roots, s = shoots, l = leaves. r s + l a b c fig. 1: phenolic compounds in leaves of vicia faba effect of maturation and uniconazole treatment. a = young leaves, b = uniconazole-treated leaves, c = nature leaves. uniconazole-induced changes of stress responses of vicia faba 131 co ntrol plan tsco ntrol plan tsco ntrol plan tsco ntrol plan ts 0 100 200 300 400 500 600 700 800 h0 untreated sds 0,1% 15 min sds 0,1% 5 hours sds 0,1% 24 hours triton 1% 5 hours sam p le / ti m esam p le / ti m esam p le / ti m esam p le / ti m e [µ m o l o [µ m o l o [µ m o l o [µ m o l o 22 22 /h/h /h/h x g f w ] g f w ] g f w ] g f w ] uni co nazo le treated p lantsuni co nazo le treated p lantsuni co nazo le treated p lantsuni co nazo le treated p lants 0 100 200 300 400 500 600 700 800 sds 0,1% sds 0,1% sds 0,1% triton 1%h0 untreated 15 min 5 hours 24 hours 5 hours sam pl e / ti mesam pl e / ti mesam pl e / ti mesam pl e / ti me [µ m o l o [µ m o l o [µ m o l o [µ m o l o 22 22 /h/h /h/h x g f w ] g f w ] g f w ] g f w ] fig. 3: ppo-activity under influence of uniconazole (uni) (20ppm) and detergents. 0,00 0,50 1,00 1,50 2,00 2,50 3,00 3,50 0 30 60 90 120 150 180 time [min] p p o -a ct vi ti y [m m o l o 2 /h x g f w ] 0,1 % sds 0,5 % sds 1,0 % sds 1,5 % sds fig. 4: time-dependence of ppo-activation of vicia faba by different sdsconcentrations. x this result underlines that v. faba phenolase is set free from the thylakiods by detergents in an active state, which is far more stable than in many other plants. the molecular basis for this enzyme stability remains to be explained. with respect to membrane stability, it is remarkable that after uniconazole treatment the activation process proceeds slower than in control plants (fig. 5), and the enzyme activity per µg of chlorophyll does not reach values of untreated plants. the activation of phenolase in leaf extracts was different in uniconazole treated samples compared to untreated control plants. the treated plants were slower in activation. in order to verify if this behaviour is due to properties of the plastids or the plastid membranes themselves, isolated plastids from a hypertonic extraction procedure were treated with 0.1 % sds directly after isolation. the phenolase activity, expressed in activity per µg chlorophyll is rising in both groups of plastids, but it is easy to be seen that plastids derived from uniconazole treated plants were slower in their activation pattern and remained at a lower activity level than the control plants (fig. 5). obviously the plastid membranes do not undergo such a fast desintegration as the plastids isolated from non-treated plants. when samples of both groups were treated by 1.5 % sds for 12 hrs, their activities finally reached the same values (data not shown). discussion uniconazole treatment leads to polyfactoral changes in plant responses to environmental stress factors. detailed descriptions on reduced electrolyte leakage or lower liberation of malondialdehyde after heat stress have been reported in rape plants (zhou and leul, 1999), the amelioration of nacl stress in peanut seedlings by a host of metabolic changes are known (muthukumarasamy and panneerselvam, 1997). leaves of v. faba, artificially dwarfed by uniconazole-p (fukuda et al., 2001) revealed many microstructural changes, e.g. the size and density of mesophyll cells. typical for all descriptions are a) the polyfactorial changes and b) the indirect action of uniconazole.fig. 2: trolox-equivalents after spraying with uniconazole (20 ppm). 132 mohamed a.g.a. bekheta, rasim sahbaz, reinhard lieberei, the direct interaction of uniconazole with cytokinins and aba are often regarded as the explanation for the widespread action on metabolic factors (flecher and arnold 1986; nishijima et al. 1997; zhou and leul, 1999; henderson et al., 2004). it seems to be more and more evident that the long-lasting effect of short uniconazole treatments is the induced change of metabolism, which obviously causes changes in membrane function and stability. so far, it is not clear, in which way the postulated membrane stabilization may occur. in order to test the impact of uniconazole on the thylakoids, we choose the vicia faba phenolase system. for a long time it is known that v. faba contains a strongly membrane integrated phenolase (swain et al., 1966), which turns into an active enzyme after thylakoid disintegration by sds. the enzyme activation has been used as a test parameter for the overall stability of the thylakoid. it could easily be shown that uniconazole treatment changed the membrane properties in that way that desintegration and concommittant phenolase activation were retarded compared to control plants. there are both a concentration dependent and a time dependent effect on the activation pattern. the phenolase test allows us to start a series of experiments to get deeper in the analyses of the molecular interaction of uniconazole with membrane physiology. recently, mascher et al. (2005) reported on the important role and stress-physiological significance of 2-aminoethanole in drought stressed barley. he used sublethal doses of the stressor paraquat as a causal agent of oxidative stress and combined this treatment with the application of 2-aminoethanol. the pretreatment with the 2aminoethanol protected the sensitive chloroplast thylakoids. the electron microscopic study revealed an expressed membrane structure protection in plants pretreated with 2-aminoethanol. according to this finding, general studies on the interaction of triazoles like uniconazole with biogenic amines and their stressphysiological significance should be intensified. they might be helpful to understand a long series of experiments which have been published in the working group of bergmann (bergmann et al., 1994 a-b,; leinhos and bergmann, 1995), who studies enhanced productivity of cereals after application of biological amines and triazoles. acknowledgements this study was supported by the dfg (deutsche forschungsgemeinschaft) german research foundation, 445agy-112/16/04. the technical supports by v. noelting, t. tumforde, d. böhm and k. puttfarken are highly appreciated. references angelton, e.a., flurkey, w.h., 1984: activation and alteration of plant and fungal polyphenoloxidases iso-enzymes in sodium dodecyl sulfate electrophoresis. phytochemistry 23, 2723-2725. aziz, a., martin-tanguy, j., larher. f., 1998: stress-induced changes in polyamine and tyramine levels can regulate proline accumulation in tomato leaf discs treated with sodium chloride. physiol. plant. 104, 195202. bekheta, m.a.e.a., 2000: physiological studies on the effect of uniconazole on the growth and productivity of vicia faba plants grown under different levels of salinity, ph.d. thesis, faculty of science, cairo university. bergmann, h., leinhos, v., machelett, b., 1994a: increase of stress resistance in crop plants by using phenolic compounds. acta horticult. 381, 390-397. bergmann, h., machelett, b., leinhos, v., 1994b: effect of natural amino alcohols on the yield of essentiell amino acids and the amino acid pattern in stressed barley. amino acids 7, 327-331. cavins, t.j., greer, l., gibson, j.l., whipker, b.e., dole, j.m., 2003: response of marguerite daisy (argyranthemum frutescens) „comet pink“ to plant growth regulators. quarterly reports on plant growth regulation and activities of the pgrsa 31, 2-7. fig. 5: ppo-activity and activitation after treatment with 0.1 % sds. 0 100 200 300 400 500 600 700 800 900 1000 0 min 10 m in 30 min 60 m in 18 h 24 h 36 h 48 h 0 min 10 min 30 min 60 min 18 h 24 h 36 h 48 h ti m e aft er sds [µ m o l o 2 /h x µ g c h l] w i t h o u t un i c o n azo le w i t h un i c o n azo l e x uniconazole-induced changes of stress responses of vicia faba 133 fletcher, r.a., arnold, v., 1986: stimulation of cytokinins and chlorophyll synthesis in cucumber cotyledons by triadimefon. physiol. plant. 66, 197201. fletcher, r.a., hofstra, g., 1988: triazoles as potential plant protectans. in: berg, d., plenpel, m. (eds.), sterol biosynthesis inhibitors: pharmaceutical and agrochemical aspects, 321-331. ellis harwood ltd., cambridge uk. fletcher, r.a., hofstra, g., gao, j.-g., 1996: comparative fungitoxic and plant growth regulating properties of triazole derivatives. plant cell physiol. 27, 367-371. fukuta, n., arai, m., yukawa, t., matsumara, o., 2001: effect of dwarfing induced by uniconazole-p on snow tolerance of the faba bean (vica faba l.). plant prod. sci. 4, 189-195. gert, m.p.n., 1995: paclobutrazole or uniconazole applied in the previous season promotes a lowering of field grown rhododendron and kalmia. j. plant growth. regul. 14, 205-210. gert, m.p.n., 1997: persistence of triazole growth retardants on stem elongation of rhododendron and kalmia. j. plant growth regul. 16, 197-203. hiron, r.w.p., wright, s.t.c., 1973: the role of endogenous abscisic acid in the response of plants to stress. j. exp. bot. 81, 769-774. henderson, j. h., li, h.c., rider, s.d., mordhorst, a.p., severson, r.j., cheng, j.c., robey, j., sung, z.r., de vries, s.c, ogas, j., 2004: pickle acts throughout the plant to repress expression of embryonic traits and may play a role in gibberellin-dependent responses. plant physiol. 134, 995-1005. hutcheson, s.w., buchanan, b.b., montalbini, p., 1980: polyphenol oxidation by vicia faba chloroplast membranes. plant physiol. 66, 11501154. kanade, r.s., paul, b., appu rao, a.g., gowda, l.r., 2006: the conformational state of polyphenol oxidase from field bean (dolichos lablab) upon sodium dodecyl sulphate and acid-ph activation. biochem. j. 395, 551-562. leinhos, v., bergmann, h., 1995: changes in yield, lignin content and protein pattern of barley (hordeum vulgare cv. alexis) induced by drought stress. j. appl. bot. 69, 206-210. lichtenthaler, h.k., 1987: chlorophylls and carotenoids – pigments of photosynthetic membranes. methods enzymol. 148, 350-382. lieberei, r., biehl, b., voigt, j., 1981: serological studies on phenolase from spinach chloroplasts. phytochemistry 20, 2109-2116. mackay, c.e., hall, j.c., hofstra, g., fletcher, r.a., 1990: uniconazoleinduced changes in abscisic acid, total amino acids, and proline in phaseolus vulgaris. pesticide biochem. physiol. 37, 74-82. mascher, r., nagy, e., lippmann, b., hörnlein, s., fischer, s., scheiding, w., neagoe, a., bergmann, h., 2005: improvement of tolerance to paraquat and drought in barley (hordeum vulgare l.) by exogenous 2aminoethanol: effects on superoxide dismutase activity and chloroplast ultrastructure. plant sci. 168, 691-698. miller, n.j., rice-evans, c., davies, m.j., gopinathan, v., milner, a., 1993: a novel method for measuring antioxidant capacity and its application to monitoring the antioxidant status in premature neonates. clin. sol. 84, 407-412. moor, b.m., flurkey, w.h., 1990: sodium dodecyl activation of a plant polyphenol oxidase. j.biol.chem. 265, 4982-4988. muthukumarasamy, m., panneerselvam, r., 1997: amelioration of nacl stress by triadimefon in peanut seedlings. plant growth regulation 22, 157-162. nishijima, t., katsura1, n., koshioka, m., yamazaki, h., mander l.n., 1997: effects of uniconazole and ga3 on cold-induced stem elongation and flowering of raphanus sativus l. plant growth regulation 21, 207214. richter, h., lieberei, r., von schwartzenberg, k., 2005: identification and characterisation of a bryophyte polyphenol oxidase encoding gene from physcomitrella patens. plant biol. 7, 283-291. smirnoff, n., 1993: the role of active oxygen response to water deficit and desiccation. new phytol. 125, 27-58. smirnoff, n., 1998: plant resistance to environmental stress. curr. opin. biotechnol. 9, 214-219. swain, t., mapson, l.w., robb, d.a., 1966: activation of vicia faba (l.) tyrosinase as effected by denaturing agents. phytochemistry 5, 469-482. tennant, d., 1975: a test of a modified line intersect method of estimating root length. j. ecol. 63, 995-1001. tikkanen, o.p., julkunen-tiitto, r., 2003: phenological variation as protection against defoliating insects: the case of quercus robur and operophthera brumata. oecologia 138, 244-251. zeevaat, j.a.d., creelmann, r.a., 1988: metabolism and physiology of abscicic acid. ann. rev. plant physiol. 39, 439-473. zhou, w.j., ye, q.f., 1996: physiological and yield effects of uniconazole on winter rape (brassica napus l.). j. plant growth regul. 15, 69-73. zhou, w.j., leul, m., 1998: uniconazole-induced alleviation of freezing injury in relation to changes in hormonal balance, enzyme activities and lipid peroxidation in winter rape. plant growth regulation 26, 41-47. zhou, w. j., leul, m., 1999: uniconazole induced tolerance of rape plants to heat stress in relation to changes in hormonal levels, enzyme activities and lipid peroxidation. plant growth regulation. 27, 99-104. addresses of the authors: dr. mohamed a.g.a. bekheta,national research centre, plant physiology department, 33, el-tahrir st.,dokki, cairo, egypt. dipl.-biol. rasim sahbaz, prof. dr. reinhard lieberei, biocenter klein flottbek, deparment of ecology and biology of useful plants, ohnhorststrasse 18, d-22609 hamburg, germany , 134 mohamed a.g.a. bekheta, rasim sahbaz, reinhard lieberei, << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false /maxsubsetpct 100 /optimize true /opm 1 /parsedsccomments true 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<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> >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice vorgabe neu journal of applied botany and food quality 80, 93 99 (2006) research institute geisenheim, 1department of wine analysis and beverage research, 2department of vegetable crops, germany identification and quantification of carotenoids in pumpkin cultivars (cucurbita maxima l.) and their juices by liquid chromatography with ultraviolet-diode array detection 1mirjam kreck, 1petra kürbel, 1michael ludwig, 2peter j. paschold, 1helmut dietrich (received april 3, 2006) summary the carotenoid pigments of different cultivars of cucurbita maxima l. (pumpkin) were investigated in pulp, peel and genuine juice by means of hplc. in dependence of the variety different concentrations and distributions of α-carotene, β-carotene, violaxanthin, neoxanthin, all-trans-lutein, zeaxanthin, lutheoxanthin and the isomers 9-cis-βcarotene and 13-cis-β-carotene were obtained. the total carotenoid content (expressed as dry weight) of the pumpkin peel depends on the variety with 12 mg/kg for „butternut“ up to 1751 mg/kg for „rouge“, whereas total carotenoid contents in the pulp differed from 17 mg/kg for „baby bear“ to 683 mg/kg for „rouge“. the variety „hokkaido“ showed high total carotenoid contents in pulp (218 mg/ kg) and peel (1048 mg/kg), whereas for „butternut“ minor concentrations in pulp (44 mg/kg) and peel (12 mg/kg) were detected. the red and orange coloured cucurbita maxima l. varieties are valuable sources of carotenoids among vegetables or vegetable juices. introduction recent research is focused on the protective properties of fruits and vegetables against diseases, such as cancer or coronary heart disease. this is due to their high content of antioxidant vitamins, such as vitamins c and e, phenolic compounds, and not at least carotenoids (burns et al., 2003). new products are being produced in this line with juice mixtures that provide increased quality (nutritive value, colour, etc.), such as high carotenoid content. carotenoids are known for a wide range of important and welldocumented biological activities. they act as potent antioxidants, as well as free radical scavengers (grassmann et al., 2002) modulate the pathogenesis of cancers (van poppel et al., 1995; giuvannucci et al., 1999) as well as coronary heart disease (krichevsky et al., 1999). various carotenoids, including α-carotene, β-carotene, and β-cryptoxanthin, possess provitamin a activity by being transformed into retinal by mammals. due to the ability of capturing free oxygen and blue light in the retina, the xanthophylls lutein and zeaxanthin are also known to provide protection against macular degeneration connected with aging (landrum et al., 2001). at present, there is no officially recommended dietary intake for carotenoids. the recommended dietary intake for vitamin a is about 1 mg/d retinal equivalent for german speaking countries and the dietary reference intake for vitamin a in north america is up to 3 mg/d (food and nutrition board, 2001). the red and orange coloured pumpkins cucurbita maxima l. are valuable sources of carotenoids among vegetables or vegetable juices, especially those varieties with orange pulp. hermann (1992) presented total carotenoid contents in cucurbita maxima l. between 1.4 and 7.6 mg/100 g fresh weight. murcovic et al. (2002) focused their research on the quantification of α-carotene, β-carotene and lutein on different varieties of the species c. pepo l., c. maxima l., and c. moschata l.. the content of the carotenoids ranged from 0.06 to 7.4 mg/100 g for β-carotene, from 0 to 7.5 mg/100 g for α-carotene and from 0 to 17 mg/100 g for lutein. hidaka et al. (1987) and arima et al. (1988) found substantial differences in the carotenoid profile of several cucurbita species as well. the purpose of this study was to investigate differences of carotenoid content and composition in pulp and peel in seven different orange or red coloured varieties of cucurbita maxima (var. „muscat“, „bischofsmütze“, „baby bear“, „butternut“, „rouge“, „neon“, „hokkaido“) grown in germany. therefore, hplc analysis was conducted in order to evaluate their carotenoid content. with regard to juice processing, tree varieties („muscat“, „rouge“, „neon“) were processed to genuine juices in a half technical scale. carotenoid contents and distributions were analysed in the genuine juices and pomace extracts. additionally chemical parameters like content of extract, sugar, minerals, total phenols as well as antioxidant capacity were investigated. materials and methods chemicals. methanol, tert-butyl methyl ether (mtbe), calcium carbonate and anhydrous sodium sulfate were purchased from sigma aldrich gmbh (taufkirchen, germany). acetone and hexane were purchased from merck (darmstadt, germany). high-purity water was prepared using a milli-q 185 plus water purification system (millipore, eschborn, germany). trans-β-apo-8´-carotenal, β-carotene, all-trans-lutein and lycopene were purchased from fluka (buchs, germany) and zeaxanthin from roth (karlsruhe, germany). preparation of samples. cucurbita maxima l. cultivars (var. „muscat“, „bischofsmütze“, „baby bear“, „butternut“, „rouge“, „neon“, „hokkaido“), cultivated at the department of vegetable crops of the research institute geisenheim, were manually peeled. peel and pulp were cut into small cubes and freeze dried (model p20-b, fa. piatowski, münchen). sample extraction was conducted according to marx et al. (2000). in dependence of the expected concentration an aliquot of the freeze dried material was mixed with solid calcium carbonate, sodium sulfate and internal standard transβ-apo-8´-carotenal (to guarantee that there where no losses of carotenoids during extraction procedure) and homogenized by an ultra turax (model t50, janke & kunkel ika labortechnik) for 2 min. aliquots were immediately extracted with acetone/hexane (1:1; v/v) until extracts were colourless (total volume: 100 ml). the organic layer was washed with water, dried with anhydrous sodium sulfate, filtered through a folded filter and evaporated to dryness in vacuo (200 mbar) at 25 °c. the dry residue was redissolved in tert-butyl methyl ether/methanol/water (90:6:4; v/v/v; 2 ml) and measured by means of hplc. all procedures were done under dim light, respectively with bay-coloured glassware. for juice and pomace extract preparation, 10 ml were extracted with acetone/hexane (1:1; v/v) until the water phases were colourless (total volume: 50 ml). following preparation steps were analogous to the preparation of pulp and peel. high-performance liquid chromatography. the chromatographic system consisted of a merck-hitachi pump model l-6200 (intelligent pump) with a gradient former and a photodiode array detector (l7450, la chrom, fa. merck). column was 250 x 4.6 mm i.d. ymc c30, 5 µm (ymc, wilmington, usa). the eluent was 81% methanol, 15% tert-butyl methyl ether, 4% water (v/v) (eluent a) and 90% tert-butyl methyl ether, 6% methanol, 4% water (v/v) (eluent b). the linear gradient programme was 100% a to 44% a / 56% b in 50 min. the flow rate was 1.0 ml/min, column temperature was set at 20 °c. identification of peaks. carotenoids were identified according to their chromatographic behaviour (retention time) on hplc and uvvis absorption spectra. by comparing both, their retention times and absorption spectra with those of obtainable authentic carotenoids and published data, respectively, we were able to identify their property. photodiode array measurements of spectral properties for the individual peaks (from 300 to 500 nm) were determined at the upslope, apex and downslope. quantification. the quantitative determination of the total carotenoid contents of pumpkin peel, pulp and juice was performed by uv-vis (davies et al.,1976). the chromatogrammes were evaluated quantitatively by relating the areas of the individual carotenoids and external calibrating curves of β-carotene and all-trans-lutein. to avoid cis/trans isomerization and epoxide-furanoid oxide rearrangement, great care was taken during the carotenoid isolation procedure. the individual carotenoid content and total carotenoid contents in pulp and peel were expressed on the basis of the vegetable dry weight. the values given in tab. 1 and 2 represent means of three independent determinations. chemical analysis. the chemical analyses were done according to the international fruit juice union (ifu) and the official collection of examination methods §35 lmbg, respectively. the soluble dry substance of the individual juice (°brix) was analysed via refraction. glucose, fructose, sucrose and the organic acids (malic acid, lactic acid) were analysed enzymatically. the volatile acid was analysed after distillation with 0.01 mol/l naoh, the ph-value and total acid content were done potentiometrically (tanner, 1979). the minerals copper, iron, zinc, sodium, calcium, potassium and magnesium were analysed via aas. total phenols were analysed at a wavelength of 720 nm (folin-ciocalteau method, (tanner, 1979)). the antioxidative capacity (teac) of the water soluble antioxidants was measured at 734 nm (uv-vis) (miller et al., 1993). fig. 1: processing technology of pumpkin juice in a half technical scale. manual pre-disintegration one step grinding (seepex-pump®) in line homogenisation (supraton®) spiral flow 90 °c, 10 min ⎝ 50 °c enzyme setting, 60 min (cellulase 100 ml/t, pectinase 100 ml/t) pomace decanter separation a juice 200 mg/l ascorbic acid 3 g/l citric acid pomace extraction 2 parts water : 1 part pomace spiral flow 50 °c filling, pasteurisation 121 °c enzyme setting, 60 min (cellulase100 ml/t, pectinase 100 ml/t) decanter separation pomace extract (b – juice) 200 mg/l ascorbic acid 3 g/l citric acid ➔ 50 °c 94 mirjam kreck, petra kürbel, michael ludwig, peter j. paschold, helmut dietrich juice processing. genuine pumpkin juices were produced in half technical scale with lots of 150 kg of three pumpkin varieties separately („rouge“, „neon“, „muscat“) (fig. 1). the processing consisted of washing, manual pre-disintegration, one step grinding with a seepex-pump®, in line homogenisation (supraton®) for a fine dispersion of the pumpkin mash, mash heating for enzyme deactivating over a spiral flow (90 °c), cooling over a spiral flow to 50 °c and immediate enzyme setting with 100 g/t cellulase and pectinase (erbslöh, geisenheim) for 60 minutes. subsequently juice and pomace were separated with a decanter (flottweg z23), after adding 3 g/l citric acid and 200 mg ascorbic acid, juices were bottled and pasteurised. besides juice production (a-juice) a pomace extraction was made. therefore, water (2:1) and enzymes (100 g/t pectinase and cellulase, fa. erbslöh, geisenheim) were added at 50 °c for 60 minutes, followed by decanter separation. then, 3 g/l citric acid and 200 mg/l ascorbic acid were added, the pomace extracts were filled and pasteurised in bottles. results and discussion carotenoids in pumpkin peel and pulp the quantitative results of all cultivars investigated for pumpkin peel are presented in tab. 1, the results for pumpkin pulp in tab. 2. the values given represent means of three independent determinations. thus, the standard deviation reflects the native variation of the carotenoid content of the samples. the wide variation observed in some cases, especially if considering very low carotenoid concentrations, may be the result of the small number of samples, the stage of ripeness and the weather conditions during growth. tab. 1 and 2 reveal the complexity of the carotenoid composition in cucurbita maxima l. cultivars. distinct differences could be observed in the different cucurbita varieties as well as pulp and peel of the same variety. the c. maxima l. var. „rouge“ was the richest in carotenoids in peel (mean 1751 mg/kg dry weight) and pulp (mean 683 mg/kg dry weight). also in the peel of „baby bear“ (mean 1070 mg/kg) and „hokkaido“ (mean 1048 mg/kg) high carotenoid concentrations were detectable. „bischofsmütze“ (mean 518 mg/kg), „muscat“ (mean 570 mg/kg) and „hokkaido“ (218 mg/kg) showed remarkable concentrations in pulp. „muscat“ and „butternut“ had means of only 135 and 12 mg/kg dry weight in the peel, respectively. for „butternut“ and „baby bear“ only 44 mg/kg and 17 mg/kg total carotenoids in the pulp could be detected. the structures of the identified carotenoids are given in fig. 2. only one carotenoid was found in all pulp and peel samples analysed, namely β-carotene. in the pumpkin peel β-carotene was the principle carotenoid in „muscat“ (61 mg/kg), „baby bear“ (403 mg/ kg), „butternut“ (8 mg/kg) and „neon“ (185 mg/kg). in the pulp of „muscat“ (263 mg/kg), „baby bear“ (17 mg/kg) and „butternut“ (22 mg/kg) it was the principle carotenoid as well, but not in „neon“. lutein was the major carotenoid in the pulp of „neon“ and „rouge“ with 115 mg/kg and 146 mg/kg respectively and the second major carotenoid in the peel of „bischofsmütze“ (69 mg/kg), „baby bear“ (376 mg/kg), „rouge“ (253 mg/kg) and „neon“ (76 mg/kg). lutein was not detected in the peel of „butternut“ and in the pulp of „muscat“, „baby bear“ and „butternut“. α-carotene appeared to be the second major carotenoid in the peel and pulp of „butternut“ (4 mg/kg; 22 mg/kg), but was neither detected in the peel of „muscat“, „bischofsmütze“, „rouge“, „neon“, „hokkaido“, nor in the pulp of „muscat“, „baby bear“, „rouge“, „neon“ and „hokkaido“. the xanthophyll α-cryptoxanthin was found in major concentrations in the peel of „bischofsmütze“ (173 mg/kg), „rouge“ (542 mg/kg) and „hokkaido“ (489 mg/kg) and in the pulp of „bischofsmütze“ (178 mg/kg), „rouge“ (98 mg/kg) and „hokkaido“ (99 mg/kg), but was not detected in the other varieties. exclusively in the pulp and peel of the varieties „bischofsmütze“, „rouge“ and „hokkaido“ also β-cryptoxanthin could be detected. high concentrations were found in the peel of „hokkaido“ (445 mg/kg) and „rouge“ (289 mg/kg). tab. 1: carotenoid content [mg/kg dry weight] of cucurbita maxima peel of different varieties. the given values represent means ± standard deviations (sd) of three independent determinations. *: unidentified carotenoid, λ max in eluent b nd: not detected carotenoids in pumpkin peel [mg/kg dry weight] ret. time pigment muskat bischofsmütze baby bear butternut rouge neon hokkaido 11.6 neoxanthin nd 5 ± 3 nd nd 108 ± 13 nd nd 12.1 violaxanthin nd 5 ± 2 nd nd 108 ± 2 nd nd 15.0 lutheoxanthin 19 ± 1 55 ± 5 218 ± 15 nd 199 ± 10 nd nd 15.4 lutein 12 ± 1 69 ± 9 376 ± 37 nd 253 ± 2 76 ± 7 89 ± 5 17.4 * λmax: 462 nm nd nd nd nd 54 ± 1 nd nd 17.8 zeaxanthin nd nd nd nd 72 ± 8 nd nd 32.6 13-cis-β-carotene nd nd 33 ± 8 nd nd nd nd 34.9 α-carotene nd nd 40 ± 7 4 ± 1 nd nd nd 35.1 * λmax: 447 nm 38 ± 2 nd nd nd nd nd nd 38.7 α-crypthoxanthin nd 173 ± 3 nd nd 542 ± 7 nd 489 ± 4 39.5 β-carotene 61 ± 2 10 ± 1 403 ± 23 8 ± 2 36 ± 13 184 ± 12 25 ± 3 40.4 9-cis-β-carotene 5 ± 2 nd nd nd 54 ± 6 325 ± 5 nd 44.1 β-crypthoxanthin nd 30 ± 1 nd nd 289 ± 4 nd 445 ± 6 46.1 * λmax: 450 nm nd nd 0 nd 36 ± 18 nd nd total carotenoid [mg/kg dry weight] 135 347 1070 12 1751 293 1048 total carotenoid [mg/kg fresh weight] 20 46 206 2 195 36 148 carotenoids in pulp, peel and juice of cucurbita maxima l. 95 several epoxycarotenoids were also encountered, namely neoxanthin, violaxanthin and lutheoxanthin in the peel and pulp of „rouge“ and in the peel of „bischofsmütze“. additionally lutheoxanthin was detected in the peel of „muscat“ (19 mg/kg) and „baby bear“ (218 mg/kg). in the varieties „rouge“ and „bischofsmütze“ two unidentified carotenoids were also detected, presenting absorption spectra in tert-butyl methyl ether with a single broad peak at 462 nm and 447 nm, respectively. however, the extend of variation demonstrated by the cucurbita maxima l. varieties analysed in this study is very capacious. the discrepancy could be due to the long period of pumpkin harvest. in contrast to other vegetables, pumpkins could be kept for weeks, but biochemical processes continue during storage (arima et al., 1988). initial products of biodegradation, such as cisand epoxycarotenoids were thus present in fluctuating, low concentrations. vitamin a value a comparison of the vitamin a values of pumpkin peels and pulps and additionally in the whole vegetables are given in tab. 3. the values are calculated from the concentrations of vitamin a-active carotenoids in re (retinal equivalent) per 100 g fresh weight according to arima et al. (1988). the vitamin a value of pumpkin varieties depend mainly on the content of β-carotene (100% activity; 0.6 µg β-carotene is equivalent to 0.1 re), α-carotene (50% activity) and β-cryptoxanthin (50% activity). „hokkaido“ appeared to be the best source of provitamin a with average concentrations of 425 re/ 100g in the pulp, 580 re/100g in the peel and 331 re/100g relating to the whole vegetable. for the variety „muscat“ also high re values can be calculated with 359 re/100 g in pulp, 152 re/100g in peel and 315 re/100g calculated for the whole vegetable. the varieties „bischofsmütze“, „butternut“ and „neon“ were low in vitamin a value, presenting averages of 93, 65 and 88 re per 100 g, respectively. conspicuous is the high vitamin a value in the peel of „baby bear“ (1356 re/100g) which depends on a high β-carotene concentration. in contrast to the values in the peel, only re values of 32 re/100g in the pulp and, therefore 159 re/100g can be determined for the whole vegetable. pumpkin juice with regard to pumpkin juice processing, three varieties („muscat“, „rouge“, „neon“) were processed in a half technical scale to genuine juices. especially high amounts of carotenes in juice are preferable. it is well known that during juice processing a big part of secondary plant metabolites get lost in the pomace. therefore the pumpkin pomace was extracted after enzyme setting and addition of water (1:2, w/w) additionally. this extract leads to a pomace extract rich in carotenes, which can be used as natural supplement for food industry. the results of the carotenoid content and carotenoid distributions of the genuine juices and pomace extracts are given in tab. 4. the juice and pomace extract of the variety „muscat“ were rich in total carotenoid content in juice (19.7 mg/l) and in pomace extract (34.0 mg/l). for the varieties „rouge“ and „neon“ only minor carotenoid contents were investigated in the juices (3.7 mg/l and 3.8 mg/l). therefore considerable concentrations could be transferred from the pomace into the pomace extract (11.0 mg/l „rouge“ and 14.1 mg/l „neon“). in all juices and pomace extracts β-carotene appeared to be the main carotenoid. in the variety „rouge“ αand β-cryptoxanthin were detectable additionally in juice and particularly in pomace extract as a result of high concentrations in the peel. lutein appeared to be detectable in the juice and pomace extracts of „rouge“ and „neon“, but not in the juice of „muscat“. this confirms the results of the carotenoid analyses in pulp and peel, due to the fact that there were no or only minor concentrations of lutein detectable. additionally to the carotenoid analyses, basic juice parameters were investigated in the genuine juices and pomace extracts. results of the juice and pomace extract analyses are given in tab. 5. sugar contents varied in dependence of the variety between 8.4 -10.3 g/l tab. 2: carotenoid content [mg/kg dry weight] of cucurbita maxima pulp of different varieties. the given values represent means ± standard deviations (sd) of three independent determinations. *: unidentified carotenoid, λ max in eluent b nd: not detected carotenoids in pumpkin pulp [mg/kg dry weight] ret. time pigment muskat bischofsmütze baby bear butternut rouge neon hokkaido 11.6 neoxanthin nd nd nd nd 49 ± 1 nd nd 12.1 violaxanthin nd nd nd nd 49 ± 4 nd nd 15.0 lutheoxanthin nd 50 ± 03 nd nd 118 ± 2 nd nd 15.4 lutein nd 50 ± 6 nd nd 146 ± 6 115 ± 8 20 ± 3 17.4 * λmax: 462 nm nd 39 ± 1 nd nd 7 ± 3 nd nd 17.8 zeaxanthin nd nd nd nd 7 ± 2 nd nd 32.6 13-cis-β-carotene 53 ± 2 nd nd nd nd nd nd 34.9 α-carotene nd 6 ± 1 nd 22 ± 1 nd nd nd 35.1 * λmax: 447 nm 254 ± 18 nd nd nd nd nd nd 38.7 α-crypthoxanthin nd 178 ± 3 nd nd 98 ± 2 nd 99 ± 2 39.5 β-carotene 263 ± 16 89 ± 4 17 ± 1 22 ± 2 132 ± 5 71 ± 6 59 ± 3 40.4 9-cis-β-carotene nd 56 ± 1 nd nd nd nd nd 44.1 β-crypthoxanthin nd 28 ± 3 nd nd 63 ± 3 nd 40 ± 3 46.1 * λmax: 450 nm nd 22 ± 1 nd nd 14 ± 1 nd nd total carotenoid [mg/kg dry weight] 570 518 17 44 683 186 218 total carotenoid [mg/kg fresh weight] 47 38 2 6 36 13 70 96 mirjam kreck, petra kürbel, michael ludwig, peter j. paschold, helmut dietrich fig. 2: structures of carotenoids detected in cucurbita maxima l. varieties; α-carotene; β-carotene; α-cryptoxanthin; β-cryptoxanthin; lutein; lutheoxanthin; neoxanthin; violaxanthin; zeaxanthin. carotenoid r1 r2 α-carotene β-carotene α-cryptoxanthin β-cryptoxanthin lutein lutheoxanthin neoxanthin violaxanthin zeaxanthin r1 r2 9 10 15 15´ 10´ 9´ ho ho ho oh ho o o oh c ho oh oh o ho o oh o ho oh glucose, 9.5 -15.9 g/l fructose and 2.3-17.6 g/l saccharose in the juices and 3.0 4.4 g/l glucose, 3.4 5.9 g/l fructose and 0.9 3.7 g/l saccharose in pomace extracts. the microbiological parameters volatile acid and lactic acid were inconspicuous in all juices and pomace extracts. ash contents were comparable in all juices (6.54 g/l6.88 g/l) and pomace extracts (2.28 mg/l 3.26 mg/l). the mineral contents in the pumpkin juices were assimilably high. potassium concentration ranged between 3682 mg/l („muscat“) and 4121 mg/l („rouge“), calcium contents laid between 156 mg/l („muscat“) and 293 mg/l („rouge“) and magnesium underlay variations between 127 mg/l („rouge“) and 186 mg/l („neon“). the total phenol contents, analysed according to the folin-method with catechin as standard, varied between 258 mg/l („muscat“) and 323 mg/l („neon“) (total phenol contents were not corrected in respect of ascorbic acid). the water soluble antioxidative capacity was determined from 2.1 mmol/l trolox („muscat“) up to 2.7 mmol/ l trolox („neon“). it has to be stressed that the values are comparable to the antioxidative capacity of carrot juice. carotenoids in pulp, peel and juice of cucurbita maxima l. 97 tab. 3: comparison of vitamin a values of different pumpkin varieties in pulp, peel and in the whole vegetable. vitamin a value variety provitamin re per 100 g fresh weight pulp peel whole vegetable cucurbita maxima var. muscat β-carotene 359 152 315 cucurbita maxima var. bischofsmütze α-carotene, β-carotene, β-cryptoxanthin 130 56 93 cucurbita maxima var. baby bear α-carotene, β-carotene 32 1356 159 cucurbita maxima var. butternut α-carotene, β-carotene 74 28 65 cucurbita maxima var. rouge α-carotene, β-carotene, β-cryptoxanthin 144 334 130 cucurbita maxima var. neon β-carotene 84 379 88 cucurbita maxima var. hokkaido β-carotene, β-cryptoxanthin 425 580 331 tab. 4: carotenoid content mg/l of cucurbita maxima l. juice and pomace extract of different varieties. the given values represent means ± standard deviations (sd) of three independent determinations. carotenoids in pumpkin juice and pomace extract [mg/l] 8° brix muscat rouge neon ret. time pigment muscat juice pomace extract rouge juice pomace extract neon juice pomace extract 15.4 lutein nd nd 0.6 ± 0.2 1.3 ± 0.2 0.7 ± 0.1 5.8 ± 0.2 35.1 * λmax: 447 nm 9.1 ± 0.5 16.4 ± 0.7 nd nd nd nd 38.7 α-crypthoxanthin nd nd 0.6 ± 0.1 2.6 ± 0.2 nd nd 39.5 β-carotene 10.6 ± 0.8 17.6 ± 0.2 1.9 ± 0.3 5.2 ± 0.4 3.1 ± 0.3 8.3 ± 0.6 44.1 β-crypthoxanthin nd nd 0.6 ± 0.2 1.9 ± 0.3 nd nd total carotenoids 19.7 34.0 3.7 11.0 3.8 14.1 *: unidentified carotenoid, λ max in eluent b nd: not detected conclusion the study was focused on carotenoids in peel, pulp and juices of different pumpkin cultivars grown in germany. pumpkin is not a very common food but its consumption increased during the last years. this study shows that pumpkin can contribute significantly to the uptake of provitamin a and carotenoids with special physiological functions, especially lutein. with regard to a rapidly growing market of functional foods worldwide, pumpkin may be considered as an interesting and valuable source of carotenoids. further studies will be focused on pumpkin juice processing with special regard to pumpkins rich in carotenoid content and high vitamin a values, respectively. acknowledgement this study was investigated within the scope of an interdisciplinary project (sps-projekt) of the research institute geisenheim. all participating persons and companies are greatfully recognized for their technical and financial support. references anonymous, 2000: referenzwerte für die nährstoffzufuhr. deutsche gesellschaft für ernährung, frankfurt/main, germany. arima, h.k., rodrigues-amaya, d.b., 1988: carotenoid composition and vitamin a value of commercial brazilian squashes and pumpkins. j. micronutrient analysis 4, 177-191. burns, j., fraser, p.d., bramley, p.m., 2003: identification and quantification of carotenoids, tocopherols and chlorophylls in commonly consumed fruits and vegetables. phytochem. 62, 939-947. davies, b.h., 1976: carotenoids. in: goodwin, t.w. (ed.), chemistry and biochemistry of plant pigments, vol. i, ed. 2, 38-165. academic press, london. food and nutrition board, 2001: dietary reference intakes for vitamin a, vitamin k, arsenic, boron, chromium, copper, iodine, iron, manganese, molybdenium, nickel, silikon, vanadium, and zink. national academy press, washington, dc. giovaaucci, e., 1999: tomatoes, tomato-based products, lycopene and cancer: a review of the epidemiological literature. j. natl. cancer inst. 91, 317331. grassmann, j., hippeli, s., elstner, e.f., 2002: plant’s defence mechanism and its benefits for animal and medicine; role of phenolics and terpenoids in avoiding oxygen stress. plant physiol. biochem. 40, 471-478. hermann, k., 1992: vorkommen, gehalte und bedeutung von inhaltsstoffen des obstes und gemüsesix. carotinoide. ind. obstund gemüseverwertung 77, 290-294. hidaka, t., anno, t., nakatsu, s., 1987: the composition and vitamin a value of the carotenoids of pumpkins different colors. j. food biochem. 11, 59-68. kritchevsky, s.b., 1999: β-carotene, carotenoids and the prevention of coronary heart disease. j. nutr. 129, 5-8. 98 mirjam kreck, petra kürbel, michael ludwig, peter j. paschold, helmut dietrich landrum, j.t, bone, r.a., 2001: lutein, zeaxanthin and the macular pigment. arch. biochem. biophys. 385, 28-40. marx, m., schieber, a., carle, r., 2000: quantitative determination of carotene stereoisomers in carrot juice and vitamin supplemented (atbc) drinks. food chem. 70, 403-408. miller, n.j., rice-evans, c., davis, m.j., 1993: a novel method for measuring antioxidant capacity and its application to monitoring the antioxidative status in premature neonates. clin. sci. 84, 407-412. murcovic, m., mülleder, u., neunteufl, h., 2002: carotenoid content in different varieties of pumpkins. j. food comp. anal. 15, 633-638. tanner, h., brunner, h.r., 1979: getränkeanalytik. verlag heller, schwäbisch hall, germany. van poppel, g., goldbohm, r.a., 1995: epidemiological evidence for βcarotene and cancer prevention. am. j. clin. nutr. 62, 1493-1503. address of the authors: dr. mirjam kreck, research institute geisenheim, dep. of wine analysis and beverage research, rüdesheimer straße 28 / p.o. b. 1154, d-65366 geisenheim, e-mail: m.kreck@fa-gm.de tab. 5: quality parameters of pumpkin juices and pumpkin pomace extracts of the varieties cucurbita maxima „muscat“, „rouge“ and „neon“. pumpkin juice pumpkin pomace extract muscat rouge neon muscat rouge neon density [20/20] 1.0317 1.0225 1.0211 1.0131 1.011 1.0087 brix [°] 7.22 5.05 4.68 3.07 2.47 1.94 conductance [µs/cm] 6010 7170 7000 2680 3880 3200 extract [g/l] 81.2 58.2 54.6 33.9 28.4 22.4 sugar free extract [g/l] 37.4 35.8 33.7 19.9 18.1 15.0 glucose [g/l] 10.3 9.7 8.4 4.4 4.4 3.0 fructose [g/l] 15.9 10.4 9.5 5.9 5.0 3.4 saccharose [g/l] 17.6 2.3 3.0 3.7 0.9 1.0 ph-value 4.41 3.97 4.31 3.79 4.33 3.77 total acid (ph 8.1, citric acid) ascorbic acid [mg/l] 184 104 92 164 325 88 lactic acid [g/l] < 0,05 < 0,05 < 0,05 < 0,05 0.12 0.05 malic acid [g/l] 3.75 3.19 1.34 1.12 1.26 0.44 volatile acid [g/l] 0.08 0.09 0.1 0.11 0.11 0.1 copper [mg/l] 0.5 0.3 0.5 0.2 0.3 0.3 iron [mg/l] 1.5 1.0 1.0 0.3 0.3 0.2 zinc [mg/l] 0.9 0.8 1.2 0.5 0.4 0.5 sodium [mg/l] 3 3 8 5 5 7 calcium [mg/l] 156 293 176 71 142 71 potassium [mg/l] 3682 4121 3955 1027 1434 1141 magnesium [mg/l] 130 127 186 51 61 76 ash [g/l] 6.54 6.6 6.88 2.28 3.26 2.63 total phenol [mg/l] 258 318 323 156 171 169 teac [mmol/l trolox] 2.1 2.3 2.7 1.1 1.5 1.5 [g/l] 3.66 3.76 3.63 2.82 2.99 3.02 carotenoids in pulp, peel and juice of cucurbita maxima l. 99 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true 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<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> >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice vorgabe neu journal of applied botany and food quality 81, 1 6 (2007) palacký university in olomouc, faculty of science, department of botany, olomouc, czech republic factors affecting protoplast isolation and cultivation of cucumis spp. gajdová, j., navrátilová, b., smolná, j., lebeda, a.* (received october 19, 2006) summary protoplasts of cucumis anguria, cucumis melo (3 accessions), cucumis metuliferus and cucumis sativus were isolated from leaves, growing apices, hypocotyls and calluses. plants were cultured on 2 concentrations of sucrose. the effect of plant culture medium, explant age and explant type on protoplast viability were investigated. the protoplasts were cultured in 3 types of culture medium and at two temperatures. optimal age range for protoplast isolation was 1-5 weeks depending on explant type and genotype. viabilities ranging between 50 % and 80 % were obtained from all explants and genotypes. increased concentration of sucrose had negative impact on viability of protoplasts. the highest level of regeneration achieved was callus, regenerated from leaf protoplasts of c. melo cv. ‘charentais’ and c. melo ‘mr-1’. the lowest regeneration capability was observed in hypocotyls. liquid lcm medium (b5 macro and microelements (1 g · l-1 cacl2), b5 vitamins with 1g · l -1 myo-inositol, 2 mg · l-1 ascorbic acid, 0.8 mg · l-1 glycine, 20 mg · l-1 glutamine, 100 mg · l-1 casein hydrolysate, 70 g · l-1 mannitol, 10 g · l-1 sucrose, 5 g · l-1 glucose, 585 mg · l-1 mes, 5.37 µmol · l-1 naa, 2.26 µmol · l-1 2,4-d, 2.22 µmol · l-1 ba) was optimal for protoplast regeneration. agarose-solidified medium caused a decrease in the number of cell divisions (used in c. melo ‘pi 124112’). cultivation at 25ºc resulted in a higher frequency of cell divisions (tested in c. metuliferus). abbreviations 2,4-d – 2,4-dichlorphenoxyacetic acid; ba – benzylaminopurine; iba – indole-3-butyric acid; ipa – indolylpropionic acid; mes – 2n-morfolin-etansulfonic acid; naa – α-naphthaleneacetic acid introduction cucurbit downy mildew (pseudoperonospora cubensis) and cucurbit powdery mildews (podosphaera xanthii, golovinomyces cichoracearum) are responsible for significant loss of cucurbit crops worldwide annually (jahn et al., 2002; lebeda and widrlechner, 2003). there are different methods used to control outbreaks of such diseases but these are all chemical based treatments which are not only sometimes inefficient but also have significant economic and environmental costs (hollomon and wheeler, 2002; lebeda and widrlechner, 2003; mcgrath, 2001; urban and lebeda, 2006). classical methods of cucumber breeding like intraspecific and interspecific hybridisation have not been reported to confer viable resistance (chen and adelberg, 2000; lebeda et al., 2007). recent studies have shown that by using protoplast fusion hybrid plants may be obtained, however these did not produce viable seeds (gajdová et al., 2004). in order to address the problems with somatic hybridisation we continued on work with previously reported accessions of wild and cultivated species of cucumis (gajdová et al., 2007). various protocols were developed and some successfully used in protoplast cultures of cucurbitaceae. many of them led to regeneration of normal plants able to form roots in soil and produce fruits (gajdová et al., 2004). despite this progress, fusion experiments are still problematic, because even when regeneration of hybrid plants was achieved, the genes of one of the parents may gradually become silenced (yamaguchi and shiga, 1993). recently several studies have reported on factors influencing protoplast yield, viability and regeneration capability, such as genotype, explant type, explant pre-treatment, presence of glycine in the enzyme solution, and culture medium composition (e.g. growth regulator concentration) (fellner and lebeda, 1998; sutiojono et al., 1998, 2002). mccarthy et al. (2001b) studied the effect of light on protoplast division, alongside temperature and agarose effects. they also demonstrated the influence protoplast density has on division rates and microcallus production (mccarthy et al., 2001b). despite improving the culture technique and increasing of the culture efficiency of protoplasts, the regeneration of plants was not always achieved (gajdová et al., 2004; mccarthy et al., 2001a, b; sutiojono et al., 1998, 2002). to improve the protocol for protoplast isolation and cultivation it is first necessary to increase the protoplast viability (the percentage of protoplasts surviving the isolation and purification procedure). second is to increase the regeneration efficiency (the percentage of protoplasts regenerating into dividing cells and subsequently into calluses or plants). the aim of this investigation was: a) to contribute towards a better understanding of regeneration from protoplasts which could then be used in subsequent somatic hybridisation; b) to find out optimal plant or callus age, plant culture media, donor plant organs, temperature and composition of culture media for cultivation and regeneration of plants. material and methods plant material and explants cucumis spp. genotypes that in previous experiments indicated good regenerative capabilities (calluses obtained) (gajdová et al., 2007) were used for this study. the following four accessions were selected for good regenerative capabilities: cucumis meloa ‘600’ (mr-1, cz 09-h40-0600), cucumis meloa ‘16’ (cv. charentais, cz 09-h40-1116), cucumis metuliferusa (cz 09-h41-0587), cucumis sativusa line sm 6514 (cz 09-h39-0768). both cucumis anguria var. anguriab (ames 23536) and cucumis melob ‘12’ (pi 124112) were selected because they have increased yield and viability of protoplasts. the plant material originated from the vegetable germplasm collection of the research institute of crop production (prague) a, department of gene bank (olomouc, czech republic), and regional plant introduction stationb (ames, iowa, usa). seeds were surface sterilised with 8 % chloramin b for 30 minutes and rinsed 3 times with sterile distilled water. the seat coat was excised and they were germinated on half strength ms (murashige and skoog, 1962) medium in petri dishes (60 mm) in the dark at 25°c. after germination, hypocotyls were detached and seedlings planted on ok medium (ms medium supplemented with 20 g · l-1 sucrose, 20 mg · l-1 ascorbic acid, 0.8 % agar, 0.049 µmol · 1-1 iba and 0.044 µmol · 1-1 ba) in plastic boxes (volume 800 ml) or erlenmeyer’s flasks (100 ml). plants were cultivated in culture room with a 16 hour day (light intensity 32 36 µmol · m-2 · s-1) and the temperature at 22 ± 2°c. they were subcultured every 3 weeks. hypocotyls were used either for protoplast isolation or for callus derivation. leaves and growing apices served as sources of protoplasts. calluses were derived from hypocotyls of in vitro plants and in the case of c. melo ‘12’ and c. sativus from leaves. leaves for callus derivation were cut into quarters, hypocotyls were cut in to several pieces and then halved and placed on msc medium (ms with 30 g · l-1 sucrose, 13.43 µmol · l-1 naa, 4.4 µmol · l-1 ba and 0.8 % agar) to induce callus growth and growing calluses were subcultured every 2 weeks. calluses were cultivated in the dark at 25°c. protoplasts isolation and culture protoplast isolation enzyme solutions for leaves, growing apices and hypocotyls contained 1% (w/v) cellulase onozuka r-10 (duchefa) and 0.25 % (w/v) macerozyme r-10 (duchefa) dissolved in pgly washing solution (debeaujon and branchard, 1992; composition: 27.2 mg · l -1 kh2po4, 101 mg · l -1 kno3, 1117.6 mg · l -1 cacl2, 246 mg · l-1 mgso4 · 7h2o, 0.16 mg · l -1 ki, 0.025 mg · l-1 cuso4 · 5 h2o, 11.5 g · l-1 glycine, 18.016 g · l-1 glucose, 0.58572 g · l-1 mes and 65.58 g · l-1 mannitol). enzyme solution containing 2% cellulase onozuka r-10, 1% macerozyme r-10 and 0.3 % driselase (fluka) was used for callus protoplast isolation. hypocotyls, growing apices and the youngest fully developed leaves were cut into fine strips or pieces and incubated in the enzyme solution for 16-17 hours in the dark at 25°c (approximately 2 ml of enzyme solution for 100200 mg of tissue). approximately 5 ml enzyme solution was used for 1 g of callus. calluses were cut into pieces and placed on a shaker (80 rpm) for 30 minutes, then in an incubator (25°c, dark) for 17 hours and finally on the shaker for 30 minutes again. the protoplast suspension was filtered through nylon mesh (72 µm), mixed with pgly solution (approximately 5 ml) and centrifuged at 100 × g for 5 minutes. after pouring off the supernatant the pellet was resuspended in 20 % (w/v) sucrose (4 ml) and overlaid with the pgly washing solution (2 ml) ensuring they did not mix. they were centrifuged at 100 × g for 10 minutes, protoplasts were isolated from the layer between sucrose and pgly using pasteur pipette. protoplasts were mixed with approximately 3 ml of pgly solution and centrifuged at 100 × g for 5 minutes. viability of protoplasts was established after purification using an olympus fluorescent microscope ‘bx60’ and fluorescein diacetate stain (larkin, 1976) and a bw filter. results were calculated by using the percentage of the protoplasts which were living; ten readings were made for each sample. protoplasts were cultured in modified liquid lcm medium (debeaujon and branchard, 1992) containing: b5 macro and microelements (1 g · l-1 cacl2), b5 vitamins with 1g · l -1 myo-inositol, 2 mg · l-1 ascorbic acid, 0.8 mg · l-1 glycine, 20 mg · l-1 glutamine, 100 mg · l-1 casein hydrolysate, 70 g · l-1 (0.38 mol · l-1) mannitol, 10 g · l-1 sucrose, 5 g · l-1 glucose, 585 mg · l-1 mes, 5.37 µmol · l-1 naa, 2.26 µmol · l-1 2,4-d, 2.22 µmol · l-1 ba, in petri dishes (diameter 40mm), approximately 1.5 ml of medium per dish, at density 105 protoplasts · ml-1. protoplast cultures were placed in the dark at 25°c for 14 days and then transferred into a culture room with 16/8 hours day/night cycles with light intensity 32-36 µmol · m-2 · s-1 at 22 ± 2°c. at this time lcm2 medium containing 3.3 µmol · l-1 ba and no mannitol (debeaujon and branchard, 1992) was added to dishes (approximately 1 ml) and protoplasts from each dish were subdivided between two dishes (using a pipette). growing microcalluses (after 4 weeks of culture) were transferred onto a solid f medium (ms macro and microelements, b5 vitamins, 10g · l-1 sucrose, 0.537 µmol · l-1 naa, 2.2 µmol · l-1 ba, 0.8% agar) (pelletier et al., 1983) and the resultant calluses were subcultured after 2-3 weeks onto a fresh medium. effect of plant culture media on viability of isolated protoplasts plants were cultured on ok medium with 30 g · l-1 of sucrose. leaves and growing apices were detached between 1563 days. protoplast isolation was carried out according to the protocol in the previous section. protoplasts were cultured in liquid lcm medium. the viability of isolated protoplasts was compared to those derived from plants grown in standard ok medium. optimal age of plants, seedlings and calluses for protoplast isolation leaves were detached from plants between 8 -107 days old, growing apices from 756 days old, hypocotyls from seedlings 4 -17 days after sowing and calluses were used for protoplast isolation 14 96 days after starting their cultivation. the viabilities of isolated protoplasts were used as criteria to find the optimal age of in vitro materials for protoplast isolation. the yield of protoplasts was usually sufficient if the viability was high. regenerative capability of protoplasts isolated from different types of explants the level of regeneration from protoplasts was investigated for all tissue types. levels of regeneration were categorised as the following: cell division (< 0.2 mm), microcallus (colonies of cells 0.22 mm which were visible by the eye) and callus (> 2 mm in diameter). results were recorded by the percentage of samples showing each level (category) of regeneration. protoplast culture technique cucumis melo ‘12’ leaf protoplasts were cultivated both in filter sterilised liquid medium and in solidified (0.6% agarose) lcm medium. the number of divisions observed after 14 days represented the regeneration efficiency when expressed as a percentage of total sample number. in cucumis anguria modified cml medium (without edamin) (colijn-hooymans et al., 1988) was compared to lcm. cml medium contained ms macro and micro elements, 25 µmol · l-1 naa and 15 µmol · l-1 ipa, 2 g · l-1 sucrose, 0.25 mol · l-1 mannitol. cucumis metuliferus leaf protoplasts were used to compare effect of culture temperature during first 14 days of cultivation. temperatures of 25°c and 27°c were compared. due to constraints of time it was not possible to carry replicates of each variable with each species within this study, future work will focus on the most responsive candidates. results and discussion effect of plant culture media on viability of isolated protoplasts it is evident that using of the culture medium with a higher concentration of sucrose (30 g · l-1) resulted in decreased protoplast viability (fig. 1). protoplasts were obtained in normal yields, and being normal in size and shape. they were however, not able to survive digestion and purification procedure. in c. melo ‘16’, c. metuliferus and c. sativus a big decrease in viability was recorded, particularly in leaf protoplasts. however, in c. melo ‘600’ an increase of viability was seen in protoplasts from growing apices. this suggests that concentration of sugar, namely sucrose in culture medium can have strong effect on subsequent isolation of protoplasts from cucumis tissue dependent on genotype. 2 gajdová, j., navrátilová, b., smolná, j., lebeda, a. there are reports about positive effect of a high concentration of sucrose (85.6 g · l-1) on somatic embryogenesis in cotyledon tissue (lou and kako, 1995) but no studies on sucrose concentration in donor plant culture medium have been reported. moreno et al. (1984) have reported increasing protoplast divisions when donor plants were cultivated on medium with yeast extract, but this medium also caused strong decrease of protoplast yield. this suggests that composition of culture medium can greatly influence protoplast physiological properties. optimal age of plants, seedlings and calluses for protoplast isolation the optimal age for protoplast isolation varies both among genotypes and explants (tab. 1). for leaves, the highest viabilities were obtained from 3-5 weeks old plants (2-3 weeks after subculture), but in some genotypes the suitable age was up to 10 weeks. plants of c. anguria and c. metuliferus grew rapidly and exhibited increased vitality in vitro for prolonged intervals (up to several months) but the optimal age for c. metuliferus was only 3-5 weeks. flowers of the c. sativus started budding after 4 weeks and plant growth decreased rapidly thereafter. plants of c. melo grew slowly from the beginning with poor rooting and leaves were rather tough in texture. despite this, these plants provided good results. colijn-hooymans et al. (1988) and debeaujon and branchard (1992) reported plant regeneration from leaf protoplasts isolated from 3 week old plants of c. melo and c. sativus. growing apices generated good protoplast viability from 1-8 weeks, depending on genotype. soft and fine grained calluses only were suitable for protoplast isolation, optimally up to 5 weeks for most genotypes, 7-14 days after last subculture. burza and malepszy (1995) reported plant regeneration from callus protoplasts used 7-10 days after subculture, and after 4-8 subcultures. hypocotyls need to be prepared within 8 days after sowing seeds. this is in line with published data from c. melo ‘green delica’ and ‘fastoso’ (sutiojono et al., 2002) where it was reported that hypocotyls older than 8 days had lignified cell walls which prevented successful isolation of protoplasts. effect of donor explant variation on protoplast viability fig. 2 shows viabilities of protoplasts which were isolated within their optimal explant age ranges for respective genotypes. differences among species and varieties and especially among explant types within one genotype are visible in the graph. in c. melo ‘12’ and c. melo ‘600’ leaf protoplasts were the most viable. all explant types of c. melo ‘16’ were suitable for protoplast isolation, in c. metuliferus growing apex and callus gave the best results using our isolation protocol. in c. sativus callus protoplasts were the most viable. different species have different requirements for cultivation and isolation, for example in c. melo ‘600’ better results were achieved for growing apices by growing plants on ok medium with higher concentration of sucrose (fig. 1). to also increase the protoplasts viability in other explant types it is necessary to optimise the culture protocols and subsequent isolation techniques for each individual genotype. for example hypocotyls of c. metuliferus were much softer than in other species so they may need a lower concentration of digesting enzymes or cutting into bigger pieces then other studied species. the isolation technique has been developed and optimised for c. melo which is why its protoplast viabilities are increased over that of the other species. the yield also differed among explant types being approximately ten times higher in leaves and growing apices than in calluses. hypocotyls gave lower yields than leaves and growing apices (unpublished data). other authors have reported differences among cucumis species and tissue types as well (gajdová et al., 2007; mccarthy et al., 2001b; sutiojono et al., 2002). fig. 1: effect of sucrose concentration on protoplast viability. data points comprised of a minimum of 16 individual explants. yerror bars represent standard deviation. 0 10 20 30 40 50 60 70 80 90 100 c. melo '16' c. melo '600' c. metuliferus c. sativus cu cumis specie s v ia b le p ro to p la st s (% ) leaf on ok medium with 20g·l¯¹ sucrose leaf on ok medium with 30g·l¯¹ sucrose growing apex on ok medium with 20g·l¯¹ sucrose growing apex on ok medium with 30g·l¯¹ sucrose tab. 1: optimal age of plants, cultivated calluses and seedlings used for protoplast isolation leaf growing apex callus hypocotyls (days after planting) (days after planting) (days of cultivation) (days after sowing) genotype tested range optimal tested range optimal tested range optimal tested range optimal c. anguria 15-74 (22) 21-74 — — — — — — c. melo ‘12’ 12-40 (51) 20-27 — — 17-96 (22) 33-34 — — c. melo ‘16’ 20-35 (4) 20-35 7-35 (7) 7-22 14-94 (10) 14-28 — — c. melo ‘600’ 8-107 (18) 30-64 8-34 (3) 8-34 18-42 (13) 35-42 4-15 (4) 4-8 c. metuliferus 22-91 (8) 22-35 — — 18-42 (12) 32-35 — — c. sativus 11-75 (16) 22-34 11-39 (6) 11-39 — — 6-17 (5) 6-8 the sample number is represented in brackets, each sample comprises of 2-4 explants for leaves, 8 explants for growing apices, 4 dishes for callus and 10 explants for hypocotyls. factors affecting protoplast isolation 3 regenerative capability of protoplasts isolated from different types of explants the highest level of regeneration was achieved using leaf protoplasts in all tested genotypes (tab. 2) despite that leaf protoplasts were not as viable as other types of protoplasts in most genotypes. calluses were regenerated from leaf protoplasts in c. melo ‘16’ and c. melo ‘600’. microcalluses were obtained in c. metuliferus and c. sativus leaf protoplasts. using the same technique calluses are also possible using c. metuliferus and c. sativus (gajdová et al., 2007). protoplasts from growing apices did not achieve this level of regeneration but had higher regeneration efficiency than hypocotyl and callus protoplasts. using growing apices as source of protoplasts has not been reported yet. hypocotyl and callus derived protoplasts exhibited very low regenerative capability. it has been reported that callus protoplasts require a different medium for regeneration (with 0.045 µmol · l-1 2,4-d and 0.913 µmol · l-1 zeatin) whereas leaf protoplasts of the same c. sativus variety had the highest regeneration rate using 25 µmol · l-1 naa and 14.8 µmol · l-1 2ip (burza and malepszy, 1995). for this study the same regeneration protocol was used for all types of protoplasts. hypocotyl protoplasts have not been fig. 2: effect of donor explant variation on viability of protoplasts. each value in the graph is based on at least 16 leaf explants, 24 growing apices, 20 hypocotyls and 12 dishes of callus. y-error bars represent standard deviation. 0 10 20 30 40 50 60 70 80 90 100 c. anguria c. melo '12' c. melo '16' c. melo '600' c. metuliferus c. sativus cucum i s species v ia b le p ro to p la s ts ( % ) leaf growing apex hypocotyl callus tab. 2: effect of plant tissue on regeneration of protoplasts genotype level of regeneration regeneration efficiency¹ (%) number of cultivated samples2 c. anguria leaf cell division 58 12 c. melo ‘12’ leaf cell division 25 28 callus no regeneration 100 15 c. melo ‘16’ leaf callus 50 2 growing apex microcallus and cell division 33 and 33 3 callus microcallus and cell division 14 and 7 14 c. melo ‘600’ leaf callus and cell division 4 and 38 26 growing apex no regeneration 100 3 hypocotyl no regeneration 100 1 callus cell division 14 14 c. metuliferus leaf microcallus and cell division 5 and 53 19 growing apex cell division 100 3 hypocotyl cell division 25 4 callus no regeneration 100 12 c. sativus leaf microcallus and cell division 25 and 25 8 growing apex microcallus 50 2 hypocotyl no regeneration 100 1 callus no regeneration 100 8 only samples with viabilities of over 40% were used for regenerative capability assessment. ¹regeneration efficiency is calculated as percentage of regenerating samples given for each level of regeneration. 2one sample consists of at least 2-4 explants for leaves, 8 explants for growing apices, 4 dishes for callus and 10 explants for hypocotyls. in cases, where two levels of regeneration were observed, the regeneration efficiencies are given respectively. 4 gajdová, j., navrátilová, b., smolná, j., lebeda, a. regenerated into plants yet in cucumis spp. (gajdová et al., 2004). sutiojono et al. (1998) have reported that leaf protoplasts had the highest regeneration potential, however cotyledon protoplasts divided slower and hypocotyl protoplasts did not divide at all. plants were regenerated from protoplasts isolated from leaves (debeaujon and branchard, 1992; orczyk and malepszy, 1985), cotyledons (roig et al., 1986; colijn-hooymans et al., 1988) and calluses (burza and malepszy, 1995) in c. melo and c. sativus. cotyledon protoplasts were mostly tetraploid and regenerated shoots were not normal however leaf protoplasts were diploid and produced normal plants (colijn-hooymans et al., 1988). regarding this, we did not use cotyledons in our study. in c. melo ‘12’ the highest regeneration efficiency was achieved with protoplasts from plants at 20-27 days old (which was also optimal age for protoplast isolation as shown in tab. 1). in all other genotypes protoplasts from explants of various age were regenerating. calluses from c. melo ‘16’ and c. melo ‘600’ were regenerated from 20-28 days old plant-derived protoplasts. regenerated calluses grew several months but organogenesis did not occur on the media used in this study. although no plant growth was observed in this study, further investigation into the callus regeneration media would likely yield plants (debeaujon and branchard, 1992; orczyk and malepszy, 1985). protoplast culture technique the number of cell divisions decreased using agarose-solidified medium in our experiment (tab. 3). interestingly, several studies showed increased plating efficiency using this technique compared to liquid culture (colijn-hooymans et al., 1988; mccarthy et al., 2001b; sutiojono et al., 1998). the decrease in number of cell divisions in our study may have been caused by heat stress when embedding protoplasts in warm agarose medium (approximately 40°c). in our experiments the lcm medium was much better for protoplast regeneration of c. anguria leaf protoplasts than cml medium (tab. 4). lcm medium has been successfully used for plant regeneration from cotyledon and leaf protoplasts in c. melo (debeaujon and branchard, 1992). similar concentrations of growth regulators (2.69 µmol · l-1 naa, 4.56 µmol · l-1 2,4-d and 2.22 µmol · l-1 ba) were used in c. sativus and leaf and cotyledon protoplast of c. melo (dabauza et al., 1991; roig et al., 1986). cml medium was successfully used for plant regeneration from leaf protoplasts of c. sativus (orczyk and malepszy, 1985; colijnhooymans et al., 1988). both temperatures used in this study had similar effects on cell division (tab. 5). no increase was seen when using 27°c. mccarthy et al. (2001a) have obtained increased rates of cell division and microcalluses in cotyledon protoplasts of c. metuliferus cultured at 30°c over those cultured at 25°c. conclusions from this study it is evident that the most important factor for obtaining the highest viability of cucumis protoplasts was donor explant age (optimal age range was mostly 15 weeks, varying according to explant type and genotype). using the plant culture medium with increased level of sucrose resulted in production of protoplasts with poor viability except with c. melo ‘600’ growing apex protoplasts. explant type was also important for viability, but there was no ‘high viability explant’ common to all genotypes. all explants were able to produce over 50 % (some over 80 %) viable protoplasts. c. melo varieties gave slightly higher protoplast viabilities than c. metuliferus and c. sativus. the regenerative capability of protoplasts was primarily dependent on source explant type, with the highest regeneration level (callus) observed in leaf protoplasts. calluses were obtained only tab. 5: effect of culture temperature on cell division tested species cell divisions in 25°c cell divisions in 27°c cucumis metuliferus regeneration number of regeneration number of efficiency¹ (%) cultivated samples2 efficiency¹ (%) cultivated samples2 75 8 63 8 1regeneration efficiency is the percentage of regenerating samples. 2each sample consisted of 8-16 leaves. tab. 3: effect of viscosity of the protoplast culture medium on cell division tested species cell divisions in liquid medium cell divisions in agarose solidified medium cucumis melo ‘12’ regeneration number of regeneration number of efficiency¹ (%) cultivated samples2 efficiency¹ (%) cultivated samples2 25 28 5 20 1regeneration efficiency is the percentage of regenerating samples. 2each sample consisted of 2-4 leaves. tab. 4: effect of the protoplast culture medium composition on cell division tested species cell divisions in lcm medium cell divisions in cml medium cucumis anguria regeneration number of regeneration number of efficiency¹ (%) cultivated samples2 efficiency¹ (%) cultivated samples2 58 12 10 10 1regeneration efficiency is the percentage of regenerating samples. 2each sample consisted of 4-8 leaves. factors affecting protoplast isolation 5 š š š in c. melo ‘16’ and c. melo ‘600’. temperature and culture medium composition affected number of cell division but there were no differences in level of regeneration obtained. this study has highlighted some of the important factors in promoting healthy regeneration of protoplasts. future work will concentrate on further optimising the growth conditions of regenerating microcalluses specific to the donor species. investigation on the effects of protoplast density in culture medium may help increase the plating efficiency. these may yield the answer in producing viable plants from protoplast fusions in the future. acknowledgements we would like to thank the ministry of agriculture of the czech republic for the co-funding of this project by a grant qf 4108 and the ministry of education of the czech republic for grant msm 6198959215 who made this research possible. technical assistance of anna zedkova is acknowledged. we would also like to thank john mitchels and alan scragg, the university of the west of england (bristol, uk) for kindly reading the manuscript. references burza, w., malepszy, s., 1995: in vitro culture of cucumis sativus l. xviii. plants from protoplasts through direct somatic embryogenesis. plant cell tiss. org. cult. 41, 259-266. chen, j.f., adelberg, j.w., 2000: interspecific hybridization in cucumis – progress, problems, and perspectives. hortscience 35, 11-15. colijn-hooymans, c.m., bouwer, r., orczyk, w., dons, j.j.m., 1988: plant regeneration from cucumber (cucumis sativus) protoplasts. plant sci. 57, 63-71. dabauza, m., roig, l.a., moreno, v., 1991: selective methods for the recovery of somatic hybrids of cucumis melo × c. metuliferus and c. sativus × c. metuliferus. cucurbit gen. coop. rep. 14, 81-84. debeaujon, i., branchard, m., 1992: induction of somatic embryogenesis and caulogenesis from cotyledons and leaf protoplast-derived colonies of melon (cucumis melo l.). plant cell rep. 12, 3740. fellner, m., lebeda, a., 1998: callus induction and protoplast isolation from tissues of cucumis sativus l. and c. melo l. seedlings. biol. plant. 41, 11-24. gajdová, j., lebeda, a., navrátilová, b., 2004: protoplast cultures of cucumis and cucurbita spp. in: lebeda, a., paris, h.s. (eds.), progress in cucurbit genetics and breeding research. proceedings of cucurbitaceae 2004, the 8th eucarpia meeting on cucurbit genetics and breeding, 441-454. palacký university in olomouc, olomouc (czech republic). gajdová, j., navrátilová, b., smolná, j., lebeda, a., 2007: effect of genotype, source tissue and media composition on cucumis and cucurbita protoplast isolation and regeneration. acta hort. 731, 89-94. hollomon, d.w., wheeler, i.e., 2002: controlling powdery mildews with chemistry. in: bélanger, r.r., bushnell, w.r., dik, a.j., carver, t.l.w. (eds.), the powdery mildews. a comprehensive treatise, 249-255. aps press, st. paul, mn. jahn, m., munger, h.m., mccreight, j.d., 2002: breeding cucurbit crops for powdery mildew resistance. in: bélanger, r.r., bushnell, w.r., dik, a.j., carver, l.w. 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urban, j., lebeda, a., 2006: fungicide resistance in cucurbit downy mildew – methodological, biological and population aspects. ann. appl. biol. 149, 63-75. yamaguchi, j., shiga, t., 1993: characteristic of regenerated plants via protoplast electrofusion between melon (c. melo) and pumpkin (interspecific hybrid, cucurbita maxima × c. moschata). jap. j. breed. 43, 173-182. address of the authors: mgr. jana gajdová, prof. dr. aleš lebeda *, rndr. bozena navrátilová, ph.d. and mgr. jitka smolná; department of botany, faculty of science, palacký university in olomouc, slechtitelu 11, 783 71 olomouc holice, czech republic. *corresponding author (ales.lebeda@upol.cz) o 6 gajdová, j., navrátilová, b., smolná, j., lebeda, a. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) 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<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> >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice art05.pmd journal of applied botany and food quality 80, 31 35 (2006) 1federal centre for breeding research on cultivated plants, institute of plant analysis, quedlinburg, germany 2philipps-universität marburg, institut für pharmazeutische chemie, marburg, germany 3institute of plant genetics and crop plant research (ipk), gatersleben, germany efficient determination of cysteine sulphoxides in allium plants applying new biosensor and hplc-ms² methods k. ziegert1, w. schütze1, h. schulz1, m. keusgen2, f. gun2, e.r.j. keller3 (received december 14, 2005) summary cysteine sulphoxide (cso) contents of 16 different accessions of garlic (allium sativum l.) and 15 varieties of onion (allium cepa l.) were measured using two different rapid analytical methods: a biosensoric approach and a newly developed hplc-ms2 technique. both methods allow quantification of naturally occurring cysteine sulphoxides present in allium plants without time-consuming sample pretreatment such as derivatisation of amino acid derivatives prior to hplc-separation. it has been found that the amount of alliin, which is the predominant cso occurring in garlic, varies between 0.2 and 2.2 g/100 g dry matter. contrary to that, isoalliin representing the main cso in onion has been detected in significantly lower amounts (0.3 to 1.25 g/100 g dry matter). introduction onion (allium cepa l.) and garlic (allium sativum l.) are economically the most important species of the genus allium. onion varieties (allium cepa) can be found in many regions of europe, asia, north america and africa. this species is one of the most important horticultural crops with an annual world-wide production of around 44 million tons (griffiths et al., 2002). it is well-known that s-alk(en)yl-l-cysteine sulphoxides (cso), which are mainly present in the bulb, act as flavour precursors generating the typical taste and odour in presence of the enzyme alliinase (e.g. dipropyl disulfide, diallyl disulfide, thiophene). fig. 1 shows the biotransformation of sulphur compounds in allium during processing of the plant raw material. the alliinase reaction initiates the production of highly reactive sulphenic acids, which condense with each other to form thiosulphinates. these compounds are mainly responsible for the flavour of fresh onion or garlic. the thiosulphinates can disproportionate into thiosulphinoates and sulphides presenting the characteristic flavour of cooked onions. steam-distilled oils obtained from various allium species such as onion, garlic or leek are widely used for flavouring food and for production of food supplements. several studies have been performed during recent years in order to evaluate potential allium wild plants and hybrids with special regard to their aroma properties as well as pharmacological value (schulz et al., 1998; schulz et al., 2000; keusgen et al., 2002; storsberg et al., 2004; schmitt et al., 2005). besides sulphur compounds, also pyruvic acid and ammonia were formed in equimolar amounts, which can be used for a rapid biosensoric determination of cysteine sulphoxides (krest and keusgen, 2002; krest and keusgen, 1999). in the latter case, alliinase has to be immobilized with an ammoniadetecting device. further on, cepaenes (α-sulphinyl disulphides) were found in aged extracts of allium species (rabinowitch and currah, 2002) acting as potent inhibitors of platelet aggregation and different enzymes (e.g. cyclooxygenase, lipoxygenase) (block et al., 1997). the formation of particular compounds depends on different reaction conditions (e.g. temperature, solvent). in an ethanolic extract, the thiosulphinate allicin can react with itself (disproportionation) forming compounds with three sulphur atoms in the molecule (ajoens), which are especially known for their antithrombotic properties. the lachrymatory factor, built from 1-propenyl-sulphenic acid, can dimerize to bisulphines and cyclic zwiebelans. as shown above, cso play an important role in the biochemistry of allium plants and significantly contribute to the health of men. therefore, efficient analytical methods are required to determine the individual content of these compounds in plant material as well as to p y r u v a te c h -c o c o o h3 s ulp h e nic a cid r-s o hn h 3 s ulp h in e (l f) r = s o+ p r op a n a l c h c h -c h o3 2 th ios u lp h ina te r-s -s o r ’ s u lp h id es r-s -s -r ’ t hios u lp hin o a tes r-s -so r ’2 ‘a j oe ne s ’ h c s o c h -s -s -c h5 3 3 5 3 5 + + + b i s-p ro p e n yl d is u lp h id e ‘c y c lic zw ie b e la n e s ’ + h o2 d im e r iza tio n c o n d e n s a tio n fo r a llic in d is p ro p o r tio n a tio n + s u lp h e n ic a c id d is p ro p o rtio n a tio n c s o r-so -c h (n h )c o o h2 2 a liin a s e ‘cep a en es ’ h c -so -c h -s -s c h5 3 3 6 3 5 fig. 1: scheme presenting the formation of sulphur compounds starting from cysteine sulphoxides coming in contact with alliinase during processing of plant material. r = methyl, propyl, 2-propenyl and 1-propenyl; lf = lachrymatory factor (rabinowitch and currah, 2002). characterise herbal products obtained from allium plant material. several hplc techniques for the determination of cso, especially of alliin, have been described in the past (ziegler and sticher, 1989; iberl et al., 1990; keusgen, 1997) but all these analysis methods generally are time consuming because of extensive sample clean-up steps necessary to separate cso from interfering matrix components. therefore, two different sophisticated methods were applied to study the cso content and distribution in various cultivars of allium cepa as well as gene bank accessions of allium sativum: a new biosensoric method based on immobilized alliinase (krest and keusgen, 2002) and a newly developed hplc-ms2 method. for the biosensoric analysis immobilized alliinase was used to initiate the degradation of cso. pyruvate and ammonia are by-products formed during this process. the amount of enzymatically formed ammonia is proportional to the level of cysteine sulphoxides and can be, therefore, used for an indirect quantification of these sulphur constituents in allium plant material, if native alliinase was inactivated during extraction (methanol, heat). the new hplc-ms2 method with selective ion monitoring (sim) was developed to more rapidly screen extracts of various allium species. the method was used for the analysis of the four main important cso in allium species: methiin, alliin, isoalliin and propiin. the special advantage of ms2-detection should be used to detect the individual analyte molecule ions and their related fragments with high selectivity also in presence of other co-eluting non-sulphur compounds. finally, we also will discuss the variation of cso found in different cultivars/accessions of a. cepa and a. sativum. materials and methods reagents alliin was obtained from carl roth gmbh & co kg (karlsruhe, germany). methiin and propiin were prepared following the procedures described earlier (keusgen, 1997; theodoropoulos, 1959; stoll and seebeck, 1951; freeman et al., 1994; koch and keusgen, 1998; krest et al., 2000). isoalliin, synthesized according to a new efficient procedure (namyslo), was generously provided by the institute of organic chemistry, clausthal university of technology, germany. alliinase was isolated, stabilized and purified according to methods developed by krest and keusgen (krest and keusgen, 2002; krest and keusgen, 1999). plant material the investigated garlic plant material was obtained from the allium collection of the institute of plant genetics and crop plant research in gatersleben, germany. the different onion varieties were cultivated by the „agrargenossenschaft calbe“, calbe, germany. the plants were grown under comparable conditions to reduce influences caused by environmental factors. the freshly harvested bulbs were directly cooled down (-80 °c) and subsequently freeze-dried in order to prevent losses of cso caused by enzymatic reactions. sample preparation hplc-ms2 about 200 mg of an exactly weighed freeze-dried sample were heated under reflux for 10 minutes in 20 ml of methanol. after that, the sample was crushed in a mortar and returned to the methanolic solution with the addition of 20 ml deionised water and heated again for 10 minutes. then the extract was filtered and the residue was washed with 3x3 ml of methanol/water (50:50). the filtrates were evaporated to dryness under reduced pressure. for hplc analysis the residue was redissolved in 2.5 ml of deionised water (theodoropoulos, 1959). from each variety two samples were taken in order to get an overview about the variability of the cso content within the analysed plant material. biosensor for biosensor analyses the freeze-dried plant material (without green parts) was pulverised in a mortar. 200 mg of this material was transferred to a test tube containing 3 ml of methanol. this enzymeinactivated solution was gently heated for five minutes in a heating block at 100 °c block temperature. then the test tube was cooled down for three minutes and the sample was heated again for another 15 minutes after addition of 3 ml of deionised water. finally, the mixture was transferred into a graduated flask (25.0 ml) and filled up with biosensor phosphate buffer (ph = 7). prior to biosensor analysis, the sample was filtered and diluted with the buffer solution (1:20, v/v). hplc-ms2 analysis the analyses were performed on an hp 1100 series hplc system (agilent) hyphenated with a bruker esquire 3000 mass spectrometer with electrospray ion source. a zorbax sb-c18 column (3 x 150 mm), particle size 3.5 µm, was used for the cso separation. sample aliquots of 1-5 µl were injected into the analytical column and the cso separation was performed using two solvents (a: deionised water, b: methanol). the following elution gradient was applied (flow rate = 0.5 ml/min). tab. 1: gradient elution profile for hplc separation of cysteine sulphoxides. step time (min.) % eluent b (methanol) 1 0.00 1.0 2 2.00 1.0 3 7.00 6.0 4 7.10 1.0 5 9.00 1.0 the optimisation steps for detection of mass fragments were carried out in the flow injection mode using a scan range of 50-400 m/z for all analysed cso. the quantification of cso was performed on the basis of the extracted ion [m+h]+ without further fragmentation within a given time window (fig. 2). the individual ms conditions were specially adapted to methiin, alliin, isoalliin and propiin. accordingly the calibration equations were established for these cso. flow-through analysis the biosensoric method is based on the alliinase reaction, the enzyme was immobilized and placed inside a flow-through cartridge. determination of free ammonia and enzymatically formed ammonia was determined by a flow-through-apparatus (mana and spohn, 2000). ammonia in equimolar amounts was obtained from cysteine sulphoxides by immobilized alliinase. the enzyme was isolated, stabilized and purified according to the methods already described earlier (krest and keusgen, 2002). the powdered garlic or onion sample was homogenized and a crude alliinase preparation was obtained by precipitation with ammonium sulphate or by ultra32 k. ziegert, w. schütze, h. schulz, m. keusgen, f. gun, e.r.j. keller filtration. ultra-filtrated alliinase was used for standard experiments. alliinase was stabilized with 10 % sucrose, 0.17 m nacl and 25 mm pyridoxal-5’-phosphate (p-5’-p) dissolved in phosphate buffer (ph 7.0, 60 mm). further purification was done by gel filtration followed by affinity chromatography. immobilization of alliinase was performed on a cona/agarose-carrier (sigma, ord.-no. c 7555). briefly, the carrier was filled into a cartridge (60 µl) and rinsed with buffer. a solution containing alliinase was then pumped through this cartridge. immobilized alliinase may be also renewed. elution of the enzyme was performed with buffer containing mannose followed by rinsing with a glucose solution and buffer. the carrier may be newly loaded with alliinase as described above. for analysis of cysteine sulphoxides, the cartridge was operated with phosphate buffer (ph 7.0, 0.02 m containing 0.17 m nacl). analysis of cysteine sulphoxides by hplc and the biosensor was repeated at least three times. given values for cysteine sulphoxides were corrected by the amount of native ammonia before alliinase reaction. results and discussion hplc-ms2 with sim has been shown to be a suitable method to detect and to quantify naturally occurring cso (methiin, alliin, isoalliin, propiin) in a. cepa and a. sativum. all four compounds could be individually detected. the newly developed method has been found to be very sensitive, robust, and reliable over a wide concentration range of the targeted compounds. the low detection limit allows to analyse cso even in concentrations below 1 µg/ml sample. the linear range could be confirmed over three concentration decades. sensitivity is sufficient even for the analysis of small allium samples even in the order of a few mg. fig. 2: the extracted ion chromatograms of cso occurring in allium cepa (black line) and allium sativum (dotted line). isolation of cysteine sulphoxide ions was performed using different time windows individually adapted to the masses of individual cso the use of ms2 detection has been proven to be particularly effective in analysing the non-volatile sulphur substances from allium extracts, because other co-eluting allium substances do not interfere with the significant key signals of the molecule ion [m+h]+. the esi interface and mass spectrometer parameter were optimised to obtain maximum sensitivity as displayed in tab. 2 and fig. 3. for calibration, pure cso standards were used in different concentration ranges related to the authentic content which is usually measured in both allium species. within these considered ranges the individual calibration curves (fig. 3) were found to be linear (tab. 2). the biosensoric method is also suitable for a simple and rapid determination of the total amount of cso. each analysis needs only less than five minutes. as shown in previous investigations with immobilized alliinase, results obtained by ammonium determination after alliinase digestion of cso are comparable with those obtained by hplc (krest and keusgen, 2002). the method is less sensitive than the hplc-ms method but concentrations below 5 µg alliin/ml can be accurately determined. samples described here in this work were diluted as least 20 times to reach the linear calibration range (about 2 µg to 100 ng/µl). so, at any rate the sensivity of the presented hplc-ms method is high enough for quality control purposes. analysis of allium sativum the cso concentration obtained for allium sativum gene bank accessions are presented in fig. 4. these data show the results of the biosensor and of the hplc method as well. tab. 2: characteristics of naturally occurring cysteine sulphoxides based on the individual molecule ions [m+h]+ cso [m+h]+ fragment ion retention time precision r.s.d. calibration range calibration linearity [m/z] [m/z] [min] (n=3) [ng/µl] (r2) methiin 151.8 88.2 1.5 0,17 0.1-20 0.9996 alliin 177.9 88.2 1.8 7.89 0.4-400 0.9994 isoalliin 177.9 88.2 2.0 6.83 0.4-400 0.9998 propiin 179.9 88.2 2.4 0.13 0.2-20 0.9996 s o o nh2 ch2 oh 177.9[m+h] + m/z r el at iv e a b un d an ce r =0.9994 2 fig. 3: hplc-ms calibration curve obtained for alliin (based on the individual signal intensity of the protonated molecule ion at m/z 177.9) and ms spectrum of a pure alliin standard determination of cysteine sulphoxides in allium plants 33 we found a high variability regarding the cso content within the measured allium sativum accessions. samples with the numbers tax 1125 and all 1288 present highest content of total cso (approx. 2.2 g/100 g dry matter) contrary to those plants with the numbers all 1549 or all 781 containing only comparatively low amounts of cso of approx. 0.23 g/100 g dry matter. the mean difference between the two analysed samples a and b (for hplc-ms²) was 0.23 g/100 g dry matter. the main cso present in a. sativum is alliin, whereas methiin was only detected in traces (< 0.1 %). mean standard deviation for the biosensor method was found to be 0.08 g/100 g dry matter. the biosensor method reflected the same trend of cso content of samples as described above for the hplc-ms² method. however, there are some significant differences in detail. we assume that inhomogeneous sample material is the reason for this behaviour. to avoid losses of cso before sample lyophilisation, cloves were only roughly cut. this causes some problems during lyophilisation. some parts of the material could not be properly dried because the sample material got amber-like at its surface preventing water evaporation in the tissue under these zones. it can be assumed that this garlic material showed a dramatically reduced alliin content. because of the limited amount of sample material, only small amounts of each accession could be analysed leading to a big influence of inhomogeneous sample material. in order to truly compare both methods with each other, the individual ratio between the results obtained by the biosensor and those obtained by hplc-ms was calculated. because only very low amounts of cso were detected in all 781, this sample was not included in the calculation. the average ratio was determined to be 0.97, demonstrating that both methods lead to nearly identical results and variations between the two series are highly randomized (as an effect of insufficient homogeneity of sample material). analysis of allium cepa the cso contents determined by both techniques in 15 varieties of a. cepa are shown in fig. 5. in agreement with the results reported in former studies (rabinowitch and currah, 2002) it has been found that the content of total cso is much lower than in the analysed a. sativum accessions (0.2 – 0.8 g/100 g dry matter). both series (biosensor and hplc) showed a rather good correlation. especially those samples, which displayed a relatively low cso concentration obtained by the biosensor (cultivars 'dacapo', 'collito', and 'aranca') were also low by hplc analysis. interestingly, values obtained by the biosensor are lower than those obtained by the hplcms² method. these differences may be explained by the fact that cso are not completely converted to ammonia by the alliinase immobilized in the biosensor. currently, further studies are under investigation aiming to get more detailed information regarding the alliinase selectivity towards individual cso in the near future. in the case of a. cepa, sample preparation in terms of drying was much easier. coarse cut pieces are rather thin and can be sufficiently freeze-dried. therefore, onion sample material is much more homogenous than that obtained from garlic bulbs applying the same method. but nevertheless, because of the good correlation of both methods, the biosensor approach as well as hplc-ms² seem to be suitable for the quality control of a. cepa material. it could be demonstrated that methods described above are rather helpful for a time-saving and effective quality control of garlic and common onion. in case of common onion, freeze drying seems to be an appropriate method for sample preparation. contrary to that, drying of garlic samples causes serious problems regarding sample homogeneity. in contrast to onion, garlic cloves consist of only one thick storage leaf covered with a rather thick skin, which makes freeze-drying more difficult. cutting in smaller pieces probably leads to more homogenous sample material, but will definitely result in lower alliin amounts because of degradation of cso at the cut surface. further research studies are necessary to overcome these problems specially related to insufficient sample homogeneity. both devices used – biosensor and hplc-ms² – are rather complex tools. however, the biosensor approach is much more inexpensive (a tenth of the price of an hplc-ms²-equipment). moreover, only the total amount of cso can be gained by the biosensor approach. as favourable point of this device, it can be fully operated by aqueous solutions. operating costs are, therefore, extremely low. on the other hand, the hplc-ms² device is a rather complex tool, which provides full information about different cso and can be also used for a broad range of further analytical tasks. therefore, this technique provides a very useful support to breeding projects as well as fast evaluation of allium genebank accessions aiming at controlling the content of individual cso. the hplc-ms method presented in this study allows to perform an effective selection of single plants and to measure simultaneously the optimal distribution and amount of all naturally occurring cso in one course of analysis. furthermore, this method can be applied also for quality control purposes in order to check the level of valuable cso in certain all 1288 tax 1125 all 1453 all 937 all 1279 tax 1337 all 1292 all 1284 all 1550 all 1540 all 290 all 1250 all 781 all 818 all 1536 all 1549 0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 1,8 2 2,2 2,4 c o n te n t of c s o ( g / 10 0 g) biosensor hplc-ms² fig. 4: total cso content in various garlic accessions analysed by biosensor and hplc-ms² methods. arenal sunskin h ybelle bonus a vanti d rago collito starito canto d acapo v aleria rx 77796 aranca barito 0 0,2 0,4 0,6 0,8 1 1,2 1,4 c on te n t o f c s o ( g /1 00 g ) biose nsor hplc-ms² fig. 5: total cso content in different allium cepa varieties analysed by biosensor and hplc-ms2 methods. 34 k. ziegert, w. schütze, h. schulz, m. keusgen, f. gun, e.r.j. keller allium species (e.g. garlic, onion, ramson) used as raw materials for the production of various phytopharmaceutical preparations. references block, e., gulati, h., putman, d., 1997: allium chemistry: synthesis of 1-[alk(en)ylsulfinyl]propyl alk(en)yl disulfides (cepaenes), antithrombotic flavorants from homogenates of onion (allium cepa). j. agric. food chem. 45, 4414-4422. freeman, f., huang, b.g., lin, r.i.s., 1994: garlic chemistry – nitric-oxide oxidation of s-(2-propenyl)cysteine and (+)-s-(2-propenyl)-l-cysteine sulfoxide. j. org. chem. 59, 3227-3229. griffiths, g., trueman, l., crowther, t., 2002: onions – a global benefit to health. phytother. res. 16, 603-615. iberl, b., winkler, g., müller, b., knobloch, k., 1990: quantitative determination of allicin and alliin from garlic by hplc. planta med. 56, 320-326. keusgen, m., 1997: tlc analysis of allium sativum constituents. planta med. 63, 93-94. keusgen, m., schulz, h., glodek, j., krest, i., krüger, h., herchert, n., keller, e.r.j., 2002: characterization of some allium hybrids by aroma precursors, aroma profiles, and alliinase activity. j. agric. food chem. 50, 2884-2890. koch, i., keusgen, m., 1998: diastereoselective synthesis of alliin by an asymmetric sulfur oxidation. pharmazie 53, 668-671. krest, i., glodek, j., keusgen, m., 2000: cysteine sulfoxides and alliinase activity of some allium species. j. agric. food chem. 48, 3753-3760. krest, i., keusgen, m., 1999: stabilization and pharmaceutical use of alliinase. pharmazie 54, 289-293. krest, i., keusgen, m., 2002: biosensoric flow-through method for the determination of cysteine sulfoxides. anal. chim. acta 469, 155-164. mana, h., spohn, u., 2000: rapid and selective determination of ammonium by fluorimetric flow injection analysis. fres. j. anal. chem. 366, 825829. namyslo, j.c. (private communication). rabinowitch, r.h., currah, l., 2002: allium crop science: recent advances. cabi publishing, new york. schmitt, b., schulz, h., storsberg, j., keusgen, m.: chemical characterization of allium ursinum l. depending on harvesting time, j. agric. food chem. 53, 7288-7294. schulz, h., krüger, h., liebmann, j., peterka, h., 1998: distribution of volatile sulfur compounds in an interspecific hybrid between onion (allium cepa l.) and leek (allium porrum l.). j. agric. food chem. 46, 5220-5224. schulz, h., krüger, h., herchert, n., keller, e.r.j., 2000: occurrence of volatile secondary metabolites in selected allium wild types. j. appl. bot. 74, 119-121. stoll, a., seebeck, e., 1951: über allium substanzen. 5. die synthese des natürlichen alliins und seiner drei optisch aktiven isomeren. helv. chim. acta 34, 481-487. storsberg, j., schulz, h., keusgen, m., tannous, f., dehmer, k.j., keller, e.r.j., 2004: chemical characterization of interspecific hybrids between allium cepa l. and allium kermesinum rchb. j. agric. food chem. 52, 5499-5505. theodoropoulos, d., 1959: synthesis of certain s-substituted l-cysteines. acta. chem. scand. 13, 383-384. ziegler, s. j., sticher, o., 1989: hplc of s-alk(en)yl-l-cysteine derivatives in garlic including quantitative determination of (+)-s-allyl-l-cysteine sulfoxide (alliin). planta med. 372-378. addresses of the authors: 1 bundesanstalt für züchtungsforschung an kulturpflanzen, institut für pflanzenanalytik, neuer weg 22-23, d-06484 quedlinburg, germany 2 philipps-universität marburg, institut für pharmazeutische chemie, marbacher weg 6, d 35032 marburg, germany 3 institut für pflanzengenetik und kulturpflanzenforschung (ipk), corrensstraße 3, d-06466 gatersleben, germany determination of cysteine sulphoxides in allium plants 35 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) 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<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> >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice vorgabe neu journal of applied botany and food quality 80, 69 81 (2006) 1institute for plant production and agroecology in the tropic and subtropics, university of hohenheim, germany 2institute of botany, university of basel, switzerland revision of entrophospora and description of kuklospora and intraspora, two new genera in the arbuscular mycorrhizal glomeromycetes 1ewald sieverding, 2fritz oehl (received february 7, 2006) summary five mycorrhizal fungal species of the glomeromycetes which were organized in the genus entrophospora are revised. they all form their spores within the hyphal stalk directly beneath or in some distance of a sporiferous saccule formed intercalary or terminally in the mycelium. based on differences respective similarities in spore morphologies and root infection characteristics only entrophospora infrequens and entrophospora baltica remain in this genus. the genus is the type genus for the new family entrophosporaceae. the other three species are organized in two new genera. kuklospora gen. nov. with kuklospora colombiana and kuklospora kentinensis (formerly entrophospora colombiana and entrophospora kentiniensis) is placed into the family of the acaulosporaceae. intraspora gen. nov. so far contains only intraspora schenckii (the former entrophospora schenckii) and is included into the family of the archaeosporaceae. the morphological differences between the genera and the distribution of these fungal species in ecosystems are discussed. introduction arbuscular mycorrhizal (am) fungi are important soil microbiological genetic resources for the functioning of terrestrial ecosystems (see overview in: van der heijden and sanders, 2002). the fungi are taxonomically organized in the recently described new phylum glomeromycota (schüssler et al., 2001). for ecosystem studies it is important that the fungi are correctly named and classified to understand the relationships between the ecosystem components. the glomeromycota have one class, the glomeromycetes (cavaliersmith, 1998). schüssler et al. (2001) named four orden. currently, seven families and nine genera are known in the glomeromycota (http://tolweb.org/tree?group=glomeromycota). of the five mycorrhizal species which were included in the genus, entrophospora r.n. ames & r.w. schneid. (ames and schneider, 1979), e. infrequens (i.r. hall) r.n. ames & r.w. schneid. was originally described by hall (1977) as glomus infrequens. the reason was that its spores showed a short ‘subtending hypha’ giving the spores a glomoid appearance. ames and schneider (1979) discovered that spores of this species are subterminally formed ‘in the stalk of a vesicle’, i.e. directly beneath a terminally or intercalary swollen hypha. such swollen structures were known from acaulospora gerd. & trappe and were called ‘vesicles’ by gerdemann and trappe (1974). they had originally been named ‘mother spore’ by mosse (1970) when she described ‘honey coloured, sessile endogone spores’ . those ‘vesicles’ were later designated ‘hyphal inflated terminus’ (janos and trappe, 1982) or ‘sporiferous saccule’ (walker et al., 1984). the later term has been widely used until today (morton and benny, 1990; stürmer and morton, 1999; oehl et al., 2006). at the site of the hyphal stalk (sensus support) of the sporiferous saccule where the spore of entrophospora is formed, the hyphal stalk is inflated by the growing spore. the spore then appears to have two hyphal attachments – proximal and distal to the saccule – with the hyphal wall forming the outer layers of the spore wall. after degrading and detaching of these outer wall layers and of the sporiferous saccule, the spore of e.g. e. infrequens remains with a short stalk which was its connection with the sporiferous saccule. the mycorrhizal status of the first species described of the genus entrophospora was not clear as formation of mycorrhizae by e. infrequens in pot cultures failed (ames and schneider, 1979). it was a few years later that pure pot cultures were obtained at the centro internacional de agricultura tropical (ciat), cali, colombia, confirming that e. infrequens forms mycorrhiza with pueraria phaseoloides (roxb.) benth. the formation of sporiferous saccules and spores inside the roots and also of arbuscles, vesicles and hyphal structures in the roots was reported (sieverding and toro, 1985). whereas pure pot cultures of three other entrophospora spp., e. colombiana spain & n.c. schenck (schenck et al., 1984), e. schenckii sieverd. & s. toro (sieverding and toro, 1987) and e. kentinensis c.g. wu & y.s. liu (wu et al., 1995b) were repeatedly established (sieverding and toro, 1985; 1987; wu et al., 1995a; fracchia et al., 2003), e. baltica blasz. (blaszkowski et al., 1998) failed to grow in pure cultures. several attempts have been started to again establish e. infrequens in pure cultures but failed over the last years. rodriguez et al. (2001) speculated that spores of e. infrequens may only be formed when the cultures are contaminated with other mycorrhizal fungi, or may form spores only in 2-3 years old pot cultures when growing in consortia with other mycorrhizal fungi. however, sieverding and toro (1985) had reported that their pure pot cultures of e. infrequens were established within nine months when they checked them for purity the first time. species described in the genus entrophospora share one feature: they form a single spore within the hyphal stalk directly beneath or in some distance of a sporiferous saccule. sporiferous saccules are known from the genera acaulospora gerd. & trappe (gerdemann and trappe, 1974) and archaeospora j.b. morton & d. redecker (morton and redecker, 2001), but spores of both genera are formed laterally on the neck (i.e. on the hyphal stalk) of the sporiferous saccule. a closer look at the type of spore formation in addition to observations of the spore wall characteristics and investigations of the root infection structures make it obvious that three of the above mentioned entrophospora spp. species are distinctly different from e. infrequens, and that they have important characteristics in common with other families of the glomeromycetes. of these three species, entrophospora colombiana and e. kentinensis share three characteristics with acaulosporaceae: their spores have three walls (stürmer and morton, 1999; oehl et al., 2006) and a ‘beaded’ wall layer on the inner wall, and they form vesiculararbuscular mycorrhizal structures in the roots which stain visibly blue with trypan blue. on the other hand, entrophospora schenckii shares feature with archaeospora trappei (r.n. ames & linderman) j.b. morton & d. redecker comb. nov. (morton and redecker, 2001): the relative thick inner wall, and the fungal structures that, except spores, do not stain or stain only weakly with trypan blue. the distinct differences justify the exclusion of e. colombiana, e. fig. 1: entrophospora infrequens: a. young spore (spore) developed in the hyphal stalk of a intercalary formed sporiferous saccule (sac). b. crushed spore with three layers of the outer spore wall (sw1, sw2, sw3) and inner wall (iw); saccule wall and parts of the outer spore wall layers staining in melzer’s reagent. detached connection (dc) and intact connection proximal (pc) to the saccule. c. broken spore with connection to the sporiferous saccule. the outer two spore wall layers (sw1-2) are continuous with the wall layers of the saccule (scw1-2). the layer sw3 is not ornamented at the connection to the saccule. pore closed by a plug (plg) formed by sw3. d. four-layered outer spore wall (sw1-4), inner wall (iw) and and rounded projections (orn) of sw3 in cross-view. e. inner wall, isolated from spore, with three layers (iw1-3). f-h. mycorrhizal fungal root structures staining blue to dark blue with trypan blue. f. saccules (sac) and spores (sp) formed in root; extraradical vesicle-like structures (vls). g. arbuscle trunks (hy) and arbuscles (arb), in process of degradation. h. vesicles (ves) and crushed spore (spore) with ornamented spore surface and saccule (sac) attached. kentinensis and e. schenckii from the entrophospora genus and the description of two new genera within other known glomeromycetes families. we include e. colombiana and e. kentinensis into a new genus in the acaulosporaceae, and e. schenckii into a new genus in the archaeosporaceae. the genus entrophospora was formerly part of the acaulosporaceae (morton and benny, 1990) because of similarities in spore ontology. however, the genus must be excluded from the acaulosporaceae not only because of spore morphological differences but also because available and published molecular biological information show that e. infrequens, the type species of 70 ewald sieverding, fritz oehl the genus, is not related to acaulospora spp. (rodriguez et al., 2001; millner et al., 2001; wubet et al., 2003). for entrophospora, we thus erect a new family in the orden diversisporales. material and methods specimen and isolates investigated we re-investigated the spore wall structure, spore formation and also mycorrhizal root colonization structures from type and isoand paratype specimen of e. infrequens, e. colombiana and e. schenckii deposited at osc herbarium in corvallis, oregon, and specimen derived from pure cultures of these species maintained on host plants pueraria phaseoloides (roxb.) benth. or brachiaria decumbens stapf at ciat in cali, colombia, between 1982 and 1986. additionally, specimen of e. colombiana, e. kentinensis (isolate tw111a originating from the type culture established by c.g. wu, taiwan) and e. schenckii were observed by fritz oehl at the international culture collection of arbuscular and vesicular-arbuscular endomycorrhizal fungi (invam), morgantown, west virginia, during his visit in 2002. specimen of e. baltica were isolated from soils that derived from high alpine elevations in the swiss alps. these were compared with the description of its type material and its online presentation (www.agro.ar.szczecin.pl/~jblaszkowski/). further specimen of e. infrequens isolated from soils from bolivia, benin and from swiss mountainous and alpine regions, and specimen of e. colombiana and e. kentinensis from soils derived from benin, tropical west africa, and those of e. schenckii from soils of alpine altitudes of switzerland were also investigated. trap and single species cultures the way of establishing the cultures at ciat, colombia, is explained in sieverding (1991) and consisted in reproducing the native mycorrhizal spore population in trap pot cultures in an acidic organic ultisol or a neutral fertile mollisol (depending on the soil ph of the sampled soil). pueraria phaseoloides was used as the trap plant. these initial cultures were sampled after about 6-9 months and of all species occurring several single species pot cultures were started by inoculating about 5-20 spores per pot. pots were checked for purity of species starting at 3-9 months. in the case of e. colombiana and e. schenckii, species started producing spores quite early and pots yielded many spores when they were checked, 3-6 months after inoculation. the culture pots of e. infrequens were only checked for purity at 9 months after inoculation and not earlier. two pure culture pots of the later species were maintained for another two months, and the soil was then stored in a cold room. whereas cultures of e. colombiana and e. schenckii were frequently renewed, and new isolates from different locations were produced from 1984 until 1986, when the project at ciat terminated, entrophospora infrequens was not intended to be reproduced again due to lack of interest in this species at that time. more recent specimen of these species were isolated from arbuscular mycorrhizal consortia in trap cultures with plantago lanceolata l., lolium perenne l., trifolium pratense l. and hieracium pilosella l. as host plants (for methodology see e.g. oehl et al., 2003; 2006) and maintained at the botanical institute of the university of basel, switzerland, or, in the case of e. schenckii, kindly provided by samar velho da silveira (porto alegre, brazil). spore wall terminology and analyses the terminology of the spore wall structure is basically adapted from invam (see homepage: www.invam.caf.wvu.edu) and from stürmer and morton (1999) with some minor modifications (oehl et al., 2006). we call invam’s ‘spore wall’ ‘outer spore wall’ (sw), the central wall ‘middle wall’ (mw), and the innermost wall (from where usually the germination starts in the glomeromycetes; spain, 1992; spain, 2003; oehl and sieverding, 2004) ‘inner wall’ (iw). the fungal structures were stained using trypan blue as described in sieverding (1991). spores were mounted in lactophenol, polyvinyl alcohol-lactic acid-lactophenol, polyvinyl alcohol-lactic acid-glycerol (pvlg; koske and tessier, 1983), in a mixture of pvlg and melzer’s reagent (brundrett et al., 1994), a mixture of lactic acid to water at 1:1, melzer’s reagent, and in water. analyses of the spore wall and mycorrhizal structures were performed with compound microscopes at 200-630 fold magnification. most photographs in figs. 1-5 were taken with a digital camera (olympus model dp70-cu) on a zeiss axioplan compound microscope. the legends and scales on the photographs of the figures were inserted with adobe photoshop 6.0. revision of entrophospora and erection of entrophosporaceae emendations of species of entrophospora entrophospora infrequens (i.r. hall) r.n. ames & r.w. schneid. emend. oehl & sieverd. (fig. 1) basionym: glomus infrequens i.r. hall, trans. br. mycol. soc. 68, 345-347. 1977. sporocarps unknown. within the extraradical mycelium and sometimes in roots hyphae swell terminally, or intercalary at a septum, and form a sporiferous saccule. a spore grows then within the stalk of the saccule flowing the content of the saccule and of the hypha into the spore. sporiferous saccules usually with a slight tapering stalk (5-9 µm in diameter). saccule hyaline, globose to subglobose, 100-180 x 120200 µm in diameter (fig. 1 a) with a bi-layered wall, 3-12 µm thick in total, that is continuous with the wall of the hyphal stalk. debris sometimes attached to the saccule surface. the saccule usually collapses after spore formation and usually is not found in field soil samples. it may stain slightly pink to pink in melzer’s reagent. spores formed by inflating the hyphal stalk of the sporiferous saccule usually directly adjacent or in 5 -10 µm distance to the saccule (figs. 1 a-c). spores are globose to subglobose, yellow-brown, orange-brown to brown, (90-)110 -140(-175) µm in diameter. spore wall consists of two walls (sw and iw). the outer spore wall (sw; fig. 1 b-d) has four layers that are together (8-)10 -15(-20) µm thick. the first and second layer (sw1 and sw2) are continuous with the wall of the stalk of the sporiferous sacccule (fig. 1 c). layer sw1 is evanescent, hyaline and 1.5 4 (6) µm thick. the second layer is more resistant to degradation than sw1, but has often completely disappeared from old spores. it is hyaline, finely laminate and 2.5-6-(8) µm thick. layers sw1 and sw2 may stain slightly pink in melzer’s reagent. the third layer (sw3) is laminate, golden yellow to orange-brown to brown, (1.5-)2.5 4(-5) µm thick and is ornamented with rounded projections, 1.5-4 µm high and 1-3 µm wide (fig. 1 c, d) that grow into sw2. the projections often have a convex central depression, 0.6 -1.2 µm deep, at the top. the spore wall layer sw3 forms a permanent pigmented part of the connection between the sporiferous saccule and the spore which remains also after degradation of the saccule (fig. 1 c). the innermost layer sw4 is very thin (about 0.5 µm), concolorous and closely adherent to sw3 and thus, usually difficult to observe even in crushed spores (fig. 1 d). the inner wall (iw) is hyaline, and 5-10 µm thick (fig. 1 e) with one revision of entrophospora 71 fig. 2: entrophospora baltica: a. young spore developed in the hyphal stalk of a sporiferous saccules (sac); saccule collapsed. spore with cicatrices formed by detached hypha (dc) and proximal (pc) connection to the saccule. b. mature spore with cicatrix (pc) formed after detaching of saccule c. crushed spore with the hyphal mantle of outer spore wall (sw) and inner wall (iw). d. broken spore showing four layers of outer wall (sw1-sw4) and three layers of inner wall (iw1-iw3). the sw2 is the mantle formed by sinuous hyphae, sw3 is the ornamented wall with warts, sw4 is difficult to concern. e. characteristic hyphal mantle (orn) with round cicatrix. prominent central finely laminated wall layer (iw2). an outer and inner wall layer (iw1 and iw3) are tightly adherent to iw2. both layers are hyaline and very thin (<0.5 µm), and thus are often hardly to detect. cicatrices: the pigmented wall layer sw3 is part of the connection between spore and saccule and continues for a short distance of 37(-10) µm into the saccules wall (fig. 1 c). after detaching of the saccule the remaining rest resembles a wide cicatrix. the spore pore at this cicatrix is about 3-7 µm wide and closed by a plug arising from sw3 (figs. 1 b-c), and by sw4. distal to the saccule, a hypha is attached that is continuous with the two outer spore wall layers. when this hypha is broken off, a cicatrix is also visible as long as sw2 is not deteriorated (fig. 1 b). germination: so far the germination structures in this species have not been detected. mycorrhizal formation: forms vesicular-arbuscular mycorrhiza (sieverding and toro, 1985; figs. 1 f-h). in some roots also hyphal coils were observed in root cells. vesicle like structures, globose to subglobose, 15-40 µm in diameter with 0.5 µm thick walls are also formed intercalary or apically on root external hypha near roots (fig. 1 f). these often collapsed in lactophenol. sporiferous saccules and spores can be formed in roots (figs. 1 f, h). fungal structures stain light blue to visibly blue in trypan blue (figs. 1 fh). specimen observed: paratype osc #39,678 and osc #42,409 deposited by ames and schneider (1979). specimen from a pure pot culture, number c-132-1 at ciat, colombia, 1984-1985, nr. 28272831 and nr. 2835 of sieverding collection, deposited at z+zt. specimen nr. 71-7101 to 71-7110 of oehl collection deriving from bolivia, benin and switzerland, deposited at z+zt. distribution: this species was first isolated from forest soils in new zealand (hall, 1977), and thereafter from a horticultural field in california and from agricultural soybean and corn fields in iowa, illinois and wisconsin (ames and schneider, 1979). there are many reports about the occurrence of this species, from acidic and neutral soils, and from many natural ecosystems and agricultural land use systems from all over the world, e.g. from colombia (sieverding and toro, 1985), brazil (maia and trufem, 1990), from mexico (oehl, unpublished), from the united states in wyoming (stahl and christensen, 1982), florida (schenck and smith, 1982) and rhode island (koske et al., 1997), from canada (boyetchko and tewari, 1993), finland (vestberg, 1995), poland (blaszkowski, 1993), germany, france and switzerland (oehl et al., 2003; 2004; 72 ewald sieverding, fritz oehl 2005), from india (sridhar and beena, 2001), from namibia (uhlmann et al., 2004) and australia (hall and abbott, 1984). we recently found it also in trap cultures inoculated with soils from tropical bolivia (oehl and pérez-camacho, unpublished data) and benin (tchabi and oehl, unpublished), and also in montainous and high alpine areas of switzerland and in the foreland of the morteratsch glacier near pontresina, engiadina, switzerland that was covered by the ice until about 30 years ago. entrophospora baltica blasz., madej & tadych, mycotaxon 68, 166-167. 1998 (fig. 2) this species does not need an emendation. it is well described (blaszkowski et al., 1998) and was presented on-line (www.agro.ar. szczecin.pl/~jblaszkowski/). we recognize the spores formed within the stalk of a sporiferous saccule (fig. 2 a), and the spores having an outer spore wall (sw) covered with a hyphal mantle with a round cicatrix visible when the saccule is broken off (fig. 2 b), and an inner wall (iw; fig. 2 c). the outer spore wall has four layers (sw1-4). the outer layers and the hyphal mantle are sw1 and sw2 (fig. 2 d). the sinuous folding of the hyphal mantle and the pigmented spore wall layer sw3 with its fine warts on the outer surface are characteristic for this species (figs. 2 c-e). layer sw4 is very thin and closely adherent to sw3 and thus hardly to detect. for the inner wall (iw) it should be noted that a very thin innermost layer (iw3) was found (fig. 2 d) that can also be observed in fig. 4 of the original description of the species (blaszkowski et al, 1998). the pore at the spore base proximal to the sporiferous saccule is closed by a plug similar as in e. infrequens. the inner wall layer of the sporiferous saccule, the hyphal mantle and the hypha distal to the sporiferous saccule may stain slightly pink to pink with melzer’s reagent in some specimen what was not mentioned in the protologue. mycorrhizal formation: unknown specimen observed: spores isolated from several locations in the swiss alps, e.g. in dolomitic soils at piz corvo (2700 m a.s.l., cantone ticino; oehl collection, slides nr. 72-7201 to 71-7204 deposited at z+zt). distribution: this species is known from northwestern poland (blaszkowski et al., 1998) and from slowenski national park (tadych and blaszkowski, 2000) in acidic to neutral soils (ph3.6-6.7) and was recently found in switzerland at several alpine elevations (e.g. at 2600 m a.s.l. at haldensteiner calanda, chur, kanton graubünden and at 2700 m a.s.l. in soils with ph 6.5-8.1 at piz corvo, cantone ticino, oehl, unpublished). it also formed part of the mycorrhizal community of forest ecosystems near valdivia in southern chile (castillo and sieverding, unpublished). emendation of genus description entrophospora r.n. ames & r.w. schneid. emend. oehl & sieverd. basionym: entrophospora r.n. ames & r.w. schneid, mycotaxon 2, 347-348. 1979. type species: entrophospora infrequens (i.r. hall) r.n. ames & r.w. schneid. emend. oehl & sieverd. basionym: glomus infrequens i.r. hall, trans. br. mycol. soc. 68, 345-347. 1977. sporocarps unknown. spores are formed by inflating the hyphal stalk directly beneath of a sporiferous saccule. spores globose to subglobose and have two walls: an outer spore wall and an inner wall. part of the outer layers of the outer spore wall are the wall layers of the hyphal stalk and the sporiferous saccule. the outer spore wall layers often degrade and are evanescent; the inner layers of the outer spore wall are more permanent and often extend for a short distance into the saccule. a plug formed by such a permanent wall layer closes the pore towards the saccule. the inner wall consists of a thick, finely laminated wall layer that might have a thin layer adherent to its outer and inner surface. none of the layers of the inner wall have a beaded appearance. fungal structures in roots stain blue with trypan blue. forming vesicular-arbuscular mycorrhizae. species remaining in the genus entrophospora entrophospora infrequens (i.r. hall) r.n. ames & r.w. schneid. emend. oehl & sieverd. basionym: glomus infrequens i.r. hall, trans. br. mycol. soc. 68, 345-347. 1977. entrophospora baltica blasz., madej & tadych, mycotaxon 68, 166167. 1998. species excluded from the genus entrophospora: entrophospora colombiana spain & n.c. schenck, mycologia 76, 693-694. 1984. entrophospora kentinensis c.g. wu & y.s. liu, mycotaxon 53, 283. 1995. entrophospora schenckii sieverd. & s. toro, mycotaxon 28, 210. 1987. erection of new family entrophosporaceae fam. nov. oehl & sieverd. type genus: entrophospora r.n. ames & r.w. schneid. (mycotaxon 2, 348. 1979) emend. oehl & sieverd. latin diagnosis: spora singulatim efformata in hypham inflatam, anguste adiacetum ad sacculum sporiferum terminalem vel intercalarem. sporae globosae vel subglobosae cum tunicis duabus: tunica exterior et tunica interior. pauciores strata exteriores sporarum coniuncta tunica hyphae et sacculi. strata interiores tunicae exterioris persistentes. tunica interior sporae subtiliter laminata et perpetua, sine strato granulato. formans structuras mycorrhizarum vesicular-arbuscularum. structurae fungarum colorantes caeruleae cum ‘trypan blue’. spore formed by swelling directly beneath and within the hyphal stalk of a terminally or intercalary formed sporiferous saccule. spores have two walls: an outer spore wall and an inner wall. layers of the outer spore wall are continuous with the wall of the stalk and the saccule. the inner layers of the outer spore wall are persistent and may form part of the connection with the sporiferous saccule. a plug closes the pore proximal to the saccule. the inner wall consists of a thick, finely laminate layer that might have a thin layer adherent to its outer and inner surface. none of the layers of the inner wall have a beaded appearance. forms vesicular-arbuscular mycorrhiza those fungal structures stain blue in trypan blue. discussion: the erection of the new entrophosporaceae family is mainly based on the distinct morphological differences between spores of its type genus, entrophospora n. ames & r.w. schneid. revision of entrophospora 73 emend. oehl & sieverd., and the two genera of the acaulosporaceae, acaulospora gerd. & trappe and kuklospora gen. novum oehl & sieverd. (see below). morton and benny (1990) included the genus entrophospora r.n. ames & r.w. schneid. into the acaulosporaceae mainly due to parallelisms in spore ontogeny and organization of spore wall structures of those species which we place into kuklospora (see below), with those of the genus acaulospora. it is, however evident that there is no known parallelism between e. infrequens or e. baltica and any of the acaulospora spp. distinct morphological differences between entrophospora and the genera of the acaulosporaceae are: i) spores in entrophospora have two walls, and spores in the acaulosporaceae have three walls. ii) in entrophospora a plug is formed to close the connection between the sporiferous saccule and the spore after the content of the saccule is emptied to form the spore; such plug is not formed in acaulospora. iii) entrophospora has a relative thick inner wall absent in acaulosporaceae. vi) most acaulosporaceae spores have a characteristic ‘beaded’ wall layer as part of the inner wall which is absent in entrophospora spores. in addition to the morphological differences, all molecular biological data generated for the phylogenetic position of entrophospora infrequens (millner et al., 2001; rodriguez et al., 2001; wubet et al., 2003) indicate that this species is not related to acaulosporaceae. hence, morphological and biochemical differences justify the exclusion of entrophospora from acaulosporaceae and the erection of the new family entrophosporaceae. both the families, entrophosporaceae and acaulosporaceae have species which form vesicular-arbuscular mycorrhiza, and the fungal structures in roots stain visibly blue with trypan blue, an important differentiating characteristic which was used by morton and redecker (2001) when they described the archaeosporaceae and paraglomeraceae those hyphal structures stain not or only weakly in trypan blue. new genus in acaulosporaceae kuklospora gen. novum. oehl & sieverd. type species: kuklospora colombiana (spain & n.c. schenck) oehl & sieverd. comb. nov. basionym: entrophospora colombiana spain & n.c. schenck, mycologia 76, 693-694. 1984. sporocarpia ignota. spora singulatim efformata subterminaliter vel intercalariter in hypham inflatam en distantiam ad sacculum sporiferum terminalem vel intercalarem. sporae globosae vel subglobosae, tribus tunicis stratis pluribus. stratum exterior tunicae exterioris coniunctum tunica hyphae et sacculi. stratum interiorem laminatum tunicae exterioris duo pora sporae occludans. stratum exterior tunica interioris granulatum, stratum medium tunicae interioris ex consuetudine colorans purpureum reagente melzeri. area germinationis in tunica interiore. formans mycorrhizas vesicular-arbusculares. structurae fungarum colorantes caeruleae cum ‘trypan blue’. sporocarps unknown. spores formed by inflating the hyphal stalk in some distance to a terminally or intercalary formed sporiferous saccule. spores have three walls: an outer spore wall, a middle wall and an inner wall. one or a few layers of the outer spore wall are continuous with the wall of the stalk and the sporiferous saccule. these layers are often evanescent. inner layers of the outer spore wall are permanent. when the connections of the hyphal stalk break off, the spore appears with two, often opposite, cicatrices which are closed by the permanent sublayers of the inner layers of the outer spore wall. the middle wall is thin and flexible. the inner wall consists of several thin layers of which the outer layer is ornamented having a characteristic ‘beaded’ appearance. the second layer of the inner wall usually stains deep purple in melzer’s reagent. the inner wall functions as germinal wall, a germination orb may be formed. forming vesicular-arbuscular mycorrhizae. the fungal structures in the roots stain significantly blue with trypan blue. etymology: greek: kuklo(κυκλος, ring), and spora (σπορα, spore), referring to the two cicatrices which resemble circular depressions and the boarders of them wedding rings on the spore surface when the hyphal connection have detached from young spores. species included into the genus kuklospora: kuklospora colombiana (spain & n.c. schenck) oehl & sieverd. comb. nov. basionym: entrophospora colombiana spain & schenck mycologia 76, 693-694. 1984. kuklospora kentinensis (c.g. wu & y.s. liu) oehl & sieverd. comb. nov. basionym: entrophospora kentinensis c.g. wu & y.s. liu mycotaxon 53, 283. 1995. notes to kuklospora colombiana (spain & n.c. schenck) oehl & sieverd. comb. nov (fig. 3) the basionym is well described in schenck et al. (1984). we want to give some additional remarks to the wall structure, applying our spore wall terminology. the spores are formed by swelling of the hyphal stalk of a terminal or intercalary formed sporiferous saccule (figs. 3 a-c). the spores have three walls (fig. 3 d-g): a threelayered outer spore wall (sw1-sw3), a thin, one to bi-layered middle wall (mw1 and mw2) and an inner wall that is three-layered (iw1iw3). on the outer spore wall and on the inner wall usually only the two outer layers are easy to detect, while the very thin closely adherent inner layers of these two walls (sw3 and iw3, respectively) are difficult to observe – they are not mentioned in the protologue. the middle and the inner wall form de novo after the pore towards the sporiferous saccule is closed. characteristic of kuklospora are the ‘beaded’ appearance of iw1 (fig. 3 g) and the dark purple reaction of iw2 in melzer’s reagent (fig. 3 f). the same characteristics have only species of the genus acaulospora. the pore at the connection to the sporiferous saccule is closed by continuous sublayers of sw2 and by the adherent, thin layer sw3. after the hypha with the sporiferous saccule is detached from the mature spore, a circular depression is seen those boarder resembles a ring (figs. 3 d-e). a second round cicatrix on the spore, opposite to the sporiferous saccule, remains when the distal hyphal connection breaks off. the outer hyaline layer of the spores frequently degrades and disappears on mature and older spores, and in such spores, in particular isolates from field samples, the cicatrices might be absent. a germination orb between iw1 and iw2 was found by spain (1992). mycorrhiza formation: forming typical vesicular-arbuscular mycorrhizae (figs. 3 h-i) with fungal root structures that stain significantly blue with trypan blue (type specimen in osc). specimen observed: isotype specimen deposited at osc, #42,497. type and isotype specimen from ciat (measured by sieverding and toro, 1985; sieverding collection). specimen from invam reference accession cl148 obtained by fritz oehl during his visit at invam in 2002. spores isolated from trap cultures inoculated with soils from benin and maintained in basel by atti tchabi, in 2005. 74 ewald sieverding, fritz oehl fig. 3: kuklospora colombiana: a. intercalary in hypha (hy) formed sporiferous saccule (sac) with stalk (stalk) b.-c. progressing spore (spore) formation by swelling in stalk in some distance from saccule (sac); hypha (hy) connected. d.-e. broken spores with outer spore wall (sw), middle wall (mw), inner wall (iw) and two round connections proximal (pc) to the saccule (sac) and distal (dc) to the hypha (hy); the connections appear like rings from where the name kuklospora derives. f. broken spore with three-layered outer spore wall (sw1-3), bi-layered middle wall (mw1-2), and two layers of the inner wall (iw1-2); iw1 is beaded (granular) and iw2 stains purple in melzer’s reagent. g. broken spore with two-layered middle wall (mw1-2) and three-layered inner wall (iw1-3). h.-i. fungal structures in root staining blue with trypan blue; intraradical hyphae (hy), arbuscles (arb) and vesicle (ves). distribution: kuklospora colombiana was first isolated from various locations in colombia (schenck et al., 1984; sieverding and toro, 1985). it was also reported from several states in brazil (balota et al., 1999; de souza et al., 2002; carrenho et al., 2002; caproni et al., 2003; dos anjos et al., 2005) and from india (mehrotra, 1998; selvam and mahadevan, 2002) and the philippines (oba et al., 2004). we recently found it in trap cultures inoculated with tropical soils from benin (tchabi and oehl, unpublished). we infrequently found this species in grasslands of lowlands and at mountainous elevations in southern germany and switzerland. in germany, k. colombiana was also isolated from an acidic sandy soil near berlin (baltruschat, pers. com.). so far, k. colombiana was only recorded from acidic soils. notes to kuklospora kentinensis (c.g. wu & y.s. liu) oehl & sieverd. comb. nov. (fig. 4) the sporogenesis of k. kentinensis was well described by wu et al. (1995b) and has well been presented on-line (http://www.tari.gov.tw/ act/sporogenesis-ektn.htm) as well. spore development is similar to k. colombiana (fig. 4 a). using our spore wall terminology the spores have three walls (fig. 4 d) : a three-layered outer wall, a thin, bi-layered middle wall and a twoto three-layered inner wall that shows the typical ‘beaded’ appearance on the outer surface (fig. 4 e-f). the beaded appearance was not mentioned in the protologue but in wu et al. (1995a) and in the internet presentation. the layer iw2 of the inner wall stains purple in melzer’s reagent (fig. 4 d-f), revision of entrophospora 75 fig. 4: kuklospora kentinensis: a. spore formed in the stalk in some distance of a terminally formed sporiferous saccule (sac). b. young broken spore with characteristic pitted surface ornamentation. c. connection of spore with hypha (dc) and sporiferous saccule (pc); three walls (sw, mw, iw); part of sw form the connection to the saccule, and part of sw closes the pore. d. broken spore with three spore walls (sw, mw, iw) and broken connections to hypha (dc) and sporiferous saccule (pc). part of iw stains dark purple with melzer’s reagent. e. broken spore with three-layered outer wall (sw1-3), middle wall (mw) and two layers of the inner wall (iw1, iw2); iw2 stained by melzer’s. f. middle and inner walls isolated from spore. middle wall two-layered (mw1-2); inner wall with characteristic ‘beaded’ ornamentation (iw1) and iw2 staining purple in melzer’s reagent. 76 ewald sieverding, fritz oehl iw3 is very difficult to concern and is not shown in the figures. a prominent feature for k. kentinensis is the characteristic pitted ornamentation on the second outer spore wall layer sw2 (fig. 4 b, c). in some spores the ornamented and pigmented sw2 extends over a distance of about 10-40(-100) µm into the connection to the sporiferous saccule (fig. 4 c). the pore proximal to the saccule is about 15-40 µm wide and is closed by sublayers of sw2; the spore pore opposite to the saccule is smaller. when the sporiferous saccule and the distal hypha are detached, the two cicatrices on the spore surface can appear ring-like. mycorrhizal formation: forming mycorrhizae with arbuscles and vesicles; the fungal structures stain blue to dark blue with trypan blue (wu et al., 1995a; 1995b). specimen observed: specimen #74-7401 (oehl collection) obtained by oehl during his visit at invam in 2002 originating from a pure culture that had been established with spores isolated from taiwanese soils and having the invam reference accession number tw111a. specimen #74-7411 to #74-7414 derived from trap cultures inoculated with agricultural soils from tropical benin (oehl collection, deposited at z+zt). distribution: kuklospora kentinensis was first known from taiwan (wu et al., 1995a; 1995b). it was also reported from brazil (caproni et al., 2003; dos anjos et al., 2005). we recently found k. kentinensis in soils from tropical benin (western africa; tchabi and oehl, unpublished). so far, it is only known from tropical areas with a wide range of soil ph (4.0-8.0). discussion of kuklospora kuklospora colombiana and k. kentinensis have morphological characteristics in common with acaulospora spp. which were discussed for the basionym of kuklospora colombiana (e. colombiana) by morton and benny (1990) so that a further discussion is not necessary. morton and benny (1990) refer to the potential parallelisms of spore formation in two different genera. considering this, k. colombiana has its sister species in a. dilatata (morton and benny, 1990), and k. kentinensis has similarities with a. scrobiculata (wu, on-line presentation: http://www.tari.gov.tw/act/sporogenesisektn.htm). the two genera can be easily distinguished by the formation of spores on the neck of a sporiferous saccule in acaulospora and by the formation of spores within the stalk of the sporiferous saccule in kuklospora. as shown above, species of the new genus kuklospora differ phylogenetically from entrophospora (e.g. millner et al., 2001) and are related to acaulospora (redecker, 2000; redecker et al., 2000; nielsen et al., 2004). kuklospora spp. are characterized by formation of vesicular-arbuscular mycorrhizal fungal structures in roots which stain blue to dark blue with trypan blue. this differentiates kuklospora from the new genus intraspora (see below). new genus in archaeosporaceae intraspora gen. nov. oehl & sieverd. type species: intraspora schenckii (sieverd. & s. toro) oehl & sieverd. comb. nov. basionym: entrophospora schenckii sieverd. & s. toro, mycotaxon 28, 210. 1987. sporocarpia ignota. sporae in solo vel in radice efformatae, singulatim in hyham inflatam, en distantiam ad sacculum sporiferum terminalem vel intercalarem. sporae globosae vel subglobosae vel oblongatae, duabus tunicis. stratum exterior sporae coniuncta tunica hyphae et sacculi; stratum interiorem tunicae exterioris duo pora sporae occludens. tunica interior sporae semiflexibilisque stratis paucioribus. formans mycorrhizam arbuscularem; vesiculae absentes vel rarae. arbusculae hyphaeque non tinguntur vel ubi tinguntur pallidae reagente ‘trypan blue’. tunica interior sporarum tinguntur caerulea vel obscura caerulea. sporocarps unknown. spores formed singly in soils and in roots, by swelling in the hyphal stalk in a short distance to a sporiferous saccule which is terminally or intercalary formed in the intraor extraradical mycelium. spores are globose to subglobose to oblong, with two walls: an outer spore wall and an inner wall. the outer layer of the outer spore wall is continuous with the wall of the hyphal stalk and the saccule. this outer layer is often evanescent on mature spores. the inner layer of the outer wall is permanent and closes the two spore pores. the inner wall of the spore is finely laminated and semi-flexible. the fungal structures in the roots do not or only very weakly stain with trypan blue. forming arbuscular mycorrhizae; formation of vesicles is rare. etymology: latin: intra (inside, within), spora (spore), referring to the spore formation within the hyphal stalk of the sporiferous saccule. species included into the genus intraspora: intraspora schenckii (sieverd. & s. toro) oehl & sieverd. comb. nov. (fig. 5). basionym: entrophospora schenckii sieverd. & s. toro, mycotaxon 28, 210. 1987. the original description of the spore morphology of i. schenckii by sieverding and toro (1987) was confirmed by fracchia et al. (2003). using our spore terminology, i. schenckii forms small spores (45-75 µm) by swelling in the hyphal stalk of a terminally or intercalary formed sporiferous saccule (figs. 5 a-b). spore formation can be in soils and also in roots. within roots the sporiferous saccule may be formed within one root cell and the spore in another cell. however, formation of sporiferous saccule and spore within one root cell appear also be possible. the spores have two walls: an outer bi-layered spore wall and an inner wall (figs. 5 c-d). the outer layer sw1 of the outer spore wall is continuous with the hyphal and saccule wall. this wall layer can readily separate in broken spores from sw2 (fig. 5c); sw1 is often absent on mature spores. spore wall layer sw2 is permanent and closes the two pores of the hyphal attachments. there, two circular depressions (cicatrices) are visible on young spores when the sw1 is still present and the connections to the stalk have detached. the inner wall is finely laminated and semi-flexible. on the outer and inner side of wall layer iw2 very thin layers (iw1 and iw3) are sometime seen (fig. 5 d); they are hard to detect in specimen mounted in pvlg but also appear on photos published by fracchia et al. (2003) although they were not described. the inner wall of the spore stains blue to dark blue in trypan blue (fig. 5 e). mycorrhizal formation: mycorrhizae formation is concluded from spores formed in the cortical cells of roots. the inner wall of i. schenckii spores formed in roots or in soil stained dark blue with trypan blue (figs. e-f) but none of the other structures stained in the studies of sieverding and toro (1985; 1987). spores or fungal structures did not stain with acid fuchsin or methylen blue. intraspora schenckii was reported by fracchia et al. (2003) to form arbuscules and seldom small vesicles in roots. in their investigation, the fungal structures stained weak with trypan blue in roots of vigna revision of entrophospora 77 fig. 5: intraspora schenckii: a. spore formed in the stalk of a sporiferous saccule (sac); hyphal connection (hy) opposite to sporiferous saccule; outer spore wall with two layers (sw1, sw2) and inner wall (iw). b. spore with broken connection to hypha (dc) and with connection to sporiferous saccule (pc); the outer layer of the outer spore wall is continuous with the the saccule wall. c. broken spore with two-layered outer wall (sw1-2) and inner wall (iw). spore had been conserved in formalin for more than 20 years, sw2 stained slightly yellow in melzer’s reagent. d. inner broken spore wall isolated from spore; three layers (iw1-3). e. part of inner wall stains with trypan blue; hyphal wall (hy) and saccule wall (sac) do not stain. f. spores formed in roots of tropical kudzu; only part of inner spore wall stains with trypan blue. 78 ewald sieverding, fritz oehl sp. but did not stain in tomato or clover. sieverding and toro (1987) described the species after culturing with p. phaseoloides (tropical kudzu). specimen observed: type specimen, osc #46,030. spores obtained from the type culture pot # c-133-8 with p. phaseoloides (sieverding collection #2858, 2863, 2871 and 2881 deposited at z+zt). specimen from estado do rio grande do sul in brazil (s. velho da silveira collection). specimen from a single species culture maintained at invam (oehl collection nr. 75-7511). specimen obtained from a trap culture inoculated with a jurassic limestone soil originating from high alpine area in switzerland (oehl collection). distribution: so far, this species was found in a neutral phosphate rich soil in cundinamarca, colombia (sieverding et al., 1985; 1987), in southern brazil (velho da silveira and oehl, unpublished), in argentina (fracchia et al., 2003), poland (blaszkowski, personal communication), india (mohankumar et al., 1988; ragupathy and mahadevan, 1993), thailand (bhadalung et al., 2005) and in the swiss alps (oehl, unpublished). discussion of intraspora intraspora schenckii is so far the only species in the new genus. it differs from the other two genera which form spores in the hyphal stalk of a sporiferous saccule firstly by the spore wall structure; i. schenckii has two walls like entrophospora spp. and not three like kuklospora. however, whereas entrophospora has a much more complex outer spore wall and the spore formation directly adherent to the saccule, intraspora has a rather simple outer spore wall and its spore formation is in some distance to the sporiferous saccule. the more important difference to entrophospora and kuklospora however is the absence or only weak staining of the root infection structures with trypan blue. such weak or faintly staining was the main characteristic described by morton and redecker (2001) for the archaeosporaceae j.b. morton & d. redecker and paraglomeraceae j.b. morton & d. redecker as ‘arbusculae hyphaeque mycorrhizarum ubi tinguntur invisibiles vel pallidae’. indeed vesicles are not or only rarely formed by i. schenckii (fracchia et al., 2003) who were able to faintly stain arbuscles in roots of vigna. the absence of a reaction of the hyphae and arbuscles to trypan blue in our studies and the high similarity of the wall structure of i. schenckii with archaeospora trappei (r.n. ames & linderman) j.b. morton & d. redecker comb. nov. (morton and redecker, 2001) led us to the conclusion that i. schenckii must be related to the archaeosporaceae. archaeospora trappei has a very similar spore wall structure (hafeel, 2004). spores of these two species separated from field soils are almost not distinguishable when the outer wall layer is detached. field collected spores of i. schenckii are also not distinguishable from field collected a. myriocarpa spain, sieverd. & n.c. schenck (schenck et al., 1986) those mycorrhizal structures also only faintly stain without vesicle formation (schenck et al., 1986) and thus, is likely to be another small-spored archaeospora species. the morphological similarities of i. schenckii with the small-spored archaeospora spp. are strong and the difference between both genera is in the sporogenesis: the spore formation in the hyphal stalk instead laterally on the hyphal neck of a sporiferous saccule. the way of such different spore formation was also used in the past to differentiate related genera (morton and benny, 1990). we interprete i. schenckii as sister species of ar. trappei as both species have almost identical spore wall structures, and mycorrhizal root infection and fungal staining characteristics in common. hence, we consider intraspora a sister genus to archaeospora and, thus, include intraspora in the archaeosporaceae. since the formation of spores in the hyphal stalk of a sporiferous saccule was not described for archaeosporaceae (morton and redecker, 2001), we here have to emend the description of this family. emendation of archaeosporaceae archaeosporaceae j.b. morton & d. redecker, emend. oehl & sieverd. type genus: archaeospora trappei (r.n. ames & linderman) j.b. morton & d. redecker. emend. spain. basionym: acaulospora trappei r.n. ames & linderman. mycotaxon 3, 566. 1976. spores monomorph or bimorph; glomoid spores formed singly or in aggregations in soils or sometimes in roots, terminally on subtending hyphae, acaulosporoid and entrophosporoid spores formed singly, in clusters or in sporocarps in soils or in roots. acaulosporoid spores formed laterally on the neck of a sporiferous saccule; entrophosporoid spores formed by swelling in the hyphal stalk of a sporiferous saccule. glomoid spores with one spore wall of one to multiple layers, acaulosporoid and entrophosporoid spores with two or more walls with each monoto multiple-layers. the fungal structures in the roots do not or only weakly stain with common dyes like trypan blue; forming arbuscular mycorrhizae; no or only very rare formation of vesicles and vesicle-like structures. acknowledgements we are thankful to the swiss national fonds programme nfp48‚ landscapes and habitats of the alps’ (‚landschaften und lebensräume in den alpen’) and the center for tropical agriculture (zil) at eth zürich for financial support on the ongoing projects. we especially thank joseph b. morton and william wheeler (west virginia state university; morgantown, usa) for giving f. oehl the opportunity to investigate, during his visit in 2002, many amf isolates maintained at invam. janusz blaszkowski (department of plant pathology, adademy of agriculture, szeczin, poland) provided type specimen of e. baltica. samar velho da silveira (departamento de horticultura e sivicultura, universidade do rio grande do sul, porto alegre, brazil) provided slides of intraspora schenckii originating from vineyards of southern brazil. atti tchabi, fabien hountondji and louis lawouin (international institute of tropical agriculture, benin) and luis rodrigo pérez-camacho (agruco, universidad mayor de san simon, cochabamba, bolivia) provided soil samples from their countries, and we acknowledge the help of sue furler, david schneider and florian haas (university of basel) in maintaining trap cultures. references ames, r.n., schneider, r.w., 1979: entrophospora, a new genus in the endogonaceae. mycotaxon 2, 347-352. balota, w.l., lopes, e.s., hungria, m., döbereiner, j., 1999: occurrence of 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(eds.), physiological and genetical aspects of mycorrhizae. proceedings of the 1st european symposium on mycorrhizae, dijon, 1-5 july 1985, 621-626. inra, service des publications, versailles, france. sieverding, e., toro, s., 1987: entrophospora schenckii, a new species in the endogonaceae from colombia. mycotaxon 28, 209-214. spain, j.l., 1992: patency of shields in water mounted spores of four species in acaulosporaceae (glomales). mycotaxon 43, 331-339. spain, j.l., 2003: emendation of archaeospora and of its type species, archaeospora trappei. mycotaxon 87, 109-112. 80 ewald sieverding, fritz oehl sridhar, k.r., beena, k.r., 2001: arbuscular mycorrhizal research in coastal sand dunes: a review. proc. nat. acad. sci. india. 71, 179-205. stahl, p.d., christensen, m., 1982: mycorrhizal fungi associated with bouteloua and agropyron in wyoming sagebrush-grasslands. mycologia 74, 877-885. stürmer, s.l., morton, j.b., 1999: taxonomic reinterpretation of morphological characters in 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kentinensis spp. nov. mycotaxon 53, 283-294. wubet, t., weiss, m., kottke, i., oberwinkler, f., 2003: morphology and molecular diversity of arbuscular mycorrhizal fungi in wild and cultivated yew (taxus baccata). can. j. bot. 81, 255-266. address of the authors: priv. doz. dr. ewald sieverding (corresponding author, sieverdinge@aol. com), institute for plant production and agroecology in the tropics and subtropics, university of hohenheim, garbenstrasse 13, d-70593 stuttgart, germany. dr. fritz oehl, institute of botany, university of basel, hebelstrasse 1, ch4056 basel, switzerland. revision of entrophospora 81 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true 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<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> >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice journal of applied botany and food quality 86, 66 70 (2013), doi:10.5073/jabfq.2013.086.010 centre for phytomedicine research, department of botany, university of fort hare, alice, south africa evaluation of the polyphenolic contents and some antioxidant properties of aqueous extracts of garlic, ginger, cayenne pepper and their mixture g.a. otunola, a.j. afolayan* (received march 22, 2013) * corresponding author † this study was conducted at the center for phytomedicine research, de partment of botany, university of fort hare, alice 5700, south africa. summary garlic (allium sativum), ginger (zingiber officinale), and cayenne pepper (capsicum frutescens) are common culinary spices that are used singly or combined in the diet of many populations of the world and there is a long-held belief of their health-enhancing properties. this study investigated the aqueous extracts each of garlic, ginger, cayenne pepper and a combination of the three for polyphenolic and antioxidant properties that might justify such claims. antioxidant activities were studied using dpph, abts, nitric oxide radical scavenging activities and reducing power assay. each of the spice extracts showed high content of phenolics, flavonoids, flavonols and proanthocyanidins, with the pepper extract exhibiting the highest concentration of each polyphenol investigated. the antioxidant activities of the spices and their mixture were concentration dependent, though positively comparable with the standards used. among the extracts, the mixture exhibited the highest antioxidant activity compared to the individual spices and standards probably due to a synergistic effect of combining the spices. the present study confirmed that the aqueous extracts of garlic, ginger and pepper exhibited significant polyphenolic content and antioxidant potentials. introduction the formation of free radicals – reactive oxygen species (ros) and reactive nitrogen species (rns), as a by-product of cellular metabolism have been implicated in the generation of oxidative stress that leads to the pathogenesis of several human diseases such as atherosclerosis, diabetes mellitus, chronic inflammation, neurodegenerative disorders, aging and certain types of cancer (valko et al., 2004). the putative protective effects of antioxidants against these deleterious oxidative-induced reactions have received increasing attention lately, especially within biological, medical, nutritional, and agrochemical fields. in addition, the efficacy of plant derived phytochemicals on human ailments related to dietary habits has gained renewed interests in the recent past. because these nonnutrient bioactive compounds are consumed in significant amounts through the diet, they may have long term physiological benefits without harmful side effects (halliwell and guteridge, 1989; hazra et al., 2008; rao et al., 2010). spices and herbs have been used to treat various diseases and ailments for thousands of years. many herbs and spices, apart from their use as aroma additives in foods, have been reported to be excellent sources of phenolic compounds with good antioxidant activities. food phenolics, especially from spices and their extracts, may therefore augment the body’s source of natural antioxidants and also prevent or delay some chemical deterioration during storage of fat-containing food systems (politeo et al., 2006). garlic, ginger and cayenne pepper have been used for thousands of years for culinary and medicinal purposes in many communities of the world. garlic has been used around the world in cooking and to treat many conditions including hypertension, infections, snakebites, reducing cholesterol levels, as antineoplastic and antimicrobial (koch and lawson, 1996; tattleman, 2005). ginger is used in cooking and has been shown to exhibit antithrombotic, antirheumatic, anti-inflammatory and cholesterol reducing activities (srivastava and mustafa, 1992; fuhrman et al., 2000). medicinal applications of capsicum has a long history dating back to the mayas and aztecs, and is best known today as an ingredient in hot sauces, as a digestive aid, in the treatment of arthritis, cough, colds and to regulate blood pressure (antonious et al., 2006; beltran et al., 2007). this study evaluated the antioxidant properties of the aqueous extracts of the three spices and their mixture comparing them with well known antioxidants in order to justify the much acclaimed medicinal potentials of these spices and to examine the effect of combining the three spices. materials and methods garlic, ginger and pepper were purchased from the ipata market in ilorin, nigeria. dpph (di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium), folin-ciocalteus reagent, sodium carbonate, abts (2,2’-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid), sulfanilic acid, vanillin, ascorbic acid (vitamin c), butylated hydroxyl toluene (bht) and rutin were purchased from sigma-aldrich chemical co (st. louis, mo, usa). all other reagents used in this study were of analytical grade. preparation of spice extracts each of the spices – garlic, ginger and pepper – were individually sorted to remove grits and dirt, washed and thinly sliced. they were dried in the oven at 60 °c for 72 h. the dried spices were individually milled into fine powder, packed into airtight plastic bottles and stored at 4°c until needed. from the powdered samples, 50 g of each spice was mixed with 1000 ml of distilled water and boiled for 10 min at 100 °c. it was allowed to cool, filtered and then freezedried (vir tis bench top k, vir tis co. gardiner, ny). the freezedried sample was reconstituted with distilled water to give desired concentrations used in this study. preparation of spice mixture extract a mixture of the ground spices was prepared by weighing equal amounts of each spice [(50:50:50 g), (w/w/w)] which were thoroughly mixed together by passing through a coffee grinder of a home blender set. fifty grams (50 g) of this mixture was mixed with 1000 ml of distilled water and boiled for 10 min at 100 °c. it was allowed to cool, filtered and then freeze-dried (vir tis bench top k, vir tis co. gardiner, ny) for 48 h. this mixture was stored in an airtight plastic bottle at 4 °c and reconstituted with distilled water to give desired concentrations as needed for the various analyses. polyphenolic and antioxidant properties 67 total phenolics the folin-ciocalteu’s reagent was used to determine the total phenolic content of the extracts (wolfe et al., 2003). 2.5 ml of folin-ciocalteu’s reagent diluted with distilled water 1:10 v/v was mixed with 0.5 ml of the spice extract and 2.0 ml (75 g/l) of sodium carbonate. the tubes were vortexed for 15 s and allowed to stand for 30 min at 40 °c to develop the color. the absorbance was read at 765 nm using a hewlett packard uv-vis spectrophotometer. samples were evaluated at a final concentration of 0.1 mg/ml. total phenolic content was expressed as mg/g tannic acid equivalent using the equation based on the calibration curve: y = 0.1216x, r2 = 0.9365; where x is the absorbance and y is the tannic acid equivalent (mg/g). total flavonoids flavonoid content was determined as described by ordoñez et al. (2006) using quercetin as standard. 0.5 ml of 2 % alcl3 was added to 0.5 ml of the spice extract, incubated at room temperature for 1 h and the absorbance measured at 420 nm. the result was expressed as mg/ g using the equation: y = 0.0255x, r = 0.9812; where x is the absorbance and y the quercetin equivalent. all determinations were in triplicates. total flavonols total flavonols was determined by the method described by kumaran and karunakaran (2007). to 2.0 ml of the spice extract was mixed 2.0 ml of alcl3 in ethanol, then 3.0 ml of sodium acetate solution (50 g/l) was added to the mixture. this was incubated at 20 °c for 150 min and the absorbance read at 440 nm. total flavonol content was calculated as quercetin (mg/g) equivalent from the calibration curve using the equation: y = 0.0255x, r2 = 0.9812, where x is the absorbance and y the quercetin equivalent in mg/g. total proanthocyanidin the method described by sun et al. (1998) was used to determine the total proanthocyanidin in the spice extracts. in a test tube, 3.0 ml of vanillin-methanol mixture (4 % v/v) was mixed with 0.5 ml of 1 mg/ml of the spice extract, 1.5 ml of hcl was added, vortexed and the solution allowed to stand at room temperature for 15 min. absorbance was read at 500 nm and total proanthocyanidin was expressed as catechin equivalent using the equation derived from the calibration curve: y = 0.5825x, r2 = 0.9277, where x is the absorbance and y the catechin equivalent in mg/g. determination ferric ion reducing power the reducing power of the spice extracts were determined according to the method described by oyaizu (1986). 1.0 ml of the extract was prepared in cold distilled water. bht and vitamin c (0.2-1.0 mg/ ml) were mixed individually with a 0.5 ml of 0.2m phosphate buffer (ph 6.6) and 0.5 ml potassium ferricyanide (1% w/v). the resulting mixtures were incubated at 50 °c for 20 min and 2. 5 ml of 10 % trichloroacetic acid added to each of them. the entire mixture was centrifuged at 3000 rpm for 10 min and 2.5 ml of the supernatant was mixed with 2.5 ml of distilled water, 0.5 ml of ferric chloride (0.1 %) was added, and the absorbance read at 700 nm. ascorbic acid and bht were used as standards for comparison. nitric oxide scavenging activity nitric oxide scavenging activity was determined by the method of garrat (1964). in a test tube, 2.0 ml of a 10 mm sodium nitroprusside prepared in 0.5 ml saline phosphate buffer (ph 7.4), was mixed with 0.5 ml of the spice extracts, bht and rutin individually at various concentrations (0.2-1.0) (rutin and bht were used as standards to compare the nitric oxide scavenging activities of the spice extracts). the mixture was incubated at 25 °c for 150 min after which 0.5 ml of the solution was mixed with 0.5 ml griess solution [(1.0 ml sulphanilic acid reagent0.33 % in 20 % glacial acetic acid at room temperature for 5 min) with 1 ml naphthylethylenediamine dichloride (0.1 % w/v)], incubated for 30 min at room temperature and the absorbance read at 540 nm. nitric oxide scavenging activity was calculated as abs (control)-abs (sample)/abs (control) x100. where abs (control) is the absorbance of no radical + methanol, and abs (sample) is the absorbance of no radical +sample extract or standard. dpph scavenging activity this was determined according to the method described by liyanapathiranan and shahidi (2005). to 0.5 ml of the extract in methanol (0.2-1.0 ml) in a test tube was added 2.5 ml of 0.5 mm methanolic solution of dpph, shaken vigorously and incubated for 30 min in the dark at room temperature. absorbance was read at 517 nm and ascorbic acid was used as positive control. dpph free-radical scavenging activity was calculated as % dpph inhibition = abs (control)-abs (sample) / x100. where abs (control) is the absorbance of dpph radical + methanol, and abs (sample) is the absorbance of dpph radical +sample extract or standard. abts radical scavenging activity the method described by re et al. (1999) was used to determine the abts scavenging activity of the spices. two stock solutions of 7 mm abts and 2.4 mm potassium persulphate v/v were mixed together, allowed to react for 12 h at room temperature in the dark and used as the working solution. this was further diluted by mixing in 1 ml of freshly prepared abts solution to obtain an absorbance of 0.706 ± 0.001 units at 734 nm using the spectrophotometer. plant extracts of different concentrations (0.2-1.0)were allowed to react with 1 ml of the abts+ and the absorbance was read at 734 nm after 7 min. percentage abts+ inhibition by the extract was calculated and compared with bht and rutin using the equation: % abts+ scavenging activity = abs (control)-abs (sample) / x 100. where abs (control) is the absorbance of abts radical + methanol, and abs (sample) is the absorbance of abts radical + sample extract or standard. statistical analysis the results were expressed as mean ± standard deviation, and were subjected to one way analysis of variance (anova) and differences between means were separated by the duncan’s multiple range test (duncan, 1955) using sas, (2002) where p < 0.05 was considered significant. results the total polyphenolic content of the spice extracts are shown in tab. 1. while the flavonoids content in garlic and ginger were not significantly different (p < 0.05) from one another, both were significantly different from the values for pepper and the mixture extracts. of the four extracts, pepper had the highest flavonoids content. the 68 g.a. otunola, a.j. afolayan total phenolics content in mg/g tannic acid equivalents of the four extracts was also highest in pepper, with the value for the mixture being significantly (p < 0.05) lower compared to the other extracts and the phenolic contents of garlic and ginger not being significantly (p > 0.05) different from each other. total flavonol content was significantly high in pepper compared to the two other extracts and the mixture as recorded in quercetin equivalents, while the same trend was observed for the proanthocyanidin content of the four extracts. in effect, pepper exhibited the highest polyphenolic content among the three spices and their mixture. the ferric reducing power is used to evaluate the antioxidant components in dietary polyphenols. fig. 1 shows the reducing power of the spice extracts compared to vitamin c and bht (which were used as standards). vitamin c activity was significantly higher (p < 0.05) than that of bht and the spice extracts. the extract of the mixture exhibited the highest reducing action of all the extracts and at all concentrations, followed by the pepper extract, with garlic showing the least activity. had an ic50 of 1.12 mg/ml, followed by ginger, garlic and pepper with ic50 of 1.21, 1.4 and 1.55 mg/ml, respectively. this could be an indication that the spices exhibited additive action in dpph sca-venging. of the two positive controls, bht had the higher scavenging property than vitamin c at concentration of 1.0 mg/ml. the extracts were also analyzed for free radical scavenging activity against abts+. the extracts exhibited significant (p < 0.05) abts scavenging activities as their concentration increased getting to the peak at concentrations of 1.0 mg/ml (fig. 3). the activities of the extracts from ginger, pepper and the mixture against abts+ were quite high and not significantly different (p < 0.05) from those of the standards vitamin c and bht at concentrations of 0.8 and 1.0 mg/ml, while that of garlic though equally high was significantly lower (p < 0.05) than the others. the ic50 was 0.04 mg/ml in ginger, 0.10 in the mixture, while for pepper and garlic were 0.45 and 0.66, respectively. tab. 1: total flavonoid, phenolic, proanthocyanidin and flavonol content of extracts of, garlic, gingers, pepper and their mixture (mg/ml) polyphenol garlic ginger pepper mixture flavonoid 4.08a 3.99b 7.05c 6.55d phenols 23.02a 22.09b 47.18c 18.97d proanthocyanidins 5.79a 3.74b 10.66c 9.4d flavonol 27.96a 32.62b 59.42c 55.34d *a-d values are means of 3 determinations ±sem. ** values along the same row with different superscripts are significantly different (p<0.05). fig. 1: reducing power of aqueous extracts of garlic, ginger, pepper and their mixture. a-f values are means of 3 determinations ± sem. bars with different colours carrying different letters are significantly different (p<0.05). vit. c = vitamin c, bht = butylated hydroxyl anisole (standards). there was a significant decrease in the concentration of the dpph+ radical due to the scavenging ability of the spice extracts (fig. 2) which was dose-responsive. it is interesting to note again that the 1, 1-diphenyl-2-picrylhydrazyl (dpph.) scavenging activity of the mixture though not as high as those of the standards (vitamin c and bht), was significantly higher (p < 0.05) than for the individual spices. the ic50 (concentration of sample needed to scavenge 50% of dpph) was calculated by linear regression of plots. the mixture fig. 2: dpph radical scavenging activities of garlic, ginger, pepper and their mixture. a-d values are means of 3 determinations ± sem. bars with different colours carrying different letters are significantly different (p<0.05). vit. c = vitamin c, bht = butylated hydroxyl anisole (standards). fig. 3: abts activities of extracts of garlic, ginger, pepper and their mixture. a-f values are means of 3 determinations ± sem. bars with different colours carrying different letters are significantly different (p<0.05). vit. c = vitamin c, bht= butylated hydroxyl anisole (standards). polyphenolic and antioxidant properties 69 the scavenging activities of the spices on nitric oxide radical were high for all the extracts (tab. 2). nitric oxide scavenging activity increased in a concentration dependent manner, with maximum inhibition exhibited at 1.0 mg/ml of the extracts. the mixture showed the highest inhibition of 90.40, followed by pepper, garlic and ginger at 87.40, 87.00 and 82.67 %, respectively. pepper and the mixture extract exhibited significantly high (p < 0.05) activity against the nitric oxide radicals at all concentrations, which were competitively comparable with the standards while garlic and ginger showed lower activities which were significantly different from one another. discussion polyphenolic compounds such as flavonoids, flavonols, proanthocyanidin and phenolics in plants have been reported to have strong antioxidant activities which help to protect cells against oxidative damage by free radicals (mccune and john, 2002; liao et al., 2008). flavonoids and phenols have been recognized to have antioxidant effects on human nutrition and health. their mechanisms of action are through scavenging, chelating and termination of free radicals (kessler et al., 2003). in this study, all the spices exhibited high phenolic and proanthocyanidin contents which have been reported to have high antioxidant activities (luximon-ramma et al., 2002). these activities probably account for some of the pharmacological effects of these spices on many diseases caused by free radicals. in addition, flavonoids and phenolic compounds are effective in preventing the formation of reactive oxygen species and protecting low density lipoprotein (ldl) from ironand coppermediated free radical production (owen and john, 2002). the reducing power of the extracts increased with increasing concentrations, with the extract from the mixture exhibiting the highest reducing power at 1.0 mg/ml among the extracts though vitamin c(the standard), showed the greatest activity. the trend observed was vitamin c>bht>mixture>pepper>ginger>garlic. deore et al. (2009) reported the reducing power of croton caudatum ethanol extracts as a function of their concentration, a trend observed in this study. the model of scavenging the stable dpph radical is a widely used method in the evaluation of the free radical scavenging ability of various compounds (ghaseni et al., 2009). fig. 3 depicts that the scavenging activities of all the extracts and the standards for dpph are dose responsive, that is, the higher the concentration, the greater the scavenging activity. high antioxidant activity of a. sativum has been reported by benkeblia (2005), but activity depends on both phenolics and sulphur compounds of the alliums. nuutila et al. (2003) and capasso (2013) in studies comparing the antioxidant properties of different allium species and antioxidant action of garlic respectively reported that the lowest antioxidant activity was in garlic (a. sativum), this is similar to the trend observed in this study between the activities of garlic, ginger, pepper and the mixture of the three spices, as garlic showed the least free radical scavenging activity with dpph, abts and the least reducing power. ginger showed almost similar activity against free radicals like garlic though in some instances it showed higher values. according to ghasemzadeh et al. (2010), ginger showed high antioxidant activity with dpph though less than that of the standard used. this is similar to our observation in the present study as ginger showed high scavenging activities with both dpph and abts. pepper (chili) on the other hand showed consistently high reactivity for reducing power, dpph, abts and nitric oxide scavenging. this reactivity was highly correlated with total flavonoids, phenolics, proanthocyanidin and other bioactive compounds (capsaicinoids, carotenoids) of the extract which was similar to the trend observed by benkeblia (2005) and bae et al. (2012), though rahiman et al. (2013) reported that capsicum frutescens seed oil exhibited the least antioxidant activity among some common home remedies with storage time. the consumption of these spices therefore, could offer protection against chronic diseases caused by free radicals and could also augment cellular defenses against oxidative damage. we report here for the first time the antioxidant activities of a mixture of garlic, ginger and pepper. interestingly, when these spices were combined, the total antioxidant activity increased which did not show any correlation with the phenolic content. the extract of the mixture exhibited the highest free radical scavenging activity with dpph, abts and nitric oxide as well as the greatest reducing power. this may be as a result of synergistic activity of the antioxidant powers of the three spices. this trend is similar to the findings of shobana and naidu (2000) who reported that a spice mix of (ginger, onion and garlic; onion and ginger; ginger and garlic) showed a cumulative inhibition of lipid peroxidation by exhibiting a synergistic property when compared with the individual spices. seah et al. (2010) also reported a similar trend with keanghleung paste (a mixture of turmeric rhizome, garlic and chili). these attributes implies that a mixture of these spices could be more effective in combating diseases than each spice used in isolation. conclusion this study has revealed that aqueous extracts of garlic, ginger and pepper singly or as a mixture, possess high polyphenolic content and high antioxidant activities in different systems providing support for their acclaimed health benefits. therefore, further study of their effectiveness in animal models of disease and oxidative stress would be undertaken to provide a useful basis for nutritional advice. tab. 2: percentage (%) nitric oxide scavenging activity of the aqueous extracts of garlic, ginger, pepper and their mixture concentration (mg/ml) garlic ginger pepper mixture rutin** bht** 0.2 80.79±0.02a 74.61±0.03b 83.58±1.05c 85.30±0.33d 82.20±1.27c 94.50±1.27e* 0.4 81.10±0.01a 75.84±0.09b 84.40±1.20c 85.40±0.10c 86.06±0.67c 96.50±1.27d 0.6 85.20±0.00a 77.24±0.04b 86.51±0.00c 86.50±0.17c 94.95±00d 97.41±1.83e 0.8 86.25±0.14a 79.84±0.04b 87.00±0.04c 87.41±0.06c 95.12±1.75d 98.22±2.42e 1.0 87.00±0.02a 82.67±0.03b 87.40±0.15a 90.40±0.10c 98.53±0.67d 98.73±2.92d *a-e values are mean of 3 determinations ± sem. **rutin and bht are standards. ***values along the same row with different superscripts are significantly (p<0.05) different. 70 g.a. otunola, a.j. afolayan acknowledgements the authors wish to thank the govan mbeki research development centre, university of fort hare, alice 5700, south africa for supporting the research. references antonius, g.f., kochhar, t.s., jarret, r.l., snyder, j.c., 2006: antioxidants in hot pepper: variations among accessions. j. environ. sci. health. b 41, 1237-1243. bae, h., jayaprakasha, g.k., jifon, j., pati, b.s., 2012: variation of antioxidant activity and the levels of bioactive compounds in lipophilic and hydrophilic extract from hot pepper (capsicum spp.) cultivars. food chem. 134, 1912-1918. beltran, j., ghosh, a.k., basu, s., 2007: immunotherapy of tumors with neuroimmune ligand capsaicin. j. immunol. 178, 3260-3264. benkeblia, n., 2005: free-radical scavenging capacity and antioxidant properties of some selected onions (allium cepa l.) and garlic (allium sativum l.) extracts. braz. arch. biol. technol. 48, 753-759. cappaso, a., 2013: the antioxidant action and therapeutic efficacy of allium sativum l. molecules. 18, 690-700. deore, s.l., khadabadi, s.s., baviskar, b.a., khadabadi, s.s., 2009: in vitro antioxidant and phenolic content of croton caudatum. int. j. chem. tech. res. 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radicals and antioxidants in normal physiological functions and human disease. int. j. biochem. cell. biol. 39, 44-84. wolfe, k., wu, x., liu, r.h., 2003: antioxidant activity of apple peels. j. agric food chem. 51, 609-614. address of the corresponding author: centre for phytomedicine research, department of botany, university of fort hare, alice 5700, south africa. e-mail: aafolayan@ufh.ac.za journal of applied botany and food quality 87, 74 79 (2014), doi:10.5073/jabfq.2014.087.011 1department of biology, abbes laghrour university, khenchela, algeria 2department of biology, mentouri university, constantine, algeria the role of osmoprotectants and antioxidant enzymes in the differential response of durum wheat genotypes to salinity a. fercha1*, h. gherroucha2 (received august 25, 2013) * corresponding author summary this study aims to investigate the importance of accumulation of osmoprotectants and activities of some key antioxidant enzymes in genotypic variation (gv) observed among durum wheat genotypes in response to increasing nacl salinity (0-200 mmol/l) at seedling stage. germination and seedling growth traits of all the genotypes were significantly decreased by salinity. mohamed ben bachir, the more salt-tolerant genotype, exhibited the lowest reduction in final germination percentage (fgp, <18%) and seedling growth (<60%, based on dry biomass), the lowest increase in proline (pro) and water soluble carbohydrates contents but the highest increase in catalase (cat) and ascorbate peroxidase (apx) activities. correlation and principal components analysis revealed that the most important variables distinguishing salt tolerant vs. salt non-tolerant genotypes were root to shoot ratio (r/s, 36.1%), cat (30.6%), apx (12.5%) and fgp (5.74%). although pro and wsc could play a key role in salt tolerance by mediating osmotic adjustment, these compounds do not seem to be significantly involved in genotypic variation (gv) for salinity tolerance in durum wheat. introduction salinity which affects more than 800 mha of world’s land area (fao, 2008), is nowadays, one of the most damaging environmental factors that limit growth and productivity of crop plants (munns and tester, 2008). in algeria, for instance, more than 3.2 mha of arable land already suffer from salinisation (benmahioul et al., 2009). overall, salinity adversely affects plant growth and development not only through imposition of osmotic stress, ion toxicity, but also by secondary effects such as oxidative stress and hormonal imbalance (ashraf et al., 2012). even though durum wheat (triticum durum desf.) is regarded as one of the most widely cultivated crops, especially in the mediterranean basin where about 75% of total durum wheat production is provided (habash et al., 2009), its production is often limited by poor seed germination and stand establishment, mainly due to drought or soil salinity (sayar et al., 2010). hence, it is imperative to develop wheat genotypes with improved salt tolerance. this is of obvious importance in the case of tetraploid wheat, which lacks the d genome, making it less tolerant to salt stress (munns et al., 2012). proper seed germination and seedling emergence are required traits to achieve a high grain yield in wheat (paulsen, 1987). therefore, it has been established that under arid conditions, the most valuable cultivars are those, which are emerging rapidly because rainfall after sowing may result in a soil crust that prevents seedling emergence (tahir, 2010). in addition, the early emerging and the fast growing crops are able to take full advantage of the soil moisture leading to better seedling establishment and grain yield. although the major variation in the coleoptile length is genetic, environmental conditions can significantly affect this trait (hakizimana et al., 2000). accordingly, identifying the physiological and biochemical attributes associated with a more vigorous and rapidly growing seedling, particularly under stress conditions will be very meaningful (rashid et al., 1999). seed germination and seedling emergence are well-regulated processes characterized by high metabolic activity mediated by reactive oxygen species (ros) in the cell (bailly, 2004). antioxidants act to scavenge ros, and therefore play an important role in the regulation of growth processes that occur during germination such as radicle protrusion or coleoptile emergence (bailly, 2004). while superoxide dismutase (sod) is the most important antioxidant enzyme synthesized in response to oxidative stress, its effectiveness was dependant on the h2o2-scavenging enzymes activity notably that of ascorbate peroxidase (apx) and catalase (cat). the combined action of these two enzymes seems to play an important role in plant growth and production since seed filling was associated with a high potential of the h2o2-detoxification, often due to apx and cat activities (costa et al., 2010). in recent years, numerous studies have been conducted to investigate whether there are differences between several durum wheat genotypes in their response to salinity (munns et al., 2012; rashid et al., 1999; sayar et al., 2010). however, to the best of our knowledge no work has been done to investigate the difference in responses to salinity of wheat genotypes used in the present study, particularly with respect to their h2o2-scavanging capacity and osmoprotectants accumulation. therefore, this study aimed to investigate the genotypic variation (gv) for salinity tolerance among three durum wheat landraces selected based on their good adaptation to drought-stressed mediterranean environments. materials and methods experimental details and treatments germination assay and growth measurements: the present investigation was performed in a randomized complete design (rcd) with two factors and three replications. certified seeds of durum wheat (triticum durum desf.) vars. mohamed ben bachir (mbb), gta-dur and waha were surface sterilized with sodium hypochlorite (5% w/v) for 3 min, washed several times against sterile water and dried in oven. thirty dried seeds were then placed in each petri dish containing two layers of whatman. no. 1 filter paper moistened with 10 ml of one of the prepared solutions (100 or 200 mmol/l nacl) or distilled water (control). seeds were germinated in darkness in a temperature-controlled chamber held at 22 ± 0.5 °c. the number of germinated seeds was counted every day until day 7, when there were no more germinated seeds. final germination percentage (fgp) was calculated at the end of the germination period (7 days). mean germination time (mgt) was calculated according to ellis and roberts (1981): mgt = ∑dn/∑n where n is the number of seeds germinated on day d, the number of days counted from the beginning of germination. final length of shoot (coleoptile) and root of seedlings seven days post-sowing were measured and used for vigor index (vi) estimation according to abdul-baki and anderson (1973): differential response of durum wheat genotypes to salinity 75 vi = fgp × sl where sl is seedling length (cm). the salt tolerance index (st) was calculated according to cano et al. (1998): st = (fw in nacl-saline solution) × 100/(fw in nacl-free solution) where fw is fresh weight. physiological and biochemical changes physiological and biochemical measurements were in general based on shoot (coleoptile) samples of three 7-day-old seedlings of uniform size from each cultivar. relative water content: shoot relative water content (rwc) was determined according to barr and weatherley (1962): rwc = (fw dw) × 100 / (tw dw) where fw is fresh weight, dw is dry weight and tw is turgid weight. proline and water-soluble carbohydrate content: quantitative determination of free proline (pro) content was performed according to bates et al. (1973). the optical density of the solution is read on a spectrophotometer at a wavelength of 528nm. the proline concentration was determined using a calibration curve of known l-proline concentration. water-soluble carbohydrates (wsc) content was determined according to dubois et al. (1956). absorbance was measured at 485nm. the wsc concentration was determined using a calibration curve of known d-glucose concentration. hydrogen peroxide and lipid peroxidation assays: h2o2 content was determined according to velikova et al. (2000). approximately 100 mg of fresh shoot samples were homogenized in 0.1% 5 ml of trichloro-acetic acid (tca), and centrifuged for 15 min at 12,000 rpm. a 0.5 ml of the supernatant is then mixed with 0.5 ml of buffer (potassium phosphate 10 mm ph 7) and 1ml of 1 m ki. the absorbance reading was taken at 390 nm. lipid peroxidation (lpo), however, was estimated as malondialdehyde (mda) according to heath and packer (1968). approximately 500 mg of fresh shoot samples were homogenized in 10 ml of 0.1% tca and the homogenate was centrifuged for 15 min at 15,000 rpm. to a 1.0 ml aliquot of the supernatant, 4.0 ml of 0.5% thiobarbituric acid (tba) in 20% tca were added. the mixture was heated at 95 °c for 30 min, and then cooled in an ice bath. after centrifugation for 10 min at 10,000 rpm at 4 °c, the absorbance of the supernatant was recorded at 532 nm on a spectrophotometer. the mda content was calculated according to its molar extinction coefficient of 155 mm-1 cm-1. h2o2-scavenging enzymes extraction: about 200 mg of fresh shoot tissues were collected from stressed and control seedlings and quickly frozen in liquid nitrogen and ground to a fine powder using a prechilled mortar and pestle. the exact weight of each powdered sample was determined before it was thoroughly homogenized in 1.2 ml of 0.2 m potassium phosphate buffer (ph 7.8 with 0.1 mm edta). the samples were centrifuged for 20 min at 15,000 rpm at 4 °c. the supernatant was removed, the pellet resuspended in 0.8 ml of the same buffer, and the suspension centrifuged for another 15 min at 15,000 rpm at 4 °c. the combined supernatants were stored on ice and used to determine antioxidant enzyme activities (elavarthi and martin, 2010). h2o2-scavenging enzymes activity assay: the activity of cat (ec 1.11.1.6) was measured according to the method of chandlee and scandalios (1984) with little modification. the assay mixture contained 2.6 ml of 50 mm potassium/phosphate buffer (ph 7.0), 0.4 ml of 15 mm h2o2 and 0.04 ml of enzyme extract. the decomposition of h2o2 was followed by the decline in absorbance at 240 nm. the enzyme activity was expressed in units mg-1 protein (u = 1 mm of h2o2 reduction min-1 mg-1 protein). the extinction coefficient of h2o2 (40 mm-1 cm-1 at 240 nm) was used to calculate the enzyme activity that was expressed in terms of mm of h2o2 per minute per gram fresh weight. apx (ec 1.11.1.11) activity was assayed using a modified method of nakano and asada (1981). apx activity was determined by measuring decreased absorbance at 290 nm due to ascorbate oxidation. the assay mixture (1 ml) contained 50 mm potassium phosphate buffer (ph 7.0), 0.5 mm ascorbate, 0.5 mm h2o2, and 10 μl of crude extract. h2o2 was added last to initiate the reaction, and the decrease in absorbance was recorded for 3 min (u = 1 mm of ascorbic acid reduction min-1 mg-1 protein). the extinction coefficient of 2.8 mm-1 cm-1 for reduced ascorbate was used in calculating the enzyme activity that gram fresh weight. the enzyme protein was estimated by the method of bradford (1976). statistical analysis for statistical analysis, the data of germination percentage were transformed by arcsine transformation. data were subjected to analysis of variance (anova) procedures, while the least significant difference (lsd) test at α = 0.05 level of significance was used to compare the differences among treatment means. correlation analysis and principal component analysis (pca) were carried out to examine the relationships between and among the factors and variables using the statistical software package statistica 8.0 (hill and lewicki, 2007). results germination assay: as shown in fig. 1a, final germination percentage (fgp) was adversely affected by nacl salinity. the decrease in fgp caused by moderate salinity (100 mm) was less than 9% in all the genotypes, whereas high salinity (200 mm) induced substantial reduction in fgp especially in the genotype waha, which has lost nearly 70% of its germination capacity. statistical analysis revealed a significant effects for both factors (salinity ‘s’ and genotype ‘g’) and their interaction (p<0.001) (tab. 1), suggesting the existence of a genetic variation. unlike the fgp, the mean germination time (mgt) was only marginally affected by moderate salinity, whereas high salinity resulted in a more pronounced increase of mgt, especially in the genotype waha. growth analysis shoot and root lengths: nacl salinity decreased more or less significantly shoot and root lengths in all genotypes in a concentration-dependent manner (fig. 1b). with the exception of the decrease in root to shoot ratio (r/s) recorded by the genotype waha at 200 mm nacl (> 40%), salinity seems to increase significantly the r/s (fig. 1c). it was noteworthy that the genotype mbb had the highest r/s. total dry weight and vigor index: among the tested wheat genotypes, the total dry weight (dw) was affected more or less to the same extent by salinity (fig. 1c). this effect was more pronounced when the concentration of nacl becomes higher. whilst root growth (particularly in genotype mbb) was much less affected, which 76 a. fercha, h. gherroucha fig. 1: effect of salinity (mm) on (a) final germination percentage (fgp) and mean germination time (mgt); (b) shoot (sl) and root lengths (rl); (c) root:shoot ratio (r/s) and total dry weight (dw); (d) salt tolerance index (st) and vigor index (vi) of durum wheat genotypes. values are means of three replicates ± se. tab. 1: f and lsd values of the effects of salinity (s), genotype (g) and the interaction between them (s×g) on the dependent variables. variation f lsd salt genotype interaction salt genotype fgp 73.53*** 12.03*** 6.45*** 0.16 0.19 mgt 55.07*** 6.95** 8.40*** 0.35 0.137 sl 107.8*** 0.60 ns 1.35 ns 4.2 ns rl 67.60*** 2.01 ns 3.69* 10.32 ns r/s 4.95* 5.50* 8.25*** 1.11 0.87 dw 83.53*** 4.57 * 0.88 ns 2.34 1.33 vi 136*** 3.43 ns 3.29* 16.01 ns st 775.9*** 18.0*** 8.24*** 30.75 7.83 rwc 22.63*** 3.33 ns 2.49 ns 7.49 ns pro 1200*** 8.27** 47.69*** 67.6 8.76 wsc 210.3*** 10.58 ns 3.41* 62.66 ns mda 16.33*** 7.62** 0.43 ns 0.34 0.23 h2o2 25.92*** 26.11*** 1.58 ns 11.96 5.83 cat 6.90** 5.93** 10.65*** 0.99 0.93 apx 13.3*** 6.55** 5.87** 8.57 6.18 *p< 0.05; **p< 0.01; ***p< 0.001; ns = not significant. explains the high r/s, high salinity (200 mm) had reduced plant biomass by 50% to 70% compared to control groups. increasing salinity induced a significant linear decline in seedling vigor index (vi) and salt tolerance index (st) of all tested cultivars (fig. 1d). physiological and biochemical parameters: relative water content: as shown in fig. 2a, rwc of wheat seedlings, especially in genotype gta (~84%) showed only slight and non-significant decrease with increasing nacl concentration. differential response of durum wheat genotypes to salinity 77 proline and water soluble carbohydrates contents: according to fig. 2a, salinity induced a significant (p<0.001) increase of proline content of all genotypes. however, genotype gta treated with 200 mm nacl showed the highest amount of proline content (up to 3-folds compared to control) (tab. 1). in all genotypes, nacl induced a significant (p<0.001) accumulation of wsc in a concentration-dependent manner (fig. 2a). while wsc content does not appear to differ significantly from one genotype to another, the existence of a significant interaction genotype × salt stress (tab. 1) gives rise to conclude that these genotypes respond differently with increasing salt level. hydrogen peroxide content and lipid peroxidation: from the fig. 3a it is apparent that the amount of h2o2 increased in response to increasing salinity. fig. 3a shows also that under both salinity levels, lipid peroxidation occurs in seedling tissues of all wheat cultivars. ascorbate peroxidase and catalase activity: unexpectedly salt stress affected the activity of apx independently of nacl concentration and differently from one genotype to another (fig. 3b). nevertheless, at 200 mm salt stress increased the activity of apx in the three genotypes with respect to their respective controls. on the other hand, salinity had not only affected differently the activity of cat, but regardless of its concentration (fig. 3b). however, at 200 mm, unlike genotype waha cat activity increased in the genotypes gta and mbb. multifactorial analysis: results of multifactorial comparison generalized over the entire set of data (432 entries) using the principal components analysis (pca), were given in fig. 4a and 4b. it is noteworthy that every single genotype was presented by 9 points (3 levels of salinity × 3 repetitions). pca results revealed that the factor 1 (first principal component or pc1) explained 63.69% of the total data variation and had positive correlation with the growth/germination performance under both stress and nonstress environments (fig. 4a and fig. 1a-d). thus this component was able to separate the genotypes according to their growth/ germination potential under the three different stress treatments. the pc 2 explained 11.04% of the total data variation. the first two pcs accounted for 74.73% of total variation. considering the projection of the variables, it appears that the mean values of proline and water-soluble carbohydrates decreased from the negative side of pc1 to its positive side (fig. 4b). this reflects the highly significant negative relationships among proline, wsc and all the growth parameters (r over to 0.8, p<0.001). pca results also indicated that the indices could discriminate the tolerant genotypes are the r/s ratio (36.1% of contributions), cat (30.6%), fgp (5.74%), which showed positive correlations with salt tolerance, and apx (12.5%), which showed a negative correlation with tolerance to salinity (fig. 4b). fig. 2: effect of salinity (mm) on (a) proline content (pro), water soluble carbohydrates (wsc) and relative water content (rwc) of durum wheat genotypes; (b) relationships of rwc with pro and wsc contents. values are means of three replicates ± se. fig. 3: effect of salinity (mm) on (a) malondialdehyde (mda) and hydrogen peroxide (h2o2) contents; (b) catalase (cat) and ascorbate peroxidase (apx) activities of durum wheat genotypes; (c) relationships between the apx activity and h2o2 and mda contents. values are means of three replicates ± se 78 a. fercha, h. gherroucha discussion it is evident from the results that nacl salinity significantly decreases the percentage of germination (fgp) and delay the germination (mgt) in all the genotypes (fig. 1a and tab. 1). in this respect, waha appears to be the most affected genotype. these results are in line with previous findings (zhang et al., 2010). salinity can affect germination of seeds either by imposing osmotic stress, which prevent water uptake, or by ion toxicity, which inhibits the metabolism of dividing and expanding cells, delaying germination and even leading to seed death (zhang et al., 2010). the first visible evidence of germination is the protrusion of the radicle through the seed coat. this is the result of cell enlargement and, to a lesser extent, cell division (haber and luippold, 1960), both of which are very sensitive to dehydration, suggesting that salinity indirectly reduces growth of seminal roots by water privatization. indeed, our results support this finding, since the decrease in germination performances was correlated to the decrease in rwc, particularly in waha, and because a rwc of about 80% may trigger the accumulation of aba that inhibit germination and radicle protrusion. as on germination, salt stress decreases almost all of growth parameters (fig. 1d) in all the wheat cultivars, which is may be attributed to either or both the osmotic and toxic effects of salinity as previously reported (rashid et al., 1999; sayar et al., 2010). in contrast to mbb, waha appears to be the most affected by salinity. increasing salinity causes the reduction of cell turgor and the rate of shoot and root elongation (munns and tester, 2008). to maintain cell turgor by osmotic adjustment (oa), plants synthesize and accumulate several kinds of compatible osmolytes, such as pro and wsc. indeed, substantial increase in pro content in all the genotypes was recorded under salt stress (p<0.001; fig. 2a). however mbb, which exhibited the highest st index, showed the lowest amount of pro. on the other hand, the strong negative correlation found between pro and rwc (r = -0.64***; fig. 2b), from one side, and since the genotype gta which exhibited the highest rwc at 200 mm nacl, showed the greatest pro accumulation from the other side, suggests that pro is involved in oa. besides oa, pro plays an important role in stabilization of enzymes/proteins and protection of membrane integrity (ashraf and harris, 2004; ashraf et al., 2012). in line with these findings, correlation analysis revealed a strong positive relationship between h2o2 and pro (r = 0.49*; fig. 3c) which corroborate recent suggestion that pro could act as an antioxidant counteracting h2o2 (ashraf et al., 2012). increase of wsc content has been considered as an adaptive response of plants to salt stress conditions (parvaiz and satyawati, 2008). wsc not only acts as an energy source, but it is also important to increase the biomass and provide the carbon backbones essential for the synthesis of numerous compounds that are involved in osmotic or anti-oxidative protection (couee et al., 2006). in agreement with this, nacl induced a significant (p<0.001) accumulation of wsc in all genotypes in a concentration-dependent manner (fig. 2a). moreover, wsc content showed a strong negative correlation with rwc (r = -0.7***; fig. 2b) and a positive correlation with h2o2 (r = 0.53**). consistent with our results, salinity induces the accumulation of h2o2 in a concentration-dependant manner (ashraf et al., 2012; lee et al., 2001). despite its role as a signaling molecule, h2o2 itself is toxic at high concentrations (costa et al., 2010). in agreement with our results (fig. 3a), salt-induced oxidative stress causes the lipid peroxidation (lpo) resulting at cellular level in membranes degradation, suggesting that salt-induced lpo occurs early in the seedling tissues of all wheat cultivars. correlation analysis also revealed the significant positive relationships among mda vs. pro (r = 0.58**) and mda vs. wsc (r = 0.43*), suggesting the involvement of pro and wsc in anti-oxidative protection. to withstand salt-induced oxidative stress, plants evolved enzymatic and non-enzymatic antioxidant defense systems. among the rosscavenging enzymes, cat and apx are primarily involved in the elimination of h2o2, and the most important antioxidant enzymes produced in different plant organelles (costa et al., 2010). our results support these aforementioned findings, showing that overall salt stress increases substantially the activity of apx in the three genotypes (fig. 3b). growing evidences suggests that rosscavenging enzymes are involved in the salt stress tolerance (ashraf et al., 2012). while salt stress may result in a rapid increase in the activity of antioxidant enzymes, high concentration or longterm of salt stress inhibit the antioxidant enzyme activities (ashraf fig. 4: multifactorial comparison of the treatments and variables using pca. data are presented for each of three replicates for each treatment. the percent variance explained by each pc is provided. treatments: t0, t3, t6: control; t1, t4, t7: 100 mm nacl; t2, t5, t8: 200 mm nacl. variables codes: fgp: final germination percentage; mgt: mean germination time; dw: dry weight; rl: root length; sl: shoot length; r/s: root to shoot ratio; st: salt tolerance index; vi: vigor index; pro: proline; rwc: relative water content; wsc: water-soluble carbohydrates; apx: ascorbate peroxidase; cat: catalase; hpx: hydrogen peroxide; mda: malondialdehyde. differential response of durum wheat genotypes to salinity 79 et al., 2012), which is partially in line with our results (fig. 3b). interestingly, lee et al. (2001) showed that the overproduction of h2o2 promotes the induction of specific apx isoforms under catalase deactivation. consistent with these findings, apx activity, but not cat, was found positively correlated with the levels of both of h2o2 (r = 0.46*) and mda (r = 0.70*) (fig. 3c). based on pca results it appears that the indices could discriminate the tolerant genotypes are the r/s (36.1% of contribution), cat (30.6%), fgp (5.74%), which showed positive correlations with st, and apx (12.5%), which showed a negative correlation with st (fig. 4b). this is in agreement with the fact that the most salttolerant cultivars are those with high germination rate, an important vigor index, able to maintain a vigorous root growth (high r/s) and showed relatively high antioxidant activity (ashraf et al., 2012; rashid et al., 1999; sayar et al., 2010). in this case, mbb met the requirement. conclusion altogether, these results revealed that salt stress affects almost all studied aspects of 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corresponding author: dr. azzedine fercha, department of biology, abbes laghrour, university, khenchela-40000, algeria e-mail: ferchazzed@yahoo.fr journal of applied botany and food quality 87, 297 (2014) buchbesprechung pharmazeutische biologie kompakt grundlagen – systematik – humanbiologie eckard leistner und sigmar-w. breckle die neue auflage des seit mehr als 30 jahren im pharmaziestudium erfolgreich eingeführten lehrbuchs von leistner und breckle richtet sich inhaltlich an der aktuellen approbationsordnung für apotheker aus und vermittelt in komprimierter und gut verständlicher weise die grundlagen aller wichtigen teilgebiete der pharmazeutischen biologie. mit unterstützung durch ein erweitertes autorenteam wurden sämtliche inhalte der auflage komplett überarbeitet und ein neues didaktisches layout konzipiert. anhand graphischer elemente ist der text übersichtlich gegliedert. darüber hinaus wurden definitionen und merkfelder farblich vom text abgesetzt, sodass sich das lehrbuch auch gut als nachschlagewerk eignet. am schluss jeden kapitels finden sich eine kurze zusammenfassung sowie einige fragen zu dem jeweils behandelten lernstoff. dieser kompakte überblick soll zur selbstkontrolle und dem kurzfristigen aufrufen der vermittelten lerninhalte dienen. zahlreiche literaturhinweise ermöglichen es zudem interessierten lesern, ein tieferes verständnis der jeweils behandelten thematik zu erlangen. in dem text sind einige praxis beispiele eingeflochten, die einen aktuellen bezug zu einzelnen arzneimitteln herstellen und deren wirkmechanismus anschaulich erläutern. in den ersten drei kapiteln des lehrbuchs wird der aufbau pround eukaryotischer zellen näher erläutert und auf die jeweiligen funktionen der einzelnen zellbestandteile eingegangen. der genetik ist ein eigenes kapitel gewidmet, das neben allgemeinen grundlagen insbesondere auf mitose und meiose eingeht. weitere kapitel beschäftigen sich mit der struktur und funktion von dna und behandeln dessen replikation sowie die transkription und synthese unterschiedlicher rna-arten. ebenso findet sich ein knapper einblick in die grundlagen der molekularbiologie und es wird exemplarisch auf die gentechnologische herstellung von insulin eingegangen. in weiteren kapiteln wird schließlich der kohlenhydratstoffwechsel, der energiestoffwechsel sowie der aufbau von fetten und fettsäuren näher behandelt. außerdem liefert das buch grundlagen zur botanischen systematik und veranschaulicht in diesem zusammenhang die bedeutung der biodiversität für die stabilität von ökosystemen. auch die kapitel, die auf viren, bakterien und pilze näher bezug nehmen, wurden in der neuen auflage grundlegend überarbeitet und es wurde als neuer bereich die humanbiologie zum bisherigen lernstoff ergänzt. in diesem zusammenhang wird in einzelnen kapiteln ein überblick hinsichtlich gewebe und haut, nervensystem, muskulatur, herzund kreislauf, blut, atmung, niere und harnwege, verdauung sowie hormone und fortpflanzung geliefert. die neue auflage des lehrbuchs von leistner und breckle ist gut lesbar und die didaktische konzeption kann insgesamt als sehr gelungen bezeichnet werden. anhand der prägnanten lehrtexte, hilfreichen begriffsdefinitionen sowie mit hilfe vieler merkkästen und abbildungen ist es auch einsteigern möglich, das gelesene schnell aufzunehmen. das buch eignet sich daher gleichermaßen zur begleitung des biologiebzw. pharmaziestudiums und zur systematischen prüfungsvorbereitung. es kann allen studenten empfohlen werden, die zu einem guten preis-leistungsverhältnis kompakte informationen hinsichtlich der biologischen grundlagen in der arzneimittellehre erhalten möchten. dr. h. schulz e-mail: hartwig.schulz@jki.bund.de bibliografie: eckard leistner und sigmar-w. breckle, pharmazeutische biologie kompakt, grundlagen – systematik – humanbiologie, wissenschaftliche verlagsgesellschaft mbh, stuttgart, 8. überarbeitete und erweiterte auflage, 740 seiten mit 445 abbildungen und 38 tabellen, preis: 47,20 €, isbn: 978-3-8047-3031-1 journal of applied botany and food quality 87, 206 209 (2014), doi:10.5073/jabfq.2014.087.029 1department of horticulture and food science, agriculture and horticulture faculty, university of craiova, craiova, romania 2 department of chemistry, sciences faculty, university of craiova, craiova, romania influence of the extraction solvent on antioxidant capacity and total phenolic in currant fruits s. cosmulescu1*, i. trandafir2, v. nour1 (received march 31, 2014) * corresponding author summary in black and red currant fruits the phenolic content and antioxidant capacity were determined. two solvent systems (methanol and ethanol) at different concentrations and two methods of extraction were used. the study was conducted using fruits of black currant and red currant for determinations. the total phenolics content of each extract was measured according to the folin-ciocalteu method. the anti-oxidant capacity of the fruit extracts was evaluated using dpph (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay. it was found that the efficiency of the solvents used for the extraction of polyphenols varied substantially. the total phenolic content was 0.88 to 4.6 gallic acid equivalents in milligrams per gram of fresh weight (mg gae /g fw). the content of phenolics was highly correlated with the anti-oxidant capacity (r = 0.97 0.98) and extracts obtained using ethanol solvents were more effective radical scavenging activities than the ones obtained using methanol solvents. fruits of red and black currant represent an abundant source of phenolics, and prove to have good anti-oxidant capacity. abbreviations: dpph: 2,2-diphenyl-1-picrylhydrazyl; gae: gallic acid equivalents; tpc: total phenolics contents; teac: trolox equivalent antioxidant capacity. introduction black currants (ribes nigrum) and red currants (ribes rubrum) are species of the genus ribes broadly cultivated domestically and commercially as a temperate climate fruit crop. the currant is highly appreciated for the food and therapeutic value of its fruits (nour et al., 2011). fruits of currant represent a source of natural antioxidant compounds. they contain high levels of phenolic compounds (eg. flavanols and anthocyanins), which substantially contribute to anti-oxidant activity (remberg et al., 2007; slimestad and soleim, 2002). the hplc analysis conducted by kapasakalidis et al. (2006) showed that delphinidin-3-o-glucoside, delphinidin-3o-rutinoside, cyanidin-3-o-glucoside, and cyanidin-3-o-rutinoside were the major anthocyanins and they constituted the main phenolic class (≈ 90%) in black currant residues that were tested. fruits extracts from various black currant and red currant cultivars effectively act as free radical inhibitors (tabart et al., 2011; wang and lin, 2000). research on anti-oxidant phenolic compounds in black currant was mainly focused on the effect of cultivars, seasonal timing and cultivation practices (tabart et al., 2006; nour et al., 2011). some studies were carried out on total phenolics of currants. borges et al. (2010) and nour et al. (2011) have determined the phenolic, anthocyanin and ascorbic acid content of currant fruits. according to lugasi et al. (2011), total polyphenol contents of white, red and black currants cultivars were 333, 192 and 533 mg/100 g, respectively. black currants have high concentrations of flavonols and phenolic acids while in red currants the concentration of phenolic acids was higher than the flavonols concentration (jakobek et al., 2007). gopalan et al. (2012) stated that black and red currants represent a good source of bioactive compounds with considerable dietetic and therapeutic impact on human health. in the present paper, total phenolics and anti-oxidant capacities in fruits of various black and red currant cultivars, which are commonly consumed in diet, were analyzed. anti-oxidant capacities of different black and red currant cultivars and extracts were compared with the aim of preparing extracts with high anti-oxidant capacity. materials and methods material the study was conducted using fruits of black currant (cv. ‘black down’, ‘bogatar’, ‘tenah’, ‘record’, ‘tinker’, ‘deea’, ‘abanos’, ‘ronix’) and red currant (cv. ‘houghton castle’, ‘abundent’, ‘rosu timpuriu’) for determinations. the study material was collected in craiova, located in southwest of romania (44°20’n, 23°49’e), at the commercial maturity stage. the climate is temperate continental and plain specific, with mediterranean influences. the average temperature range from 10 °c to 11.5 °c and precipitation level is 580 600 mm. currant fruits were harvested from 10 bushes of each cultivar. approximately 500 g of fruits were collected from each genotype. fruits were stored on ice in the field and frozen at -10 °c, taking care to avoid unripe, damaged, or overripe fruits. total phenolics content the fruits from each cultivar were homogenized using a laboratory blender. extracts were prepared using two methods and two solvents. the sample (1 g) was extracted with 5 ml 80 % methanol (variant 1), 40 % ethanol (variant 2) or 80 % ethanol (variant 3) in ultrasonic bath for 55 minutes, centrifuged for 10 minutes at 5000 rpm, and the supernatant was filtered through polyamide filter, and finally transferred into vials for analysis and stored at 4 °c. in the variant 4, the sample (1 g) was extracted with 5 ml 40 % ethanol by maceration at room temperature for four weeks. the total phenolics content of each extract was measured according to the folin-ciocalteu method, described by singleton and rossi (1965) with slight changes. each 1.0 ml of fruits extract, or 1.0 ml double-distilled water (blank), 1 ml of each standard solution was placed in a 25 ml flask and 5 ml of folin-ciocalteu reagent was added (diluted 1:10 with ultrapure water). after 2 min, 4 ml of 7.5 % (w/v) sodium carbonate solution was added and flasks were kept at room temperature (24 °-26 °c) for 2 h. the absorbance was measured at 765 nm using an evolution 600, uv-visible spectrophotometer (thermo scientific, usa) with computer control with vision pro-software (thermo scientific, usa). a standard curve was prepared by using 50, 100, 150, 200, or 250 mg/l gallic acid in 60:40 (v/v) methanol and water. gallic acid was used as reference standard and total phenolic content were expressed as mg gallic acid per gram fresh weight (mg gae/g fw). anti-oxidant activity methanol (merck, germany), dpph (merck, germany) and trolox (merck, germany) were used to determine anti-oxidant activity. the antioxidant capacity and total phenolic in currant fruits 207 tab. 1: total phenolics* content of fruits in various cultivars of black and red currant genotype total phenolic (mg gae /g fw) in different extracts** variant 1*** variant 2 variant 3 variant 4 black currant ‘black down’ 5.70±0.27dd 3.71±0.17bb 4.47±0.49bcdc 2.69±0.33ba (ribes nigrum) ‘bogatar’ 2.62±0.18ab 2.38±0.08ab 2.44±0.25ab 1.83±0.21aa ‘tenah’ 4.67±0.31cb 4.80±0.26cb 4.93±0.52db 2.84±0.25ba ‘record’ 4.14±0.38bab 4.48±0.44cb 4.69±0.44cdb 3.53±0.36ca ‘tinker’ 4.63±0.33bcb 5.72±0.38dc 5.89±0.56ec 2.81±0.16ba ’deea’ 3.05±0.21aa 4.30±0.31cb 4.04±0.42bcb 2.67±0.18ba ‘abanos’ 4.49±0.41bcb 6.81±0.48ec 6.48±0.56ec 1.91±0.09aa ‘ronix” 4.12±0.25bc 3.21±0.15bb 3.88±0.32bc 1.92±0.23aa mean 4.17±0.96 4.42±1.36 4.60±1.24 2.52±0.60 red currant ‘houghton castle’ 1.92±0.15bb 1.82±0.12bb 0.81±0.14aa 0.93±0.09aa (ribes rubrum) ‘abundent’ 2.13±0.20bb 0.96±0.16aa 1.04±0.11aa 0.88±0.10aa ‘rosu timpuriu’ 1.55±0.17ab 0.81±0.07aa 0.95±0.09aa 0.85±0.07aa mean 1.86±0.29 1.19±0.48 0.93±0.14 0.88±0.08 *average value±standard deviation. **different subscript letters within the same column indicate significant differences (p < 0.05) among cultivars. different superscript letters within the same row indicate significant differences (p < 0.05) among extraction methods. *** variant 1 (80 % methanol); variant 2 (40 % ethanol); variant 3 (80 % ethanol); variant 4 (40 % ethanol by maceration). scavenging activity of ethanolic and methanolic extracts of black and red currants against dpph radicals was established in accordance with the method described by hatano et al. (1988), with some modifications (cosmulescu and trandafir, 2012). each fruit extract (50 ml) was mixed with 3 ml of a 0.004 % (v/v) dpph methanolic solution. each reaction mixture was incubated for 30 min in darkness at ambient temperature, and the absorbance was measured at 517 nm. standards of trolox at different concentrations were used (0, 0.5, 1, 1.5, 2, 2.5 and 3.5 mm). ultrapure water was used as a blank. the radical scavenging activity (ra) against dpph radicals was assessed according to the following equation (1): ra = [(absblank – abssample) / absblank ] × 100 (1) where absblank was the absorbance of the control, and abssample was the absorbance of the fruits extract or standard solution. all assays were conducted in triplicate. anti-oxidant capacity was expressed in mmol trolox per 100 g fruits. statistical analysis data were subjected to analysis of variance (anova) using statgraphics centurion xvi software (statpoint technologies, warrenton, va, usa). differences were estimated with a multiple range test using the least significant difference (lsd) at p < 0.05. results and discussion total phenolics contents (tpc) total phenolics determined in methanol and ethanol solvents extracts of black and red currants fruits are shown in tab. 1. the concentration of phenolics in the extracts, expressed as mg gae/g fw, was dependent on the solvent and method used for extraction. the results indicated significant differences ((p < 0.05) between cultivars analyzed for each species (r. nigrum, r. rubrum) and between methods (variants). the average total phenolic contents, in the black currant, ranged from 2.52 mg gae / g fw (variant 4) to 4.6 mg gae / g fw (variant 3). the average total phenolic contents of 80 % ethanol extracts is at least 0.96 times higher than that of extract 40 % ethanol, 1.1 times higher than that of 80 % methanol extracts and 1.82 times higher than that of extracts in 40 % ethanol by maceration. results revealed that 80 % ethanol was more efficient in extracting phenolic compounds for black currants fruits. in the red currant, the average total phenolic content ranged from 0.88 mg gae / g fw (variant 4) to 1.86 mg gae / g fw (variant 1). total phenolic content of 80 % methanol extracts (variant 1) is at least 1.56 times higher than that of extracts in ethanol 40 %, 2 times higher than that of 80 % ethanol extracts and 2.11 times higher than that of extracts in 40 % ethanol by maceration. for red currants, results revealed that 80 % methanol were better solvents in extracting phenolic compounds. it follows that, depending on the species, both used solvent and extraction method are significant factors affecting total phenolic content (p < 0.05). also other authors have established the influence of different extraction solvents and techniques on the phenolic composition in berry fruits and other species (kähkönen et al., 2001; jahangiri et al., 2011; michiels et al., 2012; spigno et al., 2007). generally, solvents such as methanol, ethanol, acetone, propanol and ethyl acetate, have been commonly used for the extraction of phenolics from fresh products. ethanol is more acceptable for use in food industry. lapornik et al. (2005) indicated that ethanol and methanol extracts of red and black currants contain double amounts of anthocyanins and polyphenols compared to water extracts. the saving of polyphenols from plant materials is depending on solubility of phenolic compounds in the solvent used for the extraction process. according to cacace and mazza (2003), total phenolics in black currants increased with ethanol concentration up to a maximum at about 60 % and then decreased with further increasing in solvent concentration, irrespective of the solvent to solid ratio. thus, it could be concluded from the results shown in the tab. 1, that the efficiency of solvents, concentrations of solvent and 208 s. cosmulescu, i. trandafir, v. nour methods are strongly dependent on plant used. the results of this study show that the largest amount of total phenol content from r. nigrum was obtained using 80 % ethanol as a solvent while from r. rubrum it was obtained using 80 % methanol as a solvent. phenolics content was different and dependent on cultivars. for black currants, total phenolics varied between 1.83 mg gae g / fw in cultivar ‘bogatar’ and 6.81 mg gae / g fw in cultivar ‘abanos’. in red currants, total phenolics content variation was between 0.81 mg gae / g fw in ‘rosu timpuriu’ cultivar and 2.13 mg gae / g fw in ‘abundent’ cultivar (tab. 1). statistically significant differences (p ≤ 0.05) were found between of the analyzed cultivars. these differences may be due to genetic factors, and cultivar dependent phenolics contents in currant have been observed by other authors (djordjević et al., 2010; pantelidis et al., 2007). it can be concluded that the content of total phenolics is a differentiating factor for the currant cultivars analyzed. anti-oxidant capacity methanolic and ethanolic extracts derived from currant fruits were evaluated for their anti-oxidant capacity by the dpph radical scavenging method. trolox was used as reference standard. anti-oxidant capacity was expressed in mmol trolox/100g fw (tab. 2). significant differences were found among the free radical scavenging activities of different methods used. statistically, the interaction between plant materials and solvents significantly (p < 0.05) affected the anti-oxidant capacity. significant differences were found among the free radical scavenging activities of different methods used. average values of anti-oxidant capacity in black currants ranged from 1.16 mmol trolox / 100 g fw (variant 4) to 2.35 mmol trolox / 100 g fw (variant 1). in red currants the variation range was from 0.39 mmol trolox / 100 g fw (variant 1) up to 2.16 mmol trolox / 100 g fw (variant 4). it could be concluded from the above results that the antioxidant activities of currant fruit extracts expressed as antiradical power are statistically significant (p < 0.05) and they are affected by solvent and methods used for extraction. the highest antioxidant activity was noticed for black currant extracts by 80 % methanol in ultrasonic bath for 55 minutes, centrifuged for 10 minutes, whereas red currant extracted by 40 % ethanol maceration at room temperature for four weeks. a significant difference (p < 0.05) was found among cultivars (tab. 2) in terms of anti-oxidant capacity; for black currant from 0.82 mmol trolox / 100 g fw in ‘bogatar’ to 2.86 mmol trolox / 100 g fw in ‘abanos’; for red currant from 0.33 mmol trolox / 100 g fw to 2.33 mmol trolox / 100 g fw in ‘abundent’. cultivars of r. nigrum showed higher values for anti-oxidant capacity, compared to cultivars of r. rubrum. this is due to anthocyanins content that is higher in the r. nigrum species. borges et al. (2010) showed that black currants contained the highest levels of anthocyanins, and these were responsible for 73 % of the total antioxidant capacity, whereas vitamin c contributed by 18 %. anti-oxidant capacity of black and red currant polyphenols has already been described. black currants had the highest antioxidant capacity in the frap assay followed by blueberries, raspberries, and red currants, while the lowest value was in cranberries (borges et al., 2010). pantelidis et al. (2007) reported the lowest frap values (40.7 65.1 μmol asa / g dw) for red currant and gooseberry cultivars. anti-oxidant activities were correlated with total phenolic content (tab. 3). there is an excellent linear relationship (r = 0.84 0.98) between values for total phenolic content and anti-oxidant activity. this linear correlation suggested that phenolic compounds in currant largely accounted for its anti-oxidant capacity. good correlation (r = 0.97 0.98) was observed between anti-oxidant activity and total polyphenol content for variant 2 and variant 3, indicating that ethanol, in different concentrations, is a good solvent in obtaining an extract with high anti-oxidant activity of currants (black and red). similar results have been reported by other researchers. wang and lin (2000) found a linear correlation between total anti-oxidant capacity and phenolic content in blackberries (r = 0.961) and raspberries (r = 0.911). a highly significant correlation (0.97) was found between trolox equivalent antioxidant capacity (teac) and total phenolic content in blueberries by huang et al. (2012). tab. 2: anti-oxidant capacity* of fruits from various cultivars of black and red currant genotype anti-oxidant capacity (mmol trolox / 100 g)** variant 1*** variant 2 variant 3 variant 4 black currant ‘black down’ 2.54±0.14cc 1.12±0.09aba 2.00±0.21cb 1.16±0.13bca (ribes nigrum) ‘bogatar’ 2.15±0.15abc 0.82±0.06aa 1.16±0.16ab 1.04±0.18abab ‘tenah’ 2.45±0.26cc 2.14±0.15cc 1.80±0.13bcb 1.12±0.10ba ‘record’ 2.33±0.18bcc 2.02±0.14cb 1.91±0.14cb 1.39±0.14da ‘tinker’ 2.51±0.13cc 2.66±0.28dc 2.05±0.19cdb 1.11±0.17ba ’deea’ 2.03±0.09ac 2.06±0.19cc 1.53±0.14bb 1.25±0.14bcda ‘abanos’ 2.45±0.16cb 2.86±0.22dc 2.32±0.23db 1.35±0.06cda ‘ronix” 2.38±0.14bcd 1.41±0.17bb 1.88±0.13cc 0.87±0.09aa mean 2.35±0.22 1.88±0.70 1.83±0.36 1.16±0.19 red currant ‘houghton castle’ 0.44±0.07ba 0.82±0.07bb 0.72±0.08bb 2.17±0.22ac (ribes rubrum) ‘abundent’ 0.33±0.04aa 0.42±0.09aa 0.83±0.06bb 2.33±0.21ac ‘rosu timpuriu’ 0.40±0.03aba 0.49±0.04aab 0.59±0.04ab 1.97±0.16ac mean 0.39±0.06 0.58±0.19 0.71±0.12 2.16±0.23 *average value±standard deviation. **different subscript letters within the same column indicate significant differences (p < 0.05) among cultivars. different superscript letters within the same row indicate significant differences (p < 0.05) among extraction methods. *** variant 1 (80 % methanol); variant 2 (40 % ethanol); variant 3 (80 % ethanol); variant 4 (40 % ethanol by maceration). antioxidant capacity and total phenolic in currant fruits 209 tab. 3: correlation coefficients (r) and coefficient of determination (r2) of total phenolics and anti-oxidant capacity variant (r2) (r) variant 1 (80 % methanol) 0.71 0.84 variant 2 (40 % ethanol) 0.96 0.98 variant 3 (80 % ethanol) 0.95 0.97 variant 4 (40 % ethanol by maceration) 0.77 0.87 conclusion analysis of the total phenolic compounds and antioxidant activity of currant fruit extracts showed differences depending on solvent used and extraction method. as it was found, the extracts with higher antioxidant capacity also had higher content of phenolic compounds. it can be established that extracts obtained using ethanol solvents were more effective in radical scavenging activities, compared to extracts obtained using methanol solvents. this is convenient, because when using it in food industry, ethanol is a more suitable solvent. the results obtained are indicating that the analyzed currant species are rich sources of biologically active substances and possess real anti-oxidant activities, for use in food industry, cosmetics, and pharmaceutical industries. further research would be required for investigations on in vivo anti-oxidant activities. acknowledgement this work was partially supported by the grant number 11c /2014, awarded in the internal grant competition of the university of craiova, romania. references borges, g., degeneve, a., mullen, w., crozier, a., 2010: identification of flavonoid and phenolic antioxidants in black currants, blueberries, raspberries, red currants, and cranberries. j. agr. food 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phenolic composition of blueberry, blackberry, and strawberry in nanjing. j. zhejiang univ-sc b. 13, 94-102. jahangiri, y., ghahremani, h., abedini torghabeh, j., ataye salehi, e., 2011: effect of temperature and solvent on the total phenolic compounds extraction from leaves of ficus carica. j. chem. pharmaceut res. 3, 253-259. jakobek, l., seruga, m., novak, i., medvidovic-kosanovic, m., 2007: flavonols, phenolic acids and antioxidant activity of some red fruits. deutsche lebensm-rundsch. 103, 369-378. kähkönen, m.p., hopiaa, a.i., heinonen, m., 2001: berry phenolics and their antioxidant activity. j agr food chem. 49, 4076-4082. kapasakalidis, p.g., rastall, r.a., gordon, m.h., 2006: extraction of polyphenols from processed black currant (ribes nigrum l.) residues. j. agr. food chem. 54, 4016-4021. lapornik, b., prošek, m., wondra, a.g., 2005: comparison of extracts prepared from plant by-products using different solvents and extraction time. j. food eng. 71, 214-222. lugasi, a., hóvári, j., kádár, g., dénes, f., 2011: phenolics in raspberry, blackberry and currant cultivars grown in hungary. acta aliment. 40, 52-64. michiels, j.a., kevers, c., pincemail, j., defraigne, j.o., dommes, j., 2012: extraction conditions can greatly influence antioxidant capacity assays in plant food matrices. food chem. 130, 986-993. nour, v., trandafir, i., ionica, m.e., 2011: ascorbic acid, anthocyanins, organic acids and mineral content of some black and red currant cultivars. fruits 66, 353-362. pantelidis, g.e., vasilakakis, m., manganaris, g.a., diamantidis, g., 2007: antioxidant capacity, phenol, anthocyanin and ascorbic acid contents in raspberries, blackberries, red currants, gooseberries and cornelian cherries. food chem. 102, 777-783. remberg, s.f., måge, f., haffner, k., blomhoff, r., 2007: highbush blueberries vaccinium corymbosum l., raspberries rubus idaeus l. and black currants ribes nigrum l. − influence of cultivar on antioxidant activity and other quality parameters. acta hort. 744, 259-265. singleton, v.l., rossi, j.a., 1965: colorimetry of total phenolics with phosphomolybdic − phosphotungstic acid reagent. am. j. enol. viticult. 16, 144-158. slimestad, r., solheim, h., 2002: anthocyanins from black currants (ribes nigrum l.). j agr food chem 50, 3228-3231. spigno, g., tramelli, l., de faveri, d.m., 2007: effects of extraction time, temperature and solvent on concentration and antioxidant activity of grape marc phenolics. j. food eng. 81, 200-208. tabart, j., kevers, c., pincemail, j., defraigne, j.o., dommes, j., 2006: antioxidant capacity of black currant varies with organ, season, and cultivar. j. agr. food chem. 54, 6271-6276. tabart, j., kevers, c., evers, d., dommes, j., 2011: ascorbic acid, phenolic acid, flavonoid, and carotenoid profiles of selected extracts from ribes nigrum. j. agr. food chem. 59, 4763-4770. wang, s.y., lin, h.s., 2000: antioxidant activity in fruit and leaves of blackberry, raspberry and strawberry varies with cultivar and developmental stage. j. agr. food chem. 48, 140-146. address of the author: s. cosmulescu, v. nour, department of horticulture and food science, agriculture and horticulture faculty, university of craiova, a.i.cuza street, 13, 200585, craiova, romania i. trandafir, department of chemistry, sciences faculty, university of craiova, calea bucuresti street, 107, 200529, craiova, romania vorgabe neu journal of applied botany and food quality 80, 187-193 (2006) microbial phytopathology, institute for microbiology, friedrich-schiller-university, jena, germany diversity and adaptation of soil fungi in an ecosystem with contamination originating from a phosphate fertilizer plant ulrich terpitz, erika kothe1 (received august 18, 2006) summary in the vicinity of a former phosphate fertilizer production plant, high phosphate contents with up to 463 mg phosphate per kilogram of soil dry matter were found 13 years after closing the plant while ph is only slightly elevated at ph 7 to 8. the deposited phosphate is seen to be moved to deeper soil horizons. the effect on soil microbiota was analyzed with respect to soil respiration, fungal biomass and cultivation of benomyl resistant and ligninolytic fungi. increasing numbers and diversity of soil fungi were found with distance from the former emittent. this was confirmed by investigation of mycorrhization rates of mycotrophic, ectomycorrhizal birch trees. we tested for adaptation by growth and phosphate acquisition on soil extract media. the best growth was seen on the media containing highest phosphate concentrations showing no in vitro growth inhibition. in contrast to previous findings, however, more polyphosphate granula were seen on soil extract media from distant sites, although phosphate concentration was lowest in these media. introduction in the vicinity of jena, thuringia, germany, a former phosphate production plant has produced fertilizer from 1957 to 1990 in dorndorf/steudnitz. the plant, part of the veb chemiewerk coswig, produced with 100,000 t per year about 20 % of the phosphate fertilizer produced in the former gdr (vogler and gebauer, 1981). during the production period, high emissions of phosphate and cadmium containing dust occurred, e.g. in 1979, 3146 t of dust with high ph values of 10-11. the fertilizer plant is located in the saale valley which led to directional deposition with the direction of wind predominately north-northeast on a steep hillside of chalcerous rocks at the left bank of the saale river (görner and fröhlich, 1972). the impact on local flora was extensive (heinrich, 1984) and heavy metal contents in remaining plants was substantially elevated (anke et al., 1991). the phosphate concentrations in the soil were elevated with 1.2 g per kg soil dry matter (heinrich, 1983; langer, 2000). phosphate is an essential nutrient, which is limiting in most soil types especially since phosphates are mostly bound in inorganic apatite, or calcium, aluminum or iron phosphates. adsorption to soil particles like clay minerals, humic substances and organically bound forms further limit availability to plants and microorganisms. soil microbes and plants dissolve phosphate by acidification and especially fungi have very potent high affinity uptake systems which allow the mycorrhizal fungi, for instance, to provide phosphate for the associated symbiotic plant (beever and burns, 1980; harrison and van buuren, 1995; illmer et al., 1995; jacobs et al., 2002; jones et al., 2003; gyaneshwar et al., 2002; kothe et al., 2002; maldonadomendoza et al., 2001). filamentous fungi are forming a mycelium which is in closest contact to a large area of soil. a very impressive example for the amount of soil colonized is given by the largest living organism, a basidiomycete, armillaria ostroyae, in north america which covers 880 hectares of ground. at the same time, biomass in soils is dominated by fungi. by the close contact to soil particles, fungi are especially sensitive to soil pollution. the radioactive fallout of the tchernobyl accident, for example, led to short-time, high enrichment in fungal fruitbodies (linkov et al., 2000). heavy metal pollution and other factors of soil degradation are listed as major reasons for extinction of fungal species world-wide. with more than 90,000 species described, the eumycota are showing a high biodiversity which is threatened by pollution, especially through fertilization, changing soil ph, and increasing heavy metal concentrations (sonneborn et al., 1999; erland and taylor, 2002; chander et al., 2001a,b; kuyper, 1989). while mycorrhizal fungi generally show higher resistance to environmental stress because of the better c-source availability (cooke, 1993), it could be shown that elevated phosphate concentrations inhibit germination of arbuscular mycorrhizal fungi (de miranda and harris, 1994). however, the role of phosphate in studies of environmental stress has been widely neglected. here, we investigate the progress of restoration of a phosphate polluted ecosystems with respect to the soil microbiota, especially higher fungi, by comparing sites with increasing distance to the former plant and therefore decreasing initial pollution 13 years after closing of the phosphate fertilizer production. material and methods cultivation isolation and cultivation of fungi was performed at room temperature on complete media (czapek dox agar). czapek-dox agar (warcup, 1950) contained per liter: 30 g sucrose, 3 g nano 3 , 0.5 g mgso 4 x 7h 2 o, 0.5 g kcl, 0.01 g fe-(ii)-so 4 x 7 h 2 o, 1 g k 2 hpo 4 , 5 g yeast extract, 13 g agar-agar and 50 mg each streptomycin and chloramphenicol to minimize bacterial growth. the ph was adjusted with hcl to 4,9-5,0. for differentiation between lignin degrading fungi and for enrichment of basidiomycetes, agar with benomyl and guiacol was used. ligninguiacol-benomyl-agar (thorn et al., 1996) contained 0.5 g kh 2 po 4 , 0.2 g mgso 4 x 7 h 2 o, 0.1 g nh 4 no 3 , 0.1 g kcl, 0.02 g fe-(ii)-so 4 x 7 h 2 o, 0.05 g ca(no 3 ) 2 x 4 h 2 o, 2 g malt extract and 14 g agaragar per liter. after autoclaving, 5 ml 1m koh, 0.4 ml guaiacol, 1 g induline at (solubilized in 10ml dioxan) and 4 mg benomyl (benlate50-wp; dissolved in 2 ml acetic acid 70% ethanol 1:1 were added). antibiotics streptomycin and chloramphenicol (50 mg per liter final concentration) were used to minimize bacterial growth. to investigate influence of the three sampling sites, soil extract medium was used. the soil extract was prepared following the instructions of the german culture collection, dsmz, braunschweig (medium 80, glycerol-soil-medium: http://www.dsmz.de/ media/ med80.htm) using 100 g of 2 mm maximal sized particles. the soil extract medium contained per liter: 150 ml soil extract, 30 g sucrose, 5 g yeast extract, 13 g agar-agar and 50 mg each of streptomycin and chloramphenicol. the ph was adjusted with hcl or naoh to 7.0.1 corresponding author fig. 2: plant available phosphate at three sites with increasing distance from the former plant in three depths, (o) 0 4 cm, (x) 10 15 cm and (u) 20 25 cm from top. o x u site 1 site 2 site 3 m g p h o sp h a te /k g s o il d ry m a tt e r 0 100 200 300 400 500 microscopy and staining to stain and visualize hyphae, a sterile cover slip for microscopic slides was placed next to a growing mycelium on the agar surface and hyphae growing onto the cover slip were used. polyphosphates were stained using 0.1 % toluidine blue at ph 1 (chilvers et al., 1978; bücking and heyser, 1999). dapi (4-6-diamidino-2phenylindol) was used to stain dna (raju, 1982) and the observation was performed using a zeiss axioplan 2 fluorescence microscope. the mycorrhization rate was determined from root samples observed after washing using a zeiss stereoscope at 2,5-4-fold magnification counting mycorrhizal short roots per 100 short roots. soil characterization and respiration the soil ph was measured following the protocol of schinner et al. (1993) from three independent soil samples per horizon. phosphate was determined using ammonium hepta-molybdat (olsen and sommer, 1992) measuring the blue complex photometrically at 570 nm. two independent samples were used for each point. as a standard, na 2 hpo 4 in 0.5 m nahco 3 , ph 8.5, was used with three parallels. to correct for humic acids which are also visible at 570 nm, each filtrate was measured without addition of ascorbic acid and the values for these samples were subtracted from the sample measurements. size of soil particles was determined by sieving and sedimentation in 0.1 m na 4 p 2 o 7 (schinner et al., 1993). humic substances were determined photometrically at 570 nm (schinner et al., 1993) using myo-inosit as a standard (116 mg equivalent to 4 %, 232 mg for 8 % and 348 mg for 12 % humic substances). soil respiration was determined by titration using 40 µl 0,1 % phenolphtalein in 60 % ethanol (schinner et al., 1993) in three parallels for each measurement. fungal biomass and isolation ergosterol content was used to determine fungal biomass in soil samples after methanol extraction by hplc quantification (wheete and gandhi, 1996). ergosterol (hplc-quality, fluka, 200 µg/ml in methanol) was used as a standard. the hplc-system (dionex p580 pump, as50 autosampler, lc20 chromotography enclosure) was coupled to a uv-detector (gyntek vvd340s) and a rp18e-column (250 x 3mm, sepserv, berlin, germany). methanol was used as solvent at 0.5 ml/min. the retention time was 20 min, software chromeleon client (version 6.11) was used to determined peak volume. the isolation of saprophytic soil basidiomycetes was performed with washed soil fractions in order to eliminate resting spores (thorn et al., 1996). sieves pre-sterilized with 79 % ethanol with pore sizes of ca. 250 µm and 54 µm (bückmann gmbh, mönchengladbach) were used and after 5 min washing with tap water, soil particles in 20 ml suspension were taken from the 54 µm sieve. 5 independent plates were inoculated with 400 µl of the suspension on ligninguiacol-benomyl-agar. for investigation of polyphosphate accumulation on soil extract media, one of the dikaryotic basidiomycetes isolated was used. the strain 8-o-1 was isolated from site 2 and showed lignin degradation as indicated by guiacol staining. results plant available phosphate concentrations along a transect and in soil profiles three points along a traverse section on the hillside north of the former fertilizer plant along the left bank of the saale river were sampled 100 (site 1), 400 (site 2) and 680 m (site 3) from the former plant, respectively (fig. 1). at each site, three samples from the inceptisol soil profile were taken from topsoil at 0-4 cm (o), subsoil at 10-15 cm (x) and 20-25 cm (u) depth (fig. 1). the content in humic substances was lowest at site 1 with app. half the amount of sites 2 and 3 and the organic layer was smaller (tab. 1). the ph was slightly alkaline at all three positions. for site 1, a hill slide may have occurred, since the lower profile was very sandy and phosphate concentrations did not follow the general trend of highest concentration in the uppermost layers. plant available phosphate concentrations were shown to vary between 67 and 463 mg/kg soil dry weight. while site 1 showed highest phosphate concentrations in the lowest horizon, sites 2 and 3 had shallower gradients with highest phosphate concentrations in the uppermost samples (fig. 2). the two upper horizons at site 1 classify as low in available phosphate, the lowest horizon at site 3 shows normal phosphate contents, all other samples have excess plant available phosphate contents. tab. 1: description of sampling point soil parameters. three sites with distances of 100 to 680 m from the former phosphate fertilizer plant were used to survey a gradient of phosphate immission. site depth distance ph organic humic sand coarse fine clay to plant layer subst. silt silt [m] [cm] [%] [%] [%] [%] [%] o 7.5 1.8 1 x 100 7.9 1.38 33 2 52 13 u 7.7 0.99 65 23 9 3 o 7.0 4.3 2 x 400 7.7 2.94 30 2 49 19 u 7.9 1.87 28 4 48 20 o 7.0 3.6 3 x 680 7.5 3.1 27 1 54 18 u 7.6 1.35 17 4 55 24 188 ulrich terpitz, erika kothe fig. 1: sampling sites in the river saale valley at dordorf-steudnitz near jena, germany. inserts show the sampling sites. the former phosphate fertilizer plant is visible in all three views at increasing distance to allow visual comparison of distance from the plant. the soil profile at site 2 is shown as an example. fig. 6: polyphosphate granula content of isolate 8-o-1 grown on soil extract media from site 1 and site 3 after staining with toluidine blue. microbial biomass and activity the microbial activity was determined for all nine samples by measuring soil respiration. since respiration rates in the deeper horizons were very low, rates over 10 µg per g of dry mass are not specified (tab. 2). generally, the soil respiration was lower than in uncontaminated soils. site 1 showed elevated soil respiration in the lowest sample, u, which is unusual for normal soils. however, the highest phosphate concentrations were found in this sample which might stimulate soil respiration. fig. 4: fungal hyphae (a) and basidiomycete with clamps (b) attached to washed soil particles. tab. 2: soil respiration rates (µg/g dry soil matter) at three sites (1 through 3) with increasing distance to the former emmitent soil respiration site 1 site 2 site 3 o 9.73 ± 0.11 > 12.55 ± 0.2 > 12.43 ± 0.14 x 1.46 ± 0.16 2.29 ± 0.39 2.38 ± 0.27 u 5.15 ± 0.19 1.28 ± 0.18 0.53 ± 0.19 since soil respiration includes especially the diverse metabolic activity of bacteria, the fungal biomass was determined by ergosterol content, direct microscopy of washed soil particles and cultivation experiments. ergosterol contents showed increasing fungal biomass with distance from the emmittent (fig. 3). the cultivation of soil without a washing step generally gives higher numbers of isolates, especially of the conidiospore forming ascomycetes. this, however, does not represent fungi that have been soil fungi phosphate adaptation 189 growing in the contaminated soil, but rather the immission of spores from surrounding fields. in order to asses fungi which have developed soil mycelia, soil particles were size fractionated and washed examining a fraction of organic soil particles of 54 to 250 µm. microscopically inspected particles showed adhering mycelial growth, and clamp forming basidiomycetes could be shown at all sites (fig. 4). the remaining particles were plated on complete media and on plates containing lignin, guiacaol and benomyl. this allows the preferential growth of basidioymcetes (and to some extend zygomycetes) which are generally more resistant to benomyl. at the same time, the lignin/ guiacol shows a color reaction indicative of peroxidases activity involved in lignin decomposition, a feature of litter degrading soil basidioymcetes. for each sample, 5 replicate platings were performed. the complete medium showed the expected decrease in fungal strains from top to bottom in the profiles at all three sites with 110, 35 and 8 isolates at site 1, 105, 52 and 35 isolates from site 2 and 110, 58 and 12 isolates from site 3 for o, x and u horizons, respectively. the differences between the sites in sheer numbers on complete media are not as high as expected, if measured against the ergosterol content which was significantly lower at the contaminated site(s). however, growth on the selective plates corresponded nicely to the disturbance at site 1 with intermediate numbers at site 2 and highest number of isolates on site 3 (fig. 5). lignin degrading fungi were present at site 1 only in the uppermost horizon, at site 2 in horizons o and x, and only at site 3 all three horizons led to isolation of lignin degrading fungi. some additional filamentous fungi neither identified as zygomycetes by lack of septation and fluffy growth pattern nor as lignin degrading fungi were present at sites 2 and 3 hinting at higher biodiversity. mycorrhization and occurrence of fungal fruitbodies at locations 1 and 3, birch roots could be collected for determination of ectomycorrhiza formation rates. while at site 1 only 55 % of the short roots were mycorrhized, at site 3 79 % of short roots showed ectomycorrhiza formation upon microscopical inspection. from the fuitbodies collected, at site 1 litter decomposing fungi dominated with hygrocybe nigrescens (2 specimen), sapultario arenosa (associated with lichen) and tubaria hiemalis. only one species of ectomycorrhiza forming fungi (hebeloma spec.) was present with 2 fruitbodies. at sites 2 and 3, the ectomycorrhizal fungus hebeloma mesophaeum was present with 10 to 15 fruitbodies, and at site 3 additionally cortinarius betuletorum was found. growth on soil extract media and polyphosphate accumulation since the contamination was primarily with phosphate the ability of growth on soil extract media and the ability to accumulate phosphate from these soil extract media in hyphae grown under sterile conditions was tested. one basidiomycete isolate from site 2, upper horizon o was used for these investigations. the fungus is a dikaryon as visible by dapi staining, which does not form clamps. generally, isolates from media showed rarely clamp formation while the mycelia seen by direct microscopic observation were predominantly clamp forming basidiomycetes. soil extract media were prepared from the lowest horizon samples so as to provide a gradient of plant available phosphate. the phosphate concentration had been found to be 463, 245, and 152 mg/g soil dry matter. growth occurred on all three media. while the initial ph of 7 was unchanged in soil extract medium of site 1, the ph had dropped to 6.5 and 6.3 at sites 2 and 3, respectively. adaptation of phosphate uptake and storage at sites with high phosphate contents in order to test phosphate acquisition and storage, the staining of polyphosphate granula in hyphae grown on soil extract media was performed. polyphosphate granula were stained with toluidine blue at ph 1 to allow for specificity of the staining and counted per 100 µm hyphal length. more granules and bigger granules were observed with decreasing plant available phosphate in the extract media (fig. 6) with 6.9 granules of up to 1 µm size and 1 granule sized 1 µm or larger at site 1, 10.3 small and 1.5 larger granules at site 2 and 10.2 small and 1.9 large polyphosphate granules at site 3. thus, the phosphate storage particles were in inverse proportion to available phosphate concentrations at these phosphate eutrophic sites. discussion characterization of a traverse along the pollution gradient the soil type at the investigated area has been classified as part of the rendzina and sloped clay of the river saale valley with good water site 1 site 2 site 3 0 10 20 25 15 5 o x u o x u o x u zygomycetes lignin degrading fungi yeasts fig. 5: growth of fungi on selective media containing benomyl. washed soil particles were plated from three sites with increasing distance from the former plant in three depths, (o) 0 4 cm, (x) 10 15 cm and (u) 20 25 cm from top. zygomycetes, yeast colonies, and peroxidase producing fungi able to degrade lignin were scored. o x u g e rg o st e ro l/ g s o il d ry m a ss site 1 site 2 site 3 fig. 3: ergosterol content at three sites with increasing distance from the former plant in three depths, (o) 0 4 cm, (x) 10 15 cm and (u) 20 25 cm from top. 190 ulrich terpitz, erika kothe retention capacity and nutrient availability (heinrich, 1984; rau, 1995; seidel, 1995). the sandy lowest horizon investigated in this paper at site 1 may be due to quartz sand emission that has been mixed to the fertilizer and has shown high wind distribution (langer, 2000). this sandy layer is covered by loamy and more organic layers which may have resulted from a slide off the upper slopes. at the time of closing the plant, the flora consisted mainly of a monospecific grassland and high fluor contents led to bee killing (anke et al., 1991). the steep slopes directly situated at the northern part of the plant did show the most visible effects. eight years after closing the plant the soil was still greatly disturbed (langer, 2000). we proceeded to test the capacity of natural attenuation five years after the last investigation. by now, the flora seems regenerated, constituting a grassland ecosystem with initial stages of bush and tree growth. the microflora, however, still reflects the detrimental effects of the pollution incurred during 1957 and 1990, even after 13 years have passed since production and emission have stopped. the plant available phosphate content varied greatly as was expected among the sites along a traverse section. with increasing distance from the plant, phosphate concentrations decreased. due to a likely slide at the first site at the steep hillside, which probably has occurred during the period when due to emission there was almost no plant cover, this was best visible in the lowest horizon of the profiles. the emitted phosphate dust was of high ph 10 11, and in early investigations was observed in deeper horizons only at sites very close to the plant. at distant sites phosphate and alkalinic ph was observed only in the uppermost soil layer (heinrich, 1984). by now, also at distant sites the deeper horizons show elevated ph and higher phosphate contents which is indicative of wash-out from the uppermost layers into deeper horizons with time. the geogenic ph on the soil types formed on the chalcerous rocks of the saale valley are generally in the range of ph 6.9 to 7.2 (zorn and krause, 1999), with phosphate concentrations from 28 to 117 mg phosphate per kg soil dry matter. thus, a distinct pollution is still seen at steudnitz. the transfer of phosphates into deeper horizons is not generally observed, as mineralization specifically with calcium, and adsorption to aluminum and iron hydroxides is high and allows retention (curtin and syers, 2001; scheffer and schachtschabel, 1989). however, the extremely high contents may have facilitated phosphate deposition in deeper horizons in the soil types which are poor in organic layers at the polluted sites in steudnitz which had been additionally worsened by the lack in plant cover during high emission. the soil shows regeneration from the extremely high phosphate contents with decrease from 380 825 mg/kg four years ago (langer, 2000) to now 463 mg/kg maximum. the transect still shows correlation of high phosphate contents with distance from the former pollution source. influence of phosphate contamination on uptake and storage in hyphae since the lowest profile shows the best correlation with distance from the plant, this soil horizon was used for soil extract media to determine phosphate uptake into the fungi as visible by polyphosphate granula within the cell vacuoles. the soil extract media from the site with lowest phosphate concentration showed the highest polyphosphate granula content. this implicates that phosphate storage is fostered by depletion while in an environment of excess phosphate storage seems unnecessary and is avoided by the fungi. this is in contrast to earlier investigations, where in vitro polyphosphate accumulation on complete media showed a direct correlation between phosphate content of media and polyphosphate granula contents (beever and burns, 1980; jennings, 1995; tasaki et al., 2002). since we have used soil extract media, our investigation should reflect the natural situation while on standard media with generally lower phosphate levels tested the phosphate acquisition and storage is linked directly to phosphate availability. the low accumulation of phosphate at the polluted sites is seen as a adaptive response to the phosphate eutrophy. another explanation to higher phosphate storage at sites with low phosphate might be inferred from molecular data. phosphate acquisition need phosphate transporter proteins which are known from the yeast saccharomyces cerevisiae and from filamentous fungi including neurospora crassa and the ectomycorrhizal basidiomycetes tricholoma vaccinum and t. terreum (perron et al., 1999; versaw and metzenberg, 1995). at low phosphate concentration two high affinity transporter systems are operating, one with proton symport and the other by sodium symport. the proton symporter is highly ph dependent, with decreasing transporter activity at higher ph. the sodium symporter is almost independent of ph (see versaw and metzenberg, 1995). species growing on soils of neutral to alkaline ph show expression of the sodium symporter while the proton symporter is expressed in species typically found on acidic soils (kothe et al., 2002). however, at high phosphate concentrations, transcription of the high affinity transporters is shut down and a low affinity system is operating which uses proton symport and therefore is ph dependent. phosphate uptake therefore can be postulated to be reduced at high ph at high phosphate sites like in steudnitz. we corrected for ph effects by supplying the soil extract media at an artificially set ph of 7.0 such that the same initial rates of phosphate uptake would be provided for all three sites. the results therefore are not simply mirroring the ph dependence of the low affinity phosphate uptake system, but rather hint at an adaptive effect of high phosphate on the expression of phosphate transporters in a fungal isolate from this contaminated site. further investigation of the molecular basis of phosphate uptake in these environments is needed to substantiate this hypothesis. influence of high phosphate contents on microflora soil respiration was low at the contaminated site(s). however, the high phosphate content in the lowest horizon at site 1 allowed for higher respiration as compared to the upper profiles. the contamination also led to reduced mycorrhization which has been shown to be decreased upon contamination, especially with heavy metals (erland and taylor, 2002). the decreased soil respiration and mycorrhization as well as low biodiversity for lignin decomposing basidiomycetes and adaptive response in polyphosphate contents can be attributed to pollution. fungal biomass was determined by ergosterol content, direct microscopy of washed soil particles and cultivation experiments. ergosterol contents have been used for determination of fungal biomass (west et al., 1987). however, different taxa produce highly divergent ergosterol contents variable with species (huang et al., 1985; martin et al., 1990; nylund and wallander, 1992), cultivation (arnezeder and hampel, 1991; charcosset and chauvet, 2001; dawsonandoh, 2002) and in addition with especially zygomycetes showing low ergosterol contents in their membranes (wheete and gandhi, 1996; ruzicka et al., 2000). the ranges of ergosterol per mg fungal dry weight are given with 5-15 µg (wheete and gandhi, 1996), 0.8-11 µg (charcosset and chauvet, 2001) and 8-15 µg (davis and lamar, 1992). therefore it was proposed to use a general median of 5.1 µg per mg fungal dry weight for calculations of fungal biomass (djajakirana et al., 1996) which was proposed after comparison of 42 literature citations. even if the exact fungal biomass therefore is not easyly determined using ergosterol extraction from soil, the results are very well comparable in one study since soil texture (gong et al., 2001) and soil pollution (bajaras et al., 2002) do not influence ergosterol extraction. the examination of soil ergosterol content in the steudnitz area showed increasing contents in all depths with soil fungi phosphate adaptation 191 distance to the plant, correlating with the decreasing ph and phosphate contents. the contents are low if compared to grassland ergosterol contents of 5.52 µg ergosterol per g soil dry matter and rather are comparable with contents of (fertilized) agricultural sites of 2.14 µg/g (djajakirana et al., 1996) although the flora here constitutes a grassland community and the steep hillsite had not been used agriculturally. the determined fungal biomass using the median of 5.1 µg ergosterol per mg fungal dry mass would correspond to 200, 5 and 10 mg for o, x and u horizons at site 1, to 480, 40, and 10 mg fungal dry biomass at site 2 and to 520, 150 and 40 mg fungal biomass per kg soil at site 3, respectively. especially at the o horizon of site 1, the discrepancy between the three methods used to determine fungal biomass is obvious. total isolated strains on complete media from 400 µl washed soils suspension were scored with 110 isolates which is similar to uppermost horizons at sites 2 and 3, while ergosterol constents show intermediate levels with 200 mg/kg (with approx. 500 mg/kg at sites 2 and 3) and only 2 isolates were obtained on benomyl containing media (corresponding to 24 isolates for sites 2 and 3). this imlies predominance of benomyl sensititve, low ergosterol level containing fungi in thiese soil samples. since the use of a median ergosterol content will yield good appriximations only if the funagl biodiversity is high enough to compensate for indicidual differences in ergosterol contents, theese findings imply a substantially lower biodiveristy at site 1 where highest phosphate concentrations were present. this interpretation is substantiated by the indentification of lignin degrading fungi (50%) and zygomycetes (50 %) only at site1. at site 2, idnetification of lignin-degrading fungi (8 %), zygomycetes (70 %) and others (22 %), and at site 3 ligningdegrading fungi (25 %), zygomycetes (66 %) and others (8 %) also give the impression of lowest diversity at site 1. the isolation of fungi from all soil samples after washing to remove resting spores showed increasing diversity with distance from the plant while the numbers on complete medium varied, but did not increase generally. the selective media contained benomyl which is known to repress growth of ascomycetes stronger than growth of zygomycetes or basidiomycetes. this would lead to the conclusion that the fungal biomass at the polluted site 1 consisted more of ascomycetes as compared to the basidiomycetes that are involved in lignin degradation. lignin degrading fungal colonies were found in substantial numbers only at site 3 where lignin degraders were present in all three horizons. since lignin decomposition is an essential function of soil fungi involved in soil formation and formation of an organic layer, the lack in lignin degrading fungi accounts for the lower levels in organic substances found at sites 1 and 2. this increase in fungal biodiversity coupled to functional diversity in the ecosystem is typical for succession. the occurrence of fruitbodies and lower mycorrhization rates at sites close to the former fertilizer plant confirms this observation of successional stages in the fungal microflora, as ectomycorrhizal fungi are known to be late succession stage fungi (frankland, 1998). in order to assess fungal biodiversity, soil texture and carbon source availability must be taken into consideration. while at site 1 carbon humic substances are low, site 3 has carbon contents lower than site 2. nevertheless, site 3 shows highest fungal biodiversity and higher fungal biomass as determined by ergosterol contents. this indicates that the pollution has a higher influence on fungal biomass and diversity as compared to availability of carbon source. nitrogen availability was not investigated in the present study. however, the area is a steep hillside which has not been fertilized and not used for any type of farming which makes it unlikely that nitrogen is a meaningful parameter at this site. previous studies found no differences in nitrogen content with 0.4 to 0.6 % in top soil and 0.12 % in lower horizons (langer, 2000). the high prevalence of zygomycetes can be explained by the low content of degradable litter or degraded, organic matter which would enhance growth of ligninolytic basidioymcets. in addition, the occurrence of a grassland plant community which foms mycorrhiza with the vesicular arbuscular mycorrhizal fungi of the order glomerales might explain differences in ergosterol measurments with isolation of fungal saprotrophs. the vam fungi are strictly biotrophic and cannot be cultivated axenically on agar media with a plant partner, thus providing a reservoir of ergosterol that does not correspond with fungi isolated from soil. phosphate, albeit an essential nutrient, inhibits germination of fungal spores at high concentrations miranda and harris, 1994). thus, loss of fungal diversity as seen at this site could be attributed to the physiologically detrimental effect of the phosphate concentration itself. phosphate uptake is at least in part promoted by acidification of the substrate (olsson et al., 2002). acknowledgements we would like to thank nadin hahn for establishing ergosterol detection 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(eds.), the mycota, vol 3: biochemistry and molecular biology, 421-438. springer, berlin, germany. zorn, w., krause, o., 1999: investigation on the characterisation of plant available phosphate in thuringian calcerous soils. j. plant nutr. sci. 162, 463-469. address of the authors: prof. dr. erika kothe, insitute for microbiology, microbial phytopathology, friedrich-schiller-university, neugasse 25, d-07743 jena email: erika.kothe@uni-jena.de soil fungi phosphate adaptation 193 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends 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<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> >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice journal of applied botany and food quality 86, 99 103 (2013), doi:10.5073/jabfq.2013.086.014 1 division of germplasm evaluation, national bureau of plant genetic resources, pusa campus, new delhi, india 2 indian agricultural statistics research institute, pusa campus, new delhi, india mineral composition and their genetic variability analysis in eggplant (solanum melongena l.) germplasm m. arivalagan 1 *, r. bhardwaj 1, k.k. gangopadhyay 1, t.v. prasad 1, s.k. sarkar 2 (received july 2, 2013) * corresponding author summary eggplant, solanum melongena l. is one of the most popular and major vegetable crops grown in south asia and other parts of the world. it is an important source of plant-derived nutrients like minerals, available throughout the year and popular among the poor. thirty two morphologically diverse eggplant germplasm accessions were analyzed for macroand micro-minerals. significant differences in the mineral content among the germplasm accessions were detected. potassium and magnesium ranged from 177.19 to 274.48 mg and 6.25 to 18.34 mg/100 g fresh weight (fw), respectively. copper, iron and zinc ranged from 0.024 to 0.178, 0.170 to 0.846 and 0.073 to 0.233 mg/100 g fw, respectively. phenotypic co-efficient of variation and genotypic co-efficient of variation were high for the minerals studied except potassium. high broad sense heritability (84.44-97.07%) indicated the presence of additive gene effects. significant positive correlation were found between zinc with potassium (r=0.397), magnesium (r=0.439) and copper (r=0.409). overall, two germplasm accessions ic090785 and ic383102 have been identified as rich sources for all minerals studied, which could be utilized further in breeding programme for developing mineral-rich varieties of eggplant. introduction eggplant (solanum melongena l.), also known as aubergine, brinjal, or guinea squash, is a vegetable crop, economically important, consumed throughout the world. it is native to the south east asian region and was domesticated over 4000 years ago. current evidence suggests that eggplant is native to india, with secondary centers of diversity in other parts of southeast asia and china (swarup, 1995). the increased consumption of eggplant has been attributed to the increase in ethnic diversity and the greater awareness that diets rich in fruits and vegetables are linked to a lower mortality rate and incidence of diseases (chen et al., 2007; hasler et al., 1998). eggplant is low in calories and has various macroand micro-minerals which are beneficial for human health. potassium is the abundant mineral present in the eggplant, ranged from 200 to 600 mg / 100 g of fresh mater. eggplant is also a rich source of magnesium, calcium and iron (kowalski et al., 2003). minerals are present in all body tissues and fluids and their presence is necessary for the maintenance of certain physicochemical processes which are essential to life. every form of living matter requires these minerals for their normal life processes. deficiencies of micro-minerals are a major global health problem. more than 2 billion people in the world today are estimated to be deficient in key minerals, particularly, iron and zinc. this is particularly true in the developing world, where more than 40% of women are anaemic (who/fao, 2001). most of these people live in low income countries and are typically deficient in more than one micronutrient. deficiencies occur when people do not have access to micronutrient-rich foods such as fruit, vegetables, animal products and fortified foods, usually because they are too expensive to buy or are locally unavailable. since, eggplant is an important source of plant-derived nutrients, available throughout the year and popular among the poor; there is an urgent call to carry out extensive research efforts to know its mineral composition and identify mineral rich germplasm. although report on nutritional aspects and mineral content in the crop are available, there is no information regarding associations of mineral content and their genetic variability and heritability. therefore, to fill this knowledge gap, the present investigation was undertaken to ascertain the mineral composition of different germplasm accessions of eggplant and to find out their heritability based on phenotypic and genotypic variation, which could be useful in qualitative improvement of the eggplant rich in mineral content. materials and methods eggplant samples thirty two eggplant germplasm accessions used in the present study were obtained from national gene bank, national bureau of plant genetic resources (nbpgr), new delhi, india. seeds were sown in nursery on june, 2011 at nbpgr new area farm, pusa campus, new delhi (77.15 °e longitude and 28.64 °n latitude) in semi-arid type of climate with temperatures ranging from 20 °c to 40 °c, average rh of 50% and rainfall ranging from 550 to 850 mm. the seeds were treated with captan {cis-n [(trichloromethyl) thio]-4 cyclo hexane-1, 2-dicarboximide} to prevent soilborne fungal diseases, mainly damping-off occurring in the nursery, and sown in nursery beds. one-month-old seedlings (about 15 cm in height) were transplanted in single row, each 6 m long, with 90 cm spacing between the rows and 60 cm between plants. a basal dose of 100 kg n, 80 kg p2o5, and 60 kg k2o ha–1 was applied and top dressing with 40 kg n ha–1 was done 45 d after transplanting. irrigation and hand weeding were performed whenever necessary. five fruits per plant were harvested at marketable maturity stage. three independent samples of each accession were analyzed and each replicate was processed independently. fruits were hand rinsed under a stream of tap water to remove the dirt from fruit surface, then rinsed with double distilled water and gently blotted with paper towel to remove the water. cleaned and surface-dried fruits were sliced longitudinally into small pieces representing the whole sample, and fresh weight (fw) was recorded. the samples were dried at 65 °c in a hot-air oven (labco, india) for 72 h until constant dry weight (dw) was achieved, then ground into powder with a mechanical grinder for use in estimating mineral content. analytical methods moisture content was determined as 100 x (fw-dw / fw). mineral content was estimated according to official analytical methods (aoac, 1990) with atomic absorption spectrometer (aas, modelvarian spectra aa 220 fs, varian australia pty ltd, australia) equipped with a d2 lamp background correction system, and 100 m. arivalagan, r. bhardwaj , k.k. gangopadhyay, t.v. prasad, s.k. sarkar using an air-acetylene flame. determinations were carried out in triplicate for each independent sample of each genotype. for the analysis of minerals, 5 g of dried ground sample were calcined in a muffle furnace (labco, india) at 450 °c for 2 h. ash was moistened with double distilled water and 2-3 drops of concentrated hno3 (69%, gr grade, merck kgaa, darmstadt, germany) was added. crucibles were again kept in muffle furnace at 450 °c for 30 min to complete ashing. the ash was then dissolved in 5 ml of concentrated hcl (37% gr grade, merck kgaa, darmstadt, germany). the mixture was heated until the first vapors appeared and 10 ml of double distilled water was added immediately. then the solutions were filtered through quantitative ashless filter paper and the final volume was made up to 100 ml with double distilled water. the samples were analyzed using aas calibrated with standards (certipur grade, merck kgaa, darmstadt, germany). as-frm (food reference material) 14 (rice-2 for minerals) obtained from institute of nutrition, mahidol university, thailand was used as reference material to evaluate the analytical methods. quality control for minerals were also checked using eggplant samples spiked and not spiked with known amount of standards of minerals analyzed. the detection limits for minerals were 3 μg/100 g for k; 0.3 μg/100 g for mg; 3 μg/100 g for cu; 6 μg/100 g for fe and 1 μg/100 g for zn. statistical analyses all statistical analyses were performed using statistical analysis software (sas, 2009). analysis of variance was carried out using proc glm to determine significant differences in macroand micro-minerals among the eggplant germplasm accessions. simple linear correlation analysis was performed to indicate the measure of correlation and strength of relationship between variables. principal component analysis (pca) using correlation matrix was performed to study the relative contribution of macroand micro-minerals to the total variability in the eggplant germplasm accessions. the phenotypic variation for each trait was partitioned into genetic factors and estimated according to johnson et al. (1955): σ2p=msg/r; σ2g=(msg-mse)/r; where σ2p and σ2g are phenotypic variance and genotypic variance, respectively; msg, mse and r are mean squares of accessions, mean squares error and number of replications respectively. phenotypic (pcv) and genotypic coefficient of variation (gcv), heritability (h2) in broad sense and genetic advance (ga) were estimated according to singh and chaudhary (1985) as given below: where, σ2p: phenotypic variance, σ2g: genotypic variance, x : general mean of character, i: standardized selection differential, a constant (2.06) and σp: phenotypic standard deviation ( ). results and discussions the results revealed that there were significant differences with wide variability among the 32 eggplant germplasm accessions with respect to moisture, potassium, magnesium, copper, iron and zinc content (tab. 1). the moisture content ranged from 90.86% to 95.04% with the mean value of 92.72%. potassium: ic413648 had the highest potassium content (274.48 mg/100 g), followed by ic090940 (270.86 mg/100 g) and ic261772 (265.91 mg/100 g). the lowest amount of potassium was found in ic112744 (177.19 mg/100 g). the mean potassium content for 32 germplasm accessions was 231.83±4.08 mg/100 g. the phenotypic coefficient of variation for potassium (9.95%) was least among all the minerals analyzed. magnesium: the magnesium content averaged 12.73±0.58 mg/ 100 g, the highest being found in ic104083 (18.34 mg/100 g) followed by ic112993 (18.31 mg/100 g). the coefficient of variation for magnesium (25.65%) was second lowest among all the minerals analyzed. out of 32 germplasm accessions, 16 showed above-average values for magnesium content. copper: the copper content among the germplasm accessions studied ranged from 0.024 0.178 mg/100 g with an average of 0.093±0.01 mg/100 g. fifteen germplasm accessions showed aboveaverage mean values for copper content. highest amount of copper was found in ic545854, while lowest amount in ic104083. the coefficient of variation (44.33%) for copper was maximum than all other minerals studied. iron: the highest amount of iron was found in ic090785 (0.846 mg/100 g), followed by ic112516-1 (0.733 mg/100 g) and ic074209 (0.686 mg/100 g). the mean iron content in 32 germplasm accessions was 0.498±0.01 mg/100 g and 17 germplasm accessions showed values higher than the mean value. the coefficient of variability for iron (30.12%) was second highest among all the minerals analyzed. zinc: zinc content among the germplasm accessions ranged from 0.073 mg/100 g (ic090981) to 0.233 mg/100 g (ic383102) with an average mean of 0.158±0.01 mg/100 g. out of 32 eggplant germplasm accessions, 17 germplasm accessions contained more than the mean value. the levels of various macroand micro-minerals studied among the 32 eggplant germplasm accessions were consistent with the levels reported earlier (alvi et al., 2003; raigon et al., 2008). variability plays an important role in crop breeding programs. the extent of diversity in crop determines the limits of selection for improvement. in any crop-breeding program, it is prerequisite to have a large variation in the material at the hand of breeder. the present study revealed that there was a wide variability among the eggplant germplasm accessions with respect to their mineral content. minerals are important constituents of human diet as they serve as co-factors for many physiological and metabolic processes. potassium is the most abundant intracellular cation in the body and contributes to intracellular osmolality. enzymes involved in glycolysis and oxidative phosphorylation are potassium-dependent (haddy, 1987). magnesium is an important co-factor of many regulatory enzymes, particularly the kinases, and is fundamental in the energy transfer reactions involving high energy compounds like atp and creatine phosphate and thus muscle contraction (tillman and semple, 1988) and pregnancy-induced hypertension and preeclampsia (robert et al., 2003). adequate dietary intake of iron, eggplant genetic variability 101 tab. 1: mean values, standard error (se), least significant differences (lsd; p=0.05) and phenotypic coefficient of variation (cv %) for moisture (%), macroand micro-mineral composition (mg/100 g fresh weight) in 32 eggplant germplasm accessions studied germplasm moisture % k mg cu fe zn ic074209 91.79 247.84 7.98 0.060 0.686 0.161 ic089888 92.40 236.13 13.61 0.064 0.247 0.131 ic090053 91.84 210.29 10.76 0.129 0.498 0.159 ic090785 93.27 255.81 16.13 0.071 0.846 0.211 ic090806 92.52 220.91 11.44 0.093 0.425 0.101 ic090938 93.35 231.81 13.28 0.125 0.526 0.168 ic090940 92.85 270.86 9.29 0.088 0.560 0.161 ic090981 92.69 203.18 6.39 0.048 0.531 0.073 ic104083 94.08 236.70 18.34 0.024 0.602 0.190 ic111066-2 93.28 222.60 12.47 0.090 0.382 0.112 ic111416 92.75 228.49 11.46 0.063 0.475 0.157 ic111439 92.26 218.73 14.29 0.108 0.382 0.201 ic112516-1 93.14 221.39 6.25 0.056 0.733 0.084 ic112732 93.38 223.78 12.69 0.126 0.483 0.152 ic112744 93.54 177.19 10.81 0.115 0.389 0.132 ic112991 92.23 204.29 14.97 0.035 0.608 0.156 ic112993 90.86 212.36 18.31 0.108 0.395 0.119 ic127063 95.04 208.40 7.31 0.060 0.630 0.081 ic261772 92.82 265.91 14.42 0.051 0.296 0.154 ic261793 93.82 252.45 14.47 0.054 0.170 0.093 ic285125 92.65 250.20 12.72 0.144 0.616 0.177 ic310884 92.93 240.31 15.07 0.096 0.525 0.112 ic333527 92.08 217.24 9.93 0.078 0.404 0.165 ic354140 93.03 230.17 15.39 0.158 0.548 0.207 ic354517 92.13 242.17 13.11 0.057 0.648 0.224 ic354597 92.39 184.87 9.49 0.044 0.223 0.189 ic354604 92.67 243.89 10.76 0.108 0.405 0.188 ic354612 92.31 237.54 12.65 0.150 0.589 0.192 ic383102 92.76 258.06 16.02 0.171 0.634 0.233 ic413648 91.97 274.48 13.74 0.127 0.439 0.228 ic544884 91.18 244.78 15.72 0.107 0.464 0.140 ic545854 93.16 245.83 18.14 0.178 0.593 0.204 mean 92.72 231.83 12.73 0.093 0.498 0.158 se 0.14 4.08 0.58 0.007 0.027 0.008 lsd 0.001 7.45 0.91 0.01 0.10 0.02 pcv (%) 0.88 9.95 25.65 44.33 30.12 28.30 zinc, and copper is essential to human health. more than 2 billion people worldwide are anaemic, and this can be mainly attributed to iron deficiency. iron is an essential component of body systems involved in the utilization of oxygen. iron deficiency during childhood and adolescence impairs physical and mental development (who/fao, 2001). zinc is required for protein and carbohydrate metabolism, immune system, wound healing, growth and vision. inadequate zinc intake can result in retarded growth, delayed wound healing, reduced immune system, loss of taste sensation and dermatitis (umeta et al., 2000). the adult body contains approximately about 80 mg copper mainly stored in liver, followed by brain and muscle. cytochrome c oxidase, superoxide dismutase (sod), lysyl oxidase and tyrosine oxidase are the major enzymes which require cu for their activity. deficiency signs of copper include anemia, vascular complications, osteoporosis and neurological manifestations. the relative significance of copper was more than that of ascorbic acid in iron-deficiency anaemia (chiplonkar et al., 2003). 102 m. arivalagan, r. bhardwaj , k.k. gangopadhyay, t.v. prasad, s.k. sarkar all the significant correlations, viz. zinc with potassium, magnesium and iron (r=0.397, 0.439 and 0.429, respectively, at p < 0.05) were found to be positive (tab. 2). such correlation analysis will facilitate selection of germplasm with improved nutritional quality, as selection for one trait leads to selection of genetically correlated traits (wricke and weber, 1986). however, correlation analysis alone could not give a complete picture of interrelations because it considers only two minerals at a time, regardless of the interrelationship with other minerals in the set of data. hence, pca was carried out to understand the underlying interrelationships in the whole set of data and to select the best linear combination of minerals that explains the largest proportion of the variation in the data set. pca revealed that the first three principal components (pcs) together governed 79.27% of the total variability (tab. 3). the first principal component (pc1) accounting for 42.45% of the total variation and all the macroand micro-minerals studied had relatively positive weight on this component. pc2 and pc3 accounting for 20.80 and 15.99% of the total variation, respectively. in all the three principal components, the coefficients corresponding to iron and zinc were positive and consistent, whereas potassium is positive in first two pcs. since, iron and zinc followed by potassium in pc1 and pc2 (eigen value >1) accounted for more of the variation in the set of data than other minerals, they were the most appropriate to use in the preliminary grouping of the 32 eggplant germplasm accessions evaluated in this study. the characters like minerals are generally quantitative in nature and exhibit considerable degree of interaction with gene. thus, it becomes necessary to compute variability present in the material and its partitioning into genotypic and phenotypic effects. in the present study, the values of phenotypic coefficient of variability (pcv) were greater than the corresponding genotypic coefficient of variability (gcv) values, though in many cases the differences were very less (tab. 4). copper and iron followed by zinc showed high pcv and gcv values, while potassium showed low pcv and gcv values. the broad sense heritability for microand macrominerals was more than 84% with maximum of 97.06% for magnesium followed by potassium. the expected genetic advance as percentage of mean ranged from 19.69 to 85.96%. maximum genetic gain was observed for copper (85.96%) followed by iron (55.39%). potassium showed low genetic gain (19.69%). little difference between phenotypic coefficient of variation and genotypic coefficient of variation and high estimates of heritability (broad sense) for macroand micro-minerals indicates that the differences between accessions for mineral contents were genetic in nature. the genotypic coefficients of variation were less than that of the phenotypic coefficients of variation for all the characters indicating the influence of non-additive gene action (shukla et al., 2005). because the coefficient of variation measures the magnitude of variability present in the population, selection from population with such coefficient of variation values is very likely to be effective in improvement of the minerals studied. high broad sense heritability values indicated the predominance of additive gene action in the expression of these traits and can be improved through individual plant selection. variability alone is not of much help in determining the heritable portion of variation. the amount of gain expected from a selection depends on heritability and genetic advance in a trait. knowledge of heritability of a character is important as it indicates the possibility and extent to which improvement is possible through selection (robinson et al., 1949). however, high heritability alone is not enough to make sufficient improvement through selection generally in advance generations unless accompanied by substantial amount of genetic advance (johnson et al., 1955). the expected genetic advance is a function of selection intensity, phenotypic variance, and heritability and measures the differences between the mean genotypic values of the original population from which the progeny is selected. it has been emphasized that genetic gain should be considered along with heritability in coherent selection breeding programmes. it is considered that if a trait is governed by nonadditive gene action it may give high heritability but low genetic advance, which limits the scope for improvement through selection, whereas if it is governed by additive gene action, heritability and genetic advance would be high, consequently substantial gain can be achieved through selection. heritability estimates along with tab. 2: linear correlation (r) between moisture, macro and micro-minerals of 32 eggplant germplasm accessions studied k mg cu fe zn moisture % -0.053 -0.123 -0.147 0.135 -0.217 k 0.335 0.199 0.196 0.397* mg 0.312 -0.044 0.439* cu 0.109 0.409* fe 0.231 *significance at p=0.05 tab. 3: average range of variation and component scores of the first 3 principal components in 32 eggplant germplasm accessions studied minerals studied range mean sd first 3 principal components pc1 pc2 pc3 potassium 177.19-274.48 231.83 23.06 0.461 0.135 -0.641 magnesium 6.25-18.34 12.73 3.26 0.476 -0.452 -0.192 copper 0.024-0.178 0.093 0.04 0.443 -0.134 0.731 iron 0.170-0.846 0.498 0.15 0.222 0.871 0.110 zinc 0.073-0.233 0.158 0.05 0.561 0.033 0.067 variance % component 42.27 20.90 15.95 cumulative 42.27 63.17 79.13 eggplant genetic variability 103 genetic advance are more useful in predicting the resultant effect for the selection of the best individuals from a population. the heritability and genetic advance values were high for copper, zinc and followed by iron, suggests that these traits are under genetic control and significant improvement can be obtained for these traits. within in the range of eggplant germplasm used in this study, there exist substantial genetic variability and heritability in the minerals studied to warrant selection in the eggplant accessions for improvement. the level of genetic variability observed for different minerals would be useful for breeding program for developing mineral rich varieties of eggplant. the high genetic variance components and heritability estimates couple with significantly positive correlation could be used as selection criteria for identification of mineral rich eggplant germplasm. two germplasm accessions, ic090785 and ic383102 have been identified as rich sources for all minerals studied. hence, these two accessions could be utilized further in breeding programme for developing mineralrich varieties of eggplant acknowledgement we are grateful to director, national bureau of plant genetic resources, new delhi, india for constant support and encouragement in conducting this study. references alvi, s., khan, k.m., sheikh, m.a., shahid, m., 2003: effect of peeling and cooking on nutrients in vegetables. pak. j. nutr. 2, 189-191. aoac, 1990: official methods of the association of official analytical chemists. 15th edition. washington, dc, usa. chen, l., vigneault, c., vijaya raghavan, g.s., kubow, s., 2007: importance of the phytochemical contents of fruits and vegetables to human health. stewart posthar. rev. 3, 1-5. chiplonkar, s.a., agte, v.v., mengale, s.s., 2003: relative importance of micronutrient deficiencies in iron deficiency anemia. nutr. res 23, 1355-1367. haddy, f.j., 1987: dietary sodium and potassium in the genesis, therapy, and prevention of hypertension. j. am. coll. nutr. 6, 261-270. tab. 4: estimates of mean squares, variance components and heritability for various minerals in 32 eggplant germplasm accessions studied selection parameters mineral mean ms σ2p σ2g pcv (%) gcv (%) h2b (%) ga (%) k 231.831 1595.3450 531.7818 510.9721 9.95 9.75 96.09 19.69 mg 12.729 31.9707 10.6569 10.3440 25.65 25.27 97.06 51.28 cu 0.093 0.0051 0.0017 0.0016 44.33 43.01 94.12 85.96 fe 0.498 0.0675 0.0225 0.0190 30.12 27.68 84.44 52.40 zn 0.158 0.0060 0.0020 0.0019 28.30 27.59 95.00 55.39 ms: mean squares, σ2p: phenotypic variance, σ2g: genotypic variance, pcv: phenotypic coefficient of variation, gcv: genotypic coefficient of variation, h2b (%): broad sense heritability, ga (%): genetic advance. hasler, c.m., 1998: functional foods: their role in disease prevention and health promotion. food technol. 52, 63-70. johnson, h.w., robinson, h.f., comstock, r.e., 1955: estimates of genetic and environmental variability in soybean. agron. j. 47, 314318. kowalski, r., kowalska, g., wiercinski, j., 2003: chemical composition of fruits of three eggplant (solanum melongena l.) cultivars. fol. hort. 15, 89-95. raigon, m.d., prohens, j., munoz-falcon, j.e., nuez, f., 2008: comparison of eggplant landraces and commercial varieties for fruit content of phenolics, minerals, dry matter and protein. j. food comp. anal. 21, 370-376. robert, j.m., balk, j.l., bodnar, l.m., belizán, j.m., bergel, e., martinez, a., 2003: nutrient involvement in preeclampsia. j. nutr. 133, 1684-1692. robinson, h.f., comstock, r.e., harvey, p.h., 1949: estimates of heritability and the degree of dominance in corn. agron. j. 41, 353359. sas, 2009: statistical analysis software system, version 9.2. sas institute, cary, nc, usa. shukla, s., bhargava, a., chatterjee, a., srivastava, j., singh, n., singh, s.p., 2005: mineral profile and variability in vegetable amaranth (amaranthus tricolor). plant foods hum. nutr. 61, 23-28. singh, r.k., chaudhary, b.d., 1985: biometrical methods in quantitative genetic analysis. new delhi, kalyani, 318. swarup, v., 1995: genetic resources and breeding of aubergine (solaum melongena l.). acta hort. 412, 71-79. tillman, d.w., semple, p.f., 1988: calcium and magnesium in essential hypertension. clin. sci. 75, 395-402. umeta, m., west, c.e., haidar, j., deurenberg, p., hautvast, j., j, j.a., 2000: zinc supplementation and stunted infants in ethiopia: a randomised controlled trial. lancet. 355, 2021-2026. who/fao, 2001: human vitamin and mineral requirements. report of a joint fao/who expert consultation; geneva, switzerland. wricke, g., weber, w., 1986: quantitative genetics and selection in plant breeding. w. de gruyter, berlin, germany. address of the corresponding author: m. arivalagan, central plantation crops research institute, kerala, 671124, india e-mail: arivalagan2100@gmail.com journal of applied botany and food quality 87, 56 61 (2014), doi:10.5073/jabfq.2014.087.008 1laboratoire d’ecologie et gestion des ecosystèmes naturels, faculté des sciences de la nature et de la vie, et des sciences de la terre et l’univers 2 laboratoire des substances naturelles et bioactives (lasnabio) département de chimie, faculté des sciences, université de tlemcen, algérie 3 université de corse, umr cnrs 6134, laboratoire chimie des produits naturels, campus grimaldi, corte, france antifungal activity of essential oils of three aromatic plants from western algeria against five fungal pathogens of tomato (lycopersicon esculentum mill) s. bouayad alam1, n. gaouar benyelles1, m. el amine dib2*, n. djabou2, l. tabti1, j. paolini 3, a. muselli 3, j. costa 3 (received december 9, 2013) * corresponding author summary the antifungal effect of the essential oils from thymus capitatus l., daucus crinitus desf. and tetraclinis articulate vahl., aerial parts was evaluated in vitro against five phytopathogenic fungi of tomato (fusarium oxysporum, alternaria solani, aspergillus niger, penicillium sp1 and penicillium sp2). our results showed that among the three plant species tested, t. capitatus oil was the most potent antifungal against the fungi (inhibition of mycelial growth of 100 % at a concentration of 2 μg ml-1). furthermore, the essential oil of t. articulata was also effective against f. oxysporum, a. solani, a. niger, penicillium sp1 and penicillium sp2 with an inhibition of mycelial growth greater than 57 % at a concentration of 5 μg ml-1. d. crinitus essential oil was less effective. t. capitatus essential oil was dominated by carvacrol (69.6 %) and p-cymene (12.4 %). the isochavicol isobutyrate (44.9 %) and isochavicol 2-methylbutyrate (9.7 %) were the major compounds in d. crinitus essential oil, while the most abundant compounds in t. articulata were αpinene (32.0 %), cedrol (11.0 %) and 3-carene (9.6 %). the plant essential oils were found to be an effective antifungal against of mycelial growth and, therefore, can be exploited as an ideal treatment against disease rot of tomato or as a new potential source of natural additives for the food and/or pharmaceutical industries. introduction tomato (lycopersicon esculentum) is an important commercial crop in the world. nutritional values of tomato make it a widely accepted vegetable by consumers. nevertheless, tomato is a very perishable vegetable with a short shelf-life and high susceptibility to fungal disease. tomatoes are among the most popular fruits grown in algeria. they are of an excellent quality and are greatly appreciated for their nutritional value. furthermore, tomato production represents an important agricultural and economic activity in the country. the growing awareness of consumers concerning the relation between food and health is revolutionizing the food industry. fungal pathogens are mainly responsible for the decay of fruits and vegetables during the postharvest period (pathak, 1997). aspergillus, fusarium and penicillium are responsible for spoilage of many foods and causes decay on stored fruits damaged by insects, animals, early splits, and mechanical harvesting. apart from causing diseases in plants, many species of aspergillus, penicillium and alternaria can also synthesize mycotoxins (agrios, 1997; rojas et al., 2005). considerable interest has developed on the preservation of foods by the use of essential oils to effectively retard growth and mycotoxin production. essential oils and their main components possess a wide spectrum of biological activity, which may be of great importance in several fields, from food chemistry to pharmacology and pharmaceutics (cristani, 2007). the main aim of this work was to evaluate the antifungal properties of the essential oils of t. capitatus, d. crinitus and t. articulate against phytopathogens that cause severe diseases in tomato, such as f. oxysporum, a. solani, a. niger, penicillium sp1 and penicillium sp2. materials and methods plant materials and essential oils extraction aerial parts of d. crinitus were collected in bensekrane forest area (tlemcen province) at the flowering stage, in june 2011. the oil yield was 0.37 % (w/w). t. capitatus aerial parts were collected from beni snous in tlemcen city at the flowering stage, during june 2011 and yielded 0.52 % (w/w). t. articulata aerial parts were collected from oujlida region, tlemcen province during june 2011 and yielded 0.31 % (w/w). the plant species were stored at -18 °c after harvest. a portion (550-600 g) of material from each plant species was subjected to a clevenger-type apparatus according to the european pharmacopoeia (european pharmacopoeia, 2004). the essential oils were dried over anhydrous sodium sulfate and, after filtration, stored in sterilized amber vials at 4 °c until it was used. gas chromatography analyses were carried out using a perkin elmer clarus 600 gc apparatus equipped with a dual flame ionization detection system and 2 fused-silica capillary columns (60 m x 0.22 mm i.d., film thickness 0.25 μm), rtx-1 (polydimethylsiloxane) and rtx-wax (polyethylene glycol). the oven temperature was programmed from 60 °c to 230 °c at 2 °c/min and then held isothermally at 230 °c for 35 min. injector and detector temperatures were maintained at 280 °c. essential oils were injected in the split mode (1/50), using helium as the carrier gas (1 ml/min); the injection volume was 0.2 μl. retention indices (ri) of the compounds were determined from perkin-elmer software. gas chromatography-mass spectrometry essential oils were analyzed with a perkin–elmer turbomass quadrupole analyzer, coupled to a perkin–elmer autosystem xl, equipped with 2 fused-silica capillary columns and operated with the same gc conditions described above, except for a split of 1/80. electronic impact (ei) mass spectra were acquired under the following conditions: ion source temperature 150 °c, energy ionization 70 ev, mass range 35-350 da (scan time: 1 s). component identification the identification of the components was based on a comparison: (i) between the calculated retention indices on the polar (ri p) and apolar (ri a) columns with those of pure standard authentic compounds and literature data (jennings and shibamoto, 1980; könig et al., 2001; national institute of standards and technology, antifungal activity of oils against fungal pathogens of tomato 57 2008); and (ii) of the mass spectra with those of our own library of authentic compounds and with those of a commercial library (mc lafferty and stauffer, 1994; mc lafferty and stauffer, 1988; national institute of standards and technology, 1999). component quantification quantification of the essential oil components was carried out using the methodology reported by costa et al. (2008), and modified as follows. the response factor (rf) of 29 standard compounds grouped into 7 chemical groups (monoterpene hydrocarbons, sesquiterpene hydrocarbons, alcohols, ketones, aldehydes, esters, and others) was measured using gc (znini et al., 2011). rfs and cali-bration curves were determined by diluting each standard in hexane at 5 concentrations, containing tridecane (final concentration = 0.7 g/100 g) as an internal standard. analysis of each standard was performed in triplicate. for the quantification of the essential oil components, tridecane (0.2 g/100 g) was added as internal standard to the essential oil. the correction factor (average of the response factors from standards) of each chemical group was calculated and used to determine the essential oil component concentration (g/100 g) according to the chemical group. pathogenic fungi fusarium oxysporum, alternaria solani, aspergillus niger, penicillium sp1 and penicillium sp2 were isolated from naturally decayed tomato after storage of several weeks. these isolates were the most aggressive one in our collection and produced the largest lesions on inoculated fruit. a pure culture of these fungus were maintained on potato dextrose agar medium (pda: potato 200, dextrose 20 g and agar 15 gl-1 in distilled water at 25 °c) in the presence of a quantity of lactic acid (25 %) for stop the growth of bacteria. the plates were incubated at 25 ± 2 °c for 8 days and darkness. the developing fungal colonies were purified and identified up to the species level by microscopic examination through the help of the following references (barnett and hunter, 2006). in vitro antifungal assay the antifungal activity of the three essential oils was tested using radial growth technique (bajpai et al., 2007). appropriate volumes of the stock solutions of the oils in dimethyl sulfoxide (dmso) were added to pda medium immediately before it was poured into the petri dishes (9.0 cm diameter) at 40-45 °c to obtain two concentrations (2.0 and 5.0 μg ml-1). each concentration was tested in triplicate. parallel controls were maintained with dmso mixed with pda. the discs of mycelial felt (0.5 cm diameter) of the plant pathogenic fungi, taken from 8-day-old cultures on pda plates, were transferred aseptically to the centre of petri dishes. carbendazim was used as reference fungicide. the treatments were incubated at 27 °c in the dark. colony growth diameter was measured after the fungal growth in the control treatments had completely covered the petri dishes. percentage of mycelial growth inhibition was calculated from the formula: (i%) = [(dc-dt)/dc] x 100 (pandey et al., 1982); where dc and dt are average diameters of fungal colony of control and treatment, respectively. statistical analysis the inhibitory effect of essential oils on mycelial growth was expressed as mean ± standard error of mean (s.e.m.) and analyzed for anova and post hoc dunnet’s t-test. the separation of means was done by using the least significant difference test at p <0.05. analysis of each test was performed in triplicate. results essential oils composition a total of 26 components accounting to 99.5 % of the essential oil composition of t. capitatus were identified by comparison of their ei-mass spectra and their retention indices (ri) with those of our own authentic compound library (tab. 1). the essential oil was highly dominated by oxygenated compounds (87.1%) with high amount of aromatic terpenic components (82.6 %). however, hydrocarbons appeared also in appreciable proportion (12.4%) which monoterpene hydrocarbons are well represented (10.7 %). indeed, the main constituents of essential oil were carvacrol (69.6 %), p-cymene (12.4 %) followed by γ-terpinene (4.3 %), myrcene (2.1 %), α-terpinene (1.7 %), linalool (1.7 %) and terpinen-4-ol (1.1 %). these results were in accordance with those previously reported in literature (amarti et al., 2008; bounatirou et al., 2007; ruberto et al., 2000; tawaha and hudaib, 2012). other hand, various chemical profiles of essential oils (thymol, cavacrol or thymol/carvacrol as main components) have been reported according to geographical origins of t. capitatus (karouso et al., 2005; miceli et al., 2006). the analysis of the essential oil from the aerial parts of d. crinitus harvested in the forest of bensekrane (tlemcen) identified 30 components, which accounted for 91.3 % of the total composition. their retention indices and relative percentages are shown in tab. 1. the main components of the aerial parts oil were phenylpropanoids isochavicol esters, principally the isochavicol isobutyrate (44.9 %). the other major components identified were: isochavicol 2-methylbutyrate (9.7 %), pentadecane (5.1 %) and undecane (4.1 %) (tab. 1). this result is in according with literature data (lanfranchi et al., 2010). a total of 54 components accounting for 95.9 % of the total oil of t. articulata were identified (tab. 1). the essential oils was highly dominated by the monoterpene hydrocarbons (63.8 %) followed by oxygenated sesquiterpenes (14.7 %) and sesquiterpene hydrocarbons (10.5 %). however, the oxygenated monoterpenes appeared in small proportion (6.4 %). the most abundant compounds were α-pinene (32.0 %), cedrol (11.0 %), 3-carene (9.6 %), limonene (4.3 %), sabinene (4.3 %) and (e)-β-caryophyllene (4.0 %). ben jemia et al. (2013) have isolated and identified, by gc-ms, 66 constituents, the major constituents of the oil are: bornyl acetate (31.4 %), α-pinene (24.5 %) and camphor (20.3 %). while toumi et al. (2011) have identified, by gc/ms, more 45 compounds, with camphor(23.431,6 %), bornyl acetate (17,1-25,8 %), borneol (6.6-14,3 %), limonene (3,70-10,1 %) and α-pinene (6,5-11,3 %) were the major components of t. articulata essential oil. it was observed that the percentage of α-pinene (19.8-24.9 %) and bornyl acetate (40.2-59.2 %) for the leaves oils from two different sites in algeria were the major constituents (chikhoune et al., 2013). in addition, the percentage of cedrol and 3-carene found in our essential oil was higher than cedrol and 3-carene in previous studies. generally, the quality and quantity of components available in essential oils may be affected by several factors, such as plant genotype, geographical condition, season, and agronomic condition (gumus et al., 2010). antifungal activity of three essential oils against the development of fungi of tomato the data presented in tab. 2 show the antifungal activity of 3 plant species, belonging to 3 botanical families (lamiaceae, apiaceae and cupressaceae), against f. oxysporum, a. solani, a. niger, penicillium sp1 and penicillium sp2. the effect of plant essential oils varied according to plant species. indeed, 2 plant species out of 3 reduced 58 s. bouayad alam, n. gaouar benyelles, m. el amine dib, n. djabou, l. tabti, j. paolini, a. muselli, j. costa tab. 1: chemical compositions of aerial parts essential oils of t. capitatus, d. crinitus and t. articulate. no.a components ria b ria c ripd t. capitatus d. crinitus t. articulata identification e nonane 906 902 907 0.6 ri, ms α-thujene 932 924 1028 0.2 tr ri, ms α-pinene 936 931 1028 0.9 0.5 32.0 ri, ms α-fenchene 941 943 1039 0.6 ri, ms camphene 950 945 1071 0.2 0.3 ri, ms oct-1-en-3-ol 962 962 1441 0.5 ri, ms sabinene 973 967 1122 0.6 4.3 ri, ms β-pinene 978 972 1113 0.1 0.1 1.4 ri, ms myrcene 987 982 1160 2.1 0.6 3.3 ri, ms α-phellandrene 1002 999 1161 0.2 1.5 ri, ms 3-carene 1005 1006 1149 0.1 9.6 ri, ms α-terpinene 1008 1011 1270 1.7 ri, ms p-cymene 1015 1015 1270 12.4 0.2 0.5 ri, ms limonene 1025 1023 1201 0.9 4.3 ri, ms β-phellandrene 1023 1023 1209 1.4 ri, ms (e)-β-ocimene 1041 1037 1247 0.6 0.7 ri, ms (z)-β-ocimene 1029 1022 1234 0.6 ri, ms γ-terpinene 1051 1050 1245 4.3 1.6 0.7 ri, ms (e)-sabinene hydrate 1051 1054 1445 0.1 0.2 ri, ms nonanal 1076 1074 1403 0.1 ri, ms terpinolene 1082 1079 1281 0.2 0.4 3.2 ri, ms (z)-sabinene hydrate 1087 1084 1537 0.8 ri, ms linalool 1083 1085 1538 1.7 0.2 0.2 ri, ms undecane 1100 1098 1101 4.1 ri, ms 3-octyl acetate 1113 1107 1330 0.2 ri, ms veratol 1112 1113 1713 0.1 ri, ms camphor 1123 1124 1506 0.1 ri, ms (z)-verbenol 1027 1128 1642 0.3 ri, ms isoborneol 1143 1144 1670 0.5 ri, ms borneol 1148 1150 1688 0.3 ri, ms terpinen-4-ol 1164 1162 1591 1.1 0.1 2.0 ri, ms α-terpineol 1176 1176 1690 0.1 0.1 ri, ms octyl acetate 1188 1187 1460 2.3 ri, ms decanal 1188 1187 1483 1.4 ri, ms linalyl acetate 1239 1239 1552 0.2 ri, ms decanol 1263 1259 1729 0.1 ri, ms nonanoic acid 1263 1263 2119 0.1 ri, ms (e)-anethole 1264 1261 1815 0.1 ri, ms thymol 1266 1263 2181 0.6 ri, ms bornyl acetate 1269 1269 1562 0.7 ri, ms carvacrol 1278 1286 2193 69.6 ri, ms eugenol 1330 1329 2164 0,1 ri, ms α-terpinyl acetate 1335 1333 1686 1.8 ri, ms (e)-myrtanyl acetate 1366 1370 1479 0.1 ri, ms β-bourbonene 1386 1384 1518 0.1 ri, ms β-elemene 1389 1386 1584 0.2 ri, ms dodecanal 1389 1389 1695 3.1 ri, ms antifungal activity of oils against fungal pathogens of tomato 59 no.a components ria b ria c ripd t. capitatus d. crinitus t. articulata identification e β-funebrene 1419 1411 1591 1.6 ri, ms (e)-β-caryophyllene 1421 1416 1591 1.6 0.6 4.0 ri, ms thujopsene 1435 1427 1614 0.2 ri, ms α-humulene 1455 1448 1668 0.1 2.5 ri, ms α-acoradiene 1444 1455 1616 0.1 ri, ms β-acoradiene 1458 1459 1642 0.1 ri, ms alloaromadendrene 1462 1461 1630 0,1 ri, ms γ-curcumene 1475 1471 1680 0,3 ri, ms germacrene d 1479 1474 1700 1,3 ri, ms β-selinene 1482 1480 1703 0,1 ri, ms γ-humulene 1483 1480 1702 0.7 ri, ms pentadecane 1500 1497 1502 5.1 ri, ms δ-cadinene 1520 1511 1760 0.1 0.3 ri, ms β-elemol 1535 1533 2063 0,4 ri, ms isochavicol isobutyrate 1546 1541 2134 44.9 ri, ms dodecanoic acid 1554 1560 2474 1.1 ri, ms, ref caryophyllene oxide 1578 1567 1969 0.1 0.4 ri, ms dodecyl acetate 1585 1580 1882 2.5 ri, ms, ref globulol 1589 1577 2085 0.9 ri, ms cedrol 1595 1591 2101 11.0 ri, ms humulene epoxide ii 1602 1599 2044 0.1 ri, ms epi-cedrol 1613 1614 2141 0.2 ri, ms α-acorenol 1623 1617 2106 0.3 ri, ms γ-eudesmol 1619 1624 2198 0.1 ri, ms τ-cadinol 1633 1632 2146 0.2 ri, ms α-eudesmol 1632 1636 2211 0.3 ri, ms isochavicol 2-methyl butyrate 1651 1648 2255 9.7 ri, ms bulnesol 1665 1664 2198 0.2 ri, ms heptadecane 1700 1703 1699 3.4 ri, ms tetradecanoic acid 1761 1756 2649 3.1 ri, ms, ref cedryl acetate 1764 1750 2160 0.6 ri, ms neophytadiene 1807 1807 1918 0.4 ri, ms, ref hexadecanoic acid 1951 1949 2916 1.1 ri, ms manool 2070 2109 2684 0.3 ri, ms (e)-phytol 2114 2102 2620 1.7 ri, ms total identification % 99.5 92.0 96.5 % hydrocarbon compounds 12.4 20.5 74.5 % monoterpene hydrocarbons 10.7 5.5 63.8 % sesquiterpene hydrocarbons 1.7 1.4 10.7 % non terpenic hydrocarnon compounds 13.2 % diterpenes hydrocarbons 0.4 % oxygenated compounds 87.1 71.5 22.0 % oxygenated monoterpenes 3.8 0.3 6.5 % oxygenated sesquiterpenes 0.1 14.7 % non terpenic oxygenated compounds 0.5 14.9 0.2 % aromatic compounds 82.6 0.1 % phenylpropanoids 0.1 54.6 % oxygenated diterpenes 1.7 0.5 a order of elution is given on apolar column (rtx-1), b retention indices on the apolar rtx-1 column (ria), c retention indices on the polar rtx-wax column (rip), d retention indices on the polar rtx-wax column (rip), e ri: retention indices; ms: mass spectrometry in ei mod. 60 s. bouayad alam, n. gaouar benyelles, m. el amine dib, n. djabou, l. tabti, j. paolini, a. muselli, j. costa tab. 2: percentage of inhibition of mycelial growth at various volumes of essential oils. incubation f. oxysporum a. solani a. niger penicillium sp1 penicillium sp2 25°c ± 2 25°c ± 2 25°c ± 2 25°c ± 2 25°c ± 2 essential oil (2 μg ml-1) t. capitatus 100 ± 0.00 100 ± 0.00 100 ± 0.00 100 ± 0.00 100 ± 0.00 t. articulata 36.11± 0.08 35.12 ± 0.01 11.11± 0.11 34.56 ± 0.02 45.12± 0.06 d. crinitus 54.32± 0.21 essential oil (5 μg ml-1) t. articulata 72.22± 0.06 70.12± 0.20 57.77± 0.11 64.44± 0.12 84.44± 0.08 d. crinitus 5.55± 0.21 77.77± 0.06 mycelial growth of f. oxysporum, a. solani, a. niger, penicillium sp1 and penicillium sp2 by more than 50 %. among these plants t. capitatus, belonging to the families of lamiaceae, completely inhibited mycelial growth of tested fungus. t. capitatus essential oil produced the greatest reduction in mycelium growth with these fungi at 2 μg ml-1, with percentage reductions of 100 % (tab. 2). the second most effective essential oil with this five fungi was t. articulata essential oil, with percentage of mycelial reduction in f. oxysporum, a. solani, a. niger, penicillium sp1 and penicillium sp2 of 36.11, 35.12, 11.11, 34.56 and 45.12 %, respectively, at the same concentration (tab. 2). however, the data indicate that the percentage inhibition of mycelial growth increased with increasing concentration of essential oils for all strains tested, suggesting that the essential oil of t. articulata inhibited the growth of all strains in a dose-dependent manner. essential oil d. crinitus cause no percentage of mycelial reduction, except against penicillium sp2. this activity was more pronounced, where the percentage of inhibition increased to 54.32 % at 2 μg ml-1, reaching a maximum of 77.77 % at 5 μg ml-1, suggesting that this strain was the most sensitive to the essential oil (tab. 2). discussion in this study, the antifungal activity of essential oils of three plant species was evaluated against f. oxysporum, a. solani, a. niger, penicillium sp1 and penicillium sp2. the mycelial growth of colonies in the presence of the essential oil of t. capitatus and t. articulata showed that it effectively controlled all the fungi tested. the mycelial growth of colonies in the presence of the essential oil of t. capitatus and t. articulata showed that it effectively controlled all the fungi tested. this efficiency can be explained by the presence of active molecules that inhibited the growth of the five phytopathogenic fungi. this activity may be produced by a single major compound or by the synergistic or antagonistic effect of various compounds (deba et al., 2008). several authors have attributed the antifungal capacity of plant essential oils to the presence of components such as phenolic and terpene compounds (beuchat, 1994; davidson, 1997; nychas, 1995) indicated that mycelial growth inhibition is caused by the monoterpenes present in essential oils. these components would increase the concentration of lipidic peroxides such as hydroxyl, alkoxy and alko peroxyl radicals and so bring about cell death. however, the influence of essential oil or bioactive compounds on flavor and aroma of tomato was not investigated and further work should be conducted to purpose the use efficiency of oil components in real applications such as fumigant. in conclusion, this paper is a part of an overall study that aims to determine the antifungal activities of natural floral resources of algeria, in order to find new bioactive natural products. the essential oils of these plants studied, exhibited an interesting antifungal activity against mycelial growth. further work is necessary to explore the efficacy of these essential oils against disease rot of tomato and to exploit these oils as a new potential source of natural additives for the food and/or pharmaceutical industries. acknowledgements the authors are indebted to the ministère des affaires etrangères et européennes throughout the research program “partenariat hubert curien tassili”. references agrios, g., 1997: plant pathology. 4th ed., academic press, san diego. amarti, f., satrani, b., aafi, a., ghanmi, 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a_dibdz@yahoo.fr art15_ercisli.indd molecular characterization of autochthonous grapevine cultivars by ssr journal of applied botany and food quality 85, 224 228 (2012) 1department of horticulture, agriculture faculty, ataturk university, erzurum, turkey 2ankara university, biotechnology institute, ankara, turkey genetic characterization and relatedness among autochthonous grapevine cultivars from northeast turkey by simple sequence repeats (ssr) y. hizarci1, s. ercisli1*, c. yuksel2, a. ergul2 (received october 10, 2012) * corresponding author summary 25 autochthonous grapevine cultivars from northeast anatolia in turkey together with two well-known standard cultivars, cabernet sauvignon and merlot were fi ngerprinted using six pairs of ssr primers to assess their genetic diversity and relatedness. all six ssr primers produced successful amplifi cations and revealed dna polymorphisms that were subsequently used to assess genetic relatedness of the cultivars. a total of 52 alleles were detected with a mean value of 8.67 alleles per locus indicating allele richness. the average expected heterozygosity (he) and observed heterozygosity (ho) were 0.759 and 0.809, respectively. considering the number of alleles generated, the highest number was observed in vvs2 loci (14 alleles/locus), while the lowest in vrzag83 loci (5 alleles/ locus). the unweighted pair-group method with arithmetic mean (upgma) dendrogram constructed based on the ssr data yielded two main clusters. first cluster included only cv. kibris and the second cluster included rest of the cultivars including cabernet sauvignon and merlot. the results showed that ssr markers have proved to be an effi cient tool for fi ngerprinting grapevine cultivars and conducting genetic diversity studies in grapevine. introduction turkey, located at the junction of two main plant gene centers, has a very old and rich grapevine germplasm including over 1500 cultivars. archaeological excavations also confi rm that grapevine cultivation is a very old tradition in anatolia dating back to 4000 bc (selli et al., 2007). each grape-growing region in turkey has particular local grapevine cultivars which differ from each other in color, taste, shape, bunch density etc. there is also a wide variation in terms of synonymous cultivars in each region and correct identifi cation of these cultivars is of great importance in cultivar standardization and determination of total cultivar number (ergul et al., 2006). northeast anatolia region in turkey has older and special grapevine cultivation. there were a lot of churches in this region and grape berylliums of different shapes can be seen on the walls of churches. mcgovern (2008) stated that the region, together with other regions of the south caucasus and anatolia, is the cradle of domestication and primary viticulture in the old world. in this region, yusufeli district is the main traditional viticulture area including over 30 very old and local grapevine cultivars. this area is also very rich in terms of wild grapes (vitis vinifera ssp. sylvestris). in christian period, these cultivars were used for wine-making. however, at present, wine production is not practiced because of the conservative life style of the people in this region. hence, the cultivars previously used for wine-making are currently used in juice production. northeast anatolia region differs greatly from the other regions in turkey in terms of climatic characteristics and thus it is expected that the grape germplasm of this region would have economically important adaptive traits that can potentially be incorporated into grape breeding programs in future. during the last decade, the genomic resources that are available to the grapevine research community have increased enormously in parallel to a renewed interest in grapevine (vitis vinifera l.) germplasm resources and analysis of genetic diversity in grapes. it is well recognized that genetic variation is invaluable for crop improvement and understanding gene function, and this fact applies to grapevine as well (this et al., 2006). knowledge of the genetic diversity and relationships among the grapevine cultivars is important for recognizing gene pools, identifying pitfalls in germplasm collections, and developing effective conservation and management strategies (ergul et al., 2006). the grapevine is highly heterozygous and propagation by cuttings or layers maintains their heterozygosity. therefore, morphological classifi cations provide rough guidelines, while molecular evaluations provide further insight into the genetic structure and differentiation within and among taxa which is useful for geneticists, plant breeders, and gene bank managers (papanna et al., 2009) lots of studies have investigated the genetic variation within grapevine by using different molecular marker systems such as random amplifi cation of polymorphic dna (rapd) (benjak et al., 2005), amplifi ed fragment length polymorphism (aflp) (doulati baneh et al., 2007), inter simple sequence repeats (issr) (argade et al., 2009), single nucleotide polymorphism (snp) (pindo et al., 2008) and ssr (de mattia et al., 2009; leao et al., 2009). among these marker systems, ssrs have been widely used by researchers to determine the genetic diversity within grapevine cultivars. six so-called “core set” ssr markers (i.e., vvs2, vvmd5, vvmd7, vvmd27, vrzag62, and vrzag79) are recommended for the direct comparison of results from different laboratories (this et al., 2004). in northeast part of turkey, there are several specifi c grape germplasms and coruh valley is one of the most important grape germplasm centers of this region. there are a number of autochthonous grapevine cultivars which were used many centuries ago. therefore, it can be valuable to characterize this germplasm both morphologically and genetically. in this study, an attempt to assess the level of genetic diversity and genetic relationship among 25 autochthonous grapevine cultivars from northeast anatolia in turkey has been made. it is expected that the information presented here would be useful for selection and more effi cient utilization of this germplasm in grape breeding programs in future. material and methods plant material leaf samples of 25 autochthonous grapevine cultivars used in this study were collected from yusufeli district in northeast anatolia region in turkey. a total of 25 grapevine cultivars to gether with two reference cultivars, cabernet sauvignon and merlot were included in ssr analysis. some important ampelographic traits of these cultivars provided by the international union for the protection of new varieties of plants (upov) are presented in tab. 1. molecular characterization of autochthonous grapevine cultivars by ssr 225 dna extraction genomic dna was extracted from young leaf tissue using the wizard® genomic dna purifi cation kit (promega, madison, wi) according to the instructions provided by the manufac turer. subsequently, a rnase treatment was performed on the eluted dna samples. purity and concentration of the dna were checked both on 1% (w/v) agarose gels and by nanodrop® nd-1000 spectrophotometer. ssr analysis six ssr markers (i.e., vvs2, vvmd5, vvmd7, vvmd27, vrzag62, and vrzag79) were used in polymerase chain reaction (pcr) studies. pcr was conducted in a volume of 10 µl and contained 15 ng genomic dna, 5 pmol of each primer, 0.5 mm dntp, 0.5 unit gotaq dna polymerase (promega), 1.5 mm mgcl2 and 2 µl 5x buffer. the forward primers were labeled with wellred fl uorescent dyes d2 (black), d3 (green) and d4 (blue) (pro ligo, paris, france). reactions without dna were included as negative controls. pcr amplifi cation was performed by using the biometra® pcr system. the amplifi cation condi tions consisted of an initial denaturation step of 3 mins at 94°c, followed by 35 cycles of 1 min at 94°c, 1 min at 52-56°c and 2 mins at 72°c with a fi nal extension at 72°c for 10 mins. the pcr products were fi rst separated on a 3% (w/v) agarose gel run at 80 v for 2 hrs. the gel was then stained with ethidium bromide at a concentration of 10 mg/ml. a dna ladder (100 bp) (promega) was used for the approximate quantifi cation of the bands. the amplifi cation products were visualized under uv light, and their sizes were estimated relative to the dna ladder. for further determination of polymorphisms, the pcr prod ucts were run on ceqtm 8800 xl capillary genetic analysis system (beckman coulter, fullerton, ca). the analyses were repeated at least twice to ensure reproducibility of the results. allele sizes were determined for each ssr locus using the beckman ceqtm frag ment analysis software. in each run, cabernet sauvignon and merlot cultivars were included as reference cultivars. genetic analysis the genetic analysis program “identity” 1.0 (wagner and sefc, 1999) was used according to paetkau et al. (1995) for the calculation of number of alleles, allele frequency, expected and observed heterozygosity, estimated frequency of null alleles, and probability of identity per locus. genetic dissimilarity was determined by the program “microsat ” (version 1.5) (minch et al., 1995) using proportion of shared alleles, which was calculated by using “ps (option 1 (ps))”, as described by bowcock et al. (1994). the results were then converted to a similarity matrix, and a dendrogram was constructed with the upgma method (sneath and sokal, 1973) using the software ntsys-pc (numerical taxonomy and multiware analy sis system,version 2.0) (rohlf, 1988). results and discussion the study presents ssr analysis results of a total of 27 grapevine cultivars comprising 25 autochthonous grapevine cultivars sampled from northeast anatolia and two reference cultivars cabernet sauvignon and merlot, to determine genetic diversity and relatedness among them. as shown in tab. 2, a total of 52 alleles ranging from 5 to 14 alleles per locus with a mean value of 8.67 alleles per locus were detected. polymorphic bands were obtained with all primers. vvs2 loci were the most polymorphic among the six loci, with the highest effective number of alleles (14 alleles), followed by the loci of vrzag79 (11 alleles) and vvmd27 (9 alleles). the vrzag83 loci generated the lowest number of alleles (5 alleles) (tab. 2). the mean he and ho were determined as 0.759 and 0.809 over six loci, respectively. among the six loci, the ho values were the highest (0.889) in vvs2 loci indicating high genetic diversity while the lowest (0.593) in vrzag83 loci (tab. 2). among the six loci, only vvmd7 generated more than two alleles (tri-allelic loci) in two varieties (karul and nanebur) (tab. 3). in present study, the frequency of null alleles (r) in vvs2 and tab. 1: basic descriptive characteristics of 25 grapevine cultivars used in this study. cultivar utility berry berry color shape alvan juice dark red-violet round artvin juice green-yellow ovate beyaz istanbul table green-yellow elliptic beyaz turfanda table green-yellow ovate ciklap juice green-yellow round erik table yellow ovate gelin parmagi juice green-yellow narrow elliptic gines table dark red-violet round goh juice blue-black round hatkul juice blue-black round kara turfanda juice blue-black round karul table green-yellow narrow elliptic keci memesi juice dark red-violet narrow elliptic kibris table dark red-violet ovate kirmizi istanbul table rose ovate kiskinbur juice rose round kokulu juice blue-black round kutuk juice green-yellow ovate mandagozu juice blue-black elliptic mezarlik juice yellow round nanebur juice dark red-violet elliptic razaki table dark red-violet ovate sulu kurta juice dark red-violet round tokat table green-yellow round yag juice dark red-violet round tab. 2: number of alleles, allele range (bp), expected heterozygosity, observed heterozygosity, and probability of identity values of grapevine cultivars. loci n he ho pi r vvmd7 6 0.756 0.852 0.186 -0.0547 vvmd27 9 0.748 0.852 0.134 -0.0592 vrzag79 11 0.831 0.852 0.088 -0.0112 vvmd24 7 0.763 0.815 0.150 -0.0296 vvs2 14 0.903 0.889 0.032 0.0072 vrzag83 5 0.612 0.593 0.330 0.0119 total 52 4.613 4.853 average 8.67 0.759 0.809 n: number of alleles; ho: observed heterozygosity; he: expected heterozygosity; pi: probability; r: null allele frequencies 226 y. hizarci, s. ercisli, c. yuksel, a. ergul molecular characterization of autochthonous grapevine cultivars by ssr vrzag83 loci was positive, but these low values suggest the absence of null alleles (tab. 2). the most informative loci, with regards to the probability of identity (pi), were vvs2 and vrzag79 which generated 14 alleles (pi: 0.032) and 11 alleles (pi: 0.088), respectively, whereas the least informative locus was vrzag83 which generated 5 alleles (pi: 0.330) (tab. 2). allele sizes (bp) of 27 cultivars from six ssr loci are shown in tab. 3. the most frequent alleles were 246 (30.38%) in vvmd7, 185 (44.44%) in vvmd27, 246 (27.78%) in vrzag79, 207 (38.89%) in vvmd24, 143 (18.52%) in vvs2 and (53.70%) in vrzag83, respectively. the number of microsatellite different genotypes ranged from 8 (vrzag83) to 21 (vvs2) with an average of 13.1 and a total of 79. the genetic similarity ranged from 0.17 to 0.92 among cultivars and the cultivars kutuk and yag; kokulu and yag as well as kibris and cabernet sauvignon were found to be more distant at 0.17 similarity ratio (fig. 1). according to genetic similarity ratio, cultivars kirmizi istanbul and razaki were the closest (0.92) (fig. 1). the average similarity ratio was 0.53 indicating high genetic diversity among cultivars. to elucidate the genetic relationship among grapevine cultivars, a dendrogram generated from the upgma cluster analysis over six ssr loci classifi ed 27 cultivars into two main groups depicted in fig. 1. a single cultivar (cv. kibris) was clustered separately from the remaining cultivars. the second main cluster included 24 autochthonous cultivars and the two reference cultivars. this cluster was further divided into two subgroups. the fi rst subgroup comprised the two reference cultivars (cabernet sauvignon and merlot) and two autochthonous cultivars (cvs. mandagozu and beyaz istanbul). in this subgroup reference cultivars clustered separately from the other two cultivars. the second subgroup comprised the remaining 22 autochthonous cultivars. in this study we report for the fi rst time the use of ssr markers for assessing genetic relatedness among 25 autochthonous grapevine cultivars from northeastern turkey. the results obtained in the present study show that microsatellites can be effectively used for fi ngerprinting purposes in grapevine. in fact, all tested microsatellite primer pairs produced various levels of amplifi cations. the mean value of 8.67 alleles per locus obtained with the microsatellites that produced polymorphic amplifi cation patterns is consistent with the reported data of 4-16 alleles per locus in v. vinifera germplasms assessed with microsatellites in different countries (bowers et al., 1996; fatahi et al., 2003; martin et al., 2003; martinez et al., 2006; tangolar et al., 2009). the high levels of within-group variation and the simple genetic structure observed in the dendrogram probably suggest a complex history of development of grapevine cultivars in northeast anatolia. introduction and spread of wild and semi-domesticated grapes, especially from its native near eastern range, domestication of indigenous wild grape, natural hybridization between indigenous and introduced vines, and human selection may have contributed to this high variation. the cv. kibris was clustered separately on dendrogram. if we look at tab. 3, we can see that this cultivar has unique alleles in at least fi ve loci. this cultivar was brought to coruh tab. 3: allele sizes, in base pairs, of six microsatellites loci from 27 grapevine cultivars. cultivar vvmd7 vvmd27 vrzag79 vvmd24 vvs2 vrzag83 artvin 238:246 181:185 248:256 207:211 139:143 185:191 alvan 238:246 185:189 244:246 207:207 145:149 191:191 beyaz istanbul 236:246 185:185 244:252 207:211 135:151 185:197 beyaz turfanda 238:246 185:191 244:246 207:215 135:141 185:191 ciklap 238:246 179:185 240:246 207:215 139:151 191:191 erik 236:244 179:181 248:256 207:207 139:149 185:185 gelin parmagi 238:246 181:185 246:250 217:217 125:149 191:191 gines 236:242 183:185 246:246 207:215 133:145 191:191 goh 238:246 181:185 248:256 207:215 143:143 191:191 hatkul 238:246 179:185 244:244 205:217 123:143 191:191 kara turfanda 236:244 179:185 246:250 205:217 135:149 185:191 karul 230:238:246 179:185 238:246 201:207 135:143 187:191 keci memesi 240:246 185:189 242:246 205:217 141:145 185:191 kibris 238:238 195:195 246:248 205:211 159:159 185:191 kirmizi istanbul 236:244 181:185 248:256 207:211 137:143 185:191 kiskinbur 238:246 185:185 248:250 205:215 125:143 185:191 kokulu 238:246 179:183 234:244 205:207 123:151 185:187 kutuk 238:246 175:179 244:248 205:207 133:137 185:185 mandagozu 236:246 185:185 238:248 207:211 131:133 191:197 mezarlik 236:244 183:185 244:248 207:215 133:133 181:185 nanebur 238:242:246 179:185 240:248 207:209 139:143 185:191 razaki 236:244 181:185 248:256 207:211 139:143 185:191 sulu kurta 246:246 181:185 246:250 217:217 123:149 191:191 tokat 244:244 177:185 244:246 205:207 137:143 185:191 yag 236:244 181:185 246:250 217:217 125:149 191:191 cabernet sauvig. 236:236 175:189 246:246 207:217 139:151 197:197 merlot 236:244 189:191 258:258 207:211 139:151 191:197 molecular characterization of autochthonous grapevine cultivars by ssr 227 valley from cyprus island and due to the protective position of the island, the genetic structure of the cultivar may have been protected. based on the number of alleles generated and probability of identity values, the most informative loci were vvs2 (14 alleles per locus, pi:0.032) and vrzag79 (11 alleles per locus, pi: 0.088), whereas the least informative locus was vrzag83 generated (5 alleles per locus, pi: 0.330). previously, among the six ssr primers, the highest number of alleles was obtained from vvs2 primer (fatahi̇ et al., 2003; nuñez et al., 2004; selli̇ et al., 2007; tangolar et al., 2009) while the lowest number of alleles was detected in vrzag83 primer (sefc et al., 2000; snoussi et al., 2004; tangolar et al., 2009). microsatellites have been becoming the marker of choice for fi ngerprinting and genetic diversity studies in a wide range of living organisms. the approach described in this paper shows that microsatellite analysis is a powerful tool for characterization of grapevine cultivars as well. taken together with their co-dominant nature and reproducibility, ssr markers are very useful for the analysis of genetic diversity, genomic mapping and marker-assisted selection in many plant species compared to many other marker systems. in this study, the mean ho level was slightly higher than that of he (80.9% for ho and 75.9% for he). comparable observed heterozygosity levels were reported between 74.3-85.5% in different grape-cultivated areas in the world (bowers et al., 1996; sefc et al., 2000; fatahi et al., 2003; martinez et al., 2006; tangolar et al., 2009). such high levels of heterozygosity are commonly observed among clonally propagated, outbreeding, perennial species since they are favoured during selection and are known to confer greater adaptability, vigour and productivity on clonal varieties (aradhya et al., 1998; sefc et al., 2000). grapes, being an outbreeding species, have highly heterozygous cultivars, carry a heavy genetic load and suffer from severe inbreeding depression (olmo, 1976). our fi ndings also indicated that there was a high genetic distance value among cultivars ranging from 0.17 to 0.92. considering the environmental conditions of the region, it is expected that the grapevine germplasm in the region would have economically important adaptive traits that can potentially be incorporated into grapevine breeding programs. hence, it is expected that the results of this study will assist current grapevine breeding efforts in turkey as well as maintain the genetic integrity of the genetic resources. in summary, the gene pool of cultivated grapes surveyed in northeast anatolia has signifi cant amounts of genetic variation. in regard to germplasm management, our results show that the germplasm collection is highly variable and most variation is common to all genetic groups identifi ed. references aradhya, k.m., yee, l.k., zee, f.t., manshardt, r.m., 1998: genetic variability in macadamia. genet. res. crop evol. 45, 19-32. argade, n.c., karibasappa, g.s., patil, s.g., rao, v.s., 2009: dna profi ling and assessment of genetic relationships among important seedless grape (vitis vinifera) varieties in india using issr markers. j. plant biochem. biotech. 18, 45-51. benjak, a., ercisli, s., vokurka, a., maletic, e., pejic, i., 2005: genetic relationships among grapevine cultivars native to croatia, greece and turkey. vitis 44, 73-77. bowers, j.e., dangl, g.s., vignani, r., meredith, c.p., 1996: iso lation and characterization of new polymorphic simple sequence repeat loci in grape (vitis vinifera l.). genome 39, 628-633. bowcock, a.m., ruiz-linares, a., tomfohrde, j., minch, e., kidd, j.r., cavalli-sforza, l.l., 1994: high resolution of human evolutionary trees with polymorphic microsatellites. nature 368, 455-457. de mattia, f., lovicu, g., tardaguila, j., grassi, f., imazio, s., scienza, a., labra, m., 2009: genetic relationships between sardinian and spanish viticulture: the case of ‘cannonau’ and ‘garnacha’. j. hortic. sci. biotechn. 84, 65-71. doulati baneh, h., grassi, f., mohammadi, s.a., nazemieh, a., de mattia, f., imazio, s., labra, m., 2007: the use of aflp and morphological markers to study iranian grapevine germplasm to avoid genetic erosion. j. hortic. sci. biotechn. 82, 745-752. ergül, a., kazan, k., aras, s., cevik, v., celik, h., soylemezoglu, g., 2006: aflp analysis of genetic variation within the two economically important anatolian grapevine (vitis vinifera l.) varietal groups. genome 49, 467-495. fatahi, r., ebadi, a., bassil, n., mehlenbacher, s.a., zamani, z., fig. 1: dendrogram showing the relationships of 27 grapevine cultivars based on upgma cluster analysis of six ssr loci. 228 y. hizarci, s. ercisli, c. yuksel, a. ergul 2003: characterisation of iranian grapevine cultivars using microsatellite markers. vitis 42, 185-192. leao, p.c.s., riaz, s., graziani, r., dangl, g.s., motoike, s.y., 2009: characterization of a brazilian grape germplasm collection using microsatellite markers. am. j. enol. viticulture. 60, 517-524. martin, j.p., borrego, j., cabello, f., ortiz, j.m., 2003: characterization of spanish grapevine cultivar diversity using sequence-tagged microsatellite site markers. genome 46, 10-18. martinez, l.e., cavagnaro, p.f., masuelli, r.w., zuniga, m., 2006: ssr-based assessment of genetic diversity in south american vitis vinifera varieties. plant sci. 170, 1036-1044. mcgovern, p., 2008: the origins and ancient history of wine. http: / / w w w. m u s e u m . u p e n n . e d u / n ew / ex h i b i t s / o n l i n e _ ex h i b i t s / w i n e / winemap.html. minch, e., ruiz-linares, a., goldstein, d.b., feldman, m., cavallisforza, l.l., 1995: microsat (version 1.4d): a computer program for calculating various statistics on microsatellite allele data. stanford university medical center, stanford. nuñez, y., fresno, j., torres, v., ponz, f., gallego, f.j., 2004: practical use of microsatellite markers to manage vitis vinifera germplasm: molecular identifi cation of grapevine samples collected blindly in d.o. “el bierzo” (spain). j. hortic. sci. & biotech. 79, 437-440. olmo, h.p., 1976: grapes. in: simmonds, n.w. 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this, p., jung, a., boccacci, p., borrego, j., botta, r., costantini, l., crespan, m., dangl, g.s., eisenheld, c., ferreira-monterio, f., grando, s., ibanez, j., lacombe, t., laucou, v., magalhaes, r., meredith, c.p., milani, n., peterlunger, e., regner, f., zulini, l., maul, e., 2004: development of a standard set of microsatellite reference alleles for identifi cation of grape cultivars. theor. appl. genet. 109, 1448-1458. wagner, h.w., sefc, k.m., 1999: identity 1.0. centre for applied genetics. university of agricultural science, vienna. adrress of the corresponding author: sercisli@gmail.com journal of applied botany and food quality 87, 291 295 (2014), doi:10.5073/jabfq.2014.087.041 department of botany, faculty of veterinary science, szent istván university, budapest hungary complex chemical evaluation of the summer truffle (tuber aestivum vittadini) fruit bodies d. kruzselyi, j. vetter (received march 12, 2013) summary summer truffle (tuber aestivum vittadini) is one of the most important mycorrhizal mushrooms with underground fruit bodies. formerly the scientific investigations were focused mainly on its specific fragrant constituents. our work was concentrated on complex chemical characterization including different organic and inorganic components. summer truffle has middle crude protein, low fat-, and relatively high fiber and chitin contents; its energy level is low. distribution of protein fractions is characteristic (in % of crude protein): 40.98; 5.91; 3.85; 19.28 and 29.98 % for albumins, globulins, prolamins, glutelins and npn (non protein nitrogen), respectively. we determined soluble oligoand polysaccharides (9.00 mg/g dm and 49.9 mg/g dm, respectively), as well as the contents of phenoloids and flavonoids (2.8 mg/g dm and 0.093 mg/g dm, respectively). mineral composition is similar to other mushrooms; four macroelements (k, p, ca and mg) give 97.94 % of the all mineral content; occurrence of poisonous elements (as as, cd, v) is not characteristic. chemical nature of tuber aestivum (summer truffle) fruit bodies is very characteristic, regarding not only the occurrence of fragrant components but the classical, “usual” components, too. this rare and highly appreciated hypogaeous mycorrhizal fungus belongs to mushrooms of valuable, specific chemical composition. introduction truffles mushrooms are specific mycorrhizal fungi with hypogaeous (underground) fruit bodies (ascocarps, basidiomes). their occurrence and life was (and partly is today also) slightly unknown, “mysterious”, caused by their life-type. use of these extreme mushrooms has long history. first of all the characteristic fragrance substances and properties were and are appreciated on the market. mass of gathered truffles (wild growing and cultivated) has been showing decreasing tendency caused by different ecological, environmental etc. reasons. summer truffle (tuber aestivum vittadini) is the most frequent truffle species (or one of these) of middle europe (of carpathian basin). its habitat is wide-spreading, we have data of occurrence from different parts of europe (from great britain, russia, sweden up to spain) and from north-africa (morocco). there are data on occurrence up to 1300-1600 m in the mountains. t. aestivum occurs on different habitats of hungary mainly in the central chain of mountains but in middle and south hungary on the lowlands, too. soil requirements of summer truffle are relatively wide: best are the loamy soils with the following chemical parameters; ph: 6.1-7.4; content of organic substances 3.1-9.1 %; available p2o5 content is 200 ppm, available k2o 500 ppm (bratek, 2010). the host (mycorrhizal) partners are mostly the quercus (oak) species (q. pubescens, q. robur, q. petraea, q. cerris), beech (fagus sylvatica), birch (betula pendula), lime species (t. cordata, t. plathyphyllos), poplar (populus) species, willow (salix) species, different pines (pinus nigra, p. sylvestris etc.), hornbeam (carpinus betulus), chestnut (castanea sativa), hazelnut (corylus avellana), or other trees and shrubs. the underground fruit bodies of this truffle have 2-10 cm diameter, the color is brown to black, the outer surface (peridium) is ornamented with some pyramidal warts and transverse fine marks. inner part (gleba) has a dark brown color and a white vein structure. the yellow brown, reticulate ascospores have a network of ridges (wang and marcone, 2011). nutritional value i.e. the chemical composition of these mushrooms is partly unknown because of the non conventional occurrence and of the specific use. scientific works and investigations were and are concentrated on the volatile (fragrant) components (march et al., 2006; kiss et al., 2011; diaz et al., 2002; diaz et al., 2003; cullere et al., 2012). diaz and co-workers (diaz et al., 2003) analyzed the “aroma” of t. aestivum and t. melanosporum by a new and susceptible chemical method (head space solid-phase microextraction). eighty nine constituents were extracted and isolated, some only from t. aestivum or t. melanosporum, other molecules were found (in different quantities) in both species. most characteristic specific fragrant molecules of summer truffle are: dms (dimethylsuphide); dmds (dimethyldisulphide); methional; 3-methyl-1-butanol; 1-hexen-3-one; 3-ethylphenol (cullere at al., 2012). fragrance of t. aestivum is a chemically very complicated mix of different molecules. such works are very useful for the better level of quality control, for different food industrial products having truffles. nutritional profile (components) were analyzed mainly for desert truffle species (terfezia claveryi, tirmania species and for others: hussan and al-ruqaie, 1999) and rare for t. aestivum (saltarelli et al., 2008). number of these exact data on nutritional value is under the required limit. aims of our studies • to give exact data on “classical” chemical organic components (crude protein, -fat, -fiber, chitin); • to produce and present information on protein character based on fractionation by osborne’s method (albumines, globulines, prolamins, glutelins and npn fraction); • to extract and measure some components (soluble oligo and polysaccharides, phenoloids, flavonoids) with documented or probable biological effects; • to control the mineral composition by measurements of twenty two elements. these data can give a possibility for the complex and better evaluation of this rare and expensive truffle. materials and methods samples tuber aestivum (summer truffle) samples were gathered from six different habitats of hungary (sample no. 1: mountain mecsek / south hungary; no. 2: tápiógyörgye / hungarian lowland; no. 3: mt. bükk / bükkszentkereszt, north hungary; no. 4: szőlösardó / north hungary; no. 5: aggtelek / north hungary; no. 6: mt. bükk / szilvásvárad) in years 2007-2008. the mushrooms were transported for bot. dep. of fac. vet. sci., szent istván university (budapest, hungary), and were thoroughly cleaned, sliced, carefully dried (at 292 d. kruzselyi, j. vetter 40 c°) and pulverized. the voucher samples were deposited in the laboratory of bot. dep. all analyses were performed from these mushroom powders in triplicate. chemical analyses crude protein, -fiber, -fat and -ash contents were determined according to aoac official methods (palazzolo et al., 2011), carbohydrate content and the energy level were calculated from the aforementioned data (mattila et al., 2002). determination of chitin content was conducted according to our earlier method (vetter, 2007), glucose-amine molecules of hydrolyzed samples (20-20 mg) were measured spectrophotometrically in a reaction with 3-methyl2-benzothiazolone-hydrazone-hydrochloride (mbth). fractionation of proteins was carried out according to classical osborne method, modified and described by petrovska (petrovska, 2001). soluble oligoand polysaccharide fractions were produced by methanol: water (80:20) extraction (for oligosaccharides) and by the following boiling water extraction (for polysaccharides) and were evaluated with anthrone reaction (saltarelli et al., 2008). molecules with phenolic character were extracted with ethanol (80 %) and determined with folin reagent (dubost et al., 2007). extraction for flavonoids was made in methanol; the concentrations were measured with alcl3 reagent (sarikurcku et al., 2008). statistical evaluation of the analytical data was performed by ‘origin 4.0’ software. results and discussion data on “classical” organic components of summer truffle are summarized in tab. 1. the average crude protein content (19.11 %) seems to be a middle level among the mushrooms, it is lower than protein of agaricus or boletus species (kalac, 2009) but significantly higher than those in some wood decaying species (laetiporus sulphureus, ganoderma lucidum etc.). crude fat level (in average 2.27 % of dm) is low and something lower than fat contents in agaricus bisporus, pleurotus ostreatus or lentinula edodes (mattila et al., 2002). crude fiber content of summer truffle (22.03 % in average) seems to be higher than normally in common mushrooms (for example: in pleurotus species 11 %: del toro et al., 2006). its high fiber level can be one of the characteristic chemical properties. calculated total carbohydrate content (48.9 % dm) and total organic constituent level (92.44 % dm) indicate the dominance of organic components, the average crude ash content (7.55 %) represents the sum of different inorganic constituents. calculated energy level of tuber aestivum (1224 kj/100 g dm) is low, similar to other foods of mushroom type (mattila et al., 2002). concentrations and percentage distributions of different protein fractions (extractions of which were conducted according to their solubility’s) are presented in tab. 2. first and main component is the albumin fraction (57.8 mg/g dm that corresponds to 41 % of total crude protein content) and logically seems to be the best protein category for consumers. incidence of globulins is low (8.56 mg/g dm only) but the variability among the different samples is relatively important (lowest and highest values are 5.35 mg/g dm and 12.44 mg/dm, respectively). albumins and globulins (i.e. the heavily utilizable protein types) give together about the half (~ 47 %) of crude protein content. prolamines (and prolamine like substances) have only 3.22 and 2.47 mg/g dm contents, which fulfill for 2.27 % and 1.58 % of the crude protein content, respectively. occurrence of glutelins is remarkably high (25.7 mg/g dm), content of glutelin like substances is low (2.09 mg/g dm). united concentration of these two fractions is 19.28 % of crude protein level. last but not least: summarized contents of the six protein components present 70.02 % of crude protein. the difference between crude protein level and the sum of the protein fractions (practically the true proteins) is the npn (non protein nitrogen) rate. here this fraction is remarkable high, namely 29.98 %. evaluation and comparison of our data can be conducted based on work of petrovska (petrovska, 2001) for fifty two basidiomycetous edible mushrooms. summer truffle has significantly higher albumin, but lower globulin rates, occurrence of glutelins and of npn fractions are similar than the average data originating from petrovska’s work. concentrations of some biologically active compounds are given in tab. 3. soluble saccharides (including oligoand polysaccharides) occur in summer truffle in numerically low concentrations (in average 9.0 mg/g dm for oligo and 49.92 mg/g dm for polysaccharides). the total soluble saccharide fraction (in average 58.92 mg/g dm) is composed mainly of polysaccharides (83.18 % : 16.82 %). our data can confirm results of saltarelli (saltarelli et al., 2008) i.e. soluble saccharide-content are the highest among different tuber species (t. magnatum, t. borchii or t. melanosporum). total phenolic and flavonoid contents of t. aestivum samples are 2.8 mg/g dm and 0.093 mg/g dm, respectively. quantity of phenolics of t. aestivum seems to be markedly lower than in agaricus bisporus (dubost et al., 2007; elmastas et al., 2007) and is slightly lower than those in pleurotus ostreatus or lentinula edodes (dubost et al., 2007). total flavonoid concentration (0.093 mg/g dm) is absolutely lower than those in a desert truffle species (in tirmenia: al-laith, 2010) but similar to these values of lentinula edodes or pleurotus species (yang et al., 2002). tab. 1: different macrocomponents (crude protein, -fat, -fiber, ash, chitin, carbohydrate contents in % of dm, and energy level in kj/100 g) of tuber aestivum (mean ±sd) samples crude protein crude fat crude fibre crude ash chitin carbohydrate* organic energy* (% dm) (% dm) (% dm) (% dm) (% dm) (% dm) constituents* (kj/100 g) (% dm) tuber aestivum 1. 20.27±0.08 2.64±0.16 20.75±0.17 8.80±0.03 7.51±0.60 47.54 91.20 1231 tuber aestivum 2. 17.86±0.07 2.91±0.07 22.27±0.8 8.03±0.02 6.66±0.17 48.93 91.97 1227 tuber aestivum 3. 19.69±0.03 2.35±0.01 21.62±1.17 7.22±0.01 12.19±0.35 49.12 92.78 1240 tuber aestivum 4. 19.59±0.23 2.01±0.02 24.33±0.70 7.07±0.08 14.07±0.16 47.00 92.93 1189 tuber aestivum 5. 20.10±0.14 2.83±0.01 23.23±1.17 7.08±0.02 15.17±0.23 46.46 92.92 1219 tuber aestivum 6. 17.20±0.15 0.93±0.08 20.03±1.34 7.11±0.01 8.23±0.28 54.73 92.89 1240 tuber aestivum, 19.11±1.27 2.27±0.73 22.03±1.58 7.55±0.71 10.63±3.6 48.9±3.0 92.44±0.71 1224±19 average * calculated values chemical evaluation of summer truffle 293 twenty two elements were determined from the inorganic con stituents (tab. 4). the presented mineral composition is similar to the majority of mushrooms (kalac, 2009; kalac and svoboda, 2000; vetter, 2005; vetter et al., 2005). first (and main) mineral element is potassium (25647 mg/kg dm), second is the phosphorus (7879 mg/kg dm), third one is the calcium (3331 mg/kg dm). content of magnesium is about 1000 mg/kg dm; all other elements have lower concentration. some “microelements” as iron (230 mg/ kg dm), zinc (160.5 mg/kg dm), copper (49.3 mg/kg dm) and manganese (17.4 mg/kg dm) have lower contents, but these elements can be important for the metabolism of the consumer. level of sodium is low (148.2 mg/kg dm), but this tendency is similar to properties of other mushrooms (vetter, 2003). concentrations of some other elements (b, ba, sr and ti) are under 20 mg/kg dm level. considering the poisonous (or known as poisonous) elements: incidence of these (as, se or v) are under the detectable limits or they have detectable, but low quantities (for cd 4.11, for cr: 1.29 mg/kg dm values) i.e. the normal use of truffle has no toxicological risk for us. interesting question is the aluminum level of t. aestivum, the found concentration for total content (166.7mg/kg dm) is relatively high, but we have no information on the form(s) of aluminum in the fruit body, therefore the toxicological importance of this content is doubtful. mineral spectrum of summer truffle’s fruit bodies is characteristic: four elements (k, p, ca and mg) give 97.85%, while the all other 18 elements have 2.06% of total composition, only (fig. 1). tab. 2: different protein fractions of tuber aestivum (mg/g dm) (mean ±sd and in percent of crude protein content (%)) protein fractions (mg/g dm) sample albumins globulins prolamines prolamine-like glutelines gluteline-like substances substances tuber aestivum 1. 61.05±0.59 12.44±2.15 4.65±0.80 4.03±0.69 14.22±2.46 3.57±0.52 (%) 36.9 7.5 2.79 2.40 8.58 1.81 tuber aestivum 2. 47.7±9.16 9.32±1.79 4.06±0.78 2.76±0.53 33.70±6.47 2.34±0.45 (%) 32.98 6.44 2.80 1.91 23.3 1.62 tuber aestivum 3. 61.41±15.20 8.60±2.12 3.15±0.87 2.22±0.55 21.45±5.31 3.14±0.77 (%) 46.8 6.52 2.67 1.69 16.3 2.37 tuber aestivum 4. 62.54±17.39 5.35±1.48 2.73±0.76 2.08±0.22 25.80±7.17 1.47±0.41 (%) 47.78 4.06 2.08 0.60 19.75 1.12 tuber aestivum 5. 62.82±18.10 6.93±1.99 2.60±0.75 2.01±0.56 24.14±6.95 1.56±0.45 (%) 48.06 5.28 1.99 1.48 18.45 1.19 tuber aestivum 6. 51.48±12.49 8.77±2.12 2.14±0.50 1.72±0.42 35.0±8.49 0.49±0.11 (%) 33.36 5.66 1.33 1.12 22.68 0.29 tuber aestivum, 57.8±6.53 8.56±2.39 3.22±0.95 2.47±0.84 25.7±7.78 2.09±1.28 average tab. 3: soluble oligoand polysaccharides, total phenoloid and flavonoid contents of tuber aestivum (mg/g dm, mean ±sd) sample soluble oligosaccharides soluble polysaccharides phenolic content flavonoid content (mg/g dm) (mg/g dm) (mg/g dm) (mg/g dm) tuber aestivum 1. 8.98±0.89 29.88±2.10 2.20±0.12 0.092±0.006 tuber aestivum 2. 9.08±0.86 43.21±1.02 2.37±0.08 0.094±0.006 tuber aestivum 3. 8.16±0.33 32.41±3.77 2.64±0.16 0.097±0.004 tuber aestivum 4. 8.97±0.16 37.32±0.92 2.78±0.09 0.098±0.003 tuber aestivum 5. 9.30±0.41 43.73±0.77 3.04±0.11 0.109±0.012 tuber aestivum 6. 9.52±0.48 107.1±4.12 3.77±0.16 0.071±0.003 tuber aestivum, average 9.00±0.46 49.92±29.0 2.8±0.56 0.093±0.012 summer truffle (t. aestivum) is not only a historical, rare and expensive mycorrhizal mushroom species, but is has characteristic and specific chemical composition. we analyzed different constituents of organic and inorganic characters (excluding the fragrant molecules). six mushroom samples were analyzed for the chemical components independently. average values of certain chemical parameters were used for general characterization. summer truffle can be characterized by middle crude protein, low crude fat and high crude fiber contents. last trait seems to be one of the chemical specificities of this mushroom, in general the other (ascoand basidiomycetous) higher mushroom species have lower fiber contents (kalac, 2009). chitin level of t. aestivum is high, higher than the average of other common cultivated mushrooms (vetter, 2007). crude fat level (about 2 % of dm) is absolutely similar to values of other mushroom species. occurrence and distribution of protein fractions (based on “classical” fractionation according to osborne’s concepts) is characteristic: albumins and globulins represent together about the half of crude proteins, glutelins and glutelin-like substances give together about 20 % of crude protein content and the non protein (npn) fraction is important (about 30 %). protein distribution presented here seems to be characteristic for t. aestivum, and it is different from the common basidiomycetous mushrooms, as well as from some proteins of plant origin (petrovska, 2001). different substances of biological activity are presented in mushrooms consequently oligoand polysaccharides of different 294 d. kruzselyi, j. vetter solubility. such water soluble molecules have a probable essential role in health promoting (anti carcinogen, immune stimulating, anti viral etc.) effects of mushrooms. according to the found distribution, the main soluble saccharide component is the polysaccharide fraction (83 %), which fact supports earlier results of saltarelli (saltarelli et al., 2008). phenolic derivatives represent important group of biologically active molecules, although total their concentration in truffle is lower than those in most common mushroom species (agaricus, pleurotus etc.). mineral spectrum of truffle fruit bodies is similar to composition of other edible mushrooms, i.e. they have high potassium and fig. 1: percentage distribution of the mineral elements in summer truffle (t. aestivum) fruit bodies. tab. 4: inorganic constituents of summer truffle fruit bodies (mg/kg dm), mean ±sd tuber aestivum tuber aestivum tuber aestivum tuber aestivum tuber aestivum tuber aestivum tuber aestivum 1 2 3 4 5 6 average al 311.3±13.6 140.4±13 133.9±6.6 139.7±17.7 164.8±0.35 111±8.96 166.7±72 as <d.l.* <d.l. <d.l. <d.l. <d.l. <d.l. <d.l. b 7.19±0.57 17.22±1.32 2.43±0.03 2.68±0.54 23.76±0.49 61.91±4.0 19.2±22 ba 8.89±0.58 5.99±0.62 6.38±0.11 11.32±0.84 14.79±0.11 3.56±0.2 8.48±0.07 ca 3436±170 3535±348 3256±87 3890±336 4547±322 1324±70.4 3331±1080 cd 2.80±0.13 1.72±0.06 10.48±0.20 2.94±0.17 4.58±0.12 2.18±0.13 4.11±3.26 co <d.l. <d.l. <d.l. <d.l. <d.l. <d.l. <d.l. cr 1.87±0.30 1.51±0.13 1.16±0.35 0.97±0.22 0.95±0.06 1.31±0.07 1.29±0.35 cu 43.8±3.37 46.6±3.56 50.4±0.6 38.6±1.1 74.69±0.83 41.7±2.79 49.3±13.1 fe 344.7±6.8 168.1±13.9 158.3±4.3 153.6±13.5 419.2±1.6 138.4±13.4 230.3±120.1 k 28956±801 28113±1837 24944±108 23620±995 23321±141 24932±1656 25647±2347 mg 1352±48.8 1181±97.5 992±9.8 964±51.9 887.8±11.4 985.3±80.1 1060±173 mn 17.35±1.04 11.47±0.988 17.64±0.13 12.81±1.0 33.47±0.79 11.92±0.84 17.4±8.30 mo <d.l. <d.l. <d.l. <d.l. <d.l. <d.l. <d.l. na 193.3±11.9 127.3±17 145.7±3.0 115.4±3.5 201.9±8.5 105.7±5.11 148.2±40.6 ni 2.16±0.56 1.20±0.09 1.54±0.89 1.91±0.61 2.12±0.47 1.15±0.09 1.68±0.44 p 8192±265 7203±554 7963±100 7366±245 8714±31.7 7837±516 7879±552 se <d.l. <d.l. <d.l. <d.l. <d.l. <d.l. <d.l. sr 11.79±0.91 12.99±0.40 7.56±0.23 9.69±0.80 13.26±0.70 8.23±0.76 10.58±2.44 ti 12.2±0.19 3.30±0.28 3.05±0.04 2,49±0.30 4.30±0.08 2.52±0.21 4.64±3.76 v <d.l. <d.l. <d.l. <d.l. <d.l. <d.l. <d.l. zn 156.3±6.85 131.4±2.12 173.6±2.8 155.8±6.5 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food quality 81, 69 76 (2007) institute of food science and biotechnology, section plant foodstuff technology, hohenheim university, stuttgart, germany evaluation of the antioxidant capacity of betalainic fruits and vegetables florian kugler, florian conrad stintzing*, reinhold carle (received march 19, 2007) summary the present investigation determined total phenolics, ascorbic acid, betalain contents and the corresponding antioxidant capacities of betalain-bearing fruits and vegetables. in addition to differently coloured swiss chard petioles (beta vulgaris l. ssp. cicla [l.] alef. cv. ‘bright lights’) and hypocotyls of white, yellow, and red beetroot varieties (beta vulgaris l. ssp. vulgaris, cv. ‘albina vereduna’, cv. ‘burpee’s golden’, and cv. ‘rote kugel 2’), juices from cactus pears (opuntia ficus-indica [l.] mill. cv. ‘gialla’ and cv. ‘rossa’) and pitaya fruits (hylocereus polyrhizus [weber] britton & rose, h. undatus [haworth] britton & rose, selenicereus megalanthus [k. schumann ex vaupel] moran) were included in this study. antioxidant capacities were determined by application of the teac and frap assays, respectively, resulting in differing rankings of the commodities investigated. in both test systems, highest antioxidant capacity was shown for red beetroot extract while for the remaining samples no straightforward order could be established. introduction chlorophylls, carotenoids, anthocyanins, and betalains represent the four most important pigment classes imparting all kinds of colour shades to fruit and vegetables. according to current conception, the same pigments are considered to promote human health and wellbeing. however, scarce information is available on betalainic food crops, although knowledge on their bioactivity would allow to judge their nutritional quality more objectively. betalainic fruits and vegetables are consumed fresh or after cooking and may thus contribute to healthy nutrition. in addition, highly processed products to which betalain-containing colour preparations are added may be improved not only visually but also from a nutritional point of view. in this respect, both betaxanthins (yellow-orange betalains, fig. 1) and betacyanins (red-violet betalains, fig. 2) have recently attracted attention for their antioxidative activities which were proven to surpass even the values of classical dietary antioxidants such as ascorbic acid, rutin, and catechin (cai et al., 2003). the antioxidant properties of betacyanins are ascribed to their free phenolic group(s). additionally, the cyclic amine group of the betalamic acid moiety resembling the structure of the very strong antioxidant ethoxyquin is considered to act as hydrogen donor both in betacyanins and betaxanthins (kanner et al., 2001). other studies attributed the antioxidant potential of betalain-containing foodstuffs mainly to their polyphenolic compounds without considering the role of betalains (galati et al., 2003; 2005; kuti, 2004; pyo et al., 2004). hence, a range of differently coloured swiss chard petioles, beetroots and cactus fruits were selected to assess their potential antioxidant capacity applying the most widespread teac (trolox equivalent antioxidant capacity) and frap (ferric ion reducing antioxidant power) assays. the unpigmented representatives of the respective commodities were also included to single out the effect of the betalains. major antioxidant compounds, namely total phenolics, ascorbic acid, and betalains were determined to scrutinise their particular contribution to the total antioxidant capacity of the food crops investigated. materials and methods plant material differently coloured swiss chard (beta vulgaris l. ssp. cicla [l.] alef. cv. ‘bright lights’; chenopodiaceae; lange et al., 1999) exhibiting white, yellow, orange and red-purple petioles as well as different fig. 1: structures of dopamine-bx (= miraxanthin v; major betaxanthin in yellow swiss chard stems), glutamine-bx (= vulgaxanthin i; major betaxanthin in yellow and red beetroots), and proline-bx (= indicaxanthin; major betaxanthin in betalainic cactus pears). n h h hooc cooh n h oh oh n h h n h hooc cooh h coo nh2 o n h h hooc cooh n h coo + 11 -+ 11 glutamine-bxdopamine-bx + 11 proline-bx beetroot cultivars (beta vulgaris l. ssp. vulgaris cv. ‘albina vereduna’, cv. ‘burpee’s golden’, and cv. ‘rote kugel 2’; chenopodiaceae; lange et al., 1999) developing white, yellow, and red coloured hypocotyls, respectively, were harvested at the end of september 2005 on the experimental station for horticulture at hohenheim university. after washing the swiss chard plants, leaves were removed from the petioles and the latter immediately frozen in liquid nitrogen, sealed in plastic bags under reduced pressure, and stored at -80°c until further processing. only fully developed hypocotyls showing regular pigment distribution were selected and investigated in this study. after washing, beetroots were deprived of their hair roots, cut in quarters by longitudinal section and frozen in liquid nitrogen and subsequently treated as described for swiss chard petioles. cactus pear and pitaya fruits were purchased at the beginning of november 2005 on a local market, stored at 4°c, and processed within one week to prevent microbial spoilage. cactus pears (opuntia ficusindica [l.] mill. cv. ‘gialla’ and cv. ‘rossa’; cactaceae) were from sicily (italy). pitaya fruits from hylocereus polyrhizus [weber] britton & rose (cactaceae) with purple pulp were obtained from israel, whereas fruits from h. undatus [haworth] britton & rose (cactaceae) with white pulp were from vietnam, and fruits from the selenicereus megalanthus [k. schumann ex vaupel] moran (cactaceae) with yellow peel and white flesh from ecuador, respectively. solvents and reagents reagents and solvents were purchased from vwr (darmstadt, germany) and were of analytical or hplc grade. purified water was used throughout. cryogenic milling of swiss chard petioles and beetroot hypocotyls as described earlier (kugler et al., 2004), comminution of swiss chard petioles and beetroot hypocotyls was performed in a waring blender model 38bl41 (waring products, torrington, ct, usa). to prevent enzymatic reactions during grinding, liquid nitrogen was added. the resulting powder was directly extracted as described below. extraction of swiss chard and beetroot powder exactly 50.000 g of swiss chard and beetroot powder, respectively, were extracted by addition of 200 ml of 60% aqueous methanol (v/v) to inhibit residual enzyme activity (kugler et al., 2004). after stirring for 30 min at room temperature, the plant material was separated from the extract by filtering through a glass frit under reduced pressure and rinsing with 100 ml extraction solvent. the resulting extracts were concentrated in vacuo at 30°c and diluted to a volume of 50 ml with purified water after temperature adjustment for 15 min at 20°c in a water bath. finally, the aqueous extracts were membrane-filtered (0.45 µm; acrodisc premium filter, pall, ann arbor, mi, usa) before storage at -80°c until analysis. extraction of cactus pears and pitaya fruits cactus pears and pitaya fruits were cut in halves, and the pulp was separated from the peel before manually squeezing of the former. the obtained juice was clarified by paper filtration (macherey & nagel, düren, germany) and immediately stored at -80°c. before analysis, juice samples were thawed and membrane-filtered (0.45 µm). o oh oh oh c n h hooc cooh n h coo o oh h h ooh c c o o 15 + phyllocactin o oh oh oh c n h hooc cooh n h coo o oh h h oh betanin 15 + o ohoh oh c n h hooc cooh n h coo o oh h h o o chooc ch 3 oh hylocerenin 15 + fig. 2 fig. 2: structures of betanin (major betacyanin in red beetroot, red-purple swiss chard stems, and betalainic cactus pears), phyllocactin and hylocerenin (the latter both representing major betacyanins in fruits from h. polyrhizus). 70 florian kugler, florian conrad stintzing, reinhold carle isolation and purification of betanin from red beet betanin was isolated and purified according to a previously applied procedure (stintzing et al., 2004). photometric determination of total phenolics total phenolics were assessed by the folin-ciocalteu assay (singleton and rossi, 1965): a volume of 100 µl of sample extract or juice, respectively, was diluted to 1000 µl with purified water in a test tube prior to addition of 100 µl folin-ciocalteu reagent. for red beetroot extract, dilution to a volume of 2000 µl was required. after vortexing, the reaction was allowed to proceed for 3 min before adding 800 µl of sodium carbonate (7.5%). after repeated vortexing, the sample was allowed to stand for 60 min. absorption at 720 nm was measured in a uv-vis spectrometer (perkin-elmer, überlingen, germany) equipped with uvwinlab v 2.85.04 software (perkin-elmer instruments, norwalk, ct, usa). all determinations were performed in duplicate. based on a four point calibration, total phenolic contents were expressed as gallic acid (gae) or ascorbic acid equivalents (aae), respectively. determination of ascorbic acid ascorbic acid contents of appropriately diluted sample extracts and juices, respectively, were determined in duplicate using an enzyme test kit (r-biopharm, darmstadt, germany) based on the selective oxidation of ascorbic acid to dehydroascorbic acid and registering formazan formation at 578 nm. photometric quantification of betalains the aqueous pigment extracts were diluted with mcilvaine buffer (ph 6.0, citrate-phosphate) to obtain absorption values of 0.9 < a < 1.1 at their respective absorption maxima. measurements were performed in triplicate and the betalain content (bc) was calculated according to stintzing et al. (2003): bc [mg/l] = [(a*df*mw*1000)/(ε * l)] where a is the absorption value at the absorption maximum corrected by the absorption at 650 nm, df the dilution factor and l the pathlength (1 cm) of the cuvette. for quantification of betacyanins, the molecular weight (mw) and molar extinction coefficient (ε ) of betanin [mw = 550 g/mol; ε = 60,000 l/(mol*cm) in h 2 o; kugler et al., 2004] at their respective absorption maximum were applied. quantitative equivalents of the major betaxanthins (bx) were determined at their respective absorption maximum applying a mean molar extinction coefficient [ε = 48,000 l/(mol*cm) in h 2 o; kugler et al., 2004] and the molecular weight of glutamine-bx (vulgaxanthin i; mw = 339 g/mol) for the extracts from differently coloured swiss chard and beetroots (kugler et al., 2004; stintzing et al., 2002) and of prolinebx (indicaxanthin; mw = 308 g/mol) for coloured juices from cactus pear fruits (stintzing et al., 2002). determinations were performed with a uv-vis spectrometer (perkin-elmer, überlingen, germany) equipped with uvwinlab v 2.85.04 software (perkin-elmer instruments, norwalk, ct, usa). antioxidant capacity measurements teac assay. based on an improved teac assay (van den berg et al., 2000), radical anions of abts [2,2´-azinobis-(3-ethylbenzthiazoline)-6-sulfonate] were obtained by oxidation of the latter with peroxyl free radicals generated by thermal decomposition of abap [2,2´-azobis-(2-amidinopropane)-dihydrochloride]. the diammonium salt of abts was dissolved in a ph 7.4 phosphate-buffer medium (na 2 hpo 4 /kh 2 po 4 containing nacl) at a concentration of 20 mmol/l. a volume of 0.5 ml of this radical precursor solution was added to 100 ml of a solution of abap in phosphate-buffer (2.5 mmol/l) and incubated for 15 min at 60°c in the dark. to an aliquot of 2000 µl of the abts radical solution, 40 µl of appropriately diluted sample extract or juice, respectively, were added and mixed thoroughly. after a reaction time of exactly 6 min, absorbance was registered photometrically at 734 nm. all determinations were performed in duplicate, and for each duplicate determination a freshly pipetted blank (2000 µl abts radical solution + 40 µl of purified water) was measured. standard solutions of the vitamin e analogue trolox (6-hydroxy2,5,7,8-tetramethylchroman-2-carboxylic acid), ascorbic acid, and dopamine hydrochloride were used for calibration. antioxidant capacities were expressed as equivalents of trolox (te), ascorbic acid (aae), and dopamine (dae), respectively. frap assay. the ferric ion reducing antioxidant power assay, which is based on the reduction of the fe3+ complex of 2,4,6-tripyridyl-striazine [fe(tptz)3+] to the intensely blue coloured corresponding fe2+ complex [fe(tptz)2+], was performed as described earlier (benzie and strain, 1996; gardner et al., 2000). the frap reagent was obtained by mixing 25 ml of sodium acetate-buffer medium (ph 3.6) with 2.5 ml from a solution of tptz dissolved in diluted hydrochloric acid, and a volume of 2.5 ml from an aqueous solution of fecl 3 . to an aliquot of appropriately diluted sample extract or juice, respectively, 1500 µl of the frap reagent were added. following thorough mixing, absorbance was registered at 593 nm after a reaction time of exactly 4 min at room temperature. all measurements were performed in duplicate. standard solutions of the vitamin e analogue trolox (6-hydroxy2,5,7,8-tetramethylchroman-2-carboxylic acid), of ascorbic acid, and of dopamine hydrochloride were used for calibration. antioxidant capacities were expressed as trolox (te), ascorbic acid (aae), and dopamine equivalents (dae), respectively. in addition, the antioxidant activity of purified betanin was assessed in each assay based on a five-point-calibration (r2 > 0.997) to single out the particular contribution of betacyanins. results and discussion chenopodiaceae white, yellow, orange, and red-purple coloured swiss chard stems from the cultivar ‘bright lights’ earlier characterised (kugler et al., 2004; 2006), as well as red, yellow and white beetroot phenotypes were included in this study to investigate the impact of betalains on their overall antioxidant capacity. moreover, total phenolics, ascorbic acid, and betalain contents were assessed for correlation with teac and frap data. according to the suggestion by kim et al. (2002), concentration of total phenolics and antioxidant capacities were also calculated as ascorbic acid equivalents (aae) to facilitate comparison with well-established antioxidants (see tab. 1). the highest total phenolics value (140.7 mg gae/100 g fresh weight) was determined for the red beetroot extract, whereas red-purple coloured swiss chard petioles exhibited the lowest total phenolics concentration (31.2 mg gae/100 g fresh weight) of all investigated chenopodiaceae representatives. in beetroot tubers, winter and herrmann (1986) detected conjugates of caffeic, p-coumaric, as well as ferulic acids depending on the cultivar. in another study on phenolic compounds of red beetroot, the presence of 5,5’,6,6’-tetrahydroxy3,3’-biindolyl, ferulic acid conjugates, phenolic amides, and several flavonoids was also reported (kujala et al., 2002). glucosylated ferulic acid was found to be the clearly dominating phenolic compound in swiss chard stalks (winter and herrmann, 1986) and further antioxidant capacity of betalainic fruits and vegetables 71 derivatives of vitexin, kaempferol and isorhamnetin were reported to be present in swiss chard leaves (gil et al., 1998). it is worth being mentioned that the folin-ciocalteu reagent is not specific to phenolics, but will also react with a broad range of reducing compounds such as nitrogenous substances (ikawa et al., 2003) including dopa (3,4dihydroxyphenylalanine), and ascorbic acid (singleton et al., 1999). moreover, betanin the major betacyanin in red beetroot exhibits a nonglycosylated hydroxyl group at c6 (fig. 2) and is structurally similar to dopa. hence, it is conceivable that betanin will be assessed by the folin-ciocalteu method as supported by higher values with increasing pigment contents (tab. 1). furthermore, remarkable concentrations of free dopamine (kugler et al., 2006) and its corresponding betaxanthin (= miraxanthin v, fig. 1, kugler et al., 2004), both exhibiting aromatic ortho-dihydroxyl substitution previously found in coloured swiss chard stems, may likewise considered active. although multiple food components may contribute to an overestimation of the true total phenolics content, the folin-ciocalteu assay is reliable, widely applied and has recently been suggested appropriate for the assessment of the total reducing strength in vitro (huang et al., 2005). notwithstanding, the compositional pattern in food samples should be generally considered and the antioxidant data carefully interpreted accordingly. in the chenopodiaceae plant materials investigated in this study, no ascorbic acid could be detected, although up to 11 mg ascorbic acid/ 100 g red beetroot fresh weight and up to about 10 mg /100 g swiss chard stalk fresh weight were reported elsewhere (herrmann, 1995; pokluda and kuben, 2002). gil et al. (1998) pointed out that whole swiss chard leaves exhibited around 45 mg vitamin c/100 g fresh weight, however, exclusively as dehydroascorbic acid. it is assumed that the same applied to the samples of the present study due to inevitable ascorbic acid oxidation during extraction. therefore, ascorbic acid does not represent the major antioxidant in swiss chard or red beetroot, but rather the colourless phenolics (kujala et al., 2000; 2002; winter and herrmann, 1986) and the betalains. other electron-donors such as the catecholamine dopamine previously identified as a strong antioxidant (kanazawa and sakakibara, 2000) may also affect total antioxidant capacity. since dopamine was found only recently in swiss chard stems and beetroots ranging from 8.8 mg/ 100 g fresh weight in white swiss chard stems to 36.6 mg/100 g fresh weight in red beetroot (kugler et al., 2006), antioxidant capacities determined by applying the teac and frap assays were also expressed as dopamine equivalents (tab. 1). due to high dopamine contents (kugler et al., 2006) and considerable antioxidant responses, dopamine was suspected to markedly contribute to the total antioxidant capacities of the chenopodiaceae extracts investigated, although minor oxidative events converting dopamine into inactive dopaminequinone may still have occurred during extraction and sample work-up. based on a calibration with purified betanin, the relative contribution of betanin to the antioxidant activity in the frap assay was 3.9, 4.7, 72 florian kugler, florian conrad stintzing, reinhold carle tab. 1: total phenolics, ascorbic acid, and betalain contents as well as antioxidant capacities of extracts from differently coloured swiss chard stems and beetroot hypocotyls swiss chard stems beetroots white yellow orange red-purple white yellow red total phenolics (as gaea) 36.3 ± 0.1 50.0 ± 0.1 42.9 ± 0.3 31.2 ± 0.3 56.1 ± 0.1 41.5 ± 0.9 140.7 ± 0.1 [mg/100 g] total phenolics (as aaeb) 56.7 ± 0.2 78.3 ± 0.2 67.1 ± 0.4 48.6 ± 0.5 87.9 ± 0.2 65.0 ± 1.3 220.7 ± 0.1 [mg/100 g] ascorbic acid –c – – – – – – [mg/100 g] betaxanthins – 5.9 ± 0.0 4.1 ± 0.0 2.1 ± 0.0 – 16.5 ± 0.1 53.3 ± 0.3 [mg/100 g] betacyanins – – 2.4 ± 0.0 3.6 ± 0.0 – – 96.8 ± 0.5 [mg/100 g] total betalains – 5.9 6.5 5.7 – 16.5 150.1 [mg/100 g] teac (as ted) 287.5 ± 7.8 429.0 ± 1.7 339.5 ± 2.5 252.4 ± 3.5 428.2 ± 0.6 362.1± 6.4 1110.3 ± 22.3 [µmol/100 g] teac (as aae) 44.0 ± 1.1 64.8 ± 0.2 51.6 ± 0.4 38.8 ± 0.5 64.7 ± 0.1 55.0 ± 0.9 167.6 ± 3.3 [mg/100 g] teac (as daee) 16.1 ± 0.4 23.9 ± 0.1 19.0 ± 0.1 14.2 ± 0.2 23.9 ± 0.0 20.2 ± 0.4 61.8 ± 1.2 [mg/100 g] frap (as te) 164.5 ± 3.7 273.6 ± 2.8 211.3 ± 2.5 263.7 ± 3.2 201.0 ± 4.1 221.0 ± 4.8 985.1 ± 5.5 [µmol/100 g] frap (as aae) 29.8 ± 0.7 49.2 ± 0.5 38.1 ± 0.4 47.9 ± 0.6 36.3 ± 0.7 39.8 ± 0.9 177.8 ± 1.0 [mg/100 g] frap (as dae) 24.7 ± 0.5 40.8 ± 0.4 31.6 ± 0.4 39.7 ± 0.5 30.1 ± 0.6 33.0 ± 0.7 147.4 ± 0.8 [mg/100 g] a gae = gallic acid equivalents b aae = ascorbic acid equivalents c not detected d te = trolox equivalents e dae = dopamine equivalents and 33.7 % in orange swiss chard petioles, red-violet swiss chard petioles and red beetroot, respectively. the corresponding values in the teac assay amounted to 2.8, 5.7, and 34.1% and were thus quite similar to the frap values. the relatively small amount of betalains in swiss chard may explain the comparatively low antioxidant capacities assessed. antioxidant capacities as determined by the teac assay (as te) displayed excellent linear correlation with the respective total phenolics concentrations (as gae) [r2 = 0.9962]. accordingly, maximal teac values were determined for red beetroot (1110.3 µmol te/100 g fresh weight), yellow swiss chard stems (429.0 µmol te/100 g fresh weight) and white beetroot (428.2 µmol te/100 g fresh weight). in a previous study, pellegrini and co-workers (2003) reported a lower teac value of 521 µmol te/100 g fresh weight for beet reaching about half of the total antioxidant capacity for red beetroot found in the present investigation. additionally, pellegrini et al. (2003) determined a total antioxidant activity of 353 µmol te/100 g fresh weight for swiss chard stems, thus being in the same range from 252.4 to 429.0 µmol te/ 100 g fresh weight for red-purple and yellow coloured stems of the present study, respectively. interestingly, the same authors (pellegrini et al., 2003) found much lower teac values for carrots (44 µmol te/ 100 g fresh weight) and eggplants (110 µmol te/100 g fresh weight) containing carotenoids and anthocyanins as the predominant radical scavenging pigments, respectively. numerous publications stated excellent linear correlations between total phenolics contents and antioxidant capacities determined by electron transfer-based antioxidant capacity assays such as the teac and frap methods (huang et al., 2005). in the present work, an excellent linear correlation could be established for teac values (as te) and total phenolics (as gae) [r2 = 0.9962], with the latter exhibiting a weaker correlation with frap values (see tab. 1) [r2 = 0.9365]. although proteggente et al. (2002) observed good correlations between total phenolics and both teac and frap values, they also noted that especially teac values were highly correlated with total phenolic contents, thus supporting the higher correlation coefficient in this study. despite the mechanistic similarity of the teac and frap assays both being based on electron transfer reactions, the ph conditions in the two test systems are different, which might explain the differing rankings of the vegetables investigated (tab. 1): using the teac assay, the antioxidant activities of the investigated extracts were in the following order: red beetroot (rank 1) > yellow swiss chard stems (rank 2) > white beetroot (rank 3) > yellow beetroot (rank 4) > orange swiss chard stems (rank 5) > white swiss chard stems (rank 6) > red-purple swiss chard stems (rank 7). in contrast, when applying the frap assay, the antioxidant capacities ranked in the following order: red beetroot (rank 1) > yellow swiss chard stems (rank 2) > red-purple swiss chard stems (rank 3) > yellow beetroot (rank 4) > orange swiss chard stems (rank 5) > white beetroot (rank 6) > white swiss chard stems (rank 7). the fact that application of different assays may result in dissimilar orders of antioxidant capacities were already highlighted in a previous study (pellegrini et al., 2003) with varying rankings for a broad range of vegetable extracts applying the teac and frap assays, respectively. interestingly, in the present study, lowest frap values were found for betalain-free white swiss chard stems and white beetroot hypocotyls, whereas all betalainbearing samples were shown to possess higher frap antioxidant capacities with red beetroot displaying the maximum value of 985.1 µmol te/100 g fresh weight. noteworthy, previous investigations did not consider the betalains in betalainic plant material, but only the colourless phenolics which were exclusively held responsible for the in vitro bioactivities monitored (halvorsen et al., 2002; pellegrini et al., 2003; pyo et al., 2004). in contrast, the present findings clearly support the data by wettasinghe et al. (2002), who reported a positive correlation between antioxidant activities and pigment contents of beetroots. according to escribano et al. (1998), the betacyanins are more potent radical scavengers than the betaxanthins. this conception needs to be differentiated depending on the respective betaxanthin structures, because yellow swiss chard petioles with miraxanthin v (fig. 1) as major compound were found to be more potent than the red-purple ones with betanin (fig. 2) as the predominant pigment at the same concentration level (tab. 1). in fact, it has recently been demonstrated that betacyanins actually act as antioxidants in planta their synthesis being induced in beetroot leaves upon wounding and bacterial infiltration. it was suggested that following an oxidative burst the reactive oxygen species (ros) generated were signals for the biosynthesis of betacyanins, the latter acting as ros scavengers, thus limiting plant tissue damage (sepúlveda-jiménez et al., 2004). although it is highly conceivable that betacyanins may act as antioxidants in vivo, it has to be considered that their efficacy in the human organism will depend on their bioavailability, which has been recently shown to be quite low (netzel et al., 2005). on the other hand, poor absorption has also been reported for the anthocyanins, despite their undisputed physiological activity (clifford, 2000; stintzing and carle, 2004; ghosh, 2005). cactaceae in addition to coloured swiss chard petioles and different beetroot varieties, cactus pear and pitaya fruit juices were included in this study (see tab. 2). maximum total phenolics concentration was determined for yellow-orange ‘gialla’ juice (63.5 mg gae/100 ml juice) only slightly differing from those obtained from the red cactus pear cultivar ‘rossa’ (61.5 mg gae/100 ml juice) and from the purple pitaya fruit species h. polyrhizus (61.0 mg gae/100 ml juice). in striking contrast, the unpigmented juices from fruits of h. undatus and s. megalanthus contained only 18.2 and 22.7 mg gae/100 ml juice, respectively. according to kuti (2004), mainly quercetin and isorhamnetin conjugates and low amounts of kaempferol conjugates contribute to total phenolics of cactus pears while the pattern of phenolic compounds in pitayas has not been scrutinised yet. for fruits of tropical origin cultivated on mauritius island, luximonramma and co-workers (2003) reported total phenolics to range from 11.8 to 563.8 mg gae/100 g fresh weight for banana and red chinese guava, respectively. total phenolics for juices from cactus pears and pitaya fruits in the present study were in the same range (tab. 2). considering the high ascorbic acid concentrations of juices from the cactus pear cultivars ‘gialla’ and ‘rossa’ with 31.5 and 23.9 mg/ 100 ml juice, respectively, and comparing them with total phenolics contents calculated as aae, it was assumed that ascorbic acid markedly contributed to the total phenolics values measured. on the contrary, the juice from h. polyrhizus fruits was devoid of ascorbic acid, and only minute amounts were found in juices obtained from fruits of h. undatus and s. megalanthus, respectively (tab. 2). thus, the contribution of ascorbic acid to the total phenolics value in pitaya juices was negligible. nevertheless, the high betacyanin concentration of 46.0 mg/100 ml juice contributed distinctively as already suspected by wu and co-workers (2006). in the teac and frap assays, the particular contribution of ascorbic acid was 42.8 and 68.0% for ‘gialla’, 34.6 and 52.8% for ‘rossa’, 7.0 and 15.0% for h. undatus, and 2.6 and 6.0 % for s. megalanthus. based on a calibration with purified betanin, the particular activity of betacyanins as betanin-equivalents amounted to 0.5, 5.0, and 45.2% in ‘gialla’, ‘rossa’ and h. polyrhizus in the teac assay, respectively. the corresponding values in the frap test were slightly different with 0.8, 8.1, and 36.0%, respectively. again, the values obtained for juices from cactus fruits in the present study were consistent with the previously determined values for tropical fruits ranging from 0.8 to 142.6 mg ascorbic acid/100 g fresh weight for banana and white guava, respectively (luximon-ramma et al., 2003). antioxidant capacity of betalainic fruits and vegetables 73 as observed for the chenopodiaceae, again a good linear correlation of teac values (as te) with the corresponding total phenolics contents (as gae) [r2 = 0.9764] was registered for the cactus fruit juices (tab. 2). accordingly, the highest teac value (488.5 µmol te/ 100 ml juice) was determined for ‘gialla’ juice, followed by ‘rossa’ (457.6 µmol te/100 ml juice) and h. polyrhizus (411.6 µmol te/ 100 ml juice), respectively. less than half of the former antioxidant capacities was detected in the colourless juices from h. undatus and s. megalanthus fruits only amounting to 181.5 and 201.5 µmol te/ 100 ml juice, respectively. being in a similar range, stintzing and co-workers (2005) reported teac values for several cactus pear clones ranging from 310 to 499 µmol te/100 ml juice. by comparison with teac values obtained for extracts from mauritian fruits (luximonramma et al., 2003), antioxidant capacities of the investigated cactus fruit juices may be classified as follows: a portion of 100 ml of juice from fruits of the cactus pear cultivars ‘gialla’ and ‘rossa’ or the pitaya species h. polyrhizus ranks at a similar level as 100 g of edible parts from mango and litchi fruits (500 µmol te/100 g fresh weight), respectively. displaying lower total antioxidant capacities, juices from the pitaya fruits h. undatus and s. megalanthus rank at a level comparable to pineapple, avocado, and jamalac fruits (200 µmol te/100 g fresh weight), respectively. comparing the teac values for cactus fruit juices assessed in this study and the mean teac value for commercial blood orange juice (282 µmol te/100 ml) being rich in potent antioxidative anthocyanins (fiore et al., 2005), it appears that betalainic juices from fruits of the cactus pear cultivars ‘gialla’ and ‘rossa’ as well as from the pitaya species h. polyrhizus exert even stronger antioxidant activities whilst both the colourless juice from fruits of the pitaya species h. undatus and s. megalanthus display weaker antioxidant capacities than blood orange juice (tab. 2). only recently, red-fleshed ‘weirouge’ apples being rich in anthocyanins were assessed and exhibited total antioxidant capacities of 585 and 240 µmol te/100 g whole apple edible portion in the teac and frap assays, respectively (sadilova et al., 2006). comparing these results with the values obtained for cactus fruit juices in the present investigation, it becomes again evident that antioxidant capacity rankings strongly depend on the respective sensitivity to the particular compound class: whilst in the teac assay, 100 g of unpeeled ‘weirouge’ apple ranks best, juice from the pitaya fruit species h. polyrhizus possesses the highest total antioxidant capacity based on the values obtained by the frap assay. as has been observed for the extracts from swiss chard petioles and beetroot hypocotyls (tab. 1), frap values (as te) of cactus fruit juices only poorly correlated with the respective total phenolics concentrations (as gae) [r2 = 0.7245, linear fit]. the juice from fruits of h. polyrhizus containing phyllocactin and hylocerenin as major betacyanins (fig. 2) displaying an extraordinary betacyanin content (46.0 mg betacyanins/100 ml juice), exhibited the highest frap value (438.0 µmol te/100 ml juice), followed by ‘gialla’ (254.7 µmol te/100 ml juice) and ‘rossa’ juice (249.4 µmol te/100 ml juice), with the latter both only slightly differing in their betalain contents (tab. 2). accordingly, lowest frap values were determined for the betalain-free juices from fruits of s. megalanthus (73.9 µmol te/ 100 ml juice) and h. undatus (72.3 µmol te/100 ml juice). despite their low phenolic contents, previous studies attributed the antioxidant potential of cactus pears mainly to the presence of phenolic compounds (galati et al., 2003; 2005; kuti, 2004) and only recently, the reducing capacities of betanin and indicaxanthin from cactus pear fruits were unambiguously proven (butera et al., 2002). moreover, tesoriere and co-workers (2004) showed that the cactus pear betalains indicaxanthin and betanin may be involved in the protection of low density lipoproteins (ldl) against ex vivo-induced oxidative modifications. as mentioned before, efficacy of cactus fruit betalains in the human organism will depend on the extent of absorption. in addition to total phenolics, ascorbic acid, and betalains, further compounds present in cactus juices should be taken into account when evaluating overall bioactivity. in this regard, high proline levels were recently reported (kugler et al., 2006) in juices from the cactus pears ‘gialla’ (1476.9 mg proline/kg juice) and ‘rossa’ (1837.2 mg proline/ kg juice), as well as pitaya fruits from s. megalanthus (1803.7 mg proline/kg juice), h. polyrhizus (273.3 mg proline/kg juice), and h. undatus (254.5 mg proline/kg juice). until now, proline was mainly assumed to be accumulated in plants as osmolyte to withstand drought and high salinity (stintzing and carle, 2004). although proline itself did not exert antioxidant effects neither in the teac nor in the frap assays (data not shown), it was only recently assumed to act as ros scavenger in the fungal pathogen colletotrichum trifolii (chen and dickman, 2005). possibly, proline acts in a synergistic way with other antioxidant compounds of cactus fruit juices. further studies are retab. 2: total phenolics, ascorbic acid, and betalain contents as well as antioxidant capacities of cactus fruit juices cactus pear cultivars pitaya species ‘gialla’ ‘rossa’ h. polyrhizus h. undatus s. megalanthus total phenolics (as gaea) [mg/100 ml] 63.5 ± 1.1 61.5 ± 0.9 61.0 ± 0.8 18.2 ± 0.3 22.7 ± 0.2 total phenolics (as aaeb) [mg/100 ml] 99.5 ± 1.7 96.4 ± 1.4 95.7 ± 1.2 28.2 ± 0.5 35.3 ± 0.3 ascorbic acid [mg/100 ml] 31.5 ± 0.0 23.9 ± 0.4 –c 2.0 ± 0.1 0.8 ± 0.0 betaxanthins [mg/100 ml] 4.8 ± 0.0 3.3 ± 0.0 – – – betacyanins [mg/100 ml] 0.6 ± 0.0 5.9 ± 0.0 46.0 ± 0.3 – – total betalains [mg/100 ml] 5.4 9.2 46.0 – – teac (as ted) [µmol/100 ml] 488.5 ± 10.7 457.6 ± 4.6 411.6 ± 22.2 181.5 ± 1.6 201.5 ± 3.8 teac (as aae) [mg/100 ml] 73.6 ± 1.6 69.0 ± 0.7 63.7 ± 3.3 28.4 ± 0.2 30.5 ± 0.6 frap (as te) [µmol/100 ml] 254.7 ± 0.7 249.4 ± 4.0 438.0 ± 1.9 72.3 ± 1.9 73.9 ± 1.4 frap (as aae) [mg/100 ml] 46.3 ± 0.1 45.3 ± 0.7 79.0 ± 0.3 13.3 ± 0.3 13.4 ± 0.2 a gae = gallic acid equivalents b aae = ascorbic acid equivalents c not detected d te = trolox equivalents 74 florian kugler, florian conrad stintzing, reinhold carle quired to elucidate the role of proline as potential antioxidant. besides proline, biothiols, vitamin e, as well as carotenoids may additionally contribute to the total antioxidant capacities of cactus juices (tesoriere et al., 2005). furthermore, taurine (2-aminoethane-sulfonic acid) earlier found in cactus pear juices in a range from 32-57 mg/100 ml (stintzing et al., 1999) or 8-12 mg/kg (tesoriere et al., 2005) should be considered because militante and lombardini (2004) assumed that taurine may exert antioxidant effects. conclusions the present investigation demonstrated that despite the similar underlying mechanisms, total antioxidant capacities of betalainic samples depend on the respective assay applied. the extract from red beetroot containing large amounts of the betacyanin betanin exhibited the highest antioxidant capacity among the chenopodiaceae representatives in both test systems, whilst betacyanin-rich juice from fruits of the pitaya species h. polyrhizus showed highest antioxidant capacity within the cactaceae samples only in the frap assay. additionally, it has to be taken into account that the data obtained from antioxidant capacity assays are not only influenced by the particular methods, but also the compositional pattern of the respective crop being affected by the geographical origin as well as harvest time of the plant materials investigated (ou et al., 2002). altogether, the present study confirmed that foodstuffs containing significant amounts of betalains do not only represent colouring potential but are also excellent sources of dietary antioxidative phytochemicals which may exert beneficial effects on consumer’s health upon consumption that remain to be carefully scrutinised. acknowledgements the authors would like to thank mrs e. müssig for her excellent technical assistance and mrs m. schuster, mr. h. arnold and mr. h. fuchs (experimental station for horticulture, hohenheim university) for the cultivation of swiss chard and beetroots. we thank bruno nebelung gmbh & co. (everswinkel, germany) for delivering swiss chard and yellow beetroot seeds, carl sperling gmbh & co. kg (lüneburg, germany) for providing red beetroot seeds, and thompson & morgan ltd. 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planta med. 65, 632-635. stintzing, f.c., schieber, a., carle, r., 2002: identification of betalains from yellow beet (beta vulgaris l.) and cactus pear [opuntia ficus-indica (l.) mill.] by high-performance liquid chromatography-electrospray ionization mass spectrometry. j. agric. food chem. 50, 2302-2307. stintzing, f.c., schieber, a., carle, r., 2003: evaluation of colour properties and chemical quality parameters of cactus juices. eur. food res. technol. 216, 303-311. tesoriere, l., allegra, m., butera, d., livrea, m.a., 2004: absorption, excretion, and distribution of dietary antioxidant betalains in ldls: potential health effects of betalains in humans. am. j. clin. nutr. 80, 941945. tesoriere, l., fazzari, m., allegra, m., livrea, m.a., 2005: biothiols, taurine, and lipid-soluble antioxidants in the edible pulp of sicilian cactus pear (opuntia ficus-indica) fruits and changes of bioactive juice components upon industrial processing. j. agric. food chem. 53, 7851-7855. van den berg, r., haenen, g.r.m.m., van den berg, h., van der vijgh, w., bast, a., 2000: the predictive value of the antioxidant capacity of structurally related flavonoids using the trolox equivalent antioxidant capacity (teac) assay. food chem. 70, 391-395. wettasinghe, m., bolling, b., plhak, l., xiao, h., parkin, k., 2002: phase ii enzyme-inducing and antioxidant activities of beetroot (beta vulgaris l.) extracts from phenotypes of different pigmentation. j. agric. food chem. 50, 6704-6709. winter, m., herrmann, k., 1986: esters and glucosides of hydroxycinnamic acids in vegetables. j. agric. food chem. 34, 616-620. wu, l., hsu, h.-w., chen, y.-c., chiu, c.-c., lin, y.-i., ho, j.a., 2006: antioxidant and antiproliferative activities of red pitaya food chem. 95, 319-327. address of the authors: hohenheim university, institute of food science and biotechnology, section plant foodstuff technology, august-von-hartmann-str. 3, d-70599 stuttgart * corresponding author: dr. florian c. stintzing, wala heilmittel gmbh, dorfstrasse 3, d-73087 bad boll / eckwälden, e-mail: florian.stintzing@wala.de 76 florian kugler, florian conrad stintzing, reinhold carle << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false /maxsubsetpct 100 /optimize true /opm 1 /parsedsccomments true /parsedsccommentsfordocinfo true 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<feff0041006e007600e4006e00640020006400650020006800e4007200200069006e0073007400e4006c006c006e0069006e006700610072006e00610020006e00e40072002000640075002000760069006c006c00200073006b0061007000610020005000440046002d0064006f006b0075006d0065006e00740020006d006500640020006800f6006700720065002000620069006c0064007500700070006c00f60073006e0069006e00670020006f006300680020006400e40072006d006500640020006600e50020006200e400740074007200650020007500740073006b00720069006600740073006b00760061006c0069007400650074002e0020005000440046002d0064006f006b0075006d0065006e00740065006e0020006b0061006e002000f600700070006e006100730020006d006500640020004100630072006f0062006100740020006f00630068002000520065006100640065007200200035002e003000200065006c006c00650072002000730065006e006100720065002e> >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice journal of applied botany and food quality 88, 102 108 (2015), doi:10.5073/jabfq.2015.088.014 1 institute of genetics and physiology, hebei academy of agriculture and forestry sciences, shijiazhuang, china 2 plant genetic engineering center of hebei province, shijiazhuang, china effects of 1-methylcyclopropene on superficial scald and related metabolism in ‘wujiuxiang’ pear during cold storage manman gao1, 2 a, shuo zhou1, 2 a, junfeng guan1, 2 *, yingying zhang1, 2 (received july 31, 2014) * corresponding author a these authors contributed equally to the article. summary ‘wujiuxiang’ (pyrus bretschneideri r. × pyrus communis l.) pear often suffers from superficial scald after a long-term of cold storage. in this study, harvested ‘wujiuxiang’ pear fruits were fumigated with 1-methylcyclopropene (1-mcp) at concentrations of 0.5 μl/l and 1.0 μl/l and subsequently stored at low temperature (0 °c). the superficial scald index; flesh firmness; total soluble solids (tss) content; respiration and ethylene production rates; relative membrane permeability; contents of α-farnesene, conjugated trienols (ctols) and hydrogen peroxide (h2o2); and activities of superoxide dismutase (sod), catalase (cat), ascorbate peroxidase (apx), and lipoxygenase (lox) of the peel were investigated. the results showed that compared with control, 1-mcp reduced the index of superficial scald; maintained a higher firmness and a lower tss content; inhibited the accumulation of h2o2, α-farnesene and conjugated trienols and the increase in cell membrane permeability; and maintained a higher activity of apx, sod and cat and a lower activity of lox. these findings indicate that 1-mcp regulates the activities of h2o2-scavenging enzymes to inhibit the accumulation of h2o2 and thereby reduces cell membrane damage and inhibits the accumulation of conjugated trienols. thus, 1-mcp could decrease the incidence of superficial scald in ‘wujiuxiang’ pear. abbreviations 1-mcp, 1-methylcyclopropene; tss, total soluble solids; ctols, conjugated trienols; h2o2, hydrogen peroxide; sod, superoxide dismutase; cat, catalase; apx, ascorbate peroxidase; lox, lipoxygenase introduction ‘wujiuxiang’ (pyrus bretschneideri r. × pyrus communis l.) pear is favored for their excellent quality of flavor and exhibit a high market value. however, this fruit is highly susceptible to superficial scald, which is a physiological disorder that manifests as many irregular brown or black patches on the fruit skin after more than 90 days of cold storage and during subsequent shelf life (dong et al., 2012), resulting in reduced fruit quality and economic loss. it has been suggested that scald development in apple and pear is related with the accumulation of α-farnesene and its oxidation products, namely, conjugated trienols (ctols), which accumulate progressively in the fruit peel during storage (du and bramlage, 1993; giné bordonaba et al., 2013; whitaker, 2007). some studies have also shown that antioxidative enzyme activity and h2o2 accumulation are correlated with superficial scald development (abbasi et al., 2008; ahn et al., 2007; larrigaudiere et al., 2004; sabban-amin et al., 2011). h2o2 accumulation may cause lipid peroxidation, which may result in cell senescence and superficial scald (lu et al., 2011; rao et al., 1998; sabban-amin et al., 2011; tian et al., 2013). many postharvest methods have been established to control scald development; however, the mechanism of superficial scald development has not yet been completely understood (lurie and watkins, 2012). 1-methylcyclopropene (1-mcp), an ethylene action inhibitor, can effectively extend the shelf life and maintain a higher quality of many types of fruits, and it has been shown that 1-mcp can lower ethylene production, delay softening, prevent chlorophyll degradation, retard the decline in protein, reduce the respiration rate and volatiles and alter the titratable acidity and sugar contents (blankenship and dole, 2003; watkins, 2006). previous work has shown that 1-mcp has significant effects on inhibiting superficial scald development in ‘granny smith’ apple (zanella, 2003), ‘cortland’ and ‘delicious’ apples (lu et al., 2013), ‘rocha’ pear (isidoro and almeida, 2006), ‘anjou’ pear (argenta et al., 2003; bai et al., 2009), ‘dangshansuli’ pear (hui et al., 2011), ‘akemizu’ pear (li and wang, 2009), and ‘asian’ pear (yazdani et al., 2011). therefore, the application of 1-mcp for the control of scald development has become a promising technology and research direction. it has been shown that 1-mcp can inhibit scald development by delaying the production of α-farnesene and conjugated trienols in pear and apple fruits (ekman et al., 2004; isidoro and almeida, 2006; lu et al., 2013; yazdani et al., 2011). 1-mcp can also retain higher activities of antioxidative enzymes and inhibit h2o2 accumulation and lipid peroxidation in fruit, which may reduce scald incidence during storage (chiriboga et al., 2013a; dong et al., 2011; larrigaudiere et al., 2004; li and wang, 2009). however, research on the regulation mechanism of 1-mcp in scald development at cold storage is still relatively lacking. therefore, the objectives of this study were to investigate (1) the effect of 1-mcp on superficial scald development and (2) the possible mechanism of 1-mcp in inhibiting scald development in ‘wujiuxiang’ pear during cold storage. materials and methods materials and treatments ‘wujiuxiang’ pear fruits were harvested in jinzhou county (hebei, china) at the fruit commercial mature stage (august 29, 2010). the harvested fruits were transported to the laboratory within 2 h. approximately 750 kg fruits that exhibited uniformity of weight (average weight per fruit 289.26 g), shape and color without any visual defects were selected to take three treatments: untreated control, 0.5 and 1.0 μl/l 1-mcp fumigation. 1-mcp (rohm and haas china inc., beijing) fumigation treatments at 0.5 or 1.0 μl/l were carried out using sealed plastic containers for 24 h at 25 ± 2 °c. untreated control was sealed with air. after treatment, each group was subsequently stored at 0 °c. all experiments in this paper were conducted directly from cold storage without shelf life. peel samples were periodically collected, quickly frozen in liquid nitrogen and stored in a freezer at 80 °c. methods scald index the scald index values were monitored throughout the cold storage. the scald data are expressed as a scald index based on the percentage superficial scald in ‘wujiuxiang’ pear 103 of the fruit surface area affected (zanella, 2003), where no scald = 0, < 25 % = 1, 25-50 % = 2, and > 50 % = 3. the scald index was normalized to 100 by multiplying the values by 100/3. three replicates were measured for each treatment, and 10 fruits were included in each replicate. firmness and tss content the flesh firmness was measured on two opposite points on the equatorial line of each fruit after peel removal using a fruit hardness tester (tuopu instruments, zhejiang, china; model gy-j) mounted on a standard drill press and fitted with an 8-mm probe. the tss content (°brix) was determined with a digital refractometer (atago, japan; model pal-1) by measuring the refractive index of the juice extracted from the same fruits used to determine firmness. two opposite points per fruit were selected to determine the firmness and tss content of each treatment, and three replicates were measured for each treatment with 10 fruits per replicate. respiration rate the respiration rate was measured using an infrared carbon dioxide analyzer (kexi instruments, jiangsu, china; model hwf-1a) at 0 °c after the fruits were sealed in glass desiccators (9.35 l) for 30 min. then, a 1-ml sample of gas was withdrawn with a syringe to analyze the respiration rate. the result was expressed as mg co2 kg-1 fw h-1. three replicates were measured for each treatment with 10 fruits per replicate. ethylene production rate the pear fruits were randomly selected and sealed in glass desiccators (9.35 l) for 4 h at 0 °c. a 1-ml sample of the headspace gas was withdrawn with a gas-tight syringe from each desiccator through a septum stopper and injected into a gas chromatograph (kechuang instruments, shanghai, china; model gc-9800) that was equipped with a gdx-502 column and a flame ionization detector (fid). the column temperature was 78 °c, and the injection temperature was 120 °c. the carrier gas was n2 with a rate of 40 ml min-1, and the rate of ethylene production was expressed as μl kg-1 fw h-1. three replicates were measured for each treatment with 10 fruits per replicate. α-farnesene and conjugated trienols content the extraction and analysis of α-farnesene and conjugated trienols were performed as described by anet et al. (1972) and isidoro and almeida (2006) with some modifications. four discs (1 cm in diameter) were removed from one strip of peel taken from the equatorial portion of each pear. these discs were placed in tubes containing 20 ml of hexane for 2 h. two milliliters of the extract was filtered through a florisil column and eluted using 3 ml of hexane for measurement of the absorbance at 232 nm. the absorbance of the remaining extract was measured at 281 and 290 nm. the contents of а-farnesene and conjugated trienols per cm2 of pear peel were calculated using the molar extinction coefficients ε232 = 27,740 for α-farnesene and ε281–290 = 25,000 for the conjugated trienols. three replicates were analyzed for each treatment with 10 fruits per replicate. h2o2 content the h2o2 content was measured according to the method described by bellincampi et al. (2000) with some modifications. four grams of fresh peel were homogenized in 8 ml of 100 mm sodium phosphate buffer (ph 7.2) with 10 % (w/v) polyvinylpolypyrrolidone (pvpp). the homogenate was centrifuged at 10,000 g for 10 min at 4 °c. then, 500 μl of the supernatant fractions was added to 1 ml of assay reagent (500 μm ammonium ferrous sulfate, 50 mm h2so4, 200 mm xylenol orange, and 200 mm sorbitol) and 500 μl of 100 mm sodium phosphate buffer (ph 7.2). the absorbance at 560 nm was detected after 30 min of dark incubation. standard curves of h2o2 were obtained for each independent experiment by adding variable amounts of h2o2 to 1 ml of basal medium mixed with 1 ml of assay reagent. the data were normalized and expressed as nmol g-1 fw. three replicates were measured for each treatment with 10 fruits per replicate. relative electrolyte leakage rate the relative electrolyte leakage was used as an indicator of the cell membrane permeability and was measured using a conductivity meter (yidian scientific instruments, shanghai, china; model leici dds307) as described by wang et al. (2005) with some modifications. four discs (1 cm in diameter) were removed from one strip of peel taken from the equatorial portion of each pear. these discs were placed in tubes containing 20 ml of distilled h2o for 1 h at ambient temperature (25 °c), and the initial conductivity was then measured. the samples were boiled for 30 min to release all electrolytes, and after the temperature dropped to ambient temperature, the final conductivity was measured. the percentage of electrolyte leakage was calculated as the ratio (×100) of the conductivity measurements before and after boiling. three replicates were measured for each treatment, and 10 fruits were included in each replicate. enzymes extractions and activities sod and cat two grams of frozen peel were ground in liquid n2 in a mortar and pestle. the completely grinding powder was blended in 5 ml of 100 mm phosphate buffer (ph 7.8), 2 mm dtt, 5 % (w/v) polyvinylpolypyrrolidone (pvpp), 0.1 mm edta and 1.25 mm polyethylene glycol. the extract was centrifuged at 12,000 × g for 30 min at 4 °c. the supernatants were transferred to fresh tubes as the crude enzyme extract for the measurements of sod and cat (larrigaudière et al., 2004; zavaleta-mancera et al., 2007). the sod (ec 1.15.1.1) activity was assayed according to larrigaudiere et al. (2004) with some modifications and determined by measuring the ability of sod to inhibit the photochemical reduction of nitroblue tetrazolium (nbt). the reaction mixture was composed of 1.5 ml of 50 mm phosphate buffer (ph 7.0), 0.3 ml of 130 μm l-methionine, 0.3 ml of 750 μm nbt, 0.3 ml of 100 μm edtana2, 0.3 ml of 20 μm riboflavin, 200 μl of h2o, and 100 μl of the crude enzyme extract. the reaction was initiated by illuminating the assay mixture with 60 mmol m-2 s-1 white fluorescent light at 25 °c. after 8 min, the reaction was stopped by removing the light source. a control tube with the assay mixture and enzyme was maintained in the dark, while another tube without the enzyme was maintained in light conditions to serve as the control for the reduction of nbt by light. the absorbance of the reaction was read at 560 nm. the activity of sod is reported as the nbt reduction in light without enzyme minus the nbt reduction with enzyme. one unit of sod activity is defined as the amount of enzyme that inhibits the nbt photoreduction by 50 % under the assay conditions. the sod data are presented as units of activity per mg protein. the cat (ec 1.11.1.6) activity was assayed according to zavaletamancera et al. (2007) with some modifications. the assay mixture (3 ml) contained 50 mm phosphate buffer (ph 7.0) and 100 μl of the crude enzyme extract. the reaction was initiated by the addition of 100 μl of 0.1 m h2o2. the decomposition was followed directly 104 m. gao, s. zhou, j. guan, y. zhang by the decrease in absorbance at 240 nm every 30 s for 2 min at 25 °c. one unit of cat activity is defined as the amount of enzyme that reduces the absorbance by 0.01 in one minute. apx the extraction of the apx enzyme was performed using 100 mm phosphate buffer (ph 7.0), 2 mm dtt, and 5 mm ascorbate (zavaleta-mancera et al., 2007). the apx (ec 1.11.1.11) activity was determined by monitoring the decomposition of h2o2 at 290 nm according to the method described by zavaleta-mancera et al. (2007) with some modifications. the reaction mixture (2 ml) contained 50 mm phosphate buffer (ph 7.0), 0.1 mm edta, 0.1 mm ascorbate, 2.7 mm h2o2 and 100 μl of the crude enzyme extract. the reaction was initiated by the addition of h2o2. the decrease in absorbance due to the oxidation of ascorbate was determined at 290 nm every 30 s for 3 minutes at 25 °c. one unit of apx activity is defined as the amount of enzyme that oxidizes 1 μm ascorbate in one minute. lox the lox enzyme was extracted using 100 mm phosphate buffer (ph 7.5), 2 mm dtt, 1 mm edta, 0.1 % (v/v) triton x-100, and 1 % pvpp (axelrod et al., 1981). the lox (ec 1.13.11.12) activity was assayed according to axelrod et al. (1981) with some modifications using sodium linoleate as the substrate. the substrate was prepared as follow: 28 μl of sodium linoleate and 36 μl of tween-20 were mixed in 8 ml of oxygen-free water, and 2 m naoh was added to clear the solution. finally, the volume of the solution was set to 10 ml with distilled water. the assay mixture (3 ml) contained 2.925 ml of 200 mm phosphate buffer (ph 7.0), 25 μl of the substrate solution and 50 μl of the crude enzyme extract. the reaction was initiated by the addition of the crude enzyme extract. the absorbance of the mixture was recorded at 234 nm every 1 min for 8 minutes at 25 °c. the lox activity is expressed as δod234 min-1 mg-1 protein. the protein contents of above enzymes were determined according to the method described by bradford (1976) using bovine serum albumin as the standard. three replicates were extracted and measured for each treatment with 10 fruits per replicate. statistical analysis all of the data were analyzed by one-way analysis of variance (anova) according to treatment. all of the values are expressed as the means ± se of three replicates. differences were considered significant at p < 0.05. all of the analyses were performed with spss 12.0 software (spss inc. chicago, il, usa). results effects of 1-mcp on superficial scald index and quality during cold storage, superficial scald was observed after 120 days and was lowered by 1-mcp treatment, particularly by 1-mcp with the concentration of 1.0 μl/l (fig. 1a). these findings suggested that the 1.0 μl/l concentration of 1-mcp was more effective for reducing the scald incidence. the firmness declined in all of the treatments during the storage period, whereas the 1.0 μl/l 1-mcp-treated fruits showed higher firmness than the control (p < 0.05, fig. 1b). on the contrary, the contents of tss increased in all of the treatments, and a lower value was obtained in the 1.0 μl/l 1-mcp-treated fruits (p < 0.05, fig. 1c). this result indicated that 1-mcp could delay fruit softening and senescence. fig. 1: effects of 1-mcp on superficial scald index (a), firmness (b), total soluble solids (c), respiration rate (d), and ethylene production rate (e) in ‘wujiuxiang’ pear fruits during storage at 0 °c. the data are the means ± se of three replicates. the vertical bars represent the standard errors of the means. star represents significant difference (p < 0.05). superficial scald in ‘wujiuxiang’ pear 105 effects of 1-mcp on respiration and ethylene production rates the respiration rates of ‘wujiuxiang’ pear fruits were relatively stable during cold storage, and no peak was observed. however, the respiration rates of the 1-mcp-treated fruits were generally lower than those of the control after 60 days of storage, and this difference was particularly observed with the 1.0 μl/l 1-mcp treatment from control (p < 0.05, fig. 1d). the ethylene production rates increased steadily in the control and the 0.5 μl/l 1-mcp-treated fruits until 60 days and then declined, whereas it increased markedly slower in the 1.0 μl/l 1-mcp-treated fruit before 60 days and then remained at a lower level than that observed in the control (p < 0.05, fig. 1e). effects of 1-mcp on contents of α-farnesene and conjugated trienols in the control fruit, α-farnesene accumulated from an initial value of 6.58 nmol per cm2 to a peak of 81.56 nmol per cm2 after 60 days in storage and declined thereafter. the α-farnesene accumulation in the fruits treated with 1-mcp showed similar trends; however, it maintained a lower level with 1.0 μl/l 1-mcp than those with 0.5 μl/l 1-mcp and control (fig. 2a). the contents of conjugated trienols in all treated fruits increased at later storage period; however, it accumulated less in fruits with 1.0 μl/l 1-mcp than those with 0.5 μl/l 1-mcp and control, particularly before 105 days of storage (fig. 2b). effects of 1-mcp on h2o2 accumulation and activities of antioxidant enzymes h2o2 accumulated progressively in the peel during cold storage, especially after 60 days. compared with the control, 1-mcp could effectively inhibit the accumulation of h2o2, especially in the treatment with 1.0 μl/l (p < 0.05, fig. 3a). the sod activity in the peel steadily increased in all of the treatments until 90 days of storage and then declined. the 1-mcp-treated fruits maintained higher levels of sod activity than the control, and the 1.0 μl/l 1-mcp-treated ones exhibited the highest levels (fig. 3b). similar results were also observed with the cat and apx activities. however, the peaks of cat and apx activities occurred on 105 d and were 15 days later than that of sod activity (fig. 3c and d). fig. 2: effects of 1-mcp on the accumulation of α-farnesene (a) and conjugated trienols (b) in ‘wujiuxiang’ pear fruits during storage at 0 °c. the data are the means ± se of three replicates. the vertical bars represent the standard errors of the means. star represents significant difference (p < 0.05). fig. 3: effects of 1-mcp on h2o2 content (a) and activities of apx (b), sod (c), and cat (d) in ‘wujiuxiang’ pear fruits during storage at 0 °c. the data are the means ± se of three replicates. the vertical bars represent the standard errors of the means. star represents significant difference (p < 0.05). 106 m. gao, s. zhou, j. guan, y. zhang effects of 1-mcp on relative electrolyte leakage rate and lox activity the relative electrolyte leakage rate of the peel steadily increased in the control, reaching a value of 45.9 % after 135 days of cold storage. at the same time, it also increased in the 1-mcp-treated fruit but to a much lesser degree. however, the difference between 0.5 μl/l 1-mcp-treated fruits and the control was not significant (fig. 4a). as shown in fig. 4b, the lox activities in the control fruit increased rapidly during 105 days of cold storage and then gradually decreased. in the 1-mcp-treated fruits, the lox activities were maintained at a lower level during storage, and this was particularly true in the 1.0 μl/l 1-mcp-treated fruits. (lurie and watkins, 2012). owing to its effects on ethylene action, 1-mcp was applied to delay ripening, to maintain fruit quality during storage (blankenship and dole, 2003; watkins, 2006) and to reduce physiological disorder, including superficial scald (bai et al., 2009; hui et al., 2011; li and wang, 2009; sabban-amin et al., 2011). in ‘wujiuxiang’ pear, treatment with 1.0 μl/l 1-mcp also significantly reduced the superficial scald index, retained a higher firmness and a lower tss content, and decreased the ethylene production and respiration rates (fig. 1). this finding suggested that 1-mcp is effective for delaying fruit softening and inhibiting the incidence of superficial scald in ‘wujiuxiang’ pear. moreover, the 1-mcp concentration at 1.0 μl/l was more effective than that at 0.5 μl/l. it was interesting that scald appeared at day 120 and become more serious at day 135 in control during cold storage (fig. 1a). it suggested that scald appeared suddenly and developed quickly at late stage of cold storage in ‘wujiuxiang’ pear, which was similar to ‘bartlett’ pear (whitaker et al., 2009). it may due to ‘wujiuxiang’ pear is a hybrid cultivar from ‘yali’ (pyrus bretschneideri r.) and ‘bartlett’ (pyrus communis l.), and more like europe pear (pyrus communis l.) in postharvest storage characteristics. in fruit, the inhibition of ethylene production by 1-mcp is well known. 1-mcp could almost fully inhibit ethylene production in ‘fuji’ apple (lu et al., 2013), ‘granny smith’ apple (sabbbanamin et al., 2011), and ‘delicious’ apple (apollo arquiza et al., 2005); and delayed the increase of ethylene production in ‘bartlett’ pear (ekman et al., 2004), ‘anjou’ pear (argenta et al., 2003), and ‘conference’ pear (chiriboga et al., 2013b). in this work, compared to control, ethylene production in 1-mcp-treated ‘wujiuxiang’ pear fruits showed similar pattern with a much lower concentration (fig. 1e). it suggested that the 1-mcp could partially inhibit the ethylene action in ‘wujiuxiang’ pears. due to this, 1-mcp could not fully prevent scald development (fig. 1a), nor did it preventing firmness loss (fig. 1b), but both were less than in control. there are some possible reasons for the partial inhibition of 1-mcp on ethylene action. firstly, it is clear that 1-mcp inhibits the ethylene responses by blocking the ethylene receptor with the highly stable 1-mcpreceptor complex (sisler and serek, 1997). however, treatment with 1-mcp prior to storage could only block existing receptors, but new ethylene receptors could be formed during storage (tsantili et al., 2007). previous results showed that the transcriptional levels of pcetr1 and pcetr2 were higher in 1-mcp-treated pear fruits than those in control during cold storage (chiriboga et al., 2013b). it could be proposed that the inhibition effects of 1-mcp on ethylene fig. 4: effect of 1-mcp on relative membrane permeability (a) and lox activity (b) in ‘wujiuxiang’ pear fruits during storage at 0 °c. the data are the means ± se of three replicates. the vertical bars represent the standard errors of the means. star represents significant difference (p < 0.05). the correlation between h2o2 content and the cell membrane permeability of the peel, and between h2o2 content and scald index to reveal the relationship between h2o2 content and the cell membrane permeability of the peel, and between h2o2 content and the scald index, the correlation between these variables was calculated. the finding showed a very significant positive correlation between the h2o2 content and the relative electrolyte leakage rate of the peel (r = 0.881, p < 0.01, fig. 5), and a significant positive correlation between the h2o2 content and scald index (r = 0.782, p < 0.05, fig 5). discussion the plant hormone ethylene is considered not only the major signaling molecule that regulates most aspects of fruit ripening in climacteric fruit (pech et al., 2012), but also an important regulator in disorder development during storage, including superficial scald fig. 5: correlation between h2o2 content and the relative memebrane permeability of the peel (●), and between h2o2 content and scald index () in ‘wujiuxaing’ pear. superficial scald in ‘wujiuxiang’ pear 107 action were offset by newly translated ethylene receptors during cold storage. secondly, the ethylene biosynthetic and signal transduction pathways are differently affected by 1-mcp in different fruit cultivars (dal cin et al., 2006), harvest maturity and storage temperature (chiriboga et al., 2013b), the details of the inhibition mechanism of 1-mcp on ethylene action in ‘wujiuxiang’ pears require additonal experiments. ethylene can regulate α-farnesene accumulation in apples (ju and curry, 2000; whitaker et al., 2000) and pears (isidoro et al., 2006; li et al., 2009). our results also suggested that the accumulation of α-farnesene may be associated with ethylene production. the results showed that the productions of α-farnesene and ethylene both peaked at 60 day and that 1-mcp inhibited the productions of ethylene and α-farnesene (fig. 1e and fig. 2a). furthermore, it has been shown that α-farnesene metabolism may be a manipulating point to control scald development. the reactive conjugated trienols resulting from the oxidation of α-farnesene are believed to be directly responsible for superficial scald (du and bramlage, 1993; giné bordonaba et al., 2013; lu et al., 2013; whitaker, 2007). in our experiment, the accumulation of α-farnesene in all fruits peaked at 60 days of storage, whereas the accumulation of conjugated trienols showed an increase trend at later storage period (fig. 2), which indicates that the production of α-farnesene is followed by the accumulation of conjugated trienols. thus, it further confirmed that superficial scald was more closely associated with conjugated trienols than with α-farnesene (giné bordonaba et al., 2013; lu et al., 2013). it has been suggested that ethylene plays a fundamental role in scald development not only by regulating the α-farnesene metabolism but also by regulating the anti-oxidant system in the fruit (lu et al., 2013; sabban-amin et al., 2011). it is thought that high h2o2 concentrations, which accumulate in fruits during prolonged cold storage (dong et al., 2011; lu et al., 2011; rao et al., 1998), are often regarded as an indication of oxidative stress (apel and hirt, 2004). h2o2 together with o2may form the radical hydroxyl ·oh, which is a powerful oxidant. this product may cause many important metabolic changes, such as lipid peroxidation (tian et al., 2013), which may result in damage to the cell membrane and physiological disorders, such as superficial scald (lu et al., 2011, 2013; rao et al., 1998; sabban-amin et al., 2011). the findings showed that the accumulation of h2o2 in peel is much lower in the 1.0 μl/l 1-mcp-treated fruits than that in control during cold storage (fig. 3a). this effect was found to be associated with a lower relative electrolyte leakage rate (fig. 4a) and a lower scald index (fig. 1a), which reflected that the lipid peroxidation was reduced in the 1-mcp-treated fruit, and also, the lower h2o2 level was closely related to lower relative membrane permeability and lower scald index (fig. 5). as another indicator of lipid degradation, lox activity is closely correlated with lipid peroxidation during fruit ripening (li and wang, 2009; liu et al., 2013; singh et al., 2012). the data showed that the lox activities were kept at a low level in the 1-mcp-treated fruits (fig. 4b). therefore, this finding suggested that 1-mcp can protect the membrane integrity by inhibiting h2o2 accumulation and lipid peroxidation. in general, the h2o2 levels in plant cells are regulated by h2o2scavenging enzymes, such as apx, sod and cat (rao et al., 1996; tian et al., 2013). the lower h2o2 levels observed in the peel of ‘wujiuxiang’ pears treated with 1-mcp might be resulted from the higher activities of apx, sod and cat (fig. 3b, c, d). these results are in agreement with previous findings in ‘blanquilla’ pear (larrigaudiere et al., 2004) and ‘granny smith’ apple (sabbanamin et al., 2011). all of the results suggested that 1-mcp can re duce h2o2 accumulation by improving the activities of h2o2scavenging enzymes. previous research has indicated that the extent of oxidation of α-farnesene could be affected by anti-oxidant enzyme activities in fruit (whitaker, 2004; rao et al., 1998). in this work, the antioxidant enzyme activities were higher (fig. 3b, c, d), and the conjugated trienols were lower (fig. 2b) in the 1-mcp-treated fruits than those in the control. this finding suggested that 1-mcp inhibits the accumulation of conjugated trienols not only due to the lower α-farnesene accumulation (fig. 2a) but also the reduced oxidation reaction, which may a result of the higher anti-oxidant enzyme activities and lower h2o2 levels (fig. 3). in conclusion, these data showed that 1-mcp delayed softening and inhibited scald development in ‘wujiuxiang’ pear fruit. on the basis of these results and previous work, we propose the following possible mechanism for the inhibition of superficial scald by 1-mcp: by inhibiting ethylene synthesis and/or signal transduction, 1-mcp stimulates the activities of h2o2-scavenging enzymes to suppress the accumulation of h2o2, which results in the reduction of cell membrane damage and the lower accumulation of conjugated trienols, and thus inhibits cell death and superficial scald of peel. however, the crosstalk between h2o2 and α-farnesene metabolism remains unclear, and future work is needed. acknowledgement this research was supported by the earmarked fund for modern pear-industry technology research system of china (cars-2920). references abbasi, n.a., kushad, m.m., hafiz, i.a., maqbool, m., 2008: relationship of superficial scald related fruit maturity with polyphenoloxidase and superoxide dismutase activities in ‘red spur delicious’ apples. asian j. 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(http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 87, 256 264 (2014), doi:10.5073/jabfq.2014.087.036 institute for resistance research and stress tolerance, julius kühn-institut, groß lüsewitz, germany in vitro method for early evaluation of nitrogen use efficiency associated traits in potato annegret schum, gisela jansen (received may 21, 2014) summary the objective of the present study was to characterize various traits associated with nitrogen uptake and utilization in a range of potato cultivars. for this purpose an in vitro test system was developed which allows analyzing specific stress responses in a highly controlled environment. shoot tips were grown fixed in perforated stainless steel plates in 500 ml glass vessels in liquid culture medium at four nitrogen levels, i.e. 60, 30, 15 and 7.5 mmol l-1. at the end of a three weeks’ culture period plant developmental traits were determined and nitrogen uptake and assimilation were analyzed. reduction of nitrogen in the culture medium differentially affected morphological and physiological features. highly significant differences were found between different n-levels and cultivars as well as for genotype x nitrogen level interactions. three groups of cultivars (high, low and intermediate) were distinguished with respect to biomass production and crude protein yield under nitrogensufficient conditions of 60 mmol l-1. genotypes with a low biomass production at full nitrogen availability responded with increased root development under nitrogen deficiency stress and increased their nitrogen utilization capacity in relation to the other cultivars. introduction in recent years, the need for a sustainable exploitation of resources has attained increased attention in agricultural production. nitrogen fertilization is a key element for obtaining profitable yields but, at the same time, it implies the risk of environmental pollution. only a limited proportion of the applied nitrogen is actually taken up by plants, while as much as 60 to 70 % are lost from the plantsoil system by gaseous emissions due to soil denitrification and ammonia volatilisation as well as leaching of nitrate (e.g. raun and johnson, 1999; good et al., 2004; vos, 2009). resulting adverse environmental effects include pollution of ground-, freshand marine water as well as the release of greenhouse gases as n2o with its high impact on global warming. two strategies have been pursued in order to minimize the negative impact of nitrogen fertilizers on the environment. one approach is the development of enhancedefficiency fertilizers and optimized nutrient management (hopkins et al., 2008), while the second focuses on selection of cultivars with improved nitrogen use efficiency (nue). in this context, efficient methods for screening of germplasm are needed. commercial potato production is especially prone to lead to environmental contamination, as cultivation on sandy soils in combination with high nitrogen fertilizer rates and irrigation results in leaching of nitrate (sharifi et al., 2007). furthermore, the shallow root system of solanum tuberosum does not allow nitrogen retraction from deeper soil layers. a survey on nitrate-n contaminations of rural well water in agricultural regions of canada demonstrated that the mean no3-n concentration was in fact associated with the proportion of the area cropped to potatoes (demerchant et al., 1990). nutrient use efficiency is regarded as the second most important goal next to drought tolerance in abiotic stress improvement of crops (hirel et al., 2011). many attempts have been made in order to elucidate the genetic and physiological basis of this complex trait (reviewed for example by hirel et al., 2007; hirel et al., 2011; lea and azevedo, 2006; lea and azevedo, 2007; lightfoot, 2010). however, the current knowledge of key steps involved in the control of nutrient use efficiency from gene expression to metabolic activity and phenotypic manifestation remains incomplete (hirel et al., 2011). in spite of various experiments which have been performed with potato worldwide to identify genotypic differences in nue from an agronomic point of view, information on underlying physiological mechanisms of nutrient uptake and utilization efficiency is limited (sharifi et al., 2007). the objective of the present investigation was to characterize various traits associated with nitrogen uptake and utilization in a range of potato cultivars at an early growth stage in a highly controlled environment in order to get detailed knowledge on genetic differences in n use efficiency. for this purpose an in vitro test system was developed with the aim to possibly identify specific morphological and physiological components which are crucial for nitrogen use efficiency in solanum tuberosum l. materials and methods plant material thirteen mid-early potato cultivars (maturity group iii) developed by different breeding companies were used for this investigation, i.e. ‘afra’, ‘agria’, ‘ditta’, ‘filea’, ‘marlen’, ‘nicola’, ‘simone’, ‘solara’, ‘topas’ (europlant pflanzenzucht, germany) ‘pirol’, ‘lambada’, (norika, germany), ‘skala’ (bavaria-saat, germany) as well as ‘milva’ (dnk 1970; an old danish accession derived from the ipk (leibniz institute of plant genetics and crop plant research, germany). stock cultures of virus tested in vitro plantlets were kept in 250 ml glass vessels on gelrite solidified ms medium (murashige and skoog, 1962) without growth regulators. cultures were incubated at 18 °c in a 16-h-photoperiod and subcultured every four to six weeks. 14 days before initiation of the experiments 10 nodal sections were placed in each 250 ml culture vessel and incubated as stated above. shoot tips of approximately 1.5 2.0 cm length were excised from the newly developed sprouts and used for nitrogen stress experiments. in vitro test system for the experiments 10 shoot tips were fixed in perforated stainless steel plates and incubated in 500 ml glass vessel containing 50 ml liquid culture medium. the nitrogen levels of the media corresponded to full, 1/2, 1/4 and 1/8 of the original ms concentration (60, 30, 15, 7.5 mmol l-1), providing nh4+-n and no3--n in a ratio of 0.52. cultures were incubated at 18 °c in a 16-h-photoperiod for a period of 21 days. at that time, specific morphological traits of each individual plantlet were recorded, i.e. shoot length, number of nodes for calculating internode length, and the chlorophyll content of the youngest fully expanded leaf measured with a spad-502 chlorophyll meter (konica minolta). in addition, fresh and dry matter of both in vitro nue 257 shoots and roots were determined for each unit of 10 plantlets. the remaining nutrient solutions were frozen at -20 °c until analysis of the nitrogen withdrawal. assays were repeated in three independent time-shifted experiments each time comprising four replicates per n-variant. analysis of nitrate, ammonium and crude protein the remaining nitrogen in media at the end of the culture period was determined spectrophotometrically as described by schum and jansen (2012). the quantity of nitrate-n was roughly determined by use of mquant™ test strips (merck kgaa) and solutions were diluted as appropriate. final analysis was performed by application of a spectrophotometric method adapted from the official nitrate analysing method of soil samples of the german association of agricultural experiment and research stations (vdlufa, 2002) which determines the nitrate-n content by measuring the difference in uv-absorption at 210 nm of the original solution and after nitrate reduction by nascent hydrogen. ammonium-n was determined spectrophotometrically according to din 38406-5 of german standard methods for the examination of water, waste water and sludge (normungsausschuss wasserwesen 1983), in which nh4+ions react at a high ph-value with hypochloriteund salicylateions in the presence of sodium nitroprusside to form the salicylic acid analog of indophenols. the lowest concentration used for calibration was 0.5 mg/l in case of nitrate-n and 0.25 mg/l in case of ammonium-n, respectively. crude protein contents of shoots and roots were analyzed by near infrared reflectance spectroscopy (nir) based upon calibration by standard kjeldahl methods (schum and jansen 2012). shoots and roots of plantlets cultured for three weeks on media with four different nitrogen levels were dried at 60 °c for 24 hours. dry matter was milled in a retsch laboratory swing mill and stored in eppendorf tubes. upon calibration by application of the standard kjeldahl methods, crude protein content of samples were determined by near infrared reflectance spectroscopy (nir). reflexion spectra were measured with a multi purpose analyzer (bruker optics) using a wavelength range between 800 and 2500 nm. the device was equipped with a small flat-bottomed glass cuvette to take up the milled plant material. 150 samples of shoots and 202 samples of roots were selected in order to correlate the individual spectra with the protein amount of specific probes as determined by the kjeldahl method. the calibration provided results for predicting the protein content with r2 = 0.991 (root mean standard error of prediction rmsep = 0.85) for shoots and r2 = 0.979 (rmsep = 0.77) for roots, respectively. the minimum amount of determinable crude protein was limited by the amount of dry matter produced by the plantlets. at least 10 mg were necessary to cover the bottom of the cuvette. the lowest amounts determined were found in roots of plantlets grown in media with 1/8 n-supply with 6.3 % of dry matter. as the routine kjeldahl process does not determine nitrate and nitrite nitrogen, solely nitrogen metabolized by the plantlets into organic compounds was evaluated. stress susceptibility index ranking of cultivars was performed for selected features by computing the stress susceptibility index (ssi) introduced by fischer and maurer (1978) according to following formula: ssi = (1-ps / pc) / (1meanps / meanpc) ps = parameter determined under stress conditions (1/2n) pc = parameter determined under control conditions (1n) meanps = mean of all genotypes under stress conditions meanpc = mean of all genotypes under control conditions statistical analysis data collected from the three independent time-shifted experiments with four replicates of each variant were subjected to analysis of variance by application of the general linear model (glm) procedure of sas 9.2. results plant performance reduction of nitrogen in the culture medium differentially affected a wide range of morphological and physiological features in a genotype specific manner. highly significant differences were found between n-levels and cultivars as well as for genotype x nitrogen level interactions (tab. 1). the stepwise decrease in nitrogen supply from the 1/2 to the 1/4 and 1/8 n level is clearly reflected by a reduction in shoot and internode length, chlorophyll content of the youngest fully developed leaf as well as in fresh matter production of shoots, roots and whole plantlets (tab. 1). however, the degree of impairment caused by nitrogen deficiency stress depends on the genotype. this is indicated in tab. 1 by highlighting thresholds for a significant decline in trait performance for individual cultivars. the analysis of variance revealed significant differences between genotypes e.g. in fresh matter yield of plantlets at each individual nitrogen level as well as differential responses of individual genotypes to the reduction of available nitrogen. while cultivars ‘lambada’, ‘milva’ and ‘solara’ produced the highest amount of plant biomass under full nitrogen supply, these genotypes showed a strong and continuous decline with the reduction of nitrogen availability immediately beginning at the 1/2 n-level. in contrast, for the majority of genotypes a significant drop in fresh matter production was observed only upon further reduction of the nitrogen availability to 1/4. the two cultivars with low biomass production at a high nitrogen level (‘agria’ and ‘topas’) showed a better performance under deficiency stress conditions and fell off only at the 1/8 n level. fresh matter of cv. ‘topas’ produced at 1/4 and 1/8 of the original nitrogen concentration reached values comparable to those of the normally vigorously growing genotype ‘lambada’. the discrimination between plant parts revealed that differences in biomass production in response to nitrogen deficiency stress were generally more pronounced for roots as for shoots. the root percentage share of total biomass varies between 22 % (‘filea’) and 59 % (‘lambada’) at full nitrogen supply. upon reduction of available nitrogen two fundamentally different types of response could be distinguished between genotypes after three weeks of culture as demonstrated in fig. 1. in some cultivars root fresh matter continually decreased with increasing nitrogen deficiency stress. in contrast, other cultivars responded with a stimulation of root development upon reduction of the nitrogen supply to 1/2 of the original ms medium and were even able to sustain root proliferation at the 1/4 level. it has to be noted that in spite of the general reduction of root proliferation with decreasing nitrogen availability in ‘lambada’, this cultivar still produced the highest amount of root biomass at all n-levels as compared to the rest of the genotypes. in contrast, absolute values for root fresh matter of ‘topas’ increased under nitrogen deficiency stress in relation to the other cultivars. further analysis of biomass production in relation to nitrogen supply was based on the determined dry matter. in order to visualize type and dimension of modifications of single characters in response to nitrogen deficiency stress we followed the procedure used by ikram et al. (2012). for each evaluated trait mean values determined under nitrogen deficiency stress (1/2 n, 1/4 n, 1/8 n) were plotted on the vertical axis against values obtained at full nitrogen availability on the horizontal axis. this approach allows compiling of data from 13 genotypes and 4 nitrogen levels within one graph. further 258 a. schum, g. jansen t ra it f -v al ue s n p la nt p er fo rm an ce w it h d ec re as in g n s up pl y fo r in di vi du al t ra it s an d g en ot yp es n -l ev el (n ) g en oty pe (g ) n x g l ev el a g t o n i f i m a d i p i sk si a f so m i l a 1n 5. 84 a 6. 05 a 7. 20 a 6. 18 a 6. 68 a 6. 83 a 7. 58 a 7. 81 a 7. 98 a 9. 53 a 9. 30 a 11 .1 1a 11 .1 6a 1/ 2n 6. 83 b 7. 36 b 7. 71 a 6. 43 a 6. 22 a 7. 16 a 7. 69 a 7. 46 a 7. 96 a 8. 98 a 7. 80 b 9. 67 b 8. 83 b 1/ 4n 5. 40 a 6. 13 a 5. 62 b 4. 21 b 4. 57 b 4. 68 b 5. 43 b 5. 68 b 5. 95 b 5. 82 b 5. 05 c 6. 89 c 6. 04 c f m p la nt s [g pe r 1 0] 80 1. 70 ** * 51 .7 3* ** 7. 43 ** * 1/ 8n 3. 29 c 3. 87 c 3. 72 c 2. 48 c 3. 03 c 2. 90 c 3. 28 c 3. 67 c 3. 78 c 3. 96 c 3. 20 d 4. 73 d 4. 00 d 1n 4. 01 a 3. 77 a 5. 04 a 4. 80 a 4. 63 a 4. 16 a 4. 72 a 4. 51 a 4. 23 a 5. 56 a 6. 05 a 6. 46 a 4. 61 a 1/ 2n 4. 52 b 4. 67 b 4. 61 a 4. 18 b 4. 23 a 4. 53 b 4. 72 a 4. 55 a 4. 38 a 5. 09 a 4. 93 b 5. 83 b 4. 33 a 1/ 4n 3. 26 c 3. 61 a 3. 21 b 2. 76 c 3. 20 b 3. 08 c 3. 36 b 3. 56 b 3. 28 b 3. 58 b 3. 56 c 4. 32 c 2. 99 b f m s ho ot s [g p er 1 0 pl an ts ] 66 7. 14 ** * 26 .5 4* ** 3. 62 ** * 1/ 8n 2. 11 d 2. 25 c 2. 30 c 1. 74 d 2. 12 c 1. 96 d 2. 02 c 2. 38 c 2. 32 c 2. 57 c 2. 34 d 3. 02 d 2. 10 c 1n 1. 83 a 2. 28 a 2. 05 a 1. 38 a 2. 12 a 2. 67 a 2. 86 a 3. 30 a 3. 75 a 3. 98 a 3. 14 a 4. 65 a 6. 55 a 1/ 2n 2. 32 b 2. 70 a 3. 10 b 2. 30 b 1. 99 a 2. 63 a 2. 97 a 2. 91 a 3. 58 a 3. 89 a 2. 82 a 3. 90 b 4. 50 b 1/ 4n 2. 14 ab 2. 51 a 2. 41 a 1. 46 a 1. 37 b 1. 60 b 2. 07 b 2. 11 b 2. 68 b 2. 24 b 1. 50 b 2. 57 c 3. 05 c f m r oo ts [g p er 1 0 pl an ts ] 28 0. 91 ** * 56 .2 3* ** 8. 89 ** * 1/ 8n 1. 19 c 1. 62 b 1. 42 c 0. 74 c 0. 91 c 0. 94 c 1. 26 c 1. 29 c 1. 46 c 1. 39 c 0. 87 c 1. 71 d 1. 90 d 1n 10 .4 a 9. 0a 9. 4a 7. 5a 9. 7a 5. 7a 5. 5a 6. 3a 8. 2a 8. 8a 6. 0a 4. 7a 7. 7a 1/ 2n 11 .8 a 11 .4 a 9. 1a 7. 0a 10 .3 a 5. 9a b 5. 8a 6. 4a 9. 3a 8. 1a 5. 4a 4. 4a 7. 7a 1/ 4n 8. 1b 8. 9a 6. 0b 5. 3b 7. 9b 4. 4c 4. 1b 5. 0a 5. 6b 6. 2b 4. 1b 3. 4b 5. 3b sh oo t l en gt h [c m ] 19 6. 09 ** * 42 .5 8* ** 2. 38 ** * 1/ 8n 4. 9c 5. 3b 4. 3c 3. 9c 4. 5c 2. 9d 2. 9c 3. 4b 3. 3c 4. 1c 2. 7c 2. 8c 3. 3c 1n 1. 6a 1. 6a c 1. 5a b 1. 1a 1. 5a 0. 9a 0. 8a 1. 2a 1. 2a b 1. 5a 1, 1a 1. 0a 1. 4a 1/ 2n 1. 9b 2. 1b 1. 6a 1. 2a 1. 7a 1. 2b 1. 0a 1. 3a 1. 4b 1. 7a 1. 1a 1. 2a 1. 6a 1/ 4n 1. 7a b 2. 0a b 1. 3b 1. 2a 1. 6a 1. 1a b 0. 9a 1. 3a 1. 2a c 1. 6a 1. 0a b 1. 1a 1. 5a in te rn od e l en gt h [c m ] 29 .6 0* ** 29 .1 1* ** 1. 18 n. s. 1/ 8n 1. 5a 1. 5c 1. 3b 0. 9a 1. 0b 0. 9a 0. 8a 1. 1a 0. 9c 1. 2b 0. 9b 1. 2a 1. 1b 1n 60 .5 a 55 .4 a 53 .0 a 48 .2 a 54 .3 a 62 .3 a 56 .6 a 52 .5 a 63 .2 a 58 .2 a 52 .9 a 46 .3 a 62 .2 a 1/ 2n 60 .3 a 50 .0 b 50 .6 a 45 .4 a 52 .9 ab 57 .2 b 51 .1 b 48 .9 b 58 .5 a 48 .6 b 43 .2 b 35 .2 b 50 .5 b 1/ 4n 47 .9 b 43 .1 c 37 .7 b 29 .4 b 49 .6 b 43 .4 c 38 .7 c 30 .3 c 37 .8 b 32 .7 c 27 .6 c 26 .3 c 26 .4 c c hl or op hy ll c on te nt [s pa d v al ue s] 81 5. 67 ** * 68 .6 8* ** 8. 53 ** * 1/ 8n 46 .0 b 38 .7 d 25 .2 c 21 .7 c 40 .5 c 37 .0 d 35 .0 d 21 .5 d 30 .6 c 24 .6 d 22 .8 d 21 .2 d 14 .9 d ta b. 1 : v eg et at iv e de ve lo pm en t of 1 3 po ta to g en ot yp es i n re sp on se t o in cr ea si ng n itr og en d efi ci en cy s tr es s. d if fe re nc es b et w ee n n -l ev el s, g en ot yp es a nd c or re sp on di ng i nt er ac tio ns a re i llu st ra te d in t er m s of fva lu es . i n ad di tio n, m ea ns f or s in gl e tr ai ts a re s ho w n fo r in di vi du al c ul tiv ar s an d n -l ev el s. ( a f = ‘a fr a’ ; a g = ‘a gr ia ’; d i = ‘ d itt a’ ; f i = ‘ fi le a’ ; l a = ‘l am ba da ’; m a = ‘m ar le n’ ; m i = ‘ m ilv a’ ; n i = ‘n ic ol a’ ; p i = ‘p ir ol ’; s i = ‘s im on e’ ; s k = ‘s ka la ’; s o = ‘s ol ar a’ ; t o = ‘t op as ’) (v al ue s fo r i nd iv id ua l g en ot yp es m ar ke d w ith d if fe re nt le tte rs a re s ig ni fic an tly d if fe re nt a cc or di ng to l sd b as ed m ul tip le c om pa ri so n te st [p =0 .0 5] . t hr es ho ld s fo r a s ig ni fic an t d ec lin e in p er fo rm an ce a re hi gh lig ht ed .) in vitro nue 259 explanations are given in the captions to fig. 2 and 3. the distribution of data points in fig. 2 illustrates that both nitrogen availability as well as genotype account for the large variation in biomass production determined as dry matter of shoots (fig. 2a), roots (fig. 2b) and whole plantlets (fig. 2c). the decrease in nitrogen supply from the 1/2 to the 1/4 and 1/8 level is clearly reflected by a reduction in biomass yield after three weeks of culture, however in a varying extent according to the genotype. plotting of data from dry matter obtained from plantlets (fig. 2c) allows the identification of three clusters of genotypes. cultivars ‘lambada’, ‘milva’, ‘solara’ and ‘afra’ represent the group with highest biomass development under full and 1/2 nitrogen regime, while cultivars ‘agria’ and ‘topas’ are separated from an intermediate group by a low performance at full nitrogen availability. however, the latter show a significant decline only at the 1/8 n-level while the corresponding threshold values for ‘lambada’, ‘milva’, ‘solara’ and ‘afra’ are already reached at the 1/4 n-level. biomass allocation between different plant parts reveals an extraordinarily high contribution of roots in case of ‘lambada’ (fig. 2b), while the shoot fraction is comparatively low in ‘topas’ and ‘agria’ (fig. 2a). fig. 1: examples for differential genotype specific root response to nitrogen deficiency stress: fresh matter of roots / ten plantlets after three weeks of culture in media with different levels of nitrogen. (bars marked with different letters are significantly different according to lsd based multiple comparison test [p=0.05]; scales of y axes are different due to substantial differences in root development between cultivars.) 260 a. schum, g. jansen a dry matter shoots [mg per 10 plants] b dry matter roots [mg per 10 plants] c dry matter plantlets [mg per 10] d n withdrawel from media [%] e crude protein content shoots [%ofdm] f crude protein content roots [%ofdm] g root:shoot ratio: crude proteincontent h crude protein yield plantlets [mg] fig. 2: distribution of traits related to biomass, n-uptake, crude protein content and crude protein yield of 13 potato genotypes grown under three levels of nitrogen deficiency stress compared to full nitrogen supply. (af = ‘afra’; ag = ‘agria’; di = ‘ditta’; fi = ‘filea’; la = ‘lambada’; ma = ‘marlen’; mi = ‘milva’; ni = ‘nicola’; pi = ‘pirol’; si = ‘simone’; sk = ‘skala’; so = ‘solara’; to = ‘topas’) for each evaluated trait mean values determined under nitrogen deficiency stress (1/2 n, 1/4 n, 1/8 n) were plotted against values obtained at full nitrogen availability. this approach allows compiling of data from 13 genotypes and 4 nitrogen levels within one graph. data points representing the 1n / 1/2n relationship are marked with the cultivar’s name while corresponding data points for decreasing nitrogen availability are found in the perpendicular lines. the distribution and scattering degree of data points illustrate the impact of genotype and nitrogen availability on the modification of the evaluated characters. in addition, circles around data points indicate the nitrogen level causing a significant decline in the parameter in question for a given genotype. in this way, differences between cultivars in terms of response to nitrogen deficiency stress can be estimated by the position of specific threshold values. in vitro nue 261 a dry matter per absorbed n [mg] b protein yield per absorbed n [mg] fig. 3: distribution of traits related to nitrogen utilization of 13 potato genotypes grown under three levels of nitrogen deficiency stress compared to full nitrogen supply. (af = ‘afra’; ag = ‘agria’; di = ‘ditta’; fi = ‘filea’; la = ‘lambada’; ma = ‘marlen’; mi = ‘milva’; ni = ‘nicola’; pi = ‘pirol’; si = ‘simone’; sk = ‘skala’; so = ‘solara’; to = ‘topas’) for each evaluated trait mean values determined under nitrogen deficiency stress (1/2 n, 1/4 n, 1/8 n) were plotted against values obtained at full nitrogen availability. this approach allows compiling of data from 13 genotypes and 4 nitrogen levels within one graph. data points representing the 1n / 1/2n relationship are marked with the cultivar’s name while corresponding data points for decreasing nitrogen availability are found in the perpendicular lines. the distribution and scattering degree of data points illustrate the impact of genotype and nitrogen availability on the modification of the evaluated characters. nitrogen uptake and utilization fig. 2d 2h present data of nitrogen withdrawal and nitrogen utilization associated traits. generally all genotypes had used up the available nitrogen almost completely (93-97 %) after three weeks of culture in case of reduced n supply (1/2 n, 1/4 n, and 1/8 n). the situation is reflected by the short vertical distances between values determined for each genotype at the three levels of nitrogen reduction (fig. 2d). however, significant differences between genotypes became evident in case of full nitrogen supply. cultivars ‘agria’ and ‘topas’ are clearly separated from the rest of the genotypes due to a less efficient nitrogen uptake capacity. in contrast to the other genotypes, these cultivars have withdrawn only 75 and 71 % of the available nitrogen after three weeks of culture. highly significant differences were also determined between n-level, cultivars and genotype x nitrogen level interactions in case of crude protein related traits (fig. 2e 2h). determination of crude protein contents in dry matter of shoots and roots explicitly reflects the nitrogen availability during the growth period (fig. 2e and 2f). the decline between the two lowest n-levels (1/4 and 1/8 n), however, is less pronounced as obviously nitrogen supply in these media is immediately used up. generally, crude protein concentrations in shoots are higher as compared to roots. however, the corresponding root:shoot ratios are increasing with decreasing nitrogen availability (fig. 2g). in this context, cultivar ‘solara’ holds a special position with comparable levels of crude protein in roots and shoots at the 1/4 n and 1/8 n-level, thus reaching an extraordinary high ratio. as a rule, crude protein concentrations were low in cultivars with high biomass production and vice versa. with respect to crude protein yields of the whole plantlets three clusters of genotypes can be distinguished (fig. 2h). these groups correspond exactly to the nitrogen uptake capacity of cultivars at full nitrogen availability. ‘topas’ and ‘agria’ can be distinguished by comparatively low, ‘solara’, ‘milva’, ‘lambada’, and ‘afra’ by high protein yields. the remaining seven cultivars constitute a distinct intermediate group. plots of dry matter produced per absorbed mg of nitrogen (fig. 3a) as well as of protein yields per mg absorbed nitrogen (fig. 3b) result in defined clusters according to the n-level, illustrating that the efficiency of converting nitrogen into biomass is steadily increasing with decreasing nitrogen availability. cultivars ‘milva’, ‘afra’ and ‘lambada’ are outstanding in terms of dry matter production at all n-levels, while biomass production of the normally vigorously growing cv. ‘solara’ slightly declines upon reduction of nitrogen to 1/8 of the original concentration. ‘filea’ performs generally poor, whereas ‘topas’ and ‘agria’ in spite of their low n-uptake capacity are not the poorest in dry matter production (fig. 3a) and protein yield per absorbed unit of nitrogen (fig. 3b) under nitrogen deficiency stress. in order to compare the degree of nitrogen stress induced disturbances between cultivars, the stress susceptibility indices (ssi) were computed for dry matter and protein yield per available and per absorbed amount of nitrogen, respectively. data is shown in tab. 2 exemplarily for the 1/2 n-level. the ssi determines for each individual genotype the rate of change in a given trait between two environments relative to the mean change for all genotypes. the index is a measure for the extent of stability of a specific parameter under stress conditions, whereby high values indicate low tolerance. the high nitrogen uptake capacity of ‘lambada’, ‘solara’, ‘afra’ and ‘milva’ is reflected by low and the poor nitrogen acquisition of ‘topas’ and ‘agria’ by high ssi-values in case of productivity related to nitrogen availability. however, the latter two cultivars move to a higher ranking position when dry matter and crude protein yield are related to the amount of nitrogen actually absorbed from the culture medium. this indicates a comparatively more efficient metabolization under n limiting conditions. discussion nutrient use efficiency comprises two multifactorial components, i.e. utilization efficiency and uptake efficiency. while the first is attributed to the efficiency with which the nutrients are utilized to produce yield (agronomic efficiency) or biomass (physiological efficiency), the latter describes the effectiveness in absorbing nutrients from the surrounding environment (e.g. sattelmacher et al., 1994; ladha et al., 1998; namai et al., 2009). the experimental system used in this investigation allows the immediate measurement of nitrogen retrieval from the nutrient solution and the determination of the utilization efficiency in the sense of dry matter production and crude protein yield per mg of absorbed nitrogen within three weeks. furthermore it enables the comprehensive monitoring of root 262 a. schum, g. jansen tab. 2: ranking of cultivars according to stress susceptibility index (ssi) for dry matter (dm) and crude protein yield (py) of plantlets per available and per absorbed nitrogen, respectively, produced on media with ½ n supply within three weeks. the ssi (fischer and maurer, 1978) determines for each individual genotype the rate of change in a given trait between two environments relative to the mean change for all genotypes, whereby high values indicate low tolerance. dm per available n dm per absorbed n py per available n py per absorbed n lambada 0.52 marlen 0,71 lambada 0.77 solara 0.64 solara 0.56 topas 0.89 marlen 0.80 lambada 0.77 afra 0.66 pirol 0.91 solara 0.90 nicola 0.80 milva 0.73 lambada 0.93 milva 0.91 afra 0.91 nicola 0.86 simone 0.95 simone 0.97 marlen 0.93 skala 0.95 filea 0.95 skala 0.98 filea 1.00 marlen 1.06 agria 0.97 pirol 1.01 ditta 1.05 filea 1.06 solara 1.03 nicola 1.05 simone 1.08 simone 1.15 skala 1.05 afra 1.08 agria 1.10 ditta 1.20 ditta 1.06 filea 1.11 skala 1.11 pirol 1.28 nicola 1.07 ditta 1.14 topas 1.15 agria 1.67 milva 1.16 topas 1.24 pirol 1.17 topas 1.80 afra 1.28 agria 1.28 milva 1.33 development under standard and nitrogen deficiency conditions. phenotyping of thirteen potato cultivars grown in vitro under four nitrogen levels revealed differential responses to n deficiency stress. all considered traits related to biomass production as well as to nitrogen uptake and utilization were significantly affected both by nitrogen availability and by the genotype. with a few exceptions, significant genotype x nitrogen level interactions were also detected. vegetative development, as measured by chlorophyll content, plant height, fresh and dry matter of shoots, roots and plantlets declined in all genotypes with reduction of the nitrogen supply. however, critical threshold levels associated with the impairment of specific traits varied with the cultivar. notable differences between genotypes under all nitrogen regimes were particularly observed in root fresh matter production. while in some genotypes root fresh matter continually decreased with decreasing nitrogen availability, other cultivars intensified root development on the reduction of nitrogen supply to 1/2 of the original ms medium and were even able to sustain root proliferation at the 1/4 level. changes in the root architecture, an increased root:shoot ratio and an increase of the root surface are well known responses of plants to nitrogen deficiency stress. drew et al. (1973) demonstrated that locally restricted application of nitrate to primary roots of barley promoted a site-specific formation of lateral roots. in a further publication drew and saker (1975) provided evidence for an associated increase in absorption and transport of 15n-labelled nitrate which appeared to compensate for the deficient supply at the remaining root system. the findings of these classical experiments have been supported manifold by corresponding investigations using diverse experimental systems and different plant species. in arabidopsis such locally restricted nitrate dependent stimulation of lateral root growth has been attributed to the action of an anr1 gene encoding specific transcription factors. it was predominantly expressed in roots and was induced within 30 minutes of nitrate application (zhang and forde 1998). in addition, there is evidence for a superordinated regulation system which restricts or allows proliferation of lateral roots dependent on the overall nitrate status of the whole plant (zhang et al., 1999). the situation has been discussed in detail by lea and azevedo (2006). in our investigation plantlets were supplied with a liquid culture medium providing a homogeneous distribution and allowing a continuous uptake of nitrogen. therefore, stimulation of root development under nitrogen deficiency stress observed in several genotypes has to be interpreted as a response to the nitrogen status of the whole plant. such capacity of modifying root architecture in nitrogen limiting situations might be construed in terms of optimizing the nitrogen uptake efficiency. this view is supported by findings of sattelmacher et al. (1990) who recorded a significantly larger root system for the more n efficient genotype in a comparison of two potato cultivars differing in their nitrogen acquisition capacity. however, at this point it is not clear if such shift to preferential root proliferation upon limited nitrogen availability determined in our experimental system is a unique feature of specific cultivars. as evaluation of plantlets was generally performed three weeks after initiation of the experiments, the status of the plantlets at a particular moment in time was recorded irrespective of individual developmental rates. it is noteworthy that those cultivars which produced the highest amount of fresh matter are the ones which displayed a continuous reduction of root proliferation with decreasing nitrogen supply at the end of the three weeks’ culture period (tab. 1). therefore, further experiments are undertaken in order to elucidate the state of root development at earlier time points. analysis of the residual nitrogen in the growth media after three weeks of culture resulted in the identification of three differentially responding groups of genotypes. ‘topas’ and ‘agria’ are characterized by a low nitrogen uptake capacity. in contrast, ‘afra’, ‘lambada’, ‘milva’ and ‘solara’ are distinguished from an intermediate group by their high nitrogen uptake capacity. under the specific experimental conditions nitrogen utilization efficiency is reflected by the dry matter production and by the protein yield per absorbed n. generally, an explicit increase in nitrogen utilization efficiency with reduction of nitrogen availability was observed. these findings are in accordance with results of field and pot trials as well as with observations in natural habitats (e.g. gauer et al., 1992; ma et al., 2004; yuan et al., 2006). dry matter production per mg absorbed nitrogen at the 1n level reflects the individual n-uptake capacity of genotypes (fig. 3a, x-axis). with reduction of the nitrogen supply in the media the order of ranking is modified. while ‘afra’, ‘lambada’, and ‘milva’ are still outstanding in vitro nue 263 in terms of nitrogen utilization efficiency, the performance of ‘solara’ slightly drops. at the same time ‘filea’ turns out to be the least efficient cultivar while ‘topas’ and ‘agria’ improve their relative performance under nitrogen deficiency stress (fig. 3a, white squares). in conclusion, both genotypes characterized by a low n-uptake capacity are not the poorest with respect to n-utilization efficiency. the relative stability of the biomass production of the two cultivars under nitrogen deficiency stress is also reflected by evaluation of the ssis at the 1/2 n supply level. this finding is in accordance with the different physiological processes and pathways underlying nitrogen uptake and subsequent metabolization (e.g. good et al., 2004). the crude protein content of shoots and roots decreased with the reduction of nitrogen in the culture media and, furthermore, was significantly determined by genotype and genotype x nitrogen level interactions. the crude protein concentration in both organs was negatively correlated with the amount of biomass produced, indicating a higher utilization capacity of strong growing genotypes with immediate metabolization of acquired nitrogen for proliferation of plantlets and production of plant material (schum and jansen 2012). as a rule, the content of crude protein was lower in roots as compared to shoots. the comparatively steeper decline in shoots under nitrogen limitation resulted in increasing root:shoot protein ratios with decreasing nitrogen availability. the term crude protein comprises the total amount of organic nitrogen containing metabolites including true proteins, amines, amides, amino acids and peptides as well as nucleic acids. determination of crude protein concentrations captures the nitrogen which was metabolized into organic compounds by the plantlets. however, it does not provide any information regarding the exact composition of the fraction. nonetheless, the shift of the root:shoot ratios towards relatively higher values in roots may be a hint for reduced nitrogen transport rates into shoots under deficiency stress conditions. especially cultivar ‘solara’ stands out with almost equal levels of crude protein in roots and shoots at the 1/4 n and 1/8 n-level. the identification of tolerances against abiotic stress factors under natural conditions in a crop like potato is difficult and requires a substantial input of time and labor. in addition, results of field trials are extremely affected by variable weather conditions between years. in this investigation a highly controlled environment was chosen for evaluation of potentially genotype specific responses to different degrees of nitrogen deficiency stress. in vitro cultures allow standardizing growth conditions and facilitate the manipulation of single factors. the potential of in vitro cultures for evaluation of potato germplasm with respect to salt, heat and drought tolerance has been discussed earlier (anithakumari et al., 2011; arvin and donelly, 2008; donelly et al., 2003; khrais et al., 1998 and references cited therein). on the other hand, important agronomic traits as tuber yields cannot be assessed. furthermore, physiological conditions that differ from the natural situation have to be taken into account. in particular, plantlets are incubated under mixotrophic conditions and transpiration rates are reduced due to high humidity within the culture vessels. therefore, such an approach is intended for initial screening of germplasm and identification of divergently responding genotypes for subsequent detailed investigations in pot and field experiments. currently, attempts are made to correlate results of in vitro experiments to data obtained from potatoes grown under more natural conditions in a rain out shelter. application of the in vitro test system resulted in identification of differentially responding genotypes in terms of nitrogen acquisition and metabolization capacity under the given experimental conditions. furthermore, particular cultivars with increased root development under nitrogen deficiency stress were determined. the characterization of individual components contributing to nitrogen use efficiency in potato may help to identify promising genotypes in breeding of n efficient potato cultivars. in addition, the experimental system is suited for investigation of early physiological responses to nitrogen deficiency stress under highly controlled conditions at the proteomic level. conclusion an in vitro test system was developed which facilitates analyzing specific responses to nitrogen deficiency stress in potato within three weeks. the experimental system allows the immediate measurement of nitrogen retrieval from the nutrient solution and the determination of the utilization efficiency in the sense of biomass production and crude protein yield per mg of absorbed nitrogen. genotype dependent differences were detected with respect to various morphological and physiological traits under nitrogen limiting conditions. furthermore, the method enabled the prompt identification of genotypes responding with increased root development under nitrogen deficiency stress. this approach is helpful for initial screening of germplasm and identification of divergently responding genotypes as well as for investigation of early physiological responses to nitrogen deficiency. acknowledgements the authors thank anke schuldt, antje höxtermann-gottlob, marlies prechel, margrit jugert and jana unger for excellent technical assistance. furthermore, we greatly appreciate virus testing of in vitro material by the research group satellite collections north of the ipk department genebank (leibniz institute of plant genetics and crop plant research). references anithakumari, a.m., dolstra, o., vosman, b., visser, r.g.f., van der linden, c.g., 2011: in vitro screening and qtl analysis for drought tolerance in diploid potato. euphytica 181, 357-369. arvin, m.j., donelly, d.j., 2008: screening potato cultivars and wild species to abiotic stresses using an electrolyte leakage bioassay. j. agric. sci. technol. 10, 33-42. demerchant, g.p., maclean, a.a., milburn, p.h., richards, j.e., 1990: intensive potato production effects on nitrate-n concentrations of rural new brunswick well water. can. agr. eng. 32, 189-196. donelly, d.j., coleman, w.k., coleman, s.e., 2003: potato microtuber production and performance: a review. amer. j. potato res. 80, 103115. drew, m.c., saker, l.r., ashley, t.w., 1973: nutrient supply and the growth of the seminal root system in barley ‒ i. the effect of nitrate concentration on the growth of axes and laterals. j. exp. bot. 24, 11891202. drew, m.c., saker, l.r., 1975: nutrient supply and the growth of the seminal root system in barley ‒ ii. localized, compensatory increases in lateral root growth and rates of nitrate 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hirel, b., tétu, t., lea, p.j., dubois, f., 2011: improving nitrogen use efficiency in crops for sustainable agriculture. sustainability 3, 14521485. hopkins, b.g., rosen, c.j., shiffler, a.k., taysom, t.w., 2008: enhanced efficiency fertilizers for improved nutrient management: potato (solanum tuberosum). online crop management doi:10.1094/cm-2008-0317-01rv. ikram, s., bedu, m., daniel-vedele, f., chaillou, s., chardon, f., 2012: natural variation of arabidopsis response to nitrogen availability. j. exp. bot. 63, 91-105. khrais, t., leclerc, y., donnelly, d.j., 1998: relative salinity tolerance of potato cultivars assessed by in vitro screening. amer. j. potato res. 75, 207-210. ladha, j.k., kirk, g.j.d., bennett, j., peng, s., reddy, c.k., reddy, p.m., singh, u., 1998: opportunities for increased nitrogen-use efficiency from improved lowland rice germplasm. field crop res. 56, 41-71. lea, p.j., azevedo, r.a., 2006: nitrogen use efficiency. 1. uptake of nitrogen from the soil. ann. 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(eds.), genes for plant abiotic stress, 167-182. wiley-blackwell, ames, usa. ma, l.b., yan, w., dwyer, l.m., fregeau-rrid, j., voldeng, h.d., dion, y., nass, h., 2004: graphic analysis of genotype, environment, nitrogen fertilizer and their interactions on spring wheat yield. agron. j. 96, 169180. murashige, t., skoog, f., 1962: a revised medium for rapid growth and bio assays with tobacco tissue cultures. physiol. plant 15, 473-497. namai, s., toriyama, k., fukuta, y., 2009: genetic variations in dry matter production and physiological nitrogen use efficiency in rice (oryza sativa l.) varieties. breeding sci. 59, 269-276. normungsausschuss wasserwesen, 1983: din 38406-5: bestimmung des ammonium-stickstoffs; deutsches institut für normung e.v., deutsche einheitsverfahren zur wasser-, abwasserund schlammuntersuchung -anionen (gruppe d), berlin. raun, w.r., johnson, g.v., 1999: improving nitrogen use efficiency for cereal production. agron. j. 91, 357-363. sattelmacher, b., klotz, f., marschner, h., 1990: influence of the nitrogen level on root growth and morphology of two potato varieties differing in nitrogen acquisition. plant soil 123, 131-137. sattelmacher, b., horst, w.j., becker, h.c., 1994: factors that contribute to genetic variation for nutrient efficiency of crop plants. z. pflanzenernähr. bodenk. 157, 215-224. schum, a., jansen, g., 2012: physiological response to nitrogen deficiency stress of in vitro grown potato genotypes. acta hortic. 961, 465-472. sharifi, m., zebarth, b.j., coleman, w., 2007: screening for nitrogen-use efficiency in potato with a recirculating hydroponic system. commun. soil sci. plant anal. 38, 359-370. vdlufa (german association of agricultural experiment and research stations) 2002: book of methods i: a6.1.1.1 vos, j., 2009: nitrogen responses and nitrogen management in potato. potato res 52, 305-317. yuan, z.y., li, l.h., han, x.g., chen, s.p., wang, z.w., chen, q.s., bai, w.m., 2006: nitrogen response efficiency increased monotonically with decreasing soil resource availability: a case study from a semiarid grassland in northern china. oecologia 148, 564-572. zhang, h., forde, b.g., 1998: an arabidopsis mads box gene that controls nutrient-induced changes in root architecture. science 279, 407-409. zhang, h., jennings, a., barlow, p.w., forde, b.g., 1999: dual pathways for regulation of root branching by nitrate. proc. natl. acad. sci. usa 96, 6529-6534. address of the authors: annegret schum and gisela jansen, institute for resistance research and stress tolerance, julius kühn-institut, ot groß lüsewitz, 18190 sanitz, germany. e-mail: annegret.schum@jki.bund.de; gisela.jansen@jki.bund.de journal of applied botany and food quality 87, 147 156 (2014), doi:10.5073/jabfq.2014.087.022 1department of botany and microbiology, king saud university, riyadh, saudi arabia 2university college of agriculture, university of sargodha, sargodha-pakistan 3department of botany, university of agriculture, faisalabad-pakistan water deficit-induced regulation of growth, gas exchange, chlorophyll fluorescence, inorganic nutrient accumulation and antioxidative defense mechanism in mungbean [vigna radiata (l.)wilczek] a. afzal 3, i. gulzar 3, m. shahbaz 3, m. ashraf 1, 2* (received april 16, 2014) * corresponding author summary the study was conducted to appraise the influence of water deficit conditions on growth, yield, gas exchange characteristics, and antioxidative defense system in two mungbean [vigna radiata (l.) wilczek] lines, 97001 and 97012. the plants of both lines were grown in equal weight plastic pots for 30 days under normal natural conditions, after which time two drought regimes [control (wellwatering and 60 % field capacity)] were applied. data for various attributes were recorded after 30 days of drought application while at maturity, yield attributes were recorded. water deficit conditions caused a considerable reduction in growth attributes, net co2 assimilation rate, stomatal conductance, electron transport ratio, total phenolics, leaf ca2+ and yield attributes. imposition of water deficit conditions significantly increased leaf tocopherol contents and activity of catalase in both mungbean lines. both lines showed a considerable variation in growth attributes, the line 97001 being better in performance compared with 97012 under water deficit conditions. introduction of various abiotic stresses, drought stress holds an important position on the globe (farahani et al., 2009; kusvuran et al., 2011). the main cause of this stress is the high rate of evapo-transpiration, particularly in arid and semi-arid regions having low precipitation rate (jaleel et al., 2007; nakayama et al., 2007; shahbaz et al., 2011a, b). drought is believed to influence plants from metabolic compartments to an individual organism (choluj et al., 2004). it can also alter the morpho-physiological and biochemical attributes of plants (ali et al., 2008; rahbarian et al., 2011; shahbaz et al., 2011a). although drought stress has a marked inhibitory effect on plants at every growth and development phase, its imposition at early growth stages causes considerable reduction in both cell expansion and its elongation (shao et al., 2008; ashraf et al., 2013). drought stress mainly hampers the rate of carbon assimilation which results in decreased growth and yield of most crops (jabeen et al., 2008; mafakheri et al., 2010) as well as it significantly reduces chlorophyll fluorescence attributes (piper et al., 2007). under drought stress, a majority of plants accumulate active osmolytes in their cells, the process referred to as osmotic adjustment (afkari et al., 2009). these osmolytes support the plant’s defense mechanism to nullify the adverse effects of drought (shao et al., 2005) as these osmolytes play a pivotal role in the maintenance of water absorption and cell turgor potential under stress conditions (chaves et al., 2009; mahmood et al., 2009). osmotic adjustment is believed to occur in all plant parts like stem, leaf, root and fruit (saglam et al., 2010). under drought stress, a variety of reactive oxygen species (ros) are produced, which result in oxidative damage of cell membranes (maheswari and dubey, 2009). in order to scavenge these activated oxygen species, plants produce a number of enzymatic antioxidants including: superoxide dismutase (sod), glutathione reductase (gr), catalase (cat) and peroxidase (pod), as well as non-enzymatic antioxidants such as ascorbic acid (asa), glutathione, α-tocopherol, flavonoids and carotenoids (sgherri et al., 2000). it is reported in different studies that the oxidative cellular damage in drought stressed plants is related to the ability of their antioxidant systems (liu et al., 2009; basu et al., 2010). mungbean [vigna radiata (l.) wilzeck] is cultivated in many regions of the world because of its considerable nutritional value particularly for people encountering malnutrition (allahmoradi et al., 2011). despite this, being a leguminous plant it possesses considerable n fixing ability (nadeem et al., 2004). although mungbean crop requires less management practices (sadeghipour, 2009), sufficient availability of water is attributed for better crop productivity and vice versa (kramer and boyer, 1997). being a favorite crop of arid and semi-arid regions it must have to face drought conditions of varying intensity and time period (ladrera et al., 2007). mungbean production is adversely affected by the increase in drought prone area world-over (postel, 2000). the use of drought resistant genotypes is one of the viable means of attaining better yield under water deficit conditions (ennajeh et al., 2010). in order to develop drought resistant varieties/lines it is necessary to have the complete knowledge of plant behavior to drought stress (jaleel et al., 2009). the variability of morpho-physiological attributes under water deficit conditions helps to detect resistant genotypes/lines for better productivity under stress environment (nam et al., 2001). in view of considerable inhibitory effects of drought stress on mungbean crop the premier objective of the present investigation was to examine how far this stress regulates some key physio-biochemical attributes involved in growth and development of mungbean plant. materials and methods to assess the effect of water deficit conditions on mungbean, a pot experiment was conducted in old botanical garden, department of botany, university of agriculture, faisalabad-pakistan. two lines of mungbean (lines 97001 and 97012) were obtained from the ayub agricultural research institute, faisalabad. fifteen seeds of each line of mungbean were sown in each of the plastic pots (24.5 cm diameter and 28 cm deep) filled with equal weight dry soil. two drought stress treatments [control (well-watered i.e. normal watering as per requirement of the crop) and 60 % field capacity (fc)] were applied on 30-day old plants. the experiment was laid out in a completely randomized design with four replications of each experimental unit. the weight of each plastic pots with filled soil and the water contents present at the time of sowing were already known, therefore moisture contents present in soil of pots equal to control and 60 % fc were calculated for drought treatments. when water contents of each pot were at field capacity, then 15 seed of each line of pulse crop were sown. after 15 days of germination thinning of plants were done and 5 plants per pot were maintained. plants were irrigated normally according to their requirements till 30 days before 148 a. afzal, i. gulzar, m. shahbaz, m. ashraf treatment start. after one month of normal growth of plants, drought stress levels were maintained. after 30 days of drought treatment, two plants were harvested from each of replicate, washed with distilled water and recorded data for shoot and root fresh weights and shoot and root lengths. the samples were oven-dried at 65 °c up to their constant weight and then dry weights recorded. in addition, data for following attributes were also recorded: gas exchange characteristics a portable infra-red gas analyzer (irga) (acd lca-4 analytical development, hoddesdon, uk) was used to determine the net photosynthetic rate (a), transpiration rate (e), stomatal conductance (g s), water use efficiency (a/e), and internal co2 concentration (ci) on fully expanded leaves. following adjustments/values of the instrument were recorded/maintained during its operation: 403.3 mmol m-2 s-1 for molar flow of air, 99.9 kpa atmospheric pressure, 6.0 to 8.9 mbar water vapor pressure, 1711 μmol m-2 s-1 par, 28.4 to 27.9 °c leaf temperature and 352 μmol mol-1 ambient co2 concentration. water relation attributes a fully expanded second leaf was excised at dawn and its mid rib was used in scholander type pressure chamber (arimad-2-japan) to obtain water potential. the same leaf that used for water potential stored in freezer at -20 oc to use it for osmotic potential. after one week the frozen leaf was thawed and the sap was extracted by pressing it with glass rod. the extracted sap was used to determine the osmotic potential by using the osmometer (wescor 5520). turgor potential was calculated as the difference between water potential and osmotic potential. chlorophyll fluorescence before measurements of chlorophyll fluorescence, the leaf samples were kept in dark for 30 min by using light-exclusion clips to the surface of leaves. chlorophyll fluorescence was determined using an os5p modulator fluorometer (adc bioscientific ltd, great amwellherts, uk) according to strasser et al. (1995). determination of mineral ions the dried ground leaf or root material (100 mg) was digested with 2 ml h2so4 following the method of wolf (1982). the volume of the extract was brought up to 50 ml with distilled water, filtered and used determining mineral elements. leaf and root potassium (k+) and calcium (ca2+) contents were determined using a flame photometer (jenway, pfp-7, uk). nitrogen was determined following the kjeldahl method as described by bremner et al. (1965) while phosphorus was determined spectrophotometrically following method of jackson (1962). extraction of antioxidant enzymes antioxidant enzymes were extracted from fresh leaf samples (0.5 g each sample) in 10 ml of phosphate buffer (50 mm with ph 7.8) at 4 ºc. the homogenate was then centrifuged at 12000 × g at 4 ºc for 20 min and it was centrifuged again at 15000 × g for 10 min. the supernatant was used for determining the activities of antioxidant enzymes. superoxide dismutase (sod) the protocol described by giannopolitis and ries (1977) was used for the determination of sod activity. it was determined as the enzyme ability to inhibit photochemical reduction of nitrobluetetrazolium (nbt). the 3 ml reaction mixture consisted of 50 mm phosphate buffer of 7.8 ph, distilled water, methionine 13 mm, 50 μm nbt, 50 μl enzyme extract and 1.3 μm riboflavin. the reaction solutions were then kept under light (15 w fluorescent lamps) for 15 min at 78 μmol m-2 s-1. the absorbance of the reaction mixture was read at 560 nm with a uv-visible spectrophotometer (u2020 irmeco). one unit activity of sod was defined as the amount of enzyme required to cause 50 % inhibition of the rate of nbt photoreduction as compared to the sample that lacked the plant enzyme extract. activities of catalase (cat) and peroxidase (pod) the method described by chance and maehly (1955) was used to appraise the activities of cat and pod on protein amount basis. the reaction solution for cat contained phosphate buffer and h2o2 of 50 and 5.9 mm, respectively. addition of 0.1 ml enzyme extract to the reaction mixture initiated the reaction. after every 20 s the changes in the absorbance of the reaction mixture were observed at 240 nm. the reaction mixture for pod consisted of phosphate buffer, guaiacol, and h2o2 with molar values as 50, 20 and 40 mm, respectively, and 0.1 ml of the enzyme extract. at 470 nm, the absorbance was taken after every 20 s. the enzyme activity was assessed on protein basis, while one unit of cat considered equivalent to 0.01 units per min change in absorbance and one unit of pod defined as the 0.01 units per min change in absorbance. determination of non-enzymatic antioxidants total phenolics julkenen-titto (1985) proposed a method which was used to determine total phenolics. leaf fresh material (50 mg) was homogenized in 80 % acetone. the homogenized material was centrifuged at 10,000 × g for 10 min, removed the pellet and the supernatant was used for the determination of phenolics. then 100 μl of the supernatant were mixed with 1 ml of folin-ciocalteau,s reagent. in addition, 2.0 ml distilled water and 5 ml of 20 % na2co3 were also added. the mixture was vortexed and absorbance read at 750 nm using a uv-visible spectrophotometer (irmeco u2020). leaf ascorbic acid contents the amount of ascorbic acid in the mungbean leaves was determined following mukherjee and choudhri (1983). fresh leaves (0.25 g) were ground in 10 ml of 6 % tca. the mixture was centrifuged for 10 min at 4 ºc at 1000 × g. an aliquot of 2 ml of 2 % dinitrophenyl hydrazine solution was added to 4 ml of supernatant. one drop of thiourea (10 % thiourea prepared in 70 % ethanol) was added to the mixture, and boiled the mixture for 20 min in a water bath. the mixture was placed in ice to reduce the temperature to about 25 ºc, then added 5 ml of 80 % sulphuric acid (v/v) at 0 ºc and the absorbance read at 530 nm. the ascorbic acid content was quantified against a standard curve which was prepared by known concentrations of ascorbic acid. estimation of leaf tocopherol content the method of baker et al. (1980) was used for the determination of leaf alpha-tocopherol concentration. a mixture of 20 ml of petroleum ether and ethanol (2:1:6, v/v) was used to grind the fresh leaves (1.0 g each sample). the mixture was centrifuged at 10,000 × g for 20 min. an aliquot of 200 μl of 2 % 2, 2dipyridyl (prepared in ethanol) was added to 1 ml of supernatant, mixed the mixture and placed it in the dark for 5 min. the absorbance was read at 520 nm using a spectrophotometer. influence of water deficit conditions on mungbean 149 yield attributes data for yield attributes like number of pods per plant, number of seed per plant, seed yield per plant and 100-seed weight were collected at the maturity of the mungbean crop. statistical analysis a two-way analysis of variance (anova) of data for all attributes was calculated using the costat computer program. results imposition of water deficit conditions (normal watering and 60 % field capacity) significantly reduced shoot and root fresh and dry weights of two mungbean lines (97001and 97012) (tab. 1; fig. 1). both lines showed a significant variation in root dry weight as line 97001 performed better than 97012, while in shoot fresh and dry weights and root fresh weight both lines showed a uniform behavior under both well-watered and drought stress conditions. imposition of water deficit conditions markedly (p ≤ 0.001) reduced shoot length of the two mungbean lines i.e. 97001 and 97012 (tab. 1; fig. 1). of both lines, 97001 showed better performance in shoot length as compared to 97012. in root length, the influence of drought stress was not prominent for both mungbean lines and the lines did not show any variation in this attribute under water deficit conditions (tab. 1; fig. 1). leaf water potential increased while osmotic potential decreased prominently due to imposition of drought stress regimes (tab. 1; fig. 1). imposition of water deficit conditions caused an increase in leaf turgor pressure (fig. 1). however, both lines showed a uniform behavior with respect to water relation attributes under well-watered and drought stress conditions. various drought stress regimes proved to be non-significant for photochemical quenching (qp), co-efficient of non-photochemical quenching (qn), non-photochemical quenching (npq) and efficiency of photosystem-ii (fv/fm) in the two mungbean lines (97001 and 97012). in addition, the lines did not show any difference with respect to all these chlorophyll fluorescence attributes under various tab. 1: mean squares from analyses of variance of data for morphological, physiological, biochemical and yield attributes of mungbean [vigna radiata (l.) wilczek] when 30 day old plants were subjected to drought stress sov df shoot f. wt. shoot d. wt. root f. wt. root d. wt. shoot length root length water osmotic potential potential drought (d) 1 117.0** 2.772* 0.25* 0.024** 907.5*** 5.881ns 0.035* 0.453* lines (l) 1 7.659ns 0.336ns 0.086ns 0.009* 247.3* 1.051ns 0.002ns 0.016ns d × l 1 14.35ns 0.235ns 0.242* 0.004ns 83.26ns 1.626ns 0.0002ns 0.022ns error 12 11.68 0.466 0.045 0.002 28.83 4.027 0.004 0.050 sov df turgor a e gs a/e ci ci/ca fv/fm potential drought (d) 1 0.695** 29.00*** 0.360* 0.013* 7.777ns 17835.6** 0.144ns 0.006ns lines (l) 1 0.038ns 1.357ns 0.123ns 0.002ns 1.127ns 715.6ns 0.006ns 0.003ns d × l 1 0.019ns 0.601ns 0.240ns 0.003ns 13.20ns 1410.0ns 0.012ns 0.149ns error 12 0.053 0.366 0.065 0.002 4.965 1559.1 0.013 0.039 sov df etr npq qn qp total leaf leaf ascorbic cat phenolics tocopherols acid drought (d) 1 69.72* 0.000ns 0.001ns 0.234ns 3.597** 0.039* 0.0005ns 11.22* lines (l) 1 0.562ns 0.006ns 0.000ns 0.034ns 0.163ns 0.002ns 0.0001ns 25.49** d × l 1 1.440ns 0.000ns 0.000ns 0.730* 0.007ns 0.024ns 0.00002ns 8.390ns error 12 8.642 0.017 0.006 0.091 0.295 0.005 0.0002 2.151 sov df pod sod total soluble mda leaf k+ root k+ leaf ca2+ root ca2+ proteins drought (d) 1 0.899ns 0.291ns 4.155* 0.987ns 7.562ns 2.641ns 15.02** 1.891* lines (l) 1 13.28ns 0.238ns 0.049ns 3.822ns 9.00ns 9.766ns 5.641ns 0.141ns d × l 1 429.4** 0.025ns 0.131ns 4.077ns 0.25ns 0.016ns 5.641ns 0.141ns error 12 29.22 0.125 0.929 3.43 4.302 3.620 1.411 0.2243 sov df leaf p root p leaf n root n number of number of 100-seed seed yield pods/plant seeds/plant weight per plant drought (d) 1 2.806* 0.226ns 0.111ns 0.005ns 37.82** 5681.4** 39.74ns 12.73** lines (l) 1 0.64ns 0.331ns 0.162ns 0.011ns 0.01ns 606.4ns 34.47ns 0.022ns d × l 1 0.81ns 0.226ns 0.111ns 0.099ns 6.76ns 1113.9ns 133.7ns 0.228ns error 12 0.546 0.374 0.183 0.046 3.262 413.6 69.31 0.972 *, **, *** = significant at 0.05, 0.01, and 0.001 (probability) levels, respectively ns = non-significant 150 a. afzal, i. gulzar, m. shahbaz, m. ashraf & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & fig. 1: growth, water relations and chlorophyll fluorescence attributes of mungbean [vigna radiata (l.) wilczek] when 30 day old plants were subjected to drought stress (mean ± s.e.; n = 4). drought stress regimes (tab. 1; fig. 1 and 2). water stress caused a slight decrease (p ≤ 0.05) in electron transport ratio (etr) of both mungbean lines. the behavior of the lines was uniform under both well-watered and water deficit conditions. water deficit conditions caused a significant decrease in net co2 assimilation rate, transpiration rate, stomatal conductance and substomatal co2 concentration of the two mungbean lines (tab. 1; fig. 2). however, water use efficiency and ci/ca ratio were not altered by water deficit conditions (tab. 1; fig. 2). both lines showed a uniform behavior with respect to all the above-mentioned gas exchange characteristics. drought stress caused a slight increase (p ≤ 0.05) in leaf tocopherol contents in the two mungbean lines while it did not affect leaf ascorbic acid (tab. 1; fig. 2). total phenolic concentration decreased significantly (p ≤ 0.01) due to drought stress. both mungbean lines showed a uniform behavior in all the earlier mentioned attributes under well watered and water deficit conditions. imposition of water deficit conditions showed a non-significant effect on leaf mda content and the variation between the two mungbean lines with respect to this attribute was also non-significant (tab. 1; fig. 3). activities of superoxide dismutase and peroxidase, and amount of total soluble proteins did not change due to water deficit conditions (tab. 1; fig. 3). both mungbean lines did not show any variation under both well watered and water deficit conditions. the activity of catalase markedly (p ≤ 0.05) increased under water deficit conditions. the lines also varied prominently as line 97001 showed more catalase activity than that of mungbean line 97012. water deficit conditions did not alter leaf k+, n and root k+, p and n contents in two mungbean lines, while it caused a significant decrease in leaf ca2+ and increase in leaf p and root ca2+ in both mungbean lines under well watered and drought stress conditions (tab. 1; fig. 3). both mungbean lines showed uniform response to water deficit conditions due to root and leaf mineral elements and they did not show prominent difference in various ion contents. influence of water deficit conditions on mungbean 151 & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & fig. 2: gas exchange characteristics and antioxidants of mungbean [vigna radiata (l.) wilczek] when 30 day old plants were subjected to drought stress (mean ± s.e.; n = 4). number of pods per plant, number of seeds per plant and seed yield per plant of two mungbean lines decreased significantly (p ≤ 0.01) due to imposition of drought stress (tab. 1; fig. 4). water deficit conditions did not alter 100-seed weight prominently. both mungbean lines did not show prominent difference with respect to yield attributes under various drought stress regimes. discussion crop growth and yield is adversely affected by drought stresss (ashraf, 2010), which is ascribed to drought-induced impairment in a number of metabolic processes involved in regulation of growth and development (chaves et al., 2009). drought-induced growth reduction has been reported extensively in many crops like mungbean (zare et al., 2012), chicory (asghari et al., 2009), barley (fateh et al., 2012), wheat (shahbaz et al., 2011b) etc. in the present study, change in growth was appraised in terms of change in plant height, which decreased markedly under drought stress conditions. these results are in agreement with some previous studies on mungbean (uddin et al., 2013), chickpea (shamsi, 2010), and ocimum basilicum (alishah et al., 2006). such reduction in plant height under water deficit conditions was ascribed to reduced cell division, expansion and elongation (hussain et al., 2008). the effect of drought stress on root growth is the matter of controversy in view of a number of earlier published reports. in the present study, the root length showed a significant decrease under drought stress which does not conform to some earlier published reports on mungbean (ranawake et al., 2011) as well as on catharanthus roseus (jaleel et al., 2008). however, our results are in accordance with the findings of terzi and kadioglu (2006) for ctenanthe setosa in which a significant decrease in root length was observed. water deficit conditions alter water relation parameters in most 152 a. afzal, i. gulzar, m. shahbaz, m. ashraf & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & fig. 3: activities of antioxidants and mineral nutrients of mungbean [vigna radiata (l.) wilczek] when 30 day old plants were subjected to drought stress (mean ± s.e.; n = 4). plants. for example, imposition of water deficit stress reduced the water potential and osmotic potential of plants of melon (kusvuran, 2012), wild jujube (maraghni et al., 2011), and common bean (guler et al., 2011; zalvet et al., 2005). but in the present study, leaf water potential and osmotic potential did not change significantly by imposition of drought stress while leaf turgor potential increased significantly in mungbean plants due to drought stress. drought stress caused a marked reduction in net co2 assimilation rate (a) which is in agreement with many previous studies on different crops, e.g. wheat (kamran et al., 2009; shahbaz et al., 2011b), sunflower (vanaja et al., 2011), kidney bean (miyashita et al., 2005), grapevine (zosfi et al., 2008), and chickpea (rahbarian et al., 2011). stress-induced reduction in photosynthetic capacity is believed to be due to a number of factors including reduced leaf area, damage to photosynthetic machinery, initiation of leaf senescence before maturity, lipid peroxidation in chloroplast and changes in pigment and protein composition (anjum et al., 2011). reduction in a might also be due to reduction in rubisco carboxylation activity and regeneration of rubp (lawlor and cornic, 2002). furthermore, low leaf water contents also leads to impaired metabolic system leading to less production of photo-assimilates (lawlor and cornic, 2002) under water deficit conditions. in the present study, drought stress slightly reduced the rate of transpiration (e) and stomatal conductance (gs), but did not affect water use efficiency (a/e) in mungbean plants. these results with respect to e and gs are in agreement with many previous studies on different crops e.g. barley (fateh et al., 2012), forest plants (keenan et al., 2010), chickpea (mafakheri et al., 2010), and rice (halder and burrage, 2004) while contradictory with respect to a/e. drought stress also caused a significant reduction in internal co2 concentration (ci) and ci/ca ratio in mungbean plants as has been earlier observed in soybean (anjum et al., 2011), forest plants (keenan et al., 2010) and wheat influence of water deficit conditions on mungbean 153 (bogale et al., 2011), but in contrast, these results do not agree with those for chickpea (mafakheri et al., 2010) and rice (halder and burrage, 2004). furthermore, no significant relations were found between water deficit conditions and chlorophyll fluorescence attributes. these results are analogous to what has been reported in chickpea (khamssi et al., 2010) and olive (petridis et al., 2012). this might be due to low close relation of leaf water potential with chlorophyll fluorescence transients (jefferies, 1992) and leaf water potential might have not affected these attributes. decrease in a may also cause by the photodamage of psii, but in the present study, drought stress did not affect the efficiency of psii showing that light did not cause damage to psii in mungbean under drought stress conditions (baker and horton, 1987). decrease in a is also related to stomatal conductance as both are reduced under drought stress conditions, but these stomatal effects did not affect the efficiency of psii (fv/fm) (flexas and medrano, 2002). some coastal plants also maintained their fv/fm value even under drought stress conditions (demattos et al., 1997). in our study, drought stress did not cause any severe damage to psii, and increased photorespiration might also be a factor causing protection to psii from drought-induced adverse effects (flexas and medrano, 2002). the activity of enzymatic anti-oxidants, most specifically of superoxide dismutase (sod), did not alter due to drought stress in mungbean plants which is analogous to the findings of terzi and kadioglu (2006) in ctenanthe setosa and varga et al. (2012) in wheat. peroxidase (pod) and catalase (cat) in mungbean plants also remained unchanged under water deficit conditions which were in accordance with the findings of gamble and burke (1984) who reported a non-significant change in the activity of cat in wheat under water deficit conditions. one of the possible reasons is that cat has low affinity for ros particularly for h2o2 as compared to ascorbate peroxidase (mittler, 2002). furthermore, mda and ascorbic acid (asa) contents in mungbean plants also did not change under drought regimes. in our study, damage to membrane in the form of lipid peroxidation as shown by mda contents is not severe (non-significant due to drought stress) and this might be a main reason for the non-significant effect of drought stress on the activities of antioxidant enzymes. in addition, activities of antioxidant enzymes depend on a number of factors like plant age, drought stress duration, tolerance level of a particular species (decarvalho, 2008). drawing correlation between induction of antioxidants and degree of drought tolerance among species of a same genus or even cultivars of a same species has been found not so easy (loggini et al., 1999). these results are not in agreement with those reported for withania somnifera (jaleel et al., 2009) and tomato fruit (ghorbanli et al., 2012) under drought stress conditions but tocopherol was increased by the application of water stress, similar to that reported in canna cultivars (zhang et al., 2013). increase in tocopherol under water deficit conditions might be due to active expression of genes which are responsible for the synthesis of tocopherols (zhu, 2002; zong et al., 2009). terzi and kadioglu (2006) observed an increase in mda content during early phase of drought while a decrease in later phase in ctenanthe setosa. leaf phenolics in the present study decreased by the imposition of stress which is contrary to that found in maize (hura et al., 2008) and olive (petridis et al., 2012), while in contrast, an increase in phenolic contents was reported in horsegram (bhardwaj and yadav, 2012). water deficit conditions are believed to generally reduce nutrient uptake in plants (baligar et al., 2001) and plants with small or no reduction in nutrient uptake are considered drought resistant. however, great variation has been observed in nutrient uptake in even genotypes of a same species under drought stress conditions (shahbaz et al., 2011a, b). in the present study, drought stress had a nonsignificant effect on leaf and root n and k+ except p which decreased in mungbean plant roots under drought stress. similar results have been observed in wheat (shahbaz et al., 2011b), while in contrast, higher k+ accumulation has been reported in drought stressed common bean (zadehbagheri et al., 2012) and okra (kusvuran et al., 2011) plants. a non-significant influence of drought stress was also observed on shoot and root k+ for cenchrus ciliarus and cyanodon && & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & fig. 4: leaf and root n and yield attributes of mungbean [vigna radiata (l.) wilczek] when 30 day old plants were subjected to drought stress (mean ± s.e.; n = 4). 154 a. afzal, i. gulzar, m. shahbaz, m. ashraf dactylon (akram et al., 2008). in conclusion, water deficit conditions significantly reduced all growth attributes, net co2 assimilation rate, internal co2 concentration, ci /ca ratio, root p, total soluble proteins, phenolics and seed yield, but they markedly increased turgor potential, and tocopherol contents. acknowledgements the last author gratefully acknowledges the funding from the pakistan academy of sciences (pas)(grant no. 5-9/pas/7536). the results presented in this paper are a part of m. sc. studies of miss aneela afzal and miss iqra gulzar. references afkari, b.a., qasimov, n., yarnia, m., 2009: effects of drought stress and potassium on some of the physiological and morphological traits of sunflower (helianthus annuus l.) cultivars. j. food agric. environ. 7, 448-451. akram, n.a., shahbaz, m., ashraf, m., 2008: nutrient acquisition in differentially adapted populations of cynodon dactylon (l.) pers. and cenchrus ciliaris l. under drought stress. pak. j. bot. 40, 1433-1440. ali, q., ashraf, m., shahbaz, m., humera, h., 2008: ameliorating effect of foliar applied proline on nutrient uptake in water stressed maize (zea mays l.) plants. pak. j. bot. 40, 211-219. alishah, h.m., haidri, r., hassni, a., dizaji, a.a., 2006: effect of water stress on some morphological and biochemical characteristics of purple basal (ocimum basilium). j. biol. sci. 6, 763-767. allahmoradi, p., mokhtar, g., shayesteh, t., shahrbanu, t., 2011: physiological aspects of mungbean (vigna radiata l.) in response to drought stress. int. conf. food eng. biotechnol. 9, 272-275 anjum, s., xie, x., wang, l., saleem, m., man, c., lei, w., 2011: morphological, physiological and biochemical responses of plants to drought stress. j. afr. agric. res. 6, 2026-2032. asghari, m.t., daneshian, j., farhani, h.a., 2009: effect of drought stress and planting density on quantity and morphological characteristic of chicory (cichorium intybus l.). asian j. agric. sci. 1, 12-14. ashraf, m., 2010: inducing drought tolerance in plants: recent advances. biotechnol. adv. 28, 169-183. ashraf, m., shahbaz, m., ali, q., 2013: drought-induced modulation in growth and mineral nutrients of canola (brassica napus l.). pak. j. bot. 45, 93-98. backer, h., frank, o., deangelis, b., feingold, s., 1980: plasma tocopherol in man at various times after ingesting free or acetylated tocopherol. nutr. rep. int. 21, 531-536. baker, n.r., horton, p., 1987: chlorophyll fluorescence quenching during photoinhibition. photoinhibition 9, 145-168. baligar, v.c., fageria, n.k., he, z.l., 2001: nutrient use efficiency in plants. commun. soil sci. plant anal. 32, 921-950. basu, s., roychoudhury, a., saha, p.p., sengupta, d.n., 2010: differential antioxidative responses of indica rice cultivars to drought stress. plant growth regul. 60, 51-59. bhardwai, j., yadav, s.k., 2012: comparative study on biochemical parameters and antioxidant enzymes in a drought tolerant and a sensitive variety of horsegram (macrotyloma uniflorum) under drought stress. am. j. plant physiol. 71, 17-29. bogale, a., tesfaya, k., geleto, t., 2011: morphological and physiological attributes associated to drought tolerance of ethiopian durum wheat genotypes under water deficit condition. j. biodiv. environ. sci. 1, 2236. bremner, j.m., 1965: total nitrogen and inorganic forms of nitrogen. in: black, c.a. 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peuplement aquatiques, paris, france 5department of organic chemistry, faculty of chemistry, university of mazandaran, babolsar, iran antioxidant and antihemolytic activities of methanol extract of hyssopus angustifolius seyed fazel nabavi 1, 2, seyed mohammad nabavi 1, 2*, claire hellio 3, 4, heshmatollah alinezhad 5, mahboobeh zare 5, razieh azimi 5, robabeh baharfar 5 (received august 9, 2012) * corresponding author summary this study was designed to evaluate antioxidant and antihemolytic activities of hyssopus angustifolius fl ower, stem and leaf methanol extracts by employing various in vitro assays. the leaf extract showed the best activity in dpph (63.2 ± 2.3 µg ml-1) and h2o2 (55.6 ± 2.6 µg ml-1) models compared to the other extracts. however, fl ower extract exhibited the highest fe2+ chelating activity (131.4 ± 4.4 µg ml-1). the extracts exhibited good antioxidant activity in linoleic acid peroxidation and reducing power assays, but were not comparable to vitamin c. the stem (23.58 ± 0.7 µg ml-1) and leaf (26.21 ± 1 µg ml-1) extracts showed highest level of antihemolytic activity than the fl ower extract. introduction there are increasing evidences that the consumption of polyphenolic compounds from natural sources may lower the risk of serious oxidative injuries such as atherosclerosis, infl ammatory processes, cancer and cardiovascular diseases as a result of their antioxidant activity (surh, 2002). chelation therapy is one of the most known and reported therapeutic usages of antioxidants (grazul and budzisz, 2009). chelation therapy reduces iron-related complications and improves survival time in patients with various diseases such as thalassemia major, cancer, hiv and wilson’s disease (grazul and budzisz, 2009; hebbel et al., 1990). numerous studies have demonstrated that chelators, such as desferrioxamine, have anti-proliferative effects against different type of cancers (richardson, 2002). moreover, different synthetic chelators usage may have different side effects and therefore it remains an urgent need to identify new natural chelators with reduced side effects (porter, 1997). previous studies demonstrate the direct role of nitric oxide radical in pathogenesis of different diseases, such as cancer, infl ammation, burn, and etc. (sreejayan and rao, 1997). the antioxidant may have the property to quench no formation and prevent illnesses induced by excessive no. due to the safety and limitations of synthetic antioxidants, naturally originated antioxidants provide an interesting alternative to minimize the oxidative damage caused by no and other oxidant agents (ghasemzadeh et al., 2010). hyssopus angustifolius is one of the most famous medicinal plants from the lamiaceae family and is widely cultivated in several european countries such as russia, spain, france and italy. it is used in tea blends for its antitussive and antispasmodic properties, and, to relieve catarrh (omidbaigi, 2000). it appears that there is yet no scientifi c report about antioxidant and antihemolytic activities of methanol extracts of different parts of h. angustifolius. of h. angustifolius. of thus, this study was carried out to determine antioxidant and antihemolytic activities of methanol extracts of h. angustifolius leaf, stem and fl ower, in order to better understand the medicinal uses of this plant. materials and methods chemicals trichloroacetic acid (tca), folin ciocalteau, 1, 1-diphenyl-2picryl hydrazyl (dpph), hydrochloric acid, linoleic acid, ferrozine, butylated hydroxyanisole (bha), quercetin, ascorbic acid, hydrogen peroxide, gallic acid, sodium nitroprusside and methanol were purchased from sigma-aldrich chemical company, usa. sodium carbonate, potassium acetate, aluminium chloride (alcl3), potassium ferricyanide, sulfanilamide, n-(1-naphthyl) ethylenediamine dihydrochloride, edta and ferric chloride (fecl3) were purchased from merck compagny, germany. other chemicals were purchased at analytical grade or purer. plant material aerial parts of hyssopus angustifolius m. bieb. has been collected from veresk area (central elburz mountains latitude: 35° 54’ n, longitude: 52° 59’ e, altitude: 1900 above sea level,), iran through spring 2010. the plant was authenticated at the herbarium of department of biology in the university of mazandaran (voucher specimen no 975). extraction procedures plant powder (100 g) was placed in a soxhlet extractor and extracted with methanol (3 liter) for 8 h. the solvent was recovered by distillation in vacuo, and the residue, stored in the desiccator, was used for subsequent experiments. determination of total phenolic content briefl y, 0.5 ml of sample (1.6 mg ml-1) was incubated with 2.5 ml of folin-ciocalteau reagent (0.2 n) for 5 min and then 2.0 ml aqueous solution of sodium carbonate (75 mg ml-1) was added. the absorbance of reactions mixture was measured at 760 nm after 2 h of incubation at room temperature. total phenol content was calculated using gallic acid standard curve (akillioglu and karakaya, 2010). determination of fl avonoid content briefl y, extract (0.5 ml of a solution at 1.6 mg ml-1) was incubated with methanol (1.5 ml), aluminum chloride (0.1 ml, 10%), potassium acetate (0.1 ml, 1 m) and distilled water (2.8 ml) at room temperature for 30 min. then, absorbance of sample was recorded at 415 nm by spectrophotometer (akillioglu and karakaya, 2010). quercetin was used for calibration curve preparation. antioxidant activity of hyssopus angustifolius 199 1, 1-diphenyl-2-picryl hydrazyl (dpph) radical scavenging equal volumes of sample (25-400 µg ml-1) were added to methanol solution of dpph (100 µm). absorbance of reaction mixture was recorded at 517 nm after 15 min incubating at room temperature. the experiment was performed triplicate. vitamin c, bha and quercetin were used as standard antioxidants (villaño et al., 2007). reducing power for determination of the reducing power ability of extracts, 2.5 ml of sample (25-400 µg ml-1) was mixed with 2.5 ml of phosphate buffer (0.2 m, ph 6.6) and 2.5 ml of potassium ferricyanide (1%), and, were incubated for 20 min (50°c). then, 2.5 ml of trichloroacetic acid (10%) was added to the sample in order to stop the reaction. reaction mixture was the centrifuged (10 min, 1000 g). the upper layer of reaction mixture (2.5 ml) was mixed with 2.5 ml of distilled water and 0.5 ml of fecl3 (0.1%). finally, absorbance of the sample was recorded at 700 nm (ferreira et al., 2007). metal chelating iron ions chelating by the extracts was examined according to the method of el and karakaya (2004). sample (25-400 µg ml-1, 1 ml) was mixed with 0.05 ml of fecl2 (2 mm) and 0.2 ml of ferrozine (5 mm). after 10 min incubation at room temperature, absorbance of the sample was recorded at 562 nm. nitric oxide scavenging extract (25-400 µg ml-1) was mixed with sodium nitroprusside (10 mm) and incubated for 150 min at 25°c. then, 0.5 ml of griess reagent (1% sulfanilamide, 2% phosphoric acid and 0.1% n-(1-naphthyl) ethylenediamine dihydrochloride) was added to the reaction mixture. finally, absorbance of the sample was recorded at 546 nm (kumaran and karunakaran, 2006). hydrogen peroxide scavenging extract (2 ml, 0.1-1 mg ml-1) was mixed with 0.6 ml of 40 mm of hydrogen peroxide solution in phosphate buffer (ph 7.4). the absorbance of reaction mixture was recorded at 230 nm against a blank (gülçin et al., 2010). hemoglobin-induced linoleic acid model reaction mixture (2 ml) containing extract (25-400 µg ml-1), phosphate buffer (40 mmol l-1, ph 6.5), hemoglobin suspension (0.0016%) and linoleic acid emulsion (1 mmol l-1). reaction mixture was incubated for 45 min (37°c). then, 2.5 ml of ethanolic solution of hydrochloric acid (0.6%) was added to the sample for stopping lipid peroxidation process. the level of peroxidation was evaluated via thiocyanate method by recording the absorbance at 480 nm after adding 100 µl of fecl2 (0.02 mol/l) and 50 µl of ammonium thiocyanate (0.3 g ml-1) (bae and suh, 2007). preparation of rat erythrocytes male wistar rats (180 -220 g) were purchased from pasteur institute of iran, amol research center. the animals were anesthetized via intraperitoneally administration of ketamine (60 mg/kg) and xylazine (5 mg/kg). blood samples were collected through cardiac puncture. erythrocytes of the blood samples were isolated and further stored at -60°c. protective role of extracts against h2protective role of extracts against h2protective role of extracts against h o2 induced hemolysis protective role of the extracts against h2o2 induced hemolysis was evaluated according to the method of ajila and prasada rao (2008). different concentrations of extracts (0.5 ml, 10-100 µg·ml-1) were mixed with 2ml of erythrocyte suspension (4%) and the volume of reaction mixture was made up to 5 ml with phosphate buffered saline. after 5 min incubation at 25°c, 0.5 ml of h2o2 solution was added to the reaction mixtures. after 240 min incubation at 25°c, reaction mixtures were centrifuged (2500 g, 10 min). absorbance of the reaction mixture was recorded at 540 nm. statistical analysis of the data experimental results are expressed as means ± sd. all measurements were replicated three times. the data were analyzed by an analysis of variance (p < 0.05) and the means separated by duncan’s multiple range tests. the ic50 values were calculated from linear regression analysis. results and discussion the total phenolic content of the methanol extract of fl owers, leaves and stems were respectively of 445 ± 22.1, 663.9 ± 33.1 and 349.6 ± 18.7 mg gallic acid equivalent g-1 of extract. also, total fl avonoid contents of methanol extract of fl owers, leaves and stems were in the order of 122 ± 6.1, 131.7 ± 6.5 and 88.4 ± 4.3 mg quercetin equivalent g-1 of extract powder, respectively. leaf extract revealed better dpph radical scavenging activity (ic50 = 63.20 ± 2.32 µg ml-1) than others extracts tested, while in fe2+ chelating activity, fl ower extract displayed the highest activity (ic50 = 131.40 ± 4.43 µg ml-1). the inhibition percentage of nitric oxide radical increased concomitantly with the concentration. in this assay, the leaf extract showed the highest nitric oxide-scavenging activity tab. 1: antioxidant activities of fl owers, stems and leaves extracts of hyssopus angustifolius samples dpph free radical nitric oxide h2o2 scavenging fe2+ chelating antihemolytic scavenging scavenging activity ability activity fl ower 149.6 ± 5.5 196 ± 6.4 177.9 ± 7.8 131.4 ± 4.4 65.3 ± 2.5 leaf 63.2 ± 2.3 194.1 ± 4.1 55.6 ± 2.6 154.6 ± 7.1 26.2 ± 1 stem 197.3 ± 6 259.7 ± 7.9 123.3 ± 3.9 211 ± 9.7 23.6 ± 0.7 vitamin c 5.05 ± 0.1 21.4 ± 1.1 235 ± 9 edta 18 ± 1.5 quercetin 5.3 ± 0.2 20 ± 0.1 52 ± 2.6 there is no signifi cant difference between quercetin and leaf extract in h2o2 scavenging models (p > 0.05) and between leaf and stem extract in antihemolytic activity (p > 0.05). ic50 (µg/ml) 200 s.f. nabavi, s.m. nabavi, c. hellio, h. alinezhad, m. zare, r. azimi, r. baharfar antioxidant activity of hyssopus angustifolius (ic50 = 194.13 ± 4.12 µg ml-1 vs. quercetin 20 ± 0.01 µg ml-1). quercetin showed good no radical scavenging activity but it has carcinogenic activity (dunnick and hailey 1992), therefore search for new natural no quenching substances has been increased recently. the extracts were capable of scavenging hydrogen peroxide in a concentration dependent manner (tab. 1). vitamin c shows better activity than others. in reducing power assay, increasing absorbance of reaction mixture at 700 nm indicates an increase in reductive ability. fig. 1 shows the dose dependent reducing power of the extracts, however no signifi cant differences (p> 0.05) were observed. tested extracts showed good activity in hemoglobin-induced linoleic acid system (fig. 2) with no signifi cant differences among them (p > 0.05). in addition, extracts did not show any side effects on erythrocytes. the best antihemolytic activity was observed while assessing the leaf and stem extracts (tab. 1). any substances which can donates hydrogen or electron, can change dpph color from violet to yellow can be considered as a dpph radical scavengers and, therefore as antioxidant (nabavi et al., 2012b). previously, chapova et al. (2010) reported potent dpph radical scavenging activity for another species of hyssopus genus (hyssopus offi cinalis l.) that was higher than the results in the present study, where h. angustifolius showed a moderate dpph scavenging ability. this difference of activity may be associated with phenolic and fl avonoid contents (nabavi et al., 2012b). another assay that is related to the electron donating ability of this plant is the reducing power assay. in this assay, phenols or other electron donor compounds in the sample can reduce fe3+ to fe2+. the amount of fe2+ complex monitored through reading absorbance of sample at 700 nm (ferreira et al., 2007). fig. 1 showed the dose-response curves for the reducing power of extracts. they demonstrated very good activity, which was comparable to the vitamin c (p < 0.01). in iron ion chelating model, extracts and edta compete with ferrozine in capturing iron ion, and thus inhibited ferrozine-iron complex formation. abnormally excessive no radical production has a crucial role in the pathogenesis of numerous diseases such as sepsis and renal failure and therefore elimination of nitric oxide have therapeutic effects (shah et al., 2004). also, no scavengers inhibited chain reactions initiated by no radicals that are harmful for human health. hydrogen peroxide is not very reactive but it causes cytotoxicity through increasing of hydroxyl radicals in the cell. therefore, hydrogen peroxide removal can help to protect human health (chaudhuri et al., 2007). membrane lipids are rich in unsaturated fatty acids that are most susceptible to oxidative processes, mainly the linolenic and arachidonic acids, which are targets for lipid peroxidation (nabavi et al., 2012c). erythrocytes are known as main targets oxidative stress due to the presenting of membrane polyunsaturated fatty acids and also existence of the oxygen transport joined to hemoglobin molecules. antihemolytic activity of fl avonoids has been previously reported and the good activity of extracts may be the result of high fl avonoid content (chaudhuri et al., 2007). conclusion in conclusion, hyssopus angustifolius extracts can be used as a powerful herbal antioxidant. this activity may be associated with the presence of polyphenolic compounds. these results may explain some of the medicinal uses of hyssopus angustifolius since the excessive production of reactive oxygen species have important role in the pathogenesis of several diseases. author disclosure statement no competing fi nancial interests exist. references ajila, c.m., prasada rao, u.j.s., 2008: protection against hydrogen peroxide induced oxidative damage in rat erythrocytes by mangifera indica l. peel extract. food chem. toxicol. 46, 303-309. akillioglu, h.g., karakaya, s., 2010: changes in total phenols, total fl avonoids, and antioxidant activities of common beans and pinto beans after soaking, cooking, and in vitro digestion process. food sci. biotechnol. 19, 633-639. bae, s.h., suh, 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the corresponding author: seyed mohammad nabavi, applied biotechnology research center, baqiyatallah university of medical sciences, tehran, iran, p.o. box 19395-5487; e-mail: nabavi208@gmail.com art03 š journal of applied botany and food quality 81, 121 125 (2007) applied botany, ecology and plant physiology, agronomy department, biotechnical faculty, university of ljubljana, slovenia evaluation of selected nutritional factors in aposeris foetida (l.) less. during the harvesting period helena šircelj and franc batic (received march 6, 2007) summary aposeris foetida (l.) less. is a plant native to central european forests. in slovenia it is known as an early spring wild food. considering the total absence of studies of the leaves of aposeris regarding its use as food, the present study was made to investigate its nutritional value concerning important vitamins and antioxidants. foliar contents of ascorbic acid, α-tocopherol, γ-tocopherol, α-carotene, β-carotene, neoxanthin, violaxanthin, antheraxanthin, zeaxanthin, lutein, and chlorophyll in young edible plants during the harvesting period are reported. comparison of aposeris with supermarket lettuce analysed in our study and with numerous cultivated and wild edible plants from other studies showed that aposeris is one of the best sources of ascorbic acid, tocopherol and carotenoids. the results of our study justify its use in the early spring wild food cuisine and may stimulate consumption of this nutritious plant. introduction aposeris foetida (l.) less., (asteraceae) is a perennial herbaceous plant native to central european forests. it reminds a little of the dandelion (taraxacum officinale weber). it has about 10 cm long, lanceshaped, deeply toothed, usually glabrous leaves, always growing in a basal rosette. it flowers between may and august. the composite flowers grow individually on 10 cm to 20 cm high flower stalks. the bright yellow flower heads are 2.5 cm to 4 cm broad. the whole plant (especially the roots and to smaller extent also the mature leaves) contains white milky sap. it smells like cooked potatoes. this is why aposeris is called forest dandelion, stinking dandelion or odorous pigsalad. forest dandelion is known as an early spring wild food in slovenia. the young, soft leaves are at their best when they have just emerged and they are collected before flowering. the leaves usually emerge as early as the beginning of april. this makes the forest dandelion one of the first wild-growing plants available for the table. usually the leaves are eaten raw in salads or cooked in different dishes. after flowering the leaves become harder and the quantity of white milky sap increases. to our knowledge no studies on the nutritional value of aposeris have been made. since this plant is quite popular in the wild food cuisine in slovenia and abundant in the alpine and prealpine central european region, we conducted a study on the nutritional value of aposeris. the aim was to evaluate the contents of selected bioactive compounds in young leaves of aposeris during the harvesting period which in slovenia is in april and may. we analysed ascorbic acid, tocopherols, carotenoids and chlorophylls in leaves collected weekly from forest dandelion rosettes growing at several locations in central slovenia. the most important criterion for the selection of nutritional factors for analysis was the actual purpose of collecting wild-growing plants for culinary use; that is an intake of vitamins and antioxidants from fresh natural sources. all the selected factors represent important vitamins and antioxidants which are indispensable for normal functioning of the human body. ascorbic acid, carotenes and tocopherols are the sources of vitamins c, a and e respectively. xanthophylls lutein and zeaxanthin are crucial factors for human vision. neoxanthin and chlorophyll were found to reduce the risk of certain types of cancer (daswood, 1997; dillard and german, 2000). the present study reports foliar contents of ascorbic acid, α-tocopherol, γ-tocopherol, α-carotene, β-carotene, neoxanthin, violaxanthin, antheraxanthin, zeaxanthin, lutein, and chlorophyll in young edible aposeris foetida plants at the stage normally consumed in slovenia, and compares the nutritional value of aposeris with other cultivated and wild plants. material and methods plant material leaves of forest dandelion were collected in the way consumers do when this plant is picked for home cuisine. entire leaf-rosettes were separated with a knife from the roots and put in a plastic bag (volume 2l) until the bag was full (at least 6 plants). one bag filled with leaves represented one sample for biochemical analyses. two samples were collected from each of four selected locations in central slovenia. leaves were collected weekly (7 weeks) from the beginning of april (when the first leaves emerged) until the end of may (one week after the onset of flowering) in the morning (6:00 to 8:00 solar time). they were transported in a portable refrigerator to the laboratory (in less than 1.5 hours) where leaves were frozen in liquid nitrogen, lyophilized, ground to a fine powder and stored at -20oc in humidityproof plastic containers until analysis. before and after lyophilization samples were weighed in order to recalculate data obtained from biochemical analyses from mg per dry weight (dwt) to mg per 100g fresh weight (fwt). the foliar moisture content was 88.1 ± 2.1 g/100g fwt. in the middle of the forest dandelion harvesting period we collected also three samples (one sample comprised one plant) of soft lettuce (lactuca sativa ‘majniška kraljica’) from a supermarket. the lettuce samples were treated exactly like forest dandelion samples. analysis of chloroplast pigments chloroplast pigments (neoxanthin, violaxanthin, antheraxanthin, zeaxanthin, lutein, α-carotene, β-carotene, chlorophyll a, chlorophyll b) were determined using the methods described by pfeifhofer (1989). pigments were extracted from the dry leaf powder with ice-cold acetone. all extraction procedures were performed in dim light. acetone extracts were subjected to an hplc gradient analysis (spherisorb s5 ods-2 250 x 4.6 mm column with an s5 ods-2 50 x 4.6 mm precolumn), using the following solvents: solvent a: acetonitrile/methanol/water (100/10/5, v/v/v); solvent b: acetone/ethylacetate (2/1, v/v), at a flow rate of 1 mlmin-1, linear gradient from 10% solvent b to 70% solvent b in 18 min was applied, run time 30 min, photometric detection at 440 nm. analysis of tocopherols concentrations of tocopherols (α-tocopherol, γ-tocopherol, δtocopherol) were measured following the method reported by tausz š122 helena šircelj and franc batic et al. (2003). tocopherols were extracted from the dry leaf powder with ice-cold acetone. the acetone extracts of lyophilized leaf powder were subjected to isocratic hplc analysis (spherisorb s5 ods-2 250 x 4.6 mm column with a s5 ods-250 x 4.6 mm precolumn) using methanol as solvent. tocopherols were detected directly by fluorometry (excitation 295, emission 325). analysis of ascorbic acid the concentration of ascorbic acid was measured as reported by tausz et al. (2003). ascorbic acid was extracted from the lyophilized leaf powder with 1.5 % (w/v) metaphosphoric acid containing 1mm edta. extracts were subjected to isocratic hplc analysis (lichrosorb rp-8 250 x 4.6 mm column with a lichrosorb rp-8 50 x 4.6 mm precolumn) using methanol/water (1/3, v/v) containing 1 mm hexadecylammoniumbromide and 0.05% (w/v) sodium dihydrogen phosphate monohydrate (ph 3.6) as solvent, at a flow rate of 1 mlmin-1, run time 20 min, photometric detection at 248 nm. statistical analysis data obtained from biochemical analyses were statistically evaluated with the statgraphics programme, version 4.0. differences between sampling dates were evaluated by one-way anova. fischer’s least significant difference (lsd) was used for comparisons between means for the different sampling dates of each factor analysed. significance was accepted at p<0.05. results and discussion in this work the contents of important nutritional factors in the leaves of aposeris during the harvesting period were assessed. the analysis results of ascorbic acid, α-tocopherol, β-carotene, lutein, neoxanthin, violaxanthin, antheraxanthin, zeaxanthin and chlorophyll are presented in table 1. the foliar contents of α-carotene and γ-tocopherol were too low to contribute significantly to the nutritional value of forest dandelion (12 -112 µg/100g of fresh weight (fwt) for α-carotene and 14 70 µg/100g fwt for γ-tocopherol respectively) and are consequently not presented in detail in the table. one of the most important factors influencing the contents of above mentioned nutritional factors in plant leaves is the degree of maturity at harvest (kimura and rodriguez-amaya, 2003; foyer, 1993; munne-bosch and alegre, 2002). for this reason we sampled the leaves weekly during the entire harvesting season. statistically significant differences (p<0.05) between different dates for each factor analysed are denoted by different letters in tab. 1. ascorbic acid: the most important vitamin in vegetables for human nutrition is vitamin c. plant derived ascorbate is the major source of vitamin c in the human diet (wheeler et al., 1998). in green tissues of plants ascorbic acid is the major water soluble antioxidant. the contents of ascorbic acid change seasonally and increase with leaf age; over short time intervals the foliar ascorbate contents remain at a relatively constant level, except under stress which can affect the ascorbate pool significantly (foyer, 1993). the mean concentrations of ascorbic acid measured in aposeris leaves were between 57 and 101 mg/100g fwt (tab. 1). in the first week after leaf emergence the concentration of ascorbic acid was significantly lower compared to the last three weeks of the harvesting period (fig. 1). the highest concentration of ascorbic acid was measured in leaves collected one week before flowering (may-7). lettuce bought in a supermarket during the aposeris sampling period had only 1.1 ± 0.7 mg ascorbic acid per 100g fresh weight. sheela et al. (2004), gupta et al. (2005) and krumbein et al. (2005) reported foliar ascorbic acid contents for 57 leafy vegetables. only brassica oleracea had a higher ascorbic acid content compared to aposeris in our study. guil et al. (1997) analysed 16 wild edible plants and compared to aposeris, found similar vitamin c contents in 11 and higher content in 5 plants species. fig. 1: ascorbic acid (mg/100g fwt) in leaves of aposeris foetida l. during harvesting period. symbols represent mean ± s.d. of eight plant samples. 0 20 40 60 80 100 120 140 160 6 -a p r 9 -a p r 1 2 -a p r 1 5 -a p r 1 8 -a p r 2 1 -a p r 2 4 -a p r 2 7 -a p r 3 0 -a p r 3 -m a y 6 -m a y 9 -m a y 1 2 -m a y 1 5 -m a y 1 8 -m a y 2 1 -m a y 2 4 -m a y sampling date as c o rb ic a c id [m g /1 0 0 g fw t] tab. 1: ascorbic acid, tocopherol and chloroplast pigments (mg/100g fwt) in leaves of aposeris foetida l. during harvesting period. values are means of eight plant samples. statistically significant differences (p < 0.05) between different dates are denoted by different letters. vaz violaxanthin + antheraxanthin + zeaxanthin, e-emergence of first leaves, f-flowering. e-april-10 april-16 april-23 april-30 may-7 f-may-14 may-21 ascorbic acid 57.3 ± 11.2a 78.6 ± 33.1ab 83.0 ± 35.4ab 78.2 ± 32.0ab 101.9 ± 36.2b 98.3 ± 17.1b 100.3 ± 21.7b ααααα-tocopherol 0.41 ± 0.10a 1.87 ± 0.14b 2.22 ± 0.38bc 2.29 ± 0.32bc 2.69 ± 0.33cd 3.09 ± 0.61de 3.17 ± 0.35e βββββ-carotene 7.88 ± 0.81a 9.42 ± 1.46ab 10.29 ± 1.32bc 10.70 ± 2.14bc 11.30 ± 1.32bc 12.28 ± 2.91bc 11.64 ± 1.11c lutein 9.43 ± 0.77a 10.07 ± 0.99ab 10.35 ± 0.97abc 10.36 ± 0.69abc 11.21 ± 1.16bc 11.61 ± 2.45bc 10.82 ± 1.02c violaxanthin 4.77 ± 0.84a 6.06 ± 1.53ab 6.67 ± 1.18b 7.40 ± 1.03bc 8.18 ± 1.07bcd 8.86 ± 1.47cd 7.33 ± 0.76d antheraxanthin 0.385 ± 0.033a 0.501 ± 0.114ab 0.744 ± 0.143ab 0.583 ± 0.093bc 0.577 ± 0.123bc 0.552 ± 0.189bc 0.682 ± 0.170c zeaxanthin 0.047 ± 0.095a 0.245 ± 0.284a 0.317 ± 0.311a 0.070 ± 0.142a 0.136 ± 0.184a 0.055 ± 0.100a 0.459 ± 0.255a vaz 5.20 ± 0.82a 6.81 ± 1.72ab 7.74 ± 1.27bc 8.06 ± 0.92bcd 8.90 ± 1.22bcd 9.47 ± 1.59cd 8.47 ± 0.89d neoxanthin 3.49 ± 0.32a 4.12 ± 0.65ab 4.55 ± 0.28bc 4.27 ± 0.26bc 4.39 ± 0.32bc 4.48 ± 0.90bc 4.86 ± 0.54c chlorophyll 129.4 ± 13.6a 136.6 ± 32.8ab 144.2 ± 16.3abc 146.2 ± 9.4abc 159.2 ± 8.4bc 166.8 ± 24.4c 166.1 ± 16.7c tocopherols: in the human body tocopherols are recognized as the most important lipid soluble antioxidants, together with the tocotrienols known as vitamin e. tocopherols are only synthesized by plants and other oxygenic, photosynthetic organisms (dellapenna, 2005). the evaluation of selected nutritional factors in aposeris foetida (l.) less. 123 contents of tocopherols change diurnally and seasonally and differ between plant organs (munne-bosch and alegre, 2002). the main tocopherol in leaves of aposeris was α-tocopherol which represented approximately 98% of total tocopherols. the remainder was in the form of γ-tocopherol. the contents of α-tocopherol in aposeris leaves were between 0.32 and 3.65 mg/100g fwt (tab. 1). foliar concentrations of α-tocopherol increased significantly during the harvesting period, by approximately 670% from the first to the last sampling date (fig. 2). lettuce bought in a supermarket during the aposeris sampling period had only 0.186 ± 0.042 mg α-tocopherol per 100g fresh weight. in comparison with other studies on leafy vegetables aposeris leaves in the harvesting period contain more α-tocopherol than lettuce (burns et al., 2003; chun et al., 2006), cabbage and chinese cabbage (singh et al., 2007) and the wild leafy vegetable hummayd (rumex vesicarius) (alfawaz, 2006), but spinach leaves had approximately the same content of α-tocopherol (chun et al., 2006). ching and mohamed (2001) analysed 28 tropical leafy edible plants and 16 of them had higher α-tocopherol contents than aposeris from our study. but because foliar tocopherol content depends strongly on leaf age, a better comparison of our tocopherol results with the results from the above mentioned studies on wild plants would be possible if the leaf age or plant developmental stage at the time of plant collection were also reported in the papers. than spinach (burns et al., 2003; mangels et al., 1993). aposeris in our study had β-carotene contents higher than 56, similar to 7 and lower than 3 leafy vegetables from different parts of the world analysed in 4 different studies (gupta et al., 2005; krumbein et al., 2005; raju et al., 2007; bhaskarachary et al. 1995). mercadante and rodriguez-amaya (1990) and bhaskarachary et al. (1995) reported foliar β-carotene for 26 edible wild plants. eleven of these plants had similar and 15 lower β-carotene content than aposeris. we detected 5 xanthophylls in aposeris leaves (tab. 1). lutein was the predominant xanthophyll. the mean concentrations of lutein during the harvesting period were between 9.43 and 11.61 mg/100g fwt. the mean contents of total xanthophyll cycle pigments (vaz; the sum of violaxanthin, antheraxanthin and zeaxanthin) and neoxanthin were between 5.20 and 9.47 mg/100g fwt, and 3.49 and 4.86 mg/100g fwt, respectively. since the leaves were sampled early in the morning the xanthophyll cycle was in the epoxidised state and consequently the major cycle pigment was violaxanthin, antheraxanthin represented 810% and zeaxanthin represented only 0.6-6% of the vaz pool. the contents of xanthophylls were the lowest (significantly) in the first week after leaf emergence (fig. 3). the highest concentrations of lutein and vaz were measured in leaves collected at the beginning of flowering (may-14). the highest concentrations of neoxanthin were measured one week after the beginning of flowering (may-21). lettuce from a supermarket compared to aposeris had significantly lower contents of xanthophylls: 0.32 ± 0.09 mg/100g fwt lutein, 0.12 ± 0.04 mg mg/100g fwt neoxanthin, 0.26 ± 0.09 mg/100g fwt violaxanthin, 0.016 ± 0.006 mg/100g fwt antheraxanthin and no zeaxanthin. in published studies available on carotenoids in edible leafy vegetables data for all individual xanthophylls are rarely presented. in comparison with those few studies on common leafy vegetables in which single xanthophylls were analysed, aposeris had higher individual xanthophyll contents than lettuce, rocket, cress, chicory, collards, cabbage and chinese cabbage and similar contents as kale and spinach (kimura and rodriguez-amaya, 2003; burns et al., 2003; singh et al., 2007; humphries and khachik, 2003). comparison of our study with studies (krumbein et al., 2005; raju et al., 2007; mercadante and rodriguez-amaya, 1990; kidmose et al., 2006; wills and rangga, 1996) performed on 58 mostly less commonly cultivated and wild leafy plants showed that only 3 species had higher contents of one or more individual foliar xanthophyll namely rumex acetosella l., chenopodium album l. and commelina benghalensis l. had higher lutein and violaxanthin than aposeris from our study.fig. 2: α tocopherol (mg/100g fwt) in leaves of aposeris foetida l. during harvesting period. symbols represent mean ± s.d. of eight plant samples. 0 0,5 1 1,5 2 2,5 3 3,5 4 6a p r 9a p r 1 2a p r 1 5a p r 1 8a p r 2 1a p r 2 4a p r 2 7a p r 3 0a p r 3 -m ay 6 -m ay 9 -m ay 1 2 -m ay 1 5 -m ay 1 8 -m ay 2 1 -m ay 2 4 -m ay sampling date α -t o co p h e ro l[ m g /1 0 0 g fw t] carotenoids: carotenoids are the main lipid-soluble antioxidants of plant cells (munne-bosch and alegre, 2000). plant leaves usually contain β-carotene, lutein, violaxanthin and neoxanthin, and small amounts of zeaxanthin, antheraxanthin and α-carotene. foliar carotenoid content depends on plant species, leaf age and environmental conditions (goodwin, 1980). aposeris leaves contained two provitamin a carotenoids, β-carotene and α-carotene, the last representing less than 1% of total carotenes. the mean concentrations of β-carotene measured in aposeris leaves were between 7.88 and 12.28 mg/100g fwt (tab. 1). in the first week after leaf emergence the concentration of β-carotene was significantly lower than on other sampling dates (fig. 3). the highest concentration of β-carotene was measured in leaves collected at the beginning of flowering (may-14). lettuce bought in a supermarket during the aposeris harvesting period had only 0.27 ± 0.116 mg β-carotene per 100g fresh weight. in comparison with other studies on leafy vegetables, aposeris leaves contain significantly more β-carotene than lettuce (kimura and rodriguez-amaya, 2003; burns et al., 2003; mangels et al., 1993), cabbage, chinese cabbage (singh et al., 2007), rocket, cress and chicory (kimura and rodriguez-amaya, 2003) and more fig. 3: lutein, β-carotene, xantophyll cycle pigments (vaz = violaxanthin + antheraxanthin + zeaxanthin) and neoxanthin (mg/100g fwt) in leaves of aposeris foetida l. during harvesting period. symbols represent mean ± s.d. of eight plant samples; ■ = lutein; ● = β-carotene; ▲ = vaz; x = neoxanthin. 0 2 4 6 8 10 12 14 16 6a pr 9a pr 1 2a pr 1 5a pr 1 8a pr 2 1a pr 2 4a pr 2 7a pr 3 0a pr 3 -m a y 6 -m a y 9 -m a y 12 -m a y 15 -m a y 18 -m a y 21 -m a y 24 -m a y sampling date ca ro te n o id [m g /1 0 0g fw t] š124 helena šircelj and franc batic š š chlorophyll: chlorophylls a and b were measured in leaves of aposeris. foliar contents of total chlorophyll are presented in tab. 1. the mean concentrations of total chlorophyll were between 129 and 166 mg/100g fwt. contents of total chlorophyll were higher at the end of the harvesting period compared to earlier sampling dates (fig. 4). lettuce bought in a supermarket during the aposeris harvesting period had only 2.98 ± 1.22 mg total chlorophyll per 100g fresh weight. in comparison with other studies on leafy vegetables aposeris leaves contain significantly more chlorophyll than lettuce (burns et al., 2003) and more chlorophyll than cabbage, chinese cabbage, savoy beet and spinach (krumbein et al., 2005; negi and roy, 2000). negi and roy (2000) reported similar chlorophyll contents for amaranthus tricolor and higher for trigonella foenum graecum compared to aposeris. štajner et al. (2006) reported similar or higher chlorophyll contents compared to aposeris for 10 of 12 analysed cultivated and wild allium species. references alfawaz, m.a., 2006: chemical composition of hummayd (rumex vesicarius) grown in saudi arabia. j. food compos. anal. 19, 552-555. bhaskarachary, k., sankar rao, d.s., deosthale, y.g., reddy, v., 1995: carotene content of some common and less familiar foods of plant origin. food chem. 54, 189-193. burns, j., fraser, p.d., bramley, p.m., 2003: identification and quantification of carotenoids, tocopherols and chlorophylls in commonly consumed fruits and vegetables. phytochemistry 62, 939-947. ching, l.s., mohamed, s., 2001: alpha-tocopherol content in 62 edible tropical plants. j. agric. food chem. 49, 3101-3105. chun, j., lee, j., ye, l., exler, j., eitenmiller, r.r., 2006: tocopherol and tocotrienol contents of raw and processed fruits and vegetable in the united states diet. j. food compos. anal. 19, 196-204. dashwood, r., 1997: chlorophylls as anticarcinogens. int. j. oncology 10, 721-727. dellapenna, d.a., 2005: decade of progress in understanding vitamin e synthesis in plants. j. plant physiol. 16, 729-737. dillard, c.j., german, j.b., 2000: phytocemicals: neutraceuticals and human health. j. sci. food agric. 80, 1744-1756. foyer, c.h., 1993: ascorbic acid. in: antioxidants in higher plants. alscher, r.g., hess, j.l. (eds.), 32-58. crc press, inc.: boca raton, florida. goodwin, t.w., 1980: the biochemistry of the carotenoids. vol. 1: plants. 2nd ed. chapman and hall, london, new york. guil, j.l., rodriguez-garcia, i., torija, e., 1997: nutritional and toxic factors in selected wild edible plants. plant foods hum. nutr. 51, 99-107. gupta, s., lakshimi, a.j., manjunath, m.n., prakash, j., 2005: analysis of nutrient and antinutrient content of underutilized green leafy vegetables. lwt 38, 339-345. humphries, j.m., khachik, f., 2003: distribution of lutein, zeaxanthin, and related geometrical isomers in fruit, vegetables, wheat and pasta products. j. agric. food chem. 51, 1322-1327. kidmose, u., yang, r.-y., thilsted, s.h., christensen, l.p., brandt, k., 2006: content of carotenoids in commonly consumed asian vegetables and stability and extractability during frying. j. food compos. anal. 19, 562-571. kimura, m., rodriguez-amaya, d.b., 2003: carotenoid composition of hydroponic leafy vegetables. j. agric. food chem. 51, 2603-2607. krumbein, a., schonhof, i., schreiner, m., 2005: composition and contents of phytocemicals (glucosinolates, carotenoids and chlorophylls) and ascorbic acid in selected brassica species (b. juncea, b. rapa subsp. nipposinica var. chinoleifera, b. rapa subsp. chinensis and b. rapa subsp. rapa). j. appl. bot. food qual. 79, 168-174. mangels, a.r., holden, j.m., beecher, g.r, forman, m.r., lanza, e., 1993: carotenoid content of fruits and vegetables: an evaluation of analytic data. j. am. diet. assoc. 93, 284-296. mercadante, a.z., rodriguez-amaya, d.b., 1990: carotenoid composition and vitamin a value of some native brazilian leafy vegetables. int. j. food sci. tech. 25, 213-219. munne-bosch, s., alegre, l., 2000: the significance of β-carotene, α-tocopherol and the xanthophyll cycle in droughted melissa officinalis plants. aust. j. plant physiol. 27, 139-146. munne-bosch, s., alegre, l., 2002: the function of tocopherols and tocotrienols in plants. crit. rew. plant sci. 21, 31-57. negi, p.s., roy, s.k., 2000: effect of blanching and drying methods on βcarotene, ascorbic acid and chlorophyll retention of leafy vegetables. lebensm.-wiss. u.-technol. 33, 295-298. pfeifhofer, w., 1989: evidence of chlorophyll b and lack of lutein in neottia nidus-avis plastids. biochem. physiol. pflanzen 184, 66-51. raju, m., varakumar, s., lakshminarayana, r., krishnakantha, t.p., baskaran, v., 2007: carotenoid composition and vitamin a activity of medicinally important green leafy vegetables. food chem. 101, 1598-1605. sheela, k., nath, k.g., vijayalakshmi, d., yankanchi, g.m., patil, r.p., 2004: proximate composition of underutilized green leafy vegetables in southern karnataka. j. hum. ecol. 15, 227-229. conclusion our study showed that aposeris is an excellent source of important vitamins and antioxidants. compared to aposeris at least 20-times larger portion of supermarket lettuce is needed for the same intake of the nutrients analysed in the present study. the lowest foliar contents of vitamins and antioxidants were measured in just emerged aposeris leaves and the highest contents were measured at the beginning of flowering ± one week. because at the time of flowering the leaves became harder and the quantity of white milky sap increased, we can say that aposeris leaves were at their best one week before flowering. comparison of aposeris with cultivated and wild edible plants from different studies showed that aposeris is one of the best sources of ascorbic acid, tocopherol and carotenoids. the high foliar vitamin and antioxidant contents justify its use in the early spring wild food cuisine. the results may stimulate consumption of this nutritious plant, especially in home cuisine and in organic farms with an orientation towards tourism. acknowledgements this study was supported by research programme p4-0085-applied botany, genetics and ecology, from the ministry of science, education and sport. we are grateful to luciano cesnik, barbara janhar, polona cepon and gabrijel leskovec for their help in sample collecting and preparation of extracts. fig. 4: chlorophyll (mg/100g fwt) in leaves of aposeris foetida l. during harvesting period. symbols represent mean ± s.d. of eight plant samples. 90 110 130 150 170 190 210 6 -a p r 9 -a p r 12 -a p r 15 -a p r 18 -a p r 21 -a p r 24 -a p r 27 -a p r 30 -a p r 3m a y 6m a y 9m a y 1 2m a y 1 5m a y 1 8m a y 2 1m a y 2 4m a y sampling date ch lo ro p h yl l[ m g /1 00 g fw t] evaluation of selected nutritional factors in aposeris foetida (l.) less. 125 ú úú š singh, j., upadhyay, a.k., prasad, k., bahadur, a., rai, m., 2007: variability of carotenes, vitamin c, e and phenolics in brassica vegetables. j. food compos. anal. 20, 106-112. štajner, d, milic, n., candanovic-brunet, j., kapor, a., štajner, m., popovic, b. m., 2006: exploring allium species as a source of potential medicinal agents. phytotherap. res. 20, 581-584. tausz, m., wonisch, a., grill, d., morales, d., jimenez, m.s., 2003: measuring antioxidants in tree species in natural environment: from sampling to data evaluation. j. exp. bot. 54, 1505-1510. wheeler, g.l., jones, m.a., smirnoff, n., 1998: the biosynthetic pathway of vitamin c in higher plants. nature 393, 365-369. wills, r.b.h., rangga, a., 1996: determination of carotenoids in chinese vegetables. food chem. 56, 451-455. address of the authors: applied botany, ecology and plant physiology, agronomy department, biotechnical faculty, university of ljubljana, jamnikarjeva 101, si-1000 ljubljana, slovenia corresponding author: helena.sircelj@bf.uni-lj.si << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true 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/pagesize [907.087 680.315] >> setpagedevice journal of applied botany and food quality 87, 104 107 (2014), doi:10.5073/jabfq.2014.087.016 1 department of horticultural science, faculty of agricultural sciences, university of guilan, rasht, iran 2 hazelnut research station, astara agricultural and natural resources research center, guilan, iran evaluation of fatty acid content and nutritional properties of selected native and imported hazelnut (corylus avellana l.) varieties grown in iran fatemeh rezaei 1, davood bakhshi 1*, reza fotouhi ghazvini 1, davood javadi majd 2, mohammadreza pourghayoumi 1 (received march 5, 2013) * corresponding author summary hazelnut (corylus avellana l.) is one of the most important nuts rich in valuable nutrients. in this study, chemical composition of two iranian native varieties namely ‘pashmineh’ and ‘garche’ and four imported varieties, ‘ghafghaze’, ‘zakatala’, ‘ronde dupimont’ and ‘fertile decotard’ were investigated. the main fatty acid in hazelnut varieties were oleic (71.02 %) and linoleic acid (14.45 %). the hazelnut varieties showed oil content in a range from 53.36 % to 63.5 %; protein, 16.03-23.26 %; energy, 653.4-707.65 %; ash, 2.463.5 %; carbohydrate, 13.16-20.14 %; total phenolic content, 6.416.42 mg gae/g; antioxidant capacity, 57.17-72.38 %; oleic acid, 64.17-81.34 %; linoleic acid, 10-21.07 %; linolenic acid, 0-2 %; myristic acid, 0-0.5 %; stearic acid, 0-7.8 %; eicosenoic acid, 01.69 %; palmitic acid, 0.49-9.61 %; palmitoleic acid, 0-1.6 % and behenic acid, 0-0.25 %. introduction hazelnut is a popular nut worldwide. it is mainly distributed along the coasts of the black sea region of turkey, southern europe (italy, spain, portugal and france), and in some areas of the united states (oregon and washington). hazelnut is also grown in new zealand, china, azerbaijan, chile, georgia and iran. turkey is the world’s largest producer of hazelnut, contributing around 70.3 % to the total global production, followed by italy (11.9 %), usa (4.5 %), azerbaijan (4.2 %), georgia (3.8 %) and spain (2.5 %). other countries contribute only 2.8 % to the total global production (alasalvar et al., 2010). hazelnuts, due to their organoleptic characteristics constitute, are one of the most important raw materials for the pastry and chocolate industry. hazelnut also add flavor and texture to bakery, confectionery, cereal, salad, entrée, sauce dairy, and dessert formulation (alasalvar et al., 2003; kaleoğlu et al., 2004; oliveira et al., 2008; ozdemir and akinci, 2004). in addition, hazelnuts play a major role in human nutrition and health, because of their special composition of fat, protein, carbohydrate, vitamins, minerals and nutrients antioxidant. (alasalvar et al., 2009; garcia et al., 1994; köksal et al., 2006; oliveira et al., 2008). at the present, nutritional interest in the fatty acid composition of vegetable oils is increasing because the most important neutral lipid in most vegetable oils included in the human diet influences total fat and cholesterol absorption in the human lumen (erener et al., 2007). monounsaturated fatty acids (mufa) and polyunsaturated fatty acids (pufa) as well as minor lipid components play an important role in human nutrition. also, health diets rich in mufa, such as hazelnut oil and olive oil, decrease blood pressure and total blood cholesterol levels in human (karabulut et al., 2005). every food plant contains numerous types of natural antioxidants with different properties. the actions of antioxidants have been attributed to their ability to scavenge free radicals, thereby reducing oxidative damage of cellular biomolecules such as lipids, proteins and dna. the study of nut and kernel characteristic and nut composition helps to understand and define the relationship between internal quality and genotype, environmental and cultural factors. it provides information for culture evaluation and choice and a reference of varietal quality useful to growers, breeders and the food processing industry (cristofori et al., 2008). nut and kernel size, nut and kernel shape, percent kernel, shell thickness, low kernel defect, protein and high content of fatty acids are among the main characteristics considered in the evaluation of nut and kernel quality in hazelnut (balta et al., 2006). studies have indicated that the nutritional and chemical composition of hazelnut is affected by cultivar, ecology, harvest year, soil, irrigation and method of cultivation (açkurt et al., 1999; alasalvar et al., 2009; balta et al., 2006; caglarirmak and batkan, 2005; cristofori et al., 2008; köksal et al., 2006; oliveira et al., 2008; silva et al., 2007). recently, some studies on qualitative indices such as total phenol content, antioxidant capacity, fatty acid composition of kernel were conducted (aydin, 2002; botta, 1997; contini et al., 2008; maguire et al., 2004; ozdemir and akinci, 2004; özdemir et al., 2001; parcerisa et al., 1993; serdar and demir, 2005). unfortunately, up to date, data of fatty acid contents and nutritional properties of hazelnuts grown in iran are scarce. hence, the objective of this study is to determine the chemical composition of different hazelnut varieties growing in iran. martials and methods plant material and growth conditions the nuts of two native variety named ‘pashmineh’ and ‘garche’ and four imported hazelnut varieties including ‘ghafghaz’, ‘zakatala’, ‘ronde dupimont’ and ‘fertile decotard’ were used in this study. samples of each variety were obtained from the hazelnut research institute in astara (altitude, 22 m; latitude, 38°4 n’; longitude, 48°87 e’) located in western-guilan province of iran during the 2010 harvest season. foreign hazelnuts were provided from horticultural and agricultural experiments station, sochi, located in russia. shrubs were established with shrub training system at the spacing 4×4 meter. harvest was performed during the early august. pollinizer was ‘daviana’. chemical analysis protein, ash, total oil, carbohydrate and energy evaluation of total oil, protein and ash contents were carried out in triplicate according to aoac official methods (aoac, 1995). total oil was determined by oil extraction from five grams of dried weight of sample using diethyl ether by a soxhlet apparatus (cristofori et al., 2008; köksal et al., 2006; silva et al., 2007). protein was determined by the micro kjeldahl method. protein content was calculated as total n × 6.25 (köksal et al., 2006). ash content was determined by incineration at 600 °c (balta et al., 2006; köksal et al., 2006). carbohydrate content was quantified by calculation of the difference between total weight and other components using the following formula: carbohydrate content = 100 % (% moisture + % protein + % fat + % ash). (1) hazelnut fatty acids and nutritional properties 105 energy was expressed as kilocalories using the following formula (alasalvar et al., 2003; oliveira et al., 2008; ozdemir and akinci, 2004). energy (kcal) = 4 × (protein (g) + carbohydrate (g)) + 9 × (fat (g)). (2) determination of total phenol content total phenolics in hazelnut extracts were determined spectrophotometrically using folin–ciocalteu reagent with minor modifications as method described by oliveira et al. (2008). values of total phenolics were estimated by comparing the absorbance of each sample with a standard response curve generated using gallic acid. results are expressed as mg gallic acid equivalents (gae) on a dry weight (dw) basis (mg gae/g dw). tests were carried out for three replications. antioxidant capacity determined by dpph this spectrophotometric assay uses the stable radical dpph (1, 1 diphenyl-2-picrylhydrazyl) as a reagent. dpph radical scavenging activity was determined according to the method of du et al. (2009) with a minor modification. 50 µl of different hazelnut extract were added to 950 µl of a 6.25 × 10–5 m solution of dpph in methanol. after 30 min incubation period at room temperature (in the dark place), the absorbance was read against a blank at 517 nm. inhibition of free radicals by dpph was calculated by the following formula. % inhibition = [(ablank – asamp) /ablank] × 100 where ablank is the absorbance of control, and asamp is the absorbance of the test compound. tests were carried out for three replications. fatty acid composition of extracted oil the fatty acids composition was determined as methyl esters by gas chromatography (gc) coupled to a mass spectrophotometer, according to methods described in regulation of eec 2568/91. fatty acid methyl esters were prepared by vigorous shaking of a solution of each hazelnut oil sample in n-hexane (0.2 g in 3 ml) with 0.4 ml 2 n methanolic potassium hydroxide solution. chromatographic analysis was performed on a hewlett packard 5890n gas chromatograph equipped with a fid detector (hewlett packard, palo alto, ca, usa), using a fused-silica capillary column (30 m × 0.25 μm i.d. × 0.25 μm film thickness, hp supelco, inc., bellefonte, pa, usa). the injector and detector temperatures were maintained at 220 °c and 260 °c, respectively; the oven temperature was set at 210 °c. helium was employed as the carrier gas with a flow rate of 1 ml/ min according to the method of european regulation 2568/91 (eec, 1991). fatty acids were identified by comparing retention times with those of standard compounds (hashempour et al., 2010). sfa (saturate fatty acid), usfa (unsaturated fatty acid), mufa (monounsaturated fatty acids), pufa (polyunsaturated fatty acids) was calculated using the following equation: sfa= palmitic acid+ stearic acid + behenic acid + myristic acid (1) mufa= oleic acid + palmitoleic acid + eicosenoic acid (2) pufa= linoleic acid + linolenic acid (3) usfa=mufa + pufa (4) statistical analysis in the experiment, the design of a randomized complete block with three replications was used. the results were presented as means ± se. analysis of variance was performed by glm procedures (sas 9.1 for windows). significant differences were calculated according to lsd’s multiple range tests. differences at p < 0.05 were considered statistically significant. results and discussion nutritional properties of the six hazelnut varieties are given in tab. 1. there were significant differences among the hazelnut varieties in terms of oil, energy, protein, carbohydrate and ash. the range in hazelnut genotypes was 63.5 % (‘fertile decotard’) – 53.4 % (‘pashmineh’) in oil of the kernel, 707.7 kcal (‘fertile decotard’) – 653.4 kcal (‘pashmineh’) in energy, 23.3 % (‘pashmineh’) – 16.0 % (‘ghafghaze’) in protein and 20.1 % (‘pashmineh’) – 13.2 % (‘fertile decotard’) in carbohydrate. the highest ash of kernel was detected in ‘ghafghaze’ (3.5 %). ‘fertile decotard’ had the lowest ash of kernels (2.5 %). antioxidant capacity and total phenolic content of the hazelnut samples are shown in tab. 1. ‘garche’ had the highest (72.4 %) scavenging free radicals whereas ‘pashmineh’ had the lowest (57.2 %). kernel of ‘ghafghaze’ had the highest total phenolic content (16.4 mg gae/g) whereas ‘pashmineh’, had the lowest total phenolic content (6.4 mg gae/g). as can be seen from tab. 1, hazelnuts are a good source of energy, oil and carbohydrate. when these results were compared with the results of previous studies on hazelnut varieties, great differences were found in the contents of these analyzed compounds (köksal et al., 2006; oliveira et al., 2008; ozdemir and akinci, 2004). with respect to the nutritional values and fatty acid composition of hazelnuts, many studies have been conducted. but none of them have reported values and composition for hazelnuts grown under the ecological conditions of iran. tab. 1: some nutritional properties of hazlnuts (corylus avellana l.) varieties variety protein oil energy ash carbohydrate total phenolic antioxidant (% dry weight) (% dry weight) (% dry weight) (% dry weight) (% dry weight) content capacity mg/g (dw) (dpph %) ‘fertile decotard’ 20.9 ± 1.0ab 63.5 ± 0.6a 707.7 ± 3.6a 2.5 ± 0.2b 13.2 ± 1.2c 14.0 ± 0.3a 70.3 ± 1.4a ‘zakatala’ 20.3 ± 1.9ab 57.9 ± 2.1bc 677.7 ± 11.9bc 3.0 ± 0.4ab 18.8 ± 0.7a 13.2 ± 1.7a 67.3 ± 0.4a ‘garche’ 23.2 ± 0.9a 58.7 ± 0.4ab 680.5 ± 2.3b 3.2 ± 0.1a 14.1 ± 1.2bc 9.1 ± 1.4b 72.4 ± 0.0a ‘ghafghaze’ 16.0 ± 1.9c 60.5 ± 1.6ab 688.4 ± 8.3ab 3.5 ± 0.1a 19.9 ± 0.7a 16.4 ± 1.3a 69.2 ± 0.9a ‘pashmineh’ 23.3 ± 0.4a 53.4 ± 0.4c 653.4 ± 2.10c 3.4 ± 0.1a 20.1 ± 0.1a 6.4 ± 1.0b 57.2 ± 4.8b ‘rondedupimont’ 17.2 ± 1.4bc 61.4 ± 2.8ab 694.6 ± 13.3ab 3.3 ± 0.1a 17.7 ± 1.6ab 13.6 ± 0.2a 68.9 ± 0.1a mean in each column followed by the same letters are not significantly different at p < 0.05 according to lsd’s multiple range test. data expressed as means ± se. 106 f. rezaei, d. bakhshi, r.f. ghazvini, d.j. majd, m. pourghayoumi köksal et al. (2006) determined that some turkish hazelnut varieties such as ‘tombul’ and ‘siviri’ contain ash content 1.87-2.72 g/ 100 g and protein 11.7-20.8 g/100 g. in the present study, the highest ash content in ‘ghafghaze’ was 3.5 g/100 g which is nearly double. highest protein content was found in ‘pashmineh’ (23.3 g/100 g) which is considerably higher than their reported data. higher ash and protein content shows higher quality indices of the varieties grown in iran. it has been reported in many studies that the nut compositions of hazelnut were affected by variety, harvest year, soil, irrigation, climate and method of cultivation (açkurt et al., 1999; alasalvar et al., 2009; balta et al., 2006; cristofori et al., 2008; köksal et al., 2006; oliveira et al., 2008; silva et al., 2007). the analysis of variance indicated significant differences in fatty acid composition among varieties, as shown in tab. 2. the main fatty acids were oleic acid (c18:1) and linoleic acid (c18:2). oleic acid (c18:1) ranged from 64.2 % in ‘zakatala’ to 81.3 % in ‘fertile decotard’. linoleic acid (c18:2) showed pronounced differences among varieties, the lowest content was found in ‘pashmineh’ (10 %) and the highest in ‘zakatala’ (21.1 %). linolenic acid (c18:3) ranged from 2% in ‘zakatala’ to non-detected in ‘ronde dupimont’, ‘fertile decotard’ and ‘pashmineh’. oil of ‘pashmineh’ had the highest palmitic acid (c16:0), (9.61 %) whereas ‘ghafghaze’, had the lowest palmitic acid (0.49 %). the highest palmitoleic acid (c16:1) of oil was detected in ‘garche’ (1.6 %). ‘ronde dupimont’ and ‘pashmineh’ had the lowest palmitoleic acid of oil (0.0 %). the range of myristic acid (c14:0) of oil varied from 0.5 % (‘zakatala’) to 0.0 % in ‘ronde dupimont’ and ‘pashmineh’, and the range of stearic acid (c18:0) of oil varied from 7.8 (‘zakatala’) to 0.0 % (‘fertile decotard’). the highest behenic acid (c22:0) was in ‘ghafghaze’ (0.25 %) and it was not detected in ‘ronde dupimont’, ‘fertile decotard’, ‘zakatala’ and ‘pashmineh’. oil of ‘zakatala’ had the highest eicosenoic acid (c20:1), (1.7 %) whereas not detected in ‘ronde dupimont’, ‘ghafghaze’ and ‘pashmineh’. the analysis of variance indicated significant differences in fatty acid parameters of oil extracted among varieties, as shown in tab. 3. the main contributing saturated fatty acids for all varieties included palmitic acid (c16:0) and stearic acid (c18:0) with traces of myristic acid (c14:0) and behenic acid (c22:0). oil of ‘ronde dupimont’ had the highest saturated fatty acids (13.93 %). the main unsaturated fatty acids in the studied nuts included oleic acid (c18:1), linoleic acid (c18:2), linolenicacid (c18:3), eicosenoic acid (c20:1) and palmitoleic acid (c16:1). the range of unsaturated fatty acids of oil varied from 97.8 % (‘fertile decotard’) to 79.3 % in ‘pashmineh’. oil of ‘fertile decotard’ and ‘zakatala’, had the highest and the lowest monounsaturated fatty acids respectively (tab. 3). the highest polyunsaturated fatty acids (linoleic acid + linolenic acid) of oil were detected in ‘zakatala’ (23.1 %). ‘pashmineh’ had the lowest polyunsaturated fatty acids of oil (10 %). analysis of the fatty acid profile of the varieties indicates a high unsaturated/ saturated fatty acid ratio in ‘fertile decotard’ (56.2 %). the range of saturated / unsaturated fatty acid ratio of oil varied from 0.17 % (‘pashmineh’) to 0.02 % in ‘fertile decotard’. oil of ‘pashmineh’ had the highest saturated / monounsaturated (oleic acid + palmitoleic acid + eicosenoic acid) fatty acid ratio (0.19 %). palmitic acid is the main saturated fatty acid in hazelnuts followed by stearic acid. the major unsaturated fatty acids found in hazelnut oil are oleic acid (c18:1) and linoleic acid (c18:2) whereas linolenic acid (c18:3) exists at trace levels. the ratios of these fatty acids to each other are important to the economic and nutritional value of the hazelnuts. (koyuncu, 2004 ). köksal et al. (2006) investigated chemical composition of the 17 different hazelnut varieties grown in the black sea region of turkey and reported the following values: palmitic acid 4.72-5.87 %, stearic acid 0.86-2.49 %, oleic acid 74.2-82.8 %, tab. 2: fatty acid contents (% of total oil) of hazlnuts (corylus avellana l.) varieties variety oleic linoleic linolenic palmitic palmitoleic myristic stearic behenic eicosenoic acid acid acid acid acid acid acid acid acid ‘fertile decotard’ 81.3 ± 0.3a 14.7 ± 0.1c nd 1.6 ± 0.01c 1.3 ± 0.01b 0.11 ± 0.0c nd nd 0.5 ± 0.05c ‘zakatala’ 64.2 ± 0.2f 21.1 ± 0.1a 2 ± 0.01a 0.76 ± 0.00d 0.6 ± 0.00c 0.5 ± 0.01a 7.8 ± 0.01a nd 1.7 ± 0.00a ‘garche’ 68.2 ± 0.2e 14.9 ± 0.1b 1.63 ± 0.12b 0.65 ± 0.01de 1.6 ± 0.01a 0.3 ± 0.01b 4.1 ± 0.12c 0.24 ± 0.0b 1.3 ± 0.06b ‘ghafghaze’ 68.2 ± 0.2d 14.9 ± 0.3b 0.15 ± 0.00c 0.49 ± 0.01e 1.59 ± 0.0a 0.3 ± 0.00b 4.1 ± 0.13c 0.25 ± 0.0a nd ‘pashmineh’ 69.3 ± 0.3c 10 ± 0.1e nd 9.6 ± 0.12a nd nd 3.5 ± 0.10d nd nd ‘rondedupimont’ 74.9 ± 0.3b 11.1 ± 0.1d nd 9.3 ± 0.13b nd nd 4.6 ± 0.10b nd nd mean in each column followed by the same letters are not significantly different at p < 0.05 according to lsd’s multiple range test. data expressed as means ± se. tab. 3: summary of the important fatty acid parameters of oil extracted from hazlnuts (corylus avellana l.) varieties variety s fa usfa mufa pufa sfa/usfa usfa/sfa sfa/mufa ‘fertile decotard’ 1.74 ± 0.01e 97.8± 0.4a 83.1 ± 0.2a 14.7 ± 0.1d 0.02 ± 0.00d 56.2 ± 0.1a 0.02 ± 0.00d ‘zakatala’ 9.06 ± 0.01c 89.5 ± 0.4b 66.4 ± 0.2f 23.1 ± 0.1a 0.10 ± 0.00b 9.9 ± 0.0c 0.13 ± 0.00b ‘garche’ 5.32 ± 0.13d 87.6 ± 0.5c 71.0± 0.3c 16.5 ± 0.2b 0.06 ± 0.00c 16.5 ± 0.3b 0.07 ± 0.00c ‘ghafghaze’ 5.17 ± 0.14d 84.8 ± 0.5d 69.8 ± 0.3d 15.1 ± 0.3c 0.06 ± 0.00c 16.4 ± 0.3b 0.07± 0.01c ‘pashmineh’ 13.11 ± 0.13b 79.3 ± 0.4f 69.3 ± 0.3d 10 ± 0.1f 0.17 ± 0.00a 6.1 ± 0.0d 0.19 ± 0.00a ‘rondedupimont’ 13.93 ± 0.25a 86.0 ± 0.4c 74.9 ± 0.3b 11.1 ± 0.2e 0.16 ± 0.00a 6.2 ± 0.1d 0.19 ± 0.00a mean in each column followed by the same letters are not significantly different at p < 0.05 according to lsd’s multiple range test. sfa (saturate fatty acid), usfa (unsaturated fatty acid), mufa (monounsaturated fatty acid), pufa (polyunsaturated fatty acids). data expressed as means ± se. hazelnut fatty acids and nutritional properties 107 linoleic acid 0.03-0.08 % and linolenic acid 0.029-0.076 %. in the present study, ‘fertile decotard’ had the highest oleic acid (81.3 %), monounsaturated fatty acids (83.1 %), unsaturated fatty acid (97.8 %), and unsaturated fatty acid / saturate fatty acid (56.23 %). the highest polyunsaturated fatty acids (23.1 %), stearic acid (7.8 %), linolenic acid (2 %), linoleic acid (21.1 %), myristic acid (0.5 %), eicosenoic acid (1.7 %) was determined in ‘zakatala’ whereas the highest saturate fatty acid / unsaturated fatty acid (0.17 %), saturate fatty acid / monounsaturated fatty acids (0.19 %) and palmitic acid (9.6 %) content was in ‘pashmineh’. the highest palmitoleic acid (1.6 %), behenic acid (0.25 %) and saturate fatty acid (13.93 %) were recorded in ‘garche’, ‘ghafghaze’ and ‘ronde dupimont’, respectively. conclusion in conclusion, findings concerning nutritional composition revealed that many hazelnuts varieties grown in iran contain a high amount of carbohydrate, protein, and fatty acid contents which could be useful for future production and breeding goals. as an excellent source of monounsaturated fatty acids, hazelnuts may be beneficial, preventing from cholesterol-based atherosclerosis and ischemic cardiovascular diseases. besides their high energetic and nutritional value, hazelnuts also provide bioactive compounds such as antimicrobial and antioxidant agents, suggesting that the fruits could also be useful in the prevention of diseases in which free radicals are implicated. overall, it seems that more studies are required to decipher the role (s) of environmental factors in quality of hazelnut, as well as with the comparison of essential substances in different hazelnut varieties, researchers can introduce the varieties with high quality in future works. acknowledgements authors would like to thank dr. alireza aliakbar for his technical help, hazelnut research station in astara located in western-guilan and all farmers for their help. references açkurt, f., özdemir, m., biringen, g., löker, m., 1999: effects of geographical origin and variety on vitamin and mineral composition of hazelnut (corylus avellana l.) varieties cultivated in turkey. food chem. 65, 309-313. alasalvar, c., amaral, j.s., satir, g., shahidi, f., 2009: lipid characteristics and essential minerals of native turkish hazelnut varieties (corylus avellana l.). food chem. 113, 919-925. alasalvar, c., pelvan, e., amarowicz, r., 2010: effects of roasting on taste-active compounds of turkish hazelnut varieties (corylus avellana l.). j. agric. food chem. 58, 8674-8679. alasalvar, c., shahidi, f., liyanapathirana, c.m., ohshima, t., 2003: turkish 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sci. (prague) 32, 96-99. silva, a., santos, a., cavalheiro, j., ribeiro, c., santos, f., gonçalves, b., 2007: fruit chemical composition of hazelnut cultivars grown in portugal. j. appl. horticult. 9, 157-161. address of the authors: fatemeh rezaei, davood bakhshi*, reza fotouhi ghazvini, mohammadreza pourghayoumi, department of horticultural science, faculty of agricultural sciences, university of guilan, rasht, iran * corresponding author‘s e-mail: bakhshi-d@guilan.ac.ir davood javadi majd, hazelnut research station, astara agricultural and natural resources research center, guilan, iran journal of applied botany and food quality 88, 42 48 (2015), doi:10.5073/jabfq.2015.088.008 1 hungarian department of biology and ecology, babes-bolyai university, cluj-napoca, romania 2 plant nutrition department, cebas-csic, espinardo, murcia, spain 3 molecular biology centre, institute for interdisciplinary experimental research, babes-bolyai university, cluj-napoca, romania sodium accumulation contributes to salt stress tolerance in lettuce cultivars csaba bartha1*, laszlo fodorpataki1, maria del carmen martinez-ballesta2, octavian popescu3, micaela carvajal2 (received october 13, 2014) * corresponding author summary increasing soil salinity of irrigated agricultural areas represents a major environmental stress factor that impairs the production of many salt-sensitive crop plants. different lettuce cultivars were studied for identification of more tolerant ones, based on physiological properties. five selected lettuce cultivars were grown hydroponically, salt stress was induced by 50 mm and 100 mm of nacl. the cultivars exhibited differential reduction in shoot fresh weight. the highest sodium and free proline accumulation in the shoot of the most tolerant cultivar, associated with a moderate decrease of the root hydraulic conductance and of the leaf stomatal conductance, can be related to a better defense mechanism against osmotic stress. salt exposure increased the potassium and calcium ion content of the xylem sap, which may be important for an efficient osmotic adjustment needed to support leaf expansion. the fact that the highest amount of na+ was found in the shoot of the most tolerant cultivar, and the lowest in the most sensitive one, reflects that in lettuce na+ exclusion is not a main strategy for salt tolerance. lettuce is a good example for the case in which salinity tolerance is related not to exclusion, but to inclusion of sodium ions in the shoot system. for salt tolerant varieties the marketable yield has a higher dry biomass percentage, leaves of plants grown under high salinity are crispier, darker green and have a slight salty taste. introduction high salinity is a major stress factor that restricts crop productivity. worldwide more than 800 million hectares of land are salt-affected, most of these are located in arid and semiarid regions (fao, 2008). because the irrigated area has at least twice the productivity of rainfed land, more than one third of all crop production is coming from these salt affected regions (munns and tester, 2008). among other crop plants, the majority of the lettuce world production is increasingly being affected by salinity (fao, 2009). high concentrations of salt increase the osmotic potential, making it harder for roots to extract water (osmotic effect), and result in toxicity symptoms (ionic effect), leading to metabolic imbalance and premature senescence of leaves (munns, 2002; tester and davenport, 2003). the loss of water and the invading ions activate a concerted acclimation process that may lead to salt tolerance associated with a new steady state of growth. this acclimation includes three basic processes: restoration of turgor, regulation of ion transport across membranes, and induction of the accumulation of osmoprotectants and stress proteins. besides these processes, several secondary responses are needed to ensure salt tolerance, e. g. the scavenging of overproduced reactive oxygen species, an increase in energysupplying reactions, and the adjustment of the whole metabolism to the new situation. this is why multiple molecular and physiological changes may be observed in plants exposed to salt stress: drop of stomatal and root hydraulic conductance, osmotic adjustment, reduced growth rate, changes of the root to shoot ratio, nutritional disorders (munns and tester, 2008; rajendran et al., 2009). a rapid whole-plant response to salt stress is a decrease in stomatal conductance, caused by the osmotic effect of high nacl concen tration. the effect is very fast, but a new steady-state rate of transpiration becomes stabilized after hours, depending on the species and cultivars. in the first phase of stress reactions, the osmotic effect of the salt stress is responsible for the reduction of shoot growth (cramer, 2002), usually showing a good correlation between stomatal conductance and growth rate (james et al., 2008). to maintain turgor, plant cells have to increase the osmotic potential. energetically, the most effective way to do this in salt-rich environments is accumulating na+ and clions, but the chemical toxicity will appeared (munns and tester, 2008). another strategy is to synthesize osmotically active organic compounds (compatible solutes) which accumulate as a result of the reprogrammed metabolism (soudry et al., 2005). one of the most widespread osmoprotectants is proline, which may play multiple roles in stress tolerance. besides keeping the osmotic balance stable, it may protect enzymes and membrane lipids from degradation (ashraf and foolad, 2007), it may be a radical scavenger in the process of antioxidative defense (hong et al., 2000), and it can constitute an energy source for afterstress recovery mechanisms (hare et al., 1999). therefore, in many plants, proline accumulation can be considered an indicator of stress tolerance (hajlaoui et al., 2010; zhu et al., 2008). however, the achievement of the proper balance to maintain turgor, depends on the species, on cultivars or ecotypes, and on the environmental conditions (greenway and munns, 1980). although the ability to exclude na+ or clfrom the shoot is often a primary determinant of variability in salinity tolerance within a species, there is not necessarily an inverse relationship between shoot na+ or clconcentration and salinity tolerance. there is rather a difference in the ability of ion homeostasis maintenance in the root, in the xylem sap and in the leaves (munns and tester, 2008; rajendran et al., 2009; shabala et al., 2010). lettuce (lactuca sativa l.) has many cultivars, which are grown on extended areas and have become a much appreciated healthy food source, rich in mineral nutrients and vitamins. as most commercially grown cultivars are growing under non-saline conditions, very limited information is available about salt stress response of the different lettuce cultivars. also, the physiological and biochemical parameters that may be useful for the screening of salt tolerant varieties are not well stated, and influence of salinity on marketable yield quality of lettuce is poorly documented. the aim of this study is to compare salt stress tolerance of five, largely commercialized lettuce cultivars, and to identify more tolerant cultivars that may be suited for large-scale cultivation in areas with increasing soil salinity. materials and methods plant material and growth conditions five different cultivars of lettuce (lactuca sativa l.) were used in the experiments. these were selected from among sixteen cultivars examined for salt stress sensitivity, largely cultivated in europe. salt stress in lettuce cultivars 43 seeds of all cultivars were provided by b&t world seeds (paguignan, france). the cultivars were chosen from all of the four main lettuce convarieties (butterhead, roman, looseleaf and steam lettuce). the valdor and parella green cultivars belong to the butterhead type (lactuca sativa var. capitata), paris island is part of the roman (cos) type (lactuca sativa var. romana), salad bowl red is included in the looseleaf type (lactuca sativa var. crispa), and asparagina is a cultivar belonging to the steam lettuce type (lactuca sativa var. asparagina). the lettuce seeds were pre-hydrated with distilled water and continuously aerated for 24 h. after this, the seeds were germinated in vermiculite substrate at 20 °c and kept in dark for 3 days. on the 4th day germinated seeds were transferred under controlled conditions using a daily photoperiod of 16 h light and 8 h darkness. the relative humidity was set to 75 % during daytime and to 60 % for the night. the temperature was 23 °c during daytime and 20 °c during the dark period. a photosynthetically active radiation of 400 μmol m-2 s-1 was provided by a combination of fluorescent tubes (philips tld 36 w/83 and sylvania f36 w/gro) and metal halide lamps (osram hqi, t 400 w). on the 7th day after germination the seedlings were placed in 15 l containers with a continuous circulation of full-strength hoagland’s nutrient solution (hoagland et al., 1950). the solution was replaced every four days. salinization treatment was initiated on 21 d old seedlings, after two weeks of growth in hoagland’s solution. salt stress was induced by 50 mm and 100 mm of nacl (p. a.). the 100 mm of nacl was added in two steps, in two consecutive days. plantlets were harvested after ten days of exposure (31 days old). fresh weight (fw) of roots and shoots (stem with leaves) was recorded separately, for five plants from each cultivar and salinity combination. for dw determination, the plant material was kept at 65 °c for 5 days. the youngest fully-expanded leaves were also collected from five plants per cultivar and salinity treatment, and kept frozen at -80 °c for proline content measurements. determination of mineral elements the concentrations of na+, ca2+ and k+ were determined in plant material (roots and shoots) ground finely in a mill grinder, after drying at 65 ºc for 5 days. the samples were digested in a microwave oven (cem mars xpress, north carolina, usa), reaching 200 ºc in 20 min, and held at this temperature for 2 h. digestion of 0.1 g dw plant material was performed with 5 ml 65 % hno3, 17 ml h2o dist. and 3 ml 30 % (v/v) h2o2. for determining the sodium, potassium and calcium ion content of the xylem sap, 0.1 ml of xylem sap was diluted with distilled water to a final volume of 10 ml. the concentrations of na+, ca2+ and k+ were determined by inductively coupled plasma spectrometry (iris intrepid ii, thermo electron corporation, franklin, usa). leaf gas exchange parameters stomatal conductance (gs) was measured using a portable photosynthesis system (model lca-4, adc bioscientific ltd., hoddesdon, uk). the abaxial stomatal conductance (on the lower epidermis) was measured on the youngest fully-expanded leaves, in the middle of the daily photoperiod, under constant photon flux density, air humidity and temperature (ahmed et al., 2006). root hydraulic conductance (l0) the root hydraulic conductance of lettuce plants exposed to salt stress treatments was measured by natural exudation of detached roots. the measurements were made in the middle of the photoperiod, 10 days after the beginning of salt treatment. the aerial parts of the plants were removed and the stems were put in silicon tubes. the roots were kept in the same nutrient solution which was used for their growth. the sap that accumulated during a certain time, according to the treatment, was collected in eppendorf tubes. the roots and the tubes were weighed with a precision balance. sap flow (jv) was expressed in mg g-1 (root fresh weight) h-1. the osmotic potentials of the sap samples and of the nutrient solutions were measured using an osmometer (digital osmometer, roebling, berlin, germany). the osmotic potential difference between the xylem sap and the external solution, δψπ, was calculated from their osmolarity values. the hydraulic conductance, l0, expressed in mg (g root fw)-1 h-1 mpa-1, was calculated according to the relation l0 = jv/ δψπ, where jv = mg xylem sap h-1 g-1 root, and δψπ = ψ0 xylem sap – ψ0 solution (cabanero and carvajal, 2007). free proline content free proline content in the plant material was determined by generation of a colored product with ninhidrine (bates, 1973). 0.5 g of fresh plant material was homogenized in a pre-chilled mortar with 3 ml of 3 % sulfosalicilic acid cooled on ice. the extract was centrifuged for 10 minutes at 20 000 g, then 600 μl of 96 % acetic acid and 600 μl ninhidrine solution (containing 2.5 % w/v ninhidrine, 60 % v/v 96 % acetic acid and 40 % v/v of 6 m ortophosphoric acid) was added to 600 μl of supernatant. the samples were incubated in test tubes for 1 hour at 100 °c, and after cooling, 3 ml of toluene was added to extract the reaction product. the mixtures were stirred, and when two layers were separated, 2 ml of the upper layer was transferred in a cuvette. the concentration of the red product was determined on the base of its absorbance at 520 nm using toluene as reference. l-proline (sigma) was used for the preparation of standard curve. statistical analysis the data were analyzed statistically, using the spss 18.0 software package, by analysis of variance (anova) and by tukey’s test. significant differences were determined at p < 0.05. results upon exposure to salt stress, physiological and biochemical changes, related to disturbance in growth, water relations and mineral nutrition, occurred in the different lettuce cultivars. it is worth mentioning that under the given growth conditions, 30 days old lettuce plants may be considered well developed, having at least 12 fully extended leaves and being suitable for consumption. the high amount of nacl in the nutrient solution reduced significantly the growth rate of shoot fresh weight in all five lettuce cultivars (fig. 1, a). the most pronounced reduction, related to the control, was observed in the asparagina cultivar, at both salt concentrations (31 % decrease in the presence of 50 mm nacl and 51 % reduction with 100 mm nacl). the mildest shoot growth inhibition was registered for the paris island cultivar (24 % reduction at 50 mm nacl and 41 % reduction at 100 mm nacl). root growth was not inhibited, but it was stimulated by salt stress, except for the asparagina cultivar. in the case of salad bowl red and parella green cultivars, exposure to 50 mm sodium chloride resulted in a statistically significant increase of root fresh weight during the treatment (fig. 1, b). regarding fresh biomass, dry matter and water content of leaves, our results showed that high salinity reduces water content, and in a smaller extent it decreases fresh shoot biomass. as a consequence, the dry weight percentage of lettuce leaves increases under elevated salinity (fig. 2). in absolute values, leaf dry biomass is higher in plants exposed to 50 mm nacl than in plants grown in the presence of 100 mm sodium chloride. 44 c. bartha, l. fodorpataki, m.c. martinez-ballesta, o. popescu, m. carvajal the values of root hydraulic conductance (l0) are shown in fig. 3. there was a significant difference between the hydraulic conductance of the cultivars without salinity treatments. salt stress caused a significant l0 decrease in all cultivars. the most pronounced reduction was shown in the asparagina cultivar, for both nacl concentrations. in the case of paris island cultivar, both salt concentrations showed the same degree of reduction. in the case of parella green it was not possible to collect xylem sap from plants exposed to 100 mm nacl. the stomatal conductance (gs) of leaves decreased in all cultivars when salinity treatments were applied. the most significant difference from control was observed, with both salt treatments, in the case of aparagina and salad bowl red cultivars. in the paris island cultivar stomatal conductance decreased significantly only upon exposure to 100 mm nacl. from all cultivars, at the 50 mm salt regime, the smallest reduction was observed in paris island (fig. 4). fig. 1: effect of 50 mm and 100 mm nacl on the shoot and root fresh weight (fw) of five lettuce cultivars. data are means of five plants ± se. different letters indicate significant differences among treatments of the same cultivar, at p < 0.05, according to the tukey test. fig. 2: percentage of dry matter in the biomass of leaves of five lettuce cultivars developed under different salinity levels in the nutrient medium. vertical bars represent ± se from means (n = 5). different letters indicate significant differences among treatments of the same cultivar, at p < 0.05. fig. 3: root hydraulic conductance (l0) of five lettuce cultivars exposed to salt stress. fw – fresh weight. each value is the mean of five samples ± se. different letters indicate significant differences among treatments of the same cultivar, at p < 0.05, according to the tukey test. fig. 5 summarizes the na+, ca2+ and k+ content of the shoot of five lettuce cultivars. at both salinity levels, the highest na+ accumulation was found in paris island, while the lowest was measured in asparagina. the roots exhibited an opposite trend of sodium content as compared to shoots, the highest accumulation being achieved by asparagina (data not shown). asparagina, and in a smaller extent salad bowl red, accumulated higher amounts of na+ in the root than in the shoot, while in the case of paris island, parella green and valdor the concentration of sodium ions was higher in shoots than in roots, at both salinity levels (data not shown). calcium ion content was reduced in all cultivars as a result of salt stress. salt stress also decreased k+ concentration of all cultivars. in paris island, parella green and valdor there was no obvious difference in the k+ accumulation upon exposure to 50 mm and to 100 mm nacl. free proline level of the leaves was increased by both salt regimes in all five cultivars. a significant difference between proline concentrations of different cultivars was observed only upon exposure to 100 mm of nacl (fig. 6). the highest proline content was determined in the paris island cultivar exposed to100 mm nacl, where free proline concentration was about twenty times higher than in control plants. in the other cultivars treated with 100 mm nacl, this fig. 4: stomatal conductance (gs) in leaves of five lettuce cultivars exposed to different degrees of saltstress. bars represent means ± se, n = 5. different letters indicate significant differences among treatments of the same cultivar, at p < 0.05, according to the tukey test. salt stress in lettuce cultivars 45 increase was smaller: threefold for parella green, fourfold for as-parella green, fourfold for as-, fourfold for asparagina and valdor, and sevenfold for the leaves of the salad bowl red cultivar. no inherited salt stress resistance that could avoid growth inhibition caused by high salinity, regardless of the different cultivars. the cultivar aspargina suffered the largest shoot growth reduction at both salinity levels. this indicates that from among the five cultivars that were investigated, this is the most sensitive to salt stress in terms of biomass production. by contrast, as paris island cultivar did not significantly altered the growth after 50 mm nacl addition, may be considered more tolerant (fig. 1). root growth was less affected by salinity than leaf growth, and root elongation rate recovered remarkably well after several days of exposure to nacl. this can be related to the osmotic effect of salt stress, the impaired water supply stimulating root growth to increase water uptake surface (munns, 2002). even though the absolute chlorophyll content does not increase upon salt stress (data not shown), the lower water content of the fresh biomass confers a darker green colour to lettuce leaves. for the cv. vera of lettuce, grown hydroponically for 32 days, andriolo et al. (2005) reported that leaf number was not affected by salinity treatments, why dry biomass of leaves increased under mild salinity (up to 2.81 ds m-1 electrical conductivity, approximately equivalent to 28 mm nacl), and decreased above this salinity level. this reflects that cv. vera is most probably a salt-sensitive lettuce cultivar. in an attempt to use diluted seawater for irrigation of lettuce, turhan et al. (2014) found that dry biomass, total fresh yield and marketable fresh yield of cv. funly of lettuce, after 40 days of irrigation with 2.5 % and 5 % seawater were similar to control, but decreased in response to 10 % and 20 % seawater. they concluded that low amounts of salt (2.5 % 5 % seawater) are necessary in irrigation water to reach the optimal yield of the studied lettuce cultivar. our results showed that salt tolerant lettuce varieties may produce a slightly increased dry biomass percentage even under considerably higher salinity values. in another set of experiments, the verte de cobhain variety of lettuce was found to be salt tolerant, and upon treatment for 12 days with nacl concentrations increasing progressively up to 100 mm, displayed better growth and superior antioxidative capacity than the control grown hydroponically in hoagland’s nutrient solution (mahmoudi et al., 2010). these results, in agreement with our findings, underline the broad range of salt tolerance of different lettuce cultivars, and the fact that some degree of salinity may increase the marketable yield of this vegetable. when plants are subjected to salt stress, root hydraulic conductance (l0) is usually reduced (cabanero and carvajal, 2007; muries et al., 2011). the main cause of this reduction could be the decrease in the activity or concentration of aquaporins in the root plasma fig. 5: na+, ca2+ and k+ content of shoot in five lettuce cultivars exposed to 50 mm and 100 mm nacl. bars represent means ± se, n = 5. different letters indicate significant differences among treatments of the same cultivar, at p < 0.05, according to the tukey test. fig. 6: proline content of five lettuce cultivars exposed to salt stress. fw – fresh weight. bars represent means ± se, n = 5. different letters indicate significant differences among treatments of the same cultivar, at p < 0.05, according to the tukey test. discussion differences among cultivars in growth inhibition caused by salt stress were reported for several crop plants, e.g. for triticum monococcum (rajendran et al., 2009), for maize (hajlaoui et al., 2010), for rice (quinet et al., 2010). relatively few studies have been undertaken to investigate the effects of nacl on different lettuce cultivars. pasternak et al. (1986) found that roman lettuce types are generally more salt tolerant than iceberg types, however, mahmoudi et al. (2010; 2011; 2013) reported that for four lettuce varieties (including butterhead, romain and verte) roman type was the most sensitive to salinity. another study compared several cultivars of lettuce, and demonstrated different salt sensitivity during the germination stage (coons et al., 1990; nasri et al., 2010). in our experi-experiments, a significant reduction in shoot fresh weight of all five lettuce cultivars exposed to salt stress was found. this reflects that there is 46 c. bartha, l. fodorpataki, m.c. martinez-ballesta, o. popescu, m. carvajal membrane (carvajal et al., 2000), this effect being mainly due to the specific toxicity of na+ and cl– ions (martinez-ballesta et al., 2000). secondly, reduced hydraulic conductance may be related to the hyperosmotic stress and ionic imbalance caused by the high apoplastic concentrations of na+ and cl– (munns and passioura, 1984). due to the morphology of the lettuce (rosette type plant) it is difficult to use the scholander chamber for measuring hydraulic conductance without causing injuries to the very short stem (data not shown). the scholander pressure chamber forces xylem sap out from decapitated plants showing higher l0 than natural exudation method, since the water movement is forced to occur through the apoplast to a greater extent (fernandez-garcía et al., 2002). however, the differences between salinity treatments were maintained (lopez-perez et al., 2007). all five lettuce cultivars exposed to salt stress exhibited a certain reduction of root hydraulic conductance, but the magnitude of this decrease depends on the cultivar (fig. 3). the reduction of the xylem transport from root to shoot can be beneficial for the plant, because it prevents the accumulation of toxic levels of na+ and cl– ions in the leaves, but causing decrease of water transport. this means that for finding a proper balance, the lettuce plants can exhibit a reduced hydraulic conductivity upon salt stress ensuring a better protection to the leaves against ion toxicity, being more salt tolerant. on the other hand, certain amount of shoot na+ content may be beneficial by helping the plant to maintain turgor. again, a balance is needed to be established between the use of na+ and cl– by the plant to maintain turgor and the need to avoid their toxicity (munns and tester, 2008). taking all this parameter into account, paris island cultivar maintained a higher hydraulic conductance, which confers a more pronounced salt tolerance by enabling a better water supply of the leaves from the root. as a reaction to the osmotic component of salt stress, stomata tend to close in order to reduce water loss by transpiration (munns and tester, 2008). this impairs photosynthetic carbon uptake, but reduces moisture accumulation during storage of lettuce heads. salt stress tends to reduce stomatal conductance (gs) in a short period after exposure. the fact that stomatal conductance was less affected by salinity in the paris island cultivar (fig. 4) may be related to the other parameters (particularly with shoot fw and root hydraulic conductance) in order to show its higher salt tolerance in comparison with the other cultivars. a higher conductance enables a better carbon dioxide supply for a sustained photosynthetic assimilation, resulting in a smaller reduction of biomass production. a direct correlation between stomatal conductance and salt stress tolerance was also observed in maize cultivars (azevedo-neto et al., 2004). in our experiments, the most pronounced reduction in stomatal conductance upon salt stress was recorded in the salad red bowl lettuce cultivar, suggesting the sensitivity of the gas exchange regulation (munns and tester, 2008). all lettuce cultivars growth in saline conditions showed an in crease in na+ concentration. the highest amount of na+ was found in the cultivar paris island, and the lowest na+ concentration was determined in asparagina. therefore, two main strategies of salt stress tolerance can be considered, i. e. salt exclusion and salt sequestration, the latter one is used by lettuce cultivars. this is why the marketable biomass gets a slight salty taste. in a recent study on different cultivars of barley, shabala et al. (2010) conclude that after one week of salt treatment (320 mm nacl), shoot na+ content of the tolerant variety was about 20 % higher than in the sensitive genotype. in the first phase of the salt stress the rapidly accumulating na+ is an osmolite with low energy cost in the leaf vacuoles for the adjustment of cell turgor, and ultimately of tissue growth under the hyperosmotic stress condition imposed by salinity (munns and tester, 2008; shabala et al., 2010). salt stress disturbs the uptake of essential mineral nutrients such as k+ and ca2+, as na+ competitively inhibits k+ and ca2+ transport through membranes (zhao et al., 2007). fig. 5 shows the reduction of potassium content in shoots as a result of salt stress, and this reduction is most probably due to the competition of na+ for the same cation transporters (azevedo-neto and tabosa, 2000). we have found no correlation between k+ content and the salt tolerance of the examined lettuce cultivars. similar results were published by neocleous et al. (2014) for lettuce and azevedo-neto et al. (2004) for maize genotypes. regulation of potassium to sodium ion ratio in plant cells subjected to salt stress needs to be elucidated by further investigations. na+ also reduces the influx of ca2+ ions through the plasma membrane, and increases efflux of ca2+ from plant cells (cramer et al., 1989) this is valid for the investigated lettuce cultivars, although the dynamics of cytosolic calcium content decrement varies among the cultivars. under the influence of 100 mm nacl ca2+ content dropped to the approximately same value in all cultivars, except for valdor. no correlation could be established between calcium ion content and salt stress tolerance of the different lettuce cultivars. for lactuca sativa var. crispa, identified as moderately sensitive to salinity, it was found that dry matter ratio increased with increasing salinity in the range of 0.75-7.0 ds m-1 (approx. 7.5-70 mm nacl), calcium accumulation in leaves decreased, while the amount of potassium was unaffected (ünlükara et al., 2008). they did not distinguish between the calcium and potassium content of the xylem sap in the veins and of the parenchyma tissue in the leaf blade. they also established that the taste of lettuce was not affected by salinity, even though salt accumulated in leaves. the reduction of k+ and ca2+ content in shoots, accompanied by an increased concentration of these ions in the xylem sap (data not shown), can be explained by the pronounced decrease of the hydraulic and stomatal conductances in lettuce plants exposed to salt stress, so although the xylem sap has higher amounts of ca2+ and k+, a smaller sap volume reaches the leaves of the salt-exposed plants. the presence of 100 mm nacl in the growth medium caused a significant increase in the free proline content in all of the five lettuce cultivars (fig. 6). in the paris island cultivar the proline content increased twenty five times as compared to the control, which is a very pronounced metabolic reaction related to an effective osmoregulation with involvement of this compatible solute. similar data were presented about the involvement of proline accumulation in salt stress tolerance of some other lettuce cultivars (younis et al., 2009), however, such an increase in prolin concentration like in case of paris island was not reported before. in terms of free proline content, we obtained significantly different results (considerably higher increases) from those reported by mahmoudi et al. (2011) for two other cultivars, even though the cultivation and treatment conditions were very similar. while their one month old plants, grown hydroponically and exposed to 100 mm nacl, developed a proline content similar to control or at most two times higher, we have determined in our cultivars, under rather similar conditions, a three to twenty times increased proline concentration as a result of osmotic stress tolerance. these differences may be due to different developmental phases of the examined plants (their one month old plants had a generally lower biomass production than the ones obtained in our experiments). the higher proline accumulation of this cultivar could be a biochemical indicator of its better salt tolerance. increment of free proline concentration in plant cells subjected to osmotic stress is well documented in the literature, consequently different levels of proline content may indicate the degree of environmental stress that affects water balance of plants. the higher proline content of leaves can be related to a higher capacity to accumulate na+ ions in the shoot, as osmotic adjustment is achieved by proline accumulation in the cytosol and by sodium sequestration in the vacuole (ashraf and foolad, 2007; hasegawa et al., 2000). in summary, we concluded that from among the investigated lettuce salt stress in lettuce cultivars 47 cultivars, paris island exhibited the highest tolerance to salt stress exerted by 50 mm and 100 mm nacl in hydroponic cultures, while asparagina was the most sensitive to high salinity. the cultivars with the greater growth rate in saline solution have the higher concentrations of na+, thus suggesting that the involvement of na+ in osmotic adjustment is a contributor to the higher growth rate. stomatal clostomatal clo-stomatal closure reduces moisture accumulation during storage of lettuce. free proline content is a reliable, easy-to-determine and sensitive biochemical marker for selection of salt tolerant lettuce varieties even at an early developmental stage. salt accumulation in leaves of more salt tolerant varieties (e.g. paris island, valdor) confers a slight salty taste to the marketable yield of lettuce, while lower water content, resulting in a higher percentage of dry biomass, makes the leaves crispier and darker green, that may compensate for reduced leaf size of the plants exposed to high salinity. based on the above presented data, the paris island lettuce cultivar can be recommended for cultivation in areas affected by increased salinity. even though a controlled relation between salt concentration and salinity stress reactions can be achieved only in laboratory conditions (because in the field salt concentration cannot be stabilized around the roots and many other variables appear), in the next step field experiments should complete those undertaken in the present study. acknowledgments this work was supported by the program co-financed by the sectorial operational program “human resources development, contract posdru 6/1.5/s/3 – doctoral studies: through science toward society” and by seneca project (comunidad autónoma de la región de murcia): aprovechamiento de aguas de baja calidad para la obtención de cultivos con un alto valor nutritivo: caracterización de la respuesta a estrés nutricional (boro y salinidad) ref: (11909/pi/09). the corresponding author thank to beatriz muries bosch, maria del carmen rodriguez hernandez, cesar mota cadenas, carlos alcaraz lopez and vicente gimeno nieves from the departments of plant nutrition at cebas-csic in murcia for the technical support kindly provided during the experiments. references ahmed, s., nawata, e., sakuratania, t., 2006: changes of endogenous aba and acc, and their correlations to photosynthesis and water relations in mungbean (vigna radiata (l.) wilczak cv. kps1) during waterlogging. environ. exp. bot. 57, 278-284. andriolo, j.l., luz, g.l., witter, m.h., godoi, r.s., barros, g.t., bortolotto, o.c., 2005: 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m.s., demir, a.o., 2014: effect of different concentrations of diluted seawater on yield and quality of lettuce. chilean j. agric. res. 74(1), 5-16. ünlükara, a., cemek, b., karaman, s., ersahin, s., 2008: response of lettuce (lactuca sativa var. crispa) to salinity of irrigated water. new zealand j. crop hortic. sci. 36, 265-273. younis, m.e., hasaneen, m.n.a., tourky, s.m.n., 2009: plant growth metabolism and adaptation in relation o stress conditions., xxiv, salinity-biofertility interactive effects on proline, glycine and various antioxidants in lactuca sativa. plant omics j. 2(5), 197-205. zhao, q., ma, b.l., ren, c.z., 2007: growth, gas exchange, chlorophyll fluorescence, and ion content of naked oat in response to salinity. crop sci. 47, 123-131. zhu, j., bie, z., yana, l., 2008: physiological and growth responses of two different salt sensitive cucumber cultivars to nacl stress. soil sci. plant nutr. 54, 400-407. address of the authors: dr. csaba bartha and assoc. prof dr. laszlo fodorpataki, hungarian department of biology and ecology, babes-bolyai university, ro-400084 cluj-napoca, romania e-mail: barthacsabi@gmail.com; lfodorp@gmail.com prof. dr. micaela carvajal and dr. maria del carmen martinez-ballesta, plant nutrition department, cebas-csic, p.o. box 164, campus de espinardo-edificio 25, e-30100 espinardo, murcia, spain e-mail: mcarvaja@cebas.csic.es; mballesta@cebas.csic.es prof. dr. octavian popescu, molecular biology centre, institute for interdisciplinary experimental research, babes-bolyai university, ro-400084 cluj-napoca, romania e-mail: opopescu.ubbcluj@gmail.com journal of applied botany and food quality 86, 1 10 (2013), doi:10.5073/jabfq.2013.086.001 dipartimento di scienze agrarie, forestali e alimentari, università degli studi di torino, grugliasco (to), italy currants and strawberries as bioactive compound sources: determination of antioxidant profiles with hplc-dad/ms d. donno*, m. cavanna, g.l. beccaro, m.g. mellano, d. torello marinoni, a.k. cerutti, g. bounous (received january 18, 2013) * corresponding author summary among plant foods, berry fruit shows a high antioxidant capacity. medical research has pointed out the medicinal properties of certain pigmented polyphenols, such as flavonoids, anthocyanins, tannins and other phytochemicals, which are mainly found in the skin and seeds of the berries. the aim of this work was to contribute to the study of the nutraceutical features of some berry fruit (currants, gooseberries and strawberries). the different antioxidant compound contents of the fresh fruit of different cultivars and selections of ribes spp. and fragaria x ananassa duch have been analyzed. the fruit of 29 cultivars from 3 different species of ribes spp. and 5 strawberry cultivars have been analysed by high performance liquid chromatography coupled to a uv/vis detector and a mass detector (ms) to identify and quantify the main antioxidant compounds. as far as the ribes spp. cultivars are concerned, the presence of a high content of phenolic compounds has been confirmed, and they represent therefore an important source of antioxidant compounds. moreover, the results have shown that the considered cultivars and selections of strawberries are good sources of bioactive compounds, especially phenolic substances. the results of this study could contribute to offer new insights into the nutraceutical aspects of the considered berry fruit species. introduction the word “berry” has two meanings: one based on a botanical definition, the other on the common commercial identification. true (botanical) berries are fleshy fruit with a cartilaginous endocarp full of seeds. black currants, red currants and gooseberries are true berries because they are many-seeded epigynous berries (westwood, 1993). other berries are not real berries according to the scientific definition. blueberries, instead, can be defined as a fake epigynous berry. strawberries are not berry fruit but either are multiple fruits developed from a dialycarpous ovary (on aggregate of drupes: rubus, on aggregate of achenes: fragaria) (spichiger et al., 2002). the common term “berry fruit” includes different fruits, such as strawberry (fragaria spp.), blueberry (vaccinium spp.), currant and gooseberry (ribes spp.), raspberry and blackberry (rubus spp.). “berry fruit”, “soft fruit” and “small fruit” are synonymous terms for the above mentioned species, and they are used indifferently. in this work, the term “berry fruit” is used to indicate all the analyzed species. it is well known that a healthy diet includes the consumption of fruit and vegetables. many medical and epidemiological studies have shown an inverse relationship between fruit consumption and the incidence of coronary and heart diseases, as well as certain cancers (rice-evans et al., 1997; bub et al., 2003; vanamala et al., 2006). among plant foods, berry fruits show a high antioxidant capacity. medical research has pointed out the medicinal properties of certain pigmented polyphenols, such as flavonoids, anthocyanins, and tannins and other phytochemicals, mainly located in the skins and seeds of berries. the nutritional content and antioxidant activity are used to distinguish several berries in a new category of functional foods called “superfruits”, a rapidly-growing multi-billion dollar industry that began in 2005 and which has been identified by datamonitor as one of the top 10 food categories for growth in 2008 (food u.s.a. navigator, 2007). phenolic compounds are abundant in highly colored berry fruit, and due to their popularity and consumption, these berry fruits serve as one of the most important dietary sources of phenolics (kahkonen et al., 2001). berry fruits are reported to contain a wide variety of phenolics, including hydroxybenzoic and hydroxycinnamic acid derivatives (phenolic acids), anthocyanins, flavonols, flavanols, condensed tannins (proanthocyanidins) and hydrolyzable tannins (machiex et al., 1990). the chemical characteristics, that are, nature, size, structure, solubility, degree and position of glycosylation, the type of esterification or polymerization and conjugation of phenolics with other compounds can influence their bioavailability, absorption, distribution, metabolism and excretion in humans (aherne and o’brien, 2002; hollmann, 2001). phenolic components, as mentioned above, can range from simple molecules, such as phenolic acids, to highly polymerized compounds, such as tannins. the phenolic compounds found in berries can be divided into two principal categories: flavonoid compounds and non-flavonoid compounds, which include the so called phenolic acids, tannins and coumarins. vitamin c is also an important antioxidant berry fruit constituent. vitamin c is a highly effective antioxidant in humans, which act by lowering oxidative stress, a substrate for ascorbate peroxidase. it is also an enzyme cofactor for the biosynthesis of many important biochemicals, and it acts as an electron donor for eight different enzymes. the term vitamin c is usually used as the generic descriptor of all the components of fruit that exhibit the biological activity of ascorbic acid. in fruit, vitamin c is assumed to be the sum of the ascorbic acid (aa) and dehydroascorbic acid (dhaa) contents. these two substances are readily oxidized, especially when exposed to elevated temperatures, some divalent cations (e.g. copper and iron), oxygen, alkaline ph, light or degradative enzymes. ascorbic acid is a necessary human nutrient, and its biological functions are centered on its antioxidant properties in the biological system. furthermore, it prevents common degenerative processes (smirnoff et al., 2000). ascorbic acid is the principal biologically active form, but dehydroascorbic acid also exhibits biological activity, since it can easily be converted into ascorbic acid in the human body. therefore, it is important to measure both ascorbic and dehydroascorbic acid. the aim of this work was to contribute to the study of the nutraceutical features of some berry fruit (currants, gooseberries and strawberries), by analyzing the fresh fruit of different cultivars and selections of ribes spp. and fragaria x ananassa duch. materials and methods plant material currant and gooseberry samples of 29 cultivars from three different species of ribes spp. (tab. 1a) have been harvested at commercial 2 d. donno, m. cavanna, g.l. beccaro, m.g. mellano, d. torello marinoni, a.k. cerutti, g. bounous ripeness, according to the maturity stage of the cultivars. all the cultivars were collected from a germplasm repository in the piedmont region, italy. a protocol usually applied for raspberries (zafrilla et al., 2001) and strawberries (allende et al., 2007) has been modified for the currants and gooseberries. five strawberry genotypes (fragaria x ananassa duch.) (tab. 1b) were collected from a greenhouse on a spanish farm (huelva, andalusia, spain) under mediterranean climatic conditions. this fruit was harvested at the commercial maturity stage. three replications of 10 g of fruit were selected and frozen for each genotype in liquid nitrogen and kept at -80 °c until analyzed, while other three replications of 10 g of fruit where immediately analyzed for their vitamin c content. extraction of phenolic compounds the phenolic compounds were extracted as described by allende et al. (2007). a lyophilized powder sample (0.77 g) of each sample was homogenized with 25 ml of an extraction solution (acetone/ water/acetic acid, 70:29.5:0.5, v/v/v) using an ultra turrax (ika, staufen, germany) for 1 min on ice, and this was followed by sonication in a bath-type ultrasonic cleaner (5510e-mth, branson ultrasonic corporation, u.s.a.) for 15 min. the homogenates were then centrifuged at 3,200 rpm for 10 min in a jp selecta centronic centrifuge. the acetone was evaporated at 35 °c using a rotary evaporator under vacuum. after evaporation of the acetone, the residue was flushed through a sep-pak c-18 cartridge, previously activated with methanol, and then with water and air. the phenolic compounds were absorbed onto the column, while sugars, acids and other water-soluble compounds were eluted with 10 ml of water. the phenolic compounds were then recovered with approximately 8 ml of methanol. the methanol was evaporated at 35 °c under vacuum and recovered in 1 ml of extraction solution, filtered through a 0.45 µm nylon filter (millex hv13, millipore, bedford, ma) and directly analyzed by high performance liquid chromatography (hplc). analysis of phenolic compounds using reverse-phase hplc with diode array detector (dad) and tandem mass spectrometry (ms-ms) fifty µl samples of the extracts were analyzed using an hplc system (merck hitachi, tokyo, japan) equipped with a model l 7100 pump and a model l-7455 photodiode array uv/vis detector. the samples were injected using an autosampler (model l-7200). compound separations were achieved on a 250 mm x 4 mm i.d., 5 µm reversed phase lichrocart c18 column (merck, darmstadt, germany) with water/formic acid (h2o:ch3cooh) (95:5, v/v) (a) and methanol (b) used as mobile phases. the linear gradient started with 3 % b, at 5 min 5 % b, at 10 min 8 % b, at 15 min 13 % b, at 19 min 15 % b, at 47 min 40 % b, at 64 min 65 % b, at 66 min 98 % b, at 69 min 98 % b and at 70 min 3 % b. the flow rate was 1 ml/min, and chromatograms were recorded at 280, 320, 360 and 510 nm. anthocyanins were quantified through comparisons with an external standard of cyanidin 3-glucoside at 510 nm, flavonols as quercetin 3-rutinoside at 360 nm, hydroxycinnamic acid derivatives as chlorogenic acid at 320 nm, and flavan-3-ols as catechins at 280 nm (all these markers were from sigma, st. louis, mo). the results were expressed as mg per 100 g fresh weight. the phenolics identification was carried out considering their uv spectra, molecular weights, and their ms-ms fragments. electrospray mass spectrometric analyses were performed using an hplc system equipped with a dad detector and mass detector in series consisting of a g1312a binary pump, a g1313a autosampler, a g1322a degasser, a g1315b photodiode array detector, and an ion trap mass spectrometer equipped with electrospray ionization (esi), operating in the negative ion mode, and controlled by software (v.4.0.25) from agilent technologies (waldbronn, germany). the heated capillary and the voltage were maintained at 350 °c and 4 kv, respectively. mass scan (ms) and daughter (ms-ms) spectra were measured from m/z 100 to ~1500. collisioninduced fragmentation experiments were performed in the ion trap using helium as the collision gas, and the collision energy was set at 50 %. the mass spectrometry data were acquired in the negative mode for all of the phenolic compounds. the phenolic compounds in the fruit extracts were identified by their tab. 1a: ribes spp. cultivars ribes spp. cultivars ribes nigrum l. (black currant) ribes rubrum l. (red currant) ribes grossularia l. (goosberry) ribes x nidigrolaria (hybrids) andega augustus rokula jogranda ben lomond blanka field a jostine ben more erden ben sarek gloire de sablons black down jonker van tets black reward junifer burga red poll geant de boskoop red start invigo red win noir de bourgogne roodneus royal de naples rotet tenah rovada field a wellington werdavia tab. 1b: fragaria x ananassa l. cultivars and selections fragaria x ananassa l. 163m88 148m6 150m61 29k55 160m77 nutraceutical profiles of currants and strawberries 3 uv spectra, which was recorded by means of a diode-array detector and hplc-ms, and, wherever possible, through chromatographic comparisons with authentic pure standards. analysis of proanthocyanidins: phloroglucinol protocol the analysis of proanthocyanidins was performed as described by kennedy et al. (2001). the proanthocyanidins (pas) of 11 samples belonging to genus ribes were made to react with a solution of 0.1 n hcl in meoh, containing 5 g/l phloroglucinol and 10 g/l ascorbic acid at 50 °c for 10 min, and then combined with 1.2 ml of aqueous sodium acetate to stop the reaction. the phloroglucinol adducts were analyzed by means of reversedphase hplc. the used column was a lichrocart c18 (particle size 5µm, 250 mm x 4 mm i.d., merck, darmstadt, germany), protected by a guard column containing the same material. the method utilized a binary gradient with a mobile phase containing 1 % v/v aqueous acetic acid (mobile phase a) and methanol (mobile phase b). the eluting peaks were monitored at 280 nm. the elution conditions were 1.0 ml/min; 5 % b for 10 min, a linear gradient from 5 to 20 % b for 20 min, a linear gradient from 20 to 40 % b for 25 min. then the column was washed with 90 % b for 10 min and reequilibrated with 5 % b for 5 min before the next injection. the proanthocyanidin cleavage products were estimated using their response factors in order to calculate the apparent mean degree of polymerization (mdp), the sum of all subunits (flavan-3-ol monomers and phloroglucinol adduct, in moles) was divided by the sum of all flavan-3-ol monomers (in moles). extraction and analysis of vitamin c the ascorbic acid (aa) and dehydroascorbic acid (dhaa) contents were determined according to zapata and dufour (1992), with some modifications. the analysis was just carried out with fresh strawberry samples. 10 g of fresh fruit was homogenized for 30 s in 10 ml of the extraction solution (0.1 m citric acid, 2 mm ethylene diamine tetraacetic acid (edta) disodium salt and 4 mm sodium fluoride in methanol – water 5:95, v/v) on a ultraturrax t-25 (jauke and kundel ika, dabortechnik, staufen, germany). the homogenates were then filtered through cheesecloth, centrifuged at 10,500 rpm for 5 min at 2-5 °c, filtered through an activated c18 sep-pak cartridge (waters, milford, ma) and finally filtered through a 0.45 µm filter. then, 250 µl of 1,2-phenylenediamine dihydrochloride (opda) solution (35 mg/100 ml) was added to 750 µl of extract for dehydroascorbic acid derivatization in the fluorophore 3-(1,2dihydroxyethyl)furo[3,4-b]quinoxaline-1-one (dfq). after 37 min in darkness, the samples were analysed by means of hplc. the ascorbic and dehydroascorbic acids were evaluated using an hplc system (merk hitachi, tokio, japan), equipped with an l6000 pump coupled to a d-2500 variable-wave-length uv detector. 20 μl samples were injected into a reversed-phased kromasil 100 c18 column (250 mm × 4 mm i.d., 5 µm particle size; tecnokroma, barcelona, spain) with an ods c18 guard precolumn. the mobile phase was methanol/water (5/95, v/v), 5 mm cetrimide and 50 mm kh2po4 at ph 4.5. the flow rate was kept at 0.9 ml/min. the detector wavelength was initially set at 348 nm, and after being dfq eluted, it was manually shifted to 261 nm for ascorbic acid detection. the ascorbic and dehydroascorbic acids were identified and quantified through a comparison with the pattern areas from aa and dhaa. the aa and dhaa standards were purchased from sigma-aldrich (steinheim, germany). the vitamin c content was calculated by adding the ascorbic and dehydroascorbic acid contents, and the results were expressed as mg per 100 g fresh weight. statistical analysis all the data were subjected to one-way analysis of variance (anova) to compare the means, using spss 18.0 software (chicago, il), and significant differences between samples were calculated according to the hds tukey multiple range test at p<0.05 (probability level). the data shown are the mean values (n=3) ± standard deviation. results characterization and quantification of the phenolic compounds the combination of the data with mass spectra (ms) data allowed a tentative identification to be made of the conjugated forms, as obtained in previous studies (he, 2000; schieber et al., 2001). strawberries fifteen compounds were identified and quantified in the hplc-dad chromatograms of the strawberry selections. the molecules were classified as different phenolic compounds (tab. 2): phenolic acids (p-coumaric and ellagic acid); flavonols (quercetin, kaempferol); ellagitannins and anthocyanins (pelargonidin and cyanidin derivatives) (fig. 1). the peaks were identified by comparing the uvvisible spectra with those of the available standards. further support for this identification was obtained from their ms-ms analyses. the lower peaks, without the typical spectral characteristic of the standard, remained unidentified. the phenolic contents were different for the five strawberry selections, with a mean of 63.46 mg/g fresh fruit. the concentration ranged from 43.15 mg/100 g fresh fruit (163m88) to 75.53 mg/ 100 g (160m77), showing statistically significant differences (p<0.05) between the selections. ellagitannins the ellagitannins only showed characteristic uv spectra for an absorption maximum below 280 nm. four peaks of ellagitannins were found in all the strawberry extracts, except for the 148m6 selection which only had three peaks. peak 2 had [m−h]− at m/z 935 and the main ms2 fragments at m/z 633 and m/z 301; it was identified as galloyl-bis-hhdp-glucose. peak 4 had [m−2h]2− at m/z 935, corrsponding a mass of 1869. the ms/ms spectrum had an m− at m/z 1869 which fragmented to produce m/z 1567 (m – 302, a loss of a hexahydroxyphenoyl (hhdp) unit), 1265 (m – 302, a further loss of hhdp), 1103 (m – 162, a loss of a glucosyl group), 933 (m – 170, a loss of a gallic acid) and 631 (m – 302, a loss of hhdp). this peak was identified as sanguiin h-6 or agrimoniin, which is a dimer of casuarictin/potentillin. peak 5 had a λmax of 250 nm and, when subjected to acid hydrolysis, yielded ellagic acid. the mass spectrum of this compound was complicated. it showed a [m−2h]2at m/z 1401. the main products were m/z 2019, 1869, 1567 (a loss of hhdp), 1402, 935, 897, 633 (a loss of trihhdp-galloyl-glucose). this peak was identified as lambertianin c, which is a trimer of casuarictin/potentillin. the late-eluting ellagitannin compound (peak 9) remained unknown and requires further identification. the most abundant phenolic compounds found in strawberries were ellagitannins, with an average of 30.77 mg/100 g, and they showed statistically significant differences (p<0.05). the selections containing the highest level of total ellagitanins were 29k55 and 160m77, with 37.94 and 34.94 mg/100 g, respectively. the most important group within the ellagitanins was sanguiin-h6, which on average reached 59 % of the total ellagitannins. 4 d. donno, m. cavanna, g.l. beccaro, m.g. mellano, d. torello marinoni, a.k. cerutti, g. bounous tab. 2: phenolic compound contents of the strawberry selections. results are expressed as mg per 100 gfw (mean ± sd, n=3). values with different letters showed statistically significant differences. compound strawberry selections 148m6 150m61 160m77 163m88 29k55 anthocyanins cyanidin 3glucoside 0.88± 0.08 1.47±0.09 1.21±0.14 0.65±0.04 1.39±0.04 pelargonidin 3-glucoside 18.98±1.6 24.39±1.08 24.25±2.04 10.40±0.07 21.22±2.3 pelargonidin 3-rutinoside 2.35±0.21 1.48±0.01 1.03±0.13 0.42±0.03 0.88±0.12 pelargonidin 3acetylglucoside nd 6.40±0.28 6.36±0.21 3.11±0.11 5.37±0.61 total 22.21 b 34.28a 32.85a 14.59c 28.87a flavonols quercetin 3-glucoronide 1.03±0.02 1.18±0.06 0.56±0.02 0.83±0.01 1.71±0.11 unknown 0.40±0.05 nd nd nd nd kaempferol 3-glucoside 0.07±0.01 0.18±0.01 0.10±0.01 nd 0.13±0.02 total 1.50b 1.36b 0.66c 0.83c 1.84a ellagic acid ellagic rhamnoside 0.63±0.02 0.59±0.04 0.50±0.03 0.51±0.01 0.61±0.05 ellagic rhamnoside 0.74±0.04 0.76±0.04 0.46±0.02 0.56±0.02 0.57±0.06 total 1.37a 1.34a 0.95c 1.07bc 1.19ab hydroxycinnamic acids p-coumaric acid 4-glucoside 4.07±0.48a 4.49±0.32a 4.13±0.42a 0.33±0.04b 3.48±0.4a ellagitannins galloyl bis hhdp gluc. nd 4.18±0.94 4.28±0.18 3.29±0.29 5.11±0.73 sanguiin -h6 16.34±0.34 15.28±1.89 21.06±0.84 17.79±0.51 20.27±1.2 lambertianin c 5.59±0.04 5.57±1.07 5.44±0.55 1.64±0.06 7.84±1.13 unknown 4.15±0.32 3.56±0.56 4.15±0.21 3.61±0.06 4.72±0.05 total 26.08b 28.58b 34.94a 26.33b 37.94a total phenolic compounds 55.23b 70.07a 75.53a 43.15c 73.31a anthocyanins the anthocyanins found in strawberries were glycosides of cyanidin (λmax at 512 nm) and of pelargonidin (λmax at 495 nm). peak 6 in the dad-chromatogram had an absorption maximum at 512 nm, as well as molecular ions at m/z 447, with fragments at m/z 285 (a loss of hexose) and was identified as cyanidin 3-glucoside. three peaks (7, 8 and 14) had absorption maxima at 495 nm as well as ms fragmentation ions at m/z 269, and were identified as derivatives of pelargonidin. peak 7, with [m−h]− at m/z 431 and a subsequent loss of 162 amu (hexose), was pelargonidin 3-glucoside. peak 9, with [m−h]− at m/z 577 and a loss of 308 amu (deoxyhexosehexose), was assigned as pelargonidin 3-rutinoside. one less polar pelargonidin derivative, peak 14, had [m−h]− at m/z 473 as well as a loss of 204, and was assigned as pelargonidin 3-acetylglucoside. the total anthocyanins ranged from 14.59 mg/100 g (163m88) to 34.28 mg/100 g (150m61), with an average of 26.56, and showed statistically significant differences (p<0.05). the 150m61, 160m77 and 29k55 selections showed the highest concentrations of all studies. the lowest concentration was observed in 163m88. tab. 2 shows the single concentrations of the four major anthocyanins (i.e. cy 3-gluc, pg 3-gluc, pg 3-rut and pg 3-acetylgluc) in the five strawberry selection extracts. as far as the anthocyanins distribution is concerned, pg 3-glucoside was the predominant compound in the strawberries, representing 71 % of the total mean amount of anthocyanins, and this was followed by pg acetylglucoside, pg 3-rutinoside and finally cy 3glucoside. flavonols the flavonols detected in the strawberries were derivatives of quercetin and kaempferol. the flavonols were classified on the basis of the shape of their uv-visible spectra, with an absorption maximum at about 352 nm for quercetin glycosides and at about 344 nm for kaempferol glycosides. the shift in the uv maximum from 352 to 344 nm is due to the lack of additional hydroxyl groups attached to ring b in kaempferol, compared to quercetin. three peaks were detected in the uv-chromatogram, at tr 42, 48 and 50 min, respectively. the peak 10 had a λmax of 365 nm. the mass spectrum had an m/z 477 molecular ion that yielded an m-176 (cleavage of a glucoronosyl unit) fragment ion at m/z 301, indicating the presence of quercetin-3-glucuronide. peaks 13 was identified as a kaempferol derivative, due to its uv-spectrum and ms2 fragmentation ion at m/z 285 in negative ms mode; this peak with [m−h]− at m/z 447 lost hexose (162 amu) during fragmentation, and was assigned as kaempferol 3-glucoside. peak 15 was identified as flavonol, due to its uv-spectrum, but no further data could be obtained from lc-ms analysis for its tentative identification. the total flavonol contents ranged from 0.66 (160m77) to 1.84 (29k55) mg/100 g; the total levels were also statistically different nutraceutical profiles of currants and strawberries 5 fig. 1: chromatographic patterns of strawberry phenolic compounds obtained at 280, 320, 360 and 520 nm. peak assignment is provided on the right side. (p<0.05). quercetin 3-glucoronide was the most abundant flavonol in these strawberry samples, with a concentration of 85.5 % of the mean total flavonol content. quercetin 3-glucoronide was the only flavonol compound found in the 163m88 selection, while the 29k55 selection showed the highest values of this compound (1.71 mg/ 100 g). 6 d. donno, m. cavanna, g.l. beccaro, m.g. mellano, d. torello marinoni, a.k. cerutti, g. bounous ellagic acid ellagic acid and its glycosides were distinguished by means of their characteristic uv-visible spectra with absorption maxima at 254 and 360-368 nm, respectively. two ellagic acids were detected in the strawberry selections at 45 min and 45.4 min. peaks 11 and 12 had [m−h]− at m/z 447. the ms2 products of the ions were at m/z 301, with further fragmentation, being characteristic for ellagic acid. these peaks were identified as ellagic acid rhamnoside. the ellagic acid levels were between 0.95 (160m77) and 1.37 mg/100 g (148m6), with a mean value of 1.19 mg/100 g, and showed statistically significant differences (p<0.05). hydroxycinnamic acids the most important peak in the uv chromatogram obtained at 320 nm was for the hydroxycinnamic acids: peak 1 was identifed as a p-coumaric ester (absorption maximum at 310 nm) (tr 17 min). this peak had [m−h]− at m/z 487 with ms2 fragments at m/z 325 (loss of hexose). this compound was identified as a hexose ester (pcoumaric acid 4-glucoside). p-coumaric acid glucoside was the only hydroxycinnamic acid found in the analyzed strawberry samples; its content varied from 0.33 mg/100 g (163m88) to 4.49 mg/100 g (150m61), and showed statistical differences (p<0.05) with a mean of 3.30 mg/100 g. finally, the peaks indicated with number 3 were recognized as generic procyanidins. the identification of this class of compounds was based on the chromatographic behaviour, uv-vis spectra and a comparison with literature. quantification and identification of the single peaks were not carried out. currants and goosberries the protocol usually used for the identification of phenolics in raspberries (zafrilla et al., 2001) and in strawberries (allende fig. 3: hplc separation of anthocyanins detected at 520 nm from red currant (cv red poll). peak assignment is provided on the right side. fig. 2: hplc separation of anthocyanins detected at 520 nm from black currant (cv royal de naples). peak assignment is provided on the right side. nutraceutical profiles of currants and strawberries 7 et al., 2007) was adapted for currants and gooseberries. unfortunately, it was not possible to provide the complete chromatographic pattern of the phenolic compounds. in this study, only the anthocyanin compound data were clear and have been reported. anthocyanins the hplc analyses of 29 different cultivars of ribes spp. led to the identification of 7 anthocyanins in ribes nigrum l., 6 in ribes rubrum l., 3 in the hybrids and 2 in ribes grossularia l. ribes nigrum l. peaks 1 and 2 were identified as two delphinidins (fig. 2), the first as delphinidin 3-glucoside, with [m−] at m/z 463 and a loss of 162 amu (hexose) and the second one as delphinidin 3-rutinoside with [m−] at m/z 609 and a loss of 308 amu (deoxyhexose-hexose) upon fragmentation. peak 3 was identified as cyanidin-3-rutinoside. the mass spectrum of peak 4 revealed a small molecular ion at m/z 623 m− and a major fragment ion at m/z 315; this corresponded to petunidin 3-rutinoside (mw 623), and the fragment ion [m – 308], which originated from the loss of rutinoside moiety, resulted in petunidin (mw 317). the mass spectrum of peak 5 showed significant signals corresponding to the peonidin 3-rutinoside molecular ion at m/z 607 m− and a major fragment ion at m/z 299. peak 6 revealed m/z 609 and m/z 301. these masses corresponded to delphinidin 3-(6coumaroyl)-glucoside and its characteristic loss of m/z 308, the coumaroyl glucoside moiety, which forms delphinidin. the last identified anthocyanin component was cyanidin 3-(6-coumaroyl)-glucoside, which shows m/z 593 m− and 285, corresponding to a loss of coumaroyl glucoside. the anthocyanin content in black currant ranged from 47.03 (cv ben sarek) to 661.81 mg/100 g (cv invigo) with a mean value of 313.37 mg/100 g, and showed statistically significant differences between groups (p<0.05). the most abundant anthocyanins found in these cultivars of ribes nigrum l. were delphinidin 3-rutinoside and cyanidin 3-rutinoside with a mean value of 133.60 and 113.14 mg/100 g, respectively. ribes rubrum l., ribes grossularia l. and hybrids the first peak in the ribes rubrum l. cultivars was identified as anthocyanin, due to its uv-spectrum, but no further data were obtained for its tentative identification from the lc-ms analysis (fig. 3). peak 2 showed a shorter tr than those of the other peaks. it had a delphinidin aglycon (fragment m/z 301 m−) and a molecular weight of 595. the fragment loss was 294, which could be due to a sambubiose residue. this anthocyanin was identified as delphinidin 3-sambubioside. the other four anthocyanins detected in the ribes rubrum l. cultivars were glycosides of cyanidin (λmax at 512 nm). peak 3 was identified as cyanidin 3sophoroside. peak 4 produced a mass spectrum, with a m− at m/z 579, which yielded a fragment ion at m/z 285 (m – 294) and was identified as cyanidin 3sambubioside. peak 5 was recognized as cyanidin 3-xylosylrutinoside (cy 3xylrut), presenting a molecular weight of 725 m− and a fragment ion at m/z 285. the last anthocyanin (peak 6) was identified as cyanidin 3-rutinoside. two anthocyanin compounds were detected in cv. rokula, the only ribes grossularia l. cultivar: delphinidin 3-rutinoside and cyanidin 3-rutinoside. three anthocyanin compounds were detected in the two analyzed hybrids: delphinidin 3-glucoside, delphinidin 3-rutinoside and cyanidin 3-rutinoside. the anthocyanin content in the red currants, gooseberries and hybrids ranged from 5.74 (gooseberry, cv rokula) to 85.74 mg/100 g (red currant, cv jonker van tets), with a mean value of 33.83 mg/100 g, and showed statistically significant differences (p<0.05). the anthocyanins in the ribes rubrum l. cultivars, ranged from 14.09 (cv red win) to 85.74 mg/100 g (cv jonker van tets), with a mean value of 38.29 mg/100 g. the most abundant anthocyanin found in these cultivars of ribes rubrum l. was cyanidin 3-xylosylrutinoside, with an average value of 21.80 mg/100 g. the jogranda cultivar showed a higher anthocyanin value (32.98 mg/100 g) among the hybrids. analysis of proanthocyanidins the proanthocyanidins in the 10 analyzed cultivars of ribes spp. were procyanidins and prodelphinidin; (epi)catechin and (epi)gallocatechin were present as subunits. analysis of vitamin c strawberries statistically significant differences were found for the singular aa and dhaa contents for the five analyzed strawberry selections. the aa content ranged from 25.74 (150m61) to 30.36 (29k55) mg/100 g. the dhaa content varied from 3.03 (29k55) to 7.46 (160m77) mg/100 g. the total vitamin c content (aa+dhaa) varied from 31.74 (150m61) to 35.87 (160m77) mg/100 g, with a mean value of 33.32, but no statistically significant differences were observed (tab. 3). tab. 3: ascorbic acid (aa) and dehydroascorbic acid (dhaa) contents. results are expressed as mg per 100 gfw (mean ± sd, n=3). values with different letters showed statistically significant differences. vitamin c aa dhaa total fragaria x ananassa duch. 163m88 26.72 ± 1.10 b 6.74 ± 1.11 a 33.46 ± 2.21 a 148m6 26.38 ± 0.55 b 5.78 ± 0.59 a 32.16 ± 1.14 a 150m61 25.74 ± 1.63 b 6.00 ± 0.64 a 31.74 ± 2.27 a 29k55 30.36 ± 0.91 a 3.03 ± 0.30 b 33.39 ± 1.21 a 160m77 28.41 ± 1.69 ab 7.46 ± 0.98 a 35.87 ± 2.67 a discussion phenolic compounds strawberries the phenolic compounds found in the five strawberry selections have already been mentioned in literature, but different concentrations have been found (aaby et al., 2005). the selections that showed the highest phenolic compounds were 160m77, 29k55 and 150m61. the mean value of the phenolic compound concentration was 63.46 mg/100 g. ellagitannins were detected at 280 nm. the ellagitannins, together with anthocyanins were the most abundant phenolic compounds in the strawberries. in this study, ellagitannins were the main phenolic class in the strawberries, representing 49 % of the analyzed phenolic compounds; this result is in agreement with a previous study performed by kähkönen et al., (2001). the mean content of ellagitannins found in this research (30.77 mg/100 g) was within the range found in literature (25-59 mg/100 g) (häkkinen and törrönen, 2000; skupien et al., 2004). ellagitannins and ellagic acid have been reported to show in vitro and in vivo antitumori8 d. donno, m. cavanna, g.l. beccaro, m.g. mellano, d. torello marinoni, a.k. cerutti, g. bounous genic and antipromoting activities (larrosa et al., 2006); for this reason, there is great interest in evaluating the concentrations of ellagitannins and ellagic acid. ellagic acid is a dimeric derivative of gallic acid and is generally recognized as a hydrolytic byproduct of the release of a hexahydroxydiphenoyl (hhdp) ester group from ellagitannins, which spontaneously converts to its characteristic bislactone structure. ellagic acid was mostly present as ellagitannins, and, in this research, the relative amount of ellagic acid and its glycosides was <2 % of the total phenolics. the mean content of ellagic acid found in this study was 1.18 mg/100 g, a similar value to those previously reported for strawberries by aaby et al. (2005), but lower than the values obtained by koponen et al. (2007). this is feasible because the present extraction used a medium containing acetone, as in aaby et al. (2005), whereas koponen et al. (2007) applied acid hydrolysis, which favours the release of ellagic acid from ellagitannins esterified with glucose. the second phenolic class in the strawberries was anthocyanins, a result that is in agreement with the literature (kähkönen et al., 2001). anthocyanins are a group of flavonoids with exceptionally good antioxidant properties, which have been attributed to the phenolic hydroxyl groups attached to the ring structures (wang et al., 2002). the mean value of anthocyanins detected in this work was 26.56 mg/100 g; this value is similar, but slightly lower than that found by määttä et al. (2004) (34.66 mg/100 g). two anthocyanin glycosides, pelargonidin 3-glucoside and cyanidin 3-glucoside, are almost exclusively responsible for the red color of strawberries. in this study, pelargonidin 3glucoside was found to be the predominant pigment of the strawberries, and on average represented 75 % of the total anthocyanin content, in agreement with previous studies (määttä et al., 2004). the fruit color is affected by many ecological factors, such as light and temperature. for this reason, it is possible to find differences in the anthocyanin concentration among studies carried out under different climatic conditions. the average flavonol content found in this study in the five strawberry selections was 1.24 mg/100 g; a similar value of 2.03 mg/100 g was found in the strawberries by määttä et al. (2004). it is important to remember that the flavonoid content in strawberries (anthocyanins, flavonols, catechins and flavanones), as in any other fruit, depends on a series of factors, such as the stage of maturity, cultivar, storage conditions and analytical methods, which can readily be inferred from a comparison of different studies on this topic (mikkonen et al., 2001). quercetin 3-glucuronide has been reported to be the main flavonol in strawberry (aaby et al., 2007), and, again in this work, it has been the most abundant detected flavonol. flavonols are usually present in food as their glycosides, but are found in biological fluids also as their glucuronidated derivatives (manach and donovan, 2004). p-coumaric acid 4-glucoside was the only hydroxycinnamic acid detected in the analysed strawberry selections, with a mean value of 3.30 mg/100 g, a comparable, but slightly higher value, than formerly found by maatta et al. (2004) (2.57 mg/100 g) in fragaria x ananassa cultivars. the literature data confirm that pcoumaric acid is the most abundant aglycon in strawberries. soluble hydroxycinnamic acids mainly occur as esters in berry fruit, except in cloudberries (maatta et al., 2004); free hydroxycinnamic acids have infrequently been reported in fruit, but it is possible that free hydroxycinnamic acids are released at the late stages of ripening in cloudberry, due to environmental stress factors, or that the unbound acids are typical for these wild berries (kumpulainen and salonen, 1996). currants and gooseberries anthocyanins are mportant polyphenolic components in ribes spp. (määttä et al., 2001). the mean content of anthocyanins found in this work for the black currant cultivars was 313.37 mg/100 g; this value was lower than the content of 476.17 mg/100 g found by wu et al. (2004). the most abundant anthocyanin found in these black currant cultivars was delphinidin 3glucoside, as demonstrated by wu et al. (2004). the mean amount of anthocyanins detected in the red currant cultivars was 38.29 mg/100 g; this value was higher than that found by wu et al. (2004) (12.09 mg/100 g). the differences can be explained by the fact that the average value in this work is a mean value obtained from 13 different ribes rubrum l. cultivars, while wu et al. (2004) analyzed just one cultivar. cyanidin 3xylosylrutinoside was the most abundant anthocyanin detected in the analyzed red currant cultivars, as already found by wu et al. (2004). rokula, the only gooseberry cultivar considered in this research, showed a mean anthocyanin value of 5.74 mg/100 g, a similar value to that detected by wu et al. (2004) (5.77 mg/100 g). finally, the two hybrids showed a mean anthocyanin value of 25.55 mg/100 g, a lower value than that found by jordheim et al. (2007). it is interesting to note that the individual anthocyanins, identified in the two hybrids, reflected that these cultivars contained more anthocyanins than both parents. this result confirms what has already been observed by jordheim et al. (2007). phloroglucinol: identification of proanthocyanidins proanthocyanidins are polymeric flavonoid compounds. in this study it was possible to identify the different proanthocyanidins present in 11 cultivars of ribes spp. and the mdp (apparent mean degree of polymerization) was also found. the mdp value was 19.60, although the black currant cultivars showed a mdp of 22.42, a lower value than that found by wu et al. (2004) (47.90). this result can be considered positive, because some studies have suggested that low proanthocyanidin oligomers (dp<4) could be absorbed in the gastrointestinal tract (santos-buelga et al., 2000). dimers have been detected in human blood, after a proanthocyanidins-rich diet has been consumed, while trimers have been shown to be absorbed through the human intestinal cell line caco-2 (deprez et al., 2001). for this reason, identification of heterogenous proanthocyanidins, especially the low oligomers, are emphasized. (holt et al., 2002). analysis of vitamin c the mean value of total vitamin c (ascorbic acid + dehydroascorbic acid) contents in strawberry cultivars analyzed in this study was 33.32 mg/100 g; this value falls within the range of 23-47 mg/ 100 g detected in strawberry genotypes by tulipani et al. (2008). it has been demonstrated that strawberries are rich in vitamin c [a handful of strawberries is sufficient to cover the recommended daily allowance (rda) of vitamin c] (tulipani et al., 2008). in this study, the strawberries showed higher vitamin c values compared to the other analyzed fruit, but lower than average values of other fruit species, such as grapefruit (52 mg/100 g) or oranges (46 mg/100 g), which are well known for their high vitamin c content (proteggente et al., 2002). conclusions the aim of this work was to contribute to the knowledge of the nutraceutical features of some berry fruit species that so far have been studied less than other berry species, such as blueberry and raspberry. as far as the ribes spp. cultivars are concerned, the presence of a high phenolic compound content, especially in ribes nigrum l., has been confirmed through qualitative analysis. ribes spp. cultivars can therefore be considered as an important source of nutraceutical profiles of currants and strawberries 9 antioxidant compounds. moreover, the results have shown that the tested strawberry cultivars and selections are good sources of bioactive compounds, especially phenolics. in particular, selections 150m61, 160m77 and 29k55 have shown to possess the highest values in polyphenol contents, among the analyzed strawberry fruit selections. acknowledgements thanks are due to the department of “quality, safety and bioactivity of plant foods, food science and technology” of “cebas” (centro de edafologia y biologia aplicada del segura), murcia (spain) for their collaboration in this research. references aaby, k., skrede, g., wrolstad, r.e., 2005: phenolic composition and antioxidant activities in flesh and achenes of strawberries (fragaria ananassa). j. agric. food chem. 53, 4032-4040. aaby, k., ekeberg, d., skrede, g., 2007: characterization of phenolic compounds in strawberry (fragaria x ananassa) fruits by different hplc detectors and contribution of individual 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anthocyanins and proanthocyanidins in some cultivars of ribes, aronia, and sambucus and their antioxidant capacity. j. agric. food chem. 52, 7846-7856. zafrilla, p., ferreres, f., tomas-barberan, f., 2001: effect of pro cessing and storage on the antioxidant elleagic acid derivates and flavonoids of red raspberry (rubus idaeus) jams. j. agric. food chem. 49, 3651-3655. zapata s., dufour, j.p., 1992: ascorbic, dehydroascorbic and isoascorbic acid simultaneous determinations by reverse fase ion interection hplc. j. food sci. 57, 506-511. address of the corresponding author: e-mail: dario.donno@unito.it journal of applied botany and food quality 86, 113 125 (2013), doi:10.5073/jabfq.2013.086.016 1departamento de micologia, ccb, universidade federal de pernambuco, cidade universitaria, recife, pe, brazil 2agroscope reckenholz-tänikon research station art, ecological farming systems, zürich, switzerland paraglomus pernambucanum sp. nov. and paraglomus bolivianum comb. nov., and biogeographic distribution of paraglomus and pacispora catarina maria aragão de mello1, gladstone alves da silva1, daniele magna azevedo de assis1, juliana souza de pontes1, araeska carenna de almeida ferreira1, mariele porto carneiro leão3, helder elísio evangelista vieira1, leonor costa maia1, fritz oehl1, 2* (received april 22, 2013) * corresponding author summary paraglomus pernambucanum sp. nov. (paraglomeromycetes) was found in a tropical dry forest in the semi-arid caatinga biome of pernambuco state (ne brazil), in a cowpea and in two maize production sites. it was characterized by combined morphological and molecular analyses on the spores isolated from field soil samples. another species, pacispora boliviana (glomeromycetes), first described only by spore morphology, had been known from another semi-arid biome in southern america, the gran chaco in bolivia. we detected this fungus now also at different locations in semi-arid to semi-humid ne brazil. as for p. pernambucanum phylogenetic analyses were performed on nuclear ribosomal rna gene sequences of the lsu region. for p. boliviana, the spores for these analyses originated from a trap culture inoculated with soils from the type location. the results now revealed that also p. boliviana belongs to paraglomus. it grouped in a separate monophyletic cluster adjacent to p. pernambucanum, to p. brasilianum, p. laccatum and the type species p. occultum. thus, p. boliviana is transferred to paraglomus, as paraglomus bolivianum comb. nov. remarkably, it is the first species known in the paraglomeromycetes with pigmented spores. paraglomus pernambucanum and p. bolivianum have several features in common: e.g. bi-walled spores, and densely pitted surface ornamentations on the structural layer of the outer wall. spores of the two species can be distinguished by color and the diagnostic nature of their pitted ornamentation. the current knowledge about the global distribution of paraglomus and pacispora species is summarized and discussed. introduction arbuscular mycorrhizal (am) fungi play an important role in ecosystem functioning since they are associated with the majority of plant species, deliver essential nutrients to their symbiotic partners, and are for instance also able to suppress harmful soil-borne pathogens and pests, and may protect soils against erosion through stabilization of soil aggregates (smith and read 2008). they can also serve as bio-, soil and land use indicators (e.g. oehl et al. 2005a, 2011a, 2012). we assume that especially pacispora species might be characteristic for specific soils, land use intensity and different climates since they were found to be characteristic for soils with ph > 6.0, either in alpine areas (e.g. p. robigina; oehl and sieverding 2004, błaszkowski et al. 2008), in cultivated soils of temperate areas with lower phosphorus levels (e.g. p. dominikii; błaszkowski, 1993; oehl et al., 2005b; oehl et al., 2010), or in mediterranean and subtropical areas under semi-arid conditions subjected to naturally elevated soil ph (e.g. p. scintillans and p. franciscana; e.g. rose and trappe, 1980; bashan et al., 2007; palenzuela et al., 2008). one fungus, p. boliviana, was never found in alpine, temperate, mediterranean or subtropical areas of higher soil ph despite of extensive amf diversity studies performed in europe and northern america during the last decades (e.g. błaszkowski, 1993; koske and tews, 1987; daniell et al., 2001; jansa et al., 2002; landis et al., 2004), but so far only in the tropical, semi-arid gran chaco of bolivia (ph 6.3; oehl and sieverding, 2004), and recently also from a tropical atlantic rainforest in ne brazil (silva et al., 2012). we have lastly detected it several times in the semi-arid caatinga biome of ne brazil, which focused again our attention to this fungus. in tropical areas, however, other pacispora species were so far never reported, neither by morphological spore identifications from field soils, long-term trap culture propagations (e.g. goto et al., 2010; stürmer and siqueira, 2011; tchabi et al., 2009) nor through molecular root analyses (e.g. husband et al., 2002; öpik et al., 2010), although species of this genus could be expected in the tropics at least in semi-arid regions and soils with increased ph. pacispora boliviana has a distinct pitted ornamentation on its spore surface (sieverding and oehl, 2004). the fungus was preliminary attributed to this genus, since it forms, like all pacispora, bi-walled spores terminally on subtending hyphae. however, it was obvious from the beginning that its spore structure, and above all the structure of the subtending hyphae and the staining reaction of the wall layers in melzer’s reagent, does not fit with those characters typical for pacispora species (oehl and sieverding, 2004). however, at that time, molecular analyses of the fungus were not available, and morphologically its spores could not be attributed to any other genus within the glomeromycota, such as glomus or paraglomus (oehl et al., 2011b). glomus has only mono-walled spores, while the spore wall structure of paraglomus has not yet been fully resolved, and the genus has so far had only species without substantial spore pigmentation. when morphological analyses cannot (or not yet) resolve the relationship between different taxa, molecular analyses are urgently needed to elucidate their phylogeny (gamper et al., 2009; palenzuela et al., 2010; oehl et al., 2011d; goto et al., 2012). in the present study, we performed first molecular analyses on p. boliviana in order to clarify its phylogenetic position in relation to the other glomeromycotan fungi. more recently, an undescribed fungus, with congruent overall spore and subtending hyphae morphology as p. boliviana, was found in another tropical, semi-arid biome of southern america, i.e. in the caatinga biome of ne brazil. thus, the second objective of the present study was to carefully elaborate both the spore morphology and the phylogenetic position of this new fungus. based on the results obtained from concomitant morphological and molecular phylogenetic analyses, the new fungus is formally described hereafter. another objective was to discuss the biogeographic distribution of the two fungi, as far as this was possible at this early stage of species identification. finally, we aimed at comprehensively presenting the biogeography of the two genera, paraglomus and pacispora, in general. 114 c.m. aragão de mello, g.a. silva, d.m.a. de assis, j.s. pontes, a.c.a. ferreira, m.p.c. leão, h.e.e. vieira, l. costa maia, f. oehl materials and methods study sites the new am fungus was found in caruaru and serra talhada, pernambuco state (ne brazil), in maize (zea mays l.) fields in the semi-arid tropical caatinga biome. the sampling sites are located at 08º08’00”s and 36º02’00”w (550 m a.s.l.) and 7º59’00’’s and 38º19’16’’w (650 m a.s.l.), respectively. the climate is tropical(semi-)arid (type bs of köppen-geiger; kottek et al., 2006) with six to eight months of dry season. mean annual temperature is 2426°c, and annual rainfall is 650 and 550 mm, respectively. the maize variety sown was br 5026 ‘são josé’ (lemos et al., 1995). at these two sites, the pasture plant species sudan grass (sorghum sudanense (piper) stapf) and onion had previously been grown, respectively. more recently, the fungus was also found in a tropical caatinga dry forest where mimosa tenuifolia was the most dominant plant species and in an adjacent cowpea (vigna unguiculata) field. both locations are near petrolina, in pernambuco state (6°28’20’’-6°30’00’’s; 34°55’50-34°57’10’’w, 380 m a.s.l., mean annual temperature 26°, mean annual rainfall 550 mm). at the natural caatinga site mimosa tenuifolia was the most dominant plant species. the type location of pacispora boliviana was described in oehl and sieverding (2004). briefly, it was isolated from a degraded pasture in the semi-arid gran chaco of tropical bolivia (departamento de santa cruz de la sierra; 18°05’s; 63°20’w, 550 m a.s.l., mean annual temperature 24°, mean annual rainfall approximately 800 mm), and from trap cultures inoculated with soils from that pasture. the trap cultures were maintained under tropical temperatures in the greenhouse at the university of basel (switzerland) for eight months in 2001. in recent years, we found p. boliviana also in the semi-arid caatinga of ne brazil, e.g. in triunfo (7°50’17’’s; 38°06’06’’w), belém do são francisco (8°45’28’’s; 38°57’52’’w) and, together with the herein described new fungus, also in serra talhada (7º59’00’’s; 38º19’16’’w). all these sites are also in pernambuco state. mean annual temperature in belém do são francisco (400 m a.s.l.) is 26°c, and the mean annual rainfall is 450 mm. these values are 22°c and approximately 1200 mm, respectively, in triunfo. this isolation site has a more humid climate than it is common in the caatinga biome, with typical vegetation of an atlantic rainforest. this site profits from more frequent rainfalls due to its exposed higher altitude (650 m a.s.l.), when compared to the typical caatinga dry forest in its surrounding. soil sampling and chemical soil analyses soil samples in caruaru, serra talhada and petrolina (ne brazil) were taken in march 2011 as described by mello et al. (2012). the soils in belém do são francisco and triunfo were already taken in september and october 2008, respectively. soil samples in the gran chaco (bolivia) were taken in november 2000 as described in oehl and sieverding (2004). the soil in caruaru had a ph (h2o) of 6.4, 1.1 g kg-1 organic c and 49.4 mg kg-1 available p extracted according to nelson et al. (1953). in serra talhada, the soil had a ph (h2o) of 6.2, 0.7 g kg-1 organic c and 58.0 mg kg-1 available p, and in petrolina, ph was 5.2-6.0, organic c was 16.1 g kg-1, and available p was 22.0 mg kg-1. in belém do são francisco, ph was 6.8, organic c was 23.8 g kg-1, and available p was 16.8 mg kg-1, while ph was 6.8, organic c 39.4 g kg-1, and available p 195.0 mg kg-1 in triunfo. the bolivian soil had a ph (h2o) of 6.5; organic carbon was 26 mg kg-1, and available p (here extracted with na-acetate, see oehl et al., 2005b) was 2.3 mg kg-1. am fungal trap cultures trap cultures were established directly after sampling as described in oehl et al. (2003), tchabi et al. (2009) and mello et al. (2012). pacispora boliviana produced abundantly spores only in one of 64 trap cultures, in the rhizosphere of stylosanthes guianensis, bracharia humicicola and chromolaena odorata. the new fungus from pernambuco did not form spores in the trap cultures. using sorghum bicolor as host plant, single species cultures were initiated, that were inoculated with single or multiple (10-20) spores isolated directly from the field for the new fungus, or obtained from the trap cultures for pacispora boliviana. all these monoor multiple spore cultures failed so far. morphological analyses spores of the two fungi were separated from the soil samples by wet sieving as described by sieverding (1991). the described morphological characteristics of spores and their subcellular structures are based on observations of specimens mounted in polyvinyl alcohol-lactic acid-glycerol (pvlg; koske and tessier, 1983), in a mixture of pvlg and melzer’s reagent (brundrett et al., 1994), a mixture of lactic acid to water at 1:1, melzer’s reagent, and in water (spain, 1990). the terminology of the spore structure follows oehl and sieverding (2004) and oehl et al. (2011b, c) for species with glomoid (sensu lato and sensu stricto), paraglomoid and pacisporoid spore formation. specimens mounted in pvlg and the mixture of pvlg and melzer’s reagent were deposited at z+zt (zurich, switzerland) and urm (recife, brazil) herbaria. molecular analyses before dna extraction all spores isolated were first washed in ultrapure water and sonicated three to four times. for p. boliviana (culture code: bol 35), crude dna extracts were obtained from three single isotype spores, extracted from the trap culture that had been inoculated with field soil samples from the type location in the gran chaco. for the new fungus from pernambuco, the dna extracts were obtained from two single spores that were directly separated from the field samples of the type location in caruaru. the spores were singly placed on a slide in a drop (5-10 μl) of ultrapure water, crushed with a sterile needle. crude dna extract was used as template for a semi-nested pcr using the primers its3 (white et al., 1990) 28g2 (silva et al., 2006) and lr1 (van tuinen et al., 1998) 28g2, consecutively. pcr reactions were carried out in a volume of 50 μl, containing 75 mm tris-hcl ph 8.8, 200 mm (nh4)2so4, 0.01% tween 20, 2 mm mgcl2, 200 μm each dntps, 1 μm of each primer and 2 units of taqtm dna polymerase (fermentas, maryland, usa); cycling parameters were 5 min at 95°c (1 cycle), 45s at 94°c, 1 min at 55°c, 1 min at 72°c (40 cycles), and a final elongation of 7 min at 72°c followed the last cycle. the final amplicons (~690bp) were purified with the purelink pcr purification kit (invitrogen), sequenced directly or cloned with a clonejettm pcr cloning kit (fermentas; carlsbad, usa) following the manufacturer’s instructions and sequenced. sequencing was provided by the human genome research center (são paulo, brazil). sequence data were compared to gene libraries (embl and gen bank) using blastn (altschul et al., 1990). the new sequences deriving from the am fungi from bolivia and pernambuco were deposited in the ncbi database under the accession numbers jx122769jx122777. phylogenetic analyses the phylogeny was reconstructed by partial sequences of the lsu rrna gene. the am fungal sequences were aligned in clustalx (larkin et al., 2007) and edited with the bioedit program (hall, 1999). boletus edulis bull. and neurospora crassa shear & b.o. dodge were included as outgroup. prior to phylogenetic analysis, paraglomus pernambucanum sp. nov. 115 the model of nucleotide substitution was estimated using topali 2.5 (milne et al., 2004). bayesian (two runs over 1 x 106 generations with a burnin value of 2500) and maximum likelihood (1,000 bootstrap) analyses were performed, respectively, in mrbayes 3.1.2 (ronquist and huelsenbeck, 2003) and phyml (guindon and gascuel, 2003), launched from topali 2.5, using the gtr + g model. neighbor-joining (established with the model cited above) and maximum parsimony analyses were performed using paup*4b10 (swofford, 2003) with 1,000 bootstrap replications. biogeographic analyses a comprehensive literature and sequence data bank research was performed in order to study the biogeographic distribution of the genera paraglomus and pacispora on global scale. we included identifications performed either on the genus or on the species level, and considered for studies that were either based on spore morphology, or on the generation of species and environmental sequences. results molecular phylogeny phylogenetic analyses on the partial sequences of the lsu rrna gene place the two fungi from bolivia and pernambuco firmly, with high support values, in a monophyletic clade within the order paraglomerales next to paraglomus occultum, p. laccatum and p. brasilianum (fig. 1). taxonomy paraglomus pernambucanum oehl, c.m. mello, magna & g.a. silva sp. nov. (figs. 2-9) mycobank mb 800575 diagnosis: sporae albae ad flavo-albae, 66-95 × 62-75 mm in diametro, tunicis duabus. stratum laminatum tunicae exterioris, album ad flavo-album, 2.9-4.2 mm crassum, cum depressionibus subtilibus, 0.5-1.1 mm latis et 0.5-1.0 mm profundis ornatum, 1.3-2.4 mm in distancia. tunica interior hyalina; 1.9-3.0(-3.9) mm crassa, de novo formans et porum basae sporarum occudens. tunica hyphae confluentis cum stratis exterioribus tunicae externae, raro porum hyphae affixae occludens. in solutione melzeri tunica interior non colorans. holotypus # 37-3701: zt myc 24205. etymology: latin, pernambucanum, referring to the ne brazilian state where the species was found first. holotype: brazil, pernambuco state, caruaru (37/3701, zt myc 24205). isotypes (37-3702–37-3705 at zt myc 24206; 37-3711–373713 at urm). paratypes: brazil, pernambuco state, in the munici-paratypes: brazil, pernambuco state, in the municipalities of serra talhada and petrolina (37-3751, 37-3752, 37-3761; as zt myc 24207, 24208, 24209 at z+zt). spores are formed singly in soil. they are white to yellowish white, globose to subglobose to ovoid, 66-95 × 62-75 mm in diameter (figs. 1-4) and have an outer and an inner wall (figs. 2-3). outer wall with three layers (figs. 5-7): outer layer (owl1) evanescent to semi-persistent, subhyaline to light yellow, 0.5-0.9 mm thick, and usually tightly adherent to second layer (owl2); owl2 finely laminated, brilliant white in young mature spores to yellowish white in older spores, 2.0-2.6 mm thick, with regular, round but shallow and inconspicuous pits that are 0.5-1.1 μm in diameter and 0.5-1.0 μm deep and 1.3-2.4 μm apart; inner layer (owl3) hyaline and thin, 0.4-0.7 mm, separable under pressure but usually adherent to owl2 and then extremely difficult to observe. owl2 stains yellow in melzer’s reagent. inner wall is hyaline and three-layered (figs. 6-8). outer layer (iwl1) is 0.5-0.8 mm thick. in crushed spores it sometimes separates under light pressure from the central layer (iwl2); the central layer (iwl2) is 1.0-2.4 mm thick and the inner layer (iwl3) is very thin, 0.4-0.7 mm, flexible and may show several folds in broken spores; iwl3 is usually adherent to iwl2 and thus very difficult to observe. none of the layers stains in melzer’s reagent (fig. 9). subtending hypha (sh) is straight or recurved, 4.0-5.5 mm in diam at the spore base tapering to 3.5-4.5 mm within 6-15 mm distance from the base. it is generally slightly funnel-shaped to cylindrical (figs. 2-6), or rarely slightly constricted (fig. 9). the wall of the subtending hypha is of the same color as, and continuous with the spore wall layers owl1 and owl2, and of the same thickness, tapering to 0.5-1.1 mm within 25-90 mm distance. the length of the subtending hypha persistently remaining at the spore is often shorter (10-15 mm from the spore base), or sh is completely broken away. pore of the subtending hypha usually is open at spore base, and iw generally functions as pore closure at spore base. distribution: the new fungus was detected in maize production sites in caruaru and serra talhada, and in a tropical dry forest and a cowpea field in petrolina. all these sites are located in the semiarid caatinga biome of pernambuco state, ne brazil. specimens examined: brazil, pernambuco, caruaru (37-3701– 37-3750). brazil, pernambuco, serra talhada (37-3751–37-3760); brazil, pernambuco, petrolina (37-3761–37-3770). paraglomus bolivianum (sieverd. & oehl) oehl & g.a. silva comb. nov. basionym: pacispora boliviana sieverd. & oehl. j. appl. bot. food qual. 78:79. 2004. mycobank mb 800596 emendation: the species was well described in oehl and sieverding (2004). here it might be mentioned that the subtending hyphae sometimes have a constricted appearance when owl2 (hw2 in fig. 6a of oehl and sieverding, 2004) is constricted and owl1 already degraded. however, overall subtending hyphae shape, including owl1 respective hw1, is cylindrical to slightly funnelshaped since sh is regularly broader at spore base than at some distance from the spore (fig. 6a of oehl and sieverding, 2004). comments: the intra-specific variation between the lsu rrna gene sequences for p. pernambucanum and for p. bolivianum was around 1-2%. the sequences from p. bolivianum presented 95% of identity with those from p. pernambucanum. in the blastn analysis, the closest related species to p. bolivianum and p. pernambucanum was p. brasilianum with 93% and 92% of identity, respectively 116 c.m. aragão de mello, g.a. silva, d.m.a. de assis, j.s. pontes, a.c.a. ferreira, m.p.c. leão, h.e.e. vieira, l. costa maia, f. oehl fig. 1: phylogenetic tree of the paraglomeromycetes and archaeosporomycetes based on analysis from partial sequences of the lsu rrna gene. boletus edulis and neurospora crassa were used as outgroup. sequences are labeled with database accession numbers. support values (from top) are from neighbor-joining (nj), maximum parsimony (mp), maximum likelihood (ml) and bayesian analyses, respectively. sequences obtained in this study are in bold. (consistency index = 0.70; retention index = 0.86). paraglomus pernambucanum sp. nov. 117 figs. 2-9: paraglomus pernambucanum: figs. 2-3. crushed and uncrushed spores with outer and inner wall (ow, iw), cylindric to funnel-shaped subtending hyphae (sh) and minute pit ornamentation on ow surface. fig. 4. outer wall staining yellow in melzer’s reagent. figs. 5-6. ow and iw triple-layered (owl1-3; iwl1-3); outer surface of owl2 with minute pits. fig. 7. owl2 staining yellow in melzer’s. figs. 8-9. pore at spore base regularly open but closed by iw. 118 c.m. aragão de mello, g.a. silva, d.m.a. de assis, j.s. pontes, a.c.a. ferreira, m.p.c. leão, h.e.e. vieira, l. costa maia, f. oehl tab. 1: detection sites of paraglomus and pacispora species around the globe amf isolate ecosystem/plant species/(isolate) 1 state/region, country ph1 detected reference by2 paraglomus spp. paraglomus occultum poplar plantations iowa, usa na m walker et al. (1982) p. occultum ornamental plants in greenhouse oregon, usa na m walker et al. (1982) p. occultum na illinois, usa na m walker et al. (1982) p. occultum na the netherlands na m walker et al. (1982) p. occultum agro-ecosystems; temperate pennsylvania, usa na m franke-snyder et al. (2001) north america p. occultum sonoran desert texas & arizona, usa 7.7-7.9 m stutz and morton (1996) p. occultum boojum tree in a desert reserve baja california, mexico 7.0-8.0 m (bashan et al. 2007) p. occultum evergreen forests & pastures nicaragua & costa rica 3.9-5.6 m picone (2000) p. occultum natural ecosystems; crops like carimagua, colombia 5.0-7.0 m sieverding (1989) cassava, maize p. occultum maize pernambuco, brazil 5.5-5.7 m maia and trufem (1990) p. occultum semi-arid copper mining area bahia, brazil 6.2-7.8 m silva et al. (2005) p. occultum evergreen and deciduous forests araucanía region, chile 4.6-5.4 m castillo et al. (2006) p. occultum horticultural systems araucanía region, chile 5.5-6.1 m castillo et al. (2010) p. occultum highest elevation of plant occurrence valais, switzerland ca. 5.0 m oehl, in körner (2011) in europe, 4505 m asl! p. occultum subnival siliceous scree, 2700 m asl valais, switzerland 5.0-5.4 m oehl, unpublished p. occultum poland m błaszkowski (1989, 1993) p. occultum grasslands and crop rotation systems alsace, france; baden, 4.0-8.0, m oehl et al. (2005b, 2010) germany; basel, 5.3-7.7 switzerland p. occultum tropical forests and yam field sites benin 6.1-6.9 m tchabi et al. (2009) p. occultum namib desert namibia 7.0-8.0 m stutz et al. (1999) p. occultum forest and grassland india 7.8 m muthukumar and udaiyan (2000) p. occultum wheat golestan, iran na m sadravi (2006) p. occultum natural habitats: date palm (oasis) sultanate of oman 8.2-8.4 m al-yahya’ei et al. (2011) and native prosopis cineraria (undisturbed desert habitat) p. occultum desert ephemerals xinjiang, china 8.2 m shi et al. (2006) p. occultum (isolates from invam; ia702, iowa/florida/hawaii, usa na spseq millner et al. (2001), fl703, ha771) james et al. (2006), msiska and morton (2009) p. occultum cultivated soils of the saskatchewan, canada na spseq unpublished (jx301683-4) canadian prairies p. occultum (fj461883) (isolate from invam, cr402) costa rica na spseq unpublished p. occultum (isolates from invam; cl700, colombia na spseq millner et al. ( 2001), cl700c, cl383) turnau et al. (2001) p. occultum (isolate from invam, gr582) germany na spseq millner et al.( 2001) p. occultum (isolate from beg, beg120) alicante, spain na spseq ferrol et al. (2004) p. albidum winter wheat ohio, usa na m walker and rhodes (1981) p. albidum poplar plantations iowa, usa na m walker et al. (1982) p. albidum ‘caatinga’ dry forest bahia, brazil 6.2 m silva et al. (2005) p. albidum maize mono-cropping, crop france, germany, 5.3-7.7 m oehl et al. (2009, 2010) rotations, grasslands switzerland p. bolivianum gran chaco grasslands santa cruz, bolivia 6.5 m&spseq oehl and sieverding (2004), present study p. bolivianum ‘caatinga’ dry forest pernambuco, brazil 5.2-6.8 m present study p. bolivianum coastal ‘restinga’ forest vegetation pernambuco, brazil 5.1 m silva et al. (2012) p. brasilianum greenhouse cultures on allium porrum d.f., brasilia, brazil na m spain and miranda (1996) p. brasilianum desert ephemerals xinjiang, china 8.2 m shi et al. (2006) p. brasilianum (isolate from invam; br 105) d.f., brasilia, brazil na spseq krüger et al. (2012), p. brasilianum (isolates from invam; wv224, west virginia, usa na spseq millner et al. (2001), wv219, wv215, wv215a) msiska and morton (2009) paraglomus pernambucanum sp. nov. 119 amf isolate ecosystem/plant species/(isolate) 1 state/region, country ph1 detected reference by2 p. laccatum festuca sp. jastrzębia góra, poland. na m&spseq błaszkowski (1988b), renker et al. (2007) p. laccatum ammophila arenaria, slowinski national park, na m&spseq tadych and błaszkowski helictotrichon pubescens poland (2000), renker et al. (2007) p. laccatum grassland thuringia, germany 7.0-7.5 enseq könig et al. (2010) p. laccatum (isolate att960-3) uk na spseq krüger et al. (2012) p. lacteum central oregon desert oregon, usa 7.0-8.0 m rose and trappe (1980) p. majewskii zea mays algarve, portugal na m błaszkowski et al. (2012) p. majewskii ammophila. arenaria mallorca, spain na m błaszkowski et al. (2012) p. majewskii ammophila. arenaria karabucak-tuzla, turkey na m&spseq błaszkowski et al. (2012) p. majewskii ‘cultivated and uncultivated plants’ lubuskie, poland na m błaszkowski et al. (2012) p. majewskii ammophila. arenaria near bornholm, denmark na m błaszkowski et al. (2012) p. majewskii weeds asmara, eritrea na m błaszkowski et al. (2012) p. majewskii plantago lanceolata west pomerian, poland na m&spseq błaszkowski et al. (2012) p. pernambucanum natural caatinga, maize and cowpea pernambuco, brazil 5.2-6.4 m&spseq present study paraglomus sp. isolated from miscanthus jeonbuk, south korea na enseq lee et al. (2008) sinensis paraglomus sp. roots of panax japonicus chungbuk, south korea na enseq lee et al. (2008) paraglomus sp. isolate from invam ni116b nicaragua na spseq unpublished (fj461884) paraglomus sp. pioneer grass species miscanthus hokkaido/aichi/okinawa, 2.7-6.8 enseq an et al. (2008) sinensis in acid sulfate soils japan paraglomus sp. seminatural grasslands thuringia, germany 6.2 enseq renker et al. (2003), roots of lolium multiforum börstler et al. (2006) paraglomus sp. wetland, roots of dactylis glomerata thuringia, germany na enseq wirsel et al. (2004) paraglomus sp. semi-natural grasslands – thuringia, germany 6.2 enseq börstler et al. (2006) roots of plantago major paraglomus sp. semi-natural grasslands thuringia, germany 6.2 enseq börstler et al. (2006), hempel et al. (2007) paraglomus sp. roots of ajuga reptans yorkshire, uk na enseq unpublished (aj854100) paraglomus sp. onion (trap plant roots) england-uk na enseq unpublished (fn555262-92) paraglomus sp. arable soils – roots of zea mays basel, switzerland 4.8-5.4 enseq hijri et al. (2006) paraglomus sp. arable soils – roots of triticum basel, switzerland 5.4 enseq hijri et al. (2006) aestivum paraglomus sp. arable soils – colonized roots basel, switzerland 4.8 enseq hijri et al. (2006) paraglomus sp. aquatic macrophytes in oligotrophic the netherlands na enseq baar et al. (2011) and ultra-oligotrophic lakes paraglomus sp. organic and conventional farming the netherlands 7.4 enseq galván et al. (2009) systems – roots of allium cepa paraglomus sp. geothermal soils – roots of iceland 4.0-4.5 enseq appoloni et al. (2008) agrostis stolonifera paraglomus sp. geothermal soils – colonized roots yellowstone, usa 3.4-4.8 enseq appoloni et al. (2008) paraglomus sp. geothermal soils– roots from yellowstone,usa 4.8 enseq bunn et al. (2009) dichanthelium lanuginosum paraglomus sp. colonized roots yellowstone,usa 4.8-6.5 enseq lekberg et al. (2011) paraglomus sp. grasslands dominated by exotic california, usa na enseq unpublished (gq890654, gq890656) plant species paraglomus sp. reforestation plots on degraded south of ecuador 4.0-5.0 enseq haug et al. (2010) pasturesroots of setaria sphacelata and heliocarpus americanus paraglomus sp. roots of retama sphaerocarpa, murcia, spain na enseq alguacil et al. (2011a) psoralea bituminosa and lolium perenne paraglomus sp. seedlings grown in a heavy metal murcia, spain na enseq alguacil et al. (2011b) polluted soil paraglomus sp. roots of herniaria fruticosa and murcia, spain na enseq alguacil et al. (2012b) senecio auricula 120 c.m. aragão de mello, g.a. silva, d.m.a. de assis, j.s. pontes, a.c.a. ferreira, m.p.c. leão, h.e.e. vieira, l. costa maia, f. oehl amf isolate ecosystem/plant species/(isolate) 1 state/region, country ph1 detected reference by2 paraglomus sp. galls and roots of prunus persica aragua, venezuela 5.18 enseq alguacil et al. (2011c) paraglomus sp. ricinus communis soil guantanamo, cuba 8.7 enseq alguacil et al. (2012a) paraglomus sp. roots of dichanthium aristatum guadeloupe, 7.8 enseq jalonen et al. (2012) french antilles paraglomus sp. roots of zea mays martonvasar, hungary 5.8-6.2 enseq sasvári et al. (2011) paraglomus sp. roots of tanacetum vulgare, bohemia, czech republic na enseq unpublished (he775341-50) brachypodium pinnatum, and knautia arvensis paraglomus sp. roots of trifolium repens lombardy, italy 5.7-6.4 enseq lumini et al. (2011) paraglomus sp. roots of zea mays marche, italy 8.3-8.5 enseq borriello et al. (2012) paraglomus sp. roots of larix decidua south tyrol, italy na enseq unpublished (hm044471) paraglomus sp. roots of camellia japonica piedmont, italy 5.8 enseq unpublished (jq315319-36) paraglomus sp. pasture soil sardinia, italy 5.4-6.2 enseq orgiazzi et al. (2012) paraglomus sp. pasture soil sardinia, italy 5-6.5 enseq lumini et al. (2009) paraglomus sp. na cameroon na enseq unpublished (hq108153-9) paraglomus sp. chickpea rooting soil canada na enseq unpublished (jf340036-39) paraglomus sp. rhizosphere of tectona grandis chiang mai, thailand na enseq unpublished (jq864337) paraglomus sp. semiarid mediterranean prairies murcia, spain na enseq torrecillas et al. (2012) pacispora spp. pacispora scintillans high mediterranean dessert oregon, usa na m rose and trappe (1980) p. scintillans pot culture with plantago lanceolata pomerania, poland na spseq walker et al. (2004) inoculated with field soil from beneath triticum aestivum p. scintillans sandy heathland, beneath dactylis hessen, germany na spseq walker et al. (2004), glomerata, anthericum liliago and krüger et al. (2009) associated plants p. scintillans ancient meadow, beneath lolium dorset, uk na spseq walker et al. (2004) perenne and associated plants p. chimonobambusae bamboo garden nan-tou, taiwan na m wu et al. (1995) p. chimonobambusae trisetum grassland on serpentinite grisons, switzerland 6.1 m oehl, unpublished p. coralloidea subnival siliceous scree valais, switzerland 6.5 m oehl and sieverding (2004) p. dominikii trifolium pratense nw poland na m błaszkowski (1988a) p. dominikii trisetum grasslands grisons, switzerland 7.8 m oehl, unpublished p. dominikii organic farming systems basel, switzerland 6.4-7.5 m oehl et al. (2005b, 2010) p. dominikii pterocephalus spathulatus, andalusía, spain 8.1 m&spseq palenzuela et al. (2008) thymus granatensis p. franciscana olive tree-grasslands umbria, italy 7.0-7.5 m oehl and sieverding (2004) p. franciscana subnival calcareous screes grisons, switzerland 7.5 m oehl and sieverding (2004) p. franciscana na pomerania, poland na spseq krüger et al. (2012) p. franciscana na lower saxony, germany na spseq krüger et al. (2012) p. patagonica nothofagus forest santa cruz, argentina 5.5 m novas et al. (2005) p. robigina subnival calcareous and grisons, switzerland 7.0-7.8 m oehl and sieverding (2004) serpentinite screes p. robigina subnival screes valais switzerland 6.5 m oehl and sieverding (2004) p. robigina soldanella carpatica tatra mountains, poland na m oehl and sieverding (2004) pacispora sp. mclaughlin reserve on serpetinite california, usa na enseq schechter and bruns (2008) pacispora sp. alpine plant species (4500 m asl) tibet, china na enseq liu et al. (2011) pacispora sp. tibet plateau china na enseq unpublished (jq182767) accession numbers are given in the left column for available sequences that so far have not yet been published in peer-reviewed journals. 1 no information available. 2 m = identified by morphological analyses, spseq identified by molecular analyses, m&spseq identified by both approaches, enseq = environmental sequences deposited in public data bases. paraglomus pernambucanum sp. nov. 121 (fig. 1). environmental sequences that were closely related to the two fungi were not found in the public data bases. distribution: paraglomus bolivianum was found in a degraded pasture of the semi-arid gran chaco (bolivia, oehl and sieverding, 2004). in recent years, it was also found in the semi-arid caatinga of ne brazil, e.g. in triunfo, belém do são francisco and serra talhada, all in pernambuco state. finally, p. bolivianum was also found in a coastal ‘restinga’ forest vegetation in mataraca (paraíba state; 6°28’20’’-6°30’00’’s; 34°55’50-34°57’10’’w (silva et al., 2012). biogeography of paraglomus and pacispora spp. in tab. 1, a comprehensive summary of the biogeography of the genus paraglomus and pacispora, and their conclusively and nonconclusively identified species, is given. the identifications have been based on spore morphology, molecular analyses on formerly morphologically identified species, and on so-called environmental sequences deposited in the public data bases. it can be deduced from these results that the genus paraglomus has a worldwide distribution and occurs in many terrestrial ecosystems throughout the globe. it has been recorded from high alpine to nival areas, temperate to mediterranean up to subto inner tropic, arid to humid areas, in different soil types covering a wide spectrum of soil ph and land use intensities (tab. 1). there have been > 300 paraglomus sequences from the ribosomal gene deposited in the public data bases, from 36 countries and from many quite different ecosystems. the most widespread fungus of this genus so far might be p. occultum, followed by p. albidum, since these two species were most often identified. however, the phylogenetic tree shows at least two species identified as p. occultum, revealing current problems in the species identification of this small-spored genus. only one p. occultum clade, fixed by the isolate from the type area in iowa, can be p. occultum, while the sequence aj271713 obviously belongs to another paraglomus species (fig. 1). the genus pacispora is biogeographically more restricted to specific habitats and specific climatic zones than paraglomus (tab. 1), which confirms our assumption in the introduction. based on morphological spore identification, the genus is characteristic for higher ph soils (> 6.0) and a more restricted to specific ecosystems when compared to paraglomus. one exception might be p. patagonica which was found in soil with ph 5.5, but it was not given in novas et al. (2005) in which medium the soil ph was measured. also in the public data bases, sequences of pacispora spp. have so far been rarely deposited: from the ribosomal gene, only nine environmental sequences and 23 sequences from formerly morphologically identified species have been found. they are from usa, poland, germany, uk, spain and china, all detected from soils with ph > 6.0 (tab. 1). nevertheless, our investigation shows that the genus occurs worldwide, but none of the pacispora species appear to have a similar wide distribution as p. occultum. discussion paraglomus pernambucanum and p. bolivianum can easily be distinguished by their spore wall ornamentations, and by spore color and size. the spores of p. bolivianum are yellow brown to brown and their pits are substantially larger and deeper (oehl and sieverding, 2004) than those of p. pernambucanum, whose spores are hyaline to subhyaline. in paraglomus, there is one other species known with ornamentation on the spore wall. this is p. brasilianum whose ornamentation is labyrinthiform (spain and miranda, 1996; morton and redecker, 2001). within paraglomus, six to seven of the eight species might have two spore walls. these are p. occultum (walker, 1982), p. brasilianum (spain and miranda, 1996), p. bolivianum (oehl and sieverding, 2004), p. pernambucanum, and, according to our analyses (oehl, own observations), also p. lacteum (rose and trappe, 1980) and p. laccatum (renker et al., 2007). beneath its multi-laminated spore wall layer, p. laccatum has a separate inner wall which is difficult to observe (renker et al., 2007; oehl et al., 2011c). this might be also true for p. albidum (walker and rhodes, 1981; oehl et al., 2011c) but this need to be checked on newly isolated spores. thus, only p. majewskii might have solely one, triple-layered spore wall with a relatively thin innermost layer, which is substantially thinner than those in the other paraglomus species like p. occultum, p. brasilianum, and p. bolivianum. interestingly, p. majewskii was reported as forming a relatively distant lineage within paraglomus (błaszkowski et al., 2012), which is confirmed by our analyses. beside p. pernambucanum, there has been only one other recently described paraglomus species, with phylogenetic analyses in the original description (p. majewskii, błaszkowski et al., 2012). like p. bolivianum, p. laccatum was transferred to the genus paraglomus due to new phylogenetic analyses (renker et al., 2007) on type specimens of the former glomus laccatum (błaszkowski, 1988b). sequences on the ribosomal gene of p. occultum and p. brasilianum (formerly g. occultum and g. brasilianum) were the base for the transfer of the later two species (walker, 1982; spain and miranda, 1996) from the glomerales and glomeraceae to the paraglomerales and paraglomeraceae, respectively (morton and redecker, 2001; schüssler et al., 2001). up to date, six paraglomus species have been sequenced on the rrna or other genes (fig. 9), while for p. albidum and p. lacteum molecular phylogenetic evidence is still missing (oehl et al., 2011c). remarkably, p. bolivianum is the first species known in the paraglomeromycetes with pigmented spores. in the glomeromycota, there are currently two genera with bi-walled spores formed on subtending hyphae. this is true for all pacispora species (oehl and sieverding, 2004; oehl et al., 2001b) and for most of the paraglomus species. pacispora species form characteristic constricted to cylindrical subtending hyphae that may bear one to a few hyphal pegs, and the outermost spore wall layer is semi-persistent, while the inner wall regularly stains purple to deep purple in melzer’s reagent. in contrast, bi-walled paraglomus spores generally have slightly funnel-shaped to cylindrical subtending hyphae without hyphal pegs, and the outermost spore wall layer is rapidly degrading (‘short-lived’) and thus, can be called evanescent, while the inner wall never stains in melzer’s. remarkably, species of pacispora with ornamented spore surfaces regularly have projections on owl1 (p. scintillans, p. dominikii, p. coralloidea, p. chimonobambusae, p. patagonia) (oehl and sieverding, 2004; walker, 2008), while the paraglomus species with ornamented spore surfaces have a pitted owl2 (p. brasilianum, p. bolivianum and p. pernambucanum). our literature and data bank research strongly suggests that paraglomus species have a wider distribution than pacispora species, since they were found in many different ecosystems from the warm to very cold climates and in soils of very different soil ph (tab. 1). pacispora species are characteristic for soils with ph > 6.0, either in high alpine areas (e.g. p. robigina, oehl and sieverding, 2004; błaszkowski et al., 2008), in cultivated soils of temperate areas (e.g. p. dominikii, błaszkowski, 1993; oehl et al., 2005b; oehl et al., 2010) or in mediterranean and subtropical areas un122 c.m. aragão de mello, g.a. silva, d.m.a. de assis, j.s. pontes, a.c.a. ferreira, m.p.c. leão, h.e.e. vieira, l. costa maia, f. oehl der semi-arid conditions subjected to naturally elevated soil ph (e.g. p. scintillans and p. franciscana, rose and trappe, 1980; błaszkowski, 1993; oehl and sieverding, 2004; bashan et al., 2007). however, so far they were, to our knowledge, never found in tropical areas (e.g. sieverding, 1989; stürmer and siqueira, 2011; tchabi et al., 2008). in contrast, paraglomus bolivianum and p. pernambucanum were so far only found from tropical regions in south america, beside a single isolation site of p. bolivianum reported from southeast tibet (wang and shi, 2008). it has to be taken into account that many paraglomus species, and especially p. pernambucanum, form rather small, rapidly degrading spores that in the past might have often been difficult to identify from field samples. thus, we do not exclude that both species have, like p. occultum, a much larger distribution than known so far. acknowledgements catarina m. a. de mello 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(eds.), pcr protocols: a guide to methods and applications, 315-322. academic press, san diego. wirsel, s.g.r., 2004: homogenous stands of a wetland grass harbour diverse consortia of arbuscular mycorrhizal fungi. fems microbiol. ecol. 48, 129-138. wu, c.-g., liu, y.-s., hwuang, y.-l., wang, y.-p., chao, c.-c.,0 1995: glomales of taiwan: v. glomus chimonobambusae and entrophospora kentinensis, spp. nov. mycotaxon 53, 283-294. address of the correspondonding author: e-mail: fritz.oehl@agroscope.admin.ch vorgabe neu journal of applied botany and food quality 80, 135 137 (2006) 1institute for plant sciences, division for plant physiology, karl-franzens-university of graz, austria 2institute for plant sciences, division for botany, karl-franzens-university of graz, austria assessment of the genetic diversity of native apple cultivars in the south eastern ranges of the alps with three selected microsatellite loci stephan monschein1, martin grube2, dieter grill1 (received june 6, 2006) summary the regional diversity of native apple cultivars in parts of the south eastern ranges of the alps (styria, austria and northern parts of slovenia) was examined. as the application of conventional pomological methods to characterise cultivars may sometimes be ambiguous, we regard the application of molecular methods to be essential for thorough cultivar diversity assessments. five hundred samples were collected from different climatic and edaphic regions and analysed using three selected microsatellite loci. with this approach we were able to distinguish 190 named varieties at which we chose 50 as reference varieties. the high diversity of native races suggests that the southern alpine ranges represent a „hot spot“ of cultivar diversity. this can be attributed to historical effects and the local persistence of a traditional management practice with orchards of widely spaced and old-grown trees of various races. because these „old“ native races could harbour interesting genetic traits (pathogen resistance, taste, etc.) that will be important in future food production, measures for their conservation are overdue. our approach will not only show which local cultivars/genotypes require rapid action for their protection, but due to the international nature of our project we can also show which old and untraceable local names in different languages correspond with the same cultivars. introduction representing one the oldest and most widely cultivated temperate fruit crops, apple (malus x domestica borkh.) developed into a great diversity of cultivars. of these, only a few are grown in mass production to fit the requirements of the international market of today. in former times each country or region maintained an own local stock of cultivars (janick et al., 1996). from these, ecological types could be selected which were particularly adapted to local environments and requirements. these old varieties and land races have been used by humans since hundreds of years and therefore hold a special position in the cultural landscape heritage of any region where apple has a history of cultivation. however, for complex reasons the genetic diversity of the domesticated apples has decreased dramatically in the last few years (hokanson et al., 2001). the colline to montane elevations of the south eastern alpine borders are a perfect example for a historically rich diversity of apple cultivars. originating from styria (austria), which might be regarded as “hot-spot” of apple diversity, apple cultivation was extended towards slovenia, northern italy and parts of croatia during the times of the habsburgian empire. some of the races were better adapted than others to grow at different altitudes, to withstand high light, to resist massive damage by pathogens, etc. a tremendous number of names of these cultivars are found in the literature (rolff, 2001; hartmann, 2003; grill and keppel, 2005), or can be collected in interviews with local farmers. since there has never been a comprehensive and consistent collection of cultivars for comparison, the naming practice resulted in many confusion, especially with rarely grown cultivars. e.g. old horticultural names were sometimes replaced by informal local names, which persisted through times, or wrong determinations could result in the dissemination of shoots with wrong names. the taxonomic chaos of informal vernacular names lead to a considerable uncertainty about estimates of cultivar diversity in the south-eastern alpine ranges. because native apple cultivars represent a rich stock of exploitable genetic resources, the conservation of old and local cultivars should be seen as an important task to ensure a sustainable environment. it is clear that a general screening of the diversity of old local apple varieties is an indispensable first step before an effective conservation management can be outlined (hodgkin et al., 2001). however, and especially in the case of apple, the traditional determination methods, including measurements and objective descriptions of fruit and tree characteristics, are time-consuming and not always consistent (kenis et al., 2001). this is due to the variability in their fruit parameters (e. g. colour, shape, tightness), which is strongly influenced by ecological and physiological parameters, such as soil composition, exposition, climatic fluctuations. to complement the morphological characterisation, isoenzymes and alloenzymes have previously been included (royo and itoiz, 2004), but their patterns may again depend on environmental factors. the emergence of new pcr-based molecular markers, such as randomly amplified polymorphic dna (rapds), simple sequence repeats (ssrs), and amplified fragment length polymorphisms (aflps), has created the opportunity for finescale genetic characterizations of germplasm collections that were previously impossible (hokanson et al., 1998). an alternative method for characterisation and identification of old native apple cultivars which is independent from changing environmental influences is provided by the anlaysis of length variation at microsatellite loci. by their polymorphic nature, microsatellites became popular for various genetic approaches (genomic mapping, study of genomic instability in cancer cells, population genetics, forensics and conservation biology; shinde et al., 2003). microsatellites, or simple sequence repeats (ssrs), are short stretches of dna, consisting of tandemly repeated nucleotide units. the high levels of variation in the number of repeats are thought to arise from replication slippage, i.e. the transient dissociation of the replicating dna strands followed by misaligned reassociation (ellegren, 2004). a general method for the detection of polymorphic microsatellites is based on pcr amplification using a unique pair of primers flanking the simple sequence repeats (weber and may, 1989). as they are uniformly distributed, hypervariable, codominant and abundant in most genomes, they are interesting for studies of apple (gianfranceschi et al., 1998), and can be used to gain deeper insights in the distribution of phenotypic traits (by qtl analysis) or for an assessment of cultivar diversity. the aim of this study is to examine the genetic diversity of native apple cultivars by means of microsatellite length variations and to clarify some naming problems of apple varieties in the area of investigation. materials and methods for the estimation of the genetic diversity of native apple cultivars in the area of investigation (styria and northern parts of slovenia) a basic inquiry was carried out. we sent out a standardised questionnaire to all agricultural schools to obtain information about the cultivars composing these special cultural landscape. from these collected data we selected varieties for genetic analysis by microsatellites according to specified criteria, as follows: 1. the age of the trees (80 100 years old), 2. unclear local names, 3. special usage of the fruits (such as dried fruit), 4. occurrence at relatively high altitudes (> 1300 m) or 5. such varieties, which are not yet cultivated in a living bank of genetic resources (arboretum). varieties determined by pomological experts (s. bernkopf, linz and h. keppel, graz) were analysed as references for comparison. from the selected trees for microsatellite analyses we collected young leaves for dna extraction. the leaves were frozen in liquid nitrogen immediately after harvesting. the lyophilised leaves (hetosicc freeze dryer type cd 4, heto lab equipment, birkerod, denmark) were ground to powder by the micro-dismembrator ii (b. braun biotech international gmbh, meisungen, germany) and stored in humidity proof plastic vials at -25°c until dna extraction. the dna was isolated from 17 mg of leaf powder using the dneasy® plant mini kit (qiagen, vienna). for the genetic characterisation of the native apple cultivars by simple sequence repeats we used three different polymorphic primer combinations with high expected heterozygosity (forward and reverse primer), which represent different linkage groups (gianfranceschi et al., 1998): ch01f02, ch02c06 and ch02d12. the forward primer of each primer pair was labelled at the 5´-end with a fluorescent dye (fam, hex or ned). the amplification of the three selected microsatellite loci by pcr followed the conditions published by gianfranceschi et al. (1998). the specific amplified dna fragments (microsatellites) were run on a abi prism® 310 genetic analyser. the detected fragment lengths were calibrated by a fluorescent labelled size standard (genescan 500 rox) which was added to each sample. sizing of fragments was done with genescan®analysis software version 3.1. because lengths from each microsatellite locus was uniform within an cultivar, differences in the allelic compositions were used for characterisation and identification of old native apple cultivars. results and discussion based on the primary inquiry of the native apple cultivars by standardised questionnaires and after selection of relevant varieties (see above) we examined 500 trees by microsatellite analysis. the examined apple cultivars are characterised by their allelic composition at the three selected ssr loci. from the 500 dna-extractions, which were sampled evenly across the area of investigation, we could differentiate 190 varieties. the application of only three selected microsatellite loci can be regarded as a cost efficient method for a screening of regional apple cultivar diversity. to characterise unknown cultivars and to clarify the complex synonymy, it is necessary to compare the allelic length of reference samples of varieties with that of cultivars with dubious names. the determination of such references is a difficult task. the selection of samples, which are to become reference samples in the future was carried out in accordance of microsatellite data with morphological fruit specifications (e. g. colour, shape, tightness), and expert knowledge for local varieties (s. bernkopf, linz and h. keppel, graz). of the 190 differentiated samples we selected 50 as reference varieties (tab. 1). this reference data base will allow a secure identification of apples by their allelic composition. the allelic composition of the cultivars interpreted according to liebhard et al. (2002) as follows. in cases where only one fragment is visible, the allel can be homozygous or an null allel is involved (eg. ananasrenette at locus ch01f02). in the latter case the individual tab. 1: allelic composition of 50 reference apple cultivars (nomenclature according to grill and keppel, 2005) cultivar ssr name ch01f02 ch02c06 ch02d12 ananasrenette 183 241:249 190:198 baumanns renette 169:179 215:227 182:198 berner rosenapfel 181 233:249 198:206 boikenapfel 179 215:233 198:200 champagner renette 183 249 190:200 charlamowsky 171 205:251 176:198 coulons renette 181:183 227:237 180:210 damasonrenette 179:183 233:243:253 182:198 danziger kantapfel 169 229:251 180:198 elise rathke 169:183 249 180:190 geflammter kardinal 169:179 215:227:247 182:198:210 geheimrat dr. oldenburg 189 249 176:190 gehrers rambur 169 215:227:249 198 gelber bellefleur 173:179 233:249 194:198 goldrenette von blenheim 183 241:249:257 198 grahams jubiläumsapfel 173 249 176:198 graue herbstrenette 179:183 233:243 182:198:200 gravensteiner 181:183 215:249 198:212 großer rheinischer bohnapfel 179:183 215:249 194:198 grüner stettiner 169 215:233:255 180:194:198 hagedorn 179:183 215:251 176:198 harberts renette 183:185 241:249 180:198 haslinger 169:179:183 249 176:182:190 ilzer rosenapfel 169 259 182:198 jakob lebel 179:187:195 227:237 176:182:198 jonathan 169:179 233:249 190:198 kalterer böhmer 169 229:231 182:206 kanada renette 179 227:247 182:190:198 kardinal graf galen 169 249 176:206 karmeliter renette 179:183 257 198 klöcher maschanzker 169:181 243:249 190:198:206 kronprinz rudolf 169:183 241:249 176:198 landsberger renette 183:189 237:251 176:190 lavanttaler bananenapfel 183 249 206:210 london pepping 179 215 176:206 odenwälder 169:193 249 176:198 rheinischer krummstiel 169:181 237:249 176:198 rote schafnase 169 229:231 198:204 roter herbstkalvill 173 249 182:198 roter trierer weinapfel 169 247 180:182 sauergrauech 181:183 233:247 198 schmidberger renette 169:183 249 198:210 schöner von boskoop 183 227:237 180:194 signe tillisch 189 215:251 176 steirischer maschanzker 169:181 249 198:206 steirischer passamaner 169:171 215:231 184:198 wagener apfel 169:181 249 180:206 weißer klarapfel 181:187 215:249 178:198 weißer winterkalvill 181 249 182:198 wintergoldparmäne 183 237:249 198:210 136 stephan monschein, martin grube, dieter grill could be heterozygous and the other allel is not detectable because of mutations in the priming site and prevention of annealing. two allels represent an diploid heterozygous individual (or a triploid with null allel). three allels at the same locus of one individual could indicate a triploid heterozygous cultivar or for multilocus ssr (e. g. damasonrenette at locus ch02c06). our data confirm a high diversity of extant apple cultivars in styria and parts of slovenia. we could also resolve several nomenclatural problems with our data (tab. 2). for example the cultivar „baumanns renette“ displays the same allelic composition as the collections „kranzler“, „türken rot“, „nikolausapfel“ and „baumanova reneta“, which represent vernacular names for the regular cultivar term “baumanns renette”. a posteriori inspection of pomological characters confirmed the molecular approaches. any slight differences between the phenotypical descriptions seem to be within the range of modifications of each race. tab. 2: examples for discovered synonyms (regular cultivar names are given in bold letters, grill and keppel, 2005) cultivar ssr name ch01f02 ch02c06 ch02d12 baumanns renette 169:179 215:227 182:198 kranzler 169:179 215:227 182:198 türken rot 169:179 215:227 182:198 nikolausapfel 169:179 215:227 182:198 baumanova reneta 169:179 215:227 182:198 haslinger 169:179:183 249 176:182:190 steirischer pogatschenapfel 169:179:183 249 176:182:190 roter pogatschenapfel 169:179:183 249 176:182:190 haselapfel 169:179:183 249 176:182:190 breitschädel 169:179:183 249 176:182:190 pogacarica 169:179:183 249 176:182:190 roter herbstkalvill 173 249 182:198 himbeerapfel 173 249 182:198 erdbeerapfel 173 249 182:198 klachlapfel 173 249 182:198 roter paradiesapfel 173 249 182:198 herzapfel 173 249 182:198 klingler 173 249 182:198 tschepperer 173 249 182:198 rdeci jesenski kalvil 173 249 182:198 weißer klarapfel 181:187 215:249 178:198 butterapfel 181:187 215:249 178:198 in parallel to ssr-typing of characterised apple varieties their cultivation in an arboretum as a living bank of genetic resources is planned. this will facilitate the conservation and dissemination of genetically valuable native apple cultivars. another anticipated endeavour is a transregional alignment of the obtained microsatellite data with other research institutes. acknowledgements this research was funded by the interreg iii a programm between austia and slovenia and a “bund-bundesländer-kooperation”. we thank karin herbinger and melanie hofer for the collection of apple leaf material, herbert keppel and siegfried bernkopf for their scientific support. references ellegren, h., 2004: microsatellites: simple sequences with complex evolution. nature rev. genet. 5, 435-445. gianfranceschi, l., seglias, n., tarchini, r., komjanc, m., gessler, c., 1998: simple sequence repeats for the genetic analysis of apple. theor. appl. genet. 96, 1069-1076. grill, d., keppel, h., 2005: alte apfelund birnensorten für den streuobstbau. leopold stocker verlag, graz-stuttgart. hartmann, w., 2003: farbatlas alte obstsorten. verlag eugen ulmer, stuttgart. hodgkin, t., roviglioni, r., de vicente, m.c., dudnik, n., 2001: molecular methods in the conservation and use of plant genetic resources. acta horticulturae 546, 107-118. hokanson, s.c., szewc-mcfadden, a.k., lamboy, w.f., mcferson, j.r., 1998: microsatellite (ssr) markers reveal genetic identities, genetic diversity and relationships in a malus x domestica borkh. core subset collection. theor. appl. genet. 97, 671-683. hokanson, s.c., lamboy, w.f., szewc-mcfadden, a.k., mcferson, j.r., 2001: microsatellite (ssr) variation in a collection of malus (apple) species and hybrids. euphytica 118, 281-294. janick, j., cummins, j.n., brown, s.k., hemmat, m., 1996: apples. in: janick, j., moore, j.n. (eds.), fruit breeding, volume i: tree and tropical fruits, 1-77. john wiley & sons inc., new york. kenis, k., pauwels, e., van houtvinck, n., keulemans, j., 2001: the use of microsatellites to establish unique fingerprints for apple cultivars and some of their descendants. acta horticulturae 546, 427-431. liebhard, r., gianfranceschi, l., koller, b., ryder, c.d., tarchini, r., van de weg, e., gessler, c., 2002: development and characterisation of 140 new microsatellites in apple (malus x domestica borkh.). molecular breeding 10, 217-241. rolff, j.h., 2001: der apfel, sortennamen und synonyme. band 1. selbstverlag johann-heinrich rolff, kiefersfelden. royo, j.b., itoiz, r., 2004: evaluation of the discriminance capacity of rapd, isoenzymes and morphologic markers in apple (malus x domestica borkh.) and the congruence among classifications. genet. res. crop evolut. 51, 153-160. shinde, d., lai, y., sun, f., arnheim, n., 2003: taq dna polymerase slippage mutation rates measured by pcr and quasi-likelihood analysis: (ca/gt)n and (a/t)n microsatellites. nucleic acids res. 31, 974980. weber, j. l., may, p. e., 1989: abundant class of human dna polymorphism which can be typed using the polymerase chain reaction. amer. j. human genet. 44, 388-396. address of the authors: mag. stephan monschein (corresponding author), institute for plant sciences, division for plant physiology, karl-franzens-university of graz, schubertstraße 51, a-8010 graz, austria. ao. univ.-prof. mag. dr. martin grube, institute for plant sciences, division for botany, karl-franzens-university of graz, holteigasse 6, a-8010 graz, austria. univ.-prof. dr. dieter grill, institute for plant sciences, division for plant physiology, karl-franzens-university of graz, schubertstraße 51, a-8010 graz, austria. genetic diversity of native apple cultivars 137 v v << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) 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<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> >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice journal of applied botany and food quality 88, 202 208 (2015), doi:10.5073/jabfq.2015.088.029 1 grassland institute of animal science and technology college, china agricultural university, beijing, china 2 dlf beijing office, no. 8 beichendong st., beijing, china 3 usda-ars forage and range research lab, utah state university, logan, usa germination response of apocynum venetum seeds to temperature and water potential yuping rong1*, hongxiang li2, douglas a. johnson3 (received november 6, 2014) * corresponding author summary apocynum venetum (commonly known as luobuma or rafuma) is a shrub that is native to eurasia. it is economically important for sand fixation, forage production, honey production, and for the production of medicine, fiber and fuel. rapid and uniform seed germination is critical for successful crop establishment and vegetation restoration. the purpose of this study was to determine the germination responses of a. venetum seeds to temperature and water availability using hydrotime, thermal time and hydrothermal model analysis. seed germination was relatively high for a. venetum from 25 °c to 30 °c. the base (tb), optimum (to) and ceiling temperatures (tc(50)) of a. venetum seed germination were 16.6, 27.0 and 45.9 °c, respectively. values of base water potential (ψb(g)) shifted to zero with increasing temperature, which was reflected in the greater effect of low ψ on germination for temperatures above 30 °c. hydrotime analysis suggested that tb may not be independent of ψ, and ψb(g) may change as a function of temperature at temperatures below 30 °c. the interaction effects of ψ and temperature reduced the ability of the hydrothermal time model to predict germination performance across temperature and ψ conditions. introduction apocynum venetum is a perennial semi-shrub species that is widely distributed in the temperate regions of asia and europe, and is found in iran, afghanistan, india, russia and china. it commonly grows in barren saline soil, desert edges, riverbanks, alluvial plains and areas surrounding reservoirs and is well adapted to desert climates (thevs et al., 2012). a. ventum is of economic importance and is used in sand fixation and a forage, tea, medicine, and fiber. however, wild populations of this species are currently in danger of being overexploited (thevs et al., 2007; westermann et al., 2008). previous research on a. venetum has been conducted covering fiber extraction from its stems (gong and fu, 2001) and processing tea and medicine from its leaves (ma et al., 2003). seed germination is critical for its use in sand fixation and crop production. a. venetum can be propagated from seeds or cuttings; however, propagation by cuttings limits its use compared to propagation by seeds. studies have shown that sowing seeds in a nursery and transplanting the saplings after the second year is the best way to propagate this species (tang, 2008). temperature and soil water content are critical factors that influence seed germination in both greenhouse and field environments. in arid environments, the water required for germination is only available for short periods. consequently, successful crop establishment depends not only on rapid and uniform seed germination, but also the seed’s ability to germinate under low water availability (fischer and turner, 1978; windauer et al., 2012). knowledge of the influence of water and temperature on seed germination in a. venetum is fundamental for its use in crop production and sand stabilization. previous research was shown that seed germination of a. venetum is difficult under conditions of high soil water content, whereas high temperatures can increase its germination (liu, 2010). zhang et al. (2007) found that low concentrations of nacl (≤50 mmol/l) promote seed germination in a. venetum, whereas high nacl concentrations (≥200 mmol/l) reduce germination. however, limited research has been conducted on the interaction affects of water and temperature on the germination and seedling establishment of this economically species. population-based threshold models have been used to model the ecophysiological responses of seed germination to environmental factors (finch-savage and leubner-metzger, 2006). seed germination attributes can be quantified using the thermal time model (θt) (garcia-huidobro et al., 1982), hydrotime model (θh) (gummerson, 1986) and hydrothermal time model (θht) (gummerson, 1986). seed germination responses to temperature can be characterized using three cardinal temperatures (bewley and black, 1994): a base temperature (tb) below which germination of the seed lot does not proceed, an optimal temperature (to) at which the process occurs with the highest speed and a maximum (ceilings) temperature (tc) over which the germination process does not proceed. when temperature remains constant, but water is suboptimal, progress towards the completion of germination can be quantified in hydrotime (finch-savage and leubner-metzger, 2006). in addition, the thermal and hydrotime models can be combined to produce a hydrothermal time (htt) model (gummerson, 1986; alvarado and bradford, 2002). the objective of this study was to characterize the germination responses of a. venetum seeds to temperature and water availability using thermal time, hydrotime and hydrothermal time analysis to test the validity of these models to a. venetum seeds. materials and methods seed seeds of a. venetum were collected during october 2009 in a desert environment (41.08°n, 85.97°e, 878 m asl) of the middle tarim river in southern xinjiang, china. seeds were cleaned and stored at 4 °c in a sealed glass bottle until needed for experimantation (march 2010). the mass of 1000 seeds of a. venetum used in our study was 0.36 g. germination assays germination assays were conducted in the forage and turf grass seed laboratory at the grassland science department, china agricultural university, beijing, china. seeds were sterilized for 5 min with 10 % naclo and then washed with distilled water. seeds were placed on two layers of filter paper saturated with distilled water or polyethylene-glycol (peg) solution in glass petri dishes (90-mm inner diameter). four replicates per treatment with 50 seeds each were placed in a growth chamber under 8 h light and 16 h dark at constant temperatures of 20, 25, 30 and 35 °c (±1 °c). apocynum venetum seed germination 203 the water potential (ψ) of the germination medium was controlled by different solutions of polyethylene-glycol 6000 (peg 6000) and prepared according to michel and kaufmann (1973) so that ψ was -0.3, -0.6, -0.9, -1.2 and -1.5 mpa at the respective temperature. the actual ψ at all temperatures was measured using a vapour pressure osmometer (wescor, inc., logan, model 5100c). seeds incubated on solutions containing peg were transferred to fresh solutions every 2 d to maintain constant water potential in the germination medium. the germination (radicle protrusion) was scored daily, and the germinated seeds were removed. germination experiments were terminated when no new germination for three consecutive days was recorded in the four replicates of a treatment. germination analysis germination rates were calculated as the inverse of the time to radicle emergence. germination times for specific percentiles of the seed population (gr50) were calculated by interpolation using curves fit to the time course data. to determine the optimal germination temperature, germination rates at 30 and 35 °c were compared. if germination at 30 °c was significantly greater than germination at 35 °c, the optimal temperature was assumed to be 30 °c. the germination time course data were analyzed using repeated probit regression analysis, as described by bradford (1990) and dahal and bradford (1990, 1994). probit analyses were conducted using the proc probit routine of the sas statistical package, which employs a maximum-likelihood weighted regression method (sas 9.2). thermal time in the sub-optimal temperature range (i.e., between tb and to), the thermal time to germination at fraction g (θt1(g)) can be characterized using the following thermal time (θt1, °c d) equation: θt1(g)=(t-tb)tg (1) where t is the germination temperature, tb is the base temperature and tg is the germination time of fraction g. this equation indicates that for a given seed fraction g, θt1(g) is constant at all sub-optimal temperatures when expressed on a thermal time basis as the degrees in excess of tb multiplied by the actual time to germination. values of tb and θt were determined through repeated probit regression analysis (garcia-huidobro et al., 1982) by regressing all observed germination percentages on a probit scale versus log thermal times logθt1(g) to germination, varying the value of tb until the best fit was obtained, according to the equation: probit (g)={log[(t-tb)tg]-log[θt1(50)]} / σθt1 (2) where probit (g) is the probit transformation of cumulative germination percentage g, θt1 (50) is the median thermal time to germination, and σθt1 is the standard deviation in logθt1 among individual seeds in the population. above to, the germination rate decreases almost linearly until tc is reached, which is also known as thermoinhibition (hills and van staden, 2003). thus, in the supra-optimal temperature range, the equation used is as follows (ellis and butcher, 1988): θt2=[tc(g)-t]tg (3) germination time courses (θt2, tc) in the supra-optimal temperature range can be predicted in a similar way to those in the sub-optimal temperature range (equation 4); however, the germination rate decreases with temperature, and tc (g) varies among fractions, while the thermal time to radical protrusion is constant for all seeds: probit (g)=[log(t+θt2)/tg-logtc(50)]/σθt2 (4) where probit (g) is the probit transformation of cumulative germination percentage g, tc (50) is the median ceiling temperature to germination, and σθt2 is the standard deviation in log tc (50) among individual seeds in the population. hydortime the germination response to reduced ψ was analyzed by the hydrotime model (bradford, 1990; finch-savage and leubnermetzger, 2006) according to the equation: θh=[ψ −ψb(g)]tg (5) where θh is the hydrotime (mpa d) of the seeds required for germination, ψ is the actual water potential of the germination medium (mpa), ψb(g) is the theoretical threshold or base water potential that will just prevent the germination of fraction g and tg is the germination time (d) of fraction g. the model assumes that ψb varies among fractions of a seed population following a normal distribution with mean ψb(50) and standard deviation σψb. θh is considered constant for a seed population (bradford, 1990). the parameters in the hydrotime model were estimated according to the equation: probit (g) = [(ψ-θh/tg)-ψb(50)]/σψb (6) where ψb(50) is the median ψb, and σψb is the standard deviation in ψb among seeds within population. the parameters from the hydrotime model can be used to normalize germination time courses for the effects of reduced ψ. germination time course at any ψ can be normalized to the time course that would occur in water (0 mpa) for the seed population by multiplying the actual time to germination tg (ψ) by the factor [1-(ψ/ψ(g))] (bradford, 1990). this normalization can evaluate the ability of the model to describe the germination behavior (bradford, 1995). all normalized data from all temperatures were normalized on a common thermal time scale, using the estimated tb at 0 mpa. hydorthermal time the thermal and hydrotime models were combined to produce a hydrothermal time (htt) model to describe germination rates when temperature and ψ both varied. the htt model at the suboptimal temperature where θht is the htt constant (mpa °c d) was calculated according to the equation: θht =(t-tb) [ψ-ψb(g)]tg (7) this equation describes germination time courses at any combination of sub-optimal temperature and ψ (alvarado and bradford, 2002). the following modified hydrothermal time model was pro posed by alvarado and bradford (2002) to describe the germination timing and percentages across all t from tb to tc: θht={ψ-ψb(g)-[kt(t-to)]}(t-tb)tg (8) where [kt(t-to)] applies only when t>to and in this supra-optimal range of t; the value of ψb(g) is set equal to ψb(g)to and t-tb is set equal to to-tb. the values of kt, tb, to and θht in this model can be obtained by repeated probit regressions using germination time course data according to the equation: probit (g)=[(ψ-θht/(to-tb)tg)-kt(t-to))-ψb(50)]/σψb (9) the values of kt and to were varied for germination time courses at t>to until a fit was obtained that resulted in θht, ψb(50) and σψb values close to those obtained at or below to (alvarado and bradford, 2002). 204 y. rong, h. li, d.a. johnson results germination responses to temperature and water potential the cardinal temperature of a. venetum seeds was determined by germination in distilled water at different constant temperatures. germination of a. venetum seeds in water progressed more rapidly as t increased within the sub-optimal range (20-30 °c). in contrast, the final germination percentage in water decreased between 30 and 35 °c. the final germination percentage in the temperature range of 25 to 30 °c was relatively high with the values of ψ used in our study, which ranged from 70 to 89 %, respectively (fig. 1a). the germination rate increased with temperature in the range of 20 to 30 °c, and decreased above 30 °c. the germination rate (1/t50) was highest for seeds incubated at 30 °c (fig. 1b). as a result, the effect of the peg solutions was tested at temperatures of 20 and 25 °c, considered as sub-optimal temperatures, and 30 and 35 °c considered as supra-optimal temperatures. thermal time analysis accumulated daily germination percentages at 20 and 25 °c for distilled water were transformed to probit and regressed on logθt1(g)=log[(t-tb)tg]. the number of degree days necessary for 50 % of the seeds to germinate in the sub-optimal temperature range was 29.4 °c d (θt1) (r2=0.65). the value of tb that produced the best fit was 16.6 °c, which was taken as the minimum temperature for germination of a. venetum seeds. in the supra-optimal temperature range, the value of probit (g) was related to logtc(g)=log[(t+θt2/ tg)], and the best fit was obtained with tc(50)=45.9 °c (r2=0.87) (tab. 1). in the supra-optimal interval, the value of θt2=43.2 °c d was assumed to be constant, and the limiting factor for germination would be the distribution of tc (g) within the population. the value of tc varies among fractions of a seed population, following a normal distribution with mean tc (50) and standard deviation (σtc). similar analyses of germination data under of -0.3, -0.6, -0.9, -1.2 and -1.5 mpa were also performed (tab. 1). the r2 values indicated that the thermal model for sub-optimal temperatures occurred at lower values of ψ (r2=0.69-0.90), but tb was underestimated and θt1(50) was overestimated. tb decreased with decreasing ψ, especially below -0.6 mpa. hydrotime analysis the hydrotime model accounted for most of the variation in germination time at reduced ψ values at various temperatures (r2=0.93-0.95) (tab. 2). the predicted germination time courses at the various ψ values (fig. 2) generally fit the observed data well, except for some cases in which the predicted response deviated fig. 1: effect of temperature on the final germination (a) and germination rate (b) for a. venetum seeds incubated at various water potentials (ψ) (0– -1.5mpa). tab. 1: parameter estimates of the thermal time model describing seed germination across a range of water potentials. water potential tb θt1(50) σθt1 r2 (mpa) (°c) (°d) (°d) temperature at 20, 25 probit(g)={log[(t-tb)tg]-log[θt1(50)]}/σθt1, θt1(g)=(t-tb)tg 0 16.6 29.4 0.51 0.65 -0.3 15.9 42.6 0.57 0.69 -0.6 16.1 50.2 0.64 0.76 -0.9 12.9 89.1 0.45 0.90 -1.2 2.4 466.0 0.86 0.77 -1.5 0.3 773.1 0.90 0.86 temperature at 30, 35 probit(g)=[log(t+θt2)/tg-logtc(50)]/σθt2, θt2=[t-tc(g) ] tg water potential θt2 tc(50) σtc r2 (mpa) (°d) (°c) (°d) 0 43.2 45.9 0.12 0.87 -0.3 65.7 45.7 0.19 0.95 -0.6 103.5 48.4 0.19 0.98 -0.9 78.6 40.2 0.19 0.97 -1.2 70.0 31.5 0.20 0.95 -1.5 64.6 29.4 0.16 0.99 tb, base temperature; θt1(50), thermal time to germination of 50 % at suboptimal t; θt2, thermal time constant at supra-optimal t; tc(50), ceiling temperature to germination of 50 %; σθt1, standard deviation for θt1(50); σtc, standard deviation for tc (50). r2, coefficient of determination. considerably from actual germination. estimated values of θh, ψb(50) and σψb for various germination temperatures are shown in tab. 2. θh decreased from 14.2 mpa d at 20 °c to 4.0, 3.1 and 3.1 mpa d at 25, 30 and 35 °c, respectively. in addition, the values of ψb(50) increased as temperature increased from 20 °c to 35 °c, becoming less negative; at 35 °c, ψb(50) was -0.8 mpa. the pre dicted responses in fig. 3 are based upon the ψb(g) threshold distributions from the hydrotime model. in general, the predicted responses described the distributions of the observed cumulative germinations percentage relatively well for the treatments at 20, 25 and 35 °c and -0.3, -0.6 and -0.9 mpa, whereas the predicted values showed poor agreement with the observations at water potentials of apocynum venetum seed germination 205 -1.2 and -1.5 mpa. predicted σψb varied considerably from 1.01 to 1.95 mpa. the normalized germination curves for the hydrotime model (fig. 4) showed that the observations from various values of ψ at the various experimental temperatures merged into a common curve. when the interaction between temperature and ψb was taken into account using individual estimates of ψb(50) and σψb at sub-optimal temperatures, the observations normalized more consistently into a common curve, whereas distinct groupings of observations remained at sub-optimal temperatures (fig. 4a). however, the normalized curves revealed that observations fell into a distinct group at 35 °c, at which temperature the germination time was long and final germination values were low (fig. 4b). this indicated that the hydrotime estimates interacted with temperature, and consequently, the grouping of observations was the most profound in the seed population with the largest shift in ψb with temperature (tab. 2). hydrothermal time analysis the hydrothermal model of each sub-optimal temperature at different ψ was regressed on θht=(ψ-ψb(g))(t-tb)tg, which described the germination responses at constant sub-optimal temperatures in the ψ range of 0 to -0.6 mpa well. the values producing the best fit are presented in tab. 3. according to the hydrothermal model, tb and ψb(50) were 16.6 °c and -1.30 mpa, respectively, at sub optimal temperatures. in the supra-optimal temperature interval, θht=32.2 mpa d, predicted by the hydrothermal time model (tab. 3), was used to fit the germination data across various ψ at each t (30, 35 °c). because the distributions of ψb for various temperatures were pooled into one common distribution in the hydrothermal time model, estimates of ψb(50) and σψb in the hydrothermal time model (tab. 3) generally agreed with estimates of the hydrotime model at sub-optimal temperatures. however, the estimated values were not consistent with those of the hydrotime model at supra-optimal temperatures. discussion in the present study, we used a. venetum seeds stored for approximately 10 months, which generally exhibit a lower germinability than newly collected seeds (95 %) (hu et al., 2002b). the germination of our a. venetum seeds was 88 %, which was similar to those of hu et al. (2002a) who found that germination decreased to 81-85 % after 6 months of storage and then remained stable for more than 10 years at room temperature under sealed conditions. many studies have shown that responses of seed germination to temperature are related to the geographical and ecological distri bution of the particular species studied (grime et al., 1981; schütz tab. 2: parameter estimates of the hydrotime model (probit(g)=[(ψ-θh/tg)ψb(50)]/σψb, θh=(ψ-ψb(g))tg) describing seed germination. temperature θh ψb(50) σψb r2 (°c) (mpa d) (mpa) (mpa) 20 14.2 -1.76 1.95 0.93 25 4.0 -1.32 1.15 0.95 30 3.1 -1.19 1.01 0.93 35 3.1 -0.80 1.23 0.94 θh, hydrotime; ψb(50), base water potential for 50 % seed germination; σψb, standard deviation for ψb (g); r2, coefficient of determination. fig. 2: germination time course of a. venetum seeds for a range of water potentials at various constant temperatures. symbols indicate actual data, and lines indicate values predicted by probit analysis. 206 y. rong, h. li, d.a. johnson fig. 3: distribution of the base water potential of a. venetum seeds at various temperatures. tab. 3: parameter estimates of the hydrothermal time model describing seed germination. sub-optimal temperature (20, 25 °c): probit(g)=[ψ-(θht)/(t-tb)tg)-ψb(50)]/σψb, θht=(ψ-ψb(g))(t-tb)tg tb θht ψb(50) σψb r2 (°c) (mpa d) (mpa) (mpa) 16.6 32.2 -1.31 1.19 0.93 supra-optimal temperature (30, 35 °c): probit(g)=[ψ-(θht)/(to-tb)tg)-kt(t-to))-ψb(50)]/σψb, θht={ψ-ψb(g)-[kt(t-to)]}(to-tb)tg to θht ψb(50) σψb kt r2 (°c) (mpa d) (mpa) (mpa) (mpa °c-1) 27.0 32.2 -1.54 1.07 0.10 0.89 to, optimal temperature; θht, hydrothermal time constant; ψb(50), base water potential for 50% seed germination; σψb, standard deviation for ψb (g); r2, coefficient of determination. fig. 4: normalized time courses of the hydrothermal time model at sub-optimal temperatures (a), supra-optimal temperatures (b) and all temperatures combined (c). and rave, 1999; liu et al., 2011). the thermal requirement of seed germination also was related to the life history strategy of the species (probert, 2000). a. venetum grows in central asia and is adapted to desert climates, surviving even under extremely arid conditions (<50 mm mean annual precipitation) by exploiting groundwater to meet its water demands (chen et al., 2007; thevs et al., 2012). in our study, temperature had a marked effect on the germination of a. venetum at ψ studied, and the effect was described by the thermal time model using probit analysis (tab. 1). a. venetum seeds germinated in a relatively narrow temperature range with the final germination reaching 70-88 % in the interval of 25-30 °c. the estimated tb and θt1 were 16.6 °c and 29.4 °c d in distilled water, respectively, corresponding to a relatively high tb and low accumulated temperature during seed germination. this is consistent with some studies reporting a negative relationship between tb and θt1 (angus et al., 1981; trudgill et al., 2000). species originating from tropical regions have higher tb and lower θt1 (trudgill et al., 2005). tc was high in our study, which indicated that seeds of a. venetum are able to tolerate quite high temperatures. this germination characteristic of a. venterum is important for desert environments, which have highly variable temperatures, precipitation and soil water availability. the thermal time model did not fit the data at lower ψ (e.g., -1.2 mpa and -1.5 mpa). the low estimate of tb and considerably high θt1 (50) at low values of ψ are probably not biologically significant. the thermal time analysis in our study failed to describe germination response at the later phases of germination. one possible reason could be that maximal germination was not possible during apocynum venetum seed germination 207 the 15 days used in our study. according to hu et al. (2002a), the storage of a. venetum seeds led to lengthening the germination time period at lower temperatures, which in their study was 21 days. the parameters from the thermal time analysis can be used for production conditions for a. venetum. first, tb was estimated to be 16.6 °c; this value is quite high and precludes the sowing of these seeds in soils where the temperature does not exceed 16.6 °c. secondly, germination rate (gr50) was found to be the highest at 30 °c, demonstrating that maximum germination rate would be attained at soil temperatures approaching this value. however, our analysis also revealed that final germination percentage decreased sharply at temperatures higher than 30 °c. results from the hydrotime analysis indicated that the θh constant was reduced from 14.2 to 3.1 mpa d when the incubation temperature increased to 30 °c and then remained stable at 35 °c. seed germination was relatively low even at 20 °c, the germination rate was faster at higher temperatures (25, 30 and 35 °c), with seed germination beginning on the third day. the hydrotime analysis also indicated that ψb(50) were less negative at 30 and 35 °c than at 20 and 25 °c. according to hydrotime theory, the ψb(50) value of a seed population gives an indication of its ability to avoid stress. less negative values of ψb(50) at supra-optimal temperatures result in declines of both seed germination rate and percentage germination, eventually reaching zero at a ceiling temperature. several studies documented that changes in the dormancy state are related to changes in ψb (meyer et al., 2000; alvarado and bradford, 2002; rowse and finch-savage, 2003), with ψb(50) increasing with rising temperatures. many native species in central asia apparently have no pronounced dormancy or lack deep dormancy with species following an opportunistic strategy that allows them to germinate whenever physical conditions become suitable (wesche et al., 2006). soil water availability is critical for seed germination with higher temperatures inducing thermoinhibition (hills and staden, 2003). when the distribution of ψb overlaps with 0 mpa, the proportion of seeds with ψb larger than 0 mpa is inhibited at the given temperature (larsen et al., 2004; watt et al., 2011). in our study, ψb values above 0 mpa were observed at 35 °c (fig. 3), which is consistent with a reduction in maximum final germination percentages with temperature. at value of less than -0.6 mpa at 25 and 30 °c, values of the θh constant and σψb increased. a similar response was reported by dahal and bradford (1994) for tomato seeds, who ascribed this effect to physiological changes produced by prolonged exposure to the osmoticum. because 30 °c was found to be optimal for seed germination (i.e., gr50 was highest at this temperature), some seeds within the population probably germinated so quickly that they escaped the water stress effect of this temperature. the hydrotime model has been effective at explaining the cardinal temperatures for seed germination in potato seeds (alvarado and bradford, 2002) to investigate the effect of fluctuating temperatures on the termination of dormancy (benech-arnold et al., 2000). from a crop production standpoint, the ψb(50) values (-1.86 to -0.80 mpa) determined in our analysis suggest that seeds of a. venetum have a strong tolerance to water stress. according to alvarado and bradford (2002), the decrease of germination rate and percentage in the supra-optimal temperature range is due to an increase (less negative values) in the ψb(g) thresholds for germination. when t exceeded to, the ψb(50) of the seed population shifted to -0.8 mpa d, which was the highest value observed in our study. values of σψb ranged from 1.01 to 1.95 mpa d, which was higher than values reported in other studies and indicates that seed germination time varied substantially. this may be a survival adaptation to harsh desert environments. the extended germination time for a. venetum may avoid mass germination following suitable environment conditions by allowing a few seeds to rapidly germinate under initial favorable temperature and soil water availability, and then wait a bit longer for confirmation of safe germination conditions before the remaining seeds germinate (batlla et al., 2009; watt et al., 2011). conclusion this study characterized a. venetum seed responses to temperature and water availability through the application of thermal time, hydrotime and hydrothermal models. interactions between ψ and temperature affected the ability of the hydrothermal time model to predict germination responses across temperature and ψ conditions. results also revealed some possible limitations in seed germination that need to be considered in working with a. venetum for crop production. the narrow thermal range for seed germination and nonuniform germination in response to ψ and temperature variation are the most important characteristics during a. venetum seed germination. these factors might sufficiently delay or even prevent germination in arid and semi-arid environments. plant breeding and selection in a. venerum may be useful in modifying these responses. acknowledgments we gratefully acknowledge the key laboratory of grassland science for excellent technical assistance and gebao company in xinjiang, china for assistance with field seed collection work. financial support this work was funded by national forage production system project (cars-35) in china. conflict of interest none. references alvarado, v., bradford, k.j., 2002: a hydrothermal time model explains the cardinal temperatures for seed germination. plant cell environ. 25, 1061-1069. angus, j.f., cunningham, r.b., moncur, m.w., mackenzie, d.h., 1981: phasic development in field crops. i. thermal response in the seedling phase. field crops res. 3, 365-378. batlla, d., grundy, a., dent, k.c., clay, h.a., finch-savage, w.e., 2009: a quantitative analysis of temperature-dependent dormancy changes in polygonum aviculare seeds. weed res. 49, 428-438. benech-arnold, r.l., sa´nchez, r.a., forcella, f., kruk, b.c., ghersa, c.m., 2000: environmental control of dormancy in weed seed banks in soil. field crops res. 67, 105-122. bewley, j.d., black, m., 1994: seeds: physiology of development and germination. new york, plenum press. bradford, k.j., 1990: a water relations analysis of the seed germination rates. plant physiol. 94, 840-849. bradford, k.j., 1995: water relations in seed germination. in: kigel, j., galili, g. 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thevs, n., zerbe, s., kyosev, y., rozi, a., tang, b., abdusalih, n., novitskiy, z., 2012: apocynum venetum l. and apocynum picutm schrenk (apocynaceae) as multi-functional and multi-service plant species in central asia: a review on biology, ecology, and utilization. j. appl. bot. food qual. 85, 159-167. trudgill, d.l., honek, a., li, d., van straalen, n.m., 2005: thermal time-concepts and utility. ann. appl. biol. 146, 1-14. trudgill, d.l., squire, g.r., thompson, k., 2000: a thermal time basis for comparing the germination requirements of some british herbaceous plants. new phytol. 145, 107-114. watt, m.s., bloomberg, m., finch-savage, w.e., 2011: development of a hydrothermal time model that accurately characterizes how thermoinhibition regulates seed germination. plant cell environ. 34, 870-876. wesche, k., pietsch, m., ronnenberg, k., undrakh, r., hensen, i., 2006: germination of fresh and frost-treated seeds from dry central asian steppes. seed sci. res. 16, 123-136. westermann, j., zerbe, s., eckstein, d., 2008: age structure and growth of degraded populus euphratica floodplain forests in nw china and perspectives for their recovery. j. integrat. plant biol. 50, 536-546. windauer, l.b., martinez, j., rapoport, d., wassner, d., benech arnold, r., 2012: germination responses to temperature and water potential in jatropha curcas seeds: a hydrotime model explains the difference between dormancy expression and dormancy induction at different incubation temperatures. ann. bot. 109, 265-273. zhang, x., li, r., shi, f., 2007: effects of salt stress on the seed germination of apocynum venetum. acta scientiarum universitatis nankaiensis 40, 13-18 (in chinese with english abstract). address of the corresponding author: department of grassland science, china agricultural university, 2 yuanmingyuan west rd., 100193 beijing, china. e-mail: rongyuping@cau.edu.cn © the author(s) 2015. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 87, 46 55 (2014), doi:10.5073/jabfq.2014.087.007 1 department of medical biomaterials engineering, kangwon national university, chuncheon, republic of korea 2 department of food science and engineering, seowon university, chungju, republic of korea optimization of extraction process for enhancement of antioxidant activity of acer mono bark woon yong choi1, myung hoon jeong1, hyeon yong lee2* (received june 13, 2013) * corresponding author summary response surface methodology (rsm) was used to determine the optimum extraction condition of acer mono bark for its crude extract to have better antioxidant activities. twenty experimental conditions were set based on three key variables such as temperature, time and pressure by signalling reaction variables with 5 levels of 2, 1, 0, 1, 2 in accordance with central composite design for proceeding extraction and antioxidant tests. the optimized condition for the highest extraction yields was 13.10% at 83.48 °c, 54.36 mpa for 13.08 minutes. for dpph radical scavenging ability, an optimal condition was 92.89% at 88.50°c, 49.69 mpa for 15.08 minutes, and for sod-like activity 40.69% at 85.21°c, 53.28 mpa for 15.83 minutes. the optimized condition for total polyphenol content was 4.23 mg/g at 81.51 °c, 52.92 mpa for 14.79 minutes. the most optimized extraction condition was determined to be at 85 °c, 52 mpa for 14 minutes for considering both extraction yield and its biological activities of this plant. introduction acer mono in the aceraceae (also called the maple family) is an arbor of fallen leaves, which grows wild in 100 ~1,800 m altitude in wide areas of korea, japan, china and manchuria. however, most stu-dies conducted in accordance with acer mono have been focused on either utilizing its sap rather than tree itself (kim et al., 1991), or only cultivating various types of the trees (seiwa, 1998). particularly, in japan, there have been a wider range of studies of analyzing the saps from birch trees (terazawa et al., 1984; iguchi et al., 1985), but not investigating the biological activities of the trees such as antioxidant or anticancer activities, etc. therefore, it would be beneficial to seek a possibility of utilizing acer mono by investigating the biological activities of the tree itself since in recent years, people have been trying to lead a healthier lifestyle and improve their nutrition. however, even with the healthier lifestyle, modern people are exposed to various adult diseases, in addition to aging. active oxygen is considered as a cause of many serious diseases. a variety of factors such as normal metabolism, photochemistry reaction, drug metabolism or other abnormal cell metabolism will produce more active oxygen. the reactivity of such active oxygen is so strong that free radical reactions of which destroy lipid, protein, sugar and dna by non-selective and irreversible reactions (morita et al., 1980; morita et al., 1982), which is believed to cause cancers and many other diseases such as brain disorders, heart diseases, ischemia, arteriosclerosis, skin diseases, digestive troubles, infection, rheumatic arthritis or autoimmune diseases, etc. (halliwell and gutteridge, 1990; davies, 1995). in general, phenolic synthetic compounds such as bha (butylated hydroxyl anisol) and bht (butylated hydroxyl toluene) have been widely used for the treatment of such diseases because of their effective and economic factors as well as reliability; however, recently natural components from natural plant resources, not synthetic ones, have been more interested in applying for many areas of food, cosmetics, and pharmaceuticals, etc. (an et al., 2003; kinghorn et al., 1996; yoon et al., 2012) because excessive use of chemical substances could cause more severe toxic reactions in the liver, mucous membrane of stomach, lung, kidney and circulatory system (surh, 1993). therefore, it is necessary to develop safe and effective natural antioxidants (branen, 1975; choe and yang, 1982), and acer mono could be one of promising resources because this tree has been proved to have antioxidant activities by water and/or ethanol extractions (kwon et al., 1997; jin et al., 2008). however, there have been some difficulties in extracting the plants with those solvents through conventional extraction processes, compared to the cases for most commonly used herbs such as panax ginseng, artemisia iwayomogi, and rubus coreanus, etc. since the plants have relatively hard barks that caused to significantly reduce and limit the extraction of active components from them (senorans et al., 2003). to obtain economic and biological feasibility of extracting these plants, proper extraction processes should be developed along with optimizing these extraction parameters because this unique process would contain particular process variables such as pressure and temperature, etc., not for the heat labile substances to be more damaged during the extraction at high temperature even with high extraction efficiency under these conditions (pasquini et al., 2005; butz et al., 2004). optimization of the extraction processes has been considered to be critical not only for maintaining or improving their biological activities, but also enhancing the extraction yields because there must be very close correlation between extraction conditions and biological activities of the extracts especially under extreme extraction conditions of high pressure and high temperature for hard plants (zhang et al., 2007; saldana et al. 2002). therefore, for this case, the interactions of several factors should be carefully considered, not only one or two variables of extraction parameters. however, in most studies, the one-factor-at-time method has been most widely used for extracting natural resources, in which variables such as temperature, extraction solvent, or time are correspondingly set based on a certain variable (gontard et al., 1992). such simple methodology would not consider the cause and effect interactions among those variables, which results in limiting the reliability of the results. this process could not determine a really optimized condition to enhance the biological activities, either. on the other hand, rsm is an effective statistical analyzing method that minimizes the number of tests and evaluates reaction changes in wider ranges, in particular, when multiple independent variables interact in complexities affecting dependent variables (lee et al., 2000; myers, 1971). thus, it is able to find the most optimized condition by consecutively eliminating non significant interaction terms associated with rsm (yoon et al., 1997), which cannot be likely found by the one-factor-at-time method (wang et al., 2008; cho et al., 2004; yoon and cho, 2007). in addition, as shown in this study, optimizing the extraction process correlated to antioxidant activity has not been carried out for trees, and increasing the significance of the model by removing the nonsignificant terms either. therefore, three key extraction variables were selected as extraction time, temperature and pressure to find an optimization of extraction process of acer mono bark 47 optimized extraction process for enhancing antioxidant activity of acer mono bark by using central composite design and rsm, then a variety of extraction conditions was performed in order to obtain the most optimized condition with most significant confidence. materials and methods materials the acer mono collected in may, 2010 was provided by korea forestry research institute (kfri), korea. the trees were dried in the shade in room temperature. then, only bark parts of the plants were peeled and grinded by a grinder, then they were extracted under various conditions. extraction conditions to determine most proper extraction conditions for antioxidant activities of acer mono, 100 g of dried acer mono bark was extracted by reflux condensing extractor (pyrex, seoul, korea) with various extraction solvents such as distilled water at 100 °c, 70% ethanol with water at 85 °c, hexane at 85 °c, chloroform at 85 °c or butanol at 85 °c for 24 hours, then the extracts were concentrated by a rotary vacuum evaporator (eyela, tokyo, japan) before being freeze-dried. electron donating ability (eda) of the freeze dried extracts were estimated by measuring dpph free radical scavenge, to determine the most proper extraction solvent for this plant. after selecting the extraction solvent, three most important extraction parameters for acer mono were set as temperature for 40 ~120 °c, process time for 5 ~ 25 minutes, and pressure for 0 ~100 mpa. the ranges of these variables were chosen from the preliminary experimental results. experimental design for optimizing the extraction condition as shown in tab. 1, to determine the most optimized extraction condition, 20 experimental extraction conditions were defined by central composite design (ccd) in a combination of variables such as extraction temperature (40, 65, 80, 100 and 120 °c), time (5, 10, 15, 20 and 25 minutes) and pressure (0, 25, 50, 75 and 100 mpa) for rsm. a high pressure liquefying extractor (ilshin autoclave co., daejeon, korea) was used to maintain each extraction condition: 20 g of dried acer mono bark was extracted with water in a 1 l high pressure extraction vessel under various extraction conditions from central composite design. then, the extracts were filtered by a membrane filter paper and concentrated a rotary vacuum evaporator (rotary vacuum evaporator n-n series, eyela, tokyo, japan). after that, the concentrated liquid was freeze-dried as a powder before being used. the extraction yield (%, w/w) from each condition was estimated by the ratio of the weight of dry matter of acer mono bark after being extracted to the weight of total dry matters before extraction because higher extraction yield should contain more biologically active substances at first. to determine the most optimized model, after running the above experimental designs, non significant factors were consecutively eliminated based on p-values of curves, square, or interaction terms in proposed equations (i.e. p > 0.05) obtained from analysis of variance (anova) for re-designing the models. measurement of antioxidant activities of dpph and sod-like the electron donating effect for dpph (a,a-diphenyl-picrylhydrazyl) of each extract was measured as follows: 0.5 ml of 0.5 mm dpph liquid (abs. etoh soln.) was put into a test tube in which 1 ml tab. 1: extraction yields, dpph scavenging activity, sod-like activity and total polyphenol contents of bark of acer mono by the central composite ex perimental design for response surface analysis. independent variables response variables‡ extraction temp. extraction time extraction yields dpph (%) sod (%) polyphenol (℃) (min) pressure (mpa) (%, w/w) (mg/g) 1 40 15 50 5.67 ± 0.06 65.25 ± 0.54 32.87 ± 0.45 2.11 ± 0.04 2 60 10 25 8.41 ± 0.04 82.15 ± 1.22 33.11 ± 0.11 2.53 ± 0.06 3 60 10 75 8.16 ± 0.08 83.22 ± 0.45 33.28 ± 0.93 2.43 ± 0.06 4 60 20 25 8.55 ± 0.06 81.23 ± 1.20 33.21 ± 0.41 2.34 ± 0.02 5 60 20 75 8.47 ± 0.04 80.42 ± 0.06 34.25 ± 0.34 2.41 ± 0.14 6 80 5 50 11.15 ± 0.16 92.45 ± 0.07 36.69 ± 0.51 3.08 ± 0.08 7 80 15 0 7.49 ± 0.01 93.22 ± 1.60 37.58 ± 0.43 2.39 ± 0.21 8 80 15 50 13.42 ± 0.16 93.45 ± 0.43 41.25 ± 0.28 4.25 ± 0.08 9 80 15 50 13.24 ± 0.07 92.54 ± 0.91 39.46 ± 0.84 4.36 ± 0.02 10 80 15 50 12.42 ± 0.18 95.62 ± 0.35 42.15 ± 0.31 4.46 ± 0.21 11 80 15 50 13.56 ± 0.16 93.24 ± 0.05 40.28 ± 0.54 4.25 ± 0.10 12 80 15 50 13.28 ± 0.05 92.46 ± 1.84 41.11 ± 0.26 4.36 ± 0.06 13 80 15 50 13.33 ± 0.03 94.25 ± 1.37 40.29 ± 0.49 4.25 ± 0.02 14 80 15 100 11.25 ± 0.19 93.11 ± 0.51 38.44 ± 0.64 3.12 ± 0.04 15 80 25 50 10.28 ± 0.04 92.97 ± 1.64 38.45 ± 0.22 2.94 ± 0.08 16 100 10 25 10.24 ± 0.05 84.21 ± 0.88 37.45 ± 0.10 2.88 ± 0.01 17 100 10 75 10.38 ± 0.14 83.44 ± 2.41 37.11 ± 0.57 2.79 ± 0.04 18 100 20 25 8.36 ± 0.03 85.42 ± 1.64 37.69 ± 0.64 2.56 ± 0.05 19 100 20 75 8.42 ± 0.02 85.21 ± 2.15 37.42 ± 0.55 2.64 ± 0.03 20 120 15 50 6.89 ± 0.11 83.36 ± 1.24 31.25 ± 0.64 2.24 ± 0.02 †the number of experimental condition by central composite design. ‡results are expressed as mean±sd of data obtained from three independent experiments. exp. no† 48 w.y. choi, m.h. jeong, h.y. lee ethanol, 10 μl sample and 990 μl of 100 mm sodium acetate buffer (ph 5.5) were agitated, which remained in a darkroom for 5 minutes in order to induce responses. after that, a uv spectrometer was used to measure the concentration of the remaining radical in 517 nm (oh et al., 1996). eda (%) = (1 – as/ac)×100 (1) then, eda was calculated by equation (1), where “as” represents the optical density values of sample group (with the extract) and “ac” represents the optical density of control group (without the extract). the activity of an antioxidant, butylated hydroxyl anisole (bha) was also examined as a positive control to compare with the extracts. to confirm the antioxidant activities of dpph, the amounts of pyrogallol generated as a catalyst for conversion reaction to h2o2 have also been estimated as sod-like activity by a marklund method (blois, 1958): 0.2 ml of 7.2 mm pyrogallol and 2.6 ml tris-hci buffer (ph 8.5) were added to 0.2 ml sample in a certain concentration, which remained in 25 °c for 10 minutes. after that, a spectrophotometer was used to measure the optimal density at 420 nm to calculate the amount of pyrogallol oxidized from the response liquid. sod-like (%) = (1-as/ac)×100 (2) then, sod-like activity was calculated by equation (2), where “as” represents the optical density values of sample group (with the extract) and “ac” represents the optical density of control group (without the extract). measurement of total polyphenol contents the total polyphenol contents of the extract from each extraction condition were estimated by a method of dewanto et al. (2002), where folin-ciocaltea phenol reagent measures total phenols of polyphenolic compounds by being coloured with molybdenum blue reaction. in detail, 2 ml of 2% na2co3 was added to each of 100 μl extract and remained for 3 minutes, to which 100 μl of 50% folinciocalteu reagent was added. after 30 minutes treatment, optimal density was measured at 750 nm. a standard for phenol content in the plants, garlic acid (sigma, usa) was used to obtain a calibration curve. total polyphenol content was expressed as the weight of garlic acid (mg) in each reagent (g). statistical analysis all experiments were carried out in triplicates and the results were expressed as mean±sd. data were analysed by the generalised linear model (glm) procedure of the statistical analysis system (sas, version 9.1, sas institute, cary, nc). data were analyzed by the analysis of variance and the mean values were considered significantly different with p<0.05. the optimal extraction condition was obtained by regression analysis. results selection of the extraction solvents and measurement of extraction yields fig. 1 shows the effect of five different extraction solvents on dpph electron donating ability of acer mono, and 70% ethanol appeared to be the best extraction solvent with 84.15% of eda, compared to others. it was found that hexane or chloroform extracts had low antioxidant activities since in general non polar solvent could not effectively extract bioactive components from plant resources than polar solvents. this result was well consistent with the others reported by lee et al. (2004) that water or ethanol extracts of dulcis showed higher dpph scavenging activity than those extracted by hexane or butanol as well as high total phenol contents (wang et al., 2008). based on this result, for all other experiments, 70% ethanol was used to extract the bark of acer mono. the central composite design was represented in tab. 1, to optimize the extraction condition of increasing antioxidant activities of acer mono bark by setting 20 extraction conditions with experimental values of extraction yields, polyphenol contents and antioxidant activities. the extracts at 80 °c for 15minutes with 50 mpa pressure were appeared to have the best extraction yield as 13.56% as well as the best antioxidant activities as 95.6 % of dpph and 42.15 % of sod. the lowest yields, on the other hand, were observed at 40 °c for 15 minutes with 50 mpa pressure as 5.67%, which also resulted in low antioxidant activities. these results indicate that low antioxidant activities would be caused by low extracting active components at low temperature, but at too high temperature the antioxidant activities were also observed to be low because of degrading or breaking biologically active components. therefore, it is absolutely necessary to optimize the extraction condition for increasing biological activities especially from hard barks of the plants by considering all four response variables primarily based on extraction yield since high extraction yield could contain more biologically active substances at relatively lower extraction temperature that that of conventional hot water extraction. tab. 2 shows the results of estimating p-values of each response variable for extraction yields in the full quadratic model. p-values of constant and square in the model are 0.000 which falls into p < 0.05; however, p-values of linear and interaction terms are 0.427 (p > 0.05), which led to decrease the significance of the model. thus, these response variables were removed to determine a more optimized model, and tab. 2 is the result of re-estimating p-value after deleting interaction terms. the model equation without the interaction term was appeared to be more suitable, having p-value of the model was 0.000, which is very significant. the second order polynomial of the extraction model was determined with 0.8720 of r-square (adj) as follows: yyield = 13.0357 + 0.7812x1 – 0.6412x2 + 0.7837x3 – 7.2736x12 – 2.8386x22 – 4.4636x32 (1) in the model equation (1), p-value of the pressure term was appeared to affect the most based on p-value of 0.098, followed by temperature and time terms. it was also found that linear term of those response variables was proved to be non significant: however, on the other mean values ± sd from triplicate separated experiments are shown. mean with difference letter (a-d) within different solvents are significantly different at p < 0.05. fig. 1: scavenging effect of the extract from bark of acer mono according to the several solvents on dpph. optimization of extraction process of acer mono bark 49 tab. 2: estimation of p-value of the full quadratic model from the multi-regression analysis of extraction yields before and after deletely most non-significant values. term p-value† p-value‡ term p-value† p-value‡ constant 0.000 0.000 temp 0.105 0.099 linear 0.093 0.080 time 0.175 0.168 square 0.000 0.000 press 0.104 0.098 interaction 0.428 temp2 0.000 0.000 time2 0.002 0.001 press2 0.000 0.000 r-square (adj) 0.8720 0.8720 temp*time 0.115 temp*press 0.835 time*press 0.972 †estimated value before deletely most non-significant values based on p-value. ‡estimated value after deletely most non-significant values based on p-value. fig. 2: effect of extraction temperature, time and pressure on yields of extracts from bark of acer mono. 50 w.y. choi, m.h. jeong, h.y. lee hand, square terms of temperature, time and pressure seemed to be very significant (p < 0.05), which also supports that the polynomial equation (1) would be most proper for extraction yield. in fig. 2, effects of temperature, time and pressure on extraction yields based on each response variable are illustrated in surface and contour line plots by only reflecting linear and square terms, not interaction term. this figure also shows that pressure appeared to significantly affect to extraction yields, which is similar of the p-value results in tab. 2. measurement of antioxidant activities two different dpph radical scavenging and sod-like activities were measured to make sure the antioxidant activities of acer mono bark since these two activities represent different antioxidant action mechanisms. electron donating abilities of natural resources feature donating electrons to active radical and controlling oxidation of fat in food, and restrain aging in human body by active radical. radical scavenging reactions take an important role to prevent diseases or aging in human body (kim et al., 2001). active oxygen kinds in live bodies are derived from oxygen, which is produced by secure structured molecules such as triplet oxygen (3o2) through ultra violet rays, radioactive rays, chemical reaction, or metabolism (trush et al., 1982). the most favorable extraction condition for obtaining both high dpph and sod-like antioxidant activities was observed at 80 °c for 15 minutes with 50 mpa pressure from tab. 1 as 95.62% and 42.15%, respectively. however, it was interesting that for dpph activity, low temperature of 40 °c had the lowest antioxidant activity as 65.25% while for sod-like activity, at 120 °c the lowest activity was observed as 31.20% under same pressure of 50 mpa and extraction time of 15 min. tab. 3 are to compare the p-values of the quadratic models for two antioxidant activities before and after sequentially deleting nonsignificant terms in the model equations, to determine more accurate equations reflecting higher antioxidant activities of acer mono bark. it was interesting that interaction terms for both models of dpph radical scavenging and sod-like activities were found to be most non-significant, based on p-values such as 0.895 and 0.964, respectively. it was same pattern for extraction yield in tab. 2, but the interaction terms of the models for antioxidant activities were more non-significant than the case of extraction yield, whose trend and comparison have not been reported elsewhere even though it is necessary to improve biological activities by considering all the factors together, not separately. in tab. 3, it was clearly shown that re-designing the models by deleting non-significant variables seemed to be a proper methodology to have more suitable models by showing better regression coefficients for both antioxidant activities than the r-square values before deleting the terms as well as significant improvement of the linear terms. the re-designed polynomial equations for dpph radical scavenging and sod-like activities were obtained as follows: ydpph = 92.7893 + 4.2175x1 – 0.0375x2 – 0.1175x3 – 24.3314x12 – 2.4914x22 – 2.0364x32 (2) ysod = 40.5336 + 2.4375x1 + 0.6425x2 + 0.2900x3 – 10.8727x12 – 3.6327x22 – 3.1927x32 (3) for dpph radical scavenging activity, the extraction temperature seemed to be most effective as 0.012 (p < 0.05), followed by pressure and time that were not much significant based on p-values. such results can also be illustrated in fig. 3, in which models are shown in surface plots as expecting the effects for linear and square terms, but not for interaction term, showing that dpph radical scavenging ability was not significantly changing in accordance with time or pressure, but temperature was appeared to be the significant variable which changes the dpph radical scavenging. for sod-like activity in tab. 4, the extraction temperature also seemed to be most effective as 0.007 (p < 0.05), and followed by time and pressure that were not much significant either. however, it was interesting that two variables of pressure and time were not much non-significant for sod-like activity even though they are still higher than p-value of 0.05, compared to the cases of dpph radical scavenging activity in tab. 4. when extraction time and pressure were set at 15 minutes and 50 mpa respectively, but extraction temperature was either 40 °c or 80 °c, then extraction at 80 °c appeared to have higher sod-like activity compared to extraction at 40 °c. in addition, when extraction time and pressure were fixed at 10 minutes and 25 mpa, sod-like activity of extracts at 100 °c was 37.45% and 33.11% was at 60 °c. thus, it appears that approximately 5% was increased in sod-like activity at 100 °c compared to 60 °c. as drawn in surface and contour line plots, it was also confirmed by indicating big changes of sod-like activity indicates in accordance with temperature changes in fig. 4. it should also indicate that extraction temperature must be more important factors for enhancing antioxidant activities while extraction pressure for improving extraction yield of acer mono bark. measurement of total polyphenol contents 4.46 mg/g of the highest polyphenol content was obtained under the extraction condition of 80 °c for 15 minutes with 50 mpa, and 2.11 mg/g of the lowest concentration at 40 °c for 15 minutes with 50 mpa in tab. 1. for other studies of extracting daisy (small flowered chrysanthemum) (park et al., 1998) or mushroom (flammulina velutipes) (kim et al., 2003), the total polyphenol contents increased in using 70% ethanol, not water, which was same result with our preliminary tests for acer mono bark. that was why this study had used 70% ethanol as an extraction solvent with variables of extraction temperature, time and pressure. tab. 5 was the results of estimating p-values of the full quadratic model from the multiregression analysis of total polyphenol content before and after deleting most non-significant variable such as interaction term in the proposed model. after reconstructing the model, most suitable second polynomial was determined as follows: ypolyphenol = 4.2320 + 0.1775x1 – 0.1200x2 + 0.1775x3 – 2.3259x12 – 1.4909x22 – 1.7459x32 (4) this model seemed to be better fitting with higher regression coefficient as 0.8590 of r-square(adj) and smaller p-values (more statistically significant) than the cases of not deleting the interaction terms. it was also found that extraction pressure and temperature were considered mostly affecting the total polyphenol contents of acer mono, whereas time was considered less influenced. these results can also be found in fig. 5, in which the graphs were well fitted to the data as temperature or pressure changed, implying that extraction temperature was most significant variable for extracting polyphenols from the plant. similar results were also reported in other work that the extraction yields of polyphenol from inga edulis leaves were increased 20% by increasing the extraction temperature from 40 °c to 65.2 °c (silva et al., 2007). this result indicates that the temperature mostly affected to the total polyphenol content and that as extraction temperature increased total polyphenol content also increased. validation of the optimal extraction condition with experimental results to evaluate the predictive models, the optimal extraction conditions were determined to obtain high extraction yield with high antioxidant optimization of extraction process of acer mono bark 51 tab. 3: estimation of p-value of the full quadratic model from the multi-regression analysis of dpph radical scavenging activity before and after deletely most non-significant values. term p-value† p-value‡ term p-value† p-value‡ regression 0.000 0.000 temp 0.024 0.012 linear 0.134 0.077 time 0.982 0.980 square 0.000 0.000 press 0.943 0.936 interaction 0.895 temp2 0.000 0.000 time2 0.349 0.297 press2 0.441 0.390 r-square (adj) 0.8300 0.8620 temp*time 0.474 temp*press 0.893 time*press 0.886 †estimated value before deletely most non-significant values based on p-value. ‡estimated value after deletely most non-significant values based on p-value. fig. 3: effect of extraction temperature, time and pressure on dpph scavenging activity of extracts from bark of acer mono. 52 w.y. choi, m.h. jeong, h.y. lee tab. 4: estimation of p-value of the full quadratic model from the multi-regression analysis of sod-like activity before and after deletely most non-significant values. term p-value† p-value‡ term p-value† p-value‡ regression 0.000 0.000 temp 0.007 0.002 linear 0.037 0.013 time 0.392 0.332 square 0.000 0.000 press 0.695 0.657 interaction 0.964 temp2 0.000 0.000 time2 0.010 0.003 press2 0.019 0.008 r-square (adj) 0.8340 0.8690 temp*time 0.901 temp*press 0.663 time*press 0.822 †estimated value before deletely most non-significant values based on p-value. ‡estimated value after deletely most non-significant values based on p-value. fig. 4: effect of extraction temperature, time and pressure on sod-like activity of extracts from bark of acer mono. optimization of extraction process of acer mono bark 53 tab. 5: estimation of p-value of the full quadratic model from the multi-regression analysis of total polyphenol before and after deletely most non-significant values. term p-value† p-value‡ term p-value† p-value‡ regression 0.000 0.000 temp 0.348 0.286 linear 0.524 0.418 time 0.521 0.465 square 0.000 0.000 press 0.348 0.286 interaction 0.980 temp2 0.000 0.000 time2 0.000 0.000 press2 0.000 0.000 r-square (adj) 0.8200 0.8590 temp*time 0.804 temp*press 0.985 time*press 0.746 †estimated value before deletely most non-significant values based on p-value. ‡estimated value after deletely most non-significant values based on p-value. fig. 5: effect of extraction temperature, time and pressure on polyphenol content of extracts from bark of acer mono. 54 w.y. choi, m.h. jeong, h.y. lee activities by the ridge analysis with means of minitab release 14.12.1 (minitab inc., usa) (silva et al., 2007), and this operating condition was also used for extraction experiments as shown in tab. 7. for extraction yields, the most optimized condition was at 83.48 °c, 54.36 mpa for 13.08 minutes, having 13.10% of the maximum response with 13.24% of experimental results carried out under this condition. the maximum response of dpph radical scavenging activity was observed 92.89% at 88.50 °c, 49.68 mpa for 15.08 minutes while 40.69% of the maximum sod-like activity at 85.21 °c, 53.28 mpa and 15.83 minutes, comparing to 90.11% and 39.48% of experimental results, respectively. the optimized condition for total phenolic compounds was at 85 °c, 52 mpa for 14 minutes, having 4.23 mg/g of the maximum response of total polyphenol content by comparing with 3.94 mg/g of experimental data. the experimental data were within 95% confidence interval of the predicted values for all of independent variables, which confirms that the proposed model could be used to optimize the extraction of relatively hard plants such as acer mono bark. accordingly, antioxidant activities of acer mono bark was considered as it mostly depends on the total polyphenol content. discussion the rsm was used to optimize extraction parameters that could have high antioxidant activities along with high contents of phenolic compounds from acer mono bark. statistical analysis revealed that the effect of pressure was most significant in extraction yield whereas the effect of temperature was significant in antioxidant activities. more specifically, in considering to extract relatively harder resources of the plant, acer mono bark than other medicinal herbs, more than most commonly used variables of temperature, solvents ratio or ethanol concentration, etc. should be employed, which would be pressure response since this variable was hardly found in rsm (karacabey and mazza, 2010; liu et al., 2010). from our results, the pressure (p = 0.098) appeared to be most significant in extraction yield, followed by temperature and time, respectively. however, this was different from other works that the temperature was most tab. 6: polymonial equations calculated by response surface analysis program for extraction condition of bark of acer mono. response variables† second order polynomials‡ r2 yields yyield = 13.0357 + 0.7812x1 0.6412x2 + 0.7837x3 7.2736x12 2.8386x22 4.4636x32 0.8720 dpph ydpph = 92.7893 + 4.2175x1 0.0375x2 0.1175x3 24.3314x12 2.4914x22 2.0364x32 0.8620 sod ysod = 40.5336 + 2.4375x1 + 0.6425x2 + 0.2900x3 10.8727x12 3.6327x22 3.1927x32 0.8690 polyphenol ypolyphenol = 4.2320 + 0.1775x1 0.1200x2 + 0.1775x3 2.3259x12 1.4909x22 1.7459x32 0.8590 †dpph: dpph scavenging activity, sod: sod-like activity, polyphenol: total polyphenol. ‡x1, temperature (℃); x2, time (min); x3, pressure (mpa). tab. 7: predicted and experimented value of response variables at a given condition within the range of optimum extraction conditions. response variables independent variables† prediected value experimental value x1 x2 x3 extraction yield (%, w/w) 83.48 13.08 54.36 13.10 13.24 dpph scavenging activity (%) 88.50 15.08 49.68 92.89 90.11 sod-like activity (%) 85.21 15.83 53.28 40.69 39.48 polyphenol (mg/g) 81.51 14.79 52.92 4.23 3.94 †x1, temperature (℃); x2, time (min); x3, pressure (mpa) significant in extraction yield of roasted wheat germ (gelmez et al., 2009). for polyphenol contents with high antioxidant activities, both temperature and pressure appeared to be important, which was similar to other studies that as temperature rose under less than 150 mpa, polyphenol content increased (choi et al., 1998; murga et al., 2002). overall antioxidant activity shows that the highest antioxidant activity appeared at 80 °c, 50 mpa for 15 minutes, which was similar to the condition regarding total polyphenol content. quadratic models were used in predicting all the responses, and the optimal extraction conditions were determined based on combination all responses. the second-order polynomial models were derived by consecutively deleting most non-significant terms in the models, maintaining better significance with lower p-values of many interaction terms in the models. it was proved that this model well predicted satisfactory results, compared to the experimental data. this study would also be a standard methodology considering relationship among variables such as temperature, pressure and time to extract resources from wood substances, based on the fact that considering multiple variables is required for enhancing activity of extracts. acknowledgements this study was supported by a grant of the korean health technology r&d project, ministry of health & welfare, republic of korea. 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481-490. yoon, o.h., cho, j.s., 2007: optimization of extraction condition for hot water extracts from chrysanthemum indicum l. by response surface methology. korean j. food cookery sci. 23, 1-8. yoon, w.b., park, j.w., kim, b.y., 1997: surimi-starch interactions based on mixture design and regression models. j. food sci. 62, 555-560. young-joon surh, 1993: role of metabolic activation in drug toxicity and chemical carcinogenesis. j. pharmaceutical sci. 9, 42-51. zhang, s., ruizhan, c., changzheng, w., 2007: experiment study on ultrahigh pressure extraction of ginsenosides. j. food eng. 79, 1-5. address of the corresponding author: professor hyeon yong lee, ph.d e-mail: hyeonl@seowon.ac.kr journal of applied botany and food quality 88, 197 201 (2015), doi:10.5073/jabfq.2015.088.028 1 department of horticulture, faculty of agriculture, university of cukurova, adana, turkey 2ataturk central horticultural research institute, yalova, turkey 3 department of horticulture, faculty of agriculture, ataturk university, erzurum, turkey genetic relatedness among quince (cydonia oblonga miller) accessions from turkey using amplified fragment length polymorphisms h. topcu1, s. kafkas1*, a. dogan2, m.e. akcay2, s. ercisli3 (received february 8, 2015) * corresponding author summary among fruit species cultivated in turkey, quince shows a great deal of morphological variability and adaptability to the various environments. we attempted to study genetic relationships among 40 quince accessions using amplified fragment length polymorphisms (aflps) for future breeding programs. the accessions were previously characterized based on their pomological and yield characteristics and then the best ones were planted in a single collection in ataturk central horticultural research institute, yalova, turkey. six aflp primer combinations generated a total of 746 bands, 493 of which were polymorphic (66.1 %). resolving powers of the aflp primers ranged from 48.0 to 99.6 making a total of 421.5. unweighted pair group method with arithmetic average (upgma) clustering of the accessions showed three major clusters and ‘sapancaesme’ and ‘esme-3’ were the closest accessions with 95 % similarity. our study indicated that there is a high level of genetic diversity among quince accessions in turkey and the results of this study can be used for future cultivar breeding programs in quince. introduction the quince (cydonia oblonga miller) belongs the genus cydonia and native to warm-temperate regions including asia minor (browicz, 1972). the total quince production in the world is 596.532 tons, and turkey (135.500 tons) is the leader producer country. the other important quince producer countries are china (125.000 tons), uzbekistan (80.000 tons), morocco (46.000 tons), iran (36.500 tons), argentina (27.500 tons), azerbaijan (27.140 tons) and spain (14.000 tons) (fao, 2012). the quince is a deciduous tree, growing up to 5-8 m tall and 4-6 m wide. it has very close relationships with apples and pears, and has a pear or apple shaped pome fruits, which is bright golden yellow when mature. in most quince producer countries, they are not grown in large amounts; typically several quince trees are grown in a mixed orchard with several apples and other fruit trees (westwood, 1993). the fruits of most of quince cultivars are hard, sour and astringent in maturation time and therefore a few cultivars can be eaten raw. due to this reason, quince fruits are mainly used to make jam and jelly or they may be peeled, then roasted, baked or stewed. the flesh of the fruit turns red after cooking (silva et al., 2002; 2004). the quince fruit is also known as an important dietary source due to its antioxidant, antimicrobial and antiulcerative properties (silva et al., 2004; hamauzu et al., 2006; fattouch et al., 2007). quince trees have been used as a dwarf rootstock for pear for a long time. it forces scion to produce precocious fruits, and relatively more fruit-bearing branches, and of accelerating the maturity of the fruit (gulen et al., 1999; massai et al., 2008). earlier characterization of the quince genotypes were performed primarily based on phenotypic traits of the plants such as color, size, shape and other agronomical characters of fruits in turkey (ercan et al., 1992; ercisli et al., 1999; dumanoglu et al., 2009; kuden et al., 2009). however, information from morphological and phenotypic characteristics are not sufficient to identify quince genotypes because of environmental plasticity on it. thus, environmentally free genotypic traits are necessary for proper identification and estimation of genetic diversity among quince accessions. molecular characterization assays provide an efficient tool for the evaluation of genetic diversity in plants (halasz et al., 2010; badri et al., 2014). various types of molecular markers (rflp, rapd, ssrs, and aflp) have been successfully used to assess the levels of genetic diversity in fruit tree species (belaj et al., 2003; chen et al., 2005; halasz et al., 2006; ercisli et al., 2008; kafkas et al., 2008; kafkas et al., 2009). aflp markers are highly reproducible with overall error rates of less than 2 % (vos et al., 1995), it is suitable for high-throughput genotyping, and dna sequence information is not a prerequisite. although the aflp method has been one of the most widely used marker system to identify genetic variability in many different fruit tree species, the use of this powerful and reliable method in quince is very rare. the objective of this study is to characterize 40 quince accessions of cydonia oblonga from turkey using aflp markers, to determine whether aflp markers are appropriate to discriminate quince accessions and to have a better understanding about the variability within the turkish quince germplasm. materials and methods plant material forty quince accessions (cydonia oblonga) were used in the pre sent study (tab. 1). these accessions were previously selected for their better fruit and yield characteristics than others and vegetatively propagated, and then they were planted in the ataturk horticultural research institute in the yalova province of turkey. the pomological characteristics of accessions were published elsewhere (buyukyilmaz, 1999). dna extraction and aflp analysis genomic dna was isolated from leaf tissue by the ctab method (doyle and doyle, 1987). dna concentration was estimated by comparing band intensity with l dna of known concentrations, after 0.8 % agarose gel electrophoresis and ethidium bromide staining. dna was diluted to 50 ng μl-1 for aflp reactions. details of aflp assay, adaptor and primer sequences, pcr conditions for preselective and selective amplifications, and selective primer designation were according to vos et al. (1995). genomic dna was restricted with ecori/msei enzyme combination and double-stranded adaptors specific to each site were ligated. preselective amplifications were done with primers complementary to the adaptors with an extra selective base on each primer (ecori-a/msei-c). selective amplifications were performed using six primer combinations with three msei (m) and three ecori (e) primers (e39/m55, e39/m57, 198 h. topcu, s. kafkas, a. dogan, m.e. akcay, s. ercisli e45/m59, e42/m59, e45/m60 and e42/m60). aflp fragments were resolved using capillary electrophoresis on an abi 3130xl genetic analyzer [applied biosystems inc., foster city, calif, (abi)] with the data collection software 3.0 (abi). aflp fragments were analyzed with genescan analysis software 4.0 (abi) and the data were assembled in binary format. data analysis the ability of the most informative primer pairs to differentiate the genotypes was analyzed by calculating their resolving power (rp) according to prevost and wilkinson (1999) using the formula rp =∑ ib, where ib =1-(2 x | 0.5 p | ), and p is the proportion of the 40 genotypes containing the i band. jaccard’s similarity coefficients (jaccard, 1908) were calculated for all pair-wise comparisons among the 40 quince genotypes. a dendrogram was generated using ntsyspc version 2.11v (exeter software, setauket, ny) (rohlf, 2004) based on the un-weighted pair-group method of arithmetic average cluster analysis (upgma). results and discussion a total of 746 fragments were amplified using six primer combinations, 493 (66.8 %) were polymorphic in characterizing 40 quince accessions (tab. 2). the number of total bands produced by each primer combination ranged from 97 (e45/m60) to 156 (e42/m59) with an average of 104.1 per primer pair. the number of polymorphic fragments per primer combination ranged from 59 to 103 with an average of 69.1. among the six primer combinations tested, e42/ m59 and e45/m60 amplified the lowest and highest number of total as well as polymorphic fragments, respectively (tab. 1). the percentage of polymorphic bands varied considerably among the primer combinations. the highest polymorphism ratio (72.6 %) was observed in e39/m55 primer pair and followed by e39/m57 (69.9 %), e42/m59 (66.0 %), e42/m60 (65.7 %) and e45/m59 (60.8 %) and e45/m60 (60.8 %) (tab. 1). resolving power (rp) ranged from 48.0 (e45/m60) to 99.6 (e42/m59), with a total of 421.5. the average rp value of the primers was found to be 70.3 (tab. 1). in the present study, we obtained the highest polymorphism ratio (72.6 %) in e39/m55 primer pair and followed by e39/m57 (69.9 %) and e42/m59 (66.0 %). screening and selection of primer combinations, which detect maximum genetic variation, are vital to establish dependable genetic relationships among accessions (kafkas et al., 2009). in this study, aflp analysis successfully differentiated the quince accessions each other and it was confirmed that aflp is a powerful marker system. aflp markers have been previously used in the analysis of different fruit species for example mulberry (sharma et al., 2000), tea (kafkas et al., 2009), walnut (kafkas et al., 2005), myrtle (bruna et al., 2007) and pomegranate (jbir et al., 2008) and all these results confirmed the power of aflp technique to discriminate genotypes. although quince is known self fertile, their level of polymorphism was comparable to those of previous studies. this is an indication of the high degree of polymorphism among the accessions tested. in this study, resolving power (rp) ranged from 48.0 (e45/m60) to 99.6 (e42/m59). rp has been found to correlate strongly with its ability to distinguish between accessions. it is usually difficult to compare the value of primers used in different investigations. the use of rp might enable direct comparison of primers both within and between studies (prevost and wilkinson, 1999). a dendrogram constructed using upgma method of cluster analysis from a combined data of all primer combinations classified actab 1: the origin of quince accessions used in this study accession origin accession origin accession origin altin subasi kocaeli esme 8 sakarya esme 6 kocaeli altin yalova yalova havan yalova limon 2 sakarya bardak bursa esme 14 kocaeli viranyadevi yalova demir 1 kocaeli tekkes yalova beyaz ayva kocaeli ege-25 izmir 27-1 yalova 7-2 yalova ekmek keles bursa gordes yalova 6-3 yalova esme 2 kocaeli esme 10 sakarya 10-3 yalova sapanca esme sakarya bencikli yalova ekmek yalova yalova esme 3 kocaeli ege-22 izmir esme arifiye sakarya esme 4 kocaeli sekergevrek yalova esme 1 sakarya limon 1 sakarya 2-3 yalova esme 7 sakarya 19-2 yalova esme 5 kocaeli esme 9 sakarya esme 13 kocaeli esme 11 bilecik esme 12 bilecik limon yalova yalova tab 2: number of total and polymorphic aflp bands, percentage of polymorphic bands, and resolving powers in the dna-fingerprinting of 40 quince genotypes originating from turkey. aflp primer total bands polymorphic polymorphism resolving combinations (no.) bands (no.) (%) powers (rp) e39/m55 113 82 72.6 80.9 e39/m57 123 86 69.9 57.4 e45/m59 120 73 60.8 66.5 e42/m59 156 103 66.0 99.9 e45/m60 97 59 60.8 48.0 e42/m60 137 90 65.7 69.1 total 746 493 421.5 mean 104.1 95 66.8 70.3 genetic relatedness in quince by aflp 199 cessions into three main clusters (fig. 1). cluster i consisted of six quince accessions. the closest accessions in this cluster were ‘esme-7’ and ‘esme-9’ which were surrounded by the accessions ‘esme-12’, ‘esme-1’, ‘esme arifiye’ and ‘ekmek yalova’ respectively (fig. 1). cluster ii include only ‘10-3’ genotype. cluster iii contained 34 accessions and this cluster divided into two sub clusters. the first subcluster included 12 accessions (‘6-3’, ‘7-2’, ‘beyaz ayva’, ‘viranyadevi’, ‘limon-2’, ‘esme-6’, ‘esme-11’, ‘esme-5’, ‘2-3’, ‘sekergevrek’, ‘ege 22’ and ‘bencikli’, whereas subcluster ii contained 21 accessions (‘esme-10’, ‘gordes, 27-1’, ‘tekkes’, ‘esme-14’, ‘havan’, ‘esme-8’, ‘limon yalova’, ‘esme13’, ’19-2’, ‘limon-1’, ‘esme-4’, ‘esme-3’, ‘sapanca esme’, ‘esme2’, ‘ekmek keles’, ‘ege 25’, ‘demir-1’, ‘bardak’, ‘altin yalova’ and ‘altin subasi’). the accession ‘esme-10’ took place between two subclusters. in the subcluster ii, ‘sapanca esme’ and ‘esme-3’ were the closest accessions in this study (95.1 % similarity), with the accessions ‘esme’, ‘esme-4’, and ‘limon-1’. the pairs of accessions ‘bardak’ with ‘demir-1’ and ‘ekmek keles’ with ‘esme2’ were also close to each other with 92.8 % and 92.6 % similarity, respectively (fig. 1). the upgma dendrogram formed from aflp bands successfully separated the closely-related quince accessions (particularly ‘esme’ genotypes). it is clear that there is a large clonal variability within ‘esme’. this genotype was grown in different parts of western anatolia (tab. 1). this fact points to cultivation since remote times in quince growing areas in anatolia and finally the numerous ‘esme’ types would subsequently have been generated through accumulation of somatic mutations during the long period of mutations (ercisli, 2004). local people groups would have moved over long distances in the anatolia over time and carried with them the core of quince variation, while further ad hoc cultivation would have caused additional and location-specific variation. moreover, previous study indicating ‘esme’ genotypes showed a certain degree of morphological and phenological variability within the population of individuals (buyukyilmaz, 1999). most fruit tree species, including quince, are vegetatively propagated to maintain agronomically valuable genotypes. however, after many propagation cycles, clones accumulate phenotypic differences in agronomic traits and clonal diversity appears (orive, 2001). this diversity can then be used to select the best clones or a new improved cultivar within a given variety. a recent study of the molecular polymorphisms generated along vegetative propagation (carrier et al., 2012) through a genome-wide comparison of spontaneous grape ‘pinot noir’ clones showed that only a small number of snp and indel events are at the origin of clonal variation, while mobile elements of many families are involved in most polymorphisms, displaying the highest mutational event. in general, our results were in agreement with those of dumanoglu et al. (2009) who indicated a large genetic diversity within different ‘kalecik’ quince clones in turkey (average polymorphism ratio with 65 %) by using ssr markers. yamamato et al. (2004) used a total 118 ssr markers (developed for apple and pears) on 20 quince cultivars and they obtained relatively high discrimination capacity from 39 ssr markers. yuksel et al. (2013) conducted simple sequence repeat (ssr) analyses of 15 traditional quince (cydonia oblonga) cultivars from anatolian gene sources for molecular characterization and investigation of genetic relationships. they used eight ssr loci that previously developed from apple and pear and they found similarity ratio between 18-87 % among cultivars. in present study, in most cases, quince accessions within the same cluster did not have similar morphological fruit characteristics. for example, in the cluster i, the closest accessions ‘esme-7’ and ‘esme-9’ have different tree and fruit characteristics. ‘esme7’ has light yellow fruit skin color at maturation and esme9 has greenish yellow skin color. esme7 has oval with neck fruit shape, whereas ‘esme9’ has neck less oval fruits. the yields of these accessions are also very variable (buyukyilmaz, 1999). in addition, in the cluster i, ‘ekmekyalova’ has yellow skin color. the genotype ‘esme-1’ has medium fiber in its fruits; however the other accessions had low fiber levels. fruit weight of six accessions in cluster i varied from 350 to 396 g and yield per tree varied from 62 kg (‘esme-7’) to 110 kg (‘esmearifiye’) (buyukyilmaz, 1999). in the cluster ii, the accessions ‘sapancaesme’ and ‘esme-3’ were the closest accessions with 95 % similarity ratio. however, these two accessions had different fruit characteristics. for example, ‘sapancaesme’ had oval with neck fruit shape, however ‘esme-3’ had oval with light neck fruit shape. the other closest pairs of accessions ‘bardak’-‘demir-1’ and ‘ekmekkeles’-‘esme-2’ have also different fruit characteristics: ‘bardak’ has greenish yellow, long neck fruits while ‘demir-1’ has light yellow oval with light neck fruits. ‘bardak’ has more fiber characteristics than ‘demir-1’ (buyukyilmaz, 1999). the accessions from the same province were assigned in the different cluster. for example, the selections from esme cultivar in kocaeli province, ‘esme-1’ placed in cluster i and the other accessions (‘esme-2’, ‘esme-3’, ‘esme-4’, ‘esme-5’ and ‘esme-6’) distributed in cluster ii. likewise, the esme accessions from sakarya province were also assigned in different clusters (‘esme-7’ and ‘esme-9’ in cluster i and ‘esme-8’ and ‘esme-10’ in cluster ii (fig. 1). the closest pair of accessions in this study, ‘sapancaesme’ and ‘esme-3’, were collected from the different provinces (sakarya and kocaeli) in northwestern parts of turkey. this could be atfig. 1: dendrogram of 40 quince genotypes originating from turkey resulting from the unweighted pair-group method of arithmetic mean cluster analysis based on jaccard similarity coefficient obtained from 746 aflp markers. 200 h. topcu, s. kafkas, a. dogan, m.e. akcay, s. ercisli tributed to the unrestricted movement of planting materials from region to region, high self-pollination habit and perennial nature of the crop. on the contrary, the accessions ‘esme-11’ and ‘esme-12’ were collected from bilecik province in turkey, however they were genetically far from each other. the low genetic similarity between these accessions collected from the same region could be attributed to the vast agro-ecological diversity present within each region that can contribute to the development of genetically diverse accessions through natural selection. therefore, similarity in collection locality may not necessarily imply genetic similarity in quince, in turkey. dissimilarities in groupings using molecular markers or phenotaxonomic characteristics were also reported in strawberry (garcia et al., 2002), mulberry (orhan et al., 2007) and olive plants (hagidimitriou et al., 2005). these discrepancies were also previously reported by zamani et al. (2007), who compared data from the genetic distance matrices obtained from rapd markers and from fruit characteristics. their correlation coefficient, for comparison of morphological and rapd data, was only 23 %. these results support the view that morphological characteristics are not reliable for estimating genetic relationships among large and diverse groups of accessions, and should be used mainly for discrimination. results of the present study as well as studies by yamamato et al. (2004) in japan dumanoglu et al. (2009) in turkey, halasz et al. (2009) in hungary and bassil et al. (2011) in usa indicated the presence of genetic variation both genotypic and clonal level among quince genotypes. this confirms the importance of conservation of the turkish quince gene pool for the quince industry. since quince is a tree and a perennial crop, it demands large area and year round management which is expensive. moreover, morphological markers require evaluation over long periods of time and the present study indicated the inadequacy of morphological characters for characterization of closely related accessions. hence, genetic diversity analysis using dna-based marker techniques is recommended for cost effective and efficient conservation of quince germplasm. the results of the present study may benefit breeders in selecting the most diverse accessions to begin crossing and selection programs. this may result in increased quince growing for better fruit production. this report is also demonstrates the presence of mutations of agronomical relevance within a monoclonal cultivar of esme. references badri, j., yepuri, v., ghanta, a., siva, s., siddiq, e.a., 2014: development of microsatellite markers in sesame (sesamum indicum l.). turk. j. agric. for. 38, 603-614. bassil, n.v., postman, j.d., hummer, k.e., mota, j., sugar, d., williams, 2011: quince (cydonia oblonga) genetic relationships determined using microsatellite markers. acta hortic. 909, 75-84. belaj, a., satovic, z., cipriani, g., baldoni, l., testolin, r., rallo, l., trujillo, i., 2003: comparative study of the discriminating capacity of rapd, aflp and ssr markers and their effectiveness in establishing genetic relationships in olive. theor. appl. genet. 107, 736-744. browicz, k., 1972: cydonia miller. in: davis, p.h. 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mol. res. 12 (4), 5880-5888. zamani, z., sarkhosh, a., fatahi, r., ebadi, a., 2007: genetic relationships among pomegranate genotypes studied by fruit characteristics and rapd markers. j. hort. sci. biotechnol. 82, 11-18. addresses of the authors: h. topcu, s. kafkas, department of horticulture, faculty of agriculture, university of cukurova, 01330, adana, turkey e-mail corresponding author: skafkas@cu.edu.tr a. dogan, m.e. akcay, ataturk central horticultural research institute, yalova, 77102, turkey s. ercisli, department of horticulture, faculty of agriculture, ataturk university, 25240, erzurum, turkey journal of applied botany and food quality 88, 34 41 (2015), doi:10.5073/jabfq.2015.088.007 1uludag univ., faculty of agric., dep. of food eng., bursa, turkey 2uludag univ., faculty of agric., dep. of horticulture, bursa, turkey 3the scientific and technological research council of turkey, bursa test and analysis laboratory, bursa, turkey 4uludag univ., keles voc. high school, dep. of food tech., bursa, turkey 5uludag univ., yenisehir ibrahim orhan voc. high school, dep. of food tech., bursa, turkey 6i̇stanbul aydın univ., anadolu bil voc. high school, dep. of food tech., istanbul, turkey 7pamukkale univ., faculty of engineering, dep. of food eng., denizli, turkey 8uludag univ., faculty of science and arts, dep. of chemistry, bursa, turkey chemical and techno-functional properties of flours from peeled and unpeeled oleaster (elaeagnus angustifolia l.) yasemin sahan1, duygu gocmen1, asuman cansev2, guler celik3, emine aydin4, ayşe n. dundar5, dilek dulger6, h. betul kaplan7, asli kilci1, seref gucer8 (received september 19, 2014) summary oleaster flours were produced from two different genotypes (go1 and go2) and methods (peeled oleaster flour: pof and unpeeled oleaster flour: upof). oleaster flour samples (ofs) contained high levels of dietary fibers and micro minerals. the contents of fe, cu, b, and cr in flours obtained from oleaster fruits were higher in upof than in pof samples. palmitic acid was the major fatty acid which was followed by oleic acid and lignoceric acid. all samples contained greater amount of saturated fatty acids (sfa) as compared to mono unsaturated fatty acids (mufa) and polyunsaturated fatty acids (pufa). among seven different organic acids detected, the level of citric acid was the highest and it was followed by malic, acetic and oxalic acids. high nutritional contents of oleaster flour indicated that it is a good source of dietary fiber, micro minerals, as well as organic and fatty acids. the water solubilities (ws) and water absorption capacities (wac) of oleaster flours were adequate for their utilization. they also seem to have an improving effect on emulsion properties of albumin. these results highlighted that it is possible to use the oleaster flour in some processed foods such as bakery goods, dairy products (ice cream and yoghurt), beverages and confectionery. moreover, the oleaster flour could also be used in the preparation of low-fat, high-fiber dietetic products due to its high dietary fiber content. introduction oleaster (elaeagnus angustifolia l., russian olive) belongs to elaeagnus l. genus and elaeagnaceae family. elaeagnus angustifolia l. is a kind of shrub or tree with a height of up to 7 m and a capacity to grow under a wide range of environmental conditions (klich, 2000). this species shows a broad geographical range, existing widely in asia and europe, particularly in turkey, caucasia and central asia (aksoy and sahin, 1999). it is widely cultivated for its edible fruits in middle and east anatolia. its fruits are reddish-brown, elliptic, 9-12 mm long and 6-10 mm wide and they ripen in september. they can be consumed either fresh or in dried form. oleaster fruits are also used as diuretic, tonic, antipyretic, antidiarrheal and as a medicine against kidney disorders (to prevent inflammation or to eliminate kidney stone) in traditional turkish medicine (ayaz and bertoft, 2001; gurbuz et al., 2003), against dysentery and diarrhea in the kingdom of jordan (lev and amar, 2002) and, owing to their antiinflammatory, antinociceptive and analgesic effects, in iranian folk medicine. medicine obtained by decoction and infusion of its fruits is considered to be a good remedy against fever, jaundice, asthma, tetanus and rheumatoid arthritis (ahmadiani et al., 2000). although this species grows naturally in most parts of turkey, its fruits are of limited use in agricultural and food industry. in parallel with the increase in demand for healthy foods, functional products have been used increasingly as ingredients to improve functionality of foods by modifying their nutritive composition. cansev et al. (2011) suggested that elaeagnus angustifolia l. fruit was a valuable horticultural product thanks to its rich and beneficial nutrient composition. their study was a preliminary research for investigating the nutritional values and potential use of elaeagnus angustifolia l. species in the food industry. goncharova et al. (1993) have also reported an abundance of palmitoleic acid (16:1) in fruit skin and linoleic acid (18:0) and palmitic acid (16:0) in seeds as both phospholipids and glycolipids. oleaster flour may be obtained from dried fruits and its flour may be used as a functional ingredient in the production of bakery products, yoghurt, ice cream, infant food, chocolate, confectionery etc. thanks to its floury structure, specific taste and functional properties like dietary fibre, mineral content and phenolic compounds. however, there was no sufficient information regarding the composition of oleaster flour. therefore, the goal of our research was to examine flour from oleaster fruit and to investigate its functional properties [water absorption capacity (wac), water solubility (ws), emulsion capacity (ec) and emulsion stability (es)] and composition of minerals, organic acids and fatty acids. material and methods materials two different genotypes were used as oleaster fruit samples. these genotypes were supplied from two different regions in turkey: go1 (40o11´19.60´´n-26o06´09.16´´e) and go2 (39o34´01.48´´n 26o51´02.90´´e). the fruits had approximately the same maturity (almost reddish) with uniform shape, size and health conditions. mature fruits were randomly collected. fruit samples were dried at 50 ºc for 20 hours in a hot air oven dryer. oleaster flour was pro duced by two different methods. in the first method; skin and seeds of dry fruit samples were removed using a plastic knife, and then the fruit pulp was ground in a coffee grinder and sieved through a standard sieve (mesh size 60). the product obtained at the end of this process was named peeled oleaster flour (pof). in the second method; only seeds of dry samples were removed using a plastic knife, and then the fruit pulp and skin were ground together in a coffee grinder and sieved through a standard sieve (mesh size 60). this product was named unpeeled oleaster flour (upof). sample codes are go1-pof (genotype 1, peeled oleaster flour), go1-upof (genotype 1, unpeeled oleaster flour), go2-pof (genotype 2, peeled oleaster flour) and go2-upof (genotype 2, unpeeled oleaster flour). properties of flours from oleaster 35 methods chemical analysis oleaster flours (ofs) were analyzed for moisture (metod no: 925.40), ash (metod no: 950.49) and dietary fiber (method no: 985.29) (aoac, 1990). protein contents of samples were determined by the kjeldahl method according to aacc metod no: 4610.01(aacc 1999) using a buchi apparatus (models 430 and 320, buchi laboratoriums-techinic ag, flawil, switzerland). starch contents of oleaster flours were determined with starch (go/p) assay kit (sigma-aldrich corp.) determination of micro minerals reagents all solutions were prepared with analytical reagent grade chemicals and ultra-pure water (with 18 mω cm resistivity) generated by purifying distilled water with the tka ultra pacific and genpura water purification system (germany). suprapur hno3 (67 % v/v) was purchased from merck (darmstadt, germany). standard stock solutions containing 1000 mg/l of each element (zn, fe, cu, mn, b, cr, co, se and mo) were purchased from merck (darmstadt, germany) and used to prepare appropriate calibration standards. working standards were prepared daily in 0.3 % (v/v) hno3 and used without further purification. botanical certified reference materials (crms), cabbage iaea – 359 (austria), tea ncs zc73014-(gsb-7) (china) and strawberry lgc7162 (england), were analyzed for validation purpose. a 10 μg/l lithium, yttrium, cerium, thallium, and cobalt in 2 % hno3, cat no. 5184-3566 (agilent technologies) was used to prepare a tuning solution. 1000 mg/l standard stock solutions (merck, darmstadt, germany) were prepared in 0.3 % hno3 for internal standard solution. argon (purity: 99.9995 %, linde, turkey) was used as carrier gas. sample preparation procedure sample digestion was carried out using the millestone mls 1200 (italy) microwave digestion system, equipped with a rotor for 6 type sample vessels (polytetrafluorethylene (ptfe) tubes). before use, quartz vessels were decontaminated in a bath of 10 % hno3 (67 % v/v), then rinsed with ultra pure water, and dried in an oven at 40 °c. the samples were homogenized and subsequently around 0.5 g was weighed directly on ptfe flasks after adding 6 ml of hno3 and subjected to a digestion program: 250 w (2 min), 0 w (2 min), 250 w (6 min), 400 w (5 min) and 600 w (5 min). after cooling to room temperature, sample solutions were quantitatively transferred into 50 ml polyethylene flasks. 100 μl of internal standard solution (1 mg/l) was added and then the digested samples were diluted to 25 ml before analysis by using icp-ms and icpoes equipments. instrumentation icp-ms measurements were performed using an agillent 7500a series shield torch system icp-ms (usa). the sample solutions were pumped by a peristaltic pump from tubes arranged on a cetac asx 520 auto sampler (cetac, omaha, nebraska, usa). the isotopes 53cr, 95mo, 82se and 59co were selected as analytical masses in icp-ms standard mode. the analyses were performed at the following flow rates: (a) plasma gas of 15 l/min, (b) auxiliary gas of 0.9 l/min, and (c) sample of 0.8 ml/min. all chemical analyses were carried out in duplicate on each sample. multi-element standard solutions were used for external calibration. eight standards with standard linear regression and internal standardization were prepared at levels ranging from 0 to 200 μg/l. the calibration curve was drawn from six points including the calibration blank (sahan et al., 2007). the zn, fe, cu, mn and b determination process was performed using an inductively coupled plasma optical emission spectrometer (icp-oes) model perkin elmer 2100 with axial view (usa). the emission intensities were obtained for the most sensitive lines free of spectral interference. the analyses were performed at the following flow rates: (a) plasma gas of 15 l/min, (b) auxiliary gas of 1 l/min, and (c) sample of 0.8 ml/min. the mineral eluates were monitored at different wavelengths: 206.2 nm-zn, 238.2 nm-fe, 327.4 nm-cu and 257.6 nm-mn. all chemical analyses were carried out in duplicate on each sample. determination of fatty acid composition the fatty acids of oleaster flour were separately extracted with diethyl ether for 6 hr by using soxhlet extraction method. the extract was protected against light. the solvent was evaporated under reduced pressure and temperature and the oil was collected. the analytical methods for determination of fatty acid composition are described in regulation standard method (iso 5509:2000, 2000). fatty acids were converted to fatty acid methyl esters before analysis by shaking a solution of 0.6 g of oil and 4 ml of isooctane with 0.2 ml of 2 n methanolic potassium hydroxide. the converted fatty acid methyl esters were analyzed using a shimadzu (gc-17 a) chromatograph, equipped with a capillary column (db wax; 30 m × 0.25 mm; 0.25 μm), a split-splitless injector, and a flame ionization detector (fid). the carrier gas was nitrogen and used at a flow rate of 1 ml/ min. the temperatures of the injector, detector, and oven were held at 250, 250, and 210 °c, respectively. determination of organic acids seven organic acids (oxalic, tartaric, malic, l-ascorbic, acetic, citric, and fumaric acid) were analyzed by dionex ics 3000 ion chromatography device (ca, usa) which consists of a separation acclaim 4 x 250 mm column, a gradient pump, and ics –vwd uv detector set at 210 nm with a flow-rate of 0.6 ml/min maintaining the column temperature at 30 oc. in the mobile phase, 100 mm na2so4 (ph 2.65) was used. organic acids quantification was achieved by the absorbance value recorded in the chromatograms based on the external standards in the standards solution and the peaks were integrated using a default baseline construction technique. the chemicals for the organic acid standard solutions were purchased from supelco (usa). the measurement process repeated five times and average values were calculated based on the external standards (1200 mg/l). for the preparation of tested samples, approximately 2 g ground flour of oleaster was weighed into a 250 ml erlenmeyer flask, and 100 ml of an 5 mm h2so4 added. after adding a stirring bar, the flask was placed on a vwr adv 3500 stirrer at 175 rps for 3 hr. the extract was filtered through 0.45 μm pvdf filter (qui and jin, 2002). determination of functional properties water solubility (ws) and water absorption capacity (wac) water solubility and absorption capacity of samples were determined according to singh and singh (2003) with slight modifications. a 0.5 g flour sample was dispersed in 5 ml of distilled water and vortexed for 15 s in every 5 min. after 40 min it was centrifuged (cencom ii, selecta) at 2100 g for 10 min. supernatant was dried at 100 oc and solubility was calculated as follows: water solubility (ws) (%) = w1 / w2 * 100 where w1 is the weight of dried supernatant, w2 is the weight of sample. 36 y. sahan, d. gocmen, a. cansev, g. celik, e. aydin, a.n. dundar, d. dulger, h.b. kaplan, a. kilci, s. gucer precipitate was weighed and then dried at 100 oc. water absorption capacity was calculated as follows: water absorption capacity (wac) (%) = (w1 – w2) / w3 * 100 where w1 is the weight of wet precipitate, w2 is the weight of dried precipitate, w3 is the weight of sample. emulsion capacity (ec) and emulsion stability (es) emulsion capacity and emulsion stability values were determined according ahmedna et al. (1999) as modified by bilgi and celik (2004) with some modifications. five ml of 7 % dispersion of the oleaster flour sample (prepared with 0.05 % albumin solution) was mixed with 5 ml of corn oil and homogenized at 23 500 rpm for 1 min. then it was centrifuged at 2100 g for 30 min (cencom ii, selecta). the ratio of the height of the emulsified phase to the height of total liquid was named as emulsion capacity (%). in order to determine the value of emulsion stability, homogenized sample was incubated at 45 oc for 30 min. after that, it was allowed to stand for 10 min at room temperature. then it was centrifuged at 2100 g for 20 min (cencom ii, selecta). the ratio of the height of the emulsified phase to the height of total liquid was named as emulsion stability (%). experiments were conducted in triplicate. statistics data are presented as mean values +/standard error of 3 replicates. statistical analysis was performed by one-way analysis of variance (anova) on spss version 17.0 software for windows (usa). when significant differences were found (p ≤ 0,05), the least significant difference (lsd) test was used to determine the differences among mean values. paired t-test was carried out to compare the properties of go1-pof, go1-upof, go2-pof and go2-upof. result and discussion chemical compositions chemical compositions of oleaster flour (of) samples are presented in tab. 1. the moisture contents of the of samples in the present study varied between 18.43 and 20.20 %. the protein contents of the ofs ranged from 3.74 to 4.65 %. the highest (4.65 %) protein level was observed in go2-upof, followed by go1-upof (4.49 %), go2-pof (4.51 %) and go1-pof (3.74 %). protein content of upof samples was higher than those of the pof samples. the starch contents of the ofs obtained in this study were found to be between 13.80-43.18 %. the starch contents of the peeled oleaster flours (pofs) samples were significantly (p ≤ 0.05) higher than those of the unpeeled oleaster flours (upofs). it can be attributed to the presence of higher amount of starch in pulp than in peel. the total dietary fibre (tdf) content of the ofs ranged from 20.67 % to 30.65 %. total dietary fiber (tdf) levels of the samples. these results were lower than pumpkin flour samples (32.15 % to 36.73 %) found by aydin and gocmen (2015). the highest tdf content was found in go1-upof (30.65 %) followed by go2-upof (25.44 %). the dietary fiber values of upofs were significantly (p ≤ 0.05) higher than those of pofs. the higher tdf levels observed in upof samples are possibly related to pericarp contents. epidemiological studies suggest that dietary fibre consumption helps to reduce obesity, some kinds of cancer, cardiovascular diseases and gastrointestinal diseases. although numerous health organizations indicate the necessity of increasing fibre consumption of up to 2035 g per day, most people are unaware of the recommended dose (gomez et al., 2010). oleaster flour is a good source in tdf, it might be important from the nutrition point of view. micro minerals the micro minerals of flour samples obtained from elaeagnus angustifolia l. are presented in fig. 1. determined detection and quantification limits of elements are shown in tab. 2. according to averages level of micro minerals in all samples, fe was found to be highest (10.72 mg/kg) and it was followed by b (7.79 mg/kg) and zn (4.08 mg/kg). with regard to genotypes, generally zn, cu, mn contents of go1 samples were higher than those of go2 samples (p ≤ 0.05). these differences between genotypes may be due to growth conditions, genetic factors, soil properties and geographical variations. on the tab. 1: chemical compositions of oleaster floursa*. samples moisture* protein* starch total dietary (g/100 g) (g/100 g, db) (mg/100 g, db) fiber (g/100 g, db) go1-pof 18.99 ±1.05a 3.74±0.26c 43.18±2.26b 23.55±0.07c go1-upof 18.43±1.13a 4.49±0.17b 17.73±2.38cd 30.65±0.16a go2-pof 19.78±1.11a 4.51±0.24b 36.86±4.89b 20.67±0.21d go2-upof 20.20±0.96a 4.65±0.19b 13.80±2.13cd 25.44±0.44b a means with different superscripts in columns indicate significant difference (p≤< 0.05). *data are expressed as means ± standard deviations. fig. 1: the micro minerals of peeled (pof) and unpeeled (upof) oleaster flours from two genotypes (go1 and go2). data show mean values of three replicates +/standard error. tab. 2: performance characteristic of mineral determination methods. icp-ms recovery (%) lod mg kg-1 loq mg kg-1 i̇sotope 53cr 90.6 0.009 0.030 59co 90.1 0.006 0.020 82se 49.3 0.012 0.039 95mo 71.3 0.016 0.055 icp-oes line zn (206.200 nm) 93.8 0.3 0.8 fe (238.204 nm) 87.3 0.3 1.0 cu (327.393 nm) 100.1 0.2 0.7 mn (257.610 nm) 89.5 0.1 0.4 b (249.677 nm) 92.8 0.4 1.3 properties of flours from oleaster 37 other hand, no correlation could be found between other micro minerals and genotypes. regarding to the flour obtained from oleaster fruits, fe, cu, b, and cr contents were higher in upof than in pof samples (p ≤ 0.05). especially fe content of upof samples showed significant increase (p ≤ 0.05). our data showed that fe, cu, b, and cr accumulations were higher in fruit skin than in pulp tissues. in peel of fruit the measured concentrations of elements were higher compared to corresponding pulp and juice samples, which is in accordance with previously published results (henriquez et al., 2010 and savikin et al., 2014). conversely, no significant differences regarding co, se and mo contents were determined in all samples (p ≥ 0.05) (fig. 1). the highest content of zn was determined in go1-upof sample (5.50 mg/kg) while the lowest content of zn was determined in go2-upof (1.90 mg/kg). our results were compatible with those obtained by dolezal et al. (2001) and akbolat et al. (2008) (2.32 mg/kg). fe content was the highest (17.53 mg/kg) in go2-upof and the lowest (6.66 mg/kg) in go1-pof. our results revealed that the oleaster fruit is a good natural source of iron. dolezal et al. (2001), reported lower (5.77 mg/kg) fe content in oleaster fruit suggesting that the oleaster fruits in turkey contain greater amount of fe. the levels of cu in ofs were within the range of 2.37-6.11 mg/kg (fig. 1). our results were higher than those reported by dolezal et al. (2001), (1.73 mg/kg). we found greater cu content compared with those reported in previous studies. the boron content varied beetween 5.99 mg/kg (go2-pof) and 8.58 mg/kg (go2-upof). sungur and okur (2009) determined boron concentrations of 32 species of vegetable and 17 species of fruit in hatay, turkey. high concentrations of boron were found in thyme (10.44 mg/kg), mint (6.96 mg/kg), red cabbage (6.45 mg/kg), broad-bean (6.28 mg/kg), quince (5.41 mg/kg), pomegranate (5.27 mg/kg) and orange (4.08 mg/kg) while low concentrations of boron were found in pumpkin (0.76 mg/kg), white radish (0.97 mg/kg), plum (1.16 mg/kg) and cucumber (1.17 mg/kg). most foods had boron concentrations in the range of 1.48-3.60 mg/kg. according to our results, oleaster fruits are good natural sources of boron. hence, our findings make a significant contrubition to the limited information available in the literature. mn contents of all samples varied between 2.46 mg/kg and 4.51 mg/ kg. mn concentrations in oleaster fruit were reported as 10.15 mg/kg by dolezal et al. (2001) and 47.10-49.76 mg/kg by akbolat et al. (2008). we found lower mn content compared with those reported in previous studies. in the present study, se contents ranged from 0.025 mg/kg and 0.04 mg/kg. se levels of all samples are relatively higher than those of different fruits investigated in another study by ventura et al. (2009). additionally, we provide information with regard to levels of essential nutrients cr, co and mo in oleaster fruit for the first time in the literature. the chromium value of oleaster ranged between 0.11-0.38 mg/kg. the co content of oleaster fruits averaged 0.06 mg/kg; this value is smaller than the average co content of the fruits, including apples (35.24-39.9 μg/g), plums (45.32 μg/g), cornelians (32.45-41.03 μg/g), (hamurcu et al., 2010), red guava (3.17 μg/g), yellow guava (0.31 μg/g), and guobiroba (0.62 μg/g) (pereira et al., 2014). the mean mo levels of oleaster fruit varied between 0.03 and 0.05 mg/kg. no data have been reported on mo content in dried fruits. fatty acids nine different fatty acids were detected in pof and upof samples of two genotypes and their percentages are presented in fig. 2. the major fatty acid in all samples according to average values was palmitic acid (c16:0) (34.31 %), and it was followed by oleic acid (c18:1) (26.23 %) and lignoceric acid (c24:0) (17.47 %). linoleic acid was detected only in go2 samples (2.20 %). all samples were characterized by high amounts of saturated fatty acids (sfa) as compared to mono unsaturated fatty acids (mufa) and polyunsaturated fatty acids (pufa). limited information is available in the literature regarding the fatty acid composition of elaeagnus angustifolia. goncharova et al. (1993) reported an abundance of palmitoleic acid (16:1) in fruit skin and linoleic acid (18:0) and palmitic acid (16:0) in seeds. these differences might be explained by differences in gentypes, climatic condition and soil composition. significant differences were observed for each individual fatty acid of pof and upof samples between genotypes. palmitic and oleic acid concentrations were significantly higher in go1 samples than in go2 samples (p ≤ 0.05). conversely, palmitoleic, stearic, linoleic, arachidic acid levels in go2 samples were higher than those in go1 (p ≤ 0.05). on the other hand, palmitoleic, behenic and lignoceric acid contents were similar in all samples. in addition, levels of all fatty acids in pof samples were similar with those of upof samples in both genotypes (fig. 2.). among the pof and upof samples, go1-upof had the highest palmitic and oleic acid contents in all fatty acids (36.69 %, 28.69 %), respectively. the highest lignoceric acid level (18.02 %) was detected in go2-pof samples compared to the other samples. go2-upof samples had the highest linoleic acid content (11.59 %) among all samples (fig. 2.). oleic acid and linoleic acid were predominant unsaturated fatty acids in all samples. organic acids organic acids may have a protective role against various diseases due to their antioxidant activity (silva et al., 2004). in this study, the organic acid composition of pof and upof samples were determined by ion chromatography. the performance characteristics of organic acids are shown in tab. 3. seven different organic acids were detected and their percentages were presented in fig. 3. according to the average values of pof and upof samples, the highest levels were obtained in citric acid (16.94 mg/g) content which was followed by malic acid (15.07 mg/g), acetic acid (6.97 mg/g), oxalic acid (4.72 mg/g), tartaric acid (3.67 mg/g), fumaric acid (0.82 mg/g), and trace amounts of ascorbic acid (0.035 mg/g) (fig. 3). to the best of our knowledge, only few studies were conducted exploring the organic acid composition of oleaster (elaeagnus angustifolia l.). in the literature, citric and malic (both aliphatic) acids are the most abundant acids in fruits and vegetables (bae et al., 2014; gundogdu et al., 2014; oliveira et al., 2014; ma et al., 2015). in this study, the levels of citric acid in ofs were within the range of 13.00 26.69 mg/g (fig. 3). levels of organic acids showed significant variations (p ≤ 0.05) in two genotypes as a function of the growth con fig. 2: the fatty acids of peeled (pof) and unpeeled (upof) oleaster flours from two genotypes (go1 and go2). data show mean values of three replicates +/standard error. 38 y. sahan, d. gocmen, a. cansev, g. celik, e. aydin, a.n. dundar, d. dulger, h.b. kaplan, a. kilci, s. gucer ditions of the fruits. citric acid was the major organic acid in go2 samples and citric acid concentrations were significantly higher in go2 samples than in go1 samples (p ≤ 0.05). citric acid concentration of go2-pof sample (26.69 mg/g) was 2 times that of go1-pof sample (13.0 mg/g). additionally, its level in go2-upof was 1.3 times higher than that in go1upof sample. although citric acid contents were similar in go1-pof and upof samples, they were significantly different between go2-pof and upof samples (p ≤ 0.05). similar levels of citric acid have been reported in oleaster fruit (dolezal et al., 2001). malic acid was the predominant organic acid in go1 samples. the highest malic acid concentration (18.36 mg/g) was detected in go1pof samples while the lowest (9.29 mg/g) malic acid concentration was detected in go2-upof samples. significant differences were determined for citric acid level between genotypes (p ≤ 0.05). additionally, malic acid concentrations were significantly higher in pof samples than in upof samples (p ≤ 0.05). this could be explained by the high malic acid level of pulp in oleaster fruits (fig. 3). wang and fordham (2007) indicated that malic, quinic and citric acid were found in elaeagnus umbellata thunb. fruit and malic acid (range between 2.02 to 6.88 mg/100g of fresh mass) was the primary organic acid. among all samples, go1-pof had the highest acetic acid content (10.21 mg/g), while go2-upof had the lowest acetic acid level (4.23 mg/g). acetic acid concentrations of go1 samples were significantly higher than those of go2 samples (p ≤ 0.05). in pof samples acetic acid contents were significantly higher than in upof samples (p ≤ 0.05). oxalic acid is the simplest dicarboxylic acid and its most striking chemical impact is its strong chelating ability for multivalent cations (seabra et al., 2006). oxalic acid is usually found in green leaves. we also measured oxalic acid levels of oleaster flour (both pof and upof) the present shows that oxalic acid contents range between 4.47 mg/g and 5.06 mg/g (fig. 3). these results are much higher than in other fruits, such as fig (0.18 mg/g), cranberry (0.17 mg g-1), and blueberry (0.25 mg/g) (pande and akoh, 2010). tartaric acids are commonly found in fruits and berries (valentão et al., 2005). the highest tartaric acid level (9.67 mg/g) was detected in go2-upof samples while the lowest (1.46 mg/g) tartaric acid concentration was found in go1-upof samples. although citric acid contents were similar in go1-pof and upof samples, they were significantly different in go2-pof and upof samples (p ≤ 0.05). in addition, significant differences were observed for tartaric acid levels between the two genotypes (p ≤ 0.05). soyer et al. (2003) determined that tartaric acid level in turkish white grapes ranged between 4.98 and 7.48 g/l. these data indicate that the oleaster fruits are very rich in tartaric acid content. the levels of fumaric acid in ofs were within the range of 0.62 1.06 mg/g (fig. 3.). fumaric acid concentrations of go1 samples were significantly higher than those of go2 samples (p ≤ 0.05). in go1 samples, acetic acid content of pof was higher than that of upof samples (p ≤ 0.05). usenik et al. (2008) measured lower values for 13 sweet cherry cultivars (0.97-7.56 mg/kg). in support of the findings from previous studies, our results suggest that oleaster flour contains moderate levels of fumaric acid. ascorbic acid (vitamin c) is the most widely distributed watersoluble antioxidant in fruits and vegetables (seabra et al., 2006). although ascorbic acid was not detected in go1 samples, extremely low levels of it were detected in go2-pof and upof samples (0.01 mg/g, 0.06 mg/g, respectively). ascorbic acid levels in oleaster olives were slightly higher than those obtained in czech republic (132.5 mg/kg) (dolezal et al., 2001). similar levels of ascorbic acid have been reported in various fruits (pande and akoh, 2010). functional properties the use of flours as ingredients in food processing is dependent on its functional properties (hung et al., 1990). the functional properties directly or indirectly affect the processing applications, food quality and ultimately their acceptance and utilisation in food and food formulations (mahajan and dua, 2002). emulsification, solubility and water absorption capacity are these functional properties (adebowale and lawal, 2004). water solubility (ws) water solubility (ws) values of the ofs are given in tab. 4. the ws values of the samples varied from 90.33 to 96.01 %. the highest value (96.01 %) of solubility was observed in go2-upof, followed by go1-upof (94.79 %), go2-pof (93.28 %) and go1-pof (90.33 %). the solubility of oleaster flours differed significantly (p ≤ 0.05) with respect to the being peeled or unpeeled. the results indicated higher solubility values in the unpeeled oleaster flours (94.79, 96.01 %) than in the peeled oleaster flours (90.33, 93.28 %). the solubility was lower in peeled oleaster flour possibly due to higher starch levels (tab. 1), as cited by some authors in the case of the mango peel and kernel powders (sogi et al., 2003). on the other hand, differences in the water solubility of oleaster flours can be attributed to the dietary fiber content of unpeeled oleaster flours were higher than peeled oleaster flours (tab. 1). the water solubility levels of unpeeled oleaster flours having higher dietary fiber in our study are in accordance with those published by adrade-mahecha (2012), who also reported that the high solubility values achieved for the achira flour may be due to the presence of soluble fibers. they suggested that the solubility values for achira flour may also be influenced by the presence of some soluble components such as sugars and soluble fibers. tab. 3: performance characteristics of the determination of organic acid method. organic acid lod loq recovery (mg/100g) (mg/kg) (mg/kg) (%) oxalic acid 0.24 0.8 85.5 malic acid 2.02 6.7 79.6 l-ascorbic acid 0.23 0.8 73.2 acetic acid 1.56 5.2 78.0 citric acid 1.76 5.9 83.8 tartaric acid 1.55 5.2 77.5 fumaric acid 0.15 0.5 77.0 lod: limit of dedection loq: limit of quantification fig. 3: the organic acids of peeled (pof) and unpeeled (upof) oleaster flours from two genotypes (go1 and go2). data show mean values of three replicates +/standard error. properties of flours from oleaster 39 water absorption capacity (wac) water absorption capacity (wac) values of the ofs are given in tab. 4. the wac values of the samples were ranging between 372.74 to 430.33 %. the highest wac value (430.33 %) was obtained in go2-upof samples like water solubility. results showed that water absorption capacities of the oleaster flours were high and adequate. the wac values of upof samples were significantly (p ≤ 0.05) higher than those of the pof samples. this might be attributed to higher dietary fiber contents of unpeeled oleaster flour than those of peeled oleaster flours (tab. 1). these results are similar with those reported by sogi et al. (2013) who reported water absorption index (wai) to be higher in mango peel powder compared to mango kernel powder. koubala et al. (2013) also suggested that mango peel fibre with high hydration capacities has potential in dietary fibre-rich foods preparation. the fibers contribute markedly to the hydration properties, which is attributed to the hydroxyl groups in the fiber structure allowing more water interactions through hydrogen bonding (rosell et al., 2009). in general, soluble fibers had a high hydration ability to form a viscous solution (saura-calixto and goni, 2006). lawal et al. (2007) also reported that the higher water absorption capacity of the brazilian flour may be due to the high hydrophilicity of the cellulose present in the fibers. the main chemical compounds that enhance the water absorption capacity of brazilian flours are proteins and fibers, since these constituents contain hydrophilic parts, such as polar or charged side chains. wani et al. (2013 a, b) reported that the major chemical compositions that enhance the wac of flours are proteins and carbohydrates, since these constituents contain hydrophilic parts, such as polar or charged side chains. they suggested that the functional properties of legume flours mainly depend on proteins, carbohydrates and other components. according to kaushal et al. (2012), the high wac of taro flour can be attributed to the presence of high amount of carbo hydrates. they indicated that the flours with high water absorption may have more hydrophilic constituents, such as polysaccharides. hodge and osman (1976) reported that flour samples with high wac values contain more hydrophilic constituents. the water holding capacity of flours is a very important functional property in many different food applications (ma et al., 2011). food materials having higher wac values can act as functional ingredients. modification of viscosity and texture in formulated food can be achieved by adding ingredients with high wac, and the resulting changes are attributed to the gelling, bulking, and thickening effects arising from the wac of the ingredients (safo-kantanka and acquistucci, 1996; de escalada pla et al., 2007). water absorption capacity of thickening or functional agent used in food systems such as in bakery products is important for certain product characteristics, such as the moistness of the product, starch retrogradation, and the subsequent product staling. these agents with high water absorption capacities help reduce staling and thus increase food stability in bakery products (aziah abdul aziz et al., 2012; wani et al., 2013 a, b). wolf (1970) also indicated that flours with high water holding capacity could be good ingredients in bakery applications (e.g.,bread formulation), since a higher water holding capacity enables bakers to add more water to the dough, thus improving the handling characteristics and maintaining freshness in bread. in present study, results showed that the water solubilities (ws) and water absorption capacities (wac) of the oleaster flours were adequate for their utilization. thus oleaster flour with high ws and wac could be useful as a thickening or functional agent for food systems such as baked goods, beverages, ice cream and yoghurt. emulsion capacity (ec) in many foods, the interaction between protein and lipids adjust their ability to form stable emulsions. proteins can stabilize emulsions because of their amphiphilic nature. the emulsion properties of a protein depend on the rate at which it diffuses into the interface, on its adsorbability at the interface, and on the deformability of its conformation under the influence of interfacial tension (belitz and grosch, 1999). proteins are commonly used as emulsion forming and stabilizing agents. on the other hand, starch can not produce emulsion by itself, but might effect emulsion properties (koksel et al., 2007). therefore, in present study, effects of oleaster flours on the emulsifying properties of albumin solutions were investigated. emulsion capacity (ec) values of the ofs are given in tab. 4. emulsion capacity value of albumin solution (0.05 %) was found to be 20.96 %. emulsion capacity values of albumin solution supplemented with ofs were significantly (p ≤ 0.05) higher (46.0054.89 %) than those of the albumin solution on its own. the results indicated that the oleaster flour samples positively affected the emulsion properties of the albumin. the highest ec (54.89 %) value was detected in go2-pof samples compared to the others. emulsion capacity of albumin solution supplemented with the peeled oleaster flour samples (pofs) were significantly (p ≤ 0.05) higher than those of the unpeeled oleaster flour samples (upofs). this result was due to having lower protein contents of pofs than those of upofs (tab. 1). similarly du et al. (2014) reported that the chickpea flour with lower protein level than the other kinds of legume flour has relatively poor emulsion activity. besides protein content, starch amount of oleaster flours affected to ec values. the higher ec values of pofs can be attributed to the higher starch content of pofs than those of upofs (tab. 1). the results obtained from oleaster flour in present study corroborated well with those reported by james and norman (1979), who indicated that the differences among the emulsion activities are related to the protein contents (soluble and insoluble) and other components, such as starch, fat, and sterol contents, of the legume flours. du et al. (2014) also reported that the protein content of lentil flour was the highest among the legume flours but its emulsion activity was poor, such variation may be related to the other components in the flours. in present study, the oleaster flours seem to have an improving effect on emulsion properties of albumin. emulsion stability (es) emulsion stability and capacity are very important properties that proteins and other amphoteric molecules contribute to the development of traditional or novel foods (ma et al., 2011). emulsion stability (es) values of the samples are given in tab. 4. es values of albumin solution (0.05 %) were found to be 16.28 %. es of albumin solutions supplemented with upofs and pofs were within the range of 26.77-40.35 %. es values of albumin solution supplemented with ofs were significantly (p ≤ 0.05) higher than those of the albumin solution on its own. the results indicated that ofs did tab. 4: techno-functional properties of oleaster flour samples.a* samples water water emulsion emulsion solubility (%) absorption capacity (%) stability (%) capacity (%) soy protein -------20.96±0.16c 16.28±0.09e go1-pof 90.33 ± 0.29c 372.74 ± 0.63d 54.46± 0.03a 26.77± 0.08d go1-upof 94.79 ± 0.42ab 411.18 ± 0.23b 46.00± 0.53b 28.47± 0.11c go2-pof 93.28 ± 0.66b 395.13 ± 0.78c 54.89± 0.14a 29.63± 0.42b go2-upof 96.01 ± 0.60a 430.33 ± 0.32a 46.87± 0.23b 40.35± 0.37a a means with different superscripts in columns indicate significant difference (p≤< 0.05). * data are expressed as means ± standard deviations. 40 y. sahan, d. gocmen, a. cansev, g. celik, e. aydin, a.n. dundar, d. dulger, h.b. kaplan, a. kilci, s. gucer not have a deteriorating effect on the emulsion stability values of the albumin solution. the es values of the go2 samples were significantly (p ≤ 0.05) higher than those of go1 samples. this could be due to growth conditions, genetic factors, soil properties and geographical variations. the highest es (40.35 %) value was detected in go2-upof samples compared to the others. es values of upofs were significantly (p ≤ 0.05) higher than those of pof samples. the increase in es values is probably due to the presence of higher dietary fiber levels in unpeeled oleaster flours than those of peeled ones (tab. 1). these results were consistent with the findings of aziah abdul aziz et al. (2012), who found that the emulsifying and stabilizing agents present in mango pulp and peel are carbohydrate polymers (such as pectin), which might have increased the viscosity of the aqueous phase and reduced the tendency of the dispersed oil globules to migrate and coalesce. sharma et al. (1998) also reported that carbohydrate polymers can also stabilize emulsions by absorption at the interface and forming a coating around the dispersed particles. carbohydrates such as starch and fiber may also enhance emulsion stability by acting as bulky barriers between the oil droplets, preventing or slowing down the rate of oil droplet coalescence, as reported by aluko et al. (2009). dickinson (1994) also indicated that some types of polysaccharides can help stabilise the emulsion by increasing the viscosity of the system. the oleaster flours seem to have an improving effect on emulsion properties of albumin. these results highlight the possibility of using oleaster flour in some processed foods such as bakery products, beverages, ice cream and yoghurt. conclusion although the oleaster grows spontaneously in many regions of turkey, consumption of its fruit is limited and it is not yet used as an ingredient in food industry. the results of this study showed that oleaster flour has high nutritional contents and appropriate technofunctional properties. oleaster flours showed high levels of dietary fibers and minerals, as well as high water solubility and water absorption capacity. ofs also caused an improving effect on the emulsion stability and capacity of albumin solution. many kinds of organic and fatty acids were determined in oleaster flours. the oleaster flours obtained in the present study seem to be suitable for food products such as bakery goods, dairy products (ice cream and yoghurt), beverages, confectionary, etc. it can also be used as an alternative functional ingredient in food products which require relatively low fat and high dietary fiber content. further studies are needed to produce bakery products supplemented with oleaster flour with improved functional properties. acknowledgment the authors would like to thank the scientific and technological research council of turkey (tubitak) for their financial support to this research project (project no: tovag 110 o 060). references aacc, 1999: official methods of american association of cereal chemists, st. poul, mn, usa. abdul-hamid, a., luan, y.s., 2000: functional properties of dietary fiber prepared from defatted rice bran. food chem. 68, 15-19. adebowale, k.o., lawal, o.s., 2004: comparative study of the functional properties of bambarra groundnut (voandzeia subterranean), jack bean (canavalia ensiformis) and mucuna bean (mucuna pruriens) flours. food res. int. 37, 355-365. ahmadiani, a., hosseiny, j., semnanian, s., javan, m., saeedi, f., kamalinejad, m., saremi, s., 2000: antinociceptive and anti-inflammatory effects of elaeagnus angustifolia fruit extract. j. ethnopharmacol. 72, 287-292. ahmedna, m., prinyawiwatkul, w., rao, r.m., 1999: solubilized wheat protein isolate: functional properties and potential food applications. j. agr. food chem. 47, 1340-1345. akbolat, d., ertekin, c., menges, h.o., guzel, e., ekinci, k., 2008: physical and nutritional properties of oleaster (elaeagnus angustifolia l.) growing in turkey. asian j. chem. 20, 2358-2366. aksoy, a., sahin, u., 1999: elaeagnus angustifolia l. as a biomonitor of heavy metal pollution. turk. j. bot. 23, 83-87. aluko, r.e., mofolasayo, o.a., watts, b.a., 2009: emulsifying and foaming properties of commercial yellow pea (pisum sativum l.) seed flours. j. agric. food chem. 57, 9793-9800. andrade-mahecha, m.m., tapia-blacido, d.r., menegalli, f.c., 2012: physical-chemical, thermal, and functional properties of achira (canna indica l.) flour and starch from different geographical origin. starch/stärke 64, 348-358. aoac,1990: official methods of analysis of association of official analytical chemists, washington dc, usa. ayaz, f.a., bertoft, e., 2001: sugar and phenolic acid composition of stored commercial oleaster fruits. j. food compos. anal. 14, 505-511. aydin, e., gocmen, d., 2015: the influences of drying method and metabisulfite pre-treatment on the color, functional properties and phenolic acids contents and bioaccessibility of pumpkin flour. lwt − food sci. technol. 60, 385-392. aziah abdul aziz, n., wong, l.m., bhat, r., cheng, l.h., 2012: eva luation of processed green and ripe mango peel and pulp flours (mangifera indica var. chokanan) in terms of chemical composition, antioxidant compounds and functional properties. j. sci. food agric. 92, 557-563. bae, h., yun, s.k., jun, j.h., yoon, i.k., nam, e.y., kwon, j.h., 2014: assessment of organic acid and sugar composition in apricot, plumcot, plum, and peach during fruit development. j. appl. bot. food qual. 87, 24-29. belitz, h.d., grosch, w., 1999: food chemistry. 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thunb.). food technol. biotech. 45, 402-409. wani, i.a., sogi, d.s., wani, a.a., gill, b.s., 2013a: physico-chemical and functional properties of flours from indian kidney bean (phaseolus vulgaris l.) cultivars. lwt − food sci. technol. 53, 278-284. wani, i.a., sogi, d.s., gill, b.s., 2013b: physicochemical and functional properties of flours from three black gram (phaseolus mungo l.) cultivars. int. j. food sci. technol. 48, 771-777. wolf, w., 1970: soybean proteins. their functional, chemical, and physical properties. j. agric. food chem. 18, 969-976. address of the corresponding author: yasemin sahan, uludag university, faculty of agric., dep. of food eng. 16059, gorukle-bursa, turkey e-mail: yaseminsahan@gmail.com journal of applied botany and food quality 88, 192 196 (2015), doi:10.5073/jabfq.2015.088.027 1 julius kühn-institut (jki), federal research centre for cultivated plants, institute for resistance research and stress tolerance, groß lüsewitz, germany 2 university of rostock, faculty of agronomy, rostock, germany 3 julius kühn-institut (jki), federal research centre for cultivated plants, institute for resistance research and stress tolerance, quedlinburg, germany composition, environmental stability and potential of genetic improvement of fatty acids of lupinus angustifolius h. beyer1, h.u. jürgens1, g. jansen1, r. uptmoor2, f. ordon3 (received may 25, 2015) summary in the last decades procedures for obtaining protein isolates and concentrates derived from narrow-leafed lupins (l. angustifolius) for human nutrition have been developed. since this processes starts with defatting of seeds, lupin oil is obtained in large quantities. therefore, 50 genotypes of l. angustifolius were analysed regarding the fatty acid (fa) composition of seed oil and the environmental stability of fatty acid contents in order to get information on the application of lupin oil in the food industry. the results revealed an n-3/n-6 poly unsaturated fatty acid ratio of 0.13. furthermore, the seed oil of l. angustifolius contains rather high amounts of saturated fas (22 %). significant genotypic differences and a high heritability (h2 > 85 %) for the content of all fatty acids are suggesting that the potential for genetic improvement of fatty acid composition by breeding is given. however, coefficients of variation below 10 % for all considered traits point out that a rapid improvement in seed oil quality will be hindered by the narrow genetic base of the breeding material tested. introduction lupins belong to the legume family and have been used as a food for over 3000 years in the mediterranean (gladstones et al., 1998). since their introduction to northern europe in the 18th century, lupins are locally grown in germany (hondelmann, 1984). but because of their high seed alkaloid content they did not gain remarkable agricultural importance. with the discovery of sweet mutants of l. luteus (yellow lupin), l. albus (white lupin) and l. angustifolius (narrow-leafed lupin) at the beginning of the 1920th, lupins became fully accessible as feed and food crop (sengbusch, 1938). however, lupin cultivation remained far behind other crop plants due to limited yield and breeding progress. just in recent years lupins have been rediscovered to be used in the food industry. among the local grain legumes, lupins show a quite unique seed composition, which make them well suited for a modern human nutrition. while having a high protein and oil content, lupin grain contains minimal starch, but is very rich in dietary fibre (sipsas, 2008). functional properties of lupin derived food ingredients, like lupin protein isolates, are very promising and processes for their recovery are described in several studies (aguilera and garcia, 1989; waesche et al., 2001; sussmann et al., 2013). while great attention has been paid on the characterization of lupin protein, up to now information available about the quality of lupin oil and its potential for genetic improvement is limited. considerable amounts of lupin oil will be extracted and may be used in human nutrition, since most of the procedures for producing lupin protein isolate start with a defatting step (waesche et al., 2001). in germany, the species l. angustifolius is the most widely grown lupin species since the mid 1990s, due to their higher resistance against the fungal disease anthracnose (colletotrichum lupini) (eickmeyer, 2008). the oil content of german varieties of l. angustifolius is about 6 % (jansen and juergens, 2008). a comparative study about the oil quality of the three sweet lupin species l. albus, l. angustifolius and l. luteus has been conducted by chiofalo et al. (2012). the study has identified narrow-leafed lupins to be the less suited lupinspecies from a nutritional point of view, due to the highest content of saturated fatty acids and a low n-3/n-6 polyunsaturated fatty acid ratio. up to now, to our knowledge no studies have been undertaken to get information on the environmental stability and the potential for genetic improvement of the fatty acid profile in narrow-leafed lupins. the evaluation of variation in fatty acid contents due to genotypic, environment and genotype x environment interaction (ge interaction) will be of interest for breeders and end users. respective studies in which the influence of environment, genotype and ge interaction on the fatty acid composition was analysed, have been already carried out for l. albus seed (boschin et al., 2007; boschin et al., 2008). in this study we evaluate the oil content and the fatty acid composition of new german l. angustifolius breeding lines. we further assess the influence of environment, genotype and ge interaction on the variability in fatty acid contents and calculated heritabilities to estimate the potential for genetic improvement by breeding. materials and methods plant material whole seeds of 50 genotypes of l. angustifolius l. were analysed for oil and fatty acid content. the seed samples were provided by the saatzucht steinach gmbh & co kg (bocksee, germany) and comprised 42 advanced breeding lines and 8 german cultivars of l. angustifolius (probor, boregine, borlu, vitabor, haagena, sonate, boruta, haags blaue). the two cultivars haags blaue and boruta and 6 breeding lines belong to the restricted branching (determinate) type and the other ones belong to the branched (indeterminate) type. experimental design and characterization of environments the trials were conducted at four different sites in germany during three consecutive growing seasons (2010 2012). three sites were located in mecklenburg-western pomerania (northern germany), bornhof (53°5’n, 12°9’o), dratow (53°5’n, 12°8’o) and groß lüsewitz (54°1’n, 12°3’o) and one site was located in bavaria (southern germany), steinach (48°9’n, 12°6’o). the three sites located in northern germany were characterized by sub-acid soil ph, sandy earth (bornhof) to loamy sand (dratow, groß lüsewitz) and an annual rainfall from 558 mm (bornhof), 559 mm (dratow) to 683 mm (groß lüsewitz). steinach located in southern germany is characterized by an acid soil ph of 6.0 but loamy earth and an annual rainfall of 784 mm. the experiments were conducted in a randomized complete block design with four replications and different plot sizes. in steinach and dratow the plot size was 4.5 m2 (3.0 m x 1.5 m), environmental stability of fatty acids of l. angustifolius 193 in groß lüsewitz 4.2 m2 (2.8 m x 1.5 m) and in bornhof 10.5 m2 (7.0 m x 1.5 m). seed density of branched types was 100 seeds per m2 and of restricted branching types 120 seeds per m2. for details confer to beyer et al. (2015). for analyses of fatty acids mixed samples of replication 1 and 2 as well as 3 and 4, respectively, were used. chemical analysis samples were ground to whole seed flour by using a break mill (brabender sm3) first, followed by a falling number mill (perten laboratory mill 3100), which generate flour passing through a sieve size of 0.8 mm. each sample was analysed in duplicates. seed oil was extracted with petroleum ether (sigma-aldrich, steinheim, germany) using a dionex ase 200 accelerated solvent extractor. 5 g lupin flour was first dried in a moisture analyser (ohaus-mb35) at 130 °c and then extracted for 45 minutes at 1 500 psi at 130 °c and 3 static cycles at 130 °c and 3 static cycles of 11 ml flush volume each time. after evaporating the solvent, oil content was gravimetrically determined and expressed as weight percentage (%) on a dry matter basis. fatty acids were analysed as fatty acid methyl esters (fame); 5 μl of the extracted oil was suspended in 1 ml tert-butylmethylether/ methanol mixture (1:1 v/v), 100 μl trimethyl sulfonium hydroxide solution (tmsh, 0.15 mol/l in methanol) were added, the mixture was shaken and transferred into a vial for gc analysis. the fames were analysed by gc-fid (agilent 6890) on hp-ffap 25 m x 0.2 mm x 0.33 μm column with carrier gas h2 and a flow rate of 1 ml/min in split mode (1:100); injection volume 2 μl, temperature of injector 280 °c and detector of 250 °c. the column temperature was programmed as follows: an initial temperature of 160 °c for 1 min, with increments of 15 °c/min up to 220 °c and increments of 30 °c/min up to the final temperature of 240 °c, which was hold for 2.83 min. the single fatty acids were calibrated by using dilutions of aocs reference mixes (sigma-aldrich grain fatty acid methyl ester (47801) and low erucic rapeseed oil (o7756)) and the amounts were expressed per total fatty acid esters identified. fatty acid (fa) groups were obtained by summing up the percentage of the appropriate fas: total saturated fas (total sfa): sum of c14:0 + c16:0 + c18:0 + c20:0 + c22:0 and c24:0; monounsaturated fas (mufa): c18:1n-7 + c18:1n-9 + c20:1; polyunsaturated fas (pufa): c18:2 + c18:3. statistical analysis all statistical analyses were performed using sas software (sas 9.3, institute inc., cary nc, usa). overall means of the oil and fas were calculated and shown as least square means (lsmeans). lsmeans and variance components for the evaluated traits were estimated using a linear mixed model, which fitted random effects by the restricted maximum likelyhood (reml) method. for calculation of lsmeans the genotype (g) was considered as fixed effect and locations (l), years (y) and the various interactions between g, l and y to be random. all factors were considered to be random in the model for estimation of the variance components and their standard errors. to evaluate the relationship among oil content and the various fas, pearsons correlation coefficients were calculated based on the lsmeans values of the 50 genotypes. broad sense heritability (h2) was calculated as: h2 = σg2/ σp2 = σg2/( σg2 + σgl2/l + σgy2/y + σgly2/ly + σe2/rly) were σg2 is the genotypic variance (variance component for genotype), σp2 the phenotypic variance, σgl2 the variance component for genotype x location interaction, σgy2 the variance component for genotype x year interaction, σgly2 the variance component for genotype x location x year interaction, σe2 the variance component for the error and l, y and r are number of locations, years and replications. heritability is expressed on an entry mean basis using data from all genotypes, locations, seasons and replications. principal component analysis (pca) was carried out to characterize the influence of environment on fatty acid composition. the pca analyses were calculated based on the lsmeans of the 12 experiments (l x y). calculation of pca was carried out using the jmp tool of the sas software. results and discussion in the seed oil of 50 varieties of l. angustifolius 6 saturated fas and 5 unsaturated fas were identified and quantified (tab. 1). the major fas found were linoleic acid (30.1 42.4 %), oleic acid (28.8 39.0 %) and palmitic acid (10.0 13.2 %), accounting for more than 80 % of the total fas. the seed oil of l. angustifolius contains also significant amounts of linolenic acid, ranging between 4.2 and 6.0 %. these results are in agreement with previous investigations of german narrow-leafed lupin varieties (jansen and juergens, 2008) and also values reported by chiofalo et al. (2012), who investigated the fatty acid profile of three released cultivars of l. angustifolius. from a nutritional point of view, public health institutes, such as german nutrition society (dge), or the food and tab. 1: estimates of lsmeans, standard deviation (sd), range and relative standard deviation (cv) of oil content (%) and single fas (% of total fas) of 50 genotypes of l. angustifolius, grown in 12 field trials in germany fatty acid overall range (%) cv p-value lsmean ± sd of anova1 oil content2 6.3 ± 0.4 5.7 − 7.2 6.1 *** sfa c14:0 (myristic acid) 0.3 ± 0.0 0.2 − 0.3 9.2 *** c16:0 (palmitic acid) 11.4 ± 0.7 10.0 − 13.2 6.4 *** c18:0 (stearic acid) 6.7 ± 0.7 5.3 − 8.6 9.7 *** c20:0 (arachidic acid) 1.0 ± 0.1 0.8 − 1.2 8.4 *** c22:0 (behenic acid) 2.0 ± 0.1 1.6 − 2.3 6.6 *** c24:0 (lignoceric acid) 0.6 ± 0.1 0.7 − 8.9 8.9 *** total sfa 21.8 mufa c18:1n-9 (oleic acid) 33.8 ± 2.1 28.8 − 39.0 6.3 *** c18:1n-7 (vaccenic acid) 0.8 ± 0.1 0.7 − 1.0 10.5 *** c20:1 (gadoleic acid) 0.3 ± 0.0 0.3 − 0.4 7.7 *** total mufa 34.9 pufa c18:2n-6 (linoleic acid) 37.8 ± 2.5 30.1 − 42.4 6.7 *** c18:3n-3 (linolenic acid) 5.1 ± 0.4 4.2 − 6.0 8.1 *** total pufa 42.9 n-3/n-6 pufa 0.13 1 for genotypic differences, with *** p ≤ 0.001 2 cf beyer et al., 2015 194 h. beyer, h.u. jürgens, g. jansen, r. uptmoor, f. ordon agriculture organization (fao) have given recommendations for the daily intake of specific fas with dietary relevance. saturated fas (sfa) should account for less than 10 %, monounsaturated fas (mufa) for more than 13 % and polyunsaturated fas (pufa) for 7 10 % of the overall energy intake (deutsche gesellschaft für ernährung, 2000). special emphasis is laid on mutual proportion of n-3/n-6 pufa in the diet since our modern nutrition depends increasingly on cereals, having an unfavourable low α-linolenic acid content (n-3 fa) and excessive amounts of linoleic acid (n-6 fa) (simopoulos et al., 2002). a diet containing a ratio of 0.2 of n-3/n-6 fas is recommended by the german health authority (deutsche gesellschaft für ernährung, 2000). in this respect, our results of total saturated fatty acid content with 21.8 % in l. angustifolius revealed a high proportion in comparison to other vegetable oils, like soybean oil (15.7 %), sunflower oil (12.8 %), or rapeseed oil with contents of 8.0 % sfa (dubois et al., 2007). the analysed average n-3/n-6 pufa ratio of 0.13 in the breeding material is below the recommended threshold of 0.2, but it is comparable with the ratio of soybean oil (0.15) and distinctly higher than that of sunflower oil, which is below 0.01 (dubois et al., 2007). anova revealed significant differences between genotypes in fatty acid contents (p < 0.001), whereas the coefficient of variation below 10 % for almost all fas indicate limited genotypic variation within the breeding material tested (tab. 1). therefore, breeding for a better fatty acid composition might be restricted by the narrow genetic variation in these traits. variance components of environments (l, y and l x y), genotype (g) and genotype x environment interactions (g x l, g x y and g x l x y) were estimated for the most abundant fas via reml analysis and are shown in tab. 2. in general, the fatty acid contents were mostly influenced by the growing season (y), except palmitic acid. this indicates that fatty acid composition varies more due to changing weather conditions than to locations. since climatic conditions cannot be controlled, it will be difficult to make predictions about fatty acid composition of lupin seed oil in advance. the genotypic variance component is large in comparison to the sum of ge interaction variance components for all considered fas, indicating that the ranking of genotypes is fairly stable across environments. this also leads to high values of broad sense heritability on an entry mean basis (h2 > 0.85 for all traits), which facilitates genetic progress in improving fatty acid composition of lupin seed oil. our findings about the genotypic and environmental influences on fatty acid composition of l. angustifolius seed is in accordance with reports of boschin et al. (2008), who investigated the effect of genotype and environment on fatty acid composition of l. albus seed. boschin et al. (2008) also reported high broad sense heritability for fatty acid content depending on a large ratio of genotypic to ge interaction variance components. pearson’s correlation estimates among oil and the five main fas are given in tab. 3. oil content was significantly inversely related to the palmitic acid and linolenic acid and significantly positively correlated to the stearic acid content. the negative association between linolenic acid and fat content has also been stated by uzun et al. (2007), whereas chiofalo et al. (2012) reported a significant positive correlation between oil content and n-3 pufas in lupin seed. in our results, oleic acid has a strong negative correlation with linoleic acid. an inverse association between these two fas has already been observed in lupin seed (uzun et al., 2007; chiofalo et al., 2012) and is well documented in other oilseed crops such as soybean (bachlava et al., 2008), sesame (brar, 1982) or peanut (andersen et al., 1998). in this study oleic acid showed a negative relation to linolenic acid and a positive correlation to stearic acid. a negative relationship was also observed between stearic acid and linoleic acid as well as linolenic acid (tab. 3). a negative association between sfa and n-6/n-3 pufas was also reported by chiofalo et al. (2012), but not on a significant level. the observations suggest that selection for genotypes with high oil content and high oil quality at the same time might be difficult due to the strong negative correlation between oil content and n-3 pufas. tab. 2: variance component estimates of genotype (g), locations (l), years (y), error (e) and genotype x environment interactions, as well as heritability estimates on an entry mean basis of fas of l. angustifolius grown in 12 field trails in germany source c16:0 c18:0 c18:1 c18:2 c18:3 g 0.51 *** 0.4 *** 4.0 *** 5.5 *** 0.15 *** l 0.05 0.1 6.3 7.7 0.04 y 0.00 2.0 10.4 17.8 1.52 l x y 0.26 0.2 1.0 1.1 0.20 * g x l 0.03 *** 0.03 * 0.5 *** 0.3 *** 0.03 *** g x y 0.03 *** 0.1 *** 0.5 *** 0.7 *** 0.01 *** g x l x y 0.07 *** 0.2 *** 1.4 *** 1.6 *** 0.06 *** e 0.08 *** 0.1 *** 0.6 *** 1.0 *** 0.03 *** h2 (%) 95.1 85.4 90.8 92.3 89.4 * significant at p ≤ 0.05, ** significant at p ≤ 0.01, *** significant at p ≤ 0.001 tab. 3: correlations between oil content and fas of l. angustifolius genotypes oil c16:0 c18:0 c18:1 c18:2 c16:0 0.50 *** c18:0 0.39 ** 0.26 c18:1 0.24 0.23 0.63 *** c18:2 0.01 0.12 0.68 *** 0.91 *** c18:3 0.72 *** 0.24 0.35 ** 0.38 ** 0.18 ** significant at p ≤ 0.01, *** significant at p ≤ 0.001 principal component analysis (pca) was conducted based on the five main fas to characterize the influence of environment on the fatty acid composition (fig. 1). the first two principal components account for 94.3 % of the total variation. pca axis 1 was dominated by the strong negative association between oleic acid and the polyunsaturated fas, whereas pca axis 2 based mostly on palmitic acid and its slightly negative association to the unsaturated fas. fig. 1 revealed that environments are predominantly grouped according to the growing season and not according to locations, which is in accordance with findings of variance component analysis (tab. 2). growing season 2010 coused high amounts of stearic and oleic acid, whereas the years 2011 and 2012 resulted in high amounts of polyunsaturated fas in lupin seed oil. from a dietary point of view, seed oil of l. angustifolius could be improved concerning sfa content as well as n-3/n-6 pufa ratio. although significant differences between genotypes for all fas are present, the genotypic range is quite limited. heritability estimates on an entry mean basis are high for all considered fas, but effective selection for better fatty acid profile will be hindered by the narrow genetic base of the breeding material tested. therefore, broadening of the genetic base of l. angustifolius with respect to the fatty acid composition but also additional traits, e.g. protein content (beyer et al. 2015) is needed. environmental stability of fatty acids of l. angustifolius 195 acknowledgements this study was funded by the federal ministry of education and research (bmbf) as project 03wkbv01b, with the title “plantsprofood – food ingredients derived from blue sweet lupins.” we also thank steffen esser and stefan koch for technical assistance. references aguilera, j.m., garcia, h.d., 1989: protein extraction from lupin seeds: a mathematical model. int. j. food sci. technol. 24, 17-27. andersen, p.c., hill, k., gorbet, d.w., brodneck, b.v., 1998: fatty acid and amino acid profiles of selected peanut cultivars and breeding lines. j. food compos. anal. 11, 100-111. fig. 1: principal component biplot with scores represented by 12 trials and loadings represented by 5 fas. the dots of the scores are labeled as follows: bornhof (b), dratow (d), steinach (s) and groß lüsewitz (g) in the years 2010 (10), 2011 (11) and 2012 (12). tab. 4: eigenvectors and eigenvalues of the first two principal components for the 5 main fas in seed oil of 50 breeding lines of l. angustifolius variable eigenvectors pc1 pc2 c16:0 (%) 0.009 0.957 c18:0 (%) -0.503 0.196 c18:1 (%) -0.498 -0.209 c18:2 (%) 0.508 -0.048 c18:3 (%) 0.490 0.021 eigenvalue 3.6 1.1 cumulative 0.73 0.22 cumulative variance (%) 72.6 21.7 bachlava, e., burton, j.w., brownie, c., wang, s., auclair, j., cardinal, a.j., 2008: heritability of oleic acid content in soybean seed oil and its genetic correlation with fatty acid and agronomic traits. crop sci. 48, 1764-1772. beyer, h., schmalenberg, a.k., jansen, g., jürgens, h.u., uptmoor, r., broer, i., huckauf, j., dietrich, r., michel, v., zenk, a., ordon, f., 2015: evaluation of variability, heritability and environmental stability of seed quality and yield parameters of l. angustifolius. f. crops res. 174, 40-47. boschin, g., d’agostina, a., annicchiarico, p., arnoldi, a., 2008: effect of genotype and environment of fatty acid composition of lupinus albus l. seed. food chem. 108, 600-606. boschin, g., d’agostina, a., annicchiarico, p., arnoldi, a., 2007: the fatty acid composition on the oil from lupinus albus cv. luxe as affected by environmental and agricultural factors. eur. food res. technol. 225, 769-776. brar, g.s., 1982: variations and correlations in oil content and fatty acid composition of sesame. indian j. agric. sci. 52, 434-439. chiofalo, b., presti, v.l., chiofalo, v., gresta, f., 2012: the productive traits, fatty acid profile and nutritional indices of three lupin (lupinus spp.) species cultivated in a mediterranean environment for the livestock. anim. feed sci. technol. 171, 230-239. dubois, v., breton, s., linder, m., fanni, j., parmetier, m., 2007: fatty acid profiles of 80 vegetable oils with regard to their nutritional potential. eur. j. lipid sci. technol. 109, 710-732. eickmeyer, f., 2008: narrow-leafed lupin breeding in saatzucht steinach a private company integrated in a network of research and development. proceedings of the 12th international lupin conference, 312-314. fremantle, western australia. deutsche gesellschaft für ernährung, 2000: deutsche gesellschaft für ernährung, österreichische gesellschaft für ernährung, schweizerische gesellschaft für ernährungsforschung, schweizerische vereinigung für ernährung. referenzwerte für die nährstoffzufuhr. frankfurt/main, umschau/braus. 1. edition gladstones, j.s., atkins, c., hamblin, j., 1998: lupins as crop plants. wallingford oxon, uk: cab international. hondelmann, w., 1984: the lupin − ancient and modern crop plant. theor. appl. genet. 68, 1-9. jansen, g., juergens, h.-u., 2008: untersuchungen zur variabilität des rohfettgehaltes und des fettsäuremusters in blauen süßlupinen. mitteilungen der gesellschaft für pflanzenbauwissenschaften, vorträge für pflanzenzüchtung 20 (77), 245-246 sengbusch, r. v., 1938: the breeding of sweet lupins. herbage reviews 6, 64-71. simopoulos, a.p., 2002: the importance of the ratio of omega-6/omega-3 essential fas. biomed. pharmacother 56, 365-379. sipsas, s., 2008: lupin products − concepts and reality. proceedings of the 12th international lupin conference. fremantle, western australia, 506513. sujak, a., kotlarz, a., strobel, w., 2006: compositional and nutritional evaluation of several lupin seeds. food chem. 98, 711-719. sussmann, d., 2013: influence of different processing parameters on the isolation of lupin (lupinus angustifolius l.) protein isolates: a preliminary study. j. food process eng. 36, 18-28. uzun, b., arslan, c., karhan, m., toker, c., 2007: fat and fas of white lupin (lupinus albus l.) in comparison to sesame (sesamum indicum l.). food. chem. 102, 45-49. waesche, a., müller, k., knauf, u., 2001: new processing of lupin protein isolates and functional properties. nahrung 45, 393-395. address of the authors: helene beyer, hans-ulrich jürgens, gisela jansen, julius kühn-institut (jki), federal research centre for cultivated plants, institute for resistance research and stress tolerance, rudolf-schick-platz 3, 18190 groß lüsewitz, germany 196 h. beyer, h.u. jürgens, g. jansen, r. uptmoor, f. ordon prof. ralf uptmoor, university of rostock, faculty of agronomy, justus-von liebig-weg 6, 18051 rostock, germany prof. frank ordon, julius kühn-institut (jki), federal research centre for cultivated plants, institute for resistance research and stress tolerance, erwin-baur-straße 27, 06484 quedlinburg, germany © the author(s) 2015. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 88, 295 299 (2015), doi:10.5073/jabfq.2015.088.042 1kaunas botanical garden, vytautas magnus university, kaunas, lithuania 2 food institute of kaunas university of technology, kaunas, lithuania 3botanic garden, faculty of biology, university of warsaw, warsaw, poland investigations of anthocyanins, organic acids, and sugars show great variability in nutritional and medicinal value of european cranberry (vaccinium oxycoccos) fruit laima česonienė1*, remigijus daubaras1, ina jasutienė2, inga miliauskienė2, marcin zych3 (received march 27, 2015) * corresponding author summary the rising interest in nutraceutical-rich foods in relation to human health has resulted the increased use of cranberries in the modern diet. berries of wild european cranberry clones and cultivars show great variation in yield, colour and quantities of biologically active compounds. in the present study, we estimated the production of different phytochemical components in 40 genotypes of vaccinium oxycoccos. our main goal was to identify genotypes that have superior biochemical properties, such as high levels of anthocyanins, organic acids and easily digestible sugars. as a result, wild clones of lithuanian origin and certified cultivars were selected as the most valuable in terms of total amount of anthocyanins, organic acid, fructose and glucose concentrations. introduction native europeans, especially those from central and northeast europe, use the fruit of the european cranberry (vaccinium oxycoccos l.) for food and medicine (kardell, 1986). cranberry products, and especially cranberry juice, have long been consumed for health reasons, primarily because of their therapeutic effect on urinary tract infections (terris et al., 2001; viškelis et al., 2009). however, other nutritional and therapeutic properties of cranberries are widely acknowledged in various studies (see, e.g., borowska et al., 2009; côté et al., 2010; česonienė et al., 2011), and cranberry fruit is reported to possess a wide range of biological properties including anti-carcinogenic effects, and is useful in treating cardiovascular disorders. in general, the beneficial effect of cranberries, including v. oxycoccos and v. macrocarpon aiton, on human health has been attributed in particular to phenolic compounds (borowska et al., 2009; viškelis et al., 2009; côté et al., 2010) and organic acids (povilaitytė et al., 1998; zuo et al., 2002). furthermore, the red colour of cranberry fruit is due to the presence of anthocyanins; compounds which have important therapeutic effects, including anti-tumor, anti-ulcer, antioxidant, and anti-inflammatory properties (wang and wang, 2009). the renewed interest in anthocyanins is not only due to their health-promoting activities, but also their use as natural colourants by the food industry (lima et al., 2009). accor ding to bridle and timberlake (1997), the specific anthocyanin content of foods and beverages is an important indicator in estimating their nutritional value. similarly, the relative amounts of organic acids present can be used to distinguish between fresh and processed berries. for example, organic acids known to impart specific flavour are often used to control ph and can be used as an indicator of product quality. the use of natural organic acids is considered a good alternative to artificial compounds by the fruit-processing industry because of their preserving, antioxidant, flavouring and acidifying properties, as well as their low cost (raybaudi-massilia et al., 2009). for instance, investigations of antimicrobial activities revealed that citric acid strongly inhibited the growth of shigella dysenteriae, resulting in a 5-log reduction (in et al., 2013), and a transmission electron microscopy study showed that malic acid caused cytoplasmic disruption in bacterial pathogens, seemingly without damaging the cell membrane (raybaudi-massilia et al., 2009). according to other studies, carboxylic acids, such as malic, citric and succinic acid etc., behave as antioxidants because they also have the ability to chelate metals. therefore they are classified as preventive or synergistic compounds (pero et al., 2008). the soluble carbohydrates present in the fruit, namely glucose and fructose, contribute to the sweetness of cranberries. these sugars are important indicators of metabolic processes during fruit development. vaccinium oxycoccos clones and cultivars express great morphological diversity (gugnacka-fiedor, 1986; suda and lysak, 2001; česonienė et al., 2013), which seems to translate into differences in their production of medicinally useful phytochemicals (česonienė et al., 2011). promising results have been achieved by breeding of v. oxycoccos in russia when new productive cultivars ‘dar kostromy’, ‘krasa severa’ and ‘sazonovskaja’ cultivars were originated (makeev et al., 2000). our previous investigations corroborated big differences among v. oxycoccos clones according to berry size and productivity. consequently, the most perspective clones were selected with an average berry weight from 1.1 g to 1.3 g, which could be equal to the average weight of v. macocarpon cultivars (daubaras et al., 2004). our aim was to assess this variation by quantifying the amounts of nutritionally and medicinally important compounds, namely fructose, glucose, anthocyanins, quinic, malic and citric acids, in 40 different genotypes of v. oxycoccos, and, thus, determine the most promising genotype in terms of potential nutritional value. material and methods the species european cranberry vaccinium oxycoccos l. (syn. oxycoccus quadripetalus gilib., oxycoccus palustris pers.; ericaceae juss.), is a dwarf, woody, evergreen clonal shrub with slender, rooting stems, occasionally up to 0.8-1.0 m length, with short, usually erect, flowering shoots (jacquemart, 1997). its fruit is an over-wintering, edible berry (the cranberry), which is harvested from the wild or from cultivated varieties. plant material for the present study, we selected 40 genotypes of v. oxycoccos: 13 certified cultivars and 27 clones collected from the wild. the cultivars ‘kuresoo’, ‘soontagana’, ‘virussaare’, ‘nigula’ and ‘maima’ were of estonian origin, ‘sazonovskaja’, ‘krasa severa’ and ‘dar kostromy’ were selected from russian stock, and the remaining ‘reda’, ‘amalva’, ‘zuvinta’, ‘vaiva’, ‘vita’ and twenty seven wild clones were of lithuanian origin. all genotypes, both wild clones and cultivars, have been cultivated in the collections of kaunas 296 l. česonienė, r. daubaras, i. jasutienė, i. miliauskienė, m. zych botanical garden of vytautas magnus university under uniform ecological conditions in acid peat beds, ph 3.5-5.0. this collection is located in the central part of lithuania with typical growing season lasting 180-190 days, an average temperature -6 °c in january and +16 °c in july. an average annual rainfall is 600 mm. studied cultivars were propagated using cuttings that were planted in acid peat (ph 4.0-5.0) beds. these genotypes were selected as the most promising for further breeding purposes on the basis of a long-term monitoring programme that included phenological observations, resistance studies to fungal diseases and pests, as well as productivity studies (daubaras et al., 2004; česonienė et al., 2013). berry samples for analysis were collected at the full maturity stage (brown seeds and typical berry coloration). approximately 250-300 g berry samples were collected from a single genotype. part of the sample was frozen and later used for benzoic acid analysis. rest of berries were pressed in a conventional juicer and the compressed cakes and juices obtained were stored in a freezer (at -28 °c) prior to extraction. biochemical analyses for analyses two biological replicates, i.e. two year yields, were used. total anthocyanins (tac), malic, quinic, citric acids and sugars were investigated in berry juice, meanwhile qualitative and quantitative analysis of anthocyanins was accomplished in berry cake. the total anthocyanin content in cranberry was determined by a ph differential method. for tac measurements two dilutions of the sample were prepared namely one with potassium chloride buffer (ph 1) and the other one with sodium acetate buffer (ph 4.5), diluting by the 10 dilution factor (1 ml of juice + 9 ml of buffer). absorbance was measured using a genesy 5 spectrophotometer (usa) at a wavelength of 510 nm and 700 nm in buffers of ph 1.0 and 4.5. the tac was calculated as follows: (a510ph1 – a700ph1) – (a510ph4.5 – a700ph4.5) × mw × df × 103 e · l mw – molecular weight, for cyanidin-3-galactoside 445.2 g/mol df – dilution factor e – a molar extinction coefficient, for cyanidin-3-galactoside 41700 l – pathlength in cm a – absorbance results were expressed in terms of mg/kg of cyanidin-3-galactoside (a molar extinction coefficient 41700) (wrolstad, 1976). for qualitative analysis of anthocyanins, berry cakes were homogenized and 3 g of homogenate were extracted over a period of 1 h at room temperature with 10 ml of acidified ethanol (95 % [v/v] food grade ethanol containing 0.1 m hcl). the extracts were analyzed by hplc using a reversed phase c18 lichrospher®100 rp 18e column (125 × 4 mm, 5mm) (merck, darmstadt, germany). the eluents were (a) 4 % aqueous h3po4, and (b) 100 % hplc-grade acetonitrile (merck) (viškelis et al., 2009). chromatographic conditions were as follows: 10 % b in a at the time of injection, 14 % b in a (4 min), 16 % b in a (10 min), 30 % b in a (25 min), initial conditions (26 min). flow rate was 0.8 ml/min, 20 μl was injected. the samples were filtered through a 0.45-μm cellulose syringe filter prior to analysis. detection was performed using a diode-array detection system agilent 1200 (agilent technologies, usa) at 520 nm. chemstation software was used. determination of benzoic acid was performed according to the iso 22855 “fruit and vegetable products – determination of benzoic acid and sorbic acid concentrations – high-performance liquid chromatography method”. berries were homogenized and 5 g of homogenate were extracted for 30 min (periodically stirring) in a water bath at 70 °c with 75 ml of extraction solution. the extract was subsequently clarified with carrez i and carrez ii solutions and made up to 100 ml with extraction solution. the samples were filtered through a paper filter and a 0.45-μm cellulose syringe filter before analysis. chromatographic conditions were as follows: the eluent was a mixture of 50 parts by volume of ammonium acetate and 40 parts by volume methanol (ph adjusted to 4.5-4.6), flow rate was 0.8 ml/min, 20 μl was injected. the reversed phase zorbax eclipse xdb-c18 (5mm) 150 × 4.6 mm (agilent technologies, usa) column was used. detection was performed using a diode-array detection system agilent 1200 (agilent technologies, usa) at 235 nm. chemstation software was used. benzoic acid content mg/kg was calculated as follows: c = (v × cst × 100) / (vst × m), v – peak area of the sample vst – peak area of the standard solution cst – concentration of standard solution, 50 mg/l m – amount of berry sample used for the extraction, g for determination of malic, citric and quinic acids, 1 ml of juices was diluted with ~50 ml distilled/deionized water, clarified the solution with carrez i and carrez ii solutions and made the solution up to 50 ml with distilled/deionized water. after 5 min, the samples were filtered through a paper filter and a 0.22 μm nylon syringe filter before analysis. a standard solution of organic acid mixture was prepared by dissolving 2 g of malic (sigma-aldrich, germany), citric (sigma-aldrich, germany) and quinic (sigma-aldrich, germany) acids in 100 ml of water. standard solutions of 0.1 %, 0.5 %, and 1 % (w/v) organic acid mixture were prepared by appropriate dilution of the original standard solution. chromatographic conditions were as follows: the eluent was 20 mm na2hpo4 buffer solution (ph adjusted to 2.8 with acetic acid), flow rate was 1.0 ml/min, 20 μl was injected. the reversed phase hydrosphere c18 (5 μm, 12 nm), 150 × 4.6 i.d. (ymc co., ltd., japan) column was used. column temperature was set at 30 oc. detection was performed using a diode-aray detection system shimadzu prominence lc20ad (shimadzu corp., japan) at 220 nm and lab solutions software. quinic, citric, malic acid content were calculated from the calibration curves of each organic acid standard solutions (concentration 0.01, 0.05, 0.1 and 0.2 g/l) multiplied by the dilution factor (50). for determination of sugars, 10 ml of juices were diluted with ~80 ml of water, clarified with carrez i and carrez ii solutions, and made up to 100 ml with distilled/deionized water. after 5 min, the samples were filtered through a paper filter and a 0.22 μm nylon syringe filter before analysis. a standard solution of a sugars mixture was prepared by dissolving 0.4 g each of fructose (sigma-aldrich, germany), glucose (sigma-aldrich, germany) and sucrose (sigmaaldrich, germany) in 100 ml of distilled/deionized water. a 2 mg/ ml and a 0.4 mg/ml standard solution of carbohydrate mixture was prepared following dilution with distilled/deionized water. chromatographic conditions were as follows: the eluent was a mixture of 75 parts by volume of acetonitrile and 25 parts by volume water, flow rate was 1.2 ml/min, 20 μl was injected. the ymc-pack polyamine ii 250 × 4.6 mm, 5 μm (ymc co., ltd., japan) column was used. column temperature was set at 28 oc. detection was performed using an evaporative light scattering detector elsdltii (shimadzu corp., japan). sugar content g/kg was calculated as follows: c = (v × cst × 1000) / (vst × m), v – peak area of the sample vst – peak area of the standard solution cst – concentration of standard solution (4 mg/ml) m – amount of juice sample used for the analysis in g investigations of nutritional value of vaccinium oxycoccos 297 statistical analysis investigations were carried out in three replications for each genotype. averages of the data and standard deviations were calculated using statistica, version 7. results and discussion anthocyanin content our fruit samples clearly differed in their total anthocyanin content (tac). although the mean tac was 59.4 mg/kg, the tac values for juices of v. oxycoccos, expressed as cyanidin-3-galactoside, ranged from 12.2 mg/kg (‘virussaare‘) to 227.8 mg/kg (98-c-17). most investigated genotypes were characteristic of moderate tac values. however the wild clones 96-z-02, 98-c-17, 96-k19, and 99-z-06, were distinguished for exceptionally high tac values 100.2, 227.8, 126.2 and 103.0 mg/kg, respectively (tab. 1). these even exceeded the range reported in earlier studies from 43.3 to 96.8 mg/kg (povilaitytė et al., 1998; borowska et al., 2008). similar variation in anthocyanin content was also reported for fruit of different cultivars and clones of the large cranberry v. macrocarpon (vvedenskaya and vorsa, 2004; viškelis et al., 2009). accumulation of anthocyanins is influenced by numerous factors such as berry ripening, air temperature etc., and may also be genetically determined. interestingly, in our study, berries of clones with highest tac values had coloured pulp, although, in general, tac is mainly concentrated in the vacuoles of fruit epidermal cells, whereas the pulp is practically devoid of anthocyanin. chromatographic analysis revealed the presence of six anthocyanins (fig. 1). overall, the average anthocyanin profile was as follows: cyanidin-3-galactoside (19.3±0.53 %), cyanidin-3-glucoside (2.8±0.22 %), cyanidin-3-arabinoside (20.2±0.17 %), peonidin-3galactoside (29.6±0.48 %), peonidin-3-glucoside (8.1±0.31 %), and peonidin-3-arabinoside (19.8±0.23 %). proportions of different compounds varied between the genotypes studied. for example, berries of 96-z-13, 97-j-07, 99-z-01, 99-z-02, 99-z-05, 99-z-15 and ‘soontagana’ did not contain cyanidin-3-glucoside, whereas 99-z08 and 98-c-15a contained 10 % and 9 % of cyanidin-3-glucoside, respectively. a similar anthocyanin profile was reported for fruit of the related species v. macrocarpon (viškelis et al., 2009). its cultivars, like v. oxycoccos, differ in terms of the anthocyanin mass fraction (vvedenskaya and vorsa, 2004; borowska et al., 2009). the anthocyanin profile of the fruit provides important commercial information, since the proportion of individual anthocyanins may affect the colour stability of cranberry products, such as cranberry juice and cranberry sauce (starr and francis, 1968). the mean benzoic acid content for our study group was 52.4 mg/ kg (tab. 1). particularly high concentrations of benzoic acid were found in berry extracts of clones 98-c-15, 98-c-17, and ‘maima’ (147.0±6 mg/kg, 115.2±11 mg/kg, 129.4±2 mg/kg). our results fell well within the ranges previously reported by park et al. (2008), i.e. 10.1-240 mg/kg. the investigated genotypes varied with regard to organic acid content (tab. 1). quinic acid concentration ranged from 3.81 to 13.3 g/ kg (on the average 7.65 g/kg), malic acid from 14.1 to 43.3 g/kg (on the average 26.5 g/kg), and citric acid from 10.8 to 54.3 g/kg (on the average 33.2 g/kg). the highest concentrations of quinic acid were found in berries of clone 96-z-03 (13.3 g/kg), malic acid in clone 96-k-04 (43.3 g/kg) and citric acid in clone 96-k-19 (54.3 g/ kg). like v. oxycoccos, citric, malic, and quinic acids are the main organic acids present in the berries of v. macrocarpon (miller et al., 2009). however, viljakainen et al. (2002) reported that total acid content in v. oxycoccos juice is the highest, compared with the juices of wild berries, such as those of bilberry, lingonberry, cloudberry, red raspberry, and black crowberry. in our study cranberry, fruit accumulated, on average, 42.1 g/kg fructose and 45.1 g/kg glucose (tab. 1). the highest fructose content was detected in the estonian cultivar ‘soontagana’ (56.9±1.4 g/ kg), followed by the lithuanian clones 98-c-15 and 95-a-05, which contained 54.9±6.0 g/kg and 53.9±3.0 g/kg, respectively. berries of the cultivar ‘maima’ accumulated the largest amount of glucose (62.9±4.0 g/kg). the mean concentration of total sugar was 84.4 g/ kg. our results show that fruit of some clones may be superior in terms of sugar concentration to the berries of cultivars of v. macrocarpon, which, according to some authors, contain 45.7-80.4 g/kg of total sugars (wang and wang, 2009; povilaitytė et al., 1998). however viljokainen et al. (2002) found concentrations of fructose and glucose, respectively 20.69 g/l and 27.75 g/l. such variation may be caused by geographical and climatic conditions. the glucose and fructose contribute sweetness to cranberries. these sugars are known to be indicators of the metabolic processes that occur during ripening. for breeding purposes, it is important to select cranberry clones that accumulate significant amounts of sugars that can be rapidly absorbed by the human digestive system. furthermore, breeders seek to develop cultivars with more natural sweetness because this reduces the amount of sugar that needs to be added during processing and may thus increase the market demand for fresh fruit (trehane, 2004). conclusions our present study revealed great variation in tac and organic acids and sugar content in fruit of both cultivars and wild clones of v. oxycoccos. the wild clones, 98-c-17, 96-k-19, 96-z-02, 99-z-06 were the most promising genotypes with exceptionally high amounts of tac. because of prevailing termostable galactoside (48.9 %) and glucoside (10.9 %) conjugates berries of v. oxycoccos are perspective for processing by high temperature. of the genotypes studied so far, ‘soontagana’, ‘sazonovskaja’, ‘vaiva’ and 99-z-05 could be used to reduce supplement sugar intake by preparation of food products with cranberries. we suggest, therefore, that these genotypes should be investigated further as a source of valuable characters for future breeding programmes. references borowska, e.j., mazur, b., gadzała-kopciuch, r., buszewski, b., 2009: polyphenol, anthocyanin and resveratrol mass fractions and antioxidant properties of cranberry cultivars. food technol. biotech. 47, 56-61. bridle, p., timberlake, c.f., 1996: anthocyanins as natural food coloursselected aspects. food chem. 58, 103-109. côté, j., caillet, s., doyon, g., sylvain, j.-f., lacroix, m., 2010: analyzing cranberry bioactive compounds. crit. rev. food sci. 50, 872888. česonienė, l., daubaras, r., jasutienė, i., venclovienė, j., miliauskienė, i., 2011: evaluation of the biochemical components and chromatic properties of the juice of vaccinium macrocarpon aiton and vaccinium oxycoccos l. plant food hum. nutr. 66, 238-244. česonienė, l., daubaras, r., paulauskas, a., žukauskienė, j., zych, m., 2013: morphological and genetic diversity of european cranberry (vaccinium oxycoccos l. ericaceae) clones in lithuanian reserves. acta soc. bot. pol. 82, 211-217. daubaras, r., česonienė, l., labokas, j., 2004: phenotypic diversity of wild cranberry (vaccinium oxycoccos) in lithuania. acta hort. 663, 617620. gugnacka-fiedor, w., 1987: zmienność morfologiczna taksonów rodzaju oxycoccus hill. [morphological diversity in taxa of the genus oxycoccus 298 l. česonienė, r. daubaras, i. jasutienė, i. miliauskienė, m. zych tab. 1: total anthocyanin (tac), benzoic acid, quinic acid, malic acid, citric acid, fructose and glucose content in berry juice of 40 genotypes of vaccinium oxycoccos cultivar / genotype tac benzoic acid quinic acid malic acid citric acid fructose glucose [mg/kg] [mg/kg] [g/kg] [g/kg] [g/kg] [g/kg] [g/kg] ‘amalva’ 53.2 39.2 8.87 18.9 23.3 41.0 42.3 ‘dar kostromy’ 47.2 67.3 11.43 36.8 43.9 25.0 29.8 ‘krasa severa’ 24.2 12.2 7.62 29.1 50.3 40.7 47.2 ‘kuressoo’ 40.5 64.7 6.21 25.5 37.8 48.2 57.6 ‘maima’ 19.3 129.0 7.10 28.7 54.3 47.4 62.9 ‘nigula’ 12.9 57.7 5.42 27.9 37.1 36.2 52.4 ‘reda’ 35.2 27.0 10.00 18.5 19.1 28.9 32.4 ‘sazonovskaja’ 99.0 65.1 6.93 32.4 49.3 52.5 59.3 ‘soontagana’ 34.2 35.2 6.00 30.5 32.5 56.9 57.6 ‘vaiva’ 48.1 16.4 7.32 18.4 13.2 53.9 59.7 ‘virussaare’ 12.2 41.6 5.92 31.7 54.3 49.5 57.8 ‘vita’ 28.2 16.6 9.45 18.5 26.1 36.7 33.5 ‘zuvinta’ 32.5 39.1 11.83 25.2 43.6 33.9 52.4 95-a-03 53.5 39.5 5.96 21.0 10.8 36.4 36.1 95-a-04 43.3 64.0 6.12 22.2 12.1 35.9 37.8 95-a-07 66.5 37.2 6.01 18.0 16.9 35.2 35.1 96-k-04 98.1 64.6 10.50 43.3 41.4 50.1 49.5 96-k-19 126.2 69.3 7.36 20.6 54.3 39.5 51.3 96-z-02 100.2 45.2 6.73 14.9 25.4 28.5 29.4 96-z-03 77.1 28.4 13.30 16.9 27.1 43.6 39.5 96-z-13 55.0 78.2 9.86 22.6 40.6 40.6 37.5 97-j-04 76.1 61.7 6.93 35.7 28.4 35.5 34.4 97-j-07 44.1 39.8 9.51 43.2 38.8 54.9 43.2 98-c-15 56.3 147.0 7.61 25.3 33.6 49.5 48.7 98-c-15 a 52.7 66.3 5.52 28.7 30.0 38.3 30.0 98-c-17 228.0 115.0 8.52 32.9 36.6 49.5 48.7 99-z-01 41.8 67.4 6.67 25.8 25.9 46.1 52.5 99-z-02 36.9 17.2 6.85 24.8 33.6 41.6 42.0 99-z-04 66.4 30.5 8.71 28.0 35.3 42.5 53.0 99-z-05 73.1 74.7 9.03 37.7 36.1 52.4 54.2 99-z-06 103.0 14.3 6.22 22.8 42.2 36.3 47.5 99-z-08 52.1 65.3 8.72 32.8 21.0 36.4 42.6 99-z-11 31.4 83.6 8.40 32.7 33.7 48.3 47.0 99-z-13 62.9 17.1 7.01 25.3 27.6 29 26.6 99-z-14 78.1 27.4 6.53 17.9 27.4 43.2 47.1 99-z-15 29.6 7.1 6.25 31.6 37.5 44.3 42.9 99-z-16 74.7 36.7 6.23 27.8 30.5 39.3 44.4 99-z-17 51.1 97.0 3.81 14.1 16.4 47.6 54.2 99-z-18 64.3 37.8 8.25 24.2 42.3 43.4 38.8 nr. 184 48.2 52.9 5.23 26.0 37.6 39.8 43.9 m 59.4 52.4 7.65 26.5 33.2 42.1 45.1 sd 37.62 31.62 1.99 7.21 11.4 7.76 9.55 mmin – mmax 12.2-227.8 7.1-147.0 3.81-13.3 14.143.3 10.8-54.3 25.0-56.9 26.6-62.9 m – mean value; sd – standard deviation; mmin-mmax – range of mean values investigations of nutritional value of vaccinium oxycoccos 299 hill.]. studia societatis scientarum torunensis sectio d (botanica) 11, 1-57 (in polish). in, y.-w., kim, j.-j., kim, h.-j., oh, s.-w., 2013: antimicrobial activities of acetic acid, citric acid and lactic acid against shigella species. j. food safety 33, 79-85. jacquemart, a.l., 1997: vaccinium oxycoccos l. 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antioxidative, and antimicrobial properties of american cranberry (vaccinium macrocarpon ait.) and their press cake. j. food sci. 74, 157-161. wang, ch. y., wang, s.y., 2009: effect of storage temperature on fruit quality of various cranberry cultivars. acta hort. 810, 853-861. wrolstad, r.e., 1976: colour and pigment analyses in fruit products. agricultural experiment station bulletin 624. oregon state university. zuo, y., wang, c., zhan, j., 2002: separation, characterization, and quantitation of benzoic and phenolic antioxidants in american cranberry fruit by gc-ms. j. agric. food chem. 50, 3789-3794. address of the authors: laima česonienė, phd; remigijus daubaras, phd, kaunas botanical garden, vytautas magnus university, žilibero 6, kaunas, lt-46324, lithuania e-mail: laimac@hotmail.com e-mail: remigijusd@hotmail.com ina jasutienė, phd; inga miliauskienė, food institute of kaunas university of technology, taikos pr. 92, lt-51180, kaunas, lithuania e-mail: ina.jasutiene@lmai.lt e-mail: inga.miliauskiene@lmai.lt marcin zych, phd, botanic garden, faculty of biology, university of warsaw, aleje ujazdowskie 4, 00-478 warsaw, poland e-mail: mzych@biol.uw.edu.pl fig. 1: quantitative composition of anthocyanins in v. oxycoccos genotypes, % © the author(s) 2015. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 87, 24 29 (2014), doi:10.5073/jabfq.2014.087.004 fruit research division, national institute of horticultural and herbal science, rural development administration, suwon, korea assessment of organic acid and sugar composition in apricot, plumcot, plum, and peach during fruit development haejin bae, seok kyu yun*, ji hae jun, ik koo yoon, eun young nam, jung hyun kwon (received december 13, 2012) * corresponding author summary variation in content of organic acids and soluble sugars, and in physical characteristics was evaluated in apricot (p. armeniaca l. cv. harcot), plumcot (plum-apricot hybrid, p. salicina ⅹ p. armeniaca l. cv. harmony), plum (p. salicina lindl. cv. formosa), and peach (p. persica l. batsch cv. jinmi). the content of organic acids and sugars, as well as parameters of fruit quality (weight, dimensions, firmness, total soluble solids, and total acidity) in prunus fruits during fruit development were determined. organic acids, including oxalic acid, quinic acid, malic acid, shikimic acid, citric acid, and quinic acid, sugars, including sucrose, fructose, glucose, and sugar alcohol (sorbitol), were identified and quantified using hplc. organic acid mostly increased during the early stages of fruit growth (30 60 days after full bloom) and decreased until fruits were fully ripened. in general, plum was the highest in most organic acids compared with the other fruits, while apricot contained the lowest acid content except for citric acid. sucrose, fructose, and glucose content increased with fruit development, unlike content of sorbitol. plumcot contained the highest fructose, and peach showed the maximum content of sucrose at full maturation stages. total soluble solids averaged 17.5, 14.8, 11.9, and 10.6 ºbrix in apricot, plumcot, plum, and peach, respectively, whereas total acidity was 0.9, 1.4, 0.5, and 0.3% in four prunus cultivars at ripened stages. shikimic acid was significantly correlated with oxalic acid in apricot, plumcot, and plum, but not in peach. fructose and glucose were highly correlated in plumcot, plum, and peach. introduction edible fruit quality is influenced by organic acids and sugars, as well as by changes in color, texture, and flavor because these parameters contribute to organoleptic quality of fruits. the concentrations of organic acids and sugars have an important impact on fruit flavor and quality (borsani et al., 2009). organic acid originally occurs in mitochondria through the tricarboxylic acid cycle, but is located mainly in vacuoles due to catalytic function in the tricarboxylic acid cycle (lópez-bucio et al., 2000). in the early stages of fruit development, fruits accumulate organic acids, and thus have an acidic taste (shiratake and martinoia, 2007). during the process of fruit maturation and ripening, sugars stored in vacuoles (yamaki, 1984) generally increase in concentration along with a simultaneously decrease in organic acids, except in highly acidic fruits such as citrus (echeverria and burns, 1989). sugars are synthesized throughout the process of photosynthesis, and used for respiratory substrates and in cell wall structural (yu, 1999). therefore, total acidity and total soluble solids increase as fruits ripen. genus prunus has been commercially grown around the world. major species are stone fruits, including apricot, plum, and peach. indeed, plumcot, the interspecific hybrid of apricot and plum, has the potential for commercial production because of its early harvest season and abundant fruit production. the fruit quality of apricot, plumcot, plum, and peach is greatly influenced by the ratio of total acidity to total soluble solids at harvest time because acid and sugar concentrations mainly influence fruit taste. the major acids in prunus fruits are malic acid, citric acid, quinic acid in peach (le dantec et al., 2010), oxalic acid in plum (wu et al., 2011), and tartaric acid, ascorbic acid, shikimic acid, succinic acid, maleic acid, and fumaric acid have also been identified (flores et al., 2012). the dominant sugars in stone fruits include fructose, glucose, and sucrose, along with some individual saccharide containing stachyose (sozzi, 2004), sorbitol (cantín et al., 2009), raffinose (ledbetter et al., 2006), rhamnose (kovács and németh-szerdahelyi, 2002), arabinose, galactose, and xylose (gross and sams, 1984). organic acid and sugar profiles vary depending on the species of stone fruits, which have different qualitative traits. previous studies reported the composition of organic acids and sugars in apricot, plumcot (akin et al., 2008), plum (famiani et al., 2012), and peach (orazem et al., 2011). however, information on the profiles of organic acids and sugars is limited in commercially important prunus fruits during fruit development, maturation, and ripening. therefore, in this study, the most common and valuable cultivars of apricot, plumcot, plum, and peach were chosen and evaluated. the objective of this study was to investigate the relationship between fruit development and changes in content of organic acids and sugars during the time between full bloom and harvest. materials and methods plant materials four prunus species containing apricot (prunus armeniaca l. cv. harcot), plumcot (p. armeniaca x p. salicina l. cv. harmony), plum (p. salicina lindl. cv. formosa), and peach (p. persica l. batsch cv. jinmi) were used in this study. the ‘harmony’ plumcot cultivar was originated as a cross between ‘soldam’ plum and ‘harcot’ apricot (ji hae and kyeong ho, 2007), and the appearance of apricot, plumcot, and plum was similar except for ground color (fig. 1). four-year-old trees of apricot, plumcot, and plum, and seven-year-old peach trees were grown in the fields of the national institute of horticultural and herbal science in rural development administration, suwon, korea (37°15’n and 126°98’e). the first blossom dates in 2012 were april 20 for apricot and plumcot, april 23 for plum, and april 25 for peach. six fruits were randomly sampled at two-week intervals beginning 30 days after full blossom for all fruits, and fruit samples were collected, with three replications per fruit cultivar. when fruits started to mature (ripen), fruits were harvested at one-week intervals. the last dates for final harvest were june 26 for apricot, july 3 for plumcot, july 17 for plum, and august 24 for peach. fruit quality evaluation each fruit was immediately measured for weight, height, diameter, and flesh firmness at each harvest date. fruit diameter and height were measured by digital calliper (cd-15cpx, mitutoyo, kawasaki, japan). fruit firmness was measured by lloyd instrument ta plus plant foods 25 digital texture analyzer (ametek, west sussex, uk). firmness was expressed as maximum force (gf). total soluble solids (°brix) of fruit juice were measured using a hand refractometer pal-1 meter (atago, tokyo, japan). total acidity of final-harvest fruits was measured using an automated titrimeter (titroline easy, schott, mainz, germany). a mixture of fruit extract and water (1:4 ratio) was titrated to ph 8.1 with 0.1n naoh solution, and total acidity was expressed as percentage of malic acid. sample preparation harvested prunus fruits were transported to the laboratory for fresh weight determination. the fruits were cleaned, and seeds were removed. sections of fresh weighing about 50 g with peel were cut and blended in 40 ml using a household blender for homogenization. the homogenate was centrifuged at 1800 g for 15 min, and supernatant was filtered using whatman filter papers. the extract was then filtered through a 0.45 μm filter (acrodisc 25mm syringe filter, pall gelman laboratory, ann arbor, mi, usa) before hplc analysis. all samples were prepared in triplicate. organic acid analysis organic acid was analyzed by agilent 1100 high performance liquid chromatography (hplc) system (hewlett-packard 1100 series) containing quaternary pump, autosampler, and diode array detector with zorbax sb-aq c18 column (150 mm × 4.6 mm id, 5μm) (agilent technology, santa clara, ca. usa). chromatography separation was performed at 40°c with a flow rate of 0.4 ml/min. the mobile phase was carried out with 1% monopotassium phosphate (kh2po4, ph 2.5). the acid standards were purchased from sigmaaldrich (st. louise, mo, usa), and the detected organic acids were oxalic acid, quinic acid, malic acid, shikimic acid, citric acid, and fumaric acid. absorbance was measured at 214 nm. soluble sugar analysis sucrose, fructose, glucose, and sorbitol standards were purchased from sigma-aldrich (st. louise, mo, usa). sugars were separated and quantified by hplc analysis. the hplc system (younglin, anyang, korea) contained a quaternary pump, an autosampler, and a reflective index detector with sugar-paktm1 column (6.5 × 300 mm, waters, usa) at 90 °c with a flow rate 0.5 ml/min. the mobile phase was performed with an isocratic elution of ultrapure water for peak separation. statistical analysis data was analyzed with anova using sas statistical software. means were separated using the duncan’s multiple range test at confidence level p ≤ 0.05, and pearson correlation coefficients were calculated. data was presented as the mean ± standard deviation of triplicate. results and discussion fruit quality fruit weight, height, width, and firmness affected fruits quality. the quality parameters were measured during fruit development and maturation, and required characteristics in marketable fruit yields. changes during fruit maturation in apricot, plumcot, plum, and peach were shown in tab. 1. fruit weight, height, and width accumulatively increased from 30 days after full bloom until 65 days after full bloom in apricot, 72 days after full bloom in plumcot, 91 days after full bloom in plum, and 123 days after full bloom in peach. fruit weight rapidly and linearly increased with fruit growth and maturity. apricot was continuously larger in fruit weight, height, and width than plumcot and plum from the first onset of fruits to the end of fruit harvest. the hybrid plumcot species was in the middle position for fruit growth between apricot and plum. peach developmental changes markedly increased after 72 days after full bloom because fruit growth duration was longer than for apricot, plumcot, and plum. in general, fruit firmness linearly decreased until 65 days after full bloom, and then slightly declined after that with final growth. peach firmness was much harder than other fruits throughout the fruit development process. since firmness is associated with water content of fruits and structural rigidness of cell walls, fruit firmness decreased between the initial fruit developmental stage and harvest. total acidity and total soluble solids are also important factors in evaluation of fruit quality at harvest time. total soluble solids (°brix) dramatically increased in plumcot and apricot until final ripening stages. maximum total soluble solids were found in apricot (17.5°brix) followed by plumcot (14.8°brix), peach (12.3°brix), and plum (11.9°brix) at the final harvest stages. during fruit development and ripening, the patterns of increasing total soluble solids were related to decreased total acidity. total acidity increased between 30 days after full bloom and 44 days after full bloom, and then decreased with fruit development in apricot, plumcot, and plum, while total acidity in peach showed a gradual reduction through fruit development. the least total acidity was determined in peach (0.2%), followed by plum (0.5%), apricot (0.9%), and plumcot (1.4%). organic acid analysis oxalic acid, quinic acid, malic acid, shikimic acid, citric acid, and fumaric acid are important organic acids in prunus fruits. oxalic acid in apricot, plumcot, plum, and peach decreased from 30 days after full bloom until fruit was fully ripened at final harvest time fig. 1: fresh fruits of apricot, plumcot, and plum harvested at the stages of maturity. 26 h. bae, s. yun, j. jun, j. yoon, e. nam, j. kwon tab. 1: fresh weight, size, firmness, and quality of four prunus fruits during fruit maturity. fruits dafba weight (g) height width firmness tssb total (mm) (mm) (gf) (brix) acidity (%) apricot 30 20.7 ± 0.6 c 35.8 ± 0.1 c 33.7 ± 0.1 c 44 35.8 ± 3.0 b 40.6 ± 0.1 b 36.5 ± 0.1 c 7.2 ± 0.2 a 58 63.1 ± 4.9 a 46.7 ± 1.5 a 45.6 ± 1.1 b 3.0 ± 0.2 b 65c 72.7 ± 5.6 a 49.2 ± 1.1 a 48.7 ± 1.6 a 0.5 ± 0.1 c 17.5 ± 1.0 0.9 ± 0.1 plumcot 30 14.0 ± 0.9 c 31.1 ± 0.1 b 28.7 ± 0.1 c 44 21.1 ± 0.9 c 33.2 ± 0.1 b 30.7 ± 0.1 c 8.7 ± 0.3 a 58 45.7 ± 3.5 b 44.3 ± 1.5 a 41.7 ± 0.5 b 4.1 ± 0.3 b 65 59.1 ± 2.8 a 44.6 ± 0.7 a 45.5 ± 0.9 a 1.9 ± 0.1 c 72 c 70.3 ± 8.9 a 47.1 ± 1.4 a 47.3 ± 2.1 a 0.6 ± 0.1 d 14.8 ± 0.5 1.4 ± 0.1 plum 30 5.7 ± 0.0 e 27.0 ± 0.0 e 21.5 ± 0.1 e 44 15.9 ± 1.5 de 33.9 ± 0.2 d 28.2 ± 0.1 d 8.7 ± 0.2 a 58 30.0 ± 5.9 d 40.2 ± 2.5 c 36.7 ± 2.1 c 5.6 ± 0.4 b 65 56.1 ± 6.0 c 46.1 ± 2.7 b 46.8 ± 1.5 b 1.8 ± 0.2 c 79 81.3 ± 6.0 b 52.6 ± 1.9 a 52.9 ± 1.9 a 1.1 ± 0.1 cd 86 82.9 ± 5.2 b 52.5 ± 2.2 a 52.0 ± 1.7 a 0.5 ± 0.2 d 91c 103.2 ± 8.8 a 56.1 ± 2.1 a 54.7 ± 2.1 a 0.4 ± 0.1 d 11.9 ± 0.8 0.5 ± 0.1 peach 30 3.4 ± 0.5 f 24.4 ± 1.3 g 18.6 ± 1.1 f 44 24.0 ± 3.8 ef 41.8 ± 2.0 f 30.5 ± 2.1 e 58 39.0 ± 2.4 e 43.5 ± 1.1 f 35.3 ± 1.2 e 16.1 ± 3.3 a 72 54.0 ± 4.0 de 49.3 ± 1.7 e 42.2 ± 0.5 d 11.7 ± 1.9 a 86 80.7 ± 1.0 d 54.3 ± 0.2 d 50.4 ± 1.4 c 4.1 ± 0.5 c 100 127.3 ± 0.2 c 59.6 ± 1.1 c 60.4 ± 0.7 b 3.3 ± 0.2 cd 114 209.2 ± 27.9 b 68.4 ± 1.9 b 70.3 ± 4.5 a 2.6 ± 0.2 cd 123c 307.1 ± 10.6 a 84.2 ± 0.6 a 74.2 ± 3.2 a 1.7 ± 0.1 d 10.6 ± 0.2 0.3 ± 0 adafb: days after full bloom btss: total soluble solid clast day of fruit harvest data is means ± standard deviation. means followed by different letters in the same column for the same species are significantly different at p ≤ 0.05 (tab. 2). quinic acid, malic acid, and shikimic acid increased and then dramatically decreased after 44 days after full bloom. quinic acid is the major acid in plum at the early stage of fruit growth, and malic acid is especially abundant in plumcot during fruit maturity. concentration of citric acid in apricot rose considerably from 30 to 44 days after full bloom, and then continuously increased during fruit development and maturity. however, citric acid was much lower in plum, followed by peach, compared with plumcot and apricot. a previous study showed that malic acid, citric acid, quinic acid, and shikimic acid are major acids in peach (prunus davidiana) (wu et al., 2005). generally, concentration of organic acid is higher at early stages of fruit development than at fruit maturity. soluble sugar content sugar profiles indicated the sweet taste of prunus fruits. major sugars (sucrose, glucose, fructose, and sorbitol) were detected and concentration was determined in apricot, plumcot, plum, and peach during fruit maturation (tab. 3). sugar content was variable among fruits. due to differences in fruit growth and duration. sucrose content was the highest at the final stage of harvest in apricot apricot (1710 μg/g), plum (2828 μg/g), and peach (6761 μg/g). however, sucrose was at a peak at 58 days after full bloom in plumcot (2478 μg/g), and then declined until the final stages. sucrose in apricot and plum was detected only when fruits reached full maturation. the content of glucose and fructose was higher than sucrose and sorbitol during fruit growth. plumcot contained the highest glucose (4302 μg/g) and fructose (4374 μg/g) at the final harvest stage many studies have compared sugar content, mostly glucose, fructose, and sucrose, in berry (mikulic-petkovsek et al., 2012), mandarin (zhang et al., 2012) and peach (wu et al., 2012). the sugar composition mostly makes prunus fruits sweet taste. correlations correlation coefficients between organic acid and sugar content showed various relationships (tab. 4). individual organic acids and sugar was related positively, negatively, or insignificantly. oxalic acid and malic acid were strongly correlated with shikimic acid in apricot and plumcot. oxalic acid and quinic acid were highly related in plumcot (r = 0.89) and plum (r = 0.96). in peach, malic acid and fumaric acid were positively correlated (r = 0.93). citric acid was significantly correlated with fumaric acid only in plum (r = 0.98, p ≤ 0.01). quinic acid and malic acid were negatively correlated in plum and peach. sucrose content was correlated with glucose and fructose in apricot and peach, respectively. fructose was highly correlated with glucose in plumcot, plum, and peach (r = 0.90, r = 0.99, and r = 0.76, respectively). conclusion the content of each organic acid and sugar variable changes during fruit growth and maturity (increasing in days after full bloom) in apricot, plumcot, plum, and peach. determination of organic acid and sugar profile in main stone fruits can benefit further prunus breeding lines with particular interest of organic acid and sugar composition. plant foods 27 tab. 3: content of sucrose, fructose, glucose, and sorbitol in apricot, plumcot, plum, and peach during fruit maturation. each point indicates mean standard deviation. fruits dafba sucrose glucose fructose sorbitol apricot 30 0 3045 ± 56.1 1071 ± 20.6 1034 ± 20.3 44 0 3261 ± 142.2 1213 ± 250.4 1188 ± 164.8 58 0 2479 ± 324.2 1687 ± 185.7 1185 ± 222.3 65b 1710 ± 364.9 4142 ± 241.6 3588 ± 218.7 261 ± 152.8 plumcot 30 0 1552 ± 267.7 1010 ± 129.9 921 ± 166.0 44 0 2466 ± 244.0 1483 ± 73.6 1748 ± 141.9 58 2478 ± 155.7 2367 ± 243.0 2256 ± 164.0 2144 ± 325.5 65 1062 ± 393.6 2562 ± 245.8 3155 ± 205.5 2183 ± 214.5 72b 332 ± 113.1 4302 ± 108.8 4374 ± 114.1 237 ± 70.3 plum 30 0 1176 ± 79.6 1456 ± 58.9 217 ± 18.0 44 0 1799 ± 128.9 2067 ± 133.2 308 ± 55.4 58 0 3018 ± 201.6 3010 ± 218.3 227 ± 109.4 65 0 3123 ± 203.4 3382 ± 408.9 272 ± 44.4 79 0 3188 ± 89.7 3421 ± 98.5 516 ± 29.2 86 2242 ± 217.1 3721 ± 103.0 3693 ± 421.0 232 ± 107.5 91b 2728 ± 99.4 3432 ± 96.2 3423 ± 134.9 335 ± 15.7 peach 30 0 2103 ± 40.0 2242 ± 41.6 388 ± 8.6 44 574 ± 231.8 1795 ± 339.9 1897 ± 371.4 452 ± 66.5 58 1097 ± 463.8 1356 ± 244.3 1428 ± 238.3 393 ± 44.1 72 965 ± 156.6 1586 ± 150.7 1678 ± 245.6 631 ± 83.4 86 1542 ± 114.0 1654 ± 175.2 1570 ± 108.0 637 ± 67.2 100 3060 ± 238.9 1734 ± 191.2 965 ± 75.5 1066 ± 65.4 114 4592 ± 153.7 2223 ± 263.9 1764 ± 136.8 1344 ± 268.5 123b 6761 ± 252.8 3432 ± 246.3 2563 ± 146.9 653 ± 62.4 adafb: days after full bloom blast day of fruit harvest data is means ± standard deviation tab. 2: changes in content of organic acids in apricot, plumcot, plum, and peach during fruit maturation. fruits dafba oxalic acid quinic acid malic acid shikimic acid citric acid fumaric acid apricot 30 88 ± 0.9 230 ± 21.4 2945 ± 27.1 57 ± 0.9 236 ± 9.5 1 ± 0.1 44 54 ± 8.7 195 ±17.6 2312 ± 232.5 33 ± 0.4 1451 ± 31.8 1 ± 0.1 58 38 ± 5.0 132 ± 11.5 815 ± 73.9 17 ± 0.7 1953 ± 36.8 4 ± 0.2 65b 28 ± 2.4 232 ± 24.0 702 ± 28.4 8 ± 2.3 1240 ± 93.5 5 ± 0.5 plumcot 30 89 ± 4.1 1051 ± 35.7 3348 ± 112.9 37 ± 2.1 59 ± 9.1 2 ± 0.5 44 77 ± 13.1 1149 ±171.1 4270 ± 260.0 41 ± 1.1 289 ± 22 1 ± 0.1 58 53 ± 4.8 603 ± 25.2 3281 ± 42.6 31 ± 2.4 677 ± 52.5 1 ± 0.1 65 59 ± 9.4 583 ± 182.9 3527 ± 515.6 27 ± 4.8 996 ± 213.1 2 ± 0.3 72b 22 ± 0.8 405 ± 19.5 1987 ± 23.7 12 ± 2.8 548 ± 41 2 ± 0.1 plum 30 224 ± 5.6 2751 ± 87.8 1728 ± 39.2 68 ± 1.9 28 ± 6.8 1 ± 1.2 44 38 ± 7.6 1481 ± 92.1 2059 ± 131.6 52 ± 4.4 30 ± 0.2 1 ± 0.1 58 22 ± 4.8 563 ± 89.3 2332 ± 147.1 35 ± 2.8 31 ± 5.1 1 ± 0.2 65 28 ± 3.3 377 ± 49.8 1926 ± 31.5 23 ± 0.4 32 ± 2.8 1 ± 0.2 79 23 ± 5.2 438 ± 22.0 2040 ± 208.9 26 ± 3.0 42 ± 1.1 3 ± 0.2 86 26 ± 5.6 438 ± 74.5 1984 ± 150.8 27 ± 1.9 40 ± 8.7 3 ± 0.4 91b 224 ± 0.3 2751 ± 42.2 1728 ± 36.5 68 ± 0.4 28 ± 9.8 1 ± 0.1 peach 30 41 ± 0.9 972 ± 22.4 597 ± 15.9 26 ± 0.6 71 ± 0.6 5 ± 0.1 44 46 ± 8.0 1858 ± 223.5 398 ± 74.5 23 ± 4.7 31 ± 6.9 3 ± 0.5 58 49 ± 5.7 1633 ± 195.3 195 ± 87.3 15 ± 3.5 62 ± 13.0 0 ± 0.2 72 26 ± 2.1 2487 ± 142.9 0 16 ± 0.4 133 ± 7.2 0 ± 0.1 86 53 ± 12.0 1734 ± 66.5 216 ± 6.8 18 ± 1.2 220 ± 18.0 1 ± 0.1 100 76 ± 12.0 1163 ± 66.5 272 ± 6.8 18 ± 1.2 308 ± 54.6 2 ± 0.8 114 59 ± 9.9 481 ± 189.4 352 ± 40.8 12 ± 1.6 178 ± 12.7 2 ± 0.1 123b 51 ± 11.2 497 ± 58.8 558 ± 139.5 12 ± 0.5 126 ± 7.4 3 ± 0.5 adafb: days after full bloom blast day of fruit harvest data is means ± standard deviation 28 h. bae, s. yun, j. jun, j. yoon, e. nam, j. kwon references akin, e.b., karabulut, i., topcu, a., 2008: some compositional properties of main malatya apricot (prunus armeniaca l.) varieties. food chem. 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matrix between organic acids and sugars in four prunus cultivars. oxalic acid quinic acid malic acid shikimic acid citric acid fumaric acid sucrose fructose glucose apricot quinic acid 0.340 malic acid 0.940* 0.401 shikimic acid 0.996** 0.319 0.961* citric acid -0.804 -0.790 -0.729 -0.771 fumaric acid -0.849 -0.196 -0.960 -0.889 0.511 sucrose -0.609 0.496 -0.594 -0.643 0.018 0.727 fructose -0.330 0.770 -0.198 -0.341 -0.229 0.323 0.878 glucose -0.750 0.288 -0.761 -0.785 0.212 0.860 0.973* 0.760 sorbitol 0.479 -0.596 0.493 0.522 0.136 -0.663 -0.986* -0.885 -0.936 plumcot quinic acid 0.897* malic acid 0.800 0.769 shikimic acid 0.931* 0.896* 0.914* citric acid -0.535 -0.736 -0.150 -0.462 fumaric acid -0.290 -0.500 -0.531 -0.595 0.353 sucrose -0.293 -0.508 -0.050 -0.098 0.616 -0.222 fructose -0.926* -0.705 -0.727 -0.882* 0.350 0.285 -0.058 glucose -0.943* -0.903* -0.759 -0.956* 0.643 0.542 0.156 0.909* sorbitol 0.349 0.140 0.720 0.530 0.492 -0.355 0.596 -0.510 -0.324 plum quinic acid 0.961** malic acid -0.874* -0.816* shikimic acid 0.888** 0.972** -0.665 citric acid -0.532 -0.624 0.282 -0.700 fumaric acid -0.557 -0.666 0.290 -0.749 0.985** sucrose -0.413 -0.499 0.211 -0.560 0.953** 0.957** fructose -0.969** -0.961** 0.841* -0.904** 0.674 0.693 0.571 glucose -0.972** -0.982** 0.866* -0.927** 0.623 0.649 0.505 0.991** sorbitol -0.279 -0.413 0.127 -0.477 -0.044 0.102 -0.080 0.250 0.322 peach quinic acid -0.556 malic acid 0.204 -0.751* shikimic acid -0.197 0.238 0.342 citric acid 0.678 -0.181 -0.269 -0.315 fumaric acid 0.128 -0.581 0.931* 0.546 -0.255 sucrose 0.450 -0.710 0.306 -0.724* 0.363 0.126 fructose 0.100 -0.722 0.698 -0.279 -0.035 0.573 0.799* glucose -0.462 -0.379 0.682 0.096 -0.575 0.596 0.287 0.765* sorbitol 0.618 -0.470 -0.099 -0.528 0.706 -0.122 0.577 0.159 -0.334 *and ** indicate significant difference at * p ≤ 0.05, ** p ≤ 0.01. plant foods 29 cies. tree gen. genom. 6, 995-1012. ledbetter, c., peterson, s., jenner, j., 2006: modification of sugar 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(eds.), 135-172. springer netherlands. wu, b.h., quilot, b., génard, m., kervella, j., li, s.h., 2005: changes in sugar and organic acid concentrations during fruit maturation in peaches, p. davidiana and hybrids as analyzed by principal component analysis. sci. hort. 103, 429-439. wu, b.h., zhao, j.b., chen, j., xi, h.f., jiang, q., li, s.h., 2012: maternal inheritance of sugars and acids in peach (p. persica (l.) batsch) fruit. euphytica 188, 333-345. wu, f., zhang, d., zhang, h., jiang, g., su, x., qu, h., jiang, y., duan, x., 2011: physiological and biochemical response of harvested plum fruit to oxalic acid during ripening or shelf-life. food res. int. 44, 12991305. yamaki, s., 1984: isolation of vacuoles from immature apple fruit flesh and compartmentation of sugars, organic acids, phenolic compounds and amino acids. plant cell physiol. 25, 151-166. yu, s.-m., 1999: cellular and genetic responses of plants to sugar starvation. plant physiol. 121, 687-693. zhang, x., breksa iii, a.p., mishchuk, d.o., fake, c.e., o’mahony, m.a., slupsky, c.m., 2012: fertilisation and pesticides affect mandarin orange nutrient composition. food chem. 134, 1020-1024. address of the corresponding author: seok kyu yun, fruit research division, national institute of horticultural and herbal science, rural development administration, suwon 440-706, korea e-mail: sky0611@korea.kr journal of applied botany and food quality 86, 154 166 (2013), doi:10.5073/jabfq.2013.086.021 1 university of kiel, institute of crop science and plant breeding – grass and forage science / organic agriculture, kiel, germany 2 institute of ecological chemistry, plant analysis and stored product protection; julius kühn-institute, quedlinburg, germany different approaches to evaluate tannin content and structure of selected plant extracts – review and new aspects m. kardel1*, f. taube1, h. schulz2, w. schütze2, m. gierus1 (received july 2, 2013) * corresponding author summary tannins occur in many field herbs and legumes, providing an immense variability in structure and molecular weight. this leads to complications when measuring tannin content; comparability of different methods is problematic. the present investigations aimed at characterizing four different tannin extracts: quebracho (schinopsis lorentzii), mimosa (acacia mearnsii), tara (caesalpinia spinosa), and gambier (uncaria gambir) and impact of storage conditions. using photometrical methods as well as hplc-esi-ms, fundamental differences could be determined. quebracho, mimosa, and gambier contained 164.3, 108.2, and 169.3 g kg−1 of tannin (calculated as procyanidin c1); tara reached 647.5 g kg−1 (calculated as epigallocatechin gallate). alongside with compounds already described in the literature, several tannin molecules were found that have not been observed before in the analyzed sources. extraction with hot water provided clear advantage over treatment with acetone or methanol; the organic solvents resulted in 9.2 to 15.3 % less tannin isolation. tannin content decreased by a maximum of 1 % per year stored at room temperature compared to 4 °c, but proportions of some compounds slightly shifted. oven drying of material should be avoided. in general, the tannin extracts proved to have very diverse structures, making application of an overall standard method difficult. introduction plant tannins are polyphenols, distributed into condensed and hydrolyzable tannins with an immense structural variability, reaching high degrees of polymerization (see reviews dixon et al., 2005; mueller-harvey, 2006). this heterogeneity causes complications regarding a standardized tannin measurement (mole and waterman, 1987a, b). their role in the plants organism seems to be in the defensive system (ayres et al., 1997; nichols-orians, 1991). nutrient utilization of feeds can be derogated by high levels of tannins and other phenols (martinez et al., 2004; rubanza et al., 2005). some of them can be toxic to ruminal microbes (makkar et al., 1995; clausen et al., 1990) and even the animal itself (hervas et al., 2003). but there are beneficial aspects for herbivores, especially ruminants, in consuming small amounts of tannins, which cause increasing interest in tannins as a feed compound. tannin supplementation in ruminant feeding is aimed at increasing nitrogen use efficiency by using the tannins ability to form complexes with proteins (barry and mcnabb, 1999; cassida et al., 2000). many legumes and field herbs contain tannins, but only in low (jackson et al., 1996) and varying amounts (azuhnwi et al., 2011; theodoridou et al., 2011), causing the need to measure the present concentration with every harvest to ensure the correct tannin amount in the feed. plant tannin extracts on the other hand, are used worldwide for the industrial production of leather (for example mimosa or quebracho; romer et al., 2011) and to a lesser extend in wine making (garciaruiz et al., 2012). an amount of 33.728,465 kg of vegetable tanning extracts, tannins, salts and derivates was imported to the eu in the year 2011, mainly from argentina, with a trade value of nearly 63.5 mio. us dollar (un-comtrade, 2013). these extracts are produced industrially on a large scale. they could therefore maintain a relatively constant quality and thus provide an advantage in ruminant feeding. tannins from different sources can react very diverse regarding protein affinity (mcnabb et al., 1998; bueno et al., 2008). differences in tannin structure can occur even between similar plant species (osborne and mcneill, 2001; hattas et al., 2011) and a higher degree of polymerization is not inevitably an indicator for better protein binding ability (huang et al., 2010; theodoridou et al., 2011). therefore, when considering tannins as possible feed additives, it is essential to describe and characterize the material as comprehensively as possible to gain information about their amount and source to be added in animal feeding. this study aimed to evaluate different methods to characterize the material and possibly detect variability in tannin structures. for that purpose, different preparation methods for hplcms analysis were applied using four selected tannin extracts (quebracho, mimosa, tara, gambier). the aim was to isolate as much of the tannin as possible while keeping matrix effects to a minimum, as they may interfere with the measurement (ciric et al., 2012; ghosh et al., 2012). for comparison, a well established photometrical method was applied. the influence of different storage conditions was also accessed. material and methods description of extracts extracts of four tannin-rich plants were analyzed. being an important part of leather manufacturing, there are several companies providing industrially produced tannin extracts of plants. for the purpose of this study, extracts of schinopsis lorentzii (quebracho), acacia mearnsii (mimosa), caesalpinia spinosa (tara), and uncaria gambir (gambier) were chosen. otto dille® (norderstedt; brand of baeck & co. hamburg, germany) provided quebracho, mimosa, and tara extracts (as well as a small portion of gambier); there was no information given regarding production method or storage. the mainly used gambier extract was kindly provided by christian d. markmann gmbh (hamburg, germany). gambier leaves have been cooked in water, the tannin solution has then been spray dried and stored dry at room temperature. schinopsis lorentzii (griseb.) engler (synonym: schinopsis quebracho-colorado schltdl.) is a tree inhabiting argentina, paraguay, brazil, and bolivia. quebracho tannin extract is already an authorized additive for feedstuffs in the european union (see eu community register of feed additives). the mainly condensed tannin is extracted from the tree’s heartwood. it has been subject of many feedstuff studies, though normally a characterization of the tannin structure is not included in the investigation. in the course of the present analyses, a small piece of natural quebracho heartwood was acquired, to produce own tannin extract in the laboratory. tannin content and structure of selected plant extracts 155 acacia mearnsii de willd. (synonym: acacia mollissima auct. non willd.) is a leguminous tree known as mimosa or black wattle and occurs mainly in australia, brazil, south and east africa. the condensed tannins are extracted from the bark. it is not part of the list of authorized additives for feedingstuffs yet, but there has been some scientific research regarding its qualification for feeding already (for example carulla et al., 2005; grainger et al., 2009). caesalpinia spinosa (molina) kuntze, also a leguminous tree, occurs in south america, especially in peru. tara was chosen as a representative for hydrolyzable tannins, while the other extracts are supposed sources of mainly condensed tannins. there are fewer studies to be found regarding tara in the context of feeding (driedger and hatfield, 1972; pellikaan et al., 2011). the tannin is extracted from the tara pods. uncaria gambir (hunter) roxb. is a shrub from asia (mainly china, india, indonesia, and malaysia). it is used in the leather industry, where gambier condensed tannin is extracted mainly from leaves and small branches. in asia, it has a traditional place as a natural remedy (anggraini et al., 2011). it has been investigated as a biosorbent in waste water (tong et al., 2011), but it is largely unknown when it comes to animal feeding. no actual studies were to be found on this subject, even sole methodical approaches to the tannin structure were quite rare (taniguchi et al., 2007a, b; 2008). photometrical analyses preparations for photometrical measurements of condensed tannins (ct) and total phenols (tp) were based on the methods of terrill et al. (1992) that can be regarded as a standard in tannin measurement. 10 ml of acetone:water (70:30) were added to 100 mg of extract in a test tube. tubes were carefully vortexed and then sonicated in ice water three times for 5 min with 5 min breaks in between. the solution was then centrifuged at 4 °c with 3632 rcf (4800 rpm) for 15 min. the supernatant was collected and stored in dark bottles in the refrigerator for a maximum of two days until analysis. analyses were carried out three times. all solutions were stored at 4 °c in the refrigerator and handled cold. ct were determined modulating the butanol/hcl method (terrill et al., 1992). in tubes placed on ice, 50 μl sample solution and 200 μl of acetone:water (250 μl of acetone:water for blank value) were added to 1.5 ml of butanol : hcl (95:5; hcl concentration 37 %), the solution was then vortexed. after that, 50 μl of ferric : hcl (2 g of (nh4)fe(so4)2, filled up to 100 ml with 2 n hcl) were added, the solution was vortexed again and then placed in a hot water bath (97 100 °c). after exactly 1 hour, the samples were removed and immediately cooled in ice to stop the reaction. the cooled down samples were vortexed again before measuring the extinction at 550 nm with a biochrom libra (s32pc uv, biochrom ltd., cambridge, uk). ct content was calculated, using the following equation: ct = ext ✻ 156.5 ✻ df / w ✻ 100 / dm (1) with following representation: ct: condensed tannin content in % of dry matter ext: extinction measured at 550 nm 156.5: constant, resulting of measurements using leucocyanidin as a standard df: dilution factor (in this case 250 / 50 = 5) w: original sample weight in mg used for disintegration dm: dry matter content of the original sample in % tp were determined by modulating the folin-ciocalteu method (singleton and rossi, 1965). the amount of sample solution had to be reduced to only 5 μl added to corresponding 995 μl deionized water, due to the high concentration of phenols. for blank value, 1 ml deionized water was used. 500 μl of folin solution (1:1 deionized water and folin-ciocalteu’s phenol reagent, purchased from merck kgaa, darmstadt, germany) were added to each sample tube, which was immediately vortexed. after exactly 3 min, 2.5 ml of sodium carbonate solution (88.5 g na2co3 x h2o, filled up to 500 ml with deionized water) were added and the samples were vortexed again. after exactly 1 hour of reaction time, resting dark at room temperature, extinction was measured at 725 nm against a standard curve of tannic acid (purchased from fluka, sigma-aldrich chemie gmbh, münchen, germany). tp content was calculated, using the following equation: tp = conc ✻ 10 / w / dm ✻ 1000 (2) with following representation: conc: concentration in the sample according to standard curve 10: dilution factor to the original amount of sample solution, 50 μl w: original sample weight in mg used for disintegration dm: dry matter content of the original sample in % hplc-ms analyses extraction methods, quebracho wood, charges all analyses were performed twice. the tannin extracts were purified for analysis via hplc-ms by different extraction methods: water, water:methanol (1:1; v/v), and acetone:water:formic acid (70:29.5:0.5; v/v/v). in the first two cases, 5 ml of the respective solvent were added to 100 mg of powder, placed in an ultrasonic bath for 15 min and then held at 90 °c for 2 hours; the solution was shaken every 30 min. the sample was then centrifuged at 7 °c with 3939 rcf (4500 rpm) for 10 min. the supernatant was filtered (0.2 μm pore size) to eliminate impurities and the sample was stored dark at 5 °c. in the acetone treatment, 5 ml of acetone:water:formic acid were added to 100 mg of powder, the flask was thoroughly shaken and placed in an ultrasonic bath for 15 min. the solution was then centrifuged as described above. each supernatant was pipetted into a test tube and acetone was evacuated with a vacuum concentrator (rvc 2-18; martin christ gmbh, osterode am harz, germany) for 50 min at 40 °c. the residual solution was readjusted to 5 ml using water:formic acid (99.9:0.1; v/v) and placed in ultrasonic bath again for 5 min to redissolve some flakes that occurred during concentration. an aliquot was taken and filtered as described above. due to unexpected results of quebracho extract, a small piece of natural quebracho heartwood was acquired and a laboratory extract was produced manually. the wood was rasped by hand with a metal file and then milled in a ball mill twice for 5 min. the wood powder was treated using the different methods with the same amounts of material and chemicals as described above. an aliquot was analyzed directly, with the injection volume increased to 20 μl. the remaining solutions were instantly frozen at -28 °c and then freeze-dried in a gamma 1-16 lsc unit (martin christ gmbh, osterode am harz, germany) for 70 hours. for the water:methanol method there was not enough dried wood extract available to run two analyses, so there is only one measurement to rely on. the freeze-dried extracts were treated according to hot water method before analysis. after evaluation of the different extraction methods, following analyses were carried out using only the hot water method, which has resulted in the highest tannin measurements. aliquots were taken from each industrial tannin extract to examine if storage conditions have an impact on the tannin content. for this purpose, reasonable amounts of extracts were stored at 4 °c in the refrigerator as well as at room temperature with normal daylight on 156 m. kardel, f. taube, h. schulz, w. schütze, m. gierus a board in the laboratory for overall 1.5 years. additionally, subsamples were stored for 5 days at 60 °c in an incubator. device settings for reversed-phase-liquid-chromatography/esi-ms, a 1200 series device with diode array detector (dad) was used (agilent technologies deutschland gmbh, böbingen, germany). the dad was set to detect signals at 280, 320, and 340 nm. an accucore rp-ms (150x3.0 mm 2.6 μm solid core hplc column; thermo scientific, bellefonte pa, usa) with pre-column was used containing silica gel, followed by analyses with an ion-trap mass spectrometer (esquire3000; bruker daltonics gmbh, bremen, germany). the mass spectrometer was equipped with electrospray ionization (esi), running in negative ionization mode. the settings were: nebulizer (nitrogen): 40.0 psi, dry gas: 10.0 l/min, dry temperature: 320 °c; maximum accumulation time: 200.00 ms, scan: 100 to 1800 m/z (averages 5). all parameters were optimized by a standard tuning procedure. the scanning area was set in compromise of covering as many potential oligomers as possible while on the other hand maintaining adequate sensitivity to detect small substance amounts. water:formic acid (99.9:0.1; a) and methanol:formic acid (99.9:0.1; b) were chosen as eluents with a maximum pressure of 450 bar, fitting the following timetable: 0-5 min = 5 % b isocratic, 5-25 min = 5-80 % b linear gradient, 25-27 min = 80-100 % b linear gradient, 27-32 min = 100 % b isocratic, 32-35 min = 100-5 % b linear gradient, 35-42 min = 5 % b isocratic. all chromatographic solvents were of hplc grade. the samples were held in a rack at 10 °c during analysis. the injection volume was 10 μl for quebracho, mimosa, and gambier as well as for the laboratory quebracho wood extract. for tara samples, the injection volume was 3 μl. quantification hplc peaks of external standards were used to quantify the amount of tannins present in the extracts. beforehand, 11 different tannin standards were dissolved at 1 mg in 10 ml water:formic acid (99.9:0.1). quantification data were obtained by hplc-ms, injecting different amounts of the individual standard, resulting in a regression line for each standard. analyzed standards were: (+)catechin, (-)epicatechin, (-)gallocatechin, (-)epigallocatechin, (-)epicatechin gallate, (-)epigallocatechin gallate (all purchased from carl roth gmbh & co. kg, karlsruhe, germany); (-)catechin gallate, (-)gallocatechin gallate, procyanidin b1, b2, and c1 (purchased from sigma-aldrich chemie gmbh, münchen, germany); see tab. 1. the analyzed tannins express their highest intensity in uv/vis spectra at 280 nm; therefore this wavelength was chosen for quantification. for each sample, the uv/vis-spectrum of each peak was compared to the standards available. additionally, molecular masses recorded by the ms were checked. if the respective peak was identified as a tannin substance, it was integrated manually. using the regression equation, the peak area was converted into μg tannins. summing up all tannin peaks and setting the result off with the injected volume and the original sample weight, the total tannin concentration was calculated. for quebracho, mimosa, and gambier, procyanidin c1 was chosen as reference standard, because it fitted their mainly oligomeric condensed tannin structure best. for tara, a sufficient gallic acid derivative could not be acquired. tannic acid would have fit the molecular structure. yet because of its high molecular weight of 1701, it did not represent the majority of compounds found in the tara extract adequately. therefore, epigallocatechin gallate was considered the better reference, because here galloyl groups are the major structural component. statistical evaluation and data processing statistical evaluations were carried out using the statistical software r (r, 2012). simultaneous comparisons of means were performed using multiple contrast tests with corresponding custom contrast matrices. differences between treatments were declared significant at p < 0.05 using the tukey correction for multiple comparisons. hplc data were processed with agilent technologies chemstation software for lc 3d systems, all data from mass spectrometry were processed and displayed with bruker daltonics dataanalysis software version 3.3. molecular masses are described as [m−h]^− (m/z), unless stated otherwise. results and discussion photometrical analyses in accordance with the data communicated by the producer, tara showed a very small amount of measurable ct content, indicating its main tannin was of hydrolyzable nature. in tab. 2, the content of ct and tp is listed for each extract. due to very high tp contents, the solution had to be diluted several times. the resulting high dilution factor caused a slight overestimation. tab. 1: tannin standards with their respective molecular weight (mw), chemical formula, cas number and purity. tannin standard mw 1 chemical formula cas number purity (%) 2 (+)catechin 290.08 c15h14o6 154-23-4 ≥ 98 (-)epicatechin 290.08 c15h14o6 490-46-0 > 99 (-)gallocatechin 306.07 c15h14o7 3371-27-5 > 99 (-)epigallocatechin 306.07 c15h14o7 970-74-1 ≥ 98 (-)catechin gallate 442.09 c22h18o10 130405-40-2 ≥ 98 (-)epicatechin gallate 442.09 c22h18o10 1257-08-5 ≥ 98 (-)gallocatechin gallate 458.08 c22h18o11 4233-96-9 ≥ 98 (-)epigallocatechin gallate 458.08 c22h18o11 989-51-5 ≥ 98 procyanidin b1 578.14 c30h26o12 20315-25-3 ≥ 90 procyanidin b2 578.14 c30h26o12 29106-49-8 ≥ 90 procyanidin c1 866.21 c45h38o18 37064-30-5 ≥ 75 1 monoisotopic mass in dalton (da) 2 as communicated by producer tannin content and structure of selected plant extracts 157 all purchased standards had very similar uv/vis spectra with a maximum at 280 nm. this similarity among tannin uv spectra was already observed before (putman and butler, 1989; redeuil et al., 2009). therefore, these compounds could only be reliably identified through their retention time and ms spectrum. procyanidin b1, b2, and c1 were quite unstable in the ion trap spectrometer. therefore, adjustments were performed before starting the sample analyses to optimize the sensitivity. in order to reduce fragmentation, the voltage settings were optimized by using the automated function of the ms program. tab. 3 lists the typical masses for each standard as they occurred during normal analysis and in fragmentation of the base ion. method development hplc-ms settings negative ionization mode was selected for mass spectrometry, because prior tests revealed a higher signal intensity and more discrete peaks. ciric et al. (2012) suggested, this is caused by hydroxyl groups which are predetermined to lose a proton. formation of complex adducts (soong and barlow, 2005), reduced sensitivity (simirgiotis et al., 2012), and higher fragmentation (venter et al., 2012a) in positive mode were also reported. redeuil et al. (2009) found advantages to the negative mode as well as to the use of esi instead of atmospheric pressure chemical ionization (apci) source. they also suggested to minimize peak tailing by acidifying the methanol eluent with 0.1 % formic acid, which was also found useful in the present experiment. there are some studies preferring positive mode, reportedly due to sensitivity (liu et al., 2010) and for adduct formation with cations (jankovic et al., 2010), respectively. in general, positive mode is used primarily to detect anthocyanins and accounting for the existing literature, especially the ct measurement of quebracho turned out very low (robbins et al., 1991; benchaar and chouinard, 2009). but it seems that in most studies, the ct content of incorporated extracts was not actually measured, but instead taken from other literature or communicated by the providing industrial company (for example el-waziry et al., 2007; vasta et al., 2009). even with analyses within the given study, the numbers range widely, most likely depending on the method (beauchemin et al., 2007; bueno et al., 2008); in the latter study, quebracho extract resulted in similar ct values as the present experiment. several studies suggest a reduced color development of quebracho tannin with butanol/hcl method, because of a resistance of its bonds against acid cleavage (li et al., 2010; hattas and julkunen-tiitto, 2012). measuring quebracho tannin content with a general standard like catechin would therefore cause underestimation (vivas et al., 2004). therefore, it would cause overestimation of other tannin sources, if used as a standard itself. for mimosa, higher amounts of ct than in the present experiment (carulla et al., 2005; grainger et al., 2009) as well as lower values (bueno et al., 2008) where found in previous studies. again, there are experiments relying only on comparatively high data provided by the supplier (max, 2010; beltran-heredia et al., 2011). one measurement of total phenolics of steam-extracted mimosa was found, which was 253 g kg−1 under optimized conditions (duan et al., 2005) and equal to only condensed tannin content in the present study. as expected of a source of hydrolyzable rather than condensed tannins, measurements of tara resulted in quite low numbers. there are not many studies to be found on gambier, but measurements of total phenols are roughly comparable to previous findings (kassim et al., 2011; tong et al., 2011). even though the butanol/hcl assay can be regarded as standard procedure in tannin measurement, it is known that specificity of different tannins to this method can vary (hedqvist et al., 2000; hattas and julkunen-tiitto, 2012). apparently, even the inventors of the butanol/hcl method (swain and hillis, 1959) noticed variations in the results when measuring different tannins. logically, certain tannins are overor underestimated and therefore results not overall comparable. swain and hillis (1959) assumed the degeneration of the sample to be accelerated by light; therefore reaction tubes should be kept dark. no evidence on that could be found by porter et al. (1986), but they observed changes with temperature, age of the preparation, oxygen and water content. the best solution would be to standardize the conditions as much as possible as was tried in the present study. after optimization, the results were stable and repeatable for all standards and extracts. the four selected extracts contain varying amounts of condensed tannins and total phenols. photometrical analysis is a fast and cheap way to determine phenolic content, but the method has restrictions. a major problem consists in the lack of structural information, which makes the choice of a suitable standard impossible. hplc-ms analyses standards all standards showed high detection sensitivity and were distinguishable in their respective retention time. an adequate separation was accomplished (fig. 1). tab. 2: content of condensed tannins and total phenols for the plant extracts in g kg–1. extract condensed tannins 1 total phenols 2 quebracho (schinopsis lorentzii) 122.7 ~1000 3 mimosa (acacia mearnsii) 235.4 ~1000 3 tara (caesalpinia spinosa) 4.6 878.6 gambier (uncaria gambir) 43.1 675.1 1 determined by butanol/hcl method (terrill et al., 1992) 2 determined by folin-ciocalteu method (singleton and rossi, 1965) 3 amounts exceeding 1000 g kg–1, resulting from the extrapolation, were rounded down. fig. 1: hplc separation of all standards detected at 280 nm (regression curves achieve a coefficient of variance of r2 = 0:9995 – 0:9999; 1 gallocatechin, 2 procanidin b1, 3 epigallocatechin, 4 catechin, 5 procyanidin b2, 6 epigallocatechin gallate, 7 procyanidin c1, 8 epicatechin, 9 gallocatechin gallate, 10 epicatechin gallate, 11 catechin gallate. 158 m. kardel, f. taube, h. schulz, w. schütze, m. gierus negative mode to detect flavonols, phenolic acids, and flavan-3-ols (gavrilova et al., 2011). although there are experiments using normal-phase for hplc analyses (white et al., 2010; wallace and giusti, 2010), most research is done by reversed-phase method (e.g. vivas et al., 2004; redeuil et al., 2009; cam and aaby, 2010). in comparison, normalphase was found to give a better separation ordered by degree of polymerization (rigaud et al., 1993). but applying reversed-phase, increased elution time is also needed with higher molecular weight (putman and butler, 1989). complete peak separation could not always be achieved with the tested extracts, because of the high number of tannin peaks. especially with quebracho and mimosa, an elevation in the hplc baseline was observed, as already reported before (karonen et al., 2004; francisco and resurreccion, 2009). peak broadening may also be caused by rotational isomerism, were the units slowly rotate around the interflavonoid bond at room temperature (esatbeyoglu et al., 2010). determination of degree of polymerization was not possible with the available equipment, since it was not possible to assign a peak with several polymer masses to a specific polymer size. it is likely that oligomers were formed during handling due to oxidative polymerization or on-column reactions (putman and butler, 1989) as well as higher polymers may have been changed in their structure during the process (esatbeyoglu et al., 2010). a determination would be possible using matrix-assisted laser desorption/ionization (pasch et al., 2001) or nuclear magnetic resonance spectroscopy (thompson and pizzi, 1995), for example. extraction methods the methods selected for the present study to find the best approach for tannin extraction from the given material were based partly on literature. the extraction process was performed only once, because even though repeated extraction does increase tannin isolation (francisco and resurreccion, 2009; esatbeyoglu et al., 2010), at the same time this dilutes the solution and requires a further step of concentration for adequate analysis (tomas-barberan et al., 2001). extraction experiments showed that temperature, time, and solvent to sample ratio had much more influence on the tannin isolation efficiency than the applied technique (ultrasonic treatment, stirring, shaking, soaking) when extracting with hot water (cam and aaby, 2010). they found that extraction for longer than 120 min did not increase tannin isolation. acetone seems to be quite established in terms of tannin research (singh et al., 2009; gavrilova et al., 2011), regularly as acetone:water (7:3) (karonen et al., 2004; sivakumaran et al., 2006; esatbeyoglu et al., 2010) with different kinds of further purification. some researchers added acids as well (hosseinian et al., 2007; white et al., 2010). the second most regularly used tannin solvent is methanol, either with (xu et al., 2012) or without acid addition (sanz et al., 2010). it is possible that methanol extraction results mainly in monoand dimers while acetone extraction yields higher polymers by trend. acetone extracts can have a lower antioxidant activity (hosseinian et al., 2007). but there are also contrary results, for example sarnoski et al. (2012) hardly found any influence when comparing tannin extraction of different solvents. as known from the domestic use of tea, phenols are resolvable in hot water, and there are studies that established the suitability for laboratory use (omar et al., 2011; simirgiotis et al., 2012). so the use of water for tannin extraction could provide an easy and environmentally friendly way to investigate tannin rich plants. tab. 4 shows the tannin content as quantified after three different extraction methods for the four plant species. hot water extraction provided higher values than extraction with methanol or acetone (p < 0.005). for mimosa and gambier, no differences were observed between extraction with methanol and acetone, while for quebracho and tara acetone extraction yielded in smaller values (p < 0.0001). the adoption of methanol or acetone in the preparation process resulted in noticeable increase of interferences in the uv/vis spectra, rendering tannin identification more difficult. the utilization of acetab. 3: tannin standards with their respective ions as they occurred in ms in positive [m – h] + and negative mode [m – h] − as well as in -ms 2 mode of the main ion; most prominent peaks are bold (for mw see tab. 1). tannin standard x positive mode negative mode -ms 2 (+)catechin 139.3, 291.1 289.0 n/a (-)epicatechin 139.3, 291.1, 313.0, 602.9 288.9, 578.8 137.2, 179.0, 202.9, 205.0, 244.9, 288.9, 1120.6 (-)gallocatechin 139.3, 307.0 305.0, 610.9 125.2, 137.1, 139.2, 161.1, 164.0, 165.1, 1179.1, 204.0, 219.0, 221.0, 261.0, 304.9 (-)epigallocatechin 139.3, 307.1, 329.0, 634.9 305.0, 610.9 n/a (-)catechin gallate 123.4, 273.1, 443.0, 465.0 441.0 n/a (-)epicatechin gallate 123.3, 273.1, 443.0, 465.0 441.0 n/a (-)gallocatechin gallate 139.3, 289.1, 459.0, 481.0 457.0, 571.0 n/a (-)epigallocatechin gallate 139.3, 289.1, 459.0, 481.0 457.0, 570.9 n/a procyanidin b1 127.3, 163.2, 289.1, 579.0, 289.0, 425.0, 577.1, 599.0 125.2, 161.1, 245.0, 287.0, 288.0, 289.0, 290.0, 601.1, 1179.2, 1195.0 299.0, 407.0, 408.0, 425.0, 426.0, 427.0, 451.0, 451.9, 533.1, 559.0, 560.0, 561.0, 577.0, 577.8 procyanidin b2 139.3, 289.1, 291.1, 579.0 289.0, 425.0, 577.1 125.2, 245.0, 287.0, 289.0, 299.0, 407.0, 425.0, 426.0, 451.0, 559.0, 560.0, 577.0 procyanidin c1 123.4, 127.3, 139.3, 163.2, 865.2 286.9, 413.0, 422.9, 450.9, 533.0, 575.1, 576.1, 271.1, 289.1, 291.1, 579.0 577.0, 578.0, 695.0, 713.1, 714.0, 739.0, 847.0, 864.9 x solution of 1 mg in 10 ml; 5 μl injected twice; except for fragmentation, a threshold of 20 abundances was decided n/a not available tannin content and structure of selected plant extracts 159 tone also caused a disturbing peak at 2.5 min, overlapping several others and therefore contributing to an underestimation of the tannin content. ultrasonic treatment was found to be a valuable part of the preparation process, suspending the weighed extract in the provided solution optimally and probably releasing those tannins bond to cellulose (which is often used as a carrier in spray drying). the advantage of ultrasound was also proven by sivakumar et al. (2009). in the process of method development, test samples of quebracho and tara solutions and residue from the acetone extraction were freeze-dried and weighed to see, how much of the sample would actually dissolve. the fraction dissolvable in acetone:water:formic acid was around 85 % for quebracho samples and 74 to 80 % for tara. overall recovery of the weighed sample was 97 to 100 %. the freeze-dried centrifugation residue was sticky, most likely because of sugars (galvez et al., 1997). fig. 2 presents the ms spectra of all investigated extracts obtained by hot water extraction. differences to methanol and acetone extraction as well as tannin structures are discussed below separately for the respective tannin sources. generally, hot water method proved to possess advantages over the use of organic solvents. the industrial extracts were not completely soluble, most likely because of carrier substances like cellulose. quebracho tannin structure contrary to previous reports (vivas et al., 2004), quebracho does not contain robinetinidol, but ent-fisetinidol-4β-ol (negat. mod., m/z 289), which has been identified as monomer quebracho tannin (venter et al., 2012a). there are great variations regarding the average molecular weight of quebracho tannin from 939 (venter et al., 2012a) and 1846 (thompson and pizzi, 1995) to 2800 (makino et al., 2011). with scans up to m/z 1800, only quebracho tannins from monomer to hexamer were detectable in the present study. venter et al. (2012a) suggested that the majority of compounds would be in this range. they calculated a mean degree of polymerization (mdp) of 4.8 in conformation with previous results obtained by time-offlight mass spectrometry and thioacidolysis (vivas et al., 2004). there are only few studies measuring the tannin content of quebracho with hplc, as this normally seems to be a structural or preparative approach. tannin content as reported in the literature can roughly be compared to the current findings in quebracho (makino et al., 2011; venter et al., 2012a). quebracho extract was found to contain previously reported monomers as well as oligomers and polymers (detectable up to hexamer) fig. 2: mass spectra of quebracho, mimosa, tara, and gambier extracts (hot water method) in negative mode ([m – h]–). tab. 4: tannin content of the extracts in g kg−1 by hplc-ms analysis with different extraction methods, quantification based on procyanidin c1 (c1) and epigallocatechin gallate (eg), respectively (standard error = 1.3). plant species hot water water:methanol 1 acetone:water:formic acid 2 quebracho (c1) 164.3 a b 152.4 b b 139.1 c c mimosa (c1) 108.2 a c 101.9 b c 98.2 b d tara (eg) 647.5 a a 633.6 b a 572.9 c a gambier (c1) 169.3 a b 154.3 b b 152.9 b b 1 water:methanol (1:1) 2 acetone:water:formic acid (70:29.5:0.5) a, b different small letters indicate significant differences between methods within one extract (p < 0.005) a, b different capital letters indicate significant differences between extracts within one method (p < 0.005) 160 m. kardel, f. taube, h. schulz, w. schütze, m. gierus in the context of sulfitation products, m/z 643 (sulfited dimer) could be found in the present quebracho spectra; the mass mainly appeared together with m/z 561. the intensity in which the sulfited tetramer mass (m/z 1187) occurred was very low and comparable to the pure tetramer mass in retention times and intensity. while these compounds were sulfited by c-2 sulfitation, m/z 353 also appeared as c-4 sulfited monomer. the double-sulfited byproduct with m/z 435 suggested by venter et al. (2012b) could not be detected in the present study. the laboratory quebracho hot water extract displayed very distinctive peaks in uv/vis and ms (fig. 4), portraying quebracho tannins from monomer up to hexamer with increased intensity compared to the industrial extract. furthermore, the background in the ms seemed to be reduced in the laboratory extract. as expected, m/z 353, 643, 915, and 1187 did not rise remarkably above the normal background. neither in the industrial nor the laboratory quebracho extract, fisetinidol (m/z 273) or sulfited robinetinidol (m/z 369) could be identified. several tannin fragments mentioned by venter and coworkers (venter et al., 2012a, b) could be detected both in industrial as well as laboratory quebracho extract, including m/z 151, which was described as undetectable before. intensities were relatively low, but the industrial extract generally showed higher signals (except for m/z 409), probably indicating increased fragmentation because of the acid treatment. extraction methods quebracho tannin extraction from the industrial quebracho with hot water resulted in higher intensities for oligomers and polymers compared to methanol or acetone treatment in manually integrated ms spectra. this could be indicating hot water treatment as a more gentle method. with the laboratory wood extract, a comparable pat(venter et al., 2012a). even though in comparatively low intensities, all of them were detectable in several peaks in the present analyses; this might indicate them to be fragments of greater polymers. the monomer appeared only in very low intensity, while dimer (m/z 561) and trimer masses (m/z 833) resulted in considerably higher mass peaks. masses of tetramer (m/z 1105) and pentamer (m/z 1377) appeared in intensities equal to the monomer, but further on in the analysis run (see tab. 5). the hexamer mass (m/z 1649) was difficult to find. according to venter et al. (2012a), m/z 1667 and 1668 may indicate the hexamer via its 12c and 13c water adduct, respectively. at close inspection of a magnified ms spectrum, a peak at m/z 1667 is always detectable going along with m/z 1668 showing almost the same intensity. in contrast, a prominent m/z value at 915 appeared from about 2 min up to nearly 17 min in the chromatogram with very high intensity. the uv/vis signal shows the characteristic spectrum of a tannin (see fig. 3). fragmentation of several quebracho tannin masses (see fig. 2) was performed in negative mode with direct injection (without any hplc separation) using a cole parmer 74900 series single syringe pump (novodirect gmbh, kehl/rhein, germany). with quebracho hot water extract ms2 of m/z 561 was performed and the signals were quite stable; 557, 409, and 289 appeared as fragments. after optimization of parameters, ms2 of m/z 833 resulted in the following fragments with decreasing intensity: 681, 561, 529, 409, 289; m/z 915 was also present. with m/z 1105, fragmentation at normal scan range was extremely low in intensity and very unstable. with a scan range of m/z 800 to 1150, at least the masses 833 and 915 appeared as fragments. ms2 of m/z 915 appeared to be very unstable, which could be slightly improved by doubling the accumulation time to 400 ms. fragments included the masses 833, 681, 561, 409, 289 and therefore confirmed this compound to be a derivate of quebracho tannin. this result was verified by analyses of venter et al. (2012b), identifying this compound as sulfited trimer. sulfitation is usually performed by the industry to make quebracho extract soluble in cold water and also to enhance its performance in leather tanning. tab. 5: retention times of uv/vis peaks of quebracho (hot water extraction) at 280 nm, representing over 1 % of the tannin content, and corresponding masses in [m – h] – (most prominent masses are bold). retention time prominent masses [m – h] – % of in min total tannin amount 1.3 161 a 6.99 1.6 561 1.37 1.7 181 x 2.20 2.1 161 a, 201 b, 353 2.70 3.2 775 x 17.58 6.8 289, 643, 915 1.02 10.5 643, 915 1.63 12.0 561, 643, 915, 1187 1.88 12.8 409, 561, 643 3.29 13.3 561, 643, 915, 1187 1.28 13.5 561, 643, 915, 1187 3.06 13.9 561, 643, 915 1.68 14.1 561, 643, 915, 1187 1.51 14.3 561, 643, 833, 915, 1105, 1187 1.40 14.4 561, 643, 833, 915, 1105, 1187 1.47 14.7 643, 833, 915, 1105, 1187 2.66 14.9 561, 833, 915, 1105, 1187 7.50 15.3 561, 643, 833, 915, 1105, 1187 6.10 15.8 289, 561, 833, 915, 1105, 1187 1.13 16.3 833, 1667/8 y 4.88 16.5 289, 561, 833, 915, 1105, 1187, 1667/8 y 1.58 17.2 561, 833, 915, 1105, 1187, 1377 3.31 17.5 833, 915, 1105 3.04 17.7 833, 1105, 1187, 1377, 1649, 1667/8 y 1.22 17.9 833, 915, 1105, 1187, 1649 1.01 18.0 833, 915, 1105, 1377, 1667/8 y 2.03 a m/z described by venter et al. (2012a) and venter et al. (2012b) b m/z described by venter et al. (2012b) x undescribed m/z value y m/z 1667 and 1668 always appeared together in similar intensities fig. 3: ms spectrum of industrial quebracho extract (hot water extraction) at 15 min with corresponding uv spectrum. tannin content and structure of selected plant extracts 161 tern was obtained after freeze-drying. before freeze-drying, acetone treatment resulted in the highest recovery of tannins with 7.12 % tannin in quebracho wood compared to hot water (5.71 %) and methanol extraction (5.16 %). after the extracts were freeze-dried and the powder was redissolved for analysis, different results were obtained. freeze-dried hot water wood extract resulted in 19.11 % tannin, which was remarkably higher than results of methanol (15.37 %) or acetone treatment (14.82 %). mimosa tannin structure fewer studies than for quebracho were found as a useful basis for interpretation of the other extracts. several acacia species were accessed with hplc-ms resulting in tannin contents comparable to the present study (hattas et al., 2011). there are tannin values which are considerably higher for mimosa, but resulting from other methodical approaches (liao and shi, 2005; makino et al., 2011). drewes et al. (1966) indicated the mimosa monomer to be leucorobinetinidin (m/z 289), coupled either with catechin (dimer m/z 577) or gallocatechin (dimer m/z 593). this was confirmed by makino et al. (2011). apart from robinetinidol, fisetinidol units were also found to be attached to catechin (dimer m/z 561) (cronje et al., 1993; duan et al., 2005). the presence of these substances was confirmed by own measurements. the dimeric components with a mass difference of 16 were somehow mirrored at m/z 833, 849, and 865 as well as at m/z 1137, 1153, and 1169, indicating the according trimers and tetramers. to the trimeric group, m/z 881 was added, containing one gallocatechin and two robinetinidol units. the tetrameric group was completed by m/z 1105, 1121, and 1185. compounds like these were described before for mimosa heartwood (viviers et al., 1982; young et al., 1985) and venter et al. (2012c) recently confirmed this grouping structure. the referred pentamers (m/z 1409, 1425, 1441, 1457, 1473, and 1495) occurred sporadically with low intensities in the tested mimosa extract, but as to be seen in fig. 2, there amount adds up noticeably. undecamer masses, found in the formerly mentioned study, did not rise significantly above background level. all fragments mentioned by venter et al. (2012c) could be seen in the present mimosa mass spectra. a monomeric gallocatechin peak at 6 min (m/z 305) was found in low intensity, as well as the monomer unit m/z 289, which may be catechin or robinetinidol as suggested by venter et al. (2012c). a robinetinidol fragment at m/z 287 could also be observed. the fisetinidol monomer as well as the respective fisetinidol-containing oligomers were detected in low intensities, confirming the previous observations of their low proportion. while dimers and trimer were generally the most distinct peaks with high intensities, the signal intensity decreased with higher oligomers. therefore, the general understanding of tannin compounds in mimosa rather being of lower molecular weight (pizzi and stephanou, 1993; makino et al., 2011; venter et al., 2012c), can also be confirmed in the present study. estimates for the average molecular weight are diverse, but with a range from 1284-1507 (roux and evelyn, 1958) and 13431406 (thompson and pizzi, 1995) to 3200 (makino et al., 2011) higher than for quebracho. because robinetinidol has one additional oh group available for hydrogen bonding compared to fisetinidol, venter et al. (2012a) suspected it to be the reason for a better cold water solubility. extraction methods duan et al. (2005) evaluated steaming methods for mimosa extraction and found that too high steaming temperatures or too much water can reduce isolation efficiency. only total phenol content was observed for the optimization, so there is no information regarding influences on single tannins. in the present study, no notable differences in mass intensity could be found for m/z 289, 305, 593, and 865 between the applied extraction methods. however, for m/z 561 and 849 the hot water method resulted in slightly higher intensity. hot water and acetone extraction caused higher signal intensity of m/z 577 and 833 compared to the methanol extraction. tara tannin structure to our knowledge, hplc for estimation of tara tannin content was performed for the first time. some data resulting from other methods state 59.7 and 25.5 % tannin in the extract, respectively (galvez et al., 1997). pellikaan et al. (2011) relied on producer data reading 95.1 % tannin. concluding, the present tara tannin content can be called plausible. reported average molecular masses vary from 593 over 860 to 1736 (deaville et al., 2007; mane et al., 2007), depending on the method. most hydrolyzable tannins contain sugar as a core unit, but tara tannins are based on quinic acid instead (haslam et al., 1962; galvez et al., 1997). quinic acid linked with up to eight galloyl moieties was identified as tara tannin (mane et al., 2007), whereas depsidic and alipathic linkage can appear together (clifford et al., 2007). tara tannins from one to eight galloyl moieties were detected in the present study, while 3-galloylquinic and 4-galloylquinic acids (m/z 647, 799) were the most abundant, which confirms previous studies (clifford et al., 2007). single quinic acid (m/z 191), gallic acid (m/z 169), digallic acid (m/z 321), and glucogallin (m/z 331) were also observed in accordance with previous findings (horler and nursten, 1961; haslam et al., 1962) in several peaks during the first half of the analysis run. the peaks partly corresponded with fig. 4: extracted ion chromatogram (eic) of unsulfited (m/z 833) and sulfited trimer (m/z 915) for industrial (red) and laboratory (blue) quebracho extract. 162 m. kardel, f. taube, h. schulz, w. schütze, m. gierus galloylquinic and 2-galloylquinic acid (m/z 343, 647). with the ongoing analysis run, the smaller masses gradually disappeared, while 3-galloylquinic and 4-galloylquinic acid appeared in noticeably stronger intensity than the smaller tannins. with a retention time of about 15 min, 5-galloylquinic acid (m/z 951) appeared and reached its maximum at 16.5 min. with a delay of 0.5 min each, 6-, 7-, and 8-galloylquinic acid (m/z 1103, 1255, 1407) followed in exponentially decreasing intensity. alongside with these formerly described masses, corresponding well with 3-galloylquinic acid, unknown masses were detected at m/z 1295, 1447, and 1599 in minor intensity. apparently, they continued the mass difference of 152, strongly suggesting the addition of galloyl moieties. together with m/z 1599 the fragment m/z 1751 was observed, barely above the normal background, but consistent with all m/z 1599 peaks. since these masses are not described for tara in the literature, it is assumed that these may be compounds containing two quinic acids in the molecule, because of the obvious mass difference of 192 between 6-galloylquinic acid and the smallest unknown compound. extraction methods the extraction method had diverse effects on tara. while acetone treatment resulted in severe reduction of m/z 169, 191, 343, and 1295 compared to the hot water method; signals of m/z 1103, 1255, and 1407 increased noticeably. methanol had a negative impact on all larger masses and m/z 169, except for m/z 1447, which increased compared to water. gambier tannin structure literature is inconsistent regarding the tannin content in gambier measured by hplc. catechin values from 25 % (suharso et al., 2011) to 42-87 % are reported, depending on the solvent (kassim et al., 2011; tong et al., 2011). no value for total tannin content could be found, but gambier is supposed to contain a high proportion of monomers anyway (pizzi and stephanou, 1993). all numbers are above of what was determined in the present study, which could be attributed to different sources. nonaka and nishioka (1980) found gambiriin a1, a2, a3 (m/z 579), b1, b2, b3, and c (m/z 561) to be the major constituents of gambier tannins. others claimed gambier tannin to consist mainly of catechin (m/z 289), with small amounts of gallocatechin (m/z 305) and epigallocatechin gallate (m/z 457) (hussin and kassim, 2011; kassim et al., 2011). taniguchi et al. (2007b) as well as taniguchi et al. (2008) corrected the assumptions on the isomeric structure of gambiriin tannins, added procyanidin b1 and b3 (both m/z 577) and gambiriin d3 (m/z 561), d4, d5 (m/z 579) to the list of constituents, and proved that these dimers and chalcane-flavan dimers formed out of catechin. evelyn et al. (1960) suggested that 1530 may be the highest molecular weight occurring in gambier tannins, which corresponds with other findings of 502 as average molecular weight (thompson and pizzi, 1995). therefore, gambier can be expected to mainly consist of tannins with smaller molecular weight. all masses described above were found in the analyzed gambier extract, except for epigallocatechin gallate; the mass did not show intensities above normal background. this was also the case for uncariagambiriine (m/z 619) and gambircatechol (m/z 395) identified earlier in aqueous acetone extract of gambier (yoshikado et al., 2009). suharso et al. (2011) also determined 12 % of quercetin (m/z 447), which could only be observed in one peak at 19 min in the present study. corresponding to chalcane-flavan dimers, additional masses appeared at m/z 833, 849, and 851, most likely representing the corresponding trimers. because of the mass difference of 272, in some studies, great effort is put into the prevention of light reaching the sample solution (francisco and resurreccion, 2009). the samples of the method evaluation were stored in the (dark) refrigerator, but otherwise no precautions were taken to protect them from light. after ten days, some sample solutions were remeasured and no degradation could be observed (data not presented). for quebracho, storage conditions did not affect fragments m/z 289 and 409. storage in the oven caused a decrease in intensity for m/z 561, 643, and 833, while for the other observed masses, both oven and room storage had negative effects. but even though the reduction was significant for the room storage, the loss only accounted for less than 1 % of measurable tannins in over a year. with mimosa, the decrease was even smaller. storage conditions had no visible effect on intensities of m/z 289, 305, 593, and 833. higher storage temperature and daylight reduced intensities of the other observed masses in the order of refrigerator < room < oven. storage at room temperature and daylight had no effect on tannin masses of tara, except for a slight reduction of m/z 799, 951, and 1295. five day storage in the oven reduced m/z 343, 647, 799, and the added unit has probably another chalcane structure tetramers were found as demonstrated by m/z 1105 (very low), 1121, 1123, with counterparts to the unknown trimeric masses at m/z 1137 and 1139. a possible pentamer was observed at m/z 1377. contrary to the other extracts, with gambier higher masses showed up quite early in the analysis run. there were four comparatively intensive peaks of almost solely m/z 849 with retention times between 3.5 and 5.8 min dominating the mass spectrum. from 9 min on, monomer and oligomeric masses appeared together, with dimeric and trimeric masses clearly the most abundant. extraction methods relative to hot water, the acetone treatment resulted in higher intensities for m/z 169, 561, 579, and 1123 and methanol treatment slightly increased m/z 305 and 561. both methanol and acetone extraction lead to lower intensities for m/z 289, 833, 849, and 1377, while only methanol reduced m/z 577. influence of storage there are experiments in the literature indicating an impact of light on tannin and phenolic content of aqueous extracts (nordström, 1956; duckstein and stintzing, 2011) as well as experiments indicating that light is not an important factor (porter et al., 1986). results of the present study cannot confirm the imminent loss of phenols either, even during long term exposure (tab. 6). tab. 6: tannin content of the extracts in g kg−1 by hplc-ms analysis as influenced by different storage conditions, quantified based on procyanidin c1 (c1) or epigallocatechin gallate (eg) as calibration standard (standard error = 1.3). plant species refrigerator room 1 oven 2 quebracho (c1) 164.3 a 157.6 b 163.3 a mimosa (c1) 108.2 a 103.7 b 106.5 a tara (eg) 647.5 a 648.8 a 651.1 a gambier (c1) 169.3 a 160.1 b 163.1 b 1 room temperature around 20 °c and normal daylight exposure 2 storage at 60 °c for 5 days a, b different small letters indicate significant differences between storage conditions within one extract (p < 0.05) tannin content and structure of selected plant extracts 163 1295, while m/z 951, 1103, 1255, and 1447 actually increased. the other masses were not affected and overall, the tannin content did not change significantly. for gambier, m/z 169, 289, 305, 833, 849, and 1377 were reduced by room storage, and m/z 577, 579, and 1123 by room storage as well as in the oven. the dimeric mass 561 was found increased in samples stored at room temperature compared to storage in the refrigerator, as were m/z 289, 561, and 849 for storage in the oven. as the only extract observed, gambier indicated a significant (negative) effect of oven storage, but losses remained in the same small margin. based on these findings, stored tannin plant extracts do not necessarily need to be kept in the dark, as long as they are kept dry. also, extracts from different sources might show different sensitivities to storage conditions. losses at room conditions are neglectable, but for analytical purposes storage in a refrigerator is recommended. tannin content of individual extracts may shift between different origins within the same supplier as well as between different suppliers. conclusions 1. the photometrical methods did not give a satisfying picture of the plant materials tested. folin-ciocalteu method most likely overestimated the phenol content of the extracts. with butanol/ hcl method, ct content could be roughly measured, but may get overor underestimated, depending on the standard chosen. apart from condensed tannins there are many other phenolic compounds probably contributing to the extracts properties, which would not be taken into account by sole ct determination. therefore, an analytical solution aiming to include all contributing compounds is recommended when estimating effects of tannin extracts on protein metabolism. results differed remarkably between photometrical and hplcms measurements. since hplc-ms provides much more information on the occurring tannin structure, it is more suitable to evaluate tannin content and to detect whether or not there are reasons for overor underestimation. 2. the tested extracts were very diverse in their tannin structure and even among the condensed tannins there is no “common monomer” on which a standardized quantification could be based. from the results of hplc-ms it is obvious that there cannot be one single standard to accurately fit “tannins” in general. this group of plant compounds is very heterogeneous and photo metrical and spectroscopical reactions differ too much between different sources. therefore, it is very difficult to employ an universally applicable standard. the optimal way would be to isolate the plant individual tannins as an individual standard for every tannin source, but this would require a high amount of sample material and maybe conflict with the comparability of results. nevertheless, even one plant species would provide a large number of different tannins. 3. from the presented investigations it is evident, that there is no need for the use of organic solvents to prepare industrial tannin extracts for hplc-ms analysis. in the literature, there are many different approaches to the extraction process, often involving hazardous chemicals and several methodical steps. hot water treatment isolated more tannin from the tested extracts than preparation with methanol or acetone and also, matrix effects seemed to lessen. it is therefore suggested as a cheap and environ mentally harmless solvent. with hot water, more of the higher oligomers and polymers could be isolated in comparison to treatment with methanol or acetone. this was observed for quebracho and less obvious for mimosa and gambier. tara does not seem to react accordingly, so pro bably hydrolyzable tannins are not influenced in the same way as condensed tannins. 4. storage conditions had only minor impact on the tannin content of plant extracts. a slight reduction under the influence of light was observed, but this did not exceed 1 % in a year. depending on the plant source, some tannins may decrease when exposed to high temperatures (tested for 60 °c). therefore, oven drying of material should be avoided. 5. there are indications of greater quality differences between extracts obtained from different suppliers as well as different extracts from the same supplier. with regards to animal feeding management, a particular chosen extract should be evaluated carefully, but will most 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induction during oligomeric synthesis. j. chem. soc., perkin trans. 1, 2529-2535. address of the corresponding author: m. kardel, university of kiel, institute of crop science and plant breeding − grass and forage science / organic agriculture, hermann-rodewaldstraße 9, d-24118 kiel, germany. e-mail: mkardel@gfo.uni-kiel.de journal of applied botany and food quality 87, 265 273 (2014), doi:10.5073/jabfq.2014.087.037 1humboldt-universität zu berlin, division urban plant ecophysiology, berlin, germany 2 leibniz-institute of vegetable and ornamental crops großbeeren/erfurt e.v., department quality, großbeeren, germany survey of bioactive metabolites in selected cultivars and varieties of lactuca sativa l. under water stress ines eichholz1, nadja förster1, christian ulrichs1, monika schreiner2, susanne huyskens-keil1* (received january 22, 2014) * corresponding author summary plants respond to water stress with a variety of physiological and biochemical changes, but their response vary between species, varieties and cultivars. the present study focused on changes of bio-functional phytochemicals (phenolic compounds, chlorophyll, carotenoids, dietary fiber) in commercial cultivars and old, traditional varieties of lettuce under different water regimes (water-deficit, wellwatered and water-logged). results revealed lettuce varieties and cultivars with a different response behavior to water stress. biomass production under drought conditions was reduced significantly in old varieties (‘struwelpeter’, ‘trianon’), but in contrast, it was only affected tendentiously in the commercial cultivars (‘wiske’, ‘teodore’). carotenoid and chlorophyll contents decreased in both water extremes, while total phenols accumulated under limited water availability, predominantly found in ‘teodore’. dietary fiber content was not influenced by different water regimes in all lettuce cultivars and varieties. water stress reduced biomass production and led to a change of phytochemicals in lettuce, however, old and traditional varieties did not show a different water stress adaptation compared to commercial cultivars. introduction changing climate conditions include altered rainfall patterns with increasing severity of droughts and floods (sheffield and wood, 2008). one of the consequences of climate change is an increase in extreme weather events worldwide, e.g. water stress (drought, waterlogging). plants response to water stress conditions is accompanied by a variety of physiological and biochemical changes at cellular and whole-organism levels, thus making it a complex phenomenon (shao et al., 2009). drought stress disturbs water relation of plant and reduces leaf size, stem extension and root proliferation (farooq et al., 2009; shao et al., 2009). though, the most important effects of drought stress in respect to plant growth are the impairment of cell expansion and inhibition of photosynthesis caused by increased stomatal resistance (reddy et al., 2004). the main damage under waterlogging conditions is related to the oxygen deprivation of soils, which affects nutrient and water uptake of plants (belford et al., 1980; sairam et al., 2008). plants primarily respond with hastened leaf senescence, reduced leaf area, and inhibited growth rates resulting in a decline of biomass (promkhambut et al., 2010; cannell et al., 2008; zhou and lin, 1995). plant compounds in lettuce (lactuca sativa l.) being interesting for human diet are amongst others phenolic substances, carotenoids and dietary fiber (schreiner and huyskens-keil, 2006). water stress is known to influence biomass production as well as the composition and concentration of nutrient-providing and bio-functional plant compounds. many studies confirmed the impact of water stress on plant compounds, as reported for carotenoids (sabale and kale, 2010; loggini et al., 1999; moran et al., 1994), phenolic compounds (sabale and kale, 2010; sánchez-rodríguez and rubio-wilhelmi, 2010; alexieva et al., 2001), and dietary fibers (hemicellulose, cellulose and lignin) (tobisa et al., 1997; okuyama et al., 1995; von wilpert, 1991). while carotenoids and phenolic compounds act as protective plant compounds in plant defense mechanisms (treutter, 2010), hemicellulose, cellulose, pectin and lignin function as essential compounds for plant cell wall metabolism thus, plant growth and textural stability. furthermore, these cell wall polysaccharides as well as lignin are known to be the major components of dietary fibres (waldron et al., 2003). however, the impact of drought and waterlogging on plant metabolism is dependent on severity, time and duration of stress but also on the tolerance level of the respective plant species as well as on the cultivars or varieties (reddy et al., 2004). examining these genetically determined physiological differences may provide breeders with insight into how water stress tolerance of horticultural important species might be improved. moreover, old cultivars and varieties may be a good source of breeding material and maintain the biodiversity of cultivated plants even under changing environmental conditions. an old variety is defined as a historical variety which was never before or is not any more officially licensed (lehmann et al., 2009, lissek-wolf et al., 2009), whereas cultivars emerged from targeted breeding processes (lissek-wolf et al., 2009). recently, a pilot study was conducted in order to establish a collection of old lactuca varieties suitable for commercial utilization in local market gardens as a tool to maintain on-farm cultivation (lehmann et al., 2008). this study identified some attractive, old varieties for a “re-cultivation”. besides a huge range of different commercial cultivars, growers and consumers are gaining a growing interest in regional and traditional species and varieties as it is promoted by the campaign “indigenous voice” of the international association slow food (donati, 2005). the present study focused on changes of phytochemical compounds (phenolic compounds, chlorophyll, carotenoids, and dietary fiber) of different lettuce cultivars and varieties, i.e. commercial and old origins influenced by water stress, i.e. drought stress and waterlogging conditions. results contribute to a better understanding of the dynamics of bio-functional phytochemicals under different water stress regimes associated with climate change. furthermore, this study may result in recommendations of appropriate cultivars and varieties for climate adaptation processes. material and methods chemicals gallic acid (3,4,5-trihydroxybenzoic acid) was used as standard solution for the analysis of total phenols and was purchased from sigma aldrich, germany. folin-ciocalteu’s phenol reagent and other chemicals and reagents were obtained from merck chemicals germany. 266 i. eichholz, n. förster, c. ulrichs, m. schreiner, s. huyskens-keil plant material seedlings of two commercial cultivars (‘wiske rz’, ‘teodore rz’; seeds obtained from ryk zwaan, netherlands) and three old varieties (winter altenburger, struwelpeter, trianon; seeds obtained from vern e.v., germany) (tab. 1) were grown in 6 l pots filled with peat substrate (‘profisubstrat, topfsubstrat mit ton’; gramoflor, germany) for a period of 2 ½ months (sowing on 8th march 2010, harvest on 21st may 2010) under experimental greenhouse conditions. three weeks before harvest, plants were exposed to different water regimes (water-deficit [wd], well-watered [ww], water-logged [wl]). four replications per treatment with five plants per replication were selected randomly. during the time of treatment substrate of the water-logged treatment exhibited a soil moisture level of 61 70 %. soil moisture of well-watered pots amounted up to 49 50 %, while water-deficit pots varied from 18 25 %. used moisture levels have been selected and approved according to previous conducted experiments. the soil moisture level was measured gravimetrically for each pot at 3 days intervals and kept constant manual at the specific water regime level, respectively. within the intervals pots of wl and ww variants were watered by drip irrigation (two times a day for wl, once a day for ww), while wd variant was watered manual. tapwater was used for manual watering and for irrigation of the pots. no further fertilisation was conducted. every week reference plants of each cultivar/variety were weighed in order to calculate the water supply for each water regime. experiments were conducted as randomized block design. biomass after harvest infected or damaged leaves were removed and the plant shoots were weighed in order to determine the total aboveground biomass. a total of 30 g of fresh material was dried at 105 °c for 24 h and dry matter content calculated as the ratio between leaf dry weight and fresh weight. results were expressed in milligram per gram [mg g-1]. chemical analysis for the determination of carotenoids samples of 40 g fresh material and for analysis of total phenols and dietary fiber samples of 200 g fresh material of each treatment and cultivar/variety were frozen immediately after harvest in liquid nitrogen and stored at -20 °c. while for carotenoid assay frozen material was thawed and used, for total phenol and dietary fiber analysis frozen samples were lyophilized for 48 h (alpha 1-4, fa. christ, osterode am harz, germany), ground and stored in a vacuum desiccator. carotenoid and chlorophyll carotenoid (lutein, ß-carotene, lycopene) and chlorophyll assay was determined according to the method of goodwin (1980). frozen samples were thawed, weighed and extracted in a solvent mixture of acetone and hexane (4/5; v/v). samples were centrifuged for 10 min at 3000 rpm (heraeus christian labofuge gl, hanau, germany). supernatants were decanted and filtered (macherey-nagel, mn 260, düren, germany). this process was repeated twice. all lipophilic pigments in the hexane layer were collected and measured spectrophotometrically at 445 nm, 453 nm and 505 nm for lutein, ß-carotene and lycopene, respectively. chlorophyll content was measured at a wavelength of 663 nm for chlorophyll a and 645 nm for chlorophyll b that were subsequently summarized. results were expressed as microgram carotenoid compounds per gram dry matter [μg g-1 dm] and as milligram chlorophyll compounds per gram dry matter [mg g-1 dm]. total phenolic content for the determination of the total phenolic content 0.5 g freezedried sample were extracted in 10 ml of the cooled solvent acetone/ water/acetic acid (0.45/0.5/0.05; v/v/v) according to the method of ehlenfeldt and prior (2001). the samples were diluted in 3 ml solvent, and centrifuged for 10 min at 3000 rpm (heraeus christian tab. 1: description of the studied lettuce cultivars and varieties cultivar/variety description wiske butterhead lettuce (lactuca sativa var. capitata) quick filling short day variety medium-thick leaves and nice bottom high tolerance against tipburn and glassiness (rz seed and services, ryk zwaan) teodore butterhead lettuce (lactuca sativa var. capitata) red winter butterhead grow well in a cold greenhouse larger framed lettuce with medium thick leaves (rz seed and services, ryk zwaan) winter altenburger* butterhead lettuce (lactuca sativa var. capitata) small, rather dense butterhead lettuce, blond-green colour with slightly pink-sprinkled head surface (lissek-wolf et al., 2009) struwelpeter* leaf lettuce (lactuca sativa var. crispa) strong-green leaf lettuce, deep gashed and lobate leaves similar to oak leaf lettuce, large and open heads with compact leaves (lissek-wolf et al., 2009) trianon* romain lettuce (lactuca sativa var. longifolia) green romain lettuce, outer leaves are slightly off and are smaller than the following, which are half-upright; funnel shaped to upright; leaves are slightly bubbly, a few large flat bubbles, distinct and juicy midrib (lissek-wolf et al., 2009) * old, traditional varieties bioactive metabolites in lactuca sativa l. under water stress 267 labofuge gl, hanau, germany). supernatants were decanted and filtered (macherey-nagel, mn 260, düren, germany). this process was repeated twice. extracts were filled up to a 10 ml volume and stored at -20 °c until further analysis. total phenolic content was analyzed spectrophotometrically (sp8300, pye unicam, ok, usa) according to the folin-ciocalteu procedure (slinkard and singleton, 1977). absorbance was measured at a wavelength of 765 nm. gallic acid served as a standard. results were expressed as milligram gallic acid per dry matter [mg gae g-1 dm]. dietary fiber cellulose and lignin was analysed according to the methods of goering and van soest (1972) and aoac (1999). one gram freeze-dried sample was extracted with 100 ml acid detergent fibre (adf) reagent (n-cetyl-n, n,n-trimethyl-ammoniumbromid dissolved with 96 % h2so4) using a fibertec system (m 1020, tecator, sweden). thereafter, the solution was vacuum-filtered, washed with boiled, double distilled water until removal of the acidity and again washed with 90 % acetone. the residue was dried at 105 °c for 24 h, weighed, ash-dried at 500 °c for 24 h and weighed again to calculate adf. the dried adf residue was used for acid detergent lignin (adl) determination. cellulose content was calculated as the difference between adf and adl. the content of lignin and cellulose, respectively, were expressed as milligram compounds per gram dry matter [mg g-1 dm]. with the neutral detergent fiber (ndl) approach (van soest and goering, 1963) one gram of freeze-dried material was cooked in 100 ml of ndl mixture (titriplex iii, di-sodium borate, dodecylhydrogensulfate-na, ethylene-glycol-monoethylester) to determine the hemicellulosic cell wall fraction. afterwards, the solution was vacuum-filtered, washed with demineralized water and with 90 % acetone. the insoluble residue was dried at 105 °c for 24 h, weighed, ash-dried at 500 °c for 24 h and weighed again to calculate ndf. the hemicellulose content was obtained as the difference between ndf and adf and given as milligram per gram dry matter [mg g-1 dm]. contents of cellulose, hemicellulose and lignin were summarized as dietary fiber. results were expressed as milligram per gram dry matter [mg g-1dm]. statistics the statistical evaluation was performed using spss 13.0 (spss inc., chicago, usa 2001). significance of differences was conducted with tukey-b test (p ≤ 0.05) or in case of inhomogeneity of variances with dunnett´s t3 test (p ≤ 0.05). results and discussion biomass struwelpeter was the variety revealing the highest biomass production in all water treatments compared to the other cultivars and varieties (fig. 1). however, both of the two old varieties ‘struwelpeter’ and ‘trianon’ revealed significantly reduced biomass in the waterdeficit treatment compared to well-watered and water-logged plants. under water-deficit conditions biomass was reduced by 39 % for ‘struwelpeter’ and by 30 % for ‘trianon’ compared to the wellwatered regime. this impact of the different water regimes was also observed in the old variety ‘winter altenburger’, however only tendentiously. comparing water-deficit and water-logged treatments, ‘struwelpeter’ and ‘trianon’ revealed significant higher biomass under waterlogging conditions (+ 58 %; + 61 %, respectively), but no distinct differences were found between the well-watered and the water-logged treatment. however, most of the cultivars and varieties tended to a depletion of biomass production and thus, yield in respect to waterlogging. ongoing drought affects the growing of roots and shoots (passioura, 1983). here, closing of stomata inhibits the respiration and photosynthesis rate, and therefore, the formation of biomass (nayyar and gupta, 2006; alexieva et al., 2001) as also found in the two old lettuce varieties ‘struwelpeter’ and ‘trianon’. furthermore, inevitable consequence of drought is oxidative stress (moran et al., 1994) leading to activation of plant defense systems (loggini et al., 1999). oxidative stress causes dna damage, membrane damage, lipid peroxidation etc. (mittler, 2002). in order to protect the plant from oxidative damage substantial amount of energy and substances, e.g. secondary metabolites such as carotenoids and phenolic compounds has to be procured for plant defense and are therefore in lack for biomass production under drought stress. many studies have proven that waterlogging has a reducing impact on crop yields, e.g. in triticum aestivum (keles and öncel, 2002), trifolium pratense and trifolium repens (simova-stoilova et al., 2011), vigna sinensis and zea mays (nemat alla et al., 2002) and others. water logging also leads to an imbalance of nutrient uptake. while several minerals were depressed in their concentration e.g. nitrogen, phosphorus, potassium (belford et al., 1980), others were enhanced up to toxic concentration (shabala, 2010). such conditions facilitate the activation of oxygen, and therefore, oxidative stress. while most of studies examined severe water excess conditions, waterlogging (up to 70 % soil moisture) in this study we suppose that used experimental conditions did not lead to a permanent soil oxygen deprivation. this may implicate moderate stress in respect to waterlogging and did not influence biomass production of lettuce significantly. dry matter content of plants differed depending on lettuce variety and cultivar as well as on the water treatment (fig. 2). the cultivar wiske revealed the lowest dry matter content compared to the other cultivars and varieties. furthermore, under water-deficit regime almost all cultivars and varieties exhibited the highest contents of dry matter compared to the well-watered as well as the water-logged plants. here, except ‘wiske’ all cultivars and varieties revealed higher dry mass contents, thus reduced water contents, highest for ‘teodore’ (+ 44 %), followed by ‘trianon’ (+ 31 %), ‘struwelpeter’ (+ 28 %) and ‘winter altenburger’ (+ 22 %). no significant differences were found between well-watered and water-logged treatfig. 1: biomass [g per plant] of different cultivars and varieties of lactuca sativa spp. grown under different water regimes (water-deficit = 1825 % [wd], well-watered = 50 % [ww], water-logged = 61-70 % [wl]). data represent n ± sd. bars with different letters were significantly different (tukey-b test, p ≤ 0.05). 268 i. eichholz, n. förster, c. ulrichs, m. schreiner, s. huyskens-keil ments. comparing water-deficit and water-logged treatments all cultivars and varieties, except ‘wiske’, revealed lower dry matter contents and thus also a reduced water content under waterlogging conditions. changing sink-source relations due to drought seems to have affected the dry matter content of leaves. increasing content of dry matter is explained by higher accumulation of assimilates (marschner, 1995) that are necessary for maintenance of plant metabolism or rather activation of stress response system (roitsch, 1999). in this case, leaves are not only source but also sink organs (roitsch, 1999). thus, assumingly carbohydrates might have been accumulated under drought stress conditions, e.g. sucrose and sugar alcohols (sircelj et al., 2005), being also osmoprotectants that protect plants from extreme oxidative stress (rontein et al., 2002). waterlogging conditions reduce dry matter production (kumutha et al., 2009) and increase fresh weight of plants (nemat alla et al., 2002). however, severe water excess leads to a reduced relative water content in leaves and subsequent wilting due to inhibition of respiration and loss of atp synthesis in the roots (sairam et al., 2008). an interference of respiratory processes and breakdown of carbohydrates are common symptoms of waterlogging stress (subbaiah and sachs, 2003; das et al., 2000; zeng et al., 1999) which assumingly influenced the dry matter contents of lettuce leaves. carotenoid and chlorophyll the analysis of carotenoids revealed contents of 345 666 μg g-1 dm for ß-carotene, 370 686 μg g-1 dm for lutein and 32 59 μg g-1 dm for lycopene (tab. 2). chlorophyll content ranged from 4.0 7.6 mg g-1 dm (fig. 3). results supports findings by mou (2005), nicolle et al. (2004) and thomas and gausman (1977). ‘struwelpeter’ and ‘teodore’ grown under optimum well-watered conditions revealed the highest amounts of carotenoids (lutein, ßcarotene, lycopene) and chlorophyll compared to other cultivars and varieties. however, only for ‘teodore’ significant differences between the different water regimes were obtained. here, the waterdeficit treatment exhibited lower contents of lutein (41 %), ß-carotene (43 %), lycopene (38 %), and chlorophyll (47 %) compared to the well-watered treatment. carotenoid and chlorophyll contents did not differ between water-deficit and water-logged plants. nonetheless, ‘teodore’ exhibit significant lower contents of ß-carotene under waterlogging conditions compared to the well-watered treatment (27 %). drought conditions caused a change in the biosynthesis of plant pigments especially of chlorophylls and carotenoids (farooq et al., 2008; anjum et al., 2003). it is also known that photosynthesis of higher plants is reduced with decreasing leaf water potential (reddy et al., 2004; zlatev and yordanov, 2004; van holsteijn et al., 1977) and associated with low contents of chlorophyll pigments (mafakheri et al., 2010; arunyanark et al., 2008). the latter finding was confirmed by results of the present study where plants of the cultivar teodore grown under water-deficit conditions revealed significantly lower chlorophyll a and b concentrations compared to the other water regimes (data not shown). decompensation of chlorophyll is also induced by free radicals due to oxidative stress (reddy fig. 2: dry matter content [%] of different cultivars and varieties of lactuca sativa spp. grown under different water regimes (water-deficit = 18-25 % [wd], well-watered = 50 % [ww], water-logged = 61 70 % [wl]). data represent n ± sd. bars with different letters were significantly different (tukey-b test, p ≤ 0.05). tab. 2: lutein, ß-carotene, lycopene [μg g-1 dm] of different cultivars and varieties of lactuca sativa spp. grown under different water regimes (water-deficit = 18-25 % [wd], well-watered = 50 % [ww], water-logged = 61-70 % [wl]). data represent n ± sd. different letters in columns and rows indicate significant differences (dunett’s t3 test, p ≤ 0.05). bioactive metabolites in lactuca sativa l. under water stress 269 et al., 2004; fu and huang, 2001). although carotenoids protect chlorophyll from oxidative damage as reported by siefermanharms (1987) and foyer et al. (1994), they are highly susceptible to oxidative destruction (jaleel et al., 2009). drought stress leads to a degradation or re-organisation of carotenoid compounds, e.g. lutein, ß-carotene and neoxanthin (loggini et al., 1999; moran et al., 1994), in particular, if stress is based on the production of free radicals in the thylakoids (jaleel et al., 2009; reddy et al., 2004). moreover, reddy et al. (2004) and loggini et al. (1999) reported an increase in the pool of de-epoxidized xanthophyll-cycle components (i.e. zeaxanthin and antheraxanthin). a complex relationship between xanthophyll cycle-dependent energy quenching and formation of active oxygen species (aos) exists in photosynthetic systems of plants under drought stress (reddy et al., 2004). waterlogging has been reported to reduce photosynthetic rate (zhou and lin, 1995; yuehua, 1987), chlorophyll (kumutha et al., 2009; zhou and lin, 1995) and also carotenoid contents in plants (peng, 2012; sabale and kale, 2010; gu et al., 2009). according to das et al. (2000) carotenoids appeared less vulnerable to water excess than chlorophyll pigments. however, with the prolonging of waterlogging conditions their reduction also seems to be unavoidable, which is consistent with studies by gu et al. (2009). similar to drought, oxidative stress in photosynthetic tissue might result in the decomposition of carotenoids, which was found in the present study on the commercial lettuce cultivar teodore in the water-logged treatment. total phenol compounds the content of total phenols in lactuca sativa spp. ranged from 5.0 14.6 mg gae g-1 dm (fig. 4), which is consistent with findings by chu et al. (2002) and vinson et al. (1998). cultivars and varie-. cultivars and varieties and water treatments exhibited significant differences. the highest content of phenolic compounds was determined for the cultivar teodore. moreover, ‘teodore’ grown under the different water regimes revealed contents of total phenol which almost doubled that of the other cultivars and varieties. thus, ‘teodore’ was the only cultivar with a considerable response towards the different water regimes. plants grown under water-deficit conditions revealed higher contents of phenolic compounds (+ 11 %) than plants of the well-watered as well as of the water-logged regime. no significant differences were found between well-watered and water-logged plants. in response to stress, plants activate the defense system, like the enzymatic and non-enzymatic antioxidants. besides numerous enzymes (superoxid dismuthase, peroxidase etc.), phenolic compounds are strong antioxidants that help plants to survive stress conditions (mittler, 2002). antioxidant compounds such as phenolic compounds are able to prevent oxidative burst of plant cells and thus protect plants from damage of proteins and lipids, dna and rna structures (apel and hirt, 2004). in the present study, the commercial cultivar teodore revealed higher contents of total phenols under water-deficit conditions. the promotion of the synthesis of phenolic compounds due to drought was already documented in numerous studies (sánchez-rodríguez and rubio-wilhelmi, 2010; alexieva et al., 2001; esteban et al., 2001; ayaz et al., 2000), also for lettuce (oh et al., 2010; oh et al., 2009). waterlogging conditions did not lead to an accelerated synthesis of phenolic compounds in the lettuce cultivars and varieties studied. this might have been related to the intensity of stress applied and/ or also to the composition of phenolic substances analysed. in general, water excess is known to induce the accumulation of phenolic compounds acting as strong antioxidants (nacif de abreu and mazzafera, 2005; nemat alla et al., 2002). sabale and kale (2010) reported for coriandrum sativum a decline of flavonoids being caused by water excess, while contents of anthocyanins were enhanced. one of the main phenolic compounds in lettuce is quercetin (garcia-macias et al., 2007; dupont et al., 2000), which was probably not affected by waterlogging, as also reported for the medical herb hypericum brasiliense by nacif de abreu and mazzafera (2005). as consequence of waterlogging conditions high levels of lipid oxidation and the accumulation of antioxidative enzymes, i.e. superoxide dismutase (sod), catalase (cat) as well as peroxidases (pod) were reported (kumutha et al., 2009; zhang et al., 2007; zhou and lin, 1995). dietary fiber dietary fiber content of lettuce ranged between 130 and 170 mg g-1 dm (fig. 5). it consisted of mainly cellulose, which constitutes the highest fraction with nearly 80 % compared to hemicellulose and lignin with 10 %, respectively. no significant differences were found for lignin. for hemicellulose tendentially higher contents were determined in drought stressed plants compared to water-logged fig. 3: chlorophyll content [mg g-1 dm] of different cultivars and varieties of lactuca sativa spp. grown under different water regimes (waterdeficit = 18-25 % [wd], well-watered = 50 % [ww], water-logged = 61-70 % [wl]). data represent n ± sd. bars with different letters were significantly different (dunett’s t3 test, p ≤ 0.05). fig. 4: total phenol content [mg gae g-1 dm] of different cultivars and varieties of lactuca sativa spp. grown under different water regimes (water-deficit = 18-25 % [wd], well-watered = 50 % [ww], waterlogged = 61-70 % [wl]). data represent n ± sd. bars with different letters were significantly different (tukey-b test, p ≤ 0.05). 270 i. eichholz, n. förster, c. ulrichs, m. schreiner, s. huyskens-keil plants in the varieties and cultivars winter altenburger, struwelpeter and trianon (tab. 3). cellulose exhibited contrary results. here, plants grown under water-deficient conditions tended to lower contents than under water-logged treatment, specifically for ‘wiske’ and ‘trianon’. nevertheless, comparing the different water regimes no differences were found in the dietary fiber content (as sum of hemicellulose, cellulose, and lignin) (fig. 5). however, cultivars/ varieties differed in their content of dietary fibers. at optimal water conditions, ‘teodore’ revealed the lowest dietary fiber content compared to ‘wiske’ and ‘trianon’. in general, drought stress leads to cell wall strengthening, which is accompanied by several complex processes, such as linking phenolic components to the cell wall matrix or lignification processes (moura et al., 2010). however, lignification can differ in plant organs under drought stress conditions. while in roots, an enhanced lignin content were reported (fan et al., 2006), the lignin content in young leaves of zea mays subjected to water stress was lower than in the well-watered plants (vincent et al., 2005). sugar components as precursors of cell wall components are also necessary for the biomass production and the accumulation of defense compounds in plants. under drought stress a decreased photosynthesis may inhibit the production of sugar components in sufficient quantity. however, an increase of carbohydrates (mainly monoand disaccharides) during drought stress was reported (sircelj et al., 2005). this accumulation is achieved for example by inhibiting the cellulose synthase, an enzyme that is involved in the synthesis of cellulose (narciso et al., 2010; zhu et al., 2010). therefore, a lower proportion of cellulose under drought stress has been expected. in contrast, hemicellulose contents increased under drought stress. due to their water-storing properties within the cell wall, hemicellulose contributes to drought tolerance in plants. further, a change in hemicellulose content constitutes a direct control of the rate of expansion of plant cells under drought (iraki et al., 1989). under waterlogging conditions, photosynthetic rate and sugar accumulation in leaves decreases. an influence on dietary fiber contents, also being a product of sugar molecules, could be expected. however, most of the studies investigated changes of lignin, hemicellulose and cellulose in the subterranean plant organ root, which is directly affected by water excess (tobisa et al., 1997; galamay et al., 1992). tobisa et al. (1997) found lower contents of lignin and hemicellulose in roots of the legume aeschynomene americana by waterlogging. further, a higher content of lignin and cellulose in the plant top was determined. however, above biomass of a. americana consists of shoots, leaves and fruits with seed, while lettuce mostly forms only leaves until harvest. dietary fiber content of leaves is not affected by waterlogging in contrast to the results obtained in the present study on lettuce. conclusion depending on the sensitivity to water stress of the respective variety and cultivar, drought led to reduced biomass production, which is one key factor for producers. water stress partly affected bioactive compounds negatively, e.g. carotenoids, possibly being a decision factor for consumers. however, in the present study on different fig. 5: dietary fiber [%] of different cultivars and varieties of lactuca sativa spp. grown under different water regimes (water-deficit = 1825 % [wd], well-watered = 50 % [ww], water-logged = 61-70 % [wl]). data represent n ± sd. bars with different letters were significantly different (dunett’s t3 test, p ≤ 0.05). tab. 3: hemicellulose, cellulose, lignin [mg g-1 dm] of different cultivars and varieties of lactuca sativa spp. grown under different water regimes (waterdeficit = 18-25 % [wd], well-watered = 50 % [ww], water-logged = 61-70 % [wl]). data represent n ± sd. column and rows with different letters were significantly different (dunett’s t3 test, p ≤ 0.05). bioactive metabolites in lactuca sativa l. under water stress 271 lettuce cultivars and varieties drought stress increased the content of phenolic compounds, but did not influence dietary fiber contents, both are known to have biofunctional properties. at waterlogging conditions neither phenolic compounds nor dietary fiber were influenced in their contents. lettuce cultivars and varieties revealed different response behaviour to water stress (drought and waterlogging). in respect to biomass production, the old variety struwelpeter exhibited the highest biomass production compared to other cultivars and varieties when growing under drought as well as waterlogging conditions, although this variety also showed the greatest loss in biomass production under drought conditions. furthermore, ‘struwelpeter’ comprised the highest carotenoid content, even under both stress conditions. on the other hand, the modern cultivar teodore exhibited the highest content of total phenolic compounds, mainly under drought stress and here, also the least loss in biomass production, possibly indicating a valuable plant defense mechanism to water deficit conditions. the presented results lead to the conclusion that old and traditional varieties such as ‘struwelpeter’ did not show a different adaptation mechanism to water stress conditions in comparison to commercial cultivars, i.e. ‘teodore’. nevertheless, old varieties exhibit a high potential to evaluate adaptation processes in a changing 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zlatev, z.s., yordanov, i.t., 2004: effects of soil drought on photosynthesis and chlorophyll fluorescence in bean plants. bulg. j. plant physiol. 30, 3-18. address of the authors: dr. ines eichholz, dr. nadja förster, prof. dr. dr. christian ulrichs, dr. susanne huyskens-keil (corresponding author), division urban plant ecophysiology, humboldt-universität zu berlin, lentzeallee 55/57, 14195 berlin, germany. e-mail: susanne.huyskens@agrar.hu-berlin.de prof. dr. monika schreiner, department quality, leibniz-institute of vegetable and ornamental crops großbeeren/erfurt e.v., theodor-echtermeyerweg 1, 14979 großbeeren, germany. vorgabe neu journal of applied botany and food quality 80, 82 87 (2006) institute for food technology, plant foodstuff technology, hohenheim university, stuttgart, germany chemical quality parameters and anthocyanin pattern of red-fleshed weirouge apples e. sadilova, f.c. stintzing *, r. carle (received february 23, 2006) summary red-fleshed ‘weirouge’ apples were investigated with respect to their chemical quality parameters including anthocyanins and colour. anthocyanin concentrations were considerably higher than previously reported for common red-peeled apples. due to its high malic acid content, the cultivar ‘weirouge’ was characterised by a high colour brilliance. among the anthocyanins, cyanidin-3-maloyl-galactoside and 5-carboxy-pyrano-cyanidin-hexoside not previously found in apples were tentatively identified by hplc-ms3. highest anthocyanin and total phenolics contents were found in the peel corresponding with the respective antioxidant capacities as determined using the frap and teac assays, respectively. introduction not only in europe, apples are one of the most frequently consumed fruits, second most valued after oranges and bananas with respect to health-promoting plant secondary substances (chun et al., 2005; vinson et al., 2001). while it is well established that an increased consumption of fruit and vegetables improves overall well-being, it is both taste and colour that triggers consumers’ purchase decision. whereas apples with red peel are quite common, red-fleshed apples have still not received much attention. it is because of their remarkable anthocyanin content that red-fleshed apples such as ‘purpurroter cousin’, ‘pink pearl’ and also quite recently ‘weirouge’ are receiving increasing attention (buchter-weisbrodt, 2003). in addition to their attractive red colour, pigment degradation and oxidation was reported to be low and assumed to be due to their high acidity (buchter-weisbrodt, 2003). moreover, there is a rapidly increasing interest for potential crops for colouring food naturally (stintzing and carle, 2004). hence, coloured fruits and vegetables are attractive for both fresh fruit consumption and juice processing. ‘weirouge’ apples represent the most intensely coloured german cultivar and was bred in weihenstephan (germany) primarily for processing purposes and officially registered 1997. however, little is known about its chemical composition. therefore, the present study aimed to thoroughly investigate colour properties, anthocyanin content and further chemical quality parameters of the peel, the flesh and the whole edible portion of ‘weirouge’ apples. the anthocyanin profile was also characterised in detail by hplc-dad and mass spectrometric (ms) analyses. finally, the antioxidant potential was assessed in aqueous extracts by the frap and teac assays. materials and methods plant material and extraction red-fleshed ‘weirouge’ apples from 2005 (stoppel, kressbronn, germany) were washed, the peel dried and the core and stem removed to obtain the edible part of the fruit. at least five apples were separated into flesh and peel, while further fruits were not peeled. after slicing peels, flesh or unpeeled fruits into segments, the samples were immediately frozen in liquid nitrogen and comminuted to obtain a fine powder which was frozen at 80°c until analyses. exactly five grams of each sample were extracted with aqueous acetone (ph 1 water acidified with trifluoroacetic acid / acetone, 30/70, v/v) at a ratio of 1 part solid sample and 2 parts extraction medium. after 60 min extraction under continuous stirring, the solution was passed through a buchner funnel with a filtering paper (schleicher-schuell, dassel, germany). the so-obtained filtrate was concentrated in vacuo at 28°c until dry and re-dissolved in 5 ml of purified water. the resulting extracts were used for the determination of the total anthocyanin content, the browning index and for objective colour assessment (see below) using a uv-vis spectrophotometer equipped with uv-winlab and colour software (wincol, perkin-elmer, überlingen, germany). for the assessment of antioxidant capacities, total phenolics content and chemical quality parameters (see below), the samples were extracted with purified water and passed through a filtering paper. each extraction was repeated twice and the resulting extracts were analysed in duplicate. for the assignment of unknown anthocyanins, the scales from red onions (allium cepa l.) and the peel from blue-coloured japanese plums (prunus salicina lindl.) obtained from a local market were homogenised and extracted with aqueous acetone as described above. aqueous extracts were used for coinjection experiments and mass spectrometric analyses, respectively. antioxidant capacity the clear filtrates used for the teac (trolox equivalent antioxidant capacity; van den berg et al., 1999) and frap assays (ferric reducing capacity antioxidant power; benzie and strain, 1996), respectively. for calibration purposes, the water soluble vitamin e analogue trolox (trolox-equivalents) as well as l-ascorbic acid (vitamin c-equivalents) according to kim and lee (2004) were used. total phenolics content total phenolics content were assessed by the folin-ciocalteu method (singleton et al., 1999) and the results were expressed either as gallic acid or l-ascorbic acid equivalents. chemical quality parameters sucrose, glucose, fructose, sorbitol, ascorbic acid, citric acid and malic acid were quantified using enzymatic test kits (r-biopharm, darmstadt, germany). proline and the formol value were determined according to ifu-methods no. 49 (1983) and 30 (1984), respectively. total titratable acids were assessed by titration with 0.25 m naoh until ph 8.1 was reached and expressed as malic acid. anthocyanin content, browning index, and objective colour all samples were analysed in duplicate by uv-vis spectrophotometry (perkin elmer, überlingen, germany). based on absorption measurements covering the range from 380 to 780 nm, objective colour measurements (ciel*a*b*) were carried out in mcilvaine buffer solutions at ph 3.5. chromaticity c* [c* = (a*2+b*2)1/2] und hue angle h° [hº = arctan(b*/a*)] were calculated from a*and b*-values at d65 and an observer angle of 10°. the browning index was assessed according to giusti and wrolstad (2005). total anthocyanin content determination was based on a ph-differential method and expressed as cyanidin-3-glucoside equivalents (giusti and wrolstad, 2005) according to the following formula: c[mg/l]=a*mw*df/εm*d, with a = absorption value, mw = molecular weight of cyanidin 3-glucoside [448 g/mol], df = dilution factor, εm = molar extinction coefficient of cyanidin 3-glucoside at ph 1 [29600 l/mol*cm], and d = pathlength of the cuvette [1 cm]. the contents of individual anthocyanins were calculated considering the relative peak area ratios of the chromatogram at 520 nm after their identification by hplc-dad-ms3. anthocyanin pigment assessment by hplc-dad and lc-msn using a merck lachrom elite hplc system (merck-hitachi, darmstadt, germany) equipped with an autosampler l-2200, a pump l-2130, a diode array detector l-2450, and a jetstream column oven, anthocyanins were separated on an analytical c18 sunfire column (250x4.6 mm, 5µm; waters, wexford, ireland) equipped with a c18 pre-column (4 x 3.0 mm i.d., phenomenex, torrance, ca, usa) at a constant temperature of 25°c and a flow rate of 1 ml/min. eluent a was 5% aqueous formic acid, 100% acetonitrile was used as b. starting isocratically with 100% a for 5 min, linear gradients were followed to 10% b in 20 min, 13% at 40 min, 20% at 44 min, 25% at 50 min, and finally 100% b at 55 min before re-equilibration to initial conditions. monitoring was performed at 520 nm. using the same method, mass spectrometric analyses were carried out on an agilent series 1100 hplc system (agilent, waldbronn, germany) interfaced with a bruker model esquire 3000+ ion trap mass spectro-meter (bruker, bremen, germany) operating in the positive ionisation mode. results and discussion chemical quality parameters chemical quality parameters of the whole edible part, the peel and flesh fractions of red-fleshed ‘weirouge’ apples are compiled in tab. 1. literature values for apples (scherz and senser, 2000) are also listed for comparison. glucose, fructose and sorbitol contents are quite low. in contrast, sucrose contents are higher than expected from the literature pointing to a lower invertase activity in ‘weirouge’ apples. also ascorbic acid and proline fell behind the reference data. since the formol value of 14 exceeded the aijn value for apple juice ranging from 3 to 10, (aijn, 1996), proline can be considered a minor free amino acid in ‘weirouge’ as reported for other apple varieties (scherz and senser, 2000). the high malic acid content is worth noting because literature values were exceeded by 200 %. on the other hand, citric acid concentrations were in the mean range. exhibiting a sugar acid ratio of 7:1, ‘weirouge’ is considered a fairly acidic apple variety (tab. 1). colour properties a browning index of only 4-5% pointed to a low degree of oxidation products in ‘weirouge’. at ph 3.5, the typical ph for a broad range of foods, the l*-, c*-, and h°-values of the peel and flesh only slightly differed. the colour purity c* und the tonality h° were highest in flesh, followed by unpeeled apples and peel fractions, respectively (tab. 2). since colour stability of ‘weirouge’ has previously been reported to be high (buchter-weisbrodt, 2003), it should be examined whether this was due to the high acidity, to the high pigment concentrations or rather to a specific pigment pattern. anthocyanin content and pigment pattern for this purpose, the total anthocyanin content was determined separately in flesh, peel and unpeeled apple samples (tab. 2) on a weight basis. the anthocyanin content of the peel was 2.5 times higher than in the flesh. it needs to be considered that besides the core amounting to 14.7 %, the edible part consisted of 10.1 % and 75.2 % peel and flesh, respectively. total anthocyanin yield of the edible fractions reached 10.8 mg/100 g fresh weight thus being in the same range as reported previously for red-fleshed ‘scugog’ apples (malus pumila var. niedzwetzkyana) with 10.0 mg/100 g (mazza and velioglu, 1992). in contrast, anthocyanin concentration was found to be considerably lower in red-peeled apples ranging from 0.2 to 0.8 mg/100 g (van der sluis et al., 2001). the pigment contents in ‘weirouge’ apples were comparable to those of strawberries (15-35 mg/100 g), red currants (20-60 mg/100 g), red onions (7-21 mg/100 g), and plums (2-25 mg/ 100 g) (giusti and wrolstad, 2005). tab. 1: chemical quality parameters of ‘weirouge’ apples [mg/100 g edible portion] a chemical quality parameter ‘weirouge’ apple apples b glucose 527 ± 16 1 4 0 0 2 3 5 0 fructose 4255 ± 60 4 8 0 0 6 4 0 0 sucrose 3755 ± 33 5 4 0 2 7 8 0 sorbitol 324 ± 21 5 1 0 5 8 0 ascorbic acid 2.2 ± 0.00 3-25 citric acid 16.8 ± 0.00 9-30 malic acid 1271 ± 2.0 2 7 0 7 9 0 total titratable acids 1190 ± 0.0 c [as malic acid] proline 1.02 ± 0.00 1 0 formol value 14.0 ± 0.34 c [ml 0.1mol naoh/100 g] sugar-acid-ratiod 7 . 1 c a mean ± standard deviation b according to scherz and senser (2000) c no data available d (glucose + fructose + sucrose) / (citric acid + malic acid) quality characteristics of weirouge apples 8 3 tab. 2: colour characteristics and anthocyanin contents of ‘weirouge’ apples a parameter apple with peel apple flesh apple peel anthocyanin content 10.8 ± 0.02 8.1 ± 0.19 20.6 ± 0.57 [mg/100 g] browning index [%] 4.3 ± 0.23 4.9 ± 0.85 4.6 ± 0.35 l* (ph 3.5) 74.9 ± 0.52 74.0 ± 0.57 76.1 ± 1.31 c* (ph 3.5) 41.8 ± 1.06 44.4 ± 0.42 39.4 ± 2.89 h° (ph 3.5) 16.5 ± 0.30 18.7 ± 0.66 15.9 ± 0.70 a mean ± standard deviation according to timberlake and bridle (1971), cyanidin-3-galactoside is considered the major compound of all apple varieties, followed by cyanidin-3-glucoside, cyanidin-3-arabinoside and cyanidin-3-xyloside. in addition, the authors gave preliminary evidence of acylated structures the nature of which remained obscure. except for the acylated anthocyanins, those data were later confirmed by mazza and velioglu (1992) in red-fleshed and by vrhosek et al. (2004) in red-peeled apples, respectively. the predominant anthocyanin of red-fleshed ‘scugog’ (malus pumila) apples was cyanidin-3-galactoside (39%), followed by cyanidin-3-glucoside (27%), cyanidin-3-arabinoside (23%), and cyanidin-3-xyloside (11%), respectively (mazza and velioglu, 1992). in contrast, the relative amounts of cyanidin-3glucoside and cyanidin-3-galactoside were found to differ significantly in red-fleshed ‘weirouge’ apples as shown in tab. 3, being similar to those from m. domestica varieties. it is thus suspected that fruits from m. pumila should be easily distinguishable from those of m. domestica by the ratio of cyanidin-3-galactoside and cyanidin-3-glucoside, respectively. in agreement with earlier reports on both red-fleshed and red-peeled fruits (mazza and velioglu, 1992; timberlake and bridle, 1971; vrhosek et al., 2004; wu and prior 2005a), the dominating anthocyanin in ‘weirouge’ apples was cyanidin-3-galactoside (1) accompanied by cyanidin-3-glucoside (2), cyanidin-3-arabinoside (4), cyanidin-7-arabinoside (8), cyanidin-3-xyloside (9) and peonidin-3galactoside (5) which were assigned by comparison of specific mass spectrometric, uv-vis and retention time data from the literature (wu and prior, 2005a,b) and an own data bank (tab. 3, fig. 1). additionally, cyanidin-3-maloyl-galactoside (6) was found in apple for the first time. furthermore, a cyanidin structure with a pentose moiety (10) and a cyanidin structure (3) with an identical mass as (6) were detected. due to its short retention time and specific fragmentation pattern, (3) was assigned to a cyanidin-diglycosidic structure. while the presence of cyanidin (11) in fruits has earlier been considered an artefact of sample clean-up, anthocyanidins have also been reported as genuine compounds from beans only recently (macz-pop et al., 2006). it is worth mentioning that the predominance of malic acid is also reflected by the acylation pattern. a similar relationship was also found to apply to blackberry being rich in oxalic acid (scherz and senser, 2000a) and exhibiting an oxalyl-anthocyanin at the same time (stintzing et al., 2002). for closer assignment of the maloyl-derivative, extracts from blue-peeled plums (wu and prior, 2005a) previously reported to contain cyanidin-3-maloylglucoside were analysed by hplc-dad-ms3. cyanidin-3-maloyl-glucoside was detected at a retention time of 32.6 min with an absorption maximum at 520 nm. its specific mass spectral data matched exactly those of (6). due to its higher polarity, (6) was tentatively assigned as cyanidin-3-maloylgalactoside. from a biosynthetic standpoint, the positive quantitative correlation between the glycosides and the respective maloyl-derivative as reported previously (wu and prior, 2005a) is plausible. interestingly, a further compound (7) exhibiting identical spectroscopic properties as reported for 5-carboxy-pyranocyanidin-glucoside in red onion scales (fossen and andersen, 2003) was detected. however, closer assignment and clarification if this was the glucoside or the galactoside derivative was impossible since freshly prepared red onion extracts were devoid of this compound (data not shown). such pyranoanthocyanidin-type structures have first been found in wines (e.g., fulcrand et al., 1996; bakker et al., 1997), later in fruit juices (hillebrand et al., 2004; rein et al., 2005; schwarz et al., 2004) and only very recently as genuine fruit compounds (andersen et al., 2005; fossen and andersen, 2003). total phenolics content and antioxidant capacity the antioxidant capacities of aqueous extracts from red-fleshed apples with peel, flesh and peel fractions, respectively, are shown in tab. 4. following the same trend, absolute values for frap and teac varied considerably. this can be ascribed to different assay conditions and diverse reaction mechanisms as previously reported (prior and cao, 1999; halvorsen et al., 2002; prior et al., 2005; rechner et al., 1997). the peel exhibited the highest values and the lowest activities were found in the flesh. these findings reflect an accumulation of uvabsorbing colourless and coloured phenolics in the peel substantiating their photoprotective effect (edreva, 2005). considering the suggestion tab. 3: hplc-dad and mass spectrometric data for anthocyanins from ‘weirouge’ apples anthocyanina rt λvis-max a440 / avis-max m/z ms 2m/z ms3m/z anthocyanin contents [mg/kg] [min] [nm] [%] [m]+ [m] + [m]+ unpeeled apple apple flesh apple peel 1 cyd-3-gal 25.6 5 1 6 3 4 4 4 9 2 8 7 2 8 7 92.57 67.65 1 7 1 . 9 1 2 cyd-3-glc 27.2 5 1 5 3 7 4 4 9 2 8 7 2 8 7 0.65 0.57 1.32 3 cyd-pent-rhac 28.6 5 1 7 3 7 565, 419 2 8 7 -b 0.58 0.66 0.99 4 cyd-3-ara 28.8 5 1 6 3 3 4 1 9 2 8 7 2 8 7 2.41 1.53 5.48 5 peo-3-gal 29.7 _b _b 4 6 3 3 0 1 2 8 6 0.15 0.18 0.36 6 cyd-3-maloyl-galc 31.3 5 1 4 4 8 565, 449 2 8 7 -b 0.43 0.44 0.92 7 5-carboxy32.6 5 0 3 4 5 5 1 7 3 5 5 355, 327 0.88 0.89 1.83 pyrano-cyd-hexc 8 cyd-7-ara 34.2 5 1 6 3 6 4 1 9 2 8 7 287, 217 1.83 1.28 6.13 9 cyd-3-xyl 35.7 5 1 7 3 3 4 1 9 2 8 7 2 8 7 8.04 7.31 13.79 1 0 cyd-pent 37.3 5 1 4 3 4 4 1 9 2 8 7 2 8 7 0.28 0.22 1.70 1 1 cyanidin 41.4 5 2 2 3 6 2 8 7 2 8 7 -b 0.67 0.23 1.48 a cyd: cyanidin; peo: peonidin; gal: galactoside; glc: glucoside; ara: arabinoside; rha: rhamnosid; xyl: xyloside; hex: hexosid; pent: pentosid b not detectable c tentatively identified 8 4 e. sadilova, f.c. stintzing, r. carle of kim and lee (2004) and chun et al. (2005) the values were expressed as vitamin c-equivalents. the latter always exhibited lower values than their coresponding trolox-equivalents. halvorsen et al. (2001) reported frap-values for ‘golden delicious’, ‘granny smith’ and ‘gala’ varieties amounting to 0.15 mmol/100 g, 0.51 mmol/100 g, 0.22 mmol/100 g in the edible fruit part. in the present study a value of 0.24 mmol the trolox-equivalent per 100 g edible portion was found for ‘weirouge’. these differences may be ascribed to the specific phenolic profiles and their particular concentration governed by season, climatic conditions and intraspecific variations (van der sluis et al., 2001). the phenolic compound profile in different apple varieties has been frequently investigated, showing a high variability both from a quantitative and a qualitative perspective (keller et al., 2001; müller and treutter, 2001; ritter and dietrich, 1996; treutter, 2001; tsao et al., 2003; van der sluis et al., 2001; vrhosek et al., 2004). benzoicand hydroxycinnamic acid derivatives, dihydrochalcones, flavonols, flavan-3-ols, catechin and proanthocyanidin derivatives were detected. the phenolic profile of ‘weirouge’ apples has already been well documented with chlorogenic acid (38.02 mg/l), phloretin-2xylosylglucoside (10.48 mg/l), p-coumaroylglucoside (7.29 mg/l) and phloridzin (7.21 mg/l) constituting the major compounds in the juice (müller and treutter, 2001). hence, in the present study individual phenolics were not assessed. instead, total phenolics were quantified by the folin-ciocalteu method, considering the contribution of ascorbic acid. values both expressed as gallic acid and vitamin c-equivalents, respectively, are shown in tab. 4. the highest antioxidant potential was found in the peel paralleling total phenolics and anthocyanin contents. tsao et al. (2003) reported comparable gallic acid equivalents of 2012 mg/kg for ‘red delicious’ peel. according to chun et al. (2005), the total phenolics content amounted to 118 mg gallic acid per 100 g surmounting those of ‘weirouge’ with only 54 mg/ 100 g. by analogy, values were higher for the vitamin c-equivalent antioxidant capacity of 205 mg/100 g compared to 89 mg/100 g in ‘weirouge’ corroborating the well-known positive correlation of phenolics content and antioxidant potential (chun et al., 2005; rechner et al., 1997; vinson et al., 1998, 2001). conclusion the present study reports on chemical quality parameters of red-fleshed ‘weirouge’ apples including their anthocyanin profile. these data together with previously published phenolic compound patterns o + oh oh oh ho o-gal fig. 1: chromatogram obtained from unpeeled red fleshed ‘weirouge’ apples (peak assignment is given in tab. 3). tab. 4: antioxidant capacity [mg/kg] of edible part fractions from ‘weirouge’ apples (n=6)a parameter unpeeled apple apple flesh apple peel teac [trolox-equivalent] 1463 ± 179 911 ± 83 4879 ± 522 frap [trolox-equivalent] 599 ± 63 374 ± 82 1772 ± 237 teac [vitamin c-equivalent] 888 ± 109 563 ± 51 3004 ± 317 frap [vitamin c-equivalent] 403 ± 39 243 ± 56 1166 ± 170 total phenolics [gallic acid-equivalent] 538 ± 42 b 379 ± 22 1684 ± 184 total phenolics [vitamin c-equivalent] 784 ± 59 b 540 ± 28 2385 ± 248 a mean value ± standard deviation b corrected by the ascorbic acid content of 22 mg quality characteristics of weirouge apples 8 5 allow to classify red-fleshed apples in comparison to common nonanthocyanic fleshed fruits. anthocyanins hitherto unknown for apple cultivars could be identified by mass spectrometric detection. the ratio of cyanidin-3-galactoside and cyanidin-3-glucoside is suggested as a tool to differentiate malus pumila from m. domestica varieties. the total anthocyanin contents in ‘weirouge’ was more than ten times higher compared to other apples. the low browning index and high colour brilliance and stability cannot be ascribed to a particular pigment pattern but rather to the high acidity of the fruit stabilising anthocyanin colour (stintzing et al., 2002b). in summary, both from a nutritional and technological point of view, red-fleshed apples deserve due attention. acknowledgements the authors are grateful to mrs erika müssig for her excellent technical assistance and peter stoppel (kressbronn, germany) for supplying red-fleshed ‘weirouge’ apples. one of the authors (e.s.) gratefully acknowledges a scholarship in the framework of the academic and scientific program by the ministry of science, research and art, badenwürttemberg / germany. references a.i.j.n. code of practice for the evaluation of fruit and vegetable juices, 1996: association of the industry of juices and nectars from fruits and vegetables of the european economic community, brussels, permanent update. andersen, o.m., fossen, t., torskangerpoll, k., fossen, a., hauge, u., 2004: anthocyanin from strawberry (fragaria ananassa) with the novel aglycone, 5-carboxypyranopelargonidin. phytochemistry 65, 405-410. bakker, j., bridle, p., honda, t., kuwasno, h., saito, n., terahara, n., timberlake, c.f., 1997: identification of an anthocyanin occurring in some red wines. phytochemistry 44, 1375-1382. benzie, i.f.f., strain, j.j., 1996: the ferric reducing ability of plasma (frap) as a measure of „antioxidant power“: the frap assay. anal. biochem. 239, 70-76. buchter-weisbrodt, h., 2003: roter apfelsaft ohne fruchtbeimischung. rot im trend: sichtbar gesund. flüss. obst 70, 78-79. chun, o.k., kim, d.-o., smith, n., schroeder, d., kang, h.g., lee, c.y., 2005: daily consumption of phenolics and total antioxidant capacity from fruit and vegetables in the american diet. j. sci. food agric. 85, 1715-1724. edreva, a., 2005: the importance of non-photosynthetic pigments and cinnamic acid derivatives in photoprotection. agric. ecosys. environ. 106, 135-146. fossen, t., andersen, o.m., 2003: anthocyanins from red onion, allium cepa, with novel aglycone. phytochemistry 62, 1217-1220. fulcrand, h., cameira d o s sa n to s, p.-j., sa r n i-ma n c h a d o, p., cheynier, v., favre-bonvin, j., 1996: structure of new anthocyaninderived wine pigments. j. chem. soc. perk. trans. 1, 735-738. giusti, m.m., wrolstad, r.e., 2005: characterization and measurement of anthocyanins by uv-visible spectroscopy. in: wrolstad, r.e., acree, t.e., decker, e.a., penner, m.h., reid, d.s., schwartz, s.j., shoemaker, c.f., smith, d., sporns, p. (eds.), handbook of food analytical chemistry. pigments, colorants, flavors, texture, and bioactive food components,19-31. wiley & sons, hoboken, nj/usa. halvorsen, b.l., holte, k., myhrstad, m.c.w., barikmo, i., hvattum, e., remberg, s.f., wold, a.-b., haffner, k., baugerod, h., andersen, l.f., moskaug, j.o., jacobs jr., d.r., blomhoff, r., 2002: a systematic screening of total antioxidants in dietary plants. j. nutr. 132, 461-471. hillebrand, s., schwarz, m., winterhalter, p., 2004: characterization of anthocyanins and pyranoanthocyanins from blood orange [citrus sinensis (l.) osbeck] juice. j. agric. food chem. 52, 7331-7338. international federation of fruit juice producers. 1983: ifu method 49. zug, switzerland. international federation of fruit juice producers. 1984: ifu method 30. zug, switzerland. keller, p., streker, p., arnold, g., schieber, a., carle, r., 2001: bestimmung phenolischer verbindungen in tafelund mostäpfeln mittels hplc. flüss. obst 68, 480-483. ki m , d.-o., lee , c.y., 2004: comprehensive study on vitamin c equivalent antioxidant capacity (vceac) of various polyphenolics in scavenging a free radical and its structural relationship. crit. rev. food sci. nutr. 44, 253-273. macz-pop, g.a., rivas-gonzalo, j.c., pérez-alonso, j.j., gonzálezpa r a m á s, a.m., 2006: natural occurrence of free anthocyanin aglycones in beans (phaseolus vulgaris l.). food chem. 94, 448456. mazza, g., velioglu, y.s., 1992: anthocyanins and other phenolic compounds in fruits of red-flesh apples. food chem. 43, 113-117. müller, c., treutter, d. 2001: phenolische verbindungen in apfelsaft, apfelwein und apfelessig. mitt. klosterneuburg 51, 138-147. prior, r.l., cao, g., 1999: in vivo antioxidant capacity: comparison of different analytical methods. free rad. biol. med. 27, 1173-1181. prior, r.l., wu, x., schaich, k., 2005: standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. j. agric. food. chem 53, 4290-4302. rechner a., patz c.-d., dietrich h., 1997: beitrag zur bewertung der antioxidativen kapazität verschiedener getränke. flüss. obst 64, 6 2 6 5 . re i n, m.j., o l l i l a i n e n , v., v a h e r m o, m., y l i-kau h a l u o m a, j., heinonen, m., 2005: identification of novel pyranoanthocyanins in berry juices. eur. food res. technol. 220, 239-244. ritter, g., dietrich, h., 1996: der einfluss moderner verfahrenstechniken auf den gehalt wichtiger pflanzenphenole im apfelsaft. flüss. obst 63, 256-263. scherz, h., senser, f., 2000: souci fachmann kraut – food composition and nutrition tables. medpharm scientific publishers, stuttgart. schwarz, m., wray, v., winterhalter, p., 2004: isolation and identification of novel pyranoanthocyanins from black carrot (daucus carota l.) juice. j. agric. food chem. 52, 5095-5101. singleton, v.l., orthofer, r., lamuela-raventos, r.m., 1999: analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent. methods enzymol. 299, 152178. stintzing, f.c., carle, r., 2004: functional properties of anthocyanins and betalains in plants, food, and in human nutrition. trends food sci. technol. 15, 19-38. stintzing, f.c., stintzing, a.s., carle, r., wrolstad, r.e., 2002a: a novel zwitterionic anthocyanin from evergreen blackberry (rubus laciniatus willd.). j. agric. food chem. 50, 396-399. stintzing, f.c., stintzing, a.s., carle, r., frei, b., wrolstad, r.e., 2002b: color and antioxidant properties of cyanidin-based anthocyanin pigments. j. agric. food chem. 50, 6172-6181. timberlake, c.f., bridle, p., 1971: the anthocyanins of apples and pears: the occurrence of acyl derivatives. j. sci. food agric. 22, 5 0 9 5 1 3 . treutter, d., 2001: biosynthesis of phenolic compounds and its regulation in apple. plant growth reg. 34, 71-89. tsao, r., yang, r., young, ch. j., zhu, h., 2003: polyphenolic profiles in eight apple cultivars using high-performance liquid chromatography (hplc). j. agric. food chem. 51, 6347-6353. van den berg, r., haenen, g.r.m.m., van den berg, h., bast, a., 1999: applicability of an improved trolox equivalent antioxidant capacity (teac) assay for evaluation of antioxidant capacity mea8 6 e. sadilova, f.c. stintzing, r. carle surements of mixtures. food chem. 66, 511-517. van der sluis, a.a., dekker, m., jager, a., jongen, wim m.f., 2001: activity and concentration of polyphenolic antioxidants in apple: effect of cultivar, harvest year, and storage conditions. j. agric. food chem. 49, 3606-3613. vinson, j.a., hao, y., su, x and zubik, l., 1998: phenol antioxidant quantity and quality in foods: vegetables. j. agric. food chem. 46, 3630-3634. vinson, j.a., su, x., zubik, l., bose, p., 2001: phenol antioxidant quantity and quality in foods: fruits. j. agric. food chem. 49, 5315-5321. vrhovsek, u., rigo, a., tonon, d., mattivi, f., 2004: quantitation of polyphenols in different apple varieties. j. agric. food chem. 52, 6532-6538. wu, x., prior, r.l., 2005a: systematic identification and characterization of anthocyanins by hplc-esi-ms/ms in common foods in the united states: fruits and berries. j. agric. food chem. 53, 25892599. wu, x., prior, r.l., 2005b: identification and characterization of anthocyanins by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry in common foods in the united states: vegetables, nuts and grains. j. agric. food chem. 53, 3101-3113. address of the authors: hohenheim university, institute for food technology, plant foodstuff technology, august-von-hartmann-straße 3, d-70599 stuttga rt, germany *corresponding author: dr. f.c. stintzing, e-mail: stintzin@uni-hohenheim.de quality characteristics of weirouge apples 8 7 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb 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<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> >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice journal of applied botany and food quality 87, 196 205 (2014), doi:10.5073/jabfq.2014.087.028 agricultural and biological research division, national research center, dokki, cairo, egypt use of sesame and sorghum crops to verify the remediation of contaminated sewaged soils h. abouziena*, e. hoballah, f. abd el zaher, a. turky, s. el-ashery, m. saber, a.m. zaghloul (received november 2, 2013) * corresponding author summary at the time being, treated sewage effluent is repeatedly used in farming, particularly in newly reclaimed sandy soils, as it fortify their content of nutrients and organic matter as time goes on, despite there are significant concerns about the long-term accumulation of ptes in the soil ecosystem. pot experiment was conducted to verify the effectiveness of bioremediation of two sewaged soils, highly and marginally contaminated, with either canola (brassica napus) or indian mustard (brassica juncea) plants in the absence and presence of am inoculation, by growing sesame (sesamum indicum) and sorghum (sorghum bicolor) plants. results indicated that the vegetative parameters of both test crops did not show any sign of adverse symptoms when grown in either bioremediated sewaged soils. phytoremediation with either canola or indian mustard obviously reduced cu and zn contents in both sesame and sorghum plants and completely removed ni from soil. both sesame and sorghum plants grown in both bioremediated sewaged soils contained lower ptes, however, at varying degrees. zinc equivalent value in marginally and highly decontaminated soils after harvesting either sorghum or sesame obviously decreased compared to control soil. after phytoremediation, the dehydrogenase activity increased in all soils. generally, the efficiency of phytoremediation with canola far exceeded that with indian mustard, despite both were effective tools. abbreviation: am: arbiscular mycorrhiza, ptes: potential toxic elements, cfu: colony forming unit introduction reuse of sewage effluent in farming is well recognized as a workable environmental prospect. abouzeina et al. (2013) stated that cairo is now served by six large wastewater treatment plants which produce significant quantities of sewage effluent. the preferred option is to use it in farming, particularly on newly reclaimed desert land which is inherently deficient in organic matter, nutrients and trace elements. sewage effluent has been used in farming, since distant past, however at a restricted magnitude. early in 1531 sewage farming started in germany and thereafter progressively distributed in other parts in the world. he added that in egypt the first sewage farm was installed in 1930 at el-gabal al-asfer near cairo and sewage farms gradually disseminated later on in other regions. at the time being, sewage effluent represents one of the substantial natural water resources in egypt that should be well managed, rather than discarded. sewage effluent, however, is contaminated with enormous amounts of contaminants, for the most part ptes that mount up in sewaged soils and could effortlessly invade the food chain, with subsequent decisive adverse environmental and public health impacts. indubitably safe and sustainable reuse of sewage effluent in farming necessitates the removal of soil contaminants. abouziena et al. (2013) stated that salt et al. (1995) highlighted the concepts of phytoremediation using certain hyperaccumulator plants capable to remove ptes from soil. metal hyperaccumulators are commonly defined as plant species that can approximately accumulate 100-fold higher metal levels than common plant species (brooks, 1998). the advantages of bioremediation represent maintaining characters of the soil ecosystem after phytoremediation, visually being unobtrusive and offers the possibility the bio-recovery of ptes (sun et al., 2006; fawzi, 2008). recently it was noticed that coupling phytoremediation with other techniques such as microbial, physical and/or chemical treatment could be viable in many cases. throughout the world, over 400 species of terrestrial plants have been identified as hyperaccumulators of various ptes (baker et al., 2000; sun et al., 2006). the possibility to clean heavy metal contaminated soils with hyperaccumulator plants has shown great potential. at present, phytoremediation of metals may be approaching commercialization (midarkodi, 2007). two of the most recently studied species used in phytoremediation applications as hyper-accumulators plants are canola (marchiol et al., 2004; podar et al., 2004; ashour et al., 2006) and indian mustard (banuelos et al., 2005; hamlina and barkera, 2006; chauhan and rai, 2009; panwar et al., 2011; abouziena et al., 2012). in a pot experiment, purakayastha et al. (2008) grew five different species of indian mustard as possible accumulators of ptes in soils sewaged for more than two decades. in general, they found that the highest concentration and uptake of ptes was observed in shoots compared to roots or seeds of the different species of indian mustard. correlation analysis showed negative relation between total uptake of ptes and their available forms as well as the total concentrations of ptes. in addition, root length emerged as a powerful parameter indicating the uptake of ptes by indian mustard compared to other root parameters. microbial remediation of sewaged soils is a well welcomed technique as microorganisms perform an urgent mode in the devastation of ptes in agricultural ecosystems, and several researchers had been attempted to develop means of using microorganisms and microbial enzymes for decontaminate sewaged soils. however, as far as ptes are not biologically degradable and persist in indefinitely soil ecosystem, glick (1995) stated that once they accumulated in the soil, they inversely affect the microbial compositions, particularly plant growth promoting rhizobacteria in the rhizosphere, and their metabolic activities. he added that the elevated concentration of ptes in soils and their uptake adversely affect plant growth, symbiosis and consequently crop yields through disrupting photosynthesis, inactivating respiration, protein synthesis and/or carbohydrate metabolism. phytoremediation efficiency is often limited by many factors such as bioavailable forms of contaminants in soil, plant-root development, and the level of tolerance of the plant to each particular contaminant (pilon-smits, 2005). given the fact that there were a large number of variables in the current experiment, it is not possible to distill this information and come up with one ideal set of conditions for all ptes contaminant phytoremediation experiments. these variables include plant type, soil composition, endogenous bacteria, concentration and range of the contaminants, temperature range, and type and physiological state of bio-additives. this complexity notwithstanding, careful examination of the data suggests that certain con phytoremedatin of contaminated soil with sesame and sorghum 197 siderations might facilitate the phytoremediation of soil contaminants. ramasamy et al. (2000) concluded that the application of chelate ion exchange resin, cation − exchange resin, ferrous sulfate and silica gel, lime, gypsum, and naturally occurring clay minerals such as bentonite, zeolite and green sand had been found to immobilize ptes in contaminated soil ecosystems. in this context, the present work had been planned, to investigate the growth of sesame and sorghum in two sewaged soils, highly and marginally contaminated, previously phytoremediated with either canola or indian mustard in the absence and presence of am inoculation. materials and methods soil sampling and soil preparation in this work, two surface sewaged soil samples collected from aburawash farm previously bioremediated by both canola and indian mustard were used. some chemical and physical properties of the original soils and the kinetic study applied were previously documented in soad el-ashery et al. (2011) as shown in tab. 1. the concentrations of ptes, the distribution of these pollutants after remediation and zn equivalent as a parameter used for contamination or remediation were documented in saber et al. (2012). the two groups of soils i.e. after indian mustard and canola were separately remixed well and repacked to be used in this work. experimental in a completely randomized plot design, greenhouse experiments were conducted with three replicates and each replicate consists of five pots for growing sesame (sesamum indicum l) and sorghum (sorghum bicolor l. moench) as ptes sensitive (sumathi and umagowrieb, 2012) and tolerant (marchiol et al., 2007) crops, respectively, in the two remediated sewaged soils after either indian mustard or canola during 2011 winter season and summer 2012. seeds from each tested plants were sown on 6th june 2011 at the rate of 8 seeds in plastic pots (30 x 30 cm) filled with 4 kg of either the two contaminated sewaged soils. fifty percent of the cultivated pots were sown with seeds inoculated with 20 ml of a suspension of mycorrhyzal conidia (106 cfu) while the rest were left without mycorrhyzal inoculation. it is worthy to mention that control pots represented by the prior bioremediated soils without neither cultivation nor inoculation with am to understand rizosphere effects on the rate of ptes uptake under such conditions. all treatments including control did not receive any mineral fertilizers to eliminate reactions could be take place between ptes and applied fertilizers. the moisture content of all pots was initially adjusted to 50 % of the soil water holding capacity and kept at the same level during the experimental periods. after three weeks, the seedlings were thinned to 5 and 3 plants per pot. the plants from each pot were harvested at maturity seed production stage (harvest). soil samples were collected at initially and maturity stages, analyzed for their ptes concentrations and dehydrogenase activity. plant and its parts dry weight as well as yield and its components characteristics were determined before their content of ptes was estimated. methods microbiological methods mycorrhyzal (am) conidia: am fungi spores were extracted from soil by wet sieving and sucrose density gradient centrifugation (syliva et al., 1993). dehydrogenase activity: dehydrogenase activity in the sewaged soils was estimated initially and at harvest according the method given by skujins (1973) as μl h2 g-1 soil 24-h . chemical methods potential toxic elements (ptes): potential toxic elements in soil and plant parts were extracted by the ammonium bicarbonate dpta as described by cottenie et al. (1982) and analyzed using atomic absorption according to a.o.a.c (1984). soil quality criteria: zn equivalent model was numerically expressed for the levels of ptes toxicity in used soils according to (chumbley, 1971) using the following equation: zn concentration*1+ cu concentration *2+ni concentration*8. statistical analysis: all data were processed by microsoft excel (microsoft, 2000) and the regression of linear and nonlinear and other statistical analyses were conducted using the programs of sas release 6.12 (sas institute, 1996). results plant dry weight and seed yield/plant: dry matter biomass and seed yield of sorghum and sesame plants grown in highly or marginally contaminated sewaged soils previously bioremediated by either canola or indian mustard hyperaccumulators in the presence or absent of am inoculation are given in tab. 2 and 3. results indicated higher dry weight of roots, straw, panicle or capsules and total plant dry weight for both sorghum and sesame plants grown in contaminated sewaged soils previously bioremediated with canola plants compared to those grown in contaminated sewaged soil previously bioremediated with indian mustard either in the presence or absence of am inoculation. sorghum plants performance: the dry matter of sorghum plants and its parts were higher when grown in the highly than in the marginally contaminated sewaged soil (tab. 2). the role of am inoculation in increasing the dry matter of sorghum plants was more noticeable in the soil previously bioremediated with indian mustard than that in the soil previously bioremediated with canola plants both in marginally or highly contaminated sewaged soil. tab. 1: total pte contents in abou-rawash farm soils * (ppm oven dry basis) soil depth sewage farming landscape cd cu fe mn pb zn ni zn equivalent (cm) started since 0-30 sandy soil was 35.65 596.0 45.19 57.7 400.6 18.0 633.9 30-60 cultivated with artichoke 33.50 850.0 73.34 63.2 94.55 14.0 287.6 0-30 loamy sand soil was 14.95 111.7 758.5 160.0 48.2 73.05 18.8 458.4 30-60 cultivated with artichoke 13.45 112.1 761.0 227.0 49.3 75.05 10.0 389.2 30 30 198 h. abouziena, e. hoballah, f. abd el zaher, a. turky, s. el-ashery, m. saber, a.m. zaghloul inoculation of sorghum plants with am caused a significant increase in plant dry weight reaching 45.6 % and 10.0 % in the marginally and highly contaminated sewaged soil, respectively relative to that without am inoculation. after bioremediation with canola in association with am inoculation as shown in tab. 2, the dry weight of sorghum plants increased by 52.2 % in the marginally contaminated sewaged soil compared to that without am inoculation. it was noticed that seed yield of sorghum plants was increased by 23.6 % and 35.5 % in the marginally and highly contaminated sewaged soil respectively; this result might be ascribed to the higher concentrations of nutrients in highly contaminated soil (soad el-ashry et al. (2011). the highest grain yield of sorghum plant, which is our main concern, was harvested from the marginally contaminated sewaged soil previously bioremediated with canola without am inoculation, which was insignificantly different from plants grown in highly contaminated sewaged soil previously bioremediated with canola with am inoculation (tab. 1). sesame plants performance: results in tab. 3 indicated that phytoremediation of the sewaged soils either by canola or indian mustard exhibited a significant effect on sesame plant criteria at harvest. in most cases, inoculation with am increased dry weight of sesame plant and its parts in both marginally and highly contaminated sewaged soils. data indicated that growing sesame plants in the marginally contaminated sewaged soil previously bioremediated with indian mustard in association with am inoculation increased their yield more than three folds in terms of the dry matter of both total and separate plant organs weights. these increments were also recorded when sesame was grown in the highly contaminated sewaged soil, but at a lower level (tab. 3). seed yield of sesame plants grown after bioremediation with canola plants without am inoculation was lower by 191.7 and 29.6 % than when associated with am inoculation in the marginally and highly contaminated sewaged soils, respectively. ptes content in sorghum and sesame plants and their parts sorghum plants: sorghum plant and its parts had more or less similar cu contents whether grown in the marginally or highly contaminated sewaged soil previously bioremediated with either of the hyper-accumulators canola or indian mustard in the presence or absent of am inoculation (tab. 4). the lowest value of cu in sorghum tab. 2: plant criteria of sorghum grown in remediated sewaged soil by indian mustard or canola in the presences and absence of inoculation with am. treatments dry weight (g plant-1) grains yield (g plant-1) root straw panicle plant marginally contaminated mustard 4.8 44.5 18.0 67.3 123.04 sewaged soil mustard+ am 4.8 67.0 21.0 98.0 152.6 highly contaminated mustard 10.0 110.3 17.0 137.3 121.7 sewaged soil mustard+am 15.3 112.2 23.5 151.0 164.0 mean 8.7 83.5 19.9 113.4 129.0 marginally contaminated canola 5.8 87.7 18.5 84.3 138.1 sewaged soil canola+ am 12.8 115.9 28.3 157.0 210.0 highly contaminated canola 14.7 128.5 22.0 165.2 160.0 sewaged soil canola + am 12.5 97.6 17.9 128.0 228.7 mean 11.5 107.4 21.7 133.6 163.2 lsd at 0.05 1.6 30.2 4.0 12.9 21.4 tab. 3: plant criteria of sesame grown in remediated sewaged soil by indian mustard or canola in the presences and absence of inoculation with am. treatments dry weight (g plant-1) seeds weight (g plant-1) root straw capsules plant marginally contaminated mustard 1.2 25.0 1.1 27.3 1.5 sewaged soil mustard+ am 7.6 78.9 11.6 98.1 12.5 highly contaminated mustard 7.0 55.2 8.9 71.1 19.8 sewaged soil mustard+ am 5.1 60.0 15.0 80.1 33.5 mean 5.2 54.8 6.7 66.7 14.3 marginally contaminated canola 2.0 19.2 1.2 22.4 1.2 sewaged soil canola + am 5.4 27.2 2.5 11.7 3.5 highly contaminated canola 5.0 30.0 2.9 37.9 4.4 sewaged soil canola + am 3.4 48.4 3.1 54.9 5.7 mean 4.0 31.2 2.4 31.7 3.7 lsd at 0.05 1.1 7.6 1.9 8.1 3.4 phytoremedatin of contaminated soil with sesame and sorghum 199 seeds, however, was found in plants grown in the highly contaminated sewaged soil previously bioremediated with canola plants, as it was 75.9 % less than that of the same plants grown in soil previously bioremediated with indian mustard. this might be ascribed to that canola plants accumulated more cu in straw as previously indicated by antonious et al. (2011). results also indicated that canola hyper-phytoremediator was much more efficient in decontaminating the sewaged soil from cu than indian mustard, where the concentration of cu in sorghum grains after canola and indian mustard were 9.0 and 12.3 μg g-1 plant-1 in marginally and 3.2 and 13.3 3.2 μg g-1 plant-1 in highly contaminated sewaged soil, respectively (tab. 4). sorghum plants grown in the marginally and highly contaminated sewaged soils previously bioremediated with canola had a lower cu content in their seeds compared to those grown in soil previously bioremediated with indian mustard by 26.8 % and 74.6 %, respectively. no differences were recorded in the zn content of sorghum plants grown in the highly contaminated sewaged soil previously bioremediated with either canola or indian mustard (tab. 3). in the marginally contaminated sewaged soils, however, sorghum plants contained lower levels of zn when grown after bioremediation with canola compared to indian mustard. it is worthy to state that ni was not detected in sorghum plant or its organs after being grown in either marginally or highly contaminated sewaged soils previously bioremediated with either canola or indian mustard (tab. 4). sesame plants: data presented in tab. 5 indicated that sesame plants grown in the sewaged soil previously bioremediated with canola had lower cu contents in their stem compared to plants grown in the sewaged soil previously bioremediated with indian mustard, while a visa versa trend was noticeable in the cu content in the plant roots. the lowest cu content (6 μg/g dry weight) in sesame seeds was found in plants grown in the marginally contaminated sewaged soil previously bioremediated with canola plants, where the cu content in the seeds was less than 53.9 % compared to those plants grown in the sewaged soil previously bioremediated with indian mustard. in contrast, in the highly contaminated sewaged soils, the lowest cu content in sesame seeds was found in plants grown in the sewaged soil previously bioremediated with indian mustard causing a reduction in the cu content of seeds up to 21.% compared to plants grown in the sewaged soil previously bioremediated with canola plants. it’s worthy to mention that the concentrations of ptes studied in the roots, shoot and seeds of indian mustard plant were found to be lower than the permissible maximum limits of the recommendation of world health organization (who, 1996). data given in tab. 5 indicated that using canola to decontaminate the highly contaminated sewaged soil resulted in a decrement in the zn content of sesame seeds, which is a sensitive plant to zn, by 13.8 % compared to plants grown in the sewaged soil previously bioremediated with indian mustard. in the marginally contaminated sewaged soil canola caused a reduction in the zn content of seeds by 34.6 % compared to plants grown in the sewaged soils previously bioremediated with indian mustard. the ni content in sesame seeds, as shown in tab. 5 exhibited the same trend as sorghum plants where ni was not detected in sesame plant or its organs grown in either marginally or highly contaminated sewaged soils previously bioremediated with either canola or indian mustard. this finding means that canola and indian mustard might be considered efficient hyper-accumulators in phytoremediating sewaged soils contaminated with ni and the ni-bioremediated sewaged soil could be safely used for a sustainable growing for even sensitive crops such sesame. tab. 4: cu, zn and ni content (μg/g dm plant) in sorghum plants and its parts grown in previously bioremediated sewaged soils at harvest. treatments cu zn ni level of bioremediated root stem seed root stem seed root stem seed soil contamination crops marginally mustard 9.7 12.6 12.3 156.5 184.8 197.0 nd* nd nd canola 11.7 12.8 9.0 178.9 152.3 166.6 nd nd nd high mustard 9.7 12.6 13.3 184.8 212.2 162.3 nd nd nd canola 11.7 25.0 3.2 152.3 171.5 164.9 nd nd nd *nd: non detected (less than 0.001 μg/g). tab. 5: cu, zn and ni content (µg/g dm plant) in sesame plants and its parts grown in previously bioremediated sewaged soils at harvest. treatments cu zn ni level of bio-remediated root stem seed root stem seed root stem seed soil contamination crops marginally mustard 9.5 10.5 13.0 182.0 153.0 275.0 nd* nd nd canola 16.5 7.4 6.0 106.0 128.0 180.0 nd nd nd high mustard 9.9 15.9 12.2 236.8 306.4 187.1 nd nd nd canola 18.6 2.9 15.6 180.4 111.4 161.3 nd nd nd *nd: non detected (less than 0.001 μg/g). 200 h. abouziena, e. hoballah, f. abd el zaher, a. turky, s. el-ashery, m. saber, a.m. zaghloul zinc equivalent despite zn equivalent index is usually used in estimating the pollution of sewage sludge, in the current it was applied as a promising tools to follow the fate of ptes in sewaged soil ecosystems as recommended by hilal (1987), soad el-ashry, et al. (2011); saber et al. (2012). the zn equivalent index at harvesting of both sesame and sorghum grown in contaminated sewaged soils previously bioremediated with either canola or indian mustard is shown in tab. 6. both zn equivalent value and ni content in both marginally and highly contaminated sewaged soils after harvesting of sorghum or sesame obviously decreased compared to control soil. this finding indicated that canola and indian mustard were efficient in phytoremediating the contaminated sewaged soil from ptes, particularly ni, and hence it is recommended that the bioremediated sewaged soil might be safely used for the sustainable growing of sensitive crops in such as sorghum and sesame. in conclusion, results indicated that in the highly contaminated sewaged soil canola was significantly more effective in minimizing zn equivalent index compared to indian mustard which also exhibited a significant effectiveness. worth to mention, the same trend was observed in the marginally contaminated sewaged soil. ptes contents in the sewaged soils data in given tab. 7 indicated that bioremediation of the sewaged soil with either canola or indian mustard plants followed by growing of sesame reduced cu content in soil by more than 51 % and 40 % in the marginally contaminated sewaged soil and by 49 % and 52 % in tab. 6: zn equivalent index in remediated soils in the presence and absence of am inoculation after harvesting of sorghum and sesame crops. treatments control t1 mustard + sesame t2 canola + sesame t3 mustard + sorghum t4 canola + sorghum control 630* marginally contaminated 210 49.0 43.6 52.2 44.9 sewaged soils highly contaminated 275 61.8 54.8 66.3 56.4 sewaged soils *more details are documented in soad el-ashry et al. (2011) tab. 7: contents (μg/g dw) and reduction percent of ptes in sewaged soils previously bioremediated with either canola or indian mustard at sesame harvest. treatments cu zn ni level of bioremediator crops conc. red. % conc. red. % conc. red. % soil contamination control* 9.5 159.0 4.07 marginally canola 4.8 49.5 33.6 78.9 0.05 100 mustard 3.5 63.2 41.6 73.8 0.05 100 control* 12.0 218.0 4.07 highly canola 6.0 50.0 42.0 80.7 0.1 100 mustard 4.5 62.5 52.0 76.1 0.1 100 *without cultivation (no sesame/sorghum), conc.: concentration red.: reduction tab. 8: contents (μg/g dw) and reduction percent of ptes in sewaged soils previously bioremediated with either canola or indian mustard at sorghum harvest. treatments cu zn ni level of bioremediator crops conc.** ppm red. % conc. ** red. % conc. ** red. % soil contamination control* 9.50 159.0 4.07 canola 4.64 51.2 35.2 77.9 0.05 100 marginally mustard 5.40 43.2 41.0 74.2 0.05 100 control* 12.0 218.0 4.07 highly canola 5.80 51.7 44.0 79.8 0.1 100 mustard 6.73 43.9 52.0 76.1 0.1 100 *without cultivation, conc.: concentration red.: reduction ** concentration after canola or indian mustard and before sowing sorghum phytoremedatin of contaminated soil with sesame and sorghum 201 the highly contaminated sewaged soil, respectively. the same trend of results was achieved in both zn and ni pollutants results in tab. 8 also indicated that bioremediation of the sewaged soil with canola followed by growing sorghum reduced zn content in both marginally and highly contaminated sewaged soil by more than 77 %. bioremediation with indian mustard followed by growing sorghum or sesame, however, caused more than 73 % reduction in the zn content in both marginally and highly contaminated sewaged soils. sorghum sowing after canola plants removed 59.4 % and 18.4 % of cu and zn, respectively, from the marginally contaminated sewaged soil. the removed percent of cu and zn were much higher in the highly contaminated sewaged soil. in the highly contaminated sewaged soil the decontamination role of canola plants was more pronounced, compared to that exhibited by plants grown in the marginally contaminated sewaged soil. more or less growing sesame plants after canola or mustard plants displayed the same trend as sorghum. results indicated that ni reached a non-detectable level in the contaminated sewaged soils at sorghum and sesame harvest from both marginally and highly contaminated sewaged soils bioremediated by the two hyperaccumulators plants canola and indian mustard. kinetics of pte’s release after phytoremediation of the highly contaminated soil kinetic study used in this work to understand the rate of potential toxic elements after different remediation treatments applied as an indicator for fat of pollutants released. worth to mention that the succession in remediation for both contaminated sewaged soils was achieved through the experimental work as ni was not detected directly after phytoremediation either in soil or plant compared to control treatment. concerning zn equivalent, result showed significant minimizing in this parameter to a save level. furthermore, in all treatment the rate of cu and zn desorption from the highly contaminated soil, as shown in fig. 1, the rate processes started with speed rate trend of ptes released, followed by a decreasing order to almost steady state conditions even in control treatment. unquestionably, the rate of ptes desorption was influenced by soil type, and the concentration of sorbents found in different treatment which were already limited according to remediation treatments applied except control. according to the same parameter, it could be emphasized that phytoremediation in removing both zn and cu from the contaminated sewaged soils with canola far exceeded compared to indian mustard. their existence in both contaminated sewaged soils was noticeably diminished at sesame and sorghum harvest. the kinetic constants given in tab. 9 represents the rate constants i.e. intensity and capacity factors of cu and zn desorbed from the highly contaminated sewaged soil previously phytoremediated with either indian mustard or canola plants. in this study, elovich, mfe and horel’s models were the best fitted models in describing the kinetic data according to highly coefficient of determination (r2) and low standard error (se) compared to other models tested i.e. diffusion and 1st order models (data not shown), those models showed less conformity in describing the kinetic data. it should be mention that high values for r2 in more than one model mean that different mechanisms take place in desorption phenomena and subsequently the uptake of such ptes by growing plants used as indicators after phytoremediation. as far as the intensity factor represented by a constant of elovich equation, results pointed out that all treatments significantly decreased the rate of zn desorption from both contaminated sewaged soils. the numerical values of a, the rate of zn release as describe by elovich model, indicated that, in highly contaminated soil, grow fig. 1: kinetics of zn and cu desorption from highly contaminated soil as affected by different treatments (t1: sesame after indian mustard; t2: sesame after canola; t3: sorghum after indian mustard; and t4: sorghum after canola). ing sesame after indian mustard decreased the rate of zn desorption from 34.4 in control to 6.76 mg kg-1 min-1, the corresponding values in case of growing sesame after canola or sorghum after canola were 6.61 and 6.05 kg-1 min-1 and the best treatment under this condition was growing sesame after canola, which gave the lowest value i.e. 5.96 kg-1 min-1. results in tab. (9) also indicated that the capacity factor represents by b constant values decreased in all treatments compared to the control. furthermore, data pointed out that the lowest value, 16.93 mg kg-1, was found in sesame grow after canola, the same value was 128.10 mg kg-1 and it should be mention that the same trend was observed in both control and all other treatments applied. in mfe, data indicated that b, the capacity factor of zn desorption, decreased from 2.05 to 1.22 when growing sesame after canola. the same respective values were 1.49, 1.46 and 1.28 in cases of growing sesame after indian mustard, sorghum after indian mustard or sorghum after canola; all are significantly less than control, the same trend was observed in the same constant of cu desorption from highly contaminated soil. before viewing the results of the hoerl model, it should be mention that ptes in remediated soil move through different pathways. for example, pollutants could be transferred to cultivated plants, distributed into hardly available form etc.. horel model applied in this work describe transferring of ptes from different treated soils into cultivated plants and according to design of model increasing the rate constant value, meaning increase the rate of pollutant absorbed by plants and vice versa. this model showed high conformity to describe ptes release from the contaminated sewaged soils for the entire reaction time by having high significant r2 ranged between 0.91**-0.96** for different treatments. in zinc pollutant, increasing the intensity factor to 0.32 in t2 to be almost near the control, ensured the phytoremediation efficiency of this treatment compared to other treatments which gave higher values reached to 0.20, 0.21 and 0.29 in sesame grow after indian mustard, sorghum after indian mustard or sorghum after canola respectively. the same trend was also observed in cu desorption from the highly contaminated soil. fig. 2 represents the kinetic parameters of cu and zn desorption from marginally contaminated sewaged soil. the kinetics of all ptes 202 h. abouziena, e. hoballah, f. abd el zaher, a. turky, s. el-ashery, m. saber, a.m. zaghloul tab. 9: kinetic parameters of pte’s desorption from highly contaminated sewaged soil previously phytoremediated before growing sesame or sorghum. elovich zn cu treatment a b r2 se treatment a b r2 se control 34.40 128.10 0.86** 0.69 control 1.62 5.63 0.95** 1.59 t1 6.76 29.69 0.96** 5.63 t1 1.52 0.71 0.98** 0.77 t2 5.96 16.93 0.94** 6.31 t2 1.20 1.53 0.96** 1.28 t3 6.61 28.03 0.96** 5.79 t3 1.52 2.67 0.97** 1.18 t4 6.05 19.28 0.94** 6.14 t4 1.28 1.20 0.97** 0.87 mfe a b r2 se a b r2 se control 0.18 2.05 0.81** 0.17 control 0.51 0.78 0.92** 0.08 t1 0.13 1.49 0.93** 0.07 t1 0.25 0.38 0.95** 0.10 t2 0.19 1.22 0.87** 0.14 t2 0.24 0.23 0.94** 0.11 t3 0.14 1.46 0.91** 0.08 t3 0.20 0.54 0.95** 0.08 t4 0.18 1.28 0.87** 0.13 t4 0.25 0.29 0.94** 0.11 horel’s model a b r2 se a b r2 se control 0.34 4.39 0.86** 0.37 control 0.44 1.62 0.97** 0.04 t1 0.20 0.23 0.96** 0.04 t1 0.26 0.09 0.97** 0.06 t2 0.32 2.55 0.91** 0.24 t2 0.41 0.65 0.96** 0.11 t3 0.21 3.20 0.95** 0.06 t3 0.35 0.08 0.97** 0.05 t4 0.29 2.71 0.91** 0.18 t4 0.35 0.42 0.97** 0.09 t1: sesame after indian mustard t2: sesame after canola t3: sorghum after indian mustard t4: sorghum after canola desorbed were divided into three reaction periods; the 1st period was characterized by rapid reaction started from the beginning of esfu working and reached to about 30 min. the 2nd period showed a decline in ptes adsorption on different clay minerals and this period was about 90 min. the 3rd one, however, stayed for the rest of the reaction period, about 60 min, and was almost characterized by stability or slight increase in rate of adsorption (slow step) regardless the type of ptes. again, data indicated that the concentration of ptes in the marginally contaminated sewaged soil previously phytoremediated with either canola or indian mustard before growing of sesame and sorghum were decreased and subsequently the rate of desorption of these pollutants from the soil were took place. results also confirmed that growing of sesame after canola was the best effective treatment in diminishing ptes desorption rate. through the kinetic study it could be mention that although using of indian mustard as a phytoremediator plant significantly decreased cu and zn desorption compared to control, data indicated that canola was much more preferable to be applied under the current experimental conditions. the comparison between sesame and sorghum in their efficiency in adsorbing cu and zn from sewaged soils, results indicated that sesame after phytoremediation treatment was more effective compared to sorghum. numerically, as shown in zn desorption, the maximum value was 63 ppm when growing sesame after indian mustard, the corresponding value was 60 ppm when sorghum grow after indian mustard, the same trend was observed in the cases of growing sesame after canola or sorghum after canola. the rate constants of used models described the kinetics of zn release from the marginally sewaged soil are given in tab. 10. all used fig. 2: kinetics of zn and cu desorption from the marginally contaminated sewaged after phytoremediation. phytoremedatin of contaminated soil with sesame and sorghum 203 tab. 10: kinetic parameters of ptes desorption from the marginally contaminated sewaged previously phytoremediated before growing sesame or sorghum. elovich zn cu treatment a b r2 se treatment a b r2 se control 32.35 31.45 0.87** 0.71 control 1.30 4.42 0.95** 1.30 t1 5.41 23.75 0.96** 4.50 t1 1.20 0.67 0.97** 0.69 t2 4.88 13.54 0.94** 5.05 t2 095 1.20 0.96** 1.02 t3 5.32 22.21 0.96** 4.70 t3 1.22 2.14 0.97** 0.94 t4 4.77 15.43 0.95** 4.80 t4 1.02 0.96 0.97** 0.69 mfe treatment a b r2 se treatment a b r2 se control 0.31 1.53 0.85** 0.24 control 0.52 0.68 0.92** 0.08 t1 0.19 1.40 0.93** 0.07 t1 0.26 0.10 0.95** 0.12 t2 0.13 1.13 0.87** 0.14 t2 0.20 0.28 0.94** 0.11 t3 0.18 1.36 0.91** 0.08 t3 0.24 0.45 0.95** 0.08 t4 0.14 1.19 0.87** 0.13 t4 0.23 0.19 0.94** 0.11 horel’s treatment a b r2 se treatment a b r2 se control 0.52 3.09 0.88** 0.81 control 0.23 1.42 0.97** 0.04 t1 0.19 0.07 0.96** 0.04 t1 0.37 3.92 0.97** 0.11 t2 0.32 2.32 0.91** 0.24 t2 0.35 0.42 0.96** 0.11 t3 0.21 0.97 0.95** 0.05 t3 0.27 2.20 0.97** 0.05 t4 0.29 2.49 0.91** 0.19 t4 0.35 0.20 0.97** 0.09 t1: sesame after indian mustard t2: sesame after canola t3: sorghum after indian mustard t4: sorghum after canola models describe the kinetics of ptes studied were suitable to describe the kinetic data according to high and significant r2 and low of se. in the marginally contaminated sewaged soil, again data implied that the rate constants of horel model showed significant decrease in control followed by sesame after canola treatment and sorghum after canola or sorghum after indian mustard respectively. the lowest value was recorded when growing sesame after indian mustard means that growing sesame after canola was the best in minimizing the hazards of such contaminates and hence ensures sustainability. in mfe represented in the same table, although the same trend was observed when sesame grow after canola, data indicated that the rate of cu desorption was decreased in almost all treatments by about 50 % compared to control. the corresponding percentage values for different treatments in zn were about 70 % in sesame grown after canola and sorghum after canola. the lowest values in were found when sorghum or sesame were grown after indian mustard. the impacts of phytoremediation either alone or in combination with am inoculation, on the biological characteristics of both highly and marginally contaminated sewaged soils at sowing and maturity stages under sesame or sorghum plantation are drawn fig. 3 and 4. after sole phytoremediation in the highly contaminated sewaged soil, the dehydrogenase activity significantly increased from 22.84 to 60.23 μg g-1 soil after 24 hours under sesame and from 22.84 to 61.48 μg h2 g-1 soil after 24 hours under sorghum. phytoremediation associated with am inoculation led to slightly higher significant dehydrogenase activity that increased from 22.84 to 65.79 μg h2 g-1 soil after 24 hours and from 22.84to 63.26 μg h2 g-1 soil after 24 hours after phytoremediation, respectively with sesame or sorghum. after sole phytoremediation in the marginally contaminated sewaged soil, the dehydrogenase activity showed slight increase from 24.68 to59.45 μg h2 g-1 soil after 24 hours under sesame and from 24.68 to 60.61 mg h2/g soil after 24 hour under sorghum. phytoremediation associated with am inoculation led to slightly higher dehydrogenase activity that increased from 24.68 to 67.32 μg h2 g-1 soil after 24 hours and from 24.68 to 61.78 μg h2 g-1 soil after 24 hour after phytoremediation respectively with sesame or sorghum. it seems reasonable to conclude that during the cultivation season, indigenous rhizosphere soil microorganism flourished in the control fig. 3: sowing and final dehydrogenase activity the marginally contaminated sewaged soil under different phytoremediation treatments (μl h2 g-1 24-h) 204 h. abouziena, e. hoballah, f. abd el zaher, a. turky, s. el-ashery, m. saber, a.m. zaghloul and phytoremediate treatments of both contaminated sewaged soils due to the new well-furnished soil ecosystems and hence increased the dehydrogenase activity therein. recorded changes in dehydrogenase activity under the different phytoremediation treatments confirmed slight increases in the dehydrogenase activity in both soils due to cultivation in the presence and absence of am inoculation despite being slightly higher under the later treatments. it seems reasonable to conclude that phytoremediation did not show any adverse impacts on the biological characteristics of both highly and marginally contaminated soils. discussion the use of both hyperaccumulators plants to remove ptes from sewaged soils (phytoremediation) is expanding due to its cost-effectiveness compared to conventional methods besides its great potential. plants that absorb highly levels of a given element from soil called hyperaccumulators are now being closely investigated, both by molecular techniques and by soil/plant analyses, at the sites where they occur. the term phytoremediation generally refers to phytostabilization and phytoextraction. in phytostabilisation, soil amendments and plants are used to alter the chemical and physical state of the ptes in the soil. in phytoextraction, plants are used to remove contaminants from the soil and are then harvested for processing. plants suitable for phytoremediation should possess a highly ability to accumulate the targeted ptes, preferably in the aerial parts, can tolerate the highly accumulated ptes concentrations, characterized with a fast growth of the ptes accumulating biomass as well as with ease cultivation and harvesting (baker and brooks, 1989). however, chaney et al. (1997) argued that ptes tolerance and hyperaccumulators are more important factors than highly biomass production. recently, hyper-accumulating plants had been a subject of several phytochemical studies because unlike most other plants, milligram rather than microgram quantities of organo-metallic complexes could be separated from their plant tissues. hence, great attention is now being paid to the potential use of hyperaccumulators to bioremediate contaminated sewage soils. worthy to mention that both indian mustard and canola used as hyperaccumulators plants, before sowing sesame and sorghum in the current study, almost absorbed almost all ni found in the both contaminated sewaged soils which directly decreased the value of the zn equivalent parameter that is frequently used to define the level of ptes contamination in soils. reeves and brooks (1983) reported that indian mustard is a suitable hyperaccumulator plant in minimizing the hazards of zn in contaminated soils compared to other ptes that might be found in soil ecosystem. on the other hand, under the current experimental conditions, canola was more effective than indian mustard because it significantly accumulated higher amounts of ni, zn and cu from the sewaged soil and resulted in minimizing their amounts in the following crops (sesame and sorghum) as previously shown by turan and esringü (2007) that canola was more effective than indian mustard in the uptake of cu, cd, pb and zn. in a lysimeter experiment, agoea et al. (2009) investigated two bioremediation amendments; either compost or clay inoculated with am and streptomyces sp. followed by growing a mixture of two plant species. they found that leaching and plant uptake of ptes from the amended plots were higher than in the control, mainly because of the higher plant biomass. the inoculation of control soil with am decreased the leaching of ptes, but the effect on their uptake by plants was ptes and species dependent. the extra inoculation with streptomyces sp. increased the leaching of many ptes, and also in many cases their uptake by plants. the cumulative of ptes by plants and leaching was higher in treatments with expanded clay in the case of mn, ca, ni and pb, smaller in the case of cu. the dehydrogenase activity is repeatedly used to follow up the biological activities in the soil ecosystem. in the current work the final level of dehydrogenase activity slightly increased compared to its initial value indicating that the trailed materials have a simulative rather than inhibitive impacts on soil biomass. conclusion it might be concluded that using canola as phytoremedator was an effective tool to bioremediated contaminated sewaged soil rendering it proper for growing economic crops. acknowledgment the authors would like to express their appreciations and gratitude to the authorities of science and technology development fund (stdf) for financing the present work through the project number 1425 contracted with the national research center on bioremediation of sewaged soils. references a.o.a.c., 1984: official methods of analysis association of official analytical chemists. washington, d.c. 21 st fd. usa. abouziena, h.f., saber, m., hoballa, e.m., el-ashry, a., zaghloul, a., 2012: phytoremediation of potential toxic elements in contaminated sewaged soils by canola (brassica napus) or indian mustard (brassica juncea czern.) plants in association with mycorrhiza. j. appl. sci. res. 8, 2286-2300. antonious, g., dennis, s., unrine, j., snyder, j., 2011: ptes uptake in plant parts of sweet potato grown in soil fertilized with municipal sewage sludge. international journal of geology 5, 14-20. ashour, e.h., el-mergawi, r.a., radwan, s.m.a., 2006: efficacy of pseudomonas to phytoremediate nickel by canola (brassica napus l.). j. appl. sci. res. 2, 375-382. baker, a., brooks, r., 1989: terrestrial higher plants which hyperaccumulate metallic elements: a review of their distribution, ecology and phytochemistry. biorecovery 1, 81-126. banuelos, g., terry, n., educ, d.l., pilon-smits, e.a.h., mackey, b., 2005: field trial of transgenic indian mustard plants shows enhanced phytoremediation of selenium-contaminated sediment. environ. sci. technol. 39, 1771-1777. brooks, r.r., 1998: geobotany and hyperaccumulators. 1974. in: brooks, r.r. 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(cruciferae) as indicators of nickel and zinc. j. geochem. explor. 18, 275-283. salt, d., blaylock, m., kumar, n., dushenkov, v., ensley, b., chet, i., raskin, i., 1995: phytoremediation: a novel strategy for the removal of toxic metals from the environment using plants. biotechnology 13, 468-74. skujins, j., 1973: dehydrogenase activities in arid soils. bulletin ecology research communication 17, 235-241. sumathi, m., umagowrie, s., 2012: biotreatment to reduce the phytotoxicity of tannery effluent on arachis hypogea and sesamum indicum. int. j. curr. sci., 105-111. sun, r., zhou, q., jin, c., 2006: cadmium accumulation in relation to organic acids in leaves of solanum nigrum l. as a newly found cadmium hyperaccumulator. plant soil 285, 125-134. syliva, d., willson, d., graham, j., maddox, j., mlllner, p., morton, j., skipper, h., wright, s., jarstfer, a., 1993: evaluation of vesicular-arbuscular mycorrhyzal fungi in diverse plant and soil. soil biol. biochem. 25, 705-713. turan, m., esringü, a., 2007: phytoremediation based on canola (brassica napus l.) and indian mustard (brassica juncea l.) planted on spiked soil by aliquot amount of cd, cu, pb, and zn. plant soil environ. 53, 7-15. who, 1996: trace elements in human nutrition and health. world health organization. geneva. zaghloul, a.m., 2002: kinetics of potassium adsorption in some soils of egypt using electrical stirred flow unit (esfu). egypt. j. soil sci. 42, 463-471. address of the authors: hussein abouziena, botany department, national research center, dokki, cairo, egypt, 12622 e-mail: abouzainah@yahoo.com essam hoballah, f. abd el zaher azza turky, mohammed saber, agriculture microbiology department, national research center, dokki, cairo, egypt, 12622 soad el-ashery, alaa zaghloul, soil and water use department, national research center, dokki, cairo, egypt, 12622 art01 journal of applied botany and food quality 80, 1 5 (2006) institute for plant biology, technical university braunschweig, germany sulfur is limiting the glucosinolate accumulation in nasturtium in vitro plants (tropaeolum majus l.) lilian matallana, maik kleinwächter, dirk selmar (received august 8, 2005) summary it is well established that sulfate fertilisation significantly enhances the content of mustard oil glucosides in glucosinolate containing plants. however, with respect to tissue cultures and in vitro-plants, corresponding data are missing. in this study the influence of sulfur on the accumulation of glucosinolates was analyzed in nasturtium in vitro-plants (tropaeolum majus). the glucotropaeolin content in plants grown on standard media (ms) varied between 10 and 50 µmol/g dw, corresponding to only about 20 % to 70 % of the glucotropaeolin content in earth grown plants. a fivefold enhancement of the sulfate concentration resulted in a massive increase in the glucotropaeolin content of the in vitro-plants. a decline of sulfate in the medium leads to corresponding diminutions of the glucosinolates accumulated. these data clearly demonstrate the high impact of sulfur availability on glucosinolate biosynthesis and accumulation. introduction glucosinolates are sulfur containing natural products derived from amino acids, which are characterized by a sulphonated oxime moiety, a variable side chain, and a β-thioglucose moiety. in postmortal reactions these compounds are hydrolyzed by myrosinases to yield the unstable thiohydroximate-o-sulphonates. depending on the reaction conditions (e.g. ph, the presence of fe2+-ions, or the abundance of epithiospecifier proteins) these compounds react to a wide array of further products, the so-called mustard oils, comprising of isothiocyanates, nitriles or thiocyanates (for review see, e.g. bones and rossiter, 1996; halkier, 1999; selmar, 1999). glucosinolates and mustard oils respectively, are suggested to play an important role in the interaction of plants with their environment (for review see oleszek, 1995; zukalová and vašák, 2002; kliebenstein, 2004). they represent important flavour compounds of our food, e.g. in cabbage and other brassicacean derived vegetables. moreover they reveal various pharmacologic effects, e.g. antibacterial activity (manici et al., 1997) or cancer prevention (pintão et al., 1994; verhoeven et al., 1997). nasturtium, a traditional medicinal plant, is used to treat infections of the urinary tract (hoffmann-bohm and koch, 1994). in contrast to most other plants, t. majus contains just one type of glucosinolate (kjær et al., 1978), i.e. the benzylglucosinolate, also named glucotropaeolin. in order to create large numbers of nasturtium plants with high glucosinolate contents, which could be used for pharmaceutical purposes, numerous individual t. majus plants have been screened. the most promising should be propagated by in vitro-technology. in contrast to the soil grown nasturtium plants, the corresponding in vitro-plants of the same clone contain far less glucotropaeolin. the question arose, if this effect is due to general characteristics related to in vitro-technology, or if it is due to sulfur limitation. to enable a reliable quantification of the glucosinolate contents of tiny in vitro-plants, we developed a special determination method which permits the analyses of less than 5 mg dry matter of nasturtium plant material. we present evidence that the glucotropaeolin content in nasturtium in vitro-plants strongly is determined by the sulfur supply. materials and methods plant material to screen for high yield nasturtium varieties, numerous tropaeolum majus plants were cultivated from seeds obtained from various horticultural stores and local market-gardens. plants were grown in experimental fields with about 70 cm spacing. leaves were harvested while the plants were flowering. directly after detaching the leaves – still in the field – the plant material was shock frozen in liquid nitrogen. for in vitro-propagation, various individual plants with high glucosinolate content and high biomass production had been chosen. cultivation of in vitro-plants parts of tendrils had been sterilized repeatedly (ethanol (70%), naocl (1%) and sterile water). the sterile plant parts had been transferred to a petri dish on 0.8 % agar with ms-media (murashige and skoog, 1962), containing 0.1 ppm tdz (1-phenyl-3-(1,2,3-thiadiazol5-yl-urea) and 0.5 ppm iaa (3-indole acetic acid). cultivation was performed at 25°c and 14 hours light per day. when vital in vitroplants had been developed, they were abscised from the explant and transferred into a preserving jar (250 ml) containing 100 ml of the ms-agar described above. every month, parts of the newly grown in vitro-plants (about 0.25 to 0.3 g fw each) were transferred to new media (five explants per jar). to determine the sulfur dependence of glucosinolate accumulation, medium was modified as follows: instead of about 1.7 mm sulfate in the standard ms-medium, by increasing or decreasing the amount of mgso4, final sulfate concentrations of 8.3 mm, 0.6 mm and 0.2 mm, respectively had been achieved. extraction and quantification of glucosinolates plant material was frozen and homogenized with mortar and pestle in liquid nitrogen, and subsequently freeze dried. then 10 µl arbutin (10 mm) were added as internal standard to an aliquot of each sample. these samples (1 to 5 mg) were extracted three times with 1 ml of meoh (80 %, containing 8.5 mm ammonia acetate). extraction was boosted using ultrasonification (10 min at 50°c). after centrifugation (15 min at 10.000 x g), the supernatants were pooled and concentrated by evaporation to final volumes of about 200 to 300 µl. after the estimation of the exact volumes, water was added to yield exactly 600 µl of aqueous samples. then 340 µl ammonia acetate (42.5 mm) and 60 µl meoh were added to obtain a final volume of exactly 1 ml and to achieve the composition of the hplc eluent a (see below). hplc analysis was performed using a rp 18 column (250 x 4 mm). elution was achieved by applying a one step gradient (8 min eluent a, 3 min eluent b; a: 6 % meoh, 40 mm ammonia acetate, b: 14 % meoh, 40 mm ammonia acetate). for the detection of glucotropaeolin and arbutin, absorbance was recorded at 220 nm. in order to eliminate all undesired substances from the column, after each chromatography, a rinse cycle with 80 % meoh (10 min) and a reequilibration step (25 min, eluent a) were introduced. based on the peak areas of glucotropaeolin and the internal standard, the amounts of glucotropaeolin were calculated. results variation of glucotropaeolin content of nasturtium plants in total, in the year 2003 more than 100 individual t. majus plants have been screened for their glucosinolate content. in fig. 1, a representative selection is displayed. the content of glucosinolates varied from about 40 µmol / g dw to over 90 µmol / g dw. because of their low biomass production, the plants fl 29 and fl 07 are not suitable for bulk production for pharmaceutical purposes. consequently, the four glucosinolate-rich plants fl 31, fl 37, fl 50 and fl 08 had been chosen for in vitro propagation. unfortunately, in the corresponding in vitro-plants from fl 31, contamination by bacteria could not been eliminated so far. therefore, all experiments mentioned in this paper had been performed with the three clones fl 37, fl 50, and fl 08 (black bars in fig. 1). question arose, if this effect is due to general characteristics related to in vitro-technology, or if it is due to sulfur limitation. consequently, in vitro-cultivation was performed on media varying in their overall sulfate concentration. 0 10 20 30 40 50 60 70 80 90 fl 37 fl 50 fl 08 g lu c o tr o p a e o li n [ m o l / g d w ] soil grown plants in vitro-plants fig. 2: comparison of the glucotropaeolin content of the three nasturtium clones propagated in vitro (ms standard medium, sulfate concentration: 1,7 mm) with that of the corresponding soil grown plants. glucotropaeolin content in in vitro-plants for a reliable quantification of glucosinolates in the tiny in vitroplants, a very sensitive quantification method for glucotropaeolin had to be developed. using the method described in materials and methods, we were able to determine the glucotropaeolin content in samples of only 1 mg dw. corresponding calibration curves confirmed linearity of this method throughout a wide range of both, the amount of plant material used, and the actual glucotropaeolin concentration. the determination of glucosinolates present in the in vitro-plants had been performed at the end of the cultivation cycle, i.e. four weeks after inoculation. in general, inoculation was performed with five explants per jar with an entire biomass of about 1.3 to 1.5 g fw. after the four week cultivation period, the newly developed plants represented a total biomass of about 6.0 to 6.5 g fw, resulting in an average gain of about 5.0 g fw of all plants from one preserving jar, corresponding to about 1.0 g gain per individual plant. the glucosinolate content of the nasturtium in vitro-plants cultured under standard conditions varied between 10 and 55 µmol/g dw. these contents are significantly lower than those of the soil-grown plants (fig. 2). this effect is pronounced in fl 37 and fl 08. the impact of sulfur supply on growth and glucotropaeolin content in in vitro-plants when the sulfate concentration in the growth medium was diminished to about 0.6 mm, the development of the in vitro-plants was delayed. when the plants were harvested after the normal propagation period of four weeks, their biomass was only about 40 % of that of the plants grown on standard conditions; but the habitus of the in vitroplants growing at 0.6 mm sulfate-media appeared to be normal (fig. 3a). however, when the sulfate concentration was diminished further down to 0.2 mm, the plants showed typical sulfur deficiency symptoms, like yellowish to white leaves, and their development was strongly retarded (fig. 3a). the habitus of the in vitro-plants that are grown on media with strongly enhanced sulfate concentration (8.3 mm) was similar to that of the standard plants, however, the root development was negatively affected. characteristic for these plants grown on high-sulfur media are the abnormal proliferations of the hypocotyl accompanied by reduced root formation (fig. 3b). overall, the growth was slightly retarded, probably due to the lesser root formation. the increase of sulfate concentration in the growth medium to about 8.3 mm resulted in a massive increment of the glucotropaeolin concentration in the in vitro-plants (fig. 4). the corresponding average contents of the in vitro-plants (81 µmol/g dw for fl 37, 69 µmol/g dw for fl 50 and 58 µmol/g dw for fl 08) are very similar to the values of the original „mother“-plants (82 µmol/g dw for fl 37, 74 µmol/g dw for fl 50 and 65 µmol/g dw for fl 08). when the in vitro-plants were cultivated in media with a sulfate concentration of 0.6 mm, the glucosinolate content was only about 20 % to 40 % of content of the in vitro-plants grown under standard conditions. a further decline in sulfate concentration resulted in even lower glucosinolate contents of just 1.4 to 2.6 µmol/g dw (fig. 4). 0 10 20 30 40 50 60 70 80 90 100 g lu c o tr o p a e o li n [ m o l / g d w ] f l 3 1 f l 2 9 f l 3 7 f l 5 0 f l 0 7 f l 0 8 f l 2 2 f l 2 4 f l 4 8 f l 1 6 f l 2 8 f l 3 6 f l 4 3 f l 0 2 f l 5 1 f l 0 9 f l 0 3 f l 0 6 f l 1 2 f l 0 5 f l 1 1 f l 1 0 f l 0 4 f l 0 1 plants used for in vitro propagation fig. 1: variation in the glucotropaeolin content of 25 individual soil grown tropaeolum majus plants. the plants fl 37, fl 50 fl 08 (black bars) and fl 31 have been used for in vitro-cultivation. as in fl 31 the contamination by bacteria could not be eliminated, this clone was not suitable for further propagation. 2 lilian matallana, maik kleinwächter, dirk selmar fig. 3a: in vitro-plants of t. majus after four weeks of cultivation on ms-media containing different concentrations of sulfate. fig. 3b: close-up of a typical excrescence in the hypocotyl and root onset region in in vitro-plants grown on media with excess of sulfur (8.3 mm sulfate). in contrast to plants grown on standard media (1.7mm sulfate), the root formation is significantly diminished. 8.3 mm so 4 21.7 mm so 4 20.2 mm so 4 2-0.6 mm so 4 28.3 mm so 4 21.7 mm so 4 2glucosinolate accumulation depends on sulfur supply 3 are wetted by the growth medium. an appropriate technique is provided by the temporary immersion systems (tis), where the in vitro-plants are submerged temporary by the culture medium. in contrast to the high diffusion related restrictions in sulfate uptake for plants grown on solid media, sulfate uptake should be improved by the plants cultivated in tis, because of their large surface that has contact to the medium. consequently, the glucotropaeolin content of nasturtium in vitro-plants grown in tis should be higher than that of those cultivated on solid media. corresponding analyses of tis grown t. majus in vitro-plants are under study. acknowledgements this study was supported in part by the fachagentur für nachwachsende rohstoffe e.v. (fkz: 22018101). technical assistance by daniela kramer is greatly acknowledged. references bloem, e., haneklaus, s., peplow, e., sator, c., köhler, t., schnug, e., 2001: the effect of sulphur and nitrogen fertilisation on the glucotropaeolin content in tropaeolum majus (l.). xxxvi. vortragstagung der deutschen gesellschaft für qualitätsforschung (pflanzliche nahrungsmittel) e.v. c/o fachgebiet obstbau tum, 85350 freising, 185190. bones, a.m., rossiter, j.t., 1996: the myrosinase-glucosinolate system, its organisation and biochemistry. physiol. plant. 97, 194-208. halkier, b.a., 1999: glucosinolates. in: ikan, r. (ed.), naturally occurring glucosides, 193-223. wiley & sons, chichester, uk. hoffmann-bohm, k., koch, h.p., 1994: tropaeolum. in: hänsel, r., keller, k., rimpler, h., schneider, g. (eds.), hagers handbuch der pharmazeutischen praxis 6, 1004-1013. springer verlag, berlin. kim, s.-j., matsuo, t., watanabe, m., watanabe, y., 2002: effect of nitrogen and sulphur application on the glucosinolate content in vegetable turnip rape (brassica rapa l.). soil sci. plant nutr. 48, 43-49. kjær, a., madsen, ø., maeda, y., 1978: seed volatiles within the family tropaeolaceae. phytochemistry 17, 1285-1287. kliebenstein, d.j., 2004: secondary metabolites and plant/environment interactions: a view through arabidopsis thaliana tinged glasses. plant, cell and environment 27, 675-684. manici, l.m., lazzeri, l., palmieri, s., 1997: in vitro fungitoxic activity of some glucosinolates and their enzyme-derived products towards plant pathogenic fungi. j. agric. food chem. 45, 2768 2773 murashige, t., skoog, f., 1962: revised medium for growth and bioassay with tobacco tissue cultures. physiol. plant. 15, 473-497 oleszek, w., 1995: glucosinolates: occurrence and ecological significance. wiadomosci botaniczne 39, 49-58. pintão, a.m., pais, m.s.s., coley, h., kelland l.r., judson, i.r., 1995: in vitro and in vivo antitumor activity of benzyl isothiocyanate: a natural product from tropaeolum majus. planta med. 61, 233-236. selmar, d., 1999: biosynthesis of cyanogenic glucosides, glucosinolates and nonprotein amino acids. in: wink, m. (ed.), biochemistry of plant secondary metabolism, 79-150. crc press, sheffield. verhoeven, d.t.h., verhagen, h., goldbohm, r.a., van den brandt, p.a., van poppel, g., 1997: a review of mechanisms underlying anticarcinogenity by brassica vegetables. chemico-biological interactions 103, 79-129. wielanek, m., urbanek, h., 1999: glucotropaeolin and myrosinase production in hairy root cultures of tropaeolum majus. plant cell, tissue and organ culture 57, 39-45. zhao, f.j., whiters, p.j.a., evans, e.j., monaghan, j., salmon, s.e., shewry, p.r., mcgrath, s.p., 1997: sulphur nutrition: an important discussion the strong enhancement of glucotropaeolin content in nasturtium in vitro-plants demonstrates that synthesis and accumulation of glucosinolates clearly are limited by sulfur availability and thus confirm the results obtained on sulfur dependency of mustard oil glucoside accumulation in soil grown plants of various species (e.g. zhao et al., 1997; bloem et al., 2001; kim et al., 2002). these data point out that in the case of glucosinolates the lesser concentrations of secondary metabolites in in vitro-plants are not due to a general characteristic related to in vitro-technology, but they are a result of sulfur limitation. however, if the overall amounts of sulfur present in the media are calculated and compared with the sulfur present in the plants, or integrated within the glucosinolates, respectively, it turns out that – at least in the case of the standard medium – between 70 to 90 % of the initial sulfate concentration remained in the media. consequently, the sulfur dependency of glucosinolate accumulation is not a question of a real limitation due to the absence of residual sulfur, but must be caused by the limitation of the capacity for an effective uptake of sulfate. this assumption is underlined by the findings of wielanek and urbanek (1999), who found that the concentration of glucotropaeolin in hairy roots of t. majus is as high – or even higher – than that of leaves grown in growth chambers. it is very likely that the tremendous surface of the hairy root cultures enable a more effective sulfate uptake, although the entire sulfate concentration in the medium for the hairy roots was comparable to that used in the standard medium for the in vitro-plants used in this study. in contrast, the far lower concentration of glucotropaeolin in suspension cultures of t. majus also observed by the same authors (wielanek and urbanek, 1999) might be the consequence of the well established general feature of cell cultures to diminish or suppress the accumulation of secondary plant metabolites in dedifferentiated cells. if indeed the glucotropaeolin accumulation is limited by the ability for an effective sulfur uptake and if such uptake increases when the sulfate concentration is enhanced, the question arises, which might be the maximal concentration of glucotropaeolin that could be accumulated in t. majus by increasing the sulfate supply. unfortunately, this question cannot be answered easily with further studies on in vitro-plants, as these plants do not tolerate higher sulfate concentrations in their growth media, demonstrated by the abnormal proliferations and the reduced root development in media with enhanced sulfate concentrations. alternatively, a more effective sulfate uptake by in vitro-plants should result, when the entire plants fig. 4: glucotropaeolin content in the in vitro-plants of the selected clones fl 37, fl 50 fl 08, cultivated on growth media with different sulfate concentrations. 0 10 20 30 40 50 60 70 80 90 fl 37 fl 50 fl 8 g lu c o tr o p a e o li n [ m o l / g d w ] 8,3 mm 1,7 mm (standard) 0,6 mm 0,2 mm sulfate concentration 4 lilian matallana, maik kleinwächter, dirk selmar factor for the quality of wheat and rapeseed. soil sci. plant nutrition 43, 1137-1142. zukalová, h., vašák, j., 2002: the role and effects of glucosinolates of brassica species-a review. rostlinná výroba 48, 175-180. glucosinolate accumulation depends on sulfur supply 5 addresses of the authors: dirk selmar (corresponding author, email: d.selmar@tu-bs.de), maik kleinwächter, lilian matallana, institute for plant biology, technical university braunschweig, mendelssohnstraße 4, d-38106 braunschweig, germany << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 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<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> >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice journal of applied botany and food quality 86, 198 204 (2013), doi:10.5073/jabfq.2013.086.027 1atatürk university, faculty of agriculture, department of plant protection, erzurum, turkey 2 karamanoğlu mehmetbey university, kamil özdağ faculty of science, department of biology, karaman, turkey insecticidal effects of monoterpenes on sitophilus zeamais motschulsky (coleoptera: curculionidae) e. yildirim1*, b. emsen2, s. kordali1 (received april 2, 2013) * corresponding author summary twenty eight monoterpenes including monoterpene hydrocarbons and oxygenated monoterpenes (borneol, borynl acetate, camphene, camphor, 3-carene, carvone, 1,8-cineole, citronellal, β-citronellene, β-citronellol, dihydrocarvone, fenchol, fenchone, geranyl acetate, isomenthol, limonene, limonene oxide, linalool, linalyl acetate, menthol, menthone, myrcene, nerol, neryl acetate, α-pinene, β-pinene, terpinen-4-ol, α-terpineol), the active compounds of essential oils obtained from different plant species were tested against adults of sitophilus zeamais motschulsky under laboratory conditions. the monoterpenes were applied at contents of 10, 20 and 30 μl for liquid compounds and 10, 20 and 30 μg for solid compounds. the results show that most of the monoterpenes have significantly insecticidal effect on the tested insects. insecticidal effects of monoterpene hydrocarbons were found to be lower than those of oxygenated monoterpenes. the ketone and aldehyde and epoxide derivatives of oxygenated monoterpenes were also found to be more toxic as compared with their other derivatives. mortality percentage of s. zeamais adults, after 96th h of exposure at the maximum dose (30 μl/μg) of oxygenated monoterpenes including borneol, fenchol, linalool, menthol, terpinen-4-ol, α-terpineol (alcohols group); 1,8cineole, limonene oxide (epoxides group); camphor, carvone, citronellal, dihydrocarvone, fenchone, menthone (ketones and aldehydes group) and neryl acetate (esters group) attained 100 %. concurrently, 3-carene from monoterpene hydrocarbons showed 100 % mortality after 96th h of exposure at the maximum dose (30 μl). carvone, dihydrocarvone, fenchone, limonene oxide, menthone and terpinen4-ol from these compounds showed 100 % insecticidal effect after 48th h of exposure. among the monoterpenes tested, carvone, dihydrocarvone, menthone and terpinen-4-ol showed the strongest insecticidal activities with 100 % of mortality at all doses (96 h after treatment) and then 1,8-cineole, fenchone, linalool and limonene oxide showed stronger insecticidal activities in comparison with other monoterpenes with lethal doses (ld50) values of 1.989, 2.445, 2.445 and 3.235 μl (96 h after treatment) against the test insects, respectively. mortality rate of s. zeamais adults increased significantly (p < 0.01), as the dosage level and/or exposure time increased. based on the present results, it can be concluded that the oxygenated monoterpenes may have a potential action for control of s. zeamais adults. introduction the maize weevil, sitophilus zeamais motschulsky (coleoptera: curculionidae) is an insect that causes yield losses in storage products in turkey (yildirim, 2012). s. zeamais is present in corn storage and can destroy the entire corn harvest. it may attack the corn and makes useless the product (yildirim et al., 2012a). several insecticides have been tried for years to prevent this damage. chemical/synthetic insecticides constitute the first part of the trials. synthetic insecticides demonstrate a high degree of impact of insecticide. at the same time, they cause many negative results such as death of non-target animals, residue problems and resistance against insecticides (isman, 2006). these disadvantage sides of synthetic pesticides have created a viewpoint for the introduction of alternative pesticides. hence, in recent years research showed that natural products can be preferred by farmers to protect stored grains from insect invasion (tapondjou et al., 2005). among the natural products plant extracts, plant secondary metabolites, plant essential oils and monoterpenes are shown as botanical pesticides. one of the most important features of botanical pesticides is to perform different effects on some insects. these pesticides can be used as insect antifeedants and repellants (isman, 2006). monoterpenes, the chemical constituents of essential oils found in plants, are known biologically active compounds. these major constituents are generally isolated from essential oils in asteraceae (leonardi et al., 2013; umpierrez et al., 2013), hypericaceae (rouis et al., 2013), lauraceae (chang and chu, 2011), myrtaceae (shelton et al., 2004; webb et al., 2013), pinaceae (sadeghi et al., 2013), rubiaceae (del terra et al., 2013), rutaceae (shimada et al., 2004) and solanaceae (murungi et al., 2013) families. they constitute the main feature of many plants and keep away bacteria and fungi from plants. also, plants become attractive for insects by these metabolites. thereby some essential oils have bactericidal, fungicidal, antiparasitical and insecticidal properties (bakkali et al., 2008). at the same time, essential oils and their constituents demonstrate fumigant and topical toxicity as well as antifeedant and repellent effects (regnault-roger, 1997; shaaya et al., 1997). fumigation is a very important technique in insect pest elimination in stored products. fumigants penetrate homogenously to endpoints because of their diffusion property and they can be applied to a large amount in a short time (zettler and arthur, 2000). contact and fumigant insecticidal activities of plant essential oils and monoterpenes against stored product pests have been demonstrated (tripathi et al., 2000; lee et al., 2003; yildirim et al., 2005, 2011; abdelgaleil, 2009; wang, 2009; kordali et al., 2012; kumar et al., 2012). however, to the best of our knowledge, studies have not yet been conducted to evaluate the insecticidal activities of monoterpenes towards s. zeamais. therefore, the present research was therefore undertaken to investigate the bioactivity of the some monoterpenes against adults of s. zeamais, important stored-product insects in grain storage in turkey. materials and methods insects rearing conditions in this study, s. zeamais was obtained from storage house in tokat region, turkey. corn grains were purchased from local market and stored in a freezer at -20 °c to maintain freshness. after the corn grains were removed from the freezer, in order to prevent pre-infestation they were washed by tap water, dried and heated before using it in insecticidal effects of monoterpenes against sitophilus zeamais 199 the experiments. s. zeamais adults were reared on whole corn in the laboratory at 12-13 % moisture content in plastic box (diameter 25 cm, height 30 cm) at 64 ± 5 relative humidity, 25 ± 1 °c and l:d = 12 h:12 h in the department of plant protection, faculty of agriculture, atatürk university, erzurum-turkey. adults obtained from laboratory culture were stored in separate insect cages provided with corn. tests were also carried out under the same laboratory conditions. determination of adults’ age four to six day-old s. zeamais adults were used as the test material. in order to get adults at the same age, few grains of corn that included larvae and pupae were placed separately in petri dishes. after adult emergence, the same age adults were collected and used. individual monoterpenoids the compounds tested were borneol (fluka), bornyl acetate (sigma), camphene (fluka), camphor (fluka), 3-carene (aldrich), carvone (fluka), 1,8-cineole (sigma), citronellal (sigma), β-citronellene (fluka), β-citronellol (fluka), dihydrocarvone (alfa), fenchol (fluka), fenchone (fluka), geranyl acetate (alfa), isomenthol (alfa), limonene (fluka), limonene oxide (aldrich), linalool (fluka), linalyl acetate (fluka), menthol (fluka), menthone (fluka), myrcene (aldrich), nerol (sigma), neryl acetate (alfa), α-pinene (fluka), β-pinene (fluka), terpinen-4-ol (aldrich), α-terpineol (merck) and ddvp (2, 2-dichlorovinyl dimethyl phosphate) (erzurum, turkey). bioassays in order to test the toxicity of the monoterpenes against s. zeamais adults, 33 individuals with 33 grains of corn were placed into petri dishes (9 cm diameter). 10, 20 and 30 μl/petri dish contents of liquid compounds and 10, 20 and 30 μg/petri dish contents of solid compounds were applied with an automatic pipette on a filter paper (2 x 2 cm) attached to the upside of the petri dish. solid compounds were dissolved in ethanol (1:1, w/v). filter papers were impregnated with the appropriate amounts of the solutions (10, 20 and 30 μg/petri dish), placed on the lid of petri dishes and then kept to evaporate the ethanol. petri dishes were closed with an adhesive tape to prevent escaping of volatile compounds and kept at (23 ± 2) °c on a laboratory bench. mortality rate of the adults was determined after an exposure for 24th, 48th, 72th and 96th h. grains were divided into two and in this way insects inside the grains became visible. at the end of each trial period, it was touched to the insects with forceps and observed movements of them. it was decided that completely inactive insects were dead. petri dishes applied with sterile water and ethanol (waited for evaporating of ethanol) served as control and petri dishes applied with ddvp served as positive control. the treatments were arranged in a completely randomized design with three replications including controls. insecticidal action of monoterpenes was expressed as % mean mortality of the adults. statistical analyses differences among the insecticidal activities of the monoterpenes tested were determined according to analysis of variance (anova) test by using statistical package for social sciences (spss®, version 15.0). mortality was expressed as mean (percentage) ± standard error. differences between means were tested through duncan test and values with p < 0.01 were considered significantly different. ld50 and ld90 values at 96 h were calculated with regression analysis by probit using spss. probit analysis of dose-mortality data was conducted to estimate the ld50 and ld90 values and associated 95 % confidence limits for each treatment. results and discussion in the present study, insecticidal effects of 28 commercial monoterpenes (borneol, borynl acetate, camphene, camphor, 3-carene, carvone, 1,8-cineole, citronellal, β-citronellene, β-citronellol, dihydrocarvone, fenchol, fenchone, geranyl acetate, isomenthol, limonene, limonene oxide, linalool, linalyl acetate, menthol, menthone, myrcene, nerol, neryl acetate, α-pinene, β-pinene, terpinen-4-ol, α-terpineol) including monoterpene hydrocarbons and oxygenated monoterpenes were tested against adults of s. zeamais. the present results showed that the tested compounds have insecticidal effects on adults of s. zeamais in comparison with control groups (tab. 1). analysis of variance demonstrated that the effects of these monoterpenes on the mortality rate of s. zeamais were highly significant on the basis of both dosage rate and exposure time (p < 0.01). higher doses and longer exposure times scored maximum toxicities on s. zeamais (fig. 1 and 2). generally, oxygenated monoterpenes had higher insecticidal effect on s. zeamais as compared with monoterpene hydrocarbons. some oxygenated monoterpenes were strong toxic compounds and among them, borneol, fenchol, linalool, menthol, terpinen-4-ol (alcohols group); 1,8-cineole, limonene oxide (epoxides group); camphor, carvone, citronellal, dihydrocarvone, fenchone, menthone (ketones and aldehydes group) and neryl acetate (esters group) attained 100 % insecticidal effect. the emergence of early effects of insecticides is very important. carvone, dihydrocarvone, fenchone and menthone from ketones and aldehydes group of oxygenated monoterpenes attained 100 % of mortality at 30 μl dose after 24 h. in the monoterpene hydrocarbons, compounds that brought out the highest mortality results were 3-carene and β-citronellene. after 96th h of treatments, at 30 μl dose, 3-carene and β-citronellene achieved 100 and 90.91 % mortality, respectively. however, it was determined that there was no statistically (p < 0.01) significant difference between the 24 h results of control groups and the highest doses of five monoterpenes (bornyl acetate, myrcene, nerol, α-pinene, β-pinene) (tab. 1). as shown in fig. 1 and 2, there are positive correlations between the treatment dose and insecticidal effect; elapsed time and insecticidal effect. the highest levels of mortality percentages were achieved after 96th h of treatments at all of 10, 20 and 30 μl doses of the essential oils of carvone, dihydrocarvone, menthone (oxygenated monoterpenesketones and aldehydes) and terpinen-4-ol (oxygenated monoterpenesalcohols) (100 %), at 20 and 30 μl/μg doses of fenchol, linalool, menthol (oxygenated monoterpenes-alcohols), 1,8-cineole, limonene oxide (oxygenated monoterpenes-epoxides) and camphor, fenchone (oxygenated monoterpenes-ketones and aldehydes) (100 %), at 30 μl/ μg doses of 3-carene (monoterpene hydrocarbon), borneol, α-terpineol (oxygenated monoterpenes-alcohols), citronellal (oxygenated monoterpenes-ketones and aldehydes) and neryl acetate (oxygenated monoterpenes-esters) (100 %) (tab. 1, fig. 1 and 2). the present results showed that borneol, borynl acetate, camphene, camphor, 3-carene, carvone, 1,8-cineole, citronellal, β-citronellene, β-citronellol, dihydrocarvone, fenchol, fenchone, geranyl acetate, isomenthol, limonene, limonene oxide, linalool, linalyl acetate, menthol, menthone, myrcene, nerol, neryl acetate, α-pinene, β-pinene, terpinen-4-ol and α-terpineol had varying degrees of insecticidal activity against adults of s. zeamais as the insecticidal activity increased with increasing dose and exposure times (fig. 1 and 2). 200 e. yildirim, b. emsen, s. kordali tab. 1: percent mortality effects of twenty eight monoterpenes to adults of sitophilus zeamais treatments dose mean mortality (%)a 24b 48b 72b 96b control 1 (ethanol) 10 μl 0.00 ± 0.00 a 0.00 ± 0.00 a 2.02 ± 0.33 a 5.05 ± 0.33 a control 2 (sterile water) 10 μl 0.00 ± 0.00 a 0.00 ± 0.00 a 2.02 ± 0.67 a 5.05 ± 0.67 a ddvp (positive control) 10 μl 100.00 ± 0.00 w 100.00 ± 0.00 γ 100.00 ± 0.00 z 100.00 ± 0.00 y monoterpene hydrocarbons camphene 10 μg 0.00 ± 0.00 a 0.00 ± 0.00 a 4.04 ± 0.33 ab 6.06 ± 0.58 ab 20 μg 5.05 ± 0.67 abcd 7.07 ± 0.33 abcdef 13.13 ± 0.33 abcdefg 30.30 ± 1.15 defgh 30 μg 11.11 ± 1.20 cdefghi 17.17 ± 0.33 fghij 28.28 ± 0.88 hijk 51.52 ± 3.61 jklmn 3-carene 10 μl 12.12 ± 0.58 defghij 22.22 ± 0.67 hijkl 47.47 ± 2.03 mno 86.87 ± 1.86 tuvwxy 20 μl 57.58 ± 0.58 r 62.63 ± 0.67 rst 84.85 ± 1.15 tuvwx 97.98 ± 0.67 y 30 μl 74.75 ± 1.20 s 78.79 ± 1.53 vwxy 96.97 ± 0.58 yz 100.00 ± 0.00 y β-citronellene 10 μl 0.00 ± 0.00 a 5.05 ± 0.67 abcde 29.29 ± 0.33 ijkl 54.55 ± 1.53 klmn 20 μl 3.03 ± 0.58 ab 20.20 ± 0.33 ghijk 48.48 ± 0.58 mnop 75.76 ± 1.73 pqrstu 30 μl 16.16 ± 1.2 hijk 32.32 ± 0.33 lmno 65.66 ± 1.20 qrs 90.91 ± 1.15 vwxy limonene 10 μl 0.00 ± 0.00 a 4.04 ± 0.33 abcde 6.06 ± 0.00 abc 34.34 ± 0.88 ghi 20 μl 10.1 ± 0.33 bcdefgh 15.15 ± 1.53 efghij 15.15 ± 1.53 bcdefg 58.59 ± 1.33 lmno 30 μl 27.27 ± 0.58 mno 34.34 ± 1.86 mno 27.27 ± 1.53 hijk 78.79 ± 1.00 qrstuv myrcene 10 μl 0.00 ± 0.00 a 0.00 ± 0.00 a 4.04 ± 0.33 ab 10.10 ± 0.33 abc 20 μl 1.01 ± 0.33 a 1.01 ± 0.33 ab 7.07 ± 0.33 abcd 22.22 ± 0.67 cdefg 30 μl 3.79 ± 0.73 abc 9.09 ± 1.00 abcdefg 22.22 ± 0.67 ghij 33.33 ± 0.58 fgh α-pinene 10 μl 0.00 ± 0.00 a 0.00 ± 0.00 a 4.04 ± 0.33 ab 16.16 ± 0.88 abc 20 μl 0.00 ± 0.00 a 0.00 ± 0.00 a 7.07 ± 0.33 abcd 31.31 ± 0.88 efgh 30 μl 2.02 ± 0.33 a 6.06 ± 0.58 abcdef 17.17 ± 0.33 cdefgh 49.49 ± 0.88 jklm β-pinene 10 μl 0.00 ± 0.00 a 0.00 ± 0.00 a 3.03 ± 0.00 a 17.17 ± 0.88 abcd 20 μl 0.00 ± 0.00 a 2.02 ± 0.33 abc 8.08 ± 0.67 abcd 41.41 ± 0.88 hijk 30 μl 3.03 ± 0.00 ab 7.07 ± 0.33 abcdef 18.18 ± 1.15 defghi 53.54 ± 0.88 jklmn alcohols (oxygenated monoterpenes) borneol 10 μg 3.03 ± 0.58 ab 10.10 ± 0.67 abcdefg 30.3 ± 0.58 jkl 58.59 ± 1.76 lmno 20 μg 18.18 ± 1.00 ijkl 25.25 ± 0.88 jklm 52.53 ± 0.88 nop 89.90 ± 0.33 uvwxy 30 μg 33.33 ± 1.53 o 41.41 ± 0.88 op 73.74 ± 1.20 st 100.00 ± 0.00 y β-citronellol 10 μl 0.00 ± 0.00 a 0.00 ± 0.00 a 5.05 ± 0.33 ab 35.35 ± 1.67 ghi 20 μl 3.03 ± 0.00 ab 5.05 ± 0.33 abcde 9.09 ± 0.00 abcde 50.51 ± 1.33 jklm 30 μl 10.1 ± 0.33 bcdefgh 13.13 ± 0.88 cdefghi 18.18 ± 0.58 defghi 63.64 ± 1.53 mnop fenchol 10 μg 2.02 ± 0.33 a 20.20 ± 0.67 ghijk 51.52 ± 0.58 nop 80.81 ± 1.33 rstuvw 20 μg 16.16 ± 0.33 hijk 56.57 ± 0.88 qr 89.90 ± 0.33 vwxyz 100.00 ± 0.00 y 30 μg 30.3 ± 0.58 no 71.72 ± 0.88 tuv 100.00 ± 0.00 z 100.00 ± 0.00 y isomenthol 10 μg 0.00 ± 0.00 a 14.14 ± 0.67 defghi 44.44 ± 0.33 mn 65.66 ± 1.20 nopq 20 μg 15.15 ± 0.58 ghijk 25.25 ± 0.88 jklm 59.60 ± 0.88 pqr 82.83 ± 1.76 rstuvwx 30 μg 29.29 ± 0.33 no 38.38 ± 1.20 nop 75.76 ± 1.73 stu 94.95 ± 1.20 wxy linalool 10 μl 2.02 ± 0.67 a 55.56 ± 0.88 qr 76.77 ± 0.33 stu 95.96 ± 0.88 xy 20 μl 6.06 ± 0.00 abcde 71.72 ± 0.67 tuv 91.92 ± 0.33 vwxyz 100.00 ± 0.00 y 30 μl 15.15 ± 0.58 ghijk 88.89 ± 0.88 yzαβγ 100.00 ± 0.00 z 100.00 ± 0.00 y menthol 10 μg 1.01 ± 0.33 a 9.09 ± 0.00 abcdefg 56.57 ± 0.67 opq 79.80 ± 0.88 qrstuv 20 μg 10.10 ± 0.67 bcdefgh 59.60 ± 0.33 rs 88.89 ± 0.33 vwxyz 100.00 ± 0.00 y 30 μg 18.18 ± 0.58 ijkl 72.73 ± 0.58 tuvw 100.00 ± 0.00 z 100.00 ± 0.00 y nerol 10 μl 0.00 ± 0.00 a 0.00 ± 0.00 a 3.03 ± 0.58 a 20.20 ± 0.88 cdef 20 μl 2.02 ± 0.33 a 3.03 ± 0.00 abcd 8.08 ± 0.33 abcd 39.39 ± 0.58 hij 30 μl 8.08 ± 0.33 abcdefg 12.12 ± 0.58 bcdefgh 20.20 ± 0.33 efghij 51.52 ± 0.58 jklmn insecticidal effects of monoterpenes against sitophilus zeamais 201 terpinen-4-ol 10 μl 43.43 ± 0.33 pq 83.84 ± 0.88 xyzα 96.97 ± 0.58 yz 100.00 ± 0.00 y 20 μl 83.84 ± 0.88 t 97.98 ± 0.33 βγ 100.00 ± 0.00 z 100.00 ± 0.00 y 30 μl 96.97 ± 0.58 vw 100.00 ± 0.00 γ 100.00 ± 0.00 z 100.00 ± 0.00 y α-terpineol 10 μg 19.19 ± 0.33 jkl 25.25 ± 0.33 jklm 45.45 ± 0.58 mno 69.7 ± 1.53 opqr 20 μg 40.40 ± 0.88 p 46.46 ± 1.33 pq 76.77 ± 1.76 stu 95.96 ± 0.88 xy 30 μg 54.55 ± 1.15 r 60.61 ± 1.00 rs 90.91 ± 1.15 vwxyz 100.00 ± 0.00 y epoxides (oxygenated monoterpenes) 1,8-cineole 10 μl 13.13 ± 0.33 efghij 75.76 ± 2.52 uvwx 89.90 ± 0.88 vwxyz 98.99 ± 0.33 y 20 μl 24.24 ± 1.15 lmn 89.90 ± 1.45 zαβγ 98.99 ± 0.33 z 100.00 ± 0.00 y 30 μl 40.40 ± 1.20 p 98.99 ± 0.33 γ 100.00 ± 0.00 z 100.00 ± 0.00 y limonene oxide 10 μl 84.85 ± 1.00 t 87.88 ± 1.00 yzαβ 91.92 ± 1.20 vwxyz 96.97 ± 1.00 xy 20 μl 89.90 ± 0.33 tuv 100.00 ± 0.00 γ 100.00 ± 0.00 z 100.00 ± 0.00 y 30 μl 98.99 ± 0.33 w 100.00 ± 0.00 γ 100.00 ± 0.00 z 100.00 ± 0.00 y ketones and aldehydes (oxygenated monoterpenes) camphor 10 μg 13.13 ± 0.33 efghij 37.37 ± 1.45 nop 75.76 ± 1.73 stu 91.92 ± 1.45 vwxy 20 μg 32.32 ± 0.33 o 67.68 ± 1.45 stu 94.95 ± 0.33 wxyz 100.00 ± 0.00 y 30 μg 47.47 ± 0.33 q 80.81 ± 1.20 vwxyz 100.00 ± 0.00 z 100.00 ± 0.00 y carvone 10 μl 89.9 ± 0.33 tuv 98.99 ± 0.33 γ 100.00 ± 0.00 z 100.00 ± 0.00 y 20 μl 93.94 ± 0.58 uvw 100.00 ± 0.00 γ 100.00 ± 0.00 z 100.00 ± 0.00 y 30 μl 100.00 ± 0.00 w 100.00 ± 0.00 γ 100.00 ± 0.00 z 100.00 ± 0.00 y citronellal 10 μl 0.00 ± 0.00 a 13.13 ± 0.33 cdefghi 53.54 ± 1.86 nop 70.71 ± 1.67 opqrs 20 μl 10.1 ± 0.88 bcdefgh 28.28 ± 0.67 klmn 83.84 ± 0.88 tuvw 98.99 ± 0.33 y 30 μl 19.19 ± 0.88 jkl 41.41 ± 0.88 op 95.96 ± 0.88 xyz 100.00 ± 0.00 y dihydrocarvone 10 μl 92.93 ± 0.67 uvw 98.99 ± 0.33 γ 100.00 ± 0.00 z 100.00 ± 0.00 y 20 μl 95.96 ± 0.88 vw 100.00 ± 0.00 γ 100.00 ± 0.00 z 100.00 ± 0.00 y 30 μl 100.00 ± 0.00 w 100.00 ± 0.00 γ 100.00 ± 0.00 z 100.00 ± 0.00 y fenchone 10 μl 72.73 ± 1.15 s 82.83 ± 0.33 wxyzα 88.89 ± 0.67 vwxyz 95.96 ± 1.33 xy 20 μl 96.97 ± 0.58 vw 100.00 ± 0.00 γ 100.00 ± 0.00 z 100.00 ± 0.00 y 30 μl 100.00 ± 0.00 w 100.00 ± 0.00 γ 100.00 ± 0.00 z 100.00 ± 0.00 y menthone 10 μl 87.88 ± 0.58 tu 91.92 ± 1.2 αβγ 98.99 ± 0.33 z 100.00 ± 0.00 y 20 μl 97.98 ± 0.33 w 100.00 ± 0.00 γ 100.00 ± 0.00 z 100.00 ± 0.00 y 30 μl 100.00 ± 0.00 w 100.00 ± 0.00 γ 100.00 ± 0.00 z 100.00 ± 0.00 y esters (oxygenated monoterpenes) bornyl acetate 10 μl 0.00 ± 0.00 a 3.03 ± 0.58 abcd 37.37 ± 1.45 klm 54.55 ± 2.08 klmn 20 μl 1.01 ± 0.33 a 6.06 ± 0.58 abcdef 67.68 ± 3.38 rs 84.85 ± 2.89 stuvwxy 30 μl 7.07 ± 0.33 abcdef 15.15 ± 0.58 efghij 86.87 ± 2.03 uvwxy 94.95 ± 1.67 wxy geranyl acetate 10 μl 2.02 ± 0.33 a 3.03 ± 0.00 abcd 12.12 ± 2.00 abcdefg 43.43 ± 2.33 hijk 20 μl 7.07 ± 0.88 abcdef 8.08 ± 0.67 abcdef 39.39 ± 0.58 lm 70.71 ± 2.91 opqrs 30 μl 16.16 ± 0.88 hijk 23.23 ± 0.88 ijkl 53.54 ± 0.88 nop 88.89 ± 1.76 uvwxy linalyl acetate 10 μl 0.00 ± 0.00 a 0.00 ± 0.00 a 9.09 ± 1.00 abcde 19.19 ± 0.67 bcde 20 μl 5.05 ± 0.33 abcd 7.07 ± 0.33 abcdef 10.1 ± 0.67 abcdef 32.32 ± 1.76 efgh 30 μl 13.13 ± 0.67 efghij 20.2 ± 0.33 ghijk 21.21 ± 0.58 fghij 47.47 ± 2.33 ijkl neryl acetate 10 μl 0.00 ± 0.00 a 14.14 ± 0.33 defghi 55.56 ± 1.20 nopq 72.73 ± 0.58 opqrst 20 μl 14.14 ± 0.33 fghij 41.41 ± 2.73 op 81.82 ± 1.53 tuv 96.97 ± 0.58 xy 30 μl 22.22 ± 0.67 klm 55.56 ± 2.85 qr 92.93 ± 1.20 vwxyz 100.00 ± 0.00 y a mean ± se of three replicates, each set-up with 33 adults b exposure time (h) values followed by different letters in the same column differ significantly at p < 0.01 tab. 1 (continued) treatments dose mean mortality (%)a 24b 48b 72b 96b 202 e. yildirim, b. emsen, s. kordali the most effective ones among twenty eight monoterpenes were carvone, dihydrocarvone, menthone and terpinen-4-ol with 100 % of mortality at all doses (96 h after treatment). other compounds that had strong fumigant insecticidal efficacy were 1,8-cineole, fenchone, linalool and limonene oxide according to ld values. ld50 and ld90 values at 96th h belong to 1,8-cineole were 1.989 and 4.808 μl; values belong to fenchone were 2.445 and 6.815 μl; values belong to linalool were 2.445 and 6.815 μl; values belong to limonene oxide were 3.235 and 6.957 μl, respectively (tab. 2). some of the previous studies demonstrated that in general, the toxicity of essential oils isolated from plant samples against stored pests was mainly related to their major components. these compounds are generally described as monoterpenes (banchio et al., 2005; kordali et al., 2006; lópez et al., 2011; kumar et al., 2012) and secondary metabolites (emsen et al., 2012a, 2012b; yildirim et al., 2012a, 2012b). the results in this study suggested that the monoterpenes obtained from different essential oils might have different toxicity ratios and these differences originate from chemical constituents which is unique to them. increasing use of natural insecticides will help to decrease the negative effects like toxicity to non-target animals, residue problems (environmental pollution) and insecticide resistance of synthetic or chemical insecticides. in this context, bio-insecticides may be also effective, bio-degradable, selective and associated with little advancement of resistance in the insect population and as a result, more safe to the environment. in this study, 96th h of exposure to the maximum dose (30 μl/μg) of borneol, camphor, 3-carene, carvone, 1,8-cineole, citronellal, dihydrocarvone, fenchol, fenchone, limonene oxide, linalool, menthol, menthone, neryl acetate, terpinen-4-ol, α-terpineol was determined to cause the highest mortality rate in s. zeamais adults. these compounds may be suggested to be potential insecticidal agents for controlling the adults of s. zeamais in stored food products. obtained results and those reported earlier clearly indicated the variations in the effects of monoterpenes in regard to the stage, the tab. 2: the 96 h ld50 and ld90 values of twenty eight monoterpenes to adults of sitophilus zeamais treatments ld50 (limits) ld90 (limits) slope ± se borneol 8.992 (7.401-10.241) 18.143 (16.120-21.441) 4.204 ± 0.587 bornyl acetate 9.161 (7.098-10.775) 23.493 (20.212-29.522) 3.133 ± 0.461 camphene 28.865 (25.517-34.504) 69.510 (52.436-113.778) 3.358 ± 0.489 camphor 4.867 (0.035-7.145) 9.365 (3.753-11.235) 4.508 ± 1.985 3-carene 4.815 (1.665-6.821) 11.159 (8.737-13.468) 3.511 ± 0.950 carvone a a 0.000 ± 0.000 1,8-cineole 1.989 ( b ) 4.808 ( b ) 3.343 ± 4.259 citronellal 8.086 (6.374-9.103) 13.240 (11.953-15.908) 5.984 ± 1.255 β-citronellene 9.144 (6.397-11.192) 31.359 (25.303-46.204) 2.394 ± 0.419 β-citronellol 21.939 (19.778-24.668) 51.737 (41.793-72.794) 3.440 ± 0.439 dihydrocarvone a a 0.000 ± 0.000 fenchol 7.395 (3.015-8.649) 11.481 (10.427-16.326) 6.708 ± 2.494 fenchone 2.445 ( b ) 6.815 ( b ) 2.879 ± 1.536 geranyl acetate 11.831 (9.669-13.617) 33.658 (27.754-46.197) 2.822 ± 0.411 isomenthol 6.903 (3.994-9.039) 24.293 (20.022-34.169) 2.345 ± 0.455 limonene 15.062 (12.671-17.293) 49.265 (37.635-79.876) 2.490 ± 0.391 limonene oxide 3.235 ( b ) 6.957 ( b ) 3.854 ± 2.795 linalool 2.445 ( b ) 6.815 ( b ) 2.879 ± 1.536 linalyl acetate 35.024 (26.940-64.394) 206.010 (94.329-1766.180) 1.665 ± 0.402 menthol 7.521 (3.403-8.722) 11.599 (10.546-16.492) 6.812 ± 2.482 menthone a a 0.000 ± 0.000 myrcene 53.801 (37.031-151.354) 286.547 (115.998-4201.509) 1.764 ± 0.451 nerol 28.207 (23.108-40.568) 136.787 (75.125-569.351) 1.869 ± 0.397 neryl acetate 7.364 (5.416-8.670) 14.099 (12.524-16.709) 4.543 ± 0.838 α-pinene 32.400 (26.251-48.564) 139.764 (78.071-531.672) 2.019 ± 0.413 β-pinene 26.365 (22.376-34.051) 99.263 (63.331-254.050) 2.220 ± 0.406 terpinen-4-ol a a 0.000 ± 0.000 α-terpineol 7.680 (5.858-8.958) 14.869 (13.220-17.589) 4.467 ± 0.764 a for this monoterpene no ld values are computed because the ratios of response counts to subject counts are the same, i.e. the slope is zero b slope is not significantly different from zero. ld fiducial limits cannot be computed insecticidal effects of monoterpenes against sitophilus zeamais 203 species of insect. otherwise, ddvp which is an effective chemical pesticide was used in this study and 10 μl ddvp with a standard of pesticide applications showed 100 % insecticidal activity on s. zeamais after 24 h. however, in recent years negative effects of chemicalcontaining pesticides as ddvp were investigated. excessive usage of such pesticides causes environmental pollution (tortelli et al., 2006; kopecka-pilarczyk, 2012). moreover, it was reported that using of ddvp increased the human cancer risk (maele-fabry and willems, 2004; koutros et al., 2008). therefore, tested essential oils proved to be promising as control alternatives against stored product insects especially, s. zeamais. additionally, there is no statistically (p < 0.01) significant difference between the 24 h results of ddvp and six monoterpenes (carvone, dihydrocarvone, fenchone, limonene oxide, menthone and terpinen-4-ol) (tab. 1). when considered from this point of view, it can be considered using botanical insecticides instead of chemical insecticides which have adverse effects on animals and/or humans. furthermore, one of the reasons to prefer botanical insecticides is cheaper compared to chemical insecticides. the present study demonstrates the possibility of using the test monoterpenes particularly carvone, 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(3th ed.). erzurum: atatürk university, agriculture faculty press, no: 191. yildirim, e., emsen, b., aslan, a., bulak, y., ercisli, s., 2012a: insecticidal activity of lichens against the maize weevil, sitophilus zeamais motschulsky (coleoptera: curculionidae). egypt. j. biol. pest co. 22, 151-156. yildirim, e., aslan, a., emsen, b., cakir, a., ercisli, s., 2012b: insecticidal effects of usnea longissima (parmeliaceae) extract against sitophilus granarius (coleoptera: curculionidae). int. j. agric. biol. 14, 303-306. zettler, j.l., arthur, f.h., 2000: chemical control of stored product insects with fumigants and residual treatments. crop prot. 19, 577-582. address of the authors: dr. erol yildirim (corresponding author) and dr. saban kordali, atatürk university, faculty of agriculture, department of plant protection, 25240, erzurum, turkey bugrahan emsen, karamanoğlu mehmetbey university, kamil özdağ faculty of science, department of biology, 70200, karaman, turkey journal of applied botany and food quality 86, 180 184 (2013), doi:10.5073/jabfq.2013.086.024 1 institute for resistance research and stress tolerance, sanitz 2 institute for resistance research and stress tolerance, quedlinburg effect of n-fertilization, fungicide treatment, seed density and abiotic stress factors on the total ß-glucan content of six-rowed winter barley (hordeum vulgare l.) gisela jansen1, edgar schliephake2, doris kopahnke2, frank ordon2 (received august 12, 2013) summary in germany, the first six-rowed waxy winter barley cultivar (cv.) ‘waxyma’ was registered in 2008. besides changes in starch composition, waxy barley is rich in ß-glucans offering new applications in the food industry as ß-glucans reduce the blood cholesterol level. to investigate the stability of the ß-glucan content, cv. ‘waxyma’, three waxy breeding lines and the non-waxy cv. ‘lomerit’ were grown in field trials, shelter-, green houseand growth chamber experiments. besides this, ‘waxyma’ was grown in field trials at varying nitrogen levels and different seed rates, both with and without fungicide treatment. waxyma showed a significant increase of the ß-glucan content under optimal nitrogen fertilization and fungicide treatment, but the influence of the seed density was not significant. under shelter and greenhouse conditions, the influence of drought stress during grain filling was analyzed. the increase of the ß-glucan content under drought stress in the shelter and under rising temperatures in growth chambers was only significant for the non waxy cv. ‘lomerit’ while no influence of drought stress was observed under green house conditions. in summary, the high ß-glucan content of cv. ‘waxyma’ seems to be relatively stable with respect to growing conditions making it a suitable raw material for human nutrition. introduction barley (hordeum vulgare l.) is an old crop for the brewing and feed industries. but high amounts of ß-glucans, especially soluble ß-glucans, which are the major soluble fiber component in cereals in which barley and oat are especially rich (aman and graham, 1987), cause problems in brewing and in animal nutrition (macgregor et al., 1993). comparisons of oat and barley revealed a similar (lee et al., 1997) or even higher ß-glucan content in barley than in oat (jeroch et al., 1999; strobel et al., 2001). due to the high ß-glucan content, which is supposed to have a positive effect on human health, barley is becoming more important in human nutrition (alminger et al., 2008), also in developed countries (güler, 2003). ß-glucans are valuable fibers which can lower the blood cholesterol level and reduce the risk of coronary heart disease. therefore, efsa released a health claim for barley (efsa, 2011). wood et al. (2003) found a higher ß-glucan content in waxy barley (6.1 %, having a high amylopectin content of 95 100 % in starch) than in conventional barley (4.5 % with approximately 75 % amylopectin content in starch). the same fact, that waxy cultivars of barley generally have a higher ß-glucan content is reported e.g. in spring waxy barley cv. ‘sindy’ and cv. ‘magdalena’ compared with normal cv. ‘annabell’, ‘kinnan’, ‘iver’ and ‘tyra’ at different temperatures during grain filling (anker-nilssen et al., 2008) and in winter waxy barley cv. ‘waxyma’ compared with normal winter barley (dieckmann et al., 2009). the ß-glucan content in barley is mainly determined by the genotype, but to some extent is also influenced by environmental factors (stuart et al., 1988; zhang et al., 2001). the ß-glucan content in barley cultivars varies between 3.5 % in spain (perez-vendrell, 1996) to around 4.5 % in canada (narasimhalu, 1995) and china (zang et al., 2002). besides this, the ß-glucan content in barley is influenced by nitrogen fertilization (güler et al., 2003; jackson et al., 1994), temperature during the growing period (anker-nilssen et al., 2008) and drought stress (coles et al., 1991). in contrast to this, no impact of nitrogen fertilization on the ß-glucan content was detected in oat (saastamoinen et al., 2004). the objective of this study was therefore (i) to determine the effect of different agricultural measures (n-fertilizer, seed rate and fungicide treatment) on the ß-glucan content of the german waxy barley cultivar ‘waxyma’ in field trials and (ii) to analyze the effect of abiotic stress factors (drought stress and different temperatures during the grain filling period) on the ß-glucan content on different cultivars in shelter-, greenhouseand growth chamber experiments in order to get information on the stability of the high ß-glucan content in waxy barley which is important with respect to human nutrition. materials and methods plant material seeds of cv. ‘lomerit’ (non-waxy) and cv. ‘waxyma’ (waxy) as well as three waxy breeding lines were supplied by dieckmann gmbh & co. kg (nienstädt, germany). in order to ascertain that in all experiments with a limited number of plants analysed, i.e. in green house and growth chamber experiments, only waxy kernels are included, they were examined by using a two-wavelength colorimetric method with sodium hydroxide as solvent according to jansen et al. (2011). field trials in 2009 and 2010 field experiments with respect to the impact of nitrogen fertilization on the ß-glucan content of cv. ‘waxyma’ were carried out in bernburg, germany (northern latitude: 51.82, eastern longitude: 11.78) at the professor hellriegel institute e. v. grain samples of four replications were collected from a randomized block design. nitrogen levels of 0 kg n, 60 kg n, 120 kg n (60 kg n + 60 kg n) and 180 kg n (60 kg n + 60 kg n + 60 kg n) were applied in plots of 12.0 m² with a seed rate of 300 grains/m². the n-fertilizer was applied at bbch-code 22/23 (tillers detectable), bbch-code 31/32 (node 2 and 3 at least 2 cm above node 1 and 2, respectively) and bbch-code 45-49 (booting). in 2010 and 2011 additional field experiments concerning the influence of different intensities, i.e. seed rate, nitrogen fertilization and fungicide treatment, were conducted with cv. ‘waxyma’ in plots of 10.2 m² and 13.3 m², respectively, in sülbeck (northern latitude: 52.30, eastern longitude: 9.15) at dieckmann gmbh & co. kg. the complete experimental design is described in tab. 1. nitrogen levels of 80 kg n, 130 kg n (70/60), 180 kg n (70/70/40/0), 180 kg n (70/70/0/40) and 230 kg n (70/90/0/60), seed rates of 300 grains/m² and 230 grains/m² with and without fungicide treatment were used. the n-fertilizer was applied at bbch-code 21(beginning of tillering), bbch-code 30 (beginning of stem elongation) and bbchcode 39 (flag leaf stage, end of stem elongation) or bbch-code 49 (first awns visible). ß-glucans in barley 181 shelter experiments in 2009, the influence of drought stress on the ß-glucan content was analyzed under field conditions in a rain-out shelter. four waxy barleys (cv. “waxyma” and three waxy breeding lines) and the non waxy barley cv. “lomerit” were grown in the rain out shelter and under irrigated field conditions in single rows containing about 20 plants/ 3m in 4 replications. terminal drought stress was applied by turning off the irrigation in the shelter at bbch-code 49 (first awns visible). green house experiments the drought stress experiment in the greenhouse was carried out with three waxy breeding lines and non waxy cv. ‘lomerit’. half (waxy breeding lines, see above) and whole grains (non waxy cv. ‘lomerit’) were germinated in planting trays and after germination at 20 °c plants were vernalized for six weeks at 4 °c. experiments were conducted with one plant per pot (13 x 13 cm in standard soil with a ph of 5.8). for fertilization, 1 kg npk-fertilizer (14 % n, 16 % p2o5, 18 % k2o) /m³ soil and 50 g micro nutrient fertilizer (b, cu, fe, mn, mo, zn) /m³ soil were applied. additionally for optimal nutrient supply all plants were fertilized with a leaf micronutrient fertilizer (2 x fetrilon, 2.5 g/l), calcium ammonium nitrate (1x4 granules/pot) and a liquid iron chelate fertilizer (wuxal super, 0.2 %, 1 x 30 ml/per pot). all pots (50 pots per variant) were randomly placed in the greenhouse at 20 °c and optimally supplied with water until flowering. from flowering to harvest, plants were cultivated in a control variant (60 % water capacity of the soil) and a stress variant (30 % water capacity of the soil). growth chamber experiments the temperature stress experiment was carried out using the same genotypes as in the greenhouse experiment in 2009 in growth chambers by applying 15 °c, 20 °c and 25 °c from the beginning of heading until harvest. after germination at 20 °c plants were vernalized for six weeks at 4 °c. one plant per pot each was transferred to a 13 x 13 cm pot filled with standard soil (50 pots per line or cv. and variant) and then cultured at 20°c until stress application. for fertilization plantosan ® compact was used. sample collection grain samples were taken from field-, shelter-, greenhouseand growth chamber experiments. ears of field plots and shelter experiments were threshed by a plot combine harvester. single ears of greenhouse and growth chamber experiments were threshed by a multi-chef rot (company baumann saatzuchtbedarf). the grains were dehulled using a compressed air oat peeler (company friedrich falke maschinenund mühlenbau) and ground using a rotor speed mill pulverisette 14 (company fritsch) to pass a 0.5 mm sieve. before estimating the quality parameters of the growth chamber and greenhouse experiments random grain samples of 50 plants per variant were combined to three composite samples. the generated whole meal with a humidity of 11 to 12 % was stored at 20 °c until analysis. analysis of total ß-glucans the content of total ß-glucan was determined according to mccleary and glennie-holmes (1985). for this, a mixed linkage test kit for the measurement of 1,3 and 1,4-ß-d-glucan in cereal grains (icc standard no. 166) from megazyme international ireland was used. the ß-glucan content is given in % in dry matter of whole meal (% in dm). statistical methods to assess the effects of different treatments and abiotic stress factors on the total ß-glucan content of six-rowed winter barley a generalized linear model for the analysis of variance was applied, using the glm procedure of the software package sas (version 9.3) followed by a tukey-test (α = 0.05) for comparing means. the data sets were analyzed separately. the influence of fungicide treatment was calculated with a covariance test. results the effects of different nitrogen levels on the ß-glucan content of the waxy barley cv. ‘waxyma’ were significant. increased nitrogen levels positively affected the ß-glucan content of ‘waxyma’ both in trials conducted in bernburg in 2009 and 2010 (fig. 1) and in field experiments in sülbeck in 2010 and 2011 (fig. 2). however, applying a high amount of nitrogen (220 kg n / ha) did not increase the ß-glucan content significantly compared with nitrogen levels of 180 kg n / ha and 130 kg n / ha (fig. 2). in the trials conducted in bernburg, the ß-glucan content varied between 5.1 and 5.6 % in dry matter (dm) in 2010 and between 5.2 and 5.9 % in dm in 2009 (fig. 1). in sülbeck the mean ß-glucan content varied between 5.4 and 5.8 % in dm in the years 2010 and 2011 (fig. 2). tab. 1: experimental design of field trials in 2010 and 2011 in sülbeck, variety ‘waxyma’, four replications. seed rate fungizide total nfirst nsecond nthird nfourth n (grains/m²) treatment fertilization application application application application (kg n/ha) bbch 21 bbch 30/31 bbch 37/39 bbch 47/49 300 without 80 80 300 optimal 130 70 60 300 optimal 180 70 70 40 300 optimal 180 70 70 40 300 optimal 220 70 90 60 230 without 80 80 230 optimal 130 70 60 230 optimal 180 70 70 40 230 optimal 180 70 70 40 230 optimal 220 70 90 60 182 g. jansen, e. schliephake, d. kopahnke, f. ordon different seed rates did not influence the ß-glucan content significantly (fig. 3a). but without fungicide treatment the ß-glucan content was significantly decreased compared with fungicide treatment (fig. 3b). mean values of ß-glucan content concerning seed rate varied between 5.5 and 5.7 % in dm. the lower ß-glucan content at higher seed rate was not significant. the lower ß-glucan content (from 5.6 % to 5.3 % in dm) without fungicide treatment was significant. both experiments, under drought stress conditions in the ‘shelter’ in 2009 (tab. 2) and under drought stress conditions in the green house in 2010 (tab. 3) showed no significant influence of drought on the ß-glucan content of waxy six-rowed barley. only the ß-glucan content of the non waxy cv. ‘lomerit’ increased significantly under drought stress in rain out shelter conditions. in addition the long-term temperature experiments with different temperatures of 15 °c, 20 °c and 25 °c between heading and harvest in growth chambers also had no significant effect on the ß-glucan content of waxy barley. again, only the non waxy cv. ‘lomerit’ showed a significant increase of the ß-glucan content with increasing temperatur (tab. 4). fig. 2: response of the ß-glucan content of waxy barley cultivar ‘waxyma’ in sülbeck (means of 2010 and 2011, different lower case letters indicate significant differences in ß-glucan contents). fig. 1: response of the ß-glucan content of the waxy barley cultivar ‘waxyma’ to n fertilization in bernburg (means of 2009/2010), different lower case letters indicate significant differences in ß-glucan contents). a b fig. 3: effect of seed rate and fungicide treatment on the ß-glucan content of waxy barley variety ‘waxyma’ in sülbeck (means of 2010/2011, different lower case letters indicate significant differences in ß-glucan contents). a: response of seed rate b: response of fungicide treatment tab. 2: mean ß-glucan content in the shelter (drought stress) and in the control field in 2009. cultivar or control stress p breeding line breeding line 1 5.4 5.5 0.6078 breeding line 2 5.4 5.9 0.1651 breeding line 3 5.7 5.8 0.8275 lomerit 4.1 4.6 0.0255 waxyma 4.9 5.5 0.1073 p ≤ 0.05 significant tab. 3: variance table of the drought stress experiment in the green house (2010). source degrees of mean square f-statistic pr > f freedom variety 3 2.8883 147.88 <.0001 stress 1 0.0069 0.35 0.5588 variety*stress 3 0.0050 0.26 0.8565 pr>f = 0.05 significant tab. 4: mean ß-glucan content in growth chamber experiments at different growth temperatures. cultivar or 15 °c 20 °c 25 °c breeding line breeding line 1 5.7 ± 0.1 a 5.7 ± 0.2 a 5.6 ± 0.7 a breeding line 2 6.0 ± 0.1 a 6.2 ± 0.2 a 6.0 ± 0.4 a breeding line 3 6.0 ± 0.1 a 6.0 ± 0.1 a 6.0 ± 0.1 a lomerit 5.6 ± 0.1 a 5.6 ± 0.2 a 6.5 ± 0.4 b statistical comparison line wise (different lower case letters indicate significant differences in ß-glucan contents) ß-glucans in barley 183 discussion concerning various nitrogen levels, the differences in the ß-glucan content of the german waxy barley ‘waxyma’ were significant. the highest grain ß-glucan content was obtained using 180 kg n / ha. however, the ß-glucan content differed only between 5.2 % and 5.9 %, so that an increase due to n-fertilization amount is only 0.7 %. this is in agreement with results obtained for the effect of nitrogen on the ß-glucan content by gühler et al. (2003) and jackson et al. (1994). gühler et al. (2003) found in a two-rowed barley cultivar a variation of the ß-glucan content between 4.8 and 5.8 % using nitrogen levels between 0 and 80 kg n / ha without irrigation. jackson et al. (1994) detected a variation of the the ß-glucan content of waxy hulless barley between 6.2 and 7.6 % in response to nitrogen fertilization between 0 kg n / ha and 101 kg n / ha. in contrast to this, saastamoinen et al. (2004) reported about a trial with finnish oat cultivars in which the n-fertilization did not increase the ß-glucan content. with respect to barley, it may be concluded from our results and those from literature that nitrogen fertilization (up to 180 kg n / ha) increases the ß-glucan content in barley grains. nothing is known in literature about the influence of the seed rate and fungicide treatment on the ß-glucan content of barley. our experiments show that the seed rate and a fungicide treatment influence the ß-glucan content of the six-rowed waxy barley ‘waxyma’ only very little. furthermore, no significant influence of drought stress on the ßglucan content was observed, although the ß-glucan content was slightly higher under drought stress. similar results were obtained by perez-vendrell et al. (1996), who detected also a slightly higher ß-glucan content under low rainfall conditions. in contrast to this coles et al. (1991) revealed that in the barley variety ‘triumpf’ the grain ß-glucan contents decreases with increasing drought stress. anker-nilssen et al. (2008) reported about the influence of different temperature during grain filling on the total ß-glucan content. the grain ß-glucan varied from 4.0 % to 7.4 % and was significantly affected by temperature. furthermore, significant interaction between cultivar and temperature during grain filling was observed. the waxy cultivar ‘cindy’ showed the highest ß-glucan content of 7.4 % at 18 °c and the lowest content of 5.8 % at 9 °c. ‘tyra’, a normal two-rowed barley cultivar varied between 4.6 % at 9 °c and 4.9 % at 21 °c. these results agree with our results for the non waxy cv. ‘lomerit’ in the long term stress experiment in which we detected a significant increase of the ß-glucan content with increasing temperature. in contrast to this applying different temperature only at the beginning of heading and beginning of watery ripe (reinhardt et al., 2013), led to a significant increase of the ß-glucan content only in one breeding line in the 30 °c variant in comparison to the 10 °c variant. savin et al. (1997) even detected a lower ß-glucan content at temperatures of 27 °c and 30 °c in comparison to 21 °c during grain filling of malting barley cultivars. the ß-glucan content of german six-rowed winter waxy barley can be increased by optimal agronomic measures, but overall nitrogen fertilization, optimal seed rate, fungicide treatment and stress conditions (temperature and drought) had only a little effect on the ßglucan. the high ß-glucan content of german six-row waxy barley cultivars and breeding lines seems to be relatively stable under different agronomic practices and stress situations and thus this cultivar maybe suited to produce barley which may be beneficially used in human nutrition. acknowledgements we thank dieckmann seeds gmbh & co. kg for providing all barley test seeds and the test material of their field experiments and prof. g. kratzsch from the professor hellriegel institute e. v. for providing the grain samples of their n-fertilization trials. the excellent technical assistance of margrit jugert, maika tobien and regina schmidt is acknowledged. this work was supported by the federal ministry of food, agriculture and consumer protection via the agency for renewable resources (research project fkz: 22019308). references alminger, m., eklund-jonsson, c., 2008: whole-grain cereal products based on a high-fibre barley or oat genotype lower post-prandial glucose and insulin responses in healthy humans. eur. j. nutr. 47, 294-300. aman, p., graham, h., 1987: analysis of total and insoluble mixed-linked (1-3), (1-4)-ß-d-glucans in barley and oats. j. agric. food chem. 35, 704-709. anker-nilssen, k., sahlstrom, s., knutsen, s.h., holtekjolen, a.k., uhlen, a.k., 2008: influence of growth temperature on content, viscosity and relative molecular weight of water-soluble ß-glucans in barley (hordeum vulgare l.). j. cereal sci. 48, 670-677. coles, g.d., jamieson, p.d., haslemore, r.m., 1991: effect of moisture stress on malting quality in triumph barley. j. cereal sci. 14, 161-177. dieckmann, k., jansen, g., ahlemeyer, j., 2009: waxy winter barley – high-yielding and rich in beta-glucans. c & e spring meeting 2009 – whole grain global summit, 25.-27.03.2009, newcastle, united kingdom, cereal foods world 54, 2, s. 24 abstract. efsa panel on dietetic products, nutrition and allergies (nda), 2011: scientific opinion on the substantiation of a heath claim related to barley beta-glucans and lowering of blood cholesterol and reduced risk of (coronary) heart disease pursuant to article 14 of regulation (ec) no 1924/2006, efsa journal 9, 2471. güler, m., 2003: barley grain ß-glucan content as affected by nitrogen and irrigation. field crop. res. 84, 335-340. jansen, g., ordon, f., knopf, e., dieckmann, k., 2011: rapid methods for selecting single kernels of waxy barley. j. appl. bot. food qual. 84, 6-10. jackson, g.d., berg, r.k., kushnak, g.d., blake, t.k., yarrow, g.i., 1994: nitrogen effects on yield, beta-glucan content, and other quality factors of oat and waxy hulless barley. commun. soil sci. plant anal. 25, 3047-3055. jeroch, h., drochner, w., simon, o., 1999: ernährung landwirtschaftlicher nutztiere. stuttgart: eugen ulmer-verlag bzw. utb für wissenschaft, 205. lee, c.j., horsley, r.d., manthey, f.a., schwarz, p.b., 1997: comparisons of ß-glucan content of barley and oat. cereal chem. 74, 571-575. macgregor, a.w., fincher, g.b., 1993: carbohydrates of the barley grain. in: macgregor, a.w., bhatty, r.s. (eds.), barley: chemistry and technology, 103-104. published by the american association of cereal chemists, inc. st. paul, minnesota, usa. mccleary, b.v., glennie-holmes, m., 1985: enzymic quantification of (1-3) (1-4)-ß-d-glucan in barley and malt. j. inst. brew 91, 285-295. narasimhalu, p., kong, d., choo, t.m., ferguson, t., therrien, m.c., ho, k.m., may, k.w., jui, p., 1995: effects of environment and cultivar on total mixed-linkage ß-glucan content in eastern and western canadian barleys (hordeum vulgare l.). can. j. plant sci. 75, 371-376. perez-vendrell, a.m., brufau, j., molina-cano, j.l., francesch, m., guasch, j., 1996: effects of cultivar and environment on ß-(1,3)(1,4)-d-glucan content and acid extract viscosity of spanish barleys. j. cereal sci. 23, 285-292. reinhardt, d., jansen, g., seddig, s., eichler-löbermann, b., 2013: temperature stress during flowering time affects yield and quality parameters of waxy barley. landbauforsch, appl. agric. forestry res. 1, 7984. saastamoinen, m., hietaniemi, v., pihlava, j.-m., eurola, m., 184 g. jansen, e. schliephake, d. kopahnke, f. ordon kontturi, m., tuuri, h., niskanen, m., kangas, a., 2004: ß-glucan contents of groats of different oat cultivars in official variety, in organic cultivation, and in nitrogen ferilization trials in finland. agr. food sci. 13, 68-79. strobel, e., ahrens, p., hartmann, g., kluge, h., jeroch, h., 2001: gehalt an inhaltsstoffen von weizen, roggen und hafer bei anbau unter konventionellen und den bedingungen des ökologischen landbaus. die bodenkultur 52, 221-231. stuart, i.m., loi, l., fincher, g.b., 1988: varietal and environmental variations in (1-3, 1-4)-ß-glucan levels and (1-3, 1-4)-ß-glucanase potential in barley relationships to malting quality. j. cereal sci. 7, 61-71. wood, p.j., weisz, j., beer, m.u., newman, c.w., newman, r.k., 2003: structure of (1-3)(1-4)-ß-d-glucan in waxy and nonwaxy barley. cereal chem. 80, 329-332. zhang, g., chen, j., wang, j., ding, s., 2001: cultivar and environmental effects on (1-3, 1-4)-ß-d-glucan and protein content in malting barley. j. cereal sci. 34, 295-301. zhang, g., junmei, w., jinxin, c., 2002: analysis of ß-glucan content in barley cultivars from different locations of china. food chem. 79, 251254. address of the authors: gisela jansen, institute for resistance research and stress tolerance, rudolfschick-platz 3, 18190 sanitz edgar schliephake, doris kopahnke, frank ordon, institute for resistance research and stress tolerance, erwin-baur-strasse 27, 06484 quedlinburg journal of applied botany and food quality 87, 87 92 (2014), doi:10.5073/jabfq.2014.087.013 department of food engineering, faculty of engineering, erciyes university, kayseri, turkey physical, chemical and bioactive properties of onion (allium cepa l.) seed and seed oil hasan yalcin*, hatice kavuncuoglu (received january 14, 2014) * corresponding author summary in this study, some physico-chemical and antioxidant properties, volatile compounds and fatty acid composition of ten different onion seeds were investigated. the fatty acid composition and volatile compounds were analyzed by gas chromatography (gc) and gas chromatography-mass spectrometry (gc-ms), respectively. physicochemical analysis showed that onion seeds possessed high amount of oil (21.86 %-25.86 %) and crude protein (15.7 %-26.1 %). gc results revealed that onion seed oil was rich in linoleic acid (49.4260.66 %) which was followed by oleic and palmitic acid, respectively. there was a large number of substances such as hydrocarbons, alcohols, acids, esters and sulfur-containing compounds. it could be concluded that onion seeds can be utilized in food industry due to their physico-chemical properties and contents of oil and volatile components. introduction onion (allium cepa l.) is considered to be one of the most important agricultural products all over the world. the species belongs to the family of liliaceae and represents one of the world’s oldest cultivated products (tram ngoc et al., 2005). according to the data of 2010, turkey is number 6 at onion production in the world with annual production of 1.9 million tonnes (fao, 2010). onion has different nutritional composition depending on the individual parameters such as type, maturity level etc. (mota et al., 2010). onion seeds are also eaten, but their commercial availability is currently limited. perhaps if consumers were more acquainted with onion seeds’ nutritional and functional properties, there would be an increase in trade of this product (dini et al., 2005, dini et al., 2008a). dini et al. (2008b) studied red onion seeds and they found 10.5 % moisture, 20.4 % oil, 24.8 % crude protein in these seeds. onion bulbs represent a source of cysteine derivatives, which make them a functional food, but onion seeds contain only low concentration of these components. their presence should be important for obesity treatment because they improve glycemic control, cause decrease of food intake and induce adipose tissue cell death (lu et al., 2011; roldan et al., 2008). parry et al. (2006) determined the refractive index of onion seed oil as 1.4752. in terms of the composition of fatty acids of seed oils, 6.4-7.1 % palmitic acid (c16:0), 24.8-26 % oleic acid (c18:1) and 65.2-64 % linoleic acid (c18:2) was found in onion seeds by them. in recent years, there exists an increasing interest to find new sources of edible oils such as plant seeds due to their nutritional, industrial and medicinal importance (nehdi, 2011a). seed oils rich in bioactive constituents which have protective effects against diseases and support to improve human health are highly demanded by consumers. on the other hand, no oil is sufficient for all purposes because of different compositions of oils obtained from various sources. in terms of the increasing demand on the nutritional and functional properties of oils and scientific awareness, quality assessment of presently unused oils attract special attention (cerchiara et al., 2010; silva et al., 2009; djenontin et al., 2009; nehdi, 2011b; kesari et al., 2010; hoed et al., 2011). numerous studies related to various onion cultivars have been described in literature but those studies specially focused on the chemical and functional properties of onion seeds are very rare. therefore, the main purpose of this study is the presentation of the variety of physico-chemical properties of onion seeds with the determination of fatty acid composition and volatile compounds. onion seeds used in this study were adapted varieties in turkey. unfortunately, no literature about these turkish adapted varieties of onion seeds is available. materials and methods plant material onion seeds of 10 different cultivars, namely cv. guntan, cv. polat, cv. hasat, cv. tan8, cv. imrali kirmizisi, cv. beyaz sogan, ertan1, cv. guntan moru, cv. akgun12, and cv. kantartopu3 were used in this study. the seeds were obtained from commercial onion seed producers in balikesir (in turkey) and ataturk garden cultures central research institute in yalova (in turkey). sample extraction for antioxidant test onion seeds were powdered and 10 g defatted powder was extracted with 50 ml ethanol (1:5 w/w) for 18 hours at 25 ºc (cold extraction) in the dark. the extracts were centrifuged at 4,100 rpm for 15 minutes, then filtrated and the supernatants was collected subsequently. after filtration, the clear supernatants were evaporated under vacuum at 50 ºc. after this treatment, the dry extracts were preserved at +4 ºc. determination of moisture, ash, crude protein and oil content of the onion seed the recommended methods of the association of official analytical chemists (anon, 1990) were adopted to determine the levels of moisture, ash, crude protein and crude oil. the moisture content was determined by drying of the samples at 105 ºc to a constant weight. the ash content was detected by a laboratory furnace at 550 ºc, the temperature was increased gradually. nitrogen content was determined by using the dumas method (anon, 2000) and multiplied by the factor of 6.25 to obtain the crude protein content. crude oil was detected by the soxhlet method. crude oil was obtained by exhaustively extracting 10 g of each sample in a soxhlet apparatus using petroleum ether (boiling point range 40-60 ºc) as the extractant. each measurement was performed in triplicate and the results were averaged. determination of the fatty acid composition of the onion seed oil one hundred milligram of each of the oil samples was saponified with 100 ml 2 n koh, and 3 ml hexane was added to this mixture. the solution was strongly shaken with a vortex mixer for 1 min, and then centrifuged at 5,000 rpm for 5 min (nüve nm 110, turkey). 88 h. yalcin, h. kavuncuoglu one milliliter of the solution was put into gc vials and the injection was started immediately. gc (agilent 6890), equipped with an flame ionization detector (fid) and a 100 m × 0.25 mm i.d. hp-88 column was used. the injection block temperature was set at 250 ºc. the oven temperature was kept at 103 ºc for 1 min, then programmed from 103 to 170 ºc at 6.5 oc/min, from 170 to 215 ºc for 12 min at 2.7 oc/min, and finally, at 230 ºc for 5 min. carrier gas was helium with a flow rate of 2 ml/min; the split rate was 1/50 (anon, 2008; yalcin et al., 2012). three replications were applied to determine the fatty acid composition of each oil. determination of refractive index of the onion seed oils the refractive index of the onion seed oils were determined at 20 °c using an abbe refractometer (reichert ar 700). the measurements were performed in triplicate and the results were averaged. volatile component analysis of the onion seed analysis was performed according to the procedure described by yalcin et al. (2011) by gas chromatography mass spectrometry (agilent 7890a gas chromatography system) using a mass selective detector (agilent technologies) and hp-5ms column (60 m × 0.250 mm internal diameter; film thickness, 0.25 μm). the oven temperature was held at 40 ºc for 10 minutes, heated to 95 ºc at 3 ºc/min, heated from 95 to 210 ºc at 10 ºc/min, and finally increased to 210 ºc/min and held for 10 minutes. carrier gas was helium with a flow rate 0.5 ml/min. the voltage of electron ionization detector was 70 ev. the compounds adsorbed by the fibers were desorbed from the injection port for 15 minutes at 50 ºc in the splitless mode. the compounds were identified by comparison with spectra from the libraries flavor 2, nist 05a, and wiley7nist05 and by using internal standards (krist et al., 2006). determination of total phenolic content of the onion seed total phenol estimation in the extract was determined by the folinciocalteu colorimetric method (sagdic et al., 2011). to each tube, 2,400 μl distilled water was added and followed by 40 μl extracts and 200 μl folin-ciocalteu reagent. after 5 minutes, 600 μl sodium carbonate (20 %) and finally 760 μl distilled water were added. the solution was homogenized in a vortex mixer and incubated at room temperature for 2 hours in the dark. after incubation, absorbance of the reaction mixture was measured at 765 nm. the amount of the total phenolic compounds was expressed as gallic acid equivalents (gae) in milligrams per gram dry plant extract. the measurements were performed in triplicate and the results were averaged. determination of antiradical activity of the onion seed the dpph (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity was adapted by some modifications from the method used by tulukcu et al. (2012). 100,000 ppm solution of extracts was used as stock solution. sequences of extract concentration in methanol, that is 2,000, 1,000, 500 ppm, were prepared, and 100 μl of each concentration of extract was added to 450 μl tris-hcl and 3,500 μl 0.1 mm methanol solution of dpph. methanol was used as a control. following a 30-min incubation period at room temperature in the dark, the absorbance was measured against the blank at 517 nm. the measurements were performed in triplicate and the results were averaged. inhibition of the free radical dpph in percent (i %) was calculated using the following equation: radical scavenging activity (%) = [(a0 a1) / a0] × 100 a0 = the absorbance of the control a1 = the absorbance of the sample. statistical analysis sas statistical software (sas institute, inc., cary, nc, usa) was used for data analysis. data were subjected to one-way and twoway anova, and the comparative analyses between means were conducted by using the tukey multiple range test. results and discussion proximate analysis the proximate composition was shown in tab. 1. these values were compared to corresponding data for allium tuberosum seed (hu et al., 2006). 7.8 % moisture, 2.4 % ash, 15.8 % oil, 12.3 % crude protein was determined in allium tuberosum seeds. onion seeds which are used in our study, have high levels of ash, oil and protein, but comparatively low moisture content. the high total oil content is important for evaluation of seed oil, high content of crude protein is important for quality assessment which will be obtained from the seed pulp and high content of ash is important in that it involves higher value of inorganic material. demirkaya and sivritepe tab. 1: chemical composition of different onion seed cultivars (%) cultivar moisture ash oil crude protein polat 7.34±0.11 d 4.80±0.18 a 24.56±0.68 a, b, c 23.96±0.73 b, c hasat 9.01±0.16 b 4.38±0.37 a, b, c 21.86±1.40 c 26.16±0.33 a guntan moru 8.48±0.15 c 4.28±0.60 a, b, c, d 23.72±0.14 c 24.07±0.15 b, c beyaz sogan 8.53±0.06 c 4.68±0.13 a, b 24.47±0.63 a, b, c 22.59±0.09 d, e guntan 8.57±0.25 c 4.01±0.25 b, c, d 25.11±0.78 a, b 21.39±0.11 f, g tan8 9.79±0.02 a 3.58±0.02 d 25.38±1.12 a 22.23±0.86 e, f kantartopu3 7.17±0.06 d 3.75±0.15 d, c 25.86±0.13 a 15.70±0.16 h akgun12 7.06±0.09 d 3.68±0.04 d, c 24.14±1.15 a, b, c 20.57±0.04 g ertan1 6.49±0.05 e 3.59±0.02 d 22.29±3.65 b 23.47±0.03 c, d imrali kirmizisi 7.21±0.06 d 4.06±0.06 b, c, d 21.91±0.06 c 24.74±0.03 b mean ± standard deviation different letters indicate the statistical difference in the same column (p<0.05) physical, chemical and bioactive properties 89 tab. 2: fatty acid composition of seed oils obatained from different onion cultivars (%) fatty kantar imrali güntan akgun12 polat tan8 ertan1 hasat beyaz guntan acid topu 3 kirmizisi sogan moru c14:0 1.78±0.06 1.22±0.02 1.29±0.05 1.47±0.06 1.74±0.01 1.23±0.18 c16:0 12.2±0.09 10.79±0.04 10.89±0.03 10.52±0.05 11.08±0.12 10.83±0.21 7.27±0.03 8.95±0.09 10.24±0.01 7.23±0.15 c18:0 3.74±0.09 2.93±0.04 3.29±0.02 3.18±0.09 3.47±0.09 3.28±0.09 2.79±0.19 2.56±0.12 2.4±0.06 2.01±0.05 c18:1 28.72±0.01 27.05±0.03 27.81±0.09 27.4±0.01 26.44±0.14 28.05±0.09 31.52±0.01 26.59±0.06 28.74±0.06 28.35±0.09 c18:2 49.42±0.02 53.12±0.01 51.79±0.08 53.25±0.00 57.25±0.08 52.51±0.1 58.4±0.08 56.5±0.06 55.27±0.09 60.46±0.04 c22:1 2.94±0.01 3.43±0.09 3.57±0.05 2.87±0.03 3.03±0.08 3.67±0.08 2.23±0.02 1.32±0.04 c22:2 1.17±0.00 1.44±0.08 1.34±0.01 1.28±0.03 1.04±0.04 1.7±0.21 1.07±0.03 0.62±0.07 sat 17.72 14.94 15.47 15.17 16.29 15.34 10.06 11.51 12.64 9.24 mufa 31.66 30.48 31.38 30.27 26.44 31.08 31.52 30.26 30.97 29.67 pufa 50.59 54.56 53.13 54.53 57.25 53.55 58.4 58.2 56.34 61.08 total 99.45 99.98 99.98 99.97 99.98 99.97 99.98 99.97 99.95 99.99 mean ± standard deviation sat:total saturated fa (g/100g oil); mufa:total monounsaturated fa (g/100g oil); pufa: total polyunsaturated fa (g/100g oil). (2011) found that 24.5 % oil and 20.8 % crude protein was determined in cv. akgun12. in another study (dini et al., 2008b), chemical analysis of allium cepa l. var. tropeana (red onion) seeds showed 10.5 % moisture, 20.4 % oil and 24.8 % crude protein. we found 6.49-9.79 % moisture content in our samples, the highest level in cv. tan8. considering the oil content, samples contained high amounts of oil, the lowest level with 21.86 % in cv. hasat and the highest total oil content with 25.86 % in cv. kantartopu3. seeds contained high amounts of crude protein as well as oil. the lowest amounts of crude protein with 15.7 % was detected in cv. kantartopu3 which has the highest level in oil content. the highest amounts of crude protein with 26.16 % was determined in cv. hasat which has the lowest level of oil. fatty acid composition and refractive index tab. 2 shows the fatty acid composition of the analysed onion seed oils. onion seed oils contained about 9.24-17.72 g of saturated fatty acids, 26.44-31.66 g of monounsaturated fatty acids and 50.5961.08 g of polyunsaturated fatty acids per 100 g total fatty acids. these seed oils had high pufa contents. in these seed oils cv. guntan moru, was characterized by a high polyunsaturated/saturated (p/s) ratio of 6.6. a high ratio of p/s is regarded affirmative for the reduction of serum cholesterol and atherosclerosis and prevention of heart diseases (oomah et al., 2002; yehuda, 2001). hence, cv. guntan moru is the most suitable onion seed for the oil industry. the main fatty acid of this seed oils was linoleic acid (18:2n-6) at a level of 49.42-60.46 %. while cv. guntan moru has the highest amount of linoleic acid, cv. kantartopu3 has the smallest amount of this fatty acid. the cold-pressed onion seed oil had 64-65 % and indian onion seed oil contained 45 % linoleic acid (rao, 1994). various reports suggested that coldpressed oils present higher yield compared to oils obtained by solvent extraction (zia-ul-haq et al., 2007). in oil ındustry linoleic acid rich oils such as sunflower and soybean oils are highly demanded. the second abundant fatty acid is oleic acid (18:1n-9) which has the highest amount in cv. ertan1 (31.52%) and the lowest amount in cv. polat (26.44%). the oleic acid level is lower compared to the value (34 %) reported by rao (1994) and higher (25-26 %) than formerly reported by parry et al. (2006). linoleic acid content varies depending on the environmental conditions rather than the genetic background. as in many oily seeds, while reducing the amount of linoleic acid, an increasing amount of oleic acid has been observed in our samples. there is a complementary proportion between oleic and linoleic acids in these fatty acid rich oils (yalcin et al., 2011). in onion seed, the most abundant fatty acids are unsaturated fatty acids which comprise approximately no less than 80 % of all the fatty acid in the onion seed. saturated fatty acids were found in small amounts. palmitic and stearic acids were the most abundant saturated fatty acids but the total amount of these two fatty acids was not more than 16 %. palmitic and stearic acids exist in onion seed at a ratio of 7.23 % (cv. guntan moru), 12.2 % (cv. kantartpu3), 2.01 % (cv. guntan moru), and 3.74 % (cv. kantartopu3), respectively. the palmitic acid level is higher than the 6.4-7.1 % reported by parry et al. (2006). according to the specification described in the turkish food codex, the ratio of the linoleic acid of the cotton seed and soybean oils are 46.7-58.2 % and 48.0-59.0 %, respectively and also the limits of oleic acids for these two oils are 14.7-21.7 % and 17.0-30.0 %, respectively. the linoleic acid proportions of our onion seeds oils tab. 3: total phenolic contents and dpph scavenging capacity of onion seed cultivars cultivar total phenolic contents % inhibitions (mg gae/g dry extract) polat 2.01±0.21 f 4.59±0.71 c hasat 1.84±0.33 f 5.25±0.91 c guntan moru 2.36±0.25 f, e 13.57±0.93 b beyaz sogan 3.04±0.24 d, e 6.34±1.62 c guntan 4.14±0.40 c 8.89±4.05 b, c tan8 2.35±0.40 f, e 13.58±1.03 b kantartopu3 3.22±0.47 d, c, e 6.99±0.68 c akgun12 3.51±0.21 c, e 7.76±1.41 c ertan1 6.87±0.48 a 38.26±2.07 a imrali kirmizisi 5.35±0.23 b 4.69±0.54 c mean ± standard deviation different letters indicate the statistical difference in the same column (p<0.05) 90 h. yalcin, h. kavuncuoglu were similar to these limits. we can say that onion seed oil is similar to cotton seed and soybean oils in this regard. cotton seed oil has less amount of oleic and high amount of palmitic acid than the onion seed oil. corn oil has a similar linoleic acid content but compared with our results it has high amounts of oleic acid according to the food codex mentioned above. the refractive index values, a frequently standard parameter used for characterization of oils, ranged from 1.4555 to 1.4771. refractive index values of onion seed oils were found in the following order: 1.4555 (cv. polat) < 1.4624 (cv. hasat) < 1.4632 (cv. guntanmoru) < 1.4699 (cv. tan8) < 1.4700 (cv. ertan1) < 1.4701 (cv. akgun12) < 1.4702 (cv. guntan) < 1.4713 (cv. beyaz sogan) < 1.4727 (cv. kantartopu3) < 1.4771 (cv. imrali kirmizisi). the refractive index value of cold-pressed onion seed oils is 1.4752 reported by parry et al. (2006). total phenolic contents and dpph scavenging capacity total phenolic contents and dpph scavenging capacity of the onion seeds are shown in tab. 3. the total pheolic content of the onion seeds range from 1.84 to 6.87 mg gallic acid equivalents per gram dry extract (mg gae/g dry extract). analyses of dpph scavenging capacity showed that onion seeds have 13.58-38.26 % inhibition at concentration of 100,000 μg/g. a synthetic antioxidant, bha shows 38.26 % inhibition at a concentration of 395 μg/g. this is a prove that the presence of compounds are not reactive towards dpph (przybylski et al., 1998). it is in agreement with the results reported by gazzani et al. (1998), that the antioxidant activity of vegetables increased by boiling because of pro-oxidant activity of peroxidases which were inactivated at high temperatures. considering the total phenolic content and dpph radical scavenging activity, the antioxidant activity is low in these onion seeds. volatile components composition of the volatile components in onion seeds are shown in tab. 4. as to be seen here, a total of 48 compounds were detected in the volatile fraction of onion seeds. the highest percentage of the individual compounds is represented by 2-phenylethanol, an alcohol, with rates varying from 0.42 % to 41.17 %. only in cv. tan8 this substance was not detected and here 1-hexanol, varying from 5.85 % to 31.61 % , was detected as main component in the volatile fraction. moreover, beside these, other alcoholic substances were determined such as 1-pentanol, 2-heptanol, 2-octanol, 1-octen-3-ol, 1-heptanol, phenylmethanol, 1-dodecanol. 1-pentanol, 2-heptanol, 1-octen-3-ol and phenylmethanol were detected in all samples and 1-dodecanol was only determined in cv. hasat accounting 0.66 %. in the volatile fraction, hydrocarbons have been also identified. aromatic hydrocarbons such as p-xylene and mesitylene, ranging from 0.13 to 0.90 % and 0.10 to 0.85 %, respectively, were found. limonene as a monocyclic monoterpene varied from 0.75 to 3.17 % in the samples. the cyclic monoterpene α-phellandrene was determined in cv. akgun12, cv. imrali kirmisizi and cv. polat accounting 3.42, 0.17 and 1.43 %, respectively. a bicyclic monoterpene, pcymene was detected in all samples except cv. tan8. hexylacetate, nonylacetate, ethyloctanoate, methylhexanoate were the main esters determined in onion seeds. nonylacetate was detected only in cv. hasat with a concentration of 5.44 %. the characteristic flavor compounds occurring in the individual species of the genus allium are dominated by various biologically active organosulfur substances (gyawali et al., 2006). onion seeds in this study, contained especially carbondisulfide, 4-phenyl-2thiazolethiol, methylsulfonylmethane, and dipropyl disulfide. the most abundant carbon disulfide was determined in cv. polat achieving a concentration of 4.74 %. conclusion the higher total oil content and the fatty acid composition rich in polyunsaturated fatty acids of onion seed oil makes it desirable in terms of nutrition and the oil may be used as edible oil. however, the safety of this oil must be tested before possible applications for human consumption are discussed. furthermore, volatile aroma components have beed found in onion seed oils, which could be of special interest for the production of natural aroma extracts applying appropriate extraction techniques. acknowledgement this research was supported by erciyes university scientific research projects unit (project number is fby-11-3398). the authors would like to thank this unit, the ataturk garden cultures central research institute and the commercial onion seed 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akgun12 beyaz ertan1 guntan hasat tan8 kantarimrali guntan polat sogan topu3 kirmizisi moru carbondisulfide — 0.02 0.59 — 0.04 0.39 — — 0.12 4.74 2,2,4,6,6-pentamethylheptane 2.68 3.53 — — — — — — — — chloroform 0.53 — — — — — 0.58 0.35 0.85 0.05 2-phenylethanol 32.65 0.42 41.17 17.71 19.77 — 29.31 19.78 25.83 29.20 α-phellandrene 3.42 — — — — — — 0.17 — 1.43 p-xylene 0.61 0.13 0.29 0.35 — — 0.36 0.90 0.18 — methylhexanoate 0.60 0.38 — — 0.53 0.51 0.31 — 0.29 — limonene 1.23 0.75 0.75 0.82 2.90 — 0.56 — 3.17 2.90 acetophenone 0.58 — — 0.19 — — 0.34 — — 0.13 2-pentylfuran 2.57 4.82 0.33 1.06 2.54 3.86 1.82 1.19 1.56 1.21 1-pentanol 1.60 3.09 2.56 1.99 1.62 2.36 1.71 1.41 1.87 0.91 styrene 1.70 2.13 3.25 2.68 0.86 0.26 1.00 0.52 1.41 0.93 p-cymene 0.53 0.12 0.22 0.28 0.54 — 0.03 0.07 0.75 0.83 mesitylene 0.85 — 0.18 — — — 0.30 0.11 0.10 — 2-heptanol 0.24 0.89 0.34 0.35 1.51 1.65 0.20 0.20 0.70 0.19 1-hexanol 8.97 26.31 16.42 14.96 15.78 31.61 12.12 10.91 14.25 5.85 2-octanol 0.26 — 2.67 0.24 0.32 0.77 0.27 0.18 0.56 0.29 1-octen-3-ol 0.96 0.92 2.23 0.66 0.44 0.98 0.92 0.61 0.69 0.65 methoxycyclooctane 0.61 — — — — 0.96 — — — — 1-heptanol 0.15 1.53 — 0.93 0.95 2.82 0.38 0.36 0.73 0.15 1-anthracenamine 0.30 — 0.17 0.61 — — 0.54 0.61 0.63 0.42 (e)-2-hepten-1-ol 0.40 — — — — 1.02 1.14 0.56 — 0.08 (e)-2-octen-1-ol 0.44 3.01 0.54 1.41 — 2.68 — — 1.31 0.59 γ-hexalactone 0.15 0.27 — — 0.16 0.24 — 0.11 — — methoxyphenyloxime 0.58 1.92 1.28 1.30 0.48 0.58 0.94 1.73 0.98 1.34 hexanoic acid 0.66 1.59 0.41 1.29 1.55 0.46 1.19 1.24 1.25 0.97 phenylmethanol 0.17 0.54 1.02 0.83 0.44 0.56 0.36 0.34 0.45 0.34 naphthalene 1.16 0.23 2.55 — 0.27 0.21 0.74 0.73 0.24 0.38 n-octadecane 0.67 0.77 0.18 0.93 0.50 0.74 0.88 0.33 0.76 — icosane 0.51 0.24 0.45 0.75 1.15 0.40 0.84 0.25 0.80 0.98 toluene — 0.96 0.23 — 0.05 0.10 — 4.15 — 6.60 ethyloctanoate — 0.27 — — 0.53 0.44 — — — — hexylacetate — 0.21 — — 0.94 0.42 — — 0.87 0.65 sulfurous acid — 0.08 — 0.10 — — — — 0.12 0.15 4-phenyl-2-thiazolethiol — 0.49 0.96 — 0.36 0.42 — 0.85 — — nonanol — 2.05 1.52 0.41 0.50 0.99 0.42 0.22 0.78 0.15 1-dodecanol — — — — 0.66 — — — — — hexane — — 1.91 2.50 3.32 — 1.65 3.35 0.79 2.94 γ-nonalactone — 0.62 — — 1.00 1.25 — 0.07 — — methylsulfonylmethane — 0.12 — — 0.14 — — 0.09 — — dodecane — — — — 3.11 — — 0.88 — — dipropyl disulfide — — — 0.48 0.14 0.40 — — 0.16 — nonylacetate — — — — 5.44 — — — — — linalool — — — — 0.20 — — — 0.48 0.36 dodecanal — — — — 2.27 0.35 2.09 1.24 — — 3-methylbutanoic acid — — — — — 0.44 2.28 — — — phenol — — — 0.34 — 0.26 — — — — total % identified 65.79 58.41 82.28 53.17 70.98 58.14 63.29 53.52 63.14 65.76 92 h. yalcin, h. kavuncuoglu convective drying of onion: kinetics and nutritional evaluation. food bioprod. process. 88, 115-123. nehdi, i.a., 2011a: characteristics and composition of washingtonia filifera (linden ex andré) h.wendl. seed and seed oil. food chem. 126, 197202. nehdi, i., 2011b: characteristics, chemical composition and utilization of albizia julibrissin seed oil. ind. crop. prod. 33, 30-34. oomah, b.d., busson, m., godfrey, d.v., drover, j.c.g., 2002: characteristics of hemp (cannabis sativa l.) seed oil. food chem. 76, 33-43. parry, j., hao, z., luther, m., su, l., zhou, k., yu, l., 2006: characterization of cold-pressed onion, parsley, cardamom, mullein, roasted pumpkin, and milk thistle seed oils. j. am. oil chem. soc. 83, 847-854. przybylski, r., lee, y.c., eskin, n.a., 1998: antioxidant and radicalscavenging activities of buckwheat seed components. j. am. oil chem. soc. 75, 1595-1601. rao, p.u., 1994: nutrient composition of some less-familiar oil seeds. food chem. 50, 379-382. roldan, e., sa’nchez-moreno, c., cano, m.p., 2008: characterisation of onion (allium cepa l.) by-products as food ingredients with antioxidant and antibrowning properties. food chem. 108, 907-916. sagdic, o., silici, s., ekici, l., 2011: evaluation of the phenolic content and antiradical, antioxidant, antimicrobial activities of different floral sources of honey. int. j. food prop. 16, 658-666. silva, s.m., sampaio, k.a., taham, t., rocco, s.a., ceriani, r., meirelles, a.j.a., 2009: characterisation of oil extracted from buriti fruit (mauritia flexuosa). j. am. oil chem. soc. 86, 611-616. tram ngoc, l., chiharu, h., makoto, s., hiromune, a., koji, k., ryo, v.y., 2005: antioxidative compounds from the outer scales of onion. j. agr. food chem. 53, 8183-8189. tulukcu, e., yalcin, h., ozturk, i., sagdic, o., 2012: changes in the fatty acid compositions and bioactivities of clary sage seeds depending on harvest year. ind. crop. prod. 39, 69-72. yalcin, h., ozturk, i., hayta, m., sagdic, o., gumus, t., 2011: effect of gamma-irradiation on some chemical characteristics and volatile content of linseed. j. med. food 14, 1223-1228. yalcin, h., toker, o.s., ozturk, i., dogan, m., kisi, o., 2012: prediction of fatty acid composition of vegetable oils based on rheological measurements using nonlinear models. eur. j. lipid sci. tech. 114, 1217-1224. yalcin, h., ozturk, i., tulukcu, e., sagdic, o., 2011: influence of the harvesting year and fertilizer on the fatty acid composition and some physicochemical properties of the linseed (linum usitatissimum l.). j. consumer protection and food safety 6, 197-202. yehuda, s., 2001: physiological and behavioral functions. humana press, totowa. zia-ul-haq, m., ahmad, m., iqbal, a., ahmad, s., ali, h., 2007: characterization and compositional studies of oil from seeds of desi chickpea (cicer arietinum l.) cultivars grown in pakistan. j. am. oil chem. soc. 84, 1143-1148. demirkaya, m., sivritepe, h.o., 2011: physiological and biochemical changes occur in onion seeds during ageing. j. agr. sci 17, 105-112. address of the authors: hasan yalcin, department of food engineering, faculty of engineering, erciyes university, 38039 kayseri, turkey e-mail: hsnyalcin@hotmail.com hatice kavuncuoglu, department of food engineering, faculty of engineering, erciyes university, 38039 kayseri, turkey journal of applied botany and food quality 88, 241 248 (2015), doi:10.5073/jabfq.2015.088.035 1natural products research department, national center for radiation research and technology, atomic energy authority, cairo, egypt 2 food irradiation department, national center for radiation research and technology, atomic energy authority, cairo, egypt 3agricultural research department, nuclear research center, atomic energy authority, abou zaabal qualiobeya, egypt effect of chitosan on biochemical composition and antioxidant activity of minimally processed ‘wonderful’ pomegranate arils during cold storage ahmed abdelhamid zahran1*, ramadan attia hassanein2, ahmed taha abdelwahab3 (received august 8, 2015) * corresponding author summary developing postharvest protocols incorporating nonchemical compounds with the aim of extending shelf life and maintaining quality of fresh produce is of great importance. the potential of chitosan (irradiated and unirradiated) in preserving pomegranate arils (cv. wonderful) during cold storage was investigated in this trial. solid chitosan powder was gamma irradiated with 25 and 50 kgy doses, chitosan solutions were prepared and arils were treated prior to cold storage. initial arils quality characteristics were assessed before refrigeration at 5 oc and rh 75 % for up to 15 day, during which studied characteristics were determined trice, with a 5 day interval. different treatments were applied by immersion in one of the following for 2 min with: control (distilled water); 0.5 %, unirradiated chitosan; 0.5 %, 25 kgy irradiated chitosan; 0.5 %, 50 kgy irradiated chitosan; 1.0 %, unirradiated chitosan; 1.0 %, 25 kgy irradiated chitosan; 1.0 %, 50 kgy irradiated chitosan solutions). results revealed that chitosan treatments reduced weight loss and controlled reductions in total soluble solids, titratable acidity, vitamin c and anthocyanin contents. increased total antioxidant activity was also detected at the end of the cold storage period and was accompanied with increased total phenolic compounds. such findings suggest that chitosan can be beneficial in extending shelf life and maintaining biochemical quality of fresh cut fruits. introduction pomegranate (punica granatum l.) belongs to the punicacea family and is one of the oldest known edible fruits. it is an important and commercial horticultural fruit which originated in the middle east before it spread in the mediterranean region and subtropical areas. its fruits are of great importance because of its biological properties (antimicrobial, antioxidant, anticancer, anti-inflammatory) attributed to bioactive compounds in extracts obtained from several parts of the plant. that’s why pomegranate extracts have been used in therapeutics, such as in the prevention of infection, inflammation, cancer, among other applications, beside the nutritional value of arils. pomegranate is a non-climacteric fruit that does not ripen after harvest, and that’s why, it’s harvested when fully ripened after showing optimal organoleptic characteristics. arils, the edible parts of pomegranates make up approximately 50 % of the fruit weight and are made up of 76-85 % juice and 24-15 % seeds (varasteh et al., 2012). among the factors that affect the composition of pomegranate juice and the content of bioactive compounds are, postharvest conditions, storage, and processing, besides genetics, fruit maturity, environmental and agronomic factors (miguel et al., 2004; poyrazoglu et al., 2002). over the past few years, drastic increases in world trading of pomegranate fruits were noticed as a result of the growing awareness to its nutritional value. meanwhile, several physiological and enzymatic disorders occur during cold storage causing quality loss. never the less, loss of aril color, vitamin c, and acidity were reported, which were accompanied by reduction of acceptability in terms of freshness, juiciness, and taste (artés et al., 1998 and nanda et al., 2001). thus, the development of appropriate postharvest protocols and treatments are required to maintain quality during handling and transport of fresh produce. moreover, there has also been an increasing interest in the development of new pomegranate derived food products such as minimally processed pomegranate seeds (“ready-to-eat”) and products derived from it. that’s why, maintaining aril quality after peeling of low commercial value fruits (small size, cracked, bruised, sun-burnt, noncommercial varieties) is of great importance, since it allows the use of fruits that cannot be commercialized in the freshfruit market in spite of having arils with good quality juice and seeds. there is a growing emphasis on environmentally friendly post harvest technologies that maintain produce quality, and among those, satisfactory results have been reported for using natural compounds such as chitosan as a safe alternative to hazardous chemicals with negligible risk to human health and environment. moreover, from a biological perspective, chitosan and its oligomers are very attractive for agricultural applications. in this regard, it is well know that when chitosan is subjected to gamma irradiation, chain scissions occur through the breakage of β (1-4) glycosidic bonds bonding the glucosamine and n-acetylglucosamine units that constitute chitosan. in solid state, such scissions are mainly due to the direct effect of ionizing radiation (tahtat et al., 2012). the degradation occurs through the formation of macro radicals that produce low molecular weight polymeric chains (gryczka et al., 2009). fresh-cut products are particularly susceptible to weight loss and pathogenic activity owing to the removal of plant protective tissues, which results in reduced bioactive constituents and shelf-life reduction due to speedy deterioration. the aim of this study is to investigate the effect of edible irradiated and unirradiated chitosan coating on keeping quality of ‘wonderful’ pomegranate arils during cold storage, particularly nutritive (titratable acidity and total soluble solids) and functional properties (ascorbic acid, total anthocyanin, total antioxidant activity and total phenolic compounds). materials and methods this study was conducted during the two successive seasons of 2013 and 2014 on pomegranate cv. ‘wonderful’ grown in a commercial orchard located on cairo/alexandria desert road. fruits were harvested at commercial maturity and transported immediately to the central laboratory in the horticultural research institute, giza governorate, where intact, physically sound fruits of uniform size were selected for this investigation. after washing whole fruits in sterilized water with 200 μl l−1 sodium hypochlorite (naocl) solution using a brush, fruits were cut in half with a sharpened knife along the equator and all fruits that showed physiological disorders 242 a. abdelhamid zahran, r.a. hassanein, a.t. abdelwahab (browning or pitting of arils and internal surfaces or arils paleness) were discarded, then fruits were manually peeled and arils were separated from the husk, combined, well mixed to assure uniformity, and divided to 7 portions, one for each treatment. high molecular weight solid chitosan powder (>75 % deacetylated) obtained from sigmaaldrich was gamma irradiated with 0, 25 and 50 kgy doses at the national center for radiation research and technology (ncrrt) using a self-contained dry-storage gamma irradiator (indian gamma cell ge 4000a) that uses 60co as a radiation source. chitosan treatments/solutions were prepared according to the procedure described by jiang et al. (2005). solutions prepared were control (distilled water); 0.5 %, unirradiated chitosan; 0.5 %, 25 kgy irradiated chitosan; 0.5 %, 50 kgy irradiated chitosan; 1.0 %, unirradiated chitosan; 1.0 %, 25 kgy irradiated chitosan; 1.0 %, 50 kgy irradiated chitosan, solutions. arils were treated with one of the previous solutions by immersion for 2 min before they were dried, packed in self-sealed 250 g pp trays and refrigerated at 5 oc and rh 75 % for up to 15 day, during which studied characteris tics were determined trice, with a 5 day interval. initial aril quality evaluation was conducted right after aril extraction and before treatments and cold storage were applied (zero time). for each treatment, (3 replicates * 250 g) were used to determine weight loss percentage. juice samples required for biochemical evaluations were manually extracted from another (3 replicates * 250 g) using hand pressure and filtration through cheesecloth. • weight loss (%) was determined by measuring the difference between initial and final weight of each replicate and results were expressed as a percentage loss of initial weight. • total soluble solids (tss) (%) was determined in juice directly extracted from arils with a carl zeiss hand refractometer (aoac, 2003). • ph was measured in juice directly extracted from arils with a ph meter (model: mettler-toledo model, d6 101-sc, switzerland) (aoac, 2003). • titratable acidity (ta) (%) was calculated as citric acid percentage (dominant organic acid in pomegranates) as described in aoac (1990), where 1 ml of fruit juice was titrated with 0.1 m naoh using phenolphthalein as an indicator and the percentage was calculated as follows: ml of naoh × normality × 0.067 titratable acidity % = × 100 ml juice used • vitamin c was determined and expressed in mg ascorbic acid 100 ml-1 fruit juice, according to aoac (1990). • total anthocyanin content was determined by the phdifferential method described by lee et al. (2005) using 2 buffer systems: potassium chloride buffer, ph 1 (0.025 m), and sodium acetate buffer, ph 4.5 (0.4 m). the sample was diluted with the corresponding buffer and the absorbance was measured at 520 and 700 nm. total anthocyanin content was calculated as cyanidin-3glucoside according to the following equation: a × mw × df × 1000 total anthocyanins (mg l-1) = ε × 1 where a = (a520−a700) ph1−(a520−a700) ph 4.5; mw = 449.2 g mol-1 for cyanidin-3-glucoside; df = dilution factor; i = path length in cm; ε = 26900 molar extinction coefficient in l mol-1 cm-1 for cyanidin-3-glucoside; 1000 = conversion from g to mg. • total antioxidant activity (mg ascorbic acid eq. 100 g-1) and total phenolic compounds (mg gallic acid eq. 100 g-1) : for each sample, 5 g of arils were homogenized in 10 ml of 50 mm phosphate buffer (ph 7.8) and then centrifuged at 10 000 g for 15 min at 4 °c. the supernatant was used for total antioxidant activity (taa) and total phenolic compounds quantification in duplicate, as previously described (serrano et al., 2005). for total anti oxidant activity, l-ascorbic acid was used for the calibration curve, and the results were expressed as mg ascorbic acid eq. 100 g-1 fw. the total phenolic compounds were expressed as mg gallic acid eq. 100 g-1 fw. statistical analysis the experiment was laid out using a completely randomized block design (crbd). three replicates per treatment were evaluated for aril quality attributes. experimental data obtained was treated with analysis of variance (anova) at confidence level of 95 %, which is the procedure used for testing the differences among means of two or more treatments and the differences between means were detected using least significant difference (lsd) at p < 0.05. all data was analyzed using statistical software (mstatc 2.10, russell d. freed). results weight loss (%): results presented in fig. 1 show that like other fresh produce, pomegranate arils lost weight during cold storage. throughout the whole experiment, all chitosan treatments reduced weight loss compared to the control but differences were not always statistically significant. unirradiated 1 % chitosan was the only treatment which significantly reduced weight loss at all times. at the end of the storage period, all 1 % chitosantreated arils loss significantly less weight compared to control arils in both investigated seasons while 0.5 % chitosan treatments were significantly effective in this regard in season 2014 only. on the other hand, results reveal that irradiating chitosan generally reduced its moisture-maintaining effect, but such negative impact was statistically insignificant most of the time. tss (%): as shown in tab. 1, tss in arils generally decreased as cold storage proceeded. it was also found that chitosan treatments played a positive role in maintaining tss, though statistical significance was not always detected. in season 2013, all arils treated with 1 % chitosan and unirradiated 0.5 % chitosan recorded significantly high tss contents compared to control arils after 10 days of refrigerated storage, though this effect did not persist till the end of the storage period. contrarily, in the latter season, all chitosan treatments recorded significantly high tss contents compared to untreated control arils and this effect persisted at the end of the storage period. it was also noticed that when treatments were of statistical significance, irradiation dose effect was trivial, but still, it’s worth mentioning that irradiating chitosan reduced its effect in controlling tss decreases. ph: as show in tab. 2, chitosan treatments significantly affected ph values of minimally processed pomegranate after 15 days of cold storage. a similar effect was detected, 10 days of cold storage in season 2014 only. in all three incidents of statistical significance, different treatments showed insignificant differences in-between. the only treatment which resulted in a significantly lower ph value compared to the control in both seasons at the end of storage period was the unirradiated 1 % chitosan treatment. other treatments controlled ph increments, but its effect was statistically insignificant. ta (%): results presented in fig. 2 show that ta decreased gradually as storage proceeded. it was found that all investigated treatments controlled acidity drops compared to the control throughout the whole trial, though statistical significance was not always detected. moreover, 1 % chitosan treatments were more efficient in maintaining ta compared to 0.5 % treatments. on the other hand, irradiating chitosan had a negative effect in this regard because it led to bigger ta drops compared to unirradiated chitosan. effect of chitosan on biochemical composition and antioxidant activity of pomegranate arils 243 vitamin c: as shown in tab. 3, vitamin c reductions were recorded as storage proceeded. it was also found that all chitosan treatments controlled such reductions, though statistical significance was detected only in season 2014 after 15 days of cold storage. at that time, all treatments except the 50 kgy irradiated 0.5 % chitosan treatment, resulted in significantly high vitamin c content compared to the control. as for the effect of irradiation on chitosan, it seemed to limit its effect in controlling vitamin c reduction during cold storage. total anthocyanin content: results presented in tab. 4 show that anthocyanin content dropped as storage proceeded. it also shows that chitosan played a positive role in controlling decrements and fig. 1: effect of irradiated and unirradiated chitosan on arils weight loss (%) during cold storage. columns in the same group bearing a common letter are insignificantly different at p<0.05. tab. 1: effect of irradiated and unirradiated chitosan on arils juice tss (%) during cold storage. treatments storage period (season 2013) storage period (season 2014) 0 days 5 days 10 days 15 days 0 days 5 days 10 days 15 days control 18.2 18.0 17.0 c 15.8 17.9 17.2 16.3 b 15.5 d chitosan 0.5 % + 0 kgy 18.1 17.7 ab 16.9 17.8 17.4 a 16.3 bc chitosan 0.5 % + 25 kgy 18.0 17.6 abc 16.6 17.6 17.4 a 16.3 bc chitosan 0.5 % + 50 kgy 18.0 17.4 bc 16.3 17.6 17.3 a 16.1 c chitosan 1.0 % + 0 kgy 18.2 17.9 ab 17.2 17.9 17.6 a 16.8 a chitosan 1.0 % + 25 kgy 18.1 17.9 ab 17.2 17.8 17.6 a 16.7 ab chitosan 1.0 % + 50 kgy 18.0 18.1 a 17.0 17.6 17.6 a 16.6 ab means in the same column bearing a common letter / no letters are insignificantly different at p<0.05. tab. 2: effect of irradiated and unirradiated chitosan on arils juice ph value during cold storage. treatments storage period (season 2013) storage period (season 2014) 0 days 5 days 10 days 15 days 0 days 5 days 10 days 15 days control 3.30 3.34 3.36 3.39 a 3.22 3.25 3.32 a 3.34 a chitosan 0.5 % + 0 kgy 3.33 3.34 3.36 ab 3.24 3.26 b 3.29 ab chitosan 0.5 % + 25 kgy 3.33 3.34 3.36 ab 3.25 3.27 ab 3.29 ab chitosan 0.5 % + 50 kgy 3.34 3.34 3.37 ab 3.25 3.27 ab 3.31 ab chitosan 1.0 % + 0 kgy 3.31 3.32 3.33 b 3.23 3.25 b 3.27 b chitosan 1.0 % + 25 kgy 3.32 3.33 3.34 ab 3.24 3.26 b 3.27 b chitosan 1.0 % + 50 kgy 3.32 3.33 3.34 ab 3.24 3.26 b 3.28 b means in the same column bearing a common letter / no letters are insignificantly different at p<0.05. 244 a. abdelhamid zahran, r.a. hassanein, a.t. abdelwahab maintaining anthocyanins in arils. although this role was more pronounced when arils were treated with 1 % chitosan compared to 0.5 % treatments, but statistically significant differences were not detected. similarly, insignificant decreases in anthocyanins were recorded when chitosan was irradiated prior to aril treatments. antioxidant activity: results presented in fig. 3 show that total antioxidant activity increased as storage progressed. it also shows that all chitosan treatments resulted in increased antioxidant acti vity compared to the control. this effect was more pronounced when 1 % chitosan treatments were used, compared to 0.5 % chitosan treatfig. 2: effect of irradiated and unirradiated chitosan on arils juice ta (%) during cold storage. columns in the same group bearing a common letter are insignificantly different at p<0.05. tab. 3: effect of irradiated and unirradiated chitosan on arils juice vitamin c content (mg ascorbic acid 100 ml-1 juice) during cold storage. treatments storage period (season 2013) storage period (season 2014) 0 days 5 days 10 days 15 days 0 days 5 days 10 days 15 days control 125.2 118.4 110.0 c 100.1 118.6 110.7 102.3 95.0 d chitosan 0.5 % + 0 kgy 120.1 117.9 ab 112.8 111.9 108.5 101.1 b chitosan 0.5 % + 25 kgy 120.1 117.2 ab 111.2 112.1 106.4 99.0 bc chitosan 0.5 % + 50 kgy 119.4 115.6 b 107.1 111.1 105.3 96.4 cd chitosan 1.0 % + 0 kgy 123.3 121.8 a 116.6 116.5 111.7 107.2 a chitosan 1.0 % + 25 kgy 121.4 120.8 a 114.2 116.0 111.1 105.6 a chitosan 1.0 % + 50 kgy 121.0 121.6 a 113.5 114.4 107.5 101.4 b means in the same column bearing a common letter / no letters are insignificantly different at p<0.05. tab. 4: effect of irradiated and unirradiated chitosan on arils juice total anthocyanin content (mg cyanidin-3-glucoside eq. l-1) during cold storage. treatments storage period (season 2013) storage period (season 2014) 0 days 5 days 10 days 15 days 0 days 5 days 10 days 15 days control 312.5 301.4 296.5 c 292.0 b 318.1 307.1 300.7 b 295.3 chitosan 0.5 % + 0 kgy 306.2 301.4 abc 299.8 ab 312.0 306.6 ab 302.9 chitosan 0.5 % + 25 kgy 304.5 300.8 abc 298.9 ab 310.2 304.7 ab 299.9 chitosan 0.5 % + 50 kgy 305.1 299.5 bc 297.6 ab 307.6 303.0 ab 299.7 chitosan 1.0 % + 0 kgy 311.8 309.3 a 305.4 a 317.8 311.9 a 307.6 chitosan 1.0 % + 25 kgy 309.3 307.4 ab 305.3 a 315.1 311.8 a 305.6 chitosan 1.0 % + 50 kgy 307.5 307.0 ab 303.9 a 311.0 308.6 ab 304.1 means in the same column bearing a common letter / no letters are insignificantly different at p<0.05. effect of chitosan on biochemical composition and antioxidant activity of pomegranate arils 245 ments. moreover, irradiating chitosan prior to aril treatments proved to be beneficial in this regard. total phenols: results presented in tab. 8 show that total phenols increased as storage proceeded. all investigated treatments resulted in increased phenols but statistical significance was detected only after 10 days of cold storage in season in 2013 and 15 days of cold storage in season 2014. in these two incidents, increases resulting from 50 kgy irradiated 0.5 % chitosan were insignificantly higher from the control, unlike the other five treatments which recorded significantly high phenol contents compared to control arils. as shown, higher chitosan concentration was correlated with higher phenol contents. it was also found that irradiating chitosan was correlated with lower phenol contents compared to unirradiated chitosan. tab. 5: effect of irradiated and unirradiated chitosan on arils juice total phenols (mg eq. gallic acid 100 g-1) during cold storage. treatments storage period (season 2013) storage period (season 2014) 0 days 5 days 10 days 15 days 0 days 5 days 10 days 15 days control 90.2 91.4 94.5 e 96.0 96.4 99.0 104.0 106.2 e chitosan 0.5 % + 0 kgy 96.1 100.7 c 103.4 103.2 108.1 112.2 cd chitosan 0.5 % + 25 kgy 95.4 99.8 cd 101.6 101.9 107.8 110.8 cd chitosan 0.5 % + 50 kgy 92.6 96.8 de 98.6 100.2 104.4 108.4 de chitosan 1.0 % + 0 kgy 102.3 106.9 a 111.3 105.8 113.8 119.1 a chitosan 1.0 % + 25 kgy 101.9 106.3 ab 111.0 104.7 113.1 118.4 ab chitosan 1.0 % + 50 kgy 98.5 102.7 bc 107.6 103.0 108.4 114.5 bc means in the same column bearing a common letter / no letters are insignificantly different at p<0.05. fig. 3: effect of irradiated and unirradiated chitosan on arils juice total antioxidant activity (mg ascorbic acid eq. 100 g-1) during cold storage. columns in the same group bearing a common letter are insignificantly different at p<0.05. discussion gradual increases in arils weight loss as storage progressed agrees with results previously reported for pomegranates by caleb et al. (2013) and salama et al. (2012). this is probably due to arils respiratory activity, transpiration and some oxidation processes (ayranci and tune, 2003). maximum moisture loss in untreated control arils might be due to high rate of respiration and transpiration (abbasi et al., 2009) and the absence of the semipermeable coating formed as a result of chitosan treatments that blocks pores and lessens permeability to water vapour and gases. the positive role chitosan played in this regard was similar to its effect reported by zhelyazkov et al. (2014) who found that chitosan retarded water loss and preserved effectively the quality and extended the shelflife of fresh-cut apples. on the other hand, jinasena et al. (2011) reported that both irradiated and unirradiated chitosan treatments which showed an insignificant difference in between, significantly reduced weight loss during cold storage of banana. ibrahim et al. (2014) and quynh’ et al. (2003) also reported that irradiated and unirradiated chitosan-treated pineapple and apple fruits, respectively, maintained moister content significantly better than control fruits. expected higher viscosity of 1 % compared to 0.5 % chitosan solutions investigated in this trial most probably formed a denser coat around ails, which might be the reason for better weight maintaining properties recorded for the higher chitosan concentration. unlike our findings, ghasemnezhad et al. (2010) attributed higher apricot fruit weight loss in response to high chitosan concentrations to probable anaerobic respiration. on the other hand, expected reduced viscosity of irradiated chitosan resulting from reduced mw in response to irradiation (gryczka et al., 2009) might also explain its reduced efficiency in controlling weight loss. 246 a. abdelhamid zahran, r.a. hassanein, a.t. abdelwahab our findings are in line with results of salama et al. (2012) who reported that tss in pomegranate juice decreased as storage proceeded. meanwhile, ayhan and eştürk (2009) reported that tss remained unchanged for the first nine days of cold storage, but they affirmed tss reductions in arils afterwards. as for the effect of chitosan on tss, sritananan et al. (2005) reported that chitosan treatments did not affect soluble solid content in mangosteen fruits, which partially agrees with our results. contrarily, zhelyazkov et al. (2014) reported that chitosan coating controlled increases in soluble solid content in fresh-cut apple. moreover, our results also contradict fresh-cut nectarines tss increases in response to chitosan treatments reported by chiabrando and giacalone (2013) who found that the untreated control treatment recorded a tss decrease after 5 days of cold storage. our obtained results might be indirectly attributed to chitosan’s inhibitory effect on respiration and other bioactivities occurring in arils that consume sugars which are a main constituent of tss. accordingly, the expectedly higher inhibitory effect associated with 1 % chitosan compared to 0.5 % chitosan investigated in this trial maybe the reason for higher tss records for juice extracted from 1% chitosan-treated arils. ph increases found in this trial contradict slight ph decreases reported by ayhan and eştürk (2009) for pomegranates. such increases may be due to acids breakup with respiration. moreover, the insignificant ph differences in response to different chitosan concentrations and irradiation doses which might be due to limited effects on respiration. generally, ph change is associated with seve ral reasons, i.e.; it might be due to a change in biochemical conditions and slower respiration rate and metabolic activity (jitareerat et al., 2007) and the consumption of acids in respiration during storage. on the other hand, chitosan’s significant role in controlling ph increases was reported earlier in fresh-cut nectarines by chiabrando and giacalone (2013). contrarily, ibrahim et al. (2014) and jinasena et al. (2011) reported that chitosan’s effect on ph in banana and pineapple, respectively, was insignificant. here, it is worth mentioning that as expected, since chitosan-treated arils in this study showed less variation in titrable acidity, the associated variation in their ph was also relatively lower than the control. ta reductions found in this investigation were reported earlier for several other fruits including pomegranates (salama et al., 2012) such as pine apple (ibrahim et al., 2014) and fresh-cut nectarines (chiabrando and giacalone, 2013). in this regard, ayhan and eştürk (2009) found that arils ta decreased, especially at the third day, but they reported that it stayed constant afterwards. our results agree with the findings of caleb et al. (2013) who reported significant decreases in ta. acidity reductions may be due to the conversion of organic acids to sugars and their further utilization in respiration and metabolic processes (abbasi et al., 2009; ibrahim et al., 2014). ibrahim et al. (2014) stated that chitosan coating can develop an oxygen barrier on fruit surface leading to reduced metabolic rates and consequently, less acidity variation in chitosan-treated fruits. it is assumed that such effect is concentration dependent because better ta maintenance was always correlated with 1 % rather 0.5 % chitosan treatments. chiabrando and giacalone (2013) attributed acidity retention in response to chitosan to the expected lower respiration rate, and hence, reduced organic acids (substrates) consumption for many reactions during aerobic respiration. moreover, higher acidity values are also due to the effect of acid utilized in the film forming solution. on the other hand, although statistical significance was mostly undetectable, irradiated chitosan was found to be less efficient in maintaining aril juice ta compared to unirradiated chitosan. this contradicts what has been reported by lan et al. (2000) who stated that irradiated chitosan delayed fruits internal changes more than unirradiated chitosan. they attributed that to increased deacetylation and reduced molecular weight of irradiated compared to unirradiated chitosan (kume and takehisa, 1982). our results related to the amount of vitamin c are supported by the findings of ibrahim et al. (2014) who reported decreases in pine apple vitamin c during storage, but in their study, increases in vitamin c were recorded before the decreases occurred. they also reported a higher ascorbic acid reduction rate for uncoated fruits compared to chitosan coated fruits. the reason for high vitamin c content in chitosan treatments can be attributed to limited oxygen supply caused by the barrier effect imposed by chitosan, which causes oxidation of ascorbic acid (malundo et al., 1997). such effect was more pronounced in juice extracted from arils treated with 1 % chitosan compared to that extracted from 0.5 % chitosan-treated arils. the results also convene with the findings of jiang and li (2001) who found that ascorbic acid content decreased when longan fruit was coated with chitosan at low temperature. it is also worth mentioning that ruoyi et al. (2005) reported that 1 % chitosan associated with other treatments significantly inhibited ascorbic acid oxidases (asa-pod) and kept vitamin c in refrigerated peach fruits at a high level. on the other hand, irradiation reduced chitosan’s efficiency in controlling vitamin c reductions but statistical significance was rarely detected. this might be due to the abundance of reducing functional groups (primary –oh, secondary –oh and −nh2) of chitosan resulting from the breakdown of high mw to lower mw chitosan by irradiation which provided inappropriate conditions for the oxidation of ascorbic acid. apparent anthocyanin drops recorded in this study were similar to those reported earlier by ayhan and eştürk (2009), salama et al. (2012) and caleb et al. (2013). although 1 % chitosan and unirra diated chitosan treatments showed better anthocyanin-keeping properties compared to 0.5 % and irradiated chitosan treatments, but neither concentrations nor irradiation doses studied showed significant differences in-between. in this regard, gil et al. (1996) found that anthocyanins were insignificantly altered during the first week of cold storage and lopez-robira et al. (2005) recorded steady anthocyanin content after 13 days of refrigerated storage of early harvested pomegranates. here, it is worth mentioning that cyanidin 3-glucoside was reported to be the major anthocyanin pigment in wonderful and other several pomegranate cultivars (gil et al., 1996). on the other hand, jiang et al. (2005) observed that 2 % chitosan delayed the decrease in anthocyanin content and the increase in ppo activity in litchi fruits. it has also been demonstrated to have bene ficial effects in maintaining anthocyanin content in several fruits such as longan fruit (jiang and li, 2001) and peeled litchi fruit (dong et al., 2004). zhang and quantick (1998) reported that this might be attributed to the barrier effect the chitosan coating imposes on the surface of the produce resulting in the modification in its endogenous co2 and o2 levels, which could result in a reduced o2 supply required for the enzymatic oxidation reaction of anthocyanin. here it is worth mentioning that anthocyanin synthesis continues in harvested fruit even at low storage temperatures, and postharvest treatments may affect anthocyanin biosynthesis, degradation, or both (holcroft et al., 1998; holcroft and kader, 1999; goncalves et al., 2007). that’s why adopting protocols and procedures that enhances anthocyanin content in arils is of great importance because in addition to its colourant properties, da costa et al., 2000 reported that it exhibits a wide range of biological, pharmacological, antiinflammatory, antioxidative, and chemoprotective properties. in contrast with our general findings, varasteh et al., 2012 recorded minor anthocyanin content increases in chitosan-coated pome granates after the first 45 days of refrigerated storage. moreover, they found that chitosan treated pomegranates showed less anthocyanin content compared to control fruits at this stage. in this regard, el ghaouth et al. (1991) indicated that using chitosan coating decelerated anthocyanin synthesis in treated strawberries. miguel et al., 2004 added that increases in anthocyanin levels might be related to changes in fruit internal atmospheric conditions. such in effect of chitosan on biochemical composition and antioxidant activity of pomegranate arils 247 creases in anthocyanin concentration after harvest during cold storage have been reported for fruits other than pomegranates such as sweet cherry (goncalves et al., 2007) and raspberry (han et al., 2004). miguel et al., 2004 stated that this was correlated with the activity of the anthocyanin biosynthesis enzymes. ayhan and eştürk (2009) stated that there was a positive relationship between antioxidant activity (%) and total phenolic content, indicating the effect of polyphenols on antioxidant activity. mirdehghan et al. (2006) reported that total antioxidant activity was correlated primarily to the high levels of total phenolics and to lesser extent to ascorbic acid and anthocyanin contents (which recorded decreases as storage progressed in this trial). in this regard, ocloo et al. (2012) reported that irradiated chitosan in solutions exerted slightly faster bacterial inhibition compared to unirradiated chitosan. moreover, kumar et al. (2007) reported higher antibacterial activity and feng et al. (2008) reported higher antioxidant activity for lower molecular weight chitosan, which is in harmony with our findings. on the other hand, 1 % chitosan treatments recorded better results compared to 0.5 % treatments. in this regard, ghasemnezhad et al. (2010) reported that 0.50 % chitosan was the most effective concentration in increasing total antioxidant capa city in apricots compared to control, 0.25 and 0.75 % treatments. the authors attributed that result to high total phenolic content. this might be attributed to what ruoyi et al. (2005) reported regarding the positive effectiveness of chitosan associated with other treatments on the inhibition of polyphenol oxidase, peroxidase (pod), ascorbic acid oxidases (asa-pod) and polygalacturonase (pg) activities to some extent, in peach fruits. our results related to total phenols are also in accordance with results reported by ayhan and eştürk (2009) who found that phenols slightly increased till the 12th day before it started to decrease. they stated that the diversity in total phenolic content was probably due to changes in acidity and tss which in turn, influenced total anthocyanin content and total antioxidant activity. generally, higher phenolics were recorded for 1 % compared to 0.5 % chitosan treatments and for unirradiated compared to irradiated chitosan. in this regard, benhamou (1996) reported that chitosan has a potential of inducing phenolic contents in plants. among 0.25, 0.50 % and 0.75 % chitosan treatments, 0.50 % was reported to be the most active in increasing total phenolic compounds in apricots (ghasemnezhad et al., 2010). as for the effect of chitosan coating, irrespective of concentration (1 and 2 % dissolved in 2 % glutamic acid), it delayed changes in contents of anthocyanins, flavonoids, and total phenolics. it also delayed the increase in polyphenol oxidase (ppo) activity, and partially inhibited the increase in peroxidase activity (caro and joas, 2005; zhang and quantick, 1997). conclusion chitosan edible coating application to minimally processed pomegranate proved to be beneficial in reducing water loss and presumably, respiration and microbiological problems. integrating it with a 1 % concentration in adopted protocols used in extending shelf life of pomegranate arils seems to be promising due to it positive role in maintaining or delaying senescence of several functional compounds with antioxidant activity such as phenolic compounds, anthocyanins and ascorbic acid. it was also useful in maintaining characteristics 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technology, king mongkut’s university of technology thonburi, thailand, anonymous. tahtat, d., mahlous, m., benamer, s., khodja, a.n., youcef, s.l., 2012: effect of molecular weight on radiation chemical degradation yield of chain scission of γ-irradiated chitosan in solid state and in aqueous solution. radiat. phys. chem. 81 (6), 659-665. varasteh, f., arzani, k., barzegar, m., zamani, z., 2012: changes in anthocyanins in arils of chitosan-coated pomegranate (punica granatum l. cv. rabbab-e-neyriz) fruit during cold storage. food chem. 130 (2), 267-27. zhang, d., quantick, p.c., 1998: antifungal effects of chitosan coating on fresh strawberries and raspberries during storage. j. hort. sci. biotechnol. 73 (6), 763-767. zhelyazkov, s., zsivanovits, g., brashlyanova, b., zsivanovits, m.m., 2014: shelf-life extension of fresh-cut apple cubes with chitosan coating. bulg. j. agric. sci. 20 (3), 536-540. address of the corresponding author: ahmed abdelhamid zahran, national center for radiation research and technology, atomic energy authority 3, ahmed el-zumor st., 8th sector, madenat nasr, cairo, 29, egypt. e-mail: ahmedahszahran@outlook.com © the author(s) 2015. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). art05_thevs.indd journal of applied botany and food quality 85, 159 167 (2012) 1 institute of botany and landscape ecology, university of greifswald, germany 2 faculty of science and technology, free university of bozen-bolzano, italy 3 faculty of textile and clothing technology, niederrhein university of applied sciences, mönchengladbach, germany 4 institute of resource and environmental sciences, xinjiang university, china 5 republican research center of forestry and ornamental garndening, uzbekistan apocynum venetum l. and apocynum pictum schrenk (apocynaceae) as multi-functional and multi-service plant species in central asia: a review on biology, ecology, and utilization n. thevs1, s. zerbe2, y. kyosev3, a. rozi1, b. tang1, n. abdusalih4, z. novitskiy5 (received march 20, 2012) summary during the second half of the 20th century cotton was strongly promoted along the rivers of central asia. the irrigation agriculture resulted in wide spread soil salinization and severe water shortages within the river systems. most prominent example is the desiccation of the aral sea. the natural vegetation along the rivers of central asia is adapted to periods of water shortage, is very productive, and contains plant species with valuable utilization opportunities. we reviewed the literature about apocynum venetum l. and a. pictum schrenk, two plant species of those riparian ecosystems, which are used as fi bre and medicinal plants. a. venetum and a. pictum yield fi bres, which can be used as textiles, though the fi bres best are blended with cotton and/or chemical fi bres. though, the fi bre extraction process needs more research attention. furthermore, the literature shows that apocynum leafs are used to produce antihypertonic tea and medicine. both species grow under the arid climate of central asia without irrigation, because they exploit groundwater. furthermore, both species can withstand higher soil salinization levels than cotton. both species can be used and provide an income to local people under conditions, which are unfavourable to grow crops under irrigation. such conditions are unreliable water supply for irrigation systems and/or saline soils. 1. introduction central asia, which stretches from the caspian sea over kazakhstan, turkmenistan, and uzbekistan to northwest china (i.e. xinjiang, gansu, qonghai, inner mongolia, ningxia, shanxi, and shaanxi), and mongolia, is largely covered by steppes, semi-deserts, and deserts. there, the most productive ecosystems are part of the riparian vegetation along the rivers (thomas et al., 2006; thevs et al., 2007; 2011). but, vast areas of those productive ecosystems have been degraded due to enlarging the area for irrigation agriculture in central asia. furthermore, river runoffs have dropped and fi elds have become subject to severe soil salinization. in the aral sea basin, i.e. along the amu darya and the syr darya, planting of cotton was strongly promoted from the 1960ies onward. as a consequence the aral sea has nearly vanished (glantz, 1999). in the tarim basin, the oases area was enlarged from the 1950ies onward leading to the complete desiccation of the lakes lop nor and taitema, the former end-lakes of the kenqi and tarim river, respectively (song et al., 2000). in both basins, the lower reaches of the rivers turned into episodic river courses or fell dry completely. under those conditions, the natural riparian vegetation and the irrigation agriculture, especially along the lower reaches, suffered and suffers water shortage leading to ecological degradation and economic losses, respectively (hoppe, 1992; spoor, 1993; glantz, 1999; treshkin, 2001; zhu et al., 2006; thevs et al., 2007; westermann et al., 2008). along with the enlargement of irrigation area and periods of water shortage, soil salinization has become a major concern fig. 1: a. venetum stand, fl ood plain of the ulungush river, aleghak, altay prefecture, xinjiang, china (photo: n. thevs, sept. 2008). for farmers in the area (kuzmina and treshkin, 1997; forkusta, 2006; ibrakhimov et al., 2007). in xinjiang, gansu, and inner mongolia, there are attempts to reduce the area under irrigation and shift the land use to perennial plants, which are adapted to the local environmental conditions, e.g. fruit trees, fodder plants, or medicinal plants (gao et al., 2006). the two species apocynum venetum l. and apocynum pictum schrenk (fig. 1 and 2) are promising candidates for a sustainable land use away from irrigation. the genus apocynum comprises nine species, which are distributed in temperate regions of north america, europe, and asia. the two species apocynum venetum and apocynum pictum occur in central asia (royal botanic gardens, 2012). a. venetum and a. pictum are perennial plants with rhizomes, while the stems die yearly, i.e. life form geophytes according to (raunkiaer, 1934). new stems grow out of the roots every spring (pavlov, 1942). a. venetum and a. pictum are adapted to survive even under the extreme arid climate of the tarim basin or aral sea basin with less than 50 mm mean annual precipitation by exploiting the groundwater to cover their water demand. thus, these two species are phreatophytes (gries et al., 2003; chen et al., 2006; thomas et al., 2006). consequently, a. venetum and a. pictum provide utilization options without irrigation. the two species offer opportunities as fi bre and medicinal plants. in this paper, we present a comprehensive review on the two species apocynum venetum and apocynum pictum, focusing on biology, phytogeography, ecology, planting techniques, and utilization. we will draw conclusions on the utilization potential of these two species and point out open research questions. the term apocynum is used, when we refer to both species a. venetum and a. pictum 160 n. thevs, s. zerbe, y. kyosev, a. rozi, b. tang, n. abdusalih, z. novitskiy 2. biology of a. venetum and a. pictum 2.1 taxonomy, nomenclature, and morphology the genus apocynum belongs to the family apocynaceae, order gentiales, class dicotyledonae. today, only the two apocynum species a. venetum (fig. 1) and a. pictum (fig. 2) are distributed in central asia (royal botanic gardens, 2012) and thus reviewed in this paper. a. venetum furthermore is split into several sup-species, from which a. venetum subsp. lancifolium russanov and a. venetum subsp. scabrum russanov are distributed in central asia. the synonyms for the two apocynum species and two sub-species of a. venetum are given in tab. 1 for central asia. in the russian literature, today’s sub-species a. venetum subsp. lancifolium and scabrum are considered as species next to a. venetum (pavlov, 1942; romanovich, 1951). furthermore, a. sibiricum is a synonym of today’s a. venetum subsp. lancifolium (prozorovskii, 1932). in our review, we will only refer to the species names a. venetum and a. pictum. if there appears uncertainty regarding the species names, we use apocynum. a detailed history of the apocynum taxonomical classifi cation is provided by zhang et al. (2006a). in the russian literature, kendyr (кендыр) is used as trivial name for apocynum (berljand, 1950; romanovich et al., 1951). in china, the trivial names luobuma, translated into english as lop kendyr (or lop kendyr), is used for the genus apocynum, while luobuhongma ( ) and luobubaima ( ) refer to a. venetum and a. pictum, respectively (zhang et al., 2006a; b). a. venetum is taller and bigger than a. pictum. the former can grow as tall as 4 m, while the latter only reaches 2 m. in open vegetation, i.e. grassland, a. venetum usually grows 2 m high and each plant branches into 5-10 stems, but as an understory plant in central asian fl oodplain forests it grows up to 4 m and hardly branches off (prozorovskii, 1932; berljand, 1950; romanovich et al., 1951; zhang et al., 2006a; b). the most obvious feature to distinguish the two species is that a. venetum has opposite leafs, while a. pictum has alternate leafs (flora of china, 2011). the further morphological characteristics of the two apocynum species are given in tab. 2. the root system consists of vertical and horizontal roots and rhizomes. the vertical roots connect to the groundwater and thus secure the water supply of the plant. each autumn, buds start to grow at the upper part of the vertical rhizome, from which the new stems emerge in the following spring. nutrients are stored in the upper part of the vertical rhizome and in the entire horizontal rhizome. the horizontal rhizomes carry dormant buds (2-3 per cm), from which vertical roots and root suckers grow, when the horizontal rhizome has been cut off its mother plant. the horizontal roots of an apocynum plant can extend up to 5-6 m (berljand, 1950; romanovich et al., 1951). there are whitish to yellowish mycorrhiza knots on the roots of apocynum (berljand, 1950). vascular arbuscular mycorrhiza was described for the plant order gentiales, including apocynaceae (zhang et al., 2003). 2.2 life history the stems emerge from the rhizomes in march to april. flowering season is from june to august followed by fruit season from september to october (zhang et al., 2005). the stems show the fastest length growth before onset of the fl owering period. during fig. 2: a. pictum stand, fl ood plain of the layi river, i.e. a river branch of the tarim river, qongaral, korla, xinjiang, china (photo: n. thevs, sept. 2008). tab. 1: subspecies and synonyms of a. venetum and apocynum pictum with their distribution (royal botanic gardens, 2012). species / sub-species synonym apocynum venetum l. homotypic synonyms: trachomitum venetum (l.) woodson apocynum venetum subsp. lancifolium russanov homotypic synonyms: (distribution: siberia to china) apocynum lancifolium russanov, trachomitum lancifolium (russanov) pobed. heterotypic synonyms: nerium sibiricum medik., apocynum compressum moench, nerium antidysentericum lepech., apocynum sibiricum pall. ex roem. & schult, apocynum venetum var. microphyllum bég. & beloserky, trachomitum venetum var. microphyllum (bég. & beloserky) woodson apocynum venetum subsp. scabrum russanov homotypic synonyms: (distribution: asia to pakistan) apocynum scabrum russanov, trachomitum scabrum (russanov) pobed., trachomitum venetum subsp. scabrum (russanov) rech.f. heterotypic synonyms: apocynum venetum var. turkestanicum bég. & belosersky apocynum pictum schrenk. homotypic synonyms: (distribution: central asia to mongolia) poacynum pictum (schrenk.) bail. heterotypic synonyms: apocynum hendersonii hook, apocynum grandifl orum danguy, poacynum hendersonii (hook) woodson while luobuhongma ( ) and luobubaima ( ) refer while luobuhongma ( ) and luobubaima ( ) refer apocynum review 161 and after the fl owering period, the length growth ceases (berljand, 1950). apocynum plants reach an age of 30 years (prozorovskii, 1932). apocynum is not very prone to pests and illnesses (tang, 2008). 3. phytogeography and phytosociology a. venetum is distributed over a vast area in eurasia, i.e. from southeast europe over turkey, iran, turkmenistan, uzbekistan, kazakhstan, and southern sibiria to mongolia, northern china, and japan. in central asia, a. venetum is distributed in the fl oodplains and valleys along the rivers, e.g. amu darya, pandzh, vakhsh, syr darya, chu, talas, ili, irtysh, tarim with its tributaries, and heihe (romanovich et al., 1951). in central asia, only the subspecies a. venetum subsp. lancifolium and scabrum (tab. 1) occur. in kazakhstan, i.e. syr darya and chu river basins, a. venetum subsp. lancifolium is found, while the sub-species a. venetum subsp. scabrum is recorded in the amu darya basin (pavlov, 1942; berljand, 1950). a. pictum’s distribution is restricted to southern kazakhstan, the ili basin, xinjiang, gansu, and inner mongolia (pavlov, 1942). both species are part of the riparian vegetation along the rivers and streams in the drylands of central asia. so, they are distributed along perennial and episodic river courses, on alluvial plains, and at desert margins (pavlov, 1942; zhang et al., 2003; 2005). in china, the two apocynum species are mainly recorded in xinjiang, qinghai (caidam basin), gansu, inner mongolia, shanxi, shaanxi, hebei, jilin, and heilongjiang. within this area a. venetum occurs in the semi-arid, semi-humid, and humid part, i.e. with an annual mean precipitation of more than 250 mm. in contrast, a. pictum is restricted to the arid climate, i.e. with an annual precipitation of less than 250 mm (zhang, 2002; zhang et al., 2006a; b). only in the caidam basin with a mean annual precipitation of less than 200 mm, but at an elevation of higher than 2,600 m, a. venetum is dominant over a. pictum (tie and liu, 2006; tang, 2008). within xinjiang, a. pictum is restricted to the more arid parts, e.g. the tarim basin and the southern rim of the zhungar basin, while a. venetum occurs in the less arid northern part of the zhungar basin and the foothills of the altay mountains, as shown in fig. 1 (zhang, 2002; zhang et al., 2006a; b). all over china, the area of apocynum vegetation, natural or artifi cial, amounts to 1,330,000 ha (tang, 2008). one third to half of this area is located in xinjiang, i.e. approximately 600,000 ha (zhang et al., 2003). in the caidam basin, 91,000 ha of natural apocynum vegetation is recorded (tang, 2008). within this area, apocynum is often accompanied by nitraria spec. l. and phragmites australis trin. ex staud. the areas stated here for china as well as for xinjiang and the caidam basin do not represent apocynum dominated or even single species vegetation, but they may represent the area within which apocynum is distributed as accompanying species or dominant species (hong et al., 2002). this is further illustrated by the distribution of apocynum in aksu prefecture, xinjiang (zhang et al., 2003): apocynum is distributed along the tarim river as grassland on higher river terraces and in a mosaic with tugai forests. in aksu prefecture, the area of apocynum vegetation is 50,600 ha comprising reed and a. venetum with 17,100 ha, a. pictum with 800 ha, a. venetum and glycyrrhiza l. with 1,600 ha, tamarix ramosissima ledeb. and a. venetum with 4,400 ha, tamarix ramosissima and a. venetum and alhagi spec. gagnebin with 11,900 ha, tamarix ramosissima, a. pictum, and alhagi spec. with 3,600 ha, and populus euphratica oliv. and a. venetum with 11,200 ha. 4. ecology of a. venetum and a. pictum 4.1 climate adaptation the above-ground parts of a. venetum do not withstand frost and die off under frost. however under a snow cover, the root can withstand strong frost up to -30 °c. the seedlings do not withstand frost at all (berljand, 1950; romanovich et al., 1951). 4.2 soil conditions: water and salt apocynum is adapted to arid climate or dry periods as being a phreatophyte (berljand, 1950). the most productive sites of a. venetum are found on a groundwater not deeper than 3 m with salt contents between 1 g/l and 10 g/l (berljand, 1950; zhang, 2002). along the lower reaches of the tarim river, which had been dry from the 1970ies to 2000 (song et al., 2000), a. pictum is mainly found on groundwater levels of 4-6 m below surface, with a maximum groundwater depth of 8 m (hao et al., 2008). a. venetum in contrast, is restricted to groundwater levels not deeper than 4 m below surface (zhang, 2002). within its distribution area, apocynum forms island-like monospecies stands or dominates stands, but mostly occurs as accompanying species in various plant communities (prozorovskii, 1932; hong et al., 2002; zhang, 2002). thus, it often growths together with phragmites australis, glycyrrhiza infl ata, alhagi sparsifolia, and tamarix shrubs (zhang, 2002; hong et al., 2002; chen et al., 2006), with halophytes on salinized soils (romanovich et al., 1951), or as understory in fl oodplain forests, e.g. built up by populus euphratica (prozorovskii, 1932). a. pictum bears deeper groundwater levels than phragmites australis (thevs et al., 2008a), similar groundwater levels as glycyrrhiza infl ata (chen et al., 2006), tab. 2: morphological characteristics of a. venetum and a. pictum (flora of china, 2011). apocynum venetum apocynum pictum stem up to 4 m tall, glabrous except for infl orescences; branches and up to 2 m tall, branchlets pubescent when young, soon glabrous branchlets whitish gray, terete, fi nely striate leaves usually opposite; petiole 3-6 mm; leaf blade narrowly elliptic usually alternate; petiole 2-5 mm, rarely shorter; leaf blade oblong to narrowly ovate, 1-8 x 0.5-2.2 cm, base rounded or cuneate, to ovate, 1.5-4 x 0.2-2.3 cm, closely denticulate, granulose margin denticulate, apex acute or obtuse, mucronate flowers sepals narrowly elliptic or narrowly ovate, ca. 1.5 mm; corolla sepals ovate or triangular, 1.5-4 mm; corolla pink or purplish red, purplish red or pink; tube campanulate, 6-8 mm, granulose; often with distinct darker markings; tube basin-shaped, 2.5-7 mm; lobes 3-4 mm; disc fl eshy, 5-lobed; lobes rounded, base adnate lobes broadly triangular, 2.5-4 mm; corona inserted at base of to ovary corolla tube, lobes broadly triangular, apex long acuminate fruits follicles slender, 8-20 cm x 2-3 mm follicles slender, pendulous, 10-30 cm x 3-4 mm seeds ovoid or ellipsoid, 2-3 mm, coma 1.5-2.5 cm narrowly ovoid, 2.5-3 mm; coma 1.5-2.5 cm 162 n. thevs, s. zerbe, y. kyosev, a. rozi, b. tang, n. abdusalih, z. novitskiy but cannot exploit the groundwater as deep as populus euphratica, tamarix ramossissima, or alhagi sparsifolia (chen et al., 2006; fan et al., 2006). next to the ability to use groundwater, the stems and branches and leafs are covered with a wax layer, which reduces evaporation (romanovich et al., 1951). apocynum can withstand periods of submergence. however, prolonged submergence or water logged soil inhibits its growth (berljand, 1950). under such conditions, the majority of the roots is concentrated at the upper boundary of the reduced gleyic horizon, in order to avoid prolonged anoxic conditions (prozorovskii, 1932). apocynum is most productive on sandy (romanovich et al., 1951) and silty (prozorovskii, 1932), well drained soils (berljand, 1950). it cannot grow on clayey soils (prozorovskii, 1932). the topsoil under apocynum stands often is salinized, reaching salt contents of up to 20 %. however, 30 cm below surface the salt content drops to only 1 % and less (zhang et al., 2003). this shows that apocynum can grow on sites with a pronounced surface salinization, as long as the subsoil and the groundwater are not strongly salinized. under such conditions, fl ooding is harmful for apocynum, because the fl ood leaches salt into the subsoil, where it damages the roots. a. pictum has a stronger ability to bear salinization than a. venetum (zhang, 2002). apocynum seedlings do not tolerate salt. older plants with deeper roots reach nonor low-saline soil layers so that they can withstand saline top soils, as resulted from irrigation or natural soil salinization (berljand, 1950; zhang, 2002). the adult apocynum plants tolerate salt better than cotton (berljand, 1950). 4.3 plant nutrient status apocynum needs considerable amounts of nutrients from the soil, because it grows rather fast. from the second half of the summer onward, apocynum stores nutrients in its root system (berljand, 1950). the net primary production of vegetation dominated by apocynum in the tarim basin amounts to 0.93 t/ha (hong et al., 2002). in a. venetum leaves from xinjiang, the contents of the nutrients k, ca, and mg are 8.1 mg/g, 1.13 mg/g, and 5.01 mg/g, respectively. the na content is 6.2 mg/g (fan et al., 2006). 5. reproduction and planting techniques 5.1 natural reproduction and germination under natural conditions, apocynum predominantly recruits vegetatively through root suckers. seed germination and successful seedling establishment rarely occurs (romanovich et al., 1951). the seeds require moist, non-saline, well drained sandy to loamy sediments (romanovich et al., 1951). such sites only can be found along river banks after fl ood events. after germination, the seedlings grow slowly above ground, but invest strongly into the root system which is typical for phreatophytes. the seeds germinate easily, but the establishment of the seedlings by accessing a continuous connection to the groundwater poses a bottleneck for the generative recruitment (prozorovskii, 1932; tang, 2008). thus, the recruitment of apocynum is similar to that of populus euphratica oliv. (thevs et al., 2008b; wiehle et al., 2009). the fruits are 12-20 cm long and have a diameter of 3-4 mm. when ripe, they turn brown. each fruit contains 100-150 seeds. the seeds are 2.5 mm long with a diameter of 0.5 mm. the 1,000-grain weight of apocynum venetum ranges from 350 mg to 1,000 mg in the former soviet union (romanovich et al., 1951). in shaanxi, i.e. the more humid part of the apocynum distribution area in china the 1,000grain weight of a. venetum is 500-600 mg (hu et al., 1988). in the more arid regions of the distribution area, i.e. xinjiang and southern part of central asia, the seeds of apocynum are less than 1 mm long. there, the 1,000-grain weight of a. venetum ranges from 332 mg (bai et al., 2005) to 469 mg (berljand, 1950). the 1,000-grain weight of a. pictum was measured as 264 mg (bai et al., 2005). the seeds carry pappus-like hair. the light weight and the hair enable an anemochorous dispersion (berljand, 1950; bai et al., 2005). the seeds lose their germination ability rapidly. however, when stored at dry conditions, i.e. moisture content of 3-6 %, in air-sealed containers, 60 % of seed samples retained germination ability even after 8 years. when the moisture content exceeds 8 %, the seeds lose their germination ability within two years. under non-sealed conditions, the seeds also lose their germination ability within 2-3 years, even under a moisture content of 4 %. it is not necessary to store the seeds under dark conditions (hu et al., 1988). those results were obtained from seeds from shaanxi, which ripened under moist and hot conditions and therefore did not develop a very hard shell. a. venetum requires at least 10 °c for its germination. frost then kills saplings (prozorovskii, 1932; berljand, 1950). the infl uence of nacl solution on the germination of a. venetum and a. pictum is shown in tab. 3 (he et al., 1997; chen et al., 2007). when the nacl concentration was increased from 0.2 % to 0.4 %, the length growth of the a. venetum seedlings, measured after 38 days, decreased signifi cantly from 5.7 cm to 2.7 cm. with increasing nacl concentration, the length growth dropped further to 0.5 cm under 2 % nacl concentration. in germination experiments, the fi rst seedlings of a. venetum appear after three days, while most seedlings appear after 7 days and all seeds have germinated after 28 days. under salt concentrations around 2 %, the germination rate of a. pictum is double as high as that of a. venetum (tab. 3), corresponding with the higher ability of a. pictum to withstand salinized soil conditions than a. venetum (chen et al., 2007). 5.2 cultivation and planting techniques there are two reproduction methods for cultivation, i.e. seed propagation and vegetative propagation from root or plant parts (berljand, 1950; zhang et al., 2005). direct seeding is the most labour saving method to cultivate apocynum. but, better results are obtained, if apocynum seeds are sown in a nursery and the saplings are transplanted after the second year (berljand, 1950; tang, 2008). the most suitable seeding time is mid april to may, tab. 3: germination rate of apocynum venetum and a. pictum under different nacl concentrations. sources: (chen et al., 2007)1 and (he et al., 1997)2, = no data. nacl concen germination rate [%] tration [%] a. venetum1 a. venetum2 a. pictum2 0 86.0 0.2 87.5 0.4 96.0 91.3 0.6 100.0 91.3 0.8 87.5 0.9 85.7 1 82.5 1.6 78.0 < 1.8 89.3 96.7 2 44.0 2.1 86.7 2.5 8.6 0 3 0 apocynum review 163 in order to avoid spring frosts (romanovich et al., 1951; tang, 2008). during the fi rst year, 7-15 times irrigation with a total amount of 10,000 m³/ha to 12,000 m³/ha of water is needed for a. venetum. fields on which a. venetum is sown should have a groundwater level not deeper than 2 m. on the other hand, the soil must be well drained, in order to avoid wet conditions (romanovich et al., 1951). apocynum germinated from seeds grows 30-40 cm high until the end of the vegetation season of the fi rst year. the root system grows faster than the above ground part. after the fi rst vegetation season, the roots reach 70-75 cm deep into the soil. in the second year, the sprouts grow out of the soil in march. at the end of the second vegetation season, apocynum reaches a height of 100 cm. these stems start to yield fi bres. fruits are formed only after the second year. in the fourth year, the stems reach a height of 2 m. in the third year and afterwards, the stems can be harvested before the onset of the fl owering time. the horizontal roots penetrate the whole plantation area in the third year. the root system of a. venetum reaches a groundwater level of 1.5 m during the second year. in the third year, the roots reach a groundwater level of 2 m (berljand, 1950). for the establishment of apocynum plantations root parts can be used, too. the roots can be taken from wild apocynum stands, though over-exploitation must be avoided. root pieces of 10-15 cm length are planted 10 cm deep into the soil. the work load is high, because 60,000 to 70,000 root pieces, i.e. 1-2 t are needed per hectare. the planting time can start a bit earlier than seeding time, i.e. end of march, but spring frosts also hamper root sprouting. during the fi rst and second year, irrigation is necessary under the arid climate of central asia. water has to be applied 5-6 times per year, which sums up to 4,000 m³ to 5,600 m³ water per year. if root pieces have been planted instead of seeds, the plant development is faster. at the end of the fi rst vegetation season, the stems grow as high as 40-50 cm. at the end of the third year, the stems reach a height of 2 m (berljand, 1950). insect pests do not pose a problem in the apocynum cultivation, but the fungus septaria leaf spot damages apocynum under moist and rainy conditions (zhang et al., 2005). as the most important pests on apocynum the fungus fusarium spec. and the snail septaria spec. are considered. 6. utilization of a. venetum and a. pictum a. venetum and a. pictum are used as medicinal plant and as fi bre plant. the leaves, harvested in june/july, serve as raw material for tea and drugs, while the stems, harvested in summer or autumn, serve as raw material for the fi bre extraction. furthermore, apocynum as part of the natural riparian vegetation is grazed (zhang et al., 2003). in an experimental scale natural rubber was extracted from a. venetum. but due to its poor quality, this utilization was given up rapidly (pavlov, 1942). in this paper, we will focus on the utilization of apocynum as fi bre plant and as medicinal plant. the fi bres of apocynum are bast fi bres (fig. 3), like hemp or fl ax (romanovich et al., 1951). the fi bre bundles of apocynum are stronger, i.e. 30-40 fi bres, than the bundles of fl ax, i.e. 10-30 fi bres (pavlov, 1942). the strong bast fi bres obtained from the inner bark are used in making cloth, strings, sails, fi shing nests, and highquality paper (zhang et al., 2006b). next to the fi bres from the stem, a. venetum yields a fl oss fi bre from the seeds (zhang et al., 2006b). the fi bre quality from both apocynum species reviewed here are similar (he et al., 1997). the cultivation of a. venetum for fi bre production started as early as 1930 in the former ussr. just after world war ii, there were plans in the ussr to set aside large areas for the cultivation of a. venetum as fi bre plant. in china, apocynum fi bres are used for textiles, mainly for underwear. in china, a. venetum grows as high as 3.6 m. in the caidam basin the above ground biomass fresh weights was measured with 1798 kg/ha and 1964 kg/ha for a. pictum (tang, 2008). from a plantation near tashkent, an average yield of 5-6 t/ha with a stem density of 300,000 plants per hectare was reported (pavlov, 1942). the annual demand for apocynum fi bres of whole china has been estimated at 50,000 kg over the past years. this demand is covered by the apocynum stands in china. in contrast to that, the demand for leaves as raw material for tea and medicine cannot be covered (tang, 2008). in the fi rst year after planting, a. venetum can be harvested once during that year. afterwards, it can be harvested twice, i.e. in june before or at the beginning of the fl owering period and a second time in september (zhang et al., 2005). in contrast, (berljand, 1950) states that apocynum can be harvested after 2-3 years. only after 10 years, the productivity and yields decrease (pavlov, 1942). 6.1 fibre extraction and fi bre content after harvest, the stems are dried on the fi eld and roasted. then, the bark and woody parts have to be removed mechanically from the fi bres (berljand, 1950). in a second step, the pectin, cellulose, and lignin, in which the fi bre cells are embedded has to be digested (degumming), in order to yield the pure fi bres (wang et al., 2007). chemical degumming (wang et al., 2007) and biological (bacterial) degumming (can be applied to extract apocynum fi bres. the fi bre properties do not vary between the two degumming methods (wang et al., 2007), though bacterial degumming avoids pollution and thus is regarded superior to chemical degumming (bao et al., 2002). the fi bres can be extracted better from a. venetum compared to a. pictum, because the internodes of a. venetum are longer as the leaves and branches are opposite. but, the fi bre extraction and the dying of apocynum is more diffi cult than cotton (liu and zhou, 2002). the bark amounts for 20-22 % of the stem weight for a. venetum fig. 3: cross section of an a. venetum stem, a) pith, b) xylem, c) fi bre bundle, d) epidermis 164 n. thevs, s. zerbe, y. kyosev, a. rozi, b. tang, n. abdusalih, z. novitskiy (romanovich et al., 1951). the fi bre content of dry stems of a. venetum ranges from 10.2 % (zhang et al., 2006b) to 17 % (berljand, 1950). a. pictum has lower fi bre contents, i.e. 7.6 % as shown in tab. 4 (zhang et al., 2006b). the longest fi bres were found in the upper part of the lower half of the stem. the fi bre length correlates with the stem height (zhang et al., 2006b). the stem heights of a. venetum growing as understory in a fl oodplain forest and growing on an open site are 379.4 cm and 192.8 cm, respectively (zhang et al., 2006b). 6.2 fibre properties the apocynum fi bres resemble cotton but are stronger than cotton. in the cross-section the apocynum fi bres are round, elongated, or even triangular. the fi bres yield a pure white yarn, which is very similar to fl ax yarn. apocynum fi bres have small openings, which increase the turnover of air into the fi bre and keep the fi bre dry (han, 2006; li and li, 2006). compared to cotton, apocynum textiles keep warmer, have a higher aeration, and absorb a higher proportion of uv light (wei, 2004). the properties of apocynum fi bres are given in tab. 5. in tab. 6 and 7, the apocynum fi bre properties are compared with ramie and flax as two other bast fi bre plants and with cotton. however, between the apocynum fi bres and cotton there are considerable differences. the modulus of all apocynum fi bre from the three treatments is higher than 300 cn/dtex, while that of cotton is only 96 cn/dtex. thus, textiles from apocynum might be less soft than from cotton. furthermore, the work of rupture of the apocynum fi bres with 11.82-13.72 µj/dtex is lower than the corresponding value of cotton being 16.77 µj/dtex. apocynum fi bres thus might be less durable than cotton fi bres (wang et al., 2007). still, the apocynum fi bres extracted through the three degumming methods all can be readily processed in normal textile machinery and are suitable for blending with other commonly used fi bres (wang et al., 2007). though, the low breaking elongation, the variance in fi bre length, and the smooth surface of the apocynum fi bres may lead to diffi culties during the spinning process (pavlov, 1942; liu and zhou, 2002). therefore, apocynum fi bres usually are blended with cotton or chemical fi bres (wei, 2004). the blend ratio usually is 65 % cotton and 35 % apocynum (zhang et al., 2001). the chemical composition of apocynum is little different from other hemp fi bre. the pectin content of apocynum is 13.14 % .the water soluble content is 17.22 % it is the highest ranking among the hemp fi bre. the lignin content of is 12.14 %, higher than ramie, fl ax, abaca and sisal. the cellulose content ranges from 40.82 %, which is the lowest of all hemps, to 72 %, i.e. the same as fl ax fi bre (li and li, 2006). 6.3 hygiene and medicinal effects of apocynum fi bres apocynum fi bres show an anti-microbial effect. the inhibition rates of apocynum fi bre on staphylococcus aureus, pseudomonas aeruginosa, escherichia coli, and candida albicans is 47.7 %, 69.0 %, 56.6 %, and 40.1 %, respectively, compared to cotton fi bres. under an electron microscope, it was visible that the cell walls of the bacteria were destructed upon contact to the apocynum fi bres (lü et al., 2006). this can be attributed to the fi nding that the stem cells of apocynum contain tanning agents, which makes the fi bres resistant against microbial decomposition (romanovich et al., 1951). tab. 4: stem height, bast and fi bre contents, and fi bre lengths (mean and standard deviation) of a. venetum and a. pictum in xinjiang (zhang et al., 2006b). the asterix indicates signifi cant differences between the two means at p <= 0.05, calculated with t-test. plant parameter apocynum venetum apocynum pictum mean ± standard deviation stem height [cm] 244.1 ± 78.0 112.0 ± 22.2 * bast content of stem [%] 22.4 ± 4.4 23.7 ± 4.7 bast content of branches [%] 19.6 ± 3.5 27.3 ± 5.0 * percentage of stem bark 66.0 ± 17.1 47.0 ± 10.2 * from total bark [%] fibre content [%] 10.2 ± 2.9 7.6 ± 1.9 * fibre length of stem [mm] 36.3 ± 8.4 29.4 ± 8.0 * fibre length of branches [mm] 20.2 ± 5.9 20.8 ± 5.4 tab. 5: fibre properties of apocynum fi bres. fibre parameter size of parameter fibre length [mm] 15-25, max. 70 (prozorovskii, 1932) 50-55, min. 5, max. 120 (pavlov, 1942) 20-25, min. 10, max. 40 (li and li, 2006) average 26 (zhang et al., 2001) diameter [µm] 45 (pavlov, 1942) fineness [dtex] 4.15 (xu, 2005) 3-4 (li and li, 2006) 4.7 (zhang et al., 2001) strength [cn/dtex] 3.7-4.4 (li and li, 2006) 3-7 (zhang et al., 2001) tab. 6: comparison of fi bre properties between apocynum and the bast fi bres ramie and flax specifi cations apocynum ramie flax source fibre length [mm] 20-25 50-120 15-20 (xu, 2005) fibre diameter [µm] 14-15 20-45 12-17 (xu, 2005) fineness [dtex]1 3 4.5 2 (li and li, 2006) strength (dry) 3-7 6.5 6.3 (li and li, 2006) [cn/dtex] breaking elongation 2.5 2.3 1.8 (li and li, 2006) (dry) [%] divergence rate [%] 100 100 88 (li and li, 2006) 1 the unit tex is equivalent to 1 g/km. thus, an apocynum fi bre of 3 dtex has a weight of 0.3 g per 1 km fi bre length. tab. 7: comparison of fi bre properties of apocynum venetum (three extraction treatments) and cotton (wang et al., 2007) fibre machine bast hand bast machine bast cotton parameter pealing + pealing + pealing + chemical chemical bacterial degumming degumming degumming modulus 318.9 383.7 394.5 96.6 (cn/dtex) breaking 4.06 3.58 3.26 10.69 elongation (%) work of rupture 12.36 13.72 11.82 16.77 (µj/dtex) apocynum review 165 6.4 medicinal applications of a. venetum and a. pictum tea from a. venetum and a. pictum is used as a traditional chinese medicine, which mainly is consumed in northwest china. antihypertensive and anti-hyperlipidemic effects are attributed to apocynum tea (ma and chen, 1999). in 1977, apocynum was listed as chinese medicine in china (zhang, 2004). a. venetum leaves are rich in ash elements and minerals, such as ca, fe, and na, but differ from green tea leaves in that they contain no caffeine (yokozawa et al., 2002). the leaves of a. venetum and a. pictum have a high fl avonoid content (sakushima et al., 1978; kamata et al., 2008). aqueous a. venetum extracts showed an anti-hypertensive effect on hypertensive rats. this anti-hypertensive effect was observed after treatment with aqueous extracts from roasted and non-roasted leaves (kim et al., 2000). the anti-hypertensive effect was mainly attributed to an improved kidney function, i.e. increased excretions of urine volume and urine electrolytes. such a diuretic effect of aqueous a. venetum extracts had been reported before (qing et al., 1988). furthermore, a vascular relaxation effect after treatment of rat aortas with an a. venetum extract in an in vitro experiment was found. this vascular relaxation effect plays a role regarding the anti-hypertensive effects of apocynum tea (kwan et al., 2005). apocynum extract caused vasodilation on tissues (tagawa et al., 2004). aqueous a. venetum extracts decreased the serum total cholesterol and ldl-cholesterol levels and the atherogenic index, as well as the hepatic total cholesterol level in an experiment with hypercholesteraemic rats, but they increased the hdl cholesterol level. the decrease of cholesterol values was stronger when using roasted apocynum leaves to prepare the extract (kim et al., 1998). the leaves yield up to 5 % gum, which is used for making rubber, and a medicine used as a sedative and to treat hypertension (zhang et al., 2006a). an extract of a. venetum leaves reduced signifi cantly the immobility of rats in an forced swimming test. therefore, it was concluded that such an a. venetum extract shows antidepressant activity in the forced swimming test. (butterweck et al., 2001; butterweck et al., 2003; zhou et al., 2007). furthermore, a. venetum extracts contain anti-oxidants. the antioxidative effect was attributed to condensed tannin compounds. the radical scavenging activity of a. venetum extracts was determined to be similar to gingko biloba l. thus, a. venetum extracts may serve as protective reagents against the oxidative stress in nerve cells due to lipid peroxidation (cao et al., 2003; yokozawa and nakagawa, 2004; shirai et al., 2005). 7. conclusions the literature reviewed shows that a. venetum and a. pictum yield fi bres, which can be used as textiles, though the fi bres best are blended with cotton and/or chemical fi bres. such yarns have good aeration and warming properties due to the hollow structure of the apocynum fi bres and are mostly used to produce underwear. furthermore, the literature shows that apocynum leafs are used to produce anti-hypertonic tea and medicine. both species yield fi bres and leafs on considerable areas under the arid climate of central asia without irrigation, because they use the groundwater. furthermore, both species can withstand higher soil salinization levels than cotton. a. pictum can withstand more saline and more arid conditions than a. venetum. both species can be used and provide an income to local people under conditions, which are unfavorable to grow crops under irrigation. such conditions are unreliable water supply for irrigation systems and/or saline soils. though, a number of research questions still are open. the area, from which apocynum currently could be harvested, and the potential annual yields of fi bres and leafs are unknown. furthermore, the water consumption and water use effi ciency must be measured, in order to make sure that apocynum utilization does not consume more water than current utilizations. currently, in china mostly natural apocynum stands are harvested. these stands are not fertilized. therefore the nutrient exports due to apocynum harvest and possible stand degradations must be investigated. the current used degumming methods require longer process time (bacterial degumming), are not very environment friendly and require water resources, which are usually not available in the cultivation regions. therefore, more effi cient and environmental friendly degumming method need to be investigated. the fi bres demonstrate technological properties similar to the other cellulosic fi bres (fl ax, cotton), but with signifi cantly lower braking elongation and larger length variations. these two features disturb the classical spinning process of pure apocynum yarns and are one of the reasons the apocynum fi bres to be blended with cotton. the development of an adjusted spinning process and equipment for apocynum fi bres can allow the production of yarns with higher apocynum content, which can extend the application areas for textiles. due to its high strength, it has to be investigated if the fi bres are suitable for other than clothing applications, as for instance for natural fi bre composites. in such case the fi bres can be used directly without or after only partial degumming procedure, which will reduce their price as well. acknowledgements we thank the bauer-hollmann foundation and the helene and rudolf glaser foundation for funding personal costs within the junior research group adaptation strategies to climate change and sustainable land use in central asia (turkmenistan and xinjiang, china). furthermore, we thank the robert-bosch-foundation, the federal ministry for education and research, germany, and the german academic exchange service (daad) for funding travel costs to china and uzbekistan, respectively. references bai, l., luo, m.b., chuan, l.c., wang, s.l., a, m.n., 2005: short report on planting techniques of apocynum venetum. chinese wild plant resources 24, 65-68. bao, m.d., chen, z., liu, z.g., ren, j.p., 2002: study on biological degumming for apocynum venetum. shandong 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(eds.), watershed and fl oodplain management along the tarim river in china’s arid northwest, 77-90. shaker, aachen. address of the authors: dr. niels thevs (corresponding author), ahmedjan rozi and b. tang, institute of botany and landscape ecology, university of greifswald, grimmer strasse 88, 17487 greifswald, germany. prof. dr. s. zerbe, faculty of science and technology, free university of bozen-bolzano, universitätsplatz 5, 39100 bozen, italy. prof. dr. yordan kyosev, faculty of textile and clothing technology, niederrhein university of applied sciences, webschulstrasse 31, 41065 mönchengladbach, germany. nurbay abdusalih, institute of resource and environmental sciences, xinjiang university, 14, sheng li road, urumqi, xinjiang, china. zinovy novitskiy, republican research center of forestry and ornamental gardening, kartatal street 21, tashkent, uzbekistan. journal of applied botany and food quality 86, 133 137 (2013), doi:10.5073/jabfq.2013.086.018 1 national institute of horticultural and herbal science, rda, naju, korea 2 korean pear research organization, chonnam national university, gwangju, korea effect of sodium chloride, pgdo and arabic gum in pollen liquid diluent on suspensibility of kiwi pollen kyeong-ho lim1, sang-hyun lee2* (received may 5, 2013) * corresponding author summary this study was conducted to develop the pollen liquid diluent suitable for the artificial pollination of kiwi. the pollen of ‘matua’ kiwi was collected at 1 day before flowering. five concentrations of sodium chloride (0, 50, 150, 250, and 350 mg · l-1), four concentrations of poly (glycolide-co-p-dioxanone) (0, 7, 14, and 21 mg · l-1), and four concentrations of arabic gum (0, 150, 350, and 550 mg · l-1) were tested on an absent condition of each component in the pollen liquid diluent. twenty mg of pollen was distributed in beakers containing 10 ml of the pollen liquid diluent. suspensibility of the pollen liquid diluent was measured by the sensory evaluation and particle size analyzer. the addition of sodium chloride in pollen liquid diluent was effectible for the suspensibility improvement and the promotion of pollen growth in kiwi pollen. the kiwi pollen in pollen liquid diluent could be suspended without damage in pollen germination at low concentration of poly (glycolide-co-p-dioxanone) (pgdo), which has been known as a safe surfactant. the addition of arabic gum would be highly advantageous to the stabilization of the pollen liquid diluent without any contamination for pollen growth. kiwi fruits were set and grown well by the artificial pollination using the pollen liquid diluent. therefore, the use of the pollen liquid diluent in the artificial pollination of kiwi fruit should be an effective practice. introduction in kiwi (actinidiu deliciosa) requiring cross pollination for commercial production, artificial pollination has been developed with equipment for this process (gonzalez et al., 1998). however, effective pollination periods of kiwi flowers was quite limited with 3~4 days (sanzol and herrero, 2001), the artificial pollination, performed as hand pollination, has been formed temporarily as a heavy labor. as this reason, some growers were spraying pollens from male flowers diluted in a 10 % sucrose solution on female flowers in an attempt to improve fruit set. however, the effectiveness of this practice was incredulous. in grapefruit, pollen germination was poor both for spray and dipping treatments and less than one pollen tube per pistil reached micropyles for any of the pollen concentration used (kimura et al., 1998). pollen liquid diluent maintained pollen in a viable state while in an aqueous liquid, was already developed as named pollenaid® in new zealand and has been used by kiwi growers. however, since pollenaid® is a commercial product, some information about pollen liquid diluent has never been revealed and reported in the scientific field. the use of pollen liquid diluent could be an economically viable method for artificial pollination in kiwi production. accordingly, this study was conducted to develop the pollen liquid diluent suitable for pollination of kiwi throughout analyzing the effects of sodium chloride as a promoter, poly (glycolide-co-pdioxanone) (pgdo) as a surfactant (wang et al., 2012) and arabic gum as a stabilizer (islam et al., 1997; zhang and liu, 2011) in the pollen liquid diluent on pollen growth and pollen suspensibility. material and methods kiwi pollen flowers of ‘matua’ kiwi were collected at 1 day before flowering for the pollen collection in the experimental orchard at chonnam province fruit tree research institute, which is located in haenam, korea. flowers were brought back to the laboratory, where the anthers were collected from the flower with an anther sifter (mk600, mitsuwa, japan). roughly selected anthers by the sifter were simply put in the hopper of an anther selecting machine (kbs-6, mitsuwa, japan). finally, the anthers were automatically collected. the collected anthers were scattered on a thick black paper, then maintained for 24 h in an anther opener (m-600d, mitsuwa, japan). the anthers, thus dried, were dipped into 99.5 % acetone in a stainless steel bowl, using a sieve (100 meshes). the sieve was removed after a short period of agitation in the bowl to collect pollen grains. the supernatant of the pollen-acetone mixture was then discarded. the pollen was weighed after the volatilization of the remaining acetone, and stored at 4 ℃ until later use in the germination test. basic component of pollen liquid diluent the component of pollen liquid diluent consisted of basically 2 % of sucrose (c12h22o11=342.31), 100 mg · l-1 of boric acid (h3bo3=61.84), 70 mg · l-1 of calcium nitrate (ca(no3)2=164), sodium chloride 250 mg · l-1, arabic gum 350 mg · l-1, pgdo 14 mg · l-1 and distilled water; ph adjusted to 7.0. the concentrations of each component was tested already at various concentrations for the determination of the optimal combination and decided through a previous test. addition of sodium chloride, pdgo, and arabic gum in pollen liquid diluent five concentrations of sodium chloride (0, 50, 150, 250, and 350 mg · l-1), four concentrations of pdgo (0, 7, 14, and 21 mg · l-1), and four concentrations of arabic gum (0, 150, 350, and 550 mg · l-1) were tested on an absent condition of each component based on the basic component of pollen liquid diluent. twenty mg of pollen was distributed in beakers containing 10 ml of the pollen liquid diluent. pollen germination and pollen tube growth of the pollen liquid diluent the pollen liquid diluent was cultured for 1,2,3,4, and 5 hours in an incubator maintained at 30 ℃. all observations of pollen germination and pollen tube growth were performed at 4 hours after the distribution of the pollen in the pollen liquid diluent. fifteen μl of pollen liquid diluent was taken and put on the slide glass for the microscopic observation. 134 k.-h. lim, s.-h. lee at least 150 pollen grains were counted on 10 random microscopic fields of the optical microscope (x200, leica, germany). a pollen grain was judged to germinate when the pollen tube length was greater than the diameter of the pollen grain. pollen germination and pollen tube growth were measured using an image analyzer (imt, korea). the evaluation was carried out with a completely randomized experimental design, with five replicates. suspensibility of the pollen liquid diluent suspensibility of the pollen liquid diluent was measured by the sensory evaluation and particle size analyzer. for the sensory evaluation, the pollen liquid diluent was stirred during the observation periods and investigated the suspensibility on 5 minute interval for 20 minutes by the sensory evaluation. the appearance score of suspensibility was assessed, using a numerical interval scale from 1 to 5, with 0.1 point intervals. the scale was defined as follows: 5 = very well mixed, 4 = well mixed, 3 = mixed, 2 = minimal mixed, 1 = mass mixed. the particle size distribution in pollen liquid diluent of the pollens was determined on a particle analyzer (nanophox, sympatec gmbh, germany) based on photon cross correlation spectroscopy. the artificial pollination using the pollen liquid diluent artificial pollination was carried out on a netted plastic house for the protection of natural pollination by bees. four g · l-1 pollens of ’matua’ kiwi were placed on the basic component of pollen liquid diluent and suspended by a vortex. the suspended pollen liquid diluent was sprayed at 1, 2, 3, 4, and 5 hours after dipping, respectively. a hand-mister was used to generate the spray. in all treatments, approximately 100 ml of the pollen liquid diluent per tree was used on 5 different ‘hayward’ kiwi trees at 2 days after full bloom. fruit characteristics by the artificial pollination using the pollen liquid diluent fruit set, seed number, and fruit size were investigated from each fruit of 30 flower clusters signed on the pollination time of each tree at 30 days after pollination, when fruit set could be completely confirmed before fruit thinning process. results and discussion effects of sodium chloride in pollen liquid diluent the suspensibility of pollens in pollen liquid diluent was improved with an increase of sodium chloride concentration and an increase of dipping time (tab. 1). these results were similar to the improving effect of the suspensibility in water of dried nanofibrillated cellulose (nfc) by adding sodium chloride at different ph conditions (missoum et al., 2012). although the rate of pollen germination was depressed at 350 mg · l-1 of sodium chloride, the pollen germination rate by the addition of sodium chloride in pollen diluent solution was increased compared to the control (fig. 1a). the pollen tube length was higher at 150 and 250 mg · l-1 of sodium chloride than that of the control. however, at 50 and 350 mg · l-1 of sodium chloride, pollen tube length demonstrated no difference (fig. 1b). the promotion of pollen growth at a low concentration of sodium chloride may be considered as the permeability increase in the exine of pollen grains. however, reducing effects of the pollen growth by the high sodium chloride concentrations was confirmed in rice (zeng and shannon, 2000), tomato (yokas et al., 2008), and chickpea (samineni et al., 2011). sodium chloride (mg · l-1) tab. 1: effect of sodium chloride concentrations in pollen liquid diluent on the suspensibility by sensory evaluation of ‘matua’ kiwi pollen. pollen suspensibilityz 5 min 10 min 15 min 20 min 0 1.0 by 1.7 c 2.8 c 3.8 c 50 1.0 b 1.8 c 3.0 bc 4.0 bc 150 1.2 b 2.3 b 3.2 b 4.5 b 250 1.8 a 2.8 a 3.8 a 4.7 a 350 2.0 a 3.0 a 4.0 a 4.9 a z the scale was defined as follows: 5 = very well mixed, 4 = well mixed, 3 = mixed, 2 = minimal mixed, 1 = mass mixed. ymean separation within columns by duncan’s multiple range test at 5 % level. results are from five replicates. fig. 1: effects of sodium chloride concentrations in the pollen liquid diluent on pollen germination rate (a) and pollen tube growth (b) of ‘matua’ kiwi in vitro. vertical bars represent ± sd. results are from five replicates. replicates above consist of approximately 50 pollen grains each. the pollen liquid diluent suitable for the artificial pollination of kiwi 135 the above mentioned results indicate that the addition of sodium chloride in pollen liquid diluent is effectible for the suspensibility improvement. the optimum dipping condition without a contamination of the pollen germination in pollen liquid diluent should be announced for 20 minutes at 250 mg · l-1 of sodium chloride. effects of poly (glycolide-co-p-dioxanone) (pgdo) in pollen liquid diluent the surfactants have been commonly used in a liquid diluent for the suspensibility. we already tested five surfactants which have been known as a possibility for use in pollen liquid diluent and selected pgdo as the adaptable surfactant (data not shown). the suspensibility of kiwi pollens in pollen liquid diluent was improved with an increase of pgdo concentration. however, the progress of suspensibility in the pollen diluent solution was delayed at an initial time after pollen mixing (tab. 2). as a surfactant in pollen liquid diluent should be announced for 20 minutes at 14 mg · l-1. effects of arabic gum in pollen liquid diluent the surface dilational rheology of the pollen liquid diluent is importantly considered in the spray process of field work. for the support of suspensibility in pollen liquid diluent, arabic gum was tested at various concentrations. the suspensibility of pollens in the pollen liquid diluent was observed to gradually increase with dipping time and arabic gum concentration. pollens in the pollen liquid diluent were quickly suspended in the conditions without arabic gum at the initial time of dipping (tab. 3). for adding by arabic gum, pesticide emulsion remained stable for 7 days even at -10 ℃. this fact could be explained by a close correlation with arabic gum adsorption at the droplet surface (zhang and liu, 2011). also, the maximum of dilational modulus and dilational elasticity in the trisiloxane surfactant silwet408 was shifted toward higher concentrations when arabic gum of 1 % was added (cao et al., 2013). pollen germination rate and pollen tube growth in the pollen liquid diluent showed no difference with concentrations and dipping times by the addition of arabic gum (fig. 3). it means that the addition of arabic gum would be highly advantageous to the stabilization of the pollen liquid diluent without any contamination for pollen growth. consequently, the addition of arabic gum in the pollen liquid diluent could be considered as an effective method for the advance of surface dilational rheology in the pollen liquid diluent. pgdo (mg · l-1) tab. 2: effect of pgdo concentrations in pollen liquid diluent on the suspensibility by sensory evaluation of ‘matua’ kiwi pollen. pollen suspensibilityz 5 min 10 min 15 min 20 min 0 1.0 by 1.5 c 2.5 c 3.5 c 7 1.0 b 1.5 c 3.0 b 4.0 b 14 1.0 b 2.0 b 3.5 ab 4.5 ab 21 2.0 a 3.0 a 4.0 a 5.0 a z the scale was defined as follows: 5 = very well mixed, 4 = well mixed, 3 = mixed, 2 = minimal mixed, 1 = mass mixed. ymean separation within columns by duncan’s multiple range test at 5% level. results are from five replicates. arabic gum (mg · l-1) tab. 3: effect of arabic gum concentrations in pollen liquid diluent on the suspensibility by sensory evaluation of ‘matua’ kiwi pollen. pollen suspensibilityz 5 min 10 min 15 min 20 min 0 1.7 ay 2.5 b 3.3 b 4.0 b 150 1.7 a 2.5 b 3.5 b 4.5 ab 350 1.7 a 2.7 ab 3.5 ab 4.5 ab 550 1.8 a 3.0 a 4.0 a 4.8 a z the scale was defined as follows: 5 = very well mixed, 4 = well mixed, 3 = mixed, 2 = minimal mixed, 1 = mass mixed. ymean separation within columns by duncan’s multiple range test at 5% level. results are from five replicates. fig. 2: effects of pgdo concentrations in the pollen liquid diluent on pollen germination rate (a) and pollen tube growth (b) of ‘matua’ kiwi in vitro. vertical bars represent ± sd. results are from five replicates. replicates above consist of approximately 50 pollen grains each. the action of pgdo in pollen liquid diluent was confirmed as the formation of well-defined spherical particles during suspension polymerization in supercritical carbon dioxide (wang et al., 2012). the pollen germination rate was depressed significantly by the addition of 21 mg · l-1 of pgdo in pollen diluent solution (fig. 2a). pollen tube length was also significantly decreased at 21 mg · l-1 of pgdo compared to the control (fig. 2b). consequently, the kiwi pollens in pollen liquid diluent could be suspended without damage in pollen germination at low concentration of pgdo, which has been known as a safe surfactant. the optimum condition of pgdo for the improvement of suspensibility 136 k.-h. lim, s.-h. lee tab. 4: effects of the artificial pollination by the pollen diluent solution with dipping time on fruit set, seed number, fruit size, and l/d ratio of ‘hayward’ kiwi fruit. ymean separation within columns by duncan’s multiple range test at 5% level. results are from five replicates. replicates above consist of fruits from 30 flower clusters signed on the pollination time of each tree at 30 days after pollination. fig. 3: effects of arabic gum concentrations in the pollen liquid diluent on pollen germination rate (a) and pollen tube growth (b) of ‘matua’ kiwi in vitro. vertical bars represent ± sd. results are from five replicates. replicates above consist of approximately 50 pollen grains each. fruit size (mm) l/d ratio length diam 1 100 ay 1,019 a 51.2 a 34.2 a 1.50 a 2 100 a 1,040 a 51.6 a 34.7 a 1.49 a 3 99.0 a 987 a 49.5 a 32.5 a 1.48 a 4 97.7 a 769 b 48.8 ab 33.8 a 1.44 ab 5 96.0 a 636 bc 48.4 ab 33.7 a 1.44 ab dipping time fruit set seed number (hrs) (%) (no/fruit) fig. 4: changes of particle sizes with compositions of the pollen liquid diluent (pld) during suspended time. suspensibility analysis of the pollen liquid diluent by the particle analyzer for suspensibility analysis of the pollen liquid diluent, particle size was observed with or without sodium chloride, pgdo, and arabic gum by the particle analyzer. the pollen liquid diluent contained sodium chloride, pgdo, and arabic gum showed smaller particle size than pollen liquid diluent which was only manufactured by distilled water. the particle size was the smallest in the pollen liquid diluent without the arabic gum and higher in the pollen liquid diluent without the sodium chloride (fig. 4). these results were similar to that of sensory evaluation. our data showed that the suspensibility of pollen liquid diluent is decreased by the addition of arabic gum and increased by the addition of sodium chloride. fruit quality by artificial pollination using the pollen liquid diluent fruit set in kiwi trees, pollinated artificially by the pollen liquid diluent was shown to be more than 95 % and demonstrated no difference with dipping times. although seed number was reduced in the pollen liquid diluent suitable for the artificial pollination of kiwi 137 kiwi trees pollinated at 4 and 5 hours after dipping, fruit size was reduced a little at that time. consequently, the artificial pollination using the pollen liquid diluent should be a useful method for pollination without a weakness in fruit set and fruit growth of ‘hayward’ kiwi. conclusion the addition of sodium chloride in pollen liquid diluent was effectible for the suspensibility improvement. the low concentration of poly (glycolide-co-p-dioxanone) (pgdo) was suitable as surfactant. the addition of arabic gum related in the dilational elasticity of surfactant should be highly advantageous methods for the stabilization of the pollen liquid diluent without any contamination for pollen growth. kiwi fruits were set and grown well by the artificial pollination using the pollen liquid diluent. therefore, the pollen liquid diluent should be an effective practice in the artificial pollination of kiwi fruit performed temporarily heavy labor. references cao, c., zhang, l., zhang, x.x., du, f.p., 2013: effect of gum arabic on the surface tension and surface dilational rheology of trisiloxane surfactant. food hydrocolloids 30, 456-462. gonzalez, m.v., coque, m., herrero, m., 1998: influence of pollination systems on fruit set and fruit quality in kiwifruit (actinidia deliciosa). ann. appl. biol. 132, 349-355. islam, a.m., phillips, g.o., sljivo, a., snowden, m.j., williams, p.a., 1997: a review of recent developments on the regulatory, structural and functional aspects of gum arabic. food hydrocolloids 11, 493-505. kimura, p.h., okamoto, g., hirano, k., 1998: artificial pollination in vitis coignetiae pulliat. vitis 37, 83-86. missoum, k., bras, j., belgacem, m.n., 2012: water redispersible dried nanofibrillated cellulose by adding sodium chloride. biomacromolecules 13, 4118-4125. samineni, s., siddique, k.h.m., gaur, p.m., colmer, t.d., 2011: salt sensitivity of the vegetative and reproductive stages in chickpea (cicer arietinum l.): podding is a particularly sensitive stage. environ. exp. bot. 71, 260-268. sanzol, j., herrero. m., 2001: the ‘effective pollination period’ in fruit trees. sci. hortic. 90, 1-17. wang, t.q., hao, j.y., liu, y., gu, z.w., 2012: formation of well-defined spherical particles during suspension polymerization of biodegradable poly (glycolide-co-p-dioxanone) in supercritical carbon dioxide. rsc adv. 2, 10365-10371. yokas, i., tuna, a.l., burun, b., altunlu, h., altan, f., kaya, c., 2008: responses of the tomato (lycopersicon esculentum mill.) plant to exposure to different salt forms and rates. turk. j. agric. for. 32, 319329. zeng, l.h., shannon, m.c., 2000: salinity effects on seedling growth and yield components of rice. crop sci. 40, 996-1003. zhang, x., liu, j., 2011: effect of arabic gum and xanthan gum on the stability of pesticide in water emulsion. j. agric. food chem. 59, 13081315. address of the author: korean pear research organization, chonnam national university, gwangju 500-757, republic of korea. journal of applied botany and food quality 89, 150 155 (2016), doi:10.5073/jabfq.2016.089.018 1 the key laboratory of biomedical engineering ministry of education, department of biomedical engineering, zhejiang university, china 2 college of basic medical science, shandong university of traditional chinese medicine, jinan, china 3changshu qiushi technology co. ltd., changshu, china phytochemical compositions, antioxidant and antimicrobial activities analysis of extracts from vaccinium bracteatum thunb. leaves jin hu1, jing wang2, shouxin li3, bingxian yang1, minghua gong2, ximin li3, lin zhang1, jingkui tian1,* (received october 27, 2015) * corresponding author summary vaccinium bracteatum thunb. is an edible plant, which has been used for many food products and is also a resource of traditional chinese medicine. in this study, the antioxidant and antimicrobial activities of ethanol extracts from its leaves were investigated. to study the characteristic compositions, twelve compounds of extracts accumulated by the d-101 macroporous adsorption resin (vbe) were identified by hplc-dad and hplc-esi/ms techniques, including chlorogenic acid and its isomers, and eight flavonoid compounds. the contents of total flavonoids, orientin and isoorientin in the accumulated part were 601.4, 44.7, and 96.1 mg/g, respectively, which were far more than that in the raw materials. furthermore, the antioxidant activities were estimated by dpph, abts, and frap assays, which showed that the high content accompanied with strong antioxidant activities. besides, compared to the same type of bamboo leaves (aob), the accumulated part possesses better activities. at the last, the antimicrobial activities of vbe were assessed by a serial two-fold dilution assay, the results showed that it had good antimicrobial activities. taken together, extracts from vaccinium bracteaturn thunb. leaves have better antioxidant activities, which can be used as a natural antioxidant. introduction antioxidants, as food additive, are used to keep the foods quality during their shelf life. at the present, the most commonly used antioxidants are chemical preservatives, such as butylated hydroxyanisole (bha), butylated hydroxytoluene (bht), propyl gallate (pg), and tertbutylhydroquinone (tbhq). however, some studies showed that the chemical preservatives exhibited certain toxicity, which can affect the respiratory enzymes activity, and cause teratogenesis and carcinogenesis (wettasinghe and shahidi, 1999; luo and fang, 2008). nowadays, the demand for food additives is increasingly high, and the utilization of natural antioxidants becomes a necessary research direction. a lot of research had found that the extracts of most plants possessed antibacterial and antioxidant effects (sun et al., 2011; ryszard et al., 2004; ichikawa et al., 2001). vaccinium bracteatum thunb. is mainly distributed in the regions south of the yangtze river, which belongs to the genus vaccinium (family ericaceae). the leaves were rich in proteins, vitamins and minerals, which contained abundant vitamin c (vc), calcium, iron, selenium and manganese. with methionine (met) and histidine (his) being the highest in them, seventeen amino acids were found (chen et al., 2008). furthermore, it showed multiple advantages for wufanshu being taken as a rootstock species of blueberry, the contents of potassium, sodium, iron, phosphorus, zinc, and calcium element in fruit of wufanshu were significantly higher than those in none-grafted blueberry (xu et al., 2014). in china, a series of vaccinium bracteatum food was also made of its extracts such as vaccinium bracteatum beverage. besides, making wufan with rice was people’s custom, it had been developed into a local tourism food (hao et al., 2010). modern medical research found that it had many pharmacological activities, such as improving blood microcirculation and antiinflammatory (landa et al., 2014). in recent years, some studies showed that the main constituents of vaccinium bracteatum thunb. leaves were flavonoids, polyphenols, and diterpenes. additionally, it was reported that the flavonoids in extracts from plants had antioxidant activity (bernonville et al., 2011; chua et al., 2011; li et al., 2009), and the mechanism of antioxidant activity was related to the structure of the flavonoids and the substituents of the heterocyclic rings (kong et al., 2003). in our previous studies (li et al., 2008; zhang et al., 2009), a systematical investigation of the chemical components of vaccinium bracteatum thunb. leaves had been carried out. twenty-five compounds were isolated from the leaves, and twenty-four of them were identified. here, the antioxidant and antibacterial activities of extracts from leaves were performed. the results showed that the extracts from v. bracteratum leaves could be used as a natural antioxidant in food industry. materials and methods materials and chemicals 1,1-diphenyl-2-picrylhydrazyl (dpph) was purchased from the wako pure chemical company (osaka, japan). 2,4,6-tris(2pyridyl)-1,3,5-triazine (tptz), and 2,20-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (abts) were purchased from the tokyo chemic industry company (tokyo, japan). ethanol, methanol (hplc-grade), acetonitrile (hplc-grade), iron vitriol, ferric trichloride, sodium acetate, and potassium persulfate were obtained from the sinpharm chemical reagent company (shanghai, china). all solvents and chemicals were of analytical grade, unless otherwise specified. hplc grade water was obtained from milli-q system (millipore, billerica, ma, usa). extraction and accumulation of total flavonoids from vaccinium bracteatum thunb. leaves vaccinium bracteatum thunb. leaves were harvested in zhejiang province, china. the leaves were dried in the shade for a week, and then were ground to powder using a grinder. a portion (100 g) of powder was extracted with 70 % ethanol solution. one part of ethanol extracts was dried in a rotary evaporator, which was the raw materials part. another part of ethanol extracts was added into the column contained d-101 macroporous resin. the column was successively washed by water and 70 % ethanol. the eluted solution from the column was collected and dried, which was the macroporous adsorption resin part (vbe). all samples were packed in the bag and stored at room temperature until later analysis. the nutraceutical properties of vaccinium bracteatum thunb. leaves 151 hplc analysis the extract (25 mg) was dissolved in 10 ml of 70 % (v/v) ethanol and filtered through a 0.45 μm cellulose acetate filter before hplc analysis. the samples were analyzed by shimadzu lc-10at hplc system (shimadzu co., japan), equipped with cbm-20a triton system controller, spd-m20a detector, sil-20a auto sampler and cto-10a column oven. the analytical column was a c18 column of agilent tc-c18 (250 mm × 4.6 mm, 5 μm i.d., agilent technologies, usa) with a flow rate of 1 ml min-1 at 40 °c. the mobile phase consisted of 1 % acetic acid in water and 100 % methanol. the applied gradient program was: 0-10 min (5 % to 15 % methanol), 1040 min (15 % to 20 % methanol), 40-60 min (20 % to 30 % methanol), 60-100 min (30 % to 50 % methanol), 100-120 min (50 % to 95 % methanol), and 120-130 min (95 % methanol). 20 μl of the standard solutions and samples were used and all of the samples were analyzed in triplicate. hplc-esi/ms analysis the chromatographic separation condition was described above. an agilent 1100 analytical hplc system was used, with a g1312 binpump, g1314a variable-wavelength detector (vwd), model 7725 injector fitted with a 20 μl sample loop, and an agilent chemstation data system. lc/esi-ms analyses were performed by using the agilent hplc system described above combined with a bruker esquire 3000 plus ion trap mass spectrometer (bruker-franzen analytik gmbh, bremen, germany), which was equipped with an electrospray ionization. instrument control and data acquisition were performed by using esquire 5.0 software. the ion source temperature was 270 °c, and the needle voltage was always set at 4.0 kv. nitrogen was used as the drying and nebulizer gases at a flow rate of 10 l min−1 and a back-pressure of 30 psi. total flavonoids content the total flavonoids content of the extracts was determined by the method described in the chinese pharmacopoeia (2010 edition). 100 mg of extract was dissolved in 100 ml of 70 % (v/v) ethanol solvent. 1 ml solution was taken, being mixed with 1 ml of 5 % nano2. 6 min later, 1 ml of 10 % alcl3 was added, and then 10 ml of naoh (1m) was added after the same time. immediately, the mixture was constant volume to 25 ml and was kept in dark place to react for 15 min. with 70 % ethanol as a blank control, the absorbance (a) was measured at the wavelength of 510 nm. rutin was used as standard compound and the concentration ranged from 0.01 to 0.80 mg ml−1. the total flavonoids content was expressed as mg of rutin, which was equivalent to per g of extracts. orientin and isoorientin content the content of orientin and isoorientin in extracts was measured with hplc method. the mobile phase consisted of 0.1 % phosphoric acid in water (a) and 2 % tetrahydrofuran in methanol (b). the gradient profile was established as follows: the ratio of a and b was 77:23 (v/v) within 60 min. the flow rate was 1.0 ml min−1, and the column temperature was 40 °c. all of the samples were analyzed in triplicate. assays for antioxidant activities dpph assay the dpph assay of the extracts was determined following the method used by shimada, fujikawa, yahara, and nakamura (kazuko et al., 1992). dpph was dissolved in ethanol and the reaction solution concentration was 0.1 mm. 1 ml of the dpph reaction solution was added to 5 ml of extract solution with being shaken vigorously. the mixture reacted for 30 min in the dark at room temperature, and then was measured at a wavelength of 517 nm using a uv spectrophotometer. ascorbic acid and the antioxidant composition of bamboo leaves (aob) were used as control groups. all of the samples were analyzed in triplicate. the dpph radical scavenging activity was calculated by the following equation: scavenging activity % = [ 1 asample / ablank ] × 100 % according to different concentrations of the clearance curve, the scavenging activities were expressed as half maximal inhibitory concentration (ic50). abts assay the abts assay was based on the method of re et al. (re et al., 1999) with some modifications. abts stock solution was dissolved in deionized water to a 7 mm concentration. the abts reagent solution was generated by 5 ml of abts stock solution with 88 μl of 140 mm potassium persulfate. this solution was stored at room temperature in the dark for 12-16 h. before it was used, the abts reagent solution was diluted with pbs (ph 7.4) to an absorbance of 0.70 ± 0.02 at 734 nm. for the abts assay, 0.3 ml sample of different concentrations was added to 6 ml of diluted abts solution to react in the dark at 30 °c for 6 min, and then the absorbance was measured at a wavelength of 517 nm. ascorbic acid and aob were used as control groups. all of the samples were analyzed in triplicate. the abts radical scavenging activity was calculated by the following equation: scavenging activity % = [ 1 asample / ablank ] × 100 % according to different concentrations of the clearance curve, the scavenging activities were expressed as ic50. frap assay the frap assay was determined by the method of benzie and strain (benzie and strain, 1996) with minor modifications. the frap reagent included the following solution: 300 mm acetate buffer; 10 mm tptz in 40 mm hydrochloric acid; and 20 mm fecl3·6h2o. the frap reagent was prepared by mixing 25 ml acetate buffer, 2.5 ml of tptz solution and 2.5 ml of fecl3·6h2o solution, and then warmed at water bath to 37 °c before using. 0.3 ml of sample solutions was added into 6 ml of frap solution, and reacted for 10 min. absorbance was measured at a wavelength of 593 nm. ascorbic acid and aob were used as control groups. all of the samples were analyzed in triplicate. antimicrobial activities the antimicrobial activity of the extracts was determined by the serial 2-fold dilution method (charles et al., 1979; shadomy and espinel, 1980). three bacterial strains were used in this study: staphylococcus aureus (atcc6538), escherichia coli (8099), and candida albicans (10231). all strains were obtained from the zhejiang academy of medical sciences, china. the bacterial strains were incubated on micrococcus, nutrient, and yeast maltose (ym) media then cultured at incubator at 35 °c for 24 h. vbe was dissolved in pbs at the highest concentration (100 μg/ml), then diluted to serial two-fold dilutions. tetracycline was used as standard antibiotics. the minimum inhibitory concentration (mic) was defined as the lowest concentration that inhibited microorganism growth, which was based on the degree of turbidity visually. statistical analysis all samples were measured in triplicate. spss statistical software was used for the statistical evaluation of results, which were presented as the mean ± standard deviation (sd). statistical analysis was done by independent-samples t-test. 152 j. hu, j. wang, s. li, b. yang, m. gong, x. li, l. zhang, j. tian results and discussion hplc analysis and identification compounds by hplc–esi/ms analysis in order to get the information of the vbe compounds, the analysis of hplc and hplc–esi/ms was needed. the hplc chromatogram of the vbe was showed in fig. 1 (a), the separation of the compounds was excellent under the optimized conditions. as to the lcms information, the tic negative and the tic positive were shown in fig. 1 (b), fig. 1 (c), the tic positive intensity was obviously less than the tic negative, it was related to noise level and the compound structure, especially flavonoid compositions (petsaloa et al., 2006). compared to previous research, the compounds 1-12 in fig. 1 (a) were identified as shown in tab. 1 which include eight flavonoids: (4) orientin; (5) isoorientin; (6) hyperoside; (7) quercetin-3o-β-d-glucuronide; (8) quercetin-3-o-α-l-arabinopyranoside; (10) quercetin-3-o-α-l-rhamnoside; (11) quercetin-3-o-β-d-glucuronide methyl ester; (12) isolariciresinol-9-o-β-d-xyloside. compared with the hplc chromatogram of chlorogenic acid and the vbe, the result showed the existence of chlorogenic acid in the vaccinium bracteatum thunb. leaves in fig. 1 (d). 0 20 40 60 80 100 120 time [min] 0.00 0.25 0.50 0.75 1.00 1.25 6x10 intens . b 0 10 20 30 40 50 60 70 80 90 100 11 0 120 mi n -2 5 0 25 50 75 100 125 150 175 a 1 2 3 4 5 6 8 9 10 11 12 7 0 20 40 60 80 100 120 time [min] 0 1 2 3 4 5 7x10 intens . c 3-o-chlorogenic acid vbe d fig. 1: (a) hplc chromatography of the vbe at the 254 nm; for peak identification see tab. 1. (b) the tic negative; (c) the tic positive; (d) chromatography contrast between chlorogenic acid reference and the vbe hplc. tab. 1: identification of 12 compounds. peak retention time λmax (nm) m/z identification 1 16.4 240, 324, 443 354 5-o-chlorogenic acid 2 29.0 240, 324, 443 354 3-o-chlorogenic acid 3 30.9 240, 324, 443 354 4-o-chlorogenic acid 4 64.0 252, 266, 348 448 orientin 5 67.6 255, 269, 348 448 isoorientin 6 74.9 253, 267, 352 464 hyperoside 7 75.5 255, 354 478 quercetin-3-o-β-d glucuronide 8 77.9 247, 443 434 quercetin-3-o-α-l arabinopyranoside 9 79.0 220, 247, 342 536 10-o-trans-p-coumaroyl sandoside 10 82.9 246, 286, 321 448 quercetin-3-o-α-l rhamnoside 11 86.7 266, 344, 324 492 quercetin-3-o-β-d glucuronide methyl ester 12 90.0 248, 443 494 isolariciresinol-9-o-β-d xyloside tab. 2: the content between the raw materials and the extracts. (mean ± sd, n = 3) sample total flavonoid orientin isoorientin (mg/g) (mg/g) (mg/g) raw materials 56.3 ± 0.27 5.31 ± 0.05 9.42 ± 0.04 vbe 601.4 ± 8.50 44.7 ± 0.60 96.1 ± 0.92 the total flavonoids, orientin and isoorientin content the contents of total flavonoids, orientin, and isoorientin were compared between the raw materials and the vbe. the results are shown in tab. 2. orientin and isoorientin have a typical flavonoid structure, so we choose the two standards as index compositions for measurements. the total flavonoids content was expressed as rutin equivalents in mg/g of extracts. results indicated that the content of the total flavonoids in the extracts was 601.4 ± 8.50 mg/g, which was nearly ten times than that in the raw materials. furthermore, the contents of orientin and isoorientin in the extracts were 44.7 ± 0.60 mg/g and 96.1 ± 0.92 mg/g, respectively, which were far more than that in the raw materials. the macroporous resins d-101 had good effect in enrichment of flavonoids, which was also confirmed in many studies (fang et al., 2008; qi et al., 2007). as active ingredients, it would enhance the antioxidant activity and other activities by increasing flavonoids content. the nutraceutical properties of vaccinium bracteatum thunb. leaves 153 the antioxidant activities antioxidant activities were evaluated by three methods, and the results were displayed in fig. 2 and tab. 3. the results of dpph assay found that all samples had scavenging action. the ic50 value was 42.2 ± 1.2 μg/ml for vbe, while 449.5 ± 10.6 μg/ml for the raw materials. additionally, the results of abts assay showed that the ic50 value of the raw materials, ascorbic acid, vbe, and aob were 381.8 ± 12.4, 48.7 ± 1.0, 71.1 ± 1.1, and 104.3 ± 2.2 μg/ml, respectively. furthermore, using 0.3 mmol/l feso4 as the standard, the result of frap assay showed that the concentrations up to the same absorbance of the raw materials, ascorbic acid, vbe and aob were 327.9 ± 7.5, 28.2 ± 0.5, 65.0 ± 1.8, and 101.7 ± 1.1 μg/ml, respectively. antioxidant capacities varied among these three antioxidant assays, all the samples showed certain antioxidant ability. compared to the extracts of the raw materials, the antioxidant activity of vbe was better (p < 0.05), which also showed that the high content accompanied with strong antioxidant activities. as chemical antioxidant, ascorbic acid had the highest antioxidant ability, however, the activity was significantly higher in vbe compared to aob (p < 0.05). it is reported that all of the three bamboo extracts from different parts had antioxidant activities, which had approved the development of natural antioxidants (gong et al., 2015). besides, its main active ingredients were flavonoids, azalides, and phenolic acid, and four major flavonoids were orientin, isoorientin, vitexin and isovitexin. the vbe and aob had the same compound ingredients, but the vbe flavonoids content was much higher and the antioxidant activity of vbe was better than that of aob. it was indicated that the vaccinium bracteatum thunb. leaves had large potential to be used as natural antioxidant. the antimicrobial activities of vbe the results of antimicrobial activities were presented in tab. 4, which showed that the serial 2-fold dilution method was feasible. some researches had found that chlorogenic acid had strong antimicrobial activity (zhao et al., 2010; chakraborty and mitra, 2008), there were also many antimicrobial reports on flavonoids (mellou et al., 2005; sousa et al., 2009; sohn et al., 2004). as representative compositions of the flavonoids, there were many studies on the antimicrobial activities of orientin and isoorientin (zu et al., 2010; becker et al., 2005). based on these researches reported previously, we can infer that the vbe has good antimicrobial activity. the results obtained showed that the vbe has the inhibition to the three kinds of bacteria, and the minimum inhibitory concentration was 12.5 mg/ml with high level of antimicrobial activity. conclusions in this research, twelve compounds were identified by using hplcdad and hplc–esi/ms data analysis, which contained chloro genic acid and its isomers, and eight flavonoids. furthermore, we quantitatively analyzed the total flavonoids, orientin and isoorientin content of the extracts from vaccinium bracteatum thunb. leaves. these substances provided the basis of chemical compositions to explain the antioxidant activities. the antioxidant activities of the extracts were estimated by dpph, abts, and frap assays. the results suggested that the antioxidant activity of the vbe could be significantly stronger than the aob. finally, the antibacterial activity of the vbe was determined by a serial twofold dilution method. based on these results, the vaccinium bracteatum thunb. leaves could be a potential natural antioxidant. for further research, more works are needed to be carried out; the vbe will be added to the meat or the fried food as a readily accessible source of new natural food antioxidants. fig. 2: antioxidant activities of the raw materials, vbe, aob and ascorbic acid. (a) dpph radical scavenging assay; (b) abts radical scavenging assay; (c) frap-feso4 standard curve; (d) different concentration samples absorbance curve in frap assay. the value in the figure is average value, n = 3. 154 j. hu, j. wang, s. li, b. yang, m. gong, x. li, l. zhang, j. tian tab. 3: the antioxidant activity results. (mean ± sd, n = 3) ic50 (μg/ml) dpph abts vbe 42.2 ± 1.2** 71.1 ± 1.1** 65.0 ± 1.8** raw materials 449.5 ± 10.6** 381.8 ± 12.4** 327.9 ± 7.5** aob 105.2 ± 3.4** 104.3 ± 2.2** 101.7 ± 1.1** ascorbic acid 29.7 ± 0.5 48.7 ± 1.0 28.2 ± 0.5 a: result was expressed as the concentration of antioxidants up to the same absorbance that of 0.3 mmol/l feso4. **: significantly different at the 0.05 level (2-tailed). sample frap assay (μg/ml) a tab. 4: antimicrobial activities of the vbe the concentration of vbe (mg/ml) 50 25 12.5 6.25 3.125 1.5625 0.78125 staphylococcus aureus (atcc6538) – – – + + + + + – escherichia coli (8099) – – – + + + + + – candida albicans (10231) – – – + + + + + – +: instructions bacterial growth was positive; –: instructions bacterial growth was negative. negative control positive control bacterial strain hao, j.j., feng, j.y., ding, y.l., wang, f.s., 2010: dynamic changes of the nutritional compositions of vaccinium bracteatum leaf from different provenances. 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(chinese). chinese pharmaceutical j. 44, 1773-1776. zhao, m.m., wang, h.y., yang, b., tao, h., 2010: identification of cyclodextrin inclusion complex of chlorogenic acid and its antimicrobial activity. food chem. 120, 1138-1142. zu, y.g., liu, x.l., fu, y.j., wu, n., kong, y., wink, m., 2010: chemical composition of the sfe-co2 extracts from cajanus cajan (l.) huth and their antimicrobial activity in vitro and in vivo. phytomedicine 17, 1095-1101. address of the author: jin hu, bingxian yang, lin zhang, jingkui tian, the key laboratory of biomedical engineering ministry of education, department of biomedical engineering, zhejiang university, hangzhou 310027, china e-mail: tjk@zju.edu.cn jing wang, minghua gong, college of basic medical science, shandong university of traditional chinese medicine, jinan, 250355, pr china shouxin li, ximin li, changshu qiushi technology co. ltd., changshu, 215500, china © the author(s) 2016. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 86, 33 36 (2013), doi:10.5073/jabfq.2013.086.005 division of agricultural chemicals, indian agricultural research institute, new delhi, india plant growth inhibitory terpenes from eupatorium adenophorum leaves a. kundu*, s. saha, v. ahluwalia, s. walia (received october 15, 2012) * corresponding author summary eupatorium adenophorum is commonly known as crofton weed, growing widely throughout the northern hilly terrains. bioactive potential of e. adenophorum leaves was investigated through phytochemical study and in vitro plant growth inhibitory. essential oil was hydrodistilled from leaves and analyzed by gcms. leaf material was extracted using cold extraction process followed by evaporation of solvent under reduced pressure. five cadinene sesquiterpenes and one sterol were isolated from hexane and etoac concentrates and their structures were established spectroscopically. plant growth inhibitory activity of extractives and isolated terpenes were studied against different seeds of weed and crops. essential oil was moderate seedling growth inhibitor. among various extracts, etoac extract was found to be most inhibitory to phalaris minor seeds (ec50 117 μg ml-1). among the sesquiterpenes, 5,6dihydroxycadinan-3-ene-2,7-dione was found to be most active and inhibited both shoot and root growth of phalaris minor seed (ec50 97 μg ml-1) and polygonum plebejum (ec50 117 μg ml-1). introduction eupatorium adenophorum spreng (syn. ageratina adenophora king and robinson) is fast growing perennial herbaceous plant, belonging to the family compositae (duan, 2003). the plant flourishes abundantly forested and cultivated lands, and is widely distributed from tropical to temperate region such as america, europe, australia, south africa, india, thailand and china (auld, 1966). the plant is introduced in india in 19th century since then widely proliferating in the hilly terrain of northern, north eastern region and other lower hilly regions of southern india (borthakur, 1977). due to significant essential oil content in its aerial parts (weyerstahl et al., 1998; pala-paul et al., 2002), it is considered as a valuable raw material for perfumery industry (sharma et al., 1998; adhikari and kraus, 1994). the plant is reported to contain an array of bioactive constituents like monoterpenes, sesquiterpenes, flavonoids, phenyl propanoids and their derivatives (proksch et al., 1983). sesquiterpenoids are the major constituent of leaves and flowers (ding et al., 1999; zinghui and jingkai, 1999). cadinene sesquiterpenes were also identified in leaves (lan et al., 2006). the plant is also well known for its insect repellent activities (sood et al., 2000; li et al., 2001; mehta et al., 2002; wang, 2002; rajmohan and ramaswamy, 2007). potential molluscicidal activity of aqueous extract of the plant was reported against oncomelania hupensis (zou et al., 2009). besides, growth inhibitory activity of e. adenophorum litter was also studied on survival and growth of lantana camara (kaul and bansal, 2002). however, except some sporadic attempts on plant growth inhibitory activity (zheng and feng, 2005), detailed growth inhibition assay of cadinenes from e. adenophorum has not been studied. as far as our literature survey could ascertain, there is no report on antifungal activity of the cadinene derivatives isolated from leaves of e. adenophorum. the present study was therefore aimed to isolate and evaluate major bioactive constituents of e. adenophorum leaves for plant growth inhibitory activity against seeds of three weeds and two crops. materials and methods reagents and instruments all solvents and reagents were of the highest purity needed for each application. solvents and chemicals were purchased from sigma® (usa) and merck® india ltd. and used without further purification. materials used for column chromatography was silica gel (100200 mesh; merk specialities private ltd. mumbai, india). hsgf254 silica gel tlc plates (merck specialities private ltd. mumbai, india) were used for analytical tlc and spots were detected under uv light or by heating after spraying with 98% h2so4. preparative tlc (0.4-0.5 mm) was performed on glass plates precoated with silica gel gf254. uv and ir spectra were recorded respectively with a jasco v-650 spectrophotometer and an fts-40 infrared spectrometer with kbr pellets. nmr spectra were recorded on a brucker 400 ac (400 and 75.5 mhz) nmr spectrometer with tms as an internal standard. esi-ms/ms were measured on a thermo lc/ msd trap xct mass spectrometer (finnigan mat incos 50). plant material leaves of e. adenophorum were collected during 2009 from forest area of kangra, himachal pradesh, india. after identification of specimen, a voucher sample (ea-eaa-05-09) was deposited in the herbarium of department of botany, himachal pradesh agricultural university, himachal pradesh, india. essential oil extraction and gc-ms analysis shade dried leaf powder (5 kg) was subjected to hydrodistillation for 4 hours using a clevenger apparatus to obtain essential oil. essential oil was collected and dried over anhydrous sodium sulfate and preserved in a sealed vial at 4 °c until analysis. gc-ms analysis was carried out on a thermo fischer capillary gas chromatograph equipped with flame ionization detector (fid) for fi-detection, which is further directly coupled to the mass spectrometer system and hp-5ms capillary column (30m × 0.25 mm i.d. × 0.25 µm film thickness). temperature programming was done 60-125 °c at 2 °c/min hold time 2 min., 125-160 °c at 0.5 °c /min hold time 5 min. and 160-240 °c at 5 °c/min. injector and detector temperatures were maintained at 220 °c and 290 °c, respectively. helium was used as carrier gas with a flow rate of 1 ml/min. ion source temperature was 270 °c and mass transfer line 250 °c with split ratio 1:20. identification of the constituents of essential oil was performed by comparison of their retention times, retention indices and comparison of mass spectral fragmentation pattern. cold extraction of leaves shade dried leaves (5 kg) of e. adenophorum was extracted separately with meoh (10 l). the solvent was filtered and evaporated 34 a. kundu, s. saha, v. ahluwalia, s. walia in vacuuo at 42 °c to yield methanolic concentrates. chlorophyll was removed from methanolic concentrate (920 g) by precipitation with lead acetate (5 %). methanolic concentrate (10 g) was dissolved in aqueous ethanol (70 %), lead acetate solution (5 %) was added and kept undisturbed for 10 minutes. chlorophyll lead acetate complex precipitated was filtered to remove undesired precipitates and the filtrate was partitioned sequentially with hexane, etoac and nbuoh to obtain respective concentrates. isolation of terpenes etoac concentrate (70 g) was subjected to silica gel column chromatography (100-200 mess particle size, pre-activated at 110 °c) using eluent mixtures of etoac in hexane (20-100 %, v/v) to obtain 50 fractions. fractions 15-23 showing four major spots on tlc plates were combined and re-chromatographed using hexaneetoac (10 %) followed by preparative-tlc to give ea-1 (75 mg) and ea-2 (55 mg). fractions 37-46 showing similar spots on tlc were combined and re-chromatographed using a gradient of hexaneetoac (50-80 %) as eluent to provide 16 fractions (fr. i-fr. xvi). fraction ix-xii were combined and subjected to preparative tlc to obtain comparatively less polar ea-3 (45 mg) and more polar ea-4 (55 mg). on the other hand, hexane concentrate (50 g) was silica gel column chromatographed with the gradient of increasing hexane in etoac (0-50 %). total number of 32 fractions was collected and finally 5 sub-fractions obtained on combining the eluates based on their similar behavior on tlc. sub-fraction 1 was subjected to preparative-tlc with hexane-etoac (80 %) to obtain ea-5 (60 mg) and ea-6 (32 mg). plant growth inhibition assay seeds of three weeds phalaris minor, polygonum plebejum, chenopodium album and two field crops wheat (triticum aestivum l.) cv. hd 2329 and chickpea (cicer arietinum l.) cv. bgd72 were collected from seed technology centre, division of seed science and technology, iari, new delhi. test solutions of essential oil, extracts and terpenes were prepared using dmso (0.1 %, v/v) as the initial solvent carrier followed by diluting with distilled water to a final concentration of 25 μg ml-1. other test solutions (i.e., 50, 100, 250, 500 μg ml-1) were prepared by dilution with an aqueous solution of dmso (5 %). seeds were washed with ethanol (70 % v/v) for 2 min and surface sterilized using sodium hypochlorite (0.5 % v/v) for 2 min, followed by three washes with sterile distilled water. after sterilization seeds were stored in a refrigerator at 4 °c for 3 days before use. three layers of filter papers were put in 6 cm diameter glass petri plates, and the filter papers were impregnated with test solutions. to avoid toxic effect of organic solvent, filter paper treated with dmso solution was placed in a fume hood for 1 h to allow complete solvent evaporation. fifty seeds of each test crop were evenly placed on the moist filter paper in each petri plates. two controls (filter paper treated with 3 ml of dmso and filter paper without any treatment) were set. each treatment had three duplicates. seeds were allowed to germinate under 12 hrs light at 25 °c. the root and shoot length was measured in both treated (t) and control (c) plates (after 7 days) until all the seeds in the control petri plates were fully germinated. percentage inhibition of growth (i %) and ed50 (μg ml-1) were determined. statistical analysis experimental data were subjected to one-way analysis of variance (anova) followed by duncan’s multiple range test (dmrt) to determine significant differences among treatment means. differences were considered significant at p levels of 0.05 and 0.01. results and discussion chemical constituents light yellowish essential oil (0.90 % v/w, dry weight basis) with strong aromatic odour was hydrodistilled from leaves. gc-ms spectrum indicated total twenty six volatile compounds, representing 83.5 % of total composition. about 71.9 % sesquiterpenes and 11.3 % monoterpenic constituents were identified in essential oil (tab. 1). γ-cadinene (13.56 %) and germacrene-d (10.03 %) were the major constituents. two cadinene sesquiterpenes were isolated and purified from hexane concentrate. similarly, etoac fraction was further investigated for its phytochemical compositions. four cadinene derivatives were isolated and their structures were determined by nmr and mass fragmentation pattern. further identification of these cadinene derivatives were also done by comparison with the literature data. cadinene derivatives were iden-tified as 7-hydroxycadinan-3-ene-2-one (ea-1) (bohlmann and gupta, 1981), 5,6-dihydroxycadinan-3-ene-2,7-dione (ea-2) (zhao et al., 2009), 2-acetyl-cadinan-3,6-diene-7-one (ea-3) (baruah et al., 1994), stigmasterol (ea-4), cadinan-3-ene-2,7-dione (ea-5) (lan et al., 2008) and cadinan-3,6-diene-2,7-dione (ea-6) (lan et al., 2008) (fig. 1). copies of the original spectra are obtainable from author. plant growth inhibition activity a number of studies have been reported in literature which showed that plant allelochemicals interfere with the growth and establishtab. 1: essential oil constituents of e. adenophorum leaves. compounds rt ri content (%) cymene 9.10 1026 0.3±1.2 camphor 14.95 1100 1.4±1.1 borneol 16.20 1155 1.8±0.3 camphene 17.66 1162 2.2±0.6 verbenol 18.19 1171 0.3±0.9 bornyl acetate 23.36 1268 1.7±1.3 geranial 27.17 1294 2.2±2.6 limonene 31.35 1352 1.5±2.5 aromadendrene 32.53 1408 3.1±0.7 β-cedrene 32.94 1418 1.3±1.4 α-bergamotene 33.32 1433 0.7±2.6 β-farnesene 34.20 1445 6.7±1.0 elemene 35.56 1449 9.8±3.6 caryophyllene 35.87 1457 4.6±1.2 α-humulene 36.52 1462 2.6±1.8 germacrene-d 38.03 1464 10.0±1.1 α-curcumene 38.27 1479 0.7±0.6 bicyclogermacrene 39.66 1489 3.0±3.7 cadina-1,4-diene 40.72 1496 2.3±4.1 β-bisabolene 40.98 1503 1.5±0.4 sesquiphellandrene 42.19 1508 0.7±1.5 spathulenol 47.44 1519 1.6±1.0 γ-cadinene 50.60 1529 13.6±1.2 cadinol 53.29 1653 0.7±1.8 farnesol 58.17 1656 5.4±1.5 nerolidol 59.40 1677 3.8±0.7 rt=retention time (min) ri = relative index to n-alkanes (c9-c21) on hp-5 ms column terpenes of eupatorium adenophorum 35 ment of other crops or weeds (whittaker, 1971; ens et al., 2009). several allelopathic compounds such as catechin, juglone, apigenin, gallic acid derivatives have been discovered and investigated against various weed/crop species. our study indicated that both hexane and etoac concentrate was effective on p. minor and p. plebejum. essential oil was moderately active on shoot and root growth inhibition of weed seeds. however, seedling growth of crop seeds was not hampered. growth inhibition percentage was dose dependant and detailed lethal median concentration was depicted in tab. 2. shoot length of t. aestivum seed was also found to be inhibited by the extracts. meoh concentrate was active against p. minor and c. album. there was no growth inhibition effect observed with n-buoh extract. root and shoot growth inhibition of c. arietinum was significantly less. since, solvent concentrates were found to be active; it was investigated in details to isolate bioactive principle. among the cadinene constituents, 5,6-dihydroxycadinan-3-ene-2,7dione exhibited highest shoot and root growth inhibition against p. minor (tab. 2). similar trend was observed in growth inhibition of p. plebejum. however, c. album and c. arietinum growth was not much hampered at the same concentration. as evident from the data, growth inhibition of c. arietinum was below 20 %. 5,6dihydroxycadinan-3-ene-2,7-dione was most effective in inhibiting coleoptile growth of p. minor (ec50 97 μg ml-1) and p. plebejum (ec50 117 μg ml-1) than c. album (ec50 516 μg ml-1). it was also highly inhibitory of root growth of p. minor (ec50 190 μg ml-1). 7-hydroxycadinan-3-ene-2-one was found to be active in reducing the shoot length of p. plebejum (ec50 147 μg ml-1) and p. minor (ec50 169 μg ml-1) than c. album (ec50 601 μg ml-1). root growth of these weeds was not much affected by 7-hydroxycadinan-3-eneo o ohoh oh o o o o o ho ea-1 ea-2 ea-3 ea-4 ea-5 ea-6 o o o fig. 1: structures of terpenes isolated from e. adenophorum leaves. (ea-1), 7-hydroxycadinan-3-ene-2-one; (ea-2), 5,6-dihydroxycadinan-3-ene-2,7-dione; (ea-3), stigmasterol; (ea-4), 2-acetyl-cadinan-3,6-diene-7one; (ea-5), cadinan-3-ene-2,7-dione; (ea-6), cadinan-3,6-diene-2,7-dione tab. 2: plant growth inhibitory activity of extracts and cadinene sesquiterpenes. compounds / extracts ec50 (µg ml-1) please round the values p. minor p. plebejum c. album t. aestivum c. arietinum root shoot root shoot root shoot root shoot root shoot essential oil 375 252 468 265 636 412 841 740 755 617 hexane extract 216 161 427 186 456 517 346 226 678 521 meoh extract 346 257 527 397 338 277 457 307 735 616 etoac extract 270 117 312 190 534 435 345 155 789 630 n-buoh extract 982 790 858 690 975 757 >1000 992 >1000 876 ea-1 279 169 327 147 639 501 334 198 637 519 ea-2 190 97 200 117 435 516 291 111 601 576 ea-3 287 144 300 162 574 413 421 169 620 525 ea-4 590 437 802 679 945 583 622 480 >1000 947 ea-5 432 279 489 311 767 570 410 301 790 657 ea-6 468 333 429 353 672 520 424 361 724 580 36 a. kundu, s. saha, v. ahluwalia, s. walia 2-one. on the other hand, stigmasterol showed lowest shoot and root growth inhibition against all test species. 2-acetyl-cadinan-3,6diene-7-one was also exhibited shoot length inhibition of p. minor (ec50 144 μg ml-1). in general, shoot growth inhibition was more as compared to root growth inhibition. cadinan-3-ene-2,7-dione and cadinan-3,6-diene-2,7-dione exhibited moderate growth inhibition of test seeds. our study indicated that 5,6-dihydroxycadinan-3-ene-2,7-dione was most potential plant growth inhibitor against p. minor seeds. higher plant growth inhibitory activity of 5,6-dihydroxycadinan-3-ene2,7-dione was attributed to the fact that this compound is relatively more polar than the other isolated compounds. in general crude extracts were also active towards the weed seeds. further, the relative effect on coleoptile growth was greater than that of root growth. these results are in agreement with the previous reports (tripathi et al., 1981). furthermore, coleoptile and root growth of the test species exhibited different responses towards cadinene derivatives, confirming the contention of whittaker, 1971 that plant growth inhibitory activity depends on the nature of test species and concentration of the compounds. experimental results suggested that the cadinenes are the major constituents of e. adenophorum leaves. chemical constituents and possible growth inhibitory activity of leaves of e. adenophorum are most important examined features. in view of botanical origin of active ingredient and huge availability of the plant material makes it potential source of naturally occurring bioactive constituents. acknowledgements authors are thankful to head, division of agricultural chemicals, indian agricultural research institute, india for providing necessary facilities for carrying out the research work. references adhikari, r., kraus, w., 1994: bioactive constituents of eupatorium adenophorum spreng (banmara). j. nepal chem. soc. 13, 34-36. auld, b.a., 1966: the distribution of eupatorium adenophorum spreng on the far north coast of new south wales. new zealand sci. 102, 159161. baruah, n., sarma, j., sarma, s., sharma, r., 1994: seed germination and growth inhibitory cadinenes from eupatorium adenophorum spreng. j. chem. ecol. 20, 1885-1892. bohlmann, f., gupta, r., 1981: six cadinene derivatives from ageratina adenophora. phytochem. 20, 1432-1433. borthakur, d.n., 1977: mikania and eupatorium, two noxious weeds of ne region. indian farm. 26, 48-49. ding, z., guo, y., ding, j., 1999: chemical constituents from the flowers of eupatorium adenophorum. acta bot. yunnanica 21, 505-511. duan, h., 2003: eupatorium adenophorum spreng. chin. j. weed sci. 2, 36-38. kaul, s., bansal, g.l., 2002: allelopathic effect of ageratina adenophora on growth and development of lantana camara. indian j. plant physiol. 7, 195-197. ens, e.j., bremner, j.b., french, k., korth, j., 2009: identification of volatile compounds released by roots of an invasive plant, bitou bush (chrysanthemoides monilifera spp. rotundata), and their inhibition of native seedling growth. biol. invasions 11, 275-287. kaul, s., bansal, g.l., 2002: allelopathic effect of ageratina adenophora on growth and development of lantana camara. ind. j. pl. physi. 7, 195-197. kumar, a., 2006: evaluation of botanicals against major pathogens of rice. indian phytopathol. 59, 509-511. lan, h., jie, y., aocheng, c., yumei, l., yu, a., jiangong, s., 2006: a new sesquiterpenoid from eupatorium adenophorum spreng. chin. j. chem. 24, 1375-1377. lan, h., jing, h., maoluo, g., jiangong, s., suchada, c., hoongkun, f., ian, d.w., herman, h.s., 2008: cadinane sesquiterpenes from the leaves of eupatorium adenophorum. j. nat. prod. 71, 1485-1488. li, y., zou, h., nai, z., li, w., na, x., tang, s., yang, y., 2001: insecticidal activity of extracts from eupatorium adenophorum against four stored grain insects. entomol. knowledg. 38, 214-216. mehta, p.k., sood, a.k., parmar, s., kashyap, n.p., 2002: antifeedant activity of some plants of north-western himalayas against cabbage caterpillar, pieris brassicae (l.). j. entomol. res. 26, 51-54. pala-paul, j., perez-alonso, m.j., velasco-negueruela, a., sanz, j., 2002: spectrometry of the volatile components of ageratina adenophora spreng, growing in the canary islands. j. chromatogr. a 947, 327-331. proksch, p., witte, l., wray, v., rahaus, i., 1983: accumulation and biotransformation of chromenes and benzofurans in cell suspension culture of ageratina adenophora. planta med. 53, 488-493. rajmohan, d., ramaswamy, m., 2007: evaluation of larvicidal activity of the leaf extract of a weed plant, ageratina adenophora, against two important species of mosquitoes, aedes aegypti and culex quinquefasciatus. afr. j. biotechnol. 6, 631-638. sharma, o.p., rajinder, k.d., nitin, p.k., sharma, p.d., 1998: a review of the toxicology and biologicalproperties of the genus eupatorium. nat. toxins 6, 1-14. sood, a., mehta, p., parmar, s., 2000: evaluation of plant extracts against cabbage aphid, brevicoryne brassicae (linn.). insect environ. 6, 87-89. tripathi, r.s., singh, r.s., rai, j.p.n., 1981: allelochemical potential of eupatorium adenophorum, a dominant ruderal weed of meghalaya. proc. indian nat. acad. sci. 47, 458-465. wang, d., gaoping, z., hongzheng, y., liulin, s., 2002: anti-aphid activity eupatorium adenophorum: isolation, purification and its effect on the impact of cotton aphid acetylcholinesterase. high tech. lett.12, 400-403. weyerstahl, p., marschall, h., seelmann, i., kaul, v., 1998: constituents of the flower essential oil of ageratina adenophora (spreng) from india. flavour frag. j. 12, 387-396. whittaker, r.h., 1971: the chemistry of communities. in biochemical interactions among plants. national academy of science, washington, d.c. zhao, x., guowei, z., xuemei, n., weiqi, l., fusheng, w., shenghong, l., 2009: terpenes from eupatorium adenophorum and their allelopathic effects on arabidopsis seeds germination. j. agric. food chem. 57, 478-482. zheng, l., feng, y.l., 2005: allelopathic effects of eupatorium adenophorum spreng on seed germination and seedling growth in ten herbaceous species. acta ecol. sin. 25, 2782-2787. zinghui, d., jingkai, d., 1999: eupatoranolide, a new sesquiterpenes lactone from eupatorium adenophorum. chin. chem. lett. 10, 491494. zou, f., duan, g., xie, y., zhou, y., dong, g., lin, r., zhu, x., 2009: molluscicidal activity of the plant eupatorium adenophorum against oncomelania hupensis, the intermediate host snail of schistosoma japonicum. ann. trop. med. parasitol. 103, 549-553. address of the corresponding author: dr. aditi kundu, scientist, division of agricultural chemicals, indian agricultural research institute, new delhi-110012 journal of applied botany and food quality 88, 323 (2015) buchbesprechung hänsel/sticher pharmakognosie – phytopharmazie otto sticher, jörg heilmann, ilse zündorf das bereits seit mehr als 50 jahren bekannte standardwerk vermittelt die wissenschaftlichen grundlagen hinsichtlich pflanzlicher wirkstoffe, phytopharmazie, pflanzliche arzneimittel sowie deren wirkungen und wirksamkeit. die zielsetzung des lehrbuchs besteht insbesondere darin, angehenden apothekern ein solides basiswissen auf dem gebiet der phytopharmaka (von der pflanze zum arzneimittel, screening-methoden, herstellung und prüfung) zu vermitteln. bei der stoffauswahl liegen die schwerpunkte eindeutig in der phytochemie (biosynthese von sekundärstoffen, analytik, wirkund inhaltsstoffe) und der phytopharmakologie (rationale und traditionelle phytotherapie, tcm, klinische studien, wirkungen und nebenwirkungen). darüber hinaus werden theoretische grundlagen zu den arzneimittelmonographien (insbesondere ph.eur.8), über die herstellung und prüfung pflanzlicher arzneimitteln sowie eine anleitung zur kritischen beurteilung pflanzlicher arzneiund nahrungsergänzungsmittel geliefert. im teil a werden zunächst die phytochemischen grundlagen näher behandelt. hier wird u.a. auf die heute zur verfügung stehenden möglichkeiten zur aufklärung von biosynthesewegen näher eingegangen (z.b. tracerund isotopentechnik sowie enzymatische und molekulargenetische methoden). darüber hinaus werden die aktuellen analytischen methoden zur strukturaufklärung, aber auch screening und bioassay-methoden vorgestellt. der teil b ist den pflanzlichen arzneidrogen, arzneizubereitungen und pflanzlichen fertigarzneimittel gewidmet. auch die jeweiligen spezifikationen und qualitätsprüfungen werden in diesem zusammenhang näher behandelt. teil c beschreibt u.a. die konzeption klinischer studien, geht auf das nebenwirkungspotential pflanzlicher zubereitungen ein und beschreibt dabei auch die bedeutung der traditionellen chinesischen medizin in westlichen ländern. in teil d werden schließlich die einzelnen relevanten chemischen stoffgruppen behandelt: kohlenhydrate, lipide, isoprenoide, terpene, phenole und alkaloide. der anhang (teil e) liefert eine klassifizierung der ca. 130 im text genannten, verschiedenen pflanzenfamilien und enthält darüber hinaus ein umfangreiches sachregister. mit unterstützung durch ein erweitertes autorenteam wurden sämtliche inhalte der letzten auflage komplett überarbeitet und auf einen aktuellen wissensstand gebracht. die neue auflage des lehrbuchs von sticher, heilmann und zündorf ist gut lesbar; das preis-leistungsverhältnis kann als sehr günstig bezeichnet werden. anhand der zahlreichen abbildungen lässt sich das gelesene schnell aufzunehmen. das buch liefert aktuelles wissen aus erster hand und eignet sich daher insbesondere für studierende, apotheker und ärzte gleichermaßen. dr. h. schulz e-mail: hartwig.schulz@jki.bund.de bibliografie: hänsel/sticher, pharmakognosie phytopharmazie, wissenschaftliche verlagsgesellschaft mbh, stuttgart, 10. völlig neu bearbeitete auflage, 1013 seiten mit 629 abbildungen und 112 tabellen, preis: 79,90 €, isbn: 978-3-8047-3144-8 art11_gadze.indd properties of pomegranate (punica granatum l.) cultivars grown in croatia journal of applied botany and food quality 85, 202 206 (2012) 1 faculty of agriculture, university of zagreb, zagreb, croatia 2 institute for adriatic crops and karst reclamation, split, croatia 3 faculty of agriculture, ataturk university, erzurum, turkey physico-chemical characteristics of main pomegranate (punica granatum l.) cultivars grown in dalmatia region of croatia j. gadže1, s. voća1, z. čmelik1, i. mustać1, s. ercisli3, m. radunić2 (received march 13, 2012) * corresponding author summary consumers are increasingly expecting the fruits to be tasty and attractive while being safe and healthful. we determined the external and internal fruit quality properties of main pomegranate cultivars widely grown in croatia. of the three cultivars, ‘pastun’ produced the biggest fruits (460 g) followed by ‘konjski zub’ as 309 g and ‘ciparski’ as 341 g. cultivars exhibited a range of fl avor (from sour to sweet) and acidity (0.9 to 4.3% in juice). aril colour of ‘ciparski’ and ‘pastun’ were red while ‘konjski zub’ had light pink aril colour. ‘pastun’ had the highest soluble solids and acidity in both fruit juice and peel. ‘ciparski’ was in high for total anthocyanins content (12.8 mg of cyanidin-3,5-diglucoside equivalents per 100 g of fresh mass) while ‘pastun’ is high for total phenolics content (144.7 mg gallic acid equivalent per 100 g fresh mass) in juice. our results indicated that there were important quality differences among pomegranate cultivars grown in croatia. introduction pomegranate (punica granatum l.) is one of the oldest known edible fruits and more recently its fruits are gained more interest for its antioxidant and nutritional values that are important for human health (tezcan(tezcan(t et al., 2009; tehranifar et al., 2009; tehranifar et al., 2009; t et al., 2010). the edible part of the fruit contains considerable amounts of sugars, vitamins, polysaccharides, polyphenols and minerals (ercisli et al., 2007; ozgen et al., 2008). moreover recent clinical studies revealed its effective antimicrobial (mccarrell et al., 2008), antiviral, anticarcinogenic and anti-infl ammatory activities (viudaand anti-infl ammatory activities (viudaand anti-infl ammatory activities (v -martos et al., 2010). the cultivation of the pomegranate is mainly confi ned to semi-arid mild-temperate to subtropical climates in the main growing areas including most of mediterranean countries (stover and mercure, 2007) where pomegranate trees are particularly adapted to saline and poor soils (martinez et al., 2006). in croatia, pomegranates grow mainly in neretva valley located in east coast of adriatic and it has traditionally been consumed as fresh fruit and also processed as juice. in the country pomegranates are spread mainly as a minor fruit and traditional plantations use a few local cultivars. however more recently, there is high demand and public awareness to its cultivation and it is getting more commercialisation in the country. the fruits are usually sold in nearby local markets and supermarkets for fresh consumption. the quality of pomegranate fruits is strongly dependent on the cultivars, growing regions, climate, maturity and cultural practices (poyrazoglu et al., 2002; tehranifar et al., 2002; tehranifar et al., 2002; t et al., 2010). fruit maturity time of pomegranate is commonly determined based on external (peel colour, size) and internal (aril colour, sugar content and acidity) factors (ercisli et al., 2007; al said et al., 2009). appearance, especially red colour and size, effects the consumers behaviour and they are accepted as the most important external quality parameters for pomegranate (alighourchi and barzegar, 2009; celik and ercisli, 2009). fruit maturity occurs in general in september and october according to growing areas in croatia. there are no standard maturity indices or postharvest quality attributes to assist in the harvesting and management of postharvest fruit quality in pomegranates grown in croatia. previously, only a few published results on the limited physical properties of pomegranate cultivars have been reported in croatia (marinović and vegovegov , 2009; ugarković et al., 2009). no published data were found on their chemical and textural properties related to eating quality and processing in particular comparing pulp and juice. obtaining these data can help breeders and consumers to select genotypes with high level of desirable compounds along with better physical properties. scientifi c assessment on physical and chemical content of commercially grown pomegranate cultivars in different countries is also needed for proper evaluation on this unique fruit. therefore, one of the main objectives of this study was to characterize important external and internal quality attributes of main pomegranate cultivars grown in south dalmatia, neretva valley in croatia. in addition another important aim of the study was to compare the evaluated fruit characteristics of cultivars in order to determine which cultivars have the best quality features. materials and methods plant materials the fruits at commercially ripe stage from three main pomegranate cultivars such as ‘ciparski’, ‘konjski zub’ and ‘pastun’ were harvested from seven-year-old trees in the neretva valley in south dalmatia of croatia in october 2009. the trees were spaced 5 and 3 m between and within rows. all cultivars were grown under the same geographical conditions and took the same agronomic and cultural practices. after harvest, fruits were quickly transported in cold chain to the research laboratory at the institute for adriatic crops and karst reclamation in split. twelve pomegranate fruits sampled from each cultivar and four replicates were maintained for each analysis and each replicate includes three fruits. fruit peel and aril colour fruit peel and aril colour of pomegranates was measured on each fruit computed as means of three measurements taken from opposite sides at the equatorial region of the fruit by using chromometer (colortec-pcm, usa) and results were represented as l*, a* and b* value. chroma and hue angle were calculated by using l*, a* and b* values (perkins-veazie-veazie-v , 1992). physical properties fruits were weighed individually on balance of accuracy of 0.001 g. length and diameter of the fruit and calyx were measured with a digital vernier calliper. the measurement of fruit length was made on the polar axis of the fruit, i.e. between the apex and the end of the stem. the maximum width of the fruit, as measured in the direction perpendicular to the polar axis, is defi ned as the diameter. arils were manually separated from the fruits, and total aril content was weighed. replicate measurements of the peel thickness on the opproperties of pomegranate (punica granatum l.) cultivars grown in croatia 203 posite sides were made using a digital vernier calliper (tehranifar posite sides were made using a digital vernier calliper (tehranifar posite sides were made using a digital vernier calliper (t et al., 2010). fruit juice yield was determined by extracting the contents of replicate samples of 100 g of arils per fruit using a juice extractor. then the pulp and juice was analyzed for their chemical properties. dry matter, soluble solid, acidity, ph and ascorbic acid total dry matter (dm) was obtained by drying homogenised pomegranate arils at 105 °c until constant mass (aoac, 1995). soluble solid content (ssc, %) was measured using an abbe refractometer (a. krüss optronic, germany) calibrated against sucrose. acidity was measured according to the aoac method (1995) and expressed in g/l citric acid. ph value was measured with a ph meter (mettler toledo, switzerland). ascorbic acid (aa) was determined by the 2.6-dichloroindophenol titrimetric method according to the standard method (aoac, 1995). sugars sucrose, d-glucose and d-fructose contents were determined by enzymatic test kits (r-biopharm, france), measuring the formation of nadph at 340 nm, according to the described protocol of the kits (rosales et al., 2007). total phenolics and total fl avonoids total phenolics and total fl avonoids were determined using the folin-ciocalteu colorimetric method (ough and amerine, 1988). total phenolics were extracted from 10 g of fresh samples using 40 ml of 80 % (by volume) aqueous ethanol. the mixture was extracted (in water bath at 80 °c), kept for 20 min in inert atmosphere, and fi ltered through a whatman fi lter paper using a büchner funnel. extraction of the residue was repeated under the same conditions. the fi ltrates were combined and diluted to 100 ml in a volumetric fl ask with 80% aqueous ethanol, and the obtained extract was used for determination of total phenolics and total fl avonoids. the formaldehyde precipitation was used to determine total fl avonoids in fruit samples (kramling and singleton, 1969). total phenolics and total fl avonoids were expressed as mg of gallic acid equivalents (gae) per 100 g of fresh mass of pulp and juices. total anthocyanins the total anthocyanins content in the extract from fruits was determined by pellegrini et al. (1965). fruit anthocyanins were extracted from 2 g of fresh samples using 2 ml of 0.1 % hcl (by volume) in 96 % ethanol and 40 ml 2 % aqueous hcl (by volume). the mixture was centrifuged at 5500 rpm for 10 min. the obtained supernatant was used for the determination of total anthocyanins. the absorbance was measured at 520 nm. the molar absorbance value for cyanidin-3.5-diglucoside was used as a standard value. results were expressed as mg of cyanidin-3.5-diglucoside equivalents per 100 g fresh mass of pulp and juices. statistical analysis the experiment was a completely randomized design with four replications. data were subjected to analysis of variance (anova) and means were separated by lsd test at p<0.05 signifi cant level (sas, 1990). results and discussion physical properties physical properties of three pomegranate cultivars are shown in tab. 1. statistically signifi cant differences were recorded among cultivars (p < 0.05). average fruit mass of pomegranate cultivars ranged from 310 g (‘ciparski’) to 460g (‘pastun’). the fruit length and diameter values were between 73.3 mm (‘konjski zub’) and 83.1 mm (‘pastun’), and 79.1 mm (‘konjski zub’) and 95.3 mm (‘pastun’). with regard to fruit shape (l/d ratio), the ‘konjski zub’ is more rounded. calyx length and diameter values were 16.1 mm (‘konjski zub’) and 20.8 mm (‘pastun’) and 18.0 mm (‘konjski zub’) and 22.7 mm (‘pastun’), respectively. among cultivars, cv. ‘pastun’ had signifi cantly different physical properties than ‘ciparski’ and ‘konjski zub’ that has similar physical properties. there was signifi cant difference observed in aril ratio among the cultivars and ‘ciparski’ had the highest aril ratio (58.8%) while ‘pastun’ had the lowest one (53.6%). there were no signifi cant differences in fruit peel thickness among the cultivars that varied from 3.9 mm (‘pastun’) to 4.2 mm (‘konjski zub’) (tab. 1). in several previous studies, a wide variation was found on fruit mass of pomegranate cultivars that varied between 150 and 568 g (ercan et al., 1992; yilmaz et al., 1992; yilmaz et al., 1992; y et al., 1992; al-maiman and ahmad, 2002; kazankaya et al., 2003; ozkan, 2005; tehranifar, 2005; tehranifar, 2005; t et al., 2010). our fruit mass results are within these limits. some studies conducted on fruit length and width of pomegranate cultivars ranged from 61 to 91 and 36 to 104 mm (yilmazfrom 61 to 91 and 36 to 104 mm (yilmazfrom 61 to 91 and 36 to 104 mm (y et al., 1992; al-maiman and ahmad, 2002; kazankaya et al., 2003) which supports our fi ndings. calyx length and calyx diameter of pomegranate genotypes were found between 13.5 29.9 mm and 12.5 25.0 mm (sarkhosh et al., 2009; tehranifaret al., 2009; tehranifaret al., 2009; t et al., 2010) which in agreement with our fi ndings. these dimensions can be used to discriminate the cultivars among each other and also designing machine components and parameters for pomegranate processing. fruit peel and aril colour fruit peel and aril colour of pomegranate cultivars are shown in tab. 2. cultivar ‘pastun’ had signifi cantly higher red coloration (a value) on its peel while ‘ciparski’ had the least red coloration among the cultivars. cv. ‘ciparski’ had signifi cantly more light peel colour (l value) while ‘pastun’ had the least lightness. the chroma value (c), which represents colour intensity, was similar among the cultivars. fruit aril colour varied in all measured parameters. tab. 1: physical properties of three pomegranate cultivars grown in croatia cultivars ciparski konjski zub pastun fruit mass (g) 341b 309b 460a fruit length (mm) 75.8b 73.3b 83.1a fruit diameter (mm) 84.6b 79.1b 95.3a fruit length/diameter 0.9ns 0.9 0.9 calyx length (mm) 18.0b 16.1b 20.8a calyx diameter (mm) 20.9ns 18.0 22.7 calyx length/diameter 0.9ns 0.9 1.1 peel thickness (mm) 4.0ns 4.2 3.9 peel weight (g) 141b 129b 202a aril ratio (%) 58.8a 55.1ab 53.6b aril colour red light pink red taste sweet sour-sweet sour data are expressed as average value of four replicates. different letters in same row indicate signifi cant differences at the 5 % level by lsd test. 204 j. gadže, s. voća, z. čmelik, i. mustać, s. ercisli, m. radunić properties of pomegranate (punica granatum l.) cultivars grown in croatia red colour intensity (a value) of arils was signifi cantly higher in ‘ciparski’. cultivars ‘konjski zub’ and ‘pastun’ have signifi cantly less yellowness (b value) and less lightness (l value) compared to cv. ‘ciparski’. however, hue value (h) was signifi cantly higher in cultivars ‘konjski zub’ and ‘pastun’, but colour intensity (c) was lowest in cv. ‘konjski zub’. the visible aril colour of this cultivar was as light pink while ‘pastun’ and ‘ciparski’ have red arils. attractive red aril colour is one of the most important physical characteristics of pomegranate. chemical properties of pomegranate juice and pulp the results on the qualitative fruit traits obtained from juice and pulp in arils of pomegranate cultivars are given in tab. 3 and 4. as indicated on both tables, signifi cant statistical differences (p < 0.05) among cultivars on all searched parameters are observed (tab. 3, 4). the soluble solid content values in juice and pulp were the highest with 15.2 and 15.6 in cv. ‘pastun’ and followed by ‘ciparski’ as 14.8 and 15.1% and ‘konjski zub’ (13.1 and 15.0%). acidity of fruit juice was signifi cantly higher in ‘pastun’ (4.3 %) compared to cv. ‘ciparski’ (0.9 %) and ‘konjski zub’ (1.4 %). these cultivars were found in same order in pulp acidity. the ph value ranged from 2.6 (‘pastun’) to 3.4 (‘ciparski’) in juice and 2.9 (‘pastun’) to 4.0 (‘ciparski’) in pulp. sugar / acid ratio is an accepted main fl avor quality in most fruit species and it seems that there were modest variations on ssc and substantial variations on acidity of these cultivars. similar fi nding was reported by (ugarković et al., 2009) on pomegranates. according to acidity values, pomegranate cultivars are classifi ed as sweet (<1 %), sour-sweet (1-2 %) and sour (>2 %) (onur and kaska, 1985). therefore ‘ciparski’ is sweet, ‘konjski zub’is soursweet and ‘pastun’ is sour cultivar. sweet, sour-sweet and sour taste are commonly reported among pomegranate genotypes (sarkhosh et al., 2009; cam et al., 2009; tehranifar et al., 2009; tehranifar et al., 2009; t et al., 2010). high dark red aril colour, high ssc and relatively high acidity of pomegranate arils are considered to be a good choice for both fresh fruit and juice markets (ozgen et al., 2008). signifi cant variations in soluble solid contents (11-23 %), acidity (0.1-4.5 %) and ph (3.3-4.3) of pomegranates juices have been reported over the years by various researchers (ercan et al., 1992; yilmazyilmazy et al., 1992; al-maiman and ahmad, 2002; kazankaya et al., 2003; ozkan; 2005). there was signifi cant difference in ascorbic acid, total anthocyanins, total phenolics and total fl avonoids content in both pulp and juice of the cultivars at p<0.05 (tab. 3, 4). vitamin c values were between 18.8 (‘ciparski’) and 26.0 mg/100 ml (‘pastun’) in juice and 17.3 (‘konjski zub’) and 21.6 mg/100 ml (‘ciparski’) in pulp (tab. 3, 4). tehranifar(‘ciparski’) in pulp (tab. 3, 4). tehranifar(‘ciparski’) in pulp (tab. 3, 4). t et al. (2010) reported vitamin c in pomegranate genotypes between 9.9-20.9 mg per 100 g. total anthocyanins, total phenolics and total fl avonoids ranged from 2.5 (‘konjski zub’) to 12.8 mg of cyanidin-3.5-diglucoside equivalents per 100 g of fresh mass (‘ciparski’); 77.7 (‘konjski zub’) to 144.7 mg gae per 100 g fresh mass (‘pastun’) and 50.2 (‘konjski zub’) to 111.8 mg of gae per 100 g of fresh mass (‘pastun’) in juice and 1.5 (‘konjski zub’) to 6.8 mg of cyanidin3.5-diglucoside equivalents per 100 g of fresh mass (‘ciparski’); 104.6 (‘konjski zub’) to 179.1 mg of gae per 100 g fresh mass (‘pastun’) and 62.8 (‘ciparski’) to 87.8 mg gae per 100 g of fresh mass (‘pastun’) in pulp, suggesting that cv. ‘pastun’ had signifi cantly higher amount of bioactive content for human health than ‘ciparski’ and ‘konjski zub’ in considering both juice and pulp (tab. 3, 4). cam et al. (2009), tehranifar et al. (2009), tehranifar et al. (2009), t et al. (2010) and fawole tab. 2: fruit peel and aril colour of three pomegranate cultivars grown in croatia peel colour cultivars l* a* b* chroma hue ciparski 69.7a 10.5b 24.3a 27.3ns 63.6a konjski zub 59.6b 11.4b 25.7a 28.7 64.6a pastun 46.6c 17.0a 24.3a 30.3 54.8b aril colour ciparski 67.7a 12.1a 30.8a 33.6a 68.7a konjski zub 43.2b -4.6b 16.7b 17.5b 105.5b pastun 33.3c -6.2b 17.9b 20.2c 117.1b data are expressed as average value of four replicates. different letters in the same column indicate signifi cant differences at the 5 % level by lsd test. ns: non signifi cant. tab. 3: chemical properties of fruit juice of three pomegranate cultivars grown in croatia cultivars ciparski konjski zub pastun dry matter (%) 14.5ab 14.2b 14.8a soluble solid content (%) 14.8a 13.1b 15.2a acidity (%) 0.9c 1.4b 4.3a ph 3.4a 3.3a 2.6b vitamin c (mg/100 ml) 18.8c 20.1b 26.0a total anthocyanins (mg/100 ml) 12.8a 2.5c 10.3b total phenolics (mg gae/100 ml) 105.1b 77.7c 144.7a total fl avonoids (mg gae/100 ml) 55.9b 50.2c 111.8a sucrose (%) 0.3b 0.9a 0.2c glucose (%) 7.4a 6.4b 7.3a fructose (%) 8.3a 7.5c 8.1b data are expressed as average value of four replicates. different letters in the same row indicate signifi cant differences at the 5 % level by lsd test. tab. 4: chemical properties of fruit pulp of three pomegranate cultivars grown in croatia cultivars ciparski konjski zub pastun dry matter (%) 20.6a 15.7c 17.3b soluble solid content (%) 15.1b 15.0b 15.6a acidity (%) 0.4c 0.6b 1.8a ph 4.0a 3.7a 2.9b vitamin c (mg/100 ml) 21.6a 17.3c 20.2b total anthocyanins (mg/100 ml) 5.5b 1.5c 6.9a total phenolics (mg gae/100 ml) 124.7b 104.6c 179.1a total fl avonoids (mg ce/100 ml) 62.8c 70.3b 87.8a sucrose (%) 0.3b 0.2c 1.4a glucose (%) 7.6b 9.8a 5.7c fructose (%) 8.6b 11.1a 6.5c data are expressed as average value of four replicates. different letters along the row indicate signifi cant differences at the 5 % level by lsd test. properties of pomegranate (punica granatum l.) cultivars grown in croatia 205 et al. (2011) reported total anthocyanins in different pomegranate cultivars grown in iran, turkey and south africa were between 9.920.9; 8.10-36.9 and 16.5-26.9 mg per 100 g of juice, respectively. total phenolic contents of pomegranate cultivars reported from different countries between 14.4 and 1008 mg gae per 100 g fresh mass (ozgen et al., 2008; cam et al., 2009; tezcan et al., 2009; tezcan et al., 2009; t et al., 2009; tehranifartehranifart et al., 2010; fawole et al., 2011). their results were in agreement with our results. among the different compounds that could serve as unequivocal markers in a fruit juice product, anthocyanins and phenolic compounds are potentially the most useful because of their ubiquity, specifi city and multiplicity. phenolic compounds are important for their contribution to sensory attributes, as well as for their potential health benefi ts in fruits and vegetables. the effects of phenolic compounds on low-density lipoproteins and aggregation of platelets are benefi cial because they reduce some of the major risk factors for coronary heart disease (poyrazoglu et al., 2002). glucose and fructose were found to be dominant sugars an all analyzed cultivars in both juice and pulp. the glucose and fructose concentration were found between 6.4 (‘konjski zub’) to 7.4 % (‘ciparski’) and 7.5 (‘konjski zub’) to 8.3 % (‘ciparski’) in juice and 5.7 (‘pastun’) to 9.8 % (‘konjski zub’) and 6.5 (‘pastun’) to 11.1 % (‘konjski zub’) in pulp. sucrose was found in trace amounts and it ranged from 0.2 % in cv.’pastun’ to 0.9 % in cv. ‘konjski zub’ in juice and 0.2 % in cv.’konjski zub’ to 1.4 % in cv. ‘pastun’ in pulp (tab. 3, 4). ozgen et al. (2008) reported average 6.4 and 6.8 % fructose and glucose and negligible amount of sucrose in juice of six pomegranate cultivars from turkey. conclusions the quality of pomegranate fruits is strongly dependent on the cultivars. the physical properties of the pomegranate cultivars in this research demonstrated that the cultivar is important factor to determine fruit quality. cv. ‘pastun’ has bigger fruit size, attractive red skin colour, higher red aril coloration, high ssc and acidity, high bioactive content including ascorbic acid, anthocyanins, total phenolics and total fl avonoides. the other cultivars are also promising because they have diverse taste that is important for table consumption. references alighourchi, h., barzegar, m., 2009: some physicochemical characteristics and degradation kinetic of anthocyanin of reconstituted pomegranate juice during storage. j. food eng. 90, 179-185. al-maiman, s.a., ahmad, d., 2002: changes in physical and chemical properties during 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y , n., hizarci, y., 2007: interspecifi c variability of rapd and fatty acid composition of some pomegranate cultivars (punica granatum l.) growing in southern anatolia region in turkey. biochem. syst. ecol. 35, 764-769. fawole, o.a., opara, u.l., theron, k.i., 2011: chemical and phytochemical properties and antioxidant activities of three pomegranate cultivars grown in south africa. food bioprocess tech. doi 10.1007/ s11947-011-0533-7 if: 3.58. kazankaya, a., gundogdu, m., askin, m.a., muradoglu, f., 2003: fruit attributes of local pomegranates grown in pervari. proceedings of 4th national horticultural congress. 8-12 september antalya-turkey. 141-143 (in turkish). kramling, t.e., singleton, v.l., 1969: an estimate of the nonfl avonoid phenols in wines, amer. j. enol. viticult. 20, 86-92. marinović, i., vego, i., vego, i., v , d., 2009: pomological properties of pomegranate cultivars growing on the area of west hercegovina. pomol. croatica, 15, 37-54. martinez, j.j., melgarejo, p., 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tokat province in turkey. asian j. chem. 17, 939-942. pellegrini, n., serafini, m., colombi, b., del rio, d., salvatore, s., bianchi, m., ribereau-gayon, p., stonestreet, e., 1965: the amount of anthocyanins in red wines. bull. soc. chem. fr. 9, 2642-2649. perkins-veazie-veazie-v , p., 1992. physiological changes during ripening of raspberry fruit. hortscience. 27, 331-333. poyrazoglu, e., gokmen, a., artik, n., 2002: organic acids and phenolic compounds in pomegranates (punica granatum l.) grown in turkey. j. food comp. anal. 15, 567-575. rosales, m.a., rubio-wilhelmi-wilhelmi-w , m.m., castellano, r., castilla, n., ruiz, j.m., romero, l., 2007: sucrolytic activities in cherry tomato fruits in relation to temperature and solar radiation. sci. hortic. 113, 244-249. sarkhosh, a., zamani, z., fatahi, r., ranjbar, h., 2009: evaluation of genetic diversity among iranian soft-seed pomegranate accessions by fruit characteristics and rapd markers. sci. hortic. 121, 313-319. sas institute, 1990. sas/stat user’s guide, version 8 edition. vol. 2. cary, nc: sas institute. stover, e., mercure, e.w., 2007: the pomegranate: a new look at the fruit of paradise. hortscience. 42, 1088-1092. tehranifartehranifart , a., zarei, m., nemati, z., esfandiyari, b., vazifeshenas, b., vazifeshenas, b., v , m.r., 2010: investigation of physico-chemical properties and antioxidant activity of twenty iranian pomegranate (punica granatum l.) cultivars. sci. hortic. 126, 180-185. tezcantezcant , f., gultekin-ozguven, m., diken, t., ozcelik, b., erim, f.b., 2009: antioxidant activity and total phenolic, organic acid and sugar content in commercial pomegranate juices. food chem. 115, 873-877. ugarković, j., radunić, m., kozina, m., kozina, m., k , a., čmelik, z., 2009: characteristics of pomegranate (punica granatum l.) cultivars glavaš and paštrun. pomol. croatica. 15, 87-94. viudaviudav -martos, m., fernandez-lopez, j., perez-álvarez, j.a., 2010: pomegranate and its many functional components as related to human health: a review. comp. rev. food sci. food safety 9, 635-654. 206 j. gadže, s. voća, z. čmelik, i. mustać, s. ercisli, m. radunić yilmazyilmazy , h., sen, b., yildiz, b., yildiz, b., y , a., 1992: regional adaptation of pomegranates selected from mediterranean region. proceedings of 1st national horticultural congress. 13-16 october, izmir-turkey. 549-553 (in turkish). address of the corresponding author: e-mail: jelena.gadze@gmail.com journal of applied botany and food quality 87, 168 174 (2014), doi:10.5073/jabfq.2014.087.024 department of botany, university of fort hare, alice, south africa growth and physiological response of solanum nigrum l. to organic and/or inorganic fertilisers callistus bvenura, anthony j. afolayan* (received march 25, 2014) * corresponding author summary a pot experiment was conducted to determine the effect of fertilisers on the growth and physiological response of solanum nigrum. the experiment was laid out in a randomised complete block design with five treatments: control (t1); 100 kg n/ha (t2); 8.13 t manure/ ha (t3); 100 kg n/ha + 8.13 t manure/ ha (t4) and 50 kg n/ha + 4.07 t manure/ ha (t5). plant height, total number of leaves, chlorophyll, moisture, root: shoot ratio, leaf area and stem diameter were measured using standard growth indicator methods. fertilisers improved the yield, quality and growth of solanum nigrum. application of 100 kg n/ha produced the best total number of leaves; root: shoot ratio and stem diameter values; 100 kg n/ha + 8.13 t manure/ha produced the best chlorophyll values and 50 kg n/ha + 4.07 t manure/ha increased moisture, leaf area and plant height. the control showed signs of nutrient stress and inorganic fertiliser applied independently did not significantly affect the growth parameters. although parameters responded differently to each fertiliser treatment, applying a combination of organic and inorganic fertiliser may be a good option to poor resource farmers who may not be able to afford inorganic fertilisers. introduction humanity relies on a wide range of cultivated crops and yet only a few staple crops produce the majority of food supply in the world. notwithstanding this, the contribution of minor species should not be under estimated. researchers have focussed mainly on staple crops and paid little attention to minor species. one such specie is solanum nigrum l. (black nightshade). this plant is a short lived perennial shrub of eurasian origins which was introduced to south africa (edmonds and chweya, 1997), although no information is available on when it was introduced. while solanum nigrum is viewed as a notorious and important weed in many crops (ogg and rogers, 1989; defelice, 2003), the plant is an important wild vegetable in many african countries including south africa and has a high nutritional value, providing an important source of ca, fe, proteins, vit a and fibre among other nutrients (edmonds and chweya, 1997; husselman and sizane, 2006). solanum nigrum leaves are cooked either fresh or dry for food while ripe berries are consumed as a fruit or made into a bread spread jam. in south africa, the plant’s medicinal uses include treatment of ringworms, diarrhoea, dysentery, wounds, ulcers, fever and headache among other ailments and conditions (husselman and sizane, 2006). a preliminary survey in the eastern cape province of south africa indicated that although solanum nigrum is consumed as a vegetable especially during the rainy season and in the case of a drought, no efforts have been made to cultivate it. soils in the eastern cape province generally contain low amounts of the macronutrients n and p, while micronutrients such as b, cu, s, mn and zn are abundant (mandiringana et al., 2005). inadequate supply of n frequently results in slow growth, low protein levels and low, poor quality yield hence n is one of the most limiting elements to efficient and profitable crop production (mikkelsen and hartz, 2008). soils with low levels of nutrients need to be boosted with soil amendments in order to improve them for crop production. organic fertilisers or manures are a good option as they improve the soil structure and microbial mass (bin, 1983; dauda et al., 2008; suresh et al., 2004) as well as provide nutrients through mineralisation. however depending on the quality and source of the manure, minerals are often slowly mineralised and may not be available during the first season of application. inorganic fertilisers are the most preferred by farmers because they quickly become available to the plant after application, however, they may be toxic to soil, organisms and humans (arisha and bardisi, 1999). the present experiment was therefore conducted to evaluate the effects of organic and/or inorganic fertilisers on the growth and physiological response of solanum nigrum. yield (total number of leaves), quality (chlorophyll, moisture and root: shoot ratio) and growth indicators (height, stem diameter and leaf area) were measured to determine the effects of the fertilisers on solanum nigrum in comparison with the control to determine if successful cultivation of this wild vegetable requires the use of fertilisers and the quantities that produce favourable results under the current conditions. solanum nigrum is intended for domestication in efforts to ensure food security in the eastern cape. materials and methods this study was conducted from october to december 2012 at the university of fort hare alice campus glass house located at 32o 46' 47'' s and 26o 50' 53'' e. average temperatures were 15.62, 16.53, 18.41, 19.67 and 17.42 oc for september, october, november, december 2012 and the whole year respectively. mean annual humidity and evaporation were 63.71% and 3.32 mm respectively. the chemical properties of the soil and organic manure used in this experiment are shown in tab. 1. the soils (sandy loam) used in this study were collected from the university of fort hare research farm located at 32o 46' 47'' s and 26o 50' 53'' e at an altitude of 524 m a.s.l. the soils were dried and sieved through a 2 mm mesh sieve and packed in 5 kg black polythene bags prior to the experiment. the experiment was laid out in a randomised complete block design (rcbd) with five treatments. each treatment had 5 replicates but each replicate had 10 experimental units to ensure a sufficient number of plant samples for the duration of the trial. the treatments were: control (t1); 100 kg n/ha (t2); 8.13 t goat manure/ha (t3); 100 kg n/ha + 8.13 t goat manure/ha (t4) and 50 kg n/ha + 4.07 t goat manure/ha (t5). n was supplied in the form of n-p-k (2:3:4) and limestone ammonium nitrate (lan) fertiliser. furthermore, goat manure and n-p-k were applied at transplanting and lan fertiliser applied 4 weeks after transplanting. the goat manure was obtained from the university of fort hare research farm. ripe and mature solanum nigrum l. berries were harvested between the 3rd and 26th of april 2012 from the wild in alice. the seeds were separated from the pulp, washed, dried and kept in sealed glass bottles at ambient temperatures until further use. the seeds were planted in cavity trays in the greenhouse on the 18th of september 2012 and transplanted into the prepared polythene bags containing response of solanum nigrum to fertilisers 169 5 kg of soil on the 12th of october 2012. the seedlings had at least 6 true leaves and at this stage were about 10 15 cm tall. to measure the growth, quality and yield parameters, at least 9 plants per treatment were randomly selected, uprooted and tagged for data collection on the following parameters: plant height and number of leaves: a meter rule was used to measure the shortest distance between the upper boundary of the main photosynthetic tissues on the plant and the ground level (cornelissen et al., 2003). however, plant height was measured before the plants were uprooted. leaves formed were physically counted from each plant and the average determined. stem diameter: using a vernier calliper, stem diameter was measured about 2.5 cm above ground level (us epa pow, 2001). leaf area (la): leaf area was determined by the non destructive length × width method (saxena and singh, 1965) using the relation: la = 0.75(length × width), where 0.75 is a constant. chlorophyll: using a spectrophotometer (konica minolta spad -502 plus) the non-destructive approach method was used to determine total chlorophyll in fresh, healthy looking leaves. moisture: the method of osborne and voogt (1978) was used. about 2 g of plant samples were dried to a constant weight in an oven at 110 oc in clean and dry porcelain crucibles. using the final and initial weight of the samples, the percentage content was determined. root: shoot ratio: roots were separated from the whole plant and dried in the oven at 40 oc to a constant weight. the ratio was determined as the dry weight of the roots to the dry weight of the top of the plant (harris, 1992). this experiment was terminated when all the berries on the plant were mature and ripe (9 weeks after transplanting). statistical analysis data collected were subjected to statistical analysis using mnitab release 12. a one way analysis of variance was used to compare the means of various growth parameters among the treatments and a two way analysis of variance was used to determine the interaction between plant age (weeks after transplanting) and treatment on particular growth parameters. means were segregated using duncan’s multiple range test. the means were treated as significantly different at p < 0.05. results and discussion plant height and number of leaves there were significant differences (p < 0.05) among the treatment means on plant height and total number of leaves (tab. 2 and 3). plant height and leaf number increased with plant maturity. the height means for the duration of the trial were highest in t5 (54.33 cm) and lowest in the control (34.41 cm) while the total number of leaves were highest in t4 (174) and lowest in the control (101). however, the total number of leaves harvested throughout the trial decreased in the order 1416 (t2) > 1393 (t4) > 1150 (t5) > 804 (t1) > 713 (t3). analysis showed an interaction between plant age and fertiliser treatment on plant height and number of leaves. regression analysis with plant height and number of leaves as the dependable variables and time (plant age) as the regressor showed a coefficient of determination (r2) of 97 and 96 % respectively indicating that tab. 1: the chemical properties of the experimental soil (upper 0-30 cm depth) and organic fertiliser. soil organic fertiliser ph(kci) 6.54 7.17 bulk density (g cm-3) 1.20 ec (µs/cm) 162.05 10.75 cecsum (meq/ 100g) 12.10 available p (mg kg-1) 71 8 500 exchangeable k (mg kg-1) 406 26 000 exchangeable ca (mg kg-1) 1653 29 700 exchangeable mg (mg kg-1) 335 9 900 exchangeable acidity (cmol/l) 0.06 total cations (cmol/l) 12.10 saturated acid (%) 0 zn (mg kg-1) 10.2 172 mn (mg kg-1) 17 582 cu (mg kg-1) 5.7 54 organic c (mg kg-1) 10000 n (mg kg-1) 1400 24 800 clay (%) 17 na (mg kg-1) 1 564 fe (mg kg-1) 12 439 al (mg kg-1) 5 335 tab. 2: effect of organic and inorganic fertilisers on plant height (cm) of solanum nigrum l. cultivated in the greenhouse. plant age (weeks after transplanting) 0 3 4 5 6 7 8 9 t1 8.97±0.11 21.33±2.07a 35.33±2.08ab 36.33±1.37a 41.00±4.73a 43.33±2.25a 44.00±3.37a 45.00±6.26a t2 8.97±0.11 25.10±3.81b 40.67±5.03a 44.00±0.89b 70.00±3.23b 76.67±1.37b 77.67±1.86b 88.00±10.16b t3 8.97±0.11 20.33±0.52a 30.67±2.08b 44.00±3.23b 46.33±1.03a 51.33±6.28c 54.00±6.99c 67.17±0.93c t4 8.97±0.11 22.33±0.93ab 40.00±1.00a 49.00±0.89c 66.67±6.09b 75.33±2.73b 77.00±1.79b 80.33±4.13b t5 8.97±0.11 23.67±1.37ab 41.00±2.65a 49.67±3.14c 64.667±1.37b 75.33±1.03b 81.00±3.10b 90.33±0.52b 0 represents data collected at the time of transplanting values shown are mean ± sd means with different letters down the same column represent significant differences at p < 0.05. 170 c. bvenura, a.j. afolayan plant age had a significant effect on plant height and number of leaves. gohari and niyaki (2010) reported that excess n increases plant height and shading of lower leaves leading to auxin production which eventually stops the plant’s growth. millspaugh (1974) reported solanum nigrum plant height of between 30.48 and 60.96 cm in his study and this is lower than the results of this present study. edmonds and chweya (1997) recorded 70 cm as the maximum height of solanum nigrum in their study; however this is lower than the maximum height recorded in the present experiment (90.33 cm). comparing results obtained from different parts of the world, different agro-ecological conditions presumably produce different plant heights of the same plant. nitrogen generally stimulates vegetative growth (zhang et al., 2010) meaning the formation of more buds and a subsequent increase in the number of leaves. from this study, it is conceivable that as the height increased due the uptake of n in its nitrate form, an increase in vegetative growth as indicated by the leaves became inevitable. in addition, 100 kg n/ha produced the highest leaf yield throughout the trail while 50 kg n/ ha + 4.07 t manure/ha produced the highest average height throughout the trial. stem diameter treatment means were significantly different (p < 0.05) except in the 4th week. stem diameter increased exponentially in all the treatments between the time of transplanting and the 4th week after which it continued to increase steadily until the 9th week (tab. 4). the means for the trial period were highest in t2 (5.83 mm) and lowest in t3 (4.46 mm). however, statistical analysis showed an interaction between plant age and the fertiliser treatment on stem diameter. regression analysis with stem diameter as the dependable variable and time (plant age) as the regressor showed a coefficient of determination (r2) of 81 % indicating that plant age had a significant effect on stem diameter. ondieki et al. (2011) reported a smaller diameter (6.64 mm) in solanum scabrum as compared to the best treatment (7.35 mm) of the current study. the stem acts as a mechanical support structure on which sub-stems, leaves and fruits are formed. in this experiment, a positive increase in stem diameter due to the application of 100 kg n/ha (t2) conceivably led to the generation of more buds on which the leaf count improved as well as height of the plant. although the absence of water or subjecting a plant to a dry cycle drastically slows down stem diameter and height growth (klepper et al., 1972), an earlier report (klepper et al., 1971) proposed that stem diameter is not related to plant water status by a single-valued function but changes in stem diameter rather reflect changes in stem tissue hydration. the dynamics of stem diameter changes are generally explained by the cohesion-tension theory of the xylem and pressureflow model of the phloem while changes within the xylem and phloem thus create reversible and irreversible (growth) changes in the stem diameter (chan, 2012). water concentration in the pots was kept at field capacity throughout this present experiment. this present study showed that application of 100 kg n/ha fertiliser increased the stem of solanum nigrum. although diurnal diameter readings were not investigated, a positive growth was evident. moisture treatment means differed significantly (p < 0.05) and were consistently high throughout the experiment ranging between 75.16 and 92.08 % in the 7th and 3rd week respectively (tab. 5). the means for the duration of the trial were highest in t5 followed by t2 and least tab. 3: effect of organic and inorganic fertilisers on leaf number of solanum nigrum l. cultivated in the greenhouse. plant age (weeks after transplanting) 0 3 4 5 6 7 8 9 t1 6.00±1.03 82±11.04a 89±3.61 110±26.39ab 115±4.98a 126±2.25a 131±3.39a 145±19.62a t2 6.00±1.03 74±16.50ab 86±49.07 90±6.26a 190±20.83b 259±17.12b 349±3.39b 361±5.75b t3 6.00±1.03 62±8.31b 80±18.60 99±10.55a 103±18.85a 107±8.20a 124±9.96a 133±3.39a t4 6.00±1.03 66±9.40ab 86±16.57 124±7.17b 229±46.66b 255±52.23b 300±19.72c 327±24.57c t5 6.00±1.03 53±8.53b 71±12.94 99±14.72a 188±55.92b 236±16.27b 245±38.40d 253±19.78d 0 represents data collected at the time of transplanting values shown are mean ± sd means with different letters down the same column represent significant differences at p < 0.05. tab. 4: effect of organic and inorganic fertilisers on stem diameter (mm) of solanum nigrum l. cultivated in the greenhouse. plant age (weeks after transplanting) 0 3 4 5 6 7 8 9 t1 1.27±0.14 4.49±0.50a 4.76±0.12a 4.85±0.27a 4.96±0.45a 5.00±0.27a 5.20±0.22a 5.80±0.41a t2 1.27±0.14 5.59±0.34b 6.12±0.54b 6.38±0.45b 6.69±0.14b 6.86±0.51b 6.95±0.72b 7.35±0.06d t3 1.27±0.14 4.50±0.12a 4.64±0.18a 4.75±0.13a 4.82±0.22a 5.18±0.08a 5.24±0.52a 5.29±0.13c t4 1.27±0.14 5.20±0.35b 5.49±0.18c 5.53±0.26c 6.23±0.36b 6.41±0.26b 6.51±0.24b 6.89±0.31b t5 1.27±0.14 4.92±0.29ab 5.11±0.60ac 5.51±0.24c 6.67±0.39b 6.74±0.36b 6.91±0.15b 7.19±0.34b 0 represents data collected at the time of transplanting values shown are mean ± sd means with different letters down the same column represent significant differences at p < 0.05. response of solanum nigrum to fertilisers 171 in t1. statistical analysis showed an interaction between plant age and the fertiliser treatment on moisture content. regression analysis with moisture content as the dependable variable and time (plant age) as the regressor showed a coefficient of determination (r2) of 31 % indicating that plant age had a minimal effect on moisture content. the results of the current study are comparable with those of akubugwo et al. (2007) who reported moisture content of 84.70 % in solanum nigrum leaves. results reported by asibey-berko and tayie (1999) are also similar to the current study. these authors reported a range between 79.6 and 91.6 % in various wild vegetables including 80.4 % in solanum nigrum. hussain et al. (2010) reported a range between 85.74 and 94.42 % in a variety of wild vegetables including some solanum species (92.60 %) and these results are not very different from the current study. water constitutes about 80-95 % of the mass of a growing plant and thus plays a crucial role in the life of the plant. plants need water to maintain cell turgor pressure which is essential for physiological processes such as cell enlargement, gas exchange in the leaves, transport in the phloem and various transport processes across membranes (dainty, 1976). furthermore, turgor pressure also contributes to the rigidity and mechanical stability of non lignified plant tissues. the high water content of solanum nigrum leaves in this trial is not only a good indicator of the good quality and health status of the plant but also an indicator of its favourable nutritional status. reports have suggested that water containing foods provide about 25 % of the average water need in humans (sharp, 2007). solanum nigrum leaves would therefore be a good supplier of water to humans. all the treatments in this study generally indicated sufficient water in the leaves but 50 kg n/ha + 4.07 t manure/ha recorded the highest average water content for the trial. root: shoot ratio the root: shoot ratio decreased exponentially between the time of transplanting and the 3rd week and began to vary there after (tab. 6). there were significant differences (p < 0.05) in treatment means except in the 4th and 5th week and ranged between 0.08 and 0.28 in the 3rd week and time of planting respectively. the means for the duration of the trial were highest at t3 and least at t2 and t5. statistical analysis showed an interaction between plant age and the fertiliser treatment on the root : shoot ratio. regression analysis with the root to shoot ratio as the dependable variable and time (plant age) as the regressor showed a coefficient of determination (r2) of 23.3 % indicating that plant age had a minimal effect on the root to shoot ratio. in a study conducted on the field (bvenura and afolayan, 2013), the root: shoot ratio of solanum nigrum was found between 0.05 and 0.28 and this is not very different from the current glasshouse trial. in related species, nahar and gretzmacher (2011) as well as muthoni and musyimi (2009) reported that root: shoot ratio of some tomato cultivars and african nightshades respectively increased in response to water stress conditions. in general, most trees have a root: shoot ratio between 0.17 and 0.20 (harris, 1992). in the current study, the ratio conceivably increased in response to nutrient stress since soil moisture was kept at field capacity throughout the trial period. nutrient depletion in the control presumably led to a high root: shoot ratio while a good supply in the other treatments lowered the ratio. reduced nutrient supply increases the root: shoot ratio thereby compensating for loss in root foraging capacities (dixon, 2006). the relatively low root: shoot ratio values reported in the present study are a good indicator of the favourable conditions (nutrient and water supply) the plant was subjected to tab. 5: effect of organic and inorganic fertilisers on moisture content (%) of solanum nigrum l. leaves cultivated in the greenhouse. plant age (weeks after transplanting) 0 3 4 5 6 7 8 9 t1 88.52±0.34 98.25±0.05a 82.62±0.96a 78.75±0.13a 79.02±0.04a 75.16±0.07a 80.05±0.04a 80.32±0.02a t2 88.52±0.34 92.08±0.04b 86.78±0.09b 83.20±0.18b 85.34±0.09b 86.10±0.10b 86.36±0.11b 86.75±0.02b t3 88.52±0.34 90.50±0.45c 83.47±0.23a 80.32±0.03c 80.89±0.09c 83.54±0.04c 83.95±0.02c 85.55±0.04c t4 88.52±0.34 91.29±0.90d 86.32±2.70b 81.85±0.12d 85.10±0.13b 86.54±0.04b 86.72±0.04b 87.39±0.09d t5 88.52±0.34 89.55±0.09e 88.07±0.06b 85.75±0.11e 85.09±0.03b 86.55±0.09b 86.62±0.09b 86.47±0.06b 0 represents data collected at the time of transplanting values shown are mean ± sd means with different letters down the same column represent significant differences at p < 0.05. tab. 6: effect of organic and inorganic fertilisers on root: shoot ratio of solanum nigrum l. leaves cultivated in the greenhouse. plant age (weeks after transplanting) 0 3 4 5 6 7 8 9 t1 0.28±0.01 0.19±0.04a 0.12±0.01 0.17±0.03 0.22±0.02a 0.22±0.02a 0.22±0.01a 0.25±0.05a t2 0.28±0.01 0.08±0.02b 0.11±0.02 0.13±0.02 0.12±0.02b 0.15±0.02b 0.11±0.02b 0.12±0.02b t3 0.28±0.01 0.16±0.02a 0.15±0.02 0.20±0.03 0.19±0.03a 0.21±0.01a 0.19±0.02a 0.21±0.02a t4 0.28±0.01 0.12±0.02b 0.12±0.04 0.17±0.03 0.18±0.02a 0.16±0.02b 0.12±0.04b 0.14±0.02b t5 0.28±0.01 0.09±0.01b 0.10±0.05 0.16±0.03 0.14±0.02ab 0.13±0.02b 0.12±0.04b 0.12±0.02b 0 represents data collected at the time of transplanting values shown are mean ± sd means with different letters down the same column represent significant differences at p < 0.05. 172 c. bvenura, a.j. afolayan during its growth. furthermore, a reduction in the root: shoot ratio is almost always in response to more favourable growing conditions while an increase on the other hand indicates growth of a plant in less favourable conditions (harris, 1992). in the present study, the lowest ratio was recorded in 100 kg n/ha amended soil. leaf area leaf area increased until the 7th week in all the treatments and started to decrease (tab. 7). the treatment means were significantly different (p < 0.05) and ranged between 2.44 cm2 from the time of transplanting and 92.98 cm2 in t5. the means for the trial period were highest in t5 (90.6 cm2) and lowest in the control (48.8 cm2). statistical analysis showed an interaction between plant age and the fertiliser treatment on leaf area. regression analysis with leaf area as the dependable variable and time (plant age) as the regressor showed a coefficient of determination (r2) of 72 % indicating that plant age had a significant effect on leaf area. flowering commenced in the 4th week and berries were first noted in the 5th week. ripening was first observed in the 7th week and on the 9th week, all the berries on all the plants were mature and ripe. the results of the present study are within the range reported by muthoni and musyimi (2009). these authors reported la of 77.42 cm2 in solanum scabrum mill plants that were not subjected to water stress after 35 days (about 7 weeks). in the current study, after 7 weeks, the la ranged between 53.55 and 92.98 cm2. la estimation is critical in plant nutrition, plant-soil-water relations, plant protection measures, plant competition, respiration, light reflectance as well as heat transfer, hence it is an important parameter in understanding water and nutrient use, photosynthesis, light interception and more importantly crop growth and yield potential (mohsenin, 1986; smart 1974; williams, 1987). leaf morphogenesis usually includes three phases, which are an early phase of increasing photosynthetic rates and the leaf is actively expanding at this time, followed by a mature phase where such rates peak and lastly senescence where they decline (agüera et al., 2012). during the early phase of growth, the leaf acts a sink, receiving nutrients, however, when it reaches its full photosynthetic capacity, it becomes the main source organ of the plant (hörtensteiner and feller, 2002). senescence involves a shift from nutrient assimilation to nutrient remobilisation and recycling (guiboileau et al., 2010). in the present study application of 50 kg n/ha + 4.07 t manure/ha significantly increased la as compared to other treatments up to the 7th week when the fruits started ripening. during the 8th and 9th week, we assume that nutrients such as n were at this time being remobilised and recycled and this led to a decline in la. this study indicates that the plant went through all the growth phases successfully and 50 kg n/ha + 4.07 t manure/ha was the best treatment. chlorophyll treatment means were significantly different (p < 0.05) from the 4th week to the 6th week and ranged between 23.11 at the time of transplanting and 49.49 spad values in t2 (tab. 8). the means for the duration of the trial were highest at t4 followed by t2 and least at t1. statistical analysis showed an interaction between plant age and the fertiliser treatment on chlorophyll content. regression analysis with chlorophyll content as the dependable variable and time (plant age) as the regressor showed a coefficient of determination (r2) of 34.6 % indicating that plant age had a minimal effect on chlorophyll content. comparing the present results with similar studies conducted elsewhere, the current values are in agreement with those reported by prehl (2010). these authors reported between 30 and 40 spad units in young solanum nigrum plant leaves. masinde et al. (2009) reported higher values than the present study (between 40 and 70 spad units in solanum villosum). in a trial conducted on the field, bvenura and afolayan (2013) reported higher values between 23.11 and 60.34 spad units in solanum nigrum. different destructive and non-destructive methods of chlorophyll determination give different results. to measure the relative chlorophyll concentration in the leaves, the spectrophotometer used in this study (spad 502 plus) measures the absorbance of the leaf in two wavelengths (400-500 nm and 600-700 nm) (spad-502 plus manual, 2009). therefore, an increase in values generally indicates an increase in chlorophyll content. various authors agree that high chlorophyll values in plant cells are an indication of high n and therefore a good nutrient status of the plant. an experiment conducted to determine n status of the leaves in the treatments revealed a trend almost similar to the chlorophyll content. the average values of n were 3.08 % (t2), 5.69 % (t2), 3.45 % (t3), 4.93 % (t4) and 5.32 % (t5) while 100 kg n/ha (t2) produced the best n content compared to other treatments and this is comparable to the chlorophyll concentration. the control recorded the lowest values indicating in both chlorophyll and n readings. high chlorophyll values and n content recorded in this study are an indication of the good health status of the plant. the highest mean value for the trial period recorded was as a result of the application of 100 kg n/ha + 8.13t manure/ha. from this study, it can be concluded that different fertiliser dosages had different effects on the growth of solanum nigrum. these results further indicate that the application of fertilisers in solanum nigrum cultivation indeed improves the yield and quality of the plant while physical growth is also enhanced. more specifically, the application of 100 kg n/ha significantly boosted the total number of leaves, stem diameter and root: shoot ratio. on the other hand 100 kg n/ha + 8.13 t manure/ha significantly increased chlorophyll content and 50 kg n/ha + 4.07 t manure/ha significantly boosted moisture content, plant height and leaf area. however, the application of organic tab. 7: effect of organic and inorganic fertilisers on leaf area (cm2) of solanum nigrum l. leaves cultivated in the greenhouse. plant age (weeks after transplanting) 0 3 4 5 6 7 8 9 t1 2.44±0.33 30.74±7.32a 36.85±5.80a 40.79±2.92a 51.79±6.57a 53.55±5.67a 39.35±3.88a 37.43±5.66a t2 2.44±0.33 62.26±8.79b 62.73±8.36b 68.53±8.04b 70.61±3.45b 78.83±13.89b 70.52±4.88b 68.22±9.88b t3 2.44±0.33 28.51±14.45a 49.24±21.15ab 50.34±18.52ab 54.91±6.42ab 56.85±8.72a 45.29±8.01a 41.50±21.26a t4 2.44±0.33 43.88±1.33a 54.13±5.82ab 59.77±5.05b 62.30±11.49ab 71.90±8.61b 64.31±6.17b 62.68±4.17b t5 2.44±0.33 64.54±10.70b 65.22±10.93b 66.24±7.52b 73.71±14.32b 92.98±7.93b 90.02±25.33b 88.48±10.90c 0 represents data collected at the time of transplanting values shown are mean ± sd means with different letters down the same column represent significant differences at p < 0.05. response of solanum nigrum to fertilisers 173 tab. 8: effect of organic and inorganic fertilisers chlorophyll content (spad units) of solanum nigrum l. leaves cultivated in the greenhouse. plant age (weeks after transplanting) 0 3 4 5 6 7 8 9 t1 23.11±2.28 39.74±0.15 38.01±1.06ab 39.78±3.68a 38.28±3.86a 32.82±3.49a 24.02±1.80a 23.24±1.74a t2 23.11±2.28 39.35±1.55 41.93±3.15a 47.91±1.07b 46.16±1.87ab 37.22±3.11ab 44.15±3.07b 49.49±2.06b t3 23.11±2.28 39.17±2.42 35.55±0.87b 36.87±0.65a 38.44±3.14a 38.04±1.53ab 32.18±4.06c 25.49±2.22a t4 23.11±2.28 38.84±3.19 40.36±0.88a 48.44±2.57b 47.82±2.84b 40.58±5.42b 40.09±4.87bc 47.25±0.60b t5 23.11±2.28 36.54±1.52 40.58±0.55a 45.69±2.69b 40.28±1.00a 43.21±1.56b 41.98±1.22b 46.38±3.71b 0 represents data collected at the time of transplanting values shown are mean ± sd means with different letters down the same column represent significant differences at p < 0.05. manure alone did not positively affect the growth of the plant. the favourable results obtained from mixing organic and inorganic fertilisers are a good indicator to poor small scale resource farmers who may not be able to purchase large amounts of fertilisers required from local dealers. some of these farmers rear livestock in their back yards therefore making organic fertilisers readily available to them. the slow growth recorded in the control may be attributed to nutrient stress. acknowledgements this project was funded by the govan mbeki research and development centre of the university of fort hare. references agüera, e., cabello, p., de la mata, l., molina, e., de la haba, p., 2012: metabolic regulation of leaf senescence in sunflower (helianthus annuus l.) plants. in: nagata, t. 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zhang, j., blackmer, a.m., blackmer, t.m., kyveryga, m., 2010: fertiliser bands and dual effects of nitrogen on young corn plants. better crops 94, 17-19. address of the corresponding author: anthony j. afolayan, department of botany, university of fort hare, p bag x1314, alice, 5700, south africa e-mail: aafolayan@ufh.ac.za journal of applied botany and food quality 86, 11 15 (2013), doi:10.5073/jabfq.2013.086.002 dipartimento di scienze agrarie, forestali e alimentari, università degli studi di torino, grugliasco (to), italy improving the nutritional value of kiwifruit with the application of agroindustry waste extracts d. donno, g.l. beccaro, m.g. mellano, s. canterino, a.k. cerutti, g. bounous (received january 18, 2013) * corresponding author summary the purpose of this study was to evaluate the effects of the application of an agro-industrial waste extract (awe) on the quality of kiwifruit. the awe was obtained by extraction from apple seeds, rapeseed and rice husks and then boron (0.6 %) and zinc (1.4 %) were added. the effect of awe as a fertilizer/biostimulant on several parameters of kiwifruit quality (weight, total soluble solids, firmness, dry matter percentage, ph, titratable acidity, antioxidant capacity, ascorbic acid) was then evaluated. the application was carried out on two cultivars of actinidia deliciosa, cv. hayward and cv. green light, and also in two different cultivation environments. awe increased the fruit weight in cv. green light and in cv. hayward fruits grown in piedmont but no increase was observed in dry matter percentage for fruits of both cultivars. the most relevant effect of awe was the significant increase of the ascorbic acid (aa) content in the fruits of cv. hayward of all the tested orchards. fertilization with biostimulants shows a promising future in functional plant nutrition linked to an increase in food quality parameters. introduction the great interest in kiwifruit cultivation and consumption is also due to its nutraceutical value, its content of ascorbic acid (aa) in particular and also its long term storage capability, which makes this valuable fruit available and exportable throughout winter time in the northern hemisphere. pre-harvest treatments and storage period greatly influence the ripening physiology of the kiwifruit, and therefore the correct management of these has a significant effect on the final quality of the product. thus the current trend for local or export markets is to achieve an extended marketing period while maintaining high fruit quality standards (brigati and donati, 2007). in the global market, the quality parameters of kiwifruit, including size and shape, are essential for the product to compete effectively (hopping and simpson, 1982; galimberti, 2002). in the last few years, however, new parameters to define the final quality of the kiwifruit have been taken into consideration. nowadays the fruit is considered a “functional food”, due to its high antioxidant activity and significant aa content. kiwifruit is a food able to confer health benefits and to contribute to the prevention of diseases such as cancer and heart attacks (kaur and kapoor, 2001). this aspect could be important because many medical and epidemiological surveys have shown an inverse relationship between the consumption of fruits, rich in antioxidants and vitamins, and the incidence of coronary heart disease and certain cancers (rice-evans and miller, 1996; bub et al., 2003). in particular, aa is a strong antioxidant, along with vitamin e, beta-carotene, and many other plant-based nutrients, and blocks some of the damage caused by free radicals, which occur naturally when the human body transforms food into energy (ames et al., 1993; benzie, 2000). the broad distribution of aa throughout the plant tissues suggests this compound plays a role in a wide range of physiological phenomena. best studied among these functions is the aa role in redox processes during photosynthesis, environment-induced oxidative stress and during woundand pathogen-induced oxidative processes (noctor and foyer, 1998; davey et al., 2000; horemans et al., 2000; smirnoff, 2000, franceschi and tarlyn, 2002). to satisfy commercial demand and, recently, to implement nutraceutical positive traits, currently growth regulators (auxins, gibberellins and cytokinins) are applied to kiwifruit orchards (antognozzi et al., 1994; iwahori et al., 1988). in addition to growth regulators, micronutrients and amino acids are also distributed in the orchards to obtain better quality. the development of new fertilisers/biostimulants from natural raw materials has become the focus of recent interest for fruit crops. an indispensable requirement of sustainable fruit culture is the management of the cultures with renewable resources, lessening the need for chemical products (fertilizers, growth regulators), and thus lessening their environmental burden (tengerdy and szakacs, 1998). in this study the influence on quality parameters in kiwifruits of the application of an extract from agro-industrial vegetable by-products with bioactive compounds, with added boron and zinc, is described. the substrate for agro-industrial waste extract (awe) production was a mixture of organic waste by-products from apple seeds, rapeseed and rice husks, subjected to proteolytic and hydrolytic extraction processes. all these by-products present a high level of high value biomolecules with biological activity and many phenolic compounds – very important vegetable biochemicals involved in different physiological functions, such as fruit development (taiz and zeiger, 2006). awe was then added with boron (0.6 %) and zinc (1.4 %): these elements, added to the agro-industrial waste extracts, help to increase fruit size: in some situations this effect is accompanied by a decrease in the dry matter content (nardozza et al., 2007). taking into account the interaction phenomena between kiwifruit nutrition and quality, in this study an organic fertilizer with bioactive compounds was used in order to determine its influence on kiwifruit quality. one of the main bioactive compounds present in awe is the watersoluble phytohormonal content. it has been found that all the main phytohormones (auxins, gibberellins, cytokinins) can be extracted from vegetable organic waste by the hydrolytic process (parrado et al., 2007). apple seeds, used for the preparation of the waste extract in this study, are proven to contain high levels of growth hormones, such as gibberellins and cytokinins (luckwill et al., 1969); the chemical composition of rice husks derived from waste processing depends on the type of raw material and includes proteins and amino acids (5 % albumin, 1 % globulin, 1 % prolamin, 93 % gluten) and microelements (zemnukhova et al., 2004; juliano, 1985); finally, colza seeds contain a very high and varied amount of amino acids and microelements (szczurek, 2010). 12 d. donno, g.l. beccaro, m.g. mellano, s. canterino, a.k. cerutti, g. bounous materials and methods agro-industrial waste-extract application the main components of awe are nitrogen (peptides and free amino acids) and phytohormones (auxins, gibberellins, cytokinins), as functional molecules. some important chemical components of awe are shown in tab. 1 (awe composition is indicated by the product label). as these data show, protein is an important component. the awe was obtained from intrachem bio italia. ascorbic acid (aa) were evaluated after the storage. median weight (g) was evaluated by mettler pm460 deltarange electronic balance, while firmness (kg/cm2) was measured with a fruit pressure tester (zenith mod. ft 327). tss (°brix) were measured with a digital refractometer (dbr35 digitahi), while dry matter percentage (%) was evaluated by the difference between weighting the samples before and after 48 hours in a stove at 100 °c. ta (mmol/l) and ph were determined by titrating 10 ml of juice (rising to 100 ml final volume with milli-q water) with a solution of naoh (0.2 n), using an automatic titrator (crison titromatic 2s). extraction of antioxidant compounds for the extraction of antioxidant compounds, 10 g of fruit pulp (3 replications, each obtained by 5 fruits) were put into a 50 ml test tube and 25 ml of extraction solution (500 ml of methanol, 23.8 ml of bi-distilled water and 1.4 ml of hcl 37 %) were subsequently added to the weighed samples. after 60 minutes in the dark, the extracts were homogenized with an ultra – turrax (ikawerke mod. t25) for about 1 min and then centrifuged for 15 min at 3,000 rpm in an alc centrifuge mod. pk 120. the supernatants were recovered and transferred to small glass tubes and kept frozen at -20 °c for further analysis (slinkard and singleton, 1977). antioxidant capacity (frap assay) antioxidant capacity in fruit pulp was evaluated by frap (ferric reducing antioxidant power) assay (benzie and strain, 1999). the method is based on the reduction of the ferric (fe3+) tptz (2,4,6-tripyridyl-s-triazine) complex to its ferrous form (fe2+). the frap reagent was prepared daily by mixing 2.5 ml of tptz solution and 2.5 ml of fecl3·6h2o solution with 25 ml acetate buffer (0.3 m) and then warmed at 37 °c before using; 30 ml of sample (15 ml of extract sample and 15 ml of extraction buffer, dilution 1:2) was added to 90 μl of bi-distilled water and 900 μl of frap reagent in a 2 ml micro tube and then incubated at 37 °c for 30 minutes in a g.f.l. shaking water bath mod. 1083. absorbency at 595 nm with a uv/vis spectrophotometer (beckman mod. du® 530) was measured. standard curve was obtained using feso4·7h2o (concentration range: 100 – 1000 μmol/l) and results were expressed as mmol of fe2+ equivalents/kg of fresh fruit weight. ascorbic acid content 10 g of fruit pulp (3 replications of 5 fruits each) were put into a 50 ml test tube and 10 ml of extraction solution (0.1 m citric acid, 2 mm ethylene diamine tetraacetic acid (edta) disodium salt and 4 mm sodium fluoride in methanol – water 5:95 v/v) were then added. the extracts were homogenized with an ultra – turrax (ikawerke t25) for about 1 min and then centrifuged for 10 min at 4,000 rpm at room temperature in an alc centrifuge pk 120. the supernatants were recovered and transferred to a 15 ml test tube through filter cloth and then acidified with 4 n hcl to decrease ph solution to a value of 2.2 – 2.4 ph units (sanchez et al., 2003). acidified samples were centrifuged for 5 min at 12,000 rpm at 4 °c with an alc multi speed refrigerated centrifuge pk 121r and the supernatants were then filtered through a 0.45 μm filter (titan 2 hplc filter 17 mm ptfe membrane); polyphenolic compounds were absorbed on a c18 cartridge for solid phase extraction (waters corporation, sep-pak® c-18). then, 250 μl of opda solution was added to 750 μl of extract sample for dhaa derivatization into the fluorophore 3-(1,2-dihydroxyethyl)furo[3,4-b]quinoxalina-1one. after 37 min in the dark, the samples were analyzed by hplc – dad system (gonzáles-molina et al., 2008). tab. 1: protein, amino acids and growth regulators content of awe: means of three samples. component concentration protein (g kg−1) 980 free amino acids (g kg−1) 140 indolacetic acid (iaa) (mg kg−1) 152.68 naphtoxy acetic acid (naa) (mg kg−1) w/w) 12.51 indolbutyric acid (iba) (mg kg−1) 15.1 gibberellic acid (mg kg−1) 256.3 the application was carried out on two cultivars of actinidia deliciosa, cv. hayward and cv. green light, grown under the same agronomical and environmental conditions, in order to better understand the interaction between the treatment and the different genotypes. the trials were located in givoletto, piedmont region, north west italy. moreover, the same trials were repeated for the cultivar hayward in another cultivation environment at cisterna di latina (latium region, central italy), under the same agronomical conditions, in order to study the effect of the different pedoclimatic conditions on the results of the application. awe was applied in the ratio of 1 l ha−1, diluted in a water solution, twice, once two weeks before flowering and again one week after the fruit set. the produce was spread on the orchard by using a spray system for foliar application (chapin et al., 1993; kielland, 1994; miller and hsu, 1986). the production of plots treated with awe was compared with untreated plots. for each thesis, 25 plants in 5 sub-plots were randomly selected and 125 fruits were harvested from each plant. the fruits were harvested at conventional ripeness. sampling was carried out randomly and the fruits were immediately stored for 3 months in normal atmosphere at 1 °c and relative humidity 95 %, simulating the usual storage conditions for these fruits. any mould or alteration of the fruits was observed. reagents and standards potassium dihydrogen phosphate, sodium acetate, citric acid, hydrochloric acid, 2,4,6-tripyridyl-s-triazine (tptz) and 1,2-phenylenediamine dihydrochloride (opda) were purchased from sigma aldrich, usa, while acetic acid was purchased from fluka biochemika, switzerland. ethylene diamine tetraacetic acid (edta) disodium salt was purchased from amresco, usa, while sodium fluoride was purchased from riedel-de haen, germany. milli – q water was produced by using sartorius stedium biotech mod. arium. analytic solvent hplc grade (methanol) was purchased from extrasynthése (genay, france), while dehydroascorbic acid (dhaa), ascorbic acid (aa) and cetyltrimethylammonium bromide (cetrimide) were purchased from extrasynthése (genay, france). quality parameters median weight/fruit, total soluble solids (tss), firmness, dry matter percentage, ph, titratable acidity (ta), antioxidant capacity and agroindustry waste extracts for kiwifruit quality 13 13 an agilent 1100 high performance liquid chromatograph, equipped with a g1311a quaternary pump, a manual injection valve and a 20 ml sample loop, coupled to an agilent gi315d uv-vis diode array detector, was used for the analysis. separations of dfq and aa were achieved on a zorbax eclipse xdb – c18 column (4.6 x 150 mm, 5 mm). the mobile phase was methanol – water (5:95, v/v) containing 5 mm cetrimide and 50 mm potassium dihydrogen phosphate. the flow rate was 0.9 ml min-1 (isocratic analysis) and the detector wavelengths were 348 nm for dhaa (dfq) and 261 nm for aa detection. aa and dhaa were identified and quantified by comparison with pattern areas from aa and dhaa; the vitamin c content was calculated by adding ascorbic acid and dehydroascorbic acid values and results were expressed as mg/100 g of fresh fruit weight (gonzálesmolina et al., 2008). statistical analysis the results were subjected to analyses of variance (anova) for means comparison by using spss 15.0 software and hsd tukey multiple range tests at p<0.05. results and discussion the storage of the fruits was successful. the results of the qualitative analysis are shown in tab. 2. awe significantly increased fruit weight in cv. hayward in both locations: the median weight of the cv. hayward fruits grown in piedmont and in latium increased from 96.43 g to 101.52 g and from 96.82 g to 102.97 g, respectively. however the procedure did not increase the weight of the cv. green light fruits, suggesting a different genotype response to the treatment. moreover, the different effect of this product on cv. green light, in comparison with cv. hayward, is probably due to the fact that at the time of the treatment cv. hayward was at a more advanced phenological phase (“fruit walnut”), considering the ideal time to use it. the treatment did not significantly reduce the firmness of the fruits during post-harvest. the awe treatment did not influence dry matter percentage, tss, ta and ph of the two cultivars. the results of the nutritional parameters are shown in tab. 3. the antioxidant activity of the fruits of the two cultivars was significantly affected by the application of the product only in the case of cv. hayward, although the cv. green light fruits treated with awe had slightly higher values than the control. in piedmont the average antioxidant capacity of the fruits increased from 13.34 mmol of fe2+ equ/kgffw to 17.25 mmol of fe2+ equ/kgffw and in latium it increased from 12.33 mmol of fe2+ equ/kgffw to 16.83 mmol of fe2+ equ/kgffw. the main effect of awe was the increase in nutraceutical traits, especially the aa content. the application of phytohormones, micronutrients and amino acids from vegetal waste as nutrients is an important topic of research nowadays, and many studies show that these substances have a strong influence on many of the physiological activities of cultivated plants and plant cell cultures (taiz and zeiger, 2006; hancock et al., 2008). although the mechanisms controlling aa production and concentration are still largely mysterious, with large areas of uncertainty concerning the genetic and biochemical controls of pathway flux (hancock and viola, 2005), some studies indicate that the phloem is capable of synthesising aa from sugars (hancock et al., 2003). the treatment significantly increased the aa content in both cultivars and in both locations. for cv. hayward, in piedmont the median aa content of the fruits increased from 32.01mg/100 gffw to 41.73 mg/100 gffw and in latium it increased from 38.10 mg/ 100 gffw to 45.52 mg/100 gffw. for cv. green light the aa content of the fruits increased from 45.05 mg/100 gffw to 51.22 mg/100 gffw. fig. 1 shows an example of a chromatogram obtained in this analysis. analytic peaks were easy to detect at 348 nm (dhaa) and 261 nm (aa), because there was a good peak resolution with two different retention times: 4 minutes for dhaa and 6 minutes for aa. although further physiological and biochemical studies should be tab. 2: quality parameters in cv. green light and cv. hayward: mean values in each row for the same parameter followed by different letters are significantly different as determined by tukey’s hsd test following anova ( p ≤ 0.05). cultivar treatment fruit weight firmness dry matter content total soluble titratable acidity ph (g) (kg/cm2) (%) solids (°brix) (mol/l) (ph units) green light (latium) awe 80.05 ± 5.3 bc 2.85 ± 0.2 a 20.73 ± 0.6 a 11.56 ± 0.4 b 0.08 ± 0.1 a 3.30 ± 0.1 a control 78.51 ± 3.3 c 3.24 ± 0.2 a 19.95 ± 0.6 a 13.50 ± 0.3 ab 0.07 ± 0.1 a 3.41 ± 0.1 a hayward (latium) awe 102.97 ± 1.1 a 2.54 ± 0.1 a 21.58 ± 0.4 a 13.10 ± 0.7 ab 0.11 ± 0.1 a 3.07 ± 0.1 a control 96.82 ± 2.1 b 2.86 ± 0.1 a 21.55 ± 0.3 a 14.77 ± 0.1 a 0.12 ± 0.1 a 3.06 ± 0.1 a hayward (piedmont) awe 101.52 ± 1.1 a 2.51 ± 0.1 a 20.55 ± 0.1 a 14.17 ± 0.4 a 0.11 ± 0.1 a 2.90 ± 0.1 b control 96.43 ± 3.0 b 2.65 ± 0.3 a 19.21 ± 0.2 a 14.00 ± 0.4 a 0.12 ± 0.1 a 2.97 ± 0.1 ab tab. 3: nutraceutical parameters in cv. green light and cv. hayward: mean values in each row for the same parameter followed by different letters are significantly different as determined by tukey’s hsd test following anova ( p ≤ 0.05). cultivar treatments vitamin c antioxidant capacity (mg/100 gffw) (mmol of fe2+ equ/kgffw) green light awe 51.22 ± 3.9 a 11.34 ± 1.1 c control 45.05 ± 2.4 b 11.17 ± 0.1 c hayward (latium) awe 41.73 ± 5.4 a 17.25 ± 1.3 a control 32.01 ± 3.9 c 13.24 ± 2.1 b hayward (piedmont) awe 45.52 ± 1.9 a 16.83 ± 0.9 a control 38.10 ± 2.9 b 12.33 ± 1.1 bc 14 d. donno, g.l. beccaro, m.g. mellano, s. canterino, a.k. cerutti, g. bounous performed to establish the relationships between agro-industrial waste extract application and its influence on plant physiology and fruit quality, the awe treatment in this study, in different pedoclimatic conditions and with different genotypes, was proven to have an influence on aa content and the antioxidant activity of kiwifruit production. the raw material used for the extraction of awe has heterogeneous characteristics and a highly variable chemical composition (parrado et al., 2005), and is difficult to standardize. also the operational conditions during the formulation of the awe, such as temperature, moisture, active biomolecular composition and concentration (as enzyme/substrate ratio) and extraction time, may influence the effectiveness of awe and should be further analysed. the study of the extraction technology implementation and further developments on functional properties in agro industry by-products are promising research topics. conclusions the use of awe as fertilizer/biostimulant had positive effects on fruit weight, aa content and antioxidant capacity measured by frap. this study confirms that fertilization with vegetal waste extracts shows a promising future in functional plant nutrition linked with an increase in food quality parameters, and further development of the technologies and study of the agronomical, physiological and biochemical relationships should be carried out to develop more sustainable strategies for future agriculture. this is only a preliminary study, because other years 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r., costa, g., biasi, r., 1990: impollinazione e qualita dei frutti nell’actinidia. rivista di frutticoltura 10, 2-35. zemnukhova, l.a., tomshich, s.v., mamontova, v.a., komandrova, n.a., fedorishcheva, g.a., sergienko, v.i., 2004: composition and properties of polysaccharides from rice husk. russ. j. appl. chem. 77, 1883-1887. address of the corresponding author: d. donno, dipartimento di scienze agrarie, forestali e alimentari, università degli studi di torino, via leonardo davinci, 44 10095 grugliasco (to), italy. e-mail: dario.donno@unito.it journal of applied botany and food quality 87, 274 278 (2014), doi:10.5073/jabfq.2014.087.038 1department of food engineering, faculty of engineering and architecture, haci bektas veli university, nevsehir-turkey 2ataturk university, agricultural faculty, department of horticulture, erzurum-turkey 3corvinus university, department of genetics and plant breeding, budapest-hungary 4recep tayyip university, faculty of agriculture and natural science, pazar-rize, turkey 5department of food engineering, faculty of agriculture, ataturk university, erzurum-turkey 6 dzemal bijedic university, agromediterranean faculty, mostar, bosnia and herzegovina bioactive content and antioxidant characteristics of wild (fragaria vesca l.) and cultivated strawberry (fragaria × ananassa duch.) fruits from turkey h. yildiz1, s. ercisli2*, a. hegedus3, m. akbulut4, e.f. topdas5, j. aliman6 (received august 3, 2014) * corresponding author summary in this study, some biochemical properties (total soluble solid content, ph, acidity, antioxidant activity) and contents of biological compounds (vitamin c, total phenolics, total ellagic acid and concentration of anthocyanins) of fifteen wild strawberry accessions (fragaria vesca l.) and one commercial strawberry cultivar camarosa (fragaria × ananassa duch.) sampled in northeastern turkey were determined. antioxidant activity of fruit samples was determined by dpph (2,2-diphenyl-1-picrylhydrazyl) and frap (ferric reducing antioxidant power) assays. notable differences were found both among wild strawberries (fragaria vesca) and also between wild strawberries and cv. camarosa (fragaria x ananassa). among the strawberry accessions tested, the total phenolics ranged from 138 mg to 228 mg gallic acid equivalent per 100 g fresh fruit. the total monomeric anthocyanin content was the highest in wild accession fv-2 (53.51 mg/100 g) while the lowest in fv-6 as 25.11 mg per 100 g fresh weight. the total ellagic acid content was between 15.18 and 26.36 mg per 100 g. all wild strawberries exhibited higher antioxidant activity than cv. camarosa. thus, it can be concluded that wild strawberry is a good source of polyphenols, ellagic acid and antioxidants. introduction the garden strawberry (fragaria × ananassa duch.) is a cultivated hybrid species of the genus fragaria (collectively known as the strawberries) and it is appealing to the human senses of sight and taste due to its bright red color, juicy texture, sweetness and distinct flavor. the wild aromatic diploid species f. vesca l. (also known as woodland strawberry) is genetically not related to the octoploid f. x ananassa because f. vesca l. is not an ancestor of f. x ananassa (chandler et al., 2012). f. x ananassa is cultivated both open field and in greenhouses throughout the world. although commercial strawberry (f. × ananassa duch.) cultivation only started around the end of 1970 in turkey, the country is currently one of the biggest strawberry producers in the world after usa with an annual production quantity of 353,000 tons (fao, 2012). in turkey, there are nine different agro-climatic regions and strawberries are grown in each of those with mediterranean and subtropical climate regions being the most important producers. the diversity in climate across the country, together with the cultivation of short-day and day-neutral cultivars, and implementation of various planting practices, allow consumers to enjoy this fruit almost all year round (ozdemir et al., 2013; torun et al., 2014). fragaria vesca l., the wild strawberry, is the most extensively distributed species in the 200-2450 m a.s.l., particularly in the highlands of the black sea region of turkey and the species is characterized by considerable geographical and ecological adaptation capacity. the highly aromatic and sweet fruits were used predominantly for fresh consumption or preservation (ercisli, 2004). since this fruit is not cultivated, wild strawberry is a valuable product in turkey (ozsen and erge, 2013). fruits of wild strawberries are generally ovate, bright red, soft and aromatic. since the middle ages, wildtype strawberries were praised for their intense flavor (darrow, 2005). the aroma of wild strawberry is perceived as very aromatic and more herbaceous than those of f.×ananassa cultivars (hirvi and honkanen, 1982). therefore, f. vesca serves as a comparison with regard to flavor. zabetakis and holden (1997) hypothesis that the aroma of f. vesca may be more ‘strawberry-like’ than those of f. x ananassa. ulrich et al. (2007) and ulrich and olbricht (2013) indicated diversity of aroma patterns in wild and cultivated fragaria accessions in recent years some introduced short-day and day-neutral cultivars such as ‘fern’, ‘camarosa’, ‘festival’, ‘fortuna’, ‘rubygem’ have been widely produced in turkey, ‘camarosa’ being most common due to its high market segment (esitken et al., 2010; ozdemir et al., 2013). wild strawberries with unique aroma can suffer from poor climatic adaptation and hence they might have inadequate quality and productivity (ercisli, 2004). more recently, there is an increasing interest in colorful berry fruits including strawberry, raspberry, blackberry, mulberry, blueberry, elderberry etc. these fruits are popularly consumed not only in fresh and frozen forms but also as processed and derived products including dried and canned fruits, yogurts, beverages, jams, and jellies (seeram et al., 2006). berries provide significant health benefits because of their high levels of polyphenols, antioxidants, vitamins, minerals, and fibers (halvorsen et al., 2002; anttonen and karjalainen, 2005). it has been demonstrated that a wide diversity of phytochemical levels and antioxidant capacities exist within and across berries (moyer et al., 2002; hegedus et al., 2008). furthermore, accumulating evidence suggests that genotype has a profound influence on concentrations of bioactive compounds in berries (halvorsen et al., 2002; anttonen and karjalainen, 2005). some berries, such as strawberries, have been identified as sources of phenolic compounds like gallic and ellagic acids, which have potential cancer chemo preventive activity (xue et al., 2001). these different bioactive phenolic compounds, including flavonoids, tannins, and phenolic acids, have received considerable interest in bearing possible relations to human health. the aims of this work were to evaluate and compare some biochemical and bioactive contents in wild and cultivated strawberry fruits as well as to assess the genotypic influence on investigated characteristics in strawberry fruits. comparison of wild and cultivated strawberries in terms of bioactive content 275 material and methods plant material the research was conducted in 2012 on harvested fruits from fifteen wild strawberry (fragaria vesca) accessions and ‘camarosa’ cultivar (fragaria x ananassa) grown in the senkaya district in northeastern turkey at 40°33’43’n 42°20’47’e and 1800 m altitude. the mountainous growing area was covered by scotch pine (pinus sylvestris) forests. the climate of the sampling region has typical black sea climate (warm and humid) with an average annual humidity of 67 % and precipitation of 47 kg/m3. near the sampling region for wild strawberries, there were some commercial non-modern strawberry plantations of the ‘camarosa’ cultivar. thus there were similarities between wild and cultivated strawberries in terms of growing conditions in the study area. neither chemical treatment nor extra fertilizer was applied during experiment. irrigation was only applied to avoid damage by drought. because of high altitude, no fungicide against grey mold (botrytis cinerea pers.) was used. wild fruits were harvested per plant (per accession) and cultivated strawberry fruits were harvested from 30 plants at fully mature stage. sample preparation and determination of chemical contents about 100 g of fruit samples for each accession were frozen at -20 °c. at the time of analysis, fruits were thawed and homogenized in a standard food blender. slurries were used to determine chemical (tss, titratable acidity and ph) and bioactive content (vitamin c, total phenolic, antioxidant activity, ellagic acid, total anthocyanins). total soluble solid (tss) contents was determined by refractometer (model ra-250he, kyoto electronics manufacturing co. ltd., japan). levels of titratable acidity (ta), ph and vitamin c were determined using standard methodology (aoac, 2005). total phenolic content for the extraction and determination of total phenolic content, a single extraction procedure was designed to assay phenols (bartolome et al., 1995). for each replicate, a 3 g aliquot of slurry was transferred to polypropylene tubes and extracted with 20 ml of extraction buffer containing acetone, water, and acetic acid (70:29.5:0.5, v/v) for 2 h. after filtration, acetone was removed by rotary evaporation, after which the concentrated samples were brought to a final volume of 20 ml with deionised water. next, folin-ciocalteu’s phenol reagent and water were incubated for 8 min, followed by the addition of 7 % sodium carbonate. after 2 h, the absorbance was measured by an automated uv-vis spectro-photometer at 750 nm. gallic acid was used as standard. the results were expressed as mg gallic acid equivalents on 100 g fresh weight basis (mg gae/100 g fw). determination of antioxidant activity total antioxidant activity was estimated by two standard procedures: frap and dpph assays. the frap assay (benzie and strain, 1996) was conducted using three aqueous stock solutions containing 0.1 mol/l acetate buffer (ph 3.6), 10 mmol/l tptz [2,4,6-tris(2pyridyl)-1,3,5-triazine] acidified with concentrated hydrochloric acid, and 20 mmol/l ferric chloride. these solutions were prepared and stored in the dark under refrigeration. stock solutions were combined (10:1:1, v/v/v) to form the frap reagent just prior to analysis. for each assay laboratory duplicate, 2.97 ml of frap reagent and 30 μl of sample extract were mixed. after 10 min, the absorbance of the reaction mixture was determined at 593 nm using a spectrophotometer. the results were expressed as μmol trolox equivalent on g fresh weight basis. free radical scavenging activity was determined according to the method of suja et al. (2005) with slight modifications. fruit extract (1 ml) was added to 2 ml dpph solution (2 ml of 0.02 g/l dpph) in ethanol. the reduction of dpph was measured at 517 nm against a blank assay for 30 min. the percentage of the remaining radical in medium is calculated as the absorbance of the sample divided by that of dpph control at the same time multiplied by 100. the amount of sample needed to decrease the initial dpph concentration by 50 %, ec50, was calculated graphically. the results were expressed as μmol on g fresh weight basis. determination of total anthocyanins total monomeric anthocyanins (tma) were determined by the ph differential method (giusti and wrolstad, 2001), using a uv-vis spectrophotometer. absorbance was measured at 533 nm and 700 nm in buffers at ph 1.0 and 4.5 using a = (a533 a700) ph 1.0 (a533 a700) ph 4.5 with a molar extinction coefficient of 29,600. total anthocyanin content was expressed as mg pelargonidin-3-glucoside equivalent in 100 g fw. determination of total ellagic acid extraction and hydrolysis of ellagitannins methods were used (pineli et al., 2011). frozen strawberries (75 g) were lyophilized for 4 days in darkness. the moisture of fresh strawberries and the residual moisture of lyophilized strawberries were determined by gravimetry, after drying at 105 °c until constant weight. the results were used to convert data from dry basis to fresh basis. samples of lyophilized strawberry powder (0.5 g, for each replicate) were extracted three times in 80 % methanol (50 ml the first time, 25 ml the next two times) at 15,000 rpm for 1 min while cooled in ice bath, and vacuum filtered through whatman 1 filter paper. an aliquot of 2 ml of the combined extracts was dried under nitrogen stream and 2 ml of 2n trifluoroacetic acid (tfa) were added. hydrolysis was performed in a glycerin bath at 120 °c for 60 min. hydrolized extracts were rota-evaporated at 95 °c for 2 min, re-suspended at 1 ml of hplc grade methanol, filtered in 0.22 μm ptfe filters, and analyzed by hplc. identification and quantification of total ellagic acid was achieved using analytical reversed-phase hplc in a varian pro star system with auto sampler and ternary pump and uv-vis detector. some extracts were also analyzed in a hplc coupled with dad detector (shimadzu, spd-m10a dad, germany), in order to confirm the structures previously identified by time retention with the uv-vis detector. the column used was 250 mm x 4.6 mm, i.d., 5 μm, prodigy ods3 reversed-phase silica and elution solvents were a, water: tetrahydrofuran: tfa (98:2:0.1) and b, acetonitrile. solvent gradient was: 17 % b for 2 min, increasing to 25 % b after 5 min, to 35 % b after a further 8 min and to 50 % b after 5 min. samples were injected in duplicate. calibration was performed by injecting the external standard ellagic acid (sigma chemical co., usa) three times at five different concentrations, in the range of 0.110 μg. results were expressed as mg/100 g fw basis. statistical analysis all data were analyzed using spss software and procedures. analysis of variance tables were constructed using the least significant difference (lsd) method at p<0.01. results and discussion biochemical characteristics results related to some biochemical characteristics (tss, acidity, ph) and vitamin c of fifteen wild strawberries and cv. camarosa are given in tab. 1. remarkable significant differences among wild strawberry accessions and between the group of wild strawberries 276 h. yildiz, s. ercisli, a. hegedus, m. akbulut, e.f. topdas, j. aliman and cv. camarosa were found for most of the biochemical characteristics (tab. 1). tss and vitamin c values in the fruit of strawberry accessions were found to be different from each other at p<0.01, while ph and acidity of accessions were found not to be different from each other at p<0.01 (tab. 1). tss and vitamin c of the wild and cultivated accessions are shown in tab. 1. tss and vitamin c contents varied from 6.61 % (fv-2) to 7.51 % (fv-10) and 38.55 mg/100 g (fv-11) to 57.37 mg/100 g (fv-8) among accessions, respectively. the ph and acidity varied from 3.06 (‘fv-12’) to 3.31 (‘fv-1’) and 0.95 % (‘fv-11’) to 1.47 % (‘fv-1’) among accessions, respectively (tab. 1). our results for the strawberry fruit samples were within the previously published ranges of fruit tss, acidity, vitamin c and ph (voca et al., 2008; pineli et al., 2011; ozdemir et al., 2013; silva et al., 2013), which varied from 6.01-11.13 % for tss, 3.01-3.81 for ph, 31-85 mg per 100 g for vitamin c and 0.7-1.3 % for acidity, respectively. cordenunsi et al. (2005) reported values of vitamin c ranging from 47 to 80 mg per 100 g of fresh weight, depending on the strawberry cultivars. similar values were reported for vitamin c with 52.25 to 53.21 mg (hansawasdi et al., 2006), and with 38.4 to 72.1 mg per 100 g of fresh mass, depending on the cultivar (laugale and bite, 2006). in modern strawberry breeding, new cultivars carry high or medium sugar but low acid contents and lead to high sugar-acid ratios. the f. vesca accessions in this study are characterized by very diverse brix-acid ratios. additionally, high acid contents involve also astringency especially in combination with low sugar values. vitamin c, including ascorbic acid and dehydroascorbic acid, is one of the most important nutritional quality factors in many horticultural crops and has many biological activities in the human body. the content of vitamin c in fruits can be influenced by various factors such as genotypic differences and cultural practices etc. (lee and kader, 2000). in our study, the wild and cultivated strawberry plants found were cultivated under the same growing conditions and thus the genotypic effect seems more dominant. according to voca et al. (2008), the quantity of total soluble solids in the investigated strawberry cultivars ranged from 6.00 to 10.01 %, while laugale and bite (2006) reported values for total soluble solids from 8.4 to 11.6 %, depending on the strawberry cultivar. the soluble solid levels are used as indicator of maturity and also to determine fruit quality of strawberry. bioactive content in modern strawberry breeding, the direction recently changed and today’s strawberry cultivars are characterized by rather different patterns of secondary metabolites. bioactive compounds in strawberries and other horticultural crops can be defined as secondary plant metabolites (milivojevic et al., 2011) bioactive contents of the analyzed strawberry accessions are presented in tab. 2. we found significant differences in the level of total phenolic, total monomeric anthocyanin, total ellagic acid and antioxidant activity (characterized by using two different methods) among the assayed genotypes (p<0.01) (tab. 2). the total phenolic content of strawberry accessions ranged from 138 mg (cv. camarosa) to 228 mg gae per 100 g (fv-2) (tab. 2). results showed a genotype-dependent total phenolic accumulation in strawberry fruits and also indicated that all wild accessions had higher total phenolic contents than cv. camarosa (tab. 2). the total phenolic content of strawberry fruits grown in different countries were reported to be between 96 and 223 mg gae per 100 g fruits (voca et al., 2008; pineli et al., 2011), which is comparable to our findings. torronen and maatta (2002) determined values of total phenols in strawberry cultivars ranging from 96 mg/100 g to 133 mg/100 g of fresh weight. all these results indicate that besides other berries and small fruits (jurikova et al., 20011; jurikova et al., 2012a; jurikova et al., 2012b), strawberry fruits might also be a good source of total phenolics. the various factors such as genotype, agronomic practices, maturity level at harvest, postharvest storage, climatic and geographical locations affect the total phenolic content of horticultural plants (milivojevic et al., 2011; milivojevic et al., 2012). as indicated before, in this study genotypic effect was more dominant. the observed total monomeric anthocyanin contents greatly differed among the strawberry accessions and varied from 25.11 mg (fv-6) to 53.51 mg (fv-2) pelargonidin-3-glycoside equivalent in 100 g fw (tab. 2). this indicates that within polyphenolics strawberry fruit is also a good source for anthocyanin. a wide variation (12-44 mg per 100 g fw) in anthocyanin content was reported in strawberry cultivars previously (castro et al., 2002; voca et al., 2008; oszmianski and wojdylo, 2009; pineli et al., 2011). meyers et al. (2003) reported an average anthocyanin content of 41.4 mg/100 g in strawberry cultivars in the usa. they found great differences among the analyzed cultivars. total ellagic acid contents also varied among the accessions. when cultivars were compared, the highest total ellagic acid content was observed for fv-4 berries as 26.36 mg per 100 g and lowest in fv-7 berries as 15.18 mg per 100 g (tab. 2). pinto et al. (2008) and pineli et al. (2011) reported a significant variation among full ripe strawberry cultivars for total ellagic acid content. the values determined for ripe strawberry cultivars were between 16.6 and 19.4 mg/100 g. ellagic acid is considered the most important phenolic compound in strawberries (hakkinen et al., 1999). there is a particular interest in ellagic acid due to its potential chemoprotective, anti-inflammatory and antibacterial effects properties (vattem and shetty, 2005). the total antioxidant activities of the sixteen strawberry fruit extracts determined by dpph and frap methods are shown in tab. 2. the antioxidant capacity differed greatly among strawberry accessions for both antioxidant-determining assays (tab. 2). tab. 1: selected biochemical characteristics of fruits of wild and cultivated strawberries. accessions ssc ph acidity vitamin c (%) (%) (mg/100 g fresh fruit) fv-1 6.91ab 3.31ns 1.47ns 51.10c fv-2 6.60b 3.27 1.11 54.07b fv-3 6.67ab 3.15 1.18 43.69ef fv-4 6.78ab 3.08 1.24 50.67cd fv-5 6.86ab 3.10 1.37 49.11cd fv-6 6.70ab 3.09 0.95 45.18de fv-7 7.13ab 3.30 1.11 53.74bc fv-8 6.84ab 3.18 1.06 57.37a fv-9 7.32ab 3.21 1.01 51.06c fv-10 7.51a 3.24 1.03 45.51de fv-11 6.67ab 3.13 1.24 38.55f fv-12 7.10ab 3.06 1.20 47.44d fv-13 6.96ab 3.11 1.21 44.18e fv-14 7.34ab 3.20 1.18 41.71ef fv-15 7.42ab 3.20 1.31 44.16e ‘camarosa’ 6.90ab 3.16 1.17 38.66f means within a column followed by the same letter are not significantly different at p<0.01 ns: non significant comparison of wild and cultivated strawberries in terms of bioactive content 277 in frap assay, we found significant differences among the accessions (p<0.01). the genotype fv-2 showed the highest total antioxidant capacity (49.11 μmol per g) in frap assay. this genotype was followed by fv-8, fv-4, fv-12 and fv-13 genotypes, which had 47.62, 47.18, 46.10 and 45.01 μmol per g frap. overall, the lowest antioxidant activity was observed in ‘camarosa’ as 27.10 μmol per g (tab. 2). these results are similar to the data reported by pineli et al. (2011) who determined 24-27 μmol per g frap values. halvorsen (2002) reported frap values in wild fragaria vesca fruits between 66 and 70 μmol per g and in fragaria × ananassa fruits between 19-24 μmol per g frap value. in the dpph assay, antioxidant activity in strawberry samples varied from 7.94 μmol per g (‘fv-12’) to 11.38 μmol per g (‘fv-10’), which is consistent with previous findings in strawberry. pineli et al. (2011) reported dpph values between 10.10 and 12.83 μmol per g fruit. lower dpph value indicates higher antioxidant activity. the antioxidant activity of strawberry fruit extracts is primarily correlated with total phenolic contents of the fruit rather than any individual phenolic compound or vitamin c content (roussos et al., 2009). antioxidants from fruits and vegetables, especially with an intense coloration, are considered an important protection factor against oxidative stress and its deleterious consequences to human health (pineli et al., 2011). the hypothesized health benefits related to strawberry consumption include their role in the prevention of inflammation, oxidative stress and cardiovascular disease, certain types of cancers, type 2 diabetes, obesity, and neurodegenerative disorders (giampieri et al., 2012). conclusion this study showed marked differences in the selected biochemical and bioactive values among wild (less common) and commercial strawberry fruits. all wild material in general had higher levels of tss, vitamin c, total phenolic and total ellagic acids than cv. camarosa. therefore, fragaria vesca genotypes are one of the most valuable source with respect to examined polyphenolic compounds. it is clear that the same amount of f. vesca fruits gives a higher intake of phenolic compounds etc. than f x ananassa. considering that all accessions were grown under identical conditions and in the same location, one can clearly see the variability between genotypes, which is quite typical for strawberries. due to our findings, one can assume a high potential value of wild strawberries in terms of bioactive content and an importance for nutraceutical manufacturers. nevertheless, wild accessions should be grown in controlled conditions in successive years to ascertain their real potential and stability of the characters. acknowledgements a. hegedus is grateful for receiving a jános bolyai scholarship, hungarian academy of sciences. references anttonen, m.j., karjalainen, r.o., 2005: environmental and genetic variation of phenolic compounds in red raspberry. j. food. comp. anal. 18, 759-69. aoac, 2005: association of official analytical chemists. official methods of analysis of the association of official agriculture chemistry. 18th ed. mayland, usa. bartolome, a., ruperez, p., fuster, c., 1995: pineapple fruit morphological characteristics, chemical composition and sensory analysis of red spanish and smooth cayenne cultivars. food chem. 53, 75-79. benzie, i.f.f., strain, j.j., 1996: the ferric reducing ability of plasma (frap) as a measure of “antioxidant power”: the frap assay. anal. biochem. 239, 70-76. castro, i., goncalves, o., teixeira, j.a., vicente, a.a., 2002: comparative study of selva and camarosa strawberries for the commercial market. j. food sci. 67, 2132-2137. chandler, c.k., folta, k., dale, a., whitaker, v.m., herrington, m., tab. 2: bioactive content and antioxidant activity in fruits of wild and cultivated strawberry accessions. genotypes total phenolics total anthocyanin ellagic acid antioxidant activity (mg gae per 100 g fw) (mg per 100 g fw) (mg per 100 g fw) dpph frap (μmol per g fw) (μmol per g fw) fv-1 193bc 33.01bc 25.08ab 8.18bc 37.05fg fv-2 228a 53.51a 22.87ab 7.91bc 49.11a fv-3 170c 28.62cd 20.10bc 10.27ab 30.82jk fv-4 210ab 47.66ab 26.36a 7.96c 47.18b fv-5 201b 43.07b 17.95bc 8.11bc 43.14d fv-6 190bc 25.11d 17.05bc 9.31b 39.05ef fv-7 177bc 31.64c 15.18c 9.96ab 38.10efg fv-8 214ab 51.78ab 24.48ab 8.00bc 47.62b fv-9 207ab 39.64bc 18.91bc 8.07bc 44.09cd fv-10 187bc 29.66cd 23.36ab 11.38a 33.15i fv-11 191bc 38.41bc 18.55bc 9.67ab 37.70g fv-12 217ab 49.07ab 20.47b 7.94c 46.10b fv-13 211ab 45.18ab 19.62bc 7.98c 45.01c fv-14 195abc 45.64ab 18.15bc 8.40bc 43.18d fv-15 171c 29.37cd 21.18ab 10.84ab 31.05j camarosa 138d 36.15bc 18.56bc 11.20ab 27.10k means within a column followed by the same letter are not significantly different at p<0.01 fw: fresh weight 278 h. yildiz, s. ercisli, a. hegedus, m. akbulut, e.f. topdas, j. aliman 2012: strawberry. in: badenes, m.l., byrne, d.h. 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lee, s.k., kader, a.a., 2000: preharvest and postharvest factors influencing vitamin c content of horticultural crops. postharvest biol. tec. 20, 207-220. meyers, k.j., watkins, c.b., pritts, m.p., liu, r.h., 2003: antioxidant and antiproliferative activities of strawberries. j. agric. food chem. 51, 6887-6892. milivojevic, j., maksimovic, v., nikolic, m., bogdanovic, j., maletic, r., milatovic, d., 2011: chemical and antioxidant properties of cultivated and wild fragaria and rubus berries. j. food qual. 34, 1-9. milivojevic, j., maksimovic, v., maksimovic, jd., radivojevic, d., poledica, m., ercisli, s., 2012: a comparison of major taste-and health-related compounds of vaccinium berries. turk. j. biol. 36, 738745. moyer, r.a., hummer, k.e., finn, c.e., frei, b., wrolstad, r.e., 2002: anthocyanins, phenolics, and antioxidant capacity in diverse small fruits: vaccinium, rubus and ribes. j. agric. food chem. 50, 519-525. oszmianski, j., wojdylo, a., 2009: comparative study of phenolic content and antioxidant activity of strawberry puree, clear, and cloudy juices. eur. food res. technol. 228, 623-631. ozdemir, e., kaska, n., gunduz, k., serce, s., 2013: effects of short day conditioning, chilling and ga3 treatments to yield and fruit quality in strawberry plug transplants aiming early fruit production. not. bot. horti. agrobo. 41(1), 263-268. ozsen, d., erge, h.s., 2013: degradation kinetics of bioactive compounds and change in the antioxidant activity of wild strawberry (fragaria vesca) pulp during heating. food bioprocess tech. 6, 2261-2267. pineli, l.o., moretti, c.l., dos santos, m.s., campos, a.b., brasileiro, a.v., cordova, a.c., chiarello, m.d., 2011: antioxidants and other chemical and physical characteristics of two strawberry cultivars at different ripeness stages. j. food comp. anal. 24, 11-16. pinto, m.s., lajolo, f.m., genovese, m.i., 2008: bioactive compounds and quantification of total ellagic acid in strawberries (fragaria x ananassa duch.). food 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2014: in vitro screening of octoploid fragaria chiloensis and fragaria virginiana genotypes against iron deficiency. turk. j. agric. for. 38, 169-179. ulrich, d., komes, d., olbricht, k., hoberg, e., 2007: diversity of aroma patterns in wild and cultivated fragaria accessions. genet. res. crop evol. 54, 1185-1196. ulrich, d., olbricht, k., 2013: diversity of volatile patterns in sixteen fragaria vesca l. accessions in comparison to cultivars of fragaria ×ananassa. j. appl. bot. food qual. 86, 37-46. xue, h., aziz, r.m., sun, n., cassady, j.m., kamendulis, l.m., xu, y., stoner, g.d., klaunig, j.e., 2001: inhibition of cellular transformation by berry extracts. carcinogenesis 22, 351-356. vattem, d.a., shetty, k., 2005: biological functionality of ellagic acid: a review. j. food biochem. 29, 234-266. voca, s., dobricevic, n., dragovic-uzelac, v., duralija, b., druzic, j., cmelik, z., babojelic, m.s., 2008: fruit quality of new early ripening strawberry cultivars in croatia. food technol. biotechnol. 46 (3), 292-298. zabetakis, i.i., holden, m.a., 1997: strawberry flavor: analysis and biosynthesis. j. sci. food agric. 74, 421-434. address of the corresponding author: e-mail: sercisli@gmail.com art16_ercisli.indd journal of applied botany and food quality 85, 229 233 (2012) 1ministry of food, agriculture and livestock atatürk orman çiftliği ankara, turkey 2department of horticulture, ondokuz mayis university, samsun-turkey 3department of horticulture, ataturk university, erzurum-turkey 4ankara university biotechnology institute, ankara-turkey simple sequence repeat (ssr) analysis for assessment of genetic variability in wild cherry germplasm z. turkoglu1, s. bilgener2, s. ercisli3*, n. yildirim4 (received october 22, 2012) * corresponding author summary conservation of genetic resources is vital for future breeding programs and food security for humans. before conservation of genetic resources, it requires objective characterization and a proper assignation of individual genotypes to species. the aim of this study was to characterize of 58 prunus accessions which belong to prunus avium, prunus cerasus and prunus mahaleb by using 12 ssr markers. all twelve ssr markers produced successful amplifi cations and revealed dna polymorphisms. the number of allele per loci varied from 6 (udp96-019) to 12 (ps12a02) with an average of 9 per allele. the average of observed and expected heterozygosity was found to be 0.609 and 0.720. the allele size varied from 95 to 276 bp. the number of genotypes per allele were 7 (ucd-ch13) and 24 (udp96-005). genetic distance analysis based on ssrs divided the cherry accessions in three main groups based mainly on their species characteristics. p. cerasus genotypes had higher similarity ratio within species than p. mahaleb and p. avium. introduction sweet cherry, sour cherry and mahaleb belongs to rosaceae family, prunoideae subfamily, prunus genus and cerasus subgenus. cerasus is further divided into four groups including eucerasus, microcerasus, pseudocerasus and mahaleb. sweet cherries (prunus avium l.) and sour cherries (prunus cerasus l.) are placed in eucerasus group while mahleb (prunus mahaleb l.) is placed in mahaleb group (ercisli, 2004). asia minor in turkey is one of the origins and domestication centers for p. avium, p. cerasus and p. mahaleb (ozcagiran et al., 2005). p. avium is originated in the area between the black and caspian seas of asia minor. great morphological variation exists among p. avium, p.mahaleb and p. cerasus accessions naturally grown as wild in turkey (ercisli, 2004). this continuum of morphological characteristics makes species assignation diffi cult when considering only phenotypic traits. in turkey prunus mahaleb, wild prunus avium and prunus cerasus seedlings commonly have been used as rootstocks for both sweet and sour cherry cultivars (ercisli et al., 2011). conservation of genetic resources is vital for future breeding programs and food security for humans. before conservation of genetic resources, objective characterization and a proper assignation of individual genotypes to species is required (karp et al., 1997). among the different marker systems (morphological, biochemical and molecular), molecular markers supply more reliable tools to analyze genetic diversity in plant species (karp et al., 1997). they could be helpful by giving an accurate and unambiguous assignation of each genotype to a particular species (szikriszt et al., 2011). simple sequence repeats (ssrs or microsatellites) have become the genetic markers of choice in many plant species because they are pcr-based, highly reproducible, polymorphic, generally codominant and abundant in plant genomes (powell et al., 1996). ssr loci can be identifi ed and their alleles recognized in different genotypes of the same species because of their codominant and usually single-locus nature, and often in those of other close relatives. in other words a specifi c set of ssrs can be used in different sets of genotypes, making them particularly useful for fi ngerprinting. in general, ssrs are more transferable between species of the same genus, or between closely related genera, than between distant genera of the same family (peakall et al., 1998; zhang et al., 2005; hendre et al., 2008; luro et al., 2008). previously ssr (simple sequence repeats) markers have been successfully used on genotypes belong to different prunus genus in diversity studies (cheng and huang, 2009; guarino et al., 2009; lacis et al., 2009; gulen et al., 2010; nas et al., 2011). however, the majority of these studies have dealt with cultivars and little emphasis has been paid to wild relatives. this study has therefore sought to document indigenous knowledge related to uses of wild grown genotypes which belong to p. avium, p. cerasus and p. mahaleb. materials and methods plant material for ssr and genetic relationship studies, 37 prunus avium, 8 prunus cerasus and 7 prunus mahaleb rootstock candidates together with well-known standard rootstocks of each species, f12/1 (prunus avium l.), montmorency (prunus cerasus) and sl 64 (prunus mahaleb l.) were used. these genotypes have been previously selected from wild cherry populations as rootstock candidates in ordu region in turkey. all genotypes are maintained in a germplasm collection at the black sea agricultural research center in samsun, turkey. dna extraction genomic dna was extracted from young leaf tissue using the wizard® genomic dna purifi cation kit (promega, madison, wi) according to the instructions provided by the manufacturer. subsequently, an rnase treatment was performed on the eluted dna samples. purity and concentration of the dna were checked both on 1% (w/v) agarose gels and by nanodrop® nd-1000 spectrophotometer. ssr analysis from an initial screening, 12 ssrs were selected to check for polymorphism by capillary electrophoresis in 55 genotypes of three different prunus species (tab. 1). polymerase chain reaction (pcr) was conducted in a volume of 10 µl and contained 15 ng genomic dna, 5 pmol of each primer, 0.5 mm dntp, 0.5 unit gotaq dna polymerase (promega), 1.5 mm mgcl2 and 2 µl 5x buffer. the forward primers were “labelled” with wellred fl uorescent dyes d2 (black), d3 (green) and d4 (blue) (pro ligo, paris, france). reactions without dna were included as negative 230 z. turkoglu, s. bilgener, s. ercisli, n. yildirim molecular characterization of wild cherry germplasm by ssr controls. pcr amplifi cation was performed using the biometra® pcr system. the amplifi cation condi tions consisted of an initial denaturation step of 3 min at 94°c, followed by 35 cycles of 1 min at 94°c, 1 min at 52-56°c and 2 mins at 72°c with a fi nal extension at 72°c for 10 mins. the pcr products were fi rst separated on a 3% (w/v) agarose gel run at 80 v for 2 hrs. the gel was then stained with ethidium bromide at a concentration of 10 mg/ml. a dna ladder (100 bp) (promega) was used for the approximate quantifi cation of the bands. the amplifi cation products were visualized under uv light, and their sizes were estimated relative to the dna ladder. for further determination of polymorphisms, the pcr prod ucts were run on ceqtm 8800 xl capillary genetic analysis system (beckman coulter, fullerton, ca). the analyses were repeated at least twice to ensure reproducibility of the results. allele sizes were determined for each ssr locus using the beckman ceqtm frag ment analysis software. in each run, sl64, f12/1 and montmorency were included as reference rootstocks. genetic analysis the genetic analysis program “identity” 1.0 (wagner and sefc, 1999) was used according to paetkau et al. (1995) for the calculation of number of alleles, allele frequency, expected and observed heterozygosity, estimated frequency of null alleles, and probability of identity per locus. genetic dissimilarity was determined by the program “microsat” (version 1.5) (minch et al., 1995) using proportion of shared alleles, which was calculated by using “ps (option 1 (ps))”, as described by bowcock et al. (1994). the results were then converted to a similarity matrix, and a dendrogram was constructed with the upgma method (sneath and sokal, 1973) using the software ntsys-pc (numerical taxonomy and multiware analy sis system,version 2.0) (rohlf, 1988). results and discussion the total 12 ssrs studied amplifi ed 108 alleles, an average of 9 alleles per locus in 58 prunus accessions belongs to prunus avium, prunus cerasus and prunus mahaleb. the highest number of allele per primer was observed in ps12a02 primer as 12 alleles and followed by pchgms1, udp96-001 and udp96-005 primers (11 alleles). the primer udp96-019 gave the lowest number of alleles (6 alleles) (tab. 1). we observed an average ssr observed heterozygosity (ho) of 0.609 and the observed heterozygosity were found between 0.345 (pchgms1) and 0.890 (ucd-ch31). the average expected heterozygosity (he) was 0.720 indicating higher value than observed one (tab. 1). the probability of genetic identity (pi) was the lowest in ps12a02 locus (pi:0.089) indicating that this loci was the most informative while the locus udp96-019 (pi: 0.373) was the less informative. the allele sizes of 12 ssr locus varied from 95 to 276 bp. the number of genotypes per allele were between 7 (ucd-ch13) and 24 (udp96-005) (tab. 2). the genetic similarity measured within species ranged between 0.17-0.79 within p. avium, 0.96-1.00 within p. cerasus, 0.58-0.83 within p. mahaleb genotypes. the average similarity ratios within species in a descending order were p. cerasus (0.98)> p. mahaleb (0.72)> p. avium (0.51), respectively. the similarity ratio were 0.250.58 between p. avium accessions and f12/1; p. cerasus accessions and montmorency was 0.63 and p. mahaleb accessions and sl64 were 0.46-0.63, respectively. the average similarity ratio between p. avium-p. cerasus; p. avium-p. mahaleb and p. cerasus-p. mahaleb were 0.33; 0.007 and 0.08, respectively, indicating p. avium is more close to p. cerasus. a tree constructed from the ssr data divided the accessions into 3 main clusters according to their taxonomic classifi cation. the fi rst cluster included p. avium accessions, the second cluster included p. cerasus accessions and the last cluster included p. mahaleb accessions (fig. 1). p. cerasus accessions seem to be identical or very closely related. in contrast to p. cerasus, p. mahaleb and in particular p. avium seem to be more differentiated. the reference rootstocks were also clustered with their associated botanical species (fig. 1). the results obtained in the present study show that microsatellites could be effectively used for fi ngerprinting purposes in prunus. in the present study, 12 loci in wild prunus genotypes were assayed. the num ber of alleles per locus ranged from 6 to 12 with an average of 9 putative alleles per locus. previously, kacar et al. (2005) obtained a total of 37 alleles among 10 sweet cherry cultivars by 9 ssr primers. clarke and tobutt (2003) used 14 sweet cherry cultivars for ssr analysis and determined 2 to 7 alleles per ssr primer. in addition, vaughan and russell (2004) used 16 wild cherry accessions for molecular analysis by using 10 ssr primers and they detected 2 to 6 alleles. in fact all tested microsatellite primer pairs worked well and produced variable levels of amplifi cations. the ps12a02 locus was the most polymorphic among the twelve loci with the highest effective number of alleles (12 alleles) with the one of the lowest pi value (0.089). the udp96-019 was the less informative with the lowest allele number (6). the results showed high amplifi cation of cherry groups with plum, apricot and peach indicating a congeneric relationship within prunus species. ercisli et al. (2011) successfully used ssr markers identifi ed in other prunus species to study genetic diversity in wild sweet cherries. our results demonstrated the cross-species transferability of ssr primers developed in cultivated species to wild species in prunus for the discrimination of genotypes. previously, ps12a02 loci were found to be the most informative in other studies (downey and iezzoni, 2000; gulen et al., 2010; ercisli et al., 2011). wünsch et al. (2004) reported 7 and 11 alleles in udp96-005 and pchgms1 locus in sweet cherries. in our study, udp96-019 and ucd-ch13 gave the lowest number of alleles (6 and 7). struss et al. (2003) and zhang et al. (2008) obtained the lowest allele in ucd-ch31 loci and wünsch and hormaza (2002) reported the lowest loci in udp96-019 loci. the overall genetic diversity within the tested species was relatively low as evident from the polymorphic ratio of 21% found by ssr tab. 1: list of genetic parameters obtained with ssr used in this study locus n he ho pi r cpsct010 10 0.648 0.581 0.190 0.040 pchgms1 11 0.723 0.345 0.142 0.219 ps12a02 12 0.806 0.636 0.089 0.094 ucd-ch13 7 0.697 0.836 0.237 -0.081 ucd-ch17 8 0.861 0.418 0.068 0.238 ucd-ch21 8 0.697 0.381 0.175 0.186 ucd-ch31 8 0.773 0.890 0.146 -0.006 udap-401 8 0.729 0.636 0.177 0.053 udap-404 8 0.606 0.818 0.373 -0.131 udp96-001 11 0.807 0.545 0.084 0.144 udp96-005 11 0.850 0.818 0.072 0.017 udp96-019 6 0.440 0.400 0.370 0.028 total 133 average 13.3 0.81 0.57 n: number of alleles; ho: observed heterozygosity; he: expected heterozygosity; pi:probability of genetic identity; r: null allele frequencies molecular characterization of wild cherry germplasm by ssr 231 t ab . 2 : t he a ll el e si ze s (b p) o f p ru n u s ac ce ss io ns a t 12 s s r l oc us g en ot yp e n o p s 12 a 02 u c d -c h 17 p ch gm s1 u d a p -4 01 u c d -c h 31 u c d -c h 21 u d a p -4 04 u d p 96 -0 01 u d p 96 -0 05 c p s c t 01 0 u c d -c h 13 u d p 96 -0 19 p. a vi u m 37 15 8: 16 6 20 2: 20 2 13 8: 13 8 26 2: 26 2 13 0: 14 2 11 1: 11 9 15 4: 16 8 11 5: 12 3 10 9: 11 5 17 6: 17 6 12 2: 13 6 20 2: 20 2 38 15 8: 17 4 20 2: 20 2 13 0: 13 0 26 2: 26 2 12 4: 14 2 11 1: 11 1 15 4: 16 8 12 3: 12 3 11 5: 11 9 17 6: 17 6 12 6: 13 6 20 2: 20 2 39 15 8: 15 8 20 0: 20 0 13 0: 13 6 26 2: 26 2 13 0: 14 2 10 9: 10 9 15 4: 16 8 10 5: 12 5 12 5: 12 5 18 0: 18 0 12 6: 13 6 20 2: 20 2 40 15 8: 15 8 19 8: 19 8 13 0: 13 8 26 0: 26 6 13 0: 14 2 10 7: 10 7 15 4: 16 8 10 9: 12 3 10 9. 12 5 17 6: 17 6 12 6: 13 6 20 2: 20 2 41 15 8: 16 6 19 8: 19 8 14 2: 15 2 26 2: 26 2 13 0: 14 2 10 9: 10 9 15 4: 16 2 12 3: 13 3 11 5: 11 9 17 6: 17 6 12 6: 13 6 20 2: 20 2 42 15 8: 16 6 18 0: 19 8 13 8: 13 8 26 2: 26 6 13 0: 14 2 10 7: 11 1 15 4: 16 8 12 3: 12 3 12 5: 13 5 17 6: 17 6 12 6: 13 6 20 2: 20 2 43 15 8: 17 4 19 8: 19 8 15 2: 1 52 26 2: 26 6 13 2: 14 2 10 9: 10 9 15 4: 16 8 12 3: 12 3 10 9: 10 9 17 6: 18 0 13 6: 13 6 20 2: 20 2 44 15 4: 17 4 18 0: 20 2 13 8: 13 8 26 2: 26 6 13 0: 14 2 10 9: 10 9 15 4: 16 8 10 5: 10 5 11 9: 11 9 17 6: 17 6 12 6: 13 6 20 2: 20 2 45 15 8: 16 6 19 0: 20 4 13 6: 13 6 26 2: 26 2 13 0: 14 2 10 9: 10 9 15 4: 16 2 12 3: 12 3 10 9: 11 9 17 6: 18 0 12 2: 13 6 20 2: 20 2 46 15 8: 16 6 20 2: 20 2 13 8: 13 8 26 2: 26 2 13 0: 14 2 10 9: 10 9 15 4: 16 2 10 9: 11 5 10 9: 11 5 17 6: 17 6 12 6: 13 6 20 2: 20 2 47 15 8: 15 8 18 0: 20 0 13 8: 15 8 26 2: 26 6 12 4: 13 0 10 9: 10 9 15 4: 16 8 10 9: 12 3 10 9: 11 9 17 6: 17 6 12 6: 13 6 20 2: 20 2 48 16 0: 17 4 19 0: 19 0 14 0: 14 0 26 6: 26 6 12 4: 14 2 10 9: 10 9 16 8: 16 8 12 3: 12 3 11 9: 13 5 17 6: 17 6 13 2: 13 6 20 2: 20 2 49 15 4: 16 0 19 0: 19 0 14 0: 14 0 26 0: 26 0 12 8: 14 2 10 9: 10 9 16 8: 16 8 10 5: 12 5 11 5: 11 9 17 4: 17 8 12 6: 13 6 20 2: 20 4 50 17 4: 17 4 20 0: 20 0 14 0: 14 0 26 2: 26 6 13 2: 14 2 10 9: 10 9 15 4: 16 2 12 3: 12 3 11 5: 11 9 17 6: 17 6 12 6: 13 6 20 2: 20 2 51 15 8: 15 8 19 0: 19 0 13 0: 13 8 26 2: 26 6 13 0: 14 2 11 1: 11 1 15 4: 16 8 12 3: 12 3 10 9: 11 9 17 6: 17 6 12 6: 13 6 20 2: 20 2 52 15 8: 15 8 18 0: 20 0 13 8: 13 8 26 2: 26 6 12 4: 13 0 10 9: 11 7 15 4: 16 8 10 5: 12 3 11 9: 13 3 17 6. 18 2 12 6: 13 6 20 2: 20 2 53 15 8: 16 6 19 0: 20 4 13 0: 15 8 26 2: 26 6 13 2: 14 2 10 7: 11 9 15 4: 16 2 11 5: 12 3 13 3: 14 3 17 6: 18 0 12 2: 13 6 20 2: 20 2 54 17 4: 17 4 18 8: 19 8 13 0: 15 2 26 2: 26 2 13 0: 14 2 10 9: 10 9 15 4: 16 2 12 3: 12 3 11 9: 13 3 17 6: 17 6 12 6: 13 6 20 2: 20 2 55 15 4: 15 8 19 0: 19 0 13 8: 13 8 26 2: 26 2 13 0: 14 2 10 9: 10 9 15 4: 16 2 12 3: 13 7 10 9: 11 9 17 6: 18 0 12 6: 13 6 20 2: 20 2 56 16 6: 16 6 20 0: 20 0 13 8: 13 8 27 0: 27 0 13 0: 14 2 10 7: 11 1 15 4: 16 2 10 5: 12 5 10 9: 11 5 17 6: 18 0 12 6: 13 6 20 2: 20 4 57 16 0: 17 4 19 8: 19 8 13 8: 13 8 26 2: 27 2 13 0: 14 2 10 9: 10 9 15 4: 16 2 12 3: 12 3 11 5: 13 5 17 6: 17 6 12 6: 13 6 20 2: 20 2 58 15 8: 16 6 19 8: 19 8 13 8: 13 8 26 2: 26 2 13 0: 14 2 11 1: 11 5 15 4: 16 2 12 3: 12 3 11 5: 11 5 17 6: 17 6 12 6: 13 6 20 4: 20 4 59 15 8: 17 4 20 2: 20 2 13 0: 14 0 27 0: 27 0 13 0: 14 2 11 1: 11 1 15 4: 16 2 10 5: 12 5 11 5: 11 5 17 6: 17 6 12 6: 13 6 20 2: 20 4 60 15 8: 15 8 18 0: 18 0 13 8: 13 8 26 2: 26 2 12 4: 13 0 10 9: 10 9 15 4: 16 2 10 5: 12 3 11 5: 11 5 18 0: 18 0 12 6: 13 6 18 2: 20 2 61 15 4: 15 8 19 8: 19 8 13 8: 13 8 26 2: 26 6 13 0: 14 2 10 9: 10 9 16 2: 16 8 10 9: 11 5 11 5: 12 5 17 6: 17 6 12 2: 13 6 20 2: 20 2 62 16 2: 16 2 20 0: 20 0 13 8: 13 8 26 2: 26 2 13 2: 14 2 11 1: 11 9 15 4: 16 2 12 3: 12 3 14 3: 14 3 17 6: 17 6 13 6: 14 4 20 2: 20 4 63 15 8: 16 6 19 8: 19 8 13 8: 13 8 26 2: 26 2 13 0: 14 2 11 1: 11 9 15 4: 16 8 10 9: 12 5 11 9: 12 5 17 4: 17 6 12 6: 13 6 18 2: 20 4 64 15 8: 16 6 18 0: 20 2 13 8: 13 8 26 2: 27 2 13 0: 14 2 10 9: 10 9 15 4: 16 2 12 5: 13 7 11 5: 13 3 17 6. 17 6 12 6: 13 6 18 2: 20 2 65 15 8: 15 8 20 0: 20 0 13 0: 13 6 26 0: 27 2 12 8: 14 2 10 9: 10 9 16 4: 16 8 12 3: 12 3 11 5: 13 3 17 6: 17 6 12 6: 13 6 20 2: 20 2 66 16 0: 17 4 18 0: 20 0 14 6: 14 6 26 0: 26 6 12 8: 14 2 10 9: 10 9 15 4: 16 2 10 9: 12 3 10 9: 11 5 17 6: 18 0 12 6: 13 6 20 2: 20 2 67 15 8: 15 8 18 8: 20 4 13 8: 15 8 26 6: 27 2 13 0: 14 2 10 9: 11 9 15 4: 16 2 12 3: 12 3 13 5: 13 5 17 6: 18 0 12 6: 13 6 20 2: 20 2 68 15 4: 15 8 18 8: 20 0 13 8: 13 8 26 2: 26 6 13 2: 14 2 10 7: 11 1 15 4: 16 2 10 5: 12 5 10 9: 12 5 17 6: 18 0 12 6: 13 6 20 2: 20 2 69 15 8: 15 8 20 2: 20 2 13 8: 13 8 26 2: 26 2 13 0: 14 2 10 9: 10 9 15 4: 16 2 10 9: 11 5 11 5: 13 5 17 6: 18 0 12 2: 13 6 20 2: 20 2 70 15 8: 15 8 18 8: 20 4 13 8: 13 8 26 2: 26 2 12 4: 13 0 10 9: 11 9 16 8: 17 8 12 3: 12 3 10 9: 11 5 17 6: 17 6 12 6: 13 6 20 2: 20 2 71 15 8: 17 4 19 0: 19 8 13 8: 13 8 26 6: 26 6 13 0: 14 2 10 9: 10 9 16 8: 16 8 11 5: 12 5 11 5: 11 5 17 4: 18 2 12 6: 13 6 18 2: 20 2 72 15 8: 15 8 20 0: 20 0 13 8: 13 8 26 6: 27 2 13 0: 14 2 10 9: 10 9 15 4: 16 2 12 3: 12 3 10 9: 11 5 17 4: 17 6 12 6: 13 6 20 2. 20 2 73 15 8: 15 8 18 8: 20 0 13 0: 13 0 26 0: 26 6 13 0: 14 2 10 9: 11 9 15 4: 16 2 12 3: 13 3 11 5: 11 9 17 6: 18 0 12 6: 13 6 20 2: 20 2 232 z. turkoglu, s. bilgener, s. ercisli, n. yildirim molecular characterization of wild cherry germplasm by ssr g en ot yp e n o p s 12 a 02 u c d -c h 17 p ch gm s1 u d a p -4 01 u c d -c h 31 u c d -c h 21 u d a p -4 04 u d p 96 -0 01 u d p 96 -0 05 c p s c t 01 0 u c d -c h 13 u d p 96 -0 19 p. c er a su s 12 2 14 6: 16 0 18 0: 18 8 13 8: 16 0 26 2: 26 6 12 4: 13 2 10 3: 10 9 15 4: 16 8 99 :1 13 10 9: 11 9 17 0: 17 6 12 8: 13 6 19 0: 20 2 12 3 14 6: 16 0 18 0: 18 8 13 8: 16 0 26 2: 26 6 12 4: 13 2 10 3: 10 9 15 4: 16 8 99 :1 13 10 9: 11 9 17 0: 17 6 12 8: 13 6 19 0: 20 2 12 4 14 6: 16 0 18 0: 18 8 13 8: 16 0 26 2: 26 6 12 4: 13 2 10 3: 10 9 15 4: 16 8 99 :1 13 10 9: 11 9 17 0: 17 6 12 8: 13 6 19 0: 20 2 12 5 14 6: 16 0 18 0: 18 8 13 8: 16 0 26 2: 26 6 12 4: 13 2 10 3: 10 9 15 4: 16 8 99 :1 13 10 9: 11 9 17 0: 17 6 12 8: 13 6 19 0: 20 2 12 6 14 6: 16 0 18 0: 18 8 13 8: 16 0 26 2: 26 6 12 4: 13 2 10 3: 10 9 15 4: 16 8 99 :1 13 10 9: 11 9 17 0: 17 6 12 8: 13 6 19 0: 20 2 12 7 14 6: 16 0 18 0: 18 8 13 8: 16 0 26 6: 27 6 12 4: 13 2 10 3: 10 9 15 4: 16 8 99 :1 13 10 9: 11 9 17 0: 17 6 12 8: 13 6 19 0: 20 2 12 8 14 6: 16 0 18 0: 18 8 13 8: 16 0 26 6: 27 6 12 4: 13 2 10 3: 10 9 15 4: 16 8 99 :1 13 10 9: 11 9 17 0: 17 6 12 8: 13 6 19 0: 20 2 18 6 14 6: 16 0 18 0: 18 8 13 8: 16 0 26 2: 26 6 12 4: 13 2 10 3: 10 9 15 4: 16 8 99 :1 13 10 9: 11 9 17 0: 17 6 12 8: 13 6 19 0: 20 2 p. m a h a le b 15 2 16 2: 16 2 16 4: 16 4 18 8: 18 8 13 8: 14 6 10 0: 10 0 95 :9 5 16 8: 16 8 11 5: 11 5 11 3: 11 9 21 2: 23 2 12 2: 12 2 20 2: 20 8 15 3 15 6: 16 6 16 4: 16 4 18 8: 18 8 13 8: 14 6 10 6: 10 6 95 :9 5 16 8: 16 8 11 7: 11 7 11 3: 12 3 21 2: 23 2 12 2: 12 2 20 2: 20 8 15 4 16 2: 16 2 16 4: 16 4 18 8: 18 8 13 8: 14 6 10 6: 10 6 95 :9 5 17 0: 17 0 11 7: 11 7 11 3: 11 9 21 2: 23 2 12 2: 12 2 20 2. 20 8 15 5 14 6: 16 4 16 4: 16 4 18 8: 18 8 13 8: 14 6 12 4: 12 4 95 :9 5 17 0: 17 0 11 7: 11 7 11 3: 12 3 21 2: 23 2 12 2: 12 2 20 2: 20 8 15 6 16 4: 17 2 16 4: 16 4 18 8: 18 8 13 8: 14 6 10 0: 10 0 95 :9 5 16 8: 17 2 11 7: 11 7 11 3: 12 3 21 2: 23 2 12 2: 12 2 20 2: 21 8 15 7 16 2: 16 2 16 4: 16 4 18 8: 18 8 13 8: 14 6 10 0: 12 2 95 :9 5 17 0: 17 0 11 5: 11 5 11 3: 11 7 21 2: 23 2 12 2: 12 2 20 2: 20 2 15 8 14 6: 16 2 16 4: 16 4 18 8: 18 8 13 8: 14 6 10 0: 12 2 95 :9 5 16 8: 16 8 11 5: 11 5 11 3: 12 3 21 2: 23 2 12 2: 12 2 20 8: 20 8 s l 64 ( 18 9) 14 2: 14 2 16 4: 16 4 18 8: 18 8 13 8: 14 6 12 4: 12 4 95 :9 5 16 8: 16 8 11 9: 11 9 11 3: 11 3 20 6: 22 8 12 2: 12 2 20 8: 20 8 f 12 /1 ( 20 1) 15 6: 17 6 20 2: 20 2 13 8: 1 38 26 2: 27 2 12 4: 13 0 10 3: 10 3 15 4: 16 2 12 3: 12 3 12 7: 13 5 18 2: 18 2 12 6: 13 6 20 2: 20 2 m m ( 20 2) 15 8: 16 4 18 0: 19 2 13 8: 16 2 26 6: 26 6 12 2: 13 0 10 3: 10 9 15 4: 16 8 99 :1 13 10 3: 11 5 17 0: 17 6 12 6: 13 6 19 0. 20 2 a ll el e si ze 14 217 4 16 420 4 13 018 8 13 827 6 10 014 2 95 -1 19 15 417 8 99 -1 37 10 314 3 17 423 2 12 213 6 19 021 8 a ll el e pe r ge no ty pe 17 16 15 11 11 12 8 1 6 24 11 7 9 m m :m on tm or en cy molecular characterization of wild cherry germplasm by ssr 233 fig. 1: dendrogram of 58 prunus accessions based on upgma analysis using the genetic similarity matrix generated by the nei and li similarity coeffi cient after amplifi cation with 12 pairs of microsatellite primers. primers (struss et al., 2003) and 19% reported by zhou et al. (2002) and in cherries. we found high diversity ratio within p. avium compared to other species. a higher level of polymorphism was expected in sweet cherry due to its predominant self-incompatibility (hegedus et al., 2012). the observed and expected heterozygosities averaged over the 12 ssr loci were respectively 0.61 and 0.72 indicating higher mean values than those reported for ssrs in prunus species (aranzana et al., 2003; bouhadida et al., 2009). high allele number and high heterozygosity obtained in the present study refl ect the ability of ssr markers to provide unique genetic profi le for individual plant accessions, except in p.cerasus accessions. vaughan and russell (2004) reported he and ho values as 0.61 and 0.60 in 16 wild sweet cherry accessions by using 14 ssr locus. conclusion in conclusion, the gene pool of the prunus species surveyed in black sea and northeast anatolia has signifi cant amounts of genetic variation. in regard to germplasm management, our results show 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length polymorphism fi ngerprints. j. am. soc. hort. sci. 127, 786-792. address of the corresponding author: e-mail: sercisli@gmail.com artikel04.indd journal of applied botany and food quality 81, 126 131 (2007) 1 agricultural university of cracow, faculty of agriculture and economics, department of plant physiology, kraków poland; 2 institute of plant genetics, polish academy of sciences, poznań, poland impact of cold-induced antioxidant activity on frost resistance in androgenic festulolium genotypes e. pociecha1, a. płażek1, z. zwierzykowski2 (received march 22, 2007) summary the aim of the study was: (i) to state if selected in the field conditions androgenic festulolium genotypes are diverse in frost tolerance, and (ii) to investigate if changes in anitoxidant activity could be recognized as a physiological marker of this type tolerance. antioxidative system induced by prehardening (12°c) and hardening (2°c) temperatures was investigated in 6 androgenic genotypes generated from a festuca pratensis × lolium multiflorum (2n = 4x = 28) amphidiploid hybrid (four genotypes derived from the f 1 hybrids, and two genotypes derived from the festulolium cultivar ‘rakopan’). the electrolyte leakage, frost resistance expressed as the values of temperature causes 50% damages (lt 50 ), the activities of superoxide dismutase (sod), catalase (cat), non-specific peroxidase (px) and ascorbate peroxidase (apx) were measured. the results obtained indicated weak diversity of frost tolerance among studied androgenic genotypes. only the one genotype was chosen as the most resistant to frost, while the other genotypes demonstrated no significant differences in values of lt 50 coefficient recorded after hardening. prehardening temperature of 12°c caused an increase in cell membrane permeability in all genotypes studied. after hardening ion leakage from cells declined up to the control level. generally, cold activated sod, px and apx in leaves of the genotypes studied, and inhibited strongly cat activity. the most frost tolerant genotype was characterized by high px activity after hardening process. abbreviations: apx – ascorbate peroxidase, cat – catalase, edta – ethylenediaminetatraacetic acid, lt 50 – letal temperature causing 50% membrane cell damages, nbt – nitroblue tetrazolium, ppfd – photosynthetic photon flux density [µmol m-2 s-1], px – non-specific peroxidase, rh – relative humidity, ros – reactive oxygen species, se – standard error, sod – superoxide dismutase introduction grasses are a great important plant group to sustainable agriculture. especially at present, according to european union (eu) guidelines relating fodder quality the culture of forage grasses comes into prominence. there is a challenge of a creation of new cultivars characterized by high quality of biochemical composition and resistance to environmental stresses (humphreys et al., 1998). species within the loliumfestuca complex belong to the most important forage grasses. they offer many valuable and complementary traits, including the high productivity and forage quality of lolium, and the persistency and resistance to environmental stresses of festuca (thomas and humphreys, 1991). so, festuca species can be used as sources of genes for winter hardiness, drought tolerance as well as disease resistance. lolium and festuca species hybridise and their chromosomes pair and recombine in hybrids, thus, it is possible to transfer stress resistance genes from festuca into lolium by backcross breeding procedures (zwierzykowski et al., 1999; kosmala et al., 2006). whilst conventional plant breeding programmes provide many opportunities for combining useful traits, preand post zygotic selections limit access to all potentially useful gene combinations. in addition, many useful gene combinations in breeding programmes may remain ‘hidden’ due to the non-expression of recessive alleles, epistasis, or pleiotropy (thomas et al., 2003). androgenesis (in vitro anther culture of microspores) is a frequent component of plant breeding methodology for several crop species to a rapid achievement uniform and homogeneous genotypes. in grasses within the lolium-festuca complex androgenesis was found to be an effective way to select new gene combinations which have rarely or never been recovered by conventional backcross breeding programmes (humphreys et al., 1998; leśniewska et al., 2001). in some experiments androgenesis enhanced gene expression for complex traits, including freezing-tolerance (rapacz et al., 2005), in excess of that found in the parent genotypes. overwinter grasses should be characterized by good winter hardiness including tolerance to frost and other factors occurring during winter. frost can damage cells, tissues, whole organs, and finally lead to plant death. tolerance to low temperature depends on tolerance to dehydratation of the cell and damages of cell membranes. accumulation of low molecular compounds decreasing osmotic potential and its freezing temperature is the most common mechanism initiated during plant hardening at cold (crow et al., 1984). hardening to frost is preceded by prehardening. prehardening proceeds at day temperature of about 12°c. night temperature and light intensity during this period do not influence so significantly plant acclimation to cold. prehardening effects the frost tolerance via stimulation of photosynthesis at hardening temperature and increasing energy amount for biochemical processes by simultaneous inhibition of elongation growth. during plant hardening proceeding usually at 1-5°c the accumulation of soluble carbohydrates, a decrease in gibberellin content and an increase in growth inhibitor level were indicated (rapacz and janowiak, 1998). after cold-hardening in cell membranes many structural changes are observed. mainly there should be mentioned changes in proportion between saturated and non-saturated fatty acids, an increase of synthesis of phospholipids and a decrease in sterol content. these changes influence a decrease in membrane permeability, which protects cells against unfavourable electrolyte leakage (yoshida and uemura, 1990; skoczowski, 1999). high correlation between frost tolerance and membrane permeability in hardened plants of barley, meadow fescue and winter rape was found (płażek and żur, 2003). during cold conditions also activity of antioxidant enzymes is changed, as an effect of generation of reactive oxygen species (ros). ros traditionally considered as toxic products play also a positive role, i.e. they control programmed cell death, participate in defence response to abiotic stress and pathogens and are mediators in signal transduction pathways. so, a balance between ros accumulation and antioxidant activity is important for plant reaction to stresses (alscher et al., 1997). ros are generated as a result of disturbances on different metabolic pathways. chloroplasts and mitochondria are the most important sources generating superoxide radical (o 2 · –). at low temperature highenergy state electron is not utilised by photosynthetic co 2 fixation but is converted to reactive o 2 form. increase of ros concentration due to cold could be a result of over-reduction of the electron transport chain in mitochondria. ros cause oxidative damages to protein, nucleic acid and lipid peroxidation, and membrane deterioration (bestwick, 1998; pastori, 2000). plants are equipped with complex antioxidant systems composed by low molecular mass antioxidants and enzymes protecting against oxidative damages. essential for ros detoxification are such rosscavenging enzymes as superoxide dismutase (sod), which converts o 2 · – to h 2 o 2 , catalase (cat), ascorbic peroxidase (apx) and other peroxidases (px; mainly wall-bound ones), which decompose h 2 o 2 to h 2 o and o 2 . apx is probably responsible for the fine modulation of ros because of its low concentration. similarly, low concentration of hydrogen peroxide takes part in signal transduction involved in resistance mechanisms (mitler, 2002; vranová et al., 2002). the aim of the study was to investigate the relationship between changes in antioxidant enzyme activities during prehardening and hardening and frost tolerance of studied plants. obtained results could serve as physiological markers for breeding of new cultivars characterized by higher winter hardiness. material and methods the study was performed on six androgenic genotypes generated from the amphidiploid festuca pratensis × lolium multiflorum (2n = 4x = 28) hybrid (named festulolium) (zwierzykowski et al., 2001). four genotypes, nos. 715, 716, 729 and 768, derived from f 1 hybrid plants, and two genotypes, nos. 561 and 621, derived from the festulolium cultivar ‘rakopan’. the genotypes were chosen in the previous study conducted in the field conditions in polish breeding station in lopuszna on the basis of visual scale estimated physiological state and regrowth after two winter seasons 2002/2003 and 2003/2004 (rapacz et al., 2005). selected plants were cloned and grown in glasshouse condition at 18°c (day/night) and day light for 5 weeks (september), then were prehardened for 2 weeks at 12°c (day/night) and 10hphotoperiod at photosynthetic photon flux density (ppfd) of 250 µmol m-2 s-1 and 80% rh and next hardened for 3 weeks at constant temperature of 2°c at 8h-photoperiod at the same light intensity. the plants were fertilized weekly with hoagland’s liquid medium. analyses were performed at 18°c (control), after prehardening and hardening. electrolyte leakage five leaf disks cut to a size of 5 mm in diameter were placed in a vial containing 13 cm3 ultrapure water. they were shaken (1.7 s-1) at 20°c. conductivity was measured using ci317 conductometer (elmetron, poland) after 2 h, and the vials were stored at -50°c overnight. after thawing, the vials were shaken for 2 h and the conductivity was measured again. the measurements performed on frozen and thawed tissue represent the conductivity of the total ion content in the tissue. membrane permeability is expressed as a percentage of total electrolyte leakage. the lt 50 coefficient frost resistance of leaves was determined by lethal temperature of 50% of the leaf samples using electrolyte leakage test. leaf sections of about 15 mm long were cut from the middle part of the leaf. the sections were immediately placed on ice (to avoid excessive undercooling by ensuring ice nucleation) in plastic vials and frozen for two hours at -3, -6, -9, -12 and -15°c. the applied rate of freezing was approximately 12 k·h-1. samples were thawed for approximately 24 h at 2°c on a shaker. electrical conductance of the solution were measured using a ci317 conductometer (elmetron, poland). the index of injury was calculated according to flint et al. (1967) and lt 50 was calculated based on the linear regression fitted within the linear range of the relationship between freezing temperatures and the percentage of killed plants (at least 3 temperatures). determinations were performed in 10 replications for each freezing temperature. assay of superoxide dismutase (sod) (e.c.1.15.1.1) activity leaf samples were homogenized at 4°c with 50 mm phosphate buffer ph 7.5 and centrifuged at 16 000 x g. the resulting supernatant was used as crude extracts. the reaction mixture (final volume of 3 cm3) contains 50 mm phosphate sodium buffer (ph 7.8) with 0.1 mm edta, 13 mm methionine, 75 µm nbt, 2 µm riboflavine and 50 µl of supernatant. reaction was started by lightening with a fluorescence lamp (for 10 min.) and stopped by its switching off. spectrophotometric measurement was done at 560 nm. unlighted reaction mixture does not change colour and is treated as a control. extract volume relating to 50% inhibition of reaction is considered as an enzyme-activity unit (beauchamp and fridovich 1971). assay of catalase (ec 1.11.1.6) activity catalase activity was estimated according to aebi (1984). samples of leaves were homogenised at 4°c with 50 mm phosphate buffer (ph 7.5) and 1 mm edta and centrifuged at 16 000 x g. cat activity was assayed in reaction mixture (3 cm3 final volume) composed of 50 mm phosphate buffer ph 7.5, to which 30% (w/v) h 2 o 2 was added to reach an absorbance value in the range of 0.520-0.550 (λ = 240 nm). the reaction was started after adding to the reaction mixture 200 µl of crude extracts. cat activity was measured as the decrease in absorbance at 240 nm (using spectrophotometer lkb ultrospec ii) as a consequence of h 2 o 2 consumption. the decrease in absorbance of 0.0145 responded with 1 µmol h 2 o 2 decomposed by cat. activity of the enzyme was expressed as µmol h 2 o 2 decomposed per minute per mg of protein. the determination of cat activity was done in 5 replicates (in five leaves from different plants of each object). assay of total peroxidase (px) activity peroxidase activity was measured spectrophotometrically according to the method described by bergmeyer (1965). leaf samples were ground to a fine powder with liquid nitrogen and extracted with 50 mm phosphate buffer (ph 7.0) and 1 mm edta. the extracts were centrifuged (16 000 x g) at 4°c for 10 min and the resulting supernatants were used as the crude extracts. two cm3 of 50 mm phosphate buffer (ph 7.0) were mixed with 12 µl of 0.5% p-phenylenediamine and with 12 µl of crude extract. the oxidation of p-phenylenediamine was initiated by addition of 12 µl of buffered h 2 o 2 (0.15 cm3 of h 2 o 2 mixed with 50 cm3 of extract buffer) to the prepared mixture. the absorbance was measured at λ = 460 nm (using a spectrophotometer lkb ultrospec ii). the peroxidase activity was expressed as difference of absorbances of sample recorded at the beginning of measurement and after 1 min. assays were done in 5 replications (in five leaves from different plants of each object). assay of ascorbate peroxidise (apx) (ec 1.11.1.11.) activity ascorbate peroxidase activity was estimated spectrophotometrically according to nakano and asada (1981). sample of leaves were homogenised and extracted with 50 mm phosphate buffer (ph 7.0) and 1 mm edta then centrifuged at 16 000 x g. one cm3 of reaction mixture contained 50 mm phosphate buffer (ph 7.0), 0.25 mm ascorbic acid, 0.5 mm h 2 o 2 and 10 µl of crude extract. the absorbance was measured at λ = 290 nm (using spectrophotometer lkb ultrospec ii). the peroxidase activity was expressed as µmol of ascorbic acid · min-1·mg-1 protein. assays were done in 5 replications (in five leaves from different plants of each object). antioxidative system during hardening of festulolium 127 128 e. pociecha, a. plazek, z. zwierzykowski antioxidative system during hardening of festulolium 129 protein determination protein content was determined according to the method described by bradford (1976) using the bio-rad protein assay (munich, germany) with bovine serum albumin as a calibration standard. statistical analysis the obtained results were statistically analysed independently by analyses of variance (anova/manova) with statistica version 6.1. software. the values demonstrated in graphs are the means ± se (standard error). results cell membrane permeability and frost tolerance (lt 50 coefficient) prehardening at 12°c significantly influenced cell membrane permeability in all studied genotypes (fig. 1). in all cases prehardening caused significant increase in ion leakage from leaf cells. the highest membrane permeability after prehardening was observed in leaf cells of genotype 768. other genotypes demonstrated at this time the same level of ion leakage. after hardening membrane permeability was lower than initially found in genotypes 715, 716 and 729. in other genotypes electrolyte leakage declined to the level noted before prehardening. frost tolerance evaluated by mean of lt 50 test after prehardening increased in genotypes 561, 621, 715 and 729, while in genotypes 716 and 768 temperature of 12°c did not effect on lt 50 coefficient values (fig. 2). in these two genotypes only hardening at 2°c increased frost tolerance. in the case of genotypes 561, 621 and 715 after hardening the lt 50 values were higher comparing to values after prehardening but lower than in the control plants (before prehardening). in the case of genotype 729 lt 50 values after prehardening at 12°c did not differ from the values after hardening at 2°c. on the basis of lt 50 test genotype 561 could be recognized as the most frost resistant. sod activity prehardening at 12°c activated superoxide dismutase in leaves of only two genotypes: nos. 561 and 768 (fig. 3). hardening at 2°c increased activity of that enzyme in genotypes 561, 621 and 716. in the case of genotypes nos. 715 and 729 prehardening and hardening temperature did not affect activity of sod. the correlation between lt 50 coefficient and sod activity was found in the case of only two genotypes: 561 (r = 0.72; p < 0.05) and 716 (r = 0.97; p < 0.05). cat activity fig. 1: the electrolyte leakage from leaves of control (18°c), prehardened (12°c) and hardened (2°c) plants of androgenic festulolium. mean values of 10 replicates ± se. fig. 2: the frost resistance expressed as the values of lt 50 (temperature causing 50% of damages) of control (18°c), prehardened (12°c) and hardened (2°c) plants of androgenic festulolium. mean values of 10 replicates ± se. statistical analysis showed that the cat activity in leaves of all investigated genotypes rapidly decreased after prehardening in comparison with the control, however activity of that enzyme after hardening increased, remaining still significantly lower than the control values (fig. 4). the highest cat activity was noted in the case of control plants genotypes nos. 621 and 768. control plants of other fig. 3: the activity of superoxide dismutase (sod) in leaves of control (18°c), prehardened (12°c) and hardened (2°c) plants of androgenic festu lolium. mean values of 5 replicates ± se. (abs-absorbance). fig. 4: the activity of catalase (cat) in leaves of control (18°c), prehardened (12°c) and hardened (2°c) plants of androgenic festulolium. mean values of 5 replicates ± se. 128 e. pociecha, a. plazek, z. zwierzykowski antioxidative system during hardening of festulolium 129 genotypes demonstrated cat activity on the same level. negative correlation between lt 50 coefficient and cat activity was found in the case of two genotypes: no. 561 (r = -0.96; p < 0.05) and no. 715 (r = -0.88; p < 0.05). px activity total peroxidase activity in leaves was significantly higher after 2 weeks of prehardening at 12°c in all studied genotypes regardless of their frost tolerance (fig. 5). after hardening for 3 weeks at 2°c an increase in activity of that enzymes in comparison with the prehardened plants was observed only in two genotypes: 561 and 715, however in the case of this first one px activity was significantly higher. discussion the hardening temperature stimulates several metabolic processes that could play a important role in the plant acclimation to cold and frost. physiological background of cold acclimation involves apart from a decrease in osmotic potential mainly rebuilding of membrane structure (yoshida and uemura, 1990; uemura and steponkus, 1994). according to these authors characteristic response of plant cells to cold stress is a change in cell membrane permeability. the destabilization of membranes causes strong cell dehydratation and synthesis of abscisic acid (aba) or ros generation. bravo et al. (1998) showed correlation between aba concentration and frost tolerance of barley. carruthers and melchior (1986) demonstrated that „fluidity“ of cell membranes influences activity of membrane enzymes and receptor proteins fixed in plasmalemma. gaudet et al. (1999) supposed that the resistance mechanism for winter factors includes common physiological processes induced during cold acclimation. processes that enhance frost tolerance may also increase resistance to snow mould pathogens, which are very significant factors deciding about winter hardiness of overwintering crops. in all festulolium genotypes studied after prehardening at 12°c cell membrane permeability significantly increased and then after hardening at 2°c it returned to the control values. it is worth to add that prehardening caused the greatest ion efflux from cells, while the following temperature of 2°c did not cause membrane injuries. this result indicates the very positive role of prehardening in 12°c for acclimation to frost. the most favourable membrane rebuilding preventing ion leakage from cells during hardening was noted in plants of genotypes nos. 715, 716 and 729, so in those genotypes, which were not recognized as the most frost resistant. płażek and żur (2003) showed that cell membrane permeability in barley, meadow fescue and winter rape hardened at 5°c for 6 weeks was specific for each plant species and may change relating to time of hardening. generally, initially ion leakage from cells of these species increased, and after 6 weeks decreased significantly. in four androgenic festulolium genotypes studied prehardening increased their frost tolerance expressed as lt 50 coefficient. after following hardening the lt 50 values increased, but they were still lower than before prehardening. in two studied genotypes nos. 716 and 768 temperature of 12°c did not cause changes in frost tolerance. only at 2°c these genotypes demonstrated an increase of frost tolerance. this result could indicate a positive effect of 12°c-treatment for processes influencing resistance to frost, for example rebuilding of cell membranes and synthesis of specific proteins protecting plasmalemma and cytoplasm before ice-caused injuries, which appear during frost temperatures. similar results were obtained by rapacz (1998), which showed the positive effect of prehardening at 15°c to frost acclimation of winter rape. hardening at 2°c may stimulate ros accumulation and activation or inhibition of some antioxidant enzymes. according to some authors oxidative stress usually is connected with decrease in activity of antioxidants (scebba et al., 1999; shim et al., 2003). at prehardening temperature of 12°c scebba et al. (1999) found a decrease in sod activity, an increase in guaiacol peroxidase and no changes in cat activity, however the same authors noted an increase in activity of all antioxidants after freezing at -4°c. shim et al. (2003) reported that in rice seedlings subjected to 8°c, cat activity was reduced by low temperature and this decrease was much sever in the low-temperaturesensitive cultivar comparing to the tolerant one. oidaira et al. (2000) observed in rice seedlings exposed to low temperature a transient increase in apx activity and slow increase in sod activity. in contrast, the cat activity was not significantly affected. inhibition of cat at cold condition was observed also in maize seedling (leipner et al., 1999; pastori et al., 2000), and in meadow fescue, barley and winter rape (płażek and żur, 2003). results obtained by zhao and blumwald (1998) suggested that enzymes activated during cold acclimation, apx activity all studied genotypes demonstrated the same level of apx activity in control plants (fig. 6). the temperature of prehardening (12°c) resulted in rapid apx activity in all investigated genotypes in comparison with the control. after two weeks of hardening at 2°c also in all cases, significant decrease of that enzyme activity was observed. however, leaves of hardened plants of genotypes nos. 561, 715, 729 and 768 showed still considerably higher apx activity than the control samples. the correlation between lt 50 coefficient was found in the case of two genotypes: 561 (r = 0.95; p < 0.05) and 715 (r = 0.97; p < 0.05). fig. 6: the activity of ascorbate peroxidase (apx) in leaves of control (18°c), prehardened (12°c) and hardened (2°c) plants of androgenic festu lolium. mean values of 5 replicates ± se. (asa – ascorbate acid). fig. 5: the activity of peroxidase (px) in leaves of control (18°c), prehardened (12°c) and hardened (2°c) plants of androgenic festulolium. mean values of 5 replicates ± se. (∆ abs – difference in absorbance per min per g of protein). 130 e. pociecha, a. plazek, z. zwierzykowski antioxidative system during hardening of festulolium 131 involving in the ascorbic-glutathione cycle play a protective role against ros generated in jack pine seedlings after exposure to freezing temperatures. these authors found no changes in apx activity in roots of seedlings after 1 and 2 week-growth at 5°c but it increases after 4 week-cold. kuk et al. (2003) noted significant activation of cat and apx in cold treated rice leaves, while sod, cat, apx and glutathione reductase in roots. they concluded that increased activity of antioxidants in roots was more important for cold tolerance than the increase of their activity in shoots. results obtained by seppänen and fagerstedt (2000) suggested that although sod activity in potato hybrids may contribute to their freezing tolerance and acclimation capacity, cold-induced acclimation capacity was not related to level of that enzyme activity. according to jouve et al. (2000) the temporary rapid increase of sod after 2 days at 10°c in poplar could be due to chilling. in four androgenic festulolium genotypes studied after cold hardening activity of sod increased, while in two genotypes it remained unchanged. simultaneously, hardening caused the decrease in cat activity in all investigated genotypes. apx in the hardened plants was higher than in the control ones of four genotypes and in the others it achieved the control values. obtained data partly agreed with results of kubo et al. (1999). according to these authors chilling temperature in arabidopsis thaliana increased activity of apx, whereas it decreased cat activity and did not influence sod activity. according to asada (1992) apx is the major scavenger of hydrogen peroxide in plant cells, and its activity increases in response to various environmental stressors. ascorbate-glutathione cycle is present in almost all cellular compartments (chloroplasts, cytosol, mitochondria, peroxisome and apoplast) suggesting its crucial role in controlling the ros level. according to another author (mitller, 2002) apx and cat belong to different classes of scavengers. apx is probably responsible for the fine modulation (activity of µm range), while cat (activity of mm range) could be responsible for scavenging excess of ros during stress. it is worth to add, that cat showed its activity mainly in peroxisomes and is closely associated with photosynthesis intensity. at 2°c this process is strong inhibited. in the presented work hardening caused an increase in px activity of all genotypes. this phenomenon could be explained by involving of apoplastic peroxidases in lignification process in cell walls. obtained results indicate that activity of sod converting superoxide to hydrogen peroxide was associated with activity of total peroxidases, for which h 2 o 2 is a substrate. initially sod activity was also followed by apx activity, however after hardening at 2°c its activity significantly decreased. this result could be explained by the domination of px activity in cell walls in lignification processes over apx one. concluding, the investigation indicate that studied androgenic genotypes were not diverse in frost resistance. moreover, although the studied plant material was derived from festuca pratensis × lolium multiflorum amphidiploid hybrid (four genotypes derived from the f 1 hybrids, and two genotypes from the festulolium cultivar ‘rakopan’), the origin of these plants did not influence studied 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(eds.), the plasma membrane. structure, function and molecular biology, 239-319. springer-verlag, berlin. zhao, s., blumwald, e., 1998: changes in oxidation-reduction state and antioxidant enzymes in the roots of jack pine seedlings during cold acclimation. physiol. plant. 104, 134-142. zwierzykowski, z., lukaszewski, a.j., naganowska, b., leśniewska, a., 1999: the pattern of homoeologous recombination in triploid hybrids of lolium multiflorum with festuca pratensis. genome 42, 720-726. zwierzykowski, z., ponitka, a., ślusarkiewicz-jarzina, a., zwierzykowska, e., leśniewska-bocianowska, a., 2001: androgenesis in amphidiploid f 1 hybrids and cultivars of festulolium. zeszyty problemowe post. nauk roln. 474, 47-54 (in polish with english summary). adress of the authors: dr. ewa pociecha (corresponding author), prof. agnieszka płażek, agricultural university of cracow, faculty of agriculture and economics, department of plant physiology, podłużna 3, 30-239 kraków, poland prof. zbigniew zwierzykowski, institute of plant genetics, polish academy of sciences, strzeszyńska 34, 60-479 poznań, poland journal of applied botany and food quality 89, 11 20 (2016), doi:10.5073/jabfq.2016.089.002 school of life sciences weihenstephan, technische universität münchen 1lehrstuhl für phytopathologie, freising, germany 2professur für obstbau, freising, germany 3kompetenzzentrum obstbau bodensee, bavendorf, germany phenolic compounds in juices of apple cultivars and their relation to antioxidant activity harald schempp1, sylvia christof1, 2, ulrich mayr3, dieter treutter2, * (received september 17, 2015) * corresponding author summary apples and their juices are important sources of phenolic compounds in human diet. using the same juice processing method, we compared phenolic composition as well as antioxidant activity of juices from various table apple cultivars, grown at the same location. antioxidant activity of apple juices was estimated by application of two assays, abts cation radical decolorization and by scavenging of reactive oxygen species (ros) generated by xanthine/xanthine oxidase (o2·-, h2o2, oh·; superoxide assisted fenton reaction), as measured by inhibition of ethylene release from kmb (α-keto-γ-(methylthio)butyric acid). the apple juices differed in phenolic content and antioxidant activity, where cultivar differences were more relevant than environmental factors. furthermore, to improve antioxidant performance in the xod-test, a juice low in phenol content was supplemented with the appropriate amounts of phenols to the level of the best juice indicating that these phenolics contribute to the antioxidant activity of the apple juice. in ac cordance to literature, the phenolic compounds re presenting the main anti oxidant activity in the apple juices comprise flavan-3-ols and chlorogenic acid. abbreviations: kmb, α-keto-γ-methiol-butyric acid; abts, azobis-benzthiazolinosulfonate; ros, reactive oxygen species; oh·, oh radical; o2·, superoxide anion radical; h2o2, hydrogen peroxide; x, xanthine; xod, xanthine oxidase; apple cultivars: see tab. 1. introduction beside their nutritional value, apples and apple juices promote also health benefits due to their “bioactive” components acting as antioxidants. vitamins and phenolics represent the predominant compounds with antioxidant activity in apples (proteggente et al., 2002; paganga et al., 1999; bremner, 2000; francini and sebastiani, 2013). polyphenols of apple were also shown to prevent cardio vascular diseases (koutsos et al., 2015) and they exhibit various physiological functions (akazome, 2004; hofseth and matesic, 2011). bitsch et al. (2001) proposed apple juice to be suitable as functional food because of its influence on plasmatic antioxidant capacity. a study on pigs could show that a diet supplemented with apples decreased oxidative stress by decreasing malondialdehyde formation in the body and by decreasing dna damage in the blood cells (pajk et al., 2006). phenolics, particularly flavan-3-ols, also play a role in resistance of cultivars against the fungus venturia inaequalis causing the scab disease of apple (mayr et al., 1995; treutter et al., 1990). susceptible and resistant cultivars differ in the flavan-3ol content of their leaves (treutter et al., 1990), whereas there was no significant difference in total phenolics as measured by the folintest (picinelli et al., 1995). the aim of this study was to determine the concentrations of diverse phenolic compounds in scab resistant and susceptible apple cultivars, cultivated at the same location but harvested in two years. thus, the variation in phenolic content and antioxidant activity of apples, respectively of their juices, harvested within one year, as well as the influence of environmental conditions, comparing the results of the two years. in the lab, juices of these apples were prepared using the same method. the juices were compared for content of phenolics (hplc) and for antioxidant activity in two selected test systems. the contribution of single phenolic compounds to the antioxidant capacity was estimated with special respect to procyanidins. further more, a low level juice was supplemented with lacking phenolic compounds in order to improve the antioxidant potential. the antioxidant role of polymeric procyanidins was studied after preparation of a polymeric fraction. material and methods apple fruits were grown in the field of the “kompetenz-zentrum obstbau bavendorf”, south germany, and were harvested in autumn of the years 2001 and 2002. the weather conditions of the two years can be described as follows: mean temperature (2001: 9.1 °c; 2002: 9.7 °c), rainfall (2001: 1081 mm; 2002: 1187 mm); sun hours (2001: 1771 h; 2002: 1780 h). in spring 2001 the temperatures were higher than in 2002. the cultivars were divided in two groups: scab susceptible and scab resistant ones. the harvest dates and fruit ripeness parameters are given in tab. 1. apple juices were prepared from each 10 fully ripe and healthy fruits on laboratory scale using a mixer. the juices were frozen and stored at -20 °c. before use in 2003, aliquot amounts of methanol (1 ml/g ice) were added to the frozen juices to prevent enzymatic oxidation during thawing. the diluted juices were homogenised in a cooled (6 °c) ultra sound water bath. the homogenised extracts (1.5 ml) were concentrated to dryness, redissolved in small amounts of methanol (150 μl), centrifuged and then used for hplc analysis of phenolic compounds. quantification of phenolic compounds was performed by hplc with post-column derivatisation using the method described by treutter et al. (1989). hplc determination of phenolics the hplc equipment used consists of an autosampler (gilsonabimed modell 231), of two pumps (kontron modell 422), and a diode array detector (bio tek kontron 540). for post column derivatisation a further analytical hplc pump (gynkotek modell 300 c) and a vis-detector (640 nm, kontron detektor 432) were used. the phenolic compounds were separated using chromatographic conditions, which had been optimized for apples, according to mayr et al. (1995) on a column (250 × 4 mm i.d.) prepacked with hypersil ods, 3 μm particle size, following a stepwise gradient using mixtures of 5 % formic acid (a) and methanol gradient grade (b) with a flow rate of 0.5 ml/min. the gradient profile used is: 0-5 min, isocratic, 5 % b in a; 5-15 min, 5-10 % b in a; 15-30 min, isocratic, 10 % b in a, 30-50 min, 10-15 % b in a, 50-70 min, isocratic, 15 % b in a, 70-85 min, 15-20 % b in a, 85-95 min, isocratic, 20 % b in a, 95-110 min, 12 harald schempp, sylvia christof, ulrich mayr, dieter treutter tab. 1: properties of apple fruits harvested in 2001 / 2002 cultivar fruit flesh starch soluble solids acid juice diameter firmness yield abbreharvest [mm] [kg/cm²] [1-10] [% brix] [°oe] [mval] [g/l] [%] viations date scab susceptible cultivars summerred sr1 22.08.01 77 6.8 4.5 12.4 53 16.04 10.75 63.1 sr2 13.08.02 71 6.8 5.4 11.2 48 15.09 10.11 69.2 alkmene alk1 27.08.01 79 7.8 2.0 12.8 54 13.55 9.08 60.6 alk2 23.08.02 73 7.6 2.8 12.8 54 13.25 8.88 67.6 elstar elshof ee1 08.09.01 83 7.3 2.9 13.2 56 16.53 11.08 66.7 ee2 04.09.02 73 6.4 1.8 10.8 46 11.4 7.64 56.0 gala galaxy gg1 08.09.01 78 9.9 2.6 12.6 54 9.18 6.15 63.4 gg2 05.09.02 73 8.0 4.0 10.9 46 5.79 3.88 52.0 jonagored jg1 02.10.01 84 8.0 6.8 15.4 65 13.86 9.29 72.1 jg2 18.09.02 85 6.4 7.9 11.7 50 7.27 4.87 52.0 braeburn wellburn bw1 16.10.01 81 10.9 4.0 13.4 57 17.22 11.54 62.8 bw2 22.10.02 81 9.5 5.0 12.1 51 13.38 8.96 73.4 scab resistant cultivars santana san1 26.08.01 78 7.5 1.2 12.3 52 18.2 12.19 68.9 san2 22.08.02 79 7.1 2.0 11.9 51 16.9 11.32 52.9 gerlinde ger1 03.09.01 72 7.1 6.8 14.8 63 15.2 10.18 70.2 ger2 29.08.02 70 7.2 9.5 11.4 48 11 7.37 56.9 rebella reb1 14.09.01 70 7.4 4.3 13.7 58 14.85 9.95 59.9 reb2 04.09.02 73 6.4 7.8 12.6 54 12.72 8.52 57.0 rosana ros1 20.09.01 84 6.7 2.3 13.2 56 19.39 12.99 75.7 ros2 17.09.02 78 6.8 3.2 11.1 47 14.33 9.60 66.0 ariwa ari1 20.09.01 70 8.4 1.9 12.1 51 13.13 8.80 70.1 ari2 23.09.02 75 9.5 5.8 13.5 57 12.75 8.54 66.9 rewena rew1 30.09.01 71 6.9 3.6 12.5 53 16.89 11.32 66.0 rew2 07.10.02 78 7.4 7.0 12.7 54 14.78 9.90 67.0 topaz top1 03.10.01 78 7.5 5.6 12.6 54 18.13 12.15 75.0 top2 24.09.02 78 8.2 2.6 12.8 54 16.15 10.82 64.0 florina flo1 08.10.01 77 8.2 8.3 13.3 57 11.5 7.71 70.4 flo2 09.10.02 81 7.3 8.3 12.3 52 9.2 6.16 53.0 baujade bau1 07.11.01 79 10.5 7 14.3 61 17.03 11.41 68.2 bau2 24.10.02 76 11.2 7 13.4 57 13.76 9.22 67.8 goldrush gold1 07.11.01 77 9.5 5.8 15.7 67 15.28 10.24 73.1 gold2 22.10.02 74 10.1 7 14.3 61 13.85 9.28 71.6 20-25 % b in a, 110-140 min, 25-30 % b in a, 140-160 min, 30-40 % b in a, 160-175 min, 40-50 % b in a, 175-190 min, 50-90 % b in a. phenolic acids and flavonols were detected at 280 nm whereas the flavan-3-ols were estimated at 640 nm (treutter, 1989; treutter et al., 1994) after post column derivatisation with p-dimethylaminocinnamic aldehyde (dmaca). 6-methoxyflavone was used as internal standard for quantitative analyses. reproducibility of the method including extraction and quantification by hplc is above 90 %. the individual compounds were identified by retention times and their uv-absorbance spectra via diode array detection and by comparison with standards. these standards were either commercially available from roth and sigma (catechin, epicatechin, chlorogenic acid, phloridzin, rutin) or previously isolated from apple and ser vice tree: procyanidins b1, b2, b5, e-b5, c1, phloretin derivatives, hydroxycinnamic acids, quercetin glycosides (mayr et al., 1995; treutter et al., 1994, ölschläger et al., 2004). fractionation on sephadex-lh20 for separation of polymeric proanthocyanidins from oligomeric proanthocyanidins and low molecular weight compounds (hydroxycinnamic acids, dihydrochalcones, flavonols) a sepha dex-lh 20 co phenolic antioxidants of apple juices 13 lumn (0.7 cm diameter × 30 cm length) was used. 1.5 ml apple juice was subjected and eluted with methanol. 3 fractions were collected with 10 ml volume each. fractions were analysed by hplc. polymeric proanthocyanidins were estimated as cyanidin equivalents by boiling aliquots after adding a mixture of butanol and concentrated hcl (4:1, v/v) for 20 min. abts-radical decolorization assay the assay used here is modified, but the principle was described first by miller et al. (1993). a number of variations of this assay exist (miller et al., 1997; re et al., 1999; labrinea et al., 2004; arnao et al., 2001; cano et al., 2000; arts et al., 2004 a, b; kim et al., 2004; fischer et al., 2005). abts radicals are quite stable in the absence of a reductant, if the concentration of abts is in excess to the abts cation radical (cano et al., 1998, 2000). interestingly, trolox performs in this assay ideally concerning dose response effects (velocity and extent of abts·+ decolorization). in contrast to most phenols tested, the synthetic trolox (a water soluble vitamin e derivative) showed a straight linear correlation of decreasing abts radicals over the whole range. therefore, trolox is commonly used as a standard antioxidant to calibrate the assay and antioxidant activity is expressed in teac values (teac = trolox equi valent antioxidant concentration). the generation of abts radicals from abts, however, differ between the various assays, using either acidic manganese dioxide or potassium persulfate, or the radical generator abap (2,2´-azinobis(2-amidinopropane) di hydro chloride), or hydrogen peroxide in the presence of heme proteins (pseudo peroxidase, e.g. myoglobin). in this study abts-radicals were produced by reaction of myoglobin with hydrogen peroxide in the presence of abts at ph 6.0 and 37 °c (10 mm phosphate ph 6.0, abts 1 mm, myoglobin 2.5 μm, h2o2 500 μm). usually 5 ml aliquots were prepared and after a reaction time of 60 min the dark green solution was stored overnight at 4 °c in a refrigerator. before use, the concentration of abts-radicals was checked after dilution (λ734nm = 15 000 m-1 cm-1). in general, a 1:10 dilution in 50 mm phos phate ph 7.4 results in an absorbance of about 1.0 at λ = 734 nm. this absorbance was used as startpoint for the decolourisation assays (67 μm abts·+). a typical assay contains in 1.0 ml: 50 mm phosphate buffer ph 7.4, abts·+ 67 μm, and 20 μl of the test solution (20 μl). decolorization of the assay was measured exactly 5 minutes after starting the reaction with the test solution. for calculation of teac values, the fits in the linear range of the received dose-response plots of the different apple juices were compared to the linear equation of trolox. kmb oxidation to ethylene by ros formed via xanthine(x)/ xanthine oxidase (xod) the x/xod system is applied to produce free radicals (o2·-, ·oh) beside the main products h2o2 and uric acid. about 20 % of the electrons from xanthine are used by xod for the monovalent reduction of molecular oxygen (fridovich, 1970). under our conditions ·oh radicals are indirect products formed by the side reaction of fe2+ions with h2o2 (fenton reaction). o2·is necessary to reduce fe3+ions into fe2+-ions. trace amounts of fe3+-ions are present in the buffer solution as well as in the xod suspension, which also contains edta, an enhancer of the fenton reaction. ·oh-radicals oxidize α-keto-γ-methiol-butyric acid (kmb) under formation of ethylene, similar as described for methional (beauchamp and fridovich, 1970). under our conditions, xod converted the given xanthine into uric acid within 20-25 minutes. immediately after starting the reaction by addition of xod, the probes were gastight sealed with rubber stops and incubated for 30 min at 37 °c in a gently shaking waterbath in the dark. after that, aliquots of 1 ml of the headspace gas of the reaction tubes were withdrawn with gastight syringes and stored up to gc-quanti fication of ethylene, sticked into a rubber mat. a varian cx3300 gas chromatograph was used, equipped with a varian al2o3 column (60 cm × 1/8 inch). a typical assay contains in 2.0 ml: 100 mm phosphate buffer ph 7.4, 1 mm kmb, 500 μm xanthine, xanthine oxidase 0.04 units/ml, various diluted apple juice 100 μl/ml (solved in water – free of methanol). activity of xod was quantified by hplc analysis of xanthine and uric acid using a waters hplc, consisting of a 600-pump, 717-autosampler and 996-pda detector, column merck cartridge 125 × 4 mm, lichrospher® 60 rp-select b with precolumn 4 × 4 mm, temperature 35 °c, eluent nah2po4 50 mm isocratic for 8 min, and methanol (grad. grade) for the washing step of the column after each injection (total time, 20 min including re-equilibration to nah2po4 50 mm); flow rate 1 ml/min, sample volume 20 μl. retention times: xanthine 3.0 min, uric acid 4.0 min. for xod-activity deter mination the xod-reaction was performed in the presence or absence of the investigated compound. the enzyme reaction was started with xod and stopped after exactly 10 min by addition of 50 μl hcl10m. influence of the extracts on xod-activity was expressed by comparing amounts of uric acid produced as well as xanthine consumed with the basic reaction in the absence of antioxidants. an assay contains in a final volume of 1.00 ml: 100 mm phosphate buffer ph 7.4, 500 μm xanthine, xanthine oxidase 0.04 units/ml, and amounts of tested sample (apple juice or standard compound). results phenolic composition of cultivars phenolic profiles of the juices prepared from various apple cultivars did not differ in quality. the main hydroxycinnamic acid (hca) was identified as n-chlorogenic acid (5-caffeoylquinic acid). several minor peaks with similar uv-absorbance spectra were also calculated as chlorogenic acid and combined together into one group of hydroxycinnamic acids (hca). their concen trations (tab. 2) ranged from 94.6 mg/l (rem2) to 492.8 mg/l (eco1). an exceptionally high level was found in baujade (bau1) with 747.0 mg/l. more than 50 % of the hydroxycinammic acids are represented by chlorogenic acid which coincides with literature (treutter, 2001; kahle et al., 2005). only the cultivars alkmene, elstar elshof, remura and florina produced less. since the separation of flavanols and hca is not sufficient, the total hca level may be somewhat over estimated by flavanol impurities. the flavanols, however, were quantified more precisely by using selective post column derivatisation with dmaca and detection at 640 nm. this resulted in pure peaks which were identified as catechin, epicatechin, and the procyanidins b1, b2, b5 and c1. these known procyanidins together with some unidentified peaks at 640 nm were calculated as procyanidin b2. the most prominent flavanols in the juices were the monomeric epicatechin and its dimeric and trimeric derivatives procyanidin b2 and pro cyanidin c1. the total flavanol concentrations (tab. 2) covered a broad range from 0.3 mg/l (sr2) to 337.6 mg/l (rub2), again with the extreme 955.8 mg/l in baujade (bau1). the latter value fits into the group of cider apples which often contain much more procyanidins (guyot et al., 2003). the majority of our juices have flavanol concen trations as reported in literature from commercial juices and for table apple juices (treutter, 2001; kahle et al., 2005). it may be noted that those cultivars with total flavanols below 10 mg/l, such as summerred, santana, gerlinde, regunda, ecolette, exhibited relatively high hydroxy cinnamic acid concentrations. by that, the total phenolics are not so much reduced. those peaks showing the typical uv-absorbance of phloridzin were calculated as phloridzin and summed up to give the total dihydrochalcone value. all flavonols were calculated as rutin. the concentration of these minor components were between 4.6 mg/l (alk1) 14 harald schempp, sylvia christof, ulrich mayr, dieter treutter tab. 2: phenol content (mg/l) of apple juices (apples harvested in 2001 / 2002) estimated by hplc analysis cultivar abbrev. chloro total total total catechin epiproproproprototal total genic hydroxydhydroflavo nols catechin cyanidin cyanidin cyanidin cyanidin flavanols phenolics acid cinnamic chalcones b1 b2 b5 c1 acids a) scab susceptible cultivars summerred sr 1 187.4 256.9 15.4 6.4 0.04 0.12 0.08 0.03 0.01 0.04 0.8 279.4 sr 2 106.0 161.0 10.8 2.7 0.01 0.00 0.00 0.01 0.01 0.03 0.3 174.7 alkmene alk 1 43.3 136.2 4.6 5.5 3.1 40.2 8.5 64.4 8.6 45.4 197.8 344.1 alk 2 36.9 138.3 9.5 7.5 3.0 32.2 8.7 55.4 4.4 42.1 174.1 329.4 elstar elshof ee 1 88.1 248.1 19.3 8.6 7.0 61.8 16.2 74.6 5.5 46.6 240.0 515.9 ee 2 35.8 110.6 8.5 6.0 2.7 22.0 6.5 25.8 2.3 17.7 88.8 213.9 gala galaxy gg 1 189.6 317.6 11.9 7.4 6.2 38.8 17.1 42.7 2.6 32.9 157.8 494.6 gg 2 195.0 336.6 49.7 8.3 5.9 29.6 10.5 17.0 2.0 13.1 85.9 480.4 jonagored jg 1 216.0 360.9 23.2 15.0 2.4 36.3 9.9 72.3 5.0 52.1 220.2 619.3 jg 2 109.6 172.8 8.7 10.5 0.9 12.3 3.8 24.7 1.9 15.0 71.5 263.6 braeburn wellburn bw 1 199.3 386.2 13.3 14.1 6.2 53.7 14.6 63.6 5.1 48.7 223.6 637.3 bw 2 82.4 227.0 7.7 9.9 1.5 10.1 1.3 6.1 0.8 3.5 25.7 270.2 b) scab resistant cultivars santana san 1 154.8 260.4 13.4 11.9 0.01 0.03 0.01 0.01 0.01 0.28 0.6 286.3 san 2 158.3 249.4 15.4 8.2 0.09 0.01 0.03 1.69 0.04 0.02 2.1 275.2 ahra ahr 1 268.3 337.4 20.2 11.0 3.5 39.1 8.7 53.6 3.8 20.9 156.8 525.4 ahr 2 196.4 297.4 20.5 17.3 8.9 62.7 18.6 67.4 4.5 30.9 215.2 550.4 gerlinde ger 1 241.3 354.4 14.7 14.0 0.06 0.55 0.05 0.31 0.08 0.56 2.4 385.5 ger 2 178.9 262.2 21.2 8.2 0.02 0.16 0.01 0.04 0.02 0.02 0.6 292.2 rubinola rub 2 297.7 452.2 15.1 10.8 16.0 89.8 31.3 106.6 6.2 53.3 337.6 815.6 rub 1 352.0 481.4 20.1 15.7 4.6 55.9 18.8 100.3 4.1 46.6 265.5 782.8 remura rem 1 51.4 151.4 6.5 5.6 7.1 52.0 16.9 87.8 5.5 46.9 246.5 410.0 rem 2 31.6 94.6 4.8 5.4 4.1 29.8 6.8 35.4 1.7 18.1 109.4 214.2 rebella reb 1 74.0 154.0 18.8 21.9 0.1 3.3 0.6 7.5 0.6 7.0 24.3 219.0 reb 2 58.3 112.2 12.9 16.8 0.1 2.4 0.5 2.7 0.3 2.1 9.7 151.7 vesna ves 1 83.5 199.8 9.5 12.5 6.9 44.4 12.8 60.8 4.0 27.1 172.5 394.3 ves 2 166.0 268.3 10.9 11.9 7.2 46.1 15.4 65.2 3.5 34.9 194.3 485.2 reanda rea 1 240.3 354.1 12.0 10.0 4.8 48.2 10.0 57.3 3.6 26.1 167.8 544.0 rea 2 174.6 254.7 8.0 8.6 3.7 26.5 4.7 24.1 2.2 9.7 77.5 348.8 rosana ros 1 73.9 187.3 11.0 18.1 6.4 55.5 19.7 84.7 6.8 56.4 265.2 481.6 ros 2 48.4 105.3 16.9 9.4 2.1 16.7 4.2 16.0 1.5 9.4 55.7 187.2 ariwa ari 1 154.4 267.8 8.6 6.1 2.6 35.8 11.2 61.3 4.7 35.0 188.8 471.3 ari 2 212.8 314.2 10.7 10.3 1.6 19.3 4.1 26.8 3.1 21.8 91.5 426.7 regunde regu 1 364.4 471.9 20.4 7.2 0.06 0.34 0.08 0.61 0.03 0.07 1.4 500.8 regu 2 199.9 280.8 12.5 5.9 0.06 0.94 0.06 0.32 0.02 0.05 1.6 300.8 rewena rew 1 223.2 330.5 27.7 8.5 6.6 57.4 13.9 80.0 6.3 53.2 251.4 618.1 rew 2 157.3 254.6 8.2 11.2 5.0 40.8 8.0 47.3 4.3 37.1 164.6 438.5 topaz top 1 90.5 193.3 8.0 6.7 5.0 48.8 12.8 54.1 4.0 24.3 168.1 376.1 top 2 95.7 178.0 5.2 12.2 2.3 29.9 6.9 39.1 3.3 24.7 118.3 313.8 ecolette eco 1 329.0 492.8 19.9 10.8 0.1 2.3 0.10 1.9 0.12 0.14 5.3 528.8 eco 2 195.8 283.9 8.8 10.1 0.1 1.4 0.11 0.98 0.15 0.30 3.2 306.0 florina flo 1 98.1 297.2 13.1 8.0 5.6 51.7 15.0 69.0 5.4 49.3 221.4 539.6 flo 2 64.4 214.4 11.4 10.4 9.8 77.7 22.5 87.9 7.0 68.6 325.0 561.3 phenolic antioxidants of apple juices 15 and 49.7 mg/l (gg2) for dihydrochalcones and between 2.7 mg/l (sr2) and 27.9 mg/l (bau1) for flavonols, according to literature (treutter, 2001; kahle et al., 2005). marked differences in phenolic quantity were found between the cultivars and between the two harvest years (tab. 2). in general, the total phenol concentrations tend to be higher in 2001 than compared to 2002. this was the case in 17 out of the 26 cultivars tested. five cultivars even showed in 2002 less than 50 % of the concentration in 2001. these were elstar elshof, jonagored, braeburn wellburn, rosana and baujade. in contrast to these environmentally sensitive cultivars, 9 varieties were not affected by the season and showed only a variation within a range of 10 %: alkmene, gala galaxy, santana, ahra, rubinola, ariwa, florina, enterprise, and goldstar. antioxidant capacity abts-radical decolourisation assay for tests concerning the scavenging activity towards abts-radicals, six apple juices were selected exhibiting extreme differences in their total phenol content. the order of antioxidant activity (fig. 1) was bau1 > bau2 > jg1 > ee2 > bw2 > san1. this does not exactly represent the descending total phenolic patterns, but it fits to the descending order of flavanol amounts of the respective juices (tab. 2). the calcu lated teac values (mmol/l trolox) were listed in tab. 3. in order to identify the contribution of individual phenolics to the antioxidant activity, several standards were tested. their corresponding teac values are listed in tab. 4. this supports the assumption that flavan-3-ols play a predominant role as antioxidants of the apple juices tested. ethylene formation from kmb by x/xod apple juices are potent inhibitors of ethene formation from kmb in the x/xod test. from a first approach with dilution series of 4 selected juices (fig. 2), an assay concentration of 0.27 % (v/v) was deduced for further screening of the juices from all cultivars and harvest years (tab. 1). the corresponding inhibition of ethene formation (in %) from kmb is shown in fig. 3. the antioxidant activity correlates best with the amount of total flavanols (r2 = 0.64) and procyanidins (r2 = 0.65), followed by hydroxycinnamic acids (r2 = 0.57) and total phenolics (r2 = 0.51). none of the apple juices showed in the examined concentration an inhibition of the enzyme xanthine oxidase, as measured by hplc of the produced amounts of uric acid and consumed amounts of xanthine (data not shown), compared to the control without apple juice. a series of pure standards and defined phenolic fractions were also tested in the x/xod assay. their ic50-values were cultivar abbrev. chloro total total total catechin epiproproproprototal total genic hydroxydhydroflavo nols catechin cyanidin cyanidin cyanidin cyanidin flavanols phenolics acid cinnamic chalcones b1 b2 b5 c1 acids regine regi 1 7.5 395.1 22.3 12.1 4.4 53.2 10.2 79.1 4.7 35.3 212.7 642.2 regi 2 216.3 337.7 16.0 15.6 4.1 46.6 9.7 66.6 3.5 33.5 188.0 557.3 enterprise ent 1 268.8 387.9 12.2 6.9 10.1 29.7 13.1 30.4 1.8 13.1 105.8 512.9 ent 2 277.2 375.6 8.5 8.1 9.1 25.4 12.5 24.6 1.5 15.3 98.4 490.6 goldstar gost 1 91.2 191.5 10.0 15.8 4.9 32.0 18.2 63.9 3.3 29.9 173.0 390.3 gost 2 90.1 180.7 9.0 17.1 4.6 33.1 21.9 81.4 5.6 41.0 222.1 428.9 baujade bau 1 445.6 747.0 23.0 27.9 19.1 186.4 45.9 285.5 24.5 230.0 955.8 1753.7 bau 2 279.2 450.2 24.1 19.9 5.3 57.9 15.9 99.2 8.0 73.5 317.4 811.6 goldrush gold 1 162.7 268.8 18.2 12.1 5.0 50.7 16.4 49.6 4.5 37.3 185.6 484.7 gold 2 134.8 242.8 14.2 15.3 2.9 24.3 5.6 17.1 2.4 10.5 68.8 341.2 fig. 1: extent of reaction of abts-cation radicals with diluted apple juices. 20 μl of the appropriate dilution (as indicated on the x-axis) of the apple juice was placed to 980 μl of buffered abts-radicals (67 μm). after mixing, remaining absorbance was measured exactly after 5 min of reaction time. tab. 3: teac values (abts radical de colour isation) of selected apple juices. apple juice teac (mmol / l) baujade 2001 13.3 baujade 2002 10.8 jonagored 2001 7.5 elstar elshof 2002 3.9 braeburn wellburn 2002 2.8 santana 2001 1.9 estimated (tab. 5) and show the following order: catechin < epicatechin < procyanidin c1 < a polymeric fraction isolated from sorbus domestica < procyanidin b2 < chlorogenic acid < rutin < mixture of c1, e4, b5, e-b5. the dihydrochalcone phloridzin did not show any inhibition. as for the abts system, the flavanols were found to be most effective. however, the correlation of the concentration of 16 harald schempp, sylvia christof, ulrich mayr, dieter treutter phenolic compounds in the juices and their corresponding antioxidant capacity is not really significant, showing only a tendency. this may be due to the non-linearity of the enzymatic test system in the range of concentrations used (fig. 4). another reason for the variation may be the contribution or interference of compounds, which were not quantified in the juices. to get a more detailed view of the contribution of phenolic compounds and their extent to the antioxidant capacity of apple juices, two approaches were performed. in the first experiment, a juice poor in phenolics was supplemented with standards. the second trial focused on the role of polymeric proanthocyanidins so far not detectable by the hplc system used. tab. 4: teac values (abts radical de colour isation) of selected standards. standard compound teac (mmol / l) catechin 3.9 rutin 3.6 procyanidin b2 3.3 procyanidin c1 2.0 chlorogenic acid 1.3 tab. 5: ic50 values (x / xod mediated ethene release from kmb) of apple juices and selected standards. apple juice ic50 (% v/v) regunde 2001 0.4 gerlinde 2002 0.6 enterprise 2001 0.3 baujade 2002 0.2 standard compound ic50 (μmol / l) catechin 4.5 epicatechin 8.0 procyanidin c1 10.0 procyanidin b2 15.0 chlorogenic acid 16.0 rutin 19.0 supplementation experiment the apple juices of elstar elshof 2002 (ee2) and of baujade 2002 (bau 2) differed markedly (app. factor 10) in amounts of chlorogenic acid and of flavanols, represen ted by catechin (tab. 6). in the x/xod test final concentrations of 0.27 % apple juice were used in the assay, which means that the difference between bau 2 and ee 2 was 2.93 μm for chlorogenic acid and 7.43 μm for flavanols, respectively. to elucidate whether these components are the relevant ones responsible for the inhibition of ethene formation from kmb in the x/xod test, the missing amounts of chlorogenic acid and/or the flavanol monomer catechin were added to ee2 (0.27 %) in the assay. fig. 5 illustrates the effects on the antioxidant performance in the x/xod assay of ee2 by adapting the chlorogenic acid or the flavanol level or both of ee2 to the level of bau2. supplementation with chlorogenic acid alone improved the antioxidant activity only marginally, whereas addition of catechin highly increased antioxidant action of ee2. surprisingly, chlorogenic acid was more active in combination with catechin. although the activity of bau1 could not be achieved completely by ee2 supplemented in this way, we can deduce from the fig. 2: dose response plots of of apple juices determined in the x / xod / kmb test. the higher the antioxidant action of the apple juice, the lower is the amount of liberated ethylene from kmb. in a final aqueous (phosphate buffered 100 mm, ph 7.4) reaction volume of 2.0 ml, xanthine 500 μm is converted into uric acid by 40 mu / ml xod in gas tight sealed reaction tubes (12 ml). 1 ml gas aliquots of their headspace were quantified by gc. ethylene is liberated from kmb via the superoxide assisted fenton reaction, producing oh radicals. in the basic reaction without apple juice the amount of formed ethene from kmb was approx. 7 ± 0.35 nmol (=100 %), accumulated in 30 min at 37 °c. experiment that the flavanols of the apple juices are mainly respon sible for the antioxidant activity in the x/xod test. however, it should be noted that the catechin supplement may somewhat overestimate the contribution of flavanols to the antioxidant capacity of the juice, since catechin has a higher effect than epi catechin and its derivatives (tab. 5) in this system. preparation and antioxidant activity of a polymeric fraction of pro antho cyanidins due to their diversity (eluting as a broad unspecific increase of the baseline) and weak uv-absorbing properties, polymeric proanthocyanidins are not sufficiently separated and detected by hplc (shoji et al., 2006). they react also only weakly with dmaca (treutter, 1989). butanol/hcl treatment in the heat (boiling for 20 min) greatly enhances photometric detection by conversion of the polymers into coloured anthocyanidins. the latter can be quantified by absorbance at 540 nm, expressed as cyanidin equivalents. we found some evidence that the difference between supplemented ee2 and bau1 may be due to polymeric pro antho cyanidins. to elucidate the contribution of polymeric procyanidins to antioxidant capacity, bau2 apple juice (we assumed to contain significant amounts of polymeric pro anthocyanidins) was separated into fractions by sephadex column chromato graphy. the obtained fractions where analyzed by hplc and by photometry after boiling in butanol/hcl. antioxidant activity of the fractions was also determined in the x/xod test. three fractions were received by sephadex column separation of bau2. fraction1, containing hcas and catechins, exerted approximately 90 % of the anti oxidant activity of the original apple juice bau2, as shown in fig. 6. oligomeric proanthocyanidins and catechins represent mainly fraction2 and showed lower but still significant antioxidant activity, followed by fraction3. however, no signals were detected by hplc analysis of fraction3, although it was positive in the butanol/ hcl assay for proanthocyanidins (tab. 7). so we can deduce from this experiment that fraction3 contains polymeric proanthocyanidins performing antioxidant activity. thus, polymeric proantho cyanidins certainly contribute partly to the antioxidant per formance of the apple juice in the x/xod test. phenolic antioxidants of apple juices 17 fig. 3: inhibition of ethene formation from kmb by apple juices (final concentration in the assay 0.27 % v/v) in the x / xod test (100 % ethene formation from x / xod / kmb: 8.5 ± 0.8 nmol). the samples are arranged in the order of increasing “total flavanol concentration”. the dotted line marks the trend of increasing antioxidant activity with r2 = 0.65. fig. 4 : dose response plots of standards determined in the x / xod / kmb test. conditions see captions of fig. 2. tab. 6: flavan-3-ol and chlorogenic acid content of apple juices from ee2 and bau2 apple variety in 100 % apple juice chlorogenic acid all flavanols, (mm) calculated as catechin (mm) bau2 1.27 3.28 ee2 0.10 0.31 fig. 6: three fractions of the apple juice “baujade 2002” (bau2), received by sephadex column chromatography, were analyzed in the x/xod/ kmb test. the antioxidant activity is plotted as inhibition of ethene formation from kmb (in % of the control reaction (100 %, producing 8.7 ± 0.4 nmol ethene) and compared to bau2 (0.27 %v/v final concen tration). fraction1mainly contained hcas and catechins, fraction2 oligomeric catechins and flavanols whereas fraction3 consisted of poly meric flavanols. the fractions were also analyzed by hplc and the butanol/hcl method for proanthocyanidins (tab. 7). fig. 5: baujade 2002 (bau2) and elstar elshof 2002 (ee2) were compared in the x/xod/kmb test (apple juice: 0.27 %v/v final concen tration) for their antioxidant activity. whether their difference in antioxidant activity could be related to missing amounts of chlorogenic acid and catechins of ee2 (see tab. 6) was checked by appropriate supplementation of ee2 with either chlorogenic acid or catechin or both. 18 harald schempp, sylvia christof, ulrich mayr, dieter treutter tion, both in amount and in antioxidant activity, followed by epicatechin and procyanidin b2. the dihydrochalcone phloretin and chlorogenic acid showed comparably weak activities. in another study (imeh et al., 2002), apple fruits from 31 varieties were harvested from the same growing area, the same age and the same stage of ripeness in order to compare vitamin c content, phenol content and their relation to the antioxidant activity (abts assay). they found that antioxidant activity correlated to total phenols better as measured by the folinciocalteu test (r2 = 0.5331) than with the phenol content quantified by hplc (r2 = 0.30). this is not surprising, because the folin test is by itself a kind of antioxidant test system, utilising the reduction potential of polyphenolics and other compounds in a chemical reaction. in our study we found the best correlation for the flavan-3-ol and procyanidin content (r2 = 0.65) of the investigated apple juices, concerning antioxidant activity in the xod test. total phenol content by hplc correlated to antioxidant activity only with r2 = 0.51. in a study on four apple cultivars van der sluis et al. (2001) found the highest antioxidant activity in the cultivar showing the highest flavonoid concentration. never theless, no general correlation could be found between flavonoid concentration and antioxidant capacity. in a further analysis of 9 apple cultivars (imeh et al., 2002), differences in their antioxidant acitivity were shown in the frap test (ferric reducing antioxidant power – this test is rather strange, because in vivo reduction of free ferric ions is correlated to oxidative stress. thus, metal ions are kept safely bound to proteins, and if free, the ferroxidase caeruloplasmin prevents fe2+ mediated fenton reactions). again, neither convincing relationships to total phenols (folin-ciocalteu assay) nor to free and conjugated phenol fractions could be established. this is consistent with our findings. however, our experiments clearly show that flavan-3-ols contributed most to the antioxidant potential of the tested apple juices, whereas chlorogenic acid, dominating quantitatively, did only play a subordinate role. this is contradictory to the proposal of miller and rice-evans (1997), who stated that chlorogenic acid and phloridzin are important antioxidants in apple juices. thus, the test system applied to measure antioxidant activity is of importance for the derived results. in this study, two model systems have been selected for the measurement of antioxidant activity of apple juices. on the one hand, the popular and simple teac assay (teac = trolox equivalent antioxidant concentration) was used, quantifying the decrease of preformed dark blue green abts cation radicals, induced by antioxidant solutions as scavengers and/or reducing agents. thereby abts radicals are converted into colourless products (abts and others). the advantage of the assay is the good differentiation between phenols, which is certainly based on the restricted reactivity of the abts radical, attributing it a weak oxidant. therefore, we applied on the other hand oxidation of xanthine (x) by xanthine oxidase (xod) as a source of reactive oxygen species (ros: superoxide, hydrogen peroxide, oh radical), which is of relevance in vivo. furthermore, the ability to decrease xod activity, an oxidative stress related enzyme, could also be checked in this system as a possible mode of antioxidant action. the number of adjacent oh-groups is one of the important determinants of antioxidant activity, as observed by bors and michel (2002). one critical structural constraint for optimal antioxidant potential is the presence of a catechol group (bors and michel, 2002). structure function relationships are not always working, as for example in the abts assay: p-coumaric acid (rice-evans et al., 1996), with only one phenolic oh-group, is a much stronger anti oxidant than caffeic acid (with the catechol group). moreover, activity of the caffeic catecholic group is enhanced by methoxylation (ferulic acid). ascorbic acid (enediol structure) is surely underestimated in the abts assay, performing like trolox with only one phenolic group. thus, besides the reduction potential, dimerization and disproportionation reactions of the oxidized antioxidants play a crucial role, superimposed by the stereochemical affinity of reductant (antioxidant) and free radical, tab. 7: proanthocyanidin (pac) content of bau2 and corresponding sephadex fractions, estimated as cyanidin equivalents by absorbance at 540 nm after boiling in butanol/hcl for 20 min compound pac (mmol/l) bau2 0.83 fraction 1 0.41 fraction 2 0.29 fraction 3 0.15 discussion apples are one of the major fruits frequently consumed in germany both as fresh or processed fruits. by this, apples represent a major source of phenolics in human nutrition. although it is commonly accepted that apples are good for health, the magic ingredient of apple has not be found so far. nevertheless, the biological impact of apple, similar to that of other fruits, may be largely due to the presence of anti oxidants, where phenolics are considered to be more important than vitamin c (eberhardt et al., 2000). in general, five major phenolic groups are found in various apple cultivars: hydroxy cinnamic acids, flavan-3-ols (monomeric catechins and procyanidins), anthocyanins, flavonols, and dihydro chalcones. the extraction of phenolic compounds by a domestic juicer, as reported in this study, is not exhaustive. therefore, the values do not show the absolute content of the fruits. however, the values obtained are in the range of those published for apple juices, as reviewed by treutter (2001). during juice making (pressing, centrifugation) the easily extractable phenolic compounds from the fruit flesh are obtained whereas those in the apple peel surely remain in the residue. this is confirmed by the general phenolic profile in our juices, closely resembling those of apple fruit flesh with hydroxycinnamic acids and flavan-3-ols as the predominating groups (tsao et al., 2003, 2005; van der sluis, 2001). seasonal sensitive or insensitive apples were also described by lata et al. (2005). seasonal related differences may be due to the variable light and temperature regimes. by that, at least apple skin phenolics (awad et al., 2000; awad and de jager, 2002) were affected. this variability of concentrations of individual phenolics or groups of compounds in our study is not related to the resistance potential of the cultivars against the apple scab venturia inaequalis, which could be speculated since phenolic compounds are involved in scab defence (treutter et al., 1990; picinelli et al., 1995). furthermore, there is no relationship of juice phenolics to fruit ripeness parameters, such as flesh firmness, starch degradation, soluble solids or acid content. that is remarkable because of the generally accepted dependency of biosynthesis on accumulation of phenyl pro panoids and flavonoids to the availabiltity of carbohydrates, which was found for apple leaves (ruehmann and treutter, 2003; strissel et al., 2005; treutter 2005). it is furthermore noticeable that early ripening varieties such as ‘alkmene’ and ‘gerlinde’ are not devoid of phenolics. despite of their short periods of fruit development, the partitioning of primary metabolites towards the secondary metabolism is sufficient for the accumulation of phenolics in the fruits of the respective cultivars. it may be assumed that there are independent genetic and environmentally affected regulators of phenolic metabolism. this assumption is confirmed by the observation that some cultivars show big differences between the two years whilst others exhibit similar phenolic patterns when comparing the two harvest years 2001 and 2002. the weather conditions of the two years with respect to temperature, rainfall and sun hours showed only slight differences. lee et al. (2003) investigated the contribution of phenolic compounds to apple antioxidant capacity using methanol extracts and the abts assay. in their study, the quercetin glucosides had the highest propor phenolic antioxidants of apple juices 19 defining reaction kinetics in the in vitro test systems. nevertheless, the main apple phenolics chlorogenic acid and the derivatives of epicatechin and quercetin show the structural feature of the catecholic group. other prerequisites for flavonoids are a 2,3-double bond and 3 and 5-oh-groups adjacent to the 4-keto-structure (bors and michel, 2002; rice-evans et al., 1996). this is fulfilled by apple flavonols. however, their concentrations are quite low in the juices. thus, they do not substantially contribute to the antioxidant potential of apple juices. the results of our study indicate the flavan-3-ols and chlorogenic acid as the major antioxidants in apple juice. compared to the fresh apple fruits (skin), flavonols (especially quercetin and its glycosylated derivatives) play a minor role in apple juices due to their low content. antioxidant activity of apple juices varied as well as the content of phenols varied in quantity, but no strong correlation could be established. however, especially the catechins and procyanidins were found to be highly active in both in vitro test systems, abts decolorization and the x/xod catalyzed formation of ethylene from kmb. as reported by eberhardt et al. 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der sluis, a.a., dekker, m., de jager, a., jongen, w.m.f., 2001: activity and concentration of polyphenolic antioxidants in apple; effect of cultivar, harvest year and storage conditions. j. agr. food chem. 49, 3606-3613. van der sluis, a.a., dekker, m., skrede, g., jongen, w.m.f., 2002: activity and concentration of polyphenolic antioxidants in apple juice. 1. effect of novel production methods. j. agr. food chem. 50, 7211-7219. van der sluis, a.a., dekker, m., van boekel, m.f., 2004: activity and concentration of polyphenolic antioxidants in apple juice. 3. stability during storage. j. agr. food chem. 52, 2840-2848. address of corresponding author: dieter treutter, fachgebiet für obstbau, dürnast 2, d-85354 freising, germany e-mail: dieter.treutter@wzw.tum.de © the author(s) 2016. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 89, 176 182 (2016), doi:10.5073/jabfq.2016.089.022 1agricultural biotechnology department, agriculture college, shahid bahonar university of kerman, iran 2agriculture department, agriculture and natural science college, university of hormozgan, bandar abbas, iran 3plant protection department, agriculture college, shahid bahonar university of kerman, iran 4agriculture and natural resources research center, kerman, iran induction of resistance against powdery mildew by beta aminobutyric acid in squash r. zeighaminejad1, g.r. sharifi-sirchi2, h. mohamadi3, m.m. aminai4 (received december 1, 2015) * corresponding author summary the non-protein amino acid beta-aminobutyric acid (baba) enhances squash resistance to microbial pathogens through potentiation of the squash defense responses. in the present study, we investigated the effects of baba pretreatment on cucurbit powdery mildew di sease. three concentrations of baba (0.5, 2 and 4 mm) as priming media and distilled water as control plants were used. the results showed that the 4 mm baba was the most effective concentration to control the disease symptoms. the expression of pr-1 gene in relation to this pathogen was investigated and confirmed. guaiacol peroxidase and phenylalanine amonia-lyase enzymes activities increased in pretreated-inoculated squash plants. bases on our results and regarding the adverse effects of fungicide on the environment, use of baba as an environmentally safe agent is recommended as a valuable contribution to disease management. introduction powdery mildew, caused by podosphaera xanthii [syn. p. fusca (castagne) u. braun & shiskoff, pollacci], is a serious disease affecting the leaves, stems and fruits of squash and zucchini squash grown in greenhouses and in the field. the disease is controlled in commercial cucurbit crops by applying a high rate of fungicides (kimati et al., 1997). the extensive usage of fungicides has resulted in the development of resistant p. xanthii populations to fungicides and public concern over contamination of the environment and foods. the identification of biocompatible products for managing cucurbit powdery mildew with low animal toxicity and low potential risk to the environment would be a valuable contribution to disease management (mc grath and staniszewska, 1996). resistance can be systemically induced in plants by inoculation with non-pathogens, restricted inoculation with pathogens, or by application of chemical treatments including β-aminobutyric acid (cohen, 2002; jakab et al., 2001; sharifi-sirchi et al., 2011) salicylic acid, 2,6-dichloroisonicotinic acid (ina), benzothiadiazole (bth), probenazole and microelements (bai et al., 2010; zimmerli et al., 2001). such synthetic or natural compounds are named inducers and can induce resistance in plants (durrant and dong, 2004; ton and mauchmani, 2004; shi et al., 2007; von rad et al., 2005). physiological conditions in which plants are able to better or more rapidly mount defense responses to biotic or abiotic stresses are called the “primed state” of the plant (conrath et al., 2002). for example, cucumber plants that had been attacked by the fungus colletotrichum lagenarium combated secondary penetration attempts by rapidly depositing effective papillae at the points of attempted pathogen ingress (kuc, 1982). the non protein amino acid β-aminobutyric acid (baba) is known as a potent inducer of resistance in plants against microbial pathogens (cohen, 2002; jakab et al., 2001), nematodes (oka et al., 1999), insects (hodge et al., 2005), and abiotic stresses (jakab et al., 2005). research on the mechanisms of baba-induced resistance (baba-ir) in arabidopsis has shown that this form of induced resistance, like sar, is mostly based on priming for different pathogen-inducible defense mechanisms. in arabidopsis, baba-enhanced resistance against botrytis cinerea and pseudomonas syringae were found to have correlations with primed transcription of the sa inducible pr-1 gene, whereas induced resistance towards hyaloperonospora arabidopsidis depends on an earlier and stronger formation of callose rich papillae around the growing hyphae (yu et al., 2006; zimmerli et al., 2000, 2001). pathogenesis-related proteins consist of enzymes including chitinase, peroxidase, phenylalanine amonia-lyase and certain other proteins which accumulate in high levels following pathogen attacks. their induction has been correlated with greater resistance to subsequent pathogen attack (tuzun et al., 1989). phenylalanine ammonia lyase (pal) catalyzes the deamination of l-phenylalanine to t-cinnamic acid, which is the first step in the phenylpropanoid pathway which supplies the precursors for phenolics, lignin, furanocoumarin, phytoalexins and other downstream metabolites (tsuge et al., 2004). the activities of pal and pod may rapidly be enhanced under the influence of elicitors or pathogen attack. enhancement of pal and pod activities was reported in response to rhizoctonia solani inoculation in cowpea pretreated with sa (chandra et al., 2007). pal exhibits high reactivity to stress factors and plays a key role in the synthesis of compounds involved in plant-immunity. the purpose of this study was to evaluate the effects of the baba priming p. xanthii on cucurbit plants (cucurbita pepo l.cv. peto seed) and to analyze the expression of the pr1 gene, and gpx and pal enzyme activities during resistance performance. materials and methods biological material, growth conditions and chemicals cucurbit plants (cucurbita. pepo) were grown in plastic pots (150 mm diameter * 150 mm deep) filled with a mixture of soft mold leaves and loamy sand (5: 2, v / v) under normal greenhouse conditions (18 °c and 16 h-photoperiod). complex fertilizer was applied twice per week. all chemicals were obtained from sigma (deisendorf, germany). experiments were carried out when seedlings were in the four-leaf stage. in this stage plants were sprayed with different concentrations of baba (0.5, 2 and 4 mm). pathogen and inoculation p. xanthii obtained from “eld-grown” plants were maintained on squash plants grown in greenhouse conditions (18 °c and 16 h photoperiod). inoculum was obtained from freshly sporulation infected leaves 9-12 days after inoculation. conidia were gently brushed into 100 ml distilled water containing two drops of tween-20 and counted with the aid of a haemocytometer to give a suspension of 4 × 106 conidia ml-1. after 24 h from treatment, upper surfaces of all resistance against powdery mildew 177 the leaves were sprayed with a conidial suspension delivered by a hand sprayer. after inoculation, plants were kept in the same green house (18 °c and 16 h photoperiod) until disease development. control plants were sprayed with water and kept under similar conditions. the infection degree was assessed visually using a 0-5 scale seven days after inuculation: where 0 = no disease symptoms, 1 = less than 1/5 of the host surface covered by mycelia, 2 = 1/5 -1/3 of the surface covered by mycelia, 3 = 1/3 -1/2 of the surface covered by mycelia, 4 = 1/2 -2/3 of the surface covered by mycelia, 5 = more than 2/3 of the surface covered by mycelia. disease index of every plant was determined, according to the mathematical formula: σ a × b disease index = × 100 % n × k where a is the number of leaves with a corresponding infection degree, b is the infection degree of leaves (scale differences from 0-5), n is the total number of leaves counted in a plant, and k is the maximal value of lesion intensity (= 5 on the chosen scale) (shternshis et al., 2002). isolation of partial pr1 gene primers were designed based on the conserved sequence of the pr1 gene in cucurbitaceae family plants using the fpcr and dnaman package softwares (microsoft visual studio 6.0, visual basic 6.0 sp6 company). the primers were synthesized by the isogene company, netherlands. pcr reactions were performed in a veriti thermal cycler (applied biosystems) with a total volume of 20 μl containing 60 ng of genomic dna, 1 × pcr reaction buffer, 1.5 mmol l-1 mgcl2, 0.2 mmol l-1 dntps, 0.5 mmol l-1 of each primer, and 1 u of taq polymerase. pcr condition was set at 94 °c for 5 min, following 35 cycles of 94 °c for 1 min, 55 °c for 45 s, 72 °c for 1 min, and followed by a final extension of 5 min at 72 °c. samples were run in 1 % agarose gel in 0.5 × tris-borate-edta (tbe, ph 8.3) buffer. gels were run at voltage 100 for 35 min and visualized with ultraviolet light after ethidium bromide staining as described by sambrook and russell (2001). semi-rt-pcr for each sample, 3-week old squash leaves from three pots were harvested at the indicated time points, flash frozen in liquid nitrogen and kept at 80 °c. total rna was extracted from 0.1 g of frozen leaves purified using the rneasy plant mini kit (qiagen, germany) with additional dna clean-up using the rnase-free dnase set (fermentas, lithuania). complementary dna was synthesized from 2 μg of total rna using a first strand cdna synthesis kit (fer mentas, lithuania). the resulting first strand cdna was used for pcr. specific primers were used to amplify cdna fragments, including: f 5’-actcacctcaagact-3 (forward) and 5’-ggatgtgccaaag-3’ (reverse) for pr1 (embl: jf332040) and 5’ aagacgaacaactgcg-3’ (forward) and 5’cgaccatactcccccc-3’ (reverse) for 18s rrna. 18s rrna was used as the reference gene for normalization of gene expression levels in all samples. purification and cloning of cdna fragment pcr products were purified with an accuprep pcr purification kit according to the manufacturer’s instruction (bioneer, south korea). the purified pcr products were ligated into the ptz57r/t vector using inst/a clone™ pcr product cloning kit (fermentas, lithuania). then, recombinant plasmids were transformed into competent e. coli xl1blue cells. screening was performed based on blue-white selection method as described in sambrook and russell (sambrook and russell, 2001). transformed white colonies were selected on x-gal/iptg lb ampicilin agar plates, after 24 h of growth and confirmed by colony pcr and enzyme digestion (ecor1 and bamh1). the recombinant plasmids were extracted by accuprep® plasmid extraction kit and accuprep pcr purification kit according to the manufacturer’s instructions (fermentas, lithuania). recombinant plasmids were sequenced in both directions by extending m13 reverse and forward primers, using the automatic dna sequencer 3730xl (macrogene, korea). the sequencing data were edited using chromas software version 1.41. comparison of homologues sequences for query and drawing phylogenetic tree homology of obtained pr1 gene sequence (as query) was compared with database sequences in ncbi by nucleotide blast program (http:// blast.ncbi.nlm.nih.gov/blast/blast.cgi?program=blastn&page_ type=blastsearch&link_loc=blasthome). results of seeding were used for calculating the triangular matrix of similarity and drawing a phylogenetic tree by dnaman software version 4.02 (lynnon biosoft. 1994-98). enzyme extraction and activity determination 500 mg of leaves were homogenized in cool 50 mm potassium phosphate buffer (ph = 7.0) containing 1 % soluble polyvinilpyrolidone1 (w/v), 1 mm ethylene diamine tetra acetic acid2 and 1 mm phenylmethylsulfonyl fluoride3. all processes were carried out in ice. the homogenate was centrifuged at 20,000 × g for 20 min and the supernatant used for assay of the enzyme activity. catalase (cat) activity (ec 1.11.1.6) cat activity was determined spectrophotometrically following the decrease in absorbance of h2o2 within 30 s at 240 nm (dhindsa et al., 1981). the 3 ml reaction solution consisted of 50 mm potassium phosphate buffer (ph = 7.0), 15 mm h2o2, and 100 ml of enzyme extract. addition of h2o2 started the reaction and the decrease in absorbance was recorded after 30 s. unit of activity was taken as the amount of enzyme, which decomposes one μmol of h2o2 in one minute (ε = 40 mm-1 cm-1 for h2o2). guaiacol peroxidase (gpx) (ec1.11.1.7) gpx activity was measured using guaiacol as a substrate. reaction mixture (3 ml) contained 25 ml of enzyme extract, 2.77 ml of 50 mm phosphate buffer (ph = 7.0), 0.1 ml of 1 % h2o2 (v/v), and 0.1 ml of 4 % guaiacol (v/v). the increase in absorbance at 470 nm due to guaiacol oxidation was recorded for 3 min using an extinction coefficient of 25.5 mm-1 cm-1 (plewa et al., 1991). phenylalanine amonia-lyase (ec 4.3.1.5) phenylalanine amonia-lyase (pal) activity was determined by the modified method of cheng and breen (1991). the reaction mixture contained leaf extract (0.3 ml), 0.2 m phenylalanine solution (1 ml) and 0.05 m borate buffer (ph = 8.8, 2.7 ml). the reaction was quenched with 6 n hcl (0.1 ml). the production of cinnamic acid during 1 h at 30 ºc was measured by the absorbance change at 290 nm. one unit of enzyme represents the conversion of 1 μmol substrate to cinammic acid per min. 1 pvp 2 edta 3 pmsf 178 r. zeighaminejad, g.r. sharifi-sirchi, h. mohamadi, m.m. aminai total soluble proteins protein content was determined according to the method of bradford (1976) using bovine serum albumin as standard. measurement of chlorophyll and carotenoid contents chlorophyll a, b and carotenoid were extracted using 80 % acetone based on lichtenthaler (1987) method. the chlorophyll a, b and carotenoid contents of leaves were measured at the wave lengths 646.8, 663.2 and 470 nm by using spectrophotometry and the formulae. chl.a = (12.25a663.2 – 2.79a646.8) chl.b = (21.21a646.8 – 5.1 a663.2) car = [(1000a470-1.8 chl.a-85.02 chl.b)/198] statistical analysis all determinations were carried out in triplicate and the data subjected to analysis of variance. analysis of variance was performed using the anova procedure. statistical analyses were performed accor ding to the mstatc software. significant differences between means were determined by duncan’s multiple range tests. p values less than 0.01 were considered statistically significant (duncan, 1955). result baba-treated cucurbit plants show resistance against podosphaera xanthii in primary experiments, two approaches (root drench and foliar spray) with three doses of baba (0.5, 2 and 4 mm) were analyzed. the results showed that application of baba through root drench was not effective on p. xanthii while foliar application of baba was very effective. however, foliar spray with 0.5 mm baba was not appropriate to control p. xanthii. therefore, in other research we applied a concentration of 2 and 4 mm baba. seven days after inoculation, the powdery mildew disease index was estimated on every plant. disease index for water control plants (60) was significantly higher than those of 4 and 2 mm for baba treatment (21 and 43, respectively) (fig. 1 and 2). the concentration of 4 mm of baba showed the best control after seven days of inoculation. isolation of partial pr1 gene and its homology analysis the amplified pcr fragment of 317 bp was obtained by using forward and reverse pr1 gene primers (fig. 3). it was submitted to genbank (jf332040.1 mrna accession number cucurbita pepo pathogenesis-related protein 1 (pr1) mrna, partial cds). its deduced amino acid sequence (aea11234.1) was 114 amino acids long (fig. 3). the partial sequence of the pr1 gene in seedings of cucurbita pepo showed highest homology with the cucumis melo pr1 gene (68%). c. melo belongs to the family of cucurbitaceae (fig. 4). cluster analysis of partial sequence of pr1 in cucurbia pepo with seven pr1 genes from different plants which had homology with the pr1 gene from cucumis melo in one class (fig. 5). fig. 2: comparison of infected squash leaves exposed to powdery mildew seven days post inoculation. a; control, b; primed squash leave with 2 mm baba, c; primed squash leave exposed to 4 mm baba. a b c fig. 1: mean comparison of treatments in induced resistance of squash to powdery mildew (duncan test, p < 1 %). different letters show significant differences. standard error (se) bars are presented for each sample. fig. 3: cucurbita pepo pathogenesis-related protein 1 (pr1) mrna, partial cds (a) and partial protein (b). cucurbita_pepo 100% arabidopsis_thalian 33.1% 100% cucumis_sativus 65.2% 48.4% 100% cucumis_melo 68.0% 42.9% 74.1% 100% brassica_rapa 24.9% 63.4% 30.8% 29.7% 100% brassica_carinata 23.2% 57.3% 26.8% 24.8% 91.1% 100% eutrema_wasabi 34.8% 84.8% 49.6% 43.9% 66.3% 59.9% 100% vitis_vinifera 22.2% 30.9% 27.8% 28.5% 35.1% 37.4% 33.5% 100% fig. 4: homology matrix of cucurbita pepo partial pr1 cds and seven other plants. resistance against powdery mildew 179 fig. 5: phylogeny tree base on distance matrix of cucurbita pepo partial pr1 cds and seven other plants. the effect of baba pretreatment on the expression of rp1 gene the expression level of pr1 was evaluated by semi rt-pcr at post p. xanthii inoculation (fig. 6). pr1 gene was detected in both control and baba treated cucurbit for the six investigated time points (fig. 6-a and 6-b). there was a slight increase in pr1 expression in infected-baba-primed (4 mm) cucurbit plants compared to the infected and water-treated cucurbit plants at 12 h post infection (fig. 6-a and 6-b). cat activity: results showed that cat activity was significantly decreased after inoculation and the minimum activity of cat was observed 240 h after inoculation. however, no significant diffe rences were observed in cat activity between the treatments given at any one time point except 24 h after inoculation (fig. 7). gpx activity: as shown in fig. 7, pretreatment of plants with baba had no significant effect on gpx before inoculation, while infection of plants with p. xanthii promoted the activity of gpx in water and baba pretreated plants in comparison with non-inoculation plants. gpx showed very high activity after 168 and 240 h post inoculation. application of baba pretreatment increased the activity of 4 mm gpx after 48h post inoculation. pretreatment of plants with 2 mm baba had no significant effects on gpx activity in comparison with water treated plants (fig. 7). pal activity: result showed that pal activity was significantly increased at 24 hours after priming with both baba concentrations (2 and 4 mm). also pal activity was significantly increased in all sampling time points after inoculation in both primed and control cucurbit plants. however, pal activity was the highest at 240 hours after inoculation sampling time points in primed plants with 4 mm baba (fig. 8). fig. 7: effect baba pretreatments on catalase activity. the mean comparisons of cat activity of treated squash plants compared using the duncan method at a p < 0.01 significance level. different letters show a significant difference. z.p; time point 0. 24 pp; 24 h post priming. hpi; hours post inoculation. fig. 6: pr1 gene expression after inoculation. effect of baba treatment on the expression of pr1 during of disease development by p. xanthii. (a); pr1 gene expression pattern on infected and baba-primed (4 mm) cucurbit plants. (b); pr1 gene expression pattern on not infected control cucurbit plants. (c); balanced 18srrna expression after 35 cycle of pcr. (d); balanced total rna. z.p; time point 0. 24 p.p; 24 h post priming. 12hpi, 24hpi, 48hpi and 72hpi; hours post inoculation.   fig. 6: pr1 gene expression after inoculation. effect of baba treatment on the expression of pr1 during of disease development by p. xanthii. (a); pr1 gene expression pattern on infected and baba-primed )4 mm) cucurbit plants. (b); pr1 gene expression pattern on not infected control cucurbit plants. (c); balanced 18srrna expression after 35 cycle of pcr . (d); balanced total rna. z.p; time point 0. 24 p.p; 24 h post priming. 12hpi, 24hpi, 48hpi and 72hpi; hours post inoculation. .   antioxidant enzyme activities activities of gpx, cat and pal in infected and primed cucurbit plant leaves with p. xanthii and control plants assayed. 180 r. zeighaminejad, g.r. sharifi-sirchi, h. mohamadi, m.m. aminai ciated with an increased enzymatic activity and transcript accumulation of the pr-1 gene. in many plants, the chemical baba has been shown to enhance disease resistance and to increase salt, drought, and thermo tolerance (jakab et al., 2005; slaughter et al., 2008; ton et al., 2005; ton and mauch-mani, 2004; zimmerli et al., 2000, 2001, 2008). typically, following infection by pseudomonas syringae pv. compared to tomato dc3000, the salicylic acid (sa)dependent defense marker pathogen related gene 1 (pr1) is induced earlier and stronger in baba-treated arabidopsis (ton et al., 2005; zimmerli et al., 2000). baba does not activate a defense response directly but rather sensitizes plants to respond more quickly and strongly to biotic and abiotic stresses. this process is referred to as priming (conrath et al., 2002; conrath et al., 2006). given that accumulation of these proteins after pathogen infection correlates with induced resistance. pal activity in plant tissue might be rapidly changed under the influence of various factors, e.g. pathogen attack and treatment with elicitors. pal and pod activities were enhanced several fold in tomato roots by a biotic elicitor fusarium mycelium extract derived from fol (mandal and mitra, 2007). enhancement of pal and pod activities in sa-treated asparagus plants upon f. oxysporum f. sp. asparagi infection resulted in reinforcement of the cell wall and restricted subsequent fungal penetration and infection (he and wolyn, 2005). addition of 20 mm salicylic acid to saussurea medusa cell cultures resulted in a 7.5-fold increase in pal activity (yu et al., 2006). sa spraying on ya li pear plants increased pal and pod activities greatly and contributed to protection of pear fruits against postharvest diseases (cao et al., 2006). in the present investigation the activities of pal and pod were also increased to a great extent in the baba treated plants compared to non-baba-treated plants, probably contributing in enhanced resistance of cucurbit to podosphaera xanthii. in this study, pal activity was increased after inoculation. mauchmani and slusarenko (1996) reported that pal is involved in the synthesis of sa and precursors of lignification in arabidopsis. sar, induced biologically and chemically in plants, is associated with an ability of plants to resist pathogen attack by enhanced activation of cellular defense mechanisms (mé traux et al., 2002). the results indicate that the induced resistance observed in cucurbit against p. xanthii may be a case of sa-dependent sar. in conclusion, it seems that baba feeding does reduce susceptibility of squash plants to p. xanthii, likely due to induction of sar accompanied by increased activities of the defense enzymes pal and pod. fig. 7: effect baba pretreatments on the guaiacol peroxidase (gpx) activity. mean comparison of gpx activity of treated squash plants using duncan method at a p < 0.01 significance level. different letters show a significant difference. z.p; time point 0. 24 pp; 24 h post priming. hpi; hours post inoculation. standard bar showed differences at α = 0.5%. fig. 9: the mean comparisons of chlorophyll a and b, and carotenoid content of control and primed inoculated cucurbit plants with 4 mm baba at 240 hours post inoculation. standard bar showed diffe rences at α = 0.5%. fig. 8: effect of baba pretreatments on phenylalanine ammonia lyase (pal) activity. mean comparison of pal activity of treated squash plants using duncan method at a p < 0.01 significance level. different letters show significant difference. z.p; time point 0. 24 pp; 24 h post priming. hpi; hours post inoculation. standard bar showed differences at α = 0.5%. chlorophyll and carotenoid contents our results showed that the amount of chlorophyll a and b pigments of cucurbit plants were significantly higher at 240 hours after inoculation compared to those of controls (fig. 9). carotenoid contents of primed inoculated leaves were significantly higher than inoculated control plants and primed cucurbit plants with 4 mm baba had a higher carotenoid amount than primed cucurbit plants with 2 mm baba (fig. 9). discussion the resistance inducer baba has been shown to work mainly through priming of defense responses by sensitizing the plants to respond faster and more adequately to exposure against a given stress situation (conrath et al., 2002; conrath et al., 2006; jakab et al., 2001). in this study, we investigated the influence of baba pretreatment against p. xanthii infection and the activation of pathogenesisrelated proteins. our results confirmed that application of baba to cucurbit leaves reduced the disease index compared to plants before inoculation. we provide the first evidence that this resistance is asso resistance against powdery mildew 181 the results of this investigation show that baba may be used as a potential inducer of sar against p. xanthii. fungicide resistance and public concern over environment contamination has resulted in identification of biocompatible products for managing cucurbit powdery mildew with low animal toxicity and low potential risk to the environment. baba would be a valuable contribution to disease management. acknowledgements we would like to give special thanks to fariba nasibi, assistant professors in biology departments of shahid bahonar university of iran for their engagement and advice in entire stages of this research. we would like to thank, vice chancellor for research of shahid bahonar university of kerman, for funding this project. reference bai, w., chern, m., ruan, d., canlas, p.e., sze-to, w.h., ronald, p.c., 2011: enhanced disease 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real-time pcr. nucleic acids res. 34. zhang, z., pang, x., duan, x., ji, z.l., jiang, y., 2005: role of per oxidase in anthocyanine degradation in litchi fruit pericarp. food chem. 90, 47-52 zimmerli, l., jakab, c., metraux, j.p., mauch-mani, b., 2000: potentiation of pathogen-specific defense mechanisms in arabidopsis by betaaminobutyric acid. p. nati. a. sci. usa. 97, 12920-12925. zimmerli, l., me´ traux, j.p, mauch-mani, b., 2001: beta-aminobutyric acid-induced protection of arabidopsis against the necrotrophic fungus 182 r. zeighaminejad, g.r. sharifi-sirchi, h. mohamadi, m.m. aminai botrytis cinerea. plant physiol. 126, 517-523. zimmerli, l., hou, b.h., tsai, c.h., jakab, g., mauch-mani, b., somerville, s., 2008: the xenobiotic beta-aminobutyric acid enhances arabidopsis thermotolerance. plant j. 53, 144-156. address of the corresponding author: e-mail: sharifi-sirchi@hormozgan.ac.ir, sharifisirchi@yahoo.com © the author(s) 2016. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 87, 234 242 (2014), doi:10.5073/jabfq.2014.087.033 university of rostock, faculty of agricultural and environmental sciences, crop health, rostock, germany important maize weeds profit in growth and reproduction from climate change conditions represented by higher temperatures and reduced humidity kristian peters *, bärbel gerowitt (received may 27, 2014) * corresponding author summary climate change is predicted to result in rising temperatures and reduced precipitation during spring and summer in central europe. as a consequence, crops and weeds will be affected. our study focuses on the three weed species in maize amaranthus retroflexus, echinochloa crus-galli and setaria viridis. these weeds occur numerously in european maize fields and populations are likely to further increase. yet, there is a lack of knowledge about particular biological strategies of the weeds. our study focuses on how the weed species respond biologically to the climate change conditions. experiments were conducted in two climate chambers with a 2 °c difference in temperature and the warmer one with 13 % less humidity. emergence, development, biomass and seed production were determined of the weeds grown individually in pots and grown within maize. all tested weed species were taller during the first weeks under the climate change scenario. at later growth phases there was a trade-off between traits measured during vegetative growth and at the time when seeds were produced. to summarize the results, the weed species profited in the order e. crus-galli, s. viridis and a. retroflexus from the climate change conditions. knowledge of the weeds biological responses to the predicted conditions helps to reduce their long-term population development by targeting crop protection measures at specific growth phases of the weeds. to ensure control of the tested weed species under climate change conditions various weed management strategies are necessary. introduction climate change will result in rising temperatures (tubiello et al., 2007) and modified precipitation (robinson and gross, 2010). summer droughts will be more likely and will affect weeds in springsown crops. according to patterson et al. (1999) temperature and precipitation are the most important factors for the geographical distribution, the growth and the competitive abilities of weeds. our study focuses these two climate variables. we have chosen three important central european c4 weeds in maize: amaranthus retroflexus, echinochloa crus-galli and setaria viridis, since currently a shift of the local weed flora is occurring in favour of weeds of the c4 photosynthesis type. most of these weeds are late germinators and emerge from early summer to early autumn. thus, they are considered thermophilous in central europe and these weeds are expected to migrate further north with changes in climate conditions (walther et al., 2002). these weeds are already most numerous and most competitive in maize fields of southern europe (novák et al., 2009). they may also extend their damage potential in springsown crops, such as maize, under the predicted future conditions in central europe. therefore, our study focuses on three thermophilous c4 weeds, whose future status and biological strategies are not well understood for central european populations. amaranthus retroflexus l. (redroot pigweed) is a successful weed in maize (oveisi et al., 2013). fast growth and indeterminate flowering enable a single plant to produce up to 500,000 seeds (steckel et al., 2004). the reaction of european populations to warming and different humidity is not well studied so far (hyvönen, 2011). nevertheless, because the species produces more biomass and more seeds in a warmer climate in north america and canada (schimpf, 1977), we expect the species to develop better with warmer conditions in europe as well (kigel et al., 1977; oveisi et al., 2013). setaria viridis (l.) p. beauv. (green foxtail) is currently the most wide-spread species of the setaria genus in europe (dekker, 2003). it generally emerges after the last herbicide treatment in maize fields. thus, plants are often not affected by herbicides (mehrtens et al., 2005; beckie and tardif, 2012). a high genetic variability enables the species to grow under a great range of temperatures (dekker, 2003). plants of north-american populations produce less biomass but increase generative reproduction under warmer conditions (swanton et al., 1999). echinochloa crus-galli (l.) beauv. (barnyard grass) exhibits high phenotypic plasticity (barrett and wilson, 1981; maun and barrett, 1986). central european populations are most competitive under high temperatures, high nutrient availability and mid level humidity (otte et al., 2006). the species benefits from higher temperatures but not from dry conditions (barrett and wilson, 1981; chauhan and johnson, 2011). most studies focus on the development of weeds under various agricultural practices or on their resistance to herbicides (barrett and wilson, 1981; potvin, 1986; chauhan and johnson, 2011). with a special focus on european populations, we ascertain a lack of biological knowledge concerning the vegetative development, the development speed and the generative reproduction for the studied weeds under climate change conditions. the aim of our study is to explore how the three species perform under the predicted future conditions and which distinct biological strategies they realise. furthermore, this study attempts to assess which biological weed properties are important with climatic changes. based on these biological data, a better framework for weed management and crop protection can be devised. to achieve this, the effects of the raised temperature and less humidity on the weeds were studied combined in the experiment. materials and methods the three weed species amaranthus retroflexus, setaria viridis and echinochloa crus-galli used in the experiments were collected near göttingen, germany in 2007. seeds originated from different field populations in that area and were stored for two years until the experiments started in 2009. preliminary germination tests showed high germination rates (approx. 50-75 % germination percentage). thus, the seeds were not subjected to any special treatment. the experiment was conducted in two climate chambers, 2.15 m in width, 3.45 m in length and 2.10 m in height. temperature, humidity and light were independently adjustable. the first climate chamber had temperatures that represent the conditions of the current climate change and three maize weed species 235 tab. 1: conditions in the two climate chambers. values given after ± are the mean variations between replications. period factor chamber with current climate chamber with predicted future climate 0-2 weeks night/day temperature 7 °c/13 °c ±0.3 % 9 °c/15 °c ± 0.6 % night/day length 12/12 12/12 night/day humidity 70 %/66 % ± 1 % 58 %/52 % ± 3 % 2-6 weeks night/day temperature 9 °c/18 °c ± 0.3 % 11 °c/20 °c ± 0.6 % night/day length 14/10 14/10 night/day humidity 70 %/66 % ± 1 % 58 %/52 % ± 3 % 6-12 weeks night/day temperature 11 °c/20 °c ± 0.3 % 13 °c/22 °c ± 0.6 % night/day length 14,5/9,5 14,5/9,5 night/day humidity 70 %/66 % ± 1 % 58 %/52 % ± 3 % 12-15 weeks night/day temperature 11 °c/20 °c ± 0.3 % 13 °c/22 °c ± 0.6 % night/day length 14/10 14/10 night/day humidity 70 %/66 % ± 1 % 58 %/52 % ± 3 % 15-21 weeks night/day temperature 9 °c/18 °c ± 0.3 % 11 °c/20 °c ± 0.6 % night/day length 13/11 13/11 night/day humidity 70 %/66 % ±1 % 58 %/52 % ± 3 % climate of northern germany. the humidity (air moisture content) was raised slightly with water nozzles when compared to the second chamber that represented predicted future climate conditions (tab. 1). mean humidity was set to 68 % in the climate chamber with current conditions and was 13 % lower in the other chamber due to the disabled water nozzles. as light source ten phillips son-t agro 400 were used, which were embedded in the ceiling of each climate chamber. light levels at the centre of the chambers were 24,900 lux measured 90 cm from the ground and 120 cm below the lights. this is equal to a ppfd of approx. 400 μmol/s/m2 at the aforementioned distance. the chosen conditions were based on the a1b scenario of the ipcc (2013), which predicted an increase in temperatures of 2 °c until 2070 for central europe and less humidity during summer months. thus, daily minimum and maximum temperatures were always set 2 °c higher in the second chamber (tab. 1). the day-length was the same in both chambers, but adjusted over time to simulate an advancing season. to ensure the intended climate levels (tab. 1), temperate and humidity in the chambers were continuously adapted and monitored with data loggers 5 cm above ground and with additional air temperature sensors 1.60 m above ground. each climate chamber had two large plant tubs, 1.30 m x 1.10 m in size and a depth of 0.90 m. the bottom of each tub was equipped with a 2 cm thick layer of small stones to allow excessive water to run off through small holes at the bottom-side of the tubs. gauze was stretched above the stone layer to prevent soil from the 60 cm thick soil layer above to agglutinate at the bottom. maize seeds were sown directly into the tubs’ soil (at a depth of 3 cm) at the beginning of each replication to simulate a row typical for maize fields (80 cm row distance, 10 cm plant distance in row). these tubs were already installed for preliminary studies and thus were equipped with an established soil-bed under the two climate conditions. due to the resulting weight, they were immovable between the chambers. therefore, the tubs rested in the same climate chamber under the same conditions in time of the experiment. the tubs were fertilized with 90 g compo hakaphos® blue (equal to 135 kg n and 90 kg p per ha) before the start of each replication and again before weed seedlings were planted. for studying the emergence of the weed species and to provide seedlings for further experiments, 130 seeds of each species were sown at a soil depth of 1 cm in five germination trays in each climate chamber at the same time. the number of seedlings was counted every day (fig. 1). gdd = ( t max +t min 2 ) tb emergence sowing early-growth 0 4 12 mid-growth final-growth 21 plant tubs + m aize crop plant pots harvest harvest time [weeks] fig. 1: overview of the treatments performed in each replication in both climate chambers. in order to characterize, compare and correlate processes during emergence, various parameters were calculated (grundy, 2003). for predicting the cumulative emergence of seedlings, growing degree days (gdd) were calculated (dorado et al., 2009): where tmax and tmin are the daily maximum and minimum temperatures measured by the dataloggers in the climate chamber. tb is the base temperature for each weed. the tb values were taken from various authors: tb=4.0 °c for a. retroflexus from gardarin et al. (2009), tb=6.2 °c for e. crus-galli from guillemin et al. (2013) and tb=6.1 °c for s. viridis from gardarin et al. (2010). in order to estimate and compare the progress of seedling emergence, mean emergence time (met) and emergence rate index (eri) were calculated as follows (dorado et al., 2009): 236 k. peters, b. gerowitt where ni represents the newly emerged seedlings since the previous count, ti represents the gdd after sowing and n is the number of sampling occasions. for characterizing emergence speed, the emergence rate at mid emergence (v50) was additionally calculated (gardarin et al., 2011, modified): where m is the number of emerged seeds until mid emergence, b is a shape parameter correlated with the emergence rate at mid emergence, d0 represents the day after sowing on which first emergence was recorded, and d50 is a factor which represents the day on which 50 % the total emerged seedlings were recorded. the prediction curve of the cumulative emergence gi was fitted (gardarin et al., 2011, modified): where di represents the day of measurement. seedling height of every emerged seedling was determined after 4 weeks (bbch stage 15) (fig. 1, early-growth phase). ten randomly chosen seedlings of each species were then planted in pots in each climate chamber to study further development. pots were 30 cm in diameter and 20 cm deep. another 10 seedlings of each weed were put in the two plant tubs between the maize rows in each climate chamber. sampling and positioning of the weeds was random. both maize and weeds in the large plant tubs were harvested at the end of the mid-growth phase (fig. 1, beginning of flower onset of maize, bbch stage 53, after approx. 12 weeks). plant height and above ground dry mass were determined for each weed and maize plant. height and development stage (bbch, hess et al., 1997) were additionally determined for the weeds in the smaller plant pots. the remaining weeds in the pots were harvested at the reproduction phase (fig. 1, after 21 weeks) and their height and vegetative above ground dry mass (without seeds) were determined. panicles, tillers and seeds of all weed plants were also counted. the experiment involved three replications, which were conducted consecutively. each replication in time was set-up independently. the calculated emergence coefficients were tested for significant differences using the t-test (fig. 3). as described above, two tubs were available within each climate chamber. hence, the involved factor levels (chambers, tubs) were not arranged orthogonally, excluding classical anova approaches. instead, for comparing the biological parameters at the end of the early and mid-growth phases, as well as the reproduction phase (tab. 2, 3), linear models with random effects (lmer) were used. in these models, climate (temperature and humidity combined) was chosen as fixed factor, whereas replications in time were introduced as random factor. when tubs were involved (tab. 4), they were also introduced as random factor. each weed species was tested separately for significant differences between the climate chambers. to investigate the properties of the random coefficients of the lmer, probability values for the parameters of models fitted with lmer were calculated with a monte carlo markov chain (mcmc) approach, choosing 10,000 sample simulations of the model (baayen et al., 2008). distribution and homogeneity of variance were visually checked with the help of histograms and diagnostic plots. data were log or square root transformed before statistical analysis if conditions of normality were not met or to improve homogeneity of variances. the residual vs. fitted plots and the normal qq-plots were examined for each tested parameter according to faraway (2006). statistical analysis was carried out with the software r (ihaka and gentleman, 1997). the additional packages languager, hmisc, agricolae and lme4 were used. results seedling emergence in both climate chambers maize seedlings emerged regularly at the 11th day after sowing, whereas first weed emergence was recorded between the 9th and 13th day after sowing (fig. 2). first seedlings of amaranthus retroflexus appeared about one day earlier in the climate chamber with future conditions. only for setaria viridis a significantly different d0 was measured. seedlings emerged two days earlier under the climate change scenario. whereas the mean emergence time (met) was significantly different for a. retroflexus and s. viridis between both climate chambers (fig. 3), seedlings of eri = np i=1 ni met gi = m h 1 eln(2) ⇣ d i d0 d50d0 ⌘ b i met = np i=1 niti np i=1 ni v50 = m · b · ln(2) 2 · (d50 d0) tab. 2: comparison of the influence of predicted future conditions on plant height [cm] at the end of early-growth phase, mid-growth phase and reproduction phase of plants grown in pots for the three replications, mean and standard error (se) are given for predicted future and current climate, significant plmer-values are bold. plant height [cm] / phase early-growth phase mid-growth phase reproduction phase weed species measurement predicted current predicted current predicted current a. retroflexus mean 3.12 1.83 31.33 23.39 26.12 24.38 se 0.07 0.04 2.21 3.08 1.85 2.42 plmer 0.001 0.040 0.577 e. crus-galli mean 13.84 8,44 92.50 73.22 107.77 102.15 se 0.23 0.15 2.46 1.39 2.59 3.53 plmer 0.001 0.001 0.130 s. viridis mean 3.17 1.92 45.61 37.33 89.50 83.04 se 0.08 0.05 2.31 1.73 4.98 3.23 plmer 0.001 0.002 0.186 climate change and three maize weed species 237 tab. 4: parameters of plants grown in tubs at the end of mid-growth phase for the three replications; mean and standard error (se) are given for predicted future and current climate, significant differences lmer (plmer) are bold. weed species a. retroflexus e. crus-galli s. viridis maize factor measurement predicted current predicted current predicted current predicted current height [cm] mean 13.37 14.15 75.33 48.97 24.30 19.40 162.55 141.05 se 1.14 1.28 3.09 2.03 2.11 1.65 2.78 1.91 plmer 0.650 0.001 0.034 0.001 dry mass [g] mean 1.06 1.34 8.17 3.94 0.76 0.57 78.91 62.15 se 0.23 0.16 1.51 0.62 0.11 0.06 9.96 7.71 plmer 0.245 0.002 0.026 0.006 tab. 3: parameters with significant results of plants grown in pots at the end of reproduction phase for the three replications; mean and standard error (se) are given for predicted future and current climate, significant differences lmer (plmer) are bold. weed species a. retroflexus e. crus-galli s. viridis factor measurement predicted current predicted current predicted current panicles mean 17.50 18.35 34.96 19.12 17.50 16.42 per plant se 3.18 4.92 4.20 1.44 1.30 1.61 plmer 0.705 0.001 0.418 tillers mean 2.85 2.23 15.81 18.23 15.31 15.8 per plant se 0.43 0.40 0.81 0.76 0.91 1.39 plmer 0.035 0.030 0.870 panicles mean 6.05 6.60 2.35 1.07 1.18 1.14 per tillers se 0.52 1.12 0.29 0.08 0.08 0.10 plmer 0.663 0.001 0.458 number of seeds mean 2135.38 2654.46 4996.73 3553.58 2926.35 2401.12 per plant se 290.76 546.14 545.57 410.86 322.13 233.77 plmer 0.956 0.002 0.123 seeds mean 73.28 46.03 150.97 187.84 171.94 171.71 per panicles se 7.11 4.22 7.15 17.94 18.66 19.25 plmer 0.004 0.024 0.976 fig. 2: cumulated emergence of weed seeds over time for (a) amaranthus retroflexus, (b) echinochloa crus-galli, (c) setaria viridis for the three replications; square, diamond and circles mark readings in the climate chamber with predicted future conditions; +, x and * mark readings in the climate chamber with normal conditions; normal lines represent fitted curves under predicted future climate, dashed lines represent fitted curves under current climate. 0 5 10 15 20 25 0. 0 0. 4 0. 8 time [days] em er ge nc e [% ] (a) 0 5 10 15 20 25 0. 0 0. 4 0. 8 time [days] em er ge nc e [% ] (b) 0 5 10 15 20 25 0. 0 0. 4 0. 8 time [days] em er ge nc e [% ] (c) echinochloa crus-galli appeared nearly at the same time in both climate chambers. the time span from sowing to mid emergence (d50) differed significantly for s. viridis (fig. 3). a d50 of 13.8 days was recorded in the chamber with the predicted future conditions compared to 16.4 days in the other chamber. the d50 of e. crus-galli was reached 1.8 days earlier and the d50 of a. retroflexus was reached 1.5 days earlier under the climate change scenario (fig. 3). the mid emergence rate (v50) differed not significantly for all species between both climate chambers. however, echinochloa crus-galli had the highest v50 ratio 238 k. peters, b. gerowitt (0.8), whereas s. viridis had the lowest v50 ratio (0.1) of the tested species (fig. 3). the number of emerged a. retroflexus seedlings increased with each replication. whereas in replication one, the ratio was 41 % for current climate conditions and 50 % for the chamber with predicted future conditions after 4 weeks, the ratio was 79 % vs. 91 % in replication three (fig. 2). the emergence rate of e. crus-galli seedlings was high and differences between replications were small four weeks after sowing. a rate of 75 % was reached after four weeks (fig. 2). for setaria viridis the rate was 58 % after four weeks in both chambers (fig. 2). only e. crus-galli had a significantly different emergence rate index (eri). the seedlings emerged quicker and more uniformly in the chamber with the predicted future conditions. a. retroflexus showed no significant differences (fig. 3). development without crop competition of plants grown individually in pots early-growth phase: plants of all three species were significantly taller in the climate chamber with the predicted future conditions (tab. 2). whereas seedlings of e. crus-galli developed quite regularly, the variance of seedling height and development stage of s. viridis and a. retroflexus were more conspicuous between the different replications. the mean plant height of e. crus-galli seedlings was 14 cm in the chamber with the climate change scenario and 8 cm in the chamber with current conditions. seedlings of a. retroflexus and s. viridis were significantly smaller under current conditions: 2 cm vs. 3 cm (tab. 2). mid-growth phase: amaranthus retroflexus showed a rather undefined growth habit. most plants did not grow upright, were less elongated and branched more than plants grown under similar conditions in the field. plants were also slightly smaller in the climate chamber with current conditions (tab. 2). no significant differences in development stages between the two climate chambers were found, since first flower buds were developed after only 4 to 5 weeks in both chambers (bbch stage 71). setaria viridis grew taller under the climate change scenario (tab. 2). the average time of panicle development was the same in both climate chambers, although variance was higher in the chamber with the predicted future conditions (data not shown). echinochloa crus-galli developed panicles significantly earlier (1 to 2 weeks) under the climate change scenario. plants grown under the climate change scenario were at stage 67, whereas plants grown under current conditions were still at bbch 55 on the mean average. plant height was also increased under the climate change scenario (tab. 2). reproduction phase: we observed better growth of all species at the end of earlyand mid-growth phase under the climate change scenario. nevertheless, this relationship diminished at the end of the reproduction phase (tab. 2). amaranthus retroflexus plants still did not grow upright in both climate chambers (see above). we did not find any significant differences in plant height, panicles and seed production for a. retroflexus. the number of seeds per plant varied strongly between plants and replications. however, plants had more tillers per plant and more dry mass under the climate change scenario (tab. 3). echinochloa crus-galli developed quicker under predicted future conditions. first panicles were visible in the 11th week and flowering occurred over a longer period compared to the chamber with current conditions, which resulted in 550 more seeds/plant under the climate change scenario. the weed developed significantly more panicles with more seeds under the climate change conditions (tab. 3). echinochloa crus-galli also produced slightly less tillers under this scenario, which resulted also in slightly less vegetative dry matter (tab. 3). the species was also the tallest of the three fig. 3: parameters characterising emergence: met = mean emergence time, eri = emergence rate index, v50 = emergence rate at time of mid emergence, d50 = the calculated day on which 50% the total emerged seeds have emerged; n = 3, triangles show values under predicted future conditions, circles show values recorded under current conditions. met 7 8 9 10 11 12 13 ● ● ● predicted current predicted current predicted current a. retroflexus e. crus−galli s. viridis ● ● ● ● ● ● eri 4 6 8 10 12 14 16 ● ● ● predicted current predicted current predicted current a. retroflexus e. crus−galli s. viridis ● ● ● ● ● ● v50 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 ● ● ● predicted current predicted current predicted current a. retroflexus e. crus−galli s. viridis ● ● ● ● ● ● d50 10 11 12 13 14 15 16 17 ● ● ● predicted current predicted current predicted current a. retroflexus e. crus−galli s. viridis ● ● ● ● ● ● climate change and three maize weed species 239 weed species (tab. 3). setaria viridis produced first panicles after 17 weeks. they occurred at the same time in both climate chambers. plant height, the number of panicles and tillers were rather homogeneous after 21 weeks in both climate chambers. the weed tended to develop slightly more seeds under climate change conditions (tab. 3). vegetative dry matter was not affected (tab. 3). development with crop competition in the large plant tubs plant height and dry mass of a. retroflexus grown in the maize rows in the large plant tubs did not differ significantly between the climate chambers. both plant height and dry mass were slightly less in the chamber with the predicted future conditions (tab. 4). plants of e. crus-galli were significantly taller in the chamber with the predicted future conditions. this was also reflected by an increase in dry matter content (tab. 4). average height and dry mass of s. viridis plants differed strongly between the three replications. plants grew taller and produced also more dry mass under the climate change scenario (tab. 4). the maize plants were significantly taller when grown under the predicted future conditions. however, above ground dry mass was similar between both climate conditions (tab. 4). all weeds were significantly smaller and had significantly less dry mass when grown in competition with maize in the large plant tubs compared to plants grown individually in smaller pots (fig. 4). this was true for both climate chambers. weed plants in the tubs and the pots had the same development stage. discussion for discussing and evaluating the results, it is crucial to acknowledge that the two factors temperature and humidity were combined in our study. consequently, we link the discussion mainly to these factors and conclude for climate change. seedling emergence the growth, speed and timing of weed seedling emergence are important factors for the development and seed production of weeds in maize (sarabi et al., 2011). our results showed different emergence for all three species, which resulted in species-specific strategies to cope with the predicted future conditions. in summary, the predicted future conditions are beneficial for the tested weed species, as most of their emergence factors were enhanced under these conditions. this mainly leads to enhanced vegetative growth at later development phases (see below). the emergence results confirm that a. retroflexus seedlings typically emerge under warmer conditions later in the season (steckel et al., 2004). nevertheless, amaranthus retroflexus is also able to germinate in early spring with lower rates (baskin and baskin, 1985). as the emergence of seedlings is strongly affected by their parent plants, our findings also represent conditions the populations experienced near göttingen, in central germany (kigel et al., 1977). the number of emerged a. retroflexus seedlings almost doubled in both climate chambers in the third replication. such high emergence rates are untypical for a. retroflexus under low temperatures (guillemin et al., 2013). conditions for emergence were equal in all replications. thus, we expect the dormancy status of the weeds had changed over time of storage. seeds were stored for a total of 4 years and 2 months before the last replication started. some other weed species show lower dormancy the longer their seeds were buried (baskin and baskin, 1985). our findings that a. retroflexus changed its dormancy status over time of storage should be verified by another experiment, as dormancy changes were not reported by other authors (thompson et al., 1997; omami et al., 1999). seedlings of e. crus-galli are able to grow faster than maize once they find sufficient conditions during the first four weeks. we found that e. crus-galli seedlings were less dependent on temperature and humidity, when compared to maize seedlings. chauhan and johnson (2011) reported similar results. the resulting high v50 ratio is a good indicator for the effects of higher thermal time (gardarin et al., 2009). the relatively low emergence rate of setaria viridis seedlings confirm that the species typically germinates under warmer conditions later in the season (dekker, 2003) and not simultaneously with the sowing of maize as it was defined by the experiment. this also explains why more seedlings emerged considerably earlier under climate change conditions. current temperature conditions in northern parts of central europe are still below the considered optimum for the species (walck et al., 2011). therefore, setaria viridis will likely benefit more than the other tested weed species from higher temperatures at emergence. development without crop competition this series of measurements was performed to study various parameters of the weeds at the most important stages of their life and to fig. 4: comparison of the plant height of weeds grown in pots and tubs at the end of mid-growth phase for both climate chambers for the three replications. tubs pots 10 20 30 40 pl an t h ei gh t [ cm ] amaranthus retroflexus tubs pots 20 40 60 80 10 0 pl an t h ei gh t [ cm ] echinochloa crus−galli tubs pots 10 20 30 40 50 60 pl an t h ei gh t [ cm ] setaria viridis 240 k. peters, b. gerowitt provide answers concerning how weeds perform individually under climate change conditions. we found distinct differences between measurements taken at the time of emergence, at the end of the early and mid-growth phase and reproduction phase. the tested weeds balanced their growth and biomass allocation according to abiotic conditions and competition from neighbouring plants. their response to the predicted future conditions started to become species-specific with the end of the mid-growth phase. early and mid-growth phase: all tested weed species benefitted from the predicted future conditions at the end of the mid-growth phase. to conclude, greater plant heights under the climate change scenario suggest an increase in vegetative growth and thus, also enhanced interference capabilities of the weeds during the first weeks of their development. this may give them an advantage over single maize plants. for a. retroflexus, we observed no differences in panicle onset speed between the climate chambers, although findings by knezevic et al. (2001) and steckel et al. (2004) suggest a different relationship. e. crus-galli had an earlier onset of flowers and a longer flowering time, which increases the opportunities for outcrossing (clements and ditommaso, 2011). this is in accordance with other studies (potvin, 1986). these phenomena can also occur when seedlings emerge later in the season. thus, climate change conditions may accelerate the life cycle as described by potvin (1986) for e. crus-galli and by oryokot et al. (1997), knezevic et al. (2001) and hyvönen (2011) for a. retroflexus. this process results in more fertile seeds at the end of the reproductive stage, but probably not in more biomass. by contrast, setaria viridis develops panicles after midsummer when photoperiods are getting shorter (dekker, 2003). plants grown in the climate chambers set panicles at the same time as would plants grown under natural conditions (dekker, 2003). we observed that the earlier seedlings emerge, the longer they delay development of reproductive parts. therefore, later emerging arable plants have a shorter vegetative development period (forcella et al., 2000; dekker, 2003). similar behaviour was reported for a. retroflexus (costea et al., 2004), but was not confirmed by our experiment for this species. reproduction phase: amaranthus retroflexus plants in the climate chambers were creeping near the ground, grew sideward and tillered more, when compared to plants grown in the fields. this growth habit was likely a response on low red and far-red light levels in the climate chamber (costea et al., 2004; gimplinger and kaul, 2009). plants grown outside from seeds of the same population did show a normal growth habit (peters and gerowitt, 2014). this could explain why a. retroflexus was less competitive in comparison to the other two weed species. some a. retroflexus plants also seemed to lack senescence. unlike e. crus-galli and s. viridis, this species did not match its life cycle to the length of the season (sauer, 1967). even towards the end, some plants were still green and had not finished ripening. some plants just grew and grew. as a result, we have not found differences in seed production and we can only partly confirm with hyvönen (2011), who reported that raised temperatures enhance seed output but not overall growth. echinochloa crus-galli is better suited to the predicted future conditions because plants had more panicles and tillers under these conditions. this resulted in totally more seeds per plant (barrett and wilson, 1981). if arable cropping conditions allow, the species will likely extend its seed bank. this is especially the case under continuous maize cropping (fried et al., 2010). since we have not found any differences in vegetative biomass, we assume that e. crus-galli mainly invests the possible benefits of warmer conditions into reproduction. setaria viridis is well adapted to current and future climatic conditions and can make use of its phenotypic plasticity (dekker, 2003). we observed higher variances of plant parameters throughout the replications under the climate change scenario. since several biotypes are typical for the genus setaria, the high variance reflects genetic plasticity within the used setaria population (dekker, 2003). a future climate may select for certain ecotypes that are better suited for future conditions, thus enhancing fitness. our results are in accordance with swanton et al. (1999), who reported less biomass but increased reproductive output under warmer conditions. for s. viridis, we determined also a slight increase in seed production per plant and less dry matter content per plant in the chamber with predicted future conditions. however, higher temperature difference between the climate chambers may have been needed for significant results (douglas et al., 1985; swanton et al., 1999). in summary, although the responses were species-specific, e. crusgalli and s. viridis had higher reproductive output under the scenario with predicted future conditions. this may lead to better long-term population development, as the weeds are able to build-up their seed banks. development under crop competition this series of measurements was performed to study the effect of maize competition on the vegetative development of the weeds under climate change conditions. the broad phenotypic responses of e. crus-galli and s. viridis when grown with maize suggest that they likely will increase their success in maize when cropped under climate change conditions. plants of a. retroflexus grew even more deformed as single plants grown in the smaller pots (see above). contrarily, otte et al. (2006) reported that a. retroflexus is highly competitive even when shading occurs. it is also possible that temperatures in the climate chambers were still too low for the plants (gimplinger and kaul, 2009; hyvönen, 2011; oveisi et al., 2013). under the maize competition, s. viridis was able to balance growth to match optimally the low light levels. plants grown in the smaller pots showed no differences in habit and other phenological properties compared to plants grown together with maize. this result confirms studies of dekker (2003). however, we cannot confirm the reported result that s. viridis responds to increasing shade levels by reducing tiller production (douglas et al., 1985; dekker, 2003). the determined high phenotypic plasticity may enable the weed to adapt to a broad range of changing conditions. of the three weed species investigated, e. crus-galli was the least affected in growth and development by the shading of maize. compared to plants grown individually in the smaller pots, the weed balances the low light availability with growing upwards instead of tillering. the species is able to adapt fast to ecological factors such as shading, because of its broad genome, proliferation and crop mimicking. nevertheless, barrett and wilson (1981) also showed that high phenotypic plasticity may result in differences in tiller production of e. crus-galli. implications for crop protection from the predicted future conditions echinochloa crus-galli profited most, followed by setaria viridis. amaranthus retroflexus was the least benefiting species of the three tested weeds possibly due to the artificial conditions in the climate chambers. in detail, our study revealed that each weed species responded differently to changes in climate conditions. narrow crop rotations and continuous cropping of maize are likely in central europe in the future (mehrtens et al., 2005; weber and gut, 2005; fried et al., 2010). in subsequent years, s. viridis and climate change and three maize weed species 241 e. crus-galli will likely build up their seed banks due to better emergence and higher seed output (barrett and wilson, 1981; dekker, 2003). better growth and faster vegetative development when grown within maize under climate change conditions will also lead to better long-term population development of the weeds in the future (douglas et al., 1985). the results of our study indicate that under future climate conditions various weed management strategies are needed to ensure control of the tested weeds (olesen and bindi, 2002; peters et al., 2014). for agricultural purposes, we suggest to integrate management practices such as variations in the sowing date of maize or in the choice of cultivars that result in timely harvesting, and thus reduce the chance for the late emerging weeds to fully develop and produce seeds under climate change conditions. a challenge for maize breeding is to select for fast growing and developing cultivars, since our study revealed a trade-off between the vegetative growth of the weed species and their generative reproduction when cropped together with maize (sarabi et al., 2011). farmers themselves should avoid short crop rotations with maize alone or other late spring sown crops. continuous cropping of this type of crop will select for particularly prolific biotypes of s. viridis and e. crus-galli (barrett and wilson, 1981; dekker, 2003). furthermore, we propose that necessary herbicide treatments should be applied late in the season to cover late emerging weed cohorts with high temperature requirements, especially those of s. viridis (dekker, 2003; beckie and tardif, 2012). conclusions climate change exerts impacts on weeds during their whole life cycle. in order to get insight into underlying processes, it is necessary to determine biological parameters at the time of emergence, early growth and at the time of reproduction. as a result, climate mediated alterations in the measured biological and demographic attributes allow predictions of long-term population development of the weed species (fried et al., 2010; peters et al., 2014). biological properties and demographic data can also be used to predict the population development under future conditions and to improve bioclimatic models (peters and gerowitt, 2014). furthermore, with future climate change costly crop protection measures at different development stages of the weeds may be needed for successful weed management. in order to continue high production under future conditions, crop protection has to be adapted to the biological responses of weeds to climate change. acknowledgements this study was supported by the ministry for science and culture of lower saxony within the network kliff − climate impact and adaptation research in lower saxony. we thank ingolf gliege and martina goltermann for assisting in the experiments. references baayen, r.h., davidson, d.j., bates, d.m., 2008: mixed-effects modeling with crossed 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survey of weeds that are increasingly spreading in europe. agron. sustain. dev. 25, 109-121. address of the corresponding author: kristian peters, university of rostock, faculty of agricultural and environmental sciences, crop health, satower straße 48, d-18051 rostock, germany. e-mail: kristian.peters@uni-rostock.de journal of applied botany and food quality 89, 57 (2016) buchbesprechung kompendium phytopharmaka – qualitätskriterien und verordnungsbeispiele theodor dingermann die 7. völlig neu bearbeitete und erweiterte auflage von „kompendium phytopharmaka“ liefert eine aktuelle zusammenstellung der in deutschland zurzeit verfügbaren rationalen phytopharmaka. darüber hinaus wird in einem einleitenden kapitel auf die wichtigsten grundlagen zur herstellung und entwicklung phytopharmazeutischer produkte kurz eingegangen. das buch liefert außerdem wichtige informationen über die qualitätsmerkmale pflanzlicher arzneimittel, benennt kriterien, nach denen die wahl des jeweiligen präparats erfolgen sollte, und erläutert die aktuelle verordnungssituation. als beispiele für gut dokumentierte pharmaka, die den höchsten qualitätskriterien entsprechen, und deren wirkung anhand wissenschaftlicher studien belegt ist, listet das kompendium außerdem rund 160 präparate in 18 indikationsbereichen auf. der autor beschreibt nicht nur den einsatz von arzneipflanzen in der phyto therapie, sondern geht auch auf die unterschiedlichen verwendungen in gesundheitstees, in nahrungsergänzungsmitteln, der homöopathie, der anthroposophie und anderen alternativen therapierichtungen ein. dabei kommen auch die regulatorischen aspekte und der unterschiedliche zulassungsstatus der jeweiligen produktgruppen zur sprache. in dem vorliegenden kompendium phytopharmaka sind ausschließlich nur diejenigen präparate aufgelistet, die eigene daten zur wirksamkeit in „good clinical practice“ (gcp) konformen studien erhoben und damit ihre wirksamkeit produktspezifisch unter beweis gestellt haben. in dem anhang sind alle präparate gemäß der international classification of primary care (icpc)-nomenklatur sowie alphabetisch aufgelistet. darüber hinaus findet sich auch noch eine alphabetische sortierung der herstellerfirmen mit den von ihnen entwickelten phytopharmaka. dr. h. schulz e-mail: hartwig.schulz@jki.bund.de bibliografie: theodor dingermann, kompendium phytopharmaka ‒ qualitätskriterien und verordnungsbeispiele, deutscher apotheker verlag, stuttgart, 7. völlig neu bearbeitete auflage 2015, 165 seiten mit 41 farbabbildungen, kartoniert, preis: 14,80 €, isbn: 978-3-76926211-7 journal of applied botany and food quality 88, 97 101 (2015), doi:10.5073/jabfq.2015.088.013 1 department of nutrition and food technology, jordan university of science and technology, irbid, jordan 2 department of nutrition and food technology, al-balqa applied university, salt, jordan 3 department of food science, university of manitoba, winnipeg, manitoba, canada 4 north carolina a&t state university, food microbiology and biotechnology laboratory, greensboro, nc, united states inactivation of cronobacter sakazakii in reconstituted infant milk formula by plant essential oils anas a. al-nabulsi1*, saddam s. awaisheh2, tareq m. osaili1, amin n. olaimat3, razan j. rahahaleh2, fawzi m. al-dabbas2, lina a. al-kharabsheh2, rabin gyawali4, salam a. ibrahim4 (received september 24, 2014) * corresponding author summary this study aimed to screen the in vitro antimicrobial activity of 10 plant essential oils against 4 cronobacter sakazakii strains, and use these oils or their combination to control c. sakazakii cocktail at low (3 log10 cfu/ml) and high (6 log10 cfu/ml) contamination levels in reconstituted infant milk formula (rimf). cinnamon and fir oils were the most inhibitory to c. sakazakii strains with inhibition zone of 32 to 40 mm at 20 μl/disc (the minimum inhibitory concentrations were 0.16 and 0.625 μl/ml, respectively). the addition of each of cinnamon or fir oil at 1 % (v/v) reduced the c. sakazakii numbers in rimf by 0.7-0.8 log10 cfu/ml when inoculated with high contamination level and by 2.5-3.1 log10 cfu/ml when inoculated with low contamination level. however, the combination of cinnamon and fir oils reduced c. sakazakii numbers at both inoculum levels to undetectable levels after 3 h of incubation at 37 °c. the results of the current study indicated that a combination of cinnamon and fir oils has a potent antimicrobial activity which may potentially be used to control c. sakazakii in rimf. introduction cronobacter sakazakii is an opportunistic pathogen that may cause bacteremia, sepsis, meningitis and necrotizing enterocolitis, mainly in neonates, preterm and low birth weight infants, and immunocompromised infants and adults with case fatality rate of 10 to 80 % (nazarowec-white and farber, 1997a; yan et al., 2012). c. sakazakii has the ability to adapt several environmental stresses including chilling, heating, drying and osmotic stresses and natural antibiotics. this characteristic enables the organism to survive in foods and environments to cause foodborne illnesses (lee and jin, 2008; osaili et al., 2008). although c. sakazakii has been isolated from several food sources including dairy products, meat products, vegetable origin food, water and other foods (chap et al., 2009; friedmann, 2007; shaker et al., 2007), the c. sakazakii infection has been linked epidemiologically to the consumption of contaminated powdered infant milk formula (pimf). c. sakazakii can be inactivated during milk pasteurization; however, post-processing contamination of pimf is mainly responsible for the presence of c. sakazakii in these products. furthermore, c. sakazakii has the ability to colonize on the surfaces of rehydrated infant milk formula (rimf) preparation equipment and utensils such as brushes, bottles, and spoons (nazarowec-white and farber 1997a, b). the microbiological safety of pimf is very critical because infants are known to be particularly more vulnerable to foodborne infections (drudy et al., 2006). to reduce the risk of c. sakazakii infections, food and agriculture organization and world health organization (fao and who, 2004) recommended using hot water (≥70 °c) in reconstitution of pimf before feeding infants. however, the improper storage of rimf may permit a significant growth of c. sakazakii which has a short lag time and generation time in rimf (nazarowec-white and farber, 1997b). therefore, incorporation of effective antimicrobials may have the potential to reduce the infection risk of c. sakazakii in infants by contaminated rimf (nair et al., 2004). increasing bacterial resistance to drugs has created problems in the treatment of several diseases. since there is increased concerns regarding the safety of synthetic compounds and overuse of antibiotics as preservatives, interest in the development of effective natural antimicrobials as food preservatives has been increased recently (abee et al., 1995; nair et al., 2004). several studies investigated the inhibitory effects of natural antimicrobials, such as animal-, plant-, and microorganism-derived antimicrobials against foodborne pathogens (tajkarimi et al., 2010; lee and jin, 2008). natural antimicrobial agents derived from plant sources such as essential oils have been recognized and used for centuries in food preservation. edible, medicinal and herbal oils are potent antimicrobials against foodborne pathogens such as listeria monocytogenes, salmonella typhimurium, escherichia coli o157:h7, shigella dysenteriae, bacillus cereus and staphylococcus aureus (tajkarimi et al., 2010). however, few studies have investigated the inhibitory effects of carvacrol, thymol, eugenol and cinnamic acid (lee and jin, 2008) or trans-cinnamaldehyde (amalaradjou et al., 2009) against c. sakazakii. other individual or combined essential oils may have a potential to reduce c. sakazakii in rimf. therefore the objectives of the current study were to: i) screen the antimicrobial activity of 10 medicinal plant essential oils against 4 strains of c. sakazakii, ii) determine the minimum inhibitory concentration (mic) of cinnamon and fir oils against c. sakazakii strains, and iii) use these oils or their combination to control c. sakazakii cocktail at low (3 log10 cfu/ml) and high (6 log10 cfu/ml) contamination levels in rimf. materials and methods preparation of c. sakazakii cultures four strains of c. sakazakii isolated from infant formula in jordan were obtained from the bacterial culture collection of the department of nutrition and food technology at jordan university of science and technology: cs1; cs14; cs15 and cs19 (shaker et al., 2007; chap et al., 2009). each strain of c. sakazakii was kept in brain heart infusion broth (bhi, oxoid, basingstoke, uk) containing 20 % glycerol at -40 °c. the strains were individually maintained on bhi agar slants at 4 °c and transferred bi-weekly to maintain viability. to prepare the working cultures, one loopful of each strain was transferred to 10 ml bhi broth and incubated at 37 °c for 24 h. a volume of 100 μl of the overnight strain cultures was transferred to 10 ml bhi broth and incubated for 20-24 h at 37 °c. a cocktail of c. sakazakii strains was prepared by mixing equal proportions of each of the four strains into a sterile container and diluted with a sterile 0.1 % peptone water (oxoid, basingstoke, uk) to obtain the desired inoculum level. 98 a.a. al-nabulsi, s.s. awaisheh, t.m. osaili, a.n. olaimat, r.j. rahahaleh, f.m. al-dabbas, l.a. al-kharabsheh, r. gyawali, s.a. ibrahim medicinal plants and essential oils extraction the essential oils of 10 different medicinal plants (cinnamon, anise, eucalyptol, chamomile, fenugreek, fennel, fir, pooh, qysoom and rosemary) were obtained from a local extraction facility in amman, jordan. essential oils were extracted by using hydro-distillation procedure. the oils were kept in a sealed dark glass vial at 4 ºc until required. screening of antimicrobial activity of medicinal plant essential oils disc-diffusion method was used to determine the antimicrobial activity of the 10 essential oils against 4 c. sakazakii strains. brief ly, 20 μl of each essential oil was dispensed onto 6 mm-diameter sterile filter paper discs which were dried by air. each disc was placed on tryptic soy agar (tsa, oxoid, basingstoke, uk) plates which were previously inoculated with approximately 5.0 log10 cfu/ ml of the individual c. sakazakii strains. plates were incubated at 37 °c for 24 h and the inhibitory zones were measured in millimeter using a caliper. the synergistic activity of essential oils mixture that showed the maximum antibacterial activity (cinnamon and fir oils) was prepared by mixing equal volumes of each essential oil, and then 20 μl of the mixture was applied onto 6 mm-diameter sterile filter paper (awaisheh, 2013). determination of the minimum inhibitory concentration of cinnamon and fir oils against c. sakazakii strains mics of cinnamon or fir oils were determined using 96-wells microdilution method. a 100 μl of overnight culture containing 6.0 log10 cfu/ml of each c. sakazakii strain and 100 μl of cinnamon and fir oils dilution were added with 200 μl total volume in each well. a two-fold serial dilution was used to yield concentrations of 0.08 to 100 μl/ml essential oils. then, the sealed plates were incubated for 24 h at 37 °c and c. sakazakii growth in each well was measured by reading the respective absorbance at 600 nm using a microplate reader (elx 800, biotek, highland park, vt, usa). absolute dimethyl sulfoxide (dmso) containing essential oils added to sterile media and dmso added to the bacterial suspension were used as negative and positive controls, respectively. mic of the essential oils is the lowest dilution which inhibits the bacterial growth (awaisheh, 2013). relative antimicrobial activity of cinnamon and fir oils combination to selected antibiotics the inhibitory efficacy of cinnamon and fir oils combination at 1 % (v/v) against c. sakazakii cocktail was compared with selected antibiotics using disc-diffusion method as described above. antibiotic discs (6 mm in diameter) of 5 reference antibiotics including: amikacin (30 μg), azithromycin (15 μg), ceftriaxone (30 μg), gentamicin (10 μg), and nalidixic acid (30 μg) were obtained from arcomex medical supplies company (amman, jordan). the ratio of the inhibition zone (mm) produced by the cinnamon and fir oils combination and the reference antibiotics was used to express the relative antimicrobial activity of cinnamon and fir oils combination. inactivation of c. sakazakii in reconstituted infant milk formula model by cinnamon and fir oils or their combination commercial pimf was reconstituted according to the manufacturer’s instructions with sterile distilled water. under aseptic conditions, approximately 7.7 g pimf was reconstituted with 60 ml water and mixed using a magnetic stirrer at 350 rpm for 25 min at 100 °c until completely dissolved. a cocktail culture of 4 strains c. sakazakii by mixing equal volume from each strain at 4.0 log or 7.0 log cfu/ml was inoculated into 10 ml of rimf (control) or rimf containing either 1 % (v/v) of cinnamon oil, fir oil, or cinnamon and fir oils mixture. the samples were incubated at 37 °c for 6 h and the c. sakazakii numbers at 0, 3 and 6 h were carried out as described by osaili et al. (2010) with minor modification. a 100 μl of each sample was spread on plates of violet red bile agar (vrba, oxoid, basingstoke, uk) supplemented with 2 % glucose which were incubated at 37 °c for 24 h. statistical analysis the experiments were replicated 4 times using two samples per experiment (n=8). all data represents the mean value ± standard deviation (sd). one-way anova and multiple range tests were used to evaluate differences among means using the least significance difference (lsd) test at a significance level of p < 0.05. results and discussion antimicrobial activity of 10 medicinal plant essential oils the antimicrobial activities of essential oils obtained from 10 different plant species against 4 c. sakazakii strains were determined (tab. 1). these plants showed to have some antimicrobial properties against wide range of foodborne pathogens (awaisheh, 2013). the average zone of inhibition with 20 μl of essential oils ranged from 0.0 to 40.0 mm. cinnamon and fir oils were the strongest antimicrobials against tested c. sakazakii strains with a range of inhibition zone of 32 to 40 mm. however, chamomile and rosemary oils showed weak antimicrobial activities against c. sakazakii strains with inhibition zone range of 4.5 to 6.0 and 3.0 to 8.0, respectively. while other plant (anise, eucalyptol, fennel, fenugreek, pooh, and qysoom) essential oils did not exhibit antimicrobial activity against tested c. sakazakii strains. al-nabulsi et al. (2009) also found that herbal infant teas such as chamomile and fennel supported growth of c. sakazakii at 37 and 21 °c. the antimicrobial activity tested essential oils (except cinnamon oil) in the present study against c. sakazakii strains tab. 1: antimicrobial activity of 10 medicinal plant essential oils (20 μl/disc) against 4 c. sakazakii strains using disc diffusion method at 37 °c. inhibition zone diameter (mm) 1, 2 anise cinnamon chamomile eucalyptol fennel fenugreek fir pooh rosemary qysoom c. sakazakii cs1 0.0±0.0c 32.0±3.1a 5.0±0.2b 0.0±0.0c 0.2±0.1c 0.2±0.1c 34.0±2.1a 2.0±0.1c 3.0±0.2bc 2.0±0.1c c. sakazakii cs14 1.0±0.1c 35.0±2.9a 5.0±0.1b 0.2 ± 0.1c 0.5±0.1c 1.0±0.1c 32.0±2.1a 1.5±0.1c 3.0±0.2bc 1.5±0.1c c. sakazakii cs15 0.5±0.1c 40.0±3.4a 4.5±0.2b 0.5 ± 0.1c 1.0±0.1c 0.0±0.0c 40.0±1.8a 1.0±0.1c 4.5±1.0bc 1.0±0.1c c. sakazakii cs19 1.0 ±0.1c 38.0±2.1a 6.0±0.2b 0.5 ± 0.1c 1.0±0.1c 1.0±0.1c 36.0±2.4a 1.0±0.1c 8.0±0.4b 1.0±0.1c 1 values are mean of 4 replicates ± standard deviation 2 values with different letters in the same row are significantly different (p < 0.05). control of c. sakazakii in infant milk formula 99 c. sakazakii have not been investigated as they did not show inhibitory effect against c. sakazakii strains. however, several studies reported their inhibitory activity against other foodborne pathogens (bağci and diğrak, 1996; kon and rai, 2012). the findings of the current study are consistent with those of prabuseenivasan et al. (2006) and kon and rai (2012) who found that cinnamon oil had the strongest inhibitory activity among 21 to 35 plant essential oils against gram-negative and gram-positive bacteria. bağci and diğrak (1996) also found that essential oil from different species of fir had antimicrobial activity against bacillus spp., pseudomonas aeruginosa, l. monocytogenes, klebsiella pneumoniae, enterobacter aerogenes and s. aureus. minimum inhibitory concentration of cinnamon and fir oils against c. sakazakii strains based on the results of disc diffusion method for screening of anti microbial activity of 10 plant essential oils against c. sakazakii strains, cinnamon and fir oils showed the highest zone of inhibition and these were selected to determine their mic values against c. sakazakii strains (tab. 2). the mics of cinnamon and fir oils were 0.16 and 0.62 μl/ml, respectively, against all tested c. sakazakii strains. the mics of essential oils such as carvacrol, thymol, eugenol and cinnamic acid was found to be 1.25, 1.25, 5.0 and >5.0 mmol/l, respectively against 2 c. sakazakii strains (lee and jin, 2008). the results of current study are similar to those reported by ghosh et al. (2013) who found that mic of cinnamaldehyde was 0.263 mg/ml against c. sakazakii. similarly, kon and rai (2012) found that mic of cinnamon oil was 0.2 mg/ml against s. aureus. the mic of fir oil in the current study was slightly lower than those reported by oh et al. (2007) who found that the mics of fir oil against e. coli and staphylococcus epidermidis were 2.05 and 3.81 mg/ml, respectively. relative antimicrobial activity of cinnamon and fir oils combination to selected antibiotics the effectiveness of cinnamon and fir oils combination on inhibition of c. sakazakii cocktail was compared to the standard antibiotics (tab. 3). the cinnamon and fir oils combination showed higher antimicrobial activity against c. sakazakii than the reference antibiotics. the combination of cinnamon and fir oils significantly inhibited the growth of c. sakazakii cocktail by 38.5 mm on plates. however, ceftriaxone had the strongest inhibitory effect (20.5 mm) among antibiotics tested against c. sakazakii cocktail, while gentamicin showed the weakest activity (8.0 mm). the relative antimicrobial activity of cinnamon and fir oils combination to gentamicin, nalidixic acid, amikacin, azithromycin and ceftriaxone were 482 %, 412 %, 405 %, 385 % and 188 %, respectively. although the inhibitory effect of cinnamon and fir oil combinations against c. sakazakii was obvious, it should be noted that the concentration of the oil mixtures (20 μl, approximately 20 mg) was greater than the concentrations of the standard reference antibiotics (10 to 30 μg). previous studies have demonstrated the antimicrobial resistance of c. sakazakii to the antibiotics (al-nabulsi et al., 2011; farajnia et al., 2009; saaed and mussalam, 2011). therefore, using natural antimicrobial agents such as cinnamon and fir oils may be used as alternative to overcome emerging of antibiotics resistance thus reducing the risk associated with c. sakazakii infection. inactivation of c. sakazakii in rimf by cinnamon and fir oils or their combination the inhibitory effects of cinnamon and fir oils or their combination against a 4 strains c. sakazakii cocktail at high (6.0 log10 cfu/ml) or low (3.0 log10 cfu/ml) inocula in rimf during 6 h of incubation at 37 °c was investigated (tab. 4). in the control samples (without essential oils), c. sakazakii gradually increased from 3.1 and 6.1 log10 cfu/ml to reach 6.3 and 8.4 log10 cfu/ml, respectively after 6 h at 37 °c. the addition of 1 % cinnamon or fir oils alone reduce the viability of c. sakazakii with low inoculum level by 3.1 and 2.5 log10 reduction of c. sakazakii, respectively, compared to the control. similarly, with higher initial inoculum level (6.0 log10 cfu/ ml), cinnamon or fir oils reduced the numbers of c. sakazakii by 0.7 and 0.8 log10 cfu/ml, respectively, after 6 h. however, the c. sakazakii cells were not detected when rimf incorporated with 1 % of cinnamon and fir oils mixture after 3 h of incubation at both inoculum levels. this demonstrates that the combination of cinnamon and fir oils could be used as a practical approach to reduce the risk of c. sakazakii in rimf and to improve the safety of these consumable products. the antimicrobial effect of cinnamon oil has been well studied against e. coli o157:h7, salmonella, and l. monocytogenes (oussalah et al., 2007). but there are still limited studies on c. sakazakii strain tab. 2: minimum inhibitory concentration (mic) values of cinnamon and fir oils against 4 c. sakazakii strains at 37 °c. mic (μl/ml)1 cinnamon fir c. sakazakii cs1 0.160 0.625 c. sakazakii cs14 0.160 0.625 c. sakazakii cs15 0.160 0.625 c. sakazakii cs19 0.160 0.625 1 values are mean of 4 replicates tab. 3: relative antimicrobial activity for combination of cinnamon and fir oils (20 μl/disc) to selected antibiotics against a 4 strains c. sakazakii cocktail using disc diffusion method at 37 °c. c. sakazakii cocktail reference antibiotics (concentration (μg/disc))1 amikacin azithromycin ceftriaxone gentamicin nalidixic acid (30 μg) (15 μg) (30 μg) (10 μg) (30 μg) inhibition zone diameter 38.5±3.5a 9.5±1.5c 10.0±2.1c 20.5±2.2b 8.0±1.5c 9.0±1.4c (mm) 2, 3 relative antimicrobial 100 % 405 % 385 % 188 % 482 % 412 % activity 4 1 concentration of the oil mixtures is greater than concentrations of the standard reference antibiotics. 2 results are means of 4 replicates ± standard deviation. 3 values with different letters in the same row are significantly different (p < 0.05). 4 relative antimicrobial activity of cinnamon and fir oils mixture = (inhibition zones of cinnamon and fir oils mixture ÷ inhibition zones of antibiotic) x 100 % cinnamon and fir oils mixture (20 μl/disc)1 100 a.a. al-nabulsi, s.s. awaisheh, t.m. osaili, a.n. olaimat, r.j. rahahaleh, f.m. al-dabbas, l.a. al-kharabsheh, r. gyawali, s.a. ibrahim essential oils against c. sakazakii in rimf. the results of current study are similar to those reported by amalaradjou et al. (2009) who found that 0.5 % trans-cinnamaldehyde reduced the c. sakazakii numbers to undetectable levels (> 6 log10 cfu/ml reduction) by 4 h at 37 or 23 °c and by 10 h at 8 or 4 °c). it has been reported that mechanisms of cinnamaldehyde (the major component of cinnamon oil) action is related to inhibition of different enzymes involved in cytokinesis, action as an atpase inhibitor and/or disturb the cell membrane (hyldgaard et al., 2012). kim et al. (2009) also reported that muscadine seed extracts reduced c. sakazakii numbers (6 log10 cfu/ml) to undetectable levels by 1 h at 37 °c. osaili et al. (2009) found that c. sakazakii had lower growth in wheat-based infant follow-on formulas reconstituted with grape or apple juices compared to those reconstituted with water or milk 25 and 37 °c. the potent antimicrobial activity of these plant compounds is attributed due to the presence of several phenolic compounds, organic acids, and variety of active components in essential oils (kim et al., 2009; amalaradjou et al., 2009). the phenolic compounds may cause bacterial membrane damage and disrupt the cell wall peptidoglycan. also, the hydroxyl group in phenolic compounds may bind the active sites of enzymes and change their substrate affinity. moreover, the lipid solubility of phenolic compounds and their degree of steric hindrance may also contribute to their overall antimicrobial activity (ceylan and fung, 2004). conclusions among 10 plant essential oils, cinnamon and fir oils showed the strongest antimicrobial activity against c. sakazakii strains with a range of inhibition zone of 32 to 40 mm. the relative antimicrobial activity (inhibition zone ratio) of 1 % cinnamon and fir oils mixture compared to amikacin, azithromycin, ceftriaxone, gentamicin and nalidixic acid ranged from 188 % to 482 % against c. sakazakii cocktail. cinnamon and fir oils combination at 1 % (v/v) eliminated the c. sakazakii numbers (> 6 log10 cfu/ml reduction) by 3 h at 37 °c. fao/who (2004) recommended that rimf should be stored at room temperature for less than 2 h. it is evident from the results of this study that combination of cinnamon and fir oils caused a strong and rapid antimicrobial effect against c. sakazakii, therefore cinnamon and fir oils combination may have a potential to control c. sakazakii in rimf. however, effect of cinnamon and fir oils on the sensory attributes of rimf is required. references abee, t., krockel, l., hill, c., 1995: bacteriocins: modes of action and potentials in food preservation and control of food poisoning. int. j. food microbiol. 28, 169-185. al-nabulsi, a.a., osaili, t.m., zain elabedeen, n.a., jaradat, z.w., shaker, r.r., kheirallah, k.a., tarazi, y.h., holley, r.a., 2011: impact of environmental stress desiccation, acidity, alkalinity, heat or cold on antibiotic susceptibility of cronobacter sakazakii. int. j. food microbiol. 146, 137-143. al-nabulsi, a.a., osaili, t.m., shaker, r.r., olaimat, a.n., ayyash, m.m., holley, r.a., 2009: survival of cronobacter species in reconstituted herbal infant teas and their sensitivity to bovine lactoferrin. j. food sci. 74, 479-484. amalaradjou, m.a., hoagland, t.a., venkitanarayanan, k., 2009: inactivation of enterobacter sakazakii in reconstituted infant formula by trans-cinnamaldehyde. int. j. food microbiol. 129, 146-149. awaisheh, s.s., 2013: efficacy of fir and qysoom essential oils, alone and in combination, in controlling listeria monocytogenes in vitro and in rte meat products model. food control 34, 657-661. bağci, e., diğrak, m., 1996: antimicrobial activity of essential oils of some abies (fir) species from turkey. flav. fragr. j. 11, 251-256. ceylan, e., fung, d.y.c., 2004: antimicrobial activity of spices. j. rapid meth. autom. microbiol. 12, 1-55. chap, j., jackson, p., siqueira, r., gaspar, n., quintas, c., park, j., osaili, t., shaker, r., jaradat, z., hartantyo, s.h.p., abdullah sani, n., estuningsih, s., forsythe, s.j., 2009: international survey of cronobacter sakazakii and other cronobacter spp. in follow up formulas and infant foods. int. j. food microbiol. 136, 185-188. drudy, d., mullane, n.r., quinn, t., wall, p.g., fanning, s., 2006: enterobacter sakazakii: an emerging pathogen in powdered infant formula. clin. infect. dis. 42, 996-1002. farajnia, s., alikhani, m.y., ghotaslou, r., naghili, b., nakhlband, a., 2009: causative agents and antimicrobial susceptibilities of urinary tract infections in the northwest of iran. int. j. infect. dis. 13, 140-144. food and agriculture organization/world healthorganization, 2004: joint fao/who workshop on enterbacter sakazakii and other microorganisms in powdered infant formula, geneva, 2.-5. feb. 2004. friedemann, m., 2007: enterobacter sakazakii in food and beverages (other than infant formula and milk powder). int. j. food microbiol. 116, 1-10. ghosh, i.n., patil, s.d., sharma, t.k., srivastava, s.k., pathania, r., navani, n.k., 2013: synergistic action of cinnamaldehyde with silver nanoparticles against spore-forming bacteria: a case for judicious use of silver nanoparticles for antibacterial applications. int. j. nanomedicine 8, 4721-4731. hyldgaard, m., mygind, t., meyer, r.l., 2012: essential oils in food preservation: mode of action, synergies and interactions with food matrix components. front. microbiol. 3(12), 1-24. kim, t.j., silva, j.l., weng, w.l., chen, w.w., corbitt, m., jung, y.s., 2009: inactivation of enterobacter sakazakii by water-soluble muscadine seed extracts. int. j. food microbiol. 129, 295-299. kon, k., rai, m., 2012: antibacterial activity of thymus vulgaris essential oil alone and in combination with other essential oils. nus. biosci. 4, tab. 4: c. sakazakii numbers (log10 cfu/ml) in rimf in the presence of 1% (v/v) of cinnamon oil, fir oil, or their combination at 37 °c for 6 h.1, 2 treatments c. sakazakii numbers (log10 cfu/ml) at low level c. sakazakii numbers at (log10 cfu/ml) at high level 0 h 3 h 6 h 0 h 3 h 6 h control 3.08±0.22a 4.70±0.50a 6.26±0.40a 6.08±0.34a 7.11±0.61a 8.37±0.43a cinnamon oil 3.04±0.25a 3.00±0.19b 3.18±0.25b 6.04±0.45a 6.07±0.55b 7.67±0.51b fir oil 3.08±0.15a 3.18±0.23b 3.78±0.21b 6.04±0.50a 6.20±0.45a 7.56±0.66b cinnamon and fir oils mixture 3.05±0.12a nd nd 6.08±0.52a nd nd 1 values are means of 4 replicates ± standard deviation. 2 values with different letters in the same column are significantly different (p < 0.05). nd = not detected (detection level was < 1 log10 cfu/ml) control of c. sakazakii in infant milk formula 101 50-56. lee, s.y., jin, h.h., 2008: inhibitory activity of natural antimicrobial compounds alone or in combination with nisin against enterobacter sakazakii. lett. appl. microbiol. 47, 315-321. nair, m.k.m., joy, j., venkitanarayanan, k., 2004: inactivation of enterobacter sakazakii in reconstituted infant formula by monocaprylin. j. food prot. 67, 2815-2819. nazarowec-white, m., farber, j.m., 1997a: enterobacter sakazakii: a review. int. j. food microbiol. 34, 103-113. nazarowec-white, m., farber, j.m., 1997b: incidence, survival, and growth of enterobacter sakazakii in infant formula. j. food prot. 60, 226-230. oh, h.j., ahn, h.m., so, k.h., kim, s.s., yun, p.y., jeon, g.l., riu, k.z., 2007: chemical and antimicrobial properties of essential oils from three coniferous trees abies koreana, cryptomeria japonica, and torreya nucifera. j. appl. biol. chem. 50, 164-169. osaili, t.m., shaker, r.r., ayyash, m.m., al-nabulsi, a.a., forsythe, s.j., 2009: survival and growth of cronobacter species (enterobacter sakazakii) in wheat-based infant follow-on formulas. lett. appl. microbiol. 48, 408-412. osaili, t.m., al-nabulsi, a.a., shaker, r.r., ayyash, m.m., olaimat, a.n., abu al-hasan, a.s., qadora, k.m., holley, r.a., 2008: effects of extended dry storage in powdered infant milk formula on susceptibility of enterobacter sakazakii to hot water or ionizing irradiation. j. food prot. 71, 934-939. osaili, t.m., al-nabulsi, a.a., shaker, r.r., al-holy, m.m., alhaddaq, m.s., olaimat, a.n., ayyash, m.m., al ta’ani, m.k., forsythe, s.j., 2010: efficacy of the thin agar layer method for the recovery of stressed cronobacter spp. (enterobacter sakazakii). j. food prot. 73, 1913-1918. oussalah, m., caillet, s., saucier, l., lacroix, m., 2007: inhibitory effects of selected plant essential oils on the growth of four pathogenic bacteria: e. coli o157:h7, salmonella typhimurium, staphylococcus aureus and listeria monocytogenes. food control 18, 414-420. prabuseenivasan, s., jayakumar, m., ignacimuthu, s., 2006: in vitro antibacterial activity of some plant essential oils. bmc complem. altern. med. 6, 39. saeed, h.a., musallam, r.m., 2011: first report on enterobacter sakazakii from sudanese patients. afr. j. microbiol. res. 5, 2374-2379. shaker, r., osaili, t., al-omary, w., jaradat, z., al-zuby, m., 2007: isolation of enterobacter sakazakii and other enterobacter sp. from food and food production environments. food control 18, 1241-1245. tajkarimi, m.m., ibrahim, s.a., cliver, d.o., 2010: antimicrobial herb and spice compounds in food. food control 21, 1199-1218. yan, q.q., condell, o., power, k., butler, f., tall, b.d., fanning, s., 2012: cronobacter species (formerly known as enterobacter sakazakii) in powdered infant formula: a review of our current understanding of the biology of this bacterium. j. appl. microbiol. 113, 1-15. address of the corresponding author: anas a. al-nabulsi, department of nutrition and food technology, jordan university of science and technology, irbid, jordan e-mail: anas_nabulsi@just.edu.jo © the author(s) 2015. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). 6th international symposium breeding research on medicinal and aromatic plants (breedmap 6) in quedlinburg, germany june 19-23, 2016 preliminary program sunday, june 19, 2016 19.00 plenary lecture 19.45 get-together-party monday, june 20, 2016 9.30 to 18.40 lectures and poster session 19:30 reception given by the mayor of quedlinburg tuesday, june 21, 2016 8.30 to 18.00 lectures and workshop 19.00 symposium dinner wednesday, june 22, 2016 9.00 to 13.10 lectures and workshop thursday, june 23, 2016 8.00 to 18.00 excursions how to get there by car: coming from the b6, take exit „quedlinburg-ost"and continue on l66 towards quedlinburg. after 2km, you get to a roundabout, here follow signs to gernrode/ballenstedt (not quedlinburg). at the next roundabout (after 3km), take the 1st exit and continue on l242 towards quedlinburg. when entering quedlinburg, leave the roundabout at the 1st exit (sign „julius kühn-institute") and immediately turn right into the jki premises. by train: in front of the quedlinburg railway station, you will � nd several bus stops. take the bus numbers 10 or 31 and get o� at the stop called „moorberg" (it is behind a roundabout and there is a petrol station on your right). cross the road, turn right and you will � nd yourself at the entrance to the julius kühn institute. or take the bus numbers 32 or 318 and get o� at the stop called „moorberg". now you have to cross the roundabout. you can buy tickets on the bus. journey time is 5 minutes. „moorberg" is a request stop. so, you have to indicate that you want to get o� at the next stop. if you prefer to walk, it will take you appr. 30 minutes. by plane: the next airport is airport leipzig-halle. from airport by train via connecting stations halle/s. and halberstadt to quedlinburg railway station. for time table see http://www.bahn.de/p_en/view/index.shtml the o� cial language of the congress is english. no simultaneous translation will be provided. for more information and registration see http://breedmap6.jki.bund.de registration starts on line from october 15, 2015 we are pleased to invite you to the 6th international symposium “breeding research on medicinal and aromatic plants” (breedmap 6) at quedlinburg, germany from june 19 23, 2016. this meeting continues a series of international symposia for breeding research on medicinal and aromatic plants, which started in 1996. open to all aspects of basic and applied research in plant breeding, the conferences place emphasis clearly on medicinal and aromatic plants. this breedmap 6 symposium will be organized by the julius kuehn institute, federal centre for cultivated plants (jki) in collaboration with the leibniz institute for plant genetics and crop plant research (ipk) and the society for medicinal plant and natural product research (ga). customers all over the world are interested in products based on natural sources. there is a permanent demand for high-quality products. prerequisite are new varieties and lines of medicinal and aromatic plants with better resistance to biotic and abiotic stress, with adaptation to di� erent conditions in cultivation, and � nally with an increase of active principles. in many cases we do have a breakthrough but for a broad range of medicinal plants much can still be done in order to improve quality and quantity and to get constant high levels of active principles. the breedmap 6 symposium will present the international platform to share new results and techniques with an international audience to create new ideas and fruitful collaborations in this promising � eld. the symposium will will focus on the following main topics • next generation methods and chances for medicinal and aromatic plants • cell, tissue and organ culture, cryopreservation, endophytes • resistance breeding and new phytopathogens • improvement of organisms for bioreactors and photo bioreactors • ex situ and in situ genetic resources – protection and use by colleting practice – cultivation of new species • plant breeding and plant analytics • cbd, nagoya protocol, eu regulations • workshop a: breeding endophytes, kayser, o. (technical university dortmund, germany) • workshop b: pyrrolizidine alkaloids – a problem? marthe, f. (julius kuehn institute, quedlinburg, germany) organizers international scientif ic committee • marthe, frank (germany) • carlen, christoph (switzerland) • dudai, nativ (israel ) • kayser, oliver (germany) • kulkarni, raghavendra (india) • lohwasser, ulrike (germany) • montanari, ilio (brazil ) • németh-zámboriné, eva (hungary) • novak, johannes (austria) • shikov, alexander (russia ) • vogel, hermine (chile) • wident, mathieu (france) • wolfender, jean-luc (switzerland) keynote speaker • gosal, s.s. (punjab agricultural university, ludhiana, india) • novak, j. (university of veterinary medicine, vienna, austria) • rodeva, r. (institute of plant physiology and genetics, so� a, bulgaria) • vogel, h. (universidad de talca, chile) journal of applied botany and food quality 87, 9 15 (2014), doi:10.5073/jabfq.2014.087.002 1 college of biological sciences and technology, beijing forestry university, beijing, china 2 research institute of forestry, chinese academy of forestry, beijing, china diurnal fluctuation of volatile compounds emitted from four seasons rose (rosa damascena mill.) cultivated in beijing lina yang1, jianwu ren1*, yan wang2, qing hu1 (received july 5, 2013) * corresponding author summary four seasons rose is an old variety of rosa damascena mill. using the technique of dynamic headspace collection, the volatiles emitted from the flowers of four seasons rose were collected at five time points of a day. then, by the means of thermal-desorption cold trap/ gas chromatography/mass spectrometer technique (tct-gc/ms), constituent compounds of the volatiles were identified. after that, the daily dynamic variation of the floral scents from four seasons rose was analyzed. the results demonstrated that the daily fluctuation of volatile components changed in different weather conditions. a total of 93 volatile compounds were identified. there are the most diversity of compounds at 14:00 in a fine day, and the compounds are as many as 78, whereas only 50 of volatile compounds are detected in a rainy day. introduction rosa damascena mill., commonly known as damask rose, is indigenous to europe and middle east countries, iran and turkey (almasirad et al., 2007). some varieties are of great importance for rose oil production. rose oil is mainly used in the perfumery and cosmetics industry (aridogan et al., 2002). besides, it also finds application in the food industry as a flavor additive (mollov et al., 2007; shikov et al., 2012). because of the low oil content and the lack of natural and synthetic substitutes, rose oil is one of the most expensive essential oils in the global markets (baydar and baydar, 2005). this rose is also used worldwide for manufacture of products with diverse applications such as aroma therapeutic, antibacterial (seyhan et al., 2009), antimicrobial (basim and basim, 2003; ozkan et al., 2004), antioxidizing (achuthan et al., 2003), and anti-hiv effects (mahmood et al., 1996). anyway, fragrance is one of the most desirable traits of damask rose. the amount and composition of the rose oil distilled from the rose petals is strongly affected by the genotype, the climatic conditions, the time of rose petals harvesting, and the technology used for processing and distillation (rusanov et al., 2009). as far as genotype concerned, several damask rose varieties dominated among oil bearing roses over centuries, due to higher rose oil content and superior essential oil quality. moreover, being a hybrid, the progeny members of damask rose incline to emitting lower quality and quantity of volatile compounds (magali et al., 2007; kovacheva et al., 2010). so it seems that plantation location and harvesting time probably affect rose oil production. therefore, being raw material, the flower of oil bearing rose is necessary to be investigated completely so as to clearly understand the changing composition of floral scents emitted from damask rose flowers. in fact, damask rose flower volatiles vary at different stages of flower development, and there is fluctuation of flower volatiles were profiled at different time points during the day (rusanovir et al., 2011). so identifying the constituents of floral volatile and detecting their variations is conducive to tuning of the rose oil composition by precise flower harvesting practices is proposed. however, previous studies on constituents of volatile emissions of damask rose flower focused on the rose oil or flower extraction. due to the subjects measured indirectly acquired from flower, the measurement results were inevitably affected by processing technology or extraction method, even the storage time. here, directly collecting volatile emissions via technique of dynamic headspace collection, we detect the variation of floral volatiles emitted from an old variety of damask, named as four seasons rose, investigating variation of floral volatiles in line with flower development stages and daytime periods. given that samples are intact volatiles emitted directly from rose flowers, we hope the results could provide support for the possible applications of more precise flower harvesting in china, a new industrial plantation base of oil bearing rose. material and methods plant material four seasons rose used in this study is an old variety of damask rose, blooming in late spring to early summer, with medium pink and very fragrant flower. the plants were cultivated in botanical trial base affiliated to chinese forestry academy. the experimental site locates in western suburb of beijing, at an elevation of 150 m, and has a temperate climate. volatiles collection in order to determine the temporal variation of the floral scent over a day, volatiles were sampled from 3 full-blooming flowers (stages 6, as determined by rusanov et al. (2012)) of 4 selected plants for 30 min. once every three hours, starting at 5:00 am and ending at 6:00 pm. there were 5 time points throughout daytime, summing up to 15 samples per plant individual, 60 samples in total. trials were conducted under different weather including rainy day, cloudy day and sunny day, which were the 11th, 12th and 13th of june 2011. volatiles were collected using an improved dynamic headspace aircirculation method (gao et al., 2005). at first, three well growing flowers borne on the same node of each plant were enclosed in a loosely fitted polyester oven bag (reynolds oven bags, 482 mm × 596 mm, richmond, va, usa) , and air was extracted from the bags by vacuum pump (qc-1s air sampler, beijing municipal labor protection institute, china) at a rate of approximately 100 ml/ min. secondly, air was pumped (1.1 l/min) through a double filter consisting of charcoal and porous polymer gdx-101 and introduced purified air into the bags, balanced for 30 min. thirdly, linking a 6mm dynatherm centerless ground ss sample tube filled with 200 mg tenax-ta (mesh 60-80) to a qc-1s air sampler, a closed air circulation system was set up. finally, volatiles were trapped for 30 min via the closed air circulation system at a flow rate of 200 ml/ min. thermal desorption tubes were desorbed using a perkin elmer turbo matrix 650 automatic thermal desorber (atd). the atd was adjusted to the 10 l. yang, j. ren, y. wang, q. hu mode of 2-stage desorption. at the first stage, it was programmed to desorb the tubes at 260 °c with a 25 ml/min desorption flow, lasting 10 minutes. the desorbed compounds from the tubes were trapped in the cooled trap of tenax (set at -25 °c), then quickly heated at 40 °c/s to 300 °c, with a setting time of 5 minutes. gc-ms analysis analyses were carried out using a perkin elmer clarus 600gas chromatograph (gc) coupled to a perkin elmer clarus 600t mass spectrometer. an elite-5ms capillary column 30m × 0.32mm × 0.25μm was used to analyze the samples. the oven temperature was initially set at 40 °c for 2 min; increased at 6 °c/min to 180 °c, and held for 2min; and increased at 15 °c/min to 270 °c, maintaining at this temperature for 3 min. the carrier gas was nitrogen with a flow rate of 2.0 ml/min. the ei mass spectra of the eluted volatile compounds were obtained with an electron energy of 70 ev over a scan range of 29-500 amu. statistics and calculation identification of the volatile components was carried out by deconvolution using the amdis ver. 2.69 software (national institute of standards and technology (nist), usa) and the nist 2008 mass spectral library searched by nist ms search v2.0 software. the retention index for individual compound was determined by c10-c40 n-alkanes mixture (sigma-aldrich). using the software turbomass ver 5.4.2, the relative contents of each compound were calculated based on total ion current without standardization, by the means of the chromatographic peak area normalization method. data analysis was performed using microsoft excel (microsoft corporation). the relative content of each identified volatile compound was indicated by mean of 4 replicates plus standard error in the figs. ranging from 4 to 7. result and analysis total ion current of volatile compounds the preservation of the traditional odor and composition of the floral volatile is an ultimate prerequisite for production of high quality of rose oil. in order to preserve the authentic scent of the oil rose, monitoring the floral scent from raw material flower is of critical importance. the characteristic fragrance of damask rose results from its specific composition of volatiles and certain proportion between individual constituent compounds, and the volatile emissions change rhythmically under stable environmental conditions. for most roses, their volatile emissions reach summit during their full-blooming stage as the flower development process concerned (joanne et al., 2004). in fig. 1, 2 and 3, gc/ms total ion current of volatile compounds from four seasons rose show the relative abundance of emission constituents in different weather conditions. among them, the 5 graphs ranging from a to e represent profiles of emission constituents at different time points including 5:00 am, 8:00 am, 11:00 am, 2:00 pm and 5:00 pm, in a rainy day; from f to j, the graphs stand for the dynamics emission constituents at the same time points as above in a cloudy day; and the 5 graphs marked k, l, m, n and o, indicate the dynamics emission constituents at the same five time points in a sunny day. the analysis of chromatograms of volatile components and corresponding relative abundance at the five time points is based on net values after rejecting control air background. a total of 93 volatiles compounds were identified in daily fluctuation, in which alkanes, terpenes and aldehydes compounds are the major components. moreover, under different weather conditions, volatile composition are significant different. on the fine day, as many as 78 compounds are detected at 11:00 am, while only 50 compounds detected at the same time point on a cloudy day. diurnal dynamics of alcohols among volatile emissions alcohols among volatile emissions from four seasons rose mainly include 2-ethylhexyl alcohol, citronellol and phenylethyl alcohol, in addition, composition and amount of these group of compounds exhibit diurnal fluctuation (fig. 4). as showed in fig. 4, on a rainy day, total alcohols sharply increase in the morning, then remaining stable at noon, after that, there is a rapid rise in the afternoon; there is a small amount of citronellol at noon, and its relative content is just 0.227%. on a cloudy day, total alcohols grow swiftly in early morning, but subsequently fall down until noon, and seeing a steady soaring after noon; as for citronellol, there is a continued rise from early morning, and reaching summit at 3:00 pm, with maximal relative content of 0.741%, there followed a decline. on a sunny day, total alcohols increase rapidly from early morning, and peaking at 2:00 pm, then plunging dramatically; while citronellol rise in the morning, and reaching the peak at 9:00 am, with relative content of 0.89%. citronellol is an important contributor of pleasant floral odor of valuable, high-priced rose oils (jirovetz et al., 2005), and is highlighted in the international rose oil standard (iso 9842, 2003). on a sunny morning, harvesting of four seasons rose flower for production of rose oil probably obtains a high yielding of citronellol. so the result supports the traditional harvesting practices of rose flowers prevailing among the dominant rose oil producers in bulgaria (rusanovir et al., 2011). as regard to benzenethanol, also known as phenylethyl alcohol, its aroma is described as floral, rose-like, honey notes. on a rainy day, it increases moderately in the morning and then undergoing a fast rise after 11:00 am, however, dropping substantially after 2:00 pm; on a cloudy day, benzenethanol increases slightly, and seeing a quick rise after 3:00 pm; on a sunny day, there is a rapid increase topping at 4.572% before noon, and followed by a fast decrease afterwards. diurnal dynamics of terpenes among volatile emissions based on analysis of chromatograms in fig. 1, 2 and 3, it is found that the main terpene compounds are α-pinene, β-myrcene and d-limonene from fresh flowers of four seasons rose, and the terpenes fluctuate in daily rhythm. fig. 5 shows terpenes always grow first and then decreasing no matter what weather condition. but there is slightly difference in changing rhythms of terpenes under different weather conditions. the high peak appears at 9:00 am in rainy day, while at noon in cloudy day or sunny day. in the rainy day, cloudy day and sunny day, relative content of total terpenes reaches 40.511%, 45.491% and 16.389%, respectively. the obvious different from the other compounds is that the maximal emission occurred when sunlight is not very intensive. among terpenes of the volatiles, α-pinene, β-myrcene and d-limonene are the major component compounds. their daily changing trends are different from one another. in fig. 5, the maximal level of α-pinene was detected at the beginning of the diurnal period, and its relative abundance is as high as 10% in a rainy day, while the relative abundance remains on a low stable level on fine days. in the contrast, the fluctuation of β-myrcene relative content is consistent in three different weather conditions. β-myrcene firstly rises and then drops, peaking at noon. the maximal abundance reaches 12.201%, 17.978% and 4.887% in rainy day, cloudy day and sunny day, respectively. several minor terpenes were detected, and their amounts were very small. in fig. 6, the relative contents of three terpenes are less than 5%. but they probably play an important role in characteristic aroma. volatile profiles from four seasons rose 11 fig. 1: gc/ms total ion current chromatograms of volatile compounds from four seasons rose in rainy sabinene is a natural bicyclic monoterpene with weak turpentinelike, spicy, warm-woody aroma. it usually increases in the morning, reaching summit at noon, and declines in the afternoon. beyond expectation, the familiar major monoterpenols such as nerol and geraniol (rusanovir et al., 2011; jirovetz et al., 2005) were not detected in the flower volatiles of four seasons rose. but the oxidised monoterpenols including nerol acetate and geraniol acetate were detected on the sunny day (fig. 3). in addition, relative abundance of 0.588%, the maximum of nerol acetate was emitted at 8:00 am only on the sunny day, whereas geraniol acetate peaking at 2:00 pm, with maximal relative abundance of 0.290%. diurnal dynamics of alkanes among volatile emissions the three major hydrocarbons also show diverse accumulation patterns. while, the maximal level of pentadecane accumulation was detected at the end of the diurnal period in different weather conditions, the trace amount of heptadecane is detected only in the early morning of a rainy day. heneicosane increases moderately after noon, but it firstly declines in the morning of cloudy or sunny days. in terms of relative content in total of volatile compounds, the alkanes of flower volatile accumulation levels for the time points 2:00 p.m. and 6:00 p.m., which are out of the traditional daytime flower harvesting period are higher than those of the 5:00 a.m. to 12:00 p.m. period. given that good quality rose oil should possess a higher amount of monoterpene alcohols and a lower amount of alkanes (baser, 1992). so the results in this research support the rationality of the traditional rose harvesting, which is carried out in the morning. besides, this assay is also identified the ketones, esters, the phenyl / phenylpropanes and acids, etc. the benzene compounds including ethylbenzene and o-xylene present higher level in a cloudy day, and the maximum is 8.298% and 11.781% at noon, respectively, while the lowest level occurred on a fine day. the amounts and components of esters were little in different weather conditions. the highest content was found at 9:00am (1.099%) on fine days as compared to 12 l. yang, j. ren, y. wang, q. hu fig. 2: gc/ms total ion current chromatograms of volatile compounds from four seasons rose in cloudy day other time. there are five compounds of ketone could be detected, only 6-methyl-5-heptene-2-ketone can be measured at every time point. the other components appeared in some specific individual time points, for instance, 3,5,5-trimethyl-2-cyclohexene-1-ketone and cyclopentanone only can be found at 8:00-9:00 am and 9:0012:00, respectively. discussion the study of volatile compounds of rosa damascene showed that volatiles were emitted rhythmically, with maximum peaks coinciding with the time period after 8-10h of illumination (joanne et al., 2004), or even to the time period after 6-9h of illumination (helsper et al., 1998).the fluctuation exists not only in relative contents but in component compounds. duo to the surroundings damask rose located in changeable, the volatile components incline to varying accordingly. the aromatic components released from four seasons are most abundant at 11:00-12:00 am on sunny day. the reason probably is intensive light and high temperature, which make the volatile easier to emit. the volatile emission of many plants has a relation to light and temperature. in cold experiment, for example, orchid aroma is very little even no fragrance can be detected, while the same orchid would release a big amount of floral scent in sunny days (williams and whitten, 1983). the emission of aroma from trifolium repens l. in 20 °c is 54% higher than in 10°c (jakobsen and olsen, 1994). anyway, the diurnal regulation of scent emission is complex (hendel et al., 2007). although the daily emission of most scent compounds is synchronized, various independently evolved mechanisms control the production, accumulation and volatile profiles from four seasons rose 13 fig. 3: gc/ms total ion current chromatograms of volatile compounds from four seasons rose in sunny day release of divergent volatiles. it is noticed that there are not geraniol and nerol detected in this research, which is abnormal to some certain compared to previous studies (rusanovir et al., 2011; jirovetz et al., 2005). the reason may lie in the improper flower developmental stage for sampling of volatiles from the damask rose flower or inappropriate environmental conditions. conclusion fluctuation of flower volatiles at different time points during a day is investigated in this research. the aromatic components released from four seasons are most abundant at 11:00-12:00 am on sunny days. the daily fluctuation rhythm of volatile component compounds is different in varying weather, for instance, the relative content of terpene compounds is higher in rainy day than in a fine day. above all, it is concluded that every individual compound possibly has its own daily fluctuation rhythm in accordance with weather conditions. the results in this research suggest that there are obvious variations among different time points of flower sampling, which could provide more delicate guide for the harvesting oil bearing rose. acknowledgments we would like to thank x. fang for gc-ms analyzing, and z. baoqiang for sampling assistance, also ornamental plant center with chinese forestry academy for providing trial plot. funding in support of this study was received from the department of food science and engineering at beijing forestry university. 14 l. yang, j. ren, y. wang, q. hu fig. 6: diurnal dynamics of three minor terpenes in volatile emissions from flowers of four seasons rose fig. 4: diurnal dynamics of alcohols in volatile emissions from flowers of four seasons rose fig. 5: diurnal dynamics of terpenes in volatile emissions from flowers of four seasons rose fig. 7: diurnal dynamics of alkanes in volatile emissions from flowers of four seasons rose volatile profiles from four seasons rose 15 references achuthan, c.r., babu, b.h., padikkala, j., 2003: antioxidant and hepatoprotective effects of rosa damascene. pharm biol. 41, 357-361. almasirad, a., amanzadeh, y., taheri, a., iranshahi, m., 2007: composition of a historical rose oil sample (rosa damascena mill., rosaceae). j. essent. oil res. 19, 10-112. aridogan, b.c., baydar, h., kaya, s., demirci, m., ozbasar, d., mumcu, e., 2002: antimicrobial activity and chemical composition of some essential oils. arch. pharm. res. 25, 860-864. baser, k.h.c., 1992: turkish rose oil. perfumer and flavorist 17, 45-52. basim, e., basim, h., 2003: antibacterial activity of rosa damascena essential oil. fitoterapia 74, 394-396. baydar, h., baydar, n.g., 2005: the effects of harvest date, fermentation duration and tween 20 treatment on essential oil content and composition of industrial oil rose (rosa damascena mill.). ind. crop. 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essential oil of rosa damascena l. from china. flavour frag. j. 20, 7-12. joanne, m.p., robin, a.c., naoharu, w., hazel, s.m., colin, g.n.t., 2004: rhythmic emission of floral volatiles from rosa damascena semperflorens cv. ‘quatre saisons’. planta 219, 468-478. kovacheva, n., rusanov, k., atanassov, i., 2010: industrial cultivation of oil bearing rose and rose oil production in bulgaria during 21st century. biot. biot. equi. 24, 1793-1798. magali, c.m., frédéric, j., philippe, h., baudino, s., 2007: fragrance heritability in hybrid tea roses. sci. hortic. 113, 177-181. mahmood, n., piacente, s., pizza, c., burke, a., khan, a., hay, a., 1996: the anti-hiv activity and mechanisms of action of pure compounds isolated from rosa damascene. bioch. biophys. res. commun. 229, 73-79. mollov, p., mihalev, k., shikov, v., yoncheva, n., karagyozov, v., 2007: rose petal extracts to see strawberry pigments bloom? innov. food sci. emerg. technol. 8, 318-321. ozkan, g., sagdic, o., baydar, n.g., 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carle, r., 2012: antioxidant capacity and colour stability of texture-improved canned strawberries as affected by the addition of rose (rosa damascena mill.) petal extracts. food res. int. 46, 552-556. williams, n.h., whitten, w.m., 1983: orchid floral fragrances and male euglossine bees: methods and advances in the last sesquidecade. biol. bull. 164, 355-395. younis, a., khan, m.a., khan, a.a., riaz, a., pervez, m.a., 2007: effect of different extraction methods on yield and quality of essential oil from four rosa species. floriculture ornamental biotechnol. 1, 73-76. address of the corresponding author: e-mail: jianwur@sina.com journal of applied botany and food quality 89, 287 289 (2016), doi:10.5073/jabfq.2016.089.037 institut für pflanzenbiologie, technische universität braunschweig, braunschweig, germany case study: the difficulty of correct reference values when evaluating the effects of drought stress: a case study with thymus vulgaris jana paulsen, dirk selmar (received january 28, 2016) summary medicinal and spice plants grown under semi-arid conditions frequently contain higher concentrations of relevant secondary metabolites than plants cultivated in moderate regions. it is well established that this phenomenon is due to the impact of drought stress. however, in principle, the increase in natural product concentration could be caused by two quite different effects. firstly, it could be caused simply by a change of the reference values: typical stress-related reduced growth frequently results in a lower biomass of the stressed plants. in consequence − provided that the rate of natural product biosynthesis remains constant − this results in an enhanced concentration in the stressed plants. secondly, there is a genuine stress-related increase in the rate of biosynthesis of secondary metabolites. in the first case, the total amount of secondary metabolites remains constant, while in the second case, it increases. accordingly, a thorough differentiation requires a reliable quantification and comparison of the relevant factors. this especially accounts for the total biomass, which generally is far less in drought stressed plants than in the well-watered ones. consequently, in all studies, where data on the total biomass are lacking, it is not feasible to evaluate reliably the total amounts of secondary metabolites only on the bases of the concentrations determined. unfortunately, in most of the corresponding literature published so far, these data are missing, and thus, it is not possible to decide whether the drought stress-related increase of secondary metabolites is due to a genuine increase in biosynthesis or whether it is just due to a change of the relevant reference values, i.e., the total biomass. in this study, the relationships of these factors in well-watered and drought-stressed thyme plants are examined and discussed. introduction it is well known that spice plants grown under dry climate conditions are more aromatic than plants cultivated in moderate climates. in the same manner, medicinal plants from semi-arid areas such as the mediterranean region exhibit a greater concentration of relevant secondary metabolites than those grown in central or northern europe (kleinwächter and selmar, 2015). this phenomenon is caused by drought stress. there are two possibilities. in one case, because of water shortage, the stomata of the leaves are closed and the uptake of co2 decreases significantly. as result, the consumption of reduction equivalents (nadph+h+) for co2-fixation via the calvin cycle declines considerably, generating a large oxidative stress and an oversupply of reduction equivalents. as a consequence, all metabolic activities that consume reduction equivalents increase. accordingly, the synthesis of reduced compounds, such as isoprenoids, phenols or alkaloids, is enhanced (selmar and kleinwächter, 2013). in the second case, drought-stressed plants gene rally exhibit reduced growth resulting in a lower biomass compared to well-watered plants. in consequence − provided that the rate of natural product biosynthesis remains constant − this results in an enhanced concentration of these substances in the stressed plants. in principle, in the first case, the total amount of secondary metabolites per plant increases, whereas in the second case it remains constant. unfortunately, these coherences are much more complex, as both effects may co-occur. at an extreme, the higher rate of biosynthesis of secondary plant products could be compensated or even overcompensated by the lower biomass of the plant. as a result, the total amount of secondary metabolites will remain unchanged or will even be lower in the stressed plants, although the concentration is strongly enhanced (gershenzon, 1984; selmar and kleinwächter, 2013). accordingly, thorough differentiation between these effects requires a reliable quantification and comparison of all relevant factors, especially the total biomass that is strongly influenced by drought. in most past and current literature on secondary metabolite production these data are lacking, and the total amounts of secondary metabolites cannot be estimated. accordingly, no deduction on putative changes in the rate of biosynthesis could be made. unfortunately, in most of the corresponding literature published so far, these data are missing, and thus, it is not possible to decide whether the drought stress-related increase of secondary metabolites is only due to a genuine increase in biosynthesis or it is just due to a change of the relevant reference values, i.e., the total biomass. as consequence, solid statements on increase of biosynthetic activity require a more precise definition, because the enhancement may refer either to the biosynthesis in the entire plant (total biosynthesis) or to the specific activity, calculated per gram bio mass (specific rate of biosynthesis). this could be illustrated as follows: due to massivly reduced growth, the total amount of natural products is far lower in stressed plants in comparison to the well-watered controls. consequently, overall biosynthesis of natural products per plant is strongly reduced. since this biosynthesis is achieved by a much smaller plant, the rate of biosynthesis per gram bio mass may nonetheless increase significantly. in this paper, these complex coherences and problems are discussed and outlined on the basis of a case study involving well-watered and drought stressed thyme plants. consequences of different biomasses for the estimation of stressrelated effects thyme plants were cultivated for nine weeks under well-watered as well as under drought stressed conditions (for details see kleinwächter et al., 2015). as expected, drought stress had a strong effect on plant growth: the biomass of the stressed plants was markedly lower compared to those of the well-watered ones. at the end of this experiment, the biomass of the stressed plants accounted for only two-thirds of the biomass of the well-watered plants (tab. 1; kleinwächter et al., 2015). as assumed, the concentrations of terpenes (mg/g d.w.) are increased in the drought stressed plants compared to the well-watered controls (tab. 1), putatively due to the stress related lesser biomass. in contrast, the total amount of terpenes per plant is markedly reduced. at the first glance, drought seems to cause a decline of the overall 288 j. paulsen, d. selmar terpene biosynthesis. however, further comparison revealed that the total content of terpenes of the stressed plants accounts for 78 % of the content in the well-watered plants − a quota that is much higher than the share of the biomass (67 %). assuming that the specific rates of terpene biosynthesis (mg/g d.w.) are the same in stressed as well as in well-watered plants, the corresponding quota should be equal. obviously, the specific rates of biosynthesis change in the course of the experiment, and a more detailed evaluation with special emphasis on the specific rates of biosynthesis is required. the specific rate of terpene biosynthesis (given in milligram terpenes per gram dry weight) can be calculated from the increase in the total amount of terpenes referred to the average biomass of these plants during the particular time span. the corresponding data for such an approach are compiled in fig. 1. for a period up to three weeks, there are almost no differences in the growth of stressed and control plants, evidant by nearly the same biomass (fig. 1a). however, in the second phase of the experiment, the biomass of the stressed plants is markedly lower than that of the well watered ones. the inhibiting effect of moderate drought stress on plant growth became visible only after six weeks. in contrast, and in accordance with our previous experiments, the total content of terpenes in the stressed plants is slightly enhanced after three weeks (fig. 1c). in the same manner, also the concentration of terpenes in the stressed plants is significantly enhanced (fig. 1b). however, due to the massive growth reduction in the second phase of the experiment, time dependent increase in the total amount of terpene is also reduced. as consequence, after six weeks, the number of terpenes is nearly the same in stressed and control plants. yet, after nine weeks, it is far lower in the stressed plants. nonetheless, in all cases the concentration of terpenes is enhanced in the stressed plants. these complex coherences can easily be comprehended by the rates of biosynthesis displayed in fig. 1d. indeed, as expected, in the first phase of stress the rate of biosynthesis is markedly higher in the drought stressed plants than in well-watered ones. this enhancement is putatively due to the stress-related increase of reduction status as outlined by selmar and kleinwächter (2013). in the course of the experiment, the rates of biosynthesis decrease in either case. this obviously reflects the well known decline of terpene biosynthesis in the course of leaf maturation (e.g., vorin and bayet, 1996). in conin consequence, within the period of four to six weeks, the rates in stressed and control plants are nearly the same. however, the reduction of the biosynthetic rate in drought stressed plants proceeds in the last phase of the experiment. it is very likely that the stressed plants have – at least in part – adapted to the stress situation and the reduction status has normalized. finally, the rate of biosynthesis in thyme plants under drought stress is only 60 % that of the controls. this strong decrease may be due to putative stress-related damages. if indeed the metabolic processes and coherences outlined above are responsible for various effects in the course of the different phases of drought stress must be verified by basic experiments. however, with respect to sound estimation of stress-related changes on secon dary metabolism, especially for the evaluation of its impact on food quality, the results outlined in this paper clearly demonstrate the importance of the determination of both, concentration and total amount of the natural product of interest. only if both, the rate of plant biomass production and the biosynthetic rate of secondary metabolite production throughout growth of the plants are considered, it will be possible to deliberately apply drought stress as a means of modifying the quality of commodities derived from spice and medicinal plants. tab. 1: biomasses, concentrations and total amounts of terpenes in wellwatered (≙100 %) and drought stressed thyme plants after nine weeks of cultivation under the respective conditions (kleinwächter et al., 2015 well-watered thyme drought stressed thyme 17.45 g d.w. 11.63 g d.w. 100 % 67 % concentration 6.42 mg/g d.w. 7.44 mg/g d.w. of terpenes 100 % 116 % total amount 112 mg/plant 87 mg/plant of terpenes 100 % 78 % biomass fig. 1: biomass (a), concentration of terpenes (b) total amounts of terpenes (c) and rates of biosynthesis of terpenes (d) in well-watered and drought stressed thyme plants the difficulty of correct reference values 289 acknowledgements financial support by the german federal ministry of education and research via the “the national aeronautics and space research centre – dlr” (project no.: egy 10/001) is greatly acknowledged. we wish to thank dr. david s. seigler (university of illinois, urbanachampaign) for fruitful discussions, critical reading the manuscript, and helpful suggestions for linguistic improvements. references gershenzon, j., 1984: changes in the levels of plant secondary metabolites under water and nutrient stress. in: timmermann, b.n., steelink, c., loewus, f.a. (eds.), phytochemical adaptions to stress − recent advances in phytochemistry, 18, 273-320. plenum press, new york. kleinwächter, m., paulsen, j., bloem, e., schnug, e., selmar, d., 2015: moderate drought and signal transducer induced biosynthesis of relevant secondary metabolites in thyme (thymus vulgaris), greater celandine (chelidonium majus) and parsley (petroselinum crispum). ind. crop. prod. 64, 158-166. kleinwächter, m., selmar, d., 2015: new insights explain that drought stress enhances the quality of spice and medicinal plants: potential applications. agron. sustain. dev. 35, 121-131. selmar, d., kleinwächter, m., 2013: stress enhances the synthesis of secondary plant products: the impact of stress-related over-reduction on the accumulation of natural products. plant cell physiol. 54, 817-826. vorin, b., bayet, c., 1996: developmental changes in the monoterpene composition of mentha × piperita leaves from individual peltate trichomes. phytochem. 43, 573-580. address of the corresponding author: prof. dr. dirk selmar, institut für pflanzenbiologie, technische universität braunschweig, mendelssohnstraße 4, 38106 braunschweig e-mail: d.selmar@tu-bs.de © the author(s) 2016. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 88, 68 77 (2015), doi:10.5073/jabfq.2015.088.010 humboldt-universität zu berlin, faculty of life sciences, albrecht daniel thaer institute of agricultural and horticultural sciences, 1division biosystems engineering and 2 division urban plant ecophysiology, berlin, germany evaluation of substitutes for rock wool as growing substrate for hydroponic tomato production dennis dannehl1*, johanna suhl1, christian ulrichs2, uwe schmidt1 (received december 24, 2014) * corresponding author summary to reduce the rock wool waste, the present study is focused on the evaluation of sheep wool, cultivated sphagnum biomass and hemp, which may be used as replacement for rock wool as growing substrate for hydroponic tomato production. as such, physical and chemical properties of substrates, the plant growth, yield, fruit characteristics, as well as primary and secondary metabolites of tomatoes were considered. the marketable fruit yield of plants grown in sphagnum slabs (12.8 kg plant-1) was reduced to only a small extent compared to the yield produced by rock wool slabs (13.8 kg plant-1). sheep wool (12.3 kg plant-1) and hemp (10.4 kg plant-1), however, showed higher deviations. the lowest yield of blossom end rot (ber) fruit was produced by sphagnum. compared to this result, the ber-yield was nearly 2-fold higher caused by sheep wool. the soluble solid content in fruit ripened by the hemp material was decreased compared to those caused by the remaining substrates. furthermore, it was found that the volume of easy available water (eaw) was mainly responsible for changes in plant development. as such, a high correlation was found between eaw and: leaf area (r = 0.851); flowers (r = 0.785); lycopene (r = -0.918); ß-carotene (r = -0.997); penolics (r = -0.918); l-ascorbic acid (r = -0.848). the findings suggested that cultivated sphagnum biomass dried and pressed as slabs can be used as replacement for rock wool slabs, whereas the usage of slabs consisting of hemp and sheep wool is not suitable as growing substrate for hydroponic tomato production. introduction nowadays, not only the product quality but also the sustainable production plays a major role when foods are purchased by the consumer, where economic, social and ecological aspects are considered in the purchase decision (vermeir and verbeke, 2008). since the ecological part also includes the waste management during the production cycle, a multitude of scientists are concerned with the further development of hydroponic systems in greenhouses in order to realize a more environmentally friendly production of vegetables. the transition to closed hydroponic systems was a first step to relieve the environment, whereas substrates as growing material can contribute to the main waste flow in greenhouse production (papadopoulos and gosselin, 2007). this applies in particular to the rock wool substrate, which is the most common substrate used for the cultivation of tomato, cucumber and red pepper in hydroponic systems in a wide range of countries (benoit and ceustermans, 1995; shinohara et al., 1999; jeong and hwang, 2000; bussell and mckennie, 2004). this growing medium is often disposed after one culture period and cannot always be recycled, whereby up to 150 m3 of rock wool waste per ha are produced per year resulting in this fact that landfills threaten to become scarce (pieters et al., 1998; göhler and molitor, 2002). in the netherlands, for instance, 90 % of the used rock wool slabs are recycled and returned to the material cycle in form of new rock wool products, whereas the entire rock wool waste of the canadian greenhouse production is stored exclusively on landfills (van den bosch, 2004; papadopoulos and gosselin, 2007). furthermore, an average primary energy demand of 275 kwh is required to produce one cubic metre of rock wool, where 167 kg co2 are released into the environment (brandhorst et al., 2012). in these calculations, however, the energy expenditure for recycling processes is not considered. despite these circumstances, rock wool slabs are preferably used as a horticultural substrate in soilless systems due to numerous advantages, such as a high total pore space, as well as inert, sterile and homogeneous conditions caused by the production process of this material and the achievement of constant yields during the culti vation period (olympios, 1992; bussell and mckennie, 2004). however, the use of environmentally friendly growing substrates, which are, for example, biodegradable or can be used over several years, may reduce the waste flow under protected growing conditions (van os, 1994; pieters et al., 1998). in this context, rock wool surrogates should be used, which provide equally good results in terms of fruit quantity and quality as achieved using rock wool substrates. therefore, in recent years, various growth media were tested for their suitability as substrate for the hydroponic cultivation of vegetables. shinohara et al. (1999), for instance, found no differences in growth, yield and soluble solid content (ssc) of tomato plants, regardless of whether they were grown in rock wool, coconut fibre, bark or rice husk. the same applies if exactly the same substrates were used for further experiments. similar results were found when rock wool was compared with almond shell as growing media, whereas the ssc of tomato plants grown in the organic media was higher than that synthesized on rock wool (urrestarazu et al., 2005). furthermore, manios et al. (1995), martinez and abad (1992), and allaire et al. (2005) demonstrated that hydroponically cultivated tomato plants showed comparable yields of tomatoes when they were produced in substrates consisting of sieved pumice and peat-lite (85% : 15%, v/v), perlite and peat (85% : 15%, v/v), sepiolite and perlite (80% : 20%, v/v), sepiolite and leonardite (97% : 3%, v/v), peat and composted bark (66.6% : 33.4%, v/v) or rock wool. however, tomato yields in substrates mixed from fresh white spruce and fir sawdust (40% : 60%, v/v), as well as from white spruce and fir shavings (40% : 60%, v/v) were lower than those obtained with rock wool (allaire et al., 2005). based on these results, the present study is focused on the evaluation of cultivated sphagnum biomass, sheep wool and hemp pressed as substrate slabs, which may be used as replacement for rock wool slabs as growing substrate for hydroponic tomato production. the main objectives of this research were to analyse the physical properties of the used renewable organic substrates compared to rock wool and to find out interactions between the physical properties of these substrates and the plant development, as well as the tomato yield. furthermore, information about the influence of the physical properties of different substrates used for hydroponic systems on the accumulation of primary and secondary metabolites are scarce, although it is well known that the latter possess health-promoting properties evaluation of substitutes for rock-wool 69 for humans (dillard and german, 2000). in order to exclude negative quality effects of possible substitutes for rock wool as growing substrate for hydroponic tomato production, the present study was also conducted to investigate the effects of different growing media on the contents of valuable plant compounds, such as lycopene, ß-carotene, phenolics, l-ascorbic acid (laa), soluble solids (ssc), titratable acids (ta), as well as microand macronutrients in tomatoes. additionally, the experiments were also used to identify the impact of different substrates on fruit characteristics, such as dry matter content, fruit firmness and the occurrence of blossom end rot fruit. materials and methods experimental set-up greenhouse facility the experiments were carried out in two compartments (each 40 m2), which were integrated in a n-s oriented venlo-type glasshouse (307 m2) at the humboldt-universität zu berlin. this greenhouse consists of double glazing (16 mm) in the standing wall region and single glazing (4 mm), as well as one energy screen in the roof region and was operated by a conventional microclimatic control strategy. this means that the energy screen was closed at a global radiation of less than 3 w m-2, the floor level heating was set at a target temperature of 17 °c for day and night, the ventilation was opened above 23 °c and the co2 concentration was kept at 800 ppm during daylight hours. the co2 supply was stopped when the ventilation exceeded an opening of 10%. these processes were controlled using the application of proportional integral differences, including data obtained from different sensors to maintain the sought microclimatic conditions in the compartments of the greenhouse. the sensors were evenly distributed in the canopy and the measurements were forwarded to a central computer and recorded every 30 seconds. the microclimate conditions for each compartment are shown in tab. 1, which represent mean values from planting until the end of experiments. whereby an identical appearance of all substrate slabs was achieved. the only difference between the substrate slabs was the dry weight of them, where the highest level was caused by sheep wool (2000 g) followed by peat moss (1250 g), hemp (1200 g) and rock wool (750 g). each greenhouse compartment contained ten substrate slabs per growing media, resulting in a planting density of 2 plants m-2 and 2 tomato plants per substrate slab. before the plants were grown on high gullies, seeds were germinated in perlite, transferred to rock wool cubes (7.5 cm × 7.5 cm; cutilene®; tilburg, the netherlands) and transplanted in the prepared substrate slabs on 12th february 2013, when the first inflorescence became visible. fertigation was realized by means of a localized drip irrigation system, providing nutrient solution for 150 seconds mainly after a light summation of 560 w m-2 and a minimal overwatering of 20% after each application. to obtain the water overflow and to meet the nutrient needs of the plants, the light summation for controlling the irrigation was regularly adjusted and the nutrient solution was prepared by mixing fresh water and stock solution according to the mixture recipe of göhler and molitor (2002). physical properties of the growing media to evaluate the physical properties of the substrate slabs, 100 cm3 metal rings were slowly pushed into the prepared substrate slabs before the plants were transplanted. five samples were randomly taken from each substrate and greenhouse compartment (n = 10). subsequently, the samples were saturated with water for 24 hours as described by verdonck and gabriels (1992) and then weighed to record the initial value at a suction point of pf = 0, which is necessary to calculate the total pore space (tps) according to de boodt and verdonck (1972) as shown below. after this intermediate step, the volumetric water content of the samples was detected at dif ferent water tensions using the negative pressure method according to hartge and horn (2009), where a sandbox (sandbox, eijelkamp; giesbeek, the netherlands) were used to obtain the measurements. in this context, the water-saturated samples were placed on a layer of synthetic sand and a suction point of pf = 1.0, pf = 1.7, as well as pf = 2.0 was successively applied, in order to calculate the air volume (av), the easily available water (eaw) and the water buffer capacity (wbc) as defined by de boodt and verdonck (1972). after this procedure, the samples were dried at 105 °c until constant weight to determine the bulk density, which is the ratio of the dry weight of the substrate to the substrate volume (g cm-3). all other variables, such as tps, av, eaw and wbc, are expressed as volume percent (vol%). the tps is calculated under consideration of the weight of the water-saturated sample at the suction point pf = 0, the weight of the dried sample, the bulk density and a water density of 1 g cm-3. the av is calculated as the difference in vol% between the tps and the volumetric water content at a suction point of pf = 1.0. the eaw and the wbc are the amounts of volumetric water content released from the samples when the suction point is increased from pf = 1.0 to pf = 1.7 and from pf = 1.7 to pf = 2.0, respectively. to calculate the volume change of the substrate slabs at the end of the investigations, the slabs were weight in a dried state without plants at the beginning and the end of the experiments, where the root biomass in the slabs was not removed. the weight reduction was expressed as percent (%). assessment of crop growth and yield to determine the number of leaves and the leaf area (la) per plant six weeks after planting, a total of eight plants per growing media and greenhouse compartment (n = 16) was selected randomly. the number of leaves was noted and the leaf length and width of each substrate slabs and crop cultivation four different kinds of growing substrates were used to produce tomato plants (solanum lycopersicum l., cv. pannovy) in an open hydroponic system in which the nutrient solution was drained after one use. in this context, tubular film-wrapped rock wool slabs (cutilene®exact; tilburg, the netherlands) with the dimensions of 100 cm × 20 cm × 7.5 cm (length × width × height) were compared to slabs consisting of three different organic materials. these materials include unpurified sheep wool of different breeds of sheep (exact breeds are unknown), hemp fibre processed into insulating mats with a thickness of 18 cm (thermo-hanf® plus, hock gmbh & co. kg; nördlingen, germany) and dried sphagnum palustre biomass referred to as peat moss, which was cultivated and collected as fresh material in the netherlands. to obtain substrate slabs with the same square shape and dimensions as described for the rock wool slabs, a defined amount of each growing media was moistened and then pressed into separate plastic bags by means of a self-constructed device. subsequently, the open end of the plastic bags was welded, tab. 1: mean values of microclimatic conditions in the greenhouse during the experiments experimental temperature relative humidity co2-concenfacility (°c) (%) tration (ppm) compartment 1 21.85 80.73 526.21 compartment 2 21.96 79.92 530.49 70 d. dannehl, j. suhl, c. ulrichs, u. schmidt leaf was measured with a folding ruler. the measurements were inserted in an exponential function developed by dannehl et al. (2014) to estimate the la of each individual leaf non-destructively. subsequently, the calculated values were added up to obtain the la per plant, which was expressed as m2 plant-1. before the first leaves were removed from the tomato plants as is common in the conventional crop management, the number of flowers and formed fruit per plant of the same tomato plants as described earlier was recorded, in order to investigate the effects of different growing media on the development of fruit set. the average fruit set per plant were calculated according to the ratio of the number of formed fruit to the number of flowers and expressed as %. at the end of the experiments on 16th august 2013, the number of panicles and the plant height of the randomly selected plants were measured after the stem was cut directly above the substrate slabs. furthermore, the tomatoes were harvested, counted and weighed weekly, distinguishing between marketable fruit (> 50 g) and fruit categories (a-class ≥ 70 g; 50 g ≤ b-class < 70 g; cclass < 50 g; blossom end rot (ber) class > 20 g). after the trials were finished, the recorded data in terms of all quality classes were summed up to compare the fruit yield, fruit weight and the number of fruit per plant depending on different growing substrates. the fruit yield, fruit weight and the number of fruit were expressed as kg plant-1, g fruit-1 and number plant-1, respectively. chemical and physical analyses sampling, sample preparation and sample properties to determine the chemical composition of the unused organic substrates, ten composite samples, each containing 200 gram, were taken from five different slabs per growing media. these were dried at 75 °c until a constant weight, ground (mm 30, retsch gmbh; haan, germany) and stored until the macronutrients were analysed via inductively coupled plasma-optical emission spectrometry (icpoes) as described below. to analyse the sample properties, the primary and secondary metabolites, as well as the nutrients of tomatoes, three replicates containing 30 tomatoes (> 70 g) were randomly harvested from different plants per growing media and greenhouse compartment at a ripening stage 10, where the freshly picked tomatoes were matured at the same height in the canopy. this sampling was repeated five times during five weeks from june to july, in order to compare five dates of three biological replicates. immediately after harvesting, the fruit firmness of each tomato was measured using a shore a instrument (hhp-2001, bareiss prüfgerätebau gmbh; oberdischingen, germany) equipped with a stamp of 0.25 cm2. the firmness was measured in the range of 0 to 100, where the latter value is the measured data caused by a standardised metal disc. the investigation for each fruit was carried out non-destructively on three equidistant measuring points of the equatorial region. this means that the result caused by each growing substrate represents the average value of fruit firmness recorded over five weeks on 150 tomatoes. the fruit firmness was expressed as shore a. after this procedure, each tomato was quartered and two quarter of these tomatoes were mixed (kenwoodhb856, de’longhi deutschland gmbh; neu-isenburg, germany) to obtain a homogenous starting material of fresh tomatoes with respect to the relevant sample material. the homogenized fresh material of each replication was used to detect the contents of fruit dry matter, carotenoids, phenolics, l-ascorbic acid (laa), titratable acids (ta) and soluble solids (ssc) performed in duplicate. in this context, fresh mass and dry mass from all homogenised fruit samples were measured before and after drying in a ventilated oven for 24 hours at 105 °c, respectively. the fruit dry matter content was calculated by the ratio of the dry mass to the fresh mass and is expressed as g 100g-1 fresh weight (fw). other parts of the quartered tomatoes from the same samples were freeze-dried (christ alpha 1-4, christ; osterrode, germany) and ground (prep’line tef8100, groupe seb – tefal; offenbach, germany) to a fine powder, in order to determine the contents of specific macroand micronutrients in tomatoes. primary and secondary plant compounds the contents of primary and secondary plant compounds in the homogenized material were analysed promptly. the ta was determined using potentiometric titration with 0.05 mol l-1 naoh by ph endpoint at 8.1 according to asu-l-26.11.03-4 (1983). these analyses were carried out using a ph meter (ph526, wtw; weilheim, germany) consisting of a glass electrode (sentix 41, wtw; weilheim, germany). furthermore, the ssc was analysed using a digital refractometer (pr101, atago; karlsruhe, germany), which detects reducing sugars and other soluble solids. the results obtained for ta and ssc were converted to a 100 g fw basis and were expressed as gram citric acid and gram ssc per 100 g fw, respectively. from these data, the sugar : acid ratio was calculated. the content of laa was assayed using the enzymatic test kit (lascorbate, megazyme international ireland; bray, ireland). briefly, the tetrazolium salt mtt [3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide] was reduced by laa to a formazan compound (mtt-formazan) under the presence of phenazinium methylsulfate. mtt-formazan was determined spectrophotometrically at a wavelength of 578 nm. other reducing substances were measured in the same way after removing laa using ascorbic acid oxidase. to calculate the laa content given as mg 100 g-1 fw, the differences between both absorbance values were used. lycopene, ß-carotene and total phenolic contents were determined by spectrophotometry (model 690, gamma analysen technik gmbh; bremerhafen-lehe, germany), where the extracts of these secondary metabolites were measured at wavelengths of 505 nm, 453 nm and 765 nm, respectively. briefly, lycopene and ß-carotene contents in tomato samples were extracted using the method described by fish et al. (2002), including modifications defined by dannehl et al. (2012). afterwards, the contents of carotenoids were calculated according to nagata and yamashita (1992) and expressed as mg 100 g-1 fw. however, the phenolics were extracted as described by connor et al. (2002). to analyse the amount of phenolic compounds in the extracts, the folin-ciocâlteu method according to slinkard and singleton (1977) was applied. modifications regarding this analysis were described exactly by dannehl et al. (2011). the contents of phenolics were expressed as mg gallic acid per 100 g fw (mg gae 100 g-1 fw). detection of minerals in growing media and tomatoes to determine the easy available minerals in growing media and in tomatoes, an aliquot of 0.5 g of each dried sample was weighed into deionized containers. the microwave digestion, which was carried out as a preparation for determining the amount of minerals in the samples, was described in detail by dannehl et al. (2012). after this procedure, the analysis of the elements in the digestion solution was conducted via inductively coupled plasma-optical emission spec-inductively coupled plasma-optical emission spectrometry (icp-oes) using an icp emission spectrometer (icap 6300 duo mfc, thermo; waltham, usa). the operating conditions employed for icp-oes were 1150 w rf power and 0.55 l min-1 nebulizer gas flow, where argon was used as a plasmogen and carrier gas. the analyses were performed with a cross-flow nebulizer (mira mist, thermo scientific; cambridge, england) and from a radial (ca, k, mg, p, s) and axial (fe) view. regarding each element, a single-standard solution (carl roth gmbh; karlsruhe, ger-carl roth gmbh; karlsruhe, germany) of 1000 mg l-1 was used to prepare the reference solutions in 1.4 mol l-1 hno3. the calibration curves were generated with the evaluation of substitutes for rock-wool 71 following reference solutions: blank 1.4 mol l-1 hno3; 0.5-300 mg l-1 of k; 0-100 mg l-1 of ca; 0-50 mg l-1 of mg and p; 0-20 mg l-1 of s and fe. the respective element in the digestion solutions was measured in duplicate at the following wavelength: k = 766.5 nm; ca = 317.9 nm; mg = 279.1 nm; p = 213.6 nm; s = 182.2 nm; fe = 259.9 nm. the contents of minerals in the growing media were ex-. the contents of minerals in the growing media were expressed as g kg-1 dry weight (dw) and those contained in tomatoes as mg 100 g-1 fw, as well as mg g-1 dw. an aliquot of 0.3 g of the freeze-dried samples were used to quantify the contents of carbon and nitrogen in growing substrates and tomatoes using an elemental analyser (vario max, elementar analysensysteme gmbh; hanau, germany) and according to din-iso-10694 (1995) and din-iso-13878 (1998). the modified method was described in detail by dannehl et al. (2012). the cand n-contents in the growing media and tomatoes were expressed just as described above. statistical analysis differences in physical properties of substrates and the effects of these on the vegetative and generative plant growth, fruit yield, quality characteristics, minerals, as well as on primary and secondary plant compounds of tomatoes were evaluated using analysis of variance (anova) with spss, package version 19.0. significant differences were calculated using tukey-tests at a significance level of p < 0.05, where different small letters describe significant differences. the mean variability was indicated using standard deviation, which was illustrated by ± or bars that have both positive and negative values. in order to detect interactions between the volume of easily available water caused by different substrates and the leaf area, as well as the number of flowers and between the leaf area and the yield of marketable fruit, as well as ber-fruit, linear correlations between two variables were calculated using pearson correlation (r) at a significant level of p < 0.05. the same statistical procedure was applied for correlations between chemical elements and primary, as well as secondary plant compounds in tomatoes. results and discussion chemical composition of organic materials the analyses showed differences in chemical composition of the unused organic materials (tab. 2). in this context, the lowest levels of minerals were detected in the hemp substrate. the highest content of nitrogen (n) was found in sheep wool slabs, which was 8-fold and 26-fold higher than that found in peat moss and hemp, respectively. this was mainly caused by faeces and urine, which was contained in unpurified sheep wool. these residues were washed out by fertilization during the experiments resulting in turbid nutrient solution. thereby, it may be possible that the nutrient composition needs to be adjusted more frequently than normally required in recirculating hydroponic systems. additionally, filters should be used to avoid contaminations of the drip irrigation system, which can be transferred into the nutrient solution by the sheep wool residues. compared to peat moss and hemp, the content of potassium (k) in sheep wool was increased more than 4-fold and 9-fold, respectively. however, the highest amounts of calcium (ca), magnesium (mg) and phosphor (p) were analysed in peat moss (tab. 2). in this context, the contents of ca and p in peat moss were almost 2.5-fold higher compared to those measured in sheep wool, whereas a nearly 4-fold difference in contents of the mentioned elements were calculated in terms of hemp. even greater differences were observed with respect to the mg content, where peat moss contained a 5-fold or 13fold higher content of this mineral then that found in sheep wool and hemp, respectively. comparable results, however, were analysed regarding the carbon (c) content in sheep wool and hemp, which ranged between 452.2 and 441.3 g kg-1 dw. the same parameter was reduced to 328.9 g kg-1 dw in peat moss (tab. 2). whether these differences in chemical composition of the unused organic materials can affect plant responses will be discussed in sections below. physical properties of substrate slabs physical properties of an ideal substrate are mainly based on results found by abad and noguera (2000), abad et al. (1993), boertje (1983), de boodt and verdonck (1972) and jenkins and jarrell (1998), who primarily considered substrates for ornamental plant production. however, only minor information about ideal physical parameters of substrates used in hydroponic systems exist (allaire et al., 2005), which is why the optimum values regarding physical properties of substrates were extracted from results of the listed studies above (tab. 3). tab. 2: chemical composition of the unused organic substrates content (g kg-1 dw)[a] substrate ca k mg p n c sheep wool 5.77 54.09 1.37 1.76 127.4 452.2 peat moss 13.97 13.75 7.22 4.00 15.5 328.9 hemp 4.28 6.17 0.55 1.03 4.9 441.3 [a] the content of each element represents the mean value of five slabs per growing substrate. tab. 3: physical properties of the substrate slabs substrate total pore space[a] air volume[a] bulk density[a] easily available water buffer weight reduction[b] (vol%) (vol%) (g cm-3) water[a] (vol%) capacity[a] (vol%) (%) rock wool 97.6 ± 1.8 c 58.5 ± 5.2 b 0.06 ± 0.005 a 37.4 ± 4.5 d 0.13 ± 0.05 a + 17.0 ± 5.0 c sheep wool 92.2 ± 3.3 b 75.5 ± 3.9 c 0.09 ± 0.008 b 4.2 ± 0.9 a 0.41 ± 0.09 b 42.5 ± 1.5 a peat moss 86.0 ± 2.4 a 9.9 ± 0.8 a 0.08 ± 0.003 a 32.0 ± 2.2 c 0.21 ± 0.05 a 12.8 ± 4.1 b hemp 87.2 ± 4.4 ab 60.7 ± 6.0 b 0.12 ± 0.004 c 9.8 ± 2.2 b 0.59 ± 0.11 c 49.3 ± 5.5 a optimum value[c] 75 to > 85 10 to 30 < 0.4 20 to 30 4 to 10 30 [a] the data represent mean values of ten samples per substrate and greenhouse compartment (n = 20) collected at the beginning of the experiments, whereas [b] describes the mean values of the volume change of eight slabs including root biomass per growing substrate and compartment (n = 16) at the end of the experiments. all values were tested using tukey-test, where different small letters without square brackets indicate significant differences (p < 0.05). the symbol ± is given as standard deviation. [c] according to abad and noguera (2000), abad et al. (1993), de boodt and verdonck (1972), boertje (1983) and jenkins and jarrell (1998). 72 d. dannehl, j. suhl, c. ulrichs, u. schmidt the total pore space of the investigated substrates differed significantly from each other. as such, the highest total pore space was caused by rock wool slabs (97.6 vol%), followed by substrate slabs filled with sheep wool (92.2 vol%), hemp (87.2 vol%) and peat moss (86 vol%) (tab. 3). böhme et al. (2008) reported similar results in terms of rock wool and sheep wool slabs, where these results differed by 6.9 vol% and -4.6 vol%, respectively, compared to those calculated in the present study. however, blievernicht et al. (2012) detected that the total pore space of the peat moss substrate used for potted plants is 13.3 vol% higher than that measured in the current study, which can be explained by an increased bulk density induced by the stuffed peat moss slabs. in this context, the bulk density of the investigated substrates ranged from 0.06 g cm-3 to 0.12 g cm-3, where significantly differences between the highest value reached by hemp and those caused by the remaining substrates were observed (tab. 3). the lowest bulk density was found for rock wool and peat moss slabs, which did not differ significantly from each other. nevertheless, all growing media were in the range of the recommended optimum values regarding the total pore space (> 85 vol%) and the bulk density (< 0.4 g cm-3) as demonstrated by de boodt and verdonck (1972) and abad et al. (1993), respectively. the air volume and the easy available water are the most important physical parameters of substrates (bunt, 1976; abad et al., 1993). in the present study, the significantly highest air volume was measured in the sheep wool substrate (75.5 vol%), followed by hemp (60.7 vol%), which contained nearly the same air volume level as detected in rock wool slabs (tab. 3). the lowest value was calculated for peat moss (9.9 vol%). in this context, there is a controversial discussion on the minimum and maximum air volume in substrates used for tomato production. while bunt (1976) defined an optimum value of 10 vol%, scharpf (1997) observed only a moderate sensitivity of tomatoes when they were exposed to an air volume between 6 and 32 vol%. if all investigated substrates in terms of the air volume are suitable for tomato production will be discussed in the following sections, where optimum values will be defined depending on the plant development. furthermore, the rock wool slabs showed with 37.4 vol% the significantly highest easily available water (eaw) among all examined substrates (tab. 3). although the eaw of the stuffed peat moss slabs was significantly decreased to 32.0 vol% compared to rock wool, the eaw of both substrates were close to an ideal growing media as described by de boodt and verdonck (1972) and abad et al. (1993). sheep wool and hemp, however, did not provide sufficient amounts of eaw, which remained far below the optimum values as shown in tab. 3. the lower eaw in sheep wool slabs (4.2 vol%) was probably based on the hydrophobic character of sheep wool, which can be caused by the wool fat. under this circumstance, gruda and schnitzler (2004a) recommend to increase the irrigation frequency. an overflow between 20% and 50% should be considered after each irrigation cycle (peet and welles, 2005), which was guaranteed in the present study (data not shown). the water buffer capacity (wbc) is the amount of volumetric water content released from the substrates when the suction point is increased from pf = 1.7 to pf = 2.0, where this water content can be seen as a reserve for plants during intense plant transpiration. it was found that the wbc of organic substrates is much better than that of the inert material (tab. 3). the significantly highest wbc was calculated for hemp (0.59 vol%), followed by sheep wool (0.41 vol%), peat moss (0.21 vol%) and rock wool (0.13 vol%), where the last two values did not differ significantly. the results obtained do not correspond to the optimal values, which should be in the range between 4% and 10% (de boodt and verdonck, 1972). however, these values were recommended for growing media used for the production of ornamental plants. generally, rock wool or organic materials used as substrate slabs in hydroponic systems have a small water buffer capacity (da silva et al., 1995; urrestarazu et al., 2008). regarding rock wool slabs, benoit and ceustermans (1990) and urrestarazu et al. (2008) found a wbc of 3.5 vol% and 1.1 vol%, respectively. slabs consistent of almond shell reached a wbc of 2.6 vol%, whereas a wbc of 0.5 vol% was calculated for coir waste slabs (urrestarazu et al., 2005; urrestarazu et al., 2008). it is assumed that a small wbc of substrates does not influence the nutrient uptake of plants grown in hydroponic systems, provided that the supply of nutrient solution to the roots is continuously maintained, e.g., by sufficient irrigation frequency and under consideration of the recommended overflow as described above. the shrinkage of the substrate slabs should not be higher than 30% (abad and noguera, 2000). in the present study, however, the decomposition of the hemp and sheep wool materials was the fastest when all substrates were compared, resulting in a significantly weight reduction of 49.3% and 42.5%, respectively (tab. 3). it has to be taken into account that these results do not include the root biomass removal, whereby it is likely that the real level of weight reduction is even higher than that represented in tab. 3. in this context, it was observed that the hemp slabs were slightly lifted after two month, whereby the stability of the plants was negatively affected. this effect was more pronounced, the longer the plants were growing in this substrate. in contrast, the peat moss slabs showed a very good position stability of tomato plants during the culture period, which was based on the lower level of decomposition of this organic material. the weight of this substrate was significantly reduced by 12.8 %. nevertheless, it is obvious that the recommended optimum value is not exceed, even if the biomass would be removed. therefore, the quality of peat moss as substrate is as high as the quality of rock wool in terms of this physical parameter. crop growth and yield the leaf area development within the first six weeks showed that the highest la with 1.29 m2 plant-1 was achieved by the influence of the peat moss substrate, although the air volume was the smallest of all tested growing media (tab. 3, 4). since the impact of rock wool slabs differed only to a lesser extent compared to this result and the plant development can be negatively affected by oxygen deficiency in the root zone as reported by chérif et al. (1997), it was concluded that an air volume in a wide range from 9.9 vol% to 58.5 vol% is suitable for tomato production in hydroponic systems. however, it was found that an air volume > 60 vol% contained in hemp and sheep wool could be one reason to significantly decrease the la of tomato plants by 31% and 21%, respectively, compared to that induced by peat moss slabs (tab. 4). due to the insignificant differences in terms of the number of leaves, these reduced leaf areas were caused by a smaller la per leaf. however, an effect of the chemical composition of the different organic materials on the la development was excluded, because the nutrient solution supply was ensured regularly and the results obtained were opposite to the results demonstrated in other studies. in the present study, it was found that plants grown in peat moss slabs, which contained a much lower level of n and k than sheep wool slabs, produced the highest la. evans (1989) and tei et al. (2000), however, showed that an increase in n availability led to higher rates of photosynthesis, followed by an enhanced plant growth. the same applies to the k availability, where the tomato plant growth increased with increasing concentrations of k as shown by besford and maw (1975). rather, the mentioned results can be further explained by the volume of eaw, because a significant correlation was found between this physical property and the la (r = 0.851, p = 0.048). this result is confirmed by kirda et al. (2004), who demonstrated that a water deficit irrigation between 30% and 50% led to a slightly reduced la of tomato plants compared to a full watering situation. these evaluation of substitutes for rock-wool 73 findings are also in agreement with the observations by gruda and schnitzler (2004b), who reported from an increase in la and fresh matter of leaves, stem, as well as roots of tomato plants grown in substrates with a higher volume of eaw. from these results it can be derived that the shrinkage of the hemp and sheep wool slabs due to the decomposition of these organic substrates should not be ignored, because it might be possible that this alteration was accompanied by a decreased volume of air and eaw inside these materials resulting in a reduced la development. furthermore, it was demonstrated that the different organic and inorganic substrates did not significantly affect the plant length, fruit sets and the number of panicles per plant (tab. 4). in this context, it was concluded that no differences regarding the number of leaves of the cv. pannovy existed neither six weeks after planting nor at the end of the investigations, because heuvelink (2005) found that each panicle of other tomato cultivars is formed after the formation of three leaves. nevertheless, the number of panicles formed by plants grown in the rock wool slabs showed at least a tendency to increase by one panicle per plant compared to plants cultivated in the remaining growing substrates (tab. 4). this behaviour of panicle development could be one explanation for the highest formation of flowers on plants produced in rock wool slabs (81.6), followed by an insignificant impact by peat moss (75.5) and sheep wool (71.4), as well as an significant effect by hemp (68.4) (tab. 4). in this context, it was calculated that the number of flowers increased with an increasing volume of eaw (r = 0.785, p = 0.043), which supported the statement that the eaw contained in substrates is one of the most important physical parameter as mentioned above. furthermore, guo-jing et al. (2001) found a relation between physical parameters and the temperature behaviour inside the substrates, where growing media with a high air volume and a low volume of eaw tend to a faster warming. these physical properties were associated with the substrate slabs consisting of hemp and sheep wool. in comparison to these conclusions, it was logically assumed that the physical parameters found in rock wool and peat moss slabs led to slightly colder temperatures, which can result in a higher number of flowers as shown by phatak et al. (1966). since all growing media influenced the fruit weight regarding all weight categories to the same extent (tab. 5), the higher number of flowers caused by rock wool slabs and the fact that the number of fruit sets per plants remained unaffected depending on the different substrates were the deciding factors for the highest number of marketable fruit (161.5) matured on rock wool slabs (tab. 5). taking this result into account, the number of fruit were reduced by 5%, 14% and 24% owing to the impact of peat moss, sheep wool and hemp, respectively, where the last to variants differed significantly. while the efficiency of the number of fruit in terms of the weight classes b and c was not influenced by the substrate usage, alterations in a-class fruit production were observed. this circumstance was mainly responsible for changes in the number of marketable tab. 4: effects of different substrates on the vegetative and generative plant growth substrate leaf area[a] leaves[a] flowers[a] fruit sets per plant[a] panicles[b] plant length[b] (m2 plant-1) (number plant-1) (number plant-1) (%) (number plant-1) (m) rock wool 1.23 ± 0.12 b 18.8 ± 1.4 a 81.6 ± 9.6 b 94.9 ± 7.9 a 27.5 ± 0.96 a 6.28 ± 0.10 a sheep wool 1.02 ± 0.13 a 19.0 ± 2.3 a 71.4 ± 9.2 ab 95.2 ± 7.3 a 26.3 ± 0.96 a 6.10 ± 0.08 a peat moss 1.29 ± 0.10 b 18.8 ± 0.9 a 75.5 ± 8.9 ab 95.1 ± 7.4 a 26.3 ± 0.99 a 6.05 ± 0.25 a hemp 0.89 ± 0.09 a 18.1 ± 1.8 a 68.4 ± 5.6 a 96.2 ± 6.6 a 26.0 ± 0.82 a 6.05 ± 0.13 a the data represent mean values of eight plants per substrate and greenhouse compartment (n = 16) [a] calculated six weeks after planting or [b] at the end of the experiments. values followed by the same letter without square brackets do not differ significantly according to tukey-test (p < 0.05). values with the prefix ± indicate the standard deviation. tab. 5: fruit responses on different growing substrates fruit substrate marketable fruit a-class b-class c-class ber-class characteristic (> 50 g) (≥ 70 g) (< 70 g to 50 g) (< 50 g) (> 20 g) rock wool 13.8 ± 1.4 b 12.2 ± 1.6 b 1.6 ± 0.8 a 0.3 ± 0.1 a 1.2 ± 0.5 ab sheep wool 12.3 ± 1.2 b 11.2 ± 1.4 ab 1.1 ± 0.5 a 0.2 ± 0.1 a 1.9 ± 0.5 b peat moss 12.8 ± 1.4 b 11.3 ± 1.7 b 1.5 ± 0.7 a 0.2 ± 0.1 a 1.0 ± 0.4 a hemp 10.4 ± 1.4 a 9.1 ± 1.8 a 1.3 ± 0.6 a 0.4 ± 0.2 a 1.8 ± 0.5 ab rock wool 85.4 ± 5.6 a 89.4 ± 5.3 a 63.2 ± 3.3 a 31.0 ± 5.5 a 44.1 ± 6.8 a sheep wool 89.1 ± 6.5 a 92.5 ± 6.0 a 63.0 ± 2.6 a 34.2 ± 17.0 a 38.7 ± 2.9 a peat moss 83.4 ± 4.4 a 87.5 ± 4.2 a 61.3 ± 1.6 a 30.2 ± 10.8 a 42.8 ± 6.3 a hemp 84.1 ± 4.5 a 88.8 ± 3.2 a 62.0 ± 1.6 a 35.7 ± 14.5 a 38.1 ± 9.0 a rock wool 161.5 ± 15.8 c 136.0 ± 1.4 b 25.5 ± 12.5 a 8.8 ± 4.5 a 28.4 ± 12.7 ab sheep wool 138.9 ± 13.5 ab 121.3 ± 2.3 ab 17.6 ± 10.5 a 6.4 ± 5.4 a 48.8 ± 14.8 ab peat moss 153.4 ± 12.1 bc 129.1 ± 0.9 b 24.3 ± 10.6 a 7.8 ± 3.4 a 26.0 ± 13.8 a hemp 123.4 ± 13.1 a 101.8 ± 1.8 a 21.6 ± 10.6 a 9.4 ± 5.1 a 50.8 ± 19.5 b the data represent mean values of eight plants per substrate and greenhouse compartment (n = 16). comparisons were calculated using tukey-test, where values followed by different letters differ significantly from each other (p < 0.05). values with the prefix ± represent the standard deviation fruit yield (kg plant-1) fruit weight (g fruit-1) fruit (number plant-1) 74 d. dannehl, j. suhl, c. ulrichs, u. schmidt fruit, where the number of a-class fruit were affected in a similar manner as presented for the number of marketable fruit (tab. 5). in consequence thereof, the highest marketable fruit yield showed plants grown in rock wool slabs (13.8 kg plant-1), followed by plants produced in peat moss slabs (12.8 kg plant-1), sheep wool slabs (12.3 kg plant-1) and hemp slabs (10.4 kg plant-1). in this context, the hemp material reflected the weakest form of the used substrates, which caused a significant reduction in fruit yield compared to those obtained by the remaining growing materials. the surplus yield achieved by the other substrates was based more on changes in yield of a-class fruit than on b-class and c-class fruit, because significant differences were only calculated for the first mentioned fruit category (tab. 5). it might be possible that the higher amount of eaw and the lower air volume in rock wool and peat moss slabs were partly responsible for the higher yields in terms of these substrates, because allaire et al. (2005) found that the marketable fruit yield of tomatoes was positive related to eaw and negatively related to the air volume. these results are supported by kirda et al. (2004), who demonstrated a yield reduction based on a low water retention capacity in substrates. beside these possibilities, it was found that the leaf area correlated positively with the marketable fruit yield (r = 0.865, p = 0.044). generally, the photosynthesis can be improved by a higher photosynthetically active leaf area (engels et al., 2012). in this context, it was assumed that the lower leaf area from plants grown in sheep wool and hemp slabs led to a reduced assimilate supply, resulting in a yield decline. this hypothesis is supported by marcelis and heuvelink (1999) and heuvelink and dorais (2005), who demonstrated that higher assimilation rates promoted the formation of cucumber fruit and flowers of tomatoes. it is more likely, however, that both a higher volume of eaw and a higher leaf area can be used as explanatory approaches for changes in marketable fruit yield. regarding fruit quality, it was calculated that the significantly highest amount of ber-fruit were produced by sheep wool slabs (1.9 kg plant-1), followed by hemp slabs (1.8 kg plant-1), rock wool slabs (1.2 kg plant-1) and peat moss slabs (1.0 kg plant-1) (tab. 5). in this context, it was demonstrated that the yield of ber-fruit decreased with increasing leaf area per plant (r = -0.886, p = 0.041). a reduced leaf area and a lower volume of eaw as caused by sheep wool and hemp can retard the transpiration stream, whereby the transport of calcium to the fruit can be inhibited. while the content of the most analysed macroand micronutrients tended only to changes, the calcium content based on dry weight basis was significantly increased in fruit matured by peat moss slabs compared to those ripened by sheep wool slabs (tab. 6). the results represented the evidence that the higher the calcium content in fruit the lower the yield of berfruit (r = -0.951, p = 0.049). similar was found by guichard et al. (2001), who explained the higher proportion of ber-fruit by a lower calcium uptake due to the lower water uptake in the root zone. the firmness of tomatoes is one of the most important quality characteristic for consumers (batu, 2004), where this attribute was not affected in the present study (fig. 1). however, the dry matter in fruit produced by hemp (5.64 g 100 g-1 fw) was significantly decreased compared to rock wool (6.2 g 100 g-1 fw), sheep wool (6.3 g 100 g-1 fw) and peat moss (6.1 g 100 g-1 fw) (fig. 1). nearly the same relations apply to the ssc as well, where a high correlation between these quality characteristics was found (r = 0.819, p = 0.032). as such, the ssc differed by up to 0.3 g 100 g-1 fw between the lowest (hemp) and the highest (sheep wool) content in tomatoes, where no correlation between physical parameters of substrates and the dry matter content, as well as ssc was identified. similar results were reported by dobricevic et al. (2008) regarding comparisons between rock wool slabs and organic materials. they found that the dry matter content and the ssc in tomatoes produced by rock wool slabs showed at least a tendency to decrease compared to fruit ripened by the influence of peat, whereas the results were vice versa in consideration of coconut fiber. the same applies to the tests carried out by kowalczyk et al. (2011), who found an increase in ssc by up to 0.4 g 100 g-1 fw caused by coconut fibre. beside these results, tüzel et al. (2001) obtained different contents of ta from plants tab. 6: effects of different growing substrates on minerals in tomatoes content element rock wool sheep wool peat moss hemp c 2643.1 ± 349.3 a 2689.5 ± 151.5 a 2570.3 ± 228.9 a 2531.2 ± 154.5 a n 107.5 ± 16.8 a 113.9 ± 10.4 a 102.3 ± 7.0 a 101.9 ± 9.1 a p 30.0 ± 4.1 a 28.2 ± 1.4 a 28.8 ± 2.0 a 27.4 ± 2.5 a k 214.2 ± 36.6 a 216.6 ± 16.9 a 206.3 ± 22.2 a 200.3 ± 14.6 a ca 7.7 ± 1.4 a 7.2 ± 0.9 a 7.8 ± 1.2 a 7.0 ± 0.7 a mg 7.4 ± 1.4 a 7.5 ± 0.7 a 7.1 ± 0.9 a 7.0 ± 0.6 a s 9.6 ± 1.4 a 10.4 ± 0.4 a 9.5 ± 0.8 a 9.4 ± 0.9 a fe 0.35 ± 0.03 a 0.40 ± 0.04 a 0.41 ± 0.09 a 0.42 ± 0.12 a c 441.2 ± 2.1 ab 442.8 ± 1.9 b 440.9 ± 2.6 ab 439.0 ± 2.1 a n 17.9 ± 0.7 a 18.7 ± 0.8 a 17.7 ± 0.4 a 17.7 ± 1.2 a p 5.0 ± 0.2 a 4.8 ± 0.1 a 4.9 ± 0.1 a 4.8 ± 0.2 a k 35.6 ± 1.7 a 35.6 ± 1.1 a 35.4 ± 1.0 a 34.2 ± 0.9 a ca 1.3 ± 0.2 ab 1.0 ± 0.1 a 1.3 ± 0.1 b 1.1 ± 0.2 ab mg 1.2 ± 0.1 a 1.2 ± 0.1 a 1.2 ± 0.1 a 1.2 ± 0.1 a s 1.6 ± 0.1 a 1.7 ± 0.1 a 1.6 ± 0.1 a 1.6 ± 0.1 a fe 0.06 ± 0.01 a 0.07 ± 0.01 a 0.07 ± 0.02 a 0.07 ± 0.03 a the contents of nutrients in tomatoes represent average values of five harvest dates, where three biological replicates per harvest date and two greenhouse compartments were considered (n = 30). different small letters indicate significant differences according to the tukey-test procedure at a significance level p < 0.05. values with the prefix ± represent the standard deviation. (m g 10 0 g1 fw ) (m g g1 d w ) evaluation of substitutes for rock-wool 75 grown in different substrates, such as perlite, pumice or a mix of perlite and peat (80% : 20%, v/v), whereas no differences occurred in the present study in terms of ta and the sugar : acid ratio (fig. 1). these calculated values are in agreement with the results provided by abak and celikel (1994), who found no changes in ta neither in tomatoes grown in organic (spent mushroom compost and peat) nor in inorganic growing substrates (rock wool and volcanic tuff). the knowledge about effects of different substrates used in soilless cultures on secondary plant compounds in tomatoes is scarce. abak and celikel (1994), for instance, discovered no significant differences regarding contents of laa when diverse growing materials were compared. however, kowalczyk et al. (2011) analysed a higher laa content in tomatoes of the cultivar admiro and drw 7594, which were matured by coconut fibre instead by rock wool. referring to the present study, it was found that secondary meta bolites, such as lycopene, ß-carotene, phenolics and laa, were not significantly affected by the application of different growing substrates (fig. 2). nevertheless, it was evidenced that the eaw contained in substrates has an impact on secondary plant compounds in tomatoes. as such, a high negative correlation was calculated between eaw and: lycopene (r = -0.918, p = 0.040); ß-carotene (r = -0.997, p = 0.003); penolics (r = -0.918, p = 0.039); laa (r = -0.848, p = 0.152). except for the laa content, all correlations were significant. it might be possible that the lower volume of eaw in hemp and sheep wool slabs was responsible for a slightly increase of carotenoids and phenolics in tomatoes, which was elicited by a minor water stress. this conclusion is supported by zushi and matsuzoe (1998), who demonstrated that a soil water deficit of 50% can lead to a lycopene accumulation in tomatoes by 25% compared to non-stressed plants. sanchez-rodriguez et al. (2011) fig. 2: effects of different growing substrates on contents of carotenoids, phenolic compounds and l-ascorbic acid (laa) in tomatoes. the results represent average values of five harvest dates, where three biological replicates per harvest date and two greenhouse compartments where considered (n = 30). different small letters indicate significant differences according to the tukey-test procedure at a significance level p < 0.05. values with the prefix ± represent the standard deviation. fig. 1: effects of different growing substrates on dry matter, soluble solid content (ssc), titratable acid (ta) and firmness of tomatoes. the results represent average values of five harvest dates, where three biological replicates per harvest date and two greenhouse compartments where considered (n = 30). different small letters indicate significant differences according to the tukey-test procedure at a significance level p < 0.05. values with the prefix ± represent the standard deviation. 76 d. dannehl, j. suhl, c. ulrichs, u. schmidt showed that a moderate water stress can increase phenylalanine ammonia-lyase activity and synthesized phenolics in more tolerant tomato cultivars, e.g., cv. zarina. in the present study, it might also be possible that the higher lycopene content was a result of a lower calcium content in tomatoes as found for plants grown in hemp and sheep wool substrates, because a significant negative correlation was calculated between these variables (r = -0.990, p = 0.010). in contrast, the contents of ß-carotene, phenolics and laa remained un affected. similar results were demonstrated by fanasca et al. (2006) under a reduced concentration of calcium in the nutrient solution. under consideration of all other macroand micronutrients, no correlations were calculated in combination with the analysed secondary metabolites. conclusion sphagnum farming enables the production of a renewable organic substrate (wichmann et al., 2014). it can be recommended for practitioners that this dried peat moss material pressed as slabs (sphagnum palustre) can be used as replacement for rock wool slabs as growing substrate for hydroponic tomato production. however, the usage of slabs consisting of hemp and sheep wool is not suitable as alternative for rock wool slabs. the findings suggested that comparable results in terms of plant growth, yield, as well as primary and secondary plant compounds were obtained when rock wool slabs and peat moss slabs were considered. the advantage of peat moss slabs is that they can be used to increase the fruit quality of tomatoes, where the yield of blossom end rot fruit was reduced by approximately 17% compared to that produced by rock wool slabs. hemp and sheep wool slabs, however, promoted the formation of blossom end rot fruit to a much greater extent due to a decreased leaf area as a consequence of a lower volume of eaw, whereby the transpiration stream and therefore the accumulation of ca in fruit can be retarded. these properties and the fast decomposition of these substrates resulted in plant instability and in an inhibited plant development followed by a yield reduction. furthermore, the newly-acquired knowledge about interactions between physical properties, especially eaw and air volume, and the leaf area, flowers, as well as secondary plant compounds can be used, in order to evaluate growing media for hydroponic systems. in this context, the lower air volume contained in peat moss slabs did not negatively affect the plant development. considering peat moss and rock wool slabs, the optimum values regarding the air volume in substrates used for hydroponic systems have to be adjusted, where values in a range from 9.9 vol% to 58.5 vol% are acceptable for tomato production. the same applies for the adjustment of the optimum value in terms of eaw, which should be between 32.0 vol% and 37.4 vol% to optimize the plant development. furthermore, a wbc between 0.13 vol% and 0.21 vol% did not affect the plant development negatively. while production and recycling processes of rock wool slabs heavily pollute the environment as mentioned earlier, the peat moss slabs can be classified as an environmentally friendly growing substrate for hydroponic tomato production. this material can reduce the waste flow under protected growing conditions, because peat moss slabs belongs to organic materials and can therefore be composted environmentally friendly after using or can be used as soil additive or fertilizer under open field conditions. in this context, further investigations are necessary to proof the suitability of used peat moss as fertiliser and in what amounts fertiliser can be saved. acknowledgements we would personally like to thank all the scientists of division biosystems engineering and urban plant ecophysiology, especially stefan irrgang and armin blievernicht. references abad, m., martinez, p.f., martinez, m.d., martinez, j., 1993: evaluacio‘n agrono‘mica de los sustratos de cultivo. actas de horticultura 11, 141-154. abad, m., noguera, p., 2000: los sustratos en los cultivos sin suelo. in: urrestarazu, m. 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(ed.), mitigation of climate change in agriculture series 9 − towards climate-responsible peatlands management, 80-83. food and agiculture organization of the united nations (fao), rome. zushi, k., matsuzoe, n., 1998: effect of soil water deficit on vitamin c, sugar, organic acid, amino acid and carotene contents of large-fruited tomatoes. j. japan. soc. hort. sci. 67, 927-933. address of the corresponding author: dennis dannehl, humboldt-universität zu berlin, faculty of life sciences, albrecht daniel thaer-institute of agricultural and horticultural sciences, division biosystems engineering, albrecht-thaer-weg 3, 14195 berlin e-mail: dennis.dannehl@agrar.hu-berlin.de journal of applied botany and food quality 87, 210 219 (2014), doi:10.5073/jabfq.2014.087.030 humboldt-universität zu berlin, faculty of life science, albrecht daniel thaer-institute of agricultural and horticultural sciences, 1division biosystems engineering and 3division urban plant ecophysiology; 2institute for product quality, berlin, germany the potential of a confined closed greenhouse in terms of sustainable production, crop growth, yield and valuable plant compounds of tomatoes dennis dannehl1*, melanie josuttis2, christian ulrichs3, uwe schmidt1 (received april 20, 2014) * corresponding author summary a confined closed greenhouse (cgh) was applied to save energy and to investigate how tomatoes respond to specific microclimatic conditions. as such, new dynamic set-points for precise climate control were used in the cgh compared to those applied in a conventional greenhouse. based on the reduced ventilation frequency in the cgh, the results showed that higher levels of mean temperature, co2 concentration and relative humidity were achieved. although the light interception was increased in the cgh, these changing microclimatic conditions resulted in higher rates of photosynthesis and an associated faster crop growth. this means that the mean plant height was increased by 1.5 m, which was the decisive factor to increase the total yield by 21.4 % in relation to that produced in the conventional greenhouse. the new microclimatic environment caused by the cgh promoted the accumulation of primary and secondary plant compounds in tomatoes such as soluble solids (by 9 %), lycopene (by 22 %), ß-carotene (by 21 %), phenolics (by 8 %) and l-ascorbic acid (by 26 %) compared to conventional produced tomatoes. compared to existing greenhouse systems, the results suggested that a cgh can be used to produce tomatoes in a sustainable way, where the water use and the energy use efficiency can be improved by 71 % and 43 %, respectively. introduction generally, greenhouses have been developed to protect crops from phytosanitary problems and to extend the harvest season in order to provide the growing population with food (unfpa, 2011). additionally, these technical systems should also be used to produce food with a high quality standard, because the demand for improved quality of fruit and vegetable is increasing among consumers. one of the reasons for this amplified health awareness in society is the apparent relationship between the intake of horticultural products and numerous health benefits for consumers. these positive effects are attributed to different secondary plant compounds with antioxidant properties especially phenolic compounds, l-ascorbic acid and carotenoids, which can detoxify reactive oxygen species in the human body (bazzano et al., 2002). this process is believed to be responsible for the suppression of the occurrence of chronic diseases, e.g., human prostate cancer and gastric cancer (carr and frei, 1999; kotake-nara et al., 2001). however, these high customer and market demands are overshadowed by the future production conditions in greenhouses. among other things, the production processes in terms of fruit and vegetables require high amounts of energy for heating and freshwater for irrigation, where these traded goods are the most cost-intensive resources in greenhouse production due to the increase in fossil fuel and freshwater prices (ozkan et al., 2007; rout et al., 2008). this pricing policy is mainly based on the worldwide shortage of fossil fuels and freshwater resources (vörösmarty et al., 2000; shafiee and topal, 2009). based on these facts, scientists invested much effort into the development of agronomic approaches for using renewable energies and new irrigation strategies, in order to reduce the consumption of fossil fuels and freshwater for greenhouse production. amongst others, the solar energy is becoming used more and more for process energy in greenhouses, which can be collected in closed and semi-closed greenhouses using cold water from soil layers or rain water tanks, respectively (de gelder et al., 2012; dannehl et al., 2013b). after absorbing the excess heat using cooling fin heat exchangers installed under the roof in these greenhouses, the heat energy is stored in the aquifer or in rain water tanks used as short-term energy storage systems and can be reused in cooler periods by means of a heat pump (bot, 2001; dannehl et al., 2013b). this process results in decreasing levels of temperature and is accompanied by the dehumidification in greenhouses caused by condensation on the cooling fins (campen and bot, 2002). although the energy use efficiency (eue) and the water use efficiency (wue) can be improved using these new technologies, high amounts of energy is needed for the heat pump to collect the sensible heat and latent energy in greenhouses (dannehl et al., 2013b; dannehl et al., 2014). this applies in particular to cooling processes if the heat stored in the short-term storage tank exceeds a temperature of 35 °c in the summer period. in consideration of the cultivation period of tomatoes, it may be more effective to apply a combination of closed and semi-closed greenhouse systems including a heat pump in late winter, spring, late summer and autumn until the short-term storage tank is charged with heat. thereby, it is assumed that the greenhouse can be cooled and the required energy for the basic load for heating up the greenhouse can be covered during the year. during summer, however, a fog system combined with a semi-closed operation mode named descending fog system (descfog), which is operating without a heat pump, should be used for cooling processes (dannehl et al., 2012). this combination of operation modes was applied in the present study and is referred to as confined closed greenhouse (cgh). due to the new set-point strategies for cooling, heating, ventilation opening and co2-enrichment in the cgh, the microclimatic conditions can change in this system. in particular, it is expected that the average levels of temperature, relative humidity (rh) and co2concentration will be increased as compared to a conventionally operating greenhouse. furthermore, dannehl et al. (2013b) found a light reduction by 11 % in the closed greenhouse, which was induced by the construction parts such as cooling fins under the roof. these mentioned conditions may cause stress in plants. it is well known that high temperatures and a reduced supply of rh, solar radiation or of co2 levels could have adverse effects on tomato plants, e.g., on photosynthesis, transpiration, fruit yield and secondary plant compounds with antioxidant properties (dumas et al., 2003; camejo et al., 2005; van der ploeg and heuvelink, 2005; patane, 2011; kläring and krumbein, 2013). however, there is little information available regarding changes in plant growth, yield and secondary metabolites of tomatoes depending on a combination of higher temperatures, relative humidity and co2 concentrations during the cultivation period of tomatoes. thus, this study focussed on the effects of changing microclimatic conditions in a cgh on photosynthesis, transpiration, vegetative plant growth, fruit yield, carotenoids, phenolic compounds and l-ascorbic acid of tomatoes. furthermore, a the potential of a confined closed greenhouse 211 211 sensory analysis regarding the internal fruit quality was conducted to test the influenced by the cgh on quality parameters such as juiciness, fruitiness and sweetness. moreover, the effect of the cgh on the water consumption (wc), wue and eue was investigated, in order to estimate if plants can be produced in a sustainable way with such a greenhouse facility. materials and methods experimental set-up the experiments were conducted in two different n-s oriented venlo-type glasshouses at the humboldt-universität zu berlin. one greenhouse, operated by a conventional microclimatic control strategy as used in practice (rgh) was compared with a cgh controlled system. tab. 1 shows the structural differences between both greenhouses, which were described in more detail by dannehl et al. (2013b). in both greenhouses the energy screens were closed at a global radiation of less than 3 w m-2, in order to save energy. in the rgh, the floor level heating was set at 17 °c for day and night and the ventilation was opened above 23 °c to reduce the temperature inside the greenhouse. these processes were controlled using the application of proportional integral differences. the same control mechanisms were used in the cgh including new dynamic set-point strategies for cooling, heating, ventilation and co2 enrichment in comparison with the rgh. in this context, finned tube heat exchangers fixed under the roof of the cgh were coupled with an electrically operated heat pump and a rain water tank (300 m3) used as short-term energy storage. this system was applied to collect high amounts of sensible heat energy and latent energy while cold water (5 °c) was flowing through the finned tube heat exchangers. the mentioned energy components were generated by the incoming solar energy and plant transpiration, as well as evaporation processes, respectively. this type of energy harvesting was used, in order to reuse the stored energy if necessary and for cooling processes in the cgh. in the present study, the closed and semi-closed operation phases in the cgh alternated several times during the cultivation of tomato plants as shown in fig. 1. the closed and semi-closed operation phases from february to may and from mid-august to november were mainly used to charge the rain water tank with energy. as such, the water from this tank was used either direct without the heat pump (> 5 °c to 10 °c) or indirect with the heat pump (> 10 °c to 40 °c) for cooling processes in the cgh, which were started at an air temperature of 22 °c. while the ventilation was completely closed during the application period of the closed greenhouse, emergency ventilation above 27 °c was permitted during the semi-closed operation mode in spring and late summer, in order to avoid plant damage. as described earlier, the use of a heat pump requires high amounts of energy for cooling processes during summer. therefore, an evaporative cooling system was applied in the cgh without using a heat pump from june to mid-august (fig. 1). in this context, a high pressure fog system was installed above the plants to ensure a uniform distribution of the small droplets (10 μm) in the crop. in order to realize droplet evaporation and associated cooling processes, a coupled control for fog and ventilation was conducted using a microcontroller, which was connected between 23 °c and 27 °c measured in the plant population. to remove the energy rich water vapour from the roof region, the ventilation was opened with short time pulses and a minimum aperture (max. 10 %) at a rh of 80 % measured in the roof region. the fog system was interrupted at a rh of 80 % determined within the crop, in order to protect plants from diseases. however, the warm water stored in the rain water tank was reused either for direct heating (40 °c to > 35 °c) or for indirect heating tab. 1: greenhouse characteristics as a function of the confined closed greenhouse (cgh) and the reference greenhouse (rgh) greenhouse construction rgh cgh gross acreage [m2] 307 307 greenhouse height [m] 6.7 6.7 glazing roof single glass panes single glass panes (4 mm) (4 mm) glazing side walls double glass panes double glass panes (16 mm) (16 mm) energy screens (roof) 1 2 energy screens (side walls) no 1 finned tube heat exchangers no 16 (each 21.4 m) reversible heat pump no 1 (120 kw hp[†]; 100 kw cp[††]) high pressure fog system no 28 fog nozzles (150 bar) floor level heating yes yes tubular film blowers no yes vegetation heating no yes hp[†] and cp[††] means heating power and cooling power, respectively. fig. 1: schematic diagram for closed and semi-closed operation phases in the confined closed greenhouse system and associated energy charge and discharge processes occurring in the short-term energy storage tank. 212 dennis dannehl, melanie josuttis, christian ulrichs, uwe schmidt (35 °c to 8 °c) via a combination of the heat pump and heat exchangers mounted in the cgh, i.e., using tubular film blowers fixed under the nutrient solution gullies and a vegetation heating system installed in the plant population with a target temperature of 17 °c (day and night). this heating procedure in the cgh is accompanied by the discharge of the rain water tank (fig. 1). when the stored energy was entirely used up, the energy supply for heating processes in the cgh was realized using a floor level heating system containing district heat as used in the rgh. the co2 enrichment was applied in both greenhouses, which was kept at a level of 800 ppm during daylight hours. in this context, the co2 supply was stopped when the ventilation exceeded an opening of 10 %. to maintain the sought microclimatic conditions in the rgh and cgh, all the aforementioned set points for cooling, heating, ventilation, rh and co2 enrichment were controlled by data obtained from different sensors. the measurements were forwarded to a central control computer, where these were recorded every 30 seconds. cultivation of tomato plants and assessment of crop growth and yield a closed hydroponic system with a recirculating nutrient solution was used to produce tomato plants (solanum lycopersicum l. cv. komeett) on a net-acreage of 260 m-2 per greenhouse. these were grown on high gullies in rock-wool slabs and watered using drip irrigation, which was started mainly after a light summation of 560 w m-2 for 150 seconds. to obtain a water overflow of 40 % after each irrigation cycle, the light summation for controlling the irrigation was regularly adjusted in each greenhouse. the nutrient solution was prepared by mixing fresh water and stock solution according to the mixture recipe of göhler and molitor (2002). each greenhouse was equipped with twelve gullies, which were arranged in five double rows and two single rows with a length of 20 m. each gully contained 40 plants grown at a distance of 0.5 m from each other. the plants were transferred to the rock-wool slabs on 24th january 2012, when four leaves were formed. to calculate the leaf area index (lai) in the sixth, seventh and eighth week after planting, a total of 20 plants per greenhouse was used to measure the leaf length and width of each leaf with a folding ruler. the measurements were inserted in an exponential function developed by dannehl et al. (2013a) to estimate the leaf area of each individual leaf. the calculated area of these leaves was summed per plant, where the average leaf area regarding the evaluated plants was extrapolated to the entire plant population. this leaf area was divided by the net-acreage to obtain the lai, which is expressed as m2 m-2. at the end of the experiments on 6th november 2012, the plant height of the same plants was measured after the stem of the plant was cut directly above the rock-wool slabs. furthermore, the tomatoes were harvested and weighed weekly, as well as summed up to compare the total yield and the fruit mass per fruit in terms of both greenhouse facilities at the end of the investigations. the plant height, the fruit mass and the yield are expressed as m, g and kg m-2, respectively. determination of photosynthesis, transpiration, eue, wc and wue ten leaf cuvettes of a leaf cuvette based gas exchanged system were arranged at the same heights in the canopy of both greenhouses to measure the photosynthesis (bermonis, steinbeis gmbh & co. kg for technology transfer; berlin, germany). to ensure the same measurements points in both greenhouses, the cuvettes were transferred weekly to the first fully developed leaf of different plants. the cuvettes consist of polyethylene which is transparent for the radiation frequency from 300 nm to > 3000 nm, where the boundary layer conditions inside the cuvettes are identical compared to those of a non-influenced leaf. the photosynthesis was measured every 30 seconds as mean co2-gas exchange (geco2) during the harvest period. as such, the flow rate of the air (q), the co2 level difference between ambient air and the air in the cuvettes (diffco2), the atmospheric pressure (p), the chamber area (cha), the air temperature (t) and a constant (29.93), derived from the molar mass and the specific gas constant of co2, were used to calculate geco2 (equation 1). geco2 was calculated as follows: q × diƒƒco2 × p geco2 = (1) 29.93 × cha × tair the mean value of geco2 was generated daily in terms of the daylight hours; these values are expressed as μmol co2 m-2 s-1. furthermore, the plant transpiration was measured with the same device using the same measuring principle and calculated according to schmidt (1988). the flow rate of the air, the absolute humidity difference between the ambient air and that in the cuvettes (diffah), as well as the cha was used in equation 2 to compute the transpiration as follows: q × diƒƒah transpiration = (2) cha the mean values of the transpiration were generated daily and expressed as mg h2o m-2 s-1. the eue is defined as the amount of energy required to produce one kg of tomatoes (mj kg-1) and was evaluated on three dates of the last three weeks of the experiments. to calculate the eue for the cgh, the energy consumed (ec) for the circulation pumps (cp), for heat pump processes (hp) and for district heat (dh), the primary energy factor for electrical energy (pfee) and for district heat energy (pfdh), the reuse of the stored energy (ee) and the total yield were considered as shown in equation 3: (ecdh × pfdh) + (eccp + hp × pfee) − (ee) eue = (3) total yield the eue calculated for the rgh was determined in the same manner as described in equation 3, however, excluding the variables eccp + hp, pfee and ee. on three dates of the last three weeks of the investigations, the total water consumption (wc) consumed by the crop in each greenhouse during the cultivation period was calculated. this analysis included evapotranspiration losses and was determined using the difference between the amount of the recorded irrigation and that of the nutrient solution drained from the cultivation gullies. based on these results, the wue was calculated as a ratio between the total yield (kg m-2) and wc (m3 m-2) and is expressed as kg fresh matter per m-3. chemical analyses and sensory investigations of tomatoes three replicates containing 30 tomatoes were randomly harvested from different plants per greenhouse at the ripening stage 9. this procedure was repeated five times in five consecutive weeks from june to july to compare the average of five dates of three biological replicates. for the analyses, each sample collection per greenhouse was homogenised (kenwoodhb856, de’longhi deutschlang gmbh; neu-isenburg, germany). the homogenised fresh material of each replication was used for the determination of the contents of fruit dry matter, secondary plant compounds, titratable acids (ta) and soluble solids (ssc) performed in duplicate. fresh mass and dry mass from all fruit samples were measured before and after drying in a ventilated oven for one day at 105 °c, respectively. the fruit dry matter content was calculated by the ratio of the dry mass to the fresh mass and is expressed as g kg-1. the potential of a confined closed greenhouse 213 213 the l-ascorbic acid content (laa) was determined using the enzymatic test kit (l-ascorbate, megazyme international ireland; bray, ireland). briefly, the tetrazolium salt mtt [3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide] was reduced by laa to a formazan compound (mtt-formazan) under the presence of phenazinium methylsulfate. mtt-formazan was detected spectrophotometrically at a wavelength of 578 nm. other reducing substances were measured in the same way after removing laa using ascorbic acid oxidase. the differences between both absorbance values were used to calculate the laa content, which is expressed as g kg-1 fresh weight (fw). lycopene, ß-carotene and total phenolic contents were determined by spectrophotometry (model 690, gamma analysen technik gmbh; bremerhafen-lehe, germany). briefly, lycopene and ß-carotene contents in tomatoes were extracted using the method outlined by fish et al. (2002), where this method was modified by dannehl et al. (2011). the hexane extracts of lycopene and ß-carotene were measured at wavelengths of 505 nm and 453 nm, respectively. subsequently, the contents of carotenoids were calculated according to nagata and yamashita (1992) and are expressed as g kg-1 fw. the phenolics were extracted as described by connor et al. (2002). for the analysis of the extracts, the folin-ciocâlteu method according to slinkard and singleton (1977) was used. in this context, the modified procedure of this analysis was described exactly by dannehl et al. (2011). the results are expressed as g gallic acid per kg fw (g gae kg-1 fw). the ta content in tomatoes was determined by potentiometric titration with 0.05 mol l-1 naoh by ph endpoint at 8.1 according to asu-l-26.11.03-4 (1983). these analyses were performed using a ph meter (ph526, wtw; weilheim, germany) including a glass electrode (sentix 41, wtw; weilheim, germany). the ssc was assayed using a digital refractometer (pr101, atago; karlsruhe, germany), which detects reducing sugars and other soluble solids. the results of the analysis of ta and ssc were converted to one kg fw basis and are expressed as gram citric acid and gram ssc per kg fw, respectively. on the last date of the chemical analyses in july, a whole batch of tomatoes at the ripening stage 9 was used for a sensory investigation, in order to assess the quality of tomatoes from the perspective of consumers. in this consumer acceptance test, 100 passers-by of different origin evaluated tomatoes from the rgh and cgh regarding flavour and mouthfeel attributes, such as fruity, watery, grassy, mealy, juicy, bitter, sour, sweet, tomato-like, and peel firmness. additionally, the total quality, which includes all sensory attributes, was also assessed by the consumers. the intensity of all sensory impressions of the tomatoes was evaluated using an unstructured scale with the defined points 0 (low intensity) and 10 (high intensity). in this context, the consumers were trained over a period of 15 minutes to understand the meaning of the attributes and had to rinse their taste organ with water for neutralization. in all sensory tests, the tomatoes of each greenhouse were quartered before serving to the assessors on coded plates, where the consumers received as much tomato pieces as needed to evaluate them. these tests were performed at room temperature, 50 % rh and under artificial light conditions. statistical analysis the effects of changing microclimatic conditions caused by the rgh and cgh on crop growth, tomato yield, plant physiological responses, as well as on the water and energy balance were evaluated using spss package version 19.0. all comparisons were calculated using t-test procedure, where asterisks indicate a significant increase at a significance level *α = 0.05 and **α = 0.01. considering the harvest period, the results represent average values of 29 evaluation dates (n = 29) in terms of microclimatic conditions, transpiration rates, photosynthesis rates and fruit mass, except for the determination of lai, wc, wue and eue (n = 3), as well as for chemical analyses of primary and secondary metabolites (n = 15). to compare both greenhouse systems regarding the plant height and the total yield at the end of the experiments, twenty plants and 400 plants were used, respectively. before the flavour and mouthfeel attributes of tomatoes produced in the rgh and in the cgh were compared, a triangle test with one different and two alike tomato pieces was applied to consumers (n = 100). since a data analysis using the chi-square test showed that the number of correct perceptions of consumers was significant (α = 0.01), the t-test was used to compare sensory impressions of the consumers. results microclimatic conditions in consideration of selected days, the comparisons of microclimatic conditions between the rgh and cgh are displayed in fig. 2. at outside temperatures between 16.6 °c and 20.9 °c during the days of march, the ventilation in the rgh was opened of up to 54 %, whereas the ventilation in the cgh was completely closed due to the closed operation mode on these days. based on this fact, the temperature in the rgh was maintained at 24 °c and that in the cgh at approximately 27 °c. furthermore, the mentioned ventilation conditions led to higher levels of co2 and rh in the cgh, where maximum differences by up to 500 ppm and 22 % were observed, respectively, when compared to the rgh. however, the microclimatic conditions in both greenhouses changed depending on the warmer season in may but also due to the applied semi-closed operation mode in the cgh, which was operating with the heat pump to realize cooling processes. based on the defined set-points regarding the ventilation opening in each greenhouse, the ventilation in the cgh was kept closed for a longer period than that in the rgh (fig. 2). two consequences of this were that the rh was more than 20 % higher and the co2 concentration was increased by up to 200 ppm in the cgh compared to the rgh, where this maximum co2 difference was only one third of the difference reached in the closed operation mode. compared to the rgh, the higher levels of co2 in the cgh were accompanied by lower temperatures by up to 2.5 °c during the day. this effect was particularly based on the cooling process caused by the fined tube heat exchangers. furthermore, the semi-closed operating descfog system used for evaporative cooling in the cgh caused a lower ventilation frequency compared to that in the rgh during the summer period (fig. 2). this ventilation behaviour provoked higher levels of co2 and rh in the cgh, which were maintained at 450 ppm and 85 %, respectively. however, the co2 concentration and the rh in the rgh were greatly limited to 250 ppm and 60 % at temperature peaks between 30 °c and 33 °c measured outside during the days in july. at the same days, the temperature inside the cgh could be lowered in the range between 2.5 °c and 4.4 °c compared to the outside conditions, whereas the temperature profile within the rgh was almost equal to that recorded outside. in contrast, the temperature values in the cgh were approximately 2 °c higher compared to those obtained in the rgh during the morning and evening hours due to the descfog system and an associated ventilating at the earliest at 25 °c. the same applies to the closed operation mode used in late winter, spring and autumn, which was most pronounced with a maximum temperature difference of 2.8 °c around noon (fig. 2). considering these temperature differences between both greenhouses during the tomato cultivation, the average temperature in the cgh was significantly increased by 0.5 °c compared to the rgh (tab. 2). the average value of the rh was not significantly affected. however, the mean co2 concentration in the rgh was significantly reduced by 132 ppm versus the cgh (tab. 2), where this result was caused by the higher levels of ventilation in the rgh. 214 dennis dannehl, melanie josuttis, christian ulrichs, uwe schmidt photosynthesis and transpiration fig. 3 represents the daily mean photosynthesis and transpiration depending on different greenhouse systems during the harvest period. the photosynthesis was almost continuously higher in the cgh compared to the rgh, even though the solar radiation was reduced in the cgh described earlier. the effects of the weaker light quantity on photosynthesis were only noticeable in late april, where the photosynthesis caused by the closed operation mode in the cgh was markedly reduced by up to 100 % compared to the rgh. in contrast, the semi-closed operation mode including heat pump applied from calendar week 18 to 22 and from 34 to 39 resulted in a higher photosynthetic activity versus rgh (fig. 3). these increased levels of photosynthesis varied between 32 % and 147 %. similar differences in rates of photosynthesis were found owing to the application of the semi-closed operating descfog system in the cgh from calendar week 23 to 33, however, to a smaller extent as shown before. as such, tomato plants grown in the cgh showed a maximum increase in photosynthesis by 65 % compared to those produced in the rgh. during the last two weeks of the experiments, no differences in photosynthesis were detected when the rgh and the closed operating cgh were compared. the latter outcomes are comparable to those found as results of transpiration measurements during the last two weeks of the investigations (fig. 3). during the remaining harvest period, however, all applied operation modes in the cgh affected the plant transpiration in the same manner as shown in fig. 3 (stacked bars). this means that the rates of plant transpiration were reduced by up to 58 % by the influence of the cgh when compared with the rgh. in consideration of the entire harvest period and compared to the rgh, the average value of the photosynthesis and that of the plant transpiration achieved in the cgh was significantly increased by 21 %, as well as significantly reduced by 35 %, respectively (tab. 2). tab. 2: microclimatic conditions and plant responses of tomato plants caused by different climate control strategies characteristic rgh cgh significance relative humidity [%] 91.77 93.48 temperature [°c] 21.60 22.11 * co2 [ppm] 525.72 657.87 * transpiration [mg h2o m-2 s-1] 21.54 14.07 * photosynthesis [µmol co2 m-2 s-1] 4.39 5.31 * number of fruit [fruit m-2] 188.59 242.15 * fruit mass [g] 107.27 116.05 * fruit dry matter content [g kg-1] 60.00 60.00 wc [m3 m-2] 0.81 0.57 * wue [kg fresh matter m-3] 29.11 49.91 * eue [mj kg-1] 57.27 39.49 * the table represents average values as a result during the harvest period (n = 29), except for wc, wue and eue (n = 3), as well as fruit dry matter content (n = 15). asterisks indicate significant differences according to the t-test procedure at a significance level p < 0.05. fig. 2: comparisons of microclimatic conditions and the ventilation behaviour between the reference greenhouse (rgh) and the confined closed greenhouse (cgh) in consideration of selected days during winter, spring and summer. fig. 3: the mean daily photosynthesis (geco2) and plant transpiration caused by the reference greenhouse (rgh) and the confined closed greenhouse (cgh) during harvest time. the potential of a confined closed greenhouse 215 215 crop growth, wc, wue and eue the influences of the rgh and the cgh on lai, plant height and total yield are shown in fig. 4. eight weeks after planting, the prevailing microclimatic conditions in the cgh resulted in a significant increase in lai (2.1 m2 m-2) compared to that measured in the rgh (1.8 m2 m-2). at the end of the experiment, the average plant height of plants grown in the cgh was significantly increased by 1.56 m when compared with the rgh. this result suggested that plants produced in the cgh grew faster, whereby 28.4 % more fruit per m-2 were formed (tab. 2). compared to the average fruit mass of 107.27 g attained in the rgh, the fruit mass of tomatoes matured in the cgh was significantly increased to 116.05 g, whereas the fruit dry matter content was not affected (tab. 2). based on these plant responses, the total yield caused by the cgh was significantly increased by 21.4 % in relation to the total yield produced in the rgh (fig. 4). furthermore, the microclimatic conditions in the cgh led to a significant reduction in wc by 30 % compared to that, which was consumed by plants grown in the rgh. due to the higher wc and the lower total yield in the rgh, the wue calculated for the cgh was significantly improved from 29.11 kg m-3 to 49.91 kg m-3 (tab. 2). it was also demonstrated that the eue achieved in the cgh was significantly improved by approximately 43 % compared to the rgh as consequences of the reuse of the stored energy in the rain water tank and the higher total yield (tab. 2). cgh. however, the consumer perceived that the application of the cgh significantly intensified other flavour attributes in tomatoes such as sweet and juicy, but also the mouthfeel attribute defined as peel firmness (fig. 6). in consideration of all sensory attributes, the consumers assessed the total quality of tomatoes grown in the cgh as significant higher compared to those matured in the rgh. discussion effects of different operation modes of greenhouses on photosynthesis, crop growth, yield, transpiration, wc, wue and eue generally, the applied operation modes in the cgh can effectively be used to decrease the inside temperature at high ambient temperatures, where this process is accompanied by higher levels of rh fig. 4: crop growth and total yield of tomato plants grown in the reference greenhouse (rgh) and in the confined closed greenhouse (cgh). all displayed comparisons were calculated using t-test procedure, where asterisks indicate a significant increase at a significance level *α = 0.05 and **α = 0.01 and bars standard deviations. the data represent average values of three replications, except for plant height (n = 2) and total yield (n = 2). fruit quality and sensory investigations it was found that the investigated primary and secondary metabolites containing in tomatoes were positively affected by the cgh. this means that the contents of ssc, lycopene, ß-carotene, phenolic compounds and laa in tomatoes ripened in the cgh were significantly increased by 9 %, 22 %, 21 %, 8 % and 26 %, respectively, compared to those determined in fruit from the rgh (fig. 5). these results were achieved not only on a fresh weight basis but also on a dry weight basis; because the fruit dry matter content in tomatoes did not differ in response to the corresponding greenhouse (tab. 2). otherwise, the content of ta remained unaffected depending on the application of different greenhouse systems. furthermore, the microclimatic conditions caused by the cgh affected the intensity of some sensory attributes (fig. 6). mouthfeel attributes such as fruity, watery, grassy, mealy, bitter, sour and tomato-like were equally influenced in tomatoes, which were produced in the rgh and the fig. 6: sensory investigation regarding flavour and mouthfeel attributes of tomatoes grown in the reference greenhouse (rgh) and in the confined closed greenhouse (cgh). all attributes were tested using t-test and represent the mean values of sensory impressions of 100 passers-by, where asterisks indicate a significant increase at a significance level α = 0.05. the intensity of all sensory impressions is defined as 0 (low intensity) and 10 (high intensity). fig. 5: reference greenhouse (rgh) and confined closed greenhouse (cgh) mediated changes in contents of primary and secondary metabolites in tomatoes. bars mean standard deviations and asterisks significant differences according to the t-test procedure at a significance level *α = 0.05 and **α = 0.01. the contents represent average values of five harvest dates of three biological replicates (n = 15). 216 dennis dannehl, melanie josuttis, christian ulrichs, uwe schmidt and co2 concentration. during the late april, the combination of higher levels of co2 and a diminished solar radiation in the cgh was not sufficient to increase the rates of photosynthesis compared to the rgh. this observation is consistent with the results reported by kläring and krumbein (2013) and gomez et al. (2013), who demonstrated that lower light conditions resulted in a decreased photosynthesis. in contrast, the rates of photosynthesis of plants grown under cgh conditions were higher compared to those achieved in the rgh during the remaining month of the harvest period (fig. 3). these results were obtained, although the transmitted solar radiation in the cgh was reduced by 11 % compared to the rgh as described by dannehl et al. (2013b). therefore, it was concluded that the higher levels of co2 concentration can contribute to a compensation of the mentioned light reduction in the cgh during the light intensive season. this conclusion complies with the evidences reported by vanoosten et al. (1995) and hurd (1968), who showed a higher photosynthesis of tomato plants exposed to an elevated co2 and a reduced light environment. as reported by mortensen (1987), the enhanced rates of photosynthesis are the basis for an increased plant growth, which was also demonstrated in the present study. as such, the lai and the plant height of plants produced in the cgh were increased compared to those grown in the rgh, whereby more trusses can be formed. these plant responses can also be explained by the higher average temperature (22.11 °c) obtained in the cgh (tab. 2). this assumption is consistent with the results found by adams et al. (2001), who observed a faster plant growth and more produced trusses of tomato plants, when the temperature was increased from 21 °c to 22 °c. furthermore, it was observed that changes in microclimatic conditions in the cgh did not promote the occurrence of fungal infestation or other tomato diseases. therefore, not more plant protective agents must be used in the cgh compared to conventional production. in the present study, the promoted photosynthesis of plants exposed to the cgh resulted in an enhanced assimilates supply followed by an accelerated plant growth, whereby the total yield and the fruit mass per fruit were increased by 21.4 % and 8.2 %, respectively (fig. 4; tab. 2). similar results were found by kläring and krumbein (2013), which were attributed to higher rates of photosynthesis. since the fruit dry matter content per gram fresh weight was unaffected (tab. 2), it was concluded that the higher fruit mass per fruit was most likely caused by a higher carbon fixation within the fruit than by an increased water accumulation due to higher levels of relative humidity as demonstrated by johnson et al. (1992). comparable yield responses as described in the present study were also found depending on an elevated mean temperature and co2 concentration (kimball, 1983; reinert et al., 1997; adams et al., 2001), where these microclimatic conditions were similar to those measured in the cgh. furthermore, kläring and krumbein (2013) and marcelis et al. (2006) identified that the total yield of tomatoes depends on the light conditions, where the yields were diminished in the range between 0.54 % and 1.1 % per 1 % light reduction. these calculations do not agree with our data, because higher yields were obtained in the cgh even though the light transmission was reduced. rather, it was found that a close relationship exists between the total yield and the average value of the photosynthesis, which was calculated under consideration of the entire harvest period (tab. 2). as shown in the results section, the total yield and the average value of photosynthesis caused by the cgh resulted in a surplus of 21.4 % and 21 %, respectively. therefore, it might be possible that a 1 % increment in photosynthesis results in a 1.02 % yield increase. in this context, zelitch (1982) summarized the results of various studies, which generally showed a positive correlation between photosynthesis and yields in terms of other plant species. to date, however, no investigations were found that refer to the estimation of tomato yields in relation to the photosynthesis. in comparison of both greenhouses, the average rh tended to increase in the cgh, whereas the temporally differences in rh ranged between 20 % and 25 % in favour of the cgh (tab. 2; fig. 2). patane (2011) and leonardi et al. (2000) found that higher relative humidity conditions inhibited plant transpiration of tomato plants. other studies showed that increased co2 concentrations led to decreasing stomatal conductance and rates of plant transpiration in c3 species (waggoner and zeilitch, 1965; louwerse, 1980). these results are fully consistent with our calculations. the differences in rh and co2 concentrations described earlier resulted in a decrease in transpiration of plants grown in the cgh (by 35 %) compared to those exposed to the rgh (tab. 2). these reduced transpiration losses were crucial for the reduction of the number of irrigation cycles applied in the cgh, whereby the wc was lowered by 30 % when compared to the rgh (tab. 2). this result combined with the higher total yield improved the wue by 71 % of plants produced in the cgh. this variable was raised to a value of 49.91 kg m-3 in the cgh, whereas a wue of 45.45 kg m-3 was achieved in co2 enriched dutch greenhouses (van kooten et al., 2008). therefore, it is obviously possible to improve the wue in other greenhouse facilities when the technical refinements applied in the cgh are integrated into existing systems. furthermore, it was demonstrated that the required energy for the basic load for heating up the cgh can be covered during the year using the collected energy from february to may and from midaugust to november. in this context, it was shown that the eue in the cgh was improved by 43 % compared to the rgh, where this value was reached by the reuse of the stored energy, the energy screens and the increase in total yield (tab. 2). the data showed that 39.49 mj were necessary in the cgh to produce one kg of tomatoes. in dutch greenhouses, however, the value of the eue is much lower, where 25 mj kg-1 were calculated (elings et al., 2005). this difference is based on the warmer day and night temperatures in the netherlands, whereby less energy is required to produce tomatoes, but also on this fact that the yield production in dutch greenhouses is twice as high as shown in the present study. effects of different operation modes of greenhouses on fruit quality compared to the rgh, the contents of lycopene, ß-carotene, phenolic compounds, ssc and laa were increased in tomatoes, which were exposed to the climate conditions in the cgh (fig. 5). therefore, it was concluded that the light reduction in the cgh did not negatively influence the contents of ssc in tomatoes or at least not during the light intensive season. this result is consistent with the observations made by gautier et al. (2008) and kläring and krumbein (2013), both of whom found that shaded plants did not result in a decrease in ssc. in this context, gautier et al. (2008) reported that an increasing temperature from 21 °c to 26 °c did not affect the contents of ssc in tomatoes. that is why we excluded a temperature effect on the amount of ssc in tomatoes, which were produced with a small temperature difference occurring between the rgh and the cgh. the same applies to the influence of the rh on the ssc, because the temporally higher levels of rh in the cgh were not sufficient to dilute the ssc in tomatoes compared to those matured in the rgh (fig. 5). however, bertin et al. (2000) reported that a higher rh was responsible for lower ssc in tomatoes, where these tomatoes were matured under summer mediterranean conditions and an associated greater difference in levels of rh than in our own experiments. rather, it seems that the higher levels of co2 concentration in the cgh triggered an increase in photosynthesis followed by an enhanced supply of assimilates, whereby more content of ssc was accumulated in tomatoes. these conclusions are supported by islam et al. (2006), who found increased levels of ssc in different tomato the potential of a confined closed greenhouse 217 217 cultivars, which were grown under doubling of the co2 concentration. finally, this increase in ssc and the stronger peel firmness owing to the cgh was perceived by the consumers (fig. 6). it is assumed that the stronger peel firmness was caused by the higher mean temperature in the cgh, where riga et al. (2008) described the same phenomenon in their study. however, no changes in sourness of tomatoes were noticeable by the consumers, where this result was confirmed using the chemical analyses (fig. 5; fig. 6). the alterations in secondary metabolites were affected positively by the microclimate conditions in the cgh. regarding the effects of light deficiency on secondary plant compounds in tomatoes, there are conflicting results from different studies that give negative and positive effects. toor et al. (2006), for instance, found that the total phenol content in tomatoes varied depending on the seasonal light conditions, whereas a reduction in the mean monthly solar radiation by 10 % did not influenced the levels of phenolic compounds. this light interception is approximately equal to that measured between the cgh and the rgh, which is the reason why a light effect on phenolics was excluded in the present study. in this context, other studies showed that tomatoes accumulated more lycopene, ß-carotene and laa when they were ripened at high radiation intensities, where the solar radiation was increased by up to 97 % compared to the reference light conditions (lee and kader, 2000; gautier et al., 2005; brandt et al., 2006). however, raffo et al. (2006), kläring and krumbein (2013) and riga et al. (2008) did not observed variations in contents of carotenoids and laa in tomatoes at light differences by up to 123 %. these results and the increasing contents of secondary metabolites caused by the cgh suggest that a light reduction by 11 % in the cgh was not sufficient to diminish the secondary plant compounds in tomatoes. rather, it is assumed that the slightly higher mean temperature and the higher levels of co2 concentrations and associated higher rates of photosynthesis in the cgh (tab. 2) were mainly responsible for the accumulation of carotenoids, phenolic compounds and laa. this was concluded, because raffo et al. (2006) showed that the contents of several phenolic compounds in tomatoes were increased when the average temperature was raised by 0.5 °c. however, the laa content remained unaffected at this temperature difference. krumbein et al. (2006) demonstrated that the content of carotenoids increased with elevated mean temperatures ranging between 18°c and 22 °c. it could also be possible that the temporally higher temperatures in the rgh led to an overheating of tomatoes, followed by an inhibition of the synthesis of lycopene as shown by dumas et al. (2003). furthermore, it was found that co2 levels maintained between 550 ppm and 1000 ppm can be used to increase various phenolic compounds in strawberries and grapes (bindi et al., 2001; wang et al., 2003), whereas these higher co2 concentrations did not affect the carotenoid pattern in tomatoes (krumbein et al., 2006). nevertheless, an elevated atmospheric co2 can generally increase the photosynthetic activity as shown in the cgh. in this context, laa, phenolic compounds and carotenoids are synthesized from primary metabolites supplied through photosynthesis in plants (lee and kader, 2000; treutter, 2010). therefore, it might be possible that the higher rates of photosynthesis of plants grown in the cgh resulted in a higher carbohydrate supply, whereby more resources for carbon based secondary plant compounds were provided. this would explain the increased accumulation of secondary metabolites of tomatoes matured in the cgh compared to those ripened in the rgh. conclusion it can be recommended for practitioners that a confined closed greenhouse can be used as horticultural approach to increase fruit quantity and quality. as such, the results suggested that changes in microclimatic conditions occurred in the cgh, whereby the rates of photosynthesis, the lai, the plant height, the total yield and the accumulation of primary metabolites were increased. the same applies to the secondary plant compounds and associated contents of antioxidant activity, which most likely benefit human health. in this context, it needs to be investigated whether an individual climate factor or a general increase in photosynthesis is responsible for the accumulation of carotenoids, phenolic compounds and laa in tomatoes. furthermore, it was found that the cgh has the potential to guarantee the production of tomatoes in a sustainable way, where high amounts of water and energy can be saved. to attain all the mentioned advantages, the growers have to reduce the frequency of the ventilation 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2008: new developments in greenhouse technology can mitigate the water shortage problem of the 21st century. acta hort. 767, 45-52. vanoosten, j.j., wilkins, d., besford, r.t., 1995: acclimation of tomato to different carbon-dioxide concentrations − relationships between biochemistry and gas-exchange during leaf development. new phytol. 130, 357-367. vörösmarty, c.j., green, p., salisbury, j., lammers, r.b., 2000: global water resources: vulnerability from climate change and population growth. science 289, 284-288. waggoner, p.e., zeilitch, i., 1965: transpiration and stomata of leaves. science 150. wang, s.y., bunce, j.a., maas, j.l., 2003: elevated carbon dioxide increases contents of antioxidant compounds in field-grown strawberries. j. agric. food chem. 51, 4315-4320. zelitch, i., 1982: the close relationship between net photosynthesis and crop yield. bioscience 32, 796-802. the potential of a confined closed greenhouse 219 219 address of the authors: dennis dannehl and uwe schmidt, humboldt-universität zu berlin, faculty of life science, albrecht daniel thaer-institute of agricultural and horticultural sciences, division biosystems engineering, albrecht-thaerweg 3, 14195 berlin e-mail: dennis.dannehl@agrar.hu-berlin.de melanie josuttis, humboldt-universität zu berlin, faculty of life science, albrecht daniel thaer-institute of agricultural and horticultural sciences, institute for product quality, teltowkanalstrasse 2, 12247 berlin. christian ulrichs, humboldt-universität zu berlin, faculty of life science, albrecht daniel thaer-institute of agricultural and horticultural sciences, division urban plant ecophysiology, lentzeallee 55, 14195 berlin 190 obituary obituary prof. dr. dieter treutter on may 7, 2016, the academic community specializing in pomology and food quality lost an outstanding colleague. after studying horticultural sciences at the technical university of munich (1976 1982), dieter treutter received his ph.d. (1985) under the supervision of prof. walter feucht. in 1992, he earned his lecturer degree in pomology after submitting a postdoctoral thesis about catechins and proanthocyanidins and their impact for fruit grower and consumers at the technical university of munich. thereafter, he worked for 2 years (1996 1997) as a postdoctoral scholar at the university of salamanca at spain. he then joined the faculty at the technical university of munich (1997 1999) as assistent professor. in 1999, he was appointed associate professor at the technical university at munich. he was mainly responsible for the establishment of the horticultural sciences at the technical university of munich. after his successful and dedicated work as a chair of pomology at the technical university of munich, he served as president of the german society for quality research of plant food (dgq) for eight years (1995 2003). for his scientific impact he has been awarded with three awards and an honorary doctorate (dr. h.c.) für pomology by the corvinus university at budapest (hungary). his professional interests were mainly focused on polyphenols for food quality, plant resistance and human health. his expertise in pomology continues to be highly achnowleged in scientific journals where he served as a section editor for the journal of applied botany and food quality (jabfq). according to the web of science, he published about 175 original papers. he was a dedicated teacher in the area of pomology and food quality and the students loved him for his friendly and open personality. he will be deeply missed in the scientific community of pomology. we honor his scientific achievements in pomology and food quality and will always keep him in our memory. karl h. mühling hartwig schulz (president dgq) (editor jabfq) journal of applied botany and food quality 88, 308 313 (2015), doi:10.5073/jabfq.2015.088.044 1universidad nacional de colombia, facultad de ciencias agrarias, laboratorio de fisiología vegetal 2 universidad nacional de colombia, facultad de ciencias, departamento de biología, laboratorio de fisiología y bioquímica vegetal germination, protein contents and soluble carbohydrates during storage of sugar apple seeds (annona squamosa l.) f.e. martínez maldonado1, d.m. lasprilla1, s. magnitskiy1, l.m. melgarejo2 (received april 17, 2015) summary the aim of this study was to evaluate changes in the germination potential, protein contents and soluble carbohydrates in sugar apple (annona squamosa l.) seeds from castilla-tolima and apulo-cundinamarca, colombia during storage for 30, 60 and 120 days in a cool room, refrigeration and ambient conditions. in each period, the seed moisture contents, germination percentage (gp), mean germination time (mgt), mean germination rate (mgr), soluble protein content, and contents of sucrose, glucose and fructose were evaluated. the highest gp (> 80 %) and mgr (0.15 germinated seeds / day) and the lowest mgt (< 7 days) were reached after 60 and 120 days of storage under ambient conditions. in the three storage conditions, the protein contents declined at 60 days and 120 days in seeds from castilla and apulo, respectively. under the ambient conditions (ac), there was an increase of 72.3 % in the sucrose content, 61 % increase in the fructose content and 70 % decrease in the glucose content. the results show that this environment temperature for 60 and 120 days was best suited for maintaining short-term germination. the high gp and mgt values and low mgr value indicated a possible dormancy release that occurred naturally. introduction the sugar apple belongs to the annona genus and is widely distri buted in the wild worldwide and in colombia (martínez et al., 2013). it grows in the dry areas of the valleys in the provinces of valle, caldas, tolima, cundinamarca and santander (colombia) (guerrero and fischer, 2007), between 450 and 1,500 meters above sea level (hoyos, 1989) and requires no cold periods; meaning it develops and grows well in relatively stable temperature conditions (george and nissen, 1993). the minimum temperature is in a range of 10 to 20 °c and the maximum is 22 to 28 °c (guerrero and fischer, 2007). an important process of seed production is storage; this procedure allows growers to save seeds from one season to another. storage is also important for germplasm conservation, where one of the objectives is to maintain the viability of seeds of a wide range of species for an indefinite period (a long-term period is often considered 10 to 100 years or more) (hong and ellis, 1996). however, during storage, chemical and physiological alterations result in deterioration and loss or reduction in germination (kapoor et al., 2011). this is related to the depletion of reserves in seeds designed to provide energy for the growing embryo during storage (villela and peres, 2004; veiga et al., 1997). it has been demonstrated before that soluble carbohydrates play a key role in desiccation tolerance and seed storage. increases in the levels of sucrose, particularly in the contents of sugars of the oligosaccharide raffinose family, have been correlated with desiccation tolerance and longevity in orthodox seeds (obendorf, 1997; horbowicz and obendorf, 1994) in anonaceae, it was reported that seeds have an orthodox beha vior. lobo et al. (2007) showed that cherimoya (annona cherimola mill.) and soursop (annona muricata l.) seeds exhibit orthodox storage conditions. dornelles et al. (2002) reported that a decrease in moisture contents in sugar apple (annona squamosa l.) seeds pro motes seed vigor, indicating that they are tolerant to desiccation and are orthodox. according to villela and peres (2004), orthodox seeds can be stored at low temperatures and can withstand adverse conditions during the dormancy period when exposed to moisture and temperature conditions that are suitable for resuming their development and complete the germination process. the longevity of seeds varies greatly between species and is conditioned by the prevailing environment during the formation of fruits, seed maturation, processing, and handling of seeds after they are collected (catunda et al., 2003; santana and de cavalho, 2006). in each species and, in some cases, in different varieties, specific conservation needs may be required for maintaining their longevity, so it is important to determine the optimum conditions for seed storage in each particular case. therefore, knowledge of the effect of storage on seed physiology is important for maintaining gene banks as well as processes of propagation and conservation of species (pereira et al., 2008). the present study aimed to determine the effect of short-term storage on germination, protein and soluble carbohydrates in sugar apple seeds (annona squamosa l.) collected from two geographical areas of colombia. materials and methods the plant material was obtained from the accessions t7as180, t7as181, c2as224, and c2as225, collected in apulo (cundinamarca province) and castilla (tolima province), colombia. the seeds were obtained from ripe fruits, soft to the touch, that were disinfected with immersion for 9 minutes in 1 % sodium hypochlorite, washed with 96 % ethanol:distilled water (1:1) three times, and, finally, rinsed three times with distilled water to remove any ethanol residue that might have remained on the seeds (hou et al., 2009). storage conditions the seeds collected in apulo (cundinamarca) were stored with moisture contents between 10 and 12 %, while the moisture contents of the seeds from castilla (tolima) during the storage varied between 12 and 14 %. the seeds were stored for 0, 30, 60, and 120 d in craft paper bags under three conditions: cool room (cr) at 4 °c and rh = 79 %, refrigeration (r) at 10 °c and rh = 79 %, and ambient storage (storage at room temperature) (ac) at 19 °c and rh = 60 %; the latter corresponded to the conditions in laboratory of crop physiology, faculty of agrarian sciences, national university of colombia, bogotá, colombia. the period of storage, storage condition, and locations were arranged in a 3×3×2 factorial arrangement. seven replicates per 4 treatments were employed. for each replicate, 120 seeds were used. in total, 840 seeds per treatment were em ployed. germination changes during storage of annona squamosa l. 309 germination after each storage period, the seeds were removed to assess the germination. the seeds were sown in disinfected peat substrate klasmann® (klasmann-deilmann gmbh, germany) without nutrients at a depth equal to twice the seed length and subsequently placed in a controlled environment cabinet (sgc066, sanyo gallenkamp, leicester, uk) for 30 days at a constant temperature of 35 °c in absence of light because the germination of annona squamosa l. seeds is indifferent to light conditions (ferreira et al., 2002). observations were made every 5 days for 30 days because germinating seeds after this period could be considered dormant (baskin and baskin, 2001). germination was recorded in those seeds that had radicle protrusion. with sampling data, the following parameters were evaluated: germination percentage (gp) = 100, where n = number of ger minated seeds and ns = total number of seeds; mean germination time (mgt) = and mean germination rate (mgr) = where ni = number of germinated seeds in the ith data collection; ti = time (in days) of the ith data collection and k = time (in days) duration of the germination test. protein and soluble carbohydrate contents the protein determination was made based on the methodologies described by bradford (1976) and zor and selinger (1996), and the contents of carbohydrates sucrose, glucose, fructose were analyzed with high-performance liquid chromatography (chinnici et al., 2005; solarte et al., 2014). 1.00 g of seeds per replicate were employed. due to similarities in the germination response and protein content of plant material from two locations, the soluble carbohydrates were determined only in seeds from castilla (tolima) after two periods of storage with responses in terms of germination (0 to 120 days). to determine the content of sucrose, glucose and fructose, extraction was performed from 100 mg of macerated seeds using 1 ml of 80 % ethanol at 90 °c, which were then centrifuged 10 minutes at 6000 rpm at 4 °c and the supernatant was removed. the pellet was resuspended two more times with 1 ml of 80 % ethanol at 90 °c following the same process to obtain 3 ml of supernatant. the supernatant was dried in a speed vac and resuspended in 1 ml of distilled water (hou et al., 2009). the concentrated extract was centrifuged again at 6000 rpm for 15 minutes at 4 °c and filtered with 0.22 μm before the hplc analysis. the contents of individual sugars were measured by liquid chromatography (waters-milford, ma usa) using a 300 × 7.8 mm rezex rcm-monosaccharide column ca+ (8 %), 8 μm particle sizes and a refractive index detector 2414 (waters). the conditions of analysis were as follows: 86 °c oven temperature, 44 °c detector temperature, and a 1 ml · min-1 flow of deionized water was employed as the mobile phase. sugars were distinguished by comparing retention time and calibration curves of external standards by means of the software empower 2 2005-2008. we assessed the assumptions of normality and homogeneity of variances, and the data that showed no normality in the residuals were transformed with the arcosen function √ (pg/100) usually used in germination studies (wagner et al., 2006). anova was performed and the differences between the treatments were determined with a tukey test (p < 0.05). we used the statistical package sas (statistical analysis system), version 9.1. results and discussion seed storage of annona squamosa l. at ambient temperature and humidity (ac) for 60 and 120 d generated the best responses in the variables of seed germination from two locations (fig. 1). in both periods, the gp (fig. 1a) had similar values for both castilla-tolima and apulo-cundinamarca (80 85 % for 60 and 120 d). the maximum mgr (fig. 1b) obtained with ac storage for 60 days was for castilla-tolima (0.17 germinated seeds / d) and for 120 d for apulocundinamarca (0.18 germinated seeds / d), which was an mgr that was more than 3-fold higher than that found in the seeds without storage (0.05 and 0.06 germinated seeds / d, respectively, for castilla and apulo). the mgt values (fig. 1c) showed variation (p < 0.0001) over time with a tendency to decrease as storage progressed, resulting in lower values of mgt storage in ac for 60 and 120 days for the seeds of castilla-tolima (5.9 and 6.3 d, respectively) and apulo-cundinamarca (6.8 and 5.7 d, respectively). but, although these values did not differ from those found in cr and r at 60 days of storage, they were significantly lower than those found for seeds that were not stored (19.4 and 17 d for castilla-tolima and apulo-cundinamarca, respectively) (fig. 1c). in a. squamosa l. seeds, other authors have reported the same behavior in seed storage conditions. dornelles et al. (2002) verified a gradual increase in the mgr and pg for up to three months of storage. magalhães et al. (2009) found germination percentages of 88 % when sugar apple seeds were stored at room temperature (maximum: 25.3 °c, minimum 16.1 °c) in paper for 6 months. however, sousa (2008) reported no influence from storage for 0, 30, and 60 d on the germination of sugar apple seeds when they received no treatment to break the dormancy. in a similar study by magalhães et al. (2014), the speed and percentage of germination and seedling dry weight in response to 5 storage periods (0, 3, 6, 9 and 12 months), two storage conditions (environment and cool conditions at 6 8 °c), and two packaging types (paper bag and plastic bag) were assessed. the authors found that, with a moisture content of 8.49 %, the seed vigor was maintained during the storage and the seeds stored under ambient conditions in paper bags remained viable for six months. these results indicated that the dormancy induced by embryo immaturity that is typical for annonaceae was possibly overcome with storage. in particular, the period (60 and 120 days) and the conditions (temperature and humidity) of storage directly influenced the natural overcoming of dormancy seen in a. squamosa l. seeds, which was also indicated by dornelles et al. (2002). it has been noted that annonaceae seeds being dispersed have a small embryo, considered underdeveloped and immature (noorman et al., 1992). in the case of annona squamosa l., the embryo is rudimentary and undeveloped but differentiated with a slow growth rate, which later causes the seed to germinate after 1 to 3 months (hayat, 1963). in cherimoya and soursop seeds, it was observed that morphophysiological dormancy could also be easily overcome with storage (lobo et al., 2007). in a. crassiflora seeds, silva et al. (2007) observed that the seeds had underdeveloped embryos at the time of fruit maturity and required a period subsequent to the dispersion to terminate their development. likewise, for pinto and genu (1984), in soursop seeds, the endogenous dormancy could be completely overcome with storage. hopkinson and english (2005) found that dormancy persisted longer under cool storage than under ambient storage conditions. changes in protein content during seed storage the protein contents in seeds without storage (0 d) from both locations had similar values (9.61 and 9.92 mg / g for castilla-tolima and apulo-cundinamarca, respectively). in seeds of castilla-tolima, the protein content declined at 60 and 120 days in all of the storage conditions, presenting the lowest values at 60 days (5.21 mg / g (ac), 5.62 mg / g (cr) and 5.79 mg / g (r)). in the seeds of apulo-cundinamarca, the content significantly decreased at day 120 in all of the storage conditions (5.36 mg / g (ac), 5.90 mg / g (cr) and 7.52 mg / g (r)), as compared to the seeds without storage (9.92 mg / g) (fig. 2). the effect of time and storage conditions on the germination and 310 f.e. martínez maldonado, d.m. lasprilla, s. magnitskiy, l.m. melgarejo conservation of the physiological quality of seeds is associated with the metabolism of sugars and proteins. the protein content decreased with storage time, presenting the lowest values at 120 d, for the apulo-cundinamarca seeds and, for seeds from castilla-tolima, the lowest values between 60 and 120 days (fig. 2). this reduction in the protein content may be due to several factors that are within the increased rate of protein loss in cotyledons and embryonic axes, the damage to the protein synthesizing system, and the synthesis or activation of large quantities of proteolytic enzymes during the deterioration of seeds (bewley and black, 1994). it has been shown that the seeds of vigna radiata and cicer arietinum, under natural or artificial aging, increased protease activity (agarwal and kharlukhi, 1987); likewise, in shorea robusta seeds, increases in protease activity were recorded in this embryonic axis and cotyledons (krishna chaitanya et al., 2000). the massive loss of cell protein was also observed during storage in seeds of rice (prabhakar and mukherjee, 1980), shorea robusta (nautiyal and purohit, 1985), pisum sativum, phaseolus aureus, and citrus reticulata (samshery and banerjee, 1979). changes in sugar content during seed storage in the seeds without storage (fig. 3), a higher sucrose content (7.82 mg / g) than those of glucose and fructose was observed, which had similar levels (4.11 and 4.37 mg / g, respectively). with the cr storage for 120 days, no significant variation was detected in the levels of sucrose, although the content of glucose and fructose showed a slight decrease (3.13 and 2.69 mg / g). when the seeds were stored in r conditions, significant changes in the content of sugars were proven (p < 0,0001), a 22 % increase in sucrose levels (9.62 mg / g) with respect to the seeds without storage and a decrease in the content of the reducing sugars glucose (1.24 mg / g) and fructose (0.94 mg / g) fig. 1: germination percentage gp (a), mean germination rate mgr (b), and mean germination time mgt (c) of a. squamosa l. seeds from castilla-tolima (black lines) and apulo-cundinamarca (gray lines) under ambient conditions ac (dashed lines), cool room cr (continuous points) and refrigeration r (solid lines). germination incubation was at 35 °c and 60 % rh in wet peat. vertical bars indicate standard error (±), n = 4. a. b. c. fig. 2: soluble protein content of a. squamosa l. seeds from castilla-tolima (black lines) and apulo-cundinamarca (gray lines) for the four periods of storage under ambient conditions, ac (dashed lines), cool room cr (continuous points) and refrigeration r (solid lines). germination incubation was at 35 °c and 60 % rh in wet peat. vertical bars indicate standard error (±), n = 4. germination changes during storage of annona squamosa l. 311 were shown. the seeds stored under ambient temperature conditions (ac) showed an increase of 72.3 % in the content of sucrose (13.49 mg / g), as compared to the sugar content of the seeds without storage. the glucose content at this time in ac (1.24 mg / g) decreased by 70 % and the fructose levels (7 mg / g) increased by 61 %, as compared to the seeds without storage. the storage conditions for 120 d highly affected the sugar content in the annona squamosa l. seeds. at ambient temperature and relative humidity (ac), the sucrose levels were much higher than those observed in the seeds stored in cool room (cr) and refrigeration (r). this could be possible due to the fact that the activities of enzymes controlling the level of sucrose, such as sucrose phosphate synthase (sucrose synthesizing enzyme from fructose and glucose) and invertase (sucrose-degrading enzyme producing fructose and glucose) were decreased at low temperatures and moisture (iyare and ekwukana, 1992), which would explain the differences in the levels of sucrose. in cr at 4 °c, there was no change in the levels of reducing sugars; however, in refrigeration at 10 °c and storage at ambient condition, reductions in the glucose levels accompanied by increases in the level of sucrose were much higher than those in the seeds stored at cr. this might be because the reducing sugars were used as a substrate for the formation of sucrose (hubbard et al., 1991) for respiration and metabolism for other processes that consume energy (koch, 2004); however, when the seeds were stored at low temperatures, the levels of reducing sugars were constant (medlicott et al., 1986), as was observed in the seeds stored in cool room (cr). in this study, the glucose and fructose contents in the seeds decreased during the refrigerated storage (r), but there was a considerable increase in the levels of sucrose (23 %), as compared to the non-stored seeds. in contrast to the ac storage, the glucose levels decreased and there was a large increase in sucrose, suggesting that, in this condition, the metabolism of both the respiration and action of sucrose phosphate synthetase functioned normally (turhan and ergin, 2012). these variations in the content of sugars, such as sucrose, might also be associated with increases in solubilization reserves, as reported during the physiological maturity in other seeds with an orthodox behavior (nkang, 2002). the influence of storage temperature on the metabolism of sugars in seeds has also been recorded for other species. in caesalpinia echinata, garcia et al. (2004) found that glucose and fructose values were constant in seeds stored in paper bags for 18 months in a cool room (6 ± 1 °c and rh = 85 % ± 5 %), with a considerably decreasing temperature (25 ± 10 °c and rh = 80 % ± 15 %). at low temperatures, the sucrose content remained stable, which was also recorded for corn seeds (bernal-lugo and leopold, 1998). the results of this study contribute to the understanding of germination in annona squamosa l. seeds through the carbohydrate metabolism in seeds during storage. the best responses in the pg germination variables mgr and mgt for both locations were seen under the ambient conditions, ac, at 60 and 120 days; a condition in which the levels of sucrose and fructose increased (at 120 days). in contrast, the lowest pg and mgr and the highest mgt were detected in the non-stored seeds (0 days) and seeds stored in cr and r, where the possible limitations for metabolism by the responsible enzymes were associated with lower sucrose contents (turhan and ergin, 2012). the differences in the soluble sugars and germination capacity of a. squamosa l. seeds suggest that seed longevity is related to seed respiration rates, which may be different depending on storage conditions as indicated by garcia et al. (2004) for caesalpinia echinata lam seeds. the data indicate that the storage in cool room (4 °c) and in refrigeration (10 °c) reduced the seed quality during storage, expressed as a decrease in germination rate and, subsequently, a resulting loss of germinability; this could be due to changes in the content of soluble carbohydrates. at 4 ºc, the sucrose levels decreased or remained stable with respect to the seeds without storage (p < 0.05), resulting in limited availability of respiratory substrates for germination. another possibility is that, with the limited contents of disaccharides, the protective effect of sugars on the structural integrity of membranes and the ability of seed cells to maintain a vitrified state in the cytosol were decreased (bernal-lugo and leopold, 1998). this indicates that adequate levels of sucrose are required to maintain the viability of seeds and ensure germination during storage of a. squamosa l. seeds. sucrose and other forms of non-reducing sugars contribute to the structural stability of organelles, membranes, enzymes, and other macromolecules (obendorf, 1997; peterbauer and richter, 2001). in particular, sucrose is exceptionally effective at protecting cell membranes in systems exposed to desiccation; it is one of the best sugars for vitrification process in plant cells (bernal lugo and leopold, 1998) because it protects the structure and function of phospholipids during cell drying (leprince and buitink, 2010). conclusion the period and conditions (temperature and rh) of storage directly influenced overcoming the dormancy that was naturally present in the a. squamosa l seeds and, therefore, the physiological quality of the seeds. the best germination responses of the variables pg, vmg and tmg were present in the ambient storage conditions at 60 and 120 d, when the sucrose and fructose levels increased up to 120 d. in contrast, the pg and vmg were lower and the tmg was higher in the seeds without storage (0 d) and stored in cf and r, where the lower levels of sucrose were detected due to possible limitations for the metabolism of corresponding enzymes. seed longevity could be related to seed respiration rates that might be different at different storage conditions. adequate levels of sucrose were required for maintaining the seed viability and germination of a. squamosa during storage. acknowledgments the authors are grateful for the financial support for f.e. martínezmaldonado’s msc thesis and for the research provided by the ministerio de agricultura y desarrollo rural (madr) de colombia and universidad nacional de colombia, bogotá. references agarwal, p., kharlukhi, l., 1987: enzyme activities in seeds during storage. exp. biol. 25, 719-722. fig. 3: changes in the contents (mg / g dry weight) of sucrose, glucose and fructose in seeds of a. squamosa l. from castilla-tolima. without storage (0 days) and stored for 120 days under ambient conditions, ac (19 °c and rh = 60 %), cool room cr (4 °c and rh = 79 %) and refrigeration r (10 °c and rh = 79 %). n = 3. the anova showed a significant effect of storage on the glucose (p ≤ 0.05), fructose and sucrose (p ≤ 0.01) concentrations. means with different letters indicate significant differences according to the tukey test (p ≤ 0.05). 312 f.e. martínez maldonado, d.m. lasprilla, s. magnitskiy, l.m. melgarejo baskin, c., baskin, j., 2001: seeds. ecology, biogeography, and evolution of dormancy and germination. san diego, california. academic press. bernal-lugo, i., leopold, a., 1998: the dynamics of seed mortality. eynsham. 49, 1455-1461. bewley, j., black, m., 1994: seeds – physiology of development and germination. new york. plenum press. bradford, m., 1976: a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. anal. biochem. 72, 248-254. catunda, p., vieira, h., da silva, r., posse, s., 2003: influência do teor de água, da embalagem e das condições de armazenamento na qualidade de sementes de maracujá amárelo. rev. bras. sementes. 25, 65-71. chinnici, f., sinabelli, u., riponi, c., amati, c., 2005: optimization of the determination of organic acids and sugars in fruit juices by ion exclusion liquid chromatography. j. food comp. anal. 18, 121-130. dornelles, a., lima, a., campos, v., 2002: avaliação do potencial de armazenamento de sementes de annona crassiflora mart, annona muricata l.e annona squamosa l. anais do 17° congresso brasileiro de fruticultura, belém, brasil. ferreira, g., erig, p., moro, e., 2002: uso de ácido giberélico em sementes de fruta-do-conde (annona squamosa l.) visando á produção de mudas em diferentes embalagens. rev. bras. frut. 24, 178-182. garcia, i.s., souza, a., barbedo, c.j., dietrich, s., figueiredo ribeiro, r., 2004: changes in soluble carbohydrates during storage of caesalpinia echinata lam. seeds, an endangered leguminous tree from the brazilian atlantic forest. braz. j. biol. 66, 739-745. george, a.p., nissen, r., 1993: annonaceous. in: macrae, r., robison, r., sadla, m.i. 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dormência em sementes de pinha. revista caatinga. 21, 118-121. turhan, e., ergin, s., 2012: soluble sugars and sucrose-metabolizing enzymes related to cold acclimation of sweet cherry cultivars grafted on different rootstocks. sci. world journal 2012, 979-682. veiga, a., quimarães, r., rosa, s., von pinho, i.e., silva, i., 1997: armazenabilidade de sementes de cafeeiro colhidas em diferentes estádios de maturação e submetidas a diferentes métodos de secagem. revista brasileira de sementes. 29, 83-91. villela, f., peres, w., 2004: secagem e beneficiamento de sementes. in: ferreira, a., borguetti, r. (ed.), germinação: do básico ao aplicado, 265281. porto alegre, artmed. wagner, j., alexandre, r.s., da silva, j.r., duarte, l., da costa, j.o., bruckner, c.h., 2006: influência do substrato na germinação e desenvolvimento inicial de plantas de maracujazeiro amarelo (passiflora edulis sims f. flavicarpa deg). ciênc. agrotec. 30, 643-647. germination changes during storage of annona squamosa l. 313 zor, t., selinger, z., 1996: linearization of the bradford protein assay increases its sensitivity. theoretical and experimental studies. anal. bioch. 236, 302-308. address of the authors: f.e. martínez, universidad nacional de colombia, carrera 45 no. 26-85, bogotá, colombia; present address: corporación colombiana de investigación corpoica, km 14 vía mosquera, colombia e-mail: femartinezma@unal.edu.co d.m. lasprilla, universidad nacional de colombia, carrera 45 no. 26-85, bogotá, colombia e-mail: dmirandal@unal.edu.co s. magnitskiy, universidad nacional de colombia, carrera 45 no. 26-85, bogotá, colombia e-mail: svmagnitskiy@unal.edu.co l.m. melgarejo, universidad nacional de colombia, carrera 45 no. 26-85, bogotá, colombia e-mail: lmmelgarejom@unal.edu.co © the author(s) 2015. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 88, 228 233 (2015), doi:10.5073/jabfq.2015.088.033 1 department of agricultural sciences, alma mater studiorum university of bologna, bologna, italy 2 molecular phytopathology and mycotoxin research section, georg-august-university goettingen, goettingen, germany fusarium proliferatum and fumonisin b1 co-occur with fusarium species causing fusarium head blight in durum wheat in italy barbara amato1, katharina pfohl2, stefano tonti1, paola nipoti1, raana dastjerdi2, annamaria pisi1, petr karlovsky2, antonio prodi1 (received february 11, 2015) summary fusarium head blight caused by phytopathogenic fusarium spp. with fusarium graminearum as main causal agent is a major disease of durum wheat (triticum durum desf.). mycotoxins in wheat are dominated by trichothecenes b. fumonisins have only occasionally been reported from wheat; their occurrence was attributed to fusarium proliferatum and fusarium verticillioides. we investigated kernels of durum wheat grown in italy in 2008 2010 for colonization with fusarium spp. and for the content of fusarium mycotoxins. fungal biomass was determined using species-specific qpcr and mycotoxins were quantified by hplc-ms/ms. fusarium graminearum and fusarium culmorum were dominating fusarium species, followed by fusarium poae, fusarium tricinctum and fusarium proliferatum. no fusarium verticillioides dna was found. toxicologically relevant levels of deoxynivalenol and nivalenol but no trichothecenes a were detected. enniatins, fumonisin b1 and beauvericin were present in grain in all three years. based on these results and on the evaluation of previous published reports, we hypothesize that low levels of fumonisins commonly occur in wheat grains produced in warm climate; they may remain undetected as long as mycotoxin monitoring programs for wheat do not include fumonisins. the only relevant source of fumonisins in wheat grain appears to be fusarium proliferatum. introduction fusarium head blight (fhb) is a worldwide-occurring cereal disease complex caused by several species of the fungal genus fusarium that often cause indistinguishable symptoms. the disease leads to the reduction in grain yield, which can reach up to 70 % (parry et al., 1995). fhb also affects quality parameters such as the size of kernels, protein content, baking quality of flour and mycotoxin content, making them unsuitable and unsafe for human and animal consumption. the prevailing species in fhb in italy are f. graminearum schwabe, f. culmorum (w. g. smith) sacc. and recently f. poae (peck) wollenweb. (shah et al., 2005; pancaldi et al., 2010). infantino et al. (2012) reported that f. poae was the most prevalent species found in organic bread and durum wheat in italy. f. graminearum schwabe [gibberella zeae (schwein) petch] is the predominant fhb pathogen worldwide (xu and nicholson, 2009), producing a range of mycotoxins including type b trichothecenes nivalenol (niv) and deoxynivalenol (don). these mycotoxins disrupt cell functions by inhibiting protein synthesis via binding to the ribosome. for this reason, maximum levels of don as major trichothecene contaminating fhb-affected kernels are regulated in food and feed by european directives ec n. 1881/2006, 126/2007. f. graminearum populations comprise chemotypes that synthesizes either don or niv and their acetylated derivatives. f. poae is a pathogen of increasingly importance as a cause of fhb in countries such as argentina, canada, finland, belgium, germany, switzerland, hungary, slovakia, italy, ireland and the united kingdom (audenaert et al., 2009; vogelgsang et al., 2008; stenglein, 2009; pancaldi et al., 2010). the prevalence of f. poae in wheat reported in the last years is surprising because the species was believed to be less aggressive than other fhb pathogens (audenaert et al., 2009; xu and nicholson, 2009). symptoms induced by f. poae are different from those caused by f. graminearum, characterized by small necrotic lesions on the wheat glume (vogelgsang et al., 2008). pancaldi and colleagues (2010) emphasized the importance of f. poae in the disease, showing that the isolation frequency of the species in north italy increased from 9 % to 23 % from 1998 to 2007. moreover, in the years where the isolation frequency of f. poae was high, f. graminearum was less often isolated; these observations are consistent with the research by parry et al. (1995). fumonisins are mycotoxins occurring in maize; fumonisins levels in corn are particularly high in italy due to infection with thermo philic f. verticillioides. in wheat fumonisins have only occasionally been reported. in wheat plants artificially inoculated with f. pro liferatum kernel black point symptoms were observed and fumonisin b1 was detected (desjardins, 2007), indicating the potential of the fungus as natural contaminant in wheat fields. shephard et al. (2005) challenged the reports on fumonisins in wheat due to the occurrence of false positive signals when immunoaffinity cleanup, derivatization and fluorescence detection was used in fumonisin detection. more recent work, however, identified fumonisins in wheat unequivocally (e.g. palacios et al., 2011; busman et al., 2012). the aim of this work was to investigate the occurrence of fusarium species and their mycotoxins in durum wheat grain in italy. materials and methods experimental design a three years field trial was established in the experimental station of the university of bologna (cadriano, 44°33’4.15”n; 11°24’39.02”e), using the durum wheat cultivar simeto, which is susceptible to fhb. the plot was used for artificial inoculation with f. graminearum and f. culmorum for several years before the sampling. the field was divided in 4 plots (each plot was 3 × 3 m2) for 2008 till 2010, which served as replicates. the first evaluation of fhb symptoms − typical bleaching in the spike − was made three weeks after anthesis on a representative sample of 100 spikes/plot. disease incidence was determined by counting the number of infected spikes divided by the total number (n=100) of evaluated spikes. number of infected spikes incidence (di) = × 100 total number of evaluated spikes disease severity of every spike was determined by using an evaluation method modified by purahong et al. (2011) that categorizes the bleached spikelet area per spike in percent in eight fusarium proliferatum and fumonisin b1 in durum wheat 229 severity classes (0 2 5 -10 25 50 75 90 %). disease severity per plot (ds) was seen as the sum of infected spikes times the disease class divided by the total number of evaluated spikes in percent (including non-diseased spikes). σ number of infected spikes × disease class severity (ds) = × 100 total number of evaluated spikes kernels of every plot were harvested at maturity with a combined harvester. mycological analysis and species identification mycological analyses were performed on 25 kernels per plot in 2008 and 2009 and 50 kernels per plot in 2010. petri dishes, containing pda (potato dextrose agar, difco, usa) were set up with 10 kernels each, previously washed in water for 5 minutes, sterilized in 2 % sodium hypochlorite for 5 minutes and rinsed in sterile water. agar media with kernels were incubated at 25°c for five days in dark. all fusarium-like colonies were analyzed for their microand macroscopical features by optical microscopy (labor lux 12, leitz wetzlar, germany). species identification was performed using the synoptic keys by toussoun and nelson (1976) and leslie and summerell (2006). to confirm the belonging of a fungus to the species morphologically identified, dna extraction from mycelium was performed using a ctab (exadecyl-trimethyl-ammonium bromide) method (prodi et al., 2011). pcr specific for f. graminearum and f. poae was carried out using primer pairs fg16 f/r (nicholson et al., 1998) and fp82 f/r (parry and nicholson, 1996), respectively. amplification was done in a t3 thermocycler (biometra, göttingen, germany) under conditions described in the literature cited above. dna extraction from wheat flour and real-time pcr analysis the kernels from each plot (2 to 5 kg) were well mixed, 500 g aliquots were removed and ground. dna extraction from 1 g of wheat flour was carried out following the upscaled protocol by brandfass and karlovsky (2008). the dna was dissolved in 200 μl of buffer te (10 mm tris hcl, 1 mm edta, adjusted to ph 8.0) and diluted 50 times, 1 μl of the dilutions was used as template for real-time pcr. real-time pcr based on sybr green i chemistry was used to evaluate the level of kernel infection with fusarium spp. pcr specific for f. culmorum, f. graminearum, f. proliferatum, f. poae, f. verticillioides and f. tricinctum (tab. 1). serial dilutions of pure fungal dna as standards were used, whereas the lowest standards were used as limits of quantification, which was 28 ng/g for all fusarium species. amplification was done under conditions based on the protocols described by the authors of the primers (tab. 3) optimized for thermocycler cfx 384 (biorad, herkules, cva, usa) with 384-well plates. dna standards were quantified by comparative densitometry using known amounts of lambda phage dna (biorad multi-analyst, hercules ca, usa). samples containing dna below loq but with specific melting curve were allocated a value of ½ loq. samples with unspecific melting curves were allocated a value of zero. mycotoxin analysis wheat meal was processed, extracted and defatted as described by adejumo et al. (2007). hplc-ms/ms for the content of don and niv was carried out as described below. non-defatted acetonitrile extracts were kept at -20°c as counter samples. pcr analysis of dna extracted from the flour carried out four to six years later indicated that kernels could also be contaminated with fumonisins and depsipeptides. therefore, the counter-samples were re-analyzed for the presence of fumonisin b1, enniatins and beauvericin; because stability of these toxins in acetonitrile extracts over extended periods of time is unknown, the purpose of the analysis was to obtain a lower boundary for their concentration in kernels. samples for the analysis of beauvericin and enniatins were processed by separating acetonitrile-water supernatants from flour by centrifugation, followed by drying supernatants in vacuum and redissolving the residues in methanol/water (1:1) without defatting. samples for the analysis of fumonisin b1 were dried in vacuum, re-extracted with methanol/water (3:1) and cleaned up by spe on strong anion exchanger columns (sax, agilent, waldbronn, germany) according to shephard et al. (1990). standards for the quantification of don and niv were prepared by spiking uncontaminated wheat flour with pure mycotoxins and processing the spiked samples in the same way as unknown samples (matrix-matched standards). chromatographic separation on a reverse-phase column, ionization and quantification in multiple reaction monitoring mode of a triple quadrupole detector (trichothecenes) and an ion trap detector (the other mycotoxins) was carried out on a system described by ratzinger et al. (2009), using published mass transitions (rasmussen et al., 2012; adejumo et al., 2007). the limit of quantification (loq) was 10 μg/kg for don, 30 μg/kg for niv, 10 μg/kg for ht-2 and t-2, 5 μg/kg for das, 50 μg/kg for neo, 3 μg/kg for fumonisin b1, and 14 μg/kg for beauvericin and enniatins. limits of detection (lod) values were 3 μg/kg for don, 10 μg/kg for niv, 5 μg/kg for ht-2 and t-2, 3 μg/kg for das, 20 μg/ kg for neo, 1 μg/kg for fumonisin b1, and 5 μg/kg for beauvericin and enniatins. results fungal colonization and mycotoxin content of kernels in the years 2008 and 2010 the infection pressure was rather high while in 2009 only a limited incidence of fhb was observed (fig. 1). disease severity was highest in 2010 and very low in 2009; in spite of the relatively high incidene of fhb in 2008, on the average only 5 % of the spikes were bleached (fig. 1). colonization of wheat kernels with f. graminearum as the major causal agent of fhb was investigated by placing 100 to 200 surface sterilized kernels from each year’s harvest on agar media and counting kernels from which f. graminearum isolates were obtained. the isolation frequencies (tab. 2) matched the ranking of disease severity among years. the biomass of fusarium spp. in wheat kernels was estimated by species-specific real-time pcr analysis of dna extracted from representative samples of kernels. the analysis was carried out for the major pathogen of fhb f. graminearum and for the following species: f. culmorum, f. poae, f. proliferatum, f. verticillioides and f. tricinctum (fig. 2). f. graminearum and f. culmorum were found in high amounts in all three years (except for f. culmorum in 2009). f. poae, f. proliferatum and f. tricinctum occurred in 10 to 100 times lower amounts; no f. verticillioides was found in any sample. tab. 1: primers used for species-specific quantification of fusarium dna fungal species primer name reference f. culmorum opt18f /opt18 r schilling et al., 1996 f. graminearum fg16 f/r nicholson et al., 1998 f. poae fp82 f/r parry and nicholson, 1996 f. tricinctum tri1/tri2 kulik, 2008 f. proliferatum fp3-f/fp4-r jurado et al., 2006 f. verticillioides ver 1/2 mule et al., 2004 90 0 230 b. amato, k. pfohl, s. tonti, p. nipoti, r. dastjerdi, a. pisi, p. karlovsky, a. prodi mycotoxins don and niv were found in all three years in amounts raising toxicological concerns (fig. 3); it has to be pointed out, however, that a strong inoculum of trichothecene b producers is likely to be established at the location due to long-year inoculation experiments (see also discussion). because pcr analysis revealed the presence of fumonisin and beauvericin producer f. proliferatum and enniatin producer f. tricinctum, the counter samples kept at -20°c were used to determine lower boundaries for concentrations of these toxins in kernels. fumonisin b1 was found in all samples; four daughter ions generated from the molecular ion possessed the same m/z ratios and occurred in similar relative abundances as daughter ions of authentic fumonisin b1 standard (fig. 4). together with the retention time and m/z value of the molecular ion, these results proved convincingly that fumonisin b1 was present in durum wheat kernels. the presence of beauvericin and enniatins a1, b and b1 was supported by their retention times, the molecular ions and three daughter ions for each analyte. discussion the original goal of our investigation of fhb of durum wheat in italy was to obtain a deeper insight into the role of f. poae, which attracts increasing attention as a component of fhb complex and a source of highly toxic trichothecenes type a in general (stenglein, 2009) and in italy in particular (pancaldi et al., 2010; infantino et al., 2012). because the area sampled for our analysis was used for inoculation experiments with f. graminearum and f. culmorum in the last years, we expected a strong infection pressure of these pathogens due to inoculum originating from crop residues. real-time pcr showed that f. graminearum was the dominant fusarium species in wheat kernels (fig. 2). the presence of f. culmorum itself was not surprising. the dna levels appeared rather high, concerning the climate in emilia romagna and the displacement of f. culmorum by f. graminearum in the last decade (yli-mattila, 2010). we speculate that high levels of f. culmorum dna can be accounted for by the inoculum built after years of inoculation experiments. colonization of kernels with f. poae was negligible: we found f. poae in very few kernels by agar plating (data not shown) and the content of f. poae dna in kernels was much lower than the content of f. graminearum dna (fig. 2). in line with these results, we have not found any of trichothecenes a typically produced by f. poae (fig. 3). the presence of f. poae in durum wheat might fluctuate, fig. 1: incidence and severity of fusarium head blight on durum wheat cultivar simeto grown in a field near bologna, italy tab. 2: isolation frequency of f. graminearum from naturally infected wheat kernels 2008 2009 2010 f. graminearum 18 2 51 (n=100) (n=100) (n=200) fig. 2: fusarium dna in naturally infected durum wheat kernels fig. 4: fumonisin b1 in naturally infected durum wheat kernels identity of fumonisin b1 was confirmed by comparing authentic standard (blue) with sample extract (red) regarding retention time, m/z of the mother ion and fragmentation spectra fig. 3: deoxynivalenol and nivalenol in naturally infected durum wheat kernels fusarium proliferatum and fumonisin b1 in durum wheat 231 as shown by covarelli et al. (2015) who reported that over 20 % of fusarium isolates from durum wheat ears belonged to f. poae in 2009 but rarely any f. poae was found in the following year. f. tricinctum dna at moderate levels was found in wheat kernels in two years. although the species is rarely monitored in fhb, according to published reports it dominated fusarium microflora of wheat in syria (alkadri et al., 2013) and was one of the dominant species in barley in (nielsen et al., 2014). qpcr analysis revealed the presence of dna of f. proliferatum in all years. in year 2009, colonization of wheat kernels with f. proliferatum was comparable to the colonization with f. culmorum (fig. 2). f. proliferatum produces fumonisins, which are proven carcinogens in rodents and suspected carcinogens in humans. contamination of wheat with fumonisins is therefore highly relevant for food safety. fumonisins regularly occur in maize grown in warm climate; major fumonisin producers in maize are f. verticillioides and f. proliferatum (fandohan et al., 2003) with f. verticillioides often reported to dominate (reyes-velazquez et al., 2011). while trichothecene content in wheat has been documented in hundreds of publications, fumonisins were only occasionally detected in wheat (desjardins et al., 2007; palacios et al., 2011; busman et al., 2012). founding dna of f. proliferatum in the total dna extracted from wheat kernels motivated us to extend the analysis of mycotoxin content to fumonisins and beauvericin, which are both produced by f. proliferatum. we also analyzed the content of enniatins, which are produced by many fusarium spp. including f. tricinctum which we detected in wheat kernels in two years (fig. 2). fumonisins were detectable in wheat kernels in all three years, though the biomass of f. proliferatum was hundred times lower than the biomass of f. graminearum. in analogy to maize, the origin of fumonisins in wheat is generally attributed to f. proliferatum and f. verticillioides; the results reported by infantino et al. (2012) indicated that f. verticillioides could be the major source of fumonisins in wheat in italy. our durum wheat samples were consistently contaminated with fumonisins but we have not found any dna of f. verticillioides. f. proliferatum, which was found in the kernels consistently in all three years, appeared to be the only source of fumonisin b1. f. verticillioides is ubiquitous in italian maize fields; inoculum in form of airborn conidia likely reached wheat fields, too. we are confident that our qpcr analysis reliably distinguished between f. proliferatum and f. verticillioides. the primers targeted intergenic spacers of rrna genes, which were shown to be taxonomically informative for the differentiation between the two species (visentin et al., 2009), and they have been extensively tested before (nutz et al., 2011). even if f. verticillioides can infect wheat seeds, as reported by infantino et al. (2012), fumonisins in wheat do not seem to originate from f. verticillioides because desjardins et al. (2007) and busman et al. (2012) reported that f. verticillioides artificially inoculated into wheat did not produce any fumonisins, even thought the isolates produced large amounts of fumonisins in vitro and in inoculated maize plants. the don and niv levels in kernels reached toxicologically relevant levels in all three years. in 2008 and 2010 the amount of don exceeded by a large margin the legal limit of 1,750 μg/kg for unprocessed durum wheat established by european regulations ec n. 1881/2006 and ec n. 1126/2007. the high levels of don can likely be accounted for by inoculation experiments carried out in the field with f. graminearum in last years, which cause buildup of inoculum on crop residues. which fusarium species was the source of don and niv in our samples? we found a significant positive correlation between the concentration of niv and the amount of dna of f. graminearum in kernels. don concentration and f. graminearum dna were positively correlated, too, but the significance of the correlation was not statistically supported (data not shown). the amount of f. culmorum dna in kernels has not correlated with niv nor don. we therefore assume that the dominant source of trichothecenes in kernels was f. graminearum, which is in line with the view that f. graminearum is the major causal agent of fhb in europe (yli-mattila, 2010). the amount of don in grain exceeded the amount of niv by a factor of 4 to 10, which is in line with the observation that f. graminearum chemotypes producing don and its derivatives dominate over niv producers in wheat in north italy (prodi et al., 2009; prodi et al., 2011). high competitiveness of f. graminearum was reported by xu and nicholson (2009) in co-inoculations in vitro trials in controlled environmental conditions. wheat kernels in our samples were consistently colonized with f. proliferatum though they contained high levels of f. graminearum dna. co-inoculation experiments with f. proliferatum have not been conducted in wheat so far. to elucidate the interaction of f. proliferatum and other minor members of the fusarium complex on wheat kernels with f. graminearum, we intend to investigate individual ears and kernels for the presence of fusarium species. acknowledgements the authors would like to thanks stefano borsari for his help in the preparation of the materials for the field experiments and dr. davide pancaldi for his suggestions and comments as well as heike rollwage for technical assistance. references adejumo, t.o., hettwer, u., karlovsky, p., 2007: survey of maize from south-western nigeria for zearalenone, alphaand beta-zearalenols, tab. 3: lower boundary for the content of fumonisin b1, beauvericin and enniatins the content of fumonisins and depsipeptides was determined in samples that were stored for 4 to 6 years in acetonitrile-water at -20°c. because stability of mycotoxins under these conditions is unknown, we regard the values as lower boundaries for mycotoxin content in freshly harvested kernels. mean of four plots per year ± standard deviation is shown except for enniatin b, for which all four values are listed. limit of detection (loq) was 3 ng/g for fumonisin b1 and 14 ng/g for the other mycotoxins. “n.d.”: signal not detectable; “<loq”; positive signals too low to be quantified. 2008 2009 2010 beauvericin [ng/g] <loq <loq 48 ± 19 enniatin a [ng/g] n.d. n.d. <loq enniatin a1 [ng/g] 62 ± 9 88 ± 14 46 ± 30 enniatin b [ng/g] 3200, 3100, 73, 1900 3000, 3500, 5200, 2400 1030, 5200, 1100, 490 enniatin b1 [ng/g] 390 ± 80 510 ± 140 140 ± 110 fumonisin b1 [ng/g] 6.2 ± 3.4 5.3 ± 2.6 33 ± 18 232 b. amato, k. pfohl, s. tonti, p. nipoti, r. dastjerdi, a. pisi, p. karlovsky, a. prodi fumonisin b1 and enniatins produced by fusarium species. food addit. contam. 24, 993-1000. alkadri, d., nipoti, p., doell, k., karlovsky, p., prodi, a., pisi, a., 2013: study of fungal colonization of wheat kernels in syria with a focus on fusarium species. int. j. mol. sci. 14, 5938-5951. audenaert, k., 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pennsylvania state university press, university park. visentin, i., tamietti, g., valentino, d., portis, e., karlovsky, p., moretti, a., cardinale, f., 2009: the its region as a taxonomic discriminator between fusarium verticillioides and fusarium proliferatum. mycol. res. 113, 1137-1145. vogelgsang, s., sulyok, m., hecker, a., jenny, e., krska, r., schuhmacher, r., forrer, h.-r., 2008: toxigenicity and pathogenicity of fusarium proliferatum and fumonisin b1 in durum wheat 233 fusarium poae and fusarium avenaceum on wheat. eur. j. plant pathol. 122, 265-276. wilson, a., simpson, d., chandler, e., jennings, p., nicholson, p., 2004: development of pcr assays for the detection and differentiation of fusarium sporotrichioides and fusarium langsethiae. fems microbiol. lett. 233, 69-76. xu, x., nicholson, p., 2009: community ecology of fungal pathogens causing wheat head blight. in: annu. rev. phytopathol. annual reviews, palo alto, 83-103. yli-mattila, t., 2010: ecology and evolution of toxigenic fusarium species in cereals in northern europe and asia. j. plant pathol. 92, 7-18. address of the authors: barbara amato, stefano tonti, paola nipoti, annamaria pisi, and antonio prodi, department of agricultural sciences, alma mater studiorum univer-department of agricultural sciences, alma mater studiorum university of bologna, viale fanin 40, 40127 bologna, italy. e-mail: antonio.prodi@unibo.it katharina pfohl, raana dastjerdi, petr karlovsky, molecular phytopathology and mycotoxin research section, georg-august-university göttingen, grisebachstrasse 6, 37077 göttingen, germany. © the author(s) 2015. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 89, 49 55 (2016), doi:10.5073/jabfq.2016.089.006 1postharvest technology research laboratory, south african research chair in postharvest technology, department of horticultural sciences, faculty of agrisciences, stellenbosch university, stellenbosch, south africa 2agricultural research council – infruitec/nietvoorbij, stellenbosch, south africa 3department of horticultural sciences, faculty of agrisciences, stellenbosch university, matieland, south africa classification of ‘granny smith’ apples with different levels of superficial scald severity based on targeted metabolites and discriminant analysis asanda mditshwa1, olaniyi a. fawole1, filicity vries2, kobus van der merwe2, elke crouch3, umezuruike linus opara1* (received june 4, 2015) * corresponding author summary to study the metabolic changes in ‘granny smith’ apples with different severities of superficial scald, fruit were stored in normal refrigerated air (0 °c, 95 % rh) for 12 weeks followed by 7 d shelf-life under room conditions (20 °c, 65 % rh). fruit were graded to five groups based on scald severity and analysed for ethylene, α-farnesene and 6-methyl-5-hepten-2-one (mho) levels. reactive oxygen species (ros) were measured by confocal laserscanning microscopy on apple peel treated with fluorescent probe 2’,7’-dichlorodihydrofluorescein diacetate. ethylene production rate, α-farnesene and mho contents and ros intensity increased with increasing scald severity but declined in severely scalded fruit. malondialdehyde (mda) concentration in fruit peel, a measure of membrane damage, increased linearly (r = 0.891) with increase in scald severity. discriminant analysis was used to classify fruit by scald severity on the basis of metabolites accumulated. the stepwise model indicated that three attributes (ros, ethylene production and mda) contributed significantly (r2 ≥ 0.5) to the separation of the five scald severity indexes, with ros having the highest contri bution (partial r² = 0.961; p < 0.0001), followed by ethylene (r2 = 0.718; p < 0.0001) and mda (r2 = 0.578; p < 0.0001). introduction superficial scald is the major physiological disorder that develops after long term cold storage of apples and is associated with the disruption of tissues immediately beneath the epidermis of the fruit, with tissue browning not extending to the mesocarp of pulp (bain, 1956; sabban-amin et al., 2011; lurie and watkins, 2012). array of factors such as cultivar, seasonality, maturity, and duration of cold storage significantly influence scald development (rao et al., 1998; watkins et al., 2000; ahn et al., 2007). some apple cultivars such as ‘granny smith’ and ‘law rome’ are more susceptible (watkins et al., 1995; pechous and whitaker, 2004; jemric et al., 2006; sabban-amin et al., 2011) whilst cultivars such as ‘golden delicious’ and ‘royal gala’ are resistant (ingle, 2001; zanella et al., 2008; beuning et al., 2010). in particular, fruit maturity plays an important role in scald sus ceptibility, with scald incidence declining with advancing fruit maturity (erkan and perkmezci, 2004). there are physiological changes preceding superficial scald development. generation of reactive oxygen species or oxidative stress coupled with α-farnesene synthesis is one of the primary events leading to scald symptoms (rudell et al., 2009). moreover, conjugated trienols (ct) and 6-methyl-5-hepten-2-one (mho), both oxidised products of α farnesene, increase with scald incidence (mir et al., 1999; rowan et al., 2001; moggia et al., 2010). although concerted research efforts have been made to understand metabolic events leading to scald development, the relationship between scald severity and accumulation of metabolites linked to scald development remains unclear. the objective of this research work was to investigate the relationship between scald severity and metabolic changes in ‘granny smith’ apples. materials and methods fruit source and treatments seventeen-year old ‘granny smith’ apple trees grafted into m109 rootstock grown on a commercial orchard in grabouw (34° 12’12” s, 19° 02’35” e) were used in this study. the tree spacing was 4 × 1.5 meters giving a total of 1667 trees per hectare. all the trees were pruned to a central leader and irrigated by micro sprinklers. fruit were hand-picked at optimal maturity, and transported to the research laboratory at agricultural research council, stellenbosch and sorted to remove fruit with physical defects. the experiment was laid in a completely randomised design. uniformly sized fruit with diameter of 70 ± 2 mm and mass of 160 ± 5 g were randomly selected to provide three replications of 100 fruit each. fruit was placed in slotted and stacked high-density polyethylene plastic crates. fruit were stored in regular atmosphere (ra) at 0 °c (95 % rh) for 12 weeks followed by 7 days shelf-life at normal room conditions (20 °c, 65 % rh). at the end of shelf life, fruit were individually assessed and rated for scald severity based on the percentage of the surface area affected and sorted into five groups based on as follows: 0 = no scald, 1 = 1-25 % (slight), 2 = 26-50 % (moderate), 3 = 5175 % (high), and 4 = 76-100 % (very high) (fig. 1). for each level of scald severity, six replicates with five fruit per replicate were used for analysis. ethylene production ethylene production was measured as described by oz (2011) with slight modifications. briefly, each fruit was weighed using a mettler toledo digital balance (± 0.01g), and thereafter enclosed in 1 l airtight jar for 1 h at 20 °c. infrared ethylene analyser (ica56 ppm) was used for measurement and the results were expressed as μlkg-1h-1. headspace volatile analysis fresh peel (5 g) was weighed into a 20 ml solid phase micro extraction (smpe) glass vials. 10 μl of 3-octanol internal standard was added, and the vials were sealed. vial headspace was analysed according to mayuoni-kirshinbaum et al. (2012) and caleb et al. (2013). the vials were equilibrated for 10 min at 50 °c in the ctc autosampler incubator. after equilibration, a 50/30 μm divinylbenzene/-carboxen/-polydimethylsiloxane coated fibre was 50 a. mditshwa, o.a. fawole, f. vries, k. van der merwe, e. crouch, u.l. opara then exposed to the sample headspace for 20 min at 50 °c. after extraction, the trapped volatile compounds from the fibre coating were desorbed for 2 minutes in the injection port of the gas chromatograph operated in a splitless mode. the temperature was maintained at 250 °c for the injection. the fibre was cleaned after each sample heating for 10 min in the fibre conditioned station maintained at 270 °c. chromatographic separation of the extracted volatiles was performed on a agilent 6890n (agilent, palo alto, ca) connected through a transfer line to a agilent 5975b ms (agilent, palo alto, ca) mass spectrometer detector. the gc-ms system was equipped with a polar db-ffap column from j&w (part number 122-3263) with the following dimensions: (60 m length; 250 μm internal diameter; and 0.5 μm film thickness). helium was used as carrier gas at a flow rate of 1.3 ml min-1. the oven temperature program was as follows: initial temperature of 40 °c for 5 minutes; then ramped at 5 °c min-1 up to a final temperature of 230 °c with a final hold time of 6 minutes. the ion source and quadropole were maintained at 240 °c and 150 °c, respectively. the transfer line temperature was maintained at 280 °c. alpha-farnesene and mho were identified by a library search and quantified using abundance characteristic ion 93 and 108, respectively. generally, α-farnesene gave a single peak at 22.2 min while mho gave a peak at 16.6 min. a reading of 104 in abundance was defined as one unit and expressed as u g-1 (ju and curry, 2000). confocal microscopic analyses of ros production reactive oxygen species were determined following a method described by macarisin et al. (2007) and sabban-amin et al. (2011). the fluorescent probe 2,7-dichlorodihydrofluorescein diacetate in which dichlorodihydrofluorescein (dcf) fluorescence measurement quantifies general oxidative stress was used. 2,7-dichlorodihydrofluorescein enters cells in the diacetate form (h2dcf-da), and the acetate form (h2dcf) is hydrolyzed by intracellular esterases and then reacts with oxidants, resulting in the highly fluorescent dcf. acetate detects a broad range of oxidizing molecules rather than a single ros form, and it is efficient in localizing ros within plant cells (joo et al., 2005). immediately before microscopic analysis, slices of apple peel were cut from fruit and immediately immersed in a small petri dish containing 10 ml of 10.0 μm h2dcf-da in loading buffer (50 mm mes buffer, ph 6.5). the h2dcf-da was freshly prepared from a 20 mm stock solution in dimethyl sulfoxide (dmso). to prevent light-inducible oxidation, the slices were kept in the dark for 10 min and were thereafter transferred to a new petri dish containing loading buffer to wash off excess dye. model ix 81 inverted confocal laser-scanning microscope (fluoview 500, olympus, japan) equipped with a 488 nm argonion laser was used for sample examination and image acquisition. the fluorescent probe was excited with a 488 nm laser beam and the emission was collected through a ba 515-525 filter. for autofluorescence, a ba 660 if emission filter was used. magnification was increased by focusing the scanning laser beam onto a smaller area of the tissue. the transmitted-light images were obtained with nomarski dif ferential interference contrast (dic) optics. the relative intensity of the fluorescence signal was estimated by calculating average pixel intensity from each successive focal plane of the apple peel slice, in 5 μm steps, with mica software (multi-image analysis, cytoview, israel). the value of fluorescence intensity presented is the mean (± standard error). lipid peroxidation mda was measured by the method described by dhindsa et al. (1981) and siboza et al. (2013) with slight modifications. freeze dried and pulverised apple peel (0.1 g) was homogenised with 10 ml of ice cold 0.1 % trichloroacetic acid (tca). the homogenate was centrifuged at 10 000 rpm for 15 min at 4 °c to precipitate particulates. a 1 ml aliquot of the supernatant was thoroughly mixed with 4 ml of 20 % tca containing 0.5 % thiobarbituric acid (tba). the mixture was incubated at 95 °c for 30 min and thereafter quickly cooled in an ice bath. after centrifugation at 10 000 for 15 min at 4 °c, the absorbance of the supernatant was read at 532 nm and corrected for nonspecific absorbance at 600 nm using uv-visible spectrophotometer (thermo scientific technologies, madison, wisconsin). the concentration of mda was calculated using an extinction coefficient (∈) of 155 mm-1 cm-1. statistical analysis data was subjected to statistica 11 (statsoft inc. oklahoma, usa) for analysis of variance (anova) according to duncan’s multiple range test. graphical data presentations were performed using graphpad prism software, ver. 6 (graphpad software, inc. san diego, usa). discriminant analysis (da) was performed using xlstat, ver. 7.5.2 (addinsoft, new york, usa). fig. 1: ‘granny smith’ apples with different superficial scald severity: 0, no scald; 1, 1-25 %; 2, 26-50 %; 3, 51-75 %; 4, 76-100 %. classification of superficial scald severity in apples by metabolomics 51 results and discussion ethylene ethylene production has an influence on physiological changes associated with superficial scald (ingle, 2001). several reports have shown that ethylene is involved in regulating α-farnesene, a key volatile involved in the induction of superficial scald in apples (mir et al., 1999; ju and curry, 2000; pechous et al., 2005). in this study, ethylene production increased gradually as scald severity increased from none (0) to slight (2), and declined as scald severity progressed (fig. 2). reduced ethylene production with increasing scald severity could be an indication of its completed role in scald etiology. this observation is in agreement with previous findings by mir and beaudry (1999), who demonstrated that ethylene production increases with scald-development but reduces in severely scalded fruit. a similar trend has also been reported for ‘bartlett’ pears stored at -1 °c for 24 weeks (ekman et al., 2004; whitaker et al., 2009). severity increased. it is worth noting that the reduction in both α-farnesene and mho levels coincided with a decline in ethylene production. these results suggest that both the production and oxidation of α-farnesene may require ethylene action. in fact, previous studies focused on reducing superficial scald in apples reported a reduced α-farnesene and mho production after 1-mcp treatment, and consequently low scald incidence (ghahramani and scott, 1998; fan and mattheis, 1999; shaham et al., 2003; gapper et al., 2006; jung and watkins, 2008). recently, gao et al. (2015) has also demonstrated that 1-mcp reduces superficial scald de velopment in ‘wujiuxiang’ pears by retarding α-farnesene and ctols accumulation. mir et al. (1999) indicated that the temporary relationship between scald severity and mho may indicate that mho is not directly involved in scald development. contrary to the prevailing hypothesis linking mho and superficial scald, rupasinghe et al. (2000) reported that methyl heptenol (mhol) in ‘delicious’ apples stored at 0 °c for 17 weeks was 60 % and 20 % higher in scald-developing and severely scalded tissues, respectively. other research findings have concluded that mho production rather than its presence, is the important aspect involved in scald appearance (ju and curry, 2002; lurie and watkins, 2012). ros detection and quantification low storage temperatures trigger plant tissues to produce reactive oxygen species (ros) which are the by-products of electron flow disruption in the mitochondria (purvis et al., 1995; pinhero et al., 1997) resulting in physiological disorders such as chilling injury (lyons, 1973; sala, 1998) and superficial scald (watkins, 1995; sabban-amin et al., 2011). physiologically, ros cause the oxidative stress that consequently results in imbalances in metabolism, high respiration rate, reduced ability of biological systems to detoxify toxic metabolites (lyons, 1973). the fluorescence appearing during cold storage and shelf-life was quantified as fluorescence units related to ros levels (sabban-amin et al., 2011; pesis et al., 2012). in the current study, fluorescence intensity significantly increased with scald severity (fig. 4a-e). similarly, ros levels increased with scald severity (fig. 3); however, low fluorescence and ros levels were detected in severely scalded fruit (fig. 4e). these results have demonstrated that ros play a role in super ficial scald development in ‘granny smith’ apples. this observation is consistent with previous findings that ros are involved in scald etiology. for instance, rao et al. (1998) found scald incidence to be related to ros levels in hybrid ‘white angel × rome beauty’ apple stored for 16 weeks at 0.5 °c. moreover, hydrogen peroxide (h2o2) was reported by zubini et al. (2007) to increase with scald incidence and severity in ‘granny smith’ apples. recently, lu et al. (2014) also reported scald severity to be highly dependent on the tab. 1: headspace accumulation of α-farnesene and mho in ‘granny smith’ apples with different levels of scald severity. the data are the means ± se of three replicates with five fruits per replicate. means in each column followed by different letter(s) differ significantly according to duncan’s multiple range test (p<0.05). superficial α-farnesene mho scald severity (u g-1) (u g-1) 0 6612.09±577.18 c 56.18±6.58 c 1 11216.05±588.63 b 90.17±4.04 b 2 15286.12±355.12 a 132.04±6.38 a 3 11716.10±462.87 ab 110.01±4.99 b 4 8940.15±304.31 bc 84.13±1.46 bc fig. 2: ethylene production in ‘granny smith’ apples with different scald severity. the data are the means ± standard error (se) of six replicates with five fruits per replicate. different letter(s) on the bars mean statistical differences according to duncan’s multiple range test (p<0.05). headspace volatile analysis scald development has been strongly associated with α-farnesene accumulation in apple peel (pesis et al., 2009). in this study, headspace levels of α-farnesene increased with scald severity (tab. 1) but declined thereafter when fruit became highly scalded. this result is in agreement with previous findings which demonstrated that α-farnesene increased with scald severity in ‘law rome’ apples (watkins et al., 2000) and ‘granny smith’ apples (shaham et al., 2003; zubini et al., 2007) stored at 0 °c for up to 26 weeks. as noted in this study, zubini et al. (2007) also reported a decline in α-farnesene levels in severely scalded fruit. interestingly, gapper et al. (2006) reported a similar trend for ‘d’ anjou’ pears and concluded that afs1 transcript is mediated by ethylene. mho (a product of α-farnesene oxidation) was significantly lower in fruit with no scald and increased with the onset of scalding and up moderate severity however, after severity level 2 (26-50 %), there was a decline in mho content (tab. 1). our findings are corrobo rated by those reported in ‘granny smith’ apples by several re searchers (fan et al., 1999; mir et al., 1999; wang and dilley, 2000) who demonstrated an initial increase in mho content with onset of scald and a subsequent decline as scald incidence and 52 a. mditshwa, o.a. fawole, f. vries, k. van der merwe, e. crouch, u.l. opara accumulation of h2o2 concentration in ‘fuji’ apples stored at 0 °c for 28 weeks. our study shows that the accumulation of ros is linked to scald severity, and the low ros levels in high to severely scalded fruit could be linked to reduced reactivity of oxygen species, and hence the corresponding reductions in metabolites implicated in scald development and severity such as α-farnesene and mho. lipid peroxidation malondialdehyde (mda) is regarded to be a suitable biomarker for lipid peroxidation caused by ros which is the major cause of membrane damage in plant tissues (katsuhara et al., 2005; lu et al., 2014). unless metabolised, ros cause lipid peroxidation and eventual symptoms of damage in plant tissues. peroxidation of membrane lipids is an indication of membrane damage and electrolyte leakage under cold stress (katsuhara et al., 2005). lipid peroxidation leads to membrane damage, and consequently chilling injury symptoms (lyons, 1973). membrane damage is the primary metabolism disorder preceding superficial scald in apples (rao et al., 1998). in this study, lipid peroxidation expressed as mda concentration (fig. 5) had a significant effect on scald severity. the mda was significantly lower in fruit with scald severity index of 0 and 1. however, lipid peroxidation gradually increased with scald severity. this result is in agreement with previous findings showing the accumulation of mda in scalded apples. for instance, rao et al. (1998) reported that lipid peroxidation increases with storage time and consequently scald severity in ‘white angel × rome beauty’ apple. lu et al. (2014) also noted that mda content increases with scald incidence and severity in ‘fuji’ apples stored at 0 °c for 28 weeks. moggia et al. (2010) reported increased membrane integrity in severely scalded ‘granny smith’ apples for 6 months at 0 °c. thomai et al. (1998) also demonstrated that membrane damage increases with scald incidence and severity. the low lipid peroxidation in fruit with no scald proves the relationship between scald incidence and membrane damage. moreover, the continuous increase in lipid peroxi dation indicates that superficial scald is not only a change in symptoms but also an accumulative damage (lu et al., 2011; gao et al., 2015). discriminant analysis outcome of discriminant analysis of scald severity index and metabolic attributes is presented in fig. 6. confusion matrix showing the correct and incorrect predictions made by the model are presented in tab. 2. the confusion matrix indicated 96.76 % accuracy in classifying the five scald severity classes. four indexes were particular ly well discriminated with 100 % accuracy; however, 16.67 % confusion appeared in severely scalded fruit. the confusion between fig. 3: production of reactive oxygen species (ros), as quantified by relative intensity of dichlorodihydrofluorescein diacetate (dcf) fluorescence in peel slices of ‘granny smith’ apples with different scald severity. the data are the means ± standard error (se) of six replicates with five fruits per replicate. different letter(s) on the bars mean statistical differences according to duncan’s multiple range test (p<0.05). fig. 4: confocal laser-scanning fluorescence images of ‘granny smith’ apple peel slices. superficial scald index 0, no scald; 1, 1-25 %; 2, 26-50 %; 3, 5175 %; 4, 76-100 %. fig. 5: changes in malondialdehyde (mda) concentration of ‘granny smith’ apple peel slices as influenced by scald severity. the data are the means ± standard error (se) of six replicates with five fruits per replicate. different letter(s) on the bars mean statistical differences according to duncan’s multiple range test (p<0.05). classification of superficial scald severity in apples by metabolomics 53 scald index 0 and 4 could be explained by the fact that the tissue in severely scalded fruit was almost dead, and this presumably reduced metabolism hence the similarity in accumulated metabolites between these two groups. the stepwise model indicated that three attributes, namely; ros, ethylene production and mda contributed significantly (r2 ≥ 0.5) to the separation of the five scald severity indexes (tab. 3). amongst the contributors, ros had the highest significant (p < 0.0001) contribution with r² = 0.961, suggesting that ros could indeed be strongly linked to scald severity in ‘granny smith’ apples. this result is in agreement with rao et al. (1998), who indicated that although scald development mechanism is yet to be fully understood, the contribution of ros maybe related to the disorder. conclusion this study has demonstrated that scald severity is not directly related to some scald-associated metabolites in ‘granny smith’ apples. while increases in ethylene production, α-farnesene and mho corresponded with the onset and progression of scald severity, this relationship did not hold in high to severely affected fruit. however, the accumulation of ros leading to loss of membrane integrity corresponded strongly to the level of scald severity with the exception of severely scalded fruit which showed lower ros levels. acknowledgement this work is based upon research supported by the south african research chairs initiative of the department of science and technology and national research foundation. the authors are grateful to agricultural research council, postharvest innovation programme (phi), hortgroscience and the technology and human resources for industry programme (thrip) for financial support. thanks to howard ruiters, viole combrinck and vanessa furtuin for their technical assistance. we also thank mr lucky mokwena and ms dumisile lumkwana of central analytical facilities (caf), university of stellenbosch for their contributions to the analysis of volatiles and reactive oxygen species. references ahn, t., gopinadhan, p., murr, d.p., 2007: antioxidant enzyme activities in apple varieties and resistance to superficial scald development. food res. int. 40, 1012-1019. bain, j.m., 1956: a historical study of the development of superficial scald in granny smith apples. j. hort. sci. 31, 234-238. beuning, l., green, s., yauk, y.k., 2010: the genomic sequence of afs-1 − an alphafarnesene synthase from the apple cultivar ‘royal gala’. front. agric. china 4, 74-78. caleb, o.j., opara, u.l., mahajan, p.v., manley, m., mokwena, l., tredoux, a.g., 2013: effect of modified atmosphere packaging and 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biol. technol. 19, 17-32. zanella, a., cazzanelli, p., rossi, o., 2008: dynamic controlled atmosphere (dca) storage by the means of chlorophyll fluorescence response for firmness retention in apple. acta hort. 796, 77-82. zubini, p., baraldi, e., de santis, a., bertolini, p., mari, m., 2007: expression of anti-oxidant enzyme genes in scald-resistant ʻbelfort̓ and scald-susceptible ʻgranny smith̓ apples during cold storage. j. hortic. sci. biotech. 82, 149-155. classification of superficial scald severity in apples by metabolomics 55 address of the authors: asanda mditshwa, olaniyi a. fawole, umezuruike linus opara, postharvest technology research laboratory, south african research chair in postharvest technology, de partment of horticultural sciences, faculty of agrisciences, stellenbosch university, p/bag x1, stellenbosch 7602, south africa e-mail: opara@sun.ac.za filicity vries, kobus van der merwe, agricultural research council – infruitec/nietvoorbij, p/bag 5026, stellenbosch, 7600, south africa © the author(s) 2016. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). elke crouch, department of horticultural sciences, faculty of agrisciences, stellenbosch university, p/bag x1, stellenbosch 7602, south africa journal of applied botany and food quality 88, 109 114 (2015), doi:10.5073/jabfq.2015.088.015 1 dipartimento di scienzefarmaceutiche, via romana, università degli studi di perugia, perugia, italy 2 dipartimento di ingegneria civileed ambientale, università degli studi di perugia, perugia, italy 3 dipartimento di scienze agrarie alimentaried ambientali, perugia, italy bioactive compounds and antioxidant characterization of three edible wild plants traditionally consumed in the umbria region (central italy): bunias erucago l. (corn rocket), lactuca perennis l. (mountain lettuce) and papaver rhoeas l. (poppy) angela maurizi1, alfredo de michele1, aldo ranfa2, anna ricci2, valentina roscini3, roberto coli1, mara bodesmo2*, giovanni burini1 (received november 19, 2014) * corresponding author summary the leaves of three edible wild plants, bunias erucago l. (corn rocket), lactuca perennis l. (mountain lettuce) and papaver rhoaes l. (poppy) were analysed for their proximate composition, some nutraceutical components and total antioxidant capacity. the protein levels ranged from 2.7 to 4.1 g/100 g of the edible portion. the range of dietary fibre content was 3.8 to 6.4 g/100 g of the edible portion. the amount of ash, carbohydrate and lipid ranged from 1.7 to 1.9, 3.3 to 4.4 and 0.22 to 0.45 g/100 g of the edible portion, respectively. lipids consisted mainly of polyunsaturated fatty acids with the highest value for bunias erucago l. (71.8 % of total fatty acids) and lactuca perennis l. (70.0 %). potassium (374.0 521.0 mg/100 g) and calcium (204.8-331.8 mg/100 g) were the most representative macro-elements in the species studied. the values of vitamin e, b-carotene and total vitamin c were included in the range from 0.91 to 2.61 mg/100 g, from 1,957 to 2,631 μg/100 g and from 19.2 to 31.0 mg/100 g, respectively. our results showed that the total antioxidant capacitywhich ranges from 27.2 to 63.7 μmol te/g, according to the oxygen radical absorbance capacity (orac) method, is highly justifiable due to the high content of phenolic compounds (159-246 mg gae/100 g). introduction the use of edible wild plants had a fundamental role, linked to survival, in many past civilisations. although in recent decades their consumption has been limited, today interest in them has been rehabilitated due to their healthy properties. plants constitute an important source of active natural products that differ widely in terms of structure and biological properties. a diet rich in vegetables produces beneficial effects (kaur and kapoor, 2001) because some of their constituents counteract the action of reactive oxygen species (ros) implicated in numerous degenerative pathologies such as cardiovascular diseases, diabetes, cancer and neurodegenerative disorders (halliwell and cross, 1994). edible wild plants are a good source of nutrients and nutraceutical compounds, such as vitamin c, vitamin e (α-tocopherol), β-carotene (pro-vitamin a) and polyphenols. vitamin c, e and β-carotene have various biological functions and antioxidant activities (sies and stahl, 1995; paiva and russel, 1999). in phytochemicals, polyphenols are among the richest group and possess various biological effects including antioxidant activity (kahkonen et al., 1999). epidemiological studies suggest that diets rich in polyphenols are associated with potential neuroprotective benefits (vauzour et al., 2008), reduced incidence risk of cardiovascular diseases (chds) (who) and specific types of cancer (franceschi et al., 1997). the lipids of edible wild plants are mainly constituted by unsaturated fatty acids such as omega-3 fatty acids which are essential for normal growth and development and may play a fundamental role in many diseases (simopoulos, 2004). dietary fibre is an important fraction and shows protective effects on different diseases (kendall et al., 2010), including colorectal cancer, cardiovascular disease (theuwissen and mensink, 2008), diabetes, obesity, and diverticular disease. wild food plants provide a qualitative contribution to the traditional mediterranean diet (pieroni et al., 2005). potential health effects of these herbs are shown in several studies supported by the european commission (heinrich et al., 2006; hadjichambis et al., 2008). in recent years, many researchers have shown an increased interest in wild plants for several reasons: the renewed interest in local traditional foods (see for example guarrera et al., 2005; ghirardini et al., 2007; cornara et al., 2009; guarrera et al., 2009), the potential of these foods as nutraceuticals, and in the prevention degenerative diseases. consequently, the aim of the present study was to evaluate the chemical composition, some bioactive compounds and the antioxidant activity in three selected edible wild plants, collected in winter in their natural habitat (in umbria, central italy, on the slopes of mount subasio, in the perugia area). materials and methods collection and preparation of samples three different samples of edible wild plants, bunias erucago l. (corn rocket), lactuca perennis l. (mountain lettuce) and papaver rhoaes l. (poppy), were collected in the wild nature in february 2012, analysed with a stereomicroscope sx45, and classified according to the checklist of italian vascular flora (conti et al., 2007). all the exsiccata, of the aforementioned species are preserved in the erbario peru of the perugia university. these three species were chosen because they are among the most representative and best known in traditional folk recipes and are used both raw, in salads, and cooked, in soups, or as boiled greens. each sample was a bunch of several leaves collected from different plants to ensure a representative sample of each species. moreover, the leaves were preventively washed with distilled water to remove all foreign materials such as soil and sand. depending on the component to be determined, the analysis was carried out on the fresh or lyophilised leaves. a weighed portion (100 g) was lyophilised for 24 h (minifast 2000 edwards global headquarter, crawley, uk) and the dry weight was determined. chemical composition the moisture content of the three edible wild plants was determined by drying the leaves in an oven at 100 °c until a constant weight ac110 a. maurizi, a. de michele, a. ranfa, a. ricci, v. roscini, r. coli, m. bodesmo, g. burini cording to aoac method (aoac, 1990) was obtained. crude protein content was calculated using the conversion factor 6.25 (nx6.25) from the nitrogen content determined by the kjeldahl method (aoac, 1990). total lipid was evaluated using chloroform/methanol extraction based on the bligh and dyer procedure as described in the aoac method (aoac, 1990). ash content was determined according to the aoac method (aoac, 1990) by incineration in a muffle furnace at 500 °c for 6 h. total dietary fibre was determined by the enzymatic-gravimetric method according to the aoac method (aoac, 1995). carbohydrates were calculated by difference. according to the aoac method (aoac, 2006) iron, calcium and magnesium determination was conducted by aa-6800 model flame (airacetylene) atomic absorption spectrophotometer (aas) (shimadzu, kyoto, japan) on the ashes which were obtained from 4 g of each lyophilised sample dissolved in 10 ml of 6 n of hcl. the solution was then transferred into a 50 ml volumetric flask and the volume was adjusted with distilled water. calcium, magnesium and iron certified stock standard solutions, 1000 ppm in hno3 (carlo erba, milan, italy) were used. for calcium analysis, lanthanum chloride (5 %) was added to each sample to avoid interference with the phosphate. standard working solutions were prepared according to the user manual in order to obtain the calibration curve. sodium and potassium determination was conducted using a pfp7 flame photometer (bibby scientific, jenway, techne inc., uk) on the same solution. sodium and potassium certified stock industrial standard solutions were purchased from jenway (jenway, essex, uk). standard working solutions were prepared according to the user manual in order to obtain the calibration curve. analysis accuracy was confirmed for all the metals studied using certified standard reference material (nist-srm-1573rd, tomato leaves). phosphorus content was determined colorimetrically on 0.2 g of each lyophilised sample by reading the absorbance at 650 nm using ammonium molybdate, hydroquinone and sodium sulphide solutions according to the aoac method (aoac, 1990). polyphenols were determined by the folinciocalteu method with measurement at 760 nm with a spectrophotometer (varian uv/vis 50 cary bio model, palo alto, ca, usa). the results were expressed as mg of gallic acid equivalent (gae) per 100 g fresh weight (singleton and rossi, 1965). the assay was carried out on 250 mg of each lyophilised sample. fatty acid composition fatty acids were determined by high-resolution gaschromatography (hrgc) with a flame ionisation detector (fid) and a capillary column. fatty acid methyl esters (fames) were prepared from the lipid extract by a transesterification process catalysed with sulphuric acid (freedman et al., 1986). the fatty acid profile was analysed with a fisons instruments model hrgc mega 2 series (fisons instruments, altrincham, uk) equipped with a split-splitless injector with temperatures of the injector and detector at 300 °c. separation was achieved on a 30 m × 0.25 mm i.d. fused silica capillary column coated with a 0.25 μm film of db wax 122-7032 columns (agilent technologies, delaware, usa). helium was used as carrier gas at 1 ml/min. the column temperature was programmed from 130 °c (for a 1 min hold) to increase to 180 °c at a rate of 5 °c/min (for a 5 min hold), then to increase to 230 °c at a rate of 4 °c/min (for a 40 min hold) and finally to 250 °c at a rate of 2 °c/min (for a 5 min hold). the standard and the sample were injected in the splitless mode, and the injected volume was 0.5 μl. the results are expressed in the relative percentage of each fatty acid, calculated by internal normalisation of the chromatographic peak area. fatty acid identification was made by comparing the relative retention times of fame peaks from the samples with standards. a nucheck prep (nucheck prep, inc., mn, usa) mixture of fames standard was used. simultaneous determination by normal phase-high performance liquid chromatograph (np-hplc) of tocopherols and β-carotene tocopherols and β-carotene were determined by a modified nphplc (redi, 1999) operating with a spectra physics sp8800 model ternary pump (mountain view, ca, usa), and a rheodyne 7125 (cotati, ca, usa) sample-injection valve with a 10 l injection loop. two detectors were joined in series in order to determine, with a single run, the tocopherols (α-, β-, γand δ-tocopherol) and β-carotene. tocopherols were detected by a jasco fp-920 model, tokyo, japan fluorometric detector (fld) equipped with a light source of 150 w xenon lamp, λ ex. = 290 nm and λ em. = 330 nm while β-carotene was detected by a uv/vis detector (shimadzu spd-10 a vp model, kyoto, japan) set at 450 nm. a personal computer with appropriate software (varian star chromatograph ver. 6.00 walnut creek, ca, usa) for data acquisition and processing was used. isocratic separation was achieved on a merck hibar, lichrosorb si 60, 250 mm x 4 mm i.d., 5 m particle size column (merck, darmstadt, germany) using a mixture of n-hexane and 2-propanol (98:2, v/v) as eluent at a flow-rate of 1 ml/min. the standard stock solutions containing 500 μg/ml of each tocopherol was prepared by dissolving 50 mg of each α-, β-, γand δ-tocopherol (calbiochem a brand of emd biosciences, inc., an affiliate of merck kgaa, darmstadt, germany) in a 100 ml volumetric flask with absolute ethanol (j.t. baker, mallinckrodt baker, milan, italy). solutions containing 0.25, 0.50, 1.00, 2.00, 4.00 and 8.00 μg/ml of each tocopherol, used for the calibration curve, were prepared by appropriate dilution with n-hexane (j.t. baker, mallinckrodt baker, milan, italy). a standard stock solution, containing 250 μg/mlof β-carotene (calbiochem a brand of emd biosciences, inc., an affiliate of merck kgaa, darmstadt, germany), was prepared by dissolving 25.8 mg of β-carotene in 100 ml in a volumetric flask with n-hexane. solutions containing 0.10, 0.20, 0.40, 0.80, 1.60 and 3.20 μg/ml of β-carotene, used for the calibration curve, were prepared by appropriately diluting the stock solution with n-hexane. this solution was kept at -20 °c and made freshly every week. the extraction of tocopherols and β-carotene from the samples was carried out by the addition of 25 ml of n-hexane to 0.5 g of fresh leaves of each sample placed in a falcon 2070 50 ml polypropylene graduate conical tube (falcon, boston dickinson labware, nj, usa). the mixture was homogenised with an ultra turrax (ika ti 25, milan, italy) to obtain a fine suspension and then was centrifuged (eppendorf 5810 r model, milan, italy) at 4000 rpm for 5 min. two ml of the supernatant solution were transferred into a screw-cap tube (10 ml) and the n-hexane was evaporated under a mild stream of nitrogen at 30 °c; dry residue was dissolved in 0.5 ml of mobile phase. the resulting solution was analysed by np-hplc. the data, collected by workstation software (varian star chromatograph software version 6.00, walnut creek, ca, usa), were processed to quantify the tocopherols and β-carotene with the external standardisation method. total l-ascorbic acid determination total l-ascorbic acid (aa) determination was carried out according to the burini method (2007), based on radical oxidation of l-ascorbic acid (aa), to obtain dehydro-l-ascorbic acid (dhaa) by means of a peroxyl radical generated in situ by thermal decomposition of an azo-compound, 2,2’-azobis(2-amidinopropane) dihydrochloride (aaph). the dhaa is condensed with benzene-1,2-diamine (ophenylendiamine, opda) to form its highly fluorescent quinoxaline derivative, 3-(1,2-dihydroxyethyl)furo[3,4-b]quinoxaline-1-one (dfq) which was then separated by hplc with fluorometric detection (fld). the sample was prepared by homogenising 1 g of fresh leaves in a falcon 2070 50 ml polypropylene graduate conical tube (boston dickinson labware, nj, usa) with 35 ml of 1 % (w/v) the nutraceutical properties of three edible wild plants in umbria (italy) 111 metaphosphoric acid (mpa). the resultant mixture was transferred into a 50 ml volumetric flask and brought to volume with 1 % (w/v) mpa. an aliquot of 3 ml of each extract was filtered (sartorius 0.45-m membrane filter) and 1 ml of filtrate was analysed. total antioxidant capacity determination by hydrophilic-oxygen radical absorbance capacity (h-orac). the total antioxidant capacity by orac assay was based on the radiative fluorescence decay of the fluorescein (fluorescent probe) produced by reactive oxygen species (roo•) formed in situ by thermal decomposition of an azo-compound in the presence of an antioxidant as a reference standard (trolox) and a sample. the antioxidant compounds of the sample, acting as free radical scavengers, produce a stable signal of fluorescence that decreases more or less rapidly according to their concentration and reactivity. the data points of fluorescence intensity (fi) were summarised over time by the evaluation software. the total value of the sum fi of each sample, subtracting the total value of the sum of blank, was integrated on the calibration curve to obtain the orac value expressed as micromoles of trolox equivalents per gram (μmol of te/g). the assay was conducted with fluostar optima fluorescent microplate reader (bmg labth gmbh, germany), provided with a pump, set to λ ex. = 485 nm and λ em. = 520 nm and interfaced with a computer provided with a mars data analysis software ver. 2.00 (bmg labth gmbh, germany) for data acquisition and processing. costar 96 well black opaque plates (corning costar corporation, cambridge, ma, usa) were used. two hundred μl of 60 nm fluorescein (sigma-adrich, steinheim, germany) working solution was added to all experimental designated in the wells. in addition, blank wells received 20 μl of 75 mm phosphate buffer (ph 7.2), while standard wells received 20 μl of each 10, 20, 40 and 80 mm trolox (a vitamin e analogue hydrosoluble, 6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxyl acid) (sigma aldrich, steinheim, germany) standard solution in phosphate buffer and each sample well received 20 μl of solution as indicated below. four wells for the blank, four wells for each standard solution of trolox and four wells for the three samples for a total of 32 wells were used. the plate was then allowed to equilibrate by incubating for 30 min at 37 °c and then the fluorescence in each well was read by plate reader (2 cycles). in the next cycle, the pump was programmed to inject 60 l of 160 mm of aaph freshly prepared solution into the respective wells to start the reactions. the data were collected every 2.3 min by monitoring the fluorescence. each sample extract was obtained adding 20 ml of a ch3coch3: h2o: ch3cooh (70:29.5:0.5 v/v/v) mixture (prior et al., 2003) to 0.5 g of each fresh leaf sample in a 50 ml polypropylene graduate conical tube (falcon 2070 blue max, boston dickinson labware, nj, usa). the mixture was subjected to homogenisation (ika ti 25 ultra turrex, milan, italy) to obtain a fine suspension. after centrifuging at 4000 rpm for 5 min (eppendorf centrifuge 5810 r model, eppendorf, milan, italy) the supernatant was diluted 1:50 with phosphate buffer and the resulting solution was used for orac evaluation. statistical analysis data are presented as mean ± standard deviation (sd). each analytical determination was replicated three times except for the h-orac assay that was carried out in quadruple. the statistical analysis of the data was performed using the statistical analysis system (sas release 8.1, sas institute inc. cary, north carolina, usa). a paired student t-test was used to compare the mean values. a value of p<0.05 was considered significant. results and discussion the species analysed was characterised by moderate amounts of proteins, low amounts of lipids, considerable amounts of minerals and acceptable fibre content. for the species analysed statistically sig nificant differences among protein, lipid and dietary fibre content were observed (p<0.05). tab. 2 shows that lipids were represented mainly by such polyunsaturated fatty acids and particulary by α-linolenic acid (c18:3n-3) and linoleic acid (c18:2n-6). p. rhoeas l. had a high oleic acid content (c18:1n-9) and a higher amount of linoleic acid compared to b. erucago and l. perennis. linolenic acid was higher in b. erucago compared to p. rhoeas and l. perennis. the surprising presence of c20:5n-3 (epa) in l. perennis and in p. rhoeas was probably due to the extraction method (aoac, 1990), that allows the extraction of membrane polar lipids, important components of chloroplast. tab. 3 shows signifcant differences in the content of various minerals. it can be observed that potassium and calcium were the main minerals, followed by phosphorus, magnesium and sodium. statistically significant differences were observed in ca content for l. perennis and in mg content for b. erucago unlike the other species. iron content was important, especially in b. erucago, similar if not higher than that of meat, even though of lesser bioavailability. only a few studies have been published regarding some compositional characteristics of p. rhoeas (zeghichi et al., 2003; akrout et al., 2010) while analytical data for b. erucago and l. perennis are not available in the literature. substantial differences between our data and those reported by zeghichi et al. (2003) and akrout et al. (2010) were found in all mineral concentrations for p. rhoeas. this is probably due to different factors such as the harvest period, parts studied (leaves, stems, flowers) and to different pedo-climatic conditions where the plants grow. dietary fibre amount was satisfactory because it contributes to the satisfaction of recommended daily intake (sinu, 1996). (tab. 3). our results obtained for phenolic compounds (see tab. 4) suggest that these important bioactive components are contained in great amounts, from 159 to 246 mg gae/100 g of the edible portion. statistically significant differences between l. perennis and the other species were observed. tab. 4 shows the results of α-tocopherol and β-carotene found in the three samples analysed. the maximum values of α-tocopherol and β-carotene were found in l. perennis. 86.4±0.51 4.1±0.10 0.22±0.01 3.3±0.54 1.7±0.15a 4.3±0.23a 84.2±0.44 2.7±0.12 0.45±0.02 4.4±0.39 1.8±0.11a 6.4±0.30 87.2±0.64 3.5±0.20 0.32±0.02 3.3±0.28 1.9±0.09a 3.8±0.27a tab. 1: chemical composition of raw edible wild plants (g/100g edible weight). plant name moisture protein lipid carbohydrate ash dietary fibre bunias erucago l. (corn rocket) lactuca perennis l. (mountain lettuce) papaver rhoeas l. (poppy) a means followed by the same superscript are not significantly different at 0.05 level. 112 a. maurizi, a. de michele, a. ranfa, a. ricci, v. roscini, r. coli, m. bodesmo, g. burini tab. 2: most representative fatty acids of the raw edible wild plants (%of total fatty acids). abbreviation common name bunias erucago l. lactuca perennis l. papaver rhoeas l. (corn rocket) (mountain lettuce) (poppy) c 12:0 lauric tr. 0.49±0.11 tr. c 14:0 myristic 0.73±0.12 2.12±0.15 0.40±0.09 c 15:0 0.28±0.04 0.40±0.08 tr. c 16:0 palmitic 19.51±1.15 18.15±1.02 16.30±1.11 c 16:1n-7 palmitoleic 0.33±0.06 0.33±0.05 0.14±0.03 c 17:0 margaric 0.24±0.05 tr. tr. c 18:0 stearic 1.04±0.09 1.92±0.12 2.81±0.20 c 18:1n-9 oleic 2.41±0.20 2.77±0.22 12.12±0.62 c 18:1n-7 2.82±0.25 0.45±0.07 0.69±0.06 c 18:2n-6 linoleic 12.35±0.76 24.45±0.93 28.06±0.94 c 18:3n3 a-linoleic 59.42±1.48 45.57±1.70 37.49±1.56 c 18:3n-4 stearidonic tr. 0.27±0.04 0.20±0.04 c 20:0 arachidic 0.32±0.05 1.20±0.11 0.54±0.08. c 20:5n-3 epa tr. 1.14±0.10 0.56±0.08 tr. = trace tab. 3: mineral content of raw edible wild plants (g/100g edible weight). plant name k na ca mg fe p(*) bunias erucago l. (corn rocket) lactuca perennis l. (mountain lettuce) papaver rhoeas l. (poppy) (*) total phosphorus. a means followed by the same superscript are not significantly different at 0.05 level. 374.0±30.39a 10.9±0.77 228.5±16.79a 20.4±1.99 5.9±0.69 69.6±6.56a 440.8±38.28a,b 47.4±3.20 331.8±21.06 31.6±2.52a 3.3±0.20 47.4±4.51 521.0±33.07b 28.2±2.72 204.8±20.15a 30.7±2.84a 4.1±0.41 75.3±4.74a tab. 4: a-tocopherol, b-carotene, total ascorbic acid, total phenol content and antioxidant capacity of raw edible wild plants. in parentheses the repeatability relative to standard deviation (rsdr) is shown. component bunias erucago l. lactuca perennis l. papaver rhoeas l. (corn rocket) (mountain lettuce) (poppy) a-tocopherol, mg/100 g 0.91±0.05 (5.5) 2.61±0.14 (5.4) 1.72±0.10 (5.8) b-carotene, mg/100 g 1,957±78 (4.0) 2,631±121 (4.6) 2,176±105 (4.8) ascorbic acid, mg/100 g 31.0±0.99(3.2) 19.2±0.79 (4.1)a 21.3±1.10 (5.2)a total phenols, mg gae/100 g 159±7 (4.4) 246±15 (6.1) 188±11 (5.9) orac, mmol te/g 27.24±1.55 (5.7) 63.70±2.81 (4.4) 43.89±2.32 (5.3) a means followed by the same superscript are not significantly different at 0.05 level. a few authors have determined α-tocopherol in edible wild plants (simopoulos, 2004) by analysing the edible wild plants of crete. they found a lower value than that of our data, probably due to many different environmental factors and to the parts of the plant studied. α-tocopherol values determined in our group were similar to values found in broccoli (1.44 mg/100 g) and spinach (1.96 mg/100 g), which were the highest values of other raw vegetables studied (chun et al., 2006). fig. 1 shows the hplc of standard tocopherol solutions (channel a) and β-carotene (channel b). a good separation of α-tocopherol and the other tocopherols was obtained. fig. 2 refers, as an example, to l. perennis analysed in the same conditions. as can be seen from fig. 2, and similarly in the other two species examined, just α-tocopherol was found. tab. 5 shows the parameters relating to the linear regression, the limit of detection (lod) and the limit of quantification (loq) for tocopherols and for β-carotene. the limits of detection (lod) were evaluated, according to the federal register (1997), on the standard deviations of the response and the slope using the ratio 3.3 s/s, where s = the standard deviation of the response and s=the slope of the calibration curves of a-, b-, dand gtocopherol and b-carotene, respectively. the σ the nutraceutical properties of three edible wild plants in umbria (italy) 113 values were estimated on the calibration curves using the standard deviations of the y-intercepts of the regression lines. the limit of quantification (loq) was calculated by using the ratio 10 s/s. pre cision, considered as repeatability, was determined by analysing three replicates of all the samples analysed. the method used showed good repeatability of the relative standard deviations (rsdr), which in each instance was ≤ 5.8 % for both a-tocopherol and b-carotene. furthermore, accuracy was high considering the good recoveries obtained by spiking known amounts of tocopherols and β-carotene to the samples. recoveries were included for a-, b-, dand g-tocopherol in the ranges 99.0-102.8 %, 98.5-103.6 %, 97.1-100.2 %, 99.2102.3 % and in the range 96.8-97.9 % for b-carotene, respectively. the results of total l-ascorbic acid found in the three samples analysed are also reported in tab. 4; the contents are in the range 19.231.0 mg/100g of fresh leaves. statistically significant differences between b. erucago and the other species were observed. the content of total l-ascorbic acid was similar to the values that are found for raw celery (32 mg/100 g), raw chard (24 mg/100 g), raw cultivated asparagus (18 mg/100 g) and raw chicory (17 mg/100 g) (inran, 2000). in particular, the value obtained for p. rhoeas was comparable with that reported by sanchez mata et al., 2012. the results of orac, reported in tab. 4, show higher values (the maximum for l. perennis) compared to the values of other vegetables relevant to nutrition (ninfali et al., 2005). although orac assay allowed us to obtain lipophilic (l-orac) and hydrophilic (h-orac) antioxidant fractions (prior et al., 2003), our study was focussed on the hydrophilic fraction, which was the most important quantitative portion; indeed, in vegetables the l-orac fraction is very low compared to h-orac (wu et al., 2004) justifiable by the low content of lipids in the samples examined. the standard solutions of trolox show linearity from 10 to 80 m. the equation of the calibration curve, obtained from the sum of the fluorescence intensity versus the trolox concentrations, was y=13,392+2,524x with a good correlation coefficient (r2) of 0.9928. the repeatability relative standard deviation (rsdr) of samples was good; in each instance rsdrwas ≤ 5.7 %. the limitation of the total antioxidant capacity determination of foods lies in the fact that it is assessed in vitro far from reproducing the biological conditions of the human organism. it does not take into consideration metabolic reactions, which largely influence bioavailability of antioxidant components. to confirm the beneficial effects of the bioactive components contained in edible wild plants, it would be useful to observe in vivo plasma antioxidant capacity. a research of this kind, carried out in our laboratory on lyophilised and reconstituted red wine, produced a significant increase in plasma antioxidant capacity due to its bioactive constituents (alberti fidanza et al., 2003). plasma antioxidant capacity has been suggested as a good in vivo marker of the antioxidant status (fernandez patchon et al., 2008). conclusion in conclusion the leaves of the wild edible plants, b. erucago, l. perennis and p. rhoaes, possess important nutraceutical compounds which enhance their nutritional properties. their use in the usual diet can help providing beneficial effects to human health due to the presence of dietary fibre, vitamin c, vitamin e, β-carotene, potassium, calcium and polyphenols and to their high total antioxidant capacity. our results can also be used to expand and update the food composition database. a new, interesting property of the wild species examined is represented by the presence of an adequate concentration of antioxidant components capable of contrasting the effect of free radicals. thanks to their antioxidant properties, it would certainly be worthwhile to perform further studies of edible wild plants and to promote commercialisation campaigns, particularly in view of the growing demands by the food industry for natural antioxidants. human health in general could greatly benefit from a reconsideration of these wild plants because they represent a naturally-occurring, easy to obtain source of powerful vegetable antioxidants. acknowledgements we are grateful to prof. giovanni gigliotti, university of perugia, for his intellectual support and gina pero for her technical support. references akrout, a., el jani, h., zammouri, t., mighri, h., neffati, m., 2010: phytochemical screening and mineral contents of annual plant growing in the southern of tunisia. j. phytology 2, 34-40. alberti fidanza, a., burini, g., antonelli, g., murdolo, g., perriello, g., 2003: acute effects of lyophilized red wine on total antioxidant capacity in healthy volunteers. diabetes nutr. metab. 16, 65-71. fig. 2: typical chromatogram of l. perennis channel a: 1 =a-tocopherol (2.16 μg ml-1). channel b: 1 =b-carotene (2.20 μg ml-1). fig. 1: chromatogram of standard tocopherols (2.00 μg ml-1 of each) and 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115-126. who cardiovascular diseases (cvds). fact sheet no317. http://www. who.int/mediacentre/factsheets/fs317/en/index.html [accessed on 10 september 2009]. wu, x., beecher, g.r., holden, j.m., haytowitz, d.b., gebhardt, s.e., prior, r.l., 2004: lipophilic and hydrophilic antioxidant capacities of common foods in the united states. j. agric. food chem. 52, 40264037. zeghichi, s., kallithraka, s., simopoulos, a.p., kypriotakis, z., 2003: nutritional composition of selected wild plants in the diet of crete. world rev. nutr. diet. 91, 22-40. address of the corresponding author: mara bodesmo, dipartimento di ingegneria civile ed ambientale, università degli studi di perugia, borgo xx giugno, 06121 perugia, italy e-mail: bodesmo@hotmail.it © the author(s) 2015. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 89, 68 72 (2016), doi:10.5073/jabfq.2016.089.008 institute of plant nutrition and soil science, kiel university, kiel, germany biofortification and subcellular localization of minerals in faba bean as influenced by mg foliar application christoph-martin geilfus, neele wendler, karl-hermann mühling* (received january 12, 2016) * corresponding author summary foliar application of mg is a measure for the correction of mg deficiency in crop plants. foliar applied nutrients need to access the symplastic side where majority of physiological processes take place. to achieve an adequate uptake of the mg ions through the leaf surface, concentrations of 100-200 mm mgso4 are usually supplied. this can cause antagonistic perturbations on the subcellular distribution of ca and k cations. to test for such unintended side effects, we used the infiltration-centrifugation method to extract ions from the apoplastic and symplastic side of vicia faba leaves and quantified concentrations of mg, ca and k in dependency to the dose of the foliar fertilized mg. results show that a large fraction of mg accesses the symplast whereas the apoplastic fraction shows a concomitant increase. symplastic and apoplastic k and ca relations were only affected under conditions of high exogenous leaf supply of mg (200 mm) but did not change upon moderate mg supply (50; 100 mm). overall, results reveal the suitability of leaf fertilization to biofortify plant-based products with magnesium. introduction in plants, magnesium (mg) activates a myriad of enzymes and is important for growth and development (epstein and bloom, 2004). various environmental conditions cause magnesium deficiency and may lead to the appearance of characteristic deficiency symptoms in the form of an intervenal chlorosis that appear at older leaves of dicotyledons plant species (verbruggen and hermans, 2013; hermans et al., 2013; neuhaus et al., 2014; senbayram et al., 2016). in humans, mg has a fundamental role as co-factor for more than 300 catalytic reactions (wacker and parisi, 1968) and is especially required for energy generation and nucleic acid synthesis (institute of medicine, 1997; fawcett et al., 1999). besides being implicated in human protein synthesis, mg has also a dedicated role in the activation of the adenylate cyclase system and this system regulates cellular activities and is modulated by hormones and neurotransmitters (maguire, 1984). for human nutrition, plant-based products represent a main source of dietary magnesium, ultimately highlighting the need for plant nutritional measures to ensure adequate mg supply to growing plants in order to enrich plant-based foods with mg (white and broadley, 2009; gerendás and führs, 2013). because magnesium is the central atom in chlorophyll, green organs of the plants are rich in magnesium (institute of medicine, 1997). the foliar application of mg represents one strategy to supply mg-deficient crops rapidly with mg because, when compared to soil fertilization, the divalent cation is directly added in the vicinity of the mg-deficient tissues (jezek et al., 2015). foliar application of mg ions is in particular valuable at peak demand times and under conditions of low soil availability (fernandez and eichert, 2009). to achieve sufficient mg uptake over the leaves, mgso4 * 7 h2o at concentrations of 40 mm (reay et al., 1998), 80 mm (dordas, 2009), 200 mm (teklic et al., 2009; vrataric et al., 2006; kristek et al., 2000), or even higher (webster and drowe, 1982) have been tested. however, those studies did not evaluate the apoplastic fractioning of the foliar applied mg. the amount of mg that is trapped in the apoplastic compartment is relevant for the fertilizing effect because mg is quantitatively involved in metabolic processes that run in the cell symplast. thus, the physiological availability of the foliar applied mg is a crucial issue in leaf fertilization because it is possible that the applied mg is immobilized in the apoplastic leaf compartment and, by this means, is not immediately available for metabolic processes. as the apoplast comprises many fixed anions and is therefore supposed to serve as a storage site for cations (grignon and sentenac, 1991), the mg2+ supplied to the leaves might be partly bound apoplastically, rather than taken up into the symplast. it was one aim of this study to elucidate this topic by determining the fraction of the applied mg that accumulates in the apoplast. the second part of this study dealt with the problem associated with nutrient-nutrient interactions. the addition of mg may have side effects on the distribution of other mineral nutrients (rios et al., 2012). as demonstrated in experiments with soil or nutrient solution, the supply of the one cation can lead to a reduced uptake of others cations. this antagonistic effect was frequently reported for potassium (k) which influences the uptake of calcium (ca) and mg (e.g., miller, 1999; spear et al., 1987; ologunde and sorensen, 1982; troyanos et al., 2000). in the other way around, a high supply of mg can lead to reduced tissue concentrations of k and ca (grant et al., 1988; ologunde and sorensen, 1982; troyanos et al., 2000). based on these antagonistic effects, we hypothesize that foliar application of mg will affect leaf cation composition. for this reason it was the second aim of this study to evaluate whether foliar applied mg effects the compartmental distribution of k and ca. this study was based on vicia faba l. because the field bean has advantageous leaf anatomical features that facilitate to investigate leaf apoplastic ion relations. material and methods plant material and cultivation vicia faba l. plants of the cultivar scirocco (npz, hohenlieth, germany) were grown hydroponically in climate chambers (14/ 10 h day/night; 20 °c/15 °c; 250 μmol m-2 s-1 light intensity; and 50 % relative humidity). seedlings were placed in 0.5 mm aerated caso4 solution for one day at 25 °c and were subsequently transplanted into sterile quartz sand. after 10 days, the seedlings were transferred to 4 l plastic pots (4 plants per pot) containing one quarter-strength nutrient solution, which was raised to full concentration in a stepwise manner. nutrient concentrations as given by geilfus et al. (2015a). plants were subjected to six different groups (treatments) with four biological replications each. first group was supplied with 0.5 mm mgso4 * 7 h2o at the roots. plants from this group represent the positive control since they were sufficiently supplied with mg over the experiment. the remaining five treatments were grown in a mg-low nutrient solution containing only 0.05 mm mgso4, from which three groups were subjected to mgso4 leaf applications receiving 50 mm (2nd group), 100 mm (3rd group), or biofortification by mg foliar application 69 200 mm (4th group) mgso4, respectively. a wetting agent (0.1 % (v/v) arma; agroplanta, langenpreising-zusdorf, germany) was added to the application solutions to ensure contact with the leaf. the fifth group was not foliar fertilized with mgso4 but was sprayed with the same amount of water plus wetting agent. this group guarded the experiment against effects contributable to the water spray and the arma. the sixth group was neither foliar applied with mg nor with the wetting agent because these plants served as negative control representing plants with lack in mg. foliar application was conducted three times, starting four weeks after plants were set to the full nutrient concentration. the application was conducted using a handheld sprayer. in order to assure the application of equal amounts of 3.5 g of the fertilizer solution, the pots were placed on a balance. contamination of the nutrient solution was avoided by enwrapping the plant basis with foam. the plants were harvested 3 days after the last treatment, when plants had developed 15-16 leaves. samples were collected in a randomized manner. extraction of apoplastic and symplastic washing fluids cations were extracted by using the infiltration-centrifugation method as described by mühling and sattelmacher (1995) and shahzad et al. (2013). of each plant the 5th to 8th leaves were cut with a razor blade and washed with deionized water. subsequently, the leaf segments were placed in a 60 ml syringe infiltrated with 50 mm bacl2. bacl2 extracts apoplastically bound cations together with water soluble apoplastic ions. the solution was infiltrated into the leaves by applying a reduced pressure of ca. 20 kpa by pulling the syringe’s plunger. leaves were centrifuged for 5 minutes at 90 g and 4 °c, and the infiltration solution was collected. for the extraction of symplastic cations, leaves in which bacl2-extractable apoplastic cations had been extracted, were subsequently shock-frosted in liquid nitrogen and, after being thawed, were centrifuged again for 5 minutes at 142 g and 4 °c. samples were stored at -80 °c. determination of mg, ca and k concentrations mg2+, ca2+, and k+ in the symplastic fluid were determined by atomic absorption spectrometry (aas 5ea thermo electron s, carl zeiss, jena, germany) whereas ion chromatography (dx 300, dionex, idstein, germany) was used to determine corresponding concentration in the apoplastic washing fluids according to geilfus et al. (2015b). the latter analysis required to clear protein and chlorophyll content by means of adding chloroform to a final ratio of 1:2 (water:chloroform). samples were mixed, centrifuged and the supernatant was then retained and filtered through c-18 columns (torrance, ca, usa) for further clarification before ion analysis was started. since the quantified values represent the concentrations in the washing fluids, values were corrected about an empirical factor that estimates the ratio of gaseous volume to aqueous volume in the apoplastic space of field bean leaves (lohaus et al., 2001). determination of mg in leaf dry matter plant samples were dried for 48 h at 80 °c for dry weight measurements and then ground and ashed for 4 h at 550 °c in an oven. magnesium was extracted with 4 m nitric acid for 4 h, diluted to 0.8 m nitric acid and filtered for subsequent measurements with the atomic absorption spectrometer (aas 5ea thermo electron s, carl zeiss, jena, germany). statistics statistical analysis was carried out by using r (r version 2.11.1). for the comparison of means, multiple t-tests were adjusted according to bonferroni-holm. the homogeneity of variances and normal distribution was evaluated by boxplots, the shapiro-wilk test, and the levene test. results whole tissue mg concentration the reduction from 0.5 mm mgso4 to 0.05 mm mgso4 induced a sever mg-deficiency with the characteristic leaf intervenal chlorosis (fig. 1). with respect to whole leaf mg concentrations, a clear effect of the foliar supply became evident. low mgso4 (0.05 mm) in the nutrient solution resulted in a concentrations of only 1.3 mg mg/g dry matter (fig. 2, negative control). the foliar application raised the concentration significantly to 15 mg mg/g dry matter (200 mm mgso4 treatment), which was significantly higher than that in the positive control (3 mg/g dry matter) that was supplied with 0.5 mm mg to the roots (fig. 2). fig. 1: older leaves of vicia faba plants show mg deficiency symptoms (left; 0.05 mm mgso4). right side, leaves without deficiency (0.5 mm mgso4). fig. 2: effect of magnesium treatments on mg concentration in whole leaf dry matter. a, b, c, d: significant differences (p≤0.05) between treatments, la: leaf application (50 mm, 100 mm or 200 mm mgso4), means ±se (n=4 independent replicates). effect of mg foliar application on subcellular mg concentrations mg concentrations as quantified in the bacl2-containing washing fluid are composed of water soluble plus apoplastically bound mg and, by this means, ideally reflect the amount of leaf apoplastic mg. data clearly indicate that this mg fraction markedly increased upon mg leaf fertilization (fig. 3a) finally exceeding the apoplastic mg 70 c.-m. geilfus, n. wendler, k.-h. mühling concentrations of the positive control that received 0.5 mm mg via the roots. the same pattern was observed with respect to symplastic mg, however, absolute values were in average 1/3 higher (fig. 3b) when compared to the apoplastic fraction. nutrient-nutrient antagonistic effects between mg and sub cellular ca and k fractions the application of 200 mm mg onto the leaf decreased symplastic k concentrations whereas such an effect did not occur in the apoplast (fig. 3c-d). same treatment correlated with a decrease in apoplastic ca whereas symplastic ca concentrations remained unaffected (fig. 3e-f). concentrations of 50 or 100 mm of foliar applied mg did not modulate subcellular ca and k patterns. discussion mg foliar application fills pools of symplastic and apoplastic mg this study aimed to elucidate whether leaf foliar application of mg re-fills symplastic and apoplastic mg pools in leaves of vicia faba plants that depleted in mg. deficient plants showed pronounced intervenal chlorosis (fig. 1) and low whole leaf tissue mg concentrations (fig. 2). foliar application of mgso4 ameliorated the nutritional status of the mg-deficient leaves with regard to mg. the mg2+ concentrations in leaf dry matter (fig. 2) of plants subjected to mg2+ foliar application reached values that were markedly above the threshold value of 2 mg/g which is needed on average for an optimal mg supply (kirkby and mengel, 1976). the fertilization of mg over the leaf significantly increased mg in both, the apoplastic (fig. 3a) and symplastic (fig. 3b) compartment. such an apoplastic enrichment in mg is not the primary aim of an mg fertilization because the majority of problems that are associated with mg-deficiency are based on lack in symplastic mg. for example, mg is required for the functionality of many enzymes such as rna polymerases, carboxylases, phosphatases, glutathione synthases or plasma membrane h+-atpases (marschner, 1995; hanstein et al., 2011). the apoplast is known to act as an ion exchanger that is thought to function in storage of nutrients (bernstein and nieman, 1960). for this reason, the fact that a large fraction of the foliar applied mg is bound to the apoplast must not necessarily be associated with a permanent immobilization. a later release to symplastic sinks is possible. furthermore, apoplastic located mg plays a role for cell wall and plasma membrane stability (keegstra, 2010; pasternak et al., 2010). more importantly, this study clearly showed a remarkable accumulation of mg in the symplast (fig. 3b). it appears that the leaf application of 50 mm mg which was replicated in triplicate represents an efficient means for enriching the mg-depleted symplast rapidly with mg. from this we suggest to conduct a mg foliar application as a rapid measurement to avoid incipient mg deficien cy. intriguingly, jezek et al. (2015) reported that the foliar application of 200 mm mg increased net assimilation rate already after 2 days. we reason that such an agronomic measure is particularly advantageous at advanced developmental stages during the transition from the vegetative in the generative phase when a soil fertilization would come to late. based on the results presented here that prove the transfer of foliar applied mg into the leaf we encourage to adopt this strategy to other crop and horticultural plants with the aim to enrich plant based-foods in mg by means of agronomic biofortification. mg foliar application affects ca and k leaf concentrations in a dose-dependent manner mg deficiency often results in an increment of ca or k concentrations in the leaves (merhaut, 2007). however, with regard to k, other studies have reported a synergistic effect between mg supply fig. 3: effect of magnesium treatment on mg (a), k (b), and ca (c) concentrations in the symplastic and apoplastic leaf compartments. a, b, c: significant differences (p≤0.05) between treatments; means ±se (n=4 independent replicates). biofortification by mg foliar application 71 and k concentration (hariadi and shabala, 2004; ding et al., 2006). in our study, none of those synergistic or antagonistic effects on k+ or ca2+ concentration were observed under conditions when plants were supplied with sufficient (0.5 mm) or deficient (0,05 mm) mg over the roots (fig. 3c-f). the discrepancies between those results may be attributable to differences in the developmental state and mg supply: hariadi and shabala (2004) reported that effects on ca and k concentrations in leaves of the broad bean were induced in mg-deficient plants only at early sampling times. these effects prevailed at later sampling times only in severely deficient plants. interestingly, we found an effect of mgso4 leaf application on ca and k concentrations only in response to the 200 mm mgso4 foliar treatment. upon fertilization with 200 mm mg, the leaf apoplastic fraction of bound plus water soluble ca significantly dropped in concentration. this decrease was not associated with an concomitant increase in symplastic ca. this indicates that foliar fertilization with 200 mm mg can also negatively affect food quality of green plant organs since calcium is of utmost importance for human nutrition (sherwood, 2015). the application of mgso4 may lead to a displacement of ca from negative binding sites in the cell wall, constituted by carboxyl groups of pectins (sattelmacher et al., 1998). the displacement of ca by mg has also been shown by kirkby and mengel (1976) under ca-deficiency conditions. ca in the cell wall is important for both, the stabilizing the matrix pectins (grignon and sentenac, 1991) and the integrity of the plasma membrane (hepler and winship, 2010). thus, a loss of ca would favor a solubilization of pectins and subsequent leakage of ions across the membrane. mg has a smaller ionic radius than ca but has significantly less bond angle flexibility than other cations (kehres and maguires, 2002). thus, the displacement of ca by mg due to foliar application could have negative effects on cell wall structure and plasma membrane permeability. results further reveal an effect of the application of 200 mm mgso4 on symplastic k+ (fig. 3d). in general, the measured symplastic k concentrations that range from 66 to 100 mm are in accordance with the ranges of 40-200 mm reported by (britto and kronzucker, 2008) and 10-200 mm (marschner, 1995). the measured concen trations of apoplastic k raging from 3.6 to 5.0 mm also fit in the range reported by others (mühling and sattelmacher, 1995; mühling and läuchli, 1999). the concentration of symplastic k+ significantly decreased following the 200 mm foliar treatment. this decrease in k might be attributable to a k efflux and subsequent vascular transport to other organs in response to raising symplastic mg concentrations, as fluxes of k+ serve to counterbalance fluxes of other ions in terms of charge compensation (amtmann and blatt, 2009), an effect that could also be shown by shabala and hariadi (2005) who measured k+ efflux in high mg supply treatments of mesophyll segments. another effect of the high mg concentration on symplastic k relations might be conferred trough non-selective cation channels in the plasma membrane. these have been shown to reduce uptake of monovalent cations, such as k+, if the concentration of divalent cations, such as mg2+, is high (davenport and tester, 2000; shabala and hariadi, 2005). as we see decreases in k concentrations we reason that the fertilization of 200 mm mg over the leaf is not favorable for the production of plant-based foods − should this fertilization strategy be used in plant production − since k plays key role in a myriad of human metabolic processes (fox, 1995). conclusion the pressing question regarding leaf nutrient fertilization is whether the sprayed nutrients reach the symplast where majority of physiological processes take place. the data in this paper approve accessibility of foliar applied mg to the symplast. nevertheless considerable amount do not overcome the apoplastic site which must not necessarily be a problem since the leaf apoplastic compartment is thought to function as nutrient store. with respect to nutrient-nutrient interactions only the highest mg dose exhibited antagonistic effects on apoplastic ca or symplastic k pools whereas 50 or 100 mm mg did not interfere with the compartmental distribution of those cations. overall, our results reveal the functionality of leaf fertilization to enrich plants with magnesium. acknowledgements we thank ms. a. thießen and ms. s. thor straten for excellent technical assistance. references amtmann, a., blatt, m.r., 2009: regulation of macronutrient transport. new phytol. 181, 35-52. bernstein, l., nieman, r.h., 1960: apparent free space of plant roots. plant physiol. 35, 589-598. britto, d.t., kronzucker, h.j., 2008: cellular 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cherry rootstocks, “f.12/1” (prunus avium l.) and “colt” (prunus avium l.) × prunus pseudocerasus l.). plant soil 225, 73-82. verbruggen, n., hermans, c., 2013: physiological and molecular responses to magnesium nutritional imbalance in plants. plant soil, 368, 87-99. vrataric, m., sudaric, a., kovacevic, v., duvnjak, t., krizmanic, m., mijic, a., 2006: response of soy bean to foliar fertilization with magnesium sulphate (epsom salt). cereal res. commun. 34, 709-712. wacker, w.e., parisi, a.f., 1968: magnesium metabolism. n. engl. j. med. 278, 658-663. webster, d.h., drowe, a.d., 1982: absorption of magnesium by apple leaves following application in dilute and concentrate sprays. can. j. plant sci. 49, 225-226. white, p.j., broadley, m.r., 2009: biofortification of crops with seven mineral elements often lacking in human diets − iron, zinc, copper, calcium, magnesium, selenium and iodine. new phytol. 182, 49-84. address of the corresponding auhor: prof. dr. karl h. mühling, institute of plant nutrition and soil science, kiel university, hermann-rodewald-str. 2, 24118 kiel, germany. e-mail: khmuehling@plantnutrition.uni-kiel.de © the author(s) 2016. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 89, 58 (2016) buchbesprechung holm/herbst ‒ botanik und drogenkunde barbara eigner das von vera herbst und gabriele holm begründete lehrbuch wurde in der 10. auflage von barbara eigner aktualisiert und erweitert. das buch vermittelt kompakt und leicht verständlich die grundlagen der botanik und drogenkunde. der botanische teil liefert basiswissen zur zytologie, histologie, und morphologie. darüber hinaus wird auf die fortpflanzung, die grundlagen der vererbung und den pflanzlichen stoffwechsel näher eingegangen. der erste teil schließt mit einem kurzen einblick in die systematik der samenpflanzen. durch eine stammbaumartige gliederung der verschiedenen pflanzenfamilien werden dabei die verwandtschaftlichen beziehungen und damit die botanischen ähnlichkeiten untereinander verdeutlicht und auf diese weise ein einstieg in das komplexe thema geschaffen. der zweite teil befasst sich mit der drogenkunde und geht nach einer allgemeinen einführung zunächst auf die wichtigsten wirkstoffgruppen von medizinalpflanzen ein (kohlenhydrate, fette, isoprenoide, phenylpropanderivate, alkaloide und ätherische öle). darüber hinaus wird auf individuelle teemischungen für spezielle indikationen sowie deren zubereitung bezug genommen. schließlich finden sich auch noch einige hinweise zur verwendung pflanzlicher arzneimittel bei kindern und säuglingen sowie während der schwangerschaft und in der stillzeit. im anhang des lehrbuchs bieten drogenübersichten, sortiert jeweils nach indikationsund inhaltsstoffgruppen, dem leser einen schnellen überblick. alle drogen sind auf dem stand der aktuell gültigen arzneibücher. die autoren legen ein besonderes augenmerk auf praxisnähe und liefern viele hilfreiche tipps, die direkt für beratungsgespräche, z.b. in der apotheke, genutzt werden können. das lehrbuch ist vor allem für pharmazeutisch-technische assistenten/assistentinnen in der ausbildung sehr gut geeignet, um ihnen den einstieg in die pharmazeutische biologie zu erleichtern. dr. h. schulz e-mail: hartwig.schulz@jki.bund.de bibliografie: barbara eigner, holm/herbst ‒ botanik und drogenkunde, deutscher apotheker verlag, stuttgart, 10. aktualisierte und erweiterte auflage 2015, 261 seiten, 56 s/w abbildungen, 8 s/w tabellen, kartoniert, preis: 26,80 €, isbn: 978-3-7692-6247-6 journal of applied botany and food quality 86, 79 89 (2013), doi:10.5073/jabfq.2013.086.012 dipartimento di scienze agrarie, forestali e alimentari, università degli studi di torino medicinal plants, chemical composition and quality: may blackcurrant buds and blackberry sprouts be a new polyphenol source for herbal preparations? d. donno*, g.l. beccaro, m.g. mellano, a.k. cerutti, g. bounous (received june 18, 2013) * corresponding author summary it is well known that plants are important sources for the preparation of natural remedies as they contain many biologically active compounds: in particular, phenolic compounds are one of the most widely occurring groups of phytochemicals. some endemic species may be used for the production of herbal preparations containing phytochemicals with significant antioxidant capacities and health benefits: blackberry sprouts and blackcurrant buds are known to contain appreciable levels of phenolic compounds, including flavonols, phenolic acids and catechins. the aim of this research was to perform an analytical study of blackcurrant and blackberry bud-preparations, in order to identify the main bioactive polyphenolic compounds, to study the total polyphenolic content and to obtain a specific profile of the main polyphenols contained in these products using a high performance liquid chromatograph − diode array detector; the same analyses were performed both on the university lab preparations and on commercial preparations. different chromatographic methods were used to determine concentrations of phytochemical compounds in the preparations, allowing for quantification of statistically significant differences in their polyphenolic content both in the case of ribes nigrum and rubus ulmifolius. the assessment of chemical composition and bioactivities of the plant-derived products could help in find out new sources of natural antioxidants and other health-promoting compounds: only with the deep knowledge of the bioactive composition of plant preparations it will be possible to develop a new generation of standardized, effect-optimized, monoand multi-extract preparations. introduction plants are important sources for the preparation of natural remedies, food additives, and other ingredients, as they contain many biologically active compounds as polyphenols, vitamins (a, b6, c, e) and other very important phytochemicals. in particular, phenolic compounds are one of the most widely occurring groups of phytochemicals (dvaranauskaite et al., 2008; komes et al., 2011). in the plant kingdom, bioactive polyphenolic compounds can range from simple molecules, such as phenolic acids, to highly polymerized compounds, such as tannins. (tabart et al., 2011; tabart et al., 2012). these low molecular weight secondary plant metabolites exhibit excellent antioxidant properties. however, their particular mechanisms of action vary depending both on the structure and environment (komes et al., 2011). there is increasing evidence in the literature which indicates that secondary plant metabolites play critical roles in human health and may be nutritionally important. among the various medicinal and culinary herbs, some phenolics are ubiquitous compounds found in all plants as secondary metabolites (rababah et al., 2011; mattila et al., 2011; sakakibara et al., 2003). some endemic species are of particular interest because they may be used for the production of raw materials or preparations containing phytochemicals with significant antioxidant capacities and health benefits in the prevention of chronic diseases such as cancer, cardiovascular and neurodegenerative diseases (mattila et al., 2011; donno et al., 2012b; canterino et al., 2012); however, polyphenols may also have a negative effect on health because they form complexes with iron and therefore reduce the bioavailability of this essential element (santos-buelga and scalbert, 2000; donno et al., 2012a; moller et al., 2009). positive therapeutic effects may be related to their antioxidant activity as well their ability to regulate cellular activities of inflammation-related cells (mattila et al., 2011; tabart et al., 2012). however, since more than 5000 phytochemicals have been identified and different antiproliferation mechanisms have been reported, it is believed that synergistic or additive biological effects of multiple phytochemicals, rather than a single compound or a group of compounds, contribute to disease prevention. in this context, the diffusion and the use of herbal preparations to prevent some common diseases could be interesting (jia et al., 2012). the use of plant extracts as functional ingredients in various foods, medical and cosmetic applications is gaining growing interest among scientists, as well as among consumers and food manufacturers (komes et al., 2011). a variety of berries have been demonstrated to exhibit a broad spectrum of benefits: in particular, blackberry sprouts and blackcurrant buds are known to contain appreciable levels of phenolic compounds, including flavonols, phenolic acids and catechins (lugasi et al., 2011; cho et al., 2005). blackcurrant (ribes nigrum l.) is a shrub spontaneously growing in the cold and temperate climatic zones. the most important industrial product of black currant is fruits; however, leaves and buds, due to their characteristic chemical composition and excellent flavor, have also found some applications as a raw material for the herbal and cosmetic industries: many people use its buds as medicinal preparation for its anti-inflammatory activity and anti-dermal diseases (eczema and psoriasis) (dvaranauskaite et al., 2008). blackberry (rubus ulmifolius schott) is a rosaceae plant that grows in shrubs in temperate regions worldwide and the main importer is u.s.a., which is supplied from chile, costa rica, guatemala and mexico: sprouts have been used in traditional medicine for their many medicinal properties, as anti-inflammatory activity and antihaemorrhoids and diarrhoea activity (gudej and tomczyk, 2004; zuniga-hansen et al., 2010). in particular, bud and sprout preparations, derived from embryonic fresh plant tissues, are important therapeutic remedies, prescribed in hepatic, respiratory, circulatory and inflammatory disorders, but data on their chemical composition are lacking as, until now, phytochemical studies have principally been performed on barks, roots and root exudates, leaves, fruits and seeds (donno et al., 2012a; peev et al., 2007). the aim of this research was to perform an analytical study of blackcurrant and blackberry bud-preparations based on different genotypes, in order to identify the main bioactive polyphenolic 80 d. donno, g.l. beccaro, m.g. mellano, a.k. cerutti, g. bounous compounds, to quantify the total polyphenolic content and to obtain a specific profile of the main polyphenols contained in these products using a high performance liquid chromatograph − diode array detector; the same analyses were performed both on university lab preparations and on commercial preparations. materials and methods plant material samples of analysed species, ribes nigrum l. and rubus ulmifolius schott, were picked up in two years (2011 and 2012), in february (blackcurrant buds) and in may (blackberry young sprouts), in two germplasm repositories in turin province (italy), grugliasco (rubus ulmifolius) and san secondo di pinerolo (ribes nigrum). among the chosen species, different varieties were sampled, in order to test the genotype effect on the chemical composition of the final product (black currant: rozenthal and daniels; blackberry: kiowa, nightfall and a wild variety). buds and sprouts were used fresh to prepare herbal preparations; hplc samples were analysed after being stored for a few days at normal atmosphere (n.a.), at 4°c and 95% relative humidity (r.h.). commercial products from two different italian herbal companies were also analysed to compare their quality and to understand the effect of the utilization of different local plant materials on the final quality of the product: the two companies are located in san gregorio di catania (catania province, company 1), and predappio (forlì-cesena province, company 2). tab. 1 shows the genotypes, the sampling times and the picking sites of analyzed herbal preparations (university and company preparations). macerated sample preparation protocol the protocol of bud-preparations is detailed in the monograph “homeopathic preparations”, quoted in the french pharmacopoeia, 8th edition, 1965 (pharmaciens, 1965). bioactive compounds were extracted through a cold maceration process for 21 days, in a special solution of ethanol (95%) and glycerol, followed by a first filtration (whatman filter paper, hardened ashless circles, 185 mm ø), a manual pressing and, after two days of decanting, a second filtration (whatman filter paper, hardened ashless circles, 185 mm ø). macerated samples were then stored at n.a., at 4°c and 95% r.h. calibration standards all calibration standards were purchased from sigma aldrich (usa): chlorogenic acid, caffeic acid, ferulic acid, hyperoside, isoquercitrin, quercitrin, quercetin, gallic acid, ellagic acid, catechin and epicatechin. fig. 1 shows the chemical structures of the all detected polyphenolic compounds, divided by classes. quantitative determinations were performed using an external standard method. calibration curves in the 125 1000 mg/l range with good linearity (r2 > 0.998) for a four point plot were used to determine the concentration of polyphenolic compounds in budpreparation samples. stock solutions of cinnamic acids and flavonols with a concentration of 1.0 mg/ml were prepared in methanol: from these solutions, four calibration standards were prepared by dilution with methanol; stock solutions of benzoic acids and catechins with a concentration of 1.0 mg/ml were prepared in 95% methanol and 5% water: from these solutions, four calibration standards were prepared by dilution with 50% methanol-water. hplc analysis an agilent 1200 high performance liquid chromatograph, equipped with a g1311a quaternary pump, a manual injection valve and a 20 μl sample loop, coupled to an agilent gi315d uvvis diode array detector, was used for the analysis. the maceration solvents, ethanol and glycerol, were purchased from fluka biochemika (switzerland) and sigma aldrich (usa) respectively. analytic hplc grade solvents, methanol and formic tab. 1: species, genotype, sampling time and picking site of the analysed buds. university bud-preparations species genotype year germplasm repository ribes nigrum l. rozenthal 2011 san secondo di pinerolo 2012 daniels 2011 san secondo di pinerolo 2012 rubus ulmifolius schott nightfall 2011 grugliasco 2012 kiowa 2011 grugliasco 2012 wild variety 2011 grugliasco 2012 company bud-preparations species company year germplasm repository ribes nigrum l. company 1 2011 san gregorio di catania company 2 2011 predappio rubus ulmifolius schott company 1 2011 san gregorio di catania company 2 2011 predappio polyphenol characterization in berry fruit bud-preparations 81 81 fig. 1: chemical structures of the detected polyphenolic compounds. acid, were purchased from sigma aldrich (usa) and fluka biochemika (switzerland) respectively; potassium dihydrogen phosphate was also purchased from sigma aldrich (usa). all samples were analysed in triplicate (three repetitions for three plants for each sample), and all data are given in order to assess the repeatability of the used methods (standard deviation). two different chromatographic methods were used to analyse the macerated samples. the first method (a) was used for the analysis of cinnamic acids and flavonols; bioactive compound separation was achieved on a zorbax eclipse xdb – c18 column (4.6 x 150 mm, 5 μm), while the mobile phase consisted of methanol and a solution of 40 mm potassium dihydrogen phosphate in water. the flow rate was 1.0 ml min-1 (gradient analysis, 60 minutes) and the detector wavelength was 330 nm (peev et al., 2007; donno et al., 2012a). the second method (b) was used for the analysis of benzoic acids and catechins; bioactive molecules were separated on a zorbax eclipse xdb – c18 column (4.6 x 150 mm, 5 μm), while the mobile phase consisted of a solution of methanol/water/ formic acid (5:95:0,1 v/v/v) and a mix of methanol/formic acid (100:0,1 v/v). the flow rate was 1.0 ml min-1 (gradient analysis, 35 minutes) and the detector wavelengths were 250, 280 and 320 nm (donno et al., 2012a; moller et al., 2009). the detection limits (dl) and the quantification limits (ql) of the two chromatographic methods were calculated as the minimal concentration producing a reproducible peak with a signal-to-noise ratio greater than 3 and 10, respectively; tab. 2 shows the dl and the ql of the all considered compounds. accuracy was checked by spiking samples with a solution containing each phenolic compound in a concentration of 10 mg/ml. identification of polyphenolic compounds all single compounds were identified in samples by comparison of their retention times and uv spectra with those of standards in the same chromatographic conditions. total polyphenolic compounds (tpc) were determined as the sum of the most important classes of polyphenols present in the samples. four polyphenolic classes were considered: benzoic acids (gallic acid and ellagic acid), catechins (catechin and epicatechin), cinnamic acids (chlorogenic acid, caffeic acid and ferulic acid) and flavonols (hyperoside, isoquercitrin, quercitrin and quercetin). all results were expressed as mg per 100 g of buds fresh weight (fw). statistical analysis results were subjected to anova and t student test for mean comparison (spss 18.0 software) and hsd tukey multiple range test (p<0.05). principal component analysis (pca) was performed on the single polyphenolic concentration data. tab. 2: detection limits (dl) and quantification limits (ql) of the two used chromatographic methods for each calibration standard standard analysis method dl (mg/l) ql (mg/l) caffeic acid method a 1.23 4.11 chlorogenic acid 0.63 2.09 ferulic acid 1.01 3.37 hyperoside 0.55 1.83 isoquercitrin 0.48 1.58 quercitrin 1.07 3.57 quercetin 1.90 6.32 gallic acid method b 0.28 0.94 ellagic acid 1.88 6.27 catechin 1.75 5.85 epicatechin 1.75 5.83 82 d. donno, g.l. beccaro, m.g. mellano, a.k. cerutti, g. bounous results total polyphenolic content ribes nigrum l. regarding ribes nigrum, statistically significant differences were observed between the two varieties in both years, with a minimum tpc value of 427mg/100 gfw (rozenthal 2011) and a maximum of 834mg/100 gfw (daniels 2012); there were also significant differences in tpc in the same variety in two different years. in the commercial bud-preparations, there were statistically significant differences between the products of the two different companies with a maximum value of 856mg/100 gfw (company 2), a result similar to the mean value of cv daniels (2012 year) (fig. 2). rubus ulmifolius schott in the case of rubus ulmifolius, two statistically different groups were observed: the first one with four samples (kiowa 2011, kiowa 2012, wild variety 2011 and wild variety 2012) and the second one with two samples (nightfall 2011 and nightfall 2012); statistically significant differences were not observed between the two years in each analysed genotype: tpc ranged from a value of 211 mg/ 100 gfw (kiowa, 2012) to a value of 276 mg/100 gfw (nightfall, 2011). regarding the commercial bud-preparations, results showed statistically significant differences between the two companies with a maximum value of 384 mg/100 gfw (company 1), about 30 mg/100 gfw higher than company 2 (fig. 3). single polyphenolic profiles all data are reported in tab. 3 (blackcurrant buds) and 4 (blackberry sprouts). fig. 2: ribes nigrum: tpc in university bud-preparations (a) and company bud-preparations (b). different letters for each sample indicate the significant differences at p<0.05. fig. 3: rubus ulmifolius: tpc in university bud-preparations (a) and company bud-preparations (b). different letters for each sample indicate the significant differences at p<0.05. ribes nigrum l. blackcurrant samples showed the following polyphenolic composition: three cinnamic acids (chlorogenic acid, caffeic acid, ferulic acid), one flavonol (quercetin), one benzoic acid (gallic acid) and two catechins (catechin, epicatechin); in the cultivar daniels (2011 and 2012 years) chlorogenic acid was not detected. single polyphenolic concentration ranged from 4 mg/100 gfw (chlorogenic acid, rozenthal 2012) to 292 mg/100 gfw (catechin, daniels 2012) (fig. 4). statistically significant differences were observed among the university bud-preparation samples, while in commercial budpreparations there were significant differences only in catechin and epicatechin. multivariate analysis principal component analysis reduced variables into two principal components (82.78% of total variance) and divided samples in four groups, as the initial sample groups, confirming the statistically significant differences of anova test on tpc. pca variable graph showed a correlation among quercetin, ferulic acid, gallic acid and chlorogenic acid to pc1, while caffeic acid, catechin and epicatechin were in an intermediate position between pc1 and pc2 (fig. 5). rubus ulmifolius schott blackberry samples showed the following polyphenolic composition: two cinnamic acids (caffeic acid, ferulic acid), three flavonols (hyperoside, isoquercitrin, quercitrin) and two benzoic acids (gallic acid, ellagic acid): single polyphenolic concentration ranged from 5 mg/100 gfw (isoquercitrin, wild variety 2012) to 92 mg/100 gfw polyphenol characterization in berry fruit bud-preparations 83 83 m e t h o d a ci nn am ic a ci ds fla vo no ls (m g/ 10 0 gf w ) (m g/ 10 0 gf w ) sa m pl e na m e ch lo ro ge ni c ac id ca ffe ic a ci d fe ru lic a ci d hy pe ro si de is oq ue rc itr in qu er ci tr in qu er ce tin r ib es n ig ru m l . m ea n tu ke y h sd sd m ea n tu ke y h sd sd m ea n tu ke y h sd sd m ea n tu ke y h sd sd m ea n tu ke y h sd sd m ea n tu ke y h sd sd m ea n tu ke y h sd sd r oz en th al 2 01 1 6, 14 b 1, 56 7, 02 ab 0, 63 12 7, 51 a 10 ,7 2 nd / / nd / / nd / / 92 ,4 3 a 5, 99 d an ie ls 2 01 1 nd / / 12 ,7 3 c 2, 75 20 0, 82 b 64 ,9 7 nd / / nd / / nd / / 12 1, 66 c 11 ,4 9 r oz en th al 2 01 2 3, 94 a 1, 43 6, 07 a 1, 22 12 8, 17 a 26 ,2 1 nd / / nd / / nd / / 10 6, 28 b 13 ,1 8 d an ie ls 2 01 2 nd / / 8, 68 b 1, 00 20 5, 15 b 14 ,6 7 nd / / nd / / nd / / 12 5, 64 c 11 ,3 1 c om pa ny bu dpr ep ar at io ns m ea n tu ke y h sd sd m ea n tu ke y h sd sd m ea n tu ke y h sd sd m ea n tu ke y h sd sd m ea n tu ke y h sd sd m ea n tu ke y h sd sd m ea n tu ke y h sd sd c om pa ny 1 _b la ck cu rr an t 3, 50 a 1, 17 8, 04 a 1, 67 15 3, 03 b 11 ,2 8 nd / / nd / / nd / / 12 4, 03 a 8, 75 c om pa ny 2 _b la ck cu rr an t 3, 72 a 1, 29 8, 78 a 1, 19 17 7, 88 b 9, 81 nd / / nd / / nd / / 12 3, 95 a 17 ,3 4 m e t h o d b be nz oi c ac id s ca te ch in s (m g/ 10 0 gf w ) (m g/ 10 0 gf w ) sa m pl e na m e ga lli c ac id el la gi c ac id ca te ch in ep ic at ec hi n r ib es n ig ru m l . m ea n tu ke y h sd sd m ea n tu ke y h sd sd m ea n tu ke y h sd sd m ea n tu ke y h sd sd r oz en th al 2 01 1 16 ,7 2 a 1, 77 nd / / 12 1, 01 a 9, 42 56 ,0 5 a 4, 49 d an ie ls 2 01 1 62 ,1 9 b 5, 93 nd / / 17 9, 21 b 5, 78 55 ,7 0 a 3, 12 r oz en th al 2 01 2 20 ,4 2 a 3, 33 nd / / 24 4, 87 c 62 ,3 7 10 5, 60 b 26 ,6 1 d an ie ls 2 01 2 61 ,1 3 b 7, 27 nd / / 29 1, 78 c 55 ,6 5 14 1, 57 c 18 ,2 6 c om pa ny bu dpr ep ar at io ns m ea n tu ke y h sd sd m ea n tu ke y h sd sd m ea n tu ke y h sd sd m ea n tu ke y h sd sd c om pa ny 1 _b la ck cu rr an t 34 ,8 8 c 8, 12 nd / / 16 2, 56 a 9, 79 92 ,2 1 a 10 ,3 0 c om pa ny 2 _b la ck cu rr an t 34 ,0 3 c 4, 65 nd / / 31 8, 23 b 52 ,7 5 18 9, 74 b 7, 02 t ab . 3 : r ib es n ig ru m : s in gl e po ly ph en ol ic p ro fil e in u ni ve rs ity b ud -p re pa ra tio ns a nd c om m er ci al b ud -p re pa ra tio ns . d if fe re nt le tte rs f or e ac h sa m pl e in di ca te th e si gn ifi ca nt d if fe re nc es a t p <0 .0 5. 84 d. donno, g.l. beccaro, m.g. mellano, a.k. cerutti, g. bounous t ab . 4 : r ub us u lm ifo liu s: s in gl e po ly ph en ol ic p ro fil e in u ni ve rs ity b ud -p re pa ra tio ns a nd c om m er ci al b ud -p re pa ra tio ns . d if fe re nt le tte rs f or e ac h sa m pl e in di ca te th e si gn ifi ca nt d if fe re nc es a t p <0 .0 5. m e t h o d a ci nn am ic a ci ds fla vo no ls (m g/ 10 0 gf w ) (m g/ 10 0 gf w ) sa m pl e na m e ch lo ro ge ni c ac id ca ffe ic a ci d fe ru lic a ci d hy pe ro si de is oq ue rc itr in qu er ci tr in qu er ce tin r ub us u lm ifo liu s sc ho tt m ea n tu ke y h sd sd m ea n tu ke y h sd sd m ea n tu ke y h sd sd m ea n tu ke y h sd sd m ea n tu ke y h sd sd m ea n tu ke y h sd sd m ea n tu ke y h sd sd n ig ht fa ll 20 11 nd / / 7, 42 ab 2, 39 83 ,3 7 c 4, 40 33 ,8 8 c 2, 85 35 ,7 6 b 4, 41 19 ,7 1 a 2, 99 nd / / k io w a 20 11 nd / / 10 ,8 4 b 1, 77 52 ,6 0 b 10 ,9 0 26 ,0 0 ab 4, 53 8, 24 a 1, 83 20 ,6 1 a 3, 34 nd / / w ild v ar ie ty 2 01 1 nd / / 21 ,2 7 c 3, 58 33 ,2 0 a 6, 17 27 ,4 9 b 3, 12 5, 86 a 0, 87 31 ,9 1 b 6, 27 nd / / n ig ht fa ll 20 12 nd / / 6, 16 a 0, 82 86 ,3 3 c 3, 86 35 ,0 6 c 3, 96 34 ,0 1 b 3, 53 20 ,2 6 a 2, 87 nd / / k io w a 20 12 nd / / 9, 74 ab 1, 36 49 ,4 4 b 5, 66 21 ,6 6 a 2, 54 8, 06 a 1, 13 23 ,1 3 a 2, 40 nd / / w ild v ar ie ty 2 01 2 nd / / 22 ,7 8 c 4, 16 37 ,9 8 a 4, 46 24 ,9 7 ab 4, 17 5, 03 a 1, 25 33 ,0 8 b 3, 98 nd / / c om pa ny bu dpr ep ar at io ns m ea n tu ke y h sd sd m ea n tu ke y h sd sd m ea n tu ke y h sd sd m ea n tu ke y h sd sd m ea n tu ke y h sd sd m ea n tu ke y h sd sd m ea n tu ke y h sd sd c om pa ny 1 _b la ck be rr y nd / / 20 ,0 6 b 3, 90 42 ,6 5 a 4, 07 23 ,7 9 a 1, 00 6, 97 a 1, 80 35 ,3 0 b 4, 17 nd / / c om pa ny 2 _b la ck be rr y nd / / 22 ,2 0 b 2, 78 38 ,7 7 a 1, 76 30 ,8 8 b 4, 00 6, 89 a 1, 52 28 ,3 6 a 2, 44 nd / / m e t h o d b be nz oi c ac id s ca te ch in s (m g/ 10 0 gf w ) (m g/ 10 0 gf w ) sa m pl e na m e ga lli c ac id el la gi c ac id ca te ch in ep ic at ec hi n r ub us u lm ifo liu s sc ho tt m ea n tu ke y h sd sd m ea n tu ke y h sd sd m ea n tu ke y h sd sd m ea n tu ke y h sd sd n ig ht fa ll 20 11 17 ,1 3 ab 12 ,3 6 78 ,3 1 bc 13 ,0 4 nd / / nd / / k io w a 20 11 5, 52 a 1, 75 91 ,6 0 c 23 ,4 9 nd / / nd / / w ild v ar ie ty 2 01 1 46 ,5 4 d 20 ,5 0 70 ,3 7 ab c 18 ,9 8 nd / / nd / / n ig ht fa ll 20 12 19 ,9 2 ab c 2, 57 54 ,2 8 a 10 ,2 3 nd / / nd / / k io w a 20 12 33 ,1 1 cd 10 ,8 3 66 ,3 3 ab 11 ,5 1 nd / / nd / / w ild v ar ie ty 2 01 2 24 ,1 5 bc 6, 64 67 ,0 6 ab 13 ,5 3 nd / / nd / / c om pa ny bu dpr ep ar at io ns m ea n tu ke y h sd sd m ea n tu ke y h sd sd m ea n tu ke y h sd sd m ea n tu ke y h sd sd c om pa ny 1 _b la ck be rr y 26 ,1 6 b 4, 70 22 9, 49 b 19 ,8 8 nd / / nd / / c om pa ny 2 _b la ck be rr y 15 ,8 1 a 1, 89 20 8, 90 a 8, 67 nd / / nd / / polyphenol characterization in berry fruit bud-preparations 85 85 fig. 4: ribes nigrum: single polyphenolic profiles in university bud-preparations. fig. 5: ribes nigrum: pca individual and variable graph of university bud-preparation samples. (ellagic acid, kiowa 2011) (fig. 6). there were statistically significant differences among the university and company budpreparations samples, but in this second case differences were not observed in cinnamic acids and isoquercitrin. multivariate analysis principal component analysis was performed on all samples and it reduced the initial six groups into three groups overlapped with the genotypes: the kiowa and wild variety groups confirmed 86 d. donno, g.l. beccaro, m.g. mellano, a.k. cerutti, g. bounous fig. 6: rubus ulmifolius: single polyphenolic profiles in university bud-preparations. fig. 7: rubus ulmifolius: pca individual and variable graph of university bud-preparation samples. very similar tpc anova results. pca variable graph showed a correlation between cinnamic acids and flavonols to pc1 (54.91% of total variance) and a correlation between ellagic acid and pc2 (19.28% of total variance); gallic acid was in an intermediate position between pc1 and pc2 (fig. 7). commercial bud-preparations in fig. 8 single polyphenolic profiles showed a similar fitting between the two company bud-preparations, both in blackcurrant and blackberry: there was a substantial difference only in blackcurrant samples, with the company 2 showing an higher catechin value than company 1. polyphenol characterization in berry fruit bud-preparations 87 87 multivariate analysis pca were performed on company samples and two pc were extracted with a variance value of 92.85% of initial one: pca individual graph showed four groups similar to initial groups, but the company 1 and the company 2 data were in two very near groups; this consideration was observed both in blackcurrant and blackberry (fig. 8). discussion in the last few decades, many screening studies of different plant materials have been performed in order to find naturally occurring antioxidants for use in food or medicinal preparations, as replacements for potentially harmful synthetic additives (komes et al., 2011). it was reported that extracts from berries processing byproducts contained a high amount of phenolic compounds and possessed remarkable antioxidant activity (dvaranauskaite et al., 2008). the information on antioxidant properties of blackcurrant buds and blackberry sprouts is rather scarce. recently it was reported that buds and sprouts had an higher content in phenolics and antioxidants than fully ripened berries (peev et al., 2007; donno et al., 2012a; dvaranauskaite et al., 2008). reports on the analysis of phenolic acids (e.g. caffeic acid and its derivatives) by hplc coupled to diode array or mass detectors have been published. they describe phenolic acid determination in medicinal plants and preparations, as blackcurrant and blackberry bud-preparations (urpi-sarda et al., 2009; castro et al., 2010), according to single bioactive compound concentrations showed in this research. among other identified classes, flavonols and catechins were also selected for quantitative studies (huang et al., 2008; surveswaran et al., 2010; yi et al., 2009). the bud-preparations of the species analysed in this work are recommended by physicians to be consumed as polyphenol supplements, and further information on these compounds could be used to direct future research towards condition-specific beneficial properties associated with their therapeutic effects. berry plants with high levels of polyphenolic compounds are sought-after, especially if they have a long history of regular use that attests to their safety (donno et al., 2012a). as showed in this research, improvement of the raw plant material may be achieved by plant breeding and selection and the levels of bioactives can be increased to consistently high levels (joubert et al., 2008). it is well-known that chemical composition of secondary plant metabolites highly depends on such factors as climatic conditions, harvesting time, and plant genotype (dvaranauskaite et al., 2008; donno et al., 2012a) and the results of this research confirm this hypothesis: different species and varieties present different composition and concentrations of polyphenolic compounds, but it is also important to consider pedoclimatic characteristics of sampling sites strongly influence the presence of these molecules, as comparing the results of commercial bud-preparations. the results are highly variable depending on the genera: in ribes nigrum the sampling year influences the tpc of analysed varieties, while in rubus ulmifolius there are not differences between two years for each cultivar. fig. 8: company bud-preparations: single polyphenolic profiles and pca individual graph. 88 d. donno, g.l. beccaro, m.g. mellano, a.k. cerutti, g. bounous anova and pca results showed that the analyzed bud-preparation composition was similar in all the samples but the single compound concentrations were different; moreover, observing the chemical composition, results showed that few compounds were not detected in herbal medicines: chromatographic bioactive compound profiles could be applied in the differentiation of specific bud-preparations by other species (donno et al., 2012a; zhao et al., 2009). regarding chromatographic analysis, many good hplc methods exist for the separation and quantification of different polyphenolic groups found in various plant materials: in this research used methods showed that a good separation could be achieved by using common hplc solvents as mobile phase (methanol, water, formic acid, potassium dihydrogen phosphate solutions). the methods were sensitive and selective by using multiple wavelengths corresponding to the different uv-vis maximum absorptions of the different polyphenolic groups in buds and sprouts. the results indicated that the developed methods were feasible for comprehensive authentication and quality control of bud-preparations. certain polyphenolics can be used collectively as representative standards of a plant sample in quantification (tsao and yang, 2003), as done in this study, but these methods still allow quantification of individual compounds; such hplc data can be used as tpc for the quantification of bud or sprout phenolics. hplc methods give more information on individual compounds or groups of compounds (tpc) than the tpc by folin-ciocalteu method (gudej and tomczyk, 2004). knowledge of polyphenols molecular structure, composition and quantity is necessary to understand their role in determining potential health effects (hager et al., 2008): this study is only a preliminary research about bud-preparation chemical composition of two berryfruit species, in order to detect the most important polyphenolic classes and single compounds, but a further quantitative evaluation on the basis of their native structures with hplc coupled to mass spectrophotometry is necessary. conclusions the assessment of chemical composition and bioactivities of the plant-derived products could help in find out new sources of natural antioxidants and other health-promoting 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poirrier, p., soto, c., 2010: effect of solvent type on rubus ulmifolius (blackberry)’s phenolic antioxidant recovery. j. biotechnol. 150, supplement, 330. address of the corresponding author: dario donno, via leonardo da vinci 44, 10095 grugliasco (to), italy e-mail: dario.donno@unito.it journal of applied botany and food quality 87, 124 130 (2014), doi:10.5073/jabfq.2014.087.019 1 school of agricultural and food engineering, shandong university of technology, zibo, china 2 environment and plant protection institute, chinese academy of tropical agricultural sciences, danzhou, china responses of postharvest broccoli (brassica oleracea l. var. italica) florets to controlled atmospheres with varying co2/o2 levels at different temperature ling li1, zhengke zhang2, yanyin guo1*, binbin nian1 (received march 7, 2014) * corresponding author summary controlled atmospheres (ca) have been widely used in postharvest storage of fruits and vegetables. the gas compositions of common ca are consisted of o2, co2 and n2, postharvest storage condition with gas combination of o2 and co2 was rarely studied. in this research, the effects of ca with different co2/o2 levels, i.e. 70% o2 + 30 % co2, 60 % o2 + 40 % co2, 50 % o2 + 50 % co2, 40 % o2 + 60 % co2, 30 % o2 + 70 % co2, and air (control) on the storage life and properties of broccoli during storage were investigated at different temperatures including 0, 10 and 20 °c. results showed that the storage period of air treatments at 0, 10 and 20 °c was 21, 12, and 4d. treatments with 60 % o2 + 40 % co2 at 0 °c, 50 % o2 + 50 % co2 at 10 °c, and 40 % o2 + 60 % co2 at 20 °c maximum inhibited respiration rate and ethylene production, maintain chlorophyll and ascorbic acid content, reduced the accumulation of acetaldehyde and ethanol, and extended the storage life of broccoli florets to 49, 31, and 14d compared to 21 d at 0 °c, 12 d at 10 °c and 4 d at 20 °c in air-treated broccoli. these results indicated that an appropriate ca with o2/co2 might be a potential strategy for postharvest storage of broccoli heads, and the appropriate proportion of o2 and co2 might vary with different temperature. introduction broccoli (brassica oleracea l. var. italica) is a vegetable increasingly recognized as a nutritional source for vitamins, antioxidants, and anticarcinogenic compounds in the daily diet (podsedek, 2007). however, broccoli is a highly perishable product, its sensory quality after harvest encounters a rapid yellowing and undesirable off-flavor under inappropriate storage conditions (deschene et al., 1991). in recent years, various methods have been tested to extend the shelf life and improve postharvest quality of broccoli, such as refrigeration (toivonen, 1997), heat treatment (tetsuya et al., 2005), ethanol vapor (han, 2006), 1-methycyclopropene (1-mcp) application (yuan et al., 2010), modified atmosphere packaging (serrano et al., 2006), and controlled atmospheres (ca) (fernández-león et al., 2013). moreover, controlled atmosphere (ca) storage is also considered an effective technique for maintaining postharvest quality of broccoli (thompson, 2010), which inhibited respiration rate and ethylene production, delayed tissue turgidity, chlorophyll loss and undesirable compositional changes, reduced the incidence and severity of decay, and alleviated some physiological disorders (thompson et al., 2010; eason et al., 2007; toivonen et al., 2004; fernández-león et al., 2013). numbers of studies noted that appropriate proportions of o2 and co2 in ca condition of broccoli varied temperature, which demonstrated indicating that ca of broccoli is temperature-dependent (toivonen et al., 2004; cantwell et al., 1999; jones et al., 2006). for example, for fresh broccoli packaging storage, the recommended condition was 18.8 % o2 + 2.5 % co2 and 9.5 % o2 + 4 % co2 at 4 and 10 °c, respectively (annelie et al., 2004). in broccoli ca storage, the optimized gas concentration was 10 % o2 + 5 % co2 at 1-2 °c (fernández-león et al., 2013), and 2 % o2 + 6 % co2 at 4 °c (paradis et al., 1996). our previous study showed storage with higher concentrations of o2 and co2 (40 % o2 + 60 % co2) atmospheres at 15 °c prolonged storage life of broccoli by approximately 4 fold (guo et al., 2013). however, a combined effect of higher levels of o2/co2 and temperatures on storage life of broccoli has not been tested yet. the objective of this study was to establish the suitable ca conditions under different storage temperatures for extending storage life and maintaining quality of broccoli. materials and methods raw material two batches of broccoli florets (brassica oleracea l. var. italica) were harvested from field of shouguang vegetable station, shandong province on 12 april 2012 and 15 april 2013, respectively. healthy broccoli florets with diameter ranging from 13 to 15 cm were selected and stored in a walk-in cooler at 5 °c for pre-cooling for 4 h. experimental treatments for each temperature treatment (0, 10 and 20 °c), pre-cooled broccoli florets were divided into 6 groups with 30 broccoli heads per group, and each group divided into 3 parts for replications with 10 broccoli heads per replication. each group broccoli florets of 3 replicates were placed into three 0.5 m3 sealable plastic containers, respectively, and each group of broccoli heads was connected to a constant flow (0.05 m3 min-1) of air (control), 70 % o2 + 30 % co2, 60 % o2 + 40 % co2, 50 % o2 + 50 % co2, 40 % o2 + 60 %co2, and 30 % o2 + 70 % co2. the samples were stored at rh 80-90 % and 0, 10 and 20 °c, respectively, until the end of their storage periods. during storage, gas composition was regularly checked using an fbi-dansensor checkpoint o2/co2 (mr-07825-00, fbi-dansensor america inc.). the broccoli heads in each treatment during storage were randomly selected for evaluation of storage period and physiological attributes as described below. storage period the end of storage period of broccoli heads was designated as the time when approximately 30 % yellow coloration appeared on the surface of the product (yuan et al., 2010; xu et al., 2006) or offflavor occurred (jorge et al., 1995; dank et al., 1999). determination of respiration rate and ethylene production three broccoli heads from each treatment, were firstly kept at their storage temperature for 8 h after removal from ca conditions in order to eliminate interference of treatments on internal gases composition, and then individually sealed in a 3.86-l plastic container equipped with septa for 1 h at 0, 10, or 20 °c, according to their storage temperature. five milliliters of headspace gas was withdrawn from each container, and co2 and c2h4 levels were detected o2/co2 controlled atmospheres in broccoli 125 using a gas chromatograph (varian cp-3800, agilent technologies, lexington, ma, usa) equipped with a thermal conductivity detector (tcd, co2) and a hydrogen flame ionization detector (fid, ethylene). the temperature of column oven, tcd, fid and injector were set to 50, 130, 150 and 180 °c, respectively. respiration rate and ethylene production were expressed as mg kg-1 h-1 and μl kg-1 h-1, respectively. determination of acetaldehyde and ethanol contents acetaldehyde and ethanol levels were measured with static headspace gas chromatography (varian cp-3800, agilent technologies, lexington, ma, usa) following the method of ji et al. (2010) and the fid was set at 250 °c. acetaldehyde and ethanol contents were both expressed as mg kg-1. determination of chlorophyll content and yellowing rate chlorophyll content was measured according to moran (1982) with slight modifications. briefly, 0.1 g of broccoli florets were ground with 10 ml ethanol (95 %), and then extracted in the darkness for 24~36 h until broccoli tissue became white. the supernatant was measured at 652 nm with uv-1750 spectrophotometer (shimadzu). the total chlorophyll content was expressed as mg kg-1 fw. yellowing rate was measured according the surface yellow area of treated broccoli heads. ten broccoli heads from each treatment were pictured using canon camera (canon pc1468, canon inc., japan), and the average yellowing rate of each treatment was calculated using photoshop software (adobe photoshop cs5 software package). determination of ascorbic acid (aa) contents ascorbic acid (aa) content was analyzed with dinitrophenylhydrazine (dnph) method (terada et al., 1978). briefly, 0.5 g broccoli florets of each treatment was homogenated with 20 ml mixture acid (6 % metaphosphoric acid cantaining 2 mol l-1 acetic acid) and then centrifuged at 15,000 × g for 20 min. thereafter, the supernatant was filtrated through filter paper. one ml of filtrated supernatant was mixed with 0.05ml of 0.2 % 2, 6-dichlorophenolindophenol and then maintained at room temperature for 1 h. one ml of 2 % thiourea and 0.5 ml of 2 % dnph were added to the mixture and then heated at 60 °c for 3 h. 2.5 ml of 95 % h2so4 was added to the mixture and cooled on the ice prior to reading the absorbance at 540 nm. ascorbic acid content was expressed as g kg-1 fw. statistical analyses these experiments were conducted on a completely randomized design, and each treatment comprised three replicates. data were analyzed using spss 13.0 statistical software and significant differences among treatments were determined by least significant differences (lsd) test at p ≤ 0.05. results storage period combinations of o2/co2 prolonged the storage period of broccoli florets heads under different temperature (tab. 1). treatments of 60 % o2 + 40 % co2 at 0 °c, 50 % o2 + 50 % co2 at 10 °c, and 40 % o2 + 60 % co2 at 20 °c showed maximum efficacy, by which the storage periods of broccoli heads were extended to 49, 31, and 14 d, respectively, compared to the storage period of air treatments at 0 °c for 21 d, 10 °c for 12 d and 20 °c for 4 d. in common conditions, broccoli heads treated with higher o2 such as 70 % o2 + 30 % co2 behaved as yellow surface, while treated with higher co2 such as 30 % o2 + 70 % co2 behaved as off-flavors. respiration rate and ethylene production respiration rate in air and 70 % o2 + 30 % co2-treated broccoli heads rapidly increased with the peaks under 0, 10, and 20 °c appeared at 14, 8, and 2 d, respectively (fig. 1). among treatments, 60 % o2 + 40 % co2 at 0 °c, 50 % o2 + 50 % co2 at 10 °c, and 40 % o2 + 60 % co2 at 20 °c, strongly suppressed respiration rate, which was maintained at a relatively lower level compared to that in other treated broccoli. ethylene productions of all treatments were in parallel with the changes of respiration rate (fig. 2). broccoli treated with 60 % o2 + 40 % co2 at 0 °c, 50 % o2 + 50 % co2 at 10 °c, and 40 % o2 + 60 % co2 at 20 °c, showed a lowest and steady level of ethylene production. acetaldehyde and ethanol contents overall, acetaldehyde and ethanol contents in all broccoli florets showed a gradual increase trend with extension of storage time (fig. 3, fig. 4). differently, among all treatments, acetaldehyde and ethanol contents in broccoli florets treated with 60 % o2 + 40 % co2 at 0 °c, 50 % o2 + 50 % co2 at 10 °c, and 40 % o2 + 60 % co2 at 20 °c, showed a the lowest level during whole storage (fig. 3, fig. 4). chlorophyll content and yellowing rate chlorophyll contents of all treated broccoli florets declined during storage time, and lower temperature optimum delayed the chlorophyll loss (fig. 5). for example, during 0-14 d of storage time, the chlorophyll content of broccoli florets treated with 40 % o2 + 60 % co2 declined by 6.0 % compared to 13.8 % and 16.4 % at 0,10 and 20 °c, respectively. in general, the chlorophyll content of broccoli florets treated with 60 % o2 + 40 % co2 at 0 °c, 50 % o2 + 50 % co2 at 10 °c, and 40 % o2 + 60 % co2 at 20 °c, showed the lowest decline trend, compared with the other treatments at their respective storage temperature. yellowing rate of broccoli florets of all treatments showed a reverse trend of that of chlorophyll content (fig. 5 and 6). ascorbic acid (aa) contents changes of aa content of broccoli florets were familiar with chlorophyll content (fig. 5 and 7). among the treatments of their temperature, the least loss of aa content of broccoli florets was 60 % o2 + 40 % co2 at 0 °c, 50 % o2 + 50 % co2 at 10 °c, and 40 % o2 + 60 % co2 at 20 °c. furthermore, lower temperature could significantly delay the loss of aa content. during 0-14 d of storage time, the aa content of broccoli florets treated with 40 % o2 + 60 % co2 at 0, 10, and 20 °c, was declined by 5.7 %, 13.6 %, and 19.0 %, respectively. tab. 1: storage period of broccoli florets treated with varied o2/co2 concentration under different temperature. different little letters indicates that there is significant difference among treatments. storage period (d) treatments storage temperature (°c) 0 10 20 70 % o2 + 30 % co2 35c 8f 3e 60 % o2 + 40 % co2 49a 28b 8c 50 % o2 + 50 % co2 42b 31a 10b 40 % o2 + 60 % co2 35c 24c 14a 30 % o2 + 70 % co2 21e 20d 8c air 28d 12e 4d 126 l. li, z. zhang, y. guo, b. nian fig.1 fig.2 fig. 1: respiration rate of broccoli heads treated with varying o2/co2 levels at different temperature. vertical bars indicate the standard deviations for each treatment (n=5). air treatment was used as control. fig. 2: ethylene production of broccoli heads treated with varying o2/co2 levels at different temperature. vertical bars indicate the standard deat different temperature. vertical bars indicate the standard de-at different temperature. vertical bars indicate the standard dedifferent temperature. vertical bars indicate the standard deviations for each treatment (n=5). air treatment was used as control. discussion the optimal atmospheres proportion in ca storage of broccoli was different when storage temperature was diverse. it has been reported that the proper gas concentration was 10 % o2 + 5 % co2 at 1-2 °c (fernández-león et al., 2013), while combination of 2 % o2 and 6 % co2 was the most appropriate for broccoli preservation when stored at 4 °c (paradis et al., 1996). high o2 (30 %-80 %) has a favorable effect on preservation of fruits and vegetables, which could extend shelf life of ‘yaoshan’ pears (li et al., 2012), and high co2 could inhibit the accumulation of off-flavor substances during iceberg lettuce storage (heimdal et al., 1995). however, adverse effects also were found in other postharvest crops. for instance, 60-100 % o2 treatment accelerated the decay of chinese bay-berry, strawberry and blue berry (zheng et al., 2008), and increased the loss of green color and chlorophyll pigments (laxman et al., 2012). for high co2 treatments, studies of yasutaka (yasutaka et al., 1990) showed that treatment with 20 % o2 + 60 % co2 + 20 % o2/co2 controlled atmospheres in broccoli 127 fig.3 fig.4 n2 for 24 h significantly reduced respiration rate and ethylene production, and delayed off-flavor and decay in broccoli, tomato, and peach. however, kader (1995) pointed out that inappropriate high co2 might result in co2 injury or physiological disorder such as offflavor, which involved in the accumulation of acetaldehyde, ethanol and a tiny part of other volatiles (james et al., 2000; ke et al., 1990). present study exhibited that high proportion of o2/co2 such as 70 % o2 + 30 % co2 accelerated chlorophyll loss and yellowing. on the other side, extreme high co2 in ca condition such as 30 % o2 + 70 % co2 rapidly accumulated acetaldehyde and ethanol, indicating that inappropriate high co2 could result in off-flavor of broccoli florets. only appropriate combinations of o2 and co2 including 60 % o2 + 40 % co2 at 0 °c, 50 % o2 + 50 % co2 at 10 °c, and 40 % o2 + 60 % co2 at 20 °c, provided the most effective ways to maintain quality and reduce off-flavor, as indicated by reductions in chlorophyll loss and the acetaldehyde and ethanol. results implied that the similar proportioning with high levels of o2 and co2 in ca storage under a wide range of temperature (0-20 °c) might be an appropriate strategy for broccoli storage. in conclusion, the optimum proportion of o2 and co2 to the storage fig. 3: acetaldehyde contents of broccoli heads treated with varying o2/co2 levels at different temperature. vertical bars indicate the standard deat different temperature. vertical bars indicate the standard de-at different temperature. vertical bars indicate the standard dedifferent temperature. vertical bars indicate the standard deviations for each treatment (n=5). air treatment was used as control. fig. 4: ethanol contents of broccoli heads treated with varying o2/co2 levels at different temperature. vertical bars indicate the standard deviations for each treatment (n=5). air treatment was used as control. 128 l. li, z. zhang, y. guo, b. nian fig.5 fig.6 and of broccoli varied with temperature. the best treatments of o2/ co2 were found to be 60 % o2 + 40 % co2 at 0 °c, 50 % o2 + 50 % co2 at 10 °c, and 40 % o2 + 60 % co2 at 20 °c. these treatments could inhibit respiration rate and ethylene production, maintain chlorophyll and ascorbic acid content reduce the accumulation of acetaldehyde and ethanol, and extended the shelf life of broccoli to 49, 31, and 14, respectively. furthermore, temperature played a critical factor on the preservation period of broccoli florets, with treatment of 60 % o2 + 40 % co2 at 0 °c showed the best efficacy in extending 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yasutaka, k., akitsugu, i., reinosuke, n., 1990: respiration and c2h2 production in various harvested crops held in co2-enriched atmospheres. j. am. soc. hortic sci. 115, 975-978. yuan, g.f., sun, b., yuan, j., wang, q.m., 2010: effect of 1-methylcyclopropene on shelf life, visual quality, antioxidant enzymes and healthpromoting compounds in broccoli florets. food chem. 118, 774-781. zheng, y.h., yang, z.f., chen, x.h., 2008: effect of high oxygen atmospheres of fruit decay and quality in chinese bayberries, strawberries and blueberries. food control. 19, 470-474. address of the corresponding author: e-mail: guoyy@sdut.edu.cn. journal of applied botany and food quality 87, 131 138 (2014), doi:10.5073/jabfq.2014.087.020 1 centro de pomáceas, facultad de ciencias agrarias, universidad de talca, talca, chile 2 laboratorio de plantas aromáticas, instituto de química de recursos naturales, universidad de talca, talca, chile 3 departamento de bioquímica clínica e inmunohematología, facultad de ciencias de la salud, universidad de talca, talca, chile 4 interdisciplinary excellence research program on healthy aging (piei-es), universidad de talca, talca, chile total phenol and quercetin content and antioxidant activity in apples in response to thermal, light stress and to organic management josé antonio yuri1, 4*, amalia neira1, 4, francisco maldonado, álvaro quilodrán1, daniela simeone1, iván razmilic2, 4, iván palomo3, 4 (received february 3, 2014) * corresponding author summary flavonoids are the most abundant phenol compound group in apples, the concentration of which varies with the cultivars and climatic conditions. the objective of this study was to evaluate the effects of temperature, solar radiation, sunburn damage of the peel and the state of development of fruit on total phenol concentrations, quercetin glycosides and antioxidant activity. three assays were conducted during the 2008/09 season to evaluate aforementioned variables on these parameters. the following season, the effect of the state of development on the fruit was evaluated. sunburn increased phenol concentrations from 5.5 to 8.7 mg cae* g fw-1. in relation to the state of development of the fruit, phenol concentrations decreased from 14 to 1.3 mg cae* g fw-1 between 32 dafb to harvest, respectively. fruit that was bagged until one month before harvest had significantly higher concentrations of quercetin rutinoside (28 mg*g-1fw), galactoside (484 mg*g-1fw) and glucoside (54 mg*g-1fw) than fruit that remained bagged until harvest (6, 161 and 21 mg*g-1fw, respectively). temperature did influence phenol concentrations. this study determined that sunburn, the state of development and bagging the fruit are factors that determine phenol concentration in apples. introduction the apple (malus domestica borkh.) is a temperate-cold climate zone cultivar that has nevertheless been adapted to a wide variety of environmental conditions (feree, 2003). in chile, with its mediterranean climate, apple production is mainly concentrated between 34.5° and 38.4° latitude south (gil, 2000), covering an area of 37,200 hectares (odepa – ciren, 2007). it is recognized that apples and in particular the peel have high concentrations of phenol compounds, which result in a significant level of antioxidant activity, which is associated with reducing the risks of developing chronic non-transmissible diseases (e.g. cancer and cardiovascular illnesses) (steinmetz and potter, 1996; nakamura et al., 2008; liu, 2004). the phenol content of the fruit is influenced by a number of factors, among them the cultivar (henriquez et al., 2010; van der sluis, 2001), the state of development (labbe et al., 2010; kondo et al., 2002), tissue (peel or pulp) (yuri et al., 2009), management (conventional and organic) (asami et al., 2003; stracke et al., 2009) mineral nutrition of the plant (treutter, 2010), the agroclimatic region (yuri et al., 2009), refrigerated storage (martinez et al., 2002), light and temperature (dixon and paiva, 1995; martinez et al., 2002). flavonoids are the most abundant phenol compound group in apples (dueñas et al., 2011), among them notably are quercetin glycosides, which make a major contribution to total antioxidant activity (dueñas et al., 2011; lee et al., 2003). this study evaluated the effects of temperature, light, sunburn and the state of development of the fruit on phenol compound concentrations, quercetin glycosides and antioxidant activity in two cultivars under the agro climatic conditions of central chile. materials and methods assay 1. effect of temperature on the peel the effects of thermal stress on fruit of cv. fuji were evaluated in the 2008/09 at the panguilemo experimental station of the universidad de talca (35º 23’l.s., 71º 40’l.w., 105 m.a.s.l). the trees used were grafted in 2003 onto emla 9 rootstock planted at 4.0 m x 2.0 m. measurements were taken 150 days after full bloom (dafb). the measurements were taken from three branches of each sampled tree: the first submitted to 35 °c, the second to 45 ºc and the third as a control with ambient temperature. in the case of the first two branches, the exposure time was 5 hours, between 10:00 am and 3:00 pm. representative fruit were collected at 24 hours post-stress. to control the temperature of the treatments the branches were wrapped in plastic film for greenhouse. thermocouples were installed in the plastic wrapping and connected to a computer and thermal ventilator by means of an arduino microcontroller board. eight apples were collected for each treatment and the control (four replications with 2 fruit each) from which peel was obtained for the measurements. assay 2. sunburn effect a) different levels of damage during the commercial harvest of 2008/09 season, cv. granny smith apples, were collected from a commercial orchard located in los niches, maule region, chile (35°0’s, 71°08’w; 299 m.a.s.l.). undamaged (healthy) and different grade of damage peel fruit (mild and moderate) were collected and analyzed. b) in conventional (co) and organic orchards (oo) during the 2009/10 season apples cvs. fuji (raku raku and striped) and granny smith were collected from conventional and organic orchards located in chimbarongo, o’higgins region, chile (34° 40’s, 71° 1’w; 314 m.a.s.l.). the conventional and organic orchards were 10 km apart. the measurements were made at commercial harvest (191 and 159 dafb, respectively) using peels of healthy and moderately sunburnt fruit, with four replications with four fruit in each for each evaluated cultivar. assay 3. influence of the growth stages of the fruit healthy cvs. fuji and granny smith apples at different stages of development were collected during the 2009/10 season (25, 32, 39, 52 and 88 dafb), and commercial harvest (191 and 159 dafb, respectively), from the conventional and organic orchards described in assay 2b. the measurements were made with the entire fruit (peel and flesh). sixteen fruit were randomly selected for each stage for four replications with four fruit in each replication. 132 j.a. yuri, a. neira, f. maldonado, á. quilodrán, d. simeone, i. razmilic, i. palomo assay 4. effect of bagging in the commercial harvest of 2008/09 season, cv. fuji apples, unbagged (control), bagged until one month before harvest (commercial bagging) and bagged until harvest, were collected from a commercial orchard located in san clemente (35°30’s, 71°28’w; 83 m.a.s.l). the determinations were made with peel from fruit, with four replications of 2 apples in each replication. the apples were covered with double-layer paper bags (qingdao keentop paper enterprise ltd., china). the outer bags were blue on the outside and black on the inside while the inner bags were blue. tissue extraction assay 1: a hole punch was used to obtain disks of 95 mm2 in area and 5 mm deep until reaching one gram of peel. assays 2 and 4: 1 and 2 g of peel were obtained from the equatorial zone of the apple for assays 2 and 4, respectively. assay 3: the apples were cut into lengthwise slices until reaching one gram of tissue (peel and flesh). the seeds and core were previously removed. the tissues were frozen with liquid nitrogen, pulverized and homogenized in a mortar and pestle. the method for extraction described by coseteng and lee (1987) was used with some modification. briefly, the tissue was extracted twice with a solution of ethanol at 80 % (ethanol: water 80:20, v/v) for 10 and 5 minutes at 100 °c. subsequently, it was filtered, graduated at 10 ml with ethanol at 80 % and stored at -20 °c until use. total phenol content total phenol content was determined by the folin-ciocalteu method described by coseteng and lee (1987). briefly, 0.1 ml of the extract was mixed with 0.5 ml of the folin-ciocalteu phenol reagent (merck, darmstadt, germany). the mixture was incubated for five minutes and then 0.5 ml of sodium carbonate (na2co3; 10 %, w/v) added and incubated for 15 minutes at room temperature (20 °c). absorbance was measured at 640 nm. total phenol concentrations were expressed as mg of chlorogenic acid equivalents * g fw-1. antioxidant activity the capture of the free radical 2,2-diphenyl-1-picrylhydrazyl (dpph; fluka chemie, buchs, switzerland) was measured by the method described by von gadow et al. (1997), with modifications. briefly, 0.01 ml of each extract was used, adjusted to a final volume of 0.5 ml with 80 % ethanol. they were then mixed with 2 ml of dpph 8x10-5 m solution and incubated for 8 minutes at room temperature. their absorbance was measured at 515 nm using ethanol as blank. chlorogenic acid in different concentrations was used as a standard and the capture of the dpph free radicals was expressed as mg of chlorogenic acid equivalents * g fw-1. determination of quercetin glycosides by hplc the determination of quercetin glycosides in the samples was performed using hplc-dad merck hitachi (lachrom, tokyo, japan), equipment, which consisted of a lachrom l-7100 pump and a diode array detector, l-7455 lachrom, and a 100-5 c18 kromasil column of 259x4.6 mm with a pre-column of the same characteristics, maintained at 20 °c. briefly, 0.02 ml, previously filtered (0.45 μm filter), were injected. to identify the compounds, different standards of quercetin glycosides were used with the uv-vis spectra. the chromatogram was monitored at 300 nm. the solvents of the mobile phase were: a: formic acid 1 % in h2o quality hplc; b: acetonitrile 40 % in h2o, and c: acetonitrile. the elution used was: time 0-10 min: a (70), b (30), c (0) flow 1ml * min-1; time 45 min: a (25), b (75), c (0) flow 0.5 ml*min-1; time 52 min: a (0), b (0), c (100) flow 1 ml*min-1; and time 55 min: a (70), b (30), c (0) flow 1 ml*min-1. the results were expressed in mg equivalents of quercetin glycosides* 1 g of fw-1 (yuri et al., 2010). statistical analysis the assay was carried out with a completely random design. an anova was applied for the statistical analysis and separation of means, using the spss v15.0 computer program (spss inc., chicago, illinois). tukey’s hsd test was used to compare treatments when significant differences were found (p≤0.05). results assay 1 in cv. fuji exposure to high temperatures had no effect on total phenolic compounds concentrations and antioxidant activity, with values ranging from 4.5 to 5.3 mg cae* g-1 fw and from 3.9 to 4.7 mg cae* g-1 fw, respectively (tab. 1). quercetin glycoside concentrations in the peel of fuji apples exposed to 35 and 45 °c were not affected by the high temperature (tab. 2). tab. 1: effect of temperature in phenolic concentration and antioxidant activity in peel of fuji apple. total phenolic antioxidant activity (mg cae* g-1 fw) (mg cae* g-1 fw) initial condition 4.13 a 3.54 a 35 ºc x 5 h 4.58 a 3.96 a 35 ºc x 24 h 4.71 a 4.65 a 45 ºc x 5 h 4.00 a 3.19 a 45 ºc x 24 h 5.31 a 4.40 a significance n.s n.s statistically significant differences between treatments at p≤ 0.05 (tukeyʼs test) are expressed with *. significance: * p≤ 0.05; ** p≤ 0.01: n.s, no significance. assay 2 a tissue of cv. granny smith apples damaged by sunburn had higher concentrations of phenolics compounds (8.7 mg cae* g fw-1) than tissue of healthy apples (5.5 mg cae* g fw-1) (p= 0.001), as well as higher levels of antioxidant activity (7.4 mg cae* g fw-1) compared to healthy peel (5.2 mg cae* g fw-1) in healthy tissue, although the differences were not significant. concentrations of quercetin rutinoside, galactoside and glucoside in solar damaged tissue (tab. 3) were 6 to 20 times as high as in healthy peel, while quercetins xyloside, arabinoside and rhamnoside were 3 times as high as in healthy tissue. assay 2 b moderate sunburn significantly increased total phenol concentration (p < 0.01) in the damaged tissue of both conventionally and organically grown fruit, with values ranging from 7.3 to 16.3 mg cae* g fw-1. a similar tendency was observed with antioxidant activity, which increased until reaching ranging between 8.4 and 13.8 mg cae* g fw-1 for both management systems and cultivars (tab. 4). apple responses to thermal, light stress and organic management 133 as a result of sunburn there were increases in the concentrations of all types of quercetins in both cultivars and both management approaches (tab. 5). in cv. granny smith, quercetin rutinoside and galactoside were the quercetins that increased most, followed by glucoside, xyloside, arabinoside and rhamnoside. fuji showed a similar tendency, although with increases of lower magnitude. there were no differences between the two types of management. assay 3 in studying the evolution of total phenols and antioxidant activity during the growth stages of the fruit of cvs. granny smith and fuji, we observed that phenol concentrations increased from 9 mg cae* g fw-1 to approximately 14 mg cae* g fw-1 from 25 to 32 dafb and then began to decrease until harvest, with values ranging from 1.3 to 2.6 mg cae* g fw-1. phenol content, in contrast, increased throughout the development period, from 22-35 mg cae* fruit-1 to 287-401 mg cae* fruit-1 (tab. 6). as with total phenol concentrations, antioxidant activity increased from 25 to 32 dafb and then began to decrease until harvest (tab. 6), with values ranged between 1.5 and 2.4 mg cae* g fw-1. the antioxidant activity of the whole fruit increased throughout the growth period, from 17-32 mg cae* fruit-1 to 337-380 mg cae* fruit-1 at harvest. quercetin glycoside concentrations in both granny smith and fuji apples during fruit development increased until 32 dafb and then tab. 2: concentration of quercetin glycosides in peel cv. fuji exposed to 35 °c and 45 °c (150 dafb). quercetin type (mg* g-1 fw) rutinoside galactoside glucoside xyloside arabinoside rhamnoside initial condition 0.009 a 0.275 a 0.041 a 0.097 a 0.145 a 0.119 a 35 ºc x 5 h 0.012 a 0.398 a 0.056 a 0.139 a 0.210 a 0.158 a 35 ºc x 24 h 0.016 a 0.382 a 0.073 a 0.128 a 0.200 a 0.145 a 45 ºc x 5 h 0.012 a 0.309 a 0.052 a 0.112 a 0.167 a 0.141 a 45 ºc x 24 h 0.022 a 0.493 a 0.078 a 0.157 0.238 a 0.170 a significance n.s n.s n.s n.s n.s n.s statistically significant differences between treatments at p≤ 0.05 (tukeyʼs test) are expressed with *. significance: * p≤ 0.05; ** p≤ 0.01: n.s, no significance. tab. 3: concentration of quercetin glycosides in healthy peel and moderate sunburn damage. quercetin type (mg* g-1 fw) rutinoside galactoside glucoside xyloside arabinoside rhamnoside healthy 0.03 b 0.30 b 0.13 b 0.14 b 0.17 b 0.13 b mild sunburn 0.46 a 1.57 a 1.21 a 0.31 a 0.52 a 0.33 a moderate sunburn 0.60 a 1.94 a 1.54 a 0.31 a 0.55 a 0.34 a significance ** ** ** ** ** ** statistically significant differences between treatments at p≤ 0.05 (tukeyʼs test) are expressed with *. significance: * p≤ 0.05; ** p≤ 0.01: n.s, no significance. tab. 4: total phenol concentration and antioxidant capacity in healthy peel and moderate sunburn damage. cultivars management total phenolic antioxidant activity (mg cae* g-1 fw) (mg cae* g-1 fw) health 5.5 b 5.1 b conventional damage 16.3 a 13.8 a granny significance ** ** smith health 5.7 b 4.1 b organic damage 12.9 a 10.3 a significance ** ** health 4.8 b 5.6 b conventional damage 7.8 a 8.4 a fuji significance ** ** health 4.1 b 5.3 b organic damage 7.3 a 9.3 a significance ** ** statistically significant differences between treatments at p≤ 0.05 (tukey`s test) are expressed with *. significance: * p≤ 0.05; ** p≤ 0.01: n.s, no significance. 134 j.a. yuri, a. neira, f. maldonado, á. quilodrán, d. simeone, i. razmilic, i. palomo tab. 5: concentration of quercetin glycosides in cv. granny smith with different sunburn damage level. cultivars management rutinoside galactoside glucoside xyloside arabinoside rhamnoside health 0.004 b 0.065 b 0.015 b 0.027 b 0.062 b 0.043 b conventional damage 0.543 a 2.190 a 1.756 a 0.351 a 0.686 a 0.477 a granny significance ** ** ** ** ** ** smith health 0.003 b 0.051 b 0.013 b 0.026 b 0.049 b 0.038 b organic damage 0.527 a 2.162 a 1.574 a 0.305 a 0.593 a 0.379 a significance ** ** ** ** ** ** health 0.027 b 0.354 b 0.057 b 0.114 b 0.213 b 0.131 b conventional damage 0.251 a 1.619 a 0.470 a 0.321 a 0.542 a 0.259 a fuji significance ** ** ** ** ** ** health 0.031 b 0.337 b 0.054 b 0.104 b 0.170 b 0.090 b organic damage 0.279 a 1.500 a 0.496 a 0.244 a 0.449 a 0.218 a significance ** ** ** ** ** ** statistically significant differences between treatmeans at p≤ 0.05 (tukeyʼs test) are expressed with *. significance: * p≤ 0.05; ** p≤ 0.01: n.s, no significance. tab. 6: evolution of total phenolic concentration, total phenolic content, antioxidant activity in extracts and antioxidant activity in whole fruit from apples cvs. granny smith and fuji , from conventional and organic orchards, in different stages of development, during the 2009/2010 season. management dafb total total antioxidant activity antioxidant activity phenolic concentration phenolic content in extract in whole fruit (mg cae* g-1 fw) (mg cae* fruit-1) (mg cae* g-1 fw) (mg cae* fruit-1) 25 9.4 27 8.4 24 32 14 78 9.6 54 conventional 39 10 118 8.1 93 52 7.9 141 6.8 125 88 4.5 256 3.7 217 granny 159 2.6 401 2.4 380 smith 25 13 35 12 32 32 14 81 9.4 53 organic 39 13 143 8.4 96 52 11 172 8.2 131 88 3.7 235 3.1 197 159 2.4 439 2.1 381 25 9.4 22 7.2 17 32 14 67 9.2 46 conventional 39 11 88 9.9 83 52 8.5 138 8.6 126 88 4.7 282 2.9 212 fuji 191 1.3 326 1.5 337 25 12 26 9.6 19 32 15 62 9.9 41 organic 39 13 122 9.9 96 52 9.2 175 8.6 164 88 3.5 225 2.9 186 191 1.5 287 1.5 340 began to decrease until harvest (fig. 1 and 2). at the same time, quercetin glycoside content increased during the season, from 0.5 mg* g fw-1, until 0.5 to 6 mg* g fw-1, depending on the type. assay 4 the phenols quantity and the antioxidant activity level were slightly lower (3.9 mg cae* g fw-1 and 3.4 mg cae* g fw-1 respectively) in fruit that were bagged until harvest compared to fruit that were bagged until one month before harvest, the latter with values of 5.4 and 4.2 mg cae* g fw-1, respectively (tab. 7). the concentrations of quercetins rutinoside (28 mg*g-1 fw), galactoside (484 mg*g-1 fw) and glucoside (54 mg*g-1 fw) were significantly higher with bagging until one month before harvest compared to levels with apples that remained bagged until harvest (6, apple responses to thermal, light stress and organic management 135 fig. 2: evolution of quercetins glycosides concentration and content in whole fruit from apple cv fuji, from conventional (a and b) and organic (c and d) orchards, in different stages of development, during 2009/2010 season. curves end at harvest. q.rut, rutinoside; q.gal, galactoside; q. glu, glucoside; q. xil, xyloside; q. ara, arabinoside; q.rha, rhamnoside. fig. 1: evolution of quercetins glycosides concentration and content in whole fruit from apple cv. granny smith, from conventional (a and b) and organic (c and d) orchards, in different stages of development, during 2009/2010 season. curves end at harvest. q.rut, rutinoside; q.gal, galactoside; q.glu, glucoside; q.xil, xyloside; q.ara, arabinoside; q.rha, rhamnoside. 136 j.a. yuri, a. neira, f. maldonado, á. quilodrán, d. simeone, i. razmilic, i. palomo 161 and 21 mg*g-1 fw, respectively). the values observed in the unbagged fruit (controls) were similar to those of bagged fruit until one month before harvest (tab. 8). discussion the present study evaluated the effects of temperature, radiation, sunburn and the state of development of the fruit on total concentrations of phenols and quercetin glycosides, as well the levels of antioxidant activity in granny smith and fuji apples. no significant increases in total phenolic compounds and quercetin glycoside concentrations or levels of antioxidant activity were observed with higher temperatures. sunburn is a physiological disorder caused by the fruit being exposed to excessive levels of temperature and solar radiation (wünsche et al., 2004; racsko and schrader, 2012), which induces photo and thermal protective mechanisms in the fruit tissue, expressed as higher levels of antioxidant activity and the activity of certain enzymes (leja et al., 2003; lambers et al., 2008). this coincides with the results of this study, in which total phenol, quercetins and antioxidant activity were higher in damaged tissue. felicetti and schrader (2009) had similar results and observed that the concentrations of chlorogenic acid and quercetin glycosides were lower further away from the area of damaged tissue. yuri et al. (2010) observed that total phenol, flavonol concentrations and levels of antioxidant activity were higher in apples with sunburn in both seasons. total phenolic compounds concentration, quercetins and antioxidant activity decreased during the growth period of the both conventionally and organically grown, while the content and activity of these compounds at the level of the entire fruit presented a tendency to increase. these results concur with observations described previously by other authors (mayr et al., 1995; hamauzu et al., 1999; wang and lin, 2000). takos et al. (2006) observed that the concentration of flavonols is high during the first stages of the development of the fruit (32 dafb) and then decreases during development and maturation. renard et al., (2007) determined that the concentrations of hydroxycinnamic acids and flavanols reached their maximum value during the period of cellular division and then decreased abruptly until the end of the season. this is because the activity of phenylalanine ammonia lyase (pal) and other enzymes related to the biosynthesis of phenols reach their peak in the stage of cell division (ju et al., 1995; lister and lancaster, 1996; treutter, 2001) until 30 dafb and then decrease sharply until the stage of cellular expansion. according to renard et al. (2007), once the strong initial synthesis of phenol compounds is detained, the decrease in phenolic compounds concentrations is due to the growth of the fruit. thus, although the concentration of phenols decreases, phenol content in the fruit increases, which is in agreement with what was observed by awad et al. (2001). bagging is a used agricultural practice in some countries to protect cv. fuji fruit from pests and pathogens, as well as preventing sunburn. however, the most important motive for this practice is its positive effect on the color of the fruit. removing the bag promotes the synthesis of anthocyanins in tissue that lacks chlorophyll (chen et al., 2012). however, re-exposure to radiation increases the level not only of anthocyanins, but also of other phenol compounds. ju et al. (1995) observed that when the fruit is unbagged the activity of the pal enzyme and simple phenol concentrations of anthocyanins and flavanoids increases to levels similar to those reached by the fruit during the first development stages. this evidences that the precursor pal enzyme and some phenol compounds are dependent on light (lancaster, 1992; ju et al., 1995; ju, 1998; awad et al., 2000; oh et al., 2009). gene expression and enzyme activity related to the metabolism of phenol compounds are equally regulated by light (ju et al., 1995; takos et al., 2006; qian et al., 2013). although the fruit that were bagged until harvest did not develop red pigmentation (anthocyanins), they did have similar phenolic compounds content and levels of antioxidant activity to those of unbagged and commercially bagged fruit, which to some extent contradicts what has been indicated in terms of the need for direct solar radiation for the synthesis of phenolic compounds, opening the possibility that the these are transported to the fruit from adjacent leaves. perkins et al. (2009) and kūka et al. (2010) determined the presence of catechins, kaempferol, and quercetins in the sap of betula pendula and acer platinoides. this is supported by ju (1998), who observed that bagged apples developed simple phenols, procyanidins and quercetin glycosides. in contrast, chen et al. (2012) observed that bagging fruit reduced the concentrations of anthocyanins, hydroxycinnamic acids and flavanols in the peels of the three apple cultivars. consequently, the theme should be studied in greater amplitude and depth. tab. 8: effect of bagging in quercetin glycosides of cv. fuji. efecto del embolsado en la concentración de quercetinas glicosiladas en piel de cv. fuji. quercetin type (mg* g-1 fw) rutinoside galactoside glucoside xyloside arabinoside rhamnoside unbagged 25 a 320 ab 65 a 103 a 187 a 102 a normal bagging 28 a 484 a 54 ab 92 a 187 a 114 a bagging until harvest 6 b 161 b 21 b 61 a 122 a 82 a significance ** ** * n.s n.s n.s statistically significant differences between treatments at p≤ 0.05 (tukeyʼs test) are expressed with *. significance: * p≤ 0.05; ** p≤ 0.01: n.s, no significance. tab. 7: effect of bagging in total phenol concentration and antioxidant activity in cv. fuji peel. total phenolic antioxidant activity (mg cae* g-1 fw) (mg cae* g-1 fw) unbagged 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631-636. address of the corresponding author: dr. josé antonio yuri, centro de pomaceas, faculty of agricultural sciences, universidad de talca, talca, chile; p.o. box: 747, talca, chile e-mail: ayuri@utalca.cl journal of applied botany and food quality 87, 139 146 (2014), doi:10.5073/jabfq.2014.087.021 institute of food science and biotechnology, chair plant foodstuff technology, hohenheim university, stuttgart, germany processing and storage of innovative pasty parsley (petroselinum crispum (mill.) nym ex a. w. hill) and celeriac (apium graveolens l. var. rapaceum (mill.) dc.) products andrea kaiser*, ariane v. jaksch, klaus mix, dietmar r. kammerer, reinhold carle (received february 11, 2014) * corresponding author summary a process for the production of innovative pasty parsley and celeriac products was developed. freshly harvested plant material was blanched, processed into a paste, and subsequently heated for 3 min at 90 and 95 °c, respectively. chlorophyll stability was not affected by the thermal process due to the addition of 0.05 % (m/v) mgcl2 to the blanching water. in all products, the contents of the main phenolic compound apiin decreased, while those of the minor compound malonylapiin b increased. in parsley pastes, peroxidase (pod) and polyphenol oxidase (ppo) were fully inactivated by the heat treatment. in contrast, only complete ppo inactivation was achieved in celeriac pastes. however, since pod inactivation was incomplete, its partial reactivation during storage of celeriac pastes was observed. after 4 weeks of cold storage, the green color of the parsley pastes turned into an olive hue due to chlorophyll degradation. nevertheless, the products may be stored at -20 °c for several months. in contrast, storage of celeriac pastes at 4 and -20 °c is possible for several months without darkening. compared to conventional dried herbs and spices the products obtained by the innovative process are characterized by bright colors. pasty products are easier to handle, because lumping and dusting are avoided, thus facilitating their safe application in the food processing industry. introduction conventional herbs and spices are natural products therefore being commonly burdened with high microbial loads. furthermore, most spices are produced in tropical and subtropical countries under poor hygienic conditions. in particular, drying of the raw material is often performed by spreading the plant material on the ground without protection from animals. the hot and humid climate and often lacking risk awareness of the farmers may result in considerable hygienic and sensory problems, causing an increased number of food-borne infections and intoxications. although dried spices are microbiologically stable due to their low water activities, microbial populations may develop quickly after their rehydration in high-moisture foods. upon drying of herbs and spices deteriorative enzymes, such as peroxidases, polyphenol oxidases, proteinases, and chlorophyllases, are only inhibited, without being inactivated. during storage of spices and upon rehydration of dried foods, such enzymes may regain their activity and adversely affect color, taste, and texture properties of the food products (schweiggert et al., 2007). color is a decisive quality attribute of vegetable products. chlorophylls are the predominant pigments of green plants (schwartz and lorenzo, 1990), and the bright green color is associated with freshness of herbs like parsley (petroselinum crispum (mill.) nym ex a. w. hill), while discoloration may lead to consumers’ rejection (lanfer marquez and sinnecker, 2008). chlorophyll degradation is mainly responsible for changes or loss of color. heating results in the conversion of green chlorophylls a and b into their corresponding olive brown pheophytins due to the replacement of the mg atom by hydrogen (schwartz and lorenzo, 1990; mackinney and weast, 1940). during storage chlorophyll degradation is catalyzed by chlorophyllase (ec 3.1.1.14) and magnesium dechelatase. while chlorophyllase activity cleaves the phytol moiety, yielding water-soluble chlorophyllides, the removal of the central mg atom is accomplished by the magnesium dechelatase (kidmose et al., 2002; matile et al., 1999). brown discoloration of celeriac (apium graveolens l. var. rapaceum (mill.) dc.) has a negative impact on consumers’ acceptance. peroxidases (pod; ec 1.11.1.7) and polyphenol oxidases (ppo; ec 1.14.18.1, ec 1.10.3.2) play an important role in this process. due to decompartmentalization of individual cells, phenolic compounds and enzymes are released resulting in colored complexes, the socalled melanins (tomás-barberán and espín, 2001). since pod is the most heat stable enzyme, it has been the most common indicator of enzyme inactivation in the blanching process (vámos-vigyázó, 1981). consequently, for color stabilization, the immediate and complete inactivation of deteriorative enzymes is a prerequisite. to overcome the above-mentioned problems associated with conventional spice production, an alternative process for the production of high quality spices has recently been developed. for this purpose, the fresh plant materials (green pepper, ginger, chili, coriander and paprika) were minced and subsequently heated. in some cases, blanching prior to grinding has been shown to be advantageous. compared to conventional spices, the so obtained spice powders were characterized by brighter colors and significantly lower microbial loads due to the immediate thermal inactivation of deteriorative enzymes and reduction of microorganisms, respectively (schweiggert et al., 2005a, b). conventional herbs and spices exhibit poor sensory properties. furthermore, they are difficult to handle and dose in industrial food processing due to lump and dust formation. therefore, the development of pasty herb and spice products should be advantageous. in recent years, the demand for spice pastes has considerably increased. however, for the production of red and green chili, ginger, and coriander leaf pastes salt, organic acids, and hydrocolloids are commonly added to preserve the products (baranowski, 1985; ahmed et al., 2002, 2004). our previous studies have demonstrated blanching to be recommended as initial step for the production of pasty herb and spice products. short-time blanching resulted in the inactivation of pod and ppo, and improved color stability of parsley and celeriac (kaiser et al., 2012, 2013b). furthermore, phenolic compounds were better retained upon blanching of parsley, marjoram, celeriac, coriander leaves and fruits (kaiser et al., 2013a, b, c). the objective of the current study was to cover the entire innovative process for the production of pasty herb and spice products exemplified for parsley and celeriac. in particular, the addition of magnesium to the blanching water should avoid its leaching, and thus improve chlorophyll stability. furthermore, the impact of processing and storage conditions on color stability, chlorophyll and polyphenol retention as well as enzyme activities of innovative pasty parsley and celeriac products should be evaluated. 140 a. kaiser, a.v. jaksch, k. mix, d.r. kammerer, r. carle materials and methods raw material flat-leaved parsley (petroselinum crispum (mill.) nym ex a. w. hill cv. ‘gigante d’italia’) and celeriac (apium graveolens l. var. rapaceum (mill.) dc. cv. ‘goliath’) were cultivated in 2011 by pharmaplant arzneiund gewürzpflanzen forschungsund saatzucht (artern, germany). the fresh plant materials were processed into pastes directly after harvest. chemicals reagents and solvents were purchased from vwr (darmstadt, germany) and were of analytical or hplc grade. apiin (apigenin7-o-apiosylglucoside) was from carl roth (karlsruhe, germany). deionized water was used throughout. process for the production of pasty herb and spice products parsley amounts of 10 kg of freshly harvested parsley leaves were washed with tap water and blanched at 100 °c for 1 min in a water bath containing 0.05 % (m/v) mgcl2. the blanched material was subsequently cooled in ice-water, drained and minced for 3 min in a bowl chopper (mado garant, dornhan, germany). the resulting products were immediately heated in a hermetically sealed 3 l pilot-plant scale type el 3 reaction vessel (esco-labor, riehen, switzerland) under stirring at 90 and 95 °c, respectively, for 3 min. set temperatures were achieved after approximately 3 min. after heat treatment the pasty products were cooled to 25 °c. celeriac amounts of 15 kg of washed celeriac were manually peeled, quickly cut into 1 cm slices, and subsequently steam-blanched at 100 °c for 5 min using a stainless steel vessel. after cooling in ice-water the blanched material was minced using a grater (bucher-guyer, niederweningen, switzerland) prior to fine comminution in a k60 colloid mill (probst & class, rastatt, germany) with an aperture size of 50 μm. the resulting product was heated in the reaction vessel as described above for parsley applying the same processing conditions. unheated control samples were prepared without heating, solely by mincing parsley and celeriac, respectively, as described above. storage of pasty parsley and celeriac products aliquots of 100 g of the products were filled into polyethylene bags under reduced pressure. samples were stored at 4 and -20 °c in the dark for 12 weeks. determination of chlorophylls extraction and quantitation of chlorophylls and their degradation products were performed as earlier described (kaiser et al., 2012). determination of phenolic compounds polyphenol analyses were carried out as previously reported (kaiser et al., 2013a, b). determination of pod and ppo pod and ppo activities were determined as earlier reported (kaiser et al., 2012, 2013b). color measurement color measurements were performed using a cr-300 chroma meter (minolta, osaka, japan) as previously described (kaiser et al., 2012) and expressed as l*, a*, b*, h° and c* values. determination of dry matter (dm) contents lyophilization of the samples was carried out using a freeze-dryer (lyovac gt4 amsco finn-aqua, hürth, germany) for 72 h. prior to and after freeze-drying sample weights were measured and subsequently dry matter contents were calculated. statistical analysis tukey test, using the sas software package (sas institute, cary, nc, usa; v. 9.1), was performed to determine significant differences (α = 0.05) between independent samples. results and discussion impact of processing on the quality of pasty parsley and celeriac products pasty parsley and celeriac products were processed by blanching the washed plant material to inactivate endogenous quality deteriorating enzymes, thus enhancing color retention. subsequently, for preservation, the pasty products were heated at 90 and 95 °c, respectively, for 3 min. chlorophylls the profile of chlorophylls and chlorophyll degradation products in parsley has been reported in our previous study (kaiser et al., 2012). chlorophyll a and b represented the major compounds in the unheated control. in the sample investigated in the present study, the pheophytin a content was unexpectedly high (fig. 1a, b). release of acids from the vacuoles was enhanced due to decompartmentalization of individual cells resulting from mincing, thus probably provoking the formation of this magnesium-depleted derivative. furthermore, the replacement of the central magnesium atom by two protons may be catalyzed by magnesium dechelatase (kidmose et al., 2002). compared to the unheated control, chlorophyll a contents were significantly higher in water-blanched samples (fig. 1a, b). preliminary tests have shown that the magnesium content in water-blanched parsley after the addition of magnesium to the blanching water was higher than in unheated parsley. in contrast, the magnesium content decreased upon water-blanching without magnesium addition (data not shown). consequently, in the present study, magnesium leaching was prevented resulting in enhanced chlorophyll retention. furthermore, heating enhances the disruption of plant cells and tissues leading to an improved release, and thus better extraction of chlorophyll a, which may also contribute to higher contents upon blanching (jiménez-monreal et al., 2009). in contrast, following heat treatment, chlorophyll b contents did not significantly change. water-blanching resulted in a significant decrease of pheophytin a contents, probably due to less leaching because of magnesium addition to the blanching water. however, subsequent heating at 90 and 95 °c, respectively, led to chlorophyll degradation to the corresponding pheophytins, since chlorophyll a and b contents tended to decrease, while pheophytin a and b contents significantly increased. pheophytin b was only detected in parsley pastes heated at 90 and 95 °c, respectively, indicating that chlorophyll a is more susceptible to form pheophytin than chlorophyll b, which is in agreement with a previous report (tan and francis, 1962). innovative pasty parsley and celeriac products 141 a b c d e f dm: dry matter fig. 1: chlorophyll and pheophytin contents during processing (a, b) and 12 weeks of storage (c-f) at 4 and -20 °c of parsley pastes: (a) processing at 90 °c, (b) processing at 95 °c, (c) storage at 4 °c of the pastes processed at 90 °c, (d) storage at -20 °c of the pastes processed at 90 °c, (e) storage at 4 °c of the pastes processed at 95 °c, (f) storage at -20 °c of the pastes processed at 95 °c. columns and bars represent mean ± standard deviation (n = 2). in our previous study, heating of minced parsley resulted in a marked degradation of chlorophylls (kaiser et al., 2012). the results of the present study showed that the blanching step and the addition of magnesium to blanching prior to mincing and subsequent heating are indispensable for chlorophyll retention. similar findings irrespective of magnesium addition were reported for dried marjoram, rosemary (singh et al., 1996), mint, and basil (rocha et al., 1993) which were blanched prior to drying. in the present study, retention of chlorophylls in the parsley pastes obtained was improved compared to airdried and freeze-dried basil leaves, where chlorophyll losses could not be avoided (di cesare et al., 2003). phenolic compounds in accordance with our previous reports (kaiser et al., 2013a, b), apiin (apigenin-7-o-apiosylglucoside) was found to be the major phenolic compound both in unheated parsley and celeriac. in contrast, malonylapiin b turned out to be the predominant compound in blanched parsley and celeriac, since apiin contents decreased upon water-blanching of parsley and steam-blanching of celeriac, while malonylapiin b contents increased (fig. 2a, b; 3a, b). the latter may probably be due to enhanced extractability. therefore, only these two compounds were considered in the present investigation of polyphenol stability. compared to blanched parsley, apiin and malonylapiin b contents remained virtually unchanged in pasty parsley products independent of the heating temperature applied. in contrast, heating of the pasty celeriac products resulted in a decrease of apiin contents and an increase of malonylapiin b contents. pod and ppo activities initial pod and ppo activities of parsley were 687.7 ± 21.9 and 17.5 ± 1.0 nkat/g dm, respectively. residual pod activities ranged from 1.6 to 1.9 % after processing of pasty parsley products, coming close to complete inactivation. in contrast, ppo activity was completely ceased upon water-blanching. 142 a. kaiser, a.v. jaksch, k. mix, d.r. kammerer, r. carle a b c d e f dm: dry matter fig. 2: apiin and malonylapiin b contents during processing (a, b) and 12 weeks of storage (c-f) at 4 and -20 °c of parsley pastes: (a) processing at 90 °c, (b) processing at 95 °c, (c) storage at 4 °c of the pastes processed at 90 °c, (d) storage at -20 °c of the pastes processed at 90 °c, (e) storage at 4 °c of the pastes processed at 95 °c, (f) storage at -20 °c of the pastes processed at 95 °c. columns and bars represent mean ± standard deviation (n = 2). in unheated celeriac, pod and ppo activities amounted to 30.0 ± 2.1 and 47.7 ± 2.9 nkat/g dm, respectively. after steam-blanching, pod activity was not detectable. however, in the pastes heated at 90 and 95 °c, respectively, high residual pod activities, reaching 52 and 50 % of their initial values, respectively, were determined. it is supposed that the extraction of pod from the complex plant matrix was enhanced as a result of thermal degradation of cell walls and subcellular compartments (jiménez-monreal et al., 2009). furthermore, as previously hypothesized, during heating a novel compound exerting higher thermostability may have been formed from the heatdenatured enzyme protein comprising groups of pod which are still active (winter, 1971). however, absolute pod activities were very low. despite the presence of pod activities, enzymatic browning of the products was not observed. ppo was completely inactivated during processing suggesting enzymatic browning of celeriac to be mainly due to ppo activity as previously stated (kaiser et al., 2013b). color water-blanched parsley exhibited significantly lower a* values (greenness) and higher b* values (yellowness) compared to the unheated control (tab. 1). consequently, hue angles were higher, indicating a more intense green hue. due to blanching, tissue softening and release of intercellular air and dissolved gases may have influenced surface reflectance and depth of light penetration into tissues resulting in color enhancement (brewer et al., 1994). in our previous study, mincing prior to heating resulted in olive-green color of the products due to chlorophyll degradation (kaiser et al., 2012). in the present study, after mincing and heating at 90 and 95 °c, respectively, hue angle was comparable with that of the unheated sample which was attributed to enhanced chlorophyll retention as a result of the blanching step supported by magnesium addition. previous reports have shown that blanching prior to drying had a positive effect on color retention of marjoram and rosemary as compared to direct drying (singh et al., 1996). in contrast, significant color changes innovative pasty parsley and celeriac products 143 a b c d e f dm: dry matter fig. 3: apiin and malonylapiin b contents during processing (a, b) and 12 weeks of storage (c-f) at 4 and -20 °c of celeriac pastes: (a) processing at 90 °c, (b) processing at 95 °c, (c) storage at 4 °c of the pastes processed at 90 °c, (d) storage at -20 °c of the pastes processed at 90 °c, (e) storage at 4 °c of the pastes processed at 95 °c, (f) storage at -20 °c of the pastes processed at 95 °c. columns and bars represent mean ± standard deviation (n = 2). were not observed after microwave drying of fresh parsley compared to fresh plant material (soysal, 2004). unheated minced celeriac showed a brown color, which is ascribed to enzymatic browning as a result of decompartmentalization of individual cells (tomás-barberán and espín, 2001). after processing of celeriac pastes, lightness l* significantly increased, while chroma c* decreased, and h° values shifted towards more yellowish hues (tab. 2). consequently, the pastes obtained were characterized by a perfect white color. in agreement with our findings, a blanching step prior to air and vacuum drying, respectively, also resulted in light colored celeriac products (alibaş, 2012). impact of storage on the quality of pasty parsley and celeriac products to evaluate the storage stability of the pastes, the processed products were stored for 3 months in the dark at 4 and -20 °c. quality of the pastes was monitored considering the same parameters as for processing. chlorophylls as can be seen from fig. 1c-f, chlorophylls in parsley pastes were differently affected by various storage temperatures. during storage chlorophyll a degradation was stronger than that of chlorophyll b resulting in a marked formation of pheophytin a as reported in previous studies (schwartz and lorenzo, 1991). in pastes heated at 90 °c, chlorophyll a and b contents first significantly decreased after 6 weeks of cold storage which was accompanied by an increase of pheophytin a contents. in contrast, in the products processed at 95 °c, degradation of chlorophyll a already started after the first two weeks going along with a rise of pheophytin a contents, and chlorophyll b degradation was first observed after 4 weeks of cold storage. this indicated higher processing temperatures to cause a more pronounced 144 a. kaiser, a.v. jaksch, k. mix, d.r. kammerer, r. carle destruction of cells, thus resulting in earlier chlorophyll degradation. during storage of both parsley products, pheophytin b contents were rather low amounting to 0.09-0.44 g/kg dm. as expected, chlorophyll degradation was more pronounced at 4 °c than at -20 °c, since low storage temperatures preserve chlorophylls (lanfer marquez and sinnecker, 2008). in both parsley pastes stored at -20 °c, a slight decrease of chlorophyll a contents was observed, while chlorophyll b and pheophytin contents virtually did not change. in parsley pastes, losses during cold storage for 3 months were in the range of 44-45 % and 44-46 % for chlorophyll a and b, respectively. in contrast, in products stored at -20 °c losses amounted to 10-11 % and 11-12 % for chlorophyll a and b, respectively. in literature, different findings concerning chlorophyll degradation during storage of green herbs and vegetables were reported. when water-blanched parsley was stored at -20 and -30 °c, chlorophyll losses were insignificant (lisiewska and kmiecik, 1997). in contrast, during frozen storage at -18 °c of blanched peas and green beans for 12 and 9 months, respectively, degradation of chlorophyll was observed (gökmen et al., 2005; bahçeci et al., 2005). in blanched dill stored at -20 °c for 12 months chlorophyll losses of 9 % and 15 % for leaves and whole plants, respectively, were determined; however, during storage at -30 °c for 12 months, chlorophyll contents did not change (lisiewska et al., 2004). phenolic compounds the phenolic contents showed similar trends during storage at 4 and -20 °c for both parsley products (fig. 2c-f). during storage at 4 °c, apiin contents tended to increase, while malonylapiin contents significantly decreased. storage at -20 °c resulted in a decrease of apiin and malonylapiin b contents. it is supposed that a malonyl esterase has not completely been inactivated, thus being responsible for the degradation of malonylapiin to apiin, since malonyl esterase activity in parsley has previously been described (matern, 1983). as expected, this reaction was enhanced at 4 °c compared to storage at -20 °c. in celeriac products heated at 90 °c, apiin contents significantly increased during cold storage (fig. 3c). in contrast, malonylapiin b contents markedly declined. in pastes heated at 95 °c, a significant decrease of malonylapiin b contents was first observed after 12 weeks of storage at 4 °c (fig. 3e). apiin contents significantly increased after four weeks and then remained unchanged. storage at -20 °c did not affect apiin contents (fig. 3d, f). in contrast, malonylapiin b contents significantly decreased. although malonyl esterase activities in celery and celeriac, respectively, have so far not been reported, this enzyme is assumed to occur also in celeriac, causing the observed degradation of malonylapiin. pod and ppo activities although regeneration of pod and ppo activities during storage has previously been reported for spices, vegetables, and fruits (schweiggert et al., 2006; thongsook and barrett, 2005; holzwarth et al., 2012), in the present study, pod and ppo did not regain their activities during storage of parsley products. in contrast, regeneration of pod was observed in both celeriac products, probably due to incomplete inactivation during heat treatment (fig. 4). nevertheless, the pod activities determined were only marginal, while ppo activity was not detected during storage. thus, enzymatic browning of the celeriac products was inhibited. these findings support our assumption that mainly ppo is responsible for enzymatic browning of celeriac. color independent of processing time, color characteristics of both parsley products showed similar trends during storage (tab. 1). during 12 weeks of storage at 4 °c, h° values declined markedly, corresponding to a shift to yellowish tones. this observation goes along with major chlorophyll losses resulting in a decline of a* values. consequently, the green color turned into olive due to pheophytin formation. in contrast, hue angles only slightly increased during storage at -20 °c due to better chlorophyll retention. even though the h° values of the products stored at -20 °c were significantly lower at the end of storage than that of the unheated control, the parsley products were characterized by a bright green color. yellowness did not significantly change during storage at 4 and -20 °c. the color values of the celeriac pastes remained virtually unchanged at both storage temperatures (tab. 2). thus, the products retained their white color. enzymatic browning was prevented by complete ppo inactivation during processing of the pastes. conclusions in the present study, a process for the production of innovative pasty parsley and celeriac products possessing improved color was established. after blanching the plant material was converted into a paste and subsequently heated. compared to conventional dried herbs and spices, the so obtained pasty products are characterized by bright colors. this may be attributed to enhanced chlorophyll retention in parsley partly due to the prevention of magnesium leaching and the early ppo inactivation in celeriac, respectively, as a result of the initial heating step. complete inactivation of pod celeriac could not be achieved; however, its activities were negligible. the contents of the main phenolic compound apiin strongly decreased, while the minor compound malonylapiin b significantly increased, when processing fig. 4: residual pod activities during 12 weeks of storage of celeriac pastes, processed at 90 °c (a) and 95 °c (b). residual pod activities based on the initial pod activity in unheated celeriac. innovative pasty parsley and celeriac products 145 parsley and celeriac into pasty products. during cold storage, the green color was retained for 4 weeks, whereas the products may be stored at -20 °c for several months. in contrast, storage of celeriac pastes at 4 and -20 °c is possible for several months without color loss. furthermore, enzyme activities in celeriac were low and even ceased in parsley. pasty products are easier to handle, because lumping and dusting are avoided, thus facilitating their easy application in the food processing industry. their application is proposed for sensitive food sectors such as catering of air carriers, schools, hospitals, and residential homes for the elderly requiring especially high hygiene standards, and for their incorporation into extremely sensitive commodities (e.g. herb butter). acknowledgements we are grateful to sandra bayha for her excellent technical assistance in lab experiments. the project was supported by funds of the federal ministry of food, agriculture and consumer protection (bmelv) based on a decision of the parliament of the federal republic of germany via the federal office for agriculture and food (ble) under the innovation support program (fkz: 2815200306). references ahmed, j., shivhare, u.s., debnath, s., 2002: colour degradation and rheology of green chilli puree during thermal processing. int. j. food sci. technol. 37, 57-63. ahmed, j., shivhare, u.s., singh, p., 2004: colour kinetics and rheology of coriander leaf puree and storage characteristics of the paste. food chem. 84, 605-611. alibaş, i̇., 2012: determination of vacuum and air drying characteristics of celeriac slices. j. biol. environ. sci. 6, 1-13. bahçeci, k.s., serpen, a., gökmen, v., acar, j., 2005: study of lipoxygenase and peroxidase as indicator enzymes in green beans: change of enzyme activity, ascorbic acid and chlorophylls during frozen storage. j. food eng. 66, 187-192. tab. 2: color characteristics during processing (90 and 95 °c) and storage (12 weeks at 4 and -20 °c) of pasty celeriac products (n = 6) 90 °c 95 °c process parameter l* c* h° l* c* h° control (unheated) 49.3 ± 0.4 f 24.2 ± 0.7 a 71.8 ± 0.8 e 49.3 ± 0.4 e 24.2 ± 0.7 a 71.8 ± 0.8 e steam-blanched 62.3 ± 0.5 ab 16.8 ± 0.7 b 95.3 ± 1.2 d 63.9 ± 0.3 a 14.5 ± 0.7 b 101.4 ± 1.4 d after processing, week 0 62.8 ± 0.2 a 14.2 ± 0.3 cd 103.0 ± 0.5 bc 62.7 ± 0.2 b 12.2 ± 0.2 cd 106.1 ± 0.5 bc week 2, 4 °c 61.7 ± 0.9 bc 12.9 ± 1.0 e 104.7 ± 2.0 ab 61.7 ± 0.5 bc 11.4 ± 0.3 cde 106.0 ± 0.4 bc week 4, 4 °c 60.8 ± 0.5 cd 12.4 ± 0.6 e 106.4 ± 1.1 a 61.9 ± 0.3 bc 11.1 ± 0.3 def 107.7 ± 0.5 ab week 6, 4 °c 60.8 ± 0.6 cd 12.6 ± 0.7 e 104.6 ± 1.5 ab 60.1 ± 0.3 d 11.1 ± 0.7 def 107.1 ± 2.1 abc week 8, 4 °c 58.3 ± 0.4 e 12.6 ± 0.5 e 103.7 ± 1.0 bc 61.0 ± 0.5 cd 10.8 ± 0.3 ef 106.1 ± 1.9 bc week 12, 4 °c 60.4 ± 0.5 d 13.2 ± 0.9 de 103.2 ± 0.8 bc 60.4 ± 0.5 d 11.5 ± 0.3 cde 105.8 ± 0.8 bc week 4, -20 °c 61.7 ± 0.4 bc 14.1 ± 0.4 cd 102.7 ± 0.8 bc 60.1 ± 1.3 d 10.2 ± 1.4 f 108.8 ± 2.2 a week 8, -20 °c 61.8 ± 0.5 b 14.2 ± 0.4 cd 102.7 ± 0.4 bc 61.9 ± 0.8 bc 12.4 ± 0.4 c 105.2 ± 0.4 c week 12, -20 °c 62.1 ± 0.2 ab 14.7 ± 0.2 c 102.7 ± 0.6 c 61.6 ± 0.3 bc 12.2 ± 0.3 cd 106.8 ± 0.4 abc different letters (vertical) indicate significant differences (p < 0.05) tab. 1: color characteristics during processing (90 and 95 °c) and storage (12 weeks at 4 and -20 °c) of pasty parsley products (n = 6) 90 °c 95 °c process parameter a* b* h° a* b* h° control (unheated) -6.6 ± 0.9 e 9.8 ± 0.8 e 123.7 ± 1.6 bc -6.6 ± 0.9 e 9.8 ± 0.8 d 123.7 ± 1.6 b water-blanched -12.3 ± 0.4 i 13.5 ± 1.0 a 132.3 ± 1.2 a -11.3 ± 0.5 h 12.8 ± 0.7 ab 131.4 ± 0.8 a after processing, week 0 -9.2 ± 0.3 h 13.1 ± 0.8 ab 125.1 ± 0.8 b -8.2 ± 0.3 g 11.9 ± 0.5 abc 124.3 ± 0.3 b week 2, 4 °c -5.4 ± 0.3 d 13.5 ± 0.3 a 111.7 ± 0.7 f -5.1 ± 0.2 d 13.0 ± 0.5 a 111.5 ± 0.3 e week 4, 4 °c -4.3 ± 0.1 c 12.2 ± 0.3 bcd 109.2 ± 0.4 g -3.9 ± 0.2 c 12.3 ± 0.6 abc 107.8 ± 0.4 f week 6, 4 °c -3.2 ± 0.3 b 12.2 ± 0.3 bcd 104.2 ± 0.6 h -3.0 ± 0.2 b 11.9 ± 0.4 abc 104.4 ± 0.4 g week 8, 4 °c -2.7 ± 0.1 b 12.8 ± 0.2 ab 101.8 ± 0.4 i -2.7 ± 0.2 b 12.2 ± 0.6 abc 102.5 ± 0.9 h week 12, 4 °c -1.2 ± 0.2 a 11.3 ± 0.3 d 96.0 ± 1.1 j -1.4 ± 0.2 a 11.8 ± 0.7 bc 96.7 ± 1.2 i week 4, -20 °c -6.9 ± 0.1 ef 11.8 ± 0.3 cd 120.6 ± 0.4 e -6.6 ± 0.2 e 11.4 ± 0.5 c 120.1 ± 0.3 d week 8, -20 °c -7.9 ± 0.2 g 12.4 ± 0.4 bc 122.6 ± 0.5 cd -7.6 ± 0.3 fg 12.1 ± 0.4 abc 121.9 ± 0.3 c week 12, -20 °c -7.6 ± 0.2 fg 12.5 ± 0.3 abc 120.9 ± 0.4 de -7.0 ± 0.4 ef 12.2 ± 0.5 abc 120.9 ± 0.6 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and lipoxygenase in paprika and chili powder after immediate thermal treatment of the plant material. innovative food science & emerging technologies 6, 403-411. schweiggert, u., schieber, a., carle, r., 2006: effects of blanching and storage on capsaicinoid stability and peroxidase activity of hot chili peppers (capsicum frutescens l.). innovative food sci. emerg. technol. 7, 217-224. singh, m., raghavan, b., abraham, k.o., 1996: processing of marjoram (majorana hortensis moench.) and rosemary (rosmarinus officinalis l.). effect of blanching methods on quality. nahrung 40, 264-266. soysal, y., 2004: microwave drying characteristics of parsley. biosystems eng. 89, 167-173. tan, c.t., francis, f.j., 1962: effect of processing temperature on pigments and color of spinach. j. food sci. 27, 232-241. thongsook, t., barrett, d.m., 2005: heat inactivation and reactivation of broccoli peroxidase. j. agric. food chem. 53, 3215-3222. tomás-barberán, f.a., espín, j.c., 2001: phenolic compounds and related enzymes as determinants of quality in fruits and vegetables. j. sci. food agric. 81, 853-876. vámos-vigyázó, l., 1981: polyphenol oxidase and peroxidase in fruits and vegetables. crit. rev. food sci. nutr. 15, 49-127. winter, e., 1971: hitzebeständigkeit der peroxydase. z. lebensm. unters. forsch. 145, 3-6. address of the authors: hohenheim university, institute of food science and biotechnology, chair plant foodstuff technology, garbenstrasse 25, 70599 stuttgart, germany e-mail: a_kaiser@uni-hohenheim.de journal of applied botany and food quality 87, 249 255 (2014), doi:10.5073/jabfq.2014.087.035 1department of molecular biology, abbes laghrour university, khenchela, algeria 2laboratory of natural resources valorization, ferhat abbas university, setif, algeria 3blaise pascal university, clermont ferrand, france 4lexva analytique, beaumont, france characterization and chemosystematics of algerian thuriferous juniper (juniperus thurifera l.) azzeddine zeraib1, 2*, messaoud ramdani 2, lamia boudjedjou 2, pierre chalard 3, gille figuredo4 (received december 15, 2013) * corresponding author summary leaf essential oils (eo) of juniperus thurifera l. collected at six locates from aures mountains in algeria, were analyzed by gas chromatography (gc) and gas chromatography-mass spectrometry (gc/ ms). the main components identified were: sabinene (5.2-19.78 %), terpinene-4-ol (5.43-9.37 %), elemol (0.69-7.61 %), δ-cadinene (3.26-6.11 %). terpenoids data of our samples and those reported in other works realized by various authors were subjected to principal component analysis (pca), and unweighted pair group method with arithmetic means (upgma) cluster was carried. this analysis revealed significant differences between juniperus thurifera populations, and confirmed the clear separation of algerian populations to the european and moroccan populations. algerian thuriferous juniper is more similar to j. thurifera from moroccan populations, and different from that of essential oils obtained from european populations. introduction thuriferous juniper (juniperus thurifera l.), is an evergreen and a dioecious shrub or tree with scale leaves and bluish black berries at maturity, occurring from algeria and morocco over the iberian peninsula and the pyrenees to the french and italian alps and to corsica (gamisans et al., 1994; gauquelin et al., 1988; gauquelin et al., 2003). in algeria, the thuriferous juniper is extremely rare and only localized in the aures mountains with a number of scattered and often very large trees that are probably the remains of formerly more extensive stands (vela and schäfer, 2013). juniperus thurifera is a morphologically variable species, perhaps as a result of long-term isolation of disjunct populations. maire (1926) was the first to distinguish the north african and european populations of j. thurifera. later, based on morphometric characters such as the size of the cones and the number of seeds per cone. gauquelin et al. (1988) recognized both entities as subspecies; j. thurifera subsp. africana (maire) gauquelin in north africa, and j. thurifera subsp. thurifera in the european range of the species. of the latter, there are 3 varieties: var. thurifera on the iberian peninsula, var. gallica de coincy in the alps and var. corsicana gauquelin in corsica (gauquelin et al., 2003). the distinction of north african and european populations of j. thurifera is validated by romo and boratynsky (2007). studies based on essential oils composition, random amplified polymorphic dna (rapd) and morphometric data (adams, 1999; adams et al., 2003; barrero et al., 2004; achak et al., 2008; 2009; bahri et al., 2013; boratynsky et al., 2013), supported the clear differentiation of j. thurifera subsp. africana from morocco against the european populations, but without studying the algerian ones, which are generally empirically assimilated to the moroccan taxon. the study achieved by terrab et al. (2008), based on genetic polymorphism (aflp), has shown that the algerian population was genetically more closely related to the european than to the moroccan ones, probably due to dispersal events from europe to algeria, and the moroccan populations should be recognized as a distinct subspecies (j. thurifera l. subsp. africana (maire) romo and boratynsky. a taxonomic synthesis, completed by a brief morphological study of fruits based on herbarium samples was performed by vela and schäfer (2013), concludes with the desirable distinction of a moroccan taxon (subsp. africana) and an algerian one (juniperus thurifera var. aurasiaca véla & p. schäf). it is distinguishable as intermediate between subsp. thurifera and subsp. africana, more similar to the first one following molecular properties but sharing partial polymorphism with the second and more similar to the latter one following some morphological elements as fruit size average but with a variation amplitude partially sharing numerical values with the first. but above all, the polymorphism of algerian populations is very poorly known and would be deeply studied in the future (vela and schäfer, 2013). the aim of the present study was to characterize the algerian thuriferous juniper and sought its taxonomic status. we are based on the chemical composition of the essential oils isolated from the leaves of juniperus thurifera l. collected at different locations in aures mountains (algeria), and compare these data with other studies on the chemical variability of essential oils from european and moroccan populations. material and methods plant material leaves of juniperus thurifera l. were collected in march, 2010, from different localities around the aures mountains, in algeria (tkout1 at 1450 m of altitude, tkout2 at 1700 m, baâli at 1750 m, tizi nerrsas at 1500 m, tibhirin at 1500 m, and chelia at 1700 m of altitude). a voucher specimen is deposited in the herbarium of the laboratory of natural resource valorization, faculty of biology, farhat abbes university, setif, algeria. isolation and analysis of the essential oils the plant material was submitted to hydro distillation for 3 h, according to tumen et al. (2010). the prepared volatile oils were dehydrated over anhydrous sodium sulphate and stored in sealed glass vials at 4-5 °c prior to analysis. yield based on dry weight of the sample was calculated. the essential oils were analyzed on a hewlett-packard gas chromatograph model 5890, coupled to a hewlett-packard model 5971, equipped with a db5 ms column (30 m x 0.25 mm; 0.25 μm), programming from 50 °c (5 min) to 300 °c at 5 °c/min, with a 5 min hold. helium was used as the carrier gas (1.0 ml/min); injection in split mode (1:30); injector and detector temperatures, 250 and 280 °c, respectively. the mass spectrometer worked in ei mode at 70 ev; electron multiplier, 2500 v; ion source temperature, 180 °c; ms data were acquired in the scan mode in the m/z range 33-450. the identification of the components was based on comparison of their mass spectra with those of nist mass spectral library (nist, 2002) and those described by adams (2001) as well as on comparison of 250 a. zeraib, m. ramdani, l. boudjedjou, p. chalard, g. figuredo their retention indices either with those of authentic compounds or with literature values (adams, 2001). statistical analysis to examine the phytochemical diversity based on the content (%) of chemical constituents in essential oil among the studied six populations; these were first subjected to principal components analysis (pca) to examine the relationships among the compounds and identify the possible structure of the population. cluster analysis (upgma) was carried out on the original variables and on the manhattan distance matrix to seek for hierarchical associations among the populations. statistical analyses were carried out using statistica 8 software. results and discussion variability of algerian j. thurifera essential oils volatile oil yield of the leaves of the investigated j. thurifera populations is summarized in (tab. 1). the yield varied from 0.40 to 0.53 % in different populations of j. thurifera. maximum essential oil yield was noticed in tkout1 population (0.53 %), followed by tizi nerrsas (0.48 %), the minimum essential oils yield was noticed in chelia population (0.40 %), which is very low compared to the yield obtained by achak et al. (2008; 2009) and bahri et al. (2013) from the same species. inter-population variation of essential oil yield is quite common phenomenon and encountered earlier in several other plant species. these variations might be due to climatic conditions of the growing site, pedoclimatic variation or due to difference in the genetic makeup of the j. thurifera populations. the volatile oils of all six juniperus thurifera populations were characterized and identified by gas chromatography and gas chromatography-mass spectrometry. the relative percentages of the constituents are listed in the tab. 1. seventy nine constituents, representing 90.2-98.7 % of the total oil composition, were identified. seventeen constituents were monoterpene hydrocarbons accounting (18.640.1 %) of the eo, 17 constituents were oxygenated monoterpenes (10.3-24 %), 22 constituents were sesquiterpene hydrocarbons (15.1-23.8 %), 18 constituents were oxygenated sesquiterpenes (1836.8 %), two constituents were diterpene hydrocarbons (0.2-1.7 %), and three constituents were oxygenated diterpenes (0.5-1.8 %). the main components identified were: sabinen (5.2-20 %), terpinene-4-ol (5.4-9.4 %), elemol (0.7-7.6 %), δ-cadinene (3.3-6.1 %), linalyl acetate (0.9-6.2 %), γterpinene (2.6-3.9 %), αpinene (2.63.9 %) and myrcene (1.2-3.3 %). leaf eo of juniperus thurifera in deferent regions of morocco are rich in sabinene (12.2 % to 45.8 %), α-pinene (4 % to 17.1 %) and terpinene-4-ol (2.6 % to 16.9 %) (adams et al., 2003; roman et al., 2008; 2009; bahri et al., 2013). the major constituent of the volatile oils obtained from branches of moroccan juniperus thurifera is β-pinene (36.26 %) (mansouri et al., 2010). twenty-two compounds showed statistically significant variations among the six locations, the components identified (sabinen, α-cadinol, valencen, elemol, linalyl acetate, and linalool) show a significant variability of terpenoid (fig. 1). the principal component analysis (pca) performed on the correlation matrix of the 79 variables showed that the first three axes explained 74.3 % of the observed variation. this analysis allowed recognizing two distinct eo types based on the content of sabinene, linalyl acetate, linalool, γ-terpinene, myrcene, bulnesol, valencene, γ-eudesmol, epi-α-cadinol, epi-α-muurolol, and 4-epi-abietal. the first group was represented by four populations (tkout1, baâli, tizi nerrsas, tibhirine), located on the positive part of axis one, characterized by high concentrations of sabinene, linalyl acetate, linalool, γ-terpinene, myrcene, and bulnesol. this group is opposed to the second group, formed by two populations (chelia and tkout2), which is characterized by high concentrations of valencene, γ-eudesmol, epi-α-cadinol, epi-α-muurolol, and 4-epi-abietal (fig. 2). fig. 1: chemical variability of main compounds of juniperus thurifera (var 1 to var 79 are mentioned in the tab. 1). fig. 2: projection of algerian juniperus thurifera populations on the factor plane (1x 2). characterization and chemosystematics of algerian j. thurifera 251 tab. 1: composition of the leaf essential oils of j. thurifera from aures mountains in algeria. compound ki tk1 tk2 tz ba tb ch var1 α-thujene 924 1.2 0.8 1.1 1.5 0.8 0.5 var2 α-pinene 932 3.3 2.6 2.62 3.47 3.1 3.9 var3 fenchene 946 0.1 tr 0.1 0.1 0.1 var4 camphene 947 0.1 0.1 0.1 0.1 0,1 tr var5 sabinene 975 17.9 8.3 11.3 20.0 9.0 5.2 var6 β-pinene 979 0.3 0.3 0.3 0.3 0.3 0.4 var7 myrcene 991 2.8 1.7 2.0 3.3 1.7 1.2 var8 δ-3-carene 1011 0.2 1.5 0.9 0.5 1.0 0.8 var9 α-terpinene 1018 1.6 1.3 1.7 1.8 1.9 1.2 var10 p-cymene 1026 0.5 0.2 0.3 0.7 0.4 0.5 var11 limonene 1031 1.4 3.2 2.5 2.0 1.2 0.7 var12 β-phellandrene 1031 0.2 0.2 0.2 0.2 0.2 var13 (z)-β-ocimene 1040 0.10 var14 (e)-β-ocimene 1050 0.3 0.2 0.3 0.2 var15 γ-terpinene 1062 3.2 2.6 3.5 3.6 3.9 2.6 var16 cis-sabinene hydrate 1068 0.8 0.8 1.6 0.6 2.3 0.6 var17 terpinolene 1088 1.3 1.2 1.5 1.5 1.5 0.9 var18 linalool 1096 4.6 0.3 0.4 2.5 1.6 0.4 var19 cis-thujone 1100 0.1 0.4 0.3 0.6 var20 trans-thujone 1111 0.27 0.3 var21 cis-p-menth-2-en-1-ol 1122 0.4 0.3 0.5 0.4 0.6 0.3 var22 trans-menth-2-en-1-ol 1141 0.3 0.2 0.3 0.3 0.4 0.3 var23 terpinene-4-ol 1177 7.2 5.4 7.6 7.5 9.4 7.1 var24 α-terpineol 1195 1.6 0.6 1.1 1.1 1.1 0.9 var25 verbanone 1205 0.2 0.2 0.3 0.3 0.4 0.3 var26 trans piperitol 1209 0.1 var27 nerol 1 (80) 1224 0.3 0.2 0.2 0.2 var28 linalyl acetate 1249 6.2 1.2 3.4 3.1 3.7 0.9 var29 pregeijerene 1280 0.12 var30 bornyl acetate 1284 0.1 0.1 0.1 0.4 var31 isobutyl benzene 1287 0.3 0.3 0.3 var32 decan-2,4-dien-1-ol 1312 0.3 0.2 0.5 0.1 var33 δ-elemene 1338 0.2 0.2 0.2 0.1 0.2 var34 α-terpinene acetate 1343 0.7 0.8 1.7 0.8 1.1 0.1 var35 neryl acetate 1362 0.4 0.3 0.2 0.2 var36 geranyl acetate 1381 1.0 0.3 0.6 0.8 0.6 0.3 var37 β-elemene 1391 0.3 0.4 0.3 0.4 0.4 0.2 var38 β-caryophyllene 1419 0.9 1.1 0.9 1.0 1.2 0.5 var39 γ-elemene 1429 0.4 0.4 0.2 0.1 0.4 var40 cadina-3,5-diene 1448 0.1 0.1 0.1 0.2 0.1 var41 α-humulene 1454 0.5 1.0 0.8 0.9 1.1 0.7 var42 cis muurola-4(14),5-diene 1460 0.1 0.5 0.2 0.4 0.7 0.3 var43 cadina-1(6),4-diène 1460 0.1 0.2 0.2 0.1 var44 γ-muurolene 1477 0.3 0.6 0.5 0.5 0.5 0.5 var45 curcumene 1478 0.2 0.3 0.2 0.2 var46 germacrene-d 1480 1.9 2.7 4.4 3.1 3.3 1.6 252 a. zeraib, m. ramdani, l. boudjedjou, p. chalard, g. figuredo var47 trans muurola-4(14),5-diene 1494 0.2 0.4 0.3 0.5 0.2 0.4 var48 α-muurolene 1500 0.8 1.1 1.1 0.8 0.6 0.4 var49 β-curcumene 1516 0.5 0.2 0.7 0.2 0.4 0.2 var50 γ-cadinene 1514 0.7 1.8 1.0 1.5 1.2 1.4 var51 δ-cadinene 1523 3.3 6.1 4.1 5.6 3.9 4.1 var52 α-cadinene 1539 0.2 0.4 0.2 0.3 0.3 0.3 var53 elemool 1550 4.7 5.8 0.7 3.9 7.6 5.2 var54 germacrene-b 1561 2.5 2.3 2.0 1.2 2.6 1.2 var55 germacrene-d-4ol 1576 0.8 1.9 1.1 0.9 1.1 1.1 var56 caryophyllene oxyde 1585 0.1 1.1 0.2 var57 cedrol 1596 0.1 0.1 0.1 0.1 0.2 0.8 var58 humulene epoxyde ii 1607 0.2 0.2 0.1 0.1 var59 β-oplopenone 1608 0.5 1.2 0.7 0.6 0.5 0.9 var60 epi-cedrol 1613 0.92 var61 valencen 1619 0.5 2.5 3.1 1.4 0.9 6.6 var62 1,10-diepi-cubenol 1627 0.3 0.3 0.2 0.2 0.4 0.3 var63 1-epi-cubenol 1629 1.1 1.3 1.0 1.8 0.8 1.6 var64 γ-eudesmol 1632 1.3 2.1 2.4 1.1 2. 2.4 var65 epi-α-cadinol 1640 0.9 2.9 1.1 0.8 1.2 3.0 var66 epi-α-muurolol 1642 0.9 3.7 0.7 0.8 1.0 3.1 var67 α-cadinol 1663 6.1 13.5 10.6 5.5 9.2 15.7 var68 bulnesol 1666 3.2 1.8 0.9 1.9 2.0 0.6 var69 gurjunene 1670 0.8 1.1 0.9 0.8 1.6 var70 γ-gurjunene 1678 0.3 0.6 0.6 0.8 0.4 0.7 var71 epi-α-bisabolol 1686 0.3 0.4 0.4 0.1 0.4 0.6 var72 2-pentadecanone 1698 0.3 0.3 0.2 0.4 var73 (z,z)-farnesol 1714 0.5 0.3 0.4 0.2 1.0 0.9 var74 8-α-acetoxyl elemol 1789 0.1 0.2 0.1 var75 manoyl oxyde 1989 0.4 0.2 0.2 var76 13-epi-manoyl oxyde 1992 0.3 0.6 0.1 0.3 0.2 var77 abietatriene 2090 0.3 0.5 0.4 0.2 0.3 1.7 var78 phytol 2109 0.2 0.2 0.1 var79 4-epi-abietal 2299 0.7 1.1 0.3 0.4 0.6 1.3 monoterpene hydrocarbons 35.3 24.5 29.8 40.1 27.9 18.6 oxygenated monoterpenes 24.0 10.3 16.7 18.3 20.6 12.3 sesquiterpene hydrocarbons 15.1 23.8 21.9 20.2 20.4 19.5 oxygenated sesquiterpenes 22.4 36.2 21.7 18.0 27.7 36.8 diterpene hydrocarbons 0.5 0.7 0.4 0.2 0.4 1.7 oxygenated diterpenes 1.4 1.8 0.5 0.6 1.0 1.3 yield (%) 0.53 0.46 0.48 0.45 0.46 0.40 total % 98.7 97.3 91.0 97.4 98.0 90.2 (tk1: tkout 1, tk2: tkout 2, tz: tizi nerrsas, tb: tibhirin, ch: chelia, ba: baâli). mono and sesquiterpenoids variability reflects the heterogeneity of the genetic structure of population (dodd and poveda, 2003; lima et al., 2010; shanjani et al., 2010). hannover (1992) provides evidence that terpene chemotypes are strongly controlled by genetic factors; he also reported instances of environmental variation in terpene expression under extreme habitat conditions. a prevalence of monoterpene hydrocarbons compared to other components was noted in tkout1, baâli, tibhirin, and tizi nerrsase populations, while its reverse trend could be seen in tkout2 and chelia populations, which were dominated by sesquiterpene hydrocarbons (tab. 1). monoterpenes and sesquiterpene hydrocarbons were strongly related to chemical balance in soils (organic matter, phosphor and base saturation). the chemovariation observed appears to be environmentally determined (lesjak et al., 2013). characterization and chemosystematics of algerian j. thurifera 253 influence of environmental factors in the chemical composition of essential oils have also been reported in the genus juniperus (dodd and poveda, 2003; lima et al., 2010; shanjani et al., 2010; ložiene and labokas, 2012; lesjak et al., 2013), cupressaceae family (ottvioli, 2009), and are well known for other family (haider et al., 2004; karousou et al., 2005; cuardo et al., 2006; lei et al., 2010; djabou et al., 2012). the dendrogram based on upgma clustering (manhattan distance), shows the presence of two groups (fig. 3) that confirms result obtained from pca analyses. the first cluster is divided into two sub-groups based on the content of terpinene-4-ol, germacrene-d, α-terpinene acetate, cis-sabinene hydrate. the first sub-group formed by tkout1 and baâli, characterized by low concentration of these constituents unlike for tizi nerrsas and tibhirin populations. fig. 5: dendrogram of juniperus thurifera populations, based on manhattan similarity distance (adams, 1999: spain s3 = 2 km e ruidera); (adams et al., 2003: spain sc = consuegra, spain s1 and s2 = ruidera; morocco m1 and m2 = atlas mts, morocco tm = tizi-n-tichka, morocco om = oukaimeden; pyrenees p1 and p2 = pyrenees, france; corsica cr = corse, island); (achak et al., 2009: morocco fl = fresh leaves; morocco dl = dried leaves, ait lkak oukaimeden, atlas mts), (ottavioli, 2009: corsica cor 1-16 = corse) and (bahri et al., 2013: morocco vhd and mw= morocco) fig. 3: dendrogram of algerian juniperus thurifera populations, based on manhattan similarity distance. fig. 4: projection of juniperus thurifera populations on the factor plane (1x 2). characterization and chemosystematics leaf essential oils are extremely useful for the analyses of populational differentiation, hybridization and introgression and in assigning individual plants to a species (adams, 2010). to compare the algerian thuriferous juniper with other populations, terpenoids data of our samples and those reported in the work of adams (1999), adams et al. (2003), achak et al. (2009), ottavioli (2009) and bahri et al. (2013) were subjected to pca, and upgma cluster was carried. this analysis revealed significant differences between juniperus thurifera populations. the pca performed on the correlation matrix of the 96 variables based on the oil composition resulted in 38 factors of which the first three accounted for 47.4 % of the variance among the 39 populations, shows that j. thurifera populations are divided into three distinct groups (fig. 4). the algerian populations separated by 27.30 % of the variance in the chemical composition of essential oils. however, the moroccan populations accounted for about 12.66 % of the variance (fig. 4). the upgma based on the unweighted pair-group average distance and the city-block (manhattan) (fig. 5), has divided 39 populations of j. thurifera into two clads, confirmed the separation of the north african populations from the european populations. then, some qualitative differences in chemical composition can be deduced. the leaf essential oil composition of european populations was higher in limonene (30-75 %) and lower in sabinene (0-9,7 %) (adams et al., 2003; achak et al., 2008; ottavioli, 2009). however, the composition of the oils from algerian populations was similar to those reported for oils of j. thurifera from moroccan populations (adams et al., 2003; achak et al., 2008; bahri et al., 2013). the concentration of α-pinene and sabinene were greater in the 254 a. zeraib, m. ramdani, l. boudjedjou, p. chalard, g. figuredo oil of the moroccan populations, conversely, the concentrations of δ-cadinene and α-cadinol were greater in the oil of algerian population. should be recognized that algerian populations as sub-group distinctive from the european and moroccan populations or do they merely represent geographical interspecific variation? algerian juniperus thurifera is distinguishable as intermediate between subsp. africana and subsp. thurifera. more similar to the first one following some morphological elements as fruit size average but with a variation amplitude partially sharing numerical values with the latter (vela and schäfer, 2013). the results of the present study confirmed this similarity between moroccan populations subsp. africana and algerian populations called juniperus thuriefera var. aurasiaca véla & p. schäf. in view of the data presented in this study, it is apparent that algerian populations is not related to subsp. thurifera (sensu stricto), but to subsp. africana. however, terpenes are generally not as useful in making phylogenetic decisions because several terpenes may be controlled by a single enzyme (adams, 2010). farjon (2005) considered species based on the chemistry of terpenes analysis as based on inconclusive evidence, but dna sequence data certainly can (which is the main reason for their superiority). the study achieved by terrab et al. (2008), based on genetic polymorphism, has shown that algerian populations are distinct from the moroccan ones and more related to european populations. intraand inter-populational morphological variability throughout this vast territory fragmented (spain, pyrenees, alps, corsica and morocco) has recently been investigated by boratyński et al. (2013) and gives results congruous with genetic pattern obtained on the same whole europe/morocco (terrab et al., 2008), the algerian population excluded. finally, it is true that adding up the number of terpene differences may or may not give a good estimation of divergence. but, the number and scope of terpene differences between the algerian and european populations indicate considerable differentiation. additional research, using morphological variability, should help in elucidating these relationships. conclusions in brief, essential oils analysis carried out on six populations of j. thurifera showed both inter-population variability in their terpenoid content, with abundance of sabinene, terpinene-4-ol, elemol… the pca and upgma analysis allowed recognizing two distinct eo types based on the content of monoterpene and sesquiterpene hydrocarbons, the chemovariation observed appears to be environmentally determined. vela and schäfer (2013), proposed to call the algerian population by juniperus thuriefera var. aurasiaca véla & p. schäf, in the varietal rank, which will allow us to consider now as belonging european subset (subsp. thurifera) or moroccan subset (subsp. africana) or equal with both. in view of the results of this study, it seems that the algerian populations are much more similar to the moroccan populations, but it is still early to specify its taxonomic status. therefore, we support the proposition of vela and schäfer (2013), and the conflict between chemical and genetical data should be resolved on the morphological level. acknowledgments the authors are thankful to mr. fercha azzeddine, teacher in the department of biology, abbès laghrour university, khenchela, algeria, for the preliminary examination of this work. this study was supported, in part, by the chemistry of heterocyclic compounds and carbohydrates laboratory, higher national school of chemistry, clermont ferrand, france. and the ministry of higher education and scientific research of 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the pleistocene. molecular phylogenetics and evolution 48, 94-102. tumen, i., hafizoglu, h., pranovich, a., reunanen, m., 2010: chemical constituents of cones and leaves of cypress (cupressus sempervirens l.) grown in turkey. fresenius environmental bulletin 19 (10), 22682276. vela, e., schäfer, p.a., 2013: typification de juniperus thurifera var. africana maire, délimitation taxonomique et conséquences nomenclaturales sur le genévrier thurifère d’algérie. ecologia mediterranea 39(1), 6980. address of the corresponding author: a. zeraib, department of molecular biology, abbes laghrour university, 40000 khenchela, algeria. laboratory of natural resources valorization, ferhat abbas university, 19000 setif, algeria. e-mail: azzeraib@yahoo.fr art06_jha.indd rhizobacteria regulate paddy physiology under salinity journal of applied botany and food quality 85, 168 173 (2012) 1 n.v. patel college of pure & applied sciences, s.p. university, v.v. nagar, anand (gujarat), india 2 brd school of biosciences, sardar patel university, v.v. nagar, (gujarat), india paddy physiology and enzymes level is regulated by rhizobacteria under saline stress yachana jha1*, r.b. subramanian2 (received may 16, 2012) summary to investigate the physiological basis of salt adaptation in paddy due to inoculation with plant growth promoting bacteria, we compared their effect in paddy under saline and non-saline condition on root length, chlorophyll content, relative water content, stomatal conductance, membrane stability index and change in ascorbate peroxidase and nitrate reductase activities. in a pot experiment, the effect of endophytic and rhizospheric bacteria was studied in a local paddy rice (oryza sativa l.) variety gj-17 under salt stress. our fi ndings suggest that inoculation with pseudomonas pseudoalcaligenes and bacillus pumilus resulted in change of ascorbate peroxidase, nitrate reductase activity and plant growth parameters such as root length, chlorophyll content, rwc, stomatal conductance and membrane stability index under salinity. mixture of both pseudomonas pseudoalcaligenes and bacillus pumilus revealed better response in paddy against the adverse effects of salinity. introduction one of the most widespread agricultural problems in arid and semiarid regions is soil salinity, which make fi elds unproductive and decreases crop yield. salinity becomes a concern when an excessive amount or concentration of soluble salts occurs in the soil or water. it has been reported that salinity limits plant growth and pro ductivity (ashaf and foolad, 2007). the linkage between belowground and above ground sections of ecological system depends mainly on root system (liang peng et al., 2007). high concentration of salt in the root zone (rhizosphere) reduces soil water potential and the availability of water. as a result, reduction of the water content leading to dehydration at cellular level and osmotic stress is observed. the increased amounts of na+ and clin the environment effect the uptake of many indispensable nutrients through competitive interactions and by effecting the ion selectivity of membranes. the absorption function of root system is closely related to their morphology and activity. moreover root systems can interact with the environmental stress under the adverse situation, and adjust the system to build up adaptation responses in morphology and physiology to strengthen its survival chance. the effect of salinity on root (an et al., 2003) of plants had already been re ported. many researchers reported that with an increase in salinity there was a decrease in the de velopment of the xylem. decrease in development of xylem means decrease in loading of na+ and also essential nutrients, results in shunted growth of plants. plants in saline environment can protect themselves from na+ toxicity through restricting na+ entry; excluding na+ from root cells; sequestering na+ into vacuoles; or retrieving na+ from the transpirational xylem stream for recirculation to roots (chinnusamy et al., 2006). a strategy to acquire much water is essential for plant growth under water defi cit conditions. to overcome water defi cit, plants have developed mechanisms of physiological adaptation, such as improvement of water use effi ciency by regulation of stomatal closure, development of root system to acquire water, accumulation of osmoprotectants and control of water permeability by aquaporins (jang et al., 2004). salinity stress also decreases photosynthetic capacity due to the osmotic stress and partial closure of stomata. salinity also affects the chlorophyll pigment, both chlorophyll a and chlorophyll b decreased under salinity in rice seedlings (djanaguiram and ramadass, 2004). plants can also suffer from membrane destabilization and general nutrient imbalance. an important feature of salinity is the generation of reactive oxygen species (ros) and free radicals, such as superoxide anion radical (o2-), singlet oxygen, hydrogen peroxide (h2o2) and hydroxyl radical (ho-) which cause oxidative stress to plants. salinity promotes oxidative damage not only by direct increasing of the cellular concentration of reactive oxygen species but also by the diminution of the cellular antioxidant capacity (pinto et al., 2002). to minimize the damaging effects of ros, aerobic organisms evolved non-enzymatic defense systems (ascorbic acid, reduced glutathione, carotenoids, tocopherols, fl avonoids, alkaloids) and enzymatic protection mechanisms (superoxide dismutase, peroxidases, catalase). but in the last decade, there were number of reports on the benefi cial effects of microorganisms such as pseudomonas, bacillus, pantoea, burkholderia, rhizobium etc. in enhancing the tolerance of crops such as sunfl ower, maize, wheat, chickpea, groundnut, spices and grapes to drought, salinity, heat stress and chilling injury under controlled conditions (arshad et al., 2008). plant growth promoting bacteria are free-living soil bacteria that can either directly or indirectly facilitate rooting (mayak et al., 1999). with this aim, the objective of this study to comparatively investigate the role of plant growth promoting rhizobacteria (pgpr) alone and in combination in inoculated rice seedlings under different level of salinity stress. we hypothesise that inoculation with pgpr, alone or in com bination, can confer salinity tolerance to paddy and that such tolerance is correlated with changes in plant physiology as chlorophyll content, rwc, stomatal conductance and membrane stability index and activity of enzymes (nitrate reductase and ascorbate peroxidase) by considering that such bacteria help the plant under adverse condition of salinity. materials and methods isolation and identifi cation of pgpr certifi ed seeds of rice variety gj-17 were obtained from main rice research center, navagam, anand, gujarat. these seeds were planted in pots and maintained for forty days. microorganisms were isolated from the root tissue as well as rhizospheric soil. for isolation of endophytic bacteria from roots, fresh roots of paddy were surface sterilized with 70 % alcohol and hgcl2 for 5 min each, followed by washing with sterile distilled water. the root tissues were then homogenized in a sterile 4 % sucrose solution in mortar and pestle and the extract was used for isolation of bacteria. for isolation of rhizospheric bacteria, soil adhere with root surface were * corresponding author rhizobacteria regulate paddy physiology under salinity 169 collected and subjected to serial dilution, then both sample were plated on yema (yeast extract mannitol agar) medium. various biochemical tests were performed (data not shown here) followed by 16s rna ribo-typing to identify the isolates. the 16s rdna universal primers 8 f to 1510 r were used for amplifi cation of the dna followed by sequencing for identifi cation of isolates (jha et al., 2011b). the isolated endophytic pseudomonas pseudoalcaligenes and rhizospheric bacillus pumilus have good ability for phosphate solubilization as well as other plant growth promoting abilities (data already communicated), were selected for further studies. rice cultivation and inoculation seeds of rice variety gj-17 were washed thoroughly with distilled water followed by surface sterilization with 0.1 % hgcl2 solution for 4 min and 70 % ethanol for 10 min. the seeds were washed thoroughly with sterile distilled water and kept in a shaker for 5 6 h in autoclaved distilled water on a rotary shaker. later the seeds were transferred to petri dishes containing tryptone glucose yeast extract agar medium and incubated in dark at 30 °c to test for possible contamination. the germinated seedlings devoid of any contamination were used for inoculation experiments. to study the effect of the isolated bacteria on the physiological and biochemical parameters selected, 4 days old germinated seedlings devoid of any contamination were transferred to culture tubes containing 400 µl hoagland’s nutrient medium, 400 µl micronutrients and 1 % agar in 40 ml distilled water. before the transfer, bacterial inoculums of the isolated bacteria pseudomonas pseudoalcaligenes and bacillus pumilus were added with the medium at a concentration of 6 x 108 cfu ml-1. to obtain a mixture of both bacterial cultures, an equal volume of both the cultures were mixed in the medium to give a concentration of 6 x 10 8 cfu ml-1. the tubes were incubated at 27 °c in a 12 h light-dark cycle in a growth chamber. seven days old rice plants were carefully removed from different test tubes inoculated with the strain of bacterium, and planted in a pot. similarly the control plants (un-inoculated) were also transferred to a fresh pot. the quantity of the soil possessing the following physio-chemical properties; ph 7.79, electrical conductivity 1063 µs/cm, cec:3 cmol, organic carbon: 5500 mg kg-1 available nitrogen 200 mg dm-2, available ca: 12.1 cmol, available p205: 9.5 mg dm-2, available k20, 265 mg kg-1, fe, 3.1 mg kg-1, zn, 285 mg kg-1, mn, 3.7 mg kg-1, cu, 2.2 mg kg-1 was maintained as 5 kg per pot. rice seedlings were planted at the rate of 4 plants per transplant and 6 transplantations per pot. pots were watered at the time of transplantation of the rice seedlings. all experiments were carried in 5 replicates. maintenance of saline stress condition the saline condition was maintained at fi ve different salinity levels by adding (5g, 10g, 15g, 20g, and 25g nacl l-1) saline solution to the pots. to avoid osmotic shocks, nacl concentration was gradually increased for four consecutive days. a plastic bag was put underneath each pot to collect excess water due to drainage. this water was reapplied to the respective pot. all seedlings were grown for 5 weeks without any fertilizer treatment. the experiment was conducted in a greenhouse at 20 to 25 °c and the relative humidity 70 to 80 %. effect of pgpr on morphology and anatomy of plant root plants from each treatment after 35 days of sowing the seeds were collected carefully with plant root and cross sections of roots were examined under image analyzer microscope (carl zeiss) to analyze the effect of salinity on xylem in inoculated as well non-inoculated plant. effect of pgpr on growth parameters under different salinity level the observation on physical parameters i.e., root length, chlorophyll content, rwc, stomatal conductance and membrane stability index were recorded from thee replicate from each treatment after 35 days of sowing the seeds. total chlorophyll was extracted from fresh leaves by 80 % acetone (v/v) and its contents were determined at 663 nm and 645 nm by a hitachi u-2000 dual length spectrophotometer (arnon, 1949). stomatal conductance of plants was measured using fresh leaves by li-cor 6400. the n concentration was determined by colorimetric methods, after the kjeldahl digestion. relative water content fresh weight (fw) and dry weight (dw) of leaves of each plant were determined after counting the leaf number. leaf relative water content (rwc) was measured in the second or third youngest fully expanded leaf from top of the plant, using the following equations (schonfeld et al., 1988). rwc (%) = (fw-dw) × 100/ (tw-dw) where, fw is leaf fresh weight, dw is leaf dry weight after 24 h drying at 70 °c, and tw is leaf turgid weight after submergence in distilled h2o for 4 h. plant dry weight was determined after oven drying at 70 °c until they reached constant weight. membrane stability index membrane stability index (msi) was estimated as per sairam et al., (1997). fresh leaves (0.1 g) were taken in 10 cm³ of double distilled water in two sets. one set was subjected to 40 °c for 30 min and its electrical conductivity was recorded using an electric conductivity meter (c1). second set was kept in a boiling water bath (100 °c) for 10 min and its conductivity was also recorded (c2). membrane stability index (msi) = [1 (c1/c2)] × 100 leaf enzyme extraction and effect of pgpr on enzyme activity fresh leaves (2 g) were homogenized with a mortar and pestle at 4 °c in 4 ml of ice-cold 50 mm tris-acetate buffer ph 6.0, containing 0.1mm ethylene diamine tetraacetic acid (edta), 5 mm cysteine, 2 % (w/v) polyvinylpyrrolidone (pvp), 0.1mm phenyl methyl sulphonyl fl uoride (pmsf) and 0.2 % (v/v) triton x-100. the homogenate was centrifuged at 14,000 × g for 20 min and the supernatant fraction was fi ltered though sephadex g-25 columns equilibrated with the same buffer used for homogenisation. protein concentration was determined by taking optical density at 595 nm by spectrophotometer (hitachi-220), using bovine serum albumin as a standard (bradford, 1976). nitrate reductase activity nitrate reductase activity in leaves, roots and nodules was determined using the method of sym (1983). fresh plant ma terial (0.5 g) of the plant was homogenized in 5 ml phosphate buffer (ph, 7.0). after treating the sample extracts with 1 % sulphanilamide in 3 n hcl and 0.02 % n(1-naphthyl ethylene diamine dihydrogenchloride), the optical density was read at 542 nm on a spectrophotometer (hitachi220) and the nra was calculated from a standard curve established with nano2 concentrations and expressed in produced µmol no2 h-1 g-1 fw. all tests had been carried out in triplicate. ascorbate peroxidase activity ascorbate peroxidase activity was estimated according to the method of nakano and asada (1981). the reaction mixture consisted of 0.05 mol l-1 na-phosphate buffer (ph 7), 0.5 mmol l-1 ascorbate, 0.1 mmol l-1 edta.na2, 1.2 mmol l-1 h2o2 and 0.1 ml enzyme 170 y. jha, r.b. subramanian rhizobacteria regulate paddy physiology under salinity extract in a fi nal assay volume of 1 ml. the increase in absorption at a290 was recorded for 3 min. the concentration of oxidized ascorbate was calculated using extinction coeffi cient (= 2.8 mm cm-1). one unit of apx was defi ned as 1 mmol ml-1 ascorbate oxidized per minute. all tests had been carried out in triplicate. data analysis data was subjected to analysis of variance (anova) using a statistical computer package sas to determine whether the treatments effects were signifi cant. the treatment and variety means were separated using the least signifi cant differences (lsd) test. results identifi cation of isolated isolates biochemical and pcr amplifi cation of 16s rdna indicate that isolated organisms are p. pseudoalcaligenes and bacillus pumilus respectively having ncbi data bank accession nos. eu921258 and eu921259 respectively. effect o pgpr on plant growth parameters under salinity in control plants (without nacl), combination of both p. pseudoalcaligenes and b. pumilus enhanced the root length as well as root hair development (fig. 1), but no anatomical change was observed in the xylem vessels under salinity (fig. 2). the overall results obtained from the present study indicate that inoculation of the two pgprs viz. p. pseudoalcaligenes and b. pumilus either alone or in combination led to recovery of the plants from the saline stress (tab. 1). in control plants (without nacl) the combination of both p. pseudoalcaligenes and b. pumilus enhanced the root length to 39 %, even upto 1 % nacl root length increased by 22-29 % compared to noninoculated plants. chlorophyll content under non-saline state increased by 4.3 %, but under salinity it decreased both in inoculated as well as noninoculated plants. under salinity it decreased from 0.4-35 % in plant inoculated with both p. pseudoalcaligenes and b. pumilus in compared to pure control plants (at non-saline and non-inoculated), while in non-inoculated plants under salinity a decrease by 72 % was observed. stomatal conductance also increased by 57 % at non-saline state and 19 % at 0.5 % nacl, while a marginal decrease of 5.6 % was recorded at 1 % nacl and 65 % decreased at 2.5 % nacl in plant inoculated with both p. pseudoalcaligenes and b. pumilus in pure control plants. the concentration of n-substance was always higher in the plants inoculated with both the pgprs in saline condition and a gradual increase of 3 % in n2 concentration was recorded at 1 % nacl. in non-inoculated plants a decreased of 18 % 54 % was recorded at higher salinity state compared to plants at non-saline state. while in inoculated plants 7.6 % enhanced n2 concentration was recorded at 1 % nacl and gradually decreased upto 46 % compared plants at non-saline state. rwc (relative water content) in inoculated plants, increased by 2957 % at non saline state but under salinity, it increased by 10-14 % in plant inoculated with both p. pseudoalcaligenes and b. pumilus fig. 1: effect of inoculation of p. pseudoalcaligenes and b. pumilus on the root morphology (a) at 1.5% salinity, (b) at non saline state and (c) in non inoculated plant after 7 days of plantation. (p = p. pseudoalcaligenes, b = b. pumilus and c = control). fig. 2: (a) effect of inoculation of p. pseudoalcaligenes and b. pumilus on the root xylem (2a) at 1.5% salinity, (2b) at non-saline state, (2c) in non-inoculated at non saline state. the scale bar = 25um (micron). images are taken under 100x and the scales are calculated according to 100x magnifi cation. rhizobacteria regulate paddy physiology under salinity 171 compared to non-inoculated control. membrane stability index signifi cantly increased in inoculated plant both under non-saline as well as under salinity. msi increased by 2.5-5 % at non-saline state and 6.7-9 % under salinity in the plant inoculated with pgpr compared to non-inoculated control plants. effect of pgpr on enzymes activity nitrate reductase activity was signifi cantly (p ≤ 0.0001) inhibited by nacl treatment in non-inoculated control plant and inhibition increased progressively with an increase in nacl concentration (fig. 3). a decrease of 10-48 % was recorded in non-inoculated plants at different salinity compared to pure control (non-inoculated plants at non-saline condition). while plants inoculated with pgpr result in increased nitrate reductase activity under saline as well as non saline condition. the highest nitrate reductase activity was tab. 1: effect of pgpr on the root length, chlorophyll content, stomatal conductance, rwc and membrane stability index of paddy variety gj-17 at fi ve different levels of salinity (n = 5). % salinity treatment root length total stomatal n rwc % membrane of irrigation chlorophyll conductance [g kg -1] stability water water water content [%] [mol m-2 s-1] index 0.0 % 1. no inoculation 0.192 cd 43.3d 0.71d 19.1 d 90.2d 82.4cd nacl 2. inoculation with b. pumulis 0.211c 44.1c 0.84bc 20.4c 93 bc 84.1bc control 3. inoculation with p. pseudoalcaligenes 0.254ab 44.9ab 0.92b 23.8ab 94.7ab 84.7b 4. inoculation with b. pumulis+ 0.263a 45.2a 1.12a 24.2a 96.3a 86.3a p. pseudoalcaligenes 0.5 % 1.no inoculation 0.184cd 39.8cd 0.54cd 22.4cd 78.1cd 74.5cd nacl 2. inoculation with b. pumulis 0.197c 40.3bc 0.62bc 23.1c 81.4 bc 76.2bc 3. inoculation with p. pseudoalcaligenes 0.229b 41.2b 0.76b 26.3b 83.1b 77.1b 4. inoculation with b. pumulis+ 0.235a 43.1a 0.85a 27.3a 86.1a 79.3a p. pseudoalcaligenes 1.0 % 1.no inoculation 0.167d 36.1cd 0.36d 23.1cd 65.4 cd 59.1 cd nacl 2. inoculation with b. pumulis 0.187c 37.2c 0.45c 24.3c 67.2 bc 61.2 bc 3. inoculation with p. pseudoalcaligenes 0.231b 38.3b 0.58b 27.2b 68.7 ab 62.1 ab 4. inoculation with b. pumulis+ 0. 248a 39.2a 0.67a 29.4a 69.8 a 64.1 a p. pseudoalcaligenes 1.5 % 1.no inoculation 0.154cd 32.2cd 0.23cd 18.3cd 43.1 d 38.1 cd nacl 2. inoculation with b. pumulis 0.161c 33.3c 0.31c 19.5c 45.2 bc 39.4 bc 3. inoculation with p. pseudoalcaligenes 0.189ab 34.1ab 0.39 b 22.3ab 46.7 b 40.7 ab 4. inoculation with b. pumulis+ 0.203a 34.9a 0.42a 23.2a 49.1 a 41.6 a p. pseudoalcaligenes 2 % 1.no inoculation 0.145 c d 29.3cd 0.15d 14.4d 31.1 cd 31.0 cd nacl 2. inoculation with b. pumulis 0.151c 30.1c 0.22bc 15.3c 32.4 bc 32.4 bc 3. inoculation with p. pseudoalcaligenes 0.179b 31.3b 0.28b 17.1ab 33.8 ab 33.6 ab 4. inoculation with b. pumulis+ 0.187a 32.2a 0.31a 17.9a 35.4 a 34.1 a p. pseudoalcaligenes 2.5 % 1.no inoculation 0.113d 25.4cd 0.07d 10.2cd 21.2 cd 22.1 cd nacl 2. inoculation with b. pumulis 0.127c 26.3c 0.13bc 11.3c 22.1 bc 22.8 bc 3. inoculation with p. pseudoalcaligenes 0.149ab 27.2ab 0.18ab 13.6b 23.6 ab 23.4 ab 4. inoculation with b. pumulis+ 0.157a 27.9a 0.23a 14.5a 24.2 a 24.5 a p. pseudoalcaligenes for each parameter, values in columns followed by the same letter are not signifi cantly different at (p≤0.05). fig. 3: effect of inoculation with b. pumilus, p. pseudoalcaligenes alone and in combination on ascorbate peroxidase activity in paddy rice variety gj-17 at fi ve different levels of salinity (n = 5). 172 y. jha, r.b. subramanian rhizobacteria regulate paddy physiology under salinity recorded in the plants grown in soil having 2.5 % nacl and inoculated with both the isolates. an increase of 6-18 % was observed in the plants inoculated with the pgpr at non saline condition. the pgpr inoculated plants showed increase in nitrate reductase activity by 4-45 % in plant inoculated with b. pumilus, 5-53 % in plants inoculated with p. pseudoalcaligenes and 4-50 % in plants inoculated with both the pgpr at different salinity level compared to plants at non saline condition. ascorbate peroxidase activity showed that there is a gradual increase in activity as the concentration of nacl in the soil increased (fig. 4). the highest ascorbate peroxidase activity was observed in leaves of non-inoculated plants exposed to 2.5 % nacl. the highest of ascorbate peroxidase activity was recorded in the non-inoculated control plant leaves at highest salinity level of 2.5 % which was 40 % high compared the plants grown under at non-saline condition. inoculation of plants with pgpr resulted in 15-40 % decrease in activity compared to non-inoculated plants under non-saline condition. among the two pgpr applied, p. pseudoalcaligenes showed reduction of 9-27 % and b. pumilus showed reduction of 2-15 %, but combination of both showed reduction of 16-40 % ascorbate peroxidase activity in all treatment compared to non-inoculated control. in the present study, chlorophyll content was also signifi cantly higher in plants inoculated with p. pseudoalcaligenes and b. pumilus at non saline condition, and different level of salinity, because these isolates infl uence better root development which enhance water absorption and retention, fi ndings are accordance with han and lee (2005). decrease of chlorophyll content in non-inoculated plant under salinity has been considered to be a typical symptom of oxidative stress and may be the result of pigment photo-oxidation, chlorophyll degradation or lack of chlorophyll synthesis (smirnoff, 1993). inoculations of plants with pgpr always have higher n-concentration under saline and non-saline states as present study is supported by bashan et al. (2004). azospirillum could result in signifi cant changes in various growth parameters, such as increase in plant biomass, nutrient uptake, tissue n content, plant height, leaf size and root length of cereals (bashan et al., 2004). an interesting observation of this study was that plants inoculated with pgpr under non saline as well as at different salinity levels has greater rwc and membrane stability index which is accordance with sandhya et al. (2010). an increased level of leaf rwc in paddy under salinity suggests the role of osmoprotectants in preventing cell injury from salt stress-induced dehydration, as suggested by yancy et al. (1982). non-inoculated plants has decreased rwc and membrane stability index with increased salinity. membranes are main loci affected under water stress conditions. the lower membrane stability index refl ects the extent of lipid peroxidation, which in turn is a consequence of higher oxidative stress due to saline stress conditions. the nitrate and nitrogen nutrition is very important for plant growth (raab and terry, 1994). nitrate reductase is not sensitive to osmotic effect, but sensitive to na+ ions (gouia et al., 1994), the nitrate reductase activity (nra) was used as an indicator of the damaging effects of nacl. in present study, nitrate reductase activity increased in inoculated plant may be due to high concentration of nitrogen under saline and non-saline conditions, resulted in production of high amount of nh3. our results are accordance with the fi ndings of hamdia et al. (2002) showed that nitrate reductase activity decreased with increasing salt stress. nitrate reductase activity increased the concentration of nh3, used by a-ketoglutarate to form glutamic acid (el-komy et al., 2003). high glutamic acid concentration may be used as a sink for the synthesis of other amino acids and proteins (wang et al., 1999) or perhaps it may be directly used in osmoregulation (hsu et al., 1999). in our previous study, we also observed that these pgpr help in accumulation of osmoprotectants in paddy plants under salinity (jha et al., 2011). ascorbate peroxidases also play a vital role in plant defense against oxidative stress like superoxide reductase. ascorbate peroxidases are the key enzymes for scavenging hydrogen peroxide in chloroplast and cytosol of plant cells (asada, 1992). they catalyze the oxidation of ascorbate by hydrogen peroxide and give monodehydroascorbate radical. in present study inoculation of pgpr decreased the ascorbate peroxidase activity under saline and non-saline state are accordance with the sandhya, who reported that pseudomonas spp. treated seedlings showed decrease in ascorbate peroxidase activity, glutathione peroxidase activity, and catalase activity was higher compared to un-inoculated seedlings (sandhya et al., 2010). the decreased ascorbate peroxidase activity indicates that plants inoculated with pgpr were more relaxed under salinity. these enzymes proportions paralleled the changes in electrolyte leakage and were accordance with membrane stability function of these enzymes. inoculation probably protected the seedlings where leaves could reduce water transpiration by curling and stoma regulation. the present study showed that p. pseudoalcaligenes, an endophytic discussion water stress caused by high salinity, is one of the serious factors to limit crop productivity. osmotic stress caused by salinity is one of the major abiotic factors limiting crop productivity because it affects almost plant’s all functions. in the present study, plants inoculated with pgpr under non saline as well as at different salinity levels have greater root length and denser root hair is supported by bashan et al., 2004. bacillus is very consistent in improving different root parameters such as rooting performance, root length and dry matter content of root in mint is reported by kaymak et al. (2008). the presence of denser root hairs has increased the surface area of root, which enhance water as well as mineral uptake (raven and edwards, 2001). an improved root growth was proposed as a possible mechanism by which pgpr affects plant growth (fallik et al., 1994). salinity rapidly decreases stomatal conductance, resulted in reduced transpiration rate. stomata closure is known to be an effective mechanism for economical water utilization under salt stress and for limitation of the harmful salt ions uptake (hasegawa et al., 2000). however inoculation of pgpr increased stomatal conductance under saline and non saline state to improve leaf water potential in adverse condition, observation is supported by mia et al. 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shukla, d.s., 1997: increased antioxidant enzyme activity in response to drought and temperature stress related with stress tolerance in wheat genotypes. abstract, national seminar (issp), iari, new delhi. sandhya, v., ali, s.k.z., grover, minakshi., reddy, gopal, venkateswarlu, b., 2010: effect of plant growth promoting pseudomonas spp. on compatible solutes, antioxidant status and plant growth of maize under drought stress. plant growth regul. 62, 21-30. schonfeld, m.a., johnson, r.c., carver, b.f., mornhinweg, d.w., 1988: water relations in winter wheat as drought resistance indicator. crop sci. 28, 526-531. smirnoff, n., 1993: the role of active oxygen in the response to water defi cit and desiccation. new phytol. 125, 27-58. sym, g.j., 1983: optimization of the in vivo assay conditions or nitrate reductase in barley. j. sci. food agric. 35, 725-730. wang, h.l., lee, p.d., liu, f.l., su, j., 1999: effect of sorbitol induced osmotic stress on the changes of carbohydrate and free amino acid pool in sweet potato cell suspension cultures. bot. bull. acad. sinica 40, 219-225. yancey, p.h., clark, m.e., hand, s.c., bowlus, r.d., somero, g.n., 1982: living with water stress, evolution of osmolyte systems. science 217, 1212-1222. address of the authors: n.v. patel college of pure & applied sciences, s.p. university, v.v. nagar, anand (gujarat), india. tel.: +91-9426282152, e-mail: yachanajha@ymail.com prof. r.b. subramanian, brd school of biosciences, sardar patel university, po box 39, v.v, nagar388120 (gujarat) india, tel.: +91-2692-234402/234412, fax: +91-2692-236475/237258, e-mail: subramanianrb@gmail.com. journal of applied botany and food quality 87, 80 86 (2014), doi:10.5073/jabfq.2014.087.012 1department of agronomy, tabriz branch, islamic azad university, tabriz, iran 2department of agronomy and plant breeding, university of kurdistan, sanandaj, iran 3department of agronomy, sanandaj branch, islamic azad university, sanandaj, iran response of chickpea (cicer arietinum l.) to exogenous salicylic acid and ascorbic acid under vegetative and reproductive drought stress conditions siamak farjam1, adel siosemardeh2, hamdollah kazemi-arbat1, mehrdad yarnia1, asad rokhzadi3* (received september 23, 2013) * corresponding author summary drought stress is a critical limiting factor in plant growth. substances such as salicylic acid (sa) and ascorbic acid (aa) may enhance drought tolerance in plants. therefore this experiment was aimed to study the growth and physiological response of two chickpea cultivars (ilc482 & kurdistan) to sa and aa foliar application in four different conditions of drought stress including: control or well watered, vegetative drought stress, reproductive drought stress and complete drought stress (vegetative and reproductive drought stress). results showed that plant biomass was significantly increased through sa and aa application. sa spraying in complete drought stress condition significantly increased the proline content of leaves. foliar application of sa and aa both in well watered and water deficit treatments reduced the electrolyte leakage in leaves. generally it was concluded that sa and aa have the potential of diminishing injurious effects of drought stress and promoting crop productivity. introduction chickpea (cicer arietinum l.) is the third most important pulse crop in the world (jalota et al., 2006). in iran chickpea is the most important grain legume and more than 90% of chickpea is grown under rainfed conditions where the crop is subjected to terminal drought stress leading to high decrease in grain yield (soltani et al., 2001; anonymous, 2013). water deficiency stress can confuse plant growth through influencing cell turgor, stomatal closure and enzymatic processes which are directly under the control of water potential. improvement of drought tolerance in plants is possible in various ways such as plant breeding techniques and application of plant growth regulators. utilization of growth regulating substances including salicylic acid, ascorbic acid and jasmonic acid is easier and cheaper than the long-term and costly methods of plant breeding (el-tayeb, 2005). salicylic acid is an antioxidant compound which prohibits the activity of reactive oxygen species (ros) (hayat et al., 2010). ros such as hydrogen peroxide, hydroxyl radical and superoxide can damage mitochondria and chloroplasts. salicylic acid, as an endogenous phenolic growth regulator, adjusts the activity of antioxidant enzymes, enhances plant resistance to abiotic stresses and interferes in physiological processes such as stomatal conductance in plants (hayat et al., 2010). salicylic acid is involved in water relations of plant cells in abiotic stress conditions and it is well known that sa diminishes the impairments arisen from water deficiency in plants (hussain et al., 2009). ascorbic acid is found in the cytosol, chloroplasts, vacuoles and mitochondria of plant cells. it has a great effect on physiological processes such as cell division, plant growth and the biosynthesis of cell wall, metabolites and phytohormones. moreover ascorbic acid plays a vital role in renovation of chloroplast and mitochondrion membranes (barth et al., 2004; pavet et al., 2005; barth et al., 2006). arfan et al. (2007) reported that foliar application of salicylic acid increased grain yield and growth of spring wheat in water deficit conditions. shirani bidabadi et al. (2012) reported that banana shoot tips responded positively to the application of salicylic acid by showing significant increase in proline and chlorophyll contents under drought stress conditions. rosales et al. (2006) reported that ascorbic acid increased fresh weight of leaves and decreased the damages arised from free radicals under drought stress conditions. sa and aa can diminish the injuries in cell membranes through enhancing the antioxidant potential of plant during drought stress period (avancini et al., 2003; athar et al., 2008). in our country, there are insufficient research studies regarding the effects of plant growth regulators on agronomic and physiological characteristics of crop plants in drought stress conditions.therefore, this experiment was aimed to study the reaction of two chickpea cultivars to the application of two plant growth regulators (i.e salicylic acid and ascorbic acid) under drought stress conditions. materials and methods experimental site this study was carried out during the growing season of 2010-2011 at the agricultural research station of gerizeh, sanandaj, west of iran. this research station is located at latitude of 35° 16’ n and longitude of 47° 1’ e with an altitude of 1375 m above sea level. the long-term values of mean temperature and annual precipitation in this location are 13.4 °c and 491 mm respectively. the texture of soil was clay loam with the chemical properties of: ph 7.7, organic carbon 1.53 %, electrical conductivity 0.65 ds m-1, available p and k, 7 and 220 ppm respectively, total n 0.14 % and tnv 10 %. experiment layout, plant material and growing conditions the operations of field preparation including mouldboard ploughing and disking were done at the proper time before sowing. the trial was laid out in a split-plot factorial arrangement with randomized complete block design in four replications. four levels of drought stress including: 1. control (well watered, 2. drought stress at vegetative stage (not watered from branching to 50 % flowering stage), 3. drought stress at reproductive stage (not watered at 50 % flowering stage) and 4. complete drought stress (not watered at vegetative and reproductive stages) were compared as main plots. the factorial combinations of cultivar (with two levels) and pgr spraying (with three levels) were placed in sub-plots. two chickpea genotypes were: ilc482 cultivar (drought tolerant) and kurdistan landrace (drought susceptible) and pgr spraying levels consisted of: 1. control or spraying with distilled water 2. salicylic acid spraying at the rate of 200 ppm, and 3. ascorbic acid spraying at the rate of 100 ppm. chickpea seeds were obtained from agricultural & natural resources research center of kurdistan, iran. from sowing to the start of branching, all experimental plots were fully watered to achieve best establishment of the plants. foliar spraying of the response of chickpea to salicylic acid and ascorbic acid 81 plants was performed at the vegetative stage of fifth lateral branch emergence. experimental plots consisted of planting rows 9 m in length with 25 cm space between rows and 10 cm between plants in each row. before sowing, the chickpea seeds were treated with 2 g l-1 benomyl fungicide to prevent soil-borne diseases, then the seeds were hand-sown on 9 april 2011. irrigation of experimental plots was done through drip irrigation system. soil moisture content was measured by gravimetric method and with consideration of essential irrigation depth for chickpea, the amount of water requirement for each plot was determined. water stress treatments were exerted at relevant developmental stages and transparent plastic covers were used to prevent rain falling on stressed plots and the covers were removed after each period of rainfall to prevent heatstroke effects on plants. crop traits measurement at maturity stage, an area of 3 m2 of central rows of each plot was hand-harvested and after separating the seeds from other parts of plant shoot, the seeds were air dried and the weight of seeds was measured by digital electric scale and converted to kg per hectare as seed yield per unit area. the remaining parts of the shoot including stems, leaves and pods shell were air dried and their total weight was recorded. the total weight of shoot plant including the weight of seeds, stems, leaves and pods shell were considered as plant biomass and converted to kg per hectare as plant biomass per unit area. for determining the pods number per plant, 10 plants were randomly harvested from central rows of each plot and their pods were counted. then the total number of pods produced by 10 plants was converted to pods number per one plant. soluble carbohydrates assessment determination of soluble carbohydrates was performed by the anthrone method (yemn and willis, 1954; sanchez et al., 1998). the concentration of carbohydrates was estimated as mg g-1 using a standard curve. proline estimation estimation of proline content was performed by the ninhydrin procedure described by bates et al. (1973). proline concentration was determined from a calibration curve and calculated as mg proline g-1. cell membrane damage assay the leakage rate from the tissues of drought-stressed plants can be taken as an index of damage to plant cell membrane (sairam et al., 1997). therefore cell membrane damage was assessed by measuring the trait of electrolyte leakage (el). measurement of electrolyte leakage was done through the method described by nayyar and gupta (2006) and lutts et al. (1996). leaf samples were washed with distilled water and placed in closed vials containing 10 ml of distilled water at 25 °c on a shaker for 24 h and subsequently electrical conductivity of the solution (l1) was recorded. the same samples were then autoclaved at 120 °c for 20 min. after cooling the solution to room temperature, the final electrical conductivity (l2) was recorded. the el was calculated as: el (%) = (l1/l2)×100. statistical analysis the measured data were analyzed statistically by analysis of variance operations using the sas computer package version 9.1 (sas institute, cary, nc). means of treatments were compared by duncan’s multiple range test at the 0.05 level of significance. results and discussion seed yield effects of drought stress on seed yield were statistically significant (p ≤ 0.01). the highest rate of seed yield (952 kg ha-1) was obtained by the control (fully watered) treatment. the mean seed yield of under stress plants decreased at the ratio of 61 % as compared with control plants (tab. 1). in well watered conditions, photosynthesis rate and assimilates production are increased which consequently results in the elevation of seed yield through increasing in seed filling rate and seed weight (rezaeyan zadeh, 2008). moreover comparison between the recorded yields in vegetative and reproductive stress treatments revealed that the yield reduction in reproductive stress treatment was more pronounced compared to vegetative stress, indicating the vulnerability of chickpea yield to terminal drought stress prevailing and occurring at reproductive stage of chickpea development. terminal drought stress is considered as a primary constraint to chickpea productivity in countries such as iran, where the crop is generally sown after the main rainy season and grown on stored soil moisture (serraj et al., 2004). reduction in chickpea seed yield by means of terminal drought stress has been previously reported by other authors (behboudian et al., 2001; fallah et al., 2005). chickpea cultivars were not significantly different with respect to seed yield (tab. 1). likewise the application of plant growth regulators did not affected the seed yield of chickpea, even though the application of ascorbic acid resulted in some improvement in seed yield as compared with other treatments of foliar spraying (tab. 1), suggesting that aa can be considered as a beneficial plant growth regulator in field condition. ascorbic acid is considered as one of the plant responses in drought stress conditions which is involved in detoxification of reactive oxygen species (guo et al., 2005). pods number per plant the results showed that drought stress significantly affected the pods number per plant (tab. 1). the maximum number of pods per plant was obtained by the well watered treatment. pods number was decreased at the rate of 54 % (in vegetative stress treatment) and 58 % (in reproductive stress treatment) comparing to control treatment. whereas complete drought stress resulted in 66 % decrease tab. 1: effects of drought stress, cultivar and plant growth regulators on seed yield, pods number per plant and biomass of chickpea. treatment seed yield pods number plant biomass (kg ha-1) per plant (kg ha-1) drought stress control (well watered) 952.0 a 75.0 a 2328.8 a vegetative stress 445.0 b 34.7 b 1004.4 b reproductive stress 356.8 bc 31.4 b 815.4 bc complete stress 303.5 c 25.7 b 541.7 c cultivar ilc482 536.8 a 44.6 a 1154.5 a kurdistan 491.8 a 38.8 b 1190.7 a plant growth regulator control 485.0 a 39.4 a 1027.3 b salicylic acid 504.4 a 40.1 a 1239.2 a ascorbic acid 553.5 a 45.6 a 1251.2 a a values followed by the same letters in a group of a column are not significantly different at p ≤ 0.05 according to duncan’s multiple range test. 82 s. farjam, a. siosemardeh, h. kazemi-arbat, m. yarnia, a. rokhzadi in pods number as compared to control or fully watered treatment (tab. 1). similar results obtained by mafakheri et al. (2010) showed that drought stress had considerable effects on the number of pods in chickpea plants. in drought stress conditions, chickpea plants are confronted with reduction in the number of flowers and pods, in addition to abortion of flowers and pods and their subsequent abscission take place, consequently the number of pods per plant will be reduced. it is reported that drought stress is one of the most important factors responsible for yield reduction in chickpea which is mostly related to pod abscission. when leaves senescence is induced as the result of drought stress, the abscission of pods will take place (siddique and sedgley, 1986). there was a significant difference between cultivars regarding pods number per plant. the pods number recorded by the cultivar ilc482 was 15 % more than which recorded by the kurdistan landrace (tab. 1). in a three years study kanouni et al. (2003) declared that ilc482 cultivar with regard to its high seed yield and low fluctuations in yield across different years, may be introduced as a high yielding and compatible genotype in the region. saman et al. (2010) by studying the effects of terminal drought stress on chickpea genotypes showed that the highest number of pods per plant was recorded by ilc482 cultivar. plant biomass analysis of variance showed that the plant biomass was significantly affected by drought stress (p ≤ 0.01). the greatest amount of plant biomass was produced by control (fully watered) treatment. vegetative drought stress led to 57 % reduction in biomass as compared with control, whereas the reproductive and the complete drought stress treatments resulted in 65 and 77 % reduction in biomass compared to control respectively (tab. 1). similarly mwanamwenge et al. (1999) through studying the effects of water deficit stress during floral initiation, flowering and podding on growth traits of faba bean genotypes, declared that the most reduction in dry matter production was recorded by podding stress treatment, suggesting that the plants had more opportunity to recover from the loss of dry matter production during the earlier imposed drought stress treatments as compared with terminal drought stress. the effect of growth regulators on biomass yield was significant (p ≤ 0.05). the application of sa and aa resulted in a remarkable increase in plant biomass compared to control plants (tab. 1). the studies of zeid et al. (2009) on wheat plants indicated that the application of aa diminished the injurious effects of terminal drought stress and significantly increased root and shoot dry weight of plants. helsper et al. (1982) reported that aa application was effective in activation of carbon fixation enzymes. foliar application of sa in drought stress and non-stress conditions led to a significant increase in dry weight of susceptible and drought tolerant cultivars of sunflower (noreen and ashraf, 2008). sa adjusts the hormonal conditions of the plants and increases the levels of auxin and cytokinin in non-stress conditions. the application of sa on wheat plants increased the amounts of auxin and aba and prevented the reduction of cytokinin in drought stress conditions (shakirova et al., 2003). the application of acetylsalicylic acid (asa) and gentisic acid (gta) or other analogous of sa elevated the leaf area and dry matter in corn and soybean (khan et al., 2003). proline content the greatest rate of proline content was recorded in plants under complete drought stress, on the other hand the recorded content of proline in plants under vegetative drought stress was low similar to well watered or control plants (fig. 1). considering the fact that proline assessment in this experiment was performed at reproductive stage of the plant, the effect of vegetative drought stress has probably been ameliorated at the time of proline assessment, and accordingly proline content has been reduced and reversed to a level similar to non-stress condition. residual water resources from the preceding year in the soil are easily absorbed by chickpea roots at the beginning of growing season, besides the mean temperatures had not risen at this period of season and chickpea plants did not confront serious drought stress during vegetative phase. on the other hand, the plant at its reproductive phase encounters more drought stress conditions as the result of mean temperature elevation, solar radiation severity, evaporation increasing, expanding of leaves area and branching rate elevation. in such condition the harmful effects of drought stress on the plant could be effectively decreased by proline accumulation. in this research proline content was expressively increased in the plants under reproductive and complete drought stress conditions (fig. 1). proline accumulation in plants during drought stress has been similarly reported by other authors (hare and cress, 1997; verbruggen and hermans, 2008; szabados and savoure, 2010). inez and montagu (1995) also reported that proline accumulation in plant cells will result in preservation of turgor pressure and reduction of plasma membrane damage, hence osmotic adjustment is counted as an adaptive mechanism in drought tolerance. it is reported that proline content of leaves at both vegetative and reproductive stages in drought sensitive and tolerant chickpea cultivars may be increased (mafakheri et al., 2010). even though chiang and dandekar (1995) stated that proline accumulation resulted from drought stress conditions is more prevalent at the flowering stage than the vegetative stage. however the proline content depends on plant phenology, and the position and age of leaves. fig. 1: effect of drought stress on proline content of chickpea leaves. vertical bars indicate the standard error of means. mean values with the same letter are not significantly different at p ≤ 0.05 according to duncan’s multiple range test. the proline accumulation rate in kurdistan landrace was significantly higher than what was recorded for the ilc482 cultivar. interaction effects of drought stress and genotype on proline content of leaves showed that the rate of proline accumulation by kurdistan cultivar in all levels of drought stress was higher than ilc482 cultivar (fig. 2). moreover, complete drought stress led to a remarkable increase of proline content in kurdistan cultivar. in contrast to response of chickpea to salicylic acid and ascorbic acid 83 that, the proline content of ilc482 at the complete drought stress level remained almost constant compared to the reproductive stage drought stress (fig. 2). considering the higher tolerance of ilc482 to drought stress with having lower content of proline, it is suggested that proline may be an appropriate indicator for assessing drought tolerance in chickpea. similarly, other authors (hanson et al., 1979; premachandra et al., 1995; sundaresan and sudhakaran, 1995) have reported the occurrence of high proline accumulation in susceptible cultivars. it is reported that with increasing the duration and severity of drought stress, the proline content is increased depending on the plant species (larcher, 2001). proline is known as a beneficial solute which is accumulated in many plant species during water limitation and other stress conditions (verslues and sharma, 2010) that plays several roles such as: osmotic adjustment, osmoprotection, free radical scavenging and protection of macromolecules from denaturation (vandruscolo et al., 2007). ascorbic acid application had no effects on proline content in comparison with control at all levels of drought stress, but salicylic acid spraying resulted in a significant increase in proline concentration of chickpea leaves in complete drought stress treatment (fig. 3). shakirova et al. (2003) reported that salicylic acid application increased the accumulation of proline and preservative proteins in water deficit stress conditions. baghizadeh et al. (2009) showed that salicylic acid application accumulated more proline in the leaves of okra in comparison to ascorbic acid application. salicylic acid has a protective role and induces high proline accumulation under drought stress conditions (umebese et al., 2009). content in salicylic acid application treatment was significantly lower than control and ascorbic acid treatment (fig. 5). similarly bakry et al. (2012), through studying the effects of foliar application of sa on two flax varieties, showed that soluble sugars content in one of the studied varieties of flax plant was significantly decreased as the result of sa foliar application, compared with untreated plants, on the other hand sa spraying caused significant increases in polysaccharides and total carbohydrates. khodary (2004) and ghafiyesanj et al. (2013) also declared that exogenous application of sa on maize and wheat plants significantly reduced soluble sugar content and vice versa elevated the polysaccharides and insoluble sugars content. it has been proposed that sa treatment probably activate the consumption of soluble carbohydrates to form new cell constituents as a mechanism to promote plant growth (khodary, 2004). soluble carbohydrates means comparison of the effects of drought stress treatments on the content of soluble carbohydrates of leaves indicated that the content of soluble carbohydrates was decreased by drought stress. the lowest concentration of soluble carbohydrates was recorded in reproductive drought stress treatment (fig. 4). cruz-aguado et al. (2000) reported that soluble carbohydrates content was decreased at terminal stages of crop development in an experiment on wheat plants. in drought stress conditions, plants generally confront a decrease in carbohydrates arising from the reduction of photosynthesis rate and elevation of respiration rate (blum, 1998). soluble carbohydrates fig. 2: interaction effects of drought stress and genotype on proline content of chickpea leaves. vertical bars indicate the standard error of means. mean values with the same letter are not significantly different at p ≤ 0.05 according to duncan’s multiple range test. fig. 3: interaction effects of drought stress and plant growth regulators on proline content of chickpea leaves. vertical bars indicate the standard error of means. mean values with the same letter are not significantly different at p ≤ 0.05 according to duncan’s multiple range test. fig. 4: effect of drought stress on soluble carbohydrates of chickpea leaves. vertical bars indicate the standard error of means. mean values with the same letter are not significantly different at p ≤ 0.05 according to duncan’s multiple range test. well vegetative reproductive complete watered stress stress stress well vegetative reproductive complete watered stress stress stress 84 s. farjam, a. siosemardeh, h. kazemi-arbat, m. yarnia, a. rokhzadi cell membrane damage the highest rates of damage in cell membrane (as electrolyte leakage) were recorded by reproductive and complete drought stress treatments respectively (fig. 6). assessment of cell membrane damage was performed at reproductive stage of chickpea plants, therefore the possible damage to cell membrane under vegetative drought stress treatment, have probably been alleviated at the time of measurement and reached to a level similar to control treatment. sairam et al. (1997) similarly showed that drought stress decreased the index of membrane stability of wheat genotypes at anthesis stage. membrane damage is suggested as a consequent incident caused by reactive oxygen species (alscher et al., 1997; becana et al., 1998). water deficit conditions can result in oxidative stress leading to increasing lipid peroxidation which will be subsequently terminated to membrane injury (sairam and saxena, 2000). foliar application of pgrs both in well watered and water deficit treatments resulted in the reduction of electrolyte leakage in chickpea leaves (fig. 7). the lowest rate of electrolyte leakage was recorded by aa application in non-stressed (well watered) condition. the positive effects of sa and aa on cell membranes at vegetative stress condition was similar, however at reproductive stress condition the protective effect of aa was more remarkable than that of sa (fig. 7). in the present study, electrolyte leakage in well watered chickpea plants was dramatically decreased as the result of aa application. in non-stressed conditions, the cell membranes are usually protected from free radical injuries, but the spring sown chickpea plants in spite of receiving enough irrigation water may confront drought stress at the terminal crop growth stage because of coinciding with high temperatures. in such conditions the application of ascorbic acid may enhance the membrane stability by its antioxidant properties (khan et al., 2003; guo et al., 2005). conclusion according to the results of this study, the reduction of yield parameters by reproductive stress was more pronounced as compared to vegetative stress, indicating the susceptibility of crop growth and yield to terminal drought stress conditions. plant biomass was significantly increased through sa and aa foliar spraying. supplying the plants with sa in complete drought stress condition resulted in a high accumulation of proline in chickpea leaves. the recorded rate of proline accumulation by ilc482 cultivar in all levels of drought stress was lower than kurdistan cultivar, indicating the higher tolerance of ilc482 to drought stress. electrolyte leakage in leaves tissue as a sign of cell membrane damage was decreased by foliar application of pgrs both in well watered and drought stress treatments, even though the positive effects of aa on cell membrane stability was more expressive. it is generally concluded that sa and aa may enhance the growth of chickpea because of their protective traits, and it could be suggested that the application of these pgrs in stressed plants will resulted in diminishing the injurious effects of drought stress and consequently the promotion of crop productivity. however, more studies about their modes of action in various field conditions are needed. fig. 5: effect of plant growth regulators on soluble carbohydrates of chickpea leaves. vertical bars indicate the standard error of means. mean values with the same letter are not 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arabidopsis book vol. 8. yemn, e.w., willis, a.j., 1954: the estimation of carbohydrates in plant extracts by anthrone. biochem. j. 57, 508-514. zeid, f.a., el shihy, o., ghallab, a.e.m., ibrahim, f.e.a., 2009: effect of exogenous ascorbic acid on wheat tolerance to salinity stress conditions. arab j. biotechnol. 12, 149-174. address of the corresponding author: dr. asad rokhzadi, department of agronomy, sanandaj branch, islamic azad university, sanandaj, iran e-mail: asadrokh@yahoo.com journal of applied botany and food quality 89, 98 104 (2016), doi:10.5073/jabfq.2016.089.012 1 icar − directorate of groundnut research, junagadh, gujarat, india 2 icar − directorate of medicinal and aromatic plants research, boriavi, anand gujarat, india water deficit stress affects photosynthesis and the sugar profile in source and sink tissues of groundnut (arachis hypogaea l.) and impacts kernel quality k. chakraborty1, m.k. mahatma1*, l.k. thawait1, s.k. bishi1, k.a. kalariya1, 2, a.l. singh1 (received august 14, 2015) * corresponding author summary water deficit stress conditions disturb photosynthetic activity of plants and thereby affect further growth and the mobilization of as similates towards sink tissues. the influence of mid-season drought on sugar metabolism in both source and sink tissues and its sustained effect on kernel quality across three different habit groups of groundnut was investigated. the experiment was conducted in kharif 2012 and water deficit stress was created by withholding irrigation for 40 days between 30 -70 days after sowing under rainout shelter to simulate mid-season drought condition. imposition of water deficit stress reduced net photosynthesis rate, which significantly altered the sugar profiles in leaf. the content of glucose, fructose and sucrose decreased in the leaf tissue, whereas the content of sugar alcohol (inositol and mannitol) and trehalose increased. the sugar profile of the sink tissue (kernel) was also altered under stress but changes were slightly different. the sugar alcohol and oligosaccharides (rfos) showed significant increase, but the level of monoand di-saccharides did not show significant change. the results suggested different drought tolerance strategies in source and sink tissues. the kernel quality was also affected under stress with lower oil and higher protein content. the content of oleic acid was reduced, while linoleic acid increased resulting in a decrease of the o/l ratio and oil stability. alteration of quality traits was least in spanish genotypes, suggesting a relatively better tolerance of this group for water deficit stress. introduction groundnut is an important oil seed legume grown worldwide mostly in arid and semi arid region. over 60 % of global groundnut production is crushed for extraction of oil for edible and industrial uses, while 40 % is consumed in food uses and as seed for sowing the next season crop (birthal et al., 2010). for the food industries, nutritional composition (oil, protein, fatty acid, amino acids and sugars) of groundnut is equally important with physical and sensory characteristics. the groundnut, mostly grown as rainfed in the arid and semi-arid regions is highly vulnerable to drought stresses of varying duration and intensity due to uncertain rainfall pattern (singh et al., 2013). depending on the time of occurrence, drought has been characterized as early season, mid-season, and end-of-the-season drought. mid and end-of-the-season droughts are critical as they affect the pod yield and quality (janila et al., 2013). water deficit stress during pod-development phase is detrimental to several physiological and biochemical processes (nautiyal et al., 1991).water stress conditions disturb photosynthetic activity of plants and thereby affects further vegetative growth and the mobilization of assimilates towards storage or sink tissues. sugars in plants, derived from photosynthesis, act as substrates for energy metabolism and the biosynthesis of complex carbohydrates, providing sink tissues with the necessary resources for growth and development. responses to a specific stress can vary with the genotype, but some general reactions occur in all. under sugar depleted condition, substantial physiological and biochemical changes occur to sustain respiration and other metabolic processes (journet et al., 1986) sucrose and glucose either act as the substrates for cellular respiration or as the osmolytes to maintain cellular osmotic potential (gupta et al., 2005). sugars have also been shown to directly protect membranes and proteins in vitro, possibly by replacing water molecules and altering physical properties through the formation of hydrogen bonds (crowe et al., 1992). the production and partitioning of metabolically important non-structural carbohydrates (starch and sugar alcohols) have been reported to accumulate during drought (keller and ludlow, 1993). a linear polyhydric alcohol, mannitol, has been reported to increase in response to salt stress mostly due to the osmotic factor of salt stress than its ionic toxicity (pharr et al., 1995). expression of the mtld gene for the biosynthesis of mannitol improved tolerance to water stress in transgenic groundnut plants (bhauso et al., 2014). another important sugar alcohol which has diverse role in plant biology is myo-inositol, a six carbon cyclohexane hexitol. myo-inositol is not only required in plant growth and development, but also required as a precursor and substrate for many crucial metabolites in plants such as phytate, phosphatidylinositol, galactinol, raffinose-family oligosaccharides (rfos), ascorbate, indole acetic acid conjugate, ononitol, and pinitol. these inositol derivatives were shown to be implicated in various physiological and signal processes including plant stress adaptation (loewus and murthy, 2000; donahue et al., 2010). although, there are a few reports on the effect of drought stress on yield and kernel quality of groundnut (dwivedi et al., 1996; chakraborty et al., 2013), yet adequate information on its impact on sugar profiles of the source and sink tissues and kernel quality is not available. thus, present investigation was conducted to study the impact of mid-season drought on the sugar profile in source and sink tissues and also consequent effect on kernel quality traits. materials and methods plant material and growing condition an experiment was conducted in kharif 2012 (june-october) using 12 popular groundnut cultivars, four each from three different habit groups (spanish bunch type (sb): ak 159, drg 1, jl 286, tpg 41; virginia bunch (vb) type: gg 20, hng 10, icgs 76, kadiri 3; virginia runner (vr) type: gg 11, gg 16, csmg 84-1, somnath) at the research farm of the directorate of groundnut research, junagadh, gujarat, india. the cultivars were raised in both open field (rain-fed with protective irrigation, unstressed) and rain-out shelter (ros; imposed water deficit stress). the water deficit stress was imposed by withholding the irrigation after 30 days after sowing (das) and continued up to 70 das in the ros. samples were collected from third upper leaf in triplicate from 70 days old plants. the crop was harvested at full maturity and after curing, the kernel water deficit stress alters sugar profile in groundnut 99 samples were collected from both control and water deficit stressed plots for analysis of quality attributes. the weather condition during the study period was presented in tab. 1. due to imposition of water deficit stress by withholding irrigation for 40 days from 30-70 das, soil moisture content was reduced from 18.5 % to 10.9 % at 0-15 cm soil depth and 19.1 % to 12.3 % at 15-30 cm soil depth compared to irrigated control plot where optimum moisture level (18.5-19.1 %) was maintained throughout the crop growth period (fig. 1). these values correspond to the threshold value below which groundnut productivity is severely affected. all the cultivars studied started experiencing water deficit conditions at about 45 das, some cultivars (drg 1, kadiri 3, somnath) started a few days before. measurement of net photosynthesis rate (pn) net photosynthesis rate (pn) was measured using a portable photosynthesis system (model li-6400, li-cor, usa) between 09:3011:30 h local time. temperature was set at ambient with a stable tleaf reading. photosynthetically active radiation (par) was set at 1,650 μmol(photon) m-2 s-1 inside the cuvette, and co2 was supplied artificially to keep the concentration stable at 400 μmol m-2 s-1 inside the chamber (singh et al., 2014). oil and protein content oil and protein content of groundnut meal were determined by standard methods i.e. soxhlet and kjeldahl method, respectively. fatty acid analysis the fatty acids methyl esters (fame) of groundnut oil were prepared and analyzed by gas chromatography. in a 10 ml screw cap test tube, 200 μl oil was mixed with 3 ml hexane and kept for 1 h at room temperature with intermittent mixing using vortex. after that 3 ml of freshly prepared sodium methoxide (80 mg naoh in 100 ml methanol) was added and incubated at room temperature for 30 min. then 3 ml of 0.8 % aqueous sodium chloride was mixed with gentle shaking. solution was allowed to settle for 5 min and the upper layer of hexane containing the methyl-esters were transferred in screw capped glass vial containing 100 mg anhydrous sodium sulphate (misra and mathur, 1998). the fame (10 μl) of groundnut oil were analysed by gas chromatograph (netel india ltd., model michro 9100), using 15 % degs packed column. the oven temperature during analysis kept at 190 °c, injector temperature at 240 °c and fid detector temperature at 260 °c. carrier gas (nitrogen) flow rate was maintained at 30 ml min-1 and fuel gas (hydrogen) flow at 30 ml min-1. extraction of sugars, free amino acids and total phenolics the 500 mg of defatted flour was homogenized with 10 ml of 80 % ethanol in glass vial and kept in boiling water bath for 10 min. after that, samples were centrifuged at 5000 rpm for 10 min. extraction was repeated three times with 10 ml of 80 % ethanol and supernatants were pooled into 100 ml volumetric flasks and referred as ethanol extract hereafter. estimation of free amino acids and total phenolics the total free amino acids and total phenolics from ethanol extract were determined by using ninhydrin and folin-ciocalteu reagents respectively, as described in our earlier reports (bishi et al., 2015). briefly, for total free amino acid estimation, 0.4 ml of ethanol extract was taken in test tube. a 5 ml of ninhydrin reagent (5:12:2; 1 % ninhydrin in 0.5 m citrate buffer ph5.5: glycerol: 0.5 m citrate buffer ph 5.5) was added and mixed thoroughly. the tubes were then placed in a boiling water bath for 12 min and brought to room temperature under running water. the absorbance of the colour was read at 570 nm. the standard curve was prepared by using glycine in the range of 0-80 μg. for total phenols, one ml of ethanol extract was transferred to a test tube and evaporated till dryness. the residue was dissolved in 1.0 ml water and 0.5 ml of folin-ciocalteu reagent (1 n), was added to each test tube, mixed, and allowed to stand for 3 min. subsequently, 2 ml of 20 % na2co3 was added, mixed thoroughly and then placed in a boiling water bath for one min. after that test tubes were cooled in ice water and the colour was read at 650 nm. catechol in the range of 0-25 μg was used as the standard. tab. 1: monthly mean weather data during crop growth period (kharif 2012). figures in parenthesis under the field rainfall represent total number of rainy days during that month. month temperature (oc) relative humidity (%) evaporation (mm) rainfall (mm) max min mean max min mean june 36.5 27.0 31.7 79 50 64 234.0 84.2 (3) july 33.7 26.2 30.0 86 64 75 139.5 67.6 (6) august 32.0 25.0 28.5 91 69 80 105.4 79.5 (7) september 32.0 24.5 28.2 89 67 78 102.0 193.7 (10) october 37.0 21.5 29.2 66 30 48 186.0 0.0 (0) total 766.9 425.0 (26) fig. 1: changes in soil moisture content (w/w) at different soil depth due to imposition of water deficit stress 100 k. chakraborty, m.k. mahatma, l.k. thawait, s.k. bishi, k.a. kalariya, a.l. singh sugar profiles by ion chromatography sugars extracted in ethanol were separated by ion chromatograhy as reported in our earlier paper (bishi et al., 2013). glucose, fructose, myo-inositol, lactose, sucrose, raffinose, stachyose, and verbascose were used as standards. lactose was used as internal standard during the analysis. the concentrations of various components in the standard mixture were adjusted to such levels that a distinct peak for each was obtained in the chromatogram. ethanol extracts were membrane-filtered and an aliquot of 25 μl of samples was injected in the ion chromatograph (ics 3000 dionex, usa) equipped with amino trap column, carbopac pa10 guard column followed by carbopac pa10 analytical column. sugars were eluted from column in 150 mm naoh with a flow rate of 1 ml min-1. data integration was attained by using chromeleon software supplied with the equipment. statistical analysis all the data recorded were the mean values of at least three independent assays with three replications each. the data was subjected to analysis of variance appropriate to the experimental design. differences at lsdp=0.05 were considered statistically significant. results effect of water deficit stress on photosynthesis water deficit stress significantly reduced the rate of photosynthesis in all the genotypes; however there were enough variations observed in the genotypes of different habit group (fig. 2). in terms of percentage change in net photosynthetic rate virginia genotypes showed greater reduction compared to spanish type. at individual genotype level, hng 10 showed highest reduction (32.7 %) in photosynthesis rate followed by somnath (29.7 %) and kadiri 3 (28.1 %). this result suggested, for photosynthetic parameters relatively greater susceptibility of virginia type peanut cultivars to water deficit stress than spanish type. changes in the sugars profile in the leaf tissue imposition of water deficit stress altered the sugar profile in leaf tissues as a result of changes in the net photosynthesis as well as partitioning of the net photosynthate for production of carbohydrates (tab. 2). content of both inositol and mannitol increased in the leaf tissue under water deficit stress in all the genotypes across different habit groups. on an average the inositol content almost doubled in spanish group, whereas the increase in virginia group was about 50 %. among the genotypes jl 286 and tpg 41 showed highest increase (148 and 125 %, respectively) in inositol content under stress compared to the control plants. similarly, accumulation of mannitol in the leaf tissue also showed the increasing trend under stress. the increase was highest in sb habit group (86 %), followed by vr (46 %) and vb (33 %) group. among the genotypes, again jl 286 showed highest increase in mannitol accumulation and it increased to 521 ppm under stress from the control value of 245 ppm, whereas genotype icgs 76 showed least increase (10 %). tab. 2: sugar profiles (ppm) of groundnut leaves during water deficit stress habit cultivar inositol mannitol trehalose glucose fructose sucrose group control stress control stress control stress control stress control stress control stress ak 159 7119 7774 206 435 254 443 19613 13719 14416 10106 19926 8110 spanish drg 1 5065 10167 259 357 219 304 12103 6214 10248 4602 11914 3779 bunch jl 286 4915 12209 245 521 150 643 10732 8386 9296 6143 16699 5931 tpg 41 5022 11315 211 390 180 569 19593 11105 16662 8406 20607 13498 gg 20 5492 10970 273 347 260 192 14418 11777 9446 8987 21165 9445 viginia hng 10 7629 9694 169 293 nd nd 14198 7853 10574 5746 21284 11203 bunch icgs 76 6035 9707 281 307 440 402 13406 9070 11657 7249 22417 15442 kadiri 3 7724 11869 280 340 13 nd 13453 9536 10264 7087 20339 13406 csmg 84-1 5169 5884 307 372 369 203 9748 7411 7056 5767 12558 7586 virginia gg 11 7078 7961 251 393 nd nd 13689 7049 10265 5839 22593 11293 runner gg 16 6687 8478 316 469 243 154 10885 10694 8196 8072 18435 2263 somnath 5507 10296 191 304 119 250 13273 11490 9582 9090 17438 13692 lsd variety (v) 71.3 14.2 14.4 118.4 130.1 222.9 (p=0.05) treatment (t) 299.8 ns 27.3 450.5 282.5 78.4 v × t 100.9 20.1 20.4 167.4 184.1 315.3 nd: not detected, ns: means non-significant fig. 2: changes in net photosynthesis rate (pn) in groundnut leaves under water deficit stress water deficit stress alters sugar profile in groundnut 101 on the other hand, the content of different monoand disaccharide were reduced with imposition of stress, except trehalose (tab. 2). under stress, trehalose content in the leaf showed significant increase mostly in sb genotypes; however for virginia genotypes it remained either unchanged or even reduced in some cases, except somnath which showed almost 67 % increase. among the genotypes jl 286 and tpg 41 showed highest increase up to 643 and 569 ppm from a control value of 150 and 180 ppm, respectively. the content of other free sugar viz. glucose, fructose and sucrose reduced in all the genotypes under stress and the highest reduction was observed in sb genotypes (36, 42 and 57 % reduction, respectively for glucose, fructose and sucrose), followed by vb and vr group. changes in the sugars profile in the kernel like that of leaf sugar alcohols level, similar increasing trend was also observed in kernel under stress (tab. 3). the level of inositol was more than doubled in the kernel of spanish genotypes under stress, whereas the increase was less than half for virginia genotypes. among the genotypes jl 286 and tpg 41 showed highest increase (150 and 115 % respectively), under stress quite similar to that of leaf tissue. mannitol content in the kernel also increased significantly under stress and among different habit groups sb showed highest increase, followed by vr and vb group. the genotype jl 286 again showed highest increase mannitol content (144 %), while least increase was observed for hng 10 (14 %). trehalose content in the kernel was increased under stress only in sb group, but it was significantly reduced in both vb and vr group (tab. 3). highest increase in trehalose content was observed in jl 286 (103 %), followed by tpg 41 (69 %), while the genotype csmg 84-1 showed highest reduction (69 %). the glucose content in the kernel was increased under stress in almost all the genotypes except gg 11 and gg 16 (tab. 3). more than 75 % increase in kernel glucose content was observed in tpg 41 and somnath under stress, while in some of the genotypes like kadiri 3 and csmg 84-1, the increase was as low as 20 %. unlike that of leaf, the sucrose content in the kernel increased under stress in most of the genotypes except ak 159 and jl 286 (tab. 3). highest increase in kernel sucrose content was observed in somnath, followed by gg 11, where it was increased up to 47.6 and 65.3 mg g-1 seed weight under stress from the control value of 27.9 and 47.1mg g-1 seed weight, respectively. total raffinose family oligosaccharides (rfos) content (raffinose and stachyose) was also increased in the kernel under stress (tab. 3). on an average the sb group showed 39 % increase in rfos content under stress, whereas, it was 31 and 16 % for vr and vb group respectively. among the genotypes tpg 41 showed highest increase (84 %) in rfos content under stress, followed by jl 286 (47 %), while the genotypes icgs 76 and csmg 84-1 showed least change in rfos content when the stress was imposed. changes in kernel quality parameters imposition of water deficit stress significantly reduced the oil yield and altered different kernel quality parameters in all the genotypes (tab. 4). among different habit groups, sb showed least loss in oil content, where highest oil loss was observed in vr group. among the genotypes jl 286 showed the least reduction (1.8 %) in oil con tent whereas somnath showed the highest reduction (13.1 %). unlike oil the total protein content increased under stress, the genotype csmg 84-1 showed highest increase (23.4 %), followed by jl 286 (22.7 %). the free amino acid content was also increased under stress and the highest increase was observed in csmg 84-1, where increased up to 4.30 mg g-1 seed weight from a control value of 2.23. this increase in free amino acid content might possibly be due to increase in kernel protein content as well as stress induced breakdown of it. the total phenol content showed a mixed response under stress. although the varietal differences were significant, but no significant treatment effect was observed in the present study. changes in oil quality parameters imposition of water deficit stress altered the relative content of oleic and linoleic acid in the groundnut kernel, ultimately altering the o/l ratio and the keeping quality of the oil (fig. 3). oleic acid content tab. 3: sugar profiles (ppm) of groundnut kernels during water deficit stress habit cultivar inositol mannitol trehalose glucose sucrose rfos group control stress control stress control stress control stress control stress control stress ak 159 464 826 207 387 601 894 86 124 26768 22061 1614 1855 spanish drg 1 517 910 290 575 311 489 nd 120 22626 30043 1273 1403 bunch jl 286 320 807 300 731 751 1524 69 99 40704 34354 1867 2749 tpg 41 512 1094 577 1026 74 124 170 299 31925 40505 1440 2650 gg 20 368 698 368 424 292 110 nd 71 36569 50318 3341 4088 viginia hng 10 700 840 553 635 50 64 83 140 53065 73576 4111 5155 bunch icgs 76 694 911 505 703 211 103 nd 172 54806 57069 3667 3668 kadiri 3 670 892 611 930 57 67 158 191 45567 63218 4178 4810 csmg 84-1 996 1429 473 835 579 178 104 126 47063 65350 3777 3540 virginia gg 11 545 739 760 995 449 254 99 nd 35931 50344 2643 3863 runner gg 16 1093 1246 459 601 234 198 111 nd 51852 63565 2253 3200 somnath 347 579 660 936 313 127 78 139 27888 47652 2251 3167 lsd variety (v) 31.6 39.6 52.3 5.1 198.1 67.7 (p=0.05) treatment (t) 33.7 10.6 ns ns 548.4 105.1 v × t 44.7 55.9 74.1 7.1 280.1 95.8 nd: not detected, ns: means non-significant 102 k. chakraborty, m.k. mahatma, l.k. thawait, s.k. bishi, k.a. kalariya, a.l. singh significantly reduced in all the cultivars under water deficit stress (fig. 3a) however, highest reduction observed in virginia genotypes than that of spanish ones. the genotype kadiri 3 showed the highest reduction (12.9 %) in oleic acid content under stress, followed by hng 10 (11.3 %). linoleic acid content showed the opposite trend and was found to be increased under stress (fig. 3b). the increase was highest in hng 10 (31.4 %), followed by kadiri 3 (28.2 %), whereas ak 159 showed least change (4.5 %) under stress. with the decrease in oleic acid content and concomitant rise in linoleic acid fraction resulted in an obvious decrease in o/l ratio in the groundnut kernels in all the genotypes in the present study (fig. 3c). the genotypes hng 10 and kadiri 3 showed highest reduction in o/l ratio, which was 32.4 and 32.0 %, respectively under water deficit stress. discussion in the present study imposition of prolonged water deficit stress led to significant alternation of physiological and metabolic activities in both source (leaf) and sink (kernel) tissue in groundnut, however the impact varies across different habit groups. although groundnut is a moderately drought tolerant crop, the imposition of drought stress especially during mid or late season of crop growth significantly reduces various metabolic activities of the crop mainly due to lack of adequate water supply to the active tissue and eventual closure of stomata (devi et al., 2009). kalariya et al. (2013) also reported a 11-30 % reduction in net photosynthesis in groundnut during water deficit stress. limitation of photosynthetic activity under severe wa-ter deficit stress was also attributed to rapid degradation of thylakoid membranes in groundnut apart from stomatal constraint (lauriano et al., 2000). a decreased rate of photosynthesis in water deficit stress affects carbon delivery from source to sink tissue and its subsequent metabolism. the photosynthetic rate of leaves decreases as relative water content and water potential decreases. a reduction of the net photosynthetic rate in moisture stressed plants mainly happens through stomatal closure as a mechanism to reduce total transpiration (singh, 2004; rosas-anderson et al., 2014). as a result of reduced photosynthetic activities under water detab. 4: effect of water deficit stress on oil, protein, free amino acids and total phenol content of groundnut kernels habit group cultivar oil (%) protein (%) free amino acids (mg-1 g) total phenol (mg-1 g) control stress control stress control stress control stress ak 159 54.50 53.30 20.60 21.75 2.09 1.82 4.96 4.44 drg 1 53.10 51.90 23.50 26.05 2.55 2.85 4.55 4.95 jl 286 48.05 47.20 30.40 32.90 2.38 2.63 4.21 4.27 tpg 41 49.75 46.40 24.25 29.75 2.90 2.73 5.25 5.05 gg 20 52.15 48.00 25.70 30.20 2.95 3.80 4.87 5.90 hng 10 45.85 43.75 33.75 35.25 2.95 4.28 5.41 5.13 icgs 76 48.75 46.95 30.60 32.60 3.02 3.89 5.53 7.05 kadiri 3 46.05 43.90 33.05 35.80 3.39 5.04 7.04 6.84 csmg 84-1 48.80 45.15 27.50 33.95 2.23 4.30 4.07 5.83 gg 11 51.65 45.55 26.55 31.70 2.83 3.85 5.15 5.82 gg 16 46.30 45.25 32.90 34.50 4.13 5.63 5.22 6.52 somnath 53.20 46.25 23.15 32.65 2.41 3.69 4.77 5.00 variety (v) 0.81 1.05 0.13 0.27 lsd (p=0.05) treatment (t) 1.98 0.74 0.64 ns v × t 1.16 1.48 0.19 ns ns: means non-significant (a) (b) (c) fig. 3: changes in oleic (a), linoleic (b) acid content and o/l ratio (c) in groundnut cultivars under water deficit stress spanish bunch viginia bunch virginia runner water deficit stress alters sugar profile in groundnut 103 ficit stress, significant alteration in the sugar profile was observed in different groups of groundnut cultivars. due to lower supply of net assimilate, the carbon partitioning in the leaf tissue changed significantly. the content of readily available carbohydrates (glucose, sucrose and fructose) dropped, whereas carbohydrates necessary for stress tolerance (inositol, mannitol and trehalose) increased upon imposition of stress. similar increase in the levels of sugar alcohol, particularly the pinitol, and decrease in the levels of sucrose was observed by keller and ludlow (1993) in the leaves of pigeon pea after imposition of drought stress. morsy et al., (2007) reported higher accumulation of osmo-protectants like trehalose, inositol and mannitol in the more salt and water-deficit tolerant rice genotype, which suggested role of these organic solutes in osmo-tolerance mechanism in plants. mannitol, an important photoassimilate which participates in a wide range of physiological processes including carbon storage and translocation, regulation of the pool of the cellular reductant in plants (stoop and mooibroek, 1998), scavenging of hydroxyl radicals and serving as an osmotically active compatible solute (popp and smirnoff, 1995). in the presents study, increase in the content of inositol, mannitol and trehalose occurs at the expense of simpler carbohydrates such as glucose, fructose and sucrose content in the leaves of stressed plants. like the sugar alcohols, trehalose is also proposed as an osmoprotectant during periods of drought or water-deficit stresses (penna, 2003). this sugar possesses the unique capacity for reversible water absorption, and appears to be superior to other sugars in protecting biological molecules from desiccation-induced damage (rontein et al., 2002). adverse conditions such as heat, chilling or water stress correlate with the accumulation of high concentrations of trehalose in yeast (goddjin and van dun, 1999) and highly desiccation tolerant resurrection plants (iturriaga et al., 2000). differential responses of cultivars from different habit groups to water deficit stress implied their variable ability to tolerate stress. in the present study, spanish group of cultivars showed highest induction in accumulation of organic solute in response to external water deficit condition suggesting their superior ability to tolerate drought stress than virginia group of cultivars. reduction of sucrose content in the leaf tissue during stress condition may contribute to either higher transport towards kernels or its rapid conversion to more complex sugars for better osmo-protection. thus results from the present study suggest decrease of hexoses under stress condition is likely to be utilized in the biosynthesis of higher sucrose content in the sink tissues. in general, sucrose levels of stressed ovaries are higher or at least similar to those of non-stressed ovaries as reported in maize (schussler and westgate, 1995; zinselmeier et al., 1995). although the sugar profile of the kernel (sink tissue) changed significantly like that of leaf (source tissue), the pattern of change was found somewhat different in the present study. similar to the changes in leaf tissue, the content of sugar alcohols increased along with increase in stress induced oligosaccharide (raffinose and stachyose) content, but the level of monosaccharide and disaccharides did not show significant alteration in the kernel tissue. inositol and its derivatives are implicated in stress tolerance through various ways such as protecting cellular structures from reactive oxygen species, controlling turgor pressure or by acting as stress signaling molecules (loewus and murthy, 2000). as non-reducing carbohydrates, rfos are good storage compounds, being able to accumulate in large quantities without affecting primary metabolic processes. few previous studies reported that desiccation tolerance is strongly correlated with accumulation of rfos, primarily raffinose, stachyose, and verbascose in the seeds (horbowicz and obendorf, 1994; lin and huang, 1994). water deficit stress has dual impact on the end product synthesis in groundnut kernels. being primarily an oilseed crop, water deficit stress significantly reduced oil yield and thus altered kernel composition. the lack of adequate c-supply from the source tissue (both due to reduced photosynthesis and conversion of assimilate for biosynthesis of organic osmo-protectants) resulted in reduction in kernel oil content, but a relative increase in protein content in the present study. similarly, conkerton et al. (1989) also reported that mid-season drought reduced total oil content in groundnut. oil has negative correlation with protein content thus decrease in oil content may eventually results in increased protein content. however, we do differ from some of the previous reports that total oil and total protein were not significantly affected by mid-season drought (dwivedi et al., 1996). under drought stress, due to shortening of pod development and seed filling period alteration of oil/protein ratio in legume seeds were reported, which was mainly because of the fact that during seed filling accumulation of carbohydrate and protein were much faster than that of oil (dornbos and mcdonald, 1986; kambiranda et al., 2011). hashim et al. (1993) also observed comparatively higher percentage of linoleic acid (18:2) and lower percentage of oleic acid (18:1) in the groundnut kernels when it was grown under water deficit condition. our results also suggest that there is a shift of oleic to linoleic acid under water deficit stress resulting in reduced o/l ratio and oil stability. in conclusion, mid-season water deficit stress in groundnut significantly affects the carbohydrate composition and source and sink sugar profiles. the increase in relative proportions of stress induced complex sugars (myo-inositol and mannitol) in the leaf tissue showing the adaptive response to osmo-tolerance. on the contrary, reduction in simple sugars under stress in the leaf with subsequent translocation to sink tissue suggests a drought escape mechanism in groundnut. oleic acid content, a measure of oil stability and quality, was also decreased due to water deficit condition. the quality traits were comparatively less affected in spanish genotypes than in virginia genotypes due to water deficit stress; 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(eds.), growth and physiology of groundnut, vol. 6, 178-212. jodhpur, national research centre for groundnut (icar). singh, a.l., nakar, r.n., goswami, n., kalariya, k.a., chakraborty, k., singh, m., 2013: water deficit stress and its management in groundnuts. in: hemantranjan, a. (ed.), advances in plant physiol. 14, 370-465. stoop, j.m.h., mooibroek, h., 1998: cloning and characterization of nadp-mannitol dehydrogenase cdna from the button mushroom (aga ricus bisporus) and its expression in response to nacl stress. app. environ. microbiol. 64, 4689-4696. zinselmeier, c., westgate, m.e., schussler, j.r., jones, r.j., 1995: low water potential disrupts carbohydrate metabolism in maize (zea mays l.) ovaries. plant physiol. 107, 385-391. address of the corresponding author: maheshmahatma@gmail.com, mahesh.mahatma@icar.gov.in © the author(s) 2016. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 86, 104 112 (2013), doi:10.5073/jabfq.2013.086.015 1 department of food chemistry and nutrition, faculty of food science, corvinus university of budapest, budapest, hungary 2 department of enology, faculty of food science, corvinus university of budapest, budapest, hungary gc-ms description of the primary aroma structure of two kadarka wines considered indigenous in hungary mariann csóka1, mária amtmann1, diána ny. sárdy2, miklós kállay2, kornél korány1* (received may 17, 2013) * corresponding author summary two high quality kadarka wines of the great plains of hungary and szekszárd terroirs were thoroughly analysed by gc-ms measurements subsequent to a two step sample preparation method elaborated at our department. the goal of the work was to describe the varietal relative aroma-picture of the kadaraka species considered indigenous in hungary. the relative aromagram construction method creates the possibility of studying the scent features by chemical classes separately. the data prove that the primary aromaspectra beside certain smaller similarity differ to a reasonable extent due to the habitats of origin. the kadarka of the poorer sand soil of the great plains is more fragrant than that of the loess of szekszárd. the analytical results are supported by the organoleptic experience as well. introduction the kadarka variety of vitis vinifera l. is considered indigenous in hungary. in the centuries xvii-xixth it was the most widely spread blue grape species in the country. at the beginning of the 1800’s years more than two third of the red vine plantations consisted of it. the most characteristic attribute of kadarka wine is its piquancy. it is a highly fragrant, fruity and slim wine. unfortunately, in the middle of the last century the intensive mechanization of vine growing made its production dramatically decrease due to this cultivated variety’s capability of being merely hand-cultivated. modish „worldwide” spreading species made the case worse, thus nowadays this traditional hungarian variety almost has to be discerned again. recently kadarka as one of the hungarics (i.e. gastronomic uniquenesses) starts to live its new renaissance. the provenance of this grapevine is not known exactly. some experts originate the name from skutari what is the asia minor part of istanbul called üsküdar in turkish and skaderi in the serbian and croatian languages (újvári, 2007). certain conceptions think it derives from the environs of skhodari-lake (albania), others consider kadarka to come from asia minor (takler, 2004). it is more than likely that in the xvi. century the serbs escaping from the turks brought it into hungary through the balkan. later, having retaken the fortress of buda from the turks and serbian refugees been settled down on the depopulated lands, the serbs were the ethnic who started spreading this cultivated variety (cv.) in large scale on the emptied parts of the country. the synonyms of kadarka are „negru” in romania and „gamza” in bulgaria (janyik, 2010). after the phylloxera vine-pest in 1875 kadarka was planted in sandy soil only, thus by 2002 its ratio decreased to 1.1 % compared to the former 67 % at the beginning of the 19th century. in 2007 european danube-maros-tisza-körös region elected this wine as an euro-region wine thus hungary, romania and serbia, the three countries involved in the production agreed upon the details (registration of the producers, cultivation mode, character formation and legislation) of trade (janyik, 2010). in the last 3-5 years valuable and interesting articles of importance were published on the fragrance features of red wines considered rather „scentless” by the literature, previously. most of them is connected to the modern and fashionable spe (gómez et al., 2011 and 2012) and head-space spme sample preparation methods (veverka et al., 2012) that are rarely sensitive and selective to the varietal constituents enough, therefore are hardly sufficient for grabbing the cultivated variety’s character. the latter paper reports good results on the terpenoid distribution and volatile phenolics of the distillates of 16 red wines but spirits are not the wines themselves, brandy-distillation may seriously distort the ratios of the volatiles making them unfit for cv. description. traditional liquid-liquid extraction methods (perestrelo et al., 2006) catch all kinds of substances not only the volatile, gas chromatographically measurable ones, thus the main compounds of the wines depress those bouquet components that are important in describing the varietal scent features (ivanova, et al., 2012). our present work intends to give an overall picture of the bouquet structure of the barely known kadarka wine through the gc-ms investigation of two high quality kadarka representatives deriving from the great plains and the famous red wine producing szekszárd region of hungary. materials and methods samples two high quality (appellation d’origine vin délimité de qualité supérieure, aovdqs) kadarka wines of the 2011th year vintage have been analysed by courtesy of the excellent wineries koch in the great plains (kiskörösi kadarka) and vida (szekszárdi kadarka) in the red wine producing szekszárd region of hungary. the terroir of great plains is the largest one of hungary. its size is 27 900 hectares. at least one fourth of the hungarian wines are produced here. its climate is extreme, hard winter frosts are common and the summers are hot and dry. the soil is dominantly immune sand. among the wines deriving from here white, rosé and red ones can equally be found. they are light and mild and of table quality generally. the most well-known representative of them is the kadarka of kiskörös (eperjesi et al., 2003). the szekszárd terroir extends in the county of tolna with a size of 6000 hectares of loess soil and temperate climate. this land became famous for its kadarka wines. kadarka cv. grapevine brings outstanding quality grapes under the warm weather and brilliant aeolian soil conditions of this region. the wines of this area are flavoured and tasty and have bright colour. they are mild, piquant and pleasantly acidic. the white wines of the region are aromatic and full-bodied, but due to the warmth of the climate may suffer from the lack of acids frequently (eperjesi et al., 2003). sample preparation in the present work a two step sample preparation procedure has been elaborated at our department of food chemistry and nutrition. on the kadarka wines of hungary 105 1. liquid-liquid extraction (lle) subsequent to distillation (called „classic” further) prior to distillation 150 ml undecanol-1 internal standard (istd) of 0.8 mg/ml concentration in ethanol has been added to 500 ml wine sample (containing 100 g of analytical grade nacl to obtain „salt-out” effect) then poured into a 1000 ml round bottom flask and distilled till 100 ml condensate gathered. the operation has been repeated three times, the fractions (3 × 100 ml) were collected and fused. the unified condensates were filled into a separation funnel, 15 g of nacl were added to them and they were extracted three times with 80 ml n-pentane of special purity. then the extracts were fused and dried on anhydrous na2so4 overnight and concentrated to 1 ml end-volume by evaporation. into the gc-ms instrument 1 ml of the concentrate has been injected. 2. likens-nickerson simultaneous distillation-extraction (ln-sde) the 3 × 400 ml distillation rest of the previous step was collected, fused and completed with 300 ml distilled water that contained 60 g of analytical grade nacl. this time 450 ml undecanol-1 internal standard of the above concentration has been added to the mixture. then the sample was poured into a round bottom flask of 2000 ml, connected to an ln-sde equipment and distilled against 150 ml of n-pentane of special purity for 1.5 hours after boiling up. then the pentane extract was put into a refrigerator to freeze out the water content and the dry extract was evaporated to 1 ml endvolume. one microliter of this sample has been introduced into the gc-ms instrument. gc-ms analysis an hp 5890/ii gas chromatograph (hewlett-packard, palo alto, ca, usa) equipped with a 60 m × 0,25 mm × 0,25 μm at-wax fused silica capillary column and a 5971/a mass selective detector (msd) was used to analyse the volatile compounds of wine extracts. the initial oven temperature was 60 °c and immediately increased to 280 °c at a rate of 4 °c/min. the injector was operated in splitless mode at 270 °c, with a 100:1 split ratio at 0.1 min. delay time. the msd was run in electron impact mode (70 ev) at 280 °c. helium (4.6) was used as a carrier gas with the flow rate of 30 cm/s. the detection was performed in the 35-350 mass range at 390 mass/s scan speed. the identification of the fragrance constituents were accomplished using the nbs49k.l, wiley138.l, wiley275.l and nist05.l spectrum libraries. the identification was based on the match quality, programmed temperature retention index measurement (korány et al., 2006), and retention-time/ reference spectral data of standards if they were available. results in our former works (kovács et al., 1999; korány et al., 2000) only one step sample preparation was applied as described the above chapter „1. liquid-liquid extraction subsequent to distillation”. later the fine, balmy scent of the discarded distillation rest was discerned and decided to be subjected further investigation by ln-sde. the result is shown in fig. 1 that depicts two total ion chromatograms (tics) of the kadarka wine of szekszárd. the chromatograms clearly prove that the distillation remainder after expelling the ethanol constituent of the wine and collecting water condensate of approximately equal volume to the alcohol content, still holds cv. characterising primary compounds of importance (e.g. geranic oxide or myrcenol among many others) and substances of high scent activity, that is of low odour threshold (delta-decalactone for instance). in addition many of them (including the precedents too) can be measured only in the ln-sde fraction that we call alcohol free water-phase. fig. 1: the tics of the szekszárdi kadarka wine samples, simple distillation followed by lle extraction (upper), ln-sde extract (lower) istd istd delta-decalactone myrcenol geranic oxide 106 m. csóka, m. amtmann, d.ny. sárdy, m. kállay, k. korány the gas chromatographic separation was followed by a thorough, manual mode, compound by compound identification of the detected constituents. the number of the identified odorants were 122 in kiskörösi kadarka (great plains) and 143 in szekszárdi kadarka. the tabular statement from the amount of compounds is reported in tab. 1. the recognised components sorted into chemical classes of decreasing odour activity and species characterising significance are introduced in tab. 2 in the order of elution. instead of retention times the programmed temperature retention indices (linear retention indices in older term, lris) are used to describe the elution sequence. they were measured as discussed in one of our previous articles (korány et al., 2005). the mode of typing carries information as follows: the chemicals in boldface are present in both kadarka samples in both fractions (classic and ln-sde). compounds in „*typeface” occure only in the extracts of the classic preparation method in one wine. components of „*typing” occur in both wine samples but only in the classic phase. substances of „^letters” can be found only in the ln-sde extracts of one sample. constituents of „^typeface” appear in both wines, but only in the ln-sde phase. names of „normal” typing are present in both classic and ln-sde extracts but only in one of the wines. in the cases of „(name !)”-s the match quality is high enough (roughly 90 %) but the retention data (ptri) contradicts the chemical structure reported. in these instances of misidentification the names were kept as working designations. studying the results in the form of a table that is so complex than tab. 2 is really exhausting. therefore relative aromagrams called aroma-spectra have been constructed by measuring the ptris and calculating the relative intensities related to the undecanol-1 istd. the classification of the identified components into chemical classes creates the chance of the fast comparison of the scent constituents of different origin i.e. primary, secondary, tertiary (and/or quaternery). thus any of the important bouquet sources for example the cv. characterising, the fermentation induced, the maturation or bottle-ageing ones can equally be examined with ease by picking out the group in question. for setting an example fig. 2 depicts the total and the primary aroma-spectra of the investigated wines. while fig. 1 and 2 make the enologists capable of investigating the aroma features of any origin (i.e. primary, secondary, … etc.) with compound by compound details it is almost impossible to form an overall picture of the similarity or the measure of the dissimilarity of the examined wine samples. the block diagram-like processing of the data in tab. 2 according to the chemical classes gives the chance of the direct comparison as shown in fig. 3. discussion the detailed gc-ms analysis subsequent to the two step sample preparation suggested the fairly similar character of the kiskörösi kadarka (great plains) and the szekszárdi kadarka (red vine growing region of szekszárd) wines. inspite of the cultivated varieties’ identity significantly smaller discrepancies of the total aroma pictures were observed than presumed considering the enormous quality difference between the immune sand soil of the great plains and the mild aeolian loess of the szekszárd region. the kiskörösi and szekszárdi wines scored 7926 and 9325 points of relative intensity expressed in the undecanol-1 istd, respectively. this difference of approximately 15 % is not much if the higher fatty acid and benzene and ~relatives content of the szekszárdi kadarka is considered. the greater fatty acid level (by nearly 50 %) has relatively little effect on organoleptic features because it recruits mainly from caproic (c6), caprylic (c8) and capric (c10) acids of moderate sensory activity (burdock, 2010). in the chemical division of „benzene and ~derivative compounds” not only the b-phenylethyl alcohol content (a fermentation product) is more abundant in the szekszárdi kadarka than in the kiskörösi wine, but nine constituents of importance appear only in the latter. they are as follows: 1317, ^1,2,3-trimethylbenzene, 0.02 %; 1620, *(r*,r*)-(.+-.)-1,1'-(1,2-dimethyl-1,2-ethanediyl)bis-benzene, 0.08 %; 2046, *p-ethylguaiacol, 0.01 %; 2127, ^1-[6-hydroxy2-methyl-3-(1-methylethyl)phenyl]-ethanone, 0.06 %; 2127, ^1 tab. 1: the number of the identified compounds in kadarka wines compound group great plain szekszárd classic ln-sde in both classic ln-sde in both extracts extracts terpenes, sesquiterpenes and relatives 13 32 8 20 25 13 compounds of benzene ring 5 16 4 9 17 3 s-containing compounds 2 2 2 3 3 2 acetals 5 2 2 5 3 2 alcohols, aldehydes, ketons 10 4 2 21 4 3 furans, o-heterocycles 2 3 4 3 1 n-containing constituents 1 1 lactones 5 3 3 2 1 1 indenes 2 1 2 1 constituens of naphthalene skeleton 1 3 1 1 7 1 esters of fatty acids 23 9 8 27 8 6 fatty acids 4 4 3 7 5 3 hydrocarbons 4 1 1 2 2 2 total 74 82 34 102 81 38 on the kadarka wines of hungary 107 tab. 2: the identified components of the kadarka wines g. plains szekszárd ptri compounds area %* area %* terpenes, sesquiterpenes and relatives 1079 ^1-acetyl-4-methylbicyclo[3,1,0]hexan-3-one 0.01 1090 ^(+-)-2,6,6-trimethyl-2-vinyl-tetrahydropyran (geranic oxide) 0.1 0.06 1112 ^1,5,5,6-tetramethyl-1,3-cyclohexadiene (alpha-pyronene) 0.26 1128 ^ p-mentha-2,4(8)-diene(isoterpinolene) 0.03 0.01 1129 *3-methylene-1,5,5-trimethylcyclohexene 0.01 1175 l-limonene 0.03 0.02 1179 ^herboxide i 0.01 0.01 1200 ^cis-ocimene 0.01 1211 ^2,2-dimethyl-5-(1-methylpropenyl)tetrahydrofuran(ocimene quintoxide) 0.02 0.03 1257 ^terpinolene (d-terpinene) 0.01 0.01 1433 cis-linalool oxide 0.25 0.16 1446 ^1,2,3,4-tetrahydro-1,1,6-trimethyl-naphthalene (alpha-ionene) 0.03 1465 trans-linalool oxide 0.1 0.08 1466 *neroloxide 0.01 1541 vitispirane i 0.22 0.15 1544 vitispirane ii 0.34 0.38 1544 ^linalool 0.01 0.11 1570 ^1,4-dimethyl-3-cyclohexenyl methyl ketone 0.01 1614 *1-terpinen-4-ol 0.02 1615 ^myrcenol 0.06 0.05 1615 *3,7-dimethyl-1,5,7-octatrien-3-ol(hotrienol) 0.06 0.19 1635 ^1-p-menthen-9-al (!) 0.03 0.02 1639 ^2,3-dimethyl-bicyclo[2,2,1]hept-2-ene (santene a !) 0.03 1641 ^beta-terpineol 0.02 1660 ^1,3-p-menthadien-7-al 0.13 0.15 1661 *g-terpinene 0.03 1687 ^z-beta-ocimenol 0.2 0.12 1703 ^1,8-menthadien-4-ol 0.01 1706 ^beta-santalol 0.03 0.02 1713 (-)-alpha-terpineol 0.24 0.12 1717 ^g-terpineol(p-menth-4(8)-en-1-ol) 0.03 0.02 1781 ^(r)-(+)-beta-citronellol 0.03 0.06 1829 ^santene (b !) 0.02 1853 beta-damascenone 0.1 0.1 1865 geraniol 0.1 0.05 1969 *6-ethyl-2-methyl-delta-1-bicyclo[4.4.0]decen-8-one 0.06 2019 ^4-(1-methylethenyl)-1-cyclohexene-1-methanol (perilla alcohol) 0.01 2056 *(+/-)-trans-nerolidol 0.02 0.04 2067 ^1-p-menthen-9-al 0.09 2100 ^exo-(+-)-1,4,5-trimethyl-9-methylene-bicyclo[3.3.1]nonan-2-one 0.06 2227 ^1,4-dimethyl-7-(1-methylethyl)azulene (guaiazulene) 0.01 2327 *farnesol 0.07 0.18 2336 ^dihydro-actinidiolide 0.02 2371 ^geranic acid 0.05 compounds of benzene ring 1243 ^p-cymene 0.01 1317 ^1,2,3-trimethylbenzene 0.02 1429 ^p-cymenyl 0.01 0.01 1518 1-chloro-2-methyl-benzene 0.02 108 m. csóka, m. amtmann, d.ny. sárdy, m. kállay, k. korány 1529 benzaldehyde 0.04 0.01 1548 ^2-ethenyl-1,3,5-trimethyl-benzene (mesitylethylene) 0.01 1620 *(r*,r*)-(.+-.)-1,1’-(1,2-dimethyl-1,2-ethanediyl)bis-benzene 0.08 1758 ^2-methyl-3-(3,4,5-trimethylphenyl)-2-butene 0.01 1805 ^methyl salicylate 0.01 0.02 1842 *b-phenylethyl acetate 0.43 0.47 1868 ^p-cymen-8-ol 0.02 0.03 1880 ^2-methoxy-phenol (guaiacol) 0.02 0.02 1895 benzenemethanol 0.18 0.5 1932 benzeneethanol 9.45 11.34 2046 *p-ethylguaiacol 0.01 2127 ^1-[6-hydroxy-2-methyl-3-(1-methylethyl)phenyl]-ethanone 0.06 2167 ^eugenol 0.01 2169 ^p-ethylphenol 0.04 2172 *o-ethylphenol 0.04 2194 ^4-vinyl-2-methoxy-phenol 0.28 0.15 2235 ^1-(2,3,6-trimethylphenyl)-2-butanone 0.02 0.02 2268 ethyl 2-hydroxy-3-phenylpropanoate 0.25 2291 ^1-(2,3,6-trimethylphenyl)-3-buten-2-one 0.07 0.08 2300 ^beta-phenylethyl-2-methyl butyrate 0.01 2353 ^4-vinylphenol 0.03 0.04 s-containing compounds 1534 dihydro-2-methyl-3(2h)-thiophenone 0.04 0.08 1566 *sulfinylbis-methane 0.01 1712 ^2-thiophene carboxaldehyde 0.02 1730 3-methylthiopropanol 0.23 0.22 acetals 949 *1-ethoxy-1-methoxy-ethane 0.04 0.02 961 1,1-diethoxyethane 8.19 9.3 999 1,1-diethoxybutane 0.34 0.47 1008 *acetal-a 0.03 1085 *1-(1-ethoxyethoxy)-pentane 5.3 1069 *1,1-diethoxy-ethane 0.05 1147 ^2-ethoxy-2-methyl-propane 0.02 1286 *2,2’-[ethylidenebis(oxy)]bis-pentane 1.0 0.04 alcohols, aldehydes, ketons 971 *ethanol 0.14 0.06 971 ^isovaleraldehyde 0.05 1014 2-butanol 0.11 0.08 1058 *2-methyl-1-propanol 0.37 0.66 1101 *1-butanol 0.04 0.03 1160 ^1-pentanol 0.05 0.2 1168 *3-methyl-1-butanol 31.4 32.28 1205 *1-pentanol 0.01 1206 *3-methyl-3-buten-1-ol 0.01 1253 *3-hydroxy-2-butanone (acetoin) 0.05 1272 *3-(2,2-dimethylpropoxy)2-butanol 0.02 1275 *4-methyl-1-pentanol 0.03 1290 *3-methylpentanol 0.06 1319 *1-hexanol 1.15 0.7 1332 *3-hexen-1-ol 0.03 0.03 1357 *trans-3-hexenol 0.06 0.01 1390 *cis-2-hexen-1-ol 0.01 1437 *1-heptanol 0.06 0.04 on the kadarka wines of hungary 109 1500 *(s)-3-ethyl-4-methylpentanol-1 0.04 1554 *n-octanol 0.02 0.03 1668 *nonyl alcohol 0.02 1984 *1-dodecanol 0.03 2348 ^1-hexadecanol 0.03 2510 1-heptadecanol 0.15 furans, o-heterocycles 982 *2,4,5-trimethyl-1,3-dioxolane 0.4 0.17 1452 2-formylfuran 0.7 0.82 1463 *2,5-dimethoxy-3-n-butyl-tetrahydrofuran 0.01 0.04 1502 ^1-(2-furanyl)-ethanone (2-acetylfuran) 0.03 0.06 1580 ^5-methylfufural 0.07 0.04 n-containing constituents 1970 1-(2-cyanoethyl)-2-ethyl-4-methylimidazole 0.44 0.1 lactones 1919 cis-3-methyl-4-octanolide(cis-oaklactone) 0.33 1987 *trans-3-methyl-4-octanolide (trans-oaklactone) 0.04 2054 gamma-nonalactone 0.08 0.03 2204 delta-decalactone 0.06 0.03 2397 delta-dodecalactone 0.03 indenes 1988 ^2,3-dihydro-3,3,5,7-tetramethyl-1h-inden-1-one 0.08 1992 ^2,3-dihydro-3,3,5,6-tetramethyl-1h-inden-1-one 0.06 2133 2,3-dihydro-3,3,5,6-tetramethyl-1h-inden-1-one 0.38 0.57 constituens of naphthalene skeleton 1762 ^6-(1,1-dimethylethyl)-1,2,3,4-tetrahydro-naphthalene 0.02 0.01 1777 3,8,8-trimethildihydronaphthalene 0.19 0.04 1941 1,2,3,4-tetrahydro-1,1,6,8-tetramethyl-naphthalene 0.8 0.02 1761 ^3,4-dimethyl-4-hydroxynaphthalen-1(4h)-one 0.02 1777 1,2-dihydro-1,1,6-trimethyl-naphthalene (tdn) 0.17 1942 ^2,4-dimethyl-4-hydroxynaphthalen-1(4h)-one 0.41 2028 ^1,2,3,4-tetrahydro-4,5,7-trimethyl-1-naphthol 0.02 2070 ^1,2-dihydro-3,5,8-trimethyl-naphthalene 0.01 esters of fatty acids 959 ethylacetate 2.71 0.17 1017 *acetic acid 2-methylpropyl ester (isobutylacetate) 0.03 1032 *ethyl butanoate 0.56 0.35 1043 *ethyl 2-methylbutyrate 0.01 1054 *3-methyl-butanoic acid ethyl ester 0.02 1097 *isoamyl acetate 9.13 2.53 1132 *(e)-2-butenoic acid ethyl ester 0.01 1199 *hexanoic acid ethyl ester (ethyl caproate) 1.9 1.43 1240 *n-hexyl acetate 0.26 0.06 1311 ethyl lactate 0.5 2.8 1371 *methyl octanoate 0.01 1425 *ethyl octanoate (ethyl caprylate) 2.47 2.53 1514 ethyl 3 hydroxybutyrate 0.06 0.08 1644 butanedioic acid, ethyl methyl ester 0.08 1655 *ethyl decanoate (ethyl caprate) 1.06 0.92 1679 *isoamyl octanoate 0.02 1691 butanedioic acid diethyl ester (diethyl succinate) 1.56 9.67 1711 *ethyl 9-decenoate 0.06 1818 ethyl 4-hydroxybutanoate 0.08 1872 *ethyl dodecanoate 0.3 0.08 110 m. csóka, m. amtmann, d.ny. sárdy, m. kállay, k. korány 1926 ethyl 3-methylbutyl butanedioate 0.15 0.56 2060 *isopropyl myristate 0.02 2072 *tetradecanoic acid ethyl ester 0.18 0.05 2162 ^diethyl 2-hydroxypentanedioate 0.02 2164 *ethyl pentadecanoate 0.01 2242 *1-methylethyl ester of hexadecanoic acid 0.14 0.1 2254 hexadecanoic acid ethyl ester 0.68 0.17 2267 ^ethyl 2-hydroxy-3-phenylpropanoate 0.1 2277 *ethyl 9-hexadecanoate 0.08 0.06 2422 ethyl stearate 0.12 2439 (z)-9-octadecenoic acid ethyl ester 0.16 0.05 2443 *ethyl (e)-oleate 0.03 2476 ethyl linoleate 0.37 0.04 2526 methyl linolenate 0.4 fatty acids 1489 *acetic acid 0.06 1618 isobutyric acid 0.11 1685 butanoic acid 0.03 1717 *2-methylhexanoic acid 0.33 1722 ^2-methyl-butanoic acid (active valeric acid) 0.12 1732 ^3-methyl-butanoic acid (delphinic acid) 0.07 1897 hexanoic acid (caproic acid) 2.0 2.04 2096 octanoic acid (caprylic acid) 4.84 7.41 2298 *decanoic acid (capric acid) 2.02 2.77 2487 *dodecanoic acid 0.28 hydrocarbons 941 methylcyclohexane 0.48 0.37 2174 *cyclododecane 0.05 2349 *cyclotetradecane 0.1 0.09 2512 *cyclohexadecane 0.11 „area %*” values are the averages of 9 determinations (3 independent measurements of each wines with 3 injection of all extracts; n =3 x 3 for every wines). the standard deviatons are within + 10 % in the 0.010.10 area % range; + 5.0 8.0 % in the 0.10 1.5 area % interval; and + 3.0 5.0 % in the 1.5 15.0 area % interval, related to the mean. [6-hydroxy-2-methyl-3-(1-methylethyl)phenyl]-ethanone, 0.06 %; 2167, ^eugenol, 0.01 %; 2169, ^p-ethylphenol, 0.04 %; 2172, *oethylphenol, 0.04 %; 2268, ethyl2-hydroxy-3-phenylpropanoate, 0.25 %; 2300, ^beta-phenylethyl-2-methyl butyrate, 0.01 %). the list is dull we must admit, but clearly demonstrates the bad need of the likens-nickerson preparation step considering the organoleptic significance of the majority of the „^signed” (i.e. ln-sde extracted) components. the case is the same in the „terpenes, sesquiterpenes and relatives” class as well, where many primary compounds of importance featuring the species character occur only in the ln-sde extracts. this chemical category deserves some words. theoretically a bit more fragrant trait of the szekszárdi kadarka was expected, due to the better habitat. in fact the analysis shows the opposit. in the wine of the great plains 35 primary constituents occur with a ratio of 2.47 %, whilst 31 components of 2.33 % in the szekszárdi kadarka merely. the difference seems insignificant, but was detectable sensorically as well. the research fellows of the department of enology organised a tasting exam to evaluate the two wines. unfortunately the analysis prior to the test consumed 4.5 l of both samples and the rest was not enough for an exact sensory panel tasting experiment, barely for an organoleptic one. that is why the sensory evaluation results are missing. in spite of the virtually paltry disparity of the compound numbers and ratio data, studying the referring columns of tab. 2 the values clearly prove that kiskörösi kadarka (of the great plains) is reasonably richer in most of the scent impact primary components than the szekszárdi one. conclusion a two step sample preparation method elaborated at our department made us capable of investigating the relative aroma-picture of two high quality kadarka wines by the primary (terpenes and relatives), secondary (alcohols and other oxigenated substances, esters and fatty acids) and tertiary (mainly the benzene and relatives, lactons, … etc.) categories separately. the goal of the work was to describe the varietal (i.e. cultivated variety characterising) aroma-structures of the samples deriving from famous hungarian wineries of the two typical kadarka growing habitats, the great plains and szekszárd. the primary aroma-spectra of the wines disregarding the similarity of a small part in the pictures (circled in the right wing of fig. 2) differ substantially due to the big quality difference of the terroirs. our results show that the poor sand soil of the great plains yields a kadarka wine wealthier in terpene and relative compounds than the loess of szekszárd. the analytical data are supported fairly by the organoleptic experiences. on the kadarka wines of hungary 111 fig. 2: the total (left side) and the primary aroma-structure (right side) of the two kadarka wines fig. 3: the „comprehensive” visual representation of the volatile constituents 112 m. csóka, m. amtmann, d.ny. sárdy, m. kállay, k. korány references burdock, g.a., 2010: fenaroli’s handbook of flavor ingredients. crc press boca raton, 6th edition. eperjesi, i., kállay, m., magyar, i., 2003: in „enology” 4th edition. mezőgazdasági kiadó, budapest (in hungarian). gómez garcía-carpintero, e., sánchez-palomo, e., gonzález-viñas, m.a., 2011: aroma characterization of red wines from cv. bobal grape variety grown in la mancha region. food research international 44, 61-70. gómez garcía-carpintero, e., sánchez-palomo, e., gómez-gallego, m.a., gonzález-viñas, m.a., 2012: free and bound volatile compounds as markers of aromatic typicalness of moravia dulce, rojal and tortosí red wines. food chemistry 131, 90-98. ivanova, v., stefova, m., stafilov, t., vojnoski, b., biro, i., bufa, a., kilar, f., 2012: validation of a method for analysis of aroma compounds in red wine using liquid-liquid extraction and gc-ms. food anal. methods 5, 1427-1434. janyik, f., 2010: the comparison of kadarka clones, msc. theses, corvinus university of budapest (in hungarian). korány, k., amtmann, m., 2005: a practical, theory supported approach of linear temperature programmed gas chromatographic retention indices used in the recognition experiments of hungarian food specialities, called „hungarics”. j. food comp. anal. 18, 345-357. korány, k., amtmann, m., 2006: an experimentally supported, mathematical explanation of the gas chromatographic elution behaviour of the long-chain carbon members of the homologous series. j. food comp. anal. 19, 813-821. korány, k., mednyánszky, zs., amtmann, m., 2000: preliminary results of a recognition method visualizing the aroma and fragrance features. acta alimentaria 29, 187-198. kovács, t., kállay, m., korány, k., 1999: visualization of the gas chromatography/mass spectrometry data of muscat ottonel must and wine measurements. int. j. hort. sci. 5, 16-21. perestrelo, r., fernandes, a., albuquerque, f.f., marques, j.c., câmara, j.s., 2006: analytical characterization of the aroma of tinta negra mole red wine: identification of the main odorants compounds. analytica chimica acta 563, 154-164. takler, a., 2004: the past and present of kadarka vine in the szekszárd terroir, msc. theses, corvinus university of budapest (in hungarian). újvári, t., 2007: the significance of hungaric-vine species, msc. theses, corvinus university of budapest (in hungarian). veverka l., jelinkova, m., hron, k., balik, j., stavek, j., bartak, p., 2012: chemical markers in the aroma profiles of south moravian red wine distillates. czech j. food sci. 30, 369-376. address of the authors: mariann csóka, dr. mária amtmann and prof. kornél korány, department of food chemistry and nutrition, faculty of food science, corvinus university of budapest, h-1118 budapest, hungary dr. diána ny. sárdy and prof. miklós kállay, department of enology, faculty of food science, corvinus university of budapest, h-1118 budapest, hungary journal of applied botany and food quality 89, 243 248 (2016), doi:10.5073/jabfq.2016.089.031 department of plant eco-physiology, faculty of agriculture, university of tabriz, tabriz, iran improving amino acid composition of soybean under salt stress by salicylic acid and jasmonic acid salar farhangi-abriz, kazem ghassemi-golezani* (received june 30, 2016) * corresponding author summary a greenhouse experiment with factorial arrangement based on randomized complete block design with four replications was conducted in 2015 to investigate the effects of salicylic acid (sa) (1 mm) and jasmonic acid (ja) (0.5 mm) on protein accumulation and amino acid composition of soybean seeds under different levels of salinity (0, 4, 7, and 10 ds/m nacl, respectively). treatment of sa improved protein percentage and per seed at different stages of seed development under all salinity levels. the highest protein yield was also recorded for sa treated plants, due to higher seed yield and protein content. in contrast, treatment with ja reduced seed protein content and yield as a result of reduction in seed yield. lysine, methionine, phenylalanine, threonine, aspartic acid, glutamic acid, proline and tyrosine contents increased, but leucine and valine contents decreased with enhancing salinity. foliar spray of sa improved isoleucine, leucine, lysine, methionine, valine, alanine, aspartic acid, glutamic acid, glycine and serine contents, but application of ja increased sulfur containing amino acids such as methionine and aromatic amino acids such as phenylalanine and tyrosine in soybean seeds. treatment with sa had the greatest effect on enhancing protein quantity and quality under different levels of salinity. introduction an important aspect of agriculture is the cultivation of plants for food, fiber, biofuel, medicine and other products used to sustain and enhance human life. agriculture was the key development in the rise of sedentary human civilization, whereby farming of domesticated species created food surpluses that nurtured the development of civilization (bajpai et al., 2014; feng et al., 2014; mishra et al., 2015; nemli et al., 2015). soybean is an important agricultural crop and its seeds with 36-40 % protein content are important sources for human and animal feed (krishnan, 2005). environmental stresses such as soil salinity can limit soybean production. the reduction in soybean seed and protein yields under salinity can be attributed to a shorter seed filling period (ghassemi-golezani et al., 2010). high salinity interferes with plant growth and development and can also lead to physiological drought conditions and ion toxicity, leading to a buildup of na+ and clconcentrations in the cytosol, which can be ultimately detrimental to the cell. higher concentrations of sodium ions can also lead to a reduction in photosynthesis and production of reactive oxygen species (medhat, 2002; khalid et al., 2015). protein quality depends on the amino acid composition. soybean protein contains all the essential amino acids including isoleucine, leucine, lysine, methionine, cysteine, phenylalanine and tyrosine, threonine, tryptophan, valine, and histidine. it has been widely documented that soybean seed composition varies with environmental factors, especially during the seed filling period (wilson, 2004; carrera et al., 2009). seed filling period was correlated with the seed and protein yield in indeterminate soybean lines. wolf et al., (1982) reported that the majority of protein bound and amino acids increased as the salinity severed. karr-lilienthal et al. (2005) reported that essential, non-essential, and total amino acid contents in soybean seeds were lower in cooler zones in comparison with warmer zones. greater deposition of sulfur amino acids (methionine and cysteine) was shown at upper temperatures (wolf et al., 1982). the possible effects of plant growth regulators such as salicylic acid (sa) and jasmonic acid (ja) on protein and amino acid accumulation in oilseed crops are poorly understood. salicylic acid (sa) is an endogenous growth regulator of phenolic nature, which participates in the regulation of physiological processes in plants. sa plays an important role in the defense response to abiotic stresses in plant species (pasala et al., 2016). exogenous treatment of sa enhances plant growth and photosynthetic capa city in saline conditions (noreen et al., 2012). treatment of sa also significantly increases dry weights of root and top part of soybean (gutierrez-coronado et al., 1998), tomato (stevens et al., 2006) and maize (khodary, 2004) under saline conditions. khodary (2004) found that sa could induce salt tolerance in maize plants via accelerating their photosynthesis performance and carbohydrate metabolism. sa increases proteins inside the plant cells that improve plant ability to tolerant the salt stress (kumar et al., 1999). sa also has a positive impact on the activity of nitrate reductase (fariduddin et al., 2003), synthesis of secondary metabolites and antioxidant enzymes activity (singh and usha, 2003; eraslan et al., 2007). jasmonic acid (ja) and its methyl ester (jame) are the key molecules of the octadecanoid signaling pathway. ja is reported to be involved in diverse developmental processes, such as seed germination, root growth, fertility, fruit ripening and senescence (wasternack and hause, 2002). ja content may increase under stressful conditions such as salinity (pedranzani et al., 2007). according to kang et al., (2005) application of ja could enhance tolerances against biotic and abiotic stresses. foliar application of jasmonic acid on soybean under salt stress reduced the damaging influence of salt and improved photosynthesis and yield (yoon et al., 2009). the objective of this research is to investigate the influence of foliar application of salicylic acid and jasmonic acid on protein accumulation and amino acid composition of soybean seeds under salt stress. materials and methods experimental conditions a greenhouse experiment with a factorial arrangement on the basis of randomized complete block design with four replications was conducted in 2015 to investigate changes in protein accumulation and quality of soybean seeds (cultivar m7, a high yielding cultivar widely used in commercial level) under different nacl salinity levels (0, 4, 7 and 10 ds/m) in response to the foliar application of salicylic acid (sa) and jasmonic acid (ja). in this experiment, 48 pots, each filled with 1 kg perlite, were used. 30 seeds were sown in 3 cm depth of each pot, and all pots were then placed in a greenhouse with a day and night mean temperatures of 28 and 26 ºc, respectively. according to the treatments, tap water and saline solutions were added to the pots to achieve 100 % fc (field capacity). after seedling estab244 s. farhangi-abriz, k. ghassemi-golezani lishment, plants were thinned to keep 10 plants in each pot. the pots were weighed regularly and the losses were made up with hoagland solution (ec=1.3 ds/m). all the pots were washed every 30 days and salinity treatments were reapplied in order to avoid further increase in electrical conductivity (ec). salicylic acid (1 mm) and jasmonic acid (0.5 mm) were sprayed on plants at vegetative and flowering stages in accordance with the treatments. leaf nitrogen and sulfur contents the leaf samples were washed with deionized water at least two times to remove the dust. after oven drying at 80 ºc for 48 h, leaf samples were powdered and then nitrogen and sulfur contents were measured by a chns elemental analyzer (elementar-group, hanau, germany). protein accumulation two plants were harvested from each pot with 10 days intervals at five stages, beginning 18 days after flowering. then seeds were detached from the pods and percentages of protein for each sample were determined, using a seed analyzer (zeltex zx-50, maryland, usa). seed and protein yields per plant at final harvest. protein hydrolysis peptides and proteins completely hydrolyzed to free amino acids prior to analysis. seed samples were powdered and then 0.5 g of powder was hydrolyzed in hydrochloric acid (6 m) within glass tubes. the tubes were flushed with nitrogen and then heated at 110 ºc for 24 h. the hydrolyzed samples were rotary evaporated to dry under vacuum at 40 ºc, and then dissolved in a sodium citrate buffer (0.1 m citric acid monohydrate and 0.1 m trisodium citrate). in this way, tryptophan and cysteine were completely degraded during the hydrolysis with hydrochloric acid and glutamine and asparagine being delaminated to glutamic and aspartic acid, respectively (fountoulakis and lahm, 1998; weiss et al., 1998). gas chromatography mass spectrometry (gc–ms) analysis varian saturn 2200 gc–ms system (varian, netherland) was used with silica capillary column (30 m × 0.32 mm, 0.25 lm film thickness). one micro liter of prepared samples was separated and analyzed using gc–ms with split mode (10:1). helium was used as carrier gas (1.5 ml/min). the primary temperature of the column was 100 ºc, followed by a ramp of 10 ºc/min to 140 ºc, a second ramp of 10 ºc/min to 170 ºc, a third ramp of 15 ºc/min to 185 ºc, and finally a ramp to 230 ºc at 15 ºc/min. at each stage of programming, the temperature was held for 2, 1, 1, 2 and 5 min, respectively. the injector temperature was 250 ºc. electron impact ionization (ei) interface temperature was 250 ºc and ion source temperature was 200 ºc. statistical analysis analyses of variance of the data for different parameters were carried out, using mstatc software, and means were compared by duncan multiple range test at p ≤ 0.05. figures were drawn by the excel software. results leaf nitrogen and sulfur contents nitrogen content of soybean leaves significantly affected by salinity and hormonal applications (p ≤ 0.01). there was no significant changes in nitrogen content of leaves up to 4 ds/m nacl salinity, but thereafter it was significantly decreased with increasing salt stress. nitrogen content of soybean leaves was augmented by foliar application of sa, but it was diminished by ja treatment (fig. 1). fig. 1: changes in nitrogen content of soybean leaves under different sa linity (a) and hormonal treatments (b) different letters indicate significant difference at p ≤ 0.05. salinity and foliar application of hormones had significant effects on sulfur content of soybean leaves (p ≤ 0.01). sulfur content of leaves reduced as salinity enhanced. treatment with ja and sa increased sulfur content of leaves compared with control plants. plants with ja treatment showed the highest sulfur content in leaves (fig. 2). protein accumulation in seeds protein percentage of soybean seeds under all sa line and non-saline conditions increased with increasing seed filling period. stable value for protein percentage of all treatments was attained at about 52 days after flowering. protein percentage of all soybean seeds at different stages of seed development and sa linity levels was higher for sa treated plants, followed by control plants. the lowest protein percentage under these conditions was recorded for seeds from ja treated plants (fig. 3). protein content per seed enhanced with increasing seed filling up to 48-58 days after flowering, depending on salinity levels and hormonal applications. the highest protein content per seed under salin ity treatments was obtained 5-10 days earlier than that under non-saline condition. foliar application of sa improved protein content of soybean seeds at different developmental stages under all salinity levels. this improvement was particularly high under moderate (7 ds/m) and high salinity (10 ds/m) levels, with no noticeable differences in seed protein content of ja treated and control plants (fig. 4). fig. 2: changes in sulfur content of soybean leaves under different salinity (a) and hormonal (b) treatments different letters indicate significant difference at p ≤ 0.05. soybean amino acids affected by salinity and plant hormones 245 seed and protein yields effects of salinity and hormonal application on seed yield was significant (p ≤ 0.01). seed yield per plant decreased with increasing salinity. treatment with sa significantly improved seed yield (about 17 %), but there was no significant difference between ja treated and control plants (tab. 1). analysis of variance of the data for final harvest showed significant effects of salinity and hormonal treatments on protein percentage and yield of soybean seeds (p ≤ 0.01) interaction of salinity and hormonal application for protein yield was also significant (p ≤ 0.05). protein percentage decreased with enhancing salinity. seeds from sa and ja treated plants had the highest and the lowest protein percentages, respectively (tab. 1). protein yield per plant decreased as a result of increasing salinity. the highest protein yield was recorded for sa treated plants under saline and non-saline conditions. treatment with ja under non-saline and low saline (4 ds/m) conditions significantly reduced protein yield per plant, but there was no significant difference between ja treated and control plants under 7 ds/m and 10 ds/m salinities (fig. 5). fig. 3: changes in protein percentage of soybean (m7 cultivar) seeds in response to salinity and hormonal applications fig. 4: changes in protein content of soybean (m7 cultivar) seeds in response to salinity and hormonal applications tab. 1: means of soybean seed yield and protein percentages under different salinity and hormonal treatments treatments seed yield protein (mg/plant) (%) salinity 0 ds/m 3710 a 38.46 a 4 ds/m 3260 b 38.24 ab 7 ds/m 2840 c 37.96 b 10 ds/m 2070 d 36.25 c hormonal treatment control 2870 b 37.73 b sa 3360 a 38.92 a ja 2680 b 36.54 c different letters in each column indicate significant difference at p ≤ 0.05. essential amino acids with the exception of isoleucine (ile), all of the essential amino acids significantly affected by salinity. lysine (lys), methionine (met), phenylalanine (phe) and threonine (thr) increased, but leucine (leu) and valine (val) decreased with enhancing salinity. foliar treatment of sa improved isoleucine, leucine, lysine, methionine and valine contents in soybean seeds. however, sa had no significant effect phenylalanine and threonine contents. methionine, phenylalanine and threonine contents enhanced, but isoleucine content fig. 5: protein yields of soybean seeds per plant under different salinity and hormonal treatments different letters indicate significant difference at p ≤ 0.05. 246 s. farhangi-abriz, k. ghassemi-golezani reduced in response to ja treatment. seeds from ja treated plants did not show significant difference in leucine, lysine and valine contents, compared with those from control plants (tab. 2). non-essential amino acids increasing salinity led to the increment of aspartic acid (asp), glutamic acid (glu), proline (pro) and tyrosine (tyr) contents, but had no significant effects on alanine (ala), glycine (gly) and serine (ser) contents. all of the non-essential amino acids except proline affected by hormonal treatments. the contents of alanine, aspartic acid, glutamic acid, glycine and serine were augmented by sa application. in contrast, treatment with ja increased tyrosine content, decreased aspartic acid and glycine contents, but had no significant effect on alanine, glutamic acid and serine contents (tab. 3). discussion nitrogen is the element that plants require in large quantity. it plays a dominant role in plant metabolism as a constituent of many cell components like proteins and enzymes (hawkesford et al., 2012). reduction in leaf nitrogen content under salinity (fig. 1a) could be the result of inhibiting the activity nitrate reductase by high sodium contents (baki et al., 2000). treatment with sa promoted the activity of nitrate reductase in plants (aydin and nalbantoğlu, 2011) and increased nitrogen content of soybean leaves. however, foliar application of ja enhances ethylene synthesis and inhibits root growth (xie et al., 1998), which limits nitrogen uptake and translocation to soybean leaves (fig. 1b). decreasing sulfur content of leaves due to salinity (fig. 2a) is related with the inhibition of sulfur uptake. high salinity and drought overlaps with each other, as high salt limits water uptake by the plants. this makes it ever more difficult for the plants to acquire water as well as nutrients such as sulfur. improving sulfur uptake by sa treatment may be resulted from inhibition of ethylene synthesis and enhancing root growth by this hormone. in contrast, increasing sulfur content of leaves with ja application (fig. 2b) is related with enhancing genes expression, glutathione synthesis, and glucosinolate activities in plant organs, especially in roots (xiang and oliver, 1998; jost et al., 2005). protein is the most important constituent of soybean seeds. it is synthesized and accumulated in the seeds during filling period (ghassemi-golezani et al., 2010). de creasing protein percentage and protein content with increasing salinity (fig. 3 and 4) could be associated with the distur bance in nitrogen metabolism and inhibition of nitrate absorption. reduction in nitro gen under saline conditions is likely due to the reduc tion of absorbed water and a decrease in root permeability (strogonov et al., 1970). hobbs and muendel (1983) reported similar results for soybean seeds under moisture stress. foliar applications of sa increased nitrogen content (fig. 1b) and consequently protein percentage and protein per seed under all tab. 2: essential amino acids content of soybean seeds under different salinity and hormonal applications treatments ile leu lys met phe thr val (mg/g) (mg/g) (mg/g) (mg/g) (mg/g) (mg/g) (mg/g) salinity 0 ds/m 18.95 a 30.25 a 24.65 d 9.69 d 17.44 c 15.32 d 17.40 a 4 ds/m 18.86 a 30.15 a 25.45 c 10.25 c 17.67 c 15.71 c 16.30 bc 7 ds/m 18.92 a 29.24 b 26.56 b 10.63 b 18.12 b 16.52 b 16.36 b 10 ds/m 19.02 a 29.30 b 27.49 a 10.95 a 18.86 a 16.96 a 15.10 d hormonal treatment control 18.85 b 29.66 b 25.93 b 9.83 c 17.72 b 15.89 b 16.33 b sa 19.36 a 30.22 a 26.60 a 10.35 b 17.95 b 15.97 b 16.93 a ja 18.61 c 29.33 b 25.58 b 10.93 a 18.40 a 16.53 a 16.35 b different letters in each column indicate significant difference at p ≤ 0.05. tab. 3: non-essential amino acids content of soybean seeds under different salinity and hormonal applications treatments ala asp glu gly pro ser tyr (mg/g) (mg/g) (mg/g) (mg/g) (mg/g) (mg/g) (mg/g) salinity 0 ds/m 17.13 a 43 d 67.50 c 20.56 a 20.06 d 26.27 a 18.82 d 4 ds/m 17.12 a 44.36 c 67.66 c 20.50 a 21.13 c 26.28 a 19.51 c 7 ds/m 17.21 a 46.58 b 70.50 b 20.42 a 21.83 b 26.32 a 20.19 b 10 ds/m 17.20 a 47.11 a 72.42 a 20.57 a 22.78 a 26.31 a 21.52 a hormonal treatment control 17.03 b 45.10 b 68.88 b 20.41 b 21.46 a 26.20 b 19.64 b sa 17.43 a 46.46 a 70.58 a 21.51 a 21.42 a 26.63 a 19.89 b ja 17.03 b 44.22 c 68.87 b 19.70 c 21.47 a 26.05 b 20.50 a different letters in each column indicate significant difference at p ≤ 0.05. soybean amino acids affected by salinity and plant hormones 247 salinity levels (fig. 3 and 4). decreasing protein percentage and protein per seed by ja application under non-saline and mild salinity (4 ds/m) directly related with reduction in nitrogen content of seeds from ja treated plants (fig. 1b). little differences in seed protein content of ja treated and untreated plants under moderate and severe salinity were probably due to similar decrease in nitrogen uptake of these plants under subjected salt stresses (fig. 3 and 4). reduction in seed yield under salinity was the main reason of decreasing protein yield per plant (tab. 1, fig. 5). the negative effect of salinity on plants may provoke osmotic potential by salt, so root cells do not obtain required water from the medium (çelik and atak, 2012). consequently, the uptake of some mineral nutrients such as nitrogen dissolved in water is also restricted. higher concentrations of sodium ions are toxic to cell and inhibit the activity of essential enzymes, membrane disorganization, and osmotic imbalance, which finally can reduce protein syntheses in seeds (medhat, 2002). increasing seed and protein yields with sa treatment in soybean seeds (tab. 1, fig. 5) may be related with increasing activities of many anti-oxidant enzymes (shi et al., 2006), decreasing synthesis of ethylene (leslie and romani, 1986), improving nitrogen and sulfur uptakes (fig. 1b and 2b) and enhancing maximum efficiency of psii (ghassemi-golezani and lotfi, 2015). in contrast, treatment with ja stimulated ethylene synthesis (xie et al., 1998) and limited nitrogen uptake (fig. 1b), thereby reducing seed and protein yields under non-saline and low salinity conditions (tab. 1, fig. 5). this may also be related to allocation of nitrogen and protein to vegetative sinks (staswick, 1994). these limitations also occurred for ja untreated plants at moderate and high salinity, leading to almost similar protein yield with ja treated plants under these salinity levels (fig. 5). increasing some of essential (lysine, methionine, phenylalanine and threonine) and non-essential amino acids (aspartic acid, glutamic acid, proline and tyrosine) under salinity (tab. 2 and 3) suggests that these amino acids may be acting as the sinks for excess nitrogen when decreased growth occurring during the imposed stress. aspartic and glutamic acids were the major amino acids accumulated in seed tissues of soybean under salt stress (tab. 3). increasing aspartic and glutamic acids in the seeds may reflect the mobility of these important amino acids in the phloem (girousse et al., 1996). however, salinity has a negative impact on synthesis of leucine and valine (tab. 2). improving nitrogen content in plant leaves by sa treatment (fig. 1b) resulted in increased isoleucine, leucine, lysine, methionine, valine, alanine, aspartic acid, glutamic acid, glycine and serine content in seeds (tab. 2 and 3). foliar application of ja enhanced the phenylalanine and tyrosine (aromatic amino acids) contents of seeds (tab. 2 and 3) via stimulating shikimate pathway (herrmann and weaver, 1999; tzin and galili, 2010). however, increasing methionine (sulfur containing amino acid) synthesis in the seeds by ja treatment (tab. 2) directly related with the increment of sulfur content in plants (fig. 2b). on the other hand, allocation of more protein and nitrogen to vegetative sinks at different stages of maturity of ja treated plants (staswick, 1994) reduced some amino acids such as isoleucine, aspartic acid and glycine in soybean seeds (tab. 2 and 3). conclusions foliar application of sa enhanced protein yield of soybean seeds through increasing seed yield and protein percentage and per seed under saline and non-saline conditions. however, treatment with ja reduced seed protein yield, due to the reduction in seed yield and protein content. increasing salinity led to an increase in lysine, methionine, phenylalanine, threonine, aspartic acid, glutamic acid, proline and tyrosine contents, and a decrease in leucine and valine contents. seeds of sa treated plants had higher isoleucine, leucine, lysine, methionine, valine, alanine, aspartic acid, glutamic acid, glycine and serine contents, but seeds of ja treated plants produced more sulfur containing amino acids such as methionine and aromatic amino acids such as phenylalanine and tyrosine. application of sa was determined as a best way for enhancing protein quantity and quality 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turner, j.g., 1998: coi1: an arabidopsis gene required for jasmonate-regulated defense and fertility. science. 280, 1091-1094. yoon, j.y., hamayun, m., lee, s.k., lee, i.j., 2009: methyl jasmonate alleviated salinity stress in soybean. j. crop sci. biotechnol. 12, 63-68. address of the corresponding author: kazem ghassemi-golezani, department of plant eco-physiology, faculty of agriculture, university of tabriz, 29 bahman blvd, tabriz, east azerbaijan, iran e-mail:golezani@gmail.com © the author(s) 2016. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 89, 171 175 (2016), doi:10.5073/jabfq.2016.089.021 department of agraria, university “mediterranea” of reggio calabria, reggio calabria (rc), italy phytochemical properties and antioxidant potential from citrus bergamia, risso (bergamot) juice extracted from three different cultivars vincenzo sicari*, teresa maria pellicanò (received december 14, 2015) * corresponding author summary secondary substances occurring in plant-derived products possess biological activity and thus may protect human beings from various diseases. the following physical and nutritional properties of three bergamot cultivars (castagnaro, fantastico and femminello) were determined and compared: ph, titratable acidity, vitamin c, total flavonoids, total polyphenols, antocianyn, bioactive molecules and antioxidant capacity (abts and dpph assay). the comparison data, were found to be statistically different. in all juice samples analyzed the highest antioxidant capacity was found in castagnaro juice (64.21 % i of dpph and 1.97 % i of abts) compared to fantastico (44.48 % i of dpph and 1.83 % i of abts) and femminello (33.39 % i of dpph and 1.13 % i of abts). these differences could be attributed to the individual characteristics of these cultivars. introduction bergamot “citrus bergamia risso”, is a citrus fruit belonging to the family rutaceae, of the genus citrus. the origin of bergamot still remains mysterious. this plant has been successfully cultivated in calabria (italy) since the eighteenth century in a small area with the ideal habitat. production is mostly limited to the ionian sea coastal areas of the province of reggio di calabria in italy, to such an extent that it is a symbol of the entire city. most of the bergamot comes from a short stretch of land where the temperature is favourable. it is also grown in southern france and in the côte d’ivoire for the essential oil and in antalya in southern turkey for its marmalade. only three cultivars are commercially cultivated: “castagnaro”, femminello” and “fantastico”. formerly, femminello and castagnaro made up virtually all commercial plantings but they have largely been replaced by fantastico, a hybrid of femminello and castagnaro. the fruit is not generally grown for juice consumption, which, in contrast to that from other citrus fruits, is considered a waste product of the essential oil production; it has not found a real use in the food industries until now because of its bitter taste. in the past, bergamot was largely grown for the cosmetics and perfume industries, since its essential oil is rich in terpenes, esters and alcohols which have a characteristically intense fragrance. in recent years, the beneficial properties of bergamot juice have been raising interest and have been the subject of several recent studies, which consider the potential of its health promoting substances (kim et al., 2003; kurowska and manthey, 2004; miceli et al., 2007; pernice et al., 2009; mollace et al., 2011; impellizzeri et al., 2014; ferlazzo et al., 2015; filocamo et al., 2015). thanks to growing interest in bioactive, antioxidant compounds from alimentary source, bergamot juice has attracted the attention of the scientific research, due to its high flavonoid content. bergamot presents a unique profile of flavonoids and flavonoid glycosides in its juice, such as neoeriocitrin, neohesperidin, naringin, narirutin (sicari et al., 2015). diets rich in flavonoids reduce postischemic miocardiac damage in rats (facino et al., 1999), coronaric damage and the incidence of heart attacks in elderly man (hertog et al., 1993). the major causes of cell damage following oxidative stress are reactive oxygen species (ros). reactive oxygen species (ros) are produced as a normal product of plant cellular metabolism. various environmental stresses lead to excessive production of ros causing progressive oxidative damage and ultimately cell death (sastre et al., 2000). after ingestion as food the flavanone glycosides are metabolized by human intestinal bacterial microflora to the respective aglycone (felgines et al., 2000; matsumoto et al., 2004), which seem to possess antioxidant (hirata et al., 2005), anticarcinogenic (shen et al., 2004) and anti-inflammatory (rotelli et al., 2003) properties. flavanoids are one of the most widely studied natural antioxidants, due to their anti-inflammatory and anti-atherosclerotic properties (balestieri et al., 2003; jung et al., 2003). the wide variety and high quantity of flavonoids found in bergamot juice may offer new applications for the juice, which until now has been largely unexploited. the aim of this study was to investigate and characterize the bioactive components of bergamot juices obtained from three cultivars fantastico, femminello and castagnaro grown in calabria (italy) and their antioxidant potential. materials and methods chemicals folin ciocalteu’s reagent, abts [2, 2’-azinobis (3-ethylbenzothiazoline-6-sulfonate)], 2,2-diphenyl-1-picryl hydrazyl (dpph), 6-hydroxy-2,5,7,8-tetramethyl-2-carboxylic acid, 97 % (trolox) were purchased from sigma-aldrich chem. co. (st. louis, mo, usa). phenolic acids (gallic, vanillic, caffeic, ferulic, p-coumaric, and chlorogenic) were purchased from sigma-aldrich chem. co. (milwaukee, wi, usa). the other phenolic compounds, cyanidin 3-glucoside, narirutin, naringin, hesperidin, neohesperidin quercetin, rhamnetin and rutin (quercetin-3-o-rutinoside), were supplied by extrasynthese (genayfrance). acetonitrile, formic acid and water, all hplc grade solvents, were obtained from carlo erba reagents (milano, italia). all other reagents used were of analytical grade. samples and fruit juices extraction bergamot fruits of three different cultivars (tab. 1) were obtained from a grower in the south of italy (province of reggio calabria) in february 2015. the fruits used in the experiments are all from the same area of bova marina on the ionian coast of the province of reggio calabria, at 20 30 m.a.s.l. the trees were all between 15 20 years old and in 172 v. sicari, t.m. pellicanò full production at the time of harvest. all the trees were watered by drip-irrigation and subject to the same agricultural practices. the fruits were harvested at optimum maturity and carefully selected according to uniformity of size and shape, and subsequently transported, in a plastic crate to the laboratory. harvesting was conducted at random on 10 trees per cultivar. five (5) fruits per tree were collected, washed and wiped. juices were extracted by cutting the fruit in half and careful hand-squeezing in a commercial kitchen juicer. the juices were passed through a strainer to remove pulp and seeds. squeezed juices were centrifuged at 3000 rpm for 15 min. the supernatant was passed through a 0.45 μm filter (millipore corporation, bedford, usa) and was used directly for the final samples. the obtained juices were kept at -18 °c until analysis. chemical parameters sugar concentration, in terms of °brix, was measured by using a hand refractometer (atago co., tokyo, japan) with a scale range of 0 32 °brix. the suspended solid content was determined in relation to the total juice (%, w/w) by centrifuging 45 ml of a pre-weight sample at 2000 rpm for 20 min; the weight of settled solids was determined after removing the supernatant. the ph was measured in sample juices by ph meter (basic model 20, crison) and the total titratable acidity (ta) was assessed by titration with naoh (0.1 n) to ph 8.1 and expressed as % citric acid. the ascorbic acid content was evaluated using iodometric titration with a iodine 0,01 n solution. results are expressed in mg/100 ml of juice. total phenols, flavonoids and anthocyanins spectrophotometric determination total phenols were estimated colorimetrically by using the folinciocalteau method (singleton and rossi, 1965) and results were expressed as gallic acid equivalents. an aliquot of 500 μl of juice was mixed with 1 ml of folin-ciocalteu reagent and 10 ml of 20 % na2co3. the absorbance was measured at λ = 760 nm using a uv-vis agilent 8453 spectrophotometer (agilent technologies, italy) after 2 h in the dark. the results were expressed as gallic acid equivalents (gae) in mg/100 ml. flavonoid content was determined using alcl3 assay (yoo et al., 2014). an aliquot of 500 μl of juice was mixed with 300 μl of 5 % nano2. after 5 min, 600 μl of 10 %. alcl3 was added and finally, after a further 5 min, 2 ml of 1 m naoh was added to the mixture. the absorbance was measured at λ = 510 nm using a uv-vis agilent 8453 spectrophotometer (agilent technologies, italy). the results were expressed as rutin equivalents in mg/100 ml. anthocyanins were estimated by a ph-differential method (nielsen et al., 1991). the extract was diluted in a ph 1.0 and ph 4.5 buffer solution. absorbance was measured in a uv-vis agilent 8453 (agilent technologies, italy) spectrophotometer at 520 and at 700 nm. the ex pression a = [(a520-a700)ph1 (a520-a700) ph4.5] was used. results were expressed as μg of cyanidin 3-glucoside/ml using 29.600 molar extinction coefficient (fattahi et al., 2009). liquid chromatographic analysis of antioxidant compounds antioxidant compounds in the different bergamot varieties were determined by hplc/dad. analyses were performed on a knauer (asi advanced scientific instruments, berlin) system equipped with two pumps smartiline pump 1000, a rheodyne injection valve (20 μl), a photodiode array detector uv/vis equipped with a semi micro-cell. data processing data were carried out using clarity software (chromatography station for windows). compounds were separated in a phenomenex c18 column (250 mm × 4.6 mm, 5 μm). as la torre et al. (2006) reports, the mobile phase was a gradient prepared from formic acid in water (ph = 3, solvent a) and formic acid in acetonitrile (ph = 3, solvent b): 0.01 20.00 min 5 % b isocratic; 20.01 50.00 min, 5 40 % b; 50.01 55.00 min, 40 95 % b; 55.01 60.00 min 95 % b isocratic. the column temperature was 30 °c and the flow rate was 1.0 ml/min. samples were filtered through a 0.45 mm membrane filter before injection. the injection volume was 20 μl. peaks were monitored at 280, 254 and 365 nm. the identification and quantification of antioxidant compounds were carried out from the retention times in comparison with authentic standards. analyses were performed in triplicate. dpph test the antioxidant activity was determined using dpph free radical assay (brand-williams et al., 1995). an aliquot of 2.5 ml of 0.06 mm dpph methanolic solution was added to 50 μl of citrus juices. the absorbances at t0 and t5 were measured at λ = 515 nm using a uv-vis agilent 8453 spectrophotometer (agilent technologies, italy). the results were expressed according to the following equation: (%) inhibition = (1-af /a0) × 100 where af = absorbance dpph fruit juice at t = 5 min and a0 = absorbance dpph (control) at t = 0 min. all tests were run in triplicate and the results expressed as means ± standard deviation (sd). trolox was used as a standard antioxidant and juice activity was expressed in mm of trolox equivalents. abts radical-scavenging assay the radical scavenging capacity of the samples for the abts (2,2’-azinobis-3-ethylbenzothiazoline-6-sulfonate) radical cation was determined as described by re et al. (1999). abts was generated by mixing 7 mm of abts and k2s2o8 (potassium persulfate) 140 mm. followed by storage in the dark at room temperature for 16 h before use. the mixture was diluted (1:80) with ethanol to give an absorbance at 734 nm using the spectrophotometer. each sample was diluted, was allowed to react with fresh abts solution (900 μl), and then the absorbance was measured 6 min after initial mixing. all measurements were performed in triplicate. statistical analysis each value is the mean of three replicates. analysis of variance (oneway anova) was conducted using spss version 17.0 for windows (spss inc., chicago, il) and the tukey’s test was used to determine any significant difference among all treatments at p < 0.05. results and discussion significant differences (p < 0.05) were determined among means of the three cultivars investigated. the quality parameters, including ph, total soluble solids (tss), tab. 1: bergamot cultivars of fruit juices analyzed. species variety citrus × aurantium bergamia castagnaro c fantastico f femminello fe bioactive compounds in bergamot juice (citrus bergamia, risso) 173 tab. 4: phenolic acid contents of bergamot juice (mg ml-1). data are expressed as mean ±rsd (n = 3). letters represent significant differences between samples (p < 0.05); nd means not detected. citrus fruit gallic acid vanillic acid caffeic acid chlorogenic acid c 1.86±0.02a 1.70±0.03a 1.51±0.07a 9.37±0.09a f 1.38±0.06b 1.13±0.04b 1.38±0.06b 4.13± 0.07b fe 1.10± 0.04c 0.85±0.04c 0.65±0.1c 3.21± 0.06c ** ** ** ** data expressed as mean ±rsd (n = 3). letters represent significant differences between samples (p < 0.05). nd: not detected tab. 2: ta, tss, tss/ta ratio and ph of bergamot juice. data are expressed as mean ±rsd (n = 3). letters represent significant differences between samples (p < 0.05); ta = total acidity; tss = total soluble solids. citrus fruit tss ta ph tss/ta (g 100 ml-1) c 10.70 ± 0.62b 1.03 ± 0.03a 2.84 ± 0.07a 10.38 ± 0.32c f 9.40 ± 0.00c 0.76 ± 0.05c 2.77 ± 0.03a 12.36 ± 0.15b fe 11.27 ± 0.76a 0.87 ± 0.04b 2.79 ± 0.03a 12.95 ± 0.27a ** ** * ** total acidity (ta) and tss/ta ratio of bergamot juices were shown in tab. 2. these parameters are known to be important for the sensory characteristics of fruit (humayun et al., 2014). the total anthocyanin contents were from 0.95 μg/ml (castagnaro) to 0.65 μg/100g (femminello) and 0.67 μg/ml (fantastico). castagnaro was observed to be higher in anthocyanin content compared to other cultivars. the amounts of total phenols in analyzed juices show significant differences between different cultivars (tab. 3). the highest content of this fraction was detected in castagnaro juice (1.53 mg/ml), while the lowest values were detected in femminello (0.81 mg/ml). tab. 4 shows the phenolic acid contents of the castagnaro, fantastico and femminello bergamot juices, expressed by mean (mg/ml). bergamot juice includes gallic acid, caffeic acid, vanillic acid and chlorogenic acid. castagnaro juice showed a higher concentration of phenolic acid compared to the other cultivars (9.37 mg/ml of chlorogenic acid, 1.86 mg/ml of gallic acid, 1.70 mg/ml of vanillic acid and 1.51 mg/ml of caffeic acid). the variation of phenolic compounds in the fruits depends on many factors such as the degree of maturity at harvest, genetic differences, and environmental conditions, etc. tab. 3: flavonoids, polyphenols, anthocyanins and ascorbic acid contents of bergamot juice. data are expressed as mean ±rsd (n = 3). letters represent significant differences between samples (p < 0.05). citrus fruit flavonoid polyphenol antocianyn ascorbic total total total acid mg ml-1 mg ml-1 mg ml-1 mg ml-1 c 0.34 ± 0.02a 1.53 ± 0.07a 0.95 ± 0.03a 0.92 ± 0.05a f 0.21 ± 0.03b 1.35 ± 0.04b 0.68 ± 0.07b 0.74 ± 0.06b fe 0.19 ± 0.02b 0.81 ± 0.02c 0.65 ± 0.03b 0.61 ± 0.04c ** ** ** ** the ph of the juice samples were between 2.77 and 2.84. femminello had the highest tss value (11.27 %) whereas fantastico had the lowest value (9.40 %). castagnaro had the highest ta value (1.03 g/100 ml) followed by femminello (0.87 g/100 ml), while fantastico had the lowest value (0.76 g/100 ml). it might be presumed that the different ph values of different bergamot cultivars might be related to the variation of organic acids. the tss/ta ratio was also an important parameter, related to quality characteristics of citrus fruits, where femminello had the highest value of tss/ta (12.95), and castagnaro had the lowest value (10.38). bergamot juice is a rich source of vitamin c, which is an important antioxidant (miller and rice-evans, 1997) and the concentration of vitamin c is also a significant indicator of bergamot juice quality. changes in vitamin c could be a good indicator of enzymatic or non-enzymatic degradative reactions taking place during processing or storage of the fruit (skrede, 1996). as can be seen, castagnaro juice showed a higher concentration of total acidity and ascorbic acid compared to femminello and fantastico. concentration of ascorbic acid in cultivars castagnaro, fantastico and femminello were found to be 0.92, 0.74 and 0.61 mg/ ml respectively (tab. 3). the total anthocyanin (tac), polyphenol (tpc) and flavonoid (tfc) content among the different bergamot cultivars were statistically significant, as given in tab. 3. total flavonoids were determined using alcl3 assay (expressed as rutin equivalents), total phenolics were measured by folin ciocalteau method (expressed as gallic acid equivalents) and total antocianyns were estimated by a ph-differential method (expressed as μg of cyanidin 3-glucoside/ml). total flavonoids ranged from 0.19 mg/ ml to 0.34 mg/ml, castagnaro cultivar had a much higher content of total flavonoids than fantastico or femminello. total phenolics in bergamot juice ranged between 0.81 and 1.35 mg/ml, fantastico cultivar presents the highest value (1.35 mg/ml), while femminello had the lowest value (0.81 mg/ml). flavanones are the major flavonoid in bergamot juice. five flavanones: narirutin, naringin, hesperidin, neohesperedin and melitidin were identified in castagnaro, fantastico and femminello juices. tab. 5 shows that, of the five flavanones, naringin and neohesperedin were the most abundant in all cultivars. this agrees with the literature, which report that the most abundant components in bergamot juices are naringin, neoeriocitrin and neohesperedin (gionfriddo et al., 1996; kawaii et al., 2004; dugo et al., 2005; gattuso et al., 2006; nogata et al., 2006). gattuso et al., 2007 in a similar study, found that the cultivar with the greatest antioxidant activity was femminello, followed by castagnaro, and lastly fantastico. these results differ from those found in the present study, where the greatest antioxidant activity was found in castagnaro, followed by fantastico and then finally by femminello. these differences might be due to a number of factors, including the area from which the fruits were harvested, the age of the trees from which the fruits were chosen, the degree of ripeness at the time of harvest, the climatic conditions during the harvest year, agricultural treatments that the trees had undergone, and the storage of the fruits after harvesting. the concentration of flavanones was higher in castagnaro juice compared to fantastico and femminello. the flavones identified in all bergamot juices were rutin and diosmetin 6,8-glycoside. fantastico bergamot juice contained more flavone compounds than castagnaro and femminello. the major flavone was diosmetin 6,8-glycoside in all cultivars. the lowest concentration was detected in the femminello (313.4 mg/l), while the highest was detected in the fantastico (424.6 mg/l). flavanone glycosides account for 95 % of total flavonoids present in bergamot juice, while flavones can be found in the remaining 5 %. reactive oxygen species (ros) are closely connected to many 174 v. sicari, t.m. pellicanò pathological conditions such as inflammation, tumours, cardiovascular disease, cerebral ischemia and diabetes. dpph· and abts· are stable free radicals, which have been widely accepted as a tool for estimating free radical scavenging activities of antioxidants (krishnaiah et al., 2010). both colorimetric methods are based on the prooxidant capacity of free radicals towards easily oxidizable substrates. both methods are direct, stable, easily reproduced and widely used to determine total antioxidant activity on vegetable matrices. all samples exhibited a radical scavenging activity against both radicals in a concentration-dependent manner (tab. 6). tential of bergamot juice has been previously investigated by trovato et al., 2010, who found a noticeable effect on scavenging dpph radicals with ic50 value of 25.01 ml. conclusion the results of this study indicate the main chemical parameters content of the cultivars (castagnaro, fantastico and femminello) of bergamot fruit. bergamot juice contains several different compounds that can exert beneficial effects on human health. the aim of this study was to evaluate the qualitative and quantitative composition of a bergamot juice obtained from fruit of different cultivars harvested in province of reggio calabria (southern italy). significant differences regarding the chemical composition, nutritional value, and antioxidant activities among the bergamot cultivars were observed. from the values obtained, it can be seen that castagnaro shows a higher content of bioactive compounds and a greater antioxidant activity compared to the other two cultivars. these results may depend on the condition and treatment of the trees at the time the fruits used in the experiment were harvested. bergamot juice can be considered to be a good dietary source of nutrients and antioxidant compounds, especially flavonoids, anthocyanins and polyphenols. the correlation analysis indicated that total polyphenols contribute significantly to the antioxidant activity. the results indicated that bergamot juice has the potential to be further developed into a nutritionally interesting raw material for food and beverage applications. in particular, the castagnaro cultivar showed a considerably high nutritive value and antioxidant activity and could be chosen for functional food development that benefits human health. references balestrieri, m.l., castaldo, d., balestrieri, c., quagliuolo, l., giovane, a., servillo, l., 2003: modulation by flavonoids of paf and related phospholipids in endothelial cells during oxidative stress. j. lipid res. 44, 380-387. calabro, m.l., galtieri, v., cutroneo, p., tommasini, s., ficarra, p., ficarra, r., 2004: study of the extraction procedure by experimental design and validation of a lc method for determination of flavonoids in citrus bergamia juice. j. pharmaceut. biomed. 35, 349-363. cheng, g.w., breen, p.j., 1991: activity of phenylalanine ammonialyase (pal) and concentrations of anthocyanins and phenolics in developing strawberry fruit. j. am. soc. hort. sci. 116, 865-869. fattahi, j., fotouhi, r., bakhshi, d., aghajanzadeh, s., 2009: fruit quality, anthocyanin, and cyanidin 3-glucoside concentrations of several blood orange varieties grown in different areas of iran. hort. environ. biotech. 50, 1-5. felgines, c., texier, o., morand, c., manach, c., scalbert, a., régétab. 6: antioxidant capacity of bergamot juice. data are expressed as mean ±rsd (n = 3). letters represent significant differences between samples (p < 0.05) citrus fruit dpph abts (% i) (% i) c 64.21 ± 6.57a 1.97 ± 0.73a f 44.48 ± 9.03b 1.83 ± 0.32b fe 33.39 ± 5.99c 1.13 ± 0.11c ** ** tab. 5: flavonoid contents of bergamot juice (mg l-1). data are expressed as mean ±rsd (n = 3). letters represent significant differences between samples (p < 0.05), nd means not detected. flavones flavanones citrus fruit rutin diosmetin narirutin naringin neohesperidin melitidin narirutin 6,8-glycoside hesperidin c 28.7±0.3a 412.3±0.4a 3.41±0.16a 528.2±0.43a 554.5±0.22a 33.5±0.20b 12.4±0.1a 182±0.6a f 30.6±0.2a 424.6 ±0.3a 2.16±0.11b 370.7±0.12b 362.4±0.08b 37.1± 0.15ab 10.8±0.2b 148±0.7b fe 20.1±0.1b 313.4±0.1b 1.33±0.06c 327.4±0.09c 231.2±0.06c 37.5±0.07a 11.2±0.1b 156±0.3b ** ** ** ** ** ** ** ** phenolic compounds including anthocyanins, flavonoids, and phenolic acids are known to be responsible for antioxidant activities in fruits, and fruits with higher phenolic contents generally show stronger antioxidant activities. the cultivar influenced the dpph radical scavenging activity with a range of % i values from 64.21 to 33.39 and 44.48 % (castagnaro, femminello and fantastico samples, respectively). castagnaro was also the most effective in the abts test with % i value of 1.97 % followed by the femminello sample (% i value of 1.13 %). the results showed that castagnaro exhibited the highest antioxidant activity, while the lowest was found in femminello, which showed the lowest values for both dpph and abts radical scavenging assays (33.39 % and 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antitumor effects of flavanones in vitro and in vivo: involvement of caspase 3 activation, p21 gene expression, and reactive oxygen species production. toxicology appl. pharm. 197, 84-95. singleton, v.l., rossi, j.a., 1965: colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. am. j. enol. viticult. 16, 144-158. skrede, g., 1996: fruits. in: jeremiah, l.e. (ed.), freezing effects on food quality. dekker, new york. trovato, a., taviano, m.f., pergolizzi, s., campolo, l., de pasquale, r., miceli, n., 2010: citrus bergamia risso and poiteau juice protects against renal injury of diet-induced hypercholesterolemia in rats. phytother. res. 24, 514-519. yoo, k.m., lee, c.h., lee, h., moon, b., lee, c.y., 2008: relative antioxidant and cytoprotective activities of common herbs. food chem. 106, 929-9369. address of the corresponding author: vincenzo sicari, department of agraria, university “mediterranea” of reggio calabria, salita melissari, 89122 reggio calabria (rc), italy. e-mail: vincenzo.sicari@unirc.it © the author(s) 2016. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 89, 59 (2016) buchbesprechung food composition and nutrition tables s.w. souci, w. fachmann, h. kraut die vor mehr als 50 jahren im auftrag des bundesministeriums für landwirtschaft, ernährung und forsten durch prof. dr. s.w. souci, dr. w. fachmann, und prof. dr. h. kraut begründeten nährwert tabellen werden heute von zahlreichen fachkräften in der ernährungsberatung, der lebensmittelüberwachung und lebensmittelforschung als unverzichtbare informationsquelle für ihre tägliche arbeit genutzt. seitdem wurde die der sammlung zugrunde liegende datenbank kontinuierlich erweitert. in diesem zusammenhang wurden sowohl die jeweils aktuelle wissenschaftliche literatur ausgewertet als auch neue analysendaten der deutschen forschungsanstalt für lebensmittelchemie in freising verwendet. darüber hinaus wurden auch die sich ändernden verzehrsgewohnheiten der verbraucher sowie neue erkenntnisse der lebensmittelwissenschaften in dem werk berücksichtigt. die nunmehr vorliegende 8. auflage wurde komplett überarbeitet und erweitert. so wurden z.b. erstmalig die gehalte von phytoöstrogenen (isoflavone, lignane) in lebensmitteln mit aufgeführt. ebenso wurden die gehalte von gluten und folsäure neu aufgenommen. dem zunehmendem konsum von kräuterund früchtetees entsprechend, sind in den nährwerttabellen auch die inhaltsstoffdaten für grünen tee, matetee, fencheltee, kamillentee, malventee, pfefferminztee und rooibostee ergänzt worden. ebenfalls neu aufgenommen wurden inhaltsstoffgehalte unterschiedlicher fleischteile von rind, schwein und schaf, einigen getreideund gemüsearten sowie die lebensmittel schafgarbe, huflattich und scharbockskraut. die nährwerttabellen werden heute umfänglich in staatlichen institutionen sowie in der industrie verwendet. so dienen sie z.b. als referenzdaten für die lebensmittelüberwachung, das lebensmittel monitoring und liefern zuverlässige informationen zum nährstoffstatus der bevölkerung bei der auswertung von verzehrserhebungen. in der lebensmittelindustrie wird die datensammlung insbesondere im rahmen der qualitätssicherung und produktentwicklung eingesetzt. die einleitung sowie alle lebensmittelund inhaltsstoffbezeichnungen in den tabellen sind in deutscher, englischer und französischer sprache verfasst. das tabellenwerk wird durch einen anhang ergänzt, der die aminosäurezusammensetzung von früchten und fruchtsäften sowie die vitamin-a-inaktiven carotinoide in unterschiedlichen lebensmitteln auflistet. außerdem sind hinweise auf die verwendeten datenquellen sowie ein umfangreiches sachregister im anhang zu finden. dr. h. schulz e-mail: hartwig.schulz@jki.bund.de bibliografie: s.w. souci, w. fachmann, h. kraut, food composition and nutri tion tables, wissenschaftliche verlagsgesellschaft mbh, stuttgart, 8. überarbeitete und ergänzte auflage 2016, 1262 seiten, in deutscher, englischer und französischer sprache, hardcover, preis: 124,80 €, isbn: 978-3-8047-5072-2 journal of applied botany and food quality 89, 135 141 (2016), doi:10.5073/jabfq.2016.089.016 1key laboratory of synthetic and natural functional molecule chemistry of ministry of education, college of chemistry and materials science, northwest university, xi’an, pr china 2 lipids group, academy of state administration of grain, xicheng district, beijing, pr china the effects of different extraction methods on the physicochemical properties and antioxidant activity of amygdalus pedunculatus seed oil jun yan1, 2, yehua shen1*, yingyao wang2*, xia luan2, mimi guo2, cong li1 (received december 3, 2015) * corresponding author summary the oil extracted from amygdalus pedunculatus (a. pedunculatus) seeds is rich in nutrients. the method of oil extraction is very crucial for preserving its nutrients. the objective of the present study was to compare a. pedunculatus seed oil (apo) samples extracted by different techniques including aqueous enzymatic extraction (aee), cold-press (cp), supercritical fluid extraction (sfe), and soxhlet extraction (se). physicochemical properties and nutrients (fatty acids, triacylglycerol, polyphenol, tocopherol and phytosterol) of the oils were analyzed. antioxidant activity was measured by dpph, abts·+ radical scavenging capacity and reducing power assays. the results indicated that sfe was found to be the optimum method for apo extraction with higher nutrient contents as well as better dpph, abts scavenging capacities and reducing power. apo is beneficial to human health, and it has potential to be used in nutraceutical industries. introduction amygdalus pedunculatus (a. pedunculatus), a member of the plant family rosaceae, is a deciduous, sand-dune-stabilizing, and oil bearing shrub. it is distributed in the arid region of northwest china and mongolia. the plant shows strong tolerance to cold and drought environments and good adaptability to different types of soil moisture. a. pedunculatus has been widely used to tackle afforestation for preventing desertification in china in recent years (chu et al., 2013). it contributes significantly to local economy through its application in nutraceutical industry. however, most of a. pedunculatus seeds are wasted in the harvesting season due to lack of deep processing technique. in the last decade, apo has drawn attention of many researchers because of its significant dietetic value. high contents of mono unsaturated fatty acids (mufas) and polyunsaturated fatty acids (pufas) have been found in apo. on the other hand, apo is also rich in phenolic, tocopherol and phytosterol antioxidant compounds that have recently drawn attention of nutraceutical industry for their high functional properties (maranz et al., 2003). in the present scenario, it is important to design a suitable extraction method that not only extracts oil from a. pedunculatus seed but also preserves the nutrient content in oil. aqueous enzymatic extraction (aee) is an eco-friendly method based on simultaneous isolation process of oil and protein from seeds. the cp process has become a good substitute of solvent extraction for obtaining natural and healthy edible oil (karaman et al., 2015). supercritical fluid extraction (sfe) has gained a lot of attention because of carbon dioxide (co2) that is used as supercritical fluid in the method (crowe and white, 2003); co2 is an environment friendly, inexpensive, non-toxic, and inert solvent which allows the extraction process to be performed at low temperature and pressure (jung et al., 2012). traditional soxhlet extraction (se) uses organic solvents, and it is considered as one of the most effective extraction methods for vegetable oil seeds. although there are many reports available on physicochemical properties and antioxidant activity of oils (ni et al., 2015; parry et al., 2005) very few investigations focus on the effects of extraction methods on those attributes of oils. hence, we undertook this research work with the objective to investigate the effects of different extraction methods on physicochemical characteristics and valuable compounds (fatty acids, triacylglycerol, polyphenol, tocopherol, and phytosterol compounds) content of the extracted oil from a. pedunculatus seeds. antioxidant activity was used to compare a. pedunculatus seed oil samples, extracted by four different extraction techniques. materials and methods materials a. pedunculatus seeds were collected from yulin, shaanxi province, china and stored at 4 °c prior to analysis. alcalase2.4l was purchased from novozymes (novo, china). all reference substances for triacylglycerol (trilinolein, triolein, glycerol tripalmitate, glycerol trimyristate, and glycerol tristearate), tocopherols (α, β, γ, and δ isomers), phytosterols (cholesterol, botulin) and 1, 1-diphenyl-2picrylhydrazyl (dpph), 2, 2´-azinobis-(3-ethylbenzothiazoline-6 sulfonic acid) (abts) were purchased from sigma aldrich. aqueous enzymatic extraction (aee) process a. pedunculatus seeds were crushed by roll crusher three times. a mixture of crushed a. pedunculatus seeds (100 g) and distilled water at 1:5 (wt/vol) were taken in a reaction by gentle stirring to make slurry, the slurry ph was adjusted to 8.00 by adding 0.20 mol/l naoh and incubated at 60 °c for 0.5 h with continuous stir at 200 rpm. the protease (2 ml) was added after the ph and temperature of the slurry were adjusted to the optimal condition. then the slurry was incubated for 6 h with continuous stirring, followed by centrifugation at 4350 rpm for 15 min to obtain maximum amount of free oil which was named aeeo. cold pressing (cp) process a. pedunculatus seeds were wrapped inside four layers of filtra tion cloth and pressed using a laboratory hydraulic press (dimension: 650 l × 800 d× 1370 h mm) (national eng co., ltd., korea). the maximum pressure for mechanical hydraulic press extraction was 60 mpa and held at the pressure for 20 min. the oil was separated through centrifugation to remove any particles and was named cpo. supercritical fluid extraction (sfe) process a. pedunculatus seeds were crushed by feed crusher three times. crushed a. pedunculatus seeds were taken in a steel cylinder, equipped with mesh filters (100 μm) on both ends to protect particles from 136 j. yan, y. shen, y. wang, x. luan, m. guo, c. li being flushed out. a. pedunculatus seeds (100 g) were transferred into a 300 ml extraction vessel. the equipment used for supercritical extraction (sce) process with co2 was the sce screening system model manufactured by autoclave engineers (applied separations inc., newark, usa). the static extraction time was set for 0.5 h, the dynamic extraction time were 1.5 h with a co2 flow rate of 3 l/h. the extraction was carried out under the following conditions: pressure: 400 bar; temperature: 40 °c. the oil was separated by pressure reduction and collected in the flask and was named sfeo. soxhlet extraction (se) process the a. pedunculatus oil was extracted using the foss soxtectm system 8000 extraction unit (hilleroed, denmark), following the method of brkic et al. (2006). the oil was named seo. physicochemical properties of a. pedunculatus seed oils standard methods made by american oil chemists’ society (aocs, 1998) were used to determine phospholipid content, refractive index, color, density, free fatty acid (ffa), iodine value (iv), peroxide value (pv), and oxidative stability (osi) (aocs, 1998) of the extracted oils. fatty acid composition fatty acid composition were determined according to the method describe by mandana et al. (2013). a. pedunculatus oil after methylation was determined using gas chromatography (gc) (6890n, agilent, usa) with a capillary column (vf-23ms 30 m × 0.25 mm × 0.25 μm, agilent, usa) and a flame ionization detector (fid). triglycerides composition triglycerides composition were determined according to the method describe by shukla et al. (1983). a waters high performance liquid chromatography (hplc) column (waters corporation, usa), fitted with symmetry 300tm c185 μm (4.6 mm × 250 mm), was used to carry out chromatographic separation. polyphenol content polyphenol content was determined according to the method by parry et al. (2005). a. pedunculatus oil was determined by the folin-ciocalteu assay, the absorbances were recorded by a uv-visible spectrophotometer (shimadzu, japan) at 765 nm. we used milligram of gallic acid equivalent (gae) per g (mg gae/g) of extracted oil to express the results. tocopherol content tocopherol content was determined according to aocs method (aocs, 1998) using a hplc equipped with a fluorescence detector. the hplc system was consisted of a lichrocart @ 250-4 column (250 mm × 4.0 mm), 2695 pump and 2475 multi λ fluorescence detector (waters corporation, usa). excitation and emission wavelengths were set at 295 nm and 330 nm, respectively. the mobile phase was consisted of n-hexane/ tetrahydrofuran (1000/40 by vol.), and a flow rate of 1.0 ml/min was used. the oils were diluted in n-hexane before analysis. the tocopherol content was expressed in milligram of tocopherol per 1000 g of extracted oil. phytosterol content the content and composition of phytosterol in a. pedunculatus seed oil samples were analyzed according to an iso method (iso, 1999). agilent (6890n, agilent, usa) gc loaded with a flame ionization detector and a hp-5ms capillary column (30 m × 320 μm × 0.25 μm) was used to confirm phytosterol peak identity. betulin was used as an internal standard for quantification. phytosterol from each analyzed oil sample was identified by the relative retention time (rrt). rrt was expressed as the ratio of retention time of phytosterol to be determined and betulin. antioxidant activity scavenging of 1, 1-diphenyl-2-picrylhydrazyl (dpph) radical the antioxidant activity of a. pedunculatus seed oils extracted by four different methods was determined by a dpph assay according to brand et al. (1995). the decrease in absorbance of dpph at 515 nm was determined using a uv-vis spectrophotometer (shimadzu, japan). the radical-scavenging ability of the experimental samples was measured based on the following formula: scavenging dpph (%) = [(acont − asample)/acont] × 100, where acont and asample were the values of absorbance of blank sample and test sample at particular times, respectively. scavenging activity of radical cation 2, 2´-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (abts·+) the antioxidant activity of the extracted oils was determined using abts radical cation decolorization assay (re et al., 1999). the radical-scavenging activity of the experimental oils was expressed as percentage inhibition of abts·+ and calculated based on the following formula: scavenging abts·+ (%) = [(acont − asample)/acont] × 100, where acont and asample were the values of absorbance of blank sample and test sample at particular times, respectively. reducing power reducing power was determined according to a previously reported method by hu et al. (2008). statistical analysis the data were expressed as mean ± standard deviation, and the analysis of variance (anova) was performed using spss (version 17.0 for windows 2007, spss inc); results with p ≤ 0.05 were considered as statistically significant. results and discussion physicochemical characteristics tab. 1 lists the oil yield and physicochemical properties of oils extracted using four different methods. as evident from the results presented in tab. 1, the extraction yields of oil from the a. pedunculatus seed were 40, 41, 30, 50 g oil/100g seeds after aee, cp, sfe, se, respectively. the phospholipid contents in the oils were expressed as mg p/kg oil. the seo exhibited the highest phosphorus content, followed by aeeo and cpo. interestingly, sfeo did not have detectable quantity of phosphorus probably because of the low solubility of phospholipids. our results agreed well with previous data obtained from the perilla oil (min et al., 2012). we did not find any significant differences in the values of refractive index and density of the extracted oils. the oils extracted by aee and se methods exhibit higher color attributes than those extracted via other two methods. the level of yellow color of oil obtained from aeeo, seo, cpo and sfeo was at 31, 40, 17, and 20, respectively, while the level of red color was at 1.7 for all. this indicated that the higher the experimental temperature was, the deeper the yellowness of the the effects of different extraction methods on the apo 137 oils was. the ffa of apo extracted by cp was the highest among all. a low acidity value indicated higher stability of the oil extracted by sfe method compared to others. among the four extraction methods, the oil obtained from sfe showed the highest iv, followed by the oils extracted by se, aee and cp. compared to aee and se, oils extracted by cp exhibits the highest pv, while the oil extracted by sfe method showed the lowest pv (0.42 mmol o2/kg). the reason for lowest pv could be attributed to the presence of large amount of natural antioxidants in the extracts of supercritical carbon dioxide. apos could be preserved for a long period without deterioration because of their low pv values; oils became rancid when their peroxide value exceeds 10 mmol o2/kg (aocs, 1998). the lower the ffa and the pv value, the better. the oxidative stability of seed oil is usually poor due to its high linoleic acid content. here the stability of apo at 120 °c was expressed as induction time of oxidation that ranges from 7.0 h to 8.3 h for the studied oils. these values were greater than those measured for linseed oil (1.1 h) and olive oil (6.1 h) (wagner et al., 2000). the high oxidation induction time of apos could be ascribed to the presence of large amount of natural antioxidants such as polyphenol, tocopherols, and phytosterols and oleic acid. the osi values had clearly revealed the differences in stability among the four studied oils. we found that the sfeo had the highest oxidation induction time. the higher the iv and the oxidation induction time, the better. fatty acid composition tab. 2 presents fatty acid composition of the oils extracted by four different methods. the oleic and linoleic acids were the most abundant unsaturated fatty acids present in the oils, while palmitic acid tab. 1: physicochemical characterization of a. pedunculatus seed oil extracted using four different methods extraction method aee cp sfe se oil yield (g/100 g seeds) 40±1.2 b 41±0.8 b 30±1.1 a 50±1.3 c phospholipid (mg/kg oil) 35 ± 0.03 c 14 ± 0.04b nd a 215 ± 0.11d refractive index (25 °c) 1.5 1.5 1.5 1.5 density, 25 °c (g/cm3) 0.92 0.91 0.91 0.91 color (yellow) 31 17 20 40 color (red) 1.7 1.7 1.7 1.7 ffa (% oleic acid) 0.24 ± 0.01b 0.46 ± 0.01d 0.16 ± 0.01a 0.35 ± 0.02c pv (mmol o2/kg oil) 0.48 ± 0.04ab 0.76 ± 0.01c 0.42 ± 0.13a 0.62 ± 0.02bc iv (g i2/100 g oil) 102 ± 2.0a 98 ± 0.68a 113 ± 2.3b 103 ± 1.5a osi (120 °c, h) 7.0 ± 0.27a 7.5 ± 0.48ab 8.3 ± 0.42c 7.6 ± 0.25b values are means ± standard deviations for three preparations values given in rows followed by different superscript letters are significantly different at p ≤ 0.05 ffa: free fatty acid; iv: iodine value; pv: peroxide value; osi: oxidative stability; nd: not detect. tab. 2: fatty acid composition of a. pedunculatus seed oil obtained from different extraction methods (%) extraction method aee cp sfe se palmitic (c16:0) 1.5 ± 0.01a 2.4 ± 0.01d 1.6 ± 0.01c 1.6 ± 0.01b palmitoleic (c16:1) 0.18 ± 0.01a 0.16 ± 0.01a 0.29 ± 0.03b 0.27 ± 0.01b stearic (c18:0) 0.55 ± 0.01b 0.77 ± 0.02c 0.50 ± 0.01a 0.57 ± 0.01b oleic (c18:1) 69 ± 0.05c 71 ± 0.04d 68 ± 0.15a 68 ± 0.07b linoleic (c18:2) 28 ± 0.02b 25 ± 0.03a 29 ± 0.09c 28 ± 0.01b linolenic (c18:3) 0.12 ± 0.00a 0.13 ± 0.01a 0.20 ± 0.01b 0.62 ± 0.02c eicosenoic (c20:1) 0.22 ± 0.01ab 0.27 ± 0.01b 0.19 ± 0.01a 0.24 ± 0.02b sfa 2.1 ± 0.01 a 3.2 ± 0.01c 2.1 ± 0.02 ab 2.1 ± 0.01b usfa 98 ± 0.32b 97 ± 0.00a 98 ± 0.05b 98 ± 0.03b mufa 69 ± 0.30b 71 ± 0.04c 68 ± 0.15a 69 ± 0.05bc pufa 28 ± 0.02b 25 ± 0.04a 29 ± 0.09d 29 ± 0.03c usfa/sfa 48 ± 0.20d 30 ± 0.08a 47 ± 0.05c 46 ± 0.12b 1sfa: saturated fatty acids; 2usfa: unsaturated fatty acids; 3mufa: monounsaturated fatty acids; 4pufa: polyunsaturated fatty acids. values are presented as means ± standard deviations for three preparations values given in rows followed by different superscript letters are significantly different at p ≤ 0.05 138 j. yan, y. shen, y. wang, x. luan, m. guo, c. li ecn triglyceride tab. 3: triglycerides content (%) found in a. pedunculatus seed oil obtained from different extraction methods extraction method aee cp sfe se 42 lll 0.75 ± 0.11a 0.68 ± 0.03a 0.97 ± 0.04a 0.98 ± 0.13a 44 lol+ olno 10 ± 0.86ab 9.4 ± 0.18a 12 ± 0.76b 11 ± 0.10ab 44 llp 3.5 ± 0.03 b 3.1 ± 0.01a 4.1 ± 0.04 b 3.7± 0.01b 46 olo 29 ± 0.78b 26 ± 0.13a 30 ± 0.41b 30 ± 0.74b 46 olp 10.9 ± 0.03 b 9.6 ± 0.01a 11 ± 0.01 c 11 ± 0.01 c 48 slo+ooo 37 ± 0.04b 41 ± 0.05c 33 ± 1.2a 34 ± 0.52ab 48 slp 9.1 ± 0.01 b 10 ± 0.02 c 8.3 ± 0.02 a 8.6 ± 0.06ab ecn (equivalent carbon number) = cn (carbon number)-2db (double bond number); l= linoleic acid, o = oleic acid, s = stearic acid, p = palmitic acid, ln = linolenic acid values are presented as means ± standard deviations for three preparations values given in rows followed by different superscript letters are significantly different at p ≤ 0.05 was the principal saturated fatty acid. the content of the essential unsaturated fatty acid (usfa) in apo samples extracted by different methods was high. we also identified some more fatty acids including palmitoleic, stearic, linolenic, and eicosenoic acids, though in less quantity. usfa was major component of the total fatty acid in aeeo (98 %), sfeo (98 %), seo (98 %), and cpo (97 %). usfa exhibited excellent nutritional and physiological properties that help in preventing cancer and coronary heart disease (oomah et al., 2000). mufa was the major component of total fatty acids in apos, extracted by different methods, mainly because of the higher amount of oleic acid in apo. mufa could lower “bad” cholesterol (low density lipoproteins or ldl) and retain “good” cholesterol (high density lipoproteins or hdl) (ramadan et al., 2010). the high mufa content made apo a potential functional component of nutrition to be used in food industry. in addition, the fatty acid content and high pufa content made apo an important component of nutrition. the ratio of unsaturated to saturated fatty acids obtained from aeeo, sfeo and seo were 48, 47 and 46, respectively, while it was 30 for cpo. therefore, the method of extraction had a significant (p ≤ 0.05) effect on the fatty acid composition of the oils. triacylglycerol composition based on the total number of carbon in the acyl side chains and the equivalent carbon number (ecn), the triacylglycerol species were identified. tab. 3 shows the triacylglycerol composition of a. pedunculatus seed oil. according to the data presented in tab. 3, the oils had four types of triacylglycerol with ecn ranging from 42 to 48. triacylglycerols with ecn 48 and 46 were dominant, followed by ecn 44 and 42. we found that slo + ooo and olo (o = oleic acid, l = linoleic acid, s = stearic acid) were the major triacylglycerols representing approximately 65 % of oils obtained from different extraction methods. therefore, the major triacylglycerol molecular types were oleic and linoleic acids, indicating high unsaturation of apo. the cpo had lower contents of lll, lol + olno, llp, olo, olp (p = palmitic acid, ln = linolenic acid) compared to aeeo, sfeo, and seo. the low triacylglycerol content of cpo could be due to the shortest extraction time involved in the process. in addition cpo had the lowest linoleic acid content as revealed from the fatty acid composition (tab. 3). polyphenol composition polyphenol not only exhibit antioxidants but also have biological activities, including antiallergenic, antiviral, anti-inflammatory, and vasodilating action (pietta, 2000). fig. 1 shows the total polyphenol contents of the oils obtained from different extraction methods. as evident from fig. 1, sfeo exhibited the highest polyphenol content of 0.43 mg gaeg−1 dry weight (dw), followed by aeeo (0.39 mg gaeg−1 dw), seo (0.34 mg gaeg−1 dw), and cpo (0.26 mg gaeg−1 dw). the polyphenol contents of our studied oils fall in the middle of those of olive oil (0.07 mg gaeg−1 dw–1.3mg gaeg−1 dw) (beltan, 2007). seo, aeeo and sfeo exhibited higher total polyphenol content than cpo. this could be due to the reason that the pressure applied in cp sometimes (sporadically) failed to squeeze polyphenol out of the cell wall. the polyphenol content positively influenced oxidative stability, nutritional and health properties of apo. tocopherol composition tocopherol, an essential nutrient to human, is the most significant lipid soluble antioxidant. it plays crucial role in scavenging free radicals and preventing lipid peroxidation in biological membra nes. fig. 2 depicts tocopherol contents of apos obtained from four different extraction methods. sfeo had the highest total tocopherol content (1092 mg/kg) followed by aeeo (899 mg/kg), seo (892 mg/kg), and cpo (793 mg/kg). the tocopherol content in a. pedunculatus seed oils obtained from different extraction methods fig. 1: polyphenol content of a. pedunculatus seed oil obtained by different extraction methods [results with standard deviations shown by same letters (a-c) are not significantly different (p < 0.05)] the effects of different extraction methods on the apo 139 was higher than that of sclerocarya birrea seed oil (137 mg/kg) and olive oil (167 mg/kg 463 mg/kg) (mariod et al., 2004; ceci and carrelli, 2010). hplc results revealed γ-tocopherol (followed by δ-tocopherol, βand α-tocopherols) as the major tocopherol present in apo. sfeo contained the maximum amounts of γ-, δand β-tocopherols, followed by aeeo, seo and cpo (fig. 2). however, α-tocopherol was obtained as the lowest tocopherol isomer in solvent-extracted oils (23 mg/kg in seo) as well as in solvent free oils (24 mg/kg in sfeo, 19 mg/kg in aeeo, and 26 mg/kg in cpo). samples, followed by ∆ 5-avenasterol and campesterol. this confirmed sfe was an excellent method for accumulating phytosterol content in the extracted oil (sfeo). the high stability of sfeo and the low stability of cpo could be attributed to the fact that some of the phytosterols, such as ∆ 5-avenasterol, can function as antioxidants (kamal et al., 1992). antioxidant activity dpph is a stable radical that is often used for evaluating radicalscavenging capacity of antioxidants. as shown in fig. 3a, all the studied oils obtained from different extraction methods directly react with dpph radicals and quench them. among the studied oils, sfeo exhibited the least ic50 value (17 mg/ml), followed by cpo (25 mg/ ml), seo (39 mg/ml), and aeeo (44 mg/ml). lower ic50 value indicated more intense activity of scavenging dpph free radicals. therefore, the dpph radical scavenging activities of sfeo and cpo were higher than that of seo and aeeo. antioxidant activities of the apo might be due to the presence of phenolic and nonphenolic components. our study suggested that sfeo had better correlation with dpph radical scavenging capacity than others. moreover, the intense antioxidant activity of sfeo could be attributed to its high content of tocopherols, especially γ-tocopherol. abts radical cation was also suitable for evaluating radical-scavenging capacity of antioxidants. fig. 3b shows the effective abts radical scavenging activity of the oils obtained from different extraction methods. sfeo showed the highest ic50 value (18 mg/ml), followed by seo (18 mg/ml), aeeo (21 mg/ml), and cpo (22 mg/ ml). however, we did not find any obvious differences (p > 0.05) in the prohibiting behaviors of the oils obtained from different extraction methods. natural antioxidants have been known for breaking free radical chain reactions by providing an electron or hydrogen atom to free radicals; the assays based on reducing power are often applied to estimate the capacity of natural antioxidants to provide an electron or hydrogen atom (hu et al., 2008). fig. 3c shows the reducing power of a. pedunculatus seed oil. the reducing power increased with increasing concentration of apo. the results indicated that apos possess moderate electron donating ability that might have some connection with its antioxidant activity. fig. 2: individual tocopherol contents of apo samples extracted by different methods [results with standard deviations shown by same letters (a-c) are not significantly different (p < 0.05)] phytosterols composition the phytosterol level in seed oils is used to determine the quality of oil. phytosterols are highly valued components in health products because of their ability of lowering blood cholesterol by blocking readsorption of cholesterol from the gut (ceci and carrelli, 2010). as evident from tab. 4, individual as well as total phytosterols change depending on the extraction method. sfeo had a higher content of phytosterols than the oils obtained by other methods (p < 0.05). sitosterol was the most abundant phytosterol found in all oil tab. 4: phytosterol content (mg/kg) in a. pedunculatus seed oil obtained from different extraction methods extraction method aee cp sfe se campesterol 216 ± 12ab 178 ± 11a 287 ± 14c 268 ± 2.0bc campestanol 9.6 ± 0.54ab 7.1 ± 0.77a 8.1 ± 0.53a 14 ± 0.68b [24r]-24-methyl cholest-7-en-3β-ol 17.6 ± 0.84b 10.5 ± 0.25a 7.1 ± 0.12a tr [24s]-24-ethyl cholesta-5,25-dien-3β-ol 37 ± 0.64a 29 ± 0.93a 50 ± 0.83b 48 ± 1.93b sitosterol 2706 ± 19 a 2274 ± 24a 3766 ± 87b 3510 ± 59b sitostanol 89 ± 9.00b 49 ± 0.44a 69 ± 4.82ab 134 ± 3.34c ∆ 5-avenasterol 534 ± 4.2ab 422 ± 3.1a 698 ± 5.9c 651 ± 7.3bc stigmasta-5,24-dien-3-ol 52 ± 1.4a 46 ± 0.07a 68 ± 1.2b 70 ± 1.3b stigmast-7-en-3β-ol 30 ± 0.52a 84 ± 1.4b 43 ± 0.41a 43± 0.25a ∆ 7-avenasterol 40± 1.3a 46 ± 0.84a 50 ± 0.82a 50± 0.98a total 3730 ± 26 3145 ± 30 5043 ± 93 4787 ± 71 tr trace amount values are presented as means ± standard deviations for three preparations values given in rows followed by different superscript letters are significantly different at p ≤ 0.05 140 j. yan, y. shen, y. wang, x. luan, m. guo, c. li 05 and 2011ktcl03-04) and the national high-tech research and development projects (863 torch program 2013aa102104). references aocs, 1998: official methods and recommended practices of the american oil chemists society. methods 5th ed. champaign, il, usa: aocs press. beltrán, g., ruano, m.t., jiménez, a., uceda, m., aguilera, m.p., 2007: evaluation of virgin olive oil bitterness by total phenol content analysis. eur. j. lipid sci. tech. 109, 193-197. brand-williams, w., cuvelier, m.e., berset, c., 1995: use of a free radical method to evaluate antioxidant activity. lwt − food sci. technol. 28, 25-30. brkić, k., radulović, m., lukić, i., setić, e., 2006: application of soxtec apparatus for oil content determination in olive fruit. riv. ital. sostanze gr. 83, 115-119. ceci, l.n., carelli, a.a., 2010: relation between oxidative stability and composition in argentinian olive oils. j. am. oil chem. soc. 87, 11891197. chu, j., xu, x., zhang, y., 2013: production and properties of biodiesel produced from amygdalus pedunculata pall.. bioresource technol. 134, 374-376. crowe, t.d., white, p.j., 2003: oxidative stability of walnut oils extracted with supercritical carbon dioxide. j. am. oil chem. soc. 80, 575-578. hu, c.c., lin, j.t., lu, f.j., chou, f.p., yang, d.j., 2008: determination of carotenoids in dunaliella salina cultivated in taiwan and antioxidant capacity of the algal carotenoid extract. food chem. 109, 439-446. iso 12228, 1999: animal and vegetable fats and oils-determination of individual and total sterols contents. in: gas chromatographic method. iso, geneva. jung, d.m., yoon, s.h., jung, m.y., 2012: chemical properties and oxi dative stability of perilla oils obtained from roasted perilla seeds as affected by extraction methods. j. food sci. 77, 1249-1255. kamal-eldin, a., appelqvist, l.å., yousif, g., iskander, g.m., 1992: seed lipids of sesamum indicum and related wild species in sudan. the sterols. j. sci. food agr. 59, 327-334. karaman, s., karasu, s., tornuk, f., toker, o.s., geçgel, ü., sagdic, o., ozcan, n., gül, o., 2015: recovery potential of cold press by-products obtained from oil industry: physicochemical, bioactive and antimicrobial properties. j. agr. food chem. 63, 2305-2313. mandana, b., russly abdul, r., farah saleena, t., noranizan mohd, a., sarker, m.z.i., ali, g., 2013: supercritical carbon dioxide extraction of seed oil from winter melon (benincasa hispida) and its antioxidant activity and fatty acid composition. molecules. 18, 997-1014. maranz, s., wiesman, z., garti, n., 2003: phenolic constituents of shea (vitellaria paradoxa) kernels. j. agr. food chem. 51, 6268-6273. mariod, a., matthäus, b., eichner, k., 2004: fatty acid, tocopherol and sterol composition as well as oxidative stability of three unusual sudanese oils. j. food lipids. 11, 179-189. min, j.d., suk, hoo, y., mun, yhung, j., 2012: chemical properties and oxidative stability of perilla oils obtained from roasted perilla seeds as affected by extraction methods. j. food sci. 77, 1249-1255. ni, q., gao, q., yu, w., liu, x., xu, g., zhang, y., 2015: supercritical carbon dioxide extraction of oils from two torreya grandis varieties seeds and their physicochemical and antioxidant properties. lwt − food sci. and technol. 60, 1226-1234. oomah, b.d., ladet, s., godfrey, d.v., liang, j., girard, b., 2000: characteristics of raspberry (rubus idaeu l.) seed oil. food chem. 69, 187-193. parry, j., lan, s., luther, m., zhou, k., peter, m., whittaker, p., 2005: fatty acid composition and antioxidant properties of cold-pressed marionberry, boysenberry, red raspberry, and blueberry seed oils. j. agr. food chem. 53, 566-573. pietta, p.g., 2000: flavonoids as antioxidants. j. nat. prod. 63, 1035-1042. fig. 3: investigation of antioxidant activity of a. pedunculatus seed oil, obtained by different extraction methods, using the following in vitro assays: (a) dpph radical scavenging assay, (b) abts radical scavenging assay, and (c) reducing power measurement. conclusions this study clearly demonstrated that seed oil from a. peduncula tus, a wooden oil plant of deserts, was rich in fatty acid composition (especially, mufa and pufa), polyphenols, tocopherols and phytosterols. those valuable compound were higher than the healthy oils (olive oil and camellia oil), which are beneficial for human health. the best extraction method with regard to oil yield, valuable compound and antioxidant activity: se > cp > aee > sfe, sfe > aee > se > cp and sfe > se > cp > aee, respectively. the sfe method extracted the highest content of valuable and health promoting compounds and the oil revealed the highest antioxidant activity compared to the oil obtained by the other extraction methods (aee, cp and se). acknowledgements this research was funded by science and technology innovation project coordination of shaanxi province of china (no. 2012ktcl03 the effects of different extraction methods on the apo 141 ramadan, m.f., kinni, s.g., seshagiri, m., mörsel, j.t., 2010: fatsoluble bioactives, fatty acid profile and radical scavenging activity of semecarpus anacardium seed oil. j. am. oil chem. soc. 87, 885-894. re, r., pellegrini, n., proteggente, a., pannala, a., yang, m., riceevans, c., 1999: antioxidant activity applying an improved abts radical cation decolorization assay. free radical bio. med. 26, 1231-1237. shukla, v.k.s., nielsen, w.s., batsberg, w., 1983: a simple and direct procedure for the evaluation of triglyceride composition of cocoa butters by high performance liquid chromatography-acomparison with the existing tlc-gc method. eur. j. lipid sci. tech. 85, 274-278. wagner, k., elmadfa, i., 2000: effects of tocopherols and their mixtures on the oxidative stability of olive oil and linseed oil under heating. eur. j. lipid sci. tech. 102, 624-629. © the author(s) 2016. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). address of the authors: jun yan, yehua shen*, cong li, key laboratory of synthetic and natural functional molecule chemistry of ministry of education, college of chemistry and materials science, northwest university, no. 229 taibai north road, xi’an 710069, pr china jun yan, yingyao wang* xia luan, mimi guo, lipids group, academy of state administration of grain, no. 11 baiwanzhuang street, xicheng district, beijing, 100037, pr china e-mail: yhshen@nwu.edu.cn (y.h. shen), wyy@chinagrain.org (y.y.wang) journal of applied botany and food quality 86, 90 98 (2013), doi:10.5073/jabfq.2013.086.013 university of british columbia, michael smith laboratories, east mall, vancouver b.c., canada identification of main fine or flavour components in two genotypes of the cocoa tree (theobroma cacao l.) daniel kadow*, joerg bohlmann, wilberth phillips, reinhard lieberei (received april 3, 2013) * corresponding author summary cocoa seeds are the key raw material in chocolate manufacturing. traders separate them into bulk and fine or flavour cocoa. the latter is characterized by the presence of special aroma notes (e.g. fruity). in contrast to chocolate aroma that derives from seed endogenous components (storage proteins, carbohydrates) fine aroma has been linked to the fruit pulp but, detailed information on its molecular background is lacking. in the present study we analyzed fruit pulp and seeds of two fine or flavour cocoas (sca6, eet62) and a bulk cocoa (ccn51) using gcms. the monoterpenes β-myrcene, β-trans-ocimene, β-cis-ocimene and β-linalool were characteristic for the sca6 volatile composition. regarding eet62 the secondary alcohol 2-heptanol, its ester 2-heptanol acetate and the methylketones 2-heptanone and 2-nonanone were typical. we conclude that these molecules are main components of sca6 and eet62 fine aroma. accordingly, such components may derive from different metabolic pathways depending on the genotype. introduction the cocoa tree theobroma cacao l. is native to the tropical rain forest of the eastern andean slopes (bartley, 2005). it is a major and stable base of agricultural income for millions of farmers in africa, asia, centraland south america. its seeds are the key raw material of the multi-billion-dollar chocolate industry (motamayor et al., 2008). t. cacao is characterized by a huge genetic diversity (bartley, 2005; motamayor et al., 2008). depending on the variety traders separate cocoa seeds into bulk cocoa and fine or flavour cocoa with the latter being characterized by the presence of special aroma notes such as floral and fruity absent in bulk cocoa (ziegleder, 1990; sukha et al., 2008). fine cocoa is of higher value than bulk cocoa (international trade centre, 1991; donovan, 2006). cocoa seeds consist of two large storage cotyledons and an embryo axis enclosed by a hardened but flexible seed shell. attached to the seed shell elongated pectin rich endocarp cells form a fruit pulp (duncan and todd, 1972; figueira et al., 1993; andersson et al., 2006). the storage components in the cotyledons comprise 50 % fat, 15 % phenolics, 12 % protein, 5 % starch and 2 % sucrose on a dry weight base (reineccius et al., 1972; berbert, 1979; schmieder and keeney, 1980; kim and keeney, 1984; clapperton, 1992; voigt et al., 1993; bucheli et al., 2001; nazaruddin et al., 2001). the aqueous pulp contains 12 % monoand disaccharides, 2 % citric acid as well as further organic acids, esters, aldehydes, methylketones, secondary alcohols and terpenes (pettipher, 1986; figueira et al., 1993; quijano et al., 2010). fresh untreated cocoa seeds are characterized by an astringent taste due to the high content of phenolics especially anthocyanins (jinap et al., 2005). they do not develop any chocolate aroma during chocolate manufacturing because they do not contain the necessary aroma precursors. the latter are developed during the postharvest treatment (e.g. ziegleder and biehl, 1988) that the seeds undergo in the countries of origin. this postharvest treatment consists of fermentation and drying. during fermentation the fruit pulp surrounding the cocoa seeds is degraded by yeasts, lacticand acetic acid bacteria resulting in lactic and acetic acid formation as well as in a development of heat. the acids permeate into the seed tissue. this acidification and the heat cause seed death. subsequently, the storage proteins and carbohydrates are degraded by the respective seed enzymes yielding peptides, free amino acids and reducing sugars: the chocolate aroma precursors (reviewed by schwan and wheals, 2004; afoakwa et al., 2008). thus, the chocolate aroma derives from seed endogenous components. subsequent to fermentation the cocoa seeds are dried to a final moisture content of approximately 7 % in order to enhance storability. drying and in particular fermentation also result in a notable reduction of astringency due to enzymatic oxidation of the phenolics (kim and keeney, 1984; afoakwa et al., 2008). fermented and dried cocoa seeds are named raw cocoa. the storage cotyledons of this product are used in the following process of chocolate manufacturing. this process comprises roasting of the cotyledon tissue. during roasting peptides, free amino acids and reducing sugars undergo maillard reactions and strecker degradation leading to chocolate aroma compound development (reviewed by afoakwa et al., 2008). numerous studies have been carried out on these compounds and dominant odour-active substances such as aldehydes (e.g. 3-methylbutanal), pyrazines (e.g. 2,3-diethyl-5-methylpyrazine) and furans (e.g. 2methyl-3-(methyldithio)furan) have been identified in cocoa mass (schnermann and schieberle, 1997; schieberle and pfnuer, 1999; counet et al., 2002; taylor, 2002; granvogl et al., 2006; reineccius, 2006; reviewed by afoakwa et al., 2008). in contrast to the seed endogenous formation of the chocolate aroma precursors fine flavour notes have been linked to the fruit pulp (e.g. eskes et al., 2007). these authors evaluated the sensory properties of the fruit pulp of several cocoa genotypes including eet62 (estation experimental tropical pichilingue clone 62) a fine cocoa with fruity and floral notes as well as ccn51 (coleccion castro naranjal clone 51) a bulk cocoa lacking fine flavour. the sensory properties of the respective fruit pulps matched the descriptions given for raw cocoa of these genotypes leading eskes et al. (2007) to propose that the corresponding components are pulp derived and already present when cocoa is harvested. apparently, these components − like acetic acid − permeate into the seed tissue during fermentation and are retained throughout the drying process. it has been repeatedly suggested that monoterpenes such as linalool are part of the components or rather molecules responsible for fine flavour in cocoa (e.g. ziegleder, 1990). these authors report that fine or flavour cocoas contain higher amounts of linalool than bulk cocoa does (ziegleder, 1990). besides linalool, fermented and dried cocoa seeds also may contain the monoterpenes myrcene and ocimene (ziegleder, 1990) described as spicy and floral, respec cocoa fine aroma components 91 tively (mosciano, 1996 and 2000). hence these molecules may be important for fine flavour as well. linalool, myrcene and ocimen have been detected in the pulp of fresh ripe cacao fruits (quijano et al., 2010) furnishing further evidence to the hypothesis of eskes et al. (2007) that fine aroma components are pulp-derived. in total quijano et al., (2010) could identify 66 different components in the pulp of an undefined cacao genotype typical for columbian plantations among them methylketones such as 2-heptanone and 2nonanone as well as secondary alcohols like 2-pentanol and 2-heptanol. short-length methylketones (c5-c11) are highly potent flavour molecules (schwab et al., 2008). secondary alcohols are aroma active compounds as well with 2-pentanol and 2-heptanol being important components of passion fruit flavour (strohalm et al., 2007; schwab et al., 2008). tab. 1 summarizes those components that in the analysis of quijano et al. (2010) were contributing to 1 % or more of the total volatile amount. interestingly, 2-pentanol and 2-heptanol have also been detected in roasted cocoa powder (bonvehí, 2005). 2-heptanol also was found in dark chocolate (counet et al., 2002). moreover, counet et al. (2004) report the presence of 2-heptanone and 2-nonanone in new guinea cocoa liquor. thus, besides monoterpenes also methylketones and secondary alcohols may play a key role in fine flavour formation. however, detailed information on the molecular background of cocoa fine flavour and its genotypic plasticity is lacking. in the present study we determined the fruit pulp and unfermented seed volatile components of the cacao genotypes sca6 (scavina clone 6) − well known for its floral aroma notes in fruit pulp and raw cocoa (e.g. eskes et al., 2007) − eet62 and ccn51 using gas chromatography-mass spectrometry (gcms). monoterpenes were characteristic for the volatile composition of sca6 pulp and fresh seed tissue. in contrast, eet62 contained methylketones, secondary alcohols and their respective esters. we suggest that these molecules are a major source of fine flavour in sca6 and eet62 and conclude that fine aroma components may derive from different metabolic pathways depending on the genotype. materials and methods cocoa fruits cacao fruits of the genotypes sca6 (scavina clone 6), eet62 (estation experimental tropical pichilingue clone 62) and ccn51 (coleccion castro naranjal clone 51) were provided by the international cacao collection at catie (centro agronómico tropical de investigación y enseñanza), turrialba, costa rica. all fruits were harvested during main crop in 2012. material from three individual shipments in april, may and june was used in the experiments. fruits with differing pulp appearance e.g. with a different degree of ripeness were discarded. a total of eight fruits were analyzed per genotype. volatile extractions fruit pulp and cotyledon tissue of freshly opened fruits were separated using a scalpel and the pulp was transferred immediately into 20 ml head space vials (agilent technologies inc., mississauga, canada). the cotyledon tissue was broken into nibs and also transferred to head space vials. all vials were filled with an equal amount (~5 g) of tissue. subsequently, the samples were equilibrated to a temperature of 35 °c in a water bath. the volatiles in the head space were collected by solid phase micro extraction (spme) for 15 min using a 65 µm polydimethylsiloxane-divinylbenzene (pdms/dvb) fiber (supelco, mississauga, canada). volatile component analysis the volatile analyses were performed on an agilent 6890n gas chromatograph (agilent technologies inc., mississauga, canada) and 5975 inert xl mass spectrometer in an electron ionization (ei) mode at 70 ev. a j&w db-wax column (model number 122-7032, agilent technologies inc., mississauga, canada) with an inner diameter of 250 µm, 0.25 µm film thickness and a length of 30 m was used. the gc temperature program was: 40 °c for 3 min, 3 °c*min-1 to 100 °c, 10 °c*min-1 to 150 °c, 15 °c*min-1 to 240°c, hold 5 min with the pulsed splitless injector temperature held at 250 °c. the pulsed pressure was set at 50 psi. the carrier gas (helium) flow was adjusted to 1 ml*min-1. samples were injected manually. the volatile components were identified by comparison of their mass spectra to the reference mass spectral data base of the national institute of standards and technology ms library searches (wiley w9n08). moreover, the identification of substances referred to as major components of the volatile compositions of the three genotypes as shown in fig. 1 and 3 was verified using authentic standards. the quantity of volatile components was calculated based on the peak area and is given in percent. total peak area observed with pulp from eet62 was used as reference area in order to facilitate comparison between the genotypes as well as between fruit pulp and cotyledon tissue. all volatile component amounts are referred to the fresh weight. quantifications are based on the analyses of eight independent biological replicates (i.e. eight fruits) per clone. three technical replicates were carried out per fruit. results pulp volatile components fruit pulp of clone eet62 had the highest overall amount of volatile components when compared to sca6 and ccn51. the amount was more than 2-fold higher than in the former and four times higher in comparison to the latter (fig. 1). the volatile composition of eet62 pulp was dominated by 2-heptanol acetate which contributed tab. 1: volatile components in t. cacao pulp of an unknown genotype (from quijano et al., 2010) only components corresponding to ≥1% of the total volatile amount are shown. number compound amount [%] 1 2-methyl-3-buten-2-ol 2.6 2 2-pentanol 1.4 3 2-pentyl acetate 25.0 4 2-heptanone 1.1 5 2-heptanol 1.6 6 2-heptyl acetate 29.7 7 cis-linalool oxide 3.4 8 2-nonanone 1.5 9 linalool 11.7 10 1-phenylethyl acetate 1.8 11 isoamyl benzoate 2.3 92 d. kadow, j. bohlmann, w. phillips, r. lieberei to almost 70 % of the total volatile amount (fig. 1). in sca6 and ccn51 only 6.6 ± 1.8 and 8.3 ± 5.3 % of 2-heptanol acetate were measured, respectively. moreover, eet62 was found to contain 2.8 ± 0.5 % heptanol, 2.6 ± 0.8 % heptanone and 1.4 ± 0.5 % nonanone. regarding sca6 and ccn51 these substances contributed to less than 0.7 % of the volatiles (fig. 1). in contrast to eet62, the volatile composition of sca6 was characterized by high amounts of monoterpenes. the pulp contained 5.7 ± 1.0 % β-myrcene, 8.8 ± 1.0 % β-trans-ocimene and 5.2 ± 0.7 % β-cis-ocimene. in eet62 and ccn51 less than 0.3 % was measured regarding each of these substances. moreover, 2.3 ± 0.6 % β-linalool was detected in sca6. considerable amounts of this monoterpene were also found in eet62. its pulp contained 1.1 ± 0.5 %. in ccn51 only 0.5 ± 0.3 % β-linalool was measured (fig. 1). pulp of all three clones contained notable amounts of 2-pentanol acetate. however, with 21.7 ± 2.5 % sca6 contained approximately twice as much as eet62 and ccn51, respectively. despite of the considerable amounts of 2-heptanol acetate and 2-pentanol acetate ccn51 was characterized by a low total content of volatile components in comparison to eet62 and sca6 (fig. 1). also regarding components contributing to less than 1 % of the total volatiles grouped in the partition “others” distinct differences were observed between sca6 and eet62 on the one hand and ccn51 on the other. the former contained approximately 0.10 ± 0.06 % and 0.05 ± 0.03 % of α-farnesene as well as 0.08 ± 0.03 % and 0.09 ± 0.04 % acetophenone, respectively. no α-farnesene could be detected in the fruit pulp of ccn51. the amount of acetophenone did not exceed 0.01 %. moreover, trace amounts of perillen and 2-undecanone were found in the samples from sca6 and eet62. in ccn51 neither perillen nor 2-undecanone could be detected (fig. 2). genotype compound █ 2-pentanol acetate █ 2-heptanol acetate █ 2-heptanol █ 2-heptanone █ 2-nonanone █ β-myrcene █ β-trans-ocimene █ β-cis-ocimene █ β-linalool █ others █ dif. to reference sca 6 amount [%] 21.70 ± 2.46 6.57 ± 1.75 0.35 ± 0.11 0.52 ± 0.20 0.22 ± 0.07 5.73 ± 1.04 8.84 ± 1.04 5.21 ± 0.65 2.29 ± 0.61 5.72 42.83 eet 62 amount [%] 12.20 ± 2.05 69.89 ± 19.72 2.82 ± 0.52 2.58 ± 0.77 1.35 ± 0.45 0.23 ± 0.20 0.19 ± 0.14 0.23 ± 0.21 1.12 ± 0.48 9.41 reference ccn 51 amount [%] 10.14 ± 4.39 8.34 ± 5.28 0.29 ± 0.14 0.69 ± 0.19 0.35 ± 0.10 0.14 ± 0.11 0.19 ± 0.18 t 0.54 ± 0.33 3.78 75.52 volatile composition fig. 1: cacao fruit pulp volatiles of genotypes sca6, eet62 and ccn51 volatiles of the fresh fruit pulp were collected by spme and analyzed using gcms. quantities were calculated based on the peak area. the total peak area obtained with eet62 fruit pulp was used as reference area. all quantities are referred to the fresh weight (1g). only compounds contributing to 1% or more of the total volatile amount are shown. substances occurring in smaller quantities are grouped as “others”. the number of fruits analyzed per genotype was eight (n = 8). per fruit 3 replicates were carried out. error values are given as standard deviation. dif. to reference = difference to reference; the total peak area obtained for fruit pulp volatile components of eet62 is set as 100% (reference). in comparison to this reference area the total peak area obtained for sca6 and ccn51 fruit pulp volatile components is smaller. the difference to reference is used to complete the diagrams of sca6 and ccn51 to 100%; t = traces characteristic for the volatile composition of eet62 fruit pulp is the high amount of 2-heptanol acetate. typical also are the notable quantities of 2-heptanol, 2-heptanone and 2-nonanone. in contrast, sca6 contains high amounts of β-myrcene, β-trans-ocimene and β-cis-ocimene. 2-pentanol acetate was detected in relatively high quantities in all three genotypes. however, ccn51 contains far less volatiles than sca6 and eet62 do. cocoa fine aroma components 93 compound: acetophenone farnesene linalool perillen undecanone 0.2 0.1 0.0 a m o u n t [ % ] 0.2 0.1 0.0 0.2 0.1 0.0 0.2 0.1 0.0 0.02 0.01 0.00 0.2 0.1 0.0 0.2 0.1 0.0 4.0 2.0 0.0 0.2 0.1 0.0 0.2 0.1 0.0 * e m br yo p ul p t is su e: g e n o t y p e * n.d. n.d. n.d. n.d. n.d. n.d. fig. 2: cacao fruit pulp and cotyledon tissue volatiles of genotypes sca6, eet62 and ccn51 occurring in low amounts fresh fruit pulp as well as fresh untreated cotyledon tissue was transferred into head space vials and volatile components were collected by solid phase micro extraction. the volatile composition was analyzed using gcms. quantities were calculated based on the peak area. the total peak area obtained with eet62 fruit pulp was used as reference area. all quantities are referred to the fresh weight (1g). the number of fruits analyzed per genotype was eight (n = 8). per fruit 3 replicates were carried out. error values are given as standard deviation. n.d. = not detectable apart from the β-linaool quantities in the fruit pulp where no distinct differences between eet62 and ccn51 could be observed all substances shown occurred in significantly higher amounts in eet62 and sca6 than in ccn51. in some cases (e.g. α-farnesene) the respective components could be detected only in the fruit pulp and not in the cotyledon tissue. cotyledon volatile components like in the fruit pulp the highest overall amounts of volatile components in the cotyledon tissue were found in the eet62 samples. their total volatile content was 1.6 fold higher than in sca6 and 7.4 fold higher compared to ccn51 (fig. 3). however, in contrast to the pulp the cotyledon volatile composition of eet62 was not dominated by 2-heptanol acetate which contributed to less than 2 % of the total volatiles. remarkable were instead the 5.9 ± 1.7 % of 2-heptanol and the 6.6 ± 1.6 % of 2-heptanone. this corresponds to about twice the amount found in the fruit pulp and is about 40 times more than in sca6 and ccn51 where less than 0.2 % of 2-heptanol and 2-heptanone were measured. moreover, 0.7 ± 0.3 % 2-nonanone could be detected in the eet62 samples. in sca6 and ccn51 only trace amounts (<0.1 %) were found. 2-heptanol acetate contributed to less than 0.1 % of the total volatiles in case of ccn51 and to only 0.12 ± 0.07 % regarding sca6 (fig. 3). characteristic for the volatile composition of sca6 cotyledon tissue instead were the monoterpenes β-myrcene, β-trans-ocimene and β-cis-ocimene contributing to 2.0 ± 0.3 %, 2.4 ± 0.2 % and 0.8 ± 0.2 % of the total volatiles, respectively. in eet62 as well as ccn51 only trace amounts (<0.1 %) of these substances were found. β-myrcene could not be detected in ccn51 cotyledon tissue (fig. 3). sca6 cotyledon tissue also contained the highest amount of β-linalool. but, β-linalool contributed to only 0.10 ± 0.04 % of sca6 total volatiles (fig. 2). characteristic was further the amount of 2-pentanol acetate. with 1.3 ± 0.5 % it was 5 times higher than in the eet62 and ccn51 samples, respectively (fig. 3). moreover, 2-pentanol that was only found in traces in the pulp contributed to 3.3 ± 0.7 % of the sca6 cotyledon volatiles. however, also eet62 cotyledons contained 1.5 ± 0.5 % of this substance (fig. 3). 2-pentanone was detected in considerable amounts in the tissue of all three genotypes. but, sca6 and eet62 contained about twice the amount measured with ccn51. in general, ccn51 cotyledon tissue was characterized by a very low total amount of volatiles in comparison to sca6 and eet62 (fig. 3). in the cotyledon tissue like in the pulp there are distinct differences between the three clones regarding several of the components occurring in very small amounts. perillen for instance could only be detected in sca6. however, it contributed to only 0.006 ± 0.001 % of the total volatiles. acetophenone was found in the tissue of all three clones but, with 0.16 ± 0.04 % eet62 contained twice as much as sca6. in ccn51 tissue less than 0.01 % was measured. α-farnesene as well as 2-undecanone could neither be detected in sca6 and eet62 nor in ccn51. sensory characteristics of the pulp and cotyledon volatile components many of the components that we indentified in pulp and cotyledon tissue of eet62 and sca6 are flavoring substances with frequent use in the food and perfume industry. therefore, many of them have been evaluated individually for their sensory properties. 2-heptanol characteristic for the volatile composition of eet62 pulp and cotyledon tissue (fig. 1 and 3) for instance is described as fruity, lemon grass and floral with regard to the odour. 2-nonanone is described as fresh and sweet regarding the odour and as fruity regarding the taste. based on these descriptions 2-heptanol and 2-nonanone may be divided into the odour types citrus and fruity, respectively (mosciano, 1991c; the good scents company). odour and taste of 2-heptanol acetate dominating the pulp volatile composition of eet62 and 2heptanone present in high amounts especially in eet62 cotyledon tissue are described as fruity (mosciano, 1991b; the good scents company). in contrast, the monoterpene β-cis-ocimene typical especially for the pulp but, also for the cotyledon tissue of sca6 is described as floral regarding the odour. β-linalool characteristic for the pulp (fig. 1 and 3) is as well classified floral with regard to the odour. moreover, also the taste of β-linalool is defined floral. β-cis-ocimene and β-linalool are of the same odour type: floral (mosciano, 1996; the good scents company). the sensory properties of β-myrcene and 94 d. kadow, j. bohlmann, w. phillips, r. lieberei β-trans-ocimene typical for sca6 pulp and cotyledon tissue as well are described as citrus and sweet, respectively (mosciano, 2000; the good scents company). 2-pentanol acetate detected in considerable amounts in the pulp of all three clones whilst in the cotyledons notable amounts were only typical for sca6 is described as tropical and orange with regard to its sensory properties (mosciano, 2009; the good scents company). odour and taste of 2-pentanol only contributing to high amounts of the volatiles in eet62 and especially sca6 cotyledon tissue (fig. 1 and 3) are described as fermented and alcoholic. the substance is classified into the odour type fermented (mosciano, 2009; the good scents company). 2-pentanone detected in considerable amounts in the cotyledon tissue of all three clones is described as fruity, banana-like and sweet (mosciano, 1995). tab. 2 provides a detailed description of the sensory properties of all fruit pulp and cotyledon tissue components reported. discussion potential fine aroma components of sca6 and eet62 the individual sensory descriptions given for 2-heptanol, 2-heptanol acetate, 2-heptanone and 2-nonanone (tab. 2) match the fine aroma characteristics of eet62 reported by eskes et al. (2007). all four compounds were found to be present already in the fresh cotyledon tissue. moreover, ccn51 having no fine flavour contained significantly lower amounts especially regarding the cotyledons (fig. 1 and 3). therefore, we conclude that 2-heptanol, 2-heptanol acetate, 2-heptanone and 2-nonanone are a major source of fine flavour in eet62. interestingly, all four components have also been found in cocoa liquor (counet et al., 2004). influence on the fruity aroma notes in eet62 also may have 2-pentanone due to the relatively high content and moreover because its odour strength is rated “high” (the good scents company). however, it is also present in notable amounts in ccn51 and hence may be of only marginal importance. 2-pentanol does not match eet62 sensory properties but might be fig. 3: cacao cotyledon volatiles of genotypes sca6, eet62 and ccn51 fresh untreated cotyledon tissue was broken into nibs and volatiles were collected by spme. subsequently, volatiles were analyzed using gcms. quantities were calculated based on the peak area. the total peak area obtained with eet62 fruit pulp was used as reference area. all quantities are referred to the fresh weight (1g). only compounds contributing ≥ 0.7% of the total volatile amount are shown. substances occurring in smaller quantities are grouped as “others”. the number of fruits analyzed per genotype was eight (n = 8). per fruit 3 replicates were carried out. error values are given as standard deviation. dif. to reference = difference to reference; the total peak area obtained for fruit pulp volatile components of eet62 is set as 100% (reference). in comparison to this reference area the total peak area obtained for eet62, sca6 and ccn51 cotyledon tissue volatiles is smaller. the difference to reference is used to complete the diagrams of eet62, sca6 and ccn51 to 100%; n.d. = not detectable; t = traces characteristic for the volatile composition of eet62 cotyledon tissue are 2-heptanol, 2-heptanol acetate, 2-heptanone and 2-nonanone. also 2 pentanol occurs in notable amounts but, it is found in higher quantities in sca6. characteristic for the latter are high amounts of β-myrcene, β-transocimene and β-cis-ocimene. also 2-pentanol acetate and 2-pentanol are typical for the sca6 volatile composition. in contrast, ccn51 contains far less volatiles than sca6 and eet62 do. genotype compound █ 2-pentanol acetate █ 2-pentanol █ 2-pentanone █ 2-heptanol acetate █ 2-heptanol █ 2-heptanone █ 2-nonanone █ β-myrcene █ β-trans-ocimene █ β-cis-ocimene █ others █ dif. to reference sca 6 amount [%] 1.32 ± 0.48 3.25 ± 0.74 1.62 ± 0.57 0.12 ± 0.07 0.16 ± 0.05 0.16 ± 0.02 t 1.96 ± 0.26 2.37 ± 0.22 0.75 ± 0.15 0.53 87.74 eet 62 amount [%] 0.29 ± 0.09 1.49 ± 0.48 1.85 ± 0.58 1.45 ± 0.63 5.87 ± 1.70 6.63 ± 1.59 0.71 ± 0.34 t t t 1.22 80.43 ccn 51 amount [%] 0.25 ± 0.23 0.65 ± 0.17 0.90 ± 0.46 t 0.12 ± 0.10 0.16 ± 0.12 t n.d. t t 0.46 97.36 volatile composition cocoa fine aroma components 95 perceived differently in the presence of the other volatile components. 2-undecanone described as fruity by mosciano (1991a) may as well play role in the eet62 flavour composition. but, it was only present in trace amounts in the pulp and could not be detected in the fresh cotyledons underlining the potential importance of particularly 2-heptanol acetate, 2-heptanone and 2-nonanone for the fruity aroma notes of eet62. of importance especially regarding the floral aroma notes may further be β-linalool, β-cis-ocimene and acetophenone. however, aside from the β-linalool content of the pulp all substances are present in very small amounts and hence might be below the threshold level for sensory perception. the individual sensory description given for β-cis-ocimene and β-linalool (tab. 2) match the fine aroma characteristics of sca6 (eskes et al., 2007). with their citrus and sweet aroma notes β-myrcene and β-trans-ocimene apparently also contribute to the floral character of this genotype. together the four monoterpenes account for almost 50 % of the volatile compositions in pulp and cotyledon tissue (fig. 1 and 3) underlining their potential importance for sca6 fine aroma. moreover, ccn51 only contains trace amounts of these compounds. therefore, we suggest that β-myrcene, β-trans-ocimene, β-cis-ocimene and β-linalool – interestingly all components except β-trans-ocimene have also been found in raw cocoas (ziegleder, 1990) – are main molecules for sca6 fine aroma. hence, the potential key components of sca6 fine aroma are monoterpenes. their biosynthesis takes place via the methylerythritol 4-phosphate (mep) pathway (rodriguez-concepción and boronat, 2002; schwab et al., 2008). in contrast, the potential key components of eet62 fine aroma consist of methylketones, secondary alcohols and their respective esters. these substances derive from the fatty acid metabolism (fridman et al., 2005; schwab et al., 2008). therefore, we conclude that depending on the genotype fine aroma components derive from different metabolic pathways. besides the above mentioned monoterpenes acetophenone may further enhance the floral character of sca6. but, like discussed for eet62 the concentration of acetophenone might be below the sensory threshold level. due to its tropical and orange character as well as its abundance in the samples it seems likely that 2-pentanol acetate contributes to the sca6 dry fruit aroma note mentioned by eskes et al. (2007). 2-pentanone classified into the odour type fruity might contribute to this aroma note as well. however, in ccn51 both substances are present in comparable amounts (fig. 1 and 3). thus, they may have only marginal influence on the overall fine aroma of sca6. the high amount of 2-pentanol in the cotyledon tissue of sca6 suggests that it also plays role in the aroma composition. odour and taste are described as fermented and alcoholic but also as ripe banana (tab. 2). hence, it might be that 2-pentanol is responsible for the dry fruit character of sca6 as well. however, during fermentation it may also react with the acetic acid migrating into the cotyledon tissue resulting in the formation of additional 2-pentanol acetate. to further investigate the role of the single components in eet62 and sca6 fine flavour aroma extract dilution and aroma reconstitution trials as described for instance by hinterholzer and schieberle (1998) and buettner and schieberle (2001) may be carried out. generally, it has to be considered that when using spme the relative amount measured for a specific substance may vary depending on fibre affinity and competition with other volatiles for the fibre binding sites. however, the aim of our studies was not a quantification of the volatiles but the genotype specific comparison. in future experimental approaches precise quantification may be achieved using isotope dilution assays as described for instance by buettner and schieberle (2000). in contrast to the cocoa volatile extraction method described here the method reported by quijano et al. (2010) comprises blending of the fruit pulp using a juicer potentially resulting in enhanced compound oxidation. volatiles were extracted from the headspace tab. 2: sensory properties of t. cacao pulp and cotyledon volatiles from genotypes sca6 and eet62 the respective substances were evaluated individually. sensory descriptions are given according to mosciano (1991abc; 1995; 1996; 2000; 2009) and the good scents company. odour descriptions are shown in italics when the undiluted substance was tested (otherwise substance amount = 1-10%). descriptions cited in the text are underlined. compound odour type odour description taste description 2-heptanol acetate brown fruity, fenugreek fruity, fatty, green 2-heptanol citrus fruity, lemon grass, floral, fresh, sweet, herbal, green 2-heptanone cheesy fruity, sweet, coconut, spicy, herbal, woody fruity, coconut, cheesy, waxy, green 2-nonanone fruity fresh, sweet, green, weedy, earthy, herbal fruity, green, cheesy, dairy, buttery β-myrcene spicy balsam, peppery, terpene, spicy citrus, fruity, woody, vegetative β-trans-ocimene herbal sweet, herbal β-cis-ocimene floral floral, flower, sweet, warm, herb β-linalool floral floral, citrus, sweet, blueberry, woody, green floral, citrus, orange, lemon, waxy, woody 2-pentanol acetate herbal tropical, herbal, tropical, orange 2-pentanol fermented fermented, fusel, mild, green alcoholic, ripe banana, fusel, musty 2-pentanone fruity fruity, banana, sweet, wine, ethereal, woody fruity, banana-like, sweet acetophenone floral sweet, almond, acacia, mimosa, hawthorn, pungent bitter almond cherry pit-like, powdery perillen woody woody α-farnesene woody citrus, herbal, lavender, bergamot, neroli, myrrh, green fresh, green, vegetative 2-undecanone fruity fruity, floral, waxy, creamy, fatty fruity, waxy, ketonic 96 d. kadow, j. bohlmann, w. phillips, r. lieberei above the supernatant of pulp homogenate (quijano et al., 2010). to avoid enhanced volatile oxidation we did not apply blending. in total, quijano et al. (2010) identified 66 volatile compounds from cacao pulp. eleven of them correspond to 1 % or more of the total volatile amount and are listed in tab. 1. 2-heptanol acetate and 2-pentanol acetate together contribute to more than 50 % of the total volatiles. both substances also were detected in high amounts in our analyses (fig. 1). moreover, components occurring in relatively low quantities such as 2-pentanol, 2-heptanol, 2-heptanone and 2-nonanone (tab. 1) could be detected following our extraction protocol (fig. 1). β-myrcene, β-trans-ocimene and β-cis-ocimene contributing to less than 1 % of the total volatiles in the analyses of quijano et al. (2010) were found in quantities exceeding 5 % (fig. 1). in contrast, 1-phenylethyl acetate as well as isoamyl benzoate was detected in higher quantities by quijano et al. (2010). in our samples these substances contributed to less than 1 % of the volatiles and therefore are not shown in fig. 1. the reproducibility regarding samples taken from the same fruit (technical replicates) was very high. thus, cocoa fruit pulp volatiles may also be extracted without previous pulp juicing. that cis-linalool oxide (tab. 1) could not be detected in our analyses may be due to the avoidance of pulp juicing probably enhancing the formation of oxidation products. also 2-methyl-3-buten-2-ol could not be detected in our samples. this might be explained by genotype specific variation also observed between sca6, eet62 and ccn51 (fig. 1). fine aroma component migration during fermentation cocoa seeds fermented in the presence of aromatic annona muricata pulp revealed flavour attributes of this fruit after completion of the postharvest treatment (eskes et al., ingenic newsletter, accessed 20.12.12) proving that aroma components may permeate into the seed tissue during fermentation and that they may be retained during the drying process. the same can be expected regarding the potential sca6 and eet62 pulp fine aroma components that we reported here. however, our findings also show that these substances are already present in the unfermented seeds and that some of them even occur in higher amounts in the fresh cotyledons than in the pulp. moreover, preliminary results from fermentation-like incubations of sca6 and eet62 seeds suggest that there is no increase in the potential fine aroma component content in the seed tissue (kadow et al., unpublished data). thus, fruity and floral flavour attributes may already be fully developed in the fresh seeds. however, this does not mean that fine flavour components are not pulp derived. provided that the corresponding substances – in contrast to acetic acid that can not permeate the testa of fresh seeds (anderson et al., 2006) – are able to cross the seed shell these can migrate into the cotyledon tissue already upon fruit ripening. indeed, most of the potential fine flavour components such as 2-heptanol and β-cisocimene are insoluble or fairly soluble in water. thus, a migration with the water stream upon initiating germination as suggested for acetic acid (anderson et al., 2006) is rather unlikely. once permeated through the testa such compounds may be retained in the fat phase of the cotyledons resulting in enrichment. interestingly, with increasing temperatures as occurring during fermentation this retention of volatile components in a fat phase may be enhanced drastically (maier and kessler, 1977). moreover, fat solubility would explain the retention of the corresponding substances during the drying process. fermentation-like incubations are suitable to reassemble chocolate aroma precursor formation in cocoa seeds at lab scale (biehl and passern, 1982). however, it may be inappropriate for studying fine flavour component migration. whether the potential fine aroma components permeate through the cocoa seed testa and if a migration from the pulp to the cotyledons takes place during fruit ripening and fermentation will be in the focus of our future research. quality controls whether raw cocoa is recognized as fine flavour cocoa by traders mainly is provenance dependent since t. cacao genotypes with fine flavour attributes derive from the selections carried out by indians from several regions in centraland south america (e.g. bartley, 2005). generally, fine or flavour cocoa is produced from criollo or trinitario cocoa-tree varieties, while bulk cocoa comes from forastero trees (icco, 2011). however, with the continuous renewal of plantations and with the recent intensification of the breeding work on cocoa tree especially regarding yield and resistance major changes in the genetic structure of cocoa plantations have been taking place (aikpokpodion et al., 2006). this necessarily also has influence on the sensory attributes of the raw cocoa such as fine flavour. new genotypes may have a different fine aroma or even none. moreover, fine cocoa requires shorter fermentation time than bulk cocoa does (afoakwa et al., 2008). inappropriate fermentation may result in loss of previously present fine aroma. thus, provenience and the cacao variety planted alone cannot guarantee fine flavour quality. the genotype-specific information on the identity of potential fine flavour components reported here allows the establishment of methods for a quick qualitative and quantitative evaluation of fine flavour properties of a given raw cocoa. moreover, such evaluations could be used for fine flavour focused optimization of the postharvest treatment as well as the chocolate manufacturing processes. work on such methods is underway. selections and breeding recent breeding projects on cocoa tree such as the usda-ars project initiated in 1999 (e.g. schnell et al., 2007) are focusing on resistance and yield. indeed, there are many reports on molecular marker development for instance regarding black and frosty pod as well as witches broom resistance or tolerance (queiroz et al., 2003; risterucci et al., 2003; faleiro et al., 2006; brown et al., 2007; lima et al., 2008; lanaud et al., 2009). also regarding yield and butter fat content molecular markers have been described (schnell et al., 2005; araujo et al., 2009; marcano et al., 2009). in contrast, a development of molecular markers for genotypespecific fine aroma qualities has not been reported so far. detailed information on the molecular identity of fine aroma components will be of great help in the development of such markers. the availability of molecular markers for fruity or floral flavour attributes would significantly alleviate and accelerate breeding work aiming at the integration of such attributes into trees with favorable traits regarding resistance and yield. this would not only guarantee the maintenance of specific fine flavour qualities but could also help to increase the income of cocoa farmers since fine cocoa can achieve prices twice as high as bulk cocoa (donovan, 2006). the methylerythritol 4-phosphate (mep) pathway that monoterpenes and thus the potential key components of sca6 fine aroma derive from is localized in the plastids (rodriguez-concepción and boronat, 2002; schwab et al., 2008). methylketones apparently are synthesized by hydrolysis and subsequent decarboxylation of β-ketoacyl-acps (acyl carrier protein) by methylketone synthase (fridman et al., 2005; schwab et al., 2008). in tomato the enzyme is localized in the stroma of the plastids (fridman et al., 2005). methylketones are supposed to be the precursors of secondary alcohols such as 2-heptanol (strohalm et al., 2007; schwab et al., 2008). thus, the different metabolic pathways that the potential fine aroma components of eet62 and sca6 derive from apparently are located in the same cell compartments: the plastids. while the pathways are active in plastids, the genes encoding cocoa tree linalool synthase are localized in the nucleus on chromosome 6 (argout et al., 2011), and in general, all plant monoterpene synthases appear to be encoded in the nuclear genome cocoa fine aroma components 97 (chen et al., 2011). nevertheless, plastids may have an impact on the regulation of gene expression in the nucleus via retrograde signaling (e.g. eckardt, 2011). therefore, maternal inheritance of plastids (svab and maliga, 2007) should be taken into consideration when selecting paternal and maternal parents for breeding of fine aroma in cocoa. conclusions our study shows that cacao fruit pulp may have great quantitative and qualitative differences in the composition of volatiles depending on the genotype. among these volatiles i.e. the potential fine aroma components are monoterpenes (typical for sca6) as well as methylketones, secondary alcohols and their respective esters (typical for eet62). consequently, fine aroma components apparently derive from at least two different metabolic pathways. the migration of these fine aroma components from the fruit pulp to the cotyledon tissue seems to take place already during fruit ripening whereas the typical basic chocolate flavour is composed upon fermentation. acknowledgement we thank lina madilao for the excellent technical assistance throughout the entire project work. we also would like to thank karen reid for great organizational help, alfonso lara quesada and the entire working group of the bohlmann-lab for fruitful discussions and allan mata quirós for careful selection and preparation of the samples for shipment. this work was supported by the university of hamburg. references afoakwa, e.o., paterson, a., fowler, m., ryan, a., 2008: flavor formation and character in cocoa and chocolate: a critical review. crit. rev. food sci. nutr. 48, 840-857. aikpokpodion, p.o., kolesnikova-allen, m., schnell, r.j., motamayor, j.c., eskes, a.b., gilmour, m., 2006: genetic shift in structure of west african cacao and 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(eds.), modern methods of plants analysis, 321-390. springer-verlag, berlin, germany. ziegleder, g., 1990: linalool contents as characteristic of some flavour grade cocoas. z. lebensm. unters. forsch. 191, 306-309. address of the authors: daniel kadow* and reinhard lieberei, university of hamburg, biocenter klein flottbek and botanical garden, ohnhorststraße 18, 22609 hamburg, germany *e-mail: daniel.kadow@uni-hamburg.de joerg bohlmann, university of british columbia, michael smith laboratories, 321 2185 east mall, vancouver b.c., canada v6t 1z4 wilberth phillips, centro agronómico tropical de investigación y enseñanza (catie), 7170 cartago, turrialba 30501, costa rica journal of applied botany and food quality 88, 29 33 (2015), doi:10.5073/jabfq.2015.088.006 1 institute of pure and applied biology, bahauddin zakariya university, multan, pakistan 2 department of soil science, faculty of agricultural sciences and technology, bahauddin zakariya university, multan, pakistan 3 department of agronomy, faculty of agricultural sciences and technology, multan, pakistan biochar affects growth and biochemical activities of fenugreek (trigonella corniculata) in cadmium polluted soil uzma younis1, saeed ahmad malik1, muhammad farooq qayyum2*, m. hasnain raza shah1, ahmad naeem shahzad3, seema mahmood1 (received october 9, 2014) * corresponding author summary cadmium (cd) has no defined biological role and may enter the food chain from polluted soils. biochar has been proposed as an organic amendment to minimize the toxic effects of cd for plants grown on contaminated soils. in this study, biometric and biochemical attributes of fenugreek (trigonella corniculata) grown on artificially cadmium (cd) contaminated soil (0, 25, 50, 75 and 100 mg cd/kg soil) at three levels of cotton-sticks derived biochar (csb; 0, 3 and 5 %) were studied. data show significant decline in the growth, photosynthetic pigments (chlorophyll a, b and total, carotenoids, anthocyanin and lycopene), and physiological attributes (sub-stomatal co2 concentrations, photosynthetic and transpiration rate) in the presence of high cd concentrations (50 and 100 mg cd/ kg soil). however, the decline was reduced in the presence of csb. a steady amplification in lipid peroxidation (assessed via malondialdihyde (mda)) and ascorbic-acid assembly was noted with increasing cd. the concentration of cd in the root and shoot also decreased with increasing csb application rates from 3 % 5 %. overall, the greater production of protein, amino acids and sugar contents in response to higher application rates of csb seems to be due to alleviation in cd toxicity. thus, cotton-sticks can be safely utilized in the form of biochar as amendment with additional benefit of reducing cd bioavailability and toxicity to crop plants. introduction cadmium (cd) is a toxic element for crop plants that enters into environmental cycle through different anthropogenic activities such as electroplating, waste materials of industries, manufacturing of ni-cd batteries, plastic, pigments, as well as cement and ceramic (nriagu, 1996). entry of cd to agricultural lands and consequently in plants is through use of sewage irrigation water and sewage sludge (qadir et al., 2000). there are certain adverse effects of cd on plants when taken up by the roots in large quantity and accumulated in the aerial parts (reeves and baker, 2000) such as metabolic, physiological and restricted growth disorders (markert et al., 2003). the photosynthetic pigments are very susceptible to cd toxicity that results in disturbed photosynthetic activity (sandalio et al., 2001). moreover, the structure and functions of various membranes are destroyed by cd as it facilitates partial reduction of oxygen molecules by restricting the activity of photosynthetic electron transport which produces free radicals (dhir et al., 2004). high levels of cd also become one of major contributors in the oxidative damage as in higher plants it is engaged in lipid peroxidation. the accumulation of cd and other heavy metals in plants can be minimized by the addition of organic amendments such as biochar (a black carbon compound produced by pyrolysis of biomass). biochar can immobilize heavy metals in soil through various stabilization mechanisms (verheijen et al., 2010). biochar derived from various feedstocks can improve soil physicochemical properties (texture, structure, bulk density ph, electrical conductivity, and cation exchange capacity), biological as well as fertility status (amonette and joseph, 2009; gundale and deluca, 2007). besides, the most important use of biochar is the safe utilization of organic wastes and carbon-sequestration through reduction of greenhouse gases into atmosphere (kammann et al., 2012). due to high surface area and organic functional groups, biochar can immobilize heavy metals in soils through sorption (beesley et al., 2010; liu and zhang, 2009; uchimiya et al., 2010; zhang et al., 2013). buss et al. (2012) reported better performance and reduced uptake of cu by chenopodium quinoa after biochar application at 4 %. they reported adsorption of cu to biochar surfaces as possible mechanism of reduced cu uptake. in pakistan, most of the vegetables are grown using sewage water irrigation that poses serious threat of heavy metals accumulation (hossain et al., 2010; qadir et al., 2000). in this scenario, there is need of research to minimize the bioavailability of metals to crop plants especially vegetables. the objective of our research was to study the impact of cd and biochar on biochemical activities of fenugreek (trigonella corniculata). it was hypothesized that biochar can minimize the stress effects of cd by minimizing its bioavailability to t. corniculata. biochar was prepared using cotton-sticks because of their easy and cheap availability in the farming community. the changes in growth, chlorophyll, mda, sugar, protein, amino contents and bioaccumulation of ions and cd were investigated in tissues of fenugreek seedlings grown in metal-contaminated soil and amend with biochar. materials and methods the experiment was conducted at the botanical garden of bahauddin zakariya university multan, using a completely randomized factorial design involving fenugreek (trigonella corniculata) and two treatment factors: biochar (0, 3, and 5 % w/w) and cadmium additions (0, 25, 50, 75 and 100 mg cd/kg air dry soil using cdcl2). each treatment was replicated four times. the biochar (csb) was prepared using pyrolysis of cotton-sticks at 450 °c in vertical-silo kiln-type reactor. the physicochemical properties of the cotton-sticks derived biochar (csb) are provided in tab. 1. the soil was collected from the agricultural field of the bahauddin zakariya university and had following characteristics (texture, sandy loam; ph, 6.5; ab-dtpa extractable cd, 0.742 ppm). the plants were grown in clay pots filled with mixture of 5 kg soil and respective treatments (csb and cd). twenty seeds of fenugreek (t. corniculata) were sown and later thinned to ten seedlings per pot. the plants were irrigated on regular basis to maintain 50 % of soil’s water holding capacity. at maturity, the plants were harvested and fresh and dry biomasses (shoots and leaves) were determined. the concentration of cd in the plant samples was determined using di-acid digestion followed by measurement on atomic absorption spectrophotometer (rashid 30 u. younis, s.a. malik, m.f. qayyum, m.h.r. shah, a.n. shahzad, s. mahmood tab. 1: physiochemical properties of biochar used in the experiment ph(1:10) ec(1:10) total carbon total nitrogen hydrogen phosphorus potassium ash content volatile matter ds/m % (w/w) 9.51 1.52 46.3 1.7 3.6 0.4 1.6 15 20 1986). the total soluble protein and amino acids were determined following bradford (1976). for ascorbic acid determination, the formula of keller and schwager (1977) was used. plant malondialdihyde contents were assayed as thiobarbituric acid (tba) method and soluble sugar was determined with the anthrone reagent (cakmak and horst, 1991). the gas exchange attributes were determined by an open system lca-4 adc portable infrared gas analyzer (analytical development company, hoddeson, england). it was used for measurements of photosynthetic rate, transpiration rate (e) and sub-stomatal co2 concentration (ci). the photosynthetic pigments were determined by following the method of arnon (1949). statistical analysis the data were analyzed for statistical differences by performing analysis of variance (anova) test using statistical software package spss version 18.0. the tukey-hsd test was used to find significant differences between various treatments. results plant growth attributes the results show significant reduction in the fresh and dry biomass of fenugreek (t. corniculata) by increasing cd application rates but this retardation was less pronounced in csb treated soil (fig. 1). in the absence of cd, plants produced more fresh-biomass (32.35 g) than at 3 % csb (23.3 g) and 0 % csb (20.4 g). at all the levels of cd (25, 50 and 100 mg cd/kg soil) with csb applications at 3 % and 5 %, the plants had highest values of fresh biomass than at sole 5 % csb treatment. the plants which received 100 mg cd/kg soil had lowest biomass (13.25 g). photosynthetic pigments the effect of cd with various application rates of csb on photosynthetic pigments is depicted in tab. 2. the increase in chlorophyll (a, b, total), carotenoids and anthocyanin content was observed by increasing the csb application from 0 % to 5 %. the lycopene contents were not significantly affected by any of the csb application rate. similar results were observed at 50 and 100 mg cd/kg soil + 5 % csb for chlorophyll b, total chlorophyll, carotenoids and anthocyanin but significantly higher chlorophyll a and lycopene content were noticed at 100 mg cd/kg soil. physiological attributes the increasing application rates of cd significantly decreased the physiological attributes of plants (fig. 2). the lowest values of all the physiological attributes were observed at 100 mg cd/kg soil. however, at different combinations of cd (25, 50, and 100 mg cd/kg soil) and csb, the reduction in physiological parameters was very less which demonstrates the role of biochar in increasing photosynthetic pigments at various percentages. the maximum increase in photosynthetic rate, transpiration rate and sub-stomatal co2 concentration was observed at 5 % csb. the concentration of ascorbic acid was increased with an increase in cd application from 0 to 100 mg cd/kg soil which were significantly lower at 3 % and 5 % csb amended soil at the same cd levels. at 100 mg cd/kg soil + 5 % csb, the ascorbic acid concentration was not significantly different from plants treated with 100 mg cd/ kg soil. 3 % csb + 0 mg cd/kg soil treatment exhibited the lowest values of ascorbic acid. the concentrations of mda were also increased by increasing cd level from 25-100 mg cd/kg soil as compared to the control (0 mg cd/kg soil). by applying csb, the mda concentration was signifig. 1: effect of various application levels of cadmium (0, 25, 50 and 100 mg cd/kg soil) and cotton sticks biochar (csb, 0 %, 3 % and 5 %) on fresh and dry biomass of fenugreek (trigonella corniculata). the different letters on bars show significant differences (p ≤ 0.05) between biochar application levels within a cadmium application. fenugreek response to biochar applied in polluted soil 31 ficantly decreased at various levels of cd. a similar trend was also observed in the case of sugar concentrations at various levels of cd and biochar percentages (fig. 3). the protein concentration was increased with an increase csb application from 3 % to 5 %. however, at different concentrations of cd, the protein concentrations were decreased by increasing the cd application. the csb + cd treatments showed significant differences from all other treatments. similarly, soluble amino acids concentrations were also decreased by cd stress but this decrease was less pronounced in biochar treated soil (fig. 3). cadmium concentration in plants the application of 5 % csb significantly reduced the cd concentration in t. corniculata. whereas, in the absence of csb, cd uptake was increased from 2.55 to 3.82 mg/kg dm by increasing the cd level from 25 to 100 mg cd/kg soil (tab. 3). discussion the results of our study clearly depict the toxic effects of cd on growth of fenugreek (t. corniculata) in terms of fresh and dry biomass of plants. however, the decline in growth due to cd-toxicity was very less in plants grown in biochar-amended soil. jiang et al. (2000) also reported high reduction in growth parameters such as biomass and leaf area after application of cd to the garlic. the decline in growth in our study may be attributed due to toxic effects of cd on various biochemical attributes of the plants (fig. 2 and 3). park et al. (2011) noted an increase in dry biomass of mustard plants by 1 % application of chicken manure-derived biochar. moreover, they found a significant decrease in cd and pb uptake by plants in biochar amended soils. our results show no significant effect of higher application rates (3 % and 5 %) of csb on the dry biomass of shoots and roots in the control (cd free) treatment. however, when csb was applied along with cd-stress, the growth parameters were significantly enhanced. in our study, the photosynthetic rate of plants grown in csb treatments along with cd stress was not reduced. chlorophyll contents were comparatively higher at 3 % and 5 % csb applications with cd. decrease in pigment production by high cd could be attributed to decrease in chlorophyll biosynthesis tab. 2: effect of various application levels of cadmium (0, 25, 50 and 100 mg cd/kg soil) and cotton sticks biochar (csb, 0 %, 3% and 5 %) on photosynthetic attributes of t. corniculata. the different letters within column show significant differences (p ≤ 0.05) between biochar application levels within a cadmium application. cd treatment biochar chlorophyll a chlorophyll b total carotenoids lycopene anthocyanin application chlorophyll mg cd/kg soil (%) (mg /g fresh mass) (umol/ml) control 0 2.26 c 0.71 c 2.96 c 0.51 c 0.12 a 0.12 c 3 2.47 b 1.13 b 3.60 b 0.94 b 0.12 a 0.19 b 5 2.73 a 1.37 a 4.09 a 1.19 a 0.10 a 0.23 a 25 0 1.63 b 0.99 c 2.63 c 0.69 b 0.10 a 0.14 b 3 1.09 c 1.20 b 3.08 b 0.28 c 0.06 b 0.14 b 5 2.25 a 1.21 a 3.45 a 1.09 a 0.10 a 0.20 a 50 0 1.89 b 0.70 c 2.59 c 0.53 c 0.08 b 0.11 c 3 1.88 c 0.84 b 2.71 b 0.68 b 0.07 b 0.14 b 5 2.08 a 1.18 a 3.25 a 0.89 a 0.09 a 0.18 a 100 0 1.74 a 0.44 a 2.18 b 0.46 b 0.09 a 0.10 b 3 1.67 c 0.52 a 2.19 b 0.11 c 0.08 c 0.10 b 5 1.73 b 0.88 a 2.61 a 0.62 a 0.09 b 0.13 a fig. 2: effect of various application levels of cadmium (0, 25, 50 and 100 mg cd/kg soil) and cotton sticks biochar (csb, 0 %, 3 % and 5 %) on photosynthetic rate, transpiration rate and sub-stomatal co2 concentrations of fenugreek (trigonella corniculata). the different letters on bars show significant differences (p ≤ 0.05) between biochar application levels within a cadmium application. p ho to sy nt he tic ra te (μ m ol /m 2 / se c) 32 u. younis, s.a. malik, m.f. qayyum, m.h.r. shah, a.n. shahzad, s. mahmood fig. 3: effect of various application levels of cadmium (0, 25, 50 and 100 mg cd/kg soil) and cotton sticks biochar (csb, 0 %, 3% and 5%) on biochemical attributes (ascorbic acid, malondialdihyde, sugar, amino acids and protein) of fenugreek (trigonella corniculata). the different letters on bars show significant differences (p ≤ 0.05) between biochar application levels within a cadmium application. by restriction of important enzymes (δ aminolevulinic acid dehydratase and protochlorophyllide reductase) involved in the production of chlorophyll. similarly, cd destructs the supply of mg and fe that are essential for chlorophyll manufacturing (kupper et al., 1996) and that would decrease photosynthetic rate, transpiration and sub-stomatal co2 concentrations. the transport and bioaccumulation of metals in the plants could be affected by many factors such as light, temperature, transport mechanism, ionic strength and anoxic conditions (john et al., 2009). high production of mda is an indication of oxidative stress by heavy metals. an increased level of lipid peroxidation (high mda production) at high concentrations of cd can be an expression of metal toxicity due to the generation of alkoxy and peroxy radicals that cause oxidative damage to biological membranes through destruction of polyunsaturated fatty acids (dhir et al., 2004). in our study, high production of amino acids and proteins was promoted by csb application under toxic levels of cd in tissues that indicates the activation of defensive mechanism against oxidative damage. production of such compounds is an evolutionary comeback of plants to stress which has been observed in diverse groups of organisms (chen et al., 2001; wu et al., 1995). the observed variations in the analyzed parameters clearly depict that increasing levels of cd affect the growth and chlorophyll production in fenugreek (t. corniculata). moreover, the enhanced production of mda showed oxidative damage as a result of cd toxicity. biochar derived from cotton sticks has shown the capability to a c b b c a a a b a a b 0 0.2 0.4 0.6 0.8 1 1.2 0 25 50 100 a sc or bi c a ci d (m g g1 ) mg cd kg-1 soil 0 % csb 3 % csb 5 % csb b b a a a a c b c a b c 0 0.5 1 1.5 2 2.5 0 25 50 100 m d a ( µg g -1 ) mg cd kg-1 soil 0 % csb 3 % csb 5 % csb b c c b a a b a c b a a 0 0.5 1 1.5 2 2.5 3 3.5 0 25 50 100 s ug ar ( m g g1 ) mg cd kg-1 soil 0 % csb 3 % csb 5 % csb a c a b c b c a b a b c 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 0 25 50 100 a m in o a ci ds ( m g g1 ) mg cd kg-1 soil 0 % csb 3 % csb 5 % csb b b b c c c c b a a a a 0 5 10 15 20 25 0 25 50 100 p ro te in ( m g g1 ) mg cd kg-1 soil 0 % csb 3 % csb 5 % csb fenugreek response to biochar applied in polluted soil 33 tab. 3: effect of various application levels of cadmium (0, 25, 50 and 100 mg cd/kg soil) and cotton sticks biochar (csb, 0 %, 3 % and 5 %) on concentrations of cadmium in t. corniculata. the different letters within column show significant differences (p ≤ 0.05) between biochar application levels within a cadmium application. cd treatment biochar %age cadmium concentration mg cd/kg soil (mg cd/kg dry mass) root shoot 0 0.199 a 0.166 b control 3 0.133 c 0.173 a 5 0.143 b 0.129 c 0 2.553 a 2.613 b 25 3 1.679 c 2.697 a 5 1.743 b 0.455 c 0 2.521 b 2.031 b 50 3 2.546 a 2.885 a 5 1.777 c 1.480 c 0 3.823 a 3.032 a 100 3 3.029 b 2.964 b 5 1.698 c 1.299 c sequester metals in the rhizosphere and to inhibit its transfer to the aerial parts of plants. protein and amino acids appeared to play a key role in the defense against cadmium toxicity under biochar application. therefore, biochar can be a choice in situations where irrigation water contains appreciable amounts of cadmium as well as for utilization in abandoned soils contaminated with cd. acknowledgments financial support from higher education commission pakistan under scheme ‘indigenous scholarship program’ is highly acknowledged.” references amonette, j.e., jospeh, s., 2009: characteristics of biochar: microchemical properties. in: lehmann, j., joseph, s. (eds.), biochar for environmental management science and technology, 33-43. earthscan, london. arnon, d.i., 1949: copper enzymes in isolated chloroplasts: polyphenoloxidases in beta vulgaris. plant physiol. 24, 1-15. beesley, l., moreno-jimenez, e., gomez-eyles, j.l., 2010: effects of biochar and greenwaste compost amendments on mobility, bioavailability and toxicity of inorganic and organic contaminants in a multi-element polluted soil. environ. pollut. 158, 2282-2287. bradford, m.m., 1976: a rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein-dye binding. anal. biochem. 72, 248-284 buss, w., kammann, c., koyro, h., 2012: biochar reduces copper toxicity in chenopodium quinoa willd. in a sandy soil. j. environ. qual. 40, 1157-1165. cakmak, i., horst, w.j., 1999: effect of aluminium on lipid peroxidation, siiperoxide dismutase, catalase, and peroxidase activities in root tips of soybean (glycine max). physiol. plant. 83, 463-468. chen, c.t., chen, l.m., lin, c.c., kao, c.h., 2001: regulation of proline accumulation in detached rice leaves exposed to excess copper in plants. plant sci. 160, 283-290. dhir, b., sharmila, p., saradhi, p., 2004: hydrophytes lack potential to exhibit cadmium stress induced enhancement in lipid peroxidation and accumulation of proline. aquat. toxicol. 66, 141-147. gundale, m.j., deluca, t.h., 2007: charcoal effects on soil solution chemistry and growth of koeleria macrantha in the ponderosa pine/douglasfir ecosystem. biol. fertil. soils. 43, 303-311. hossain, m.k., strezov, v., chan, k.y., nelson, p.f., 2010: agronomic properties of wastewater sludge biochar and bioavailability of metals in production of cherry tomato (lycopersicon esculentum). chemosphere. 78, 1167-1171. jiang, w., liu, d., hou, w., 2000: hyperaccumulation of cadmium by roots, bulbs and shoots of garlic (allium sativum l.). biores. technol. 76, 9-13. john, r., ahmad, p., gadgil, k., sharma, s., 2009: cadmium and leadinduced changes in lipid peroxidation, antioxidative enzymes and metal accumulation in brassica juncea l. at three different growth stages. arch. agron. soil sci. 55, 395-405. kammann, c., ratering, s., eckhard, c., müller, c., 2012: biochar and hydrochar effects on greenhouse gas (carbon dioxide, nitrous oxide, and methane) fluxes from soils. j. environ. qual. 41, 1052-1066. keller, t., schwager, h., 1977: air pollution and ascorbic acid. eur. j. for. pathol. 7, 338-350. kupper, h., kupper, f., spiller, m., 1996: environmental relevance of heavy metal substituted chlorophylls using example of water plants. j. exp. bot. 47, 259-266. lehmann, j., gaunt, j., rondon, m., 2006: bio-char sequestration in terrestrial ecosystems – a review. mitig. adapt. strat. glob. change. 11, 403-427. liu, z., zhang, f.s., 2009: removal of lead from water using biochars prepared from hydrothermal liquefaction of biomass. j. hazard. mater. 167, 933-939. markert, b., breure, a.m., zechmeister, h.g., 2003: bioindicators and biomoniters-principles, concepts and apllications. elsevier, amsterdam. nriagu, j.o., 1996: a history of global metal pollution. science, 272, 223230. park, j.h., choppala, g.k., bolan, n.s., chung, j.w., chuasavathi, t., 2011: biochar reduces the bioavailability and phytotoxicity of heavy metals. plant soil 348, 439-451. qadir, m., ghafoor, a., murtaza, g., 2000: cadmium concentration in vegetables grown on urban soils irrigated with untreated municipal sewage. environ. dev. sustain, 2, 11-19. rashid, a., 1986: mapping zinc fertility of soils using indicator plants and soils-analyses. phd dissertation, university of hawaii, hi, usa. reeves, r.d., baker, a.j.m., 2000: metal-accumulating plants. in: raskin, i., ensley, b.d. (eds.), phytoremediation toxic metals: using plants clean up environment, 193-229. john wiley and sons, new york. sandalio, l.m., dalurzo, h.c., gomez, m., romero-puertas, m.c., delrio, l.a., 2001: cadmium-induced changes in the growth and oxidative metabolism of pea plants. j. exp. bot. 52, 2115-2126. uchimiya, m., lima, i.m., klasson, k.t., chang, s., wartelle, l.h., rodgers, j.e., 2010: immobilization of heavy metal ions (cuii, cdii, niii, and pbii) by broiler litter-derived biochars in water and soil. j. agric. food chem. 58, 5538-5544. verheijen, f., 2010: biochar application to soils: a critical scientific review of effects on soil properties, processes and functions. jrc scientific and technical reports, ispra, italy. wu, j.t., chang, s.j., chou, t.l., 1995: intracellular proline accumulation in some algae exposed to copper and cadmium. bot. bull. acad. sinica. 36, 89-93. zhang, x., wang, h., he, l., lu, k., sarmah, a., li, j., bolan, n.s., pei, j., huang, h., 2013: using biochar for remediation of soils contaminated with heavy metals and organic pollutants. environ. sci. pollut. res. 20, 8472-8483. address of the corresponding author: e-mail: farooq.qayyum@bzu.edu.pk journal of applied botany and food quality 89, 212 216 (2016), doi:10.5073/jabfq.2016.089.027 1council for agricultural research and economics (crea), research centre for fodder crops and dairy productions (flc), lodi, italy 2 crea, research unit for mediterranean agro-pastoral systems (aam), sanluri, italy 3 indena s.p.a., qc/r&d laboratories, settala (milan), italy detection and exploitation of white lupin (lupinus albus l.) genetic variation for seed γ-conglutin content p. annicchiarico1*, m. romani1, s. barzaghi1, b. ferrari1, a.m. carroni2, p. ruda2, e. de combarieu3, l. pagni3, v. tedesco3 (received february 10, 2016) * corresponding author summary the seed γ-conglutin protein fraction of white lupin has particular pharmacological interest, but its industrial production is hindered by low content in the seed. this study provides an unprecedented assessment of genotypic and environmental variation for seed con tent and production of γ-conglutin, exploring also the ability of near-infrared spectroscopy (nirs) to predict seed γ-conglutin content. significant (p < 0.01) genetic variation for seed γ-conglutin content emerged among ten genotypes (cultivars or breeding lines) across three environments (range: 1.59-2.02 %) and five genotypes in other two environments (range: 1.47-1.80 %). genotype variation was found also for seed protein content and γ-conglutin proportion on total protein, the latter trait having higher impact than the former on genotype variation for seed γ-conglutin content. the production of γ-conglutin per unit area was affected also by genotype yielding ability beside genotype seed γ-conglutin content. no genotype × environment interaction was detected for any γ-conglutin trait. nirsbased prediction based on cross-validations was only moderately accurate for seed γ-conglutin content (r2 = 0.66), while being accurate for seed protein content (r2 = 0.95). in conclusion, breeding for higher seed γ-conglutin content is feasible using data from very few test sites and, to some extent, nirs-based predictions. introduction greater cultivation of rain-fed cool-season grain legumes could improve the sustainability of european agriculture in many respects (nemecek et al., 2008). white lupin (lupinus albus l.) is a traditional food legume with good potential as a high-protein feed crop in several european regions (lucas et al., 2015), owing to its moderately high grain yield, outstanding seed protein content, and good content of essential amino acids (papineau and huyghe, 2004; sujak et al., 2006; annicchiarico, 2008). in addition the white lupin seed contains 8-12 % of oil with excellent nutritional characteristics (boschin et al., 2008), and possesses various properties that make it particularly useful as an ingredient of functional, nutraceutical or healthy foods (arnoldi, 2005; duranti et al., 2008). the γ-conglutin protein fraction has particular interest in this respect, owing to its proved ability to control glycaemia via interaction and binding with insulin and its insulin-mimetic properties (bertoglio et al., 2011; lovati et al., 2012). a patent, which awaits commercial exploitation, has been granted for the pharmacological use of γ-conglutin for the treatment of type ii diabetes (morazzoni and duranti, 2004). large-scale production of white lupin seed γ-conglutin is hindered by its modest content in the seed, which represents only about 4-5 % of the total protein (duranti et al., 2008). optimizing its industrial production requires information on the extent of genetic, environmental, and genotype × environment interaction effects that may influence its content. earlier work on other seed quality traits of white lupin, such as oil, tocopherol or alkaloid contents, revealed that the relative size of these effects may vary largely depending on the specific trait (annicchiarico et al., 2014), with implications for crop improvement strategies and optimal production environments. despite its practical importance, we found no published information on the extent of genetic and environmental variation for seed γ-conglutin of white lupin. if exploitable genetic variation existed, it would be important to know whether it is associated mainly with overall protein content or with the proportion of γ-conglutin on total protein. only the latter case would prompt selection programs to specific analyses of genotype seed γ-conglutin content (which are more expensive than protein content analyses). in that case, nearinfrared spectroscopy (nirs) procedures could be explored as a means to reduce the costs of seed γ-conglutin analyses for plant breeding material or seed lots from different production environments. nirs has been already used to predict seed coat proportion in intact seeds of lupin (alomar et al., 2010) and contents of amino acids and protein in the seed (biston et al., 1983; kaffka, 1988). the main objective of this study was generating information on the extent of genetic and environmental variation for γ-conglutin in white lupin seed. in addition we report preliminary information on the relationship of seed γ-conglutin with total protein content and the ability of nirs procedures to predict seed γ-conglutin content. materials and methods plant material and cultivation seven elite breeding lines (7-50, 685, 722, b-17, b-20, mb-29 and mb-38) issued by a white lupin breeding program for climaticallycontrasting italian regions that is described in annicchiarico et al. (2011), and three commercial varieties (lucky, rex, rumbo), were grown in three autumn-sown field experiments with the aim to gather preliminary information on the genetic variation for seed γ-conglutin. the experiments were performed in the subcontinentalclimate site of lodi (northern italy) in the seasons 2011-12 and 2012-13, and the mediterranean-climate site of sanluri (sardinia) in 2012-13. climate and soil characteristics of these test environments are reported in tab. 1. white lupin underwent low-temperature stress during winter in lodi and terminal drought stress mainly in sanluri (featuring modest spring rainfall), in agreement with the general climatic features of the sites. soil characteristics, including active lime < 0.5 % and ph < 7.5, were always suitable for lupin cultivation (papineau and huyghe, 2004). these experiments adopted a randomized complete block design with three replications, plots of 3 m2 size, and 20 and 30 germinating seeds/m2 in 2011-12 and 201213, respectively. in these and following experiments, the seed was inoculated with nppl histick (becker underwood) before sowing. seed γ-conglutin analyses were carried out for each genotypeenvironment combination on a seed sample of 50 g that was obtained by bulking same amounts of seed from each experiment replicate. white lupin variation for seed γ-conglutin content 213 two breeding lines that displayed high and moderate seed γ-conglutin content in these experiments (lines 7-50 and mb-38, respectively), and three varieties (lucky; multitalia; rumbo), were grown in autumn-sown field experiments in lodi and sanluri in the season 2013-14. to further enhance the environmental differences between testing sites, the evaluation in sanluri was performed in a field whose soil active lime and ph (tab. 1) were definitely higher than those required for optimal lupin growth (papineau and huyghe, 2004), unlike lodi. grain yield in lodi was limited by biotic stresses (particularly fusarium spp.). rainfall and temperature patterns were relatively favourable in both locations (tab. 1). these experiments adopted a randomized complete block design with four replications, plots of 6 m2 size, and 30 germinating seeds/m2. genotype grain yields were assessed in all field replicates, whereas seed γ-conglutin and protein contents were assessed on two replicates per experiment using 50 g seed samples. chemical and nirs analyses lupin seed samples were ground to 1 mm particle-size in three grinding steps using a “pulverisette 14” variable speed rotor mill (fritsch gmbh, idar-oberstein, germany) with different sieve rings. the γ-conglutin content of each ground sample was assessed by indena’s proprietary hplc-uv method, whose details cannot be disclosed completely. a single, exhaustive extraction of γ-conglutin from the lupin flour was obtained in a tris-glycine buffer (extraction solvent/flour ratio: 200). after centrifugation, the supernatant was analyzed twice on a reverse phase hplc, using an apolar stationary phase butyl silicagel and an acetonitrile/water/tfa gradient. detection was carried out by ultraviolet at 210 nm. quantification was carried out against a γ-conglutin reference standard. this method was assessed for linearity (0.010.4 mg/ml), precision (relative standard deviation < 4 %), and stability of the sample solution (up to 48 hours). seed protein content was determined as described by kjeldahl (1883). nirs analyses were performed on 46 seed samples, which included: (i) 30 genotype-environment combinations of experiments in the seasons 2011-12 and 2012-13; (ii) ten genotype-environment combinations of experiments in 2013-14 (using seed of the first field replicate); (iii) six additional seed samples relative to other breeding lines that were evaluated only in lodi in 2011-12 or 2013-14. two ground seed replications of each sample were analyzed in petri dishes (re-blending and re-loading the dish between measurements), averaging results of the two measurements. we used a ftnir spectrometer (nirflex n500, büchi, italy) in the whole nirs range (10,000-4,000 cm-1). nirs spectra were collected in reflectance mode, adding 64 scans with a 4 cm-1 resolution. spectra were converted in absorbance mode before data analysis. statistical analysis genotype data of seed γ-conglutin from lodi and sanluri in the seasons 2011-12 and 2012-13 underwent an analysis of variance (anova) aimed to compare the genotype means across the three environments and the environment means, using genotype × environment interaction as the error term. plot data of seed γ-conglutin and protein contents, proportion of γ-conglutin on total protein, and grain and γ-conglutin dry matter (dm) yield from lodi and sanluri collected in the season 2013-14 underwent an anova aimed at assessing genotype, environment and genotype × environment interaction effects and comparing genotype means across environments. genotype γ-conglutin dm yield on a plot basis was computed as the product of plot yield by genotype seed γ-conglutin content on the site. data from these environments were also used for simple and multiple regression analyses of seed γ-conglutin content as a function of protein content and/or γ-conglutin proportion of total protein. all anova and regression analyses were performed using statistical analysis software (sas institute inc., cary, nc, usa). nirs calibrations to quantify the seed contents of γ-conglutin and protein were calculated using the software pls toolbox (eigenvector research inc, manson, wa, usa). we used different spectra pretreatments to improve nirs calibration performances. owing to the limited number of available samples, each calibration curve was assessed through cross-validations that used 11 % of the spectra for predictions, repeating the procedure nine times using contiguous data blocks. results significant (p < 0.01) genetic variation among ten genotypes emerged for mean seed γ-conglutin content across three test environments in the cropping seasons 2011-12 and 2012-13. genotype values ranged from 1.59 % to 2.02 %, both exhibited by breeding lines. control varieties displayed moderate to low values (1.60 -1.83 %). tab. 1: climate and soil characteristics, and mean white lupin grain yield and seed γ-conglutin content, for two test locations (lodi, northern italy; sanluri, sardinia) in different growing seasons. lodi sanluri variable 2011-12 2012-13 2013-14 2012-13 2013-14 rainfall nov-feb (mm) 124 365 427 266 432 rainfall mar-jun (mm) 222 598 270 148 163 lowest winter temp (°c) -17.0 -11.3 -5.7 -2.3 0.2 mean temperature nov-feb (°c) 2.4 5.0 4.9 10.8 16.1 mean temperature mar-jun (°c) 15.7 14.2 16.6 11.2 16.6 soil active lime (%) a ~0 ~0 ~0 0.3 2.6 soil ph 6.2 6.3 6.2 7.4 8.1 grain yield (t/ha) b 6.41 5.10 3.30 6.24 1.15 seed γ-conglutin content (% w/w) b 1.96 1.64 1.73 1.88 1.52 a according to drouineau (1942). b averaged across 10 genotypes in 2011-12 and 2012-13, and five genotypes in 2013-14. 214 p. annicchiarico, m. romani, s. barzaghi, b. ferrari, a.m. carroni, p. ruda, e. de combarieu, l. pagni, v. tedesco on average, the environment of lodi 2012-13 displayed lower seed γ-conglutin content (1.64±0.05 %) than lodi 2011-12 (1.96±0.05 %) and sanluri 2012-13 (1.88±0.05 %) (p < 0.05). one breeding line (7-50) with high seed γ-conglutin content (1.95 %; tab. 2), and a second line (mb-38) with moderate γ-conglutin content (1.72 %), were selected for further testing in lodi and sanluri in 2013-14 along with three commercial varieties. the anova performed on data from the season 2013-14 detected no genotype × environment interaction for every γ-conglutin trait, namely, seed γ-conglutin content, proportion of γ-conglutin on total protein, and γ-conglutin yield per unit area. in contrast, genotype × environment interaction (p < 0.05) was detected for seed protein content and grain yield. large variation for seed γ-conglutin content of genotypes across environments was found also in this data set (p < 0.01), with values ranging from 1.47 % in multitalia to 1.80 % in the line 7-50 (tab. 2). the ranking of four genotypes for seed γ-conglutin content across two environments in 2013-14 was identical to that across three environments in 2011-12 and 2012-13 (tab. 2), confirming the high consistency of genotype responses across environments for this trait. significant (p < 0.01) genotype variation across environments of the season 2013-14 emerged for all other traits. line 7-50 achieved topvalues of seed γ-conglutin content via high values of both seed protein content and proportion of γ-conglutin on total protein (tab. 2). the variety rumbo exhibited low seed γ-conglutin content because of low proportion of γ-conglutin on total protein, since its protein content was the highest one (tab. 2). results of simple and multiple regression analyses, which are summarized in tab. 3, confirmed that: (i) seed γ-conglutin content of the genotypes was largely determined by the proportion of γ-conglutin in the protein fraction (with 81 % of genotype variation explained by this variable); (ii) additional information on seed protein content had some value for predicting seed γ-conglutin content (as showed by its significance in the multiple regression model). the production of γ-conglutin per unit area of the genotypes was also influenced by genotype grain yielding ability besides seed γ-conglutin content. the variety lucky and the line 7-50, which combined high values of both variables, were top-ranking for γ-conglutin yield (tab. 2). on average, the unfavorable cropping environment of sanluri in 2013-14 exhibited, in comparison to lodi in the same season, not only almost three-fold lower grain yield but also lower seed γ-conglutin content (tab. 4). the latter response was the result of distinctly lower seed protein content, while the proportion of γ-conglutin on total protein did not differ between environments (tab. 4). a nirs-based calibration curve for seed γ-conglutin content was established, which held five latent variables and used a standard normal variate and a gap segment 2nd derivative as pretreatments to process the spectra. it displayed moderate predicting ability based on cross-validations (r2 = 0.66, with a root mean square error of 0.14). predictions for individual observations tended to shrink the range of γ-conglutin content relative to observed values (fig. 1). a nirsbased calibration curve for seed proteins content, which used four latent variables and a standard normal variate plus savitzky-golay 1st derivative algorithm as pretreatments, achieved much higher prediction (r2 = 0.95, with a root mean square error of 0.81). discussion our study provides an unprecedented assessment of the extent of genotypic and environmental variation for seed content and production of seed γ-conglutin in white lupin. its indications, while being valuable in the absence of other information, cannot be considered as conclusive, owing to the fairly limited sample of genotypes and test environments they are based upon. our findings indicate that white lupin selection for high seed content of γ-conglutin is feasible, on the basis of (i) the large genetic variation detected for this trait, and (ii) the consistency of genotype differences across environments. the latter result is supported by the lack of genotype × environment interaction in the anova for every γ-conglutin trait, and the consistent ranking of four genotypes for seed γ-conglutin content across two sets of environments. the consistency of genotype responses for seed γ-conglutin content facilitates genotype selection, which may rely on results from very few test environments, and ensures that selection gains are maintained in tab. 2: mean values over two climatically-contrasting italian locations of seed γ-conglutin and protein contents (in % w/w) measured by hplc and kjeldahl methods, respectively, and grain and γ-conglutin dry matter (dm) yield, for five white lupin genotypes. genotype γ-conglutin protein γ-conglutin grain dm γ-conglutin dm γ-conglutin % a % a % on protein a (t/ha) a (kg/ha) a % b 7-50 1.80 a 34.15 b 0.053 a 2.35 b 42.4 ab 1.95 a lucky 1.73 ab 32.50 c 0.053 a 2.77 a 47.8 a 1.83 ab mb-38 1.61 bc 33.77 b 0.048 ab 1.84 c 29.6 c 1.72 ab rumbo 1.51 c 35.83 a 0.042 b 1.59 c 24.0 d 1.60 b multitalia 1.47 c 32.15 c 0.046 b 2.59 ab 38.1 b se (df) c 0.04 (8) 0.34 (8) 0.002 (8) 0.10 (24) 1.8 (24) 0.09 (18) column means with different letter differ at p < 0.05 according to duncan’s test. a over two environments (lodi and sanluri, 2013-14). b over three environments (lodi 2011-12 and 2012-13; sanluri, 2012-13). c se, standard error of genotype mean; df, associated degrees of freedom. tab. 3: ability of simple and multiple linear regressions models to explain seed γ-conglutin content variation of five white lupin genotypes across two climatically-contrasting italian environments as a function of seed protein content and/or γ-conglutin proportion on total protein. model variables r2 p a γ-conglutin proportion on total protein 0.81 0.05 seed protein content 0.01 0.05 γ-conglutin proportion on total protein 0.99 0.05 / 0.05 + seed protein content a p level significance of explanatory variables in the model. white lupin variation for seed γ-conglutin content 215 different cropping environments. other seed quality traits of white lupin displayed less favourable responses, for example tocopherol content, whose genetic improvement proved hindered by high genotype × environment interaction (annicchiarico et al., 2014). the scope for selecting for higher seed γ-conglutin content is reinforced by the possibility to achieve values of γ-conglutin proportion on total protein that exceed the 4-5 % range reported in literature (duranti et al., 2008), as in the case of the genotypes 7-50 and lucky (5.3 %; tab. 2). our nirs calibration results, although preliminary, suggest that nirs-based predictions for seed γ-conglutin content are not sufficiently accurate to substitute completely for chemical analyses. however, nirs data could be exploited in preliminary selection stages for higher seed γ-conglutin content, performing the costly chemical analysis for this trait only on breeding lines promoted to final selection stages. we verified by earlier work that nirs calibration for seed γ-conglutin content based on spectra recorded from whole grain samples, which are exploitable also for selection of individual seeds, are characterized by too modest predicting ability to be of practical use (r2 = 0.27; ferrari et al., 2015). nirs prediction based on ground seed samples can be used in breeding programs to select genotypes on the basis of seed γ-conglutin content of their bulked progeny seed. the greater dependency of seed γ-conglutin content of lupin cultivars from γ-conglutin proportion on total protein than from seed protein content, which emerged from regression analyses, has practical implications for breeding programs. it suggests that selection for higher protein content, which could reliably be performed by nirs measurements, may have limited value for increasing seed γ-conglutin content. our results show that high γ-conglutin production per unit area, which is the ultimate target for industrial production of γ-conglutin, is largely affected also by high grain yielding ability of the genotypes. therefore, breeding strategies aimed to improve crop yields, e.g., by exploiting innovative plant types, stress tolerance, global genetic resources and adaptation to specific climatic conditions (huyghe, 1997; annicchiarico et al., 2010), remain of utmost importance even when targeting higher yield of this protein fraction. the current information on environmental variation for γ-conglutin traits is limited. its results for the season 2013-14 suggest that a favourable environment may increase the crop production of γ-conglutin not only via higher grain yield but also via higher seed γ-conglutin content that derived, in this case, from higher seed protein content. results for three relatively favourable cropping environments in the seasons 2011-12 and 2012-13 failed to reveal distinct and/or consistent differences between the subcontinental-climate site of lodi and the mediterranean-climate site of sanluri. in conclusion, this study indicates that white lupin seed content of γ-conglutin can be enhanced through plant breeding. selection costs can be reduced by adopting very few test sites because of limited genotype × environment interaction, and by relying on nirs-based predictions in early stages of selection. acknowledgements the results of this study were generated by the projects “white lupin for functional foods” funded by indena and “plant genetic resources – fao treaty” funded by the italian ministry of agricultural and forestry policies. references alomar, d., mera, m., errandonea, j., miranda, h., 2010: prediction of seed coat proportion in narrow-leafed and yellow lupins by near-infrared reflectance spectroscopy (nirs). crop pasture sci. 61, 304-309. annicchiarico, p., 2008: adaptation of cool-season grain legume species across climatically-contrasting environments of southern europe. agron. j. 100, 1647-1654. annicchiarico, p., boschin, g., manunza, p., arnoldi, a., 2014: quality of lupinus albus l. (white lupin) seed: extent of genotypic and environmental effects. j. agric. food chem. 62, 6539-6545. annicchiarico, p., harzic, n., carroni, a.m., 2010: adaptation, diversity, and exploitation of global white lupin (lupinus albus l.) landrace genetic resources. field crops res. 119, 114-124. annicchiarico, p., manunza, p., proietti, s., 2011: white lupin tolerance to winter cold, terminal drought and soil lime: patterns of genetic variation and their exploitation in breeding for southern europe. in: nagatab. 4: mean values over five white lupin genotypes of seed γ-conglutin and protein contents (in % w/w) measured by hplc and kjeldahl methods, respectively, and grain and γ-conglutin dry matter (dm) yield, for two contrasting cropping environments (lodi, northern italy; sanluri, sardinia) in the season 2013-14. environment γ-conglutin protein γ-conglutin grain dm γ-conglutin dm % % % on protein (t/ha) (kg/ha) lodi 1.73 a 37.69 a 0.046 a 3.30 a 52.2 a sanluri 1.52 b 29.68 b 0.051 a 1.15 b 18.9 b se (df) a 0.05 (2) 0.36 (2) 0.002 (2) 0.12 (6) 2.4 (6) column means with different letter differ at p < 0.05. a se, standard error of genotype mean; df, associated degrees of freedom. fig. 1: scatter plot of measured vs. predicted seed γ-conglutin content (in % w/w) for 46 genotype-environment combinations. prediction based on a near-infrared reflectance spectroscopy calibration curve, using cross-validations (continuous line = regression line; broken line = 1:1 line) 216 p. annicchiarico, m. romani, s. barzaghi, b. ferrari, a.m. carroni, p. ruda, e. de combarieu, l. pagni, v. tedesco nowska, b., kachlicki, p., wolko, b. (eds.), lupin crops – an opportunity for today, a promise for the future, 99-103. international lupin association, canterbury. arnoldi, a., 2005: optimized processes for preparing healthy and added value food ingredients from lupin kernels, the european protein-rich grain legume. aracne, rome. bertoglio, j.c., calvo, m.a., hancke, j.l., burgos, r.a., riva, a., morazzoni, p., ponzone, c., magni, c., duranti, m., 2011: hypoglycemic effect of lupin seed gamma-conglutin in experimental animals and healthy human subjects. fitoterapia 82, 933-938. biston, r., dardenne, g., sindic, m., dardenne, p., wathelet, b., sousa, b. de, 1983: near infra-red spectroscopy as a rapid and accurate technique for estimating lupinus seed protein and vicia faba seed amino acids and protein. in: thompson, r., casey, r. (eds.), perspectives for peas and lupins as protein crops, 247-255. martinus nijhoff, the hague, netherlands. boschin, g., d’agostina, a., annicchiarico, p., arnoldi, a., 2008: effect of genotype and environment on fatty acid composition of lupinus albus l. seed. food chem. 108, 600-606. drouineau, g., 1942: dosage rapide du calcaire actif du sol. ann. agron. 12, 411-450. duranti, m., consonni, a., magni, c., sessa, f., scarafoni, a., 2008: the major proteins of lupin seed: characterisation and molecular properties for use as functional and nutraceutical ingredients. trends food sci. tech. 19, 624-633. ferrari, b., barzaghi, s., annicchiarico, p., romani, m., pagni, l., de combarieu e., 2015: development of a nir calibration to select white lupin genotypes with high gamma-conglutin fractions. in: capraro, j., duranti, m., magni, c., scarafoni, a. (eds.), developing lupin crop into a major and sustainable food and feed source, 100. university of milan, milan. huyghe, c., 1997: white lupin (lupinus albus l.). field crops res. 53, 147-160. kaffka, k.j., 1988: determination of amino acids in lupine by near infrared reflectance spectroscopy. acta alimentaria 17, 3-11. kjeldahl, j., 1883: neue methode zur bestimmung des stickstoffs in organischen körpern. z. anal. chem. 22, 366-383. lovati, m.r., manzoni, c., castiglioni, s., parolari, a., magni, c., duranti, m., 2012: lupin seed γ-conglutin lowers blood glucose in hyperglycaemic rats and increases glucose consumption of hepg2 cells. br. j. nutr. 107, 67-73. lucas, m.m., stoddard, f., annicchiarico, p., frías, j., martínezvillaluenga, c., sussmann, d., duranti, m., seger, a., zander, p., pueyo, j.j., 2015: the future of lupin as a protein crop in europe. front. plant sci. 6, 705. morazzoni, p., duranti, m., 2004: the use of lupin conglutin for the treatment of type ii diabetes. patent wo2004071521. nemecek, t., von richthofen, j.-s., dubois, g., casta, p., charles, r., pahl, h., 2008: environmental impact of introducing grain legumes into european crop rotations. eur. j. agron. 28, 380-393. papineau, j., huyghe, c., 2004: le lupin doux protéagineux. editions france agricole, paris. sujak, a., kotlarz, a., strobel, w., 2006: compositional and nutritional evaluation of several lupin seeds. food chem. 98, 711-719. address of the authors: p. annicchiarico, m. romani, b. ferrari, s. barzaghi, council for agricultural research and economics (crea), research centre for fodder crops and dairy productions (flc), viale piacenza 29, 26900 lodi, italy e-mail: paolo.annicchiarico@crea.gov.it a.m. carroni, p. ruda, crea, research unit for mediterranean agro-pastoral systems (aam), podere ortigara, 09025 sanluri, italy l. pagni, e. de combarieu, v. tedesco, indena s.p.a., qc/r&d laboratories r&d, via don minzoni 6, 20090 settala (milan), italy © the author(s) 2016. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 90, 58 67 (2017), doi:10.5073/jabfq.2017.090.009 1university of gastronomic sciences, pollenzo, italy 2 estonian literary museum, tartu, estonia the disappearing wild food and medicinal plant knowledge in a few mountain villages of north-eastern albania andrea pieroni 1 *, renata sõukand 2 (received october 31, 2016) * corresponding author summary in recent years, an increasing number of ethnobotanical investigations have focused on the documentation of folk plant knowledge systems in mountainous areas of the balkans, as this area is considered a very important reservoir of bio-cultural heritage. an ethnobotanical field study was carried out among (gheg) albanians living in eight villages of north-eastern albania. the field survey was conducted by interviewing 45 local, elderly informants, who retain folk plant knowledge. sixty-three wild food and medicinal folk taxa and approx. 150 plant reports, as well as other domestic remedies, were recorded and represent a crucial portion of the local cultural heritage related to tra ditional food, medicinal, and veterinary practices; approximately one-third of the reports were not previously recorded in albania or kosovo. among these findings, the uncommon, yet abandoned utilizations of wild pears to produce home-made vinegar, unripe wild apples, and grapes as starters/yeasts for baking, and a few unripe wild fruits, as well as beech cambium and sedum album leaves as yogurt starters deserve further in-depth food technological and nutraceutical investigation. the fact that the most interesting findings are represented by obsolete and past practices and that most of the selected villages were chosen expressly because of their disadvantaged economic conditions and, in a few cases, remarkable geographical isolation, demonstrates that even in remote areas of se europe ethnobotanical knowledge is vanishing. nevertheless, this study supports the idea that territories which are less economically advantaged may retain more ethnobotanical knowledge than other, more “de veloped” ones. initiatives aimed at revitalizing traditional practices of wild food and medicinal plant use may be crucial in the study area for implementing rural development programs focusing on local food resources and associated small scale trade. keywords: albania; wild food plants; ethnobotany introduction in recent years a number of field studies have explored the ethnobotany of pastoralist, mountainous areas of diverse western balkan countries, with the aim of recording a specific portion of their traditional environmental knowledge (tek), which is represented by the folk perceptions, knowledge, practices, and beliefs concerning wild food and medicinal plants (redžić, 2006 and 2007; jarić et al., 2007 and 2015; šarić-kundalić et al., 2010 and 2011; menković et al., 2011; mustafa et al., 2012a, 2012b, and 2015; rexhepi et al., 2013; savikin et al., 2013; pieroni and quave, 2014 and references therein; zlatković et al., 2014; quave and pieroni, 2015; pieroni et al., 2017). the inland part of the balkan peninsula represents a tremendous reservoir of folk knowledge on wild plants, due to both its rich bio diversity and ethno-religious-cultural diversity. the mountainous territories of albania, in particular, due to their isolation and historical vicissitudes of the 20th century, and the fact that they still largely rely upon small-scale agro-pastoral economies, may present a crucial arena for recording ethnobotanical knowledge of wild species. local wild food and medicinal plants have been shown in recent studies to be one of the pillars of the subsistence economy of the local communities of mountainous areas in albania, and folk knowledge and practices concerning botanicals are not only important for understanding local perceptions and uses of plants, but also for providing baseline data that could be employed in projects aimed at developing programs for a sustainable valorization of the local flora. the main goal of the present study, therefore, was to document folk food and medicinal uses of wild plants in a few isolated mountainous municipalities of ne albania, which represent the most economically disadvantaged communities according to the most recent albanian census (instat, 2012) and also the most isolated ones. in addition, a further objective of this study was to compare the collected data with the findings of previous ethnobotanical surveys conducted in albania and kosovo and to identify new plant uses and reports of potential interest to the small-scale specialty food arena and herbal markets. materials and methods study area fig. 1 shows a map of the study area in ne albania and the eight visited villages: six inhabited by muslim (gheg) albanians: arrën (1094 meters above sea level [masl]), radomirë (1247 masl), çidhen (609 masl), grykë-nokë (685 masl, kalis (557 masl), and çajë (1306 masl); one inhabited by catholic (gheg) albanians: domgjoni (649 masl); and one inhabited by “albanicized” (muslim) macedonians: dovolan (615 masl). each of the eight villages nowadays has a permanent population between approx. 100 and 500 inhabitants. most of the visited villages were selected for two reasons: 1) they were included in former municipalities which were among the most economically disadvantaged in albania (taking as reference the number of tvs and washing machines per household [instat, 2012]); and 2) they are located – with the exception of dovolan – in remote mountainous areas, which are currently accessible (arrën, çidhen, grykë-nokë, kalis) or were so until the last decade (radomirë, çajë, domgjoni) only by using four-wheel drive vehicles and which remain isolated for several weeks during the snowy winters, due to the lack of a proper road infrastructure. field study the field study was carried out during the summer 2016. study participants were identified among elderly individuals who were engaged in agro-pastoral activities and still retained traditional en vironmental knowledge. in-depth open and semi-structured interviews were then conducted with 45 selected participants (28 men and 17 women between the ages of 45 and 85 years), with the majority of disappearing ethnobotanical knowledge in remote villages of ne abania 59 the informants, however, above 65 years of age. study participants were asked about traditional (past and present) uses of wild plants in the food and medicinal domains, pertaining to both humans and animals, as well as the utilization of other possible domestic medicinal remedies. to elicit these data, informants were requested to share their knowledge via one or more of the following four approaches: 1. to freelist wild plants for food and medicine they currently use or have used during their lives; 2. to quote remedies based on wild plants or other domestic ingredients they have used for treating specific pathologies (listed by the researchers); 3. to show all wild species used for food and medicine they have stored at home; 4. while on short walks around their houses and pastures, to show all wild plants they know and have used. specifically, the local name(s) of each reported taxon, plant part(s) used, and in-depth details about their manipulation/preparation and food or medicinal use(s) were recorded. study participants were asked to report current uses considered “traditional”, i.e. considered part of the perceived cultural heritage, as well as uses they could recall from their childhood, which may no longer be exploited. interviews were conducted in the albanian language with the help of an interpreter. informed consent from all participants was verbally obtained prior to conducting interviews and ethical guidelines prescribed by the international society of ethnobiology (ise, 2008) were strictly followed. during the interviews, informants were always asked to show the reported plants (fresh or dried) or to describe the plants and their ecology (if the plants were not available). voucher specimens of all the species that were shown in the current study had been collected during previous fieldwork conducted in the neighboring areas of gora and gollobordo (pieroni et al., 2014a; quave and pieroni, 2015) and are stored at the herbaria of the university of camerino (came, italy), university of pristina (prn, kosovo), and emory university (geo, usa). the taxonomic identification followed the official flora of albania (paparisto et al., 1988; qosia et al., 1992 and 1996; vangjeli, 2000), while the bo tanical nomenclature and family assignments followed the plant list (2013), and the angiosperm phylogeny group iv (stevens, 2016), respectively. local plant names were transcribed following the rules of standard albanian. data analysis the collected field data were compared with the available ethnobotanical literature of albania (pieroni et al., 2005, 2011, 2014a, 2014b, 2015, and 2017; pieroni, 2008; quave and pieroni, 2014 and 2015) and kosovo (sejdiu, 1984; mustafa et al., 2012a, 2012b, and 2015; pieroni et al., 2017). results and discussion a total of sixty-three wild food and medicinal folk taxa and approx. 150 plant reports, as well other domestic remedies, were recorded in the study area. wild food plants and wild teas tab. 1 presents the plant-based wild foods and teas mentioned by the informants. in the category “teas” we included all those infusions or decoctions locally prepared and drunk as recreational beverages, as well those drunk and ingested in order to obtain a specific, perceived therapeutic effect. fig. 1: map of the study site and visited villages. 60 a. pieroni, r. sõukand tab. 1: wild food plants and their traditional uses recorded in the study area (a few cultivated and semi-domesticated plants used in uncommon ways are also included) botanical taxon, family, used parts, and recorded local name(s) traditional food use and eventually similar use(s) voucher specimen code(s) perceived medicinal value previously recorded (treated disease) among albanians in albania and kosovo allium triquetrum l. (amaryllidaceae) (?) purris, pras e egër filling for pies, esp. white maize flour-based pies partially aerial parts (fresh) (peta, ljaknur, ljakrur) amaranthus retroflexus l. (amaranthaceae) llaboda filling for pies yes leaves (fresh) artemisia absinthium l. (asteraceae) pelindov tea: stomach-ache, diarrhea yes aerial parts asplenium trichomanes l. and possibly also fir guri, fir i egër tea: kidney stones yes ceterach officinarum willd. (aspleniaceae) aerial parts (dried) came26293 (a. trichomanes) cornus mas l. (cornaceae) thona, thana, consumed raw or dried: stomach-ache, ulcers; partially fruits (fresh and dried) dreniladov tea: diarrhea; compote: diarrhea; syrup: came-26279 cardiotonic, diarrhoea; fermented and distilled prn-23/pz/2013 in raki: cardiotonic, diuretic, blood depurative, anti-hypertensive; fermented in water a few weeks to obtain vinegar (uthull): cold crataegus monogyna jacq. and possibly morrisi consumed raw as a snack* yes other crataegus spp. (rosaceae) fruits (fresh) came26280 fagus sylvatica l. (fagaceae) ahu ground, mixed together with corn flour no wood for making bread* came-26249 fragaria vesca l. (rosaceae) dreza, lulestrydhe consumed raw as a snack yes fruits (fresh) came26247 hordeum vulgare l. (poaceae) elbi bread* yes fruits (dried) hypericum perforatum l., (hypericaceae) lulë kuqë tea: recreational yes flowering aerial parts geo-020051 prn-36/pz/2013 juglans regia l. (juglandaceae) arra consumed fresh and dried, as a snack, often yes seeds and kernels together with honey: restorative; tea (kernel): came26238 cough juniperus communis l. (cupressaceae) dëllinjë distilled to obtain raki: digestive; yes cones (fresh and dried) fermented beverage (prepared by leaving the came26253 cones in water); tea: kidney problems and cough malus sylvestris (l.) mill. (rosaceae) molla e egër unripe fruits were added to dough as a yeast/ partially unripe fruits (fresh)/ starter during the process of making bread, fruits (fresh and dried) when sourdough was not available*; came26288 ripe fruits, fermented in water a few weeks, to produce vinegar (uthull), which is used for treating cold; oshaf: dried fruits are boiled and both the cooked fruits and the resulting liquid ingested matricaria chamomilla l. (asteraceae) kamomili tea: stomach-ache, digestive yes flowering tops (dried) came26235 origanum vulgare l. (lamiaceae) çaj, çaj i egër, tea: recreational/panacea, sore throat, cough, yes flowering aerial parts (dried) çaj mali, çaj bjeshke head-ache geo-020049 prn-52/pz/2013 disappearing ethnobotanical knowledge in remote villages of ne abania 61 plantago major l. (plantaginaceae) bar preme, bar premi, tea: diuretic yes inflorescences (dried) bar premti geo-020043 prn-54/pz/2013 primula veris l. (primulaceae) lule aguliçe tea: diuretic, cough partially flowering tops (dried) geo-020060 prn-56/pz/2013 prunus avium (l.) l. and p. cerasus l. qershi tea: diuretic yes (rosaceae) fruit peduncles (dried) came26240 (p. avium) came26298 (p. cerasus) prunus cerasifera ehrh. (rosaceae) kaisi, kaisi e egër, fermented and distilled in raki: yes fresh fruits kumbull e egër cardiotonic, blood depurative came26266 pyrus communis l. (rosaceae) dardhë tea: diarrhea no bark pyrus pyraster (l.) buirgsd. (rosaceae) dardhë e egër compote; oshaf: dried, then boiled (both partially fruits (fresh and dried) the cooked fruit and the resulting liquid ingested); came26244 home-made vinegar (uthull): cold rosa canina l. (rosaceae) kaça tea: sore throat, cough, flu; boiled in water yes pseudofruits (dried) shipunkadov and slightly fermented to obtained a gassy, came26237 sour beverage (lënge) rubus ulmifolius schott (rosaceae) manaferra (fruits) consumed raw as a snack; fermented partially fruits/young shoots and distilled in raki/ came26310 (young shoots) tea: diarrhea in children rubus ideaus l. (rosaceae) mjedra, njetra consumed raw as a snack yes fruits (fresh) rumex acetosa, r. acetosella l., and uthulla, liakra e bjeske consumed raw as a snack: filling for pies partially r. pulcher l. (polygonaceae) leaves (fresh) came26243 (r. acetosa) rumex patientia l. and possibly other elqeta, lepjeta, lepjeta filling for pies yes rumex spp. (polygonaceae) e egër, leqeta, liakra e leaves egër, lakneshta, liqet came26285 salvia officinalis l. (lamiaceae) sherbela tea: sore throat, cough, liver protective, partially leaves (dried) cardiotonic satureja montana l. (lamiaceae) trumza tea: recreational yes aerial parts sideritis raeseri boiss. & heldr. and çaj, çaj të bardhë, tea: recreational/panacea, sore throat, cough yes possibly s. scardica griseb., lamiaceae çaj fushe flowering aerial parts (dried) came26281 (s. raeseri) solanum lycopersicum l. (solanaceae) domate soup no unripe fruits taraxacum officinale weber (asteraceae) qumstore filling for börek (peta) no leaves came26289 thymus ongicaulis c.presl (lamiaceae) lisën seasoning; tea: different ailments yes aerial parts came26272 triticum monococcum l. (poaceae) tep mixed with maize flour for baking bread* no grainà flour urtica dioica l. (urticaceae) hitha, hithal filling for pies or consumed boiled; yes leaves (fresh) and roots tea: rheumatisms; tea (roots): colitis came26262 62 a. pieroni, r. sõukand vaccinium myrtillus l. (ericaceae) borovnica, consumed raw as a snack; tea: cough, blood partially fruits (fresh and dried) qershia e egër depurative, cardiotonic; fermented beverage geo-020040 prn-87/pz/2013 vitis vinifera l. (vitaceae) rrush unripe fruits are added to dough as a yeast/starter partially unripe fruits (fresh)/fruits (fresh) during the process of making bread, when sourdough is not available*; ripe fruits are consumed fresh in winter for counteracting cold and flu; sometimes they are fermented in water a few weeks to naturally obtain vinegar (uthull): vinegar is heated with hot stones to eliminate possible alcohol and drunk as a digestive; ripe fruits can be fermented and distilled in raki, which is drunk for treating stomach-aches and digestive complaints zea mays l. (poaceae) kolomoç tea: diuretic yes stigma unidentified taxon aerial parts (fresh) bar miseli tea: cardiotonic no (?): identification based only on plant description and folk name; *: past use; dov: folk name remembered and recorded in the “albanicized” macedonian village of dovolan; plants and uses mentioned by more than 50% of the informants are reported in bold. forty-five folk wild taxa were recorded, for which we documented local name(s), taxonomic family, voucher code(s), used plant part(s), local food or herbal use(s), and their correspondence with the en tire ethnobotanical literature of albanians living in albania and kosovo. approximately one-fifth of the recorded folk reports were not previously recorded in albania and kosovo. among the most uncommon uses, the following are worth mentioning: a soup made from unripe tomatoes; the use of taraxacum in pies, which is commonly used in the folk cuisines of slavic populations in the balkans but not much among gheg albanians (łuczaj et al., 2013; pieroni et al., 2014a); the production of home-made vinegar starting from wild pears; and the obsolete (past) uses of beech wood for making bread as well as unripe wild dog apples and grapes as starters/yeasts for baking bread. the most commonly cited wild food and herbal plants mentioned by the study participants (in bold in tab. 1) included cornus mas (fruits, processed in many ways), origanum vulgare (flowering aerial parts, in teas), and urtica dioica and rumex patientia (leaves, both considered very important wild leafy vegetables). plant-based yogurt starters tab. 2 indicates the domestic ingredients (mainly plant-based) that the informants remembered to have been used as home-made yogurt starters (i.e. starters used in the absence of previously produced yogurt). the entire set of mentioned plants listed in the table have never been recorded for such utilizations in the most globally inclusive economic botanical literature (hedrick, 1972; facciola, 1998) and therefore deserve further investigation. in particular, the use of a few unripe wild fruits, as well as beech (fagus sylvatica) cambium and fresh sedum album leaves, as yogurt starters warrant further in-depth food technological and nutraceutical evaluation and investigation. moreover, the fact that all the mentioned uses refer to forest and mountain products, which were probably available during the transhumance period spent in the higher alpine pastures during summer, recalls traces of archaic pastoralist food customs. external plant remedies for curing humans and plant remedies used for treating animals tab. 3 and 4 present the external plant remedies pertaining to humans mentioned by the informants (as well as those few other domestic, external remedies arising from animal sources) and domestic (mainly plant-based) ethnoveterinary remedies, respectively. as in the previous tables, folk names and exact detailed, local, traditional uses are reported. those ingredients and reports that were mentioned by more than half of the study participants are highlighted in bold type. the majority of the recorded plant reports, especially those devoted to animal health, were used in the past and have been abandoned nowadays. comparison with albanian and kosovar ethnobotanies most of the recorded wild food and medicinal plant uses had been previously documented in other mountainous areas of albania and kosovo, although a few reports found in this study are novel. among the food plant uses, these new reports include all reports related to species utilized as (past) yogurt starters, as well as the uses of ground beech wood and emmer wheat flour – mixed with maize flour – for making bread, unripe tomatoes for preparing a specific sour soup, and dandelion leaves, as (bitter) filling for home-made börek. these reports can be explained by the permanence of archaic plant utilizations in the study area, which may be linked to its long history of isolation and famine and its ancestral pastoralist economy, which has left traces that possibly disappeared much earlier in other mountainous areas of albania and kosovo. in the medicinal domain, we recorded a few interesting “unknown” plant uses, which should be maybe further investigated pharma cologically, such as fresh garlic for externally treating eye inflammations, fresh crushed nettle leaves for externally treating cuts, buttermilk for externally treating snake bites, as well as walnut teas, juniper berries, dwarf elderberry roots and elderberry branches for externally treating various skin problems in animals. all these reports demonstrate an interesting permanence of a few still living or remembered ethnoveterinary treatments. the last tra ces of uncommon folk veterinary practices recorded in this study, as well as the past uses of wild plants as yogurt starters, attest an original bulk of tek in ne albania that shows the remarkable pastoralist attitudes and skills of the study communities. tek in mountainous areas of albania: quo vadis? the study sites were selected because of their disadvantaged economic situation and isolation, however, a relevant portion of the practiced tek recorded in the present study refers only to recent disappearing ethnobotanical knowledge in remote villages of ne abania 63 tab. 2: traditional, domestic yogurt starters mentioned and used in the study area in the past ingredient (botanical taxon, family, and used parts) recorded local name(s) similar use previously recorded among albanians in albania and kosovo plants fagus sylvatica l. (fagaceae) see tab. 1 no cambium fragaria vesca l. (rosaceae) see tab. 1 no unripe fruits hypericum perforatum l. (hypericaceae) see tab. 1 no fresh aerial parts malus sylvestris mill. (rosaceae) see tab. 1 no unripe fruits prunus cerasifera ehrh. (rosaceae) see tab. 1 no unripe fruits prunus domestica l. (rosaceae) see tab. 1 no unripe fruits rumex acetosa l., r. acetosella l., and r. pulcher l. (polygonaceae) see tab. 1 no fresh leaves sedum album l. (crassulaceae) rrushi i egër, rrush guri, rrushi uiku no fresh leaves vitis vinifera l. (vitaceae) see tab. 1 no unripe fruits other ingredients ants milingonë yes clarified butter tylënë no rain shi no yogurt (sour) buttermilk dhallët yes tab. 3: traditional external remedies recorded in the study area remedy (botanical taxon, family, used parts, recorded local name(s) traditional preparation and similar use(s) and voucher specimen code(s)) medicinal value/treated disease previously recorded among albanians in albania and kosovo plants achillea millefolium l. (asteraceae) bar premi crushed and topically applied on cuts as an yes fresh leaves haemostatic and cicatrizer came26294 allium cepa l. (amaryllidaceae) qepë crushed, mixed with salt, and topically applied yes bulb for curing bruises allium porrum l. (amarylidaceae) prash instilled in the ear for treating ear inflammations yes leaf juice allium sativum l. (amaryllidaceae) hudhra crushed and applied on the eye region no fresh bulb for treating inflammations cornus mas l. (cornaceae) see tab. 1 fermented into vinegar (uthull); applied on snake partially fresh fruits bites* or applied to the head for treating lice; fermented and distilled into raki, which is topically applied for disinfecting cuts, treating tooth-aches, eye inflammations, and ear-aches malus sylvestris (l.) mill. (rosaceae) see tab. 1 fermented into vinegar (uthull); this is applied on partially fresh fruits snake bites* or applied to the head for treating lice nicotiana tabacum l. (solanaceae) duhan topically applied on cuts as a haemostatic yes dried leaves 64 a. pieroni, r. sõukand plantago lanceolata l. (plantaginaceae) bar premti topically applied as a cicatrizer yes fresh leaves came26284 plantago major l. (plantaginaceae) see tab. 1 topically applied as a suppurative yes fresh leaves prunus cerasifera ehrh. (rosaceae) see tab. 1 fermented and distilled into raki; this is topically partially fresh fruits applied for disinfecting cuts, treating tooth-aches, eye inflammations, and ear-aches prunus domestica l. (rosaceae) kumbull see prunus cerasifera partially fresh fruits sempervivum tectorum l. (crassulaceae) bar veshe instilled in the ear for treating ear inflammations yes leaf juice geo-020035 prn-71/pz/2013 solanum lycopersicum l. (solanaceae) domate considered a mosquito repellent no fresh leaves urtica dioica l. (urticaceae) see tab. 1 crushed and topically applied for no fresh leaves treating cuts and bruises vitis vinifera l. (vitaceae) see tab. 1 juice is instilled in the ear for treating ear yes juice of the fresh shoots and fresh fruits inflammations; fresh fruits are fermented into vinegar (uthull) and this is applied on snake bites* or to the head for treating lice; the fresh fruits are fermented and distilled into raki and this is topically applied for disinfecting cuts, treating tooth-aches, eye inflammations, and ear-aches diverse tree species burned into ash (fain), which in then boiled in yes wood water and topically applied, as an anti-lice agent and for treating cuts* other ingredients bear fat dhjam ariu extracted and stored; as an alternative, slices of meat yes and fat are dried as pastrma; these are heated before use and the resulting melted fat is topically applied for treating burns cow milk qumështi i lopës topically applied to treat eye inflammations yes curdle djathi i bardhë topically applied to treat eye inflammations yes egg vezë the yolk is topically applied on eye inflammations no hare ljepur the meat is fried and consumed for treating no gunshot wounds* hen anus bytha e pulës topically applied on snake bites, which “sucks out” yes the poison; the hen dies and is thrown away afterwards* human urine urinë topically applied on wounds yes leather lëkurë rubbed on cuts, as an haemostatic; burned, partially the resulting smoke is considered a snake repellent snake (not specified) gjarpër macerated in oil for several months in the sun, no the resulting extract is topically applied for treating burns*; snake head topically applied on the spot where the same snake had bitten* yogurt (sour) buttermilk see tab. 2 topically applied on snake bites no woman milk tomël i gruas topically applied to relieve eye and yes ear inflammations* *: past uses; plant remedies and related uses mentioned by more than 50 % of the informants are reported in bold. disappearing ethnobotanical knowledge in remote villages of ne abania 65 tab. 4: ethnoveterinary remedies recorded in the study area remedy (botanical taxon, family, recorded local name(s) traditional preparation and medicinal value/ similar use(s) and used parts) treated disease previously recorded among albanians in albania and kosovo plants hordeum vulgare l. (poaceae) see tab. 1 boiled in water; the vapours are inhaled by the animal no fruits for curing respiratory problems* juglans regia l. (juglandaceae) see tab. 1 tea (unripe pericarps): externally applied for treating no unripe pericarps and kernels cow horn infections and skin cuts; decoction (unripe kernels): instilled in the ear for treating ear-aches juniperus communis l. (cupressaceae) see tab. 1 externally applied on wounds* no cones nicotiana tabacum l. (solanaceae) see tab. 3 decoction: in external washes as an anti-parasitic no stems pinus spp. (pinaceae) pisha topically applied on wounds and broken bones* yes resin prunus cerasifera ehrh. (rosaceae) see tab. 1 fermented and distilled into raki; this is given to partially fresh fruits animals for treating diverse internal complaints salvia officinalis l. (lamiaceae) see tab. 1 tea: cough; rheumatisms partially leaves sambuculus ebulus l. (adoxaceae) kigël inserted into the animal’s ear for treating various no roots diseases (goats, sheep); decoction: in external washes came26254 to disinfect the skin (all animals)* sambuculus nigra l. (adoxaceae) shtog mixed (aerial parts) with butter or (bark) with butter no branches and bark and lime water, topically applied for treating burns prn-69/pz/2013 tilia platyphyllos scop. (malvaceae) lule blini tea: respiratory problems* no flowers came26241 ulmus minor mill. and possibly other vidh decoction: topically applied as a cicatrizer yes ulmus spp., ulmaceae bark came26308 vitis vinifera l. (vitaceae) see tab. 1 fermented and distilled into raki; this is given to partially fresh fruits animals for treating diverse internal complaints zea mays l. (poaceae) see tab. 1 boiled and given as food for treating internal no fruits gastro-intestinal problems other ingredients bees wax dyll bletesh topically applied for treating eye inflammations no egg see tab. 3 fried and applied on cuts or ingested raw for no counteracting bruises hen anus see tab. 3 topically applied on snake bites, which “sucks out” the poison; the hen dies and is thrown away afterwards* yes mouton fat dhjam deleje burned, topically applied for treating wounds no yogurt (sour) buttermilk see tab. 2 given to constipated animals no *: past uses. past folk plant uses which therefore indicates that it is remarkably eroded. the ethnobotanical heritage of the region is under threat for many reasons, but the key factor is the urbanization and migration process, which 25 years after the fall of communism is still on-going in rural and mountainous areas of albania. young and middle-aged community members still migrate to tirana or western countries for work and they are more and more detached from traditional agropastoral activities, thus interrupting the oral transmission of tek, and subsequently the daily practice of dealing with the surrounding plant environment, which ultimately affects the permanence of ethnobotanical knowledge. the elderly portion of the population, which remains in the mountain 66 a. pieroni, r. sõukand villages, is still well connected to the traditional lifestyle, but they also highly rely upon remittances from relatives. on the other hand, it is precisely these mountainous areas, with their pristine environments, which could become of strategic importance for the development of sustainable eco-tourism activities and the small-scale trade of local herbal and wild food plants in the region. in fact, internal rural and mountainous areas in albania, as a consequence of the political and economic developments of the country during the last century, have been largely unaffected by industrialization and still offer an amazingly rich bio-cultural landscape. conversely, the fact that the aforementioned social changes contribute to the disappearance of tek has to be of great concern, as eco-tourism and small-scale chains of specialty foods and herbal products can only be put in place upon the permanence of local folk knowledge systems. conclusions this study contributes to the documentation of vanishing traditional environmental knowledge and research on the influence of isolation and economic disadvantage on the preservation of tek. despite the number of past studies conducted within albania, the present research still provided several folk plant uses that had never been previously reported. the plants and folk plant uses mentioned by the informants in this field study offer an important set of data, and, we hope inspirations, which could be used to promote a germane re-vitalization and valorization of the tek still retained by the visited local communities. acknowledgements special thanks are due to all the study 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10.1016/j.jep.2013.02.006. sejdiu, s., 1984: fjalorth ethnobotanik i shqipes. rilindja, prishtina. the plant list, version 1.1, 2013: http://www.theplantlist.org/ (accessed 21.10.2016). stevens, p.f, 2016: angiosperm phylogeny website. version 13. http://www. mobot.org/mobot/research/apweb/ (accessed 21.10.2016). vangjeli, j.r., b., mullaj, a., paparisto, k., qosia, x., 2000: flora e shqipërisë 4. akademia e shkencave e republikes se shqipërisë, instituti i kërkimeve biologjike, tirana. zlatković, b.k., bogosavljević, s.s., radivojević, a.r., pavlović, m.a., 2014: traditional use of the native medicinal plant resource of mt. rtanj (eastern serbia): ethnobotanical evaluation and comparison. j. ethnopharmacol. 151, 704-713. doi: 10.1016/j.jep.2013.11.037. address of the corresponding author: e-mail: a.pieroni@unisg.it © the author(s) 2017. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 87, 220 226 (2014), doi:10.5073/jabfq.2014.087.031 mped research center, department of botany, university of fort hare, alice, south africa mineral uptake in solanum nigrum l. cultivated on fertiliser amended soils of the eastern cape, south africa callistus bvenura, anthony j. afolayan* (received april 23, 2014) * corresponding author summary a considerable interest has been manifested in the cultivation of wild vegetables to combat the ever increasing hunger and micronutrient deficiencies especially in children from the developing world. solanum nigrum, one of the popular wild vegetables consumed in the eastern cape was cultivated on sandy loam soils to determine the effect of fertilisers on cu, fe, mn and zn uptake in relation to its growth stages. five treatments (control; 100 kg n/ha; 8.13 t manure/ha; 100 kg n/ha + 8.13 t manure/ha and 50 kg n/ha + 4.07 t manure/ha) were arranged in plots in a randomised complete block design with five replicates. cu (mg/kg) remained variably high throughout the trial, ranging between 7.20-23.50 on the field and 5.30-21.40 in the glasshouse. fe (mg/kg) also remained variably high, ranging between 213-766 on the field and 178-523 in the glasshouse. mn (mg/kg) increased with increasing plant maturity and ranged between 85-222 on the field and 64-215 in the glasshouse, but zn (mg/kg) decreased with plant maturity and ranged between 33-78 on the field and 16-74 in the glasshouse. the results indicate that solanum nigrum has the potential to supply the recommended daily micronutrient intake values throughout all its growth stages. introduction there is no doubt that one of the causative factors to low nutritional status of food plants is poor soil fertility. plants obtain their nutrients from the soil on which they are cultivated. the replenishment of nutrients to low yielding soils through the addition of fertilisers often leads to better performance of food plants. nutrient uptake and composition of food plants has for a long time been a subject of interest among researchers. food plants play a critical role in human life by supplying the much needed nutritional elements as well as boosting the human immune system. however, most food plants are currently based on a limited number of crops and these comprise about 103 plant species which contribute 90 % of national per capita supplies of food plants (prescott-allen and prescott-allen, 1990). although wild vegetable plants were an important part of traditional agricultural systems, their consumption has over the years declined in south africa due to their association with poverty and poverty foods, degree of urbanisation, modernisation of agriculture, distance to fresh produce markets and season of the year, among other factors (flyman and afolayan, 2007; jansen van rensberg et al., 2007). according to modi et al., (2006), the poor utilisation of wild vegetables may be associated with a lack of knowledge about how to access quantities that can satisfy daily human food requirements. nevertheless, in many parts of the world, including south africa, the use of wild vegetables is not negligible (misra et al., 2008). one of the major health problems affecting children in south africa is micronutrient deficiency and these have been well documented in south africa particularly for fe, zn and cu (faber and wenhold, 2007; voster, 2010). the rich nutritional value of wild vegetables has also been documented by many authors (edmonds and chweya, 1997; flyman and afolayan, 2007; odhav et al., 2007; lewu and mavengahama, 2010). faber and wenhold (2007) suggested the cultivation of wild vegetables as one of the strategies that can be adopted to address micronutrient malnutrition. wild vegetables usually grow as volunteer plants alongside conventional crops in agricultural fields during the planting season and are mostly viewed as weeds that must be removed usually by mechanical or chemical means. although they are viewed as weeds, the cultivation of wild vegetables has been documented in south africa for some species, for example, amaranthus and brassica (jansen van rensberg et al., 2007; husselman and sizane, 2006). however, agronomic data required to cultivate wild vegetable plants is scanty. a preliminary survey of wild vegetables consumed in the eastern cape province of south africa, where the current study was conducted indicated that s. nigrum is one of the popular wild vegetables gathered from the wild for food and consumed by the province’s rural populace (bvenura and afolayan, 2014). this wild vegetable was therefore cultivated in the glasshouse and on the field under different concentrations of organic and/or inorganic fertilisers with a view to determine the wild vegetable’s response to these fertilisers and also determine the best fertiliser option for its cultivation as well as the best time to harvest the leaves for extraction of the micronutrients; zn, fe, cu and mn. materials and methods the experimental site the experiment was conducted in the glasshouse and on the field at the university of fort hare, alice campus, south africa between september and december 2012. the study area falls under 32o 47’ s and 26o 50’ e and 535 m a.s.l. and is within a semi arid ecological zone with an average annual rainfall of approximately 575 mm in summer; mean daily temperatures of 22.5 oc during the day and 18.8 oc at night while during the winter the temperature is about 13.6 oc during the day and less than 10.3 oc at night (marais and brutsch, 1994). according to the south african system of soil classification, the soils are deep alluvial; of the oakleaf form (oa) and belong to the jozini series and are texturally sandy loam (soil classification working group, 1991). according to the soil map of the world, the soils are eutric fluvisols (fle) (fao-unescoisric, 1988). the properties of the soil, used for this experiment are shown in tab. 1. agronomic practices ripe and mature s. nigrum berries were harvested between the 3rd and 26th of april 2012 from the wild in alice. the seeds were separated from the pulp, washed in distilled water and dried at room temperature on the laboratory bench for 2 h and kept in sealed bottles until further use. the extracted seeds were planted in cavity trays in the glasshouse and later transplanted to the field when they were mineral uptake in solanum nigrum l. 221 about 6 weeks old. for the glasshouse trial, the seedlings were transplanted into prepared polythene bags containing 5 kg of soil. the soil used in the glasshouse was obtained from the field where the field trial was conducted to ensure consistency in soil properties from the two trials. the organic fertiliser (goat manure) used in this experiment was obtained from the university of fort hare animal farm while the inorganic fertilisers (npk [2:3:4] and limestone ammonium nitrate [lan]) were purchased from a local fertiliser dealer. the properties of the organic fertiliser used for the experiment are shown in tab. 1. ing the samples in a dust free, forced-draft oven at 40 oc to a constant weight. the samples were then ground to a powder using a mortar and pestle and passed through a 2 mm sieve. the samples were kept in plastic val containers and stored in a refrigerator at 4 oc till when needed. the first data were collected on the day of transplanting followed by 3 weeks after transplanting after which data were collected on a weekly basis. the experiment was terminated in the 9th week for the glasshouse experiment and 12th week for the field experiment when all the berries on the plant were mature and ripe. mineral analysis the agrilasa (2008) method of determining mineral elements in plant samples was followed. about 0.5 g of finely ground vegetable samples was placed in dry, clean digestion tubes and 5 ml of the digestion mixture comprising 1 part hcio4 + 2 parts hno3 added. this mixture was digested at 230 oc on a digestion block for 70 min, allowed to cool down and made up to 100 ml volume with distilled water. the concentration of cu, fe, mn and zn was then determined using the inductively coupled plasma optical emission spectrometer (icp oes). statistical analysis data of the nutrient concentrations of various treatments were subjected to statistical analysis using mnitab release 12. a one way analysis of variance was used to compare the means of various nutrient concentrations among the treatments and a two way analysis of variance used to determine the interaction between plant age (weeks after transplanting) and treatment on nutrient accumulation in the plant. means were segregated using duncan’s multiple range test. the means were treated as significantly different at p < 0.05. results and discussion copper (cu) cu exponentially increased from the time of transplanting to the 3rd week on the field, after which it varied, but was highest in the 11th week in t3 (tab. 2a). treatment means significantly differed (p < 0.05) and ranged between 7.20 and 21.60 mg/kg in the week of transplanting and the 4th week respectively. the means for the duration of the trial were highest in t1 (16.2 mg/kg) and lowest in t4 (15.5 %). statistical analysis showed an interaction between plant age and the fertiliser treatment on cu uptake. regression analysis with cu as the dependable variable and time (plant age) as the regressor showed a coefficient of determination (r2) of 25.7 % indicating that plant age had a minimum effect on cu. in the glasshouse, cu exponentially increased from the time of transplanting to the 3rd week after which it decreased (tab. 2b). treatment means significantly differed (p < 0.05) and ranged between 5.30 and 21.40 mg/kg in the 9th and 4th week respectively. the means for the trial period were highest in t3 (13.6 mg/kg) and least in t1 (9.50 %). statistical analysis showed an interaction between plant age and the fertiliser treatment on cu. regression analysis with cu as the dependable variable and time (plant age) as the regressor showed a coefficient of determination (r2) of 12.7 % indicating that plant age had a minimum effect on cu uptake. the field experiment showed higher concentrations of cu than the glasshouse experiment. according to shorrocks and alloway (1988), the availability of cu for uptake by plants is determined by soil ph and organic matter content among other factors. low ph enhances the absorption of cu from the soil. the higher concentration of cu on the field may be due to high organic matter concentrations tab. 1: the chemical properties of the experimental soil (upper 0-30 cm depth) and organic fertiliser soil organic fertiliser ph(kci) 6.54 7.17 bulk density (g cm-3) 1.20 ec (µs/cm) 162.05 10.75 cecsum (meq/ 100g) 12.10 available p (mg kg-1) 71 8 500 exchangeable k (mg kg-1) 406 26 000 exchangeable ca (mg kg-1) 1653 29 700 exchangeable mg (mg kg-1) 335 9 900 exchangeable acidity (cmol/l) 0.06 total cations (cmol/l) 12.10 saturated acid (%) 0 zn (mg kg-1) 10.2 172 mn (mg kg-1) 17 582 cu (mg kg-1) 5.7 54 organic c (mg kg-1) 10000 n (mg kg-1) 1400 24 800 clay (%) 17 na (mg kg-1) 1 564 fe (mg kg-1) 12 439 al (mg kg-1) 5 335 experimental design the experiments were laid out in a randomised complete block design (rcbd) with five treatments and five replicates. the treatments were: control (t1); 100 kg n/ha (t2); 8.13 t manure/ha (t3); 100 kg n/ha + 8.13 t manure/ ha (t4) and 50 kg n/ha + 4.07 t manure/ ha (t5). nitrogen was supplied in the form of npk and lan fertilisers. the organic fertiliser (goat manure) and npk were applied at transplanting and lan fertiliser applied 4 weeks after transplanting. these fertilisers were applied in the top 5-7 cm of soil depth by mixing with a spade in plots measuring 3 m × 2 m on the field. in the glasshouse, the experiment was laid out as described in the field except that each treatment had 5 replicates but each replicate had 10 experimental units to ensure a sufficient number of plant samples for the duration of the trial. each replicate consisted of one s. nigrum plant in a polythene bag containing 5 kg of soil. data collection the third youngest fully expanded leaves (jones et al., 1971) were collected from the shoots by uprooting the whole plant; washed in distilled water to remove sediments and other impurities before dry222 c. bvenura, a.j. afolayan tab. 2a: effect of organic and inorganic fertilisers on cu (mg/kg) of solanum nigrum l. cultivated in the field plant age (weeks after transplanting) 0 3 4 5 6 7 8 9 10 11 12 t1 7.20±0.18 17.20±0.18a 15.70±0.18a 18.20±0.18a 16.90±0.36 12.40±0.18a 16.80±0.36a 19.80±0.18a 17.40±0.18a 18.70±0.18a 18.00±0.18a t2 7.20±0.18 16.60±0.18b 16.10±0.09b 17.80±0.18b 14.50±0.18a 14.30±0.27b 12.70±0.18b 17.40±0.36b 19.50±0.18b 16.40±0.18b 21.50±0.18b t3 7.20±0.18 16.70±0.09b 21.60±0.27c 15.60±0.27c 16.60±.027 16.60±0.27c 12.70±0.18b 15.60±0.27c 20.60±0.27c 23.50±0.18c 8.10±0.18c t4 7.20±0.18 18.80±0.36c 17.90±0.09d 17.10±0.09d 16.90±0.09 15.40±0.18d 14.40±0.18c 19.00±0.09d 17.70±0.18a 15.00±0.09d 10.93±0.23d t5 7.20±0.18 17.00±0.18ab 17.60±0.27d 16.10±0.09e 16.80±0.18 15.00±0.09e 16.50±0.45a 17.70±0.27e 19.50±0.09b 15.60±0.27e 14.10±0.09e 0 indicates readings taken at the time of transplanting values shown are mean ± s.d. means with different letters down the same column represent significant differences at p < 0.05. tab. 2b: effect of organic and inorganic fertilisers on cu (mg/kg) of solanum nigrum l. cultivated in the glasshouse plant age (weeks after transplanting) 0 3 4 5 6 7 8 9 t1 7.20±0.18 17.60±0.27a 13.70±0.18a 12.80±0.18a 6.90±0.09a 5.80±0.18a 6.10±0.18a 5.60±0.27a t2 7.20±0.18 16.20±0.18b 17.90±0.18b 12.50±0.18a 9.00±0.18b 5.50±0.18a 6.20±0.18a 5.50±0.18a t3 7.20±0.18 20.40±0.36c 15.10±0.09c 15.50±0.18b 15.40±0.18c 12.70±0.18b 10.90±0.09b 11.50±0.27b t4 7.20±0.18 18.20±0.18d 14.00±0.18a 11.50±0.45c 7.60±0.18a 6.20±0.18a 7.00±0.18c 5.30±0.27a t5 7.20±0.18 19.70±0.18e 21.40±0.18d 17.00±0.27d 13.10±0.09d 11.30±0.27c 7.30±0.18c 9.20±0.18c 0 indicates readings taken at the time of transplanting values shown are mean ± s.d. means with different letters down the same column represent significant differences at p < 0.05. and therefore slightly lower soil ph as compared to the glasshouse. in another species, atta et al. (2010) observed that cu was highest during the first stages or the final stages of h. sabdariffa growth depending on the ecotype. however, the concentration of cu in their study was at least 600 % higher than in the current study. on the other hand, morillo et al. (1997) reported lower concentrations of cu as compared to the current study but in a. gayanus. these results indicate that s. nigrum has the potential to provide more cu than the needed daily recommended values according to new zealand and australian standards (nhmrc, 2005) at any growth phase of the plant if children and adults respectively consume about 150 g and 300 g of the vegetable. under field conditions, s. nigrum may best be harvested in the final stages of growth while under glasshouse conditions, the early growth stages would be ideal as the micronutrient is at its peak during these stages of growth. iron (fe) fe uptake differed significantly (p < 0.05) and ranged between 213 and 766 mg/kg at the time of transplanting and the 5th week respectively (tab. 3a). the means for the trial period were highest in t3 (528 mg/kg) and lowest in t5 (440 mg/kg). statistical analysis showed an interaction between plant age and the fertiliser treatment on fe. regression analysis with fe as the dependable variable and time (plant age) as the regressor showed a coefficient of determination (r2) of 37.7 % indicating that plant age had a minimum effect on fe uptake. results of the glasshouse experiment showed lower means which ranged between 178 and 523 mg/kg in the 4th and 5th week respectively except in t1 (tab. 3b). the means for the duration of the trial were highest in t1 (526 mg/kg) and lowest in t5 (239 mg/kg). statistical analysis showed an interaction between plant age and the fertiliser treatment on fe. regression analysis with fe as the dependable variable and time (plant age) as the regressor showed a coefficient of determination (r2) of 0.9 % indicating that plant age had a very minimum effect on fe uptake. fe concentration was observed to be constant on the field but slightly decreased as the plant aged in the glasshouse experiment. the observation here is different from the report of morillo et al. (1997) in a. gayanus. in the same way, flyman and afolayan (2008) observed variations in fe concentration on m. balsamina and v. unguiculata. according to graham and stangoulis (2003), solubility of fe is very low particularly in the presence of moderate oxygen and acidic conditions. the high organic matter content on the field and the continuos release of minerals to the soil may be attributed to the differences reported in comparison with the glasshouse in the current study. the variations in concentration between the field and the glasshouse concentration of fe in s. nigrum remained more than sufficient to supply the required human daily average intake across all age groups and gender according to the new zealand and australian standards (nhmrc, 2005). however, harvesting the plant for maximum fe content would be best during the middle stages of the plant’s growth cycle when fe will be at its peak. furthermore, the concentration range reported in the current study is higher than what odhav et al. (2007) reported in wild uncultivated s. nigrum leaves. a portion of about 150 g and 300 g of cultivated and cooked s. nigrum therefore has the potential to supplement a starch rich diet of poor rural communities at all stages of growth in children and adults respectively in view of the fact that fe is one of the most prevalent forms of micronutrient malnutrition in the world (fao, 2004). mineral uptake in solanum nigrum l. 223 manganese (mn) mn concentration increased as the plant matured and ranged between 86 and 159 mg/kg in the 4th and 10th week respectively (tab. 4a). the means for the trial period were highest in t2 (123 mg/kg) and lowest in t1 (104 mg/kg). statistical analysis showed an interaction between plant age and the fertiliser treatment on mn. regression analysis with mn as the dependable variable and time (plant age) as the regressor showed a coefficient of determination (r2) of 71.1 % indicating that plant age had a significant effect on mn. similarly, the glasshouse means also differed significantly (p < 0.05) and ranged between 64 and 215 mg/kg in the 6th and 9th weeks of respectively (tab. 4b). the means for the trial were highest in t5 (144 mg/kg) and lowest in t1 (83 mg/kg) and this was lower than what was reported on the field except for t5 which was higher in the glasshouse. statistical analysis showed an interaction between plant age and the fertiliser treatment on mn. regression analysis with mn as the dependable variable and time (plant age) as the regressor showed a coefficient of determination (r2) of 65.8 % indicating that plant age had a significant effect on mn. statistical analysis shows that there is a relationship between the age of the plant and mn concentration. as the plant matured in age, the concentration of mn also increased. this observation is in line with the report of flyman and afolayan (2008) and atta et al. (2010) but at variance with that of morillo et al. (1997). the continuous increase of mn in s. nigrum leaves in the present study is an indication of the continuous release of the mineral from the soil for uptake by the plant. according to mcgrath et al. (1994), during the summer season, the relatively high decomposition rate of organic matter releases mn in the soil solution for possible uptake by plants and this is a possible reason why concentrations were higher on the field than in the glasshouse where organic matter content is expectedly low. once taken up into plant tissues, mn becomes immobile and this is possibly why the mineral continued to increase in the leaves (mcgrath et al., 1994). instead of being reassigned to the reproductive parts of the plant, the element remained immobile in the leaves of s. nigrum. the results of this work further indicate the ability of s. nigrum to provide mn needed to supply the required human daily intake according to new zealand and australian standards (nhmrc, 2005) at all stages of the plant’s growth if children and adults respectively consume 150 and 300 g of the cooked leaves. in addition, results of this study indicate that in order to harness the maximum amount of the mineral, leaves of mature plants should be harvested. zinc (zn) zn exponentially increased on the field between the time of transplanting and the 3rd week and decreased until week 12 (tab. 5a). the treatment means significantly differed (p < 0.05) and ranged between 33 and 78 mg/kg in the 12th and 3rd week respectively. the means for the duration of the trial were highest in t5 (62 mg/kg) and least in t1 (59 mg/kg). statistical analysis showed an interaction between plant age and the fertiliser treatment on zn. regression analysis with zn as the dependable variable and time (plant age) as the regressor showed a coefficient of determination (r2) of 59.4 % indicating that plant age had a fairly significant effect on zn. in the glasshouse, zn also increased between the time of transplanting and the 4th week after which it decreased (tab. 5b). the treatment means significantly differed and ranged between 19 and 74 mg/kg in the 9th and 4th week respectively. the means for the trial were highest tab. 3a: effect of organic and inorganic fertilisers on fe (mg/kg) of solanum nigrum l. cultivated in the field plant age (weeks after transplanting) 0 3 4 5 6 7 8 9 10 11 12 t1 213±2.68 426±2.68a 348±3.58a 591±0.89a 562±1.79a 475±4.47a 439±2.25a 629±1.79a 408±3.58a 570±1.79a 435±4.47a t2 213±2.68 624±3.58b 246±1.79b 766±2.68b 403±2.68b 480±1.79a 374±1.79b 521±0.89b 483±2.68b 540±1.79b 750±1.79b t3 213±2.68 514±1.79c 300±1.79c 587±1.79a 678±1.79c 669±3.58b 548±1.79c 556±2.68c 667±1.79c 677±1.79c 403±2.68a t4 213±2.68 371±0.89d 406±2.68d 617±1.79c 395±4.47b 575±4.47c 671±0.89d 396±2.68d 567±1.79d 578±1.79a 375±4.47c t5 213±2.68 305±4.47e 499±1.37e 655±4.47d 366±2.68d 499±0.89d 414±1.79e 501±0.89b 466±2.68b 567±1.37a 356±3.58c 0 indicates readings taken at the time of transplanting values shown are mean ± s.d. means with different letters down the same column represent significant differences at p < 0.05. tab. 3b: effect of organic and inorganic fertilisers on fe (mg/kg) of solanum nigrum l. cultivated in the glasshouse plant age (weeks after transplanting) 0 3 4 5 6 7 8 9 t1 213±2.68 430±1.79a 299±4.03a 337±6.26a 246±1.79a 325±4.47a 327±1.79a 433±2.68a t2 213±2.68 320±1.79b 323±2.68b 212±5.37b 255±4.47b 254±1.79b 302±1.79b 256±2.68b t3 213±2.68 447±1.79a 340±1.79b 265±4.47c 296±2.68c 363±2.68c 269±4.03c 304±3.58c t4 213±2.68 260±1.79c 523±2.68c 178±3.58d 195±4.47d 239±1.79d 224±1.79d 258±7.16b t5 213±2.68 217±1.79d 432±1.79d 191±0.89d 179±3.14e 230±3.58d 209±8.05e 239±0.89b 0 indicates readings taken at the time of transplanting values shown are mean ± s.d. means with different letters down the same column represent significant differences at p < 0.05. 224 c. bvenura, a.j. afolayan tab. 4a: effect of organic and inorganic fertilisers on mn (mg/kg) of solanum nigrum l. cultivated in the field plant age (weeks after transplanting) 0 3 4 5 6 7 8 9 10 11 12 t1 88.67±3.14 101±0.89a 89±3.14a 90±3.58a 95±2.68a 108±7.16a 102±1.79a 85±4.47a 110±4.47a 137±2.68a 143±2.68a t2 88.67±3.14 116±4.47b 87±3.58a 121±2.68b 110±2.68b 139±4.03b 153±2.68b 114±6.26b 156±2.68b 129±4.03b 134±1.79b t3 88.67±3.14 109±4.93c 86±2.68a 90±4.47a 95±1.79a 124±1.79c 89±4.03c 86±2.68a 114±6.26a 132±1.79b 222±0.89c t4 88.67±3.14 103±1.37d 104±3.58b 104±0.52c 101±0.89c 126±2.68c 132±1.79d 127±6.26c 159±4.03b 121±0.89c 154±3.58d t5 88.67±3.14 106±1.79cd 112±5.37c 108±2.68c 92±1.79a 131±0.89d 136±5.37d 111±1.79b 127±1.79c 149±4.03d 154±2.68d 0 indicates readings taken at the time of transplanting values shown are mean ± s.d. means with different letters down the same column represent significant differences at p < 0.05. tab. 4b: effect of organic and inorganic fertilisers on mn (mg/kg) of solanum nigrum l. cultivated in the glasshouse plant age (weeks after transplanting) 0 3 4 5 6 7 8 9 t1 89±3.14 88±3.58bc 78±3.58a 73±0.89a 64±1.79a 79±4.03a 79±4.47a 114±6.26a t2 89±3.14 83±2.68c 108±3.58b 88±1.79b 103±2.25b 101±0.89b 128±3.58bc 148±1.79b t3 89±3.14 94±1.79b 79±4.03a 90±4.47b 118±2.68c 125±1.79c 125±4.47c 167±6.26c t4 89±3.14 86±2.68bc 98±3.58c 78±3.58a 90±4.47d 102±1.79b 138±5.37b 147±1.79b t5 89±3.14 117±13.42a 153±2.68d 119±4.47c 160±4.47e 164±3.58d 134±3.58b 215±1.79d 0 indicates readings taken at the time of transplanting values shown are mean ± s.d. means with different letters down the same column represent significant differences at p < 0.05. in t2 (49 mg/kg) and least in t5 (37 mg/kg) and these values were lower than those reported on the field. statistical analysis showed an interaction between plant age and the fertiliser treatment on zn. regression analysis with zn as the dependable variable and time (plant age) as the regressor showed a coefficient of determination (r2) of 81.7 % indicating that plant age had a very significant effect on zn. zn was found to be gradually decreasing as the plant matured. this observation is in line with the report by afolayan and flyman (2008). however, atta et al. (2010) reported variations in zn concentration in h. sabdariffa as it grew older while morillo et al. (1997) observed no change in zn concentration in a. gayanus. according to kabata-pendias (2001), about 75 % of total zn taken up by plants is stored in the shoots of young plants whereas about 20-30 % occurs in the shoots of old plants. this phenomenon is a possible explanation to the decline in zn concentration with advancing plant maturity in the present study. furthermore, kabatapendias (2001) proposed that the roots often contain more zn than the shoots. the results of the current study indicate that the minimum value of zn reported in the present study (19 mg/kg in the glasshouse) can potentially supply only 51.8 % of the daily recommended human intake values of the mineral in women according to the new zealand and australian standards (nhmrc, 2005), however, the maximum value reported (78 mg/kg in the field) can potentially supply about 213 % of the recommended daily human intake in women. although the values reported in this trial declined with increasing plant maturity, the concentrations have the potential to supply the daily recommended human intake of the mineral across all age groups and gender up to the 12th week on the field while in the glasshouse, the supply is adequate across all age groups and gender up to the 5th week and begins to vary. the optimum time for harvesting s. nigrum leaves for zn would therefore be during early stages of its growth. conclusion this study revealed that solanum nigrum is a high micro mineral yielding wild vegetable that can be cultivated and incorporated into the diets of marginalised poor rural communities whose diets are mainly starch based. the best fertiliser to apply and best time to harvest the plant leaves for food may be a challenge as different minerals respond differently to different fertiliser options, therefore it is critical to recommend the best fertiliser and time of harvesting based on specific micronutrient requirements. however, 50 kg n/ ha + 4.07 t manure/ha increased the concentration of zn on the field and mn in the glasshouse while 8.13 t/ha goat manure increased the uptake of mn as well as fe on the field and cu in the glasshouse. in addition, 100 kg n/ha increased the concentration of ca and zn in the glasshouse while the field and glasshouse controls increased the concentration of cu and fe respectively. cu and fe from both the field and glasshouse varied up and down throughout the trail while mn concentration increased steadily but zn decreased. in general, the nutrient values recorded indicate the ability of the wild vegetable to supply the molarity of recommended daily mineral intakes of micronutrients at all stages of the plant’s growth if children consume about 150 g of the cooked vegetable and adults consume 300 g. however, in order to exploit the maximum potential amounts of the minerals, the results indicate that for cu and zn, the leaves are best harvested during the early stages of growth and the middle stages for fe while the final stages of the plant’s life cycle would be ideal for mn. mineral uptake in solanum nigrum l. 225 tab. 5a: effect of organic and inorganic fertilisers on zn (mg/kg) of solanum nigrum l. cultivated in the field plant age (weeks after transplanting) 0 3 4 5 6 7 8 9 10 11 12 t1 60±3.00 78±1.67 68±8.37 66±5.86 62±2.51a 67±3.35 60±4.67ab 56±3.45 43±3.35a 39±4.18ab 42±6.69 t2 60±3.00 76±1.53 72±4.00 66±2.00 66±3.00ab 69±10.00 59±4.00ab 53±5.00 59±4.00b 37±1.00a 43±3.00 t3 60±3.00 75±5.00 74±2.00 64±4.00 71±1.53b 59±4.00 56±3.00ab 56±2.00 47±2.00a 47±2.00b 33±3.00 t4 60±3.00 78±4.00 71±2.52 65±5.00 70±5.00b 70±6.00 52±4.00b 53±3.00 49±9.00ab 45±6.00ab 37±2.00 t5 60±3.00 77±2.00 73±3.00 67±6.51 73±3.00b 67±5.00 63±3.00a 61±2.00 57±4.00b 41±3.00ab 39±3.51 0 indicates readings taken at the time of transplanting values shown are mean ± s.d. means with different letters down the same column represent significant differences at p < 0.05. tab. 5b: effect of organic and inorganic fertilisers on zn (mg/kg) of solanum nigrum l. cultivated in the glasshouse sodium plant age (weeks after transplanting) 0 3 4 5 6 7 8 9 t1 60±2.68 59±7.15a 48±3.58a 46±5.37 26±4.93a 16±5.37a 23±4.00a 19±2.68a t2 60±2.68 72±1.78b 74±3.58b 46±2.68 46±2.68c 32±3.58a 32±4.00ab 33±2.68b t3 60±2.68 59±8.05a 38±3.58c 37±3.58a 34±3.58b 21±1.79b 23±3.00a 24±3.58c t4 60±2.68 68±0.52b 53±2.68a 50±4.47 41±3.14bc 33±2.68b 34±4.00b 34±3.58b t5 60±2.68 63±2.68ab 60±4.47d 46±2.25 39±4.03b 29±3.57b 32±3.51b 31±1.79b 0 indicates readings taken at the time of transplanting values shown are mean ± s.d. means with different letters down the same column represent significant differences at p < 0.05. acknowledgements we thank the govan mbeki research and development center of the university of fort hare for funding this project. references atta, s., diallo, a.b., bakasso, y., sarr, b., saadou, m., glew, r.h., 2010: micro-element contents in roselle (hibiscus sabdariffa) at different growth stages. afr. j. food agric. nutr. dev. 10(5), 26152628. bvenura, 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m.v., afolayan, a.j., 2008: effect of plant maturity on the mineral content of the leaves of momordica balsamina l. and vigna unguiculata subsp. j. food qual. 31(5), 661-671. graham, r.d., stangoulis, j.c.r., 2003: trace element uptake and distribution in plants. j. nutr. 133, 1502-1505. jansen van rensburg, w.s., van averbeke, w., slabbert, r., faber, m., van jaarsveld, p., van heerden, i., wenhold, f., oelofse, a., 2007: african leafy vegetables in south africa. water sa. 33, 317-326. jones, j.b. 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(eds.), soil testing and plant analysis, 3rd ed. soil science society of america. segoe rd., madison, wi 53711, usa, sssa book series no. 3. kabata-pendias, a., 2001: trace elements in soils and plants, 3rd ed. crc press, llc. khader, v., rama, s., 2003: effect of maturity on macromineral content of selected leafy vegetables. asia pac. j. clin. nutrition 12(1), 45-49. lewu, f.b., mavengahama, s., 2010: wild vegetables in northern kwazulu natal, south africa: current status of production and research needs. sci. res. essays 5(20), 3044-3048. marais, j.n., brutsch, m.o., 1994: the ehlers system of assessing the suitability of temperature regime of a region for crop production. paper presented at 1994 sashs congress, nelspruit, south africa. mcgrath, s.p., chang, a.c., page, a.l., witter, e., 1994: land application of sewage sludge: scientific perspectives of heavy metal loading limits in europe and the united states. environ. rev. 2, 108-118. misra, s., maikhuri, r.k., kala, c.p., rao, k.s., saxena, k.g., 2008: wild leafy vegetables: a study of their subsistence dietetic support to the inhabitants of nanda devi biosphere reserve, india. j. ethnobiol. ethnomed. 4, 15. modi, m., modi, a.t., hendricks, s., 2006: potential role for wild vegetables in household food security: a preliminary case study in 226 c. bvenura, a.j. afolayan kwazulu-natal, south africa. afr. j. food agric. nutr. dev. 6(1), 1-13. morillo, d.e., caraballo, i., faria-marmoll, j., mcdowell, l.r., 1997: effect of plant age and n and p fertilisation on mineral composition of andropogon gayanus. conference proceedings xviii igc 1997 winnipeg, manitoba, id no. 684: available online at: http:// bit.ly/15ibp8z. [accessed 24 july 2013]. nhmrc, 2005: nutrient reference values for australia and new zealand: including recommended dietary intakes. canberra, australia. odhav, b., beekrum, s., akula, u., baijnath, h., 2007: preliminary assessment of nutritional value of traditional leafy vegetables in kwazulu-natal, south africa. j. food comp. anal. 20, 430-435. prescott-allen, r., prescott-allen, c., 1990: how may plants feed the world? conserv. biol. 4(4), 365-374. shorrocks, v.m., alloway, b.j., 1988: cu in plant, animal and human nutrition. cda publication, tn35. soil classification working group, soil and irrigation research institute (south africa), 1991: soil classification: a taxonomic system for south africa. a report on a research project conducted under the auspices of the soil and irrigation research institute. 2nd edition. department of agricultural development, pretoria, south africa. vorster, h.h., 2010: the link between poverty and malnutrition: a south african perspective. health sa gesondheid. 15(1), 435/6. address of the corresponding author: anthony j. afolayan, mped research center, department of botany, university of fort hare, p bag x1314, alice, 5700, south africa. e-mail: aafolayan@ufh.ac.za journal of applied botany and food quality 88, 255 258 (2015), doi:10.5073/jabfq.2015.088.037 1department of biotechnology, suleyman demirel university, isparta, turkey 2 department of horticulture, ataturk university, erzurum, turkey 3west mediterranean development agency, isparta, turkey harvest and postharvest quality of sweet cherry are improved by pre-harvest benzyladenine and benzyladenine plus gibberellin applications fatih ali canli1, murat sahin1, sezai ercisli2*, ozgur yilmaz1, nurettin temurtas1, mustafa pektas3 (received june 12, 2015) * corresponding author summary this study was carried out to evaluate the effects of pre-harvest benzyladenine (ba) and ba plus gibberellin (ga4+7) treatments on fruit quality attributes of ‘0900 ziraat’ cherry at harvest and after cold storage. ‘0900 ziraat’ cherry trees were sprayed with ba (50, 100, and 150 mg·l-1) and ba + ga4+7 (12.5, 25, and 50 mg·l-1) when fruit was at their straw-yellow color stage. all of the treated fruit were significantly firmer than control fruit. fruit treated with 25 and 50 mg·l-1 ba + ga4+7 and 50 and 150 mg·l-1 ba had significantly higher soluble solids content (ssc) than untreated fruit. sweet cherry trees treated with the optimum concentration of ba + ga4+7 (50 mg·l-1) yielded fruit with 15.17 % greater weight, 9.0 % higher firmness and 13.6 % higher ssc. additional samples were harvested, placed in plastic bags, and stored at 4 °c for 30 days. at the end of the cold storage period, fruit treated with 25 and 50 mg·l-1 ba + ga4+7 and 50 and 150 mg·l-1 ba were significantly firmer than the control. 50 mg·l-1 ba + ga4+7 -treated fruit had higher ssc than untreated ones. in conclusion, fruit treated with the optimum dose of ba + ga4+7 (50 mg·l-1) were larger and firmer than untreated fruit at harvest and this concentration had the best effects. most of the treated fruit maintained a superior firmness and quality to control fruit during cold storage. introduction horticultural plants including fruits, vegetables and grapes have long been valued as part of a nutritious and tasty diet and there is increasing scientific awareness that fruits including sweet cherry plays important role for human nutrition and health (bacvonkralj et al., 2014; rop et al., 2014). large and firm sweet cherry fruits are preferred by both consumers and producers (whiting and ophardt, 2005). producers always seek for solutions to avoid lower returns associated with cherries harvested during the peak period when cherry supplies are overly abundant. pgrs can be used to increase fruit size and firmness, to delay maturity (canli and orhan, 2009) and to improve after storage quality of fruit crops (canli et al., 2009). the effects of gibberellic acid (ga3) on fruit quality of cherry fruit are well documented. ga3-treated sweet cherry fruit were larger, heavier (clayton et al., 2006; canli and orhan, 2009; zhang and whiting, 2011a) and firmer than untreated fruit (kappel and macdonald, 2002; canli and orhan, 2009). pre-harvest ga3 applications were also effective in delaying maturity (kappel and macdonald, 2002; webster et al., 2006) and improving cold storage quality and shelf life (einhorn et al., 2013) of sweet cherry fruit. ga3 reduced pedicel browning (einhorn et al., 2013) and improved resistance to surface pitting disorder (einhorn et al., 2013) during the storage period. ga3-treated fruit were also firmer than the untreated fruit at the end of the cold storage period (ozkaya et al., 2006; zhang and whiting, 2011b). ga3 treatments resulted in variable responses in some of the fruit quality attributes such as pedicel length, ssc, and fruit cracking. in contrast to results of canli and orhan (2009), few researchers have reported that it also increased the pedicel length of the cherry fruit (horvitz et al., 2003). an increase in ssc, as a response to ga3 application was reported by some researchers (lenehan et al., 2006; canli and orhan, 2009), but contrary to these results, there were not always changes in ssc (kappel and macdonald, 2002). as in the examples of pedicel length and ssc, the response of fruit cracking to ga3 treatment was also irregular and complex (looney, 1996; usenik et al., 2005). the variable responses of sweet cherry fruit to ga3 applications are possibly caused by ecological and environ mental factors such as location (canli and orhan, 2009), humidity, temperature, precipitation, water status, light, and nutrition (facteau et al., 1985) or by the utilization of different cultivars (usenik et al., 2005) and by application time and application doses (kappel and macdonald, 2002). the final fruit size is mostly determined by the cell division and enlargement in the early phases of fruit development (bohner and bangerth, 1988). while the cell expansion is stimulated mainly by endogenous gas, the cell division in a young fruit is promoted primarily by endogenous cytokinins. ba is the first discovered synthetic compound with cytokinin activity and it is very efficient in promoting cell division (buban, 2000). ‘0900 ziraat’ is a self-incompatible, low-cropping, and a late maturing sweet cherry variety, which constitutes about 12 % of the world cherry trade. although ga3 is currently used to improve the fruit size and quality, and also to delay maturity in high-cropping and selffertile cherry varieties in north america and most other parts of the world (kappel and macdonald, 2002), the effects of ba and ba + ga4+7 on fruit quality attributes of cherry at harvest and after a cold storage period have not been studied yet. therefore, the objective of this study was to determine if a single pre-harvest application of ba or ba + ga4+7 will improve the fruit quality and the cold storage quality of ‘0900 ziraat’ cherry, which constitutes an important portion of the cherry trade in the world. materials and methods plant material and experimental site the experiments were carried out in two consecutive years using 9-year-old ‘0900 ziraat’ cherry trees planted at 7 × 7 m and grown on mazzard rootstocks in kayi (lat. 37°49’12.59”n, long. 30°29’51.65”e, altitude 1097 m), isparta, turkey. plant growth regulator (pgr) applications ‘0900 ziraat’ cherry trees were treated with a single application of 50, 100 or 150 mg·l-1 ba [exilis (20 g·l-1 ba); fine agrochemicals, worcester, uk] and 12.5, 25, or 50 mg·l-1 ba+ga4+7 [perlan (18 g·l-1 ga4+7 and 18 g·l-1 ba), fine agrochemicals, worcester, uk] using a handgun applicator when the fruit were at their straw 256 f.a. canli, m. sahin, s. ercisli, o. yilmaz, n. temurtas, m. pektas yellow color stage of development on a non-windy day in the afternoon. all treatments also had a surfactant [tween-20 (polyethylene glycol sorbitan monolaurate ); sigma-aldrich, st. louis, mo]. the experiments were conducted in the same trees each year. harvest and data collection when fruit reached their maturity [determined by hedonic taste analysis of local farmers, size, color (at ideal harvest color of the variety determined by the naked eye of the experienced local farmers), ssc and firmness], samples of 120 fruit/tree for each application were harvested and fruit quality parameters were evaluated in terms of: fruit weight, stone weight, pedicel length, ssc, firmness and ph. fruit weight fruit weight measurements were taken in groups of ten fruit using a digital balance (model sba 51, sensitivity 0.01 g; scaltec instruments, goettingen, germany), then weights of all 12 groups were added together to find the total weight of each replication. the total weight of each replication was divided by 120 to find the mean fruit weight for each replication. pedicel length the pedicel length of each of 120 fruit/tree was determined using a digital caliper (absolute 500-196-20; mitutoyo, aurora, il). fruit firmness fruit firmness of each of 120 fruits per tree samples was measured on two sides of the equatorial region at the fruit’s maximum width using a fruit texture analyzer (model ft 001; gullimex, alfonsine, italy) equipped with a 4.94 mm diameter probe. seed weight after fruit weight, pedicel length and fruit firmness data were collected, stone of each fruit was taken out. stone weight measurements were taken in groups of ten seeds using the digital balance described above. then, weights of all 12 groups were added together to find the total weight of each replication. the total weight of each replication was divided by 120 to find the mean stone weight for each replication. ssc and fruit ph after fruit weight, pedicel length, fruit firmness and stone weight data were collected, fruits of each replication were divided in to 12 groups (each group containing 10 fruits). each of these groups was mashed to obtain fruit juice, then ssc and fruit ph measurements were taken for each of 12 groups for all replications. the values of all 12 groups were added together to find a total value for each replication. the total value of each replication was divided by 12 to find the mean ssc and fruit ph values for each replication. the fruit ph was determined with a digital ph meter (model ph 330; wtw, weilheim, germany). fruit soluble solids concentration was measured using a refractometer (model n.o.w. 507-1; brix scale of 0 to 32; nippon optical works, tokyo). cold storage experiment in the second year of the experiment, additional samples of 120 fruit/ tree were harvested, placed in 1.5 kg capacity polyethylene bags with perforation. each bag had four holes (1 cm in diameter) at the midsections of each side. cherries were stored at 4 °c for 30 days. the gaseous composition and the relative humidity inside the bags were not measured in this study. firmness, ssc and surface pitting rate of fruit were evaluated after 4 weeks of cold storage. experimental design and statistical analysis the experimental design was a completely randomized design with three single-tree replicates for each treatment since the soil type and the trees were uniform throughout the orchard. homogeneity tests were performed to see if the data from two separate years could be combined. the data were homogeneous over two years for each parameter tested [the differences (d) between the highest standard deviation (std dev1) and the lowest std dev2 were within the acceptable limits (std dev1std dev2 = d, and d ≤ 4std dev2)], thus the data of 2 years were combined and subjected to analyses of variance (anova). the means were separated using tukey range test (version 9.0; sas institute, cary, nc). data from cold storage experiment were also subjected to analyses of variance (anova), and then the means were separated using tukey range test. results and discussion the effects of pre-harvest plant growth regulator (pgr) applica tions on quality of ‘0900 ziraat’ cherry fruit at harvest and after cold storage were evaluated. the effects of pgr applications on fruit weight, pedicel length (tab. 1), fruit firmness and ssc (tab. 2) were significant, but stone weight, fruit weight/stone weight (tab. 1) and fruit ph (tab. 2) were not affected by the applications. some of these tab. 1: effects of pre-harvest benzyladenine (ba) and ba + gibberellin (ga4+7) applications on fruit weight, stone weight and pedicel length of ‘0900 ziraat’ sweet cherry (n=120)a. treatment concentration fruit weight stone weight fruit weight / pedicel length ( mg·l–1) (g)b (g)b stone weight (g)b (mm)b control 0 7.71 by 2.24 3.44 4.82 b ba + ga4+7 12.5 8.36 ab 2.27 3.68 4.80 b ba + ga4+7 25 8.19 ab 2.20 3.72 4.97 ab ba + ga4+7 50 8.88 a 2.33 3.81 4.99 ab ba 50 8.04 ab 2.62 3.06 5.00 ab ba 100 7.62 b 2.48 3.07 4.95 ab ba 150 8.35 ab 2.25 3.71 5.09 a atrees were treated at straw-yellow color stage of the fruit. the control spray was composed of water and tween-20. bmeans within a column followed by different letters are significantly different at p ≤ 0.05 by tukey’s hsd test. the effects of pre-harvest benzyladenine and benzyladenine plus gibberellin applications on quality of sweet cherry fruits 257 differences were also maintained during the 30 days of cold storage when most of the treated fruit retained a superior quality to untreated fruit. pre-harvest pgr applications significantly affected fruit firmness, ssc and surface pitting during cold storage (tab. 3). fruit size data were summarized in tab. 1. fruit treated with 50 mg·l-1 ba + ga4+7 were significantly heavier than the control fruit and the heaviest fruits were obtained from 50 mg·l-1 ba + ga4+7 application. although the differences were not statistically different, most of the other ba and ba + ga4+7 applications yielded heavier fruits than the control (tab. 2). when compared to untreated ‘0900 ziraat’ cherry trees, 50 mg·l-1 ba + ga4+7 treated trees yielded fruit with 15.17 % greater weight. similarly, ga3 treatments increased fruit size of cherries about 10 % to 15 % (kappel and macdonald, 2002; usenik et al., 2005; lenehan et al., 2006; canli and orhan, 2009). a significant increase of fruit size in fruit crops is one of the most common and important effects of pre-harvest ga3 applications (canli and orhan, 2009) and gibberellins plus benzyladenine treatments (usenik et al., 2005; stern et al., 2007). when compared to untreated control fruit, only 150 mg·l-1 ba treatment significantly increased fruit pedicel length (tab. 2). sweet cherry fruits with long pedicels are preferred by consumers (canli and orhan, 2009). this is the first report that benzyladenine treatments can be used to increase the pedicel length of cherry fruit. lower concentrations of ba or ba + ga4+7 applications did not affect pedicel length. horvitz et al. (2003) reported an increase in pedicel length of cherry fruit as a response to pre-harvest ga3 applications. however, there are other reports that pedicel length is not always increased by ga3 applications and the responses to ga3 application were variable (canli and orhan, 2009). no significant differences were observed between untreated fruit and pgr treated fruit with respect to fruit ph (tab. 2). all ba and ba + ga4+7 treatments significantly increased fruit firmness when compared with untreated fruit (tab. 2). in agreement with our results, pre-harvest sprays of ba also increased fruit firmness in pear (stern et al., 2007; canli et al., 2009). one of the most consistent effects of ba, ga, and ba plus ga combinations is also to increase firmness in fruit crops (usenik et al., 2005; stern et al., 2007; canli and orhan, 2009). the pre-harvest treatments of ga3 increased fruit firmness in cherry (kappel and macdonald, 2002; usenik et al., 2005; canli and orhan, 2009). when compared with untreated control fruit, most of the ba and ba + ga4+7 treatments increased the ssc of fruit except the 12.5 mg·l-1 ba + ga4+7 and 100 mg·l-1 ba treatments (tab. 2). an increase in ssc as a response to ga application was also reported by other researchers (basak et al., 1998; lenehan et al., 2006). however, the responses to ga application were complex and variable and there were not always changes in ssc (facteau et al., 1985b; kappel and macdonald, 2002; horvitz et al., 2003). when fruits were evaluated for quality parameters after the cold storage period, the firmness of the fruits treated with 25 and 50 mg·l-1 ba + ga4+7 and 50 and 150 mg·l-1 ba were still significantly higher than the untreated control fruit. similarly, ga3 treated cherry fruit maintained a superior firmness to control fruit during cold storage (clayton et al., 2006; ozkaya et al., 2006). ga3 sprays also reduced pedicels browning in cold stored cherries (ozkaya et al., 2006). surface pitting devaluated the appearance of the cherry fruit, reflecting irregular formed sunken areas (clayton and biasi, 2003). no significant differences were observed between control and pgr treated fruit with respect to surface pitting (tab. 3). on the other hand, cherries treated with 100 and 150 mg·l-1 ba were significantly less susceptible to pitting than 12.5 and 50 mg·l-1 ba + ga4+7 and 50 mg·l-1 ba treatments. in addition, when compared to untreated control fruit, the pre-harvest application of 100 mg·l-1 ba reduced surface pitting disorder of ‘0900 ziraat’ cherry about 5 % after 4 weeks of cold storage; but the difference was not statistically significant (tab. 3). ga3 treatments improved resistance to surface pitting disorder in cherry (einhorn et al., 2013). similarly, pre-harvest ga3 applications reduced the pitting in cold-stored ‘van’, but had no effects on surface pitting in ‘lambert’ cherries (loney and lidster, 1980). uncertainty continues regarding the effects of gas on pitting of cherries. to the best of our knowledge, this is the first report on the effects of ba on postharvest surface pitting of cherries. at the end of the cold storage period, ssc content of 50 mg·l-1 ba + ga4+7 -treated fruit was still significantly higher than that of control fruit. in general, most of the treated fruit had superior firmness and quality than control fruit at the end of the cold storage period. similarly, pear fruits with high pre-cold storage period firmness were confirmed to have longer postharvest life and higher postharvest quality (calvo and sozzi, 2004; yehia and hassan, 2005). conclusion a single pre-harvest application of 50 mg·l-1 ba + ga4+7 increased the size and firmness of ‘0900 ziraat’ cherry. the ba alone or in combination with ga4+7 treatments showed a good potential for imtab. 2: effects of pre-harvest benzyladenine (ba) and ba + gibberellin (ga4+7) applications on firmness, soluble solids concentration (ssc) and ph of ‘0900 ziraat’ sweet cherry fruit (n=120)a. treatment concentration firmness ssc ph ( mg·l–1) (n) (%) control 0 9.30 cb 17.22 c 3.75667 ba + ga4+7 12.5 9.96 ab 16.57 d 3.81267 ba + ga4+7 25 9.70 b 17.99 b 3.76455 ba + ga4+7 50 10.14 a 19.57 a 3.77467 ba 50 9.91 a 18.51 b 3.78800 ba 100 10.05 a 17.00 cd 3.74933 ba 150 10.05 a 18.35 b 3.75000 atrees were treated at straw-yellow color stage of the fruit. the control spray was composed of water and tween-20. bmeans within a column followed by different letters are significantly different at p ≤ 0.05 by the tukey’s hsd test. tab. 3: effects of benzyladenine (ba) and ba + gibberellins (ga4+7) applications on firmness, soluble solids content (ssc), and surface pitting of ‘0900 ziraat’ sweet cherry fruit after cold storage (n=120)a. treatment concentration firmness ssc surface ( mg·l–1) (lbf)13 (%) pitting (%) control 0 1.54 d b 17.04 bc 17.29 ab ba + ga4+7 12.5 1.59 cd 16.28 d 24.25 a ba + ga4+7 25 1.74 a 17.17 bc 21.86 ab ba + ga4+7 50 1.70 ab 18.53 a 24.52 a ba 50 1.68 abc 17.62 b 23.56 a ba 100 1.63 bcd 16.68 cd 12.20 b ba 150 1.75 a 16.92 c 15.96 b atrees were treated at straw-yellow color stage of the fruit. the control spray was composed of water and tween-20. bmeans within a column followed by different letters are significantly different at p ≤ 0.05 by the tukey’s hsd test. 258 f.a. canli, m. sahin, s. ercisli, o. yilmaz, n. temurtas, m. pektas proving sweet cherry fruit storability by maintaining fruit firmness during the cold storage. these results are the first report on the effects of ba and ba + ga4+7 on fruit quality and shelf life of cherry and particularly useful for sweet cherry due to the relatively low application costs of the pre-harvest pgr treatments. applications that led to high level of fruit firmness demonstrated to be more promising to improve postharvest life and quality. references basak, a., rozpara, e., grzyb, z., 1998: use of bioregulators to reduce sweet cherry tree growth and to improve fruit quality. acta hortic. 468, 719-723. bacvonkralj, 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(eds.), cherries: crop physiology, production and users, 279-298. cab international, wallingford, uk.. ozkaya, o., dundar, o., kuden, a., 2006: effect of preharvest gibberellic acid treatments on postharvest quality of sweet cherry. j. food agric. environ. 4, 189-191. rop, o., ercisli, s., mlcek, j., jurikova, t., hoza, i., 2014: antioxidant and radical scavenging activities in fruits of 6 sea buckthorn (hippophae rhamnoides l.) cultivars. turk. j. agric. for. 38, 224-232. stern, a.r., doron, i., ben-arie, r., 2007: plant growth regulators increase the fruit size of ‘spadona’ and ‘coscia’ pears pyrus communis in a warm climate. j. hortic. sci. biotechnol. 82, 3-7. usenik, v., kastelec, d., stampar, f., 2005: physicochemical changes of sweet cherry fruits related to application of gibberellic acid. food chem. 90, 663-671. webster, a.d., spencer, j.e., dover, c., atkinson, c.j., 2006: the influence of sprays of gibberellic acid (ga3) and aminoethoxyvinylglycine (avg) on fruit abscission, fruit ripening and quality of two sweet cherry cultivars. acta hortic. 727, 467-472. whiting, m.d, ophardt, d., 2005: comparing novel sweet cherry crop load management strategies. hortscience 40, 1271-1275. yehia, a.t., hassan, h.s.a., 2005: effect of some chemical treatments on fruiting of “le conte” pears. j. appl. sci. res. 1, 35-42. zhang, c., whiting, m.d., 2011a: improving ‘bing’ sweet cherry fruit quality with plant growth regulators. sci. hortic. 127, 341-346. zhang, c., whiting, m.d., 2011b: pre-harvest foliar application of prohexadione-ca and gibberellins modify canopy source-sink relations and improve quality and shelf-life of ‘bing’ sweet cherry. plant growth regul. 65, 145-156. address of the corresponding author: e-mail: sercisli@gmail.com © the author(s) 2015. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 90, 52 57 (2017), doi:10.5073/jabfq.2017.090.008 1dep. of field crops, faculty of agriculture, ege univ., bornova, i̇zmir, turkey 2 directorate of plant protection research institute, bornova, i̇zmir, turkey 3 dep. of plant protection, faculty of agriculture, ege univ., bornova, i̇zmir, turkey 4 genomic services australia, darley circle bull creek wa, australia identification of resistance to eurygaster integriceps put. on some bread wheat genotypes fatma aykut tonk1,5, ekrem kaya2, deniz i̇ştipliler1, emre i̇lker1, ferit turanlı3, muzaffer tosun1, erkan yılmaz2, firdevs ersin3, ebru savran takak3, mehmet çakır4 (received may 30, 2016) * corresponding author summary sunn pest (eurygaster integriceps put.), is one of the most important pests of wheat in eastern europe including turkey, west and central asia. its damage on leaves, stems, spikes and grains reduce the baking quality of flour made from damaged grains. in this study, some wheat genotypes from turkey and icarda were evaluated for the pest resistance. the genotypes were planted in a randomized block design using hill plots in nylon mesh screening cages in wheat growing season of 2011-2012 and 2012-2013. sunn pest population was collected from çanakkale province, where the pest was intensely found in recent years. the plants of each hill plots were infested with one male and one female sunn pest adults. the results with 12.5 % sucking damage showed that the genotypes from icarda had higher resistance than the landraces from turkey to sunn pest. especially, the genotypes ic3 and ic4 from icarda and tr7 from turkey with respect to their sed and dsed values were found the most promising genotypes resistant to sunn pest for future breeding programs. abbreviations icarda, international center for agricultural research in the dry areas; nss, number of sucking symptoms; dr, damage ratio; tgw, thousand grain weight; sed, sedimentation; dsed, delayed sedimentation; ipm, integrated pest management; anova, analysis of variance; dgw, damaged grain weight; udgw, undamaged grain weight keywords sunn pest, bread wheat, resistance, sedimentation, delayed sedimentation introduction sunn pest (eurygaster integriceps put., hemiptera; scutelleridae), is one of the most important pest of wheat in eastern europe including turkey, west and central asia. sunn pest adults overwinter in mountains under the plants such as oak, wild liquorice and echinacea, and they migrate to wheat fields when it gets warm in spring. it can cause damage on leaves, stems, spikes and grains (lodos, 1961, 1982; el bouhssını et al., 2007; trıssı et al., 2006; popov et al., 1996). typical feeding behavior of sunn pest is piercing and cutting tissues and injecting digestive enzymes through salivary canal to predigest food. after predigestion, food is ingested through the alimentary channel for further digestion (cohen, 2000; boyd et al., 2002). during the predigestion, the insect injects an enzyme complex into the grains, which reduces the baking quality of flour made from damaged grains. the damage of the pest causes break down of gluten rapidly by powerful proteolytic enzymes. if the insects feed on as little as 2 to 5 % of the grain, the entire lot may be rendered unacceptable for baking purposes due to the baking quality of the flour being damaged (lodos, 1982; harırı et al., 2000). sunn pest has been one of the most harmful pests in turkey since 1927. a serious outbreak occurred during 1987-89 in the thrace region and thousands of hectares of wheat were damaged. the wheat growing areas in the central anatolian region infested by sunn pest were stated 0.3 million ha and the cost of chemical treatment was about 2 million dollars in 1996 (anonymous, 1996; kınacı et al., 1998). in 2014, sunn pest management was carried out in 6.460.213 ha of wheat production area of turkey. the cost of this management in 22 provinces of turkey was 1.7 million dollars (özkan and babaroğlu, 2015). it is also estimated that a total of 40 million dollars is spent per year for sunn pest control in turkey and neighbors syria, iran and iraq (javahery, 1995). the government of turkey has extensively supported the wheat producers to use the chemical control programs since 1928 but this support was terminated in 2008. currently, chemical control is still continued in certain areas of the marmara, the southeast and the central anatolian regions. as a result of these chemical applications, the biological control agents that could control the pest were negatively affected. nowadays, integrated pest management (ipm) programs have been implemented to control this pest especially in the southeast anatolia and other regions where the pest is a major problem (kınacı et al., 1998; el bouhssını et al., 2007; koçak and kılınçer, 2002; kıvan, 2005; canhılal et al., 2007; keçecı et al., 2007; el bouhssını et al., 2009; gözüaçık et al., 2010). the resistance studies, which are extremely safe in terms of human health and environment, and constitute an important part of ipm strategies, are carried out against sunn pest in both turkey and many other countries of the world. the development of varieties that show moderate to high resistance should be seriously considered because it can reduce chemical applications. besides, certain studies have suggested that high quality bread wheat cultivars were more resistant to the damaging effects of insect proteinase than low quality cultivars (every et al., 1997; najafı mırak et al., 2008). durıc et al. (2014), reported the reduction in quality traits (protein content, sedimentation value, modified sedimentation value, wet gluten content and energy test) in accordance to the number of infested grains. a number of cultivated wheat and wild relatives have been identified as being sources of resistance to this pest at the vegetative stage at icarda (el bouhssını et al., 2007; el bouhssını et al., 2009). some of these resistance accessions were stated to originate from afghanistan and tajikistan (el bouhssını et al., 2009). identification of resistant genotypes and using them in wheat production or transferring resistance genes to cultivated genotypes and minimizing the chemical applications are effective and economically important control strategies against sunn pest. the aims of this study were firstly to determine whether there were any differences between the infested and the non-infested sunn pest cages in terms of observed wheat traits, and secondly to identify sources of resistance to eurygaster integriceps in wheat 53 tab. 2: average monthly rainfall (mm) and temperature (ºc) in 2011-12 and 2012-13 growing seasons of wheat in the experimental site climate years november december january february march april may june total and average 2011-12 0.0 140.5 127.7 128.2 34.7 105.0 86.6 19.9 642.6 2012-13 56.9 218.2 252.5 187.0 56.8 30.2 43.7 27.1 872.4 lya 109.7 137.9 112.2 99.7 82.9 46.4 25.4 7.5 621.7 2011-12 11.1 10.7 6.8 7.6 11.3 17.5 20.5 27.3 14.1 2012-13 16.4 10.7 9.4 11.2 14.0 17.3 22.7 25.7 15.9 lya 13.8 10.5 9.0 9.2 11.8 16.1 21.0 26.0 14.7 lya: long year average resistance against sunn pest in bread wheat landraces of turkey and a set of international wheat lines with known resistance to the pest biotype in syria from international center for agricultural research in the dry areas (icarda) under artificial infestation. materials and methods plant material and field screening seven bread wheat landraces from sunn pest prevalent areas of turkey and four wheat lines from icarda, which were identified as resistant to sunn pest (el bouhssını et al., 2007; el bouhssını et al., 2009), were used for resistance screening (tab. 1). the pest population was collected from çanakkale province located at northwest part of turkey, where the pest intensely caused damage to wheat crop in recent years. in the first year it should be determined whether sunn pest causes any notable damage to the bread wheat genotypes in the infested nylon mesh screening cages compared to the control cage. therefore, four landraces originated from the central and east anatolia were obtained from the national gene bank in izmir, turkey and seven resistant lines from icarda were planted in a randomized block design in wheat growing season of 2011-2012. the experiment was conducted with three infested and one control cages. in 2012-2013 wheat growing season, the same genotypes were planted in three infested cages without control cage in order to identify the responses of the wheat genotypes to sunn pest. in both seasons, the genotypes were grown under natural rainfall conditions. the average monthly rainfall and temperature during 2011-2012 and 2012-2013 growing seasons of wheat are shown in tab. 2. in both growing seasons, the genotypes were planted using hill plots. every hill contained 15 seeds and the distance among the hills was 50 cm in each cage. the genotypes were infested with the pest in three cages only for the first year, one cage was allocated for the control having no pest in field crops department of faculty of agriculture, ege university in i̇zmir, turkey. the sizes of the mesh screening cages were 2 by 2.2 meters with height of 2.5 meters. insecticide application (alphacypermethrin, 100 g/l) was done to clean up the cages from other pests thirty days before infestation of sunn pest for all cages. the plants of each hill plot were infested with two adults (one male and one female) at the time of the insects’ migration to wheat fields in april, and allowed to feed on the plants. the sucking damage ratio was predicted by number of sunn pest adult/m2 (kılıç, 1988; özkan and babaroğlu, 2015). measured traits before harvest, each of the experimental cages were sprayed with dimethoate (400 g/l) to kill the remaining sunn pest and the hill plots were separately harvested. one hundred kernels representing each hill plot were randomly selected and checked under magnifying glass; damaged and undamaged kernels were separated and weighted then recorded as damaged grain weight (dgw) and undamaged grain weight (udgw), respectively. the feeding punctures on damaged kernels for each genotype caused by sunn pest were counted and recorded as number of sucking symptoms (nss). kernels with at least one puncture site were considered as damaged grain. to calculate thousand grain weight (tgw), one hundred kernels were counted four times, weighted and average value was multiplied with ten for each genotype. the damage ratio (dr) was calculated with the following formula: dr (%) = dgw/(dgw+udgw)*100 gluten quality was analyzed in flour sample for each genotype using the zeleny sedimentation (sed) (aacc, 2000) and delayed zeleny sedimentation (dsed) test (greenaway et al., 1965). swelling of the gluten fraction of flour in lactic acid solution affects the rate of sedimentation of a flour suspension in the lactic acid medium. for two parallel analyses, 3.2 × 2 = 6.4 g of weighted flour samples previously milled in a suitable laboratory mill were placed in a graduated cylinder, the reagents were added and mixed thoroughly. after mixing with all reagents, the cylinder was left to stand for exactly 5 min and the volume of sediment was read. delayed sedimentation values were recorded after two hours. the expected damage ratio was calculated according to technıcal ınstructıon of agrıcultural protectıon (2008). statistical analyses a paired t-test was used to compare the mean of the infested and the control cages for each trait in the first year of the study. the analysis tab. 1: origin of wheat genotypes used in the study no. ic/tr no. entry name type origin 1 ic1 icbw209272 t. aestivum afghanistan 2 ic2 ig139883 t. aestivum afghanistan 3 ic3 ig139814 t. aestivum afghanistan 4 ic4 ig139753 t. aestivum afghanistan 5 tr1 tr 46774 t. aestivum turkey 6 tr2 tr 38895 t. aestivum turkey 7 tr3 tr 62808 t. aestivum turkey 8 tr4 tr 63314 t. aestivum turkey 9 tr5 tr 63410 t. aestivum turkey 10 tr6 tr 46826 t. aestivum turkey 11 tr7 tr 56095 t. aestivum turkey rain distribution (mm) average temperature (°c) 54 f. aykut tonk, e. kaya, d. i̇ştipliler, e. i̇lker, f. turanlı, m. tosun, e. yılmaz, f. ersin, e. savran takak, m. çakır of variance (anova) was performed using totemstat software (açıkgöz et al., 2004) for all measured traits with combining the average data of the genotypes obtained from each infested cage in two years. probability was accepted at the p ≤ 0.01 and p ≤ 0.05 levels. the means were compared using fisher’s least significant differences (lsd) at the p ≤ 0.05 level. results sunn pest population density was calculated as 5 overwintered adults/ m2 in infested cages (2 adults * 11 hill plots = 22 adults for 4.4 m2). the expected damage ratio was equal to 12.5 % sucking damage in each of the infested cages. the results from the control and the infested cages were found statistically different for all measured traits except dsed in 20112012 growing season (tab. 3). unexpectedly, some grains with sucking symptoms were observed in the control cage. this damage could be the result of the insects such as stink bugs at low population level remaining after insecticide spraying in the control cage. the highest t value was found for the dr and the cages significantly differed for this trait. the tgw in the control cage were higher than the mean of infested cages and according to t test, the difference between them was statistically significant. although both of the sed and dsed values were higher in the control cage than the mean of infested cages, the genotypes in the infested and the control cages did not statistically differed for dsed (tab. 3). all these findings indicated that this population level of sunn pest significantly damaged the wheat plants and therefore this level of damage through infestation would be suitable to differentiate the wheat genotypes for response to sunn pest. the anova showed that the differences between the years were found significantly important for all traits except nss and dr (tab. 4). also, the differences among the genotypes were significant for tgw, sed and dsed. however, genotype × year interaction was significant for only dsed trait (tab. 4). in the infested cages, the values of the genotypes in each year and the mean of two years for the measured traits are separately shown in fig. 1a-e. the nss values of the genotypes were higher in 20112012 than 2012-2013. in both years, the nss of ic genotypes were lower than tr genotypes. for the nss, the genotypes tr6 (23.27 %) and ic3 (25.93 %) had the lowest values while tr3 (56.23 %) had the highest nss for two years mean (fig. 1a). in parallel with the nss, the dr values of the genotypes were found higher in the first year than the second year. the higher dr values of tr genotypes than the ic genotypes could be seen especially from the first year results (fig. 1b). the lowest dr mean measured was for tr6 (15.60 %) and ic3 (16.10 %) while the highest dr obtained for tr5 (31.13 %) (fig. 1b). when the tgw of the genotypes was analyzed, the first year values of all genotypes were higher. the tgw of ic genotypes had higher values than tr genotypes. the highest and the lowest mean values were observed for ic2 (48.11 g) and tr5 (30.04 g), respectively (fig. 1c). the sed of all genotypes are viewed in fig. 1d. in contrast to nss, dr and tgw, the sed values of all genotypes in 2011-2012 were detected higher than 2012-2013. the sed of ic genotypes had higher values than tr genotypes. the highest sed values were measured for ic1 (21.54 ml) and ic3 (23.66 ml) while the lowest value was obtained from tr7 (11.66 ml) (fig. 1d). unlike the other traits, the mean square of genotype × year interaction was found statistically significant for the dsed (tab. 4). therefore, the genotypes were grouped separately for each year. while, all genotypes were placed in the same group during first year, they were classified in different groups in the second year of the study. in the second year, ic4 (16.00 ml) and tr7 (15.00 ml) had the highest dsed values whereas tr1 (6.50 ml) and tr5 (6.33 ml) had the lowest values (fig. 1e). the obtained results revealed that the nss, dr and tgw values of the genotypes in 2011-2012 were higher than those of 2012-2013. therefore, the sed and dsed values of the genotypes in 2011-2012 were lower compare to 2012-2013 (fig. 1a-e). the mean tgw, sed and dsed of ic genotypes were identified more than tr genotypes. in contrast, the nss and dr of tr genotypes were higher than ic genotypes. the mean of nss value were detected as 32.59 % for ic genotypes and 42.65 % for tr genotypes. the dr means were 20.64 % and 25.15 % for ic and tr genotypes, respectively. the mean of sed value were found 19.60 ml for ic and 14.37 ml for tr genotypes. the mean of dsed values obtained from the ic and tr genotypes were 8.92 ml and 7.42 ml, respectively (fig. 1f). discussion a number of studies on resistance screening against sunn pest were conducted on vegetative stage of wheat genotypes. seven accessions originated from afghanistan and tajikistan with this kind of retab. 3: results of t test for the genotypes in the control and the infested cages in 2011-2012 traits infested cage control cage t value nss (%) 55.28 6.00 8.35** dr (%) 31.14 4.47 9.16** tgw (g) 50.07 53.28 -2.08* sed (ml) 11.43 15.72 -3.21** dsed (ml) 5.56 7.63 -1.34 *, ** : significant at p ≤ 0.05 and p ≤ 0.01, respectively. nss: number of sucking symptoms, dr: damage ratio, tgw: thousand grain weight, sed: sedimentation, dsed: delayed sedimentation. tab. 4: analysis of variance for measured traits mean of squares df nss (%) dr (%) tgw (g) sed (ml) dsed (ml) year 1 21318.8 4718.00 7785.8** 1849.5** 420.3** genotype 10 860.9 1361.74 287.1** 107.6** 21.1** genotype × year 10 825.63 170.90 4.5 17.2 13.1* error 54 582.3 103.54 18.2 14.2 5.0 *, ** : significant at p ≤ 0.05 and p ≤ 0.01, respectively. nss: number of sucking symptoms, dr: damage ratio, tgw: thousand grain weight, sed: sedimentation, dsed: delayed sedimentation. source of variation resistance to eurygaster integriceps in wheat 55 fig. 1: effects of sunn pest on the bread wheat genotypes in 2011-2012 and 2012-2013 growing seasons: (a) nss, number of sucking symptoms, (b) dr, damage ratio, (c) tgw, thousand grain weight, (d) sed, sedimentation, (e) dsed, delayed sedimentation, (f) the mean values of two years for ic and tr genotypes. (a) (b) (c) (d) (e) (f) 56 f. aykut tonk, e. kaya, d. i̇ştipliler, e. i̇lker, f. turanlı, m. tosun, e. yılmaz, f. ersin, e. savran takak, m. çakır sistance reported by el bouhssını et al. (2009). however, trıssı et al. (2006) emphasized that feeding by sunn pest at vegetative stage does not affect the bread-making quality of the grain in wheat. therefore, economic damage of sunn pest actually occurs in generative stage of wheat plant. grain damage of sunn pest at generative stage in wheat was investigated in a few studies in turkey and other countries (kınacı et al., 1998; dızlek and i̇slamoğlu, 2010; durıc et al., 2014). in accordance with previous studies, at generative stage of wheat five sunn pest per m2 infestation level resulted in significant differences for all traits except dsed between the infested and the control cages. trıssı et al. (2006) stated that protein, sedimentation and damaged kernel values for the infested plot at six sunn pest individuals per m2 density were significantly lower than those of the control wheat plot. the reason of the absence of significance for dsed is that in contrast to the tr genotypes, the dsed values of the ic genotypes did not varied between the infested and the control cages. for the sed and dsed traits, the variation between the infested and the control cages can be attributed to the effect of proteolytic enzyme secreted by sunn pest, which caused significant reduction in gluten level in damaged grains (lodos, 1982; harırı et al., 2000). in turkey, 2 % sunn pest sucking damage ratio was determined as maximum limit in wheat commodity (özkan and babaroğlu, 2015). according to kılıç (1988), the economic threshold for sunn pest control is 10 nymphs/m2 and 0.8 overwintered adults/m2 (özkan and babaroğlu, 2015). in this study determining the resistant genotypes, the sucking damage ratio (12.5 %) obtained was higher than the economic threshold level because of high population density (5 sunn pest adults/m2). similar to our results, kahraman et al. (2011) reported the highest rate of sucking damage as 11.7 % in greenhouse and the lowest as 2.3 % in the field. due to a very high pest pressure, the means of nss and dr values were found very high in both years (fig. 1a). the genotypes from icarda, which were reported as resistant against sunn pest at vegetative stage (el bouhssını et al., 2007; el bouhssını et al., 2009), appeared to be more resistant to sunn pest compared to the tr genotypes at generative stage of wheat (fig. 1f). especially the genotypes ic3 and ic4 with noticeably lower mean values for nss and dr draw the attention and showed higher mean values for sed and dsed. however, amongst tr genotypes, the genotype tr6 did not show higher values for sed and dsed although nss and dr values were lower like in ic genotypes (fig. 1a-e). the reason of these differences could be due to a lack of gluten quality or the number of gluten allele in these two genotypes. fatehı et al. (2008) investigated the correlation of hmw subunits and the percent of damaged seed by sunn pest and reported that 7+8 and 2+12 alleles have significant positive correlation while 7+9 and 12 alleles have significant negative correlation with the percent damaged seed. in the present study, the sed values of the genotypes were generally higher in less damaged kernels, which is consistent with the results of previous studies (karababa and ozan, 1998; dızlek and i̇slamoğlu, 2010). similarly, canhılal et al. (2005) revealed a strong negative relation between sedimentation values and percentage of damaged kernels. the nss, dr and tgw values of the genotypes in the year 20112012 were higher compared to year 2012-2013. these differences can be explained with the average amount of monthly rainfall in april and may of two years (tab. 2). the april and may received the average rainfall of 105.0 and 86.6 ml, respectively in 2012, and 30.2 and 43.7 ml, respectively in 2013 in experimental area. the higher rainfall of these two months in 2012 may extended the milk stage due to the longer grain filling period. as a result, the tgw values of all genotypes were higher in this year (fig. 1c). the extending milk stage caused more sucking damage on grain by the 2-5th stage nymphs and new generation of adults of sunn pest on wheat (özkan and babaroğlu, 2015). more sucking damage contributed in increasing the number of damaged kernel (nss) and therefore the percentage of damaged kernel weight in the total grain weight (dr) rose during the first year. however, the reduction in the amount of gluten in the damaged grains, because of proteolytic enzyme of sunn pest, caused the decrease in the sed and dsed values of the genotypes. in case of nss and as a result dr, the values for some genotypes e.g. tr1 differed significantly during two years. it must be because of the differences between the numbers of damaged grain by sunn pest in two years. the kernels with at least one puncture site were considered as damaged grain. therefore, the pest damaged more kernels of the genotypes in the first year compared to the second year because of the longer flowering and grain filling period. in contrast, the pest damaged fewer kernels but with more puncture sites during second year due to shorter flowering and grain filling period. besides, the damage by sunn pest in wheat kernel is determined by the sed and dsed test (dızlek and ıslamoglu, 2015). also it has been emphasized that the higher the difference between the values of sed and dsed, the higher the damage caused by sunn pest will be (elgun et al. 1998; ozkaya and ozkaya, 2005; dızlek and ıslamoglu, 2015). in conclusion, the extension of the flowering and grain filling period can fairly increase the damage of sunn pest at generative stage in wheat. therefore, the resistance of wheat genotypes to sunn pest may vary among years. the ic genotypes, known resistant to sunn pest in the vegetative period, also exhibited resistance in the generative period of wheat. in general, these genotypes were identified to be more resistant than the tr genotypes originated from sunn pest prevalent areas of turkey. the genotypes ic3 and ic4 from icarda and tr7 with noticeable values for dsed can be a promising source of germplasm for resistance to sunn pest in next breeding programs. the resistance sources identified in the study provides opportunities towards establishing a base to a much broader study of understanding genetic and physiological bases of resistance against sunn pest in wheat. furthermore, the genotypes identified here can be used by plant breeders for the introgression of resistance genes into susceptible wheat cultivars. acknowledgments we would like to thank dr. mustapha el bouhssini from icarda (international center for agricultural research in the dry areas) for providing seeds of resistant lines. references aacc, 2000: approved methods of the american association of cereal chemists, method 38-10.01, method 38-12.02, method 56-60.01. 10th ed., the association, st. paul. açıkgöz, n., i̇lker, e., gökçöl, a., 2004: computer evaluations of biological research. ege university press, totem, no: 2, izmir, turkey. anonymous, 1996: sunn pest control, agriculture and village. ministry of agriculture and rural affairs, publication no. 106 (turkish). boyd, d.w., cohen, a.c., alverson, d.r., 2002: digestive enzymes and stylet morphology of deraeocoris nebulosus (hemiptera: miridae), a predacious plant bug. ann. entomol. soc. am. 95, 395-401. canhılal, r., kütük, h., kanat, a.d., i̇slamoğlu, m., el-harameın, f., el-bouhssını, m., 2005: economic threshold for the sunn pest, eurygaster integriceps put. 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(in turkish, with english summary). popov, c., barbulescu, a., vonıca, i., 1996: population dynamics and management of sunn pest in romania. in: miller, r.h., morse, j.g. (eds.), sunn pest and their control in the near east. fao plant product. and protec. paper 138, 47-59. technıcal ınstructıon of agrıcultural protectıon, 2008: tarım ve köyişleri bakanlığı, tarımsal araştırmalar genel müdürlüğü. i. cilt hububat. başak matbaacılık, 282, ankara (in turkish). trıssı, a.n., el-bouhssını, m., i̇brahem, j., abdulhaı, m., parker, b.l., reıd, w., el-harameın, f.j., 2006: effect of egg parasitoid density on the population suppression of sunn pest, eurygaster integriceps (hemiptera; scutelleridoe), and its resulting impact on bread wheat grain quality. j. pest sci. 79, 83-87. doi: 10.1007/s10340-005-0116-3. address of the corresponding author: e-mail: fatma.aykut@ege.edu.tr © the author(s) 2017. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 88, 170 176 (2015), doi:10.5073/jabfq.2015.088.024 1 erciyes university, faculty of engineering, department of food engineering, kayseri, turkey 2 yildiz technical university, faculty of chemical and metallurgical engineering, department of food engineering, istanbul, turkey natural food colorants and bioactive extracts from some edible flowers okan bayram1, osman sagdic2, lutfiye ekici1* (received june 6, 2014) * corresponding author summary consumers’ interest in natural coloring has been growing in parallel with their consciousness of food-health relationship. anthocyanin based colorings bear antioxidative features which makes this group of colorings attractive. in this research, heat stabilizations and some bioactive properties of anthocyanin-based extracts (abe) obtained from corn poppy, tulip, rose and roselle, were determined. while the highest amount of phenolic substance was determined in the tulip (113.8 mg gallic acid equivalents (gae) g-1dry extract), the highest amount of anthocyanin is in the corn poppy (405.2 mg cyanidin3-glucoside g-1 dry extract). among the extracts, the corn poppy has been determined to have the highest antiradical capacity with 55.9 μg ml-1, and the tulip, with a level of 63.4 mg of ascorbic acid equivalent (aae) g-1 dry extract, has been determined to have the highest antioxidant activity. while there has been no antimicrobial effect of corn poppy extract observed on any microorganism, roselle extracts have been found to display high antimicrobial activity. heat stability of abes was investigated in buffer solution ph 3.5. the flowers have high bioactive activities. introduction edible flowers originate from a wide variety of plants different in form, color and flavor. such flowers are used in many countries to enhance the color, appearance and nutritive value of foods (kilic and sahin, 2009).the petals of some flowers are used in the production of salads, desserts and ice cream as well as in food decoration. for instance, marigold (calendula officinalis), crocus (crocus sativus), violet (viola odorata) and dandelion (taraxacum officinale) are used in certain drinks and salads (mlcek and rop, 2011). the most common edible flowers in turkey are saffron (crocus sativus), rose (rosa damascena), lavender (lavandula angustifolia), violet (viola odorato), roselle (hibiscus sabdariffa),verbena (verbena officinalis), corn poppy (papaver rhoeas), pumpkin (curcubita pepo) and borage (borago officinalis) (kilic and sahin, 2009). in addition to their beauty, recent studies on their nutritive quality are an important factor in increasing flower consumption in food sector (mlcek and rop, 2011). color, one of the most important organoleptic features of edible plants, varies depending on the carotenoid and anthocyanin content (friedman et al., 2007). anthocyanins, a subgroup of flavonoids, gives flowers and fruits colors varying from red to blue (longo and vasapollo, 2006; ekici, 2011). natural or synthetic colors have been in use for centuries to render the color, one of the most important visual features, more attractive. however, synthetic coloring is known to affect health adversely (kammerer et al., 2007). therefore, natural colorants and anthocyanin in particular have been used increasingly in food in recent years. this interest in the use of anthocyanin as a coloring agent results from their capability to make the color more appealing and their high solubility in water as well as their beneficial effects on health (ekici, 2011). taken up in this study are the bioactive characteristics of the petals of corn poppy (papaver rhoeas), tulip (tulipa gesneriana), rose (rose damascene) and roselle (hibiscus sabdariffa). corn poppy, the petals of which are used in making the traditional sorbet, is included in the family papaveraceae (ekici, 2014). it is effective on the intestinal and urinary system problems as well as with the common cold (kultur, 2007; zeybek and zeybek, 2002), bronchitis, diarrhea (soulimani et al., 2001). tulip, a member of the family liliaceae, has different varieties (tulip), two of which are endemic in turkey (mlcek and rop, 2011). the dark color tulips in particular are reported to have high antioxidant activity (sagdic et al., 2013). the rose is a sweet smelling plant of the rosa variety of the rosaceae family (shikov et al., 2012) and has more than 200 varieties with over 18.000 cultivar (gudin, 2000). it is known to possess antioxidative (kim et al., 2003; schieber et al., 2005; wang et al., 2006) and antibacterial properties (ozkan et al., 2004). roselle, rich in antioxidants and used for strengthening the immune system, is consumed by different cultures (chang et al., 2006). this study aims to determine (1) total phenolic, total anthocyanin, antiradical, antioxidant and antimicrobial properties of the flowers, and (2) thermal degradation kinetics of anthocyanin based extracts (abe) at 70, 80 and 90 °c. material and methods in this research the petals of corn poppy, red tulip, rose and roselle are studied. wildly growing corn poppies were picked in the botanical garden locality of erciyes university and their petals separated from their roots and stems. while using poppy’s petals, the black part of petal which is close to the capsule of flower, an alkaloid called ‘thebaine’ should be seperated. after the removal the black sections on the petals, the washed samples were dried at ambient temperature. red tulip and rose were obtained from istanbul nursery and landscaping corporation. red tulip and roses were also harvested, washed with tap water and dried at ambient temperature. roselle, however, was dry when obtained from the kayseri market. having been dried at ambient temperature, the petals were put into colored nylon bags and stored at room temperature until extraction. extraction of abe extraction has been carried out as described by ersus and yurdagel (2007). a mixture of ethanol (merck, darmstadt, germany) and pure water (1:1) acidified with 0.01 % hydrochloric acid (hcl, merck, darmstadt, germany) was used as solvent for the extraction of anthocyanin pigments. the sample-solvent ratio in the extraction process was set to be 1:19 (weight/volume). the samples were extracted for 2 hours at 35 °c in a shaking-water bath, filtered and the extract was dried at 50 °c in a rotary evaporator (buchi, rotavapor r-200, switzerland). nitrogen gas was pressed on the dried extracts placed in tubes to prevent adverse effects by oxygen, and the extracts were kept at -18 °c until use (vestel ft 280, istanbul, turkey). natural food colorants and bioactive extracts from some edible flowers 171 determination of total phenolic content the total phenolic content (tpc) of abe’s has been determined using a modified version of the method developed by singleton and rossi (1965). briefly, 40 μl of sample were taken in test tubes. 2.4 ml of distilled water, 200 μl of folin-ciocalteu reagent, 600 μl sodium carbonate (20 %) and 760 μl distilled water were added respectively. the tubes were mixed and allowed to stand at the dark for 2 hours. the absorbance of the samples has been determined with spectrophotometer (varian cary 100 conc uv-visible, america) at 765 nm wavelength. the tpc of the samples were calculated as gallic acid equivalent (gae) g-1 dry extract. determination of total anthocyanin total anthocyanin content (tpc) of samples were determined by means of ph differential method (fuleki and francis, 1968). quantities of anthocyanin were determined in cyanidin-3-glucoside (mw=449.2, molar absorbance, ε=26.900) for corn poppy, rose and roselle, and in pelargonidin-3glucoside (mw=433.2, molar absorbance, ε=22.400) for tulip materials. determination of antioxidant activity the antioxidant activity (aa) of abe’s has been determined at 695 nm according to the method by prieto et al. (1999). a sample amount of 0.4 ml was mixed with 4 ml of the reagent solution (0.6 m sulfuric acid, 28 mm sodium phosphate and 4 mm ammonium molybdate). the samples were incubated in a water bath at 95 °c for 90 min. for calculations, a series of standard solution has been prepared out of ascorbic acid within 0-1 mg ml-1 and, with the help of the curve obtained, the antioxidant activities of the samples was presented in mg ascorbic acid equivalent (aae) g-1 dry extract. determination of antiradical capacity the sentence “the antiradical capacities (ac) of abe’s have been determined with method of brand-williams et al. (1995).” was changed as “the antiradical capacities (ac) of abe’s have been determined with method of brand-williams et al. (1995).” briefly, abes were diluted with an ethanol-water (1:1) mixture. then 4000 μl 0.1 mm l-1 dpph (sigma st. louis, mo, america) were added to 200 μl of the sample. the absorbance levels of the samples, kept in the dark for 30 minutes, were measured at 517 nm. the ac of the samples have been calculated using the following equation, %i=100 × (1-as/ac) where %i stands for dpph inhibited by the sample, as: for the absorbance of the sample, ac : for the absorbance of the control. the amount of extract required for the inhibition of 50 % of dpph (μg ml-1) was calculated. determination of the antimicrobial activities of abes by agar diffusion method the antimicrobial activity of abes was determined using the method of agar diffusion (sagdic et al., 2003). in this study, 13 microorganisms containing 11 bacteria: staphylococcus aureus atcc 29213, staphylococcus aureus atcc 25923, enterobacter cloasea atcc 13047, salmonella typhimurium atcc 14028, escherichia coli atcc 23897, escherichia coli 0157:h7 atcc 33150, yersinia enterocolitica atcc 27729, pseudomonas aeruginosa atcc 27853, listeria monocytogenes atcc 7644, bacillus cereus atcc 33019, bacillus subtilis atcc 6630, and two yeasts: saccharomyces cerevisiae bc 5461 and candida albicans atcc 1223 were used. extracts with concentrations of 1, 2.5, 5 and 10 % were prepared with ethanol: water (without acid). for agar diffusion method, 4 small wells with 4 cm in diameter were drilled with a cork bore into fresh feeding locations on petri boxes kept in the fridge for an hour. then 40 ml of the extracts prepared in above-mentioned percentages were placed into the wells. y. enterocolitica and yeasts were incubated at 27 ºc for 18-24 hours and other microorganism at 35 ºc for the same time period. the diameter of the forming inhibition zones were measured in mm. determination of thermal stability of abes the effects of the heating process on the abes were determined in buffer solutions with a ph of 3.5 (ekici, 2011). abes containing 4 mg anthocyanin (poppy: 0.10 g, red tulip: 2.92 g; rose: 0.34 g and roselle: 0.38 g) were added to 100 ml of sodium citrate buffer solution prepared immediately before the analysis. the samples taken every 30 min beginning from the minute zero were subjected to either a 420 min (70 and 80 ºc) or 180 min (90 ºc) heating. for this purpose, 15 ml of samples in colored buffer solutions were placed in experimental tubes and immersed in a water bath (memmert wb-22, germany) adjusted to the respective temperature. the temperatures of the sample in the tube were measured, and the moment when it reached the desired temperature was taken as starting point. when reaching the respective time point, the samples were immediately cooled to ambient temperature, and tac analysis was completed. calculation of kinetic parameters it has been determined in previous studies that the degradation reactions are suitable for the kinetic model of the first degree (sagdic et al., 2013; cemeroglu et al., 1994). for this reason, kinetic coefficients have been determined using the equation no.2 derived by taking the integral to the differential equation no.1, which defines the reaction appropriate for the kinetics of the first degree (sagdic et al., 2013; kirca et al., 2004). -(dc/dt) = k c (1) ln (c/c0) = -k t (2) where c0 stands for the initial concentration of anthocyanin, c is the concentration of anthocyanin during the ‘t’ time, k is the constant for reaction rate, t time (in hour). the calculation of the constant rate the losses of anthocyanin for each temperature exerted was plotted on the axis “y” and the times on the axis “x” and a linear curve has been obtained in a graphic with a semi-logarithmic scale. linear regression analysis was applied to the curve to derive its equation. again, use was made of the slope of this curve and the equation no.3 use was taken into consideration for the calculation of reaction speed constant (sagdic et al., 2013; kirca et al., 2004). k = slope × 2.303 (3) calculation of activation energy dependency of the reaction on temperature was determined by calculating activation energy (ea) using the arrhenius equation (ekici, 2011; sagdic et al., 2013; kirca et al., 2004). k = k0×expea/rt (4) for the calculations the formula shown in equation no.5 derived by taking the logarithm of equation no.4 was used ln k = [(ea/r) × (1/t)]+ ln k0 (5) 172 o. bayram, o. sagdic, l. ekici for this purpose, the logarithm of the speed constants of reaction (k) was placed on the axis “y” of a graphic with arithmetic scale and the (kelvin) equivalent (1/t) of the temperatures were placed on the axis “x” and thus, a linear curve was derived. this curve called arrhenius graphic underwent regression analysis. activation energy was calculated by multiplying the slope of the curve obtained by the gas coefficients (sagdic et al., 2013; kirca et al., 2004). calculation of half-life (t1/2) half-life is the period of time needed for 50 % of the studied compound to get last, and it was calculated on the basis of the equation no.6 for reactions developing according to the kinetic model of the first degree (kirca et al., 2004). t1/2 = -ln (0.5) × k-1 (6) statistical analysis all the stages of the study were repeated. for the assessment of the data, one way and two way variance analyses, and the method of duncan multiple comparison was used. for analyses, use has been made of sas (version 8.2) statistical program (sas, 2000). results and discussion the yields of extracts obtained from edible flowers are given in tab. 1. the extracts yields in diminishing order are red tulip, roselle, rose and poppy. in tab. 1 the bioactive properties tpc, tac, aa and ac are also presented. the highest tpc was determined in red tulip (113.8 mg gae g-1 dry extract), followed by rose (43.1 mg gae g-1 dry extract) while the tpc of the poppy has been found to be (29.0 mg gae g-1 dry extract) and roselle (9.8 mg gae g-1 dry extract). conforti et al. (2009) reported that the poppy contains phenolic substance of 72 mg chlorogenic acid equivalent g-1 extract. in another study on the phenolic profile of the poppy its total phenolic content was found to be 31 mg 100 g-1 (trichopoulou et al., 2000). we determined the roselle tcp as 9.8 gae g-1 dry extract. mohdesa et al. (2010) have demonstrated that the total phenolic content of extract obtained from the petals of roselle (hibiscus sabdariffa l.) with 80 % methanol and water was 2.9 mg gaeg-1 dry extract and 1.9 mg gae g-1 dry extract (mohd-esa et al., 2010). the tpc of the red tulip has been found to be 113.8 mg gae g-1 dry extract. this value is consistent with the data in the literature as sagdic et al. (2013) have reported the tpc of the claret red, orange-red, pink, violet and yellow tulips to be 126.6, 113.8, 73.7, 80.5 and 2.6 mg gae g-1 dry extract, respectively. youwei et al. (2008) on the other hand, has found the tpc yellow and red tulips to be 0.5 mg g-1 catechin equivalent (ce) and 0.3 mg ce g-1, respectively (youwei et al., 2008). with a tac of 405.2 mg cyanidin-3-glucoside equivalents kg-1 dry extract, poppy extracts was found to have the richest content of anthocyanin among the species investigated. poppy extracts was followed by the rose, tulip and roselle with dry extracts of 322.6 mg cyanidin-3-glucoside equivalents kg-1 dry extract, 236.5 mg pelargonidin-3-glucoside equivalents kg-1 dry extract and 175.2 mg cyanidin-3-glucoside equivalents kg-1 dry extract, respectively. in a study on 5 different colored tulips, it has been detected that while yellow tulip does not contain anthocyanin, claret red, orange-red, pink and violet tulip have a combined anthocyanin ranging from 839.1 to 236.5 mg pelargonidin-3-glucoside equivalents kg-1 dry extract (sagdic et al., 2013). although tac of extracts obtained from edible flowers is within a wide range, it should be emphasized that this is an expected case. indeed, the anthocyanin compositions of raw materials and their bioactive contents are affected by variety, strain, maturity, the conditions in which they are raised and climate. in studies on different varieties, it is deemed natural that the secondary metabolites synthesized under genetic controls, namely phenolics, and accordingly their anthocyanin contents should be found different (ekici, 2011). aa of the abe have been determined through the method of phosphomolybdenum and the results were presented in tab. 1. with 63.4 aae g-1 dry extract, the red tulip has been determined to had the highest antioxidant activity in this study. as for the least antioxidant activity, it was roselle extract (20.0 aae g-1 dry extract). in general, an increase in antioxidant activities seems to depend on the increase in tpc (tab. 1). it is known that the antioxidant activities of phenolic compounds are proportional to the number of the hydroxyl group to be able to enter a reaction. the studies on the flavonoids containing hydroxyl in 3’, 4’ and 5’ positions in the ring b in different model systems revealed that they display a lot higher antioxidant activity then do their counterparts containing are hydroxyl only in the same ring (ruberto et al., 2007). as can be seen in tab. 1, with a dry extract level of 405.2 cyanidin-3-glucoside equivalents g-1 dry extract the poppy possesses the highest amount of anthocyanin followed by tulip and roselle. the amounts of extracts required for the abes to inactivate 50 % of dpph radicals appear in tab. 1 as ic50 (μg ml-1). since ic50 is the quantity of extract that provided inhibition by 50 %, the flowers with high inhibition possessed a lower ic50. the ic50 of poppy, which has the highest ac, was found to be 55.9 μg ml-1. conforti et al. (2009) found in their study that the ic50 of the poppy was 49.0 μg ml-1, which agrees with our findings. the results of antiradical analysis suggested that ac was not correlated with tpc. it was demonstrated that poppy extract, which had a relatively low tpc (29.0 mg gaeg-1 dry extract) among the samples, displayed the highest level of antiradical activity (55.9 μg ml-1). it was found that the ac of tulip extract, despite having a tpc of 113.8 mg gaeg-1 dry extract, was much lower (159.8 μg ml-1) than that of the poppy. the phenolic and dpph results may not be correlates (piljac-zegarac et al., 2009). additionally, it was argued it was not right to correlate ac to tpc only (makris et al., 2008). it was reported that the ic50 values of tulips in different colors were in the 62.46-159.8 μg ml-1 range. in the same study, antioxidant activities were found within the range of 23.9-48.7 mg aae g-1 dry extract (sagdic et al., 2013). the microbial activities of the abes of edible flowers the inhibitory effect of abes from poppy, tulip, rose and roselle, had inhibitory effects on a total of 13 different microorganisms, two tab. 1: yields and bioactive properties of the abes obtained from edible flowers poppy red tulip rose roselle tpc 29.0c±0.5 113.8a±0.3 43.1b±0.3 9.8d±0.6 tac 405.2a±1.3 236.5c±3.7 325.6b±2.3 175.2d±1.0 aa 39.1c±0.1 63.4b±1.0 53.6b±1.5 20.0d±0.0 ic50 55.9d±1.1 159.8b±2.8 103.2c±0.3 167.5a±2.1 yield (%) 40.9 58.7 44.2 45.7 tpc: total phenolic content (mg gaeg-1 dry extract), tac: total anthocyanin content (mg pelargonidin-3-glucoside equivalents kg-1 dry extract for red tulip, mg cyanidin-3-glucoside equivalents kg-1 for poppy, rose and roselle), aa: antioxidant activities (mg aae g-1 dry extract), ic50: antiradical capacity (μg ml-1), for each property; ab: means in different capital letters in the same row compare flower type and show significant differences at p< 0.05. natural food colorants and bioactive extracts from some edible flowers 173 of which were yeasts. results are presented in tab. 2. we found that the ethanol water (1:1) did not have any inhibitory effect on microorganisms. the antimicrobial activities of abes rose generally with increasing concentrations. none of abes showed antimicrobial effects on s. cerevisiae and c. albicans. similarly, sagdic et al. (2013) had reported that tulip extracts do not affect on yeasts. roselle was found to have the highest antimicrobial activity. in the current study, it was observed that the abes of the poppy flowers had no inhibitive effect on any microorganism, whereas the abes of red tulip flowers had inhibitive effects on bacteria only in concentra tions of 10 % (see tab. 2). rose extracts, however, have displayed inhibitive effect on all microorganisms except escherichia coli o157:h7 and escherichia coli. in general, the flowers with the exception of poppy, can be said to have high antimicrobial activities. in another study, the examination of tamarix gallica leaves and flowers had revealed that, in general, flower extracts exhibited high antimicrobial activity compared to leaf extracts. in the same study, while m. luteus was determined to be the most susceptible bacterium, e. coli was reported to be the most resistant bacterium (ksouri et al., 2009). koncic et al. (2010) had found that the diluted extracts of moltkia petraea had no antimicrobial effects on fungi (c. albicans and aspergillus niger), gram-positive (b. subtilis and s. aureus) and gram-negative (e. coli and p. aeruginosa) bacteria. the abes included in the study, were in general somewhat more effective in gram positive bacteria, which was in agreement with the data in the literature. it was reported, that phenolic compounds generally had higher antimicrobial effect on gram-positive bacteria (katalinic et al., 2010). the cell membranes’ polysaccharide structure of gram-negative bacteria were thought to be an important factor in these bacteria’s resistance to chemical stress. the reaction rate constants of abes in the buffer solution the degradation kinetics of the buffer solutions with a ph of 3.5 were determined following its coloring with different abes of 4 mg anthocyanin100 ml-1. results are shown in tab. 3 and fig. 1. it was reported that the degradation of anthocyanin depending on the temperature during the storage process was primarily fit for the reaction kinetics (sagdic et al., 2013; cemeroglu et al., 1994). the reaction rate constant of the poppy anthocyanins were 0.20 × 10-3, 0.60 × 10-3 and 1.10 × 10-3 min-1 for 70, 80 and 90 ºc, respectively. the group of anthocyanins in poppy was the most heat resistant among the investigated (tab. 3). the most degraded group when subjected to heating was roselle anthocyanin and its degradation rate constants for 70, 80 and 90 ºc were 1.84 × 10-3, 2.99 × 10-3 and 10.80 × 10-3 min-1, respectively. wang and xu (2007) reported that the degradation rate constants of blackberry juice for 70, 80 and 90 ºc were 1.32 ×10-3, 2.47 × 10-3 and 3.94 × 10-3 min-1, respectively. in this study, it was determined that poppy anthocyanin, in particular, possessed a much greater stability than those of blackberry, in contrast to roselle and rose, which posed weak stability. the increase in temperature shortened the half-life (t½) while increasing the degradation rate constant, which was consistent with the data in literature. in a multitude of studies it was observed that as k rises, depending on the increases in temperature, t½ diminished (sagdic et al., 2013; wang and xu, 2007; kirca et al., 2003). while t½ of blackberry for temperatures of 70, 80 and 90 ºc was 8.8, 4.7 and 2.9 hours, respectively, its activation energy was found to be 58.95 kj mol-1 (wang and xu, 2007). the t½ of sour cherry juice for 60, 70 and 80 ºc was reported to be 54.3, 22.5 and 8.1 hours (cemeroglu et al., 1994). in current study for the effect of temperature on the stability of anthocyanin to be determined, the activation energy tab. 2: antimicrobial activities of abes obtained from different flowers (inhibition zone diameter, mm) 10 9.8±1.4 16.2±3.0 9.0±1.8 14.8±3.1 7.2±0.5 20.8±2.6 12.3±2.8 9.1±2.4 7.3±0.2 5 2 1 10 14.0±0.7 14.0±0.0 18.0±1.4 18.3±15.5 12.8±0.5 14.0±0.0 13.8±1.5 37.3±4.2 14.0±0.8 5 12.0±0.7 11.0±0.0 12.2±0.5 15.5±0.6 9.3±0.5 11.5±1.4 9.8±1.3 28.3±1.9 10.3±1.5 2 10.0±0.7 6.8±1.0 9.8±1.5 12.3±0.5 7.5±1.0 9.5±0.7 7.0±0.0 25.3±2.1 9.0±1.2 1 8.0±0.7 5.0±0.0 7.0±0.0 10.8±0.5 6.5±2.4 7.0±0.0 3.5±0.6 22.0±0.0 7.0±1.4 10 15.0±1.2 17.0±0.8 14.0±1.2 19.8±1.0 15.3±1.0 16.5±1.0 18.5±1.0 23.5±1.0 16.0±1.2 20.3±0.0 14.3±1.3 5 11.5±0.6 14.3±0.5 10.0±0.0 17.8±1.5 12.0±1.4 9.0±2.0 15.8±1.3 17.0±1.2 12.5±0.6 15.8±0.7 10.3±1.0 2 8.5±0.6 10.0±1.4 7.5±1.0 13.3±1.9 7.8±0.5 5.5±0.6 12.5±1.0 13.5±0.6 10.0±0.0 13.5±0.7 6.8±0.5 1 5.5±0.6 6.8±2.1 5.0±0.0 9.8±0.5 5.3±0.5 7.5±0.6 9.3±0.5 22.0±1.2 6.0±0.0 20.0±0.0 5.0±0.0 poppy extracts did not exhibit inhibitive effect on all microorganisms. red tulip, rose and roselle extracts did not exhibit inhibitive effect on s. cerevisiae bc 5461 and c. albicans atcc 1223. -: not effective % c on ce nt ra ti on e . c ol i o 15 7: h 7a t c c 3 31 50 l . m on oc yt og en es a t c c 7 64 4 s. t yp hi m ur iu m a t c c 1 40 28 s. a ur eu s a t c c 2 59 23 e . c lo ac ae a t c c 1 30 47 y . e nt er oc ol iti ca a t c c 2 77 29 b . c er eu s a t c c 3 30 19 b . s ub til is a t c c 6 63 0 p . ae ru gi no sa a t c c 2 78 53 s. a ur eu s a t c c 2 92 13 e . co li a t c c 2 38 97 r os el le r os e r ed tu lip 174 o. bayram, o. sagdic, l. ekici tab. 3: kinetic parameters of abes obtained from different edible flowers at ph 3.5 buffer solution during heating at 70, 80 and 90 ºc flowers temperature k x 10-3 t1/2 ea (ºc) (min-1) (h) (kj mol-1) 70 0.20 25.1 poppy 80 0.60 8.4 88.53 90 1.10 4.6 70 1.50 7.79 red tulip 80 3.68 3.12 76.00 90 6.45 1.81 70 0.92 12.5 rose 80 3.68 3.1 114.13 90 8.29 1.4 70 1.84 6.3 roselle 80 2.99 3.9 69.79 90 10.8 1.1 k: reaction rate constant, t1/2: half-life value, ea: activation energy fig. 1: degradation of anthocyanins of edible flower extracts at ph 3.5 buffer solution. a: rosa, b: poppy, c: roselle, d: red tulip calculated for poppy, red tulip, rose and roselle was reported, to be 88.53, 76.00, 114.13 and 69.79 kj mol-1, respectively. similarly, sagdic et al. (2013) reported that the activation energy levels of tulips in different colors range from 68.69 to 76.00 kj mol-1. we found that the sample with the highest degradation rate constant was the poppy anthocyanin, which might result from the difference in anthocyanin composition. in fact, the difference in anthocyanin degradations may result from the anthocyanin composition of the samples (wang and xu, 2007). furthermore, fischer et al. (2013) reported that intermolecular and intramolecular complex formations are very important mechanisms of copigmentation contributing to enhanced anthocyanin stability. degradation of anthocyanin changes depending on the storage temperature and extraction conditions (prieto et al., 1999), and it was reported repeatedly that anthocyanin loss accelerates depending on the increase on the temperature of heating process (ekici, 2011; sagdic et al., 2013; kirca et al., 2003; harbourne et al., 2008). conclusion in this research, the antimicrobial properties of some edible flowers along with their bioactive features were studied. it was ascertained that roselle extracts contain less tpc and tac than poppy, rose and tulip extracts. the lowest aa and ac were found in roselle extracts. findings show that red tulips possessed extremely high tpc. as for natural food colorants and bioactive extracts from some edible flowers 175 poppy, it had both the highest tac and ac. while it was found that the abes derived from roselle possess antimicrobial activity, the poppy extracts was determined not to be effective on any microorganisms used in the study. the findings of anthocyanin degradation kinetic of extracts revealed that the most resistant to heating process was the poppy anthocyanin. among the materials studied, the tulip petals only cannot be used for food yet. sagdic et al. 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dna deoxyribonucleic acid; dw dry weight; eo essential oil; issr intersimple sequence repeat; pa proazulene; pca principal coordinate analysis; rapd random amplified polymorphic dna; upgma unweighted pair group method with arithmetic averages introduction achillea species are well known medicinal plants having an important role both in folk medicine and in the modern phytotherapy. main indications include loss of appetite, bloating, flatulence, minor menstrual spasms and wound healing (final community herbal monograph, 2011). the majority of the drug is still obtained from wild collection. the genus achillea consists of six sections and appr. 140 species (guo et al., 2005), which are allogamous, herbaceous perennials in the northern hemisphere. the presence of spontaneous hybrids, alloand autopolyploids, aneuploids, and phenocopies results in a big cytological, morphological and chemical variability not only at species but also at intraspecific level. therefore, taxonomic evaluation, identification of species and systematic classification has been a difficult task for decades. with regard to species identification earlier studies were obviously focusing on morphological traits. although the majority of characteristics proved to be extremely variable, some of them like fruit size (dabrowska, 1977), shape of leaflets and rayflorets (rauchensteiner et al., 2002), and pollen morphology (akyalcin et al., 2011) have been defined as stable ones at least in the investigated taxa. cytological studies ascertained that in some species different caryotypes are present which, however, do not necessarily show connection with morphological traits (dabrowska, 1977; daniheka and rotreklová, 2001). phytochemical parameters represent a valuable part of taxonomic evaluation of yarrow species. based on the pharmacopoeial require ments, most frequently, the presence of chamazulene in the essential oil has been in the focus of investigations (tétényi et al., 1962; oswiecimska, 1962; michler et al., 1992) although flavonoids may have taxonomic importance, as well (valant, 1978). it is wide ly accepted now that chamazulene is a characteristic of the members of millefolium group; however, the presence of azulenes is not a universal phenomenon for all of these species. earlier, a firm connection between chromosome number of the species and the potential for accumulation of chamazulene was supposed. recently, it has been accepted that the production of proazulenes in polyploids may depend on the chemism of the parent/original diploid species (kästner et al., 1992; ma et al., 2010). by the development of analytical methods evaluation of a wider range of oil components and their enantiomers provided a more complex approach in identification and systematics (orth, 2000; rauchensteiner at al., 2002; radulovic et al., 2007). the use of molecular markers in the systematic studies of yarrow was introduced from the 1990th. wallner et al. (1996) proved the applicability of rflp and pcr based fingerprinting methods for characterisation of micropropagated achillea clones. investigations by nrits and plastid trnl-f dna sequences revealed phylogenetic connections: differentiation patterns of achillea s.l. in time and space (guo et al., 2004), although this method could not assure a well established separation of a. millefolium aggr. in the work of guo et al. (2005) characterization was realized by aflp markers and more recently, ma et al. (2010) demonstrated the ongoing introgression of diploid progenitor and tetraploid progenies in the a. millefolium complex by analysing single copy nuclear genes and aflp markers. besides taxonomic studies, several publications appeared in the last years about rapd, issr (ebrahimi et al., 2012; farajpour et al., 2011; gharibi et al., 2011) and aflp (rahimmalek et al., 2009) assessment of yarrow species. characteristically, the authors compared populations from different geographical locations for conservation purposes without describing the phytochemical values of the plant material. these studies are focusing on indigenous species (a. santolina, a. tenuifolia, a. eriophora, etc.), not found at the international market and official therapies. it can be established that practice-oriented investigations on economically important taxa of yarrow are scarce up to now. the goal of our investigations was to look for reliable, relatively simple methods for differentiation/determination of intraspecific taxa of a. collina, the most frequently collected and cultivated species. according to our knowledge, molecular genetic study of intraspecific diversity of this species has not been published till now. 106 k. inotai, z. györgy, s. kindlovits, g. várady, é. németh-zámbori materials and methods plant material eleven achillea collina becker accessions have been included into the investigation. three of them are officially registered cultivars, two ones are cultivated commercial materials without special denomination and six accessions originate from wild populations (tab. 1). for comparative studies, seven other species of different ploidy level were used as control. among them a. ptarmica belongs to section ptarmica (dc.) w. koch. all other species are members of the section achillea and except a. crithmifolia and a. filipendulina of the a. millefolium agg. (guo et al., 2004). seeds of accessions nr. 1-2 were obtained from the maintainers of cultivars, nr. 3 derived from own maintenance breeding, nr. 4-5 were obtained from farmers, 6-11 were collected from wild populations of hungary and accessions nr. 12-18 were obtained from the genebank of the national botanical garden, vácrátót. the species identity has been controlled according to the morphological traits and through checking the ploidy level by flow cytometry. plants were grown from seeds in climatic chambers in 2014. in 5-6 weeks they reached a 4-5 leafy stadium when bulk sampling (kraft and säll, 1999) from 10-15 plants/accession was carried out for pcr trials as well as checking of chromosome numbers by flow cytometry (fig. 1) according to the modified method of galbraith et al. (1983). after 2 months, the seedlings were planted into open field plots in three replicates at our experimental station in budapest. at flowering stage, representative bulk samples with max. 20 cm of shoots were taken from each plot for essential oil extraction. the plant material was dried at room temperature and stored at +4 °c until distillation. tab. 1: list of the investigated achillea accessions accession species origin of population nr. sign /chromosome nr.*/ sect. achillea agg. a. millefolium s.l. 1 c1 a. collina becker (4x) german variety ‘proa’ 2 c2 a. collina becker (4x) slovakian variety ‘alba’ 3 c3 a. collina becker (4x) hungarian variety ‘azulenka’ 4 c4 a. collina becker (4x) cultivated commercial plant material, gyula 46° 38’ 50.2” n/ 21° 16’ 42.3” e 5 c5 a. collina becker (4x) cultivated commercial plant material, földes 47° 17’ 22.8” n/ 21° 21’ 47.8” e 6 cw1 a. collina becker (4x) wild collected population, aszód 47° 39’ 12.1” n/ 19° 29’ 3.6” e 7 cw2 a. collina becker (4x) wild collected population, csörötnek 46° 56’ 59.3” n/ 16° 22’ 14.7” e 8 cw3 a. collina becker (4x) wild collected population, diósd 47° 24’ 29.8” n/ 18° 56’ 36.5” e 9 cw4 a. collina becker (4x) wild collected population, mikóújfalu 46° 3’ 13.5” n/ 25° 50’ 6.7” e 10 cw5 a. collina becker (4x) wild collected population, nagymaros 47° 47’ 16.9” n/ 18° 57’ 14.9” e 11 cw6 a. collina becker (4x) wild collected population, remeteszőlős 47° 33’ 23” n/ 18° 55’ 44.6” e 12 asp a. asplenifolia vent. (2x) genebank of vácrátót bot.garden 14 dis a. distans walds. et kit. (6x) genebank of vácrátót bot.garden 16 mil a. millefolium l. s.s. (6x) genebank of vácrátót bot.garden 17 pan a. pannonica scheele (8x) genebank of vácrátót bot.garden excl. agg. a. millefolium 13 cri a. crithmifolia walds. et kit. (2x) genebank of corvinus university 15 fil a. filipendulina lam. (2x) genebank of vácrátót bot.garden section ptarmica 18 ptr a. ptarmica l. (2x) genebank of vácrátót bot.garden the accessions are maintained as living genebank collection at the research station of the faculty of horticulture, corvinus university, budapest. essential oil extraction and analysis the essential oil content was measured from the dried drug in triplicate, by hydrodistillation in a clevenger-type apparatus according to the hungarian pharmacopoeia vii (millefolii herba). after 3 hours of distillation, n-hexane was added to take up the essential oil. after evaporation of the hexane, the collected extract was stored in a cool place. the essential oil content was calculated as ml/100 g dried plant material. water content of the drug was determined by heating 4 g of the drug at 105 °c for 3 hours. the proazulene content in the essential oil samples was determined by spectrophotometric method at 608 nm as described in the european pharmacopoeia vii (millefolii herba) in triplicate and calculated as a percentage of the dry weight expressed as chamazulene. dna isolation one young leaf from each of 10-15 plants of each accessions was collected and ground together with liquid nitrogen. genomic dna was extracted from these bulk samples of fresh young leaves by dneasy plant mini kit (qiagen, bioscience, hungary). dna concentration and quality was assessed using nanodrop (bioscience, hungary) and visually checked on 1 % agarose gel. out of the primarily screened 17 rapd and 13 issr primers only 11 rapd and 12 issr primers produced clear, reproducible and scorable bands, thus, the investigations have been carried out by these ones. evaluation of achillea accessions 107 rapd analysis 11 rapd primers have been used, the optimum annealing temperature was determined for each one individually (tab. 2). amplification reactions were performed in 12 μl volume containing 15-25 ng genomic dna, 1 μm primer, 6 μl of 2× gotaq hot start green master mix (promega), 3 mm mgcl2 and nuclease free water. pcr amplification was performed in a supercycler sc-200 thermocycler (kyratec) under the following conditions: 2 min at 95 °c, followed by 35 cycles of 30 s at 94 °c, 1 min at specific annealing temperature, 1 min at 72 °c and a final extension for 7 min at 72 °c. amplified dna fragments were separated in a 1.5 % agarose gel (seakem le agarose, lonza) at 100 v for 90-120 min in 1× trisacetate edta (tae) buffer (ph 8.0) and stained by 1 % (w/v) ethidium bromide. the pcr products were visualized under uv light by alphaimager ep imaging system (cell bioscience). the 100 bp ladder (promega) was used as a molecular weight size marker. issr analysis issr analysis has been performed with 12 primers (tab. 2), the optimum annealing temperature was determined for each one individually. pcr reactions were carried out in a volume of 12 μl containing 15-25 ng genomic dna, 2 μm of primer, 6 μl of 2× gotaq hot start green master mix (promega), 3 mm mgcl2 and nuclease free water. the supercycler sc-200 (kyratec) was programmed as follows: an initial cycle of 3 min at 95 °c, followed by 35 cycles each consisting of 30 s at 94 °c, 45 s at specific annealing temperature, 45 s at 72 °c and final extension of 7 min at 72 °c. fragment separation and visualization was performed as above. statistical analysis the results of essential oil and proazulene analysis were evaluated by one-way anova using the ibm spss statistics 22 program. the pairwise comparisons of the variances were made by the tukey post hoc test. amplified dna fragments were scored visually for presence (1) or absence (0) of homologous bands and the results were summarized in microsoft excel table. popgene version 1.32 (yeh and boyle, 1997) was used to estimate number of polymorphic bands, percentage of polymorphic bands, nei’s (1973) gene diversity (h) and shannon’s information index (i) (lewontin, 1972) for dominant marker data. genetic relatedness among genotypes was studied by upgma (unweighted pair group method with arithmetic averages) cluster analysis and principal coordinate analysis (pca) using past software (hammer et al., 2001). results and discussion essential oil and proazulene content the essential oil content of the examined accessions varied between 0.002 (a. distans) and 0.365 (a. collina cw4) ml/100 g (tab. 3). the fig. 1: histograms of relative fluorescence intensity of octoploid (a. pannonica), hexaploid (a. millefolium), tetraploid (a. collina) and diploid (a. filipendulina) achillea species (from left to right), (codes of accessions as in tab. 1). tab. 2: the tested rapd and issr primers rapd sequence annealing no. of primer name temp. (°c) bands opa-20 5’-gttgcgatcc-3’ 39 10 opg-18 5’-ggctcatgtg-3’ 38 14 opb-11 5’-gtagacccgt-3’ 38 14 opa-02 5’-tgccgagctg-3’ 35 13 opg-13 5’-ctctccgcca-3’ 43 13 m2 5’-acaacgcctc-3’ 41 13 g11 5’-tgcccgtcgt-3’ 48 14 seg1 5’-aggggtcttg-3’ 35 15 seq2 5’-gggtttaggg-3’ 35 9 seq3 5’-gacagacagg-3’ 35 15 seq4 5’-cgaagctacc-3’ 35 10 total no. of bands (rapd) 140 number of polymorphic loci 136 percentage of polymorphic loci 97.14 % issr sequence annealing no. of primer name temp. (°c) bands 818 5’-cccccccaaaaaaag-3’ 47 10 825 5’-aaaaaaaacccccccct-3’ 49 14 849 5’-ggggggggttttttttc-3’ 49 14 cag5 5’-cccccaaaaaggggg-3’ 49 15 ctc4rc 5’-cccccccccttttr-3’ * 50 18 issr1 5’-cacacacacacacacagt-3’ 51 21 issr2 5’-gagagagagagagagag-3’ 49 10 issr3 5’-gtgtgtgtgtgtgtgtc-3’ 49 12 issr4 5’-acacacacacacacactg-3’ 51 21 issr5 5’-agtgagtgagtgagtg-3’ 45 18 issr6 5’-gatagatagatagatagata-3’ 47 14 issr7 5’-tcttcttcttcttcttct-3’ 45 15 total no. of bands (issr) 188 number of polymorphic loci 183 percentage of polymorphic loci 97.34 % total no. of bands (rapd+issr) 328 number of polymorphic loci 319 percentage of polymorphic loci 97.26 % 108 k. inotai, z. györgy, s. kindlovits, g. várady, é. németh-zámbori accumulation levels of each species are in the range of data mentioned in other investigations (németh, 2005). evaluating the studied populations with giving consideration to the accumulation level of the essential oil, the tukey test distinguished 5 subsets at p = 0.05 significance level. among them both a. distans having the lowest content (0.01 ml/100 g) and a. crithmifolia having the highest one (0.42 ml/100 g) represent distinct subsets. on the other hand, the largest homogenous subset includes all of the a. collina accessions, besides a. crithmifolia, a. pannonica and a. filipendulina. taking into account only the a. collina accessions, the differences are much lower; significant differences were proven only for a. collina wild growing population cw2 and another wild growing population cw4 compared to all of the other ones. these accessions show the two extreme values of the essential oil content: cw4 (mikóújfalu, transylvania) produced the highest content (0.327 ml/100 g) and the genotype cw2 (csörötnek, west-hungary) the lowest one (0.135 ml/100 g). in other wild collected accessions (in central hungary) concentrations between these extreme values were detected. however, each a. collina sample could surpass the requirements of the european pharmacopoeia. in our previous study on 23 hungarian wild populations of this species we detected also significant (up to four-fold) differences among the samples, however, no connection between the geographical location and the level of the essential oil content could be ascertained (németh et al., 2007). the essential oil content of the selected cultivars and the cultivated genotypes was much more similar to each other, within a range of 0.2 and 0.3 ml/100 g without significant difference among them. according to the recent chemotaxonomic conception, among the investigated species, proazulenes are only accumulating in a. collina and a. asplenifolia (kastner et al., 1992; rauchensteiner et al., 2002). this has been ascertained by our results. the distilled oil of the other species had a yellowish colour indicating the lack of azulenes, while the samples of the mentioned two species each showed a blue colour of different intensity. the concentration of proazulenes prescribed by the european pharmacopoeia viii is 0.1 % which, however, was exceeded only by half of the samples. besides the sample of a. asplenifolia it was the case in three wild growing accessions and in two selected cultivars of a. collina (tab. 3). the lowest proazulene content (0.020 %) was detected in a wild growing a. collina population (cw2), while significantly the highest values (0.135 % and 0.148 %) were also measured in accessions of wild origin (cw3 and cw5, respectively). among the cultivated genotypes, the registered cultivars ‘proa’ (c1) and ‘azulenka’ (c3) showed significantly the highest proazulene contents (0,110 % and 0.133 %, respectively). both cultivated populations (c4 and c5), furthermore the cultivar ‘alba’ (c2) and the sample from remeteszőlős (cw6) have each statistically similar proazulene contents (0.074-0.079 %). in previous hungarian investigations the proazulene content of the wild growing populations was between 30 % and 67 % (németh et al., 2007). although these are area percentages detected by gc method, therefore difficult to compare with the present data, significant differences among accessions were detected in both studies. other trials on european wild yarrow populations a. collina has been rarely evaluated. in germany, michler et al. (1992) de scribed large differences among populations concerning the presence of proazulenes. genetic markers in the rapd analysis 140 bands were detected of which 97.14 % was polymorphic. the numbers of obtained bands were between 9 and 15, the average was 12.36 polymorphic bands/primer. it is higher than obtained with a. santolina, a. tenuifolia (ebrahimi et al., 2012) and with a. millefolium (farajpour et al., 2011), (tab. 2). based on the pca of rapd markers, segregation of the taxonomically most distant a. ptarmica from the accessions of a. collina is obvious while samples of a. crithmifolia and a. filipendulina are between them (fig. 2). these latter diploid species both belong to sect. achillea, however, are less closely related to the polyploid members of the a. millefolium agg. (guo et al., 2004). a. collina genotypes c3 (hungarian cultivar ‘azulenka’) and cw6 (wild collected genepool from remeteszőlős, central hungary) show the largest distances from the other ones while genotypes cw1, cw3 and c5 show the closest linkage to each other. the mentioned pattern, however, may not reflect geographical or genetic connection. in case of the studied genotypes, it could be established, that rapd markers tend to distinguish primarily among species and less characteristically among intraspecific populations of a. collina. using the issr markers, the total number of detected bands was 188, about 30 % more than in case of rapd primers. the percentage of the polymorphic bands reached 97.34 % exceeding the values of other related studies with a. millefolium (farajpour et al., 2012; gharibi et al., 2011). the mean number of polymorphic bands/primer was 15.25, ranging from 10 to 21, also higher than in rapd analysis (tab. 2). principal coefficient analysis of the studied samples shows a clear separation of a. ptarmica (fig. 3) which reflects well the fact that it is a member of sect. ptarmica and taxonomically the less related species with all the other ones. this is a similar result as that in the analysis with rapd markers. a well defined group is formed by four of the accessions of a. collina of wild origin and another one by the cultivated accessions of this species. cw2 which is a population of wild origin shows a larger separation from all of the other a. collina accessions and especially from the other wild growing ones. a single tab. 3: essential oil and proazulene content of the examined accessions (codes of accessions as in tab. 1) accession essential oil content proazulene content code (ml/100 g dw) (% d.w.) mean standard mean standard deviation deviation c1 0.248 c,d 0.082 0.105 b,c 0.029 c2 0.273 c,d 0.097 0.075 b 0.035 c3 0.290 c,d 0.051 0.173 b,c 0.052 c4 0.236 c,d 0.940 0.078 b 0.039 c5 0.248 c,d 0.108 0.074 b 0.043 cw1 0.202 b,c 0.145 0.061 a,b 0.018 cw2 0.135 a,b,c 0.088 0.021 a 0.005 cw3 0.235 c,d 0.141 0.135 c 0.057 cw4 0.365 d,e 0.183 0.106 b,c 0.038 cw5 0.317 c,d 0.212 0.148 c 0.086 cw6 0.199 c,d 0.103 0.079 b 0.075 asp 0.249 c,d 0,038 0.136 c 0.014 dis 0.485 e 0,044 mil 0.005 a 0,001 pan 0.198 b,c,d 0,026 cri 0.159 a,b,c 0,062 fil 0.189 a,b,c 0,009 ptr 0.059 a,b 0,011 different letters represent statistically different subsets according to the tukey test at p = 0.05 evaluation of achillea accessions 109 fig. 2: patterns of relationships among the investigated achillea accessions revealed by principal component analysis based on rapd data (codes of accessions as in tab. 1) fig. 3: patterns of relationships among the investigated achillea accessions revealed by principal component analysis based on issr data (codes of accessions as in tab. 1) wild growing accession of a. collina (cw4) and other species of the sect. achillea form a further, larger group. in this pattern, species of the a. millefolium agg. do not reflect a close relationship with each other. the most characteristically separated wild growing population cw2 (western hungary) and cw4 (transylvania) are located to larger distances (200-400 km) from the central populations (cw1, 3, 5 and 6). according to the results, issr markers proved to be appropriate first of all for the separation of a. collina accessions while the relationships of other species inside the section are less specific. a joint evaluation of rapd and issr analysis revealed a good separation of achillea species (fig. 4.). similarly to the results of both rapd and issr markers separately, a. ptarmica and the members 110 k. inotai, z. györgy, s. kindlovits, g. várady, é. németh-zámbori tab. 4: genetic distances matrix of the investigated achillea accessions based on rapd and issr data (codes of accessions as in tab. 1) cw2 c3 c2 c1 c4 c5 cw6 cw1 cw5 cw3 cw4 dis pan fil cri asp mil ptr cw2 1.00 c3 0.40 1.00 c2 0.38 0.30 1.00 c1 0.44 0.36 0.30 1.00 c4 0.46 0.28 0.28 0.29 1.00 c5 0.42 0.35 0.26 0.30 0.23 1.00 cw6 0.45 0.44 0.36 0.36 0.34 0.26 1.00 cw1 0.47 0.41 0.35 0.36 0.33 0.30 0.27 1.00 cw5 0.47 0.45 0.38 0.43 0.32 0.33 0.36 0.30 1.00 cw3 0.46 0.39 0.32 0.39 0.27 0.30 0.30 0.27 0.28 1.00 cw4 0.44 0.42 0.45 0.44 0.38 0.38 0.38 0.36 0.39 0.32 1.00 dis 0.50 0.46 0.44 0.43 0.46 0.47 0.46 0.42 0.43 0.42 0.39 1.00 pan 0.48 0.46 0.45 0.46 0.49 0.49 0.48 0.48 0.50 0.50 0.40 0.25 1.00 fil 0.62 0.57 0.61 0.65 0.65 0.68 0.68 0.63 0.66 0.60 0.55 0.55 0.59 1.00 cri 0.65 0.64 0.64 0.62 0.69 0.64 0.57 0.54 0.58 0.62 0.64 0.56 0.61 0.54 1.00 asp 0.47 0.46 0.48 0.43 0.48 0.50 0.49 0.50 0.52 0.41 0.47 0.44 0.45 0.45 0.55 1.00 mil 0.50 0.47 0.49 0.53 0.51 0.59 0.54 0.49 0.50 0.46 0.45 0.38 0.46 0.53 0.51 0.37 1.00 ptr 0.63 0.63 0.59 0.56 0.63 0.62 0.56 0.59 0.72 0.62 0.59 0.57 0.59 0.57 0.60 0.52 0.53 1.00 fig. 4: patterns of relationships among the investigated achillea accessions revealed by principal component analysis based on rapd and issr data (codes of accessions as in tab. 1) of sect. achillea are the most characteristically distinguishable from each other. the accessions of the two related species a. crithmifolia and a. filipendulina (section achillea, exc. a. millefolium agg.) are situated closer to each other in the pca analysis than to any of the accessions in agg. a. millefolium. the studied genotypes of a. distans and a. millefolium seem to be closely linked with each other. aflp profiles of these two species were also hardly distinguishable from each other (guo et al., 2005) being two related polyploids of the a. millefolium agg. the species a. pannonica, a. distans and a. millefolium gave overlapping patterns and could not be clearly separated based on essential oil markers, either (rauchensteiner et al., 2002). accessions of a. collina represent another group. by the joint evaluation of rapd and issr markers, cultivated and wild growing ac evaluation of achillea accessions 111 cessions do not seem to separate characteristically from each other, except the geographically more distant cw2 and cw4 which are situated in the pca coordinate system characteristically far away. in coincidence with the above mentioned, the largest genetic distance coefficients were proven between a. ptarmica and the members of sect. achillea (genetic distances between 0.52 and 0.72), (tab. 4). distances of the species inside the section achillea are in most cases below 0.5. the genetic distances of cw2 originating from western hungary are the largest to any of the other wild originated accessions exceeding 0.4. on the other side, similarity is highest (genetic distance 0.27 both) between the populations cw1 cw3 and cw1 cw5 where geographical distances of the original locations are 52-55 km. nei’s genetic distances among the cultivars are also relatively low, between 0.23 and 0.36. the values of the shannon diversity index (h) (lewontin, 1972) reflect similar results. this value calculated for all of the accessions in this study is 0.5209 while taking into account only the a. collina accessions it shows a lower value: 0.4098. the intraspecific diversity seems to be higher among the wild growing accessions (h = 0.3793) than among the cultivars (h = 0.3028). based on the investigated markers, it can be established, that the studied rapd provided a more specific approach for distinction of species while the used issr and combined rapd and issr primer evaluation enabled an informative evaluation among a. collina accessions, as well. nevertheless, the separation of populations based on the investigated molecular markers does not reflect any connection with their essential oil and proazulene contents. according to the results of this study, a common origin of the cultivated populations might be anticipated. ‘proa’ is the first selected cultivar of a. collina. it is a german cultivar, which has been on the market since 1973. compared to this, ‘alba’, a slovakian cultivar was registered almost twenty years later in 1992. as no relevant information on the original genetic background of these selections is available, a relationship cannot be excluded. besides, even in case of their different origins, during the decades of their cultivation in central europe, a stepwise reduction of the genetic divergence might be hypothesized. it is supported by the fact that the two accessions from commercial cultivation seem to be closely related to them, as well. ‘azulenka’, however, a recently (2013) registered hungarian cultivar which has been selected from a wild population of central hungary shows the lowest similarity with the formerly mentioned taxa. genetic relationships of the accessions collected from the wild show a connection with their original geographical habitats. this is similar to the findings of iranian authors in case of some other yarrow species (farajpour et al., 2011; gharibi et al., 2011) although the geographic distances of the studied populations were much larger than in 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characterisation and long-term monitoring of micropropagated clones. plant. cell rep. 15, 647-652. yeh, f.c., yang, r.c., boyle, t.b.j., ye, z.h., mao, j.x., 1997: popgene, the user-friendly shareware for population genetic analysis. molecular biology and biotechnology centre, university of alberta, canada. addresses of the authors: éva németh-zámbori, katalin inotai, sára kindlovits, department of medicinal and aromatic plants, szent istván university, h-1118, budapest, villányi str. 29-43., hungary e-mail: zamborine.nemeth.eva@kertk.szie.hu e-mail: inotai.katalin@kert.szie.hu e-mail: kindlovits.sara@kertk.szie.hu zsuzsanna györgy, department of genetics and breeding, szent istván university, h-1118, budapest, ménesi str. 44., hungary e-mail: gyorgy.zsuzsanna@kertk.szie.hu györgy várady, institute of enzymology, research centre for natural sciences, hungarian academy of sciences, 1117 budapest, magyar tudósok körútja 2., hungary e-mail: varady.gyorgy@ttk.mta.hu © the author(s) 2016. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 88, 139 144 (2015), doi:10.5073/jabfq.2015.088.020 1department of microbiology, university of agriculture, cracow, poland 2 department of forest pathology, university of agriculture, cracow, poland species composition and molecular assessment of the toxigenic potential in the population of fusarium spp. isolated from ears of winter wheat in southern poland katarzyna wolny-koładka1*, anna lenart-boroń1, piotr boroń2 (received january 4, 2015) * corresponding author summary the aim of this study was to identify the species composition and to evaluate the prevalence of genes involved in the synthesis of the following mycotoxins: deoxynivalenol, nivalenol and fumonisins in the population of fusarium spp. isolated from ears of winter wheat in southern poland. all fungal isolates were identified by the species-specific pcr or sequencing of the translation elongation factor. significant differences were observed in both abundance and species composition of the collected strains between two years of studies. a total of 304 ear samples were processed and 107 fusarium strains belonging to 10 species: f. graminearum, f. culmorum, f. sporotrichioides, f. poae, f. avenaceum, f. oxysporum, f. verticillioides, f. equiseti, f. tricinctum and f. cerealis were isolated in 2012 and 2013. numerous presence of mycotoxin-biosynthesis pathway genes was detected in the examined material, which evidences the potential toxicity of the analyzed fusarium fungi. introduction fungi from the genus fusarium are extremely diverse group of microorganisms, both in terms of physiological and morphological characteristics. their adaptability caused that they are currently considered as ones of the most important pathogens worldwide. plant diseases caused by fusarium spp. are global and contribute to measurable economic loss. fusarium spp. cause, among others, seedling blight, root rot, leaf spot and fusarium ear blight (leslie and summerell, 2006). another extremely important aspect is the negative effect of secondary metabolites produced by fusarium spp. on human and animal health (wolny-koładka, 2014). apart from aspergillus spp. and penicillium spp., fungi from the genus fusarium are considered as ones of the most toxinogenic microorganisms worldwide. mycotoxins produced by fusarium spp. exhibit phytotoxic, zootoxic and antibiotic activity (chełkowski, 1985; tate, 2000). secondary metabolites of fusarium spp. have mutagenic, teratogenic and estrogenic properties, and while accumulating in the organism they become the cause of chronic multiorgan diseases (pławińska-czarnak and zarzyńska, 2010). fungi from the genus fusarium produce mycotoxins not only during growing season, but also during harvesting, transport and storage (creepy, 2002). the ability to produce mycytoxins is a strain-specific feature and is not characteristic for the entire species (tan and niessen, 2003). among the most frequently produced mycotoxins by the genus fusarium there are deoxynivalenol (don), nivalenol (niv), zearalenon (zea) and fumonisins (fum). numerous genes are involved in the mycotoxin synthesis, among which the following systems were distinguished: tri (deoxynivalenol, nivalenol), pks (zearalenone) and fum (fumonisins) (alexander et al., 1999; ward et al., 2002; alexander et al., 2004; fanelli et al., 2013). the potential toxicity of fungi can be inferred from the detection of particular genes involved in the biosynthetic pathway of mycotoxins. currently, the most relevant methods in the detection of toxinogenic strains include the molecular biology techniques. these methods are based on polymerase chain reactions with the use of specific primers and allow both rapid identification of strain species as well as their potential to produce mycotoxins (niessen, 2007). the aim of this study was the molecular identification of fungi from the genus fusarium isolated from winter wheat ears in southern poland in 2012 and 2013. moreover, the presence of the key genes involved in the biosynthetic pathways of deoxynivalenol, nivalenol and fumonisins was assessed in the studied fusarium spp. strains, in order to indicate the potential ability for the production of these mycotoxins. materials and methods fungal isolation from winter wheat ears was conducted during two growing seasons of 2012 and 2013. the ears were randomly collected into sterile bags from the same fields in both years in southern poland and after collection transported to the laboratory, in order to isolate the fusarium strains. eight ears were collected per each field and one ear represented a single sample. a total of 304 ear samples were analyzed, 152 each year. the material was surface sterilized with 1 % sodium hypochlorite and then rinsed in sterile distilled water and placed onto potato-dextrose agar (pda). the plates were incubated at 24 oc for 7 days and then mycelium fragments were subcultured onto subsequent petri dishes with pda until homogeneous cultures were obtained (kowalska, 2011). the initial identification of fungi was conducted using the diagnostic manuals: gilman (1957), domsch et al. (1980), marcinowska (2003). macroscopic evaluation of the cultures was also conducted, taking into account the structure and pigmentation of the mycelium. subsequently all isolates qualified as the genus fusarium were counted. prior to dna extraction fusarium spp. isolates were cultured on pda for 7 days. afterwards, mycelia were scraped into eppendorf tubes with a sterile spatula. the mycelium was ground with 2 mm wolfram beads using retsch mm 400 mixer mill (30 hz, 3 min). total dna was extracted from mycelium of each isolate (about 100 mg wet weight) using genomic mini ax plant dna extraction kit (a&a biotechnology, gdynia, poland) according to the manufacturer’s instructions. the quality and quantity of dna obtained was assessed using nanodrop spectrophotometer (thermo scientific, usa). molecular identification of the collected fusarium spp. isolates was conducted based on the species-specific pcr assay using previously published primer pairs for f. poae, f. oxysporum, f. graminearum, f. sporotrichioides, f. culmorum, f. proliferatum and f. verticillioides (tab. 1). pcr reactions were performed for all isolates and sterile deionized water served as negative control. the reaction mixtures of a total volume of 25 μl contained 10×pcr buffer, 1.5 mm of mgcl2, 0.3 μm of each primer, 0.2 mm of dntps, 1 u of taq dna polymerase (thermo scientific fermentas, canada) and approximately 25 ng of fungal template dna. pcr amplification was carried out in the biorad t100 thermal cycler (biorad, usa) using temperature profiles described by nicholson et al. (1998), mishra et al. (2003), mulè et al. (2004a), demeke et al. (2005), koncz et al. (2008), and rahjoo et al. (2008). the pcr products were visualized 140 k. wolny-koładka, a. lenart-boroń, p. boroń by 1×tbe electrophoresis in ethidium bromide-stained 1 % agarose gel. the strains which did not give any positive result in the pcrs, were subjected to sequencing of ef-1α gene. this enabled the unequivocal identification of all doubtful strains as it provided the exact information about one of the most frequently used fusarium barcode sequence. a standard pcr reaction was used to amplify the ef-1α gene of 62 samples whose identification based on speciesspecific pcr was unsuccessful or gave uncertain results, for instance lack of band, very weak/not sharp band or multiple weak bands. the primer pair ef1 and ef2 (tab. 1) was used in pcr reactions performed as given earlier using the temperature profile described by amatulli et al. (2010). the pcr products were visualized by 1×tbe electrophoresis in ethidium-bromide-stained, 1 % agarose gel and then purified using clean up purification kit (a&a biotechnology, poland), according to the manufacturer’s instructions and sequenced in 3500 series sequencer (applied biosystems, usa). sequencing data were edited with chromas (technelysium, australia) and used as a query against national centre for biotechnology information (ncbi) using blast network services and a fusariumid database (http:// isolate.fusariumdb.org/). the potential of fusarium spp. isolates to produce fumonisin and trichothecenes was determined by the pcr-based molecular analyses using the tri7f and tri7donr, tri13nivf and tri13r, and fum1f and fum1r specific primer pairs (tab. 1) which target the mycotoxin-synthesis pathway genes: tri7don (deoxynivalenol), tri13niv (nivalenol) and fum1 (fumonisin), respectively. the pcrs were performed after completed species identification for all isolates belonging to the species that could be the potential producers of given mycotoxins. the reaction mixtures of a total volume of 25 μl contained 10×pcr buffer, 2 mm of mgcl2, 0.4 μm of each primer, 0.2 mm of dntps, 0.75 u of taq dna polymerase (thermo scientific fermentas, canada) and approximately 50 ng of fungal template dna. sterile deionized water served as negative control. pcr amplification was carried out in the t100 thermal cycler (biorad, us) according to temperature profiles described by lenc et al. (2008) and yazeed et al. (2011). the pcr products were visualized by 1×tbe electrophoresis in ethidium-bromide-stained, 1 % agarose gel. results in total 107 strains of fusarium spp. were isolated during two years of studies. the frequency of isolation of fusarium spp. from winter wheat ears was found at the level of 27.63 % in 2012 and 42.76 % in 2013. the two-stage identification with the use of speciesspecific pcr and ef-1α region sequencing allowed to determine the systematic position of all isolates. the obtained nucleotide sequences were submitted in the genbank database with the following accession numbers: kj947314 – kj947343; km025393 – km025423 and km052630 – km052646. there were significant differences in the species composition and the prevalence of strains between the two seasons of the study, as presented in tab. 2. based on the results of species-specific pcr and ef-1α region sequencing it can be stated that f. graminearum (fig. 1) was the predominant species in 2012, while in 2013 f. poae was the predominant one. the prevalence of tri7don, tri13niv and fum1 genes also varied over the analyzed period (tab. 2). 32 out of 107 identified fusarium isolates were capable of the production of the tested mycotoxins. in 2012, 20 out of 40 isolates (50 %) possessed the examined genes, while in 2013 the 381-445 bp-long band was observed in 12 out of 64 isolates (18.75 %), which evidenced the presence of tri7don only. discussion the conducted studies included 107 strains from the genus fusarium isolated from the ears of winter wheat growing in southern poland. the systematic position of all species was detected using speciesspecific pcr and sequencing of ef-1α region. additionally, the tab. 1: list of primer sequences, expected dna fragment length and sources of primers. primer 5’ – 3’ sequence product length (bp) target sequence reference fspo-r cagcgcacccctcagagc f. poae fsp-f cgcacgtatagatggacaag ~ 400 f. poae, jurado et al., 2005 f. sporotrichioides fsp-r gtcagaagagacgcatccgcc ~ 400 f. sporotrichioides jurado et al., 2005 fgr-f gttgatgggtaaaagtgtg ~ 500 f. graminearum jurado et al., 2005 fgr-r ctctcatataccctccg ver1 cttcctgcgatgtttctcc 578 f. verticillioides mulè et al., 2004b ver2 aattggccattggtattatatatcta fof1 acataccacttgttgcctcg ~ 340 f. oxysporum mishra et al., 2003 for1 cgccaatcaatttgaggaacg c51f atggtgaactcgtcgtggc ~ 570 f. culmorum nicholson et al., 1998 c51r cccttcttacgccaatctcg ef1 atgggtaagga(a/g)gacaagac ~ 700 ef-1 α gene o’donnell et al., 1998 ef2 gga(g/a)gtaccagt(g/c)atcatgtt fum1 f ccatcacagtgggacacagt 183 fum1 gene bluhm et al., 2004 fum1 r cgtatcgtcagcatgatgta tri7f tgcgtggcaatatcttcttcta 381-445 tri7don gene chandler et al., 2003 tri7donr gtgctaatattgtgctaatattgtgc tri13nivf ccaaatccgaaaaccgca 312 tri13niv gene chandler et al., 2003 tri13r ttgaaagctccaatgtcgtg species composition and toxigenic potential of fusarium in southern poland 141 were identified in the conducted study: f. poae, f. graminearum, f. sporotrichioides, f. avenaceum, f. equiseti, f. oxysporum, f. tricinctum, f. verticillioides, f. culmorum and f. cerealis. research carried out in lithuania confirm the frequent prevalence of these particular species in cereal grains in central-eastern part of europe (mačkinaitė et al., 2006). in the study conducted in 2000-2002 by narkiewicz-jodko et al. (2005), two species: f. culmorum (14-36 %) and f. poae (3-37 %) were identified to be the most prevalent. the share of other species was much smaller: f. avenaceum (2-19 %) and f. solani (1-4 %). the studies conducted in northern europe and asia indicate the presence of geographical differences in the occurrence of different fusarium species (yli-mattila, 2010). it was found that in northern europe (scandinavia, finland and north-western russia) the predominant species that contaminate cereals include: f. avenaceum, f. arthrosporioides, f. tricinctum, f. poae, f. culmorum, f. graminearum, f. sporotrichioides and f. langsethiae (levitin, 2001; kosiak et al., 2003; yli-mattila et al., 2004; stępień et al., 2008; nicolaisen et al., 2009). less frequently occurring species tab. 2: species composition, prevalence of each fusarium species isolated from ears of winter wheat and the occurrence of the examined mycotoxin pro duction genes no. year 2012 year 2013 species number tri7don tri13niv fum1 species number tri7don of isolates of isolates 1. f. poae 1 f. poae 32 4 2. f. graminearum 27 16 f. graminearum 19 8 3. f. sporotrichioides 3 f. sporotrichioides 8 4. f. avenaceum 2 5. f. equiseti 2 6. f. oxysporum 2 f. oxysporum 2 7. f. tricinctum 1 8. f. verticillioides 3 3 9. f. culmorum 3 1 f. culmorum 1 10. f. cerealis 1 total 42 16 1 3 total 65 12 total isolates 107 fig. 1: pcr detection of fusarium graminearum using species-specific primer set: fgr-f/fgr-r; m – dna size marker generuler dna ladder mix (thermoscientific, canada); 1, 2, 5 – positively identified f. graminearum isolates; 3, 4 – negatively identified f. graminearum isolates. fig. 2: pcr detection of don production potential indicated by the presence of tri7don marker tri7f/tri7donr. m – dna size marker generuler dna ladder mix (thermoscientific, canada); 1 – negatively identified presence of tri7don marker; 2, 3, 4 – positively identified presence of tri7don marker. conducted analysis allowed to determine which of the collected isolates possessed the biosynthesis pathway genes included in the production of deoxynivalenol, nivalenol and fumonisins. in 2013 the percentage of fungal isolations from wheat ears was greater than in 2012. as shown by other authors, the rate of winter wheat infection is variable and depends on many factors, which were not analyzed in this study, including meteorological conditions, fertilization type, forecrop and the applied plant protection agents (narkiewicz-jodko et al., 2005). jaczewska-kalicka (2002) in the studies conducted over several years, found significant differences in the incidence of fusarium root rot in wheat. in 1999 this disease was detected in 34 % of the examined plants, in 2000 − 27 % and in 2001 − 54 %. in the studies by narkiewicz-jodko et al. (2005) fungi from the genus fusarium were isolated from winter wheat ears with a frequency from 2 % to 37 %. in addition to the changes in the frequency of isolation of fusarium spp., the differences in the species composition of the isolated strains were also noted. in 2012 the frequency of isolation of f. poae was 2.4 %, whereas in 2013 it was already 49.2 %, while the frequency of isolation of f. graminearum decreased from 64.3 % to 29.2 % and the remaining species were rarely detected. ten species 142 k. wolny-koładka, a. lenart-boroń, p. boroń include: f. equiseti, f. torulosum and f. oxysporum (kosiak et al., 2003). in western siberia f. avenaceum, f. sporotrichioides and f. poae were the most frequently isolated species, while in eastern siberia f. acuminatum and f. avenaceum were the predominant ones. cereals grown in eastern russia were most frequently infested by two species: f. graminearum and f. poae (levitin, 2001, 2004). common prevalence of f. graminearum in southern poland is confirmed by other authors, who indicate that this species is the predominant one in this part of europe (yli-mattila, 2010). rapid spread of f. graminearum both within the entire european continent and outside europe has been observed in recent years (nicholson et al., 2003; waalwijk et al., 2003; fredlund et al., 2008; ylimattila et al., 2009; yli-mattila and gagkaeva, 2010). the presence or absence of tri7don, tri13niv and fum1 which are the biosynthesis pathway genes of the selected mycotoxins produced by the genus fusarium was verified based on pcr reactions. it was estimated that the tri7don gene, which participates in the biosynthesis pathway of deoxynivalenol in fusarium spp. was the most frequently detected. within two years of the study the presence of tri7don was found in 24 strains of the species f. graminearum and in 4 strains of f. poae. three strains of the species f. verticillioides possessed the fum1 gene and 1 strain of f. culmorum possessed the tri13niv gene. f. graminearum can produce numerous mycotoxins, including deoxynivalenol, nivalenol and zearalenone (marasas et al., 1984), but no moniliforminproducing strains have been found so far (farber et al., 1988). in our study, the greatest number of mycotoxin biosynthesis genes were detected within the group of f. graminearum strains. f. poae is the producer of fusarin x, deoxynivalenol, nivalenol, t-2 toxin, diacetoxyscispenol, monoacetoxyscirpenol, beauvericin and enniatin a, b, b1 (chełkowski, 1985; ewards, 2002), nevertheless it is considered as little toxic or not producing toxins at all. this has been confirmed by the results of our research, as within numerous f. poae isolates the presence of don gene was identified only in 4 cases. the presence of fum1 gene was found in three (100 %) strains of f. verticillioides, which can produce significant amounts of fumo nisins (gelderblom et al., 1988). it appears paradoxical that the literature data indicate that f. verticillioides is not capable of trichothecene production (marasas et al., 1984; mirocha et al., 1990), although at the same time it possesses genes encoding functional enzymes in the biosynthetic pathway of trichothecenes (kimura et al., 2003). f. culmorum was the last species, in which the examined genes were found. within the collected isolates there was one that possessed the tri13niv, which is one of the nivalenol biosynthesis pathway gene. apart from nivalenol (chełkowski, 1985), f. culmorum can produce moniliformin (scott et al., 1987), deoxynivalenol (marasas et al., 1984), fusarin c (farber and sanders, 1986), zearalenone (marasas et al., 1984) and many other toxic compounds (leslie and summerell, 2006). the multitude of mycotoxins produced by f. culmorum is an important virulence factor of this species, affecting the spread of fusarium head blight (fhb) (snijders and perkowsji, 1990; miedaner and reinbrecht, 2001). according to muthomi et al. 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(eds.), current advances in molecular mycology, 107-138. new york, nova science publishers. address of the corresponding author: katarzyna wolny-koładka, department of microbiology, university of agriculture in cracow, mickiewicza ave 24/28, 30-059 cracow, poland e-mail: k.wolny@ur.krakow.pl © the author(s) 2015. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 90, 106 114 (2017), doi:10.5073/jabfq.2017.090.013 1department of plant anatomy and cytology, maria curie-skłodowska university, lublin, poland 2department of mathematical and statistical methods, poznań university of life sciences, poland 3department of vegetable crops and medicinal plants, university of life sciences, lublin, poland 4botanical garden of maria curie-skłodowska university, lublin, poland effect of environment fluctuations on biomass and allicin level in allium sativum (cv. harnas, arkus) and allium ampeloprasum var. ampeloprasum (ghg-l) dorota tchórzewska1*, jan bocianowski2, agnieszka najda3, agnieszka dąbrowska4, krystyna winiarczyk1 (received september 6, 2016) * corresponding author summary climate variables such as temperature and precipitation are the major abiotic environmental factors determining the yields in crop plants. given the observed trends in climate change, it is important to carry out analyses aimed at description and selection of plant species characterised by not only the best performance traits but also the best adaptation to climate changes. this study focused on phenologicalmorphological-biochemical investigations, comparing allium sativum with a. ampeloprasum var. ampeloprasum ghg-l. we present analyses of economically important traits (biomass and allicin levels) in garlic and ghg-l grown in ecological system and effect of environment fluctuations on these traits. comparative analysis of the biomass and allicin level in the underground part of garlic and ghg-l revealed not only substantial differences between the species and cultivars, but also great impact of the climate variables on these traits. it was found that garlic and ghg-l cultivated in adverse conditions, exhibited lower yielding rates, but the content of allicin was inversely proportional to the biomass. it should be emphasised that, irrespective of the climate fluctuations, ghg-l produced higher biomass and exhibited higher allicin level than garlic grown in the same conditions, indicating that ghg-l is well adapted to adverse climate changes. keywords: allium sativum; allium ampeloprasum var. ampeloprasum; allicin; temperature; drought; precipitation; morphological traits. abbreviations: dad – diode array detector; dw – dry weight; hplc – high-performance liquid chromatography; is – internal standard; lsds – least significant differences; pca – principal component analysis; s.d. – standard deviations. introduction changing environmental conditions are essential for the process of evolution, as they can contribute to expansion of some species and extinction of others. seasonal fluctuations in air temperature and precipitation rates are part of a natural cycle of growth and reproduction in plants. these climate variables exert an impact on the transpiration rate, which determines plant growth and productivity through the drought stress effect (pelter et al., 2004). currently, trends towards an increase in the mean annual temperature are observed in europe (christensen and christensen, 2007) and additionally, periodic precipitation deficits, with lower water availability than plants’ requirements, are one of the most frequent environmental stresses (bray, 1997; kalbarczyk and kalbarczyk, 2014). these atmospheric fluctuations can contribute to loss of crop plant yields, which is of particular importance in view of the human population growth worldwide (bita and gerats, 2013). plants representing the genus allium are widely used by humans, due to their medicinal properties determined by the presence of nume rous sulphur compounds, especially allium sativum (garlic) cultivars have been grown for hundreds of years at all latitudes (block, 2010). one of the most important and best-known bioactive sulphur compounds is allicin, the content of which may account for up to 70 % of all thiosulfinates present in garlic (block, 1992; lawson, 1998; ariga and seki, 2006). allicin is beneficial to human health due to its antimicrobial, anticancer, antiinflammatory, antithrombotic, and antiatherosclrotic activities (block, 1985, 1992; koch and lawson, 1996; singh and singh, 2008). the centuries-long cultivation of the species in varying environmental conditions has resulted in emergence of hundreds of garlic cultivars, whose traits have been conserved through vegetative propagation (simon and jenderek, 2003). therefore, it can be stated that a majority of currently grown a. sativum cultivars are a result of spontaneous point mutations rather than genetic recombinations taking place via sexual reproduction (volk et al., 2004). interestingly, such plants differ from each other, sometimes to a substantial degree, in their phenological and morphological traits (pooler and simon, 1993; pardo et al., 2007; volk and stern, 2009). clove wrapper colour, bulb size, yield, and flavour of garlic are greatly dependent on the growth environment (waterer and schmitz, 1994; volk and stern, 2009), which indicates high plasticity of the species in relation to the environment. particularly noteworthy is the process of a. sativum flowering, which is extremely sensitive to changing environmental conditions. pooler and simon (1994) showed that appropriate day length and temperature are essential factors initiating formation of inflorescence shoots. similarly, other authors emphasise a significant impact of specific temperature and photoperiod regimes on the florogenesis and bulbing processes (mathew et al., 2011). therefore, it can be concluded that garlic is a labile plant responding to varying environmental conditions especially by changes in flowering processes, but the morphological cha racteristics of vegetative organs might be affected as well. it should be noted, that the ability of species to adapt to environmental changes highly depends on genetic heterozygosity, which facilitates generative propagation. given the advantages of sexual reproduction, which ensures unlimited genetic fluctuations, extensive investigations are carried out to elucidate the causes of garlic sterility (kononkov, 1953; novak, 1972; konvicka, 1973; etoh and simon, 2002; kamenetsky et al., 2004; winiarczyk, 2009; shemesh mayer et al., 20013, 2015). although studies on generatively reproducing a. sativum ecotypes from central asia and caucasia (etoh, 1986; etoh et al., 1988; etoh and simon, 2002) have resulted in emergence of fertile lines (inaba et al., 1995; jenderek, 1998; kamenetsky et al., 2003, 2005), there are no reports about a wider use of this type of plants. furthermore, the emergence of fertile lines does not guarantee a possibility of mass production, since fertile garlic grown in a different climate gradually loses its ability to produce seeds (etoh et al., environment fluctuations on biomass and allicin level in garlic and great headed garlic 107 1988). therefore, irrespective of the ongoing research on overcoming garlic sterility, it is important to search for new a. sativum cultivars, that will be best adapted to changing environmental conditions and have the best economically important traits. the climate changes observed currently in europe reflected in a constantly increasing trend in the mean monthly temperatures and decreased precipitation rates, justify the need for systematic analyses, aimed at description and selection of garlic cultivars with not only the best performance traits, but also the best adaptation to climate changes. so far, there have been no reports of the impact of the environment on such performance traits in garlic as yielding and the content of bioactive compounds in the underground parts of the plant. this paper focuses on the so far undescribed relationships between climatic factors, biomass growth, and allicin content in a. sativum and a. ampeloprasum var. ampeloprasum. the study was carried out on garlic cultivars commonly grown in eastern europe, i.e. harnas and arkus, and a. ampeloprasum var. ampeloprasum – great headed garlic (ghg-l) described recently. ghg-l belongs to a separate clade within the genus allium; it is phylogenetically related to a. sativum, which was described by najda et al. (2016). the analyses carried out for the first time, were performed in an ecological cultivation system in the natural environment. the investigations highlight both the climatic-phenological-morphological-biochemical relationships in garlic and ghg-l and the degree of their adaptation and productivity to changing climatic conditions. materials and methods plant materials and growth conditions the experimental material included garlic (a. sativum cultivars harnas and arkus) obtained from krakowska hodowla i nasiennictwo ogrodnicze polan and a. ampeloprasum var. ampeloprasum (ghg-l – genbank number: kt809295-kt809296) deposited in the collection of the botanical garden of maria curie-skłodowska university in lublin under catalogue number 105/2013. all experiments were performed at the botanical garden of maria curie-skłodowska university in lublin situated in the nw part of the town, latitude 51º 16’n and longitude 22º 30’e. the terrain is highly diversified with altitudes from 217.0 m to ca. 178.0 m a. s. l. brown soils formed on loess (33 % of fractions < 0.02 mm) with 2.72 % humus content and 7.1 alkaline ph in 1 mol kcl · dm-3 prevail in the area (index seminum 2014 hortus botanicus universitatis marie curie-skłodowska). the experiment was established in a univariate randomised block design with 4 replications. after drying, single cloves of a. sativum (both cultivars) and ghg-l were planted in autumn 01.10.2013 and 2014. thus, no herbicide, fungicide, or any chemical inputs were incorporated, and manual weeding methods were used in each case before and throughout the investigated plant cultivation periods. data on weather conditions (temperature and rainfall) prevailing during the growing seasons 2013/14/15 as well as the multiyear means (19512005) are presented in fig. 1; numerical data are presented in tab. s1 (supplementary data). phenology and morphology observations of the developmental phases and morphological traits of the analysed species were carried out in the period between clove germination and anthesis of single flowers in the inflorescence. the length of the inflorescence shoot was measured from the base, and the inflorescence composition was analysed after growth and maturation of the analysed species (15.08.2014 and 2015). morphological observations of the underground parts were conducted in the postharvest period (30.07.2014 and 2015). approximately 50 plants from each species were analysed. macroscopic images were taken with a nikon d300 camera equipped with an af micro nikkor 60 mm objective. single flowers from the inflorescences (arkus and ghgl) were examined under a stereoscopic microscope olympus szx16 equipped with a dp 72 camera. biochemical analyses the analysis of the allicin content in the underground parts of a. sativum (both cultivars) and ghg-l were performed immediately after the harvest period. for evaluating the allicin content, we used the methods of lawson et al. (1991) and baghalian et al. (2005). this analysis was performed in the following steps: distilled water was added to 5 g of fresh cloves of a. sativum cultivar harnas and arkus fig. 1: meteorological data from 2013/2014/2015 and multiyear average (1951-2005) rainfall (mm) and temperature (oc). the meteorological station in lublin litewski square (index seminum 2013, 2014, and 2015 hortus botanicus universitatis marie curie-skłodowska). 108 d. tchórzewska, j. bocianowski, a. najda, a. dąbrowska, k. winiarczyk and a. ampeloprasum var. ampeloprasum (10 ml per g), mixed, kept for 10 min at room temperature, and centrifuged at 14000 rpm for 5 min. next, 600 μl of the supernatant was added to methanol (1:1 v/v). the hplc method with butyl parahydroxybenzoate as an internal standard (is) was used for quantification of allicin. the 1 mg of is corresponds to 8.65 mg of allicin. the hplc conditions were as follows: lachrom-merck type equipped with a diode array detector dad (l–7450), quaternary pump (l–7100), degasser (l–7612), injection loop 20 μl, thermostat (l–7360), rheodyne injector, steel column lichrospher 100 rp c 18 (250 mm × 4 mm dimensions) filled with the stationary phase (dp = 5 μm). the mobile phase was methanol: water (50 % v/v) at a flow rate of 0.8 ml/min. peaks were detected at 254 nm and recorded by a lachrom–merck recorder model l–7210. the content of allicin was calculated by the d–7000 hplc system manager program. each sample was measured at least twice. values were expressed as mg allicin 100g-1 dw. statistical analysis two-way analysis of variance (anova) was performed in order to verify the zero hypotheses on a lack of differences between species, between years as well as the hypothesis on a lack of an effect of species × years interaction of the values of the observed traits, i.e. bulb size, bulb weight, clove size, clove weight, and allicin. for individual traits, mean values and standard deviations (s.d.) were calculated. moreover, fisher’s least significant differences (lsds) were also estimated at the significance level α = 0.05. the results were also analysed using multivariate methods. the possibility of graphic distribution of the species in the particular years of the study described by the bulb size, bulb weight, clove size, and clove weight together was obtained with the use of the principal component analysis. principal component analysis (pca) is a mathematical procedure that uses orthogonal transformation to convert a set of observations of possibly correlated variables into a set of values of linearly uncorrelated variables called principal components (nowosad et al., 2016). all the analyses were conducted using the genstat v. 17 statistical software package. results climate parameters the investigations were conducted in the growing seasons of 2013/14 and 2014/15, which were characterised by substantial fluctuations in the climate variables (temperature and precipitation). the precipitation rates in the analysed period were considerably lower in 2015 than in 2014. the differences ranged from 80 % to 120 %, as noted in may (2014 175.7 mm/2015 101.7 mm), june (2014 62.7 mm/2015 16.3 mm), or july (2014 50.0 mm/2015 21.9 mm). the meteorological data from the two analysed seasons were compared with the multiyear sum means. the observations of the weather variables indicate that 2014 and 2015 were warmer by 3.1 °c and 2.7 °c, respectively, than the multiyear temperature means. in turn, the atmosphe ric precipitation rates in the analysed seasons were highly varied. the total precipitation sum in 2014 was by 166.5 mm higher and that in 2015 by 84 mm lower than the multiyear totals (fig. 1). detailed numerical meteorological data are presented in tab. s1 (supplementary data). in the period of intensive biomass growth and accumulation of reserve substances in the allium species, the average air temperature in 2014 was comparable to that in 2015, and the precipitation rate in 2014 was substantially higher than that in 2015. a comparison of the two seasons with the multiyear means revealed a growing trend in air temperature; in turn, precipitation varied widely with clear periodic droughts in 2015. phenology analyses in this study, we analysed a. sativum cv. harnas and arkus as well as a. ampeloprasum var. ampeloprasum ghg-l, plants exhibiting genetic uniformity, investigated by our research team. the material used for reproduction (cloves) was considered to be of high initial quality based on participant surveys. a. sativum had been cultivated for many years in the botanical garden and the yields were monitored every year. harnas, arkus, and ghg-l have to be exposed to low temperatures before the growing season. single cloves of each species were planted in autumn (2013 and 2014) and thus vernalised. in both 2014 and 2015, the first leaves appeared in spring between march 1 and 15 (1-6.03. harnas and arkus, 9-15.03. ghg-l). the inflorescence shoot in harnas, arkus, and ghg-l was formed in late may and early june in 2014 and 2015 (1.05-7.05. harnas, 1.05-5.05. arkus, 20.05-27.05. ghg-l). the observations of the development of the inflorescence shoot in the analysed species showed that anthesis proceeded in the period from 25.07 to 5.08 in arkus and from 2.07 to 12.08 in ghg-l, whereas no anthesis was noted in the harnas cultivar in 2014 and 2015. the development of the aboveground part of the investigated species was synchronous in 2014 and 2015 (fig. 2). fig. 2: period of formation of the first leaf, inflorescence shoot, and anthesis in the species analysed in 2014 and 2015. a. sativum cv. harnas (h), arkus (a), a. ampeloprasum var. ampeloprasum ghg-l (g). morphology analyses the comparative analysis consisted in morphological observations of the aboveground parts in terms of the foliage attitude, leaves width, initiation of the inflorescence, and formation of its components (flowers and bulbils). the underground part was assessed for its shape and the size of the bulb and its components – cloves and bulbils. the a. sativum cultivars, i.e. harnas and arkus, and ghg-l had scaly assimilating leaves, which initially did not differ in their shape and attitude. in contrast, they clearly differed in the width of the leaf blade, which was substantially broader in ghg-l from the beginning of the growth of the aboveground part. furthermore, after emergence of a greater number of leaves, the foliage attitude in ghg-l changed from erect to semi-erect (fig. 3a, e, i). a permanent and characteristic feature of genus allium species is the amount of biomass (number of leaves) indispensable for formation of the inflorescence shoot. prior to the emergence of the inflorescence shoot, harnas and arkus produced 7 leaves, while ghg-l had from 8 to 11 leaves. there were also differences in the shape of the inflorescence shoot; in a. sativum, it was initially curved, but it slightly straightened during the inflorescence maturation, reaching a length of 1000-1200 mm in harnas and 1200-1500 mm in arkus (fig. 3b, f). in turn, the inflorescence shoot in ghg-l was erect from the beginning to the end of its growth and reached a considerable length of 1500-1700 mm (fig. 3j). the inflorescence in the harnas and arkus cultivars and in ghg-l was initially covered by a broad transformed bract called the spathe (fig. 3c, g, k). after spathe dehiscence, an umbel-like inflorescence was visible; in harnas and arkus, it was composed of tiny single flowers and numerous bulbils (fig. 3d, h). in contrast, ghg-l formed numerous flowers but no bulbils in the inflorescence (fig. 3l). the perianth of the flower was green in all the analysed species. during the maturation period until the anthesis stage, it changed colour into purple in arkus and ghg-l. in turn, there was no anthesis in harnas environment fluctuations on biomass and allicin level in garlic and great headed garlic 109 and the colour of the perianth remained green until the end of the vegetation period. in the analysed plants, the underground part, i.e. the bulb composed of cloves, did not differ in its shape of the base and distribution of cloves. harnas, arkus, and ghg-l had a flat shape of the bulb base (fig. 4a, d, g) and radial distribution of bulb cloves (fig. 4b, e, h). the plant differed in the bulb colour (harnas, ghg-l – yellowish and arkus – purple), the number of bulb cloves (harnas 6-15, arkus 5-8, ghg-l 3-11), as well as the presence of bulbils on the peduncles in ghg-l (fig. 4g – small picture) and the absence of bulbils in the harnas and arkus bulbs. tab. 1 presents a summary of the morphological traits of the analysed species. the analysis of the underground biomass was based on measurements of the size of the bulbs and cloves (fig. 4a, c – bar) and the weight of the bulbs and cloves of the analysed species. the data obtained in 2014 indicate that the diameter/weight of bulbs in both harnas (503.2 mm/36.13 g) and arkus (437.9 mm/21.91 g) was significantly lower than that in ghg-l (821.4 mm/95.25 g). this trend was also observed in 2015 (harnas – 436.4 mm/27.65 g; arkus – 366.6 mm/18.82 g; ghg-l – 691.1 mm/84.65 g). similarly, the values of the diameter/weight of cloves in the analysed harnas and arkus cultivars were lower than in ghg-l: harnas – 179.8 mm/2.838 g; arkus – 207.1 mm/3.86 g; ghgl – 296.7 mm/18.722 g in 2014. while in 2015, harnas – 126.0 mm/ 2.658 g; arkus – 175.6 mm/3.817 g; ghg-l – 272.4 mm/14.064 g. the results of the analysis of variance indicated that the main effects of the species and years as well as the effect of the species × years interaction were statistically significant for all observed traits (tab. s2, supplementary data). the multidimensional analysis of the tested traits comparing the species and years in terms of bulb size, bulb weight, clove size, and clove weight simultaneously is shown in fig. 5a, b, c, and d. numerical data are presented in tab. s3 (supplementary data). ghg-l produced greater biomass amounts than garlic; furthermore, the diameter/weight of bulbs and cloves was shown to be gretaer in 2014 than in 2015 in all the species. the multivariate analysis of variance (anova) allowed discarding the tested hypotheses about the lack of multi-trait variability between the species, years and species × years interaction (p < 0.001). individual traits are of different importance and have a different share in the joint multivariate variability. a study on the multivariate variabili ty for treatments also includes identification of the most important traits in the multivariate variation of treatments. principal components analysis pca is a statistical tool making it possible to solve this problem. it showed that the analysed a. sativum cultivars formed separate groups in relation to ghg-l, and their variability ranges did not overlap. all traits with the exception of the clove size, which differentiated the cultivars best, were correlated with the first principal component (pc1). in turn, the clove size was correlated with the second principal component (pc2). pc1 explained as much as 97.67 % of the variability, whereas pc2 explained only 2.23 % of the total variability of the analysed morphological traits (fig. 6). allicin level analyses the phenological and morphological analyses were extended with biochemical assays, which revealed the allicin level in the underground part (cloves) of a. sativum cv. harnas and arkus as well as a. ampeloprasum var. ampeloprasum ghg-l grown in an ecological cultivation system. the analyses showed differences in the content of fig. 3: morphology of the aboveground part of a flowering plant of a. sativum cv. harnas: a, b – inflorescence shoot, c – inflorescence partially covered by a spathe, d – inflorescence devoid of a spathe and a single flower and single topset; cv. arkus: e, f – inflorescence shoot, g – inflorescence partially covered by a spathe, h – inflorescence devoid of a spathe and a single flower during anthesis and single topset; a. ampeloprasum var. ampeloprasum ghg-l: i, j – inflorescence shoot, k – inflorescence partially covered by a spathe, l – inflorescence devoid of a spathe and a single flower during anthesis. 110 d. tchórzewska, j. bocianowski, a. najda, a. dąbrowska, k. winiarczyk this substance between garlic and ghg-l. particularly noteworthy is the fact that there were differences in the allicin level between the vegetation seasons in all the analysed species. the cloves of the harnas cultivar exhibited lower allicin content in 2014 (on average 1.262 g 100g-1 dw) than in 2015 (1.813 g 100g-1 dw). similarly, arkus had lower amounts of allicin in 2014 (1.081g 100g-1 dw) than in 2015 (1.335 g 100g-1 dw). the same tendency in the allicin level in cloves was noted in ghg-l (2014 – 1.343 g 100g-1 dw, 2015 – 2.097 g 100g-1 dw). the results of the analysis of variance indicated that the main effects of the species and years as well as the effect of the species × years interaction were statistically significant for the allicin level (tab. 2 and 3). to sum up, the allicin content was lower in 2014 than in 2015 in all the analysed species. noteworthy, ghg-l was found to be richer in allicin in comparison with the garlic cultivars in both growing seasons. discussion climate variables, e.g. air temperature and atmospheric precipitation are major abiotic factors affecting plant growth and development. they induce ontogenetic development of plants, in particular bulb plants, which can be observed upon vernalisation thereof (kamenetsky and okubo, 2012). crop plants have been observed to have relatively low adaptability to climate variables, such as periodic drought; hence, the genetic yield potential cannot be fully exploited in changing weather conditions (bray, 1997). therefore, the search for species able to adapt efficiently to adverse environmental conditions is desirable. plants representing the genus allium are widely distributed in cultivation and used as a valuable source of bioactive compounds; therefore, maintenance of the high level of yielding is extremely vital. this can be achieved by production of new cultivars with better adaptation to climatic variables or by selection of existing varieties that can adapt to climate fluctuations. since, a. sativum has lost the capability of sexual reproduction, many researchers have focused their attention on the impact of the environment on the formation of generative organs in this plant, and on the process of gametogenesis. this research is important in view of restoration of the capability of generative reproduction and, consequently, effective production of new varieties (inaba et al., 1995; jenderek, 1998; etoh and simon, 2002; kamenetsky et al., 2003, 2004, 2005; shemesh mayer et al., 20013, 2015). on the other hand, there is no detailed information on the relationship between the climate impact and garlic yielding and the content of bioactive substances such as allicin. these investigations focus on assessment of biomass and the level of allicin in two species, garlic and ghg, in response to adverse climate changes. the a. sativum cultivars harnas and arkus analysed in this study are widely grown in central and eastern europe. in turn, fig. 4: morphology of the underground part of a. sativum and a. ampeloprasum var. ampeloprasum. single bulb (the site of the bulb diameter measurement bar): a – harnas, d – arkus, g – ghg-l (bulbils located on a peduncle small picture). cross section across the bulb shows the distribution of cloves: b – harnas, e – arkus, h – ghg-l. single cloves (the site of the clove diameter measurement bar): c – harnas, f – arkus, i – ghg-l. tab. 1: morphological traits of a. sativum (cv. harnas, arkus) and a. ampeloprasum var. ampeloprasum (ghg-l). is – inflorescence shoot. morphological traits harnas arkus ghg-l leaf width narrow narrow broad foliage attitude erect erect semi-erect leaves before is (number) 7 7 8-11 shape of is curved curved erect length of is (mm) 1000-1200 1200-1500 1500-1700 flower colour green purple purple bulbils in the inflorescence present present absent bulb colour yellowish purple yellowish shape of bulb base flat flat flat distribution of bulb cloves radial radial radial number of bulb cloves 6-15 5-8 3-11 bulb with bulbils absent absent present environment fluctuations on biomass and allicin level in garlic and great headed garlic 111 fig. 5: box-and-whisker diagram of the values of a bulb size (mm), b bulb weight (g), c clove size (mm), d clove weight (g); classified by the a. sativum cv. harnas, arkus and a. ampeloprasum var. ampeloprasum ghg-l and the years of the study. fig. 6: principal components analysis (pca) – grouping coordination scheme of a. sativum and a. ampeloprasum var. ampeloprasum ghg-l cultivars in relation to morphological features. 112 d. tchórzewska, j. bocianowski, a. najda, a. dąbrowska, k. winiarczyk the a. ampeloprasum var. ampeloprasum ghg-l is cultivated noncommercially in eastern poland. ghg-l is phylogenetically related to a. sativum but, as we have shown recently, has higher nutritional values than garlic (najda et al., 2016). given the significant climatic differences in the two growing seasons, the periods of 2013/2014 and 2014/2015, the biomass and level of allicin were analysed to focus more light on the relationship between climate fluctuations and biomass production among these two species. the species analysed during the two seasons were grown in an ecological cultivation system in the same area. the seasons were characterised by fluctuations in average temperatures and clearly different values of rainfall. compared to the previous 54 years, higher values of the mean air temperature (by 3.1 °c) and higher mean precipitation rates (by 166.5 mm) were noted in 2014. in contrast, in 2015, the mean air temperature was by 2.7 °c higher, but particularly noteworthy, the mean precipitation rates were by 84 mm lower than the multiyear means. these data indicate a constant increase in the air temperature and occurrence of substantial periodic water shortages, which was also reported from europe (marsz, 2005; bita and gerats, 2013; kalbarczyk and kalbarczyk, 2014). thus, these two vegetative seasons are very suitable to compare the analyses with respect to biomass production by the analysed species. the first stage of our study was the comparative phenological analysis of a. sativum cv. harnas, and arkus and a. ampeloprasum var. ampeloprasum ghg-l. it showed that some periodic phenomena in the development of the analysed species, such as formation of the first leaf and the inflorescence shoot, took place synchronically in arkus and harnas. in contrast, the first leaf and the inflorescence shoot in ghg-l were formed at a later period than in garlic, and anthesis lasted longer than in arkus. in harnas, there was no anthesis stage in the inflorescence. however, no significant phenological differences were observed between the analysed cultivars in both growing seasons (2014 and 2015). another step was the morphological analysis of the aboveground parts of the analysed plants. these observations indicate that harnas and arkus produce assimilating leaves with the same size, shape, and number, and inflorescence shoots with a similar shape, size, and similar constituents. in turn, ghg-l differed from garlic in its size, habit, and inflorescence composition, as it produced numerous flowers but no bulbils. these differences are associated with the fact that ghg-l resembles leek in its aboveground parts, with which it is phylogenetically related (as well as with garlic) (najda et al., 2016). it can be stated that the environmental variables (temperature and rainfall) had no effect on the morphological traits of the aboveground parts of harnas, arkus, and ghg-l during the two analysed growing seasons. it can be explained by the fact that, in the initial stages of analysed plant development, winter soil water resources were sufficient for effective growth of the aboveground parts and the periodic precipitation shortages did not produce a negative effect, indicating that initial water resources are important at first stages of the aboveground parts of the analysed plants. the subsequent comparative analysis of the underground parts revealed that such morphological traits as the shape of bulbs and distribution of cloves were identical in all the analysed species, which however differed in the colour and number of cloves in the bulb. additionally, ghg-l had characteristic bulbils arranged on peduncles, which were not found in harnas and arkus. based on the analysis of the two vegetation seasons, it can be concluded that the climate fluctuations had no impact on the aforementioned features of the underground parts in harnas, arkus, and ghg-l, as is formed at early stage of plant formation. these observations are consistent with previous reports (volk and stearn, 2009), whose authors conclude that such traits as the colour and shape of bulbs and arrangement of cloves in the bulb are species specific and independent of environmental parameters. it has been found that the yields determined as diameter and weight of bulbs and cloves in the analysed plants revealed differences between the cultivars harnas, arkus, and ghgl. noteworthy, the differences between harnas and arkus were inconsiderable, whereas ghg-l was characterised by a significantly greater diameter and bulb and clove weight, compared with garlic. ghg (great headed garlic, synonym “elephant garlic”) is known for the large size of its underground bulb and is cultivated at all latitudes as a garlic substitute (figliuolo et al., 2001; bohanec et al., 2005; hirschegger et al., 2006, 2010; lanzavechia, 2009; najda et al., 2016). it should be emphasized that the economically important trait, i.e. the bulb size, ranks ghg in the first place among the plants from the genus allium. the comparative analysis of the yielding level in the examined plants in both vegetation seasons showed noteworthy dependence. significant differences were observed between the cultivars. in 2015 with water shortage, all the plants produced bulbs with a smaller diameter and weight than in 2014, which implies that the unfavourable climate conditions led to a decrease in the yields of all the analysed cultivars. it should also be emphasised that, irrespective of the lower yields, ghg-l was characterised by greater biomass than that of garlic in both growing seasons and produced the highest yield. our observations are in line with other authors, which reported that garlic yielding depends not only on the genetic traits of the species, but largely on climatic variables (waterer and schmitz, 1994; volk and stern, 2009; wang et al., 2014). the yielding efficiency is especially important in crop plants, but an equally important trait in garlic is the content of bioactive compounds, e.g. allicin. the presented comparative analysis of this compound in the bulb (cloves) revealed intriguing results, which has never been reported for garlic species, ecologically cultivated in the natural environment. the level of allicin contained in the cloves differed not only between cultivars harnas, arkus, and ghg-l, but interesting dependence was noticed between the studied seasons, the level of allicin was higher in all the plants analysed in 2015 with seasonal droughts, although their yield was lower than that in 2014. these results are consistent with previous observations carried out in isotab. 2: mean squares from analysis of variance for allicin. source of variation number of degrees mean squares of freedom year 1 1.23343*** species 2 1.16741*** year × species 2 0.05522*** residual 24 0.00008 *** p<0.001 tab. 3: mean values and standard deviations (s.d.) of allicin levels (dry mass) in raw a. sativum cv. harnas, arkus and a. ampeloprasum var. ampeloprasum ghg-l (g 100g-1 dw). year 2014 2015 species mean s.d. mean s.d. harnas 1.262 0.00608 1.813 0.00841 arkus 1.081 0.00576 1.335 0.0092 mean for cv. 1.172 1.574 ghg-l 1.685 0.00394 2.097 0.01626 mean for all species 1.343 0.262 1.748 0.3259 lsd0.05 year: 0.0069; species: 0.0085; year × species: 0.01198 environment fluctuations on biomass and allicin level in garlic and great headed garlic 113 lated systems, which showed a correlation between increased allicin content and decreased bulb weight, which was associated with modulation of the metabolism of these plants by humidity and temperature (bloem et al., 2011). furthermore, lee et al. (2013) have shown a correlation between the activity of γ-glutamyl transpeptidase (ggt), i.e. an enzyme involved in the metabolism of sulphur compounds and the temperature and humidity, which may be a direct factor contri buting to the increase in the allicin content in garlic heads. additionally, a close relationship between the growing conditions (fertilisation) and the sulphur content in plants from the genus allium has also been reported (wang et al., 2014). thus, allicin level may represent a sensitive indicator of metabolic changes upon fluctuating growth conditions. conclusion this paper presents for the first time an analysis of economically important traits in garlic and ghg-l grown in an ecological cultivation system, without human interference. the comparative analysis of the development of the aboveground and underground parts of each species included an important yield-determining element, i.e. environment fluctuations during the two vegetation seasons. analysed plants grown in adverse climatic conditions (periodic droughts) were cha racterised by lower mass yields, but the allicin content was inversely proportional to the increase in biomass. in the adverse soil moisture conditions in 2015, garlic cv. harnas exhibited a substantial increase in the level of allicin contained in the underground bulbs. it should be emphasised that, irrespective of the climate fluctuations, ghg-l had a significantly higher biomass and a higher level of allicin than garlic grown under the same conditions. the correlations between the bulb size and the allicin content in harnas, arkus, and ghg-l in both growing seasons are presented in fig. 7. as already reported in earlier studies and according to the data presented in this paper, it can be concluded that a. ampeloprasum var. ampeloprasum – ghg-l represents much better characteristics from the agricultural and biochemical/nutritional point of view. thus, greater popularisation of ghg and introduction thereof to large-scale cultivation should be considered as an alternative to traditional garlic, especially in regions with significant climate fluctuations. acknowledgements d.t. – conceived the study, interpreted the data, wrote the manuscript, j.b. – statistical analyses, a.n. – biochemical analyses, a.d. – provided materials, k.w. – conceived the study, interpreted the data. reference ariga, t., kumagai, h., yoshikawa, m., kawakami, h., seki, t., sakurai, h., hasegawa, i., etoh, t., sumiyoshi, h., tsuneyoshi, t., sumi, s., iwai, k., 2002: garlik-like but odorless plant allium ampeloprasum mushuu-ninniku. j. jpn. soc. hortic. sci. 71, 362-369. doi: 10.2503/jjshs.71.362. ariga, t., seki, t., 2006: antithrombotic and anticancer effects of garlicderived sulfur compounds: a review. biofactors 26, 93-103. doi: 10.1002/biof.5520260201. baghalian, k., ziai, s.a., naghavi, m.r., badi, h.n., khalighi, a., 2005: evaluation of allicin content and botanical traits in iranian garlic (allium sativum) ecotypes. sci. hortic. 103, 155-166. doi: 10.1016/j.scienta.2004.07.001. bita, c.e., gerats, t., 2013: plant tolerance to high temperature in changing environment: scientific fundamentals and production of heat stresstolerant crops. front. plant sci. 4, 1-18. doi: 10.3389/fpls.2013.00273. block, e., 1985: the chemistry of garlic and onions. sci. am. 252, 114-119. block, e., 1992: the organosulfur chemistry of the genus allium. implications for the organic chemistry of sulfur. angew. chem. 31, 1135-1178. block, e., 2010: garlic and other alliums: the lore and the science. royal society of chemistry, cambridge. bohanec, b., jakse, m., sesek, p., havey, m.j., 2005: genetic characterization of an unknown chinese bulbous leek-like accession and its relationship to similar allium species. hortscience 40, 1690-1694. bray, e.a., 1997: plant responses to water deficit. trends in plant science 2, 48-54. doi:10.1016/s1360-1385(97)82562-9. christensen, j.h., christensen, o.b., 2007: a summary of the prudence model projections of changes in european climate by the end of this century. clim. change 81, 7-30. doi: 10.1007/s10584-006-9210-7. etoh, t., 1986: fertility of the garlic clones collected in soviet central asia. j. jpn. soc. hortic. sci. 55, 312-319. etoh, t., noma, y., nishitarumizu, y., wakamoto, t., 1988: seed productivity and germinability of various garlic clones collected in soviet fig. 7: correlations between the bulb size and allicin level of the a. sativum cv. harnas and arkus and a. ampeloprasum var. ampeloprasum ghg-l in the years of the study. graphic signs: black droplet – allicin level (number of droplets corresponds to the allicin content), bulb size – yields. 114 d. tchórzewska, j. bocianowski, a. najda, a. dąbrowska, k. winiarczyk central asia. mem. fac. agric. kagoshima univ. 24, 129-139. etoh, t., simon, p.w., 2002: diversity, fertility and seed production in garlic. in: rabinowitch, h.d., currah, l. 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10.1007/s10681-014-1097-1. waterer, d., schmitz, d., 1994: influence of variety and cultural practices on garlic yields in saskatchewan. can. j. plant. sci. 74, 611-614. doi: 10.4141/cjps94-110. winiarczyk, k., 2009: badania embriologiczne bezpłodnych ekotypów allium sativum l. wydawnictwo umcs, lublin, 112. volk, g.m., henk, a.d., richards, c.m., 2004: genetic diversity among u.s. garlic clones as detected using aflp methods. j. amer. soc. hort. sci. 129, 559-569. volk, g.m., stern, d., 2009: phenotypic characteristics of ten garlic cultivars grown at different north american locations. hortscience 44, 12381247. address of the corresponding author: dorota tchórzewska, department of plant anatomy and cytology, maria curie-skłodowska university, akademicka 19 street, 20-033 lublin, poland. e-mail: dorota.tchorzewska@poczta.umcs.lublin.pl © the author(s) 2017. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). supplementary material i tab. s1: meteorological data from 2013/2014/2015 and multiyear average (1951-2005) rainfall (mm) and temperature (oc). the meteorological station in lublin litewski square (index seminum 2013, 2014, and 2015 hortus botanicus universitatis marie curie-skłodowska) 3     meteorological data from 2013/2014/2015 and multiyear average (1951-2005) rainfall (mm) 74   and temperature (oc). the meteorological station in lublin litewski square (index seminum 75   2013, 2014, and 2015 hortus botanicus universitatis marie curie-sk�odowska) 76   77   month decade rainfall (mm) temperature (oc) 2013/14 2014/15 long-term 2013/14 2014/15 long-term 1 0 0 19.4 7.5 11.1 9.8 2 4.4 1 14.6 9.3 13.6 8.2 x 3 1 24.6 12.3 13.3 4.2 6 1 56.6 2.6 13.7 8.9 9.9 4.7 2 5.4 13.7 12 4.1 4.8 2.2 xi 3 18 6.4 11.4 3.2 -1.6 0.6 1 13 3.9 9.9 0 -2.6 -0.6 2 3.2 19.9 9.9 0.7 4.2 -1.5 xii 3 0 32.7 13.9 3.8 -1.2 -2 1 18.5 24.6 7.3 4 -1.3 -3.5 2 45.2 4.9 6 -0.4 3.2 -3.7 i 3 7.1 17.3 8.3 -1.4 1 -3.7 1 4.4 2.9 7.7 -1.5 -1.4 -3.3 2 7.7 0 9.1 3 0.1 -2.8 ii 3 0.2 4.5 8.1 2.7 4.5 -2.1 1 3.8 9.9 8.4 3.3 3.4 -0.7 2 19.7 15.7 7.4 6.2 3.8 0.4 iii 3 24.9 30.2 10.1 8.8 6.5 3 1 3.2 12.7 14.2 7.4 4.4 5.9 2 15.3 2.8 12.3 7.9 8.9 6.9 iv 3 26.2 25.3 14.1 14.1 12.5 9.6 1 32.7 26.8 16.6 10.6 13.1 11.6 2 88.4 8.9 18.3 12.9 13.4 13.6 v 3 72.5 76.2 23.5 17.4 13.3 13.7 1 11.2 11.1 20.8 17.4 19.1 16 2 3 0.1 21.2 16 17.9 16.3 vi 3 63.9 0 23.8 15.4 17.1 17.1 1 14.2 0.4 23.5 19.4 21.4 17.4 2 38.4 23.5 25.7 19.8 19.8 18.2 vii 3 30.6 19.7 29 21.8 20.7 18  78   table s2 79   ii supplementary material 4     mean squares from analysis of variance for the observed traits of the studied species 80   trait source of variation species year species × year residual d.f. 2 1 2 335 bulb size m.s. 7456640*** 1342025*** 68301*** 6413 d.f. 2 1 2 274 bulb weight m.s. 297118.6*** 8660.8*** 974.5* 407.9 d.f. 2 1 2 592 clove size m.s. 956818*** 258135*** 13966** 2308 d.f. 2 1 2 692 clove weight m.s. 11790.41*** 288.64*** 363.45*** 22.32 * p<0.05; ** p<0.01; *** p<0.001 81   82   table s3 83   mean values and standard deviations (s.d.) for the observed traits of a. sativum cv. harnas, 84   arkus, and a. ampeloprasum var. ampeloprasum ghg-l 85   86       bulb size bulb weight year 2014 2015 2014 2015 species mean s.d. mean s.d. mean s.d. mean s.d. harnas 503.2 73.89 436.4 51.73 36.13 13.52 27.65 8.17 arkus 437.9 72.2 366.6 58.06 21.91 11.11 18.82 7.52 ghg-l 821.4 147.83 691.1 162.59 95.25 34.57 84.65 35.48 lsd0.05 s: 14.58; y: 11.52, s×y: 21.00 s: 3.07; y: 2.425, s×y: 4.423   clove size clove weight year 2014 2015 2014 2015 species mean s.d. mean s.d. mean s.d. mean s.d. harnas 179.8 62.81 126.0 36.37 2.838 1.479 2.658 1.351 arkus 207.1 39.41 175.6 25.38 3.86 1.964 3.817 1.843 ghg-l 296.7 65.77 272.4 52.61 18.722 11.673 14.064 8.873 lsd0.05 s: 8.73; y: 6.9, s×y: 12.58 s: 8.73; y: 6.9, s×y: 12.58 87    88   89   tab. s2: mean squares from analysis of variance for the observed traits of the studied species tab. s3: mean values and standard deviations (s.d.) for the observed traits of a. sativum cv. harnas, arkus, and a. ampeloprasum var. ampeloprasum ghg-l journal of applied botany and food quality 91, 226 231 (2018), doi:10.5073/jabfq.2018.091.030 department of horticulture, faculty of agriculture, urmia university, urmia, iran 24-epibrassinolide enhanced the quality parameters and phytochemical contents of table grape mohammadreza asghari*, rana rezaei-rad (submitted: february 24, 2017 accepted: october 6, 2017) * corresponding author summary enhancing the nutritional quality of fruits using safe and environmental friendly methods has become one of the most important targets in modern fruit production systems. brassinosteroids, a new group of phytohormones with positive roles on human health, have been shown to modulate a wide range of plant activities and enhance fruit quality in some crops. this study was conducted to examine the effect of 24-epibrassinolide (ebl), a synthetic brassinosteroid, on quality attributes and some active bio-compounds of ‘thompson seedless’ table grapes. grape vines and bunches were sprayed with ebl (at 0, 3 and/or 6 μmol l-1) at three different stages (4 weeks after full bloom, at veraison stage and one day before harvest). as a novel finding in seedless grapes, exogenous ebl substantially enhanced soluble solids content, total organic acids, antioxidants, phenolics and ascorbic acid levels in treated berries. also the activity of catalase and polyphenol oxidase enzymes was increased. there was no significant difference between the two tested brassinosteroid concentration levels in most cases. ebl showed a good potential for enhancing table grape phytonutrients, nutritional quality and phytochemical contents and can be introduced as a safe compound to be used in table grape production programs. keywords: brassinosteroid, polyphenol oxidase, phenolic compounds, phytochemicals, total antioxidant activity, ‘thompson seedless’ introduction few fruits have garnered as much attention in the health research projects as grapes. in addition to being rich in some important biocompounds necessary for human health including antioxidants, phenolics and anthocyanins, part of the reason for the importance of grapes may be their widespread presence in diets worldwide and profound economic importance. grapes are cultivated in almost all countries and are consumed in different kinds of foods including fresh fruit, raisins, vinegar, juices, wines, seed oil and also medicinal and cosmetic products (eyduran et al., 2015). the health benefits of fresh grapes are mainly related to their phenolic compounds such as gallic acid, catechin, anthocyanins and resveratrol and a wide variety of procyanidins. these phytochemicals have been reported to have a wide range of pharmacological effects, including anti-carcinogenic, anti-atherogenic, anti-inflammatory, antimicrobial and antioxidant activities (luan et al., 2013; harindra-champa, 2015). improving the quality including phytonutrients, phytochemicals, antioxidants and vitamins of fruit not only results in enhanced marketability, nutritional and health improving properties of the berries, but also increases the quality of byproducts. different quality parameters including nutritional properties are determined by genetic, environmental factors and cultural activities (harindra-champa, 2015). for many years, the use of chemicals, including biocides against biotic stresses and inorganic nutrients for supplying essential elements for the plants, has been the main strategy to enhance crop quality during production. different chemicals have been used to control diseases and disorders of crops and maintain the quality during hand ling and storage operations. however, for food safety issues and environmental concerns, recently the use of chemicals in food production systems has highly been restricted and organically produced foods, or foods with the least chemical residues, are preferred by the consumers (asghari and soleimani-agdam, 2010; romanazzi et al., 2016). it is well known that different plant growth regulators (pgrs) and phytohormones are the main players in different plant growth processes and new findings show that the use of different pgrs may be considered as an effective strategy to manage the plant growth and enhance the crop quality during growth stages (vardhini and anjum, 2015). while there is a little evidence about the effects of new plant growth regulators and phytohormones, the role of some classic pgrs such as ethylene, abscisic acid and gibberellins on yield, qua lity and responses of different table grape cultivars against different stresses has been widely studied. according to recent studies, among different phytohormones brassinosteroids (brs) may have more crucial roles in development and ripening of table grapes (symons et al., 2006; harindra-champa, 2015; işçi and gökbayrak, 2015). brassinosteroids are plant-specific polyhydroxylated derivatives of 5α-cholestane, structurally similar to cholesterol-derived animal steroid hormones. recent studies indicate the positive roles of brs in human health including inhibition of herpes simplex virus type 1 (hsv-1) and arenavirus, measles, junin and vesicular stomatitis virus replication in cell culture (esposito et al., 2011). brassinosteroids regulate the expression of specific plant genes and complex physio logical responses related to different growth and defense mechanisms, cell division and enlargement, nutrient uptake, antioxidant systems, co2 enrichment, fruit quality and stress related responses (divi and krishna, 2009; choudhary et al., 2012; asghari and zahedipour, 2016). studies show that with the onset of ripening the concentration of natural brs is increased in table grapes, indicating a positive role for these phytohormones in berry development and ripening (rees et al., 2012; harindra-champa, 2015). according to the findings of symons et al. (2006), exogenous brs may enhance skin colora tion and sugar accumulation and promote ripening process in table grapes, while brassinazole (an inhibitor of br biosynthesis) significantly delays fruit ripening. enhancing the natural defense systems of plants against pathogens and abiotic stresses is one of the most important and unique roles of brs in plants. increase in yield and quality of some horticultural crops has been reported after treatment with brs and according to the reports the effects of brs depend on plant growth stage, environmental conditions and br concentrations (divi and krishna, 2009; vardhini and anjum, 2015). however, the positive effects of brs on table grape production is not limited to fruit ripening. a wide range of different genes and enzymes may be regulated or affected by brs. some evidence in dicate that brs may enhance the synthesis of phytonutrients and activate resistance of grapes against different stresses. according to the findings of zhu-mei et al. (2013), external ebl has been shown to enhance the resistance related phytochemicals and subsequent resistance of ‘zicuiwuhe’ table grape seedlings against chilling stress. the authors reported that ebl treatment has increased the gene 24-epibrassinolide enhanced the quality of table grape 227 expression and enzyme activity of some antioxidant and anti-stress enzymes. the positive effects of ebl on maintaining fruit quality, enhancing postharvest life and decreasing grey mold extension in table grapes has been reported by liu et al., (2016). treatment of strawberry seedlings with ebl simultaneously enhanced some growth parameters and disease resistance systems of strawberry plants, acting as a growth-promoting and relatively stress-mediating agent at low concentrations while strongly enhancing stress resistance mechanisms at higher doses (asghari and zahedipour, 2016). the role of brs in increasing leaf photosynthesis rate and improving the capacity of grape vines under stress conditions has also been reported by wang et al. (2015). the important roles of brs in promoting plant growth and enhancing natural stress related phytochemicals make it an appropriate candidate for organic crop production systems. ‘thompson seedless’ is one of the most important table grape cultivars cultivated and exported worldwide. it is considered for relatively large berry size, high soluble sugars and capacity for both fresh consumption and processed to raisins, vinegar and wines. however, reports about brs influencing the phytochemicals and different antioxidant fractions in grapes are few and there is no information about the effects of exogenous brs on fruit quality parameters. on the other hand, in most of the studies on table grapes and other fruit crops, the effect of single spray with exogenous brs have been studied. it has been well demonstrated that the effect of exogenous phytohormones on plant physiological traits and fruit quality is mostly dependent on crop species, phytohormone concentration, and the time and number of applications (işçi and gökbayrak, 2015). since a few days after application, the exogenous phytohormones are inactivated by plant cells, then it seems that in order to achieve the best results the hormone application should be repeated several times during plant and fruit growth stages. in this study, we examined the effect of spraying exogenous ebl at 3 successive stages on ‘thompson seedless’ table grape quality parameters and some natural phytonutrient contents. materials and methods treatment of vines and bunches with ebl: the experiment was performed on 12-year old own rooted grapevines (vitis vinifera l. cv. thompson seedless) planted at 2 m × 3 m spacing, trained on cordon system in a commercial vineyard located in urmia, iran. the orchard was trained according to standard cultural practices inclu ding guyot-training and drip irrigation. nine vines (three vines per treatment) were chosen and uniform cultural practices were adopted according to recommendations (mahindra, 2010). in order to de termine the effects of exogenous ebl on fruit quality indices, vine canopy and bunches were sprayed with ebl solutions at 3 different growth stages: 1) four weeks after full bloom, 2) veraison stage, and 3) one day before harvest. ebl was obtained from sigma (st. louis, mo, usa) and different concentrations of solutions (0, 3 and 6 μmol l-1) were applied. appropriate amount of ebl to reach the desired concentration was dissolved in ethanol and then made to volume with distilled water. final concentration of ethanol in each solution was about 0.1% (1 ml ethanol in 1000 ml of ebl solution) and the same concentration of ethanol was added to distilled water used for treating control vines. table grapes were harvested at commercial maturity (when the control berries reached 18.5 °brix) and immediately transferred to postharvest laboratory. grape clusters were selected for uniformity of size, shape, color and freedom from blemishes and subjected to quality analysis. determination of total soluble solids (tss), ph, total acidity (ta) and ascorbic acid (aa) content: all chemicals were purchased from sigma (sigma-aldrich co. germany). berry tss, ph and ta were determined according to the method described by ayala-zavala et al. (2007). 40 berries from each replicate, two berries from the shoulder, 2 from the middle and 1 from the bottom of each bunch, were wrapped in cheesecloth and squeezed with a hand press. tss was determined at 20 °c with an atago dbx-55 refractometer (atago co. ltd., tokyo, japan). ph was evaluated by a ph-meter (az-8601, china). ta was determined by diluting each 5 ml aliquot of grape berry juice in 95 ml of distilled water and then titrated to ph=8.2 using naoh (4 g l-1). aa content was determined according to the method described by ballentine (1941). the berry juice was extracted by pressing the berries and filtered using a muslin cloth, 5 ml of juice was added to 1 ml of 10% potassium iodide (ki) and 2 ml of 2 n sulfuric acid and the resulting solution was titrated with 0.01 n iodate until the starch was formed. 1 ml of 0.01 n iodate corresponds to 0.88 mg of aa. evaluation of catalase (cat) and polyphenoloxidase (ppo) enzymes activity, total phenolics content (tpc) and total anti oxidant activity (taa): all enzyme extract procedures for whole berry flesh were conducted at 25 °c. cat activity was measured according to beers and sizer (1952) with slight modifications. the reaction mixture consisted of 2.5 ml sodium phosphate buffer (50 mmol l-1, ph 7.0), 0.2 ml h2o2 (1%) and 0.3 ml enzyme. the decomposition of h2o2 was measured by the decline in absorbance at 240 nm. the specific activity was expressed as u mg-1 protein, where one unit of catalase converts 1 mol of h2o2 per min. the activity of ppo enzyme was determined using the method described by pizzocaro et al. (1993). enzyme activity was assayed by determining the rate of increase in absorbance at 420 nm and 25 °c. the reaction mixture contained 0.5 ml of enzyme extract and 2.5 ml of buffered substrate (100 mmol l-1 sodium phosphate, ph=6.4, and 50 mmol l-1 catechol). the linear section of the activity curve as a function of time was used to determine the ppo activity (u mg-1 protein min-1). the unit for the ppo activity was defined as a change of 0.001 in absorbance at the conditions of the assay. tpc of the whole berry extracts was determined by folin-ciocalteu method and was expressed as mg gallic acid kg-1 on a fresh weight basis. for extraction, 1 gr of berry sample was homogenized with a solution composed of methanol/hcl (v/v 2:28). then, the mixture was centrifuged at 10000 g and 4 °c for 10 min. the supernatant was used for the assay of total phenolics content. for the assay 0.1 ml of this extract was mixed with 0.5 ml of folin-ciocalteu reagent and 7 ml distilled water. after incubating for 2 min at room temperature in the dark, 1 ml of sodium carbonate saturated solution was added and the samples were incubated again for 2 h at room temperature. the absorbance of mixture was read at 760 nm using a spectrophotometer (model, analytik jena specord 200, germany) and the sample phenolics content was expressed as mg gallic acid 100 g-1 dry weight (dw) (plessi et al., 2007). taa of berry juice was determined by ferric ions reducing antioxidant power assay (frap) according to benzie and strain (1996) with slight modifications. the stock solutions included 5 ml of a 10 mmol l-1 tptz (2, 4, 6-tripyridyl-s-triazine) with 40 mmol l-1 hcl plus 5.41 ml of fecl3 (20 mmol l-1) and 50 ml of phosphate buffer, (0.3 mol l-1, ph=3.6) and was prepared freshly and warmed at 37 ºc. berry extracts (150 ml) were allowed to react with 2.85 ml frap solution and the absorbance of reaction mixture at 593 nm was measured spectrophotometrically after incubation at 37 ºc for 10 min. for construction of calibration curve five concentrations of feso47h2o (1000, 750, 500, 250, 125 μmol l-1) were used to obtain the calibration curves. the values were expressed as the concentration of antioxidants having a ferric reducing ability equivalent to that of 1 mmol l-1 feso4. (y = 0.0009 × 0.0275, r2 = 0.995). 228 m. asghari, r. rezaei-rad statistical analysis the experiment was conducted as a completely randomized design with 3 ebl levels and 3 replicates (3 vines). 5 bunches were harvested from each vine (replicate) for quality analysis and bulked prior to analysis. the data were analyzed by repetitive measures analysis of variance using sas (v 9.3, sas institute inc., usa) package and means were compared by duncan’s multiple range test. differences at p≤ 0.05 were considered significant. results tss, ph, ta and aa content: as shown in fig. 1a, berries from vines sprayed with ebl had a significantly higher tss than the control (p≤0.05) and a substantial increase in tss content of treated berries was recorded. there was no significance difference between the two levels of ebl. ph value of the berry juice was decreased as the result of treatment with ebl and the effect was concentration dependent (p≤0.05) (fig. 1b.). as shown in fig. 1c, total organic acid content of the berries from treated vines was higher than the control and there was no significant difference between the two ebl levels (p≤0.05). according to the data shown in fig. 1d, ebl treatment had a significant effect on aa content of ‘thompson seedless’ grape berries (p≤0.05) at the lower ebl concentration, but not for the higher one. also the difference between ebl concentrations was not statically significant. cat and ppo enzymes activity, total phenolics and total antioxidants contents: cat and ppo, as important antioxidant and antistress enzymes in plants, were significantly affected by pre-harvest ebl treatment (p≤0.01). as shown in fig. 2a, b, the activity of these enzymes was significantly enhanced in response to ebl treatment. with increase in ebl concentration, the effect of phytohormones on cat and ppo was not significantly increased. as shown in fig. 2c, d, ebl spray effectively enhanced fruit tpc and taa (p≤0.01). while ebl in a concentration dependent manner enhanced the total phenolics content it was more effective on enhancing tpc and taa at 3 μmol l-1 (significant diffe rence between treatments for tpc, while not for taa). discussion some bioactive compounds of the berries like phenolics, stilbenes and antioxidants are main factors determining the quality of both fresh and processed products of grapes (xia et al., 2010). increase in berry phenolics and other antioxidants are not only important for fresh table grapes but also directly affects the quality of byproducts. also high quality berries have high storage capacity and are suitable for export (jaakola, 2013). increase in tss and ta of the berries results in enhanced organoleptic property and nutritional quality. there are contradictory reports about the effect of brs on aa content in plants and harvested crops. for example, exogenous br has been reported to increase the aa levels in cultured cells of chorispora bungeana under stress conditions (liu et al., 2009). in contrast, br treatment has been shown to decrease aa content in tomatoes and strawberry fruits (hayat et al., 2012; asghari and zahedipour, 2016). according to our data, exogenous ebl enhanced the aa content of grape berries. brs may enhance the synthesis of ascorbic acid in plant cells by enhancing the activity of l-galacton-1, 4-lacton dehydrogenase (l-galdh). this enzyme catalyzes the last step of ascorbic acid biosynthesis. according to the findings of debolt et al. (2006), ascorbic acid is also used as a precursor for the synthesis of tartaric acid in grapes and the rate of ascorbic acid conversion to tartaric acid is increased during berry ripening. since brs play roles in signaling pathways of other plant hormones involved in ripening process, it has been demonstrated that brs are the latest phytohormones implicated in the control of table grape berry ripening and anthocyanin accumulation (luan et al., 2013). effects of ebl on enhancing fruit quality parameters and phytochemical contents including antioxidants, phenolics, ascorbic acid fig. 1: effect of ebl treatment on total soluble solid (tss) content (a), ph (b), total acidity (ta) (c) and ascorbic acid (aa) content (d) in ‘thompson seedless’ table grape. different lowercase letters indicate the significance difference between the means (r = 5) according to duncan’s multiple range test (p ≤ 0.05). 24-epibrassinolide enhanced the quality of table grape 229 (vitamin c) and soluble sugars is mainly due to effects on photosynthesis reaction. exogenous brs may substantially enhance the photosynthesis activity in different plants. brs may enhance the net photosynthesis rate by enhancing chlorophyll and carotenoid development and playing crucial roles in gene expression and enzyme activity of some important photosynthetic enzymes such as rubisco (vardhini and rao, 1998; yu et al., 2004; vardhini and anjum, 2015; asghari and zahedipour, 2016). in addition, the role of brs in increasing the absorption and transport of co2 in leaves and enhancing stomatal conductance has been reported in our pre vious studies on strawberry plants (asghari and zahedipour, 2016). increase in photosynthesis rate results in enhanced carbohydrate production. carbohydrates produced during photosynthesis reaction are not only used as precursors for different structures and bio-compounds during normal growth and development of the cells, but also modulate the growth and metabolic processes in plants via mediating gene expression and enzymes activity (koch, 1996). reactive oxygen species (ros) and free radicals are produced during normal cell metabolism and should immediately be removed after production. chloroplasts, mitochondria and peroxisomes are the main sites of free radical and ros generation during photosynthesis, respiration and photorespiration. also free radicals and ros are produced during normal metabolisms in human cells. ros and free radicals, in the absence of antioxidant systems, are able to destroy the living cells by creating the oxidative burst. different stresses, metabolic activities and physical and chemical conditions of the cells, such as antioxidant activity and ph, may substantially affect the oxidative burst (metwally et al., 2003). plant and human cells protect themselves against free radicals and ros using different antioxidant systems, including enzymatic antioxidants such as cat, superoxide dismutase (sod), peroxidase (pod), and non-enzymatic ones such as ascorbic acid and phenolics. in fact, the production and activity of different antioxidants are necessary for suppressing oxidative damage in cells. cat is one of the most important antioxidants scavenging h2o2. elevated levels of h2o2 are toxic to the cells and cat immediately converts this molecule to h2o and o2, leading to protection of cells from h2o2 damage (gayatridevi et al., 2013). effect of brs on enhancing the antioxidant systems in plant cells and activating plant resistance against oxidative damage caused by pathogens and environmental conditions such as drought, salinity, heavy metal, high temperature and chilling stresses in some plants has been well demonstrated (vardhini and anjum, 2015). brs have been shown to enhance total antioxidant capacity and some antioxidant fractions in some plants including ‘zicuiwuhe’ table grape seedlings and some harvested crops (zhu et al., 2010; zhu-mei et al., 2013). phenolics are important bio-chemicals in foods. grapes are rich in different important phenolic compounds such as, catechins, epicate chins, procyanidins, proanthocyanidins, viniferones, quercetin, kaempferol, myricetin, isorhamnetin, caffeic acid, coumaric acid, ferulic acid and gallic acid, all providing the human body with antioxidant, anticancer, anti-inflammatory and anti-aging benefits (paredes-lopez et al., 2010). in addition to acting as powerful antioxidants, these compounds have been shown to play roles in a series of plant and fruit physiological processes including growth, color development and anti-stress responses (asghari and zahedipour, 2016). exogenous application of some phytohormones and pgrs have been reported to affect the metabolism and biosynthesis of phenolics in plants (luan et al., 2013). brs enhance production of phenolic compounds and consequent resistance against biotic and abiotic stresses in different plants and harvested crops by increasing gene expression and enzyme activity of phenylalanine ammonia lyase (pal), the main enzyme responsible for production of phenolics, and polyphenol oxidase (asghari and zahedipour, 2016; gao et al., 2016). ppo (ec 1.10.3.1.) is a copper-containing enzyme catalyzing the oxidation of o-diphenols to o-diquinones. the main function of quinones in plants seems to be the mediation of resistance induction against pathogens and unfavorable conditions giving plants the ability of surviving and maintaining productivity under stress conditions. these compounds have been shown to have freeradical scavenging capacity, anti-coronary, anti-cancer, antivirus, antioxidant and anti-inflammation activities, prevent metabolic di fig. 2: effect of ebl treatment on catalase (cat) enzyme activity (a), polyphenoloxidase (ppo) enzyme activity (b), total phenolics content (tpc) (c) and total antioxidant activity (taa) (d) in ‘thompson seedless’ table grape. different lowercase letters indicate the significance difference between the means (r = 5) according to duncan’s multiple range test (p ≤ 0.01). 230 m. asghari, r. rezaei-rad seases and protect umbilical vascular endothelial cells in human body cells (paredes-lopez et al., 2010; xia et al., 2010). as an important resistance related enzyme, ppo improves the resistance of fruits and plants against pathogens and pests by oxidizing phenolic compounds to quinines. increase in ppo activity during growth stages not only results in high quality grapes but also is crucial for establishment of an efficient resistance network in plants and fruits, making the fruit more resistant against postharvest losses (yoruk and marshal, 2003; zhu-mei et al., 2013). because phenolic compounds have antifungal and antibacterial effects, therefore, enhancing ppo activity and increasing total phenolics content of fruit with brs may help producing fruit with no further need for the use of chemical biocides. conclusions exogenous brassinosteroid substantially enhanced ‘thompson seedless’ berry quality attributes, natural phytochemicals, antioxidants and biochemical compounds. increase in fruit biochemical content is of most important priority for food scientists. according to the data from this study we may conclude that ebl at 3 and 6 µmol l-1, enhances berry quality indices and promotes health saving benefits of table grapes by enhancing phenolic componds biosynthesis and accumulation, ppo and cat enzymes activity and total phenolics, different antioxidant fractions, total antioxidant capacity and ascorbic acid content. increased tss and total acidity and decreased ph as well as increased antioxidants, phenolics and ascorbic acid content of berries in an organic production system, without further need for the use of chemicals, results in enhanced nutritional property and medicinal quality of fresh berries and its byproducts. interestingly, ebl acted as a growth enhancing, photosynthesis promoting and resistance mediating agent. therefore, the increase in fruit quality parameters and phytonutrient contents is not at the expense of reduced crop yield and because of decreasing the need for disease control, the cost of ebl spray is economically acceptable. increase in different phytochemicals and bio-compounds such as phenolics, total antioxidant capacity, antioxidant enzymes and ascorbic acid not only enhances the nutritional quality, which is very important for the consumers, but also promotes the fruit storage life. since ebl treatment had no adverse effect on crop yield (unpublished data), we could recommend exogenous brassinosteroid treatment as a safe cultural activity for enhancing table grape quality, safety and nutritional property. since no significant difference was seen between the two ebl levels in most cases, then 3 μmol l-1 is recommended for use in commercial scales. acknowledgements the authors wish to thank vice chancellor of research in urmia university for supporting this work. conflict of interest: the authors declare that they have no conflict of interest references asghari, m., 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journal of applied botany and food quality 86, 172 179 (2013), doi:10.5073/jabfq.2013.086.023 portland state university, department of biology, portland, usa effect of mycorrhizal colonization and light limitation on growth and reproduction of lima bean (phaseolus lunatus l.) jess a. millar, daniel j. ballhorn* (received july 3, 2013) * corresponding author summary plants can respond with sink stimulation of photosynthesis when colonized with fungal or bacterial root symbionts, compensating costs of carbohydrate allocation to the microbes. however, constraints may arise under light limitation when plants cannot extensively increase photosynthesis. we hypothesize that under such conditions the costs for maintaining the symbiosis outweigh the benefits, ultimately turning the mutualist microbes into parasites, resulting in reduced plant growth and reproduction. using lima bean (phaseolus lunatus) as an experimental plant, we applied two levels of light (full light, 75% shading) and microbial inoculation (sterile soil, mycorrhizal fungi) and quantified both vegetative and generative plant traits. as expected, shaded plants produced less vegetative biomass and seeds than non-shaded plants. however, individual seeds were significantly heavier in shaded plants and required less time for germination. while under both light conditions mycorrhizal plants showed a significantly reduced belowground biomass, mycorrhizal fungi neither enhanced overall plant performance in terms of total biomass and seed production nor resulted in measurable costs in either light condition. our study suggests that mycorrhizal colonization neither provided benefits to lima bean plants grown under full light, nor created costs when photosynthesis was limited. introduction mutualistic interactions between plants and microbes are an important component in the determination of plant diversity and ecosystem productivity. one of the most important groups of plantassociated microbial mutualists are mycorrhizal fungi (smith and read, 1997). the symbiosis between plants and mycorrhizal fungi is extremely widespread and ancient in the plant kingdom. root colonization with mycorrhizal fungi occurs in >80% of all plant species (smith and read, 1997) and has been observed in fossils dating back 400 million years ago (remy and taylor, 1994). mycorrhizal fungi colonize the host plant’s roots, form extensive networks and participate in the acquisition of phosphorus (p) in the case of arbuscular mycorrhizal fungi (amf) and nitrogen in ectomycorrhizal fungi (emf) (smith and read, 1997). due to their critical impact on plant growth and species composition these microbial symbionts are considered keystone species in terrestrial ecosystems (lodge et al., 1996). root colonization with mycorrhizal fungi generally has positive effects on plant growth (chalk et al., 2006) and mycorrhizal inoculation is frequently applied to increase crop plant productivity in agricultural systems (li et al., 2000, 2004; ortas et al., 2003; ortas, 2010). positive effects of mycorrhiza on plants include increases in height (hayman, 1986; hoeksema et al., 2010; safapour et al., 2011), biomass (vejsadova et al., 1993; mathur and vyas, 2000; ramana et al., 2010), shoot:root ratio (gavito et al., 2000; veresoglou et al., 2012), production of flowers (dodd et al., 1983; carey et al., 1992), and yield in crop plants such as phaseolus vulgaris, glycine max, and triticum aestivum (vejsadova et al., 1993; bethlenfalvay et al., 1997; abdel-fattah, 1997; li et al., 2005; ramana et al., 2010; safapour et al., 2011). there is an extensive body of literature on the effects of mycorrhizal fungi in a broad range of plant families including legumes (barea and azconaguilar, 1983; yang et al., 1994; olsen et al., 1999a; 1999b; liu et al., 2003; scheublin and ridgway, 2004; ortas, 2008; muleta, 2010) but a detailed understanding of costs and benefits arising from the mycorrhizal symbiosis under different abiotic conditions is often lacking. legumes are both important components in many terrestrial ecosystems and crop plants of world economic importance. due to their association with another group of microbial symbionts – nitrogen-fixing rhizobia – legumes critically determine the productivity and species composition of ecosystems (sprent and sprent, 1990) and according to their key function in global nitrogen cycles legumes are considered ecosystem engineers (carney and matson, 2005; van der heijden et al., 2008). in addition to their high impact on natural ecosystems, legumes are of high relevance in agro-ecosystems. legumes critically enhance the sustainability of agroforestry systems (muleta, 2010) or pastures (haystead et al., 1988) and some legume crop plants are of world economic importance (glycine max, pisum sativum, phaseolus vulgaris, phaseolus lunatus). although in most cases clearly beneficial for the plants, the associations with mycorrhizal fungi also incur costs as the microbial symbionts may consume up to 16% of photosynthetically-fixed carbon, which otherwise could be allocated to growth and reproductive functions (kaschuk et al., 2009). however, recent research demonstrated that plants can compensate for this cost through sink stimulation of photosynthesis, which is thought to be an adaptation to take advantage of the nutrient supply provided without compromising the total amount of photosynthates available for plant growth and development (mortimer et al., 2008). in natural ecosystems, light availability is often a variable resource due to competition among plant species and, depending on cultivation method, also shows strong variation in agricultural systems (chirko et al., 1996). while sink stimulation of photosynthesis is generally an efficient strategy to compensate for costs of carbohydrate allocation to microbial root symbionts, the question arises as of how plants respond to mycorrhizal inoculation under light-limited conditions when photosynthesis cannot be increased easily. the interacting effects of mycorrhizal colonization and light limitation on plant vegetative growth have been studied previously (smith and read, 1997), however, the impact of mycorrhiza on plant reproduction and the actual crop yield have received much less attention. one study found maize plants exposed to lower irradiance had a smaller percent mycorrhizal infection and smaller shoot weight, which was equated to yield (daft and el-giahmi, 1978). another study showed that mycorrhizal infection did not affect pisum sativum biomass; however, increased light resulted in enhanced growth once the plant reached the flowering stage (reinhard et al., 1994). unfortunately, these studies did not elucidate mycorrhizal fungi’s effects on seed production. analyzing the effects of mycorrhizal mycorrhiza under light limitation 173 colonization on actual seed set is of high importance due to two reasons: first, it is a much more precise fitness measure than plant biomass as mycorrhizal symbionts might lead to resource allocation and reduce plant biomass while increasing seed set. second, for many agricultural plants, seed yield is of larger interest than vegetative parameters (smith and smith, 2011; ronsheim, 2012). in the present study we used lima bean (fabaceae: phaseolus lunatus) as experimental plant. lima bean represents an emerging model plant commonly used in studies on indirect and direct plant defense against herbivores (ballhorn et al., 2008; 2009; 2010), and also bacterial (yi et al., 2009) and fungal pathogens (ballhorn et al., 2010). furthermore, this plant is one of the most economically important phaseolus species cultivated for food (fofana et al., 1999; alves et al., 2008; bonifácio et al., 2012). to better understand the concerted effects of mycorrhizal colonization and light availability, we exposed mycorrhizal and mycorrhiza-free lima bean plants to two levels of light. we hypothesized that mycorrhizal fungi provide benefits for their host plant under full light regimes, but that reduced light availability might increase the costs of the symbiosis and shift a beneficial interaction to a detrimental one. to our knowledge, this study is the first to analyze the interactive effects of mycorrhizal fungi and light availability in lima beans in order to uncover potential costs of these microbes when photosynthesis is limited. materials and methods plants lima bean plants (phaseolus lunatus cv. henderson) were grown from seeds (american meadows inc., williston, vt). plants were cultivated in a greenhouse with light regime of 13:11 light:day. light in the greenhouse was provided by a combination (1:1) of hqi-bt 400 w (osram) and rnp-t⁄lr 400 w (radium) lamps. temperature was 27:19 °c (i.e. temperature of 27 °c in the light period and 19 °c in the dark period) and we maintained an air humidity of 70-80%. plants were grown in containers (one plant per pot) with 12 cm in diameter in sunshine mix #1, lc1 (sungro horticulture®, bellevue, wa) 175 g per pot and were watered daily. experimental setup in a full factorial split plot design with two whole plots we applied four treatments, 15 replicates each (60 plants total) including two levels of light (full light, 75% shading) and two levels of mycorrhizal colonization (with and without mycorrhizal fungi). light availability was measured at noon on a sunny day (+ additional lighting) and at table height was an average of 525 μmol photon s-1 m-2 in full light and an average of 138 μmol photon s-1 m-2 (li-250 light meter; li-cor biosciences, lincoln, nebraska, usa) under the shade tent (205 cm × 113 cm × 113 cm). experimental plants were inoculated with commercial mycorrhizal inoculum [powder inoculant, bio organics™, la pine, oregon (glomus aggregatum, g. etunicatum, g. mosseae, g. clarum, g. deserticola, g. monosporus, gigaspora margarita, paraglomus brasilianum, rhizophagus irregularis), 10 cc (8 g) per plant] when they had developed completely unfolded primary leaves. plants were not inoculated with any other microbes (such as rhizobia) to avoid uncontrolled cross-effects between fungal and bacterial root symbionts. plant biomass and reproduction over the experimental period of 14 weeks we measured plant height, leaf number, and initial number of seed pods. at the end of the experiment we evaluated the above and belowground biomass, the final number of seed pods, and determined the shoot:root ratio. to obtain belowground biomass, plant root systems were carefully washed until all potting soil was removed. above and belowground parts of plants were dried in an oven (incumax™ cv250 convection oven, amerex instruments, inc., lafayette, ca) at 70 °c for 5 days until constancy of weight. seeds produced per plant were counted and weighed. to assess viability of seeds they were germinated by placing them between four 24 cm × 45 cm wet papers towels in the dark at 25 °c. number of days to germination was recorded for each seed. microscopic analysis of mycorrhizal colonization mycorrhizal colonization was evaluated by taking 1 g of fresh root samples, from each plant, from 4 separate locations of the washed roots. root segments were placed into histocassettes (vwr, west chester, pa). all root samples were cleared with 10% koh, acidified in 2% hcl, stained with 0.05% trypan blue solution, and preserved in lactoglycerol (phillips and hayman, 1970). roots were cut into 1 cm sections and at least 40 cm of roots from each plant were placed on a single microscope slide with lactoglycerol. microscopic observations were conducted using an amscope fm320 trinocular microscope in both 100x and 400x magnification. roots were examined for mycorrhizal structures that intersected the microscope eyepiece crosshair at 100 random points using the magnified intersections method (mcgonigle et al., 1990). the presence or absence of mycorrhizal structures at 100 intersects was used to calculate percent root length colonization by mycorrhizal fungi per plant. data analysis the effects of ‘mycorrhizal inoculation’ and ‘light availability’ on plant traits were assessed with 2-way anova split-plot analysis with ‘light availability’ as a blocking factor, using the glm procedure in sas. square root transformation was performed on several plant growth parameters (number of leaves, buds, flowers, and seed pods) to control for normality in tests. ‘mycorrhizal inoculation’ and ‘light availability’ were set as fixed effects in all tests. number of inflorescences per plant was set as a covariate in the 2-way anova for number of seed pods at a defined time point (6 weeks after cultivation). all tests were performed in sas version 9.2. results light availability the colonization of experimental plants with mycorrhizal fungi was not affected by light availability (tab. 1). light availability significantly affected all considered vegetative plant traits including plant height (f=144.59, p<0.001), number of leaves (l: f=38.93, p<0.001), total biomass (f=103.48, p<0.001), above(f=70.10, fig. 1: percent mycorrhizal colonization. mf = mycorrhizal fungi; values shown are means + se; n = 15 plants per treatment 174 j.a. millar, d.j. ballhorn p<0.001) and belowground biomass (f=290.16, p<0.001), as well as shoot:root ratio (f=4.99, p=0.034; tab. 1). plants growing under full light were significantly taller, had more leaves, more total biomass, more aboveand belowground biomass than plants cultivated under shaded conditions (fig. 2). light availability also significantly affected all generative plant traits analyzed, such as the initial number of pods per plant (l: f=127.88, p<0.001), the final number of pods (l: f=34.28, p<0.001), the number of pods dropped before maturation (f=129.94, p<0.001) as well as the number of seeds (l: f=202.15, p<0.001; tab. 2). plants growing under full sun had more initial pods, more final pods, and more seeds, but also dropped more pods than shaded plants (fig. 3). seed quality parameters were significantly affected by light conditions including total seed weight (f=43.56, p<0.001), average seed weight (f=10.72, p=0.002), and days to germination (f=54.13, p<0.001), but not the percentage of seeds that germinated (tab. 3). total and average seed weight were higher for shaded plants than for full light controls and seeds from plants under shaded conditions germinated significantly quicker than seeds from plants growing under full light conditions (fig. 4). am fungal colonization microscopical analyses revealed successful mycorrhizal colonization in the inoculated group, whereas the control group showed little to no mycorrhizal fungi (fig. 1). am fungi influenced far fewer traits than light availability. specifically, mycorrhizal fungi significantly affected belowground biomass (f=9.44, p=0.005) and the shoot:root ratio of plants (f=23.49, p<0.001). plants inoculated with mycorrhizal fungi had significantly lower belowground biomass than controls and accordingly a higher shoot:root ratio (fig. 2). all other vegetative plant traits that we analyzed were not significantly changed in response to mycorrhizal colonization including plant height, number of leaves, aboveground biomass and total biomass (tab. 1). reproductive plant traits, which were significantly affected by am fungal colonization, were the number of initial pods (f=7.38, p=0.011) and the number of dropped pods (f=12.14, p<0.001). plants with mycorrhizal fungi had less initial pods and dropped less pods than plants grown without the symbiotic partner (fig. 3). however, the total number of final pods and total seed numbers did not show significant variation between mycorrhizal and non-colonized control plants (tab. 2). inoculation with mycorrhizal fungi significantly affected the average seed weight (f=11.80, p=0.002) as well as the days to germination (f=5.27, p<0.022). inoculated plants had significantly higher average seed weight per plant than controls, but seeds of these plants required more time to germinate (fig. 4). in contrast, the total seed weight and the percent of seeds that germinated were not altered by mycorrhizal fungi (tab. 3). interacting effects of ‘light availability’ and ‘mycorrhizal fungi’ the interaction of ‘light availability’ and ‘mycorrhizal fungi’ (m×l) did not significantly affect any of the vegetative plant parameters assessed (tab. 1). of the reproductive plant traits analyzed, the number of initial pods (f=5.37, p=0.028) and the number of dropped pods (f=10.28, p=0.003) were significantly affected by the interaction of ‘light availability’ and ‘mycorrhizal fungi’, while the number of final pods and the number of seeds per pod were not affected tab. 1: summary of anova results of the effects of mycorrhizal fungi and light availability on vegetative plant traits. effects height total leaves aboveground belowground total biomass shoot:root biomass biomass df f p f p f p f p f p f p mycorrhiza (m) 1 2.78 0.107 1.43 0.242 1.20 0.283 9.44 0.005* 0.10 0.750 23.49 <0.001* light (l) 1 144.59 <0.001* 38.93 <0.001* 70.10 <0.001* 290.16 <0.001* 103.48 <0.001* 4.99 0.034* m × l 1 0.40 0.534 0.67 0.419 0.61 0.387 3.13 0.088 0.10 0.757 1.17 0.288 * p-values are significant (p<0.05) fig. 2: effects of mycorrhizal fungi and light availability on vegetative plant and biomass traits. plant height (a) and number of leaves per plant (b) were determined at the end of the cultivation period. total plant aboveground biomass (c), belowground biomass (d), total biomass (e) and plant shoot:root ratio (f) were determined on dry weight basis. mf = mycorrhizal fungi; values shown are means + se; n = 15 plants per treatment mycorrhiza under light limitation 175 tab. 2: summary of anova results of the effects of mycorrhizal fungi and light availability on reproductive plant traits. effects total initial pods total final pods total dropped pods total seeds df f p f p f p f p mycorrhiza (m) 1 7.38 0.011* 0.05 0.821 12.14 0.002* 1.14 0.294 light (l) 1 127.88 <0.001* 34.28 <0.001* 129.94 <0.001* 202.15 <0.001* m × l 1 5.37 0.028* 1.54 0.225 10.28 0.003* 1.14 0.294 * p-values are significant (p<0.05) fig. 3: effects of mycorrhizal fungi and light availability on seed production. number of initial pods per plant (a) was determined 6 weeks after planting. final number of pods (b), dropped pods (c), and total seed production per plant (d) was determined at the end of the cultivation period. mf = mycorrhizal fungi; values shown are means + se; n = 15 plants per treatment tab. 3: summary of anova results of the effects of mycorrhizal fungi and light availability on seed traits and viability. effects total seed weight average seed weight percent seed germination days to germination df f p f p f p f p mycorrhiza (m) 1 1.93 0.178 11.80 0.001* 0.52 0.470 5.27 0.022* light (l) 1 543.56 <0.001* 10.72 0.002* 3.20 0.081 54.13 <0.001* m × l 1 0.42 0.526 0.46 0.498 0.52 0.470 4.93 0.027* * p-values are significant (p<0.05) fig. 4: effects of mycorrhizal fungi and light availability on weight and viability of seeds. total weight of seeds per plant (a) and average seed weight (b) were determined on dry weight basis. percent seeds germinated per plant (c) and days to seed germination per plant (d) were determined over an 8-day germination period. mf = mycorrhizal fungi; values shown are means + se; n = 15 plants per treatment for (a); for (b), (c), and (d), n = 27 seeds per treatment for shaded plants, n = 129 seeds for inoculated, no shade plants, and n = 147 seeds for control, no shade plants(tab. 2). the only seed trait to be significantly affected by the interacting term of both variables was the days to germination (f=4.93, p=0.027), while all other seed traits were not significantly altered (tab. 3). discussion even though the benefits of root-associated microbes are well studied in many cases, it remains widely elusive as to now the outcome of this mutualism is affected by external abiotic conditions. in our study we quantitatively analyzed the effects of mycorrhizal fungi and light availability on growth and reproduction of lima bean. we hypothesized enhancing effects of growth and reproduction by mycorrhizal fungi under full light whereas we expected reduced plant development in mycorrhizal plants under shaded conditions due to constraints for plants to support both growth and the mycor176 j.a. millar, d.j. ballhorn rhizal partner when photosynthesis is limited. studies in the past have shown decreases in plant growth in lower light conditions, mainly seen as decreased plant biomass (daft and el-giahmi, 1978; bethlenfalvay and pacovsky, 1983; tester et al., 1986; pearson et al., 1991). thus, under such conditions the costs for maintaining the mutualism with mycorrhizal fungi may outweigh the benefits, which ultimately turns the mutualistic microbes into parasites that exploit resources and reduce host fitness (bethlenfalvay and pacovsky, 1983; reinhard et al., 1993). contrary to our expectations, under full light, mycorrhizal fungi did not significantly enhance plant performance. inoculated plants did not produce more biomass than the respective controls (fig. 2), and mycorrhizal plants actually had significantly less pods than controls (fig. 3). however, in our study the significantly negative effects of mycorrhizal fungi disappeared on the level of actual number and weight of seeds, which showed no significant differences between the treatments (figs. 3 and 4). these overall neutral effects of mycorrhizal fungi on plant reproductive structures observed in our study are in line with a recent meta-analysis looking at various legume species including cicer arietinum, lens culinaris, phaseolus vulgaris, pisum sativum, and vicia faba. in these plants, mycorrhizal fungi were found to have no effect on crop yield (kaschuk et al., 2010). in our study the only factor affecting biomass and seed production was light availability. under reduced light, plants produced less biomass and less seeds compared to plants growing under full light conditions. there are several possible explanations for the limited impact of mycorrhizal fungi on plant growth and reproduction as observed in the present study. one possibility is that mycorrhizal fungi may play a less important role for grain legumes than for other plant species in general (kaschuk et al., 2010) or that lima bean in particular is not adapted to form a symbiosis with mycorrhizal fungi. even though lima bean represents an important crop plant, to the best of our knowledge there is no information available on the colonization of lima bean plants with mycorrhizal fungi under laboratory or field conditions. on the other hand, studies on a closely related plant species (snap bean, phaseolus vulgaris) showed positive effects of mycorrhizal fungi on growth and nutrient uptake (hacisalihoglu et al., 2005; ciftci et al., 2010). another possibility that could explain the limited impact of mycorrhizal fungi on lima bean observed in our study is that the commercial inoculum we used may not have contained fungal strains that provide a benefit to lima bean plants. although we could show mycorrhizal colonization of the plants roots and formation of haustoria microscopically, this does not necessarily mean the fungi efficiently provided nutrients for their host plant. furthermore, the rate of mycorrhizal colonization was relatively low compared to other plant species. however, this might be due to sampling roots towards the end of the plants’ life cycle, which was required, as we needed to collect data on seed production of the same individual plants. at earlier stages of plant development, the rate of mycorrhizal colonization likely might have been higher as colonization rates decrease with plant age, as has been reported for hordeum vulgare, secale cereale and triticum aestivum (dodd and jeffries, 1986; boswell et al., 1998; li et al., 2005). furthermore, together with our microscopic proof of successful colonization, the observation that mycorrhizal colonization of lima bean resulted in a significantly increased shoot:root ratio compared to the respective controls supports a good matching quality of plants and fungi rather than limited compatibility. a relatively lower root biomass in mycorrhizal plants compared to uncolonized conspecifics is a common phenomenon and indicates an efficient transport of minerals and water via mycorrhizal fungi (kothari et al., 1990). thus, the increased shoot:root ratio in mycorrhizal lima bean plants we observed here suggests an actual interaction between both partners. in addition to effects of mycorrhizal fungi on aboveand belowground biomass of the lima bean plants themselves, we also considered effects of mycorrhizal inoculation on germination and viability of produced seeds, that is, on the next generation of host plants. compared to plants growing under full light conditions, shaded plants produced overall significantly fewer but heavier seeds. nevertheless, within each light treatment, mycorrhizal inoculation had no significant effect on the total seed weight produced per plant (fig. 4a). mycorrhizal colonization, however, increased the average seed weight with heaviest seeds in shaded plants (fig. 4b); in plants grown in full light and in shade, this increase was significant. increases in average seed weight due to mycorrhizal inoculation has been described previously for various plants such as triticum aestivum and abutilon theophrasti (lu and koide, 1994; karagiannidis and hadjisavva-zinoviadi, 1998). while the underlying mechanisms are little understood, changes in resource allocation within the plant are likely as mycorrhizal fungi represent a significant carbon sink (mathur and vyas, 2000; chalk et al., 2006; kaschuk et al., 2009). based on the results of our study we cannot make predictions on whether the observed effects of mycorrhizal colonization on seeds are positive or negative for plants in nature; however, other studies showed that changes in seed traits can have a far reaching impact on plant fitness. seed weight and size have been identified as plant traits strongly influencing the dispersal, establishment, survival and growth of seedlings (harper et al., 1970; harper, 1977; westoby, 1998; weiher et al., 1999; leishman et al., 2000; moles and westoby, 2004), particularly at early seedling stages (leishman et al., 2000; coomes and grubb, 2003). fenner and thompson (2005) showed that large seeds had an increased probability of establishment under detrimental conditions. generally seedlings developing from large seeds cope better than those of smaller seeded species under competition (parrish and bazzaz, 1985; rees, 1995), drought, nutrient limitation (lee and fenner, 1989; jurado and westoby, 1992; leishman and westoby, 1994) and depth of seedling emergence (gulmon and url, 1992; peterson and facelli, 1992; vázquezyanes and orozco-segovia, 1992). in line with our study, deep shade has also been identified as a factor selecting against small seed size (grime and jeffrey, 1965; leishman and westoby, 1994). thus, development of larger seeds under shaded conditions and the increases in average seed weight in response to mycorrhizal colonization as we observed in our study might represent fitness relevant parameters. beyond changes in seed weight, seeds produced by mycorrhizal and non-mycorrhizal plants showed differences regarding germination time (tab. 3). while seeds from mycorrhizal parent plants took equal time to germinate compared to mycorrhiza-free controls when plants were grown in full light, seeds produced by colonized plants under shaded conditions germinated significantly later than seeds produced by mycorrhiza-free plants (fig. 4). whether these changes were due to variation in the thickness of seed coats determining water intake as the first step of the germination process or whether the observed variation is due to different enzymatic activities in seeds derived from the different treatments remains elusive and, again, it is difficult to predict if these changes in germination time increase or decrease plant fitness as this depends on the specific environmental conditions. in general, the benefits to shorter germination time in plants is variable, however in large-seeded plants shorter germination time has been demonstrated to enhance growth and fecundity (verdú and traveset, 2005). conclusions mycorrhizal fungi play a key role for plant performance and productivity in natural and agricultural ecosystems yet their effects on actual seed production in interdependence with variable abiotic conditions remains elusive in many cases. thus, as mycorrhizal symbiosis holds mycorrhiza under light limitation 177 great potential to improve sustainable crop production (plenchette et al., 2005) there is an urgent need to functionally study this almost ubiquitous interaction and analyze the effects of environmental factors on the symbiosis (ortas, 2012). using lima bean (phaseolus lunatus) as an experimental plant, we hypothesized that for plants under light limitation the costs for maintaining the symbiosis outweigh the benefits of the fungal partner, ultimately turning the beneficial microbes into parasites, resulting in reduced plant growth and reproduction. contrary to our expectations, we found that mycorrhizal colonization neither provided benefits in terms of increased biomass and total seed number and total seed weight to plants grown under full light, nor created costs under shaded conditions. mycorrhizal colonization did, however, significantly increase the average seed weight in both light treatments with heaviest seeds in shaded plants and lengthened germination time of seeds produced by shaded plants. our study shows that the effects of mycorrhizal symbionts go beyond mere effects on plant biomass production as they significantly alter plant reproductive traits. results of our study suggest that even though 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(lhs) plant ecology strategy scheme. plant soil 199, 213-227. yang, x.h., luo, x.s., liu, r.j., 1994: effect of vesicular-arbuscular mycorrhiza on yield and quality of watermelon (in chinese). fruit science 11, 1994. yi, h.-s., heil, m., adame-alvarez, r.m., ballhorn, d.j., ryu, c.-m., 2009: airborne induction and priming of plant defenses against a bacterial pathogen. plant physiol. 151, 2152-2161. address of the authors: department of biology, portland state university, 1719 sw 10th ave, portland, or 97201, usa e-mail: ballhorn@pdx.edu journal of applied botany and food quality 90, 232 237 (2017), doi:10.5073/jabfq.2017.090.029 1university of craiova, faculty of horticulture, department of horticulture and food science, romania 2university of craiova, faculty of sciences, department of chemistry, romania bioactive compounds and antioxidant activity of hot pepper fruits at different stages of growth and ripening mira elena ionică1, violeta nour1*, ion trandafir2 (received january 17, 2017; accepted may 1, 2017) * corresponding author summary the evolution of some bioactive compounds and antioxidant activity has been investigated during fruit growth and ripening of five pepper cultivars: ‘dracula’, ’pintea’, ‘pepperone’, ‘bulgarian carrot’ (c. annuum) and ‘christmas bell’ (c. baccatum var. pendulum). highperformance liquid chromatography was used to quantify the content of capsaicin in the fruit in order to determine the pungency level of analyzed peppers. pepper fruits were collected at five stages of growth and ripening. dry matter, soluble solids, ascorbic acid, total phenolics, including total flavonoids, capsaicin content and anti oxidant activity were determined at each stage. there were major differences among the cultivars in the accumulation of the bioactive compounds in the fruit during their growth and ripening, although the quantitative accumulation pathway of various components had a similar trend during phenophases. antioxidant activity and ascorbic acid content increased during growth and ripening of hot peppers, the highest levels being found in the last stage of ripening. the pattern of variation of total flavonoid content was cultivar dependent. in most cultivars, an important increase of the total phenolic and total flavonoid content was observed in the last stage of ripening. capsaicin content recorded a maximum level in f3 or f4 depending on cultivar, and decreased afterwards until the complete ripening of the pepper fruits. ‘dracula’ cultivar was classified as “non-pungent” (fruits are not spicy) while ‘pintea’ was classified as “highly pungent”, the other analyzed cultivars having an average level of pungency. keywords: hot pepper, antioxidant activity, phenolics, flavonoids, capsaicin introduction hot pepper was part of ancient human diet in the american continent since 7500 years bc. its south american origin is supported for all species of capsicum genus, which probably occurred in the region between southern brazil and bolivia. hot pepper has a sweet-pungent flavor, with pungency ranging by cultivar. peppers are good sources of phytochemicals such as polyphenols, among them flavonoids, and carotenoids (alvarez-parrilla et al., 2010) that are known to present high antioxidant activity (materska and perucka, 2005). phenolic compounds from pepper contribute to nutritional quality and fruit taste (ornelas-paz et al., 2010). the substance that gives peppers pungency is capsaicin that is found in very small quantities in sweet pepper and in quantities of tens and hundreds of times higher in hot peppers. as the content of capsaicin in peppers increases, so does its pungent taste, which triggers an increase in their antioxidant level (usman et al., 2014). therewith capsaicin controls the appetite and raises the body temperature (ludy and mattes, 2011). pugency is measured with the dilution test and expressed as scoville organoleptic scale designed by wilbur scoville in 1912. recognized as the most pungent pepper (world-record for hottest chili of 2012) is trinidad moruga scorpion, 2.000.000 scoville units higher than any other species. recognized as the less spicy peppers are pimento, peperoncini and banana pepper cultivars, with only 100-500 scoville units. in addition to capsaicin, chili peppers’ taste is given by their content of essential oils. capsaicin and dihydrocapsaicin represent about 7590% of capsaicinoids in peppers, compounds that also have other properties and biological effects, such as chemopreventive (chanda et al., 2004) or antioxidant properties (zhuang et al., 2012). the epidermal cell of the placenta seems to be the accumulation site of capsaicinoids in peppers (barbero et al., 2014). capsaicinoids are found only in the capsicum genus and their amounts varies in different pepper varieties depending on cultural practices, environmental conditions, storage conditions, etc. (usman et al., 2014). literature mentions that dry matter and soluble solids content of pepper and different compounds in pepper, such as ascorbic acid and capsaicinoids vary during growing and ripening of the fruit (barbero et al., 2014; martinez et al., 2007). this work was conducted to investigate the evolution of some bioactive compounds and antioxidant activity of five pepper varieties grown in dolj county, romania during fruit growth and ripening. this work also aims to estimate the evolution and the level of capsaicin in fruit and to determine the pungency level of analyzed peppers. materials and methods plant material fruits of five pepper cultivars (capsicum annuum and capsicum baccatum): ‘dracula’, ‘pintea’, ‘pepperone’, ‘bulgarian carrot’ (c. annuum) and ‘christmas bell’ (c. baccatum var. pendulum), were collected at five stages of development and ripening i.e. f1-14 days after flowering, f2-20 days after flowering, f3-30 days after flowering, f4-40 days after flowering, f5-50 days after flowering (physiological maturity) from dabuleni, dolj county, romania and analyzed in terms of chemical characteristics and antioxidant activity. the selection of the fruits was based on the number of days after flowering. dabuleni is a sandy area in the south of romania close to the danube river. it has a strong continental climate with mild mediterranean influence and an average temperature of 11 °c. the region gets severe drought from july to september and average rainfalls in may and june. the orchard management was consistent with cultural practice (10-12 watering sessions with 300-350 m3/ha, 100-100-50 npk fertilization, breaking the tip of plant, pests and diseases control). the planting was carried out in sandy soil, with the experiment being set up as randomized block design in 3 replicates with 30 plants per cultivar. from each cultivar 10 fruits in 3 replicates were collected in order to perform chemical analysis. evolution of bioactive compounds during growth and ripening of hot peppers 233 analytical methods several analyses have been performed: dry matter, soluble solids, ascorbic acid, total phenolic and total flavonoid content, capsaicin content and antioxidant activity. the dry matter content was determined by removing water from the sample in an oven at 105 °c and the result was expressed in percentages. the soluble solids were measured with a digital refractometer (hanna instruments, woonsocket, usa) from the juice pressed from the fruit, the results being expressed in percentages. ascorbic acid content ascorbic acid was extracted and analyzed by reversed phase hplc. fresh pepper homogenate (5 g) was mixed and diluted with 2% hcl. after 20 minutes the solution was centrifuged at 4200 rpm for 10 min. the supernatant was filtered through 0.2 mm pore size filter. the ascorbic acid in the sample test solution was separated by reversed phase chromatography on a 250 mm × 4.6 mm i.d., 5 μm particle hypersil gold aq analytical column, of which was detected by absorbance and quantified with external calibration graph. the detector was set at λ=254 nm. this setting was chosen since ascorbic acid has its maximum optical absorbance close to 254 nm. the hplc analysis was performed with a surveyor thermo electron system (thermo electron corporation, san jose, ca, usa) comprising a vacuum degasser, surveyor plus lcpmpp pump, surveyor plus asp autosampler and diode array detector with 5 cm flow cell. integration, data storage and processing were performed by chrom quest 4.2 software. the determinations were made in isocratic conditions, at 10 °c, using a mobile phase made of 50 mm phosphate solution filtered through a polyamide membrane (0.2 μm) and degassed in a vacuum. the flow rate of the mobile phase was 0.7 ml/min for all the chromatographic separations. the volume injected was 5 μl for either prepared sample or standard solution. the results were expressed in mg kg-1 fresh weight (fw). all reagents were acquired from sigma-aldrich, germany and ultrapure water was obtained from a milli-q water purification system (tgi pure water systems, usa). total phenolic content total phenolic content was assessed by using the folin-ciocalteu phenol reagent method (singelton and rossi, 1965). folin-ciocalteu reagent (2 n, merck), gallic acid (99% purity, sigma-aldrich), anhydrous sodium carbonate (99% sigma-aldrich) were used. one g dried pepper homogenate was extracted with 15 ml ethanol in an ultrasonic bath for 60 min at ambient temperature. after extraction, the samples were centrifuged for 5 min at 4200 rpm and supernatants were filtered through polyamide membranes with pore diameter of 0.45 μm and stored at a temperature of -20 °c. hundred μl of each pepper ethanolic extract were mixed with 5 ml of distilled water and 500 μl of folin-ciocalteu reagent. after 30 sec to 8 min, 1.5 ml of sodium carbonate (20% v/v) was added. the reaction mixture was diluted with distilled water to a final volume of 10 ml. the preparation of the standard solution of gallic acid followed the same procedure. the absorbance at 765 nm of each mixture was measured on a varian cary 50 uv spectrophotometer (varian co., usa) after incubation for 30 min at 40 °c and results were expressed in mg gallic acid (gae) kg-1 dry weight (dw). antioxidant activity the antioxidant activity (aox) was measured in the ethanolic extract using the dpph (2,2-diphenyl-1-picrylhydrazil) assay. ethanol (merck, germany), dpph (2,2-diphenyl-1-picrylhydrazil) (sigmaaldrich, germany) and trolox (merck, germany) were employed. the extraction of samples was made according to the same protocol described for total phenolic content. the free radical scavenging ability of the extracts against dpph free radical was evaluated as described by oliveira et al. (2008), with some modifications. each ethanol pepper extract (50 μl) was mixed with 3 ml of a 0.004% (v/v) dpph methanolic solution. the mixture was incubated for 30 min at room temperature in the dark and the absorbance was measured at 517 nm on varian cary 50 uv-vis spectrophotometer. the dpph free radical scavenging ability was calculated in refe rence to trolox (6-hydroxy-2,3,7,8-tetramethylchroman-2-carboxylic acid), which was used as standard reference to convert the inhibition capability of each extract solution to the mmol trolox equivalent antioxidant activity/l. the radical was freshly prepared and protected from light. a blank control of methanol/water was used in each assay. all assays were conducted in triplicate and results were expressed in mmol trolox kg-1 dw. total flavonoid content total flavonoids content was assessed by the spectrophotometric method described by mohammadzadeh et al. (2007) based on the color reaction of this class of compounds with the ions of al (iii). pepper homogenate (1 g dry matter) was extracted with 15 ml ethanol in an ultrasonic bath for 60 min at ambient temperature. after extraction, the samples were centrifuged for 5 min at 4200 rpm and supernatants were filtered through polyamide membranes with a pore diameter of 0.45 μm and stored at -20 °c. 0.5 ml filtrate was placed in a polyethylene test tube, together with a 0.1 ml 10% aqueous solution of aluminum nitrate, 0.1 ml aqueous 1 m sodium ace tate, 4.3 ml methanol, mixed well and left to react for 40 min at room temperature. after completion of the reaction, the absorbance of the mixture was measured at 415 nm on a varian cary 50 uv-vis spectrophotometer (varian co., usa). the quantification of flavonoids was carried out on the basis of a calibration curve using quercetin as standard reference in the range of 0-100 mg/l. the results were expressed as mg of quercetin equivalents kg-1 dw. capsaicin content identification and quantification of capsaicin were carried out by hplc following the method described by zhuang et al. (2012), with some modifications. for sample preparation, fresh matter (pulp transformed into a paste by a disintegrator) was dried in an oven at 55 °c to a constant weight and the dried matter was ground finely in a blender (polytron). an amount of 1 g of the dried matter was extracted with 20 ml of ethanol 95%. after 30 min, the extract was kept in an ultrasonic bath for 60 min at 60 °c and filtered through a 0.2 mm pore size filter. hplc-dad analysis was performed on a finnigan surveyor plus system (thermo electron corporation, san jose, ca, usa) coupled with diode array detector (pda5p with cell flow in 5 cm). the separation was performed using a hypersil gold aq column (250 × 4.6 mm) with a particle size of 5 μm. capsaicin was detected at 278 nm and quantified (mg kg-1 dw) using standard compound. the determinations were made in isocratic conditions at 25 °c, using a mobile phase made of 50 mm acetonitrile solution (water:acetonitrile 57:43) filtered through a polyamide membrane (0.2 μm) and degassed in a vacuum. the flow rate of the mobile phase was 1 ml min-1 for all the chromatographic separations. the volume injected was 5 μl for either prepared sample or standard solution. for device control, data acquisition and processing, chrom quest 4.2 software was used. statistical analysis significance of differences between cultivars was determined by performing a one-way anova test using statgraphics centurion xvi software (statpoint technologies, warenton, va, usa). 234 m.e. ionică, v. nour, i. trandafir results and discussion the data obtained concerning the dry matter, soluble solids and ascorbic acid content of the hot peppers are presented in tab. 1. the dry matter content (%) in hot peppers followed an upward trend during growth and ripening except the f2 (20 days after flowering) where a small absolute decrease was noticed. however, the dry matter content varied little in immature stages but showed high increase in ripening stages with a maximum in the physiological maturity, corresponding to the ripening process of fruits, which was manifested through the synthesis of metabolites with complex molecular structure. the same changes were reported by niklis et al. (2002). there were also differences among cultivars, the highest content of dry matter being found in the ‘pepperone’ cultivar while the ‘pintea’ cultivar registered the lowest content. the differences among cultivars were maintained during all the stages of growth and ripening. regarding the soluble solids content (%), it was found that it had the same upward trend during growth and ripening except the ‘pintea’ cultivar which registered a small decrease in f2 and ‘bulgarian carrot’ cultivar which registered the same decrease in f3. the highest rate of accumulation of soluble substances was found in the last sta ges with similar values to those found by dorji et al. (2005). dorji et al. also found that the highest changes in soluble solids happened in the last stages of ripening and in the firm red stage, respectively. there were differences among cultivars: the highest content of soluble solids being found in the ‘bulgarian carrot’ (9.82%) followed by ‘pepperone’ (9.65%), ‘dracula’ (8.4%), ‘pintea’ (8.1%) and ‘christmas bell‘ (7.40%). in all cultivars the ascorbic acid content was positively correlated with the dry matter and the soluble solids content, which is in accordance with the data presented by niklis et al. (2002). considering that the glucose is the precursor in the ascorbic acid synthesis (davey et al., 2000; niklis et al., 2002) it is understandable why there is a positive correlation between dry matter, soluble solids and ascorbic acid content. the highest accumulation of ascorbic acid was recorded between the first phenophases (f1-f2). in this period in all cultivars high plant growth rates were observed. the data obtained are comparable to data presented in the literature, the upward trend being also described by kumar and subba tata (2009) and martinez et al. (2005). there were significant differences among cultivars, the highest content of ascorbic acid being found in the ‘bulgarian carrot’ cultivar (1606.47 mg kg-1 fw) and the lowest content in the ‘pintea’ (1161.35 mg kg-1 fw) cultivar. according to the classification of hot pepper cultivars depending on the content of ascorbic acid described by khadi et al. (1987) and simonne et al. (1997), the analyzed cultivars fall into the category of an average content of ascorbic acid (1010-2000 mg kg-1 fw) even if analyzing in terms of species, chilli is considered a species with high ascorbic acid content. the results obtained on the content of phenolics, total flavonoids, and antioxidant activity (aox) in hot peppers are shown in tab. 2. antioxidant activity is an important parameter to establish the health functionality of a food product and there are many methods used for its measurement (kaur and kapoor, 2001). aox increased during growth and ripening of the hot peppers, the highest levels being found in the last stage (f5). there were differences among cultivars, the highest aox level being found in the ‘pintea’ cultivar (20.04 mmol trolox kg-1 dw) and the lowest in the ‘dracula’ cultivar. the diffe rences between cultivars were also reported by zhuang et al. (2012), who mentioned some pepper extracts as effective electron donors. hot peppers contain several flavonoids in different forms including glycosides (bae et al., 2012). the data presented in tab. 2 show important variations in the content of phenolics and total flavonoids in hot peppers. the total phenolic content increases during growth and ripening with the highest level in the last stage of ripening, the results being in accordance with the data presented by zhuang et al. (2012) and deepa et al. (2007). righetto et al. (2005) mentioned that the content of phenolics is tab. 1: dry matter, soluble solids and ascorbic acid content in hot peppers during growth and ripening. different letters within the same row indicated significant differences (p < 0.05) among cultivars dracula pintea pepperone bulgarian carrot christmas bell dry matter (%) f1 10.11±1.41c 11.38±1.09b 11.80±1.41ab 12.39±1.36a 12.53±1.62a f2 9.99±1.19c 10.65±1.17bc 10.50±1.29b 10.12±1.11bc 12.13±1.49a f3 10.86±1.75c 11.47±1.37b 13.31±1.73a 10.57±1.26c 12.73±1.52ab f4 15.96±2.01b 14.17±1.73bc 15.42±2.15b 18.00±2.52a 13.66±1.77c f5 17.43±2.26b 16.16±2.02c 18.71±2.6a 18.09±2.48a 17.53±2.38ab soluble solids (%) f1 4.45±0.53b 5.45±0.65a 4.70±0.56ab 3.72±0.40c 3.86±0.41c f2 4.67±0.61ab 4.37±0.48c 4.54±0.49b 5.10±0.58a 4.66±0.51ab f3 5.64±0.69bc 5.82±0.62b 7.10±0.91a 4.57±0.54c 6.00±0.72ab f4 7.77±0.93ab 4.80±0.52d 7.60±0.98b 8.32±1.05a 6.66±0.86c f5 8.40±1.09b 8.10±1.03b 9.65±1.32ab 9.82±1.27a 7.40±0.96c ascorbic acid (mg kg-1 fw) f1 170.32±15.40a 152.80±12.80b 169.85±14.20ab 173.62±13.60a 105.43±90.80c f2 1186.54±108.70a 1102.38±97.30d 1188.21±99.10a 1178.89±86.70b 1158.40±109.20c f3 1104.62±100.40c 1123.19±104.10bc 1191.05±110.50b 1289.21±116.90a 1259.47±114.90a f4 1211.20±109.60c 1149.52±98.30de 1176.23±105.80d 1297.58±113.70b 1393.62±121.40a f5 1383.09±118.70c 1161.35±106.10d 1387.11±120.80bc 1603.57±147.50a 1446.47±137.30b evolution of bioactive compounds during growth and ripening of hot peppers 235 affected by the type and cultivar of pepper, maturity and cultural conditions which are in accordance with the data presented in tab. 2, the highest content in phenolics (f5) being found in the ‘pintea‘ cultivar (8331.27 mg gae kg-1 dw) and the lowest in the ‘bulgarian carrot’ cultivar (2964.25 mg gae kg-1 dw). the total flavonoids content presented different variation patterns from cultivar to cultivar but 30 days after flowering (f3) a decrease is recorded as compared with f1 in all cultivars. except for ‘bulgarian carrot’ cultivar, total flavonoids content grew strongly in the last stage of ripening. howard et al. (2000) and medina-juarez et al. (2012) found the same variation associated to pepper maturity, cultivar and growing conditions. in the last stage of ripening (f5) the total flavonoids content was significantly higher in the ‘pepperone’ cultivar (7666.095 mg quercetin kg-1 dw) followed by ‘pintea’ and ‘dracula’. the data regarding the capsaicin content of the hot pepper are presented in tab. 2. in the early days after flowering (14 days) the capsaicin was found in peppers in very small amounts (undetectable in the ‘bulgarian carrot’ and ‘christmas bell’ cultivars). during growth and ripening, the fruit content of capsaicin increased to a maximum level that was recorded depending on the cultivar precocity in f3 (30 days after flowering) in: ‘dracula’, ‘pintea’ and ‘pepperone’ (early cultivars) either f4 (40 days after flowering) in ‘bulgarian carrot’ and ‘christmas bell’. after reaching this peak, the capsaicin content decreased until the full ripening of the pepper fruits. barbero et al. (2014) mentioned that the maximum relative content of capsaicin is reached on day 20 of fruit maturation, earlier than other capsaicinoids, and between day 40 and 50 of maturation the relative content of capsaicin represented only 52% of the total capsaicinoids. the same trend of capsaicin accumulation in fruits was reported by estrada et al. (2000) and kirschbaum-titze et al. (2002). there are major differences between cultivars regarding the content of capsaicin, the highest level being found in the ‘pintea’ cultivar (2229.81 mg kg-1 dw) with an equivalent pungency of 35676.96 shu followed by the ‘bulgarian carrot’ and ‘pepperone’. the lowest level of capsaicin content was found in the ‘dracula’ cultivar (24.85 mg kg-1 dw) with an equivalent pungency of 397.68 shu. similar differences between cultivars were reported by usman et al. (2014), vera-guzmán et al. (2011) and gnayfeed et al. (2001). to express the pungency level of the studied cultivars, capsaicin content (grams of capsaicin per grams pepper dry weight) was converted to scoville heat units by multiplying it by the coefficient of the heat value (1.6 × 107) (tilahun et al., 2013). the data obtained are shown in tab. 3. scoville score analysis showed that the ‘dracula’ cultivar is classified as ‘non-pungent’ (fruits are not spicy) while ‘pintea’ is classified as ‘highly pungent’, the other analyzed cultivars being classified as ‘moderately pungent’. although the ‘pintea’ cultivar registered the highest capsaicin content and the highest level of pungency, it is very far from the first places on the scoville scale (scoville units between 200,000 and 300,000 − capsicum chinense, while very spicy peppers from thailand only reach 100,000). tab. 2: phenolics, total flavonoids, capsaicin content and antioxidant activity in hot peppers during growth and ripening. different letters within the same row indicated significant differences (p < 0.05) among cultivars dracula pintea pepperone bulgarian carrot christmas bell antioxidant activity (mmol trolox kg-1 dw) f1 5.26±0.49c 6.94±0.58a 5.95±0.53bc 7.08±0.68a 6.24±0.60b f2 7.47±0.61b 8.64±0.611a 7.36±0.71b 8.89±0.86a 7.14±0.68c f3 9.01±0.88c 18.03±1.74a 11.58±1.08b 11.85±1.94b 6.91±0.67d f4 9.49±0.90c 20.04±1.99a 12.55±1.92bc 13.87±1.26b 8.44±0.82d f5 10.51±0.97cd 19.56±1.73a 17.50±1.67b 17.23±1.66b 11.55±1.09c total phenolics (mg gae kg-1 dw) f1 3074.32±284.12b 3210.27±301.23a 3087.00±287.65ab 3299.95±301.11a 2768.23±265.34c f2 3358.95±297.25b 3253.23±298.73bc 3462.78±300.87b 3779.28±369.30a 2878.89±279.61c f3 3450.00±300.13cd 4965.69±405.87a 3525.13±312.65c 3758.11±358.49b 3267.67±311.42d f4 3645.58±315.09d 7026.63±691.45a 4435.05±403.12c 6476.10±609.28b 3248.35±311.73d f5 5146.45±463.67c 8331.27±786.70a 6433.51±503.84b 2964.25±275.33c 3744.84±350.22d total flavonoids (mg quercetin equivalents kg-1 dw) f1 1896.90±176.55c 1853.85±171.33c 2218.33±199.76bc 4532.89±432.05a 2415.15±230.17b f2 1422.61±139.68d 1996.33±183.74c 2286.09±202.11b 4569.75±408.17a 2001.43±198.67b f3 1868.20±183.15b 1819.65±167.95bc 1849.44±169.81b 3397.92±311.26a 1774.00±163.50c f4 1632.97±156.70d 2726.10±222.56a 2340.39±201.54b 2382.73±202.88b 1844.23±187.34c f5 5461.93±497.78b 5247.27±497.88bc 7666.09±700.80a 1435.57±122.86d 2848.81±256.40c capsaicin (mg kg-1 dw) f1 4.98±0.45c 37.57±3.28b 45.16±5.03a 0 0 f2 5.19±0.49c 705.90±56.91a 429.55±40.89b 11.10±0.98d 38.07±3.77e f3 24.85±2.89e 2229.81±220.37a 777.49±75.69b 438.46±39.69c 126.78±11.59d f4 16.89±1.56e 1814.86±179.65a 405.90±38.45c 990.37±93.15b 187.90±17.73d f5 18.64±1.72e 1797.52±17.05a 501.07±48.71c 656.13±62.99b 30.46±2.86d 236 m.e. ionică, v. nour, i. trandafir however we can say that the fruits of ‘pintea’ cultivar are swifter than the mexican jalapeno or the italian peperoncino cultivars that barely reach a score of 500-5,000 scoville units (mathur et al., 2000). bae, h., jayaprakasha, g.k., jifon, j., patil, b.s., 2012: variation of antioxidant activity and the levels of bioactive compounds in lipophilic and hydrophilic extracts from hot pepper (capsicum spp.) cultivars. food chem. 134(4), 1912-1918. doi: 10.1016/j.foodchem.2012.03.108 barbero, g.f., ruiz, a.g., liazid, a., palma, m., vera, j.c., barroso, c.g., 2014: evolution of total and individual capsaicinoids in peppers during ripening of the cayenne pepper plant (capsicum annuum l.). food chem. 153, 200-206. doi: 10.1016/j.foodchem.2013.12.068 chanda, s., erexson, g., riach, c., innes, d., stevenson, f., murli, h., bley, k., 2004: genotoxicity studies with pure trans-capsaicin. mutat. res. genet. toxicol. environ. mutagen. 557(1), 85-97. doi: 10.1016/j.mrgentox.2003.10.001 davey, m.w., montagu, m.v., inzé, d., sanmartin, m., kanellis, a., smirnoff, n., fletcher, j., 2000: plant l-ascorbic acid: chemistry, function, metabolism, bioavailability and effects of processing. j. sci. food agr. 80(7), 825-860. doi: 10.1002/(sici)1097-0010(20000515)80:7<825::aid-jsfa598>3.0. co;2-6 de jesús ornelas-paz, j., martínez-burrola, j.m., ruiz-cruz, s., santana-rodríguez, v., ibarra-junquera, v., olivas, g.i., pérezmartínez, j.d., 2010: effect of cooking on the capsaicinoids and phenolics contents of mexican peppers. food chem. 119(4), 1619-1625. doi: 10.1016/j.foodchem.2009.09.054 deepa, n., kaur, c., george, b., singh, b., kapoor, h.c., 2007: antioxidant constituents in some sweet pepper (capsicum annuum l.) genotypes during maturity. lwt-food science and technology 40(1), 121129. doi: 10.1016/j.lwt.2005.09.016 dorji, k., behboudian, m.h., zegbe-dominguez, j.a., 2005: water relations, growth, yield, and fruit quality of hot pepper under deficit irrigation and partial rootzone drying. sci. hort. 104(2), 137-149. doi: 10.1016/j.scienta.2004.08.015 estrada, b., bernal, m.a., díaz, j., pomar, f., merino, f., 2000: fruit development in capsicum annuum: changes in capsaicin, lignin, free phenolics, and peroxidase patterns. j. agric. food chem. 48(12), 62346239. doi: 10.1021/jf000190x gnayfeed, m.h., daood, h.g., biacs, p.a., alcaraz, c.f., 2001: content of bioactive compounds in pungent spice red pepper (paprika) as affec ted by ripening and genotype. j. sci. food agr. 81(15), 1580-1585. doi: 10.1002/jsfa.982 howard, l.r., talcott, s.t., brenes, c.h., villalon, b., 2000: changes in phytochemical and antioxidant activity of selected pepper cultivars (capsicum species) as influenced by maturity. j. agric. food chem. 48(5), 1713-1720. doi: 10.1021/jf990916t juárez, l.á.m., quijada, d.m.m., sánchez c.l.d.t., águilar, g.a.g., meza, n.g., 2012: antioxidant activity of peppers (capsicum annuum l.) extracts and characterization of their phenolic constituents. interciencia: revista de ciencia y tecnología de américa 37(8), 588-593. kaur, c., kapoor, h.c., 2001: antioxidants in fruits and vegetables – the millennium’s health. int. j. food sci. tech. 36(7), 703-725. doi: 10.1111/j.1365-2621.2001.00513.x khadi, b.m., goud, j.v., patil, v.b., 1987: variation in ascorbic acid and mineral content in fruits of some varieties of chilli (capsicum annuum l.). plant foods hum. nutr. 37(1), 9-15. doi: 10.1007/bf01092295 kirschbaum-titze, p., hiepler, c., mueller-seitz, e., petz, m., 2002: pungency in paprika (capsicum annuum). 1. decrease of capsaicinoid content following cellular disruption. j. agric. food chem. 50(5), 12601263. doi: 10.1021/jf010527a kumar, o.a., tata, s.s., 2009: ascorbic acid contents in chili peppers (capsicum l.). not. sci. biol. 1(1), 50. doi: 10.15835/nsb.1.1.3445 ludy, m.j., mattes, r.d., 2011: the effects of hedonically acceptable red pepper doses on thermogenesis and appetite. physiol. behav. 102(3), 251-258. doi: 10.1016/j.physbeh.2010.11.018 martínez, s., curros, a., bermúdez, j., carballo, j., franco, i., 2007: the composition of arnoia peppers (capsicum annuum l.) at different tab. 3: the pungency of the hot peppers cultivar maximum content scoville units pungency of capsaicin shu mg kg-1 dw dracula 24.85±2.89 397.68 non-pungent (0-700 shu) pintea 2229.81±220.37 35676.96 highly pungent (25000-70000 shu) pepperone 777.49±75.69 12439.92 moderately pungent (3000-25000 shu) bulgarian carrot 990.37±93.15 15846 moderately pungent (3000-25000 shu) christmas bell 187.90±17.73 3006.48 moderately pungent (3000-25000 shu) conclusions there are major differences among cultivars in the accumulation of the bioactive compounds in the fruit during their growth and ripening, although the quantitative accumulation pathway of various components had a similar trend during phenophases. the dry matter content varied little in immature stages but a large increase was observed at ripening stages with a maximum at the physiological maturity. the soluble solids content had the same upward trend during growth and ripening and the ascorbic acid content was positively correlated with the dry matter and the soluble solids content. antioxidant activity increased during growth and ripening of hot peppers, the highest levels being found in the last stage of ripening. although the pattern of variation of total flavonoid content was affected by the cultivar, a lower value was recorded 30 days after flowering (f3) as compared with f1 in all cultivars. also, in most cultivars, an important increase of the total phenolic and total flavonoid content was observed in the last stage of ripening. cultivar’s greatest influence on the accumulation of bioactive compounds was observed regarding the content of capsaicin that was found in peppers in very small amounts (undetectable in the ‘bulga rian carrot’ and ‘christmas bell’ cultivars) in f1. during growth and ripening, fruit content of capsaicin increased to a maximum level (f3 or f4), then declined until the full ripening of the pepper fruits. scoville score analysis showed that the ‘dracula’ cultivar is classified as “non-pungent” (fruits are not spicy) while ‘pintea’ is classified as “highly pungent”, the other analyzed cultivars having an average level of pungency. acknowledgement this work was supported by a grant of the romanian national authority for scientific research and innovation, cncs/cccdi – uefiscdi, project number pn-iii-p2-2.1-bg-2016-0019, within pncdi iii. references alvarez-parrilla, e., de la rosa, l.a., amarowicz, r., shahidi, f., 2010: antioxidant activity of fresh and processed jalapeno and serrano peppers. j. agric. food chem. 59(1), 163-173. doi: 10.1021/jf103434u evolution of bioactive compounds during growth and ripening of hot peppers 237 stages of maturity. int. j. food sci. nutr. 58(2), 150-161. doi: 10.1080/09637480601154095 martínez, s., lópez, m., gonzález-raurich, m., bernardo alvarez, a., 2005: the effects of ripening stage and processing systems on ascorbic acid content in sweet peppers (capsicum annuum l.). int. j. food sci. nutr. 56(1), 45-51. doi: 10.1080/09637480500081936 materska, m., perucka, i., 2005: 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netto, f.m., carraro, f., 2005: chemical composition and antioxidant activity of juices from mature and immature acerola (malpighia emarginata dc.). food sci. technol. int. 11(4), 315-321. doi: 10.1177/1082013205056785 scoville, w.l., 1912: note on capsicums. j. am. pharm. assoc. 1(5), 453454. doi: 10.1002/jps.3080010520 simonne, a.h., simonne, e.h., eitenmiller, r.r., mills, h.a., green, n.r., 1997: ascorbic acid and provitamin a contents in unusually colored bell peppers (capsicum annuum l.). j. food comp. anal. 10(4), 299-311. doi: 10.1006/jfca.1997.0544 singleton, v.l., rossi, j.a., 1965: colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. am. j. enol. vitic. 16(3), 144-158. tilahun, s., paramaguru, p., rajamani, k., 2013: capsaicin and ascorbic acid variability in chilli and paprika cultivars as revealed by hplc analysis. j. plant breed. genet. 1(2), 85-89. usman, m.g., rafii, m.y., ismail, m.r., malek, m.a., latif, m.a., 2014: capsaicin and dihydrocapsaicin determination in chili pepper genotypes using ultra-fast liquid chromatography. molecules 19(5), 6474-6488. doi: 10.3390/molecules19056474 vera-guzmán, a.m., chávez-servia, j.l., carrillo-rodríguez, j.c., lópez, m.g., 2011: phytochemical evaluation of wild and cultivated pepper (capsicum annuum l. and c. pubescens ruiz & pav.) from oa xaca, mexico. chil. j. agr. res. 71(4), 578. doi: 10.4067/s0718-58392011000400013 zhuang, y., chen, l., sun, l., cao, j., 2012: bioactive characteristics and antioxidant activities of nine peppers. j. funct. foods 4(1), 331-338. doi: 10.1016/j.jff.2012.01.001 address of the authors: mira elena ionica, violeta nour: university of craiova, faculty of horticulture, department of horticulture and food science, 13 a.i.cuza street, 200585, romania e-mail: vionor@yahoo.com ion trandafir: university of craiova, faculty of sciences, department of chemistry, 107 calea bucuresti street, 200529, romania © the author(s) 2017. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 88, 152 163 (2015), doi:10.5073/jabfq.2015.088.022 1college of quartermaster technology, jilin university, changchun, china 2laboratory of nutrition and functional food, jilin university, changchun, china optimization of ultrasound-assisted extraction of polyphenols from maize filaments by response surface methodology and its identification lv lingzhu1, wei lu1, chen dongyan1, liu jingbo2, lin songyi2, ye haiqing1, yuan yuan1* (received february 16, 2015) * corresponding author summary maize filaments (mf) are the outer thread-like part of corn, which are widely used in traditional and official medicine. besides, mf polyphenols are important secondary metabolites in mf which have their significant biological activities. however the existing mf is underutilized. this study aimed to raise the comprehensive utilization of mf and to increase the economic value and benefit of mf. in the current study, central composite design (ccd) was first used to investigate the effect of process variables on polyphenols contents from mf by ultrasound-assisted extraction (uae). results showed that the obtained optimal uae conditions were as follows: extraction power of 520.01 w, ethanol concentration of 61.08 %, and a solvent-to-material ratio of 26.83 ml/g for polyphenols extraction. these experimental values under optimal conditions were consistent with the predicted values with polyphenols content of 7.1±0.015 mg/g. sixteen phenolic compounds, including gallic acid, catechin, epicatechin, hyperoside were identified in mf polyphenols extractions by hplc-ms/ms method. antioxidant activity assays demonstrated that mf phenols had excellent scavenging ability for abts radicals, •oh, dpph radicals and •o2-, and 42.56 ± 1.24 % of lipid oxidation inhibition. introduction maize filaments (mf), as an indispensable part of corn, have high pharmaceutical values, as they are mild diuretic, urinary demulcent, which passes stones and gravel from the kidneys and the urinary bladder; these filaments function against benign prostatic hyperplasia, cystitis, gout, chronic nephritis, and similar ailments (british herbal pharmacopoeia, 1996; czygan and maydis, 1997; maksimović et al., 2005; stagos et al., 2012). in previous studies, a series of phenolic compounds such as rutin, quercetin, epicatechin, vanillic acid, benzoic acid, syringic acid, and gallic acid have been identified and isolated from mf (chen et al., 2010; pedreschi and cisneros-zevallos, 2007). phenols (hydroxybenzenes) and polyphenols (containing two or more phenol groups) are ubiquitous in plant foods. phenols have been traditionally considered as “anti-nutrients” because these substances could reduce the digestibility of proteins and subsequently increase fecal nitrogen excretion (alzueta et al., 1992; longstaff and mcnabb, 1991). pharmacological investigations have shown that phenolic compounds have an important function in human health (vijaya kumar reddy et al., 2010), including anti-platelet and antithrombotic actions (lu et al., 1999), anti-cancer activity (sun et al., 2011; stagos et al., 2012), inhibition of hela cell multiplication (sun et al., 2010), and antioxidant activity (gladine et al., 2007; lee et al., 2010; wang et al., 2012). conventional extraction methods such as maceration, boiling, refluxing, heating, and soxhlet extraction are used for extraction of biologically active polyphenols from various plants. however, these conventional extraction methods are generally time-consuming and low efficient of the yield of extract (li et al., 2005; ma et al., 2008). in the recent years, novel extraction techniques such as ultrasoundassisted extraction (uae), microwave-assisted extraction (mae), accelerated solvent extraction (ase), and supercritical fluid extraction (sfe) have been developed to increase efficiency of the extraction process with respect to time and solvent consumption as well as the extraction yield and active compound concentration (yang and zhai, 2010; liu et al., 2011; skalicka-woźniak et al., 2011; pan et al., 2012). among these methods, uae method is advantageous for polyphenols extraction due to the process simplicity, low working temperatures (from 40 to 60 oc, high recovery rate, low oxidation loss, high energy-saving efficiency, and high extraction yield (pan et al., 2012)). the mechanism of uae involves mechanical and cavitation efficacies which can result in disrupting the cell wall, reducing the particle sizes, and enhancing the mass to transfer across the cell membrane (hossain et al., 2012). phenols and polyphenols very soluble in a variety of solvents. the most common solvents are aqueous mixtures with methanol, ethanol and acetone. methanol and acetone have toxic effects on the human body, besides, the production cost is high, so ethanol is always used as a extraction solvent. response surface methodology (rsm) is a modelling tool used to optimize the extraction conditions by evaluation of multiple parameters and their interaction effects based on quantitative data. therefore rsm can not only effectively optimize complex extraction procedures statistically, but can also reduce the number of experimental trials (brachet et al., 1999). rsm has been often used to optimize uae of phenolic compounds from different plant sources (liu et al., 2011; skalicka-woźniak et al., 2011; hossain et al., 2012). li et al. (2015) have evaluated an ionic-liquidbased mae and uae method for the extraction of 2,4-dihydroxy 7-methoxy-1,4-benzoxazin-3-one and 6-methoxy-benzoxazolin-2one from etiolated maize seedlings. however, there are no published reports on the uae of polyphenols from mf as well as on use of rsm for its optimization of uae. the aim of the present work was to use rsm to establish the optimum parameters of uae of mf polyphenols and their identification. three factors with three level central composite designs were used to optimize and investigate the effects of ultrasound, ethanol concentration, and solid-to-liquid ratio of uae on the maximum yield of polyphenols from mf. hplc-ms/ms method was used to identify the main phenol compounds in mf extracts obtained by uae. materials and methods plant materials the plant materials with poaceae female flowers used in this experiment were mature corn silk of zea mays l. exhibiting a regular growth pattern, which were gathered from corn fields in october 2012. collected corn silks were dried in a shaded and well-ventilated ultrasound-assisted extraction of polyphenols and identification 153 place, homogenized by a blender (ds-1, shanghai precision instruments co., ltd, shanghai, china), and sieved through a 100 meshes sieve. the powder was stored in sealed polyethylene bags at 4 oc. chemicals and reagents absolute ethanol and sodium bicarbonate were purchased from beijing chemical works (beijing, china). pyrogallic acid was purchased from the tianjin guangfu fine chemical research institute. folin-ciocalteu (fc reagent) was purchased from shanghai sunny chemical industry tech co., ltd. deionized water was purified by milli-q system (millipore corp., bedford, ma, usa). all of the organic solvents used in the study were of analytical grade. uae of polyphenols from mf mf powder (1.000 g) was placed in a capped tube and mixed with ethanol. extraction was performed using an ultrasonic cell crushing apparatus [jy92-2d, 20 khz to 25 khz, 1800 w (max), ningbo new cheese biological technology co., ltd., zhejiang, china]. after extracting, the sample was centrifuged at 4000 rpm/min for 15 min to collect the supernatant. the mf powder was extracted twice with the same method, and the supernatant of the three extracts were collected together into a 50 ml dark volumetric flask. the sample solution was evaporated using the re-52a-type rotary evaporator apparatus enrichment (re-52aa, shanghai yarong-biochemical instruments factory, shanghai, china), and then dried with the vacuum freeze-drying system (alpha 1-2, martin christ gmbh germany). rsm experimental design for optimal extraction conditions prior to rsm design, the single factors influencing the extraction yields have been conducted and suitable ranges of each independent variable have been obtained. the results showed that three main factors affect the extraction efficiencies, including the power of ultrasound (w, x1), ethanol concentration (%, x2), and solvent-tomaterial ratio (ml/g, x3). in the rsm design, these factors were used as independent variables for optimizing extraction conditions. according to the single factors experiments, we determined the variables and their ranges were as following, extraction power of ultrasound (400 w to 600 w), ethanol extraction concentration (50% to 70%), and solvent-to-material ratio (20:1 ml/g to 40:1 ml/g). temperature was not considered in this study since the sample was processed at 35 oc to avoid the degradation of temperature-sensitive polyphenols compounds. in this study, the experiments were performed based on a central composite design (ccd). the coded values of the experimental factors and their levels in ccd were shown in tab. 1. the complete design was performed randomly and consisted of twenty combinations, including six replicates at the central point (tab. 2). the data from ccd were analyzed by multiple regressions to fit the following quadratic polynomial model (prakash et al., 2013): (1) where y is the response, xi and xj are variables (i and j range from 1 to k), β0 is the model intercept coefficient, βj, βjj, and βij, are the interaction coefficients of linear, quadratic, and second-order terms, respectively, k is the number of independent parameters (k = 4 in this study), and ei is the error (prakash et al., 2013). determination of polyphenols polyphenols were determined by folin-ciocalteu (fc) procedure according to the method of hagerman et al. (2000) with slight modifications. in brief, 1.0 ml of polyphenol extract was mixed with 2.5 ml of fc reagent. then 2.5 ml of sodium bicarbonate (10 %, w/v) was added to the mixture. the volume was increased to 25 ml by adding distilled water. the solution was allowed to stand for 1 h at room temperature and the absorbance was measured by spectrophotometry at 785 nm. the results were expressed as μg gallic acid equivalent per g sample. polyphenols content can be calculated using the following equation: polyphenolyield (mg/g) = c × n × 25 x 10-3/m (2) where c is the sample fluid concentration of the polyphenols calculated based on gallic acid standard curve, μg/ml ; n is the diluted amount; and m is the weight of mf powder, g. antioxidant activity analysis of mf polyphenols extractions the antioxidant activity of mf polyphenols extractions was evaluated by scavenging abts radical, hydroxyl radical (•oh), dpph radical, and superoxideanion (•o2-), which were followed by the method of yuan et al. (2013). the lipid oxidation inhibition assay of mf polyphenols extraction was measured using the similar method of muñiz-márquez et al. (2013). identification of the main polyphenols compounds by hplcms/method this analysis was conducted according to method described by wang et al. (2008). a waters e2695-tqd system (waters corporation, usa), consisting of waters e2695 hplc system (including vacuum degasser, binary pump, autosampler, column oven), waters tq detector (tqd) and waters 2489 ultraviolet/visible light detector were used for separation and identification of polyphenols compounds. chromatographic separations were carried out using a waters symmetry c18 column (4.6 × 150 mm, 5 μm). the mobile phase was constituted of 0.1 % glacial acetic acid in water (solvent a) and methanol (solvent b). the gradient conditions used was run as described hereafter: from 0 to 10.0 min, a linear gradient from 30 % solvent b to 45 % solvent b; from 10.0 to 12.0 min, a linear gradient from solvent b at 45 % to 70 %; from 12.0 to13.0 min, solvent b at 70 % to 0 %; and from 13.0 to15.0 min, a linear gradient of solvent b from 0 % to 30 %. the column was reconditioned at 30 % solvent b from 15.0 to 20.0 min. the column temperature was set at 40 oc with a solvent flow rate of 0.3 ml/min. a total of 5 μl of sample was injected into the column for hplc-ms/ms analysis. the detection was carried out on a waters tqd mass spectrometer from waters (waters corporation, usa) equipped with an electrospray ionization source in positive mode. the ionization parameters were: nitrogen sheath gas flow, 10 / min; spray voltage, 30 psi; capillary temperature, 300 oc; capillary voltage, 4000 v; and tube lens voltage, 80 v. the full scan mass spectrum in the range of m/z 30 to 3000 amu was measured.   5     suitable ranges of each independent variable have been obtained. the results showed that three main 99   factors affect the extraction efficiencies, including the power of ultrasound (w, x1), ethanol 100   concentration (%, x2), and solvent-to-material ratio (ml/g, x3). in the rsm design, these factors were 101   used as independent variables for optimizing extraction conditions. according to the single factors 102   experiments, we determined the variables and their ranges were as following, extraction power of 103   ultrasound (400 w to 600 w), ethanol extraction concentration (50% to 70%), and solvent-to-material 104   ratio (20:1 ml/g to 40:1 ml/g). temperature was not considered in this study since the sample was 105   processed at 35 oc to avoid the degradation of temperature-sensitive polyphenols compounds. in this 106   study, the experiments were performed based on a central composite design (ccd). 107   the coded values of the experimental factors and their levels in ccd were shown in tab. 1. the 108   complete design was performed randomly and consisted of twenty combinations, including six 109   replicates at the central point (tab. 2). the data from ccd were analyzed by multiple regressions to fit 110   the following quadratic polynomial model (prakash et al., 2013): 111   (1) 112   where y is the response, xi and xj are variables (i and j range from 1 to k), β0 is the model intercept 113   coefficient, βj, βjj, and βij, are the interaction coefficients of linear, quadratic, and second-order terms, 114   respectively, k is the number of independent parameters (k=4 in this study), and ei is the error 115   (prakash et al., 2013). 116   determination of polyphenols 117   polyphenols were determined by folin-ciocalteu (fc) procedure according to the method of 118   hagerman et al. (2000) with slight modifications. in brief, 1.0 ml of polyphenol extract was mixed 119   with 2.5 ml of fc reagent. then 2.5 ml of sodium bicarbonate (10%, w/v) was added to the mixture. 120   the volume was increased to 25 ml by adding distilled water. the solution was allowed to stand for 1 121   h at room temperature and the absorbance was measured by spectrophotometry at 785 nm. the results 122   were expressed as µg gallic acid equivalent per g sample. polyphenols content can be calculated using 123   the following equation: 124   (2) 125   where c is the sample fluid concentration of the polyphenols calculated based on gallic acid standard 126   curve, µg/ml ; n is the diluted amount; and m is the weight of mf powder, g. 127   antioxidant activity analysis of mf polyphenols extractions 128   the antioxidant activity of mf polyphenols extractions was evaluated by scavenging abts radical, 129   hydroxyl radical (•oh), dpph radical, and superoxideanion (•o2 -), which were followed by the method 130   of yuan  et al. (2013). the lipid oxidation inhibition assay of mf polyphenols extraction was measured 131   using the similar method of muñiz-márquez et al. (2013). 132   tab. 1: levels of variables for the experimental design. symbols independent variables -1 0 +1 x1 ultrasound power (w) 400 500 600 x2 ethanol concentration (%) 50 60 70 x3 solvent-to-material ratio (ml/g) 20:1 30:1 40:1 154 lv l., wei l., chen d., liu j., lin s., ye h., yuan y. tab. 2: central composite design (ccd) plan in coded value and results of experiment treatment factors yield of polyphenols (mg/g) x1 x2 x3 power(w) ethanol concentration (%,v/v) solid-liquid ratio (ml/g) experimental predicted 1 -1 1 1 5.7768 5.8460 2 -1 -1 -1 5.8448 6.1089 3 0 1.6818 0 5.7072 5.9020 4 0 0 -1.6818 7.3070 7.2700 5 1 -1 -1 6.2346 6.4154 6 1 1 1 6.4925 6.4784 7 -1 1 -1 5.8459 5.7979 8 0 0 0 7.9686 7.9974 9 0 0 0 7.9789 7.9974 10 0 0 0 7.9812 7.9974 11 1.6818 0 0 6.4789 6.4016 12 -1.6818 0 0 5.8884 5.6121 13 0 0 0 7.9777 7.9974 14 0 0 0 7.9914 7.9974 15 0 -1.6818 0 5.7080 5.1596 16 1 1 -1 6.9063 6.7742 17 0 0 1.6818 6.6761 6.3595 18 -1 -1 1 4.9399 5.3220 19 1 -1 1 4.9866 5.2846 20 0 0 0 8.0257 7.9974 statistical analysis the experimental results of the response surface design were analyzed by design-expert 8.0.6 software (trial version, stateease inc., minneapolis, mn, usa). the modeling was started with a quadratic model including linear, squared, and interaction terms. significant terms in the model for each response were found by analysis of variance (anova) and the significance was judged by the f-statistic calculated from the data. the experimental data was evaluated with various descriptive statistical analyses such as p value, f value, degrees of freedom (df), sum of squares (ss), and mean sum of squares (mss) to reflect the statistical significance of the developed quadratic mathematical model. after fitting the data to the models, the generated data were used for plotting response surfaces and contour plots. p-values ≤ 0.05 were considered as statistically significant. all of the experiments were conducted in triplicate unless specified otherwise. results fitting the response surface models uae was conducted in 20 runs to study the effect of different variables on the amount of polyphenols extracted from mf. the polyphenols responses of the experimental design were presented in tab. 2. the decoded values of independent variable for the experiment were also presented. polyphenols content of mf extract varied from 4.3183 to 7.0206 mg per g dried mass based on different extraction conditions. multiple regression analysis and pareto analysis of variance (anova) were used to check the adequacy and fitness of the developed models and results of anova were given in tab. 3. considering the single factor test results, the response surfaces analysis results, and the experimental results, we summarized the test data result of linear regression and binomial fitting analysis in tab. 3. the results showed that the response of worth rate (y) model was highly significant. an item ultrasound power (x1) and ethanol concentration (x2) was significant and solvent-to-material ratio (x3) was extremely significant. the second order items were reached extremely significant level. to calculate the polyphenols content in mf (y) encoded by independent variables ultrasound power (x1), ethanol concentration (x2), and solvent-to-material ratio (x3), we used the quadratic multi nomial regression equation as follows: (3) the anova results and regression coefficients were presented in tab. 3 and tab. 4, indicating that the contribution of the quadratic model was significant (p < 0.05). the ‘‘fitness’’ of the model was investigated using the lack-of-fit test (p > 0.05), indicating the suitability of models to predict variations accurately (prasad et al., 2011). the f value of the model was 25.89 and f statistical analysis shows that the model of “prob > f” return value was < 0.05, indicating that the model was significant. the proposed item is not significant. in these test results, x1, x2, x3, x12, x22, and x32 were significant. y = 34.11660 + 0.057147x1 + 0.80761x2 + 0.12396x3 + 0.000146625x1x2 + 0.001828x2x3 0.0000616296x12 0.00763692x22 0.00366157x32 ultrasound-assisted extraction of polyphenols and identification 155 tab. 3: results of analysis of variance of the primarily established quadric regression mode. source squares df square value prob > f remark model 15.85 9 1.76 25.89 <0.0001 ** x1 0.58 1 0.58 8.48 0.0155 * x2 0.51 1 0.51 7.50 0.0209 * x3 0.77 1 0.77 11.28 0.0073 ** x1x2 0.17 1 0.17 2.53 0.1429 x1x3 0.05 1 0.05 0.67 0.4332 x2x3 0.27 1 0.27 3.93 0.0756 x12 5.47 1 5.47 80.46 <0.0001 ** x22 8.41 1 8.41 123.54 <0.0001 ** ** x32 1.93 1 1.93 28.40 0.0003 residual 0.68 10 0.07 lack of fit 0.68 5 0.14 3.63 0.16 pure error 0.001565 5 0.00031 cor total 16.53 19 df means degrees of freedom; * showed significant difference, p< 0.05; * * showed very significant difference, p< 0.01; although x3, x12, x22, and x32 were very significant, the interactive items were not significant. this result shows that the effect of the three factors on the polyphenols extraction yield was summarized as follows: ultrasonic power > solid-to-liquid ratio > ethanol concentration. effect of extraction parameters on polyphenols content the response surface and contour plots of the effects of extraction parameters on the polyphenol content were presented in fig. 1 and fig. 2. as shown in fig. 1a, the efficiency of ultrasound power on the polyphenols was evaluated and the results showed that the yield increased with the increase of ultrasound power. it can be con cluded that maximum polyphenols extraction could be achieved when the ethanol concentration and ultrasonic power were 61.08% and 520.01 w, respectively. the polyphenols yield increased with an increase of ethanol concentration from 43 % to 61.08 %, while decreased when the ethanol concentration was above 61.08 %. as for the effect of ultrasound power on the polyphenols yield, the yield increased with prolonged the extraction power from 400 to 520.01 w, and decreased after 520.01 w. the interaction effect of extraction power and solvent-to-material ratio on the polyphenols was presented in fig. 1b. the increase extraction yield of polyphenols was observed with an increase solventto-material ratio from 13 to 26.83. further increase of solvent-tomaterial ratio resulted in decrease of the polyphenol content. the interaction effect of ethanol concentration and the solvent-tomaterial ratio on the polyphenols was also presented in fig. 1c. it was found that maximum polyphenols yield is achieved when the ethanol concentration was 61.08 % and the solvent-to-material ratio was 26.83 ml/g. ethanol volume fraction and the material liquid have significant interaction effect, the phenolic yield was first increased with the increase of the ethanol volume fraction and solidliquid ratio, and then decreased. verification of predictive models an optimum uae conditions for obtaining maximum mf polyphenols content in the extract were determined. all the factors and responses with the respective high-limit and low-limit experimental region have to satisfy the criteria defined for the optimum operations were stated in tab. 1. as shown in tab. 2, the predictive values matched well with experimental values obtained using optimum extraction parameters, which were confirmed with a good regression coefficient (r2 = 0.9588). applying the methodology of desired function, the optimum level of various parameters was obtained and it indicated that extraction power of 520.01 w, ethanol concentration of 61.08 % and solvent-to-material ratio of 26.83 ml/g given a phenol content of 7.1±0.015 mg/g. these optimize conditions could be considered as optimum as well as feasible conditions. antioxidant activity of mf polyphenols extractions the antioxidant activity of mf polyphenols extractions was evaluated using five assays and the results were shown in tab. 5. the abts radical scavenging ability, •oh-scavenging ability, dpph radical scavenging ability, •o2scavenging ability and lipid oxidation inhibition increased with increased mf polyphenols extractions concentration. the ic50 values of various antioxidant assays were used to evaluate the antioxidant activity of mf polyphenols extractions concentration. the ic50 values of mf polyphenols extractions concentration for the abts radical, •oh, dpph radical and •o2 were 0.7228, 0.301, 12.79, and 0.010 mg/ml, respectively. lower ic50 values indicated that mf polyphenols extractions had excellent antioxidant properties. among the assays, the best inhibiting capacity of mf polyphenols was observed in •o2(0.010 mg/ml). in lipid oxidation inhibition assay, the general ability of extracts to prevent lipid oxidant was tested, and results showed 42.56 ± 1.24 % of lipid oxidation inhibition. the results showed that the polyphenols compounds extracted from the mf also have ability to inhibit lipid oxidation. 156 lv l., wei l., chen d., liu j., lin s., ye h., yuan y.   14     412   413   414   415   fig.1: response surface for the effect of independent variables on polyphenols extraction yield. 416   (a) response surface graph showing interaction between ultrasonic power (x1/w) and 417   fig. 1: response surface for the effect of independent variables on polyphenols extraction yield. (a) response surface graph showing interaction between ultrasonic power (x1/w) and ethanol concentration (x2/%); (b) response surface graph showing interaction between ultrasonic power (x1/w) and solvent-to-material ratio (x3/ml/g); (c) response surface graph showing interaction between ethanol concentration (x2/%) and solvent-to-material ratio (x3/ml/g). fig. 2: contour plots for the effect of independent variables on polyphenols extraction yield. (a) contour plot graph showing interaction between ultrasonic power (x1/w) and ethanol concentration (x2/%); (b) contour plot graph showing interaction between ultrasonic power (x1/w) and solvent-to-material ratio (x3/ml/g); (c) contour plot graph showing interaction between ethanol concentration (x2/%) and solventto-material ratio (x3/m/g).   16     422   423   424   425   fig. 2: contour plots for the effect of independent variables on polyphenols extraction yield. 426   (a) contour plot graph showing interaction between ultrasonic power (x1/w) and ethanol 427   ultrasound-assisted extraction of polyphenols and identification 157 tab. 4: significance test table of regression coefficient. std. dev. 0.2608 r-squared 0.9588 mean 5.8034 adj r-squared 0.9218 c.v. % 4.4945 pred r-squared 0.6887 press 5.1468 adeq precision 13.4740 tab. 5: antioxidant activity of mf phenol extractions by scavenging free radicals. free concentration of scavenging ic50 radical mf phenol extractions rate (mg/ml) (mg/ml) 0.5333 44% 0.7999 52% 1.066 62% 1.33 76% 0.083 7% 0.16 18% 0.25 38% 0.333 59% 5 13% 10 23% 15 87% 20 94% 0.05 36% 0.01 54% 0.02 72% 0.03 80% dpph 0.72 abts 0.30 •oh 12.79 •o2 0.01 hplc-ms/ms analysis and identify the main polyphenols in mf extractions the hplc-ms/ms chromatograms of mf extraction were given in fig. 3. sixteen phenolic compounds, including gallic acid, catechin, epicatechin, hyperoside were identified according to the method of hplc-ms/ms. the chemical structures of sixteen phenolic compounds were provided in tab. 6. discussion in the current study, ultrasonic extraction and rsm design were successfully used to optimize the extraction conditions of polyphenols from mf, and optimal extraction conditions were also established. the best extraction conditions were extraction power of 520.01 w, ethanol concentration of 61.08%, and solvent-to-material ratio of 26.83 ml/g. from the statistical analysis, we learned that ultra sonic power was the most significant factor influencing the extraction yield. the yield increased to 3.56% with the increase of extraction power from 400 to 520.01 w. the results might due to the collision of molecules when the ultrasound power increased, thus increased the release of phenolic compounds. besides, the increases of the ultrasound power may lead to the increase of temperature together with the degradation of the phenols during oxidation at high temperature (spigno and de favari, 2009). the ethanol concentration was another important factor. the polyphenols content increased to 18.07 % when the ethanol concen tration increased from 43 % to 61.08 %. the findings obtained from our study were in good agreement with bucić-kojić et al. (2011), where the total phenolics yield from lyophilized fig fruits increased to 54.17 % when ethanol concentration increased from 50 to 80 %. although pure water is the most polar solvent of all solvents, it did not yield the best results with respect to extract and polyphenols content. this phenomenon might be attributed to the higher viscosity of water than that of the other solvents, which is a matter of mass transfer. as the polyphenols yield depended largely on the polarity of solvents and compounds, a single solvent might not be effective for the extraction of a bioactive compound. hence, a combination of ethanol with water was more effective in extracting phenolic compounds than ethanol alone (markom et al., 2007). sixty percent of ethanol showed the greatest yield of polyphenols, as a result of lower viscosity and altering the plant structure by swelling the matrix, which enabled the solvent to more completely penetrate the leaves. accordingly, water is acting as the plant swelling agent, while ethanol is believed to disrupt the bonding between the solutes and plant matrices. therefore, the mixture of water and ethanol as solvent agent exhibited the best performance to extract phenolic among all the extractants used. another explanation might be the high dielectric constant of water, which leads to increase polarity indices of ethanol in aqueous solution (sahin and saml, 2013). in the present study, the increase extraction yield of polyphenols was observed with the increase solvent-to-material ratio from 13 to 26.83. this was probably due to the fact that more solvent could enter plant cells while more phenolic compounds could permeate into the solvent under the higher solvent-to-material ratio conditions (prasad et al., 2009). many plants have been examined have strong antioxidant ability (vijaya, et al., 2010; wang et al., 2012). in the present study, mf total phenolic extractions exhibited strong in vitro antioxidant ability and could be considered as a good source of natural antioxidant compounds, which was in agreement with the study of mohammad et al. (2008), they showed that mf have potent antioxidant ability and might exert a protective effect against oxidative damage. besides, the antioxidant activity of mf polyphenols extractions increased with the increase of its concentration. sixteen phenolic compounds, including gallic acid, catechin, epicatechin, hyperoside were identified in mf polyphenols extractions by hplc-ms/ms method, which was the first time uae polyphenols from mf were identified. it can be concluded that the chemical properties of the phenolic compounds vary significantly because these compounds have different hydroxyl groups and glycoside.the number of identified phenolic compounds is higher that previously reported by chen et al. (2010), they used hplc technology to identify eight phenolic compounds in sweet corn silk, including gentisidc acid, rutin, quercetin, epitatechin, syringic, catechin, gallic acid and caffeic acid. pedreschi et al. (2007) analysed phenolic compounds of andean purple corn and found some major phenolic compounds similar to our study, such as protocatechuic acid. however, there was some dissimilarity in the composition of phenolic acids, including vanillic acid and p-coumaric acid, which might be due to the different maize varieties and experimental methods. therefore, it is concluded that uae is an efficient and credible method for the extraction of polyphenols from mf. the isolated polyphenols possess strong antioxidant activity. sixteen phenolic compounds were firstly identified in mf extracts by hplc-ms/ms method. all results can provide scientific reference of analysis and utilization of natural plant resources. 158 lv l., wei l., chen d., liu j., lin s., ye h., yuan y.   17     concentration (x2/%); (b) contour plot graph showing interaction between ultrasonic power 428   (x1/w) and solvent-to-material ratio (x3/ml/g); (c) contour plot graph showing interaction 429   between ethanol concentration (x2/%) and solvent-to-material ratio (x3/m/g). 430   431   432   433   434   435   (1) (2) 436   437   (3) (4) 438   439   440     18     (5) (6) 441   442   443   (7) (8) 444   445   446   (9) (10) 447   448   449   11 12 450   451   452   453   ultrasound-assisted extraction of polyphenols and identification 159   18     (5) (6) 441   442   443   (7) (8) 444   445   446   (9) (10) 447   448   449   11 12 450   451   452   453     19     454   13 14 455   456   457   15 16 458   459   460   fig. 3. hplc-ms/ms chromatograms of mf polyphenols extract and analysis mass spectrum of 461   the sixteen phenolic compounds. 462   ms: (1) benzoic acid; (2) gentisic acid; (3) epicatechin; (4) b2 benzoic acid; (5) syringic acid; (6) 463   catechin; (7) catechin gallate; (8) quercetin; (9) quercetin-3-o-rhamnoside; (10) rutin;(11) 464   chlorogenic acid; (12) gallic acid; (13) gallic acid-6 hydroxy diphenyl-pyran glucose; (14) ellagic acid; 465   (15) epicatechin trimer; (16) epicatechin tetramer. 466    467    468   469   470   471   472   473   474   160 lv l., wei l., chen d., liu j., lin s., ye h., yuan y.   19     454   13 14 455   456   457   15 16 458   459   460   fig. 3. hplc-ms/ms chromatograms of mf polyphenols extract and analysis mass spectrum of 461   the sixteen phenolic compounds. 462   ms: (1) benzoic acid; (2) gentisic acid; (3) epicatechin; (4) b2 benzoic acid; (5) syringic acid; (6) 463   catechin; (7) catechin gallate; (8) quercetin; (9) quercetin-3-o-rhamnoside; (10) rutin;(11) 464   chlorogenic acid; (12) gallic acid; (13) gallic acid-6 hydroxy diphenyl-pyran glucose; (14) ellagic acid; 465   (15) epicatechin trimer; (16) epicatechin tetramer. 466    467    468   469   470   471   472   473   474   fig. 3: hplc-ms/ms chromatograms of mf polyphenols extract and analysis mass spectrum of the sixteen phenolic compounds. ms: (1) benzoic acid; (2) gentisic acid; (3) epicatechin; (4) b2 benzoic acid; (5) syringic acid; (6) catechin; (7) catechin gallate; (8) quercetin; (9) quercetin-3-o-rhamnoside; (10) rutin;(11) chlorogenic acid; (12) gallic acid; (13) gallic acid-6 hydroxy diphenylpyran glucose; (14) ellagic acid; (15) epicatechin trimer; (16) epicatechin tetramer. acknowledgements this work was supported by jilin province science and technology development plan project (20130206098sf) and the youth scientific innovation leading talent and team building project of jilin province (20140519014jh). accordingly, the authors gratefully acknowledge the funds support. references alzueta, c., trevino, j., ortiz, l., 1992: effect of tannin from fava beans on protein utilisation in rats. j. sci. food agric. 59, 551-553. british herbal pharmacopoeia, 1996: corn silk, british herbal medicine association. brachet, a., mateus, l., cherkaoui, s., christen, p., gauvrit, j.y., lanteri, p., veuthey, j.l., 1999: application of central composite designs in the supercritical fluid extraction of tropane alkaloids in plant extracts. analusis 27, 772-778. bucić-kojić, a., planinić, m., tomas, s., jokić, s., mulić, i., bilić, m., velić, d., 2011: effect of extraction conditions on the extractability of phenolic compounds from lyophilised fig fruits (ficus carica l.). pol. j. food nutr. sci. 61, 195-199. czygan, f.c., maydis stigma, 1997: teedrogen und phytopharmaka, wissenschaftliche verlagsgesellshaft mbh, stuttgart, in: m. wichtl (ed.), 362. chen, z.y., xu y.j., yin,y., liu, x.m., li, x.q., 2010: analysis of polyphenol composition of different parts of ear of sweet corn. food sci. 31, 235-238. gladine, c., morand, c., rock, e., bauchart, d., durand, d., 2007: plant extracts rich in polyphenols (perp) are efficient antioxidants to prevent lipoperoxidation in plasma lipids from animals fed n-3 pufa supplemented diets. anim. feed sci. tech. 136, 281-296. hagerman, a., harvey-mueller, i., makkar, h.p.s., 2000: quantification of tannins in tree foliage-a laboratory manual, 4-7. fao/iaea, vienna. hemwimol, s., pavasant, p., shotipruk, a., 2006: ultrasound-assisted extraction of anthroquinones from roots of morinda citrifolia. ultrason. sonochem. 13, 543-548. hossain, m.b., brunton, n.p., patras, a., tiwari, b., o’donell, c.p., 2012: optimization of ultrasound assisted extraction of antioxidant compounds from marjoram (origanum majorana l.) using response surface methodology. ultrason. sonochem. 19, 582-590. longstaff, m., mcnabb, j.m., 1991: the inhibitory effects of hull polysaccarides amd tannins of field beans (vicia faba l.) on the digestion of amino acids, starch and lipid, and on digestive activities in young chicks. brit. j. nutr. 65, 199-216. lu, t.l., ye, d.j., mao, c.q., liu, x.s., 1999: studies on antiplatelet aggregation and antithrombosis action of total flavonoids of common burreed (sparganium stoloniferum). chin. tradit. herbal drugs. 30, 439-441. lang, q., wai, c.m., 2001: supercritical fluid extraction in herbal and natural product studies-a practical review. talanta. 53, 771-782. li, h., chen, b., yao, s.z., 2005: application of ultrasonic technique for extracting chlorogenic acid from eucommia ulmodies oliv. 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acid-6 hydroxy diphenyl-pyran glucose 14 0.0026 320.19 322.19 302.00 ellagic acid 15 3.487 867.7 869.70 865.00 epicatechin trimer 16 8.277 1149.09 1151.09 1153.30 epicatechin tetramer tion of ultrasound-assisted extraction of polysaccharide from cucurbita moschata. carbohydr. polym. 92, 2018-2026. pedreschi, r., cisneros-zevallos, l., 2007: phenolic profiles of andean purple corn (zea mays l.). food chem. 100, 956-963. prasad, k.n., chun, y., yang, e., zhao, m., jiang, y., 2009: effects of high pressure on the extraction yield, total phenolic content and antioxidant activity of longan fruit pericarp. innov. food sci. emerg. 10, 155-159. prasad, k.n., hassan, f.a., yang, b., kong, k.w., ramanan, r.n., azlan, a., ismail, a., 2011: response surface optimisation for the extraction of phenolic compounds and antioxidant capacities of underutilized mangifera pajang kosterm, peels. food chem. 128, 1121-1127. pan, g.y., yu, g.y., zhu, c.h., qiao, j.l., 2012: optimization of ultrasound-assisted 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mingdao university, peetow, changhwa county, taiwan ultrasonication-enhanced microbial safety of sprouts produced from selected crop species kai ying chiu (received august 17, 2014) summary seed sprouts are susceptible to microbial contamination; therefore, use of an effective sanitization treatment on these products is of great importance. this study investigated the effects of ultrasonication, heating and chemical washing on sanitization of nine crop seeds and the microbial safety of sprouts produced from decontaminated seeds. most of the sanitization treatments improved seed germination (except dry heating), reduced microbial loads on produced sprouts, and increased sprouts yield. however, only ultrasonication treatment reduced both the total aerobic counts and total coliforms counts to < 3 log 10 cfu g-1 level on produced alfalfa, mung bean pea and radish sprouts. the sprouts produced from ultrasonication-treated alfalfa, mung bean, pea and radish seeds also outyielded their respective non-treated sprouts by 88, 25, 73 and 56 %. therefore, ultrasonication can be used as a seed sanitization treatment for producing alfalfa, mung bean, pea and radish sprouts. introduction there has been an increasing interest in consuming various seed sprouts worldwide because of their high nutrient value and widespread availability. however, consumption of fresh and uncooked sprouts might impose a safety risk, because of their potential for microbial growth during the sprouting process. among the sprouts available in the market, alfalfa and mung bean sprouts are most often implicated in outbreaks of food-borne diseases (cerna-cortes et al., 2013; taban et al., 2013). the source of microbial contamination on sprouts is mainly from the micro-biological load of the seeds. the sprouting process also provides suitable conditions for propagation of microorganisms if they are present in the seeds. therefore, sanitization procedures for reducing the possible contamination of pathogenic microbes are generally performed on the selected seeds prior to sprouting (peñas et al., 2008). many chemicals, such as aqueous cio2, ozone and organic acids, have been studied for their efficacies of reducing microbial loads in seed sprouts (kim and song, 2009; scouten and beuchat, 2002; sikin et al., 2013). however, the application of chemical disin fectants in food processing is always concomitant with health concerns. heating treatment is also applied extensively to inactivate microbes (rajkovic et al., 2010; sikin et al., 2013). but the efficacy of heating treatment depends on the treatment temperature and duration, and it often leads to the loss of nutrients, development of undesirable colors and deterioration of organoleptic properties of the treated food (oms-oliu et al., 2012). the germination of heattreated seeds is also occasionally decreased to an unacceptable level (bari et al., 2009; chiu and sung, 2014). ultrasonication has long been used in many industrial sectors as an effective tool for surface cleaning (farmer et al., 2000). it has also been used as an alternative to heat processing in the food industry (awad et al., 2012; de são josé et al., 2014). however, only limited research data are currently available on the efficacy of using ultrasonication to decontaminate the seeds (yaldagard et al., 2008). the ultrasonication treatment was reported to reduce the level of microbial load on alfalfa and broccoli seeds to some extent, but the germination percentages of treated seeds were decreased to below 85 % (kim et al., 2006). on the other hand, ultrasonication treatment was reported to have satisfactory results for reducing the microbial loads of pea seeds and improving their germination percentage and sprout growth (chiu and sung, 2014). it appears that the efficacy of ultrasonication on seed sanitization is species-dependent. the objective of this study was to investigate the effects of ultra sonication on the sanitization and germination of seeds of alfalfa (medicago sativa), mung bean (vigna radiate (l.) r. wilczek), adzuki bean (vigna angularis), pea (pisum sativum l.), soybean (glycine max (l.) merr.), peanut (arachis hypogaea l.), radish (raphanus sativus l.), purple cabbage (brassica oleracea capitata f. rubra) and cauliflower (brassica oleracea botyritis) (medicago sativa). several other sanitization treatments, including heating and chemical sanitizer, were also tested and compared with ultrasonication. the effects of these seed treatments for inactivating microbes on the subsequently produced sprouts were also examined. knowledge of these variations would help identify the potential value of ultrasonication on safety control of produced sprouts. materials and methods seed materials the commercially-produced seeds of alfalfa, mung bean, adzuki bean, pea, soybean, peanut, radish, purple cabbage and cauliflower were purchased from a local market and stored at 4 ℃ until treatments were applied. seed sanitization treatments for aqueous chemical treatments, four chemical solutions were prepared and evaluated for their effectiveness in inactivating microbes on the tested crop seeds: naocl (1 g l-1), vinegar (350 ml l-1), ethanol (350 ml l-1), nacl (10 g l-1). the experiments were performed by dispersing 20 g of seed samples in 200 ml of respective chemical solutions for 15 min at 25 ℃. for heating treatments, the seed samples of each variety were soaked in a 55 ℃ hot water bath (wet heating) for 15 min or in a 70 ℃ oven (dry heating) for 72 h. the ultrasonication treatment was performed by dispersing 20 g of seed samples in 200 ml of double-distilled water in an ultrasonic bath (4ht-1014-6, crest ultrasonics, trenton, nj, usa) without mechanical agitation while applying a power input of 40 khz for 1 min at 25 ℃. the water in the ultrasonic tank was degassed for 40 min prior to the treatment. a sub-sample of non-treated seeds was prepared by soaking the seeds in double-distilled water at 25 ℃ for 15 min, and was used as a control. seed germination and sprout production fifty seeds (three replicates) subjected to various decontamination treatment were germinated. the treated seeds were placed on two pieces of moistened filter paper in a sterilized plastic tray. the plastic trays were sprinkled with distilled water every 6 h. seeds were ultrasonication seed treatment 121 germinated in a growth chamber (es4-1s, saint tien co. ltd, kaoshiung city, taiwan) in the dark at 20 ℃ for 6 days. germination was counted daily for 6 days; with seeds recorded as germinated when radicles were protruding and visible (2 mm in length), and mean germination time (mgt) was calculated (chiu et al., 2006). a sub-sample of the treated seeds (10 g) from each sanitization treatment was also germinated under the same conditions for 6 days (the growth period generally used for commercial-scale production of seed sprouts in taiwan), and the growing sprouts were collected for hypocotyl and radicle lengths and sprout fresh weight measurements. microbial analyses microbial analyses were performed on the seeds that have received sanitization treatments and on the sprouts produced from the treated seeds. in each test, the treated seeds or their sprouts (25 g) were added to 225 ml of 8.5 g l-1 nacl solution, then the solution was homogenized and diluted (a six-fold dilution) with distilled water. resultant solution was surface-deposited on a sanita-kun cultural medium sheet (chisso corporation, tokyo, japan) containing a transparent cover film, an adhesive sheet, a layer of nonwoven fabric and a water-soluble compound film designed for detection of aerobic microorganisms (morita et al., 2003). the sheets were kept in a temperature-controlled incubator according to the manufacturer’s instructions for counting of total aerobes (the compound film containing a red color chromogen generated from 2,3,5-triphenyltetrazolium chloride by microbial metabolism), total coliform (the compound film containing an indigo blue color chromogen generated from β-glucuronidase of e. coli) or total mould (the compound film containing a blue or indigo blue chromogen color generated from esterase of mould). all the sheets were incubated at 35 ℃ for 48 h, 35 ℃ for 24 h and 25 ℃ for 5 days for total aerobes, e. coli and mold counting, respectively. microbial counts were expressed in colonyforming units per g fresh weight ((log 10 cfu g-1 fresh weight). experimental design a randomized complete block design with three replicates was used to evaluate the effects decontamination on the tested seeds. the results were analyzed using the statistical analysis system (sas institute, raleigh, n.c., u.s.a.) for two-way analyses of variance (anova) to determine differences in the germination, sprout growth and microbial parameters among the tested crop seeds that have been subjected to each treatment. differences among treatments were studied with duncan’s multiple range test and differences at p < 0.05 were considered to be significant. results and discussion effects of seed sanitization treatments on microbial loads of tested crop seeds seeds usually contain high microbial loads, ranging from 3 to 6 log 10 cfu g-1 (peñas et al., 2009). in this study, the initial total aerobic counts determined from the non-treated control seeds of tested crop species, prior to sprouting, ranged from 1.82 (adzuki bean seeds) to 4.71 (radish seeds) log 10 cfu g-1 fresh weight (tab. 1). the seeds of radish, cauliflower and purple cabbage of brassicaceae family had higher total aerobic counts than alfalfa, adzuki bean, mung bean, pea, peanut and soybean seeds of family fabaceae. it appears that the pod wall tissues of fruit pod presented in fabaceae species may act as a physical barrier, which can affect the infection and propagation of microorganisms during seed development. the total aerobic count values obtained from the examined alfalfa, mung bean, soybean and radish seeds are lower than the values of the alfalfa seeds (4.01 log 10 cfu g-1) reported by soylemez et al. (2001), the mung bean seeds (4.4 log 10 cfu g-1) reported by gabriel (2005), the soybean seeds (4.6 log 10 cfu g-1) reported by molinos et al. (2009) and the radish seeds (6.3 log 10 cfu g-1) reported by bang et al. (2011). the nontreated control seeds also exhibited various levels of total coliforms counts (1.61 3.76 log 10 cfu g-1 fresh weight) and total mould counts (1.69 3.58 log 10 cfu g-1 fresh weight). the observed microbial contaminations on the tested crop seeds were expected because the selected seeds were obtained from the plants grown on open fields without special measures. as shown in tab. 1, most of the sanitization treatments were able to substantially reduce the microbial loads on the selected crop seeds. from the decontaminated seeds the measured total aerobic bacterial counts, total coliforms counts and total mould counts ranged between 0.53 and 3.82, between 0.17 and 3.32, and between 0.36 and 3.18 log 10 cfu g-1 fresh weights, respectively. the lowest average of total aerobic count (0.89 log 10 cfu g-1 fresh weight) across all the tested seeds was obtained from dry heating-treated seeds, followed by ethanol-treated (1.25 log 10 cfu g-1 fresh weight) and ultrasonication-treated (1.93 log 10 cfu g-1 fresh weight) seeds (tab. 1). the dry heating also effectively reduced the total coliforms counts and total mould counts to the lowest levels (averages of 0.58 and 0.65 log 10 cfu g-1 fresh weights across all the examined species, respectively) comparing to other sanitization treatments. these results suggest that the dry heating is the most effective seed sanitization approach comparing to other treatments. however, the ultrasonication treatment also reduced the total aerobic counts, total coliforms counts and total mould counts to 1.93, 1.55 and 1.51 log 10 cfu g-1 (average of all the tested crop species) on the treated seeds. these values were significantly lower than the data obtained from the nontreated control seeds (tab. 1). effects of seed sanitization treatments on germination of tested crop seeds the effects of seed sanitization treatments on the germination responses of tested crop seeds are shown in tab. 2. among the nontreated control seeds, the germination percentages ranged from 30.80 to 100 %, with an average of 75.47 % across all the tested species. the seeds of alfalfa, mung bean, pea, peanut, soybean, radish and cauliflower generally had an acceptable germination percentage. however, extremely lower germination percentages were obtained from the purple cabbage (30.80 %) and adzuki bean seeds (34.47 %) (tab. 1). these results suggest that some germination enhancement treatments for these two crop seeds should be taken prior to producing their sprouts (chiu et al., 2006). the germination percentages of de-contaminated seeds ranged between 6.57 and 100 % (tab. 2). most of the sanitization treatments (except dry heating) improved seed germination, among which the ultrasonication treatment raised the germination percentage of tested seeds to 93.56 % (the average of all the tested crop species) (tab. 2). both naocl and ethanol treatments also elevated the germination percentage of tested crop species to 89.97 and 87.82 %, respectively. it appears that the sanitization treatments (i.e., ultrasonication, chemical washings and wet heating) may suppress the incidence of the seed-borne microorganisms (tab. 1) that could damage the growing embryos during seed germination (nwachukwu and umechuruba, 2001). on the other hand, the lowest germination percentage was obtained from dry heating treated-seeds, with average of 61.1 % germination across all the tested crop species, with the poorest germination percentage of 6.5 % obtained from adzuki bean seeds (tab. 2). this result reconfirms that the dry heating is in some cases (except the radish and cauliflower seeds) harmful to the viability of treated seeds (bari et al., 2009), even though it is an effective seed sanitization treatment (tab. 1). 122 k.y. chiu ta b. 1 : e ff ec ts o f s ee d de co nt am in at io n tr ea tm en ts o n th e m ic ro bi al lo ad s of v ar io us te st ed c ro p se ed s. a lf al fa m un g be an a dz uk i b ea n p ea so yb ea n p ea nu t r ad is h p ur pl e ca bb ag e c au lifl ow er m ea n to ta l a er ob ic b ac te ri al c ou nt (l og 10 c f u g -1 fr es h w ei gh t) c on tr ol 3. 91 ± 0 .2 1a 2. 77 ± 0 .1 7a 1. 82 ± 0 .1 2a 2. 67 ± 0 .1 7a 3. 28 ± 0 .2 1a 3. 32 ± 0 .2 1a 4. 71 ± 0 .2 9a 4. 58 ± 0 .2 9a 4. 43 ± 0 .2 8a 3. 56 ± 0 .9 7a n ao c l ( 1 g/ l ) 2. 91 ± 0 .2 4b 2. 69 ± 0 .1 5a 1. 73 ± 0 .1 1a 1. 72 ± 0 .1 1b 1. 98 ± 0 .1 2c 2. 15 ± 0 .1 3c 3. 32 ± 0 .2 1b c 3. 65 ± 0 .2 3b 3. 82 ± 0 .2 4b 2. 70 ± 0 .8 0b w et h ea ti ng (5 5 ℃ ) 2. 57 ± 0 .2 1c 2. 05 ± 0 .0 8c 1. 30 ± 0 .0 4b 1. 33 ± 0 .0 5c 2. 65 ± 0 .1 1b 2. 57 ± 0 .1 1b 3. 07 ± 0 .1 3c 2. 68 ± 0 .1 1c 2. 32 ± 0 .1 1d 2. 26 ± 0 .5 7c d ry h ea ti ng (7 0 ℃ ) 0. 87 ± 0 .0 8e 0. 66 ± 0 .0 3e 0. 81 ± 0 .0 6d 0. 84 ± 0 .0 4d 1. 08 ± 0 .0 5e 0. 96 ± 0 .0 4f 0. 98 ± 0 .0 4f 0. 83 ± 0 .0 4f 0. 92 ± 0 .0 5f 0. 89 ± 0 .1 2e v in eg ar (3 50 m l /l ) 1. 59 ± 0 .1 3d 1. 26 ± 0 .0 7d 0. 76 ± 0 .0 4d 1. 22 ± 0 .0 6c 1. 62 ± 0 .0 9d 1. 62 ± 0 .0 9d 1. 92 ± 0 .1 0e 1. 78 ± 0 .0 9e 1. 62 ± 0 .0 9e 1. 48 ± 0 .3 4d e th an ol (3 50 m l /l ) 1. 32 ± 0 .1 1d 0. 91 ± 0 .0 5e 0. 53 ± 0 .0 3e 1. 21 ± 0 .0 6c 1. 19 ± 0 .0 6e 1. 32 ± 0 .0 7e 1. 78 ± 0 .0 9e 1. 62 ± 0 .0 9e 1. 44 ± 0 .0 8e 1. 25 ± 0 .3 6d n ac l ( 10 g /l ) 3. 62 ± 0 .1 8a 2. 44 ± 0 .1 2b 1. 11 ± 0 .0 7c 1. 71 ± 0 .1 1b 2. 65 ± 0 .1 7b 2. 32 ± 0 .1 5b c 3. 65 ± 0 .2 4b 3. 82 ± 0 .2 5b 3. 21 ± 0 .2 1c 2. 76 ± 0 .9 2b u lt ra so ni ca ti on 2. 54 ± 0 .2 2c 1. 81 ± 0 .1 1c 1. 22 ± 0 .0 6b c 1. 32 ± 0 .0 7c 2. 07 ± 0 .1 1c 1. 88 ± 0 .1 0d 2. 34 ± 0 .1 3d 2. 28 ± 0 .1 2d 2. 12 ± 0 .1 3d 1. 93 ± 0 .4 4c to ta l c ol if or m s co un t ( lo g 10 c f u g -1 fr es h w ei gh t) c on tr ol 3. 13 ± 0 .1 6a 1. 66 ± 0 .1 0b 1. 61 ± 0 .1 0a 2. 19 ± 0 .1 4a 3. 76 ± 0 .1 3a 2. 71 ± 0 .1 7a 2. 79 ± 0 .1 5a 2. 33 ± 0 .1 4a 2. 36 ± 0 .1 3a 2. 55 ± 0 .6 8a n ao c l ( 1 g/ l ) 2. 66 ± 0 .2 2b 1. 31 ± 0 .0 8c 1. 48 ± 0 .0 9b 1. 67 ± 0 .1 0b 3. 32 ± 0 .2 7b 2. 03 ± 0 .1 3b c 1. 66 ± 0 .1 0b c 1. 86 ± 0 .1 2c 2. 01 ± 0 .1 4b 2. 14 ± 0 .8 9b w et h ea ti ng (5 5 ℃ ) 2. 48 ± 0 .2 0c 1. 87 ± 0 .0 8a 1. 04 ± 0 .0 4c 0. 83 ± 0 .0 3d 1. 33 ± 0 .0 5d 1. 92 ± 0 .0 8c 1. 91 ± 0 .0 8b 2. 12 ± 0 .0 9b 2. 12 ± 0 .0 9b 1. 72 ± 0 .5 1c d ry h ea ti ng (7 0 ℃ ) 0. 69 ± 0 .0 6d 0. 49 ± 0 .0 2g 0. 21 ± 0 .0 1g 0. 17 ± 0 .0 2f 0. 85 ± 0 .0 4e 0. 41 ± 0 .0 2e 0. 46 ± 0 .0 1d 0. 54 ± 0 .0 2f 0. 87 ± 0 .0 4e 0. 58 ± 0 .2 1e v in eg ar (3 50 m l /l ) 0. 95 ± 0 .0 8d 0. 71 ± 0 .0 4e 0. 61 ± 0 .0 3e 0. 84 ± 0 .0 4d 1. 41 ± 0 .0 7d 1. 34 ± 0 .0 7d 1. 64 ± 0 .0 9c 1. 41 ± 0 .0 7d 1. 41 ± 0 .0 7d 1. 14 ± 0 .3 6d e th an ol (3 50 m l /l ) 0. 61 ± 0 .0 4d 0. 55 ± 0 .0 3f g 0. 48 ± 0 .0 3f 0. 61 ± 0 .0 3e 1. 17 ± 0 .0 6e 0. 44 ± 0 .0 2e 0. 46 ± 0 .0 1d 1. 13 ± 0 .0 6e 1. 25 ± 0 .0 6d 0. 74 ± 0 .3 2e n ac l ( 10 g /l ) 2. 86 ± 0 .1 9a b 1. 11 ± 0 .0 7d 1. 04 ± 0 .0 7c 1. 64 ± 0 .1 1b 2. 81 ± 0 .1 9c 2. 18 ± 0 .1 4b 2. 89 ± 0 .0 9a 2. 06 ± 0 .1 4b c 1. 81 ± 0 .0 9c 2. 07 ± 0 .7 0b u lt ra so ni ca ti on 2. 61 ± 0 .2 2b c 0. 67 ± 0 .0 4e f 0. 85 ± 0 .0 5d 1. 04 ± 0 .0 6c 3. 12 ± 0 .1 7c 1. 29 ± 0 .0 7d 1. 63 ± 0 .0 8c 1. 51 ± 0 .0 7d 1. 37 ± 0 .0 6d 1. 55 ± 0 .7 6c t ot al m ou ld c ou nt (l og 10 c f u g -1 fr es h w ei gh t ) c on tr ol 3. 43 ± 0 .1 8a 2. 08 ± 0 .1 3a 1. 69 ± 0 .1 1a 2. 37 ± 0 .1 5a 3. 58 ± 0 .2 2a 2. 94 ± 0 .1 8a 3. 52 ± 0 .2 2a 3. 19 ± 0 .2 0a 3. 15 ± 0 .2 0a 2. 94 ± 0 .6 8a n ao c l ( 1 g/ l ) 1. 87 ± 0 .1 5d 1. 64 ± 0 .1 0c 1. 42 ± 0 .0 9b 1. 08 ± 0 .0 7c 2. 07 ± 0 .1 3c 1. 38 ± 0 .0 9c 1. 54 ± 0 .1 0d 1. 48 ± 0 .0 9d 1. 28 ± 0 .0 8c 1. 55 ± 0 .3 0d w et h ea ti ng (5 5 ℃ ) 2. 51 ± 0 .2 1c 1. 94 ± 0 .0 8b 1. 15 ± 0 .0 5c 1. 02 ± 0 .0 4c 1. 83 ± 0 .0 8d 2. 17 ± 0 .0 9b 2. 35 ± 0 .1 0c 2. 33 ± 0 .1 0c 2. 20 ± 0 .0 9b 1. 92 ± 0 .5 0c d ry h ea ti ng (7 0 ℃ ) 0. 76 ± 0 .0 7e 0. 55 ± 0 .0 3e 0. 43 ± 0 .0 2f 0. 36 ± 0 .0 2e 0. 94 ± 0 .0 4f 0. 62 ± 0 .0 3d 0. 66 ± 0 .0 3e 0. 65 ± 0 .0 3e 0. 89 ± 0 .0 4d 0. 65 ± 0 .1 9f v in eg ar (3 50 m l /l ) 0. 82 ± 0 .0 7e 0. 92 ± 0 .0 5d 0. 59 ± 0 .0 3e 0. 69 ± 0 .0 4d 1. 04 ± 0 .0 6e f 1. 45 ± 0 .0 8c 1. 75 ± 0 .0 9d 1. 55 ± 0 .0 8d 1. 49 ± 0 .0 8c 1. 14 ± 0 .4 0e e th an ol (3 50 m l /l ) 0. 88 ± 0 .0 6e 0. 69 ± 0 .0 4e 0. 50 ± 0 .0 3e f 0. 83 ± 0 .0 4d 1. 27 ± 0 .0 7e 0. 58 ± 0 .0 3d 0. 87 ± 0 .0 5e 0. 87 ± 0 .0 5e 0. 98 ± 0 .0 5d 0. 83 ± 0 .2 2f n ac l ( 10 g /l ) 3. 15 ± 0 .2 1b 1. 62 ± 0 .1 1c 1. 06 ± 0 .0 7c 1. 66 ± 0 .1 1b 2. 75 ± 0 .1 8b 2. 23 ± 0 .1 5b 3. 18 ± 0 .2 1b 2. 73 ± 0 .1 8b 2. 34 ± 0 .1 5b 2. 33 ± 0 .7 2b u lt ra so ni ca ti on 2. 04 ± 0 .0 8d 1. 01 ± 0 .0 5d 0. 87 ± 0 .0 5d 1. 02 ± 0 .0 5c 2. 38 ± 0 .1 3c 1. 42 ± 0 .0 8c 1. 74 ± 0 .0 9d 1. 68 ± 0 .0 9d 1. 54 ± 0 .0 8c 1. 51 ± 0 .4 8d r es ul ts a re m ea ns o f t hr ee d et er m in at io ns ± s d . m ea ns in th e sa m e co lu m n fo llo w ed b y th e sa m e le tte r a re n ot s ig ni fic an tly d iff er en t a t l sd te st p < 0. 05 . ultrasonication seed treatment 123 ta b. 2 : e ff ec ts o f s ee d de co nt am in at io n tr ea tm en ts o n th e ge rm in at io n pe rc en ta ge , m ea n ge rm in at io n tim e of te st ed s ee ds a nd th e fr es h w ei gh ts o f p ro du ce d cr op s pr ou ts . a lf al fa m un g be an a dz uk i b ea n p ea so yb ea n p ea nu t r ad is h p ur pl e ca bb ag e c au lifl ow er m ea n g er m in at io n pe rc en ta ge (% ) c on tr ol 93 .5 2 ± 2. 32 bc 97 .1 4 ± 2. 41 a 34 .4 7 ± 2. 11 d 81 .6 7 ± 2. 33 d 78 .0 8 ± 2. 59 c 84 .0 4 ± 2. 11 c 99 .3 3 ± 0. 94 a 30 .8 0 ± 0. 50 d 80 .2 0 ± 1. 31 d 75 .4 7 ± 24 .0 8b n ao c l ( 1 g/ l ) 98 .0 1 ± 2. 01 ab 99 .3 3 ± 0. 94 a 94 .9 1 ± 3. 68 a 89 .4 9 ± 2. 22 bc 97 .8 2 ± 1. 96 a 99 .3 3 ± 0. 94 a 99 .3 3 ± 0. 94 a 32 .9 9 ± 0. 41 c 98 .4 7 ± 1. 23 a 89 .9 7 ± 20 .4 5a w et h ea ti ng (5 5 ℃ ) 91 .0 1 ± 3. 36 c 97 .6 8 ± 2. 60 a 89 .9 0 ± 3. 72 a 88 .9 0 ± 2. 54 bc 80 .4 0 ± 2. 63 bc 83 .4 6 ± 2. 48 c 99 .6 7 ± 0. 47 a 32 .0 8 ± 0. 95 cd 94 .1 7 ± 2. 36 b 84 .1 4 ± 19 .4 7a b d ry h ea ti ng (7 0 ℃ ) 70 .8 6 ± 3. 20 d 77 .7 4 ± 0. 97 b 6. 57 ± 0 .3 0e 76 .3 4 ± 2. 20 e 42 .3 6 ± 1. 56 d 57 .4 2 ± 1. 98 d 99 .3 3 ± 0. 94 a 24 .3 4 ± 0. 61 e 91 .6 8 ± 1. 57 bc 60 .7 4 ± 29 .2 8c v in eg ar (3 50 m l /l ) 93 .6 1 ± 1. 92 bc 99 .3 3 ± 0. 94 a 79 .2 2 ± 3. 23 b 88 .5 9 ± 2. 53 c 78 .3 6 ± 2. 24 c 82 .5 7 ± 1. 69 c 99 .3 3 ± 0. 94 a 36 .8 0 ± 0. 60 b 98 .7 4 ± 1. 20 a 84 .0 6 ± 18 .6 1a b e th an ol (3 50 m l /l ) 99 .3 3 ± 0. 94 a 99 .1 4 ± 1. 02 a 92 .5 2 ± 3. 43 a 93 .5 2 ± 2. 32 ab 81 .0 0 ± 2. 36 bc 88 .9 0 ± 1. 45 b 99 .6 7 ± 0. 47 a 35 .7 0 ± 0. 58 b 99 .6 7 ± 0. 47 a 87 .8 2 ± 19 .4 0a n ac l ( 10 g /l ) 94 .6 1 ± 2. 70 ab c 98 .5 4 ± 1. 60 a 66 .1 4 ± 3. 77 c 88 .6 9 ± 2. 53 bc 84 .5 0 ± 2. 07 b 91 .6 8 ± 2. 30 b 99 .6 7 ± 0. 47 a 32 .1 0 ± 0. 52 cd 89 .5 0 ± 1. 53 c 82 .8 3 ± 20 .3 4a b u lt ra so ni ca ti on 99 .3 3 ± 0. 94 a 96 .9 8 ± 1. 21 a 93 .4 3 ± 2. 72 a 96 .9 4 ± 1. 64 a 99 .6 7 ± 0. 47 a 99 .3 3 ± 0. 94 a 99 .3 3 ± 0. 94 a 58 .6 0 ± 0. 96 a 98 .4 0 ± 0. 80 a 93 .5 6 ± 12 .5 7a m ea n ge rm in at io n ti m e (d ay s) c on tr ol 1. 43 ± 0 .0 8b c 3. 13 ± 0 .2 0a 5. 26 ± 0 .3 3b c 3. 96 ± 0 .2 5a b 2. 82 ± 0 .1 8b c 4. 62 ± 0 .2 9a 2. 06 ± 0 .1 3a 6. 24 ± 0 .3 9a 3. 48 ± 0 .2 2a 3. 67 ± 1 .4 6a b n ao c l ( 1 g/ l ) 1. 39 ± 0 .1 1c d 2. 15 ± 0 .1 3c 5. 63 ± 0 .3 5b 3. 83 ± 0 .2 4a bc 1. 98 ± 0 .1 2f 3. 58 ± 0 .2 2c 1. 68 ± 0 .1 0b c 6. 06 ± 0 .3 8a b 2. 88 ± 0 .1 8b c 3. 26 ± 1 .6 2b c w et h ea ti ng (5 5 ℃ ) 1. 70 ± 0 .1 4a b 2. 73 ± 0 .1 1b 4. 76 ± 0 .2 0c d 3. 54 ± 0 .1 5c d 2. 51 ± 0 .1 0d e 3. 87 ± 0 .1 6b c 2. 01 ± 0 .0 8a 6. 09 ± 0 .2 5a b 3. 23 ± 0 .1 3a b 3. 36 ± 1 .3 0a bc d ry h ea ti ng (7 0 ℃ ) 1. 75 ± 0 .1 5a 3. 14 ± 0 .1 4a 6. 34 ± 0 .2 9a 4. 18 ± 0 .1 9a 4. 41 ± 0 .2 0a 5. 01 ± 0 .2 3a 2. 03 ± 0 .0 9a 6. 32 ± 0 .2 9a 3. 23 ± 0 .1 5a b 4. 03 ± 1 .5 8a v in eg ar (3 50 m l /l ) 1. 25 ± 0 .1 0c d 2. 34 ± 0 .1 2c 4. 74 ± 0 .2 5d 3. 86 ± 0 .2 1a bc 3. 07 ± 0 .1 6b 3. 88 ± 0 .2 1b c 1. 89 ± 0 .1 0a b 5. 88 ± 0 .3 1a b 2. 98 ± 0 .1 6b c 3. 33 ± 1 .3 7a bc e th an ol (3 50 m l /l ) 1. 34 ± 0 .1 0c d 2. 43 ± 0 .1 3b c 4. 78 ± 0 .2 5c d 2. 71 ± 0 .1 4e 2. 73 ± 0 .1 5c d 3. 48 ± 0 .1 8c 1. 78 ± 0 .0 9b 5. 62 ± 0 .3 0b c 2. 74 ± 0 .1 5c 3. 08 ± 1 .3 0b c n ac l ( 10 g /l ) 1. 27 ± 0 .0 8c d 2. 11 ± 0 .1 4c 4. 55 ± 0 .3 0d 3. 73 ± 0 .2 5b c 2. 36 ± 0 .1 6e 4. 12 ± 0 .2 7b 2. 02 ± 0 .1 3a 5. 98 ± 0 .3 9a b 3. 09 ± 0 .2 0b 3. 25 ± 1 .4 1b c u lt ra so ni ca ti on 1. 09 ± 0 .0 9e 1. 86 ± 0 .1 0d 3. 98 ± 0 .2 1e 3. 16 ± 0 .1 7d e 1. 86 ± 0 .1 0f 3. 28 ± 0 .1 8c 1. 54 ± 0 .0 8c 5. 19 ± 0 .2 8c 2. 69 ± 0 .1 4c 2. 75 ± 1 .2 4c s pr ou t f re sh w ei gh t ( g 10 g -1 s ee ds ) c on tr ol 78 .4 ± 4 .1 cd 10 2. 1 ± 6. 4b cd 16 7. 6 ± 9. 5e 20 3. 5 ± 9. 8c 60 .5 ± 3 .8 d 32 .8 ± 2 .1 e 72 .1 ± 4 .5 d 21 .2 ± 1 .3 bc 40 .3 ± 2 .5 d 76 .5 1 ± 51 .1 7d n ao c l ( 1 g/ l ) 94 .4 ± 7 .7 b 10 5. 6 ± 6. 6b cd 22 1. 1 ± 9. 9a b 29 2. 7 ±8 .3 b 82 .5 ± 5 .1 bc 74 .2 ± 4 .6 c 82 .1 ± 5 .1 bc 22 .4 ± 1 .4 bc 66 .4 ± 4 .1 b 11 6. 28 ± 8 0. 83 ab cd w et h ea ti ng (5 5 ℃ ) 72 .3 ± 5 .9 d 96 .7 ± 4 .0 cd 16 8. 3 ± 6. 9e 22 6. 9 ± 9. 3c 76 .8 ± 3 .2 c 58 .4 ± 2 .4 d 76 .2 ± 3 .1 cd 21 .8 ± 0 .9 bc 54 .6 ± 2 .2 c 94 .5 0 ± 60 .3 1b cd d ry h ea ti ng (7 0 ℃ ) 72 .9 ± 6 .4 d 93 .8 ± 4 .3 d 12 2. 5 ± 5. 6f 21 1. 6 ± 9. 7c 42 .5 ± 1 .9 e 28 .4 ± 1 .3 e 70 .6 ± 3 .2 d 19 .8 ± 0 .8 c 42 .8 ± 1 .8 d 78 .3 0 ± 56 .3 8c d v in eg ar (3 50 m l /l ) 48 .2 ± 3 .8 e 97 .3 ± 5 .2 cd 21 2. 2 ± 8. 9b c 31 4. 6 ± 16 .7 b 78 .6 ± 4 .2 c 78 .9 ± 4 .2 c 89 .4 ± 4 .7 b 22 .9 ± 1 .2 bc 57 .9 ± 3 .1 c 11 1. 44 ± 8 8. 27 ab cd e th an ol (3 50 m l /l ) 95 .9 ± 7 .0 b 11 4. 3 ± 6. 1b 19 3. 7 ± 9. 8c d 32 7. 5 ± 17 .4 ab 92 .1 ± 4 .9 b 70 .6 ± 3 .8 c 88 .6 ± 4 .5 b 23 .1 ± 1 .1 b 59 .8 ± 3 .2 c 11 8. 77 ± 8 6. 77 ab c n ac l ( 10 g /l ) 86 .3 ± 5 .7 bc 10 4. 4 ± 6. 9b cd 22 2. 3 ± 8. 9a b 32 2. 7 ± 10 .8 ab 10 7. 3 ± 7. 1a 87 .2 ± 5 .8 b 75 .2 ± 4 .9 cd 21 .4 ± 1 .4 bc 55 .9 ± 2 .7 c 12 1. 52 ± 8 9. 24 ab u lt ra so ni ca ti on 14 7. 7 ± 6. 0a 12 7. 8 ± 6. 8a 24 3. 9 ± 9. 7a 35 2. 7 ± 8. 9a 11 5. 6 ± 5. 1a 10 7. 8 ± 5. 4a 11 2. 8 ± 5. 8a 42 .8 ± 2 .2 a 78 .2 ± 4 .2 a 14 6. 28 ± 9 1. 02 a r es ul ts a re m ea ns o f t hr ee d et er m in at io ns ± s d . m ea ns in th e sa m e co lu m n fo llo w ed b y th e sa m e le tte r a re n ot s ig ni fic an tly d iff er en t a t l sd te st p < 0. 05 . 124 k.y. chiu the mean germination times (mgt) of tested crop seeds are also shown in tab. 2. among the non-treated control seeds, the calculated mgts ranged between 1.43 and 6.24 days, with an average of 3.67 days across all the tested species. among the tested species, alfalfa seeds had the shortest mgt, followed by radish seeds, soy bean seeds and mung bean seeds, while the longest mgt was obtained from purple cabbage seeds (tab. 2). treating the seeds with sanitization treatments (except dry heating) improved their mgt, among which the ultrasonication treatment showed the greatest improvements in mgt (average of 2.74 days across all the crop species) (tab. 2). similar improvements in mgt were also observed in the ultrasonication-treated chick peas, wheat, melon and broccoli seeds (goussous et al., 2010; kim et al., 2006). on the contrary, the dry heating treatment resulted in an increase in mgt (an average of 4.05 days across all species), suggesting that the sprouting processing of treated seeds were slowed down to some extent (tab. 2). yilddirim et al., (2011) reported that the ultrasonication enhanced germination speed was attributable to the large and rapid oscillations in bubble size that lead to disruption of plant cell walls, thereby increasing water diffusion rate by the seed. recent study of da silva and dobránszki (2014) further indicated that the ultrasonication increased the organogenesis, possibly resultant from the enhanced activities of antioxidant enzymes such as superoxide dismutase and catalase that protected cell membranes against stress caused by sound irradiation. these enhanced antioxidant enzymes were reported as one of the main reasons for priming-improved germination speed (chiu et al., 2006). thus, the ultrasonication may be beneficiary to germinating seeds by activating the antioxidant enzymes that protecting the seeds from possible soaking injury during imbibition. in this regard, a detailed physiological and biochemical examinations should be conducted on ultrasonication-treated seeds in the future. effects of seed sanitization treatments on microbial loads of pea sprouts tab. 3 presents the results of microbial loads on the sprouts produced from the control and decontaminated seeds. while the microbial loads of control seeds were kept at relatively low levels prior to sprouting (tab. 1), microbial populations increased substantially during sprouting. the sprouts produced from non-treated control seeds exhibited high levels of total aerobic bacterial counts, ranged from 5.74 (radish seeds) to 12.75 log 10 cfu g-1 fresh weights (purple cabbage seeds) (tab. 3). they also exhibited high levels of total coliforms counts (ranged between 3.42 and 7.35 log 10 cfu g-1 fresh weight) and total mould counts (ranged between 3.78 and 8.12 log 10 cfu g-1 fresh weight) (tab. 3). the increased microbial populations are due to the microbes present on the seeds and the favorable environmental conditions (e.g., water activity, temperature) in which they were grown ghandi and matthews, 2003). the extremely higher total aerobic bacterial counts obtained from nontreated control purple cabbage (12.75 log 10 cfu g-1 fresh weights and adzuki bean (11.81 log 10 cfu g-1 fresh weights) sprouts were not anticipated (tab. 3). these results are believed to be associated with the poor germinations (30.80 and 34.47 %, respectively) and longer mgt (6.24 and 5.26 days, respectively) of these two crop species (tab. 2). the organic residues left within the seeds will be further hydrolyzed and utilized by the rapidly propagated micro organisms if their fail to germinate. as a result, an extremely higher microbial population was observed on the purple cabbage or adzuki bean sprouts. as shown in tab. 3, most of the seed sanitization treatments were able to reduce the microbial loads of produced sprouts, but each with different magnitude of reduction. from the decontaminatedseeds produced sprouts the measured total aerobic bacterial counts, total coliforms counts and total mould counts ranged from 2.01 to 11.99, from 1.21 to 9.23, and from 1.64 to 9.94 log 10 cfu g-1 fresh weights, respectively. microbial sanitization using ultrasonication has been investigated for application to a range of liquid foods (chandrapala et al., 2012). it is also a safe and clean technology with high potential for quality and safety improvements of seeds and sprouts (chemat et al., 2011; chiu and sung, 2014). in this study, the ultrasonication appeared to be the most effective approach for reducing the total aerobic counts, total coliforms counts and total mould counts because their counts were reduced to lowest levels (averages of 3.53, 2.41 and 2.55 log 10 cfu g-1 fresh weights respectively, across all the examined species) comparing to other sanitization treatments (tab. 3). it is generally accepted that the total coliforms populations should be kept at the level below 3 log cfu g-1 on the raw and prepared vegetables (including salad vegetables) (stannard, 1997). same standard is also applied to the salad vegetable in taiwan. based on this scale, only the soybean sprouts produced from ultrasonicationtreated seeds are not suitable for consumption of raw-seed sprouts, and the other crop sprouts are acceptable (tab. 3). however, if a standard of < 3 log cfu g-1 on total aerobic bacterial counts is also considered, only ultrasonication-treated alfalfa, mung bean, pea and radish sprouts are acceptable (tab. 3). some of other seed sanitization treatments are also capable of reducing both total aerobic bacterial counts and coliforms counts to < 3 log cfu g-1, but their applications are limited to pea (naocl, wet heating), and radish (vinegar, ethanol) sprouts (tab. 3). these results indicate that a further sanitization procedure for the sprouts produced from decontamina ted alfalfa, mung bean, adzuki bean, soybean, peanut, purple cabbage and cauliflower seeds should be performed if the produced sprouts are consumed raw in salads and sandwiches. growth and yield of sprouts the effects of seed sanitization treatments on the sprouts yields were also evaluated based on the produced sprout fresh weight (tab. 3). the yields of sprouts produced from the non-treated control seeds were in the range of 40.4 to 203.5 g 10 g seeds-1, with an average of 83.2 g 10 g seeds-1 across all the species. the averages of sprout yields (across all species) produced from the seeds that have re ceived sanitization treatments ranged between 78.3 and 146.3 g 10 g-1 seeds (tab. 2). the sprout yield differences were noticeable among the applied treatments. the highest average of yield increase was obtained from the ultrasonication-treated seeds, with 78 % more sprout yield than the average of non-treated control seeds. the lowest average of yield increase was obtained from the wet heating-treated seeds, with 14 % more sprout yield than the average of non-treated control seeds. these results re-confirm that the higher microbial loads on germinating seeds may have a negative impact on subsequent growth of sprouts (nwachukwu and umechuruba, 2001). conclusion the present study demonstrated that most of sanitization treatments (except dry heating) were able to improve or at least maintain the germination responses of tested crop seeds, decrease the seeds and sprouts microbial loads and enhanced sprouts growth. a single ultrasonication seed treatment can reduce the total coliforms counts to < 3 log 10 cfu g-1 fresh weight on nearly all the tested sprouts (except soybean). however, if both total aerobic bacterial counts and total coliforms were required to exhibit < 3 log 10 cfu g-1 fresh weight level, only the sprouts produced from ultrasonication-decontaminated seeds of alfalfa, mung bean, pea and radish seeds were acceptable. both naocl and wet heating treatments were also capable of reducing the total aerobic bacterial counts and total coliforms ultrasonication seed treatment 125 ta b. 3 : e ff ec ts o f s ee d de co nt am in at io n tr ea tm en ts o n th e m ic ro bi al lo ad s of v ar io us te st ed c ro p sp ro ut s. a lf al fa m un g be an a dz uk i b ea n p ea so yb ea n p ea nu t r ad is h p ur pl e ca bb ag e c au lifl ow er m ea n to ta l a er ob ic b ac te ri al c ou nt (l og 10 c f u g -1 fr es h w ei gh t) c on tr ol 6. 31 ± 0 .3 3a 8. 03 ± 0 .4 9b 11 .8 1 ± 0. 74 a 8. 42 ± 0 .5 3a 6. 12 ± 0 .3 8b 7. 89 ± 0 .4 9b 5. 74 ± 0 .3 6a 12 .7 5 ± 0. 80 a 8. 17 ± 0 .5 1a 8. 38 ± 2 .3 6a n ao c l ( 1 g/ l ) 4. 04 ± 0 .3 3c 3. 13 ± 0 .2 0e 5. 98 ± 0 .3 7b 2. 62 ± 0 .1 6d 4. 68 ± 0 .2 9c d 6. 02 ± 0 .3 8d e 3. 75 ± 0 .2 3b 7. 89 ± 0 .4 9d 6. 16 ± 0 .3 8b 4. 93 ± 1 .6 2c w et h ea ti ng (5 5 ℃ ) 3. 26 ± 0 .2 7d e 5. 11 ± 0 .2 1d 6. 27 ± 0 .2 6b 2. 44 ± 0 .1 0d e 4. 98 ± 0 .2 0c d 6. 89 ± 0 .2 8c d 3. 71 ± 0 .1 5b 9. 43 ± 0 .3 9c 5. 27 ± 0 .2 2c 5. 23 ± 1 .9 7b c d ry h ea ti ng (7 0 ℃ ) 5. 89 ± 0 .5 1b 9. 78 ± 0 .4 4a 11 .9 9 ± 0. 55 a 6. 17 ± 0 .2 8b 8. 12 ± 0 .3 7a 9. 12 ± 0 .4 1a 3. 74 ± 0 .1 7b 10 .3 8 ± 0. 47 b 6. 01 ± 0 .2 7b 7. 89 ± 2 .5 2a v in eg ar (3 50 m l /l ) 4. 25 ± 0 .3 4c 7. 96 ± 0 .4 2b c 6. 65 ± 0 .3 5b 6. 74 ± 0 .3 6b 6. 04 ± 0 .3 2b 5. 98 ± 0 .3 2e 2. 68 ± 0 .1 4c 7. 97 ± 0 .4 2d 6. 07 ± 0 .3 2b 6. 05 ± 1 .6 0b e th an ol (3 50 m l /l ) 3. 87 ± 0 .2 8c d 4. 69 ± 0 .2 5d 6. 12 ± 0 .3 3b 3. 01 ± 0 .1 6c d 4. 39 ± 0 .2 3d 4. 87 ± 0 .2 6f 2. 57 ± 0 .1 4c 6. 69 ± 0 .3 6e 4. 32 ± 0 .2 3d 4. 52 ± 1 .2 6c d n ac l ( 10 g /l ) 5. 14 ± 0 .3 4b 7. 25 ± 0 .4 8c 6. 06 ± 0 .4 0b 3. 4 ± 0. 22 c 5. 03 ± 0 .3 3c 7. 04 ± 0 .4 6c 3. 82 ± 0 .2 5b 9. 67 ± 0 .6 4b c 6. 47 ± 0 .4 3b 5. 99 ± 1 .8 2b u lt ra so ni ca ti on 2. 78 ± 0 .1 1e 2. 02 ± 0 .1 1f 4. 71 ± 0 .2 5c 2. 01 ± 0 .1 2e 3. 48 ± 0 .1 9e 4. 59 ± 0 .2 5f 2. 42 ± 0 .1 3c 5. 86 ± 0 .3 1e 3. 87 ± 0 .2 1d 3. 53 ± 1 .2 7d t ot al c ol if or m s co un t ( lo g 10 c f u g -1 fr es h w ei gh t) c on tr ol 3. 43 ± 0 .1 8b c 5. 11 ± 0 .3 2a 7. 35 ± 0 .4 6a 5. 35 ± 0 .3 3a 6. 87 ± 0 .4 3a 4. 87 ± 0 .3 0a 3. 42 ± 0 .2 1a 5. 79 ± 0 .3 6b 4. 58 ± 0 .2 9a 5. 21 ± 1 .3 0a n ao c l ( 1 g/ l ) 3. 42 ± 0 .2 8b c 2. 79 ± 0 .1 7b c 3. 99 ± 0 .2 5c 1. 89 ± 0 .1 2e 5. 04 ± 0 .3 1c 3. 77 ± 0 .2 4c 1. 97 ± 0 .1 2c d 4. 01 ± 0 .2 5d 3. 19 ± 0 .2 0c 3. 35 ± 0 .9 6b c w et h ea ti ng (5 5 ℃ ) 3. 18 ± 0 .2 6c d 2. 64 ± 0 .1 1c d 3. 97 ± 0 .1 6c 1. 21 ± 0 .0 5f 3. 27 ± 0 .1 3e 4. 08 ± 0 .1 7b c 2. 09 ± 0 .0 9c 4. 65 ± 0 .1 9c 3. 25 ± 0 .1 3c 3. 13 ± 0 .9 9c d ry h ea ti ng (7 0 ℃ ) 2. 22 ± 0 .1 9e 4. 84 ± 0 .2 2a 7. 07 ± 0 .3 2a 4. 12 ± 0 .1 9b 5. 74 ± 0 .2 6b 5. 28 ± 0 .2 4a 1. 65 ± 0 .0 8e f 9. 23 ± 0 .2 2a 4. 25 ± 0 .1 9a b 5. 03 ± 2 .4 2a v in eg ar (3 50 m l /l ) 2. 17 ± 0 .1 7e 4. 88 ± 0 .2 6a 3. 18 ± 0 .1 7d e 2. 31 ± 0 .1 2d 4. 02 ± 0 .2 1d 4. 09 ± 0 .2 2b c 1. 98 ± 0 .1 1c d 4. 14 ± 0 .2 2c d 3. 92 ± 0 .2 1b 3. 41 ± 0 .9 8b c e th an ol (3 50 m l /l ) 1. 63 ± 0 .1 2f 2. 35 ± 0 .1 2d e 3. 57 ± 0 .1 9c d 1. 47 ± 0 .0 8f 3. 56 ± 0 .1 9d e 3. 67 ± 0 .1 9c 1. 45 ± 0 .0 8f 4. 08 ± 0 .2 2d 3. 08 ± 0 .1 6c 2. 77 ± 1 .0 0c d n ac l ( 10 g /l ) 4. 02 ± 0 .2 7a 3. 18 ± 0 .2 1b 4. 55 ± 0 .3 1b 2. 98 ± 0 .2 0c 3. 07 ± 0 .2 0e 4. 35 ± 0 .2 9b 2. 97 ± 0 .2 0b 5. 55 ± 0 .3 7b 4. 06 ± 0 .2 7b 3. 86 ± 0 .8 4b u lt ra so ni ca ti on 2. 58 ± 0 .1 1d 1. 98 ± 0 .1 1e 2. 87 ± 0 .1 5e 1. 31 ± 0 .0 7f 3. 35 ± 0 .1 8e 2. 86 ± 0 .1 5d 1. 79 ± 0 .1 0d e 2. 79 ± 0 .1 5e 2. 13 ± 0 .1 1d 2. 41 ± 0 .6 4d to ta l m ou ld c ou nt (l og 10 c f u g -1 fr es h w ei gh t ) c on tr ol 4. 48 ± 0 .2 4a 5. 58 ± 0 .3 5b 8. 12 ± 0 .5 1b 5. 65 ± 0 .3 5a 6. 42 ± 0 .4 1b 7. 23 ± 0 .4 5a 3. 78 ± 0 .2 4a 6. 78 ± 0 .4 2b 5. 42 ± 0 .3 4a 5. 95 ± 1 .3 1a n ao c l ( 1 g/ l ) 2. 68 ± 0 .2 2e 3. 18 ± 0 .2 0d 4. 78 ± 0 .3 0e 2. 14 ± 0 .1 3d e 3. 51 ± 0 .2 2d e 3. 61 ± 0 .2 2d 1. 89 ± 0 .1 2d 4. 69 ± 0 .2 9d 3. 02 ± 0 .1 9e 3. 29 ± 0 .9 7d e w et h ea ti ng (5 5 ℃ ) 3. 12 ± 0 .2 6c 4. 11 ± 0 .1 7c 4. 95 ± 0 .2 0e 2. 75 ± 0 .1 1c 5. 71 ± 0 .2 3c 5. 36 ± 0 .2 2c 2. 79 ± 0 .1 1b 6. 62 ± 0 .2 7b 4. 17 ± 0 .1 7c 4. 37 ± 1 .2 8b c d ry h ea ti ng (7 0 ℃ ) 4. 12 ± 0 .3 6b 5. 31 ± 0 .2 4b 8. 98 ± 0 .4 1a 2. 33 ± 0 .1 1d e 9. 94 ± 0 .4 7a 7. 84 ± 0 .3 6a 2. 61 ± 0 .1 2b 9. 23 ± 0 .2 0a 5. 03 ± 0 .2 3a b 6. 25 ± 2 .9 0a v in eg ar (3 50 m l /l ) 2. 21 ± 0 .1 8e 6. 91 ± 0 .3 7a 5. 13 ± 0 .2 7d e 3. 44 ± 0 .1 8b 6. 99 ± 0 .3 7b 5. 07 ± 0 .2 7c 2. 27 ± 0 .1 2c 5. 69 ± 0 .3 0c 3. 64 ± 0 .1 9d 4. 60 ± 1 .7 1b c e th an ol (3 50 m l /l ) 3. 14 ± 0 .2 3c 4. 27 ± 0 .2 3c 5. 78 ± 0 .3 1d 1. 99 ± 0 .1 2e f 4. 22 ± 0 .2 2d 5. 45 ± 0 .2 9c 2. 29 ± 0 .1 3c 4. 75 ± 0 .2 5d 4. 27 ± 0 .2 3c 4. 03 ± 1 .2 5c d n ac l ( 10 g /l ) 3. 78 ± 0 .2 5b 5. 05 ± 0 .3 3b 6. 87 ± 0 .4 1c 2. 42 ± 0 .1 6c d 5. 37 ± 0 .3 5c 6. 45 ± 0 .4 3b 3. 62 ± 0 .2 4a 6. 87 ± 0 .4 5b 4. 81 ± 0 .3 2b 5. 03 ± 1 .4 7b u lt ra so ni ca ti on 2. 55 ± 0 .1 0d e 2. 14 ± 0 .1 1e 2. 86 ± 0 .1 5f 1. 64 ± 0 .0 9f 3. 01 ± 0 .1 6e 2. 93 ± 0 .1 6e 1. 88 ± 0 .1 0d 2. 94 ± 0 .1 6e 2. 96 ± 0 .1 6e 2. 55 ± 0 .5 0e r es ul ts a re m ea ns o f t hr ee d et er m in at io ns ± s d . m ea ns in th e sa m e co lu m n fo llo w ed b y th e sa m e le tte r a re n ot s ig ni fic an tly d iff er en t a t l sd te st p < 0. 05 . 2 . 126 k.y. chiu counts to < 3 log 10 cfu g-1 fresh weights on pea sprouts, and the sprouts produced from ethanol-treated radish seeds also were acceptable at < 3 log 10 cfu g-1 fresh weights level. however, the ultrasonication treated seeds produced greater sprouts yield than other sanitizations-treated seeds, which gave an average of 78 % sprouts yield increase (across of all the tested crop species) comparing to non-treated seeds. therefore, ultrasonication treatment can be used as a microbial control treatment for alfalfa, mung bean, pea and radish sprouts production. references awad, 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of plant products, department of crop sciences, faculty of agricultural sciences, georg-august university of goettingen, goettingen, germany impact of fusarium spp. infection of bread wheat (triticum aestivum l.) on composition and quality of flour in association with eu maximum level for deoxynivalenol marie kreuzberger, sasithorn limsuwan, kai eggert, petr karlovsky, elke pawelzik (received march 7, 2014) summary contamination of grain with mycotoxins such as deoxynivalenol (don) is the major threat after fusarium head blight (fhb) infection of wheat; however technological quality can also be impaired. the european union has established maximum levels (ml) of don for wheat grain and foodstuffs. the composition (starch, gluten proteins) and quality (protein content, sedimentation value, wet gluten, water absorption, mixing properties of dough, baking volume) of 72 flour type 550 samples from two years either fulfilling or exceeding ml of 0.75 mg kg-1 were investigated. don content of flours ranged widely from below limit of quantification to 11.84 mg kg-1. aside from a slight loss of loaf shape in flours highly exceeding ml, no negative effect on composition and quality of flour was observed in flours exceeding ml compared to those fulfilling ml. a significant decrease in total glutenin and lmw-gs content did not correlate with any quality trait. hence, if flours fulfill ml for don, reduced technological quality due to fhb is not significant. introduction wheat is one of the most important staple foods worldwide. together with rice and maize, it provides about 60 % of the world’s food energy intake (fao, 2014). in germany, winter wheat covers about 44 % of the total cereal production area (statistisches bundesamt, 2013), while each citizen consumes about 71 kg wheat flour (statistisches bundesamt, 2013) as well as 85 kg bread and bakery products per year (belv, 2009). due to its great importance in the human diet, especially as bread wheat, the production of wheat with a minimum content of contaminants is of major interest. fusarium head blight (fhb) of wheat is a worldwide-occurring disease with tremendous economic importance. during the 1990s, fhb of wheat and barley caused about $3 bn losses due to reduced yield, grain and seed quality in the united states (windels, 2000). fusarium graminearum is generally described as the predominant specie causing fhb (osborne and stein, 2007). in europe, deoxynivalenol (don), its acetylated derivates (3-adon, 15-adon), and zearalenon (zen) are the most frequently detected mycotoxins in wheat and wheat products (bottalico and perrone, 2002). the toxicological effects of these mycotoxins on mammals range from anorexia to impaired reproduction (pestka, 2010; zinedine et al., 2007). therefore the european union has established maximum levels (ml) of don and zen for cereals and cereal products, including foodstuffs produced from wheat (commission regulation (ec) no. 1881/2006). accordingly, unprocessed wheat, cereal flour, bread and wheat-based foods for infants and young children must not contain more than 1250, 750, 500, and 200 as well as 100, 75, 50, and 20 μg kg-1 for don and zen, respectively. aside from the toxicological risk resulting from mycotoxin contamination of wheat, wheat quality may be impaired since fhb infection may influence grain components, such as starch and proteins (siuda et al., 2010) and subsequently end-use quality traits, as pasting properties (wang et al., 2005a) and baking performance (lancova et al., 2008). wang et al. (2005b) found the compo sition of gluten proteins, which contribute considerable to the baking properties of wheat, altered as a result of fhb. a distinct reduction in the content of both total glutenin and high-molecular-weight glutenin subunits was detected in wheat after serious artificial infection. as a result, the dough quality was impaired and consequently, an unsatisfactory bread quality was observed. biochemical changes in grain composition and subsequent changes in wheat quality traits caused by fhb might result from both fungal enzymes and impaired synthesis of grain components. it is assumed that fusarium ssp. secrete enzymes, such as carbohydrases and proteases, during the invasion of the kernel thus degrading starch, cell wall components, and gluten proteins (eggert et al., 2011; dexter and nowicki, 2003; pekkarinen et al., 2000). fusarium infection might also lead to an incomplete accumulation of kernel constituents through the mechanical blocking of vascular bundles by fungal mycelium (goswami and kistler, 2004; kang and buchenauer, 2000; ribichich et al., 2000) or thorough impaired synthesis of grain components due to the presence of mycotoxins (eriksen and pettersson, 2004; casale and hart, 1988). this study investigates how wheat quality is affected if ml of don for flour of 0.75 mg kg-1 is fulfilled or exceeded. both flour composition and common quality parameters of wheat, including ultimate criterion of baking volume, are investigated. ml for don was used because zen content of flour samples was insignificant. the study uses flour type 550 which is the most processed bread flour in ger many. flour was milled from naturally fusarium infected grain. earlier studies, investigating the relationship of fusarium infection and wheat quality, mainly investigated only severely artificially infected samples (gartner et al., 2008; prange et al., 2005) or un processed grain (lancova et al., 2008; wang et al., 2005a, b). others compared several kernel classes ranging from lightly to severely fusarium damaged grain (siuda et al., 2010; boyacioglu and hettiarachchy, 1995; bechtel et al., 1985). the often cited study of nightingale et al., 1999 demonstrated impaired wheat quality in in fusarium damaged wheat that comprised 88.0-113.0 mg kg-1 don. severely fusarium damaged kernels and highly don contaminated wheat and wheat products are suitable to demonstrate a potential fusarium damage on grain but rather lack practical relevance since a grain lot is a mixture of all kernel classes, removal of severely and moderately infected kernels by cleaning procedures is possible to a certain degree (seitz et al., 1986), and maximum level of don is a legal criterion of exclusion. therefore earlier studies might have overestimated the impact of fhb on baking quality wheat within the legal range of don. material and methods plant material flour type 550 were gained from fusarium infected wheat grain harvested in a field experiment carried out from 2007 and 2009 at two locations (torland, gladebeck) near goettingen, in the south of lower saxony, germany. natural variation of don within the samples resulted from the experimental factors including pre-crop (wheat, maize, sugar beet), cultivar (highly/hardly susceptible against 178 m. kreuzberger, s. limsuwan, k. eggert, p. karlovsky, e. pawelzik fhb), and fungicide treatment (strobilurin, triazole, chlorathalonil applied during tillering). a complete description of the field experiment is given in kreuzberger (2011). two common german bread wheat cultivars were cultivated, alike in quality parameters (according to federal office of cultivar variety) but differing in susceptibility against fhb; ritmo shows high susceptibility against fhb (fhb+), centrum is hardly susceptible against fhb (fhb-). grain was harvested by combine harvester (farmliner, deutz-fahr, cologne, germany) at a grain moisture content of 14.5-16 %. post-harvest treatment thirty-six bulk samples of always six kg were created per year within each factor treatment of the trial. samples were cleaned once with the standard settings of a/s rationel kornservice, esbjerk, denmark. after one month of storage two kg of each bulk sample were tempered for 12 h at ambient temperature to gain a moisture content of 15.5 %. after that grain was milled into flour with an extraction rate of 75 % and an average mineral content of 0.55 % in dry matter, also known as flour type 550 (buhler laboratory mill type mlu 202, uzwil, switzerland; quadrumat senior 220/380, brabender, duisburg, germany). mineral content of flour was determined slightly modified according to icc standard no 104/1. five g of flour were dried until constant weight and incinerated in a muffle furnace for 4 h at 900 °c. flour samples were kept in air-tight bottles (pe) at 4-8 °c in the dark until analysis. quantification of deoxynivalenol don determination was performed by lc-ms/ms. five g of flour were extracted with 50 ml acetonitrile-water (84:16, v/v). the extraction solvent was replaced with methanol-isopropanol-water (80:5:15, v/v/v) during the so-called acetonitril crisis in 2008/2009. the extracts were cleared, defatted, dried, dissolved in methanolwater (1:1, v/v) and filtered as previously described (adejumo et al., 2007) and injected into a c18 column (kinetex, 50 x 4.6 mm i.d., 2.6 μm; phenomenex, ca, usa), equipped with a pre-column containing the same c18 material. a gradient of solvent a (5 mm acetic acid in water containing 5 % acetonitrile) and solvent b (5 mm acetic acid in methanol) was used as the mobile phase. don was detected by tandem mass spectrometry (varian 1200l ms/ms system (varian, inc. ca, usa) equipped with a triple quadrupole mass spectrometer, two prostar 210 liquid chromatographic pumps, a 410 autosampler, and a 500 ms ion trap mass spectrometer with esi interface). the triple quadrupole settings (adejumo et al., 2007) and mass transitions (klotzel et al., 2006) were previously described. calibration curves were prepared by spiking certified analytical standards into blanks of flour, which were then processed in the same way as the samples (matrix-matched standards). the limit of detection (lod: 0.015 mg kg-1) and quantification (loq: 0.10 mg kg-1) were determined based on the signal-to-noise ratio. don content was based on dry matter of flour. determination of flour composition starch starch content was determined according to icc standard no. 123/1 and measured by polarimetric method (polarimeter type v drna, zeiss ag, jena, germany). protein was precipitated with wolframatophosphoric acid. starch content was calculated with the rotation angle of the filtrate at 589 nm. each measurement was repeated twice. gluten proteins extraction of gliadins and glutenins from 100 mg of flour was conducted according to wieser (2000). before each extraction step, solvent and flour or sediment, respectively, were vortexed for two minutes. albumins/globulins and gliadins were extracted at room temperature with magnet stirring (variomag multipoint 15, h+p labortechnik ag, oberschleissheim, germany). extraction of glutenins was performed for 20 min at 60 °c (thermomixer comfort, eppendorf ag, hamburg, germany) at 750 rpm. suspensions were centrifuged (centrifuge 5804r, eppendorf, hamburg, germany) at 20 °c for 15 min at 7500 rpm. each sample was extracted twice. protein extracts were stored at -20 °c until measurement. for quantification of protein fractions, gliadin and glutenin standards were prepared from commercially available gluten from wheat (sigma-aldrich chemie gmbh, steinheim, germany) as described by eggert et al. (2011). protein content of isolated gliadin and glutenin was determined by c/n analyser (vario max cn elementar analysensysteme gmbh, hanau, germany). protein content of gliadin and glutenin standard were 93.41 % and 100.00 % of dry matter, respectively. gliadin and glutenin standards were solved and determined accordingly wieser (2000). quantification of gliadin and glutenin fractions followed with slight modification the description of wieser et al. (1998) and eggert et al. (2010). for the rp-hplc, a perfectsil 300 c8 300 x 4.6 column (machery-nagel, dueren, germany) was used. mobile phases were a = 0.1 % trifluoroacetic acid (tfa) (merck, darmstadt, germany) in h2o (v/v) (chromanorm, vwr, fontenay-sous-bois, france) and b = 0,1 % tfa in acetonitrile (v/v) (hipersolv, chromanorm, vwr; leuven, belgium). all solvents were of hplc grade. 100 μl of the aliquots of gliadin and glutenin were injected. the gradient conditions for separation and analysis of protein fractions were as followed: a, 100 76 % in 0 5 min; 76 50 % in 5 50 min; 50 10 % in 50 54 min; 10 100 %, 54 59 min. flow rate was 1 ml min-1; and the column temperature were set at 50 °c. gliadin sub fractions were separated into w5-, w1,2-, a-, g-gliadins, whereas glutenins (gs) were separated into wb-gs, hmw-gs (high molecular weight glutenins), and lmw-gs (low molecular weight glutenins). for the quantification of the protein fractions gliadin and glutenin standards were used. in order to eliminate the effect of starch accumulation and the adverse relationship with protein content in grain, quantity of gliadin, glutenin as well as subfractions were calculated as percentage of total protein in flour type 550 (% protein). total protein content was on average 11.7 %, 10.2 %, and 10.4 % 2007, 2008, and 2009, respectively. hplc system (mz analysentechnik, mainz, germany) consisted of autosampler (jasco as-2051 plus intelligent sampler), oven (jasco co-2060 plus intelligent column thermostat), degaser (jasco dg-2080-54 4-line-degaser), pump (jasco pu-2080 plus intelligent hplc pump), gradient mixer (jasco lg-2080-04 s), and detector (jasco md-2015 plus multiwavelength detector). the chromatograms were analyzed by jasco chrompass chromatography data systems version 1.8.6.1. determination of quality parameters protein content nitrogen content was determined according to dumas principle from 100 mg dried flour by c/n analyzer (vario max cn elementar analysensysteme gmbh, hanau, germany). protein content was calculated according to icc standard no. 105/2 (n x 5.7). sedimentation value and wet gluten content sedimentation value and wet gluten content were determined according to icc standard no. 151 and icc standard no. 106/2, respectively. water absorption and mixing properties of dough water absorption and mixing properties (dough development time, dough stability, degree of softening) were obtained according to flour composition and baking quality of wheat associated with eu maximum level for don 179 26 pre-crop sb sb ww ww m m d o n (m g kg -1 ) 0 2 4 6 8 10 12 14 fhbfhb+ 2007 pre-crop sb sb ww ww m m 0 2 4 6 8 10 12 14 fhbfhb+ d o n (m g kg -1 ) 2009 precrop *** cultivar *** precrop x cultivar *** precrop *** cultivar *** precrop x cultivar *** a b modified icc standard no. 115/1 using a valorigraph (type fqa-205, metefem, budapest, hungary) in 2007 and farinograph (brabender®, no. 901368, type 810 105 001, duisburg, germany) in 2009. farinograms were analyzed with brabender® farinograph version 4.0.3. each measurement was repeated three times. microbaking test dough was prepared with 50 g flour (corrected to 14 % moisture content), 1 % sucrose, 1 % fat, 1.5 % salt, 5 % fresh yeast, and 0.002 % l-ascorbic acid based on flour weight. water was added according to determined water absorption. dough was mixed for 2 min at 30 °c, proofed for 20 min at 30 °c in a water saturated atmosphere (ecocell 55, mmm medcenter ein richtungen gmbh, munich, germany), separated into five dough balls of equal weight and relaxed for further 3 min. in order to remove air bubbles, dough balls were formed with a noodle machine (model atlas 150, marcato, italy) and rolled into a log shape. dough pieces were further proofed for 45 min. baking was performed for 12 min at 240 °c (electric furnance, aeg haustechnik, nuernberg, germany) according to kieffer et al. (1993). baking volume was measured after loaves had cooled by rapeseed displacement. statistical analysis data were analyzed separately for 2007 and 2009 with sigmaplot version 13.0, systat software gmbh, erkrath, germany. to describe factors influencing variation of don content two-factorial analysis of variance (anova) was conducted with raw data considering precrop (p) and cultivar (c) as main factors. assumptions were normal distribution and homogenicity of variance checking residuals with q plot. to investigate the difference in quality between flours fulfilling and exceeding ml flour type 550 samples were divided into two groups independently of experimental factors: flours fulfilling ml of don for flour (≤ 0.75 mg kg-1), and flours exceeding this level (> 0.75 mg kg-1). statistical significance (p < 0.001) between the two groups was calculated with two-tailed t test if raw data was normally distributed and exhibited equal variance. if these assumptions were not given whitney-rank sum test was applied. results don content of flours ranged from 0.31 to 11.84 mg kg-1 and from < loq to 8.42 mg kg-1 in 2007 and 2009, respectively. aside from year, most of the total variation in don content of flour type 550 basically resulted from the experimental factors pre-crop and cultivar (fig. 1). the average don level was significantly higher in 2007 than in 2009. in both years don content was maximized after pre-crop maize in the strongly susceptible cultivar, followed by pre-crop wheat and sugar beet. flour from the hardly susceptible cultivar contained in both years on average about 76 % less don than the strongly susceptible cultivar. however, there was inter action between pre-crop and cultivar. the categorization of flour samples into the two groups of fulfilling or exceeding ml led to different group sizes within the two years. in 2007 only about 30 % of the samples fulfilled ml, in 2009 over 50 % of the samples fulfilled the limit (fig. 2). don content ranged from 0.31 to 0.75 mg kg-1and from 0.79 to 11.84 mg kg-1 in the two groups in 2007. in 2009 don content ranged from < loq (0.10) to 0.73 mg kg-1 and from 0.85 to 8.42 mg kg-1. starch content of flours exceeding ml was about 2.5 % increased compared to flours fulfilling ml in 2009 (fig. 3). this effect was not visible in 2007. other studies observed either no effect (wang et al., 2005a) or found starch content decreased after severe fusarium infection of grain (siuda et al., 2010; matthaus et al., 2004; pawelzik et al., 1998). protein content did not differ between flours fulfilling or exceeding ml in both years (fig. 3). some studies also confirm that protein content was not affected by fusarium infection (terzi et al., 2007; prange et al., 2005; dexter et al., 1996; wang et al., 2005b; seitz et al., 1986). in contrast, other studies observed an increase (e.g. siuda et al., 2010; matthaus et al., 2004; boyacioglu and hettiarachchy, 1995) or a decrease of protein content after severe fusarium infection of grain (e.g. gartner et al., 2008; prange et al., 2005; nightingale et al., 1999). total gluten content did not differ between flours fulfilling or exceeding ml in both years (fig. 3). however, gluten composition differed in both years (fig. 4). content of total gliadin (fig. 4 a) and gliadin subfractions (w5, w1,2, a) showed no significant difference between the two groups in both years (not shown). that gliadins are not significantly influenced by fhb of wheat is supported by the fig. 1: distribution of don in flours type 550 milled from wheat cultivated after pre-crop sugar beet (sb), winter wheat (ww), and maize (m) divided by cultivar hardly (fhb-) and highly (fhb+) susceptible against fhb in 2007 (a) and 2009 (b). results of two-way anova. box plots show 25-75% percentile, median (solid line), and mean (dotted line). 180 m. kreuzberger, s. limsuwan, k. eggert, p. karlovsky, e. pawelzik d o n (m g kg -1 ) 0 1 2 3 4 10 20 ** 2007 ≤0.75 (12) >0.75 (24) ≤0.75 (22) >0.75 (14) 2009 fig. 2: don contamination of flour type 550 in 2007 and 2009 grouped into fulfilling (≤ 0.75) and exceeding (> 0.75) eu maximum level of don (mg kg-1) for flour. in breaks − number of samples per group. significant difference between groups within year is indicated through * (p < 0.001). box plots show 25-75 % percentile, median (solid line), and mean (dotted line). fig. 3: starch (a), protein (b), and total gluten (c) content of flour type 550 samples in 2007 and 2009 grouped into fulfilling (≤ 0.75) and exceeding (> 0.75) eu maximum level of don (mg kg-1) for flour. significant difference between groups within year is indicated through * (p < 0.001). box plots show 25-75 % percentile, median (solid line), and mean (dotted line). results of e.g. dexter et al. (1996) and prange et al. (2005) where don levels where similar or even exceeded don of this study. on the contrary, wang et al. (2005b) and eggert et al. (2010) observed a slight increase in total gliadin content and gliadin subfractions in severely fusarium infected kernels of one wheat cultivar. this could only be observed for g-gliadin in 2007 in flours exceeding ml. in 2009 this effect was not visible (not shown). in contrast, in both years content of total glutenins was significantly reduced in flours exceeding ml compared to flours fulfilling ml (fig. 4 b). the quantitative difference was about 20.6 and 10.8 % in 2007 and 2009, respectively. this effect basically resulted from the lower lmw-gs content of these flours (fig. 4 c). in 2007 and 2009 lmw-gs was about 22.4 and 11.7 % lower in flours exceeding ml. consequently gliadin-to-glutenin ratio was significantly increased in the samples exceeding ml (not shown). content of hmw-gs was slightly decreased in flours exceeding ml in both years, however this difference was not significant. there was also no significant difference in wb-gs (not shown). other studies also detected a decrease of total glutenin however this was mainly due to decreased hmw-gs content (eggert et al., 2010; wang et al., 2005b). they also observed an increase of lmw-gs which was contrary to the results of this study. on the contrary prange et al. (2005) did not find an effect in flours that contained similar high don levels as flours investigated in this study. wieser and kieffer (2001) demonstrated that a changed gluten composition, more specifically a wider gliadin-to-glutenin or lmw-gs-to-hmw-gs, leads to impaired dough, gluten, and baking properties. since in this study the quantitative change in gluten composition did not correlate with any of the quality parameters it can be regarded as insignificant. sedimentation value and water absorption of flours did not show any difference between the two groups in any year (fig. 5 a, c). these results are confirmed by a few studies (e.g. siuda et al., 2010; gartner et al., 2008; dexter et al., 1996). on the other hand, several authors reported a decrease of sedimentation value (gartner et al., 2008; terzi et al., 2007; wang et al., 2005b; boyacioglu and hettiarachchy, 1995) and at least a slight increase of water absorption with increasing intensity of fusarium infection (wang et al., 2005b; pawelzik et al., 1998). wet gluten content was significantly increased about 14 % in flours exceeding ml compared to flours fulfilling ml in 2007 (fig. 5 b). this effect was former reported by siuda et al. (2010). in 2009 this effect was not visible which is supported by the findings of dexter et al. (1996). other studies found wet gluten content reduced (boyacioglu and hettiarachchy, 1995; meyer et al., 1986) in artificially fusarium infected samples. doug softening was slightly increased in flours exceeding ml in both years; however this effect was not significant (fig. 6 a). further to ta l g lu te n (% ) 0 60 70 80 90 100 p ro te in (% ) 0 10 12 14 16 s ta rc h (% ) 0 5 72 74 76 78 80 82 *2007 ≤0.75 >0.75 ≤0.75 >0.75 2009 ≤0.75 >0.75 ≤0.75 >0.75 ≤0.75 >0.75 ≤0.75 >0.75 2007 2009 2007 2009 a b c flour composition and baking quality of wheat associated with eu maximum level for don 181 dough mixing properties (dough development time, dough stability) did not differ between the two groups (not shown). these results are confirmed by the study of dexter et al. (1996). however, other studies reported a decrease of dough development time (wang et al., 2005b; nightingale et al., 1999; pawelzik et al., 1998), and dough stability (wang et al., 2005b; nightingale et al., 1999; pawelzik et al., 1998) with increasing intensity of fusarium infection. gartner et al. (2008) observed cultivar-depended an increase, a decrease as well as no impact of fusarium infection on dough stability; also an increase of dough softening after fusarium infection was observed. microbaking test revealed significant differences in baking volume between the years (fig. 6 b). baking volume ranged from 360 to 460 ml 100 g-1 flour and from 240 to 290 ml 100 g-1 flour in 2007 and 2009, respectively. in 2007 baking volume was significantly increased in flours exceeding ml. the difference made up about 44 ml 100 g-1 flour between the two groups. in 2009 no difference was detectable. an increase of baking volume after fusarium infection of wheat is a typical observation (lancova et al., 2008; gartner et al., 2008; prange et al., 2005; wang et al., 2005b; dexter et al., 1996). it was observed that loaves prepared from flours highly exceeding ml in 2007 partly lost their shape during leavening. after baking, these loaves appeared wider and flatter (fig. 7). in these samples dough was already sticky and wet during handling for rapid mix test and therefore could not be brought into the common shape. discussion agronomic practices influence don contamination of flour type 550 certainly warm and humid weather conditions during wheat anthesis enhanced the epidemic spread of fhb and subsequent don formation in grain (de wolf et al., 2003) and led to extraordinarily high don content of flour in both years. over 50 % of the total samples of two years exceeded ml. in 2007, 50 % of the flours exceeding ml trespassed ml of 0.75 mg kg-1 about factor 3.7, in 2009 about factor 2.9 (fig. 2). the wide range of don content of flours resulted from to the experimental design of the field trial where grain was harvested. the trial combined agronomic practices such as pre-crop, cultivar choice and fungicide treatment that were all known to influence (reduce or enhance) fhb in wheat. the effect of such agronomic measures on don content of grain was already investigated thoroughly by e.g. beyer et al. (2006) and schaafsma et al. (2001). the present study demonstrates that the effect of pre-crop and cultivar is still observable in processed grain (fig. 1) even though one could expect that a cleaning step and milling of flour smoothes them (lancova et al., 2008). as in other studies the combination of prefig. 4: total gliadin (a), total glutenin (b), lmw-gs (c), and hmw-gs (d) content of flour type 550 samples in 2007 and 2009 grouped into fulfilling (≤ 0.75) and exceeding (> 0.75) eu maximum level of don (mg kg-1) for flour. significant difference between groups within year is indicated through * (p < 0.001). box plots show 25-75 % percentile, median (solid line), and mean (dotted line). lm w -g s (% p ro te in ) 0 5 10 15 20 25 30 * * h m w -g s (% p ro te in ) 0 2 4 6 8 10 to ta l g lia di n (% p ro te in ) 0 40 50 60 70 to ta l g lu te ni n (% p ro te in ) 0 10 20 30 40 * * 2007 ≤0.75 >0.75 ≤0.75 >0.75 2009 2007 ≤0.75 >0.75 ≤0.75 >0.75 2009 2007 ≤0.75 >0.75 ≤0.75 >0.75 2009 2007 ≤0.75 >0.75 ≤0.75 >0.75 2009 a b c d 182 m. kreuzberger, s. limsuwan, k. eggert, p. karlovsky, e. pawelzik fig. 5: sedimentation value (a), wet gluten content (b), and water absorption (c) of flour type 550 samples in 2007 and 2009 grouped into fulfilling (≤ 0.75) and exceeding (> 0.75) eu maximum level of don (mg kg-1) in flour. significant difference between groups within year is indicated through * (p < 0.001). box plots show 2575 % percentile, median (solid line), and mean (dotted line). crop maize with the highly susceptible cultivar led to the highest don contamination of flour. again the high risk coming from precrop maize enhancing toxin accumulation in wheat grain is emphasized as reported before (e.g. dill-macky and jones, 2000; beck and lepschy, 2000). high don contamination of flour is not necessarily associated with reduced technological quality aside from mycotoxin contamination of grain and processed cereal, many studies reported on the negative impact of fhb infection of wheat on grain composition, indirect quality traits and baking performance (gartner et al., 2008; wang et al., 2005 a, b; nightingale et al., 1999). however studies assessed fusarium damage of wheat and the investigated material differently. this may limit the comparability of the results of this study with former studies and could also explain contradictory results regarding the effect of fhb on wheat composition and quality. the majority of studies investigated hand-picked kernel fractions with differing degree of fhb damage (siuda et al., 2010; nightingale et al., 1999; dexter et al., 1996; bechtel et al., 1985). don levels of the investigated samples there often highly exceeded those analyzed in this study (nightingale et al., 1999). samples with very high don contamination might be suitable to reveal what impact fhb has on the wheat kernel, yet they are not so much of practi cal relevance in bread processing due to legally set percentage of fusarium damaged kernels in grain and ml for don (dexter and nowicki, 2003; commission regulation (ec) no 824/2000, commission regulation (ec) no. 1881/2006). therefore this study in w at er a b so rp ti o n ( % ) 0 50 55 60 65 70 w et g lu te n ( % ) 0 20 30 40 50 60 * s ed im en ta ti o n v al u e (m l) 0 75 80 85 90 95 100 2007 2009 ≤0.75 >0.75 ≤0.75 >0.75 2007 2009 ≤0.75 >0.75 ≤0.75 >0.75 2007 2009 ≤0.75 >0.75 ≤0.75 >0.75 a b c b ak in g vo lu m e (m l 1 00 g fl ou r -1 ) 0 200 300 400 500 600 * d ou gh s of te ni ng (f u ) 0 50 100 150 200 250 2007 ≤0.75 >0.75 ≤0.75 >0.75 2009 a b 2007 ≤0.75 >0.75 ≤0.75 >0.75 2009 fig. 6: dough softening (a) and baking volume (b) of flour type 550 samples in 2007 and 2009 grouped into fulfilling (≤ 0.75) and exceeding (> 0.75) eu maximum level of don (mg kg-1) for flour. significant difference between groups within year is indicated through * (p < 0.001). box plots show 25-75 % percentile, median (solid line), and mean (dotted line). flour composition and baking quality of wheat associated with eu maximum level for don 183 vestigated flour from cleaned grain. the still wide range of don levels in flour made a categorization into flours either fulfilling or exceeding ml possible. even though grain was cleaned in a onestep procedure, don contamination of most flours clearly exceeded ml and they were therefore unsuitable for further processing and consumption. nevertheless it enabled an investigation whether there was an association between composition and quality of flours where don levels were in and out the legal range. baking volume is the complex result of the interaction of various flour components, mainly starch and protein, with water and is the ultimate criterion of wheat to be categorized into wheat quality classes (according to federal office of plant variety). starch content, starch properties, protein content and protein composition, more specifically composition and content of gluten protein, are all influenced by the complex interaction of environment and genotype (williams et al., 2008; wieser and kieffer, 2001). even though there were slight differences in starch content and glutenin composition in flours highly exceeding ml (fig. 3, fig. 4), baking volume of these flours was not negatively affected. in 2007, baking volume was even increased (fig. 6). moreover, indirect quality parameters such as sedimentation volume and protein content (fig. 5, fig. 3), which are commonly determined as predictors for baking performance, did not differ between flours fulfilling or exceeding ml indicating that there was no loss of quality to be expected from highly don contaminated flours. the effect of fusarium infection on baking volume depends on various factors, such as growing season, infection severity, protein content and protein quality (meyer et al., 1986). dexter et al. (1996) explained the increase in baking volume after fusarium infection with an improved viscoelastic balance of gluten. however, overall flours in this study produced very low baking volume not meeting baking wheat quality as one might have expected. obviously, experimental conditions (e.g. n fertilization) did not meet the demands of the two wheat cultivars to develop their potential as bread wheat. very high don contamination of flour is associated with impaired dough handling and loss of loaf shape due to sticky and gluey properties dough handling was difficult in flours highly exceeding ml in 2007, resulting in a loss of loaf shape (fig. 7). however, this was only visible in flours of the highly susceptible cultivar after pre-crop maize where don content highly exceeded ml ranging from 3.18 to 11.84 mg kg-1. loss of shape can also be seen as a loss of quality. however, this is not a commonly measured quality trait. since dough mixing properties such as dough development time and dough softening did not differ between the two groups (fig. 6), it became obvious that the dough mixing procedure according to icc standard must not correspond with dough consistency during further handling. wang et al. (2005b) also reported an unfavorable dough consistency and handling problems of severely fusarium infected grain. the plastic deformation of dough in this study, which was already visible after the first leavening, was probably due to enzymatic activity during dough processing (nightingale et al., 1999; pawelzik et al., 1998). proteolytic acti vity may result from endoproteases secreted into the kernel by fusarium spp. (pekkarinen and jones, 2002; pekkarinen et al., 2000). conclusion summarizing, except from the loss of shape in 2007, there was no significant loss of technological quality measurable in flours within the range of don content of this study (up to 11.84 mg kg-1). don content of flours did not correlate negatively with a significant loss of quality measurable with the common indirect quality parameters or baking test. loss of loaf shape was only visible in flours highly exceeding ml and therefore anyway unsuitable for further processing and consumption. since don content of 0.75 mg kg-1 is the criterion of exclusion for further processing of flour, it can be concluded that technological quality of flours fulfilling ml is not significantly impaired by fhb of grain and that don content must exceed ml manyfold before changed flour composition and reduced quality due to fhb infection of grain becomes measurable in flour type 550 with the common methods. acknowledgements this study was conducted within the frame of the forschungsverbund agrarund ernährungswissenschaften niedersachsen (faen) joint project 3 “quality-related plant production under modified basic conditions: mycotoxins in the context of production, quality and processing”, funded by the ministry of science and culture of lower saxony, germany. the authors acknowledge the work of dr. r. goedecke, department of crop sciences, general plant patho logy and crop protection, university of goettingen, germany as well as the colleagues of the institute for sugar beet research, goettingen, germany for design and maintenance of the field trial. the authors also thank ulrike hill for technical assistance. references adejumo, t.o., hettwer, u., karlovsky, p., 2007: survey of maize from south-western nigeria for zearalenone, alphaand beta-zearalenols, fumonisin b-1 and enniatins produced by fusarium species. food addit. contam. 24, 993-1000. bechtel, d.b., kaleikau, l.a., gaines, r.l., seitz, l.m., 1985: the effects of fusarium graminearum infection on wheat kernels. cereal chem. 62, 191-197. beck, r., lepschy, j., 2000: ergebnisse aus dem fusarium-monitoring 1989-1999-einfluss der produktionstechnischen faktoren fruchtfolge und bodenbearbeitung. in: schriftenreihe der bayerischen landesanstalt für bodenkultur und pflanzenbau. 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2007: review on the toxicity, occurrence, metabolism, detoxification, regulations and intake of zearalenone: an oestrogenic mycotoxin. food chem. toxicol. 45, 1-18. address of the authors: dr. marie kreuzberger, julius kühn-institut, federal research centre for cultivated plants, institute for biosafety in plant biotechnology, 06484 quedlinburg, germany. e-mail: marie.kreuzberger@jki.bund.de dr. sasithorn limsuwan, betagro science centre co., ltd., klong nueng, klong luang, pathumthani 10120, thailand. e-mail: jjoy1117@yahoo.com © the author(s) 2015. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). dr. kai eggert, leibniz-institut für pflanzengenetik und kulturpflanzen forschung (ipk), molecular plant nutrition, 06466 stadt seeland, germany. e-mail: eggert@ipk-gatersleben.de prof. dr. petr karlovsky, molecular phytopathology and mycotoxin research, department of crop sciences, faculty of agricultural sciences, georg-august university of goettingen, 37075 goettingen, germany. e-mail: pkarlov@gwdg.de prof. dr. elke pawelzik, quality of plant products, department of crop sciences, faculty of agricultural sciences, georg-august university of goettingen, 37075 goettingen, germany. e-mail: epawelz@gwdg.de journal of applied botany and food quality 88, 87 96 (2015), doi:10.5073/jabfq.2015.088.012 1 laboratory of pomology, department of crop science, agricultural university of athens, athens, greece 2 pomology institute, hellenic agricultural organization ‘demeter’, naousea, greece influence of rain cover on respiration, quality attributes and storage of cherries (prunus avium l.) mina kafkaletou1, miltiadis v. christopoulos1*, maria-eleni ktistaki1, thomas sotiropoulos2, eleni tsantili1 (received december 2, 2014) * corresponding author summary the effects of rain cover on tree yield, fruit cracking and quality of two cherry (prunus avium l.) cultivars, ‘adriana’ and ‘noire de meched’ (‘nm’), were investigated. cherry quality was determined at harvest and after storage at 1 oc in air for up to 21 d. rates of co2 production and o2 uptake were determined at weekly intervals during and after storage. a taste panel rated quality attributes of ‘nm’ after 14 d storage. the results showed that ‘adriana’ was completely, but ‘nm’ moderately resistant to cracking, while covering prevented the symptom. respiration rates showed immediate and continuous cooling requirements of all fruit. in ‘adriana’, covering reduced weight loss during the first 7-d storage, and promoted the peel colour (pc) and total soluble solids subsequently. under different environmental conditions, pc and total phenolics were advanced in un covered ‘nm’ at harvest and after storage. in both cultivars, covering did not affect negatively the yield, nor did it significantly the fruit weight, firmness, titratable acidity, ph value, total antioxidant activity, stem browning and resistance to stem removal. ‘adriana’ and ‘nm’ retained good quality for 21 and 14 d storage, respectively. introduction sweet cherries are very popular fruit and the first fresh temperate ones of the year with a high economical impact in cherry-producing countries. additionally, there has been an increasing consumption of fresh cherries due to consumer awareness of their benefits on health (mccune et al., 2011). these effects are mainly attributed to their content of phenolic antioxidants (kris-etherton et al., 2002), serotonin and melatotin (gonzales-gomez et al., 2009). however, fresh cherries are exposed to the market for a relatively short period of time, while they cannot be stored for longer than few weeks. besides, they are susceptible during transport. additionally, the cherry cultivation has a major problem, the fruit cracking, due to the rapid increase in water absorption either through the fruit skin or the root system, both occurring after rainfall (balbontin et al., 2013). cracking susceptibility increases with advanced maturation. it also depends on many cultivar characteristics, rootstock, fruit set, amount of rainfall, and other factors (measham et al., 2009; measham et al., 2014; simon, 2006). under unfavourable conditions cracking could result in a great loss of production, reaching up to 90 % (christensen, 1996), while cracked cherries are used only for processing provided that they are not affected by fungus (simon, 2006). reduction of the problem includes the establishment of plastic sheets that cover the tree canopy (rain cover) for at least three weeks before harvest (meland et al., 2014), preharvest sprays with hydrophobic and other compounds or other practices (balbontin et al., 2013; meland et al., 2014). none of the treatments can eliminate or even guarantee the result. however, the most effective treatment seemed to be the expensive technique of tree covering. rain cover has been studied mainly in northern europe, but changes in microclimate under cover exhibited inconsistent effects on delayed or advanced ripening (borve et al., 2003). the aim of this work was to use a kind of typical rain covers in order to investigate their interactive effect (temperature, humidity, solar radiation) on cracking, as well as quality attributes in undamaged cherries in two less studied cultivars. the selected cultivars were ‘adriana’ and ‘noire de meched’, being one mid-early and one mid ripening, respectively. there is not much information in the literature concerning quality characteristics of these cultivars in comparison to many others. resistance to cracking is among the main targets for cherry cultivation. ‘adriana’ originates from italy and is described as resistant to cracking, while ‘noire de meched’ originates from iran, belongs to ferrovia group (sansavini and lugli, 2008) and also showed a high cracking resistance (greco et al., 2008). on the contrary, according to vercammen and vanrykel (2014) ‘noire de meched’ showed moderate susceptibility to this symptom. thus, there is a global tendency to evaluate the less studied cherry cultivars in situ (balbontin et al., 2013). the present evaluation was carried out on fruit from trees grown in northern greece, the major cherry cultivation area in greece, where rainfall still remains a serious problem. the tree yield was also determined since the trees were relatively young with increasing yield that is usually inversely related to fruit size. fruit size in turn could affect the cracking percentage. in contrast to the principle that storage is not applied to early cultivars the present study kept the fruit under low temperature in order to cover the time required for the fruit to reach the consumer at distant markets and to get the opportunity to examine the quality changes in detail. emphasis was given on respiration rates since they reflect on the rate of quality deterioration and therefore of cooling requirements and storability. materials and methods source and handling of fruit experiments were conducted in macedonia, greece on a property located approximately 52 m above the sea (lat. 40o 41' n; long. 22o 9' e). rain cover was used on cherry trees (prunus avium l.) of the cultivars ‘adriana’ and ‘noire de meched’. all trees were grafted on gisela 5 rootstock, planted at distances of 3.5 m x 1.5 m apart in 2006 and trained as palmette. trees were permanently covered by a white high density polyethylene film of light transparency equal to or greater than 85 % (helios, italy) for approximately three weeks before harvest. uncovered trees served as controls. the rain covering system resembled to the three-wire one (meland et al., 2014). the construction had a frame of wooden poles spaced at 10 meters apart and three overhead wires running the length of the row. the side wires were positioned up to 0.5 m lower than the top wire. the top of the tree was kept at a distance of 0.5 m from the top wire in the begging of the season. the overhead covers could slide back and forth to open and close on three wires down the row and were removed after harvest. the yield and fruit cracking percentage for each treatment (covered or uncovered trees) were estimated at harvest from 2 replicates of 7 trees each in the case of ‘adriana’ and from 3 replicates of 5 trees each in the case of ‘noire de meched’. the yield and cracking were measured in both cultivars in two experimental years, 2009 and 2011. fruit of ‘adriana’ in 2009 and ‘noire 88 m. kafkaletou, m.v. christopoulos, m.-e. ktistaki, t. sotiropoulos, e. tsantili de meched’ in 2011, without cracking at harvest, selected at random from 3 replicates of 5 trees each from both treated and controls and were transferred to the laboratory. upon arrival, fruit from each treatment were mixed and then divided into three lots. for each treatment, cherries attached to stems and free from visual defects were sorted out at random and placed in pots in groups of ten each. then, the pots were placed at 1 oc with 95 % rh, according to a completely randomised design. respiration was measured either at 1 oc or at 20 oc, as denoted, while all the rest determinations at 20 oc after temperature equilibration. on each sampling day, for each treatment and at each temperature, further determinations were carried out on fruit of three pots (three replicates), apart from 10-fruit weight that evaluated on twelve pots. first samples were collected at random prior to storage at 1 oc and corresponded to samples after harvest (day 0 of storage). determinations on fresh fruit weight loss before storage the initial weights of all fruit samples were recorded, while on each sampling date the weights of three samples per treatment were recorded after temperature equilibration at 20 oc. respiration rates on each sampling day, respiration gases were measured on the same replicates. measurements were carried out according to tsantili et al. (2003) after some modifications. in particular, headspace co2 and o2 were analysed by the same injection of 0.5 ml in a gas chromatograph (hewlett packard 5390 series ii) with a thermal conductivity detector and he as the carrier gas at a flow rate of 0.33 ml s-1. the instrument was equipped with two successive columns, one of porapaq q (50-80 m) for co2 and one of molecular sieve (40-60 m) for o2, both of 300 cm x 0.2 cm i.d. a commercial standard consisted of 2 % co2 and 2 % o2 in n2 was used for instrument calibration and a laboratory computing integrator (hewlett packard 3395) for results calculation. gas samples were taken from 0.5 l sealed jars after 1 h incubation. results are expressed in nmol kg-1 s-1 at the corresponding temperature, while respiration quotient (rq) and q10 (for o2, between 0 oc and 20 oc) were calculated. peel colour peel colour was measured on the two opposite sides of each fruit with a minolta chromatometer (cr-300; minolta, ahrensburg, germany) according to tsantili et al. (2007) and expressed in l*, ho and c*. stem browning stem browning (sb) was measured visually. stems were divided into four classes, depending on their length with brownish colour: no brown corresponded to 4/4 green; slightly brown, ≤ 1/4 brownish; low brown, 1/4 1/2 brownish; brown, > ½ brownish. resistance to stem removal resistance to stem removal (rsr) was measured by a chatillon traction-penetrometer (j. chatillon and sons inc.) with a scale of 0-1.0 kg (± 0.01) and according to tsantili et al. (2007). results are presented in newtons (n) after division into four classes: 0-3 n, 3-6 n, 6-9 n and > 9 n. fruit firmness fruit firmness was measured according to tsantili et al. (2007). it was recorded as the peak force to penetrate one side of unpeeled fruit using a conical probe of 5 mm diameter x 5 mm height, mounted on a bench chatillon df1s 50 penetrometer (j. chatillon and sons inc., new york, usa). the speed of the probe was at 0.42 mm s-1. total soluble solids total soluble solids (tss) of flesh was estimated in each fruit separately by an atago 8469 (atago co. ltd., tokyo, japan) hand refractometer. ph ph was measured by a ph-meter (jenway 3310; jenway ltd., dunmow, uk). titratable acidity titratable acidity (ta) was measured by titration of 10 g sap from unpeeled fruit to ph 8.2 with 0.1 m naoh. extraction of phytochemicals the extraction procedure and determinations of total phenolics (tp) and total antioxidant activity (taa) were carried out according to tsantili et al. (2010) after some modifications. slices of 10 fruit were placed immediately at 80 oc after sampling. frozen tissue of approximately 10 g was homogenised with cold 80 % acetone (v/v) in deionised water (4 l kg-1 tissue) using an ultra-turrax (t 25, ika labortechnik, germany). the homogenate was placed in an ultra sonic ice bath for 15 min and then filtered in the dark through # 1 whatman paper. the residue was filtered twice with 15 ml of acetone (80 %), while the whole procedure was conducted at 4 oc. then the filtrate was recovered at 38 oc under n2, brought to 30 ml with water and kept at 80 oc until analysis. determination of total phenolics the tp concentration was determined by the folin-ciocalteu method (singleton et al., 1999). in particular, 0.2 ml of diluted extract was added into a tube containing 0.2 ml of folin-ciocalteu reagent and 2.6 ml of deionised water. after stirring, the tube was allowed to stand at 20 oc for 6 min. then, 2 ml of na2co3 (7 %, w/v) were added, the tube was incubated at 20 oc for 90 min. absorbance was measured at 750 nm with a spectrophotometer (heλios gamma 7 delta, spectronic unicam, uk) versus a blank. the results were expressed as gallic acid equivalents (gae) on a fresh weight basis and according to the corresponding calibration curves. determination of total antioxidant activity the taa was determined according to dpph (brand-williams et al., 1995). diluted extract (with deionised water) of 0.1 ml was added into a tube containing 3.9 ml dpph solution (2,2-diphenyl-1picryhydrazyl, 60 μm in meoh). absorbance decrease was measured at 515 nm after incubation in the dark at 20 oc for 30 min versus a blank. the results were expressed as mmol of trolox (6-hydroxy2,5,7,8-tetramethlchroman-2-carboxylic acid) equivalents (te) on a fresh weight basis and according to the corresponding calibration curves. sensory evaluation the panel consisted of 14 people (7 men and 7 women, aged 20-58), all untrained, but familiar with cherries consumption. to avoid any bias, a minimum of information was given. each panelist individually evaluated two groups of 10 cherries each, uncovered and raincovered, in succession. the groups were blind labelled with 4 digit codes randomly. during the evaluation each panelist had a glass of room temperature water to rinse his palate. for each evaluated characteristic the rating was based on a three-point scale (1: low inten rain cover and cherry quality 89 sity; 2: medium intensity; 3: high intensity). the evaluated attributes were: size, peel colour, flesh colour, stem browning, texture (firmness), resistance to stem removal (rsr), easiness of stone removal, sweetness, sourness, overall taste and overall acceptance. statistical analysis the covering effect at harvest, the year effect and their interaction on yield and fruit cracking percentage were estimated by a two-way analysis of variance. where denoted, some means are followed by their standard error. the effects of covering, storage days at 1 oc and their interaction on weight loss (wl), colour parameters, firmness, tss, ta, tss/rta, ph, tp and taa, as well as, on respiration (co2 production rates, o2 uptake and rq) at each temperature were analyzed by a two-way factorial analysis of variance. the effect of temperature, covering, storage days at 1 oc and their interactions on respiration data were also analyzed by a three-way analysis of variance after excluding data at harvest. when needed, data were properly transformed to meet the assumptions of normality and homoscedacity. presented data are back transformed. comparison of means between the above variables was based on lsd (a = 0.05). data for sb and rsr were evaluated by c2 tests. the effect of covering on weight of fruit, seeds and stems, and on each characteristic evaluated by the taste panel was statistically estimated by twosample comparisons (independent samples). the statistical analyses were made using jmp 7.0.1. (sas institute, cary, nc, usa). results and discussion tree yield and fruit cracking in the particular orchard, the commercially estimated harvest season for ‘adriana’ ranged between 1 june and 13 june, while for ‘noire de meched’ between 9 june and 19 june. in the present study, yield and cracking in ‘adriana’ were estimated on 1 june, 2009 and 10 june, 2011 and in ‘noire de meched’ on 10 june, 2009 and 15 june, 2011 (tab. 1). the yield averaged approximately 20.1 kg per tree in ‘adriana’ and approximately 17.6 kg in ‘noire de meched’, in 2009 (tab. 1). covering did not affect the yield in ‘noire de meched’, whereas increased the yield in ‘adriana’, but only in 2009 and at the limits of significance. sotiropoulos et al. (2014) found that covering protected all four studied cultivars from cracking in a threeyear study without decreasing the productivity of trees trained to the open vase system. in 2011, here, the presented averaged yield in both covered and uncovered trees increased by approximately 1.7fold and 2.3-fold in ‘adriana’ and ‘noire de meched’, respectively. however, the yield increase was expected during growing of the relatively young trees. cracking was not practically observed in ‘adriana’. in contrast, ‘noire de meched’ showed similar cracking levels in both studied years in the uncovered trees, reaching approximately 20 %, in average (tab. 1). covering exhibited a great beneficial effect on ‘noire de meched’, eliminating cracking in 2009 and reduced it largely to 3 % in 2011. precipitation, during the last 22 days before harvest of ‘adriana’ and ‘noire de meched’, reached 68 mm and 62 mm, respectively, in 2009, while 63 mm and 31 mm, respectively, in 2011. the observed cracking in this study was of stem-end type, as described by simon (2006). the present results agreed with other studies in the case of ‘adriana’ (greco et al., 2008; sansavini and lugli, 2008). however, in ‘noire de meched’, the present results agreed with those suggested by vercammen and vanrykel (2014), but not with those by greco et al. (2008) who found an almost immune behaviour of this cultivar to cracking. the disagreement between studies could be ascribed, at least partially, to the variation of the symptom among years in relation to weather conditions and/or cultivation practices. according to lane et al. (2000) the threshold of absorbed water at which cracking starts is connected to cracking susceptibility. however, there is no apparent relation of cracking susceptibility to stomata number (christensen, 1972) and pore areas per stomata (as one of the factors that could influence water absorption) since stomata were not functional during ripening (peschel et al., 2003). interestingly, the last research group found that ‘adriana’ had the smallest number of stomata per fruit and pore area in cheek region among eight studied cultivars. research approaches, such as gene tab. 1: effects of rain cover on tree yield and fruit cracking (%) in ‘adriana’ and ‘noire de meched’, in 2009 and 2011. cultivar ‘adriana’ ‘noire de meched’ orchard (uncovered or or) rain covered orchard (rc) orchard (uncovered or or) rain covered orchard (rc) attribute 1/6/2009 10/6/2011 1/6/2009 10/6/2011 10/6/2009 15/6/2011 10/6/2009 15/6/2011 yield (kg per tree) 19.59 31.59 20.76 32.10 18.1 41.1 17.2 40.23 n 2 (x 7) 3 (x 5) lsd(0.05)a 0.89 4.56 ptrb * ns py *** *** ptr x y ns ns cracking (%) 1.9 2.3 0.9 2.8 17.7 23.3 0.11 3.4 n 2 (x 7) 3 (x 5) lsd(0.05) 2.1 5.4 ptr ns *** py ns ns ptr x y ns ns a lsd(0.05), least significant difference at a = 0.05. b ptr, covering treatment; py, year; x, interaction. ns, not significant. *, significant at p < 0.05. ***, significant at p < 0.001. 90 m. kafkaletou, m.v. christopoulos, m.-e. ktistaki, t. sotiropoulos, e. tsantili expression of expansins and of the enzyme β-galactosidase that are related to cell wall extension in cultivars with different cracking susceptibility seemed to be promising to investigate the mechanisms of cherry cracking (balbontin et al., 2013). respiration in ‘adriana’, covering exhibited no effect on respiration gases (fig. 1) after harvest, as also confirmed by two-sample comparisons. in particular, the rates of co2 production were approximately 278 nmol kg-1 s-1 and 361 nmol kg-1 s-1 in controls and covered fruit, respectively, while those of o2 uptake were 11.1 nmol kg-1 s-1 and 361 nmol kg-1 s-1, respectively (fig. 1a and c). during keeping fruit at 1 oc, both respiration rates decreased dramatically, being by 6-fold lower on day 7 than at harvest, in average, and remained at similarly low levels up to day 14. under the lower temperature, there were significant, but small increases in both gases in uncovered fruit on day 21 (tab. 2). after storage, ‘adriana’ showed similar trend of changes between covered and uncovered fruit for respiration rates of both gases at 20 oc. the reduction of co2 production at 20 oc in covered fruit on day 7 and the increase in co2 in controls on day 21 were the only significant changes in comparison to fruit at harvest. in ‘noire de meched’, the rates of co2 production and o2 uptake in controls and treated fruit at harvest were between 250 nmol kg-1 s-1 and 278 nmol kg-1 s-1, in average (fig. 1b and d), while covering had no effect on them. at 1 oc, the rates of both gases averaged by 5.1-fold lower on day 7 than at 20 oc after harvest and remained almost stable up to the end of storage. after storage, however, the increased co2 production rates at 20 oc were advanced by storage days in both controls and treated fruit, whereas o2 uptake decreased in all fruit at 20 oc on day 14. the significant effects of storage days and temperature, but not of covering on respiration gases in both cultivars are shown in tab. 2. the present results are in general agreement with those of gas exchange rates measured at 0 oc and 20 oc (wang and long, 2014) or of co2 production rates at 20 oc after storage at 0-1 oc (diaz-mula et al., 2012; tsantili et al., 2007). in the last study, inexplicable increases were observed at 20 oc towards the end of storage, being similar to these results. at harvest, rq averaged 0.93 and 0.98 in ‘adriana’ and ‘noire de meched’, respectively (tab. 3). covering did not show any significant effect on rq at harvest on both cultivars (by two-sample comparisons) and on ‘adriana’ cherries during or after storage. on the contrary, treatment resulted in increased rq values in the covered ‘noire de meched’ fruit during and after storage in comparison to controls (tab. 3). significant increases were also observed progressively during and after storage, resulting in values of 1.06 and 1.37 in ‘adriana’ and ‘noire de meched’, respectively, averaged for both temperatures at the end of the experiment. the higher temperature used reduced the rq values in ‘adriana’, but did not affect those ‘a dr ia na ’ ‘n oi re d e m ec he d’ ture (oc) fig. 1: effects of rain cover on respiration rates in ‘adriana’ in 2009 and ‘noire de meched’ cherries in 2011 before, during and after storage. (a) and (b), co2 production rates; (c) and (d), o2 uptake rates; (a) and (c), ‘adriana’; (b) and (d), ‘noire de meched’. or, orchard uncovered; rc, rain covered orchard. vertical bars correspond to lsd at a = 0.05. (a) bars, 1 oc, including data at harvest; (b) bars, 20 oc, including data at harvest; (c) bars, all data apart from those at harvest. tab. 2: probabilities of the effects of rain cover treatment (ptr), storage time (pst), temperature (pte) and their interactions on co2 production and o2 uptake rates and on rq in ‘adriana’ and ‘noire de meched’ cherries at harvest at 20 oc, during storage at 1 oc and after storage at 20 oc. cultivar tempera probabilitiesa attribute co2 o2 rq ptr ns ns ns 1 pst *** *** *** ptrxst ** ** ns ptr ns ns ns 20 pst *** ** *** ptrxst ns ns ns ptr ns ns ns pst *** *** *** pte *** *** ** 1 & 20 ptrxst ns ns ns ptrxte ns ns ns pstxte *** ns * ptrxstxte ns ns ns ptr ns ns ns 1 pst *** *** *** ptrxst ns ns ** ptr ns ns ns 20 pst *** ** *** ptrxst ns ** ** ptr ns ns * pst * *** *** pte *** *** ns 1 & 20 ptrxst * ** ns ptrxte ns * ns pstxte ns ** ns ptrxstxte ns ns ** a ptr, covering treatment; pst, storage time; pte, temperature; x, interaction. ns, not significant. *, significant at p < 0 .05. **, significant at p < 0.01. ***, significant at p < 0.001. rain cover and cherry quality 91 in ‘noire de meched’ (tab. 3). wang and long (2014) found the rq to be almost constant when measured at 0 oc, 10 oc and 20 oc after harvest, with all values being between 0.60 and 0.70 in ‘bing’ and ‘sweetheart’ cherries. differences in rq could be attributed to different cultivars, maturity state at harvest and experimental conditions. in the case of ‘noire de meched’, the highly increased rq values to approximately 1.37 after 14 d storage (at both 1 oc and 20 oc, in average) is of interest and could be largely attributed to oxidation of organic acids (beaudry et al., 1992) and mainly of malate (tsantili et al., 2007; usenik et al., 2008). by contrast, rq values equal to 1 indicated the sugars as respiration substrates, while lower than 1 the lipids (fonseca et al., 2002). the present study also calculated the q10 values from rates of o2 uptake since o2 is less soluble in fruit sap than co2. on day 7, the averaged q10 values (rate of o2 at 20 oc/rate of o2 at 1 oc) were 2.8 and 3.0 for ‘adriana’ and ‘noire de meched’, respectively. these values are relatively high and denoted the necessity of keeping the fruit at low temperature, as also suggested by wang and long (2014) for similar q10 values calculated from co2 production rates measured after harvest. objective measurements of quality attributes of fruit in ‘adriana’ controls and treated fruit with cover, the initial values of fruit weight, colour parameter c*, firmness, ta, tss/ta, ph, tp and taa averaged approximately 8.5 g, 26.5, 4.0 n, 0.63 g kg-1, 21.6, 3.97, 0.66 g kg-1 and 4.73 mmol kg-1, respectively and they were not affected by the covering treatment after harvest (tab. 4). results showed that ‘adriana’ cherries have a satisfactory fruit weight as a mid-early ripening cultivar (sansavini and lugli, 2008), adequate firmness, but relatively high ta and low tss at early harvest. in covered fruit, the value of the colour parameter ho was slightly lower and of tss higher than controls, indicating a tendency of earlier ripening that became significant, however, only after storage (tab. 4). a clear effect of covering on earlier ripening was observed in some early ripening cultivars, due to the increased temperature in a completely enclosed tunnel (balmer and blanke, 2008; schmitzeiberger and blanke, 2012). in this study, the meteorological data logging system, measuring at three positions from 7.00 am to 8.30 pm during the last 22 days before harvest, recorded some differences between the uncovered and covered orchard. in 2009, covering reduced the averaged mean of solar radiation from 391 w m-2 in controls to 360 w m-2 in covered fruit and relative humidity (rh) from 59.5 % to 55.7 %, whereas increased that of temperature from 24.4 oc to 25.3 oc and the averaged maximum temperature from 27.8 oc to 29.8 oc. consequently, ‘adriana’ is a cultivar practically resistant to cracking, at least in the studied area, with no need for covering. in ‘adriana’, storage increased the ratio of tss/ta, tp concentration and taa, lowered l*, c* and ta values, with the effects being significant, while had no effect on ho, firmness and tss in both controls and treated cherries (tab. 4). the changes in ta resulted in fruit less acid and more agreeable than after harvest with a value of tss/ta equal to or greater than 25 towards the end of storage. a value of tss/ta greater than 25 is considered critical for eating quality (schmitz-eiberger and blanke, 2012), but this contrasts the findings of kappel et al. (1996) that the optimum tss/ta should be 15-20. however, it is well known that consumer preference is influenced by many factors. here, it is worth noting that when tss/ta was greater than 25 the rq value was the maximum observed and ta the lowest in the case of ‘adriana’, implying that organic acids were the respiration substrates. in covered and control ‘noire de meched’ fruit, the values of fruit weight averaged 12 g (per fruit) and of fruit seed 0.67 g (per seed) at harvest and there was no effect of covering on them (tab. 5). the fruit weight of this cultivar, here, was, in agreement with that found by vercammen and vanrykel (2014) and much higher than in other study (usenik et al., 2008). an averaged weight of 12 g is considered ideal for cherry fruit, according to kappel et al. (1996). the different cultivation practices could influence these attributes to a great extent. in this work, firmness, tss, ta, tss/ta, and ph values in ‘noire de meched ’averaged 3.62 n, 16.39 obrix, 0.79 g kg-1, 20.68 and 3.53, respectively, in all fruit at harvest (tab. 5). the values were independent of the covering treatment and storage apart from ph that increased gradually in stored fruit. the present results showed that ‘noire de meched’ had higher weight and tss value and lower ho and ph values than ‘adriana’ at harvest, being more attractive by comparison. in ‘noire de meched’, ho was the only colour parameter significantly reduced by covering and not by storage (tab. 5). in 2011, however, the meteorological data system showed that covering reduced the averaged mean radiation from 268 w m-2 in controls to 227 w m-2 in covered fruit, whereas increased the corresponding relative humidity from 70.9 % to 72.9 %, and the temperature from 22.7 oc to 23.3 oc and the averaged maximum temperature from 26.3 oc to 27 oc during the last 22 days before harvest. it seems that when temperature difference between covered and uncovered fruit is small then other factors become more important to affect ripening attributes. indeed, in 2011 the weather was cooler and cloudier than in 2009, and the higher solar radiation adriana (2009) ‘noire de meched’ (2011) 0.20 0.11 0.19 0.17 tab. 3: effects of rain cover on respiratory quotient (rq) values in adriana’ and ‘noire de meched’ cherries at harvest, during and after storage. cultivar temperature (oc) treatmenta storage days (d) lsd(0.05)b 0 7 14 21 1 or 0.88 0.92 1.31 1.07 rc 0.98 0.82 1.33 1.03 20 or 0.88 0.78 1.11 1.06 rc 0.98 0.70 1.08 1.10 1 & 20 or & rc 0.16 1 or 1.05 0.95 1.40 rc 0.91 1.25 1.33 20 or 1.05 1.09 1.28 rc 0.91 1.08 1.46 1 & 20 or & rc 0.18 a or, orchard (uncovered); rc, rain covered orchard. b lsd, least significant difference at a = 0.05. 92 m. kafkaletou, m.v. christopoulos, m.-e. ktistaki, t. sotiropoulos, e. tsantili tab. 4: effects of rain cover on quality attributes in ‘adriana’ cherries at harvest and after storage, in 2009. treatment orchard (uncovered or or) rain covered orchard (rc) storage days (d) storage days (d) probabilitiesc attribute 0 7 14 21 0 7 14 21 lsd(0.05)b ptr pst ptrxpst weight of 10 fruit 82.50 82.54 ns (g)a weight loss (%)a 2.47 3.72 5.06 1.79 3.33 4.92 0.66 * *** ns l* 32.73 33.30 32.45 31.22 32.44 31.53 31.73 30.86 1.28 * * ns ho 16.6 16.79 15.19 13.23 15.15 13.58 14.13 12.4 2.50 * ns ns co 27 28.07 24.65 21.09 26.01 22.62 22.31 19.68 4.49 ns ** ns firmness (n) 3.92 4.07 4.08 3.70 4.08 4.14 3.89 3.64 0.58 ns ns ns tss (obrix) 13.40 13.06 13.51 13.56 13.93 14.13 14.43 14.13 0.57 *** ns ns ta (g kg-1) 0.65 0.58 0.59 0.48 0.61 0.61 0.57 0.52 0.06 ns *** ns tss/ta 20.54 22.69 22.77 27.93 22.67 23.36 25.09 27.57 3.29 ns *** ns ph 4.01 3.79 3.95 4.05 3.94 3.84 3.92 4.00 0.09 ns *** ns tp (g kg-1 )d 0.73 0.59 0.69 0.91 0.59 0.76 0.84 0.79 0.21 ns * * taa (mmol kg-1)e 4.83 5.20 4.82 6.21 4.64 6.04 5.78 6.27 1.24 ns * ns awhole fruit including stems. standard error (sen=6) was equal to 0.94 and 0.95 for or and rc, respectively. the probability of the covering effect was estimated by comparison between the two samples. b lsd(0.05), least significant difference at a = 0.05. c ptr, covering treatment; pst, storage time; x, interaction. d tp, total phenolics. e taa, total antioxidant activity. ns, not significant. *, significant at p < 0.05. **, significant at p < 0.01. ***, significant at p < 0.001. tab. 5: effects of rain cover on quality attributes in ‘noire de meched’ cherries at harvest and after storage, in 2011. treatment orchard (uncovered or or) rain covered orchard (rc) storage days (d) storage days (d) probabilitiesd attribute 0 7 14 0 7 14 lsd(0.05)c ptr pst ptrxpst weight of 10 whole fruit (g)a 120.16 119.33 nse weight of 10 seeds (g)b 6.73 6.71 nse weight of 10 stems (g) 1.51 1.04 1.06 1.67 1.12 1.03 0.20 ns *** ns weight loss of 10 whole fruit (%)a 2.11 4.43 1.91 4.17 0.78 ns *** ns l* 31.11 30.27 29.68 31.25 31.07 30.61 1.17 ns ns ns ho 14.35 14.09 13.15 15.23 16.00 15.20 2.38 * ns ns co 20.60 18.85 17.03 20.03 16.72 14.17 4.15 ns * ns firmness (n) 3.64 4.02 3.92 3.60 3.83 3.65 0.32 ns * ns tss (obrix) 16.07 17.01 17.12 16.72 16.63 14.98 1.24 ns ns * ta (g kg-1) 0.75 0.71 0.77 0.84 0.77 0.73 0.11 ns ns ns tss/ta 20.88 23.63 21.85 19.48 20.95 20.03 3.69 ns ns ns ph 3.49 3.75 3.77 3.57 3.68 3.81 0.28 ns * ns tp (g kg-1)f 0.76 0.76 0.67 0.61 0.63 0.62 0.05 *** * * taa (mmol kg-1)g 5.07 4.46 5.39 4.79 4.99 4.55 0.58 ns ns * a whole fruit including stems. standard error (sen=6) was equal to 9.18 and 11.16 for or and rc, respectively. b standard error , se (n = 9) was equal to 0.10 and 0.11 for or and rc, respectively. c lsd(0.05), least significant difference at a = 0.05. d ptr, covering treatment; pst, storage time; x, interaction. e probability of the covering effect was estimated by comparison between the two samples. f tp, total phenolics. g taa, total antioxidant activity. ns, not significant. *, significant at p < 0 .05. **, significant at p < 0.01. ***, significant at p < 0.001. rain cover and cherry quality 93 in open orchard became important for an advanced peel development. therefore, the effects of rain cover depend on the weather conditions, including the covering period (borve and meland, 1998). weight loss remains a major deteriorating factor in stored fruit. in ‘adriana’, the observed loss was reduced by the covering treatment significantly on day 7 (tab. 4). by contrast, in ‘noire de meched’ covering treatment did not exhibit any significant effect (tab. 5). storage time advanced fruit weight losses in both cultivars, while in ‘noire de meched’ the rate was slightly higher, by comparison, but did not exceed the level of 4.5 % at the end of the experiment. fruit respiration and water diffusion differences in cultivars are related to different cuticular permeability to water, regulating the postharvest quality (lara et al., 2014). it has been observed that fruit under covers could be softer than controls (schmitz-eiberger and blanke, 2012). the present results for both studied cultivars contrasted this statement. covering of open vase cherry trees also exhibited no adverse effect on fruit quality (sotiropoulos et al., 2014). here, firmness was not influenced by covering in both cultivars, but reduced by storage days only in ‘noire de meched’. however, all stored fruit in both cultivars retained satisfactory firmness values. in ‘adriana’, covering had no effect on tp and taa. taa in cherries depends mainly on tp and secondly on ascorbic acid since its con tribution to taa is much less than 15 % (wang et al., 1996). however, the covering effects are usually unpredictable since the combined effect is more critical than each factor separately (schmitzeiberger and blanke, 2012). in this study, however, tp and taa in ‘adriana’ increased progressively after storage (tab. 4). this significant effect of storage time, here, was not distorted after expressing the results of tp and taa on a dry weight basis in order to eliminate the involvement of weight loss. it seemed that ripening could be continued during storage. alternatively, the low temperature effect would be a possible explanation for the increased antioxidants in stored ‘adriana’ fruit, as observed in other cases (tsantili et al., 2010). in ‘noire de meched’, the advanced colour development (lower ho) in open orchard agreed with the higher tp concentration (0.76 g gae kg-1) in comparison to covered fruit (0.61 g kg-1 ), as shown in tab. 5 since the red colour in cherries is mainly ascribed to anthocyanins (goncalves et al., 2007). the present tp results are very similar to those in usenik et al. (2008) study determined in the same cultivar. storage time showed a significant, but minor effect on reduced tp in uncovered fruit on day 14. taa in this cultivar was not affected by covering or storage. differences in the pattern of changes between tp and taa are often observed in stored fruit and ascribed to different antioxidant activity of each phenolic compound (riceevans et al., 1996) that could increase or decrease during storage (tsantili et al., 2010). stem quality stem condition is critical for the cherry quality (linke et al., 2010). in particular, the green colour of stem is related to fruit freshness or to stored cherries of high quality. in the case of ‘adriana’, stem browning appeared only towards the end of storage to a relatively and similarly limited extent in all fruit (fig. 2a). in ‘noire de meched’ browning was practically observed at the end of the experiment (fig. 2b). on day 14, in all stored fruit a percentage of approximately 25 % remained entirely green, whereas the half of the stems developed a light brownish colour covering more than the half of their length. results showed the significance of storage on browning. indeed, stem browning are connected to water loss and chlorophyll degradation (harb et al., 2006). the present study measured the changes in the stem weight in ‘noire de meched’ during the experiment in 2011. the stem weight averaged 0.157 g in all fruit at harvest, was not affected by covering treatment, but significantly reduced after storage (tab. 5). according to linke et al. (2010), stems showed higher water and co2 losses due to low resistance to vapour transfer. after harvest, there is a high rate of water loss, but water transfer occurs from the fruit body to the stem that buffers this decline initially until the situation results in decreased vapour transfer conductance, respiration and photosynthetic activity. additionally, the chlorophyll degradation is promoted during aging, which means that browning is promoted if fruit are harvested at a later maturity state. here, differences in stem browning between ‘noire de meched’ and ‘adriana’ could be attributed to genetic or exogenous factors. concerning the rsr, zhao et al. (2013) concluded that stem retention force is not related to fruit quality and varies with genotype and year. in ‘adriana’, rsr was reduced by advanced storage time, but the limited and cumulative data did not permit to distinguish any significant effect of covering or storage on it (fig. 3a). these results resembled to those observed in other cultivar stored under similar conditions (tsantili et al., 2007). however, it has to be mentioned that rsr in ‘adriana’ was very high, as observed subjectively. by contrast, in ‘noire de meched’, a small percentage of the stems (approximately 10 %) showed rsr values between 0 and 3 n in covered fruit after harvest, while this class did not exist in uncovered fruit (fig. 3b). storage, however, increased the frequency of stems with low rsr, as previously observed (tsantili et al., 2007). quantitative descriptive analysis the three-point scale was considered appropriate for the untrained panel. the mean sensory scores obtained from the visual evaluation were 2.61, 2.51, 1.42 and 1.47 for size, peel colour, flesh colour and stem browning. the score for size was the highest among attributes, while no difference in size was observed between treated and control cherries, as also measured objectively. the panelists could distinguish the difference in peel colour between covered (2.21) and uncovered (2.71) fruit, rating higher the cherries that had developed fig. 2: effects of rain cover on frequency of classes of stem browning (sb) in ‘adriana’ and ‘noire de meched’ cherries after harvest and storage. (a), ‘adriana’; (b), ‘noire de meched’. each column represents 30 cherries. or, orchard uncovered; rc, rain covered orchard. sb classes: no brown, slightly brown (up to ¼ of stem length brownish), low brown (up to ½ of stem length brownish), brown (more than ½ of stem length brownish). data were analyzed by c2 tests. in a, when data were divided into three classes (no brown, slightly brown, and low brown + brown), p > 0.05. in b, due to small numbers in some classes, data were divided into two classes (no brown and all the rest) and showed p < 0.001. partial comparisons between or day 0 and or day 14, and between rc day 0 and rc day 14 showed the significant storage effect (p < 0.001 and p < 0.05, respectively), while the covering effect after 14 d storage (or day 14 and rc day 14) was not significant (p > 0.05). 94 m. kafkaletou, m.v. christopoulos, m.-e. ktistaki, t. sotiropoulos, e. tsantili fig. 3: effects of rain cover on frequency of classes of resistance to stem removal (rsr) in ‘adriana’ and ‘noire de meched’ cherries after harvest and storage. (a), ‘adriana’; (b), ‘noire de meched’. each column represents 30 cherries. or, orchard uncovered; rc, rain covered orchard. rsr classes: 0-3 n, 3-6 n, 6-9 n, > 9 n. data were analyzed by c2 tests. when data were divided into two classes, 0-6 n and > 6 n, (due to small numbers in some classes), p > 0.05 in both a and b. when data were divided into three classes (0-6 n, 6-9 n, > 9 n), all dual comparisons in both a and b were not significant (p > 0.05). fig. 4: quantitative descriptive profile presented in a spider-web chart for sensory evaluation of ‘noire de meched’ cherries by 14 panelists at 20 oc after 14 d storage at 1 oc. or, orchard uncovered; rc, rain covered orchard. the evaluated attributes were: size, peel colour, flesh colour, stem browning, texture (firmness), resistance to stem removal (rsr), easiness of stone removal, sweetness, sourness, overall taste and overall acceptance. the num-the numbers indicate the intensity of each attribute (1: low intensity; 2: medium intensity; 3: high intensity). the difference between means for each attribute was carried out by two-sample comparisons and the significance (p) is denoted within the parentheses. each number in parenthesis next to each mean corresponds to se. a redder or darker red colour (fig. 4) in agreement with the objective measurement. the difference in peel colour was the only significant attribute among the sensory ones observed. consumers in greece prefer dark red cherries with a sweet taste. the scores for flesh colour and sweetness were relatively low and of sourness high. most of the attributes tested were not related to preference, at least directly. however, attributes intensities underestimated or overestimated could have been affected by the panel preference. additionally, the panelists found that the stone was removed easily from the flesh and the rsr was moderate, while stem browning did not seem to be a serious defect. scores for texture (firmness), overall taste and overall acceptance were 2.31, 2.42 and 2.42, respectively, indicating that the good quality of ‘noire de meched’ could be maintained at 1 oc for 14 d. conclusions ‘adriana’ was, indeed, an almost completely resistant to cracking cultivar at least when cultivated in northern greece. due to expenses covering should not be applied to this cultivar although exhibited no adverse quality effect. the cherries were of medium weight, but had low tss content and relatively high ta at an early harvest. ‘adriana’, although a mid early ripening cultivar, retained a firm texture and attached green stems during storage at 1 oc for time long enough to cover transport and exposure to distant markets, while the tss/ta ratio could increase gradually, but considerably by storage time. alternatively, the cultivar could be harvested at an advanced maturity state. ‘noire de meched’, a mid ripening cultivar, was moderately suscep rain cover and cherry quality 95 tible to cracking, while rain cover was necessary to cope with the symptom without any adverse effect on quality. the cherries were very large (12 g), firm, with satisfactory values of tss/ta ratio and tss for cherry good quality. fruit could be stored for 2 weeks, retaining their good quality attributes according to panelists and to objective determinations apart from a moderate stem browning. panelists distinguished the redder peel colour of the uncovered fruit, being the only significant difference observed between treated and controls. also, covering did not affect respiration rates. however, in both cultivars the q10 values (0-20 oc) of respiration showed the immediate and continuous cooling requirements of the harvested fruit. acknowledgements thanks are due to the greek organization of agricultural assurance for funding the experiment. references balbontin, c., ayala, h., bastias, r.m., tapia, g., ellena, m., torres, c., yuri, j.a., quero-garcia, j., rios, j.c., silva, h., 2013: cracking in sweet cherries: a comprehensive review from a physical, molecular, and genomic perspective. chilean j. agric. res. 73, 66-73. balmer, m., blanke, m.m., 2008: cultivation of sweet cherry under cover. acta hortic. 795, 479-484. beaudry, r.m., cameron, a.c., shirazi, a., dostal lange, d., 1992: modified-atmosphere packaging of blueberry fruit: effect of temperature on package oxygen and carbon dioxide. j. am. soc. hortic. sci. 117, 436-441. borve, j., meland, m., 1998: rain cover protection against cracking of sweet cherries. ii. the effects on fruit ripening. acta hortic. 468, 455458. borve, j., skaar, e., sekse, l., meland, m., vangdal, e., 2003: rain protective covering of sweet cherry trees − effects of different covering methods on fruit quality and microclimate. horttechnology, 13, 143148. brand-williams, w., cuvelier, m.e., berset, c., 1995: use of a free radi-: use of a free radiuse of a free radical method to evaluate antioxidant activity. lebensm. wiss. technol. 28, 25-30. christensen, j.v., 1972: cracking in cherries. iv. physiological studies of the mechanism of cracking. acta agric. scand. 22, 153-162. christensen, j.v., 1996: rain-induced cracking of sweet cherries: its causes and prevention. in: webster, a.d., looney, n.e. 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efficacy of using rain protective plastic films against cracking of four sweet cherry (prunus avium l.) cultivars in greece. int. j. agric. innov. res. 2, 2319-1473. tsantili, e., karaiskos, g., pontikis, c., 2003: storage of fresh figs in low oxygen atmosphere. j. hortic. sci. biotech. 78, 56-60. tsantili, e., rouskas, d., christopoulos, m.v., stanidis, v., akrivos, j., 2007: effects of two pre-harvest calcium treatments on physiological and quality parameters in ‘vogue’ cherries during storage. j. hort. sci. biotech. 82, 657-663. tsantili, e., shin, y., nock, j.f., watkins, c.b., 2010: antioxidant concentrations during chilling injury development in peaches. postharvest biol. technol. 57, 27-34. usenik, v., fabcic, j., stampar, f., 2008: sugars, organic acids, phenolic composition and antioxidant activity of sweet cherry (prunus avium l.). food chem. 107, 185-192. vercammen, j., vanrykel, t., 2014: testing of sweet cherry varieties in belgium. acta hortic. 1020, 265-270. wang, h., cao, g., prior, r.l., 1996: total antioxidant capacity of fruits. j. agric. food chem. 44, 701-705. wang, y., long, l.e., 2014: respiration and quality responses of sweet cherry to different atmospheres during cold storage and shipping. postharvest biol. technol. 92, 62-69. 96 m. kafkaletou, m.v. christopoulos, m.-e. ktistaki, t. sotiropoulos, e. tsantili zhao, y., athanson, b., whitting, m., oraguzie, n., 2013: pedicel-fruit retention force in sweet cherry (prunus avium l.) varies with genotype and year. sci. hortic. 150, 135-141. address of the corresponding author: lab of pomology, department of crop science, agricultural university of athens, iera odos 75, botanikos, greece, gr 118 55. e-mail: miltchrist@aua.gr/ miltchrist@yahoo.gr © the author(s) 2015. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 88, 264 273 (2015), doi:10.5073/jabfq.2015.088.039 ministry of agriculture key laboratory of tropical fruit biology, south subtropical crops research institute, chinese academy of tropical agricultural sciences, zhanjiang, guangdong, china evaluation of 28 mango genotypes for physicochemical characters, antioxidant capacity, and mineral content s. shi, x. ma*, w. xu, y. zhou, h. wu, s. wang (received may 13, 2015) * corresponding author summary mango germplasm remains underutilized due to the limited knowledge of its quality properties. the objective of this study was to analyze and compare the physicochemical characters, antioxidant capacity, and mineral content of 28 mango genotypes, in order to assess useful information for the utilization of mango genetic resour ces in china. all the genotypes were grown under the same geographical conditions and with the same standard cultural practices. the results showed that there were significant differences among the genotypes in all studied traits. potassium, calcium, manganese, and iron were the dominant mineral components; sucrose and/or fruc tose were the dominant sugars; and malic and citric acid were the dominant organic acids. variation in sugars (glucose, 15.37-218.20 mg·g-1 fresh weight [fw]; fructose, 39.42-327.67 mg·g-1 fw; and sucrose 26.32-472.69 mg·g-1 fw), total phenolic compounds (13.69-82.65 mg gallic acid·100 g-1 fw), and total carotenoids (10.91-71.21 μg·g-1 fw) was significant among the genotypes. the total antioxidant potency composite index varied among the genotypes (6.12-81.39) and was significantly correlated with total phenolic compounds, but not with total carotenoids. overall, the results demonstrated that the physicochemical characteristics, antioxidant capacity, and mineral content in mango are genotype-dependent. introduction mango [mangifera indica l. (anacardiaceae)] is a widely grown horticulture crop in many tropical and subtropical countries and is the fifth largest fruit industry in the world after citrus, banana, grape, and apple. it is popularly known as “the king of tropical fruits” for its succulence, different flavors and aromas, delicious taste, high carotenoid content, and high pro-vitamin a value (tharanathan et al., 2006). china is the second-largest mango producer after india, in terms of production, marketing, and consumption. the total acreage planted to mango is approximately 129,000 ha, distributed in hainan, guangxi, yunnan, sichuan, and guangdong provinces, and the total annual production is approximately 906,000 t (wu et al., 2014). in china, the dry-hot valley of the jinsha river is the most suitable area for mango cultivation, especially for late production that commands high prices. approximately 40 % of the mango production in china occurs in this area (chen, 2013). china is the center of the natural distribution of wild mango germplasm (ni et al., 2008); however, mango breeding industry in china is not as well developed as that in america, australia, and india. most of the cultivars that used in china were introduced from australia, america, and india prior to 1980, except for some new cultivars that were released by our group (he et al., 2006). germplasm resources are the basis of genetic improvement and cultivar development. due to the long history of cultivation, extensive geographical distribution, and intense selection, there are more than a thousand mango cultivars worldwide. during the past decades, the chinese academy of tropical agricultural sciences has collected and conserved more than 300 mango genotypes representative of worldwide genetic variability, in order to produce new cultivars with better traits such as high soluble solid content, early-ripening, latematuring, middle size, red peel, resistance to anthracnose, and higher yield performance. however, mango germplasm remains under utilized due to the limited knowledge of quality properties. the quality of mango greatly depends on fruit physical properties, such as shape, vertical diameter, cross section, weight, stone weight, pulp recovery, pulp fibrosis, and fruit color, and on chemical properties such as total soluble solids, titratable acidity, total sugars, vitamin c, taste, and aroma. many studies have focused on the physicochemical and nutritional properties, such as phenolic compounds (rocha ribeiro et al., 2007; manthey and perkins-veazie, 2009), sugars (medlicott and thompson, 1985), organic acids (valente et al., 2011; liu et al., 2013), antioxidant capacity (mahattanatawee et al., 2006; kim et al., 2010), fruit aroma (pino and mesa, 2006; pandit et al., 2009), and carotenoids (pott et al., 2003). however, studies on the comprehensive and systematic evaluation of fruit quality attributes and the relationships among pomological traits are limited, particularly for mango germplasm in the china. the objective of this study was to analyze and compare the physicochemical characters, antioxidant capacity, and mineral content of 28 mango genotypes from the south subtropical crops research institute of the chinese academy of tropical agricultural sciences, in order to assess useful information for the utilization of mango genetic resources in china. materials and methods plant materials and quality properties twenty-eight genotypes grown in the orchards of the south subtropical crops research institute (sscri) in zhanjiang, china were used in this study (tab. 1). all the genotypes were grown under the same geographical conditions and with the same standard cultural practices. for each genotype, four replicates (consisting of five fruits each) were carried out and used for analysis. fruits were incubated at 25 °c for ripening, which was ascertained for each cultivar by conventional indices such as ripening period after harvest, change in skin color, smell, and softness to touch. ripe fruits were washed, drained, and dried with paper towels, and fruit weight was immediately measured on a balance with accuracy of 0.01 g. the pulp was manually separated from the fruit and cut into small pieces to obtain homogeneous samples. a 300-g fruit pulp sample was homogenized in a blender, and stone weight, ph, total soluble solids (tss), and titratable acidity (ta) were evaluated. the remaining flesh sample was immediately ground in liquid nitrogen and stored at -70 °c until use. the ph values were determined from the juice of each sample with a digital ph meter (dl 25, mettler toledo, greifensee, switzerland). tiratable acidity (ta) was expressed as a percentage of malic acid, and total soluble solids (tss) were measured with a digital refractrometer (atc-20e, atago, tokyo, japan). evaluation of mango germplasm 265 mineral content dry fruit material (0.5 g) was heated at 550 °c in a muffle furnace for 4-5 h. the resultant ash was dissolved in 2 ml of 30 % (v/v) hno3 and distilled water was added until the total volume was 50 ml. calcium (ca), zinc (zn), magnesium (mg), iron (fe), manganese (mn) and copper (cu) were measured using a hitachi z-8000 atomic absorption spectrometer, and potassium (k) was determined using a flame photometer (410 corning) according to the association of analytical communities (aoac, 1995). sugars and organic acids sugars and organic acids were determined by high-performance liquid chromatography (hplc) (lc-20a, shimadzu corp., kyoto, japan). a 2-g flesh sample was mixed and homogenized with 10 ml distilled water, incubated at 37 °c for 30 min, and then centrifuged at 5000 × g for 10 min. the supernatant was collected and evaporated to dryness at 75 ºc in a water bath. the residue was dissolved with 5 ml distilled water and filtered before analysis. analysis of sugars was carried out using an amino column (250 mm × 4.6 mm; kromasil, bohus, sweden) with a flow rate of 1.0 ml·min-1 at 35 °c. for the mobile phase, acetonitrile and twice distilled water (70:30 v/v) were used along with a refractive index detector as described by liu et al. (2006) with some modifications. organic acids were extracted from a 2-g fruit sample mixed with 8 ml of 0.2 % metaphosphoric acid. the reaction mixture was centrifuged at 4,000 × g for 10 min and 1 ml of the supernatant was used for further analysis. the elution system consisted of 0.2 % metaphosphoric acid running isocratically with a flow rate of 1 ml·min-1. the organic acids were eluted through a venusil xbp-c18 column (250mm × 4.6mm) and detected at 210 nm. sugars and organic acids were identified and calculated using the corresponding external standards. carotenoids, total phenolic compounds, and antioxidant capacity the total amount of carotenoids was determined as described by zhao et al. (2013). carotenoids were extracted from 1-g flesh using an ethanol:acetone solution (1:3 v/v) at room temperature for 60 min. after centrifugation at 4,000 × g for 10 min at 4 °c, the supernatant was collected, and its absorbance was measured in a spectrophotometer at 450 nm. the total carotenoid content was calculated using the extinction coefficient of β-carotene, e1% = 2592. a 50-g flesh sample was homogenized and extracted in 200 ml of ethanol: acetone (7:3 v/v) for 1 h at 37 °c as described by lee and wicker (1991). the extract was filtered through whatman no. 41 paper and rinsed with 50 ml of ethanol: acetone (7:3 v/v). extraction of the residue was repeated under the same conditions and the two filtrates were combined. the combined extract was used to determine total phenol and antioxidant activity. the amount of total phenols was determined following the folinciocalteu colorimetric method (rapisarda et al., 1999). absorbance was measured at 765 nm and total phenols were expressed as mg·l-1 of gallic acid equivalents (gae). gallic acid standard solutions were prepared with concentrations ranging between 0 and 1000 mg·l-1. the antioxidant capacity was evaluated using 2,2-diphenyl-1-picrylhydrazyl (dpph), 2,2-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid) (abts), ferric reducing antioxidant power (frap), super oxide radical scavenging activity (srsa), and metal chelating capacity (mcc). dpph scavenging capacity was measured as described by masuda et al. (1999) with some modifications. a 25-μl extract sample was mixed with 2 ml of 62.5 μm dpph methanol solution and 30 min later the absorbance was measured at 517 nm. results were expressed as trolox equivalent antioxidant capacity. abts assay was performed as described by re et al. (1999). a 25-μl extract sample was mixed with 2 ml abts solution and 30 min later the absorbance was measured at 734 nm. the radical-scavenging activity of the test samples were expressed as trolox equivalent antioxidant capacity. frap was assayed as described by benzie and strain (1996). a 20-μl extract sample was mixed with 1.8 ml 2,4,6-tris(2pyridyl)-s-triazine (tptz) reagent consisted of 0.3 m acetate buffer (ph 3.6), 10 mm tptz, and 20 mm ferric chloride, and the absorbance of the colored product was measured at 593 nm. the antioxidant activity was expressed as μmol trolox equivalents. srsa was assayed as described by chen and yen (2007) with a minor modification. a 50-μl extract sample was mixed with 1 ml of 0.1 m phosphate buffer (ph 7.4) containing 150 μm nitro blue tetrazolium, 60 μm n-methylphenazinium methylsulfate, and 468 μm nicotinamide adenine dinucleotide phosphate and then incubated for 8 min at 25 ºc. absorbance was measured at 560 nm. the superoxide anion scavenging activity was calculated as follows: scavenging activity (%) = (1-absorbance of sample/absorbance of tab. 1: origin and parentage of the mango genotypes asseyed genotype origin parentage genotype origin parentage yuexi no. 1 china carabao kensinton australia brooks hongmang no. 8 australia unknown lippens america haden saigon vietnam unknown bambaroo india unknown irwin america lippens krs australia unknown guangxi no. 4 china india 901 × yingzui xiaoji china unknown mallika india dashehari × neelum edward america haden × carabao sijimang thailand unknown zihua china ok-rung lilley america unknown guixiang china golock × neelum carabao philippines unknown jinshui china zihua glenn america unknown renong no.1 china unknown nam dok mai america unknown vandyke america unknown tommy america haden xiangjiao thailand unknown lianmang china chance seedling valencia pride america haden aimang china chance seedling yingzui china unknown 266 s. shi, x. ma, w. xu, y. zhou, h. wu, s. wang control) × 100. mcc was determined as described by dinis et al. (1994). a 1-ml mango extract in 2.8 ml distilled water was mixed with 50 μl of 2 mm fecl 2·4h2o and 150 μl of 5 mm ferrozine. the mixture was shaken for 10 min and then fe2+ was measured by monitoring the formation of ferrous ion-ferrozine complex at 562 nm. the metal chelating capacity was calculated as follows: scavenging activity (%) = (1-absorbance of sample/absorbance of control) × 100. the overall antioxidant potency composite (apc) index was calculated as described by seeram et al. (2008): antioxidant index score = (sample score/best score) × 100. statistical analysis all samples were prepared and analyzed in triplicate. analysis of variance (anova) were performed with sas 9.0 (sas corp., cary, nc, usa). correlation coefficients (r) were determined by pearson correlation matrix method also using sas 9.0. probability values of p < 0.05 and p < 0.01 were considered statistically significant and very significant, respectively. the eigenvectors, contribution rate, and accumulative contribution rate in principal component analysis were processed using xlstat (addinsoft, paris, france) software package. results and discussion quality properties tab. 2 presents the fruit mass, stone mass, edible ratio, ph, soluble solids, and titrable acidity of each mango genotype. valencia pride had the highest fruit mass (771.73 g) and stone mass (60.22 g), whereas yuexi no. 1 had the lowest fruit mass (138.06 g) and stone mass (16.71 g). the edible ratio ranged from 67.27 % in xiaoji to 83.96 % in nam dok mai. we also found a significant positive correlation between fruit mass and edible ratio (r = 0.594, p < 0.001), suggesting that the bigger-fruit genotypes also had a higher edible ratio. these results were in agreement with those reported by shi et al. (2011). mango acidity that assessed by ph or titratable acid was the highest in guixiang, regardless the method. the ph values ranged between 3.35 and 5.09. the highest titratable acidity content was identified in guixiang (2.35 g·100 g-1), whereas the lowest tab. 2: fruit mass (g), stone mass (g), edible ratio (%), ph, total soluble solids (tss, °brix), and titratable acidity (ta, % citric acid) of 28 mango genotypes. genotype fruit stone edible ph tss ta tss/ta mass mass ratio yuexi no. 1 138.06k 16.71i 70.94hi 4.09c-f 9.40jk 0.98c-e 9.56mn hongmang no. 8 336.01c-g 33.20b-e 79.00a-e 3.75f 15.10b-e 1.71b 8.85n saigon 227.97h-k 34.77bc 71.99g-i 3.59d-f 14.13d-f 0.36h-l 39.39e-g irwin 266.22f-j 22.04d-i 78.82a-f 3.53d-f 10.87ij 0.85de 12.71k-n guangxi no. 4 205.84h-k 21.42e-i 77.17b-g 4.33b-f 16.27a-c 0.30i-l 54.98cd mallika 377.24b-e 31.32b-f 80.75a-c 3.62d-f 15.67a-d 0.74e-g 21.31i-m sijimang 211.31h-k 22.76d-i 71.56g-i 4.08c-f 15.27b-e 0.23kl 67.48b lilley 227.51h-k 25.89b-i 73.34e-h 4.20b-f 12.47f-i 0.23kl 54.98cd carabao 225.50h-k 21.38e-i 78.57a-f 3.93c-f 17.27a 1.12c 15.46j-n glenn 420.06bc 31.99b-f 80.92a-c 4.33b-e 12.20f-i 0.44h-k 27.67g-j nam dok mai 274.24f-j 17.96hi 83.96a 5.09ab 17.40a 0.14l 120.16a tommy 422.11b-e 37.41b 82.04ab 4.87a-c 13.47e-g 0.38kl 35.44bc lianmang 242.43g-k 29.20b-h 74.50d-h 5.54a 16.47a-c 0.23h-l 71.59ef aimang 178.77jk 20.31f-i 70.96hi 4.03c-f 17.00ab 0.89c-e 19.04h-l kensinton 398.13b-e 33.37b-e 78.62a-f 4.04c-f 14.73c-e 1.79b 8.21n lippens 348.02b-f 27.70b-i 79.20a-e 4.21b-f 11.67g-i 0.34i-l 34.71f-h bambaroo 298.67e-i 30.49b-g 75.76c-h 3.66d-f 13.27e-h 0.54g-j 24.49h-l krs 353.07b-f 37.08b 77.87b-f 3.37ef 13.60e-g 1.87b 7.28n xiaoji 144.69k 24.97c-i 67.27i 3.91c-f 13.60e-g 0.29j-l 46.34de edward 445.48b 26.32b-i 79.99a-d 4.25b-f 14.60c-e 0.56f-j 26.30h-k zihua 188.46jk 27.37b-i 73.03f-h 4.27b-f 7.60k 1.02cd 7.42n guixiang 207.97h-k 25.15c-i 75.37c-h 3.35d-f 11.47hi 2.35a 4.88n jinshui 186.35jk 19.18h-g 78.62a-f 3.66d-f 8.60k 0.94c-e 9.15mn renong no. 1 405.61b-d 35.02bc 80.49a-c 4.31b-f 11.27i 0.48g-k 23.39h-l vandyke 307.02d-h 30.38b-g 77.31b-g 4.47b-d 14.77c-e 0.61f-h 24.33h-l xiangjiao 201.19i-k 27.90b-i 71.78g-i 4.43b-d 13.33e-h 1.66b 8.02n valencia pride 771.73a 60.22a 78.40a-f 3.95c-f 11.67g-i 0.80d-f 14.55k-n yingzui 214.12h-k 31.53b-f 70.51hi 4.86a-c 16.00a-d 0.56f-i 28.69g-i different superscript letters indicate significant differences across genotypes at p < 0.01. evaluation of mango germplasm 267 in nam dok mai (0.14 g·100 g-1). these results were in agreement with those reported by vasquez-caicedo et al. (2002). the highest tss (17.00-17.40 %) were found in kensinton, carabao, and nam dok mai, whereas the lowest in zihua and jinshui (7.60-8.60 %). it is widely believed that the tss/ta ratio is widely applied as the most reliable parameter to evaluate mango fruit quality, and the typical values for high quality mango fruits range between 23 and 50 (vaquez-caicedo et al., 2005). in this study, 11 (39.3 %) mango genotypes fell within this range, suggesting high quality. mineral content the mineral concentration of mango genotypes is shown in tab. 3. mineral content differed widely and significantly among the genotypes. potassium (k), magnesium (mg), and calcium (ca) were the dominant minerals in mango pulp. k, an essential mineral for controlling the salt balance in human tissues, is the most abundant mineral in mango fruits. lippens had the highest k content [32.80 g·kg-1 dry weight (dw)], while kensinton had the lowest (7.81 g·kg-1 dw). mg is the second most abundant element in mango fruits, which is required by many enzymes, especially sugar and protein kinases that catalyze atp-dependent phosphorylation reactions (gorinstein et al., 2001). banana had the highest mg content (816.98 g·kg-1 dw) and carabao had the lowest (312.76 g·kg-1 dw). ca is the third most abundant mineral and in this study ranged between 146.77 g·kg-1 dw in saigon and 640.82 g·kg-1 dw in renong no. 1. ca concentration is closely related to cell structure and membrane stability and associated with the physiological diseases of mango. our results showed that renong no.1, xiaoji, and jinshui had the highest ca content and can be used as potential donors for developing ca-rich mango varieties. trace elements (i.e., fe, mn, cu, and zn) are essential regulators of cell redox homeostasis, because they are co-factors for several antioxidant enzymes as well as contributors to signal transduction (lu et al., 2014). among the different mango genotypes, the highest content was found for mn (12.69-103.48 mg·kg-1 dw), followed by fe (8.84-16.49 mg·kg-1 dw), zn (5.17-12.12 mg·kg-1 dw), and cu (3.84-7.18 mg·kg-1 dw). guangxi no. 4 had the highest content of fe and cu, while xiaoji had the highest content of mn and zn and considerable amounts of fe and cu. our study is one of the few to focus on mineral content as part of mango quality. while mineral content is influenced by many factors, tab. 3: minerals content (mg·kg-1) of 28 mango genotypes referred to dry matter (dm) content. genotype k ca mg fe mn zn cu yuexi no. 1 14504.25c-d 205.62j 606.10e-h 13.05b-d 16.95m 9.08bc 6.54ab hongmang no. 8 10449.68g-j 230.89ij 806.98ab 11.38c-g 23.90jk 5.53f-h 5.10d-h saigon 11823.86d-h 146.77k 489.07kl 11.00c-g 22.37kl 6.23e-g 4.24f-i irwin 9424.95h-j 347.53f 535.58ij 10.29d-g 22.07kl 3.91h 4.66e-i guangxi no. 4 8206.01ij 454.95d 472.10lm 16.49a 28.54hi 6.69d-g 7.18a mallika 9462.35h-j 216.78j 449.17mn 10.50d-g 36.81e 5.52f-h 4.67e-i sijimang 9614.98h-j 292.68g 614.04e-h 12.78b-e 18.14m 7.36c-f 5.14d-g lilley 11559.55d-i 456.62d 583.34h 13.58a-d 54.30d 5.59f-h 5.16d-g carabao 9188.43h-j 298.43g 312.76p 11.64c-g 34.00ef 6.54d-g 3.93i glenn 14641.88c-d 250.13i 632.98d-f 10.28d-g 29.21g-i 8.22cd 3.95i nam dok mai 14112.69b-f 255.53hi 552.86i 12.00c-g 12.69n 6.45d-g 4.27g-i tommy 17063.11b 331.21f 610.92e-h 10.19d-g 31.91f-h 6.01fg 5.17d-f lianmang 10365.92g-j 169.07k 433.90n 15.78ab 21.85kl 5.90fg 3.84i aimang 11299.45d-j 281.61gh 778.06b 11.49c-g 25.54i-k 9.08bc 4.71e-i kensinton 7814.45j 502.46c 601.31f-h 11.23d-g 58.32c 7.10d-g 5.63cd lippens 142798.9a 330.48f 442.69mn 10.18d-g 27.21ij 5.20g-h 4.45f-i bambaroo 14284.10b-e 356.25f 656.56d 8.84g 20.15ml 7.95c-e 7.00a krs 13541.25c-g 285.67g 599.50gh 12.66b-f 26.49ij 7.91c-e 5.49c-e xiaoji 9689.88h-j 628.71a 808.14ab 14.35a-c 103.48a 12.12a 6.18bc edward 10580.14g-j 294.87g 515.28jk 11.13c-g 17.58m 10.49ab 4.33f-i zihua 8998.00h-j 409.56e 635.62de 10.53d-g 36.81e 5.52f-h 4.67e-i guixiang 11017.39e-j 444.81d 629.18d-g 9.28e-g 57.87c 5.53f-h 3.90i jinshui 16486.78bc 544.45b 740.09c 13.55a-d 65.52b 11.62a 4.07i renong no.1 11579.99d-i 640.82a 389.84o 11.56c-g 29.23g-i 5.17gh 4.34f-i vandyke 10767.57f-j 361.40f 603.90e-h 9.02fg 32.59fg 5.81fg 4.74e-i xiangjiao 14471.75c-d 336.53f 816.98a 11.62c-g 60.58c 8.04c-e 4.42f-i valencia pride 9688.39h-j 232.69ij 538.20ij 11.91c-g 17.87m 5.63f-h 5.19d-f yingzui 10131.47g-j 256.34hi 547.37i 11.48c-g 27.19ij 6.31e-g 5.81b-d different superscript letters indicate significant differences across genotypes at p < 0.01. 268 s. shi, x. ma, w. xu, y. zhou, h. wu, s. wang such as environmental conditions, agricultural practices, and water quality, the present study demonstrated that in mango fruits, the accumulation of k, ca, mg, fe, mn, zn, and cu is primarily dependent on genotype. sugars and organic acids the organoleptic quality of fruits greatly depends on the content and composition of sugars in fruits. three soluble sugars were detected and quantified in mango flesh. the concentration of glucose, varied significantly between 15.37 and 218.20 mg·g-1 fw; of fructose between 39.42 and 327.67 mg·g-1 fw; and of sucrose between 26.32 and 472.69 mg·g-1 fw (tab. 4 ). based on the concentration of fructose and sucrose, mango varieties were classified into 2 groups. the first group included 16 genotypes (saigon, guangxi no. 4, mallika, sijimang, lilley, nam dok mai, tommy, lianmang, lippens, bambaroo, xiaoji, edward, renong no. 1, vandyke, valencia pride, and yingzui) with higher sucrose than fructose concentration. the two genotypes with the highest sucrose content were nam dok mai and lianmang that was 3.13 and 2.17 times higher than fructose, respectively. the second group included 12 genotypes (yuexi no. 1, hongmang no. 8, irwin, carabao, glenn, aimang, kensinton, krs, zihua, guixiang, jinshui, and xiangjiao) with higher fructose than sucrose concentration. however, liu et al. (2013) found that sucrose predominated in mango varieties. this difference between the two studies could be attributed to the different mango genotypes and number of samples used in the experiments. it is concluded that the content and composition of sugars in mango fruit is genotype-depended. total sugars ranged between 138.38 and 698.12 mg·g-1 fw. the highest total sugars were found in edward (698.12 mg·g-1 fw) and aimang (671.38 mg·g-1 fw), while the lowest in vandyke (138.38 mg·g-1 fw) (tab. 4). in the present study, statistically significant differences were found in the individual organic acids and the total acid content among the 28 genotypes. total acids ranged between 12.91 and 57.08 mg·g-1 fw (tab. 4). the three genotypes with the highest acid content were edward, guixiang, and hongmang no. 8. in all mango genotypes, malic acid was the dominant organic acid, which ranged between 4.31 and 41.74 mg·g-1 fw, followed by citric acid that ranged between 0.59 and 20.63 mg·g-1 fw (tab. 4). these results are in agreement with previous studies (tovar et al., 2001). the high malic acid content genotype, edward, and the three high tartaric acid content genotypes (hongmang no. 8, krs, and kensinton) were distinct from all other genotypes. oxalic acid content ranged between 1.98 and 6.17 mg·g-1 fw (tab. 4) and two genotypes, saigon and guangxi no. 4, had the highest content. previous studies showed that the content of tartaric acid is genotype-depended. liu et al. (2013) found no tartaric acid in any mango samples, while medlicott and thompson (1985) identified a low concentration of tartaric acid in keitt. in this study, five genotypes (sijimang, lilley, cacarbao, glenn, and zihua) had no tartaric content; 23 genotypes had a low tartaric content that ranged between 0.07 and 5.58 mg·g-1 fw; and 2 genotypes (vandyke and nam dok mai) had high tartaric content (tab. 4). in addition, previous studies also found (tovar et al., 2001; liu et al., 2013) that mango fruit contains trace levels of succinic and a-ketoglutaric acid, in addition to the high levels of ascorbic and fumaric acid. carotenoids, total phenolic compounds, and antioxidant capacity carotenoids are the main pigments in mature mango fruit. considerable variation was found in total carotenoids among the 28 mango genotypes (tab. 4). the highest total carotenoid content (68.3471.21 μg·g-1 fw) was found in tommy, vandyke, bambaroo, and zihua, while the lowest in lianmang, kensinton, vanlencia pride, and renong no.1 (10.91-16.67 μg·g-1 fw). the mean values of total carotenoids did not differ significantly from a previous study in 60 mango cultivars (10.52-50.24 μg·g-1 fw; zhao et al., 2013), but the maximum values of total carotenoids were higher in the present study. phenolic compounds are considered the most important antioxidants of plant materials. mean values of the total phenolic content varied between 13.69 and 82.65 mg gallic acid·100 g-1 fw. a relatively high total phenolic content was found in aimang and lianmang, whereas low values were found in lippens and valencia pride (tab. 4). the antioxidant capacity of fruits is an important indicator of health promoters, and a number of methods have been adapted to assess antioxidants. our results showed that there were significant differences in antioxidant capacity among the 28 genotypes (tab. 5). the range of dpph was 807.17-2963.89 μm trolox, of abts 237.231573.07 μm trolox, of frap 8607.88-58298.18 μm trolox, of srar 2.50-93.33 %, and of mcc 1.87-26.17 %. tab. 5 shows the rank order of mango genotypes according to the apc index. the apc index showed significant variation (6.12-81.39) among the 28 mango genotypes. aimang and lianmang had the highest apc indices, while lippens and tommy the lowest. therefore, aimang and lianmang had a stronger antioxidant capacity than other mango genotypes, which is a potential trait for further utilization and improvement. correlation analysis was used to explore the relationships among the different antioxidant variables measured in all mango extracts (tab. 6). frap, abts, and dpph were significantly correlated with total polyphenols (p < 0.01, frap, r = 0.629; abts, r = 0.624; dpph, r = 0.741). no significant correlation was found between mcc and srsa and total polyphenols. the absence of correlation between the two assays and total polyphenols was probably due to a diverse sensibility of mcc and srsa assays for hydrophilic antioxidants. some research (perez-jimenez and saura-calixto, 2005) suggested that carotenoids may also contribute to the antioxidant activity of carotenoid-rich fruits. however, no significant relationship was observed between carotenoids and antioxidant capacity determination methods. therefore, total polyphenols are probably the major contributor of the antioxidant capacity in mango fruits. pca of genotypes pca was used in order to understand the underlying interrelationships and to select the best linear combination of measured traits that explains the largest proportion of variation. the results showed that more than 80 % of the observed variability was explained by 9 components (tab. 7). the results indicated that variation in mango fruit quality was multi-directional, in accordance with previous results demonstrating that mango fruit quality is influenced by multiple traits (pradeepkumar et al., 2006). pc1 represented 21.24 % of the variation and pc2 represented 16.70 % of the variation. fig. 1 shows the score scatter plot of pca of all mango samples. fig. 1 as a total shows the relationships between genotypes (scores) and variables (loadings). the positive values for pc1 indicated genotypes with a higher content of citric acid and glucose and higher titratable acidity (guixiang, kensinton, and hongmang no. 8), while the negative values indicated genotypes with higher ph, ssc/ta, and sucrose content (nam dok mai and guangxi no. 4). the positive values for pc2 indicated genotypes with higher total phenolic content and antioxidant capacity (xiaoji, lianmang, and aimang), while the negative values indicated genotypes with higher fruit weight, stone weight, and edible rate (valencia pride and lippens). conclusions mango quality is defined as the conjunction of good appearance and inherent qualities to the consumable product. in china, mango evaluation of mango germplasm 269 ta b. 4 : c ar ot en oi ds (μ g· g1 ) , t ot al p he no lic s (m g· 10 0 g1 ) , t ot al s ug ar s (m g· 10 0 g1 ) , a nd o rg an ic a ci ds (m g· 10 0 g1 ) o f 2 8 m an go g en ot yp es . g en ot yp e c ar ot en oi ds to ta l fr uc to se g lu co se su cr os e to ta l o xa lic ta rt ar ic m al ic c itr ic to ta l ph en ol ic s su ga rs ac id ac id ac id ac id ac id s y ue xi n o. 1 42 .9 0c -f 48 .6 9d e 19 6. 45 cg 12 3. 48 de 29 .2 3i j 34 9. 15 hj 3. 44 di 4. 13 ab 13 .0 6m n 9. 70 g 30 .3 3f h on gm an g n o. 8 27 .5 2e -k 26 .4 5l m 24 9. 16 bc 18 6. 69 ab 39 .7 5h -j 47 5. 61 eh 2. 57 gi 3. 34 ab 30 .5 5d e 20 .6 3a 57 .0 8a sa ig on 30 .8 1d -k 33 .8 0h -j 14 8. 71 fj 60 .9 8f -i 42 6. 20 ab 63 5. 89 ab 6. 17 a 4. 18 ab 15 .7 9k l 7. 17 hi 33 .3 1e f ir w in 20 .2 3h -k 24 .8 9m n 22 1. 06 be 13 5. 77 ce 89 .5 8h 44 6. 41 fh 3. 32 di 1. 34 ab 21 .1 8i j 7. 39 h 33 .2 2e f g ua ng xi n o. 4 37 .2 4d -h 32 .2 4h -k 15 8. 87 fj 29 .1 7i j 29 3. 07 f 48 1. 11 eh 5. 79 ab 0. 07 b 15 .0 8k l 3. 72 kl 24 .6 7g m al lik a 46 .2 6b -e 35 .4 1h i 15 1. 99 fj 14 1. 90 ce 18 4. 49 g 47 8. 39 eh 2. 44 hi 2. 82 ab 32 .1 2c d 9. 18 g 46 .5 5b si jim an g 25 .6 9e -k 37 .4 3g h 12 8. 48 hj 69 .8 9f -i 36 2. 30 ce 56 0. 66 bf 4. 29 bg 0. 00 b 24 .5 8j 7. 28 h 36 .1 5c -e l ill ey 57 .9 6a -c 35 .9 6h i 16 8. 97 dj 46 .3 3h -j 42 9. 35 ab 64 4. 65 ab 4. 41 bf 0. 00 b 5. 01 qr 3. 49 l 12 .9 1i c ar ab ao 34 .6 4d -j 69 .0 8b 27 5. 36 ab 19 8. 88 ab 14 2. 25 g 61 6. 50 ad 5. 70 ab 0. 00 b 13 .9 7l m 14 .8 2e 34 .4 9d -f g le nn 50 .7 0a -d 35 .7 5h i 20 7. 57 cf 10 2. 87 ef 17 6. 88 g 48 7. 32 dg 3. 34 di 0. 00 b 11 .2 8n 7. 84 h 22 .4 6g h n am d ok m ai 30 .0 2d -k 37 .5 9g h 12 1. 12 j 28 .6 5i j 47 2. 69 a 62 2. 46 ac 3. 50 di 5. 53 a 12 .2 8m n 1. 86 m 23 .1 8g h to m m y 68 .3 4a 19 .2 1o 13 5. 00 hj 49 .8 7h -j 29 0. 92 f 47 5. 79 eh 3. 59 di 2. 41 ab 9. 20 o 3. 84 kl 19 .0 4h l ia nm an g 10 .9 1k 81 .5 1c d 13 7. 22 gj 73 .9 9f -h 45 8. 26 a 66 9. 47 ab 4. 29 bg 3. 68 ab 28 .1 1f 4. 49 jl 40 .5 7c a im an g 60 .5 2a -c 82 .6 5f g 27 5. 79 ab 21 8. 20 a 17 7. 39 g 67 1. 38 ab 3. 58 di 3. 17 ab 22 .6 1h i 9. 47 g 38 .8 2c d k en si nt on 15 .2 6i -k 54 .5 0c 32 7. 67 a 21 7. 82 a 43 .4 8h -j 58 8. 98 ae 2. 67 fi 2. 34 ab 7. 21 p 17 .7 0c 29 .9 3f l ip pe ns 42 .0 3c -g 13 .6 9p 12 4. 26 ij 71 .3 6f -i 39 1. 24 bc 58 6. 87 ae 2. 33 hi 0. 26 b 16 .6 1k 4. 07 jl 23 .2 7g h b am ba ro o 70 .2 9a 27 .2 3k -m 15 5. 54 fj 48 .3 5h -j 29 5. 45 f 49 9. 34 cg 3. 87 ch 0. 43 b 4. 31 r 5. 05 j 13 .6 7i k r s 31 .9 0d -k 29 .4 2j -m 22 6. 90 bd 17 6. 77 bc 39 .6 1h -j 44 3. 27 hf 2. 40 hi 2. 56 ab 6. 25 pq 19 .4 9b 30 .7 1f x ia oj i 65 .6 7a b 78 .9 9a 12 4. 52 ij 65 .7 3f -i 37 7. 12 bd 56 7. 37 af 4. 79 ad 3. 29 ab 34 .0 9b 4. 13 jl 46 .3 0b e dw ar d 30 .1 8d -k 81 .1 4a 18 5. 98 dh 16 5. 25 bc 34 6. 88 cf 69 8. 12 a 2. 56 gi 2. 88 ab 41 .7 4a 6. 29 i 53 .4 7a z ih ua 71 .2 1a 43 .3 7f 16 4. 18 ej 77 .3 8f -h 60 .6 2h -j 30 2. 18 j 5. 40 ac 0. 00 b 23 .6 5g h 9. 49 g 38 .5 4c d g ui xi an g 22 .0 5f -k 31 .2 8i -l 20 7. 07 cf 16 2. 19 bd 26 .3 2i j 39 5. 58 gj 3. 48 di 2. 59 ab 32 .9 1b c 16 .4 4d 55 .4 1a ji ns hu i 36 .7 1d -i 27 .7 0k -m 14 1. 06 gj 10 2. 10 eg 74 .3 8h -j 31 7. 54 ij 2. 97 ei 1. 91 ab 16 .2 2k 9. 63 g 30 .7 3f r en on g n o. 1 16 .6 7h -k 28 .8 1j -m 11 6. 48 j 59 .4 6g -i 31 0. 72 ef 48 6. 66 dg 3. 22 di 4. 27 ab 7. 79 op 3. 54 l 18 .8 1h v an dy ke 69 .6 2a 37 .3 9g h 39 .4 2k 15 .3 7j 83 .5 9h i 13 8. 38 k 4. 97 ad 5. 58 a 11 .4 9n 0. 59 n 22 .6 3g h x ia ng jia o 20 .7 4g -k 44 .8 9e f 25 0. 25 bc 17 0. 16 bc 22 .0 3j 44 2. 45 fi 4. 50 ae 1. 35 ab 11 .4 4n 13 .8 2f 31 .1 0f v al en ci a pr id e 13 .4 5j k 20 .0 5n o 16 0. 32 fj 14 0. 59 ce 17 7. 87 g 47 8. 78 eh 1. 98 i 0. 89 ab 28 .8 4e f 6. 32 i 38 .0 2c -e y in gz ui 34 .7 0d -j 36 .3 0g -i 18 3. 76 di 76 .9 6f -h 33 1. 03 df 59 1. 75 ae 4. 89 ad 0. 32 b 19 .9 4j 4. 59 jk 29 .7 3f d iff er en t s up er sc ri pt le tte rs in di ca te s ig ni fic an t d iff er en ce s ac ro ss g en ot yp es a t p < 0 .0 1. 270 s. shi, x. ma, w. xu, y. zhou, h. wu, s. wang tab. 6: correlation coefficients between antioxidant content and antioxidant capacity determined by ferric reducing antioxidant power (frap; μm trolox), 2,2-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid) (abts; μm trolox), 2,2-diphenyl-1-picrylhydrazyl (dpph; μm trolox), metal chelating capacity (mcc; %), and superoxide radical scavenging activity (srsa; %). tpc tc frap mcc abts srsa dpph tpc 1 tc 0.030ns 1 frap 0.671** -0.336 ns 1 mcc 0.320 ns -0.245 ns 0.518** 1 abts 0.623** 0.036 ns 0.571** 0.454* 1 srsa -0.073 ns 0.004 ns 0.023 ns 0.067 ns 0.278 ns 1 dpph 0.696** -0.022 ns 0.491** 0.214 ns 0.524** -0.195 ns 1 note: tc: total carotenoids; tpc: total phenolics; ns: non-significant; *: level of significance (* p < 0.05, ** p < 0.001). tab. 5: antioxidant capacity in pulp extract of 28 mango genotypes determined by ferric reducing antioxidant power (frap; μm trolox), 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (abts; μm trolox), 2,2-diphenyl-1-picrylhydrazyl (dpph; μm trolox), metal chelating capacity (mcc; %), superoxide radical scavenging activity (srsa; %) and antioxidant index score (apc; %). genotype frap mcc abts srsa dpph apc index rank yuexi no. 1 37188.92b-d 23.83ab 1573.07a 26.67j-m 2419.36b-d 69.80 4 hongmang no. 8 15801.16l-n 20.33b-e 953.07e 69.17b-d 1008.69kl 45.21 21 saigon 23282.55h-k 21.25b-e 1342.44a-c 25.83i-m 1241.20f-h 47.52 20 irwin 11969.22no 22.58a-d 291.14f 19.17j-n 1823.78i-k 32.27 25 guangxi no. 4 14773.55m-o 1.83j 279.16f 11.67l-n 1682.79fg 13.23 26 mallika 22408.60i-l 14.42f 965.05e 93.33a 1281.51i-k 50.98 16 sijimang 35133.70c-f 10.75gh 1570.07a 66.67b-e 1287.71i-k 56.39 11 lilley 22206.92i-l 14.25fg 1303.50a-c 52.50e-h 1495.42g-i 48.92 18 carabao 41107.29bc 26.17a 1558.09a 15.00k-n 2318.53c-e 69.59 5 glenn 19681.11k-m 13.17fg 1189.69c-e 62.50c-e 1239.65i-k 45.11 23 nam dok mai 27143.30g-j 12.33fg 1570.07a 50.00e-h 1689.18f-h 54.57 13 tommy 9760.34no 2.50j 237.23f 9.17l-n 1388.65h-j 7.86 27 lianmang 53246.55a 21.42b-e 1555.10a 57.50c-g 2400.68b-d 80.55 2 aimang 31820.37c-f 23.17ab 1573.07a 83.33ab 2630.85bc 81.39 1 kensinton 29918.81e-h 21.92b-e 1258.58b-d 46.67e-i 2182.12de 62.74 7 lippens 8607.88o 8.67hi 258.19f 2.50n 827.10l 6.12 28 bambaroo 20420.61j-m 18.25e 1024.95de 65.00b-e 965.28kl 45.12 22 krs 25597.08g-k 20.58b-e 1195.68c-e 46.67e-i 1307.86i-k 50.86 17 xiaoji 29381.00e-i 13.17fg 1546.11a 49.17e-h 1990.38ef 58.41 9 edward 58298.18a 22.83a-c 1564.08a 5.83mn 2963.89a 77.85 3 zihua 36910.41b-d 23.17ab 1567.08a 28.33i-l 1535.72ef 61.22 8 guixiang 28900.80f-i 23.17ab 1201.67c-e 74.17a-c 1064.49a 58.13 10 jinshui 23023.25h-k 20.08b-e 1471.23ab 40.83f-i 1535.72g-i 54.38 14 renong no.1 23292.16h-k 18.92de 1357.42a-c 38.33g-j 1259.80j-l 48.72 19 vandyke 15205.72m-o 19.17c-e 1558.09a 15.00k-n 2346.44b-d 53.67 15 xiangjiao 36132.50b-e 18.83de 1570.07a 19.17j-n 2674.61ab 65.95 6 valencia pride 42682.32b 20.67b-e 446.89f 33.33h-k 807.17l 39.05 24 yingzui 23090.48h-k 6.00i 1390.36a-c 60.83c-f 2604.11bc 55.89 12 different superscript letters indicate significant differences across genotypes at p < 0.01. evaluation of mango germplasm 271 tab. 7: eigenvectors and percentages of accumulated contribution of principal components (pc). pc1 pc2 pc3 pc4 pc5 pc6 pc7 pc8 pc9 fruit weight -0.056 -0.601 -0.508 0.023 0.259 -0.207 -0.466 0.004 0.093 stone weight -0.014 -0.548 -0.314 -0.038 0.263 -0.058 -0.487 -0.280 -0.066 ph -0.548 0.378 -0.278 0.176 -0.155 -0.067 -0.203 0.097 -0.024 ssc -0.146 0.414 -0.498 -0.314 -0.190 0.353 -0.114 0.179 0.052 edible rate -0.172 -0.568 -0.391 0.177 -0.052 -0.069 -0.068 0.482 -0.011 carotene -0.259 0.099 0.601 0.074 0.076 0.227 -0.102 -0.058 0.559 fructose 0.731 -0.008 -0.146 -0.424 -0.435 -0.048 -0.045 0.109 0.069 glucose 0.889 -0.072 -0.287 -0.203 -0.139 -0.085 -0.014 0.114 0.143 surcose -0.762 0.370 -0.308 -0.182 0.196 -0.046 0.112 0.039 0.030 total sugar -0.142 0.405 -0.574 -0.515 -0.037 -0.118 0.105 0.153 0.136 ta 0.858 -0.298 0.090 -0.018 -0.149 0.071 0.034 0.072 -0.166 ssc/ta -0.709 0.428 -0.276 -0.086 0.088 0.175 0.080 0.215 -0.177 total phenolic 0.380 0.799 -0.092 0.007 -0.007 -0.161 -0.104 0.071 0.278 k -0.308 -0.333 -0.009 -0.105 0.020 -0.328 0.491 0.181 0.400 ca 0.003 0.023 0.598 -0.040 0.193 -0.418 -0.012 0.322 -0.311 mg 0.394 0.133 0.521 -0.131 0.314 0.257 -0.193 0.145 0.152 fe -0.156 0.517 -0.024 -0.339 0.059 -0.277 -0.096 -0.047 -0.503 mn 0.270 0.233 0.578 -0.151 0.312 -0.268 0.073 0.227 -0.195 zn 0.263 0.527 0.318 -0.088 0.302 -0.235 -0.253 0.233 0.238 cu -0.160 0.031 0.418 -0.484 -0.035 0.070 -0.455 -0.245 0.037 frap 0.375 0.541 -0.461 0.188 0.089 -0.294 -0.165 -0.240 -0.041 mcc 0.721 0.061 -0.150 0.446 -0.096 -0.048 0.044 -0.073 -0.031 abts 0.312 0.705 0.053 0.378 -0.035 0.161 -0.038 0.109 -0.069 srsa 0.256 0.121 -0.077 -0.301 0.328 0.665 0.017 0.138 -0.122 dpph 0.227 0.706 -0.069 0.196 -0.338 -0.188 -0.202 0.039 0.190 oxlic acid -0.271 0.514 0.311 0.071 -0.373 0.104 0.150 -0.431 -0.159 tartaric acid 0.046 0.203 -0.162 0.550 0.201 0.296 -0.149 0.378 -0.122 malic acid 0.312 0.243 -0.361 -0.002 0.647 -0.051 0.271 -0.299 0.106 citric acid 0.858 -0.210 -0.044 -0.222 -0.180 0.107 0.087 0.075 -0.139 total acid 0.643 0.193 -0.324 -0.014 0.466 0.060 0.265 -0.209 -0.007 eigen value 6.371 5.009 3.665 1.969 1.900 1.585 1.399 1.350 1.267 contribution rate (%) 21.238 16.698 12.217 6.562 6.333 5.284 4.664 4.501 4.224 cumulative percentage (%) 21.238 37.936 50.153 56.715 63.048 68.332 72.996 77.497 81.721 germplasm resources are valuable gene pools of resistance, performance, and functional constituents that used in genetic improvement programs. in the present study, we analyzed the physicochemical characters, antioxidant capacity, and mineral content of 28 mango genotypes. considerable variation was found in all measured traits among the genotypes. since all the genotypes were grown under the same environmental conditions and with the same standard conditions of irrigation, fertilization, and disease control, trait variation corresponded to the genetic diversity of the genotypes. the content and composition of sugars and acids in mango fruits was genotype-depended. sucrose and/or fructose were the dominant sugars, while malic and citric were the dominant organic acids in mango fruits. edward showed the highest content of total sugars and organic acid content, whereas vandyke and lilley had the lowest values, respectively. aimang had the highest total phenolic content, and zihua had the highest total carotenoid content. aimang and lianmang had significantly higher apc indices than the other mango genotypes. among all studied genotypes, valencia pride showed the highest fruit mass, edible ratio, and total sugar content, traits preferred by the processing industry. however, edward, aimang, and zihua were the most suitable genotypes for fresh consumption, due to their better biochemical properties and related health benefits. our work provided information on the physicochemical characters, antioxidant capacity, and mineral content of 28 mango genotypes. however, mango fruit quality is affected by many factors, such as geographic region, climate, soil characteristics, and cultivation techniques. therefore, further studies are needed to investigate the genotype by environment interactions and identify superior genotypes suitable for fresh consumption, processing, or breeding research. 272 s. shi, x. ma, w. xu, y. zhou, h. wu, s. wang acknowledgements this research was supported by the special fund for crop germplasm resources protection (2015nwb049), the agro-scientific research for the public interest (no. 201203092), and the fundamental research funds of the national nonprofit research institute for the south subtropical crops research institute, catas (1630062013003 and1630062014008). references aoac, 1995: official methods of analysis, 15th edn. association of official analytical chemists. arlington. benzie, i.f.f., strain, j.j., 1996: the ferric reducing ability of plasma (frap) 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(http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 90, 147 153 (2017), doi:10.5073/jabfq.2017.090.018 1ardahan university, faculty of engineering, food engineering department, ardahan, turkey 2 çukurova university, faculty of agriculture, department of horticulture, adana, turkey 3 i̇nönü university, faculty of agriculture, department of horticulture, malatya, turkey determination of s alleles in paviot × levent apricot progenies by pcr and controlled pollination zehra tugba murathan1*, salih kafkas2, bayram murat asma3 (received july 31, 2015) * corresponding author summary in this study, the sexual incompatibility of paviot and levent apricot parents and 89 f1 (paviot × levent) progenies was determined by self-pollination experiments and s-allele-specific polymerase chain reaction (pcr) technique. according to the self-pollination and isolation analyses under field conditions, it was found that the paviot genotype is self-compatible (sc), whereas the levent genotype is self-incompatible (si). it was determined that, of all the progenies, 55 had a fruit set below 5% and were self-incompatible, whereas 34 had a fruit set over 5% and were self-compatible. the pcr-based techniques showed that, in parallel to the data obtained from the field studies, 55 f1 progenies did not have sc allele, whereas 34 progenies involved sc allele. there were scs2 alleles in the paviot genotype and s20s52 alleles in the levent genotype. it was determined that there were s2s20, s2s52, scs20, and scs52 alleles in 89 f1 progenies and the distribution of the four alleles in the progenies was found to be as follows: 35.9% s2s20, 25.8% s2s52, 23.6% scs20, and 14.6% scs52. f1 progenies nos. 41, 46, 86, and 89 should be used as pollinators in further breeding studies. keywords: apricot, paviot, levent, self-incompatibility, pcr introduction apricot is one of the most important fruit types grown under mild temperature conditions in the world. it is a delicious fruit owing to its strong flavor and sugar-organic acid balance (gurrieri et al., 2001). some european and mediterranean countries such as turkey, spain, italy, france, and greece have many local types of apricots, and these countries contribute to more than 75% of the total apricot production in the world (leccese et al., 2010). apricot belongs to prunus species of the rosaceae family (ozbek, 1978). it was reported that there are two genes that control the gametophytic self-incompatibility in prunus species. one of these genes is s-ribonuclease (s-rnase) related to the stylus and the other one is the s-haplotype-specific f-box protein gene (sfb) related to pollen (kao and tsukamoto, 2004; qiao et al., 2004; mcclure, 2006). similar to the incompatibility, the functional capability of pollen is ascribed by a series of genes (s1, s2, s3, s4, … ,sn [multiple allele series]). the diploid stylus typically involves two different s genes, and each pollen grain carries one of the two genes. these gene regions code s-rnase protein, which makes the incompatible species to reject their own pollen (ebert et al., 1986; mcclure et al., 1989). the glycoproteins that have this ribonuclease activity define s specificity in the pistil. if the pistil has the same gene as that of the pollen, s-rnase shows a cytotoxic effect on the pollen tubes and prevents the growth of the tubes, which leads to incompatibility (roalson and mccubin, 2003; goldraij et al., 2006). in this case, either the tip of the pollen tube that progresses through the stylus swells or the tube end explodes (heslop-harrison, 1975). some physiological studies indicated that rna degrades within 12-45 h followed by an incompatible pollination (mcclure et al., 1990). the breeding experiments are divided into the following two main groups: conventional and biotechnological. the biotechnological breeding includes molecular marker-assisted selection and genetic transformation methods. this breeding yields the results more rapidly than the conventional one (bassi, 2006). in the apricot species obtained from north america and spain, initially, seven s alleles (s1-7) were defined by using molecular techniques (alburquerque et al., 2002). later, nine more alleles (s8-16) were defined through nephge and polymerase chain reaction (pcr) methods (halasz et al., 2005). the existing s alleles were then detected in the apricot species obtained from china, north america, europe, turkey, and tunisia (egea and burgos, 1996; halasz et al., 2005; zhang et al., 2008; milatovic et al., 2010; halasz et al., 2010; lachkar et al., 2013). the main objective of all breeding experiments is to improve pro ductivity. the productivity depends on the factors such as environ mental adaptability and self-incompatibility. most of the selfincompatible apricot varieties cannot be used in breeding programs as they result in irregular fruit set and require a pollinator (zhebentyayeva et al., 2012). therefore, it is very important to know the incompatibility among the species that are used as parents in breeding experiments. sexual incompatibility can be found in many commercially cultivated fruit species. in order to ensure the fruit set in these species, there is a necessity for cross-pollination by the wind or insects and pollinator species (badanes et al., 2000). the present study aimed to determine the sexual incompatibility and to reveal the heredity of sexual incompatibility in 89 f1 (paviot × levent) progenies by the field and laboratory experiments. materials and methods plant material the research materials were obtained from the apricot collection orchard affiliated to i̇nönü university. this area has a continental climate with latitude: altitude 977 m, 38˚20’20.23 n and longitude 38˚26’26.56 e. in this study, 89 f1 progenies (paviot × levent) were used. f1 progenies were obtained through the artificial pollination performed under the scope of the tubitak-togtag project in 2003. the hybridization was carried out to obtain the progenies having the desired characteristics such as paviot’s big fruits, orange color of fruit peel, and resistance against plum pox: levent’s late blooming features. the leaf samples of each plant were stored at 4 °c after lyophilization. fruit yield was determined as the mean fruit quantity of per apricot tree (kg/tree). for each genotype, weighting was done for every 10 fruits using a 0.05-g digital balance. the brix° degree of the fruit juice from 10 fruits was determined by digital refractometry (asma and ozturk, 2005). pollination tests in the pollination tests, conducted during 2009-2011, from each progeny, three different branches, each of which having approxi 148 z.t. murathan, s. kafkas, b.m. asma mately 300 flowers, were selected. the first branch was labeled and left to open pollination. the second branch was bagged using double-layered cheese cloth to prevent cross-pollination and to ensure self-pollination a week before the anthesis. the third branch was emasculated and left open, after that, they were artificially pollinated for two or three times with their own pollen, which had been collected and dried a day before. approximately after 70-80 days, the fruit set rates were determined. at the end of the isolation and artificial pollination, the progenies having the fruit set less than 5% were evaluated to be self-incompatible, and the others having more than 5% were considered self-compatible (faust, 1998). dna extraction, s allele pcr analysis, and dna sequencing for dna isolation from the leaf samples, the ctab (cetyl trimethyl ammonium bromide) protocol developed by doyle and doyle (1987) was used with minor modifications (kafkas and perltreves, 2001). the concentration of dna in the samples was determined by comparing with λ-dna that was quantified by the gel electrophoresis. to determine s alleles, pcr was conducted using the primer combinations designed for the first and second introns of s-rnase genes and developed by tao et al. (1999), romero et al. (2004) and vilanova et al. (2005) as listed in tab. 1. each pcr reaction in 25 μl contained 75 mm tris-hcl (ph 8.8), 20 mm (nh4)2so4, 2 mm mgcl2, 0.1% tween 20, 100 μm datp, 100 μm dttp, 100 μm dgtp, 100 μm dctp, 0.2 μm of each primer, 1.0 unit of taq dna polymerase, and 50 ng of dna. for pcr amplification, the samples were pre-denatured at 94 °c for 3 min, followed by 35 cycles with denaturation for 45 s at 94 °c, annealing for 45 s at 54 °c or 58 °c, and extension for 60 s at 72 °c. for the final extension step, the samples were kept at 72 °c for 10 min. the pcr products were separated by electrophoresis on a 2% or 3% agarose gel with 0.5× tbe (tris-borate-edta) depending on the band size and were visualized under uv light by staining after with ethidium bromide. at the same time, the amplification products were analyzed by capillary electrophoresis using an abi prism 3130xl automatic dna sequencer (applied biosystems). the dna sequencing of the pcr products was commercially performed following sanger’s method at medsantek, istanbul, turkey. the s alleles of the parents were determined by comparing the sequences using blast with those available at the national center for biotechnology information (ncbi) database. duncan’s test (1955) was used for the significance control (p < 0.05) following variance analysis (anova). results and discussion pollination tests the fruit set was 70% for the paviot genotype and 45% for the levent genotype left open to the pollination. the fruit set of 51% was observed in the paviot genotype, and no fruit set was found for the levent genotype left to closed pollination during the harvest period in 2009. similarly, in the self-pollination branches, the fruit set rate was detected to be 52% in the paviot genotype and 1.5% in the levent genotype. in the isolation and self-pollination experiments, the fruit set rate in the 56 f1 genotypes was below 5% (tab. 2). faust (1998) suggested that the verities having a fruit set rate less than 5%, where self-pollination has been conducted, can be defined as selfincompatible, whereas those having a fruit set more than 5% can be defined as self-compatible. asma (2008) conducted the isolation and self-pollination experiments and reported that the fruit set rate of the levent apricot genotype was below 5% and this genotype was self-incompatible. thus considering the results of our study, it can be affirmed that paviot genotype is self-compatible and the levent genotype is self-incompatible; 55 f1 progenies are self-incompatible while 34 are self-compatible (tab. 2). similar results were obtained from the field studies of different cultivars in recent years. askin (1989) reported that fruit set in tokaloglu and sam apricot cultivars that do not yield fruit regularly in the aegean region was 0.46% and 0.65%, respectively, and these species were self-incompatible. bolat and guleryuz (1994) reported that the fruit set rate was higher in the case of cross-pollination than self-pollination in hasanbey cultivars. paydas et al. (2001) determined that 25 of the 62 apricot cultivars cultivated in the malatya province were selfcompatible, while gulcan et al. (2006) determined that 32 of the 70 apricot genotypes cultivated in adana and malatya provinces were self-compatible. according to self-pollination studies conducted on katy, harcot, and jiguang hybrids by wu et al. (2011), fruit set rates were determined to be as follows: 19.68% for katy × harcot, 15.45% for harcot × katy, 7.78% for katy × jiguang, and 16.75% for jiguang × katy. in self-pollination studies of harcot and chuanzhihong cultivars, fruit set rates were 0.57% and 0%; these rates were 11.29% and 22.87% in cross-pollination experiments (harcot × chuanzhihong and chuanzhihong × harcot), and both cultivars were reported to be self-incompatible (gu et al., 2013). s allele pcr analyses and dna sequencing at the end of pcr studies conducted with the prut2, src-f, and src-r primer combinations for the amplification of the first intron region of the apricot s-rnase, a band of 353-bp was found in the paviot genotype (fig. 1). in previous studies, the cultivars that showed the 353-bp band were reported to be self-compatible when this primer combination was used (vilanova et al., 2005). in addition, a band of 328-bp in the paviot genotype and 420-bp in the levent genotype were found. in the pcr experiment, conducted with pruc2f and pruc4r primer combination for the amplification of the second intron region of s-rnase, no band was amplified for the paviot genotype, and two bands of approximately 1400 and 2100 bp were detected for the levent genotype. the analysis of the alleles included in f1 genotypes showed that the 420-bp band in the gel obtained through prut2srcf-srcr combination in the levent genotype was the same to the 1400-bp band found in pruc2f-c4r combination. the comparison of the nucleotide sequence obtained from the srcfsrcr primer combination in parents and the current apricot s allele sequences in the ncbi database indicated that s allele sequences of tab. 1: primers used to determine s-alleles of genotypes primers primer design base reference number src-r ggc cat tgt tgc aca aat tg 20 vilanova et al., 2005 src-f ctc gct ttc ctt gtt ctt gc 20 romero et al., 2004 prut2f gtt ctt gct ttt gct ttc ttc 21 tao et al., 1999 pruc4r gga tgt ggt acg att gaa 21 tao et al., gcg 1999 pruc2f ctt tgg cca agt aat tat tca 24 tao et al., aac 1999 statistical analysis the data are presented as means (n = 3) ±standard deviations (s.d.). all statistical analyses were performed using spss 15.0 software. self-incompatibility of apricot 149 tab. 2: mean comparison of fruit set percentage after open, isolated and self-pollination in f1 progenies progenies open isolated self progenies open isolated self pollination pollination pollination pollination pollination pollination (%) (%) (%) (%) (%) (%) paviot 70a 51a 52a p×l 45 16.9d 0.9d 2.5cd levent 45bc 0 0 p×l 46 24.2cd 40.5ab 34.6b p×l 01 30cd 0 0 p×l 47 47.5b 10.4c 10.9c p×l 02 25cd 12.2c 25.3b p×l 48 28.5cd 20bc 40.2ab p×l 03 52.7b 1.2d 0 p×l 49 15.5d 0 3.2cd p×l 04 50b 1.1d 0 p×l 50 17.4d 0 1.2d p×l 05 27.1cd 0 0 p×l 51 38.5bc 6.3cd 14.6bc p×l 06 30.7cd 23bc 17bc p×l 52 26.5cd 16bc 16.1bc p×l 07 20cd 0 0 p×l 53 32.1c 6cd 1d p×l 08 38c 3cd 2d p×l 54 52.2b 0 4.1cd p×l 09 45bc 0 2d p×l 55 25cd 12.3c 10.8cv p×l 10 13.9d 20bc 30b p×l 56 15.9d 0 0 p×l 11 25cd 25.9b 19.1bc p×l 57 39.6bc 12c 19.7bc p×l 12 36.9c 0 1.5d p×l 58 15.4d 14.2bc 40.6ab p×l 13 46.7bc 45ab 29.1b p×l 59 15d 1d 0 p×l 14 41.6bc 15.2bc 35ab p×l 60 21cd 0 0 p×l 15 34.5c 2.3cd 0 p×l 61 20.1cd 0 0 p×l 16 59.5ab 16.7bc 46.5ab p×l 62 14.5d 6.7cd 4.9cd p×l 17 30.4cd 2.7cd 1.6d p×l 63 11d 5.8cd 5cd p×l 18 7.4de 1.9d 0 p×l 64 11.8d 0 0 p×l 19 13.3d 17.5bc 24.4b p×l 65 14.1d 15.2bc 14.3bc p×l 20 37.1c 1.3d 0 p×l 66 28.9cd 0 0 p×l 21 31.4cd 0 0 p×l 67 21.6cd 0 0 p×l 22 26.7cd 1.5d 1.4d p×l 68 11d 0 0 p×l 23 30cd 1.5d 0 p×l 69 17.6d 0 0 p×l 24 42.6bc 6.9cd 17.5bc p×l 70 13.4d 5cd 6.7cd p×l 25 21.3cd 16.7bc 10c p×l 71 17.1d 6.2cd 6.4cd p×l 26 10.7d 16bc 14.7bc p×l 72 13.2d 0 0 p×l 27 20.8cd 13.8c 20.5bc p×l 73 9.9de 0 2.2cd p×l 28 50.6b 12.9c 16.5bc p×l 74 11.3d 1d 0 p×l 29 11.3d 0 1.1d p×l 75 10.9d 0 0 p×l 30 11.9d 3.2cd 1.7d p×l 76 7de 0 0 p×l 31 25cd 0 0 p×l 77 8.9de 0 0 p×l 32 22cd 0 1d p×l 78 12.1d 7.6c 6.5cd p×l 33 29.1cd 0 1.7d p×l 79 9.1de 0 3cd p×l 34 16.4d 5.8cd 6.7cd p×l 80 11.4d 1d 1d p×l 35 50b 0 0 p×l 81 15d 0 1d p×l 36 15.5d 0 0 p×l 82 9.9de 7.2c 6.6cd p×l 37 15.6d 9.4c 20.9bc p×l 83 10.1d 0 0 p×l 38 34.7c 0 0 p×l 84 12.5d 0 0 p×l 39 10.9d 4.7cd 4.4cd p×l 85 15.9d 0 0 p×l 40 36.2bc 20.5bc 19.9bc p×l 86 19.8cd 4.5cd 5.7cd p×l 41 54.3b 46.3ab 34.2b p×l 87 5.7e 0 0 p×l 42 41.7bc 0 0 p×l 88 12.3d 0 0 p×l 43 17.8d 1.1d 0 p×l 89 9.1de 7.2c 6.9cd p×l 44 34c 12.9c 11.8c data followed by different letters are significantly different from each other (p < 0.05) according to duncan’s test. 150 z.t. murathan, s. kafkas, b.m. asma paviot and levent genotypes show homology with the sc (353 bp), s2 (328 bp), s52 (1400 bp), and s20 (2100 bp) allele sequences of prunus armeniaca available at genbank (romero et al., 2004; vilanova et al., 2006; zhang et al., 2008; jiang et al., 2010). halasz et al. (2010) reported that there are s6s19 alleles in the levent genotype so this apricot genotype is self-incompatible. in the present study, the pcr bands obtained for the levent genotype were sequenced bidirectionally using the primers designed with the first and second intron regions and the obtained dna sequences were compared with the allele sequences available in genbank. at the end of this study, the presence of s20s52 allele was found in the levent geno type. similarly, yilmaz et al. (2013) reported that there was no sc allele in the levent genotype and this genotype was self-incompati ble. in a study conducted with 63 wild apricot genotypes in erzin can, it was reported that the local apricot cultivars cultivated in the eastern region of turkey mostly do not carry sc allele (halasz et al., 2013). in parallel with the results obtained under field conditions, it was found that 55 of the 89 f1 progenies did not carry the sc allele, and these progenies were self-incompatible (tab. 3). it was found that 55 f1 progenies carried s2s52 and s2s20 allele pairs, and these plants were self-incompatible. the distribution in f1 progenies of the alleles detected in paviot (scs2) and levent (s20s52) parents was as follows: 35.9% for s2s20, 25.8% for s2s52, 23.6% for scs20, and 14.6% for scs52. burgos et al. (1997) reported that self-compatibility alleles are dominant over incompatible alleles. in the present study, one fig. 1: s alleles determined through the use of pru t2, srcf and srcr primer combination in parents and f1 progenies tab. 3: s genotypes of paviot × levent f1 progenies progenies alleles progenies alleles progenies alleles progenies alleles paviot scs2 p×l 22 s2s20 p×l 45 s2s52 p×l 68 s2s20 levent s20s52 p×l 23 s2s52 p×l 46 scs20 p×l 69 s2s20 p×l 01 s2s52 p×l 24 scs20 p×l 47 scs52 p×l 70 scs20 p×l 02 scs20 p×l 25 scs52 p×l 48 scs20 p×l 71 scs20 p×l 03 s2s20 p×l 26 scs52 p×l 49 s2s52 p×l 72 s2s20 p×l 04 s2s20 p×l 27 scs52 p×l 50 s2s52 p×l 73 s2s20 p×l 05 s2s52 p×l 28 scs20 p×l 51 scs52 p×l 74 s2s20 p×l 06 scs52 p×l 29 s2s20 p×l 52 scs20 p×l 75 s2s52 p×l 07 s2s52 p×l 30 s2s20 p×l 53 s2s52 p×l 76 s2s20 p×l 08 s2s20 p×l 31 s2s20 p×l 54 s2s52 p×l 77 s2s20 p×l 09 s2s52 p×l 32 s2s20 p×l 55 scs20 p×l 78 scs20 p×l 10 scs52 p×l 33 s2s52 p×l 56 s2s20 p×l 79 s2s20 p×l 11 scs20 p×l 34 scs20 p×l 57 scs52 p×l 80 s2s20 p×l 12 s2s52 p×l 35 s2s52 p×l 58 scs20 p×l 81 s2s20 p×l 13 scs20 p×l 36 s2s20 p×l 59 s2s20 p×l 82 scs52 p×l 14 scs20 p×l 37 scs20 p×l 60 s2s20 p×l 83 s2s52 p×l 15 s2s20 p×l 38 s2s20 p×l 61 s2s52 p×l 84 s2s52 p×l 16 scs52 p×l 39 s2s52 p×l 62 s2s52 p×l 85 s2s20 p×l 17 s2s20 p×l 40 scs20 p×l 63 scs20 p×l 86 scs52 p×l 18 s2s20 p×l 41 scs20 p×l 64 s2s52 p×l 87 s2s20 p×l 19 scs52 p×l 42 s2s20 p×l 65 scs52 p×l 88 s2s52 p×l 20 s2s20 p×l 43 s2s52 p×l 66 s2s52 p×l 89 scs20 p×l 21 s2s20 p×l 44 scs20 p×l 67 s2s20 self-incompatibility of apricot 151 incompatible allele and one sc allele were found in 34 f1 progenies, but they were self-compatible; in other words, sc allele was found dominant over the incompatible allele. tab. 4 shows the pomological features of f1 progenies observed in 2011. f1 progenies nos. 2, 3, 15, 17, 18, 20, 21, 22, 23, 34, 41, 46, 51, 52, 53, 54, 56, 62, 63, 68, 69, 72, 83, 86, and 89 had high fruit yield. but the fruit weight and the total soluble solid content of some of these progenies were low. the genotypes that can be used in breeding experiments should be self-compatible and have good pomological properties. f1 progenies nos. 41, 46, 86, and 89 had both high fruit yield, fruit weight, and total soluble solid content and they were also found to be self-compatible. the f1 progeny no. 84 was self-incompatible although the quality was high in pomological characters. conclusion the sexual incompatibility of paviot and levent apricot parents and 89 f1 (paviot × levent) progenies was determined by self-pollination studies and s-allele-specific polymerase chain reaction (pcr). the fruit set rate was high as cross-pollination was allowed in the branches left open to pollination. no fruit set was found due to the incompatible fertilization in the isolated and self-pollinated branches in some progenies. of the progenies, 55 were determined to be selfincompatible. in conclusion, it is recommended that f1 progeny nos. 41, 46, 86, and 89 should be used as pollinators in further breeding experiments, as these progenies have high quality in pomological terms and they are self-compatible. the obtained results will be useful in the selection of parents in apricot breeding studies and these results will be useful for the selection of genotype in new apricot orchards. tab. 4: fruit characteristics of f1 genotypes profruit kernel brix° profruit kernel brix° profruit kernel brix° genies weight weight (%) genies weight weight (%) genies weight weight (%) (g) (g) (g) (g) (g) (g) p×l 01 27.6± 2.5c 3.1± 0.2b 16.0± 1.1b p×l 31 31.6± 2.4bc 3.3± 0.2b 18.0± 1.0a p×l 61 24.9± 3.1c 3.3± 0.2b 18.5± 1.2ab p×l 02* 23.1± 2.2c 2.6± 0.2b 16.0± 1.0b p×l 32 18.2± 1.9d 2.4± 0.2b 16.0± 1.4b p×l 62* 24.9± 3.1c 3.3± 0.2b 18.5± 1.2ab p×l 03* 24.0± 2.8c 2.2± 0.2b 18.0± 0.9ab p×l 33 33.5± 2.4bc 3.5± 0.3b 17.0± 1.0b p×l 63* 29.9± 2.5bc 2.8± 0.2b 14.0± 0.5c p×l 04 30.2± 2.3bc 4.3± 0.3a 18.0± 0.6ab p×l 34* 39.4± 2.7b 3.4± 0.2b 18.0± 1.2ab p×l 64 22.9± 2.0c 2.7± 0.2b 23.0± 1.2a p×l 05 44.2± 4.5b 3.3± 0.3b 19.0± 1.2a p×l 35 31.5± 2.8bc 3.0± 0.3b 18.0± 1.4ab p×l 65 35.7± 2.9bc 4.1± 0.3a 20.0± 1.5a p×l 06 20.0± 2.0c 2.7± 0.2b 18.0± 1.5ab p×l 36 28.3± 2.1c 3.0± 0.2b 18.0± 1.1ab p×l 66 26.9± 2.2c 2.9±0.3b 19.0± 1.3a p×l 07 18.8± 1.6d 3.4± 0.3b 18.0± 1.2ab p×l 37 44.9± 2.8b 4.1± 0.3a 21.0± 0.8a p×l 67 34.5± 2.5bc 3.2± 0.3b 15.0± 1.1b p×l 08 25.4± 2.3c 3.1± 0.2b 21.0± 1.1a p×l 38 35.8± 2.2bc 3.5± 0.2b 20.0± 0.6a p×l 68* 24.9± 2.6c 2.8±0.4b 16.0± 1.1b p×l 09 24.8± 2.1c 2.5± 0.3b 19.0± 1.3a p×l 39 32.7± 2.1bc 3.7± 0.2ab 19.0± 0.9a p×l 69* 18.1± 1.8d 2.4±0.6b 12.0± 1.0d p×l 10 20.2± 2.0c 2.4± 0.1b 18.0± 1.1ab p×l 40 18.4± 1.9d 2.6± 0.2b 14.0± 0.5c p×l 70 39.3± 3.9bc 3.8± 0.3ab 21.0± 1.1a p×l 11 49.8± 3.7b 3.4± 0.1b 20.0± 1.4a p×l 41** 51.8± 2.2ab 4.3± 0.4a 20.0± 0.8a p×l 71 40.1± 3.0b 3.8± 0.3ab 18.0± 1.0ab p×l 12 22.3± 2.5c 2.1± 0.1b 22.0± 1.2a p×l 42 40.6± 2.4b 2.5± 0.1b 19.0± 0.6a p×l 72* 20.6± 1.5c 2.3± 0.2b 16.0± 1.0b p×l 13 21.5± 3.1c 2.6± 0.1b 14.0± 0.6c p×l 43 30.4± 2.1bc 3.1± 0.2b 13.0± 0.5c p×l 73 27.4± 2.6c 2.8± 0.2b 18.0± 1.1ab p×l 14 23.5± 2.4c 2.1± 0.1b 16.0± 0.7b p×l 44 39.4± 3.9b 3.0± 0.2b 19.0± 0.9a p×l 74 47.3± 3.8b 4.3± 0.3a 19.0± 1.0a p×l 15* 30.6± 2.8bc 3.9± 0.3ab 20.0± 1.3a p×l 45 36.9± 2.5bc 3.1± 0.1b 20.0± 0.6a p×l 75 23.6± 2.5c 2.7± 0.2b 17.0± 1.4b p×l 16 18.1± 1.9d 3.0± 0.2b 21.0± 1.2a p×l 46* 59.3± 4.8ab 2.5± 0.2b 18.0± 0.8ab p×l 76 33.6± 3.7bc 3.4± 0.3b 17.0± 1.5b p×l 17** 33.5± 2.5bc 3.8± 0.3ab 22.0± 1.4a p×l 47 27.8± 2.9c 3.2± 0.2b 18.0± 0.5ab p×l 77 25.7± 2.5c 2.1± 0.2b 15.0± 1.3b p×l 18* 31.1± 2.3bc 3.2± 0.2b 17.0± 1.4b p×l 48 70.5± 4.7a 4.6± 0.3a 14.0± 0.6c p×l 78 21.2± 2.6c 2.6± 0.2b 16.0± 0.8b p×l 19 42.6± 3.6b 3.7± 0.2ab 22.0± 1.2a p×l 49 32.9± 2.5bc 3.7± 0.2ab 16.5± 0.8bc p×l 79 23.6± 2.2c 2.8± 0.2b 18.0± 0.6ab p×l 20* 30.5± 2.9bc 3.5± 0.2b 19.0± 1.1a p×l 50 31.4± 2.2bc 3.2± 0.2b 19.0± 1.5a p×l 80 34.8± 2.1bc 3.0± 0.2b 17.5± 0.9ab p×l 21* 34.8± 2.4bc 3.4± 0.2b 22.0± 1.0a p×l 51* 21.6± 2.1c 2.9± 0.2b 16.0± 0.6b p×l 81 45.6± 4.9b 4.3± 0.3a 18.0± 1.0ab p×l 22* 22.2± 2.1c 2.5± 0.1b 19.0± 1.5a p×l 52* 23.5± 2.9c 3.4± 0.2b 17.0± 0.5b p×l 82 30.6± 2.8bc 2.5± 0.1b 16.0± 1.2b p×l 23* 14.6± 1.9d 1.6± 0.1c 20.0± 1.5a p×l 53* 40.3± 2.1b 3.4± 0.3b 22.0± 0.9a p×l 83* 65.0± 5.5a 4.6± 0.3a 14.0± 1.2c p×l 24 35.7± 2.5bc 3.9± 0.2ab 17.5± 1.1ab p×l 54* 19.0± 1.4d 2.1± 0.1b 19.0± 0.9a p×l 84 59.0± 4.9ab 4.4± 0.3a 19.0± 1.1a p×l 25 29.4± 1.4bc 2.7± 0.2b 18.0± 1.4ab p×l 55 24.1± 1.6c 2.1± 0.1b 17.0± 0.5b p×l 85 37.0± 2.5bc 3.4± 0.2b 16.0± 1.0b p×l 26 40.3± 3.8b 3.9± 0.2ab 17.0± 1.6b p×l 56* 48.2± 1.5b 3.0± 0.1b 20.0± 1.1a p×l 86* 62.0± 5.5a 4.5± 0.3a 20.0± 1.2a p×l 27 32.9± 2.3bc 2.9± 0.2b 17.0± 1.2b p×l 57 46.3± 3.6b 3.9± 0.1ab 19.0± 1.0a p×l 87 43.0± 5.2b 3.8± 0.2ab 15.0± 0.8b p×l 28 38.1± 3.6bc 3.9± 0.2ab 19.0± 1.5a p×l 58 34.0± 3.2bc 3.3± 0.3b 15.0± 1.4b p×l 88 33.3± 2.6bc 3.3± 0.2b 16.0± 0.7b p×l 29 18.1± 1.8d 2.2± 0.2b 21.0± 1.5a p×l 59 31.6± 3.5bc 3.0± 0.3b 16.5± 1.2b p×l 89** 55.7± 4.5ab 4.2± 0.3a 19.0± 1.8a p×l 30 39.1± 2.6b 4.1± 0.3a 23.0± 1.6a p×l 60 23.8± 3.2c 2.2± 0.1b 20.0± 1.5a *: high yield; **: very high yield values are means ± standard deviation (sd) of three replications. data followed by different letters are significantly different from each other (p < 0.05) according to duncan’s test. 152 z.t. murathan, s. kafkas, b.m. asma acknowledgments this research was supported by a grant (no. 2010/12) from i̇nönü university scientific research project unit. references alburquerque, n., egea, j., pérez-tornero, o., burgos, l., 2002: genotyping apricot cultivars for self-(in)compatibility by means of rnases associated with s aleles. plant breeding 121, 343-347. doi: 10.1046/j.1439-0523.2002.725292.x. askin, a., 1989: biological studies in some apricot fruit varieties that are not regularly fruit set in the aegean region, phd thesis, ege university, izmir/turkey. asma, b.m., 2008: determination of pollen viability, germination ratios and 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(eds.), fruit breeding, handbook of plant breeding, springer, berlin, heidelberg. © the author(s) 2017. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). address of the authors: zehra tugba murathan, ardahan university, faculty of engineering, food engineering department, ardahan, turkey e-mail: ztugbaabaci@hotmail.com salih kafkas, çukurova university, faculty of agriculture, department of horticulture, adana, turkey bayram murat asma, i̇nönü university, faculty of agriculture, department of horticulture, malatya, turkey journal of applied botany and food quality 91, 155 162 (2018), doi:10.5073/jabfq.2018.091.021 1 school of biology and basic medical sciences, soochow university, suzhou, china 2 college of food science and nutritional engineering, china agricultural university; national engineering research center for fruit & vegetable processing; key laboratory of fruit & vegetable processing, ministry of agriculture, beijing, china characterization of physico-chemical and bio-chemical compositions of selected four strawberry cultivars xiamin cao1*, yongtao wang2, xiaojun liao2, xiaosong hu2 (submitted: july 24, 2017 accepted: may 11, 2018) * corresponding author summary the physico-chemical and bio-chemical compositions of ‘hongyan’, ‘tiangxiang’, ‘tongzi ι’ and ‘zhangji’ strawberries in china were analyzed. their values were ph 3.42 to 3.73, titratable acidity 0.63 to 0.79 g/100 g fw, total soluble sugars 5.26 to 8.95 g/100 g fresh weight (fw), ascorbic acid 21.38 to 42.89 mg/100 g fw, total phenolics 235.12 to 444.73 mg/100 g fw, pectin 82.84 to 96.13 mg/ 100 g fw, total organic acids 874.30 to 1216.27 mg/100 g fw, individual phenolics other than anthocyanins 7.60 to 12.18 mg/ 100 g fw, free amino acids 13.35 to 32.66 mg/100 g fw, monomeric anthocyanins 4.47 to 47.19 mg/100 g fw, antioxidant capacity of dpph 14.14 to 18.87 and frap 7.97 to 10.54 equal to mg/100 g ascorbic acid, polyphenol oxidase (ppo) activity 0 to 0.42 abs/min, peroxidase (pod) activity 0.17 to 0.34 abs/min and pectin methyl esterase (pme) activity 0.012 to 0.018 ml/min. ‘tongzi ι’ was most suitable for food processing due to the highest titration acidity, total phenolics, pectin, total organic acids, monomeric anthocyanins, antioxidant capacity of ·dpph and frap with lower ppo, pod and pme activity. introduction strawberry which is cultivated almost all over the world is a soft and juicy, sweet-sour and palatable berry with bright-red color, and also an important commercial fruit with good processing potential. the common quality characteristics of strawberries for consumer acceptance are appearance (uniform bright red color, size and shape), firmness, and flavor perceived by the combination of taste and smell (aroma) senses (gunnessa, kravchukb, nottinghamc, d’arcyb and gidleya, 2009). the attractive color of strawberry is mainly due to the presence of abundant anthocyanins. polyphenol oxidase (ppo) and peroxidase (pod) are demonstrated to be the color-related enzymes and play a key role in color degradation due to enzymatic browning and decreasing anthocyanins. pectin is a criti cal structure component of the cell wall (riahi and ramaswamy, 2013), which is easy to be de-esterified by pectin methyl esterase (pme) and converted into low-methoxy pectin or pectin acid, thereby influence the texture and firmness of strawberry. the sugars (fructose, glucose and sucrose) (skupień and oszmiański, 2004) and organic acids (watson, wright, mcburney, taylor and linforth, 2002) give strawberry its characteristic taste, while more than 360 volatile compounds distinguish its aroma (gunnessa et al., 2009). according to häkkinen et al. (2000), strawberries exhibit high antioxidant capacity compared to other fruits due to its high content of phenolic compounds. ascorbic acid is another plentiful component in strawberry, which is known as a powerful reducing agent that plays a key role in human nutrition. antioxidant capacity of strawberry can participate in the prevention of cancer, cardiovascular and other chronic diseases (oszmiajski and wojdyio, 2009). at present, foods are assumed the status of being “functional”, that is they should be capable of providing additional physiological benefit, such as preventing or delaying onset of chronic diseases, as well as meeting basic nutritional requirements (skupień et al., 2004), so strawberry will be one of the most popular summer fruits, consumed fresh, conserved or in manufactured products along with the unique, highly desirable taste and flavor. in the late 1980s, strawberry cultivars were introduced into china and great efforts were made to extend the use of strawberries, which accelerated the development of strawberry industry. according to the statistical data by the ministry of agriculture, china, the total planting area of strawberry in china increased up to 20 million hectares and the strawberry output totaled 2.442 million tons in 2015. in china, some strawberry cultivars come from european countries, usa and japan, but most of the cultivars are self-bred. in general, the cultivars are classified into two categories for fresh consumption and food processing. cultivars with large fruits, bright red color, good taste, moderately sweet/sour ratio and abundant aromatic compounds are suitable for fresh consumption, while high ascorbic acid and anthocyanins contents, fleshy hard and great acidity are in flavor of food processing. though the planting areas are booming and the outputs of strawberry in china are increasing year by year, there is still lack of evaluation of the physico-chemical and biochemical compositions closely related to the quality of strawberries. the aim of the present work is to analyze and to compare physico-chemical and bio-chemical compositions of four strawberry cultivars in china, hoping to give useful information for the best use of the strawberry cultivars, for both technological research and processing practice. materials and methods plant materials strawberries (‘hongyan’, ‘tiangxiang’, ‘tongzi i’, ‘zhangji’) grown tianyi bio-engineering company in changping county (beijing, china) were harvested at commercial maturity in march 18th, 2015. the berries were individually frozen by liquid nitrogen and packed in aluminum foil bags, then stored at -80 °c until analyzed within 4 months. weight of individual fruits average weight of individual fresh fruits was determined with a scale (ey-300a, panasonic, japan). 100 individual strawberries were used for average. measurement of the ph values the ph value measurement was carried out by an orion 868 ph meter (thermo orion, usa) with a combined ph electrode at 25 °c. the meter was calibrated with commercial buffer solutions at ph 6.8 and 4.0. extraction and determination of pectin for extracting and determination of soluble pectin, the method described by peng et al. (2008) was used. 5 g strawberry puree was mixed with 100 ml 95% ethanol and cooked in boiling water for 156 x. cao, y. wang, x. liao, x. hu 30 min, this process was repeated for 3 times. the mixture was filtered using a bush funnel by vacuum, the residues were collected and mixed with 20 ml water and heated in 50 °c for 30 min to dissolve soluble pectin, then filtered using quantitative filter paper (xinxing 202, hangzhou special paper co. ltd.). the residues were washed 3 times again by distill water, the filtrate was transferred into a flask and was made up to 50 ml by adding distill water. 6 ml 0.0125 mol/l sodium tetraborate (dissolved in h2so4) and 1 ml galacturonic acid solution or sample (diluted 10 times with water) were added into a glass tube with plug, and cooled in an ice cold bath. the cooled mixture was boiled for 5 min in boiling water bath and then cooled again immediately. 0.1 ml 0.15% n-phenylphenol (dissolved in 0.5% naoh) was added into the above-mentioned tube and was mixed by shaking for 30 s on an oscillator. after 5 min reaction, samples were evaluated at 520 nm by spectrophotometer (uv-726, shanghai precision & scientific instrument co. ltd., shanghai, china). results were expressed as milligrams of galac turonic acid per 100 g of fw. extraction and hplc analysis of organic acids the organic acids in strawberry were extracted as described in an earlier study with some modifications (liu et al., 2014). 50 g straw berry purees were mixed with 100 ml distilled water and heated at 50 °c for 30 min, then centrifuged using 12000 rpm/min for 15 min at 4 °c, and filtered through two-layer cheese-cloth. the supernatant was collected. the analytical column was an alltech alltimatm c18, (4.6×250 mm i.d, 5 μm particle size) from waters. the organic acids were separated by the methods described by gao et al (2014). the mobile phase was an isocratic solvent system consisting of 97% of 0.01 mol/l dipotassium phosphate (ph=2.55) and 3% methanol, the flow rate was 0.5 ml/min at 30 °c. the detection was carried out at 210 nm in absorbance mode. results were expressed as milligrams per 100 g of fw. determination of titratable acidity the titratable acidity (ta) was determined via titration with 0.1 m naoh, set to reach ph 8.1 by an automatic titrimeter (751 gpd titrino, metrohm, switzerland). ta value was analyzed in triplicate and expressed as citric acid equivalents, since citric acid is the predominant acid found in strawberries (watson et al., 2002). the ta of strawberry was calculated using the following formula. where, c was naoh concentration (0.1 m), m was the weight of strawberry for extraction (50 g), v2 was the volume of naoh used (ml), v1 was the volume of sample used (50 ml organic acid extracts), v0 was the volume of extracts (ml), k was conversion factor of citric acid (0.07). the result was expressed as g/100 g of citric acid equivalent fw. extraction and hplc analysis of sugar the sugars in strawberry were extracted by the same method as organic acids. the sugars were detected by the method described by wang et al. (2006) and were performed on asahipak nh2p-50 4e (4.6×250 mm i.d, 5 μm particle size) equipped with asahipak nh2p-50g 4a (4.6×10 mm i.d, 5 μm particle size) as guard column. 50 mg/l calcium disodium salt of ethylene diamine tetraacetic acid (edta) was used as mobile phase for separating sucrose, glucose and fructose. in the separation systems, the solvent flow rate was 0.5 ml/min at 90 °c and aliquots of 60 μl were injected. it was detected by a differential refraction detector (rid-2301, knauer co. ltd., german). the sugars were expressed as grams per 100 g of fw. extraction and hplc analysis of ascorbic acid for extracting and analyzing ascorbic acid in strawberry, the me thod of tiwari et al. (2009) were used with some modifications. 50 g strawberry purees were mixed with 250 ml 2.5% metaphos phoric acid and incubated at 4 °c for 2 h, then centrifuged using 12000 rpm/min for 15 min at 4 °c, and filtered through two-layer cheese-cloth, the supernatant was collected. the ascorbic acids were performed on the same column as organic acids. the mobile phase was an isocratic solvent system consisting of 95% of 50 mm monopotassium phosphate (ph=3.0) and 5% acetonitrile, the flow rate being 1 ml/min. the analyses were conducted at ambient temperature. the detection was carried out at 245 nm in absorbance mode. results were expressed as milligrams per 100 g of fw. extraction and hplc analysis of monomeric anthocyanins 250 g frozen strawberries were homogenized with a juice extractor (jy-610, joyoung co., ltd. shandong, china) to obtain strawberry purees for further analysis. anthocyanin was extracted as earlier described by cao et al. (2012) with some modifications. 50 g straw berry purees were mixed with 100 ml 0.1% hcl in methanol and kept 30 min at 4 °c, then centrifuged at 12000 rpm/min for 10 min at 4 °c (hitachi himac cr21g, japan), the insoluble plant material was re-extracted with 100 ml solvent. the supernatants were col lected and mixed for hplc analysis. chromatographic separation was performed on a venusil c18 column (250 mm×4.6 mm i.d, 5 μm particle size) equipped with a 5 μm c18 guard column both from agela (usa). the separation of anthocyanins was as described by garc et al. (1997) with mobile phases consisting of (a) formic acid/ water (5:95, v: v) and (b) methanol. the detection was carried out at 520 nm for anthocyanins at 30 °c with the flow rate at 1 ml/min. anthocyanins were dissolved in 0.1% hcl. results were expressed as milligrams per 100 g of fw. extraction and hplc analysis of individual phenolic com pounds other than anthocyanins the extraction of soluble individual phenolic compounds other than anthocyanins was presented in a previous study with some modi fications (cao et al., 2011). 50 g strawberry purees were mixed with 100 ml ethyl acetate and stirred with a magnetic stirrer for 30 min, and the ethyl acetate phase was collected. the extractions were performed by repeated vigorous vortexing of samples with ethyl acetate (3×100 ml). the combined ethyl acetate extracts were evaporated to dryness at 40 °c with a rotary evaporator (sencq r-501, shenshun biotechnology co., shanghai, china), then dis solved in 10 ml methanol. the separation of individual phenolic compounds other than anthocyanins was as described by cao et al. (2011) with some modifications. the mobile phases were (a) formic acid/acetonitrile (2.5:97.5, v: v) and (b) formic acid/water (2.5:97.5, v: v). the detection was carried out at 280 nm and 320 nm for phenolic compounds in absorbance mode with the flow rate being 1 ml/min at 30 °c, and aliquots of 20 μl were injected. the gra dient elution was as follows: 0 to 5 min, 5% a; 5 to 15 min, 5-13% a; 15-20 min, 13% to 30% a; 20 to 25 min, 30% a; 25 to 28 min, 30% to 45% a; 28 to 32 min, 45% b; 32 to 35 min, 45% to 90% b; 35 to 40 min, 90% b; 40 to 45 min, 90% to 5% b. results were expressed as milligrams per 100 g of fw. determination of total phenolics total phenolics were determined using folin-ciocalteau method described by cao et al. (2012) with some modifications. 125 μl 100-folds diluted phenolic compounds extracts were mixed with 2 ml folin-ciocalteau reagent (previously diluted 10-fold with distilled water) and set 1 hour at room temperature away from light. c × v2 × k v0 ta(%) = × × 2 × 100% v1 m composition of four selected strawberry cultivars 157 then 1.8 ml of sodium carbonate (105.99 g/mol) were added to the mixture and reacted for 15 min, the mixture was measured at 765 nm by spectrophotometer (uv-726 shimadzu, shanghai, china), immediately. results were expressed as mg gallic acid/100 g fw. extraction and hplc analysis of faas the analysis of free amino acids (faas) in strawberry were extrac ted using the method as earlier described. 50 g strawberry purees were mixed with 100 ml of solvent (0.1% hcl in 95% ethanol) and stirred with a magnetic stirrer for 30 min and then centrifugation (12000 rpm/min for 15 min at 4 °c), filtered and collected super natant, the insoluble plant material was re-extracted two times with 0.1% hcl in 80% ethanol. the residual ethanol was removed in a rotary evaporator at 40 °c. the volume of the extracts was accurately recorded. chromatographic analysis method of faa was proposed by agela, it was performed on a venusil-aa column (4.6×250 mm i.d, 5 μm particle size) equipped with a c18 guard column both from agela, usa. the standards of 20 kinds of aas and norleucine (nle) were dissolved in hcl (0.1 mol/l). for derivatisation, 200 μl standards or samples were mixed with 20 μl nle, 100 μl 1 mol/l triethylamine/acetonitrile (1.4:8.6, v/v), 100 μl 0.1 mol/l pitc/ acetonitrile (1:80, v/v) and shaking for 30 s on an oscillator, after 1 h reaction away from light, 400 μl n-hexane was added and shook for 30 s, after 10 min setting, the underlayer solution was obtained for analysis. the mobile phase was consisted of (a) 0.1 mol/l sodium acetate (ph=6.5)/acetonitrile (93:7, v/v), (b) 80% acetonitrile (diluted with water), the flow rate was 1 ml/min at 40 °c with detected at 254 nm. results were expressed as milligrams per 100 g of fw. the sugars and organic acids were separated using a knauer hplc system (knauer co. ltd., german) equipped with a pump (k1001) connected a refractive index detector (rid, k-2301) or uv detector (k-2501) and a 20 μl loop. ascorbic acid, anthocyanins, phenolic compounds and free amino acids were separated using a shimadzu liquid chromatograph (shimadzu co. japan) equipped with a prominence uv/vis detector (spd-20av), an auto sampler (sil-20a) and a column oven (cto-20a). prior to injection, all the samples were filtered through a 0.45-mm millipore membrane filter. quantification was performed based on external standards of known concentrations. determination of antioxidant activity the antioxidant activity was studied through the evaluation of the free-radical scavenging-effect on dpph radical according to procedure described by odriozola-serrano et al. (2009) and ferric reducing/antioxidant power (frap) according to the method developed by benzie et al. (1996). dpph assay the reaction started by adding 100 μl vc solution or the diluted strawberry juice to the cuvette containing 4 ml 0.14 mol/l methanol solution of the free radical (dpph). the mixture was set away from light for 15 min, and then the absorbance was measured at 765 nm with a uv-726 spectrophotometer (shanghai precision & scientific instrument co. ltd., shanghai, china). frap assay the freshly prepared ferric ion reducing antioxidant power (frap) solution contained 25 ml 0.3 mol/l acetate buffer (ph 3.6) plus 2.5 ml 10 mmol/l tripyridyltriazine(tptz) (dissolved in 40 mmol/l hcl) and 2.5 ml 20 mmol/l ferric chloride (fecl3·6h2o). 4 ml frap solution at 37 °c was mixed with 100 μl ascorbic acid solution or diluted strawberry juice. ten minutes later, the ferric reducing ability of strawberry juice was measured by monitoring the increase of absorbance at 593 nm with an uv-726 spectrophotometer (shanghai precision & scientific instrument co. ltd., shanghai, china) and the frap solution was used as blank. determination of enzyme activity extraction and determination of ppo and pod the extraction of ppo and pod was described by garcia-palazon et al. (2004). 100 g strawberries were mixed with 20 ml extraction solution and 4% polyvinyl polyvinylpyrrolidone (pvpp) and pulped for 2 min, then extracted for 4 h at 4 °c. the mixture was centrifuged at 12000 rpm/min for 10 min at 4 °c, and the supernatant was collected and analyzed for enzyme activity. the extraction solution was 0.2 m phosphate buffer (ph=6.5) for ppo and pod. ppo and pod activity were determined by a spectrophotometric method (garcia-palazon et al., 2004) with some modifications. the reaction mixture for ppo was 0.5 ml extract and 2.5 ml 0.07 m catechol (o-diphenol) in 0.5 m sodium phosphate buffer (ph=6.5) solution. the reaction mixture for pod contained 2.2 ml 1.0% (v/v) guaiacol (dissolved in 0.2 m phosphate buffer, ph 6.5), 0.2 ml of 1.5% h2o2, and 0.8 ml extract. the increase in absorbance at 420 nm for ppo and 470 nm for pod, respectively, was monitored at intervals of 0.1 s-1 immediately after incubation with a cary 50 spectrophotometer (varian co. ltd., california, usa), which was equipped with a peltier thermostatted cell holder, a water pump (varian co. ltd., california, usa) to keep temperature at 30±0.1 °c and an in-built electromagnetic stirring to mix up the substrate and the extracts. prior to measurement, a preequilibrium at 30 °c of the substrate solution as well as the extracts by the peltier thermostatted cell holder was obtained. the slope of the very first linear part of the reaction curve was taken as ppo and pod absolute activity (abs/min). extraction and determination of pme pme was extracts by a previous method described by zhi et al., 2008. the composition of the extraction buffer was as follows: 0.2 m tris-6 m hcl buffer (ph 8.0) (containing 0.1 m nacl). the ratio of buffer volume (ml) to strawberry puree (g) was 2:1.the mixture was incubated at 4 °c for 12 h, then centrifuged using 12000 rpm/min at 4 °c for 15 min, filtered and collected supernatant as enzyme extracts for further analysis. the pme activity was detected by ph automatic potentiometric titrator (751 gpd titrino, metrohm, switzerland). 5 ml pme extracts were added into 40 ml 0.1 g/l pectin (ph=7.5), and automatically added 0.05 m naoh to keep the ph value at 7.5, the reaction temperature was 30 °c. the volume of naoh was recorded every 30 s. slope of liner (volume-time) was obtained for calculating the pme activity using the formula. statistical analysis all the experiments were performed in triplicate. the data were analyzed using the statistical program for social sciences (spss 12.0) software for analysis of variance, duncan’s test. the significance was established at p ≤ 0.05. results and discussion basic physico-chemical characteristics as shown in tab. 1, the average weight of individual fruits of ‘hongyan’ berries was the highest and of ‘tianxiang’ berries was the smallest. sugar content is an important taste attribute for straw slope × cnaohpme activity = vsample 158 x. cao, y. wang, x. liao, x. hu berries and is highly correlated with consumer acceptance (crespo et al., 2010). ‘hongyan’ berries were the richest in sugars and had approximate 1.5 times reducing sugars as ‘tongzi i’ and ‘zhangji’. skupień et al. (2004) determined similar reducing sugars content (5.74~9.09 g/100 g fw) in different strawberry cultivars. however, the sucrose in this cultivars was remarkable lower than 3.16~7.25 g/ 100 g fw as was reported by skupień et al. (2004). this great dif ference resulted from various cultivars, seasons, origins, agricultural practices, as well as the differences in the extraction procedures used between this and other studies; the greater solubility of sucrose in methanol as compared to fructose and glucose has already been pointed out by others (crespo et al., 2010; giné et al., 2009). apart from the sugars, acids within the fruit are part of the soluble solids pool and are also important contributors to strawberry taste and flavor (crespo et al., 2010). there were significant differences of ph and ta among the four cultivars. the ph value was 3.42~3.73 in the four cultivars strawberries. ‘tongzi i’ had the highest ta content (0.79 g citric acid/100 g) and ‘zhangji’ the lowest (0.63 g citric acid/100 g). the ta content of the cultivars studied was in concurrent with the content of total organic acids in tab. 3. the ratio of total soluble solids/titration acidity is related to flavor quality for various fruits, especially for strawberry flavor and determines the optimum time for strawberry harvesting, because it is considered to be an index of quality. however, perkins-veazie et al. (1995) reported that the ratio of total soluble sugars to organic acid is a major parameter of strawberry taste and may be more important for quality and perceived sweetness by a sensory panel than total soluble solids alone. ‘hongyan’ had the highest total soluble sugars/titration acidity ratio than other cultivars. the ascorbic acid is also regarded as an important health-related indicator of fruit quality and its content in fruits causes by a balance between synthesis and degradation. the in situ synthesis occurs in strawberries from d-galacturonic acid, a component of cell wall pectins during fruit ripening and the final concentration in the fruits depends on the expression of the gene galur as well as the availability of the substrate d-galacturonic acid (agius et al., 2003). in the present study, the content of ascorbic acid ranged from 21.38 mg/100 g in ‘zhangji’ to 42.89 mg/100 g in ‘hongyan’. masny et al. (1999) estimated similar levels of ascorbic acid in different strawberry cultivars ranging from 25.7 to 53.2 mg/100 g, but skupień et al. (2004) reported a higher level ranging from 54 to 87 mg/100 g. the ascorbic acid in ‘hongyan’ is significantly higher (up to 2-folds) compared to ‘zhangji’, suggesting advantages of the former in terms of better resistance to oxidizing agents during processing. the total phenolics content in this study was from 235.12 mg/100 g in ‘zhangji’ to 444.73 in ‘tongzi i’, which was similar to 230~ 340 mg/100 g as reported by aaby et al. (2005), the difference in total phenolics in the studied cultivars could be attributed to the wide range of anthocyanins and other phenolic compounds content as discussed in the following. pectin ranged from 82.84 to 96.13 mg/ 100 g and was least in ‘zhangji’. pectin is one of the most complex natural plant biopolymers, which are identified as critical structure components of the cell wall (sara et al., 2012), since they provide cell-to-cell adhesion and mechanical strength (riahi et al., 2013). in this study it was shown that cultivars with higher pectin content were more suitable for processing. organic acids the contents of organic acids in the studied cultivars were shown in tab. 2. citric acid was the most abundant acid, contributing 73.5~84.7% in total organic acids. previous studies also found citric acid to be a dominating acid in different strawberry cultivars. the amount of citric acid was 0.64~0.93 g/100 g in this study, similar to 0.6~1.7 g/ 100 g in fully ripe berries (sturm, koron and stampar, 2003). the following two major organic acids were malic acid and oxalic acid, which contributed 9.5 to 21.7% and 4.5 to 7.9% in total organic acids, respectively. however, skupień et al. (2004) reported that malic acid is the most abundant acid and constituted 56% in total organic acid in elsanta berries. the minor organic acids in this work were tartaric acid (0.82~1.35 mg/100 g), acetic acid (0.11~ 0.15 mg/100 g) and fumaric acid (0.19~0.73 mg/100 g). sturm et al. (2003) reported that tartaric acid was 0.05~0.18 g/100g and fumaric acid was 0.4~1.7 mg/100 g. lactic acid and succinic acid were not observed in four cultivars, but skupień et al. (2004) detected 0.12~ 0.25 mg/100 g succinic acid in strawberries. there was a significant difference in total organic acids among the four cultivars. the summation of total organic acids determined by hplc was higher than ta estimated by titration method for each cultivar in this study. ‘tongzi i’ showed the highest acidity and ‘zhangji’ the lowest regardless of these methods. individual phenolic compounds other than anthocyanins quantitative results of individual phenolic compounds other than anthocyanins in four cultivars were presented in tab. 3. the tab. 1: basic physico-chemical characteristics of selected four cultivars of strawberries. characteristicsa ‘hongyan’ ‘tianxiang’ ‘tongzi i’ ‘zhangji’ average weight of individual fruit (g) 25.79±3.812b 19.78±2.00a 22.91±2.61ab 22.045±1.91a ph 3.70±0.015c 3.42±0.015a 3.52±0.021b 3.73±0.012c ta (g/100 g fw expressed as citric acid) 0.65±0.013a 0.73±0.015b 0.79±0.023c 0.63±0.031a sucrose (g/100 g fw) 0.046±0.0027b 0.059±0.0029c 0.015±0.0015a 0.75±0.042d glucose (g/100 g fw) 4.25±0.23d 3.25±0.18c 2.36±0.11a 2.73±0.16b fructose (g/100 g fw) 4.65±0.25d 3.75±0.16c 2.88±0.19a 3.13±0.14b total soluble sugars (g/100 g fw) 8.94±0.48d 7.06±0.34 c 5.25±0.31 a 6.61±0.33b tss/ta ratio 13.80±0.82d 9.69±0.32b 8.28±0.52a 10.46±0.88c ascorbic acid (mg/100 g fw) 42.89±0.65c 30.92±0.79b 22.76±0.88a 21.38±0.73a tp (mg gallic acid/100 g fw) 366.53±6.53c 313.39±8.54b 444.73±4.58d 235.12±4.86a pectin(mg galacturonicacid/100 g fw) 95.51±1.85c 92.19±3.54b 96.13±5.28c 82.84±2.66a a data were presented as the means ±standard deviations (n=6). a-d: values with different superscript letters within one row were significantly different (p≤0.05). composition of four selected strawberry cultivars 159 summation of individual phenolic compounds other than antho cyanins significantly reduced in the order of ‘tongzi i’, ‘hongyan’, ‘tianxiang’ and ‘zhangji’, closely corresponding to the decreasing order of tp in tab. 1. the summation of the phenolic compounds other than anthocyanins was considerably lower than 45.20~ 84.62 mg/100 g (calculated as total phenolic compounds without hydrolysis) estimated by skupień et al. (2004) and 42.1~54.4 mg/ 100 g proposed by hikkinen et al. (1999). this was possibly due to that the achenes were not broken and discarded after centrifugation process, since there were abundant phenolic compounds in achenes, especially ellagic acid and ellagic acid glycosides (aaby et al., 2005). strawberries contained 1% achenes on a fresh weight basis, however, they contributed to about 11% of total phenolics (aaby et al., 2005). catechin varied from 1.85 to 4.31 mg/100 g depending on the cultivars in this study. aaby et al. (2005) reported 6.2~9.0 mg/100 g catechin in strawberries. ellagic acid, ρ-coumaic acid and p-hydroxybenzoic were three predominant soluble phenolic acids. ellagic acid was the highest, followed by ρ-coumaric acid and p-hydroxybenzoic in four culti vars. studies carried out by odriozola-serrano et al. (2009) and häkkinen et al. (1999) also showed that ellagic acid and ρ coumaric acid are predominant phenolic acids in strawberries. the free ellagic acid in strawberries (without achenes) was 2.77 to 3.94 mg/100 g, which fell in the range of 0.54 to 5.91 mg/100 g in different strawberry cultivars detected by simirgiotis et al. (2009), but the range increased up to 60.00 to 151.38 mg/100 g after acid hydrolysis in strawberries since ellagic acid is a hydrolytic product of ellagitannins. in this work, we quantified the soluble phenolic acids without acid or alkali hydrolysis. the ρ-coumaric acid content was the highest (0.66 mg/100 g) in ‘tongzi i’ and lowest (0.41 mg/100 g) in ‘tianxiang’, which was lower than in previous studies. mattila et al. (2006) showed it was 4.4 to 6.3 mg/100 g ρ-coumaric acid in different cultivar strawberries in finland, and skupień et al. (2004) detected 15.46 to 42.22 mg/100 g ρ-coumaric acid in fresh strawberries of six cultivars grown in northwest poland. reasons for the high variation may lie in differences in the analytical methodology used and in the natural variation of sample material. the p-hydroxybenzoic (0.31 to 1.06 mg/100 g) was significantly dif ferent in four cultivars, and was lower than in the strawberry proposed by mattila et al. (2006). the p-hydroxybenzoic in ‘hongyan’ was 3.4-folds in ‘tianxiang’. other two soluble phenolic acids in strawberries were ferulic acid and caffeic acid. there were 0.41 to 0.67 mg/100 g ferulic acid and 0.13 to 0.15 mg/100 g caffeic acid. ferulic acid in ‘tongzi i’ was the highest and in ‘zhangji’ the lowest. apart from ‘zhangji’, the other three cultivars showed no difference in caffeic acid content. the content of ferulic acid and caffeic acid in this study was not in agreement with the study of mattila et al. (2006) who reported of 0.25 to 0.32 mg/100 g ferulic acid and 0.14 to 0.42 mg/100 g caffeic acid in strawberries from finland. regarding the flavonols, kaempferol, the main flavonol in four cultivars, was 0.44 to 0.68 mg/100 g, which was the lowest in ‘ting xiang’ and the highest in ‘tongzi i’. myricetin and quercetin ranged from 0.35 to 0.40 mg/100 g and 0.39 to 0.62 mg/100 g, respectively. the summations of kaempferol, myricetin and quercetin as total flavonols were significantly different in the four cultivars, which were 1.19 mg/100g in ‘zhangji’ and 1.86 mg/100 g in ‘hongyan’. tab. 2: organic acids (mg/100 g fw) in selected four cultivars of strawberries. organic acidsa ‘hongyan’ ‘tianxiang’ ‘tongzi i’ ‘zhangji’ oxalic acid 82.61±0.80c 54.08 ±0.55b 89.75±0.25d 39.48±0.13a tartaric acid 1.35±0.058b 1.30±0.02b 0.99±0.019a 0.82±0.013a malic acid 104.60±1.34b 93.08±0.37a 195.98±2.69c 189.80±15.54c acetic acid 0.11±0.0066b 0.14±0.0046c 0.11±0.0075a 0.15±0.0029c citric acid 851.81±25.38b 826.51±37.63b 930.19±47.96c 644.47±20.10a fumaric acid 0.31±0.011b 0.73±0.0029d 0.43±0.0010c 0.19±0.0026a total 1040.80±37.47c 975.30±38.59b 1216.27±50.94d 874.30±35.80a a data were presented as the means ±standard deviations (n=6). a-d: values with different superscript letters within one row were significantly different (p≤0.05). tab. 3: individual phenolic compounds (mg/100 g fw) in selected four cultivars of strawberries. individual phenolic compoundsa ‘hongyan’ ‘tianxiang’ ‘tongzi i’ ‘zhangji’ catechin 1.95±0.054b 2.61±0.17c 4.31±0.29d 1.85±0.076a ellagic acid 3.35±0.23b 3.42±0.054b 3.93±0.15c 2.77±0.082a ß-hydroxybenzoic 1.06±0.046c 0.31±0.022a 0.75±0.039b 0.88±0.044b ρ-coumaric acid 0.63±0.075c 0.41±0.026b 0.66±0.022c 0.35±0.016a ferulic acid 0.55±0.037b 0.43±0.030a 0.67±0.043c 0.41±0.029a caffeic acid 0.15±0.0012b 0.14±0.00091b 0.13±0.00068a 0.14±0.0016b kaempferol 0.65±0.019b 0.44±0.029a 0.68±0.023b 0.44±0.014a quercetin 0.62±0.036d 0.47±0.030b 0.53±0.023c 0.39±0.0051a myricetin 0.59±0.036c 0.38±0.010a 0.53±0.041b 0.35±0.039a total 9.55±0.73c 8.61±0.35b 12.18±0.85d 7.59±0.38a a data were presented as the means ±standard deviations (n=6). a-d: values with different superscript letters within one row were significantly different (p≤0.05) 160 x. cao, y. wang, x. liao, x. hu the content of kaempferol, myricetin and quercetin in this study was similar to or slightly lower than previous studies (häkkinen and törrönen, 2000; häkkinen et al., 1999). häkkinen et al. (2000; 1999) reported the contents of quercetin and kaempferol in straw berry were 0.52 to 0.7 and 1.18 to 1.8 mg/100 g, respectively. the content of phenolic compounds may vary according to the strawberry cultivars, geographical origin, harvesting seasons, growing condition as well as to different extraction/hydrolysis and analytical methods used (odriozola-serrano et al., 2009). free amino acids the free amino acids in four cultivars were summarized in tab. 4, asparagine (asn), glutamine (gln), aspartic acid (asp), glutamic acid (glu) and alanine (ala) were the five most prominent free amino acids. there were significant differences in the total free amino acids among four cultivars, which were 13.35~32.66 mg/100 g. there were few studies on the content of free amino acids in strawberry. gallander (1979) reported that the total amino acids were 160 370 mg/100 ml in strawberry juice after hydrolyzing the protein, and the content of free amino acids was 79-87% of total amino acids in different cultivars. a small difference in the numbers of major amino acids in four cultivars was found. the most abundant free amino acids was asn, contributing 44.96% (‘tianxiang’) and 49.60% (‘tongzi i’) of the total free amino acids. asn in ‘tianxiang’ was 14.68 mg/100 g and 2.3 times higher than in ‘zhangji’. the content of asn varied from 218 to 75 mg/100 ml in early harvest and late harvest strawberry juice (gallander, 1979). other major free amino acids greater than 1 mg/100 g in four cultivars were similar, and in the order were serine (ser), glu, asp, gln and ala in ‘hongyan’, gln, ser, glu and asp in ‘tianxiang’, gln, ser, glu, asp and ala in ‘tongzi i’, ser and glu in ‘zhangji’. the minor free amino acids lower than 1 mg/100 g in four cultivars exhibited significant differences. cys teine (cys) was not found in the studied four cultivars, methionine (met) and isoleucine (ile) were absent in ‘tongzi i’ and ‘zhangji’ while present less than 0.2 mg/100 g in ‘hongyan’ and ‘tianxiang’. monomeric anthocyanins anthocyanins contributed well for the attractive color, affected con sumer acceptance and preference, and also had great relations with antioxidant capacity. strawberries are reported to owe two types of anthocyanidin pigments, derivatives of bright red pelargonidin and dark red cyanidin, and the major anthocyanins in strawberries were pelargonidin-3-glucoside (pg-3-glu), pelargonidin-3-rutinoside (pg3-rut) and cyanidin-3-glucoside (cy-3-glu) (da silva et al., 2000). in this study, pg-3-glu was predominant in four cultivars, contributing 67.46~89.41%. skupień et al. (2004) reported that pg-3-glu contributes 78~92% as the predominant pigment in six cultivars. as shown in fig. 1, ‘tongzi i’ was the highest and ‘zhangji’ the lowest in three major monomeric anthocyanins and total anthocyanins, respectively. in a previous study, the total anthocyanins were 20~60 mg/100 g in fresh strawberry (da silva et al., 2000). the total anthocyanins and pg-3-rut in ‘hongyan’ and ‘tianxiang’ were similar, pg-3-glu in ‘tianxiang’ (23.82 mg/100 g) was significantly higher than in ‘hongyan’ (19.48 mg/100 g), but cy-3-glu in ‘tianxiang’ was significantly lower than in ‘hongyan’. antioxidant capacity differences in the profile and contents of these related antioxidant compounds reported above could contribute to the differences in tab. 4: free amino acids (mg/100g fw) in selected four cultivars of strawberries. faasa ‘hongyan’ ‘tianxiang’ ‘tongzi i’ ‘zhangji’ asp 1.37±0.033b 1.75±0.027d 1.44±0.0038c 0.89±0.018a glu 1.741±0.0951c 1.99±0.062d 1.46±0.048b 1.01±0.0057a asn 9.72±0.45b 14.68±0.23d 11.56±0.071c 6.41±0.073a gln 1.11±0.069 b 4.14±0.068 d 2.26±0.051 c 0.78±0.036a ser 2.35±0.016c 2.72±0.054d 1.97±0.084 a 1.15±0.037a gly 0.044±0.00031b 0.13±0.0065d 0.095±0.0024c 0.031±0.0034a his 0.41±0.012b 0.92±0.039c 0.44±0.022b 0.33±0.034a arg 0.30±0.016a 0.59±0.058c 0.39±0.0034b 0.054±0.12c thr 0.31±0.025a 0.56±0.0080c 0.38±0.014b 0.26±0.023a ala 1.03±0.057c 0.31±0.033a 1.40±0.033d 0.59±0.027b pro 0.046±0.0012b 2.22±0.14c 0.011±0.0028a 0.014±0.0030a tyr 0.13±0.0075b 0.22±0.013c 0.096±0.0060a 0.078±0.0037a val 0.064±0.0024c 0.14±0.0095d 0.048±0.0031b 0.014±0.0035a met 0.030±0.0013b 0.18±0.011c nd nd ile 0.18±0.024b 0.15±0.0031c nd nd leu 0.037±0.0031a 0.37±0.018b 0.43±0.0066c 0.36±0.0052b phe 0.26±0.019c 0.044±0.0047a 0.10±0.00099b 0.10±0.0046b trp 0.33±0.0071c 0.27±0.0030b 0.33±0.015c 0.13±0.0055a lys 0.93±0.049b 0.92±0.045b 0.89±0.054b 0.73±0.058a total 20.39±0.66b 32.66±0.38d 23.31±0.32c 13.35±0.38a a data were presented as the means ±standard deviations (n=6). a-d: values with different superscript letters within one row were significantly different (p≤0.05). nd, not detected. composition of four selected strawberry cultivars 161 antioxidant activity. the antioxidant capacity of dpph and frap in four cultivars was shown in fig. 2, there were significant differences in the antioxidant capacity of ·dpph and frap among four culti vars. ‘tongzi i’ had the highest antioxidant capacity of ·dpph and frap, while ‘zhangji’ was the lowest, which closely corresponded to the content of anthocyanins and other phenolic compounds. ‘tian xiang’ was the second and ‘hongyan’ the third in the antioxidant capacity of ·dpph, but the two cultivars exhibited similar antioxidant capacity of frap. the antioxidant capacity of ·dpph and frap was not consistent with the ascorbic acid content, since the antioxidant capacity of ‘hongyan’ was lower than ‘tongzi i’, while its ascorbic acid content was higher. kalt et al. (1999) found that phenolics and anthocyanins were both strongly correlated to the antioxidant capacity of fruits, whereas the ascorbic acid content and antioxidant capacity were inversely correlated. in general, the antioxidant potential of strawberry could be due to synergistic actions of com plicated bioactive compounds, and it would be difficult to define the contribution of these bioactive compounds to antioxidant activity. predominantly located in the chloroplast thylakoid membranes, and its phenolic substrates are mainly located in the vacuoles, but upon any cell-damaging treatment, the enzyme and substrates may come into contact, leading to rapid oxidation of phenols and contributing significantly to quality loss (jiang et al., 2004). the results in this study implied that it was easy to turn brown in ‘hongyan’ due to its highest ppo activity during the storage and processing, and there was no browning catalyzed by ppo in ‘tianxiang’. pod activity was similar in ‘hongyan’, ‘tianxiang’ and ‘tongzi i’, but was significantly higher in ‘zhangji’. the involvement of pod in browning was reported by many researchers (jiang et al., 2004; chisari et al., 2007), although it is limited by the availability of electron acceptor compounds such as superoxide radicals, hydrogen peroxide, and lipid peroxides. pod is also responsible for the offflavors and texture loss as well as color changing. higher pod activity in ‘zhangji’ led to easier quality loss during storage or processing than other cultivars. pme is an enzyme found in all species of higher plants. pme catalyzes the de-esterification of the methyl group of pectin and converts it into low-methoxy pectin or pectic acid. pme activity like ly plays an important role in strawberry fruit softening and viscosity of products decreasing (riahi et al., 2013). the pme activity was low in four cultivars, it was the least in ‘tianxiang’ and ‘tongzi i’, and the highest in ‘zhangji’. fig. 1: major monomeric anthocyanins (mg/100 g fw) in selected four cultivars of strawberries. fig. 2: ·dpph and frap values in selected four cultivars of strawberries. enzyme activity as shown in fig. 3, there were significant differences in ppo activity in four cultivars, which decreased in the order of ‘hongyan’, ‘zhangji’ and ‘tongzi’, and it was not observed in ‘tianxiang’. in plants, ppo is conclusions the present study demonstrated that different strawberry cultivars presented different physico-chemical and biochemical compositions, which are important factors for appraising the characterization of strawberry cultivars with respect to their nutritional value and potential use for different products. ‘tongzi ι’ was most suitable for food processing due to the highest titration acidity, total phenolics, pectin, total organic acids, monomeric anthocyanins, antioxidant capacity of ·dpph and frap with lower ppo, pod and pme activity. acknowledgements this work is supported by national natural science foundation of china 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zhang, z.h., liao, x.j., wang, z.f., 2006: kinetic analysis of non-enzymatic browning in carrot juice concentrate during storage. eur. food res. technol. 223, 282-289. zhi, x., zhang, y., hu, x.s., wu, j.h., liao, x.j., 2008: inactivation of apple pectin methylesterase induced by dense phase carbon dioxide. j. agric. food chem. 56, 5394-5400. address of the author: xiamin cao, school of biology and basic medical sciences, soochow university, suzhou, china © the author(s) 2018. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 90, 346 353 (2017), doi:10.5073/jabfq.2017.090.043 1chemical research laboratory, institute of agriculture, lithuanian research centre for agriculture and forestry, akademija, kėdainiai district, lithuania 2department of drug chemistry, faculty of pharmacy, lithuanian university of health sciences, kaunas, lithuania 3department of analytical and environmental chemistry, vilnius university, vilnius, lithuania 4department of grass breeding, institute of agriculture, lithuanian research centre for agriculture and forestry, akademija, kėdainiai district, lithuania young herbaceous legumes – a natural reserve of bioactive compounds and antioxidants for healthy food and supplements bronislava butkutė1*, raimondas benetis2, audrius padarauskas3, jurgita cesevičienė1, audronė dagilytė2, lukas taujenis3, hiliaras rodovičius2, nijolė lemežienė4 (received december 8, 2016; accepted october 12, 2017) * corresponding author summary young plants of clover (trifolium pratense l. and t. medium l.), medick (medicago sativa l. and m. lupulina l.), sainfoin (onobry chis viciifolia scop.) and milkvetch (astragalus glycyphyllos l. and a. cicer l.), were investigated for total contents of phenolics, flavonoids, isoflavones, condensed tannins and triterpene saponins as well as their extracts for antiradical and ferrous ion chelating activity. the impact of two sample drying methods on the aforementioned characters was compared. the phytochemical concentrations were higher in the freeze-dried legumes; however, antioxidant activities were generally higher of oven-dried samples. both the composition of health promoting phytochemicals and antioxidant properties were strongly species-dependent. among the species tested, trifolium spp. were most abundant in isoflavones, medicago spp. – in saponins and o. viciifolia – in tannins. plants of t. medium and o. viciifolia were rich in tpc. the extracts of t. pratense, o. viciifolia and a. cicer possessed significant antiradical activity; the extracts from astragalus spp. proved to be promising chelators of ferrous ion. we concluded that young perennial legumes could be considered as potential candidates for the development of nutraceuticals and functional food ingredients to accommodate the need for a particular bioactive component or property. key words: perennial fabaceae; phenolic compounds; saponins; antioxidant properties; drying methods introduction nowadays, the live question is not only to eat for survival, but to be aware of what we eat and know that the food will provide the opportunity to enjoy the quality of life for longer (muzquiz et al., 2012). the increasing occurrence of non-infectious diseases such as cancer or cardiovascular diseases might be caused by nutritional and lifestyle habits. legumes play an important role in the traditional diets of many regions of the world and are used both in staple and functional foods (prati et al., 2007). though forage legumes are not common for human consumption, there is evidence that mankind in all ages and in various countries has been using young plants, leaves, or flowers of various perennial legumes in food and phytotherapy (butler, 1995; redžić, 2010). according to barrett (1990), humans have been using leaves of total 88 genera with 290 species in fabaceae, including 63 genera and 205 species in the sub-family faboideae as vegetables: raw, steamed, boiled, fried or cooked mixed in with other foods. at present, leaves, flowers and seeds of alfalfa (m. sativa l.) and red clover (t. pratense l.) are sold as bulk pow dered herb, capsules, and extracts in health food stores or different online shops. potential health benefit of legume consumption is associated with the presence of different phenolic compounds like isoflavones, coumestans, tannins and other phytochemicals, for instance saponins, in their plant materials (prati et al., 2007; muz quiz et al., 2012). the diversity and complexity of the phytochemical composition of plants of fabaceae family may explain their polyvalent pharmacological activity. proanthocyanidins (condensed tannins) constitute an important group of natural polyphenols. they occur naturally in many plants, including legumes. many of their biological effects of nutritional interest derive from antibacterial and antioxidative properties providing protection against radical mediated injury and cardiovascular disease (cos et al., 2004). other health-promoting components specific to legumes are isoflavones: they have come into focus of interest due to several reports about their positive effects on human health, in particular prevention of hormone-dependent cancers, cardiovascular diseases, osteoporosis, adverse menopausal manifestations and age-related cognitive decline (pilšáková et al., 2010). saponins are exceptional plant metabolites because of their invaluable pharmaceutical properties (cheok et al., 2014). clinical studies have suggested that saponins affect the immune system in ways that help to protect the human body against cancers, and also lower cholesterol levels, blood lipids, cancer risks, and blood glucose response (shi et al., 2004). there are many biological activities associated with the total phenolics and individual groups of metabolites, preeminently their antioxidant and anticarcinogenic properties (campos-vega et al., 2010). numerous findings emphasize that dietary antioxidants are useful radioprotectors and play an important role in preventing many human diseases (cancer, atherosclerosis, diabetes and others) (fang et al., 2002). therefore, there is an increasing trend towards finding alternative sources of valuable phytochemicals due to their diverse potentialities in food industry and pharmaceutical applications (barreira et al., 2016). to our knowledge, no comprehensive studies have been done on phytochemicals combined with antioxidant activities of perennial legumes of branching stage. isoflavones have been quantitatively studied only in a limited number of species of these legumes before. sample preparation and drying methods are important factors to preserve stability of natural bioactive compounds and their antioxidant properties. a review of the existing researches, done by abascal et al. (2005) indicates that freeze-drying has unanticipated and significant effects on the constituent profiles of medicinal plants. many authors argue that freeze-drying proved to preserve more the quality of the plant materials, including antioxidant activity, phenolics and other bioactive substances (pinela et al., 2011; orphanides potential of young forage legumes for healthy food 347 et al., 2013). however, conflicting evidence has been found by que et al. (2008) who suggest that hot air-dried pumpkin flour exhibited higher reducing power, free radical scavenging and metal chelating activities than freeze-dried flour. esparza-martínez et al. (2016) reported that drying at high temperatures shows higher antioxidant activity than at low temperatures. little information exists on the influence of drying methods on the concentration of phytochemicals and antioxidant properties of plant material of perennial legume species. thus, the aims of this study were to quantify bioactive compounds in 7 species of perennial legumes from the sub-family faboideae, cut at branching stage, and to assess in vitro antioxidant activity of plant extracts as well as to evaluate the effects of two drying methods on the phytochemical profile and properties. materials and methods plant material the 7 species chosen for the study represent legumes from the following genera: red and zigzag clovers, lucerne, black medick, sainfoin, liquorice and cicer milkvetches (tab. 1). the germplasm collection was established in a field trial in 2014 in the central lowland of lithuania (55°23 4́9˝n; 23°51́ 40˝e), at the institute of agriculture, lithuanian research centre for agriculture and forestry. the soil of the experimental site is endocalcariepihypogleyic cambisol. the seeds of the entries were sown in a single row (2.5 m long with 0.5 m spacing between plants) in 4 replications. no herbicides were applied in the collection nursery. the plant material represents whole aerial parts collected at the plant branching stage. the samples were washed thoroughly with tap water, rinsed with distilled water and blotted on filter paper. then each of the samples was divided into two subsamples. one subsample was pre-dried in an oven at 105 °c for 15 minutes to rapidly stop metabolism and then oven-dried at 65±5 °c for 24 hours. the other subsample was freeze-dried. sublimation /lyophilisation was performed in a sublimator 3×4×5 (zirbus technology gmbh, germany), the condenser temperature was -85 °c, and the vacuum was 2 × 10-6 mpa, the samples were frozen at -40 °c in a laboratory freezer, and then left in a freeze-drier for 72 hours. both ovenand freeze-dried samples were ground to pass a 1 mm screen. reagents and chemicals folin-ciocalteu phenol reagent (2n), gallic acid monohydrate (≥98.0%), sodium carbonate (anhydrous, 99.5-100%), 3-(2-pyridyl)5,6-diphenyl-1,2,4-triazine-4 ,́4̋-disulfonic acid monosodium salt (ferrozine, 97%), methanol (≥99.9%), acetone (≥99.8%), hexane (≥97.0%), acetic acid (≥99.7%), sulphuric acid (95-98%), vanillin (4 hydroxy-3-methoxybenzaldehyde, ≥97%), catechin hydrate (≥98%), biochanin a (≥98%), daidzein (≥98%), formononetin (≥99%), genistein (≥98%), oleanolic acid (≥97%) were purchased from sigmaaldrich chemie gmbh (steinheim, germany). a stable dpph (2,2-diphenyl-1-picrylhydrazyl) free radical (95%) and iron(ii) chloride (anhydrous, 99.5%) were supplied by alfa aesar gmbh & co kg (karlsruhe, germany). acetic acid (100%), aluminium chloride hexahydrate (≥95%), hexamethylene tetramine (≥99%) and rutin trihydrate (≥95%) were obtained from carl roth gmbh + co. kg (karlsruhe, germany). lc-ms grade methanol, acetic acid and formic acid were obtained from fluka (sigma-aldrich). ethanol 96.3% (v/v) was purchased from stumbras ab (kaunas, lithuania). double-deionized water with conductivity lower than 18.2 mω was purified by using a milli-q direct 8 water purification system (millipore, bedford, ma, usa). preparation of extracts the assays for total phenolics, flavonoids and antioxidant properties revealed that the best results in the recovery of the bioactive properties were achieved when extraction was performed with 70% (v/v) aqueous ethanol through ultrasonic agitation at 50 °c for a 15 min period of sonication (data not shown). about 0.25 g (precision ±0.0001 g) of plant material was sonicated with 25 ml of the 70% (v/v) aqueous ethanol for 15 min using an ultrasonic bath elmasonic s40h (elma schmidbauer gmbh, germany). the suspension was filtered and supernatant was adjusted to 25 ml. two replicate extractions were performed for each plant sample. extraction procedure for condensed tannins was carried out as follows: 500 mg of plant sample was extracted with 5 ml acetone/ water mixture (70/30, v/v) containing 0.5% (m/v) of ascorbic acid by vortexing for 1 h. ascorbic acid was added to prevent tannin oxidation during the extraction. then the sample was centrifuged at 3000 × g for 15 min and resulting supernatant was filtered through 0.20 μm nylon filter. three ml of hexane was added to 1 ml of fine solution to remove chlorophyll. the aqueous layer was separated and taken for spectrophotometric analysis. both acid hydrolysis and extraction of isoflavones were performed in a single step. the representative amount of sample (250 g) was extracted with 10 ml of methanol/water (8:2, v/v) containing 2 m hcl using sonication for 30 min at room temperature before being hydrolysed at 80-85 °c for 1.5 h. the extracts were filtered through a 0.2 μm nylon syringe filter and analyzed. for the hydrolysis and extraction of triterpene saponins, 0.100 g of plant material was treated with 10 ml of 2 m hcl in methanol/water (1:1 v/v) under reflux for 8 h. methanol was removed under vacuum and the aglycones were extracted with ethyl acetate (2 × 5 ml). the combined organic phase was evaporated to dryness, the residue dissolved in 5 ml of methanol then filtered through 0.2 μm nylon syringe filter and analyzed. tab. 1: list of germplasm collection of the perennial species from the sub-family faboideae (fabaceae) studied. species (tribea) entry nob cultivar (cv.) or entry notation originc wild ecotype (we) red clover, t. pratense (trifolieae) 31 cv. sadūnai tpr lithuania zigzag clover, t. medium (trifolieae) 2148 we tme lithuania lucerne, m. sativa (trifolieae) 2097 cv. malvina msa lithuania black medick, m. lupulina (trifolieae) 10 cv. arka mlu lithuania sainfoin, o. viciifolia (hedysareae) 28 cv. meduviai ovi lithuania liquorice milkvetch, a. glycyphyllos (galegeae) 13 we agl latvia cicer milkvetch, a. cicer (galegeae) 71 we aci lithuania a tribes are indicated according to lewis et al. (2005); b entry no in catalogue of institute of agriculture, lithuanian research centre for agriculture and forestry; c country where seeds were collected. 348 b. butkutė, r. benetis, a. padarauskas, j. cesevičienė, a. dagilytė, l. taujenis, h. rodovičius, n. lemežienė total phenolics total phenolic contents (tpc) in the plant extracts were determined spectrophotometrically by the folin-ciocalteu method using gallic acid as a reference. one ml of standard solutions of gallic acid at different concentrations or appropriately diluted extract was assayed with 5 ml 0.2 n folin-ciocalteu reagent and, after 5 min, 4 ml of sodium carbonate (7.5%, w/v) solution was added to the mixture and shaken. after a 60 min period of incubation at room temperature, the absorbance was determined at 765 nm using a uv/vis spectrophotometer spectronic genesys 2 (spectronic instruments, usa). quantification was done on the basis of the standard curve of gallic acid (solution of gallic acid in 96% (v/v) ethanol, 11-350 μg ml-1). the concentration of tpc was expressed in mg of gallic acid equivalents (gae) g of dry mass (mg gae g-1 dm). total flavonoids analysis of total content of flavonoids (tfc) in extracts was performed by a colorimetric assay, based on complexation with al(iii). one ml aliquot of 70% (v/v) ethanolic plant extract (10 g l-1) was added to a 25 ml volumetric flask containing 10 ml of 96% (v/v) ethanol. then, 0.5 ml of 33% acetic acid, 1.5 ml 10% alcl3 and 2 ml 5% hexamethylenetetraamine solutions were pipetted into the flask and made up with distilled water. the absorbance was read at 407 nm after 30 min at 20 °c versus the prepared blank. blank samples were prepared from 1 ml of plant extract, 10 ml of 96% (v/v) ethanol and 0.5 ml of 33% acetic acid and diluted to 25 ml with distilled water. the absorbance of a reference solution, which was prepared by using 1 ml of rutin solution instead of plant extract, was measured simultaneously. standard rutin solution was prepared by dissolving 0.05 g of rutin in 100 ml of 96% ethanol. samples were analyzed in three replications. tfc was expressed as milligrams of rutin equivalents (re) per g of dry mass (mg re g-1 dm). condensed tannins condensed tannins (ct) were performed using vanillin assay. 10 μl of aqueous sample was incubated with 2 ml of 1.8 m sulphuric acid solution in methanol, 2 ml of 10 g l-1 vanillin so lution in methanol and 990 μl pure methanol for 5 minutes. the absorbance was measured at 500 nm using a pg instrument uv-vis spectrophotometer t60 (oasis scientific inc., usa). a solution of (+)-catechin in methanol (1 g l-1) was used as the standard. the ct concentration was expressed as mg of catechin equivalents (ce) per g of dry mass (mg ce g-1 dm). the limit of ct quantification (loq) was 3.2 mg ce g-1 dm. total triterpene saponins saponin aglycones were analyzed on a 1290 infinity uplc system equipped with a 6410 triple quadrupole mass spectrometer (agilent technologies, usa). the atmospheric pressure chemical ionization source was operated in the negative ion mode and ms data acquisition was performed in the selected ion monitoring mode. data were acquired and processed using the masshunter software (agilent). the acquity uplc beh shield c18 (2.1 × 100 mm, 1.7 μm) column (waters) was employed for the separations. the mobile phase was composed of (a) water and (b) methanol both containing 0.25% (v/v) formic acid. the gradient elution program was as follows: 0-15 min, 40-100% b linear; 15-17 min, 100-40% b linear; 17-22 min, 40% b isocratic. the column temperature was maintained at 30 °c, the mobile phase flow rate was 0.25 ml min-1, and the injection volume was 5 μl. the total amount of saponin aglycones was measured using internal calibration with oleanolic acid. the limit of quantification was 0.25 mg g-1 dm. quantification of isoflavones the quantification of the four isoflavones (daidzein, genistein, and their 4’-methylated derivatives, formononetin and biochanin a) was performed by ultra-performance liquid chromatography (uplc) using a waters acquity uplc system (waters, milford, ma) equip ped with diode array detector (dad). data were collected and managed using the hystar 3.2 software (bruker). analytes in the extracts were identified according to our recently published procedure (lemežienė et al., 2015; taujenis et al., 2015) and by comparing the retention times with those of the corresponding standards. quantification was performed by external calibration and results were expressed in mg per 1 g of the dry material (mg g-1 dm). the limits of quantification defined as the concentration resulting in a signal of ten times the noise level were 0.15 mg l-1 (0.006 mg g-1 dm) for biochanin a and formononetin, 0.20 mg l-1 (0.008 mg g-1 dm) for genistein, and 0.25 mg l-1 (0.010 mg g-1 dm) for daidzein. the sum of four isoflavones was presented in the current study. dpph radical-scavenging activity the ability to scavenge the stable free 2,2-diphenyl-1-picrylhydrazyl radical (dpph) was determined spectrophotometrically. the solution of dpph in 96% (v/v) ethanol (6 × 10-5 m) was prepared daily before measurements. to initiate the antioxidant reaction an aliquot of 50 μl of plant extracts (10 g l-1), i.e. an aliquot containing bioactive compounds from 0.5 × 10-3 g of plant material was transferred into a test tube containing 2 ml of dpph solution (containing 0.12 μmol of pure dpph). the mixture was incubated for 30 min (until the reaction reached the steady state plateau) in the dark at room temperature. the decrease in absorbance due to the scavenging of dpph was monitored with a spectrophotometer at 515 nm. the absorption of a blank sample containing the same amount of 70% (v/v) ethanol and dpph solution was determined before each analysis. radical scavenging capacity of plant extracts was calculated as a percentage of dpph inhibition as follows: dpph(%) = [(ac as) × 100] / ac, where ac is the absorption of blank sample and as is the absorption of the solution with the analyzed extract (t = 30 min). finally the results were recalculated as μmoles of dpph free radicals scavenged by extract from 1 g of plant material (on a dm basis): dpph(μmol g-1) = (a × dpph%) / (m × 100), where a is μmoles of pure dpph in the aliquot, and m – plant material mass (g) in the extract volume used for the test. fe2+ chelating efficacy the ferrous ion-chelating (fic) potential of legume extracts was monitored spectrophotometrically by measuring ferrous ironferrozine complex formation by its absorbance at 562. an aliquot of 50 μl of 2 mm fecl2 solution (containing 0.1 μmol fe2+) was added to a 1 ml of 70% (v/v) ethanolic plant extract (10 g l-1). one ml of extract contained fe2+-chelating compounds from 0.01 g of plant material. after 5 min, the reaction was initiated by the addition of 0.2 ml 5 mm ferrozine solution. the mixture was vigorously shaken and left to stand at room temperature for 10 min. the absorbance of the solution was thereafter measured at 562 nm. one ml of 70% (v/v) ethanol was used instead of the sample for the control. fic capacity was calculated as amount of fe2+ μmoles bound by chelating agents in extract from 1 g of plant material (on a dm basis): fic(μmol g-1) = (a × fic%) / (m × 100), where a is fe2+ μmoles in the aliquot of fecl2 solution, m – plant material mass (g) equivalent to the volume of extract used for the test. fic % is a percentage calculated by using the formula: fic(%) = [(ac as) × 100] / ac, where ac is the absorbance of the reaction mixture with blank and as is the absorbance of the reaction mixture with the plant extract. potential of young forage legumes for healthy food 349 all bioactive compounds and antioxidant properties were analyzed in triplicate. statistical analysis statistical analysis of the results was performed by using software statistica 7.0 for windows (statsoft inc., usa). t-test for dependent samples was performed to determine the significance of the drying method on the concentration of bioactive compounds and properties tested for the individual legume entry. one-factor repeated-measures anova followed by duncan’s test was carried out to test for simple main differences among legume entries separately for the drying method. significance of differences was defined at the 1% level (p < 0.01). results effects of drying method on bioactive compound concentrations and antioxidant activity means of bioactive characters for all species dried by a different method and probability values of t-test revealed that concentrations of bioactive compounds and properties of plant extracts responded differently to sample drying method subject to both entry and bioactive character (tab. 2). total mean values of freeze-dried and oven-dried samples for total phenolic and flavonoid contents were very close; however, according to the probability values, the drying method significantly affected the concentration of total phenolics for t. pratensis and a. glycyphyllos, and total flavonoid content for m. sativa, a. glycyphyllos and a. cicer. all four individual isoflavones quantified as well as their sum were tightly reliant on the drying method for t. medium. generally, almost all legume species statistically significantly differed in the sum of isoflavones except for m. lupulina and a. glycyphyllos. the impact of the sample drying method as a factor was statistically significant on the concentrations of individual isoflavones for one or two species only. the mean values of condensed tannins, triterpene saponins and isoflavones (individual and sum) for freeze-dried samples were variably higher than those for oven-dried samples. at the same time means of antioxidant activities clearly showed an opposite direction: extracts of oven-dried legumes on average displayed higher potential as free radical scavengers and ferrous ion chelators than those of freeze-dried ones. both dpph radical scavenging and ferrous ion chelating capacity (fic) of extracts of t. pratensis and m. lupulina were under the significant influence of the drying method. bioactive compounds the amount of total phenolic, flavonoid contents (tpc and tfc, respectively) and capacity of their ethanolic extracts to scavenge free radicals as well as to chelate ferrous ions varied depending on the legume species (tab. 3, fig. 2) for both sample sets freeze-dried and oven-dried. in plant materials of different perennial legumes of branching stage, tpc ranged from 7.94 to 40.9 mg gae g-1 for freeze-dried samples and from 7.68 to 42.0 mg gae g-1 for ovendried samples (tab. 3). the highest tpc values (>40 mg gae g-1) were observed in the plants of t. medium and o. viciifolia. m. sativa and m. lupulina were the poorest among the legumes tested for tpc. the tfc varied in the similar ranges both in oven-dried and freezedried sample sets (3.69 41.9 and 3.42 36.9 mg re g-1, respectively). the results clearly indicated that the richest source of total flavonoids was o. viciifolia containing tfc more than twice above the mean for the investigated plants (15 mg re g-1, tab. 2). plant material of t. medium also had a noticeably higher content of total flavonoids than the mean and significantly higher than t. pratense, medicago spp. and astragalus spp. had (tab. 3). plants of a. cicer were the least rich in tfc than other species. generally, significant differences both in tpc and tfc were observed between species of the same genera (trifolium, medicago and astragalus), except for tpc in medicago species. quantifiable concentrations of proanthocyanidins (ct) were de termined in sainfoin (11.2 and 13.3 mg ce g-1 for ovenand freeze-dried plants respectively), in other legume species, vanillin assay showed only ct traces (<loq). m. sativa and m. lupulina were the only two species of perennial legumes which exhibited the quantifiable concentrations of triterpene saponins (7.71 and 7.88 mg g-1 for oven-dried and 9.02 and 8.59 mg g-1 for freeze-dried samples, respectively). using uplc-ms (spectra not shown) in the other plant species studied there were found also a few compounds attributable to saponins (two in clover entries and one in sainfoin and milkvetch species); however, the total concentrations of these tab. 2: significance of the drying method effect on bioactive characters (separately for each legume entry) and mean values of phytochemical concentration and bioactivity for ovenand freeze-dried samples. bioactive character probability values meana for tpr tme msa mlu ovi agl aci odb fdc total phenolic content 0.036d 0.562 0.380 0.065 0.929 0.001 0.094 23.74 23.69 total flavonoid content 0.184 0.687 0.0003 0.689 0.230 0.005 0.021 15.12 15.07 formononetin 0.055 0.032 0.413 0.075 0.081 0.240 0.002 3.36 3.69 biochanin a 0.155 0.007 0.0003 0.057 0.057 0.253 0.395 3.01 3.65 daidzein 0.048 0.004 0.113 0.146 genistein 0.197 0.004 0.423 1.04 1.26 sum of isoflavones 0.010 0.009 0.028 0.077 0.020 0.477 0.046 11.2 13.3 condensed tannins 0.283 6.85 7.92 triterpene saponins 0.313 0.559 7.79 8.81 dpph 0.008 0.283 0.675 0.007 0.116 0.120 0.037 72.2 59.5 fic 0.00001 0.0004 0.622 0.002 0.192 0.015 0.839 7.32 6.77 a units of mean values for character – in material and methods. b oven-dried. c freeze-dried. d probability levels of differences in bold are statistically significant at p < 0.05. 350 b. butkutė, r. benetis, a. padarauskas, j. cesevičienė, a. dagilytė, l. taujenis, h. rodovičius, n. lemežienė compounds were lower loq. differences between medicago species in total ts content were not significant. although the impact of drying methods on the ts and ct levels was not statistically significant, concentrations of the metabolites indicated the advantage of the freeze-drying method to retain these bioactive compounds. t. medium and t. pratense of the branching stage were characterised by isoflavone abundance. according to the total amount of four isoflavones averaged for both drying sets, the species ranked as follows: t. medium >> t. pratense >> o. viciifolia ≥ a. cicer = m. sativa = m. lupulina ≥ a. glycyphyllos. the rank for non-clover species slightly differed between the ovenand freeze-dried sample sets. generally, freeze-drying proved to be the method of plant material preparation, preserving a higher isoflavone concentration than oven-drying, except for o. viciifolia and m. sativa. moreover, qualitative uplc-dad chromatograms of non-hydrolysed extracts of red clover showed that drying conditions affected the composition of isoflavones and their conjugates (fig. 1). freeze-dried samples had higher levels of formononetin and biochanin a aglycones and lower levels of their glucoside malonates than oven-dried samples. antioxidant properties the results of dpph quenching showed the presence of radical scavenging compounds in all investigated legume species. comparison of antiradical activity of ethanolic extracts obtained from different legumes revealed that their radical scavenging capacities were highly variable (one gram of plant material eliminated from 12.98 to 174.88 μmol dpph) (fig. 2a). the extracts of t. pratense and a. cicer exhibited the highest antiradical activity (102.47 and 136.73 μmol g-1, respectively for freeze-dried samples and 174.88 and 129.00 μmol g-1, respectively for oven-dried samples). despite the relatively high phenolic content in t. medium and moderate level in a. glycyphyllos (tab. 3), fairly low activity against dpph was distinctive for the extracts of the above-mentioned species (36.91 and 17.58 μmol g-1, on average for ovenand freeze-dried samples of respective legume). extracts of m. sativa possessed the weakest scavenging properties among the analyzed plants. overall, the dpph radical scavenging assay showed higher antioxidant activity of most extracts prepared from the legume samples which were oven-dried at 65 °c combined with pre-drying at 105 °c compared with freezedried samples. ferrous ion chelating (fic) assay exhibited that extracts from raw materials of all the investigated species were able to capture fe2+ ions (fig. 2b). it was determined that fic values in extracts strongly differed subject to species of legume plants tested and ranged from 3.61 to 9.30 μmol of bound fe2+ per chelating agents in g of freeze-dried plant materials and from 5.22 to 9.29 μmol g-1 of ovendried ones (fig. 2b). according to the average fic potential of the ovenand freeze-dried samples, the species fell in the following rank: a. cicer (9.29 and 9.30 μmol g-1) = a. glycyphyllos (8.50 and 9.19 μmol g-1) > m. sativa (7.17 and 7.26 μmol g-1) > t. medium (6.66 and 5.17 μmol g-1) > m. lupulina (5.22 and 6.19 μmol g-1) ≥ o. viciifolia (5.29 and 4.97 μmol g-1) ≥ t. pratense (6.09 and 3.61 μmol g-1). tab. 3: contents of total phenolic (tpc), total flavonoid (tfc), condensed tannin (ct), triterpene saponin (ts) and sum of isoflavones in the ovenand freezedried perennial legumes. values are means ± sd. bioactive character tpr tme msa mlu ovi agl aci oven-dried tpc (mg gae g-1) 37.1±0.29d 42.0±1.88e 7.68±0.98a 8.10±0.07a 40.9±1.91e 13.2±0.97b 17.2±0.93c tfc (mg re g-1) 12.4±0.68c 22.5±1.70d 9.50±0.51b 5.28±0.44a 41.9±0.45e 10.6±0.20bc 3.69±0.07a ct (mg ce g-1) <loq <loq <loq <loq 11.2±1.42 <loq <loq ts (mg g-1) <loq <loq 7.71±0.687 7.88±0.908 <loq <loq <loq sum of isoflavones (mg g-1) 18.3±0.43b 28.5±0.46c 0.258±0.014a 0.212±0.005a 0.283±0.005a 0.203±0.013a 0.223±0.016a freeze-dried tpc (mg gae g-1) 31.9±0.13d 40.9±0.30e 7.94±0.17a 8.27±0.04a 40.8±0.06e 17.1±0.03b 18.9±0.02c tfc (mg re g-1) 11.0±0.68bc 22.8±1.02d 12.7±0.45c 5.90±0.05ab 36.9±2.24e 12.8±0.08c 3.42±0.14a ct (mg ce g-1) <loq <loq <loq <loq 13.3±1.11 <loq <loq ts (mg g-1) <loq <loq 9.02±1.014 8.59±0.944 <loq <loq <loq sum of isoflavones (mg g-1) 19.8±0.19b 34.5±0.82c 0.208±0.007a 0.233±0.015a 0.256±0.004a 0.211±0.011a 0.256±0.005a mean values of variable followed by the same letter are not significantly different among species (in the row) at the p < 0.01 level by duncan’s multiple range test computed separately for differently dried sample set. fig. 1: uplc-dad profiles of isoflavones and their conjugates of nonhydrolysed extracts of differently dried red clover of branching stage. a – oven-dried; b – freeze-dried. peaks: 1 – daidzein glucoside malonate; 2 – genistein glucoside malonate; 3 – formononetin glucoside; 4 – formononetin glucoside malonate; 5 – genistein; 6 – biochanin a glucoside; 7 – biochanin a glucoside malonate; 8 – formononetin; 9 – biochanin a. potential of young forage legumes for healthy food 351 discussion effect of drying method on antioxidant activity and bioactive substances disagreement on the effect of drying method on both antioxidant activity and bioactive substances has been found in literature (que et al., 2008; pinela et al., 2011; orphanides et al., 2013; esparzamartínez et al., 2016). the chosen legume species have not been studied before or studied only fragmentary for the aspect of drying method influence on phytochemical profile and antioxidant properties of plant material. the results on this issue obtained in our study are in line with those of sang et al. (2014) who revealed that the differences between total phenolic content and antioxidant activity of the differently dried malaysian forage legume leaves were genera-specific. however, we observed higher differences among species in bioactivity response to drying conditions. the antioxidant activity of plant material of trifolium species showed a stronger response to drying method than other legume species. this may be related to the transformation of the phytochemicals, both investigated and not investigated in the current study, including distinctive compounds for clovers – isoflavones. antioxidant and biological activities of isoflavones have been known to depend on the concentrations and structures of isoflavones and their derivatives (andres et al., 2015). in regard to these compounds, swinny and ryan (2005) revealed that oven-drying promotes decarboxylation of the malonyl glucosides to the acetyl glucosides, however, our results did not show such oven-drying effect on decarboxylation of the malonyl glucosides. this contradiction could be related to different oven-drying conditions, used by swinny and ryan (2005) (100 °c) and in the current study (65 °c in combination with short sample pre-drying at 105 °c). yuan et al. (2009) disclosed that the decarboxylation of glycosides malonates occurred at a very low rate for the first period of 20 min when the soybeans were heated at 110 °c, and an inconsiderable change in isoflavone profile was ob served while the temperature was lower (90 °c). even small alteration in the drying conditions may affect the composition and properties of the plant materials. for instance, swinny and ryan (2005) found that immediate freeze-drying of red clover samples after being collected inhibited the conversion of the isoflavone glycosides to the aglycones; whereas tsao et al. (2006) demonstrated that freezedried samples contained mainly the aglycones of isoflavones, when samples were kept at -5 °c for a few days before freeze-drying. in our study, samples were put in a sublimator within a few hours after being washed and blotted up. such delayed procedure probably led to higher levels of aglycones through partial hydrolysis of conjugates, than in oven-dried samples (fig. 1). short and prompt sample predrying at 105 °c used before oven-drying in mild conditions (65 °c) as a factor retaining antioxidant properties of plant material should not be dismissed. in summary, sample preparation could be one of the causes of variation in the biological activity of isoflavones as well as of other phytochemicals, therefore the choice of drying conditions is important for particular plant use in food and phytotherapy. our results highlighted the need for additional studies on this matter. phytochemical composition and antioxidant properties to our knowledge, this is the first interand intra-generic phyto chemical characterisation of the plant materials of young perennial legumes. the findings of our study revealed that the concentrations of isoflavones, triterpene saponins, and condensed tannins in perennial legumes and antioxidant properties are taxon-dependent features. although trifolium and medicago are classified in the same tribe, the quantitative phytochemical compositions of plant materials as well as the antioxidant activities of their ethanolic extracts differed radically between the species of the two genera. under the tested set of features, the differences between the species of the same genus were smaller, but obvious. scanty information was found in the literature on the question concerning both bioactive substances and properties of the legume species studied. with regard to red clover, our results on tpc and dpph radical scavenging are in agreement with those obtained by khorasani esmaeili et al. (2015) when phenolics extraction with methanol from in vivo grown red clover plants was used. however, tfc in our study was lower than that in the work mentioned above. ince et al. (2012) confirmed also that sainfoin is a good source of phenolics, and its methanolic extracts exhibited high dpph radical scavenging activity. in the recent study of karimi et al. (2013), both tpc content and dpph inhibition percentage of m. sativa leaf ex tract were found to be much higher, compared with the findings presented here; total flavonoids concentration matched that observed in our work. admittedly, it is not appropriate to directly compare the results of our study with the scarce literature data owing to the differences in the plant genotype, maturity, plant parts, pedoclimatic dissimilarity, peculiarities in assay conditions, units of data ex pression, etc. in addition, no specific quantitative data have been found on bioactive properties in other species involved in the current study. one of the most substantial attributes of sainfoin – the ct content in our study was higher than that reported by theodoridou et al. (2011) (3.66-6.40 g kg-1 dm); though, according to azuhnwi et al. fig. 2: mean of dpph radical scavenging (a) and ferrous ion chelating activities (b) of ethanolic extracts from the perennial legumes. mean values (columns of the identical color) labeled by the same letter for bioactive character are not significantly different among species at the 0.01 level by duncan’s multiple range test computed separately for differently dried sample set; error bars indicate ± sd. 352 b. butkutė, r. benetis, a. padarauskas, j. cesevičienė, a. dagilytė, l. taujenis, h. rodovičius, n. lemežienė (2011), the concentration of ct in 15 swiss accessions of sainfoin was considerably higher and varied within a wide range of 47-80 g kg-1 dm. summarized data of numerous studies showed that ct in most legumes including t. pratense and m. sativa are located in genera tive plant organs, like flowers, seeds or seed coats (piluzza et al., 2014). furthermore, piluzza et al. (2014) noticed that plant tannin content is often difficult to compare between laboratories because of the different methods or standards used for quantification. among the legume entries discussed in this paper only isoflavones in red clover have been quantified fairly in detail (tsao et al., 2006; oleszek, 2007; lemežienė et al., 2015). the concentration of total isoflavones we found in red clover (19.6 mg g-1) agrees with the range reported by the mentioned authors, except for the results published by lemežienė et al. (2015). such mismatch has resulted from the dissimilarities in plant maturity. the total mean content of isoflavones in different extracts from m. sativa (2.29-8.39 mg kg-1) that was observed by rodrigues et al. (2014) proved to be noticeably lower than we found (0.209 mg g-1, tab. 2). reliable data on isoflavone quantification in other species of plants tested in our work are not available currently, only sporadic descriptive information can be found in the literature. generally, the concentration of triterpene saponins in our study was in agreement with the report of tava et al. (1999), who found that total saponin concentration in m. sativa varied from 2.3 to 10 mg g-1 dm depending on the cultivar and harvesting time. however, regarding the saponin concentration in black medick, górski et al. (1984) found 2.5-3-fold higher values in cv. renata (21-25 mg g-1) than we determined in cv. arka (7.98 mg g-1). in contrast to the low concentrations of tpc, tfc and inhibiting activity against dpph, the extracts from two species of the genus medicago were found to possess moderate activity in fic assay. this may be related to the presence of genus-specific bioactive compounds – saponins. joshi et al. (2013) revealed that saponins in all concentrations exhibited high fic values. extracts of a. glycyphyllos exhibited the highest ferrous ion chelation potential. the high fic capacity of the extracts from astragalus species suggests that they contain higher amounts of ligands to compete with ferrozine and their extracts could prevent the generation of chemically reactive molecules containing oxygen, also known as reactive oxygen species (ros). currently, synthetic compounds are used in the chelation therapy and they have certain side-effects (sudan et al., 2014). therefore, chelation of metal ions by natural phytochemicals from fabaceae plants, particularly from astragalus species can prove to be of therapeutic importance. generally, it is recognized that among important forage crops there are accessions, including species studied in the current work, with relatively high bioactive and antioxidant properties, which have the potential to be used for the development of new functional, medici nal food and nutraceuticals (kroyer, 2004; prati et al., 2007; ionkova, 2008; ince et al., 2012; karimi et al., 2013). the bioactive characteristics (substances and antioxidant properties) observed in legumes in our studies showed that each of the investigated species exhibited distinct and exceptional value. perennial legumes are not common for human consumption; however, there is potential to include young legume plants as functional food ingredients to accommodate the need for a particular bioactive component/ property or use them in nutraceuticals and supplements production. at this point, the present work is an initial phase for the forethought of further aspects of investigation on studying species including preclinical and epidemiological studies. conclusions bioactive compounds and properties of legumes respond differently to sample drying method: freeze-drying retained higher concentra tion of phytochemicals, and antioxidant activity in most cases was higher of the oven-dried samples. plants of the seven fabaceae species of branching stage contain a considerable amount of bioactive compounds, and their extracts exhibit significant antioxidant activity; however, all properties vary significantly among the investigated species. plant materials of t. medium and o. viciifolia are abundant in total phenolics. the extracts of t. pratense, o. viciifolia and a. cicer possess significant antiradical activity; the extracts from astragalus species prove to be promising chelators of ferrous ion. o. viciifolia contains the highest concentrations of total phenolics, total flavonoids and condensed tannins among the species tested. perennial legumes, particularly trifolium, are important sources of isoflavones. medicago species contain saponins. hence, all these findings proved that perennial legumes of branching stage merit to be considered as a potential source of valuable bioactive compounds for medicinal application and for functional components in foods which may increase the intake of potentially health-promoting phy tochemicals. acknowledgements this study was funded by a grant (no. sve-06/2014) from the research council of lithuania and was partly supported by the long-term research program “biopotential and quality of plants for multi-functional use” implemented by lithuanian research centre for agriculture and forestry. references abascal, k., ganora, l., yarnell, e., 2005: the effect of freeze-drying and its implications for botanical medicine: a review. phythoter. res. 19, 655-660. andres, s., hansen, u., niemann, b., palavinskas, r., lampen, a., 2015: determination of the isoflavone composition and estrogenic activity of commercial dietary supplements based on soy or red clover. food funct. 6, 2017-2025. azuhnwi, b.n., boller, b., martens, m., dohme-meier, f., ampuero, s., günter, s., kreuzer, m., hess, h.d., 2011: morphology, tannin concentration and forage value of 15 swiss accessions of sainfoin (onobrychis viciifolia scop.) as influenced by harvest time and cultivation site. grass forage sci. 66, 474-487. barreira, j.c.m., visnevschi-necrasov, t., pereira, g., nunes, e., oliveira, m.b.p.p., 2016: phytochemical profiling of underexploited fabaceae species: insights on the ontogenic and phylogenetic effects over isoflavone levels. food res. int. in press. doi: 10.1016/j.foodres.2016.07.009 barrett, r.p., 1990: legume species as leaf vegetables. in: janick, j., simon, j.e. 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effect of plant development during first and second growth cycle on chemical composition, condensed tannins and nutritive value of three sainfoin (onobrychis viciifolia) varieties and lucerne. grass forage sci. 66, 402-414. tsao, r., papadopoulos, y., yang, r., young, j.c., mcrae, k., 2006: isoflavone profiles of red clovers and their distribution in different parts harvested at different growing stages. j. agric. food chem. 54, 57975805. yuan, j.p., liu, y.b., peng, j., wang, j.h., liu, x., 2009: changes of isoflavone profile in the hypocotyls and cotyledons of soybeans during dry heating and germination. j. agric. food chem. 57, 9002-9010. address of the corresponding author: bronislava butkutė, chemical research laboratory, institute of agriculture, lithuanian research centre for agriculture and forestry, instituto al. 1, lt58344 akademija, kėdainiai district, lithuania e-mail: brone@lzi.lt © the author(s) 2017. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 90, 315 322 (2017), doi:10.5073/jabfq.2017.090.039 1research center for food safety and nutrition, key lab of urban agriculture (south), bor s. luh food safety research center, school of agriculture and biology, shanghai jiao tong university, shanghai, china 2 zhenjiang institute of agricultural sciences in hilly area of jiangsu province, jiangsu, china 3 department of nutrition and health, yihs institute of biomedical technology co. ltd., jiangsu vocational college of medicine, jiangsu, china phytochemical composition, antioxidant capacity and ace-inhibitory activity of china-grown radish seeds bingjun qian1, 3†, yueping pan2†, zhan cai1, pu jing1* (received april 25, 2017; accepted august 7, 2017) * corresponding author † these authors made equivalent contributions to the manuscript. summary the seeds of nine china-grown radish cultivars were analyzed for their phytochemical composition, antioxidant properties and aceinhibitory activity. radish seeds contained 36.87-43.06% (w/w) oils, whereas 64.55-69.26% of the fatty acids were monounsaturated and 20.33-25.11% were polyunsaturated. the levels of δ-tocopherol (552.24-670.31 μg/g seed oils) and lutein (4.82-8.95 μg/g seed oils) differed in cultivars. the nine cultivars varied in total phenolics, flavonoids, and free phenolic acids, but not in proanthocyanidins. seed extracts of hybrid #63, tou xin hong, and hybrid #72 showed stronger dpph radical scavenging capacity, orac, and frap than others (p<0.05). the yanzhi #2 extracts exhibited a strong aceinhibitory activity, which was positively correlated with vanillic acid contents (r = 0.890, p = 0.001). it provides evidence on developing value-added utilization of radish seeds or seed fractions such as oil and flour as nutraceuticals or functional food ingredients. keywords: radish seeds; phenolics; flavonoids; antioxidants; ace-inhibitory activity introduction radish (raphanus sativus l.) is a root vegetable crop that is native to europe and eastern asia (muminović et al., 2005). the consump tion of radish has increased during the last decade due in part to recognition of their nutritional values and antioxidant properties (lugasi et al., 1998; vitória et al., 2001). it reported that radish consumption could reduce the risk of lung and colorectal cancers (martinez-villaluenga et al., 2010), and minimize genotoxicity and cytotoxicity (hassan et al., 2011). the radish seeds have gained considerable attention of countries such as brazil, turkey and poland, due to their high levels of unsaturated fatty acids (uluata and özdemir, 2012; ávila and sodré, 2012; kaymak, 2015) and other antioxidant compounds, such as phenolic acids and flavonoids (pająk et al., 2014). so, the extracts from the radish seeds or seed flour could be potent functional food ingredients. a recent study reported that total seed oil (43%, w/w) of turkey-grown radish consists of 62% monounsaturated fatty acids (mufas) and 17% polyunsaturated fatty acids (pufas) and radish seed oil contains significantly high levels of tocopherols (uluata and özdemir, 2012). compared to selected seeds including mung bean, broccoli, and sunflower seeds, radish seeds in poland contain higher levels of total phenolics and flavonoids (pająk et al., 2014). additionally, radish seeds contain functional proteins with in vitro antifungal activity (terras et al., 1992). about 17.5 million people die each year from cardiovascular di seases (cvds), an estimated 31% of all deaths worldwide (http:// www.who.int/cardiovascular_diseases/en). hypertension and chronic inflammation, both of which can be caused by oxidation in vivo, are regarded as risk factors of cvd. many studies showed that the secondary metabolites from plants are the potential compounds for preventing cvd, possible attributable to their antioxidant and angiotensin-converting enzyme (ace)-inhibitory activity (vasanthi et al., 2012; balasuriya and rupasinghe, 2011). loizzo et al. (2007) isolated six flavonoids with ace-inhibitory activity from ailanthus excelsa (roxb), of which kaempferol-3-o-β-galactopyranoside possesses the highest activity with an ic50 value of 260 μmol/l. ojeda et al. (2010) isolated and identified two ace inhibiting anthocyanins, delphinidin-3-o-sambubioside and cyanidin3-o-sambubioside, from the aqueous extract of hibiscus sabdariffa with ic50 of 84.5 and 68.4 g/ml, respectively. most of the researches have been targeted at bioactive compounds from natural resources. it is almost universally accepted that the phytochemicals are the new alternatives for replacing the drugs to avoid the adverse side effects initiated by long use of the ace inhibitors, such as captopril and lisinopril. there are several radish cultivars grown in different regions of china. white round, korean white, japan white, and white pink are the radish cultivars with high consumption, and tou xin hong, xin lin mei, and yanzhi #2 are the radish cultivars whose roots are rich in pigments we have investigated (jing et al., 2012), and hybrid #63 and hybrid #72 are new hybrid radish cultivars which have been created by prof. pan. however, few studies have evaluated their phytochemical compositions and antioxidant activities in seeds. therefore, the objective of this study was to investigate the levels of fatty acids, tocopherols, carotenoids, total phenolics, flavonoids, and proanthocyanidins in the seeds of these nine radish cultivars and to evaluate the antioxidant capacity and ace-inhibitory properties of these radish seeds. materials and methods radish seeds and chemicals the nine radish cultivar were cultivated with conventional plantation measure, and their seeds were harvested by zhenjiang institute of agricultural sciences in hilly area of jiangsu province and donated to this experiment. standards of supelco 37 component fame mix, α, β, γ, δ-tocopherols, β-carotene, lutein, cryptoxanthin, and zeaxanthin, 2,4,6-tripyridyl-s-triazine(tptz),6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox), folin-ciocalteu reagent, 2,2´-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (abts), and 2,2-diphenyl-1-picryhydrazyl radical (dpph), angiotensin-converting enzyme (ace , ec 3.4.15.1) from rabbit lung, hippuryl-his-leu (hhl), and sinapic, vanillic, syringic, and ferulic 316 b. qian, y. pan, z. cai, p. jing acid were purchased from sigma-aldrich (st. louis, mo, usa). gallic acid, (±)-catechin hydrate, cyanidin chloride were purchased from aladdin (shanghai, china). all other chemicals and solvents were of the highest commercial grade and used without further purification. all other chemicals were purchased from sinopharm chemical reagent (shanghai, china). extraction of radish seeds the oils and antioxidant extracts were prepared according to the previous description by jing et al. (2012). radish seeds were ground into powder with a standard household coffee grinder until they could pass through a 40-mesh screen. five grams of radish seed powder were extracted in 80 ml hexane for 6 h in a soxhlet device. the hexane in the oil was evaporated using a rotary evaporator (rotavapor re-52, yarong inc., shanghai, china) at reduced pressure. the oils were collected and stored at -20 ºc until analysis. each sample was analyzed in three technical repeats. the radish seed flour (rsf) after oil extraction was then dried overnight under hood and weighed, where the weight loss was equal to the mass of extracted oils. one gram of the flour was shaking in water bath with 10 ml of 50% acetone at room temperature for 2 h. then, the extracts were centrifuged at 3000 g for 8 min. the prepared extracts were kept in the dark at 4 ºc for further analysis of antioxidants, such as phenolic acid, flavonoids, and proanthocyanidins. each sample was analyzed in three technical repeats. analysis of fa composition by gas chromatography (gc) fatty acid methyl esters (fame) were prepared by the koh– methanol method (jing et al., 2012) prior to analysis of gc. briefly, dissolving 20 mg oil in 2 ml iso-octane followed by another addition of 0.2 ml of 1 mol/l koh–methanol. after a reaction at room temperature for 5 min, 2 ml of iso-octane and approximately 3 ml water were successively added. then, removing the supernatant and washing the residues with 3 ml water twice. the upper iso-octane layer was collected and subjected to fatty acid analysis using gas chromatography (shimadzu gc-2010), which was equipped with an omegawax column (30 m × 0.25 mm with a 0.25 μm film thickness) from supelco (bellefonte, pa, usa) and a flame ionization detector (fid). the carrier gas was helium, and the flow rate was set as 1.0 ml/ min. oven temperature-rising program was set as follows: initially 50 ºc for 2 min, increasing to 220 ºc at 30 ºc/min and holding at 220 ºc for 20 min, followed by heating from 220 to 260 °c at 10 °c/ min and holding at 260 °c for 10 min. each sample was analyzed in three technical repeats. the various fatty acids were identified according to the standards mixture (supelco 37 component fame mix, supelco, pa, usa). the concentration of each fatty acid was expressed as a percentage of total area of all fatty acid peaks. determination of tocopherol contents of oil from seed by ultra performance liquid chromatography (uplc) the separation samples were prepared by dissolving about 200 mg oil in 10 ml methanol/tetrahydrofuran (thf) (1:1, v/v) prior to analysis of tocopherol contents in seed oil. the samples were separated on a waters acquity uplc-c18 column (100 mm × 2.1 mm, 1.7 μm particle size) using an acquity ultra performance liquid chromatography (uplc) system with a tuv detector (waters, milford, usa) according to a previous method (jing et al., 2012). elution was performed at a flow rate of 0.3 ml/min with a binary gradient (a mobile phase of water was used as solvent a, and acetonitrile was used as solvent b) of solvent b in a going from 80% to 99% over 5 min followed by 99% for 5 min. tocopherols were monitored at 294 nm and identified according to the uplc retention time with those of tocopherol standards. the standard curves were constructed for quantification. each sample was analyzed in three technical repeats. analysis of carotenoid compositions by ultra performance liquid chromatography (uplc) the separation samples prepared was also loaded on c-30 ymc carotenoid column (ymc, wilmington, nc; 150 × 4.6 mm, 5 μm particle size) using the acquity ultra performance liquid chromatography (uplc) system equipped with a tuv detector according to a previous method (jing et al., 2012). the mobile phase was methanol/methyl tertiary butyl ether (mtbe)/h2o (81:15:4, v/v/v) as solvent a and mtbe/methanol (91:9, v/v) as solvent b. elution was performed at a flow rate of 1 ml/min with a binary gradient of solvent b in a going from 0% to 50% over 30 min, 100% for next 10 min, and 0% for next 5 min. carotenoids were monitored at 450 nm and identified by comparing the uplc retention time with standard (compounds containing β-carotene, lutein, cryptoxanthin, and zeaxanthin), and then quantified based on standard curves. each sample was analyzed with three technical repeats. determination of total phenolic contents (tpc) of radish seed flour extract total phenolics of radish seed flour extract were measured using a modified folin-ciocalteu method (waterhouse, 2001). briefly, 50 μl of radish seed flour extract samples, gallic acid dilutions (standards), or water blank was added into each tube, respectively, which was filled with 3 ml of water and 250 μl of folin-ciocalteu reagent in advance. the sample were mixed well and placed at ambient temperature for 10 min. then, 750 μl of 20% (w/v) na2co3 solution was added into each test tube and mixed well prior to reaction at ambient temperature for 2 h. absorbance was read at 765 nm using a l5s uv/vis spectrophotometer (shanghai analytical instrument, china). each test was performed in triplicate. total phenolics were calculated as gallic acid equivalents (gae) per gram of radish seeds based on a gallic acid standard curve. analysis of phenolic acid composition by high performance liquid chromatography (hplc) soluble free phenolic acid compositions in each radish seed were analyzed with a previously reported procedure (jing et al., 2012). for quantitative analysis of phenolic acid composition in antioxidant extracts of radish seeds, the extracts were loaded on a zorbax eclipse xdb-c18 column (250 mm × 4.6 mm, 5 μm, agilent technologies, palo alto, ca, usa) using agilent 1260 infinity hplc system. phenolic acids were separated at a flow rate of 1 ml/min with a bi nary gradient (mobile phase of formic acid/h2o (0.1:99.9, v/v) was used as solvent a and mobile phase of formic acid/acetonitrile (0.1:99.9, v/v) was used as solvent b) with going from 0% to 7% over 5 min; from 7% to 25% b over 40 min; from 25% to 45% over 10 min. phenolic acids were identified by comparing the retention time and spectrum of peaks in the samples to that of the standards under the same hplc conditions. each sample was analyzed in three technical repeats. quantification of each phenolic acid was determined using external standards and total area under each peak. determination of total flavonoid contents (tfc) of radish seed flour extracts the tfc of radish seed flour extracts were determined according to a description by jing et al. (2012). thirty microliters of sample were added into a well of a 96-well plate and mixed with 180 μl of water followed by 10 μl of a 5% nano2 solution was added and allowed phytochemical composition and antioxidant capacity of radish seeds 317 to stand for 6 min. subsequently, 20 μl of a 10% alcl3 solution was added and allowed to stand for 6 min. finally, 60 μl of 4% (w/v) naoh solution were added into the mixture to stop the reaction for 15 min. absorbance was measured at 510 nm using a microplate reader (infinite f200 pro; tecan, switzerland). each test was performed in triplicate, and total flavonoids were calculated as catechin equivalents (cae) of radish seed based on a catechin standard curve. determination of total proanthocyanidin content (tpcc) of radish seed flour extracts the tpcc of radish seed flour extracts were determined using the hcl/ n-butanol assay (porter et al., 1985). briefly, 0.5 ml of seed flour extract was added into test tubes containing 6 ml of a 95% solution of n-butanol/hcl (95:5, v/v) and mixed well. subsequently, 0.2 ml of a solution of 2% (w/v) nh4fe(so4)2 in 2 mol/l hcl was added into the mixture and incubated for 40 min at 90 ºc. the absorbance was measured at 550 nm in l5s uv/vis spectrophotometer. tpcc was expressed as mg of cyanidin equivalents (cye) per gram of radish seeds. evaluation of antioxidant capacity by chemical assays dpph radical scavenging capacity briefly, 100 μl of 0.2 mmol/l dpph solution was mixed with 100 μl of radish seed flour 50% acetone extracts at different concentrations to initiate the reactions in each well of a 96-well plate. absorbance at 515 nm was determined after 40 min of reaction in a microplate reader (infinite f200 pro; tecan, switzerland). the blank contained only 200 μl of solvent, and the control consisted of 100 μl of solvent and 100 μl of 0.2 mmol/l dpph. the dpph radical-scavenging activity in the extracts was expressed as micromoles of trolox equivalents per gram of seed. oxygen radical absorbance capacity (orac) assay determination of orac of the studied compounds was performed according to the previous description (jing et al., 2014). same as samples tested, trolox standards were dissolved in 50% acetone. other reagents were prepared in 75 mmol/l phosphate buffer (ph 7.4). briefly, 30 μl of 20 μmol/l extracts (or 50% acetone for blank control) were mixed 225 μl fluorescein (81.63 nmol/l) in each well of a 96-well plate. subsequently, the plate was covered and incubated at 37 °c for 20 min. to start reaction, 25 μl of 0.36 m 2,2´-azobis(2amidinopropane) hydrochloride (aaph) were added and mixed well with above mixture in each well. the fluorescence was recorded with 5 min internal for 2 h (ex/em: 485/538 nm) at 37 °c in a microplate reader (infinite f200 pro; tecan, switzerland). a concentrationarea under the curve (auc) stand curve of trolox was constructed under the same experimental conditions and used for calculation of equivalents. results are expressed as micromoles of trolox equi valents per gram of radish seed. reactions were conducted in triplicate. the ferric reducing ability of plasma (frap) assay the frap assay was determined based on the reduction of fe3+tptz to a blue coloured fe2+-tptz (benzie and strain, 1996) with some modification. three hundred millimoles per liter of acetate buffer (ph 3.6), 10 mmol/l tptz and 20 mmol/l fecl3 ·6h2o were mixed together in a ratio of 10:1:1 (v/v/v) to produce the frap reagent. then, 3 ml of frap reagent was added into the test tube containing 100 μl of sample or standards and 300 μl of water and mixed well by vortexing followed by an incubation at 37 °c for 30 min. absorbance at 593 nm was measured using l5 uv spectrometer. trolox was used as standard for comparison and adequate dilution of sample was performed. results were reported as trolox equivalents (te) per gram of radish seed. reactions were conducted in triplicate. determination of angiotensin i converting enzyme (ace) inhibitory activity the ace inhibitory activity was measured according to a descrip tion of kurita et al. (2010). twenty five microliters of prepared antioxidant extracts were pre-incubated with 50 μl of substrates (4.7 mmol/l hippuryl-his-leu in 400 mmol/l potassium phosphate buffer (ph 8.3) containing 600 mmol/l nacl) at 37 °c. then 50 μl of 0.025 u/ml ace solution were added followed by an incubation at 37 °c for 40 min. aliquots (25 μl) of the reaction mixture were added into each well containing 150 μl of 0.3 mol/l naoh and 10 μl of 2% o-phthalaldehyde in methanol of a 96-well microplate. finally, the reaction was terminated by adding 20 μl of 3 mol/l hcl and kept at ambient temperature for 10 min. the fluorescence generated was recorded at excitation and emission wavelengths of 355 and 460 nm, respectively, in a microplate reader (infinite f200 pro; tecan, switzerland). for the control and blank, water was added instead of samples and ace, respectively. the inhibition rate was calculated as the following: fs fbace inhibitory activity % = (1 ) × 100 fc fb where fs is the fluorescence intensity of the test sample in the presence of the reaction mixture, fb is the fluorescence intensity of test sample in the absence of ace, and fc is the fluorescence intensity of buffer in the absence of the test sample. each sample was analyzed in three technical repeats. statistical analysis all results are expressed as the mean±sd. univariate anova among means of chemical contents, antioxidant activities, or ace-inhibitory activities in nine radish seeds were performed by least significant difference (lsd) test in general linear model at the level of 0.05. correlation among means was determined using a two-tailed pearson correlation test. statistics was analyzed using spss (version 14.0, spss inc., chicago, il, usa). results and discussion total phenolics, flavonoids, and proanthocyanidins in radish seeds the total phenolics, flavonoids, and proanthocyanidins levels in ra dish cultivars are shown in tab. 1. total phenolic levels varied from 9.15 mg (in hybrid #63) to 14.54 mg (in white pink) gallic acid equivalents (gae)/g dry mass (dm). among the cultivars, white pink seeds had the highest total phenolic levels (14.54 mg gae/g dm; p<0.05). in this study, the total phenolic levels obtained were higher than those previously reported in radish seeds of 6.7 or 6.1 mg gae/g dm (pajak et al., 2014; aguilera et al., 2015) and in pomegranate seeds of 1.29-2.17 mg gae/g dm (jing et al., 2012). the differences in results could be attributed to the species, cultivars, growing conditions, genotypes, and extraction methods. the results revealed that the tested radish seeds had high levels of total phenolic compounds. the total flavonoids in radish seeds varied from 0.511.36 mg catechin equivalents (cae)/g dm (tab. 1). tou xin hong seeds contained the highest level of total flavonoids (1.36 mg cae/g dm) followed by white pink and yanzhi #2 (~1.28 mg cae/g dm; p>0.05). it has been reported that radish leaves contain >200 mg cae/g dm flavonoids (kim et al., 2014), which is considerably higher than the level present in radish seeds. total proanthocyanidin levels 318 b. qian, y. pan, z. cai, p. jing china-grown radish seed oils consisted of 89% (w/w) unsaturated fatty acids, composing of 64.55-69.26% mufa and 20.33-25.11% pufa in tab. 2. all were higher than those grown in turkey (uluata and özdemir, 2012). the predominant mufas were erucic (c22:1, 34.53-40.32%) and oleic (c18:1, 16.20-21.30%) acids that were slightly higher than or comparable to the previous reports by uluata and özdemir (2012) or kaymak (2015), who found 22.1-36.4%, or 40.83% erucic and 12.6-23.9%, or 19.08% oleic acids in radish seed oils, respectively. the levels of linoleic (c18:2, 10.14-14.28%) and linolenic (c18:3, 8.40-11.17%) acids as major pufas were comparable to those as 10.09% and 7.02% for each in previous literatures (uluata and özdemir, 2012; kaymak, 2015). tocopherols and carotenoids tocopherols and carotenoids were detected in seeds oils of tested radish cultivars in tab. 3. the δ-tocopherol varied from 552.24 670.31 μg/g seed oils, whereas α-, γ-, or β-tocopherols were not detected. the level of δ-tocopherol in china-grown radish seeds was greater than those in turkey-grown radish seeds reported as 545.67 μg/g seed oils by uluata and özdemir (2012), who also found α-, γ-, and β-tocopherols as 12.41-28.66 μg/g seed oils in turkey radish seeds. tou xin hong (670.31 μg/g seed oils) and white pink (656.60 μg/g seed oils) contained greater amount of δ-tocopherol than other seven cultivars in this study (p<0.05). among the carotenoids, only lutein was detected with levels ranging from 4.82-8.95 μg/g seed oils. hybrid #63 seeds among all cultivars contained the greatest amount of lutein (p<0.05), comparable to one in soybeans of 1.6-14.8 μg/g seed oils (kanamaru et al., 2006). free phenolic acid composition the sinapic, vanillic, syringic, and ferulic acid were found in the seed flours of nine tested cultivars in tab. 4. a study reported that gallic, tab. 1: total phenolics, flavonoids, and proanthocyanidins in radish seeds cultivars phenolics flavonoids proantho cyanidins (mg gae/g) (mg ce/g) (mg cye/g) white round 11.60 ± 0.25ab 0.77 ± 0.10abcd 1.15 ± 0.10a korean white 9.34 ± 0.59ab 0.68 ± 0.06abcd 1.12 ± 0.35a japan white 9.84 ± 0.53ab 0.86 ± 0.07bcd 0.82 ± 0.17a hybrid #63 9.15 ± 0.94a 0.51 ± 0.07a 0.90 ± 0.01a hybrid #72 9.93 ± 0.25ab 0.90 ± 0.17cd 0.79 ± 0.21a white pink 14.54 ± 2.76c 1.28 ± 0.14e 0.87 ± 0.14a tou xin hong 10.79 ± 0.61ab 1.36 ± 0.10e 1.10 ± 0.23a xin lin mei 10.51 ± 0.23ab 0.61 ± 0.06abc 0.74 ± 0.24a yanzhi #2 12.21 ± 0.81bc 1.28 ± 0.10e 1.11 ± 0.26a values are expressed as mean ± sd (n=3). different letters within a column represent significant differences (p<0.05). gae: gallic acid equivalents; ce: catechin equivalents; cye: cyanidin equivalents tab. 2: fatty acid composition and total oil content of radish seeds cultivars percentage of fatty acids (%) total oil (g/100g 16:0 16:1 18:0 18:1 18:2 18:3 20:0 20:1 20:2 22:0 22:1 22:6 24:0 sfa mufa pufa seeds) white round 4.82 0.18 1.71 19.32 14.08 9.46 1.15 9.76 0.58 0.93 35.44 0.67 1.90 10.51a 64.70a 24.79a 38.65cd 0.12 0.01 0.12 0.14 0.35 0.14 0.02 0.11 0.05 0.02 0.52 0.10 0.13 0.45 0.17 0.20 0.67 korean white 4.67 0.17 1.81 20.70 10.68 8.40 1.37 11.44 0.48 1.15 36.95 0.77 1.40 10.40a 69.26b 20.33b 40.90abc 0.11 0.02 0.23 0.23 0.22 0.11 0.05 0.31 0.02 0.10 0.47 0.02 0.14 0.31 0.10 0.02 0.90 japan white 4.50 0.15 1.58 16.20 14.28 9.24 1.15 7.86 0.64 1.27 40.32 0.95 1.86 10.37a 64.55a 25.11a 43.06a 0.15 0.02 0.12 0.08 0.25 0.20 0.22 0.20 0.03 0.23 0.53 0.01 0.02 0.22 0.03 0.02 0.30 hybrid #63 4.62 0.15 1.97 21.77 12.02 10.40 1.36 9.72 0.47 1.18 34.53 0.60 1.21 10.34a 66.17c 23.49a 37.28d 0.05 0.04 0.03 0.31 0.31 0.22 0.13 0.31 0.01 0.15 0.22 0.04 0.21 0.41 0.04 0.06 0.26 hybrid #72 4.70 0.16 1.71 18.40 13.76 8.57 1.21 8.55 0.56 1.22 38.50 0.90 1.74 10.58a 65.61ac 23.79a 39.75bc 0.02 0.01 0.08 0.14 0.24 0.32 0.05 0.25 0.12 0.20 0.30 0.11 0.23 0.32 0.15 0.07 0.70 white pink 5.11 0.27 1.69 19.36 12.93 8.41 1.25 9.76 0.56 1.06 36.61 0.77 1.62 10.73a 66.00c 22.67ab 42.82a 0.16 0.13 0.07 0.23 0.15 0.15 0.07 0.32 0.01 0.02 0.51 0.03 0.02 0.14 0.86 0.27 0.28 tou xin hong 4.88 0.18 1.59 19.98 13.67 8.60 1.03 8.93 0.52 0.91 36.94 0.75 2.04 10.45a 66.03c 23.54a 36.87d 0.05 0.11 0.05 0.23 0.41 055 0.23 0.41 0.03 0.12 0.45 0.12 0.14 0.22 0.09 0.05 0.96 xin lin mei 4.70 0.19 1.91 21.30 10.14 11.17 1.36 9.80 0.44 1.17 35.79 0.67 1.36 10.50a 67.08d 22.42ab 40.24bc 0.08 0.08 0.13 0.13 0.52 0.62 0.14 0.35 0.02 0.23 0.38 0.23 0.05 0.12 0.12 0.07 0.49 yanzhi #2 4.72 0.17 1.67 19.55 13.75 9.32 1.14 9.84 0.57 0.93 35.81 0.70 1.84 10.30a 65.37a 24.34a 41.19ab 0.06 0.05 0.22 0.24 0.63 0.45 0.06 0.51 0.06 0.05 0.39 0.21 0.21 0.43 0.14 0.06 0.45 oils were extracted for 6 h from 40-mesh ground seeds by soxhlet extraction. data expressed as mean and standard deviation (n=3). sfas: saturated fatty acids. mufas and pufas represent monounsaturated fatty acids and polyunsaturated fatty acids, respectively. different letters within a column represent significant differences (p<0.05). ranged from 0.74-1.15 mg cyanidin equivalents (cye)/g dm (tab. 1). similar levels have been reported in pomegranate seeds of 0.68 1.82 mg cye/g dm (jing et al., 2012). total oil levels and fatty acid composition the fatty acid compositions of radish seed oils are shown in tab. 2. total oil levels (36.87-43.06 g/100 g seeds) in dry china-grown radish seeds were higher than or comparable to those (22.6-37.9 g/100 g seeds or 42.64 g/100 g seeds) in turkey-grown radish seeds reported by uluata and özdemir (2012) or kaymak (2015), repectively. the phytochemical composition and antioxidant capacity of radish seeds 319 protocatechuic, caffeic, p-coumaric, and ferulic acids were detected as free phenolic acids in radish seeds (pajak et al., 2014). ferulic acid was the predominant free phenolic acid (33.40-47.59 μg/g seeds) in tested radish seeds, much greater than those in radish seeds var. flamboyant 2 (ferulic acid, 21 μg/g seeds) (pajak et al., 2014). yanzhi #2 contained the highest levels of vanillic acid (18.77 μg/g seeds, p<0.05) among all tested seeds. antioxidant capacity more than one radical system was applied to investigate the radical scavenging capacities of radish seed extracts and results are shown in fig. 1. the 50% acetone extracts of radish seed flour (rsf) were used to quench dpph radical in the testing system. the dpph radical scavenging capacity of rsf from 14.84-26.35 μmol te/g seeds in fig. 1a, was much greater than those in previous studies reported as 12 μmol te/g (pajak et al., 2014) and 1.4 μmol te/g (aguilera et al., 2015). among all tested cultivars, the hybrid #63 showed the highest levels (p<0.05). the oxygen radical absorbance capacity of rsf extracts was determined using the orac assay. fig. 1b shows the orac values of rsf from nine tested cultivars. tou xin hong had the high orac value (36.32 μmol te/g seeds), followed by white round, white pink, hybrid #72, and yanzhi #2. hybrid #63 exhibited the lowest value (16.67 μmol te/g seeds) among all cultivars (p<0.05). the reducing power of rsf extracts were evaluated using the frap test. fig. 1c shows that hybrid #72 exhibited the higher frap value as 1.04 μmol te/g seeds than other cultivars that showed no differences. generally, the frap values of nine radish cultivar seeds were lower than those reported as 30.6 μmol te/g dw seeds (aguilera et al., 2015). positive correlations were found between total phenolics and flavonoids (r = 0.679, p = 0.044), proanthocyanidins and phenolic acids (r = 0.754, p = 0.019), flavonoids and orac (r = 0.668, p = 0.049). wolfe et al. (2008) also described that total phenolics from common fruits were significantly correlated to orac values (r = 0.761, p<0.05). in this study, we only evaluated the content of free phenolic acids, while majority of them may be present in radish seeds as a bound forms (i.e, esters and glycosides), which might be a main reason for lack of correlation between free phenolic acids and antioxidant activities. it is easy to understand that as the phenolics and flavonoids can quench or prevent the formation of reactive oxygen species and reactive nitrogen species. therefore, plant phenolics and flavonoids are critical in preventing various diseases associated with oxidative stress, such as cardiovascular diseases (manach et al., 2005). plant phenolics could reduce oxygen radicals and increase the bioavailability of no to promote vasodilation (schewe et al., 2008). additionally, plant flavonoids could promote rna expression of endothelial nitric oxide synthase to increase the level of no attributing to decreasing blood pressure (vasanthi et al., 2012). so, it suggested that the extracts of rsf exhibited a potential role in promoting cardiovascular health. ace-inhibitory activity angiotensin i converting enzyme (ace) catalyzes the conversion of angiotensin i into angiotensin ii, a strong vasoconstrictor that increases blood pressure (skeggs et al., 1956). many compounds were reported to exert both of antioxidant activity and ace-inhibitory activity (chopra et al., 1992; pihlanto et al., 2008). therefore, the nine china-grown radish seeds were evaluated for their inhibitory effect on ace, based on 1-ml extracts obtained from 2 mg of seed flour. yanzhi #2 inhibited 100% of ace activity, followed by hybrid #63 (40.16%; fig. 2). additionally, yanzhi #2 still exhibited 34.72% of ace inhibitory activity following a five-fold dilution, which remained higher than the ace-inhibitory activity of other extracts with the exception of hybrid #63 (p<0.01). positive correlations were found between vanillic acid and acetab. 3: tocopherol and lutein contents of radish seed oils1 cultivars δ-tocopherol (μg/g oils) lutein (μg/g oils) white round 562.24 ± 61.27a 7.90 ± 0.34c korean white 579.92 ± 15.90a 6.20 ± 0.08b japan white 585.15 ± 47.90a 8.55 ± 0.35d hybrid #63 591.55 ± 46.92a 8.95 ± 0.31e hybrid #72 610.16 ± 32.94ab 8.29 ± 0.10cd white pink 656.60 ± 35.20b 5.98 ± 0.08b tou xin hong 670.31 ± 25.78b 8.31 ± 0.13d xin lin mei 578.25 ± 14.88a 7.55 ± 0.12c yanzhi #2 552.24 ± 25.97a 4.82 ± 0.06a 1oils were extracted for 6 h from powder of radish seeds by the soxhlet method. δ-tocopherol was quantified by hplc. results are expressed as μg/g oils. values are expressed as mean±standard deviation (n = 3). different letters within a column represent significant differences (p<0.05). tab. 4: free phenolic acids in radish seeds1 cultivars sinapic vanillic syringic ferulic total white round 3.78±0.14a nd 4.87 ± 0.45ab 47.59 ± 1.01e 56.24 ± 1.57e korean white 3.62±0.45a 1.99 ± 0.28b 6.94 ± 0.70a 47.75 ± 1.86e 61.35 ± 2.98e japan white 3.23±0.13a 1.32 ± 0.08b 1.90 ± 0.26ab 34.47 ± 1.02c 40.92 ± 1.49c hybrid #63 3.00±1.11a nd nd 20.18 ± 1.73a 23.18 ± 1.84a hybrid #72 3.22±0.15a 0.46 ± 0.01b 3.03 ± 0.25ab 27.91 ± 1.65b 34.62 ± 2.06b white pink 2.82±0.30a 1.29 ± 0.05b 1.54 ± 0.13b 33.40 ± 1.42c 39.05 ± 1.90b tou xin hong 3.65±0.12a 2.02 ± 1.43b 3.73 ± 0.12ab 40.43 ± 2.43d 49.83 ± 2.81d xin lin mei 3.52±0.22a 1.25 ± 1.25b 4.29 ± 0.56ab 35.70 ± 2.31cd 41.24 ± 3.21c yanzhi #2 nd 18.77 ± 1.65a 4.55 ± 0.43ab 46.77 ± 3.20e 70.09 ± 5.28f 1 sinapic, vanillic, syringic, and ferulic stand for sinapic, vanillic, syringic, and ferulic acids. results expressed as micrograms of individual standard per gram of radish seeds. data expressed as mean±standard deviation (n = 3). different letters within a column represent significant differences (p<0.05); nd, not detected. 320 b. qian, y. pan, z. cai, p. jing inhibitory activity (r = 0.890, p = 0.001). there were no correlations between antioxidant activity and ace-inhibitory activity, or with vanillic acid. another report also described the similar results, in which intake of flavonoid-rich apple peel extract did not cause significant differences in serum and lung ace activity at week eight but reduced blood pressure after 5 weeks of treatment in spontaneously hypertensive rats (shr) possibly through endogenous antioxidant pathways (balasuriya et al., 2015). the ic50 of vanillic acid for ace-inhibitory activity was about 8 mmol/l, which linear correlated to the molecular docking score between phenolic compounds and testicular ace (al shukor et al., 2013). the ace-inhibitory activity of vanillic acid was proposed to be charge-charge interactions with the zinc ion in the active site of ace (al shukor et al., 2013). therefore, vanillic acid might be responsible for the ace-inhibitory activity of radish seeds but not for their antioxidant activities like phenolics and flavonoids (schewe et al., 2008; vasanthi et al., 2012). certainly, as mentioned in the introduction, some flavonoids and anthocyanins also had ace inhibitoring activity (loizzo et al., 2007; ojeda et al., 2010). on the other hand, compounds with sulfhydryl groups are effective as free radical scavengers and as ace 6     fig. 2. 0 10 20 30 40 50 60 70 80 90 100 110 a c e in hi bi to ry a ct iv it y (% o f co nt ro l) radish seeds a c d c b bc a a a a fig. 2: ace inhibitory activities of antioxidant extracts of radish seeds. ace inhibitory activity was measured from 1-ml extracts obtained from 2 mg of seed flour except for yanzhi #2 (5d), which had a five-fold dilution. tests were conducted in triplicate. vertical lines represent standard deviations. columns marked with different letters are significantly different (p<0.05). 6     fig. 1. a) dpph radical scavenging capacity (µmol te/g) a a c b b b a b b a b a a b b ab a a a a b a a a a a a c) frap (µmol te/g) b) orac (µmol te/g) a fig. 1: antioxidant activities of radish seeds: a) dpph· radical scavenging capacity values; b) orac values; c) frap. results expressed as μmol trolox equivalents (te)/g rsf. tests were conducted in triplicate. vertical lines represent standard deviations. columns marked with different letters are significantly different (p<0.05). phytochemical composition and antioxidant capacity of radish seeds 321 inhibitors (terras et al., 1992). so, it could not be overlooked that the ace inhibitory activity of the extracts in this study was also associated with some other phytochemicals. conclusions the results of this study confirmed that radish seeds are good sources of unsaturated fatty acids, tocopherols, lutein, and antioxidants. radish cultivars may differ in seed composition and health properties; seed composition might be dependent on the geographical location. this study provides important information for developing valueadded utilization of radish seeds or seed fractions such as oil and flour as nutraceuticals or functional food ingredients. acknowledgments this study was funded by the national key r&d program of china (grant no. 2016yfd0400200) and the national nature science foundation of china (grant no. 31371756). conflict of interest authors declare no conflict of interest. references aguilera, y., herrera, t., benitez, v., arribas, s.m., de pablo all., esteban, rm., martin-cabrejas, m.a., 2015: estimation of scavenging capacity of melatonin and other antioxidants: contribution and eva luation in germinated seeds. food chem. 170, 203-211. doi: 10.1016/j.foodchem.2014.08.071. al shukor, n., van camp, j., gonzales, g.b., staljanssens, d., struijs, k., zotti, m.j., raes, k., smagghe, g., 2013: angiotensinconverting enzyme inhibitory effects by plant phenolic compounds: a study of structure activity relationships. j. agric. food chem. 61, 1183211839. doi: 10.1021/jf404641v ávila, r.n.d.a., sodré, j.r., 2012: physical-chemical properties and ther mal behavior of fodder radish crude oil and biodiesel. ind. crop. prod. 38, 54-57. doi: 10.1016/j.indcrop.2012.01.007 balasuriya, b.w.n., rupasinghe, h.p.v., 2011: plant flavonoids as angiotensin converting enzyme inhibitors in regulation of hypertension. funct. food. health dis. 5, 172-188. balasuriya, n., rupasinghe, h.p.v., sweeney, m., mccarron, s., gottschall-pass, k., 2015: antihypertensive effects of apple peel extract on spontaneously hypertensive rats. pharmacol. 6, 371-376. doi: 10.5567/pharmacologia.2015.371.376 benzie, i.f., strain, j.j., 1996: the ferric reducing ability of plasma (frap) as a measure of “antioxidant power”: the frap assay. anal biochem. 239, 70-76. chopra, m., beswick, h., clapperton, m., dargie, h.j., smith, w.e., mcmurray, j., 1992: antioxidant effects of angiotensin-converting enzyme (ace) inhibitors: free radical and oxidant scavenging are sulfhydryl dependent, but lipid peroxidation is inhibited by both sulfhydryl and nonsulfhydryl-containing ace inhibitors. j. card. pharmacol. 19, 330-340. doi:10.1097/00005344-199203000-00005 hassan, a.m., alam, s.s., abdel-aziem, s.h., ahmed, k.a., 2011: benzo-a-pyrene induced genotoxicity and cytotoxicity in germ cells of mice: intervention of radish and cress. j genet. eng. biotech. 9, 65-72. doi: 10.1016/j.jgeb.2011.05.006 jing, p., ye, t., shi, h.m., sheng, y., slavin, m., gao, b.y., liu, l.w., yu, l.l., 2012: antioxidant properties and phytochemical composition of china-grown pomegranate seeds. food chem. 132, 1457-1464. doi: 10.1016/j.foodchem.2011.12.002 jing, p., zhao, s.j., lu, m.m., cai, z., pang, j., song, l.h., 2014: multiple-fingerprint analysis for investigating quality control of flammulina velutipes fruiting body polysaccharides. j. agri. food chem. 62, 1212812133. doi: 10.1021/jf504349r kanamaru, k., wang, s., abe, j., yamada, t., kitamura, k., 2006: identification and characterization of wild soybean (glycine soja sieb. et zucc.) strains with high lutein content. breeding sci. 56, 231-234. doi: 10.1270/jsbbs.56.231 kaymak, h.c., 2015: profile of (n-9) and (n-7) isomers of monounsaturated fatty acids of radish (raphanus sativus l.) seeds. j. am. oil chem. soc. 92, 345-351. doi: 10.1007/s11746-015-2600-0 kim, m., kim, e., kwak, h.s., jeong, y., 2014: the ingredients in saengshik, a formulated health food, inhibited the activity of alpha-amylase and alpha-glucosidase as anti-diabetic function. nutr. res. pract. 8, 602-606. doi: 10.4162/nrp.2014.8.5.602 kurita, i., maeda-yamamoto, m., tachibana, h., kamei, m., 2010: antihypertensive effect of benifuuki tea containing o-methylated egcg. j. agri. food chem. 58, 1903-1908. doi: 10.1021/jf904335g loizzo, m.r., said, a., tundis, r., rashed, k., statti, g.a., hufner, a., menichini, f., 2007: inhibition of angiotensin converting enzyme (ace) by flavonoids isolated from ailanthus excelsa (roxb.) 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macedonian academy of sciences and arts, skopje, macedonia 4 department of biology, faculty of arts and science, al-mergeb university, al-khoms, libya the in vitro antioxidative and cytotoxic effects of selected salvia species water extracts ana alimpić 1*, nikola kotur 2, biljana stanković 2, petar d. marin 1, vlado matevski 3, najat beleed al sheef 4, sonja duletić-laušević 1 (received october 10, 2014) * corresponding author summary the current paper presents antioxidant and cytotoxic activities and total phenolic and flavonoid content of the selected species of genus salvia (lamiaceae) growing wild in macedonia (s. jurisicii košanin, s. amplexicaulis lam., s. ringens sibth. & sm.) and libya (s. fruticosa mill. and s. lanigera poir.). crude water extracts, obtained from aerial parts, were yielded from 6.50 to 14.32 %. total phenolic content was the highest in water extracts of s. amplexicaulis and s. ringens (226.30 and 189.01 mg gae/g, respectively), while the flavonoids were the most abundant in s. jurisicii extract (32.36 mg qe/g). antioxidant activities of extracts were measured using dpph, abts and frap assays. s. amplexicaulis and s. ringens extracts showed the strongest antioxidant activity, measured using dpph (14.21 and 23.44 μg/ml, respectively) and abts assays (2.91 and 2.42 mg aae/g, respectively). in frap assay, s. amplexicaulis and s. fruticosa extracts exhibited strongest activity (1406.73 and 1191.51 μmol fe(ii)/g). water extract of s. amplexicaulis and s. ringens performed the strongest cytotoxic activity against k562 cells (151.07 and 173.06 μg/ml, respectively). based on these findings, it can be concluded that s. amplexicaulis and s. ringens water extracts could be considered as possible source of antioxidant and cytotoxic agents. introduction the genus salvia (lamiaceae) represents an enormous and cosmopolitan assemblage of nearly 1000 species worldwide of which 36 were found in europe (hedge, 1972) and 10 in libya (jafri and el-gadi, 1985). the representatives of salvia genus are widely cultivated and used in flavoring and folk medicines. sage and rosemary from the lamiaceae family were shown to have similar patterns of phenolic compounds and the antioxidant activity had been attributed mainly to carnosic, rosmarinic acid and their isomers. additional classes of active compounds include terpenoids, flavonoids and other phenolic acids (lu and foo, 2001). majority of these compounds, excluding terpenoids, are water-soluble and present in aqueous extract obtained using common techniques of extraction (tiwari et al., 2011). the genus salvia was the research topic of numerous chemical, medicinal and pharmacological studies. species of salvia showed diverse biological activities of plant material and/or isolated essential oil/extracts due to the presence of large number of different compounds. the antioxidant (couladis et al., 2003; janicsák et al., 2010; orhan et al., 2013; alimpić et al., 2014), cytotoxic (fiore et al., 2006; kamatou et al., 2005; janicsák et al., 2007; abu dahab et al., 2012), antimicrobial (kamatou et al., 2005; hawas and el-asnari, 2006), antiinflammatory (kamatou et al., 2005; 2010), antimalarial (kamatou et al., 2005), anticholinesterase (şenol et al., 2010, orhan et al., 2012; 2013), etc. effects were reported. s. jurisicii košanin, perennial herb inhabiting arid habitats, is an endemic species in the central part of republic of macedonia (hedge, 1972). it was previously investigated for antioxidant activity (janicsák et al., 2010). s. amplexicaulis lam. is a perennial plant, distributed on balkan peninsula (hedge, 1972). it was investigated for vasodepressor (ulubelen, 2003), antioxidant, and neurobiological activity (orhan et al., 2012) of extracts. s. ringens sibth. & sm. is a hardy herbaceous perennial plant, distributed in south and eastern parts of balkan peninsula (hedge, 1972). total acetone extract and some isolated compounds showed cytotoxic activity against hela cells (janicsák et al., 2007), while ethanol extracts performed strong antioxidant activity (couladis et al., 2003). s. lanigera poir. is perennial herb with thick woody rootstock dis tributed in north africa, from northern egypt and arabia, to the south of turkey and iran (jafri and el-gadi, 1985). there are a few reports on phenolic composition, antimicrobial and cytotoxic activity of s. lanigera extracts (hawas and el-asnari, 2006; shaheen et al., 2011). s. fruticosa mill. (s. triloba l.) is subshrub distributed in canary islands and north africa. it is sometimes used for flavouring tea and cultivated as an ornamental plant (jafri and el-gadi, 1985). jordanian s. fruticosa ethanol extract performed significant cytotoxic activity (abu-dahab et al., 2012). the objective of this study was to investigate antioxidative and cytotoxic activity as well as phenolic/flavonoid contents in water extracts of selected macedonian and libyan salvia species. material and methods plant material aerial parts of the salvia species were collected in flowering period from their natural populations at localities in macedonia and libya and were identified by prof. p.d. marin and prof. v. matevski. plant material was dried and kept in shadow at room temperature for further processing. total sample for each species consisted of at least 20-50 individuals (about 500 g of dry material, depending of species). voucher specimens were deposited in the herbarium of the institute of botany and botanical garden “jevremovac”, faculty of biology, university of belgrade. collection data and voucher numbers of investigated salvia species are given in tab. 1. preparation of water extracts extracts were prepared of whole aerial plant parts using classic maceration procedure. dry plant material (5 g), randomly taken 116 a. alimpić, n. kotur, b. stanković, p.d. marin, v. matevski, n.b. al sheef, s. duletić-laušević from whole collected sample of each species, was grounded in small pieces (2-6 mm) and extracted by 50 ml of boiling distilled water (10 % w/v). extraction was performed during 24 h at room temperature. the mixture was exposed to ultrasound 1 h before and after 24 h-maceration. subsequently, extracts were filtered through a paper filter (whatman no.1) and evaporated under reduced pressure by the rotary evaporator (buchi rotavapor r-114). the obtained crude extracts (tab. 2) were stored in the fridge at +4 °c for further experiments. as a control. bha, bht and ascorbic acid were used as positive controls (standards). each blank, samples and standards’ absorbances were measured in triplicate. absorbance of the reaction mixture was measured after 30 min in the dark at room temperature at 517 nm using the jenway 6305uv/vis spectrophotometer. the decrease of absorption of dpph radical at 517 nm was calculated using equation: inhibition of dpph radical (%) = [(ac-as)/ac] * 100% where ac is the absorbance of control (without test sample) and as is the absorbance of the test samples at different concentrations. ic50 values (µg/ml) (concentrations of the test samples and standard antioxidants providing 50% inhibition of dpph radicals) were calculated from dpph absorption curve at 517 nm. abts assay abts assay is performed according to procedure of miller et al. (1993) with some modifications. fresh abts+ solution was prepared 12-16 hours before use by dissolving abts in 5 ml of 2.46 mmol potassium-persulfate to obtain concentration of 7 mmol/l and stored in the dark at room temperature. the abts+ solution was dissolved by distilled water to achieve an absorbance of working solution 0.700 ± 0.020 at 734 nm. 50 μl of test samples (1 mg/ml) and/or standards (0.1 mg/ml) were mixed with 2 ml of diluted abts+ solution and incubated for 30 min at 30 ºc. absorbance was recorded at 734 nm using jenway 6305uv/vis spectrophotometer. distilled water was used as blank. bha and bht dissolved in methanol were used as standards. abts activity was calculated from ascorbic acid calibration curve (0-2 mg/l) and expressed as ascorbic acid equivalents per gram of dry extract (mg aae/g). ferric-reducing ability of plasma (frap) assay the frap assay was performed according to benzie and strain (1996) procedure with slight modifications. frap reagent was prepared freshly to contain sodium acetate buffer (300 mmol/l, ph 3.6), 10 mmol/l tptz in 40 mmol/l hcl and fecl3*6h2o solution (20 mmol/l), i.e. in proportion 10:1:1(v/v/v), respectively. working frap solution was warmed to 37 °c prior to use. 100 μl of test sample (500 μg/ml) were added to 3 ml of working frap reagent and absorbance was recorded at 593 nm after 4 min using the tab. 1: collection data and voucher numbers of examined salvia species salvia species collection data voucher no. locality date geographical data s. jurisicii štip n 41°50.324' košanin (macedonia) 11.7.2011 e 22°08.289' beou; 16674 alt. 420 m s. amplexicaulis pletvar n 41°22.169' lam. (macedonia) 12.7.2011 e 21°39.180' beou; 16673 alt. 1010 m s. ringens krivolak n 41°53.33' sibth. & sm. (macedonia) 2.7.2012 e 21°41.08' beou; 16675 alt. 229 m s. lanigera zintan n 31°55.50' poir. (libya) 22.3.2010 e 12°14.54' beou; 16880 alt. 694 m s. fruticosa biadda n 32°45.59' mill. (libya) 10.3.2010 e 21°44.30' beou; 16702 alt. 624 m tab. 2: the yield, total phenolic and flavonoid content of salvia water extracts. values are presented as mean ± standard deviation. water extracts extract yielda total phenolic total flavonoid contentb contentc s. jurisicii 9.50 81.03 ± 0.216 32.36 ± 0.731 s. amplexicaulis 14.32 226.30 ± 1.179 17.87 ± 0.089 s. ringens 6.50 189.01 ± 1.699 22.64 ± 0.898 s. lanigera 7.32 58.47 ± 0.200 17.18 ± 0.544 s. fruticosa 7.62 67.68 ± 0.001 21.73 ± 0.163 a % of weight of dry plant material b mg gae/g dry extract c mg qe/g dry extract evaluation of antioxidant activity antioxidant activity was evaluated using three spectrophotometric assays: dpph, abts, and frap. stock solutions of dry extracts were prepared in the distilled water in concentration of 1000 μg/ ml (w/v). dpph assay for evaluation of antioxidant activity of extracts, 2,2-dyphenyl-1 picrylhydrazyl (dpph) free radical scavenging method (blois, 1958) with slight modifications was used. stock extract solution was diluted with methanolic solution of dpph (40 μg/ml) to adjust the final volume of reaction mixture (2000 μl) of the test tube. methanol was used as a blank, while methanol with dpph solution was used antioxidative and cytotoxic effects salvia water extracts 117 jenway 6305uv/vis spectrophotometer. blank was prepared to contain distilled water instead of extract. ascorbic acid, bha and bht dissolved in methanol in concentration 0.1 mg/ml were used as standards. the same procedure was repeated for standard solution of feso4 * 7h2o (200-1600 μmol/l) in order to construct calibration curve. frap values of sample was calculated from standard curve equation and expressed as μmol fe (ii)/g dry extract). determination of total phenolic content the total phenolic content was measured using spectrophotometric method (singleton and rossi, 1965). the reaction mixture was prepared by mixing 0.2 ml of extract solution in concentration of 1 mg/ml, 1 ml of 10 % folin-ciocalteu reagent and 0.8 ml of 7.5 % na2co3. blank was prepared to contain distillated water instead of extract. absorbance was recorded at 740 nm after 2 h incubation at room temperature using jenway 6305uv/vis spectrophotometer. phenolic content in samples was calculated from standard curve equation and expressed as gallic acid equivalents (mg gae/g dry extract). determination of flavonoid concentration flavonoid concentrations of samples were measured spectrophotometrically according to procedure of park et al. (1997). the reaction mixture was prepared by mixing 1 ml of extract solution in concentration 1 mg/ml, 4.1 ml of 80 % ethanol, 0.1 ml of 10 % al(no3)3 x 9 h2o and 0.1 ml 1m ch3cook. blank was prepared to contain 96 % ethanol instead of extract. after 40 min of incubation at room temperature, absorbance was measured at 415 nm using jenway 6305uv/vis spectrophotometer. concentration of flavonoids in samples (mg/ml) was calculated from standard curve equation and expressed as quercetin equivalents (mg qe/g dry extract). cytotoxic assay − mtt assay to assess cytotoxic effect of sage water solutions, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt) assay was performed (drakulić et al., 2012). it is a colorimetric assay which detects conversion of yellow tetrazolium salt into purple formazon. the conversion is catalyzed by cellular enzymes and its rate represents measure of cells viability. k562 cells are human immortalised myelogenous leukemia line. k562 cells are of the erythroleukemia type. the line is derived from a cml patient in blast crisis. k562 cells were maintained in essential minimal medium (mem) supplemented with 10 % fcs. cells were treated in 96 well plates for 48 h with 50, 100, 150, 200, 300 and 400 μg/ml sage water extracts in mem. after addition of mtt solution (0.5 mg/ml), plate was incubated for additional 3 h. acidified isopropanol was added to dissolve tetrazolium salts. absorbance was measured at 620 nm. statistical analysis all experimental measurements were carried out in triplicate and are expressed as average of three measurements ± standard deviation. pearson’s correlation coefficients were calculated between on one hand total phenolics and flavonoids and on the other hand cytotoxic and antioxidant assays and interpreted according to taylor (1990). calculations and constructing of the charts were performed using the ms office excel, 2007. results and discussion the yield of extract, total phenolic and flavonoid content the yields were expressed as percent of dry extract of dry plant material. the yields of obtained water extract were ranged from 14.32 % for s. amplexicaulis to 6.50 % for s. ringens extract (tab. 2). considering the uniform extraction procedure applied in this study, variations in extract’s yields can be attributed to differences of plant material (species). in some previous studies researchers have observed that type of plant material, choice of extraction solvent and extraction procedure affects composition, activity and possible future use of the obtained extract (tiwari et al., 2011). şenol et al. (2010) obtained that yield of methanolic extract of 55 salvia taxa varied between 2.88 and 13.41 % and our results are in agreement with these findings. total phenolic and flavonoid contents of water extracts were eva luated spectrophotometrically and expressed as gallic acid equivalents/g dry extract and flavonoids concentrations as quercetin equivalents/g dry extract. as can be seen in tab. 2, the highest amount of phenolics was measured in water extracts of s. amplexicaulis and s. ringens (226.30 and 189.01 mg gae/g, respectively), while the content of phenolics in the other extracts was lower than 100 mg gae/g. on the contrary to phenolics, flavonoids were the most abundant in s. jurisicii extract (32.36 mg qe/g). when the obtained results are compared to the values for total phenolic content of camellia sinensis green tea (140.11 mg gae/g) and ginkgo biloba (140.18 mg gae/g) standardized extract (stanković et al., 2010), it can be seen that s. amplexicaulis and s. ringens have higher values. our results are in agreement with the literature data for ethanol extracts of fourteen turkish salvia species (57.10-218.09 mg gae/g for total phenols and 8.29-108.78 mg qe/g for flavonoids) obtained by orhan et al. (2013). stagos et al. (2012) found total phenolic content of 267 and 190 mg gae/g for methanolic and water extract of s. fruticosa from greece, respectively. as many researchers reported, amount of extracted phenolics and flavonoids depends on selection of extraction solvent. evaluation of antioxidant activity oxygen sometimes can be fatal for the organisms although eukaryotic organisms cannot exist without it, which is known as oxygen paradox. a free radical is any species capable of independent existence that contains one or more unpaired electrons. reactive oxygen species (ros) may damage important cellular molecules such as dna, proteins and lipids, causing cancer, cardiovascular, neurodegenerative and other diseases (halliwell, 2006). antioxidant activities of salvia species water extracts measured using dpph, abts and frap assays are presented in tab. 3. dpph and abts assays were applied to examine scavenging activity of extracts, while frap assay measured ability of extracts to reduce fe (iii) to fe (ii) ion. water extract of s. amplexicaulis showed markedly the strongest antioxidant activity in all three assays. tab. 3: dpph, abts and frap activity of salvia water extracts. water extracts dpph assay abts assay frap assay (ic50, μg/ml) (mg aae/g) (μmol fe(ii)/g) s. jurisicii 212.24 1.15 ± 0.062 290.14 ± 6.880 s. amplexicaulis 14.21 2.91 ± 0.019 1406.73 ± 8.055 s. ringens 23.44 2.42 ± 0,019 615.44 ± 6.720 s. lanigera 230.87 1.77 ± 0.085 79.13 ± 5.255 s. fruticosa 48.11 1.98 ± 0.005 1191.51 ± 8.109 positive controls bht 17.94 2.75 ± 0.021 445.34 ± 5.772 bha 13.37 2.82 ± 0.011 583.72 ±5.255 ascorbic acid 5.11 / 180.81 ± 8.607 118 a. alimpić, n. kotur, b. stanković, p.d. marin, v. matevski, n.b. al sheef, s. duletić-laušević 200 μg/ml), followed by s. fruticosa and s. lanigera (ic50 200 400 μg/ml) and s. jurisicii (> 400 μg/ml). some of the species examined in this study were partially investigated before for their cytotoxic activity. ethanol extract of s. fruticosa collected in jordan showed lower ic50 values (17.43-38.91 μg/ ml) against breast cancer cell lines measured by sulphorhodamine b assay (abu-dahab et al., 2012) than those in our study. water extract of libyan s. lanigera in this study was less active than several extracts of egyptian s. lanigera (ic50 values from 9.83 to over 100 μg/ml), especially obtained by acetone (shaheen et al., 2011). janicsák et al. (2007) reported on cytotoxic activity of bulgarian s. ringens extract and its isolated components. the literature data on cytotoxic activity of s. ringens, s. jurisicii and s. amplexicaulis were not available. previous studies have demonstrated that south african and jordanian salvia species showed ic50 values from approximately 20 to above 100 μg/ml (kamatou et al., 2005) and from 90 to 400 μg/ml (fiore et al., 2006), respectively. our findings are in accordance with previous reports. correlation between cytotoxic and antioxidant activities and total phenolic and flavonoid content pearson’s correlation coefficients were calculated between total phenolics and flavonoid content of the salvia water extracts and their antioxidant and cytotoxic activities (tab. 4). in this study, interpretation of correlation coefficients according to taylor (1990) was chosen. on the contrary to abts and frap activity, cytotoxic and dpph activity of extracts were negatively correlated to phenolic and positively to flavonoid content because of presenting of results as ic50 values (tab. 4). correlation between total phenolic and flavonoid contents was negative (data not presented). it was previously reported that water soluble flavonoids (mostly anthocyanins) have no antimicrobial significance and water soluble phenolics were only important as antioxidant compounds (tiwari et al., 2010). however, obtained results indicated that biological activities of extracts were negatively correlated to flavonoid content in extracts. previously, ben farhat et al. (2014) reported on negative correlation of some flavonoids from salvia officinalis extracts and antioxidant activity evaluated by dpph, abts and frap assays. our findings are in agreement with various studies which showed that antioxidant activity of rosemary and sage, both belonging to the family lamiaceae, is mostly manifested by presence of the phenolic acids (rosmarinic and carnosic acids and their derivates) and then terpenoids, flavonoids and other phenolic acids (lu and foo, 2001; kamatou et al., 2010; orhan et al., 2012). tab. 4: linear correlation coefficients (r) of cytotoxic and antioxidant activities versus total phenolic and flavonoid content of salvia water extracts total phenolic content flavonoid content cytotoxic activity -0,9514c 0,4886b dpph activity -0,7338c 0,3248a abts activity 0,8394c -0,6972c frap activity 0,5512b -0,3169a according to taylor (1990): a r ≤ 0.35 weak correlation; b 0.36 < r < 0.67 moderate correlation; c 0.68 < r < 1 strong correlation fig. 1: cytotoxic activity of water extracts of tested salvia species tested against k562 cell line conclusions based on these findings, it can be concluded that some of the examined extracts could be taken into consideration as possible antidpph activity of extracts was assessed as good (ic50<30 μg/ml) for s. amplexicaulis and s. ringens, as moderate 30<ic50<80 μg/ ml) for s. fruticosa and as poor (ic50>80 μg/ml) for s. jurisicii and s. lanigera according to kamatou et al. (2010). s. amplexicaulis water extract showed the best dpph radical scavenging activity (14.21 μg/ml), higher than activity of the synthetic antioxidant bht (17.94 μg/ml) and commercially used green tea (20.62 μg/ml) (stanković et al., 2010) and very close to bha (13.37 μg/ml). besides, water extract of s. amplexicaulis performed stronger activity than ethanol and methanol extracts (28.74 and 21.28 μg/ml, respectively) as previously reported by alimpić et al. (2014). s. fruticosa water extract showed lower dpph activity than those measured by stagos et al. (2012) for methanolic and water s. fruticosa extracts, 22 and 16 μg/ml, respectively. janicsák et al. (2010) previously found low antioxidant activity of s. jurisicii aqueous-methanol extract (191.2 μg/ml), as confirmed in this study. in abts assay, s. amplexicaulis and s. ringens showed the strongest activity (> 2 mg aae/g), while other extracts were quite weaker than aforementioned (tab. 3). some researchers preferred to express abts activity by ic50 value (kamatou et al., 2010; stagos et al., 2012), and more frequently using standard equivalents (trolox, ascorbic acid, etc.). in this study, the ascorbic acid calibration curve was chosen for presentation of obtained results. li et al. (2008) measured wide range of abts activity of selected chinese medicinal plants (0.97-265.43 μmol trolox/g dry plant material). s. fruticosa showed activity of 13 μg/ml for methanolic and 29 μg/ml for water extract (stagos et al., 2012). s. amplexicaulis followed by s. fruticosa water extract showed frap activity over 1000 μmol fe(ii)/g. in contrast, s. lanigera water extract performed frap activity lower than 100 μmol fe(ii)/g (tab. 3). benzie and strain (1996) suggested expressing of results using fe(ii) calibration curve. some researches preferred to present results by ic50 values or as trolox equivalents. the lacking of a unique form of presenting results makes comparison of results obtained in various studies rather complicated. li et al. (2008) measured frap ability of methanol extracts of 45 chinese medicinal plants, and results ranged as 1.23-453 μmol fe(ii)/g. ethyl-acetate extracts of selected turkish salvia species exhibited quite low frap activity, whereas methanol extracts were highly active (orhan et al., 2012). cytotoxic activity of extracts cytotoxic activity of extracts was tested using mtt assay against k562 cell line over 48 h. obtained results, expressed as ic50, are presented in fig. 1. all tested water extracts exhibited cytotoxic effect on k562 cells. among examined salvia species, s. amplexicaulis and s. ringens showed the strongest antioxidant activity (ic50 < antioxidative and cytotoxic effects salvia water extracts 119 oxidant and cytotoxic agents. the results showed that certain species have optimal ratio of the yield, total phenolic and flavonoid content and performed antioxidant and cytotoxic activities, such as macedonian s. amplexicaulis, s. ringens and libyan s. fruticosa. taking into account non-toxicity of the water as extraction solvent, their application in prevention and treatment of some free radical caused disorders such as cancer, cardiovascular and neurodegenerative diseases could be proposed. future studies will provide data on qualitative composition of phenolics and other possible biologically active components of the water extracts (research in progress). acknowledgments authors are grateful to the ministry of education, science and technological development of serbia for financial support (project no. 173029 and 41004). references abu-dahab, r., afifi, f., kasabri, v., majdalawi, l., naffa, r., 2012: comparison of the antiproliferative activity of crude ethanol extracts of nine salvia species grown in jordan against breast cancer cell line models. pharmacogn. mag. 8, 319-324. alimpić, a., oaldje, m., matevski, v., marin, p.d., duletić-lausević, s., 2014: antioxidant activity and total phenolic and flavonoid contents of salvia amplexicaulis lam. extracts. arch. biol. sci. 66, 307-316. ben farhat, m., chaouch-hamada, r., sotomayorc, j.a., landoulsi, a., jordán, m., 2014: antioxidant potential of salvia officinalis l. residues as affectedby the harvesting time. ind. crop. prod. 54, 78-85. benzie, i.f., strain, j.j., 1996: the ferric reducing ability of plasma (frap) as a measure of „antioxidant power“: the frap assay. anal. biochem. 239, 70-76. blois, m.s., 1958: antioxidant determinations by the use of a stable free radical. nature 181, 1199-1200. couladis, m., tzakou, o., verykokidou, e., harvala, c., 2003: screening of some greek aromatic plants for antioxidant activity. phytother. res. 17, 194-195. drakulić, d., krstić, a., stevanović, m., 2012: establishment and initial characterization of sox2-overexpressing nt2/d1 cell clones. genet. mol. res. 11, 1385-1400. fiore, g., nencini, c., cavallo, f., capasso, a., bader, a., giorgi, g., micheli, l., 2006: in vitro antiproliferative effect of six salvia species on human tumor cell lines. phytother. res. 20, 701-703. halliwell, b., 2006: reactive species and antioxidants. redox biology is a fundamental theme of aerobic life. plant physiol. 141, 312-322. hawas, u., el-ansari, m., 2006: flavonoids and antimicrobial activity of salvia lanigera poir. jasmr, 1, 159-167. hedge, i.c., 1972: salvia l. in: tutin, t.g., heywood, v.h., burges, n.a., valentine, d.h., walters, s.m., webb, d.a. 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(purslane) extracts display antioxidant and hypoglycaemic effects vincenzo sicari1*, monica rosa loizzo2, rosa tundis2, antonio mincione1, teresa maria pellicanò1 (submitted: november 3, 2017; accepted: february 7, 2018) * corresponding author summary purslane (portulaca oleracea l.) is a member of the family por tulacaceae. due to its many health benefits, it is listed in a world health organization database. the aim of this work is to investigate the purslane extracts for their chemical profile and bioactivity. in this study, two different solvents (meoh/h2o and etoh) were applied to fresh and dried leaves. the extracts were analysed using hplc-dad. phenolic acids (caffeic acid, p-coumaric acid and ferulic acid) and flavonoids (apigenin, kaempferol, luteolin, quercetin, isorhamnetin, kaempferol-3-o-glucoside and rutin) were identified in all samples. quercetin and p-coumaric acid were the most abundant compounds. total antioxidant activity was measured by using the abts and dpph tests, and the ferric reducing antioxidant power (frap) assay. hypoglycaemic properties were investigated via the inhibition of carbohydrate-hydrolysing enzymes, α-amylase and α glucosidase. fresh hydroalcoholic purslane extract exhibited the highest radical scavenging potential in both abts and dpph test (ic50 values of 52.86 and 66.98 μg/ml, respectively), whereas dried hydroalcoholic purslane extract showed the highest α-glucosidase inhibitory potential (ic50 value of 45.05 μg/ml). collectively these data show the health properties of this widely consumed salad plant. keywords: portulaca oleracea, phenols, hplc-dad, antioxidant potential, carbohydrate hydrolysing enzymes. introduction purslane (portulaca oleracea) is an annual succulent plant of the family portulacaceae. originally, from india, today it has spread throughout the world’s temperate zones, including italy. the plant, which can grow to a height of 35 cm, has reddish-brown, prostrate, fleshy, branching stems, and light-green, fleshy, oval leaves. although often considered a weed, all the aerial parts can be eaten, especially as a salad plant. purslane is rich in vitamins (uddin et al., 2014), protein, carbohydrates and minerals (uddin et al., 2014; mohamed and hussein, 1994) such as calcium, iron, magnesium, potassium, zinc and sodium (aberoumand, 2009). apart from is alimentary use, purslane has been traditionally used as a medicinal plant. it has anti-inflammatory and analgesic properties (lee et al., 2012; zhou et al., 2015; rafieian-kopaei and alesaeidi, 2016; meng et al., 2016), anti cancer activity (zhou et al., 2015; lee et al., 2014; ahangarpour et al., 2016) and antioxidant activity (lim and quah, 2007; siriamornpun and suttajit, 2010; erkan, 2012). this plant can be used externally for various skin complaints, such as eczema, ulcers and acne, and to give relief from insect bites. it can also be used for coughs, bronchitis and fever (zhang et al., 2002). due to its many health benefits, it is listed in a world health organization database. moreover, purslane is an excellent vegetable source of omega-3 fatty acids (uddin et al., 2014; liu et al., 2002; simopoulos et al., 2005) since 100 g of purslane leaves contain around 350 mg of α-linolenic acid. several works reported the presence of flavonoids as main bioactive purslane constituents (erkan, 2012; xu et al., 2006; zhu et al., 2010). in recent decades, the role of foods in disease prevention and treatment has been increasingly recognised. the role played by reactive oxygen species (ros) in the pathogenesis of several diseases including diabetes mellitus (dm) has been clarified (alfadda and sallam, 2012; tundis et al., 2016; loizzo et al., 2017). to prevent complications, the stabilization of blood glucose levels in dm patients is crucial (mai and chuyen, 2007). several therapeutic approaches may be used to achieve this objective: stimulating insulin release, increasing the amount of glucose transporters, inhibiting gluconeogenesis, reducing the absorption of glucose or decreasing the post-prandial hyperglycaemia (kim et al., 2005). this last approach could be obtained by inhibiting carbohydrate hydrolysing enzymes α-amylase and α-glucosidase, using acarbose, voglibose and miglitol (rios et al., 2005; loizzo et al., 2016; loizzo et al., 2017). however, these drugs are characterized by several gastrointestinal side effects including abdominal discomfort, flatulence, bloating, and diarrhoea. for these reasons natural sources are being investigated to provide new hypoglycaemic drugs (rios et al., 2005; tundis et al., 2010). both fresh and dried samples are used in medicinal plants studies. in most cases, dried samples are preferred considering the time needed for experimental design. purslane is highly perishable in the fresh state; it has the shortest shelf life among fruits and vegetables due to its high metabolic reactions, which lead to loss quality. therefore, the aim of this work is to investigate and compare the chemical com position, antioxidant and hypoglycaemic properties of dried and fresh purslane leaves. materials and methods chemicals and reagents all reagents were of analytical grade and were purchased from sigma-aldrich s.p.a. (milan, italy). acarbose from actinoplanes spp. was obtained from serva (heidelberg, germany). hplc solvents were obtained from vwr international s.r.l. (milan, italy). sample, extraction and analysis procedure purslane plants were bought from a supermarket in reggio calabria (italy) in november 2016. leaves were manually separated from the stems and divided into two groups: fresh and dried (35 °c for 48 h). samples were subsequently homogenized in a commercial blender and subjected to different extraction procedures: a) meoh:h2o (80:20 v/v) and b) 100% etoh, in ika ultra-turrax t25 and centrifuged for 10 min at 5000 rpm, after which the supernatant was filtered through a 0.45 mm millipore filter (gmf whatman) before analysis. for fresh leaves, yields (%) of 15.3 and 8.2 were obtained for hydroalcoholic and etoh extracts, respectively, while for dried leaves, yields (%) of 10.7 and 6.6 were obtained for hydroalcoholic and etoh extracts, respectively. 40 v. sicari, m.r. loizzo, r. tundis, a. mincione, t.m. pellicanò extraction of bioactive compounds and rp-hplc/dad analysis rp-hplc/dad analyses of all samples were obtained as reported by sicari et al. (2017) using a knauer (asi advanced scientific instruments, berlin) system equipped with two pumps smartline pump 1000, a rheodyne injection valve (20 μl) and a photodiode array detector uv/vis equipped with a semi micro-cell. compounds were separated on a knauer rp-c18 (250 mm × 4.6 mm, 5 μm). the chromatographic method used was a gradient elution of solvent a (water/formic acid, 99.9:0.1 v/v) and b (acetonitrile/formic acid 99.9:0.1 v/v). the gradient was used as follows: 0.01-20.00 min 5% b isocratic; 20.01-50.00 min, 5-40% b; 50.01-55.00 min, 40-95% b; 55.01-60.00 min 95% b isocratic. the column temperature was 30 °c and the flow rate was 1.0 ml/min. the injection volume was 20 μl. peaks were monitored at 254, 330 and 305 nm. the identification and quantification of antioxidant compounds were carried out from the retention times in comparison with authentic standards. data processing data were carried out using clarity software (chromatography station for windows). all analyses were performed in triplicate and the results were expressed as mg/kg of leaves. total phenolic content (tpc) the total phenolic content of the extracts was determined as described by singleton et al. (1999). an aliquot of 350 μl of extract was mixed with 1 ml of folin-ciocalteau reagent and 10 ml of 20% na2co3 solution. absorbance was measured at λ = 760 nm using a uv-vis agilent 8453 spectrophotometer (agilent tech nologies, italy) after 2 h in the dark. the results were expressed in milligram gallic acid equivalents per 100 g (mg/100 g) weight of the sample. all samples were analysed in triplicate. dpph radical scavenging activity assay dpph radical scavenging activity of p. oleracea was determined according to the technique previously described (loizzo et al., 2016) at 517 nm using a uv-vis jenway 6003 spectrophotometer. the dpph radical scavenging activity was calculated as follows: [(a0-a1/a0) × 100], where a0 is the absorbance of the control and a1 is the absorbance in the presence of the sample. ascorbic acid was used as positive control. abts radical scavenging activity assay as reported by loizzo et al. (2016) the abts radical cation solution was mixed with potassium persulphate and left in the dark for 12 h. the abts solution was diluted with methanol to an absorbance of 0.70 ± 0.05 at 734 nm. after addition of sample (1-1000 mg/ml in methanol) to the abts solution, absorbance was measured after 6 min. ascorbic acid was used as positive control. ferric reducing activity power (frap) assay the frap assay is based on the redox reaction that involves tptz (2,4, 6-tripyridyl-s-triazine)-fe3+ complex. frap reagent was pre pared as previously described (loizzo et al., 2015). extracts were dissolved in methanol and tested at 2.5 mg/ml. bht was used as control. carbohydrate hydrolysing enzymes inhibition study a starch solution, α-amylase (ec 3.2.1.1) solution and colorimetric reagent were prepared. both control and juice were added to starch solution and left to react with enzyme at room temperature for 5 min (loizzo et al., 2014). the absorbance was read at 540 nm. the enzyme inhibition (%) was obtained by the following equation: in the α-glucosidase inhibition test a maltose solution, α-glucosidase solution (ec 3.2.1.20) and o-dianisidine (dian) solution were prepared (loizzo et al., 2014). a mixture of juice maltose solution and enzyme were left to incubate at 37 °c for 30 min. then perchloric acid was added and mixture was centrifuge. the supernatant was collected and mixed with dian and pgo and left to incubate at 37 °c for 30 minutes. the absorbance was read at 500 nm. the α-glucosidase inhibition (%) was calculated by using the following equation: relative antioxidant capacity index (raci) calculation relative antioxidant capacity index (raci) is an integrated approach to evaluate the antioxidant capacity generated from different in vitro tests (sun and tanumihardjo, 2007). for the calculation, standard scores were used with no unit limitation and no variance among methods. data obtained from tpc, abts, dpph and frap tests were used to calculate raci values for purslane samples. statistical analyses excel software (office 2007) was used to calculate the means and the standard deviation. statistical analysis was carried out using spss software for windows (spss inc., elgin, il, u.s.a.) 15.0 version. the means of all parameters were examined for significance using anova with tukey test to determine any significant difference between the treatments at p < 0.05. further multivariate analysis was performed using principal component analysis (pca). all samples were analysed in triplicate. results and discussion identification and quantification of the phenolic compounds present in p. oleracea both fresh and dried purslane leaves were analysed by hplc-dad to determine their bioactive compounds and the effect of drying on selected phenolic compounds. tab. 1 reported the chemical profile of both fresh and dried purslane leaves extracts. quercetin, apigenin, luteolin, kaempferol, isorhamnetin, kaempferol-3-o-glucoside and rutin were the flavonoids (erkan, 2012; zhu et al., 2012), and caffeic, p-coumaric, and ferulic acids were the phenolic acids found in all extracts (siriamornpun and suttajit, 2010; erkan, 2012; yang et al., 2009). among identified flavonoids, quercetin was the most abundant compound with values in the range 16.01-6.02 mg/kg, followed by rutin (6.17-4.12 mg/kg) and kaempferol (3.25-1.85 mg/kg). apigenin, luteolin, isorhamnetin, kaempferol-3-o-glucoside were found in smaller quantities (tab. 1). the level of flavonoids depends on the portion of the plant, and is normally higher in the root, followed by the stem and leaves (xu et al., 2006). p-coumaric acid was the main phenolic acid with the highest value in hydroalcoholic fresh leaf extract (20.53 mg/kg) followed by etoh dried sample (18.77 mg/ kg). the same trend was found for ferulic acid. wide investigations have shown that p-coumaric acid and quercetin exhibit various bioactivities, including antioxidant, anti-inflamma tory and anti-cancer activities, in addition to mitigating atherosclerosis, oxidative cardiac damage, diabetes and many other biological actions (lim, 2007; dkhil et al., 2011; pei et al., 2015). in addition, quercetin is a versatile antioxidant known to possess protective abili% inhibition = 100 [maltose] test [maltose] control x 100 ± s.d. % inhibition = 100 [glucose] test [glucose] control x 100 ± s.d. bioactivity of portulaca oleraceae extract 41 ties against tissue injury induced by various drug toxicities (anand david, 2016). the efficiency of extraction varies according to the polarity of the solvent, ph, temperature, extraction time, and composition of the sample. when extraction time and temperature are the same, the solvent used and the composition of sample were shown to be the most important parameters. in the present study, extraction was performed on samples of purslane leaves using two different solvents: methanol/ water (80/20 v/v) and ethanol (100%). ethanol is known as a good solvent to extract phenol, as well as being safe for human consumption. methanol is considered to be more efficient when extracting phenols of lower molecular weight (dai and mumper, 2010). huang et al. (2007) identified isorhamnetin, quercetin and kaempferol with values of 2.8, 1.3 and 1.1 mg/100 g, respectively. more recently, apigenin, bergapten, caffeic acid, p-coumaric acid, ferulic acid, scopoletin, quercetin, and quercetin-3-o-rhamnoside, were quantified in p. oleracea from different localities (ai et al., 2015). xu et al. (2006) identified mirycetin and luteolin. antioxidant potential in this study, two in vitro tests, dpph and abts, were used to screen the radical scavenging activity of p. oleracea extract. the total phenolic content and ferric reducing power was also tested. the different methods for measuring the radical scavenging potential can give different results according to which specific free radical is being used as a reactant. dpph is often used to test how far compounds can act as free radical scavengers or hydrogen donors, and to quantify antioxidants in complex systems. the procedure which inhibits the production of the abts radical cation did not involve a substrate (antolovich et al., 2002). the ferric thiocyanate method determines the quantity of peroxide, the main product of lipid oxidation, which is produced in the initial stages of oxidation. in this test, hydroperoxides formed from linoleic acid added to the reaction mixture, oxidized in air during the experiment, were indirectly measured. antioxidants may be reductants and inactivation of oxidants by reductants are redox reactions in which one reaction species is reduced when the other is oxidized (apak et al., 2004). total phenols were calculated and expressed as gallic acid/100 g of extract. the highest value was found in fresh leaves hydroalcoholic extract (565 mg/100 g) followed by fresh leaves ethanol extract (488.04 mg/ 100 g) (tab. 2). a concentration-dependent activity was observed for all tested purslane extracts. fresh purslane leaf ethanolic extract exerted the greatest dpph radical scavenging activity with ic50 value of 52.86 mg/ ml. this extract was followed by fresh leaf hydroalcoholic extract (ic50 value of 53.92 mg/ml) (tab. 2). a similar trend was also seen in abts·+ radical scavenging ability where the ic50 values of 66.98 and 72.60 mg/ml were found for f and f1 samples, respectively. generally, a promising ferric reducing ability was observed. in particular, fresh leaf hydroalcoholic extract showed a frap value comparable to the positive control bht (56.11 vs 63.2 mm fe (ii)/g). the raci calculation was used to integrate the antioxidant capacity generated from different in vitro methods of each samples. for this calculation, the mean of standard scores was used, taken from the raw data of the different antioxidant tests. any differences between units and in variances in the raw data did not influence the raci. stepwise regression between raci and different tests demonstrated the following: 1) each test was selected as a significant variable with no one applied method being removed, 2) each method had the same weight in building raci, and 3) the regression was highly significant (r = 1, p < 0.001). based on raci the following order of antioxidant ability was found: dried leaves (etoh) > dried leaves (meoh:h2o) > fresh leaves (meoh:h2o) (fig. 1). this trend plainly showed that dried leaves, independently of which solvent was used for extraction, had the highest antioxidant potential. recently, youssef et al. (2014) reported the radical scavenging potential of purslane leaves fresh and under different drying procedures (hot-air drying, microwave drying and freeze-drying). fresh purslane leave extracts showed values of 53.23% for dpph and 147.78 μmol trolox for abts per 100 g dry weight. all the examined methods of drying significantly lowered the antioxidant capacity of the sample. analysis of data demonstrated that among investigated dried sample, hot air dried and freeze-dried purslane leaves retained a better antioxidant capacity independently by the temperature applied while microwave procedure drastically reduced the antioxidant potential of tab. 1: quantification (mg/kg) of selected phenols of purslane leaves extracts. meoh:h2o etoh (80:20 v/v) fresh leaves dried leaves fresh leaves dried leaves flavonoids apigenin 0.29 ± 0.05a 0.11 ± 0.03c 0.17 ± 0.05b 0.09 ± 0.01c kaempferol 3.25 ± 0.15a 2.03 ± 0.12c 2.75 ± 0.15b 1.85 ± 0.14d luteolin 0.55 ± 0.02a 0.03 ± 0.08d 0.32 ± 0.02b 0.23 ± 0.03c quercetin 16.01 ± 0.33a 6.02 ± 0.03d 11.11 ± 0.33c 14.14 ± 0.30b isorhamnetin 0.36 ± 0.01a 0.08 ± 0.03c 0.27 ± 0.01b 0.25 ± 0.07b kaempferol-3-o-glucoside 0.63 ± 0.05a 0.23 ± 0.08c 0.55 ± 0.01b 0.53 ± 0.08b rutin 6.10 ± 0.12b 4.12 ± 0.04d 5.11 ± 0.10c 6.17 ± 0.12a phenolic acids caffeic acid 7.35 ± 0.08a 3.48 ± 0.08d 6.33 ± 0.24b 5.58 ± 0.88c p-coumaric acid 20.53 ± 0.46a 11.03 ± 0.15d 16.44 ± 1.18c 18.77 ± 1.2b ferulic acid 9.62 ± 0.41a 4.12 ± 0.28d 7.53 ± 0.88c 9.27 ± 1.01b sign. ** ** ** ** values are mean ± sd of three sample seed oils, analyzed in triplicate. different letters indicate significant differences. differences were evaluated by one-way analysis of variance (anova) test completed with a multicomparison tukey’s test. **p<0.01 compared with the positive control. 42 v. sicari, m.r. loizzo, r. tundis, a. mincione, t.m. pellicanò the matrix. these modifications in terms of bioactivity could be related to the different phytochemical content in investigated samples. however, the generation and accumulation of antioxidants during food dehydration may cause antagonistic or synergistic effects with each other or with other compounds present in the sample. the complex interactions influencing the functional properties of food during drying require further research (hsu et al., 2003; di scala et al., 2011; loizzo et al., 2013; lòpez et al., 2013). the methanol extracts edible fresh parts of thirteen p. oleracea from malaysia were examined for their phytochemical content and antioxidant activity by using the dpph radical scavenging method and frap assay (alam et al., 2014). the ic50 values ranged from 2.52 to 3.29 mg/ml for dpph test, and for 7.39 to 104.2 μmol te/g dw for frap assay. these results are better than our data in terms of both radical scavenging activity and ferric reducing power. differently, similar dpph radical scavenging results were obtained with airdried powered of iranian p. oleracea. in fact, the n-hexane, dichloromethane, chloroform, ethyl acetate and methanol extracts showed ic50 values in the range from 62.9 to 91.0.8 mg/ml for ethyl acetate and dichloromethane extracts, respectively (salehi et al., 2013). the influence of area of collection on the phytochemical content and antioxidant potential of this plant species was confirmed also by silva et al. (2014) that investigated leaves, flowers and stems of p. oleracea from different area of portugal. results revealed that in the dpph assay, samples from vendas novasr reached the 50% inhibition rate in lower concentrations than plants from tavira. recently, hydroalcoholic extracts of the aerial parts of p. oleracea from bulgaria (pob) and greece (pog) were studied for their radical scavenging activity and ferric reducing power (gevrenova et al., 2016). both purslane extracts revealed a similar radical scavenging potential with ic50 values of 1.98 and 2.00 mg/ml, and 0.88 and 0.92 mg/ml in dpph and abts test for pob and pog, respectab. 2: total phenols content, radical scavenging activity and ferric reducing potential of purslane leaves extracts. total phenols dpph abts frap (mg gae/100 g) (ic50 mg/ml) (ic50 mg/ml) (mm fe(ii)/g) meoh:h2o (80:20 v/v) fresh leaves 565.07 ± 3.23 52.86 ± 0.8 66.98 ± 1.9 54.35 ± 0.5 dried leaves 244.17 ± 4.04 53.92 ± 1.3 72.60 ± 1.7 56.11 ± 3.9 etoh fresh leaves 488.04 ± 1.54 55.92 ± 1.1 85.91 ± 1.9 45.14 ± 3.3 dried leaves 260.19 ± 2.07 56.87 ± 1.3 89.46 ± 2.3 36.22 ± 3.2 positive control ascorbic acid 2.0 ± 0.9 1.7 ± 0.8 bht 63.2 ± 2.8 data are expressed as means ± s.d. (n = 3). dpph radical scavenging activity assay: one-way anova ***p<0.0001 followed by a multicomparison dunnett’s test: ***p<0.01 compared with ascorbic acid. antioxidant capacity determined by radical cation (abts+): one-way anova ***p<0.0001 followed by a multicomparison dunnett’s test ***p<0.01, *p<0.05 compared with ascorbic acid. ferric reducing antioxidant power (frap): one-way anova ***p<0.0001 followed by a multicomparison dunnett’s test **p<0.01 compared with positive control. fig. 1: relative antioxidant capacity index of purslane leaves samples. 0,47 -0,40 0,54 0,67 fresh leaves (meoh/water) 12*3456/7*)8 dry leaves (meoh/water) 12*3456/7*)8 fresh leaves (etoh) 1;7348 dry leaves (etoh) 1;7348 r a c l v al u es bioactivity of portulaca oleraceae extract 43 tively. however, considering that all measured values are greater than those are found for the calabrian extracts, our sample showed a most promising antioxidant potential. the sample from bulgaria showed two-time highest ferric reducing ability respect pog with value of 0.16 mm trolox equivalent. carbohydrate hydrolysing enzymes inhibition following our research interest in starch hydrolase inhibitors from edible plants we have investigate purslane extracts against α-amylase and α-glucosidase enzymes. these extracts inhibited carbohydratehydrolysing enzymes depending upon their concentration. generally, α-glucosidase enzyme was most sensible since the ic50 values are in the range from 45.05 to 195.01 mg/ml for fresh leaf hydroalcoholic extract and fresh leaf ethanol extract, respectively. on α-amylase dried leaves, hydroalcoholic extract showed the highest inhibitory activity (ic50 value of 488.49 mg/ml) (tab. 3). all these values are greater than those for the positive control acarbose. our values are in agreement with those reported by salehi et al. (2013), which found an ic50 value of 93.2 μg/ml for p. oleracea methanol extract against α-glucosidase. previously, the effect of p. oleracea were screened in vivo in rats with type 2 dm. results clearly evidenced that the extract reduced body weight, improved impaired glucose tolerance, attenuated hyperinsulinemia and elevated insulin sensitivity. the mechanism might be related to improved lipid metabolism and decreased free fatty acids (lan and fuer, 2003). successively, el-sayed (2011) studied the effect of p. oleracea seeds in thirty type-2 dm patients. patients were split into two groups, one received 5 g of seeds two times a day, while the other received 1.5 mg of metformin daily. the treatment caused a significant drop in total cholesterol, low-density lipoprotein and serum levels of triglycerides and a rise in high-density lipoprotein. other effects included modifications of liver transaminase, total and direct bilirubin, body weight and body mass index, fasting and post-prandial blood glucose, and insulin. in the metformin group similar effects were obtained. tab. 3: carbohydrate hydrolysing enzymes of purslane extracts. assay α-amylase α-glucosidase (ic50 mg/ml) (ic50 mg/ml) meoh:h2o (80:20 v/v) fresh leaves 902.74 ± 3.8 195.01 ± 1.7 dried leaves 640.01 ± 3.5 45.05 ± 1.1 etoh fresh leaves 774.02 ± 3.7 132.88 ± 2.1 dried leaves 488.49 ± 3.5 138.51 ± 2.3 positive control acarbose 50.0 ± 0.9 35.5 ± 1.2 data are expressed as means ± s.d. (n = 3). α-amylase: one-way anova ***p<0.0001 followed by a multicomparison dunnett’s test: ***p<0.01 compared with acarbose. α-glucosidase: one-way anova ***p<0.0001 followed by a multicomparison dunnett’s test: ***p<0.01 compared with acarbose. 6 fig. 2: pca loading plot (pc1 vs pc2) for the first and second principal components. dry leaves (etoh) fresh leaves (etoh) fresh leaves (meoh/h2o) dry leaves (meoh/h2o) pc2: 33,8 % pc1: 61,24 % fig. 2: pca loading plot (pc1 vs pc2) for the first and second principal components. principal component analysis pca showed that the two principal components accounted for 95.12% of total variance, with pc1 for 61.24% and pc2 for 33.88% of total variance. the first principal component (fig. 2) shows strong correlation with apigenin, kaempferol, luteolin quercetin isorhamnetin, kaempferol-3-o-glucoside, rutin, caffeic acid, p-coumaric acid, ferulic acid, total phenols, α-glucosidase and a lower correlation with α-amylase, dpph, and abts test. this suggests that these thirteen variables are grouped together. in addition, from the analysis of variable loads, it was seen that the pc1 has a negative correlation with frap. the second principal component is strongly correlated with apigenin, kaempferol, frap and α-amylase, while it is strongly negatively correlated with dpph and abts. total phenols (tpc) 44 v. sicari, m.r. loizzo, r. tundis, a. mincione, t.m. pellicanò is positively correlated with both pc1 and pc2. the significant correlations obtained support the hypothesis that phenolic compounds contribute significantly to the total antioxidant capacity (cai et al., 2004; djeridane et al., 2006; sicari et al., 2016; sicari et al., 2016a). fig. 3 shows that pc1 positive correlation with hydroalcoholic extracts obtained from the fresh leaves and was characterized by the presence of total phenols, flavonoid compounds, phenolic acids, α-amylase, α-glucoside and a low value of abts and dpph. more over, values of the original variables are greater than those of the ethanolic extracts obtained from fresh and dried leaves, respectively. methanol is the most suitable solvent in the extraction in polyphenolic compounds from plant tissue, due to its ability to inhibit the action of polyphenol oxidase that causes the oxidation of polyphenols (yao et al., 2004; lim and quah, 2007). in addition, the different relationships between the antioxidant activity and the total phenolic content can be due to many factors; in fact the total phenolic content does not incorporate all the antioxidants. also, it must be taken into account the synergism between the antioxidants in the extracts that makes the antioxidant activity not only dependent on the concentration, but also on the structure and the interaction between the antioxidants (piluzza and bullitta, 2011). the ethanolic extract obtained from fresh leaves has a positive correlation with pc1, while the same obtained from the dried leaves shows low positive correlation and was characterized by assay of antioxidant activity (dpph and abts). hydroalcoholic extracts (dried leaves) were grouped at the negative side of pc1 (showing low flavonoid compounds, phenolic acids, total phenols and high frap values). conclusions in this work, extracts from fresh and dried leaves of p. oleracea were tested to evaluate the most efficient process in terms of extracting bioactive molecules. was carried out a qualitative analysis of phenolic compounds present in the leaves of purslane examined by using lc-dad by comparison with standard and literature data. analysis of results revealed that fresh hydroalcoholic purslane extract exhibited a promising radical scavenging activity. a great difference was observed in hypoglycaemic whereas dried hydroalcoholic purslane extract exhibited the highest α-glucosidase inhibitory potential. therefore, the results of this paper, with the addition of further stu dies, will allow this plant with a large amount of biomolecules useful for 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pharmacological effects. bio. med. res. int. id 925631, 11 pages. zhu, h.b., wang, y.z., liu, y.x., xia, y., tang, t., 2010: analysis of flavonoids in portulaca oleracea l. by uv-vis spectrophotometry with comparative study on different extraction technologies. food anal. method. 3, 90-97. © the author(s) 2018. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). address of the corresponding author: e-mail: vincenzo.sicari@unirc.it journal of applied botany and food quality 90, 280 287 (2017), doi:10.5073/jabfq.2017.090.035 1department of plant physiology, university of agriculture of krakow, kraków, poland 2institute of plant physiology, polish academy of sciences, kraków, poland effects of zearalenone and 24-epibrassinolide on the salt tolerance of selected monocotyledonous crop plants agnieszka płażek1*, maria tatrzańska2, maciej maciejewski2, michał dziurka2, franciszek dubert2 (received may 19, 2017; accepted august 28, 2017) * corresponding author summary salinity has an increasing impact on crop production worldwide. contemporary agricultural practices increasingly use plant biostimulants that protect plants against various environmental stresses. the aim of the work was to investigate whether such stimulants as 24-epibrassinolide (epi) and zearalenone (zen) may alleviate effects of salinity in bread and durum wheat, maize, and sorghum plants. plants were grown in glasshouse, in pots filled with perlite under continuous salinity stress (120 mm of nacl). four-week-old plants were treated with the stimulants. the plant responses to salinity were determined analyzing the following parameters: fresh and dry weights of plants, water content, electrolyte leakage, proline, abscisic acid, and the soluble carbohydrate contents in the leaves. the positive effect of zen on the studied parameters was more frequently observed than in the case of epi. zen increased the root mass of both wheat species, as well as the stem and root masses of sorghum. this stimulant improved water relations in bread and durum wheat. both stimulators increased the content of soluble carbohydrates. zen elevated significantly the abscisic acid content in sorghum plants as well as it increased strongly the proline level in all studied plant species. zen was more effective in alleviation salinity disorders than epi. keywords: abscisic acid; cereals; 24-epibrassinolide; proline; salinity; soluble carbohydrates; zearalenone introduction more and more agricultural areas worldwide are experiencing soil salinity problems. in poland, located in central europe, long-lasting drought periods and the progressive reduction in the groundwater level have been observed. many crops require irrigation, which results in an increase in soil salinity (walter et al., 2011). according to the central statistical office in poland, an unprecedented drought which caused huge crop losses, was recorded in 2015. thus, soil salinity will most likely affect regions that have not previously experienced problems with this environmental stress. in addition to natural conditions, salinity is also evoked by improper irrigation and fertilization. in most cases high soil salinity is the result of salt accumulation over long cultivation periods and deforestation (brini et al., 2009). salinity causes osmotic stress and ion toxicity stress due to the pre sence of sodium (na+) and chloride (cl−) ions (munns and tester, 2008). a high na+ concentration mainly disturbs the osmotic ba lance and results in an inhibition of water uptake by the plant. the toxic influence of na+ may be manifested by the premature death of leaves, disturbances in the cell membrane’s structure or functions, and the inhibition of enzymes taking part in many metabolic processes mainly in photosynthesis (mitsuya et al., 2003). the plant tolerance responses to salt stress involves: the exclusion of salt ions, the controlled uptake of ions by roots and transport into leaves, ion compartmentation, the synthesis of specific osmolytes, and changes in membrane structure, as well as the activation of antioxidant defense system (munns and tester, 2008). the most important criterion indicating salt tolerance is biomass production (munns and james, 2003). salt stress causes stomatal closure which limits co2 assimilation. lower rates of photosynthesis disturb the energy balance between light and dark phases, which stimulates the generation of reactive oxygen species. furthermore, oxygen radicals disturb the photochemical processes in thylakoids and can evoke strong photoinhibition. thereby, damage to the membrane structure as a result of salinity causes strong membrane depolarization and membrane lipid peroxidation (yasar et al., 2006). the sequestration of na+ and cl− in vacuoles affects cell osmotic pressure, which should be balanced with the accumulation of specific compounds, such as proline and glycine betaine (flowers et al., 1977; elsayed et al., 2014). osmotic potential is regulated mainly by soluble monoand oligosaccharides. raffinose and other raffinose family oligosaccharide (rfo) members are recognized as signaling compounds in stresses, such as drought, salinity, and chilling (elsayed et al., 2014). disaccharide trehalose, in addition to regula ting osmotic pressure under various stresses may serve as a protectant of enzymes and membranes, may elevate salinity stress by decreasing the rate of ion leakage and lipid peroxidation as well as increasing the potassium k+ / na+ ratio (zeid, 2009). plant responses to osmo tic stress involving stomatal closure and the regulation of cell water potential may be controlled by abscisic acid (aba), which is often called the osmotic stress hormone. aba-dependent signaling under salt and drought stresses is well documented (zhu, 2002; zhang et al., 2006; shinozaki and yamaguchi-shinozaki, 2007). contemporary agricultural practices increasingly use plant biostimulants that enhance the heights and qualities of yields as well as protect plants against various environmental stresses (ranawake et al., 2013). to minimize the stress effects exogenous plant hormones are often applied (biesaga-kościelniak et al., 2003; janeczko et al., 2003; kościelniak et al., 2011). the most successful this mitigation are brassinosteroids (brs), plant steroidal hormones that play roles in a broad spectrum of developmental and physiological processes (yuan et al., 2010). many studies have demonstrated brs ability to enhance plant tolerance to salinity, heavy metal stress, high and low temperature stress, and pathogen attacks (houimli et al., 2010; janeczko et al., 2010; yuan et al., 2010; kościelniak et al., 2011). talaat and shawky (2013) showed the alleviating effects of 24-epibrassinolide (epi) (fig. 1), which occurred by enhancing the antioxidant system, in wheat plants under salt stress. another plant regulator, zearalenone (zen), also shows positive effects in plant protection against unfavorable environmental factors. zen was isolated for the first time from a fungal gibberella zeae culture by stob et al. (1962). zen [2,4-dihydroxy-6-(10-hydroxy6-oxo trans-1-undecenyl)-benzoic acid lactone] (fig. 2) is an f2toxin produced by fungi belonging to genus fusarium. zen possesses estrogenic activity by competing with animal hormones to bind to estrogen receptors. plants infected by fusarium fungi producing zen demonstrate chromosomal damage and disturbances in chlorophyll zearalenone alleviates salt effects on cereals 281 synthesis and photosynthesis processes (kumar and sinha, 1995; kościelniak et al., 2011). however, zen is an endogenous regulator that at hormone concentrations controls plant development. zen has a positive role in vernalization processes (filek et al., 2010) and the stimulation of ear, pod and seed numbers in wheat and soybean (biesaga-kościelniak et al., 2006). this study investigated whether epi or zen can mitigate the effects of salinity in monocotyledonous crop plants, such as bread wheat (triticum aestivum l.), durum wheat (triticum durum desf.), maize (zea mays l.), and sorghum (sorghum bicolor l. moench). mentioned species belong to the most important crops in all of the world. plants were grown under continuously salinity stress (120 mm of nacl), while seeds were sown directly into saline perlite. the plant’s responses to salinity stress were determined on the basis of following parameters: fresh and dry weights (fw and dw) of stems and roots, tissue hydration, cell membrane permeability, proline and aba contents, as well as the quality and quantity of soluble sugars in the leaves. materials and methods plant material and experimental design the experiment was conducted under glasshouse conditions (2024 °c day; 18 °c night) from the beginning of june until the end of august at day light (50°04́10̋ n, 19°50́40̋ e). the study was performed on t. aestivum cv. banderola, t. durum cv. komnata, z. mays cv. król, and s. bicolor cv. cukrosorgo. the experiment was a completely randomized design with five replicates for each treatment. seeds were sown in 22 cm × 22 cm × 22 cm (v = 10 dcm3) pots filled with perlite. in the experiment perlite was used instead a soil to maintain controlled salinity level. for both wheat species there were 12 plants per pot, and for maize and sorghum there were 4 plants per pot. all of the plants were treated daily with 120 mm nacl solution supplemented with hoagland medium (hoagland et al., 1950). the molar concentration of hoagland’s medium was low in comparison with that of the nacl solution; therefore, we also analyzed the influence of only nacl. the salinity level used in the experiment was determined in an earlier our study. non-treated with salt solution plants were the control, while plants growing in saline perlite untreated with epi or zen were used as the saline control. four week-old plants were sprayed with 40 cm3 per pot with the solution of the plant regulator epi (2 mg dm−3) or zen (2 mg dm−3). two mg of 24-epibrassinolide (olchemim, olomouc, czech republic) or zearalenone (fermentek, jerusalem, israel) were dissolved in 1 cm3 of 50% ethanol and next filled up with distilled water to 1 dm3. control plants were sprayed with distilled water containing 0.05% ethanol. the applied doses of both regulators were based on previous experiments (biesaga-kościelniak et al., 2003; janeczko et al., 2003). four weeks after the stimulator application, plant samples were collected for analyses. measurement of fw, dw, and relative water content (rwc) the fw of aboveground plant parts (shoots) and roots were determined. next, their dws were measured after drying for 48 h at 70 °c. finally, the ratio of the fw of shoots (s) to the whole plant weight (wp) was calculated according to the formula: s × 100 / wp. the rwc of shoots and roots was calculated as (fw-dw) / dw and expressed as g h2o g−1 dw. analyses were performed in 15 replicates (15 plants) for each plant species and treatment. electrolyte leakage ion leakage, which indicates the plasma membrane’s integrity level, was measured in the 3rd upper, well-expanded leaves. leaf discs (ø = 1 cm) were washed in distilled water, put into plastic vials containing 13 cm3 deionized water and shaken for 24 h (50 rpm) at 20 °c. next, ion conductivity was measured (el1) using a conductometer (ci 317, elmetron, poland). then, the samples were frozen at -80 °c for 24 h. after thawing, they were shaken again for 24 h, and the total ion leakage (el2) was measured. membrane permeability was expressed as a percentage of el2, (el1 × 100 / el2). the measurements were performed in 10 replicates for each plant species and treatment. proline content the free proline content in leaves was determined as described by bates et al. (1964). samples were homogenized in 3% (w/v) sulfosalicylic acid to precipitate proteins, and centrifuged at 14,000 × g for 10 min. the reaction mixture contained 2 cm3 glacial acetic acid, 2 cm3 ninhydrin reagent (2.50% w/v ninhydrin in 60% v/v 6 m phosphoric acid) and 2 cm3 of supernatant. the incubation lasted for 1 h at 90 °c. after stopping the reaction with ice, 4 cm3 of toluene was added and mixed by vortexing. the upper toluene phase was decanted into a glass cuvette, and the absorbance was measured using an ultrospec 2100 pro spectrophotometer (biosciences amersham, sweden) at λ = 520 nm. the concentration was assayed using proline as the calibration standard. each assay was performed in five replicates, with five leaves from different plants for each treatment. the content of proline was expressed as μg g−1 dw. aba content from five plants of each species and treatment the 3rd upper, wellexpanded leaves were collected. lyophilized samples were pulve rized then extracted with 1 cm3 of bieleski buffer (bieleski, 1964). extracts were evaporated, re-dissolved in 1 m formic acid, cleanedup on oasis mcx spe cardridges (waters, ireland) and used for high-performance liquid chromatography (hplc) analyses according fig. 2: the structure of zearalenone (wikipedia public domain) fig. 1: the structure of 24-epibrassinolide (https://www.chemsrc.com/en/ cas/78821-42-8_1009735.html) 282 a. płażek, m. tatrzańska, m. maciejewski, m. dziurka, f. dubert to żur et al. (2015). a chromatograph coupled to a triple quadruple mass spectrometer (6410 triple quad lc/ms, agilent, usa) controlled by masshunter software was used. quantization was based on calibration curves obtained for the pure standards of aba, taking into account the recovery of the internal standard ([2h6]aba). phytohormone standards were obtained from olchemim (olomouc, czech republic), while other chemicals were from sigma-aldrich. soluble carbohydrate content sugars were analyzed according to the method reported by janeczko et al. (2013). approximately 10 mg of freeze-dried and pulverized samples were extracted in 1 cm3 of ultrapure water by shaking for 15 min at 30 hz (mm 400, retsch, germany). then, samples were centrifuged for 5 min at 21,000 × g (universal 32r, hettich, ger many). supernatants were collected, diluted with acetonitrile 1:1 (v/v), and filtered (0.22 μm nylon membrane, costar spin-x, corning, usa). samples were analyzed by hplc (agilent 1200). an esa coulochem ii electrochemical detector with a 5040 analytical cell (esa, usa) coupled to an analog-to-digital converter (agilent, china) was used. the separation of soluble sugars (glucose, fructose, sucrose, maltose, trehalose, and raffinose) was achieved on a rcx10 (7 μm; 250 × 4.1 mm) column (hamilton, usa) at a flow rate of 1.5 cm3 min–1 in gradient mode. the injection volume was 0.01 cm3, and the column temperature was 40 °c. pulse amperometric detection was employed (analytical potential of 200 mv; oxidizing potential of 700 mv, and reducing potential of -900 mv, with reference to a palladium electrode) on a gold electrode. statistical analyses all of the data were analyzed with statistica 10.0 software (statsoft, ok, usa) using the manova method to evaluate statistical differences between treatments. the percentage data were transformed according to the formula arcsin √x. all data were presented as means ± standard errors (ses). the differences between means were additionally analyzed according to duncan’s multiple range test at p < 0.05. results visual symptoms of salinity effect, fw, dw and water relations among the studied plant species, the most visible symptoms of the applied salinity level (120 mm nacl) were observed on maize plants, which demonstrated slight leaf drying and a reddening of the stems. leaf blades of both bread and durum wheat dried at the ends, while sorghum plants underwent the drying of older leaf blades. all of the salt treated plants showed strong growth inhibition. the most sensitive to salinity were plants of maize and sorghum demonstrating a decrease in the fresh weights of shoots by 75% and 85%, respectively, while bread and durum wheat were more tolerant showing 47% decline of shoot fw (tab. 1). the applied salinity affected the considerable decrease in shoot dw of all studied plant species, but maize and sorghum demonstrated greater reduction (67%) of this parameter than bread and durum wheat (~30%). fresh weights of roots of bread and durum wheat, as well as of maize and sorghum decreased by 35%, 39%, 38% and 56%, respectively, while dry weights by 61%, 58%, 29% and 28%, respectively. these data demonstrate that in the case of maize and sorghum the salinity affected more the shoots than tab. 1: influence of 24-epibrasinolid (epi) and zearalenone (zen) on fresh (fw) and dry weight (dw) of shoots (s) and roots (r), as well as percentage participation of shoots’ fw to whole plant (wp) fresh weight of triticum aestivum, t. durum, zea mays and sorghum bicolor plants grown at 120 mm of nacl. means (n=15) ± se within a column for each plant species marked with the same lowercase do not differ significantly (multiple range duncan’s test; p < 0.05). species/regulator fw of s [g] dw of s [g] fw of r [g] dw of r [g] s / wp [%] triticum. aestivum cv. banderola control 42.08±4.05a 5.08±0.60a 5.02±0.45b 2.82±0.25a 89.3±5.1a saline control 22.30±3.35b 3.66±0.29b 3.26±0.29c 1.10±0.91b 87.2±5.0a epi 13.37±2.05c 2.76±0.33b 3.04±0.23c 1.01±0.82b 81.5±5.1a zen 22.02±3.30b 4.33±0.45a 10.29±0.93a 2.83±0.24a 68.1±3.8b triticum. durum cv. komnata control 30.64±3.95a 4.57±0.55a 7.90±0.95a 4.21±0.37a 79.5±6.8a saline control 16.24±1.95b 3.16±0.38b 4.82±0.58b 1.77±0.21c 77.1±6.9a epi 10.62±1.17c 2.76±0.33b 5.53±0.66b 1.82±0.20c 80.7±8.1a zen 11.12±1.45c 4.33±0.52a 7.12±0.85a 2.39±0.28b 57.4±5.2b zea mays cv. król control 423.10±21.15a 43.57±3.49a 29.32±2.35a 4.45±0.36a 93.5±7.5a saline control 105.75±2.29b 14.38±1.15b 18.18±1.45c 3.16±0.25b 85.3±6.8b epi 106.85±5.34b 14.47±1.16b 22.26±1.78b 3.66±0.31b 82.8±6.6b zen 90.73±4.53c 12.92±1.03b 19.81±1.58b 3.19±0.26b 82.1±6.6b sorghum bicolor cv. cukrosorgo control 227.87±20.51a 13.45±1.21a 21.29±1.56a 2.44±0.22a 91.5±8.3a saline control 34.18±3.08c 4.44±0.39c 9.37±1.04b 1.76±0.16b 77.9±7.0b epi 28.68±2.58c 4.03±0.36c 7.67±0.99c 1.89±0.17b 78.9±7.0b zen 46.56±4.19b 7.46±0.99b 10.59±2.51b 2.51±0.23a 81.5±7.3b control – plants untreated with nacl and stimulants; saline control – plants grown at 120 mm nacl zearalenone alleviates salt effects on cereals 283 tab. 3: the influence of 24-epibrassinolide (epi) and zearalenone (zen) on percentage electrolyte leakage (el) [%] from the leaf cells of triticum aestivum, t. durum, zea mays and sorghum bicolor plants grown in perlite at 120 mm nacl. means (n=10) ± se in the rows for each species marked with the same lowercase do not differ significantly (multiple range duncan’s test; p < 0.05). species/cultivar control saline control epi zen triticum. aestivum cv. banderola 3.01±0.29c 5.33±0.48b 7.15±0.64a 5.91±0.42b triticum. durum cv. komnata 3.63±0.32c 7.02±0.63b 6.63±0.53b 10.31±0.91a zea mays cv. król 6.84±0.75d 16.15±1.78a 12.69±1.39b 9.16±0.83c sorghum bicolor cv. cukrosorgo 4.53±0.36b 7.97±0.72a 8.35±0.92a 9.76±1.07a control – plants untreated with nacl and stimulants; saline control – plants grown at 120 mm nacl roots. salinity decreased s/wp ratios also only in the case of maize and sorghum. on the basis of these data maize and sorghum could be recognized as more salt sensitive than both studied wheat species. neither epi nor zen relieved the visual symptoms of salt stress in the wheat species, however in the case of bread wheat zen increased dw of shoots and roots to the level of salt untreated plants (control) and enhanced two-fold fw of roots compared with these control plants. similar effect of zen was observed in dw of shoot and fw of roots of durum wheat. thus s/wp ratios of both wheat species under zen impact was significantly lower when compared with that of the both control and the epi-treated plants. the epi decreased the fw of bread and durum wheat’s shoots compared with the saline control plants, but the s/wp ratios of plants under epi influence did not differ considerably from that of the saline control. zen and epi alleviated the visual symptoms of salinity mainly in sorghum and maize plants, respectively, but only in the case of sorghum zen increased the fw and dw of shoots as well as dw of roots compared with the saline control. however, this effect did not influence the s/wp ratio. maize plants responded to the zen treatment by decreasing the fw and dw of the shoots, however, the s/wp ratio was similar for saline control and epi treated plants. the studied plant species differed considerably in natural (in control plants) tissue hydration (tab. 2). maize and sorghum plants of c4 photosynthesis type demonstrated ca. 7-fold and two-fold, respectively, higher rwc values of shoots than bread and durum wheat. salt stress decreased rwc values of shoots and roots of both wheat species by 30% and 50%, respectively. in maize plants rwc of shoots decreased by 27%, while rwc of roots did not change, similarly as in the case of the sorghum plants. the most visible reduction of this parameter at 120 mm of nacl was observed in sorghum shoots (60%). the impact of applied stimulants on rwc changes in shoots and roots was specific for each plant species. zen decreased the rwc of shoots in all studied plant species, while epi in bread wheat and sorghum. this stimulant increased rwc value of shoots in maize plants to that of the control plants which were not grown under salt stress. zen increased the rwc values in the roots of bread and durum wheat, while in sorghum it declined the roots’ rwc, compared to the saline control. generally, epi did not change root rwc of studied plants compared with that of the saline control, with exception of the sorghum roots, where it decreased the values of this parameter. electrolyte leakage (el) maize plants demonstrated the highest el from leaf cells under 120 mm nacl; it amounted to 236% comparing to the control plants (tab. 3). it was three times greater than that noted in bread wheat plants and more than two times greater than those in durum wheat and sorghum. epi significantly increased the el in bread wheat leaves, while in maize it caused a 27% reduction of the el, in relation to that of the saline control. in other plants this br did not considerably change the membrane permeability in comparison with that of the tab. 2: influence of 24-epibrassinolide (epi) and zearalenone (zen) on relative water content (rwc) in shoots and roots of triticum aestivum, t. durum, zea mays and sorghum bicolor plants grown at 120 mm of nacl. means (n=15) ± se within a column for each plant species marked with the same lowercase do not differ significantly (multiple range duncan’s test; p < 0.05). species/regulator rwc of shoots rwc of roots [g h2o g–1 dw] [g h2o g–1 dw] triticum. aestivum cv. banderola control 7.39±0.88a 3.94±0.47a saline control 5.10±0.57b 1.97±0.24c epi 3.84±0.42c 2.03±0.25c zen 4.08±0.44c 2.64±0.32b triticum. durum cv. komnata control 5.58±0.67a 3.51±0.39a saline control 3.96±0.41bc 1.72±0.18c epi 4.05±0.42b 1.59±0.14c zen 3.39±0.29c 2.54±0.21b zea mays cv. król control 38.25±3.42a 4.95±0.54a saline control 27.92±3.35b 4.75±0.52a epi 37.74±4.51a 5.08±0.48a zen 19.91±2.38c 5.21±0.62a sorghum bicolor cv. cukrosorgo control 16.78±2.01a 4.10±0.38a saline control 6.71±0.80b 4.51±0.41a epi 5.51±0.66c 3.06±0.27b zen 5.24±0.65c 3.21±0.28b control – plants untreated with nacl and stimulants; saline control – plants grown at 120 mm nacl saline control. zen increased the el in the leaves of durum wheat, while the ion outflow in maize leaves was decreased under zen treatment, however it was still higher than in the salt-untreated plants. proline content salt stress decreased proline content in the leaves of bread and durum wheat by 33%, while in sorghum and maize leaves proline amount increased comparing to the plants grown under non-salt stress conditions (tab. 4). sorghum plants grown at 120 mm of nacl showed higher proline contents in the leaves than the other studied plant spe284 a. płażek, m. tatrzańska, m. maciejewski, m. dziurka, f. dubert tab. 5: the influence of 24-epibrassinolide (epi) and zearalenone (zen) on abscisic acid (aba) content [ng mg–1 dw] in the leaves of triticum aestivum, t. durum, zea mays and sorghum bicolor plants grown in perlite at 120 mm nacl. means (n=5) ± se in the rows for each plant species marked with the same lowercase do not differ significantly (multiple range duncan’s test; p < 0.05). species/cultivar control saline control epi zen triticum. aestivum cv. banderola 0.26±0.03c 0.37±0.05a 0.33±0.03b 0.31±0.02b triticum. durum cv. komnata 0.18±0.01d 0.24±0.03c 0.36±0.04b 0.45±0.05a zea mays cv. król 0.23±0.02c 0.34±0.04a 0.26±0.03b 0.32±0.03a sorghum bicolor cv. cukrosorgo 0.15±0.01c 0.19±0.03b 0.22±0.02b 0.44±0.04a control – plants untreated with nacl and stimulants; saline control – plants grown at 120 mm nacl tab. 4: the influence of 24-epibrassinolide (epi) and zearalenone (zen) on free proline content [μg g–1 dw] in the leaves of triticum aestivum, t. durum, zea mays and sorghum bicolor plants grown in perlite at 120 mm nacl. means (n=5) ± se in the rows for each species marked with the same lowercase do not differ significantly (multiple range duncan’s test; p < 0.05). species/cultivar control saline control epi zen triticum aestivum cv. banderola 3.9±0.35b 2.6±0.25c 4.1±0.38b 15.1±1.42a triticum durum cv. komnata 3.1±0.28b 2.1±0.17c 3.3±0.29b 19.8±1.91a zea mays cv. król 2.3±0.15c 2.7±0.18b 2.4±0.21c 4.7±0.38a sorghum bicolor cv. cukrosorgo 3.1±0.28b 3.4±0.31ab 2.5±0.18c 3.9±0.32a control – plants untreated with nacl and stimulants; saline control – plants grown at 120 mm nacl cies. treatment with zen increased the amount of proline in all of the studied plants, with the exception of sorghum. the largest effect of zen was observed in the case of durum wheat, in which this regulator increased the proline content 9-fold in relation to the saline control plants and 6-fold compared to the salt-untreated plants. in the case of bread wheat and maize, the proline amount in the leaves was 6 times and 1.7 times higher, respectively, after the zen treatment than in the saline control plants. in the wheat species, the epi effect was much lower than that of zen; however, epi increased the proline content in the leaves of both wheat species to the level noted in the control plants, non-treated with the salt. in maize leaves the epi treatment did not change the proline level in comparison with that in the both control plants, while in the sorghum leaves, under this br influence the proline content was the lowest. aba content among the control plants, the highest aba level was noted in bread wheat leaves (tab. 5). salinity enhanced considerably content of this hormone in the leaves of all studied plant species. the zen increased evidently the aba content in the leaves of durum wheat and sorghum by 1.9-fold and 2.3-fold, respectively, compared to that of the saline control. zen and epi reduced the aba content in the leaves of bread wheat, while epi decreased its amount in the maize leaves to that of the non-salt treated plants. soluble carbohydrate contents salinity affected the total soluble carbohydrate content in the leaves of all studied plant species (tab. 6). it increased by 26% in bread wheat, 14% in durum wheat, 24% in maize, whereas by 26% in sorghum. under salt impact the increase of glucose, fructose and maltose amount was noted in all studied plants, while sucrose amount enhanced only in maize leaves. raffinose level did not change under salinity impact, whereas salinity increased trehalose amount only in the sorghum leaves. in most cases, epi and zen significantly influenced particular sugar contents in the leaves of the studied species. generally, each plant species responded specifically to the regulator applications. in the case of bread wheat, epi and zen increased all type of sugars without raffinose and trehalose in comparison with the saline control. in durum leaves, the glucose and fructose levels were greater after epi and zen treatments than that of the saline control. additionally, in this plant species zen increased the sucrose level 5-fold. the both regulators decreased the maltose content compared with the saline control level, while epi also decreased the sucrose content. in maize plants, epi considerably increased the fructose, sucrose, and trehalose amounts, while zen elevated only trehalose content. in sorghum leaves, the zen treatment increased the sucrose and maltose contents, which were 3.6 and 5 times, respectively, higher than that of the saline control. raffinose was detected only in maize plants under the zen influence. both regulators’ effects on particular sugars influenced the total sugar amount. epi treatment significantly enhanced the total soluble sugar content in the leaves of bread wheat and maize compared to the both control plants. similar effect of the zen treatment was observed in bread and durum wheat leaves, as well as those of sorghum in which total sum of soluble sugars increased 2.4-fold compared to the control (untreated with salt) and 1.8-fold compared to the saline control plants. discussion in the most sensitive plant species, high salinity causes premature leaf senescence, necrosis or the reddening of stems, which could be the result of a phosphorus metabolism disturbance (sonneveld and kreij, 1999). in the present investigation, we observed similar symptoms on the studied plants. as demonstrated in our previous study, a salinity level greater than 100 mm nacl causes significant reductions in the fw and dw of bread and durum wheat shoots (płażek et al., 2013). reductions in the fw and dw of shoots and roots of barley under rising salinity levels were observed also by el-tayeb (2005). treatments with epi or zen did not alleviate the visual symptoms of zearalenone alleviates salt effects on cereals 285 salinity on bread and durum wheat plants, but sorghum plants treated with zen did not show visible symptoms of salt stress, while epi evoked the same effect in maize plants. in many cases, the stimulators used did not restore the value of the studied parameters under salinity stress to that determined in the control plants not treated with salt. however, in many times we have observed positive effects of zen and epi on investigated physiological and biochemical indicators. zen increased the root masses of both wheat species and maize. additionally, in sorghum, it considerably improved the fw and dw values of the stems. in our experiment, all studied plants at 120 mm nacl demonstrated lower hydration level in the shoots and roots compared to the control plants untreated with salt, which could be the effect of a very low water potential in soil caused by the high ion concentration. a sign of salt tolerance is the stable maintenance of the rwc. however, toxic effect of nacl, which appears at high salt concentrations, results in a high water influx through damaged membranes and could increase rwc values (flowers et al., 1977; munns and tester, 2008). in the investigation, applied stimulants resulted in unique responses of each plant species. zen decreased the rwc in the cells of leaves of both bread and durum wheat, but in roots increased the rwc, compared to the saline control plants. this result indicate that positive effect of this regulator may be to reduce the transport of na+ ions between roots and shoots. a similar effect was observed by el-tayeb (2005) in barley seedlings treated with salicylic acid. the mechanism of plant tolerance to salinity is related to salt ion excretion, control of root ions’ uptake and transport to leaves, ion compartmentalization as well as osmolyte synthesis (munns and tester, 2008). in contrast to zen, epi increased the rwc in the shoots of maize plants. in general, this exogenously applied hormone was less active than zen. this effect may seem strange when compared with the results obtained by other researchers. yuan et al. (2010) demonstrated that epi increased the rwc, stomatal conductance, net photosynthesis rate, antioxidant enzyme activities and aba concentration of tomato plants under drought stress. el from the plant cells is a physiological indicator of cell membrane damage from salt stress (ashraf and harris, 2004). kathar and kuhad (2000) observed an increase in the el from bread wheat leaf cells under the influence of applied nacl at 0-200 mm. salinity in the same nacl range caused considerable el from rhizomes of miscanthus × giganteus (płażek et al., 2014). in our previously investigation (płażek et al., 2013) a salinity of 125 mm nacl caused a two-fold increase in ion leakage from leaves of the bread wheat ‘banderola’ and durum wheat ‘komnata’. in the present experiment, ‘banderola’ showed the lowest el value from leaf cells of both control plants compared with those of other plant species in the study. epi applications evoked different responses in the studied plants in this respect. leaves of bread wheat showed a greater ion efflux after epi application, while maize plants underwent a significant el reduction comparing to the saline control plants. a 30% reduction in the membrane permeability, as an alleviating salt stress effect of epi in pepper plants, was observed by houimli et al. (2010). zen stabilized cell membrane permeability under salt stress only in maize. many plants accumulate proline as a protective osmolyte under saline conditions (flowers et al., 1977). khatkar and kuhad (2000) as well as khan et al. (2003) observed a large increase in the proline tab. 6: the influence of 24-epibrassinolide (epi) and zearalenone (zen) on soluble carbohydrates’ content [mg g–1 dw] in the leaves of triticum aestivum, t. durum, zea mays and sorghum bicolor plants grown in perlite at 120 mm nacl. means (n=5) ± se within a column for each species marked with the same lowercase do not differ significantly (multiple range duncan’s test; p < 0.05); lod – limit of detection. species/cultivar glucose fructose sucrose maltose raffinose trehalose total sum of soluble sugars triticum aestivum cv. banderola control 9.43±0.81c 5.25±0.60d 1.12±0.09c 0.43±0.05d < lod 0.04±0.01a 16.27±1.11c saline control 12.67±1.10b 7.39±0.62c 1.08±0.09c 0.57±0.06c < lod 0.05±0.01a 21.96±1.76b epi 16.60±1.38a 11.60±0,99a 2.81±0.22b 1.76±0.15b < lod 0.04±0.01a 32.81±2.62a zen 11.94±1.06b 8.95±0.80b 7.25±0.63a 3.60±0.31a < lod 0.05±0.01a 31.79±2.86a triticum durum cv. komnata control 9.52±0.73c 6.61±0.51c 2.35±0.21b 0.79±0.07b < lod 0.05±0.01a 19.32±1.35c saline control 10.87±0.77b 8.21±0.77b 2.32±0.21b 0.98±0.09a < lod 0.05±0.01a 22.43±1.72b epi 12.74±1.11a 9.99±0.83a 1.02±0.09c 0.68±0.07b < lod 0.04±0.01a 24.43±1.72b zen 12.71±1.09a 9.48±0.80a 11.94±0.95a 0.53±0.06c < lod 0.04±0.01a 34.70±2.15a zea mays cv. król control 4.26 ±0.39b 1.65±0.14c 16.55±1.54b 0.17±0.02b < lod 0.01±0.0b 22.64±2.08c saline control 6.61±0.58a 3.01±0.25b 19.63±1.53b 0.28±0.03a < lod 0.01±0.0b 29.54±2.09b epi 7.50±0.69a 5.74±0.49a 23.41±1.75a 0.29±0.03a < lod 0.04±0.01a 36.98±3.04a zen 3.93±0.30b 1.55±0.09c 21.97±1.69ab 0.12±0.01c 0.09 0.03±0.0a 27.69±2.26c sorghum bicolor cv. cukrosorgo control 7.14±0.60c 2.64±0.25c 7.86±0.61b 0.29±0.03d < lod 0.02±0.0c 17.95±1.61c saline control 11.49±1.00a 5.28±0.47a 6.75±0.55c 0.41±0.04c < lod 0.06±0.01a 23.99±1.95b epi 9.17±0,88b 4.07±0.39b 8.99±0.72b 1.35±0.12b < lod 0.02±0.0c 23.60±1.94b zen 10.99±1.08a 5.99±0.54a 24.47±2.01a 2.12±0.19a < lod 0.03±0.0b 43.60±2.87a control – plants untreated with nacl and stimulants; saline control – plants grown at 120 mm nacl 286 a. płażek, m. tatrzańska, m. maciejewski, m. dziurka, f. dubert content in wheat plants grown under moderate salinity. in an earlier investigation (płażek et al., 2013) control plants of durum cultivars, non-treated with salt stress, were characterized by a two-fold higher proline content compared with the bread wheat cultivars, but 125 mm nacl salinity did not increase the proline content in the leaves of both wheat species. in the present experiment, in the leaves of both wheat species under 120 mm nacl a decline of proline content was observed. epi significantly increased the proline amount in the leaves of both bread and durum wheat, but this was lower than after the zen treatment, which increased the level 5.8-fold in bread wheat leaves and 9.4-fold in durum leaves. talaat and shawky (2013) showed that epi alleviated the inhibition of wheat productivity in saline soil by increasing proline accumulation. especially high proline synthesis levels under salt stress were observed in m. × giganteus leaves (płażek et al., 2014). at a salinity level of 150 mm nacl, the level of free proline was 17 times as high as that of the control. some authors have reported that proline cannot be used as biochemical indicator of salt tolerance (aziz et al., 1998; ashraf and harris, 2004). aziz et al. (1998) described a negative relationship between the proline content in the leaves and the salt tolerance of tomato plants. a key role of aba in drought and salt tolerance as a long-distance signaling phytohormone is well documented (zhu, 2002; zhang et al., 2006; yuan et al., 2010). moreover, the role of aba in drought and salt stress is two-fold: water balance and cellular dehydration tolerance. in the studied plants, an increase in aba, as an effect of the zen treatment, was noted in durum wheat and sorghum plants. yuan et al. (2010) stated the amelioration of the drought stress in tomato seedlings by the epi-induced elevation of endogenous aba concentrations. the accumulation of soluble carbohydrates in plants has been widely reported as a response to salinity or drought (ashraf and harris, 2004). ashraf and tufail (1995) found higher concentration of sugars in salt tolerant lines of sunflower than in less tolerant lines. generally, in the studied plant species grown at 120 mm nacl, an increase in the total sum of soluble sugars, and in particular mono and disaccharides, under epi and zen treatments was observed. a similar effect occurred with epi, which increased the soluble carbohydrate content in grains of wheat, was noted by janeczko et al. (2010). trehalose is a disaccharide that plays a special role in plants under osmotic stress (zeid, 2009). this sugar accumulates at very low concentrations, which was corroborated by our investigation. according to garg et al., (2002), trehalose levels remain well below 1 mg g−1 fw, and its accumulation correlates with the higher soluble carbohydrate levels and elevated photosynthesis efficiency under stress and nonstress conditions. zeid (2009) found that maize seedlings grown at high salinity, and treated with exogenous trehalose, demonstrated a stabilization of the plasma membranes, decreases in ion leakage and the lipid peroxidation rate, as well as an increase in the k+ / na+ ion ratio in the leaves. in the described experiment epi and zen eleva ted the amount of trehalose only in maize leaves of plants grown at 120 mm nacl. this effect was observed along with a simultaneous decrease in ion leakage from the leaf cells. conclusions in bread and durum wheat as well as in maize and sorghum plants, zen had a greater influence on the studied parameters than epi. the zen-alleviating effects on salinity stress could be the result of the improved root mass, which is a typical effect of plant tolerance response to soil drought. in this plant species zen caused a reduction of ion transport between roots and shoots. moreover, this stimulator increases the abscisic acid, proline as well as soluble carbohydrate content in plant leaves, the compounds which play key roles in defence reactions to osmotic stress. acknowledgment the study was supported by the polish ministry of science and higher education no. 813/n-cost/2010/0 (within cost action fa0901). references ashraf, m., harris, p.j.c., 2004: potential biochemical indicators of salinity tolerance in plants. plant sci. 166, 3-16. doi: 10.1016/j.plantsci.2003.10.024 ashraf, m., tufail, m., 1995: variation in salinity tolerance in sunflower (helianthus annum l.). j. agron. crop sci. 174, 351-362. doi: 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cultures. plant cell rep. 34, 47-62. doi: 10.1007/s00299-014-1686-4 address of the corresponding author: department of plant physiology, university of agriculture of krakow, podłużna 3, 30-239 kraków, poland e-mail: rrplazek@cyf-kr.edu.pl © the author(s) 2017. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 89, 163 170 (2016), doi:10.5073/jabfq.2016.089.020 1suleyman demirel university, agriculture faculty, horticulture department, isparta, turkey 2 eğirdir fruit research institute, isparta, turkey variability of phenolic composition and tocopherol content of some commercial almond cultivars adnan nurhan yildirim1*, fatma yildirim1, bekir şan1, mehmet polat1, yılmaz sesli2 (received january 20, 2016) * corresponding author summary the phenolic (gallic acid, catechin, caffeic acid, chlorogenic acid, epicatechin, ferulic acid, kaempferol, naringenin, and p-coumaric acid) and tocopherol contents (α, β, γ, and δ) of some commercially significant almond cultivars were determined in the research. wide variations in phenolic and tocopherol contents were detected among the cultivars. the highest content among the phenolic substances was obtained for catechin, with the average values of 27.35 mg kg-1 in 2008 and 39.87 mg kg-1 in 2009. the highest catechin content was recorded in the cultivar ‘ferraduel’ in both years, with the values of 117.59 mg kg-1 in 2008 and 145.86 mg kg-1 in 2009. the highest content among the tocopherols was obtained in α-tocopherol, and the average values were detected as 312.29 mg kg-1 in 2008 and 467.31 mg kg-1 in 2009. the highest α-tocopherol contents were determined as 899.49 mg kg-1 and 945.41 mg kg-1 in the cultivar ‘supernova’ in both years, respectively. in the research, α-tocopherol turned out to be the major tocopherol. introduction almond is the species with the highest economic value among the nut fruits cultivated worldwide (venkatachalam and sathe, 2006). its high contents of oil, proteins, minerals, fiber, sterols, phenolics and tocopherols (α, β, δ, and γ) (beyhan et al., 2011; kirbaşlar et al., 2012; venkatachalam and sathe, 2006; yada et al., 2011) and its different ways of consumption and uses (e.g. uncooked, roasted, blanched, the chocolate and cream cake industry, and the pharmaceutical and paint industries) are among the features which highlight almond (esfahlan and jamei, 2012; socias i company, 2008; vijeratne et al., 2006). in addition, it is effectively used to reduce the risk of cardiovascular and other chronic diseases (28 to 100 g day-1); to decrease the level of ldl cholesterol in blood; to reduce the effect of many types of cancer such as lung, pancreas, stomach, and colon; to nourish the brain and nervous systems; and to fight obesity and diabetes for a better life (ahmad, 2010; bolling et al., 2010; davis and iwahashi, 2001; esfahlan et al., 2010; jenkins et al., 2002). furthermore, the richness of almond oil in antioxidants also allows it to be utilized as a natural softener and a skin rejuvenator today by beauticians, massage specialists, and aromatherapists (ahmad, 2010). natural antioxidant substances are compounds which are comprised of carotenoids, ascorbic acid, phenolics, and tocopherols that are found in the fruits, leaves, roots, seeds, and oils of plants and that provide them with their characteristics such as color, bitterness, and astringency (isfahlan et al., 2010; kasnak and palamutoğlu, 2015; nizamlioğlu and nas, 2010). phenolics and tocopherols are widely used in the food industry for the stability of oils, the preservation of the quality of food, the extension of their shelf life, and their healthy consumption (kirbaşlar et al., 2012; korekar et al., 2011; sang et al., 2002; takeoka and dao, 2003). moreover, they have a feature of scavenging the free radicals which induce the oxidative degradation of lipids, proteins, and nucleic acids (esfahlan and jamei, 2012; isabelle et al., 2010; izaddost et al., 2013; kirbaşlar et al., 2012; mishra et al., 2010). phenolics play a significant role in such physiological events as growth and reproduction, and in developing the defense mechanism against pathogens and predators in plants (balasundram et al., 2006). tocopherols (vitamin e) are lipophilic antioxidants with isomers as α, β, γ, and δ that are synthesized particularly by the leaves and seeds of plants (szymanska and kruk, 2008). by inhibiting lipid peroxidation, they enhance the biological activity of the body and protect the skin against external effects like ultraviolet rays (di mambro et al., 2003). although almond is known to be a fruit which is rich in biochemical contents, no sufficient information is available about the potential of commercial almond cultivars in terms of these characteristics (moayedi et al., 2011), for the genetic traits of species and cultivars directly affect biochemical contents. besides, the maturity state of fruits, the growing season, soil properties, temperature and the duration of storage of products also have indirect effects on biochemical contents in plants (balasundram et al., 2006; garcia-pascual et al., 2003; habila et al., 2012; ismail et al., 2004; kodad et al., 2006; kodad et al., 2011; milbury et al., 2006; piironen et al., 1986). in this study, it was aimed to determine the variations of some commercial almond cultivars grown at isparta, turkey in terms of phenolic and tocopherol contents. in addition, the studied traits were subjected to principal component analysis for the classification and discrimination of almond cultivars. materials and methods plant material the research was conducted at eğirdir fruit research institute in 2008 and 2009. the cultivars ‘cristomorto’, ‘d. largueta’, ‘ferraduel’, ‘ferragnes’, ‘ferrastar’, ‘glorieta’, ‘lauranne’, ‘masbovera’, ‘nonpareil’, ‘picantili’, ‘sonora’, ‘supernova’, ‘texas’, ‘tuono’ and ‘yaltinski’, grafted on almond seedling planted in 2003, were used in the study. the trees were irrigated by using a drip irrigation system and supported with the standard fertilizers ammonium nitrate, potassium sulphate, phosphoric acid, zinc sulphate, and borax. the study was set up according to a completely randomized design with 3 replications, and one tree was used in each replication. the mature fruits were collected at an approximate amount of 1 kg from different places of the trees, dried at room temperature for about 20 days, and then crushed by hand. almond kernels obtained from each cultivar were ground into powder and placed in plastic bags. the samples were preserved at -80 °c until extraction. hplc analysis of phenolics the samples (5 g) were extracted in 25 ml of methanol for 16 hours in shaker incubator operated at 40 °c. the samples were passed through membrane filters with 0.45 μm pore size. extracts were then evaporated at 40 °c under vacuum until they dried. extracts were re164 a.n. yildirim, f. yildirim, b. şan, m. polat, y. sesli dissolved in 2 ml of methanol, and then 20 μl of samples was injected into high performance liquid chromatography (hplcshimadzu). phenolics were determined using hplc device with a diode array detector (dad, λ max = 278 nm) by the modified procedure of caponio et al. (1999). 20 μl of the extracts were injected into hplc device equipped with an agilent eclipse xdb-c 18 (250 × 4.60 mm 5 μm) column operated at 30 °c. the elution solvents were solvent a: 3 % acetic acid and solvent b: 100 % methanol. the gradient as follows: 7 % in b as initial condition, 28 % in b for 20 min, 25 % in b for 8 min, 30 % in b for 7 min, 30 % in b for 15 min, 33 % in b for 10 min, 42 % in b for 2 min, 50 % in b for 8 min, 70 % in b for 3 min, 80 % in b for 2 min, 100 % in b for 5 min and 7 % in b for 1 min. the flow rate was 0.8 ml min-1. peak identification was performed according to the standards (gallic acid, catechin, chlorogenic acid, caffeic acid, epicatechin, p-coumaric acid, ferulic acid, o-coumaric acid, rutin, hesperidin, cinnamic acid, quercetin, naringenin, luteolin, kaempferol) (fig. 1). phenolic quantities were explained as mg kg-1 dw. hplc analysis of tocopherols two g from ground kernels were taken and oil was extracted in a soxhlet apparatus with 100 ml of hexane as a solvent. the solvent was evaporated at 40 °c under vacuum using a rotary evaporator and the oil was taken (bligh and dyer, 1959). tocopherols (α, γ, δ, and β) were analyzed by hplc with a rf10axl fluorescence detector (295 nm). 10 μl of the oil was injected into the hplc device equipped with luna silica column (250 × 4.6 mm, 5 μm particle size). the conditions were as follows: mobile phase flow rate, 1.2 ml/min; mobile phase mixture, heptane: thf (95:5) (v/v). peak identification was performed according to standards (fig. 2). the quantity of tocopherols was determined according to peak area and expressed in mg kg-1 oil. statistical analysis all experiments were performed with three replications. the data were subjected to analyze of variance (anova) using minitab software (minitab inc.) at 5 % significance level. the means were separated by tukey’s test. results and discussion phenolic contents in the research, the phenolic contents of the almond cultivars are presented in tab. 1. wide variations were detected among the cultivars in terms of phenolic contents. the analysis of variance showed that the effects of cultivars, years and the cultivars × years interaction were significant for all phenolic compounds except p-coumaric acid (p < 0.05). the highest content among the phenolics was obtained for catechin, with 27.35 mg kg-1 in 2008 and 39.87 mg kg-1 in 2009. the highest catechin content was recorded in the cultivar ‘ferraduel’ in both years, with 117.59 mg kg-1 in 2008 and 145.86 mg kg-1 in 2009. the gallic acid contents ranged from 1.22 mg kg-1 (ferraduel) to 3.26 mg kg-1 (nonpareil) in 2008 but from 0.67 mg kg-1 (glorieta) to 3.03 mg kg-1 (picantili) in 2009. the highest caffeic acid content was determined as 2.53 mg kg-1 in the cultivar ‘ferraduel’ in 2008 but as 2.80 mg kg-1 in cultivar ‘sonora’ in 2009. although varying by year, no chlorogenic acid could be detected in some cultivars. the chlorogenic acid content was found to be in the range 0.47 mg kg-1 (texas)-6.16 mg kg-1 (ferraduel) in 2008 and in the range 0.92 mg kg-1 (lauranne)-7.00 mg kg-1 (sonora) in 2009. the highest epicatechin content was detected in the cultivar ‘ferraduel’ in both years (21.07 mg kg-1 in 2008 and 27.57 mg kg-1 in 2009). ferulic acid, kaempferol, naringenin and p-coumaric acid contents were found to be rationally less than the other phenolic contents. additionally, it was established that these phenolics varied very slightly by year and were more stable. the highest ferulic acid content was recorded in the cultivar ‘ferraduel’ (0.48 mg kg-1) in 2008 and in the cultivar ‘yaltinski’ (0.52 mg kg-1) in 2009, whereas the lowest ferulic acid content was detected in the cultivar ‘texas’ in both years. kaempferol could not be determined in most cultivars in either year. it was found to be in the range 0.05 mg kg-1 (sonora)-0.21 mg kg-1 (yaltinski) in 2008 and in the range 0.04 mg kg-1 (ferragnes)-0.12 mg kg-1 (nonpareil and picantili) in 2009. naringenin ranged from 0.05 mg kg-1 (supernova) to 1.82 mg kg-1 (yaltinski) in 2008 and from 0.11 mg kg-1 (glorieta) to 3.23 mg kg-1 (ferrastar) in 2009 but could not be determined in the cultivar ‘d. largueta’ in either year. no statistical difference in p-coumaric acid was found among the cultivars. p-coumaric acid was detected to range from 0.04 mg kg-1 (masbovera, glorieta, and cristomorto) to 0.13 mg kg-1 (d. largueta and fig. 1: hplc chromatograms of phenolic standards and extracts of ferraduel kernel. 1; gallic acid 2; catechin 3; chlorogenic acid 4; caffeic acid 5; epicatechin, 6; p-coumaric acid 7; ferulic acid 8; o-coumaric acid 9; rutin 10; hesperidin 11; cinnamic acid 12; quercetin 13; naringenin 14; luteolin 15; kaempferol phenolic and tocopherol composition of some almond cultivars 165 ferraduel) in 2008 and from 0.03 mg kg-1 (glorieta) to 0.38 mg kg-1 (d. largueta) in 2009. the phenolic contents were found to vary by year. this variation was significant in some cultivars but more stable in some of them. in previous studies, it was reported that such factors such as fungi, bacteria, pests, air and light might be effective on the occurrence of this difference, along with the environmental factors in the region of cultivation, cultivation techniques, the maturity state of fruits, the soil properties in the region, and the genetic traits of species and cultivars (balasundram et al., 2006; esfahlan and jamei, 2012; garciapascual et al., 2003; habila et al., 2012; ismail et al., 2004; kodad et al., 2006; kodad et al., 2011; piironen et al., 1986; rice-evans et al., 2006). bolling et al. (2010) reported that the phenolic contents varied by cultivar and that the catechin content ranged from 0.5 mg kg-1 to 3 mg kg-1, the epicatechin content from 0.3 mg kg-1 to 0.7 mg kg-1, the kaempferol content from 0.2 mg kg-1 to 1.4 mg kg-1, and the naringenin content from 0.2 mg kg-1 to 0.8 mg kg-1. milbury et al. (2006) detected a large number of phenolics in many almond cultivars and determined lower contents of catechin (0.09-0.4 mg kg-1), epicatechin (0.03-0.1 mg kg-1), naringenin (0.002-0.02 mg kg-1), and kaempferol (0-0.02 mg kg-1) than the results presented in this paper. similarly, bartolome et al. (2010) obtained lower kaempferol and naringenin contents than our results. nut fruits are rich in phenolic compounds (yada et al., 2011), and such phenolic compounds as catechin, p-coumaric acid, caffeic acid, and ferulic acid are also prevalent in kernels. it is reported that the catechin content of almond kernel is quite high, while its ferulic acid content is low (rice-evans et al., 2006; sang et al., 2002). these results are also supported by the findings obtained from our study. tocopherol contents in the research, the tocopherol contents of the almond cultivars are presented in tab. 2. statistically significant differences in tocopherol contents occurred among the cultivars (p < 0.05). the analysis of variance showed that the effects of cultivars, years and the cultivars × years interaction were significant for all tocopherols (p < 0.01). the highest content among the tocopherols was obtained in α-tocopherol, and the average values were recorded as 312.29 mg kg-1 in 2008 and as 467.31 mg kg-1 in 2009. the highest α-tocopherol content was determined in cultivar ‘supernova’ in both years (899.49 mg kg-1 and 945.41 mg kg-1, respectively). the highest β-tocopherol content in the research was again found in cultivar ‘supernova’ in both years (10.53 mg kg-1 and 7.64 mg kg-1, respectively). when the γ-tocopherol content of the almond cultivars was evaluated, the highest content was recorded in cultivar ‘supernova’ (57.24 mg kg-1) in 2008 but in cultivar ‘picantili’ (77.87 mg kg-1) in 2009. in the research, the lowest values among the tocopherols were obtained in δ-tocopherol and varied between 0.10 mg kg-1 (masbovera) and 2.29 mg kg-1 (supernova) in 2008 and between 0.10 mg kg-1 (masbovera) and 2.86 mg kg-1 (d. largueta) in 2009. in the research, the average tocopherol concentrations were determined to be higher in 2009 than in 2008. it is thought that this difference might be due to the ecological factors which vary by years (kodad et al., 2006; korekar et al., 2011). furthermore, it was established that the α-tocopherol was the predominating tocopherol which is in agreement with findings of kodad et al. (2006) who reported that the α-tocopherol content was ten times higher than the δand γ-tocopherol contents and that this feature might be used in almond breeding studies. kornsteiner et al. (2006) and zacheo et al. (2000) reported that α-tocopherol was the dominant tocopherol in nut fruits, particularly almond. yang (2009) stated that the α-tocopherol content was 439.5 mg kg-1 and that the γ-tocopherol content was 12.5 mg kg-1. kodad et al. (2011) reported that the tocopherol contents varied by years and cultivars and that, in parallel with our results, the α-tocopherol content ranged from 210.9 mg kg-1 to 553.4 mg kg-1, the γ-tocopherol content from 4.64 mg kg-1 to 14.92 mg kg-1, and the δ-tocopherol content from 0.2 mg kg-1 to 1.02 mg kg-1. socias i company et al. (2014) stated that the tocopherol contents varied by years and the region of cultivation and that the α-tocopherol content ranged from 437.83 mg kg-1 to 488.89 mg kg-1, the γ-tocopherol content from 13.17 mg kg-1 to 26.23 mg kg-1, and the δ-tocopherol content from 0.65 mg kg-1 to 1.39 mg kg-1, which were lower than our results. almond is the fruit with the highest tocopherol content among nut fruits (kodad et al., 2011; yang, 2009; izaddost et al., 2013). this feature ensures that the fruits can be stored without becoming rotten for a long period of time, that the oils they contain remain stable for a long period of time, and that the nutritional content is not impaired without adding any synthetic additive (sang et al., 2002; miraliakbari and shahidi, 2008). this is because tocopherols have a high antioxidant quality retention time (min) fig. 2: hplc chromatograms of tocopherols standards and extracts of ferraduel kernel. 166 a.n. yildirim, f. yildirim, b. şan, m. polat, y. sesli ta b. 1 : ph en ol ic c om po un ds o f a lm on d cu lti va rs (m g kg -1 d w ). c ul ti va rs g al lic a ci d c at ec hi n c af fe ic a ci d c hl or og en ic a ci d 20 08 20 09 20 08 20 09 20 08 20 09 20 08 20 09 c ri st om or to 1. 46 ±0 .1 7 de fa 0. 73 ±0 .1 5 cd b 12 .5 8± 2. 40 fg b 43 .8 1± 7. 26 da 0. 53 ±0 .0 3 fb 2. 13 ±0 .5 1 bc a n d 1. 12 ±0 .0 1 e d . l ar gu et a 1. 27 ±0 .2 7 ef b 1. 43 ± 0. 27 bc a 31 .1 8 ±2 .8 2 bc a 21 .0 8 ±4 .1 8 hb 1. 50 ± 0. 29 bc da 0. 97 ±0 .1 9 ef b 2. 08 ±0 .3 8 ca 1. 19 ±0 .1 3 de b fe rr ad ue l 1. 22 ±0 .4 0 fa 0. 78 ±0 .2 3 cd b 11 7. 59 ± 1. 35 ab 14 5. 86 ±2 .1 7 aa 2. 53 ± 0. 40 ab 2. 57 ±0 .6 9 ab a 6. 16 ±0 .2 7 aa 4. 67 ±0 .0 2 bb fe rr ag ne s 2. 34 ±0 .2 1 bc a 1. 53 ± 0. 26 bb 31 .0 3± 1. 21 bc b 57 .8 6 ±0 .4 1 ca 1. 23 ± 0. 29 cd eb 1. 80 ±0 .5 2 cd a 1. 73 ±0 .3 5 cd b 1. 83 ±0 .0 2 cd a fe rr as ta r 2. 10 ±0 .4 0 cd a 1. 70 ± 0. 23 bb 24 .2 2 ±3 .8 7 c db 45 .9 8± 1. 42 da 0. 43 ±0 .0 2 fb 1. 26 ±0 .8 1 de a 0. 88 ±0 .0 9 ef gb 0. 93 ±0 .0 8 ea g lo ri et a 1. 67 ±0 .4 0 cd ef a 0. 67 ± 0. 29 db 10 .6 7 ±1 .3 0 f gb 21 .4 0± 1. 98 ha 0. 73 ±0 .0 3 ef b 1. 00 ±0 .2 9 ef a 1. 30 ±0 .1 4 de f n d l au ra nn e 1. 59 ±0 .6 1 de fa 1. 33 ± 0. 52 bc db 29 .6 6 ±0 .4 8 bc b 30 .3 7± 2. 12 fg a 1. 83 ±0 .2 3 bc b 2. 40 ± 0. 34 ab ca n d 0. 92 ±0 .0 3 e m as bo ve ra 1. 73 ±0 .5 2 cd ef a 1. 05 ± 0. 54 bc db 9. 40 ±2 .1 5 ga 5. 19 ±1 .5 4 jb 0. 57 ±0 .0 5 fa 0. 50 ±0 .0 4 fb 1. 50 ±0 .4 0 cd e n d n on pa re il 3. 26 ±0 .7 0 aa 1. 37 ± 0. 29 bc db 6. 77 ±2 .7 5 g b 20 .2 5± 2. 56 ha 0. 57 ±0 .0 7 fb 1. 00 ±0 .4 4 ef a n d n d pi ca nt ili 2. 83 ±0 .5 2 ab b 3. 03 ±0 .2 7 aa 8. 15 ±1 .8 0 gb 13 .7 2 ±2 .1 8 ıa 0. 45 ±0 .0 6 fb 0. 87 ±0 .0 8 ef a 0. 62 ±0 .0 6 fg hb 2. 09 ±0 .8 8 ca so no ra 3. 10 ±0 .5 2 aa 1. 70 ± 0. 37 bb 34 .6 8 ±2 .0 8 bb 41 .8 7 ±1 .7 0 de a 1. 87 ±0 .5 8 bb 2. 80 ±0 .5 2 aa 2. 95 ±0 .2 8 bb 7. 00 ±0 .0 1 aa su pe rn ov a 1. 60 ±0 .5 8 cd ef a 1. 27 ± 0. 38 bc db 19 .4 6± 2. 91 de b 73 .3 4± 2. 64 ba 0. 47 ±0 .0 8 fb 2. 56 ±0 .7 8 ab a 1. 23 ±0 .4 6 de f n d te xa s 1. 65 ±0 .4 5 cd ef a 1. 60 ±0 .2 3 bb n d 12 .2 3± 1. 22 ı 0. 43 ±0 .0 7 fb 0. 59 ± 0. 06 fa 0. 47 ±0 .0 4 gh n d tu on o 2. 10 ±0 .2 3 cd a 1. 19 ± 0. 29 bc db 16 .3 8 ±1 .5 1 ef b 28 .3 1± 2. 28 ga 1. 00 ±0 .1 1 de fb 2. 67 ± 0. 64 ab a 1. 63 ±0 .7 1 cd n d y al tin sk i 2. 00 ±0 .4 2 cd ea 0. 99 ±0 .2 9 bc db 31 .1 6 ±0 .8 1 bc b 36 .7 2± 3. 20 ef a 1. 03 ±0 .2 3 de fa 1. 00 ±0 .0 7 ef b 1. 46 ±0 .6 5 cd e n d m ea n 1. 99 ±0 .4 3 1. 36 ±0 .3 1 27 .3 5± 1. 96 39 .8 7± 2. 46 1. 01 ±0 .1 7 1. 61 ±0 .3 9 1. 69 ±0 .2 6 2. 47 ±0 .0 8 l sd 0. 65 97 6. 45 8 0. 57 55 0. 64 70 e ac h va lu e is e xp re ss ed a s m ea n ±s ta nd ar t d ev ia tio n, m ea ns fo llo w ed b y di ff er en t c ap ita l l et te rs (y ea rs ) i n th e ro w a re s ig ni fic an tly d iff er en t ( p< 0. 05 ). m ea ns fo llo w ed b y di ff er en t s m al l l et te rs in th e co lu m ns (c ul tiv ar s) a re s ig ni fic an tly d iff er en t ( p< 0. 05 ). n d: n ot d et ec te d. phenolic and tocopherol composition of some almond cultivars 167 ta b. 1 : p he no lic c om po un ds o f a lm on d cu lti va rs (m g kg -1 ). c ul ti va rs e pi ca te ch in f er ul ic a ci d k ae m pf er ol n ar in ge ni n p -c ou m ar ic a ci d 20 08 20 09 20 08 20 09 20 08 20 09 20 08 20 09 20 08 20 09 c ri st om or to 2. 53 ±0 .5 2 de b 21 .0 7± 0. 16 ba 0. 16 ±0 .0 7 eb 0. 30 ±0 .0 1 ca n d n d 0. 14 ±0 .0 3 cb 0. 26 ±0 .0 3 ea 0. 04 ±0 .0 2 n d d . l ar gu et a 6. 27 ±0 .8 4 ba 6. 23 ±0 .8 1 fg b 0. 47 ±0 .0 8 aa 0. 39 ±0 .0 2 bb n d n d n d n d 0. 13 ±0 .0 1 0. 38 ±0 .0 4 fe rr ad ue l 21 .0 7± 0. 40 ab 27 .5 7± 0. 81 aa 0. 48 ±0 .0 9 aa 0. 41 ±0 .1 0 bb 0. 07 ±0 .0 2 bc b 0. 09 ±0 .0 2 ab a 0. 15 ±0 .0 4 cb 0. 23 ±0 .0 5 ea 0. 13 ±0 .0 6 n d fe rr ag ne s 3. 60 ±0 .9 5 cb 10 .2 0± 0. 69 da 0. 30 ±0 .0 9 ba 0. 25 ±0 .0 7 db n d 0. 04 ±0 .0 1 bc 0. 12 ±0 .0 4 cb 0. 15 ±0 .0 7 ea 0. 06 ±0 .0 2 n d fe rr as ta r 1. 63 ±0 .0 8 ef gb 4. 97 ±0 .1 9 ha 0. 23 ±0 .0 1 cd n d n d 0. 07 ±0 .0 1 b 0. 06 ±0 .0 1 cb 3. 23 ±0 .0 9 aa 0. 06 ±0 .0 2 0. 05 ±0 .0 1 g lo ri et a 0. 93 ±0 .0 6 gh b 3. 17 ±0 .7 8 ıa 0. 27 ±0 .0 6 bc a 0. 25 ±0 .0 9 db n d n d 0. 14 ±0 .0 3 ca 0. 11 ±0 .0 3 eb 0. 04 ±0 .0 2 0. 03 ±0 .0 1 l au ra nn e 2. 60 ±0 .4 0 de b 7. 10 ±0 .8 1 fa 0. 25 ±0 .0 5 cd b 0. 37 ±0 .0 5 ba n d 0. 05 ±0 .0 2 b 0. 07 ±0 .0 1 cb 0. 17 ±0 .0 5 ea 0. 06 ±0 .0 2 0. 06 ±0 .0 2 m as bo ve ra 1. 50 ±0 .5 5 fg ha 0. 90 ±0 .0 8 kb 0. 22 ±0 .0 6 db 0. 30 ±0 .0 7 ca n d n d 0. 15 ±0 .0 5 cb 0. 21 ±0 .0 6 ea 0. 04 ±0 .0 1 0. 04 ±0 .0 2 n on pa re il 1. 03 ±0 .0 9 gh b 5. 93 ±0 .8 7 ga 0. 30 ±0 .0 4 ba 0. 25 ±0 .0 7 db 0. 11 ±0 .0 1 bb 0. 12 ±0 .0 3 aa 1. 45 ±0 .0 3 aa 0. 77 ±0 .0 4 db 0. 06 ±0 .0 1 0. 05 ±0 .0 2 pi ca nt ili 1. 10 ±0 .0 9 gh b 1. 20 ±0 .8 7 ka 0. 22 ±0 .0 6 db 0. 50 ±0 .0 7 aa 0. 08 ±0 .0 3 bc b 0. 12 ±0 .0 4 aa 0. 78 ±0 .0 2 bb 2. 07 ±0 .0 6 ba 0. 06 ±0 .0 3 0. 06 ±0 .0 1 so no ra 3. 37 ±1 .1 9 cd b 8. 10 ±1 .0 4 ha 0. 26 ±0 .0 9 bc db 0. 39 ±0 .0 8 ba 0. 05 ±0 .0 1 cb 0. 06 ±0 .0 1 ba n d 0. 16 ±0 .0 4 e 0. 10 ±0 .0 3 0. 10 ±0 .0 2 su pe rn ov a 2. 10 ±0 .9 5 ef b 11 .6 7± 1. 73 ca 0. 21 ±0 .0 7 d n d n d n d 0. 05 ±0 .0 1 c n d 0. 05 ±0 .0 1 n d te xa s 0. 53 ±0 .0 7 hb 0. 97 ±0 .0 7 ka 0. 11 ±0 .0 9 fa 0. 13 ±0 .0 4 eb n d n d 1. 74 ±0 .3 9 aa 1. 41 ±0 .2 8 cb 0. 11 ±0 .0 1 0. 07 ±0 .0 3 tu on o 2. 37 ±1 .0 1 ef a 1. 30 ±0 .0 9 kb 0. 24 ±0 .0 6 cd a 0. 16 ±0 .0 6 eb n d 0. 06 ±0 .0 1 b 0. 11 ±0 .0 2 cb 0. 12 ±0 .0 2 ea 0. 10 ±0 .0 3 n d y al tin sk i 2. 53 ±0 .9 2 de a 2. 20 ±0 .0 7 jb 0. 47 ±0 .0 4 ab 0. 52 ±0 .0 6 aa 0. 21 ±0 .0 3 aa n d 1. 82 ±0 .0 4 aa 1. 16 ±0 .1 0 cb 0. 10 ±0 .0 2 0. 05 ±0 .0 1 m ea n 3. 54 ±0 .5 4 7. 51 ±0 .6 0 0. 28 ±0 .0 6 0. 32 ±0 .0 5 0. 10 ±0 .0 07 0. 08 ±0 .0 1 0. 52 ±0 .0 4 0. 77 ±0 .0 6 0. 08 ±0 .0 2 0. 09 ±0 .0 2 l sd 0. 89 29 0. 04 46 1 0. 04 53 3 0. 38 49 0. 13 9 e ac h va lu e is e xp re ss ed a s m ea n ±s ta nd ar d de vi at io n, m ea ns f ol lo w ed b y di ff er en t ca pi ta l le tte rs ( ye ar s) i n th e ro w a re s ig ni fic an tly d iff er en t (p <0 .0 5) . m ea ns f ol lo w ed b y di ff er en t sm al l le tte rs i n th e co lu m ns (c ul tiv ar s) a re s ig ni fic an tly d iff er en t ( p< 0. 05 ). n d: n ot d et ec te d. 168 a.n. yildirim, f. yildirim, b. şan, m. polat, y. sesli ta b. 2 : to co ph er ol c on te nt s of a lm on d cu lti va rs (m g kg -1 o il) . c ul ti va rs a lp ha -t oc op he ro l b et ato co ph er ol g am m ato co ph er ol d el ta -t oc op he ro l 20 08 20 09 20 08 20 09 20 08 20 09 20 08 20 09 c ri st om or to 19 5. 67 ±1 1. 16 fb 58 6. 28 ±1 7. 40 ea 1. 80 ±0 .9 8 fg b 5. 99 ±2 .0 5 bc a 11 .2 6± 4. 14 hf b 43 .0 3± 7. 27 da 0. 17 ±0 .0 1 cd eb 1. 30 ±0 .4 2 bc a d . l ar gu et a 29 1. 12 ±1 8. 60 de b 35 2. 58 ±1 7. 70 ha 2. 88 ±1 .1 9 db 5. 44 ±1 .2 2 cd a 15 .3 2± 1. 79 fg b 42 .9 7± 2. 14 da 0. 41 ±0 .0 6 cd eb 2. 86 ±0 .2 1 aa fe rr ad ue l 30 3. 07 ±1 9. 30 db 70 4. 52 ±4 .2 8 ca 2. 42 ±0 .3 8 de fb 4. 99 ±0 .6 9 da 15 .8 0± 0. 21 fb 41 .8 5± 0. 99 de a 0. 23 ±0 .0 3 cd eb 1. 18 ±0 .0 7 bc a fe rr ag ne s 30 0. 94 ±1 .4 0 db 51 2. 16 ±2 1. 30 fa 3. 06 ±0 .7 2 cd b 4. 01 ±0 .6 4 ea 13 .3 3± 1. 54 gh b 25 .5 8± 1. 22 ha 0. 39 ±0 .0 4 cd eb 0. 58 ±0 .0 7 de a fe rr as ta r 29 9. 77 ±1 5. 45 da 23 1. 64 ±1 1. 20 db 2. 73 ±0 .4 5 de b 2. 89 ±0 .4 7 fa 19 .2 9± 1. 36 eb 27 .9 8± 1. 66 ga 0. 57 ±0 .0 9 cd a 0. 55 ±0 .0 9 de fb g lo ri et a 26 7. 81 ±8 .0 6 eb 34 2. 43 ±2 3. 80 ha 2. 24 ±0 .6 4 de fb 5. 11 ±0 .3 6 da 5. 87 ±1 .7 8 kb 16 .3 1± 1. 88 ja 0. 40 ±0 .0 3 cd ea 0. 27 ±0 .0 8 ef b l au ra nn e 26 0. 23 ±1 8. 40 ea 14 6. 75 ±1 5. 44 kb 3. 74 ±0 .6 7 ca 1. 99 ±0 .0 8 gb 16 .7 9± 0. 91 fa 8. 78 ±1 .5 0 lb 1. 17 ±0 .0 6 cd ea 0. 49 ±0 .0 2 ef b m as bo ve ra 19 1. 31 ±6 .9 1 fa 16 5. 60 ±2 5. 20 hb 2. 87 ±0 .4 0 db 3. 17 ±0 .4 6 fa 8. 00 ±0 .8 9 jk b 14 .0 4± 0. 88 ka 0. 10 ±0 .0 2 eb 0. 10 ±0 .0 1 fb n on pa re il 37 6. 89 ±2 5. 50 ca 35 0. 65 ±6 .7 3 hb 2. 52 ±0 .7 2 de fb 4. 17 ±0 .6 8 ea 27 .4 5± 1. 30 cb 28 .1 4± 1. 08 ga 0. 29 ±0 .0 2 cd eb 0. 96 ±0 .1 5 cd a pi ca nt ili 19 3. 69 ±2 4. 80 fb 26 2. 49 ±1 3. 11 ıa 1. 96 ±0 .6 7 ef gb 3. 57 ±0 .9 6 ef a 40 .1 4± 2. 29 bb 77 .8 7± 1. 93 aa 0. 60 ±0 .0 3 cb 1. 43 ±0 .6 0 ba so no ra 54 6. 95 ±2 2. 90 ba 40 2. 24 ±2 5. 10 gb 9. 23 ±0 .5 4 ba 6. 25 ±0 .5 8 bb 21 .6 8± 1. 00 da 20 .8 0± 2. 46 ıb 1. 28 ±0 .0 5 bb 1. 29 ±0 .0 7 bc a su pe rn ov a 89 9. 49 ±2 4. 10 ab 94 5. 41 ±2 2. 40 aa 10 .5 3± 0. 62 aa 7. 64 ±0 .4 7 ab 57 .2 4± 2. 01 aa 56 .4 6± 1. 91 bb 2. 29 ±0 .1 2 aa 1. 40 ±0 .1 7 bc b te xa s 22 3. 11 ±3 9. 40 fb 79 3. 51 ±4 1. 60 ba 1. 97 ±0 .4 6 ef gb 7. 06 ±0 .7 0 aa 9. 73 ±2 .1 9 ıjb 49 .4 9± 1. 79 ca 0. 12 ±0 .0 4 de b 1. 10 ±0 .2 1 bc a tu on o 13 6. 33 ±2 .8 5 gb 67 3. 85 ±2 1. 00 da 0. 73 ±0 .0 9 hb 5. 48 ±0 .5 7 bc da 6. 42 ±1 .8 0 kb 40 .2 0± 2. 25 ea 0. 45 ±0 .0 8 cd eb 1. 07 ±0 .6 8 bc a y al tin sk i 19 8. 07 ±9 .9 2 fb 53 9. 50 ±2 3. 00 fa 1. 27 ±0 .5 2 gh b 2. 78 ±0 .6 7 fa 9. 94 ±1 .7 8 ıjb 31 .4 1± 1. 73 fa 0. 11 ±0 .0 2 de b 0. 33 ±0 .0 7 ef a m ea n 31 2. 29 ±1 6. 58 46 7. 31 ±1 9. 28 3. 33 ±0 .6 0 4. 70 ±0 .7 1 18 .5 5± 1. 67 34 .9 9± 2. 05 0. 57 ±0 .0 5 0. 99 ±0 .1 9 l sd 30 .0 3 0. 73 68 2. 18 0. 40 54 e ac h va lu e is e xp re ss ed a s m ea n ±s ta nd ar d de vi at io n, m ea ns f ol lo w ed b y di ff er en t c ap ita l l et te rs ( ye ar s) in th e ro w a re s ig ni fic an tly d iff er en t ( p< 0. 05 ). m ea ns f ol lo w ed b y di ff er en t s m al l l et te rs in th e co lu m ns (c ul tiv ar s) a re s ig ni fic an tly d iff er en t ( p< 0. 05 ). phenolic and tocopherol composition of some almond cultivars 169 (esfahlan et al., 2010; izaddost et al., 2013; yada et al., 2011). to reveal the variables (phenolics and tocopherols) causing differences among the almond cultivars, multivariate analysis was used in the research. the characteristics with the eigenvalues greater than 1 are evaluated as descriptive in pca (shin et al., 2012). the results of the principal component analysis (tab. 3) indicate that the first 4 components accounted for 71.72 % of the total variability ob served. the shares of components pc1, pc2, pc3, and pc4 in the total variance were 28.49 %, 18.07 %, 14.15 %, and 11.02 %, respectively. pc1 showed 8 variables with higher scores (values ≥ 0.50) with respect to phenolic components (catechin, chlorogenic acid, caffeic acid, and epicatechin) and tocopherols (α, β, γ and δ). the highest contribution of pc2 corresponded to chlorogenic acid and ferulic acid as well as to α, β, and γ tocopherols. the separation along pc3 was primarily due to variations in gallic acid, naringenin, and kaempferol. the highest contribution of pc4 corresponded to p-coumaric acid. these results support the relevance of catechin, caffeic acid, epicatechin, and p-coumaric acid as discriminant parameters to differentiate almond varieties. similar results were reported in previous pca applications to almond research (garrido et al., 2010; bartolome et al., 2010; maestri et al., 2015; barreira et al., 2012; shin et al., 2010; colic et al., 2012; kodad et al., 2013). in plants and agri-industrial by-products: antioxidant activity, occurence, and potential uses. food chem. 99, 191-203. barreira, j.c.m., casal, s., ferreira, i.c.f.r., antonio peres, a.m., jose, a.p., oliveira, m.b.p.p., 2012: supervised chemical pattern recognition in almond (prunus dulcis) portuguese pdo cultivars: pcaand lda-based triennial study. j. food agr. food chem. 60, 9697-9704. bartolome, b., monagas, m., garrido, i., gomez-cordoves, c., martin-alvarez, p.j., lebron-aguilar, r., urpi-sarda, m., llorach, r., 2010: almond (prunus dulcis (mill.) d.a. webb) polyphenols: from chemical characterization to targeted analysis of phenolic metabolites in humans. arch. biochem. biophys. 501, 124-133. beyhan, ö., aktaş, m., yilmaz, n., şi̇mşek, n., gerçekçioğlu, r., 2011: determination of fatty acid composition of some important almond (prunus amydalus l.) varieties selected from tokat province and eagean region of turkey. j. med. plants res. 5, 4907-4911. bolling, b.w., dolnikowski, g., blumberg, j.b., chen, c.y.o., 2010: polyphenol content and antioxidant activity of california almonds depend on cultivar and harvest year. food chem. 122, 819-825. caponio, f., alloggio, v., gomes, t., 1999: phenolic compounds of virgin olive oil: influence of paste preparation techniques. food chem. 64, 203209. chandrasekara, n., shahidi, f., 2011: effect of roasting on phenolic content and antioxidant activities of whole cashew nuts, kernels, and testa. j. food agr. food chem. 59, 5006-5014. colic, s., rakonjac, v., zec, g., nikolic, d., aksic, m.f., 2012: morphological and biochemical evaluation of selected almond (prunus dulcis (mill.) d.a. webb) genotypes in northern serbia. turk. j. agric. for. 36, 429-438. davis, p.a., iwahashi, c.k., 2001: whole almonds and almond fractions reduce aberrant crypt foci in a rat model of colon carcinogenesis. cancer lett. 165, 27-33. di mambro, v.m., azzolini, a.e.c.s., valim, y.m.l., fonseca, m.j.v., 2003: comparison of antioxidant activities of tocopherols alone and in pharmaceutical formulations. int. j. pharmaceut. 262, 93-99. esfahlan, a.j., jamei, r., 2012: properties of biological activity of ten wilg almond (prunus amygdalus l.) species. turk. j. biol. 36, 201-209. esfahlan, a.j., jamei, r., esfahlan, r.j., 2010: the importance of almond (prunus amygdalus l.) and its by-products. food chem. 12, 349-360. garcia-pascual, p., mateos, m., carbonell, v., salazar, d.m., 2003: influence of storage conditions on the quality of shelled and roasted almonds. biosyst. eng. 84, 201-209. garrido, i., urpi-sarda, m., monagas, m., gomez-cordove, c., martin-alvarez, p.j., llorach, r., bartolome, b., andreslacueva, c., 2010: targeted analysis of conjugated and microbialderived phenolic metabolites in human urine after consumption of an almond skin phenolic extract 1-3. j. nutr. 140, 1799-1807. habila, n., inuva, h.m., aimola, i.a., agbaji, a.s., ladan, z., shangodare, r., williams, i.s., odjobo, o.b., ogabiela, e., 2012: variation of fatty acids and vitamin e composition in seed oils of some plant species. j. plant stud. 1, 55-60. isabelle, m., lee, b.l., lim, m.t., koh, w.p., huang, d., ong, c.n., 2010: antioxidant activity and profiles of common fruits in singapore. food chem. 123, 77-84. isfahlan, a.j., mahmoodzadeh, a., hassanzadeh, a., heidari, r., jamei, r., 2010: antioxidant and antiradical activities of phenolic extracts from iranian almond (prunus amygdalus l.) hulls and shells. turk. j. biol. 34, 165-173. ismail, a., marjan, z.m., foong, c.w., 2004: total antioxidant activity and phenolic content in selected vegetables. food chem. 87, 581-586. izaddost, m., imani, a., piri, s., bagiri, a.m., 2013: oil content, major fatty acids composition, γ-tocopherol, β-tocopherol and nut characteristics of almond at time of harvest. j. basic appl. sci. res. 3, 201-205. jenkins, d.j., kendall, c.w., marchie, a., parker, t.l., connellly, p.w., qian, w., 2002: dose response of almonds on coronary heart disease risk factors: blood lipids oxidized low-density lipoproteins, lipotab. 3: principle component analysis of almond varieties. variable principle component 1 2 3 4 gallic acid -0.391 -0.071 0.544 0.034 catechin 0.728 0.450 -0.181 -0.282 chlorogenic acid 0.500 0.603 0.095 0.058 caffeic acid 0.743 0.343 -0.003 0.024 epicatechin 0.768 0.431 -0.162 -0.210 p-coumaric acid 0.128 0.068 0.403 0.760 ferulic acid 0.237 0.561 0.388 0.276 naringenin -0.304 0.022 0.635 -0.433 kaempferol 0.079 0.383 0.728 -0.280 alpha tocopherol 0.680 -0.554 -0.079 -0.297 beta tocopherol 0.661 -0.588 0.100 -0.028 gamma tocopherol 0.501 -0.540 0.449 -0.255 delta tocopherol 0.649 -0.466 0.364 0.323 eigenvalue 3.988 2.530 1.981 1.542 percent variation 28.487 18.072 14.147 11.017 cumulative 28.487 46.559 60.706 71.723 percent variation acknowledgements we thank to research and application center of suleyman demirel university for 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antioxidant activity of minor components of tree nut oils. food chem. 111, 421-427. mishra, n., dubey, a., mishra, r., barik, n., 2010: study on antioxidant activity of common dry fruits. food chem. toxicol. 48, 3316-3320. moayedi, a., rezai, k., moini, s., keshavarz, b., 2011: chemical compositions of oils from several wild almond species. j. am. oil chem. soc. 88, 503-508. nizamlioğlu, n.m., nas, s., 2010: the phenolic compounds in vegetables © the author(s) 2016. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 90, 43 51 (2017), doi:10.5073/jabfq.2017.090.007 1 botany and microbiology department, faculty of science, alexandria university, egypt 2 botany department, faculty of science, kafr el sheikh university, egypt 3 botany and microbiology department, faculty of science, damanhour university, egypt role of cellular nadp+/nadph ratio in the acclimative mechanism of two common bean cultivars toward salt stress ghada saber mohamed ismail1*, awatif saad ali2, eman mohammad mustafa eldebawy3, nabil el-sayed saber1 (received july 31, 2016) * corresponding author summary this study is aimed to evaluate the adaptive mechanism in two common bean (phaseolus vulgaris l.) cultivars, cv. bronco and cv. paulista subjected to different concentrations of nacl (0, 25, 50 and 75 mm). salt treatments resulted in a significant decrease in the fresh and dry biomasses of shoots and roots as well as leaf relative water content (rwc) in both bean cultivars. salt stress determines a diversion of plant metabolism towards the synthesis of phenolics, proline and trigonelline (trg) components together with an increase in the nadp+/nadph ratio. this increase was accompanied with a significant increase in glucose-6-phosphate dehydrogenase (g-6-pdh) and l-phenylalanine ammonia-lyase (pal) activities in both bean cultivars. furthermore, increasing nacl levels induced oxidative stress measured in terms of malondialdehyde and h2o2 contents. salt stress triggered an increase in guaiacol peroxidae (gpx) activity in both bean cultivars, whereas polyphenol oxidase (ppo) activity increased only in bronco plants. the results are discussed in the light of possible roles and regulation of cellular redox potential (nadp+/ nadph) in the maintenance of acclimative mechanism in the two common bean cultivars grown under nacl stress. introduction salinity is one of the most important abiotic constraints that adversely affect plant growth and development throughout the world. it exerts negative consequences on plant growth, protein biosynthesis and photosynthetic pigments and triggers secondary stresses like oxidative, ionic and osmotic imbalances (morari et al., 2015). in addition, salinity increases the production of reactive oxygen species (ros) leading to oxidative damage and hence cell death (apel and hirt, 2004). however, to maintain the level of ros under tight control, plants develop complex defense mechanisms composed of enzymatic and non enzymatic systems (kaya et al., 2013). some enzymatic antioxidants have been reported to increase in response to salinity such as catalase (cat), guiacol peroxidase (gpx) and polyphenol oxidase (ppo) (kavas et al., 2015). the non enzymatic antioxidants include tocopherols, carotenoids and phenolic phytochemicals with the latter serve as phenolic antioxidants due to their ability to donate hydrogen from the hydroxyl groups positioned along their aromatic ring to terminate ros and other biomolecules (balasundram et al., 2006). several studies showed that increasing water deficit and salt stress resulted in an increase of phenolic compounds in different plant species (ali and ismail, 2014; abhilash-joseph et al., 2015). synthesis of compatible solutes (e.g. proline, glycine betaine, trigonelline) is one of the most prominent response mechanisms triggered upon salt stress which contribute to stabilize protein structures (cardi et al., 2015). trigonelline (trg; 1-n-methylnicotinic acid) is a member of alkaloids, derived during de novo and salvage of pyridine nucleotides in plants (matsui et al., 2007). many legumes produce trg also as a secondary metabolite such as fenugreek (hassanzadeh et al., 2011). it has been reported that glucose-6-phosphate dehydrogenase (g6-pdh) is a key enzyme for maintenance of redox potential in cells via oxidative pentose phosphate pathway, oppp (farhud and yazdanpanah, 2008; scharte et al., 2009). yu et al. (2004) have stated that the oxidative stress and h2o2 accumulation induced by the elicitors under environmental stresses could stimulate g-6-pdh activity and enzymes of glutathione cycle. redox reactions are the fundamental metabolic processes through which cells convert and distribute the energy that is necessary for growth and maintenance. most stress conditions that impose constraints on plant growth and development involve adjustments in redox state (buchanan and balmer, 2005). scheibe et al. (2005) reported that in plant stress reactions, nadph is particularly important as a branch point between ros-promoting and ros-processing metabolism. cytosolic nadph provides the reductant for production of ros through nadph oxidases as well as the protection of cells against ros by transferring of oxidized glutathione to reduced form via glutathione reductase activity (farhud and yazdanpanah, 2008). phenylalanine ammonia-lyase (pal) is considered the branch point enzyme between primary (shikimate pathway) and secondary (phe nylpropanoid) metabolism. it can be induced by various biotic and abiotic stresses, which result in the accumulation of secondary metabolites such as phenolics, flavonoids, phytoalexins and lignin involved in defense response to protect the plants from stress (kelij et al., 2013). common bean (phaseolus vulgaris l.) is widely used as protein source with highly nutritive value in human nutrition in many parts of the world including egypt. it is also a valuable leguminous crop worldwide because of its ability to fixate atmospheric nitrogen into the root nodules in a symbiotic interaction with soil rhizobia. the present study aimed to investigate the regulatory role of nadp+/ nadph ratio in the response of two cultivars of phaseoulus vulagaris l. (cv. bronco and cv. paulista) to salinity. the changes in some growth parameters, proline, trg and pyridine nucleotides [(nadp(h)], total phenolics, alkaloids and flavonoids as well as gpx , ppo, pal and g6pdh activities were followed. materials and methods plant material, growth conditions, and treatments common bean (phaseoulus vulagaris l.) seeds (cv. bronco and cv. paulista) were obtained from the agricultural research center, giza, egypt. they were surface sterilized with 2.5 % sodium hypochlorite for 10 min, rinsed with distilled water, and soaked for 24 h at 25 °c in aerated water. to choose suitable concentrations of nacl, a preliminary experiment was conducted where 20 seeds allocated at random in petri dishes (15 cm diameter) containing filter paper moistened with 20 ml of 0, 10, 25, 50, 75, and 100 mm nacl, covered by lid, and incubated at natural environmental conditions (photoperiod of 44 g.s.m. ismail, a.s. ali, e.m.m. eldebawy, n. el-sayed saber 16 h/8 h light/dark, 28/23±2 °c light/dark temperature; light intensity ppfd, 23 μmol m-2s-1) for 3 days. the germination percentage was calculated as a standard of radicle emergence and the specified concentration of nacl was determined as 25, 50 and 75 mm nacl. seeds of uniform size were sterilized as previously mentioned and they were transferred to plastic pots (15 cm in diameter, 20 cm length with a hole at the bottom) filled with fixed amount of previously acidwashed quartz sand. twenty seeds were germinated in each pot and the pots were placed under natural environmental conditions (a 16-h photoperiod, 28/23±2 °c light/dark temperature and light intensity of about 23 μmol m-2s-1). the water holding capacity was kept at 80 % during the whole experimental period. the pots were irrigated using half strength modified hoagland solution (epstein, 1972) supplemented with 0, 25, 50 and 75 mm nacl every two-day interval throughout the whole experiment period. after 21 days, homologous plants were harvested, washed thoroughly from adhering soil particles, gently plotted, dissected to shoots and roots and quickly saved for estimation of the various growth parameters and chemical analyses. all chemical analyses were performed on roots and leaves. experimental methods growth parameters the roots and leaves were separated and taken for determination of fresh (fm) and dry biomass (dm). leaf relative water content (rwc) was determined as described by silveira et al. (2003) based on the following equation: [(fm dm) / (tm dm)] × 100 where fm is the leaf fresh mass, dm is leaf dry mass (after drying at 80 °c for 48 h) and tm is the turgid mass of leaves (after soaking in water for 4h at room temperature). estimation of photosynthetic pigments the photosynthetic pigments were determined after grinding of leaf samples with acetone following the method described by metzner et al. (1965). estimation of lipid peroxidation, h2o2 and proline contents hydrogen peroxide content was determined according to the method of velikova et al. (2000). lipid peroxidation was monitored by spectrophotometric determination of malondialdehyde (mda) using thiobarbituric acid (tba) as described in valentovič et al. (2006). the content of mda was determined using the extinction coefficient of 155 mm-1 cm-1. proline was estimated by the method of bates et al. (1973). samples were homogenized in 10 ml 3% (w/v) sulfosalicylic acid, and proline was assayed by the acid ninhydrin method. the absorbance was measured spectrophotometrically at 520 nm and proline was calculated based on μm g-1 dm. extraction and determination of total phenolic compounds, falvonoids and alkaloids contents total phenolic contents of leaves and roots of bean plants were determined using the modified folin-ciocalteu spectrophotometric method (mcdonald et al., 2001). total flavonoids were extracted and determined by the colorimetric methanolic aluminium chloride method of luximon-ramma et al. (2002). the total alkaloids content in the plant samples were extracted and determined by the method described by singh et al. (2004). high-performance liquid chromatographic (hplc) analysis trigonelline for estimation of trg, samples were homogenized in 80 % methanol (zheng and ashihara, 2004). the homogenate was centrifuged at 3000 × g for 15 min at 4 °c. the supernatant was filtered through a 30-μm syringe filter, and 50 μl of the filtrate was analyzed using a hplc system (perkinelmer series 200 lc and uv/vis detector 200 lc, usa) equipped with a 5-μm column (spheri-5 rp-18; 220 × 4.6 mm; brownlee). the solvent used was a mixture of methanol:water (30:70 v/v) as mobile phase run isocracticaly with a flow rate of 1.0 ml/min. the detector was set at 265 nm for the integration of peak areas after calibration with the external standard and the results were expressed as μg g-1 dm. nadp+ and nadph for the analysis of adenine and pyridine nucleotides by hplc, samples (0.3g) were subjected to either acid extraction using 0.6 m perchloric acid (for measurement of atp, adp, nicotinamide adenine dinucleotide phosphate (nadp+) and nicotinamide adenine di nucleotide (nad+) or alkaline extraction using 0.5 m potassium hydroxide (for measurement of nadph and nadh). the extract was centrifuged at 10,000 × g at 4 °c for 10 min followed by neutralization with either 0.5 m koh or 1 m kh2po4 and re-centrifugation at 10,000 × g at 4 °c for 10 min to remove the precipitate. twenty μl of the resulting supernatants were used for hplc analysis, as described by caruso et al. (2004) using the previously mentioned hplc system. the mobile phase consisted of potassium phosphate 100 mm (solvent a) at ph 6.0 and methanol (solvent b) (10 % a 30 % b) up to 15 min and (30 % a 10 % b) for 5 min at flow rate of 0.8 ml/min, uv/vis detector set at 260 nm for the integration of peak areas after calibration with the external standard and the results were expressed as μg g-1 dm. enzymes assay l-phenylalanine ammonia-lyase (pal, ec 4.3.1.5) l-phenylalanine ammonia-lyase was extracted according to the method of morello et al. (2005). samples (300 mg) were homogenized in chilled 0.05 m potassium phosphate buffer (ph 6.6) containing 0.2 g of triton x-100. the homogenate was centrifuged at 5,000 × g for 15 min and the supernatant was used for enzyme activity assay. the pal activity was assayed according to the method of chen et al. (2006). the cinnamic acid yield was estimated by measuring the absorbance at 290 nm using spectrophotometer (jenway, 6305, uk). one unit of enzyme activity equals the amount of pal that produced 1 μmol of cinnamic acid per hour. the amount of trans-cinnamic acid synthesized was calculated using its extinction coefficient (9630 m-1) (kagale et al., 2004) and the results were expressed as μmol cinnamic acid h-1g-1 dm. glucose-6-phosphate dehydrogenase activity (g-6-pdh, ec 1.1.1.49) activity of g-6-pdh was assayed according to the method of ashihara and komamine (1976). fresh plant material (0.5 g) was homogenized in 0.05 m tris-hcl buffer ph 7.0. the homogenate was centrifuged at 5,000 × g for 20 min at 4 °c. g-6-pdh activity was calculated using the extinction coefficient of the nadph (6.22 mm-1 cm-1) and was expressed in mmol nadph min-1 g-1 dm. antioxidant enzyme assays for estimation of antioxidant enzymes activity, frozen plant tissues were homogenized in ice-cold 0.1 m potassium phosphate buffer (ph 6.8) containing 0.1 mm edta. the homogenate was centrifuged at 15,000 g for 20 min at 4 °c. after centrifugation, the super natant was used for determination of guaiacol peroxidase (gpx, ec 1.11.1.7) and polyphenol oxidase (ppo, ec 1.10.3.1) activities. gpx was assayed according to the method of urbanek et al. (1991) where guaiacol oxidation to tetraguaiacol was followed for 5 min at the adaptive mechanism of salt stress in two common bean cultivars 45 470 nm. gpx activity was calculated using the extinction coefficient of 26.6 mm-1 cm-1 and was expressed as μmol guaiacol g-1 min-1. polyphenol oxidase (ppo, ec 1.10.3.1) was assayed following the method described by kumar and khan (1982), where the formation of the purpurogallin was followed at 495 nm. ppo activity was expressed in u min-1 g-1 dm. statistical analysis each experiment was repeated at least three times. values were expressed as means ± standard error (se). the data of all experiments were analyzed using spss-16 statistical software. mean difference comparison of treatments was done by anova and the least significant differences (lsd) at level of p ≤ 0.05. results growth parameters increasing the salinization of nutrient solution with nacl resulted in a significant reduction of fm and dm of shoots and roots of both tested common bean cultivars, which was in parallel with a significant decrease in rwc of leaves (tab. 1). the reduction was more obvious in paulista than in bronco, for example the rwc of leaves of 50 mm nacl-stressed bronco and paulista plants were 70 % and 47 % respectively, compared to controls. photosynthetic pigments the total pigment content significantly decreased with increasing nacl level. this decrease was mainly related to chl a especially at high nacl level. on the other hand, it was clear that the trend of carotenoids content was greatly different according to the cultivar type (tab. 2). in cv. bronco, carotenoids content was significantly increased with increasing salinity levels, while in cv. paulista they significantly decreased at moderate and highest salinity levels. although, variation trend of carotenoids content was noted, the carot./ chl.a+b increased significantly by increasing salinity levels in the nutrient medium, particularly in cultivar paulista lipid peroxidation, h2o2 and proline contents increasing nacl concentrations in the nutrient medium of bean plants resulted in a significant accumulation of h2o2 in the leaves and roots of bean cultivars in response to salinity levels (fig. 1). at highly-stressed bronco plants, the h2o2 concentration in the leaves and roots was 2.6-and 3.0-fold of untreated plants, respectively. the corresponding values in paulista plants were 4.0-and 5.1-fold respectively. parallel to changes in h2o2, increasing nacl concentrations in the nutrient medium resulted in a significant increase of endogenous mda in the leaves and roots of bean plants. in 75 mm nacl-treated 21-d old bronco plants, mda content in the leaves and roots was 3.3-and 6.6-fold of control respectively. the corresponding values in paulista plants were 5.6and 11.3-fold respectively (fig. 1). data in fig. 2 clearly demonstrated that proline content significantly increased with increasing nacl concentrations in the rooting nutrient medium; this increase was markedly greater in the leaves and roots of cv. bronco than in the paulista one. total phenolic, falvonoids and alkaloids contents there was a significant increase of phenolic compounds in the leaves and roots of both cultivars in response to salt stress; the accumulation in the cv. paulista was greater than in the cv. bronco (tab. 3). at high nacl level in nutrient medium, the total phenolics content of leaves and roots of bronco plants was 2.7 and 4.2-fold of unstressed plants, respectively. in paulista plants these values were 5.5 and 9.9-fold the control, respectively. the same trend was observed for total flavonoid tab. 1: changes in fresh and dry biomasses of roots and shoots as well as leaf relative water content (rwc) of bean plants (cv. bronco and cv. paulista) in response to salinity in response to salinity. values are the means of 3 independent replicates ± se; means followed by different letters are significantly different at p ≤ 0.05 according to the least significant difference (lsd). bean cultivar treatment fm dm rwc roots shoot roots shoot leaves cv. bronco control 381a±4.4 551a±3.1 86a±1.5 119a±2.2 80.1a±1.4 25 mm nacl 301a±3.5 466ab±3.6 77a±1.7 93a±1.1 73.9b±0.76 50 mm nacl 138b±3.8 242b±4.1 62b±1.5 66b±1.6 56.0c±0.98 75 mm nacl 71b±1.0 118c±2.1 35c±0.87 49b±1.7 41.3d±0.72 cv. paulista control 321a±3.2 439a±6.0 82av±1.4 99a±2.1 85.3a±1.16 25 mm nacl 141b±3.2 209b±4.4 60b±0.87 76a±1.4 69.1b±0.85 50 mm nacl 68c±1.1 94c±1.6 41b±0.93 46b±0.87 39.8c±0.5 75 mm nacl 28d±1.1 56d±1.1 19c±0.55 29b±0.79 25.2d±0.52 tab. 2: change in photosynthetic pigments (mg g-1 dm) in the leaves of bean plants (cv. bronco and cv. paulista) in response to salinity. chl.: chlorophyll; carot.: carotenoids. values are the means of 3 independent replicates ± se; means followed by different letters are significantly different at p ≤ 0.05 according to the least significant difference (lsd). treatment cv. bronco cv. paulista chl.a chl.b carot. cart./(a+b) chl.a chl.b carot. cart./(a+b) control 98.03a±1.8 40.17a±0.68 19.59b±0.33 0.144d 94.03a±1.1 42.35a±0.44 24.18a±0.39 0.177c 25 mm nacl 72.75b±1.5 39.58a±1.03 21.11a±0.45 0.188c 88.89b±1.49 32.74b±0.62 23.30a±0.36 0.196c 50 mm nacl 59.01c±1.26 26.14b±0.38 24.68c±0.65 0.290b 38.01c±0.9 19.36c±0.40 19.25b±0.26 0.336b 75 mm nacl 46.60d±1.02 11.99c±0.44 24.69c±0.38 0.421a 20.24d±0.33 10.56d±0.23 14.98c±0.32 0.486a 46 g.s.m. ismail, a.s. ali, e.m.m. eldebawy, n. el-sayed saber content. as far as alkaloid content is concerned, increasing nacl in the nutrient solution resulted in a significant increase of alkaloids content in leaves and roots of cv. bronco only, compared to corresponding control (tab. 3). conversely, moderate and higher nacl levels led to significant decrease of alkaloids in leaves and roots of paulista plants compared to corresponding control. trigonelline, nadp+/ nadph ratio in cv. bronco, there was a significant increase in trg content in the leaves and roots in response to increasing nacl concentration. the opposite trend was observed in cv. paulista. at 75 mm nacl, the increase in trg content in the leaves and roots of bronco plants amounted 126 % and 59 %, respectively compared to control. in cultivar paulista, trg content decreased by 31 % and 27 % in the leaves and roots of highly nacl-stressed plants, respectively (fig. 2). the nadp+ and nadph content (tab. 4) in the leaves and roots of bronco and paulista plants showed variable changes in response to cultivar and nacl concentrations. however, the ratio of nadp+/ nadph in the leaves and roots of both bean cultivars was markedly increased with increasing nacl levels; the increase in paulista was lower than that in the bronco plants. the nadp+/nadph ratio in the leaves and roots of unstressed bronco plants increased from 0.347 and 0.626 to 1.546 and 1.745 respectively, in 75 mm naclstressed plants. in paulista plants these values increase from 0.334 and 0.640 to 0.938 and 1.506 respectively. enzyme activities the activity of pal enzyme significantly increased in response to increasing nacl level (tab. 5). the highest increase was recorded in the leaves and roots of 75 mm nacl-stressed bronco plants and recorded 4.9and 2.8-fold of corresponding control. the correspon ding values in paulista plants were 2.7-and 2.2-fold respectively. glucose-6-phosphate dehydrogenase activity increased significantly in the leaves and roots of cv. bronco and cv. paulista with increasfig. 1: changes in hydrogen peroxide (h2o2) content (μmol h2o2 g-1 dm) and malondialdehyde (mda) content (μmol g-1 dm) in the roots and leaves of bean plants (cv. bronco and cv. paulista) in response to salinity. values are the means of 3 independent replicates ± se; means followed by different letters are significantly different at p ≤ 0.05 according to the least significant difference (lsd). tab. 3: changes in total phenolic (mg gallic acid eq · g-1 dm), flavonoids (μg g-1 dm) and alkaloids (μg g-1 dm) contents in the roots and leaves of bean plants (cv. bronco and cv. paulista) in response to salinity. values are the means of 3 independent replicates ± se; means followed by different letters are significantly different at p ≤ 0.05 according to the least significant difference (lsd). bean cultivar treatment total phenolics total flavonoids total alkaloids roots leaves roots leaves roots leaves cv. bronco control 557d±8.7 441d±3.8 69d±1.80 574d±8.1 55d±0.81 106d±0.96 25 mm nacl 1228c±7.2 821c±10.6 85c±1.50 643c±6.8 64c±0.84 139c±1.42 50 mm nacl 1870b±18.0 1087b±16.8 97a±1.80 893a±6.8 95b±1.35 202b±1.71 75 mm nacl 2356a±34.2 1175a±23.6 102a±1.90 812b±8.2 108a±1.36 231a±2.36 cv. paulista control 371d±5.3 393d±4.6 53c±1.30 327d±3.2 49a±0.66 97a±1.16 25 mm nacl 1484c±21.60 1294c±20.8 78ab±2.10 533c±3.6 51a±0.78 99a±1.21 50 mm nacl 2186b±22.0 1510b±18.0 80ab±2.70 641a±4.7 37b±0.46 81b±1.15 75 mm nacl 3676a±43.4 2142a±51.4 86a±1.80 580c±4.1 33b±0.71 73b±1.07 fig. 1 the adaptive mechanism of salt stress in two common bean cultivars 47 ing salinity levels of the nutrient medium (tab. 5); the activity in the former was greatly lower than in the latter. the highest increase was recorded in the leaves of 75 mm nacl-stressed paulista plants; 4.5fold in compare to control. increasing the salinization of nutrient solution with nacl resulted in a significant increase in gpx activity in the two tested bean cultivars (fig. 3). it is noteworthy that gpx activity in the leaves and roots of nacl-stressed paulista plants was greater than in bronco plants. the gpx in the leaves of 50mm nacl-stressed paulista plants was 2.9fold of unstressed plants. the corresponding value for bronco plants was 1.6-fold. ppo activity in the leaves and roots of bronco plants was significantly increased in response to nacl treatment (fig. 3). in contrast, the ppo activity in the leaves and roots of paulista plants decreased significantly at high salinity level. the decrease of ppo activity in 75 mm nacl-stressed paulista leaves and roots was 67 % and 73 % respectively, compared to control. discussion the drastic effect of nacl on the growth and leaf rwc of both bean cultivars, particularly paulista plants might be attributed to increasing ros generation and hence disturbance of plasma membrane integrity and water uptake as well as osmotic imbalance. srivastava et al. (2015) have reported that salt stress stimulate the formation of ros leading to oxidative stress. in agreement with this view, there was a significant increase in h2o2 and mda contents in salt stressed paulista plants more than in bronco one revealing the sensitivity of paulista plants to salt stress. similar observations were recorded for several plant species such as arachis hypogaea (kavas et al., 2015). in the present study, the content of photosynthetic pigment significantly declined in both bean cultivars in response to salt stress except carotenoid content, which was increased only in bronco plants. these observations were accompanied by a significant increase in h2o2 and mda contents suggesting an enhancement of oxidative stress causing a destructive effect on the photosynthetic machinery. tab. 4: changes in nadp+, nadph contents (μm) and nadp+/ nadph ratio in the roots and leaves of bean plants (cv. bronco and cv. paulista) in response to salinity. treatment cv. bronco roots leaves nadp+ nadph nadp+ / nadph nadp+ nadph nadp+ / nadph control 5.853 9.343 0.626 13.633 39.210 0.348 25 mm nacl 35.246 33.646 1.048 15.432 22.617 0.682 75 mm nacl 5.414 3.102 1.745 5.448 3.523 1.546 treatment cv. paulista nadp+ nadph nadp+ / nadph nadp+ nadph nadp+ / nadph control 36.442 56.968 0.640 9.080 27.174 0.334 25 mm nacl 5.432 5.867 0.926 3.678 9.749 0.377 75 mm nacl 5.767 3.618 1.594 12.922 13.780 0.938 fig. 2: changes in proline (μg g-1 dm) and trigonelline content (μg g-1 dm) in the roots and leaves of bean plants (cv. bronco and cv. paulista) in response to salinity. values are the means of 3 independent replicates ± se; means followed by different letters are significantly different at p ≤ 0.05 according to the least significant difference (lsd). fig. 2 48 g.s.m. ismail, a.s. ali, e.m.m. eldebawy, n. el-sayed saber the suppression in photosynthetic pigments content has been reported to be related to the inhibition of specific enzymes responsible for their synthesis and induction of some degradative enzymes such as chlorophyllase (fang et al., 1998) as well as destruction of the photosynthetic machinery (yamane et al., 2008). in response to increasing oxidative damage by generated ros due to increasing salinity levels in the nutrient medium, both bean cultivars have increased the car/chl a+b ratio, particularly in cultivar paulista, denoting a development of an adaptive mechanism for removing ros and reducing the possibility of further damage of photosynthetic apparatus. it was obvious that carotenoids decreased in cultivar paulista in response to salinity level. but when it is compared as a ratio from total chlorophyll it was found increased, which denotes an initial development of adaptive mechanism in this cultivar. de pascale et al. (2001) reported that carotenoids react with lipid peroxidation product to terminate chain reactions, while loggini et al. (1999) suggested that carotenoids directly react with singlet oxygen. free proline is primarily localized in the cytosol and plastids (kavi kishor and sreenivasulu, 2014). both proline and trg are low molecular-weight quaternary qac accumulated in cells of higher plants in response to osmotic stress and acting as osmoprotectant and ros scavengers (cardi et al., 2015). there was a significant accumulation of proline and trg in the leaves and roots of cv. bronco in response to salinity while in cv. paulista, increasing nacl levels lead to a significant decrease in trg content in both roots and leaves. these results might reveal the involvement of proline and trg (as osmolyte) in mediating osmotic adjustment by preventing the dehydration of protoplasm, protecting sub-cellular structures and membranes as ohscavenger and stabilizing protein (demiral and turkan, 2006). several authors have reported the accumulation of proline and trg in several plant species grown under salinity conditions (dawood et al., 2014) while other authors did not observe any appreciable increase in free proline content in response to salinity stress (taffouo et al., 2009). rivero et al. (2004) reported that the accumulation of proline in salt-stressed plant organs might be due to enhancement of nadph-pyrroline-5carboxylate synthetase. in tab. 5: changes in glucose -6-phosphate dehydrogenase (g-6-pdh) (μmol g-1 dm min-1) and phenylalanine ammonia-lyase (pal) activities (μmol cinna mic · g-1 dm min-1) in the roots and leaves of bean plants (cv. bronco and cv. paulista) in response to salinity. values are the means of 3 independent replicates ± se; means followed by different letters are significantly different at p ≤ 0.05 according to the least significant difference (lsd). bean cultivar treatment g-6-pdh pal roots leaves roots leaves cv. bronco control 1.76c±0.03 1.87b±0.03 35.92c±1.00 33.29d±2.01 25 mm nacl 1.84bc±0.04 2.59b±0.10 42.46c±0.50 90.78c±1.04 50 mm nacl 2.65ab±0.05 4.78a±0.08 75.40b±0.40 109.12b±1.00 75 mm nacl 3.48a±0.07 4.82a±0.10 98.98a±1.00 162.15a±2.04 cv. paulista control 1.69d±0.10 1.77b±0.11 31.98d±1.02 38.85c±2.01 25 mm nacl 5.30c±0.10 3.98b±0.20 40.32c±2.02 86.78b±1.01 50 mm nacl 5.88bc±0.12 6.45a±0.50 58.92b±2.02 90.30b±1.90 75 mm nacl 6.54ab±0.31 7.98a±0.15 69.96a±1.03 105.58a±2.02 fig. 3: changes in guaiacol peroxidase (gpx) activity (μmol guaiacol min-1 g-1 dm) and polyphenol oxidase (ppo) activity (u min-1 g-1 dm) in the roots and leaves of bean plants (cv. bronco and cv. paulista) in response to salinity. values are the means of 3 independent replicates ± se; means followed by different letters are significantly different at p ≤ 0.05 according to the least significant difference (lsd). 1 cv . bronco ro ot le av es 0 50 100 150 200 250 g p x a ct iv ity (µ m ol g ua ia co l m in -1 g -1 d m ) cv . paulista ro ot le av es 0 50 100 150 200 250 control 25 mm nacl 50 mm nacl 75 mm nacl cv . bronco ro ot le av es 0.0 0.5 1.0 1.5 2.0 p p o a ct iv ity ( u m in -1 g -1 d m ) cv . paulista ro ot le av es 0.0 0.5 1.0 1.5 2.0 control 25 mm nacl 50 mm nacl 75 mm nacl d c b a c b a a c b a a d c b a c b ab a c b b a a a a c a a b c fig. 3 the adaptive mechanism of salt stress in two common bean cultivars 49 the present study, the accumulation of proline and trg in bronco plants was accompanied by a higher rwc and lower h2o2 and mda contents compared to cv. paulista, which might reveal the role of proline and trg as osmoregulator and antioxidant compound i.e. cv. bronco might be a salt-adaptive cultivar. in this respect hare et al. (1998) noted that proline and trg biosyntheses maintain appropriate nad+/nadh and nadp+/nadph ratios compatible with metabolism. data recorded in this study demonstrated that nadp+/ nadph ratio in the leaves and roots of nacl-stressed bronco was higher than those in paulista plants revealing the consumption of nadph as h-donner in proline and trg biosynthesis. this might reflect the role of cytosolic nadp+/nadph in the development of an adaptive mechanism via biosynthesis of osmoregulators. nadph is a key cofactor in cellular redox homoeostasis. thus, the switch of nadp+/nadph might make a sense in particular situation during subjection of both bronco and paulista plants to salt stress, when metabolism shifts to synthesizing defense compounds, such as phenolics, proline and trg. moreover, the enhancement of adaptive mechanisms against generated ros in salt-tolerant bronco plants might indicate that nadph acts as a cofactor for other antioxidant enzymes, such as glutathione reductase (farhud and yazdanpanah, 2008). furthermore, nicotinic acid (na) that is produced during pyridine nucleotide cycle in plants is known as a precursor of trg biosynthesis (matsui et al., 2007). khanam (2007) reported that when trg accumulated, it is considered as a detoxifying agent for excess na and nicotinamide released from nad cycle. scharte et al. (2009) concluded that the increase in nadph might induce the activity of nadph-oxidase enzyme and hence increasing ros generation. thus, in addition to its role as an osmoregulator the increase in trg content in salt-adaptive bronco plants could be related to detoxification as well as shifting off suppression of growth caused by excess na during nad cycle, whereas in salt-sensitive paulista plants the decrease in trg content might be attributed to increased nadph-oxidase activity and increase in h2o2 content (fig. 1). osmoregulation is not the sole response to salinity, as this stress is also counteracted by generation of secondary metabolites and enhancing several antioxidant enzymes. total phenolics and flavonoids significantly increased in both bean cultivars in response to salinity level (tab. 3). these results were associated with an enhancement of gpx activity revealing the adaptive role of these secondary metabolites against salt stress. furthermore, the activity of ppo in bronco plants was significantly increased in response to salinity while it decreased in plants of cv. paulista. these observations might be explained by a marked reduction of generated ros in nacl-stressed bronco plants using phenolic compounds as h-donors to shift off the inhibitory effect of generated h2o2 and the toxic effect of accumulated phenolics and hence improve the growth more than in paulista plants; in other words cv. bronco may be a salt-tolerant or salt-acclimative bean cultivar. the increase in total phenolics and flavonoid contents is in conformity with the findings in vicia faba (dawood and el-awad, 2015). in addition, increased gpx and ppo activities in response to salinity has been reported by many authors (bian and jiang, 2009; dawood et al., 2014). it has been reported that upon exposure to salt, the cytosolic g6pdh activity may increase in order to support the syntheses of cofactors (as reducing power, nadph) or intermediates (as phenolics) involved in the tolerance mechanisms (dal santo et al., 2012). the cytosolic g6-pdh activity significantly increased in the leaves and roots of both bean cultivars in response to salinity. these results denote the enhancement of oppp to generate the precursors of phenolic compounds via shikimate and phenylpropanoid pathways. moreover, it was clearly demonstrated that pal activity in the leaves and roots of both bean cultivars subjected to different concentrations of nacl increased significantly compared to the control. in accordance with these results, many authors have shown a positive correlation between pal and total phenolics content in various plants grown under salt stress, such as salvia sp. (valifard et al., 2015). furthermore, the enhancement of g6-pdh activity in the salt-treated bean plants might reflect the increase in nadp/nadph ratio under salt stress due to the involvement of produced na cofactor (nadph) in the biosynthesis of proline and trg. conclusions the salt-tolerance ability of two common bean cultivars namely bronco and paulista has been compared. the findings reveal better regulation of cytosolic nadp/nadph ratio in bronco plants, in which the enhancement of g6-pdh activity under salt stress resulted in the generation of antioxidant components, such as phenolics, falvonoids and trg as well as osmoregulatory components (proline and trg) and hence protect the plasma membrane integrity and keep water conservation. thus, the varied response of cv. bronco and cv. paulista to studied nacl levels might be related to their ability to maintain a cellular redox homoeostasis. references abhilash-joseph, e., radhakrishnan, v.v., mohanan, k.v., 2015: variation in 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of taxus chinensis. plant sci. 167, 329-335. doi: 10.1016/j.plantsci.2004.03.031. zheng, x.q., ashihara, h., 2004: distribution, biosynthesis and function of purine and pyridine alkaloids in coffea arabica seedlings. plant sci. 166, 807-813. doi: 10.1016/j.plantsci.2003.11.024. © the author(s) 2017. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). address of the authors: ghada saber mohamed ismail, nabil el-sayed saber, botany and microbiology department, faculty of science, alexandria university, egypt e-mail: ghada5f@yahoo.com awatif saad ali, botany department, faculty of science, kafr el-sheikh university, egypt eman mohammad mustafa eldebawy, department, faculty of science, damanhour university, egypt journal of applied botany and food quality 91, 79 87 (2018), doi:10.5073/jabfq.2018.091.011 1department of forestry and range management, bahauddin zakariya university, multan, pakistan 2instituto de biología, universidad nacional autónoma de méxico, méxico, distrito federal, mexico 3department of chemistry, university of central punjab, multan campus, multan, pakistan 4department of agronomy, university of agriculture faisalabad, pakistan 5department of plant breeding and genetics, bahauddin zakariya university, multan, pakistan 6institute of food science and nutrition, bahauddin zakariya university, multan, pakistan drought affects size, nutritional quality, antioxidant activities and phenolic acids pattern of moringa oleifera lam. wasif nouman1*, mark e. olson2, tehseen gull3, muhammad zubair1, shahzad maqsood ahmad basra4, muhammad kamran qureshi5, muhammad tauseef sultan6, mehak shaheen1 (submitted: january 13, 2018; accepted: february 23, 2018) * corresponding author summary to observe variation in growth performance, antioxidant activities, and nutritional quality of moringa oleifera we exogenously applied benzyl amino purine (bap), ascorbic acid, and moringa leaf extract (mle) to moringa plants at three field capacity levels, 100, 75, and 40% in a completely randomized design with three replications. we observed a decrease in growth, chlorophyll a and b, total phenolic contents, antioxidant activities, crude protein, and mineral contents of moringa leaves at 100 and 40% field capacity in comparison with 75% field capacity. bap best improved growth performance of moringa plants, improving shoot length, root length, number of leaves and photosynthetic pigments, followed by mle at 75% field capacity, while moringa plants showed reduced growth at 40% field capacity which was increased by bap and mle foliar application. maximum contents of gallic acid, p-coumaric acid and sinapic acid were found in moringa leaves when the plants were sprayed with ascorbic acid while p-hydroxybenzoic acid and caffeic acid were maximally increased under 75% field capacity when the plants were subjected to bap followed by mle. the lowest and highest crude protein, calcium, potassium, magnesium, and phosphorous contents were recorded under 40 and 75% field capacity, with mle improving these contents under both conditions. it can safely be concluded that moringa plants showed retarded growth under 100 and 40% field capacity, and that the effects of deficit in nutritional quality were mitigated by applying bap and mle. among these two plant growth regulators, mle can be preferred being a natural source. keywords: ascorbic acid; benzyl amino purine; rp-hplc; mo ringa leaf extract introduction moringa oleifera lam., moringaceae, is grown in contexts as diverse as an agricultural crop, for living fences, and as livestock fodder for its multiple attractive attributes. moringa has multiple compounds with strong antioxidant potential and is a rich source of crude protein, minerals, and polyphenolics (nouman et al., 2016; olson et al., 2016). its various parts are used in folk practices against an array of ailments (e.g. anwar et al., 2007). plant scientists mostly focus on moringa leaves because they not only are a source of potentially useful compounds but leaf extracts are used as growth regulators which have been tested on various crops and rangeland grasses (nouman et al., 2012a, b, 2014a; yasmeen et al., 2013). moringa is tolerating typical seasonal conditions of a dry tropical and subtropical environment. abiotic stress affects plant metabolism, resulting in impaired plant growth resulting in reduced crop yield and economic loss (mahajan and tuteja, 2005). the nutritional and medicinal importance of moringa demands improvement in its production, especially under stress conditions, given that a large percentage of world agriculture is facing stress. moringa is a promising crop under stressful conditions given evidence that it can provide acceptable yields despite marginal conditions. for example, in one study, moringa growth behavior, nutritional quality, and antioxidant system tolerated salinity up to 8 ds m-1 with only slight reductions in biomass production and nutritional quality (nouman et al., 2012a). moreover, there is evidence that moringa tolerance to saline conditions can be enhanced by the application of plant growth regulators as seed priming agents (nouman et al., 2014a). in addition to salinity, drought is another abiotic stress that causes significant decrease in yield (mahajan and tuteja, 2005). in addition to yield decrease, drought also results in changes in nutritional quality of plants and bioactive compounds such as phenolic acids (farooq et al., 2009). with increasing climate change, drought events are becoming more erratic. therefore, we need crops that can deal well with drought, rather than requiring abundant irrigation. moringa is a suitable option which is known to be drought-tolerant. moringa is well known for its nutritional quality and antioxidant compounds (anwar et al., 2007). these contents vary with different cultural practices, changing climatic conditions, soil nutrients availability, and soil types (nouman et al., 2013, 2014b; olson et al., 2016). drought stress is no exception, with water availability to plants strongly influencing nutrient uptake and the production of plant biomass and its nutritional quality and phenolic acid composition. for example, saline conditions, which comprise a form of drought stress that is an osmotic factor of salinity, can cause a significant decline in nutritional quality of moringa leaves (nouman et al., 2014a). moreover, variation in phenolic acids under abiotic stress conditions has been reported in different plants such as tithonia diversifolia, solanum lycopersicum and triticum aestivum (ali and ghada 2014; chakhchar et al., 2016; sampaio et al., 2016), but there is no study available illustrating the variation in these contents in moringa leaves under drought stress. such data would be useful because they would help identify currently low-yield agricultural lands that could be made much more productive if planted with moringa, provided that a means could be identified of obtaining acceptable yields of phenolic acids on these lands. phenolic acids not only help mitigating environmental stress; phenolic acid-rich plants are also used as therapeutic herbal medicines, with moringa (leaves, flowers, roots, green pods, and seeds) being used in traditional medicine (anwar et al., 2007; pavarini et al., 2012; miranda et al., 2015). so, it is imperative to study variation in nutritional attributes and phenolic acid composition under varying abiotic stress conditions. 80 w. nouman, m.e. olson, t. gull, m. zubair, s.m.a. basra, m.k. qureshi, m.t. sultan, m. shaheen plants mitigate drought stress through bioactive compounds such as phenolic acids, flavonoids, isothiocyanates, etc., whose quantities can be enhanced by addition of plant growth regulators. different plant growth regulators have been used to mitigate drought stress in different plants, especially agricultural crops (farooq et al., 2009), but regulators have not been tested for moringa under water deficit. enhanced abiotic tolerance in moringa plants under saline conditions has been reported for moringa plants with the assistance of plant growth regulators (nouman et al., 2014a). so, it can be hypothesized that moringa growth, nutritional quality, antioxidant activities, and composition of phenolic acids can also be affected under drought. because moringa is used as a food and fodder crop and medicinal plant in different regions including drought prone areas, the variation in its nutritional attributes, antioxidant potential, and phenolic acids requires study. here, we investigated variation in growth behavior, nutritional quality, enzymatic antioxidant activities, and the com position of phenolic acids under varying levels of drought stress. materials and methods seed material to test the growth performance, nutritional quality, antioxidant activities and composition of phenolic acids of moringa oleifera leaves under drought conditions, moringa plants were grown in the greenhouse of the department of forestry and range management, bahauddin zakariya university, multan, pakistan. we collected seeds from locally grown moringa plant on the campus of bahaud din zakariya university, multan, pakistan in may 2013. experimental layout the experiment was laid out in a two factor (drought stress and plant growth regulators) factorial completely randomized design with three replications under greenhouse conditions (temp: 34±3 ºc, 14 h day light, 26±3 ºc, 10 h night with 28±4% average humidity). moringa seeds were sown in earthen pots filled with 3 kg of soil (clay loam), sand, and green manure (1:1:1). we planted five seeds in each pot, keeping three plants. when plants reached an age of two weeks, three field capacity levels (100, 75, and 40%) were induced and maintained for two months till the end of the experiment. field capacity i.e., 100% was considered as half of the soil saturation capacity. exogenous foliar application of benzylaminopurine (bap, 50 mg l-1), ascorbic acid (50 mg l-1), and moringa leaf extract (mle, 3.3%) were carried out, in addition to a control (no spray) and water spray to investigate the efficacy of these plant growth regulators to mitigate drought stress. water spray was used as a positive control. plant vigour evaluation the growth performance of moringa plants was noted on the day of harvest. for this, shoot and root lengths, number of leaves and roots were noted. the roots emerging from the main tap root were counted as number of roots. chlorophyll and total phenolic contents chlorophyll a and b contents were quantified following nagata and yamashta (1992), while total phenolic contents were quantified following singleton and rossi (1965) as revised by waterhouse (2001). antioxidant enzyme assay to determine the variation in the activities of these enzymatic antioxidants, 1 g of fresh moringa leaves was ground in 10 ml of 50 mm cooled phosphate buffer of ph 7.8, filtered through whatman no. 1 filter papers and centrifuged at 15000 rpm for twenty minutes at 4 ºc. the supernatant was removed with a micropipette and stored in opaque eppendorf tubes. the supernatant was used to determine sod activity at 560 nm with a uv spectrophotometer (uv4000, o.r.i. germany) following giannoppolotis and ries (1977). assay ingredients (1 ml of 1.3 μm riboflavin, 500 μl of 13 mm methionine, 500 μl of 75 nm edta and 950 μl of 50 mm phosphate buffer of ph 7.8) were pipetted into a quartz cuvette followed by 50 μl of enzyme extract, and 1 ml of 50 μm nbt. after introdu cing the nbt, the cuvette was immediately placed under a fluorescent lamp (30 w) for 5 minutes until blue formazane was produced by nbt photo-reduction. in addition, one cuvette was placed in a dark chamber containing the same assay ingredients except the enzyme extract for the same duration. after 5 minutes, the absorbance was recorded at 560 nm. one unit of sod was defined as the amount of enzyme required to cause 50% inhibition at the rate of nbt reduction at 560 nm in comparison with tubes having no enzyme extract. to determine cat activity, 2 ml phosphate buffer of ph 7.0 and 900 μl of 5.9 mm h2o2 were combined in a quartz cuvette placed in a uv spectrophotometer (uv-4000, o.r.i. germany). after placing in the spectrophotometer, 100 μl enzyme extract was introduced into the cuvette and recording of absorbance at 240 nm was imme diately started. the absorbance was noted every 20 seconds for 5 mi nutes or until constant reading. cat activity was measured as units (μmol of h2o2 decomposed per minute) per mg of protein (chance and maehly, 1955). pod activity was also determined using the protocol of chance and maehly (1955) with minor modifications. 2 ml of 50 mm phosphate buffer of ph 7.0, 400 μl of 20 mm guaiacol and 500 μl of 40 mm h2o2 were introduced into a cuvette and placed in uv spectrophotometer. then enzyme extract was added to the cuvette while in the spectrophotometer. the recording of absorbance reading at 470 nm was immediately started and was noted after every 20 seconds for 5 minutes or until constant reading. pod one unit activity was defined as the change of 0.01 absorbance unit per minute per mg of protein. crude protein and mineral analyses moringa leaf samples were dried at 60 ºc for 70 hours until constant weight and ground to pass a 2 mm sieve. the samples were digested by using 10 ml of nitric acid (hno3) and 5 ml of perchloric acid (hclo4) (aoac, 2003). potassium (k) contents were measured using a flame photometer (jenway pep-7, jenway, uk). calcium (ca), magnesium (mg), and phosphorous (p) contents were measured using an atomic absorption spectrophotometer (model: z-8200, hitachi, japan). crude protein (cp) contents were determined according to aoac (2003) guidelines. for this, dried and grinded moringa leaves (5 g) were digested in sulfuric acid (h2so4) with a mixture of k2so4: cuso4: feso4 (10: 05: 01) with micro kjeldhal’s apparatus for nitrogen digestion, distillation and quantification. separation and quantification of phenolic acids by reverse phase high performance liquid chromatography (rp-hplc) phenolic acids are bioactive compounds which indicate plant response to drought stress. to study the variation in the selected phenolic acids (p-coumaric acid, caffeic acid, p-hydroxybenzoic acid, gallic acid and sinapic acid) of moringa leaves under drought by rp-hplc, methanolic extracts were prepared. these phenolic acids were selected due to their reported nutraceutical activity. extraction / hydrolysis hydrolysis of sample extracts followed nuutila et al. (2002) with slight modification. for this, 1000 mg of crude extract was mixed in 10 ml of 50% aqueous methanol (1.2 m hcl) in a refluxing flask at 80 °c for 2 h. after refluxing, the extract was allowed to cool to drought effects on moringa oleifera 81 room temperature and the volume adjusted to 10 ml with methanol and filtered through 45 μm (millipore) before injecting into an rphplc column. chromatographic system and conditions used for phenolic acids analysis phenolic acids analysis was performed on an agilent 1200-series hplc system (agilent technology germany) equipped with a gradient model lc (g1312b) binary pump system, auto sample injection (g1367b), degasser (g1379), a uv-vis detector (g513c), and column oven (g1316b). separation of individual phenolic acids was done on a hypersil cold c18 column (250×4.6mm, 5 μm particle size) (thermo fisher scientific inc., massachusetts, united states). a nonlinear gradient system consisting of solvent a (acetonitrile: methanol 70:30) and solvent b (water with 0.5% glacial acetic acid) at a flow rate of 1ml/min was used for elution. the gradient programming used for the separation of phenolic acids included: 10-15% a from 0 to 5 min; 15 to 20% a from 5 to 18 min; 20-40% a from 18-40 min and maintained at 40% a from 40-45 min; 40-10% a from 45 to 50 min and kept at 10% a from 50-55 min). detection of the targeted phenolic acids was made at 280 nm. the analytes were identified by matching the retention time and spiking samples with pure standards and quantification was based on an external standard calibration method. the selected phenolic acids were identified at different retention times (fig. 1). gallic acid was identified at 3.96 min (retention time) while p-coumaric acid, caffeic acid, p-hydroxybenzoic acid and sinapic acid were identified at 11.97, 12.44, 19.04 and 22.13 min (retention time), respectively. statistical analysis the replicated data were pooled and a crd two factor factorial design was used for analysis of variance (anova) using statistix 8.1. a significance level of p<0.05 was used for determining differences among the interaction of field capacity levels and foliar application of plant growth regulators. the differences among the obtained mean values of above mentioned parameters were compared and computed by using tukey’s hsd test considering 5% probability level (steel et al., 1997). results plant vigour evaluation exogenously applied pgrs significantly improved seedling vigour of moringa plants. bap and mle were the treatments that most improved moringa shoot length at 75% field capacity (24.16 and 22.00 cm, respectively) while at 40% field capacity, mle was most effective. (tab. 1). in general, moringa plants grow best in areas of low water availability. perhaps as a result, the plants grew less in height under 100% field capacity. moringa plants that were not subjected to foliar application (control) had varied root length, with the maximum being recorded at 75% field capacity followed by 100 and 40% levels. the results showed that moringa can endure 75% field capacity with no reduction in root length, but under 40% field capacity level, a significant decrease was observed. a similar trend was observed in moringa leaf score, bap maximally increased leaves of moringa plants at 75% field capacity followed by mle (35.11 and 29.56, on average respectively) while no significant improvement was recorded at 40% field capacity. moreover, maximum roots were counted in moringa plants grown at 40% field capa city when the plants were exogenously applied with ascorbic acid (tab. 1). chlorophyll and total phenolic contents it was noted that bap and mle performed best in improving chlorophyll a and b contents. moringa leaves showed variation in expressing chlorophyll a contents under different field capacity levels which were further improved when subjected to foliar application of plant growth regulators. maximum chlorophyll a contents were observed when moringa plants were sprayed with bap and mle at 75% field capacity level (28.87 and 26.73 μg g-1, respectively) followed by ascorbic acid (23.47 μg g-1) at the same field capacity. a similar trend was observed in chlorophyll b contents (tab. 2). moreover, maximum total phenolic contents were recorded when moringa plants were sprayed with bap or mle at 75% field capacity level (651.67 and 639.08 μg g-1, respectively). at 40% field capacity level, mle performed well in improving total phenolic contents (621.09 μg g-1) which were 57% more than untreated plants at 40% field capacity (tab. 2). fig. 1: a typical rp-hplc chromatogram of mixture of phenolics acid standards. peak identification: 1: gallic acid (rt 3.96), 2: p-hydroxyl benzoic acid (rt 11.97), 3: caffeic acid (rt 12.44), 4: p-coumeric acid (rt 19.04), 4: sinapic acid (rt 22.13) 82 w. nouman, m.e. olson, t. gull, m. zubair, s.m.a. basra, m.k. qureshi, m.t. sultan, m. shaheen enzymatic antioxidant activities the antioxidant enzymes sod, pod, and cat were significantly affected by varying field capacity and foliar application of plant growth regulators. an increase in sod activity was observed in moringa plants with decrease in field capacity while the plants subjected to foliar application of plant growth regulators improved sod activities in moringa plants. maximum sod activity was observed in bap applied moringa plants (418.55 unit mg-1 protein) at 75% field capacity, with lower levels (390.91 unit mg-1 protein) under appli cation of ascorbic acid (fig. 2). moreover, maximum pod activities (1260.93 units mg-1 protein) were recorded at 40% field capacity when moringa plants were exogenously applied with ascorbic acid followed by mle (1067.55 units mg-1 protein) (fig. 3). catalase activity showed varying trends with decreasing field capacity. maximum cat activity was observed at 75% field capacity, followed by 100 and 40% field capacity levels. at 75% field capacity, bap and mle maximally improved cat activity followed by ascorbic acid (125.16, 112.60 and 97.60 unit mg-1 protein, respectively). the lowest cat activity was recorded at the lowest field capacity in untreated moringa plants (fig. 4). crude protein and mineral analyses moringa plants were also analyzed for their nutritional quality. maximum crude protein (cp) contents were recorded at 75% field tab. 2: effect of foliar application of plant growth regulators on chlorophyll a, chlorophyll b and total phenolic contents of moringa leaves under different field capacity levels. treatments chlorophyll a (μg g-1) chlorophyll b (μg g-1) 100% 75% 40% 100% 75% 40% control 18.67±0.47 d-f 20.03±0.35 c-e 16.37±0.51 ef 5.17±0.53 fef 6.67±1.54 d-f 3.43±0.75 f water 18.97±0.32 d-f 20.83±0.46 cd 17.20±0.25 d-f 5.23±0.50 ef 8.33±0.39 b-e 4.77±0.23 ef bap 20.13±0.71 c-e 28.87±0.29 a 17.97±2.01 d-f 7.30±0.32 c-e 16.70±0.49 a 7.33±1.42 c-e ascorbic acid 19.17±0.22 de 23.47±0.29 bc 15.13±2.42 f 5.90±0.81 ef 10.67±0.59 bc 6.30±0.90 ef mle 19.00±0.19 d-f 26.73±0.53 ab 21.10±0.89 cd 6.23±0.36 ef 11.20±1.14 b 10.03±1.33 b-d total phenolic contents (μg g-1) 100% 75% 40% control 363.53±16.35 c 431.67±14.26 bc 394.64±13.82 bc water 370.19±28.12 bc 455.38±19.69 c-e 418.34±14.28 bc bap 567.97±18.07 ab 651.67±36.53 a 557.6±21.78 a-c ascorbic acid 462.79±15.74 b-e 562.04±20.55 a-c 490.93±14.73 a-c mle 495.38±19.31 a-c 639.08±13.36 a 621.09±23.10 a means not showing same letters differ significantly at 5% probability level, critical value for comparison for chlorophyll a: 3.91, critical value for comparison for chlorophyll b: 3.65, critical value for comparison for total phenolic contents: 198.68 tab. 1: effect of foliar application of plant growth regulators on shoot length (cm), root length (cm), number of leaves and roots of moringa seedlings under different field capacity levels. treatments shoot length (cm) root length (cm) 100% 75% 40% 100% 75% 40% control 11.24±0.60 e 15.28±0.83 b-e 12.33±0.65 de 5.06±0.47 de 6.21±0.47 b-e 4.62±0.18 e water 11.61±1.23 de 15.40±0.93 b-e 12.88±1.25 de 5.49±0.77 c-e 6.21±0.18 b-e 4.77±0.07 e bap 17.36±0.30 a-e 24.16±0.60 a 17.99±1.83 a-e 8.23±0.81 a-c 10.11±0.94 a 6.21±0.47 b-e ascorbic acid 16.26±1.76 b-e 18.29±1.82 a-e 14.28±2.05 c-e 6.93±0.61 b-e 7.37±0.31 a-e 5.63±0.53 c-e mle 18.58±2.57 a-d 22.00±1.21 ab 20.19±0.58 a-c 7.66±0.71 a-d 8.52±1.51 ab 6.64±0.35 b-e leaves roots 100% 75% 40% 100% 75% 40% control 16.44±1.16 d-f 25.44±1.16 a-d 13.44±1.19 gh 9.44±0.83 f 13.22±1.21 d-f 21.56±1.77 a-e water 19.33±3.06 c-f 27.67±1.47 a-c 13.78±0.54 ef 14.11±1.80 c-f 20.11±0.54 a-e 25.11±2.65 ab bap 28.78±4.53 a-c 35.11±1.19 a 16.56±1.83 d-f 11.44±0.14 ef 17.78±2.52 b-f 23.89±4.91 a-d ascorbic acid 21.22±4.01 b-e 26.56±3.83 a-d 11.22±0.98 ef 17.78±0.59 b-f 23.33±0.63 a-d 29.44±2.14 a mle 24.00±0.85 b-d 29.56±3.25 ab 9.67±1.03 f 12.56±2.81 ef 18.00±0.24 b-f 24.33±5.89 a-c means not showing same letters differ significantly at 5% probability level, critical value for comparison for shoot length: 7.16, critical value for comparison for root length: 2.80, critical value for comparison for leaves: 10.15, critical value for comparison for roots: 10.67 drought effects on moringa oleifera 83 fig. 2: effect of foliar application of plant growth regulators on superoxide dismutase activity in moringa leaves under different field capacity levels: means not showing same letters differ significantly at 5% probability level, critical value for comparison: 88.42 fig. 3: effect of foliar application of plant growth regulators on peroxi dase activity in moringa leaves under different field capacity levels: means not showing same letters differ significantly at 5% probability level, critical value for comparison: 247.91 fig. 7: effect of foliar application of plant growth regulators on potassium content (mg kg-1) in moringa leaves under different field capacity levels: means not showing same letters differ significantly at 5% probability level, critical value for comparison: 439.29 fig. 6: effect of foliar application of plant growth regulators on calcium content (mg kg-1) in moringa leaves under different field capacity levels: means not showing same letters differ significantly at 5% probability level, critical value for comparison: 703.19 fig. 5: effect of foliar application of plant growth regulators on crude protein (%) in moringa leaves under different field capacity levels: means not showing same letters differ significantly at 5% probability level, critical value for comparison: 4.88 fig. 4: effect of foliar application of plant growth regulators on catalase activity in moringa leaves under different field capacity levels: means not showing same letters differ significantly at 5% probability level, critical value for comparison: 25.49 capacity when the plants were subjected to mle foliar application (29.77 mg kg-1). bap foliar application was found effective at 75 and 100% field capacities (28.68 and 26.62 mg kg-1, respectively) (fig. 5). maximum calcium contents were exhibited by bap and mle at 75% field capacity level followed by ascorbic acid at same level (3951.87, 3917.6 and 3322.76 mg kg-1, respectively) while the lowest calcium contents were found in moringa plants grown at 40% field capacity (fig. 6). maximum potassium contents were observed at 75% field capacity with foliar application of mle > bap > ascorbic acid (3122.47, 2203.76 and 2014.29 mg kg-1). at 100% field capacity, maximum potassium contents were found in moringa plants sprayed with mle > ascorbic acid > bap (fig. 7). mle and ascorbic acid foliar applications were equally effective in improving magnesium contents of moringa leaves at 75 and 100 field capacity levels while a drastic reduction in these contents was recorded at 40% field capacity (fig. 8). in the case of phosphorous, bap was the best foliar application for improving phosphorous contents followed by mle at 75% field capacity (2787.48 and 2707.79 mg kg-1, respectively). these both treatments were also found at 100% field capacity (fig. 9). separation and quantification of phenolic acids by rp-hplc as per hplc analysis, five phenolic acids (gallic acid, p-coumaric acid, caffeic acid, p-hydroxybenzoic acid and sinapic acid) were 84 w. nouman, m.e. olson, t. gull, m. zubair, s.m.a. basra, m.k. qureshi, m.t. sultan, m. shaheen identified and quantified in the moringa leaves. a significant varia tion in these selected phenolic acids was observed in moringa leaves with decrease in field capacity and with application of foliar plant growth regulators. bap mostly increased p-hydroxybenzoic and caffeic acid content (25.62 and 26.48 μg g-1, respectively), whereas higher amount of gallic, p-coumaric and sinapic acids was recorded when moringa plants were sprayed with ascorbic acid (20.93, 19.06 and 12.35 μg g-1, respectively). as a conclusion, increase in p-hydroxybenzoic acid and caffeic acid can be associated with bap exogenous application while maximum content of gallic acid, p-coumaric acid and sinapic acid were associated with foliar application of ascorbic acid. mle application ranked 3rd in improving or maintaining selected phenolics (tab. 3). as a result of correlation matrices, p-hydroxybenzoic acid, gallic acid and sinapic acid contents were found as positively correlated with shoot length while gallic acid showed a positive correlation with root length. moreover, p-coumaric acid was positively correlated with number of roots, and activities of enzymatic antioxidants (tab. 4). discussion study of the stress physiology of plants examines factors such as salinity, heat, cold, and drought, which affect agricultural yield, nutritional quality, antioxidant systems, and phenolic compounds. in the present study, we found that growth of moringa plants was affec ted at 100, 75 and 40% field capacity which was mitigated by applying plant growth regulators, especially in the case of root growth and expansion. prolific and extended root growth is a plastic response to fig. 9: effect of foliar application of plant growth regulators on phosphorous content (mg kg-1) in moringa leaves under different field capacity levels: means not showing same letters differ significantly at 5% probability level, critical value for comparison: 848.75 fig. 8: effect of foliar application of plant growth regulators on magnesium content (mg kg-1) in moringa leaves under different field capacity levels: means not showing same letters differ significantly at 5% probability level, critical value for comparison: 3382.0 water deficit allowing plants to uptake water and nutrients from deep soil (nguyen et al., 1997; kavar et al., 2007). root extension and proliferation under drought stress has been noted in moringa seedlings, as well as in crops as diverse as tea, onion, and cotton (farooq et al., 2009; nouman et al., 2014a). increase in number of roots at 75 and 40% field capacity might involve lower osmotic pressure under stress conditions which trigger root elongation in search of water and nutrients (hsiao and xu, 2000). similar reactions of moringa plants have been previously reported under saline conditions (nouman et al., 2012a, 2014a). decline in number of leaves and increase in number of roots under 40% field capacity likely represents an adaptive plastic allocation pattern that reduces transpiration loss through leaves with a compensating water absorption surface area by proliferated roots (jefferis and rudmik, 1991; houle et al., 2001). in the present study, foliar application of synthetic and natural plant growth regulators improved moringa tolerance to drought conditions. among these, bap and mle were effective in inducing drought tolerance. bap and mle have been previously reported as effective plant growth regulators to improve plant survival and vigour under stress conditions (ali et al., 2011; nouman et al., 2012b, 2014a) whereas this was the first time that they were tested to investigate the response of moringa plants under water deficit with the application of plant growth regulators, especially moringa leaf extract. moreover, rivas et al. (2012) and arauja et al. (2016) reported that moringa plants can withstand under water stress conditions while the variation in its chlorophyll contents, nutritional quality and phenolic acid contents were studied in the present investigation. the growth behavior of plants can be linked to chlorophyll content and plant survival under stress conditions might be attributed to the existence of total phenolic contents which serve as antioxidants. decrease in chlorophyll content under water deficit is a common phenomenon that can be compensated by using plant growth regulators (bijanzadeh and emam, 2010; din et al., 2011). drought tolerant plants have higher chlorophyll content, mitigating drought stress, with chlorophyll content being improved by exogenous application of plant growth regulators (farooq et al., 2009). in the present investigation, a similar improvement in chlorophyll content was observed under stress conditions when plants were sprayed with mle and bap increase in chlorophyll content by the application of plant growth regulators has been previously reported in moringa plants under saline conditions (nouman et al., 2014a). it can be concluded here that moringa plants show higher chlorophyll content under low field capacities and these can be further improved with the exogenous application of mle and bap. in addition to chlorophyll content, antioxidant enzymes are another factor that affects plant tolerance in stressful conditions. plants produce reactive oxygen species, which affect plant vigour and results in a lower nutritional quality. enzymatic antioxidants enable plants to resist such oxidative damage. higher antioxidant activities have been reported in drought tolerant plants in comparison to intole rant ones (lum et al., 2014). in the present study, a similar trend was observed. with increasing water deficit, sod, pod, and cat activi ty increased, further increased by plant growth regulators especially bap, ascorbic acid and mle. previously, an increase in antioxidant activity in moringa plants under saline conditions was reported (nouman et al., 2014a). because antioxidants help buffer environmental challenges, they help to maintain nutritional quality despite stress. nutrient uptake, antioxidant compounds i.e., phenolic acids and water availability are intimately related, and this relationship might shift under stress conditions. plant growth regulators can assist plants in mitigating drought stress enabling regulation of phenolic acids (garg, 2003). the present investigation showed that moringa plants can even perform better at 75% field capacity than at 100% drought effects on moringa oleifera 85 field capacity with the application of plant growth regulators. we observed a significant increase in moringa leaf mineral and crude protein contents. based on findings regarding phenolic acid composition, it can be predicted that phytochemicals of moringa leaves vary with changes in water availability and application of plant growth regulators. under stress conditions, changes in phenolic acids have been previously reported in different plants like solanum lycoper sicum and salvia officinalis (dixon and paiva, 1995; bettaieb et al., 2011; ali and ghada, 2014). the present investigation sup ports the finding that with moringa leaves exhibited lower phenolic acids at 100% field capacity while an increase was recorded with decrease in field capacity. moringa leaves exhibited maximum phenolic acid contents when grown at 75% field capacity while the least quantities were recorded at 100% field capacity. the results of the present study also suggest that plants exhibit varying amount of phenolic acids under different field capacity which could improve antioxidant system to mitigate stress conditions. moreover, the change in phenolic acids can be correlated with growth behavior of m. oleifera. in addition, a positive correlation was found among the selected phenolic acids and sod, pod and cat activities. similarly, shoot length, number of leaves and root length were positively correlated with gallic acid (0.99, 0.79 and 0.82, respectively). these findings support the notion that phenolic acids protect plant tissues against oxidative damage inhibiting decrease in nutritional quality of moringa leaves under abiotic stress (ali et al., 2014). caffeic acid, p-hydroxybenzoic acid and p-coumaric acid and are considered as potential antioxidants due to their polyhydroxy nature (natella et al., 1999; sgherri et al., 2004). by studying correlation among these phenolic acids, growth parameters and enzymatic antioxidant activities, p-hydroxybenzoic acid and caffeic acid were positively correlated with sod and cat activities while p-coumaric acid was significantly correlated with sod and pod activities. as activities of enzymatic antioxidants and composition of phenolic acids increased under unfavorable growing conditions of moringa, it can be conclu tab. 3: effect of foliar application of plant growth regulators on selected phenolic acids (μg g-1) of moringa leaves under different field capacity levels. treatments p-hydroxybenzoic acid caffeic acid 100% 75% 40% 100% 75% 40% control 12.75±0.48 b 16.11±0.97 ab 14.76±0.62 ab 8.83±0.33 d 14.70 cd 13.72±0.42 cd water 15.48±0.67 ab 16.89±0.66 ab 16.06±0.88 ab 10.80±0.34 cd 16.84±0.59 cd 16.66±0.65 a-d bap 21.81±1.01 ab 29.74±1.23 a 25.32±0.97 ab 21.34±0.88 a-d 29.93±0.93 a 28.17±0.92 ab ascorbic acid 23.65±1.24 ab 24.97±0.98 ab 21.70±1.28 ab 20.04±0.76 a-d 24.05±1.24 a-d 22.60±1.36 a-c mle 13.54±0.57 ab 28.01±1.01 ab 23.48±1.19 ab 16.31±1.03 b-d 21.46±0.79 a-d 16.20±0.74 b-d gallic acid p-coumaric acid 100% 75% 40% 100% 75% 40% control 8.78±0.29 c 10.36±0.37 c 9.19±0.35 bc 6.37±0.21 f 7.13±0.29 f 8.20±0.28 d-f water 8.89±0.21 c 12.60±0.40 c 10.07±0.40 bc 6.27±0.28 f 7.33±0.31 ef 8.63±0.32 c-f bap 11.94±0.39 bc 17.58±0.57 a-c 14.29±0.56 a-c 12.08±0.39 c-e 12.87±0.59 b-d 13.52±0.48 bc ascorbic acid 20.18±0.64 ab 23.76±1.11 a 18.86±0.60 a 17.76±0.55 ab 19.24±0.51 a 20.18±0.79 a mle 16.42±0.62 a-c 14.71±0.45 bc 14.71±0.49 a-c 7.47±0.28 ef 9.35±0.31 c-f 10.28±0.39 c-f sinapic acid 100% 75% 40% control nd 5.83±0.21 c-e 4.05±0.16 ef water nd 5.80±0.20 c-e 4.92±0.19 d-f bap 7.16±0.29 b-e 9.68±0.37 b-d 9.98±0.36 bc ascorbic acid 11.03±0.34 ab 15.60±0.46 a 10.43±0.43 bc mle 7.03±0.27 b-e 9.26±0.41 b-d 7.93±0.29 b-e means not showing same letters differ significantly at 5% probability level, critical value for comparison for p-hydroxybenzoic acid: 16.69, critical value for comparison for caffeic acid: 13.17, critical value for comparison for gallic acid: 8.74, critical value for comparison for p-coumaric acid: 4.90, critical value for comparison for sinapic acid: 5.04. tab. 4: correlation among growth parameters (shoot length, number of leaves, root length, number of roots), enzymatic antioxidant activities (superoxide dismutase (sod), peroxidase (pod) and catalase (cat)) and the selected phenolic acid contents in moringa leaves. phenolic acids shoot length leaves root length roots sod pod cat p-hydroxybenzoic acid 0.92** 0.44 ns 0.49 ns 0.45 ns 0.73* 0.20 ns 0.88* caffeic acid 0.83* 0.26 ns 0.31 ns 0.62 ns 0.85* 0.38 ns 0.78* gallic acid 0.99** 0.79* 0.82* 0.01 ns 0.35 ns 0.25 ns 0.99* p-coumaric acid 0.17 ns 0.52 ns 0.47 ns 0.99** 0.97** 0.93** 0.09 ns sinapic acid 0.88* 0.35 ns 0.40 ns 0.54 ns 0.79* 0.29 ns 0.84* 86 w. nouman, m.e. olson, t. gull, m. zubair, s.m.a. basra, m.k. qureshi, m.t. sultan, m. shaheen ded that moringa expedites its antioxidant activities improving the phenolic acids to mitigate unfavorable conditions. keeping in view the findings of the present study, moringa plants can grow better under low water availability areas where other crops are difficult to cultivate. it can be suggested to farmers that it is preferable to cultivate moringa as fodder for their livestock under water deficit areas rather to leave the land as fallow. by these means, the farmers cannot only bring their abandoned land under cultivation but they can also have fodder for their livestock being rich in essential nutrients and further can be used as nutraceutical as phenolic rich source. conclusion it can safely be concluded here that moringa can be cultivated as a fodder crop in areas facing water shortage and even the tolerance can be improved by the exogenous application of bap and mle. among these both, mle is more suitable for farmers as it is economical, easily available and environment friendly being directly extracted from moringa leaves while bap is synthetic plant growth regulator which is relatively expensive than mle. acknowledgement the authors acknowledge bzu multan-pakistan and papiit it200515, unam, mexico for research support. references ali, e.m.a., ghada, s.m.i., 2014: tomato fruit quality as influenced by salinity and nitric oxide. turk. j. bot. 38, 122-129. ali, z., basra, s.m.a., munir, h., mahmood, a., yousaf, s., 2011: mitigation of drought stress in maize by natural and synthetic 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(http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 89, 60 67 (2016), doi:10.5073/jabfq.2016.089.007 1humboldt-universität zu berlin, faculty of life sciences, division urban plant ecophysiology, berlin, germany 2humboldt-universität zu berlin, faculty of life sciences, division biosystems engineering, berlin, germany 3egerton university, department of crops, horticulture and soils, egerton, kenya impact of direct-electric-current on growth and bioactive compounds of african nightshade (solanum scabrum mill.) plants elisha otieno gogo1,3, susanne huyskens-keil1*, anja krimlowski1, christian ulrichs1, uwe schmidt2, arnold opiyo3, dennis dannehl2 (received december 16, 2015) * corresponding author summary production of indigenous african leafy vegetables such as african nightshade (solanum scabrum mill.), whose high nutritional and medicinal value is well documented is still limited due to insufficient preharvest techniques. electric current is known to improve quality in food crops. therefore, in the present study, the effects of direct-electric-current (dc) on growth and characteristic bioactive and health promoting compounds were evaluated in different morphological sections, i.e., leaves and stems of african nightshade cv. olevolosi. six weeks old plants were exposed to different dc applied with a voltage of 8 and 16 v, 10 h/day for 12 days. non-treated plants served as control. plant growth, primary and secondary plant compounds were evaluated. applying dc increased leaf fresh (11.5-14.4 %) and dry (12.1-24.2 %) weight as well as marketable leaves (29.1-55.3 %). biosynthesis of chlorophylls and carotenoids was enhanced by increased dc. furthermore, dietary fibre fractions such as hemicellulose was promoted (23.3-45.3 %) by dc applications, while cellulose and lignin remained unaffected. minerals accumulated with increasing dc. alteration of cell membrane permeability due to dc may have enhanced physiological processes leading to the improved growth and acceleration of bioactive compounds in african nightshade leaves. introduction african leafy vegetables such as african nightshade (solanum scabrum mill.) have the potential to contribute to poverty alleviation and nutritional security because of their ease to grow, minimum production input requirement, and being highly rich in minerals, vitamins, fiber and bioactive compounds (kamga et al., 2013). recently, attention is being directed to these vegetables because of their high contents in bioactive phytochemicals (mibei et al., 2012). these chemicals possess strong antioxidant properties and have been implicated in prevention of diseases such as cancer, arteriosclerosis, diabetes and aging (voutilainen et al., 2006). furthermore, diets rich in micronutrients and antioxidants are strongly recommended as supplements in medicinal therapy for fighting hiv/aids (friis et al., 2002). as a result of changing lifestyle and eating habits, the protection against diseases such as cancer and diabetes are on the rise. consequently, these circumstances are leading to increasing demands for high quality and healthy food products (oniang’o et al., 2008). during postharvest processes, high electric fields were used to destroy plant cells in order to increase the bioavailability of consumed secondary plant compounds in processed food products (gonzalez and barrett, 2010). moreover, the application of electricity is also known to stimulate plant growth (black et al., 1971; costanzo, 2008), and has been used to protect plants from insect pests and diseases and in weed management (diprose et al., 1984). however, information in terms of the effect of weak direct-electric-current (dc) treatments on growth, yield and quality of leafy vegetables are scarce or just currently being investigated. internal electric fields of plants are affected by the externally applied electric fields, already early being reported (scott, 1967), influencing crop physiology. it is assumed that electric fields accelerate mass transfer by affecting cell membrane permeability properties (black et al., 1971; adeomowaye et al., 2002). recently, different intensities of dc in the range of 200 to 1800 ma were applied to roots of radish plants (raphanus sativus l.) and garden cress (lepidium sativum l.) during growth, whereby primary and secondary bioactive compounds (phenols, carotenoids), without visible damages to the plants were increased (dannehl et al., 2009; dannehl et al., 2012). previous studies during postharvest pro cesses also noted that dc intensities between 100 and 500 ma with varied application times between 15 and 60 min resulted in the accumulation of carotenoids, phenolic compounds and an increase in antioxidant activity in tomatoes (solanum lycopersicum l.) (dannehl et al., 2011). however, plant tissue is characterized by a heterogeneous structure and the uptake, as well as the availability of nutrients into plant cells are determined by the direction of the current flow (ben-ammar et al., 2011). cell size, cell size distribution, and cell orientation may have an impact on the effectiveness of the applied electric current (ben-ammar et al., 2011). therefore, the present study focused on the investigation of effects of dc on growth parameters, as well as primary (structural carbohydrates, mineral composition) and secondary bioactive (chlorophyll, carotenoid pattern) compounds in different morphological sections, i.e. leaves and stems of african nightshade (solanum scabrum mill.) cv. olevolosi during plant growth. additionally, the content of heavy metals in african nightshade was determined in order to examine a possible accumulation of toxic elements by dc, which might be caused by electrolysis of the metal electrodes used. materials and methods experimental site and plant material seeds of african nightshade (solanum scabrum mill.) cv. olevolosi were procured from avrdc (the world vegetable center, eastern and southern africa arusha, tanzania). the african nightshade plants were grown in the experimental station under greenhouse conditions at humboldt-universität zu berlin (berlin-dahlem, germany). the experiment was conducted from 30.06.2014 until 19.08.2014. the weekly mean values of the microclimatic conditions are presented in fig. 1. average air temperature ranged between 15 °c to 27 °c (night and day), while the average relative humidity was recorded ranging between 60 % and 85 %. daily light quantity ranged from 150 wm -2 to 300 wm -2. electric current affects quality of african nightshade plants 61 experimental set up and dc treatments all plant boxes were equipped with stainless steel plates (enstandard: x5crni18-10; thyssenkrupp nirosta gmbh; krefeld, germany) with the dimensions of 90 cm × 8 cm × 0.1 cm (length × width × thickness), which were used as electrodes. to minimize the surface area of the box acting as conducting surfaces and thus altering the electric field pattern and distribution of current, the electrodes were centrally placed within each box. a part of the horticultural substrate (profi-substrate + ton + fe, gramoflor gmbh & co., vechta, germany) was first added to about 2 cm before the plate was placed into each box and more substrate was added to about 10 cm mark. five seedlings were transplanted (3 weeks after sowing) in each box at a spacing of 20 cm apart. a power supply (voltcraft, vlp 1303 pro, hirschau, germany) with an output of 0-30 v was integrated in this system. to close the electric circuit, wires were fixed at the stainless steel plate and at the top of each plant. as such, the dc flow was directed from the horticultural substrate to the top of the plant. the experiment was carried out in a completely randomized design with 3 replications. excluding non-treated plants, the established plants were exposed to an electrical voltage of 8 and 16 v applied ten hours a day for 12 days. output dc was monitored and recorded three times a day (8 am, noon and 6 pm) on a one-day interval from the start of the experiment until harvesting. irrigation was done manually early in the morning on a daily basis ensuring an equal amount of water was applied to each treatment. determination of growth parameters plant height, total leaf number, stem diameter and number of primary shoots were determined on three central plants from each box at a 3-days interval from the beginning of the treatments until harvesting (12 days). after harvest, the leaves and stems of each plant per treatment were separated. leaves per plant were used to determine leaf area (leaf area meter-3100, bachofer, germany) of all plants, fresh weight, and amount of marketable and non-marketable leaves. non-marketable leaves in this context were leaves which were considered not sellable due to discoloration. the plant compartments (stems and leaves) were used for further chemical analysis as described below. bioactive primary and secondary plant compound analysis three replicates of leaves and stems from freshly harvested plants of the control and each dc treatment were used for analysis of each of the phytochemical compound. one part of the harvested plants was immediately used for the determination of the dry matter. the other part of the harvested plant material was shock-frozen with liquid nitrogen and kept at -20 °c for subsequent analysis of chlorophylls and carotenoids. the remaining samples were freeze-dried for 48 h (christ alpha 1-4, christ; osterode, germany), ground and mixed to a fine homogenized powder and stored in a desiccator for further analysis such as structural carbohydrates, minerals elements and heavy metals. determination of dry matter, chlorophylls and carotenoids to determine the dry matter, approximately 30 g fresh material per sample was placed in a drying oven at 105 °c for 24 h until constant weight was achieved. subsequently, the dry weight of each sample was measured in order to calculate the dry matter by the ratio of the dry weight to the fresh weight of the samples. the results were expressed as % dry matter. the dry matter of each sample was further used to calculate all phytochemicals on a dry weight basis. the extraction and determination of the chlorophylls and carotenoids content in african nightshade was conducted according to the method described by goodwin and britton (1988). an aliquot of 0.5 g fresh material was homogenized with acetone/hexane (4:5, v:v) (ultra-turrax t 25, jahnke & kunkel, ika-labortechnik, staufen, germany) for 1 min at 18,000 rpm, and afterwards centrifuged for 10 min (4000 rpm). this procedure was carried out twice. the resulting supernatants were collected in a 25 ml volumetric flask and filled up with acetone/hexane (4:5, v:v). three replications of the representative sample were measured twice with a spectrophotometer (uv-vis spectrophotometer, uv-mini-1240, shimadzu, japan) at wavelengths of 450 nm (total carotenoids), 453 nm (β-carotene), 505 nm (lycopene), 445 nm (lutein), as well as 663 nm and 645 nm for chlorophyll a and b, respectively. carotenoids and chlorophylls were calculated as described by nagata and yamashita (1992) and expressed as mg g-1 dm. inductively coupled plasma-optical emission spectrometry (icpoes) analysis for the determination of mineral elements (n, p, c, k, ca, mg, na, zn, fe) and heavy metals (cr, cd, pb and ni), the icp-oes analysis was applied for leaves and stems from each treatment in duplicates of three replications. for the microwave digestion, 0.2 g of each freeze dried sample was weighed into deionized containers. an aliquot of 5 ml hno3 (65 %) and 3 ml h2o2 (30 %) were added. the containers were then packed and placed into a microwave (mars xpress, cem; north carolina, usa) and digested according to the following program: step 1, 20 min to reach 200 °c; step 2, 5 min at 200 °c; step 3, 1 min to reach 210 °c; step 4, 5 min at 210 °c; step 5, 1 min to reach 220 °c; step 6, 5 min at 220 °c; and step 7, 30 min to cool down to room temperature. the resultant solution was trans ferred to 50 ml volumetric flasks using distilled water and finally filtrated into plastic flasks. thereafter, the analysis of the elements in the digestion solution was conducted via icp-oes with an icp emission spectrometer (icap 6300 duo mfc, thermo; waltham, usa). the operating conditions employed for icp-oes were 1150w rf power, 0.55 l min-1 nebulizer gas flow with argon employed as plasmogen as well as carrier gas and performed with a cross-flow nebulizer (mira mist, thermo scientific; cambridge england), in addition to radial (ca, mg) and axial (fe, zn, cu, al, cd, cr, ni, pb) view. for each element, a single-element standard solution (roth, karlsruhe, germany) of 1000 mg l-1 was used to prepare the reference analytic solutions in 1.4 mol l-1 hno3. the calibration curves were established with the following reference solutions: blank 1.4 mol l-1 hno3; 1-200 mg l-1 of ca; 0.5-50 mg l-1 of mg; 0.510 mg l-1 of al; 0.5-5 mg l-1 of cu, zn, and fe; 0.01-1 mg l-1 of cd, cr, ni, and pb. the elements in the solutions were analyzed in duplicate at wavelength as follows: ca at 317.9 nm, mg at 279.0 nm, fe at 259.9 nm, zn at 213.8 nm, cu at 324.7 nm, al at 369.1 nm, cd at 214.4 nm, cr at 267.7 nm, ni at 231.6 nm and pb at 220.3 nm. the con tents of macroand micronutrients, as well as heavy metals in leaves and stems of african nightshade were expressed as mg g-1 dm. fig. 1: microclimate condition during greenhouse production of african nightshade plants. 62 e.o. gogo, s. huyskens-keil, a. krimlowski, c. ulrichs, u. schmidt, a. opiyo, d. dannehl determination of carbon and nitrogen content carbon and nitrogen analysis were determined using three replicates of freeze-dried leaves and stems per treatment using an elemental analyzer (vario max, elementar analysensysteme gmbh; hanau, germany) according to din-iso-10694 (1995) and din-iso-13878 (1998). as such, an aliquot of 0.3 g of sample material was weighed into individual crucibles and catalytically combusted at 900 °c with pure oxygen. the combustion products and helium (as the carrier gas) passed through specific adsorption columns at a temperature of 830 °c to separate carbon and nitrogen. based on the differences in thermal conductivity of these elements, carbon and nitrogen were determined successively with a thermal conductivity detector. each analysis was performed twice and the results were calculated using glutamic acid as the standard reference. the results obtained were expressed as mg g-1 dm. determination of structural carbohydrates content cellulose, hemicellulose and lignin were analyzed according to goering and van soest (1972), van soest et al. (1991) and aoac (1999). one gram freeze-dried sample was extracted with 100 ml acid detergent fiber (adf) reagent (n-cetyl-n,n,n-tri methyl-ammoniumbromid dissolved with 96 % h2so4) using a fibertec system (m 1020, tecator, sweden). thereafter, the solution was vacuum-filtered, washed with boiled, double-distilled water until removal of the acidity and again washed with 90 % acetone. the residue was dried at 105 °c for 24 h, weighed, ash-dried at 500 °c for 24 h and weighed again to calculate adf (acid detergent fiber). the dried adf residue was used for acid detergent lignin (adl) determination. cellulose content was calculated as the difference between adf and adl. the contents of lignin and cellulose were expressed as mg g-1 dm. using the neutral detergent fiber (ndf) approach (van soest and goering 1963), one gram of freeze-dried material was cooked in 100 ml of ndf mixture (titriplex iii, di-sodium borate, dodecylhydrogensulfate-na, ethylene-glycol-monoethylester) to determine the hemicellulosic cell wall fraction. the solution was subsequently vacuum-filtered, and washed with demineralized water and with 90 % acetone. the insoluble residue was dried at 105 °c for 24 h, weighed, ash-dried at 500 °c for 24 h and weighed again to calculate ndf. the hemicellulose content was obtained by subtracting adf from ndf and expressed as mg g-1 dm. statistical analysis the impact of different dc treatments on plant growth and bioactive compounds, minerals and heavy metals in african nightshade was evaluated using analysis of variance (anova) with the statistical program sas (version 10). proc-univariate procedure was used to check for normality of data and all comparisons of the mentioned parameters were calculated using tukey-tests at a significance level of p < 0.05. the same significance level was used for the calculation of differences between the electric current flows when a voltage of 8 and 16 v was applied using fisher’s t-test. mean values labeled with different small letters indicate significant differences. the mean variability is shown as standard deviation. results changes in electrical current flow during african nightshade production results obtained from the voltmeter readings indicated that different voltage applications led to significant differences in dc flows during the experiment (fig. 2). doubling the voltage resulted in a maximum dc flow of 172 μa through plants, whereas a maximum flow of 73 μa was found at 8 v. there was a periodic fluctuation of the current flow up to 56 μa from one day to another at a higher electrical voltage (16 v), while the current flow at a lower voltage intensity (8 v) remained relatively constant after 6 days of electrical treatments. effect of different electrical voltages on growth of african nightshade plants as expected, growth of african nightshade plants increased with time. however, different electrical voltages did not influence plant height (tab. 1a), leaf number (tab. 1b), stem diameter (tab. 1c), and primary shoots (tab. 1d). furthermore in terms of leaf area (la), both dc treatments did not differ significantly from non treated plants (fig. 3). however, lower voltage (8 v) reduced la by 12.4 % compared to the higher voltage (16 v). effect of different electrical voltages on fresh and dry weight of african nightshade plants different electrical voltages had a significant influence on fresh weight of leaves, but not on stems (fig. 4a). in terms of leaf fresh weight, the values indicated a significant increase by 11.5 % and 14.4 % at 8 v and 16 v treatments, respectively, when compared to the control plants. however, the reverse effect was found in the case of stem fresh weight, where this parameter decreased with increasing voltages. the control stems had the highest fresh weight with 58.2 g followed by those treated with a voltage of 8 v (53.9 g) and 16 v (49 g). in terms of leaves, similar results were found on a dry weight basis (fig. 4b). thus, the same increase pattern was observed, where only the voltage treatment with 16 v caused a significant increase in leaf dry weight by 24.2 % compared to the control leaves. on the other hand, a lower electrical voltage (8 v) tendentiously increased stem dry weight by 5.8 % while doubling voltage tended to reduce stem dry weight by 2.9 % compared to the control. different voltage applications significantly influenced both marketable and non-marketable leaves harvested (fig. 4c). a lower voltage application (8 v) increased marketable leaves by 55.3 %, while a higher voltage (16 v) increased marketable leaves by 29.1 % compared to plants grown under non-treated conditions. regarding non-marketable leaves, the control had the highest amount with 5.7 leaves/plant, whereas number of leaves of dc at 8 v and 16 v was significantly reduced by 77.2 % and 64.9 %, respectively. no significant differences were observed between 8 v and 16 v. effect of different electrical voltages on chlorophyll and carotenoid contents of african nightshade plants varying responses of leaves and stems of african nightshade plants were noted due to different voltage treatments. generally, a voltage fig. 2: changes in current flow on african nightshade plants during production as influenced by dc intensity. means (± standard deviation) followed by the same letter are not significantly different according to t-test (p < 0.05). electric current affects quality of african nightshade plants 63 tab. 1: effect of different electrical voltages on growth parameters of african nightshade plants. days after treatment application dc 0 4 8 12 a. plant height (cm) control 41.4 ± 3.4 g 49.3 ± 3.6 f 58.4 ± 2.3 bcd 65.2 ± 3.0 ab 8 v 41.0 ± 3.5 g 52.1 ± 5.1 def 60.9 ± 5.6 bc 68.8 ± 5.5 a 16 v 40.6 ± 1.2 g 50.7 ± 1.8 ef 56.9 ± 3.2 cde 64.2 ± 4.2 ab b. leaf number (no./plant) control 10.3 ± 0.3 e 12.3 ± 0.9 d 13.4 ± 0.7 bc 14.9 ± 0.7 a 8 v 10.2 ± 0.2 e 12.4 ± 0.5 cd 13.6 ± 0.4 b 14.8 ± 0.8 a 16 v 10.4 ± 0.8 e 12.8 ± 0.5 bcd 13.7 ± 0.7 b 14.8 ± 0.8 a c. stem diameter (mm) control 9.6 ± 0.7 cd 10.0 ± 0.4 abcd 10.6 ± 0.7 abc 10.9 ± 1.0 a 8 v 9.2 ± 0.3 d 9.8 ± 0.4 bcd 10.4 ± 0.8 abcd 10.5 ± 0.7 abc 16 v 9.3 ± 0.6 d 9.8 ± 0.4 bcd 10.1 ± 0.5 abcd 10.1 ± 0.4 abc d. primary shoots (no./plant) control 4.9 ± 0.8 de 5.4 ± 0.8 bcde 6.1 ± 0.4 abcd 6.9 ± 0.4 ab 8 v 4.1 ± 1.0 e 5.1 ± 1.3 de 6.7 ± 0.9 abc 7.6 ± 0.9 a 16 v 4.2 ± 1.0 e 5.3 ± 1.0 cde 6.8 ± 0.8 abc 7.6 ± 0.8 a mean values (± standard deviation) within a growth variable followed by the same letter are not significantly different according to tukey-test (p < 0.05). fig. 4: effect of different voltage applications on fresh (a) and dry (b) weight and marketable and non-marketable leaves (c) of african nightshade plants. mean values (± standard deviation) within a specific variable followed by the same letter are not significantly different according to tukey-test (p < 0.05). fig. 3: effect of different voltage applications on leaf area of african nightshade plants. mean values (± standard deviation) followed by the same letter are not significantly different according to tukey-test (p < 0.05). of 16 v resulted in higher chlorophyll and carotenoid contents in african nightshade leaves, while a lower voltage treatment reduced both chlorophyll and carotenoids content. leaves had higher chlorophyll and carotenoid contents compared with stems. compared to the control, a voltage of 8 v significantly reduced the contents of lutein (15.6 %), β-carotene (16.2 %), total carotenoids (16.0 %) and chloro phyll a (15.5 %) in african nightshade leaves, whereas lycopene and chlorophyll b were not statistically different from control and a higher voltage treatment (tab. 2a). on the other hand, com pared to the control, a higher voltage of 16 v increased lutein (4.6 %), β-carotene (4.4 %), total carotenoids (4.5 %) and chlorophyll a (4.0 %) contents of african nightshade leaves, however, only tendentiously. regarding african nightshade stems, an increase in voltage treatments resulted in a significant increase in almost all carotenoids and chlorophylls (tab. 2b). when compared with the control plants, mainly the highest voltage treatment (16 v) increased lutein, 64 e.o. gogo, s. huyskens-keil, a. krimlowski, c. ulrichs, u. schmidt, a. opiyo, d. dannehl tab. 2: effect of voltage applications on chlorophyll and carotenoid contents (mg/g dm) of african nightshade plants. dc lutein β-carotene lycopene total carotenoids chlorophyll a chlorophyll b a. leaves control 2.0 ± 0.15 a 2.1 ± 0.16 a 1.5 × 10-1 ± 0.01 a 8.2 ± 0.62 a 16.9 ± 1.52 a 7.5 ± 0.74 a 8 v 1.7 ± 0.24 b 1.7 ± 0.24 b 1.5 × 10-1 ± 0.02 a 6.9 ± 0.97 b 14.3 ± 1.58 b 6.9 ± 0.54 a 16 v 2.1 ± 0.10 a 2.2 ± 0.10 a 1.6 × 10-1 ± 0.01 a 8.6 ± 0.40 a 17.6 ± 0.70 a 7.6 ± 1.01 a b. stems control 1.0 × 10-1 ± 0.01 b 1.0 × 10-1 ± 0.01 b 7.9 × 10-3 ± 0.00 a 4.1 × 10-1 ± 0.03 b 7.8 × 10-1 ± 0.07 b 4.1 × 10-1 ± 0.02 b 8 v 1.1 × 10-1 ± 0.01 ab 1.1 × 10-1 ± 0.01 b 8.4 × 10-3 ± 0.00 a 4.4 × 10-1 ± 0.04 b 8.6 × 10-1 ± 0.09 ab 4.8 × 10-1 ± 0.01 ab 16 v 1.2 × 10-1 ± 0.01 a 1.3 × 10-1 ± 0.00 a 8.7 × 10-3 ± 0.00 a 5.0 × 10-1 ± 0.02 a 9.5 × 10-1 ± 0.05 a 5.0 × 10-1 ± 0.02 a mean values (± standard deviation) within a specific variable and plant part followed by the same letter are not significantly different according to tukey-test (p < 0.05). β-carotene, total carotenoids, chlorophyll a and b contents in stems by 20.0 %, 30.0 %, 22.0 %, 21.8 %, 22.0 %, respectively, whereas lycopene was affected to a smaller extent (10.0 %). plants treated with a voltage of 8 and 16 v did not differ significantly from each other when bioactive compounds such as lutein, lycopene and chlorophylls were considered. however, doubling the voltage led to an increase in ß-carotene by 18.2 % and total carotenoids by 13.6 % in stems compared with the lower voltage (8 v) applied on african nightshade plants. effect of different electrical voltages on structural carbohydrates of african nightshade plants there was a similar response to different voltage in terms of structural carbohydrates in leaves and stems of african nightshade plants. a higher content of structural carbohydrates was obtained in stems compared to leaves. in leaves, only the treatment of 16 v resulted in a significant increase in hemicellulose (40.0 %) content compared to the non-treated plants. in consideration of both dc treatments, no significant differences were found (fig. 5a). however, neither 8 v nor 16 v influenced cellulose and lignin content in leaves significantly. similar results were obtained in stems. the hemicellulose content was significantly increased by 52 % and 38.7 % when 8 v and 16 v were applied, respectively, compared to the control (fig. 5b). cellulose and lignin contents in stems were not significantly affected by different voltages. effect of different electrical voltages on mineral compounds and heavy metals in african nightshade plants voltage treatments differently affected minerals and heavy metals of african nightshade plants, in both leaves and stems. a general increase in voltage resulted in an increase in most of the mineral elements and heavy metals analyzed. generally, a higher content of n, c, ca, mg, fe, zn, ni, and cd was observed in leaves while stems had higher k, na, pb and cr contents. in leaves, a voltage of 8 v significantly influenced the contents of ca, pb and cr which were increased by 20.2 %, 42.1 %, and 52.7 %, respectively, compared to the contents observed in control leaves (tab. 3a). a voltage of 16 v evoked nearly the same accumulations, except for the content of ca which was increased by 27.5 % when compared to the non-treated plants. a significant difference between lower and higher voltages occurred only when the content of ca was considered. this was significantly increased by 6.1 % when leaves were exposed to 16 v. in stems, however, a voltage of 16 v significantly increased the contents of mg (15.6 %), zn (244.4 %), ni (113.2 %), cd (36.8 %) and cr (288.5 %) compared to the control. no significant difference between elements was achieved when control plants and plants exposed to 8 v were considered (tab. 3b). significant differences between both voltage treatments were only demonstrated in terms of na, zn and ni. the content of these elements in stems grown under 16 v conditions was increased by 31.4 %, 167.0 % and 105.5 %, respectively. however, different voltages were not able to influence the contents of n, c, p, k, ca, mg, fe and pb in stems of african nightshade plants significantly. discussion results from the present study have demonstrated that weak voltages may be used to improve growth and bioactive compounds in african nightshade plants. the different voltages had a significant influence on current flow through african nightshade plants. this was in creased with increasing voltage. this circumstance could be attrifig. 5: effect of different voltage applications on different structural carbohydrates on leaves (a) and stems (b) of african nightshade plants. mean values (± standard deviation) within a specific variable followed by the same letter are not significantly different according to tukey-test (p < 0.05). electric current affects quality of african nightshade plants 65 buted to varying resistance caused by african nightshade plants as a result of changes in growth and development with time. on the other hand, changes in water supply could result in variation in electrical conductivity followed by changes in electrical current flows. even though different voltages did not affect the plant growth (leaf number, plant height, stem diameter and number of primary shoots) of nightshade plants, la was tendentiously enhanced when a voltage of 16 v was applied compared to the control. however, kareem (1999) successfully demonstrated that a higher electric current (110 to 220 v) at varying time (10 to 30 s) was able to improve leaf length, leaf width and stem length of cowpea (vigna unguiculata (l.) walp.), guinea corn (sorghum bicolor (l.) moench) and groundnut (arachis hypogaea l.), when seeds were treated using wet (seeds soaked in water inside aluminum cup connected to live wire) and dry (seeds with empty aluminum cup connected to live wire) application methods. kotaka and krueger (1968) found that varying ion densities improved growth of barley (hordeum vulgare l.), oats (avena sativa l.) and lettuce (lactuca sativa l.). these studies suggest that increases in ion density as a result of voltage effects may enhance the availability of nutrients in plant cells as observed in the current study. this phenomenon may result in improved physiological processes such as photosynthesis, whereby more photosynthates can be accumulated in leaves leading to a higher la as tendentiously observed in the present study at the 16 v treatments. okamura et al. (2011) showed that growth of radish and thale cress (arabidopsis thaliana (l.) heynh.) was more promoted when seeds were treated with 10.0 kv/m dc than with 2.5 kv/m dc. it is also important to note that plant tissue is characterized by a heterogeneous structure with a high degree of anisotropy (ben-ammar et al., 2011). cell size, cell size distribution, and cell orientation have an impact on the effectiveness of electric currents on crops (ben-ammar et al., 2011). these influencing factors could be taken into consideration to explain the reason why some growth parameters were not affected by different electric voltages as observed in the present study. in the current study, the use of dc increased leaf fresh and dry weight of african nightshade plants. dc was considered to affect visual damage (yellowing) of plants, therefore, marketable and nonmarketable leaves were recorded. it was found that dc reduced the amount of non-marketable leaves. murr (1963) observed tip burn to orchard grass seedlings (dactylis glomerata l.) due to electric fields (40 kv/m) effect manifested by dark brown spots. further, microscopic examination revealed chloroplast damage. however, the strength of electric field has an influence on the extent of plant damage. the results in terms of dry weight were corroborated by ward (1996) who found that weak direct electric current (6 v) using wires as electrodes increased this parameter in greenhouse produced tomatoes. electrical fields are reported to play a vital part in crop physiology (scott, 1967). for example, an electrogenic proton pump helps in the regulation of cytoplasmic ph, active transport of mineral ions, hormones or organic metabolites (spanswick, 1981). in this context, the externally applied voltages in the present study resulted in an increase in contents of mineral elements (e.g. mg, ca and zn) and structural carbohydrates (hemicellulose) in a bid to cope with electric stress (gall et al., 2015). this circumstance can promote physiological processes of african nightshade plants. for instance, ca plays a major role in root system development associated with a better water and nutrient uptake (picchioni et al., 2001) while mg and zn is involved in chlorophyll synthesis, which is important for photosynthesis (hu and sparks, 1991). the latter evidence could be the reason for a higher accumulation of carbohydrates as caused by increasing voltages in the present study, especially observed for hemicellulose content in leaves and stems. hemicellulose facilitates cell wall thickening by reinforcement of secondary wall and hence reduction in water loss through transpiration (gall et al., 2015). these plant responses were jointly responsible and sufficient, in order to increase fresh and dry weight in leaves as well as marketable leaves as observed on dc treated plants. furthermore, the increase in mineral elements (e.g. mg and zn) in dc treated plants enhanced chlorophyll formation (green pigment), thus preventing yellowing (chlorosis) leading to reduced non-marketable leaves as reported in the present study. as indicated earlier, a shift in consumer eating habits has led to increasing demand for high quality food products. in the present study, application of a voltage of 16 v through the whole plant significantly increased contents of chlorophyll a and b, as well as lutein, β-carotene and total carotenoids in stems and tendentiously in leaves of african nightshade plants. these plant bioactive compounds have antioxidant potential and are therefore very important in maintaining human health. for instance, carotenoids have been reported to possess provitamin a activity (tang, 2010); a precursor of vitamin tab. 3: effect of voltage applications on mineral element and heavy metal content (g/kg dm) of african nightshade plants. a. leaves b. stems element control 8 v 16 v control 8 v 16 v n 29.60 ± 3.20 a 27.10 ± 3.40 a 28.60 ± 4.30 a 7.40 ± 0.80 a 6.79 ± 0.60 a 8.34 ± 1.30 a c 425.50 ± 2.30 a 421.00 ± 3.80 a 424.40 ± 3.10 a 410.70 ± 5.80 a 413.10 ± 5.30 a 409.00 ± 4.80 a p 6.13 ± 0.39 a 5.73 ± 0.54 a 6.02 ± 0.37 a 3.46 ± 0.18 a 3.6 ± 0.22 a 3.86 ± 0.22 a k 33.35 ± 0.82 a 32.20 ± 3.17 a 32.56 ± 4.68 a 40.72 ± 2.68 a 39.37 ± 3.26 a 41.42 ± 3.64 a ca 21.56 ± 0.16 c 25.91 ± 0.77 b 27.49 ± 3.28 a 12.38 ± 1.02 a 12.90 ± 0.57 a 14.10 ± 1.18 a mg 3.12 ± 0.39 a 3.25 ± 0.39 a 3.07 ± 0.41 a 1.60 ± 0.05 b 1.76 ± 0.26 ab 1.85 ± 0.11 a na 1.10 × 10-1 ± 0.00 a 0.90 × 10-1 ± 0.00 a 0.97 × 10-1 ± 0.00 a 2.38 × 10-1 ± 0.03 ab 2.10 × 10-1 ± 0.02 b 2.76 × 10-1 ± 0.03 a fe 9.04 × 10-2 ± 0.00 a 8.83 × 10-2 ± 0.00 a 9.05 × 10-2 ± 0.00 a 3.75 × 10-2 ± 0.00 a 4.14 × 10-2 ± 0.00 a 4.26 × 10-2 ± 0.00 a zn 3.53 × 10-2 ± 0.00 a 3.73 × 10-2 ± 0.00 a 3.98 × 10-2 ± 0.00 a 2.35 × 10-2 ± 0.00 b 3.03 × 10-2 ± 0.00 b 8.09 × 10-2 ± 0.00 a ni 0.00 ± 0.00 a 1.19 × 10-6 ± 0.00 a 1.15 × 10-6 ± 0.00 a 0.53 × 10-6 ± 0.00 b 0.55 × 10-6 ± 0.00 b 1.13 × 10-6 ± 0.00 a pb 3.40 × 10-4 ± 0.00 b 4.83 × 10-4 ± 0.00 a 4.57 × 10-4 ± 0.00 ab 3.63 × 10-4 ± 0.00 a 4.10 × 10-4 ± 0.00 a 4.79 × 10-4 ± 0.00 a cd 4.87 × 10-4 ± 0.00 a 5.47 × 10-4 ± 0.00 a 5.43 × 10-4 ± 0.00 a 2.34 × 10-4± 0.00 b 2.58 × 10-4 ± 0.00 ab 3.20 × 10-4 ± 00.0 a cr 3.47 × 10-4 ± 0.00 b 5.30 × 10-4 ± 0.00 a 5.10 × 10-4 ± 0.00 a 5.2 × 10-4 ± 0.00 b 9.90 × 10-4 ± 0.00 ab 20.2 × 10-4 ± 0.00 a mean values (± standard deviation) within a row and plant part followed by the same letter are not significantly different according to tukey-test (p < 0.05). 66 e.o. gogo, s. huyskens-keil, a. krimlowski, c. ulrichs, u. schmidt, a. opiyo, d. dannehl a, essential for the promotion of general growth, maintenance of visual function, regulation of differentiation of epithelial tissues and embryonic development (underwood and arthur, 1996). in addition, carotenoids have been reported to be necessary for the preven tion of cardiovascular diseases and also have anticarcinogenic effects (voutilainen et al., 2006). just like carotenoids, chlorophylls are important for health promotion such as enhancing oxygen uptake in the blood, which can increase energy, relieve fatigue and improve many blood disorders (ferruzzi et al., 2001). regarding electrophysiology, studies involving the use of intermittent-directelectric-current (idc) between 200 and 1800 ma applied to plant roots increased phytochemical compounds in one week old garden cress sprouts compared to the control (dannehl et al., 2012). previously, the same group of researchers demonstrated that different intensities of idc (100 to 500 ma) with varied application times (15 to 60 min) during postharvest resulted in accumulation of carotenoids, phenolic compounds and an increase in antioxidant activity in tomatoes (dannehl et al., 2011). similarly, they also reported that application of idc (200 to 1000 ma) to roots of radish plants during growth resulted in an increase in phenolic compounds in radish tubers as well as in roots without visible damages to the plants (dannehl et al., 2009). another report indicates that the application of dc (10v/12.5 μa) resulted in an increase in peroxidase activity; an indicator of stress in spinach leaves (spinacia oleracea l.) (montavon et al., 1988). this stress response could be initiated when the plant recognizes a stimulus at the cellular level, as a result of the activity of specific ion channels such as voltage-gated ion channels embedded in a cell plasma membrane (gaspar et al., 2002). thus, perturbation of internal electric field as a result of applied dc could induce stress mediated changes in the electrochemical proton gradient or the cytoplasmic and vacuolar ph, among others. this in turn could result in an increase in biosynthesis of primary and secondary metabolites in order to rectify this plant state. in the present study, the higher contents of chlorophylls and carotenoids as a result of applied dc confirm this assumption. in addition, dc improved content of hemicellulose, an important structural carbohydrate necessary in food digestion. however, dc did not significantly influence cellulose and lignin content. stress induction by dc may result in various enzymatic activities, e.g. cell-wall bound peroxidases (dymek et al., 2014). this could lead to biosynthesis of structural carbohydrates such as hemicellulose in african nightshade plants as observed in the present study, in order to enhance plant resistance to stress. however, the enzymatic signaling pathways of various structural carbohydrates are compartment-specific or even cell-specific (dymek et al., 2014). this would explain why some structural carbohydrates were affected (hemi cellulose) while others were not (cellulose and lignin). majority of the world population currently suffer from ‘hidden hunger’ (e.g. lacking various mineral elements, vitamin a and fiber content) in the diet (biesalski, 2013). this is exacerbated by frequent consumption of food either lacking or having low content of such key mineral elements or phytochemicals. the present study revealed that dc treatments through the whole plant result in an increase in certain mineral elements such as mg, ca and zn in african nightshade plants. however, other elements, for example, n, c, p, k and fe were not significantly affected. an increased nutrient uptake of mg and ca was also found in greenhouse produced tomato treated with 6 v dc (ward, 1996) while black et al. (1971) reported an significant increase in k, ca and p content in tomato when a much lower dc of 15 to 30 μa was applied. as such, additional potential induced from external electric field in plants is reported to raise pressure on the membrane due to attraction between opposite charges on both sides of the membrane, whereby a faster transport of ions is induced simultaneously (zimmerman et al., 1974). this could cause an accumulation of elements, as this hypothesis is supported by increased contents of minerals as observed in the present study. this rapid influx of mineral ions through cation channels imbedded in the plant membrane can induce stress in plants, which can promote the synthesis of secondary metabolites as well (dannehl et al., 2012). in order to ensure reversible membrane permeability and associated plant vitality, critical electric field strength must be taken into consideration, which is dependent on plant species, as well as cell geometry and size (heinz et al., 2002). dc also increased the content of heavy metals, i.e. cr, ni, cd and pb of african nightshade plants, assumingly due to an electron transport between both electrodes used in the present study. similar results were reported by dannehl et al. (2012) when dc (200 to 1800 ma) was applied to garden cress. however, the observed results are still within acceptable thresholds regulated by law for human consumption according to european food safety authority (efsa, 2008). conclusion and recommendation in general, the findings of this study have demonstrated that the application of weak dosages of dc (8 and 16 v) can improve growth, and quality in terms of health protecting plant compounds of african nightshade plants. plant specific carotenoids (ß-carotene and lutein), chlorophyll a and b, as well as characteristic mineral elements such as mg, ca and zn and structural carbohydrates (hemicellulose) in african nightshade were promoted by dc. the results showed that dc induced a defense response of plant cells and may have altered the cell membrane properties and hence physiological processes. farmers of african leafy vegetables in kenya commonly practice small-scale farming or kitchen gardening, where they cultivate vegetables in small plots or in pots, especially for home consumption. therefore, the findings of the study may be beneficial to such farmers. this technology is also applicable in areas where electricity is absent or unstable which in most cases is the situation in rural areas as farmers can use alternative power sources, e.g. batteries, solar cells, or dynamos. furthermore, emerging technologies such as dc could be used either as stand-alone or in synergistic combination with other technologies, in order to satisfy the increased consumer demand for high quality and healthy food products. as such, dc applications provide an alternative to enhance food security. however, it remains a need to adjust the intensity of dc and duration of exposure to various crop species and varieties for optimization of yield and quality. furthermore, it has to be investigated in detail if such technologies are worthwhile in terms of economic aspects. acknowledgement this study is part of the project horticultural innovations and learning for improved nutrition and livelihood in east africa (hortinlea) being funded by the german federal ministry of education and research (bmbf) and the german federal ministry of economic cooperation and development (bmz) within the framework of the program globe – global food security. we gratefully acknowledge the financial support of bmbf and bmz. the authors thank susanne meier and sigrun witt for conducting an excellent job in the laboratories. references ade-omowaye, b.i.o., rastogi, n.k., angersbach, a., knorr, d., 2002: osmotic dehydration of bell peppers: influence of high intensity electric field pulses and elevated temperature treatment. j. food eng. 54, 35-43. aoac, 1999: official methods of analysis. 14th ed. washington: association of official analytical chemists. ben-ammar, j., lanoisellé, j.l., lebovka, n., van hecke, e., electric current affects quality of african nightshade plants 67 vorobiev, e., 2011: impact of a pulsed electric field on 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14, 881-899. adresses of the authors: dr. susanne huyskens-keil, prof. dr. dr. christian ulrichs, msc anja krimlowski, humboldt-universität zu berlin, faculty of life sciences, division urban plant ecophysiology, lentzeallee 55-57, 14195 berlin, germany e-mail: susanne.huyskens@agrar.hu-berlin.de prof. dr. uwe schmidt, dr. dennis dannehl, humboldt-universität zu berlin, faculty of life sciences, division biosystems engineering albrechtthaer weg 3, 14195 berlin, germany dr. arnold opiyo, msc elisha otieno gogo, egerton university, department of crops, horticulture and soils, p.o. box 536, 20115 egerton, kenya © the author(s) 2016. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 89, 217 222 (2016), doi:10.5073/jabfq.2016.089.028 1suleyman demirel university, faculty of agriculture, department of field crops, isparta, turkey 2suleyman demirel university, faculty of agriculture, department of agricultural biotechnology, isparta, turkey mobilization of seed reserves during germination and early seedling growth of two sunflower cultivars sabri erbaş1*, muhammet tonguç2, yaşar karakurt2, arif şanli1 (received february 17, 2016) * corresponding author summary the present study was carried out to determine the mobilization of seed storage components of sunflower seeds during germination and early seedling development. two sunflower cultivars (duet cl and tr 3080) were used as plant materials. seeds were germinated for 120 h and samples were taken every 24 h. the total chlorophyll and carotenoid contents of the germinating seeds of both cultivars significantly increased till 96 h, and then decreased. while the total soluble and reducing sugar contents were decreased during the first 24 h, their amounts increased significantly afterwards. the total protein contents of the germinating seeds of tr 3080 and duet cl decreased from 48.1 % and 40.9 % to 35.5 % and 28.4 %, respectively. however, their free amino acid contents were steadily increased during germination and early seedling growth. the oil contents of duet cl and tr 3080 started to decrease significantly after 72 h and dropped to 41.3 % and 40.2 %, respectively, at the end of the study. free fatty acid contents of the seeds increased until 72 h, but decreased thereafter. while, oleic acid contents of the cultivars decreased during the germination period, their linoleic acid contents increased. these results suggest that significant changes occur in the pigment, sugar, lipid and protein metabolisms during germination and early seedling growth period of sunflower. introduction sunflower (helianthus annuus l.) belongs to asteraceae family and is one of the significant oil seed crops in the world. sunflower seeds contain 35-50 % oil, 20-30 % protein, 20-40 % carbohydrate, 20-30 % husk and 4-6 % ash (weiss, 2000). sunflower oil is rich in unsaturated fatty acids (80-90 %). commercial sunflower oil containes 5 fatty acids including palmitic acid (c16:0), stearic acid (c18:0), oleic acid (c18:1), linoleic acid (c18:2) and linolenic acid (c18:3). recently, the development of sunflower cultivars with high oleic acid content (< 85 %) further increased its industrial use (weiss, 2000). moroever, the remaining meal after the removal of oil from seeds is rich in tryptophan and it is important for animal feed (balasaraswatti and sadasivam, 1997). in general, there are two substantial changes take place in the plants after pollination and fertilization: one is the increase in seed volume and the other is the change in biochemical and physiological characteristics. the former involves the division, expansion and differentiation of seed cells and the latter involves the changes in seed metabolites such as storage lipids, proteins, carbonhydrates and other seed storage metabolic substances (satyanarayana et al., 2011). sucrose synthesized during maturation of oil seeds is the source of carbon for triacylglycerol (tag) synthesis (bewley and black, 1994). macromolecules accumulated in seeds are used as an energy source for early seedling development and seed germination. germination begins with water uptake by the seed (imbibition) and the emergence of embryonic axis, usually the radicle, through the structures surrounding it (bewley et al., 2013). when seed germination begins, starch and proteins are converted to sugars and amino acids within the starch granules and protein storage vacuoles, by diastase and protease enzymes, respectively (wilson, 2006). tags are hydrolysed by lipases, enzymes catalyzing the hydrolytic cleavage of the fatty acid ester bonds, to yield glycerol and free fatty acids (theimer and rosnitschek, 1978). free fatty acids enter the glyoxysome for conversion to oxaloacetic acid (oaa), passes into the mitochondrion, and ultimately into the cytosol for conversion to sucrose, which then transported as an energy source from cotyledons to the growing axis of seedling (graham, 2008). almost all tags present in oleiferous seeds are lost during seed germination and seedling development (muto and beevers, 1974; yaniv et al., 1998; rabiei et al., 2007; kim et al., 2011; tonguç et al., 2012). although there are studies regarding the physiology and mobilization of sunflower seed oil during germination (balasaraswatti and sadasivam, 1997; rosales et al., 1998; moya et al., 2000; munshi et al., 2007; rabiei et al., 2007), little is known about the changes in other seed reserves during the germination. the objective of the present study was to investigate the mobilization of seed reserve molecules during the germination and early seedling growth of sunflower. materials and methods two sunflower cultivars, tr 3080 and duet cl were used as plant materials. tr 3080 is a high linoleic acid and duet cl is a high oleic acid cultivar. seeds were surface sterilized with 2 % sodium hypochlorite solution for 5 min, and then washed with distilled water several times to remove residual sodium hypochlorite. seeds were germinated in 9 cm petri dishes on 2 sheets of filter paper moistened with distilled water. petri dishes were incubated at 25 °c under alternating light and dark periods (12 h light/dark periods). 25 seeds were placed in each petri dish, and 3 replications were used for the analysis of each parameter. seeds were germinated for 120 h, and the germinated seeds were removed from the incubator at every 24 h for analysis. the cotyledons from germinating seeds and growing seedlings were separated and dried for at least 24 h at 50 °c or until they reached to a constant weight (tonguç et al., 2012). the germination percentage of duet cl and tr 3080 were 96.2 and 98.5 %, respectively. non-germinated or unevenly germinated seeds were discarded. for all analysis, the seeds were dehulled, and only the grinded dried cotyledons were used. the hull content of both cultivars was 23.5 %. the total protein content was determined using the defatted and dehulled seeds. total chlorophyll and total carotenoid contents were determined from fresh cotyledons. the dry matter content of fresh cotyledons (2 g) was determined gravimetrically, by drying to a constant weight at 105 °c [dry matter (%) = dry weight / fresh weight × 100] (aoac, 1990). ash content of samples was determined following the method of aoac (1990). total chlorophyll content of fresh cotyledons (0.25 g) was deter218 s. erbaş, m. tonguç, y. karakurt, a. şanli mined according to kirk and allen (1965) with the following equation: total chlorophyll (mg g-1) = [(0.0202 × a645) + (0.00802 × a663) × 10] / sample weight. for total carotenoids content of fresh cotyledons, 10 ml of acetone-hexane (4:6) solvent was added on 0.25 g sample and the mixture was homogenized. two phases were separated, and the aliquots were taken from the upper phase and read on a spectrophotometer (pg instruments) at 480, 645 and 663 nm wavelengths. total carotenoid content was calculated according to miller et al. (1993) using the following equation: total carotenoids (mg g-1) = a480 + (0.114 × a663 – 0.638 × a645). total soluble and reducing sugars were extracted as described (tonguç et al., 2012). the total soluble sugar content was determined by the phenol sulfuric acid assay (dubois et al., 1956) and the reducing sugar content was determined following the somogyi (1952) method. glucose was used as standard and the results were expressed as mg g-1 dry weight. the protein extraction was performed according to the method of larson and beevers (1965) and the total protein content was determined as described (hartree and lowry, 1995). bovine serum albumin was used as standard. amino acids were extracted according to noctor et al. (2007). total free amino acid content was determined with the ninhydrine method as described by lee and takahashi (1966). standard curve was prepared using l-valine as standard. the oil content and fatty acid composition were determined using nuclear magnetic resonance (nmr, brükermqone) and gas chromatography (gc, perkin elmer auto system xl), respectively. the oil samples (50-100 mg) were obtained with cold extraction and converted to its fatty acid methyl esters (fame) as described by marquard (1987). the conditions for gc analysis were as follows: capillary column, mn ffap (50 m × 0.32 mm i.d., film thickness, 0.25 m), oven temperature kept at 120 °c for 1 min and programmed to 250 °c at a rate of 6 °c min-1, and then constant at 240 °c for 15 min, total run time 60 min, injector temperature 250 °c, detector (70 ev) temperature 260 °c, flow rate for helium 40 ml min-1, split ratio 1/20 ml min-1, injection volume 0.5 μl. the free fatty acid content of lipids was determined colorimetrically according to the method of lowry and tinsley (1976). samples (1 g) were extracted with hexane and the extracted lipids (4 μl) were dissolved in benzene (5 ml). cupric acetate-pyridine reagent (1 ml) was added and the samples were vortexed for 90 s. the mixture was then centrifuged for 3 min at 1500 g. 3 mls of supernatant were used to determine the free fatty acid contents of the samples. the total free fatty acid content of samples (%) was determined as oleic acid equivalents. the experiment was set up according to a completely randomized experimental design with 3 replications. the data were subjected to the analysis of variance (anova) using sas (2009) program (inc sas/stat user’s guide release 7.0, cary, nc, usa). differences among means were determined using least significant difference (lsd) test. results and discussion plants use seed coat to protect embryo from the environmental effects until seeds germinate and seed coats are not used as seed storage compartment. therefore; seed coats of the sunflower cultivars were removed prior to analysis. mobilization of the main reserves such as starch, oil and protein in seed storage tissues occurs following the completion of germination to supply nutrients for the growing seedling until it develops true leaves. reserve mobilization differs among plant species during germination depending on the accumulated reserves (bewley et al., 2013). in the present study, breakdown and mobilization of different molecules were investigated. the germination period, cultivar and the germination period × cultivar interaction were significant (p < 0.01) for dry matter, total chlorophyll, total soluble and reducing sugar contents during the study period (tab. 1). while the germination period and cultivar effects were significant (p < 0.01) for ash, total protein and free fatty acid contents, the germination period × cultivar interaction was not significant for these parameters. for free amino acid and oil contents, the germination period and the gemination period × cultivar interaction were significant at 1 %, and the cultivar effect was significant at the 5 % level of significance. for the total carotenoid content of the cotyledones during the germination, the germination period and the cultivar × germination period interaction were significant (p < 0.05), but the cultivar effect was not significant (tab. 1). the dry matter contents of both cultivars decreased gradually throughout the germination period. at 0 h, the dry matter contents of tr 3080 and duet cl were 95.2 % and 95.7 %, respectively. at 120 h, the dry matter contents decreased to 36.8 % and 27.3 %, respectively. the highest dry matter loss in the cotyledons was observed at the end of the first 24 h period (fig. 1a). the dry matter loss in germinating seeds depends on the germination period. a negative correlation was reported between the dry matter loss and tab. 1: analysis of variance (anova) results for the examined parameters during germination and early seedling growth in sunflower cotyledons (f values). sources of variation dm (mg g-1) ac (%) tcc (mg 100 g-1) tc (mg 100 g-1) tss (mg g-1) germination period 764.5** 6.12** 617.2** 258.8* 564.9** cultivar 123.6** 22.7** 37.9** 2.8 180.4** germination period × cultivar 7.63** 2.15 6.6** 2.7* 61.2** coefficient of variation (%) 3.4 6.8 4.8 7.4 5.4 rs (mg g-1) pc (%) faa (%) oc (%) ffa (%) germination period 1478.8** 47.6** 225.7** 235.3** 16.5** cultivar 139.6** 157.1** 5.65* 4.8* 108.2** germination period × cultivar 164.1** 1.7 4.5** 5.1** 0.5 coefficient of variation (%) 4.7 5.0 10.5 1.6 10.7 *: p<0.05, **: p<0.01 dm (mg g-1): dry matter, ac (%): ash content, tcc (mg 100 g-1): total chlorophyll content, tc (mg 100 g-1): total carotenoid content, tss (mg g-1): total soluble sugar, rs (mg g-1): reduced sugar, pc (%): total protein content, faa (%): free amino acid content, oc (%): oil content, ffa (%): free fatty acid content. reserve mobilization during germination of sunflower seeds 219 seed germination period, due to the water uptake and excessive respiration process in the germinating seeds (tarasevičienė et al., 2009). the dry matter loss with water uptake throughout the germination period was reported in seeds of different species including linseed (wanasundara et al., 1999), cotton (joshi and doctor, 1975) and tobacco (koiwai and matsuzaki, 1990). the ash contents of the cultivars fluctuated during the germination period (fig. 1b). at 0 h, duet cl and tr 3080 had 4.99 % and 4.19 % ash contents, while their ash contents increased to 5.54 % and 4.88 %, respectively, at 24 h, thereafter decreased till 72 h but the ash content of the cultivars increased again during the last 48 h period of the study. the ash contents of the sunflower cultivars also increased during the germination period. increased ash content might be related to the increase in the relative proportion of minerals due to the loss of other seed storage reserves (borek et al., 2006). similar results were reported in amaranth (colmenares de ruiz and bressani, 1990) and sorghum seeds (elmaiki et al., 1999). however, in contrast to our findings, taraseviciene et al. (2009) reported that the ash content of broccoli seed remained constant during the germination. at the beginning of germination, duet cl had a total chlorophyll content of 7.4 mg 100 g-1 and tr 3080 had 13.9 mg 100 g-1. total chlorophyll contents of the cotyledons of both cultivars increased from 0 to 96 h, but decreased thereafter. at the end of the study period, the total chlorophyll contents were 187.9 and 199.2 mg 100 g-1 for tr3080 and duet cl, respectively (fig. 1c). the changes in total carotenoid contents of the cultivars were similar to the changes in total chlorophyll contents during the germination period. chlorophyll synthesis in plants ceases after the emergence of leaves and depletion of reserves (harris et al., 1986). total chlorophyll contents of both cultivars increased till 96 h but thereafter their chlorophyll contents decreased when the first true leaves of seedlings developed. as observed in the present study, bush and grunwald (1972) reported an increase during the first 6 days of the germination but thereafter a decrease in the chlorophyll content of tobacco seeds. tr 3080 and duet cl had total carotenoid contents of 4.2 μg 100 g-1 and 1.5 μg 100 g-1 respectively, at 0 h. similar to the changes in the chlorophyll content, the total carotenoid contents of cotyledons increased up to 96 h and reached to their highest levels (52.3 μg 100 g-1 for tr 3080 and 47.6 μg 100 g-1 for duet cl), but thereafter they started to decrease (fig. 1d). chlorophyll is sythesized in cotyledons with the start of germination in dicot seeds. carotenoids are essential components of the photosynthetic machinery in plants and play significant roles in during germination and the restriction of free radical induced membrane deterioration and seed ageing (calucci et al., 2004). the synthesis of carotenoids in seeds starts with the germination of seeds under light conditions (von lintig et al., 1997). similar results were also observed during the germination of tobacco seeds (bush and grunwald, 1972). the total soluble and reducing sugar contents of both cultivars showed similar trends during the study period (fig. 1e, f). the total soluble and reducing sugar contents of the cultivars were 7.3 mg g-1 and 1.8 mg g-1 for tr3080 and 11.0 mg g-1 and 1.5 mg g-1, for duet cl respectively, at 0 h. the total soluble and reducing sugar contents of the cultivars decreased during the first 24 h, and thereafter both total soluble and reducing sugars contents increased. the total soluble and reducing sugar contents of the cultivars at the 120 h was determined to be 28.6 mg g-1 and 6.4 mg g-1 for tr3080 and 44.4 mg g-1 and 10.4 mg g-1 for duet cl, respectively. the carbonhydrate source used throughout the germination period in sunflower seed is glucose (nascimento do et al., 1994). at the first 24 h of germination, total soluble and reducing sugars contents decreased as they are the first reserves to be mobilized during the early germination period. sugars generally used as the initial energy sources and supplied with the hydrolyses of complex carbonhydrates and the synthesis from triglycerides in oilseed crops at the begin ning of germination (tonguç et al., 2012). the free fatty acid contents of both cultivars increased slightly at the first 24 h (fig. 1j). after 24 h germination period, tss and rs contents significantly increased indicating an accelerated conversion of reserves to sugars along with the development of cotyledeons with chlorophyll synthesis for starting photosynthesis. the total protein content of tr 3080 (48.1 %) was higher than that of duet cl (40.9 %). the total protein contents of both cultivars decreased throughout the study period. the total protein content of tr 3080 showed a high rate of drop between 24 and 72 h, but the protein content of the duet cl demonstrated a high rate of decrease between 0 and 48 h. at the end of 120 h, the higher protein degregation was observed in duet cl (30.6 %) as compared to the tr 3080 which showed a 26.2 % reduction with respect to the total protein content at 0 h (fig. 1g). the free amino acid contents of both cultivars steadily increased during the study period. the free amino acid content was 0.59 % for tr 3080 at 0 h, and increased to 5.07 % at 120 h. the free amino acid content of duet cl increased from 0.28 % to 5.62 % during the same period. at the first 24 h, tr 3080 had higher free amino acid content as compared to duet cl, but the free amino acid content of duet cl was higher at the other time periods (fig. 1h). starch, hemicellulose, or tags in cotyledons are the alternative sugar sources for the growing axis during the germination, but amino acids are not used to produce sugars (balasaraswathi and sadasivam, 1997). free amino acid contents of the seeds were very low at the beginning of the study. storage proteins are proteolysed by different enzymes in different parts of the seed with imbibition and studied comprehensively in vetch and buckwheat seeds (bewley et al., 2013). the total protein content of cotyledons dramatically decreased at the 24 and 48 h, and thereafter continued to decrease at a slower pace during the study period. at the same time, the free amino acid contents of cultivars increased significantly indicating the start of storage protein mobilization to produce free amino acids for the syntheses of protein components of the growing cells. the amino acids used for protein synthesis and assembly are obtained from the reserves in the cotyledons (bewley et al., 2013). balasaraswathi and sadasivam (1997) reported that globulins and albumins were the storage proteins used in the initial period of germination in sunflower. the reductions in total protein contents during the germination and initial seedling development have been reported in different species including sterculia urens (satyanarayana et al., 2011), soybean (kim et al., 2011) and safflower (tonguç et al., 2012). the oil contents of both cultivars did not show a significant change during the 48 h germination period, but their contents started to decrease after 48 h. the highest reduction in oil content was observed after 72 h for both cultivars. the oil contents of the cultivars decreased from 52.4 % and 51.7 % to 40.2 % and 41.3 % for tr3080 and duet cl, respectively (fig. 1i). the free fatty acids contents of extracted oils from the developing cotyledons were 0.71 % for tr 3080 and 0.49 % for duet cl at the beginning of the study. their contents increased to 1.10 % and 0.80 %, at 72 h, respectively. then, the free fatty acids content of tr 3080 decreased to 0.84 %, at 120 h. the free fatty acid content of duet cl did not change between 72 and 96 h, but afterwards it decreased to 0.57 % (fig. 1j). the oil contents of the cultivars slightly increased at the beginning of the study but thereafter their oil contents decreased throughout the germination and seedling development. the most dramatic decrease in oil content was observed after 72 and 96 h. it was reported that oils were not used as an energy source till 1-3 days of germinating seeds of many plant species including safflower (tonguç et al., 2012), castor bean (muto and beevers, 1974), sinapis and crambe (yaniv et al., 1998) and were hydrolyzed later on during the germination 220 s. erbaş, m. tonguç, y. karakurt, a. şanli fig. 1: change in dry matter content (a), ash content (b), total chlorophyll content (c), total carotenoid content (d), total soluble sugar content (e), reduced sugar content (f), total protein content (g), free amino acid content (h), oil content (i) and free fatty acid content (j) of cultivars during germination and seedling growth. to ta l c hl or op hy ll co nt en t ( m g 10 0 g1 ) d ry m at te r c on te nt (% ) to ta l c ar ot en oi ds c on te nt (μ g 10 0 g1 ) reserve mobilization during germination of sunflower seeds 221 period. the lipid mobilization is regulated either by the factors influencing lipase activity directly or by the action of a thiole protease processing oleosin which enhances the organelle tag matrix accessibility to lipolytic actions (babazadeh et al., 2012). lipase activities of seeds in several plant species, including rapeseed, were not active at the beginning of germination period and their activities rapidly increased afterwards (rosales et al., 1998). the free fatty acid contents of cotyledons increased till 72 h, and then decreased. this may be due to the increase in the hydrolysis of oil, and then the conversion of free fatty acids to sucrose and their mobilization to the growing embriyonic axis. it has been reported that the biosynthe-iosynthesis of membrane lipids contributes significantly to the cell development and the rapid growth of growing embriyonic axis (bewley and black, 1983; munshi et al., 2007; satyanarayana et al., 2011). the changes in fatty acid compositions (%) of cultivars during the study period were given in tab. 2. the palmitic acid content slightly decreased at the end of the study period for both cultivars, however; the stearic acid contents of the cultivars increased during the same time period. the oleic acid contents of cultivars steadily decreased, while their linoleic and linolenic acid contents increased during the germination period. at 0 h, tr 3080 had an oleic acid content of 19.98 %, and duet cl had an oleic acid content of 89.95 %. after 120 h, their oleic acid contents decreased to 18.33 % and 85.19 %, respectively. linoleic acid contents increased from 67.06 % to 69.12 % for tr 3080 and from 1.77 % to 5.75 % for duet cl. (tab. 2). cultivars have a high oleic and linoleic acid contents, therefore palmitic and stearic acid variations were not significant in the study, which was reported by other researchers for tobacco (bush and grunwald, 1972), mustard and crambe (yaniv et al., 1998) and sunflower (rabiei et al., 2007) seeds with low palmitic and stearic acids. morever, moya et al., (2000) reported that the changes in these fatty acids in mutant sunflower seeds with high palmitic and stearic acid contents were not significant for the study period. however, these fatty acids in cotton and soybean seeds were started to be utilized from the beginning of the germination and their contents decreased throughout the germination period (joshi and doctor, 1975; joshi et al., 1973). during germination, the decrease of the oleic acid content and increase of the linoleic acid content may depend on the increased desaturase activity. it is known that oleic acid content decreased and linoleic acid content increased in seeds with high oleic or linoleic acid contents during germination (bush and grunwald, 1972; joshi et al., 1973; rosales et al., 1998; moya et al., 2000; rabiei et al., 2007). however, the changes in these fatty acids in mustard and crambe with low palmitic and stearic acid, and rapeseed with erusic acid were not significant (yaniy et al., 1998). linolenic acid content of cultivars slightly increased suggesting either the desaturation of fatty acids or the increase in relative amount of linolenic acid due to the breakdown of other fatty acids. the dry matter content continually decreased during germination and early seedling development of sunflower cultivars, while the photosynthetic activity increased with the development of the cotyledons and leaves up to 96 h. oil is utilized only at the later stages of seedling development. the breakdown of oil in the cotyledons coincided with an increase in total soluble and reducing sugar contents. the changes in the oil composition are marked by the increases of unsaturated linoleic and linolenic acids. a significant mobilization was also observed in total proteins which showed a decrease in the cotyledons associated with an increase in the free amino acid content. the results suggest that lipids, proteins, carbohydrates and color components contribute significantly to the seed germination and early seedling growth and development in studied sunflower cultivars. acknowledgements the authors are very grateful to dr. yalçın kaya (trakya university, engineering faculty, genetic and bioengineering department) and may agro seed corporation for providing sunflower seeds used in the study. references aoac, 1990: official methods of analysis. 15th ed. section. association of official analytical chemists, washington d.c. babazadeh, n., poursaadat, m., sadeghipour, h.r., hossein zadeh colagar, a., 2012: oil body mobilization in sunflower seedlings is potentially regulated by thioredoxin h. plant physiol. biochem. 57, 134142. 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fatty acids cultivars 0 h 24 h 48 h 72 h 96 h 120 h palmitic acid tr 3080 5.59 5.27 5.19 5.59 5.33 5.40 (c16:0) duet cl 3.25 3.24 3.22 3.24 3.11 3.12 stearic acid tr 3080 5.77 5.44 5.36 5.67 5.34 5.83 (c18:0) duet cl 2.78 2.85 2.73 2.76 2.80 2.86 oleic acid tr 3080 19.93 19.88 19.30 19.17 19.15 18.33 (c18:1) duet cl 89.95 89.83 89.50 88.48 86.36 85.19 linoleic acid tr 3080 67.06 67.77 67.83 68.11 68.13 69.12 (c18:2) duet cl 1.77 1.85 2.29 3.00 4.68 5.75 linolenic acid tr 3080 0.34 0.34 0.35 0.38 0.40 0.41 (c18:3) duet cl 0.22 0.22 0.23 0.26 0.59 0.66 222 s. erbaş, m. tonguç, y. karakurt, a. şanli chem. 52, 4274-4281. colmenares de ruiz, a.s., bressani, r., 1990: effect of germination on the chemical composition and nutritive value of amaranth grain. cereal chem. 67, 519-522. dubois, m., gilles, k.a., hamilton, j.k., 1956: colorimetric method for determination of sugars and related substances. anal. chem. 28, 350356. elmaki, h.b., babiker, e.e., tinay, a.h.e., 1999: changes in chemical 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y., 2012: changes in seed reserve composition during germination and initial seedling development of safflower (carthamus tinctorius l.). turk. j. biol. 36, 107-112. von lintig, j., welsch, r., bonk, m., giuliano, g., batschauer, a., kleinig, h., 1997: light-dependent regulation of carotenoid biosyn thesis occurs at the level of phytoene synthase expression and is mediated by phytochrome in sinapis alba and arabidopsis thaliana seedlings. plant j. 12, 625-634. wanasundara, p.k.j.p.d., wanasundara, u.n., shahidi, f., 1999: changes in flax (linum usitatissimum l.) seed lipids during germination. j. am. oil chem. soc. 76, 41-48. weiss, e.a., 2000: oilseed crops, 2nd edition, blackwell sci. ltd., victoria, australia. wilson, k.a., 2006: mobilization of storageproteins in dicots. in: black, m., bewley, j.d., halmer, p. (eds.), encyclopedia of seeds. science, technology and uses, 672-674. cabi, wallingford. yaniv, z., shabelsky, e., schafferman, d., granot, i., kipnis, t., 1998: oil and fatty acid changes in sinapis and crambe seeds during germination and early development. ind. crops prod. 9, 1-8. address of the authors: sabri erbaş, arif şanli, suleyman demirel university, faculty of agriculture, department of field crops, isparta, turkey muhammet tonguç, yaşar karakurt, suleyman demirel university, faculty of agriculture, department of agricultural biotechnology, isparta, turkey © the author(s) 2016. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 91, 120 125 (2018), doi:10.5073/jabfq.2018.091.017 1university of craiova, faculty of horticulture, department of horticulture and food science, romania 2 university of craiova faculty of agriculture, department of agricultural and forestry technologies, romania 3 university of craiova, faculty of sciences, department of chemistry, romania 4 university of agronomic sciences and veterinary medicine of bucharest, faculty of horticulture, department of bioengineering of horticultural systems, romania analysis of nutritional composition and antioxidant activity of sweet potato (ipomoea batatas l.) leaf and petiole maria dinu1, rodica soare2*, cristina băbeanu3, gherghița hoza4 (submitted: february 9, 2018; accepted: march 20, 2018) * corresponding author summary two cultivars of sweet potatoes, pumpkin and chestnut, were plan ted in the didactic field of the faculty of horticulture, craiova, and biochemical determinations were carried out from the blades and petioles of the leaves. this study highlights the antioxidant properties of these species, which are not well known in romania, to encourage consumption of the vegetative parts of the plant, as well as use of the leaves in the future to develop natural antioxidants. the chlorophyll and carotene content in the blade and petiole were determined, highlighting the large amounts of petiole, depending on the cultivar. catalase (cat), peroxidase (pox), and vitamin c content, as well as total dry matter (tdm), reducing sugars, phenolic compounds, and antioxidant activity, were also measured. the petioles exhibited high antioxidant activity, with 62.186 μmol te/g fw in pumpkin and 95.168 μmol te/g fw in chestnut. the cultivars also exhibited differences at the blade level, with high values recorded for the chestnut cultivar. positive correlations between total polyphenols and reducing sugars (r = 0.89), antioxidant activity and reducing sugars (r = 0.48), and antioxidant activity and phenolic compounds (r = 0.53) were found. this study demonstrates that sweet potato leaves are a potential, inexpensive and beneficial source of natural antioxidants. key words: ipomoea batatas l., catalase, peroxidase, polyphenols introduction regular consumption of vegetables and fruits reduces the risk of cardiovascular diseases (otaki et al., 2009). in vegetables and fruits, the main constituents with antioxidant properties are polyphenols, which have a beneficial effect in contributing to the prevention of these diseases. several studies have shown that polyphenols from various foods, such as red wine, green tea, and chocolate, can reduce bad cholesterol levels (kalkan et al., 2004). antioxidants reduce bad cholesterol levels and stimulate the production of good cholesterol. thus, vitamin c and flavonoids such as beta carotene or lycopene, which are found in vegetables and fruits, play an especially important role. different varieties of sweet potatoes are consumed worldwide. in japan, the tuberous roots are commonly eaten and used to make spirits. a brazilian variety called “simon no. 1” is popularly known as a drug (nagai et al., 2011). they are also an important source of starch, sugar, and alcohol. the sweet potato is one of the top 15 agricultural crops and can be used as raw material for many industrialized products, given its composition and agricultural potential. in addition, the young leaves can be consumed (antonio et al., 2011). the leaves of sweet potatoes are consumed as vegetables in tropical areas, especially in southeast asia (villareal et al., 1982). because the plants grow quickly in moist conditions, these leaves can be harvested several times a year. various research studies have recently been carried out to improve the production of leaves and petioles in the varieties known as “elegant summer” and “suioh” (nagai et al., 2011). several studies have demonstrated that sweet potato leaves inhibit hiv replication, mutagenicity, diabetes, and cancer cell proliferation (yoshimoto et al., 2002). in romania, as in other european countries, consumption of sweet potato leaves is not common because this species is cultivated in small spaces. according to šlosár et al. (2016), the main european producers of sweet potatoes are spain and italy. because they can be harvested several times a year, the annual yield of sweet potatoes is much higher than that of many other vegetables grown for the leaves. they can be prepared alone or mixed with other species, consumed fresh or dried, and stored for later use. their nutritional content differs according to the environmental, harvest, and storage conditions, as well as the cultivar. this study aims to highlight the nutrient-rich content of the leaf blade and petiole in the pumpkin and chestnut sweet potato cultivars, as well as to make the consumer aware of their beneficial role in human health. due to their diverse biochemical composition and tolerance to diseases and pests (the plants do not require much care), consumption should certainly be encouraged for people of all ages. material and methods the experiment was conducted in the didactic field of the faculty of horticulture, university of craiova, romania (latitude 44°19’ north latitude and longitude 23°48’ east), on reddish-brown preluvosoil with a ph of 7.5 and a humus content of 1.9%. the biological material consisted of two cultivars of sweet potato originating in south korea: pumpkin and chestnut. the crop was formed from cuttings obtained by forcing tuberous roots in a hot greenhouse. the cuttings were then root-planted by turn on may 25, 2016, and may 15, 2017, in 30 cm high beds. the distance between rows was 70 cm, and the distance between plants was 40 cm, resulting in 33.333 pl/ha. the plants were placed in three randomized blocks, with 30 plants per block. it should be noted that, in addition to basic fertilization with 20 t/ha of compost, fertilization was performed in different phases. to con trol the weeds, the field was hoed at the beginning of the vege tation phase, after which the vegetative growth of the sweet potato covered the soil between the plants, and no further intervention was needed. irrigation was carried out in 10 watering sessions, with 300350 m3/ha. from each cultivar, 10 leaf petioles were harvested in three repetitions for chemical analysis. composition and antioxidant activity of sweet potato 121 analytical methods several analyses were carried out: chlorophyll a and b content, carotenes, enzymatic activity (catalase and peroxidase), vitamin c content, total dry matter (tdm), reducing sugars, polyphenols, and antioxidant activity. determination of total chlorophylls and carotenes the weighed samples, having been placed separately in 95% acetone (50 ml per gram), were homogenized with braun mr 404 plus for 1 minute. the homogenate was filtered and centrifuged using a hettich universal 320/320r centrifuge at 2500 rpm for 10 minutes. the supernatant was separated, and the absorbencies at 400-700 nm were read on a cary 50 spectrophotometer. chlorophyll a showed maximum absorbance at 662 nm, chlorophyll b at 645 nm, and total carotene at 470 nm. the amounts of these pigments were calculated according to the following formulas (nagata and yamashita, 1992): ca = 11.75 a662 2.350 a645; cb = 18.61 a645 3.960 a662 cx + c = 1000 a470 2.270 ca 81.4 cb/227 ca = chlorophyll a; cb = chlorophyll b; cx + c = total carotene determination of vitamin c content a sample consisting of 5-10 g of the blades and petioles of sweet potato leaves, previously ground with quartz sand, was placed in a 100 ml 2% hydrochloric acid solution. it was stirred, and after sedimenting, it was filtered into a dry glass. a 10-ml aliquot was added to a glass berzelius beaker containing 30 ml of distilled water, 5 ml of 1% potassium iodate, and a 1-ml solution of starch. it was then titrated with potassium iodate n/250 and stirred until it turned bluish, using the method described in elgailani et al. (2017). the ascorbic acid concentration was calculated using the following equation: vitamin c mg % = 352. n.f / g, where: n = ml used for titration; f = the factor of the potassium iodate n/250; g = sample weight in grams. determination of tdm the tdm was determined by oven drying at 105 °c to constant mass (iso 751: 2000). enzyme assays fresh tissue was homogenated with 0.1 m phosphate buffer, ph 7.0 (1:20 w/v), containing 0.1 mm edta. homogenates were centrifuged for 15 minutes at 10,000 rpm, and the supernatants were used for enzyme assay. total soluble peroxidase (pox) activity was assayed by measuring the increase in a436 due to guaiacol oxidation, and activity was expressed as δa/min/g fresh weight (fw). catalase (cat) activity was assayed through the colorimetric method at 570 nm, and the results were expressed as mmol h2o2/min/g at 25 °c (băbeanu et al., 2010). reducing sugars using its the method described by miller (1959), reducing sugars were extracted in distilled water (1:50 w/v) for 60 minutes at 60 °c. a colorimetric assay was then performed with 3,5-dinitrosalicylic acid, using glucose as the standard. absorbance was recorded at 540 nm using a thermo scientific evolution 600 uv-vis spectro photometer with vision pro software. total phenolic content the amount of total phenolic compounds in the leaf blade and petiole of sweet potato extract was determined colorimetrically with folin-ciocalteu reagent using the method described by singleton and rossi (1965), with some modifications. briefly, each probe was prepared like this: to 0.1 ml extract (1 g fw + 10 ml 60% metha nol) add 0.9 ml ultrapure water and 5 ml reactive folin-ciocalteu (diluted 1:10 with ultrapure water). after two minutes, 4 ml of a 7.5% sodium carbonate solution were added, and the samples were kept in the incubator at room temperature for two hours. after that, the absorbance of probes was measured at 765 nm by using an evolution 600 double beam scanning, uv-visible spectrophotometer, pc controlled, using vision pro software. a standard curve was prepared by using 50, 100, 150, 200, and 250 mg/l solutions of gallic acid in methanol and water (60:40 v/v). gallic acid was used as the reference standard, and the results (total phenolic content) were expressed as gallic acid equivalents (gae) and as mg/g fw. antioxidant activity antioxidant activity was measured in the ethanolic extract using the dpph (2,2-diphenyl-1-picrylhydrazyl) assay. ethanol (merck, germany), dpph (2,2-diphenyl-1-picrylhydrazyl) (sigma-aldrich, germany), and trolox (6-hydroxi-2,3,7,8-tetramethylchroman-2carboxylic acid) (merck, germany) were employed. the samples were extracted according to the same protocol described for total phenolic content. the free-radical scavenging ability of the extracts against the dpph free radical was evaluated as described by oliveira et al. (2008), with some modifications. each ethanol extract of sweet potato leaf and petiole (50 ml) was mixed with 3 ml of a 0.004% (v/v) dpph methanolic solution. the mixture was incubated for 30 minutes at room temperature in the dark, and the absorbance was measured at 517 nm on a varian cary 50 uv-vis spectrophotometer. the dpph free-radical scavenging ability was calculated in reference to trolox, which was used as a standard reference to convert the inhibition capacity of each extract solution to the mmol trolox equivalent (te) antioxidant activity/l. the radical was freshly prepared and protected from light. a blank control of methanol/water was used in each assay. all assays were conducted in triplicate, and results were expressed in mmol trolox kg-1 dw. statistical analysis the statistical significance of differences between variants was determined with variance analysis using anova and the statgraphics centurion xvi program (statpoint technologies, warrenton, va, usa) and by calculating the limit differences, lsd ≤ 0.05% (lsd = least significant difference). in addition, correlation coefficients (r) between chemical compounds analyzed in sweet potato leaves were calculated. results and discussion the variable climate in southwest romania, where the summers are dry and warm, offers favourable conditions for the cultivation of sweet potatoes. the sandy or sandy loam soil allows good ventilation for the roots, which need oxygen and do not tolerate excessive humidity (dinu et al., 2015). during the sweet potato’s vegetation period, the average temperature is 20.33 °c, and the average precipitation is 269.38 (diaconu et al., 2016). in romania, sweet potatoes are beginning to be cultivated, and they could be a good addition to the range of less widely cultivated species. depending on the cultivar and the environmental conditions under which the crop was planted, the nutrient content of sweet potato leaves is comparable to that of spinach. 122 m. dinu, r. soare, c. băbeanu, g. hoza sweet potato leaves are an excellent source of chlorophylls and carotenes. as tab. 1 indicates, fresh sweet potato leaves of the pumpkin cultivar had a chlorophyll a content of 8.22 mg/100 g fw in the blade and 10.8 mg/100 g fw in the petiole. in the chestnut cultivar, the chlorophyll content was 4.66 mg/100 g fw in the blade and 6.78 mg/100 g fw in the petiole. the chlorophyll content in the petiole was significantly higher than in the blade. differences were also found between cultivars. tab. 1: chlorophyll and total carotene content in sweet potato blade and petiole. cultivar specification chlorophyll a chlorophyll b carotene (mg/100 g fw) (mg/100 g fw) (mg/100 g fw) pumpkin blade 8.22b 8.32b 25.05a petiole 10.8a 10.35a 16.39d chestnut blade 4.66d 6.82d 23.80b petiole 6.78c 7.73c 16.60c lsd ≤ 0.5 0.48 0.16 0.18 note: different letters within the same row indicate significant differences (p ≤ 0.05) between cultivars. similar differences in chlorophyll b content exist between the blade and the petiole, as well as between cultivars. in pumpkin, the chlorophyll b concentration was 8.32 mg/100 g fw in the blade and 10.35 mg/100 g fw in the petiole. the chestnut cultivar recorded 6.82 mg/100 g fw of chlorophyll b in the blade and 7.73 mg/100 g fw in the petiole. not much is understood about chlorophyll’s effect on health. it is known only that it is a primary photosynthetic pigment that de termines the green colour of plants. some reports suggest that it protects organisms against some cancers linked to mutant dna by preventing proliferation of the dna. because these chlorophylls are found at different concentrations in many edible plants, they can be associated with certain protective effects observed in diets rich in green vegetables (fahey et al., 2005). furthermore, chlorophyll is present in those plants at much higher concentrations than other „phytochemicals”, similar with determinations reported by zikalala (2014) on baby spinach. the carotene content in the blade varied from 25.05 g/100 g fw in the pumpkin cultivar to 23.8 g/100 g fw in the chestnut cultivar. the content in the petiole was 16.39 g/100 g fw in pumpkin and 16.6 g/100 g fw in chestnut. the high carotene content in the blade helps to capture the luminous energy that is transferred to the plant during photosynthesis. additionally, the carotene from the sweet potato leaves supplies a variety of nutrients that cannot be supplied by other vegetables. the amounts of carotenes found in this study were significantly higher than those found by odongo et al. (2015), who reported a beta carotene content of 0.53 ± 0.03 mg/100 g dry weight for sweet potato leaves, spk 004 variety. these results demonstrate that the carotene concentrations in the blade and petiole of sweet potato leaves are higher than in the sweet potato’s tuberous root. ellong et al. (2014), in a study of eight sweet potato cultivars, observed that the doses of carotenes in sweet potato roots had values ranging from 0.50 to 1.47 mg/100 g. this study also determined the enzymatic activity of catalase (cat) and peroxidase (pox). this enzymatic antioxidant system plays a key role in the scavenging and regulation of the reactive oxygen species (ros) generated in aerobic metabolic processes. the enzymatic leaf activity caused by cat and pox had different values between cultivars and is presented in tab. 2. in the pumpkin tab. 2: enzymatic activity and vitamin c content of sweet potato leaves. no. cultivar cat1 pox2 vitamin c (mmol h2o2/ (δa/min/g fw) (mg/100 g fw) g fw/min) 1. pumpkin 3.94a 1680ns 5.96a 2. chestnut 1.15b 1665ns 3.56b lsd ≤ 0.5 0.17 24.81 0.20 note: different letters within the same row indicate significant differences (p ≤ 0.05) between cultivars. 1cat = catalase, 2pox = peroxidase. cultivar, activity due to cat was 3.94 mmol h2o2/g fw/min, while activity in the chestnut cultivar was low at only 1.15 mmol h2o2/g fw/min. since both cultivars were obtained under the same crop conditions and from the same area, this difference can be attributed to the cultivar. cat catalysis is the reduction of h2o2 generated during photo synthesis to h2o and molecular oxygen. cat and pox have different affinities for ros and appear to have different cellular roles in h2o2 scavenging. cat does not need a reductant to scavenge h2o2, making it reducing power-free, whereas pox needs a reductant. cat has a lower affinity for h2o2 (in the mm range) than pox (in the μm range) (cruz de carvahlo, 2008). enzymatic activity caused by pox did not vary greatly from one cultivar to another, measuring1.680 δa/ min/g fw in pumpkin and 1.665 δa/min/g fw in chestnut. the measurements of vitamin c content in sweet potato leaves shows levels of 5.96 mg/100 g fw in pumpkin and 3.56 mg/100 g fw in chestnut. the difference between cultivars may be because the pumpkin cultivar’s leaves are more intensely green than those of the chestnut cultivar. research has demonstrated that spinach leaves with an intensely green color have higher vitamin c concentrations than light-colored spinach leaves (edenharder et al., 2001). the zikalala study (2014) indicated that vitamin c concentration was 4.46 mg/g in spinach, suggesting that sweet potato leaves can replace spinach in various preparations. according to ishiguro et al. (2004), the average vitamin c content of sweet potato leaves was 7.2 mg/ 100 g fw, higher than the values recorded in this study. the total dry matter for the blade, presented in tab. 3, ranged from 16.0% for pumpkin to 18.5% for chestnut; for the petiole, it ranged from 12.3% for pumpkin to 16.0% for chestnut. high values were observed in both cultivars. in comparison with other vegetables, this study found that the tdm content in sweet potato leaves was higher than the 10.0% to 12.8% values in some vegetable species reported by asaolu (2012). this indicates that sweet potato leaves contain more dry matter and, therefore, more nutrients, and should be considered for human consumption. reducing sugars in the blade ranged from 23.40% in the pumpkin cultivar to 25.68% in chestnut. in the petiole, the sugars were higher than those in the blade, at 43.07% for pumpkin and 51.91% for chestnut. the differences between the values in the blade and the petiole are significant both within the same cultivar and between the two cultivars. the sweet potato is high in reducing sugars due to the high levels of sugars that appear in the leaves during photosynthesis. polyphenol content. sweet potato leaves are a source of polyphenolic compounds. these biologically active compounds have multiple actions, including antioxidation, antimutagenicity, antiinflammation, and anticarcinogenesis. sweet potato leaves contain more poly phenols than any other commercial vegetables, such as spinach (islam, 2007). polyphenol content in the blade was 2.598 mg/g fw for the pumpkin cultivar and 2.250 mg/g fw for the chestnut cultivar. there were composition and antioxidant activity of sweet potato 123 no significant differences between cultivars. this study found higher polyphenol levels than those obtained by ghasemzadeh et al. (2012) on the leaves of six cultivars in malaysia. the quantities in the petiole were double those accumulated in the blade, but again, no significant differences were observed between cultivars. truong et al. (2007), in a study of three sweet potato cultivars, claimed that the phenols from the leaves of this species were approximately 8, 16, and 18 times higher, respectively, than those found in the root coat, in the entire root, and in the root pulp. they also reported that the phenolic-compound content ranged between 1,123.6 and 1,298.1 mg chlorogenic acid/100 g fw; equivalent values expressed in gallic acid are 587 mg gae/100 g fw and 623 mg gae/100 g fw, respectively. the total polyphenolic content in the leaves of pumpkin and chestnut was 2.5 mg/g fw and 2.2 mg/g fw. according to islam et al. (2002), who evaluated 1,389 genotypes for total phenol content, these varieties can be classified as average polyphenolic accumulators, which have a content of less than 5 g. those with a content greater than 9 g are high content accumulators (cha equivalent per 100 g dry matter). the sweet potato leaves used in this study (as well as the petioles) had a higher total phenol content than the iranian olive pulp studied by hajimahmoodi et al., (2008). they argued that this pulp is a source of natural antioxidants with the highest level of total polyphenols, approximately 2.997 ± 0.361 g gae/100 g. however, the chestnut petiole had a polyphenol content of 4.736 mg /g fw, and the pumpkin had a content of 5.384 mg/g fw. the antioxidant activity of the two sweet potato cultivars varied greatly between the blade and petiole within the same cultivar, as well as between cultivars. the highest values were recorded in the leaf petiole, with 62.186 μmol te/g fw in pumpkin and 95.168 μmol te/g fw in chestnut, while the blade content was 42.290 μmol te/g fw in pumpkin and 62.688 μmol te/g fw in chestnut. it is notable that the petiole developed greater antioxidant activity than the blade. this result is also supported by jeng et al. (2015), who found that the blade had lower antioxidant activity than the petiole of sweet potato. the values for blade antioxidant activity recorded in this study are higher than those obtained by truong et al. (2007) and ghasemzadeh et al. (2012). this activity in sweet potato leaves is equivalent to that recorded by zambrano-moreno et al. (2015) in eggplants, in both conventional and organic culture. the high level of capturing activity in sweet potato leaves was also demonstrated in the study by yang et al. (2005), in which ipomea batatas had the highest dpph radical-capture activity of 23 vegetable species consumed in taiwan. the high antioxidant activity correlates well with the polyphenol content. in both the pumpkin and chestnut cultivars, polyphenol content was greater in the leaf petiole than in the blade. this higher content correlates positively with intense antioxidant activity, indicating intense activity in this part of the leaf. several other studies have also demonstrated the positive relationship between high levels of phenols (e.g., phenolic acid) and antioxidant activity. bertoncelj et al. (2007) established that in seven types of honey originating in slovenia, increased levels of phenolic com pounds were correlated with high antioxidant activity. céspedes et al. (2008), in a study of the mature fruits of aristotelia chilensis (mol) stuntz (elaeocarpaceae), found that high antioxidant activity was strongly correlated with total polyphenol content, and thus the fruits could be used as antioxidant, cardioprotective, and nutraceu tical sources. garcia-alonso et al. (2004) analyzed 28 different species of fruits and found no significant correlation between anti oxidant activity and flavonol content. the authors therefore argue that the antioxidant activity of the analyzed fruits cannot be attributed only to their flavonol content; it must also involve the actions of various antioxidant compounds present in fruits, as well as possible synergistic and antagonistic effects that are still unknown. tab. 4 presents the correlations between tdm, reducing sugars, total polyphenols, and antioxidant activity. the results reveal a positive correlation between total polyphenols and reducing sugars (r = 0.89), between reducing sugars and antioxidant activity (r = 0.84), and between antioxidant activity and total polyphenols (r = 0.53). according to sun et al. (2014), the correlation coefficient between antioxidant activity and total polyphenol content was the highest (r = 0.76032), and ghssemzadeh et al. (2012) found that antioxidant activity was strongly correlated with total polyphenol content (r2 = 0.827). tab. 3: biochemical composition of sweet potato leaves. no. specification tdm reducing sugars total polyphenols antioxidant activity (%) (%) (mg/g fw) (μmol te/g fw) (dpph) 1. blade 16.0b 23.80b 2.598c 42.290d petiole 12.3c 43.07a 5.384a 62.186c 2. blade 18.5a 25.68b 2.250d 62.688b petiole 6.0b 51.91a 4.736b 95.168a lsd ≤ 0.5 0.92 16.09 0.17 0.46 note: different letters within the same row indicate significant differences (p ≤ 0.05) between cultivars. tab. 4: correlation coefficients (r) between chemical compounds analyzed in sweet potato leaves. specification reducing total antioxidant sugars (%) polyphenols activity (mg/g fw) (μmol te/g fw) (dpph) tdm 0.49 0.82 0.03 (%) reducing sugars 0.89** 0.84** (%) total polyphenols 0.53 (mg/g fw) note: *p 5% = 0.58, **p 1% = 0.71 conclusions this study demonstrates the importance of sweet potato leaves (both the blade and petiole) as a natural source of antioxidants. the two 124 m. dinu, r. soare, c. băbeanu, g. hoza sweet potato cultivars used in this study had the highest carotene content in the blades, with 25.05 mg/100 g fw in pumpkin and 23.80 mg/100 g fw in chestnut. the capacity to scavenge free radicals is due to the presence of total polyphenols, which were present in significant amounts in the leaf petiole. these polyphenols also produced high antioxidant activity in pumpkin at 62.186 μmol te/g fw, and an even higher activity level in chestnut at 95.168 μmol te/g fw. the significant correlations between carotenes and antioxidant activity and between total polyphenols and antioxidant activity suggest that the antioxidant activity of blade and leaf petiole can be attributed mainly to phenolic compounds. because sweet potato leaves could be used in the food, medicine, and agriculture industries as a source of natural antioxidants, the research effort to confirm the antioxidant activities of ipomoea batatas 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di-, and tricaffeoylquinic acid derivatives isolated from sweet potato (ipomoea batatas l.) leaf. biosci. biotechnol. biochem. 66, 2336-2341. doi: 10.1271/bbb.66.2336 address of the authors: dinu maria, faculty of horticulture, university of craiova, a.i.cuza street, no. 13, craiova, 200585, romania e-mail: dinumariana@hotmail.com soare rodica, faculty of agriculture, university of craiova, libertatii street, no. 19, craiova, 200333, romania e-mail: soarerodi@yahoo.com (*corresponding autor) băbeanu cristina, faculty of sciences, university of craiova, calea bucu rești, no. 107, craiova, 200478, romania e-mail: cbabeanu@yahoo.com hoza gheorghița, faculty of horticulture, university of agronomic sciences and veterinary medicine of bucharest, 59 mărăști street, 011464 bucharest, romania e-mail: hozagh@yahoo.com © the author(s) 2018. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 89, 113 125 (2016), doi:10.5073/jabfq.2016.089.014 1icar-national institute of abiotic stress management, baramati, india 2 department of botany, aligarh muslim university, aligarh, u.p., india 3 department of biochemistry and physiology of plants, university of bielefeld, bielefeld, germany 4 department of plant breeding and genetics, bihar agricultural university, bihar, india can plant bio-regulators minimize crop productivity losses caused by drought, salinity and heat stress? an integrated review pasala ratnakumar1*, md iqbal raja khan2, paramjit singh minhas1, mahummad ansar farooq3, rafat sultana4, tasir s. per2, pallavi p. deokate1, nafees ahmad khan2, yogeshwar singh1, jagadish rane1 (received september 23, 2015) * corresponding author summary plant bio-regulators (pbrs), are biochemical compounds stimulates plant growth and productivity when applied, even in small quantities at appropriate plant growth stages. these are being extensively used in agriculture to enhance the productivity particularly in horticultural crops but are not as prevalent in field crops. their central role in plant growth and development is through nutrient allocation and sourcesink transitions while most of the pbrs stimulate redox signaling under abiotic stress conditions. since climate change and degrading natural resources are projected to amplify the stresses, particularly soil moisture deficit, high temperature and soil salinity, pbrs are likely to play a crucial role in plant growth regulation. however, the utility of pbrs to enhance crop productivity under stresses induced by abiotic factors needs critical evaluation. research efforts so far have centered on the crop and agro-ecosystem specificity, optimal doses and schedule of their application for optimizing crop yields under stress conditions. these efforts are being complemented by investigations on genes and gene regulatory network at molecular level to tailor crop plants for climate resilience. in addition to complying with regulation governing use of bio-chemicals, issues related to crop yield losses in case of excessive doses as well as their impacts on soil health are being addressed. in this review, prospects and pathways of pbrs are thrashed out as an emerging stress alleviating technology for crop production in harsh agro-ecosystems, specifically those featured by drought, heat and salinity stress. introduction understanding the adaptive responses of plants is of paramount importance particularly when grown under harsh environments. a series of experiments conducted in the past have clearly established that plant growth regulators and hormones play a vital role in determining growth, development and productivity of crops. this fundamental knowledge served as basis for a number of biochemi cal as plant growth promoters in crop production. this tends to be the integral part of modern agriculture, which is often affected by adverse environmental factors such as drought, high temperature and salt stress. in addition, recent surge in low external input sustainable agriculture (leisa) demands enhanced intervention of modern plant biology that can integrate knowledge of plant responses to environmental factors at molecular, cellular, whole plant as well as cropping system levels. through leisa, the plant stress tolerance can be improved with an exogenous use of stress alleviating chemicals (wahid and shabbir, 2005; wahid et al., 2007b; farooq et al., 2009). plant bioregulators (pbrs) are powerful tools for maximizing yield and quality, and increasing net income to farmers. pbrs have been used from many years to alter the behavior of agricultural and horticultural crops for the economic benefit of the grower. control of vegetative vigor, stimulation of flowering, regulation of crop load, reduction of fruit drop, and delay or stimulation of fruit maturity and ripening are best examples of regulation with the exogenous applications. novel pbrs with possible benefits for fruit growers are continually being made available by industries. in addition, research is in progress to find new uses for bioregulator products that have been made available for specific uses. every aspect of plant growth and development is controlled by plant hormones and these serve as key integrators of exogenous (environmental) and endogenous (developmental) cues. the classes of phytohormones are auxins, gibberellins (ga), cytokinins, ethylene, abscisic acid (aba), brassinosteroids (bs), salicylic acid (sa), and jasmonates, with strigolactones representing a relatively new addition (stamm et al., 2011). stress may induce common responses such as enhancement of plant hormones. for instance, wounding can induce increased production of ethylene, auxin, and aba. since many kinds of stresses including water, salt, and temperatures, induce aba synthesis since aba is considered as a plant stress hormone. it regulates several important aspects of plant growth and development. recent studies have demonstrated a pivotal role for aba in modulation at the gene level of adaptive responses for plants in adverse environmental conditions. aba is also involved in several other physiological processes such as stomatal closure, embryo morphogenesis, development of seeds, and synthesis of storage proteins and lipids, germination, leaf senescence, and defense against pathogens. nevertheless, aba acts as a mediator in controlling adaptive plant responses to environmental stresses. in several instances, it has been implicated in signal transduction at the single-cell level. other than plant hormones, exogenous pbrs such as inorganic, organic chemicals and booster’s application will also have strong impact on plant adaptation to abiotic stress either independently or synergistically with one another (srivastava et al., 2016). many pbrs influence with the signaling pathways of one another thereby promoting plant tolerance to abiotic stress. various researchers established the interlinking of redox signaling pathway among different pbrs to accomplish the goal of tolerance (srivastava et al., 2016). the coordination of the responses triggered by the multiple stimuli is controlled by a network of intricate signal transduction pathways. this network has many signaling components, and a small number of highly interconnected components which are central for the functioning of the network (hetherington and woodward, 2003). for instance, in many studies nitric oxide (no) has been found to mediate the action of stomatal closure of aba (garcia-mata and lamattina, 2007). pbrs also promote the uptake and metabolism of nutrient elements as well. among the stress alleviating compounds, thiourea is one of the important molecule with two functional groups; ‘thiol’ is important to oxidative stress response and ‘imino’ partly fulfills the nitrogen requirement. apart from thiol compounds other chemi114 p. ratnakumar, m.i. raja khan, p.s. minhas, m.a. farooq, r. sultana, t.s. per, p.p. deokate, n.a. khan, y. singh, j. rane cals including plant growth regulators − 6-benzyladenine (ba), prohexadione-calcium (pro-ca), n-2-chloro-4-pyridinyl-n-phenylurea (cppu), 2,4-dichlorophenoxyacetic acid (2,4-d), 3,5,6-trichloro-2pyridyloxyacetic acid (3,5,6-tpa), and amino ethoxy vinyl glycine (avg), dithiothreitol (dtt), potassium nitrate (kno3), thiourea (tu), salicylic acid (sa), silicon (si) products were used, to a small extent, to screen for their ability to increase agricultural yield without compromising quality. stressful environments interrupt the balance between generation and utilization of reactive oxygen species (ros) leading to toxicity by enhancing production of ros such as superoxide radicals (o2−), hydrogen peroxide (h2o2), hydroxyl radicals (oh−) etc. in plants thereby creating a state of oxidative stress in them (panda et al., 2003a, b). this increased ros level in plants cause oxidative damage to bio-molecules such as lipids, proteins and nucleic acids, thus altering the redox homeostasis (smirnoff, 1993; gille and singler, 1995; srivastava et al., 2016). when applied exogenously at suitable concentrations pbrs enhance the efficiency of antioxidant system, upregulates osmolytes and enhance the expression of stress responsive gens in plants (srivastava et al., 2016; knorzer et al., 1999) (fig. 1) and we also proposed pbrs for sustainable agriculture by integrating redox signaling as a possible unifying mechanism (srivastava et al., 2016). in the current scenario, various kinds of pbrs such as tu, sa, si, no, h2o2, hydrogen rich water, bs and polyamines (pa) etc., have been tested for enhancing the plant stress tolerance as well as the crop yield (jisha et al., 2013; srivastava et al., 2016). most of these pbrs treatments are applied either for seed priming or as foliar spray. seed priming is a pre-sowing treatment that partially hydrates seeds without allowing the radicle emergence. the seed priming may be induced alone or in combination with the foliar application of pbrs at the time or early flowering or grain filling stage. the mechanism of seed priming mediated action is not well understood, but it may act in two ways. first, seed priming sets in motion germination-related activities (e.g. respiration, endosperm weakening, and gene transcription and translation, etc.) that facilitate the transition of quiescent dry seeds into germinating state and lead to improved germination potential. secondly, priming imposes abiotic stress on seeds that represses radicle protrusion but stimulates stress responses e.g. accumulation of late embryogenesis abun dant (leas), potentially inducing cross-tolerance (chen and arora, 2013). these two strategies together also impose a ‘memory’ in seeds, which can be recruited upon a subsequent stress-exposure and may mediate a greater stress-tolerance in the subsequent generation (pastor et al., 2013). the specific influence of exogenous pbrs on crop plants has already been integrated into the crop production systems; e.g. the use of chlormequat chloride (cc) in cereals crop growth, ethephon (eth) for influencing the development and maturity of various crops and mepiquat chloride in cotton. several such pbrs also may be useful in overcoming the production constraints. foliar application of synthetic auxin − naphthal acetic acid (naa) to enhance apple flower (cline, 2006a; harley et al., 1958; mcartney et al., 2007; stover et al., 2001) and also documented as flower-promoter in several tree fruit crops (cowgill and autio, 2009; schwallier, 2006; washington state university extension, 2009). eth has also enhanced flowering in bearing apple trees (cline, 2006a). the pbr prohexadione-calcium (p-ca), an inhibitor of gibberellic acid (ga) biosynthesis reduces shoot extension in several fruit tree species (rademacher et al., 2006). exogenous cytokinin application of benzyladenine (ba) has effectively improved lateral branching of nursery trees (hrotko et al., 1997) by limit blindwood and improves canopy development. plant bio-regulators (pbrs) the most commonly used pbrs, include various plant hormones (farooq et al., 2015; ratnakumar et al., 2015a, b; srivastava et al., 2016) thus, affect plant’s ability to respond to its environment (tab. 1 and 2). they interact with specific target tissues to cause physiological responses, such as growth and development. each response is often the result of two or more hormones acting together. among them, many hormones can be synthesized in the laboratory, therefore increasing the quantity of hormones available for commercial applications. for instance, ga is widely used as pbrs. likewise, sa is another important plant hormone that regulates a wide range of metabolic and physiological responses in plants such as seed germination, seedling establishment, cell growth, respiration, stomatal closure, senescence-associated gene expression, responses to abiotic stresses, basal thermo-tolerance, nodulation in legumes, and fruit yield. hence they are used as pbrs for enhancing plant growth and yield (vlot et al., 2009; vicente and plasencia, 2011) (tab. 2) and for particular stress alleviation (tab. 1). however, judicious use of sa dose is required as the excess concentration proves fig. 1: schematic representation of abiotic stress tolerance mechanism induced by plant bio-regulators (pbrs) in holistic level; impact on antioxidative system, up-regulation of osmolytes and gene activation. owing to their ability to modulate ros level, upregulation of osmolytes and activation of stress responsive genes, pbrs help to establish redox homeostasis which either avaoids or minimizes the plant ability to combat stress induced redox imbalance. plant bio-regulators to minimize crop productivity losses 115 tab. 1: drought, heat stress and salinity induced plant tolerance by plant bio-regulator (pbrs) viz., sa (salicylic acid), tu (thiourea) and pgpr (plant growth promoting rhyzobactria). the indication marks ‘+’ represents the positive and ‘-’ represents negative responses. stress plant pbrs studied parameters response reference drought wheat sa content of ascorbate and glutathione + kang et al. (2013) wheat sa moisture content, dry matter accumulation, + singh and usha (2003) carboxylase activity of rubisco, sod and total chlorophyll barley sa aba content in leaves + bandurska and stroinski (2005) wheat sa stomatal regulation, maintaining leaf chloro+ anosheh et al. (2012) phyll content, increasing water use efficiency, and stimulating root growth salvia officinalis sa remobilization of stored food + abreu and munne-bosch (2008) phaseolus vulgaris pgpr production of iaa, cytokinins, antioxidants + figueiredo et al. (2008) and acc deaminase wheat sa chlorophyll pigments and chlorophyll a/b ratio moharekar et al. (2003) wheat thiourea individual grain weight + sahu and singh (1995) heat arabidopsis sa oxidative stress + alonso-ramĺrez et al. (2009) mustard sa h2o2 content and cat activity + and dat et al. (1998) agrostis stolonifera sa pox activity and cat activity + and larkindale and huang (2004) cicer arietinum sa protein and proline contents induction of + chakraborty and various stress enzymes viz. pox and apx tongden (2005) wheat sa proline content + khan et al. (2013) salt wheat sa iaa and cytokinin levels, proline content, + shakirova et al. (2003) aba accumulation, sod and pox activity wheat sa osmotic potential, shoot and root dry mass, + kaydan et al. (2007) k+/na+ ratio and photosynthetic pigments content barley sa photosynthetic rate, membrane stability + el tayeb (2005) b. juncea sa growth, photosynthetic parameters and + yusuf et al. (2008) activities of enzymes (nitrate reductase, carbonic anhydrase, cat, pox and sod), proline content mungbean sa antioxidant system + khan et al. (2014) tomato sa activation of aldose reductase and ascorbate tari et al. (2002, 2004), peroxidase, accumulation of osmolytes such + szepesi et al. (2005) as proline toxic for the plant growth (donovan et al., 2013). exogenous application of cytokinin, preferably kinetin, to the foliage of plants has also been shown to increase crop productivity. the application of low concentrations of potassium together with cytokinin provides synergistic effect (us patent, 2012). similarly, early application of trehalose has also shown to enhance the health and vigor of plant resulting in better production of sugar content (us patent, 2013). the application of plant sterols and steroid hormones such as brassinosteroids (bs) are essential for plant growth, reproduction and responses to various abiotic and biotic stresses. the use of these bs (vriet et al., 2012) has also been proposed as a promising strategy for crop improvement. biotic agents apart from chemical based pbrs, various biotic agents (tab. 2) have also been used for the same purpose. nagaraju et al. (2012) have used trichoderma harzianum isolates to enhance the plant growth and resistance of sunflower towards downy mildew disease. many bacteria have been found to not only promote the growth of plants but also protect the plants against various abiotic and biotic stress agents including flooding, drought, salts, metals, organic contaminants, wilting and pathogens (glick, 2014). effects of pbrs on crop growth salicylic acid (sa) and other salicylates, thio-urea and other thiol compounds, kno3 and other nitrites, are known to affect various physiological and biochemical activities of plants and may play a key role in regulating plant growth and productivity (arberg, 1981). studies have provided conflicting evidences regarding role of sa in flowering. however, some demonstrations argue the role of sa in flowering. in sa-deficient arabidopsis initiation of flowering failed when irradiated with uv-c and substantial flowering occurred when grown under non-stress condition than wild-type plants (shulaev et al., 1995). siz1, a sumo e3 ligase, negatively regulates flowering via an sa-dependent pathway (jin et al., 2008). it was reported that the dry matter accumulation was significantly enhanced in brassica juncea, with sa foliar application. however, higher concentrations of sa had an inhibitory effect. in sugarcane ga3 increased 116 p. ratnakumar, m.i. raja khan, p.s. minhas, m.a. farooq, r. sultana, t.s. per, p.p. deokate, n.a. khan, y. singh, j. rane inter-nodal elongation, while glyphosate, cepa and other regulators increase the deposition of sucrose, diquat and 2-chloroethylphosphonic acid (cepa) inhibit flowering, and paraquat desiccates leaves just prior to harvest to facilitate leaf removal or burning (kossuth, 1984). ga increased the rate of cell division and stimulation of vegetative growth (arteca, 1995). leaf area was found to increase significantly after kno3 application probably due to promoting role of potassium (k) in plant growth. generally the essential element k has a great regulatory role within plant cells and organs such as, activating on enzymes, osmosis regulation and photosynthesis and loading and unloading of sugars in phloem (osmosis regulation and photosynthesis). dcpta delay the natural senescence of mature leaves thereby contributing to enlarged leaf canopies and improved carbon assimilation per unit leaf area. flowering is another important parameter is directly related to yield and productivity of plants. it is well known that sa application induces flowering in a number of plants (cleland and ajami, 1974). however, the rigorous mechanism of flower inducing property of sa is not been explored. kumar et al. (2000) studied the cumulative effect of sa in combination with ga, kinetin, naa, ethral and chloro chloro chloride (ccc), and found a synergistic effect between sa and ga on flowering as compared to other combinations of hormones. likewise, ethylene modulates plant growth and development under normal conditions and is also a key feature in the response of plants to a wide range of stresses. ethylene is synthesized in plants in response to various stresses typically in response to the presence of metals, organic and inorganic chemicals, cold or heat stress, drought or flood, ultraviolet light, insect and nematode damage, phyto-pathotab. 2: chemical-based pbrs and biotic agents, their mode of action in crop plants and orchards. pbrs type of plant mode of action references chemical based pbrs tu, sa, si, no, h2o2, crop plants improves plant stress tolerance srivastava et al., 2016 hydrogen rich water, and crop yield brassinosteroids and polyamines chlormequat chloride (cc); cereals, cotton crop growth, development and jisha et al., 2013; ethephon (eth) maturity of various crops srivastava et al., 2016 and mepiquat chloride naphthal acetic acid (naa) apple and several enhance flower and known as harley et al., 1958; and eth tree fruit crop flower promoter mcartney et al., 2007; stover et al., 2001; cline, 2006a prohexadione-calcium (p-ca), several fruit tree species inhibitor of gibberellic acid (ga) rademacher et al., 2006 biosynthesis reduces shoot extension benzyladenine (ba) nursery trees improved lateral branching hrotko et al., 1997 trehalose enhance the health and vigor of plant us patent, 2013 production of sugar content brassinosteroids (bs) plant growth, reproduction vriet et al., 2012 and responses potassium (k) plant cell osmosis regulation and photosynthesis arteca, 1995 aminocyclopropane-1-carboxylate transcription of genes that encode plant robison et al., 2001 (acc, the precursor of ethylene) defensive/protective proteins thio-urea (tu) wheat grain yield sahu et al., 2006 brassinosteroids (bs) hypocotyl cell elongation involves a wang et al., 2012b microtubule regulatory protein, microtubule destabilizing protein 40 thio-urea (tu) wheat improved productivity and enhanced sahu and singh, 1995 grain weight sa and snp (nitric oxide source) promoted fe uptake, translocation and kong et al., 2014 activation; modulated the balance of mineral elements; and protect fe deficiency biotic agents pseudomonas asplenii phragmites australis seeds improved germination and protected bashan et al., 2008 the plants purple phototrophic bacteria (ppb) stevia rebaudiana increased the growth and also yield wu et al., 2013 of stevioside (st) pgprs (plant growth promoting induce the production of auxin or steenhoudt and rhizobacteria) inhibit ethylene synthesis or vanderleyden, 2000 mineralization of nutrients trichoderma harzianum sunflower plant growth and resistance nagaraju et al., 2012 downy mildew pseudomonas fluorescens promote plant growth glick, 2014 plant bio-regulators to minimize crop productivity losses 117 gens (both fungi and bacteria), and mechanical wounding (glick, 2014). a small fraction ethylene synthesis, which consumes the existing pool of 1-aminocyclopropane-1-carboxylate (acc, the precursor of ethylene) in stressed plant tissues, is believed to be responsible for initiating the transcription of genes that encode plant defensive/ protective proteins (robison et al., 2001). however, higher level of ethylene production following synthesis by the plant of additional acc in response to a stress is generally injurious to plant growth and usually involved in initiating senescence, chlorosis and leaf abscission (glick, 2014). there has been a major increase in the utilization of brs in agricultural applications as a mean to boost crop productivity and stress tolerance. crosstalk and interactions between brs and other plant growth regulators occur through either the modification or intersection of their primary signaling cascades and function to regulate a large and diverse array of biological processes (choudhary et al., 2012). aba regulates both stressand non-stress-related plant responses by acting as a signal molecule. interactions between brs and aba regulate the expression of many genes that govern several biological processes, such as seed germination, stomatal closure and plant responses to environmental stresses. interaction between soil bacterium may be negative, positive or neutral and sometimes the effect of soil bacterium changes according to the conditions of soil. for instance, when the availability of chemical nitrogen fertilizer is abundant in soil, a nitrogen fixing bacterium is useless for the plants. similarly many bacteria are useful for the plants only under environmental stress conditions and are unlikely to have any beneficial effect under optimal conditions (glick et al., 2007). it is important to mention here that, not all strains of a particular bacterial genus or species promote plant growth due to varied genetic makeup and metabolic capabilities. some strains of pseudomonas fluorescens, for instance may actively promote plant growth while other strains of this species have no measurable effect on plants (glick, 2014). plant growth-promoting bacteria may facilitate plant growth and development either directly or indirectly. the indirect effect on promotion of plant growth occurs by preventing harmful effects of plant pathogens and direct role of the pgbrs involves either acquisition of mineral nutrients or modulation of plant growth by alteration of phytohormones such as auxin, cytokinin and ethylene (glick, 2012). bacteria control the level of ethylene production in some plants through the auxins they produce; the most commonly observed mechanism employed by bacteria that diminish levels of ethylene production is via the activity of bacterial 1-aminocyclopropane-1-carboxylate deaminase (acc deaminase). bacteria of different origins expressing acc deaminase activity (leading to a decrease in acc levels and thus in ethylene production) can stimulate plant growth even in soils containing phytotoxic concentrations of cadmium. it has been found that most of the plant growth-promoting rhizobacteria isolated from graminaceae grasses growing in a meadow polluted with heavy metals exhibited acc deaminase activity, which resulted in plant growth promotion (dell’amico et al., 2005). some of the abiotic stresses whose effects can be ameliorated in this way include temperature extremes, flooding, drought, metals and metaloids, hypoxia, salt and organic contaminants (glick, 2012). foliar spray and rhizosphere irrigation with purple phototrophic bacteria (ppb) on a herb stevia rebaudiana native to certain regions of south america increased the growth and also yield of stevioside (st), one of the steviol glycosides, which is 300 times sweeter than cane sugar and non-calorific, and has specific immunomodulatory activities (wu et al. 2013). germination sa is known to improve germination, plant growth, rate of trans piration, stomatal regulation, photosynthesis, ion uptake and transport in plants (metwally et al., 2003; khodary, 2004; he et al., 2010; khan et al., 2014, 2015). low doses of sa have potential to improve seed germination and seedling establishment under different abiotic stress conditions (rajjou et al., 2006; alonso-ramírez et al., 2009). lee et al. (2010) reported the role of sa in seed germination under salinity. they found that sa promotes germination under high salinity by modulating antioxidant activity in arabidopsis. further, they suggested that, sa was not essential for germination under normal growth conditions, but under saline conditions it promotes seed germination by reducing oxidative damage. similarly, hanieh et al. (2013) determined the effect of pre-sowing treatment of sa on seed germination of sweet pepper and found that, pre-sowing seed treatment of sa caused better germination percentage and faster growth rate. proteomic analyses showed that two sods are induced by sa in arabidopsis germinating seeds, which might contribute to an enhanced antioxidant capacity (rajjou et al., 2006). for instance, about 0.5 mm sa treatment for 24 h caused a strong up-regulation of translation initiation and elongation factors, proteases, and two subunits of the 20s proteasome. this supports the hypothesis that sa improves seed germination by promoting the synthesis of proteins that are essential for germination, and the mobilization or degradation of seed proteins accumulated during seed maturation (rajjou et al., 2006). br-related mutants (det2-1 and bri1-1) showed in creased sensitivity to the inhibitory effects of aba during seed germination in comparison with wild type plants (steber and mccourt, 2001). however, primary root and hypocotyl elongation assays in the sax1 mutant revealed hypersensitive responses to aba, as well as auxin and ethylene (ephritikhine et al., 1999). in phragmites australis inoculation of seeds with pseudomonas asplenii improved germination and protected the plants from growth inhibition (bashan et al., 2008). root growth prolific root system is important for stress tolerance and improved water uptake particularly under abiotic stress conditions in order to harvest better crop yield (farooq et al., 2009; flowers, 2004) under abiotic stress conditions. normally sensitive genotypes have poor prolific roots than susceptible genotypes. corresponding to this, a pronounced increment in root growth of sensitive wheat varieties to salt and high temperature stress was reported with the application of tu and that strongly correlate to grain yield (sahu et al., 2006). thus, in view of tu being water soluble, readily absorbable in the tissues that enhance the plant, it has ability to ameliorate stress effects under field conditions. superiority of synthetic growth hormone naa over other bio regulators attributed its unique role in delaying senescence process, hastening root and shoot growth and setting more fruits (wein et al., 1989). keithly et al. (1990) reported that photosynthate partitioning in dcpta-treated (foliar application) sugar beet plants appeared to be balanced between the demands of plant growth and taproot sucrose accumulation supply. increased taproot weight of 30 μm dcptatreated taproots resulted in an 81 % increase in sucrose yield. sa increased the level of cell division within the apical meristem of seedling roots of wheat plants (sakhabutdinova et al., 2003). similarly, applications of 10-8 and 10-6 m sa increased root length (sanmiguel et al., 2003). the exogenous application of sa to plants results in an interference with the ion transportation and absorption in the membranes of root cells (harper and balke, 1981). in soybean plants treated with 10 nm, 100 μm, and up to 10 mm sa, root growth increased up to 45 % (shakirova et al., 2003). the influence of bss on pin aux efflux carriers, which control mitotic activity and cell differentiation, suggests a possible mechanism that contributes to bs-mediated root growth through regulation of aux distribution (choudhary et al., 2012). bss were unable to an tagonize the et effects on hypocotyl growth in the etiolated fer-2 118 p. ratnakumar, m.i. raja khan, p.s. minhas, m.a. farooq, r. sultana, t.s. per, p.p. deokate, n.a. khan, y. singh, j. rane mutant, indicating fer-dependent bs effects on et-induced growth responses (deslauriers and larsen, 2010; cheung and wu, 2011). br-mediated hypocotyl cell elongation involves a micro tubule regulatory protein, microtubule destabilizing protein 40 (mdp40) (wang et al., 2012b). in some plants, the treatment of seeds or cuttings with non-pathogenic bacteria, such as agrobacterium, alcaligenes, bacillus, pseudomonas, streptomyces, etc., induces root formation (esitken et al., 2003). pgprs (plant growth promoting rhizobacteria) might induce the production of auxin or inhibit ethylene synthesis or mineralization of nutrients (steenhoudt and vanderleyden, 2000). desbrosses et al. (2009) investigated the pgpr-arabidopsis interaction to establish the signaling pathways involved in controlling plant development and observed an ethylene-independent and auxin-independent mechanism, regulating the elongation of root hair in arabiopsis. nutrient uptake, mobilization and translocation application of naa (20 ppm) produced mean maximum yield of 17.60 and 47.05 q ha-1 of seed and straw of fenugreek, respectively and was found to be superior to other bio-regulators like ga. n, p and k content. their uptake in seed and straw were also the maximum with 20 ppm naa treatment which was n: 5.50, p: 8.14, k: 2.20 and n: 5.81, p: 12.86, k; 1.94 percent high over water sprayed in seed and straw of fenugreek, respectively. it was also documented that hormone application increases physiological and metabolic activities of the plant as a result more uptake of nutrients by plants from the soil (nickell, 1982). it was also perceived that higher concentration of bs (i.e. 0.5 ppm) increases yield, npk content and uptake over ga, due to homo-brassinosteroid synergistic interaction with the endogenous auxin. (crane, 1944). the favorable effect of plant growth regulators in enhancing the yields and nutrient uptake was also reported in cotton (patel, 1992) and green gram (takahashi, 1994), respectively. nitric oxide (no) or its donor has been shown to mediate chlorophyll increase, fe availability and antioxidant enzymes (zhang et al., 2012). several models suggest that redox signalling through no and ros is enhanced by sa in a self-amplifying process. however, the relationship between no, sa, and ros in the activation of defense genes and/or induction of host cell death is not clearly defined (zottini et al., 2007). sa and snp (nitric oxide source) promoted fe uptake, translocation and activation; modulated the balance of mineral elements; and protected fe deficiency induced oxidative stress (kong et al., 2014) (tab. 2). little information is available on the role of brs under nutrient deficient conditions. wang et al. (2012) reported that brs play a negative role in regulating iron (fe)-deficiency-induced ferric reductase (fro), expressions of csfro1 (transcripts encoding fro) and csirt1 (fe transporter), as well as fe translocation from roots to shoots. in wheat, the mobilization flow rate of sucrose to grain determines the rate at which carbohydrate can accumulate in the ear (jenner and rathjen, 1972). sahu and singh (1995) reported that improved productivity of wheat under soil and foliar treatments of thio-urea (tu) was mainly due to enhanced grain weight. metabolic transport of sucrose to grains via effects on phloem loading was enhanced with application of tu. mobilization of dry matter (reserves) from leaves to grains increased in wheat with tu spray at tiller stage. improvement in harvest index under tu treatments lends further credence to the role of tu in improving dry matter partitioning to grains (sahu and solanki, 1991). reduced grain yield with 20 kg/ha soil-applied tu compared with 10 kg/ha treatment might be attributed to inhibitory effects perhaps on phloem transport of sucrose (uppal, 1986) the role of tu as a thiol compound and growth-regulating chemical for improving the productivity of field crops such as wheat and brassica were studied extensively (srivastava, 2010; sahu, 2006; niasm, 2013) (tab. 1). inoculation of rhizobacteria increased uptake of nutrient elements like ca, k, fe, cu, mn and zn by plants through stimulation of proton pump atpase (mantelin and touraine, 2004; kumar et al., 2014). in crop plants, mineral elements uptake also increased in combined inoculants of bacillus and microbacterium (karlidag et al., 2007). pgprs might increase the nutrient uptake of plants through organic acid production and decreasing the soil ph in rhizosphere. pbrs induced plant adaptation and mitigation under drought, salinity and heat stress drought exposure of plants to water stress leads to serious physiological and biochemical dysfunctions including reduction in turgor, growth, photosynthetic rate, stomatal conductance and damages of cellular components (janda et al., 2007). sa has significant role in controlling abiotic stresses including drought and salinity stress. sa has high potential for improving stress tolerance in agriculturally important crops. its utility, however depends on various factors like concentration of sa applied, mode of application and the stage of plant growth. at low concentrations sa has been found to alleviate abiotic stress and at higher concentrations it induces oxidative stress (vicente and plasencia, 2011; miura and tada, 2014). higher tolerance to drought stress was also observed in the plants raised from the grains soaked in aqueous solution of acetyl salicylic acid (hamada, 1998; hamada and al-hakimi, 2001). it has been suggested that sa-induced drought tolerance is associated with an enhanced antioxidant system (horváth et al., 2007; mutlu et al., 2009; zhou et al., 2009). however, very little is known about the molecular mechanisms of sa-induced drought or other abiotic tolerances in higher plants. a few studies have shown that abiotic tolerance induced by sa could be related to the altered expression of the genes encoding osmotin, pathogenesis-related proteins, and heat shock proteins (ding et al., 2002; kim and delaney, 2002; clarke et al., 2004). to explore the molecular mechanisms involved an effort was made by kang et al. (2013). they found that treatment with 0.5 mm salicylic acid (sa) significantly alleviated growth inhibition induced by drought in wheat seedlings by significantly increasing the content of ascorbate (asa) and glutathione (gsh) due to enhanced transcription of gst1, gst2, glutathione reductase (gr), and mono-dehydro ascorbate reductase (mdhar) genes (kang et al., 2013). treatment with sa increased drought tolerance of common bean (phaseolus vulgaris) and tomato (solanum lycopersicum) plants (senaratna et al., 2000). exogenously applied sa has also been reported to modulate activities of intracellular antioxidant enzymes sod and pod and increase plant tolerance to environmental stresses (sakhabutdinova et al., 2004; senaratna et al., 2000). application of sa alleviated adverse effects of drought stress in wheat plants by increasing improving stomatal regulation, maintaining leaf chlorophyll content, increasing water use efficiency, and stimulating root growth (anosheh et al., 2012). leaf senescence is a highly regulated physiological process, allowing the remobilization of stored food from the older leaves to the rest of the plant, during stressful conditions and sa involved in the promotion of drought-induced leaf senescence in salvia officinalis plants (abreu and munne-bosch, 2008). the results reported by singh and usha (2003) revealed that the wheat seedlings subjected to drought stress when treated with sa generally exhibited higher moisture content and also higher dry matter accumulation, carboxylase activity of rubisco, sod and total chlorophyll content compared to the untreated plants. exogenous application of sa also alleviated the damaging effects of water deficit on cell membranes of barley plants and concomitantly increased aba content in leaves, which might have contributed to the enhanced tolerance of plants to water scarcity (bandurska and stroinski, 2005). besides providing tolerance to plant bio-regulators to minimize crop productivity losses 119 plants against drought stress, the exogenous application of sa was also found to be effective in providing resistance to the plants against the excessive water stress as was observed in cell suspensions prepared from the fully turgid leaves of sporobcdus stapfianus (ghasempour et al., 2001). the impact of thiourea in improving productivity of wheat (sahu and singh, 1995), showed individual grain weight significantly improved under soil and foliar treatments of tu. it is a well-established fact that sa potentially generates a wide array of metabolic responses in plants and also affects the photosynthetic parameters and plant water relations. however, contrary to these observations, a reduction in chlorophyll content was observed in plants pre-treated with sa (anandhi and ramanujam, 1997; pancheva et al., 1996). moharekar et al. (2003) reported that sa activated the synthesis of carotenoids and xanthophylls and also enhanced the rate of deep oxidation with a concomitant decrease in chlorophyll pigments and chlorophyll a/b ratio in wheat. however in contrast, the transpiration rate decreased significantly in phaseolus vulgaris and commelina communis after the foliar application of sa. that decrease in transpiration rate was attributed to the fact that sa induced the closure of stomata (larque-saavedra, 1978, 1979). sa was also shown enhance photosynthesis and growth of soybean (c3 plant) and corn (c4 plant) under greenhouse conditions (khan et al., 2003). sa also has capacity of osmotic adjustment by maintaining low mda contents and decreased na+/k+ ratio in leaves (fayez and bazaid, 2014). brassinosteroids enhance tolerance to drought and cold stress by modulations of the expression of droughtand cold-stress marker genes (kagale et al., 2007). however, the exact biochemical link between the brassinosteroids -signal cascade and stress tolerance remains a mystery. both soil and foliar treatments of thio-urea (tu) increased the number of ears and grains/ear, indicating an improved storage capacity. in this context, it is noteworthy that di-thiothreitol, a thiol containing two -sh groups, stimulated carbon dioxide assimilation in the dark up to five-folds (werdan et al., 1975). because of its cytokine-like activity, tu might have also delayed leaf senescence (halmann, 1990). in maize, foliar spray of tu increased both canopy photosynthesis and photosynthetically active leaf surface during grain filling (sahu et al., 1993). chloroplasts isolated from mature leaves of 30 μm dcpta-treated plants, as compared with that of controls, showed a 23 % increase in the total soluble protein to chlorophyll ratio. this parallels to an observed increase in activated ribulose 1, 5-bisphosphate carboxylase/oxygenase (rubisco) activity in vitro per unit chlorophyll. the rubisco activity increased 87 % per dm2 leaf area of 30 μm dcpta-treated plants. increased rubisco activity largely accounted for increase in net photosynthesis in dcptatreated plants. however, intense sink demand for photosynthate and the delayed leaf senescence of older leaves may increase net-carbon assimilation in mature (source) leaves of dcpta-treated plants. tolerance to drought stress is enhanced in pgpr inoculated plants (figueiredo et al., 2008). plants achieve this tolerance either due to the production of iaa, cytokinins, antioxidants and acc deaminase. reports are also available regarding role of pgpr in conferring resistance to water stress in plants such as tomatoes and peppers under water deficient conditions (aroca and ruiz-lozano, 2009). more efforts are needed to investigate the mechanistic approach of pgpr in eliciting tolerance to different stresses. this would improve our understanding of induced systemic tolerance to water stress in modern agriculture. salinity high salinity induces serious metabolic perturbations in plants, as it generates ros which disturb the cellular redox system in favor of oxidized forms thereby creating an oxidative stress that may damage dna, inactivate enzymes and cause lipid peroxidation (smirnoff, 1993) (tab. 1). the results of srivastva et al. (2009) recommend that tu treatment maintains the integrity and functioning of mitochondria in seeds as well as seedlings exposed to salinity. thus, tu has the potential to be used as an effective bioregulator to impart salinity tolerance under field conditions, and might prove to be of high economic importance by opening new avenues for both basic and applied research. however, most of the literature indicates that exogenous application of sa to the stressed plants can potentially alleviate the toxic effects, generated by salinity. an enhanced tolerance against salinity stress was observed in wheat seedlings raised from the grains soaked in sa (hamada and al-hakimi, 2001). similar observations were also made in tomato plants raised from the seeds soaked in sa and was presumed to be due to the enhanced activation of some enzymes viz. aldose reductase and ascorbate peroxidase and to the accumulation of certain osmolytes such as proline (tari et al., 2002, 2004; szepesi et al., 2005). accumulation of large amounts of osmolytes (proline) is an adaptive response in plants exposed to stressful environments (rai, 2002). wheat seedlings accumulated large amounts of proline under salinity stress which was further increased with sa applied exogenously, thereby alleviating the deleterious effects of salinity (shakirova et al., 2003). the exogenous application of sa prevented the lowering of iaa and cyto kinin levels in salinity stressed wheat plants resulting in the betterment of cell division in root apical meristem, thereby increasing growth and productivity of plants (shakirova et al., 2003). it is widely reported that the pre-treatment with sa resulted in the accumulation of aba which might have contributed to the pre-adaptation of seedlings to salinity stress since; aba induces the synthesis of a wide range of anti-stress proteins, thereby providing protection to the plants. further, the treatment also lowered the level of active oxygen species and therefore the activities of sod and pox were also lowered in the roots of young wheat seedlings (shakirova et al., 2003). these findings indicate that the activities of these antioxidant enzymes are directly or indirectly regulated by sa, thereby providing protection against salinity stress (sakhabutdinova et al., 2004). exogenous application of sa enhanced the photosynthetic rate and also maintained the stability of membranes, thereby improved the growth of salinity stressed barley plants (el tayeb, 2005). the damaging effects of salinity were also alleviated by exogenous application of sa in arabidopsis seedlings (borsani et al., 2001). kaydan et al. (2007) observed that pre-sowing soaking treatment of seeds with sa positively affected the osmotic potential, shoot and root dry mass, k+/na+ ratio and contents of photosynthetic pigments (chlorophyll a, b and carotenoids) in wheat seedlings, under both saline and nonsaline conditions. the loss of growth, photosynthetic parameters and the activities of enzymes (nitrate reductase and carbonic anhydrase) as a result of salinity stress in b. juncea was revived when sa was sprayed to the foliage, at 30 days stage. further the activities of various antioxidant enzymes (cat, pox and sod) were increased with a concomitant increase in proline content (yusuf et al., 2008). sa enhanced plant salt tolerance in terms of improving all measured growth parameters, photosynthetic efficiency and enhanced the antioxidant enzyme contents in response to nacl and/or sa treatment providing a synergistic interaction (ismail, 2013; khan et al., 2014). in both abscisic acid (aba) levels increase in both drought and high salinity conditions, along with gene expression changes (zeller et al., 2009). intensive crosstalk of aba with different signaling pathways produces abundance of proteins and secondary messengers that act as regulators or modulators of aba responses (christmann et al., 2006). both aba and ethylene responses integrate at the level of della proteins under salt stress and under conditions of high salinity and vegetative growth of plants occurs through activation of aba and ethylene signaling. these two pathways eventually result in a della-mediated inhibition of growth and a delay in flowering, respectively, which ultimately promotes plant survival (achard 120 p. ratnakumar, m.i. raja khan, p.s. minhas, m.a. farooq, r. sultana, t.s. per, p.p. deokate, n.a. khan, y. singh, j. rane et al., 2006). this della-mediated growth control and plant survival under high salt conditions was shown to be due to an increased accumulation of enzymes that detoxify reactive oxygen species (ros) (achard et al., 2008). bs crosstalk with sa regulates plant responses to abiotic stress. exogenously applied brs could not confer salt stress tolerance in the sa-insensitive npr1-1 mutant in arabidopsis (divi et al., 2010). this supports the idea that br-induced salt tolerance in arabidopsis partially depends on npr1, a master regulator of the sa-mediated defense signaling pathway (divi et al., 2010). in brassica juncea salt stress tolerance increased with exogenous applications of bs and sa. the combined application of bs and sa was most effective in alleviating the salt stress when compared with their individual treatments (hayat et al., 2012). other pgbrs which enhnce salt tolerance in plants include mycorrhizal associations of plants. different mechanisms are employed by arbuscular mycorrhizal fungi to enhance salt tolerance of host plants like enhancing nutrient acquisition (p, n, mg and ca) (azcon and atrash, 1997; giri and mukerji, 2004; sheng et al., 2009), inhibition of high uptake of na and cl and their transport to plant shoots (daei et al., 2009), improving water uptake (ruiz-lozano and azcon, 2000), accumulating of proline and polyamines (evelin et al., 2009; ibrahim et al., 2011) and also by increasing some of enzymatic antioxidant defense system (sod and cat) (farooq et al., 2015; wu et al., 2010) (fig. 2). arbuscular mycorrhizal association also improve osmotic adjustment, which helps in maintenance of the leaf turgor pressure, which in turn affects photosynthesis, transpiration, stomatal conductance and water use efficiency (juniper and abbott, 1993). under saline condition, mycorrhizal inoculation of three different arbuscular mycorrhizal fungi, glomus mosseae, g. deserticola and gigaspora gergaria significantly increased growth responses, nutrient contents, acid and alkaline phosphatases, proline and total soluble protein of wheat plants compared to non-mycorrhizal ones (abdel-fattah and asrar, 2012). foliar application of moringa (moringa oleifera) leaf extract (mle; 30 times diluted), benzyl amino purine (bap; 50 mg l-1) and hydrogen peroxide (h2o2; 120 μm) at tillering, jointing, booting and heading growth stages decreased the shoot na+ and clcontents, with simultaneous increase in shoot k+ contents (yasmeen et al., 2013). heat stress deviation from optimum temperature results in serious perturbations in plant growth and development. this may be due to membrane disruptions, metabolic alterations and generation of oxidative stress (mittler, 2002). both salinity and high temperatures have a common facet of oxidative damage (wahid et al., 2007a). looking at the structure of thio-urea, both ‘imino’ and ‘thiol’ functional groups has great implications in abiotic stress tolerance. with foliar spray, amino group provides a ready source of nitrogen and thiol has a great role in alleviating oxidative stress damage on the physiologically more important mesophyll tissue. it was evident from the results that foliar applied thio-urea was effective in improving salt(6 -11 %) and high temperature tolerance (4 -10 %) of wheat varieties. however, sa plays a key role in providing tolerance against temperature stress. sa stimulates alternative respiratory pathway in mitochondria by inducing expression of alternative oxidase, the terminal electron acceptor of the alternative respiratory pathway and releases unused potential energy as heat (vlot et al., 2009). sa has been found to provide protection against heat stress in plants (karlidag et al., 2009; khan et al., 2013). exogenous application of sa also partially reverses the inhibitory effect of oxidative (0.5 mm paraquat) and heat stress (50 oc for 3 h) on seed germination (alonso-ramírez et al., 2009). jasmonic acid also acts with sa, resulting basal thermotolerance in arabidopsis thaliana (clarke et al., 2009). a foliar spray of lower concentrations of sa conferred heat tolerance to mustard. further this treatment accompanied with hardening at 45 °c for 1 h enhanced the h2o2 level and this also reduced the cat activity, thereby increasing the potential of plants to withstand the heat stress (dat et al., 1998). a similar response was observed in potato plantlets, raised from the cultures, supplemented with lower concentrations of acetyl salicylic acid (lopez-delgado et al., 1998). larkindale and huang (2004) pointed out the enhanced heat tolerance in plants of agrostis stolonifera, pre-treated with sa enhanced the protection of plants from oxidative damage. these authors further reported that the pre-treatment with sa had no effect on pox activity, whereas, the cat activity was shown to decline as, compared to control. moreover, the treatment enhanced the activity of enzyme ascorbate peroxidase. contrary to this, an enhanced activity of cat and sod was observed in heat stressed plants of poa pratensis, after the treatment with sa (he et al., 2005). in a study carried out by chakraborty and tongden (2005) and (tab. 1), it was reported that the application of sa in cicer arietinum reduced heat stress induced membrane injury and enhanced the protein and proline contents significantly with a concomitant induction of various stress enzymes viz. pox and apx. fig. 2: cellular level representation of scavenging ros (reactive oxygen species) induced by plant bio-regulators (pbr like tu: thiourea; sa: salicyclic acid; pas: polyamines); through enhancing the antioxidative system (e.g. sod: superoxide dismutase; cat: catalase), maintains redox homeostasis and reduced cell damage and deterioration. plant bio-regulators to minimize crop productivity losses 121 knowledge gaps carrying forward scientific leads from lab to farmer’s field the use of pbrs is a unique facet of biotechnology and a new approach of manipulating plant biochemistry for enhancing productivity and quality. although, there is a wide range of pbrs that are commonly used to improve plant growth, development, defense and productivity, the molecular mechanisms of their effects still remain to be fully elucidated. in addition, their commercialization depends upon several factors such as their stability under the field conditions, inertness, cost-effectiveness, ease of application and versatility towards different stresses. from the practical point of view, both endogenous and exogenous substances can be regarded as pbrs if they exert an influence on the growth and development of the plant, in low concentrations and without having any biocidal or nutritive action. since the environmental conditions before, during and after application of pbr may influence its effect on a given plant, it is critical to develop specific protocols for each pbr before it can widely be commercialized. in fact, changing environmental conditions in the field causing fluctuations of multiple parameters simultaneously such as temperature, humidity makes it difficult to utilize the results obtained from controlled experiments into practical applications for growers. in addition, plants face multiple stresses under farmer’s field conditions and combinations of pgrs may be needed to achieve significant impact on growth and productivity. another aspect which needs to be thoroughly explored is the economic feasibility of commercializing thepbrs from the point of view of agrochemical industry and the customers/users which are the smallholder farmers. plant growth regulator as integral part of management of crop plants under stress under stressful conditions, crop management is a key component for optimizing inputs and reducing yield losses. plant growth regulators play a vital role in determining growth, development and productivity of crops. advances in aba signaling science have already allowed refined development of crop management techniques for reducing irrigation input (wilkinson et al., 2012). ethylene physiology is accessible to manipulation via crop management. syngenta and -dow agosinces have developed a new crop management technique to reduce crop ethylene perception and reduce stress-induced grain yield losses, based on novel applications of 1-mcp (1methylcyclopropene). liquid foliar applications of ‘invinsa’ (agrofresh inc., usa), which produces gaseous 1-mcp, can act on a range of ethylene-associated process. crop management using plant-growthpromoting rhizobacteria (pgprs), as either seed treatments or soil additions, that reduce plant ethylene accumulation under stress by metabolizing the ethylene precursor at the root-soil interface, also increase yields of legumes in the field (belimov et al., 2009). the extent of stress-ethylene production may be genotype-dependent (balota et al., 2004). this provides scope for genetic or management modulation of water use and carbon gain in field crops. quality control and biosafety aspects for impending bioregulatory technologies while working with any pbr, the first step is to optimize its exact dose and mode of application. initially, an optimum dose can be derived either from pot based experiment or pilot scale field data and then the same can be tested using multi-location field trials in different agro-climatic zones for at least three calendar years. during these trials, various modes of pbr application such as seed soaking, soil applied and foliar spray or combination of these can be optimized for maximum crop yield with minimum input of pbrs. once, the exact dose and mode of application is optimized, there are many quality controls (qc) and biosafety aspects which need to be addressed before recommending any pbr for field application. the first qc is to check whether the pbr application can lead to any toxicity in human or other animals. since, most of the pbrs are chemical based, it is difficult to say that they are completely safe for human consumption. this is because the term “safe” can be explained only with respect to particular dosage. thus, at least, the dose at which any pbr is being applied onto the field should be non-toxic. this can be explained in a much better way by taking the example of thiourea which is the most widely used pbr for different crops. the iarc (international agency for research in cancer) has categorized thiourea in group-3 which states that the evidence of carcinogenicity is inadequate and is limited to animal testing and hence, is not classifiable as probable carcinogen for humans. on the basis of animal testing, “lowest-observed-adverse-effect-level” (loael) and “no-observed-adverse-effect-level” (noael) are derived as 27.5 and 6.88 mg thiourea per kg body weight per day, respectively. the concentration of thio-urea which is normally used for field studies is in the range of 5-10 mm (approximately 250 g/hectare). since, the foliar application is preferred mode of application, we can assume ~50 % uptake of thiourea by plants. with this estimate, one hectare of land having ~1,000,000 wheat plant (standard recommendation from department of agriculture, queensland univeristy; http:// www.daff.qld.gov.au) will have ~0.25 mg of thiourea/plant. this is the maximum carry over concentration which is more than 2000fold less than the naoel limit. additionally, thiourea degradation through plant peroxidases will further reduce its residual concentration. thus, taking these calculations into account, field application of thiourea in the range of 5-10 mm can be considered as safe. towards this endeavor, yet another important aspect is accumulation potential of thiourea in soil and air. owing to its very low vapour pressure, the significant adsorption of thiourea on airborne particles is not expected. due to its solubility in water (137 g/litre at 20 °c), the washout from the atmosphere by wet deposition (fog, rain, snow) is assumed to be significant. from water solubility and vapour pressure data, a henry’s law constant in the range of 5.58 × 10-9 to 8.44 × 10-9 pa. m3/mol can be calculated, indicating that thiourea is not expected to volatilize from aqueous solutions. based on these data, the hydrosphere is expected to be the main target compartment for this compound. in hydrosphere, thiourea did not undergo any significant ion exchange or other sorption process and remains in the soil solution as neutral thiourea. although, thiourea is hydrolytically stable and from uv spectrum it also appears that direct photolysis of thiourea in air or water is not expected. however, thio-urea may get photo-oxidized by hydroxyl radicals with a half-life of 2.4 h. in hydrosphere, the specific rate constants for the reaction of thiourea with hydrated electrons and hydroxyl radicals are given as 3.0 × 10+9/mol per second (ph 6.4) and 4.7 × 10+9/mol per second (ph 7). based on hydroxyl radical concentration of 1 × 10-16 mol/ litre in water, a half-life of 17 days can be calculated which is again depended upon the nature of soil microorganism. cultures of different fungi isolated from soil behave differently with respect to thiourea degradation. for instance, aspergillus glaucus, penicillium citrinum and trichoderma viride took up around 30 -50 % of initial thiourea concentration at 0.1 g/litre even after long incubation periods of 46 to 106 days and converted not more than 15 -17 % of thiourea sulfur to sulfate; however, the same concentration was completely removed within 7 days by penicillium rugulosum. based on these data on soil sorption, biodegradation in soil and calculated koc value; accumulation of thiourea in geosphere is less likely and hence, thiourea based pbrs can be assumed to cause minimum damage to soil ecosystem. apart from human toxicity, residual grain concentration and ecological imbalance, there is yet another qc which is most important before any pbr is released for field application. this is to evaluate the effect of pbr application on grain nutritional composition. thus, along with the yield, almost all the field trials with any pbr 122 p. ratnakumar, m.i. raja khan, p.s. minhas, m.a. farooq, r. sultana, t.s. per, p.p. deokate, n.a. khan, y. singh, j. rane should be accompanied with the analysis of grain nutritional profile and only after confirming the minimal change, their application should be recommended. in recent years, although a significant progress in pbr based research has been seen but, most of these are limited with either yield data or underlying molecular mechanism. for most of the pbrs, information about human toxicity, residual grain concentration, nutritional imbalance and ecological bio-safety are not available and hence, they did not get the approval to be used in farmer’s field. thus, there is a great need to give equal attention to these issues so as to increase the chance of converting more and more pbr based research into actual technology. way forward ample of research investigations have been carried out to estimate efficacy of the pbrs under drought, saline and high temperature conditions in crops. many of such studies depended on pot culture or single location or season experiment. 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institute of abiotic stress management, baramati-413 115, pune, maharashtra, india e-mail:pratnakumar@gmail.com © the author(s) 2016. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 90, 140 (2017) buchbesprechung phytotherapie – rationale empfehlungen für die behandlung helmut brinkmann, klaus wißmeyer, beatrice gehrmann, wolf-gerald koch, claus tschirch die 2. völlig neu bearbeitete auflage des „kitteltaschenbuches“ phytotherapie hat zusätzlich zu den in der 1. auflage bereits dar gestellten 100 drogen-profilen weitere 26 profile neu aufgenommen und deren bewertung unter berücksichtigung der monografien der kommission e aktualisiert. grundlage für die auswahl der aufgelisteten phytopharmaka sind hauptsächlich die positivmonografien der kommission e; darüber hinaus wird auf die monografien der escop und der who hingewiesen. die empfehlungen der autoren stützen sich generell auf den ergebnissen rationaler, evidenzbasierter und valider studien. dabei wird auch den teerezepturen unter berücksichtigung von standardzubereitungen mit garantierter qualität ein besonderer stellenwert eingeräumt. zu jeder indikation werden beispielhaft einige bewährte fertigarzneimittel aufgeführt und die entsprechenden verordnungsempfehlungen in kurzform mitgeliefert. in diesem zusammenhang werden auch die deklaration des jeweiligen droge-extrakt-verhältnisses, das verwendete extraktionslösemittel, die empfohlene dosierung sowie die unterschiedlichen darreichungsformen (flüssig / halbfest / fest) des pro dukts genannt. die autoren bevorzugen bei ihrer auswahl grundsätzlich die verordnung von monopräparaten; es finden sich aber dennoch auch hinweise auf sinnvolle kombinationspräparate, bei denen alle komponenten jeweils einen beitrag zur gewünschten wirksamkeit leisten. nahrungsergänzungsmittel sind in dem taschenbuch nicht mit aufgenommen worden, da sie rechtlich nicht als arzneimittel gelten. das taschenbuch liefert nicht nur medizinstudenten und heilpraktikern sondern auch hausärzten eine übersichtliche orientierung für die verordnung von arzneipflanzen und phytopharmaka. bibliografie: helmut brinkmann, klaus wißmeyer, beatrice gehrmann, wolfgerald koch, claus tschirch, phytotherapie – rationale empfehlungen für die behandlung, wissenschaftliche verlagsgesellschaft mbh, stuttgart, 2. völlig neu bearbeitete auflage 2016, 286 seiten, preis: 14,80 €, isbn: 978-3-8047-3602-3 dr. h. schulz e-mail: hartwig.schulz@julius-kuehn.de journal of applied botany and food quality 89, 235 242 (2016), doi:10.5073/jabfq.2016.089.030 1chiang mai university, department of plant science and soil science, faculty of agriculture, chiang mai, thailand 2 queen sirikit botanic garden, chiang mai, thailand chemical composition and comparison of genetic variation of commonly available thai garlic used as food supplement s. sommano1*, n. saratan2, r. suksathan2, t. pusadee1 (received april 2, 2016) * corresponding author summary in order to classify true garlic cultivars, comparisons of oil composition and genetic of three garlic cultivars (allium sativum l.) commonly used for essential oil production in the northern thai market [viz., thai (th), chinese (ch) and pingpong (pp) cultivars] were carried out. garlic essential oils were obtained by hydrodistillation and microwave hydrodistillation which were then analysed for chemical components by gas chromatography-mass spectrometry. the rapd data suggests similarity (>95 %) of the three cultivars in chemical compositions, and the major compounds are trisulphide, di-2-propenyl, the disulphide, di-2-propenyl, and the trisulphide, methyl 2-propenyl. sulphur-containing compounds (rf = 0.18-0.2) were detected by thin-layer chromatography (tlc) with ninhydrin staining reagent. the essential oil of ch from hydrodistillation and microwave hydrodistillation showed the highest alliin content. the rapd analysis of the three garlic cultivars presents 45 fragments. a dendrogram shows genetic similarity between the garlic cultivars. the th and the ch showed similarity value as 0.93, while the pp was classified as a different cluster. though there was considerable similarity between the chemical and the genetic profiles of the th and the ch, the ch demonstrated high potential as an ingredient in food supplement products due to its high alliin content. abbreviations: cas, chemical abstracts service; ch, garlic cv. chinese; gc-ms, gas chromatography-mass spectrometry; mog, garlic essential oil from microwave hydrodistillation; og, garlic essential oil from hydrodistillation; pp, garlic cv. pingpong; th, garlic cv. thai; tlc, thin layer chromatography. introduction besides being an important spice in many cultures, garlic (allium sativum l.) is also well known for its excellent medicinal properties (velíšek et al., 1997). the major components found in garlic cloves are sulphur-containing compounds such as allicin, alliin, ajoene, diallyl disulphide, dithiin and s-allylcysteine. these compounds are responsible for the pungent smell and taste of garlic (gafar et al., 2012). among those, allicin is the most abundant (70 % of the overall thiosulphates) present in fresh garlic which can be activated upon mechanical crushing (miron et al., 2004). this compound possesses powerful antibiotic, antimicrobial, anticancer and other properties. essential oil of garlic also contains a number of sulphides such as diallyl disulphide and dilly trisulphide; however, allicin can be completely eliminated during thermal or chemical extraction processes (block, 1985). food supplement products made from garlic are available in plenty, including, for example, garlic essential oil capsules, dehydrated garlic powder, garlic essential oil macerate and aged garlic extract (amagase et al., 2001; gafar et al., 2012). the challenge for food processors is to control the products in terms of quality, and, thus, at the starting point, searching for a suitable raw material is vitally important (velíšek et al., 1997). by dealing with major fresh garlic suppliers in thailand, we identified that there was a minimum of three garlic cultivars in the market. these types include locally grown cultivars, such as ‘thai’ and ‘pingpong’. the latter is named after its colour and shape. the ‘chinese’ cultivar is said to be imported through the thai-burmese border; however, its real origin is unknown. classification of ‘true’ variety of garlic can be done by using different techniques including morphological comparison and chemical analyses such as volatile components, dna fingerprints and contents of bioactive compounds (trirongjitmoah et al., 2015). in this study, we compared three garlic specimens generally available in thai markets based on their morphology, bioactive components, volatile profiles of essential oils and variation in their dna fingerprints. the aim was to classify the sources of raw materials and to control for quality the final products produced by thai food producers. materials and methods plant material garlic bulbs were supplied by chiang mai harvest co., ltd., the major garlic supplier from chiang mai, thailand. three garlic cultivars, commonly known as (1) garlic cv. thai -(th), (2) garlic cv. chinese -(ch) and (3) garlic cv. pingpong -(pp) were examined for morphology and stored in commercial warehouse conditions until use. extraction of garlic essential oil essential oil of garlic was extracted by hydrodistillation from fresh garlic cloves (200 g) with 1 l of water for 3 h. the essential oil was collected from a clevenger trap and dried over anhydrous sodium sulphate. the yield of the oil obtained (og) was calculated as a percentage (v/w) (sowbhagya et al., 2009). the same amount of garlic was also used for garlic essential oil extraction using microwave hydrodistillation and gravity unit at 700 w without the addition of water (sommano et al., 2015). the yield of the extract (mgo) was also accessed. analysis of chemical composition of garlic essential oil by gas chromatography-mass spectrometry (gc-ms) the oil was diluted in hexane (0.01%) prior to injection onto a gcms system (bruker, scion sq 436-gc). the gas chromatographic column was restek (rxi®-5sil ms, 30 m, 0.25 mmid, 0.25 um). the column was maintained at 45 °c for 1 min, and then heated to 245 °c at 4 °c min-1 using helium as the carrier gas (2 cm3/min) (weinberg et al., 1993). the mass spectrometer was run in electron ionization mode (ei 70 ev); source temperature, 100-325 °c; quadrupole temperature, 90 °c; mass scan range, m/z 1-1200 da; and scan rate, up to 14,000 da s-1, in a complete scan 236 s. sommano, n. saratan, r. suksathan, t. pusadee acquisition mode. the comparison of ms fragmentation patterns with those from the national institute of standards and technology (nist) ms 98 library was performed. the relative peak areas (ras) of a single compound were expressed in relation to the total peak area of the identified compounds. cluster analysis was performed based on 75 chemical compositions of garlic essential oil using the statistical package xlstat version 2015.5.01.23234 software. the coefficients of genetic similarity for all pair-wise comparisons were computed using jaccard’s coefficient of similarity and then the distance matrix was subjected to cluster analysis using the unweighted pair group method with arithmetic mean (upgma) to produce a dendrogram. identification of sulphur-containing compounds by thin layer chromatography separation of chemical compositions from essential oil (5-10 μl) was achieved by thin layer chromatography (tlc silica gel 60 f254, merck) with glacial acetic acid:propanol:water:ethanol (20:20:20:20) as the mobile phase. sulphur-containing compounds were visible after spraying the tlc plates with ninhydrin reagent (belemkar et al., 2013; keusgen, 1997). the retention factor (rf) is the result of the distance travelled by the compound divided by the distance travelled by the mobile phase. analysis of alliin content by spectrophotometry the alliin content was analysed using the spectrophotometry method with some adaptations (miron et al., 2002). briefly, garlic essential oil (4 μl) was incubated with 1.1 × 10-4 m of mercaptopyridine (4mp) in 50 mm sodium phosphate buffer, ph 7.2, containing alliinase. the decrease in the optical density (od) at 324 nm was determined after 30 min of incubation at room temperature. the alliin concentration was calculated using the following equation: [alliin] = δa324 × dilution × [εm]-1, where δa324 = [od without extract] − [od with extract] εm = molar extinction coefficient at 324 (4mp) = 19,800 dna analysis total genomic dna was extracted using the ctab (hexadecyltrimethylammonium bromide) method with some modification (doyle and doyle, 1987). into the ground sample, 1000 μl of the extraction buffer [100 mm tris–hcl ph 8.0, 20 mm edta (ethylenediaminetetracetate) ph 8.0, 1.4 mm nacl and 4 % (w/v) ctab] was added and the sample was incubated at 65 °c for 1 h. thereafter, the sample was further extracted with 600 μl chloroform:isoamyl alcohol [24:1 (v/v)] and centrifuged at 13,000 rpm for 10 min. the resultant supernatants were transferred to a new microcentrifuge tube. the air-dried pellet was re-suspended in 50 μl te buffer (10 mm tris–hcl ph 8.0, 1 mm edta and ph 8.0). the dna samples were stored at -20 °c prior to rapd analysis. dna quantification dna was quantified by using nano-drop spectrophotometer (nd1000, spectrophotometer). for re-quantification, the extracted dna was run on 1 % agarose gel electrophoresis using 1× tbe buffer at 5-8 v/ml for 30 min and visualised under blook led transilluminator (genedirex, taiwan) by staining with maestrosafetm (maestrogen, usa). the dna solution was diluted with sterile distilled water to a concentration of 50 ng/μl for pcr analysis and kept in -20 °c until use. rapd-pcr protocols for rapd analysis of the genomic dna, 10-base primers from operon technologies (alameda, usa) and ubc (university of british columbia, canada) were chosen (tab. 1). a total of 10 primers were screened. the polymerase chain reaction (pcr) was adjusted to 10 μl containing 8 μl of onepcrtm plus (genedirex, taiwan), 1 μl of 1 μm rapd primer and 1 μl of 10 ng genomic dna. all the reactions were carried out on a flexcycler2 thermal cycler (analytik jena, germany) using the following profile: 1 cycle, 94 °c, 4 min; 40 cycles, 94 °c, 30 s; 37 °c, 30 s; 72 °c, 60 s; 1 cycle, 72 °c, 10 min. the sample was separated in a 1.5 % agarose gel in 1× tbe buffer. the samples were run at 70 v for 90 min. the gels were then visualised using the blook led transilluminator (genedirex, taiwan). tab. 1: sequences of oligonucleotide primers used for rapd analysis primer sequence number of number of percent name score bands polymorphic poly bands morphism opa08 gtgacgtagg 8 7 87.5 ubc106 aggagtcgga 6 4 66.6 ubc120 agacccttgg 7 7 100 ubc155 ctggcggctg 8 3 37.5 ubc184 caaacggcac 6 5 83.3 ubc215 tcacacgtgc 5 2 40 ubc237 cgaccagagc 5 0 0 ubc275 ccgggcaagc 5 4 80 statistical analysis the extractions were performed in triplicate. the data were analysed by using the ibm spss statistical software version 22. the means were subjected to comparison by using the one-way analysis of variance (anova) and the ducan’s post hoc multiple comparisons at the 99 % confidence level. the banding pattern for each primer was scored as diallelic (1 = band present, 0 = band absent), and stored in an excel (microsoft) spreadsheet file in the form of a binary matrix. in order to assess the genetic differentiation between the three garlic accessions, eight rapd markers were analysed using the statistical package xlstat version 2015.5.01.23234 software. the coefficients of genetic similarity for all the pair-wise comparisons were computed using the jaccard’s coefficient of similarity, and then the distance matrix was subjected to cluster analysis by using the unweighted pair group method with arithmetic mean (upgma) to produce a dendrogram. results and discussion garlic bulb is solitary with globose to applanate-globose shapes depending upon the variety. in general, garlic is 2-6 cm in diameter, consisting of several bulblets. the bulblets are covered with white to purple tunic and underneath the tunic are layers of scales which are held together by the basal plate. the basal plate is located at the bottom of the bulb (wu et al., 1998). morphological differences of the thai garlic bulbs used in this experiment are shown in tab. 2 and fig. 1. the major chemical components of garlic are sulphur-containing compounds which are measured in the forms of cysteine sulfoxides [i.e., alliin (1)] and non-volatile γ-glutamylcysteine peptides (>82 %) (sendl, 1995). the representatives of this group are as shown in fig. 2: thiosulphinates [i.e., allicin (2)], ajoenes [i.e., e-ajoene (3) chemical and genetic variations of thai garlic bulbs 237 fig. 1: garlic bulb and bulblets of a. sativum l. cv. thai (a-b), cv. chinese (c-d) and cv. pingpong (e-f). scale bar = 5 cm. tab. 2: the morphological descriptions of three garlic cultivars cultivars bulb weight shape colour of no. of clove size weight garlic flavour (cm) (g) tunic bulblets (cm) (g) th 3.7–4.3 14.78–20.85 round pinkish white 21–23 1×2–2.3 0.79–1.33 ++ ch 1.8–2.5 7.91–12.87 oval opaque 4–11 0.9-2.1×1.9-3.0 0.68–2.35 +++ white–purple pp 5–5.5 32.23–51.33 round pinkish 12–14 1.3-1.7×3.2-3.5 2.28–5.97 ++ white–purple and z-ajoene (4)], vinyldithiins [i.e., 2-vinyl-(4h)-1,3-dithiin (5) and 3-vinyl-(4h)-1,2-dithiin (6)] and sulphides [i.e., diallyl disulphide (7) and diallyl trisulphide (8)]. among those, diallyl sulphides (57 %), allyl methyl (37 %) and dimethyl monoto hexasulphides (6 %) are most commonly found in garlic essential oil (cerella et al., 2011). essential oil from garlic specimens of the th, ch and pp was analysed for the chemical profiles by gc-ms. all the tested samples consisted of similar major chemical components, which were trisulphide, di-2-propenyl (40.9745.17 %), disulphide, di-2-propenyl (cas) (16.95-25.59 %) and trisulphide, methyl 2-propenyl (cas) (13.41-14.43 %) (tab. 3). these results are in line with the findings of the work reported by lalla (2013). the garlic essential oil from a. sativum for. pekinense makino had disulphide, di-2-propenyl, 32.82 %, and trisulphide, di-2-propenyl, 29.12 %, and essential oil of a. sativum var. jam nagar gave trisulphide, di-2-propenyl, as high as 60 %, and disul phide, di-2-propenyl, 13.07 % (pyun and shin, 2006; sowbhagya fig. 2: the chemical structures of sulphur-containing compounds found in garlic. 238 s. sommano, n. saratan, r. suksathan, t. pusadee ta b. 3 : c he m ic al p ro fil es o f g ar lic e ss en tia l o il an al ys ed b y g c -m s n o. c om po un ds % o f t ot al c om po un ds o f g ar lic e ss en ti al o il t h 1 c h 1 p p 1 m or oc co c hi ne se e gy pt ia n m ex ic an fo r. p e tu ni si an va r. va r. or ig in 2 or ig in 3 or ig in 3 or ig in 3 ki ne ns e or ig in 5 ja m n ag ar 6 sa ti vu m 7 m ak in o4 1 1, 2d itr ia cy cl ope nt an e 0. 83 ±0 .0 5a 0. 85 ±0 .1 1a 0. 81 ±0 .1 4a 2 1pr op en e, 3, 3th io bi s (c a s) 0. 74 ±0 .0 6a 0. 45 ±0 .0 6a 1. 39 ±0 .1 2b 3. 93 3 d is ul ph id e, m et hy l 2 – p ro pe ny l ( c a s) 2. 07 ±0 .2 0a 1. 63 ±0 .1 0a 4. 12 ±0 .1 4b 1. 71 4 3c hl or oc ro to na lde hy de 1. 28 ±0 .4 6a 0. 97 ±0 .2 2a 0. 99 ±0 .3 8a 5 e th ox yc yc lo he xy ld im et hy ls ila ne 6 a lly l t hi ol 0. 15 0. 21 0. 14 7 m et hy l a lly l s ul ph id e 0. 64 1. 21 3. 15 8 d im et hy l d is ul ph id e 0. 23 0. 41 0. 88 1 9 d ia lly l s ul ph id e 4. 25 3. 69 6. 96 2. 30 1. 20 10 m et hy l a lly l d is ul ph id e 2. 23 5. 37 15 .2 5 1. 70 11 a lly l m et hy l s ul ph id e 12 d im et hy l d is ul ph id e 13 d ia lly l s ul ph id e 14 a lly l m et hy l d is ul ph id e 15 3, 3t hi o bi s1pr op en e 0. 87 16 2, 4d im et hy l t hi op he ne 0. 03 17 2, 5d im et hy l t hi op he ne 0. 06 18 m et hy l c is -p ro pe ny l d is ul ph id e 0. 13 19 m et hy l t ra ns -p ro pe ny l d is ul ph id e 0. 24 20 n ,n ’d im et hy l t hi ou re a 1. 46 21 tr is ul ph id e, d im et hy l ( c a s) 0. 80 ±0 .1 7a 0. 58 ±0 .1 1a 0. 85 ±0 .1 4a 0. 18 1. 3 1. 44 0. 51 22 d is ul ph id e, d i2pr op en yl (c a s) 14 .7 4± 3. 72 a 16 .0 0± 0. 66 a 18 .0 6± 4. 74 a 14 .3 0 32 .8 2 13 .0 7 23 d ia lly l d is ul ph id e 2. 19 ±0 .9 0a 0. 92 ±0 .1 0a 1. 60 ±0 .4 4a 0. 46 24 d ia lly l d is ul ph id e 1. 64 ±0 .1 1a 1. 25 ±0 .1 1a 3. 19 ±0 .4 0b 25 m et hy l i so pe nt yl d is ul ph id e 19 .0 0 26 m et hy l a lly l d is ul ph id e 27 d ip ro py l d is ul ph id e 0. 11 28 d ia lly l d is ul ph id e 29 tr an spr op en yl p ro py l d is ul ph id e 0. 30 30 tr is ul ph id e, m et hy l 2 -p ro pe ny l ( c a s) 11 .3 4± 1. 37 a 13 .1 4± 0. 17 a 11 .1 2± 1. 54 a 10 .8 8 7. 40 31 a lly 1 m et hy l t ri su lp hi de 32 m et hy l p ro py l t ri su lp hi de 0. 15 33 1, 3, 5tr ith ia ne 0. 26 34 3m et hy l t hi o 1pr op en e 0. 03 35 4, 5d im et hy l t hi az ol e 0. 02 36 1m et hy l t hi o1pr op en e 0. 12 37 3v in yl -1 ,2 -d ith ia ne 1. 46 1 d at a ar e ex pr es se d as m ea n ± sd o f t ri pl ic at e hy dr od is til la tio n (o g ) e xt ra ct s. m ea ns in th e sa m e ro w w ith d iff er en t l et te rs (a –b ) a re s ig ni fic an tly d iff er en t ( p <0 .0 1) , a n o va , d uc an , i b m s ps s st at is tic s. 2 g ar lic es se nt ia l o il of m or oc co o ri gi n (d o u ir i e t a l., 2 01 4) 3 g ar lic e ss en tia l o il of c hi ne se , e gy pt ia n an d m ex ic an o ri gi n (s h a a t h a nd f l o r e s, 1 99 5) 4 g ar lic e ss en tia l o il of f or . p ek in en se m ak in o of s eo ul ( py u n a nd sh in , 2 00 6) 5 g ar lic e ss en tia l o il of t un is ia n or ig in (d z ir i e t a l., 2 01 4) 6 g ar lic e ss en tia l o il of j am n ag ar v ar ie ty o f i nd ia (s o w b h a g y a e t a l., 2 00 9) 7 g ar lic e ss en tia l o il of s at iv um v ar ie ty o f i ra n (k h a r a z i, 20 05 ). d at a w as c om pa re d w ith ou t c on si de ra tio n of d iff er en t g c m s co nd iti on s fr om d iff er en t r ep or ts . chemical and genetic variations of thai garlic bulbs 239 38 2v in yl -1 ,3 -d ith ia ne 3. 42 39 3v in yl -[ 4h ]1, 2di th iin 2. 00 ±0 .4 3a 1. 83 ±0 .0 9a 1. 43 ±0 .2 2a 2. 76 1. 99 40 d ia lly l t ri su lp hi de 41 2v in yl -[ 4h ]1, 3di th iin 1. 64 1. 85 42 2v in yl +h ) 1 ,3 -d ith iin 5. 87 43 1, 2, 3t hi ad ia zo le ,5 -m et hy l(c a s) 2. 12 ±0 .1 0a 2. 42 ±0 .3 0a 1. 71 ±0 .2 3a 44 3v in yl -1 ,2 -d ith ia cy cl oh ex -5 -e ne 4. 60 ±0 .5 4a 4. 61 ±0 .2 4a 3. 44 ±0 .1 3a 45 2, 5d im et hy lth ia zo le 0. 01 46 tr is ul ph id e, d i2pr op en yl 21 .5 9± 11 .0 5a 42 .5 7± 2. 60 a 14 .0 3± 8. 44 a 46 .5 2 29 .1 2 60 .0 0 47 is ob ut yl is ot hi oc ya na te 1. 57 ±0 .3 0a 1. 28 ±0 .2 0a 1. 73 ±0 .1 6a 48 3, 5d ie th yl -1 ,2 ,4 -t ri th io la ne 1. 00 49 5m et hy l1, 2, 3, 4te tr at hi acy cl oh ex an e 0. 72 ±0 .0 9b 0. 45 ±0 .1 1b 50 d is ul ph id e, m et hy l 2 -p ro pe ny l ( c a s) 0. 90 ±0 .2 1b 0. 64 ±0 .1 5b 51 1, 2d ih yd ro cy cl ob ut ab en ze ne 0. 64 ±0 .2 1a 1. 00 ±0 .2 7a 0. 50 ±0 .1 1a 52 d ia lly l d is ul ph id e 4. 34 ±0 .8 1a 4. 74 ±1 .1 0a 4. 01 ±1 .0 2a 28 .6 27 .4 5 42 .4 6 53 tr an spr op en yl m et hy l d is ul ph id e 0. 40 54 su l ph u r ,m o l .(s 8) 1. 15 ±0 .1 8a 1. 28 ±0 .1 3a 0. 87 ±0 .1 6a 55 a lly l p ro py l d is ul ph id e 0. 57 0. 74 0. 28 56 c 6h 10 s 2 (t en t i d ) 0. 91 1. 92 0. 03 57 m et hy l a lly l t ri su lp hi de 6. 77 16 .8 2 10 .3 6 58 d im et hy l t ri su lp hi de 0. 20 59 d ia lly l d is ul ph id e 29 .1 0 60 m et hy l a lly l t ri su lp hi de 10 .4 0 61 2v in yl -1 .3 -d ith ia ne 3. 90 62 1. 4d im et hy l t et ra su lp hi de 0. 40 63 d ia lly l t ri su lp hi de 50 .9 2 35 .3 0 12 .5 2 37 .3 0 3. 20 64 e ug en ol 0. 40 65 α -c ar yo ph yl le ne 0. 30 66 α -g ua ie ne 1. 00 67 a ro m ad en dr en e 1. 70 68 α -b is ab ol en e 2. 10 69 γc ad in en e 4. 30 70 d i2pr op en yl te tr as ul ph id e 5. 01 71 d ia lly l t et ra su lp hi de (t en t i d ) 0. 74 0. 70 1. 09 6. 35 3. 00 72 1, 4d im et hy l t et ra su lp hi de 0. 28 73 1, 2d ith ia ne -4 -o ne 0. 05 ta b. 3 : c he m ic al p ro fil es o f g ar lic e ss en tia l o il an al ys ed b y g c -m s (c on tin ue d) n o. c om po un ds % o f t ot al c om po un ds o f g ar lic e ss en ti al o il t h 1 c h 1 p p 1 m or oc co c hi ne se e gy pt ia n m ex ic an fo r. p e tu ni si an va r. va r. or ig in 2 or ig in 3 or ig in 3 or ig in 3 ki ne ns e or ig in 5 ja m n ag ar 6 sa ti vu m 7 m ak in o4 1 d at a ar e ex pr es se d as m ea n ± sd o f t ri pl ic at e hy dr od is til la tio n (o g ) e xt ra ct s. m ea ns in th e sa m e ro w w ith d iff er en t l et te rs (a –b ) a re s ig ni fic an tly d iff er en t ( p <0 .0 1) , a n o va , d uc an , i b m s ps s st at is tic s. 2 g ar lic es se nt ia l o il of m or oc co o ri gi n (d o u ir i e t a l., 2 01 4) 3 g ar lic e ss en tia l o il of c hi ne se , e gy pt ia n an d m ex ic an o ri gi n (s h a a t h a nd f l o r e s, 1 99 5) 4 g ar lic e ss en tia l o il of f or . p ek in en se m ak in o of s eo ul ( py u n a nd sh in , 2 00 6) 5 g ar lic e ss en tia l o il of t un is ia n or ig in (d z ir i e t a l., 2 01 4) 6 g ar lic e ss en tia l o il of j am n ag ar v ar ie ty o f i nd ia (s o w b h a g y a e t a l., 2 00 9) 7 g ar lic e ss en tia l o il of s at iv um v ar ie ty o f i ra n (k h a r a z i, 20 05 ). d at a w as c om pa re d w ith ou t c on si de ra tio n of d iff er en t g c m s co nd iti on s fr om d iff er en t r ep or ts . 240 s. sommano, n. saratan, r. suksathan, t. pusadee fig. 3: the dendrogram of the chemical composition of garlic oil obtained from a. sativum l. cv.thai (th), cv. chinese (ch) and cv. pingpong (pp), of morocco origin (douiri et al., 2014), var. jam nagar (sowbhagya et al., 2009), for. pekinense makino (pyun and shin, 2006), var. sativum (kharazi, 2005), egyptian origin, chinese origin, mexican origin (shaath and flores, 1995) and tunisian origin (dziri et al., 2014) derived by upgma from the similarity matrix of 75 chemical compositions. fig. 4: the dendrogram of three garlic cultivars, cv. thai (th), cv. chinese (ch) and cv. pingpong (pp), derived by upgma from the similarity matrix of 75 chemical compositions. et al., 2009). garlic essential oils of different origins (viz., chinese, egyptian, mexican and tunisian) also contained two major compounds, diallyl trisulphide (12.52-50.92 %) and diallyl disulphide (28.6-42.46 %) (shaath and flores, 1995; dziri et al., 2014). cluster analysis of 75 chemical compositions of garlic essential oil from the three cultivars and eight references divided garlic into three groups on a dendrogram (fig. 3). group 1 includes garlic cv. ch, cv. th, of morocco origin (douiri et al., 2013), cv. pp, var. jam nagar (sowbhagya et al., 2009), and for. pekinense makino (pyun and shin, 2006). group 2 includes garlic var. sativum (kharazi, 2005) and group 3 includes garlic of egyptian, chinese, mexican (shaath and flores, 1995) and tunisian origin (dziri et al., 2014) (fig. 3). the th, ch and pp were classified into the same group where the ch and the th showed 0.997, while the pp gave 0.981 similarity values (fig. 4). identification of sulphur-containing compounds from the three cultivars was achieved on tlc. staining these compounds with ninhydrin revealed the compounds with rf to be in the range of 0.180.2 in all the tested cultivars and extraction methods (tab. 4). this result is in agreement with the report by belemkar (2013), which suggested the rf of sulphur-containing compounds as being about 0.2, and those compounds were identified as diallyl disulphide, diallyl sulphide, alliin and thiosulphinate. however, the oil extracted by the hydrodistillation of the pp showed no evidence of the presence of such compounds. the chemical component of garlic essential oil varied, depending largely upon the method of extraction: for example, allylsulphides were apparent in hydrodistillated oil, while solvent extraction using ethanol at the ambient temperature gave thiosulphinates, and temperatures below 0 °c were suitable for alliin and amino acid (li et al., 2010; sendl, 1995). as one of the heat stable compounds found in garlic, the alliin content was analysed using spectrophotometry (miron et al., 2002). the alliin content of the oil extract by hydrodistillation of the three cultivars was in the range of 40.0-90.0 mg/100 ml, while the content was less in the oil extracted using microwave-assisted apparatus (15.0-60.0 mg/100 ml) (tab. 4). in any case, the ch gave the highest alliin content among the tested cultivars. variations in the alliin content among the cultivars and because of the types of processing as well as products have also been observed in previous studies (bhandari et al., 2014; velíšek et al., 1997). alliin is a bioactive compound that shows high anti-human colon cancer anti-stomach cancer activities and stimulates peripheral blood cell immune functions (khanum et al., 2004; salman et al., 1999). in addition, several reports have illustrated its health benefits including biological activities such as anti-platelet aggregatory effect, in addition to decreasing of systolic blood pressure, lipid peroxides, hl-60 human leukaemia uric acid, blood glucose, total lipid, triglyceride and cholesterol, glycemic controlling, antioxidant system modulation of red blood cell and anti-atherogenic effect (yun et al., 2014; anwar and meki, 2003; liu et al., 2005; wu et al., 2001; jain and konar, 1978). in this study, rapd-pcr fingerprints were generated from three garlic cultivars (cv. th, cv. ch and cv. pp). ten randomly designed 10-mer oligonucleotide primers were initially used for screening the dna samples to obtain reproducible rapd fingerprints. out of the ten primers tested, only eight primers provided consistent well-resolved and reproducible band patterns, and were therefore selected for further analysis.the total number of fragments observed among the three garlic cultivars based on the rapd analysis with eight polymorphic primers was 45 fragments (fig. 5). a dendrogram based on the similarity matrix generated with the rapd primers is presented in fig. 6. the dendrograms at a similarity value of 0.93 grouped the th and the ch together, while the pp was separated into different clusters with 0.31 genetic similarity. tab. 4: quantitative and qualitative comparisons of sulphur-containing compounds in garlic bulbs thai, chinese and pingpong cultivars garlic time of % yield sulphur-containing compounds alliin content essential extraction retention factor (rf) (mg 100/ml) oil (min) th ch pp th ch pp th ch pp og 120 0.36 ± 0.01b 0.39 ± 0.02b 0.45 ± 0.02a 0.20 0.19 88.70 ± 0.20b 91.53 ± 0.11a 40.76 ± 0.20c mog 45 0.22 ± 0.02a 0.21 ± 0.01a 0.16 ± 0.01b 0.19 0.20 0.18 15.04 ± 0.29c 60.43 ± 0.18a 22.91 ± 0.09b all data are expressed as mean±sd of triplicate measurements. means in the same row with different letters (a–c) are significantly different (p<0.01), anova, ducan, ibm spss statistics. chemical and genetic variations of thai garlic bulbs 241 it is noteworthy that the alliin content of the garlic essential oil from the ch was higher than that of the th from both hydrodistillation and microwave hydrodistillation. the pp of garlic was genetically classified in clusters different from the th and the ch. the extraction yield and the alliin content of the pp were also significantly different from the other two cultivars.in summary, garlic cv. th and cv. ch were similar in chemical and genetic profiles. however, considering the higher content of bioactive alliin, the ch of garlic can be considered as the potential cultivar that can be used as raw material in food supplements and for further study. acknowledgements the financial support for this research and development was provided, together with the 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(ed.), food flavors: generation, analysis and process influence, proceedings of the 8th international flavor conference, 2025-2037. developments in food science. vol. 37. elsevier science. sommano, s., kerdtongmee, p., chompoo, m., nisoa, m., 2015: fabrication and characteristics of phase control microwave power for jasmine volatile oil extraction. j. essent. oil res. 27, 316-323. sowbhagya, h.b., punmima, k.t., florence, s.p., rao, a.g.a., srinivas, p., 2009: evaluation of enzyme-assisted extraction on quality of garlic volatile oil. food chem. 113, 1234-1238 trirongjitmoah, s., juengmunkong, z., srikulnath, k., somboon, p., 2015: classification of garlic cultivars using an electronic nose. com put. electron. agric. 113, 148-153. velíšek, j., kubec, r., davídek, j., 1997: chemical composition and classification of culinary and pharmaceutical garlic-based products. z. lebensm. unters. forsch. a 204, 161-164. © the author(s) 2016. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). weinberg, d.s., manier, m.l., richardson, m.d., haibach, f.g., 1993: identification and quantification of organosulfur compliance mar kers in a garlic extract. j. agric. food chem. 41, 37-41. wu, c.c., sheen, l.y., chen, h.-w., tsai, s.-j., lii, c.-k., 2001: effects of organosulfur compounds from garlic oil on the antioxidation system in rat liver and red blood cells. food chem. toxicol. 39, 563-569. wu, z., raven, p.h., hong, d., missouri botanical garden, 1998: flora of china. illustrations. beijing st. louis, science press, missouri botanical garden. yun, h.-m., ban, j.o., park, k.-r., lee, c.k., han, h.-s.b., hong, j.t., 2014: potential therapeutic effects of functionally active compounds isolated from garlic. pharmacol. ther. 142, 183-195. address of the corresponding author: chiang mai university, department of plant science and soil science, faculty of agriculture, chiang mai 50200, thailand e-mail: sarana.s@cmu.ac.th journal of applied botany and food quality 90, 76 82 (2017), doi:10.5073/jabfq.2017.090.011 department of agraria, university of reggio calabria, salita melissari feo di vito, italy antioxidant potential of extra virgin olive oils extracted from three different varieties cultivated in the italian province of reggio calabria vincenzo sicari (received november 8, 2016) summary in this study, the physicochemical properties and bioactive compounds of olive oils from cultivars “roggianella”, “sinopolese” and “ottobratica”, grown in the province of reggio calabria (italy) have been evaluated. polyphenols are a large family of compounds found in fruits and vegetables, which exhibit strong antioxidant activity by scavenging different families of reactive oxygen species (ros). dialdehydic form decarboxymethyl oleuropein aglycon, hydroxytyrosol acetate, dialdehydic form oleuropein aglycon, pinoresinol, 1-acetoxypinoresinol, tyrosol and vanillic acid were the main phenolic compounds in all samples analyzed. pinoresinol was the most abundant compound in the lignin fraction. in all oil samples analyzed the highest antioxidant capacity was attributed to roggianella oil (36.85 % i of dpph and 4.07 % i of abts) compared to ottobratica (27.37 % i of dpph and 2.52 % i of abts) and sinopolese (18.33 % i of dpph and 1.72 % i of abts). the main characteristics of the roggianella cultivar were a very high concentration of total phenols (530 mg/kg of gallic acid) and α-tocopherol (211 mg/kg). keywords: olea europaea; extravirgin olive oil; phenolic compounds; antioxidant activity. introduction the olive tree (olea europaea l.) is one of the most important crops in mediterranean countries, especially italy, spain and greece. extra virgin olive oil shows very interesting nutritional and sensorial properties (visioli and galli, 1998; huang et al., 2008; urpi-sarda et al., 2012). extra virgin olive oil resists oxidation due to its composition. apart from the saponifiable fraction, composed mainly of triglycerides, with a low polyunsaturated fatty acid content, it contains a group of antioxidant molecules made up mainly of polyphenols and tocopherols (coetesi et al., 1995; tsimidou, 1998; visioli and galli, 1998; esti et al., 1998; velasco and dobarganes, 2002; lavelli and bondesan, 2005; bendini et al., 2007; sicari et al., 2009; sicari et al., 2010; giuffrè et al., 2010). the polar phenolic molecules of virgin olive oil belong to different classes: phenolic acids, phenolic alcohols, lignans, secoiridoids and flavonoids (angerosa and di giovacchino, 1996; brenes et al., 1999; owen et al., 2000a; owen et al., 2000b; gomez-alonzo et al., 2002; lavelli and bondesan, 2005). of the antioxidant compounds, it is also important to consider the tocopherols. these are compounds whose concentration strongly depends on the variety of the plant. usually, values found in extra virgin olive oil range from 100 mg to 300 mg/kg. the concentration of phenols in virgin olive oil is strongly affected by agronomic factors, such as the area of origin, the cultivar, the stage of fruit ripening and also by several agronomic procedures and the technological, operative conditions of the oil extraction process (morellò et al., 2004; bruni et al., 1994; servili et al., 2004; cerretani et al., 2005; inglese et al., 2009; suthawan and alyson, 2012; di vaio et al., 2013). for this reason, differences in the chemical make-up of the oil are inevitable, especially as regards the minor polar compounds (antioxidants). phenolic compounds are responsible for the bitterness and pungency of extra virgin olive oil (angerosa and di giovacchino, 1996; andrewes et al., 2003; kalua et al., 2005; kalua et al., 2006). the dialdehydic form decarboxymethyl ligustroside aglycon (p-hpeaeda) is the compound which is most responsible for the pungent taste (beauchamp et al., 2005; kalua et al., 2005). these compounds are related to the health benefits associated with the consumption of olive oil (visioli and galli, 1995; visioli et al., 1995; visioli et al., 1998; petroni et al., 1994; manna et al., 1999; tuck and hayball, 2002; psaltopoulou et al., 2004; franconi et al., 2006). in the present work, the chemical-physical properties, tocopherols, total phenols and the phenolic fraction of three italian cultivar extra virgin olive oils has been evaluated. materials and methods plant material and growing areas selected the study was carried out on the three different cultivars (ottobratica, sinopolese and roggianella), grown in the experimental orchards belonging to the calabrian regional government, located near gioia tauro in the province of reggio calabria, italy. the agronomic processes carried out were the same for each cultivar: fertilizing, spraying for parasites, and pruning. the olives were harvested by hand in mid-october 2015 directly from the plant, and immediately taken to the laboratory, where the oil was extracted within 12 hours of harvesting, using a laboratory “mini 30” made by agrimec (firenze, italy). after crushing and malaxing of the olive paste, oil extraction was performed at room temperature by means of a pressure system. the olive paste was mixed for 30 min, and then a pressure of 200 atm was applied for 40 min. the oil was separated from the water (by centrifuge), then filtered through a paper at room temperature and analyzed immediately. reagents gallic acid (ga), p-hydroxyphenyl-ethanol, vanillic acid, caffeic acid, p-coumaric acid, ferulic acid, sinapic acid, o-coumaric acid, apigenin, luteolin and oleuropein were from extrasynthese (france). α, β and γ-tocopherol was from sigma-aldrich (italy). all reagents used of hplc or analytical grade from carlo erba (milan, italy). peroxide, free acidity and rancimat test quality parameters (peroxide value, acidity value and uv absorption characteristic) were evaluated following the methodology proposed by reg. (cee) 2568/91, and later modifications of the reg. (cee) antioxidant compounds in extra virgin olive oils 77 2003. oxidative stability was carried out using a rancimat apparatus. the temperature was maintained at 110 °c during analysis, and airflow was 12 l/h. the analyses were carried out in triplicate. tocopherol analysis the tocopherol contents were estimated with the iupac 2.432 method (iupac, 1987). analytical hplc was conducted using a knauer liquid chromatography (asi advanced scientific instruments, berlin germany) and a uv-vis a diode array detector (dad). a normal phase column lichrosphere si60 (250 mm length, 4.6 mm i.d. and 5 μm particle size) was used with an injection volume of 20 μl and a flow rate of 1.0 ml/min. the absorbance was measured at 295 nm. the results were expressed as mg of tocopherol per kg of oil. tocopherols were quantified by an external standard method. determination of total phenols and phenol content by hplc the total phenols were determined using the folin ciocalteu’s reagent method (singleton and rossi, 1965). briefly, 10 ml of sample were taken from the solution and placed into a flask of 100 ml, to which were added 50 ml of ultrapure water, 5 ml of folin ciocalteu’s reagent, 20 ml of na2co3 (15 % in water) and finally made up to 100 ml with ultrapure water. the flask was left in the dark for 2 hours and then the solution was subjected to spectrophotometric analysis at 750 nm, with a perkin elmer apparatus (model lambda 2). phenolic compounds in different olive varieties were determined by hplc (pirisi et al., 2000). the hplc analysis was performed using a knauer instrument (asi advanced scientific instruments, berlin germany), and a uv-vis diode array detector (dad). the column used was a nova-pak c-18 with the following features: 300 mm length, 3.9 mm i.d., and 4 μm particle size (waters – italy). the injection volume was 10 μl of the phenolic solution. two mobile phases were used, the first (a) water/acetic acid (98:2, v/v) and the second (b) methanol/acetonitrile (1:1, v/v), with a flow rate of 1 ml/ min. the gradient used was as follows: 0-25 min 95 % a/5 % b; 25-35 min 70 % a/30 % b, 35-40 min 60 % a/40 % b, 40-50 min 52 % a/ 48 % b; 50-55 min 30 % a/70 % b; 55-60 min 0 % a/100 % b; 60 65 min 95 % a/5 % b. the monitoring wavelength was 280-320 nm. the identification of each compound was based on a combination of retention time and spectral matching. antioxidant activity total antioxidant activity was estimated by abts (radical sca venging) and dpph (free radical scavenging activity). the radical scavenging capacity of the samples for the abts (2,2’-azinobis3-ethylbenzothiazoline-6-sulfonate) radical cation was determined as described by re et al. (1999). abts (7 mm) and k2s2o8 (140 mm) were mixed and stored in the dark at room temperature for 16 h before use. the mixture was diluted (1:80) with ethanol to give an absorbance at 734 nm using a spectrophotometer. for each olive oil sample, a diluted methanol solution of the sample (100 μl) was allowed to react with fresh abts solution (900 μl), and then the absorbance was measured 6 min after initial mixing. all measurements were performed in triplicate. radical scavenging activity was studied using 1,1-diphenyl-2 picrylhydrazyl free radical (dpph) as described by blois (1958), with some modifications: for each olive oil, 1.5 ml was mixed with 1.5 ml of a 0.2 mm methanolic dpph solution. after an incubation period of 30 min at 25 °c, the absorbance at 515 nm was measured. the free radical-scavenging activity of each solution was then calculated as percentage inhibition. all measurements were performed in triplicate. statistical analysis all parameters analyzed were carried out in triplicate. the results were reported as mean values of three repetitions and standard deviation. the data were subjected to analysis of variance (one-way anova) using spss 15.0 for windows (spss, chicago, il, usa). separation of the means was obtained using tukey’s test, and significant difference was defined as (p ≤ 0.05). results the mean values obtained for the different parameters analyzed in the fruit are shown in tab. 1 and 2. the analyzed olive oils (tab. 1) showed good values for the regulated physicochemical parameters evaluated. free acidity is measured as free fatty acids expressed as a percent of oleic acid and should be ≤ 0.8 %; peroxides are expressed as milliequivalent of free oxygen per kilogram of oil ≤ 20 meq o2/ kg. the quality parameters did not exceed the limits established for the best commercial quality olive oil, designated as “extra virgin olive oil category”, by cee (1991/2568) and later modifications of the cee (2003). the lowest values of acidity were those of the cultivar sinopolese (0.28 %), while those of ottobratica and roggianella were 0.31 and 0.44 %, respectively. as shown in tab. 1, the oils showed peroxide values of 3.85, 4.32 and 5.03 meq o2/kg of oil in roggianella, ottobratica and sinopolese, respectively. the amounts of total phenols in analyzed oils show significant differences between different cultivars (tab. 1). the highest content of tab. 1: levels of total phenols, tocopherols, antioxidant activity and rancimat test of extra virgin olive oil from three calabrian cultivars sinopolese roggianella ottobratica sign. p<0.05 acidity (av) (% oleic acid) 0.28±0.03 b 0.44±0.01a 0.31±0.12 b ** peroxide (pv) (m eq o2/kg) 5.03±0.14 b 3.85±0.56 a 4.32±0.15 b ** total phenols (mg/kg of gallic acid) 367.43±7.07 b 530.14±8.41a 417.74±5.68 c ** α-tocopherol (mg/kg) 145.21±4.5 b 235.51±7.67 a 177.59±8.04 b * (β + γ) tocopherols (mg/kg) 29.44 b 51.26 a 37.16 c ** total tocopherols (mg/kg) 174.65 286.77 214.75 dpph° (% i) 18.33±2.83 a 36.85±3.25 c 27.37±2.09 b ** abts° (% i) 1.72±0.03 b 4.07±0.14 a 2.52±0.02 b * rancimat 110 °c (h) 18.76 27.45 21.33 ** data are expressed as mean ± sd from three replications. values having different superscripts are significantly (p < 0.05) 78 v. sicari this fraction was detected in roggianella oil (530 mg/kg), while the lowest value was detected in sinopolese (367 mg/kg). the results are in agreement with other mediterranean oil olives (franconi et al., 2006; esti et al., 1998). the α-tocopherol content had a mean value of 145.21 mg/kg, 235.51 mg/kg and 177.59 mg/kg for sinopolese, roggianella and ottobratica, respectively (tab. 1). these results are similar to those described by aguilera et al. (2005); allalout et al. (2009); escuderos et al. (2009); nakbi et al. (2010). tab. 1 also shows the values obtained from the rancimat test, expressed in hours. the longest time before induction was for the roggianella cultivar (27.45 h), followed by ottobratica and sinopolese (21.33 h and 18.76 h, respectively). these results agree with those from the literature (aparicio et al., 1999; baccouri et al., 2008; dabbou et al., 2010). tab. 1 shows the antioxidant activity determined by dpph and abts assay. a positive correlation was observed between the antioxidant activity and the concentration of total polyphenols and α-tocopherol. the extra virgin olive oil from the roggianella cultivar had a higher antioxidant activity than the oils from ottobratica and sinopolese cultivars. five classes of compounds were identified by phenolic hplc analy sis: phenolic acids, phenolic alcohols, lignans, secoiridoids and flavonoids (montedoro, 1972; montedoro et al., 1992; mateos et al., 2001) (tab. 2). anova analysis showed significant differences between the phenolic contents of different cultivars (tab. 2). the major phenolic compounds (tab. 2 and fig. 1) identified in the olive oils were secoiridoids (dialdehydic form decarboxymethyl oleuropein aglycon (3.4-dhpea-eda) and lignans (pinoresinol, 1-acetoxypinoresinol). the secoiridoidic derivatives of oleuropein and ligstroside have been described previously (montedoro et al., 1993; owen et al., 2000b). the major phenolic acids were vanillic acid, 4-hydroxybenzoic acid, p-coumaric acid p-coumaric acid, ferulic acid and cinnamic acid. the concentration of total phenolic acids was 49.92 mg/kg in ottobratica oil, and 37.63 and 31.99 mg/kg in roggianella and sinopolese, respectively. of the phenolic acids, cinnamic acid showed the greatest concentration in all samples analyzed, while vanillic acid and p-coumaric acid show the highest concentration in oil from ottobratica cultivar. the concentration of these compounds is largely affected by agronomic and technological conditions of virgin olive oil production (servili and montedoro, 2002). the main phenolic alcohols were hydroxytyrosol (3.4-dhpea) and tyrosol (p-hpea) as reported in the literature by selvaggini et al. (2006) and bendini et al. (2007). their concentration is generally low in freshly pressed oil, but this increases with oil storage due to the effect of hydrolysis of secoiridoid compounds (montedoro et al., tab. 2: phenolic compounds in extra virgin olive oil obtained from the cultivars sinopolese, roggianella and ottobratica compounds sinopolese mg/kg roggianella mg/kg ottobratica mg/kg sign. p<0.05 vanillic acid 8.44±0.71b 7.05±0.37b 14.17±0.89a ** 4-hydroxybenzoic acid 0.91±0.17b 0.65±0.04b 1.76±0.25a ** p-coumaric acid 7.27±0.25b 7.18±0.40b 14.39±2.30a ** o-coumaric acid 1.24±0.15b 2.48±0.38a 0.65±0.07b ** ferulic acid 2.51±0.40b 0.48±0.15c 4.60±0.60a ** cinnamic acid 11.62±0.94b 19.79±1.64a 14.35±1.31b ** hydroxytyrosol (3,4-dihydroxyphenyl-ethanol) 2.57±0.47a 3.03±0.34a 3.28±0.22a n.s. tyrosol (p-hydroxyphenyl-ethanol) 12.95±0.95b 10.62±0.39c 15.39±1.09a ** (+) 1-acetoxypinoresinol 18.14±0.23b 24.45±0.56a 23.02±1.41c ** (+) pinoresinol 58.87±2.32b 51.31±0.79c 88.49±3.55a ** dialdehydic form decarboxymethyl oleuropein aglycon 50.43±2.51b 48.91±2.41b 58.72±2.23a ** (3,4 dhpea-eda) dialdehydic form decarboxymethyl ligustroside aglycon 7.46±0.34b 8.57±0.25a 7.98±0.14b ** (p-hpea-eda) hydroxytyrosol acetate 21.36±0.50b 32.99±1.23a 7.31±1.03c ** oleuropein aglycon 0.21±0.03c 0.72±0.10a 0.53±0.01b ** dialdehydic form oleuropein aglycon 8.20±0.33b 20.53±0.55a 4.77±0.19c ** dialdehydic form ligstroside aglycon 1.78±0.12a 1.93±0.14a 1.56±0.28a n.s. apigenin 0.90±0.13b 1.97±0.18a 0.24±0.03c ** luteolin 1.54±0.51b 2.74±0.19a 1.13±0.25b ** phenolic acids (mg/kg) 31.99 37.63 49.92 phenolic alcohols (mg/kg) 15.52 13.65 18.67 lignans (mg/kg) 77.02 75.76 112.31 secoiridoids (mg/kg) 89.44 113.65 80.87 flavonoids (mg/kg) 2.44 4.71 1.37 total poliphenols hplc (mg/kg) 216.41 245.40 263.14 data are expressed as mean ± sd from three replications. values having different superscripts are significantly (p < 0.05) different antioxidant compounds in extra virgin olive oils 79         tab. 1: levels of total phenols, α-tocopherol, antioxidant activity and rancimat test of extra virgin olive oil from three calabrian cultivars 1992). in this current study the hydroxytyrosol content was 2.57, 3.03 and 3.28 mg/kg for sinopolese, roggianella and ottobratica, respectively (tab. 2). the tyrosol content ranged from 10.62 mg/kg in roggianella to 15.39 mg/kg in ottobratica, with an intermediate value in sinopolese (12.95 mg/kg). the content of 3.4-dhpea-eda ranged from 48.91 mg/kg in roggianella oil to 58.72 mg/kg in ottobratica oil. sinopolese oil shows an intermediate content of 50.43 mg/kg. total secoiridoids are present in a higher concentration in oil from roggianella, with a value of 113.65 mg/kg. lower values were obtained in sinopolese and ottobratica: 89.44 and 80.87 mg/kg, respectively. olive oil from roggianella cultivar had a high concentration of hydroxytyrosol acetate (32.99 mg/kg) compared to sinopolese and much higher than ottobratica (21.36 and 7.31 mg/kg), respectively. gordon et al. (2001) and ammendola et al. (2011), have shown that hydroxytyrosol acetate is a more powerful antioxidant than α-tocopherol or hydroxytyrosol, the latter being less soluble in a lipid substrate. studies on olive polyphenols have revealed the importance of the lipophilicity of antioxidants on cell uptake and membrane crossing, and on the substrate to be protected (membrane constituents or ldl) (grasso et al., 2007). pinoresinol and 1-acetoxypinoresinol are major component of the phenolic fraction of olive oil (owen et al., 2000b). they are common components of the lignan fraction. the content of pinoresinol was 51.31, 58.87 and 88.49 mg/kg in roggianella, sinopolese and ottobratica, respectively (tab. 2). the content of 1-acetoxypinoresinol ranged from 18.14 mg/kg in sinopolese to 24.45 mg/kg in roggianella. ottobratica oil had an intermediate concentration 23.02 mg/kg. lignans are an important part of the phenolic fraction of olive oil because they may play a major role in the health promo ting effect of the mediterranean diet (setchell et al., 1981; axelson et al., 1982; owen, 2000b). the flavonoids analyzed included luteolin and apigenin. in all oils analyzed, the greatest concentration was in oil from the roggianella cultivars (1.97 mg/kg of apigenin and 2.74 mg/kg of luteolin). rovelli et al., (1998), reported that flavonoids such as luteolin and apigenin were phenolic components of virgin olive oil. these results confirm this chemical class as a minor constituent of the phenolic fraction as previously described for other virgin olive oils (garcia et al., 2002; mulinacci et al., 2005). discussion the concentration of phenolic compounds is an important factor when evaluating the quality of virgin olive oil because of their involvement in its resistance to oxidation, and its sharp bitter taste (andrewes et al., 2003; esti et al., 1998). the variability of the minor fractions of olive oil cannot always be correlated to definite factors (fiorino and nizzi grifi, 1991), which causes difficulty in reliably identifying the genetic and geographic origin of any single extravergin olive oil. one of the factory that greatly influence the composition of the minor fractions, in particular the phenolic fraction, is the fruit’s degree of ripeness. this means that all the many factors which influence fruit ripening will each have an effect on the quality on the oil (inglese et al., 2009; fiorino and nizzi grifi, 1991). the various cultivars may show different reactions to environmental and agronomic factors. this means that environmental factors, which vary from year to year, can have a notable effect on the com position of the oil from any particular cultivar (lo curto et al., 2001; fiorino and ottanelli, 2004; lombardo et al., 2008; tura et al., 2007; salvador et al., 2001; sweeney et al., 2002; d’imperio et al., 2007). thus, although the three cultivars considered in the present work were all found in the same geographical area, and had undergone the same agronomic processes, each of them had, no doubt, reacted differently to the same environment. this interaction between cultivar and environment will have caused differences in the antioxidant content of the olive oils studied. as it is widely reported in the literature, the phenolic compounds content varies according to degree of ripeness (fiorino et al., 1991; youssef et al., 2009; sicari et al., 2009; giuffrè et al., 2010). olives which are not completely ripe give an oil with a higher content of phenolic compounds compared to oil from fruit which have reached full ripeness. since trees of the sinopolese and ottobratica cultivars grow very tall, a uniform harvest was not possible. often, the first fruit that fall, when using a mechanical vibrator, are those which are ripest and grow on the outermost branches of the tree. on the other hand, the small size of roggianella trees allowed a harvest from each part of the tree, including less mature fruit. the present work has not considered the olive maturity index to avoid creating an overly complex model. the aim of the present work was to study the antioxidant profile of extra virgin olive oil extracted fig. 1: gallic acid (std) (1), hydroxytyrosol (2), tyrosol (3), vanillic acid (4), 4-hydroxybenzoic acid (5), p-coumaric acid (6), ferulic acid (7), o-coumaric acid (8), dialdehydic form decarboxymethyl oleuropein aglycon (9), oleuropein aglycon (10), cinnamic acid (11), dialdehydic form decarboxymethyl ligustroside aglycon (12), (+) pinoresinol (13), (+) 1-acetopinoresinol (14), luteolin (15), dialdehydic form oleuropein aglycon (16), apigenin (17), hydroxytyrosol acetate, dialdehydic form ligstroside aglycon (18). 80 v. sicari from three different cultivars. of the three cultivars, roggianella showed the greatest oxidative stability. this may be explained by the fact that the secoiridoids are present in greater quantity in oil from roggianella than in oil from the other two cultivars. furthermore, the tocopherol content (α, β e γ), is also higher in oil from roggianella compared to oil from the other cultivars. this work confirmed that cultivar is the most important factor influencing the antioxidant profile of the olive oil. conclusions this research confirmed that oxidative stability in virgin olive oil is correlated mainly to polyphenols and tocopherols. the quality of extra virgin olive oil is linked to its phenolic content. phenolic compounds influence not only its shelf-life but also its health and sensorial properties. the cultivar used to produce virgin olive oil is an important factor in determining its content of antioxidant molecules. from the study of the data obtained in this work, it can be noted that roggianella, ottobratica and sinopolese varieties all produced high quality olive oil. roggianella oil showed a higher antioxidant activity compared to ottobratica and sinopolese oils. in particular, the extra virgin olive oil extracted from roggianella cultivar olives had a higher content of 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cardiovascular diseases 5, 306-314. visioli, f., galli, c., 1998: olive oil phenols and their potential effects on human health. j. agric. food chem. 46, 4292-4296. doi: 10.1021/jf980049c. youssef, n.b., zarrouk, w., carrasco-pancorbo, a., ouni, y., seguracarretero, a., fernández-gutiérrez, a., daoud, d., zarrouk, m., 2009: effect of olive ripeness on chemical properties and phenolic composition of chétoui virgin olive oil. j. sci. food agric. 90, 199-204. doi: 10.1002/jsfa.3784. address of the author: vincenzo sicari, department of agraria, university of reggio calabria, salita melissari feo di vito e-mail: vincenzo.sicari@unirc.it © the author(s) 2017. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 88, 127 133 (2015), doi:10.5073/jabfq.2015.088.018 1department of plant breeding & genetics, university college of agriculture, university of sargodha, pakistan 2 department of experimental design and bioinformatics, warsaw university of life sciences, warsaw, poland 3 department of agronomy, warsaw university of life sciences, warsaw, poland 4 international potato center (cip), lima, peru multitraits evaluation of pakistani ecotypes of berseem clover (trifolium alexandrinum l.) under full-irrigation and water restriction conditions muhammad mubashar hussain1, saeed rauf1, jakub paderewski2, ikram ulhaq1, dorota sienkiewicz-paderewska3, philippe monneveux4 (received january 20, 2015) summary berseem clover (trifolium alexandrinum l.) is an important forage crop in pakistan and many ecotypes are grown across the country. its yield is however frequently affected by insufficient irrigation due to unavailability of water. in the present study, twenty pakistani ecotypes of berseem clover have been evaluated in lysimeters under full irrigation and water restriction conditions. in the full irrigation treatment soil humidity was maintained at field capacity, while in the water restriction treatment water was only supplied after severe wilting and to maintain humidity in the deep profile of the soil. assessed traits included forage yield, calculated as the sum of the biomass harvested at 70 and 110 da days after emergence, and morpho-physiological traits. significant effects of water restriction were noted on yield, leaf gas exchange parameters, canopy temperature and osmotic adjustment. most morpho-physiological traits had higher broad sense heritability than forage yield, both under full irrigation and water restriction conditions. water restriction increased genetic and phenotypic variability and heritability of most traits under study. under these conditions forage yield was positively associated to leaf temperature and recovery rate index and, under full irrigation, to net photosynthetic rate, canopy depression temperature and leaf area. the possible use of these traits as indirect selection criteria in berseem clover breeding programs is discussed. some ecotypes with favorable traits such as high forage yield potential, good adaptation to water restriction and aptitude to multiple harvesting have also been identified. introduction trifolium alexandrinum l. (berseem clover or egyptian clover) is one of the most important leguminous forages in the mediterranean region and the middle-east (sardana and narwal, 2000; el bably, 2002; iannnucci, 2002; de santis et al., 2004). it contributes to soil fertility and improves soil physical characteristics (graves et al., 1996) and its forage is superior to grasses in protein and mineral contents (laghari et al., 2000). berseem clover originated in syria and was introduced into egypt in the 6th century (hannaway and larson, 2004), india in the 19th century and pakistan, south africa, the usa and australia in the 20th century (heuzé et al., 2014). it is grown in the semi-arid regions of the world under pure stand and crop mixture (martinello and iannucci, 1998; vasilakoglou and dhima, 2008). limitation of water supply is a major production constraint for this crop (iannucci et al., 2000; lazaridou and koutroubas, 2004). studies concerning the evaluation of tolerance to water restriction are scarce in berseem clover. iannucci et al. (2000) reported a significant reduction in total dry weight, plant height and proline content due to water stress treatments. lazaridou and tsiridis (2004) noted a 75 % decrease in biomass, leaf area and transpiration rate as a result of water restriction. evaluation and selection of berseem clover germplasm in a specific environment have generally concerned small sets of ecotypes and have been based on a single or a reduced number of traits (iannucci et al., 2000; lazaridou and koutroubas, 2004). the present experiment compared the yield of a large set of berseem clover ecotypes originated from different regions of pakistan under full irrigation and water restriction conditions. as in the semi-arid environment and under limited irrigation the berseem plant has often to rely on moisture stored in a deep profile of soil to maintain its growth, the water restriction treatment consisted in maintaining humidity only in the deep profile of the soil. the ability of the different ecotypes to utilize the stored soil moisture content and maintain its yield under these specific water restriction conditions was evaluated using a multiplicity of traits including canopy temperature (and its response to variation in air temperature), gas exchange para meters, osmotic potential, chlorophyll and carotenoid contents, water use efficiency, recovery rate index and wilting rate index, as well as calculating a drought resistance index on the basis of comparisons between the two treatments. the genetic variability and heritability of the assessed traits and their relation with yield were also examined, in order to identify potential criteria for the further selection of ecotypes under stored moisture stress. materials and methods plant material twenty pakistani berseem clover ecotypes were provided by the national agriculture research council and the forage research institute of faisalabad, pakistan. out of them, 17 originated from punjab, one from balochistan, one from khyber pakhtunkhwa and one from singh provinces (tab. 1). experimental conditions the experiments were conducted in lysimeters of 60 cm diameter and 40 cm depth. field conditions are known to exploit full yield potential of the ecotypes but make difficult to ensure uniform moisture storage and avoid confounding factors such as soil heterogeneity or presence of multiple stress factors (araus and cairns, 2014). conversely, the use of lysimeters facilitates better control in the application of uniform treatments (masuka et al., 2012). each lysimeter was filled with 50 kg of dry soil with an equal quantity of silt and loam. soil fertility was increased by adding 5 % of organic matter. the field capacity of the soil, measured using the gravi metric method, was 14 % by weight. the seeds were inoculated before sowing with 5 μl of rhizobium bacterial suspension (rizobium leguminosarum bv. trifolii). the experiment was conducted in a completely randomized design with three replications and two factors, i.e. ecotypes (20) and water regimes (2). the two contrasting water regimes consisted in a full irrigation and a water restriction regime. in the full irrigation regime, soil was flooded when the moisture level fell below the field capacity of the soil, while the water restriction regime was created by irrigating the crop when severe wilting (90 %) 128 m. huassain, s. rauf, j. paderewski, i. ulhaq, d. sienkiewicz-paderewska, p. monneveux tab. 1: list of ecotypes of berseem clover (trifolium alexandrium l.) used in the study number ecotype origin source 1. l-94 punjab, pakistan forage research institute, faisalabad, pakistan 2. anmol punjab, pakistan forage research institute faisalabad, pakistan 3. p-209 punjab, pakistan forage research institute faisalabad, pakistan 4. berseemqueta balochistan, pakistan national agriculture research council, pakistan 5. sandal bad punjab, pakistan forage research institute faisalabad, pakistan 6. agaiti punjab, pakistan forage research institute sargodha, pakistan 7. sb-12 punjab, pakistan forage research institute faisalabad, pakistan 8. berseem peshawar kpk, pakistan national agriculture research council, pakistan 9. berseem tandojam sindh, pakistan national agriculture research council, pakistan 10. pachati punjab, pakistan forage research institute sargodha, pakistan 11. pak berseem punjab, pakistan national agriculture research council, pakistan 12. punjab berseem punjab, pakistan national agriculture research council, pakistan 13. chenab punjab, pakistan national agriculture research council, pakistan 14. samarkand punjab, pakistan national agriculture research council, pakistan 15. l-48 punjab, pakistan national agriculture research council, pakistan 16. sk-1 punjab, pakistan national agriculture research council, pakistan 17. super berseem punjab, pakistan forage research institute faisalabad, pakistan 18. p-22 punjab, pakistan national agriculture research council, pakistan 19. sb-10 punjab, pakistan national agriculture research council, pakistan 20. sandal bar punjab, pakistan forage research institute faisalabad, pakistan was observed for each ecotype, through a 3 cm diameter pipe allowing storing the moisture in the lower profile of the soil (30-40 cm). the quantities of water supplied to each lysimeter were 16.5 l in the full-irrigation regime (11 irrigations) and 4.5 l in the water restriction regime (3 irrigations). the lysimeters were covered with a plastic tunnel to avoid chilling stress or damage due to frost during the night and maintain the optimum temperature for growth (25±3 °c) during the day. relative humidity was around 40 % and photon flux density was 600 μmol m-2 s-1 during the peak photosynthesis hour. air temperature and humidity in the tunnel were registered at regular intervals using a digital thermometer and a hygrometer. thinning was carried out to keep the plant population close to 100 plants per lysimeter. during the entire crop growth cycle, weeds were removed manually and no herbicide or pesticide was applied. the different ecotypes were regularly evaluated for insect and disease, and no attacks were identified during the entire crop cycle. soil and plant trait measurements a soil sampler with a coring cylinder of 3.8 × 10 cm was used to collect soil samples at various depths (10-40 cm) in each lysimeter. the samples were quickly transported to the laboratory without exposure to air. the soil sample was oven-dried at 60 °c (ed-115, binder, tuttlingen, germany) to determine soil moisture content at constant weight. all ecotypes were harvested at 70 and 110 dae. for each harvest, fresh biomass (fb) was measured on a digital balance (gw 6202, sartorius, germany). plants were then dried in a heating oven (ed115, binder, tuttlingen, germany) at 70 °c for 72 hours to determine dry biomass (db). forage yield was estimated as the sum of biomass of the two harvests (krenzer et al., 1992). leaf area (la) was measured on well expanded leaves at 70 days after emergence (dae), using a leaf area meter (ci-202, camas, usa). canopy temperature was assessed at 60 dae using a hand-held infrared thermometer (model ir-aht, chino co., tokyo, japan) at a uniform height (1.5 m), angle (60°) and distance between the thermometer and the target. measurements were made in windless conditions during the afternoon. each measurement was repeated three times. the air temperature was subtracted from the canopy temperature to determine the canopy temperature depression (ctd). net photosynthesis rate (pn), transpiration rate (tr), leaf temperature and ambient air temperature were measured at 68 dae on 10 days old leaves at the top of canopy around noon with a gas exchange apparatus (ci-340, camas, usa). each measurement was repeated three times. leaf temperature depression (ltd) was measured by subtracting the temperature of ambient air around the leaf from the leaf temperature. chlorophyll contents were measured using the acetone extraction method. collected samples (0.5 g of fresh leaf) were dissolved in 15 ml of acetone. the leaf extract was centrifuged at 8000 rpm for 5 minutes and absorbance was assessed at 663 nm, 645 nm and 470 nm using a uv-vis spectrophotometer (uv 2600, schemadzo, japan). chlorophyll a (chla), chlorophyll b (chlb) and carotenoids (car) were calculated according to hiscox and israelstam (1979) as chla = 11.75a663 – 2.35a645, chlb = 18.61a645 – 3.96a663 and car = (1000a470 – 2.27 chla – 81.4 chlb)/227 witha663, a645 and a470 being the absorption values at 663, 645 and 470 nm, respectively. osmotic adjustment (oa) was determined for each ecotype by subtracting the osmotic potential under full irrigation from the osmotic potential under water restriction, after irrigation and recuperation of full turgor, as described by babu et al. (1999). osmotic potential was measured using a vapor pressure osmometer (vapro 5520, wescor, utah, usa). at 70 dae and after irrigation leaf samples were collected in both treatments when 80 % of plants recovered from wilting. the leaf samples were pressed in the eppendorf tube to extract the cell sap which was centrifuged at 8000 rpm for five minutes to collect the supernatants. 10 μl of supernatants were used for the measurement of osmotic potential (in mpa) in both regimes. evaluation of berseem germplasm against drought stress 129 the water used by the plant (wu) was estimated as the product of transpiration rate (tr) by leaf area (lazaridou and noitsakis, 2003). transpiration was asses on fully expanded leaves at the top of canopy with a hand-held photosynthesis system (ci-340 4845 nw, camas, wa, usa) between 10:00 and 11:00 am. plant water use efficiency (wue) was calculated according to lazaridou and koutroubas (2004) as the ratio of above-ground dry biomass to the water transpired by the plant. drought resistance index was calculated as dri = (ys/yn) / (ms/mn) where ys was the biomass of an ecotype under stress (water restriction) conditions, yn the biomass of this ecotype under non-stress (full irrigation) conditions, ms the mean biomass yield of all ecotypes under stress and mn the mean yield of all ecotypes under non-stress conditions. a recovery rate index was calculated for each ecotype as rri = [(r1/t1) + (r2/t2) + (r3/t3)] / r where r1 was the number of plants which recovered from wilting the first day after restoring water in the lower profile of the lysimeter, r2 the number of plants which recovered from wilting during the second day, r3 the number of plants which recovered from wilting during the third day and r the final recovery after 3 days. t1, t2 and t3 had values of 1, 2 and 3, respectively. a wilting rate index was also calculated for each ecotype as wri = (w1/ t1+w2/t2+w3/t3) / % w where w1 was the number of plants showing symptoms of wilting 10 days after restoring the soil moisture content, w2 and w3 being the number of plants that showed symptoms of wilting 2 and 3 days after the first measurement, respectively, and w the total number of wilted plants wilting after 3 days. t1, t2 and t3 had values of 1, 2 and 3, respectively. statistical analyses the data were subjected to the analysis of variance using the cropstat 7.2 software. genotypic and phenotypic coefficients of variation were estimated as gcv % = √genotypic variance/overall average of the ecotypes × 100 and pcv % = √phenotypic variance/ overall average of the ecotypes × 100, respectively. pcv % and gcv % reflected the variation in the germplasm at phenotypic and genotypic levels. the phenotypic variation is the sum of genotypic variance (σ2g)+ environmental variance (σ2e). genetic purity of each of the ecotypes (inbreeding coefficient = 1) was maintained for seve ral generations by growing them in isolation. therefore variation within ecotypes was considered environmental which was used to estimate overall σ2e. variation between ecotypes was used to estimate the phenotypic variance (σ2p). genotypic variance was estimated by subtracting the environmental variance from the phenotypic variance. broad sense heritability (h2) was calculated according to allard (1960) as h2= (σ2g/ σ2p) × 100. a stepwise selection procedure based on the akaike information criterion for multiple regression models (venables and ripley, 2002) was conducted to determine the traits associated with fresh and dry biomass. the plant trait values were used to describe multiple regression models for fresh forage yield (sum of first and second harvest) and dry forage yield (sum of first and second harvest), both measured in the water restriction treatment. calculation of regressions and stepwise selection were carried out using the ‘lm’ and ‘step’ procedures of the r software (r 2013), respectively. a genotype plus genotype by environment (gge) analysis (yan and kang, 2003) was carried out to analyze the fresh and dry biomass in the first and second harvests for the two treatments. both fresh and dry biomasses were arranged into a two-way genotype-by-combination of the harvest number and the water regime. a biplot analysis was carried out on traits showing positive influence on yield in order to select promising ecotypes. the traits were standardized before the analysis in accordance with different scales of the chosen variables. the biplot calculations were made using the ‘scale’ and ‘svd’ procedures of the r software (r 2013). results as shown by the average pattern of water removal in different layers at various intervals (tab. 2) moisture content was higher in upper layers in the full irrigation treatment, compared to the water restriction treatment. in this last one, moisture content was maintained higher in the lower layers than in the upper ones. significant effects due to ecotypes, water regimes and interaction (ecotypes × water regimes) were noted for all traits, except carotene contents which showed insignificant differences due to water treatments (tab. 3). water restriction effects were particularly drastic on fresh forage yield (ffy), canopy temperature depression (ctd) and transpiration rate (tr) which reduced 160, 79 and 61 %. water restriction increased the phenotypic and genotypic coefficients of variation for all traits except leaf area. high phenotypic and genotypic coefficients of variation were noted in both treatments except for leaf area. the broad sense heritability of ffy, leaf area (la), chloro phyll b (chlb) and carotene content (car) was less under water restriction conditions. most physiological traits had a higher heritability than forage yield. osmotic adjustment (oa) had higher phenotypic and genotypic coefficients of variation than drought resistance index (dri), recovery rate index (rri) and survival rate index (sri) (tab. 4). dri and sri had higher heritability than water use efficiency (wue), oa and rri. tab. 2: moisture gradients in soil layers of lysimeters under two regimes at 60 days after emergence of plants. moisture content (%) full irrigation water restriction 24 h 48 h 72 h 24 h 48 h 72 h 10 17 14 10 5 5 4 20 14 10 8 7 6 6 30 10 8 7 10 9 7 40 5 6 7 15 12 10 soil depth (cm) the biplot analysis realized on biomass retained 88 % of the variation for fresh biomass (fig. 1) and 80 % for dry biomass (fig. 2). in both treatments, the evaluated ecotypes showed differential performance across the two harvests. under full irrigation conditions, the highest fresh biomass was noted in ‘sb-10’ and ‘l-94’ for the first harvest and in ‘berseem tandojam’, ‘pachati’ and ‘agaiti’ for the second harvest. the highest dry biomass was noted in ‘sb-10’ and ‘l-94’ for the first harvest and in ‘sb-10’ and ‘sandal bar’ for the second harvest. under water restriction, the highest biomass was noted in ‘sb-12’, ‘berseem queta’ and ‘p-22’. the best fitted models for fresh biomass under water restriction and full irrigation conditions were respectively fb (fresh biomass) = 6.63 – 0.02 chla + 0.8 ltd – 0.17pn + 3.18 dri + 0.84 wue + 0.03 rri – 0.01 sri (r2 = 0.4) and fb = 15.07 + 0.06 la + 2.09 ltd (r2 = 0.1). the best fitted model explaining dry biomass under water restriction and full irrigation conditions were db (dry biomass) = 1.560 + 0.26 ltd + 0.73 dri + 0.21 wue (r2 = 0.32) and db = 2.45 + 0.01 la + 0.13 pn – 1.02 e + 0.42 ctd (r2 = 0.47), respectively. the biplot analysis realized on the traits associated to yield under water restriction (ie, dri, ltd, rri and wue) showed a negative association between wue and ltd, and between dri and rri (fig. 3). the ecotypes ‘anmol’, ‘agaiti’ and ‘sb-12’ had the high est wue (and the lowest ltd values). conversely, the ecotypes ‘super’ and ‘chenab’, followed by ‘samarkand’, ‘l-48’, ‘sk-1’, ‘p130 m. huassain, s. rauf, j. paderewski, i. ulhaq, d. sienkiewicz-paderewska, p. monneveux tab. 3: mean, analysis of variance, genotypic coefficient of variation (gcv%), phenotypic coefficient of variation (pcv%) and heritability (h2) for fresh forage yield (ffy), dry forage yield (dfy), canopy temperature depression (ctd), leaf temperature depression (ltd), net photosynthesis rate (pn) and transpiration rate (tr) of berseemclover under full irrigation and water restriction conditions. plant traits mean mean sum of squares‡ gcv% pcv% h2 s full water g t g × t full water full water full water irrigation restriction irrigation restriction irrigation restriction irrigation restriction ffy† (g) 26.85 10.31 21.67** 4104.15** 11.73** 22.65 23.52 43.22 46.33 0.52 0.51 dmy† (g) 4.79 2.98 0.88* 49.27** 84.63** 25.21 27.87 47.41 51.58 0.53 0.54 ctd (°c) 2.80 1.56 0.52* 50.70** 0.34* 21.94 42.03 28.91 52.57 0.76 0.80 ltd (°c) 2.20 1.51 0.52* 16.65** 1.26** 18.64 41.05 23.3 49.51 0.80 0.83 la (cm2) 125.92 112.67 637.98** 4957.92** 381.39** 8.19 4.87 12.47 11.23 0.66 0.43 chla (mg kg-1) 90.23 67.44 1637.60** 15258.72** 1510.28** 27.31 31.72 31.27 35.8 0.87 0.89 chlb (mg kg-1) 17.41 26.92 112.56** 2582.44** 45.10* 23.02 27.19 28.02 35.31 0.82 0.77 carotene 19.84 19.39 85.10** 19.14ns 128.42** 30.44 31.94 43.44 46.38 0.70 0.69 pn (μmolm-2 s-1) 8.01 5.61 70.78** 247.25** 17.95** 40.69 62.67 51.36 74.22 0.79 0.84 tr (mmolm-2s-1) 1.74 1.08 0.34* 12.93** 0.25* 14.90 28.07 21.43 34.64 0.70 0.81 † ffy and dmy were sum over two harvests ‡ g = variation due to ecotypes with df = 19; t = water treatment df = 1 and g×t = ecotypes × water regimes with df = 19 ** p ≤ 0.01, *p ≤ 0.05 fig. 1: gge biplot of fresh biomass in the first (1) and second (2) cuts under water restriction (s) full irrigation (w) for twenty ecotypes of berseem clover (trifolium alexandrinum l.). 22’, ‘sb-10’, ‘sanadal bar’, ‘pak berseem’ and ‘punjab’ had high ltd and low wue values. the highest dri and lowest rri values were observed in ‘berseem peshawar’, ‘agati’, ‘sandal bad’, ‘super’, ‘pachati’, ‘l-48’, ‘berseem tandojam’ and ‘sanadal bar’. the ecotypes ‘l-94’, ‘sb-10’, ‘p-209’, ‘p-22’, ‘anmol’ and ‘punjab’ had the highest rri and lowest dri values. the biplot analysis realized on the traits associated to yield under full irrigation (ie, pn, la and ctd) showed a positive association between pn and la (fig. 4). those traits were poorly correlated to ctd. the ecotype ‘sb-10’ had high pn and la values. ‘p-209’, ‘p-22’, ‘sanadal bar’, ‘samarkand’ and ‘pachati’ showed high pn values, while ‘berseem peshawar’, ‘berseem queta’, ‘sandal bad’, ‘berseem tandojam’, ‘l-48’, ‘l-94’, ‘anmol’ and ‘punjab’ had low pnvalues. the ecotypes ‘p-209’, ‘p-22’, ‘sandal bar’, ‘samarkand’, ‘l-94’ and ‘sk-1’ had the highest la while ‘punjab’, ‘berseem peshawar’, ‘agaiti’, ‘sb-12’ and ‘chenab’ had the lowest. ‘punjab’, ‘chenab’, ‘pak’, ‘agaiti’, ‘sb-12’ and ‘pachati’ had high ctd values. contrastingly, the ecotypes ‘l-94’, ‘sandal bad’, ‘anmol’, ‘berseem queta’, ‘berseem tandojam’,’p-209’ and ‘l-48’ had low ctd values. pn was not associated to ctd and la. ‘pachati’ and ‘pak berseem’ combined high ctd and pnvalues, while ‘p-209’, ‘p22’, ‘sanadal bar’, ‘samarkand’ and ‘sk-1’ had high la and pn values. the ecotypes ‘berseem peshawar’, ‘berseem queta’, ‘berseem tandojam’ and ‘l-48’ had lower ctd, pn and la values. discussion after having used yield as an exclusive breeding objective, many breeders progressively replaced this empirical approach by indirect tab. 4: mean, analysis of variance, genotypic coefficient of variation (σ2g), phenotypic coefficient of variation (σ2p) and broad sense heritability (h2) for osmotic adjustment (oa), drought resistance index (dri), water use efficiency (wue), recovery rate index (rri) and survival rate index (sri) under water restriction conditions. plant traits mean ecotypes σ2g σ2p h2 mean sum of square oa 0.23 0.14** 71.34 85.31 0.81 dri 1.08 0.32** 36.18 37.40 0.94 wue 1.43 1.01** 39.39 42.93 0.84 rri 44.75 1136.01** 48.35 60.43 0.78 sri 83.58 5195.23** 50.38 52.41 0.92 **p ≤ 0.01) evaluation of berseem germplasm against drought stress 131 selection (jackson et al., 1996), based on the selection for ‘secondary traits’ or plant characteristics that provide additional information about how the plant performs under a given environment (lafitte et al., 2003). any trait to be used as a surrogate of yield in the evaluation or selection process has to be genetically variable, highly heritable, genetically associated with yield, easy, inexpensive and fast to observe or measure, non-destructive and stable over the measurement period (edmeades et al., 1997). in the present study, all measured traits had high genotypic and phenotypic coefficients of variation. moreover, water restriction increased the variability of various traits as previously observed by iannucci et al. (2000), fig. 2: gge biplot of dry biomass in the first (1) or second (2) cut under water restriction (s) andfull irrigation (w) for twenty ecotypes of berseem clover (trifolium alexandrinum l.). fig. 3: gge biplot for standardized leaf temperature depression (ltd), drought resistance index (dri), recovery rate index (rri) and water use efficiency (wue) for twenty ecotypes of berseem clover (trifolium alexandrinum l.) under water restriction. ltd, dri, rri, and wue were averaged across replications for each combination of genotype-by-trait. the first principal component axis (pc1) retains 41 % and the second 31 % of sum of squares. el-bably (2002) and lazaridou and koutroubas (2004). phy siological traits had higher broad sense heritability than yield. finally, an association was noted between forage yield and ltd and rri under water restriction conditions, suggesting the use of these traits as indirect selection criteria for yield under stress in berseem clover. under full irrigated conditions, yield was positively associated to pn, canopy temperature depression (ctd) and leaf area. gas exchange measurements which provide simultaneous information about ltd and pn appears provide useful prediction of forage yield. the use of leaf gas exchange traits has been proposed for the selection of sunflower (kalyar et al., 2013). these measurements however require expensive equipment and are slow, stable over the measurement period. the use of the recovery index consequently appears as a good option for the prediction of forage yield under water restriction. similarly, under full irrigated conditions, leaf area and canopy temperature depression could represent good candidates for indirect selection. leaf area is strongly reduced by water stress in berseem clover (martiniello and texeira da silva, 2011) and is closely associated to yield in these conditions (ahmed, 2006). ctd is a highly integrating trait resulting from the effects of several biochemical and morpho-physiological features acting at the root, stomata, leaf, and canopy levels. it is useful mainly in hot and dry environments, with high vapor pressure deficits (tuberosa, 2012). in wheat, significant genetic gains in yield have been reported in response to direct selection for ctd in these environments (brennan et al., 2007). the mean sum of square (mss) due to ecotypes × water regimes was high when compared with mss due to ecotypes for traits such as ffy and dmy (tab. 3), suggesting differential performance of ecotypes over water regimes (desmarais et al., 2013). this was confirmed by the magnitude of genetic variation (gcv%) which increased under water restriction, particularly for ctd, ltd and pn, indicating that this treatment successfully discriminated the ecotypes for their tolerance. gge biplot analysis of multiple harvests under water regimes showed that the biomass of the first cut is not always a good indicator of biomass of the second cut. some ecotypes like ‘sb-10’ from punjab, however, showed stable biomass across cuts. as the development of high-yielding and stable ecotypes across multiple harvests is a major breeding objective of forage breeding (chakroun et al., 1990; krenzer et al., 1992), these ecotypes should be recommended for cultivation. the relationships observed between traits under water restriction conditions suggest that ecotypes with a higher ltd may recover well from drought stress. ltd is an important index of drought avoidance, transpiration and root growth under drought stress. it has been noted earlier that this trait positively correlated with biomass in various forage grass species (acuna et al., 2011; merewitz et al., 2014). it also appears that simultaneous selection for ltd and dri or for rri and wue might be difficult. however, simultaneous selection may be practiced to some extent for ltd and rri or wue and dri. under full irrigated conditions, simultaneous selection for pn and ctd or pn and la may be possible for the selection of highyielding ecotypes under irrigated conditions. selection for low la might lead to higher ctd. kalyar et al. (2013) also showed that a higher temperature depression was negatively related with leaf area in irrigated sunflower and suggested that reduced leaf area could participate in leaf cooling. a large variation has been reported in berseem clover for the adaptation to multiple cuts (graves et al., 1996; putnam et al., 1999; ross et al., 2003; ranjbar, 2007) and the knowledge of productivity across cutting regimes is of primary importance in breeding programs (juskiw et al., 2000). among the tested ecotypes, ‘sb-10’ (from punjab) showed the highest productivity. this ecotype was also characterized a good aptitude for double harvesting in irrigated conditions. this aptitude that highly depends harvesting management 132 m. huassain, s. rauf, j. paderewski, i. ulhaq, d. sienkiewicz-paderewska, p. monneveux (abdel-gawad, 1993; geweifel and rammah 1990; iannucci et al., 2000) should however be confirmed under in different dates of harvest. under water restriction conditions, the highest forage yield was noted in ‘sb-12’ (punjab), ‘berseem queta’ (balochistan) and ‘p-22’ (punjab). these ecotypes could be recommended to farmers according to their needs and demand and for further use as progenitors in breeding programs. references abdel-gawad, k.i., 1993: effect of cutting management on forage, protein and seed yields of berseem clover (synthetic 79 var.). zagazig j. agric. res. 20, 67-75. acuña, h., inostroza, l., tapia, g., 2011: strategies for selecting drought tolerant germplasm in forage legume species. in: mofizur rahman, i.m, (ed.), water stress, 277-300. intech. ahmed, m.a.s., 2006: response to three methods of recurrent selection in a khadawari bersim (t. alexandrinum l.) population. alex. j. agric. res. 51, 13-23. allard, r.w., 1960: principles of plant breeding. johan wiley and sons inc, new york. araus, j.l., cairns, j.e., 2014: field high-throughput phenotyping: the new crop breeding frontier. trends plant sci. 19(1), 52-61. babu, r.c., pathan, m.s., blum, a., nguyen, h.t., 1999: comparison of measurement methods of osmotic adjustment in rice cultivars. crop sci. 39, 150-158. brennan, j.p., condon, a.g., van ginkel, m., reynolds, m.p., 2007: an economic assessment of the use of physiological selection for stomatal aperture-related traits in the cimmyt wheat breeding programme. j. agric. sci. 145, 187-194. chakroun, m., taliaferro, c.m., mcnew, r.w., 1990: genotype environment interactions of bermudagrass forage yields. crop sci. 30, 49-53. de santis, g., iannucci, a., dantone, d., chiaravalle, e., 2004: changes during growth in the nutritive value of components of berseem clover (trifolium alexandrinum l.) under different cutting treatments in a mediterranean region. grass for. sci. 59, 378-388. des marais, d.l., hernandez, k.m., juenger, t.e., 2013: genotypeby-environment interaction and plasticity: exploring genomic responses of plants to the abiotic environment. annual rev. ecol. evol. syst. 44, 5-29. edmeades, g.o., bolaños, j., chapman, s.c., 1997: value of secondary traits in selecting for drought tolerance in tropical maize. in: edmeades g.o., bänziger, m., mickelson, h.r., peña-valdivia, c.b. 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(eds.), optimal forage systems for animal production and the environment, 344-347. grassland sci. europ., vol 8. martiniello, p., iannucci, a., 1998: genetic variability in herbage and seed yield in selected half-sib families of berseem clover, trifolium alexandrinum l. plant breed. 117, 559-562. martiniello, p., teixeira da silva, j.a., 2011: physiological and bioagronomical aspects involved in growth and yield components of cultivated forage species in mediterranean environments: a review. europ. j. plant sci. biotech. 5, 64-98. masuka, b., araus, j.l., das, b., sonder, k., cairns, j.e., 2012: phenotyping for abiotic stress tolerance in maize. j. int. plant biol. 54, 238249. merewitz, e., belanger, f., warnke, s., huang, b., bonos, s., 2014: quantitative trait loci associated with drought in creeping bentgrass. crop sci. 54, 2314-2324. putnam, d.b., williams, w.a., graves, w.l., gibbs, l., peterson, g., 1999: the potential of berseem clover varieties for california. in: proceeding of 29th california alfalfa symposium. university of california coop. extension, 108-119. r 2013: r, project for statistical computing: a language and environment for statistical computing. r foundation for statistical computing, vienna, austria. ranjbar, g.a., 2007: forage and hay yield performance of different berseem clover (trifolium alexandrinum l.) genotypes in mazandaran conditions. asian j. plant sci. 6, 1006-1011. ross, s.m., king, j.r., o’donavan, j.t., izaurralde, r.c., 2003: seed© the author(s) 2015. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 88, 78 86 (2015), doi:10.5073/jabfq.2015.088.011 1institute of pure and applied biology, bahauddin zakariya university, multan pakistan 2institute of molecular biology and biotechnology, bahauddin zakariya university, multan pakistan 3department of botany and microbiology, king saud university, riyadh, saudi arabia 4department of agronomy, muhammad nawaz shareef university of agriculture, multan pakistan photosynthetic capacity of canola (brassica napus l.) plants as affected by glycinebetaine under salt stress ayesha khalid1, habib-ur-rehman athar1, zafar ullah zafar1, ahmad akram1, kausar hussain1, hamid manzoor2, fahad al-qurainy3, muhammad ashraf3,4 (received july 16, 2014) summary salinity causes reduction in growth and severe losses of crop productivity by affecting various biochemical and physiological processes including photosynthesis. plants sense and adapt to salt stress by modulating different physiological processes including accumulation of osmoprotectants. glycinebetaine (gb) is an important osmoprotectant and found to have ameliorative effects on the growth of plants by altering ion homeostasis, photosynthetic and antioxidant capacity. in this study, it was attempted to reveal how gb improves photosynthetic activity and salt tolerance of two canola cultivars which differ in degree of salt tolerance. two cultivars (dunkled and cyclone) of canola (brassica napus l.) were grown under non-saline or saline (150 mm nacl) conditions. glycinebetaine (100 mm) was foliarly applied to both non-stressed and salt stressed plants of both canola cultivars at the vegetative growth stage. salt stress reduced growth of both canola cultivars, however, cv. dunkled was superior to cv. cyclone. foliar application of gb improved leaf relative water contents (rwc), osmotic potential and proline accumulation in salt stressed plants. the chlorophyll fluorescence transient (cft) remained unchanged at o and j phase while at i and p phase it was affected by salt stress in both cultivars that was ameliorated by gb application. the positive values of k band after 1000 μs under salt stress revealed the reduced efficiency of oxygen evolving complex (oec). the gb application enhanced electron transport chain and decreased heat dissipation under salt stress. this effect was more in cultivar cyclone as compared to dunkled. furthermore, a considerable proteomic variation was noted in canola cultivars after application of gb under both saline and non-saline condition. the results suggested that exogenous foliar application of gb ameliorated the adverse effects of salt stress on both cultivars of canola by osmotic adjustment, growth improvement, increased light absorption by reaction centers, efficient energy trapping and enhanced electron transport chain in both cultivars of canola. introduction abiotic stresses like salinity, drought, high and low temperature severely affect the crop productivity which strains more on food insecurity (ashraf and harris, 2005; ashraf et al., 2008). it was reported by many researchers that crop yield losses due to salinity and drought is 80 % along with actual losses. high salinity causes both ionic and osmotic stresses (zhang et al., 2009; ahuja et al., 2010) by modifying the plant cell plasma membrane, lipid and protein composition, and ultimately impairing optimal growth and development (fujii and zhu, 2009; rodríguez-milla and salinas, 2009). salt induced growth and yield reduction are associated with many factors i.e reduced photosynthetic metabolism, leaf chlorophyll content and photosynthetic capacity (athar et al., 2009; athar et al., 2015), diversion of energy in the processes of osmotic adjustment and ion exclusion and nutritional imbalance. salt induced reduction in photosynthesis is associated with the partial stomatal closure and/or the non-stomatal limitation which is involved in the dark enzymatic processes of co2 assimilation e.g. the decrease in rubisco activity and content, or pi-regeneration capacity (ashraf and harris, 2013). plant salt tolerance is correlated with accumulation of osmoprotectants in chloroplasts such as glycinebetaine and proline (ashraf and harris, 2004). glycinebtaine (gb), a quaternary ammonium compound, stabilizes the psii structure and maintains its activity under saline conditions (chen and murata, 2011). it also maintains the association of rubisco activase with rubisco and inhibits the production of ros under stress conditions (murata et al., 2007). moreover, gb protects the membrane from high concentrations of na+ and cl(hanson and bendixen, 1995). some crops are accumulating gb in high quantities like sugar beet, some accumulate it in moderate amounts and some crops are non accumulator of gb such as canola. so, for a non accumulator crop, exogenous foliar application of this osmoprotectant acts as an alternate shotgun approach to ameliorate growth reduction under salt stress (mäkelä et al., 1996). exogenous foliar application of gb improves growth, survival and tolerance in a wide variety of gb accumulator and non accumulator plants. in our earlier studies, it is reported that exogenous foliar application of gb ameliorated the salt induced adverse effects in canola (athar et al., 2009) by improving the photosynthetic capacity (athar et al., 2015). it has been reported that salt stress en hances the photoinhibition of psii by damaging the d1 protein (chen and murata, 2011). chlorophyll a fluorescence measurement has been one of the most used techniques for providing rapid insights in the ability of a plant to tolerate environmental stresses such as salt stress (kalaji and guo, 2008; kalaji et al., 2011). in addition, it also facilitates the understanding to what extent salt stress has affected the structural and functional stability of the photosynthetic apparatus (baker, 2008; stirbet and govindjee, 2011). of various chlorophyll fluorescence techniques, the chlorophyll a fast fluorescence transient measures the photochemical performance of ps-ii, reflecting the time course of photochemistry. based on the theory of energy flow in thylakoid membranes, strasser et al. (1995) have developed the jip test which has been found to be very sensitive to stress induced changes in psii even before visible symptoms appear on the leaves. keeping in view the above mentioned reports, the present study aimed to assess how exogenous foliar application of gb ameliorates the adverse effect of salt stress on photosynthetic capacity in two cultivars of canola. material and method seeds of two canola (brassica napus l) cultivars (cv. dunkled and cv. cyclone) differing in salinity tolerance were obtained from ayub agriculture research institute, faisalabad, pakistan. the experiment was conducted at the botanic gardens of bahauddin zakariya university multan, pakistan, with 10/14 light/dark period at 8001000 mmol m-2 s-1 ppfd, a day/night temperature cycle of 26/15 °c and 60±5 % relative humidity. before experimentation, 500 seeds glycinebetaine protects psii under salt stress 79 of each canola (brassica napus l.) cultivar were surface sterilized in 5 % sodium hypochlorite for 5 minutes. seeds were sown in 32 plastic pots (28 cm diameter and 32 cm depth), each of which filled with 10 kg thoroughly washed river sand. plastic pots have drainage holes covered with a piece of muslin cloth at the bottom. all pots were irrigated with 2 l of full strength hoagland’s nutrient solution. the seeds were allowed to emerge for one week and then thinned to 8 plants per pot initially. after a period of further one week, plants were thinned to 5 plants per pot of uniform size and placed equidistantly. four weeks after the start of the experiment, pots were irrigated with full strength hoagland’s nutrient solution containing 0 or 120 mm nacl. salinity level was developed by the addition of nacl stepwise in aliquots of 40 mm every day until the appropriate treatment was attained. varying concentrations of gb [0 or 100 mm in 0.1 % tween-20 solution] (~5 ml per plant and ~1.5 l for whole sub-experiment) applied as a foliar spray to plants of both canola cultivars growing in non-saline or saline conditions. the control (0 mm gb) plants were sprayed with the same volume of 0.1 % tween-20 solution. the ph value of the sprayed solution was maintained at 6.5 to ensure the maximum penetration into the leaf tissue and to avoid the leaf injury. plants were sprayed in the evening to avoid drying of solution on leaves and allowing maximum gb solution penetration into the leaf tissue. hoagland’s nutrient solution containing (0 or 120 mm nacl) was replaced every week to replenish nutrients. however, treatment solution was applied in excess to each pot so as to flush through all the salts previously present in the sand and to ensure the desired salt level. after four weeks of gb treatment (2 months old plants), plants were harvested, washed with distilled water, blotted dry and separated into shoots and roots, and data for fresh biomass recorded. however, canola plants growing in 120 mm nacl solution were washed in cold 140 meq/l lino3 solution, isotonic with the corresponding salt treatment. however, 1 mm ca(no3)2 .4h2o was added in lino3 solution to maintain membrane integrity and to avoid marginal changes in ion content during washing. these plants were then oven-dried at 65 oc for 72 h and dry biomass recorded. however, before harvest, relative water content (rwc), leaf proline, chlorophyll contents, and chlorophyll fluorescence parameters were measured. leaf relative water contents (rwc): a fully developed and young leaf from each plant was taken and fresh weight of each leaf recorded. all leaf samples were dipped in distilled water for six hours and measured the turgid weight of each sample leaf. then all the sample leaves were dried in oven at 70 °c to measure dry weights. rwc= [l fresh wt. – l dry wt.]/[l turgid wt. – l dry wt.]*100 leaf osmotic potential: third leaf from top was taken from each cultivar and was frozen at -20 °c for seven days, after which time the frozen leaf material was thawed and the cell sap was extracted with the help of a disposable syringe. the extracted sap was directly used for the determination of osmotic potential using an osmometer (wescor 5500, usa). chlorophyll content: a fully developed and young leaf from each plant (usually third leaf from top) was used to measure chlorophyll contents using a portable chlorophyll meter (minolta spad-502, japan). chlorophyll fluorescence: the data for chlorophyll fluorescence were recorded following nomenclature by tsimilli-michael and strasser (2008), stirbet and govindjee (2011) and the literature available on the websites of manufacturers of chlorophyll fluorescence meters. fast chlorophyll a fluorescence transients were recorded on third leaf by using portable fluorescence meter, fluorpen fp 100 (photon system international, czech republic). before taking measurements, leaves of each genotype/cultivars were dark adapted for 30 minutes by wrapping the aluminium foil to the leaf surface to ensure complete oxidation of photosynthetic electron transport chain or psii with open reaction centers (rcs). the fluorescence emission was induced in a 4 mm diameter area by exposing to a saturating actinic light at the intensity of 3000 μmol m-2 s-1. the fast chlorophyll fluorescence kinetics was measured from 10 μs to 2 s and the settings of fluorpen was as: fo (initial fluorescence) as o (20 μs) when all the reaction centers are open, l (150 μs), k (300 μs), j (2000 μs) and i (30000 μs) are the intermediate and p (500000 μs) as the maximum fluorescence (fm). the original (without normalization) chlorophyll a fluorescence transients of different treatments were plotted. for detailed analysis of the whole digitized fluorescence kinetics, different normalizations, calculation of kinetic differences or ratios as well as time-derivatives were undertaken. the original ojip transients were double normalized between the two fluorescence extreme o (fo) and p (fm) phases and the variable fluorescence between op expressed as vop (vop = (ft-fo)/(fm-fo) was determined. similarly, chlorophyll a fluorescence transients were double normalized between fo (30 ms) and fk (300 ms) expressed as vok [vok = (ft-fo)/ (fk-fo)] to reveal the possibility of fluorescence rise at an early step at about 300 ms. further the chlorophyll a fluorescence transients were double normalized between fo and fj expressed as voj [voj = (ft-fo)/(fj-fo)]. the o-i phase was evaluated by double normalization of fluorescence transients between fo and fi expressed as voi (<1) [voi = (ft-fo)/(fi-fo)]. the i-p phase was evaluated following two approaches: (i) normalization of fluorescence transients voi between the time range of 30-300 ms expressed as voi or pi (>1) [voi = (ft-fo)/(fi-fo)] and (ii) transient normalisation to the time range of 30-200 ms expressed as vip [vip = (ft-fi)/(fm-fi)]. moreover, differences in transients with respect to a reference were calculated as l-band (δvok = vok (salt stress) -vok(control)) and k-band (δvoj = voj (salt stressed) -voj(control)). similarly, δvoi and δvip were calculated for the detailed analysis of chlorophyll fluorescence kinetics. other phenomenological and biophysical parameters were also calculated using jip-test as described by tsimilli-michael and strasser (2008) that give information about the structural and functional state of ps ii. protein extraction: fresh leaves (0.5 g) were ground in 4 ml of cooled phosphate buffer (50 mm, ph 7.8). the homogenous mixture was centrifuged at 15000 g at 4 °c for 20 minutes and the supernatant used for protein analysis. sodium azide was added to avoid any type of growth in protein sample. sodium dodocyl sulphate polyacrylamide gel electrophoresis (sds-page): one dimensional gel electrophoresis (e-vs10-sys, germany) was performed on 12 % polyacrylamide to visualize the protein samples according to the standard procedure (laemmli, 1970). after electrophoresis, gel was stained with coomassie brilliant blue r-250 dye for approx. one hour. the corresponding gel was visualized after de-staining process and the sample protein bands were compared with the bands of the protein marker (invitrogen, 69079-3). statistical analysis of data: the data obtained from all parameters are described as mean value ± se. by using the statistical software costat, data were subjected to three way analysis of variance (anova). the mean values were compared with least significance difference (lsd) test following snedecor and cochran (1989). 80 a. khalid, h.r. athar, z.u. zafar, a. akram, k. hussain, h. manzoor, f. al-qurainy, m. ashraf result a marked inhibitory (p<0.001) effect of salt stress was observed on fresh and dry weights of shoots and roots of both canola cultivars. cultivar dunkled had significantly (p<0.001) higher shoot fresh and dry weights than those of cv. cyclone under both normal and saline conditions (fig. 1a; 1b). however, cultivars did not differ in root fresh and dry weights. gb application caused a significant (p<0.001) increase in shoot fresh and dry weights of both cultivars. foliar application of gb significantly increased (p<0.001) root fresh and dry weights of both cultivars under normal conditions, whereas under saline condition gb application slightly improved root fresh weight of cv. dunkled only (fig. 1c; 1d). salt stress reduced the leaf osmotic potential (more negative) in both canola cultivars and exogenous foliar application of gb cause a further decrease in leaf osmotic potential of both canola cultivars. in addition, impact of salt stress and gb application on leaf osmotic potential of both cultivars was the same (fig. 2a). relative water content (rwc) decreased in salt stressed plants of both canola cultivars. cultivar dunkled had greater rwc values than cv. cyclone under normal conditions, while the reverse was true under saline conditions (fig. 2b). exogenous application of gb reduced rwc in non-stressed plants of cv. dunkled and salt stressed plants of cv. cyclone only. chlorophyll content measured as spad units decreased due to imposition of salt stress in cv. dunkled only. exogenous foliar application of gb did not change the chlorophyll contents of cv. dunkled under both non-saline and saline conditions. however, application of gb reduced the chlorophyll contents of cv. cyclone under saline conditions (fig. 2c). chlorophyll a fluorescence transients of dark adapted leaves plotted on logarithmic time scale clearly showed differential impact of salt stress and exogenous application of glycinebtaine on o-j-i-p phases of two canola cultivars (fig. 3). the fluorescence rise at o-j and j-i phases was significantly decreased in cv. dunkled due to salt stress, whereas in cv. cyclone such a decrease in fluorescence was found at o-j phase only. foliar application of gb did not change amplitude of fluorescence at o-j phases of both canola cultivars (fig. 3). however, exogenous gb application compensated the loss of fluorescence at o-j phase by increasing fluorescence level at j-i and i-p phases in salt stressed plants of both canola cultivars. in addition, in nonstressed plants of cv. cyclone it remained almost unaffected (fig. 3a; 3b). since changes in the ojip shape are translated to changes of the structural and functional parameters of psii, chlorophyll a fluorescence transients were expressed as ft/fo so that differences concerning the fo values would not interfere with the other differences as well as it decreased heterogeneity among replicates (fig. 3c; 3d). chlorophyll a fluorescence transients normalized by fo (ft/fo) showed typical o, j, i and p steps. moreover, it also showed that salt stress and application of gb increased fv in both canola cultivars which are almost equivalent to increase in primary photochemistry (jpo). to avoid any interference and heterogeneity due to different fo and fm values, all fluorescence transients were doubly normalised and presented as relative variable fluorescence, which are plotted on log time scale (fig. 3e; 3f). transients showed that exogenous application of gb caused a dip in fluorescence at j-i phase in salt stressed plants of both cultivars. fig. 1: shoot and root fresh and dry weight of two cultivars of canola (brassica napus l.) when three week old plants were subjected to foliar application of glycinebetaine and salt stress for further two weeks. fig. 2: osmotic potential, relative water content and chlorophyll content of two cultivars of canola (brassica napus l.) when three week old plants were subjected to foliar application of glycinebetaine and salt stress for further two weeks. control salt control salt dunkled cyclone control salt control salt dunkled cyclone control salt control salt dunkled cyclone glycinebetaine protects psii under salt stress 81 in order to validate the observed differences, a semi-quantitative evaluation was performed using the differences of suitably normalised transients exhibited by stressed and non-stressed plants and expressed as op, l and k bands. l and k bands are usually hidden among the o-j-i-p steps of the original transients. the difference kinetics of dvop (fig. 4a) of gb applied plants of both cultivars under non-saline or saline conditions showed a negative bands in the o-i region (and more in j-i region, photo-electrochemical quenching) of the ojip transient, which indicates that processes from exciton trapping to pq reduction are faster than in respective control plants. however, negative band of dvop of gb treated plants of cv. dunkled was more negative than thin in gb treated plants of cyclone under both non-saline and saline conditions. moreover, amplitude of difference kinetics of dvop in gb treated plants of cv. cyclone at o-i region was greater than that in salt stressed plants of cv. cyclone, whereas in cv. dunkled such difference in amplitudes is not observed (fig. 4a). to reveal changes in chlorophyll fluorescence at each step, difference kinetics at l and k steps were also calculated and presented as δvok (l band, energetic connectivity among psii units) and δvoj (k band, antenna size or oec activity), respectively (fig. 4b; 4c). negative amplitude of l-band of gb treated plants of both cultivars under saline or non-saline condition indicated gb treated plants had greater energetic connectivity among psii units than in control plants. however, amplitude of l band in cv. cyclone applied with 100 mm gb was higher than in plants of cv. dunkled applied with 100 mm gb under non-saline conditions (fig. 5b), whereas under saline conditions cultivars did not differ. difference kinetics as δvoj (k band) revealed that a negative band appeared at 300 μs in gb treated plants of both canola cultivars under both nonsaline and saline conditions (fig. 4c). cultivar difference in amplitude of negative k-band due to foliar application of gb is only visible under non-saline condition, where cv. dunkled presents lower amplitude of the l band. changes in i-p phase have been measured by two normalization methods, which is associated with the electric trans-thylakoid potential generated by the proton pump fuelled by cyclic electron transport (cet) in psi (processes related with the electron flow from reduced pq (pqh2) to psi end electron acceptors i.e. fd and nadp) (strasser et al., 2010; vredenberg, 2011). exogenous application of gb increased the amplitude of i-p curve in both canola cultivars under non-saline conditions, whereas under saline conditions amplitude is lower in both canola cultivars (fig. 5a; 5b). this suggests that gb increased the pool of electron carriers of psi end to be reduced from electrons coming from pq in both canola cultivars. moreover, i-p phase measured as vip = [(ft-fi)/(fm-fi)] showed that (fig. 6c; 6d) salt stress reduced the rate constant of cv. dunkled and exogenous application of gb improved the rate constant in cv. dunkled. however, such changes in rate constant due to gb application are fig. 3: chlorophyll flourescence with and without normalization of two cultivars of canola (brassica napus l.) when three week old plants were subjected to foliar application of glycinebetaine and salt stress for further two weeks. 82 a. khalid, h.r. athar, z.u. zafar, a. akram, k. hussain, h. manzoor, f. al-qurainy, m. ashraf not observed in cv. cyclone (fig. 5c; 5d). salt stress induced changes in biophysical parameters derived from the chlorophyll fluorescence curve of both cultivars of canola are presented as radar plot (fig. 6a; 6b). all data of these parameters were normalized to the reference control and each variable at the reference control was standardized by giving a numerical value of 100. salt stress decreased the variable fluorescence at step j (vj) of both canola cultivars. exogenous foliar application of gb did not affect vj of both canola cultivars. however, salt stress and gb application did not change the vi (relative variable fluorescence at step i) in both cultivars (fig. 6a; 6b; tab. 1). among ratios of chlorophyll fluorescence, fv/fm (maximum primary yield of photo chemistry of psii) remained unaffected due to salt stress and gb application in both canola cultivars, fv/fo and fm/fo more sensitive parameters for photosynthetic capacity reduced due to salt stress in both cultivars of canola and exogenous application of gb improved both these parameters in both canola cultivars (fig. 6a; 6b; tab. 1). however, such improvement in fv/fo and fm/fo due to gb application was more in cv. dunkled under non-saline conditions, whereas the same was true for cv. cyclone under saline conditions. salt stress reduced the rate of primary photochemistry (mo) of both canola cultivars. however, exogenous application of gb slightly improved in cv. dunkled only (fig 6a; 6b). similarly, total pq pool (area), multiple turnover of pq reduction and oxidation (sm) and number of qaredox turnover until fm (n) reduced in cv. cyclone only and gb application did not change these biophysical parameters (fig. 6a; 6b; tab. 2). the derived specific energy fluxes such as absorbance flux per active reaction center (abs/rc), trapped energy flux per reaction center (tro/rc), electron transport flux (further than qa-) per reaction center (eto/rc) and dissipation energy flux per reaction center (dio/rc) reduced due to salt stress in both canola cultivars and foliar application of gb did not affect these jip test parameters in both cultivars (fig. 6a; 6b; tab. 4). the relationship between log piabs and relative electron transport yield can be considered as plant’s ability to transform light energy into chemical energy (nadh) which is subsequently directed into metabolic reactions in the biochemical process of photosynthesis (hermans et al., 2003). in this respect our result suggests that the plants that were sprayed with gb under salinity stress have higher ability to convert light energy into chemical energy which can be used further to reduce co2 to carbohydrate. the higher ability to convert light energy into chemical energy is observed in cultivar cyclone (fig. 7). comparative protein expression of two canola cultivars was performed using one-dimensional sds-page (fig. 8), which shows that 22, 56 and 100 kda water soluble protein expression increased salt stressed and exogenously applied plants of cv dunkled, whereas in cyclone expression of 22 and 56 kda proteins was low. discussion to overcome salt-induced reduction in growth of canola, exogenous application of glycinebetaine (gb) is considered to be an alternative strategy (athar et al., 2009; abbas et al., 2010) and this has been verified in the present study. these inhibitory effects of salt stress on canola cultivars were mainly due to salt induced osmotic stress and toxic effects of na+ accumulation (data not shown) along with reduced photosynthetic rate as has been found earlier (ulfat et al., 2007; athar et al., 2015). salt stress and gb application decreased the leaf osmotic potential, representing an important factor which enhances the salt tolerance. similar results were reported by raza et al. (2006) that foliar application of gb reduced the osmotic potential in salt stressed plants of wheat which favor the osmotic adjustment. the main function of foliar applied gb is to promote photosynthetic capacity in canola cultivars. however, this improving effect of gb on photosynthetic capacity is cultivar specific and due to the cultivar’s genetic potential (yang and lu, 2005; chen and murata, 2008), but its impact on the exact site of the photosynthetic machinery is still not clear. in a semi-quantitative evaluation, impact of salt stress and exogenous application of gb on different sites of photosynthetic machinery of salt tolerant and sensitive canola cultivars, whole transients of ojip, suitably normalized transients and differences of normalized transients of stressed and non-stressed plants are measured. whole transients data from the present study suggest that salt stress reduces the rate of primary photochemistry (fluorescence at o-j phase changes) and photo-electrochemical quenching (j-i phase changes) in both canola cultivars and exogenous gb application improved photosynthetic activity by compensating rate of reduction at psi end electron acceptors in salt tolerant cultivar dunkled (fluorescence changes at i-p phase). these results are further supported by fo normalized transient and transients of relative variable fluorescence between fo and fm (fig. 3). to further confirm the results, whole difference kinetics and difference kinetics at each step was performed (fig. 4). in l-band, the low values of florescence under saline conditions show that energetic connectivity loss to some degree is because of reaching to the verge of salinity acclimation. in k-band, no positive peak till 1000 s μs shows the ability of both cultivars of canola to resist the imbalance in number of electrons at donor and acceptor side of photosystem ii fig. 4: δvop, δvok, δvoj of two cultivars of canola (brassica napus l.) when three week old plants were subjected to foliar application of glycinebetaine and salt stress for further two weeks. glycinebetaine protects psii under salt stress 83 which is induced by salinity. however, an increased k-band from 1000-2000 μs under saline condition reveals that the performance of oec (oxygen evolving complex) reduced due to imbalance in eflow from the oxygen evolving complex to the reaction centers and in the direction of acceptor side of ps ii. however, an attenuated k-band from 1000-2000 μs under gb foliar spray in saline condition suggesting the ability of both cultivars of canola to resist the imbalance in number of electrons at donor and acceptor sides of psii. o-i part indicates the kinetic properties for oxidation/reduction of pq pool. the negative δvoi peaks recorded under salinity stress indicate that salinity-stressed plants of both cultivars of canola had the ability to maintain the pq reduction rate and gb does not ameliorate this process under salinity stress to greater extent. the i-p phase which is the last phase of fluorescence transient indicates the change in electrons currents from the pqh2 to electron acceptor of ps i. increase in positive values of i-p phase indicates higher pool of the ps i’s electron acceptor. a decrease in i-p phase on salt treatment and again rise under gb foliar spray might be a part of acclimation process of both cultivars of canola in which the plants endeavor to lessen the salinity effects by maintaining the pq pool size. however, salinity induced reduction in i-p phase is due to the sudden decline in leaf water status that might reach to the threshold level of salinity acclimation. chlorophyll fluorescence at different steps of ojip and ratios of chlorophyll fluorescence are potential indicator of psii efficiency. salt stress significantly decreased the minimum level of fluorescence (fo) reveals increase excitation energy transfer from the antenna to the rc, leading to low fo. in contrast, kalaji et al. (2011) found that imposition of salt stress reduced fo of two syrian barley landraces after 7 days of salt stress. exogenous foliar gb application also decreased the fo under non saline condition also reveals increase transfer efficiency of energy absorbed from antenna chl.a to psii reaction center. on the other hand, gb application increased the fo under saline condition reveals reduced energy transfer efficiency from the antenna chl a to psii reaction center. salt stress also reduces the maximal fluorescence (fm) reveals the salt stress reduces the transfer of electrons to the acceptor side of ps ii. it induces changing in the rate of qa reduction. gb protects the psi so the foliar application of gb protects the rate of oxidation of plastoquinone by electron transfer to psi. abs/rc which indicates the antenna size of active rcs which is estimated as the ratio of total absorbance of photons by chlorophyl molecules to total active reaction centers that decreased in plants those are treated by foliar application of glycinebetaine due to increased in size of antenna of active reaction centers. among the jip parameters, pi is the most sensitive parameters. this parameter gives information about fluorescent changes that associated with antenna conformational changes and energy fluctuations. therefore with the help of pi, the vitality of the plants is estimated with high resolution. the foliar application of glycinebetaine under saline condition increased the pi value and the relationship between et0/abs and log piabs which demonstrates that in natural environment plants have high efficiency to utilize the par which reduce co2 to carbohydrate. similar results are reported by shine and guruprasad (2012) that shows that pre-sowing magnetic field exposure of seed increased the pi and the relationship between log piabs (=df). the analysis of polypeptide banding obtained on sds-page have shown that a 14-day treatment of gb and nacl resulted in the change of the leaf water soluble proteins in comparison with that of control leaves. both substances have induced the accumulation and synthesis of many different proteins inside the leaf tissue. out of the three observed protein bands, the 100 kda protein was significantly present in treated plants as compare to control. maslenkova et al. (1992) reported the higher concentrations of many polypeptides in barley seedlings as a result of salinity and jasmonic acid treatments. they have observed a substantial enrichment of one polypeptide with an apparent molecular mass of 55 to 57 kda. the exact function of these induced proteins is still unclear for us and it is very difficult at this stage with this minimal data to say clearly about the identification and characterization of these water soluble proteins. however, it can be assumed that the same proteins present in control sample, have shown higher expression in leaf tissues of the treated plants. it is therefore of immense interest to go for further characterization of these important proteins either through mass spectrometry or molecular structure determination through nmr/x-ray crystallography. fig. 5: vip and voi of two cultivars of canola (brassica napus l.) when three week old plants were subjected to foliar application of glycinebetaine and salt stress for further two weeks. 84 a. khalid, h.r. athar, z.u. zafar, a. akram, k. hussain, h. manzoor, f. al-qurainy, m. ashraf tab. 1: mean squares from analysis of variance of data for vj, vi, fv/fo, fv/fm and fm/fo in leaves of two cultivars of canola (brassica napus l.) treated with or without foliar spray of gb to salt stressed and non stressed condition. source of variance df vj vi fv/fo fv/fm fm/fo salt 1 0.026*** 6.125*** 7.998*** 0.0084*** 7.998*** gb 1 4.205ns 3.001*** 0.641** 7.801*** 0.641** cultivar 1 2.101ns 1.125*** 0.116ns 1.25ns 0.116ns salt* cultivar 1 2.88ns 1.8ns 0.111ns 1.361** 0.111ns salt*gb 1 3.781ns 0.0029*** 0.0028ns 2.812ns 0.002ns gb*cultivar 1 0.0035*** 1.512ns 0.3676* 1.8e-5ns 0.3676* salt*gb*cultivar 1 0.0010* 1.012ns 0.185ns 3.125*** 0.185ns error 24 0.0042 2.99 1.25 2.465 0.0524 ns = non-significant; *,**,*** significant at 0.05, 0.01 and 0.001 probability gb = glycinebetaine, salt = salt stress vj, = relative variable fluorescence at phase j of fluorescence transient curve, vi = relative variable fluorescence at phase i of fluorescence transient curve, fv/fo = maximum primary yield of photochemistry, fv/fm = maximum yield of photochemistry of psii. fig. 6: radar plot of jip test parameters of two cultivars of canola (brassica napus l.) when three week old plants were subjected to foliar application of glycinebetaine and salt stress for further two weeks. references abbas, w., ashraf, m., akram, n.a., 2010: alleviation of salt-induced adverse effects in eggplant (solanum melongena l.) by glycinebetaine and sugarbeet extracts. sci. hort. 125, 188-195. ahuja, i., de vos, r.c., bones, a.m., hall, r.d., 2010: plant molecular stress responses face climate change. trends plant sci. 15, 664-674. ashraf, m., 2004: some important physiological selection criteria for salt tolerance in plants. flora-morphology, distribution, functional ecology of plants 199, 361-376. ashraf, m., ali, q., 2008: relative membrane permeability and activities of some antioxidant enzymes as the key determinants of salt tolerance in canola (brassica napus l.). environ. exp. bot. 63, 266-273. ashraf, m., athar, h.r., harris, p.j.c., kwon, t.r., 2008: some prospective strategies for improving crop salt tolerance. in: donald, l.s. (ed.), adv. agron., vol. 97, 45-110. academic press. ashraf, m., harris, p., 2004: potential biochemical indicators of salinity tolerance in plants. plant sci. 166, 3-16. ashraf, m., harris, p.j., 2005: abiotic stresses: plant resistance through breeding and molecular approaches. food products press. ashraf, m., harris, p.j.c., 2013: photosynthesis under stressful environments: an overview. photosynthetica 51, 163-190. athar, h.u.r., ashraf, m., wahid, a., jamil, a., 2009: inducing salt tolerance in canola (brassica napus l.) by exogenous application of glycinebetaine and proline: response at the initial growth stages. pak. j. bot. 41, 1311-1319. athar, h.u.r., zafar, z.u., ashraf, m., 2015: glycinebetaine improved photosynthesis in canola under salt stress: evaluation of chlorophyll fluorescence parameters as potential indicators. j. agron. crop sci. doi: 10.1111/jac.12120 baker, n.r., 2008: chlorophyll fluorescence: a probe of photosynthesis in vivo. annu. rev. plant biol. 59, 89-113. chen, t.h.h., murata, n., 2008: glycinebetaine: an effective protectant against abiotic stress in plants. trends plant sci. 13, 499-505. chen, t.h.h., murata, n., 2011: glycinebetaine protects plants against abiotic stress: mechanisms and biotechnological applications. plant, cell environ. 34, 1-20. fujii, h., zhu, j.-k., 2009: an autophosphorylation site of the protein kinase sos2 is important for salt tolerance in arabidopsis. molecular plant 2, 183-190. hanson, b., bendixen, w., 1995: drip irrigation controls soil salinity under row crops. calif. agric. 49, 19-23. hermans, c., smeyers, m., rodriguez, r.m., eyletters, m., strasser, r.j., delhaye, j.p., 2003: quality assessment of urban trees: a comparative study of physiological characterisation, airborne imaging and glycinebetaine protects psii under salt stress 85 tab. 3: mean squares from analysis of variance of data for φpo, ψo, φeo, φdo, φpav in leaves of two cultivars of canola (brassica napus l.) treated with or without foliar spray of gb to salt stressed and non stressed condition. source of variance df φpo ψo φeo φdo φpav salt 1 0.0091*** 0.0269*** 0.0358*** 0.0091*** 1073.8*** gb 1 7.1253* 4.205ns 0.001* 7.125** 501.882*** cultivar 1 1.4878ns 2.101ns 3.445ns 1.487ns 225.234*** salt* cultivar 1 1.4028ns 2.88ns 4.575ns 1.4028ns 493.506*** salt*gb 1 2.5312ns 3.781ns 2.587ns 2.531ns 1452.05*** gb*cultivar 1 5.3628** 0.003*** 0.0036*** 5.3628** 133.861** salt*gb*cultivar 1 5.6640* 0.0010* 0.0013* 3.1878* 745.047*** error 24 5.6281 1.754 1.926 0.0013 247.93 ns = non-significant; *,**,*** significant at 0.05, 0.01 and 0.001 probability gb = glycinebetaine, salt = salt stress φpo= maximum quantum yield of primary photochemistry, ψo= capacity of ps ii to transfer trapped excitation, φeo= quantum yield of electron transport, φdo= quantum yield of dissipation energy, φpav= performance indux tab. 2: mean squares from analysis of variance of data for area, fix area, sm, ss and n in leaves of two cultivars of canola (brassica napus l.) treated with or without foliar spray of gb to salt stressed and non stressed condition. source of variance df area fix area sm ss n salt 1 2.50* 4.069ns 107083.4*** 0.054*** 261404.0*** gb 1 4.98** 4.177ns 53670.7*** 4.27ns 434800.2*** cultivar 1 1.89ns 2.466ns 31302.4*** 0.002* 2039.8ns salt* cultivar 1 1.26** 3.083ns 55587.0*** 4.27ns 384013.7*** salt*gb 1 5.89** 1.156*** 60786.1*** 0.007*** 104372.5*** gb*cultivar 1 1.82ns 6.131ns 4017.64* 2.82ns 95882.2*** salt*gb*cultivar 1 4.71ns 1.590** 40273.9*** 1.015ns 923.9ns error 24 1.325 4.094 13294.1 0.010 32840.7 ns = non-significant; *,**,*** significant at 0.05, 0.01 and 0.001 probability gb = glycinebetaine, salt = salt stress area= size of reduced plastoquinone pool or the area above the chl. fluorescence between fo and fm, sm=multiple turnover number, ss=single turnover number, n= number of qaredox turnover until fm. tab. 4: mean squares from analysis of variance of data for abs/rc, tro/rc, eto/rc and dio/rc in leaves of two cultivars of canola (brassica napus l.) treated with or without foliar spray of gb to salt stressed and non stressed condition. source of variance df abs/rc tro/rc eto/rc dio/rc salt 1 0.8613*** 0.3224*** 0.005ns 0.1297*** gb 1 0.006ns 7.8125e-5ns 0.0011ns 0.0048** cultivar 1 0.0411** 0.0192** 0.0032ns 0.0040** salt* cultivar 1 0.0136ns 0.0047ns 7.2e-5ns 0.0023* salt*gb 1 0.0716*** 0.0438*** 0.0080* 0.0033* gb*cultivar 1 5e-5ns 0.0017ns 0.0144** 0.0023* salt*gb*cultivar 1 0.0037ns 1.5313e-4ns 0.0023ns 0.0023* error 24 0.0038 0.0023 0.0012 4.5569e-4 ns = non-significant; *,**,*** significant at 0.05, 0.01 and 0.001 probability gb = glycinebetaine, salt = salt stress abs/rc = absorbance flux per reaction center, tro/rc = trapped energy flux per reaction center, eto/rc = electron transport flux per reaction center, dio/rc = dissipation energy flux per reaction center. 86 a. khalid, h.r. athar, z.u. zafar, a. akram, k. hussain, h. manzoor, f. al-qurainy, m. ashraf on site fluorescence monitoring by the ojip-test. j. plant physiol. 160, 81-90. kalaji, h.m., bosa, k., kościelniak, j., hossain, z., 2011: chlorophyll a fluorescence − a useful tool for the early detection of temperature stress in spring barley (hordeum vulgare l.). omics: j. integrative biol. 15, 925-934. kalaji, m., guo, p., 2008: chlorophyll fluorescence: a useful tool in barley plant breeding programs. photochemistry research progress. nova science publishers, inc., new york, 439-463. laemmli, u.k., 1970: cleavage of structural proteins during the assembly of the head of bacteriophage t4. nature 227, 680-685. mäkelä, p., mantila, j., hinkkanen, r., pehu, e., peltonen-sainio, p., 1996: effect of foliar applications of glycinebetaine on stress tolerance, growth, and yield of spring cereals and summer turnip rape in finland. j. agron. crop sci. 176, 223-234. maslenkova, l.t., m.t.s., popova, l.p., 1992: changes in the polypeptide patterns of barley seedlings exposed to jasmonic acid and salinity. plant physiol. 98, 700-707. munns, r., tester, m., 2008: mechanisms of salinity tolerance. annu. rev. plant biol. 59, 651-681. murata, n., takahashi, s., nishiyama, y., allakhverdiev, s.i., 2007: photoinhibition of photosystem ii under environmental stress. biochimica et biophysica acta (bba)-bioenergetics 1767, 414-421. raza, s.h., athar, h.-u.-r., ashraf, m., 2006: influence of exogenously applied glycinebetaine on the photosynthetic capacity of two differently adapted wheat cultivars under salt stress. pak. j. bot. 38, 341-351. rodríguez-milla, m.a., salinas, j., 2009: prefoldins 3 and 5 play an essential role in arabidopsis tolerance to salt stress. mol. plant 2, 526534. snedecor, g.w., cochran, w.g., 1989: statistical methods (8th edition). iowa state university press, ames. strasser, r.j., srivastava, a., govindjee, 1995: polyphasic chlorophyll a fluorescence transient in plants and cyanobacteria. photochem. photobiol. 61, 32-42. strasser, r.j., tsimilli-michael, m., qiang, s., goltsev, v., 2010: simultaneous in vivo recording of prompt and delayed fluorescence and 820-nm reflection changes during drying and after rehydration of the resurrection plant haberlea rhodopensis. biochim. biophys. acta 1797, 1313-1326. stirbet, a., govindjee, 2011: on the relation between the kautsky effect (chlorophyll a fluorescence induction) and photosystem ii: basics and applications of the ojip fluorescence transient. j. photochem. photobiol. b: biol. 104, 236-257. tsimilli-michael, m., strasser, r.j., 2008: experimental resolution and theoretical complexity determine the amount of information extractable from the chlorophyll fluorescence transient ojip. in: allen, j., gantt, e., golbeck, j., osmond, b. (eds.), photosynthesis. energy from the sun, 697-701. springer netherlands. ulfat, m., athar, h.u.r., ashraf, m., akram, n.a., jamil, a., 2007: appraisal of physiological and biochemical selection criteria for evaluation of salt tolerance in canola (brassica napus l.). pak. j. bot. 39, 1593-1608. vredenberg, w., 2011: kinetic analyses and mathematical modeling of primary photochemical and photoelectrochemical processes in plant photosystems. biosyst. 103, 138-151. yang, x., lu, c., 2005: photosynthesis is improved by exogenous glycinebetaine in salt-stressed maize plants. physiol. plant. 124, 343-352. zhang, l., tian, l.-h., zhao, j.-f., song, y., zhang, c.-j., guo, y., 2009: identification of an apoplastic protein involved in the initial phase of salt stress response in rice root by two-dimensional electrophoresis. plant physiol. 149, 916-928. address of the authors: institute of pure and applied biology, bahauddin zakariya university, multan pakistan fig. 8: comparative analysis of protein banding pattern of the two canola cultivars. lanes 1-4, dunkled; lanes 5-8, cyclon. lane 1: control, lane 2: gb (100 mm), lane 3: salt (150 mm), lane 4: gb + salt. similar sample arrangement is for cyclon cultivar in lanes 5-8. molecular weights of the marker proteins are given in kda. fig. 7: ability to produce nadph of two cultivars of canola (brassica napus l.) when three week old plants were subjected to foliar application of glycinebetaine and salt stress for further two weeks. © the author(s) 2015. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/3.0/). journal of applied botany and food quality 89, 270 278 (2016), doi:10.5073/jabfq.2016.089.035 1* department of botany, pmas arid agriculture university rawalpindi, pakistan drought mitigation potential of azospirillum inoculation in canola (brassica napus) maimona saeed1*, noshin ilyas, roomina mazhar, fatima bibi, nazima batool (received december 9, 2015) * corresponding author summary azospirillum is considered to be a most effective plant growth promoting rhizobacteria (pgpr), which is responsible for various modifications in plants to cope with stress conditions. therefore, the present research was planned to evaluate the effect of azospirillum lipoferum (gq 255949) inoculation on growth, biochemical, yield attributes of canola grown under drought conditions. two different modes of inoculation were used; i.e., inoculation of seeds directly and exposure of planted seed in the rhizosphere. drought stress was imposed at flowering stage. azospirillum seed inoculation was helped mitigate stress effects by improving germination percentage up to 12.49 %. root area was increased up to 18.5 % and 11.38 % with seed and rhizosphere inoculation in drought stress respectively. chlorophyll contents and water potential were increased 12.21 %, and 11.0 % in seeds inoculated under drought conditions. superoxide dismutase activity was decrease up to 24.6 % and 12.5 % in seed and rhizosphere inoculated plants under well watered conditions. seed inoculation was most effective, as number of seeds per pod and seed weight per plant was significantly increased up to 25 %, and 14.28 % as compared to the control. in conclusion, azospirillum can mitigate deleterious effects of drought stress in canola under water deficiency conditions. key words: pgprs, canola, drought introduction drought stress is scarcity of available water in soil, which is necessary for optimum growth and reproduction of plants (lichtfouse, 2010). reduced water supply is one of the most important factors which is responsible for reduction in agricultural productivity (mahmood et al., 2009; ashraf, 2010). deficiency of water causes injurious effects on plants by reducing growth, decreasing nutrient intake and changing water status of plants (ali and ashraf, 2011; shahbaz et al., 2011a). reduced water supply also causes a decline in leaf development and can alter stomatal conductance (qaderi et al., 2006). in addition to morphology, physiological processes, inhibited by water stress are photosynthesis, cell turgidity and cell growth (tahir et al., 2007). in short, every feature of plant growth is affected by water stress including anatomy, morphology, biochemistry, plant physiology and yield (jones et al., 2003; hafiz et al., 2004). canola (brassica napus l.) oil productivity stands third after soybean and palm oil crops in the world. it produces as much as 14.7 % of the vegetable edible oil and has a high content of unsaturated fatty acids (yasari et al., 2008). compared to cereal crops, canola is ranked fifth after wheat, maize, rice and cotton (cardoza and stewart, 2003). canola oil is a premium cooking oil that has less than 2 % erucic acid and is low in saturated fatty acids. it also is rich in mono – poly unsaturated fatty acids which helps to decrease cholesterol level (carvaloh et al., 2006; omidi et al., 2010). drought is one of the important stress factor which is responsible for reducing production of canola in semi-arid regions of the world. almost, 17 to 70 % yield reductions have been recorded due to drought (nasri et al., 2007). plant growth-promoting rhizobacteria (pgpr) are able to promote growth and yield of plants under stress conditions. inoculation of these microorganisms provides higher crop yield without interfering with natural processes in the ecosystem (thakore, 2006). during the last two decades, various bacteria (i.e., azotobacter sp., azospirillum sp., acetobacter sp., bacillus, pseudomonas sp.) are used for plant growth promotion under various prevailing biotic and abiotic stresses (turan et al., 2006). azospirillum is one of the very effective plant growth promoting rhizobacteria (pgpr) which act as a general root colonizer to improve crop growth and yield up to 5 to 30 %. azosprilium offer inexpensive and easy application while providing minerals, and phytophormones as well as fixed nitrogen, and reduce the synthesis of ethylene thereby increasing yield (yasari et al., 2008). azospirillum spp inoculation can improve tolerance to water stress, improve the growth of plants in arid and semiarid regions (ilyas and bano, 2010). various studies have documented the role of azospirillum in improving growth and yield of canola (yasari et al., 2008; baniaghil et al., 2013). the impact of water deficit conditions on plant physiology has been studied for many years. the present study was conducted in order to evaluate the effect of azospirillum inoculation on growth and yield of canola. further, physiological and biochemical responses of canola under drought stress and the role of azospirillum in mitigation of drought stress in canola were also studied. materials and methods to evaluate the effect of azospirillum inoculation on growth and yield parameters of canola plants under drought stress a pot experiment was conducted in a green house, department of botany, pir mehr ali shah arid agriculture university, rawalpindi. two canola varieties (i.e., rainbow and con ii) were collected from the national agricultural research center (narc), islamabad. seeds of both varieties were surface sterilized in 0.1 % mercuric chloride solution for 5 minutes, then washed thoroughly with distilled water. inoculum was prepared by inoculating 24 h old culture of azospirillum in lb media and kept for 72 h on a shaker, then centrifuged at 10,000 rpm for 10 min. the pellet was diluted to 100 ml with distilled water and the supernatant was discarded. optical density was calculated to be one. six hour sterilized seeds were soaked in the inoculum and the heat killed inoculum was prepared by autoclaving the inoculum. six seeds per pot were sown from 19 november 2013 to 10 february 2014. for rhizosphere inoculation live cultures were added into soil after sowing the sterilized seeds. heat killed bacteria also were inoculated onto seeds. each treatment was replicated three times. plant drought stress was imposed at the flowering stage by restricting water for ten days along with a control for each inoculation. germination analysis for germination analysis under drought conditions, inoculated and uninoculated seeds were placed in a set of petri plates, which were potential of azospirillum for drought 271 provided with moist filter paper. seeds were monitored daily for mean daily germination. the seed was considered germinated when its radical emerged 5 mm. un-inoculated seeds were sown in another set of plates. following parameters of germination were evaluated. germination percentage for each treatment, germination percentage was estimated by using the following formula: germination % = total seeds germinated / total no of seeds planted × 100 germination index using the following formula (ista, 2005), germination index was calculated. germination index = n/d where, d = days after planting, n = no of seedlings emerged on day‘d’ promptness index by using the method of (noreen et al., 2007) promptness index (p.i) was determined. p.i = nd2 (1.00) + nd4 (0.75) + nd6 (0.50) + nd8 (0.25) where (nd2… nd8) are number of emerging seedlings on day 2, 4, 6, 8. seedling vigor index the method of abdulbaki and anderson (1973) was used to determine the seedling vigor index. seedling vigor index = germination % × seedling length (mm) plant growth parameters after washing root and shoot length were measured for three plants for each treatment. physiological parameters a scholander pressure chamber was used for determination of leaf water potential (scholander et al., 1965). chlorophyll content was determined by following the method of bruinsma (1963). biochemical parameters proline content was determined by using a spectrophotometer (bates et al., 1973). soluble sugar was determined by following dubois (1951). for protein determination, an extract of plant samples was prepared by homogenizing 0.2 g of fresh leaf material in 4 ml of sodium phosphate buffer (ph 7) and then centrifuged. in separate test tube 0.5 ml of leaf extract and 0.5 ml distilled water and 3 ml of coomassie bio red dye were mixed. the reaction mixture was placed undisturbed for 5 minutes and the absorbance recorded at 595 nm (bradford, 1976). antioxidant enzyme assay superoxide dismutase for determination of superoxide dismutase, 10 ml sodium phosphate buffer was used to grind 0.5 g leaf material. the solution was allowed to settle and then 0.1ml of extract was added to another set of test tubes along with 0.1 ml riboflavin and 3 ml phosphate buffer. this reaction mixture was placed under a fluorescent lamp for 8 min to start reacting. the same reaction mixture was prepared for the dark reaction in another set of tubes. absorbance for both sets of tubes was recorded at 560 nm wavelength (giannopolitis and ries, 1977). sod μg/ml = absorbance of sample × k value × dilution factor / weight of the sample yield parameters: various yield parameters of canola plants were analysed such as pod length, number of seeds per pod, number of pods per plant and seed weight per plant, estimated after harvesting. treatments t0 well watered and control t1 seed inoculated and well watered t2 rhizosphere inoculated and well watered t3 heat killed-inoculated and well watered t4 drought stress and un-inoculated t5 seed inoculated and drought stress t6 rhizosphere inoculated and drought stress t7 heat killed-inoculated and drought stress statistical analysis the software used for statistical analysis was statistix 9.1. a two way anova with a factorial block design was carried out for all treatments. each treatment had three replicates results germination parameters the effect of plant inoculation with azospirillum lipoferum (accession no. gq255949) on germination of two canola varieties was significant (p ≤ 0.05). drought stress decreased the germination percentage up to 37 % as compared to well-watered conditions (tab. 1). seed inoculation, with bacteria resulted in a 10 % increase in germination under well-watered conditions and a 12.49 % increase under water stress. heat killed inoculums had no significant effect on germination percentage under both water regimes. results from the germination index (tab. 1) showed a similar effect of drought stress. reduction in germination index was improved by treating the seeds with azospirillum. after inoculation under both well watered and drought conditions an 8.91 % and 12.28 % increase was observed in plants relative to their respective controls. effect of the heat killed inoculum remained insignificant under both control and drought conditions. a significant increase in promptness index was encountered in plants treated with azospirillum inoculum under both well watered conditions and under a water shortage (tab. 1). there was an increase of 20.87 % in seedling vigour index that was recorded in plants inoculated under well watered conditions as compared to un-inoculated plants. a similar trend of inoculation was observed in plants grown under drought conditions; a 12.4 % and 4.16 % increase in seedling vigour index with both modes of inoculation was observed. there was a significant difference observed for all the parameters of germination in both canola varieties (tab. 1). effect of inoculation on growth parameters the effect of bacterial inoculation on growth parameters of plants was significant (p ≤ 0.05) when compared to control plants in both seed and rhizosphere inoculations. a decrease of 10 % was observed in shoot length for drought exposed plants as compared to control plants (fig. 1a). with a restricted water supply, both seed and rhizosphere inoculations resulted in a 7.2 % and 4.5 % increase in shoot 272 m. saeed, n. ilyas, r. mazhar, f. bibi, n. batool tab. 1: effect of azospirillum inoculation on germination parameters of two canola varieties under drought stress. treatments seedling vigor index promptness index germination index germination % v1 v2 v1 v2 v1 v2 v1 v2 t0 780±5h 749.91±4i 4.5±0.1f 4.5±0.1g 1.57+ 0.1d 1.57±0.1d 78.57±0.9e 71.42±1e t1 685.68±5f 650±4h 2.25±0.2e 2.25±0.1e 1.14±0.2ab 1±0.1bc 57.14±0.9d 50±1d t2 942.81±5b 903.55±4g 6.25±0.2b 6±0.1d 1.71±0.1ab 1.71±0.1bc 85.71±1b 78.57±0.9c t3 824.98±5a 785.62±4e 4.75±0.1a 3.25±0.1c 1.57±0.1a 1.57±0.1ab 78.57±0.9a 71.42±0.9b t4 771.36±5k 742.82±4d 3.25±0.1i 2.5±0.1c 1.28±0.1d 1.14±0.2ab 64.28±0.9f 57.14±0.9b t5 714.25±5j 700±4c 2.5±0.1h 2.25±0.2b 1.14±0.2cd 1±0.1a 57.14±0.9e 50±1a e f c d e ab c a h f g g h e f f g d e f b c d a b c a b c a b a b a where, to= un-inoculated and well watered, t1= un-inoculated and drought exposed, t2= seed inoculated and well watered, t3= seed inoculated with heat killed inoculums and well watered, t4= seed inoculated and drought exposed, t5= seed inoculated with heat killed inoculums and drought exposed. and v1=rainbow, v2=con ii. length respectively. data regarding shoot fresh and dry weight also showed the same results with different modes of inoculation (fig. 1b and 1c). analysis of data from well watered and drought exposed plants treated with azospirillum showed a significant difference between inoculated and control plants. seed inoculation resulted in a 23.68 % increase in root area compared to controls treated under well watered conditions. even rhizosphere inoculation resulted in a 18.09 % increase in root area compared to regularly watered plants. a similar result was observed in drought exposed plants, where a 18.5 % and 11.38 % increase in area treated with seed and rhizosphere inoculations respectively. the increase of 9.72 % in root area was observed with a heat killed inoculum compared to control plants grown under drought conditions. rainbow variety showed a better response towards treatments compared to con ii. physiological parameters drought stress caused a 29.7 % decrease in water potential for the leaf in both canola varieties (fig. 2a). inoculation tended to reduce the drought effect resulting in a 11.01 % and 8.26 % increase in water potential with seed and rhizosphere inoculation under drought stress conditions. effect of heat killed inoculation was not significant unfig. 1a: effect of azospirillum inoculation on shoot length of two canola varieties (v1 and v2) grown under drought stress. where, to = un-inoculated and well watered, t = un-inoculated and drought exposed, t2 = seed inoculated and well watered, t3 = rhizosphere inoculated and well watered, t4 = seed inoculated with heat killed inoculums and well watered, t5 = seed inoculated and drought exposed, t6 = rhizosphere inoculated and drought exposed, t7 = seed inoculated with heat killed inoculums and drought exposed. and v1 = rainbow, v2 = con ii. fig. 1b: effect of azospirillum inoculation on shoot fresh weight of two canola varieties (v1 and v2) grown under drought stress. where, to = un-inoculated and well watered, t = un-inoculated and drought exposed, t2 = seed inoculated and well watered, t3 = rhizosphere inoculated and well watered, t4 = seed inoculated with heat killed inoculums and well watered, t5 = seed inoculated and drought exposed, t6 = rhizosphere inoculated and drought exposed, t7 = seed inoculated with heat killed inoculums and drought exposed. and v1 = rainbow, v2 = con ii. der both well watered and drought conditions. the effect of drought stress on photosynthetic pigments was more pronounced compared to other parameters. considerable variation was observed in chloro phyll content of well watered and water stressed plants. water deficit stress resulted in a 23.31 % decrease in the total chlorophyll content of water stressed plants as compared to well watered plants (fig. 2b). plants with seed and rhizosphere inoculation showed a 20.28 % and 9.52 % increase of total chlorophyll in leaves compared to controls grown one under well watered conditions. a similar trend was seen in drought exposed plants, where a 12.21 % and 5.98 % increase was seen in seed and rhizosphere inoculated plants grown under water deficit conditions. seed inoculation with a heat killed inoculum did not show any significant difference in plants grown under both well watered and water deficit conditions. effect on compatible solutes a considerable amount of osmolytes was accumulated in water stressed plants. in contrast to well watered plants a 23.29 % proline potential of azospirillum for drought 273 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 b b f c d e b a b b a b b c d e c c d e c d c d e c d e v1 v2 0 5 10 15 20 25 30 t0 t1 t2 t3 t4 t5 t6 t7 to ta l c hl or op hy ll co nt en t ( µg /m l) treatments v1 v2 e d e g g f e f e e c d b c b c b b a b b a increase was observed in drought treated plants (fig. 3a). seed and rhizosphere inoculation allowed the plants to maintain a high proline level up to 10.34 % and 8.62 % under water stress compared to plants without inoculation. a similar increase of 21.72 % in soluble sugar content was observed in drought exposed plants (fig. 3b). inoculation effects remained significant under limited water availability where a 10.69 % and 7.28 % increase was observed in sugar content of seed and rhizosphere inoculated plants respectively. results revealed a 36.67 % decrease in soluble proteins in drought imposed plants (fig. 3c). inoculation reduces the injurious effect of drought with seed inoculation resulting in a 10.02 % and 9.48 % decrease in well watered and drought conditions in contrast to their respective controls. results were found to be similar with rhizosphere inoculation, with a 12.63 % decrease in well watered and a 10.70 % decline in stressed plants. response of both varieties was significant following inoculation. rainbow showed a better response compared to con ii. an increase in super oxide dismutase (fig. 4a) was observed in plants grown under drought stress compared to normally irrigated plants. super oxide dismutase activity was 35.8 % increased in drought exposed plants compared to control plants. data for azospirillum inoculation showed a significant decrease in super oxide dismutase in inoculated plants as compared to untreated plants. seed and rhizosphere inoculation showed a 24.6 % and 12.5 % decrease in super d e f b c d e f g f g d e d e f d e f c d e a b c d a b c d a b c a b a b c a b c a g fig. 1c: effect of azospirillum inoculation on shoot dry weight of two canola varieties (v1 and v2) grown under drought stress. where, to = un-inoculated and well watered, t = un-inoculated and drought exposed, t2 = seed inoculated and well watered, t3 = rhizosphere inoculated and well watered, t4 = seed inoculated with heat killed inoculums and well watered, t5 = seed inoculated and drought exposed, t6 = rhizosphere inoculated and drought exposed, t7 = seed inoculated with heat killed inoculums and drought exposed. and v1 = rainbow, v2 = con ii. a b a f g c d e f g c d e f g c d e f c d e f c d e f g f g e f g c d e f g d e f g b c d b c d e a b c fig. 1d: effect of azospirillum inoculation on root surface area of two canola varieties (v1 and v2) grown under drought stress. where, to = un-inoculated and well watered, t = un-inoculated and drought exposed, t2 = seed inoculated and well watered, t3 = rhizosphere inoculated and well watered, t4 = seed inoculated with heat killed inoculums and well watered, t5 = seed inoculated and drought exposed, t6 = rhizosphere inoculated and drought exposed, t7 = seed inoculated with heat killed inoculums and drought exposed. and v1 = rainbow, v2 = con ii. fig. 2b: effect of azospirillum inoculation on total chlorophyll content of two canola varieties(v1 and v2) grown under drought stress. where, to = un-inoculated and well watered, t = un-inoculated and drought exposed, t2 = seed inoculated and well watered, t3 = rhizosphere inoculated and well watered, t4 = seed inoculated with heat killed inoculums and well watered, t5 = seed inoculated and drought exposed, t6 = rhizosphere inoculated and drought exposed, t7 = seed inoculated with heat killed inoculums and drought exposed. and v1 = rainbow, v2 = con ii. fig. 2a: effect of azospirillum inoculation on water potential of two canola varieties (v1 and v2) grown under drought stress. where, to = un-inoculated and well watered, t = un-inoculated and drought exposed, t2 = seed inoculated and well watered, t3 = rhizosphere inoculated and well watered, t4 = seed inoculated with heat killed inoculums and well watered, t5 = seed inoculated and drought exposed, t6 = rhizosphere inoculated and drought exposed, t7 = seed inoculated with heat killed inoculums and drought exposed. and v1 = rainbow, v2 = con ii. 274 m. saeed, n. ilyas, r. mazhar, f. bibi, n. batool oxide dismutase activity compared to un-inoculated plants under well watered conditions. a similar trend of inoculation was observed in drought exposed plants where a 45 % and 43 % decrease was observed following seed and rhizosphere inoculation relative to their respective controls. a significant difference was recorded in both varieties. response of rainbow was much better as compared to con ii. yield parameters data for yield parameters differs significantly between well watered and drought exposed plants. drought stress caused a 36.08 %, 9.41 %, and a 41.42 % reduction in the number of seeds per pod, number of pods per plant and seed weight per plant as compared to control. a significant difference was observed in azospirillum inoculated plants as compared to un-inoculated plants. the best outcome of inoculation for number of seeds per pod was seen in droughtexposed plants where a 25 % and 16.67 % increase was observed with two different modes of inoculation (fig. 5a). data regarding to number of pods per plant (fig. 5b) reveals a 9.41 % decrease in yield in drought exposed plants compared to control plants. seed and rhizosphere inoculation resulted in a 6.07 % and 4.88 % increase in yield compared to control plants grown under well watered conditions. in drought conditions, only a 3.52 % and 2.79 % increase was observed following seed and rhizosphere inoculation. while in the case of seed weight per plant seed and rhizosphere inoculation resulted in a 14.14 % and 6.06 % increase in well watered compared to control plants. in drought exposed plants, seed and rhizosphere inoculation resulted in a 14.28 % and 7.14 % increase with respect to control plants (fig. 5c). b a e d d e c n l m k j hi g f a a a a a a a a a a a a a a a a fig. 3c: effect of azospirillum inoculation on soluble protein of two canola varieties (v1 and v2) grown under drought stress. where, to = un-inoculated and well watered, t = un-inoculated and drought exposed, t2 = seed inoculated and well watered, t3 = rhizosphere inoculated and well watered, t4 = seed inoculated with heat killed inoculums and well watered, t5 = seed inoculated and drought exposed, t6 = rhizosphere inoculated and drought exposed, t7 = seed inoculated with heat killed inoculums and drought exposed. and v1 = rainbow, v2 = con ii. fig. 3a: effect of azospirillum inoculation on proline content of two canola varieties (v1 and v2) grown under drought stress. where, to = un-inoculated and well watered, t = un-inoculated and drought exposed, t2 = seed inoculated and well watered, t3 = rhizosphere inoculated and well watered, t4 = seed inoculated with heat killed inoculums and well watered, t5 = seed inoculated and drought exposed, t6 = rhizosphere inoculated and drought exposed, t7 = seed inoculated with heat killed inoculums and drought exposed. and v1 = rainbow, v2 = con ii. fig. 3b: effect of azospirillum inoculation on soluble sugar of two canola varieties (v1 and v2) grown under drought stress. where, to = un-inoculated and well watered, t = un-inoculated and drought exposed, t2 = seed inoculated and well watered, t3 = rhizosphere inoculated and well watered, t4 = seed inoculated with heat killed inoculums and well watered, t5 = seed inoculated and drought exposed, t6 = rhizosphere inoculated and drought exposed, t7 = seed inoculated with heat killed inoculums and drought exposed. and v1 = rainbow, v2 = con ii. fig. 4a: effect of azospirillum inoculation on super oxide dismutase of two canola varieties (v1 and v2) grown under drought stress. where, to = un-inoculated and well watered, t = un-inoculated and drought exposed, t2 = seed inoculated and well watered, t3 = rhizosphere inoculated and well watered, t4 = seed inoculated with heat killed inoculums and well watered, t5 = seed inoculated and drought exposed, t6 = rhizosphere inoculated and drought exposed, t7 = seed inoculated with heat killed inoculums and drought exposed. and v1 = rainbow, v2 = con ii. a b a d e f c d c d a b b c a b i g h h i f g e g h d e f f g d e bc abc e e a b a ab c a b c a b c a b c a b c a b c b c a b c c d bc a potential of azospirillum for drought 275 0 10 20 30 40 50 60 to t1 t2 t3 t4 t5 t6 treatments n 0 of p od s pe r pl an t v1 v2 c c d e a b a f g g d e f e f c d e f b b a b a b a a t7 was observed in seed inoculated plants compared to un-inoculated plants. results of seed inoculation were also more pronounced compared to rhizosphere inoculation and use of a heat killed inoculum. similar effects of bacterial inoculation on the germination parameter have been reported in cereals such as sorghum (raju et al., 1999), maize (gharoobi et al., 2012), wheat (bangash et al., 2013) and in sunflower (zaefizadah et al., 2011). this improvement may be due to synthesis of hormones, and increased activity of certain enzymes like amylase which speed up starch assimilation. seed vigor index was also improved by an increase in synthesis of auxin by germinating seedlings when inoculated with bacteria (bharathi et al., 2004). the most immediate effect of drought stress is a reduction in growth (shalhevet et al., 1995) and a number of morphological processes adversely affected by water deficit (dickin and wright, 2008). during the present study there was a decrease in shoot length (fig. 1 a) that was observed in drought exposed plants compared to well watered plants because of a reduction in cell division and elongation. a similar reduction in growth parameters was observed by ashraf et al. (2013) and shafi et al. (2009). shoot fresh weight and dry weight showed a significant decrease under stress conditions. this reduction in biomass was due to activity of metabolic enzymes being used under stress conditions (hong and ji-yun, 2007; xu et al., 2008), a change in metabolism (chimenti et al., 2006) and inhibition in cell division (arshad et al., 2008). previously reduction in biomass was reported by ashraf et al. (2013). plants with azospirillum inoculation showed an increase in shoot length and biomass (fig. 1-3 a). seed inoculation produced maximum results followed by inoculation of the rhizosphere, and use of a heat killed inoculation compared to controls. phosphate solublization, production of auxin, the fixation of nitrogen and enhanced nutrient intake are likely responsible for better shoot length and shoot weight (bashan and holguin, 1997; bashan et al., 2004). data looking at leaf water potential indicates a considerable decrease in water potential under drought stress. inoculation tends to reduce drought effects. increase in water potential was observed in seed inoculated plants, although it was not significantly different from control plants (fig. 1 b). similar decreases in water potential under drought stress and its mitigation by azospirillum was reported by ilyas and bano (2010). karimi and mohseni (2013) reported a similar decline in chlorophyll content in soybean cultivars grown c c d e a f g g d e f e f c d e f b b a b a b a a a b fig. 5c: effect of azospirillum inoculation on seed weight per plant of two canola varieties (v1 and v2) grown under drought stress. where, to = un-inoculated and well watered, t = un-inoculated and drought exposed, t2 = seed inoculated and well watered, t3 = rhizosphere inoculated and well watered, t4 = seed inoculated with heat killed inoculums and well watered, t5 = seed inoculated and drought exposed, t6 = rhizosphere inoculated and drought exposed, t7 = seed inoculated with heat killed inoculums and drought exposed. and v1 = rainbow, v2 = con ii. fig. 5a: effect of azospirillum inoculation on number of seeds per pod of two canola varieties (v1 and v2) grown under drought stress. where, to = un-inoculated and well watered, t = un-inoculated and drought exposed, t2 = seed inoculated and well watered, t3 = rhizosphere inoculated and well watered, t4 = seed inoculated with heat killed inoculums and well watered, t5 = seed inoculated and drought exposed, t6 = rhizosphere inoculated and drought exposed, t7 = seed inoculated with heat killed inoculums and drought exposed. and v1 = rainbow, v2 = con ii. b c d e a b c d e b c d e a b c d e a b c d e a b c d a b c a b c a b c a b a b c a d e e c d e b c d e fig. 5b: effect of azospirillum inoculation on number of pods per plant of two canola varieties (v1 and v2) grown under drought stress. where, to = un-inoculated and well watered, t = un-inoculated and drought exposed, t2 = seed inoculated and well watered, t3 = rhizosphere inoculated and well watered, t4 = seed inoculated with heat killed inoculums and well watered, t5 = seed inoculated and drought exposed, t6 = rhizosphere inoculated and drought exposed, t7 = seed inoculated with heat killed inoculums and drought exposed. and v1 = rainbow, v2 = con ii. discussion drought stress causes a significant reduction in germination percentage, germination index and seedling vigor index when compared to control plants. decline in germination components of water stress is due to less water intake by the seed coat and inhibition of water intake at the initial stage of growth (turk et al., 2004; bahrami et al., 2012) or reduction in external water potential. another reason for the reduction in germination is a delay in hydrolysis of storage compounds in the endosperm and cotyledon, or slow transport of water to the developing embryo axis (ayaz et al., 2000). under drought stress, enzymatic activity slows down and subsequently a decrease in the germination percentage. reduction in water absorption decreases cell turgidity, division, and radical length (zaefizadah et al., 2011). bacterial inoculation is an effective strategy to enhance germination, improve seedling emergence and respond to external environmental factors (lugtenberg et al., 2002). in the present study, a considerable increase in germination parameters (tab. 1) 276 m. saeed, n. ilyas, r. mazhar, f. bibi, n. batool under drought stress. the causal agent behind the reduction of chlorophyll content may be a decline in pigment synthesis due to disruption of macro-aggregates of chl a, chl b, or production of reactive oxygen species (ros) (smirnoff, 1993). there was a significant increase in chlorophyll content of plants with azospirillum inoculation under drought stress compared to un-inoculated plants. seed inoculation showed the best result. a considerable decrease in the chlorophyll content of leaves was observed under water stress in canola plants (kauser et al., 2006). in the present study analysis of data regarding chlorophyll content showed a tremendous decrease in chlorophyll content of drought exposed plants compared to well watered conditions (fig. 2). production and accumulation of proline and soluble sugars is a common response in plants exposed to water deficit stress, which plays a role in protection of the cell membrane and macromolecule structure under stress conditions (prado et al., 2000). similar findings were reported by din et al. (2011) in canola plants. azospirillum inoculation improves proline and soluble sugar contents as a result of water influx increases in plant cells (fig. 1-2 c). nosheen et al. (2011) also reported the same result following inoculation of canola. the reason for this increase was the breakdown of polysaccharides, which help to stabilize cell turgor (nazarli et al., 2011). among compatible solutes soluble proteins are quite important for the manifestation of drought stress. soluble protein content was decreased in plants exposed to drought stress (fig. 3 c). these findings were supported by good and zaplachinski (1994), who observed a reduction in soluble protein content in brassica napus under stress. the decline in soluble protein contents was due to reduction in photosynthesis, absence of raw materials for protein synthesis that cause a decline in or completely stop the process (mohammadkhani and heidari, 2008). in the present research, a similar decline was observed in protein content, but inoculation of azospirillum mitigated the effects of stress by reducing proportions of plants with which it decreases without inoculation under stress conditions. the water deficit condition is associated with production of reactive oxygen species (ros) in the chloroplast, mitochondria and peroxisome leading to oxidative stress. activation of antioxidant enzyme systems is a common response in plants to reduce oxidative stress (foyer and noctor, 2003). plants depend on activities of super oxide dismutase (sod) and consequently on the activities of other antioxidant enzymes (alscher et al., 2002). in the present study, tremendous increase in super oxide dismutase was noticed in drought exposed plants (fig. 1). our results are consistent with those of abedi and pakniyat (2010). data for inoculated plants showed a significant reduction in super oxide dismutase under both well watered and drought conditions. seed inoculation has shown a better response as compared to the rhizosphere and heat killed inoculation with bacteria. heat killed inoculation did not show any significant difference in both water regimes. bacterial inoculation decreased the level of sod by stimulating the intake of nitrogen and phosphorous, which interacted with carbohydrates as non-enzymic antioxidants. these substances utilize less energy and increase speedily compared to enzymic compounds (dai et al., 2009). drought imposes a negative impact on performance of canola. however this negative effect on yield depends on stage, duration of stress and ability of the plant to cope with stress (trotel-aziz et al., 2000). drought stress reduced the yield and yield component in the canola plant (birunara et al., 2011). yield parameters of both plant varieties tested were badly affected by drought stress, resulting in a reduction in yield components like number of pods per plant, number of seeds per plant, and seed weight per plant encountered under water deficit conditions compared to well watered conditions (fig. 1-3 e). reduction in photosynthesis under water deficit conditions caused pod abortion, consequently a decrease number of pods produced (diepenbrock, 2000). decline in seed weight is associated with a decrease in the number of seeds per plant as well as number of seeds per pod. water deficit had a direct impact on size of sink, decreased storage capacity of source and subsequently caused a reduction in seed weight (shiranirad et al., 2013). effects on seed treated in the rhizosphere and with a heat killed inoculation were not very effective compared to direct seed treatment with the bacteria. this may be due to ability of the azospirillum to develop roots, facilitate nutrient and water uptake, displace pathogenic bacteria and help in nitrogen fixation (okon and itzigsohn, 1995). from the present research, we conclude that azospirillum lipoferum (accession no. gq255950) was able to mitigate the adverse effects of drought stress by improving the morphological, physiological and biochemical aspects of the plant. among the different modes of inoculation, effects of seed inoculation were more pronounced compared to seed treated in the 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different sub-cultivars of durum wheat during germination. adv. environ biol. 5, 74-80. address of the corresponding author: maimona saeed, department of botany, pmas arid agriculture university rawalpindi, pakistan e-mail: maimonasaeed6@gmail.com © the author(s) 2016. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 90, 191 196 (2017), doi:10.5073/jabfq.2017.090.024 1department of horticulture, faculty of agriculture, harran university, şanlıurfa, turkey 2department of horticulture, institute of natural and applied sciences, harran university, şanlıurfa, turkey 3department of horticulture, faculty of agriculture, atatürk university, erzurum, turkey effects of dormancy-breaking treatments on seed germination and seedling growth of pistacia khinjuk stocks using as rootstock for pistachio trees izzet acar1*, halil yasar2, sezai ercisli3 (received january 18, 2017; accepted april 15, 2017) * corresponding author summary this study was carried out to determine the effects of different dormancy-breaking treatments including stratification, sulphuric acid scarification, dehulling (removing the mesocarp and exocarp from the nut) and gibberellic acid (ga3) on seed germination and seedling development of two different pistacia khinjuk genotypes (a and b) using as rootstock for pistachio cultivars. seed dormancy-breaking treatments were shelled (control), shelled + ga3, dehulled, dehulled + ga3, sulphuric acid scarification and sulphuric acid scarification + ga3 applications in the present experiment. the seeds of both genotypes were stratified at 4 ºc for 50 days after the dormancybreaking treatments. stratified seeds were sown in the vials filled with peat in the greenhouse to determine the germination percentage. plantlets were transplanted to plastic containers to determine the vegetative growth. the highest germination rate was obtained from sulphuric acid scarification in both p. khinjuk genotypes. in p. khinjuk-a seedlings, the highest stem growth was obtained from scarification and dehulled applications, whereas the poorest development was observed from dehulled + ga3 application. the best growth in the p. khinjuk-b seedlings was obtained from scarification + ga3 application. the effect of the dehulled application on the root development of p. khinjuk-a seedlings was better than the other applications; however the effect of dormancy-breaking applications on root development of p. khinjuk-b seedlings was found to unsteady. scarification increased the number of leaves in both genotypes. as a result, dormancy-breaking applications have been found to be effective on seed germination and seedling growth of p. khinjuk. it was determined that ga3 applications negatively affected both seed germination and root, stem and leaf growth of p. khinjuk-a. keywords: pistacia khinjuk, stratification, scarification, ga3, seed germination, seedling growth introduction horticultural plants including pistachio have been an indispensable part of human life for ages. ever since ancient times, their fruits, seeds, even roots and branches have been used to meet personal and social needs such as severing food, curing diseases and beautifying the planet (sahin et al., 2002; ercisli, 2009; erturk et al., 2010; hricova et al., 2016; yazici and sahin, 2016; zorenc et al., 2016). pistacia genus is a member of the anacardiaceae family and consists of at least eleven species (zohary, 1952). except for p. mexicana and p. texana, which originated in the usa and mexico, all other species are distributed mainly within the mediterranean region, western and central asia and the middle east (esmail-pour, 2001). turkey has a large population of pistacia species and seven species, namely p. vera, p. terebinthus, p. khinjuk, p. atlantica, p. mutica, p. palaestina and p. lentiscus are present and distributed in different regions of turkey (atli et al., 2001). the main pistachio rootstock used in turkey is p. vera, and followed by p. khinjuk, p. terebinthus and p. atlantica. pistacia khinjuk stocks. is an irano-turanian species and widely distributed in iran, iraq, syria, turkey, afghanistan and pakistan. its leaves vary greatly in shape, size and leaflet number not only from tree to tree, but even on the same tree and even on the same branch (karimi et al., 2012). it has been postulated that p. khinjuk has four varieties, var. po pulifolia boiss., var. glabra engl., var. microphylla boiss. and var. macrocarpa zoh. (zohary, 1952). p. khinjuk trees are distributed at siirt, hakkari, gaziantep, adıyaman and bitlis provinces in turkey and their trees may grow up to 10 m in height. (bilgen, 1973; tekin et al., 2001). although able to withstand some of the harshest weather conditions, it is sensitive to the fungus phytophthora spp. (banihashemi, 1995) but has moderate resistance to root-knot nematodes (farivar-mehin, 1995). there are two types of p. khinjuk in nature having large fruits (a) and small fruits (b). fruits of pistacia species consist of a nutmeat (kernel) enclosed in a thin, hard shell (endocarp) surrounded by a fleshy hull (mesocarp and exocarp) (ferguson et al., 2005). the hulls of p. khinjuk (a) fruits are oily and are mainly used for soap making in siirt province of turkey. the seedlings of p. khinjuk form a smooth body. although the p. vera and p. atlantica seedlings are taller, the seedling diameters at the site of budding do not grow as much as p. khinjuk. the base of the seedling and its budding point grow faster than the other rootstocks, thus they can reach to suitable budding thickness more quickly (tekin et al., 2001). it was determined that p. atlantica and p. khinjuk rootstocks were better than p. vera rootstock in terms of tree vigour and crown formation of budded cultivars in dry conditions (ulusarac, 1992). among the pistacia species used as rootstocks for pistachio, it was determined that p. khinjuk is the best beneficiary of the soil nitrogen. graft compatibility is well with pistachio cultivars, and no swelling or growth differences at the budding area (atli et al., 2001). pistachio trees have significant potential for arid and semi-arid areas having suitable climatical conditions in the world. areas suitable for pistachio production have long, hot, dry summers and moderate winters (ferguson et al., 2005). pistachio cultivars are extremely difficult to propagate clonally on their own roots, and therefore rootstocks offer a simple method for propagate the pistachio cultivars. rootstocks have been used for propagating temperate fruit trees for more than 2000 years. rootstocks can influence scion vigour, cropping, fruit quality, climatic adaptability, and susceptibility to pests and diseases. until the mid 19th century, almost all of deciduous fruit tree rootstocks were raised from seed of fruits collected from indigenous wild populations. usually, the seedling rootstocks and the fruiting clones grafted on them were of the same botanical genus and species (webster, 1995). because the seeds of pistacia species are surrounded by a hard scle192 i. acar, h. yasar, s. ercisli rotic endocarp that makes it difficult to germinate, the germination rate in these species is low (isfendiyaroglu and ozeker, 2002). various chemical solutions are used to stimulate seed germination. gibberellic acid (ga) is one of the growth regulators which can be used to partially or fully replace the required period of cold moist stratification in a number of plant species (baskin and baskin, 1998). scarification and cold stratification were found to improve the seed germination in pistacia spp. (ak et al., 1995). seed scarification favours significantly the germination process, therefore it involved the fast inhibition of the tegument of seeds and the entry of water in the reserves that allows the fast exit of the root and the starting of the metabolic reactions of the embryo and the cotyledons (ahoton et al., 2009; chebouti-meziou et al., 2014). there are many researches dealing with stratification, acid scarification and ga3 application on seed germination of different pistacia species (abu-qaoud, 2005; ak, 1988 and 1990; ak et al., 1995; chebouti-meziou et al., 2014; crane and forde, 1974; kafkas and kaska, 1997; piotto, 1995; rahemi and baninasab, 2000). but, no detailed study has so far been reported about the effects of dormancy-breaking treatments on seed germination and seedling growth of pistacia khinjuk. the objective of this study was to determine the effect of dormancy-breaking treatments on seed germination and seedling growth of pistacia khinjuk stocks using as rootstock for pistachio trees. so the study is testing two types of dormancy: physical dormancy where seeds are impermeable to water (hence the need for scarification) and physiological dormancy where the embryo is prevented from growing (hence ga3), and a combination of the two types. materials and methods plant material this experiment was carried out in the greenhouse of the harran university, faculty of agriculture, department of horticulture located in the şanlıurfa province of turkey. dried seeds of both pistacia khinjuk genotypes having large fruits (a) and small fruits (b) were obtained from siirt province of turkey in 2015. seeds were collected from the trees of pistacia khinjuk (a) and (b) genotypes at maturity, dried for 7 days in room conditions and stored in refrigerator (+4 °c, 70-80% relative humidity) for 4 months. the seeds were used to determine the seed germination and seedling development in respect to dormancy-breaking treatments in the research. treatments six different dormancy-breaking treatments were applied to the seeds. treatments were a) shelled (seeds with hull), b) shelled + ga3 application, c) dehulled (seeds without hull), d) dehulled + ga3 application, e) sulphuric acid scarification and f) sulphuric acid scarification + ga3 applications. treatments were; · shelled (control): seeds (with hull) soaked for 24 h in the tap water and then stratified in perlite for 50 days in refrigerator (dark conditions, +4 °c, 70-80% relative humidity). · shelled + ga3: seeds (with hull) soaked in the 500 ppm ga3 for 24 h and then stratified in perlite for 50 days in refrigerator (dark conditions, +4 °c, 70-80% relative humidity). · dehulled: seeds soaked for 24 h in the tap water and then removed their hulls (mesocarp and exocarp). dehulled seeds (without hull) were washed and stratified in perlite for 50 days in refrigerator (dark conditions, +4 °c, 70-80% relative humidity). · dehulled + ga3: seeds soaked for 24 h in the tap water and then removed their hulls. dehulled seeds soaked in the 500 ppm ga3 for 24 h (ak, 1990; kose, 2001), and then stratified in perlite for 50 days in refrigerator (dark conditions, +4 °c, 70-80% relative humidity). · sulphuric acid scarification: seeds were immersed in sulphuric acid for 90 min and then washed and soaked for 24 h in the tap water. scarified seeds were stratified in perlite for 50 days in refrigerator (dark conditions, +4 °c, 70-80% relative humidity). · sulphuric acid scarification + ga3: seeds were immersed in sulphuric acid for 90 min and then washed and soaked for 24 h in the tap water. scarified seeds soaked in the 500 ppm ga3 for 24 h and then stratified in perlite for 50 days in refrigerator (dark condi tions, +4 °c, 70-80% relative humidity). (ak, 1988; baninasab and rahemi, 2001). stratified seeds were sown in the vials filled with peat in the greenhouse conditions (temperature 25-35 and 15-20 °c during day and night respectively) to determine the germination percentage. then, plantlets were transplanted from vials to plastic containers (20 cm × 30 cm in size) filled with 2:1:1, peat, soil, and perlite mixture and irrigated at 4-day irrigation intervals to determine the vegetative growth. vegetative properties were determined in terms of stem and root growth, and some leaf characteristics of the seedling. for this purpose, seedling diameter, seedling height, stem fresh and dry weight; the number of lateral roots, primary root length, fresh and dry root weight; and number of leaves per plant and leaf chlorophyll contents were measured. leaf chlorophyll contents were determined as chlorophyll content index (cci) by using portable chlorophyll meter (ccm-200 plus, apogee instruments, inc., logan, ut) (van den berg and perkins, 2004). statistical analyses the experimental design was completely randomized design with 3 replications, and 25 plants were used for each dormancy-breaking treatment in each replication of both pistacia khinjuk a and b geno types. data were analysed using minitab 17 software (pa, usa, minitab inc.). means was separated by duncan’s multiple range test at p≤0.05. the relationship between morphological characteristics of the seedlings was evaluated through a simple pearson correlation analysis. results and discussion the results obtained from the present study confirmed that dormancy-breaking treatments had an important effect on seed germination and seedling growth of p. khinjuk genotypes. seed germination in the present study, there were significant differences among the treatments for germination rates of both p. khinjuk genotypes (fig. 1). it was known that the exogenous application of various stimulants and inhibitors could affect the plant growth regulators levels and rates in the seeds and thus increases the germination rates (mehanna et al., 1985; baninasab and rahemi, 2001). scarified seeds were reached the highest germination, and followed by dehulled and scarification+ga3 applications in p. khinjuk-a and b, respectively. shelled + ga3 had the lowest germination percentage in both genotypes (fig. 1). data obtained from all treatments were tested for pearson correlations, and the interaction of seed germination and dormancy-breaking treatments were presented in tab. 3. there was a significant positive relationship between the seed germination and dormancy-breaking treatments and number of leaves (p≤0.01); and between the seed germination and seedling diameter and primary root length (p≤0.05) (tab. 3). kafkas and kaska (1997) were obtained the highest seed germination from the stratification and the lowest germination from the directly sown treatment in p. khinjuk types. it was stated that there were not significant differences between the scarified and scarified + prechilled seeds of p. lentiscus (piotto, dormancy-breaking treatments affects seed seed germination and seedling growth of pistacia khinjuk 193 1995). besides, seeds soaking with 1000 ppm ga3 for 24 h did not increase the germination percentages in p. atlantica and p. vera (ak et al., 1995). on the other hand, ak (1990), mentioned that the increases in stratification periods were found positively correlated with the germination percentages of p. vera and p. khinjuk seeds. pipinis et al. (2014) reported that, in non-stratified seeds of cotinus coggygria, acid-scarified+ga3 treatment improved germination significantly, whereas in stratified seeds, no significant differences were observed in the germination percentages of ga3 treated and untreated seeds. furthermore, the concentration of ga3 was not found to affect germination. abu-qaoud (2005) obtained the highest germination rate from p. palaestina acid scarified+cold stratified seeds as 60%, on the other hand the germination rate of scarified seeds and scarified+ga3 applied seeds were 13.3% and 34% respectively in p. lentiscus. gunes et al. (2013) reported that mechanical scarification and sulfuric acid treatment were the most successful treatments for enhancing seed germination in carob. stem growth of p. khinjuk seedlings obtained results showed that seedling stem growth, number of leaves per seedling and leaf chlorophyll content index (cci) was signi ficantly different between p. khinjuk genotypes and dormancybreaking treatments (tab. 1). simple correlations between the genotypes, dormancy-breaking treatments and morphological properties of p. khinjuk a and b seedlings were presented in tab. 3. in p. khinjuk-a seedlings, the best seedling growth was obtained from acid scarification and dehulled applications, whereas the poor development was from dehulled + ga3 application. the highest seedling diameters, seedling height, stem fresh and dry weights, number of leaves and chlorophyll content were determined for scarification. although high values were obtained from the acid scarification, it was observed that these values decreased when ga3 application was added. the lowest results from seedling diameters, seedling height, stem fresh and dry weights, number of leaves and chlorophyll content were achieved in dehulled + ga3 application (tab. 1). according to kafkas and kaska (1997), ga3 treated seedlings of p. khinjuk types had the biggest stem diameter and followed by stratification treatment, and 92% of b-56a1 and 88% of b-5602 types seedlings were reached to budding stage 6 month after the sowing. qureshi et al. (2016) reported that 30 days cold stratification and 100 ppm ga3 was feasible procedure for forcing seed germination and to extend the period of propagation in walnut. rahemi and baninasab (2000) reported that ga3 application during and after stratification significantly increased the length, trunk diameter, internode length, leaf area and fresh and dry weight of seedlings of both pistacia mutica and pistacia khinjuk species. however, application of ga3 after stratification was more effective on seedling growth of p. mutica. ga3 applied at higher concentrations (500 and 750 ppm) increased the rate of growth, but growth malformations were clearly evident in seedlings of p. khinjuk. ga3 at 250 ppm enhanced seedling growth fig. 1: effects of dormancy-breaking treatments on seed germination of pistacia khinjuk seedlings. the letters over the columns indicate different groups determined by duncan’s test (p≤0.05). capital letters indicate p. khinjuk-a, and small letter indicate p. khinjuk-b. tab. 1: effects of dormancy-breaking treatments on stem growth, leaf number and leaf chlorophyll content index of pistacia khinjuk seedlings. treatments seedling diameter seedling height stem fresh weight stem dry weight number of leaves chlorophyll content (mm) (cm) (g) (g) index (cci) pistacia khinjuk-a seedlings shelled (control) 6.59 b* 19.83 bc 3.60 b 2.56 b 11.61 bc* 54.49 a shelled+ga3 6.32 bc 14.61 d 2.57 c 1.82 b 9.50 cd 49.03 a dehulled 7.26 a 21.11 b 5.02 a 3.59 a 13.55 b 57.53 a dehulled+ga3 4.75 d 11.97 e 1.33 d 0.92 c 8.95 d 40.16 b scarification 7.36 a 23.61 a 4.93 a 3.60 a 17.78 a 55.79 a scarification+ga3 5.79 c 18.53 c 3.16 bc 2.10 b 11.45 bc 54.81 a lsd (p≤0.05) 0.67 2.38 0.98 0.74 2.03 7.82 pistacia khinjuk-b seedlings shelled (control) 5.54 a 15.10 b 1.63 1.10 9.33 d 50.09 bc shelled+ga3 5.16 b 15.03 b 2.02 1.41 9.33 d 67.63 a dehulled 5.52 a 14.47 b 2.09 1.46 14.00 ab 56.75 ab dehulled+ga3 4.93 b 18.20 ab 2.03 1.39 11.67 c 43.61 c scarification 4.37 c 17.50 ab 1.64 1.29 15.00 a 47.93 bc scarification+ga3 5.21 b 21.07 a 2.23 1.53 12.67 bc 51.84 bc lsd (p≤0.05) 0.28 3.48 ns ns 1.45 11.03 *the letters following the numbers indicate different groups determined by duncan’s test (p≤0.05) 194 i. acar, h. yasar, s. ercisli of p. khinjuk. in p. khinjuk-b, the highest seedling diameters were observed for control and dehulled applications and followed by scarification+ga3 while high in seedling height, stem fresh and dry weights in scarification+ga3. leaf number and chlorophyll content were high in scarification and shelled+ga3, respectively (tab. 1). the effect of the ga3 application was unstable in growth of p. khinjuk-b seedlings. there was a significant negative relationship (p≤0.01) between the genotypes and seedling diameter, stem fresh and dry weights. dormancy-breaking treatments had a positive and significant correlation with seedling height (p≤0.05) and number of leaves (p≤0.01). the seedling diameter was an important factor in the budding and grafting of pistachio seedlings. seedling diameter had a positive and significant correlation with seedling height, stem fresh and dry weight (p≤0.01), and number of leaves and leaf chlorophyll content (p≤0.01). seedling height was highly correlated (p≤0.01) with stem fresh and dry weight and number of leaves. also stem fresh and dry weights were correlated with number of lateral root, root fresh and dry weight, number of leaves (p≤0.01) and leaf chlorophyll content (p≤0.05) (tab. 3). tab. 2: effect of dormancy-breaking treatments on root growth of pistacia khinjuk seedlings. treatments number of lateral roots primary root length (cm) root fresh weight (g) root dry weight (g) pistacia khinjuk-a seedlings shelled (control) 4.67 c* 35.74 b 8.51 b 6.52 b shelled+ga3 6.67 b 38.95 ab 4.42 d 3.47 cd dehulled 8.66 a 48.19 a 12.15 a 9.21 a dehulled+ga3 4.33 c 30.64 b 2.67 e 1.96 d scarification 7.00 b 47.95 a 9.96 b 7.52 b scarification+ga3 3.00 d 46.68 a 6.40 c 4.77 c lsd (p≤0.05) 1.31 8.78 1.49 1.63 pistacia khinjuk-b seedlings shelled (control) 2.67 bc 50.74 a 3.64 c 2.69 c shelled+ga3 4.00 b 39.98 d 4.35 a 3.42 a dehulled 6.00 a 39.42 d 4.02 b 3.12 b dehulled+ga3 5.67 a 43.68 c 3.12 d 2.37 d scarification 2.67 bc 46.87 b 3.12 d 2.30 d scarification+ga3 2.33 c 49.52 a 4.04 b 3.18 ab lsd (p≤0.05) 1.36 1.96 0.31 0.25 *the letters following the numbers indicate different groups determined by duncan’s test (p≤0.05) tab. 3: pearson correlations between genotypes, dormancy-breaking treatments, seed germination and seedling growth properties of p. khinjuk genotypes. morphological genotreat seed seedling seedling stem stem primary number root root number characteristics types ments germidiameter height fresh dry root of lateral fresh dry of nation weight weight length roots weight weight leaves treatments 0.000ns seed germination 0.083ns 0.773** seedling diameter -0.618** -0.195ns -0.026ns seedling height -0.197 ns 0.344* 0.376* 0.550** stem fresh weight -0.584** 0.057ns 0.223ns 0.866** 0.779** stem dry weight -0.584** 0.056ns 0.234ns 0.849** 0.763** 0.982** primary root length 0.246ns 0.271ns 0.384* 0.177ns 0.491** 0.279ns 0.249ns number of lateral roots -0.450** -0.211ns 0.046ns 0.651** 0.182ns 0.558** 0.580** -0.157ns root fresh weight -0.600** -0.049ns 0.194ns 0.842** 0.667** 0.923** 0.930** 0.216ns 0.574** root dry weight -0.586** -0.058ns 0.182ns 0.853** 0.679** 0.940** 0.937** 0.248ns 0.549** 0.983** number of leaves -0.050ns 0.457** 0.628** 0.372* 0.630** 0.517** 0.545** 0.340* 0.321ns 0.480** 0.473** chlorophyll content 0.061ns -0.175ns -0.058ns 0.375* 0.306ns 0.415* 0.370* 0.247ns 0.118ns 0.415* 0.431** 0.168ns *: p≤0.05, **: p≤0.01, ns: not significant dormancy-breaking treatments affects seed seed germination and seedling growth of pistacia khinjuk 195 root growth of p. khinjuk seedlings dormancy-breaking treatments were significantly affected the root growth of both genotypes seedlings. the effect of the dehulled application on the root development of p. khinjuk-a seedlings was better than the other applications. in p. khinjuk-a, the best root growth was obtained from seedling of dehulled seeds. ga3 addition had a negative effect on primary root length, root weights and lateral root number except for shelled application. the lowest root growth was observed for shelled and dehulled + ga3 applications (tab. 2). dormancy-breaking applications’ effect on root development of p. khinjuk-b seedlings was found to unsteady. number of lateral roots was higher in dehulled and dehulled + ga3 applications than the other treatments. while maximum root length was obtained from shelled and scarification + ga3 applications, the maximum root fresh and dry weight obtained from shelled + ga3. the effect of the ga3 application was also unstable in root growth of p. khinjuk-b seedlings (tab. 2). rahemi and baninasab (2000) reported that generally, application of ga3 was more effective on the growth of shoots than on roots. the small or insignificant effect of ga3 on root growth may be due to the synthesis of ga3 in roots of many species of plants, and a lower level of ga3 may be required for optimum root growth than in shoots (bugbee and white, 1984). there was a significant negative correlation (p≤0.01) between the genotypes and number of lateral root and root fresh and dry weights (tab. 3). primary root length was correlated with number of leaves (p≤0.05); number of lateral root was correlated with root fresh and dry weight (p≤0.01); root fresh and dry weights were correlated with number of leaves and leaf chlorophyll content (p≤0.01), significantly. as a conclusion, dormancy-breaking applications have been found to be effective on seed germination and seedling growth of p. khinjuk. it was determined that ga3 applications negatively affected both seed germination and root, stem and leaf growth, as known to the contrary. references abu-qaoud, h., 2005: effect of scarification, gibberellic acid and stratification on 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(in turkish). ulusarac, a., 1992: selection of rootstock for pistachio cultivars. pistachio research inst. pub. gaziantep, turkey. webster, a.d., 1995: rootstock and interstock effects on deciduous fruit tree vigour, precocity, and yield productivity. new zeal. j. crop hort. sci. 23, 373-382. yazici, k., sahin, a., 2016: characterization of pomegranate (punica granatum l.) hybrids and their potential use in further breeding. turk. j. agric. for. 40, 813-824. doi:10.3906/tar-1604-120 zohary, m., 1952: a monographical study of the genus pistacia. palestine j. bot. jerusalem ser. 5. zorenc, z., veberic, r., stampar, f., koron, d., mikulic-petkovsek, m., 2016: changes in berry quality of northern highbush blueberry (vaccinium corymbosum l.) during the harvest season. turk. j. agric. for. 40, 855-864. doi:10.3906/tar-1607-57 address of the corresponding author: e-mail: izzetacar@harran.edu.tr © the author(s) 2017. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 88, 1 4 (2015), doi:10.5073/jabfq.2015.088.001 1julius kühn-institut (jki), institute for resistance research and stress tolerance, ot groß lüsewitz, sanitz, germany ²julius kühn-institut (jki), institute for resistance research and stress tolerance, quedlinburg, germany effects of growing system and season on the alkaloid content and yield of different sweet l. angustifolius genotypes g. jansen1, h.-u. jürgens1, e. schliephake2, s. seddig1, f. ordon2 (received august 18, 2014) * corresponding author summary nine varieties and two breeding lines of sweet lupinus angustifolius were cultivated under organic and conventional conditions in northern germany in growing seasons 2010, 2011 and 2012. the alkaloid content was significantly influenced by the growing system and year and also by genotype. the variety ‘vitabor’ and the breeding line ‘bo 083521ar’ revealed a very low alkaloid content in all years and cropping systems, while ‘sanabor’, ‘borlu’ and ‘boregine’ had a higher content. in the years 2010-2012 significantly lower alkaloid contents (475 μg g-1) were found under organic conditions than under conventional conditions (615 μg g-1). the mean alkaloid level of all varieties and breeding lines in organic farming was highest in 2010 (640 μg g-1) and lowest in 2012 (364 μg g-1), depending on temperature during the seed filling period. investigations on the yield of l. angustifolius revealed significant effects of the genotype, the year and the growing system. introduction in the past the use of lupins for feed and food purposes was limited because of their high alkaloid content. alkaloids are toxic and in particular unlimited feeding of bitter seeds resulted in a fatal disease called lupinosis (hondelmann, 1984, 1996). therefore, the interest in lupins free from alkaloids was steadily increasing (hondelmann, 1984). on the one hand this can be achieved by debittering on a large scale and on the other hand by breeding (hondelmann, 1996). at the beginning of the 20th century v. sengbusch (v. sengbusch, 1930) identified some sweet grains of lupins (l. luteus and l. angustifolius) which were the starting point for breeding sweet lupins. this opened new possibilities for using sweet lupins because of their high protein content (jansen and balko, 2012) without quantitative restrictions in feed and as food ingredients due to their functional properties (wäsche et al., 2001). this largely broadened the original use of bitter genotypes of lupinus species for soil improvement because of their long taproot (clements et al., 1993) and ability to fix nitrogen due to symbiosis with the bacterium rhizobium (lindemann and glover, 2003). due to this the so-called ‘old world’ species l. albus, l. luteus and l. angustifolius as well the ‘new world’ type l. mutabilis achieved some agricultural importance (hondelmann, 1984). currently in germany only l. angustifolius is cultivated, mainly due to its resistance to anthracnose (colletotrichum lupini, ruge-wehling et al., 2008). because of the advantageous features mentioned above, especially because of its ability to nitrogen fixation, l. angustifolius is of special importance in organic farming, in which the use of herbicides, pesticides, and mineral fertilizers is not allowed. legumebased crop rotations are an option to supply nitrogen in organic farming systems to enhance soil fertility (maeder et al., 2002). in this respect temple et al. (1994) compared conventional, low input and organic farming concerning yield, weed biomass and percent n in leaves of different crops. for dry beans (legumes) no significant differences were detected between cropping systems in all years. for lupins it is known that the alkaloid content, which is of essential importance for food and feed is influenced by many environmental factors, i.e. differences in fertilizer application (ciesolka et al., 2005 and gremigni et al., 2003), soil ph (jansen et al., 2012) and ambient temperatures during seed filling (jansen et al., 2009). the objective of this study was therefore to assess the effect of (i) the genotype, (ii) the growing system (organic versus conventional farming) and (iii) the year on the alkaloid content and yield of german l. angustifolius genotypes. material and methods plant material and cultivation the seeds of all l. angustifolius cultivars and breeding lines were supplied by the seed company saatzucht steinach gmbh and co. kg, bocksee, germany. field experiments were carried out in four random replications with a plot size of 9.6 m² under organic and conventional farming conditions in groß lüsewitz (near rostock, mecklenburg-western pomerania, germany, northern latitude: 54.071955, eastern longitude: 12.321031). determinate types (‘boruta’ and ‘haags blaue’) were sown with a sowing rate of 120 seeds m-2 and branched types (‘boregine’, ‘borlu’, ‘hagena’, ‘probor’, ‘sanabor’, ‘sonate’, ‘vitabor’, ‘bo 083517ar’and ‘bo 083521ar’) with 100 seeds m-2 in march/april in 2010, 2011, 2012 and were harvested at maturity in july/august of the respective season. the nine varieties and two breeding lines were registered or bred, respectively, between 2001 and 2008. under organic conditions the following crop rotation was applied: potato, spring cereals, legumes, winter cereals, grass clover (twice). no manure, chemical seed treatment and chemical or mechanical weed controlwas conducted. conventional cultivation was done according to good agricultural practice with the same crop rotation. the location groß lüsewitz is characterized by a loamy sand with a medium soil water holding capacity, ph value of 5.8, long-term mean (1972 2007) rainfall of 686 mm and mean temperature of 8.3 °c. meteorological data for the years 2010 2012 were supplied by the university of rostock, faculty of agriculture and environmental science, institute for environmental engineering, field of hydrology. sample collection yields were estimated at a moisture content of 11 % for each replication separately. in a next step seeds of each replication per genotype were mixed and pre-crushed using a laboratory break mill sm 3 brabender® (brabender® gmbh & co. kg, duisburg, germany) and after that ground to pass a 0.8 mm sieve with a falling number laboratory mill 3100 (perten instruments, hägersten, sweden). all whole meal samples were stored at 20 °c until analysis. analysis of alkaloids the alkaloids in whole meal samples were analyzed by an agilent 7890a gas chromatograph equipped with a flame ionization de tector (fid) on agilent j&w ultra 1 column ( 25 m, 0.20 mm id, 2 g. jansen, h.-u. jürgens, e. schliephake, s. seddig, f. ordon 0.33 μm film thickness) and identified in the mass spectrometer agilent 5975c according to wink et al. (1995) and bermudes torres et al. (2002). this method was described in detail by jansen et al. (2009). analyses were carried out in duplicate with variation coefficient lower than 4 %. the alkaloid content was calculated as the sum of the main alkaloids which are angustifoline, isolupanine, lupanine, and 13-hydroxylupanine in narrow leafed lupins. statistical analysis the statistical analyses were conducted using the software package sas (version 9.3) for windows programs (sas institute, inc., cary, nc, usa). a generalized linear model for the analysis of variance (anova) was applied, using the glm procedure and a tukey test (α = 0.05) for comparing means (genotype, year and cultivation system). results the field experiments with eleven sweet l. angustifolius over a period of three years showed significant differences in the alkaloid content between genotype and year. the influence of the growing system was significant, too (tab. 1), but no significant interaction between genotype and growing system was found. tab. 1: results of the anova for alkaloid content. source degrees p-value (2010-2012) of freedom genotype 10 <.0001 year 2 <.0001 growing system 1 0.0012 genotype*growing system 10 0.9185 the alkaloid content of different genotypes, years and cultivation systems is listed in tab. 2. the variety ‘vitabor’ and the breeding line ‘bo 083521 ar’ showed the lowest alkaloid content in all years and growing systems. ‘vitabor’ varied between 69 μg g-1 and 158 μg g-1 in organic cultivation and between 127 μg g-1 and 341 μg g-1 in conventional farming. the breeding line ‘bo 083521 ar’ varied between 71 μg g-1 and 151 μg g-1 and between 127 μg g-1 and 225 μg g-1, respectively. ‘boregine’, a well known variety with high yield showed high alkaloid contents between 308 μg g-1 and 1108 μg g-1 in organic systems and between 445 μg g-1 and 1312 μg g-1 under conventional conditions. the varieties ‘sanabor’ and ‘borlu’ were characterized by a very high alkaloid content in seeds, comparable to the content in seeds of ‘boregine’. fig. 1 and tab. 2 show that higher alkaloid contents were detected in the conventional growing system. fig. 1: mean alkaloid content of eleven lupin genotypes cultivated under organic and conventional conditions (ls-means with the same letter are not significantly different). the year and the associated different mean temperature during the growing season had an effect on the alkaloid content of the investigated sweet narrow-leaved lupins. the investigated eleven lupins accumulated a higher amount of alkaloids at higher temperatures (fig. 2). in the growing seasons 2010 2012 all genotypes, with the exception of ‘haagena’, showed significant differences in yield between the growing systems, i.e. conventional and organic farming (fig. 3). the highest yield, both in organic and conventional farming was obtained by the cultivar ‘sonate’. in general the yield of the lupins cultivated under organic conditions over the three years was significantly lower (2.96 to 4.02 t ha-1) compared to the conventionally cultivated lupins (3.96 to 4.56 t ha-1). tab. 2: alkaloid content [μg g-1] of nine varieties and two breeding lines of organic (o) and conventional (c) cultivated sweet narrow-leafed lupins in three years. genotype cultivation year 2010 cultivation year 2011 cultivation year 2012 mean value 2010-2012 o c o c o c o c sanabor 942 1204 572 846 753 777 756 942 borlu 1058 1273 489 751 508 667 685 897 boregine 1108 1312 599 1027 308 445 672 928 haagena 864 1075 421 796 653 557 646 809 sonate 774 696 726 720 482 578 661 665 bo 083517 ar 655 833 438 733 283 435 459 667 probor 707 838 472 792 266 527 482 719 haags blaue 398 410 322 448 375 307 365 388 boruta 220 189 455 594 209 286 295 356 bo 083521 ar 151 187 71 225 94 127 105 180 vitabor 158 341 82 170 69 127 103 213 variability of yield and alkaloid content in l. angustifolius 3 the results of anova for yield of lupins cultivated under conventional and organic conditions are given in table 3. significant effects of the genotype, the year, and the cropping system were observed as well as respective significant interactions. (tab. 1), by the analyses of 11 genotypes of sweet narrow leaf lupins during 3 years. several studies compared quality parameters and the effects of organic and conventional growing systems, but in general only small and partially inconsistent differences were found. possible effects on human health of organic and conventional food are examined in many studies, e.g. by brand and mølgaard (2001) as well winter and davis (2006). in general only small and partially inconsistent differences between organically and conventionally produced foods were detected. only for nitrate and vitamin c a tendency to lower and higher contents, respectively was detected under organic conditions (brand and mølgaard, 2001). nevertheless, further investigations on nutritional important plant contents will be necessary to provide more answers to the question, whether organic or conventional cultivation can enhance the nutritional value. results of our study show that under organic conditions all l. angustifolius genotypes exhibited significantly lower alkaloid contents than under conventional conditions in all years. the reason for this may be that a higher n-content in the soil leads to higher concentrations of alkaloids. gholamhosseinpour et al. (2011) stated that nitrogen is a constituent of the alkaloids which plays an important role in the synthesis of these ingredients. therefore, increasing nitrogen supply leads to an increase in the alkaloid content. this has been shown for many species, e.g. lupins (lupinus succulentus, johnson et al., 1987), datura (datura innoxia mill., al-humaid, 2004), periwinkle (catharanthus roseus l., gholamhosseinpour et al., 2011) and for the total glycoalkaloid content in potatoes (mondy and munshi, 1990). however, also contradictory results are reported e.g. for datura (ruminska and gamal, 1978) and potatoes (hamouz et al., 2005). ruminska and gamal (1978) have shown that there is no remarkable influence of nitrogen fertilizer on the alkaloid content in datura innoxia mill. hamouz et al. (2005) found, that growing under organic conditions did not influence the glycoalkaloid content of potatoes. due to these contradicting results new investigations with respect to the alkaloid content in relation to the growing system, i.e. organic versus conventional were conducted. in 2012 the nh4-n-level at groß lüsewitz was equal in soil samples of both cropping systems (0.1 mg 100g-1, 0-30 cm), but the no3-nconcentration in soil samples derived from conventional cultivation reached a level of 0.3 mg 100g-1 compared to organic soil samples with only 0.1 mg 100g-1. it is known that the alkaloid content is influenced by a lot of environmental factors, such as temperature. higher ambient temperatures during the seed filling led to a higher content of alkaloids (jansen et al., 2009) in seeds of 6 older varieties of l. angustifolius registered between 1998 and 2004. as expected, in the present study the different temperatures during the growing period from 2010 to 2012 also had a significant influence on the alkaloid content (fig. 3). furthermore, genotypic differences were also observed in older studies conducted under organic conditions (jansen et al., 2005) only. as already mentioned the threshold for the alkaloid content for animal nutrition is 0.05 % and for human nutrition 0.02 % (jansen et al., 2009) in germany, while it is 200 mg kg-1 for contaminants and natural toxicants in australia and new zealand (abbott et al., 2003). alkaloid contents above this level were also detected by muquiz et al. (1994) in genotypes of sweet lupinus albus l. from different countries and locations. reinhard et al. (2006) detected up to 2120 μg g-1 in seeds and flour of “sweet” l. angustifolius genotypes. small, but significant differences in yield between organic (3.53 t ha-1) and conventional (4.25 t ha-1) growing systems were found for 9 varieties and 2 new breeding lines. similar results were obtained fig. 3: mean yield of l. angustifolius genotypes over a period of three years in organic and conventional growing systems cultivated lupins (all varieties had significantly different ls-means-values, with the exception of ‘haagena’). fig. 2: relationship between mean alkaloid content of eleven lupin geno types cultivated under organic conditions and mean temperature from flowering to pod ripening (pearson correlation coefficient r = 0.9997, p = ≤ 0.05). tab. 3: results of the anova for yield. source degrees f-statistic pr > f of freedom growing system 1 244.65 <.0001 genotype 10 8.47 <.0001 year 2 230.47 <.0001 genotype*year 20 5.87 <.0001 genotype*growing system 10 4.06 <.0001 genotype*growing system*year 20 4.13 <.0001 discussion differences in the alkaloid content of sweet l. angustifolius are well known. in the descriptive variety list published by the federal plant variety office (bundessortenamt, 2011) the variety ‘vitabor’ is characterized by a very low alkaloid content, compared to the other tested varieties. this is also confirmed by the results of our work 4 g. jansen, h.-u. jürgens, e. schliephake, s. seddig, f. ordon in four years field trials for pisum sativum and vicia faba and in one year field trials of l. angustifolius (jansen and seddig, 2007). in conclusion the alkaloid content and yield of different narrowleafed lupins is significantly influenced by the genotype, the growing season and the growing system (organic vs. conventional). significantly lower alkaloid contents and lower yields were found in organic farming compared to conventional farming. however, due to the lower alkaloid content and the only slightly reduced yield, which holds especially true for the cultivar ‘haagena’, l. angustifolius is well suited for organic farming. acknowledgements the authors thank c. leesch, m. jugert and b. lembke for excellent technical assistance. references abbott, p., baines, j., fox, p., graf, l., kelly, l., stanley, g., tomaska, l., 2003: review of the regulations for contaminants and natural toxicants. food control 14, 383-389. al-humaid, a.i., 2004: effects of compound fertilization on growth and alkaloids of datura plants. j. plant nutr. 27 (12), 2203-2219. bermudez torres, k., robledo quintos, n., barrera necha, l.l., and wink, m., 2002: alkaloid profile of leaves and seeds of lupinus hintonii c.p. smith, z. naturforsch. 57 c, 243-247. brandt, k., mølgaard, j.p., 2001: organic agriculture: does it enhance or reduce the nutritional value of plant foods? j. sci. food agric. 81, 924-931. bundessortenamt, 2011: beschreibende sortenliste: getreide, mais, ölfrüchte, leguminosen (großkörnig), hackfrüchte (außer kartoffeln). deutscher 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lachman, j., dvřák, p., pivec, v., 2005: the effect of ecological growing on the potatoes yield and quality. plant soil environ. 51 (9), 397-402. hondelmann, w., 1996: die lupine − geschichte und evolution einer kulturpflanze. landbauforsch völkenrode, special issue 162, 1-248. hondelmann, w., 1984: the lupin − ancient and modern crop plant. theor. appl. genet. 68, 1-9. jansen, g., balko, c., 2012: leguminosen unter stress-stabilität von ertrag und qualität. das blatt 4, 48-51. jansen, g., jürgens, h.-u., flamme, w., 2005: influence of location and cultivar on selected quality parameters of ecologically produced lupins for animal feeding. landbauforsch völkenrode, special issue 290, 1-10. jansen, g., jürgens, h.-u., ordon, f., 2009: effects of temperature on the alkaloid content of seeds of lupinus angustifolius cultivars. j. agron. crop sci. 195, 172-177. jansen, g., jürgens, h.-u., schliephake, e., ordon, f., 2012: effect of the soil ph on the alkaloid content of lupinus angustifolius. int. j. agron vol. 2012, doi: 10.1155/2012/269878. jansen, g., seddig, s., 2007: organically and conventionally cultivated legumes – comparison of yield and selected quality parameters. landbauforsch völkenrode, special issue 314, 41-53. johnson, n.d., liu, b., bentley, b.l., 1987: the effects of nitrogen fixation, soil nitrate, and defoliation on the growth, alkaloids, and nitrogen levels of lupinus succulentus (fabaceae). oecologia (berlin) 74, 425431. lindemann, w.c., glover, c.r., 2003: nitrogen fixation by legumes. http://aces.nmsu.edu/pubs/a/a129.pdf, cooperative extension service, college of agriculture and home economics, accessed 23.09.2014. maeder, p., fliessbach, a., dubois, d., gunst, l., fried, p., niggli, u., 2002: soil fertility and biodiversity in organic farming. sci 31, 16941697. mondy, n.i., munshi, c.b., 1990: effect of nitrogen fertilization on glycoalkaloid and nitrate content of potatoes. j. agric. food chem. 38, 565567. muzqiz, m., cuadrado, c., ayet, g., de la cuadra, c., burbano, c., osagie, a., 1994: variation of alkaloid components of lupin seeds in 49 genotypes of lupinus albus l. from different countries and locations. j. agric. food chem. 42, 1447-1450. reinhard, h., rupp, h., sager, f., streule, m., zoller, o., 2006: quinolizidine alkaloids and phomopsins in lupin seeds and lupin containing food. j. chromatogr. a, 1112, 353-360. ruge-wehling, b., dieterich, r., thiele, c., eickmeyer, f., wehling, p., 2008: resistance to anthracnose in narrow-leafed lupin (lupinus angustifolius l.): source of resistance and development of molecular markers. j. kulturpflanzen 61, 62-65. ruminska, a., el gamal, s., 1978: effect of nitrogen fertilization on growth, yield and alkaloid content in datura innoxia mill. acta hort. 73, 173-179. sengbusch, r. von, 1938: bitterstoffarme lupinen iii. züchter 10 (4), 9195. temple, s.r., somasco, o.a., kirk, m., friedmann, d., 1994: conventional, low-input and organic farming systems compared. california agric. 48 (5), 14-19. wäsche, a., müller, k., knauf, u., 2001: new processing of lupin protein isolates and functional properties. nahrung/food 45 (6), 393-395. wink, m., meissner, c., witte, l., 1995: patterns of quinolizidine alkaloids in 56 species of the genus lupinus. phytochem. 38, 139-153. winter, c.k., davis, s.f., 2006: organic foods. j. food sci. 71 (9), 117124. address of the authors: g. jansen, h.-u. jürgens, s. seddig, julius kühn-institut (jki), institute for resistance research and stress tolerance, ot groß lüsewitz, rudolfschick-platz 3, 18190 sanitz, germany e-mail: gisela.jansen@jki.bund.de e. schliephake, f. ordon, julius kühn-institut (jki), institute for resistance research and stress tolerance, erwin-baur-straße 27, 06484 quedlinburg, germany journal of applied botany and food quality 90, 1 9 (2017), doi:10.5073/jabfq.2017.090.001 1laboratory of methods and analysis techniques (lmta), national institute of research and physico-chemical analysis (inrap), sidi thabet, ariana, tunisia 2department of ecology, faculty of sciences of tunis, el manar, tunis, tunisia maturational effects on phenolic constituents, antioxidant activities and lc-ms / ms profiles of lemon (citrus limon) peels nadia mcharek1*, belgacem hanchi1, 2 (received september 12, 2016) * corresponding author summary methanol extracts from peels of lemon (citrus limon (l.) burm) fruits collected at three maturity stages (immature, half-mature and mature) were analyzed for their total phenolic contents (tpc) and antioxidant activities. irrespective to peel parts analyzed (albedo or flavedo), a declining trend was observed for tpc during maturation. by using dpph, abts and frap assays, it was found that all extracts had strong antioxidant activities. as maturation progress, the antioxidant activity of both peel parts decreased. significant relationships between antioxidant capacity and tpc were found, indicating on the one hand, that phenolic compounds are the major contributors to the antioxidant properties, and that the three methods have similar predictive capacity for antioxidant activity of lemon peel, on the other hand. analysis by hplc-dad-ms/ms revealed that flavedo and albedo extracts haves similar phenolic profiles and allowed the identification of hesperidin, rutin, eriocitrin, diosmin and hyperoside, as the main compounds. introduction plants particularly those of the horticulture section are used by people for food, either as edible products or for culinary ingredients, medicinal products, and ornamental and aesthetic purposes. they represent a genetically very diverse group and play a major role in modern society end economy. fruits and vegetables are an important component of traditional food, but possess also a central role in healthy diets of modern urban population (kaczmarska et al., 2015; mlcek et al., 2015; wojnicka-poltorak et al., 2015). citrus (rutaceae family) plants are the most important fruit tree crop in the world, with an annual production of approximately 1.02 hundred million tons (hwang et al., 2012). the fruits are the most popular for consumers throughout the world owing to their pleasant flavors and nutritional value (qiao et al., 2008). they are mainly used for dessert, juice and jam production. they are also credited with a long list of medicinal uses including antioxidant, antimicrobial, antifungal, anti-inflammatory, antitumoral and hypoglycemic, among others (di vaio et al., 2010; espina et al., 2011; tripoli et al., 2007). these functional properties were attributed to a plethora of bioactive ingredients including vitamin c, dietary fibres, folic acid, essential oils and flavonoids (senevirathne et al., 2009). within the genus citrus, lemon (c. limon) has received particular attention and it is considered as a consolidated source of functional ingredients namely vitamin c, minerals, carotenoids, limonoids and flavonoids (gonzalez-molina et al., 2008). the latter components usually obtained from the peel, are of particular interest due to their intriguing biological properties, such as antioxidant, antiinflammatory, anticancer, chemopreventive, cardioprotective and neuroprotective activities (benavente-garcia and castillo, 2008; hwang et al., 2012). previous phytochemical studies have revealed that lemon flavonoids are mainly composed of flavanones, flavones, and polymethoxyflavones (gil-izquierdo et al., 2004). hesperidin, disometin and luteolin, to which a single or multiple functional properties are ascribed, are reported as the main lemon flavonoids (hwang et al., 2012). however, as commonly happens in many plant species, the phenolic constituents are particularly prone to quantitative and qualitative variations depending on genotype, plant part analyzed, environmental conditions, cultural practices and also on the stage of maturity of the fruit (del rio et al., 2004; gonzalez-molina et al., 2008; kawaii et al., 1999) changes occurring during the fruit maturation stages can affect the nutritional value and health properties of the lemon fruit; hence it is important to determine the optimum developmental stage having the maximum functional properties. in the specific case of lemon, the effect of fruit maturation has received little attention, and the only report available showed that the maturation process was associated with deep quantitative changes in flavonoid constituents (del rio et al., 2004). however, the latter study was limited to the characterization and quantitation of these constituents without considering their biological activity, which was ultimately affected by the type and quantity of flavonoids. this knowledge is, however, crucial to define the possible application areas for the rational use of lemon peels. in tunisia, the area under citrus cultivation was estimated to be about 19.250 ha with an annual production of 230.000 tons, covering the fresh fruit market, agro-food industry and the exportation demands (hosni et al., 2010). in food and agro-food industries, considerable amounts of wastes or by-products such as peels, seeds and pulps are produced. these by-products, however, could represent an interesting source of dietary fibres, phenols and essential oils, among others (garau et al., 2007). consequently, there is a considerable emphasis on the recovery and upgrading to higher value and useful products of these by-products. despite their socio-economic relevance, comprehensive phyto-chemical investigations on phenolic constituents of tunisian citrus species are scanty. bearing this in mind, the present study intends to: (a) investigate the evolution of total phenolic contents in relation to peel parts (flavedo and albedo) and during fruit maturation, (b) characterize the main flavonoids by lc-dad-ms2 and (c) to evaluate the antioxidant activity of lemon fruit from tunisian origin, which has not yet been reported. materials and methods plant materials fruits of c. limon were randomly collected from healthy trees cultivated in the experimental station of the institut national de recherche en génie rural, eaux et forêts, oued souhil, nabeul, tunisia (latitude 36°27’53”n; longitude10°42’24”e). the fruits were picked up at three different stages of maturity based on their color, fruit size and weight in (fig. 1). the fruits were cut on six equal portions and the flesh was removed. the flavedo and albedo layers 2 n. mcharek, b. hanchi were carefully separated, dried at 40 °c in a forced air oven type venticell 55 (mmm medcenter, einrichtungen gmbh, gräfelfing, germany) to constant weight, crushed with a laboratory grinder type blender lb20e (waring laboratory, torrington, usa) and subsequently essayed for their polyphenols analysis. with slight modifications. briefly, 500 μl of appropriately diluted extract were added to 5 ml of freshly diluted 10-fold folin-ciocalteu reagent, and the mixture was neutralized with 4 ml of a sodium carbonate solution (1m). the reaction mixture was kept in the dark for 15 min, and its absorbance was measured at 765 nm against a prepared methanol blank using a jasco v-630 uv-vis spectrophotometer (tokyo, japan). gallic acid was used as the standard, and results were expressed as milligram of gallic acid equivalents (mg gae /g extract). antioxidant activity 2,2-diphenyl-1-picrylhydrazyl (dpph) assay the radical scavenging activity of the extracts against dpph free radical was measured by using an aliquot of 0.2 ml of each extract that was mixed with 0.8 ml of methanol and 2 ml of a daily prepared methanol dpph solution (0.1 mm). the mixture was shaken gently and left to stand at room temperature in the dark for 1 h. thereafter, the absorbance was read at 515 nm. dpph antiradical scavenging activity was extrapolated from a standard curve and expressed as micromole of trolox per gram of extract (μm te/g extract). abts (azinobis (ethylbenzothialzoline 6-sulphonic acid)) assay the total antioxidant activity of the samples was measured by abts radicals according to the method of re et al. (1999), slightly modified. briefly, the radical cation abts+ was produced by reacting 7 mm abts aqueous solution with 2.4 mm potassium persulfate in the dark for 12-16 h at room temperature. the bluegreen abts solution was diluted in ethanol to give an absorbance of 0.7 at 734 nm prior to assay. the diluted abts solution (2850 μl) was mixed with 150 μl of sample extracts or trolox standard. the mixture was left to stand at room temperature in the dark for 15 min, and then the absorbance was measured at 734 nm. trolox was used as positive control, and results were expressed as micromole of trolox per gram of extract (μm te/g extract). ferric reducing antioxidant power (frap) assay the reducing power is determined by the method described by benzie and strain (1996), with slight modifications. stock solutions of 300 mm acetate buffer, 10 mm tptz (2,4,6-tripyridyl-striazine in 40 mm hcl), and 20 mm fecl3. 6h2o were prepared. the working frap reagent was prepared by mixing the stock solutions in a 10:1:1 ratio. the solution was maintained at 37 °c and ph 3.6. then, 150 μl of the sample was mixed with 2850 μl of the working solution and left standing at room temperature for 30 min in the dark. absorbance of the mixture was then measured spectrophotometrically at 593 nm. the change in absorbance was calculated and related to the standard curve generated with trolox. results were expressed as milligram of trolox equivalents per gram of extract (mg te/g extract). lc-dad-ms2 analysis the chromatographic separation and mass spectrometric analyses of phenolics contained in the methanolic extracts were carried out on an agilent 1100 series hplc systems equipped with a diodearray detector (dad) and a triple quadrupole mass spectrometer type micromass autospec ultima pt interfaced with an esi ion source. separation was achieved using a superspher®100 (12.5 cm × 2 mm i.d, 4 μm, agilent technologies, rising sun, md) at 40 °c. the samples (20 μl) were eluted through the column with a gradient mobile phase consisting of a (0.1 % acetic acid) and b (acetonitrile) with a flow rate of 0.3 ml/min. the following multi-step linear solvent gradient was employed: 0-5 min, 2 % b, 5-60 min, 40 % b, fig. 1: color, diameter and weight of lemon fruits at different maturity stages chemicals and reagents folin-ciocalteu reagent (fcr), gallic acid, quercetin, 2,2-diphenyl1-picrylhydrazyl (dpph), trolox and fecl3 were purchased from sigma-aldrich inc. (steinheim, germany). solvents of analytical and hplc grade were purchased from carlo erba reactif-cds (val de reuil, france). water was purified on a milli-q system from millipore (bedford, ma, usa). samples preparation the extraction procedure adopted for this work is based on a simple maceration in methanol. this solvent has been largely used for the investigation of phenolic compounds in citrus fruits, as it provides better extraction yields and higher efficiency compared to other solvents (ma et al., 2008; mokbel and suganuma, 2006). moreover, this procedure is sufficient enough to make comparative study on antioxidant activities of different samples. indeed, the obtained methanolic extracts can be handled without further fractionation to perform in vitro assays and generate good quality analytical data, especially when using chromatographic techniques hyphenated with mass spectrometry (lc-ms or lc-ms2). in the present study, samples of ground powder (0.25 g) in triplicate were extracted with 100 ml of methanol (labscan, dublin, ireland) for 8 h under orbital agitation (gfl 3005; dominique dutscher, brumath, france). the extracts were filtered through ptfe membrane, 0.45 μm pore size (supelco, bellfonte, pa) and the extraction was repeated 3 times. the combined filtrates were stored at 4 °c until used. total phenolic content (tpc) total phenolics were determined with the folin-ciocalteu assay according to the procedure reported by mcdonald et al. (2001), maturational effects on lemon peels 3 60.1-80 min, 100 % b, 80.1-110 min, 2 % b, and 85.1-100 min, 2 % b. dad detection was performed in the 250-550 nm wavelength range and the mass spectra were recorded in negative ion mode, under the following operating conditions: capillary voltage, 3.2 kv; cone voltage, 60 v; probe temperature, 350 °c; ion source temperature, 120 °c. the spectra were acquired in the m/z range of 130-750 amu. identification of phenolic compounds was based on detection in multiple reaction monitoring (mrm) mode. all the retention times and spectral data (uv and ms spectra and mrm transitions) were compared with authentic standards and data reported from literature (anagnostopoulou et al., 2005; barreca et al., 2011; nogata et al., 2006) to confirm identification. statistical analysis all measurements were carried out in triplicate and the results were presented as mean values ± sd. statistical analyses were performed using a one-way analysis of variance anova test and the significance of the difference between means was determined by duncan’s multiple range test. correlations among data obtained were calculated using pearson’s correlation coefficient. the spss 18 (chicago, illinois, usa) and microsoft excel package were used to perform statistical analysis. results and discussion total phenol contents fruit development is a dynamic process commencing with pollination and fertilization, followed by intensive cell division and expansion, with concurrent seed development and maturation. fruit maturation typically involves tissue softening and changes in flavor, texture and color, usually caused by a series of concerted biochemical and physiological processes (birtic et al., 2009; hosni et al., 2011). during the maturation of lemon fruit, the most noticeable change is the gradual decrease of total phenolic content in both flavedo and albedo peel (fig. 2). the total phenolic contents appear to follow predictable patterns over fruit maturation, occurring at the highest levels at the immature stage (189.77 and 176.31 mg gae/g extract in flavedo and albedo, respectively), and declining until full maturation (51.71 and 61.08 mg gae/g extract in flavedo and albedo, respectively). such dynamic of total phenol accumulation in flavedo and albedo tissues is consistent with that observed in c. myrtifolia (barreca et al., 2011), suggesting a spatio-temporal regulation of these valuable compounds. the decline in total phenolic contents with advancing fruit maturity has previously been reported in citrus limon (del rio et al., 2004), punica granatum (fawole and opara, 2013), malpighia emarginata (lima et al., 2005) and lycopersicon esculentum (ilahy et al., 2011). all these studies attribute the decrease in total phenol contents during the maturation process to the oxidation of polyphenols by polyphenoloxidase. usually, the observed trend was concomitant to enhanced chlorophyll degradation and a carotenoid accumulation resulting in a significant increase of yellow color. because lemon is a nonclimacteric fruit whose ripening is regulated by abscissic acid (aba), it may be suggested that the transition from immature (green) to mature (yellow) is mediated by this phytohormone. support to this assumption is recently given by palma et al. (2011) and ren et al. (2010), who reported accumulation of aba during the maturation of citrus fruit. this behavior is similar to that observed in some citrus fruits such as satsuma mandarin, valencia orange and lisbon lemon (kato et al., 2004). from the molecular point of view, the massive accumulation of carotenoids (mainly lycopene) was mainly due to a significant up-regulation of the gene expressions namely phytoene desaturase (pds) and ζ-carotene desaturase (zds) during fruit maturation, resulting in enhanced lycopene synthesis (schweiggert et al., 2011). from quantitative standpoint, our obtained values were different from those reported in some citrus species. for example, the albedo layer of argentinean c. aurantium was found to contain variable amounts (234-322 mg gae/g extract) of total phenolic contents, depending on the extraction procedure (zapata zapata et al., 2012). similarly, ghanem et al. (2012) have compared the total phenolic content in three tunisian citrus species, and have reported values varying from 29.11 and 24.51 mg cafeic acid equivalent/g dw in c. reticulata and c. limon, respectively, to 18.99 mg cafeic acid equivalent/g dw in c. sinensis. in another comprehensive study from mauritius, ramful et al. (2011) compared the total phenol content in 20 varieties belonging to 11 citrus species and reported values ranging from 406.3 to 1694 μg gae/g fresh weight. in general, the observed discrepancy between results may be due to the genetic makeup, origin, peel part analyzed, stage of maturity, environmental conditions, samples preparation and analysis. on the other hand, the high concentration of phenolic compounds in citrus peel was primarily ascribed to their protective role against uv radiations, pathogens and predators (barros et al., 2012). polyphenols are known as potent antioxidant; thereby any changes in their contents may have strong influence on their biological activities. in this direction, the flavedo and albedo extracts at different stages of fruit maturity were evaluated for their antioxidant activities. fig. 2: total phenolic contents (mg gae /g extract) at different stages of maturity. different letter(s) on bar indicate statistically significant differences (p<0.05). 4 n. mcharek, b. hanchi antioxidant activity scavenging activity on dpph radicals the dpph radical is a stable radical with absorption band at 515528 nm. it loses this absorption when accepting an electron or a free radical species, which results in a visually noticeable discoloration from purple to yellow. because it can accommodate many samples in a short period and is sensitive enough to detect active ingredients at low concentrations, it has been extensively used for screening antiradical activities of extracts (dziri et al., 2012). fig. 3 shows that irrespective to the peel parts, the scavenging activity on dpph radicals of all extracts was the highest (55.91 and 56.76 μm te/g extract for flavedo and albedo, respectively) at the immature stage, and then decreased gradually to reach the lowest values (23.07 and 26,04 μm te/g extract for flavedo and albedo, respectively) at the full maturation stage. the hydrogen-donating ability of flavedo and albedo extracts is most likely attributed to their higher total phenolic contents. a strongly significant (p<0.01) positive correlation (r = 0.986) was detected between total phenolic contents and the dpph radical scavenging activity, supporting thereby our hypothesis and confirm previous reports that free radical scavenging activity can be related to phenolic content (dudonne et al., 2009). scavenging activity on abts radicals for abts radicals scavenging activity (fig. 4), the rank order among the extracts was similar to dpph results. the immature flavedo and albedo peel extracts showed the highest antioxidant activities (87.21 and 82.01 μm te/g extract for flavedo and albedo, respectively), followed by the extracts from the half-mature fruits (54.02 and 57.77 μm te/g extract for flavedo and albedo, respectively), while the extracts from the full mature fruits exhibited the lowest antioxidant capacities (32.28 and 50.52 μm te/g extract for flavedo and albedo, respectively). as for the dpph assay, significant (p<0.01) linear correlation (r = 0.851), was found between abts assay and total phenolic content, confirming the relationships between phenolic compounds concentration in plant extracts and their radical scavenging activity. similar to the results reported herein, xu et al., 2008 evaluated the abts radical scavenging capacity of 15 varieties of chinese citrus and found that the antioxidant activity was associated with their higher total phenolic contents. another point to be considered is that abts values are significantly higher than the dpph values. the lower values determined by the dpph assay may be due to color interference by carotenoids (λmax= 400-500 nm) and anthocyanins (λmax= 470-580 nm) with dpph fig. 3: dpph radicals scavenging activity of lemon flavedo and albedo extracts (in μm te/g extract) and its correlation with total phenolic contents. different letter(s) on bar indicate statistically significant differences (p<0.05); ** significance at p<0.01. fig. 4: abts radicals scavenging activity of lemon flavedo and albedo extracts (in μm te/g extract) and its correlation with total phenolic contents. different letter(s) on bar indicate statistically significant differences (p<0.05); ** significance at p<0.01. flavedo a lbedo maturational effects on lemon peels 5 chromagen (λmax= 515 nm). these results were in good agreement with previous works on other species such as guava (thaipong et al., 2006) and potato (teow et al., 2007). ferric reducing antioxidant power (frap) results of frap assay are shown in fig. 5. the trend for the ferric ion reducing activities of the flavedo and albedo peel extracts did not vary markedly from their dpph and abts scavenging activities. in this study, extracts from the immature fruits possessed the highest ferric reducing capacity (241.71 and 297.11 mg te/g extract for flavedo and albedo, respectively), while the lowest capacity was exhibited by extracts from full mature fruits (58.06 and 70.44 mg te/g extract for flavedo and albedo, respectively). these results were in good agreement with those of barreca et al. (2011) who reported the strong antioxidant activity of c. myrtifolia peel extracts, with the albedo extracts being the most powerful when compared with flavedo ones. alike the dpph and abts assays, the ferric reducing capacity was significantly (p<0.01) correlated with the total phenolic contents (r = 0.918), which indicates that the reducing power of flavedo and albedo extracts were mainly attributable to their phenolic compounds. these studies were in line with those reported for some citrus species such as c. sinensis, c. reticulata, c. latifolia, and c. limettioides (barros et al., 2012), where the ferric reducing power was found to be tightly associated with higher total phenolic contents. in contrast, a very weak ferric reducing power was found in c. reticulatae pericarpium and c. reticulatae viride pericarpium from taiwan (su et al., 2008). such divergences can be due to different cultivars/varieties, origin, extraction methods and the analytical procedures (ramful et al., 2011). in any case, total phenolic contents in lemon peels can be used as indicator in assessing the antioxidant activity since they showed high correlations with all assays (dpph, abts, and frap). also, high correlations between dpph and abts (r = 0.938, p<0.01), dpph and frap (r = 0.973, p<0.01), and abts versus frap (r = 0.879, p<0.01) were also found (fig. 6). these results indicate that flavedo and albedo extracts had comparable activities in all the three assays, and are consistent with the view that the three assays share a similar mechanistic basis, especially transfer of electrons from the antioxidant to reduce an oxidant, as proposed by clarke et al. (2013). additionally, the integral lemon peel (flavedo + albedo) may be considered as valuable source of antioxidant phenolic compounds, because of their ability to inhibit free radicals formation and to suppress the formation of the fenton reaction, and hence, impede the formation of a highly reactive hydroxyl radical (pichaiyongvongdee and haruenkit, 2009). bearing in mind that the antioxidant activity of plant extracts is directly related to their chemical composition and the structural conformation of their phenolic compounds (jang et al., 2010), it is of interest to characterize the phenolic constituents of both flavedo and albedo extracts. fig. 5: ferric reducing antioxidant power (frap) of lemon flavedo and albedo extracts (in mg te/g extract) and its correlation with total phenolic contents. different letter(s) on bar indicate statistically significant differences (p<0.05); ** significance at p<0.01. fig. 6: coefficients of determination between dpph, abts and frap; ** significance at p<0.01. 6 n. mcharek, b. hanchi identification of phenolic components by lc-dad-ms/ms the typical phenolic profile of lemon peel (flavedo part) is depicted in fig. 7a. data of the retention time (tr), uv, mass spectra and mrm transitions of the identified compounds are given in tab. 1. as can be seen, 5 components (peaks 4-8) were unambiguously identified by comparing their retention time tr, uv, ms spectra and mrm transitions with those of reference standards (fig. 7b). peak 4 (tr = 28.58 min, λmax : 286 nm) exhibited [m-h]base ion at m/z 595 and fragmentation ion at m/z 151. by comparing its tr, uv spectrum, molecular mass and fragmentation pattern with the reference standard, this compound was identified as eriocitrin (eriodictyol7-o-rutinoside) (barreca et al., 2011). peak 5 (tr = 31.01 min; λmax : 256-354 nm) showed a [m-h]ion at m/z 609 with a base peak at m/z 301 indicative of the quercetin aglycone. this compound was assigned as rutin (quercetin-3-o-rutinoside) by comparison of retention time, uv spectrum and mass spectrometric data with rutin reference standard (hosni et al., 2013). peak 6 (tr = 31.55 min; λmax: 256-354 nm) showed a pseudo-molecular ion [m-h]at m/z 463 and a fragment [m-h-162] ion at m/z 301 (quercetin aglycone) due to the elimination of hexose unit (162 amu). this compound was identified as quercetin-3-o-galactoside (hyperoside) by comparison of the retention time, uv, mass spectra and specific transitions with authentic standard (albouchi et al., 2013; de brito et al., 2007). peak 7 (tr = 35.11 min; λmax: 286 nm) exhibited [m-h]base ion at m/z 609 and a fragment [m-h-308] ion at m/z 301. coelution with a known standard, along with comparison with literature data (barreca et al., 2011), led to the identification of peak 7 as hesperidin, frequently reported as the main components of lemon. peak 8 (tr = 36.18 min; λmax: 253-346 nm) exhibited [m-h]base ion at m/z 607 and a fragment [m-h-308] ion at m/z 299. this fragmentation pattern is similar to that observed for the diosmin reference standard and literature data (nogata et al., 2006) . unfortunately, for the remaining components; peak 1 (tr = 23.8 min; λmax: 349 nm, [m-h]609), peak 2 (tr = 25.49 min; λmax: 271-349 nm; [m-h]593), peak 3 (tr = 27.4 min; λmax: 271-247 nm, [m-h] 479) and peak 9 (tr = 36.9 min; λmax: 361 nm, [m-h]56=651), the uv spectrum and mass spectra are not sufficient to elucidate their structure and they are still unidentified. previous studies have shown that hesperidin and eriocitrin, a flavanone glucosides, were the major flavonoids in the pulp of lemon (c. limon) and lime (c. aurantiifolia) (peterson et al., 2006). the former compounds have also been reported as the most prominent flavanone in c. sinensis and the interspecific hybrid c. reticulata × c. sinensis (goulas and manganaris, 2012) and c. latifolia (da silva et al., 2013). the flavone glycoside, diosmin and the flavonol rutin have been frequently reported in c. limon (baldi et al., 1995), c. sinensis (anagnostopoulou et al., 2005), c. reticulata, c. tankan, and c. grandis (wang et al., 2008). in contrast, the occurrence of hyperoside in lemon was reported herein for the first time. fig. 7: typical lc-ms/ms chromatogram of lemon peel methanolic extracts (a) and mrm chromatogram of the identified components (b) (peak assignments are given in tab. 1. maturational effects on lemon peels 7 conclusion the antioxidant activity of the identified components has previously been reported. for example, hesperidin was found as the main contributor to the antioxidant activity of peel and juice of different citrus species (di majo et al., 2005). according to the latter authors, the potent antioxidant and free radical scavenging activity of hesperidin may be ascribed to the hydroxyl group at the position 3’. the scavenging activity of hesperidin on dpph radicals, hydroxyl radicals, hydrogen peroxide, superoxide anions and its reducing power has been evidenced three years later by yi et al. (2008). for eriocitrin, the first evidence for its antioxidant activity has been reported by miyake et al. (1997), who successfully isolated this component from lemon fruits and proved its antioxidant activity by using the linoleic acid system. six years later, the same group of researchers have reported the protective effects of eriocitrin against oxidative damages caused by acute exercise-induced oxidative stress (minato et al., 2003), in another study from poland, sroka et al. (2005) have reported that eriocitrin possess strong scavenging activity against dpph radicals and hydrogen peroxides. in contrast, they found a very weak antioxidant activity of the flavone diosmin, supporting the previous results by castillo et al. (2000). the latter authors attributed the weaker antioxidant activity of diosmin to the methylation of the 4’-hydroxyl group in the b-ring, which significantly reduces the antioxidant of this component, via the reduction of its electron-donating ability. the glucolsylated flavonols, rutin and hyperoside have been found to possess strong antioxidant activity because of its adjacent dihy droxy groups on the b-ring (anagnostopoulou et al., 2005; rainha et al., 2013; yang et al., 2008). taken together, it appears that the strong antioxidant activity of lemon peel can be attributed to specific phenolic components acting alone or in combination. taking into consideration the complexity of the antioxidant mechanism, which includes i) single electron transfer from the antioxidant to the radical leading to indirect h-abstraction (rojano et al., 2008), ii) direct hydrogen atom transfer from the antioxidant to the radical (mayer and rhile, 2004), iii) sequential proton-loss electron transfer (klein and lukeš, 2007) and iv) metal chelating (gülçin et al., 2010), it is difficult to get deeper insight into the antioxidant mechanism of lemon peels and to ascertain the exact contribution of their individual phenolic compounds. nevertheless, the results of this study could be helpful to find possible nutritional, cosmeceutical and pharmaceutical outlets for the discarded fruit parts. acknowledgement this work was supported by the tunisian ministry of higher education and scientific research and by the tunisian-french “comité mixte de coopération universitaire” (cmcu) network # 13g0904. the authors also thank the “institut national de recherche en génie rural, eaux et forêts − tunisia” for providing the plant material. references albouchi, f., hassen, i., casabianca, h., hosni, k., 2013: phytochemicals, antioxidant, antimicrobial and phytotoxic activities of ailanthus altissima (mill.) swingle leaves. s. afr. j. bot. 87, 164-174. doi: 10.1016/j.sajb.2013.04.003. anagnostopoulou, 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2012: enzymatic maceration of albedo layer from sour orange (citrus aurantium l.) with protopectinase-se and measurement of antioxidant activity of the obtained products. lwt − food sci. technol. 45, 289-294. doi: 10.1016/j.lwt.2011.08.009. address of the corresponding author: e-mail: nadya.mcharek@gmail.com © the author(s) 2017. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 91, 1 7 (2018), doi:10.5073/jabfq.2018.091.001 1departamento de biologia, centro de ciências naturais e exatas, universidade federal de santa maria, santa maria, brazil 2institute for global food security, queen’s university belfast, david keir building, belfast, northern ireland 3departamento de fitotecnia, centro de ciências rurais, universidade federal de santa maria, santa maria, brazil chemical properties and protective effect of rosmarinus officinalis: mitigation of lipid peroxidation and dna-damage from arsenic exposure farias, g.j.1,2*, frescura, d.v.1, boligon, a.a.1, trapp, c.k.1, andriolo, l.j.3, tedesco, b.s.1, bernardy, k.1, schwalbert, r.1, del frari, k.b.1, carey, m.2, nicoloso, t.f.1 (received march 7, 2017; accepted october 12, 2017) * corresponding author summary recent studies have implicated dietary factors in the cause and prevention of important diseases, with strong evidence that plant’s compounds can protect against these diseases. moreover, food security and environmental contamination are topics in focus at the moment. in this view, contamination by arsenic (as) has received much attention as well as some spices with medicinal properties. among these plants, the use of rosmarinus officinalis l. has demonstrated antioxidant properties besides being used for circulatory disorders. therefore, we measured the mitotic index of allium cepa l. and characterized the antioxidant effects to determine the capacity of r. officinalis to ameliorate arsenic-induced dna damages. r. officinalis extract showed no mutagenic effects and exhibited antimutagenic potential, reducing the dna damages, anaphase-telophase bridges and micronuclei chromosome aberrations that result from treatment with the arsenic. additionally, reduction in arsenic-induced lipid peroxidation was also observed. key words: arsenate, cytogenetic, heavy metal, medicinal plant, rosemary. introduction rosmarinus officinalis l. is a species of the lamiaceae family native from the mediterranean region, popularly known as rosemary (ferrari et al., 2011). according to anvisa (2010), the extracts of rosemary leaves can be used for combating circulatory disorders, and is recommended as an antiseptic and for cicatrization. among the chemical constituents of rosemary, the flavonoids and phenolic acids are the two major groups of phenolic compounds (cunha et al., 2012). these substances are known for their anti oxidant, anti-inflammatory, antitumor and estrogenic properties, suggesting the role of certain phenolic compounds in the prevention of coronary heart diseases and cancer (espín, 2001). chronic arsenic (as) toxicity from ingestion of contaminated drinking water and food has been reported in many countries and is an environmental problem of colossal proportions with a wide range of deleterious health impacts, including hyperpigmentation, keratosis, skin and internal cancers, and vascular diseases (national research council, 2001; meharg and zhao, 2012). the present study aimed to evaluate the concentration of phenolic compounds of r. officinalis extracts grown off the ground and its effects on arsenic-induced dna damage and oxidative stress through allium cepa test and biochemical analysis. materials and methods plant cultivation onion cultivation onion plantlets (allium cepa l.) were obtained from universidade federal de santa maria (ufsm), rs, brazil. the plantlets were transplanted to plastic trays containing substrate and cultivated in a greenhouse with shading (50%), at a spacing of 10 cm and a density of 100 per m of surface seedlings. these plants were administered with three daily irrigations (15 min each) with complete nutrient solution containing (mg l-1): 155.90 n; 46.40 p; 5271.00 s; 123.00 ca; 30.00 mg; 253.60 k; (mmol l-1) 2622.00 b; 133.00 na; 277.00 mo; 2274.00 zn; 636.00 cu; 6501.00 mn and (mg l-1) 1.20 fe. at the end of the cycle, the plants were collected and the produced bulbs were subsequently used for the allium cepa test and biochemical analyses. rosemary cultivation the rosemary plants were grown in a 115 m2 polyethylene shelter, covered with polyethylene additive anti-uv 200 mm thick, at ufsm, rio grande do sul, brazil. polypropylene vessels (2.8 dm3) filled with sand with one plant per pot were used for this experiment. during the cultivation four daily irrigations with nutrient solution were performed to keep the water level in the vessels. the nutrient solution used had the following ionic composition in mmol l-1: 11.02 no3-; 2.32 nh4+; 0.8 h2po4-; 6.0 k+; 1.75 ca2+; 1.25 mg2+ and 1.25 so42-. micronutrients were supplied in concentrations in mg l-1, 0.03 mo; 0.26 b; 0.06 cu; 0.50 mn; 0.22 zn; through a stock solution fe was separately provided at a concentration of 1.0 mg l-1 in the chelate form. the ph was maintained between 5.5 and 6.0. the leaves of 10 plants were harvested at 160 days after planting (dap). these leaves were dried under the shade during 5 days at room temperature before the preparation of extracts and essential oil extraction. preparation of aqueous extracts the extracts were prepared in the laboratory of cytogenetic and plant genotoxicity of ufsm, by infusion of the leaves at 5 and 20 g l-1, using distilled water as liquid extractor. the infusions were obtained by boiling distilled water at 100 °c and pouring water on whether the chopped plant material to facilitate the action of water. after mixing, the container remained capped for 15 minutes. these extracts were filtered and, after reaching room temperature, were analyzed by high performance liquid chromatography with diode arrangements detection (hplc-dad) for identification and quantification of the phenolic compounds, and used for the a. cepa test. 2 g.j. farias, d.v. frescura, a.a. boligon, c.k. trapp, l.j. andriolo, b.s. tedesco, k. bernardy, r. schwalbert, k.b. del frari, m. carey, t.f. nicoloso extraction of volatile oil of rosemary for the extraction of essential oil composite samples of 30 g were prepared from plants collected in four replications. the extraction of the essential oil of the leaves was carried out by hydrodistillation method in a clevenger apparatus for 3 hours, and stored at -4 °c until the chromatographic analysis and evaluation of the effect on the cell cycle through a. cepa test. high performance liquid chromatography the analysis by high performance liquid chromatography (hplcdad) was performed on hplc system (shimadzu, kyoto, japan) with auto injector (sil 20a) equipped with reciprocating pumps (shimadzu lc-20at) connected to the degasser (dgu 20a5) with integrator (cbm 20a) detector uv / vis diode array (spd-m20a) and lc software solution 1.22 sp1. the analysis were performed in reversed phase under gradient conditions using c18 column (4.6 mm × 150 mm) packed with 5μm diameter particles. the mobile phase was composed of: water containing acetic acid 2% (a) and methanol (b), and composition gradient was: 5% b for 2 min, 25% b to 10 min, 40, 50, 60, 70 and 80% (b) every 10 min, following the method described by coelho et al. (2013), with slight modifications. the mobile phase extracts were filtered through a 0.45 μm membrane filter (millipore) and then degassed in a soni cation bath. the flow rate was 0.9 ml min-1, injection volume of 50 l and wavelengths were 285 nm for carnosic acid, 325 nm for caffeic, chlorogenic and rosmarinic acid, and 365 nm for quercetin, rutin and kaempferol. chromatographic analysis of the volatile oil of rosmarinus officinalis gas chromatography (gc-fid) the gas chromatography (gc) analyses were carried out using an agilent technologies 6890n gc-fid system, equipped with db-5 capillary column (30 m × 0.25 mm; film thickness 0.25 mm) and connected to an fid detector. the injector and detector temperatures were set to 280 °c. the carrier gas was helium, at a flow rate of 1.3 ml min-1. the thermal programmer was 50-300 °c at a rate of 5°c/min. two replicates of samples were processed in the same way. component relative concentrations were calculated based on gc peak areas without using correction factors. the injection volume of the oil was 1 μl (verma et al., 2010). gas chromatography-mass spectrometry (gc-ms) gc-ms analyses were performed on an agilent technologies autosystem xl gc-ms operating in the ei mode at 70 ev, equipped with a split/splitless injector (250 °c). the transfer line temperature was 280 °c. helium was used as carrier gas (1.3 ml min-1) and the capillary columns used were an hp 5ms (30 m × 0.25 mm; film thickness 0.25 mm) and an hp innowax (30 m × 0.32 mm i.d., film thickness 0.50 mm). the temperature program was the same as that used for the gc analyses. the injected volume was 1 μl of the volatile oil. identification of the components identification of the constituents was performed on the basis of retention index (ri), determined with reference of the homologous series of n-alkanes, c7-c30, under identical experimental conditions, comparing with the mass spectra library search (nist and wiley), and with the mass spectra literature date adams (1995). the relative amounts of individual components were calculated based on the cg peak area (fid response). cytogenetic analysis cytogenetic analysis of meristematic cells obtained from a. cepa radicles were used to evaluate morphological and structural cell modifications and determine the mitotic index. nineteen groups of five bulbs were placed in distilled water, constituting 19 treatments with 5 replicates each. the following treatments were used: to check the individual effect of each solution; t1: distilled water, as a negative control; t2: r. officinalis oil 0.8%, t3: r. officinalis oil 3%, t4: r. officinalis extract 5 g l-1, t5: r. officinalis extract 20 g l-1, t6: arsenic (100 μm), to check the effect of r. officinalis prior to arsenic exposure; t7: r. officinalis oil 0.8% (24 h) + arsenic (24 h), t8: r. officinalis oil 3% (24 h) + arsenic (24 h), t9: r. officinalis extract 5 g l-1 (24 h) + arsenic (24 h), t10: r. officinalis extract 20 g l-1 (24 h) + arsenic (24 h), t11: arsenic (24 h) + r. officinalis oil 0.8% (24 h), to check the effect of r. officinalis after arsenic exposure; t12: arsenic (24 h) + r. officinalis oil 3% (24 h), t13: arsenic (24 h) + r. officinalis extract 5 g l-1 (24 h), t14: arsenic (24 h) + r. officinalis extract 20 g l-1 (24 h), to check the effect of r. officinalis use concomitantly with arsenic exposure;t15: arsenic with r. officinalis oil 0.8%, t16: arsenic with r. officinalis oil 3%, t17: arsenic with r. officinalis extract 5 g l-1, t18: arsenic with r. officinalis extract 20 g l-1, t19: ethanol to check alcohol effect. for the analysis of cell division, approximately 2 cm of onion radicles were collected and hydrolyzed in 1 n hcl for 5 min, followed by staining with 2% acetic orcein in accordance with guerra and souza (2002). the meristematic region was crushed using a small glass rod, and the material was placed onto a coverslip. the slides were observed under the microscope at 40 × magnification. subsequently, the total cell count (interphase and cell division) was obtained, and the mitotic index (mi) was calculated based on the percentage of dividing cells. a total of 1000 cells were counted per bulb, totaling 4000 cells per group for each treatment and each species studied. biochemical parameters for the biochemical assays, the root samples were collected, immediately placed in liquid nitrogen and pulverized to a fine powder using a porcelain mortar. the level of lipid peroxidation was estimated using a tbars assay (thiobarbituric acid reactive substances assay) according to the method of beleid el-moshaty et al. (1993). the h2o2 concentration in the roots was determined according to loreto and velikova (2001). the activity of peroxidase (pod, ec 1.11.1.7) nonspecific present in the extract was determined according zeraik et al. (2008), using guaiacol as substrate. the oxidation of guaiacol (a tetraguaiacol) was measured by the increase in absorbance at 470 nm (chance and maehly, 1955). statistical analysis the experiments were performed using a randomized design. the analyses of variance were computed on statistically significant differences determined based on appropriate f-tests. the results are presented as the means ± sd of at least three independent replicates. the mean differences were compared using scott-knott (p<0.05). results and discussion chromatography and cytogenetic analysis r. officinalis is an aromatic plant popularly known as rosemary which has important biological properties, especially due the phenolic and the volatile constituents, such as carnosol, carnosic acid and rosmarinic acid present in the extract of rosemary and α-pinene, bornylacetate, camphor and eucalyptol present in the essential oil of this species (ribeiro-santos et al., 2015; arranz et al., 2015). protective effect of rosmarinus officinalis to arsenic exposure in allium cepa 3 also, minor constituents in rosemary may have biological functions, mainly because possible synergistic effects among their compounds (ribeiro-santos et al., 2015). however, studies have demonstrated that there are significant variations in rosmarinic and carnosic acid content according to the harvest season and site of rosemary cultivation (yesil-celiktas et al., 2007; frescura et al., 2013). the compound found in highest amounts in aqueous extracts of r. officinalis was rosmarinic acid (tab. 1). the antioxidant activity has been attributed to this acid in the literature, reducing numerous events deleterious to the organism, such as formation of reactive oxygen species, lipid peroxidation and dna fragmentation (simões et al., 2001; izzo and capasso, 2007). besides, makino et al. (2000), suggest that rosmarinic acid in lamiaceae herbs is a promising agent to prevent mesangioproli ferative glomerular diseases. there are many studies demonstrating the in vitro activity of rosemary diterpenes, such as carnosic acid, carnosol, and rosmanol, in anticancer activity (reviewed by petiwala and johnson, 2015). furthermore, carnosic acid is described as the active ingredient responsible for antimicrobial activity exhibited by the extract from the leaves of r. officinalis (bernardes et al., 2009) and has clinical application for diseases affecting the outer retina, including agerelated degeneration, in which oxidative stress is probably one factor that contributes to disease progression (rezaie et al., 2012). the rutin has been cited having beneficial effects in reducing symptoms of impaired lymphatic and venous vessels (pathak et al., 1991). the quercetin offers many benefits of health promotion, including improved cardiovascular health, reducing the risk of cancer and many others (lakhanpal and rai, 2007). still, kaempferol and quercetin have protective action against pancreatic hypertrophy and hyperplasia (rawel et al., 2002). the major compounds of the volatile oil were monoterpenes corresponding to 64.6% of 91.4% (tab. 2) in agreement with published literature (ribeiro et al., 2012). interestingly, the two most abundant compounds, 1,8-cineole and camphor in rosemary oil were described as insecticidal constituents against the cabbage looper, trichoplusia ni, showing synergistic interactions and more effective control when used together (tak and isman, 2015). the results demonstrate that both volatile oil in concentrations of 0.8 and 3% and aqueous extracts of r. officinalis in concentrations of 5 and 20 g l-1, have no mutagenic effect as compared to arsenic treatments (tab. 3), and show slightly different effects from control treatment, consistent with the results of other studies using different techniques (amin and hamza, 2005). this is an important foundation for future studies including human cell lines to ensure safety of volatile oil and aqueous extracts of r. officinalis as a phytomedicine. genotoxicity of arsenic compounds has been observed in a variety of cultured human cells (yih and lee, 1999; yih et al., 2005), animals (patlolla and tchounwou, 2005) and arsenic-exposed human populations (horvathova et al., 2003). in the present study, r. officinalis (both volatile oil and aqueous extract) reduced arsenicmediated chromosome aberrations, especially the occurrence of micronucleus in allium cepa (tab. 3; fig. 1). when added after 24 h exposure to as, the aqueous extract was able to completely reverse the damage caused by exposure to as. when used concomitant, there was no occurrence of chromosomal aberrations (tab. 3; fig. 1). some studies have reported the antimutagenic effects of r. officinalis in plants (horvathova et al., 2014), and human cell lines (wang et al., 2012); but to our knowledge, there were no studies evaluating the effect of r officinalis in to repair the dna damage caused by exposure to as. higher plants present characteristics that make them excellent genetic models to assess environmental pollutants, being frequently tab. 1: concentration of phenolic compounds, mg g-1, in extracts of r. officinalis, prepared with two concentrations (5 g of leaves l-1 and 20 g of leaves l-1). phenolic compounds r. officinalis extract 5 g l-1 20 g l-1 rosmarinic acid (mg ml-1) 5.99 26.30 chlorogenic acid (mg ml-1) 2.30 8.45 carnosic acid (mg ml-1) 1.83 5.09 caffeic acid (mg ml-1) 1.33 4.87 canpferol (mg ml-1) 0.61 2.40 quercetin (mg ml-1) 1.45 5.21 rutin (mg ml-1) 0.73 5.71 tab. 2: chemical composition of rosmarinus officinalis volatile oil. compounds ria rib concentration % α-pinene 936 939 11.15 α-camphene 954 953 4.52 β-pinene 980 980 3.29 β-myrcene 991 991 8.18 α-phellandrene 1003 1005 0.25 p-cymene 1026 1026 0.44 1.8 cineol 1037 1033 16.78 camphor 1143 1143 21.33 δ-terpinene 1065 1062 1.28 linalool 1098 1098 2.54 isoboneol 1156 1156 2.74 borneol 1165 1166 1.82 menthol 1173 1173 0.94 terpin-4-ol 1178 1177 0.51 pinocarvone 1160 1162 1.11 naphthalene 1178 1179 0.34 α-terpineol 1189 1189 1.70 piperitol <cis-> 1195 1193 0.93 verbenone 1205 1204 7.20 pulegone 1237 1237 0.15 eugenol 1356 1356 0.73 α-copaene 1376 1376 0.01 caryophyllene 1417 1418 1.34 aromadendrene 1440 1439 0.48 α-muurolene 1498 1499 0.07 α-bisabolene 1504 1504 0.46 α-cadidene 1538 1538 0.25 caryophyllene oxide 1580 1581 0.83 total identified (%) 91.37 relative proportions of the essential oil constituents were expressed as percentages. aretention indices experimental (based on homologous series of n-alkane c7-c30). bretention indices from literature (adams, 1995); m = monoterpenes; s = sesquiterpenes. m s 4 g.j. farias, d.v. frescura, a.a. boligon, c.k. trapp, l.j. andriolo, b.s. tedesco, k. bernardy, r. schwalbert, k.b. del frari, m. carey, t.f. nicoloso used in monitoring studies. however, this feature is not only due to the sensitivity to detect mutagens in different environments, but also to the possibility of assessing several genetic endpoints, which range from point mutations to chromosome aberrations in cells of different organs and tissues (grant, 1994). the most common abnormality caused by the arsenic exposure in this study was the occurrence of micronuclei, which was already reported by other studies (yi and si, 2007; banerjee et al., 2013). banerjee et al. (2013) reported the association between micronuclei frequency in urothelial cells from men, women, smoker group and non-smokers group, and arsenic content in cooked rice. they indicated a strong positive correlation of mean urinary arsenic with mean cooked rice arsenic content among the tested groups. the detection of micronuclei has been considered by many authors as an efficient and simple way to analyze the mutagenic effect promoted by chemicals (mekki, 2013). micronuclei results from damage in the parental cells as acentric fragments or lagging chromosomes that fail to incorporate into either of the daughter nuclei during telophase in cell mitosis (fernandes et al., 2007) being easily observed in daughter cells as a similar structure to the main nucleus, but in a reduced size. thus, micronuclei arise from the development of some ca, for instance, chromosome breaks and losses. moreover, micronuclei may still derive from other processes as polyploidization, in which they originate from the elimination of exceeding dna of the main nucleus in an attempt to restore the normal conditions of ploidy (fernandes et al., 2007). oxidative stress r. officinalis, as others lamiaceae species, contains flavonoids and phenolic acids (coelho et al., 2013). phenolic compounds can slow down oxidative reactions in biological systems. the study of phenolic compounds is also associated with important antioxidant activity of these compounds, suggesting that diseases caused by oxidative reactions in biological systems can be delayed by the intake of natural antioxidants found in plants such as r. officinalis (simões et al., 2001). the exposure to as causes oxidative stress, as can clearly be evidenced by tbars formation, increased content of h2o2 and pod activity. in the present study, the application of r. officinalis extract reduced the tbars concentration in cells exposed to arsenic, as well as the h2o2 concentration regardless the moment of exposure (tab. 4). on the other hand, the volatile oil did not alter tbars or h2o2 concentration under the 0.8% dose when used only 24hs before arsenic exposure (tab. 4). wang et al. (2012) assessed the comparative antibacterial and anticancer activities of r. officinalis essential oil as well as its compounds, being the three main components 1,8-cineole, α-pinene and β-pinene. even though r. officinalis essential oil exhibited the strongest antibacterial and cytotoxic activities towards sk-ov-3, ho-8910 and bel-7402 human tumor cell lines. it is important to point that even though in this study the r. officinalis essential oil didn’t show high antioxidant activity, it is well known its beneficial proprieties to human health (posadas et al., 2009). the pod activity was enhanced by as exposure as compared to the others treatments (tab. 4). among the treatments with r. officinalis, the concomitant exposure to as and r. officinalis resulted in a higher activity as compared to r. officinalis prior or after as exposure (tab. 3). however, in general terms this activity of the r. officinalis extracts was not enough to prevent dna damage in a higher level as compared to treatments with r. officinalis extracts being used after as exposure (tab. 4). tab. 3: effect of rosmarinus officinalis oil and extract on mitotic index and chromosomal aberrations in root tip cells of a. cepa exposed to arsenic. treatments mitotic index total micronuclei chromosomal anaphasic (%) abnormalities breakage and lost telophasic (%) chromosomes bridges t1 distilled water* 5.37 a 0.25 c 0 0 1 t2 r. officinalis oil 0.8% 5.00 b 0.50 c 1 0 1 t3 r. officinalis oil 3% 3.15 d 0.75 c 3 0 0 t4 r. officinalis extract 5 g l-1 1.37 f 0.50 c 1 0 1 t5 r. officinalis extract 20 g l-1 0.80 g 0.75 c 2 0 1 t6 arsenic 4.27 c 3.50 a 10 0 4 t7 r. officinalis oil 0.8% (24 hs) + arsenic (24 hs) 1.15 f 2.00 b 7 0 1 t8 r. officinalis oil 3% (24 hs) + arsenic (24 hs) 0.20 h 1.50 b 5 0 1 t9 r. officinalis extract 5 g l-1 (24 hs) + arsenic (24 hs) 0.20 h 1.00 c 3 0 1 t10 r. officinalis extract 20 g l-1 (24 hs) + arsenic (24 hs) 0.10 h 0.75 c 1 0 2 t11 arsenic (24 hs) + r. officinalis oil 0.8% (24 hs) 3.95 c 2.00 b 6 1 1 t12 arsenic (24 hs) + r. officinalis oil 3% (24 hs) 1.00 g 0.25 c 1 0 0 t13 arsenic (24 hs) + r. officinalis extract 5 g l-1 (24 hs) 1.00 g 0.50 c 1 0 1 t14 arsenic (24 hs) + r. officinalis extract 20 g l-1 (24 hs) 0.95 g 0.00 c 0 0 0 t15 arsenic with r. officinalis oil 0.8% 4.10 c 2.00 b 5 1 2 t16 arsenic with r. officinalis oil 3% 2.35 e 0.35 c 1 0 0 t17 arsenic with r. officinalis extract 5 g l-1 0.75 g 0.50 c 2 0 0 t18 arsenic with r. officinalis extract 20 g l-1 0.20 h 0.00 c 0 0 0 t19 ethanol 4.25 c 0.50 c 2 0 0 different low letters show significant differences among the treatments. *negative control protective effect of rosmarinus officinalis to arsenic exposure in allium cepa 5 conclusion this study demonstrated that the r. officinalis extract exerted no mutagenic effects and showed antimutagenic potential, reducing the dna damage and lipid peroxidation resulting from treatment with as exposure. references adams, r.p., 1995: identification of essential oil components by gas chromatography/mass spectroscopy. illinois usa: allured publishing corporation. amin, a., hamza, a.a., 2005: hepatoprotective effects of hibiscus, rosmarinus and salvia on azathioprine-induced toxicity in rats. life sciences. 77, 266-278. doi: 10.1016/j.lfs.2004.09.048 anvisa, 2010: resolução-rdc no 10, de 9 de março de 2010. dispõe sobre a notificação de drogas vegetais junto à agência nacional de vigilância e dá outras providências. retrieved february 2015, from http://189.28.128.100/dab/docs/legislacao/resolucao10_09_03_10.pdf banerjee, m., banerjee, n., bhattacharjee, p., mondal, d., lythgoe, p.r., martínez, m., pan, j., polya, d., giri, a.k., 2013: high arsenic fig. 1: a b: allium cepa cells during normal anaphase (t1 distilled water a and t5 rosmarinus officinalis extract b); c d allium cepa cells during interphase exhibiting micronuclei (t6 treated with arsenic (24 h); and t7 r. officinalis oil 0.8% (24 h) + arsenic (24 h)); e: allium cepa cells exhibiting anaphase chromosome bridges (t6 treated with arsenic); f: allium cepa cells during normal anaphase (t18 arsenic with r. officinalis extract 20 g l-1). tab. 4: levels of h2o2, tbars and pod in cells obtained from allium cepa radicles exposed to arsenic and rosmarinus officinalis oil and extract. treatments tbars (mda nm g-1 fw) h2o2 (um g-1 fw) pod (umol min-1 mg fw) t1 distilled water* 0.567 d 1.602 c 22.217 d t2 r.officinalis oil 0.8% 0.587 d 2.203 c 26.728 d t3 r. officinalis oil 3% 0.564 d 1.456 c 28.984 c t4 r. officinalis extract 5 g l-1 0.498 d 1.467 c 26.728 d t5 r. officinalis extract 20 g l-1 0.476 d 2.202 c 19.961 d t6 arsenic 1.876 a 10.267 a 55.173 a t7 r. officinalis oil 0.8% (24 hs) + arsenic (24 hs) 1.654 a 8.467 a 36.428 c t8 r. officinalis oil 3% (24 hs) + arsenic (24 hs) 1.132 c 7.233 a 29.661 c t9 r. officinalis extract 5 g l-1 (24 hs) + arsenic (24 hs) 1.109 c 5.533 b 31.917 c t10 r. officinalis extract 20 g l-1 (24 hs) + arsenic (24 hs) 1.187 c 4.822 b 36.428 c t11 arsenic (24 hs) + r. officinalis oil 0.8% (24 hs) 1.543 b 8.467 a 38.684 b t12 arsenic (24 hs) + r. officinalis oil 3% (24 hs) 0.765 d 6.875 a 31.917 c t13 arsenic (24 hs) + r. officinalis extract 5 g l-1 (24 hs) 0.708 d 4.076 b 29.661 c t14 arsenic (24 hs) + r. officinalis extract 20 g l-1 (24 hs) 0.698 d 4.897 b 29.554 c t15 arsenic with r. officinalis oil 0.8% 1.106 c 8.467 a 34.172 c t16 arsenic with r. officinalis oil 3% 0.706 d 5.876 b 43.195 b t17 arsenic with r. officinalis extract 5 g l-1 0.654 d 4.035 b 43.194 b t18 arsenic with r. officinalis extract 20 g l-1 0.698 d 4.876 b 40.706 b different low letters show significant differences among the treatments. *negative control. 6 g.j. farias, d.v. frescura, a.a. boligon, c.k. trapp, l.j. andriolo, b.s. tedesco, k. bernardy, r. schwalbert, k.b. del frari, m. carey, t.f. nicoloso in rice is associated with elevated genotoxic effects in humans. sci. rep. 3, 1-8. doi: 10.1038/srep02195 bernardes, w.a., lucarini, r., souza, m.g.m., tozatti, m.g., silva, m.l.a., filho, a.a.s., martins, c.h.g., cunha, w.r., 2009: ácido carnósico: um diterpeno isolado de rosmarinus officinalis com potencial antibacteriano frente à bactérias bucais. in: 32a reunião anual da sociedade brasileira de química. anais... fortaleza: sociedade brasileira de química. chance, b., maehley, a.c., 1995: assay of catalase and peroxidases. methods in enzymology. 11, 764-775. doi: 10.1016/s0076-6879(55)02300-8 coelho, a.p.d frescura, v.d.-s., mambri, a.p., augusti boligon, a., tedesco, s.b., 2013: avaliação dos compostos fenólicos e potencial genotóxico e antiproliferativo do extrato de echinodorus longiscapus arech. enciclopédia biosfera. 9, 2698-2709. beleid el-moshaty, f.i., pike, s.m., novacky, a.j., sehgal, o.p., 1993: lipid peroxidation and superoxide production in cowpea (vigna unguiculata) leaves infected with tobacco ringspot virus or southern bean mosaic virus. physiol. mol. plant pathol. 43, 109-119. doi: 10.1006/pmpp.1993.1044 espín, j.c., soler-rivas, c., wichers, h.j., 2000: characterization of the total free radical scavenger capacity of vegetable oils and oil fractions using 2,2-diphenyl-1-picrylhydrazyl radical. j. agric. food chem. 48, 648-656. doi: 10.1021/jf9908188 fernandes, t.c.c, mazzeo, d.e.c., marin-morales, m.a. 2007: mechanism of micronuclei formation in polyploidizated cells of allium cepa exposed to trifluralin herbicide. pest. biochem. physiol. 88, 252259. doi: 10.1016/j.pestbp.2006.12.003 ferrari, g.n., suguino, e., martins, a.n., mello, s.c., minami, k., 2011: alecrim (rosmarinus officinalis l.). piracicaba: esalq. 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fatibello-filho, o., leite, o.d., 2008: desenvolvimento de um spot test para o monitoramento da atividade da peroxidase em um procedimento de purificação. química nova. 31, 731734. protective effect of rosmarinus officinalis to arsenic exposure in allium cepa 7 address of the corresponding author: departamento de biologia, centro de ciências naturais e exatas, universidade federal de santa maria, santa maria, rs 97105-900, brazil e-mail: fariasjuliagomes@gmail.com © the author(s) 2018. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). vab-liquidation-bekanntmachung vereinigung für angewandte botanik association for applied botany ohnhorststr. 18, d-22609 hamburg tel: 040/42816-349; fax: 040/42816-565 e-mail: helmut.kassner@uni-hamburg.de haspa (blz 200 505 50) kontonummer: 1127214144 # iban de 86 20050550 11272-14144 bic/swift-code: haspdehhxxx entsprechend dem bürgerliches gesetzbuch ist bei auflösung eines vereins eine offizielle veröffentlichung erforderlich. dieser pflicht kommt die vab mit folgendem text nach: bekanntmachung der liquidation der vereinigung für angewandte botanik e.v. (vab), eingetragen beim amtsgericht hamburg im vereinsregister vr 20777 auf dem registerblatt vr 20777 notariell bestätigt und vom amtsgericht am 31.01.2017 eingetragen ist: “die mitgliederversammlung vom 31.08.2015 hat die auflösung des vereins beschlossen.” als liquidatoren sind benannt: “dr. kassner, helmut, hamburg, *18.04.1951 prof. dr. selmar, dirk, braunschweig, *11.04.1955” “die liquidatoren vertreten gemeinsam.” die liquidatoren fordern hiermit eventuelle gläubiger auf, ihre ansprüche an die vab anzumelden (§50 (1) bgb bekanntmachung des vereins in liquidation). die bekanntmachung erfolgt in dem vereinseigenen journal of applied botany and food quality und gilt mit dem ablauf des zweiten tages nach veröffentlichung als bewirkt. hamburg, 30.03.2017 gez. dr. helmut kassner, schatzmeister, liquidator prof. dr. dirk selmar, präsident, liquidator journal of applied botany and food quality 90, 330 338 (2017), doi:10.5073/jabfq.2017.090.041 1department of morphology and systematic of plants, institute of botany and botanical garden “jevremovac”, faculty of biology, university of belgrade, serbia 2department of plant physiology, institute for biological research “siniša stanković“, university of belgrade, serbia 3institute for medicinal plant research “dr. josif pančić”, belgrade, serbia 4,5department of biology, faculty of natural sciences and mathematics, ss. cyril and methodius university, institute for biology and macedonian academy of sciences and arts, skopje, macedonia laserpitium ochridanum: antioxidant, antimicrobial and anti-quorum sensing activities against pseudomonas aeruginosa ksenija s. mileski1*, ana d. ćirić2, jovana d. petrović2, mihailo s. ristić3, vlado s. matevski4,5, petar d. marin1, ana m. džamić1 (received june 6, 2016; accepted august 23, 2017) * corresponding author summary this study shows laserpitium ochridanum essential oil composition, its antifungal potency, and antioxidant, antimicrobial and anti quorum sensing activities of different extracts. monoterpene hydrocarbons (40.9%) were the most abundant group of constituents in the oil. sabinene (22.8%), viridiflorol (14.7%) and α-pinene (11.40%) were the main components of the oil. the ethanolic extract had the highest antioxidant capacity in dpph and abts assays and it was the richest in phenolic contents. microdilution method revealed the strongest antibacterial activity of ethanolic extracts in comparison to other tested extracts and streptomycin. essential oil of l. ochridanum evidenced the best antifungal potential against used micro mycetes. results of an anti-quorum sensing activity assay indicated high affection of aqueous extract in reduction of pao1 pyocyanin production (18.07%). used samples possessed slight reduction of twitching and swimming motility. this study shows for the first time anti-quorum sensing activity of l. ochridanum against pseudomonas aeruginosa pao1, as well as its significant antioxidant potential. introduction the genus laserpitium l. (apiaceae) comprises about 30 species, mostly biennial and perennial plants, widely distributed from the canary islands to siberia and iran (nikolić, 1973). it is characterized by ternate or several times pinnate leaves, white, yellow or pinkish petals (tutin, 1968). laserpitium ochridanum micevski is rare and an endemic perennial plant, up to 40-60 cm with white colour of the petals which can be found only at the national park mt. galičica (fyrom), at 1600-2000 m a.s.l. (micevski, 2005). different parts of some widely distributed laserpitium species (e.g. l. siler, l. latifolium) have been used as traditional herbal medicines in europe. they are usually used as tonics for strengthening and refreshing, for treating toothache, as diuretics, for treating gastro intestinal disorders, heart and liver dysfunctions, pulmonary tuberculosis, rheumatism and topically in pruritic dermatomycoses, as well as for sleep disorder and major depression in taiwan (popović et al., 2013; yi-lin chen et al., 2015). in earlier studies, sesquiterpene lactones were found as the main secondary metabolites in laserpitium extracts (appendino et al., 1987, 1993; đermanović et al., 1996). recently, it was published that sesquiterpene lactones mainly belong to the class of guajanolides (popović et al., 2013). however, monoterpene hydrocarbons were predominant compounds in the essential oil (eo) of laserpitium species (baser and duman, 1997; chizzola et al., 1999; chizzola, 2007; petrović et al., 2009; tirillini et al., 2009; popović et al., 2010, 2013, 2014). litarature data showed that different laserpitium species possessed antibac terial, antifungal, cytotoxic, anticancer, antinociceptive and antiedematous activities (petrović et al., 2009; tirillini et al., 2009; popović et al., 2010, 2013, 2014). lately, the interest in studying different pathogens is rapidly incrising, because of their resistance towards synthetic antibiotics or antimycotics. it is known that the pathogenic, gram-negative bacillus pseudomonas aeruginosa is a major cause of nosocomial infections, bronchopneumonia, septic shock and wound infections. this opportunistic bacterium forms populations with distinctive density-dependant behavour. by antiquorum sensing (anti-qs) agents, growth of p. aeruginosa can be weaken and some of its pathologically significant virulence factors, such as production of biofilm, swarming motility, pigment and antibiotic production, can be reduced (soković et al., 2014; sepahi et al., 2015). some medical plants possess anti-qs activity and can be considered as potential anti-quorum agents (al-hussaini and mahasneh, 2009; koh et al., 2013; sepahi et al., 2015). the aim of this study was to define the chemical composition of l. ochridanum essential oil and to determined antioxidant, anti microbial and anti-qs activities of its extracts. to the best of our knowledge l. ochridanum crude extracts were assayed for the first time for their antioxidant potency combined with total phenolic and flavonoid contents. also, no anti-qs activity of this species has been reported to date. material and methods solvents and chemical reagents solvents and chemicals that were provided for performing the ex periments were of analytical grade. organic solvents were procured from zorka pharma, šabac, serbia. gallic acid, 3-tert-butyl4-hydroxyanisole (bha), 2,2-diphenyl-1-picrylhydrazyl (dpph), folin-ciocalteu phenol reagent, potassium acetate and aluminum trinitrate nonahydrate were obtained from sigma-aldrich co., st louis, mo, usa. sodium carbonate anhydrous was purchased from centrohem d.o.o, stara pazova, serbia. potassium peroxide sulphate and l(+)-ascorbic acid were obtained from fisher scientific uk ltd., loughborough, leicestershire, uk. 2,2’-azino-bis(3-ethyl benzothiazoline-6-sulfonic acid) (abts) and quercetin hydrate were purchased from tci europe nv, binnenveldsweg, belgium. malt-broth (mb), tryptic soy broth (tsb), mueller-hinton agar (mh), luria-bertani medium (lb) (1% w/v nacl, 1% w/v tryptone, 0.5% w/v yeast extract) and malt agar (ma) were obtained from the institute of immunology and virology, torlak (belgrade, serbia). streptomycin (sigma-aldrich s6501 st. louis, mq, usa), ampi cillin (sigma-aldrich a9393 st. louis, mq, usa) and dimethyl sulfoxide (dmso) (sigma aldrich, st. louis, mq, usa) were used in these study. antimicotic diflucan (containing 50 mg fluconazole) was obtained from pfizer pgm, pocesur-cisse, france. biological activities of laserpitium ochridanum 331 plant material plant material was collected during the flowering stage, at mt. galičica, the national park in republic of macedonia (fyrom) in july, 2013 (gps: n 40°56́30 ,̋ e 20°49́34̋). it was determined as laserpitium ochridanum micevski by one of the authors (prof. vlado s. matevski). a voucher specimen (bu16778) is deposited at the herbarium of the institute of botany and botanical garden “jevremovac”, faculty of biology, university of belgrade, serbia. isolation of the essential oil the dark blue eo of l. ochridanum was obtained from 200 g of dry aerial parts by 3 h of hydrodistilation using a clevenger type appa ratus. the yield of the oil was 0.11% for herbal parts (w/w-dry bases). the essential oil obtained was preserved in sealed vials at 4 ºc prior to further analysis. preparation of plant extracts dried, ground plant material (10 g) was treated with 200 ml of methanol, ethanol and distilled water to obtain different extracts. the ultrasonic extraction procedure was performed during 24 h in the dark; the extracts were exposed to ultrasound for the first and the last hour of extraction and subsequently filtered through a whatman filter paper no 1. methanolic and ethanolic extracts were subjected to solvent evaporation under reduced pressure at maximum temperature of 40 ºc. the frozen aqueous extracts were lyophilized, reduced to a fine dried powder and mixed to obtain homogenous samples. the dried and crude extracts were measured, packed in glass bottles, and stored at 4 ºc until subjection to subsequent analysis. obtained yields of l. ochridanum extracts were 0.597 g for methanolic (me), 1.323 g for ethanolic (ee) and 0.983 g for aqueous extracts (ae). gas chromatography–flame ionization detector (gc–fid) and gas chromatography–mass spectrometry (gc–ms) qualitative and quantitative analysis of the essential oil was performed using gc and gc-ms methods. the gc analysis of the oil was carried out on a gchp-5890 ii apparatus, equipped with splitsplit less injector, attached to an hp-5 column (25 m × 0.32 mm, 0.52 μm film thickness) and fitted to fid. carrier gas flow rate (h2) was 1 ml/min, split ratio 1:30, injector temperature was 250 °c, detector temperature 300 °c, while column temperature was linearly programmed from 40 to 240 °c (at rate of 4 °/min.). the same analy tical conditions were employed for gc-ms analysis, where a 1800c series ii gcd system equipped with hp-5ms column (30 m × 0.25 mm, 0.25 μm film thickness) was used. the transfer line was heated at 260 °c. mass spectra were acquired in ei mode (70 ev), in m/z range 40-400. the identification of the individual eo components was accomplished by comparison of retention times with standard substances and by matching mass spectral data with those of the wiley 275 mass spectra library. confirmation was performed using amdis software and literature (adams, 2007). quantitative analyses were based on area percents obtained by fid. analyses of total phenols and total flavonoids total phenolic content (tpc) the spectrophotometric method described by singleton et al. (1999) with some modifications was applied for recording total tpcs of all tested l. ochridanum extracts, using folin-ciocalteu reagent and ga as a standard. after preparing a 10% folin-ciocalteau reagent, the mixtures of 1000 μl of this solution and 200 μl of extracts solutions (1 mg/ml) were left to react for 6 min. after short incubation, 800 μl of 7.5% sodium carbonate solution was added and thus prepared solution was allowed to stand for 2 h at room temperature in the dark. the absorbance was measured at 736 nm versus a blank sample. total phenols were calculated from the ga calibration curve (10-100 mg/l). data were expressed as milligrams of ga equivalents per gram of dry plant extract. the values were presented as means of triplicate analysis. total flavonoid content (tfc) measurements of tfcs of l. ochridanum extracts were based on the method described by park et al. (1997) with slight modification. an aliquot of each extract solution (1 ml) was mixed with 80% ethanol, 10% aluminium nitrate nonahydrate and 1 m potassium acetate. absorption readings at 415 nm using a spectrophotometer were taken after 40 min. against a blank sample consisting of a 0.5 ml 96% ethanol instead of the tested sample. the tfcs were determined from the qe standard curve (10-100 mg/l). results were expressed as mg of qe equivalents/g of dry extract. generally, all measurements were done in triplicates. antioxidant capacity dpph assay series of eo and extracts solutions in appropriate solvents, with concentrations of 0.25-2 μl/ml for eo and 0.025-0.2 mg/ml for extracts were subjected for examination of free radical scavenging activity by dpph assay. this spectrophotometric procedure described by blois (1958), was performed to evaluate the quantity of tested solutions needed to reduce 50% of the initial dpph radical concentration. 0.2 ml of each dilution was mixed with 1.8 ml of dpph methanol solution (0.04 mg/ml). the absorbance was recorded at 517 nm after 30 min. of dark incubation at room temperature. bha and ascorbic acid were used as reference standards and methanol as a blank. the corresponding percentage of inhibitions of each sample was calculated from obtained absorbance values by using following equation: percentage (%) of inhibition = (ac-as)/ac × 100 tested concentrations of eo and extracts which decrease absorption of dpph solution for 50% (ic50) were obtained from the curve dependence of absorption of dpph solution on 517 nm from concentration for each tested solution and used standards. abts assay the procedure of miller and rice-evans (1997) with slightly mo difications was followed for determination of in vitro abts radicalscavenging potency. before usage, 5 ml of the mixture of 2.46 mm potassium persulphate and 19.2 mg of abts was allowed to react in the dark for 12-16 h at room temperature to obtain abts+ solution. 100-110 ml of distilled water was added to 1 ml of abts+ solution to adjust an absorbance of 0.7 ± 0.02 units at 734 nm. the mixtures of 2 ml of diluted abts·+ solution and 50 μl of each tested extract solution were incubated for 30 min. at 30 ºc and the absorbance was determined spectrophotometrically at 734 nm, using water as a blank. for every experiment a fresh abts+ solution was prepared. the results were expressed from an ascorbic acid calibration curve (0-2 mg/l) in mg of ascorbic acid equivalents/g of dry extract. tests were carried out in triplicate and all measurements were expressed as average of three analyses ± standard deviation. evaluation of antimicrobial properties microorganisms and culture conditions the antimicrobial activity of all investigated samples was tested using pure control strains obtained from the mycological laboratory, department of plant physiology, institute for biologycal research 332 k.s. mileski, a.d. ćirić, j.d. petrović, m.s. ristić, v.s. matevski, p.d. marin, a.m. džamić “siniša stanković”, belgrade, serbia. the microorganisms included following bacterial strains: bacillus cereus (food isolate), listeria monocytogenes (nctc 7973), micrococcus flavus (atcc 10240) and staphylococcus aureus (atcc 6538), enterobacter cloacae (human isolate), escherichia coli (atcc 35210), pseudomonas aeruginosa (atcc 27853), and salmonella typhimurium (atcc 13311). the following micromicetes were used: aspergillus fumi gatus (atcc 9197), aspergillus niger (atcc6275) aspergillus ochraceus (atcc 12066), aspergillus versicolor (atcc 11730), candida albicans (atcc 10231), penicillium funiculosum (atcc 10509), penicillium ochrochloron (atcc 9112) and trichoderma viride (iam 5061). dilutions of bacterial inocula were cultured on solid mh medium, while micromycetes were maintained on solid ma medium. the cultures were subcultured once a month and stored at +4 °c for further usage (booth, 1971). microdilution method for determination of antimicrobial activity of l. ochridanum oil and extracts, the modified microdilution technique described by hanel and raether (1998) was applied. the assay was performed by sterile 96-well microtiter plates, by adding pure eo or dilutions of tested extracts (in 5% dmso) into corresponding medium tsb and ma for bacteria and fungi, respectively. to achieve the concentration of 1.0 × 108 colony forming units (cfu)/ml for bacterial strains, 100 μl of overnight cultures were mixed with 900 μl of medium in eppendorf. fungal inocula were prepared by washing spores with sterile 0.85% saline solution (containing 0.1% tween 80 (v/v)). the microbial cell suspensions were adjusted with sterile saline to a concentration of approximately 1.0 × 106 cfu/ml for bacteria and 1.0 × 105 cfu/ml for fungi in a final volume of 100 μl per well. the microplates were incubated for 24 h at 37 °c for bacteria and for 72 h at 28 °c for fungi. the lowest concentrations of tested samples completely inhibiting the growth of used pathogens were defined as minimum inhibitory concentrations (mics). the minimum bactericidal/fungicidal concentrations (mbcs, mfcs) were determined as the lowest concentrations with no visible growth after serial sub cultivation, indicating 99.5% killing of the original inoculums (hanel and raether, 1998). in addition, bacterial growth was determined by a colorimetric microbial viability assay, based on reduction of an 0.2% p-iodonitrotetrazolium violet color (int) aqueous solution (i 8377-sigma aldrich, st. louis, mq, usa) and compared with positive control for each bacterial strain (clsi, 2009; tsukatani et al., 2012). two replicates were done for each sample. the solution of synthetic standard streptomycin with concentration of 1 mg/ml 5% dmso was used as positive control for bacteria, while the fluconazole solution (antimicotic diflucan containing 50 mg fluconazole) at concentration of 2 mg/ml 5% dmso was included for fungi. sterilized distilled water containing 0.02% tween 80 and 5% dmso was used as negative control. preparation of stock solutions of plant extracts for antimicrobial tests different quantities of stock solutions of l. ochridanum extracts, dissolved in 5% dmso (20 mg/ml) were tested against various pathogenic microorganisms. anti-quorum sensing activity of extracts bacterial strains, growth media and culture conditions in this study, pseudomonas aeruginosa pao1 from the institute for biological research “siniša stanković”, belgrade, serbia, was used. bacteria were routinely grown in luria-bertani (lb) medium with shaking (220 rpm) and cultured at 37 °c. biofilm formation considering the results obtained in antimicrobial assay and low yields of isolated eo and me, further anti-qs analyzes were continued with ethanolic and aqueous extracts of l. ochridanum. the samples (0.5, 0.25, 0.125 of mics, respectively) were tested on biofilm forming ability on polystyrene flat-bottomed microtitre 96 well plates as described by spoering and lewis (2001); drenkard and ausubel (2002), with some modifications. in brief, 100 μl of overnight culture of p. aeruginosa (1.0 × 108 cfu/ml) was added to each well of the plates in the presence of 100 μl subinhibitory concentrations (submic) of l. ochridanum samples (0.5, 0.25 and 0.125 mic) or 100 ml medium (control). after incubation for 24 h at 37 ºc, each well was washed twice with sterile pbs (ph 7.4), dried, stained for 10 min with 0.1% crystal violet in order to determine the biofilm mass. after drying, 200 μl of 95% ethanol (v/v) was added to solubilise the dye that had stained the biofilm cells. the excess stain was washed off with distilled water. after 10 min, the content of the wells was homogenized and the absorbance at λ = 620 nm was read on a sunrise™ tecanelisa reader. the experiment was done in triplicate and repeated two times and values were presented as a mean values ± se. twitching and flagella motility after growth in the presence or absence of submics of l. ochri danum ethanolic and aqueous extracts, streptomycin and ampicil lin, the cells of p. aeruginosa pao1 were washed twice with sterile pbs and re-suspended in pbs at 1.0 × 108 cfu/ml (od of 0.1 at 660 nm). in brief, the cells were stabbed into a nutrient agar plate with a sterile toothpick and incubated overnight at 37 °c. the plates were then removed from the incubator and incubated at room tem perature for two more days. colony edges and the zone of moti lity were measured with a light microscope (o’toole and kolter, 1998a, b). submics of extracts (0.5 mics) were mixed into 10 ml of molten lb medium and poured immediately over the surface of a solidified lb plate as an overlay. the plate was point inoculated with an overnight culture of pao1 once the overlaid agar had solidified and incubated at 37 °c for 3 days. the extent of swimming was determined by measuring the area of the colony (sandy and foongyee, 2012). the experiment was done in triplicate and repeated two times. the colony diameters were measured three times in different direction and values were presented as a mean values ± se. inhibition of synthesis of p. aeruginosa pao1 pyocyanin the flask assay was used to quantify the inhibitory activity of the l. ochridanum against p. aeruginosa pyocyanin production. overnight culture of the bacillus pao1 was diluted to od600 nm 0.2. then, 0.5 mics of tested extracts dissolved in 5% of dmso (1.25 mg/ml for ee and 12.50 mg/ml for ae), were added to the bacteria (5 ml) and incubated at 37 °c for 24 h. the treated culture was extracted with chloroform (3 ml), followed by mixing the chloroform layer with 0.2 m hcl (1 ml). absorbance of the extracted organic layer was measured at 520 nm using a shimadzu uv1601 spectrophoto meter (kyoto, japan) (sandy and foong-yee 2012). the experiment was done in triplicate and repeated two times. the values were expressed as ratio (od520/od600) × 100. statistical analysis three samples were used and all the assays were carried out in triplicates. the results are expressed as mean values and standard deviation (sd). the results were analyzed using one-way analysis of variance (anova) followed by tukey’s hsd test with a = 0.05. this analysis was carried out using spss v. 18.0 program. biological activities of laserpitium ochridanum 333 results essential oil composition referring to the results presented in tab. 1, fifty nine components were identified in l. ochridanum eo. monoterpene hydrocarbons were the most abundant group of compounds (40.87%), followed by oxygenated sesquiterpenes (24.14%), oxygenated monoterpenes (15.27%) and sesquiterpene hydrocarbons (13.17%). the dominant compounds of the eo were sabinene (22.8%), viridiflorol (14.7%) and α-pinene (11.4%) (tab. 1). total phenolic contents according to the results obtained for tpc in l. ochridanum extracts (tab. 2), phenols were present from 111.28 to 141.30 mg ga/g of dry extract for methanolic and ethanolic extract, respectively. in tested extracts, tfc ranged from 21.38 to 67.69 mg qe/of dry extract for aqueous and ethanolic extract, respectively. in general, greater variation in tested extracts was recorded in flavonoid contents. the highest phenolic and flavonoid concentrations were measured in ee of l. ochridanum (tab. 2). antioxidant activity the results of obtained antioxidant activity for l. ochridanim are listed in tab. 2. in dpph test, used extracts exhibited similar anti oxidant activity, stronger than bha, but lower activity compared to ascorbic acid. still, the strongest radical scavenging activity was recorded for ee (0.113 ± 0.002 mg/ml), which was in accordance with the highest measured total phenolic and flavonoid contents. eo of l. ochridanum showed the lowest antioxidant potency compared to all other samples. results obtained by the abts test showed that the ae was the most effective agent in concentration of 2.172 ± 0.005 mg ascorbic acid/g of dry extract. according to the obtained results this sample had slightly lower antioxidant capacity than standard qe (2.749 ± 0.004 mg ascorbic acid/g of dry extract). antimicrobial properties antibacterial activity the results presented in tab. 3 indicate that l. ochridanum extracts exhibited moderate antibacterial activity. the ee was the strongest in bactericidal activity (mbcs = 1.00-5.00 mg/ml), while the lowest potency had ae (mbcs = 11.00-14.00 mg/ml). both, methanolic and ethanolic extracts were more effective compared to streptomycin, but all extracts, including aqueous, showed stronger inhibitory activity on l. monocytogenes and e. cloacae than used antibiotic. tab. 1: chemical composition of eo of l. ochridanum aerial parts. compounds kie kil % α-thujene 919.1 924 0.32 α-pinene 924.8 932 11.36 thuja-2,4(10)-diene 944.9 953 0.17 sabinene 965.6 969 22.76 β-pinene 974.7 974 0.80 myrcene 985.7 988 0.69 n-octanal 997.2 998 0.39 α-terpinene 1009.4 1014 1.02 p-cymene 1018.0 1020 0.35 β-phellandrene 1020.9 1025 0.96 γ-terpinene 1051.3 1054 1.93 cis-sabinene hydrate 1057.3 1065 0.56 n-octanol 1069.3 1063 3.86 terpinolene 1080.7 1086 0.51 6-camphenone 1088.1 1095 0.59 trans-sabinene hydrate 1097.0 1098 0.29 6-camphenol 1113.5 1111 0.43 α-campholenal 1119.3 1122 0.56 trans-pinocarveol 1132.4 1135 0.85 trans-sabinol 1135.5 1137 0.40 trans-verbenol 1139.6 1140 1.79 terpinen-4-ol 1171.0 1174 3.86 thuj-3-en-10-al 1178.9 1181 0.31 α-terpineol 1186.8 1186 0.28 myrtenal 1188.8 1193 0.37 myrtenol 1192.1 1194 0.54 verbenone 1204.0 1204 0.19 octanyl acetate 1207.5 1211 1.26 isobornyl acetate 1277.8 1283 0.50 α-terpinyl acetate 1343.0 1346 0.21 cyclosativene 1366.2 1369 0.27 α-copaene 1370.2 1374 1.12 daucene 1376.8 1380 0.69 β-cubebene 1381.1 1387 0.23 β-elemene 1383.4 1389 0.43 (e)-caryophyllene 1409.0 1417 1.62 trans-α-bergamotene 1427.0 1432 0.18 α-humulene 1443.5 1452 0.66 (e)-β-farnesene 1450.2 1454 0.19 cis-muurola-4(14),5-diene 1460.6 1465 0.20 γ-himachalene 1467.9 1468 0.81 dauca-5,8-diene 1471.4 1471 1.96 ar-curcumene 1475.5 1479 0.19 β-selinene 1487.0 1489 1.73 cis-eudesma-6,11-diene 1488.0 1489 0.83 bicyclogermacrene 1494.9 1500 0.44 β-bisabolene 1500.6 1505 0.12 δ-cadinene 1514.3 1522 0.70 α-calacorene 1546.1 1544 0.80 spathulenol 1570.3 1577 2.65 caryophyllene oxide 1573.4 1582 1.16 viridiflorol 1588.9 1592 14.71 1-epi-cubenol 1627.5 1627 0.54 β-cedren-9-one 1631.6 1630 2.44 β-eudesmol 1649.4 1649 0.44 α-bisabolol 1679.3 1685 2.20 chamazulene 1721.9 1730 3.04 neophytadiene (isomer ii) 1829.5 1830 1.02 incensole acetate 2181.9 2184 0.52 total 100.00 number of constituents 59 monoterpene hydrocarbons 40.87% oxygenated monoterpenes 15.27% sesquiterpene hydrocarbons 13.17% oxygenated sesquiterpenes 24.14% others 6.55% kie = kovats (retention) index experimentally determined (amdis) kil = kovats (retention) index literature data (adams, 2007) 334 k.s. mileski, a.d. ćirić, j.d. petrović, m.s. ristić, v.s. matevski, p.d. marin, a.m. džamić b. cereus and s. aureus (mbcs = 1.00-11.00 mg/ml) were the most sensitive bacteria, while e. coli and m. flavus (mbcs = 5.00->14.00 mg/ml) proved to be the most resistant strains. antifungal activity the results obtained for the antifungal activity of investigated samples are presented in tab. 4. the eo of this species had the strongest activity in inhibition of micromycetes growth (mfcs = 0.55-2.20 mg/ml) and it was similar to the activity of applied fluconazole (mfcs = 0.03-1.50 mg/ml). among the investigated extracts, the ee showed the highest activity (mfcs = 5.00 mg/ml) on all used fungal strains, exept for a. niger (mfcs =18.00 mg/ml) (tab. 4). the most resistant micromycetes were a. niger and a. fumigatus, while the most sensitive strains were a. versicolor, p. ochrochloron and p. funiculosum. fungi t. viride and p. ochrocloron were more sensitive to l. ochridanum oil (mfc = 1.10 mg/ml), than to fluconazole (mfc = 1.50 mg/ml) (tab. 4). standards / / qe 2.75 ± 0.004a tab. 2: tpc, tfc and antioxidant activity of l. ochridanum extracts and eo (means ± sd). l. ochridanum extracts/ total phenolic contents antioxidant activity tpc 1 mg/ml tfc 1 mg/ml dpph abts 1 mg/ml (mg ga/g of de) (mg qe/g of de) (ic50 = mg/ml) (mg ascorbic acid/g of de) me 111.28 ± 0.005c 31.31 ± 0.010b 0.12 ± 0.011b 1.63 ± 0.009b ee 141.30 ± 0.013a 67.69 ± 0.018a 0.11 ± 0.002b 1.56 ± 0.004b ae 125.30 ± 0.010b 21.38 ± 0.004c 0.12 ± 0.000b 2.17 ± 0.005a eo / / 1.88 ± 0.009c / bha 0.13 ±0.012b ascorbic acid 0.03 ± 0.008a indicated letters mean significant difference (p < 0.05) eo tab. 3: antibacterial activity of l. ochridanum extracts in mg/ml (means ± sd). me ee ae streptomycin mic mbc mic mbc mic mbc mic mbc b. cereus 0.40 ± 0.06a 1.00 ± 0.03a 0.50 ± 0.02a 1.00 ± 0.00a 6.00 ± 0.02a 11.00 ± 0.01a 1.50 ± 0.01a 2.50 ± 0.06a m. flavus 4.00 ± 0.02c 5.00 ± 0.02b 2.00 ± 0.01ab 5.00 ± 0.00b 10.00 ± 0.01b >14.00 ± 0.03b 2.50 ± 0.00a 5.00 ± 0.00b l. monocytogenes 3.00 ± 0.04c 4.00 ± 0.07b 0.50 ± 0.00a 2.00 ± 0.10a 5.00 ± 0.02a 11.00 ± 0.01a 15.00 ± 0.10c 20.00 ± 0.02c p. aeruginosa 3.00 ± 0.00c 4.00 ± 0.05b 0.50 ± 0.02a 2.00 ± 0.01a 5.00 ± 0.00a 11.00 ± 0.02a 2.50 ± 0.07a 5.00 ± 0.01b e. coli 4.00 ± 0.01c 8.00 ± 0.03c 4.00 ± 0.02b 5.00 ± 0.01b 10.00 ± 0.00b >14.00 ± 0.01b 2.50 ± 0.04a 5.00 ± 0.03b e. cloacae 2.00 ± 0.01b 4.00 ± 0.01b 1.00 ± 0.10a 4.00 ± 0.02b 5.00 ± 0.10a 11.00 ± 0.01a 10.00 ± 0.02b 20.00 ± 0.00c s. typhymurium 2.00 ± 0.00b 3.00 ± 0.05b 1.00 ± 0.06a 2.00 ± 0·05a 10.00 ± 0.01b >14.00 ± 0.02b 2.50 ± 0.01a 5.00 ± 0.01b s. aureus 0.40 ± 0.10a 1.00 ± 0.07b 1.00 ± 0.05a 2.00 ± 0.03a 5.00 ± 0.07a 11.00 ± 0.00a 2.50 ± 0.05a 5.00 ± 0.00b indicated letters mean significant difference (p < 0.05) l. ochridanum/ bacteria l. ochridanum/ fungi tab. 4: antifungal activity of l. ochridanum extracts and eo in mg/ml (means ± sd). me ee ae eo fluconazole mic mfc mic mfc mic mfc mic mfc mic mfc c. albicans 8.00 ± 0.02c 12.00 ± 0.01b 6.00 ± 0.10c 12.00 ± 0.05b 12.00 ± 0.01b 14.00 ± 0.03a 0.55 ± 0.00b 1.10 ± 0.02b 0.02 ± 0.05a 0.03 ± 0.03a t. viride 3.00 ± 0.00a 8.00 ± 0.02a 5.00 ± 0.05b 14.00 ± 0.07b 10.00 ± 0.01a 12.00 ± 0.02a 0.55 ± 0.07b 1.10± 0.03b 1.00 ± 0.02d 1.50± 0.02d p. ochrochloron 6.00 ± 0.03b 10.00 ±0.10a 4.00 ± 0.02a 5.00 ± 0.05a 10.00 ± 0.00a 14.00 ± 0.00a 0.28 ± 0.05a 1.10 ± 0.01b 1.00 ± 0.00d 1.50 ± 0.02d p. funiculosum 3.00 ± 0.01a 8.00 ± 0.05a 4.00 ± 0.00a 5.00 ± 0.00a 12.00 ± 0.07b 14.00 ± 0.02a 0.55 ± 0.05b 2.20 ± 0.01c 0.25 ± 0.01b 0.50 ± 0.00b a. fumigatus 6.00 ± 0.00b 14.00 ± 0.01b 6.00 ± 0.10c 14.00 ± 0.20b 12.00 ± 0.10b 14.00 ± 0.02a 0.55 ± 0.10b 2.20 ± 0.07c 0.50 ± 0.02c 1.00 ± 0.00c a. versicolor 6.00 ± 0.03b 8.00 ± 0.20a 4.00 ± 0.01a 5.00 ± 0.03a 10.00 ± 0.20a 12.00 ± 0.07a 0.28 ± 0.02a 0.55± 0.00a 0.13 ± 0.00a 0.50 ± 0.01b a. ochraceus 6.00 ± 0.02b 19.00 ± 0.03c 4.00 ± 0.03a 5.00 ± 0.02a 10.00 ± 0.00a 14.00 ± 0.05a 0.55 ± 0.10b 1.10 ± 0.02b 0.50 ± 0.05c 1.00 ± 0.03c a. niger 8·00 ± 0.01c 19.00 ± 0.05c 6.00 ± 0.00c 18.00 ± 0.05c 12.00 ± 0.01b 18.00 ±0.10b 1.10 ±0.07c 2.20 ± 0.00c 0.25 ± 0.03b 1.00 ± 0.10c indicated letters mean significant difference (p < 0.05) biological activities of laserpitium ochridanum 335 anti-qs activity evaluation biofilm formation tab. 5 presents the effects of l. ochridanum extracts on p. aeruginosa pao1 biofilm formation. the samples were tested at 0.5, 0.25 and 0.125 of mic values. l. ochridanum extracts showed significant difference in terms of anti-biofilm formation activity. l. ochridanum ee showed dose dependant inhibitory activity, reducing from 8.63% to 63.88% of biofilm formation, where the best result was obtained in the presence of 0.5 mic of the extract. results revealed that l. ochridanum ee reduced biofilm formation more effectively than both antibiotics allowing formation of pao1 in the range from 36.12% to 91.37%. contrary, the tested submic concentrations of ae did not show any suppression of p. aeruginosa biofilm formation (tab. 5). in the presence of commercial antibiotics streptomycin and ampicillin, biofilm formation occurred in narrower range, with slightly stronger biofilm inhibition recorded for streptomycin. twitching and flagella motility in addition, the ethanolic and aqueous extracts of l. ochridanum reduced the twitching and flagella motility activity of p. aeruginosa (tab. 6 and fig. 1). as presented in tab. 6, the color of the colony ranged from white, through light green to green. used extracts changed the color and diameter of treated colonies to a certain ex tent. in the presence of extracts, colonies where white and larger (14.00 mm and 17.67 mm for ee and ae, respectively) in comparison to colonies treated with antibiotics (tab. 6). the green colony of p. aeruginosa with streptomycin, had minimal growth (11.00 mm) and completely reduces protrusions. also, the most reduced flagella in size, shape and number were in colony with streptomycin (fig. 1). inhibition of synthesis of p. aeruginosa pao1 pyocyanin submics of l. ochridanum samples were tested for inhibition of p. aeruginosa pigment production and both extracts showed substantial activity in pigment synthesis inhibition. the affection was observed by the reduction of the green pigmentation of the samples, tab. 5: effects of l. ochridanum extracts on biofilm formation of p. aeruginosa pao1 (%). biofilm formation* 0.5 mic 0.25 mic 0.125 mic (% ± se) (%± se) (% ± se) ee 36.12 ± 1.73 60.82 ± 1.05 91.37 ± 0.42 ae n.d. n.d. n.d. ampicillin 69.16 ± 0.65 56.46 ± 0.46 92.16 ± 0.37 streptomycin 49.40 ± 0.46 70.97 ± 0.36 88.36 ± 0.42 *biofilm formation values were calculated as: ((mean a620 control wellmean a620 treated well)/mean a620 control well) × 100. − values are expressed as means ± se. − n.d. not determinate l. ochridanum/ standards tab. 6: effects of l. ochridanum extracts on twitching and flagella motility of p. aeruginosa (pao1). l. ochridanum extracts/ colony diameter flagella diameter colony colour colony edge standards (mm ± se) (μm) ee 14.00 ± 2.65 40-160 white rare flagella ae 17.67 ± 5.51 16-80 white tiny flagella streptomycin 11.00 ± 1.00 24-56 green tiny flagella ampicillin 13.33 ± 5.03 16-56 green regular flagella p. aeruginosa (pao1) 12.00 ± 1.00 56-80 light green regular flagella fig. 1: light microscopy of colony edges of p. aeruginosa in twitching motility, grown in the presence or absence of l. ochridanum extracts and commercial antibiotics. the colonies from the bacteria grown with extracts in concentration of 0.5 mic (a-b). the colony with ee was with moderately reduced protrusions (a); in the presence of ae colony formed almost regular protrusions (b); p. aeruginosa colony in the presence of streptomycin (0.5 mic) with reduced protrusion (c); p. aeruginosa colony in the presence of ampicillin with regularly formed protrusions (d); p. aeruginosa produced a flat, widely spread, irregularly shaped colony in the absence of extracts and commercial antibiotics (e); magnification: (a-d) × 100. 336 k.s. mileski, a.d. ćirić, j.d. petrović, m.s. ristić, v.s. matevski, p.d. marin, a.m. džamić compared to the coloration of the control pao1 sample (fig. 2). the strongest inhibition of pigment’s production was detected for l. ochridanum ae. in the presence of tested concentrations of extracts, pyocyanin was less produced (23.46% and 18.07% for ethanolic and aqueous extracts, respectively), than by control strain (141.55%). all extracts were better in prevention of pigment production regarding to applied antibiotics (fig. 2). screening results indicated that ee and ae samples showed some potential of anti-qs activity. l. ochridanum ee demonstrated an inhibition at the initial stage of biofilm formation in the manner of different tested concentrations. that is of great importance since bacteria form biofilms as a protection against host’s immune system and as a factor of antibiotic resistance (soković et al., 2014). it was clearly indicated that tested concentrations of l. ochridanum extracts were more effective on p. aeruginosa pigment production than those of applied antibiotics. this green, toxic pigment acts as a virulence factor in bacteria, so reduction of its production is crucial for increasing the effectiveness of host defense. while ae notably demonstrated the best activity in suppression of pyocyanin synthesis, it did not show any reduction of colony formation at the tested submics. opposite results obtained for ae in these two assays could be associated with possible different mechanisms responsible for its activity. at this point, there is no sufficient data to highlight the exact method of qs inhibition. a few potential modes of action have been proposed, many of them to interfere with the qs system such as inhibition of biosynthesis of auto-inducer molecules, inactivation or degradation of the auto-inducer, interference with the signal receptor and inhibition of the genetic regulation system. due to complex phytochemistry of plant extracts, different compounds could be associated with specific effects linked to the qs system, so the difference in the activities of l. ochridanum aqueous sample suggest less polar nature of l. ochridanum active compounds in reduction of p. aeruginosa biofilm formation. the importance of plant extracts preparation should be also considered (adonizio et al., 2006; adonizio, 2008: glamočlija et al., 2015). in the research of adonizio et al. (2008), where aqueous extract of schefflera actinophylla, with no anti-qs activity, was used as a negative control, it was concluded that plant extracts differentially affect biofilm formation. inhibition of swarming and twitching motility of pao1 by l. ochridanum samples was achieved in moderate extent with better results obtained for ee. both types of motilities are important in the initial stages of biofilm formation of p. aeruginosa (o’toole and kolter, 1998b). conclusions this study established the chemical characterization of l. ochridanum eo and provided new data concerning antioxidant and anti-qs activities of crude extracts/eo. the presence of phenolic compounds and previously reported sesquiterpene lactones mostly contribute to biological potency of l. ochridanum extracts. among all extracts, ee possessed the best potency in evaluated antioxidant and antibacterial activities. the eo revealed strong antifungal potential. l. ochridanum showed promising anti-qs effectiveness that was sufficient for the reduction of biofilm formation and pyocyanin production. to establish the application of this species in various pharmaceutical, dietary or alternative medicine branches, further research is needed especially concerning that the exact mechanistic interactions with the qs system should be resolved. acknowledgements the authors are grateful to the ministry of education, science and technological development of the republic of serbia for financial support grants no. 173029 and 173032. references adams, r.p., 2007: identification of essential oil components by gas chromatography/mass spectrometry, 4th ed. carol stream, il, usa: allured publishing corp. adonizio, a.l., downum, k., bennett, b.c., mathe, k., 2006: antidiscussion the chemical compositions of eos of different laserpitium spe cies have been previously reported (baser and duman, 1997; chizzola et al., 1999; chizzola, 2007; petrović et al., 2009; tirillini et al., 2009; popović et al., 2010, 2014). results obtained for l. ochridanum eo revealed sabinene, viridiflorol and α-pinene as the most dominant compounds (tab. 1). recent studies on l. ochridanum herb oil showed sabinene and α-pinene as the major components, which is in accordance with our results. in contrary, limonene, which was the main constituent of fruit oil, was not detected in our sample (popović et al., 2014, 2015). our results revealed that ee of l. ochridanum was the richest in phenols and flavonoids among all extracts. also, ee had the highest dpph radical scaven ging activity, while ae was the strongest agent in abts radical scavenging assay. in general, all extracts possessed comparable activity to synthetic antioxidants. in contrary, eo showed low anti oxidant capacity. results obtained in a microdilution assay, indicated that grampositive and gram-negative bacteria showed similar sensitivity to l. ochridanum extracts. according to the literature data, l. ochridanum chloroform extract of roots and rhizomes exhibited antimicrobial activity against some food spoilage microorganisms (popović et al., 2015). in comparison with those results, it can be concluded that methanolic and ethanolic extracts of l. ochridanum in our study showed higher antimicrobial potency. we found that extracts indica ted stronger antibacterial than antifungal potential (tab. 3 and 4). eo of l. ochridanum showed similar inhibitory effect as fluconazole (tab. 4). also, l. ochridanum eo was more effective than l. garganicum eo in growth inhibition of the most resistant micromycete a. niger (tirillini et al., 2009). inhibition of bacterial qs offers new strategies for the treatment of bacterial infections. anti-qs agents can interfere with the bacterial communication system and can disrupt the pathogenicity process of bacteria (sepahi et al., 2015). therefore, l. ochridanum extracts were submitted for anti-qs screening for the first time since it is of great importance to discover new potential anti-qs agents. the 0 20 40 60 80 100 120 140 160 ampicillin pa01 p ro du ct io n of p yo ci an in (% ) l. ochridanum ethanol l. ochridanum aqueous streptomycin fig. 2: reduction of pyocyanin production of p. aeruginosa pao1 by l. ochridanum extracts streptomycin and ampicillin tested at submics (mg/ml). biological activities of laserpitium ochridanum 337 quorum sensing activity of medicinal plants in southern florida. j. ethnopharmacol. 105, 427-435. doi: 10.1016/j.jep.2005.11.025 adonizio, a.l., 2008: anti-quorum sensing agents from south florida medicinal plants and their attenuation of pseudomonas aeruginosa pathogenicity. fiu electronic theses and dissertations 13, 14-21. al-hussaini, r., mahasneh, a.m., 2009: microbial growth and quorum sensing antagonist activities of herbal plants extracts. molecules 14, 3425-3435. doi: 10.3390/molecules14093425 appendino, g., cravotto, g., nano, g.m., 1993: sesquiterpene lactones from laserpitium gallicum. phytochemistry 33, 883-886. appendino, g., valle, g.m., gariboldi, p., 1987: sesquiterpene lactols from laserpitium halleri. phytochemistry 26, 1755-1757. baser, k.h.c., duman, h., 2011: composition of the essential oil of laserpitium petrophilum boiss. et heldr. j. essent. oil res. 9, 707-708. doi: 10.1080/10412905.1997.9700818 blois, m.s., 1958: antioxidant determinations by the use of a stable free radical. nature 181, 1199-1200. doi: 10.1038/1811199a0 booth, c., 1971: fungal culture media. in: norris, j.r., ribbons, d.w. 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(eds.), flora europaea, 2nd ed, 368-370. cambridge, cambridge university press. © the author(s) 2017. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). address of corresponding author: institute of botany and botanical garden “jevremovac”, faculty of biology, university of belgrade, studentski trg 16, 11000 belgrade, serbia e-mail: ksenija.mileski@bio.bg.ac.rs journal of applied botany and food quality 88, 186 191 (2015), doi:10.5073/jabfq.2015.088.026 1 pear research station, national institute of horticultural and herbal science, naju, korea 2 functional food research center, chonnam national university, kwang-ju, korea antioxidant and whitening activities of five unripe pear cultivars sun-hee yim1, seung-hee nam2* (received march 18, 2015) * corresponding author summary the present study was the first evaluation of the arbutin content, antioxidant activity, and whitening function of the unripe pears of five major korean pear cultivars. unripe pears were investigated 30 days after florescence for possible utilization as a whitening ingredient, instead of being thrown away for thinning out. among the five cultivars tested, gamcheonbae and manpungbae had significantly higher total phenolics and arbutin contents, while niitaka had lower values of both total phenolics and arbutin. for whitening activity related to tyrosinase and cellular melanin formation, manpungbae also showed the strongest tyrosinase inhibition (4.9 %), and achieved 74 % reduction of the cellular melanin compared to nontreated cells. these results indicate that unripe pears, especially the manpungbae cultivar, could be useful for application as a possible natural whitening additive with high arbutin content and excellent whitening activity. introduction pear (pyrus spp.) is one of the most widely consumed fruits in the world. it is typically eaten fresh, and is often found in processed foods such as juice, puree, jellies, and jams. for many years, pears have been used not only as one of the most common edible fruits, but also as an herbal medicine for relieving cough, embellishing lung, eliminating constipation, and relieving alcoholism (cui et al., 2005). various phenolic compounds have been found in pear fruits, including arbutin, chlorogenic acid, rutin, and procyanidins (ferreira et al., 2002; chen et al., 2007; lee et al., 2011). in particular, arbutin (4-hydroxyphenyl β-d glucopyranoside) is widely used as a whitening agent in cosmetic products (sugimoto et al., 2004). arbutiwas also reported to inhibit the activity of mushroom tyrosinase, the biosynthesis of b16 mouse melanoma, and hmv-ii human melanoma cells (chakraborty et al., 1998; nihei and kubo, 2003; sugimoto et al., 2004). arbutin was found to be the most abundant phenolic compound in pear fruit (zhang et al., 2006), and has also been used as a specific marker to evaluate the authenticity of pear products (branca et al., 2000). pear was reported to contain approximately 0.1 ~ 1 % arbutin and chlorogenic acid, although the values vary depending on the cultivars and the ripening of the fruit (cui et al., 2005; lee et al., 2011). several studies showed similar results in that the phenolic compounds and antioxidant activities of pears differed between cultivars and reduced with maturity (cui et al., 2005; zhang et al., 2006). oriental pear cultivars (chinese or asian) have a tendency to contain higher amounts of phenolic compounds including arbutin and chlorogenic acid than occidental pears cultivars, although oriental pear cultivars were also found to have variation in the arbutin level depending on the cultivar (galvis sanchez et al., 2003; cho et al., 2011a; li et al., 2012; 2014). among maturing stages, arbutin content was the highest in unripe pear before and after florescence, and rapidly decreased by 70 % at 40 days after florescence (cui et al., 2005; zhang et al., 2006). large amounts of unripe pears are usually discarded due to natural disasters, poor cultivation techniques, or the thinning out of superfluous fruits. thinning out is an especially high contributor, with 90 % of unripe pears thrown away to obtain high quality pears. therefore, is necessary to find out how these unripe fruits could be utilized to produce new food ingredients, such as functional food ingredients, instead of wasting them. so far, the previous studies have mostly focused on phenolic functional compounds and the physiological activities of pears between different cultivars or different parts of the pear during fruit development (galvis sanchez et al., 2003; li et al., 2012; 2014). in the present study, we investigated the possible utilization of five unripe pear cultivars at a certain developmental stage (30 days after florescence) as a whitening ingredient. this stage was chosen for investigation since most unripe pears are thrown away 30 days after florescence due to thinning out, even though they have peak arbutin level or antioxidant activity (cui et al., 2005; zhang et al., 2006). the total phenolics and arbutin contents of the five unripe pear cultivars were measured, and further functional characterization was carried out with respect to the antioxidant activities, and inhibitory effects on tyrosinase as well as cellular melanin formation. materials and methods reagents folin-ciocalteu’s phenol reagent, 1,1-diphenyl-2-picrylhydrazyl (dpph), 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (abts), α-tocopherol, trolox, sodium chloride, tannic acid, quercetin, and ascorbic acid were purchased from sigma aldrich-fluka (germany). acetonitrile and formic acid (98 % purity) were of hplc grade, and were also purchased from sigma aldrichfluka (germany). all other solvents and chemicals were of analytical grade. sample preparation five cultivars of asian pear (pyrus pyrifolia) were chosen for this study based on the highest produced in south korea. these included gamcheonbae, manpungbae, chuwhangbae, hanareum, and niitaka varieties, obtained from an orchard at the naju national pear experimental station, in chonnam, korea. pear fruits of the five cultivars were collected 30 days after full bloom. seven to ten fruits from each selected standard pear trees were used for the experiments, with uniformity in size and no defects. the pears were washed, rapidly cut into thin slices, and lyophilized by freeze-dryer (fd8512, ilshin, korea). the samples were then further grinded by pulverizer (fm681c, hanil electric., korea) and finally stored at -20 °c in poly ethylene bags until further analysis. the frozen material of each pear cultivar (10 g) was previously homogenized in a moulinex stirrer and then extracted with 80 % ethanol by the soxhlet extraction method at 60 °c for 6h. the extracts were then filtered through whatman no 1 filter paper. the residues were extracted again with 80 % ethanol using the same method as mentioned above. the extracts were then combined and evaporated to dryness under vacuum. the whitening activities of five unripe pear cultivars 187 dried extract was prepared at the concentration of 0.1 g/ml of the freeze-dried pear in methanol. the methanol solution was used for analysis of the total phenolics, flavonoids, and antioxidant capacity. total phenolics and total flavonoids contents the total phenolic content in the extracts of the five pear cultivars was determined using folin-ciocalteu’s, reagent as described in cui et al. (2005). the absorbance of each sample was measured at 765 nm with a uv/visible spectrophotometer (jp/u-3900, hitachi, japan.) after incubating at 25 °c for 2 h. total phenolic content was calculated from a calibration curve, using tannic acid as the standard (160-960 μg per 100 g of dry weight). the flavonoid content was measured by the aluminum chloride colorimetric method, which was described by boo et al. (2009). quercetin was used as the standard. the flavonoid content was determined at 506 nm with a spectrophotometer. the data were expressed as milligram quercetin equivalents /100 g dry weight, and the calibration curve ranged from 40 to 840 μg. quantification of arbutin by hplc analysis the pear samples were filtered through 0.22-μm membrane filters and analyzed by an hplc system. the separation of arbutin was performed on ace c18 columns (4.6×250 mm, advanced chromatography technologies, aberdeen, uk). the solvent system used was a gradient of water and formic acid (19:1, v/v) (a) and methanol (b), starting with 5 % methanol. a gradient was installed to obtain 15, 25, 30, 35, 45 and 50 % b at 3, 13, 25, 35, 39, and 42 min, respectively. the absorbance was measured at 280 nm with a photodiode array (pda) detector (spd-m20a, shimadzu, japan) at a solvent flow rate of 1 ml/min. finally, arbutin was eluted at 25 min. arbutin standard (mg/ml) was dissolved in 80 % ethanol. this solution was diluted to the appropriate concentrations for construction of a calibration curve. the calibration curve was constructed by plotting the peak areas vs. the concentration of the standard. dpph and abts radical scavenging activity dpph (2, 2-diphenyl-1-picrylhydrazyl) radical scavenging activities of the pear extracts were determined by the method of blios (1958). to 0.25 ml of sample (1 mg /ml), 0.8 ml of 0.15 mm dpph· (methanol) was added, and shaken vigorously, followed by incubation at room temperature for 30 min in the dark. the control sample was prepared with 80 % ethanol instead of pear extract. the decrease in absorbance in presence of dpph· was measured at 517 nm using a spectrophotometer (jp/u-3900, hitachi, tokyo, japan). ascorbic acid was used as a reference compound. radical scavenging activity was expressed as % inhibition of dpph· radicals using the following equation: dpph radical scavenging activity (%) = 1(sample absorbance / control absorbance) × 100 abts radical scavenging activity of the pear extracts was measured by the abts cation decolorization assay, as described by re et al. (1999) with some modifications. the abts radical cation (abts+·) was produced by reaction of 7 mm stock solution of abts with 2.45 mm potassium persulfate, which was allowed to stand in the dark at room temperature for 12 h -16 h before use. the abts+· solution was diluted with methanol to give an absorbance of 0.7 ± 0.01 at 734 nm. pear extracts (1 mg/ml) were allowed to react with 2 ml of the abts+· solution for 1 min, after which the absorbance were measured at 734 nm. trolox was used as the reference compound. radical scavenging activity was expressed as % inhibition of abts radicals, using the following equation: abts radical scavenging activity (%) = (1sample absorbance / control absorbance) × 100 in vitro tyrosinase inhibition assay tyrosinase inhibitory activity was determined by spectrophotometry as described by masamoto et al. (1980), with minor modifications. in brief, the incubation mixture (total volume = 190 μl) consisted of 50 u/ml mushroom tyrosinase in 140 μl phosphate buffered saline (pbs) (ph 6.8), and 50 μl of l5 mm l-dopa (l-β-3, 4-dihydroxyphenyl alanine) solution, in pbs, with or without pear extracts. the mixture was incubated at 37 °c for 10 min, after which the absorbance was measured at 515 nm using a benchmark microplate reader (bio-rad laboratories, hercules, ca, usa). tyrosinase inhibition rate was expressed as % inhibition of tyrosinase using the following equation: tyrosinase inhibition rate (%) = (1sample absorbance / control absorbance) × 100 cell viability and inhibition of melanin synthesis cell viability was assessed by the standard mtt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, with a slight modification (shibata et al., 2006). b16f10 mouse melanoma cells (1 × 105 cells/well) were seeded in a 96-well microtiter plate and allowed to adhere completely to the plate overnight. the next day, new media containing pear extracts at dosages of 10 to 500 μg/ml dosages was added to the plate, which was then incubated at 37 °c in a co2 incubator. after a total of 72 h of incubation, the media was removed and 50 μl of mtt solution (1.0 mg/ml) was added to each well. after further incubation for 4 h, the formazan was solubilized in dimethyl sulfoxide (dmso) and the absorbance was measured at 450 nm (reference at 630 nm) using an mr-96a microplate reader. dmso at a toxic concentration of 5 % v/v was used as a negative control (cao et al., 2007). each treatment was performed in triplicate, and each experiment was repeated three times. the release of extra-cellular melanin was measured according to the method of makpol et al. (2009). in brief, melanoma cells were seeded in 24-well tissue culture plates at 1 × 105 cells/ml and incubated for 24 h. next, α-msh (melanocyte stimulating hormone, 0.1 μm) was added, and the cells were treated with 100 μg/ml doses of the pear extracts for a total of 72 h incubation. after washing twice with phosphate buffered saline, cells were dissolved in 1 ml of 1 n naoh. for measurement of the melanin content, 100 μl aliquots of solution were then placed in 96-well plates, and the absorbance was measured at 450 nm using a microplate reader. inhibition of melanin synthesis was expressed as the percentage of melanin content in the cells treated with pear extract to that of untreated melanoma cells. statistical analysis all experiments were carried out with three replicates and results were expressed as mean ± standard deviation. the data were analyzed by one-way anova, and the means of different groups were compared with the duncan’s multiple range test (dmrt) using the spss version 17.0 statistical software package. values of p < 0.05 were considered as significant in all cases. results and discussion total phenolics and total flavonoids contents the total phenolics and flavonoids contents of the five cultivars of asian pear (pyrus pyrifolria) examined herein are presented in tab. 1. the pears examined included five major korean major cul188 s.-h. yim, s.-h. nam tivars: gamcheonbae, manpungbae, chuwhangbae, hanareum, and niitaka. the samples were collected in the early growth stage, 30 days after florescence, for analysis of the functional compounds since the phenolic contents in pear fruit were reported to be the highest before and after florescence, with rapid decrease from 40 70 days after florescence (cui et al., 2005; zhang et al., 2006). the five pear cultivars tested were found to have total phenolic con tents ranging from 255 + 2.64 to 367 + 9.78 mg/100g fresh weight (fw), while the flavonoid contents were between 26.5 + 0.82 and 36.6 + 1.45 mg/100g fw. among the species tested, gamcheonbae and manpungbae had significantly higher total phenolics. niitaka, the representative cultivar with above 70 % domestic production, had a lower value for total phenolics. the flavonoid content followed a similar trend to that of total phenolics, with higher values of 36.6 + 1.45 mg/100 g fw in gamcheonbae and manpungbae, but lower values of 26.5 + 0.82 mg/100 g fw in niitaka. interestingly, total phenolics and flavonoids of manpungbae showed higher content in the unripe fruits, but lower content in the ripe fruits compared to the other cultivars (data not shown). this is because manpungbae has a relatively heavy weight than other cultivars, at more than 800 g, resulting in lower levels of polyphenols due to the larger fruit volume. with respect to total phenolic content, the values obtained (tab. 1) were comparable with the contents in other kinds of fruit, such as guava (179 mg/100 g fw), banana (51 mg/100 g fw), and peach (112-126 mg/100 g fw) (veberic and colaric, 2008; arranz et al., 2009). the amounts in the five pears (255-367 mg/ 100 g fw) were similar to those of other fruits reported in many works (du et al., 2009; salta et al., 2010). this pattern agreed with a previous report concerning phenolic content among similar pear cultivars, but 1.7-3.2 times higher content was found in the ripe fruit, in that study compared to the unripe pears in the present study (zhang et al., 2006). besides pears, unripe peach (hong et al., 2006) and mandarin orange fruit (kang et al., 2005) also showed higher levels of polyphenols than the ripe fruits, with variation of polyphenol contents among cultivars. this could be due to several factors. since polyphenols are important protective agents in plants, high levels of polyphenols in unripe fruits are required to prevent chemical damage and invasion by damaging organisms (zhang et al., 2006). the polyphenols in fruit react with other substances and accumulate in different forms as the fruit matures (choi and lee, 2013). this may be the reason why rapid maturity of the fruit results in decreased polyphenol content (choi and lee, 2013). quantification of arbutin by hplc analysis arbutin (4-hydroxyphenyl β-d glucopyranoside) is the main phenolic constituent in pear fruit. arbutin has gained wide use as a whitening agent to prevent unnecessary spots and freckles through inhibitions of melanin synthesis and tyrosinase activity (maeda and fukuda, 1996). the arbutin contents in ethanol extracts of the five pears were determined by hplc/dad and shown in tab. 1. manpungbae, hanareum, and nikita showed the higher levels of arbutin at 40-50 mg/100 g fw, but gamcheonbae displayed lower levels at 26-27 mg/100 g fw. at the early growth stage, arbutin contents among cultivars seemed to be positively correlated with the trend of phenolic contents, except for gamcheonbae, which contained relatively lower amounts of arbutin. this agrees with the report that nikita contained greater arbutin concentration than other cultivars, including chuwhangbae and hosui (zhang et al., 2006). a number of studies have reported that arbutin content varies greatly among pear cultivars, ranging from 5.0-40 mg/100 g fw (li et al., 2012), and also among pear parts, at 11-530 mg/100 g fw (cui et al., 2005; zhang et al., 2006). in addition, comparison of the arbutin levels of oriental pears and occidental pears, during different growth stages, or in different parts of fruit was also carried out (cui et al., 2005). the mean concentration of arbutin in the oriental pear cultivars was twice as high as the occidental pear cultivars, at 16.4 mg/100 g fw compared to 8.3 mg/100 g fw. during the growth stages of yali pear, the arbutin levels were higher in the unripe fruit at 992 mg/100 g fw right after florescence, but rapidly decreased to approximately 200 mg/100 g fw 40 days after florescence before further dropping to 40 mg/100 g fw at maturity (cui et al., 2005). among the parts in the pear fruit, the concentration of arbutin was reported to be the highest in the peel (120 mg/100 g fw), at 3-5 times greater levels than found in the core and 10-45 times greater than in the flesh (cui et al., 2005; zhang et al., 2006; li et al., 2014). dpph and abts radical scavenging activity antioxidant activities of the five pear extracts were evaluated by dpph· or abts+· radical scavenging activity (tab. 2). the antioxi-2). the antioxi-). the antioxidant activity of pear ethanol extract was determined for 100 mg/ml concentrations. for each methods, dpph· can only be dissolved in organic media (especially in alcohols), not in aqueous media, which causes limitation in interpreting the role of hydrophilic antioxidants. in contrast, abts+· can be solubilized either in aqueous or organic media, allowing interpretation of the role of the hydrophilic and lipophilic antioxidants. in terms of dpph· scavenging activity, the five pear cultivars did not show any significant differences (p > 0.05), with dpph bleaching abilities of 77.4 + 2.134 % to 82.5 + 3.97 %. it was reported that the antioxidant capacities of pear extracts were similar among cultivars, ranging from 79.3 % to 92.0 % (zhang et al., 2006). among the cultivars examined herein, nikita had lower antioxidant activity, while manpungbae and chuwhangbae displayed higher activity. this result was similar with the study of zhang et al. (2006), in that the antioxidant activity of nikita was lower than that of chuwhangbae in the unripe fruits. pear cultivars showed similar values tab. 1: total phenolics, total flavonoids, and arbutin contents of five unripe pear cultivars. cultivars total phenolics total flavonoids arbutin mg/100 g fw mg/100 g fw mg/100 g fw gamcheonbae 354 ± 6.17 b z 35.2 ± 0.97 a 26.8 ± 0.64 c z manpungbae 367 ± 9.78 a 36.6 ± 1.45 a 53.9 ± 0.72 a chuwhangbae 258 ± 2.64 d 32.5 ± 0.87 b 27.1 ± 1.06 c hanareum 304 ± 5.92 c 29.7 ± 0.54 c 50.1 ± 2.84 a niitaka 255 ± 3.75 d 26.5 ± 0.82 c 40.6 ± 0.97 b z different letters within columns indicate significant differences of the means, as determined by duncan’s multiple range test at p < 0.05. tab. 2: dpph and abts radical scavenging of five young pear cultivars. cultivars dpph radical scavening abts radical scavening % % gamcheonbae 80.9 ± 3.38 a z 67.7 ± 6.23 bc manpungbae 82.5 ± 3.97 a 74.2 ± 2.99 a chuwhangbae 81.8 ± 2.46 a 70.4 ± 4.77 ab hanareum 77.4 ± 2.13 a 66.3 ± 5.19 bc niitaka 77.4 ± 1.09 a 65.5 ± 3.42 c z different letters within columns indicate significant differences of the means, as determined by duncan’s multiple range test at p < 0.05. whitening activities of five unripe pear cultivars 189 when the antioxidant capacities per serving (100 g) were compared (galvis sanchez et al., 2003). the free radical scavenging activity of the pear cultivars increased linearly with increase in the sample concentration (data not shown). in comparison to other fruits, the pear fruits showed relatively higher antioxidant activity. chinese plum (lee et al., 2008) and peach (kim et al., 2009) were reported to have antioxidant activities of 80.9 % and 76.8 %, respectively, at the same concentration of 100 mg/ml sample. the antioxidant activity of loquat greatly differed between parts, with 91.8 % in the leaf, 5.0 % in the seeds, and 14.2 % in the flesh at the concentration of 500 mg/ml sample (park et al., 2008). with regard to the abts+· radical scavenging activity, the five pear cultivars examined showed similar values, ranging from 65.5 + 3.41 % to 74.2 + 2.99 % antioxidant activity, though some differences were observed among cultivars (p < 0.05). for the five pear cultivars examined herein, the antioxidant capacities determined by the two methods showed good agreement. chuwhangbae and manpungbae presented the strongest abts+· bleaching activities (70.4 + 4.77 % or 74.2 + 2.99 %), respectively, followed by gamcheonbae, hanareum, and nikita at 67.7 + 6.23 %, 66.3 + 5.19 % and 65.5 + 3.42 %, respectively (tab. 2). manpungbae, with its high total phenolics, flavonoids, and arbutin contents, exhibited significantly higher antioxidant abilities than nikita when assayed by the dpph and abts+· methods. therefore, it can be deduced that total phenolics and arbutin contents make a major contribution to the antioxidant capacity of the pears. a number of studies have reported that phenolic compounds, including arbutin and chlorogenic acid, are the main phytochemicals responsible for the antioxidant capacity of vegetables and fruits (du et al., 2009; salta et al., 2010). phenolic compounds in pears made much greater contribution to the antioxidant capacity than vitamin c (galvis sanchez et al., 2003). in vitro tyrosinase inhibition assay tyrosinase is a key enzyme that catalyzes the initial steps in the formation of the pigment melanin. it catalyzes two major reactions, including the hydroxylation of tyrosine, and oxidation of l-dopa (sanchez-ferrer et al., 1995). tyrosinase is responsible for the coloring of the skin, hair and eyes in animals, as well as for the molting process in insects, fruits and vegetables (wang et al., 2006). application of tyrosinase-inhibiting agents may be the least invasive procedure for maintaining skin whiteness. recently, natural substances such as fruits and vegetables have been in increased demand as new agents for depigmenting, cosmeceutical, and skin lightening purposes (aburjai and natsheh, 2003). accordingly, the inhibitory effects of the ethanol extracts of pear were evaluated on mushroom tyrosinase activity herein (fig. 1). the five pear cultivars exhibited dose-dependent inhibition of tyrosinase based on the examination of three different concentrations from 10-100 μg/ml. for the 100 μg/ ml pear ethanol extracts, manpungbae displayed the strongest inhibitory activity at 4.9 ± 0.7 %, followed by hanareum, chuwhangbae, and niitaka, at 3.5 ± 0.4 %, 2.9 ± 1.0 %, and 2.8 ± 0.7 %, respectively. gamcheonbae showed significantly lower inhibition than the other four cultivars, at 2.0 ± 0.7 %. phenolic compounds are good inhibitors of tyrosinase activity in melanoma cells. there are also reports that phenolic compounds may be used as depigmenting agents, since they have a similar chemical structure to tyrosine, the substrate of tyrosinase (boissy and manga, 2004). manpungbae showed high tyrosinase inhibition due to its high phenolic content, while nikita displayed lower inhibition with low phenolic contents among the five cultivars. in terms of the mushroom tyrosinase inhibition of fruits, unripe pear extracts had relatively inhibition (up to 4.9 %) compared to those of other fruitextracts. although unripe peach and cucumber extract showed slightly higher inhibition at 4.6-8.5 % with a dose of 100 μg /ml (kim et al., 2012; yang and boo, 2013), other fruits required much higher doses to obtain good activities. for example, trifoliate orange and kiwi fruits showed 1.9-11.5 % and 6.8-14.4 % inhibition with doses of 2 mg/ml (lee et al., 2010; park et al., 2008), while loquat fruits showed 16 % inhibition at a dose of 4 mg/ml (park et al., 2008). cell viability and inhibition of melanin synthesis we measured the cytotoxicity of the pear extracts for 10-500 μg/ml dosages. as shown in tab. 3, the five pear extracts showed relatively low cytotoxicity, with cell viabilities of 78.6 + 6.74 98.8 + 10.36 % for the 500 μg/ml sample. this low cytotoxicity shows promise for possible clinical usefulness. fig. 1: the inhibition of mushroom tyrosinase in vitro among different pear cultivar extracts. results are means + sd (n=3). the inhibitory effects of five pears cultivars on tyrosinase were measured at three concentrations: 10, 50, and 100 μg/ml. tab. 3: cell viability and melanin systhesis inhibition by pear ethanol extracts. cultivars dose μg/ml melanin content* 10 50 100 500 % gamcheonbae 96.9 ± 1.61 a z 92.9 ± 6.10 a 90.4 ± 3.37 ab 88.8 ± 9.12 ab 62.9 ± 7.51 a manpungbae 90.9 ± 3.60 a 93.4 ± 13.49 a 93.5 ± 7.17 a 98.8 ± 10.36 a 16.9 ± 6.42 b chuwhangbae 90.3 ± 3.82 a 81.5 ± 5.59 a 77.6 ± 12.27 b 78.6 ± 6.74 b 25.8 ± 8.96 b hanareum 81.6 ± 6.03 b 85.8 ± 7.26 a 85.9 ± 4.99 ab 92.7 ± 7.45 ab 63.4 ± 11.23 a niitaka 95.2 ± 6.70 a 91.6 ± 9.14 a 86.1 ± 7.66 ab 81.8 ± 8.80 b 58.9 ± 9.00 a z different letters within columns indicate significant differences of the means, as determined by duncan’s multiple range test at p < 0.05. * inhibition of melanin synthesis was expressed as the percentage of melanin content in the cells treated with pear extract (100 μg/ml) to that of untreated melanoma cells (64.8 ± 9.03 % melanin content) 190 s.-h. yim, s.-h. nam to investigate the effects of the five pear extracts on melanogenesis, b16f10 mouse melanoma cells were treated with pear extracts (100 μg/ml) and compared with untreated controls. the acid in soluble fraction was prepared from the cells, and the amount of melanin was quantitatively measured by the method of makpol et al. (2009). the cellular melanin content was reduced by the addition of pear extract to the medium, as shown in tab. 3. melanin syn-3. melanin syn-. melanin synthesis was significantly inhibited by manpungbae (16.9 + 6.42 %), and chuwhangbae (25.8 + 8.96 %), displaying 74 % and 60 % reduction, respectively, compared to the levels in the non-treated cells (64.8 + 9.03 %). the gamcheonbae, hanareum, and niitaka pear extracts showed only slightly lower content of cellular melanin compared to the control, ranging from 58.9 + 9.00 % to 63.4 + 11.23 %, failing to meet significance at p < 0.05. thus, these results indicate that the pear extracts had an inhibitory effect on melanogenesis at non-cytotoxic concentrations (100 μg/ml). among the five cultivars (tab. 1-3), arbutin content and whitening effects were the high-3), arbutin content and whitening effects were the high-), arbutin content and whitening effects were the highest in manpungbae, but the lowest in gamcheonbae. here, it can be suggested that gamcheonbae may possess different functional compounds rather than those involved in whitening activity, since it showed relatively high total phenolics and flavonoid contents (tab. 1-3). although many papers have studied the inhibitory effects of various fruit or plant extracts on melanin synthesis, the extracts from unripe pear in this study showed a more prominent reduction of the melanin content, with 74 % inhibition, compared to the untreated control at a concentration of 100 μg/ml. kojic acid, as a strong whitening agent, showed only 44.7 % reduction of the melanin formation at 100 μg/ ml (kim et al., 1998). hippophae rhamnoides l. fruit and ginseng extract showed 46 % and 27 % decreased melanin synthesis, respectively, compared to controls at the same concentration (ko et al., 2012; hwang and choi, 2006). extracts of persimmon leaves and gardenia fruit had similar effects, with 46.7 % and 55.9 % reduced melanin formation, respectively, by high doses of 10 mg/ml (cho et al., 2011b; kwak et al., 2004). taken together, the test findings indicate that pear extract efficiently inhibited tyrosinase and melanin biosynthesis in mouse melanoma cells, suggesting use as a possible natural whitening additive. conclusions the findings of this study could be summarized as follows: 1. five unripe pear cultivars were measured for total phenolics and arbutin content, and further characterized with respect to antioxidant activities, and inhibitory effects on tyrosinase activity and cellular melanin formation. 2. cultivars included manpungbae, gamcheonbae, chuwhangbae, hanareum and niitaka, which were harvested 30 days after florescence. 3. total phenolic and flavonoid contents of the five pear cultivars ranged from 255 + 2.64 to 367 + 9.78 mg, and from 26.5 + 0.82 to 36.6 + 1.45 mg per 100 g fresh weight, respectively. among the five cultivars tested, gamcheonbae and manpungbae had significantly higher total phenolics and flavonoid contents, while niitaka had lower total phenolics and flavonoids. 4. quantification of the arbutin content by hplc/dad analysis revealed manpungbae to have the highest levels (53.9 + 0.72 mg/ 100 g fw), while chuwhangbae and gamcheonbae had only half as much. 5. with regard to the antioxidant capability by abts or dpph radical scavenging, nikita showed lower activity (65 % or 77 %), while manpungbae was the highest (74 % or 83 %). 6. to examine whitening activity, pear extracts of five cultivars were tested for inhibitory effects on tyrosinase in vitro, and on cellular melanin formation. manpungbae showed the strongest tyrosinase inhibition, at 4.9 ± 0.7 %. the pear extracts showed relatively low cytotoxicity, with cell viability ranging from 79~96 % at concentrations of 500 μg/ml. melanin formation (pears extract of 100 μg/ml) was significantly inhibited by manpungbae (16.9 %), and chuwhangbae (25.8 %), resulting in 74 % and 60 % reduction compared to the level in non-treated cells (64.8 %). 7. these accessions 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agrisciences, czech university of life sciences prague, czech republic ethnobotanical review of wild edible plants used in the czech republic k. simkova1, z. polesny1* (received october 15, 2014) * corresponding author summary this paper is the literature survey of wild food plants used within the present borders of the czech republic. thirty-seven freely available publications documenting the culinary use of wild plants were examined. the use of 175 vascular plant species (approximately 5% of native and naturalized flora of the czech republic), 3 lichens and 1 bryophyte has been reported. for each species listed, plant parts used, use category and mode of consumption are given. rosaceae, asteraceae and ericaceae were the most represented botanical families. the most frequently reported categories of plant uses include green vegetables (e.g. urtica dioica, glechoma hederacea, rumex spp., taraxacum sect. ruderalia), seasonings (e.g. juniperus communis, viola spp.), wild fruits (e.g. rubus idaeus, rosa canina), and beverages (e.g. cornus mas, sambucus nigra). the structure of the most commonly used wild food taxa is similar to those used in other central european countries like poland, slovakia or hungary. this review highlights the traditional knowledge of wild edible plants which were used in the czech republic since 16th century onwards with an attempt to document diversity of plant species and discuss the current potential of the forgotten plants used in the past. introduction the czech republic is small landlocked country in central europe with area 78,866 km2 and some 10.5 million inhabitants. its boundaries 2,290 km in length are surrounded by very heterogeneous landscape. bohemia, the western part, consists of a basin drained by the labe (elbe) and the vltava (moldau) rivers. moravia, the eastern part of the country, is drained mainly by the morava and odra (oder) rivers. both parts are largely surrounded by low mountains. the climate in the czech republic is mild and transient between oceanic and continental with continental character of the climate increasing to the east, due to prevailing western air flow and position towards the atlantic ocean. a characteristic feature of the climate is distinctly marked by regular alternation of four seasons. czech territory belongs to the central european region, the intersection of current spread of plant species, which implies a great diversity of nature. because of the rugged topography, the country covers a variety of biotopes within relatively small area. wild plant species have been collected by people for various purposes such as food, medicine and social uses. it is a challenge for ethnobotanists who try to gather and record this knowledge. the collected data are important from a cultural perspective as they conserve traditional wisdom and also because of the increasing interest in the use of wild edible plants in our postmodern society. during the last two decades, growing interest in wild edible plants has led to many local ethnobotanical studies carried out in european countries to preserve the traditions of wild food use. such studies were performed for example in poland (łuczaj, 2008; łuczaj, 2010a; łuczaj and szymański, 2007), slovakia (łuczaj, 2012), on the iberian peninsula (tardío et al., 2006; bonet and vallès, 2002; tardío et al., 2005; pardo-de-santayana et al., 2005; tardío and pardo-de-santayana, 2008), in italy (guarrera et al., 2006; nebel et al., 2006; leonti et al., 2006; pieroni et al., 2005), estonia (kalle and sõukand, 2012), bosnia and herzegovina (redzic, 2006), hungary (dénes et al., 2012), sweden (svanberg, 2009) and cyprus (della et al., 2006). these studies have shown that the continent has a rich and varied culture associated with the gastronomic use of wild plants. although the czech republic is not poorer in traditional use of wild plants compared with other parts of europe, no such comprehensive review has been undertaken yet. the authors believe that this review could extend the scientific knowledge on traditional use of wild food plants in europe previously summarized in the paper of łuczaj et al. (2012). this literature survey cover wild plant species used for food including beverages with the exception of plants used in the form of the herbal infusions or decoctions, which were drunk almost exclusively for medicinal purposes. materials and methods thirty-seven publications dealing with czech food history, gastronomy, ethnography and botany were surveyed. all information summarized in this review refers to use of wild edible plants within the boundaries of recent czech republic, based on literature sources providing relevant information since the beginning of the modern period (16th century) onwards. for data collection and analysis we have used combination of methods applied in similar previous studies of tardío et al. (2006), łuczaj and szymański, 2007, łuczaj (2010a), and kalle and sõukand (2012). literature sources surveyed are listed in the tab. 1. the same sources, indicated with a reference numbers (rn), are also referred in the tab. 2. for each publication, main topic, geographical area, and number of plant species reported are given. most of the sources cover the whole country, except for ten publications with regional focus. among those, nine sources cover bohemian regions (rn 8, 9, 10, 14, 18, 19, 33, 37, 23), and one source was focussed on east moravia (26). additionally, one book (7) includes whole european area. all records of using any parts of plant species as food or drink were considered excluding species used for preparation of herbal teas having exclusively medicinal applications (korbelář and endris, 1985). all data were grouped into alphabetically sorted botanical families (tab. 2), where latin name, standard czech name, folk name(s), plant part(s) used, use category, number of reports, mode of use and reference number(s) are provided. contrary to tardío et al. (2006) information on collecting season were not included as it was rarely referred in the sources studied and the information obtained usually falls into 3 categories, i.e. green plant parts collected in spring (march june), fruits collected in their ripening time (july october), and underground parts collected mostly in both seasons mentioned (łuczaj and szymański, 2007). in the present study one report was considered one mention of a species use in particular food category and literature source. 50 k. simkova, z. polesny definition of “wild species” being considered in this study in compliance with similar studies (tardío et al., 2006; łuczaj and szymański 2007; kalle and sõukand, 2012) the term “wild plants” in this review refers to non-cultivated species gathered in the field without intended cultivation, including alien spontaneously occurring taxa. in accordance with the methodical approach of tardío et al. (2006) and kalle and sõukand (2012), plants which wild forms are sometimes cultivated (corylus avellana l., rubus idaeus l.), species which parts reported in the sources surveyed are usually not eaten (humulus lupulus l.), as well as the species cultivated solely for non-food purposes (e.g. aesculus hippocastanum l.) were considered because they are known to be feral and they could be gathered from the wild. use categories considering previous studies of tardío et al. (2006) and łuczaj (2012), eight main categories of food uses were established to classify wild edible plants in this review. plant species whose aerial parts like leaves and shoots were contab. 1: references consulted, with their reference number (rn), main topic, and number of species from each source included in the database. rn reference cited main topic research area no. of species reported 1 beranová (1997) food history whole country 9 2 beranová (2001) cookbook whole country 24 3 beranová (2005) food history whole country 77 4 fialová (1958) food history whole country 5 5 fialová (1989) cookbook whole country 9 6 fialová and styblíková (1983) cookbook whole country 14 7 gumowska (1994) cookbook europe 2 8 hajný (1912) ethnographic (food) nymburk 2 9 jakouběová (2000) cookbook central bohemia 10 10 jakouběová (2009) ethnographic (folklore) central bohemia 24 11 janalík and marhold (2003) cookbook whole country 7 12 janča and zentrich (1994-1999) herbarium (medical) whole country 11 13 janků-sandtnerová and janků (2007) cookbook whole country 10 14 kaizl (1944) ethnographic (food) east bohemia 19 15 karlík (2007) cookbook whole country 13 16 kubátová (2003) ethnographic (food) whole country 1 17 lánská (1990) edible plant guide whole country 35 18 marhold (2008) cookbook east bohemia 28 19 novotná et al. (2005) ethnographic (food) south bohemia 5 20 polívka (1900-1904) plant encyclopaedia whole country 59 21 rettigová (2005) cookbook whole country 8 22 rodovský z hustiřan (1975) cookbook whole country 10 23 roubal (1902) ethnographic (plants) west bohemia 2 24 rozmarová (1938) cookbook whole country 18 25 skopová (2009) cookbook whole country 12 26 štika (1980) ethnographic (food) east moravia 18 27 trachtová (1902) cookbook whole country 14 28 triwaldová (1909) cookbook whole country 8 29 úlehlová-tilschová (1937) healthy nutrition whole country 18 30 úlehlová-tilschová and goldhammer (1970) ethnographic (food) whole country 14 31 úlehlová-tilschová (2011) ethnographic (food) whole country 66 32 úlehlová-tilschová (2000) cookbook whole country 17 33 vrabec and smotlacha (1982) cookbook south bohemia 5 34 winter (1892) food history whole country 79 35 zíbrt (2000) food history whole country 24 36 zíbrt (2012) food history whole country 26 37 zuntová (2005) ethographic (food) south bohemia 2 wild food plants of the czech republic 51 sumed raw, boiled or fried were placed in the category green vegetables (veg). fruits eaten raw or preserved in the form of jams and jellies were categorized as wild fruits (fru). plants whose bulbs, rhizomes, roots, and tubers, consumed raw as a snack or added to boiled dishes were placed into the category of subterranean parts (sub). seasoning (sea) category covered plants added in small amounts to dishes. flowers and their nectar used as a snack or added to dishes in larger quantities were placed in the category of flowers (flo). plants used for making non-alcoholic beverages (bev), home-made liqueurs, beers and other alcoholic beverages (bevliq) or used as a coffee and cacao substitutes (bevoth) were also considered. plants with use as preservative additives or rennet substitutes were included in preservatives (pre). finally, there were categories for other uses such as oils (othoil), flours (othflo) and non-specified use (oth) considering making vinegar or honey. plant species identification in the literature sources containing data on wild edible plants, the species were mostly reported under their folk names (only in 3 reference sources latin names of plants were given). no herbarium specimens in the cited works were available to verify the proper taxonomic identification. nevertheless, according to łuczaj and szymański (2007), we tried to validate the identification using generally available floras and plant guides (polívka, 1900-1904; kubát, 2002; skalická et al., 2012, rystonová, 2007). accordingly, a list of taxa was created using a modified code for credibility of identification previously defined by łuczaj (2010b). in case of plant record did not allow for taxonomic identification down to species level, even though it comprises two or more very common species, a taxon was identified down to genus level. in the consequence, only commonest species of such genus occurring in the czech republic, which we personally witnessed being collected, have been mentioned in the results. in case when plant identification credibility was very low or the taxon was impossible to determine, according to kalle and sõukand (2012) such record was left aside and it was not included in the plant list provided in the tab. 2. latin plant names and authority were adjusted according to ‘flora europaea’ database (http://rbg-web2. rbge.org.uk/fe/fe.html.) and cross-checked in the ‘tropicos’ − botanical information system of the missouri botanical garden (www. tropicos.org). czech standard names were adopted from the current checklist of vascular plants of the czech republic (kubát, 2002; danihelka et al., 2012) and the flora of the czech republic. results in total 179 wild edible plants used for various food purposes were documented in the czech lands since the 16th century (tab. 2). they include 175 vascular plant species (approximately 5 % of native and naturalized flora of the czech republic), 3 lichens, and 1 bryophyte. eighty-three per cent of the plants reported in this study were classified according to danihelka et al. (2012) as autochthonous and seventeen per cent as allochthonous species. from the total number, 150 plants were identified down to species level. twenty-nine plants could only be identified down to genus level. fig. 1: botanical families with the greater number of species cited for the major categories. 52 k. simkova, z. polesny all species belong to 57 botanical families, most represented by asteraceae (19 species), rosaceae (14) and brassicaceae (11). the fig. 1 shows botanical families with the greater number of species cited for the major categories. the reported species are consumed in a variety of ways (eaten raw, cooked or fried, ground into flour or pressed into oil). considering all food categories, the most important species according to the number of reports were: rubus idaeus (52), sambucus nigra l. (44), rosa canina l. (38), junniperus communis l. (33), vaccinium myrtillus l. (30), viola spp. (29), vaccinium vitisidaea l. (26), urtica dioica l. (25), fragaria vesca l. and rumex spp. (22 each). some species were included in more than one category such as sambucus nigra, fragaria vesca and viola spp. which were classified in 5 categories. therefore, the total number of plants and their related uses was 284, higher than the number of species (179). seven per cent of all plants across all food categories were reported as children’s snacks, including mostly species already mentioned in the category of fruits. the use of some wild plants as children’s snack food could be a relic of general use of these plants by adults when they were young. additionally we would like to mention that 7 % of all use reports are considered as preparation during time of famine. green vegetables green vegetables including edible weeds constitute the largest category with 74 species recorded. it includes plants whose green parts such as leaves, stems and stolons are eaten raw or after special preparation (cooking, stewing and frying) excluding seasonings. among species in this category urtica dioica (urticaceae) shows the highest number of reports (25), even though the most represented families were asteraceae (12 species), brassicaceae (10) and polygonaceae (5). beside urtica dioica, also glechoma hederacea l. (19 reports), rumex spp. (16), taraxacum sect. ruderalia (10) and atriplex spp. (10) were frequently reported species. glechoma hederacea, known under the folk name kundrlátek was used for making soups, vegetable dishes or added to scrambled eggs. plants of the genus rumex were consumed raw or used as potherb or they were chewed frequently by children and shepherds against thirst. lepidium spp. leaves were used occasionally as filling for traditional czech doughnuts. wild fruits excluding species used solely as a seasoning, twenty-six species were recorded in this category. more than one third of these species belongs to the family rosaceae (10 species) and ericaceae (4 species). most wild fruits of these families complete with species sambucus nigra (16 reports) and rubus caesius l. (14) were consumed as a snack or made into preserves (jams, jellies, compotes) and were consumed especially during the winter months. fruits of corylus avellana were added to pastry and confectionary. sweet sauce called žahúr made of vaccinium myrtillus fruits mixed with milk and honey (stoličná, 1997) was used as topping for dumplings or crumpets. beverages this category covers 3 subcategories, alcoholic beverages (13% of all use reports), non-alcoholic (8 %) and others (2 %). most plants in this category are fruit species. the most remarkable species for making beverages are rubus idaeus (16 reports), fragaria vesca (10), cornus mas (9) and rosa canina (9), largely prepared as juices or alcoholic beverages. flowers of taraxacum sect. ruderalia and bellis perennis as well as rhizomes of acorus calamus were used for making syrups. fruits of juniperus communis were used to make the distillate jalovcová which taste resembles slovak borovička. brewing has been very popular in the czech republic since ancient times. for homemade beers, people occasionally used hop substitutes from wild plants such as rhizomes of acorus calamus and geum urbanum l., leaves of salvia pratensis l. and tanaceum vulgare l. and flowers of origanum vulgare l. coffee or cocoa substitutes were used mostly in times of increasing prices of these exotic commodities during 18th and 19th centuries. most popular species used for this purpose was cichorium intybus l., whose roasted and ground roots were used as coffee substitute. roots of scorzonera hispanica l. were used in the same way as well as seeds of rosa canina and astragalus glycyphyllos l., fruits of quercus robur and unspecified parts of vicia sativa l. (most probably seeds). seasonings out of 31 wild plants used as seasoning juniperus communis (24 reports) and viola odorata, were the most frequently reported species. berry-like cones of j. communis were mostly used for seasoning game. very popular were seasoning spring soups made of the leaves of viola spp., fragaria vesca, achillea ptarmica and vaccinium myrtillus. flowers this category was recorded for 11 species. sambucus nigra (16 reports) was most frequently reported species. its fried dough coated inflorescences was typical rural spring dish called kosmatice. interestingly, flowers of robinia pseudoacacia and trifolium spp. were used in the same way but they were not as popular as sambucus nigra. viola and centaurea genera were used for colouring foodstuffs in blue (e.g. pudding) or decorating easter eggs. candied flowers, usually of viola and centaurea species or bellis perennis, were preserved by a coating with crystallised sugar. flower receptacles or flower buds from carlina acaulis l., fagus sylvatica, betula pendula, tilia spp., and populus spp. (probably p. alba l. or p. tremula l.) were eaten raw or boiled in vegetable dishes. underground parts the use of underground plant parts (roots, rhizomes, bulbs and tubers) was recorded for 30 species. however, the frequency of citation for individual species did not exceed 4 reports. acorus calamus rhizomes were preserved with sugar. roots of campanula rapunculoides l., phyteuma spicatum l. were eaten raw in fresh salads. rhizomes of arum maculatum l. and tubers of chaerophyllum bulbosum were used in boiled dishes. bulbs of galanthus nivalis l. and leucojum vernum l. were usually dried and ground into porridges. underground parts of scorzonera hispanica, cyperus esculentus l., onopordum acanthium l., chenopodium bonus-henricus l., sagittaria sagittifolia l. and of several species of the genus arctium (a. tomentosum mill., a. lappa l. and a. minus bernh.) were used for preparation of salads and other vegetable dishes. preservatives a few species were identified to be used in this category. fruiting branches of juniperus communis were used to preserve game. except for j. communis, all other species in this category, i.e. fragaria vesca, galium verum, rumex spp. and urtica dioica (leaves), and fruits of corylus avellana and quercus robur were used as rennet substitute. wild food plants of the czech republic 53 other uses this category includes oils, flours, vinegars and ‘honey’. the term ‘honey’ in the present study refers to home-made boiled down herbal syrup. honey was traditionally prepared from flowers of hypericum spp. (most probably hypericum perforatum l.) and taraxacum sect. ruderalia. the oil was extracted from fruits of corylus avellana, fagus sylvatica and quercus robur and from the seeds of alliaria petiolata (m. bieb.) cavara et grande, prunus spinosa and sambucus nigra. the family of the poaceae was best represented with 6 species. seeds of digitaria sanguinalis (l.) scop, glyceria fluitans (l.) r. br., echinochloa crus-galli (l.) p. b., setaria viridis (l.) p. b. subsp. viridis and milium effusum l., as well as rhizomes of elymus repens (l.) gould were ground into flours. other species, whose many different parts as rhizomes (pteridium aquilinum (l.) kuhn, arum maculatum, calla palustris l.), bulbs (leucojum vernum, galanthus nivalis), leaves (urtica dioica), inflorescences (narrow spikes of typha latifolia l.) flowers (flower buds of quercus robur, trifolium spp.), fruits (quercus robur, aesculus hippocastanum, trapa natans), seeds (amaranthus retroflexus, persicaria spp., fallopia convolvulus (l.) á. löve) and even inner bark of betula pendula, were used particularly in times of famine. they were mixed with cereal flour to make bread dough. only exceptionally bread dough was prepared solely by using flours made from wild plant species. sometimes for these purposes were also used non-vascular plants as lichens cladonia rangiferina (l.) weber ex f. h. wigg., cetraria islandica (l.) ach. and evernia prunastri (l.) ach. and bryophyte sphagnum palustre l. six species (artemisia vulgaris, betula pendula, prunus spinosa, rubus idaeus, viola spp. and salix spp.) were used to make a vinegar. discussion comparison with other countries the presented list of wild edible plants includes 175 vascular species (5 % of the czech flora). in comparison with poland, which was classified as herbophobic country with 112 species recorded (3.7 % of polish flora) (łuczaj, 2008), or with 106 species documented in slovakia (3 % of slovak flora; łuczaj, 2012), our study indicates that the czech people could be considered herbophylic. unfortunately, it was not possible to compare the use of wild food plants in the czech republic with other neighbouring countries, i.e. austria and germany, as they lack similar ethnobotanical reviews. comparing our study with south european countries, czech republic should be classified as herbophobic. for example, only in alt empordà region (catalonia, spain) with the area of 1,358 km2 parada et al. (2011) found 211 wild food plants. lentini and venza (2007) recognized in sicily, an island 3 times smaller than the czech republic, 188 wild edible species (6.2 % of the flora). in bosnia-herzegovina, smaller country than the czech republic was listed around 10% of flora (redzic, 2006). summarizing our results the czech republic should be classified as herbophobic. according to łuczaj and szymański (2007) two reasons could be responsible for this contrast. one factor is that czech flora is poorer than in mediterranean countries, thus the choice of species is poorer as well. czech flora has 3,557 species, compared to around 6,700 species in italy (conti et al., 2005) or 7,000 in spain (tardío et al., 2005). on the other hand in two small regions in cyprus, with a flora around 2,000 species, the use of 78 species as wild food plants was recorded (della et al., 2006). therefore, this indicates higher significance of culinary habits, the other factor. in the mediterranean basin, people use wild plants mainly as appetizers, spices or ingredients of omelettes, salads and beverages. in countries as the czech republic, slovakia or poland people use wild plants more as staple food (guarrera et al., 2006; leonti et al., 2006; tardío et al., 2005). another explanation is that in central european countries vegetable could be easily cultivated because of large proportion of arable land. the mountainous countries of the mediterranean basin are mostly covered by semi-arid pastures. therefore, cultivation of field crops is more difficult and wild plants were used instead of cultivated vegetable (łuczaj, 2008). the use of wild fruits of rosaceae and ericaceae families is nearly identical in all european countries (dénes et al., 2012; kalle and sõukand, 2012; łuczaj, 2012; łuczaj and szymański, 2007; svanberg, 2009; tardío et al., 2006). with decreasing price of sugar in the early 20th century, european people have found enthusiasm in collecting wild fruits and turning them into jams or pasteurized compotes and it became a part of everyday cuisine (łuczaj, 2012). the proportion of families in other food categories in the czech republic context it is the most similar to slovakia, poland and spain (łuczaj, 2012; łuczaj and szymański, 2007; tardío et al., 2006). if we focus on non-vascular plants as cetraria islandica and cladonia spp., both were used in estonia and in bosnia and herzegovina. analogous to this study, there was report of using them for making bread ingredient during times of famine (kalle and sõukand, 2012; redzic et al., 2010). antinutritive properties and toxicity some of the wild plans consumed commonly in the czech republic might have some toxic effects as shown in the following examples. the most poisonous plants could be considered bryonia dioica jacq. as its roots contain bryodin, a ribosome-inactivating protein, which inhibits protein synthesis (stirpe et al., 1986). special attention is also paid to salix spp., as it contains salicylates (ruuhola et al., 2003), considered by many people as a natural source of aspirin. therefore people with known aspirin allergy should not use any willow bark products (vlachojannis et al., 2011). chenopodium spp., rumex spp. (guil et al., 1996), polygonum spp., oxalis acetosella (krčálová, 2009) and persicaria spp. (keshavarzi and mosaferi, 2012) contains oxalic acid, which gives plants their acrid flavour. this chemical agent in large quantities can lock up some of the nutrients in the food. cooking the plant will reduce its content of oxalic acid (savage et al., 2000). in addition, people with kidney disease predisposition should be aware of including these plants in their diet (palaniswamy et al., 2004). pregnant women should avoid artemisia vulgaris, levisticum officinale, tanacetum vulgare, berberis vulgaris, capsella bursa-pastoris, cichorium intybus, mentha spp. (ernst, 2002), glechoma hederacea (pfaf, 2013) and humulus lupulus (zanoli and zavatti, 2008) as they may stimulate the uterus to contract and induce abortion. problems with liver damage or liver cancer could be associated with consumption of borago officinalis (dodson and stermitz, 1986), symphytum officinale l. (ernst, 2002), senecio vulgaris l. (cao et al., 2008) and echium vulgare (el-shazly et al., 1999) as they contain toxic pyrrolizidine alkaloids which can have a cumulative effect upon the liver (prakash et al., 1999). in case of pteridium aquilinum, there are a lot of reports connect with the possible health risks. the huge quantity of spores released by large areas is suggested to be implicated in stomach cancers (rasmussen et al., 2013). also substance (thermolabile thiaminase) in the leaves of bracken deprives the body of vitamin b1 (vetter, 2009). on the other hand this substance could be damaged by cooking. seeds of prunus spinosa produce cyanogenic glycosides (kumarasamy et al., 2003), which is readily detected by its bitter taste. however, when cyanogenic plants are eaten slowly or over a period of time there may be no harmful effect of cyanide poisoning (jones, 1998). furocoumarins in the most of the plants of the family apiaceae as angelica spp. (skopalová, 2008), daucus carota, pastinaca sativa, heracleum sphondylium l. and the genus hypericum (pathak et al., 1962) cause phytophoto54 k. simkova, z. polesny dermatitis so their consumption increase skin sensitivity to sunlight. among other poisonous species could be mentioned fresh roots of acorus calamus (bjornstad et al., 2009) because surface coats calcium oxalate crystals, microscopic double needles. when plants are eaten fresh, crystals cause unpleasant sensation like formication sense in mouth, tongue and throat (paull et al., 1999). solanum dulcamara overdose may paralyse the central nervous system, slow heart, low temperate, dilated pupils, delirium and even death (smith et al., 2008). bulbs of galanthus nivalis (berkov et al., 2008) and leucojum vernum (forgo and hohmann, 2005) are source of toxic alkaloids. must not be forgotten species which have edible parts but other parts of plant are poisonous as sambucus nigra bark containing nigrin b, a two-chain ribosome-inactivating protein (battelli et al., 1997), berberis vulgaris with toxic bark, robinia pseudoacacia which only wholesome parts are flowers or ribes uva-crispa with toxic leaves (pfaf, 2013). to sum up, a lot of more or less toxic species exist amongst the wild edibles. fortunately, most of toxic agents could be destroyed by cooking or drying as mentioned above, nonetheless we must be careful about the quantities (tardío et al., 2006). according to rivera-núñez and obón de castro (1993) wild food plants play a major role as the preventive medications through nutritional habit considered as healthful (rightly or not) and a significant part in the healing repertory. future perspective of wild food plants in humankind history culinary habits were never static. during the communist era, czech people were focused mostly on collecting wild fruits. nowadays during the last few years we can observe a slow revival in the use of other wild plants. for example in many health food shops now we can find alternatives to coffee from cichorium intybus or from acorns. until today a few species remain as a common ingredient in household kitchens, mostly seasoning as seeds of carum carvi and juniper ‘berries’ (juniperus communis). interestingly, recently consumption of wild plants is being enlarged by people living in cities (bonet and vallès, 2002). in croatia, several respondents mentioned that there is a demand for vegetable mixtures of wild plants by young health-oriented people like vegetarians etc. (łuczaj et al., 2013). therefore, modern agriculture should turn profit and through agricultural and rural development policies encourage the conception of profit activities, such as the controlled harvesting of weedy herbs. also they should start with re-introduction of old and archaic crops and start development of agroand eco-tourism and farmer’s market (turner et al., 2011). recently, particular studies have been elaborated with the aim to use wild species as vegetable crops, i.e. silene vulgaris (moench) garcke and sinapis arvensis (sojka, 2012), taraxacum sect. ruderalia and urtica dioica (ptáček, 2011), all considered wild plants in this paper. this aspect has been seriously considered by fao (2009) stating that “nutrition and biodiversity converge to a common path leading to food security and sustainable development” and “wild species and intraspecies biodiversity have key roles in global nutrition security.” according to this report, around one billion people use wild foods (including wild animals) in their diet. it is obvious that wild foods form a significant part of the global food basket. conclusion the study shows that there is an urgent need for conserving traditional knowledge on wild edible plants as in the czech republic many of wild plants were used. however, from the wide spectrum of wild plant products documented by our research, people still collect mainly wild fruit species, and especially mushrooms, which were not included in this study. recently, the traditional wisdom only survives in the memories of the elderly, thus it is in danger of disappearing. therefore, there is still need for further ethnobotanical research in the ethnographic archives as well as in the field to identify whole spectrum of wild gathered edible plants. this review attempts to analyze the information from literature sources to complete data on trato complete data on tra-complete data on traditional use of wild food plants in europe and thus promote further research on forgotten useful plants as potential new food sources. acknowledgement this research was supported by the czech science foundation (grant number 521/09/p589) and the internal grant agency of the faculty of tropical agrisciences of the czech university of life sciences prague (grant numbers iga ftz 20135122 and iga ftz 20145025). references battelli, m.g., citores, l., buonamici, l., ferreras, j.m., de benito, f.m., stirpe, f., girbés, t., 1997: toxicity and cytotoxicity of nigrin b, a two-chain ribosome-inactivating protein from 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(eds.). praha, dauphin. zuntová, a., 2005: co se vařilo před šedesáti lety. in: šafr, p., taureová, i. (eds.). jak se dříve vařívalo. české budějovice, jihočeské museum. address of the corresponding author: department of crop sciences and agroforestry, faculty of tropical agrisciences, czech university of life sciences prague, kamycka 129, prague 6-suchdol, 16521, czech republic; e-mail: polesny@ftz.czu.cz wild food plants of the czech republic 57 ta b. 2 : l is t o f w ild p la nt s pe ci es u se d fo r f oo d pu rp os es in th e c ze ch r ep ub lic . r ef er en ce n um be rs a re g iv en in th e ta b. 1 . f am ily a nd s pe ci es st an da rd c ze ch n am e c r. * f ol k na m e p ar ts u se d † u se c at eg or ie s ‡ m od e of u se r ef er en ce s (n o. o f r ep or ts ) l ic h e n s c l a d o n ia c e a e c la do ni a ra ng ife ri na (l .) du to hl áv ka s ob í o so bí li še jn ík th al lu s o t h flo (1 ) gr ou nd in to fl ou r [3 5] w eb er e x f. h . w ig g. du ri ng fa m in e pa r m e l ia c e a e c et ra ri a is la nd ic a (l .) a ch . pu kl éř ka is la nd sk á o liš ej ní k th al lu s o t h flo (3 ) gr ou nd in to fl ou r [3 , 1 4, 3 5] du ri ng fa m in e e ve rn ia p ru na st ri (l .) a ch . vě tv ič ní k sl ív ov ý n m ec h tr nk ov ý th al lu s o t h flo (1 ) gr ou nd in to fl ou r [3 5] du ri ng fa m in e b r y o p h y t e s sp h a g n a c e a e sp ha gn um p al us tr e l . ra še lin ík č lu nk ol is tý o th al lu s o t h flo (2 ) gr ou nd in to fl ou r [3 , 3 5] du ri ng fa m in e va sc u l a r p l a n t s a d o x a c e a e sa m bu cu s ni gr a l . be z če rn ý o be zi nk y in flo re sc en ce fl o (1 6) fr ie d do ug ht c oa te d flo w er s [1 , 2 , 3 , 1 5, 1 7, 1 9, 2 2, 2 4, 2 5, or a dd ed to p or ri dg es 29 , 3 0, 3 1, 3 2, 3 4, 3 5, 3 6] fr ui t fr u (1 6) so up s, c hu tn ey s, fo r m ak in g [4 , 5 , 6 , 8 , 1 0, 1 7, 2 0, 2 2, 2 4, ja m s/ je lli es 27 , 2 8, 2 9, 3 0, 3 1, 3 2, 3 6] b e v (7 ) ju ic es [2 , 6 , 1 4, 1 7, 2 4, 2 5, 3 2] se ed o t h oi l ( 1) pr es se d in to o il [3 ] a c o r a c e a e a co ru s ca la m us l . pu šk vo re c ob ec ný o pi ši šv or rh iz om e se a (4 ) n s [3 , 9 , 1 7, 3 6] su b (3 ) rh iz om es p re se rv ed w ith s ug ar [2 0, 3 1, 3 6] b e v (1 ) sy ru ps [3 6] b e v liq (6 ) us ed fo r m ak in g sp ir its o r [2 , 1 0, 1 7, 2 0, 2 4, 3 6] ad de d in to b ee rs a l is m a ta c e a e sa gi tta ri a sa gi tti fo lia l . ší pa tk a st ře lo lis tá o ča pí c ap a rh iz om e su b (1 ) us ed in v eg et ab le d is he s [2 0] a m a r a n t h a c e a e a m ar an th us b lit um l . la sk av ec h ru bo ze l o bl ít le af v e g (2 ) st ea m ed le av es in v eg et ab le [2 0, 3 1] di sh es a m ar at hu s re tr ofl ex us l . la sk av ec o hn ut ý o am ar an t se ed o t h flo (3 ) gr ou nd in to fl ou r [3 ] du ri ng fa m in e a tr ip le x pa tu la l . le be da ro zk la di tá o šp en át le af v e g (2 ) ra w in s al ad s [2 0, 2 3] a tr ip le x sp p. le be da o šp en át le af v e g (1 0) so up s, s te am ed in v eg et ab le [2 , 3 , 1 4, 1 5, 1 8, 2 4, 2 9, 3 1, di sh es 32 , 3 6] c he no po di um a lb um l . m er lík b ílý o le be dn ík le af v e g (1 ) ra w in s al ad s [2 0] c he no po di um b on us -h en ri cu s l . m er lík v še do br o ch ře st ov ý šp en át le af v e g (3 ) ve ge ta bl e di sh es [1 2, 2 0, 3 5] 58 k. simkova, z. polesny a m a r y l l id a c e a e a lli um s ch oe no pr as um l . pa ži tk a po bř ež ní o šn itl ík st em se a (1 ) n s [1 7] a lli um u rs in um l . če sn ek m ed vě dí o di vo ký č es ne k w ho le p la nt se a (3 ) n s [3 , 1 7, 3 1] g al an th us n iv al is l . sn ěž en ka p od sn ěž ní k o sn ěh ov ka bu lb o t h flo (1 ) gr ou nd in to fl ou r d ur in g [3 5] fa m in e su b (1 ) dr ie d an d gr ou nd in to p or ri dg es [1 4] le uc oj um v er nu m l . bl ed ul e ja rn í o bl ed ni vk a bu lb o t h flo (1 ) dr ie d an d gr ou nd in to fl ou r [3 5] du ri ng fa m in e su b (1 ) dr ie d an d gr ou nd in to p or ri dg es [1 4] a pi a c e a e a eg op od iu m p od ag ra ri a l . br šl ic e ko zí n oh a o , n br zl ic e, ja ro us le af v e g (5 ) ra w o r s te am ed [3 , 1 2, 2 0, 3 1, 3 5] a ng el ic a ar ch an ge lic a l . an dě lik a lé ka řs ká o dě he l a nd ěl ik a le af se a (1 ) n s [3 ] b er ul a er ec ta (h ud s. ) c ov ill e po to čn ík v zp ří m en ý n be rl a úz ko lis tá le af v e g (1 ) ra w [2 0] b un iu m b ul bo ca st an um l . bu lv uš ka h líz na tá o bu lk a, tu be r su b (1 ) ea te n ra w o r r oa st ed [3 1] ze m sk ý ka št an c ar um c ar vi l . km ín k oř en ný o ch le bo vé k oř en í le af se a (1 ) se as on in g so up s [3 1] n s se a (6 ) n s [1 1, 1 7, 2 2, 3 4, 3 5, 3 6] d au cu s ca ro ta l . s ub s. c ar ot a m rk ev o be cn á pr av á n m rk vo us ro ot su b (1 ) ro ot s, ra w a s a sn ac k [3 5] h er ac le um s ph on dy liu m l . bo lš ev ní k ob ec ný o km ín s vi ňs ký le af v e g (2 ) us ed in s ou ps [3 , 3 5] c ha er op hy llu m b ul bo su m l . kr ab ili ce h líz na t o kr ab ili ce b ul va tá tu be r su b (2 ) bo ile d or ro as te d [3 1, 3 5] le vi st ic um o ffi ci na le w . d . j . k oc h lib eč ek lé ka řs ký o , n m ag íč ko , v op ic h le af se a (4 ) n s [1 0, 1 1, 1 8, 3 2] v e g (4 ) us ed in s ou ps [1 4, 1 8, 3 1 33 ] rh iz om e se a (1 ) dr ie d as a s ea so ni ng [1 8] n s b e v liq (1 ) fo r m ak in g sp ir its [1 0] p as tin ac a sa tiv a l . pa st in ák s et ý o dř en ka ro ot su b (1 ) n s [3 5] p im pi ne lla s pp . be dr ní k o be dř in ec le af v e g (4 ) us ed ra w in s al ad s [3 , 2 0, 3 1, 3 5] se a (4 ) n s [3 , 1 2, 1 7, 2 9] a r a c e a e c al la p al us tr is l . ď áb lík b ah en ní o di vo ká k al a rh iz om e o t h flo (1 ) n s [2 0] a ru m m ac ul at um l . ár on p la m at ý o bl áz iv ec rh iz om e su b (2 ) bo ile d [2 0, 3 5] o t h flo (2 ) gr ou nd in to fl ou r [3 , 2 0] du ri ng fa m in e a sp a r a g a c e a e a sp ar ag us o ffi ci na lis l . ch ře st lé ka řs ký n šp ar gl le af v e g (1 ) yo un g le av es u se d in [3 5] ve ge ta bl e di sh es p ol yg on um s pp . ko ko ří k o ď áb lík n s b e v liq (1 ) fo r m ak in g sp ir its [3 4] a sp l e n ia c e a e p hy lli tis s co lo pe nd ri um (l .) n ew m an je le ní ja zy k ce lo lis tý o by lin a st ud no vá le af v e g (1 ) us ed in s ou ps [3 6] a st e r a c e a e a ch ill ea p ta rm ic a l . ře bř íč ek b er tr ám o pe rs án le af se a (3 ) se as on in g in s ou ps [2 , 3 , 1 1] f am ily a nd s pe ci es st an da rd c ze ch n am e c r. * f ol k na m e p ar ts u se d † u se c at eg or ie s ‡ m od e of u se r ef er en ce s (n o. o f r ep or ts ) wild food plants of the czech republic 59 v e g (7 ) us ed in v eg et ab le d is he s [2 , 3 , 1 7, 1 8, 2 5, 3 1, 3 6] a nt he m is a rv en si s l . rm en ro ln í o ho řk á tr áv a le af se a (1 ) n s [3 ] a rc tiu m s pp . lo pu ch o ba bá k, b ej lí ro ot su b (1 ) ve ge ta bl e di sh es [3 ] a rt em is ia v ul ga ri s l . pe ly ně k če rn ob ýl o če rn ob ýl st em se a (4 ) n s [3 , 9 , 1 7, 1 8] b e v liq (2 ) fo r m ak in g sp ir its [2 4, 3 4] o t h (1 ) vi ne ga r [2 ] o le af v e g (2 ) ve ge ta bl e di sh es [3 , 3 1] o flo w er se a (3 ) n s [1 8, 2 0, 3 1] b el lis p er en ni s l . se dm ik rá sk a ob ec ná o , n ch ud ob ka le af v e g (8 ) yo un g le av es u se d in s al ad s [2 , 3 , 1 0, 1 3, 1 7, 2 7, 3 1, 3 5] or s ou ps d ur in g sp ri ng flo w er fl o (3 ) ra w , p re se rv ed w ith s ug ar [3 , 1 7, 3 6] b e v (1 ) fo r m ak in g sy ru ps [1 7] c ar du us a ca nt ho id es l . bo dl ák o be cn ý o ch ab lá k, je ža te c le af v e g (1 ) ra w le av es u se d in s al ad s [3 5] c ar lin a ac au lis l . pu pa va b ez lo dy žn á o bo dl áč ek flo w er fl o (4 ) ra w re ce pt ac le s as a c hi ld re n’ s [1 2, 2 7, 2 6, 3 1] sn ac k fo od , v eg et ab le d is he s c en ta ur ea s pp . ch rp a o ch rp in a, flo w er b e v (1 ) fo r m ak in g ju ic es , s yr up s [2 ] m od rá če k fl o (4 ) pr es er ve d w ith s ug ar , [1 , 3 , 3 4, 3 6] co lo ur in gs se a (1 ) n s [2 2] c ic ho ri um in ty bu s l . če ka nk a ob ec ná o , n ci ko ri e, š tě rb ák le af v e g (4 ) yo un g le av es e at en ra w [1 8, 2 1, 3 1, 3 5] or a dd ed to s ou ps ro ot su b (1 ) us ed ra w in s al ad s, p re se rv ed [3 5] in to s ug ar b e vo th (7 ) gr ou nd in to c of fe e su bs tit ut es [2 , 1 4, 1 5, 2 0, 2 6, 3 1, 3 5] n s b e v liq (1 ) n s [3 4] m at ri ca ri a ch am om ill a l . he řm án ek p ra vý o vo ňa v rm en flo w er b e v liq (1 ) flo w er s fo r m ak in g sp ir its [3 4] o no po rd um a ca nt hi um l . os tr op es tr ub il o ko st ro pe s le af v e g (1 ) le av e bu ds u se d in s al ad s [2 0] flo w er v e g (1 ) yo un g re ce pt ac le s us ed in s al ad s [2 0] ro ot su b (1 ) yo un g ro ot s us ed in s al ad s [2 0] p et as ite s hy br id us (l .) p. g ae rt n. , de vě ts il lé ka řs ký o po db ěl le af v e g (1 ) us ed in s ou ps , v eg et ab le d is he s [3 5] b . m ey . & s ch er b. p ic ri s hi er ac io id es l . ho řč ík je st řá bn ík ov itý o le af v e g (1 ) us ed in s ou ps , v eg et ab le d is he s [3 5] sc or zo ne ra h is pa ni ca l . ha dí m or d šp an ěl sk ý o če rn ok oř en ro ot su b (1 ) ra w in s al ad s [2 0] b e vo th (1 ) gr ou nd in to c of fe e su bs tit ut es [2 0] se ne ci o vu lg ar is l . st ar če k ob ec ný o bu ře na , t er an ka le af v e g (1 ) so up s, v eg et ab le d is he s [3 5] si ly bu m m ar ia nu m (l .) g ae rt ne r os tr op es tř ec m ar iá ns ký o ja te rn í s em ín ko flo w er se a (1 ) flo w er s as a s ea so ni ng [2 0] so nc hu s ol er ac eu s l . m lé č ze lin ný o , n m líč í le af v e g (2 ) ra w , s ou ps [1 8, 3 5] ta na ce um v ul ga re l . vr at ič o be cn ý o , n ci cv ár , k op re tin a le af se a (3 ) n s [3 , 2 2, 3 4] b e v liq (1 ) su bs tit ut e fo r h op s in [2 0] be er b re w in g f am ily a nd s pe ci es st an da rd c ze ch n am e c r. * f ol k na m e p ar ts u se d † u se c at eg or ie s ‡ m od e of u se r ef er en ce s (n o. o f r ep or ts ) 60 k. simkova, z. polesny ta ra xa cu m s ec t. r ud er al ia k ir sc hn er , pa m pe liš ka o , n sm et án ka le af v e g (1 0) ra w , s ou ps , v eg et ab le d is he s [3 , 6 , 1 0, 1 5, 1 7, 1 8, 2 0, h . o llg aa rd e t š tě pá ne k 29 , 3 1, 3 2] flo w er o t h (3 ) bo ile d flo w er s w ith s ug ar [1 0, 1 7, 1 8] to m ak e a ho ne y b e v (1 ) flo w er s fo r m ak in g sy ru ps [1 8] b e v liq (3 ) w in es [1 0, 1 7, 1 8] tr ag op og on p ra te ns is l . ko zí b ra da lu čn í o ko zi br ad ka le af v e g (1 ) n s [2 0] rh iz om e su b (3 ) ch ild re n’ s sn ac k fo od [2 6, 3 1, 3 5] b e r b e r id a c e a e b er be ri s vu lg ar is l . dř iš ťá l o be cn ý o be rb er ka le af v e g (2 ) ra w [1 7, 3 5] fr ui t fr u (6 ) ch ild re n’ s sn ac k fo od , [1 2, 1 7, 2 4, 2 9, 3 1, 3 2] ja m s/ je lli es b e v (2 ) ju ic es [2 , 3 ] b e t u l a c e a e b et ul a pe nd ul a r ot h bř íz a bě lo ko rá o bř íz a br ad av ič na tá tr un k b e v (3 ) sa p dr un k fr es h du ri ng s pr in g [1 , 3 , 2 0] ba rk o t h flo (1 ) in ne r b ar k gr ou nd in to fl ou r [1 4] du ri ng fa m in e o t h (1 ) vi ne ga r [3 6] le af fl o (1 ) le av e bu ds [3 5] c or yl us a ve lla na l . lís ka o be cn á o fr ui t fr u (1 2) us ed ra w o r a dd ed to p as tr y [2 , 6 , 1 3, 1 6, 1 8, 1 9, 2 4, 2 7, an d co nf ec tio na ry 29 , 3 1, 3 2, 3 6] o t h oi l ( 1) fr ui ts p re ss ed in to o il [3 1] pr e (1 ) re nn et s ub st itu te s [3 6] b o r a g in a c e a e a nc hu sa o ffi ci na lis l . pi lá t l ék ař sk ý o sl áz a le af v e g (2 ) ra w y ou ng le av es [3 1, 3 5] b or ag o of fic in al is l . br ut ná k lé ka řs ký o , n ba re č le af v e g (2 ) us ed in s ou ps , v eg et ab le [1 2, 1 8] di sh es , p ic kl es e ch iu m v ul ga re l . ha di ne c ob ec ný o ko ňs ký o ca s le af v e g (1 ) yo un g le av es in s ou ps [1 8] p ul m on ar ia o ffi ci na lis l . pl ic ní k lé ka řs ký o le af v e g (3 ) ra w , v eg et ab le d is he s [3 , 1 7, 3 1] sy m ph yt um o ffi ci na le l . ko st iv al lé ka řs ký o če rn ý ko ře n le af v e g (2 ) ve ge ta bl e di sh es [3 , 3 5] rh iz om e su b (1 ) n s [3 5] b r a ss ic a c e a e le pi di um s pp . ře ři ch a o le af v e g (9 ) ra w , s ou ps , c on fe ct io ne ry [2 , 1 8, 2 0, 2 1, 2 4, 2 7, 2 9, (fi lli ng d ou gh nu ts ) 31 , 3 5] se a (2 ) [2 , 5 ] tu be r se a (1 ) dr ie d as a s ea so ni ng [1 8] a lli ar ia p et io la ta (m . b ie b. ) če sn áč ek lé ka řs ký o če sn ač ka se ed o t h oi l ( 1) n s [2 0] c av ar a et g ra nd e b ar ba re a vu lg ar is w .t . a ito n ba rb or ka o be cn á o le af v e g (3 ) ra w , v eg et ab le d is he s [3 , 1 8, 3 1] b un ia s sp p. ru ke vn ík o le af v e g (2 ) so up s, v eg et ab le d is he s [3 , 3 1] f am ily a nd s pe ci es st an da rd c ze ch n am e c r. * f ol k na m e p ar ts u se d † u se c at eg or ie s ‡ m od e of u se r ef er en ce s (n o. o f r ep or ts ) wild food plants of the czech republic 61 c ap se lla b ur sa -p as to ri s (l .) m ed . ko ko šk a pa st uš í t ob ol ka o ba bí k ap sa le af v e g (2 ) st ea m ed y ou ng le av es [3 1, 3 5] st em se a (1 ) n s [3 ] c ar da m in e pr at en si s l . ře ři šn ic e lu čn í o le af v e g (2 ) ra w , s ou ps [3 , 3 5] le pi di um d id ym us l . vr an ož ka p od vo jn á n le af v e g (2 ) ra w [3 1, 3 5] n as tu rt iu m o ffi ci na le w .t . a ito n po to čn ic e lé ka řs ká o če rn ý pe př le af v e g (2 ) ra w [2 0, 3 1] r ap ha nu s ra ph an is tr um l . ře dk ev o hn ic e o bl ej sk av a le af v e g (2 ) ba sa l l ea ve s in [3 , 3 1] ve ge ta bl e di sh es si na pi s ar ve ns is l . ho řč ic e po ln í o bl ej sk av ic e le af v e g (3 ) so up s, v eg et ab le d is he s [2 0, 3 1, 3 5] si sy m br iu m o ffi ci na le (l .) sc op . hu le vn ík lé ka řs ký o kl uk ov ka le af v e g (2 ) ra w s to lo ns in s al ad s [3 1, 3 5] c a m pa n u l a c e a e c am pa nu la r ap un cu lo id es l . zv on ek ře pk ov itý o ro ot su b (2 ) us ed ra w in s al ad s [2 0, 3 5] le af v e g (1 ) us ed ra w in s al ad s [2 0] c am pa nu la r ap un cu lu s l . zv on ek ře pk a o ro zp on ka ro ot su b (1 ) n s [3 5] p hy te um a or bi cu la re l . zv on eč ní k hl av at ý n ře pk a hl av at á le af v e g (1 ) us ed ra w in s al ad s [2 0] p hy te um a sp ic at um l . zv on eč ní k kl as na tý n ře pk a kl as na tá le af v e g (1 ) us ed ra w in s al ad s [2 0] ro ot su b (2 ) us ed ra w in s al ad s [2 0, 3 5] c a n n a b a c e a e h um ul us lu pu lu s l . ch m el o tá či vý o le af v e g (1 ) st ol on s us ed in s ou ps , [1 7] eg g di sh es c a r y o ph y l l a c e a e si le ne v ul ga ri s (m oe nc h) g ar ck e si le nk a na dm ut á o bě he n le af v e g (1 ) us ed in s ou ps , v eg et ab le d is he s [3 5] st el la ri a sp p. pt ač in ec o ha dí p us a le af v e g (1 ) ra w [3 5] c o r n a c e a e c or nu s m as l . dř ín ja rn í o dř ín ov é ja hů dk y fr ui t fr u (4 ) ra w , j am s/ je lli es [3 , 1 7, 2 9, 3 1] b e v (1 ) ju ic es [1 7] b e v liq (9 ) w in es , s pi ri ts [1 7] c or nu s sa ng ui ne a l . sv íd a kr va vá o kr va vý p ru t fr ui t fr u (1 ) n s [3 ] c r a ss u l a c e a e h yl ot el ep hi um m ax im um (l .) h ol ub ro zc ho dn ík v el ký o ko zí z el í le af v e g (1 ) ra w in s al ad s [2 0] se du m a lb um l . ro zc ho dn ík b ílý o bě lo ro zc ho dn ík le af v e g (1 ) ra w in s al ad s [3 5] se du m r efl ex um l . ro zc ho dn ík s ka ln í o pa ne tř es k le af v e g (1 ) so up s [2 0] se du m s pp . ro zc ho dn ík le af v e g (1 ) ra w in s al ad s [3 1] se m pe rv iv um te ct or um l . ne tř es k st ře šn í n ne tř es k ze dn í le af v e g (1 ) ra w in s al ad s [1 7] b e v (1 ) fo r m ak in g ju ic es [1 7] c u c u r b it a c e a e b ry on ia d io ic a ja cq . po se d dv ou do m ý o di bl ík , o se ch ro ot su b (1 ) n s [3 5] c u pr e ss a c e a e ju ni pe ru s co m m un is l . ja lo ve c ob ec ný o bo le rá z, b ří n br an ch pr e (3 ) fr ui tin g le af y br an ch es to [1 , 3 , 3 6] pr es er ve m ea t f am ily a nd s pe ci es st an da rd c ze ch n am e c r. * f ol k na m e p ar ts u se d † u se c at eg or ie s ‡ m od e of u se r ef er en ce s (n o. o f r ep or ts ) 62 k. simkova, z. polesny fr ui t se a (2 4) se as on in g (m os tly g am e) [1 , 2 , 3 , 4 , 5 , 6 , 9 , 1 0, 1 1, 1 3, 17 , 1 8, 2 0, 2 1, 2 2, 2 4, 2 7, 2 8, 29 , 3 0, 3 1, 3 3, 3 4, 3 6] b e v liq (6 ) sp ir its [2 , 1 7, 2 0, 2 4, 2 9, 3 4] c y pe r a c e a e c ar ex s pp . os tř ic e o ps ár ka , t uř ic e st em v e g (1 ) st al ks a s a ch ild re n’ s [2 6] sn ac k fo od c yp er us e sc ul en tu s l . šá ch or je dl ý o ga lg án p la ný tu be r su b (1 ) us ed ra w , f ri ed o r b ak ed [3 5] d e n n st a e d t ia c e a e p te ri di um a qu ili nu m (l .) k uh n ha si vk a or lič í o ha si na rh iz om e o t h flo (3 ) gr ou nd in to fl ou r d ur in g fa m in e [3 , 1 4, 3 5] e r ic a c e a e va cc in iu m m yr til lu s l . br us ni ce b or ův ka o če rn á ja ho da le af se a (1 ) se as on in g in s ou ps [1 8] du ri ng s pr in g fr ui t fr u (2 2) fr ui ts , r aw a s a ch ild re n’ s [2 , 3 , 6 , 7 , 9 , 1 0, 1 3, 1 4, 1 5, sn ac k fo od , s ou ps , j am s/ je lli es 17 , 1 8, 1 9, 2 0, 2 4, 2 5, 2 6, 2 8, 29 , 3 0, 3 1, 3 2, 3 6] b e v (4 ) ju ic es [2 , 6 , 3 1, 3 2] b e v liq (3 ) w in es a nd s pi ri ts [1 5, 1 8, 2 4] va cc in iu m o xy co cc os l . kl ik va b ah en ní n kl ik va ž or av in a fr ui t fr u (2 ) fo r m ak in g ch ut ne ys [1 7, 2 0] va cc in iu m u lig in os um l . vl oc hy ně b ah en ní o ba ži nn á bo rů vk a fr ui t fr u (2 ) n s [2 0, 3 1] va cc in iu m v iti sid ae a l . b ru sn ic e br us in ka o če rv en á bo rů vk a fr ui t fr u (2 4) fo r m ak in g fr ui t s ou ps , [3 , 4 , 5 , 6 , 7 , 9 , 1 0, 1 1, 1 3, 1 7, ja m s/ je lli es 18 , 1 9, 2 0, 2 1, 2 4, 2 5, 2 7, 2 8, 29 , 3 0, 3 1, 3 2, 3 3, 3 6] b e v (1 ) ju ic es [2 ] b e v liq (1 ) sp ir its [2 4] fa b a c e a e a st ra ga lu s gl yc yp hy llo s l . ko zi ne c sl ad ko lis tý o dř ez ov ec se ed b e vo th (1 ) gr ou nd in to c of fe e su bs tit ut es [1 4] la th yr us tu be ro su s l . hr ac ho r h líz na tý o ha lu ch a, o ře ší tu be r su b (3 ) ch ild re n´ s sn ac k fo od [2 6, 3 1, 3 5] la th yr us v er nu s (l .) b er nh . hr ac ho r j ar ní o hr ac ho r l ec ha tu be r su b (1 ) ch ild re n´ s sn ac k fo od [3 5] m ed ic ag o sa tiv a l . to lic e se tá n v oj tě šk a le af v e g (1 ) us ed in s ou ps a nd v eg et ab le [3 5] di sh es r ob in ia p se ud oa ca ci a l . tr no vn ík a ká t o ak át flo w er fl o (1 ) fr ie d flo w er s [1 8] tr ifo liu m s pp . je te l o ču dl ek , d ět el flo w er fl o (3 ) bo ile d or fr ie d; c hi ld re n’ s [2 4, 3 1, 3 5] sn ac k fo od b e v liq (1 ) fo r m ak in g sp ir its [2 4] o t h flo (3 ) gr ou nd in to fl ou r d ur in g [3 , 1 4, 3 5] fa m in e vi ci a sa tiv a l . vi ke v se tá o le af v e g (1 ) so up s [3 ] se a (1 ) n s [3 ] n s b e vo th (1 ) gr ou nd in to c of fe e su bs tit ut es [1 4] f am ily a nd s pe ci es st an da rd c ze ch n am e c r. * f ol k na m e p ar ts u se d † u se c at eg or ie s ‡ m od e of u se r ef er en ce s (n o. o f r ep or ts ) wild food plants of the czech republic 63 fa g a c e a e f ag us s yl va tic a l . bu k le sn í o bu či na flo w er fl o (1 ) flo w er b ud s [3 5] fr ui t fr u (2 ) fr ui ts (b ee ch nu ts ) r aw o r d ri ed [2 0, 3 1] o t h oi l ( 4) pr es se d in to o il [3 , 2 0, 2 6, 3 1] q ue rc us r ob ur l . du b le tn í o do ub í le af b e v liq (1 ) sp ir its [3 4] flo w er o t h flo (1 ) flo w er b ud s gr ou nd in to [3 5] flo ur d ur in g fa m in e fr ui t b e vo th (6 ) gr ou nd in to c of fe e or [2 , 1 4, 2 0, 2 6, 3 1, 3 5] ca ca o su bs tit ut es o t h oi l ( 1) pr es se d in to o il [3 ] o t h flo (4 ) fr ui ts (a co rn s) g ro un d in to [3 , 1 4, 3 1, 3 5] flo ur d ur in g fa m in e pr e (1 ) re nn et s ub st itu te s [3 6] g e n t ia n a c e a e g en tia na lu te a l . ho ře c žl ut ý n en ci án n s se a (1 ) n s [3 ] g r o ss u l a r ia c e a e r ib es u va -c ri sp a l . sr st ka a ng re št o fr ui t fr u (1 ) ra w in m ea t d is he s [3 1] h y pe r ic a c e a e h yp er ic um s pp . tř ez al ka o zd ěš ní k fr ui t o t h (1 ) bo ill ed w ith s ug ar to [1 0] m ak e a ho ne y l a m ia c e a e g le ch om a he de ra ce a l . po pe ne c ob ec ný o , n ku nd rl át ek , le af v e g (1 9) so up s, v eg et ab le d is he s, [2 , 3 , 5 , 6 , 1 0, 1 3, 1 7, 1 8, 2 0, op en ec eg g di sh es 21 , 2 5, 2 7, 3 0, 3 1, 3 2, 3 3, 3 5, 36 , 3 7] la m iu m s pp . hl uc ha vk a o cu cá če k flo w er se a (1 ) flo w er s as a s ea so ni ng [3 ] m en th a pu le gi um l . po le j o be cn á o bl eš í m át a n s se a (1 ) n s [3 ] m en th a sp p. m át a n ba lš án le af se a (1 ) n s [3 ] o ri ga nu m v ul ga re l . do br om ys l o be cn á o or eg án o flo w er b e v liq (1 ) su bs tit ut e fo r h op s in b ee r [2 0] br ew in g p ru ne lla v ul ga ri s l . če rn oh lá ve k ob ec ný o če rn oh lá vk a n s v e g (1 ) us ed in s ou ps a nd v eg et ab le [3 5] di sh es sa lv ia p ra te ns is l ša lv ěj lu čn í o ba bí b ru ch le af b e v liq (2 ) su bs tit ut e fo r h op s in b ee r [2 0, 3 4] br ew in g or fo r m ak in g sp ir its th ym us s pp . m at eř íd ou šk a o dé m ut st em se a (7 ) dr ie d flo w er in g sh oo ts a s a [2 , 3 , 1 1, 1 7, 3 0, 3 1, 3 6] se as on in g b e v liq (1 ) ad de d in to s pi ri ts a s fla vo r [1 0] ly t h r a c e a e tr ap a na ta ns l . ko tv ic e pl ov ou cí o vo dn í o ře ch fr ui t fr u (5 ) ra w [3 , 2 0, 3 1, 3 5, 3 6] o t h flo (2 ) fr ui ts g ro un d in to fl ou r [2 0, 3 1] m a lv a c e a e a lth ae a of fic in al is l . pr os ku rn ík lé ka řs ký o le af v e g (1 ) us ed in s ou ps a nd v eg et ab le [3 5] di sh es f am ily a nd s pe ci es st an da rd c ze ch n am e c r. * f ol k na m e p ar ts u se d † u se c at eg or ie s ‡ m od e of u se r ef er en ce s (n o. o f r ep or ts ) 64 k. simkova, z. polesny m al va a lc ea l . sl éz v el ko kv ět ý o sl éz lé či vý le af v e g (1 ) so up s an d ve ge ta bl e di sh es [3 5] m al va n eg le ct a w al lr. sl éz p ře hl íž en ý n bo ch ní čk y fr ui t fr u (4 ) im m at ur e fr ui ts ra w a s [1 2, 2 6, 3 1, 3 5] a ch ild re n’ s sn ac k fo od m al va s pp . sl éz o n s se a (2 ) n s [3 , 3 6] m al va s yl ve st ri s l . sl éz le sn í o bo ží k ol áč ky le af v e g (1 ) so up s an d ve ge ta bl e di sh es [3 5] ti lia s pp . líp a o n s b e v liq (2 ) sp ir its [1 0, 3 4] flo w er fl o (1 ) bo ile d flo w er b ud s [3 5] fr ui t fr u (1 ) fr ui ts a s a ch ild re n’ s [2 6] sn ac k fo od o n a g r a c e a e e pi lo bi um a ng us tif ol iu m l . vr bk a úz ko lis tá o ch m ýř í, pe jř í ro ot su b (1 ) so up s [3 ] o en ot he ra b ie nn is l . pu pa lk a dv ou le tá o no čn í h vě zd a ro ot su b (4 ) fir st y ea r r oo t e at en ra w a s a [1 2, 2 0, 3 1, 3 5] ch ild re n’ s sn ac k fo od o r c h id a c e a e d ac ty lo rh iz a m aj al is (r ch b. ) pr st na te c m áj ov ý n vs ta va č ší ro lis tý tu be r su b (1 ) fo rm er ly u se d as fo od [3 5] p. f . h un t e t s um m er h. o rc hi s sp p. vs ta va č o di vo ká o rc hi de j tu be r su b (1 ) fo rm er ly u se d as fo od [3 5] o x a l id a c e a e o xa lis a ce to se lla l . šť av el k ys el ý o za je čí je te l le af v e g (3 ) ra w , v eg et ab le d is he s, [3 , 2 6, 3 1] ch ild re n’ s sn ac k fo od pa pa v e r a c e a e p ap av er r ho ea s l . m ák v lč í o oh ní če k n s b e v liq (1 ) sp ir its [3 4] pl a n ta g in a c e a e p la nt ag o sp p. jit ro ce l o ba bí u ch o le af v e g (2 ) fr ie d or u se d in s al ad s, s ou ps [3 , 1 7] se a (1 ) n s [1 7] b e v liq (6 ) sp ir its [3 4] po a c e a e d ig ita ri a sa ng ui na lis (l .) sc op . ro si čk a kr va vá o pr os o kr va vé se ed o t h flo (2 ) gr ou nd in to fl ou r [3 , 3 5] e ch in oc hl oa c ru sga lli (l .) p. b . je ža tk a ku ří n oh a o bé r p la ný se ed o t h flo (1 ) gr ou nd in to fl ou r [3 5] e ly m us r ep en s (l .) g ou ld pý r p la zi vý o , n pe jř rh iz om e o t h flo (4 ) gr ou nd in to fl ou r d ur in g fa m in e [3 , 1 4, 2 6, 3 5] g ly ce ri a flu ita ns (l .) r . b r. zb lo ch an v zp lý va vý o vr ab čí p ro so se ed o t h flo (2 ) gr ou nd in to fl ou r [3 , 3 5] m ili um e ffu su m l . pš en íč ko ro zk la di té o pr os íč ko se ed o t h flo (1 ) gr ou nd in to fl ou r [3 5] se ta ri a vi ri di s (l .) p. b . s ub sp . v ir id is bé r z el en ý pr av ý o bá r se ed o t h flo (1 ) gr ou nd in to fl ou r [3 5] po ly g o n a c e a e b is to rt a of fic in al is d el ar br e rd es no h ad í k oř en o be ra ní o ca s le af v e g (2 ) ra w y ou ng le av es [1 8, 2 0] us ed in s al ad s f al lo pi a co nv ol vu lu s (l .) á . l öv e op le tk a ob ec ná o hr uš tič ka se ed o t h flo (1 ) gr ou nd in to fl ou r d ur in g fa m in e [3 ] p er si ca ri a sp p. rd es no o ho řč ák se ed o t h flo (8 ) gr ou nd in to fl ou r d ur in g fa m in e [3 ] r um ex a ce to sa l . šť ov ík k ys el ý o , n ky se la nd a le af v e g (2 ) us ed ra w in s al ad s, s ou ps [1 7, 2 0] an d ve ge ta bl e di sh es f am ily a nd s pe ci es st an da rd c ze ch n am e c r. * f ol k na m e p ar ts u se d † u se c at eg or ie s ‡ m od e of u se r ef er en ce s (n o. o f r ep or ts ) wild food plants of the czech republic 65 r um ex a qu at ic us l . šť ov ík v od ní o sl ad ký li st le af v e g (1 ) ra w in s al ad s [2 0] r um ex c ri sp us l . šť ov ík k ad eř av ý o sl ad ké li st í le af v e g (1 ) ra w in s al ad s [2 0] r um ex s pp . šť ov ík o st em v e g (2 ) le af y st em s ch ew ed b y ch ild re n [2 3, 2 6] an d sh ep he rd s ag ai ns t t hi rs t le af v e g (1 4) us ed ra w , i n so up s, v eg et ab le [2 , 3 , 9 , 1 0, 1 5, 1 8, 2 7, 2 8, 2 9, di sh es , e gg d is he s 30 , 3 1, 3 2, 3 5, 3 6] b e v (3 ) sy ru p [1 , 3 , 3 6] b e v liq (2 ) sp ir its [1 0, 3 4] pr e (1 ) re nn et s ub st itu te s [3 1] se ed o t h flo (1 ) se ed s gr ou nd in to fl ou r [3 5] du ri ng fa m in e po ly po d ia c e a e p ol yp od iu m v ul ga re l . os la di č ob ec ný o rh iz om e su b (1 ) ra w a s a ch ild re n’ s sn ac k fo od [3 1] po r t u l a c a c e a e m on tia fo nt an a l . zd ro jo vk a po to čn í o ko zl ík č er ve ný n s v e g (2 ) ra w in s al ad s [3 1, 3 5] p or tu la ca o le ra ce a l . šr uc ha z el ná o , n ku ří n oh a, le af v e g (5 ) us ed in v eg et ab le d is he s [3 , 1 5, 2 0, 2 9, 3 5, 1 5] po rt ul ák se a (1 ) n s [2 9] pr im u l a c e a e c yc la m en p ur pu ra sc en s m ill . br am bo ří k na ch ov ý o br am bo ří k tu be r su b (1 ) bo ile d [2 0] ev ro ps ký p ri m ul a ve ri s l . pr vo se nk a ja rn í n pe tr kl íč flo w er b e v (2 ) fo r m ak in g sy ru ps [3 , 3 6] r o sa c e a e a ro ni a m el an oc ar pa (m ic hx .) e lli ot t te m no pl od ec č er no pl od ý n ar on ie č er ná fr ui t fr u (1 ) fo r m ak in g ja m s/ je lli es [1 2] b e v liq (1 ) n s [2 5] a lc he m ill a vu lg ar is l . ko nt ry he l o st ro la lo čn ý o al ch em ilk a n s se a (1 ) n s [3 ] c ra ta eg us s pp . hl oh o hl oh ož í flo w er b e v liq (1 ) sp ir its [1 0] fr ui t fr u (1 ) ra w [3 ] b e v liq (3 ) sp ir its [3 4] f ra ga ri a ve sc a l . ja ho dn ík o be cn ý o ja ho dn íč ek le af se a (8 ) se as on in g in s ou ps [6 , 1 0, 1 5, 1 8, 2 5, 2 7, 3 1, 3 5] du ri ng s pr in g pr e (1 ) re nn et s ub st itu te s [3 6] fr ui t b e v liq (2 ) sp ir its [1 7, 2 4] b e v (1 ) sy ru ps [1 7] fr u (1 0) ea te n ra w a s a ch ild re n’ s [5 , 6 , 9 , 1 0, 1 7, 1 8, 2 4, 2 6, 3 1, sn ac k fo od , j am s/ je lli es 32 ] g eu m u rb an um l . ku kl ík m ěs ts ký o ku kl ic e rh iz om e se a (1 ) dr ie d as a s ea so ni ng [1 7] b e v liq (2 ) ad de d to b ee rs [1 7, 2 0] p ot en til la a ns er in a l . m oc hn a hu sí o hu sí k ví tk o rh iz om e su b (1 ) n s [3 ] p ru nu s sp in os a l . tr nk a ob ec ná o sl iv oň fr ui t fr u (6 ) ea tin g ra w a ft er fr os ts , c hi ld re n’ s [9 , 1 0, 1 7, 2 6, 3 1, 3 6] sn ac k fo od , u se d in fr ui t s ou ps f am ily a nd s pe ci es st an da rd c ze ch n am e c r. * f ol k na m e p ar ts u se d † u se c at eg or ie s ‡ m od e of u se r ef er en ce s (n o. o f r ep or ts ) 66 k. simkova, z. polesny b e v liq (3 ) fo r m ak in g w in es , s pi ri ts [1 7, 2 4, 3 1] o t h (5 ) vi ne ga r [2 , 3 , 2 2, 3 4, 3 6] se ed o t h oi l ( 1) oi l f ro m s ee ds [3 1] r os a ca ni na l . rů že š íp ko vá o m er he le c fr ui t fr u (2 5) fr ui ts fo r m ak in g so up s, 21 , 5 , 6 , 4 , 1 8, 1 1, 3 1, 3 0, 2 9, ch ut ne ys , j am s/ je lli es , 32 , 1 , 3 , 3 5, 3 6, 1 4, 2 2, 1 9, 9 , pr es er ve d w ith s ug ar 28 , 1 5, 2 4, 1 3, 2 7, 1 0, 1 7 b e v (1 ) n s [1 7] b e v liq (9 ) fo r m ak in g w in es a nd s pi ri ts [1 0, 1 4, 1 5, 1 7, 1 8, 2 4, 2 5, 3 1, 34 ] se ed b e vo th (3 ) gr ou nd in to c of fe e su bs tit ut es [1 4, 3 1, 3 5] r ub us c ae si us l . os tr už in ík je ži ní k o fr ui t fr u (1 4) ea te n ra w a s a ch ild re n’ s [3 , 6 , 1 0, 1 2, 1 3, 1 7, 2 0, 2 2, sn ac k, fo r m ak in g ja m s/ je lli es 24 , 2 6, 2 9, 3 1, 3 2, 3 4] b e v (3 ) ju ic es [2 4, 2 9, 3 2] b e v liq (4 ) w in es [1 0, 2 0 24 , 3 1] r ub us c ha m ae m or us l . os tr už in ík m or uš ka o m or uš ka fr ui t fr u (1 ) n s [3 ] kr ko no šs ká r ub us id ae us l . os tr už in ík m al in ík o m al in a fr ui t fr u (2 7) fr ui ts , e at en ra w , f or [1 , 2 , 3 , 4 , 5 , 6 , 9 , 1 0, 1 2, 1 3, m ak in g so up s 15 , 1 7, 1 8, 2 0, 2 1, 2 2, 2 4, 2 5, 26 , 2 7, 2 8, 2 9, 3 0, 3 1, 3 2, 3 4, 36 ] b e v (1 6) ju ic es [2 , 3 , 4 , 1 0, 1 4, 1 7, 2 0, 2 1, 2 4, 25 , 2 7, 2 8, 2 9, 3 1, 3 2, 3 6] b e v liq (6 ) w in es a nd s pi ri ts [2 , 1 0, 1 7, 2 4, 2 8, 3 6] o t h (3 ) vi ne ga r [2 , 2 4, 2 8] r ub us n em or os us h ay ne e t w ill d. os tr už in ík h aj ní o fr ui t fr u (1 ) n s [2 0] sa ng ui so rb a of fic in al is l . kr va ve c to te n o kr va ve c le af v e g (3 ) us ed ra w in s al ad s [2 0, 3 1, 3 5] so rb us s pp . je řá b o , n fr ui t fr u (8 ) ea te n ra w , c hi ld re n´ s sn ac k [6 , 1 7, 1 8, 2 4, 2 5, 3 0, 3 1, 3 2] fo od , m ak in g ja m s/ je lli es b e v (1 ) ju ic es [1 7] b e v liq (5 ) w in es a nd s pi ri ts [1 0, 1 5, 1 7, 2 4, 3 1] r u b ia c e a e g al iu m o do ra tu m (l .) sc op . sv íz el v on ný n m ař in ka v on ná st em b e v (1 ) n s [1 7] g al iu m v er um l . sv íz el s yř iš ťo vý o m ař en a le af pr e (1 ) re nn et s ub st itu te s [2 0] sa l ic a c e a e p op ul us s pp . to po l o le af fl o (1 ) le av e bu ds [3 5] sa lix s pp . vr ba o ba rk o t h (1 ) vi ne ga r [3 6] sa pi n d a c e a e a ce r ps eu do pl at an us l . ja vo r k le n o kl en ka tr un k b e v (1 ) sa p dr un k fr es h du ri ng s pr in g [2 0] a ce r pl at an oi de s l . ja vo r m lé č o m lé č tr un k b e v (1 ) sa p dr un k fr es h du ri ng s pr in g [2 0] a ce r sp p. ja vo r o tr un k b e v (1 ) sa p dr un k fr es h du ri ng s pr in g [3 ] a es cu lu s hi pp oc as ta nu m l . jír ov ec m aď al o ko ňs ký k aš ta n fr ui t o t h flo (2 ) dr ie d fr ui ts g ro un d in to fl ou r [1 4, 3 5] f am ily a nd s pe ci es st an da rd c ze ch n am e c r. * f ol k na m e p ar ts u se d † u se c at eg or ie s ‡ m od e of u se r ef er en ce s (n o. o f r ep or ts ) wild food plants of the czech republic 67 sc r o ph u l a r ia c e a e ve rb as cu m s pp . di vi zn a o ba bí k no t n s b e v liq (2 ) sp ir its [1 0, 3 4] ve ro ni ca b ec ca bu ng a l . ro zr az il po to čn í o po to čn ík le af v e g (3 ) us ed ra w in s al ad s, [3 , 1 8, 3 1] ve ge ta bl e di sh es b e v liq (1 ) sp ir its [3 4] so l a n a c e a e p hy sa lis a lk ek en gi l . m oc hy ně ž id ov sk á o ži do vs ká tř eš eň fr ui t fr u (1 ) n s [3 ] so la nu m d ul ca m ar a l . lil ek p ot m ěc hu ť o fr ui t fr u (1 ) n s [3 ] t y ph a c e a e ty ph a la tif ol ia l . or ob in ec š ir ok ol is tý o ci gá ra in flo re sc en ce o t h flo (1 ) na rr ow s pi ke s gr ou nd in to [3 ] flo ur d ur in g fa m in e u l m a c e a e u lm us g la br a h ud s. jil m d rs ný o jím el le af v e g (1 ) n s [3 5] u r t ic a c e a e u rt ic a di oi ca l . ko př iv a dv ou do m á o , n ži ha vk a le af v e g (2 0) yo un g st ea m ed le av es u se d in [3 , 5 , 6 , 8 , 1 0, 1 3, 1 4, 1 7, 1 8, so up s, v eg et ab le d is he s, 25 , 2 6, 2 7, 2 8, 2 9, 3 0, 3 1, 3 2, eg g di sh es 33 , 3 5, 3 7] b e v liq (2 ) sp ir its [1 0, 3 4] o t h flo (1 ) ad de d to b re ad d ur in g fa m in e [1 4] pr e (2 ) re nn et s ub st itu te s [2 8, 3 1] va l e r ia n a c e a e va le ri an el la lo cu st a (l .) b et ck e ko zl íč ek p ol ní če k o , n ja rn í s al át le af v e g (8 ) ra w u se d in s al ad s, s ou ps a nd [3 , 1 7, 2 0, 2 7, 2 9, 3 0, 3 1, 3 5] ve ge ta bl e di sh es n s o t h flo (1 ) gr ou nd in to fl ou r d ur in g fa m in e [3 5] va le ri an a of fic in al is l . ko zl ík lé ka řs ký n ba ld ri án n s se a (1 ) n s [3 ] v io l a c e a e vi ol a sp p. vi ol ka o le af se a (1 3) se as on in g in s ou ps [2 , 3 , 6 , 1 0, 1 5, 1 7, 1 8, 2 5, 2 7, du ri ng s pr in g 30 , 3 1, 3 2, 3 5] rh iz om e se a (2 ) n s [2 2, 3 6] b e v liq (1 ) w in es [2 ] flo w er b e v (6 ) flo w er s fo r m ak in g sy ru ps [1 , 2 , 3 , 2 4, 3 4, 3 6] fl o (5 ) co lo ur in gs , p re se rv ed [2 , 3 , 2 2, 3 4, 3 6] w ith s ug ar o t h (1 ) vi ne ga r [2 ] † n s – no n sp ec ifi ed * b ot an ic al n am e id en tifi ed u si ng : o – o bv io us c om m on n am e un iv er sa lly u se d in a la rg e ar ea ; n – id en tifi ed u si ng c om pa ra tiv e an al ys is o f f ol k na m es . ‡ u se d ca te go ri es : v e g – g re en v eg et ab le a nd e di bl e w ee ds (a er ia l p ar ts ra w , b oi le d or fr ie d) ; f r u – fr ui ts (r aw o r i n pr es er ve s) ; s u b – s ub te rr an ea n pa rt s (r hi zo m es , r oo ts , b ul bs a nd tu be rs ) a s a sn ac k or a dd ed to bo ile d di sh es ; s e a – s ea so ni ng ; f l o – fl ow er s ( t he ir n ec ta r u se d as a s na ck o r fl ow er s ad de d to d is he s in la rg er q ua nt iti es ); b e v – n on -a lc oh ol ic b ev er ag es ; b e v liq – a lc oh ol ic b ev er ag es ; b e vo th – o th er b ev er ag es (c of fe e an d ca ca o su bs tit ut es ); p r e – p re se rv at iv es in cl ud in g re nn et s ub st itu te s; o t h oi l – o ils ; o t h flo – fl ou rs ; o t h – o th er u se s f am ily a nd s pe ci es st an da rd c ze ch n am e c r. * f ol k na m e p ar ts u se d † u se c at eg or ie s ‡ m od e of u se r ef er en ce s (n o. o f r ep or ts ) journal of applied botany and food quality 88, 209 214 (2015), doi:10.5073/jabfq.2015.088.030 1department of analytics and control of medicines, faculty of pharmacy and biochemistry, university of zagreb, zagreb, croatia 2 department of pharmaceutical botany, faculty of pharmacy and biochemistry, university of zagreb, zagreb, croatia analysis of aucubin and catalpol content in different plant parts of four globularia species miranda sertić1, maja crkvenčić2*, ana mornar1, kroata hazler pilepić2, biljana nigović1, željan maleš2 (received may 7, 2015) * corresponding author summary iridoids are plant secondary metabolites that are gaining more scientific interest due to the wide range of their observed biological activities such as neuroprotective, anti-inflammatory, immuno modulatory, hepatoprotective and cardioprotective. the presence and content of aucubin and catalpol, two iridoid glucosides frequently present in iridoid-containing plants, was studied in methanolic extracts of leaves, flowers, woody stems and underground parts of four globularia l. species, including the medicinal plant globularia alypum l., using a specific and reliable hplc-dad-esi/ms method. aucubin was found in all four species, while catalpol was found only in g. alypum and g. punctata. flowers contained the highest amounts of investigated iridoids, with catalpol content reaching 1.6 % in g. punctata flowers. comparing to the medicinal plant g. alypum, related species contained higher amounts of investigated iridoids, which makes them interesting candidates for further biological activity investigations. introduction medicinal plants produce various phytochemicals that are responsible for their preventive and/or healing properties. along with different phenolic compounds, such as phenolic acids, flavonoids and tannins, which represent the largest part of plant secondary metabolites (dai and mumper, 2010), more attention considering biological activity and possible applications of plant extracts is recently given to iridoid-containing plants (viljoen et al., 2012; west et al., 2014). iridoids are a large group of monoterpenoid compounds (andrzejewska-golec, 1995), whose role in plants is mainly defence (wink, 2003). they possess a wide range of biological activities, such as neuroprotective, anti-inflammatory, immunomodula tory, hepatoprotective, cardioprotective, anticancer, antioxidant, antimicrobial, hypoglycaemic, hypolipidemic, choleretic, antispasmodic and purgative (tundis et al., 2008). however, the distribution of iridoids is with few exceptions limited to the plant orders lamiales, gentianales and cornales (jensen, 1991), which makes these compounds interesting as potential chemotaxonomic markers (taskova et al., 2006). globularia l. is a small angiosperm genus recently included in the plantaginaceae family (albach et al., 2005). one of the members of this genus, namely globularia alypum l., is a medicinal plant traditionally used in mediterranean countries for some of the aforementioned activities attributed to iridoid compounds (carrió and vallès, 2012; boudjelal et al., 2013; bousta et al., 2014). its hypoglycaemic, anti-ulcer and anti-inflammatory activity were confirmed by different studies (zennaki et al., 2009; fehri and aiache, 2010; amessis-ouchemoukh et al., 2014b). recent findings suggest that related species, such as g. cordifolia l. and g. meridionalis (podp.) o. schwarz, could also have potentially useful biological activities (tundis et al., 2012a; sipahi et al., 2014). the amount and type of iridoids present in these species might serve as an indicator of their potential health benefits, especially since these compounds were recognized as one of the main secondary metabolites of globularia (chaudhuri and sticher, 1981; kirmizibekmez et al., 2003; tundis et al., 2012b). aucubin is the most widespread compound belonging to the group of iridoid glycosides (andrzejewska-golec, 1995). aucubin is also a precursor of catalpol (rønsted et al., 2000), one of the main secondary metabolites of g. alypum (chaudhuri and sticher, 1981), whose presence frequently accompanies the presence of aucubin in species of the plantaginaceae (taskova et al., 2006). having in mind that medicinal use of different plant parts of g. alypum has been reported (carrió and vallès, 2012; bousta et al., 2014), one of the aims of the present study was to investigate the content of aucubin and catalpol in leaves, flowers and woody stems of the medicinal plant to see if they could be connected to its recorded medicinal applications. moreover, the study included three related species from the same genus, namely g. cordifolia, g. meridionalis and g. punctata, to try to predict their medicinal potential based on the comparison of the found amounts of investigated iridoids and known uses of the medicinal plant. underground parts of the three less investigated species were also included in the analysis, while g. alypum was harvested without underground parts, due to its near threatened status in croatia. the analysis was performed using a rapid method coupling high-performance liquid chromatography with diode array detector and electrospray ionization/mass spectrometry (hplc-dad-esi/ms), which was optimized and validated in our laboratory. materials and methods chemicals aucubin was obtained from fluka chemie ag (buchs, switzerland) and catalpol from sigma-aldrich co. (st louis, mo, usa). hplcgrade acetonitrile and formic acid were obtained from merck (darmstadt, germany). ultrapure water used in the mobile phase and for the preparation of sample and standard solutions was obtained by a milli-q water 140 purification system (millipore, bedford, usa). methanol was obtained from t.t.t. d.o.o. (sveta nedelja, croatia) and was of analytical grade. plant material aerial and underground plant parts of g. cordifolia, g. meridionalis and g. punctata were collected during the flowering period in may 2012 and g. alypum in march 2013 from natural populations growing in croatia and bosnia and herzegovina. geographical coordinates for localities of g. alypum, g. cordifolia, g. meridionalis and g. punctata were: 42° 30´ 50˝ n/18° 19´ 07˝ e, 43° 21´ 41˝ n/17° 48´ 20˝ e, 44° 31´ 41˝ n/15° 08´ 38˝ e and 45° 23´ 36˝ n/13° 51´ 20˝ e, respectively. voucher specimens are deposited in the her barium of the department of pharmaceutical botany, faculty of pharmacy and biochemistry, university of zagreb, croatia (voucher no. 16 020, 16 030, 16 043, 16 051). the identity of each species was verified by prof. kroata hazler pilepić, department of pharma210 m. sertić, m. crkvenčić, a. mornar, k. hazler pilepić, b. nigović, ž. maleš ceutical botany, faculty of pharmacy and biochemistry, university of zagreb, croatia. preparation of sample and standard solutions dry powdered plant parts (leaves, flowers, woody stems and underground parts) (2.5 g) of each of the four globularia species were extracted twice with methanol (25 ml) in a bandelin sonorextm super ultrasonic extractor (30 min, room temperature) (bandelin electronic gmbh & co. kg, berlin, germany), filtered and their volume was adjusted to 50 ml with methanol. prior to analysis the extracts were diluted two times with water and filtered through an acrodisc® ghp syringe filter (diameter 26 mm, pore size 0.20 μm) (gelman, ann arbor, usa). stock solutions of aucubin and catalpol were prepared by dissolving each of the standards separately in water (0.2 mg/ml). all stock solutions were stored in a refrigerator at 4 °c for no longer than four weeks and were stable during that time. working standard solutions used for method optimization and validation were prepared daily by serial dilutions with water just before measurements. hplc-dadesi/ms analysis of aucubin and catalpol contents of aucubin and catalpol were analyzed by agilent 1100 series lc-msd trap system (agilent technologies, waldbronn, germany). separation was performed on a zorbax sb-c18 column (250 mm x 4.6 mm i.d.; 5 μm particle size) (agilent technologies, waldbronn, germany) under isocratic elution conditions using a mixture of acn:h2o:hcooh (5:95:0.1, v:v:v) as the mobile phase, with a flow rate 0.5 ml/min, 25.0 °c column temperature and uv detection at λ 210 nm. prior to use, the mobile phase was filtered through a cellulose nitrate filter (diameter 47 mm, pore size 0.45 μm) (sartorius, göttingen, germany). electrospray ionization (esi) was performed in negative ionization mode. nitrogen was used both as drying gas (5.0 l/min) and as nebulizer gas (15.0 psi). electrospray source temperature was optimized at 325 °c and the capillary voltage was set at 3.5 kv. the uv detection was set at λ 210 nm. the full scan mass spectra were acquired over a range m/z 100 – 500. data acquision and processing were done using chemstation for lc 3d and lc/msd trap v.5.2 software. all samples were injected in triplicate (10 μl, splitless mode). the contents of aucubin and catalpol were calculated from calibration curves obtained by the use of ms detector and the results were presented as mg/g dry plant material. method validation the method was validated for linearity, precision, accuracy and limits of detection (lod) and quantification (loq) according to guidelines of international conference on harmonization (ich, 2005). linearity was evaluated by least squares linear regression using seven different concentrations of standard solutions (10, 25, 50, 75, 100, 150 and 200 μg/ml). lod and loq were evaluated by using the same dilutions of standard solutions as for linearity and were defined as the signal-to-noise ratio equal to 3:1 and 10:1, respectively. accuracy of the method was determined using three different known concentrations (25, 50 and 100 μg/ml) of aucubin and catalpol, each analyzed individually three times. the intraand inter-day precision of the method was validated with 100 μg/ml standard solution of aucubin and catalpol. for intra-day precision measurements were conducted six times within the same day, while for inter-day precision measurements were performed three times a day on three consecutive days. relative standard deviation (rsd) for retention time and rsd for peak area, defined as the percentage ratio of standard deviation to the mean, were taken as measures of precision. all data were evaluated using microsoft office excel 2007 (12.0.6683.5002). statistical analysis the results are presented as means ± standard deviations of triplicate measurements. exceptionally, reported retention times of aucubin and catalpol were based on six different analyses. a two-way analysis of variance (anova) followed by bonferroni post-hoc test was carried out to determine significant differences between results. significance level α was set at 0.05. the statistical analysis was carried out using graphpad prism 5.03 for windows (graphpad software, san diego, usa). results and discussion in the present study, amounts of aucubin and catalpol in different plant parts of g. alypum, g. cordifolia, g. meridionalis and g. punctata were evaluated using a specific and reliable hplc-dad-esi/ ms method. the method provided good separation of analytes, with retention times being 7.56 ± 0.01 min for catalpol and 12.23 ± 0.01 min for aucubin (n = 6). the presence of aucubin and catalpol was determined by comparison of their retention times together with uv and mass spectra with those of standard compounds. final quantification of analytes was based on the results obtained from the ms detector, which in comparison to a diode array detector provided better selectivity with obtained good sensitivity of the used method. after testing electrospray ionization in both positive and negative mode, much higher peaks were observed in negative ionization mode, similar as in some previous studies considering iridoid glycosides (li et al., 2008; deng et al., 2013). the observed fragmentation pattern of aucubin was similar to that of catalpol (tab. 1). deprotonated pseudo-molecular ions [m – h]– were present at m/z 345 for aucubin and at m/z 361 for catalpol. also, characteristic fragment ions of [m – h – 162]– due to loss of a dehydroglucose residue were observed at m/z 183 for aucubin and m/z 199 for catalpol. a similar fragmentation pattern was observed previously (kumar et al., 2013). in the mass spectrum of aucubin, an additional peak was present at m/z 137, most likely obtained by loss of glucose and co [m – h – 180 – 28]–. peaks at m/z 381 and 383 observed for aucubin and at m/z 397 and 399 for catalpol, having a height ratio of about 3:1, indicated the presence of chlorine adducts (zhu and cole, 2000). the major ion observed for both compounds was a formic acid adduct of the pseudo-molecular ion [m – h + 46]–. the formation of adducts with formic acid present in the mobile phase was reported previously for some other iridoid glycosides (li et al., 2008; deng et al., 2013). high peaks appearing at m/z 443 and 459 might be a result of adduct formation with phosphoric/sulfuric acid as possible residual contamination of the used system, leading to a rise of pseudo-molecular ion mass for approximately 98 da (tong et al., 1999). tab. 1: peak identification for aucubin and catalpol mass spectra m/z auc cat peak identification 136.9 [m – h – glucose – co]– 182.8 198.8 [m – h – dehydroglucose]– 344.9 360.9 [m – h]– 381.0 396.9 [m + cl]– 391.0 406.9 [m – h + hcooh]– 442.9 458.9 [m – h + h3po4/h2so4]– auc, aucubin; cat, catalpol. aucubin and catalpol content of globularia 211 the developed method was validated for linearity, limits of detection (lod) and quantification (loq), accuracy, intraand inter-day precision. validation parameters are presented in tab. 2. the calibration curves for both analytes were linear over the concentration range used (10 – 200 μg/ml) (r > 0.99). the recovery values were between 98.11 % and 102.09 % indicating excellent accuracy of the method. the results of the analysis showed that aucubin was present in all four investigated species, while catalpol was only found in g. alypum and g. punctata (tab. 3). in a similar study done on different plantago species, aucubin was also found to be more frequently present than catalpol (jurišić et al., 2004), which could be connected with the fact that aucubin is a biosynthetic precursor of catalpol (rønsted et al., 2000). our results confirm the presence of aucubin in g. alypum, which was earlier reported by wieffering (1966). this disproves the claims of chaudhuri and sticher (1981), reporting its presence to be questionable in g. alypum, which were probably based on the fact that aucubin was never isolated from this species. previous studies primarily reported aucubin and its derivatives as typical for g. cordifolia (kirmizibekmez et al., 2003), while for g. alypum mostly catalpol and catalpol derivatives were observed (chaudhuri and sticher, 1981; amessis-ouchemoukh et al., 2014a). when comparing plant parts of g. punctata it can be seen that flowers were richest in both aucubin and catalpol, followed by leaves (p < 0.05) (fig. 1). the same iridoid distribution pattern was observed in all investigated species (tab. 3). recently, aerial parts of g. meridionalis were reported to contain higher amounts of iridoids than underground parts (tundis et al., 2012a), as well. similar observations were reported in other species belonging to the plantaginaceae family, such as linaria dalmatica (l.) p. mill. (jamieson and bowers, 2010). our findings and cited data are in accordance with the crucial ecological roles of secondary metabolites for plants, especially when having in mind that organs important for survival and reproduction usually have more potent compounds and higher amounts thereof (wink, 2003). tab. 2: validation parameters for the applied hplc-dad-esi/ms method (linearity, limits of detection (lod) and quantification (loq), accuracy, intra and inter-day precision) validation parameters aucubin catalpol linearity range (μg/ml) 10-200 10-200 regression equation y = 23618x – 249491 y = 13700x – 25381 r 0.993 0.995 lod (μg/ml) 1 2.5 loq (μg/ml) 3 7 low 102.09 98.11 accuracy (%) medium 100.87 101.32 high 101.21 99.48 intra-day precision rsd (%) for retention time 0.51 0.72 rsd (%) for peak area 3.87 3.25 inter-day precision rsd (%) for retention time 0.54 0.87 rsd (%) for peak area 4.92 4.85 lod, limit of detection; loq, limit of quantification; rsd, relative standard deviation. tab. 3: aucubin and catalpol contents in different plant parts of four globularia species content/(mg/g dry plant material)aucubin g. alypum g. cordifolia g. meridionalis g. punctata leaves 0.58 ± 0.01 0.96 ± 0.03 1.53 ± 0.03 0.77 ± 0.01 flowers 0.92 ± 0.00 1.65 ± 0.02 2.00 ± 0.01 1.29 ± 0.01 woody stems 0.58 ± 0.01 0.59 ± 0.00 0.61 ± 0.01 0.66 ± 0.01 underground parts n.m. 0.81 ± 0.00 0.76 ± 0.01 0.57 ± 0.01 content/(mg/g dry plant material)catalpol g. alypum g. cordifolia g. meridionalis g. punctata leaves 1.79 ± 0.03 nd nd 2.52 ± 0.06 flowers 12.58 ± 0.03 nd nd 15.97 ± 0.63 woody stems 1.47 ± 0.02 nd nd 0.51 ± 0.01 underground parts n.m. nd nd 0.66 ± 0.01 values are means ± sd, n = 3; n.m., not measured; nd, not detected. 212 m. sertić, m. crkvenčić, a. mornar, k. hazler pilepić, b. nigović, ž. maleš in our study higher amounts of aucubin were found in leaves and flowers of the three related species comparing to those of g. alypum (p < 0.05). catalpol concentrations found in g. alypum and g. punctata were generally higher than aucubin concentrations. they were especially high in flowers, with the content reaching almost 1.6 % in g. punctata. a similar relationship between aucubin and catalpol was observed in rehmannia glutinosa (gaertn.) dc., one of the fundamental herbs used in traditional chinese medicine (won et al., 2010; xu et al., 2012). catalpol content was also seen to be higher than aucubin content in some plantago species where both iridoids were present (jurišić et al., 2004). from an ecological point of view these observations could be explained by the greater toxicity of catalpol to generalist herbivores (nieminen et al., 2003). according to different ethnobotanical studies flowers of g. alypum are usually used for treatment of diabetes, digestive disorders and eczema (boudjelal et al., 2013; bousta et al., 2014). the use of leaves and aerial parts of the same plant was reported for similar indications (merzouki et al., 2000; hammiche and maiza, 2006), but also for some additional purposes, such as treatment of arterial hypertension (carrió and vallès, 2012; sari et al., 2012). based on data from previous studies considering biological activities of iridoids and the results presented in this study, it can be assumed that some of these activities could be attributed to aucubin and/or catalpol. however, it should be noted that this species contains a large number of compounds with medicinal potential (amessisouchemoukh et al., 2014a). recently, the study of merghache et al. (2013) confirmed hypoglycaemic and hypolipidemic activity of globularin, a cinnamoyl ester of catalpol isolated from g. alypum. in g. punctata both investigated iridoids were present in higher amounts than in g. alypum, which makes it an interesting plant for further analysis, especially due to the fact that it is a well-distributed species of the genus (tutin et al., 1972). however, one should bear in mind that besides genetic predisposition, other factors (internal and external) could have also contributed to the production of secon dary metabolites in these plants (jamieson and bowers, 2010). for example, observed differences might be a consequence of different plant age (andrzejewska-golec, 1995) and/or reaction to damage caused by insect herbivores and pathogenic microorganism infections (marak et al., 2002). it wasn’t possible to eliminate all of these factors, due to the fact that harvesting was done from wild populations. however, all samples were collected in the same phenological period (flowering). fig. 1: chromatograms of g. punctata leaves, flowers, woody stems and underground parts obtained by diode array detector (arrows indicating the presence of catalpol and aucubin, respectively). aucubin and catalpol content of globularia 213 conclusions comparing aucubin and catalpol content of g. cordifolia, g. meridionalis and g. punctata to amounts of these iridoids found in the medicinal plant g. alypum, it can be assumed that the three species might have some potential for medicinal use. certainly, possible biological activity investigations in the future should be accompanied by more detailed analyses of their chemical composition. these studies are currently in progress. so far the results show a greater similarity between the phytochemical composition of g. punctata and g. alypum than that between g. cordifolia/g. meridionalis and g. alypum. with regard to its wider distribution in europe, g. punctata shows the highest potential for further investigations. acknowledgements this research was supported by the ministry of science, education and sports of the republic of croatia (project no. 006-00611171239 and 006-0061117-1240). we would like to thank s. maslo for his help in collecting plant material from mostar. references albach, d.c., meudt, h.m., oxelman, b., 2005: piecing together the “new” plantaginaceae. am. j. bot. 92, 297-315. amessis-ouchemoukh, n., abu-reidah, i.m., quirantes-piné, r., rodríguez-pérez, c., madani, k., fernández-gutiérrez, a., segura-carretero, a., 2014a: tentative characterisation of iridoids, phenylethanoid glycosides and flavonoid derivatives from globularia alypum l. 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zennaki, s., krouf, d., taleb-senouci, d., bouchenak, m., 2009: globularia alypum l. lyophilized methanolic extract decreases hyper glycemia and improves antioxidant status in various tissues of streptozotocin-induced diabetic rats. j. complement integr. med. 6, article 34. zhu, j., cole, r.b., 2000: formation and decompositions of chloride adduct ions, [m + cl]−, in negative ion electrospray ionization mass spectrometry. j. am. soc. mass spectrom. 11, 932-941. address of the corresponding author: maja crkvenčić, department of pharmaceutical botany, faculty of pharmacy and biochemistry, university of zagreb, schrottova 39, hr-10000 zagreb, croatia. e-mail: mcrkvencic@pharma.hr © the author(s) 2015. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 91, 219 225 (2018), doi:10.5073/jabfq.2018.091.029 1 department of plant production, soil science and agricultural engineering, school of agricultural and environmental science, university of limpopo, south africa 2 risk and vulnerability science centre (rvsc), university of limpopo, south africa seasonal effect on moringa oleifera gaseous exchange and water use efficiency under diverse planting densities moshibudi paulina mabapa1,2,*, kwabena kingsley ayisi2, irvine kwaramba mariga1 (submitted: may 21, 2018; accepted: july 21, 2018) * corresponding author summary the study on moringa oleifera was conducted over twelve months during 2014 -2015 to evaluate the impact of the growing season and varying planting densities on biomass yield and physiological attributes under dryland conditions. trial was established at densities of 5000, 2500, 1667 and 1250 plants ha-1, with eight replicates. the increase in planting density led to an increase in biomass production. the monthly and seasonal data collected showed significant differences in net photosynthetic rate, transpiration, sub-stomatal co2 and stomatal conductance. however, planting densities of m. oleifera had no significant effect on all the gaseous exchange parameters measured. the results further revealed that the amount of carbon dioxide assimilated by the tree is not attributable to photosynthetic and transpiration rates as well as stomatal conductance. under water shortage condition and high temperature, m. oleifera used an adaptation strategy by reducing stomatal conductance and transpiration and hence increasing water use efficiency. moringa oleifera thus has the ability to sequester carbon even under water stress conditions. the tree can therefore be recommended for planting at a relatively high density of 5000 plants ha-1 in many parts of limpopo province where temperatures are favorable for improved farmers’ livelihoods as well as for climate change mitigation. keywords: biomass; climate change; monthly; moringa oleifera; physiological response; planting density introduction moringa oleifera is a highly valued tree that is widely distributed in many countries of the tropics and subtropics. it is considered as one of the most useful trees, since almost every part of the tree is a valuable source of food, medication and industrial purposes (morton, 1991; foidl et al., 2001). moringa oleifera is a fast growing plant tolerant to harsh climatic and environmental conditions where many agricultural plants would not survive, requiring only 400 mm of annual rainfall (morton, 1991; reyes-sánchez, 2006; pandey et al., 2011). moringa oleifera trees can play a significant role in mitigating adverse effects of climate change, due to its ability to capture carbon (daba, 2016). it also has the potential to improve the income and livelihood of smallholder farmers as well as nutritional needs of rural communities (mabapa et al., 2017; babu, 2000; moyo et al., 2011). through observations, south africa and other countries globally are already affected by severe negative consequences of climate change. this is particularly evident in the smallholder farming sector that relies on natural resources for production. best practices of agriculture can contribute towards climate change mitigation and adaptation by adopting various agricultural management practices that could minimize adverse effects of unpredictable rainfall patterns as well as other extreme weather conditions (hulme, 2005; pareek, 2017). pareek (2017) reported that various agricultural adaptation management practices are available to attenuate the effects of climate change on crop production. this include broader agronomic management strategies such as altering planting densities, row spacing, planting time as well as introducing new germplasms that are resistant to heat and drought stress. there is no information on literature regarding carbon sequestration through m. oleifera trees as influenced by varying plant densities in the south african context. it is known from the literature that m. oleifera has been described as a tree that can be grown in the drier ecological zones (asante et al., 2014; seresinhe and marapana, 2011). however, no quantitative information is available in south africa about the m. oleifera’s gaseous exchange response such as stomatal conductance, stomatal co2, transpiration and photosynthetic rate to planting density over the different seasons. this study was therefore established to evaluate the seasonal response of m. oleifera in terms of biomass yield and gaseous exchange under varying planting densities as a contribution towards climate change mitigation. materials and methods study location the study was conducted over twelve months to cater for four seasons, summer (december february), autumn (march may), winter (june august) and spring (september november) during 2014 – 2015, at ntl baraka eco-farming organic farm (23°57.691’s and 30°35.205’e), situated in eiland. the farm is situated about 50 km on the eastern side of tzaneen in the limpopo province. the area is a tropical region and receives about 429 mm of rain per annum, with most rainfall occurring during mid-summer. the annual rainfall data were derived from the total monthly values for eiland averaged in the past 7 years. the area received the lowest average rainfall (<0.5mm) in june and july; the highest in december and january of more than 120 mm in the past 7 years. the monthly temperature distribution shows that, the average maximum monthly temperatures for eiland could rise above 31 °c, while minimum temperatures could range between 7 to 25 °c, during winter and summer seasons, respectively. experimental design, planting and management a 60 m length and 40 m breadth demarcated area was prepared by conventional tillage using disk ploughing, followed by two disk harrowing and manual digging of shallow holes for planting. the experiment was laid out in a two factor (months and plant spacing) factorial randomized complete block design (rcbd) with eight replicates. the blocks were divided into four plots where treatments were placed. the treatments consisted of 12 months: may’14, june’14, july’14, august’14, september’14, october’14, november’14, december’14, january’15, february’15, march’15 and april’15 as well as four levels of intra-row spacing: d1= 1 m, d2= 2 m, d3= 3 m and d4= 4 m, with a uniform inter-row spacing of 2 m, giving total populations of 5000, 2500, 1667 and 1250 plants ha-1, respectively. 220 m.p. mabapa, k. kingsley ayisi, i. kwaramba mariga untreated seeds of m. oleifera were used for planting by placing 2 seeds per hole at a depth of 2 cm during december 2013. each plot measured 4 m × 12 m and plots were separated from each other by 2 meters walkways. irrigation was applied for four hours twice a week using a micro jet irrigation system until the tenth week to encourage good tree establishment, after which the study was allowed to run under rainfed conditions. eight weeks after trial establishment, plants were thinned out to the right densities, retaining only the healthier plants during the thinning process. prior to data collection, the whole experiment was uniformly cut at a height of 50 cm aboveground and all foliage was removed but not weighed. leaf gaseous exchange and water use efficiency measurements leaf gaseous exchange measurements were measured monthly on a fully-expanded leaf on the abaxial side of three selected leaves per experimental unit, using a portable photosynthesis system (adc bio scientific, uk). monthly measurements were averaged for each season to come up with seasonal data. the photosynthetic rate (a), stomatal conductance (gs), transpiration rate (e) and sub-stomatal co2 (ci) were simultaneously determined for each species using a non-destructive method. all the measurements were carried out under steady-state conditions in full sun between 10:00 am and 14:00 pm (clifford et al., 1997). the instantaneous water use efficiency (wue) which is defined as the ratio of photosynthetic capa city to the rate of transpiration was calculated for each density as a/e (field et al., 1983; rivas et al., 2013). plant biomass determination leaf biomass was collected once from a net plot of 12 m2 during harvesting in april 2015. the leaf yield (kg) was determined by separating the leaves from the petiole. the fresh leaf biomass was shade-dried at room temperature for 72 hours and later weighed using a battery-operated top loading weighing balance (radwag, w/ c6/12/c1/r model). statistical analysis data were subjected to analysis of variance using statistix 10.0 to compare the response of m. oleifera planting density on measured variables. a significance level of p<0.05 was used for determining differences among the interaction of months and plant spacing on leaf gaseous exchange and water use efficiency. the differences among the obtained mean values of measured parameters were compared and computed by using tukey’s hsd test considering 5% probability level (gomez and gomez, 1996). where significant f-values from density treatment effects were observed on biomass yield, means were separated by least significant difference (lsd) at probability level of 0.05 (gomez and gomez, 1996). correlation and regression analyses were performed using both statistix 10.0 and microsoft excel, to determine the relationship between gaseous exchanges. results weather parameters of the study area data on weather parameters measured during the study and anomaly in rainfall distribution are presented in tab. 1 and fig. 1. rainfall dropped during summer season (november february) of 2014/15, whereby the rainfall was below 100 mm as compared to the past two years during which the amount exceeded 200 mm. the temperatures fig. 1: total monthly rainfall (mm), average maximum (tx) and minimum (tn) temperatures collected at eiland from during the production seasons. tab. 1: total monthly rainfall (mm), mean minimum and maximum temperatures recorded between may 2014 and april 2015 at the study site. month/year total monthly min temp max temp rainfall (mm) (oc) may’14 0.00 10.29 28.06 june’14 0.00 6.29 26.23 july’14 0.51 6.19 24.79 august’14 8.38 8.31 26.41 september’14 0.51 12.37 30.15 october’14 25.91 15.26 29.48 november’14 13.97 18.99 30.39 december’14 95.49 20.40 30.94 january’15 17.26 21.39 31.91 february’15 99.31 21.06 32.41 march’15 12.18 19.37 32.47 april’15 41.15 16.65 29.42 gaseous exchange and water use efficiency of moringa oleifera 221 tab. 2: leaf gaseous exchange parameters and instantaneous water use efficiency of m. oleifera as influenced by month and planting density. treatments/factor transpiration rate (e) stomatal conductance (gs) photosynthetic rate (a) sub-stomatal co2 (ci) wue inst mmol m-2 s-1 mol m-2 s-1 μmol m-2 s-1 vpm μmol mol-1 month/year may’14 2.88abc 0.15b 6.73bc 276.91ab 2.33cde june’14 3.49a 0.09d 6.73bc 220.75bcd 1.91de july’14 2.53bc 0.07de 10.65a 133.38f 4.37a august’14 2.46bc 0.07de 9.04ab 152.12ef 3.72ab september’14 3.14ab 0.05fg 4.31cde 246.22abcd 1.29e october’14 2.10cd 0.19a 8.96ab 270.41abc 4.29a november’14 2.19cd 0.05ef 5.54cd 190.81def 2.68bcd december’14 2.65abc 0.07de 5.85cd 207.31cde 2.38bcde january’15 1.47de 0.03g 3.46de 195.53def 3.40abc february’15 1.06e 0.04fg 1.76e 297.03a 2.16cde march’15 3.28ab 0.13c 8.98ab 229.03bcd 2.55bcde april’15 1.39de 0.06ef 4.667cde 215.50bcde 4.2659a significance *** *** *** *** *** density (plants ha-1) 5000 2.29 0.08 6.57 221.75 3.24 2500 2.51 0.09 6.30 224.69 2.71 1667 2.30 0.08 6.09 213.10 2.83 1250 2.46 0.09 6.60 218.79 3.00 significance ns ns ns ns ns interaction (month × density) significance levels: *p<0.05, ** p<0.01, *** p<0.05, ns: not significant. means with different letters are statistically significant. increased during summer season with the maximum temperature exceeding 32 oc (fig. 1). the drought that was experienced during 2015 had a significant influence on rainfall anomaly. the accelerated heat accompanied by moisture deficit led to below average rainfall anomaly mainly during the year 2015 as compared with the years 2013 and 2014, in which the rainfall anomaly was above average in many instances, mainly during the rainy season (fig. 2). monthly gaseous exchange and water use efficiency of m. oleifera as influenced by planting density the monthly data collected showed highly significant differences in all gaseous exchange parameters and water use efficiency (tab. 2). however, planting density of m. oleifera had no effect on photo synthetic rate, transpiration rate, sub-stomatal co2 and stomatal conductance as well as water use efficiency (tab. 2). fig. 2: rainfall (mm) anomaly at eiland as compared to long term average rainfall from 2009 to 2015. ns ns ns ns ns 222 m.p. mabapa, k. kingsley ayisi, i. kwaramba mariga seasonal effect on m. oleifera gaseous exchange the seasonal effect significantly influenced all the measured gaseous exchange parameters (fig. 3). photosynthetic rate, stomatal conductance and transpiration rate were reduced during summer season, whilst the sub-stomatal co2 concentration increased during the same season. furthermore, the photosynthetic rate and stomatal conductance were higher during winter season when temperatures were low. no significant differences in transpiration were found during winter, autumn and spring seasons, while sub-stomatal co2 was found not to change during the three seasons except in the winter season. correlations and regression of m. oleifera gaseous exchange parameters seasonal relationships between gaseous exchange are presented in fig. 4. transpiration rate and photosynthetic rate showed a strong positive relationship. a similar relationship was observed between stomatal conductance and photosynthetic rate as well as between stomatal conductance and transpiration rate (fig. 4). however, substomatal co2 had a negative relationship with photosynthetic rate, transpiration rate and stomatal conductance. biomass production of m. oleifera as influenced by planting density tab. 3 shows the effect of planting density on biomass production at harvest in april 2015. the planting density had a significant influence on both total and leaf biomass yields, where increasing planting density led to an increase in biomass yield. leaf and total biomass yields had no significant difference at the densities of 1667 and 1250 plants ha-1. leaf biomass yield increased by 118.1 and 63.0 kg ha-1 at densities 5 000 and 2500 plants ha-1, respectively, relative to a population of 1250 plants ha-1. on the other hand, total biomass yield increased by 93.2 and 55.5 kg ha-1 at densities 5000 and 2500 plants ha-1, respectively relative to a population of 1250 plants ha-1. discussion weather parameters of the study area during data collection between 2014 and 2015, the study area experienced extreme drought and high temperatures. a total rainfall of 432 mm was received during this period. it was observed from the 2014 and 2015 summer season, that eiland received below average rainfall with the situation worsening during 2015 where the rainfall anomaly was greater than 100 mm below average. these data show the magnitude of abiotic stress experienced in the study area. study by anjum et al. (2011), reported that environmental abiotic stresses, mainly drought, salinity and extreme temperatures could impair plant growth and development and as such limit the productivity of many agricultural crops. monthly gaseous exchange and water use efficiency of m. oleifera as influenced by planting density the effect of planting density on gaseous exchange was not significant during the study period. however, there was noticeable significant effect on monthly gaseous exchange parameters. this might be due to variable monthly temperatures and rainfall which influenced fig. 3: (a) photosynthetic rate (a), (b) transpiration rate (e), (c) sub-stomatal co2 (ci) and (d) stomatal conductance (gs) as affected by season irrespective of planting density. means with different letters are statistically significant. gaseous exchange and water use efficiency of moringa oleifera 223 the response of m. oleifera trees. findings from this study revealed the survival mechanism utilized by m. oleifera to tolerate harsh environmental and climatic conditions. it was observed that m. oleifera does not rely on moisture from immediate rainfall; instead, the tree absorbs and stores water within the roots and other succulent tissues to utilize it when there is water deficit in the soil. this was evident because when rainfall was low or in deficit, m. oleifera still exhi bited high transpiration rate which was influenced by high temperature. however, when temperature continues to rise under moisture shortage, m. oleifera showed adaptation strategy by also reducing the transpiration rate. these results concur with findings by rivas et al. (2013) who reported that m. oleifera can maintain a high leaf relative water content (rwc) under low soil moisture which helps to maintain cellular physiological processes and growth. another study conducted on drought resistant bread wheat cultivars showed that, under drought stress conditions, the cultivars had more rwc (keyvan, 2010). fig. 4: seasonal linear regression for m. oleifera gaseous exchanges. tab. 3: biomass yield (kg. ha -1) production of m. oleifera under different densities at eiland during the harvest in april 2015. density dry leaf yield total dry biomass yield (plants ha-1) (kg ha-1) (kg ha-1) 5000 447.91a 1250.6a 2500 338.80b 1006.6ab 1667 263.13bc 805.2bc 1250 205.33c 647.3c p value 0.000 0.001 cv% 23.79 28.18 significance (0.05) ** ** significance levels: *p<0.05, ** p<0.01, *** p<0.05, ns: not significant. means with different letters are statistically significant. 224 m.p. mabapa, k. kingsley ayisi, i. kwaramba mariga plants use several physiological mechanisms that contribute to their drought tolerance. the first mechanism utilized by most plants, is through decreased stomatal conductance. when the resistance to h2o is greater than that to co2, there is an increase in the water use efficiency (rivas et al., 2013; galle et al., 2011). photosynthetic rate was reduced when there was water deficit in the soil and the plant itself and these response negatively affected water use efficiency (tab. 1). low moisture experienced until november 2014 and in january 2015 with increased temperature decreased stomatal conductance, photosynthetic rate and the extent of sub-stomatal co2. gaseous exchange activities, mainly photosynthetic rate, are among the primary processes to be negatively affected by drought and higher temperatures (chaves, 1991). similar findings were reported by frosi et al. (2017) on the response of two tropical evergreen species to drought stress. they found that gaseous exchange under severe drought stress showed a significant decrease with the average values of 0.01 (mol m-2s-1), 0.66 (mmol m-2s-1) and 0.4 (mmol m-2s-1) for stomatal conductance, photosynthetic rate and transpiration rate, respectively. rivas et al. (2013) also reported a drop in gaseous exchange due to water stress as compared to irrigated m. oleifera plants. when plants encounter water deficit, they respond by lowering stomatal conductance in order to reduce water loss which eventually results in decreased photosynthetic rate (munjonji et al., 2016). seasonal effect on m. oleifera as influenced by gaseous exchange this study revealed a significant influence of season on gaseous exchange of m. oleifera. the higher temperatures that were experienced during summer led to a decrease in photosynthetic rate, stomatal conductance, transpiration rate, while lower temperatures decreased the concentration of sub-stomatal co2. under mild water stress, plants showed small decline in stomatal conductance which is a protective mechanism against stress, by allowing water saving and improving plant water use efficiency (chaves et al., 2009). water use efficiency increased in stressed plants compared to irrigated m. oleifera plants. however, overtime, a rapid recovery from stress was observed on rehydrated plants through increased stomatal conductance (gs), net co2 assimilation (pn), transpiration (e) and decreased water use efficiency (rivas et al., 2013). a nursery study on irrigation interval revealed that irrigating m. oleifera seedlings at 8 days interval had a negative effect on gaseous exchange; however, the internal co2 concentration displayed significantly higher values as compared to shorter intervals (wafa, 2015). the long taproot of m. oleifera and its succulent tubers enhances the drought resistance properties of the tree (jagadheesan et al., 2011). succulent plants often resist moderate water stress by storing water in their succulent tissues and utilizes it during the drought stress period (jagadheesan et al., 2011). findings from this study showed that higher temperature favored assimilation of carbon dioxide by m. oleifera tree (tab. 1). results from current study concur with findings by ndubuaku et al. (2014) who reported that the higher rate of sub-stomatal co2 concentration is an indication that m. oleifera is able to partition carbon into different parts of the plants. no literature is available on seasonal effect of m. oleifera on gas eous exchange. however, results from this study showed that higher temperatures, which prevail mainly in summer months, had a negative effect on transpiration, photosynthetic rate and stomatal conductance, but favored the accumulation of carbon dioxide in three out of four seasons of the year. during winter season, there was an excessive leaf fall and less soil moisture and this significantly affected carbon assimilation and plant growth. under water stress conditions, plants generally produce a lower number of leaves, with general reduction in their size (jagadheesan et al., 2011). muhl et al. (2011) conclu ded that higher temperatures favor growth of m. oleifera, while low temperature leads to thickening of leaves which is symptomatic of an adaptation against temperature stress, resulting in reduced growth. furthermore, jagadheesan et al. (2011), reported that m. oleifera is sensitive to prolonged water stress which may result in a prominent decrease in cellular growth. nevertheless, the tree is still able to produce satisfactory yields and high nutritional content from the leaves under such condition. correlation of m. oleifera gaseous exchange parameters the findings from this study showed a strong positive relationship between photosynthetic rate and transpiration rate as well as stomatal conductance. sub-stomatal co2 showed a moderate to weak negative relationship with photosynthetic and transpiration rates as well as stomatal conductance. therefore, the amount of carbon dioxide assimilated by the tree cannot be determined by photosynthetic rate and transpiration rate as well as its stomatal conductance. the results from this study are in line with findings by araújo et al. (2016), who reported that the impairment of net co2 assimilation rate cannot be attributed to stomatal effects, since stresses did not reduce the intercellular co2 availability. this study further showed a positive relationship between measured stomatal conductance and photosynthetic rate, whereby, the increase in stomatal conductance led to an increase in photosynthetic rate. this relation was mainly influenced by higher temperature and stored moisture in the plant. similar results were reported by muhl et al. (2011) who found out that the reduction in leaf stomatal conductance leads to a decrease in net photosynthetic rate. a study by rivas et al. (2013), further reported that m. oleifera can maintain high leaf relative water content even under drought stress and this helps the plant to maintain physiological processes and growth. leaf biomass production of m. oleifera as influenced by planting density the effect of planting density on biomass was evident whereby the highest planting density of 5000 plants ha-1 produced the highest total leaf dry biomass yield of 1251kg ha-1, with the leaf biomass yield of 448kg ha-1, relative to lower planting densities of 2500, 1667 and 1250 plants ha-1. several authors have reported similar findings, where increase in planting density led to an increase in dry matter yield (foidl et al., 2001; sánchez et al., 2006; basra et al., 2015). planting density of 250 000 and 500 000 plants ha-1 established under dryland conditions had a total dry matter yield of 7.6 and 8.1 tons ha-1, with leaf yield of 4.6 and 4.9 tons ha-1 (sánchez et al., 2006). conclusions in conclusion, this study showed that m. oleifera can survive harsh climatic and environmental conditions such as high temperatures and moisture deficit which commonly occur under the tropical climates. the study further revealed that planting density has no influence on various gaseous exchange parameters of m. oleifera tree. under soil water deficit and high temperature, m. oleifera uses an adaptation strategy by reducing stomatal conductance and transpiration, there by increasing the water use efficiency. the results also revealed that m. oleifera plant has the ability to sequester more carbon in its succulent parts throughout the growing seasons. therefore, m. oleifera can be recommended for planting at a higher density of 5000 plants ha-1 in many parts of limpopo province that experiences high temperatures, as the potential crop to contribute to climate change mitigation and nutritional security given its many nutritional attributes. gaseous exchange and water use efficiency of moringa oleifera 225 acknowledgments we thank ms nomsa ngwenya for providing the seeds and allowing us to conduct the study at her farm. this project was funded by nrf-thuthuka; nrf-rvsc and vlir-iuc-university of limpopo. references anjum, s.a., xie, x.y., wang, l.c., saleem, m.f., man, c., lei, w., 2011: morphological, physiological and biochemical responses of plants to drought stress. afr. j. agric. res. 6, 2026-2032. doi: 10.5897/ajar10.027 araújo, m., santos, c., costa, m., moutinho-pereira, j., correia, c., dias, m.c., 2016: plasticity of young moringa oleifera l. plants to face water deficit and uvb radiation challenges. j. photochem. photobiol. b. 162, 278-285. doi: 10.1016/j.jphotobiol.2016.06.048 asante, w.j., nasare, i.l., tom-dery, d., ochire-boadu, k., kentil, k.b., 2014: nutrient composition of moringa oleifera leaves from two agro ecological zones in ghana. afr. j. plant. sci. 8, 65-71. doi: 10.5897/ajps2012.0727 babu, s.c., 2000: rural nutrition interventions with indigenous plant foods − a case study of vitamin a deficiency in malawi. biotechnology, agron. soc. environ. 4, 169-179. basra, s.m.a., nouman, w., usman, m., nazli, z.e.h., 2015: biomass production and nutritional composition of moringa oleifera under different cutting frequencies and planting spacings. int. j. agric. biol. 17, 1055-1106. doi: 10.17957/ijab/15.0076 chaves, m.m., 1991: effects of water deficits on carbon assimilation. j. exp. bot. 42, 1-16. doi: 10.1093/jxb/42.1.1 chaves, m.m., flexas, j., pinheiro, c., 2009: photosynthesis under drought and salt stress: regulation mechanisms from whole plant to cell. ann. bot. 103, 551-560. doi: 10.1093/aob/mcn125 clifford, s.c., kadzere, i., jones, h.g., jackson, j.e., 1997: field comparisons of photosynthesis and leaf conductance in ziziphus mauritiana and other fruit tree species in zimbabwe. trees. 11, 449-454. doi: 10.1007/pl00009685 daba, m., 2016: miracle tree: a review on multi-purposes of moringa olei fera and its implication for climate change mitigation. j. earth sci. clim. change. 7, 366. doi: 10.4172/2157-7617.1000366 foidl, n., makkar, h.p.s., becker, k., 2001: the potential of moringa oleifera for agricultural and industrial uses. in: what development potential for moringa products? 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(slu acta universitatis agriculturae sueciae, uppsala). rivas, r., oliveira, m.t., santos, m.g., 2013: three cycles of water deficit from seed to young plants of moringa oleifera woody species improves stress tolerance. plant physiol. biochem. 63, 200-208. doi: 10.1016/j.plaphy.2012.11.026 sánchez, n.r., ledin, s., ledin, i., 2006: biomass production and chemical composition of moringa oleifera under different management regimes in nicaragua. agrofor. sys. 66, 231-242. doi: 10.1007/s10457-005-8847-y seresinhe, t., marapana, r.a.u.j., 2011: goat farming systems in the southern province of sri lanka: feeding and management strategies. world. j. agric. sci. 7, 383-390. wafa, e.a.a., 2015: effect of irrigation interval on physiological and growth parameters of moringa oleifera and moringa peregrina seedlings [masters dissertation, university of khartoum (uofk)]. address of the author: university of limpopo, private bag x1106, sovenga, 0727 e-mail: paulina.mabapa@ul.ac.za © the author(s) 2018. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 89, 1 10 (2016), doi:10.5073/jabfq.2016.089.001 laboratory of pomology, department of crop science, agricultural university of athens, athens, greece oil content and composition in relation to leaf photosynthesis, leaf sugars and fruit sugars in maturing koroneiki olives – the mannitol effect on oil mina kafkaletou, eleni tsantili* (received june 21, 2015) * corresponding author summary in koroneiki olive tree, leaf photosynthesis, and sucrose, glucose, fructose and mannitol concentrations in leaves and fruit were in vestigated at fruit maturity index (mi) 1.1, 3.8 and 6.9, along with oil accumulation and composition, total phenolics (tp) and total antioxidant capacity (tac) in fruit during a fully productive season in experiment 1 (i). the effect of mannitol treatment at 50 and 100 mg l-1, applied in mid-october, on oil content and composition, tp and tac were investigated in fruit harvested 25 d after treatment, at an average mi of 3.4, in experiment 2 (ii). in i, in leaves net photosynthesis, and sucrose, glucose and fructose concentrations decreased, but mannitol increased by advancing mi. in fruit, however, concentration of all sugars decreased apart from fructose, which increased. oil content (% dw), already high initially at mi 1.1, increased slowly thereafter, exhibiting decreases in oleic acid (ol) and increases in linoleic (ll). tp and tac decreased at mi 3.8, remaining stable afterwards. in ii, increasing mannitol concentration promoted oil accumulation and ol in oil and reduced ll slightly, indicating an acceleration of olive metabolism. practically, mannitol could be applied to hasten the harvest of olives, so as to avoid adverse winter conditions. introduction olive (olea europaea l.) trees are cultivated in mediterranean climates. they can grow under unfavourable conditions, such as on arid areas and hilly lands, where other fruit trees cannot grow. modern olive cultivation has introduced dense plantations with ir rigation and fertilization regimes, resulting in increased yield and better quality of the products, olives and olive oil (loumou and giourga, 2003). in the mediterranean basin, olives remain among the most important species environmentally and economically (conde et al., 2008). the european union is the leading producer and consumer of olive oil, producing 73 % and consuming 66 % of the world’s olive oil. the data indicate that the european union has a problem with a surplus of 1 or 2 million olive trees (economic analysis of the olive sector, 2012). the growing popularity of olive oil is attributed to its high nutritional value, mainly due to unsaturated fatty acids (fa) and antioxidants (visioli and galli, 2002). olive oil has a balanced composition of fas, with a high content of oleic (ol) acid (80 %) that has beneficial effects on human health and contributes to the resistance of olive oil to oxidation (conde et al., 2008). the olive belongs to the few plants that synthesize both polyols and oligosaccharides. in particular, it is capable of synthesizing the polyol mannitol as well as oligosaccharides of the raffinose family, raffinose and stachyose, all these being the end products of leaf photosynthesis, which along with sucrose are transported from the leaf to the fruit through the phloem (flora and madore, 1993; sanchez and harwood, 2002). recently, research focusing on both yield and quality of olive oil has been increasing. studies including photosynthesis in leaves, being the first step of oil biogenesis, have mainly referred to photosynthetic parameters in relation to fruit load (proietti, 2000) or to leaf sugars and oil content in fruit (proietti, 2003). considering that the olive tree has an alternate fruit-bearing habit, fruit load in connection with fruit quality or oil content/quality is another area attracting much attention (bustan et al., 2011; lavee and wodner, 2004). there is also a great number of studies on fruit harvested at different developmental stages in relation to oil composition (anastasopoulos et al., 2011; bustan et al., 2011), but the respective findings were not usually associated with photosynthesis and its products. the objective of the present work was to study some successive steps throughout the oil biosynthesis chain, such as leaf photosynthesis rate, major sugar concentrations in leaves and fruit, oil content and fa composition in fruit during the late developmental stages, as a very first step to studying sugars and oil. it was desirable to monitor all these steps concomitantly during olive maturation in a fully fruit-bearing year, with emphasis on mannitol as playing an important role in oil biosynthesis. mannitol concentration in fruit was indeed positively correlated with oil content (issaoui et al., 2008; marsilio et al., 2001). however, these results conflicted with observations by others, who claimed that the correlation of oil with fruit mannitol is rather negative (nergiz and engez, 2000; nergiz and ergonil, 2009). an early study (wodner et al., 1988) suggested that fruit mannitol concentration might indicate the potential for oil biosynthesis and suggested a positive effect of mannitol application on oil content. in consequence, any relation between oil and fruit mannitol was also of interest to us, and mannitol being a transportable sugar in tissue, a preharvest treatment with mannitol was also considered. koroneiki was the cultivar studied because it is the main olive cultivar grown in greece and produces olive oil of exceptional quality, being fruity with an aroma of leaves and grass, enriched with notes of green apple and some astringency (anastasopoulos et al., 2011). materials and methods source and handling of fruit self-rooted olive (olea europea l. cv. koroneiki) trees grown on the experimental orchard at the agricultural university of athens (latitude 37o 58΄56΄΄, longitude 23o 42΄47΄΄) were used during two consecutive years and corresponded to experiments 1 (i) and 2 (ii) during the first and second year, respectively. sevenand nine-yearold trees, spaced at 4 × 2.5 m and trained as vase, were used in i and ii, respectively. all trees used were in fully productive seasons. in i, leaf photosynthesis was measured on six leaves per tree and on six trees on 5 november, 19 november and 22 december 2010. on the same dates, approximately 250 leaves and 350 fruit were also harvested from the same six trees. in ii, trees were sprayed once with 50 mg l-1 or 100 mg l-1 mannitol (fluka, sigma-aldrich chemmie gmbh, steinheim, germany) or with water (controls) until run-off at the green olive maturation stage on 17 october 2011. tween-20 at 0.05 % (v/v) was added to all solutions. in ii, approximately 350 olives from three trees per measurement of mannitol concentration were harvested on 10 november 2011. in both experiments, all 2 m. kafkaletou, e. tsantili harvested samples, macroscopically free of disorders and diseases, were transferred to the laboratory. on all harvest dates in i and ii, a subsample of olives (three groups of 100 olives each) was used for the estimation of maturity index (mi) according to hermoso et al. (1991). in i, mean mis for the three replicates and the correspon ding ses of the means for the harvested olives were 1.1 (± 0.09), 3.8 (± 0.26) and 6.9 (± 0.08) on the three successive harvest dates, respectively, corresponding to green, cherry and black olives, respectively. in ii, mis for harvested controls and olives treated with 50 and 100 mg l-1 mannitol were 3.3 (± 0.185), 3.5 (± 0.23) and 3.5 (± 0.22), respectively. on each sampling day, sampling and sorting of leaves or fruit were all carried out according to a completely randomized design. harvested leaves and fruit were stored at -80 oc for a short time, freeze-dried and stored at -80 oc again until analyses. leaf photosynthetic parameters photosynthetic parameters were measured on leaves near fruit on fruit-bearing shoots and selected randomly from the outer part of the canopy at a height of 1.5 m from the ground. four measurements per leaf were carried out around 11.00 am – 1.00 pm on cloudless days with a portable photosynthesis system (li-6400, li-cor, lincoln, ne, usa). the system operated at a steady light intensity (1200 μmol photons m-2 s-1), provided by an led light source, and co2 concentration (400 μl l-1). the air flow in the chamber was 300 ml min-1. leaf net photosynthesis (pn), stomatal conductance (gs), substomatal co2 concentration (ci) and transpiration rate (e rate) were expressed as the rate of moles of co2 assimilation per leaf surface unit (μmol m-2 s-1), the rate of moles of h2o exiting per leaf surface unit (mol m-2 s-1), the moles of co2 per mole air (μmol mol air-1) and the rate of moles of h2o exiting per leaf surface unit (mmol m-2 s-1), respectively. extraction and determination of soluble sugars sucrose, glucose, fructose and mannitol in both leaves and fruit were determined on powdered (with mortar and pestle) samples (roussos et al., 2010). briefly, 50 mg of powdered tissue were extracted with 2 ml water (hplc grade) in a microwave apparatus at 400 w for 2 min, thrice. the extractions were centrifuged at 5000 × g for 6 min, thrice. the combined supernatants were filtered through a nylon syringe filter (0.2 μm pore size) before hplc analyses. the separation of sugars was achieved with a hamilton hc-75 cation exchange column, calcium form, (bonaduz, switzerland) at 80 oc, operating with a water 510 isocratic pump with a flow of 0.6 ml min-1 and connected to a refractive index detector in an hp 104 7a hplc (hewlett-packard, waldbronn, germany) system. peaks were identified and quantified with standards using a data processing system (peak simple 3.25), and expressed on a dry weight basis (μmοl g-1 dw). moisture and oil content in fruit fruit slices from approximately 60 olives were dried at 60 oc for 3 days and then at 105 oc for 3 h. weights before and after drying were used for the expression of results on a dry weight basis, minimizing the fluctuations due to different moisture content in samples. oil percentage was estimated according to tsantili (2014). the total oil content of fruit was determined from 5 g of dried sample extracted for 6 h with 50 ml petroleum ether (b.p. 40-60 oc) using a soxhlet apparatus. extraction of oil for fa composition a cold-pressing method with a laboratory screw-press device was used for oil extraction (christopoulos and tsantili, 2015). chilled (at 4 oc) fruit slices (50 g) were compressed between two parallel stainless steel plates of 10 × 5 cm pre-cooled at 4 oc and each at a torque of 30 n m-1. the recovered oil was used for analyses after clarification by centrifuging at 5000 × g for 3 min. fatty acid composition the fa composition of the oil samples was determined by gas chromatography of fatty acid methyl esters (fames). esterification was conducted in tubes (12 × 100 mm) with teflon-lined screw cap. one hundred milligrams of oil and 1 ml 0.5 n methanolic potassium hydroxide solution were added to the tube, the headspace was flushed with n2 and the tube was heated in a water-bath at 90 °c for 10 min. after cooling the tube smoothly, 1 ml 14 % (w/v) methanolic boron trifluoride was added. the heating step was then repeated, followed by cooling at room temperature. one millilitre of deionized water and 0.5 ml hexane were added and fames were extracted by vigorous shaking for 1 min. the tubes were then centrifuged at 2,500 × g for 5 min and the top hexane layer was transferred to a vial for gc analysis (christopoulos and tsantili, 2015). fas were analyzed by injecting 1 μl fame into a glc (hp 5890 series ii, hp, usa) equipped with a split/splittless injector (ratio 50:1), a flame ionization detector and a capillary column (db-23, j &w scientific, uk; 60 m length × 0.25 mm i.d., 0.25 μm film thickness), according to christopoulos and tsantili (2015) after some modifications. the carrier gas was helium. the injector and detector temperatures were 270 oc and 280 oc, respectively. the oven temperature program was as follows: isotherm at 190 oc for 30 min, increase to 230 oc at a rate of 10 oc min-1 and isotherm at 230 oc for 5 min. the identification and quantification of peaks were carried out with fame standards (glc-20, supelco, uk; me93, larodan fine chemicals, sweden), and the results were expressed as % (w/w) in oil. extraction for total phenolics (tp) and total antioxidant capacity (tac) frozen olive slices were powdered with a mortar and pestle in liquid nitrogen before phenolic extraction. a quantity of 500 mg of powdered tissue was added to 80 % v/v acetone in deionized water (1 ml 100 mg-1 tissue) and the mixture was placed in an ultrasonic ice-bath for 15 min. the samples were then centrifuged at 4000 × g for 5 min. the extraction was repeated thrice and the combined supernatants were used for tp and tac (tsantili, 2014). determination of tp and tac a modified folin-ciocalteu method was used for the tp determination (tsantili et al., 2010). specifically, 0.2 ml of diluted olive fruit extract was added to a tube containing 2.6 ml of water and 0.2 ml of folin-ciocalteu reagent. the tube was stirred and allowed to stand at room temperature for 6 min. then, 2 ml (7 % w/v) of sodium carbonate was added to the mixture. after 90 min, absorbance was measured at 750 nm versus a blank. the results were expressed as gallic acid equivalents on a dry weight basis (μmοl g-1 dw). tac was estimated according to brand-williams et al. (1995). a volume of 0.1 ml of extract diluted with deionized water was added to a tube containing 3.9 ml 2,2-diphenyl-1-picryhydrazyl solution (0.06 mm in methanol). after 30 min incubation at room temperature, the decrease in absorbance was measured at 515 nm versus a blank. the results were expressed as trolox acid (6-hydroxy-2,5,7,8,tetramethylchroman-2-carboxylic acid) equivalents on a dry weight basis (μmοl g-1 dw). statistical analyses the significance of the effect of sampling date or mi in i and of mannitol concentration treatment in ii on the measured variables were oil and sugars in koroneiki olives 3 estimated by one-way analyses of variance (anova). the presented ses in tables and figures were calculated from the residual variances. photosynthetic parameters were estimated on three replicates of 12 leaves each, whereas the remaining determinations were on three replicates of 60 leaves or fruit each. principal component analyses and pair wise correlations were applied to get an overview of the main variation in the data and interpret variable relationships among the variables determined in i. data analyses were conducted using jmp 7.0.1 (sas institute, cary, nc, usa). results and discussion photosynthetic parameters the pn was at 17.1 μmol co2 m-2 s-1 (fig. 1a) on the first measuring date, but decreased gradually to 14.8 μmol co2 m-2 s-1 on the third date, with the seasonal change being significant (p < 0.05) and affected by decreases in temperature, while the light intensity was stable (1200 μmol photons m-2 s-1), as described earlier in materials and methods. during measurements, the temperature was 22.3 oc, 20.8 oc and 16.3 oc on the first, second and third date, respectively, while the corresponding rh was 32.4 %, 37.2 % and 46.2 %. pn on the last date (22 december) was rather high and might be attributed to the relatively high temperature for the winter season. in an earlier study, the leaves of the koroneiki olive cultivar were found to have the highest pn among olive cultivars, while pn in all studied cultivars was lower during summer and late autumn (end of november) than during spring and early autumn (hagidimitriou and pontikis, 2005). however, in arbequina and maurino, pn, measured monthly from july to november, was highest in mid november (proietti et al., 2012). in this study, the values of gs ranged between 0.186 and 0.173 mol m-2 s-1, while those of ci between 246 and 205 μmol mol air-1 (fig. 1b and 1c, respectively), but the changes in both parameters were not significant (p > 0.05). the e rate also did not significantly decrease during the season (fig. 1d), although the effect of date was of borderline significance (p = 0.0503). in another olive study (proietti et al., 2012), gs decreased and ci and e rate increased, but determinations were carried out for many months, allowing the opportunity to exhibit large changes, in contrast to the present results determined only within 1.5 months. stomatal density was lowest in koroneiki among five cultivars studied (hagidimitriou and pontikis, 2005), providing an adequate justification of the high resistance of koroneiki to drought. in the present results, this could be associated with almost stable gs when pn decreased, indicating non-stomatal limitation for leaf photosynthesis. the pairwise correlation among photosynthetic parameters showed non-significant relations (tab. s1). in contrast to this, a positive linear relation between pn and gs was found in control coratina olive leaves measured on young trees early in summer (angelopoulos et al., 1996). major soluble sugars in leaves and fruit in leaves, the initial concentrations of sugars were 31.93, 218.1, 33.9 and 140.4 μmol g-1 dw for sucrose, glucose, fructose and mannitol, respectively (fig. 2a, 2c, 2e, 2g). sucrose, glucose and fructose decreased gradually, resulting in final concentrations reduced by 1.64-, 1.22and 1.17-fold, respectively, in comparison to the initial ones. in contrast, mannitol concentration in leaves gradually rose, reaching a final level of 1.34-fold higher than the initial value, while the pn decreased by 1.2-fold within the same time period. the seasonal effect was significant for all sugar changes in leaves (fig. 2). in another study on koroneiki, leaf glucose and fructose were at higher and sucrose at lower levels than here, while the leaf mannitol level was similar to the present results (roussos et al., 2010). however, the experimental conditions were different from this study since the trees were very young and growing in pots. a detailed study on carbohydrate allocation to vegetative tissues and roots in barnea olive tree in a fully production year showed that in leaves, mannitol and fig. 1: net photosynthesis (pn), stomatal conductance (gs), intracellular co2 concentration (ci) and transpiration rate (e rate) in response to seasonal development in koroneiki leaves, in 2010. a, pn; b, gs; c, ci; d, e rate. bars without numbers correspond to standard deviations; bars with numbers to standard errors from the residual variance (anova) of three replicates, d.f. = 6. in a, p < 0.05, whereas in b, c and d, p > 0.05 in all of them. 4 m. kafkaletou, e. tsantili sucrose concentrations were highest in february, but declined thereafter, exhibiting fluctuations (bustan et al., 2011). in november, leaf mannitol and sucrose were approximately 236 and 117 μmol g-1 dw, respectively, with both sugars being much higher than found here. according to the findings in barnea, mannitol is the predominant carbohydrate in leaves, and this agrees with the present results for december, but disagrees with the sugars determined earlier here, when glucose was found at higher levels than mannitol. indicatively, in other olive studies, the molar ratio of glucose/mannitol was 1.9 (flora and madore, 1993) or 1.2 (cataldi et al., 2000) in olive fig. 2: sucrose, glucose, fructose and mannitol, in leaves and fruit, in response to seasonal development and fruit maturity indices (mi) in koroneiki olive fruit, in 2010. a, c, e and g, leaves; b, d, f and h, fruit. bars without numbers correspond to standard deviations; bars with numbers to standard errors from the residual variance (anova) of three replicates, d.f. = 6. in e, p < 0.05, in d, p < 0.01, whereas in a, b, c, f, g and h, p < 0.001 in all of them. oil and sugars in koroneiki olives 5 plant extracts. indeed, the present concentration ratio of leaf glucose/mannitol ranged from 1.55 to 0.94 at mis from 1.1 to 6.9, respectively. glucose and mannitol in koroneiki leaves did remain the major sugars during the whole experiment, although exhibiting an opposite pattern of changes. significant relationships were observed among all leaf sugars, being negative between mannitol and sucrose, and glucose and fructose, but positive for the remaining relationships (tab. s1). in leaves, mannitol showed the strongest, but negative correlation with sucrose (r = -0.923). additionally, significant correlations were found among photosynthetic parameters and sugars, with that between pn and leaf mannitol being the strongest and negative (r = -0.821), followed by positive correlation between pn and leaf sucrose (r = 0.804) (tab. s1). in other words, leaf mannitol increased at reduced photosynthesis late in the season and in contrast to decreasing leaf sucrose. it is well known that mannitol and sucrose or raffinose saccharides are direct photosynthetic products and comprise the transportable forms of carbon. in particular, mannitol is synthesized in the cytosol and transported to vacuoles rapidly, and indeed, in the mesophyll close to the co2 fixation site (flora and madore, 1993). it is synthesized in mature leaves from mannose-6-phosphate, translocated via the phloem to sink tissues, stored or oxidized to mannose, and used for energy supply or a carbon source (conde et al., 2008). additionally, it has a protective role in plants, being a compatible solute and an oxygen radical scavenger (shen et al., 1997; lo bianco et al., 2011), coping with abiotic or biotic stress. in general, soluble sugars might protect plant tissues from stress, either directly when they exist at high concentrations (van den ende and valluru, 2009) or due to their signalling role, resulting indirectly in production of reactive oxygen species scavengers (bolouri-moghaddam et al., 2010). indeed, mannitol was found to increase the capacity of chloroplasts to scavenge radicals and in contrast to sucrose, glucose and fructose, it does not repress photosynthesis or result in any harmful effect even at high concentrations (bolouri-moghaddam et al., 2010; shen et al., 1997). consequently, the mannitol increase in koroneiki leaves in december might be attributed to a protective role against subsequent winter conditions. in this study, the seasonal effect was considered as an integrated factor influencing fruit, reflecting mainly the interaction between fruit state and weather conditions. fruit load does not seem to affect sugar allocation between leaves and fruit, apart from some extreme cases, such as severe fruit thinning, when soluble sugar accumulation in leaves and fruit was observed (bustan et al., 2011; haouari et al., 2013). in fruit in our study, the concentrations of sucrose, glucose, fructose and mannitol at mi 1.1 were 75.81, 271.81, 4.67 and 37.28 μmol g-1 dw, respectively (fig. 2b, 2d, 2f, 2h). during the growing season, sucrose, glucose and mannitol decreased, whereas fructose increased. decreases by 1.21-, 1.19and 1.66-fold in sucrose, glucose and mannitol, respectively, and an increase by 1.79-fold in fructose were observed between the first and third date. however, the sugar changes, albeit significant, occurred mainly at mi 3.8, and the levels remained almost stable thereafter. the sum of these sugars, expressed per dw, decreased (by 1.21-fold) during fruit development, and glucose remained the predominant sugar in fruit during the entire experiment. these decreases in sugar and glucose predominance in fruit both agreed with other work on three olive cultivars studied from august to december (wodner et al., 1988). the main difference between the two studies lies in the absolute glucose concentrations, being high in koroneiki and low in the other cultivars. in contrast to these results, mannitol concentration increased during maturation in hojiblanca olives (marsilio et al., 2001), reaching levels close to those in green koroneiki fruit, whereas the minimum sucrose level in koroneiki fruit was higher than the minimum in hojiblanca. the differences could be ascribed to compensation between mannitol and sucrose in fruit, depending on maturity stage, cultivar or environment. moreover, in black olives, mannitol varied between 21 in duro and 99 μmol g-1 dw in thasos (marsilio et al., 2001), with mannitol in koroneiki being within this range. it should be noted that koroneiki, being a late ripening cultivar with long oil-filling period (parvini et al., 2015), possibly utilizes mannitol for longer periods than other cultivars. indicatively, koroneiki fruit exhibited significant and positive correlations between mannitol and sucrose or glucose, but negative between fructose and mannitol, and sucrose and glucose (tab. s1). relations between sucrose and fructose (r = -0.975) and between mannitol and glucose (r = 0.935) were among the strongest observed. among the relations of fruit sugars with leaf sugars, fruit mannitol with leaf sucrose exhibited a highly significant positive relation, with the highest correlation coefficient observed (r = 0.907; tab. s1). taking into consideration the systems of sugar loading into the phloem and unloading and the complexity of sugar interconversions and meta bolism, it would not be possible to gain detailed information about sugar transport. moreover, fruit photosynthesis occurs at the green stage (blanke and lenz, 1989) and contributes to fruit carbon economy. in the fruit gas phase, co2 concentration rises not only due to fruit mitochondrial respiration of the photoassimilates imported from the leaves via the phloem, but also due to the prevention of co2 diffusion by cuticle impermeability. in green olives, particularly, it was shown that the photosynthetic products contribute to some extent to oil synthesis (sanchez, 1995). oil content in this study, oil content was 54.2, 60 and 64.71 % (w/w dw) at mi 1.1, 3.8 and 6.9, respectively, and this gradual increase was affected significantly by advancing mi (tab. 1). however, the majority of oil accumulation seemed to take place at mi <1.1. this observation was in general agreement with other studies on olive cultivars (wodner et al., 1988), where approximately 80 % of the oil content was accumulated until fruit reached 50 % black stage. additionally, koroneiki exhibited approximately 8.3 % mannitol in fruit and an already high oil accumulation at mi 1.1, in accordance with the proposal that fruit mannitol indicates the potential for oil accumulation (wodner et al., 1988). in other studies, the maximal oil content was observed at different mi, with differences depending on cultivar (bodoira et al., 2015; dag et al., 2014). the main oil constituents, triacylglycerols, are formed from the fas synthesized in the plastids with glycerol-3-phosphate via the kennedy pathway. fa biosynthesis, concisely, requires acetyl-coa as a precursor, which can be formed from carbohydrates via glycolysis in the plastid, as well as, from pyruvate in the mitochondria that hydrolyzed to acetate, transported to plastid and forms acetyl-coa. (sanchez and harwood, 2002). in this work, the oil percentage exhibited significant and relatively strong correlations with all determined sugars in both leaves and fruit. oil accumulation exhibited particularly strong and negative correlations with leaf sucrose (r = -0.979) and fruit mannitol (r = -0.953), while being positively correlated with leaf glucose (r = 0.937) and leaf mannitol (r = 0.915) (tab. s1). the negative correlation of fruit mannitol and oil accumulation during the late stages of fruit development here agrees with the findings of two studies (nergiz and engez, 2000; nergiz and ergonil, 2009), while not necessarily disagreeing with others (marsilio et al., 2001; wodner et al., 1988). wodner et al. (1988) reported that the high levels of fruit glucose in uovo di piccione and of fruit fructose in manzanillo at the beginning of august, followed by their sharp decrease, might be responsible for the rapid oil accumulation in those cultivars. the discrepancy of results between studies might be attributed to different experimental conditions, cultivars and season. the significant and negative correlation between oil percentage and 6 m. kafkaletou, e. tsantili pn (r = -0.757; tab. s1) was of interest, and might be associated with the late season that the experiment took place. cherbiy-hoffmann et al. (2015) showed that shading applied at the beginning of phase iii of fruit development reduced oil accumulation, directly confirming the necessity for sunlight and photosynthesis during olive maturation. in this study, the increase in leaf mannitol might reflect either the increased requirement of leaves for mannitol to cope with seasonal change and stress, or ‘reduced transport necessity/ability’ of mannitol to fruit after the completion of oil accumulation. indeed, it was postulated that leaves have an important role as carbohydrate storage organs, while exhibiting prolonged photosynthetic activity to store adequate levels of reserves for tree survival under the unpredictable mediterranean climatic conditions (bustan et al., 2011). oil composition the oil composition is very important for human nutrition. at mi 1.1, the values of palmitic (pa), palmitoleic (po), stearic (st), ol, vaccenic (va), ll, linolenic (ln), arachidic (ar) and gondoic (go) were 11.4, 1.2, 2.32, 73.43, 2.53, 6.70, 1.1, 0.49 and 0.4 % (w/w in oil), respectively (tab. 1). pa decreased at mi 6.9, while ol decreased at mi 3.8 and remained almost stable thereafter. po showed no consistent changes, but ll exhibited a continuous increase that was highly significant. the remaining acids st, va, ln, ar and go were fairly constant. all these observations were confirmed statistically (tab. 1). at the end of the experiment, pa and ol were reduced by 1.1and 1.03-fold, respectively, while ll increased by 0.64-fold in comparison to initial levels. at mi 1.1, the values of saturated fatty acids (sfa), mono-unsaturated fatty acids (mufa), poly-unsaturated fatty acids (pufa), unsaturated fatty acids/saturated fatty acids (ufa/sfa) and linolenic acid/linoleic acid (ω-6/ω-3) were 14.22, 77.55, 7.8, 6 and 6.09, respectively, but significant changes occurred, resulting in sfa and mufa values reduced by 1.08and 1.04-fold, respectively, whereas pufa, ufa/sfa and ω-6/ω-3 increased by 1.48-, 1.09and 1.43-fold, respectively, by the end of the experiment. these changes during fruit maturation were in general agreement with another study on koroneiki (anastasopoulos et al., 2011). here, ol was already at 73 % of total fas early in november. ol then decreased to approximately 70 %, indicating that the most active oil biosynthesis occurred early during fruit development, in agreement with other studies (bodoira et al., 2015). the decrease in ol at the two last sampling dates resulted in an apparently negative correlation of ol with oil percentage (tab. s1). ol was also significantly positively correlated with fruit glucose, sucrose and mannitol, and leaf glucose and sucrose, with the strongest correlation being with fruit glucose (r = 0.955) (tab. s1). the main fas in koroneiki oil were found to be ol and ll (tab. 1), as expected. in general, stearoyl-acp δ9-desaturase in the plastids and oleoyl-acp δ9-desaturase in the endoplasmic reticulum are tab. 1: content of individual fatty acids (fa), total saturated (sfa), mono-unsaturated (mufa), poly-unsaturated fa (pufa) and total unsaturated fa (ufa), on the unsaturation degree (ufa/sfa) and ω-6/ω-3 ratios in oil, as well as, on oil content, total phenolics concentration (tp) and total antioxidant capacity (tac) in the flesh in koroneiki olive fruit, in response to seasonal development and fruit maturity indices (mi) in 2010. units date – mi seb p c 5/11 – mi 1.1 19/11 – mi 3.8 22/12 – mi 6.9 palmitic c16:0 % 11.41 ± 0.10 a 11.73 ± 0.28 10.20 ± 0.21 0.12 *** palmitoleic c16:1 n-9 % 1.20 ± 0.06 1.39 ± 0.05 1.19 ± 0.07 0.03 * stearic c18:0 % 2.32 ± 0.04 2.42 ± 0.08 2.41 ± 0.16 0.06 ns oleic c18:1 n-9 % 73.43 ± 0.46 70.14 ± 0.66 70.75 ± 0.50 0.31 *** vaccenic c18:1 n-11 % 2.53 ± 0.06 2.67 ± 0.09 2.47 ± 0.10 0.05 ns linoleic c18:2 n-9,12 % 6.70 ± 0.39 9.09 ± 0.34 10.38 ± 0.13 0.18 *** linolenic c18:3 n-9,12,15 % 1.10 ± 0.05 1.16 ± 0.04 1.19 ± 0.05 0.03 ns arachidic c20:0 % 0.49 ± 0.01 0.53 ± 0.04 0.54 ± 0.04 0.02 ns gondoic c20:1 n-11 % 0.40 ± 0.02 0.41 ± 0.01 0.41 ± 0.01 0.01 ns sfa cv:0 % 14.22 ± 0.07 14.67 ± 0.35 13.15 ± 0.39 0.18 ** mufa cv:1 % 77.55 ± 0.45 74.61 ± 0.70 74.83 ± 0.41 0.31 *** pufa cv:n (n≥1) % 7.80 ± 0.41 10.25 ± 0.38 11.57 ± 0.13 0.19 *** ufa 85.35 ± 0.04 84.85 ± 0.33 86.40 ± 0.39 0.17 ufa/sfa 6.00 ± 0.03 5.78 ± 0.16 6.57 ± 0.22 0.09 ω-6/ω-3 6.09 ± 0.29 7.85 ± 0.09 8.75 ± 0.40 0.17 *** oil % 54.28 ± 0.59 60.03 ± 1.32 64.71 ± 0.97 0.58 *** tp μmol g-1 208.00 ± 10.24 136.63 ± 6.00 140.63 ± 4.68 4.25 *** tac μmol g-1 161.66 ± 2.29 104.00 ± 8.26 107.66 ± 4.25 3.19 *** a numbers are means ± standard deviation b standard error from the residual variance (anova) of three replicates, d.f. = 6. c probabilities. ns, not significant. *significant at p < 0.05. **significant at p < 0.01. ***significant at p < 0.001. % ** o il fl es h d w oil and sugars in koroneiki olives 7 responsible for ol and ll synthesis, respectively (sanchez and harwood, 2002). during the late developmental stages, ll increases at expense of ol were possibly due to oleoyl-δ9-desaturase, while stearoyl-δ9-desaturase seemed to be still active for ol synthesis (banilas et al., 2005). indeed, in this work, ll was positively related to oil percentage (r = 0.979), but negatively to ol. as mentioned earlier, ll contributes to oil oxidation, which is undesirable and thus, these increases should be avoided. changes in sfa, mufa, pufa, ufa/sfa could be largely attributed to ol and ll changes. the ufa/sfa ratio along with the ol percentage value belong to the criteria for the characterization of oil quality. koroneiki exhibits a high proportion of ol, being higher than in other cultivars (parvini et al., 2015). additionally, decreases in the ratio of mufa/pufa were connected with oil deterioration during advanced fruit development and/or in relation to field temperature increases (dag et al., 2014). here, mufa/pufa decreased gradually, being 9.94, 7.27 and 6.46 at mi 1.1, 3.8 and 6.9, respectively, indicating a deterioration at advanced maturation. the ratio of ω-6/ω-3 was included into the present results since it is considered important for diseases prevention or therapy (simopoulos, 2008). total phenolics (tp) and total antioxidant capacity (tac) at mi 1.1, determinations of tp and tac were 208 μmol g-1 dw and 161.66 μmol g-1 dw, respectively (tab. 1). both variables reduced considerably at mi 3.8 and remained almost stable thereafter. at mi 6.9, tp and tac were reduced by 1.47and 1.5-fold, respectively, against the initial values. these reductions during ripening complied with other studies (morello et al., 2005). here, tp was positively and strongly related to tac, as expected (tsantili, 2014). both tp and tac showed highly significant and positive correlations with fruit sucrose, glucose and mannitol, but negative with fruit fructose (tab. s1). these correlations could be explained by the antioxidant character of sugars, as mentioned earlier. it is also noted that tac exhibited a highly significant, positive and strong relationship with ol (r = 0.973), but a negative one with ll (r = -0.903), suggesting the prevention of ll increases. principal component analysis overview the pca was performed for all 24 variables determined and already presented in fig. 1, fig. 2 and tab. 1. it showed two interpretable components, explaining together 76.05 % (eigenvalue 3.55) of the total variation (fig. 3). in score plot, all fruit were separated according to their mi, in green, red and black olives. correlations among the variables presented in tab. s1 are summarized in the load plot, in two main groups, g1 and g2, the left-side and the right-side groups, respectively. particularly, g1 includes ci, leaf mannitol, fruit fructose, oil %, ll, ln, ar and go, whereas g2 includes pn, leaf fructose, leaf sucrose, leaf glucose, fruit mannitol, fruit sucrose, fruit glucose, ol, tp, tac and e rate. positive correlations are shown among the variables in each separate group, whereas negative ones among variables between groups. the mannitol effect the previous experiment confirmed the close relationship among sucrose and glucose in both leaf and fruit, and oil content. however, the reduction of fruit mannitol in parallel with oil accumulation was not a definitely expected observation. it is known that the higher mannitol in fruit, the more energy is released from mannitol degradation that could be available for oil synthesis via acetyl-coa (marsilio et al., 2001). considering the transportable character of mannitol (conde et al., 2008) in conjunction with the above observations, the authors proceeded to preharvest treatments with mannitol. indeed, in ii, the oil percentage increased progressively by advanced mannitol concentration, being higher by 1.043and 1.076-fold in fruit treated with the lower and higher mannitol concentration, respectively, in comparison with controls (tab. 2). additionally, ol, st and mufa increased, whereas ll, pufa and ω-6/ω-3 were lowered by increasing mannitol concentration. also, the ratio of mufa/ pufa increased from 7.43 in controls to 8.41 and 8.97 by mannitol at 50 and 100 mg l-1, respectively. these changes seemed to conform to the retention of oil quality in mannitol treated olives. values of pa, po, va, ln, ar, go, sfa and ufa/sfa were not affected significantly by the treatment. however, in ii, mi of controls and fig. 3: principal component analysis (pca) in koroneiki olive tree according to 24 variables (related to leaf photosynthetic parameters and sugars, and fruit sugars, oil content, fatty acids and total antioxidants) affected by seasonal growth maturity index, in 2010 (a and b). in pca: a, score plot; b, load plot. in a: mi, maturity index; circle, green olives at mi 1.1; squares, red olives at mi 3.8; rhombus, black olives at mi 6.9. in b: dagger indicates the position of each variable in load plot. numbers in parentheses correspond to the percentage of the total variance explained by each component. l, leaf; f, fruit. 8 m. kafkaletou, e. tsantili treated olives with the lower and higher mannitol concentration were 3.3, 3.5 and 3.5, respectively, whereas the treatment effect was not significant (p > 0.05; se = 0.212). besides, mannitol concentration reduced tp and tac in fruit, and this could be attributed rather to an effect of mannitol on promoting the olive metabolism. the current results seemed to be promising for increasing the oil accumulation, while no quality oil deterioration seemed to occur. conclusions this study was a first attempt to investigate different steps in the chain of oil accumulation during fruit maturation in koroneiki olives. being aware of the long chain and the complexity of various steps, the study was focused on photosynthesis, as the initial step, on changes in concentrations of the major sugars in leaves and fruit, and on oil accumulation and composition in green, green-cherry and black olives from early november up to late december. according to results, leaf sucrose, glucose and fructose decreased during fruit maturation, while leaf mannitol increased, with glucose and mannitol remained the major sugars in leaves on all sampling dates. in fruit, glucose and sucrose were the major sugars, even after their decreases during fruit maturation. however, fruit fructose increased, whereas fruit mannitol decreased. preharvest treatment with mannitol resulted in increased oil accumulation without decreasing the oil quality, as estimated by fa analysis. the mannitol effect seemed to be related to an accelerated metabolism. koroneiki exhibits a long oil accumulation period. thus, mannitol treatment seemed to be promising for increasing the oil accumulation earlier than usual and thus, harvest would be completed before the unfavourable winter conditions that make the harvest hard and deteriorate the oil quality. however, the positive mannitol treatments raised questions concerning the physiology of exogenous mannitol uptake, transport and utilization. consequently, further studies are needed to investigate the mannitol effects from the aspect of basic research and of applied, as well. references anastasopoulos, e., kalogeropoulos, n., kaliora, a.c., koun touri, a., andrikopoulos, n.k., 2011: the influence of ripening and crop year on quality indices, polyphenols, terpenic acids, squalene, fatty acid profile, and sterols in virgin olive oil (koroneiki cv.) produced by organic versus non-organic cultivation method. int. j. food sci. technol. 46, 170-178. angelopoulos, k., dichio, b., xiloyannis, c., 1996: inhibition of photosynthesis in olive tress (olea europea l.) during water stress and rewatering. j. exp. bot. 47, 1093-1100. banilas, g., moressis, a., nikoloudakis, n., hatzopoulos, p., 2005: spatial and temporal expressions of two distinct oleate desaturases from olive (olea europaea l.). plant sci. 168, 547-555. tab. 2: effect of preharvest mannitol treatment on content of individual fatty acids (fa), total saturated 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0.31 ns mufa cv:1 % 72.19 ± 0.63 73.62 ± 0.96 74.44 ± 0.74 0.46 * pufa cv:n (n≥1) % 9.71 ± 0.16 8.75 ± 0.49 8.29 ± 0.53 0.25 * ufa 81.89 ± 0.47 22.37 ± 0.53 82.73 ± 0.29 0.26 ns ufa/sfa 4.61 ± 0.19 4.79 ± 0.25 4.84 ± 0.09 0.10 ns ω-6/ω-3 9.58 ± 0.70 9.20 ± 0.78 7.29 ± 0.85 0.45 * oil % 52.30 ± 1.18 54.55 ± 1.35 58.73 ± 0.85 0.66 *** tp μmol g-1 230.44 ± 10.48 172.22 ± 4.63 184.12 ± 9.65 4.99 *** tac μmol g-1 146.75 ± 3.09 99.44 ± 8.37 128.31 ± 4.93 3.40 *** a numbers are means ± standard deviation. b standard error from the residual variance (anova) of three replicates, d.f. = 6. c probabilities. ns, not significant. *significant at p < 0.05. **significant at p < 0.01. ***significant at p < 0.001. % o il fl es h d w oil and sugars in koroneiki olives 9 blanke, m.m., lenz, f., 1989: fruit photosynthesis. plant cell environ. 12, 31-46. bodoira, r., torres, m., pierantozzi, p., taticchi, a., servilli, m., maestri, d., 2015: oil biogenesis and antioxidant compounds from ‘arauco’ olive 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(eds.), plant lipid metabolism, 564-566. kluwer academic publications, the netherlands. sanchez, j., harwood, j.l., 2002: biosynthesis of triacylglycerols and volatiles in olives. eur. j. lipid sci. technol. eur. j. lipid sci. technol. 104, 564-573. shen, b., jensen, r.g., bohnert, h.j., 1997: mannitol protects against oxidation by hydroxyl radicals. plant physiol. 115, 527-532. simopoulos, a.p., 2008: the importance of the omega-6/omega-3 fatty acid ratio in cardiovascular disease and other chronic diseases. exp. biol. med. 233, 674-688. tsantili, e., 2014: quality attributes and their relations in fresh black ripe ‘kalamon’ olives (olea europea l.) for table use – phenolic compounds and total antioxidant capacity. int. j. food sci. technol. 49, 657-665. tsantili, e., shin, y., nock, j.f., watkins, c.b., 2010: antioxidant concentrations during chilling injury development in peaches. postharvest biol. technol. 57, 27-34. van den ende, w., valluru, r., 2009: sucrose, sucrosyl oligosaccharides, andoxidative stress: scavenging and salvaging? j. exp. bot. 60, 9-18. visioli, f., galli, c., 2002: biological properties of olive oil phytochemicals. crit. rev. food sci. nutr. 42, 209-221. wodner, m., lavee, s., epstein, e., 1988: identification and seasonal changes of glucose, fructose and mannitol in relation to oil accumulation during fruit development in olea europea (l.). sci. hortic. 36, 47-54. address of the corresponding author: laboratory of pomology, department of crop science, agricultural university of athens, 75 iera odos, 118 55 athens, greece e-mail: etsantili@aua.gr © the author(s) 2016. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). 10 m. kafkaletou, e. tsantili ta b. s 1: p ai rw is e co rr el at io ns a m on g ph ot os yn th et ic p ar am et er s, s ug ar s in le av es a nd f ru it, o il co nt en t, in di vi du al f at ty a ci ds ( fa ), to ta l p he no lic s co nc en tr at io n (t p) a nd to ta l a nt io xi da nt c ap ac ity ( ta c ) in o liv e fr ui t o f d iff er en t s am pl in g da te s – m at ur ity in de x( m i) , i n 20 10 # . 1 t ab . s 1: p ai rw is e co rr el at io ns a m on g ph ot os yn th et ic p ar am et er s, s ug ar s in le av es a nd f ru it, o il co nt en t, in di vi du al f at ty a ci ds ( fa ), to ta l p he no lic s co nc en tr at io n (t p) an d to ta l a nt io xi da nt c ap ac ity ( t a c ) i n ol iv e fr ui t o f d if fe re nt s am pl in g da te s – m at ur ity in de x( m i) , i n 20 10 . pn g s c i e ra te su c l g l u l fr u l m a n l su c f g l u f fr u f m a n f % o il pa po st o l v a l l l n a r g o t p g s 0. 29 8n s c i -0 .4 90 n s -0 .4 10 n s e ra te 0. 47 8n s 0. 64 4n s -0 .3 69 n s su c l 0. 80 4* * 0. 08 7n s -0 .6 92 * 0. 53 3n s g l u l 0. 58 1n s 0. 33 1n s -0 .6 94 * 0. 78 0* 0. 87 6* * fr u l 0. 72 5* 0. 15 9n s -0 .3 05 n s 0. 66 8* 0. 81 3* * 0. 81 9* * m a n l -0 .8 21 ** 0. 05 1n s 0. 51 1n s -0 .4 95 n s -0 .9 23 ** * -0 .7 62 * -0 .8 13 ** su c f 0. 62 5n s 0. 57 4n s -0 .7 72 * 0. 84 4* * 0. 77 5* 0. 87 7* * 0. 59 8n s -0 .6 53 n s g l u f 0. 66 2n s 0. 26 6n s -0 .4 94 n s 0. 75 0* 0. 78 6* 0. 77 3* 0. 64 1n s -0 .7 21 * 0. 81 7* * fr u f -0 .6 62 n s 0. 45 1n s 0. 76 8* -0 .7 55 * -0 .8 68 ** -0 .8 57 ** -0 .5 96 n s 0. 69 0* -0 .9 75 ** * -0 .8 44 ** m a n f 0. 75 0* 0. 18 5n s -0 .5 25 n s 0. 75 6* 0. 90 7* ** 0. 87 2* * 0. 82 5* * -0 .8 75 ** 0. 83 6* * 0. 93 5* ** -0 .8 63 ** % o il -0 .7 57 * -0 .1 44 n s 0. 64 8n s -0 .6 63 n s -0 .9 79 ** * 0. 93 7* ** -0 .8 69 ** 0. 91 5* ** -0 .8 19 ** -0 .8 34 ** 0. 85 2* * -0 .9 53 ** * pa 0. 65 5n s -0 .2 52 n s -0 .3 29 n s 0. 12 1n s 0. 73 4* 0. 53 3n s 0. 75 0* -0 .8 19 ** 0. 27 1n s 0. 24 4n s -0 .3 16 n s 0. 52 2n s -0 .6 87 * po 0. 12 2n s -0 .2 35 n s 0. 10 4n s -0 .3 81 n s -0 .0 19 n s -0 .1 43 n s 0. 17 3n s -0 .1 26 n s -0 .3 67 n s -0 .5 56 n s 0. 38 8n s -0 .2 91 n s 0. 08 3n s 0. 63 1n s st -0 .2 09 n s -0 .1 60 n s 0. 05 1n s -0 .5 15 n s -0 .2 67 n s -0 .4 06 n s -0 .3 17 n s 0. 32 6n s -0 .3 33 n s -0 .6 18 n s 0. 27 7n s -0 .4 39 n s 0. 35 4n s 0. 04 4n s 0. 42 2n s o l 0. 52 2n s 0. 38 5n s -0 .6 34 n s 0. 74 1* 0. 71 6* 0. 77 8* 0. 49 0n s -0 .5 77 n s 0. 86 6* * 0. 95 5* ** -0 .8 76 ** 0. 84 7* * -0 .7 65 * 0. 07 7n s -0 .6 67 * -0 .5 83 n s v a 0. 31 1n s -0 .0 20 n s -0 .1 10 n s -0 .1 57 n s 0. 23 1n s 0. 16 2n s 0. 40 8n s -0 .2 08 n s -0 .1 66 n s -0 .1 07 n s 0. 18 7n s 0. 00 7n s -0 .1 95 n s 0. 54 1n s 0. 61 5n s -0 .0 52 n s -0 .1 73 n s l l -0 .7 79 * -0 .2 21 n s 0. 71 7* -0 .6 62 n s -0 .9 67 ** * -0 .9 13 ** * -0 .7 90 * 0. 88 0* * -0 .8 55 ** -0 .8 93 ** 0. 88 6* * -0 .9 55 ** * 0. 97 9* ** -0 .5 82 n s 0. 20 4n s 0. 40 6n s -0 .8 52 ** -0 .1 70 n s l n -0 .4 38 n s -0 .1 19 n s 0. 58 4n s -0 .3 96 n s -0 .6 19 n s -0 .6 38 n s -0 .4 26 n s 0. 57 9n s -0 .5 23 n s -0 .7 12 * 0. 49 6n s -0 .6 24 n s 0. 64 5n s -0 .2 69 n s 0. 29 0n s 0. 70 3* -0 .7 40 * -0 .3 32 n s 0. 73 5* a r -0 .5 07 n s -0 .3 32 n s 0. 51 4n s -0 .5 41 n s -0 .7 44 * -0 .5 97 n s 0. 49 2n s -0 .6 23 n s -0 .6 99 * 0. 65 0n s -0 .6 26 n s 0. 69 5* -0 .2 81 n s 0. 26 1n s 0. 63 8n s -0 .7 17 * -0 .3 26 n s 0. 72 8* 0. 67 7* -0 .5 20 n s g o -0 .5 28 n s -0 .0 23 n s 0. 10 9n s -0 .2 38 n s -0 .5 20 n s -0 .3 67 n s -0 .4 65 n s 0. 40 3n s -0 .2 62 n s -0 .6 12 n s 0. 36 4n s -0 .4 93 n s 0. 48 2n s -0 .2 26 n s 0. 26 0n s 0. 58 7n s -0 .5 16 n s -0 .3 23 n s 0. 52 6n s 0. 50 3n s 0. 81 3* * t p 0. 66 4n s 0. 48 8n s -0 .7 01 * 0. 83 5* * 0. 77 8* 0. 83 6* * 0. 58 3n s -0 .7 11 * 0. 96 8* ** 0. 90 6* ** -0 .9 49 ** * 0. 88 0* * -0 .8 23 ** 0. 25 1n s -0 .4 46 n s -0 .4 98 n s 0. 92 2* ** -0 .1 96 n s -0 .8 77 ** -0 .6 30 n s -0 .6 38 n s -0 .3 63 n s t a c 0. 65 7n s 0. 46 4n s -0 .6 22 n s 0. 82 8* * 0. 79 0* 0. 84 5* * 0. 61 5n s -0 .6 83 n s 0. 93 9* ** 0. 95 9* ** -0 .9 36 ** * 0. 90 9* * * -0 .8 39 ** 0. 21 5n s -0 .5 28 n s -0 .5 39 n s 0. 97 3* ** -0 .1 31 n s -0 .9 03 ** * -0 .6 86 * -0 .7 12 * -0 .4 84 n s 0. 97 7* **  p n, n et p ho to sy nt he si s; g s, st om at al c on du ct an ce ; c i, su bs to m at al c ar bo n di ox id e co nc en tr at io n; e r at e, t ra ns pi ra tio n ra te ; su c , su cr os e; g l u , gl uc os e; f r u , fr uc to se ; m a n , m an ni to l; l , le af ; f , f ru it; p a , p al m iti c; p o , p al m ito le ic ; s t , s te ar ic ; o l , o le ic ; v a , v ac ce ni c; l l , l in ol ei c; l n , l in ol en ic ; a r , a ra ch id ic ; g o , g on do ic . # pn , n et p ho to sy nt he si s; g s, st om at al c on du ct an ce ; c i, su bs to m at al c ar bo n di ox id e co nc en tr at io n; e ra te , t ra ns pi ra tio n ra te ; s u c , s uc ro se ; g l u , g lu co se ; f r u , f ru ct os e; m a n , m an ni to l; l , l ea f; f , fr ui t; pa , p al m iti c; po , p al m ito le ic ; s t, s te ar ic ; o l , o le ic ; v a , v ac ce ni c; l l , l in ol ei c; l n , l in ol en ic ; a r , a ra ch id ic ; g o , g on do ic . n s, n ot s ig ni fic an t. *s ig ni fic an t a t p < 0 .0 5. ** si gn ifi ca nt a t p < 0 .0 1. ** *s ig ni fic an t a t p < 0 .0 01 . journal of applied botany and food quality 88, 10 15 (2015), doi:10.5073/jabfq.2015.088.003 1 department of food engineering, faculty of engineering, osmaniye korkut ata university, osmaniye, turkey 2 department of plant protection, faculty of agriculture and natural sciences, usak university, usak, turkey effects of sunn pest (eurygaster maura l. heteroptera; scutelleridae) sucking number on physical and physicochemical characteristics of wheat varieties halef dizlek*1, mahmut islamoglu2 (received december 5, 2014) * corresponding author summary in this study, we analysed the effects of sunn pest sucking number (0, single and double) on some physical and physicochemical characteristics of wheat varieties. we obtained that sucking number on the kernel had effects on wheat and some varieties (zenit and ziyabey-98) are more sensitive than others. considering six different wheat varieties, for the same wheat variety, two number of sucks caused a decrease in thousand kernel weight of up to 2.8 %, hectoliter weight up to 2.2 %, zeleny sedimentation test up to 7.1 %, delayed zeleny sedimentation test up to 37.5 %, dry gluten content up to 8.6 % and gluten index value up to 58.9 % rather than one number of suck. these decrements were statistically significant in 16 of 42 evaluations in seven quality analyses of six different wheat varieties. in addition, sunn pest sucking ratio (0 % and 3 %) and sunn pest sucking number, cause to decline in the analyses of our findings. this was significant (p < 0.05) in 14, respectively 25 of the total 42 evaluations of variety×quality interactions. for these reasons, kernels with double sucking should be considered as two damaged kernel instead of one damaged kernel during classification of the sunn pest damaged wheat mass. abbreviations sp: sunn pest; ws: wheat stinkbug; tkw: thousand kernel weight; hw: hectoliter weight; zst: zeleny sedimentation test; dzst: delayed zeleny sedimentation test; wgc: wet gluten content; dgc: dry gluten content; giv: gluten index value. introduction cereal crops are grown on 218.458.858 hectare throughout the world, and 713.217.069 tons of grains were produced in 2013. wheat, with the 7.772.600 hectare growing area and 22.050.000 ton annual production, takes the first place in field crops in turkey (fao, 2014), and it retains its features of having the feedstock of human beings basic nutrients. especially from north africa to central asia, it accounts for half of the total dietary calories (reynolds et al., 2008) and is an essential dietary component in many countries. physicochemical (technological) features of wheat were affected dramatically by cultivar genetic properties, weather conditions, soil features, fertilizing and agronomical applications, cereal diseases and harmful insects in both vegetative and storage periods (koksel and sivri, 2002; altenbach et al., 2002; torbica et al., 2007). certain species of sap-sucking insects (eurygaster, aelia, nysius huttoni, chlorochroa sayi and stodiplosis mosellana gehin, etc.) feeding on wheat may damage the grain in different countries (karababa and ozan, 1998). in turkey, the eurygaster integriceps put. sunn pest (sp) (hemiptera: scutelleridae) and aelia spp. wheat stinkbug (ws) (heteroptera; pentatomidae) are the most important insects of cereals, which cause serious damage almost every year especially wheat and barley (lodos, 1982). sp and ws feed grains at different stages of development (ravan et al., 2009). overwintered adults of the sp and ws attack the leaves and stems of young, succulent wheat and barley plants causing them to wither and die prior to spike formation. they also feed at the base of the spike during the early growing period, resulting in grayish white spikes without kernels (called white spikes). fourth and fifth nymph instars and new-generation adults of the sp and ws feed grains (lodos, 1982; memisoglu and ozer, 1992). yield losses are estimated at 50-90 % in wheat and 20-30 % in barley. in addition to direct yield reduction during feeding, they excrete abundantly digestive secretions with proteolysis enzyme activities. these secretions become active at a suitable temperature, moisture and a certain time. when flour is processed into dough by kneading with water, and suitable temperature and moisture are provided, enzymes are activated and degrade gluten proteins. hence, dough softens and its elasticity decreases, so further procession by hand or machine gets difficult. moreover, gas absorption capacity of dough during fermentation decreases, and swelling as well as other quality characteristics of the bread such as crumb structure and texture are affected (hariri et al., 2000; vaccino et al., 2001; koksel et al., 2002; kinaci and kinaci, 2004; aja et al., 2004; ozberk et al., 2005; diraman et al., 2013). for years, the sp has reduced both wheat yield and quality in turkey and its neighboring countries (karababa and ozan, 1998). in turkey, thousands of dollars are spent annually struggling with the sp; in 2014, an area of about 636.000 hectare were made sp control (general directorate of food and control, 2014). according to the calculations of the turkish ministry of food, agriculture and livestock, in 2011, the national economy contributed about 370 million dollars to the fight against the sp in 22 provinces. however, farmers lost about 20 million dollars due to the damage sp caused to coarse wheat (union of turkey agriculture chambers, 2014). characterizing wheat masses correctly is possible by evaluating the wheat kernels with sp and ws sucking properly. however, different methods are used to determine the rate of sucked wheat kernels in turkey (dizlek and islamoglu, 2010). in the analysis based on the weigh, sucked kernels are chosen and then weighed. later, they are proportioned with the first weight of kernel mass (albayrak, 1999). in the physical analysis, sucked kernels are chosen and counted by either inspection or binocular and proportioned with healthy kernels to find % sucking ratio. however, in both methods only sucked kernel was considered, sucking number on the kernels were not regarded (dizlek and islamoglu, 2010). these cases have caused misleading results in evaluating the sucked kernel analysis. because the kernels are exposed to more sp and ws damage, they have more proteolysis activity and thus a stronger decline is observed in the quality of flour products (kretovich, 1944; anon., 1983; lorenz and meredith, 1988b; koksel et al., 2002; dizlek et al., 2008). dizlek et al. (2008) determined that increase in infection ratio (1/4, 2/4, 3/4 and 4/4) in sp damaged wheat (golia) caused a decrease (p < 0.05) in physical characteristics of wheat very quickly, so infection ratio is a very important factor for insect damage in wheat. therefore, the cereal industry should not only take damaged kernel ratio into consideration but also consider infection ratio of insect damaged wheat. effects of sunn pest sucking number on wheat 11 karababa and ozan (1998) reported that an allowable ratio of damaged kernels can be increased with high quality varieties. therefore, technological effects of bug damage should be investigated on a wide range of turkish cultivars. in this study, we categorized the single sucked kernels (one sucking point on a kernel) and double sucked kernels (two sucking points on a kernel) separately. we investigated the effects of sp (eurygaster maura) sucking number (0, 1 and 2) on the properties of wheat varieties grown in turkey. the physical characteristics of wheat (thousand kernel weight [tkw], hectoliter weight [hw]) and physicochemical characteristics of wheat flour (zeleny sedimentation test [zst], delayed zeleny sedimentation test [dzst], wet and dry gluten content [wgc and dgc, respectively] and gluten index value [giv]) were evaluated. materials and methods materials six wheat varieties (golia, panda, adana-99, ziyabey-98, ceyhan-99 and zenit) were harvested ten days after the harvest time instead of being harvested on the right time. thus, these varieties were exposed to more sp sucking damage and more than one sucking was formed on the kernels. meanwhile, one hundred sp were collected from each wheat field and their varieties were determined. on average, two kilograms from each variety of wheat was taken to the laboratory. methods in order to comprise the groups of wheat, damaged and undamaged by sp were identified by using binocular (sivri and koksel, 2000; koksel et al., 2002; olanca et al., 2008). sp damaged kernels were grouped according to the damaging degrees as single or double sucked kernels and the kernels not included in these groups were discarded. in the present study, 3 % sucking ratio which deteriorated the technological (bread and pasta) characteristics of the wheat was selected (kosmin, 1933; el-haramein et al., 1984; matsoukas and morrison, 1990; koksel et al., 2002; sivri et al., 2004; kacar et al., 2008). then, 3 sucked grains (single sucked kernels for the single sucked group and double sucked grains for the double sucked group) were added to a kernel mass including 97 healthy grains for each group. this procedure was repeated numerously to obtain sufficient amount of samples of each wheat varieties. thus, in every wheat variety three different wheat groups including kernels with control (without sp sucking), single sp sucking 3 % and double sp sucking 3 % were formed. great care was taken in choosing almost the same sized damaged kernels to form groups, whereas the weak and broken kernels were eliminated. physical and physicochemical analyses physical and physicochemical analyses were carried out for the blended healthy and sp damaged wheat kernels. according to uluoz (1965) tkw and hw of the wheat groups were assigned. prepared blended wheat kernels were milled with a laboratory type mill (yucebas, izmir, turkey) including four rolls to determine some important physicochemical characteristics of wheat flours. flour samples were objected to zst (aacc method 56-60.01; aacc, 2000), dzst (greenaway et al., 1965), wgc and dgc (aacc method 38-10.01; aacc, 2000) and giv (aacc method 38-12.02; aacc, 2000). standard hydration time (5 min) in zst was extended to 120 min in dzst. data analyses analyses were carried out in three replicates. analyses of variance (anova) were conducted by using the spss procedures (spss, version 18.0 for windows, spss inc., chicago, usa). when a significant difference was found among treatments, duncan’s multiple range tests were performed to determine the differences among the mean values (p < 0.05). results and discussion sp species collected in the research areas were identified. it was determined that 98 % of them were eurygaster maura l. (heteroptera: scutelleridae) and 2 % were eurygaster integriceps in the town of karaisalı (37º 05’ n 35º 08’ e). in another study, it was determined that 97 % of them were eurygaster maura and 3 % were eurygaster integriceps (sayan, 2010). the effects of different sucking numbers on the tkw and hw are given at tab. 1. sp sucking numbers on the tkw caused different variations result on wheat varieties. in this context, sp sucking numbers were determined not to effect on the tkw in golia (f2,6 = 0.364, p = 0.709) and zenit (f2,6 = 0.389, p = 0.694). the effects of the sucking numbers on the tkw were found important in panda (f2,6 = 17.898, p = 0.003) and adana-99 (f2,6 = 17.064, p = 0.003). in ceyhan-99, for damaged samples by sp, number of sucking (single and double) on kernel was not effective on the tkw, however sucking ratios (0 % and 3 %) were found to be effective (f2,6 = 10.597, p = 0.011). tkw was affected significantly from the number of sucking on kernel (f2,6 = 99.214, p = 0.000) for ziyabey-98. tab. 1: the effects of different sp sucking numbers on physical characteristics (tkw and hw) of wheat varieties. variety sp sucking ratio (%)tkw 1 (g) 3 hw 2 (kg) sp sucking numbers 0-no sucking (control) 30.20±0.40a 4 84.56±0.16a golia 3-single sucking 29.84±0.37a 83.61±0.30b 3-double sucking 30.29±0.40a 83.80±0.29b control 43.40±0.17a 84.00±0.30a panda 3-single sucking 43.54±0.20a 83.85±0.40ab 3-double sucking 42.33±0.34b 83.21±0.34b control 43.56±0.92a 87.44±0.22a adana-99 3-single sucking 43.49±0.55a 86.58±0.14b 3-double sucking 43.04±0.49b 84.69±0.20c control 39.92±0.37a 83.78±0.40a ziyabey-98 3-single sucking 39.51±0.34b 83.56±0.26a 3-double sucking 39.27±0.25c 82.19±0.19b control 38.97±0.22a 84.86±0.21a ceyhan-99 3-single sucking 38.45±0.12b 85.07±0.27a 3-double sucking 38.39±0.10b 84.17±0.10b control 46.86±0.32a 85.09±0.30a zenit 3-single sucking 46.60±0.20a 85.21±0.31a 3-double sucking 47.02±0.44a 85.14±0.30a 1 thousand kernel weight 2 hectoliter weight 3 calculations based on the dry matter basis. 4 mean values in the table in the same column in same wheat followed by different superscript with lowercase are significantly different p < 0.05. the same situation was observed in hw of the samples. sp sucking numbers were not effective on hw in zenit (f2,6 = 0.120, p = 0.889) and panda (f2,6 = 4.373, p = 0.067). in golia, although sp sucking ratio was effective on hw, number of sucking, whether single 12 h. dizlek, m. islamoglu sucking or double sucking, had no influence on hw (f2,6 = 11.627, p = 0.009). in adana-99, the number of suckings on kernels have effects on hw (f2,6 = 169.955, p = 0.000). an important result is seen in the tab. 1 for ziyabey-98 (f2,6 = 25.506, p = 0.001) and ceyhan-99 (f2,6 = 15.715, p = 0.004). although researchers reported that two or three sucking were acceptable as only one sucking (atli et al., 1988a), the results in the tab. 1 indicated that every single sucking on a kernel has effect on hw. in terms of sucking number effects on hw, while differences were observed between wheat varieties, some of them were affected lesser or never. these differences between varieties were thought to be derived from genetic differences. similarly, it was reported that there were variations in yield and quality losses caused by sp damage in wheat varieties (kinaci et al., 1998; sivri et al., 2002; kinaci and kinaci, 2004). tkw and hw are important quality parameters for determining the suitability of wheat for milling (karababa and ozan, 1998). tkw is positively correlated with flour yield (lee et al., 1983). soundness of the grain also directly influences flour yield and quality. according to critchley (1998), the tkw of sp damaged wheat can be 8-22 % lower than that of the undamaged kernel. hariri et al. (2000) detected that the average reduction in tkw due to sp damage was about 24 % in bread wheat and 20 % in durum wheat samples. in this study, considering 6 different wheat varieties together (tab. 1), two number of sucks on kernel cause decrease in tkw till 2.8 % and hw till 3.2 % compared to one number of suck on kernel or undamaged control sample. zs, dzs, wgc, dgc and gi tests were carried out for the determination of the effect of sp sucking number damage on the physicochemical characteristics of wheat flour samples. in these analyses especially dzst, as improved by greenaway et al. (1965), is used for the detection of sp damage in wheat flour. the effects of different sucking numbers on the zst, dzst, wgc, dgc and giv are given in tab. 2. it was found that while sucking ratios and sucking numbers on the kernels did not affect zst in the varieties of adana-99 (f2,6 = 0.627, p = 0.874), ziyabey-98 (f2,6 = 0.750, p = 0.512) and ceyhan-99 (f2,6 = 0.750, p = 0.514) on the contrary to golia (f2,6 = 42.000, p = 0.000) and zenit (f2,6 = 57.000, p = 0.000) varieties were affected by these factors significantly (p < 0.05). in panda wheat variety, number of sucking on kernel have relative effects on zst (f2,6 = 3.814, p = 0.085). examining the dzst, in golia (f2,6 = 124.000, p = 0.000), panda (f2,6 = 74.000, p = 0.000), ziyabey-98 (f2,6 = 148.000, p = 0.000) and ceyhan-99 (f2,6 = 75.000, p = 0.000) except of adana-99 and zenit varieties, it was determined that the effects of sucking numbers (no, single or double) on the dzst values were significant (p < 0.05). sedimentation tests (zst and dzst) were done to determine whether the wheat was exposed to sp damage, or to what extent it was. as expected, zst and dzst values were found to decrease when the sp damage increase in wheat varieties. it was reported that how much the value in dzst is lower than the value in zst means more sp-ws damage (elgun et al., 1998; ozkaya and ozkaya, 2005). as zst and dzst values are considered together (tab. 2), it is observed that gluten quality in control samples is improved. the reason is that control samples include no damaged kernels and therefore, temperature (30 ºc), at which flour slurry (flour + solution of brome phenol blue) are kept in dzst, increase strength of gluten network structure. differences between the varieties in terms of the resistance against sp damage were found with panda being the tab. 2: the effects of different sp sucking numbers on physicochemical characteristics (zst, dzst, wgc, dgc and giv) of wheat (flour) varieties. variety sp sucking ratio (%) zst 1 (ml) 6 dzst 2 (ml) 6 wgc 3 (%) dgc 4 (%) giv 5 (%) -sp sucking numbers 0-no sucking (control) 29.0±0.5a 7 36.0±1.0a 31.12±0.50a 10.90±0.50a 99.9±0.1a golia 3-single sucking 28.0±0.0b 29.0±1.0b 31.40±0.80a 11.10±0.50a 99.9±0.1a 3-double sucking 26.0±0.5c 25.0±0.5c 30.96±0.50a 10.70±0.50a 99.9±0.1a control 32.0±0.5a 40.0±1.0a 30.54±1.00a 10.38±0.13a 99.9±0.2a panda 3-single sucking 31.5±0.5ab 36.0±0.5b 30.92±0.97a 10.20±0.15ab 99.7±0.3a 3-double sucking 31.0±0.3b 33.0±0.5c 30.48±0.88a 10.00±0.20b 99.9±0.1a control 37.0±1.0a 42.0±1.0a 27.36±1.01a 9.36±0.36a 99.8±0.2a adana-99 3-single sucking 37.0±1.0a 35.0±1.0b 27.85±0.97a 9.56±0.36a 99.7±0.3a 3-double sucking 37.0±1.5a 35.0±1.0b 27.28±1.00a 9.38±0.33a 99.9±0.2a control 23.0±1.0a 24.0±1.0a 28.24±1.00a 9.54±0.09a 98.6±1.0a ziyabey-98 3-single sucking 22.0±1.0a 16.0±1.0b 28.01±0.90a 9.08±0.08b 99.6±0.4a 3-double sucking 22.5±1.0a 10.0±1.0c 28.38±0.87a 9.18±0.16b 94.7±0.2b control 36.5±1.0a 45.0±1.0a 26.36±0.91a 9.50±0.10a 99.9±0.0a ceyhan-99 3-single sucking 37.0±1.0a 40.0±1.0b 26.32±1.00a 9.32±0.14ab 99.9±0.1a 3-double sucking 36.0±1.0a 35.0±1.0c 26.02±0.04b 9.20±0.12b 99.9±0.1a control 20.5±0.5a 20.5±1.0a 35.12±0.92a 12.84±0.15a 93.7±2.7a zenit 3-single sucking 19.0±0.0b 9.0±0.0b 35.32±1.01a 12.91±0.21a 68.6±2.6b 3-double sucking 18.0±0.0c 9.0±0.0b 34.84±0.98a 11.80±0.05b 28.2±1.2c 1 zeleny sedimentation test 2 delayed zeleny sedimentation test 3 wet gluten content 4 dry gluten content 5 gluten index value 6 adjusted to 14 % moisture basis. 7 mean values in the table in the same column in same wheat flour followed by different superscript with lowercase are significantly different p < 0.05. effects of sunn pest sucking number on wheat 13 most resistant variety followed by ceyhan-99, golia and adana-99. ziyabey-98 and zenit varieties were sensitive to sp damage and even though 3 % sp damage caused an obvious decline in gluten quality in these two varieties. in general, number of sucking on kernel was not effective on wgc values of wheat varieties (tab. 2). there were no differences (p > 0.05) between single sucking and double sucking of the wgc of five wheat varieties (golia; f2,6 = 392.000, p = 0.692, panda; f2,6 = 171.000, p = 0.847, adana-99; f2,6 = 286.000, p = 0.761, ziyabey-98; f2,6 = 0.105, p = 0.902 and zenit; f2,6 = 0.174, p = 0.844), limited difference occurred only in ceyhan-99 (f2,6 = 14.389, p = 0.005) variety. sp and the other insects had the same damaging effect on cereals protein quality rather than the protein content as the results in many other conducted studies also suggest (lorenz and meredith, 1988a, 1988b; atli et al., 1988a, 1988b; every et al., 1990; rosell et al., 2002; perez et al., 2005). sp sucking ratio and sp sucking number on the grain affected dgc much more than wgc values (tab. 2). in ziyabey-98 (f2,6 = 13.040, p = 0.007) sucking ratio and in zenit (f2,6 = 50.331, p = 0.000) sucking number were found significant (p < 0.05) on dgc values. while dgc of golia (f2,6 = 0.480, p = 0.641), adana-99 (f2,6 = 0.297, p = 0.754), ceyhan-99 (f2,6 = 4.664, p = 0.060) and panda (f2,6 = 4.096, p = 0.076) varieties were not affected by sp sucking ratio or sp sucking number on the grain, dgc of other varieties were affected by sp sucking ratio or sp sucking number on the grain to a limited extent. a possible reason for higher wgc and dgc of zenit may be its descent from a durum wheat variety. the giv is defined as the percentage of wet gluten remaining on the sieve after automatic washing in a salt solution and centrifugation; it is a fast method to analyze gluten characteristics, indicating whether the gluten is weak, normal or strong. it is also used for the detection of gluten quality (perten, 1990). the gi method can be used to determine sp damage in flour, since sp damaged wheat is composed of proteolytic enzymes which weaken the gluten bonds. this results in a significant decrease of the giv (aja et al., 2004). examining the giv in tab. 2, while sp sucking ratio or sp sucking number on the grain effects on the gluten quality were found significant in zenit (f2,6 = 334.515, p = 0.000) and ziyabey-98 (f2,6 = 50.275, p = 0.000) varieties, no difference was found between gluten qualities, in terms of giv, in the other varieties (golia; f2,6 = 0.470, p = 0.740, panda; f2,6 = 0.923, p = 0.447, adana-99; f2,6 = 0.562, p = 0.597, and ceyhan-99; f2,6 = 0.814, p = 0.742). as the level of sp sucking number in wheat increased, in general, zst, dzst, wgc, dgc and giv decreased (considering six different wheat varieties together (tab. 2), two number of sucks on kernel cause decrease in zst till 12.2 %, dzst till 58.3 %, wgc till 2.1 %, dgc till 8.6 % and giv till 69.9 % compared to one number of suck on kernel or undamaged control sample). however, these decreases were not statistically significant (p > 0.05) for adana-99, ziyabey-98 and ceyhan-99 varieties in zst; golia, panda, adana-99, ziyabey-98 and zenit varieties in wgc; golia and adana-99 varieties in dgc; golia, panda, adana-99 and ceyhan-99 in giv. the dzst is the most important indication of the sp damage level of wheat kernels. in this study, dzst values decreased dramatically with the increase of sp sucking ratio and sp sucking number in all wheat varieties (tab. 2). these results showed a clear decreasing in sedimentation tests especially dzst and gluten quantity and quality which can be attri buted to the effect of enzyme injected to grains by sp (tab. 2; kretovich, 1944; every et al., 1989). it was observed that sp damage affected the protein quality of wheat more than its protein quantity. this data was supported by some early studies (johnson and miller, 1953; greenaway et al., 1965; redman, 1971; lorenz and meredith, 1988a, 1988b; atli et al., 1988a, 1988b; every et al., 1990). kinaci and kinaci (2004) showed that the damage caused by sp pierced grain affects the tkw, protein content and zst value depending on the variety and grain type. six wheat varieties used in this study shows that wheat quality was significantly (p < 0.05) affected by sp sucking ratio and sp sucking number. however, there were variations derived from the genetically differences between varieties in terms of the flour qualities. ziyabey-98 and zenit varieties were determined to be affected excessively. it was observed that these results were in accordance with the findings of kinaci et al. (1998), karababa and ozan (1998), sivri et al. (2002), kinaci and kinaci (2004), dizlek et al. (2008). as expected, the control sample had higher results than the sp damaged samples for all physical and physicochemical quality parameters (tab. 1 and 2). dizlek et al. (2008) reported that each wheat variety was affected from sp damage differently and wheat varieties cannot be damaged by sp at the same degree. atli et al. (1988a) reported that there were differences between the methods applied for determining the sucked kernels by sp and these differences were originated from that amount of the sucked kernels given on the base of weight and percentage. although researchers reported that two or three sucking were able to be acceptable as only one sucking, they indicated that every single sucking on a kernel contains enzyme spoiling the quality. single sucked and double sucked kernels are considered in the same group in practice. results in tab. 1 and tab. 2 show that number of sucking in wheat grain play an important role in quality characteristics of wheat varieties (the results of this study clearly demonstrated that the quality parameters of wheat varieties and its flours were influenced by the increasing sucking number of sp damaged kernels in wheat samples). for this reason, double sucking kernel has to be handled in different groups than single sucking kernel. conclusions the findings of this study could be summarized as follows: 1. sp sucking ratio (0 % and 3 %) and sp sucking number (0, single and double), cause to decline in the analyses of our findings. and they were significant (p < 0.05) in 14 and 25 through total 42 evaluations pertaining to variety × quality interactions, respectively. 2. the investigated factors were affected on the wheat varieties and some varieties were more sensitive to sp damage than others. 3. quality losses caused by sp damage appear in both physical and physicochemical measurements. with observing and evaluating the data together (tab. 1 and 2) a general decline in quality at limited level was observed. carrying out research with more sp damaged grains (≥ 5 %) would be more useful to obtain better results. 4. in general, while the levels of sp sucking number in kernel increased, tkw, hw, zst, dzst, giv, wgc and dgc values of wheat varieties decreased. 5. gluten quality (dzst and giv) of the samples was affected higher than gluten quantity (wgc, dgc, tkw and hw) by sp sucking number. 6. sp sucking number on the grains affected the quality in some varieties especially zenit and ziyabey-98 significantly. the classification of the damaged grains should be adepted and single sucked grains and double sucked grains should not be evaluated in the same way. it is concluded that double sucked grains can be dealt with properly as two number single sucked grains. 7. in order to conduct the sp sucking analysis correctly, sucking number on a kernel must be taken into consideration as well as the sucking ratio. this case will the industry on handling sp damaged wheat and to elect additives used in preparing wheat blends and/or making bread. 14 h. dizlek, m. islamoglu references aacc, 2000: approved methods of the american association of cereal chemists. method 38-10.01, method 38-12.02, method 56-60.01. 10th ed. the association, st. paul. aja, s., perez, g., rosell, c.m., 2004: wheat damage by aelia spp. and erygaster spp.: effects on gluten and water-soluble compounds released by gluten hydrolysis. j. cereal sci. 39, 187-193. albayrak, t., 1999: a research on the determination of insect (eurygaster spp. and aelia spp.) damage grain ratio in wheats incoming to konya two wheat mill operators. msc. selçuk university, konya. altenbach, s.b., kothari, k.m., lieu, d., 2002: environmental conditions during wheat grain development alter temporal regulation of major gluten protein genes. cereal chem. 79, 279-285. anonymous, 1983: counteracting suni bug damage to wheat flour baking quality. icarda highlights 82, 17-18. atli, a., koksel, h., dag, a., 1988a: effect of sunn pest damage on bread wheat quality and its determination. in: proceedings of the 1st international sunn pest symposium, 1-19. 13-17 june 1988, tekirdag. atli, a., kocak, n., koksel, h., ozan, a.n., aktan, b., karababa, e., dag, a., tuncer, t., dikmen, b., ozkan, s., 1988b: the effects of damaged grains by sunn pest (eurygaster spp.) and wheat stinkbug (aelia spp.) on quality of bread wheat. tarm printing, ankara. critchley, b.r., 1998: literature review of sunn pest eurygaster integriceps put. 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(eds.), the gluten proteins, 425428. viterbo. address of the authors: halef dizlek, department of food engineering, faculty of engineering, osmaniye korkut ata university, 80000 osmaniye, turkey e-mail corresponding author: hdizlek@osmaniye.edu.tr mahmut islamoglu, department of plant protection, faculty of agriculture and natural sciences, usak university, 64200 usak, turkey e-mail: mahmut.islamoglu@usak.edu.tr journal of applied botany and food quality 89, 126 134 (2016), doi:10.5073/jabfq.2016.089.015 1 department of agronomy, institute of agronomy and plant breeding, justus liebig university giessen, germany 2 institute for animal nutrition and functional plant compounds, university of veterinary medicine, vienna, austria content and composition of essential oil of four origanum vulgare l. accessions under reduced and normal light intensity conditions marzieh shafiee-hajiabad1, johannes novak2, bernd honermeier1 (received june 11, 2015) * corresponding author summary the variation in the chemical composition and content of the essential oil was examined in four origanum vulgare accessions (ov): 1origanum vulgare l. ssp. vulgare (=ovu), 2origanum vulgare l. ssp. hirtum (link) letswaart (=ohi), 3origanum vulgare l. ssp. viride (boiss.) hayek (=ovi) and 4origanum vulgare l. ssp. viride (boiss.) hayek × o. majorana l. (=oxm), growing under reduced (26 %) and normal light intensity. altogether, 64 compounds re presenting 98.95 % of the total oil were identified. reduced light had a minor effect on the composition of essential oil. it decreased the content of p-cymene in ohi and increased the sabinene content in oxm herb samples. the essential oil of ovu in both samples was mainly composed of trans-sabinene hydrate, β-caryophellene and germacrene d. the major components of essential oil of ohi were thymol and carvacrol followed by γ-terpinene and p-cymene. herb samples had a considerably higher amount of essential oil than leaf samples. in herb extracts of ovi, cymyl compounds such as p-cymene, thymol and γ-terpinene were dominant. oxm was characterized by several monoterpenes with low concentrations including γ-terpinene, sabinene cis-b-ocimene and carvacrol methyl ether. the results of the current study suggest a chemical toleration of the evaluated accessions to the applied light reduction. furthermore, a full investigation of essential oil profiles of origanum vulgare accessions is presented. abbreviations eo: essential oil; nli: normal light intensity; ohi: origanum vulgare l. ssp. hirtum (link) letswaart; ovi: origanum vulgare l. ssp. viride (boiss.) hayek; ovu: origanum vulgare l. ssp. vulgare; oxm: origanum vulgare l. ssp. viride (boiss.) hayek × o. majorana l.; rli: reduced light intensity. introduction within the genus origanum, ietswaart (1980) distinguished six subspecies of o. vulgare l. (ov): 1ssp. hirtum (link) ietswaart, 2ssp. vulgare, 3ssp. virens (hoffmannsegg et link) ietswaart, 4 ssp. viride (boissier) hayek, 5ssp. gracile (kock) ietswaart and 6ssp. glandulosum (desfontaines) ietswaart based on the morphological criteria. however, as ietswaart (1980) established the revision of origanum, ov accessions were not extensively investigated regarding their chemical characteristics. with regard to the bulk of research addressing the eo content and composition in ov accessions, it is proposed that these parameters can also be used for the discrimination of the ov plants (d’antuono et al., 2000; novak et al., 2003a; skoula and harborne, 2004), therefore, a full investigation about eo profiles of all ov accessions is recommended. skoula et al. (1999) proposed that origanum species are rich in either sabinyl compounds or cymyl compounds but never in both. ohi is an oil-rich accession with high quality eo, due to high concentrations of carvacrol and/or thymol in its eo. other main compounds in eo of ohi include γ-terpinene, p-cymene (baranauskienė et al., 2013; johnson et al., 2004; novak et al., 2003a). in ohi plants two main chemotypes: carvacrol type (baranauskienė et al., 2013; johnson et al., 2004; skoula et al., 1999; tibaldi et al., 2011) and thymol type (jerkovic et al., 2001; russo et al., 1996) have been reported. moreover, an intermediate type containing both thymol and carvacrol with/without high content of p-cymene and γ-terpinene has also been identified (d’antuono et al., 2000; milos et al., 2000). ovu and ovi are poor sources of volatiles (baranauskienė et al., 2013; lukas et al., 2013). ovu is rich in acyclic compounds and sesquiterpenoids while the eo of ovi is rich in either acyclic compounds (mainly linalool and linalyl acetate) and sesquiterpenoids or in cymyl-compounds (mainly γ-terpinene, carvacrol and/or thymol together with other related compounds). sabinyl-compounds (mainly sabinene and cis-/trans-sabinene hydrate acetate) can be found mainly in ovi (skoula and harborne, 2004). nevertheless, the eo of ovu plants from lithuania have been observed with essentially high concentration of sabinene, β-ocimene, β-caryophyllene and germacrene d (baranauskienė et al., 2013). the expression of eo compounds in origanum plants is principally influenced by the genotype (azizi et al., 2012). additionally, the environmental factors including season (grausgruber-gröger et al., 2012; johnson et al., 2004), light intensity (chang et al., 2008; tibaldi et al., 2011) and photoperiod (circella et al., 1995; dudai et al., 1992) can influence the composition of essential oil in lamiaceae plants. moreover, it has been observed that the growing conditions such as harvest time (baranauskienė et al., 2013), spatial distribution (de falco et al., 2013) and post-harvest conditions (tibaldi et al., 2011) changed the composition and content of eo in origanum plants. furthermore, composition and content of eo vary noticeably among plant organs at different development stages. however, flowering parts of dalmatian sage (salvia officinalis l.) had considerably higher eo compared to leaves and stems. moreover, the concentration of monoterpene and sesquiterpene fractions of eo varied among these organs (perry et al., 1999). in young developing leaves of sweet basil (ocimum basilicum l.) the content of eo was higher than in mature leaves. besides, different composition of eo was observed in the young and mature leaves (werker et al., 1993). actually, in the lamiaceae family the density and distribution of glandular trichomes, which are responsible for eo secretion, is higher on reproductive organs and young leaves compared to mature leaves (werker, 1993). the effect of light intensity on the content and composition of eo in several lamiaceae plants was reported. light irradiance influences the content and composition of eo through alteration in photosynthesis, physiological, and morphological processes of plants (figueiredo et al., 2008). sage (salvia officinalis) and thyme (thymus vulgaris) under 15 %, 27 %, 45 % and 100 % of full sunlight conditions showed different content and profile of eo. the sage plants under 45 % of full sunlight had the highest content of eo with higher reduced light effect on essential oil of o. vulgare accessions 127 level of (+)-thujanone and decreased level of camphor. in thyme the highest content of eo with thymol and myrcene concentrations occurred in full sunlight (li et al., 1996). in the closely related species o. syriacum the composition of essential oil was influenced by the effects of temperature, photoperiod, and light intensity. higher light intensity enhanced the relative content of p-cymene, while the content of phenolic monoterpenes, for example thymol and γ-terpinene, was generally decreased (dudai et al., 1992). although ov plants are native to the warm climate of mediterranean regions with high solar irradiance (ietswaart, 1980), these plants are also cultivated in temperate climate conditions with lower light intensities and sunny days similar to those in germany (honer meier et al., 2013). therefore, it is important to discover if light intensity can influence the eo properties of oregano plants. in our previous publication the effect of light reduction on the physical properties of glandular trichomes was evaluated (shafiee-hajiabad et al., 2015). in this paper four relevant ov accessions were studied under reduced light intensity (rli) and normal light intensity conditions (nli) regarding their eo content and composition. on the other hand, a full profile of eo in two types of samples: herb and first developed leaf (third leaf) of these accessions will be presented. materials and methods experimental design plant material and design: in 2012, four origanum accessions, provided by the national german genebank (ipk gatersleben), were investigated in the research station of rauischholzhausen, justus liebig university of giessen, germany. a pot experiment was conducted with four accessions (tab. 1) and two light intensities: reduced light and normal light intensity as the treatments including four replications. each pot (one plant per pot) was considered as one replication. daily between 9 and 11 a.m. near to the pots by the use of a light meter (eblx4, hartman & braun ag, frankfurt germany). the mean daily light intensity during the plants growth cycle (may to august) inside and outside of the wire-house was calculated (43761 lux and 59067 respectively). the wire-house reduced the light intensity by an average of 26 %. the climate parameters of the research station did not vary considerably inside and outside of the wire-house. during the growth period of origanum plants a mean air temperature of 15.1 °c and a mean air humidity of 78.4 % was measured. the soil used in this experiment was prepared according to the method described in shafiee-hajiabad et al. (2015). gc-ms analyses dried plant material was immersed in dichloromethane and extracted for 30 min in an ultrasonic water bath (lukas et al., 2009). the extracts were separately prepared from the third leaves (30 mg/ml) and the rest of herb (300 mg/10 ml). the extracts were filtered through cellulose wadding in a pasteur pipette and analyzed by gas chromatography coupled with gas chromatography mass-spectrometer (gc/ ms) [hp 6890 coupled with a hp 5972 msd (hewlett-packard, palo alto, ca, usa)]. for quantitative analyses, biphenyl 30 (mgl-1) was used as internal standard. gc-ms was used for identification of the compounds with the following conditions: db-5ms capillary column (30 m * 0.25 mm i.d.; 0.25 μm film thickness; agilent); helium as carrier gas (average velocity 42 cm s-1); injector temperature 250 °c, split ratio 1:1; temperature program: 60 °c for 4 min, rising to 180 °c at 3 °c min, and finally holding at 320 °c for 5 min. retention indices (ri) of the sample components were determined on the basis of homologous n-alkane hydrocarbons (retention index standard for gas chromatography, sigma, vienna, austria) under the same conditions. the composition was obtained by peak area normalization, and the response factor for each component was considered to equal 1. the compounds were identified by comparing their retention indices (adams, 2007) and the mass spectra to published data (mclafferty, 1989). statistical analyses a completely randomized experimental design was conducted as a two-factorial pot experiment. the analysis of variances was carried out using the ibm spss program version 20 on all parameters. a two-way between-groups analysis of variance was conducted to explore the impact of accessions and light intensity on the composition and content of eo in herb and leaf samples. data of two different samples (herb and leaf) of the investigated accessions were analyzed separately in two-way analysis of variance (anova). duncan’s multiple-range test was applied for the calculation of the significant differences among and between the groups at a probability level (p < 0.01). lsd values were calculated and used for the mean comparison of different treatments within one factor. all data were reported as means (mg per g dry matter) ± standard error. results effect of light intensity on content and composition of eo the applied treatment during the growth of ov accessions led to alteration in the content of some chemical compounds, but many of these changes were not found to be statistically significant. in particular, the content of carvacrol in leaf samples was higher under nli (0.2 mg/g) than rli (0.08 mg/g) condition. conversely, thymol content was lower under nli in both sample types, although in both cases this effect was not statistically relevant (tab. 2). an interaction effect between accessions and light intensity was observed on sabitab. 1: subspecies and origin of investigated origanum accessions no. subspecies accession no. origin 1 origanum vulgare l. ssp. vulgare ori 07 /79 unknown (ovu) 2 origanum vulgare l. ssp. hirtum ori 34 /03 usa (link) letswaart (ohi) 3 origanum vulgare l. ssp. viride ori 29 italy (ovi) 4 origanum vulgare l. ssp. viride ori 35 italy (boiss.) × o. majorana (oxm) the seeds were sown on 17th april 2012 and plantlets with three leaves were transferred to pots on april 24th 2012. the first developed leaf (third leaf from the apex) and herb were used as sample. the collection of leaf samples was before harvest on july 4th 2012. in order to take the leaf samples, four representative leaves (third leaf from the apex) from each pot were collected and mixed. in the case of herb samples, the main stems were separated and a mixture of the rest was considered as herb samples. both sample types were dried at 39 °c for 48 hours in an air-circulating oven. light reduction: the pots were arranged from the north-west to south-east direction under normal light intensity (nli) and reduced light intensity (rli) conditions. the light reduction was applied by using a wire-house (a woven metal net with 16.7 × 16.7 mm square mesh and 1.86 mm wire diameter). the light intensity was measured 128 m. shafiee-hajiabad, j. novak, b. honermeier ta b. 2 : t he a ve ra ge c on ce nt ra tio n of a ll de te ct ed c he m ic al c om po un ds (m g/ g) in e xt ra ct s of h er b an d le af s am pl es o f o ri ga nu m v ul ga re a cc es si on s (o vu : s sp . v ul ga re , o hi : s sp . h ir tu m , o vi : s sp . v ir id e, o xm : s sp . vi ri de × m aj or an a) b y us in g g c /m s, r l i: re du ce d lig ht in te ns ity , n l i: n or m al li gh t i nt en si ty , h : h er b sa m pl e, l : l ea f s am pl e. a cc es si on s l ig ht c on di tio n sa m pl e o vu o hi o vi o xm r l i n l i n r. c om po un ds k i a h l h l h l h l h l h l h l 1 sa bi ne ne 96 9 0. 04 9 0. 07 9 0. 00 2 0. 00 3 0. 00 7 0. 09 5 0. 18 1 0. 02 4 0. 05 3 0. 04 9 0. 08 2 0. 03 6 0. 06 7 2 1o ct en -3 -o l 97 9 0. 06 8 0. 06 3 0. 11 0 0. 01 1 0. 00 2 0. 03 1 0. 11 6 0. 06 2 0. 03 9 0. 04 8 0. 05 2 0. 05 5 0. 04 5 3 3o ct an on e 98 3 0. 03 1 0. 00 1 0. 00 2 0. 00 9 0. 00 1 0. 00 8 0. 00 8 4 m yr ce ne 99 0 0. 00 6 0. 00 3 0. 20 7 0. 00 6 0. 02 2 0. 01 9 0. 01 6 0. 04 7 0. 06 5 0. 02 3 0. 06 0 0. 01 4 0. 06 3 0. 01 9 5 α -t er pi ne ne 10 21 0. 00 5 0. 00 6 0. 00 3 0. 00 3 0. 00 3 6 ρc ym en e 10 24 0. 02 3 0. 00 6 1. 19 6 0. 05 4 0. 35 2 0. 18 1 0. 11 2 0. 04 1 0. 45 7 0. 05 8 0. 38 4 0. 08 3 0. 42 1 0. 07 1 7 βph el la nd re ne 10 29 0. 02 1 0. 00 3 0. 00 3 0. 00 8 0. 00 2 0. 00 2 0. 00 1 0. 00 5 0. 00 2 8 ci sβo ci m en e 10 37 0. 06 0 0. 14 3 0. 01 7 0. 07 1 0. 12 5 0. 03 3 0. 18 0 0. 04 0 0. 13 1 0. 05 1 0. 09 3 0. 04 5 0. 11 2 9 tr an sβo ci m en e 10 50 0. 01 9 0. 05 1 0. 06 6 0. 01 5 0. 03 4 0. 01 2 0. 08 8 0. 00 9 0. 04 5 0. 04 8 0. 04 1 0. 02 8 0. 04 3 1 0 γte rp in en e 10 59 0. 03 6 0. 01 9 1. 64 5 0. 06 3 0. 19 3 0. 29 3 0. 14 1 0. 38 8 0. 53 7 0. 18 5 0. 47 1 0. 19 6 0. 50 4 0. 19 1 1 1 ci ssa bi ne ne h yd ra te 10 70 0. 01 6 0. 03 1 0. 00 5 0. 02 3 0. 00 6 0. 02 0 0. 00 2 0. 01 8 0. 00 1 0. 01 9 0. 00 2 1 2 tr an ssa bi ne ne h yd ra te 10 98 0. 40 0 0. 15 1 0. 02 8 0. 00 8 0. 00 2 0. 15 6 0. 02 8 0. 05 9 0. 05 2 0. 10 7 0. 04 0 1 3 l in al oo l 10 96 0. 00 2 0. 00 5 0. 01 2 0. 00 7 0. 00 5 0. 00 8 0. 00 7 1 4 o ct ad ie na l < (2 e .4 e )> 11 16 0. 00 9 0. 00 2 0. 00 2 0. 00 2 1 5 al lo -o ci m en e 11 28 0. 03 1 0. 05 6 0. 00 2 0. 03 5 0. 05 5 0. 01 3 0. 07 5 0. 01 6 0. 05 5 0. 02 4 0. 03 8 0. 02 0 0. 04 7 1 6 b or ne ol 11 69 0. 02 8 0. 00 8 0. 00 6 0. 00 7 1 7 te rp in en -4 -o l 11 77 0. 00 1 0. 02 9 0. 00 8 0. 00 7 0. 00 8 1 8 α -t er pi ne ol 11 88 0. 00 7 0. 00 6 0. 00 1 0. 00 2 0. 00 4 0. 00 1 0. 00 3 0. 00 1 0. 00 3 0. 00 1 1 9 ci sd ih yd ro ca rv on e 11 92 0. 00 5 0. 00 1 0. 00 1 0. 00 1 2 0 tr an sd ih yd ro ca rv on e 12 00 0. 00 3 0. 00 1 0. 00 1 2 1 t hy m ol m et hy l e th er 12 35 0. 01 4 0. 00 3 0. 11 2 0. 06 7 0. 00 6 0. 03 6 0. 02 4 0. 02 9 0. 01 1 0. 03 3 0. 01 8 2 2 c ar va cr ol m et hy l e th er 12 44 0. 00 1 0. 02 6 0. 04 6 0. 02 9 0. 10 8 0. 13 9 0. 04 6 0. 05 1 0. 04 5 0. 03 3 0. 04 5 0. 04 2 2 3 t hy m oq ui no ne 12 52 0. 13 2 0. 01 6 0. 00 1 0. 03 3 0. 04 1 0. 03 7 2 4 tr an ssa bi ne ne h yd ra te a ce ta te 12 56 0. 02 2 0. 01 1 0. 00 6 2 5 tr an sa ne th ol e 12 84 0. 02 1 0. 01 1 0. 00 4 0. 00 3 0. 00 8 0. 00 2 0. 00 4 0. 00 3 0. 00 6 2 6 t hy m ol 12 90 0. 07 2 0. 03 1 4. 51 9 0. 13 5 0. 38 7 0. 40 1 0. 02 7 1. 31 7 0. 16 5 1. 18 5 0. 11 8 1. 25 1 0. 14 2 2 7 c ar va cr ol 12 99 0. 01 0 3. 65 1 0. 47 8 0. 00 6 0. 05 9 0. 09 2 0. 93 3 0. 08 1 0. 92 9 0. 20 4 0. 93 1 0. 14 3 2 8 d ih yd ro ca rv eo l a ce ta te 13 07 0. 03 0 0. 01 5 0. 00 7 2 9 m en th -1 -e n7al < 3ox oρ> 13 33 0. 00 1 0. 00 1 tr ac e 3 0 α -t er pi ny l a ce ta te 13 46 0. 00 1 0. 00 2 0. 00 1 tr ac e tr ac e 3 1 t hy m ol a ce ta te 13 52 tr ac e 0. 00 1 0. 00 1 3 2 c ar va cr ol a ce ta te 13 71 0. 00 2 0. 00 8 0. 02 5 0. 01 3 reduced light effect on essential oil of o. vulgare accessions 129 3 3 βb ou rb on en e 13 88 0. 08 2 0. 09 2 0. 00 1 0. 02 3 0. 03 5 0. 03 2 0. 02 9 0. 03 5 0. 01 6 0. 03 2 3 4 βe le m en e 13 90 0. 00 4 0. 02 2 0. 01 8 0. 00 2 0. 11 4 0. 00 9 0. 05 8 0. 00 4 3 5 βc ar yo ph yl le ne 14 19 0. 23 4 0. 28 6 0. 20 8 0. 00 7 0. 02 7 0. 03 3 0. 07 3 0. 13 5 0. 11 7 0. 08 1 0. 06 8 0. 09 9 3 6 βc ed re ne 14 20 0. 02 2 0. 00 7 0. 00 8 0. 00 5 0. 00 6 0. 00 3 0. 00 7 3 7 βc op ae ne 14 32 0. 02 2 0. 00 2 0. 00 7 0. 00 3 3 8 βh um ul en e 14 38 0. 00 5 0. 00 3 0. 00 2 0. 03 7 0. 00 1 0. 01 8 0. 00 1 3 9 a ro m ad en dr en e 14 41 0. 00 3 0. 14 4 0. 03 7 0. 00 1 0. 00 2 0. 01 9 0. 00 1 4 0 ci sm uu ro la -3 .5 -d ie ne 14 50 0. 00 8 0. 00 2 0. 00 2 0. 01 2 0. 00 7 4 1 α -h um ul en e 14 54 0. 02 6 0. 02 7 0. 02 2 0. 00 1 0. 00 3 0. 00 9 0. 01 4 0. 01 1 0. 00 2 0. 00 7 0. 00 8 0. 00 9 4 2 al lo a ro m ad en dr en e 14 60 0. 00 7 0. 00 8 0. 00 2 0. 00 1 0. 00 3 0. 00 1 0. 00 2 4 3 c ar yo ph yl le ne 9 -e pi -( e) 14 66 0. 00 1 0. 00 2 tr ac e 4 4 γh im ac ha le ne 14 82 0. 00 1 0. 00 6 0. 00 1 0. 04 5 0. 02 3 4 5 g er m ac re ne d 14 85 0. 17 9 0. 26 8 0. 01 8 0. 02 6 0. 13 4 0. 06 0 0. 13 4 0. 07 7 0. 03 0 0. 10 5 4 6 ci sβg ua ie ne 14 93 0. 00 1 0. 00 1 0. 00 1 4 7 b ic yc lo ge rm ac re ne 15 00 0. 00 5 0. 00 2 0. 00 2 0. 00 4 0. 00 2 0. 00 2 0. 00 1 0. 00 1 0. 00 2 4 8 tr an str an sα -f ar ne se ne 15 05 0. 00 2 0. 00 3 0. 00 3 0. 00 4 0. 02 0 0. 00 8 0. 00 3 0. 00 5 0. 00 2 0. 00 6 4 9 βb is ab ol en e 15 05 0. 00 4 0. 08 8 0. 00 4 0. 02 2 0. 01 7 0. 01 0 0. 00 7 0. 02 9 0. 00 8 0. 03 3 0. 00 6 0. 03 1 0. 00 7 5 0 γc ad in en e 15 13 0. 00 2 0. 00 2 0. 00 1 0. 00 1 0. 00 1 5 1 en do -1 -b ou rb on an ol 15 20 0. 00 2 0. 00 1 0. 00 1 5 2 γ c ad in en e 15 23 0. 00 4 0. 00 2 0. 00 2 0. 00 1 0. 00 2 5 3 tr an sγb is ab ol en e 15 31 0. 00 2 0. 00 4 0. 00 1 0. 00 2 0. 00 1 5 4 t hy m oh yd ro qu in on e 15 55 0. 00 9 0. 00 2 0. 00 2 0. 00 2 5 5 g er m ac re ne d -4 -o l 15 75 0. 06 4 0. 12 8 0. 00 1 0. 00 7 0. 00 5 0. 03 1 0. 01 6 0. 03 5 0. 01 9 0. 04 8 0. 01 8 0. 04 2 5 6 sp at hu le no l 15 78 0. 00 4 0. 00 2 0. 00 1 0. 00 2 0. 00 2 0. 00 2 5 7 c ar yo ph el le ne o xi de 15 83 0. 02 6 0. 02 0 0. 00 8 0. 01 6 0. 00 2 0. 02 0 0. 01 5 0. 00 1 0. 01 8 0. 00 1 5 8 g lo bu lo l 15 90 0. 00 2 0. 00 1 tr ac e 5 9 h um ul en e ep ox id e ii 16 08 0. 00 2 0. 00 2 0. 00 1 0. 00 1 0. 00 1 6 0 al lo -a ro m ad en dr en e ep ox id e 16 41 0. 00 3 0. 00 1 0. 00 1 6 1 ep iα -m uu ro lo l 16 42 0. 00 1 tr ac e 6 2 14 -h yd ro xy -9 -e pi -( e) -c ar yo ph yl le ne 16 69 0. 00 1 tr ac e 6 3 g er m ac ra -4 (1 5) , 5 ,1 0( 14 )tr ie n1α -o l 16 86 0. 01 1 0. 00 1 0. 00 3 0. 00 3 0. 00 3 6 4 o pl op an on e 17 40 0. 00 6 0. 00 1 0. 00 2 0. 00 2 0. 00 2 un kn ow n 0. 11 9 0. 01 5 0. 00 2 0. 00 3 0. 00 2 0. 02 9 0. 04 0 0. 00 2 0. 03 4 0. 00 1 to ta l c on te nt o f e o 1. 52 8 1. 47 4 12 .4 35 0. 74 9 1. 38 4 1. 30 7 0. 81 7 1. 71 9 4. 23 1 1. 31 2 3. 85 1 1. 31 2 3. 99 6 1. 31 2 a k ov át s re te nt io n in di ce s ca lc ul at ed a ga in st c 8– c 10 n -a lk an es o n no np ol ar d b -5 c ol um n. *: t he c on ce nt ra tio n w as lo w er th an 0 .0 00 9 m g/ g; -: it w as n ot d et ec te d 130 m. shafiee-hajiabad, j. novak, b. honermeier nene and p-cymene contents in herb. rli decreased the content of sabinene only in oxm plants. the content of p-cymene increased in ohi under rli condition. ovi plants, however, showed an opposite trend; although, it was not statistically significant (fig. 1). effect of accessions on eo content and composition of herb samples the investigated accessions showed different profiles of eo both in herb and leaf samples. in total, 64 compounds were identified in herb and leaf extracts of ov accessions (tab. 2). the mean concentrations of twenty compounds, with higher concentrations than 0.05 mg/g, were analyzed statistically. the results of the mean comparisons of eo compounds in herb samples are presented in tab. 3. in the herb ovu, trans-sabinene hydrate was the main compound (26 % of the total eo content). it was followed by β-caryophyllene and germacrene d (15.3 % and 11.7 % of the total eo content respectively). further six compounds namely β-bourbonene, thymol, 1-octen-3-ol, germacrene d-4-ol, cis-βocimene and sabinene were detected with relevant concentrations of more than 0.05 mg/g. another thirty compounds were identified with a lower concentration of 0.05 mg/g in herb samples (tab. 2). in the means comparison of chemical compounds, octen-3-ol <1->, cis-β-ocimene, trans-sabinene hydrate, allo-ocimene, β-bourbonene, germacrene d and germacrene d-4-ol were significantly higher in ovu compared to other accessions (tab. 3). compared to ovu a different composition of eo was found in ohi. thymol and carvacrol together reached around 65 % of total compounds (4.5 and 3.6 mg/g representing 36.3 % and 29.4 % of total content of eo). two further compounds namely γ-terpinene and pcymene were detected with higher concentrations than other compounds (tab. 2). in herb extracts of ovi, p-cymene and thymol were the dominant compounds (representing 31.3 % and 26.9 % of total content eo respectively) followed by γ-terpinene, thymol methyl ether, and cis-βocimene. in the means comparison of the main compounds of ovi herb samples cis-β-ocimene and thymol methyl ether had higher concentrations compared to other accessions (tab. 3). tab. 3: main chemical compounds in herb samples of origanum vulgare accessions detected by using gc/ms (s.e.: standard error of the mean; ovu: ssp. vulgare, ohi: ssp. hirtum, ovi: ssp. viride, oxm: ssp. viride × majorana) compounds mean ± s.e (mg/g) lsd (α<0.01) ovu ohi ovi oxm 1-octen-3-ol 0.068±0.13ab 0.11±0.13a 0.011±0.13b 0.031±0.13b 0.052** myrcene 0.006±0.016b 0.207±0.016a 0.022±0.017b 0.016±0.016b 0.62** cis-β-ocimene 0.060±0.014a 0.017±0.014 b 0.055±0.014a 0.033±0.014 b 0.054* γ-terpinene 0.036±0.155b 1.645±0.155a 0.193±0.155b 0.141±0.155b 0.61** trans-sabinene hydrate 0.40±0.098a 0.028±0.098b 0±0.0104b 0±0.098b 0.38* allo ocimene 0.031±0.007a 0.002±0.007b 0.035±0.008b 0.013±0.007b 0.029* thymol methyl ether 0.014±0.014b 0±0.014b 0.112±0.015a 0.006±0.014b 0.056** carvacrol methyl ether 0.001±0.011c 0.026±0.011bc 0.046±0.011b 0.108±0.011a 0.043** thymoquinone 0±0.022b 0.132±0.022a 0.016±0.023b 0.001±0.022b 0.086** thymol 0.072±0.665b 4.519±0.665a 0.387±0.706b 0.027±0.665b 2.65** carvacrol 0.010±0.666b 3.651±0.666ab 0.006±0.706b 0.059±0.666b 2.65** β-bourbonene 0.082±0.012a 0.001±0.012b 0.08±0.013b 0.023±0.012b 0.047** aromadendrene 0±0.015b 0.144±0.015a 0.003±0.016b 0±0.015b 0.058** germacrene d 0.179±0.011a 0±0.011b 0.013±0.011b 0.026±0.011b 0.042** β-bisabolene 0.004±0.01b 0.088±0.01a 0.022±0.011b 0.010±0.01b 0.041** germacrene d -4-ol 0.064±0.007a 0±0.007b 0.001±0.007b 0.005±0.007b 0.026** total content of eo 1.528±0.704b 12.435±0.704a 1.384±0.747b 0.818±0.704b 2.81** values within rows followed by the same letter (a–c) do not differ statistically (duncan test). ** at α <0.01; * at α <0.05; lsd: least significant difference fig. 1: effects of accession and light intensity on the chemical compounds of essential oil in herb samples (s.e.: mean standard error of mean, ovu: origanum vulgare ssp. vulgare, ohi: origanum vulgare ssp. hirtum, ovi: origanum vulgare ssp. viride, oxm: origanum vulgare ssp. viride × majorana), rli: reduced light intensity, nli: normal light intensity rli nli rli nli rli nli rli nli reduced light effect on essential oil of o. vulgare accessions 131 no dominant compound was identified in either herb or leaf samples of oxm. the herb samples were characterized by several monoterpenoid compounds namely γ-terpinene (22.6 % of the total eo content), sabinene (10.5 %), cis-β-ocimene (10.5 %), carvacrol methyl ether (8.1 %), 1-octen-3-ol (6.7 %), and carvacrol (5.4 %) (tab. 3). effect of accessions on eo content and composition of leaf samples the mean comparisons of the main eo compounds in leaf samples are reported in tab. 4. the same pattern of eo composition was observed in ovu leaf samples. however, the concentration of germacrene d-4-ol, cis-β-ocimen, trans-β-ocimen in leaf samples were about two times greater than in herb samples (tab. 2). in ovu plants, the total content of eo was very low in both sample types (1.53 and 1.47 mg/g respectively) (tab. 3 and fig. 2a). in contrast to the herb samples of ohi, a higher concentration of carvacrol and a lower concentration of thymol were detected in leaf samples (0.48 and 0.13 mg/g, representing 63.8 % and 18 % of total content of eo). the content of eo was notably higher in herb samples compared to leaf samples (12.43 and 0.79 mg/g respectively) (tab. 2 and fig. 2b). in ovi leaf samples six compounds had higher concentrations compared to other accessions: myrcene, p-cymene, cis-β-ocimene, thymol methyl ether, thymol and β-bisabolene (tab. 4). in contrast to the herb in leaf extracts of ovi, a lower content of p-cymene and a higher content of thymol (representing 19.6 % and 30.7 % of total eo content, respectively) was detected (fig. 2c). in addition, γ-terpinene (20.8 %) reached a relatively large concentration followed by cis-β-ocimene, thymol methyl ether and allo-ocimene (tab. 2). interestingly, it was observed that the concentration of thymol in ovi leaf samples was higher even than in ohi samples. in leaf samples of oxm, γ-terpinene (17.3 %) had a greater concentration than p-cymene (13.7 %). it was followed by carvacrol methyl ether (13.2 %) and sabinene (7.8 %) (tab. 4). interestingly, the leaf samples had superior eo content in comparison to herb samples (tab. 2 and fig. 2d). discussion effect of light intensity on content and composition of eo sabinene is one of the final products of the sabinyl pathway (novak et al., 2010). different investigations showed the effect of abiotic factors on this pathway and its final products. novak et al. (2010) found out that by increasing the temperature from 18 to 26 °c, the concentration of sabinene increased linearly from 3.9 % to 5.3 %. in origanum majorana l. sabinene, cis-sabinene hydrate and transsabinene hydrate were produced in larger quantities in the plants grown under longer days, whereas the content of terpinenes de creased with increasing day length (circella et al., 1995). de falco et al. (2013) observed ovu plants grown in single-rows were rich in sabinene. as single-row plants received higher light intensities compared to binate-rows, it can be assumed that the lower light intensity in binate-rows decreased sabinene content in eo, confirming our results. moreover, in our samples, trans-sabinene hydrate (15.02 min) appeared earlier than linalool (15.11 min) (tab. 2). according to adams (2007), however, trans-sabinene hydrate (ki: 1098) should appear after linalool (ki: 1096) in the chromatogram. in the process of cymyl compounds biosynthesis, p-cymene is formed by aromatisation of γ-terpinene (lukas et al., 2010; poulose and croteau, 1978). afterwards, thymol and carvacrol are synthesized by hydroxylation of p-cymene by two hypothetical hydroxylases. it is proposed that the alteration in the composition of cymyl compounds, as a reaction to different environmental conditions, is mediated by carvacrol hydroxylase and thymol hydroxylase (novak et al., 2010). accordingly, when light intensity was reduced, the activity of carvacrol hydoxylase was diminished. therefore, in ohi under a rli condition an accumulation of p-cymene and smaller concentration of carvacrol (rli: 0.24, nli: 0.71 mg/g, data not presented) was observed. confirming our results, johnson et al. (2004) observed in ohi plants decreased p-cymene concentration and increased carvacrol concentration during spring to summer time. specifically, in ohi plants the 50 %-shade treatment modified the profile of eo. the eo of the plants under full light treatment was mainly composed of 4-terpineol, γ-terpinene, carvacrol, p-cymene and α-terpinene. moreover, tab. 4: main chemical compounds in leaf samples of origanum vulgare accessions detected by using gc/ms (s.e.: standard error of the mean; ovu: ssp. vulgare, ohi: ssp. hirtum, ovi: ssp. viride, oxm: ssp. viride × majorana) compounds mean ± s.e (mg/g) lsd (α<0.01 ovu ohi ovi oxm sabinene 0.079±0.023b 0.002±0.025b 0.007±0.023 b 0.181±0.023a 0.094** 1-octen-3-ol 0.063±0.017ab 0±0.018b 0.002±0.017b 0.116±0.017a 0.067** myrcene 0.003±0.008b 0.006±0.008b 0.019±0.008ab 0.047±0.008a 0.031** ρ-cymene 0.006±0.02b 0.054±0.02b 0.181±0.02a 0.041±0.02b 0.08** cis-β-ocimene 0.143±0.032a 0±0.035b 0.125±0.032a 0.18±0.032a 0.098** trans-β-ocimen 0.051±0.014ab 0±0.015b 0.034±0.014b 0.088±0.014a 0.041** trans-sabinene hydrate 0.151±0.036a 0±0.039b 0.008±0.036b 0.002±0.036b 0.15* thymol methyl ether 0.003±0.012b 0±0.013b 0.067±0.012a 0±0.012b 0.051** thymol 0.031±0.062b 0.135±0.067b 0.401±0.062a 0±0.062b 0.25** β-bourbonene 0.092±0.009a 0±0.01b 0±0.009b 0.035±0.009b 0.036** germacrene d 0.268±0.033a 0±0.036b 0.018±0.033b 0.134±0.033b 0.137** β-bisabolene 0±0.004b 0.004±0.005ab 0.017±0.004a 0.007±0.004ab 0.017* germacrene d -4-ol 0.128±0.015a 0±0.016b 0.007±0.015b 0.031±0.015b 0.06** values within rows followed by the same letter (a–d) do not differ statistically (duncan test) ** at α <0.01; * at α <0.05; lsd: least significant difference 132 m. shafiee-hajiabad, j. novak, b. honermeier in the 50 %-shade treatment this composition changed to γ-terpinene, 4-terpineol, carvacrol, p-cymene and allo cymene. in the reduced light condition, the concentration of p-cymene increased and the concentration of sabinene decreased (tibaldi et al., 2011). effect of accessions on eo content and composition of herb and leaf samples in all accessions, the extracts of herb samples were characterized by higher biochemical diversity compared to the extracts received from the leaf samples (tab. 2). this property refers to the presentation of different plant organs, including leaves, flowers and young stems in one herb sample. furthermore, the ontological stage of plant organs in herb samples can influence the composition of eo (johnson et al., 2004; raduðienë et al., 2005; tibaldi et al., 2011). ovu plants have been found to be rich in acyclic compounds and sesquiterpenoids (skoula and harborne, 2004). azizi et al. (2012) analyzed the eo composition of ovu and found that two sesquiterpenes: β-caryophyllene and germacrene d composed more than 60 % of eo. other main components included terpinene-4-ol (6.7 %), spathulenol (4.4 %) and γ-terpinene (3.9 %). they could not identify any sabinene, 1-octen-3-ol, ocimene compounds or germacrene d-4ol which were detected in the ovu plants of the current experiment. besides, the concentration of trans-sabinene hydrate was very low in their experiment. however, these compounds were identified by de falco et al. (2013) and baranauskienė et al. (2013) who intensively investigated the eo of ovu. furthermore, in 374 individuals of ovu plants from austria the monoterpene fraction was mainly made up of sabinyl-compounds (primarily sabinene and cis-sabinene hydrate) and/or cymyl-compounds (primarily p-cymene, γ-terpinene, and carvacrol) usually accompanied by smaller amounts of bornyl-compounds and acyclic compounds (lukas et al., 2013). in the current study, ovu plants were rich in sabinyl compounds (trans-sabinene hydrate and cis-sabinene hydrate) and sesquiterpenoids (β-caryophyllene and germacrene-d). each of the other compounds, such as 1-octan-3-ol, β-ocimene (an acyclic compound) and thymol (a cymyl-compound), had a concentration of more than 5 %. therefore, these plants are comparable to the mixed-type of austrian ovu plants (lukas et al., 2013) and with ovu plant from lithuania which were analyzed by raduðienë et al. (2005). according to our results, it can be proposed that the investigated ohi accession is an intermediate type with high content of p-cymene and γ-terpinene. these results confirm the findings of azizi et al. (2012) who investigated the ohi accession from the same source. moreover, the greater density of glandular trichomes on the inflorescences of ov plants generates the superior eo content in this organ compared to other plant’s parts (werker et al., 1985). the existence of flowers in herb samples should be the explanation for higher eo content in these samples. in the previous study, a higher density of peltate glandular trichomes was reported in ohi leaf samples when compared with other accessions (shafiee-hajiabad et al., 2015). the result of the current study showed no difference in eo content in leaf samples of all accessions. particularly, in the case of ohi not only the content of each compound but also the total content of eo was very low in leaf samples compared to herb samples (tab. 4). fig. 2: composition of essential oil in herb (h) and third leaf (l) samples of origanum vulgare accessions ovu: origanum vulgare ssp. vulgare ohi: origanum vulgare ssp. hirtum ovi: origanum vulgare ssp. viride oxm: origanum vulgare ssp. viride × majorana a b c d compound lsd 0.01 octen-3-ol <1-> 0.01 myrcene 0.039 p-cymene 0.156 cis-b-ocimene 0.07 -terpinene 0.048 thymoquinone 0.05 thymol 1.503 carvacrol 1.521 ß-caryophellene 0.088 aromadendrene 0.03 germacrene d -4-ol 0.03 rest total eo 1.717 compound lsd 0.01 octen-3-ol <1-> 0.01 myrcene 0.04 p-cymene 0.16 cis -ocimene 0.07 -terpinene 0.05 thymoquinone 0.05 thymol 1.50 carvacrol 1.52 ß-caryophellene 0.09 aromadendrene 0.03 germacrene d -4-ol 0.03 rest total content of eo 1.71 reduced light effect on essential oil of o. vulgare accessions 133 since peltate glandular trichomes, the primary location for eo secretion, are formed on the epidermal surfaces of plants and because the leaves of ohi are thicker than ovi (shafiee-hajiabad et al., 2014), it is possible that the leaf samples of ohi involve more parenchyma than epidermal tissue. as a consequence, less peltate glandular trichomes were presented in the ohi leaf samples; therefore less eo was detected in leaf samples of ohi compared to other accessions. however, in the herb samples a considerably higher amount of eo was detected. it was also observed that the composition of eo changed among different organs (werker et al., 1985) as well as among different peltate glandular trichomes of one leaf (johnson et al., 2004). therefore, different profiles of eo in herb and leaf samples can be expected. in contrast to our results, eo composition analysis of ovi plants by azizi et al. (2012) showed that thymol (62.5 %) was the dominant compound, and other four compounds: β-caryophyllene, thymol methyl ester, carvacrol methyl ester, as well as β-bisabolene had concentrations between 3.5 to 4.9 %. moreover, ovi plants native to crete were found to be rich in sabinyl compounds, acyclic compounds and sesquiterpenoids (skoula et al., 1999) whereas in ovi plants from argentina the main compound was carvacrol (farías et al., 2010). in ovi plants from turkey, the sesquiterpenoid compounds composed the main fraction of eo (caryophyllene oxide, caryophyllene, spathulenol and caryophyllenol ii: 41.8 %), followed by linalool (8.3 %), 1,8-cineol (8 %), while p-cymene had a low concentration (4.1 %) (koldaş et al., 2014). in iranian ovi plants, the monoterpene fraction constitutes 52.4 % of the oil with the main components such as linalyl acetate (20.1 %), sabinene (13.4 %), γ-terpinene (5.6 %), trans-ocimene (3.6 %), and cis-ocimene (3.4 %) (afsharypuor et al., 1997). in the case of oxm, our results showed enhanced concentrations of monoterpenes and a low concentration of the sesquiterpene fraction. in contrast, azizi et al. (2012) found that carvacrol (22.5 %), germacrene d (24.8 %), β-caryophyllene (9.8 %), carvacrol methyl ester (9.7 %) and terpinene-4-ol (4 %), as the main compounds in oxm. the eo composition of oxm investigated in this study is somehow similar to eo profile of “green spanish” clone (o. vulgare ssp. viridulum) from argentina (farias et al., 2010). generally, bicyclic sabinyl monoterpenes (cis-sabinene hydrate, cis-sabinene hydrate acetate, trans-sabinene hydrate and sabinene) are the main eo compounds of marjoram (origanum majorana l.). while p-cymyl compounds are characteristic for oregano (origanum spp.), and they are completely absent in the cultivated chemo variety of marjoram (novak et al., 2004). this accession, which is rich both in sabinyl compounds and cymyl compounds, shows an interesting composition of eo. furthermore, thymoquinone was detected in the herb samples of all investigated accessions (tab. 2). however, its concentration was considerably higher in ohi compared to other accession (tab. 3). this compound is an interesting consistent of eo which was reported previously in the genus origanum (russo et al., 1996; skoula et al., 1999). it exhibits promising effects against inflammatory diseases and cancer by enhancing the anticancer potential of clinical drugs, while reducing unwanted side effects (schneider-stock et al., 2014). in conclusion, the current study found that light intensity made small changes to the eo composition. accordingly, it can propose that the biosynthetic pathway of these compounds is mostly genetically influenced and most likely in order to have relevant changes in the eo profile, more intensive light reduction or light difference is required. however, the effect of applied light reduction on macro and micromorphological parameters was previously studied (shafiee-hajiabad et al., 2015). besides, minor changes of eo composition under two light intensities reveal the genotype stability of these plants. since for the industry of 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(ed.), oregano: the genera origanum and lippia. taylor and francis e-library, new york. tibaldi, g., fontana, e., nicola, s., 2011: growing conditions and postharvest management can affect the essential oil of origanum vulgare l. ssp. hirtum (link) ietswaart. ind. crops prod. 34, 1516-1522. werker, e., 1993: function of essential oil-secreting glandular hairs in aromatic plans of lamiacea-a review. flavour fragrance j. 8, 249-255. werker, e., putievsky, e., ravid, u., dudai, n., katzir, i., 1993: glandular hairs and essential oil in developing leaves of ocimum basilicum. ann. bot. 71, 43-50. werker, e., putievsky, e., ravid, u., 1985: the essential oils and glandular hairs in different chemotypes of origanum vulgare l. ann. bot. 55, 793-801. address of the authors: marzieh shafiee-hajiabad, bernd honermeier, depatment of agronomy, institute of agronomy and plant breeding, justus liebig university giessen, schubertstr. 81, d-35392 giessen, germany e-mail: marzieh.shafiee-hajiabad@agrar.uni-giessen.de e-mail: bernd.honermeier@agrar.uni-giessen.de johannes novak, institute for animal nutrition and functional plant compounds, university of veterinary medicine, veterinaerplatz 1, a-1210 vienna, austria e-mail: johannes.novak@vetmeduni.ac.at © the author(s) 2016. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 89, 202 207 (2016), doi:10.5073/jabfq.2016.089.025 university of perugia, department of pharmaceutical sciences, section of food science and nutrition, perugia, italy seasonal variations in antioxidant compounds of olea europaea leaves collected from different italian cultivars francesca blasi, eleonora urbani, maria stella simonetti, claudia chiesi, lina cossignani* (received january 20, 2016) * corresponding author summary the objectives of this research were to study the best conditions for phenol extraction from olea europaea l. leaves and to evaluate the content of phenolics and the antioxidant activity by in vitro assays in olive leaves harvested in different periods from four different italian cultivars. the results showed that leaves/solvent ratio and temperature showed a significant and positive correlation with phenol extraction yield. in all harvesting periods the phenol content of dolce agogia samples was higher (p ≤ 0.05) with respect to moraiolo, leccino and frantoio samples. the results of analysis by high-performance liquid chromatography coupled with diode array detector (hplc-dad) showed that dolce agogia had the highest concentrations of hydroxytyrosol and oleuropein. moreover the highest contents of bioactives have been found in olive leaves harvested in december and march. in these months, corresponding to olive harvest and pruning of olive trees, leaves represent a waste product and this interesting result could be useful in the production of nutraceuticals. introduction the olive tree, olea europaea l., is one of the most important fruit tree in mediterranean countries. the olive fruit, its oil, and the leaves of the olive tree have a rich history for nutritional, medicinal, and ceremonial purposes (ghanbari et al., 2012). it is known that natural foods and food-derived antioxidants such as phenolic phytochemicals have interesting biological properties (quideau et al., 2011). in recent years, interest in olive leaves has grown among scientists because they contain many potentially bioactive compounds with health benefits (el and karakaya, 2009). therefore, the demand of whole olive leaf and olive leaf extract has increased for use in food, additives and functional products (lafka et al., 2013). thus, olive leaf extract is now a popular nutraceutical, a dietary supplement manufactured in liquid or capsule form (hayes et al., 2011; de bock et al., 2013). the most abundant biophenol in olive leaves is oleuropein (ole), followed by hydroxytyrosol (ht). olive leaves have the highest antioxidant and scavenging power among the different parts of the olive tree, in fact ole content ranges between 1 % and 14 % (japón-luján et al., 2006). accordingly, in vitro and in vivo studies have documented the antioxidant, hypolipidaemic, and especially hypotensive, anticancer and cardioprotective properties of ole (hassen et al., 2014). also the ht shows interesting biological properties (hu et al., 2014). the chemical composition of olive leaves varies depending on several conditions such as origin, proportion of branches on the tree, climatic conditions, storage conditions, and moisture content (sabry, 2014). several studies have investigated the phenolic com position of olive leaves (pereira et al., 2007; abaza et al., 2011; salah et al., 2012; stamatopoulos et al., 2014). some authors studied the influence of the solvent type on the extraction of phenolic compounds and the antioxidant properties of the extracts obtained from chétoui olive leaves (abaza et al., 2011). other authors studied the phenolic composition and biological activities of olive leaves from different varieties grown in tunisia (salah et al., 2012). a high content of polyphenol and flavonoid was detected in all varieties and ole was the major compound. also, olive leaf extract exhibited a good antioxidant activity. it has been reported that the combination of steam blanching process and multistage extraction (after optimization) is advantageous since it provides short extraction times (≤ 30 min) at moderate operation temperatures (40 °c) (stamatopoulos et al., 2014). recently, talhaoui et al. (2015) analyzed by high performance liquid chromatography-diode array detector-time-of-flight-mass spectrometry (hplc-dad-tofms) leaves from six olive cultivars collected at four different times. this research concerns the evaluation of antioxidant fraction in olive leaves from four different italian cultivars (dolce agogia, moraiolo, leccino, frantoio), harvested at four different periods (december 2013; march, june and september 2014). firstly, experimental design methodology was used to optimize operating conditions of the phenol extraction. then total phenolic (tp) content and antioxidant properties by two scavenging activity methods, dpph (2,2 diphenyl-2-picrylhydracyl hydrate) and abts [(2,2’-azinobis(3ethylbenzothiazoline-6-sulfonic acid) diammonium salt], have been determined on olive leaf extracts. moreover, to quantify ole and ht a chromatographic method, hplc with diode array detector (dad), was developed. this study deepens the knowledge on antioxidant compounds of olive leaves harvested in different periods from some characteristic olive cultivars of central italy, also for the purpose to highlight the best period of the year to harvest olive leaves for using as source of bioactives. materials and methods olive leaf samples olive leaves from four different olea europaea l. cultivars (dolce agogia, moraiolo, leccino and frantoio) were collected from an olive orchard located in the area of perugia (umbria, central italy). the selected cultivars, grown under the same agronomic and environmental conditions, were some of the most widespread cultivars in central italy. the trees had not been irrigated and no phytosanitary treatments had been applied in the last year. leaves were randomly harvested in different times (december 2013; march, june and september 2014) from five trees of each cultivar. branches with both young and mature leaves were collected at the operator height around the whole perimeter of each tree. the collected samples were stored in plastic bag until arrival at the laboratory, where they were immediately separated from the branches. then the leaves were dried in a ventilated oven for 72 h at 40 °c and stored away from light and humidity until extraction. extraction and isolation of phenols the optimization of phenol extraction conditions was carried out by modde 5.0 experimental design software (umetrics ab, umeå, sweden). the following factors were considered: etoh%, seasonal variations of olive leaf phenols from italian cultivars 203 leaves/solvent (l:s) ratio and temperature. the solvent composition ranged from 0 to 100 % etoh in h2o and the temperature from 20 to 60 °c. the l:s ratio, indicated as 1, 2 and 3, referred respectively to 0.7, 1.7 and 2.7 g of dried leaves in 75 ml of solvent. tp content, determined as reported in a following paragraph and expressed as milligrams of gallic acid equivalent (mg gae) in total extract, was selected as response. d-optimal design including three replicated center points was employed and a total of 14 experiments was obtained. the extractions were conducted in random order using the experimental conditions indicated in the worksheet (tab. 1). the model was then fitted using multiple linear regression analysis. 2.7 g of dried leaves from each tree were added with 75 ml of a mixture h2o/etoh (40:60, v/v) and homogenized using a homogenizer (oster, model 869-50r, usa). after that the mixture was kept under magnetic stirring for 30 min at 60 °c and filtered through a whatman filter paper in a büchner funnel. all extractions were carried out in duplicate. the obtained extracts were subjected to the analytical determinations, reported in the following paragraphs. determination of phenolic compounds the tp content was determined spectrophotometrically according to the method of singleton and rossi (1965) and expressed as milligrams of gallic acid equivalent per gram (mg gae/g) of dried leaves. a calibration curve was built using gallic acid as standard (97.5-102.5 % titration, sigma, st. louis, mo, usa), in the range 0.0025-0.0125 mg/ml. 0.5 ml of folin-ciocalteu reagent (sigma), 2.0 ml of 20 % na2co3 and 6.5 ml of deionized h2o were added to leaf diluted extract (1.0 ml). the solution was mixed, incubated in the dark for 90 min and then the absorbance was measured at 750 nm. all determinations were carried out in duplicate. dpph assay the dpph free radical-scavenging activity of olive leaf extracts was determined according to the method described by assimopoulou et al. (2005) with some modifications. in brief, a 0.06 mmol/l solution of dpph• (sigma) in etoh was prepared and left for 1 h in the dark at 4 °c. an aliquot of each extract (0.1 ml) was added to 3.9 ml of dpph• solution and vortexed. the samples were left for 30 min in the dark, and then their absorbance was measured at 517 nm. the percentage of antioxidant activity (aa %) was calculated using the following formula: aa% = (absc – abss / absc) × 100 where: absc is the absorbance of the control solution (containing only dpph•) abss is the absorbance of the dpph• solution mixed with sample. all determinations were carried out in duplicate. abts assay the abts assay was determined by earlier reported method (tawaha et al., 2007) with slight modifications. abts radical cation (abts•+, sigma) was produced by reacting abts solution with k2s2o8. an aqueous solution of 7 mmol/l abts was prepared and used to dissolve 2.45 mmol/l k2s2o8. the mixture was left in the dark at room temperature for 12-16 h and then it was diluted with h2o until the absorbance reached 0.70 ± 0.02 at 734 nm. after addition of 4 ml of diluted abts•+ solution to 60 μl of each extract appropriately diluted, the reaction was left in the dark at room temperature for 6 min. after that the absorbance was measured at 734 nm. the antioxidant capacity of each sample was expressed as milligrams trolox equivalents per gram (mg te/g) of dried leaves. the calibration line was built using (±)-6-hydroxy-2,5,7,8tetramethylchromane-2-carboxylic acid standard (trolox, 97 %, sigma) at different concentrations (from 0.1 to 0.5 mg/ml). all determinations were carried out in duplicate. instrumentations spectrophotometric measurements to determine total phenolic content and antioxidant activity were performed using a jasco 7850 uv-vis spectrophotometer (jasco inc., easton, md, usa). hplc analysis of olive leaf phenols was performed using a shimadzu gt154 system equipped with a thermo spectra series pump, an agilent zorbax ods column (5 μm particle size, 3.0 × 150 mm i.d., agilent tab. 1: worksheet and response for d-optimal design exp. no. run order factors response etoh% l:s ratio temperature (°c) tp (mg gae) 1 12 0 1 20 31.7 2 6 100 1 20 17.0 3 3 40 2 20 88.3 4 2 0 3 20 76.1 5 1 100 3 20 42.6 6 11 40 1 40 49.7 7 4 100 2 40 40.0 8 8 0 1 60 38.3 9 9 100 1 60 29.6 10 10 0 3 60 110.9 11 13 80 3 60 196.2 12 5 60 2 40 106.7 13 14 60 2 40 11.0 14 7 60 2 40 97.7 a l:s ratio (indicated as 1, 2 and 3) referred respectively to 0.7, 1.7 and 2.7 g of dried leaves in 75 ml of solvent. 204 f. blasi, e. urbani, m.s. simonetti, c. chiesi, l. cossignani technologies italia, milano, italy) and a spectra system uv6000lp dad (thermo separation products, san jose, ca, usa). detection was performed on-line using the dad in the wavelength range 200700 nm. separation was achieved by a gradient elution, according to ortega-garcia and peragon (2010). the solvents were water (ph adjusted to 3.1 with 2 ml/l acetic acid) (a) and meoh (b). the samples were analyzed by gradient elution at a flow rate of 0.8 ml/ min. the elution conditions were: initial a-b (90:10); in 5 min a-b (70:30); for 5 min a-b (70:30); in 5 min a-b (60:40); in 2.5 min a-b (50:50); in 2.5 min a-b (40:60); in 2.5 min a-b (30:70); in 3.5 min a-b (0:100); for 4 min a-b (0:100). the column was re-stabilized to the initial conditions for 15 min before the next injection. an injection volume of 20 μl was used. the chromatograms were acquired and the data handled using xcalibur software version 1.2 (finnigan corporation, san jose, ca, usa). calibration lines were built using ole (≥ 90 %) and ht (≥ 98 %) reference compounds (extrasynthese, genay cedex, france) and the least square method was used to calculate the regression equations. all analyses were carried out in duplicate. statistical analysis the results of the chromatographic analysis and the antioxidant assays are expressed as the mean value and standard deviation (sd) of the five samples of each cultivar. microsoft excel 2007 (microsoft corporation, redmond, wa, usa) was used for data analysis. results and discussion optimization of extraction conditions to study the influence of the selected variables on tp content, an experimental design was carried out by entering three factors (etoh%, l:s ratio and temperature) and one response (tp, expressed as (mg gae), by selecting the screening objective and by choosing the d-optimal experimental design. the experimental design reported acceptable values regarding the percentage of the variation of the response explained by the model (r2 = 0.85) and the percentage of the variation of the response predicted by the model (q2 = 0.61). fig. 1 shows the coefficient plot of the considered factors (etoh%, l:s ratio, temperature). it can be observed that the response was positively influenced by l:s ratio, temperature and their interaction (l:s*temp), while etoh% influenced to a lesser extent the response and exhibited an inverse correlation. fig. 2 shows the prediction plots of tp content as a function of the single factors, etoh% (a), l:s ratio (b) and temperature (c), maintaining fixed the values of the other two factors. the results confirmed that etoh% had little influence on the tp content with respect to the other two variables, in fact l:s ratio and temperature showed a significant and positive correlation on extraction yield. in fig. 3 the two-dimensional contour plot showing the tp content as a function of temperature and l:s ratio, maintaining fixed the percentage of etoh at 60, is reported. it can be noted that the highest tp values were obtained when the l:s ratio was 3 (2.7 g of dried leaves in 75 ml of solvent) and the temperature was 60 °c. on the base of the obtained results, the conditions selected for the extraction of olive leaf samples, from different cultivars in different harvesting periods, were the following: ratio 3, 60 °c and 60 % etoh in h2o. the composition of the extraction mixture was chosen because the extract had a better workability. etoh l:s temp l:s*temp tp , m g g a e fig. 1: coefficient plot of the considered factors (etoh, ethanol %; l:s, l:s ratio; temp, temperature) for total phenol (tp) response. 220 160 100 40" 0 50 100 1 2 3 20 40 60 etoh l:s ratio temperature tp , m g g a e ! 20 40 60 temperature l: s r at io 3 2 1 fig. 3: two-dimensional contour plot showing the total phenol content as a function of l:s ratio and temperature, when the percentage of etoh was fixed at 60. l:s ratio values have been described in tab. 1. fig. 2: prediction plots of the considered factors: etoh% (l:s ratio 3 and temperature 60 °c); l:s ratio (etoh 60 % and temperature 60 °c); temperature (etoh 60 % and l:s ratio 3) for total phenol (tp) response. l:s ratio values have been described in tab. 1. phenol content and antioxidant activity fig. 4 shows the phenol content of all the considered samples determined by folin-ciocalteu method. the mean values of tp in olive leaf extracts ranged from 40.9 mg gae/g for frantoio (december harvesting) to 66.6 mg gae/g for dolce agogia (june harvesting). phenol content increased from december to march (p ≤ 0.01) and from march to june (p ≤ 0.01), in fact the highest tp values (p ≤ 0.05) were found in june for all cultivars. phenol content showed marked variations with plant growth, in fact probably the phenol storage in the leaves is a timedependent regulated process, according to the life cycle of olive leaves (phenological phase). this hypothesis has been confirmed by other authors (brahmi et al. 2012; brahmi et al. 2015). in the seasonal variations of olive leaf phenols from italian cultivars 205 ca ba aa aa aab bb ab cbc bac ab ac ab aab ac ba aab harvesting period of march the phenol content of dolce agogia samples was higher (p ≤ 0.05) with respect to moraiolo, leccino and frantoio. results comparable to those presented in this research have been reported by alzweiri and al-hiari (2013) for an olive leaf methanolic extract (40 mg gae/g). on the contrary, abaza et al. (2011) reported lower values (from 16.52 to 24.93 mg gae/g) for the second main variety (chétoui) cultivated in the north of tunisia. salah et al. (2012) reported instead higher values (from 73.05 of sevillane to 144.19 mg gae/g of limouni variety) for different tunisian varieties in respect to those reported in this study. ahmad-qasem et al. (2014) studied the phenol content of spanish olive leaves (variety serrana) dried with different methods and they found significant differences. in fact they reported the highest values (59.0 mg gae/g) for samples dried using hot air and the lowest (36.3 mg gae/g) for freeze dried samples. in order to obtain data about the antioxidant capacity of olive leaf extracts, two different in vitro assays, dpph and abts, have been performed. free-radical scavenging potential of samples was first tested by dpph assay and the results are shown in fig. 5a. generally, it can be observed that all olive leaf extracts exhibited high radical scavenging activity even if it is well known that antioxidant capacity is influenced by several factors, among which harvesting period and cultivar (brahmi et al., 2015). a similar trend was obtained for each cultivar, according to the biological cycle of olive leaves, in fact antioxidant activity was the highest in march, when the leaves had completed their growth, while it decreased slightly or strongly (p ≤ 0.05) until september, when started the ripening of the fruit. the lowest values had been found for all cultivar samples harvested in december (from 40.9 of frantoio to 67.1 % of dolce agogia), while olive leaves from leccino cultivar harvested in march exhibited the highest activity (86.1 %). the results reported by abaza et al. (2011) showed that the dpph radical scavenging activity increased with the extract concentration (at 0.5 mg/ml the activity was 59.74 %, using 70 % etoh). also lafka et al. (2013) studied the influence of the extraction solvent on olive leaf dpph antioxidant activity and reported the lowest value (18.8 %) for n-propanol and the highest (55.0 %) for etoh. fig. 5b shows the results of the radical scavenging potential of samples tested by abts assay. the values of antioxidant activity ranged from 44.8 mg te/g (179.0 μmol te/g) for leccino leaves harvested in september to 99.8 mg te/g (398.7 μmol te/g) of dolce agogia leaves harvested in december. moraiolo samples showed a significant decrease (p ≤ 0.05) from december to march, to june, to september and the values ranged from 88.4 to 53.6 mg te/g. with regard to dolce agogia samples, significant differences between december and june and between december and september (p ≤ 0.05) have been found. as regards leccino and moraiolo cultivar significant differences (p ≤ 0.05) had also been observed between december and september, march and june, march and september, june and september. the antioxidant activity determined by abts showed significant differences also between moraiolo and frantoio and between moraiolo and leccino (p ≤ 0.05) with regard to september harvest. not always the trend of abts antioxidant activity was similar to that observed for dpph results and this occurrence could be due to the different qualitative composition of phenols in the leaves, as already suggested by brahmi et al. (2013). the values of antioxidant activity determined by abts assay were lower than those reported by abaza et al. (2011) which found values in the range 629.871064.25 μmol te/g. even higher values for antioxidant activity (1.80-4.36 mmol te/g) have been reported by altiok et al. (2008) which studied the isolation of polyphenols from the extracts of olive leaves by adsorption on silk fibroin. on the contrary ahmad-qasem et al. (2014) reported very lower values (4.5-7.5 mg te/g) for abts assay than those reported in this research. ba aa cb abb ba ca da aa ba aab aba bab cb ab ba abab b aa ba ca ca aa bb cb cb ba cb ab dc cb ac bb dc a fig. 4: total phenol content (mg gae/g dried leaves, mean values) in dolce agogia (da), moraiolo (m), leccino (l), frantoio (f) olive leaves harvested at december (dec), march (mar), june (jun) and september (sep). error bars indicate sd of the means (n = 5). means with different capital letters indicate significant differences (p ≤ 0.05) among months within the same olive cultivar. means with different lower case letters indicate significant differences (p ≤ 0.05) among olive cultivars within the same month. ba aa cb abb ba ca da aa ba aab aba bab cb ab ba abab b aa ba ca ca aa bb cb cb ba cb ab dc cb ac bb dc a fig. 5: antioxidant activity determined by (a) dpph assay (aa%, mean values) and (b) abts assay (mg te/g, mean values) in olive leaves harvested in different periods from different cultivars. error bars indicate sd of the means (n = 5). means with different capital letters indicate significant differences (p ≤ 0.05) among months within the same olive cultivar. means with different lower case letters indicate significant differences (p ≤ 0.05) among olive cultivars within the same month. abbreviations of olive cultivars and harvesting periods have been reported in fig. 4. 206 f. blasi, e. urbani, m.s. simonetti, c. chiesi, l. cossignani hplc-dad analysis among the compounds detected by hplc-dad particular attention has been given to ht and ole, the main phenol compounds in olive leaves. fig. 6a shows the content of ht in olive leaf extracts for the considered samples. the ht content varied considerably considering both the cultivars and the harvesting periods. the values ranged from 1.5 mg/g of leccino in september to 7.0 mg/g of dolce agogia in december. in all considered cultivars the content was higher in december than in june and september (p ≤ 0.05) and it was also higher in march than in june. it was observed a significant difference (p ≤ 0.05) of ht content between december and march in dolce agogia and between june and september in leccino samples. generally, talhaoui et al. (2015) reported lower values for ht content (sum isomers), comparable only with leccino values. fig. 6b shows the ole content in the considered samples and the results showed that it was very different among cultivars and harvesting periods. dolce agogia exhibited the highest values in each period, while the lowest was found in frantoio cultivar. in december the ole content significantly decreased (p ≤ 0.05) from dolce agogia (91.8 mg/g) to frantoio (9.7 mg/g). the same trend was showed in september from dolce agogia (80.2 mg/g) to frantoio (2.8 mg/g). savournin et al. (2001) studied the ole content in leaves from 14 different olive cultivars and reported values ranging from 9.04 of aglandau to 13.66 % of picholine cultivar and their data were comparable to that reported in this research. in another study instead, tayoub et al. (2012) reported lower values for syrian varieties harvested at spring and fall. the ole concentration, indeed, ranged between 5.6 and 9.2 mg/g in spring samples and the highest content was found in jlott variety. in fall leaf samples the concentration ranged between 4.3 to 8.2 mg/g and the highest content was found in the same variety. generally, talhaoui et al. (2015) reported lower values of ole content (sum isomers) for six spanish cultivars. the results obtained for olive leaf samples have been elaborated in order to highlight possible correlations between the antioxidant activity measured by in vitro assays and ht and ole content. as an example, fig. 7 shows the significant positive correlations obtained for ole content and dpph value, in all the considered harvesting periods. moreover, for samples harvested in september it was observed that ht content well correlated with dpph value (r2 = 0.8354), abts value (r2 = 0.8079) and ole content (r2 = 0.9422). in conclusion, in this study the results of phenolic content and aa ab bb bb aa ba ca bca ab bb aab ccd aa bb ac bbd a b ab bb cb bb aa ba ba aba ba ac cbc dbc ac bb cc acd fig. 6: hydroxytyrosol (a) and oleuropein (b) contents (mg/g, mean values) in olive leaves harvested in different periods from different cultivars. error bars indicate sd of the means (n = 5). means with different capital letters indicate significant differences (p ≤ 0.05) among months within the same olive cultivar. means with different lower case letters indicate significant differences (p ≤ 0.05) among olive cultivars within the same month. abbreviations of olive cultivars and harvesting periods have been reported in fig. 4. aa ab bb bb aa ba ca bca ab bb aab ccd aa bb ac bbd a b ab bb cb bb aa ba ba aba ba ac cbc dbc ac bb cc acd fig. 7: correlation plots between oleuropein content (mg/g) and dpph value (aa%) of olive leaf samples from the considered cultivars harvested in different periods. seasonal variations of olive leaf phenols from italian cultivars 207 antioxidant activity of olive leaves, from different cultivars harvested in different times of year, have been reported. significant differences in ht and ole contents among the cultivars have been observed. dolce agogia showed the highest values, while frantoio the lowest ones. in december and march the highest contents of bioactive compounds were found and this result was interesting because olive leaves represent a waste product in these periods, corresponding to olive harvest and pruning of olive trees. the results of this research give information on the optimization of olive leaf phenol extraction useful in the production of nutraceuticals. acknowledgements the authors are grateful to prof. franco famiani and mirco boco of the university of perugia for allowing the harvesting of olive leaves in the olive orchard of university of perugia. the authors thank giuseppa verducci for her support in the chemical analysis. references abaza, l., youssef, n.b., manai, h., haddada, f.m., methenni, k., zarrouk, m., 2011: chétoui olive leaf extracts: influence of the solvent type on phenolics and antioxidant activities. grasas y aceites. 62, 96104. ahmad-qasem, m.h., cánovas, j., barrajón-catalán, e., carreres, j.e., micol, v., garcía-pérez j.v., 2014: influence of olive leaf processing on the bioaccessibility of bioactive 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perugia, department of pharmaceutical sciences, section of food science and nutrition, via san costanzo, 06126, perugia, italy e-mail: lina.cossignani@unipg.it © the author(s) 2016. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 89, 21 28 (2016), doi:10.5073/jabfq.2016.089.003 1botany department, faculty of agriculture, fayoum university, fayoum, egypt 2irrigation department, centro de edafología y biología aplicada del segura (cebas-csic), murcia, spain 3botany department, national research centre, dokki, giza, egypt 4biology department, faculty of sciences, albaha university, albaha, suadi arabia growth, heavy metal status and yield of salt-stressed wheat (triticum aestivum l.) plants as affected by the integrated application of bio-, organic and inorganic nitrogen-fertilizers mostafa m. rady1*, oussama h. mounzer2, juan josé alarcón2, magdi t. abdelhamid3, saad m. howladar4 (received september 4, 2015) * corresponding author summary efforts have been made to use the integrated application of bio-, organic and inorganic nitrogen (n)-fertilizers to decrease waste accumulation, and to minimize nutrient losses and yield contamination with heavy metals for human nutrition and health. therefore, a field experiment was conducted to assess the effect of integrated applications of organic manures, bio-fertilizer and/or mineral-n fertilizers on growth, yield, some chemical constituents and shoot and yielded grain heavy metal contents of wheat (triticum aestivum l. cv. sakha 93) plants grown under salinity stress (ece = 7.84 ds m-1). results showed that, the treatment comprised of ⅓nh4no3 (55 kg n ha-1) + cerealine (bio-fertilizer; 4 kg ha-1) + cattle manure (10 t ha-1) was found to be most effective, producing the best status of growth characteristics, osmoprotectants concentrations, essential nutrient contents, shoot heavy metal concentrations, and grain yield and its content of heavy metals compared to the all other treatments. the treatment comprised of cerealine (4 kg ha-1) + cattle manure (20 t ha-1) was occupied the second order. we can recommend to use the integrated treatment of ⅓nh4no3 (55 kg n ha-1) + cerealine (biofertilizer; 4 kg ha-1) + cattle manure (10 t ha-1) effectively in saline soils to improve wheat growth and yield with minimum contents of heavy metals for human health and nutrition. introduction wheat (triticum aestivum l.) is the most important cereal crops used for human nutrition in most countries worldwide. it is a moderately salt-tolerant crop (maas and hoffman, 1977), and is often grown on newly-reclaimed saline soils in egypt. on the other hand, the cultivation of wheat will be restricted or even prevented in such soils if their salinity reached about 10 ds m-1 due to the severe reduction in wheat growth and productivity. salinity stress is one of the major problems of agriculture in arid and semiarid regions. salt stress affects plant physiology due to the osmotic and ionic stresses at both the cellular level and whole plant. it generates a physiological drought by affecting the plant water relations (munns, 2002). the accumulation of toxic salts in the leaf apoplasm leads to dehydration, loss of turgor and the death of cells and tissues. photosynthesis is one of the most severely affected processes during salinity stress, mediating by decreased levels of chlorophyll (rady and mohamed, 2015) and the inhibition of various enzymes, including rubisco (soussi et al., 1998). in addition, lipid peroxidation and the antioxidant system of the plant have been reported to be stimulated by salt stress (rady et al., 2013; semida and rady, 2014). all these, and other altered processes lead to poor plant growth and productivity. saline soils have low fertility and a poor structure with a high bioavailability of heavy metals (acosta et al., 2011) to be available for plant roots. heavy metal bioavailability can be minimized via biological immobilization and stabilization methods using a range of organic fertilizers (ofs), such as cattle manure (cm) and poultry manure (pm). the beneficial effects of ofs, which are sourced locally, are an increase soil quality, total c and n content of the soil and soil-waterholding capacity, alteration of soil ph, elevation of soil humus and soil aggregation, and water infiltration properties (domagala-swiatkiewicza and gastol, 2013; yagioka et al., 2014; abdou and mohamed, 2014; bobul’ska et al., 2015), and a reduction in the bioavailability of heavy metals (angelova et al., 2013). although some studies have reported that organic farming tends to reduce plant growth and yield (jansen et al., 2015), an important one of the ofs, cm application was found to increase the growth and yield of many crops without any undesirable impacts on the environment (suthar, 2009; rady, 2011). poultry manure (pm) has long been recognized as perhaps the most desirable of these ofs because of its high nitrogen content. the application of ofs and other organic amendments to farmland is an economical and environmentally sustainable mechanism for increasing crop production and reducing bioavailability of heavy metals (younis et al., 2015). cerealine (bio-fert) is a commercial product of biofertilizer contains azospirillum sp. for decreasing high doses of chemical fertilizer, the integrative effect of bio-fert, ofs and chemical fertilizers is a promising tool to increase wheat productivity, get cleaner product with low undesirable high doses of heavy metals and other pollutants (sushila et al., 2000). different strategies are being employed for attaining optimum growth and productivity under saline conditions. one of them is the proper management of nitrogen (n) fertilizer (rady, 2012). to alleviate deleterious salinity effects such as a reduction in growth and yield, and an increase of bioavailability and plant absorption of heavy metals, different n-forms are being used. in view of these reports, it was postulated that the integrated application of bio-, organicand mineral-n fertilizer could alleviate the adverse effects of salt stress on the growth and productivity of wheat. thus, the major objective of this study was to examine the extent to which n-forms could ameliorate the effects of salt stress on growth, ion accumulation, heavy metal uptake, and yield of wheat crop. materials and methods plant material, treatments and growth conditions a field experiment was conducted at the experimental farm of the faculty of agriculture, fayoum university, egypt. the main characteristics of the experimental soil, organic manures and irrigation water (southeast fayoum; 29° 17´n; 30° 53´e) used in this study were determined according to jackson (1973) and wilde et al. (1985) and are shown in tab. 1. healthy seeds of wheat (triticum aestivum l. cv. sakha 93) were obtained from the crop research institute, the agricultural research center, giza, egypt, and were sown on 15 november 2009 and final harvest was done on 20 may 2010. seeds were washed with distilled 22 m.m. rady, o.h. mounzer, j.j. alarcón, m.t. abdelhamid, s.m. howladar tab. 1: physical and chemical properties of experimental soil, organic manures and irrigation water. soil analysis organic manure analysis property values property cattle poultry sand% 76.8 1m3 wt. (kg) 738 498 silt% 18.1 moisture% 70.0 66.7 clay% 5.1 ph 7.50 6.34 texture sandy loam ec (dsm-1) 5.76 5.21 *ph 7.68 *o.c.% 34.0 43.6 ec (ds m-1) 7.84 *o.m.% 47.6 69.8 total n% 0.63 c/n ratio 23:1 22:1 total p% 0.36 total n% 1.47 1.99 total k% 1.25 total p% 0.50 0.78 caco3% 11.1 total k% 1.32 1.52 fe (ppm) 16.9 caco3% 2.86 3.26 mn (ppm) 8.4 fe (ppm) 946 1928 zn (ppm) 7.2 mn (ppm) 429 876 cu (ppm) 6.4 zn (ppm) 146 230 pb (ppm) 12.8 cu (ppm) 68.2 98.2 cd (ppm) 13.6 pb (ppm) 12.2 31.2 ni (ppm) 11.9 cd (ppm) 5.91 18.0 cr (ppm) 9.04 ni (ppm) 7.13 16.9 se (ppm) 6.17 cr (ppm) 5.81 17.3 co (ppm) 4.96 se (ppm) 4.95 13.0 co (ppm) 5.01 11.9 *ph (1:1 suspension), o.c.% = organic carbon %, o.m.% = organic matter%. water, sterilized in 1% (v/v) sodium hypochlorite for approx. 2 min, washed thoroughly again with distilled water, and left to dry at room temperature (25 ºc) for approx. 1 h before sowing. fourteen treatments in a randomized complete block design were used with three replications, generating 42 plots. the area of each plot was 10.5 m2 (3 m × 3.5 m). the distance between plots was 1 m from all sides to avoid treatments overlapping. the treatments imposed in the present investigation were detailed in tab. 2. the experiment was designed to examine the influence of n-form (organic, bioand/or inorganic) fertilizer on growth, heavy metal uptake, nutrients concentration and yield of wheat plants grown at salinity with ec = 7.84 ds m-1. pm and cm were added to soil before sowing, during soil preparation. they spread on the soil surface, and then mixed into the soil-surface layer. ammonium nitrate and urea quantities according to treatments were added before sowing. a quantity of 360 kg ha-1 of single super phosphate (15.5 % p2o5) was added before sowing as recommended dose. cerealine is a commercial product of biofertilizer contains azospirillum sp. produced by general organization of agriculture, egypt, and was added before sowing at rate of 4 kg ha-1. all other agricultural practices for wheat production were conducted according to the recommendations of the egyptian ministry of agriculture and land reclamation. plant sampling, and growth and yield measurements at fourteen weeks after sowing, plants were sampled to study the growth characteristics, and to perform the chemical analyses in leaves or shoots. at harvest, yield parameters were estimated. the following growth and yield parameters were measured: plant height (cm), number (no.) of leaves plant-1, leaf area plant-1 (cm2), no. of tillers plant-1, shoot dry weight (dw) plant-1 (g), 1000-grain weight (g) and grain yield ha-1 (ton). chemical analyses at fourteen weeks after sowing, the fifth leaf or shoot of four se lected plants were collected from each experimental unit for chemical determinations. the concentrations of chlorophylls and carotetab. 2: components of the 14 treatments used in the present experiment treatment nitrogen (n) source amount of n source ha-1 amount of n ha-1 t1 (control)* not found not found not found t2 (recom.)** ammonium nitrate (nh4no3) 475 kg (33.5 % n) 160 kg t3 (recom.)** urea (nh2-co-nh2) 350 kg (46 % n) 160 kg t4 poultry manure (pm1) 10 t 199 kg t5 cattle manure (cm1) 20 t 294 kg t6 bio-n (cerealine) 4 kg ……… t7 ½nh4no3 + pm2 237.5 kg (33.5 % n) + 5 t 80 kg + 99.5 kg*** t8 ½nh4no3 + cm2 237.5 kg (33.5 % n) + 10 t 80 kg + 147 kg*** t9 ½nh4no3 + cerealine 237.5 kg (33.5 % n) + 4 kg 80 kg + ……… t10 ½nh2-co-nh2 + pm2 175 kg (46 % n) + 5 t 80 kg + 99.5 kg t11 ½nh2-co-nh2 + cm2 175 kg (46 % n) + 10 t 80 kg + 147 kg t12 ½nh2-co-nh2 + cerealine 175 kg (46 % n) + 4 kg 80 kg + …….. t13 cerealine + cm1 4 kg + 20 t ……. + 294 kg t14 ⅓nh4no3 + cerealine + cm2 158.3 kg + 4 kg + 10 t 55 kg + …... + 147 kg * the control treatment was not received any n-fertilizer. ** the recommended dose of n for wheat from either ammonium nitrate or urea as the egyptian ministry of agriculture and land reclamation. *** calculated from the tab. 1 (analyses of cattle and poultry manures). effect of n form on wheat productivity and its quality 23 noids of the fresh upper fifth leaf were extracted by acetone 80 %, and were then measured colorimetrically using the method of arnon (1949). the concentration of free proline was determined colorimetrically by using acid ninhydrin reagent with the sulfosalicylic acid (3 %)-extract as outlined by bates et al. (1973). the concentration of total free amino acids was determined according to muting and kaiser (1963). the concentration of total soluble sugars was determined in ethanolic extract with freshly-prepared anthrone reagent [150 mg anthrone + 100 ml of 72 % (v/v) sulphuric acid] according to irigoyen et al. (1992). the contents of nutrients were determined in digested samples according to jackson (1973). the n content was measured using the semi-micro kjeldahl method (bremner, 1965). the p content was determined colorimetrically by the molybdenum blue method (chapman and pratt, 1961). the contents of k and na were assessed using flame photometry (jackson, 1973). the ca content, and the all tested heavy metals (pb, cd, ni, cr, se, co, zn and cu) contents were determined using a perkin-elmer model 3300 atomic absorption spectrophotometer (chapman and pratt, 1961). statistical analysis the data were subjected to the analysis of variance (anova) appropriate to the randomized complete block design according to the procedure outlined by gomez and gomez (1984). the significant differences among treatments were compared using the fisher’s least-significant difference test (lsd) at p ≤ 0.05. results influences of n-fertilizer form on plant height, no. of leaves plant-1, leaf area plant-1, no. of tillers plant-1, shoot dw plant-1 of wheat grown under saline soil conditions are shown in tab. 3. the treatment comprised of ⅓nh4no3 (55 kg n ha-1) + cerealine (4 kg ha-1) + cattle manure (cm2; 10 t ha-1) was found to be most effective, producing the highest values of the all mentioned growth characteristics compared to the all other treatments. the treatment comprised of cerealine (4 kg ha-1) + cattle manure (cm1; 20 t ha-1) was occupied the second order. the treatment of the recommended mineral-n dose of ammonium nitrate or urea was in a parallel line with each of the treatments of ½urea (80 kg n ha-1) + pm2 (5 t ha-1), ½urea (80 kg n ha-1) + cm2 (10 t ha-1) and ½urea (80 kg n ha-1) + cerealine (4 kg ha-1) in the third order compared to the other treatments, including the control. fig. 1 shows the influences of n-fertilizer forms on the concentrations of total chlorophyll, total carotenoids, free proline, total soluble sugars and free amino acids in wheat leaves under saline soil conditions. the treatments comprised of ⅓nh4no3 (55 kg n ha-1) + cerealine (4 kg ha-1) + cattle manure (cm2; 10 t ha-1) and cerealine (4 kg ha-1) + cattle manure (cm1; 20 t ha-1) were found to be most effective, producing the highest values of the all aforementioned parameters, occupying the first and the second orders, respectively compared to the all other treatments. tab. 4 shows the effects of n-fertilizer forms on nutrient contents (nitrogen; n, phosphorus; p, potassium; k, calcium; ca and sodium; na), and k/na and ca/na ratios in shoots of wheat plants grown under saline soil conditions. the treatment comprised of ⅓nh4no3 (55 kg n ha-1) + cerealine (4 kg ha-1) + cattle manure (cm2; 10 t ha-1) was found to be most effective, producing the highest values of the contents of n, p, k and ca and the ratios of k/na and ca/na, and the lowest values of na contents compared to the all other treatments. the treatments comprised of cerealine (4 kg ha-1) + cattle manure (cm1; 20 t ha-1) and ½nh4no3 (80 kg n ha-1) + cattle manure (cm2; 10 t ha-1) were occupied the second and third orders, respectively compared to the other treatments, including the control. the effects of n-fertilizer forms on the concentrations of heavy metals (lead; pb, cadmium; cd, nickel; ni, chromium; cr, selenium; se, cobalt; co, zinc; zn and copper; cu) in shoots and yielded grains of wheat plants grown under saline soil conditions are shown in tab. 5 and 7. the treatments comprised of ⅓nh4no3 (55 kg n ha-1) + cerealine (4 kg ha-1) + cattle manure (cm2; 10 t ha-1) and cerealine (4 kg ha-1) + cattle manure (cm1; 20 t ha-1) were found to be most effective, producing the lowest values of the concentrations of all heavy metals, except some fluctuations compared to the all other treatments. on the other hand, the treatment of ½urea (80 kg n ha-1) + poultry manure (pm2; 5 t ha-1) was found to generate the highest concentration values of all heavy metals, followed by all treatments with urea either solely or combined with poultry manure, or cattle manure, and even with cerealine. n-fertilizer form effects on the yield parameters (1000-grain weight and grain yield ha-1) of wheat plants grown under saline soil conditions are shown in tab. 6. the treatments comprised of ⅓nh4no3 (55 kg n ha-1) + cerealine (4 kg ha-1) + cattle manure (cm2; 10 t ha-1) and cerealine (4 kg ha-1) + cattle manure (cm1; 20 t ha-1) were found to be most effective, producing the highest values of the 1000-grain weight and grain yield ha-1, occupying the first and the second orders, respectively compared to the all other treatments. discussion the salinity of soil (ece = 7.84 ds m-1; tab. 1) may affect the plant metal uptake through the toxic effects of the na+ and clions. the tab. 3: effect of n-fertilizer form applications on plant height, number (no.) of leaves plant-1, leaf area plant-1, and shoot dry weight (dw) plant-1 of wheat grown under saline soil conditions treatments plant height no. of leaves leaf area shoot plant-1 dw plant-1 (cm) plant-1 (cm2) (g) t1 58.4e* 5.11e 20.1g 6.1e t2 78.4c 6.89c 37.8cde 11.5c t3 78.4c 6.79c 36.7cde 11.3c t4 76.9cd 6.68c 35.3de 11.1c t5 77.1cd 6.61c 34.9e 11.0c t6 70.7d 5.98d 30.3f 9.9d t7 80.1c 6.95c 37.9cd 11.6c t8 78.4c 6.78c 36.9cde 11.3c t9 81.6bc 7.09bc 38.8c 11.8bc t10 77.8c 6.66c 35.5de 11.1c t11 80.1c 6.73c 35.7de 11.2c t12 80.0c 6.96c 37.8cde 11.6c t13 87.8b 7.63b 41.8b 12.7b t14 96.3a 8.37a 46.6a 14.0a *values are means (n = 9), and mean values in each column followed by a different lower-case-letter are significantly different by fisher’s least-significant difference test (lsd) at p ≤ 0.05. t1: control (without any n-fertilizer); t2: recommended n as ammonium nitrate (160 kg n = 476 kg nh4no3 ha-1 with 33.5 % n); t3: recommended n as urea (160 kg n = 348 kg ha-1 urea [(nh2)2co] with 46 % n); t4: poultry manure (pm1 = 10 t ha-1); t5: cattle manure (cm1 = 20 t ha-1); t6: bio-n (cerealine at rate of 4 kg ha-1); t7: ½nh4no3+pm2 (pm2 = 5 t ha-1); t8: ½nh4no3+cm2 (cm2 = 10 t ha-1); t9: ½nh4no3+cerealine; t10: ½urea+pm2; t11: ½urea+cm2; t12: ½urea+cerealine; t13: cerealine+cm1; t14: ⅓nh4no3+cerealine+cm2. 24 m.m. rady, o.h. mounzer, j.j. alarcón, m.t. abdelhamid, s.m. howladar na+ ions may release heavy metals from the sediment to the soil solution, thereby increasing the heavy metal accumulation in the plant organs (tab. 5 and 7). in arid regions including egypt, soils and irrigation water are characterized by high salinity levels that may aggravate the problem of heavy metal pollutions. in addition, high levels of chloride ions-containing irrigation water might increase soil heavy metal mobilization through the formation of metalchloride complexes (mclean and bledsoe, 1992; ghallab and usman, 2007). they also reported that the consequences of soil salinity can increase heavy metal solubility and availability, depending on the composition of soil solution and its dominant inorganic ligands. the soil applications of organic manures have been shown to improve soil fertility, and to reduce salt stress and the bioavailability of heavy metals to plant roots (weggler et al., 2004; ok et al., 2011). it has been also reported that the application of organic residues to heavy metal-contaminated soils increased soil organic matter content, improved the chemical and biological properties, and decreased the heavy metal bioavailability to plant roots (ok et al., 2011). moreover, the application of organic manures to heavy metalcontaminated soil reduced the metal toxicity and consequently increased soil microbial activities (usman et al., 2013). in the present study, unlike the poultry manure the addition of cattle manure (cm) as a full alternative or a partial alternative to mineral fertilizers [i.e., the treatment comprised of ⅓nh4no3 (55 kg n ha-1) + cerealine (4 kg ha-1) + cattle manure (cm2; 10 t ha-1) and the treatment comprised of cerealine (4 kg ha-1) + cattle manure (cm1; 20 t ha-1)] was significantly increased wheat growth characteristics (tab. 3), nutritional status (tab. 4), photosynthetic pigments and osmoprotectants (fig. 1) and grain yield (tab. 6), and significantly decreased the concentrations of heavy metals in plant shoots and yielded grains (tab. 5 and 7). the most interesting finding is that these treatments were found to produce higher grain yield than average grain yield productivity of egypt (6.452 t ha-1) in 2009, compared to 7.85 and 6.66 t ha-1 of these two treatments, respectively. our results showed that the addition of cm caused a significant increase in shoot mineral nutrients (tab. 4). this finding may be attributed to the release of nutrients with the decomposition of this organic manure (wang et al., 2014). results showed also that soil salinity caused increases in the bioavailability of heavy metals, as indicated by a significant increase in the wheat shoot and grain heavy metal concentrations in the control and the treatments contained inorganic n fertilizers and/or poultry manure (tab. 5 and 7). this was observed in the treatment comprised of urea (inorganic n) + poultry manure (t10) which resulted in the highest heavy metal concentrations in wheat plants. this increase in plant heavy metal concentration may be due to poultry manure application that has shown to increase the soil concentration of heavy metals (hanč et al., 2008; arroyo et al., 2014). in addition, the frequent use of chemical fertilizers such as urea and pesticides in common agricultural fields can also cause an increase in the concentration of heavy metals in soil and water (hadi et al., 2014) and, consequently, in plants grown on this soil. however, the addition of cm significantly decreased shoot and grain heavy metal concentrations of wheat plants. these results are in accordance with those of ahmad et al. (2011), noting that farm manure amendment significantly decreased cd and pb content in wheat grains. the lower heavy metal avail ability in the farmyard amended soils could be mainly related to the heavy metal immobilization through sorption, chelation, and sequestration by the solid and soluble organic matter. indeed, soil heavy metal availability can be decreased by adding the organic materials, particularly cm through heavy metal transformation from more readily available forms to less available forms such as fractions associated with organic materials, carbonates, or heavy metal oxides, causing a reduction in heavy metal uptake by plants (ok et al., 2011; bolan et al., 2003). narwal and singh (1998) observed that the cd, cu, ni and zn in soils decreased with increasing the addition of farmyard manure and peat that correspond with our results by soil application with cm (tab. 5 and 7). soil organic matter may contribute to the adsorption and immobilization of heavy metals, restricting its solubility and bioavailability. therefore, organic manures such as cm may have an important role in improving the quality and minimizing the metal toxicity of contaminated soils under heavy metals and salinity stress (usman, 2015). it has been speculated that the application of organic manure to metal-contaminated soil may create a good environmental condition for mitigating the toxicity induced by high heavy metal concentrations (usman et al., 2013). the application of cm in combination with a partial inorganic fertilization and/or bio-fertilization (cerealine) found to significantly increase wheat growth characteristics (plant height, number of fig. 1: effect of n-fertilizer form applications on the leaf concentrations of total chlorophyll, total carotenoids, free proline, total soluble sugars, and amino acids in wheat plants grown under saline soil conditions. *mean values are presented in form of vertical bars. bars with a different lower-case-letter are significantly different by fisher’s leastsignificant difference test (lsd) at p ≤ 0.05. t1: control (without any n-fertilizer); t2: recommended n as ammonium nitrate (160 kg n = 476 kg nh4no3 ha-1 with 33.5 % n); t3: recommended n as urea (160 kg n = 348 kg ha-1 urea [(nh2)2co] with 46 % n); t4: poultry manure (pm1 = 10 t ha-1); t5: cattle manure (cm1 = 20 t ha-1); t6: bio-n (cerealine at rate of 4 kg ha-1); t7: ½nh4no3+pm2 (pm2 = 5 t ha-1); t8: ½nh4no3+cm2 (cm2 = 10 t ha-1); t9: ½nh4no3+cerealine; t10: ½urea+pm2; t11: ½urea+cm2; t12: ½urea+cerealine; t13: cerealine+cm1; t14: ⅓nh4no3+cerealine+cm2. effect of n form on wheat productivity and its quality 25 tab. 5: effect of n-fertilizer form applications on the concentrations of heavy metals [lead (pb), cadmium (cd), nickel (ni), chromium (cr), selenium (se), cobalt (co), zinc (zn), and copper (cu)] in shoots of wheat plants grown under saline soil conditions treatments pb cd ni cr se co zn cu (mg kg-1 dw) t1 2.85g* 4.16f 2.51g 3.27gh 1.26bcd 2.25f 138i 55.2ij t2 3.86e 5.61d 3.40e 4.41e 1.20de 3.06d 182f 71.6f t3 6.24c 8.96b 5.49c 7.12c 1.25bcde 4.94b 229bc 92.1bc t4 7.38b 9.80a 6.56b 8.39b 1.12e 5.95a 238b 94.8ab t5 2.64gh 3.82fg 2.33gh 3.01h 1.41a 2.11fg 132i 52.6jk t6 2.85g 4.13f 2.50g 3.45g 1.25bcde 2.24f 151h 60.3hi t7 5.75d 8.13c 5.00d 6.70d 1.20de 4.55c 212de 84.3de t8 3.48f 5.05e 3.05f 4.00ef 1.37abc 2.75e 164g 65.1gh t9 3.45f 4.99e 3.10f 3.94f 1.26bcd 2.73e 165g 66.2g t10 7.83a 9.98a 6.82a 9.12a 1.18de 6.03a 256a 98.7a t11 5.70d 8.24c 4.97d 6.58d 1.30abc 4.47c 202e 80.4e t12 5.88d 8.53c 5.16d 6.67d 1.24cde 4.66c 218cd 87.0cd t13 2.52h 3.68g 2.19h 2.86h 1.30abc 1.96g 117j 47.4k t14 2.02i 3.21h 1.81i 2.33i 1.38ab 1.59h 101k 40.8l *values are means (n = 9), and mean values in each column followed by a different lower-case-letter are significantly different by fisher’s least-significant difference test (lsd) at p ≤ 0.05. t1: control (without any n-fertilizer); t2: recommended n as ammonium nitrate (160 kg n = 476 kg nh4no3 ha-1 with 33.5 % n); t3: recommended n as urea (160 kg n = 348 kg ha-1 urea [(nh2)2co] with 46 % n); t4: poultry manure (pm1 = 10 t ha-1); t5: cattle manure (cm1 = 20 t ha-1); t6: bio-n (cerealine at rate of 4 kg ha-1); t7: ½nh4no3+pm2 (pm2 = 5 t ha-1); t8: ½nh4no3+cm2 (cm2 = 10 t ha-1); t9: ½nh4no3+cerealine; t10: ½urea+pm2; t11: ½urea+cm2; t12: ½urea+cerealine; t13: cerealine+cm1; t14: ⅓nh4no3+cerealine+cm2. tab. 4: effect of n-fertilizer form applications on the contents of various elements [nitrogen (n), phosphorus (p), potassium (k), calcium (ca), and sodium (na)] and the ratios of ca:na and k:na in shoots of wheat plants grown under saline soil conditions treatments n p k ca na ca/na k/na (%) (%) (%) (%) (%) ratio ratio t1 1.85h* 0.24i 1.53h 0.13h 0.30a 0.43h 5.1g t2 2.29defg 0.42gh 2.20efg 0.23e 0.22c 1.05de 10.0def t3 2.26efg 0.42gh 2.16fg 0.21f 0.24b 0.88fg 9.0ef t4 2.24fg 0.40h 2.12g 0.19g 0.24b 0.79g 8.8f t5 2.24fg 0.42gh 2.14g 0.21f 0.22c 0.95ef 9.7def t6 2.22g 0.40h 2.11g 0.19g 0.24b 0.79g 8.8f t7 2.42cd 0.51c 2.41bc 0.25cd 0.18e 1.39c 13.4c t8 2.46c 0.51c 2.40bc 0.26c 0.18e 1.44c 13.3c t9 2.35cdefg 0.46ef 2.29cdef 0.24de 0.22c 1.09d 10.4d t10 2.38cdef 0.48de 2.33cde 0.25cd 0.22c 1.14d 10.6d t11 2.39cde 0.50cd 2.36bcd 0.25cd 0.18e 1.39c 13.1c t12 2.30defg 0.44fg 2.22defg 0.24de 0.22c 1.09d 10.1de t13 2.68b 0.55b 2.49b 0.28b 0.14f 2.00b 17.8b t14 2.91a 0.60a 2.71a 0.31a 0.10g 3.10a 27.1a *values are means (n = 9), and mean values in each column followed by a different lower-case-letter are significantly different by fisher’s least-significant difference test (lsd) at p ≤ 0.05. t1: control (without any n-fertilizer); t2: recommended n as ammonium nitrate (160 kg n = 476 kg nh4no3 ha-1 with 33.5 % n); t3: recommended n as urea (160 kg n = 348 kg ha-1 urea [(nh2)2co] with 46 % n); t4: poultry manure (pm1 = 10 t ha-1); t5: cattle manure (cm1 = 20 t ha-1); t6: bio-n (cerealine at rate of 4 kg ha-1); t7: ½nh4no3+pm2 (pm2 = 5 t ha-1); t8: ½nh4no3+cm2 (cm2 = 10 t ha-1); t9: ½nh4no3+cerealine; t10: ½urea+pm2; t11: ½urea+cm2; t12: ½urea+cerealine; t13: cerealine+cm1; t14: ⅓nh4no3+cerealine+cm2. 26 m.m. rady, o.h. mounzer, j.j. alarcón, m.t. abdelhamid, s.m. howladar leaves plant-1, leaf area plant-1 and shoot dry weight plant-1; tab. 3), positively reflecting in yield parameters. this finding agrees with earlier reports of baiyeri and tenkouano (2007) and ndukwe et al. (2011) that animal manure is a valuable source of crop nutrients and organic matter, which can improve the soil biophysical condi tions making the soil more productive and sustainable for plant growth. growth and yield improvements in plants grown in soil amended with a combination of bio-, organic and inorganic fertilizers might be due to nutrient availability, particularly n, p and p ions (maman and mason, 2013) that agreed with our results obtained under salt stress (tab. 4). organic manures not only provide nutrients by making them more available for plants but also add to the most important constituent of the soil humus, which provides excellent substrate for plant growth. this could be attributed to the fact that the nutrients in the organic fertilizers were released gradually through the process of mineralization (mehedi et al., 2012), maintaining optimal soil levels over prolonged periods of time. some of the organic substances released during the mineralization may act as chelates, which help in the absorption of essential ions and other micro-nutrients (lobo et al., 2012). they added that, organic materials could have formed complex (or chelate), preventing the precipitation of p, reduced the p-sorption capacity of the soil, enhanced p availability, improved p-recovery or resulted in better utilization by plants. organic materials add carbon into the soil, provides substrate for microbial growth, and subsequent microbial activity. the turnover resulting from the decomposition of organic materials improves c and n mineralization rates, and enzyme activities, which affect nutrient cycling and availability to the plants (smith et al., 1993). in addition, vanlauwe et al. (2002) reported that the combination of organic and inorganic fertilizers result into synergy and improved conservation and synchronization of nutrient release and crop demand, leading to increased fertilizer efficiency and higher growth and yields. the combined treatment of cm (10 t ha-1) + ⅓nh4no3 tab. 6: effect of n-fertilizer form applications on the 1000-grain weight and grain yield of wheat plants grown under saline soil conditions treatments 1000-grain weight (g) grain yield ha-1 (ton) t1 29.5d* 3.18g t2 43.9bc 5.40cdef t3 43.6bc 5.32cdef t4 42.7bc 5.26def t5 42.4bc 5.23ef t6 41.0c 5.11f t7 44.5bc 5.68cde t8 44.3bc 5.55cdef t9 44.9bc 5.80c t10 44.2bc 5.63cde t11 44.1bc 5.53cdef t12 44.4bc 5.74cd t13 45.3b 6.66b t14 49.9a 7.85a *values are means (n = 9), and mean values in each column followed by a different lower-case-letter are significantly different by fisher’s least-significant difference test (lsd) at p ≤ 0.05. t1: control (without any n-fertilizer); t2: recommended n as ammonium nitrate (160 kg n = 476 kg nh4no3 ha-1 with 33.5% n); t3: recommended n as urea (160 kg n = 348 kg ha-1 urea [(nh2)2co] with 46% n); t4: poultry manure (pm1 = 10 t ha-1); t5: cattle manure (cm1 = 20 t ha-1); t6: bio-n (cerealine at rate of 4 kg ha-1); t7: ½nh4no3+pm2 (pm2 = 5 t ha1); t8: ½nh4no3+cm2 (cm2 = 10 t ha-1); t9: ½nh4no3+cerealine; t10: ½urea+pm2; t11: ½urea+cm2; t12: ½urea+cerealine; t13: cerealine+cm1; t14: ⅓nh4no3+cerealine+cm2. tab. 7: effect of n-fertilizer form applications on the concentrations of heavy metals [lead (pb), cadmium (cd), nickel (ni), chromium (cr), selenium (se), cobalt (co), zinc (zn), and copper (cu)] in harvested grains of wheat plants grown under saline soil conditions treatments pb cd ni cr se co zn cu (mg kg-1 dw) t1 1.31de* 1.40de 1.89fgh 1.18e 0.93c 0.38g 141efg 56.3efg t2 1.36de 1.48d 1.99def 1.24de 0.87cd 0.42f 150ef 59.8e t3 4.26c 2.34c 4.63c 2.58c 0.71e 0.58c 194c 77.5c t4 6.12b 2.99b 6.02b 3.16b 0.64f 0.79b 212b 84.6b t5 1.20e 1.26e 1.75hi 1.04f 1.00b 0.33h 128h 51.4g t6 1.30de 1.38de 1.85fghi 1.18e 0.92c 0.38g 140fg 56.1efg t7 1.42d 1.56d 2.09d 1.30d 0.81d 0.47d 168d 67.2d t8 1.30de 1.39de 1.84ghi 1.19de 0.92c 0.38g 141efg 56.0efg t9 1.33de 1.43de 1.92efg 1.20de 0.90c 0.40fg 144ef 57.6e t10 7.10a 3.52a 7.31a 3.48a 0.58f 0.91a 244a 94.9a t11 1.36de 1.49d 1.98defg 1.24de 0.87cd 0.43ef 152e 60.7e t12 1.40d 1.55d 2.07de 1.29de 0.82d 0.46de 170d 68.1d t13 1.20e 1.24e 1.72i 1.04f 1.02b 0.32h 131gh 52.1fg t14 1.01f 0.93f 1.53j 0.91g 1.11a 0.27i 114i 46.2h *values are means (n = 9), and mean values in each column followed by a different lower-case-letter are significantly different by fisher’s least-significant difference test (lsd) at p ≤ 0.05. t1: control (without any n-fertilizer); t2: recommended n as ammonium nitrate (160 kg n = 476 kg nh4no3 ha-1 with 33.5% n); t3: recommended n as urea (160 kg n = 348 kg ha-1 urea [(nh2)2co] with 46% n); t4: poultry manure (pm1 = 10 t ha-1); t5: cattle manure (cm1 = 20 t ha-1); t6: bio-n (cerealine at rate of 4 kg ha-1); t7: ½nh4no3+pm2 (pm2 = 5 t ha-1); t8: ½nh4no3+cm2 (cm2 = 10 t ha-1); t9: ½nh4no3+cerealine; t10: ½urea+pm2; t11: ½urea+cm2; t12: ½urea+cerealine; t13: cerealine+cm1; t14: ⅓nh4no3+cerealine+cm2. effect of n form on wheat productivity and its quality 27 + cerealine was found to significantly increase wheat growth and productivity and significantly decrease plant and grains heavy metals concentrations. these results show that it is important to choose the suitable n source and its suitable amount to sustain the agricultural productivity, by minimizing the concentrations of heavy metals in the edible parts for human nutrition and health. conclusions the higher growth and yield, and the lower plant and grain heavy metal contents obtained as a result of soil application with cm in addition to bioand inorganic fertilizers are an indication that integrated use of cm, bioand mineral fertilizers for salt-affected soil is advantageous over the use of others. integration of cm as organic fertilizer with bioand inorganic fertilizers could therefore be recommended as a best option in increasing fertilizer use efficiency and provision of a more balanced supply of essential nutrients and a lower bioavailability of heavy metals under salt stress to obtain economical wheat grain yield with minimum heavy metal content for human nutrition and health. acknowledgment the authors are grateful to the faculty of science, albaha university, albaha, saudi arabia for laboratory assistance. references abdou, m., mohamed, m.a.h., 2014: effect of plant compost, salicylic and ascorbic acids on mentha piperita l. plants. biol. agric. hortic. 30, 131-143. acosta, j.a., jansen, b., kalbitz, k., faz, a., martinez-martinez, s., 2011: salinity increases mobility of heavy metals in soils. chemosphere. 85, 1318-1324. ahmad, h.r., ghafoor, a., corwin, d.l., aziz, m.a., sabir, m., 2011: organic and inorganic amendments affect soil concentration and accumulation of cadmium and lead in wheat in calcareous alkaline soils. commun. soil sci. plant anal. 42, 111-122. angelova, v.r., akova, v.i., artinova, n.s., ivanov, k.i., 2013: the effect of organic amendments on soil chemical characteristics. bulg. j. agric. sci. 19, 958-971. arnon, d.i., 1949: copper enzymes in isolated chloroplasts, polyphenoloxidase in beta vulgaris. plant physiol. 24, 1-5. arroyo, m.d., hornedo, r.m., peralta, f.a., almestre, c.r., sánchez, j.v.m., 2014: heavy metals concentration in soil, plant, earthworm and leachate from poultry manure applied to agricultural land. rev. int. contam. ambie. 30, 43-50. baiyeri, k.p., tenkouano, a., 2008: manure placement effects on root and shoot growth and nutrient uptake of ‘pita 14’ plantain hybrid (musa sp. aaab). afr. j. agric. res. 3, 13-21. bates, l.s., waldren, r.p., teare, i.d., 1973: rapid determination of free proline for water stress studies. plant soil. 39, 205-207. bobul’ska, l., fazekasova, d., angelovicova, l., kotorova, d., 2015: impact of ecological and conventional farming systems on chemical and biological soil quality indices in a cold mountain climate in slovakia. biol agric hortic. 31, 205-218. bolan, n.s., adriano, d.c., duraisamy, p., mani, a., 2003: immo bilization and phytoavailability of cadmium in variable charge soils. iii. effect of biosolid compost addition. plant soil. 256, 231-241. bremner, j.m., 1965: total nitrogen. in: black, c.a. 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mrady2050@gmail.com oussama h. mounzer, juan josé alarcón, irrigation department, centro de edafología y biología aplicada del segura (cebas-csic), campus universitario de espinardo, 30100 espinardo, apartado 164, murcia, spain magdi t. abdelhamid, botany department, national research centre, 33 el bohouth st., dokki, giza, 12622, egypt saad m. howladar, biology department, faculty of sciences, albaha university, 65441 albaha, suadi arabia journal of applied botany and food quality 88, 22 28 (2015), doi:10.5073/jabfq.2015.088.005 1 cebra centre for energy technology brandenburg e.v., cottbus, germany 2 brandenburg university of technology cottbus-senftenberg, chair of soil protection and recultivation, cottbus, germany effects of nitrogen and phosphate fertilization on leaf nutrient content, photosynthesis, and growth of the novel bioenergy crop fallopia sachalinensis cv. ‘igniscum candy’ laurie anne koning1, 2, maik veste1, 2, *, dirk freese2, stefan lebzien3 (received october 12, 2014) * corresponding author summary the aim of the study was to determine the effects of nitrogen and phosphate fertilization on the growth performance of the novel bioenergy crop fallopia sachalinensis cv. ‘igniscum candy’ (polygonaceae). in a controlled pot experiment various nitrogen (0, 50, 150, 300 kg n ha-1) and phosphate (20, 40, 80 kg p ha-1) fertilizer amounts were applied to measure the effect on the biomass, plant height, leaf area, and leaf nutrient (n and p) content. furthermore, the ecophysiological processes of chlorophyll content, chlorophyll fluorescence, and gas exchange were measured. the application of nitrogen correlated positively with biomass production, while phosphate fertilization did not show a significant effect on plant growth or ecophysiological parameters. the leaf nitrogen contents were significantly correlated with the nitrogen applications, while the leaf phosphate contents did not show a correlation with the p fertilizations, but increased with the leaf nitrogen contents. a significant linear correlation between n-tester chlorophyll meter values and chlorophyll contents as well as with leaf nitrogen contents could be determined. under the influence of the nitrogen fertilization, net photosynthesis increased from 3.7 to 6.6 μmol m-2 s-1. the results of this experiment demonstrated that nitrogen fertilization has an overall positive correlation with leaf nitrogen content, photosynthesis, and growth of the bioenergy crop fallopia sachalinensis var. igniscum candy. introduction renewable energy production from biomass is of growing importance worldwide and will increase in the next decades (kaltschmitt et al., 2009). biomass can be utilized in diverse manners, such as for fuel, heating, and power. in germany, maize is currently dominating agricultural biomass production for use in biogas plants because of its high methane production (schittenhelm et al., 2008; vetter et al., 2009; franck, 2012). high biomass production, biomass quality, optimal energy yield of the harvested biomass and existing harvesting technology are key factors for the selection of new plant species for bioenergy production. to avoid monospecific biomass production based on maize, other species need to also be considered. the major second generation bioenergy crops include e.g., miscanthus giganteus (lewandowski and schmidt, 2006), panicum virgatum (guretzky et al., 2011), sida hermaphrodita (borkowsky and molas, 2012; franzaring et al., 2014) and silphium perfoliatum (vetter et al., 2009; gansberger et al., 2014; franzaring et al., 2014). in this context, knotweed species (fallopia spp., syn. reynoutria spp.) can play important roles in the production of biomass for bioenergy in the near future (seppälä, 2013). the new cultivars igniscum basic and igniscum candy have been cultivated from wild forms of fallopia sachalinensis hybrids for use as bio energy crops (lebzien et al., 2012). igniscum basic is intended for use in combined heat and power plants, while igniscum candy can be harvested up to two times during the growing season for biogas production. these cultivars can be alternatives to maize, adding to the portfolio of bioenergy crops. as members of a pioneer species, the cultivars are able to grow in a wide range of habitats (adachi et al., 1996) and are characterized by high biomass production (pude and franken, 2001; veste et al., 2011). knotweed species have proven themselves to achieve comparable biomass outputs to miscanthus. already in the third year of an experiment in which fallopia x bohemica and miscanthus x giganteus were grown under field conditions, yields were 24.2 mg dm -1 and 21.2 mg dm ha-1, respectively (pude and franken, 2001). other reported biomass production has ranged from 13.2 to 25 mg dm ha-1 depending on the particular knotweed species, soil, climate and planting density (vetter et al., 2009; strašil and kára, 2010). it is crucial to understand plant responses to combinations of water and nutrient availability for the development of sustainable plant production systems. nitrogen is the most common limiting factor in plant production systems. as a consequence, it is standard to add nitrogen fertilizer to a crop. but, the uptake and biomass production is crop specific e.g., switchgrass (panicum virgatum) has con sistently high biomass yields and relatively low nutrient removal rates, while giant reed (arundo donax) with its high biomass production needs increased fertilization rates over time (kering et al., 2012) and silphium perfoliatum requires 130-160 kg n ha-1 (aurbacher et al., 2012; gansberger et al., 2014). furthermore, there are environmental concerns about the losses of nitrogen due to leaching and gaseous emissions (denitrification, ammonia volatilization and nox emission), which are influenced by fertilization, soil type, crop species and management practices (paustian et al., 1990; hellebrand et al., 2008; cameron et al., 2013). the benefit of growing second generation bioenergy crops, like miscanthus, sida, silphium and igniscum, is their ability to store nutrients in their rhizomes over the winter months, which enables them to grow back strong in the spring (pude and franken, 2001; heaton et al., 2009). this feature lowers the required nitrogen application in the early growing season. furthermore, there can be a reduction of greenhouse gas emissions depending on the crop management (hudiburg et al., 2014). the other major limiting factor in plant production systems is phosphorus. only around 15 % of the phosphate fertilizer applied to a soil is taken up by a plant from the soil solution immediately after fertilizing and the remainder relatively quickly converts to an insoluble form in the non-labile fraction (mengel and kirkby, 2001). the development of second generation bioenergy crops and optimization of their nitrogen use efficiencies will help fulfill the sustainability criteria for biomass in the production of biofuels (erisman et al., 2010). proper development of crop production systems based on specific crop and environmental quality information can lead to maximum economic returns and reductions of negative environmental consequences. the objective of our study was to investigate the relationship between nutrient supply, the ecophysiological processes and biomass production. materials and methods experimental conditions for plant cultivation one-year-old igniscum candy saplings grown in plastic trays were obtained from conpower rohstoffe gmbh and cultivated in indi influence of nitrogen and phosphate on igniscum 23 vidual 7 l plastic pots in the greenhouse of the brandenburg university of technology cottbus-senftenberg under semi-controlled conditions. each pot was filled with 7 kg of silty sand with a texture of 67.7 % sand, 27.0 % silt, and 5.3 % clay. soil nitrogen content was 0.6 0.9 g n kg-1dry soil and phosphate content was 0.3 g p kg-1dry soil. the average soil ph was 7.8. every pot received 200 ml of a modified hoagland’s solution (without the basic n and p content; hoagland and arnon, 1950) as a basic fertilizer. after sixty days of the estabishment of the young plant in the greenhouse, the plants were cut down to allow a resprouting from the rhizomes; this event marked the start of the experiment. nitrogen and phosphate fertilizers were applied to the soil surfaces. twelve treatments with ten plants each involving every possible combination of commonly obtained nitrogen (calcium ammonium nitrate) and phosphate (superphosphate) pellet fertilizers at rates of 0, 50, 150 and 300 kg n ha-1 and 20, 40 and 80 kg p ha-1, respectively, were evaluated in the experiment. volumetric soil moisture was measured with a frequency domain reflectometry probe (�m300, �oil moisture �ensor delta�t de�(�m300, �oil moisture �ensor delta�t devices, cambridge, uk) and was kept just below the field capacity for sandy soils (14 % soil moisture). temperature in the greenhouse ranged between 20 °c and 25 °c. natural light was supplemented by high-pressure mercury lamps to provide photosynthetic photon flux density (ppfd) of 350�450 μmol m-2 s-1 at plant level for 10 hours per day. the pots were spatially sorted by treatment, while the treatments themselves were randomized in the greenhouse. twice a week the pots were shifted in order to avoid possible spatial effects on the plants. measurements of chlorophyll contents leaf chlorophyll contents were assessed with a hand-held chlorophyll meter and through chemical analysis. relative indices for the chlorophyll contents of leaves were obtained with the n-tester chlorophyll meter from yara (yara international asa, oslo, norway) that is based on leaf transmittance of the wavelengths 650 nm (red) and 940 nm (ir) (netto et al., 2005). in the standard mode the measurements of 30 leaves is averaged. another mode offered by the device is to sample individual leaves. this individual leaf mode produces a relative index result comparable to results obtained with �pad chlorophyll meters from konica minolta. readings were taken in the middle portion of fully expanded leaves on the day of harvest. five plants from each treatment were assessed in the standard mode while every single plant in the experiment was assessed in the individual mode. for comparison between both modes, we calculated the chlorophyll index values of the yara-n-tester (ynt) follow the formula: ynt = 15*chl – 90, where ynt is the relative n-tester index value and chl is the chlorophyll meter value of a single leaf (yara gmbh, dülmen, germany, f. brentrup, personal communication). the same leaves that were assessed in the n-tester individual mode were subjected to chlorophyll a and b extraction with 80 % acetone and analyzed spectrophotometerically with a lambda 2 uv/ vis spectrophotometer to determine total chlorophyll contents (perkin elmer, norwalk, usa) after lichtenthaler (1987). chlorophyll fluorescence for the measurements of in vivo photosynthesis on harvest day, a pam (pulse amplitude modulation) fluorescence system (mini� pam heinz walz gmbh, effeltrich, germany; veste et al. (2000) with a 6 mm diameter standard fibre optic was used. the fibre optic was fixed at a distance of 10 mm from the leaf surface with a leaf clip holder at an angle of 60°. �tandard routine programs within the pam were used to determine effective quantum yield of photosystem ii (φp�ii; (f´m – f´t) * f´m-1) in the presence of light on a photosynthesizing sample (for nomenclature of fluorescence signals used see schreiber et al., 1994; von willert et al., 1995). all chlorophyll fluorescence parameters were instantly calculated, displayed on a lcd screen and stored on an internal data logger. photosynthetic photon flux density (ppfd) was determined with a calibrated light sensor connected with the leaf clip holder. an external halogen lamp was used to illuminate the leaf sample with ppfd ≥1100 μmol m-2 s-1 for 2 minutes prior to pam measurements. the linear electron transport rate (etr) for photosystem ii can be calculated by multiplying the effective quantum yield of p�ii (φp�ii) and the incident ppfd (etr = φp�ii * ppfd * α * 0.5; see schreiber et al., 1994; von willert et al., 1995). the leaf absorption for light (α) was set to 0.84 (veste et al., 2000). leaf co2 gas exchange the gas exchange of fully expanded leaves was measured with a compact minicuvette system (cm� 400, heinz walz gmbh, effeltrich, germany; midgley et al., 1997) on the harvest day. the gas exchange measurements were conducted at the same leaves as the chlorophyll fluorescence measurements (10 leaves, 5 plants). the gas exchange measurements were carried out under ambient co2 concentrations (veste and herppich, 1995). illumination was set to a constant ppfd ≥1100 μmol m-2 s-1 with an external halogen lamp to ensure a light-saturated photosynthesis. air temperature in the cuvette was 25 °c and water vapor pressure deficit (vpd) of 17.4 mpa pa-1 was set with a cooling trap to correspond to the climatic conditions in the greenhouse. changes of co2 concentration were determined with an infra-red gas analyzer (binos 100-4p, rosemount, hanau, germany). net co2 exchanges were calculated using the standard software diaga� 2.0 (heinz walz gmbh, effeltrich, germany) and co2 flux was expressed on the projected leaf area (von willert et al., 1995). biomass the plants were harvested 173 days after the application of nitrogen and phosphate fertilizers. the fresh matter (directly after harvest) and dry weight (oven drying at 60 °c) of every plant was obtained for the biomass assessment. the harvesting procedure was carried out as follows in order to accommodate the various leaf assessments made preand post-harvest: i) the leaves on which individual ntester measurements (one leaf per plant) had been made were cut from each plant at the base of the leaves (petioles considered stem components and not leaf components) and stored in a freezer in individual plastic freezer bags. these leaves would later be subjected to a chemical analysis of chlorophyll content in the laboratory. ii) the five plants from each treatment which had been used in the eco� physiological assessments, which included: n-tester in the standard and individual modes, chlorophyll fluorescence, and co2 gas exchange were harvested in three steps. the first step was to acquire a representative mixed leaf sample for the chemical analysis of nitrogen and phosphate contents in the laboratory by cutting off ten mature leaves from halfway up the plants and packaging them in paper bags. the second step was to cut off the rest of the leaves and package them in a paper bag for the biomass assessment. the third step was to cut down the stem to just above the soil surface and package it in a paper bag for the biomass assessment. iii) the other five plants in each treatment were individually harvested for the biomass assessment by cutting off the leaves and cutting down the stems, then packaging the two components separately in paper bags. analysis of nitrogen and phosphorus contents the representative mixed leaf samples (n=10) obtained in step ii of the harvesting procedure were oven-dried at 60 °c and ground up for nutrient analysis. the carbon and nitrogen contents were analyzed 24 l.a. koning, m. veste, d. freese, �. lebzien with a cns element analyzer (vario el iii, elementar analysensysteme gmbh, hanau, germany). the phosphorus contents of the samples were determined using a lambda 2 uv/vis spectrophotometer (perkin elmer, norwalk, u�a) following the din en i�o 6878 method. statistics the relationships between the treatments and the independent variables were statistically assessed in the r software suite (r development core team, 2008) and grapher 2.0 (golden software inc, golden, usa) with linear regression analysis by applying the pearson correlation and accepting significance at p<0.05. results and discussion leaf nitrogen and phosphorus contents in a linear regression analysis, the nitrogen contents of the leaves were significantly correlated with the nitrogen treatments in a pearson correlation (r2= 0.55) (fig. 1). in contrast to this strong relationship, no correlation existed between the leaf phosphorus contents and the phosphate treatments (r2= 0.01) (fig. 2). a linear regression analysis of the leaf phosphorus and nitrogen contents demonstrated a weak positive trend (r2= 0.16) (fig. 3). the nitrogen contents of the igniscum candy leaves in the greenhouse experiment compared well with the results of wild fallopia x bohemica in belgium, both having mean values ranging from mid 1 % to mid 2 % nitrogen (herpigny et al., 2011). the nitrogen contents of igniscum candy leaves from field trial sites in germany with various management regimes (maturely rooted crops planted in 2008 and sampled in 2010 and 2011) were higher than the greenhouse leaves, having mean values ranging from 2.8 % to 3.1 % (veste, unpublished data). base nutrient levels in the respective soils may be a factor in the variance of nitrogen content (mengel and kirkby, 2001). the starting soil condition in the greenhouse experiment before fertilizer was applied averaged 0.06 0.09 % n. soil analysis of the sites where fallopia was found in belgium also showed the soil to be poor in nitrogen (mean of 0.11 0.19 % n) and no additional fertilizer or management was applied to these wild plants before sampling took place. the amount of influence of the p fertilizer in this experiment is inconclusive. all three p treatments (20 p, 40 p, and 80 p) delivered similarly uneventful results for nutrient contents and biomass. a plot of the p content in the leaves against the n treatments showed no significant trends (r2=0.01 and r2=0.07, respectively), suggesting that nutrient uptake was not influenced by the p fertilization rates. a plot of dry matter against the p treatments also produced a lack of trend (r2=0.00) (data not shown). phosphorus uptake is not well understood (schachtman et al., 1998). an investigation into the availability of phosphorus in the soil or a biological analysis of the phosphorus distribution throughout the entirety of the plants (e.g. veneklaas et al., 2012) may have shed more light on the phosphorus uptake by igniscum. chlorophyll content previous studies have shown that the chlorophyll contents of leaves correlates strongly with the nitrogen contents of leaves (gianquinto fig. 1: relationship between applied nitrogen fertilizer and leaf nitrogen content at various phosphate levels (20, 40, 80 kg p ha-1). linear regression y = 0.001477 x + 0.692778, r2 = 0.3429) fig. 2: relationship between applied phosphate fertilizer and leaf phosphate content at various nitrogen levels (0, 50, 150, 300 kg n ha-1). linear regression y = -6.071* 10-5 x + 0.0787, r2 = 2.752 * 10-5) fig. 3: relationship between leaf nitrogen content and leaf phosphate content at various nitrogen levels (0, 50, 150, 300 kg n ha-1). linear regression y = 0.019396 x + 1.06893, r2 = 0.474 influence of nitrogen and phosphate on igniscum 25 et al., 2004; samborski, 2009) and based on this relationship, handheld devices measuring the chlorophyll in living plants can reflect the nitrogen status and physiological activity of plants (bullock et al., 1998; netto et al., 2005). the relative indices measured by the n�tester were in significant linear correlation to the chlorophyll contents of the leaves (r2=0.65) (fig. 4) and the nitrogen contents of the leaves (r2=0.52) (fig. 5). as expected, a significant linear correlation also existed between the chlorophyll contents of the leaves and the nitrogen contents of the leaves (r2=0.42) (not shown). the theory behind the n-tester was supported by the current research through strong positive trends in the relationships between the chlorophyll and nitrogen contents as well as the n-tester results and nitrogen contents. between the different nitrogen treatments (fig. 6b). even though photosynthesis depends on phosphate containing compounds and phosphate efficiency should influence photosynthetic activity (veneklaas et al., 2012), we did not find any relation between the photosynthesis parameters and the phosphate fertilization. however, co2 exchange rates and electron transport rates showed a high variance within each treatment. the measured net photosynthesis of fallopia is lower compared to other perennial bioenergy crops or highly productive annual crops. mantovani et al. (2014) measured mean co2 exchange rates of 7.5 to 9.7 μmol m-2 s-1 for igniscum in a lysimeter experiment. for the perennial sida hermaphrodita, which is considered a highly productive bioenergy crop, mean net photosynthesis rates from 6 �17 μmol m-2 s-1 were measured (franzaring et al., 2014; veste et al., 2014), while annual crops like sunflowers or tomatoes reach net photosynthesis rates of 12 � 24 μmol m-2 s-1 and 18 � 26 μmol m-2 s-1, respectively (breckle et al., 2003). in general, the photosynthesis and related physiological processes are directly linked to the leaf nitrogen content (evans, 1983; sage et al., 1987; lawlor et al., 2001). in our experiment a linear relationship between the leaf n content and the net co2 exchange could be shown (fig. 7a), while the electron transport rate showed no correlation with the leaf n content (fig. 7b). however, a clear linear influence of nitrogen leaf content on photosynthesis could be observed under various field conditions in germany (veste et al., 2011). furthermore, nitrogen also alters leaf morphology (lawlor, 2001). since co2 exchange rates are area related, leaf morphological changes in response to nitrogen needs to be taken into account to understand the plant co2 uptake. hereby, the leaf area demonstrated a strong positive trend over the increasing n treatments (fig. 8c) and increased total carbon uptake. it needs to be considered that nutrient deficiency fig. 4: relationship between yara n-tester values and chlorophyll content (linear regression y = 0.4924 x + 11.871, r2 = 0.645) at nitrogen levels (0, 50, 150, 300 kg n ha-1). fig. 5: relationship between leaf nitrogen content and yara n-tester value (linear regression y = 384.918 x + 14.2766, r2 = 0.518) at various nitrogen levels (0, 50, 150, 300 kg n ha-1). leaf photosynthesis the mean net photosynthesis rates varied between 3.7 and 6.6 μmol m-2 s-1 and increased (r2=0.251) with increasing nitrogen application (fig. 6a), while the electron transport rates showed no changes fig. 6: influence of the applied nitrogen fertilizer on net co2 exchange (a) and electron transport rate (b). (a) linear regression y = 0.00216963 x + 1.133749, r2 = 0.2013), (b) linear regression y = 0.022849 x + 56.83, r2 = 0.004). 26 l.a. koning, m. veste, d. freese, �. lebzien might also affect the carbon source-sink interrelation. understanding the carbon-sink interrelation as well as carbon allocation is important for understanding the growth performance of plants in relation to their photosynthesis (körner, 2013; fatichi et al., 2014). in the case of the studied fallopia, the formation of the underground rhizome to store carbon and nutrients during the establishment of young plants is such an example of carbon and nutrient flows into different sink organs. however, in mature established plants the interrelation between aboveground and belowground organs is important to understand biomass production under different nutrient supply und harvest regimes (adachi et al., 1996; li et al., 1998; aravindhakshan et al., 2011) and needs further investigations in herbaceous perennial bioenergy plants. biomass production the dry weight, plant height, and leaf area (fig. 8) all had quadratic relationships with the nitrogen treatments (r2=0.32, 0.34, 0.32, respectively). the peaks of the parabolic curves, or the point of optimum yield for dry weight, were reached around 170 kg n ha-1. a complete lack of correlation (r2=0.00) existed between the dry weight and phosphate treatments (not shown). our experiment showed that increasing nitrogen fertilization to young knotweed plants leads to higher biomass yields, as shown previously by schmitt (1994). the strongest effect of the nitrogen supply was from 100 to 150 kg kg n ha-1, which could also be shown in a lysimeter experiment (mantovani et al., 2014) and is in the same range as observed for silphium perfoliatum (gansberger et al., 2014). the positive effects of the nitrogen fertilization on biomass, plant height and leaf area showed a general decline between the 150 and 300 kg n ha-1 treatments, possibly due to nitrogen uptake entering the zone of luxury nutrient consumption (westerman, 1990). pude and franken (2001) observed an increase in biomass production with up to 200 kg n ha-1, while on agricultural fields of the �oester börde (northrhine west� falia, germany) the growth response to nitrogen fertilization was indifferent (veste et al., 2011). as shown in fig. 8c, the leaf area of the igniscum plants in this experiment ranged between 50 and 100 cm2. consequently, plants in the current experiment did not reach their full leaf area potentials between 150 and 350 cm2 (herpigny et al., 2011) or 300 cm2 (bailey and bimova, 2009) as described for f. x bohemica. just as the plants in the greenhouse did not reach as high of nitrogen contents nor produce as high of relative indices with the n�tester as the plants in field trials, the small size of the leaves could likely be contributed to the more limited greenhouse conditions as compared to the outdoor sites (weih and nordh, 2005). conclusions the results of this experiment demonstrated that nitrogen fertilization has an overall positive correlation with leaf nitrogen content, photosynthesis, and growth of the bioenergy crop fallopia sachalinensis var. igniscum candy. the existence of significant trends in the influence of phosphate fertilizer on igniscum candy could not be demonstrated and requires further investigation. fig. 8: influence of the applied nitrogen fertilizer on aboveground biomass (a), plant height (b) and total leaf area (c) at various phosphate levels (20, 40, 80 kg p ha-1). (a) biomass (n = 10, r2 = 0.32), (b) plant height (n = 10, r2 = 0.34), and (c) leaf area (n = 5, r2 = 0.32). fig. 7: relationship between leaf nitrogen content and net co2 exchange (a) and electron transport rate (b) at various phosphate levels (20, 40, 80 kg p ha-1). (a) linear regression y= 2.518 x + 2.378, r2 = 0.251), (b) linear regression y= 13.173 x + 48.117, r2 = 0.134). influence of nitrogen and phosphate on igniscum 27 acknowledgement thanks to the students christian halke and mirko milke for their help with the harvest as well as mandy turski, philipp lange and thomas fischer (btu cottbus-senftenberg) for their support with the chemical analyses. thanks to yara research centre hanninghof, dülmen, germany for providing the n�tester. the research was funded by conpower rohstoffe gmbh, planegg, germany. thanks to the two reviewers for their helpful recommendations for improvements to the manuscript. references adachi, n., terashima, i., takahashi, m., 1996: nitrogen translocation via rhizome systems in monoclonal stands of reynoutria japonica in an oligotrophic desert on mt fuji: field experiments. ecological research 11 (2), 175-186. aravindhakshan, s.c., epplin, f.m., taliaferro, c.m., 2011: switchgrass, bermudagrass, flaccidgrass, and lovegrass biomass yield response to nitrogen for single and double harvest. biomass bioenerg. 35, 308-319. aurbacher, j., benke, m., formowitz, b., glauert, t., heiermann, m., herrmann, c., idler, c., kornatz, p., nehring, a., rieckmann, c., rieckmann, g., reus, d., vetter, a., vollrath, b., wilken, f., willms, m., 2012: energiepflanzen für biogasanlagen. regionalbroschüre niedersachsen. fachagentur nachwachsende rohstoffe e.v. 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brandenburg university of technology cottbus-senftenberg, chair of soil protection and recultivation, konrad�wachsmann�allee 6, 03046 cottbus, germany dirk freese, brandenburg university of technology cottbus��enftenberg, chair of �oil protection and recultivation, konrad�wachsmann�allee 6, 03046 cottbus, germany �tefan lebzien, enagra biomasse gmbh, auf der grub 1, 54472 monzelfeld, germany journal of applied botany and food quality 89, 208 211 (2016), doi:10.5073/jabfq.2016.089.026 1“babeş-bolyai” university, faculty of chemistry and chemical engineering, cluj-napoca, romania effects of storage temperature on the total phenolic content of cornelian cherry (cornus mas l.) fruits extracts bianca moldovan1, anamaria popa1, luminita david1* (received december 11, 2015) * corresponding author summary cornelian cherry (cornus mas l.) fruits are an important source of bioactive compounds, especially phenolics and anthocyanins which exhibit a high antioxidant activity and contribute to their numerous health benefits. the aim of this study was to evaluate the effect of storage temperature on the stability of polyphenolic compounds of cornelian cherry (cornus mas l.) fruits extracts, stored under darkness at 2 °c, 22 °c, 55 °c and 75 °c respectively. for all analysed conditions, a first-order reaction kinetics was established for the degradation process of polyphenols. the degradation rate constants of the phenolic compounds at 2 °c and 22 °c presented comparable values (4.79 and 4.88 × 10-3 day-1). the phenolics stored at 75 °c showed the lowest stability, with half-life value (t1/2) and reaction rate constant (k) of 8.79 days and 78.76 × 10-3 day-1, respectively. the temperature dependence of the polyphenols degradation rate constants was expressed by calculating the activation energy ea (33.43 kj mol-1) and the temperature coefficients q10 of the process. the cornelian cherry (cornus mas l.) fruits extract can be stored at least 2 months at room temperature without significant loss of their bioactive compounds. introduction fruits and vegetables are the main source of natural antioxidants, nutrients which play an important protective role against harmful free radicals which are responsible for cellular oxidation reactions and oxidative stress (scalzo et al., 2005). polyphenols and flavonoids are the most abundant dietary antioxidants (aruoma, 1998). the long term consumption of antioxidants-rich fruits plays an important role in human health by offering protection against oxidative stress related diseases, such as aging, vascular, heart and neurodegenerative diseases, cancer and diabetes (smith et al., 2000; tsuda et al., 2003;vauzour et al., 2014). lately, the consumers’ interest to particularly polyphenolic-rich fruits increased. among these, cornelian cherry fruits gained a great importance. cornelian cherry (cornus mas l.) is a species of dogwood widely grown in central and south-eastern europe and asia. the deep red shinny fruit is spherical or oval shaped, one stone drupe, sweet-sour in taste but astringent when unripe. cornelian cherries are edible fruits that can be eaten fresh, dried, pickled or used to produce different drinks, gels and jams. these fruits are rich in ascorbic acid (vitamin c), sugar, organic acids, flavonoids, tannins and other bioactive compounds among which polyphenols were found in significant amounts (david and moldovan, 2015). these compounds, known for their high antioxidant activity, are responsible for the wide range of biological properties of these fruits, among which the antibacterial, anti-histamine, anti-inflammatory, anti-microbial and antimalarial properties were reported (celik et al., 2006; vareed et al., 2006). due to all these health benefits, there is lately an increased interest on the physical and chemical properties, total phenolics and ascorbic acid content and antioxidant activity of cornelian cherries (pantelidis et al., 2007; popovic et al., 2012). cornelian cherries extracts can be employed as functional ingredients in different nutraceutical products. so, the investigation of the thermal stability of the bioactive compounds of the extracts is of great interest. the characteristics and properties of the extracts must not be essentially affected by temperature elevation, which often occurs during industrial processing, or by storage conditions. the effect of thermal treatments on fruit extracts and juices has been previously reported (wang and xu, 2007; hillmann et al., 2011; moldovan et al., 2011) and first order kinetics degradation was found for anthocyanins (hernandez-herreros and frutos, 2011; moldovan et al., 2012) while this tendency was not so clear for other polyphenolic compounds. to the best of our knowledge, no kinetic data were published regarding the degradation kinetics of cornelian cherry fruits polyphenols. the purpose of this study was to determine the stability of the polyphenols from cornelian cherries extracts during storage at different temperatures. therefore, the precise determination of the degradation kinetic parameters of the polyphenols present in these fruits is essential for predicting quality losses of nutritional values related to both storage and heat processing of foods. to these end, extracts were stored at 2 °c, 22 °c, 55 °c and 75 °c. the investigated temperatures were chosen in order to assure a high compa tibility with processing techniques required to extend the shelf-life of food products, as thermal processing is the most effective treatment used to preserve food. material and methods plant material fruits of cornelian cherry (cornus mas l.) were obtained from a local market in cluj-napoca, romania in august 2014. the specimen was identified and authenticated by prof. emeritus m. tamas, department of botany, faculty of pharmacy, “iuliu hatieganu” university of medicine and pharmacy cluj napoca, romania. 45 uniform ripened fruits at consume maturity stage were chosen. skin colour (dark red) was the main criterion for maturity of fruits. the fruit mass ranged from 1.6 to 1.8 g with an average flesh: fruit ratio of 82 %. samples of 15 fruits in 3 replications were frozen and stored in polyethylene bags at -18 °c until used for the extraction of polyphenols. chemicals and reagents all reagents and solvents were of analytical grade and purchased from merck (darmstadt, germany). distilled water was used for analyses and was obtained using a typdp1500 water distiller (techosklo ltd. czech republic). preparation of fruits extract the stones of 15 frozen cornelian cherries were removed and the fruits were milled. ten grams of milled fruits were transferred to an erlenmeyer flask and homogenised with 100 ml of cold acetone. the stability of phenolics from cornelian cherries 209 extraction was carried out for 1 h at room temperature. the obtained mixture was vacuum filtered through whatman no. 1 paper. the acetone was totally removed under vacuum and a concentrated extract was obtained and further analyzed to determine the total phenolic content as well as the variation of this parameter in different storage conditions in order to establish the degradation kinetics. determination of total phenolic content (tpc) the total phenolic content was assessed using the folin-ciocalteu reagent (singleton et al., 1999). six ml folin-ciocalteu reagent were mixed with 1 ml of diluted extract (dilution factor = 256) and allowed to react for 5 min in the dark. after that, 4.8 ml of na2co3 solution (0.7 m) were added and the resulted solution was kept in the dark for 2 h at room temperature (22 °c). the absorbance of the mixture was measured at 765 nm (using an uv-vis perkin elmer lambda 25 double beam spectrophotometer), against a blank sample. the total phenolic content was calculated with reference to a gallic acid (ga) calibration curve (range 0 100 mg ml-1), and expressed as mg ga equivalents l-1 extract. each measurement was performed in triplicate. degradation studies the thermal degradation of the phenolic compounds from cornelian cherry fruits extract was investigated at four different temperatures: 2 °c, 22 °c, 55 °c and 75 °c. the fruit extract was divided into 5 ml portions and kept in closed vials in order to prevent solvent evaporation. the samples were stored in the dark at 2 °c (in refrigerator) and at room temperature (22 °c) for 2 months, in a thermostatic water bath, preheated at 55 °c for 30 days and at 75 °c, respective ly (± 1 °c) for 10 days. samples were analyzed for the total phenolic content at 0, 10, 20, 30, 40, 50 and 60 days for storage at 2 and 22 °c, at 0, 5, 10, 15, 20, 25 and 30 days for storage at 55 °c and at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days for storage at 75 °c. changes of the total phenol content were used to evaluate the stability of the polyphenolic compounds in the investigated extracts. all experiments were carried out in three replicates. data analysis all data presented are mean values ± standard deviations of three independent experiments (n = 3). data obtained were statistically analyzed by one-way variance analysis (anova) using the statistical package xlstat release 10 (addinsoft, paris, france). statistical significances were considered acceptable at p < 0.05. results and discussion the determined total phenolic content of the cornelian cherry fruit extract was 1137.78 ± 21.44 mg l-1 expressed as gallic acid equi valents. the stability of polyphenols from the extract during sto rage at different temperatures (2 °c, 22 °c, 55 °c and 75 °c) was evaluated based on changes in their concentration. tab. 1 shows the kinetic parameters (kinetic rate constants and the half-life values) determined for the thermal degradation of the polyphenolic compounds. previously reported data showed that storage and thermal degradation of polyphenols from various sources is described by simple first-order reaction kinetics (wang et al., 2008; li et al., 2012). the degradation kinetics of polyphenols can be described by following equation (1): ln[tpc] = ln[tpc0] – kt (1) where: [tpc] = total phenolic content, mg l-1 at time t; [tpc0] = initial total phenolic content, mg l-1; k = reaction rate constant, days-1; t = reaction time, days. the half-lifes of polyphenols from the investigated extracts during storage can be calculated using equation (2): t1/2 = – ln 0.5/k (2) where: t1/2 = half-life (days); k = reaction rate constant (days-1). according to equation (1) a series of k at different temperatures can be obtained by plotting the changes in the total polyphenolic content of the aqueous extract obtained from cornelian cherries as a function of time (fig. 1). high values of coefficients of determination (r2, 0.93-0.98) were obtained for all linear regressions, confirming that the degradation process of these bioactive compounds, at temperatures from 2 °c to 75 °c, indeed followed first-order reaction kinetics. the degradation of polyphenols from cornelian cherries extract was investigated. storage time and temperature had a significant effect (p < 0.05) on total phenolic content. during storage of cornelian cherries extracts at 2 °c the level of phenolic compounds slightly decreased. we observed losses of 3.1 %, 6.9 %, 11.9 % and 22.6 % after 10, 20, 30 and 60 days of storage, respectively. storage of extracts at room temperature (22 °c) resulted in no significant change of the degradation rate constant (p = 0.9), the lost of phenolic compounds being 25.4 % after 60 days of storage. stability of polyphenolic compounds appears to be greatly affected at temperatures higher than 50 °c. in the case of thermal treatment on cornelian cherry fruits extracts at 55 °c and 75 °c, significant decrease can be observed in total phenolic content values. this is consistent with results presented in previous studies on polyphenols degradation (kalt, 2005; kyi et al., 2005). the decrease of the polyphenolic content of the investigated extracts at high temperatures may be due to increased oxidation of these bioactive components. the rates of these oxidation reactions increase as temperature increases. the degradation rate of polyphenols stored at 55 °c was 8 times faster as compared to degradation at refrigerated storage (at 2 °c) while, at 75 °c, the degradation rate was 16 times higher (tab. 1). the activation energy normally describes the required energy to reach the transition state of a reaction. this energy can be determined from the kinetic rate constants by using an arrhenius model (eq. 3): k = k0e-ea/rt (3) where: k = rate constant, day-1; k0 = frequency factor, day-1; ea = activation energy, kj mol-1; r = universal gas constant (8.314 j mol-1 k-1); t = absolute temperature, k. fig. 2 presents the plot of the degradation rate constants of the polyphenols from each extract as function of storage temperature. the slope of ln k vs. 1/t was used to determine the ea value of the polyphenols degradation reactions. the calculated ea value was 33.43 kj mol-1 (r2 = 0.915), comparable to that determined by other authors, who reported a value of 30.324 tab. 1: effect of temperature on the kinetic parameters of polyphenols de gradation in cornelian cherries extracts temperature/oc k × 10-3/(day-1)* t1/2/days 2 4.79 (0.9379) 144.67 22 4.88 (0.9501) 142.01 55 38.86 (0.9693) 17.83 75 78.76 (0.9858) 8.79 *numbers in parentheses, r2, are the determination coefficients. 210 b. moldovan, a. popa, l. david kj mol-1 for the degradation of polyphenols from cocoa beans (kyi et al., 2005). the temperature coefficient q10 is another parameter which, in addition to activation energy, provides information related to temperature influence on the degradation rate of polyphenols. q10, defined as the change of degradation rate upon a temperature increase of 10 k, can be calculated by the following equation (4) (moldovan and david, 2014): (4) where: q10 = the temperature coefficient, k-1; k1,2 = rate constant, day-1 at temperature t1,2, k. tab. 2 presents the q10 values calculated for the temperatures applied in this study. the highest q10 value (q10 = 1.875) was obtained for storage at temperatures between 22 and 55 °c indicating that degradation of phenolics was stronger affected by temperature within the range 2255 °c than between 55-75 °c (q10 = 1.423). the lowest temperature coefficient value (q10 = 1.009), determined for storage temperatures 2-22 °c, proves that increasing temperature from 2 °c to 22 °c has no influence on the degradation process of these bioactive compounds from cornelian cherries extracts (tab. 2). the results obtained in this study regarding the degradation kinetics parameters of the polyphenols from cornelian cherry fruits extracts are comparable to the literature data reported for the degradation process of phenolics from other sources such as hibiscus and cocoa beans, although the degradation process of the total phenolics from fruits was less investigated. perez-ramirez et al. (2015) reported a half-life value of 17.2 days for the degradation of the polyphenols from hibiscus sabdariffa l. beverages stored at 50 °c, value in agreement with our results (t1/2 = 17.8 days at 55 °c). comparing the loss of total phenolics from cornelian cherry fruits extracts during thermal processing and storage to that of anthocyanins isolated from the same fruits, extensive anthocyanin degradation occurred under the same heating and storage conditions. previous studies (moldovan and david, 2014) reported accelerated anthocyanin degradation, the half-life values for this process being 2.4 fold higher at 2 °c, 4.3 fold higher at 22 °c and 25 fold higher at 75 °c, as compared to degradation of total phenolics, indicating a higher stability of cornelian cherry polyphenols. this fact may be related to the degradation pathway of cornelian cherry anthocyanins. the major anthocyanins in cornelian cherries are cyanidin3-o-galactoside, pelargonidin-3-o-galactoside and delphinidin-3o-galactoside (vareed et al., 2006). the a and b rings of these anthocyanins can degrade into other phenolic derivatives, such as p-hydroxibenzoic acid, protocatechuic acid or phloroglucinaldehyde (sadilova et al., 2007). a) b) c) fig. 1: degradation kinetics of polyphenols during storage at (a) 2 °c and 22 °c; (b) 55 °c; (c) 75 °c (vertical lines represent sd, n = 3) fig. 2: arrhenius plot for polyphenols degradation in cornelian cherries extracts tab. 2: q10 and ea values of polyphenols’ degradation at different storage temperatures ea/kj mol-1* ko /day-1 q10 2-22 °c 22-55 °c 55-75 °c 33.43 (0.915) 7.39·103 1.009 1.875 1.423 *number in parentheses, r2, is the determination coefficient. a) b) c) a) stability of phenolics from cornelian cherries 211 conclusions the present study evaluated the influence of the temperature and storage on the degradation kinetic parameters of phenolics from cornelian cherries extracts. phenol content was stable at temperatures up to 22 °c. temperature increase determined higher rate constants of the degradation process. the extracts can be stored during 2 months at room temperature, without significant decrease of their total phenolic content. the bioactive compounds made responsible for the health promoting effects of cornelian cherry fruits were not substantially affected even after excessive long time heating. it should be highlighted that cornelian cherry fruits could be an interesting source of nutraceuticals, considering the high stability to storage and temperature of their nutritional parameters. acknowledgements this work was supported by the ministry of education and scientific research, romania as a part of the research project no. 147/2011 pn-ii-pt-pcca-2011-3-1-0914. references aruoma, o.i., 1998: free radicals, oxidative stress, and antioxidants in human health and disease. j. am. oil chem. soc. 75, 199-212. celik, s., bakirci, i., sat, i.g., 2006: physico-chemical and organoleptic properties of yogurt with 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21, 207-213. singleton, v.l., orthofer, r., lamuela-raventós, r.m., 1999: analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent. meth. enzymol. 299, 152-178. smith, m.a.l., marley, k.a., seigler, d., singletary, k.w., meline, b., 2000: bioactive properties of wild blueberry fruits. j. food sci. 65, 352-356. tsuda, t., horio, f., uchida, k., aoki, h., osawa, t., 2003: dietary cyanidin 3-o-ß-d-glucoside-rich purple corn color prevents obesity and ameliorates hyperglycemia in mice. j. nutr. 133, 2125-2130. vauzour, d., kerr, j., czank, c., 2014: plant polyphenols as dietary modulators of brain functions. in: polyphenols in human health and diseases, 357-370. london, uk: academic press, elsevier. vareed, s.k., reddy, m.k., schutzki, r.e., nair, m.g., 2006: anthocyanins in cornus alternifolia, cornus controversa, cornus kousa and cornus florida fruits with health benefits. life sci. 78, 777-784. wang, w.-d., xu, s.-y., 2007: degradation kinetics of anthocyanins in blackberry juice and concentrate. j. food. eng. 82, 271-275. wang, r., zhou, w., jiang, x., 2008: reaction kinetics of degradation and epimerization of epigallocatechin gallate (egcg) in aqueous system over a wide temperature range. j. agric. food chem. 56, 2694-2701. address of the corresponding author: luminita david, department of chemistry, faculty of chemistry and che mical engineering, “babes-bolyai” university, 11, a. janos str., 400028, cluj-napoca, romania e-mail: muntean@chem.ubbcluj.ro © the author(s) 2016. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 91, 163 170 (2018), doi:10.5073/jabfq.2018.091.022 1tea research institute, guangdong academy of agricultural sciences, guangdong provincial key laboratory of tea plant resources innovation & utilization, guangzhou, china 2college of horticulture, south china agricultural university, tianhe district, guangzhou, china optimization of brewing conditions in epigallocatechin-3-gallate (egcg) extraction from jinxuan summer green tea by response surface methodology limin xiang1,#, shunshun pan1,2,#, xingfei lai1, lingli sun1, zhigang li1, qiuhua li1,*, yahui huang2,*, shili sun1,* (submitted: december 11, 2017; accepted: june 12, 2018) * corresponding author # these authors contributed equally to this work summary the extraction conditions of epigallocatechin-3-gallate (egcg) from jinxuan summer green tea and antitumor activity against human gastric cancer sgc-7901 cells of the green tea extracts were investigated. on the basis of a single factor experiment, box-behnken design and response surface methodology were employed to optimize the hot water extraction conditions. the optimal extraction conditions for egcg were determined as: extraction temperature of 85 °c, extraction time of 34 min, water-tea ratio of 41 ml/g, a solution of ph 6, and extraction twice. under these conditions, the experimental extraction yield value of egcg was 33.82 mg/g, which was not significantly different in comparison to predicted values. the results indicated that the regression models were suitable for the egcg extraction from jinxuan summer green tea. the summer green tea extract prepared under the optimal conditions had a higher antitumor activity against human gastric cancer sgc-7901 cells than the green tea extract made with traditional tea brewing method. keywords: response surface methodology, summer green tea, egcg, gastric cancer introduction camellia sinensis tea is the second most widely consumed nonalcoholic beverage in the world (dalar and konczak, 2013). tea is usually classified into unfermented (green tea), slight-fermented (white tea), semi-fermented (oolong tea), fermented (black tea), and post-fermented (dark green tea) forms (zhao et al., 2014). among them, green tea is one of the most popular beverages consumed worldwide. and its consumption is increasing as a result of accumulated scientific evidence on its beneficial effects for human health. the epidemiological studies have suggested that daily consumption of green tea will help to prevent cancer, cardiovascular diseases, dental decay, obesity, diabetes, and improve the immune system (kao et al., 2006; khan and mukhtar, 2007; vuong et al., 2013; wheeler and wheeler, 2004). the aforementioned health benefits of green tea have been closely associated with tea catechins, purine alkaloids and theanine. tea catechins which usually account for 30% of the dry weight of the green tea are associated with various health benefits, including antioxidant activity, antitumor activity, and antiaging activity (khan and mukhtar, 2007; savic et al., 2016). epigallocatechin-3-gallate (egcg) has been considered as the most abundant and active constituent in green tea and may account for up to 50% of the catechins. it is usually used as a quality indicator (gadkari et al., 2015; khan and mukhtar, 2007; perva-uzunalić et al., 2006). a number of studies have reported that egcg has various biological, physiological, and pharmaceutical activities, such as prevention of obesity, hyperglycemia, insulin resistance, hypercholesterolemia, and hepatic steatosis (chen et al., 2011; hiningerfavier et al., 2009; liu et al., 2012; sae-tan et al., 2011). additio nally, it has been reported to have excellent anticancer and antioxidant activities (garbisa et al., 2001; singh et al., 2011; xu et al., 2010). hence, egcg may be extensively applied in the fields of medication, cosmetics, beverages, and foodstuffs (pavlovic et al., 2015). owing to its great potential as a health promoter, the extraction of egcg from green tea is considered to be an important research work for its wide use in a variety of health and food products (zhang et al., 2012). the extraction efficiency of the tea constituents and quality of obtained extracts have been directly influenced by several extraction conditions, including brewing temperature, extraction time, the ratio of solvent to tea, tea particle size, the ph of the brewing solution and the number of times the same sample is extracted (castiglioni et al., 2015; hajiaghaalipour et al., 2016; saklar et al., 2015). thus, the extraction efficiency can be improved by optimizing each of these factors during the extraction process. the conventional single factor experiment for performing optimization is very time consuming and the interactions among multiple parameters are not considered in this method (d’archivio et al., 2016). response surface methodology (rsm) is a helpful statistical technique which is useful for developing, improving and optimizing processes. it can minimize the number of experiments and provide sufficient information for a statistically acceptable result. rsm has become more and more attractive in process optimization, which has been successfully applied in the extraction of biologically active constituents from plant sources (kong et al., 2010; savic et al., 2015; savić et al., 2013). therefore, the aim of the present study was to extract egcg using the rsm with a box-behnken design (bbd) to optimize the extraction conditions and obtain maximum egcg from summer green tea (the lower grade green tea). according to the results of a single factor experiment, extraction temperature, extraction time and water-to-tea ratio were selected as independent variables for a box-behnken design. meanwhile, the number of times the tea is extracted and the ph of the brewing solution were set as fixed values. the chemical compositions of the lower grade green tea extract was analyzed, identified and quantified using an hplc (high performance liquid chromatography) gradient type elution system and authentic standards. additionally, the lower grade green tea extract was tested in vitro for its antitumor activity against human gastric cancer cells. materials and methods materials the summer green tea of the jinxuan variety (camellia sinensis), harvested in june 2013, was kindly provided by guangdong minghuang tea co., ltd. (guangdong, china). the dried green tea was ground by a disintegrator of fw100 (taisite co., ltd, tianjin, china) and then passed through a 40-mesh sieve. the tea power was kept in airtight bags at -20 °c until use. the conventional chemical composi164 l. xiang, s. pan, x. lai, l. sun, z. li, q. li, y. huang, s. sun tions of green tea used in brewing trails were determined according to the gb/t of chinese standard (gb/t 8313-2008 and gb/t 83142013). the data analysis for the green tea used in brewing trails is shown in tab. 1. deionized water was prepared using a millipore zmqs5001 system (millipore, bedford, ma, usa). standards of c (catechin), cg (catechin gallate), ec (epicatechin), ecg (epicatechin gallate), egc (epigallocatechin), egcg (epigallocatechin-3-gallate), gc (gallocatechin), gcg (gallocatechin gallate) and caffeine were purchased from sigma-aldrich (st. louis, mo, usa). all solvents used for hplc analysis were hplc grade and were purchased from thermo scientific (waltham, ma, usa). all other reagents were of analytical grade. (1) where y represents the predicted extraction yield of egcg, xi and xj represent the independent variables. β0, βi, βii and βij represent the regression coefficient for intercept, linearity, quadratic and interactive terms, respectively. the data were analyzed using design expert program (7.0.0 version, stat-ease inc., minneapolis, minnesota, usa). additional confirmation experiments were subsequently conducted to verify the validity of the statistical experimental design. tab. 1: the composition of green tea leaves (g/100 g dry matter). green tea g/100 g dry matter* tea polyphenols 27.33 ± 0.25 amino acids 4.58 ± 0.05 caffeine 4.74 ± 0.07 soluble sugars 3.42 ± 0.04 soluble protein 0.64 ± 0.01 * values are mean ± standard deviation of three measurements egcg extraction three grams of pretreated green tea powder was extracted with distilled water using a designed temperature, time, brewing solution ph, water-tea ratio and the extraction times in a hhs thermostat waterbath (aohua co., ltd, changzhou, china). following extraction, the mixtures were centrifuged at 8000 rpm for 10 min by the centrifuge (hc-3018, anhui ustc zonkia scientific instruments co., ltd, anhui, china). the supernatant was collected and transferred into a 250 ml measuring flask for determination of egcg content. each extraction was performed in three replicates. samples (the extracts) were stored at -20 °c until hplc analysis. single-factor design for egcg extraction the extraction yield of egcg depends on the factors, including temperature, extraction time, brewing solution ph, water-tea ratio and the extraction times. the single-factor design was used to determine the preliminary range of extraction factors. single factor experiment was used to study the effect of different factors on the extraction yield of egcg. for single factor experiment, one factor was changed in a certain range while all other factors were kept constant. the extraction parameters were temperature (50, 60, 70, 80 and 90 °c), time (10, 20, 30, 40 and 50 min), water-tea ratio (10, 20, 30, 40 and 50 ml/g), times (once, twice, thrice, and four times), and brewing solution ph (4, 5, 6, 7 and 8), of which the single factor experiment was investigated in this paper, respectively. response surface optimization designs according to the results of single-factor experiment, three major factors and their appropriate ranges were finally determined for a three-level factorial box-behnken design (bbd) in order to investigate the relationship between process variables and the extraction yield of egcg as the response value (tab. 2). the whole design comprising 17 experimental runs were carried out in a randomized order as shown in tab. 3. the following second-order polynomial equation was used to explain the behavior of the system: tab. 2: the uncoded and coded levels of independent variables used in the rsm design. levels independent variable symbol low (-1) middle (0) high (+1) temperature (°c) x1 50 70 90 time (min) x2 20 40 60 water-tea ratio (ml/g) x3 10 30 50 determination of tea catechins content the tea catechins in extract were quantified by hplc using an agilent 1200 series hplc system equipped with a uv-detector and an analytical workstation (agilent co., ltd, santa clara, ca, usa). the separation was performed on an agilent zorbax eclipsexdb-c18 column (4.6 mm × 150 mm, 5 μm; agilent technologies, wilmington, de, usa). the solvents used were: (a) acetonitrile/acetic acid/ methanol/water (1:0.5:2:96.5, v/v/v/v), and (b) acetonitrile/acetic acid/methanol/water (10:0.5:20:69.5, v/v/v/v). the following linear gradient elution was used: 0-30 min, a from 72.5% to 20%; 30 35 min, a from 20% to 72.5%. the column temperature was 28 °c. the flow rate was 1 ml/min. the detection wavelength was 280 nm and the injection volume was 10 μl. quantification was carried out with the external standard method. calibration plot was constructed by tea catechins concentrations versus each plotting peak areas. the concentrations of tea catechins in extracted samples were obtained by linear calibration curve. determination of antitumor activity of green tea extracts preparation of green tea extracts for cell viability assay in order to evaluate the antitumor activity of green tea infusions brewed with different methods, traditional tea brewing method (ttbm) and improved tea brewing method (itbm) were chose to prepare tea infusion. different brewing methods to prepare tea infusion are mentioned below: (1) traditional tea brewing method (ttbm): tea infusions were prepared by placing 1 g of tea in 50 ml of distilled water at boiling temperature (100 °c) twice, and 3 min each time. (2) improved tea brewing method (itbm): tea infusions were prepared by placing 1 g of tea in 41 ml of distilled water (ph 6) at 85 °c twice, and 34 min each time. the tea infusions were freeze-dried. the concentrated green tea extracts were stored at -20 °c for assay. cell viability assay human gastric cancer cell sgc-7901 was obtained from shanghai institute of cell biology (shanghai, china) and maintained in an atmosphere of 5% co2 and 95% air at 37 °c in dmem medium supplemented with 10% fetal bovine serum (fbs, gibco/brl, gaithersburg, md, usa). cell viability assay was determined by egcg enrich brewing method of green tea 165 mtt assay as described with some modification. briefly, sgc-7901 was seeded at a density of 2 × 104 cells per well in 96-well plate, grown for 24 h, and then treated with different concentration of green tea extracts for 24 h. after incubation, 20 μl mtt (5 mg/ml in pbs) was added to each well and the cells were incubated at 37 °c for 4 h. the supernatants were discarded and 150 μl dmso was added to each well to dissolve the precipitate and the absorbance of the well was measured at 570 nm with a microplate reader (thermo scientific multiskan go, vantaa, finland). results were expressed as percentage of cell viability (%), assuming control cells as 100%. statistical analysis all experiments were performed in triplicate and centered. analysis of variance (anova) was performed by anova procedure. p < 0.05 and p < 0.01 were regarded as significant and very significant, respectively. the optimal extraction conditions were established based on regression analysis and plotting of three-dimensional (3d) response surface plots. results and discussion single-factor experiment of egcg extraction effect of temperature on the content of egcg extraction the effect of temperature on the egcg extraction yield using an extraction time of 30 min, a solution ph of 6, water-tea ratio of 30 ml/g and extraction once, is shown in fig. 1a. it demonstrates that the extraction temperature significantly affected the egcg extraction yield. the egcg yield increased significantly as the temperature increased from 50 to 70 °c. this could be explained by the fact that the rise in temperature can decrease solvent viscosity and surface tension, increase the solubility and diffusion rate of egcg (kong et al., 2010; vuong et al., 2010; vuong et al., 2011). how ever, beyond 70 °c, the egcg yield decreased, suggesting that the higher extraction temperatures induced the concurrent decomposition of the compounds. this result was in agreement with several previous studies (kong et al., 2010; vuong et al., 2011). therefore, a temperature of 70 °c was chosen as the central point for the rsm. tab. 3: box-behnken design of factors with experimental and predicted values. egcg content (mg/g) run x1temperature (°c) x2time (min) x3water-tea ratio(ml/g) experimental predicted 1 70 (0) 40 (0) 30 (0) 32.5 31.0 2 70 (0) 60 (+1) 10 (-1) 10.6 10.8 3 50 (-1) 20 (-1) 30 (0) 21.8 24.4 4 50 (-1) 60 (+1) 30 (0) 23.4 24.4 5 90 (+1) 40 (0) 50 (+1) 31.5 32.7 6 70 (0) 40 (0) 30 (0) 32.3 31.0 7 90 (+1) 60 (+1) 30 (0) 30.2 27.1 8 50 (-1) 40 (0) 10 (-1) 13.8 12.1 9 70 (0) 40 (0) 30 (0) 28 31.0 10 70 (0) 40 (0) 30 (0) 32.6 31.0 11 70 (0) 60 (-1) 10 (-1) 19.5 10.8 12 90 (+1) 40 (0) 10 (-1) 13.4 15.4 13 50 (-1) 40 (0) 50 (+1) 28 25.6 14 70 (0) 40 (0) 30 (0) 30.7 31.0 15 70 (0) 20 (-1) 50 (+1) 29.4 28.7 16 70 (0) 60 (+1) 50 (+1) 30.2 31.1 17 90 (+1) 20 (-1) 30 (0) 33.7 32.2 fig. 1: effect of extraction temperature (a), time (b), water-tea ratio (c), brewing solution ph (d), and extraction times (e) on the extraction yield of egcg 166 l. xiang, s. pan, x. lai, l. sun, z. li, q. li, y. huang, s. sun effect of time on the content of egcg extraction the egcg was extracted at extraction time varying from 10 to 50 min and a temperature of 70 °c, a solution ph of 6, water-tea ratio of 30 ml/g and extraction once. as shown in fig. 1b, a positive effect was caused by a longer extraction time. when extraction time changed from 10 min to 30 min, the egcg yield increased from 20.91 ± 1.30 to 23.42 ± 0.74 mg/g due to the increase in reactive sites (chen et al., 2017). however, beyond 30 min, the egcg yield decreased, suggesting that prolonging extraction time may expose the egcg to oxidative degradation (kong et al., 2010). thus, an optimal extraction time of 30 min was chosen as the central point for the rsm. effect of water-tea ratio on the content of egcg extraction the effect of water-tea ratio on egcg yield was investigated under fixed conditions (a temperature of 70 °c, an extraction time of 30 min, a solution ph of 6 and extraction once). fig. 1c exhibits that the yield of egcg significantly increased from 14.57 ± 0.71 to 23.17 ± 0.88 mg/g when the ratio rose from 10:1 to 30:1 ml/g because of the increasing contact surface area between tea powders and water, and the increase of the solid-liquid concentration gradient as well as the diffusion rate in the extraction (yang et al., 2017). however, further increase in the ratio did not significantly improve the extraction yield (p > 0.05), which is in agreement with several previous studies (felkai-haddache et al., 2016). thus, a water-tea ratio of 30:1 ml/g was selected as the central point for the rsm. effect of ph on the content of egcg extraction the egcg was extracted at solution ph varying from 4 to 8 and a time of 30 min, a temperature of 70 °c, water-tea ratio of 30 ml/g and extraction once. as shown in fig. 1d, ph level showed a positive effect on the egcg extraction ratio, which reached at 24.04 ± 0.71 mg/g at ph = 6 and then decreased rapidly. this is due to the fact that egcg was quite stable in acidic solutions but its stability decreased when the ph of the extraction solution rose from ph 4 to 8 (vuong et al., 2013). consequently, a solution ph of 6 was selected as the central point for the rsm. effect of extraction times on the content of egcg extraction the effect of the extraction times on egcg yield was investigated under fixed conditions (a temperature of 70 °c, an extraction time of 30 min, a solution ph of 6 and water-tea ratio of 30 ml/g). fig. 1e showed that the extraction ratio increased significantly with the increasing number of extraction times at first. however, the extraction ratio increased slightly with the increasing number of extraction times after extraction twice. this could be explained by the fact that most of egcg had been dissolved out in the first two extraction cycle. and this tendency was also in accordance with previous studies (yang et al., 2017). thus, the number of extraction times was fixed as twice in the rsm. optimization of egcg extraction by response surface designs according to the above results of single factor experiment, the three major parameters (extraction temperature, time and water-tea ratio) and their ranges for egcg extraction were determined and adopted for box-behnken design with response surface methodology with the consideration of cost, energy and solvent consumption. model fitting tab. 3 showed the design matrix and the experimental and predicted values of the response. according to the multiple regression analysis, the fitted second-order polynomial model for egcg in coded variables was shown in the following eq. (2). y = -35.2775 + 1.0123x1 + 0.2983x2 + 1.0528x3 0.0032x1x2 + 0.0024x1x3 + 0.0061x2 x3 0.0059x120.004x220.018x32 (2) where y represents the egcg extraction ratio, x1, x2 and x3 re present the experimental value of extraction temperature, time, and water-tea ratio, respectively. in order to determine whether the quadratic model is significant, it is necessary to run anova analysis. the significance of each coefficient was measured using f-values and p-values. the smaller the p-value and the greater the f-value, the more significant the corresponding coefficient (gan and latiff, 2011; hu et al., 2016). the regression coefficients of the linear, quadratic and interactive parameters are shown in tab. 4. lack of fit was also given in tab. 4 in order to confirm model fit. the p-value of the model was less than 0.001, which indicated that the fitting model is adequate to describe the experimental data. f-value for the lack of fit was insignificant (p > 0.05) imply that the model is valid. among all variables, the linear of x1 and x3, and the quadratic of x32 exhibited significant effects (p < 0.05), whereas the others had negligible effects on the egcg extraction ratio (p > 0.05). the linear variable of water-tea ratio had the highest influence on the egcg content to its p-value below 0.001, followed by the linear effect of extraction temperature. however, extraction time had no remarkable effect (p > 0.05). besides, the quadratic effect of water-tea ratio has the significant influence on the content of egcg (p < 0.001). it could be concluded that water-tea ratio played the most important role in the egcg extraction among three independent variables, followed by extraction temperature and extraction time. tab. 4: anova of the regression model for the prediction of mlp ex tractions. source sum df mean f value p-value of squares square prob > f model 851.82 9 94.65 12.85 p < 0.001 x1 59.41 1 59.41 8.07 p < 0.05 x2 12.5 1 12.5 1.7 p > 0.05 x3 477.4 1 477.4 64.82 p < 0.001 x1x2 6.5 1 6.5 0.88 p > 0.05 x1x3 3.8 1 3.8 0.52 p > 0.05 x2 x3 23.52 1 23.52 3.19 p > 0.05 x12 23.2 1 23.2 3.15 p > 0.05 x22 10.75 1 10.75 1.46 p > 0.05 x32 218.12 1 218.12 29.62 p < 0.001 residual 51.55 7 7.36 lack of fit 36.21 3 12.07 3.15 p > 0.05 pure error 15.35 4 3.84 total 903.37 16 analysis of response surface plot and contour plot the three-dimensional response surface plots and two-dimensional contour plots were applied to visualize the interactive effects of the independent variables on the egcg yield (fig. 2). the surface response plots of the model were created by changing two variables within their test range and keeping the other variable at their central level, which is the best way of showing the effect of the independent variable on the content of egcg (felkai-haddache et al., 2016; guo et al., 2016). the 3d response surface and 2d contour plots are the graphical representations of regression equation. through these plots, it is easy to judge interactions between two variables and deter egcg enrich brewing method of green tea 167 mine optimum ranges for independent variables (dahmoune et al., 2014; zhang et al., 2011). the variables of temperature/time, temperature/water-tea ratio, and time/water-tea ratio collaboratively worked in a nonlinear synergistic manner to the content of egcg (fig. 2a, 2b, and 2c). as it can be found from fig. 2a and fig. 2c, the response curve of extraction time was smooth. it indicated that the extraction time is less significant than other two effects. in fig. 2b and fig. 2c, water-tea ratio has the huge influence on the content of egcg. with the increase of water-tea ratio from 30 to 41 ml/g, the content of egcg increased to a definite value and subsequently maintained stable or decrease. the content of egcg was not found to be significantly affected by an enhancement of the extraction temperature at a fixed water-tea ratio and extraction time, shown in fig. 2a and fig. 2b. this suggested that the change in extraction temperature had an alternative influence on the content of egcg. model verification by applying the regression analysis to eqs. (1) and (2), the predic ted optimized conditions were concluded as follows: x1= 85.42 °c, x2 = 34.22 min, and x3 = 40.79 ml/g. based on eq. (2), the predicted fig. 2: three-dimensional response surfaces and contour plots for the effect of variables on the extraction yield of egcg maximum extraction yield of egcg was 34.54 mg/g. for convenient operation in actual experiments, the verification experiment was performed under predicted optimal conditions with slightly modification as follows: temperature of 85 °c, time of 34 min, water-tea ratio of 41 ml/g, a solution ph of 6 and extraction twice. under these conditions, the experimental extraction yield value of egcg was 33.82 mg/g, which was no significant difference to predicted value. therefore, the results revealed the accuracy and adequacy of the response model in the optimization of extraction process of egcg from the lower grade green tea (lim et al., 2017; yang et al., 2017). effect of brewing conditions on the content of individual cate chins the concentrations of eight main tea catechins in green tea extract made with two brewing methods were quantified by hplc (tab. 5). the hplc profile is shown in supplementary materials 1. compared with the ttbm group, the contents of all kinds of tested catechins in itbm group were much higher, especially for the contents of c, cg, gcg, egcg. additionally, the contents of gallated catechins and total catechins in itbm group were significantly higher than ttbm group (p < 0.001 and p < 0.05, respectively). 168 l. xiang, s. pan, x. lai, l. sun, z. li, q. li, y. huang, s. sun antitumor activity gastric cancer is one of the most serious malignant tumors, which is the leading cause of cancer-related death in most countries. casecontrol studies have suggested that green tea consumption has protective effect of against gastric cancer (xu et al., 2010; zhu et al., 2007). it is reported that egcg suppresses tumor growth by inhi biting proliferation and inducing apoptosis through a variety of me chanisms. in this study, we investigated cell growth inhibition effect of above green tea extract made with two different brewing methods on human gastric cancer sgc-7901 cells. cells were treated with 1.5, 2.5 and 3.5 mg/ml of green tea extract for 24 h and cell viability was determined using the mtt assay. as shown in fig. 3, green tea extract had significant growth inhibition effect on gastric cancer cells tab. 5: catechin contents of green tea extract brewing with ttbm and itbm (mg/l). catechins ttbm itbm gc 163.34±0.54 164.43±1.69 egc 256.06±6.05 256.31±4.81 c 28.58±0.83 36.11±0.76** ec 68.8±5.28 72.5±2.15 cg 4.15±0.67 13.68±0.79** ecg 1.59±0.16 3.14±0.12 gcg 41.93±3.46 52.66±2.87* egcg 281.98±16.80 340.07±13.77* gallated catechins 329.65±21.56 409.55±17.1** total catechins 846.43±25.63 938.92±25.95* ttbm, traditional tea brewing method. itbm, improved tea brewing method. values are presented as means ± sd (n = 3). * p < 0.05, ** p < 0.01 versus ttbm group. in a dose-dependent manner with a maximum loss at concentration of 3.5 mg/ml. moreover, the decrease of the cell viability exposed to green tea extract made with itbm was more significant than that of ttbm, which was consistent with the results of hplc analysis. this indicated that the antitumor activity of green tea extract was due to the cumulative effects of tea catechins (cooper et al., 2005). conclusions in the present study, box-behnken design based on the response surface methodology was successfully employed to optimize the egcg extraction from summer green tea. the optimal extraction conditions for egcg were: temperature of 85 °c, time of 34 min, water-tea ratio of 41 ml/g, a solution ph of 6, and extraction twice. water-tea ratio was also found to have the major influence on the yield of egcg. under these conditions, the experimental extraction yield value of egcg was 33.82 mg/g. furthermore, the summer green tea extract prepared under the optimal conditions had the higher antitumor activity against human gastric cancer sgc-7901 cells. the results indicated that the rich and valuable resource of summer green tea can be utilized much better by using the optimal extraction conditions (zhang et al., 2012). it is worth mentioning, however, that from the point of view of industrial application, future work need to be done to establish the pilot-plant trials of egcg extraction and to claim the mechanism of the healthy benefits of summer green tea benifits in human body or food industry (dahmoune et al., 2014). acknowledgements this work was financially supported by the fund for guangdong provincial key laboratory of tea plant resources innovation and utilization (no. 2008a060301004), the construction of industrial technology innovation platform for black tea of guangdong big leaves species, the earmarked fund for modern agro-industry technology research system (no. 2016lm2151), the science and technology planning project of guangdong province (no. 2016b090918118), and the president foundation of guangdong academy of agricultural sciences (grant no. 201534 and 201720). references castiglioni, s., 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sci. emerg. 12, 18-25. doi: 10.1016/j.ifset.2010.12.003 zhang, x., xu, f., gao, y., wu, j., sun, y., zeng, x., 2012: optimising the extraction of tea polyphenols, (-)-epigallocatechin gallate and theanine from summer green tea by using response surface methodo-logy. int. j. food sci. technol. 47, 2151-2157. doi: 10.1111/j.1365-2621.2012.03082.x zhao, z.y., huangfu, l.t., dong, l.l., liu, s.l., 2014: functional groups and antioxidant activities of polysaccharides from five categories of tea. ind. crop. prod. 58, 31-35. doi: 10.1016/j.indcrop.2014.04.004 zhu, b.-h., zhan, w.h., li, z.r., wang, z., he, y.l., peng, j.s., cai, s.r., ma, j.p., zhang, c.h., 2007: (-)-epigallocatechin-3-gallate inhibits growth of gastric cancer by reducing vegf production and angioge nesis. world j. gastroentero. 13, 1162-1169. doi: 10.3748/wjg.v13.i8.1162 170 l. xiang, s. pan, x. lai, l. sun, z. li, q. li, y. huang, s. sun address of the corresponding author: tea research institute, guangdong academy of agricultural sciences, dafeng road 6, tianhe district, guangzhou 510640, china e-mail: sunshili@tea.gdaas.cn (s. s.); tearesearch123@163.com (y. h.); liqiuhua@tea.gdaas.cn (q. l.) © the author(s) 2018. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 87, 296 (2014) buchbesprechung vitamin d – die heilkraft des sonnenvitamins uwe gröber und michael f. holick das seit der erstauflage im jahr 2012 jetzt in einer verbesserten und korrigierten 2. auflage vorliegende buch beschreibt die wichtigsten wissenschaftlichen forschungsergebnisse zum thema vitamin d in einer allgemein verständlichen form. anhand ausgewählter fallbeispiele wird dabei immer wieder der hohe therapeutische stellenwert, der von den autoren als „sonnenvitamin“ bezeichneten verbindung, hervorgehoben. in diesem zusammenhang werden dem leser zahlreiche empfehlungen für eine optimale versorgung, die sich aus aktuellen erkenntnissen der vitamin-d-forschung ableiten, präsentiert. die autoren gehen davon aus, dass in deutschland bis zu 90 % der bevölkerung in allen altersklassen nicht ausreichend mit vitamin d versorgt sind. sie stützen ihre hypothese insbesondere auf die beobachtung, dass gesundheitsbewusste menschen die sonne aufgrund eines potentiellen hautkrebsrisikos im zunehmenden maße meiden. da vitamin d jedoch in der haut aus cholesterin nur in gegenwart von sonnenlicht (uv-b-strahlung) gebildet werden kann, sehen die autoren hierdurch einen risikofaktor für mehrere zivilisationskrankheiten wie brustkrebs, herzinfarkt, schlaganfall, diabetes mellitus, multiple sklerose oder morbus crohn. in den insgesamt acht kapiteln gehen die autoren u.a. auf die biosynthese von vitamin d, auf effektive präventionsmaßnahmen sowie auf unerwünschte wechselwirkungen mit verschiedenen medikamenten, die einen vitamin d-mangel auslösen können (wie z.b. antiepileptika, glucocorticoide, krebsmedikamente und johanniskraut) näher ein. auch auf die positiven auswirkungen einer kontrollierten vitamin d-supplementierung bei bluthochdruck und tuberkulose wird hingewiesen. schließlich werden noch die einzelnen vitamin d-formen vorgestellt und empfehlungen für einen optimalen 25-(oh)-vitamin d-spiegel im blutserum (40-60 ng/ml) abgegeben. das buch fasst die wichtigsten erkenntnisse aus den wissenschaftlichen studien der vergangenen 10 jahren in verständlicher form zusammen und kann daher als spannende und gleichzeitig lehr reiche lektüre einer breiten leserschicht uneingeschränkt empfohlen werden. es liefert nicht nur ernährungswissenschaftlern und medizinern, sondern auch medizinischen laien eine ausgezeichnete möglichkeit, umfassende informationen über den aktuellen kenntnisstand zum thema vitamin d zu erhalten. im anhang sind die zitate der wichtigsten wissenschaftlichen fachartikel, auf die sich die autoren beziehen, aufgeführt, sodass es den lesern möglich ist, in einzelfällen mehr hintergrundinformationen zu den jeweiligen forschungsergebnissen zu erfahren. darüber hinaus sind in der anlage mögliche wechselwirkungen mit arzneimittel-therapien bei unterschiedlichen erkrankungen tabellarisch aufgelistet. dr. h. schulz e-mail: hartwig.schulz@jki.bund.de bibliografie: uwe gröber und michael f. holick, vitamin d – die heilkraft des sonnenvitamins, wissenschaftliche verlagsgesellschaft mbh, stuttgart, 2. durchgesehene und korrigierte auflage, 302 seiten mit 155 farbigen abbildungen und 17 tabellen, preis: 39,80 €, isbn: 9783-8047-3177-6 journal of applied botany and food quality 91, 47 55 (2018), doi:10.5073/jabfq.2018.091.007 1department of food and nutrition, suwon women’s university, gyeonggi, republic of korea 2food analysis research center, suwon women’s university, gyeonggi, republic of korea 3department of microbiology and molecular biology, chungnam national university, daejeon, republic of korea nutritional compositions in roots, twigs, leaves, fruit pulp, and seeds from pawpaw (asimina triloba [l.] dunal) grown in korea jin-sik nam1,2, 3, hye-lim jang2*, young ha rhee3* (submitted: august 6, 2017; accepted: february 11, 2018) * corresponding author summary pawpaw (asimina triloba l.) roots, twigs, leaves, fruit, and seeds were analyzed for their nutritional compositions. seeds exhibited significantly higher levels of crude protein, lipid, fiber, and dietary fiber than those of the other parts. sucrose in fruit was 9321.24 mg%, which was the highest among the samples. the total essential amino acid to total amino acid ratio was highest in the leaves, and the leaves contained the highest amount of potassium. the calcium content ranged between 8.15-153.41 mg%. oleic and linoleic acids in seeds were 5905.11 and 8045.56 mg%, respectively, which were the highest among the pawpaw parts. the highest amount of linolenic acid was measured in the leaves, and β-carotene, vitamin c, and vitamin e were also the most abundant in the leaves. these results suggest that every part of pawpaw is a good source of an important food item. additionally, this study provides basic data for improving the sitological value of pawpaw. abbreviations: aoac, association of analytical chemists; hplc, high performance liquid chromatography; gaba, γ-aminobutyric acid; icp, inductively coupled plasma spectrometer; fid, flame ionization detector; aa, amino acid; eaa, essential amino acid; naa, nonessential amino acid; ufa, unsaturated fatty acid; sfa, saturated fatty acid. introduction asimina triloba [l.] dunal (family: annonaceae) is one species of asimina, usually known as pawpaw. it is widely distributed from the southeastern areas of united states (florida) to the eastern areas of canada (ontario). although pawpaw is sometimes confused with papaya (carica papaya), it is an entirely different species (levine et al., 2015). papaya is a tropical plant grown in tropical regions, but pawpaw can grow well in tropical regions as well as in humid microthermal climates. in korea, pawpaw trees have approximately been cultivated since 2012 to the present day, and areas for cultivation have been on the rise as use of pawpaw as a food has increased. almost all parts of pawpaw trees are attracting considerable attention for their economic value due to their broad use across diverse areas such as nutritional and medicinal fields. in terms of its medicinal properties, pawpaw roots, twigs, and seeds contain a large amount of acetogenins, which are cancer cell inhibitors (mclaughlin, 2008). indeed, the extracts obtained from pawpaw twigs are used in a commercial product for the prevention of cancer (i.e., paw paw cell-reg). in addition, many researchers have studied the isolation and identification of acetogenins from pawpaw twig extracts (gu et al., 1999; mclaughlin, 2008). mccage et al. (2002) demonstrated that herbal shampoo products that included pawpaw twig extracts could effectively remove head lice. further, essential oil generated from pawpaw leaves has exhibited anticancer activity against breast and lung cancer cell lines (farag, 2009). moreover, pawpaw leaves are rich in various bioactive compounds such as phenolic acids and flavonoids (pande and akoh, 2010). accordingly, various uses of pawpaw leaves have been employed, including infusing herbs in water. pawpaw fruit has a sweet and sour taste like a mixture of pineapple, banana, and mango, and is popular ly used as an ingredient for jam, wine, ice cream, and puree (pomper and layne, 2005). it is also a good source of amino acids, minerals, and vitamins (templeton et al., 2003). brannan et al. (2015) reported that pawpaw fruit contains a large amount of procyanidins, which have antioxidant effects, and kobayashi et al. (2008) demonstrated that pawpaw fruit exhibits antioxidant activity. despite the importance of pawpaw as a nutritional and medicinal material, relatively little attention has been given to the nutritional composition of the roots, twigs, leaves, fruit, and seeds of the a. triloba variety of pawpaw grown in korea. to this end, the objective of the present study was to investigate and compare the nutritional compositions of different parts of pawpaw cultivated in korea. on the basis of these results, we seek to provide basic data for improving the sitological value of pawpaw and promote further studies on the nutritional compositions of other plants and their various components. materials and methods samples and reagents pawpaw trees (a. triloba) grown in identical conditions were purchased from a farm situated on the edge of okchon, south korea in september 2016. the climate of okchon is as follows: an average annual temperature of 13.0°c, an average relative humidity of 66.7%, wind velocity of 1.9 m/s, and total annual rainfall of 1458.7 mm. roots, twigs, and leaves were collected from 140-150 pawpaw trees aged 2 years old (height, 1-1.5 m). the fruit (length, 8-12 cm; weight, 150-300 g) was obtained from 25-30 pawpaw trees aged 8 10 years old. the roots, twigs, leaves, and fruit were washed dozens of times with water until all extraneous substances were removed. the samples were then drained and the fruits were peeled; seeds were then collected from the pawpaw fruit. all samples were cut into pieces and chopped using a chopper (tc-8b; techon co., ltd., bucheon, korea) and stored at -70 °c until further use. free sugar standards (fructose, glucose, sucrose, maltose, and lactose), organic acid standards (malic acid, citric acid, oxalic acid, acetic acid, formic acid, tartaric acid, and succinic acid), an amino acid standard solution, fatty acid methyl ester, β-carotene, vitamins b1, b2, b3, b5, b6, and b12, ascorbic acid, and α-, β-, γ-, and δ-tocopherol were purchased from sigma chemical co. (st. louis, mo, usa). mineral standards were obtained from accustandard (new haven, ct, usa). determination of proximate composition and dietary fiber content the proximate composition of the samples including moisture, crude ash, crude protein, crude lipid, and crude fiber was analyzed using 48 j.-s. nam, h.-l. jang, y.h. rhee procedures from the association of official analytical chemists (aoac, 2005). the moisture content was analyzed using a dry oven (of-22; jeio tech, daejeon, korea) at 105 °c, and the crude ash content was analyzed with a muffle furnace (jsmf-140t; jsr inc. laboratory, north ringwood, victoria, australia) at 550 °c until a constant weight was attained. the crude protein content was determined by the kjeldahl method using a kjeltec® 2300 analyzer unit (foss tecator ab, höganäs, sweden). the crude lipid content was measured by ether extraction with a soxtec system (soxtec 1043; foss tecator ab, höganäs, sweden), and the crude fiber content was determined using an automatic fiber extractor (fiwe 6; velp scientifica, usmate, italy). the carbohydrate content was calculated as the weight of the entire sample less the moisture, crude ash, crude protein, and crude lipid. determination of the dietary fiber content was conducted by an enzymatic-gravimetric method using a dietary fiber extraction instrument (1023 filtration module; foss tecator co., hillerød, denmark). determination of free sugar and organic acid contents the free sugar and organic acid contents were determined using high-performance liquid chromatography (agilent 1100 series; agi lent technologies, palo alto, ca, usa). five grams of samples were blended with 10 times its weight of distilled water (w/v) and centrifuged at 8,000 rpm for 20 min (supra-21k; hanil, incheon, korea). the supernatant was transferred into a 50 ml volumetric flask and filtered through a 0.45 μm membrane filter (millipore, bedford, ma, usa). the filtrates were then used to analyze the free sugar and organic acid contents. to analyze the free sugars, 10 μl of each sample was injected into the hplc system equipped with a carbohydrate analysis column (4.6 × 250 mm, waters co., usa). the free sugars were separated using an isocratic elution at a flow rate of 1.0 ml/min and a mobile phase of 80% acetonitrile. the elution peaks were detected using a refractive index (ri) detector. to analyze the organic acids, aliquots (20 μl) of each sample were injected into the hplc system equipped with shodex rs pak kc-811 column (8.0 × 300 mm; shodex, tokyo, japan). the organic acids were separated using an isocratic elution at a flow rate of 1.0 ml/min and a mobile phase of 0.1% h3po4. the elution peaks were detected using an ultraviolet (uv) detector at 210 nm. determination of amino acid content the amino acid contents were determined using an amino acid analyzer (biochrom 30; pharmacia biotech, stockholm, sweden). samples for analysis of 18 amino acids were hydrolyzed with 6 n hcl at 110 °c for 22 h in test tubes filled with nitrogen. the hy drolysate was concentrated on a rotary evaporator (r-210; buchi, flawil, switzerland) and diluted with 50 ml of distilled water in cluding 10 ml of dilution buffer. samples for analysis of taurine and γ-aminobutyric acid (gaba) were prepared identically by the same pre-treatment method used for the free sugar and organic acid analysis. each sample was filtered through a 0.45 μm membrane filter (millipore) and loaded onto a cation-exchange column (11 ± 2 μm). the flow of the ninhydrin reagent (ph 3.20-6.45) was set to 25 ml/h, and the sample injection volume was 20 μl. determination of mineral content each sample was dissolved in a hno3/h2o2 mixture (9:1) and digested for 30 min using a microwave digestion system (ethos tc digestion lab station 5000; milestone inc., monroe, ct, usa). after digestion, the solution from each sample was passed through a filter paper (no. 5a; whatman international ltd., maidstone, uk). an inductively coupled plasma (icp) spectrometer (perkin elmer co., shelton, ct, usa) was used to measure the contents of seven elements (ca, cu, fe, k, mg, na, and zn). the operating conditions of the icp instrument were as follows: 1.4 kw reflected power, 10 l/ min argon plasma gas flow rate, 0.2 l/min auxiliary gas flow rate, and 0.55 l/min nebulizer gas flow rate. selenium was determined by icp-mass spectrometry; these analysis conditions were as follows: 1.4 kw reflected power, 9.6 v lens voltage, 18 l/min plasma flow, 1.5 l/min auxiliary gas flow rate, and 0.92 l/min nebulizer gas flow rate. determination of fatty acid content and composition fatty acids were measured by the method of jang et al. (2016) with some modification. ten grams of each sample were extracted with ether, and the extracted fat (25 mg) was dissolved in 0.5 n naohmethanol (2 ml) and converted to its fatty acid methyl esters using 14% bf3-methanol (2 ml). the fatty acid contents were deter mined by gas chromatography (agilent 6890n/5975 msd series; avondale, pa, usa) coupled with an sptm 2560 column (100 m × 0.25 mm; supelco inc., bellefonte, pa, usa). the column oven temperature was programmed as follows: initial temperature was 170 °c (held for 15 min); increased to 180 °c at 1 °c/min (held for 15 min); further increased to 245 °c at 3 °c/min with a final holding time of 13 min. the injector and detector (fid, flame ionization detector) temperatures were 250 °c and 285 °c, respectively. helium was used as a carrier gas at a flow rate of 0.75 ml/min. the fatty acid contents were calculated using a fid conversion factor. determination of β-carotene content β-carotene was determined by the aoac method (2005). samples (1 g) were dissolved in 30 ml of ethanol and 1 ml of 10% of pyrogallol. then, 3 ml of koh was added to the sample solution and the mixture was boiled under reflux for 30 min. the sample was allowed to cool to 20 °c, and 30 ml of distilled water were added in order to collect the ether layer. the ether layer was then dehydrated by na2so4 and evaporated. n-hexane (20 ml) was added to the dehydrated product, which was used as the test solution. the test solution was injected into an agilent 1100 series hplc system coupled with a nova-pak silica column (3.9 × 150 mm; waters co., milford, ma, usa). the mobile phase consisted of n-hexane and isopropyl alcohol (99:1, v/v) at a flow rate of 1.0 ml/min with an injection volume of 20 μl. the β-carotene content was expressed as μg per 100 g of fresh weight. determination of vitamin b complex content each sample (1 g) was weighed and extracted by 20 ml of a 75 mm ammonium formate solution (ph 7.0) for 1 h. the extracts were centrifuged at 3,000 rpm for 15 min, and the supernatant was fil tered through a 0.45 μm membrane filter (millipore). the filtrate was injected into an hplc-tandem mass spectrometer (agilent 1200 series; agilent technologies, santa clara, ca, usa) equipped with a luna c18 column (3.0 × 150 mm; phenomenex, torrance, ca, usa). the mobile phase a was 5 mm ammonium formate in distilled water, and mobile phase b was 5 mm ammonium formate in methanol. the gradient system was programmed as follows: 0 min (10% b); 0-8 min (45% b); 8-15 min (10% b). the flow rate was 0.3 ml/ min, and the temperature was 35 °c. the content of vitamin b was expressed as μg per 100 g of fresh weight. determination of vitamin c content the sample (5 g) was extracted adding 5% meta-phosphoric acid (50 ml) for 40 min and filtered through a 0.45 μm membrane filter (millipore). the filtrate was injected into an agilent 1200 series hplc system equipped with a shiseido capcell pak c18 column (4.6 × 250 mm; shiseido, tokyo, japan). the mobile phase was 0.05 m nutritional composition of different organs from pawpaw (asimina triloba) 49 kh2po4 and acetonitrile (99:1, v/v), and the flow rate was 0.9 ml/ min. the eluate was detected using a uv detector at 254 nm. the content of vitamin c was expressed as mg per 100 g of fresh weight. determination of vitamin e content sample preparation for vitamin e determination was performed in the same way as that for β-carotene. the samples were analyzed using an agilent 1100 series hplc system equipped with a novapak silica column (waters co.). the mobile phase was n-hexane and isopropyl alcohol (99:1, v/v), and the flow rate was 0.5 ml/min. the content of vitamin e was expressed as mg per 100 g of fresh weight. statistical analysis all experiments were performed in triplicate, and the results were analyzed using the statistical package for social sciences version 10.0 (spss inc., chicago, il, usa). for multiple comparisons of normally distributed data, parametric one-way analysis of variance was used. the significance of differences between data was calculated by duncan’s multiple range test, with p<0.05 being considered as statistically significant. results and discussion proximate composition and dietary fiber the proximate composition and dietary fiber content of different parts of pawpaw tree (a. triloba) differed significantly (p<0.05, tab. 1). the moisture content these various parts ranged from 28.48% (seeds) to 84.98% (roots). the highest value of crude ash was observed in the leaves, which was significantly different from the other pawpaw tree parts (p<0.05). pawpaw seeds exhibited significantly higher levels of crude protein, crude lipid, crude fiber, carbohydrate, and dietary fiber than those of the roots, twigs, leaves, and fruit (p<0.05). in particular, the dietary fiber content of seeds (46.77%) was more than 15 times the dietary fiber of fruit (3.03%). fruit had significantly lower levels of crude ash, crude protein, crude lipid, crude fiber, and dietary fiber than those of the other parts (p<0.05). this result is consistent with that of a previous study of papaya (carica papaya), in which the fruit pulp showed a low crude protein compared to the leaves and seeds (nwofia et al., 2012). however, many reports proposed that all the proximate composition and dietary fiber content may differ according to several factors such as growing season, cultivars, and climatic conditions (jang et al., 2011; nwofia et al., 2012; rohloff et al., 2015). therefore, the present study suggests that the proximate composition and dietary fiber content of pawpaw tree can depend on their different parts, as well as these other factors. free sugars the free sugar content in various parts of pawpaw tree is given in tab. 2. fructose, glucose, and sucrose showed the highest levels in the fruit at 1691.35 mg%, 2148.20 mg%, and 9321.24 mg%, respectively. these free sugars in the fruit had levels higher than at least twice and up to 16 times the free sugars contained in the roots, twigs, leaves, and seeds. in particular, the sucrose content in the fruit was the highest among the free sugars, which was ~9% of the fruit weight. maltose and lactose were not detected in any part of pawpaw tree. fructose, glucose, and sucrose were observed as the main free sugars of pawpaw, and their amounts differed between parts. components (% fresh weight) tab. 1: proximate composition and dietary fiber content in various parts of asimina triloba. roots twigs leaves fruit seeds moisture 84.98±0.11a 61.86±0.38d 75.47±0.04c 79.14±0.03b 28.48±0.11e crude ash 1.24±0.02b 0.90±0.04d 1.51±0.02a 0.38±0.01e 1.01±0.02c crude protein 1.97±0.02d 2.20±0.08c 5.18±0.01b 1.51±0.14e 9.18±0.04a crude lipid 0.75±0.02d 0.93±0.02c 1.38±0.01b 0.36±0.02e 16.68±0.09a crude fiber 6.37±0.02c 23.45±0.35b 5.77±0.04d 2.47±0.03e 38.63±0.06a carbohydrate 11.05±0.14e 34.12±0.25b 16.47±0.04d 18.61±0.11c 44.66±0.21a dietary fiber 10.06±0.23d 30.80±0.26b 11.81±0.30c 3.03±0.12e 46.77±0.77a results are presented as mean±sd (n = 3). means with different letters in the same row are significantly different by duncan’s multiple range test (p< 0.05). components (mg% fresh weight) tab. 2: free sugar content in various parts of asimina triloba. roots twigs leaves fruit seeds fructose 193.20±12.70d 331.68±36.53c 740.75±79.77b 1691.35±29.66a 238.58±29.27d glucose 207.45±27.79d 430.34±76.84c 660.33±120.06b 2148.20±74.14a 131.76±20.66d sucrose n.d. n.d. n.d. 9321.24±122.38a 1067.61±47.91b maltose n.d. n.d. n.d. n.d. n.d. lactose n.d. n.d. n.d. n.d. n.d. results are presented as mean±sd (n = 3). means with different letters in the same row are significantly different by duncan’s multiple range test (p< 0.05). n.d. = not detected. 50 j.-s. nam, h.-l. jang, y.h. rhee sweetness is closely related to free sugar, and its intensity depends on the free sugar composition. lee et al. (2013) showed that the free sugar content of tropical fruit was consistent with the trend of °brix. in addition, fructose has a sweetness ~70% higher than sucrose, and the sweetness of glucose is only ~50% of the sweetness of sucrose (moskowitz, 1970). according to anderson (1986), the free sugar content depends on climate conditions including temperature, rainfall, relative humidity, and the amount of light. increases of sucrose and sucrose synthases are related to metabolic adaptations by low temperature, and water stress was been shown to increase the free sugar level in leaves (ackerson, 1981; guy et al., 1992). therefore, it is expected that the free sugar content and composition of pawpaw tree may differ according to the cultivation environment and that the intensity of sweetness by the free sugars and their composition will vary between pawpaw constituents. organic acids malic acid and citric acid were the most abundant organic acids in all samples, with their highest levels measured in pawpaw roots. however, oxalic acid, acetic acid, formic acid, tartaric acid, and succinic acid were not detected in the roots (tab. 3). the twigs and leaves only contained malic acid, citric acid, and formic acid. the malic acid and citric acid levels in the twigs were higher than those in the leaves; however, the formic acid content in the twigs was lower level than that in the leaves. fruit contained various types of organic acids such as malic acid, citric acid, oxalic acid, acetic acid, and formic acid, and their content was 30.62, 8.65, 37.17, 61.59, and 43.29 mg%, respectively. the major organic acids identified in the seeds were malic acid, citric acid, and oxalic acid, and, in particular, citric acid, which was the most abundant organic acid measured at 66.27 mg%. organic acids including malic acid, citric acid, oxalic acid, and succinic acid play an important role in the tricarboxylic acid cycle, which is the metabolic pathway to produce energy viz. oxidation of pyruvate in the mitochondria of animals and plants (yang et al., 2014). malic acid participates in photosynthesis, and citric acid and oxalic acid have been reported to be involved in diverse metabolic pathways such as the transportation of cations, detoxification of metal substances, and resistance of anaerobic stress in roots (mucha et al., 2005). in addition, organic acids found in the metabolites of plants or various plant products are important indicators of biological and physiological metabolism and fermentation processes (chung et al., 2000). organic acids have also been widely used as food additives in the production of fruit and vegetable beverages, drinks, and juices (pereira et al., 2013). finally, the addition of these acids to food imparts distinct sensory characteristics such as color, flavor, and aroma and affects their tissue and biological stability (chung et al., 2000). therefore, it is likely that each part of pawpaw trees can be effectively utilized on account of their distinct organic acid content and composition. amino acids the amino acid contents in pawpaw tree are displayed in tab. 4. the contents of the total amino acid (aa), total essential amino acid (eaa), total nonessential amino acid (naa), and total extra amino acid of the seeds were significantly higher than those of the roots, twigs, leaves, and fruit (p<0.05). however, the eaa ratio of seeds to aa was the third largest (leaves > twigs > seeds > fruit > roots). the contents of aa, eaa, naa, total extra amino acid, and the ratio of fruit to aa were conspicuously lower than those of the other parts (p<0.05), which is consistent with previous studies that found that most of the amino acids contained in the seeds of okra (hibiscus esculentus) and marula (sclerocarya birrea) had higher levels than those in the fruit (glew et al., 1997). most of the amino acids were identified evenly in all samples, but taurine was not detected in all parts of pawpaw. the twigs, leaves, and seeds contained methionine, but the roots and fruit did not. tryptophan and arginine were not detected in the fruit, and proline was not detected in the twigs and leaves. in addition, cysteine was observed only in the seeds. the main amino acids detected in the pawpaw roots and fruit were proline, accounting for more than 38% and 25% of the entire amino acid profile, respectively. aspartic acid, glutamic acid, and histidine were the major amino acids detected in the twigs, and leucine, aspartic acid, and glutamic acid were main amino acids found in the leaves. aspartic acid, glutamic acid, and arginine were the major amino acids measured in seeds; glutamic acid had the highest level at 1396.27 mg%. overall, it is clear that the amino acid content and composition in pawpaw tree vary according to the parts of the tree. however, the most prolific amino acids of pawpaw, except in fruit, were aspartic acid and glutamic acid, the contents of which closely matched in the different tree sections. aspartic acid and glutamic acid have an umami taste, and their mixture creates a tremendous synergic effect (dermiki et al., 2013). proline was observed as the most plentiful amino acid in roots and fruit. this amino acid is closely related to radical scavenging activity; previous in vitro stu dies found that it contributed to free radical scavenging (kaul et al., 2008). therefore, these results suggest that the distinct components of pawpaw tree have various nutritional and functional properties. components (mg% fresh weight) tab. 3: organic acid content in various parts of asimina triloba. roots twigs leaves fruit seeds malic acid 297.09±3.67a 159.79±0.87b 74.42±0.98c 30.62±1.92d 31.25±0.55d citric acid 252.03±2.74a 113.53±0.45b 48.91±1.88d 8.65±0.23e 66.27±0.82c oxalic acid n.d. n.d. n.d. 37.17±0.34b 56.73±0.40a acetic acid n.d. n.d. n.d. 61.59±0.92 n.d. formic acid n.d. 41.48±0.67b 201.80±1.55a 43.29±1.59b n.d. tartaric acid n.d. n.d. n.d. n.d. n.d. succinic acid n.d. n.d. n.d. n.d. n.d. results are presented as mean±sd (n = 3). means with different letters in the same row are significantly different by duncan’s multiple range test (p< 0.05). n.d. = not detected. nutritional composition of different organs from pawpaw (asimina triloba) 51 minerals tab. 5 presents the mineral content of pawpaw tree according to its different parts. potassium was identified as having the highest level in all tissue; its content in roots, twigs, leaves, fruit, and seeds was measured as 215.21, 193.98, 376.93, 239.36, and 289.17 mg%, respectively. the pawpaw leaves contained the highest amount of potassium, which contrasts the reported potassium content (128.33 mg/ 100 g) in celosia argentea l. leaves (usunomena and samuel, 2016). typically, minerals such as magnesium, sodium, and zinc were detected at a significantly higher level in roots than in the other pawpaw parts (p<0.05), and the mineral content (except potassium) were remarkably lower in fruit than in the other parts (p<0.05). the calcium content ranged from 8.15 mg% to 153.41 mg% and exhi bited the lowest and highest levels in the fruit and twigs, respective ly. the calcium content of the pawpaw leaves was also high at 141.47 mg%, which is higher than the calcium content (123.40 mg/ 100 g) reported in kale (agarwal et al., 2017). copper was only observed in pawpaw seeds, and there was little or no selenium detected in any of the samples. plant roots not only provide support to a plant, but also play an important role in water and mineral transport. some minerals absorbed from roots are transported to the twigs or leaves, whereas others are stored in the roots. this transport and storage of minerals in plants are known to differ according to the growth season (mitchell, 1936). accordingly, it is thought that the distribution of minerals in pawpaw tissues may depend on factors like these. fatty acids the unsaturated fatty acid (ufa) content was higher than the saturated fatty acid (sfa) content in all samples, and the difference between ufa and sfa was more pronounced in pawpaw seeds. in contrast, the difference between the two contents in fruit was negligible (tab. 6). significant differences were observed for the ratio of total ufa to total sfa in different pawpaw tissues, with the highest ratio found in seeds and the lowest in twigs (p<0.05). various sfas such as caproic (c6:0), caprylic (c8:0), capric (c10:0), and lauric (c12:0) acids were only identified in fruit, which is consistent with a previous report for tropical fruit pulps (coimbra and jorge, 2012). the primary fatty acids included palmitic acid (c16:0), stearic acid (c18:0), oleic acid (c18:1), and linoleic acid (c18:2, n-6), which were significantly higher in the seeds (p<0.05). in particular, the contents of oleic and linoleic acids contained in the seeds were 5905.11 and essential amino acid nonessential amino acid extra amino acid tab. 4: amino acid content in various parts of asimina triloba. amino acids (mg% fresh weight) roots twigs leaves fruit seeds threonine 46.14±1.23cd 61.43±6.27c 170.05±8.96b 23.86±2.62d 278.95±28.69a valine 34.84±5.27c 45.10±4.61c 142.60±3.90b 24.09±2.97c 288.97±27.94a methionine n.d. 22.00±9.00c 81.06±7.93a n.d. 70.85±3.57b isoleucine 23.17±3.23c 29.24±3.65c 98.89±11.53b 12.69±0.23c 208.20±19.02a leucine 70.61±5.44c 85.57±5.10c 319.13±18.98b 37.63±2.99d 618.03±26.85a phenylalanine 44.56±1.00c 54.05±3.19c 186.43±11.18b 27.74±9.09c 269.22±31.32a lysine 58.49±7.22c 76.29±4.22c 224.15±19.50b 29.69±2.86c 377.95±53.90a tryptophan 12.81±0.06d 21.24±1.43c 54.82±2.24b n.d. 81.28±4.06a total essential amino acid 290.61±14.24cd 394.92±15.39c 1277.14±50.19b 155.71±18.59d 2193.45±180.37a aspartic acid 108.60±5.61c 129.84±9.32c 377.76±35.85b 47.31±2.85d 916.53±14.65a serine 78.90±3.25c 86.72±6.19c 219.74±19.80b 35.01±2.75d 453.11±48.66a glutamic acid 117.83±4.56c 113.99±9.26c 391.55±29.92b 67.68±4.17c 1396.27±79.28a proline 531.95±10.98b n.d. n.d. 163.25±13.14c 553.79±19.15a glycine 54.44±3.55c 66.45±4.59c 213.49±17.31b 29.08±1.59c 463.64±54.54a alanine 52.68±0.40c 67.23±4.50c 223.80±18.37b 67.14±4.96c 429.05±6.62a cystine n.d. n.d. n.d. n.d. 50.63±5.15 tyrosine 25.05±1.31c 34.61±8.35c 126.52±6.44b 16.34±0.97c 252.18±30.65a histidine 93.47±15.84d 116.89±3.12c 165.65±10.24b 44.03±5.69e 236.86±20.16a arginine 30.42±5.91c 46.96±4.48cd 214.67±18.80b n.d. 754.99±35.72a total nonessential amino acid 1093.36±11.04c 662.68±49.61d 1933.20±153.62b 469.83±16.95d 5507.05±290.43a taurine n.d. n.d. n.d. n.d. n.d. gaba 7.78±0.12e 25.54±0.44c 39.61±0.71b 9.85±0.18d 41.90±0.46a total extra amino acid 7.78±0.12e 25.54±0.44c 39.61±0.71b 9.85±0.18d 41.90±0.46a total amino acid 1391.76±25.38c 1083.15±64.81c 3249.95±203.90b 635.38±28.79d 7742.41±470.12a total eaa /total aa (%) 20.87±0.64e 36.49±0.84b 39.33±0.90a 24.46±2.13d 28.31±0.64c results are presented as mean±sd (n = 3). means with different letters in the same row are significantly different by duncan’s multiple range test (p< 0.05). n.d. = not detected. gaba = γ-aminobutyric acid; eaa = essential amino acid; aa = amino acid. 52 j.-s. nam, h.-l. jang, y.h. rhee tab. 5: mineral content in various parts of asimina triloba. roots twigs leaves fruit seeds ca 99.74±2.12c 153.41±11.25a 141.47±2.66b 8.15±0.29e 57.51±1.09d cu n.d. n.d. n.d. n.d. 0.57±0.08 fe 2.77±0.65c 5.16±0.20b 6.25±0.22a 0.29±0.02e 2.13±0.12d k 215.21±1.73d 193.98±5.75e 376.93±6.36a 239.36±3.00c 289.17±5.74b mg 140.24±0.78a 87.55±4.59c 97.69±1.14b 10.93±0.18d 93.77±2.07b na 49.79±0.47a 8.52±0.47b 0.92±0.06c n.d. n.d. zn 1.28±0.07a 0.61±0.02c 0.48±0.03d n.d. 0.98±0.04b se tr tr tr n.d. n.d. results are presented as mean±sd (n = 3). means with different letters in the same row are significantly different by duncan’s multiple range test (p< 0.05). n.d. = not detected. tr = trace. minerals (mg% fresh weight) tab. 6: fatty acids component in various parts of asimina triloba. roots twigs leaves fruit seeds c6:0 n.d. n.d. n.d. 1.61±0.19 n.d. c8:0 n.d. n.d. n.d. 9.73±0.51 n.d. c10:0 n.d. n.d. n.d. 1.43±0.15 n.d. c12:0 n.d. n.d. n.d. 1.53±0.02 n.d. c13:0 n.d. n.d. 3.00±0.00 n.d. n.d. c14:0 n.d. n.d. n.d. 6.00±0.24a 4.70±0.06b c15:0 n.d. 4.00±0.00b n.d. n.d. 5.36±0.08a c16:0 70.33±0.58d 89.33±0.58c 158.00±1.00b 25.36±0.84e 917.11±4.61a c16:1 n.d. 4.00±0.00c 4.00±0.00c 6.51±0.08b 137.85±1.28a c17:0 n.d. 3.00±0.00b 2.00±0.00c n.d. 11.16±0.33a c18:0 7.00±0.00d 10.00±0.00c 19.67±0.58b 4.79±0.27e 373.13±2.33a c18:1 46.00±1.00b 92.33±0.58b 57.33±9.45b 48.02±1.83b 5905.11±54.30a c18:2, n-6 140.33±0.58b n.d. 143.00±1.73b 9.79±0.37c 8045.56±76.80a c20:0 2.67±0.58c 3.00±0.00c 5.00±0.00b n.d. 31.37±0.37a c18:3, n-3 19.33±0.58d 73.33±0.58c 401.33±5.69a 18.59±0.65d 233.63±1.92b c20:1 2.00±0.00c n.d. 2.67±0.58b n.d. 48.55±0.46a c22:0 n.d. 4.00±0.00b n.d. 0.85±0.06c 7.03±0.10a c20:3, n-3 3.00±0.00n.s. n.d. 3.00±0.00 n.d. n.d. c22:1, n-9 n.d. 3.33±0.58 n.d. n.d. n.d. c20:4, n-6 n.d. n.d. 2.33±0.58 n.d. n.d. c24:0 n.d. 5.00±0.00b 9.33±0.58a 0.68±0.04c 4.69±0.06b c24:1 n.d. 3.00±0.00 n.d. n.d. n.d. total sfa 80.00±0.00d 118.33±0.58c 197.00±1.73b 51.98±1.93e 1354.56±6.65a total ufa 212.67±1.53c 176.00±1.73cd 613.69±4.16b 82.90±2.88d 14375.96±134.77a total ufa/total sfa 2.63±0.02c 1.49±0.01e 3.12±0.01b 1.59±0.00d 10.61±0.08a results are presented as mean±sd (n = 3). means with different letters in the same row are significantly different by duncan’s multiple range test (p< 0.05). n.d. = not detected. n.s. = not significant. sfa = saturated fatty acid; ufa = unsaturated fatty acid. fatty acids (mg% fresh weight) nutritional composition of different organs from pawpaw (asimina triloba) 53 8045.56 mg%, respectively, which was more than 120 times and 820 times the oleic and linoleic acids contained in the fruit, respectively. the highest linolenic acid (c18:3, n-3) content was measured in the leaves (401.33 mg%). the contents of linolenic acid in other pawpaw parts were in the following order: seeds (233.63 mg%) > twigs (73.33 mg%) > roots (19.33 mg%) > fruit (18.59 mg%). the content of arachidonic acid (c20:4, n-6) in the leaves was 2.33 mg%, and it was not found in the roots, twigs, fruit, or seeds. in addition to its antihypertensive effects, oleic acid has been known to be effective in reducing low density lipoprotein cholesterol and increasing high density lipoprotein cholesterol (terés et al., 2008). cunnane and anderson (1997) and ruthig and meckling-gill (1999) speculated that mild skin enlargement and hair loss were found in rats with linoleic acid deficiency, and pan et al. (2012) reported that linolenic acid, a plant-derived omega-3 (n-3) fatty acid, reduces the risk of cardiovascular disease. arachidonic acid protects the brain from oxidative stress and activates syntaxin-3, which is involved in the growth and repair of nerve cells (darios and davletov, 2006). in this regard, it was previous found that the mental deve lopment index was improved in infants with arachidonic acid, and supplementation of arachidonic acid has been shown to be effective in reducing symptoms and delaying progression in the early stages of alzheimer’s diseases (birch et al., 2000; calderon-garciduenas et al., 2004). consequently, it is expected that pawpaw leaves and seeds rich in ufa such as oleic acid, linoleic acid, linolenic acid, and arachidonic acid will elicit beneficial physiological effects. vitamins β-carotene was the major vitamin in pawpaw tree (tab. 7). pawpaw leaves contained the highest level of β-carotene (3931.35 μg/100 g) among all samples, which is much higher than the β-carotene content (1957-2631 μg/100 g) measured in corn rocket, mountain lettuce, and poppy (maurizi et al., 2015). the β-carotene content contained in the twigs was similar to that in the fruit (84.18 μg/100 g versus 82.82 μg/100 g, respectively). pawpaw roots exhibited a remarkably lower level of β-carotene (7.06 μg/100 g). in the twigs, vitamin b1 (thiamine) was measured in the highest amount (at 96.30 μg/100 g). the vitamin b1 content in the other pawpaw parts were in the following order: roots (53.35 μg/100 g) > seeds (50.09 μg/100 g) > leaves (10.60 μg/100 g), and it was not observed in the pawpaw fruit. the vitamin b2 (riboflavin) content contained in pawpaw twigs, leaves, and seeds were 85.36, 51.54, and 82.73 μg/100 g, respectively, and none was detected in the roots and fruit. vitamin b3 (niacin) was only detected in the roots, and vitamin b12 (cyanocobalamin) was not detected in any of the samples. vitamin b5 (pantothenic acid) was observed in all samples, and vitamin b6 (pyridoxine) was detected in all samples except seeds; its content varied according to the specific tissue. the vitamin c (ascorbic acid) and vitamin e (tocopherol) contents in leaves were 3.70 mg/100 g and 4.04 mg/100 g, respectively, which are significantly higher level than those of roots, twigs, fruit, and seeds (p<0.05). β-carotene, a precursor of vitamin a, is known to be a functional compound with antioxidant, anticancer, and antiaging properties and plays a useful role in the retina of human eye (jang et al., 2016). vitamins c and e also have antioxidant effects on account of their ability to reduce reactive oxygen species, and an antihypertensive effect on account of their action on redox-sensitive vascular tissue (chen et al., 2001). as a result of this study, we identified that abundant levels of β-carotene, vitamin c, and vitamin e were contained in the leaves of pawpaw. consequently, pawpaw leaves are expected to be a useful material for producing functional foods. conclusions the present study investigated the nutritional compositions in different parts of pawpaw (a. triloba) tree. specifically, the roots, twigs, leaves, seeds and fruit were found to contain an abundance of essential nutrients such as amino acids, minerals, and vitamins required in the body. however, their amounts showed considerable variation tab. 7: vitamin content in various parts of asimina triloba. vitamins roots twigs leaves fruit seeds 7.06±0.10d 84.18±0.17b 3931.35±12.85a 82.82±1.50b 70.45±1.53c 53.35±3.23b 96.30±0.93a 10.60±0.59c n.d. 50.09±2.59b n.d. 85.36±2.42a 51.54±2.52b n.d. 82.73±8.77a 15.94±8.26 n.d. n.d. n.d. n.d. 5.64±0.91d 136.09±1.82a 114.14±1.07b 13.29±0.64c 6.71±2.99d 28.60±0.54a 12.78±0.98b 2.20±0.13d 10.38±1.92c n.d. n.d. n.d. n.d. n.d. n.d. 0.59±0.05e 0.80±0.03d 3.70±0.02a 0.98±0.06c 1.29±0.08b 0.19±0.01d 0.61±0.08c 4.04±0.47a tr 1.28±0.05b results are presented as mean±sd (n = 3). means with different letters in the same row are significantly different by duncan’s multiple range test (p< 0.05). n.d. = not detected. tr = trace. β-carotene (μg/100 g fw) b1 (thiamin) (μg/100 g fw) b2 (riboflavin) (μg/100 g fw) b3 (niacin) (μg/100 g fw) b5 (pantothenic acid) (μg/100 g fw) b6 (pyridoxine) (μg/100 g fw) b12 (cyanocobalamin) (μg/100 g fw) c (ascorbic acid) (mg/100 g fw) e (tocopherol) (mg/100 g fw) 54 j.-s. nam, h.-l. jang, y.h. rhee among the distinct pawpaw parts. high levels of amino acids and fatty acids were measured in the seeds, and minerals and vitamins were found to be rich in the pawpaw roots and leaves. overall, these results provide basic data for improving the sitological value of pawpaw and will promote further studies on the nutritional compositions of other plants and their components. references ackerson, r.c., 1981: osmoregulation in cotton in response to water stress ii. leaf carbohydrate status in relation to osmotic adjustment. plant physiol. 67, 489-493. doi: 10.1104/pp.67.3.489 agarwal, a., raj, n., chaturvedi, n., 2017: a comparative study on proximate and antioxidant activity of brassica oleracea (kale) and spinacea oleracea (spinach) leaves. int. j. adv. res. biol. sci. 4, 22-29. doi: 10.22192/ijarbs.2017.04.04.004 anderson, j.w., 1986: dietary fiber in nutrition management of diabetes. in: vahouny, g.v., kritchevsky, d. 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accepted: march 29, 2018) * corresponding author summary mulberry leaves have been widely used to produce various health products. in this paper, an efficient procedure of ultrasound-assisted hydroalcohol-acid extraction (uahae) was established to estimate the total flavonoids content in mulberry leaves by quantifying their resulting aglycones (quercetin and kaempferol) using hplc-dad. effective hydrolysis of glycosides to aglycones was achieved in an ethanol-hcl-water (7/2/1, v/v/v) solution at 75 °c by ultrasound (40 khz) for 60 min. the average contents of quercetin and kaempferol were 7.12 mg/g and 2.13 mg/g, respectively, in the variety of cultivar mc308, and their recoveries were 105.48% and 105.81%, respectively. the total flavonoid content of most varieties was between 4-10 mg/g, accounting for 80% in 86 species of mulberry leaves. with the increase in leaf maturity, the general trend of the change in flavonoid content was a decrease at first and then an increase. the highest total flavonoid content in commercial mulberry tea (ct 2) is 4.76 mg/g, and the dpph radical scavenging activities were similar to the distribution of total flavonoids measured by uahae in three types of mulberry tea. these finding provide a basis for industrial-scale manufacturing of edible medicinal mulberry leaf products. key words: mulberry leaves; total flavonoids; aglycones; quercetin; kaempferol; ultrasound; hydrolysis introduction mulberry (morus alba l.) is an industrial crop with a high economic value (zhang et al., 2016 ). its fruit, leaves, branches and roots are good sources of bioactive compounds, and it has a long history of use in traditional chinese medicine. mulberry leaf is the main product of the mulberry tree, accounting for 64% of the ground part of the tree (wang et al., 2005). it is nutritious and nontoxic and is usually used as feed for silkworms in most asian countries. some researchers have found that mulberry leaves are rich in phenylpropanoids (hunyadi et al., 2012), flavonoids (wanyo et al., 2011), polysaccharide (thirugnanasambandham et al., 2015), alkaloids (sugiyama et al., 2016) and many other bioactive compounds. as a herbal medicine, extracts of mulberry leaves possess medicinal benefits against obesity (lim et al., 2013), diabetes (ren et al., 2015), atherosclerosis, hypertension (lee et al., 2011) and tumours (yang et al., 2012). because of their significant bioactivities, mulberry leaves have been widely used to produce various health products, such as beverages, health food and mulberry-leaf tea (zhang et al., 2016 ). flavonoids and flavonoids glycosides are the most important components in mulberry leaves. the total flavonoid content accounted for 1.0-3.0% of the mulberry leaf dry weight, which is higher than in other plants (li et al., 2012). the major type of flavonoid in mulberry leaves is glycoside, with mainly quercetin and kaempferol aglycones (tab. 1). kim et al. identified nine flavonoids from mulberry leaves, kaempferol-3-o-β-d-glucopyranoside; kaempferol-3 o-(6”-o-acetyl)-β-d-glucopyranoside; quercetin-3-o-(6”-o acetyl)-β-d-glucopyranoside; quercetin-3-o-β-d-glucopyranoside; kaempferol-3-o-α-l-rhamnopyranosyl-(1-6)-β-d-glucopyranoside; quercetin-3-o-α-l-rhamnopyranosyl-(1-6)-β-d-glucopyranoside; quercetin-3-o-β-d-glucopyranosyl-(1-6)-β-d-glucopyranoside; quercetin-3,7-di-o-β-d-glucopyranoside; and quercetin (kim et al., 1999). kayo et al. purified and identified two new flavonoids from a butanol extract of mulberry leaves, isopentenyl flavanone and iso pentene flavanone glycoside (doi et al., 2001). rutin, hesperetin, quercetin, kaempferol, naringenin, hydroxyflavin (chang et al., 2016) and kaempferol (yang et al., 2012) have also been isolated from mulberry leaves. many researchers have confirmed that flavonoids extracted from mulberry leaves possess anti-hyperlipidemic (kojima et al., 2000), anti-diabetic (kobayashi et al., 2010), anticancer (deepa et al., 2013), hepatoprotective (kalantari et al., 2009), and anti-alzheimer’s disease (niidome et al., 2007) activities as well as have a synergistic effect on regulating the blood sugar level with alkaloids and polysaccharides in mulberry leaves. the level of total flavonoids in mulberry leaves is conventionally estimated by a colorimetric method using rutin as a standard. how o oh o oh or2 r1o oha o oh o oh or2 r1o b tab. 1: selected flavonoids with quercetin and kaempferol aglycones isolated from mulberry leaves r1 = h, r2 = h quercetin chang et al., 2016 r1 = h, r2 = glc quercetin 3-o-β-d-glucopyranoside (isoquercitrin) kim et al., 1999 r1 = h, r2 = 6’’-acetyl-glc quercetin 3-o-(6’’-o-acetyl)-β-d-glucopyranoside kim et al., 1999 r1 = h, r2 = rha-glc quercetin 3-o-β-d-rutinoside (rutin) chang et al., 2016 r1 = glc, r2 = h quercetin 7-o-β-d-glucopyranoside wang et al., 2013 r = glc, r2 = glc quercetin 3,7-di-o-β-d-glucopyranoside kim et al., 1999 r1 = h, r2 = h kaempferol yang et al., 2012 r1 = h, r2 = glc kaempferol 3-o-β-d-glucopyranoside (astragalin) kim et al., 1999 r1 = h, r2 = rha-glc kaempferol 3-o-β-d-rutinoside wang et al., 2013 novel estimation method of total flavonoids in mulberry leaves 115 ever, due to the interference of other coexisting substances, such as chlorogenic acid and protocatechuic acid, in medicinal herbs and extracts, the total flavonoids determined by the colorimetric method is higher than the actual value. furthermore, most flavonoids standards are not commercially supplied, and there are difficulties in measuring the content of various flavonoids in mulberry leaves. after oral administration, flavonoid glycosides will be transferred to their aglycones by the action of hydrolytic enzymes and microorganisms. therefore, the effective components of oral flavonoid glycosides are their aglycones (liu et al., 2007). ultrasound-assisted hydroalcohol-acid extraction (uahae) was de veloped in this study to estimate the content of flavonoids in mulberry leaves and mulberry tea. quercetin and kaempferol, the major aglycones in mulberry leaves, were quantitatively detected by high performance liquid chromatography with a diode array detector (hplc-dad). then, the total levels of the two aglycones were used to estimate or express the total flavonoids and their bioactivities in biosamples, especially mulberry leaves and tea. in this study, the flavonoid contents of 86 varieties of mulberry leaves and 3 species of mulberry leaves tea were examined by uahae. materials and methods chemicals and materials different species of mulberry leaves were picked from the mulberry garden of soochow university in may 2016. then the mulberry leaves were freeze-dried on a lyophilizer (modulyod, thermo elec tron corporation, usa). the resulting powder was stored at -4 °c and further used for the following experiments. commercial tea 1 of mulberry leaves (ct1) and mc792 mulberry leaves were provi ded from shandong sericulture institute. commercial teas 2 and 3 of mulberry leaves (ct2 and ct3) were purchased from hangzhou qingxin and anhui cunyutang co. ltd. (china). rutin and chlorogenic acid were purchased from tci shanghai co. ltd (china). quercetin, 5, 6-dihydroxycoumarin, isoquercitrin, astragalin and kaempferol standards were purchased from sigma-aldrich co. ltd. all other chemicals and solvents were of analytical grade, except those used for hplc analysis, such as acetonitrile and methanol, which were of hplc grade. to optimize hydrolysis conditions the extraction procedure was measured using zhao’s method (zhao et al., 2016) with some modifications. 250 mg of powder was suspended in 10 ml of an ethanol-hcl-water (v/v/v: 7/2/1) solution and kept at 75 °c and 40 khz (ultrasonic bath jiemeng instruments, china) for different times for the hydrolysis. for comparison, 250 mg of powder was suspended in 10 ml of an ethanol-water (v/v: 7/3) solution and kept at 75 °c and 40 khz for one hour. then, the supernatant was used in the following analysis. reproducibility analysis a 250-mg sample powder [leaf of mulberry cultivar 308# (shortly mc308), from middle position of the branch] was suspended in 10 ml of an ethanol-hcl-water (v/v/v: 7/2/1) solution and kept at 75 °c and 40 khz for 1 h. five repeated extractions of the samples were measured by hplc-dad to calculate the relative standard deviation (rsd). recovery assay known levels of quercetin and kaempferol were added to the sample solution. then, the mixture was extracted. the levels of quercetin and kaempferol were quantitatively detected by hplc-dad. the recoveries of the two aglycones were calculated by the linear equation produced from the standard curves. hplc-dad analysis hplc was performed with a shimadzu hplc system (shimadzu, japan), which consisted of a pump (lc-20at), diode array detector (dad, spd-m20a), c18 column (agilent 250 × 4.6 mm) and lc-solution system manager program. the mobile phase was methyl alcohol-water-acetic acid at a ratio of 500/500/0.4 (v/v/v). the flow rate was 1 ml/min, and the eluate absorbance was monitored at 370 nm using a scanning range of 200 nm to 600 nm. the injection volume of the extract was 10 μl. hplc analytical condition: mobile phase solvent a: acetonitrile, and solvent b: acetic acid / water (4:96, v/v). the flow rate was 1.0 ml/ min. the gradient for separation was 95% solvent b at 0 min, 25% solvent a and 75% solvent b at 40 min, 40% solvent a and 60% solvent b at 50 min, 47% solvent a and 53% solvent b at 60 min, and 60% solvent a and 40% solvent b at 65 min. the eluate absorbance was monitored at 370 nm using a scanning range of 200 nm to 600 nm. the injection volume of the extract was 10 μl. determination of various samples mulberry leaves and tea were collected and immediately turned into powder through freeze-drying. different leaf positions were collected from husang 32. the mulberry branches were divided into 1-10 from apical to basal. the extraction method was uahae. assay of total flavonoids by colorimetric method for this section, 0.4 ml of the 70% ethanolic plant extract was mixed with 4.6 ml of ethanol. then, 0.3 ml of 5% nano2 (for 5 min), 0.3 ml of al(no3)3 (for 6 min), 4 ml of 4% naoh and 0.4 ml of 70% ethanol were added to the mixture solution. the mixture was incubated for 10 min at room temperature. then, the absorbance was measured at 510 nm (hitachi uv-3000, japan). the flavonoid content of the extracts was compared against the rutin standard calibration curve, which was plotted at 0, 0.01, 0.02, 0.04, 0.06 and 0.08 mg/ml. dpph assay the 70% ethanolic extracts of cultivar lu-792 and three commercial mulberry teas (ct1, ct2 and ct3) were concentrated in a vacuum and freeze-dried (thermo, america). the dpph radical scavenging activities of these extracts were measured using a previously reported method (zhao et al., 2014). statistical analysis the data are expressed as the mean ± sd. comparisons were made using one-way anova with origin 9.1 software. p< 0.05 was considered statistically significant. results and discussion comparison of hydroalcoholic and hydroalcohol-acid extraction high performance liquid chromatography (hplc) was used for qualitative and quantitative research. the chromatogram showed several peaks; these peaks were identified by comparing the retention times of various standards (fig. 1). chlorogenic acid, rutin, isoquercitrin, and astragalin were identified from hydroalcoholic extraction, but hardly any quercetin of kaempferol was detected. however, high levels of quercetin (52.88 min) and kaempferol (57.50 min) were identified from hydroalcohol-acid extraction, and the levels of ru116 j.-g. zhao, y.-q. zhang tin, isoquercitrin, and astragalin significantly decreased. this result means that flavonoid glycosides transformed to their resulting aglycones (quercetin and kaempferol). on a c18 reverse-phase column, quercetin and kaempferol showed good retention behaviour. the concentrations and area under the chromatographic peak showed a good linear relationship. the linear regression equations for the two standard samples were as follows: quercetin, y= -149539.64 + 2100898.95x, r2 = 0.9954, and kaempferol, y = -136852.43 + 2674291.47x, r2 = 0.9963. the levels of the two aglycones, quercetin and kaempferol, detected by hplc-dad were individually calculated by two linear regression equations in the following experiment. optimisation of acid hydrolysis different extraction times (10, 30, 50, 60, and 70 min) were investigated to obtain all of the flavonoid aglycones. the levels of the quercetin and kaempferol aglycones were approximately 6.6 mg/ml and 1.9 mg/ml with the increase in extraction time from 10-70 min (40 khz ultrasonic). the maximum extraction levels of flavonoid aglycones were 6.91 ± 0.16 mg/ml and 2.06 ± 0.05 mg/ml at 60 min, and the flavonoid concentration decreased with a longer extraction time. at 30 and 50 min, the levels of the quercetin and kaempferol aglycones were also high, but the higher error line indicated that hydrolysis was not stable. therefore, the best extraction condition was to suspend 250 mg of the sample powder in 10 ml of an ethanolhcl-water (v/v/v: 7/2/1) solution and kept at 75 °c and 40 khz for 60 min. reproducibility of aglycone extraction by uahae-hplc the mulberry leaf sample was extracted five times; then, these samples were measured by hplc-dad (tab. 2). according to the individual linear equations, the levels of quercetin and kaempferol were calculated to be an average of 7.12 mg/g and 2.13 mg/g with relative standard diversity (rsd) values of 6.44% and 6.86%, respectively. these data indicate that this method for measuring the contents of quercetin and kaempferol from mulberry leaves has good reproducibility. recovery of aglycone extraction by the uahae method to evaluate the accuracy of this analytical method, a recovery study of the extracts from mulberry leaves was performed by adding known levels of quercetin and kaempferol standards. the mean recovery was 105.48% for quercetin and 105.81% for kaempferol (tab. 3). recovery percentages higher than 100% could be explained by the interference of the sample matrix. the relative standard deviation (rsd) determined the accuracy and stability of the methods. the rsd of the average recovery was 2.99% for quercetin and 2.62% for kaempferol. the recovery test showed that the uahae method for the hydrolysis and extraction of quercetin and kaempferol from mulberry leaves had very good precision. the content of total flavonoids at different leaf positions the mulberry branches were divided into several parts, from apical to basal (p1-p10). the total flavonoid levels of different mulberry leaves from the same branch also exhibited significant differences. at position 1 (p1), the highest level of total flavonoid content was 13.86 mg/ml (fig. 2). then, a significant decrease was observed from p2 to p5 (6.37 mg/g). from p5 to p7, the total flavonoid content increased to 9.63 mg/ml, and no significant differences were observed until p10. with the increase in leaf maturity, the general trend of the change in flavonoid content was a decrease at first and then an increase. the same results were found in bilberry leaves. a previous study reported that higher levels and increased expression of flavonoid pathway genes were observed in bilberry apical leaves exposed to direct sunlight compared to basal leaves that received limited sunlight (jaakola et al., 2004).the increase in the flavonoids from p5 to p7 may be due to synthesis and accumulation in basal leaves (martz et al., 2010). the difference between leaf positions may result from fig. 1: hplc chromatogram of hydroalcoholic (black line) and hydroalcohol-acid (blue line) extracts. hydroalcoholic extraction: extracted by ethanol-water (v/v: 7/3) solution. hydroalcohol-acid extraction: extracted by ethanol-hcl-water (v/v/v: 7/2/1) solution tab. 2: reproducibility for the determination of the two aglycones by the uahae method aglycones repeated measurements (mg/g) means (mg/g) ± sd rsd (%) quercetin 7.73 7.19 6.96 7.26 6.48 7.12 0.46 6.44 kaempferol 2.35 2.13 2.08 2.16 1.94 2.13 0.15 6.86 experimental materials: the mulberry leaf variety is mc308. novel estimation method of total flavonoids in mulberry leaves 117 different accumulation and allocation due to a variety factors that are responsive to the interaction between intrinsic, soil nutrient content and, environmental stimuli, including light, pathogens, temperature (vagiri et al., 2015). the content of total flavonoids in different mulberry varieties the content of total flavonoids was estimated by the sum of querce tin and kaempferol. as shown in tab. 4, the content of total flavonoids in different mulberry leaves ranged from 2.25-14.33 mg/g, which had a broad range for different varieties of mulberry leaves. this result also suggested that the total flavonoids had significant genotypic differences in different mulberry leaves. the minimum content of total flavonoids was found in liuye (2.25 mg/g) of all tested varieties. in the lu-10 variety, the maximum flavonoid content (14.33 mg/g), was six-fold higher than in liuye. the distribution of flavonoids in mulberry leaves also showed a normal distribution (fig. 3). most varieties were distributed between 4-10 mg/g, accounting for 80% of the total determination. additionally, 16% of mulberry leaves were higher than 10 mg/g, and several of the highest varieties were lu-9, g60, lu10, indicating that they were potential resources for new product development. tab. 3: recoveries for the extraction of the two aglycones by the uahae method aglycones sample content dosage theoretical measured recovery rsd (mg/g) (mg) value (mg/g) value (mg/g) (%) (%) quercetin 7.72 3.87 7.73 8.16 ± 0.24 105.48 ± 3.15 2.99 kaempferol 2.34 1.17 2.34 2.48 ± 0.07 105.81 ± 2.77 2.62 experimental materials: the mulberry leaf variety is mc308. fig. 2: the content of total flavonoids at different leaf positions 1-10: from apical to basal of the branch tab. 4: total flavonoids in 86 varieties of mulberry leaves mulberry total flavonoids mulberry total flavonoids mulberry total flavonoids mulberry total flavonoids cultivars (mg/g) cultivars (mg/g) cultivars (mg/g) cultivars (mg/g) mc206 5.76 t1 8.61 lu-10 14.33 g60 13.71 mc302 11.74 t10 10.68 lu-11 7.86 boluo 6.18 mc303 8.56 t2 7.48 lu-12 5.19 canzhuan4 7.53 mc304 7.2 t3 6.84 lu-14 8.3 duohu 9.38 mc307 5.39 t4 7.78 lu-2 5.47 fengweiandu 5.45 mc308 9.61 t5 10.91 lu-3 6.61 fuyang 8.83 mc403 6.89 t6 11.78 lu-4 6.44 gaogan 2.96 mc406 7.12 t7 10.03 lu-5 4.08 gui 94 5.87 mc842 6.06 t8 9.56 lu-6 7.1 gui d50 3.98 mc843 5.47 t9 11.31 lu-7 7.26 gui y50 11.57 mc3071 5.73 beidong2 8.78 lu-8 7.94 hetian 8.53 d05 10.05 beidong 3 8.99 lu-9 12.87 heiyou 5.99 g04 9.98 beidong 7 11.26 no1 6.88 hongding 7.04 hongqiao7 6.39 kanghan6 4.65 wolong 8.73 hongpimao 8.53 hongpi 7.61 jiuwenlong 7.68 husang4 8.77 rizhou 9 6.86 bei l6 10.12 guapiao 4.38 husang7 5.77 sha2 6.92 liuye 2.25 husang 1 8.66 huangbansang 5.66 suzhou 4.24 bingwu 8.94 husang32 6.38 nand 7 9.32 tang10 6.75 baipi 5.05 no3 8.23 niujin 5.2 yu71-1 5.48 wuhai 8.48 nand1 9.07 nong 10.51 yuantouheye 4.25 wulong 5.67 nand2 5.79 qingpiguapiao 5.27 nand 3 5.38 qingpiwasang 5.16 nand 4 4.49 118 j.-g. zhao, y.-q. zhang total flavonoids in mulberry tea mulberry leaf preparations are commonly used in commercial be verages (especially mulberry tea) and health foods. because mul berry leaf tea does not contain tannic acid and caffeine, it cannot cause an increase in blood pressure or nervous excitation. it has become a popular functional beverage in some countries, such as china, japan, and korea. the quantitative analysis results revealed substantial diversity in the total flavonoid contents in different species of mulberry tea. we measured three commercial teas of mulberry leaves (ct1, ct2, and ct3 (dark colour) ) by the traditional colori metric method and uahae. the total flavonoid content measured by the traditional colorimetric method was 26.14 mg/ml in ct1; however, the total flavonoid content measured by the uahae me thod was 3.15 mg/ml (fig. 4). it was also found in mulberry tea that the contents measured by the traditional colorimetric method were much higher than those measured by uahae. recently, guo et al. had doubts about the traditional colorimetric method, which uses rutin as a standard to estimate the total flavonoid levels in bio samples. their results showed that non-flavonoids, such as caffeic acid, protocatechuic aldehyde, and chlorogenic acid, had a maximum absorption at 510 nm (guo et al., 2002). chlorogenic acid (sánchezsalcedo et al., 2015) and protocatechuic acid (wu et al., 2013) have been identified from mulberry leaves, and these compounds react with nano2, al(no3)3, and naoh with a maximum absorption at 507 nm. anna also found that the procedure in the presence of nano2 in alkaline medium seemed to be specific for rutin, luteolin and catechins, but phenolic acids also exhibited considerable absorbance at 510 nm. (pękal et al., 2014). these reasons can lead to an increase in the total flavonoid content measured by the colorimetric method. commercial mulberry leaf ct1 tea is made from lu-792. the content of total flavonoids in lu-792 is 8.74 mg/g; however, 3.15 mg/g is observed in ct1. for the lu-792 ethanolic extract, the ic50 value of the dpph radical scavenging abilities was 227.45 ug/ ml, which was significantly better than that of ct1 (470.24 μg/ml). drying is one of the most critical processes in tea manufacturing, but this process may damage the antioxidant and nutritional qualities of the product (wanyo et al., 2011). kumar et al. found that a long drying time may cause degradation in food quality. in addition, this method results in undesirable qualities of the product, such as a dark colour and decreased antioxidant activity (praveen kumar et al., 2005). flavonoids have been demonstrated to have good antioxidant activity (parhiz et al., 2015). to evaluate the relationship between the flavonoid content measured by the uahae method and antioxidant acti vity, tea extracts were used to analyse the dpph scavenging property. as shown in fig. 4, the ct2 extract showed the maximum content (4.76 mg/g), while ct3 had the lowest content (2.13 mg/g). the ic50 values of the dpph radical scavenging abilities of each mulberry tea extract were 470.24 μg/ml, 338.44 μg/ml and 651.39 μg/ml. the total flavonoid content of ct2 (59.84 mg/g) measured by the traditional colorimetric method was higher than lu-792 (54.45 mg/g), but the content measured by uahae was reversed in the two samples. the ic50 values of dpph radical scavenging abilities of lu-792 (227.45 μg/ml) were lower than ct2 (338.44 μg/ml), which means that uahae more accurately reflects the actual flavonoid content. the radical scavenging activities were similar to the distribution of total flavonoids measured by uahae in three types of mulberry tea. conclusion mulberry leaves have become a health product, especially mulberry tea, in some countries, such as china, japan, and korea. the established uahae method is efficient for flavonoid extraction and quantification in mulberry leaves and tea. the optimal hydrolysis of flavonoid glycosides and extraction of the resulting aglycones occur in an ethanol-hcl-water (7/2/1, v/v/v) solution at 75 °c (40 khz ultrasonic) over 1 h. the validated method is precise and stable. in this study, mulberry leaves of 86 varieties and mulberry leaf tea of 3 species were examined for their flavonoid content. these findings will provide a basis for better industrial-scale manufacture of mulberry tea and for a wide range of similar food products. compliance with ethical standards funding the authors gratefully acknowledge the earmarked fund (cars-18-zj0502) for china agriculture research system (cars) and a project funded by the priority academic program development of jiangsu higher education institutions, p. r. china. conflict in interests jin-ge zhao declares that he 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yao, x.h., 2016: ultra sound extraction of polysaccharides from mulberry leaves and their effect on enhancing antioxidant activity. carbohyd. polymer 137, 473-479. zhao, j.g., zhang, y.q., 2016: a new estimation of the total flavonoids in silkworm cocoon sericin layer through aglycone determination by hydrolysis-assisted extraction and hplc-dad analysis. food nutr. res. 60. zhao, j.g., yan, q.q., xue, r.y., zhang, j., zhang, y.q., 2014: isolation and identification of colourless caffeoyl compounds in purple sweet potato by hplc-dad–esi/ms and their antioxidant activities. food chem. 161, 22-26. address of the author: jin-ge zhao, yu-qing zhang, silk biotechnology laboratory, school of biology and basic medical sciences, soochow university; rm702-2303, no. 199, renai road, dushuhu higher edu. town, suzhou 215123; pr china e-mail: sericult@suda.edu.cn © the author(s) 2018. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 90, 10 17 (2017), doi:10.5073/jabfq.2017.090.003 universidade federal de lavras, programa de pós-graduação em botânica aplicada, departamento de biologia, setor de botânica estrutural, laboratório de anatomia vegetal, lavras, brasil anatomical and physiological modifications in water hyacinth under cadmium contamination fabricio josé pereira1*, evaristo mauro de castro1, marinês ferreira pires1, cynthia de oliveira1, moacir pasqual1 (received february 19, 2016) * corresponding author summary the pollution of water bodies with heavy metals is generating increasing concern worldwide, and among those heavy metals, cadmium is one of the most toxic elements released into the environment. the present study aimed to evaluate the anatomical and physiological modifications adopted by the water hyacinth (eichhornia crassipes) under cadmium contamination. the plants were grown in hoagland solution in a greenhouse at five cadmium levels: 0.00, 3.5, 7.0, 14.0, and 28.0 μm. the net photosynthesis, stomatal conductance, transpiration rate, ci/ca ratio, antioxidant system enzymes activity, and anatomical traits in plant roots and leaves were evaluated. the plants exhibited increased photosynthesis, stomatal conductance, transpiration, and ci/ca ratios in all treatments containing cadmium. antioxidant system enzymes displayed increased activity in the roots and leaves of plants treated with cadmium. plants exhibited higher stomatal density and spongy parenchyma thickness under cd contamination. the anatomical traits of the roots exhibited no evidence of toxicity or improved vascular system traits. thus, eichhornia crassipes demonstrated an ability to tolerate cd by adopting changes in the anatomy, gas exchange and antioxidant system. keywords: eichhornia crassipes. phytoremediation. antioxidant system. ecological anatomy. ecophysiology. introduction recent anthropogenic activities such as industrialization, mining, and agricultural practices have led to the high production and accumulation of toxic elements in the soil, food chains, and aquatic environments (gratão et al., 2005). cadmium (cd) is a toxic element that can negatively influence plant growth and development and is released into the environment by power stations, heating systems, metallurgical industries, and vehicular traffic (benavides et al., 2005). phytoremediation is a cleaning process that can be used to remove toxic elements from the soil and water (gratão et al., 2005). cadmium phytoremediation has been the subject of recent studies because it is a relatively inexpensive and clean process; however, some studies have shown evidence of high cd toxicity in plants, thus making the identification of species that hyperaccumulate cd difficult (mangkoedihardjo, 2008; krämer, 2010). native macrophyte species are used in the phytoremediation of contaminated water. the water hyacinth (eichhornia crassipes) is a native brazilian macrophyte that belongs to the family pontederiaceae and can potentially be used in phytoremediation systems. this species has shown a potential ability to phytoremediate chromium that had accumulated at high levels (faisal and hasnain, 2003). oliveira et al. (2001) studied potential cd hyperaccumulation in e. crassipes and found that this species has a high capacity to uptake cd, with greater accumulation observed in the roots and accumulation occurring proportionally to exposure time and element concentration. this species may accumulate about 80 % of the cadmium in the solution, depending on the concentration of this element (mishra et al., 2007; wu et al., 2008). absorbed cd e. crassipes is readily associated to low molecular weight complex and further a high molecular weight complex, containing pc2, pc3 and pc4 phytochelatins is developed as the final storage cd form (wu et al., 2008). however, chelation cd in e. crassipes may also be related to s-containing compounds and vacuolar accumulation (puzon et al., 2008). this species also exhibited a significant ability to accumulate cu and zn and was able to remove these metals from biofertilizer solutions (mondardo et al., 2006). the e. crassipes is able to hyperaccumulate arsenic (dhankher et al., 2002; pereira et al., 2011) and lead (gonçalves júnior et al., 2008). physiological traits of plants exposed to cd are generally altered. for example, schützendübel et al. (2001) found that cd concentrations of 50 μm stimulated superoxide dismutase at the expense of catalase and peroxidases, promoting h2o2 accumulation in necrotic pinus sylvestris. pietrini et al. (2003) reported a 30 % inhibition in chlorophyll content, photosynthesis, and rubisco activity as well as stimulated antioxidant system activity in phragmites australis plants exposed to cd; these alterations enabled the maintenance of chloroplast structure at the cd levels evaluated and protected the photosynthetic system of this species. in e. crassipes plants, cd decreased chlorophyll content of leaves (mishra et al., 2007). the antioxidant system in e. crassipes plants was stimulated by the increased enzymatic activity of catalase, superoxide dismutase, and guaiacol peroxidase in the presence of cadmium; furthermore, the increases in activity occurred proportionally to cd concentrations (odjegba and fasidi, 2007). however, e. crassipes show cd stimulated superoxide dismutase in roots, but decreased the catalase, glutathione peroxidase, glutatione reductase and peroxidase activities (vestena et al., 2011). pistia stratiotes plants exposed to cd also showed anatomical and reproductive modifications (silva et al., 2013). the anatomy of e. crassipes showed plasticity in the presence of textile industry effluents, with reductions in cell size in the leaf tissue observed (mahmood et al., 2005). moreover, e. crassipes plants developed enhanced leaf and root structure under arsenic and lead contamination, increasing stomatal density, mesophyll thickness, number of xylem vessels between another favorable anatomical modifications (pereira et al., 2011; pereira et al., 2014). cadmium is capable of influencing the foliar anatomy of alternanthera philoxeroides and polygonum ferrugineum, resulting in reduced amounts of aerenchyma and crystal idoblasts, thicker mesophyll, and increased numbers of glandular trichomes, although with no effect on photosynthesis (souza et al., 2009). given the possible effects of cd on the anatomical and physiological traits of plants with phytoremediation potential, the present study aimed to evaluate the effect of cd on these traits in e. crassipes. materials and methods water hyacinth (eichhornia crassipes mart.) plants were collected from a set of ponds in alfenas, mg, brazil, that were apparently the cadmium tolerance of water hyacinth 11 free of cadmium contamination and that originated from a spring located approximately 60 meters away. the collected plants were washed with running water and grown in a greenhouse in hoagland and arnon nutrient solution (hoagland and arnon, 1940) at 40 % ionic strength for 30 days to obtain clonal generations acclimatized to the greenhouse and endogenously cadmium-free. to obtain uniform experimental units, plants were selected based on the number of leaves, the absence of stolons, the viability of daughter plants, and size. six-l polypropylene vases containing 4 l of hoagland and arnon nutrient solution at 20 % ionic strength supplemented with increasing cadmium levels (0.0, 3.5, 7.0, 14.0, and 28.0 μm) were used to grow the plants under the treatment conditions. the cadmium levels corresponded to the control (0 mg l-1 concentration) and the treatment levels corresponded to 100, 200, 400, and 800 times the maximum concentration permitted, starting at 3.5 μm, determined as the maximum concentration permitted by conama resolution no. 357 (wolff et al., 2009). the plants were kept under these conditions for 20 days. after 15 days, the plant gas exchange characteristics were evaluated using an infrared gas analyzer (irga), li-6400 model (li-cor biosciences, lincoln-usa) and a 6 cm2 cuvette with a red/blue led light source (li-6400-02b). the stomatal conductance (gs), transpiration rate (e), photosynthetic rate (a), and the ratio between internal and external carbon (ci/ca) were evaluated. to measure these variables, fully expanded leaves from five plants per treatment were selected, starting at 10 a.m., when the photosynthetic photon flux density was fixed in the device chamber at 1000 μmol m2 s-1, the pump flow was 500 μmol s-1, the vapor pressure deficit was 2.6 kpa. the measurements were conducted only in pathogen-free leaves. the anatomical analyses were performed on daughter plants with fully developed leaves and roots grown during the 20-day experimental period. the daughter plants were fixed in a solution of formaldehyde, acetic acid, and 70 % ethanol (f.a.a. 70) for 72 hours and then stored in 70 % ethanol until further analysis. paradermal leaf sections were obtained using steel blades on abaxial and adaxial sides. next, the sections were cleared with 50 % sodium hypochlorite, rinsed in distilled water twice for 10 minutes, stained with 1 % safranin solution, and mounted on slides with coverslips with 50 % glycerol (johansen, 1940). the 2-cm leaf fragments taken from the median region of leaves. in roots, the maturation zone region, 2 cm away from the root apex, was sampled. all of the sections were performed using a lpc table microtome. sections were cleared with sodium hypochlorite, rinsed in distilled water twice for 10 minutes, stained with safrablau solution (1 % safranin and 0.1 % astra blue at 7:3 ratio), and mounted on slides with coverslips with 50 % glycerol. the slides were photographed under an olympus bx 60 microscope (olympus, tokyo, japan) coupled to a canon a630 digital camera (canon inc., tokyo, japan). the uthscsa-imagetool software was used for image analysis and the following parameters were measured: abe = abaxial epi dermis thickness, ade = adaxial epidermis thickness, mf = mesophyll thickness, pp = palisade parenchyma thickness, sp = spongy parenchyma thickness, pp/sp ratio = palisade and spongy parenchyma ratio, dv = distance between vascular bundles in the mesophyll, bsc = diameter of the bundle sheath cells, pae = proportion of aerenchyma in the leaves (area/area), ns = number of stomata per field: nc = number of epidermal cells per field; pol = polar diameter of the stomata; equ = equatorial diameter of the stomata, sd = stomatal density (stomata per mm2), fun = stomatal functionality (ratio pol/equ), si = stomatal index, par = proportion of aerenchyma in the cortex, cc = diameter of cortical cells, ep = epidermis thickness, ed = endodermis thickness, cvi = vascular system vulnerability index (carlquist vulnerability index), mp = diameter of pith parenchyma cells, ex = exodermis thickness, and phl = phloem thickness. the cvi was calculated according to the method described by carlquist (1975) and the proportions of the root aerenchyma area were calculated according to the method described by pereira et al. (2008). twenty days after the onset of the experiment, leaf and root samples were collected in the morning for biochemical analysis and then were frozen in liquid nitrogen and preserved in a freezer at -80 ºc until further analysis. to obtain the analyzed enzymes, protein was extracted from 0.5 g of roots or leaves to which were added 2.0 ml of extraction buffer consisting of 1.924 μl of 0.1 m potassium phosphate buffer at ph 7, 20 μl of 0.1 m edta [ethylenediaminetetraacetic acid], 8 μl of 0.5 m dtt [dithiothreitol], 16 μl of 0.1 m pmsf [phenylmethanesulfonyl fluoride], and 40 mg of pvpp [polyvinylpolypyrrolidone], as adapted from the method described by bor et al. (2003). after homogenization, the enzyme extract was centrifuged at 14,000 g for 20 minutes at 4 ºc. the supernatant was then collected and used to determine the activity of the following enzymes: ascorbate peroxidase (apx), catalase (cat), and superoxide dismutase (sod). sod activity was assessed according to the method proposed by giannopolitis and ries (1977). ascorbate peroxidase (apx) activity was evaluated by adding 160 μl of 0.1 m potassium phosphate buffer ph 7, 100 μl of 0.005 m ascorbic acid, 100 μl of 0.005 m h2o2, and 605 μl of distilled water to 35 μl of the enzyme extract, as adapted from the method described by nakano and asada (1981). enzymatic activity was determined by monitoring decreasing absorbance at 290 nm for 2 minutes and calculated based on the extinction factor of 2.8 mm-1cm-1. to evaluate catalase (cat) activity, we added 35 μl of enzyme extract to 160 μl of 0.1 m potassium phosphate buffer at ph 7, and then added 125 μl of 0.01 m h2o2 dissolved in buffer and 680 μl of distilled water, as adapted from the method described by madhusudhan et al. (2003). enzyme activity was determined by monitoring decreasing absorbance at 240 nm for 1 minute and calculated based on the extinction factor of 36 mm-1cm-1. the experiment consisted of a completely randomized design with five treatments and five replications with the experimental units composed by one tray with one e. crassipes plant. the data were subjected to analysis of variance and comparison of means by scottknott test at p<0.05 or regression analysis using sisvar statistical software. results for all cd levels applied, the e. crassipes plants experienced changes in gas exchange characteristics (fig. 1). photosynthesis increased proportionally to cd concentrations on nutrient solutions (fig. 1a). the stomatal conductance increased with higher concentrations of cd, however, dropped under concentrations higher than 14 μm (fig. 1b). the transpiration rate increased proportionally to cd concentrations (fig. 1c) and the ci/ca ratio were increased with higher cd levels, but were decreased under concentrations higher than 7 μm (fig. 1d). e. crassipes leaves display uniseriate epidermis with reduced cuticle on both sides. the palisade parenchyma consists of three to four layers of cells facing the adaxial side. the spongy parenchyma is composed by large aerenchymal chambers filled with trabeculae formed by bundles of parenchyma cells; parenchyma occurs in some of the chambers, and idioblasts can be observed. the vascular bundles are collateral, have developed bundle sheath, and exhibit the xylem toward the adaxial side in some cases and towards the abaxial side in other cases. sclereids and idioblasts containing raphides in the palisade and spongy parenchyma can be seen, and the leaves are amphistomatic display uniseriate epidermis with reduced cuticle on both sides. the palisade parenchyma consists of three to four layers of cells facing the adaxial side. the spongy parenchyma is composed 12 f.j. pereira, e.m. de castro, m. ferreira pires, c. de oliveira, m. pasqual by large aerenchymal chambers filled with trabeculae formed by bundles of parenchyma cells; parenchyma occurs in some of the chambers, and idioblasts and angular collenchyma can be observed. the vascular bundles are collateral, have developed sheathes with some bundles, and exhibit the xylem toward the adaxial side (the palisade parenchyma) in some cases and towards the abaxial side in other cases. sclereids and idioblasts containing raphides in the palisade and spongy parenchyma can be seen, and the leaves are amphistomatic (fig. 2). changes in anatomical traits in e. crassipes leaves were observed in the different treatments; in particular, all tissues from treated leaves were thicker than those of control leaves (fig. 2). the epidermis of the abaxial side was already 99.57 % thicker at the first cd concentration (3.5 μm); the thickness increased another 12.17 % at the 14 μm concentration then decreased at the 28 μm concentration (tab. 1). the thickness of the epidermis increased up to 124 % on the adaxial side in all treatments containing cd and was significantly higher than in the control group, although not significantly different between cd levels (tab. 1). mesophyll thickness increased by 154.65 % in treatments containing cd and did not differ between cd levels (tab. 1 and fig. 2). palisade parenchyma was 138.20 % thicker at the 3.5 and 7 μm levels, increased further in thickness fig. 1: gas exchange characteristics of e. crassipes plants grown under different cadmium levels. a = photosynthetic rate, b = stomatal conductance, c = transpiration rate, d = ci/ca ratio. the bars represent standard error. fig. 2: cross-sections of leaves from e. crassipes grown under different cadmium levels. ade = adaxial epidermis, abe = abaxial epidermis, pp = palisade parenchyma, ae = aerenchyma, vb = vascular bundle. a = 0.0; b = 3.5; c = 7.0; d = 4.0 and e = 28.0 μm of cadmium. bars = 100 μm a b c d e the cadmium tolerance of water hyacinth 13 by 26.59 % at 14 μm, and then decreased again at the 28 μm concentration. spongy parenchyma thickness increased by 168.98 % in all treatments with cd but did not differ between the different levels (tab. 1). the palisade/spongy parenchyma ratio remained unchanged between the different treatments (p = 0.29; mean value was 0.25). the distance between the vascular bundles increased by 53.60 % at the 7 and 14 μm cd levels then decreased again at the 28 μm concentration. the proportion of aerenchyma in leaves was the same in all plants, including treated and control plants (p = 0.26; mean value was 27.8 %). in the paradermal section, changes in stomatal traits were observed only on the abaxial side of e. crassipes leaves. the stomatal density increased by 14.29 % at the 7 μm concentration but remained unchanged at higher levels (tab. 1). cadmium treatment did not result in changes in stomata size on the abaxial side, as shown by the polar and equatorial diameter values (tab. 2); in addition, there were no changes in stomatal functionality or in stomatal index on this leaf side. on the adaxial face, there were no significant changes in stomatal characteristics (tab. 2). the anatomical structure of e. crassipes roots shows uniseriate epidermis and exodermis formed by a layer of thick-walled cells. the cortex can be divided into three regions: the outer cortex, comprising three layers of large elongated parenchyma cells with few intercellular spaces, the median cortex, which exhibits aerenchymal chambers bounded by trabeculae of elongated parenchymal cells, and the inner cortex, composed of six layers of thin-walled isodiametric parenchymal cells and the endodermis. the intercellular spaces of the inner cortex cells are larger than those present in the outer cortex, and idioblasts are sometimes found among these cells; the inner cortex ends with the endodermis which displays small tabular-shaped cells. the vascular cylinder is composed of large metaxylem vessels alternating with phloem, pericycle was composed of one layer of cells, and the pith parenchyma in found in the center with isodiametric cells; some smaller metaxylem vessels may be associated with larger vessels or may form new metaxylem clusters. there were no changes in the number of aerenchyma in roots treated with cd (p = 0.52; mean value was 14.4 %). however, there was an 18.12 % reduction in the diameter of cortical cells at the 14 μm cd concentrations that remained unaltered at the 28 μm concentration (tab. 3). there were no significant changes in epidermis, endodermis, and exodermis thicknesses in cd-treated plants (tab. 3). the cvi decreased by 54.78 % in treated plants at the 7 μm concentration and remained unaltered at the higher levels; this decrease was related to a higher number of metaxylem vessels in the treated plants (fig. 3). the phl increased by 36.15 % only at the 28 μm concentration (tab. 3). tab. 1: leaf tissue characteristics in cross-sections of leaves of e. crassipes grown under different cadmium levels (μm) cd (μm) abe (μm) ade (μm) mf (μm) pp (μm) sp (μm) dv (μm) 0.0 09.4±0.7c* 13.1±1.4b 320.4±31.7b 062.9±9.6c 260.3±39.3b 104.0±10.1b 3.5 18.7±1.7b 24.5±3.8a 815.8±33.8a 149.8±18.8b 700.2±63.4a 118.9±8.9b 7.0 18.9±2.5b 25.5±2.7a 761.1±46.7a 142.9±9.5b 618.3±56.9a 151.4±11.2a 14.0 21.0±1.8a 27.0±2.6a 752.5±39.0a 181.0±2.3a 591.6±42.7a 159.8±24.9a 28.0 16.8±1.6b 29.3±2.2a 784.7±67.6a 141.6±24.6b 660.9±92.9a 124.4±9.6b *means followed by the same letter in the column do not differ (scott-knott, 5 %). data is shown as mean ± standard deviation. abe = abaxial epidermis thickness, ade = adaxial epidermis thickness, mf = mesophyll thickness, pp = palisade parenchyma thickness, sp = spongy parenchyma thickness, pp/sp = palisade and spongy parenchyma ratio, dv = distance between vascular bundles in the mesophyll, pae = proportion of aerenchyma in the leaf (area/area) tab. 2: anatomical characteristics of paradermal leaf sections of e. crassipes grown under different cadmium levels abaxial side cd (μm) ns nc pol (μm) equ (μm) sd fun si (%) 0.0 8.40±0.5b* 63.2±1.8b 43.7±1.7a 21.2±0.6a 107.9±7.0b 2.06±0.1a 13±1.0a 3.5 8.00±0.5b 61.4±1.6b 47.9±5.6a 23.5±1.5a 107.9±7.3b 2.04±0.2a 14±0.9a 7.0 9.40±0.4a 61.2±3.4b 48.4±3.2a 20.7±3.4a 120.7±10.7a 2.36±0.3a 15±0.7a 14.0 9.00±1.0a 63.4±7.4b 52.9±5.3a 23.9±1.4a 115.6±12.8a 2.21±0.2a 14±1.8a 28.0 9.60±0.5a 69.2±4.2a 46.9±5.4a 25.9±5.8a 123.3±9.7a 1.87±0.4a 14±0.6a adaxial side cd (μm) ns nc pol (μm) equ (μm) sd fun si (%) 0.0 8.20±0.4a* 65.0±2.2a 46.3±2.6a 24.8±1.5a 105.3±5.7a 1.87±0.2a 13±0.3a 3.5 8.60±0.5a 61.4±1.7a 46.3±3.0a 27.0±1.6a 110.5±7.3a 1.72±0.1a 14±0.7a 7.0 9.40±1.1a 64.4±5.5a 46.6±1.4a 23.7±2.1a 120.8±14.7a 1.97±0.1a 15±1.8a 14.0 9.80±2.0a 66.0±6.5a 42.3±6.6a 23.6±3.3a 125.9±26.3a 1.79±0.1a 15±2.2a 28.0 10.60±1.9a 67.4±5.7a 46.1±4.0a 25.4±1.3a 136.2±25.0a 1.82±0.1a 16±1.7a *means followed by the same letter in the columns do not differ (scott-knott, 5 %). data is shown as mean ± standard deviation. ns = number of stomata per field; nc = number of epidermal cells per field; pol = polar diameter of the stomata; equ = equatorial diameter of the stomata, sd = stomatal density (stomata per mm2); fun = stomatal functionality (pol/equ ratio); si = stomatal index 14 f.j. pereira, e.m. de castro, m. ferreira pires, c. de oliveira, m. pasqual the antioxidant system of e. crassipes changed with cadmium treatment (fig. 3). cat activity in e. crassipes leaves increased by 141.60 % at the 3.5 μm concentration and decreased under higher cd concentrations (fig. 3a). however, cat activity in roots was not modified by cd (p = 0.84). apx activity in the e. crassipes leaves increased proportionally to the cd levels, but its activity decreased in roots with under higher cd concentrations (fig. 3b). sod activity increased proportionally to cd levels in roots of e. crassipes (fig. 3c) but its activity in leaves was not affected (p = 0.12). discussion the responses of the photosynthetic system of e. crassipes under the influence of cd were different from those reported in the literature for sensitive species. according to pietrini et al. (2003), the presence of cd reduced the photosynthetic rate of phragmites australis plants by up to 30 %. in the presence of cd, the deposition of reactive oxygen species such as h2o2 that can accumulate in cells was observed by schützendübel et al. (2001); in these cases, reactive oxygen species can be harmful to the photosynthetic system by damaging chloroplast membranes, changing their structure and reducing photosynthetic efficiency (stoeva and bineva, 2003). thus, in plants exposed to cd, reduced photosynthetic rates are likely associated with damaged chloroplast membranes due to oxidative stress. in the present study, e. crassipes plants did not exhibit inhibited photosynthesis rates (fig. 1) but actually it increases under cd influence. likewise, increased photosynthesis has been recently reported to species under low cd concentrations (jia et al., 2015; pereira et al., 2016). this ability of some species to maintain photosynthesis in the presence of cd may be related to the activation of the antioxidant system, as the activities of apx, cat, and sod in leaves of this species increased with increasing cd concentrations (fig. 3). this modification may have enabled the maintenance of the chloroplast structure in e. crassipes leaves, thus preventing the inhibition of the photosynthetic rate. high cd concentrations may reduce the chlorophyll concentration in e. crassipes, but at lower concentrations this element may increase chlorophyll content in this species (mishra et al., 2007) and in robinia pseudoacacia (dezhban et al., 2015). this shows that the response of e. crassipes to cd may be related to the concentrations of this element in the solution. likewise, at the 28 μm or higher concentrations, stomatal tab. 3: anatomical characteristics of cross-sections of roots from e. crassipes grown under different cadmium levels cd (μm) cc (μm) ep (μm) ed (μm) cvi ex (μm) phl (μm) 0.0 115.9±8.2a 26.32±2.5a 19.51±1.6a 2.30±0.3a 39.58±3.5a 34.54±5.1b 3.5 108.0±5.5a 23.83±1.7a 18.59±0.7a 2.19±0.4a 31.04±3.6a 33.70±1.3b 7.0 109.8±15.4a 27.06±2.4a 17.86±1.2a 1.36±0.1b 41.03±1.7a 33.17±3.8b 14.0 099.4±1.9b 24.82±2.2a 18.09±1.9a 1.31±0.2b 39.50±2.2a 38.41±2.6b 28.0 094.9±8.4b 27.99±3.6a 19.64±2.4a 1.04±0.1b 38.26±6.5a 45.16±4.3a *means followed by the same letter in the columns do not differ (scott-knott, 5 %). data is shown as mean ± standard deviation. cc = diameter of cortical cells, ep = epidermis thickness, ed = endodermis thickness, cvi = vascular system vulnerability index (carlquist vulnerability index), ex = exodermis thickness, phl = phloem thickness fig. 3: activity of the antioxidant system enzymes of e. crassipes plants grown under different cadmium levels. a = catalase activity in leaves, b = ascorbate peroxidase activity in roots and leaves c = superoxide dismutase activity in roots. bars indicate standard error. the cadmium tolerance of water hyacinth 15 conductance and ci/ca ration tended to decrease (fig. 1), which may be related to the decreased activities of apx and cat and the unchanged activity of sod (fig. 4). these altered enzymatic activities may have led to h2o2 accumulation in the chlorenchymal cells and thereby a reduction in photosynthesis. these data are evidence of the relationship between the antioxidant system of this species and the maintenance of the photosynthetic rate in the presence of cd. while the photosynthetic rate can be regulated by different factors, radiation and co2 availability are the main factors that can increase photosynthesis when in higher quantities (zhou and han, 2005). radiation was standardized for all treatments at the time of measurement; the increased of the photosynthetic rate is therefore likely associated with higher co2 availability in the leaves. this hypothesis is corroborated by the alterations in leaf anatomy that promoted uptake of this gas. increased stomatal density on the abaxial side may be a factor contributing to higher co2 uptake under the experimental conditions, as this trait can increase when plants are under certain types of stress (souza et al., 2010; pereira et al., 2014) and under cd contamination (pereira et al., 2016). the absence of changes in stomatal functionality in plants from all treatments enabled the maintenance of the functional characteristics of e. crasssipes stomata under cd stress. another anatomical factor that contributed to increased co2 uptake was the increased mesophyll and spongy parenchyma thickness in all treatments containing cd. this increase in thickness may have enabled a higher capacity for co2 retention in the leaves, as co2 retention is one of the basic functions of the spongy parenchyma and aerenchyma. these effects were also described to schinus molle trees under cd contamination, increasing its leaf photosynthesis (pereira et al., 2016). thicker leaf mesophyll under cd contamination was also reported to arachis hypogaea (shi and cai, 2009). the unaltered proportion of leaf aerenchyma is an interesting result, as this tissue conserves the space for retaining gases (co2 and o2), thus contributing to photosynthesis and respiration in the leaves of this species. these results are similar to those observed in other aquatic macrophytes (souza et al., 2009) even though a lower number of leaf aerenchyma was found under conditions of cd stress. increased palisade thickness can promote a more efficient use of incident radiation in photosynthesis. other findings that corroborate the hypothesis that increased photosynthesis of e. crassipes is associated with co2 uptake are the observed increases in gs and in the e ratio in the cd-treated plants. an increase in these parameters results in favored co2 flow into the leaf, thus improving the uptake of this gas; this enhanced ability to uptake co2 may also be related to increased stomatal density, which promoted higher gs and thereby increased transpiration. these results, combined with the maintained stomatal functionality and stomata size, indicate that a stress response, similar to that seen under conditions of water stress in which stomatal functionality tends to increase and stomatal diameter to decrease, occurred in the plant (souza et al., 2010). thus, co2 uptake and retention inside the leaves was favored, which may have increased the photosynthetic rate of the e. crassipes in the presence of cd. in aquatic or wetland environments, the percentage of aerenchyma in the roots is an essential component of the physiology of plant roots, and this percentage can grow when plants are grown under these conditions (pereira et al., 2008; pereira et al., 2010). the fact that this tissue remained unchanged in e. crassipes roots in the presence of cd is a favorable factor. the endodermis, epidermis, and exodermis are considered apoplastic barriers to substance flow in the roots (ribeiro et al., 2015). as these layers did not increase in the presence of cd, a flow of metal to the shoots may have been promoted that can result in reduced photosynthesis which may explain why photosynthesis was inhibited at the highest cd concentration (benavides et al., 2005; mangkoedihardjo, 2008). the characteristics of the vascular system of e. crassipes roots were modified to increase their efficiency under conditions of stress. the cvi is a parameter that, when reduced, may indicate higher water conductivity in the roots, thus reducing embolism (carlquist, 1975); this parameter typically decreases when the plants are under stress (pereira et al., 2010). thus, reduced cvi may indicate improved water conductivity in e. crassipes, which may have promoted increased nutrient flow and enhanced survival under these conditions; in addition, transpiration is not an issue because e. crassipes is an aquatic species. the thickening of the phloem observed at the highest concentration of cd also contributed to the improvement of the vascular system. as this tissue is related to photoassimilation transport, it may allow better root system development and improved food in daughter plants. the increased activity of the antioxidant system enzymes in e. crassipes corroborates the findings of schützendübel et al. (2001), pietrini et al. (2003), and odjegba and fasidi (2007), who found increased activity of these enzymes in plants subjected to cd. however, vestena et al. (2011) reported reduced activity for the antioxidant system enzymes of e. crassipes under cd contamination. this different response may be related to the experimental conditions, in their work, vestena et al. (2011) carried out experiments under growth chamber conditions, with lower radiation intensity and different cd concentrations were applied. the high sod activity in the root that occurred at the expense of apx and cat corroborates the results of schützendübel et al. (2001), who reported the same behavior in pinus sylvestris plants. this alteration in enzymatic activity may have resulted in h2o2 accumulation in cells and led to oxidative stress. however, these effects are not evident in e. crassipes, as no signs of toxicity were observed at the levels of cd studied. the evaluated characteristics are evidence of the potential use of e. crassipes in cd phytoremediation systems. the anatomical and physiological traits observed allowed us to elucidate the mechanisms by which this species demonstrate potential for hyperaccumulation and phytoremediation of several toxic elements, as reported by dhankher et al. (2002), oliveira et al. (2001), mondardo et al. (2006), and gonçalves júnior et al. (2008). the absence of negative effects of cd on physiology and anatomy of e. crassipes may be related to efficient vacuolar sequestration (puzon et al., 2008) or the association of this element to phytochelatins (wu et al., 2008), leading to a inactive form of this element. thus, the e. crassipes is promising for use in cd phytoremediation; indeed, this plant appears to commit significant resources to photosynthesis to meet the requirements for energy and carbon for daughter plants, which may promote its capacity for vegetative reproduction and high population growth. conclusion e. crassipes plants exhibit physiological modifications in the presence of cadmium that contribute to maintaining photosynthetic rates and increasing the activity of antioxidant system enzymes. changes in anatomical characteristics of e. crassipes promote higher co2 uptake and retention in leaves which may allow for higher photosynthesis. the changes in root anatomy of e. crassipes do not exhibit signs of toxicity. antioxidant system enzymes are more active in the leaves than in the roots of e. crassipes in the presence of cd. acknowledgements the authors thank cnpq (conselho nacional de desenvolvimento científico e tecnológico [national counsel of technological and 16 f.j. pereira, e.m. de castro, m. ferreira pires, c. de oliveira, m. pasqual scientific development]), capes (coordenação de aperfeiçoamento de pessoal de nível superior [coordination for the improvement of higher education personnel]), and fapemig (fundação de amparo à pesquisa do estado de minas gerais [minas gerais state research foundation]) for funding and research grants awarded to complete the present study. references benavides, m.p., gallego, s.m., tomaro, m.l., 2005: cadmium toxicity in plants. braz. j. plant physiol. 17, 21-34. doi: 10.1590/s1677-04202005000100003 bor, m., özdemir, f., türkan, i., 2003: the effect of salt stress on lipid peroxidation and antioxidants in leaves of sugar beet beta vulgaris l. and 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cd-binding complex in water hyacinth (eichhornia crassipes) when exposed to cd. j. agric. food. chem. 56, 5806-5812. doi: 10.1021/jf8011272 © the author(s) 2017. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). zhou, y.m., han, s.j., 2005: photosynthetic response and stomatal behaviour of pinus koraiensis during the fourth year of exposure to elevated co2 concentration. photosynthetica 43, 445-449. doi: 10.1007/s11099-005-0071-5 address of the corresponding author: fabricio josé pereira, universidade federal de lavras, departamento de biologia, caixa postal 3037, campus universitário, cep: 37200-000, lavras, minas gerais, brasil e-mail: fabriciopereira@dbi.ufla.br journal of applied botany and food quality 91, 304 309 (2018), doi:10.5073/jabfq.2018.091.039 1 department of biology, portland state university, portland, or 2 environmental science and management program, portland state university, portland, or quantitative effects of soil salinity on the symbiosis of wild lima bean (phaseolus lunatus l.) and bradyrhizobium in costa rica daniel j. ballhorn1*, emily r. wolfe1, jess tyler2, wren ronan1, scott sands-gouner1, curran shaw1, mehmet a. balkan1, stefanie kautz1 (submitted: february 16, 2018; accepted: june 5, 2018) * corresponding author summary global climate change and local anthropogenic activities are increasing soil salinization with permanent negative effects on agricultural and ecosystem productivity. while salt stress is known to affect plant performance, its effects on the association with key microbial plant symbionts, such as legume-associated nitrogen-fixing rhizobia, are less understood. in this field study conducted in costa rica (punta renas), we used sympatrically-occurring wild lima bean (phaseolus lunatus l.) and bradyrhizobium to quantify biomass production of unfertilized rhizobial (r+) and fertilized rhizobia-free (r-) plants at different levels of experimentally manipulated salinity in native soil. in response to salt stress, nodulation was significantly reduced even at slightly increased salt levels. plants growing at soil salinity levels of 2, 4, 6, and 8 ms/cm showed a mean reduction of nodules by 60.22, 76.52, 83.98, and 92.5% compared to the controls. similarly, we also observed a significant decline in plant biomass at elevated salinity. however, biomass accumulation of rplants was significantly less impacted compared to r+ plants, suggesting that the plant-microbe symbiosis is more salt-sensitive than the plant host itself. we suggest that the search for more salt-tolerant, crop plant-compatible rhizobial strains may provide a sustainable approach to maintain agricultural productivity on low to moderately saline soils. introduction human activities have increased the prevalence of soil salinization in arable land, with significant detrimental impact on crop plant productivity (singleton et al., 1982; schofield et al., 2001). soil salinization is a wide-spread form of land degradation that affects between 20-30% of agricultural land (li et al., 2014; singh, 2015), and occurs when excess salts accumulate in the upper layers of otherwise productive soils (rojas et al., 2016). salt deposition in soils can occur naturally through erosion, periodic flooding, saltwater intrusion, and leaching; however, human activities including intensive irrigation, deforestation, and industrial emissions also introduce a significant amount of salt into environments (rengasamy, 2006). elevated salt levels can be difficult to ameliorate in soils, and if unchecked, soil is irreparably degraded, causing ecosystem disturbances and reducing crop yield (cabot et al., 2014; birgé et al., 2016). salt stress manifests incrementally, causing difficulties with plant water and nutrient uptake, thus inhibiting biomass accumulation and fruit production, root hair growth, and causes premature leaf senescence (tu, 1981; kaushal and wani, 2016). plant physiology is directly altered by unfavorable shifts of ionic balances and osmotic stress (borsani et al., 2003; ballhorn and elias, 2014). certain plant families have evolved adaptations to this osmotic stress through specific traits such as salt excreting glands or suites of xerophobic traits (barhoumi et al., 2007). other plants show a relatively broad range of salt tolerance, but these levels of tolerance vary considerab ly among plant families and species (yadav et al., 2011). in addition to plant intrinsic factors, the interaction with plant-associated microbes may affect the salt tolerance of a given species. for example, sheng et al. (2008) found that the salt tolerance of zea mays improved when inoculated with the arbuscular mycorrhizal species glomus mosseae. similarly, certain soil microbes have been shown to confer salt tolerance by emitting volatile organic compounds that regulate plant stress responses (yang et al., 2009). in nature, most vascular plants form complex associations with multiple microbial symbionts − including bacterial and fungal endophytes, mycorrhizal fungi, and nitrogen-fixing bacteria. these interactions can complicate studies on host plant salt stress tolerance through the added symbiont response and host-symbiont interaction. legumes (fabaceae) play critical roles in determining the productivity, sustainability, and diversity in many terrestrial ecosystems due to their association with nitrogen-fixing rhizobia (crews and peoples, 2004; barea et al., 2005; valiente-banuet and verdú, 2007; peoples et al., 2009). at the same time, this plant family includes some of the globally most important crop plants such as soybeans, alfalfa, and common bean (singleton et al., 1982; zahran, 1999; peoples et al., 2009). in both crops and wild legumes several factors have been identified as having significant impacts on the belowground rhizobial symbiosis, and thus the vigor of the host plant. these include abiotic factors such ph, soil moisture, and salinity (boonkerd and weaver, 1982; graham, 1992) as well as biotic factors such plant disease and herbivory (zahran, 1999; heath and lau, 2011). by focusing on soil salinity as one of the most detrimental abiotic factors on plant productivity, research has shown that the establishment and maintenance of rhizobial symbiosis is impacted through reduced root-hair infection and nodule development, which subsequently results in reduced nitrogen-fixation capacity and plant growth (zahran and sprent, 1986; delgado et al., 1994; yanni et al., 2016). indeed, this phenomenon has been observed in several important crop plant species. in soybean (glycine max), 26.6 mm nacl was sufficient to inhibit nodulation (singleton and bohlool, 1984), while fertilized chickpea plants (cicer arietinum) were still able to nodulate when treated with 75 mm nacl (lauter et al., 1981). despite using salt-tolerant strains of rhizobium and brady rhizobium, delgado et al. (1994) corroborated that salinity decreased nodular dry weight in soybean, as well as in pea (pisum sativum), faba bean (vicia faba), and common bean (phaseolus vulgaris). in alfalfa, serraj and drevon (1998) attributed impaired plant growth following salinity treatments to inhibited nitrogenase activity. while matching the salt tolerances of both the rhizobial and crop plant strains may help optimize the symbiosis (bouhmouch et al., 2005), the observed negative effects of soil salinity on the bacterial symbiosis, and thus the symbiotic nitrogen-fixation, raise the question of whether or not supplementing legume host plants with additional nitrogen may alleviate the negative effects of saline soils. although evidence exists that legumes relying exclusively on symbiotically-fixed n supplies are more sensitive to salinity than soil salinity effects on symbiosis of wild lima bean with bradyrhizobium 305 those grown with additional or only mineral nitrogen, quantitative information on these interactions is scarce. overall, much unknown variation exists on the effects of soil salinity on the legume-rhizobia symbiosis (rao et al., 2002; bouhmouch et al., 2005) – which is surprising given its vast impact on productivity, stability, and diversity of natural and agricultural ecosystems (ballhorn et al., 2011, 2013). in our study, we used wild lima bean (phaseolus lunatus l.) as an experimental plant to analyze quantitative effects of soil salinity on rhizobial nodulation and plant performance. while cultivated genotypes of lima bean are grown as important grain legumes in the americas (broughton et al., 2003), wild lima bean represents an emerging model plant to study the chemical ecology of plant-herbivore and plant-microbe interactions (ballhorn et al., 2011, 2013). although lima bean has been extensively studied for its interactions with the biotic and abiotic environment, the tolerance of this species and its rhizobial symbiosis to saline soils is unknown. here, we focused on the symbiosis between costa rican wild lima bean and a bradyrhizobium strain (genbank accession id mf871645) both naturally occurring at the field site (puntarenas, pacific coast), and tested quantitative effects of soil salinity on unfertilized rhizobial plants vs. fertilized rhizobia-free plants. specifically, we aimed to answer the following questions: 1) how salt tolerant is wild lima bean and its association with a native rhizobial strain? 2) do observed ne gative effects of soil salinity on plant growth result primarily from direct effects on the host plant, or from effects on the microbial symbiosis? and 3) does the addition of mineral nitrogen fertilizer alle viate the negative consequences of the declining symbiosis in saline soils? materials and methods field collection and processing of soil five hundred kilograms of soil were collected at a natural lima bean site in puntarenas, costa rica at a depth of 0 – 30 cm corresponding to the major rooting depth of wild lima bean at this location. the soil was sieved to remove rocks and large organic debris and mixed thoroughly in an 8-horsepower concrete mixer for 30 min (honda). the container of the concrete mixer was cleaned with a wire brush, 0.5% bleach solution, and was washed with sterile water prior to use [autoclaved in a bench top sterilizer (benchmark scientific bioclave 16™); 1260 mbar, 121 °c, 30 min)]. after mixing, the soil was separated into ten 50 kg batches and each batch was sampled three times for conductivity analysis (cdb-387 conductivity meter, omega) according to the saturated paste extract method. after that, 2 kg of soil per batch were removed and unprocessed sea salt from a local salt producer (solar salinas) was added until the electrical conductivity (ec) of the subsamples were adjusted to 2, 4, 6 and 8 ms/ cm. from the amount of salt used per 2 kg soil, the amount of salt required for adjusting the remaining 48 kg of soil to the different conductivity levels was calculated and added. subsequent conducti vity measurements were conducted to confirm the desired salt concentration. additional salt or soil was added in case re-adjustment was necessary. subsequently, soil was autoclaved in two identical benchtop sterilizers (see above) in batches of 5 kg (1260 mbar, 121 °c, 1 h). these batches were tested for variation in conductivity again but no further adjustment was required. preparation of plants the autoclaved, differently saline soil mixtures were added to bleachsterilized 2-gallon plastic pots while still hot and pots were covered with sterile aluminum foil. per salt treatment (ec: 1.4, 2, 4, 6, 8 ms/ cm) pots were split into two groups and planted with pre-germinated sterile lima bean seeds collected from the natural local population. to produce sterile seedlings, surface-sterilized seeds were germinated in sealed sterile petri dishes (12 cm diameter) on autoclaved paper towels. when the radicle had reached a length between 0.1 and 0.5 cm, seedlings were transplanted to the pots in a sterile hood by punching a small hole in the aluminum foil cover and inserting the radicle. the aluminum foil remained on the pots for the experimental period to reduce spontaneous rhizobial colonization. plants were watered and fertilized through a silicone tube with attached bacterial filter; sterile water and fertilizer solutions were applied with a syringe at 50 ml per day. rhizobial inoculation plants were either inoculated with rhizobia (r+, n=12 plants per salinity treatment group) and unfertilized, or maintained rhizobiafree (r-, n=12 plants per salinity treatment group) and fertilized daily during the cultivation period with a 5.3 mmol l-1 cano3 solution made with sterilized water, a concentration previously used to compare nitrate-fed plants to those with nitrogen-fixing rhizobia (kiers et al., 2006). for rhizobial inoculation, a local bradyrhizobium strain (genbank accession id mf871645) was isolated from wild plants, cultivated in liquid medium (yeast extract), and 10 ml of bacterial inoculum were applied to experimental plants (107 cells per ml) 5 days after planting the seedling. the bacterial solution was applied next to the plant with a pipette. this rhizobial strain is maintained in the ballhorn lab and has been demonstrated to successfully colonize lima bean plants under field and laboratory conditions. also, this strain was able to effectively fix nitrogen in preliminary experiments in which chloro tic, rhizobia-free lima bean plants turned dark green after inocu lation and successful nodulation with this strain. destructive harvest and processing of plant material after a cultivation period of eight weeks, plants were destructively harvested and all nodules (visible under a stereo microscope at 6× magnification) were counted. to determine plant aboveand belowground biomass production, root and shoot portions were separated, roots were carefully washed, and all soil particles removed. plant samples were dried in an oven at 72 °c for three days (amerex instruments, incumax cv250) and dry weight was determined with a fine balance (mettler toledo, xa deltarange®). data analysis statistics were performed in r version 3.3.1 with an α = 0.05. plant treatment differences within each soil electrical conductivity (ec) level were determined using two sample t-tests that assumed equal variance. to analyze differences in responses to soil ec within plant treatments, one-way analysis of variance (anova) and pairwise tukey’s hsd post-hoc tests were used. shapiro-wilk and levene’s tests were used to determine if assumptions of anova were met. data were not transformed because only a small subset of treatment × response groups did not meet these assumptions. accession number information about the bradyrhizobium strain used in this study is available through genbank under the accession number mf871645. results our study sought to identify quantitative effects of soil salinity on a natural legume-rhizobia symbiosis using costa rican wild lima bean and a local bradyrhizobium strain as the experimental system. experimentally elevated soil salinity levels resulted in a significant quantitative variation in nodulation (f4,55=113, p<0.001). the num306 d.j. ballhorn, e.r. wolfe, j. tyler, w. ronan, s. sands-gouner, c. shaw, m.a. balkan, s. kautz ber of nodules on plants with rhizobia (r+) significantly decreased with increasing salinity; more nodules were produced at the control ec level (1.4 ms/cm) than at plants growing at any other ec level (p<0.001; fig. 1). numbers of nodules produced by plants growing in soil with an ec of 4 and 6, and 6 and 8 ms/cm, respectively, were similar. levels except between 6 and 8 ms/cm. in r+ plants, aboveground biomass also differed significantly across all salt treatments (f4,55=83.5, p<0.001), as well as between plants growing in control soil (1.4 ms/ cm) and all other ec levels; however, differences were insignificant between plants grown in 2 and 4, 4 and 6, and 6 and 8 ms/cm soil (p>0.05). fig. 1: number of nodules in rhizobia-inoculated plants. plants were grown in soils with different electrical conductivity (ec) and nodules were harvested and counted of 8 weeks of plant cultivation. bars are means (±sd), stars represent statistical difference based on t-tests within ec treatment groups (significance: ** p<0.01, *** p<0.001). lower case letters show significant differences among experimental plants according to post-hoc analysis (tukey’s hsd, p<0.01) after one-way anova. total biomass production of both rhizobial (r+) and non-rhizobial (r-, nitrogen fertilized) plants was similar at the control ec level (1.4 ms/cm), but decreased in response to elevated soil salinity (f4,55=173, p<0.001; f4,55=131, p<0.001). when treated with salt, r+ plants (5.77 ± 2.78 g) produced significantly less biomass than the corresponding rplants (8.24 ± 2.57 g) overall (t=5.07, df=117, p<0.001). at each ec level above the control (1.4 ms/cm), total biomass was significantly lower for r+ plants than for rplants (twosample t-test; 2: t=15.4, df=22, p<0.001; 4: t=11.8, df=22, p<0.001; 6: t=6.24, df=22, p<0.001; 8: t=4.00, df=22, p<0.001; fig. 2). r+ plants were more sensitive to changes in the soil electrical conductivity as total biomass accumulation suffered at all ec levels when compared to the control ec level, except between 2 and 4, and 6 and 8 ms/cm (tukey hsd, p<0.001). in rplants supplied with nitrogen fertilizer, total biomass did not differ between 1.4 and 2 ms/cm, but differences were significant between 1.4 and 4, 6, and 8 ms/cm. when looking at aboveand belowground biomass separately, we observed significant differences depending on symbiotic state (r+ vs. r-) and salt treatment. aboveground biomass decreased for both r+ and rplants with increasing soil ec (f4,115=128, p<0.001; fig. 3). plants without rhizobia produced significantly more aboveground biomass than r+ plants at 2, 4, and 8 ms/cm (two-sample t-test; 2: t=7.96, df=22, p<0.001; 4: t=3.99, df=22, p<0.001; 8: t=3.03, df=22, p<0.01). in rplants, aboveground biomass differed significantly across salt treatments (f4,55=214, p<0.001) and between all ec fig. 2: total biomass of plants with or without rhizobia. plants were grown in soils with different electrical conductivity (ec) and total plant biomass (dry weight) was determined after 8 weeks of cultivation. bars are means (±sd), lower (rhizobia) and upper case letters (fertilizer, no rhizobia) show significant differences among experimental plants according to post-hoc analysis (tukey’s hsd, p<0.01) after one-way anova. fig. 3: aboveand belowground plant biomass. plants with and without rhizobia were grown in soils with different electrical conductivity (ec) and plant above and below biomass (dry weight) was determined after 8 weeks of cultivation. bars are means (±sd), stars represent statistical difference based on t-tests within ec treatment groups (significance: ** p<0.01, *** p<0.001). lower (rhizobia) and upper case letters (fertilizer, no rhizobia) show significant differences among experimental plants according to post-hoc analysis (tukey’s hsd, p<0.01) after one-way anova. soil salinity effects on symbiosis of wild lima bean with bradyrhizobium 307 belowground biomass increased through 4 ms/cm in rplants, and was significantly higher in rplants than in r+ plants (t=9.04, df=105, p<0.001) overall, particularly at all ec levels above the control (two-sample t-test; 2: t=14.5, df=22, p<0.001; 4: t=13.9, df=22, p<0.001; 6: t=11.1, df=22, p<0.001, 8: t=4.22, df=22, p<0.001; fig. 3). plants with rhizobia did not respond differently to soil salinity levels between 2 and 4, or 6 and 8 ms/cm; however, plants grown in control soil showed significantly different belowground biomass from all other ec levels. plants without rhizobia showed similar belowground biomass when growing in soil with ec of 2 and 4, or 2 and 6 ms/cm, but had significantly different belowground biomass at all other ec levels when compared to control soil. overall, root-to-shoot ratio decreased significantly with increasing ec within each plant treatment (r+: f4,55=5.75, p<0.001; r-: f4,55=50.5, p<0.001), and only differed between plant treatments at the 4 ms/cm (t-test; t=2.26, df=22, p<0.05) and 6 ms/cm ec levels (t-test; t=12.03, df=22, p<0.001). in plants with rhizobia, the ratios at the control level were significantly different from those at 4, 6, and 8 ms/cm, which were all similar (fig. 4). ratios at the control level and 2, and 2 and 4 to 8 ms/cm were likewise similar. plants grown without rhizobia showed root-to-shoot ratios differing between all ec levels except for 1.4 to 2, and 4 to 8 ms/cm (fig. 4). additio nally, root-to-shoot ratios increased with ec, except for 8 ms/cm in rplants, where it decreased significantly. factors following recognition of legume-produced flavonoids (cár denas et al., 2000). however, elevated salinity decreases growth of rhizobia and interactions with legume root-hairs (tu, 1981), reducing nodulation and consequently decreasing the nitrogen-fixing capacity of inoculated host plants. here, nodule production decreased by over 60% between the 1.4 (control) and 2 ms/cm treatment, and by over 90% between control plants and plants growing at 8 ms/cm, indi cating high sensitivity of the symbiosis to even small increases in salinity. since rhizobia and host plant genotypes vary in their relative salt tolerances, it is possible that the particular bradyrhizobium strain (isolated from wild lima bean plants growing about 700-1000 meters inland) used in this study may be less salt-tolerant than other regional and more coastal strains, but the significant reduction in nodulation is still remarkable. while some information on negative effects of salinity on nodule and biomass production in legume hosts is available (lauter et al., 1981; singleton and bohlool, 1984; delgado et al., 1994; serraj and drevon, 1998; bouhmouch et al., 2005; yanni et al., 2016), future studies might seek to further define particular tolerance ranges. total biomass was reduced significantly in r+ plants at already 2 ms/cm, unlike rplants that did not show significant changes in biomass production until 4 ms/cm. differences between r+ and rtreatments may have been driven by the opposite trends in belowground biomass, which actually increased slightly with increasing salinity for rplants, while belowground biomass in r+ plants consistently decreased. our study shows that even low (2-4 ms/cm) salinization negatively affects a natural legume-rhizobial symbiosis. while we do have a good understanding of plant-rhizobia symbioses in many host plant systems – in particular crop plants – in other systems detailed information on the factors driving these interactions is lacking. in-depth research is still required to determine the impact of abiotic and biotic factors on the establishment, maintenance, and quantitative expression of this interaction. such knowledge is important to estimate the impact of changing environmental conditions on natural and agricultural ecosystems. this study shows that the sensitivity of the symbiosis to environmental stressors such as soil salinity critically determines the performance of host plants growing on nonnitrogen supplemented soils. our data point to future research possibilities in the breeding of legumes and their symbionts for higher salt tolerance; otherwise, in the absence of these optimized symbioses, our findings suggest that additional nutrient supplements are required to maintain productivity under increasing salinity. however, additional fertilization is not always a viable option for farmers (sanginga, 2003), nor is it always benign for the environment. runoff from fertilized fields can induce eutrophication of neighboring freshwater ecosystems (smith et al., 1999; hirel et al., 2007), while weese et al. (2015) found that excessive n-addition to soils decreased rhizobia n-fixation capacity over time. rhizobia vary in their salt sensitivity, with many strains being inhibited only at levels well above what a plant could tolerate (bouhmouch et al., 2005). lauter et al. (1981) showed that rhizobia growth is only moderately inhibited at nacl levels of 250mm. this implies that the plant-rhizobia interaction rather than the sensitivity of rhizobia themselves is a major limiting factor in crop salt tolerance. legumes are major crops and forage plants throughout the world, and the symbiosis with rhizobia is crucial to their success in agriculture (de araujo et al., 2016). according to the food and agriculture organization of the united nations (2008), over 6% of arable land is currently considered saline or sodic. problems have continued to arise due to outdated agricultural practices (crop rotations, irrigation practices, fertilizers, etc.) and many have been late to adopt eco-friendly policies to maintain sustainable growing conditions (riley and barber, 1970; tu, 1981). better knowledge of the complex factors driving the plant-rhizobia symbiosis is required to establish, maintain, and protect natural and agricultural ecosystems. fig. 4: root:shoot ratio for plants with and without rhizobia. bars are means, stars represent statistical difference based on t-test within salinity treatment group (significance: * p<0.05, *** p<0.001). lower (rhizobia) and uppercase letters (fertilizer, no rhizobia) show significant differences among experimental plants according to post-hoc analysis (tukey’s hsd, p<0.01) after one-way anova. discussion the aim of this study was to quantify the response of a natural plant-rhizobia symbiosis to a gradient of soil salinity. we found that rhizobia-free but nitrogen-supplemented plants (r-) significantly outperformed plants with rhizobia (r+) at elevated soil ec levels, indicating that either the natural symbiosis or bradyrhizobium per formance is impaired – or both. rhizobia colonize legume roothairs through signaling interactions involving the production of nod 308 d.j. ballhorn, e.r. wolfe, j. tyler, w. ronan, s. sands-gouner, c. shaw, m.a. balkan, s. kautz such future studies on the effects of salt stress on plant-rhizobia interface should include analysis of biochemical and molecular parameters on both sides of the interaction. conclusions in this study, we showed that the symbiosis between bradyrhizobium and wild lima bean interactions displays sensitivity to even low level salt stress. rhizobial inoculation is standard practice for growing legumes and the application of nitrogen can be lessened with appropriate combination of host plants and rhizobial strains. our study suggests that the salt tolerance of such interactions is an important factor which needs to be considered for the development of sustainable agriculture approaches. acknowledgements funding by the national science foundation (nsf) to djb (grants ios 1457369, 1656057, and deb 1501420) is gratefully acknowledged. conflicts of interest the authors declare that they have no conflicts of interest. references de araujo, a.s.f., de almeida lopes, a.c., teran, j.c.b.m.y., palkovic, a., gepts, p., 2016: nodulation ability in 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rhizobia in extensive fields. agric. ecosyst. environ. 232, 119-128. doi: 10.1016/j.agee.2016.07.006 zahran, h.h., 1999: rhizobium-legume symbiosis and nitrogen fixation under severe conditions and in an arid climate. microbiol. mol. biol. rev. 63, 968-989. zahran, h.h., sprent, j.i., 1986: effects of sodium chloride and polyethy lene glycol on root-hair infection and nodulation of vicia faba l. plants by rhizobium leguminosarum. planta 167, 303-309. doi: 10.1007/bf00391332 address of the corresponding author: daniel j. ballhorn, e-mail: ballhorn@pdx.edu © the author(s) 2018. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 89, 315 321 (2016), doi:10.5073/jabfq.2016.089.043 1department of food technology, pir mehr ali shah, arid agriculture university rawalpindi, pakistan 2department of agricultural sciences, university of haripur, pakistan 3department of botany, pir mehr ali shah, arid agriculture university rawalpindi, pakistan 4college of agriculture, bahauddin zakariya university, bahadur campus layyah, pakistan photo-induced changes in quality attributes of potato tubers during storage kashif sarfraz abbasi1, tariq masud1, abdul qayyum2*, asif ahmad1, ayaz mehmood2, yamin bibi3, ahmad sher4 (received august 7, 2016) * corresponding author summary the retail display of potato tubers is carried out in supermarkets under additional light sources to impart aesthetic value and consu mer’s attention, however, is associated with potato greening and associated disorders. the objective of this study was to identify a most appropriate light source for potato variety ’lady rosetta‘ along with photo-induced changes in different quality parameters. potato tubers were placed for 27 days at ambient storage (25 ± 2 oc) under different light sources i.e. blue, fluorescent, green, mercury and red along with dark storage, which also served as normal control. in general, quality parameters, such as sugars, chlorophyll, total glycoalkaloids, increase while attributes, such as starch and ascorbic acid decrease during the storage period. the initial increase followed by final decline has been observed in parameters, such as total phenolic contents and radical scavenging activity. the results showed maximum retention of different quality attributes in dark potato storage. amongst different light sources mercury and green light retained appreciable retention of different quality parameters with non-significant difference estimated between them in most of the studied parameters. storage of potato under fluorescent, red and blue light proved to be precarious due to skin discoloration. overall results revealed tuber sensitivity to different colored light along with their potential storage stability in the retail markets. keywords: potato; light; quality attributes; storage stability introduction the worldwide consumption of potatoes is ascribed to its lusciousness and high nutritive value (rytel et al., 2005). it produces more dry matter per hectare than the major cereal crops, such as wheat, rice, etc. (gravoueille, 1999) thus considered as an important staple crop in most parts of the world. potatoes are a rich source of carbohydrate with starch being the key ingredient or the main form, which serves as an inexpensive source of energy (lachman et al., 2001). in addition, it is also considered as good source of high quality protein such as lysine (friedman, 2004). four of the six main vitamins required in our daily diet are present in potatoes, which highly indicates the nutritious importance of the crop. the predominant vitamins found in potatoes are thiamin, riboflavin, niacin and an antioxidant ascorbic acid. ascorbic acid is vulnerable to heat and light, thus its loss during storage is considered as a major index of quality deterioration (burlingame et al., 2009). in addition, potatoes are the second most imperative source of vitamin b6 for the elderly who are particularly at risk of chronic diseases. vitamin b6 influences hormonal synthesis, erythrocyte production, immune mo dulation and central nervous system functions. in addition, it is also important for treatment of various chronic diseases, such as sickle cell anemia, asthma, and cancer (kolasa, 1993). potato is low cholesterol, high potassium food with significant antioxidants potential and thus capable of protecting human beings against cardiovascular diseases and cancer (lachman et al., 2000). polyphenols, in addition, are the most common dietary antioxidants (lachmann et al., 2008) being efficient as reducing agents, metal chelators and reactive oxygen species quenchers in a biological system. it is impossible to prevent potato tubers from light exposure during the marketing chain and commercial processing. the response, however, presents remarkable changes in the physiology of the potato tuber and deteriorates its quality by cold sweetening, sprouting or tuber greening due to excessive chlorophyll accumulation and formation of toxic steroidal glycoalkaloids (sengul et al., 2004). glycoalkaloids are present as alpha-solanine and alpha-chaconine in marginal tuber layers. however, with the increased amassing of the compounds, significant economic losses coupled with food safety hazards are observed. presently, high glycoalkaloids (ga) intake is considered as grave food safety concern and with the increasing demand for fresh and processed potatoes the threat has increased manifold (percival, 1999). excessive glycoalkaloids accumulation leaves a bitter taste, if the concentration exceeds a specific level, with upper safe intake limit for a human is 20 mg 100 g-1 f.w (nema et al., 2008). excessive glycoalkaloids intake results in diarrhea, vomiting, fever, rapid pulse, colic pain, low blood pressure, gastro enteritis, and neurological disorders (slanina, 1990). although photo-induced chlorophyll formation in potatoes is not a serious threat for human body, but green potatoes appear to be highly unattractive in comparison to normal red skinned or white skinned potatoes. the unappealing green color of the potatoes reduce their market value and according to research carried out by british potato council, £ 6 million loss has been borne by the grower due to its green pigmentation (anon., 1998). changes in the quality attributes of potato tubers under different colored light sources have been an important point of interests for the retailers. the present study was designed to identify the best light source along with the transition in the vital attributes of potato variety ‘lady rosetta’ at ambient temperature storage. materials and methods potato variety ’lady rosetta‘ was harvested from potato research institute, sahiwal (punjab, pakistan). the experimental material was shifted to the postharvest technology laboratory, department of food technology pmas-arid agriculture university rawalpindi, pakistan. potatoes were washed, sorted and graded into the homogenous lot before they were subjected to the different analytical trials. the selected tubers were cured for one week at a temperature between 15-20 oc. potato tubers were placed under different light sources in specially designed cabinets. the trial was divided into the following set of treatments: 316 k.s. abbasi, t. masud, a. qayyum, a. ahmad, a. mehmood, y. bibi, a. sher t1 = blue light (bl) t2 = fluorescent light (fl) t3 = green light (gl) t4 = mercury light (ml) t5 = red light (rl) t6 = dark (d) potatoes were placed under the lamp of 20 w of different light sources maintained at a distance of 1 m during the complete storage duration (27 days) except in t6. all the tubers were barred exposed to different light sources and rotated at 24 hours interval to ensure maximum skin exposure. 1 kg of potato tubers was placed in each replication, and 3 kg tubers were maintained in each treatment for per day analysis. in total, 30 kg of potatoes were left in each treatment and were subsequently subjected to different physicochemical analyses at three days interval. all the potatoes were stored at temperature and relative humidity maintained around 25 ± 2 oc and 70 ± 5 % respectively before being subjected to different analytical estimations. glucose: glucose estimation was carried out by glucose test strips imported from snack food association, (arlington, virginia usa) along with the color chart expressing glucose percentage. the test strip color change was correlated with the color chart, and the values were expressed in percentage. the tests were conducted in triplicate. total sugar: lane and eynon titration method using fehling’s solution was employed for the estimation of reducing sugar (rs), nonreducing sugar (nrs) and total sugars (ts) contents as reported in aoac (1990) method no. 925.35. ten grams sample was diluted with 100 ml water, stirred thoroughly to dissolve all suspended particles, subsequently filtered in 250 ml volumetric flask. 100 ml of prepared solution was transferred into conical flask along with 10 ml of diluted hcl and boiled for 5 minutes. the resultant solution was cooled and neutralized with 10 % naoh and made up to volume in 250 ml volumetric flask. the solution was titrated against fehling’s solution, and readings were recorded up to brick red end point. the readings were calculated as under: total sugar (%) = 4.95 (factor) × 250 (dilution) × 2.5 / weight of sample × titre × 10 × 100 starch: starch estimation was carried out by making the tuber sugar free by the repeated extraction with 80 % isopropanol. tubers were dried at 70 oc and the starch was hydrolyzed by 60 % perchloric acid. the glucose was estimated spectrophotometrically at 620 nm by using anthrone reagent as described by kumar et al. (2005). ascorbic acid: ascorbic acid (aa) estimation was carried out by the titrimetric method by employing 2, 6, dichlorophenol indophenol dye (redox dye) as explained in aoac (1990) method no. 967.21. 10 g representative sample was taken in a beaker and made up to volume with 100 ml 3 % phosphoric acid and filtered. 10 ml of filtrate was titrated with the standard dye solution till pink end point. the ascorbic acid contents were quantified as under: aa (mg 100 g-1 f.w) = dye factor × titration × volume made up / weight of sample × volume of filtrate × 100 dye standardization: 5 ml of standard ascorbic acid solution was diluted with 5 ml of metaphosphoric acid (3 %) and titrated with dye solution till pink color endures for 10 seconds. dye factor was estimated (mg aa / ml of dye) as: dye factor (d.f) = 0.5 / titration. chlorophyll: chlorophyll extraction from different tuber samples was carried out by spectrophotometric grade 99.5 % acetone (sigmaaldrich) and subsequent quantification was done in spectrophoto meter (ce-2021, 2000 series cecil instruments cambridge, england) as illustrated by percival (1999). 5 g representative (selected from each side of potato tuber) lyophilized tissues were ground to a fine powder with the help of mortar and pestle. the sample was transferred to test tubes followed by extraction with 10 ml of acetone. the extract was vortexed then stored at 4 oc for 24-72 hours. after storage, chilled extracts were again vortexed and centrifuged for 15 min at around 2500 × g. the supernatant was collected for chlorophyll determination. chlorophyll content was measured in 1 cm cuvettes at a647 and a664.5 using spectrophotometer (ce-2021, 2000 series cecil instruments cambridge, england). total chlorophyll concentrations were expressed as mg 100 g-1 d.w from the following equation: total chlorophyll = 17.90 (a647) + 8.08 (a664.5) total glycoalkaloids: the total glycoalkaloids (tga) determination was carried out by the method described by grunenfelder et al. (2006). ground lyophilized potato tissue (500 mg) was extracted in 10 ml of 80 % ethanol at 85-90 oc for 25 minutes. the extract was filtered and reduced to 3-5 ml on rotary evaporator at 50 oc. each extract was rinsed twice with 3 ml of 10 % (v/v) acetic acid and then centrifuged at 10,000 × g for 30 minutes at 10 oc. the ph of the supernatants was adjusted at 9.0 with nh4oh. the extract was refluxed at 70 oc for 25 minutes followed by overnight storage at 4 oc temperature. the extract was similarly centrifuged as earlier, after discarding the supernatants the resulting pellets were dissolved in 0.5 ml of 7 % (v/v) phosphoric acid and stored at -20 oc. the total glycoalkaloids were estimated by adding 200 μl of extract in 1 ml of 0.03 % (w/v) in concentrated phosphoric acid. the contents were allowed to settle for 20 minutes, and absorbance was measured at 600 nm. tga concentrations were quantified based on α-solanine (sigma-aldrich) standard curve using a ce-2021, spectrophoto meter (cecil instruments cambridge, england) and expressed as mg tga 100 g-1 d.w. total phenolic contents: total phenolic contents (tpc) estimated as gallic acid equivalent (gae) were carried out by folin-ciocalteu (fc) assay as explained by lachmann et al. (2008) with few modifications. tubers randomly selected were freeze dried and then extracted with 80 % ethanol. 2 g extract was quantitatively converted into 100 ml volumetric flask and adjusted with 80 % ethanol. in 5 ml of the sample slightly diluted with distilled water, 2.5 ml of fc and 7.5 ml of 20 % solution of sodium carbonate were added. contents were allowed to settle for 2 hours and absorbance was measured at 765 nm using a ce-2021, spectrophotometer (cecil instruments cambridge, england). total phenolic contents were quantified by standard calibration curve derived from the absorbance of known gallic acid concentration (10-100 ppm). results were articulated as mg gae 100 g-1 d.w. radical scavenging activity (rsa): antioxidant activity was mea sured as radical scavenging activity (rsa) using the method described by singh and rajini (2004) that involves electron transfer reaction based assay by employing free radical 2, 2-diphenyl-1picrylhydrazyl (dpph). five mg of freeze-dried potato extract was incubated with 1.5 ml of dpph solution (0.1 mm in 95 % ethanol). the reaction mixture was properly shaken and allowed to stand for 20 minutes at ambient temperature. the absorbance of the resultant mixture was determined at 517 nm against blank. the radical scavenging activity was determined as a decrease in the absorbance of dpph using the following equation: radical scavenging activity (%) = 1 asample 517 nm / acontrol 517nm × 100 statistical analysis: date obtained as the mean of three replications were statistically analyzed by two-factor factorial in completely randomized design (crd), and treatments and storage intervals mean comparisons were carried out by duncan multiple range test using m-stat-c statistical software as described by steel et al. (1997). photo-induced storage of potato tubers 317 results and discussion the general trend was an increase in glucose contents in all treatments except under dark storage. the treatment means exhibited a maximum increase in fluorescent and red lights and were found at par statistically (α-0.05). minimum glucose retention was demonstrated in dark followed by green and mercury lights. storage interval means showed minimum glucose contents at the start and maximum at the end of storage period. the interaction between treatments and storage intervals revealed a significant increase in glucose contents during the last week of storage under blue, fluorescent and red lights (fig. 1). the increase in glucose contents in tubers initiated early under blue, fluorescent and red illuminations and remained two folds than the rest of treatments by the end of 3rd week. green and mercury lights retained moderate increase in glucose contents during most of the storage period. glucose contents recorded at the end of storage in the dark were found lower than that recorded in fluorescent and red lights by the end of 2nd week and found lowest throughout the storage period. the glucose contents accumulation in potato tubers is associated with the starch hydrolysis during the storage period (sonnewald, 2001) and accelerated during tuber stress due to exposure to high energy illuminations (percival, 1993). the greater increase in glucose contents during storage under fluorescent, red and blue lights as compared to other treatments might be due to the increased relative degradation of starch contents. the results were also in close confirmation with the findings of chen and setter (2003) who reported decreased glucose contents in potato tubers under dark storage. total sugar accumulation in potato tubers under different illuminations progressively increased during storage. this increase was found in consistence with the trend observed in glucose contents. the treatment means showed maximum sugar accumulation in fluorescent followed by storage under blue and red lights (α-0.05). storage interval means showed a non-significant increase in sugar content during 1st week followed by a prominent increase during the rest of storage period. the interaction between treatments and storage intervals showed maximum sugar accumulation under fluorescent light while minimum estimated in dark at the end of the trial (fig. 2). in general, total sugar increased under all illuminations, however, the increase was highly significant under fluorescent, blue and red lights. in contrast, a steady increase was observed under mercury and green lights. storage under dark at the end of storage period retained lowest sugar contents than that estimated under all other illuminations. in terms of their sugar accumulation blue and red illumination were found same during most of the storage period. continuous exposure of potato tubers to different light sources caused sugar accumulation due to starch hydrolysis mediated through tuber stress (percival, 1999). the initial increase in sugar contents in response to different illuminations during storage has also been reported by olson (1996). dale et al. (1993) reported elevated sugar contents in potato exposed to continuous illumination as compared to those placed under dark. the moderate sugar contents identified under green and mercury lights might be due to lesser starch hydrolysis owing to their low energy spectrums, which have also been confirmed by nema et al. (2008). data related to starch contents in response to different illumination revealed non-significant initial increase followed by a progressive decline with the increase in storage period. the treatment means showed minimum starch contents in blue, fluorescent and red lights and were found similar (α-0.05). dark storage retained maximum starch contents at the end of storage followed by green and red lights. the storage interval means showed maximum starch content during 1st week storage and then declined significantly by the end of sto rage period. the interaction between treatments and storage intervals showed minimum starch contents in blue, fluorescent, and red lights on last day storage (fig. 3). in general, after an initial increase all the treatments experienced starch depletion in response to different illuminations. however, the decrease was highly prominent in red (17.90 %), blue (17.94 %), and fluorescent (18.10 %) lights as compared to other treatments. the minimum decline in starch contents was reported in the dark (18.59 %) followed by green (18.42 %) and merfig. 1: effect of different light sources on glucose. vertical bars show ±se of means (n=3). the interaction between storage duration and light sources was significantly different at p ≤ 0.05. fig. 2: effect of different light sources on total sugar. vertical bars show ±se of means (n=3). the interaction between storage duration and light sources was significantly different at p ≤ 0.05. fig. 3: effect of different light sources on starch (%). vertical bars show ±se of means (n=3). the interaction between storage duration and light sources was significantly different at p ≤ 0.05. 318 k.s. abbasi, t. masud, a. qayyum, a. ahmad, a. mehmood, y. bibi, a. sher cury (18.27 %) illuminations at the end of storage. the initial increase in starch contents might be due to the tendency of freshly cure potato to accumulate dry matter primarily in the form of starch (kaul et al., 2010). the metabolism of carbohydrate contents during post-harvest storage is very important both in table and processing potato varieties (herrman et al., 1996). the quality of potato tubers keeps on changing due to depletion of starch and formation of corresponding sugars during storage (nourian et al., 2003). nema et al. (2008) reported that continuous exposure of tuber to high energy wavelengths cause physiological stress characterized by increased rate of respiration followed by starch depletion. maximum starch depletion in potato under different high energy illuminations (fluorescent, blue and red) during the present study confirmed the findings reported above. the general decline in starch content in different treatments might be attributed to the consumption of respiratory substrate (starch) during post-harvest storage which has also been reported by abbasi et al. (2015). the general trend showed reduction in ascorbic acid (aa) contents in response to different illuminations during the storage time. the treatment means showed a significant difference between them with maximum and minimum aa retention in dark and fluorescent light, respectively (α-0.05). the storage interval means showed a significant difference in aa contents with maximum retention at the start and minimum recorded at the end of the trial. the interaction between treatments and storage intervals showed appreciable retention of aa in dark followed by green during most of the storage period. significant reduction in aa contents was observed in fluorescent and red lights after one-week illumination (fig. 4). in the present study, exposure of potato tubers to different illuminations caused a significant reduction in aa. the reduction was highly significant in fluorescent light after mid storage i.e. 21.97 mg 100 g-1 which lasted up to 17.8 mg 100 g-1 by the end of one-month storage. by the end of one-month storage tubers, exposure to blue and red lights retained 19.13 mg 100 g-1 and 19.73 mg 100 g-1 aa contents, respectively. moderate reduction of aa contents also reported in the case of green (21.70 mg 100 g-1) and mercury (21.40 mg 100 g-1) illuminations during the same storage period. dark storage of potato retained maximum aa contents and the eventual decline observed was only around 22.37 mg 100 g-1. ascorbic acid is considered as important dietary antioxidant vitaminand known to decline during post-harvest storage period in fruits and vegetables (lee and kader, 2000) due to oxidation (piga et al., 2002), photodegradation (leskova et al., 2006) or utilization as a respiratory substrate (kader, 2002). owing to its high degree of sensitivity it is considered as an imperative index of quality in fruits and vegetable during storage and food processing (ozkan et al., 2004). in addition, aa depletion has been associated with reduced nutritional quality; therefore their assured stability during storage has been a major concern of the post harvest technologists (abbasi et al., 2016 a). dale et al. (2003) reported that aa contents decrease in horticultural products due to light exposure, heat; low relative humidity and prolonged storage time, hence, are critical considerations in optimizing suitable post-harvest storage conditions. similar results were reported in the present study by maximum retention of aa in dark storage as compared to different illuminations. chlorophyll contents continue to increase significantly during the storage time in response to different light exposures. the treatment means showed maximum chlorophyll accumulation under fluorescent followed by red and blue lights (α-0.05). considerable chlorophyll contents were estimated in red followed by restrained contents identified under green and mercury lights. chlorophyll contents remained at a minimum level under dark storage. the interaction between treatments and storage intervals demonstrated minimum contents witnessed in most of the treatments by the end of 1st week storage, whereas maximum contents were estimated in blue, fluorescent and red lights by the end of last week (fig. 5). chlorophyll contents increased progressively under fluorescent, blue and red illuminations and showed steady increase under dark during the storage period. the contents started to increase in high energy illuminations within 1st week of storage particularly in fluorescent light, i.e. 1.283 mg 100 g-1. in contrast, lowest chlorophyll contents were estimated in the dark i.e. 0.721 mg 100 g-1 during the same storage period. a similar trend continued with maximum contents that were recorded under fluorescent light i.e. 3.64 mg 100 g-1. this significant linear increase in the chlorophyll contents was more than six folds over the complete storage period under fluorescent light. the response of potato to exposure to red (3.219 mg 100 g-1) and blue (3.231 mg 100 g-1) illuminations accumulated considerable chlorophyll contents as compared to mercury (1.99 mg 100 g-1) and green (1.66 mg 100 g-1) lights at the end of storage. in general, all different kind of illuminations caused an increase in chlorophyll contents at erratic pace with no indication of termination during the complete trail. exposure of potato tubers in response to light caused the formation of chlorophyll in cortical parenchyma due to the conversion of amyloplast into chloroplast (pavlista, 2001).the extent of greening in retail outlets emphasizing the development and realization of the appropriate light source to maintain desirable quality attributes in fig. 4: effect of different light source on ascorbic acid. vertical bars show ±se of means (n=3). interaction between storage duration and light sources was significantly different at p ≤ 0.05. fig. 5: effect of different light sources on chlorophyll. vertical bars show ±se of means (n=3). the interaction between storage duration and light sources was significantly different at p ≤ 0.05. photo-induced storage of potato tubers 319 potato tubers. percival (1999) compared the response of different tuber varieties to various light sources and reported irrespective of variety maximum chlorophyll accumulation under fluorescent and sodium illuminations as compared to mercury light and dark. grunenfelder et al. (2006) exposed different colored potato varie ties to fluorescent lights of similar intensity and found discoloration of periderm in all of them due to a significant increase in chlorophyll contents. the present study demonstrated significant chlorophyll accumulation in potato tubers due to various illuminations. our results concluded that the replacement of fluorescent light with green or mercury illuminations in retail displays, might cause a reduction in potato greening as also reported by researchers above. dark potato storage demonstrated appreciable tuber quality due to minimum chlorophyll accumulation and thus might be recommended for prolonged tuber storage, which has also been concluded by different researchers like nema et al. (2008) and machado et al. (2007). data related to total glycoalkaloids (tga) showed progressive increase under different illuminations during storage. the treatment means showed a significant difference between their tga contents with maximum retention in fluorescent followed by blue and red lights, while minimum tga contents were identified under dark storage (α-0.05). the storage interval means showed minimum tga contents during 1st week and then progressively increased until the end of storage. a significant interaction was observed between treatments and storage intervals with highest tga contents estimated in fluorescent during last week (fig. 6). the tga contents increased throughout the storage period irrespective of treatments, however, the rate of increase in tga contents was higher in fluorescent, blue and red light illuminations. the increase in tga contents under fluorescent light was maximum (70.40 mg 100 g-1 d.w) and estimated around 8-folds as compared to the 2.5-folds increase in the dark by the end of storage period. the increase in tga contents under fluorescent illumination at the end of storage surpassed the safe limits described for human intake i.e. 20 mg 100 g-1 f.w (mensinga et al., 2005). in general, percentage increase in tga within different sto rage interval was found highest in last week. minimum tga accumulation was identified in the dark (18.90 mg 100 g-1 d.w) followed by mercury (28.03 mg 100 g-1 d.w) and green (30.93 mg 100 g-1 d.w) illuminations. tga contents estimated in the present study regarding solanine equivalent are affected by different illuminations and are also associated with the elevated chlorophyll contents. grunenfelder et al. (2006) studied the increase in tga and chlorophyll contents under fluorescent light and concluded parallel but independent de velopment of both compounds. machado et al. (2007) compared tga accumulation in potato tubers under different illuminations during postharvest storage. she found maximum and minimum tga contents under fluorescent light and dark respectively as also observed in the present study. another investigation carried out by percival et al. (1999) observed light-induced tga accumulation in potato tubers under four different illuminations. he declared maximum contents under fluorescent and sodium lights and minimum under dark. our results in the present study also confirmed the previous finding as steady tga contents in the dark as compared to significant increase under fluorescent light. the trial also exhibited a parallel association between chlorophyll and glycoalkaloids accumulations in potato tubers. the process is however found independent due to increased tga contents in blue to red and green to mercury lights having less corresponding chlorophyll contents during the same storage period. total phenolic contents (tpc) increased steadily during the storage period in response to different illuminations. however, the increase is followed by a considerable decline in blue, fluorescent and red lights. in general non-significant difference in treatment means was observed except in mercury and dark storage (α-0.05). the storage interval means, however, showed significant difference with maximum and minimum tpc identified at the end and the start of the experiment. highly significant interaction was observed between treatments and storage intervals after the mid storage period (fig. 7). total phenolic contents increased during the start of sto rage in all the treatments, and the initial rise was found indepen dent of different illuminations. in general non-significant difference was observed in most of the treatments till 9th day storage. tpc contents significantly increased in all the treatments by the end of 2nd week storage, which was followed by progressive decline in fluorescent, blue and red illuminations during the last week of storage. tpc increased continuously under green, mercury and dark storage with no sign of cessation during the storage period. however, the rate of tpc increase during the 1st half of storage was found slower as compared to high energy illuminations (blue, fluorescent and red lights). total phenolic contents showed varied retention under different illuminations during the storage period. light is considered as an important factor causing the biosynthesis of phenolic compounds facilitated by the activity of phenylalanine ammonia-lyase enzymes (lewis et al., 1998). in addition, it is also known to initiate the anthocyanin and chlorogenic biosynthesis pathways contributing to the total phenolic contents in potato tubers (griffiths, 1995). the swift initial increase in tpc under different light sources and steady tpc fig. 6: effect of different light sources on total glycoalkaloids. vertical bars show ±se of means (n=3). the interaction between storage duration and light sources was significantly different at p ≤ 0.05. fig. 7: effect of different light sources on total phenolic content. vertical bars show ±se of means (n=3). interaction between storage duration and light sources was significantly different at p ≤ 0.05. 320 k.s. abbasi, t. masud, a. qayyum, a. ahmad, a. mehmood, y. bibi, a. sher under dark partially confirmed the findings reported by different researchers above. these results, however, negated the investigation reported by reyes and zevallos (2003) who reported no considerable effect of light on the tpc accumulation in purple-fleshed potato tubers. the possible reason might be due to the varietal difference and lack of their comparative study under different illuminations. tubers placed under green and mercury lights retained appreciable tpc at the end of storage probably due to lack of tuber stress and moderate respiration rate. the decline in tpc contents under high energy illuminations like fluorescent and blue at the end of storage might be attributed to the tuber stress due to high metabolic rate, increased rate of respiration causing an eventual decline in tpc contents. radical scavenging activity (rsa) estimated as % inhibition of dpph showed slight initial increase followed by gradual decrease under all illuminations except in the dark. treatment means showed that dark storage maintained maximum activity followed by green and mercury lights, while minimum rsa activity was shown under fluorescent followed by blue lights (α-0.05). storage interval means showed maximum activity till 2nd week and then steadily declined till the end. the interaction between treatments and storage intervals was initially found less significant and then became highly significant by the end of storage period. the maximum activity was expressed in green and dark during 3rd and 2nd week storages, respectively (fig. 8). in general rsa activity exhibited initial increase till mid storage period and finally declined to form a sort of para bolic curve. however, the rate of increase and decrease in activity was considerably affected by different illuminations. radical scavenging activity increased during the first week and attained maximum by the second week in blue (45.43 %), fluorescent (45.80 %) and red (47.57 %) lights followed by progressive decline afterward. the trend remained same at variable pace in green (48.73 %), mercury (47.33 %) and red (49.47 %) where they showed maximum activity by the end of the third week followed by a steady decline onwards. overall results revealed maximum retention of rsa in dark storage (45.57 %) followed by green (43.60 %) and mercury (42.77 %) lights at the end of storage period. it is eventually observed that, except in dark storage, potato tubers placed under different illuminations lost considerable rsa activity after the completion of storage period. rsa activity corresponds to the antioxidant potential retained by the potato tubers and is associated with the presence of different functional components, such as phenolic compounds, carotenoids, ascorbic acid and tocopherols (abbasi et al., 2016 b). in the present study, storage under different illuminations conferred diverse effects on these antioxidant components as attributed to the parallel accumulation of phenolics and depletion of ascorbic acids. the prolonged exposure of potato tubers to high energy illuminations might confer tuber stress consequently resulted in the loss of phenolic substrate due to the increased polyphenol oxidase activity (bryant, 2004). the significant retention of rsa activity at the end of storage under dark as compared to dif ferent illuminations might be due to the appreciable retention of dietary antioxidants, such as ascorbic acid, total phenolic contents etc. hejtmankova et al. (2009) and lachmann et al. (2008) have also expressed this significant correlation between rsa activity and these functional components. conclusion it is concluded that during thirty day storage (25 ± 2 oc) under different illuminations (blue, fluorescent, green, mercury, red, dark), potato tubers presented variable response regarding their investigated quality parameters. best results were identified in potato tubers kept under dark with minimum loss of quality parameters. storage under different illuminations showed maximum retention of quality para meters in green and mercury lights with better storage life. tuber stability was found highly susceptible to fluorescent light with least retentions of quality parameters. storage under red and blue light also resulted in decreased storage life with the loss of quality attributes studied. references abbasi, k.s., masud, t., qayyum, a., khan, s.u., ahmad, a., mehmood, a., farid, a., jenks, m.a., 2016a: transition in tuber quality attributes of potato (solanum tuberosum l.) under different packaging systems during storage. j. appl. bot. food qual. 89, 142-149. abbasi, k.s., masud, t., qayyum, a., khan, s.u., abbas, s., jenks, m.a., 2016b: storage stability of potato variety lady rosetta under comparative temperature regimes. sains malay. 45, 677-688. abbasi, k.s., masud, t., ali, s., khan, s.u., mahmood, t., qayyum, a., 2015: sugar-starch metabolism and antioxidant potential in potato tubers in response to different antisprouting agents during storage. potato res. 58. 361-375, doi: 10.1007/s11540-015-9306-4. anon., 1998: potato industry digests, british potato council. u.k. aoac (association of analytical chemists), 1990: official methods of analysis. 15th ed., virginia 22201, arlington, usa. bryant, p., 2004: optimising the postharvest management of lychee (litchi chinensis sonn.). a study of mechanical injury and desiccation.ph.d thesis.university of sydney. burlingame, b., mouille, b., charrondiere, r., 2009: nutrients, bioactive non-nutrients and anti-nutrients in potatoes – a critical review. j. food comp. anal. 22, 494-502. doi: 10.1016/j.jfca.2009.09.001. chen, c.t., setter, t.l., 2003: response of potato tuber cell division and growth to shade and elevated co2. ann. bot. 91, 373-381. doi: 10.1093/aob/mcg031. cip (international potato center), 1997: annual report, 239-264. dale, m.b.f., griffiths, d.w., bain, h., todd, d., 1993: glycoalkaloid increase in solanum tuberosum on exposure to light. ann. appl. biol. 123, 411-418. dale, m.f.b., griffiths, d.w., todd, d.t., 2003: effects of genotype, environment, and postharvest storage on the total ascorbate content of potato (solanum tuberosum) tubers. j. agri. food chem. 51, 244-248. doi: 10.1021/jf020547s. friedman, m., 2004: analysis of biologically active compounds in potatoes (solanum tuberosum), tomatoes (lycopersicon esculentum) and jimson weed (datura stramonium) seeds. j. chromatogr. a. 1054, 143-155. doi: 10.1016/j.chroma.2004.04.049. fig. 8: effect of different light sources on radical scavenging activity (%). vertical bars show ±se of means (n=3). the interaction between storage duration and light sources was significantly different at p ≤ 0.05. photo-induced storage of potato tubers 321 gravoueille, j.m., 1999: utilization en la alimentacion humana (use in the human feeding). in: rouselle, p., robert, y., crosnier, j.c. 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seed and ware storage conditions at tigonikenya. proceedings of the triennial symposium, international society for root crops (istrc)-ab, lilongwe, malawi, 515-579. address of the corresponding author: e-mail: aqayyum@uoh.edu.pk © the author(s) 2016. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 91, 135 144 (2018), doi:10.5073/jabfq.2018.091.019 1laboratory of ornamental plants and vegetable crops, faculty of agriculture and biotechnology, utp university of science and technology, bydgoszcz, poland genetic resources and selected conservation methods of tomato dariusz kulus1* (submitted: march 21, 2018; accepted: april 18, 2018) * corresponding author summary tomato is one of the most popular vegetable crops. however, over time, the species has suffered a strong genetic diversity reduction and domestication bottlenecking. this growing trend is known as the genetic erosion. the human intervention on the genetic erosion intensification is high and has severe implications on the future programmes of management and use of s. lycopersicum biodiversity. the wild tomato species (especially accessions originating from the andes to mesoamerica) harbour many valuable genes, which have been lost among the cultivated ones. therefore, there is an increasing interest to mine new alleles from the interspecific gene pool of lycopersicon section. sustainable genetic diversity management constitutes a basis for crop improvement, classification and protection. moreover, conservation of plant genetic resources is crucial to food security, as well as pharmaceutical industry. there are a few strategies developed which address the preservation of tomato genetic resources. in situ and ex situ conservation are the two main complementary methods of biodiversity protection. the aim of this review is to summarise the most recent information about tomato genetic resources, genetic erosion phenomenon, as well as some traditional and modern preservation strategies. key words: biodiversity; dna library; genetic erosion; solanum lycopersicum l.; sustainable management; in vitro tissue culture introduction the importance of tomato the cultivated tomato (solanum lycopersicum l.) is a dicotyledo nous perennial or annual plant, which belongs to the large nightshade family – solanaceae (van eck, 2018). the solanaceae family is the third most important plant taxon, consisting of 96 genera and over 3000 species (koo et al., 2008). nowadays, tomato is the 2nd most important vegetable (after potato) and the 7th among most important crops in the world. it is grown on approximately 5 million hectares from the tropics to within a few degrees of the arctic circle (fentik, 2017). the global tomato production is around 130 million tons, of which 88 million are destined for the fresh market and 42 million are processed (eurofresh, 2017). the species is of enormous economic value reaching billions of dollars (van eck, 2018). due to its high consumption level, it is an important source of mineral salts, vitamins, bioflavonoids and carotenoids (flores et al., 2017). bhowmik et al. (2012) reported that consuming tomatoes can decrease the risk of various conditions, such as cancer, osteoporosis, neurodegenerative diseases and cardiovascular problems. moreover, due to the presence of chlorine and sulphur, tomato has a detoxification effect in the body, helps to replace skin cells and in sun burns (capel et al., 2017). the species importance is reflected by the enormous number of research on all aspects of the crop. since 2000, over a thousand publications related to tomato studies have been published annually (google scholar, 2018). overtime, tomato has been adapted to different growing systems by selection and adjustment of a limited set of traits (the 100 tomato genome sequencing consortium, 2014). application of modern biotechnological tools, such as sequencing technologies, genomic selection and multi-omic analysis, provided good results in the breeding of (a)biotic stress resistant s. lycopersicum plants with amended characteristics (uluisik et al., 2016; fentik, 2017; zhu et al., 2018). development of genetic linkage maps and gwas (genome-wide association studies) made it possible to learn the chromosomal locations of qtl (quantitative trait) genes for improving yield and other complex features, such as: fruit abscission, size, flavour, texture and colour (kulus, 2018). site-directed mutagenesis using genetic approaches, e.g. crispr/cas9 system, also can provide a wealth of resources for crop breeding, as well as for biological research by inducing precise mutation in the first and later generations (ito et al., 2015; pan et al., 2016). for example, crispr/cas9-induced targeted mutagenesis and gene replacement were used to produce long-shelf life tomato lines, parthenocarpic plants and other (ueta et al., 2017; wang et al., 2017; yu et al., 2017). unfortunately, the reduced genetic basis that resulted from extensive inbreeding has impeded tomato improvement (the 100 tomato genome sequencing consortium, 2014). consequently, despite its great meaning, progress in breeding systems and development of highly efficient superior cultivars, tomato suffers a strong genetic basis reduction and domestication bottlenecking. genetic erosion is now a common phenomenon reported in numerous crops, both locally and globally (hoban et al., 2014). it might have severe implications on the future programmes of management and use of s. lycopersicum genetic resources. there fore, to enlarge the genetic variation, breeders recently focus on introgression of genes from wild relatives into the elite cultivars, but so far, this has been quite limited and more actions are required (the 100 tomato genome sequencing consortium, 2014). the aim of this review is to summarise the most recent information about tomato genetic resources, their meaning, genetic erosion phenomenon and development of molecular markers (from isozymes to next generation sequencing) in evaluating tomato genetic diversity, as well as some selected traditional (field collections and seed banks) and modern (in vitro slow-growth banks and dna libraries) in situ and ex situ preservation methods. genetic diversity of tomato genetic diversity estimates constitute a basis for future strategies for crop improvement, sustainable use, classification and conservation. the obtained knowledge can then be applied to increase the genetic variation in base populations by crossing cultivars with a high level of genetic distance and for the introgression of exotic germplasm (labate and robertson, 2012). the genetic diversity in plant species is dependent on the mating system, the domestication history, ecological factors, and the size of the sample being analysed (mazzucato et al., 2008). the lycopersicon section began its initial radiation 7 million years ago (robertson 136 d. kulus and labate, 2006). this small monophyletic clade comprises se veral species, of which only s. lycopersicum was domesticated from s. lycopersicum var. cerasiforme, although s. pimpinellifolium is also casually planted for consumption (giovannoni, 2018). the latter one is the closest relative of the cultivated tomato; genome sequences of both of the above-mentioned tomatoes showed only a 0.6% nucleotide divergence (gerszberg et al., 2015). still, there are 15 more wild species of tomato, including s. arcanum, s. cheesmaniae, s. chilense, s. chmielewskii, s. corneliomulleri, s. galapagense, s. habrochaites, s. huaylasense, s. jugandifolium, s. lycopersicoides, s. neorickii, s. ochranthum, s. pennellii, s. peruvianum, s. sitiens (anderson et al., 2010). since the mid-20th century, approaches based on controlled hybridisation allowed crossing between cultivated and wild tomato compartments (bauchet et al., 2017). however, there are some serious crossability barriers with s. peruvianum and s. chilense that are quite difficult to overcome and require biotechnological methods even in successive generations (beddows et al., 2017). currently the tomato germplasm is distributed throughout the following categories of: modern cultivars, obsolete cultivars, commercial breeding stocks (breeding lines), genetic stocks (e.g. mono somics, alien addition lines), landraces, heirloom varieties or primi tive varieties and wild forms of cultivated species, obtained from spontaneous mutations, natural outcrossing or recombination of pre existing genetic variation (grout and crisp, 1995). in total, more than 10,000 morphologically different tomato cultivars and forms exist (gerszberg et al., 2015). the typical turnover time of com mercial cultivars is approximately five years (bai and lindhout, 2007). cultivated tomato is autogamous, whereas its wild relatives, such as s. peruvianum, s. pennellii or s. hirsutum, are often facultative or obligate out-crossers (kochieva et al., 2002). this gametophytic incompatibility system contributes in greater genetic diversity (the 100 tomato genome sequencing consurtium, 2014). according to miller and tanksley (1990), more genetic variation can be found within a single accession of one self-incompatible tomato species than among all accessions of any one of the self-compatible species, estimated at 75% versus 7%. as for cultivated tomato, old introductions and locally developed cultivars (landraces) in the 1970s present substantially greater genetic variability than the ones produced during the 1990s (mazzucato et al., 2008). landraces usually have a higher number of rare alleles and a lower number of private alleles when compared to contemporary cultivars (corrado et al., 2014), although carelli et al. (2006) observed, that the frequency of rare alleles was similar in brazilian commercial and landrace accessions. the high level of genetic variability within landraces is related to their plasticity (hassan et al., 2013). the genetic structure of tomato over time, several attempts have been made to circumscribe the genetic structure of tomato (kobayashi et al., 2014), including sequencing the genomes of s. arcanum, s. habrochaites, s. lycopersicum, s. pennellii and s. pimpinellifolium (the tomato genome consortium, 2012; the 100 tomato genome sequencing consortium, 2014). all tomato species are diploid with 24 acrocentric to metacentric chromosomes (2n = 2x = 24), except for two natural tetraploid populations of s. chilense (bauchet and causse, 2012). they are considered to have stable genomes in which speciation has evolved primarily by genic changes rather than large-scale chromosomal rearrangements, however, small inversions have been also reported (anderson et al., 2010). genetic variation within tomato species occurs both: intraspecific − within cultivated tomato, and interspecific − between wild species (bauchet and causse, 2012). domestication; i.e. selection of beneficial alleles; has evolved a number of morphological and physiological traits which distinguish cultivated tomato from its wild ancestors. this phenomenon is known as the ‘domestication syndrome’ and includes a more compact growth habit, increased earliness, reduction/loss of seed dispersal, and fruit shape diversity (paran and van der knaap, 2007; sauvage et al., 2017). leaf variation and fruit colour observations are the easiest ways for distinguishing wild tomatoes (which have a low intraspecific phenotypic diversity) from cultivated ones (zhou et al., 2015). those traits are often controlled genetically by a relatively small number of loci on a limited number of chromosomal regions with major phenotypic effect (frary and doganlar, 2003). moreover, morphological characters of tomato can be altered by environmental factors and management practice. therefore, despite domesticated accessions show a range of morphological diversity in comparison to wild species, a strong loss of molecular diversity throughout the whole genome of cultivated tomato is observed (van deynze et al., 2007). genetic erosion – causes and solutions in tomato, the level of genetic diversity in the cultivated gene pool is substantially lower than in other crops and represents a narrow range of the species original variability due to domestication bottle necking which took place in europe 400 years ago (bhattarai et al., 2016). this is because only few tomato seeds were brought back from mexico to europe. those impoverished resources were then re-introduced to america (bauchet and causse, 2012). in the following years, genetic diversity protection was neglected in official policy (hoban et al., 2014). consequently, miller and tanksley (1990) claim that less than 5% of the available species genetic variation exists in modern tomato cultivars. restricted habitat is another issue. most of the tomato wild relatives are endemic in narrow geographical regions; their habitats are usually isolated valleys where they were adapted to particular micro climates and soil types (albrecht et al., 2010). a common pheno menon in wild tomato species is founding of populations by a small number of highly homozygous individuals via seed dispersal, which causes a severe genetic bottleneck (nakazato and housworth, 2011). moreover, due to a drastic reduction of their natural habitats, some wild species, e.g. s. pimpinellifolium, are now endangered (bauchet and causse, 2012). as for the landraces, despite the superior flavour, yield stability in low input agricultural systems, great value for niche markets and sustainable farming, their cultivation is currently limited to gardens for personal consumption and in small-size farms for local markets. the lack of information about the origin and the relationship of landraces are the main limiting factor for their application (corrado et al., 2014). this is unfortunate, since those genetic resources, once lost, are very difficult to reconstitute (coste et al., 2015). as for the commercial tomato cultivars, due to the evolution of highly mechanised farming systems, most of them are the f1 hybrids (kwon et al., 2009). consequently, hybrid sterility is frequently observed. moreover, under selection for research and breeding purposes, the initial already narrow genetic basis of the tomato is even more restricted by the development of superior but few cultivars, which replace more numerous, but less productive vintage and regional varieties (sahu and chattopadhyay, 2017). for example, patil et al. (2010) observed high levels of pair wise similarity (mean = 0.838) within 17 tomato cultivars. also zhou et al. (2015) noticed the high similarity coefficient of the 29 cultivated tomatoes (0.845) by applying est-ssr (expressed sequence tag) markers. this low variability within the species was confirmed by other biochemical and molecular markers (cebolla-cornejo et al., 2013; shirasawa et al., 2015; sahu and chattopadhyay, 2017). on the other hand, managing tomato genetic resources 137 according to sim et al. (2012), the long history of crossing with wild relatives, breeding for various market classes and selection of distinct ideotypes for different production systems has broadened the genetic diversity in contemporary germplasm relative to vintage and landrace germplasm, although similar reports are sparse. a fair conclusion was delivered by labate and baldo (2005), who claim that genetic variation in domesticated s. lycopersium is unevenly dispersed, with rare islands of polymorphism originating from introgression. for example, kwon et al. (2009) observed that the commercial tomato cultivars pic (polymorphism information content) was ranging from 0.21 to 0.88. similarly, 19 azerbaijan genotypes had a genetic similarity ranging from 0.188 to 1.000 (sharifova et al., 2013). nevertheless, the reduction of genetic diversity at important loci can limit improvements in the future (muños et al., 2011). the increasing application of plant breeders rights also has negative implications for plant genetic diversity. seed companies sell crop cultivars under strict legal protection (tripp and heide, 1996). there are about a dozen tomato-breeding companies which are the main players in the world market. for example, in india private seed companies control 90% of the tomato seed market (patil et al., 2010). this leads towards narrowing of the biological diversity, especially in secondary centres of diversity (cebolla-cornejo et al., 2013). at the same time, much of the wild tomato biodiversity is still untouched in the andes. in the 1940s, breeder dr. charlie rick ob served during his expeditions, that this disbanded gene pool is valuable in searching for new genes, breeding for hybrid vigour and in analysing the taxonomy and evolution of the genus (bai and lindhout, 2007). also research conducted by williams and st. clair (1993) showed a much higher diversity in andean s. pimpinellifolium and s. lycopersicum var. cerasiforme populations (where first domestication of tomato took place) than among mesoamerican cultivars (from where tomato was introduced to europe). those fin dings were supported by recent high density snp (single nucleotide polymorphism) genotyping performed by blanca et al. (2015). they pointed ecuadorian and peruvian accessions to represent a pool of unexplored variation. conservation and sustainable use of those wild tomato relatives would benefit from an understanding of genetic diversity and relationships within and between populations, as well as for sampling of the populations for genes of interest (albrecht et al., 2010). seed mixing and pollen contamination can be performed to increase variation within the tomato (cebolla-cornejo et al., 2013). for example, the sexually compatible wild relatives, such as s. pimpinellifolium or s. habrochaites, can be used as donors of useful genes for salt tolerance during seed germination (sifres et al., 2011), resistance genes to clavibacter michiganensis subsp. michiganensis, which causes bacterial canker of tomato, and the pathogen oidium neolycopersici, responsible for powdery mildew (passam et al., 2007). trichome characteristics from s. cheesmanii and s. pennellii affect the behaviour of an aphid myzus persicae or resistance to infection by parasitic giant dodder (cuscuta reflexa) (krause et al., 2017). the mi gene found in s. peruvianum is controlling nematode resistance, while invulnerability to whitefly (bemisia spp.) has been identified in wild populations of s. lycopersicum var. cerasiforme. the jointless j2 allele was already introgressed from s. cheesmanii into many processing cultivars, allowing a large scale mechanical harvest of tomato fruits (bauchet et al., 2017). good fruit quality traits (i.e. soluble solids, sugar and β-carotene content, as well as aromatic fragrance) have also been detected in s. cheesmanii and s. peruvianum, even though the fruits of those species are not usually consumed by humans (zhou et al., 2015; zhang et al., 2016). conservation of genetic resources it is predicted, that the world human population will increase by 50% before 2050. at the same time the surface of arable land is progressively decreasing. tomato characteristics must satisfy the constantly growing consumer’s preference. it also must be suitable for post harvest handling and marketing, even over large distances (passam et al., 2007). consequently, dramatic increase in crop production will be required (wang et al., 2009). plant genetic resources are crucial to food security, as well as pharmaceutical industry (edesi et al., 2017). therefore, it is essential to collect, preserve, evaluate and exchange the genetic variability of tomato (petrović and dimitrijević, 2012). preserving genetic diversity in plants has been performed for over a century (tanksley and mccouch, 1997). to achieve this task, the international treaty for plant genetic resources for food and agriculture was made within the framework of the food and agriculture organization of the united nations in 2004. its main objectives are the conservation and sustainable use of plant genetic resources for food and agriculture production (wang et al., 2009). there are a few fundamental strategies developed which address the conservation of plant genetic resources (fig. 1). fig. 1: there are two complementary systems of germplasm conservation; in situ and ex situ conservation. in situ conservation can refer to on site (in nature) preservation of wild species and on farm conservation of landraces and cultivated genetic resources. dna libraries, seed collections, in vitro conservation and low-temperature storage are under the “umbrella” of ex situ gene banks. 138 d. kulus in situ conservation in situ conservation is used for both on site preservation of wild species and on farm preservation of landraces and cultivated genetic resources. arguments in support on traditional cultivation and sto rage of genotypes include the importance of recognising the roles of environmental factors and the possibility to evolve under natural conditions through crossing with wild or weedy relatives (tripp and heide, 1996; corrado et al., 2014). on the other hand, protection in situ is laborious and expensive since it requires large areas, nur sing, agrotechnology, and it is threatened with loss due to (a)biotic stresses. the tomato hosts over 200 species of pests and pathogens that can cause significant financial losses (bai and lindhou, 2007). the most common diseases of tomato crops include: bacterial scab, spot and wilt, as well as fungal diseases, such as powdery mildew. other main diseases are leaf spot, early blight, leaf mould and wilts. changes in insect’s biotype and disease resistance are becoming a continuing threat to in situ collections (chaudhry et al., 2010). pathogens have to be controlled via chemical compounds, which are not always fully-effective, harmful to the environment and require compliance with chemical-use laws (bai and lindhou, 2007). furthermore, the in situ strategy is climate-dependent; tomato is a warm season plant, requiring high light intensity and a minimum temperature of 10oc to grow (liza et al., 2013). as a result, other conservation strategies are also explored. ex situ conservation tomatoes are well represented in ex situ (off-site) working and informal collections. they are used for research and commercial purposes at the global level, with a range of dozens of thousands of accessions located in seed gene banks (mostly), in vitro laboratories and cryobanks, as well as in the form of dna libraries. conventional gene banks the possibility of maintaining genetic resource in gene banks is attracting great attention. they are mostly based on collections of seeds (or more rarely pollen) stored; in plastic bags, culture jars or cryovials; in controlled conditions and periodically regenerated. gene banks are an important source of publicly available genetic material for plant breeding programmes and other research activities (labate et al., 2009). it is estimated that over 83,000 accessions of the wild and cultivated species of lycopersicon section are maintained in germplasm banks located in over 120 countries, ranking 1st among vegetable species collected (bauchet and causse, 2012). the most important ones include facilities at the cm rick tomato genetics resource center (tgrc), university of california in davis, u.s. (with a large collection of open-pollinated cultivars) and at the united states department of agriculture (usda) or plant genetic resources unit at geneva (pgru), u.s., as well as laboratories in the international board for plant genetic resources (ibpgr) network (sharifova et al., 2013). the universidade estadual do norte fluminense in rio de janeiro (brazil), has a tomato gene bank with accessions that have been maintained for nearly 50 years (gonçalves et al., 2008). the mexican government recently established a national genetic resources center (cnrg) as a component of a long-term strategy for conservation and sustainable use of plant biodiversity (arizaga et al., 2016). the world vegetable center (wvc), in tainan (taiwan) maintains one of the largest collections of lycopersicon germplasm. most of the collection (60%) was harvested from old world regions. in the netherlands, the botanical and experimental garden maintains the most extensive ex situ plant collections of non-tuberous solanaceae species (bgard, 2018). the european cooperative programme for plant genetic resources tomato database maintains passport information of more than 20,000 accessions of numerous tomato species. the tomato collection of european solanaceae database is composed of about 7,000 domesticated and wild lines of s. lycopersicum (eu-sol, 2018). this biological material was provided by international gene banks and by donations from private collections. the accessions were established and phenotyped, accompanied by an ad hoc database. large collections of tomato germplasm are also conserved in russia (vir), japan (nias), peru (dhuna), cuba (inifat) and norway (svalbard global seed vault) (foolad, 2007). other countries with great numbers of stored tomato accessions are: bulgaria, canada, china, colombia, france, germany, hungary, the philippines and spain. they conserve mainly s. lycopersicum germplasm. large germplasm collections are typically duplicated at a second backup location in case the primary collection is lost due to mechanical breakdowns, natural disasters or political disturbance. for example, the usda and tgrc accessions are backed up at the national centre for genetic resources preservation (ncgrp) at fort collins, colorado (89 and 95% of the collections, respectively) (robertson and labate, 2006). the management of tomato germplasm collections requires harvesting representative samples and performing two related activities; long-term storage of high-quality biological material, and regeneration of plants with further selection to replenish seed stocks. therefore, most gene banks maintain also field collections (robert son and labate, 2006). gene banks include also various monogenic stocks and a large pool of tomato mutants, which were either spontaneous or induced by irradiation or chemically (shalaby and el-benna, 2013). for example, tgrc maintains an isogenic tomato ‘mutation library’ containing a total of 13,000 m(2) families (bai and lindhout, 2007). a miscellaneous mutant population is a fundamental resource for exploring gene function (menda et al., 2004). tilling (target induced local lesion in genomes) is a mutagenesis method to generate chemically, via 0.5 1.0% ems (ethylmethane sulphonate), or fast-neutron induced point mutations in genomes. unlike genetic transformation, mutagenesis is random, cost effective and is not submitted to gmo regulation. moreover, the technique allows to rapidly transfer interesting mutations into cultivars that can improve important agrono mic traits in tomato (shirasawa et al., 2015). collections of tomatoes carrying artificially induced genetic variants, with more than 3,000 phenotype alterations catalogued, are publicly available and can be accessed in the solanaceae genome network website (minoia et al., 2010; okabe et al., 2011). traditional gene banks are useful with plant species, which produce orthodox seeds that have a low water content or will survive drying (and optional freezing) during ex-situ conservation. otherwise, seeds must be often regenerated and the samples replaced with new ones. prior to storage, tomato seeds are dried to a level of 5±1% in a room that is maintained at 20% relative humidity at 4-5 oc. working and active collections are stored at 5 oc. longer storage of base collections is possible after (deep)freezing of seeds or pollen (-20; -80 oc). the longevity of such samples depends also on the type of storage container. it should be air-tight but not vacuum-sealed to avoid seed damage. regeneration of plants is conducted once the danger of frosts has passed (robertson and labate, 2006). further selection efficiency can be improved by genetic markers that are associated, through linkage or pleiotropy, with genes or qtls that control the trait(s) of interest (foolad, 2007; kulus, 2018). germplasm banks, like any other facility, have some drawbacks. one of the main concerns of breeders is how to quantify the degree of variability of stored tomato plant resources, especially that during regeneration cross pollinating plants can be contaminated by foreign pollen. furthermore, storage and regeneration steps need consider managing tomato genetic resources 139 ably large numbers of plants and seeds, due to the intermittent viability testing to monitor the progress of viability decrease within the stored accession. to prevent genetic drift, wild or cross-pollinated tomato species require more seeds for storage and regeneration (over 50 regenerated plants per variety), than cultivated taxa (25 plants) (robertson and labate, 2006). the costs of characterising and cataloguing gene bank material are also considerable. high-quality seed production requires constant laborious monitoring for diseases and pests, and timely application of pesticides followed by costly seed processing (separating of seeds from skin and pulp, washing, drying, etc.). the lack of coordination and conflicting passport data is a another drawback for an efficient s. lycopersicum germplasm management (bauchet and causse, 2012). moreover, despite the exchange of germplasm should be free to all those who wish to use them, there is a risk that countries which host important international collections may deny another country access to them, due to political or religious reasons (tripp and heide, 1996). therefore, modern biotechnology-based methods, which can help to overcome some of these problems, will be more frequently implemented for tomato genetic resources conservation. nowadays, there is a possibility of storage under slow-growth in vitro conditions or of cryopreserving tissues in liquid nitrogen (-196 oc) (fao, 2014; coste et al., 2015). in vitro slow-growth storage the development of in vitro culture technique has opened possibilities for various applications after its inception in the 1930s (parmar et al., 2012). tissue banks can be used for medium-term preservation of pathogen-free staple crops and further selection and production of stress-tolerant, high-quality plants (al-abdallat et al., 2017). by reducing the temperature and light intensity in the growth room, it is possible to reduce the growth pace of tomato shoots and the number of subcultures required to one per two-three years. besides the temperature and light regimes, the success of tomato slow-growth in vitro system varies with the nutrient media, concentration and combination of growth regulators and osmotic agents, as well as genotype and explant related factors (liza et al., 2013; bhushan and gupta, 2017). the selected for the purpose of slow-growth biological material cannot be contaminated or excessively hydrated. in this case, source material should be cultured de novo, and the invalid one; immediately eliminated (fao, 2014). schnapp and preece (1986) were the first to develop an in vitro slow-growth of tomato; i.e. decline of shoot length, rooting and callogenesis rating; when the ms (murashige and skoog, 1962) nutrient salts level was lowered to 25%, 50%, or 75% compared to full strength medium and when sucrose was supplied at 0.05%. recently, in vitro preservation of transgenic tomato lines overexpressing the stress-responsive transcription factor slareb1 was studied (al-abdallat et al., 2017). by adding 200 300 mm sucrose into the ms medium, a reduction in tomato plantlets length, leaf number and rooting was reported. the increased levels of sucrose induce an osmotic stress that inhibits the growth of microshoots and extends the subculturing interval due to the restricted cell osmotic potential, reduction of water availability and decrease of cell expansion and division. the application of aba (abscisic acid) was even more effective when compared with sucrose treatment. severe growth retardation phenotypes were observed when microshoots were cultured on ms media supplemented with 8 or 12 μm aba (al-abdallat et al., 2017). a serious drawback of tissue banks, is the fact that tomato is susceptible to somaclonal variation occurrence (poopola, 2015). a number of factors, viz. pre-existing genetic dissimilarities uncovered during in vitro tissue differentiation, stress due to the unnatural in vitro culture conditions, medium components and explant isolation, as well as selection for specific genotypes during plant regeneration stimulate genetic variation in regenerated microshoots (rzepka-plevneš et al., 2010; ali et al., 2017). according to gavazzi et al. (1987), regeneration from in vitro culture leads to a higher number of tomato mutations than application of the chemical mutagen. in vitro-produced s. lycopersicum plants may contain several monogenic mutations with morphologically visible variability, e.g. lethality, altered leaf morphology, absence of anthocyanin, reduced chlorophyll content, variegation and dwarfing (bulk et al., 1990). a second commonly observed variation is tetraploidy, and the third is plants ste rility (ali et al., 2017). therefore, tissue culture system is not desirable for long-term pre servation. it can be used, however, as a source of explants for cryopreservation (storage at cryogenics; at -196 oc to -140 oc). in vitro culture is used as a preparatory phase prior to storage in ln (liquid nitrogen), as well as for recovery phase after rewarming. preservation of tissues in dewar flasks is power-independent (eco-friendly) and cost-efficient, as ln is inexpensive. moreover, explants are stored in a small volume (2-ml cryovial per sample, comprised of even 50 tomato seeds), protected from contamination, and require very little maintenance (except for controlling the level of ln, as it is rapidly evaporating). over time several slowand fast-cooling cryopreservation techniques were developed (kulus and zalewska, 2014). however, limited data on tomato seeds, pollen and shoot tips cryopreservation are yet available (coste et al., 2015; al-abdallat et al., 2017; halmagyi et al., 2017). dna libraries at the molecular level, the tomato gene pool can be stored in the form of dna libraries: genomic or complementary dna (cdna). a genomic library contains dna fragments, generated by a specific endonuclease, that represent the entire genome of an organism. as for the cdna libraries, mrna from a specimen are extracted and then cdna is prepared in a multistep reaction catalysed by reverse transcriptase enzyme. therefore, cdna libraries contain only the coding regions (ests) of expressed genes with no introns or regulatory regions. the produced fragments are ligated into vector molecules and transferred into host cells (wu et al., 2006). the development of artificial chromosome vectors (bacterial – bac, or yeast – yac) and the relative ease of manipulating dna libraries has led to the widespread use and acceptance of this approach. bacs are more efficient since they do not have the technical problems observed with yac libraries, such as easier purification. the further development of binary-bac (bibac) vectors established a new method for dna management, plant transformation and gene identification. this technology allows to immediately introduce very large fragments of dna, intact, into the plant genome via agrobacterium tumefaciens-mediated plant transformation (hamilton et al., 1999). moreover, since linkage map distances are not simply related to physical distances, artificial chromosomes are used in physical mapping to determine the locations of markers on chromosomes and in map-based cloning of agronomically important genes and qtls (koo et al., 2008). as for tomato, the first binary-bac vector library was constructed by hamilton et al. (1999) (cornell university, u.s.). large insert genomic libraries, containing approximately 4.6 haploid nuclear genomic equivalents, were constructed for solanum lycopersicum ‘mogeor’ and s. pennellii la716. the s. lycopersicum dna library has an average insert size of 125 kb and is comprised of 42,272 individual colonies stored as frozen cultures in a 384-well format (108 plates). the s. pennellii genomic library has an average insert size of 90 kb and is comprised of 53,760 individual clones (140,384well plates). in 2003, qu et al. constructed large-insert genomic libraries of tomato, based on a new tac (transformation-competent 140 d. kulus artificial chromosome) vector. the library contains 96,996 clones (28.3-38.5 kb insert size) and has 3.18 haploid genome equivalents. tac vectors proved to be useful to rapidly isolate important genes in tomato. a detailed physical map of s. pennellii genomic regions including two genomic libraries (in a form of bac/cosmid clones) was screened with 104 collocated markers from five selected genomic regions by kamenetzky et al. (2010). this gave a genome-wide map of a nondomesticated tomato species, which covers 10% of the physical distance of the selected regions corresponding to approximately 1% of the wild tomato genome. to accelerate the progress of tomato genomics studies, recently a full-length cdna library has been established for the cultivar ‘micro-tom’ (kobayashi et al., 2014). development of molecular markers in evaluating tomato genetic diversity knowledge about genetic distances is essential for optimum organi sation of gene banks, and for identifying parental combinations which produce progenies with maximum genetic diversity. genetic variation can be investigated via various methods, e.g. through morphological and biochemical traits, pedigree analysis or by molecular markers (corrado et al., 2014). because the tomato phenotype can be easily altered by environmental factors, thus quantification of genotypic variation by phenotypical markers, despite being intuitive and practical, is not always possible. molecular markers are recognised as a more reliable method for fingerprinting the accessions in gene banks (zhou et al., 2015). in the past three decades, various molecular markers (including isozymes, seed proteins and pcr-based markers) were employed, either individually or in combination, for genetic diversity analyses in tomato (ohyama et al., 2017; sharifova et al., 2017; chaudhry et al., 2018). development of isozymes allowed for the first evaluation of wild tomato germplasm (rick and fobes, 1975). however, isozyme marker scarcity and their low polymorphism was a serious limitant. the narrow genetic base of tomato cultivars makes it difficult to distinguish them via many contemporary genetic markers (zhou et al., 2015). commonly used identification methods, based on the amplification of a limited number of pre-selected barcoding regions, are often inapplicable due to dna degradation, low amplification success or low species discriminative power of selected genomic regions (raime and remm, 2018). the lack of sufficient marker systems in the tomato was a bottle neck in genetic and linkage studies for many years. fortunately, nowadays, specific dna fragment-based markers can identify tomato cultivars despite high levels of mono morphism and low polymorphism information content values (labate and baldo, 2005). for s. lycopersicum, (gata)4, (ccta)4 and (ggat)4 oligonucleotide motifs are suitable for the differen tiation of cultivars and breeding lines that are otherwise difficult to distinguish (garcía-martínez et al., 2013). moreover, (gata)4 fingerprinting generated hierarchical classifications consistent with the history of tomato cultivation (kaemmer et al., 1995). in 2005, tam et al. found that ssap (sequence-specific amplification polymorphism) is more corresponding for inferring overall genetic variation and relationships, while microsatelites (short tandem repeats; ssrs) have the capability to detect specific genetic relationships. recently, ssrs have become the most popular source of tomato genetic markers owing to their multi-allelic nature, high reproducibility, wide dispersion in eukaryotic genomes, and co-dominant inheritance. due to variations in repeat copy number; possibly caused by slippage during replication; they show a higher level of polymorphism than any other genetic marker (ohyama et al., 2017). for example, bredemeijer et al. (2002) documented, that despite stms (sequence tagged microsatellite site) polymorphism in tomato was relatively low (the number of alleles per locus ranged from 2 to 8), though, more than 90% of 508 analysed popular tomato cultivars had different microsatellite profiles. unfortunately, microsatellite development is expensive and time consuming, so other markers are also used. ruiz et al. (2005) compared srap (sequence-related amplified polymorphism) and ssr marker systems in order to ana lyse the genetic variability of some traditional tomato cultivars of spain (including commercial cultivars, local cultivars and a few wild species). srap is a reliable and simple pcr-grounded dominant marker system, designed to detect mostly coding sequence polymorphisms. the system is based on a combination of two types of pri mers. the forward primer amplifies exonic regions, while the reverse primer amplifies intronic and promoters regions. in the study by ruiz et al. (2005), both marker types (ssr and srap) sorted out the cultivars from different groups, but ssr failed to distinguish some of those classified within the same group. this can be explained by the fact, that despite microsatellites regions present higher variability than other genomic regions, the srap system has a higher multiplexing ratio and analyses a much higher number of loci. therefore, this system can also be recommended for evaluating tomato genetic diversity. the finding of snps as bi-allelic molecular markers, first described in ests (labate and baldo, 2005) then in non-coding tomato sequences (labate et al., 2009), delivered access to a high level of polymorphism as they exhibit less homoplasy than markers based on fragment-size. snps are highly abundant in plants (they represent even 90% of the genetic variation in any species), and can be assayed cost-effectively (van deynze et al., 2007). it should be mentioned tough, that snps can be divided in two clusters: snps of which both forms are present in the wild tomato relatives and in domesticated ones (originating from common ancestors), and snps unique for the domesticated tomato (originating from after the domestication event) (víquez-zamora et al., 2013). according to the 100 tomato genome sequencing consortium (2014), in wild tomato species the number of snps exceeds 10mm, i.e. 20-fold higher than found in most of the cultivated accessions. therefore, screening of snps through de novo sequencing is inefficient within cultivated tomato, as the sequencing error rate is over ten-fold higher than the polymorphism rate. fortunately, the development of dna microarray me thod, ests and cos (conserved orthologous set) data; the number of which reaches now several hundred thousand; has made it possible to discover putative snps in silico, prior to experimental verification (sim et al., 2012; ohyama et al., 2017). as sequencing costs continue to decline, researchers are developing new technologies, e.g. genotyping by sequencing (or next generation sequencing; ngs). the availability of high-throughput sequencing tools, accompanied by the development of array-based genotyping platforms, has provided unparalleled ability to determine genome diversity across entire clades, at both the structural and genotype level by rapid scoring of several thousand markers in parallel (the 100 tomato genome sequencing consortium, 2014; liu et al., 2018). for example, the 152 snps (obtained via custom-made illumina snp-panel) were able to clearly distinguish 75 landraces from a set of 25 contemporary italian varieties (corrado et al., 2014). víquezzamora et al. (2013) identified a set of 6,000 snps and 5,528 of them (1,980 originated from 454-sequencing, 3,495 from illumina solexa sequencing and 53 were additional known markers) were used to evaluate tomato germplasm at the level of species, varieties and segregating populations. such genotyping techniques have enabled high-density molecular map construction and genome-wide association analysis (celik et al., 2017). another novelty, based on plastid genome sequences, was described by raime and remm (2018), who identified over 800 solanum lycopersicum specific dna k-mers (32 nucleotides in length) from 42 different chloroplast genome regions. the chloroplast dna is high copy and has a small, gene rally stable and mechanical breakdown resistant, circular form as compared to nuclear dna (kim et al., 2015). moreover, the chlo managing tomato genetic resources 141 roplast genome is endemic to plants and may help to bypass dna contamination from chloroplast-free organisms (dong et al., 2014). therefore, plastid genome sequencing can be considered as a valuable tool allowing for rapid identification of plant taxa directly from raw sequencing reads without aligning, mapping or assembling the reads. bioinformatic data extrapolation can increase the efficiency of molecular markers discovery (pailles et al., 2017). the development of nanotechnologies in the so called “post-genomics” era opens new perspectives in terms of genetic diversity management, toward conservation and survey of large populations (muños et al., 2011; giovannoni, 2018). the use of computer simulations and multi variate statistical algorithms (such as: principal component analysis, canonical variable analysis, clustering methods, etc.) is an important strategy to quantify the degree of (dis)similarity or number of (rare) alleles in plant genetic resources (gonçalves et al., 2008). after previous cloning and sequencing, genetic markers from database sequences (e.g. blast, embl or genbank) can be screened in silico and utilized to create diversity profiles in tomato, especially for cultivars with limited genetic variation and in other accessions of the lycopersicon section (yang et al., 2014; shirasawa et al., 2016). conclusions despite new donor segments have been introduced from more variable related species into the cultivated tomato germplasm, typically of self-pollinated crops, the species severely lacks genetic diversity (shirasawa et al., 2015). the conservation and utilisation of crop biodiversity is of particular importance, especially to the least developed countries, where modern plant breeding has had much less success. the polytechnic of agriculture and cattle husbandry at the uni versity of manabi (ecuador) is conducting a project aiming at collecting, characterising and conserving the genetic resources of wild tomato species (including rare and endangered accession). the pro ject involves characterisation of in situ environmental conditions in which plants grow, as well as ex situ morphological description and molecular analysis of collected specimens. moreover, the establishment of a wild tomato seed cryobank is planned (zevallos et al., 2014). projects, such as the one described above, are necessary for plant biodiversity protection, and thus, efficient future breeding and food security. however, the maintenance, characterisation and management of all accessions within a collection is economically demanding. according to corredo et al. 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open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 88, 134 138 (2015), doi:10.5073/jabfq.2015.088.019 1 igdir university, agricultural faculty, department of horticulture, igdir-turkey 2ataturk university, agricultural faculty, department of horticulture, erzurum, turkey 3 oregon state university, agricultural faculty, department of horticulture, corvallis, oregon-usa 4sakarya university, pamukova vocational school, sakarya-turkey 5 igdir university, agricultural faculty, department of animal science, biometry genetics unit, igdir-turkey 6 igdir university, agricultural faculty, department of agricultural economy, igdir-turkey organic acids, sugars, vitamin c, antioxidant capacity, and phenolic compounds in fruits of white (morus alba l.) and black (morus nigra l.) mulberry genotypes s.p. eyduran1, s. ercisli2, m. akin3, o. beyhan4, m.k. gecer1, e. eyduran5, y.e. erturk6 (received january 26, 2015) * corresponding author summary mulberries (morus spp.) are historically grown in particular microclimatic regions in eastern anatolia, including aras valley. in the valley, mulberries are one of the ancient crop and used for several purposes by local people. the aim of the present study was to first time evaluate organic acids, sugars, vitamin c, antioxidant capacity (teac assay, trolox equivalent antioxidant capacity). and phenolic compounds of the historical black and white mulberry genotypes growing in aras valley in turkey. results showed that, species and geno-types strongly influenced the chemical content and antioxidant capa-city (p<0.05). malic acid was the main organic acid in all genotypes and ranged from 1.130 to 3.040 g/100 g. among sugars, fructose and glucose are predominant and were between 4.057 and 7.700 g/100 g and 5.337 and 9.437 g/100 g in all mulberry genotypes, respectively. the black mulberry genotypes showed remarkably higher antioxidant capacity determined by teac assay (10.167 to 14.400 μmol te/g) compared to white mulberry genotypes (6.170 to 9.273 μmol te/g). chlorogenic acid and rutin were the main phenolic compounds. introduction mulberries are widely spread on tropical, subtropical and temperate zones of asia, europe, america, and africa, which indicate their high adaptation capacity to different environmental conditions (ercisli and orhan, 2007). turkey is one of the important diversity centers of mulberries with a long cultivation history dating back to 400 500 years ago. the most popular mulberry species with edible fruits grown in turkey are morus nigra, morus rubra, morus alba and morus laevigata (ozgen et al., 2009; yildiz, 2013). the high genotypic diversity for mulberries in turkey (kafkas et al., 2008) is especially attributed to its long period generative propagation in turkey in the past. although white mulberry (morus alba) is the main cultivated specie in turkey with share 95 % of the total production. more recently there is a growing interest on black and white mulberries and potential benefits on human health associated with its higher phytochemical content and antioxidant properties (ercisli and orhan, 2007; ercisli and orhan, 2008; koyuncu et al., 2014; natic et al., 2015; sanchez et al., 2014; sanchez salcedo et al., 2015). since ancient times, mulberry has been cultivated in some microclimatic areas particularly in eastern anatolia including aras valley. the plant is also widely grown in western anatolia as well as mediterranean side of turkey. each growing area has own special genotypes, which greatly differ each others (ercisli, 2004). traditionally, mulberry fruits have been processing into several product including mulberry juice, molasses, jam, vinegar and some very special products such as ‘mulberry pestil’, ‘mulberry kome’, etc. in turkey. all of these products have significant marketing value due to its nutritive features and distinct aroma characteristics (erturk and gecer, 2012). there are big differences between black (morus nigra) and white (morus alba) mulberry trees and particularly fruits. morus nigra is one of the most difficult fruit species for vegetative propagation and it has also slow growing characteristics. plant habit of this species is more compact and free of pest of diseases due to thick leaves and high phenolic contents in its tissues (ercisli and orhan, 2008). black mulberry fruits are mainly processed into juice and have also high fresh consumption value. mulberry fruits are harvested several times in a growing period (four to seven times in a year, according to altitude of orchards). the first harvested fruits are sent to market for fresh consumption, while later harvested fruits have increased their sugar content during fruit development and are destined for processing purposes. black and red mulberry fruits have been used in small quantities for jam industry (akbulut et al., 2006). in turkey 95 % of mulberry trees belongs to white mulberry (morus alba) and 70 % of white mulberry fruits processed into mulberry molasses, whereas 10 % processed into ‘mulberry kome’ and 3 % processed into ‘mulberry pestil’. approximately 4 % dried and 5 % consumed as fresh of white mulberries (ercisli, 2004). the other species cannot be used to obtain above products. however, fruits of morus nigra, are purple to black in color, juicy and with pleasant acidic flavor (ercisli and orhan, 2008; ozgen et al., 2009). together with fresh consumption, fruits of black mulberry are utilized in syrup, marmalade, ice-cream, vinegar and vine industries (gundogdu et al., 2011). fruits of morus nigra are very rich source of phenolics due to their high content in anthocyanins (aramwit et al., 2010). phenolic compounds prove anti-aging properties attributed to their antioxidant activity by scavenging free radicals. in addition, many studies suggest that phenolics help reducing the risk of coronary heart diseases and some cancer types (rodriguez-mateos et al., 2014). black mulberries are important for use in folk medicine as a remedy for diabetes, arthritis, hypertension, anemia and moth lesions due to its high phytochemical content (bae and suh, 2007; kostic et al., 2013). organic acids and sugars are another important components of the fruits, characterizing their organoleptic properties. the flavor, which is a very significant criterion in fresh market, is generally characterized by the ratio of organic acids and sugars. in addition, organic acids are probable antioxidants with multi-purpose usages in pharmacology (soyer et al., 2003). besides environmental conditions, genotype is a prominent factor in the determination of fruit chemical composition and antioxidant capacity (hegedus et al., 2010; mikulic-petkovsek et al., 2012; rop et al., 2014; jurikova et al., 2014). in the last decade, several bioactive content of mulberries from eastern anatolia 135 studies have been reported on the definition of the organic acids, sugars, and phenolic compounds of black, white, and red mulberries growing naturally at different part of turkey for promoting quality of the produced products in mulberry industry (koyuncu, 2004; ercisli and orhan, 2008; ozgen et al., 2009, gundogdu et al., 2011; orhan and ercisli, 2014). to our knowledge, no available data are found on the definition of the phytochemical characteristics of mulberries grown in aras valley-located eastern anatolia in turkey. hence, the objective of this study was to investigate the organic acids, sugars, vitamin c, antioxidant capacity, and phenolic compounds of the historical morus nigra (black mulberry) and morus alba (white mulberry) genotypes growing wildly in melekli district of igdir province in turkey. the phytochemical definition of the reachable genotypes for proving a distinguishable improvement in mulberry industry will be a notable reference to similar further studies. materials and methods study area eastern anatolia region is located in the easternmost part of turkey. it is bounded by turkey’s central anatolia region on the west, its black sea region on the north, its southeast anatolia region and iraq on the south, and with iran, azerbaijan, armenia, and georgia on the east. since most of the region is far from the sea, and has high altitude, it has a long winter and short summer periods. during the winter, it is very cold and snowy, during summer the weather is cool in the highlands and warm in the lowlands. the region has the lowest average temperature of all turkish regions (-25 °c) and sometimes temperature drop below -40 °c in winter. the summer average is about 20 °c. the region’s annual temperature difference is the highest in turkey. however, some areas such as igdir province in the region have milder climate and accepted as microclimate. plant material three black mulberry (morus nigra ) genotypes (76-igd-1, 76-igd-2 and 76-igd-3) and two white mulberry (morus alba) genotypes (76igd-4 and 76-igd-5) were used as material. mulberry trees found in melekli district (latitude 39° 56' 47 n, longitude 44° 5' 52 e, and 856 meters above sea level) belong to igdir province, located in aras valley, eastern anatolia region in turkey. fruits (berries) for analyses were collected from different branches of one mulberry plant (tree) per genotype because there was only one plant (tree) per genotype in natural growing area. in turkey mulberry trees are generally found in natural growing areas and there were no similar genotypes. a total of 100 fruits (berries) were sampled per genotype to ensure good representative of the genotypes to make average sampling, and the berries of those mulberry genotypes were taken during commercial ripe stage. the berries were stored at -20 oc (ozgen et al., 2009) until the extraction and analysis of organic acids, sugars, vitamin c, antioxidant capacity, and phenolic compounds. analysis of organic acids succinic, citric, malic, fumaric and tartaric acid composition of mulberries were analyzed by modified method of bevilacqua and califano (1989). fruits were mashed within a cheesecloth and the obtained sample juices were stored at (-20 °c) until processed. 5 ml sample juice and 20 ml 0.009 n h2so4 were mixed (heidolph silent crusher m, germany), and homogenized using a shaker (heidolph unimax 1010, germany) for 1 hour, after which centrifuged for 15 min at 15000 rpm. supernatants were firstly filtrated with coarse filter, then twice with 0.45 μm membrane filter (millipore millexhv hydrophilic pvdf, millipore, usa), and run through sep-pak c18 cartridge. hplc device was adjusted to 214 and 280 nm wavelengths, on agilent package program (agilent, usa) with aminex column (hpx 87 h, 300 mm x 7.8 mm, bio-rad laboratories, richmond, ca, usa) was used for the determination of organic acids. the results are presented as g/100 g fresh mass. extraction and determination of sugars modified method of melgarejo et al. (2000) was used for sugar content determination. samples of 5 ml were centrifuged at 12000 rpm for 2 min at 4 °c, than filtrated in sep-pak c18 column. μbondapaknh2 column with 85 % acetonitrile liquid phase with refractive index detector (ir) was used for the hplc device. calculations of the sugar concentrations were according to the prepared fructose and glucose standards. the results are presented as g/100 g fresh mass. analysis of ascorbic acid (vitamin c) ascorbic acid content was determined according to the method suggested by cemeroglu (2007). samples of 5 ml were mixed with % 2.5 (w/v) metaphosphoric acid (sigma, m6285, 33.5 %), and centrifuged at 6500 rpm for 10 min at 4 °c. 0.5 ml of the solution was completed to 10 ml with % 2.5 (w/v) metaphosphoric acid. supernatants were passed through 0.45 μm membrane filter. ascorbic acid detection was made with hplc device using c18 column (phenomenex luna c18, 250 x 4.60 mm, 5 μ) and the temperature was adjusted to 25 °c. mobile phase consisted of ultra distilled water with 1 ml/min flow rate at 2.2 ph acidified with h2so4. dad detector was used and spectral measurements were made at 254 nm wavelength. different levels of l-ascorbic acid (sigmaa5960) (50, 100, 500, 1000, and 2000 ppm) were used for ascorbic acid readings. results are presented as mg/100 g fresh mass. determination of trolox equivalent antioxidant capacity (teac) trolox equivalent antioxidant capacity (teac) analysis was performed with abts formulated with potassium persulphate dissolved in a buffer (ozgen et al., 2006). for longer stability, abts+ was diluted with 20 mm sodium acetate buffer with ph 4.5, at 734 nm wavelength, 0.700 ± 0.01. spectrometric assay was done by mixing 20 μl of the samples with 3 ml abts+ for 10 min, and absorbance measurements were done at 734 nm wavelength. the results are presented as μmol te/g fresh mass. analysis of phenolic compounds phenolic compounds viz. gallic acid, catechin, chlorogenic acid, caffeic acid, p-coumaric acid, o-coumaric acid, ferulic acid, vanillic acid, rutin, syringic acid, and quercetin were analyzed with the modified method of rodriguez-delgado et al. (2001). fruit juice samples were mixed in a ratio of 1:1 with distilled water and centrifuged for 15 min at 15000 rpm. after filtration with coarse filter paper and twice with 0.45 μm membrane filter (millipore millex-hv hydrophilic pvdf, millipore, usa), the supernatants were injected to hplc. agilent 1100 (agilent) hplc system on a dad dedector (agilent. usa) was used. chromatographic separation was performed with 250 x 4.6 mm, 4 μm ods column (hichrom, usa). as a mobile phase, solvent a methanol:acetic acid:water (10:2:28) and solvent b methanol:acetic acid:water (90:2:8) with flow rate 1 ml/ min and 20 μl injection volume were used for spectral measurements at 254 and 280 nm. results are presented as mg/g fresh mass. statistical analysis five replicates including 20 fruits per replicate were used. descriptive statistics of organic acids, sugars, vitamin c, antioxidant capacity, and phenolic compounds extracted from 2 morus alba and 3 morus 136 s.p. eyduran, s. ercisli, m. akin, o. beyhan, m.k. gecer, e. eyduran, y.e. erturk nigra genotypes were represented as mean ± se. the phytochemical characteristics were statistically analyzed with one-way anova with five replicates. duncan test determined significant differences between the evaluated genotypes. all statistical evaluations were performed using spss 20. results and discussion tab. 1 shows the results of organic acids, tab. 2 shows sugars, vitamin c and antioxidant capacity and tab. 3 shows phenolic compounds of 2 white and 3 black mulberry genotypes collected from melekli district in igdir province. anova indicated that the genotype had a major influence on all parameters under evaluation (p<0.05). organic acids as shown in tab. 1, among organic acids, malic acid was the predominant one for all the mulberry genotypes in the study. it was followed by citric acid, succinic acid, tartaric acid, and fumaric acid, respectively. the malic acid levels in fruits were between 1.130 (‘76igd-5’, white mulberry) and 3.040 g/100 g (‘76-igd-3’, black mulberry). black mulberries had the highest level in citric acid (1.033 mg/l) compared to white mulberries (tab. 1). sanchez et al. (2014) from spain and ozgen et al. (2009) and gundogdu et al. (2011) from turkey reported that malic and citric acids were main organic acids in mulberry fruits. the results indicated that the fumaric acid contents were detected at the lowest values for all black and white mulberry genotypes. koyuncu (2004) and mikulic-petkovsek et al. (2012) found that fumaric acid was found to be lowest level among the organic acids in black mulberry fruits. the organic acids contained in fruits are one of the important factors influencing fruit flavor and are of vital importance to human health. several studies emphasized the importance of organic acids such as malic, citric and tartaric acids which are important in prevention and elimination of kidney stones (penniston et al., 2007). malic acid possesses many health related benefits such as boosting immunity, maintaining oral health, reducing the risk of poisoning from a build-up of toxic metals, promoting smoother and firmer skin and help reducing symptom of fibromyalgia (abraham and flechas, 1992). sugars main sugars as glucose and fructose contents of mulberry samples were also investigated. we found higher glucose content than fructose in fruits of all mulberry genotypes. the genotype ‘76-igd-4’ (white mulberry) had the highest glucose (9.437 g/100 g) and fructose (7.700 g/100 g) content (tab. 2). the lowest glucose (5.337 g/ 100 g) and fructose content (4.057 g/100 g) was determined in ‘76igd-5’ (white mulberry) genotype (tab. 2). the glucose contents of the genotypes were in descending order ‘76-igd-4’ (white mulberry) > ‘76-igd-3’ (black mulberry) > ‘76-igd-2’ (black mulberry) = ‘76igd-1’ (black mulberry) > ‘76-igd-5’ (white mulberry). however this order was ‘76-igd-4’ (white mulberry) > ‘76-igd-3’ (black mulberry) > ‘76-igd-1’ (black mulberry) > ‘76-igd-2’ (black mulberry) = ‘76-igd-5’ (white mulberry) for fructose content (tab. 2). the ratio of glucose/fructose were 1.500 in ‘76-igd-2’ (black mulberry) > 1.311 in ‘76-igd-5’ (white mulberry) > 1.225 in ‘76-igd-4’ (white mulberry) > 1.196 in ‘76-igd-3’ (black mulberry) > 1.098 in ‘76-igd-1’ (black mulberry) (tab. 2). sanchez et al. (2014) re-reported that the predominant sugar was fructose (~61 %) followed by glucose (~39 %), while sucrose was presented only at trace level in mulberry fruits sampled from spain and they also found big genotypic differences among clones for different sugars. ozgen et al. (2009) declared that fructose and glucose contents of 14 black and red mulberry genotypes ranged from 4.86 to 6.41 g/100 ml, and 5.50 to 7.12 g/100 ml, respectively. in a more comprehensive study, mikulic-petkovsek et al. (2012) suggested that fructose and glucose were predominant sugars for 25 wild or cultivated berry species and they reported that glucose and fructose contents were found 3.68 tab. 1: organic acids (g/100 g fw) content of black and white mulberry genotypes genotypes citric acid tartaric acid malic acid succinic acid fumaric acid 76-igd-1 (black) 0.877±0.018b 0.147±0.007d 1.210 ±0.015d 0.280±0.015c 0.010± 0.000d 76-igd-2 (black) 0.733±0.009c 0.223±0.015c 2.713±0.048b 0.360±0.006b 0.057±0.012c 76-igd-3 (black) 1.033±0.032a 0.297±0.017b 3.040±0.056a 0.117±0.007d 0.107±0.003ab 76-igd-4 (white) 0.687±0.022c 0.153±0.009d 2.103±0.028c 0.267±0.009c 0.100±0.006b 76-igd-5 (white) 0.480±0.021d 0.430±0.006a 1.130±0.023d 0.437±0.012a 0.123±0.003a cv % 4.9 7.9 3.2 6.1 13.8 means with different letter in same column were statistically significant (p<0.05). tab. 2: sugar (g/100 g fw), vitamin c (mg/100 g fw) and antioxidant capacity (μmol te/g fw) of black and white mulberry genotypes genotypes glucose fructose teac vitamin c 76-igd-1 (black) 6.173±0.052c 5.620±0.071c 11.297±0.047b 10.123±0.023e 76-igd-2 (black) 6.247 ±0.088c 4.163 ±0.035d 14.400±0.025a 16.293± 0.039b 76-igd-3 (black) 8.573±0.061b 7.167±0.037b 10.167±0.043c 12.040±0.055d 76-igd-4 (white) 9.437±0.048a 7.700±0.070a 6.170±0.061e 18.220±0.136a 76-igd-5 (white) 5.337±0.075d 4.057±0.052d 9.273±0.038d 13.400±0.081c cv % 1.6 1.7 0.8 1.00 means with different letter in same column were statistically significant (p<0.05). bioactive content of mulberries from eastern anatolia 137 and 3.99 g/100 g for black mulberry grown naturally in central slovenia, and that the concentration of fructose sweeter than glucose or sucrose was important for consumers willing sweeter fruit. the present genotypes illustrated wide variability for the sugars, which may be ascribed to genetic factors, cultural applications, and ecological conditions (light, temperature, and humidity etc.) as also referred in the previous studies (gundogdu et al., 2011). vitamin c and antioxidant capacity we found vitamin c content between 10.123 and 16.293 mg/100 g for black mulberry genotypes under the investigation (tab. 2). in the earlier work conducted on the northeast anatolia region of turkey, ercisli and orhan (2008) reported vitamin c contents of black mulberry genotypes varied from 14.9 to 18.8 mg/100 ml. ercisli et al., (2010) reported the average vitamin c content in black and purple mulberries as 20.79 and 18.87 mg per 100 ml, respectively. fruit species can be classified into three groups (low, moderate and high) in terms of their vitamin c content (karacali, 2000) and mulberries are generally placed within the moderate vitamin c content group. lale and ozcagiran (1996) reported that vitamin c content in black and purple mulberries was 16.6 and 11.9 mg/100 ml. results of statistical assessments made for antioxidant activity (teac assay) are presented in tab. 2. as depicted in tab. 2, the analysis results of variance illustrated that the genotype is of big importance in characterizing teac (p<0.05) that changed between 6.170 and 14.400 μmol te/g fw and black mulberries had higher than white mulberries. the highest contents for teac were statistically enabled by ‘76-igd-2’, followed by ‘76-igd-1’ > ‘76-igd-3’ > ‘76-igd-5’ > ‘76-igd-4’ (tab. 2). gundogdu et al. (2011) reported that black mulberry had highest teac values than white mulberry. phenolic compounds in our study, we found different levels of gallic acid, catechin, chlorogenic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, o-coumaric acid, vanillic acid, rutin, and quercetin in fruits of black and white mulberry genotypes (tab. 3). chlorgenic acid and rutin were the main phenolics for mulberry fruits. our results are consistent with the findings of gundogdu et al. (2011) and natic et al. (2015). gundogdu et al. (2011) reported that the amount of gallic acid, catechin, chlorogenic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, o-coumaric acid, vanillic acid, rutin and quercetin in mulberry fruits were 0.150, 0.075, 3.106, 0.131, 0.103, 0.129, 0.064, 0.134, 0.036, 1.423, and 0.113 mg/g for black mulberries, and 0.215, 0.037, 0.119, 0.133, 0.049, 0.047, 0.033, 0.015, 0.008, 1.111, and 0.015 mg/g for white mulberries, respectively. memon et al. (2010) used white mulberry fruits in three extraction techniques (sonication, magnetic stirring, and homogenization) and found that gallic acid (5.81, 4.32, and 3.57 mg/100 g), vanillic acid (4.57, 3.95, and 3.70 mg/100 g), chlorogenic acid (20.47, 17.03, and 24.45 mg/ 100 g) and syringic acid (9.19, 6.31 and 8.48 mg/100 g) were main phenolic compounds in the fruits of morus alba (white mulberry). for the fruits of morus nigra (black mulberry), these values were 5.81, 1.72, and 4.21 mg/100 g for gallic acid, 2.31, 2.25, and 1.63 mg/ 100 g for vanillic acid, 4.41, 5.43, and 7.44 mg/100 g for chlorogenic acid, 1.81, 1.63 and 1.59 mg/100 g for syringic acid and 8.66, 2.27 and 4.89 for p-coumaric acid. the difference may be ascribed to the use of different extraction methods. gundogdu et al. (2011) argued that, within the phenolic compounds, chlorogenic acid had the highest content for black mulberry (morus nigra), which was in agreement with the first two black mulberry genotypes, but not consistent with the predominant content (rutin) for the last black mulberry accession (tab. 1). conversely, in the present study, chlorogenic acid was also determined to be the predominant phenolic compound for two white mulberry genotypes. the difference may be due to genetic factors, and non-genetic factors (temperature, humidity, light, etc.), and cultural applications (hegedus et al., 2008; gundogdu et al., 2011). conclusion we have characterized for the first time the chemical attributes of white and black mulberry genotypes from igdir province located eastern anatolia in turkey. organic acids, sugars, vitamin c, antioxidant activity, and phenolic spectrum varied significantly within and between white and black mulberry genotypes. high antioxidant activity was found in particular in black mulberry genotypes. the study highlighted that the chemical properties of mulberry fruits (berry) were strongly affected by genotype. this study also increases our knowledge about variation of phytochemical properties including organic acids, sugars, vitamin c, antioxidant activity and phenolic spectrum in mulberry fruits and may be useful to producers, breeders and processors. tab. 3: phenolic compounds (mg/g fw) of black and white mulberry genotypes 76-igd-1 76-igd-2 76-igd-3 76-igd-4 76-igd-5 cv % (black) (black) (black) (white) (white) catechin 0.085±0.002a 0.046±0.002c 0.066±0.001b 0.032±0.002d 0.070±0.002b 5.0 rutin 0.820±0.004b 1.365±0.198a 1.238±0.004a 0.750±0.002b 0.925±0.004b 15.0 quercetin 0.119±0.002b 0.067±0.001d 0.137±0.003a 0.101±0.003c 0.045±0.001e 4.2 chlorogenic acid 2.339±0.004b 1.641±0.004c 0.759±0.005e 2.667±0.004a 0.980±0.005d 0.4 ferulic acid 0.036±0.002d 0.023±0.001e 0.063±0.002c 0.110±0.003b 0.141±0.003a 5.2 o-coumaric acid 0.034±0.001d 0.129±0.003a 0.043±0.001c 0.062±0.002b 0.026±0.001e 5.3 p-coumaric acid 0.111±0.004b 0.062±0.002c 0.035±0.001d 0.065±0.002c 0.127±0.003a 5.9 caffeic acid 0.114±0.002c 0.094±0.002d 0.158±0.004a 0.094±0.003d 0.134±0.001b 3.7 syringic acid 0.119±0.001a 0.053±0.001d 0.085±0.002b 0.115±0.004a 0.060±0.001c 4.3 vanillic acid 0.035±0.001c 0.011±0.001e 0.062±0.002b 0.074±0.002a 0.017±0.001d 6.5 gallic acid 0.122±0.003b 0.410±0.004a 0.105±0.003b 0.206±0.004b 0.214±0.083b 30.5 means with different letter in same row were statistically significant (p<0.05). 138 s.p. eyduran, s. ercisli, m. akin, o. beyhan, m.k. gecer, e. eyduran, y.e. erturk references abraham, g., flechas, j., 1992: management of fibromyalgia: rationale for the use of magnesium and malic acid. j. nutr. med. 3, 49-59. akbulut, m., cetin, c., coklar, h., 2006: determination of chemical content and minerals in mulberry cultivars. proceedings of 2nd national berry symposium, 14-16 september 2006, tokat-turkey, 176-180. aramwit, p., bang, n., 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accepted march 27, 2017) * corresponding author summary much interest is currently concentrated on phenol compounds and antioxidants of wine. the aim of this study was to characterize and evaluate controlled designation of origin (cdo) and typical geographical indications (tgi) red wines from central italy and to evaluate possible modifications after one year of storage. the total phenol content and antioxidant activity by orac method were determined, while phenolic qualitative and quantitative profiles were evaluated by hrgc-fid or hplc-dad. all wines showed a good content of total phenols and an obvious antioxidant effect. after a one-year storage in the bottle, a significant decrease (p<0.05) of the orac values was observed for tgi wines. interesting correlations between phenol and orac values for cdo wines were found. it can be confirmed that one-year storage in the bottle has not significantly affected the quality of the wines analyzed, in particular the cdo category. keywords: chromatography, orac, phenol, red wine, storage. introduction red wine is an important source of phenol compounds, natural anti oxidants known for their biological properties and associated with the prevention of oxidative damage (becker et al., 2004). several clinical studies and epidemiological studies have shown a correla tion between the intake of phenol compounds from red wine and a decrease in the incidence of many diseases (arranz et al., 2012). many studies have been carried out over the years and numerous analytical techniques have been developed to analyze phenolic compounds of grapes and wine (lorrain et al., 2013; khoddami et al., 2013). complete analysis of phenolic compounds requires analytical techniques such as hrgc and hplc associated with uv-vis and diode array detectors (dad) often coupled to other detection systems. a broad bibliography is now available for the study of red or white wines produced in various parts of the world (granato et al., 2011; tourtoglou et al., 2014; ivanova-petropulos et al., 2015) although few results relate to the wines of central italy. sato et al. (1996) investigated varietal differences in the phenol content of thirty-one wines from various sources, including a sangiovese umbrian sample from 1982. versari et al. (2007), characterized by uvvis spectrophotometry, the color components and polymer pigments of commercially available red wines, including a merlot-umbrian sample. recently, esti et al. (2010) made an explorative sensory study of grechetto’s wine tasting, some of which were produced in umbria. the region of umbria (central italy) plays an important role in the production of red wines, including montefalco sagrantino known worldwide. to the best of our knowledge, there are no works that focus solely on phenolic composition and antioxidant capacity of umbrian wines. in this research, twelve umbrian red wines, six cdo (controlled designation of origin) and six tgi (typical geographical indication) samples were characterized. total phenol content, antioxidant capacity, hrgc and hplc phenol profiles were determined. the same analyzes were repeated after one year of storage in the bottle to evaluate possible variations. materials and methods wine samples commercial wines, six cdo and six tgi, all collected in 750 ml bottle of cork (eight bottles for each wine) were collected from wineries in the province of perugia, the capital of umbria region (central italy). all wines were produced with varieties of vitis labrusca l. grape varieties. wine samples were selected to obtain a broad sampling of vine varieties and production area. all the wine samples were purchased at the market of umbrian wines in 2010, where the wines are available on the market. it should be noted that the year of release for consumption does not correspond to the same vintage for different wines, on the individual production rules. the samples tested in this work are shown in tab. 1, while some chemical parameters determined according to the official methods described in commission regulation (cee) no 2676/90 of 17 september 1990 are shown in tab. 2. wines were produced according to italian legislation. the wines were stored in the dark at 10 ± 1 °c until analysis. the samples, representative aliquots from four bottles, were tested shortly after opening. the same wines stored in unopened bottles at 10 ± 1 °c were analyzed in 2011, one year after marketing. chemicals and reagents rutin, kaempferol-3-o-glucoside, isorhamnetin-3-o-glucoside, kaempferol, rhamnetin and isorhamnetin were obtained from extrasythese (genay, france). cis-resveratrol was from cayman chemical company (ann arbor, mi, usa). the other phenol compounds were purchased from sigma-aldrich (milan, italy). they were stored in the dark at temperature recommended by the producer. folin & ciocalteu’s phenol reagent, bstfa [n,o-bis(trimethylsilyl)trifluoracetamide] and trolox (6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxyl acid) were purchased from sigma-aldrich. all analytical grade solvents and reagents were from j.t. baker (deventer, netherlands), carlo erba (milan, italy), panreac (barcellon, spain) and supelco (bellafonte pa, usa). hplc-grade acetonitrile and water were purchased from j.t. baker. determination of total phenol content the tp content was determined spectrophotometrically according to the method of singleton and rossi (1965), with slight modifications (blasi et al., 2016). the wine samples were diluted (1:20 or 1:30, v/v) with distilled water. diluted red wine (1.0 ml), 20% sodium carbonate (4.0 ml) and folin & ciocalteu’s reagent (1.0 ml) were mixed and brought up to a volume of 20 ml with distilled water. 198 g. lombardi, l. cossignani, l. giua, m.s. simonetti, a. maurizi, g. burini, r. coli, f. blasi the solution was mixed, kept at 25 °c and incubated at the dark for 90 min and then the absorbance was measured at 750 nm using a jasco 7850 uv-vis spectrophotometer (jasco inc., easton, md, usa). the tp content, expressed as milligrams of gallic acid equi valent per liter of wine (mg gae/l), was determined using a calibration curve built using 15% ethanol gallic acid as standard solution (2.5-12.5 mg/l) that was analyzed in the same mode of wine samples. extraction and derivatization of phenol compounds phenols were extracted from red wine according to the method of minuti et al. (2006). briefly, sodium metabisulphite (20 mg) and sodium chloride (20 mg) were added to the red wine (1 ml) and then ethyl acetate (3 ml × 3) was used for the extraction. after anhydrification with na2so4, the extracts were evaporated to dryness under nitrogen stream at 25 °c. the solid residue was dissolved in acetone (50 μl) and derivatized with bstfa (100 μl). the obtained solution was held in the dark, at room temperature, for 1 h. then the volume was made up to 1 ml. high-resolution gas-chromatography analysis high-resolution gc analyses were carried out using a perkin-elmer autosystem apparatus (norwalk, ct, usa), equipped with a split/ splitless injection port, interfaced to a flame ionization detector (fid). the separation was obtained using the hp1-ms fused silica capillary column (30 m × 0.25 mm i.d., 0.25 μm f.t.). the injector and detector temperature was 300 and 320 °c, respectively. the initial oven temperature was 120 °c, held for 3 min and raised to 320 °c at 5 °c/min; the final temperature was held for 15 min. carrier gas (he) flow rate was 1 ml/min; the injection volume was 1 μl with a split ratio of 1:50. a standard solution in acetone, suitably derivatized as reported in the previous paragraph, containing twenty compounds (vanillin, trans-cinnamic acid, tyrosol, veratric acid, vanillic acid, homovanillic acid, hydroxytyrosol, 3,4-dihydroxyphenil acetic acid, homogentisic acid, syringic acid, p-coumaric acid, gal lic acid, ferulic acid, caffeic acid, sinapinic acid, cis-resveratrol, trans-resveratrol, epicatechin, catechin, fisetin), was used to identify and to quantify the analytes. calibration curves were obtained by three injections of four different concentrations ranging from 2.5 to tab. 1: characteristics of umbrian (central italy) red wines code wine name grape variety production area vintage year wines of controlled designation of origin (cdo) cdo1 sangiovese colli martani sangiovese (85% min.) bettona 2007 ciliegiolo (43°0‘48.96“n-12°29‘11.04“w) cannaiolo nero montepulciano merlot cdo2 óscano colli del trasimeno gamay colle umberto 2009 sangiovese (43°07‘59.99“n-12°21‘59.98“w) cdo3 rosso colli perugini sangiovese sant’enea 2008 merlot (43°06‘43.56“n-12°23‘19.68“w) cabernet sauvignon cdo4 montefalco rosso sangiovese 65% montefalco 2007 merlot 20% (42°55‘59.99“n-12°35‘60“w) sagrantino 15% cdo5 baccio del rosso colli del sangiovese castiglione del lago 2008 trasimeno trasimeno gamay (43°06‘59.98“n-12°03‘00“w) cdo6 montefalco sagrantino sagrantino 100% montefalco 2007 (42°55‘17.98“n-12°38‘31.99“w) wines of typical geographical indication (tgi) tgi1 sangiovese umbria sangiovese (85% min.) bettona 2009 and other red grapes (43°0‘48.96“n-12°29‘11.04“w) typical of umbria tgi2 campiglione rosso merlot colle umberto 2009 cabernet sauvignon (43°07‘59.99“n-12°21‘59.98“w) sangiovese gamay tgi3 garbino dell’umbria sangiovese sant’enea 2009 cabernet sauvignon (43°06‘43.56“n 12°23‘19.68“w) merlot tgi4 umbria rosso sangiovese 50% montefalco 2009 merlot 50% (42°55‘59.99“ n-12°35‘60“w) tgi5 corio rosso sangiovese (80% min.) castiglione del lago 2009 gamet (43°06‘59.98“n-12°03‘00“w) cabernet tgi6 rosso sangiovese marsciano 2009 cabernet (42°91‘66.67“n-42°55‘12.20“w) antioxidant properties of italian red wines 199 40 mg/l. the least square method was used to calculate the regression equations. high-performance liquid chromatography analysis of phenol compounds before hplc-dad analysis, wine samples (100 ml) were de-alcoholized and dried under vacuum at 40 °c. the residue was diluted in distilled water (50 ml) and then filtered through a 0.20 μm nylon syringe filter (corning incorporated, corning, germany). the hplc analyses were performed using a shimadzu gt-154 system equipped with a thermo spectra series pump, an hypersil gold column (5 μm particle size, 250 × 4.6 mm i.d., thermo scientific, rockford, il, usa) and a spectra system uv6000lp dad. detection was performed on line in a range of wavelength between 240 and 390 nm. the chromatograms were acquired and the data handled using xcalibur software version 1.2 (finnigan corporation 1998-2000, san jose, ca, usa). the solvents were (a) acetonitrile and (b) water/acetic acid (40:1, v/v). the samples were analyzed by gradient elution at a flow rate of 0.8 ml/min. the elution conditions were: 0-15 min at 15% a; 15-30 min from 15 to 35% a; 30-40 min from 35 to 55% b; 40-44 min from 55 to 100% a with re-equilibration of the column at 44-47 min 100% a to 15% a and 47-60 min at 15% a. a standard solution solubilized in water/acetic acid/methanol (8:2:90, v/v/v), containing ten compounds (rutin, ethyl gallate, quercetin-3β-d-glucoside, kaempferol-3-o-glucoside, isorhamnetin-3-o-gluco side, myricetin, quercetin, kaempferol, isorhamnetin, rhamnetin), was used to identify and to quantify the analytes. calibration curves were obtained by three injections of five different concentrations ranging from 0.5 mg/l to 60 mg/l. antioxidant capacity the total antioxidant capacity was assessed using oxygen radical absorbance capacity (orac) method as reported by maurizi et al. (2015). the assay was conducted with fluostar optima fluorescent microplate reader (bmg labth gmbh, germany), provided with a pump, set at wavelengths of excitation and emission at 485 and 520 nm respectively, and interfaced with a computer provided with a mars data analysis software ver. 2.00 for data acquisition and processing. costar 96 well black opaque plates (corning costar corporation, cambridge, ma, usa) were used. the values were expressed as micromoles of trolox equivalents per liter of wine (μm te/l). the orac values were calculated using the following formula: orac = [ct × (aucs – auc0) × k]/(auct – auc0) where: aucs, area under curve in the presence of wine sample auc0, area under curve in the presence of blank auct, area under curve in the presence of trolox ct, trolox concentration k, dilution factor statistical analysis all analytical determinations were carried out in triplicate and the results were expressed as mean value and standard deviation (sd). student’s t test (type: 3, heteroscedastic; tails: 2), calculated using excel 2003 (microsoft corporation, redmond, wa, usa), was used to evaluate the differences between the results obtained for the different wine categories in different years. the probability of p<0.05 was considered statistically significant. correlation analyses have been performed using microcal origintm, version 5.0 (microcal software inc., northampton, ma, usa). results and discussion total phenol content and antioxidant capacity fig. 1 shows the tp content of red wines analyzed in 2010 and after one-year storage (2011). the present study established that in 2010 the highest concentration of phenol compounds was detected in cdo6 wine (3684 mg gae/l), even if tgi wines showed tp values (from 1341 to 2436 mg gae/l) comparable with those of cdo ca tegory (from 1968 to 3684 mg gae/l). in fact there were no significant differences (p>0.05) between the tp content of cdo and tgi wine categories. after one-year storage in bottle, a slight decrease of tp content was observed in all samples, with the exception of cdo3 tab. 2: some chemical parameters of umbrian red wines analyzed in 2010 and after one-year storage code total acidity volatile acidity total so2 free so2 ph total dry alcohol content (g/l) (g/l) (mg/l) (mg/l) extract (g/l) (% vol) 2010 2011 2010 2011 2010 2011 2010 2011 2010 2011 2010 2011 2010 2011 wines of controlled designation of origin (cdo) cdo1 5.17 5.32 0.35 0.34 86.60 96.00 25.60 32.00 3.41 3.49 25.15 26.30 12.30 12.34 cdo2 5.02 5.10 0.41 0.56 76.80 70.40 25.60 16.00 3.55 3.63 27.50 27375 12.65 12.52 cdo3 5.55 5.17 0.58 0.54 64.00 38.40 38.40 19.20 3.73 3.79 30.60 30.50 14.15 14.11 cdo4 5.62 5.55 0.57 0.60 51.20 57.60 19.20 19.20 3.67 3.71 32.95 32.80 13.83 13.65 cdo5 4.42 4.27 0.42 0.45 89.60 89.60 25.60 44.80 3.91 3.99 31.00 31.00 13.54 13.83 cdo6 4.95 5.02 0.32 0.41 76.80 70.40 25.60 19.20 3.67 3.71 29.70 29.55 13.83 13.79 wines of typical geographical indication (tgi) tgi1 5.10 5.17 0.60 0.63 64.00 76.80 19.20 9.60 3.60 3.65 26.70 28.25 13.65 13.61 tgi2 5.40 5.32 0.53 0.55 70.40 83.20 25.60 32.00 3.63 3.66 31.93 33.10 13.93 14.02 tgi3 5.25 5.55 0.66 0.76 64.00 57.60 25.60 25.60 3.67 3.67 31.50 32.15 14.91 15.59 tgi4 4.95 5.17 0.32 0.40 70.40 108.8 44.80 51.20 3.66 3.67 29.40 29.70 13.56 13.56 tgi5 5.17 5.40 0.28 0.26 102.40 102.40 38.40 51.20 3.55 3.54 29.30 28.70 14.11 14.11 tgi6 5.25 5.40 0.41 0.38 70.40 70.40 38.40 25.60 3.59 3.58 27.75 29.85 12.48 12.73 200 g. lombardi, l. cossignani, l. giua, m.s. simonetti, a. maurizi, g. burini, r. coli, f. blasi and cdo6 samples. the decrease could be due to the transformation of phenol compounds into condensed forms, but also to the enzyma tic activity from residual microorganisms of wine (monagas et al., 2006). no significant differences (p>0.05) in tp content of the two wine categories were also found in 2011. neither tp content obtained in the two years was different significantly (p>0.05). the results obtained in this paper for tp content were in accordance with those reported by other authors. for example, sato et al. (1996) found a value of 2282.5 ppm for a sangiovese umbrian sample, while milano et al. (2009) reported a value of 2550 mg gae/l for an umbrian red wine (assisi rosso 2006, a cdo wine produced in umbria). all wine samples tested in 2010 showed an evident antioxidant effect (fig. 2), evaluated by orac assay, with values ranging from 22382 μm te/l to 43801 μm te/l for cdo and from 21555 μm te/l to 37111 μm te/l for tgi wine category. no significant differences (p>0.05) between orac values of cdo and tgi wines were found. after one year, a significant (p<0.05) decrease of orac values was observed for all samples (with the exception of cdo6 sample). as regards the correlation between the tp content of red wines and their antioxidant capacity (fig. 3), positive results have been obtained for all wines and in particular for cdo category, in both years. in literature, conflicting and confused data exist about the correlation between the tp content and antioxidant capacity in wines (arnous et al., 2002; katalinić et al., 2004). phenol profile by chromatographic analyses in order to determine the polyphenol composition of wine samples, the analysis was initially performed by hrgc-fid (tab. 3 and 4). the most abundant compounds in both wine categories were tyrosol, gallic acid, epicatechin, and catechin. it was interesting to note that there were no significant differences (p>0.05) between the content of the single compounds in cdo and tgi wines analyzed in 2010. after one-year storage only syringic acid content changed significantly (p<0.05) between cdo and tgi wines. as regards cdo category, it was verified a significant difference (p<0.05) in the content of some phenol acids, but also of cis-resveratrol and fisetin. as regards tgi category, it was verified a significant difference (p<0.05) also fig. 1: total phenol (tp) content (mg gae/l) of umbrian red wines analyzed in 2010 and after one-year storage. error bars represent the sd of the mean values (n = 3). fig. 2: orac values (μm te/l) of umbrian red wines analyzed in 2010 and after one-year storage. error bars represent the sd of the mean values (n = 3). antioxidant properties of italian red wines 201 fig. 3: correlations between tp content (mg gae/l) and orac value (μm te/l) obtained for all wines and cdo or tgi categories, in 2010 and after oneyear storage. (a1), 2010 – all wines; (a2), 2011 – all wines; (b1), 2010 – cdo wines; (b2), 2011 – cdo wines; (c1), 2010 – tgi wines; (c2), 2011 – tgi wines; tp, total phenol; orac, oxygen radical absorbance capacity; cdo, controlled designation of origin; tgi, typical geographic indication.         fig. 3   (a1) r2 = 0.84059 (b1) r2 = 0.93506 (c1) r2 = 0.54647 (a2) r2 = 0.89433 (b2) r2 = 0.89791 (c2) r2 = 0.52124 in the content of homogentisic acid. as regards the comparison with previous literature data, milano et al. (2009) found values much lower for ferulic acid (0.16 mg/l), gallic acid (22.71 mg/l) and trans-resveratrol (1.24 mg/l), while reported much higher concentrations for caffeic and cinnamic acids, catechin and epicatechin. in order to quantify some flavonols (myricetin, quercetin, kaempferol, rhamnetin/isorhamnetin, rutin), both as aglycones and glycosides, an hplc-dad analysis was performed (tab. 5 and 6). the most abundant compounds were ethyl gallate for cdo wines and quercetin-3β-d-glucoside for tgi wines, while the most represented flavonol was quercetin, both in cdo (on average 6.78 mg/l) and tgi (on average 4.35 mg/l) wines. in all wines, the glycosidic form of quercetin, kaempferol and isorhamnetin had always a higher concentration (p<0.05) than the respective aglycones, both in wines analyzed in 2010 and in 2011. the results obtained in this study showed a high variability in the phenol composition in wine samples, although 202 g. lombardi, l. cossignani, l. giua, m.s. simonetti, a. maurizi, g. burini, r. coli, f. blasi tab. 3: content§ of phenol compounds in red wines analyzed by high-resolution gas chromatography in 2010 phenols cdo1 cdo2 cdo3 cdo4 cdo5 cdo6 tgi1 tgi2 tgi3 tgi4 tgi5 tgi6 (mg/l) vanillin 2.4±1.8 1.6±0.5 1.6±0.1 3.3±0.1 1.8±0.2 1.8±0.4 2.4±0.3 2.0±0.5 1.4±0.0 0.9±0.1 2.2±0.7 3.5±2.0 trans-cinnamic acid 1.5±0.1 1.3±0.0 9.6±1.4 7.8±5.2 12.4±0.2 10.0±0.7 5.7±0.4 1.5±0.0 1.0±0.1 5.6±0.5 9.7±1.4 6.5±1.0 tyrosol 21.9±1.8 30.6±2.0 27.1±4.2 21.6±1.2 21.2±6.3 26.6±2.2 10.2±1.0 25.5±3.6 13.0±0.4 20.7±1.9 26.9±3.5 10.7±0.7 veratric acid 1.7±0.2 1.7±0.0 2.1±0.2 2.1±0.3 1.9±0.2 2.3±0.1 1.7±0.1 1.5±0.1 1.7±0.0 1.1±0.1 2.3±0.3 1.4±0.1 vanillic acid 2.3±0.1 3.1±0.1 4.7±0.4 4.1±0.1 5.8±0.1 4.0±1.0 3.6±0.2 2.7±0.2 4.4±0.1 3.6±0.3 3.3±0.8 5.1±0.3 homovanillic acid 2.6±4.6 3.8±0.3 1.2±0.0 4.9±0.3 1.2±0.2 1.4±0.1 1.3±0.0 3.9±0.5 3.0±0.8 1.1±0.1 4.6±0.5 1.2±0.1 hydroxytyrosol 3.2±0.2 3.8±0.1 3.3±0.4 4.3±0.1 3.8±0.3 4.3±0.4 2.7±0.1 3.3±0.3 2.7±0.1 1.4±0.0 4.0±0.4 3.3±0.2 3,4-dihy acid 1.6±2.2 1.5±0.0 1.4±0.0 1.8±0.2 1.5±0.1 1.3±0.0 1.3±0.1 1.7±0.1 1.4±0.0 1.5±0.0 2.0±0.1 1.5±0.1 homogentisic acid 1.6±2.3 1.8±0.1 1.4±0.1 1.4±0.1 2.3±0.3 1.4±0.0 1.2±0.1 1.7±0.1 1.5±0.0 2.0±0.2 1.5±0.0 1.4±0.1 syiringic acid 3.4±0.2 4.2±0.1 4.4±0.3 4.1±0.2 4.3±0.2 5.3±0.2 3.0±0.0 4.0±0.2 3.9±0.0 3.1±0.4 4.3±0.0 3.8±0.2 p-coumaric acid 6.5±0.4 6.6±0.1 5.5±0.6 7.7±0.4 8.9±0.6 8.0±0.4 5.6±0.2 5.4±0.8 4.9±0.3 5.8±0.5 7.8±0.4 7.4±0.5 gallic acid 51.3±4.6 13.9±1.7 40.6±5.5 52.5±3.5 34.5±1.3 53.1±3.6 28.3±2.2 36.5±5.1 18.9±1.8 32.1±3.8 38.9±1.5 29.9±4.2 ferulic acid 3.3±0.7 1.9±0.2 2.0±0.3 2.2±0.6 1.7±0.1 2.5±0.1 1.8±0.2 1.8±0.0 2.2±0.2 2.0±0.1 2.1±0.7 1.7±0.1 caffeic acid 6.4±0.4 7.3±0.2 6.0±0.5 12.8±0.8 7.3±0.6 15.7±0.9 5.4±0.3 6.5±0.6 17.0±1.7 9.9±0.9 8.9±4.2 9.1±1.0 sinapinic acid 2.8±0.0 3.6±0.7 2.2±0.0 2.4±0.2 2.3±0.1 4.4±0.2 2.2±0.1 3.6±0.7 2.5±0.0 2.2±0.1 2.4±0.2 2.6±0.3 cis-resveratrol 1.7±0.2 2.6±0.1 2.5±0.2 1.5±0.1 3.2±0.1 1.3±0.0 1.5±0.0 2.1±0.2 2.1±0.1 1.2±0.1 2.2±0.6 2.0±0.1 trans-resveratrol 2.1±0.5 2.9±0.3 2.9±0.3 1.8±0.0 3.7±0.2 1.2±0.0 1.8±0.1 2.1±0.2 2.2±0.1 1.2±0.0 2.0±0.2 3.0±0.3 epicatechin 25.0±2.2 15.2±1.0 40.5±5.1 37.1±2.3 30.3±1.8 16.3±1.1 26.4±0.9 32.4±4.9 41.9±2.8 40.8±2.6 34.9±4.2 21.5±3.2 catechin 25.7±2.3 28.7±4.3 33.1±4.9 42.3±2.6 35.5±2.0 33.4±1.0 22.4±1.0 33.7±5.1 35.1±2.4 16.9±1.2 38.5±5.8 29.7±3.2 fisetin 5.3±2.1 2.9±0.2 6.6±6.0 2.7±0.0 2.9±0.2 3.5±0.8 4.6±1.6 3.2±0.3 3.1±0.6 2.7±0.1 3.0±0.0 3.2±0.1 tab. 4: content§ of phenol compounds in red wines analyzed by high-resolution gas chromatography in 2011 phenols cdo1 cdo2 cdo3 cdo4 cdo5 cdo6 tgi1 tgi2 tgi3 tgi4 tgi5 tgi6 (mg/l) vanillin 3.2±0.1 2.2±0.0 1.9±0.0 2.7±0.1 0.9±0.0 3.3±0.2 3.1±0.1 1.7±0.0 0.9±0.0 2.6±0.1 2.1±0.0 0.6±0.0 trans-cinnamic acid 1.3±0.0 1.5±0.0 0.9±0.0 1.8±0.1 1.0±0.0 1.7±0.0 1.5±0.0 1.0±0.0 1.1±0.0 1.2±0.1 1.1±0.0 0.9±0.0 tyrosol 24.9±0.2 30.8±0.9 22.8±0.3 16.4±0.4 28.0±0.3 25.8±0.9 12.5±0.4 19.2±0.0 8.3±0.0 19.5±0.3 28.0±0.2 20.7±0.3 veratric acid 1.6±0.0 1.1±0.0 1.5±0.0 0.7±0.0 1.4±0.0 0.9±0.0 0.7±0.0 0.8±0.0 1.0±0.0 2.2±0.0 1.7±0.1 1.0±0.0 vanillic acid 1.1±0.0 4.9±0.6 2.6±0.1 7.1±0.3 0.1±0.0 4.6±0.4 0.4±0.0 12.8±0.2 1.5±0.1 16.0±0.4 1.4±0.2 2.4±0.0 homovanillic acid 1.7±0.0 1.9±0.0 1.8±0.0 1.9±0.0 2.1±0.0 1.8±0.0 1.6±0.0 1.9±0.0 1.5±0.0 1.6±0.0 1.9±0.0 2.0±0.0 hydroxytyrosol 3.3±0.0 3.9±0.0 2.7±0.1 3.3±0.1 3.4±0.1 5.9±0.4 2.9±0.1 2.4±0.2 1.7±0.0 2.6±0.1 3.7±0.2 2.7±0.0 3,4-dihy acid 1.4±0.0 1.3±0.0 1.2±0.0 1.3±0.0 1.3±0.0 1.5±0.0 1.2±0.0 1.2±0.0 1.3±0.0 1.2±0.0 1.3±0.0 1.3±0.0 homogentisic acid 1.3±0.0 0.8±0.0 2.4±0.0 1.0±0.0 1.4±0.0 1.0±0.1 1.0±0.0 1.9±0.0 1.0±0.0 0.7±0.0 1.3±0.0 1.0±0.0 syiringic acid 2.3±0.0 3.0±0.0 2.7±0.0 2.0±0.0 2.9±0.0 4.0±0.2 1.5±0.1 1.8±0.0 1.4±0.0 1.4±0.0 2.7±0.1 2.4±0.0 p-coumaric acid 8.5±0.2 8.9±0.4 5.1±0.2 6.7±0.1 8.9±0.1 7.8±0.2 9.9±0.3 7.8±0.0 4.9±0.0 8.4±0.1 9.9±0.5 4.7±0.1 gallic acid 67.2±1.0 43.0±0.4 38.1±0.0 43.7±2.0 34.3±0.6 54.8±3.0 41.4±1.0 32.2±0.6 11.4±0.0 37.8±0.4 35.3±0.0 38.0±0.3 ferulic acid 3.6±0.1 3.7±0.0 3.1±0.1 3.4±0.0 3.1±0.0 3.6±0.0 3.6±0.0 3.5±0.0 6.6±0.0 4.3±0.1 3.5±0.0 2.8±0.0 caffeic acid 9.3±0.1 10.0±0.3 6.4±0.2 11.2±0.2 8.8±0.1 14.4±1.0 7.2±0.1 17.5±0.0 10.5±0.1 23.3±0.4 8.5±0.1 10.5±0.1 sinapinic acid 3.9±0.0 4.2±0.1 4.2±0.1 4.4±0.2 4.4±0.1 4.2±0.1 4.1±0.2 5.9±0.1 2.9±0.0 4.1±0.1 4.3±0.2 3.5±0.0 cis-resveratrol 1.7±0.0 2.6±0.0 0.4±0.0 1.0±0.0 3.0±0.0 0.6±0.0 0.2±0.0 0.3±0.0 0.3±0.0 0.1±0.0 0.4±0.1 0.3±0.0 trans-resveratrol 2.9±0.0 2.8±0.0 1.2±0.2 1.0±0.0 3.5±0.0 3.2±0.0 1.7±0.1 0.9±0.0 1.5±0.2 0.6±0.0 2.0±0.0 1.5±0.0 epicatechin 28.7±0.3 31.0±1.3 28.5±0.0 30.3±0.2 33.3±0.4 35.0±1.1 35.0±1.0 21.2±1.0 31.9±0.5 27.3±0.7 32.8±0.1 29.9±0.1 catechin 25.7±0.4 28.7±0.0 22.9±0.2 42.3±0.0 35.5±0.3 33.4±0.7 26.2±0.7 20.7±0.9 23.9±0.4 27.6±0.8 34.4±2.6 26.0±0.1 fisetin 12.5±0.1 12.0±0.0 11.3±0.0 11.8±0.1 12.6±0.2 12.0±0.2 11.9±0.0 11.5±0.1 11.2±0.0 11.2±0.0 12.3±3.0 11.5±0.1 § mean values ± sd, n=3 3,4-dihy acid, 3,4-dihydroxyphenilacetic acid antioxidant properties of italian red wines 203 coming from the same region. it is known that the qualiand quanti tative phenol composition of grape and wine shows a great variability in relation to environmental, viticultural, enological or storage practices (ivanova-petropulos et al., 2012). simonetti et al. (1997) analyzed ten red italian wines and reported the myricetin content ranging from 0.6 mg/l (squinzano) to 9.6 mg/l (cabernet sauvignon), while in this study the value changed between 3.2 and 7.2 for cdo wines and from 2.3 to 5.1 mg/l for tgi wines, analyzed in 2010. with regard to rutin, it was detected only in some samples as also reported by simonetti et al. (1997). rhamnetin was never detected in umbrian wine samples. after one-year storage, a slight decrease of single phenol content was observed, even if only the concentration of quercetin changed significantly (p<0.05) in doc wine category. in conclusion, this is the first study of phenol content, qualitative composition and antioxidant capacity in wines produced in central italy. the results confirmed the good quality of cdo and tgi umbrian red wines, in fact there were no significant differences between wine categories, both as regards tp content, orac value and phenol composition. generally, one-year storage in bottle did not affect significantly the quality of the analyzed wines, in particular for cdo category. the relevance of these results is evident when considering that some umbrian red wines, as montefalco sagrantino, are appreciated all around the word. a broader examination of umbrian red wine is actually in progress. acknowledgments this work was funded by the fondazione cassa di risparmio di perugia (project 2009.020.0004“i vini umbri: speciazione della frazione polifenolica per via hplc-ms e/o hrgc-ms ed aspetti salutistici correlati”). references arnous, a., makris, d.p., kefalas, p., 2002: correlation of pigment and flavanol content wine antioxidant properties in selected aged regional wines from greece. j. food comp. anal. 15, 655-665. doi: 10.1006/jfca.2002.1070 arranz, s., chiva-blanch, g., valderas-martínez, p., medinaremón, a., lamuela-raventós, r.m., estruch, r., 2012: wine, beer, tab. 5: content§ of phenol compounds analyzed by high-performance liquid chromatography in 2010 phenols cdo1 cdo2 cdo3 cdo4 cdo5 cdo6 tgi1 tgi2 tgi3 tgi4 tgi5 tgi6 (mg/l) rutin 3.1±0.4 13.3±0.6 3.0±0.1 3.5±0.3 0.1±0.0 5.7±0.3 1.8±0.2 7.0±0.2 0.1±0.0 1.1±0.0 0.1±0.0 3.8±0.1 ethyl gallate 14.2±0.7 16.3±0.3 16.7±0.8 20.9±1.1 21.6±1.0 19.6±1.0 7.0±0.3 7.5±0.1 3.8±0.0 8.7±0.9 8.0±0.2 7.9±0.1 quercetin-3-β-d12.4±0.6 13.5±0.2 10.8±0.7 16.5±0.8 15.9±0.6 14.5±0.5 16.8±0.3 12.3±0.3 6.4±0.2 20.7±1.3 19.0±1.0 15.3±0.1 glucoside kaempferol-3-o0.5±0.1 0.8±0.1 2.0±0.1 0.7±0.1 1.8±0.0 0.9±0.0 0.1±0.0 0.3±0.0 0.1±0.0 0.3±0.0 1.0±0.0 0.6±0.2 glucoside isorhamnetin-3-o2.1±0.4 3.1±0.1 2.6±0.1 1.1±0.0 1.3±0.0 2.5±0.1 1.4±0.1 0.1±0.0 0.2±0.0 0.1±0.0 2.4±0.1 2.0±0.3 glucoside myricetin 3.2±0.2 7.2±0.1 5.1±0.1 6.2±0.4 4.8±0.4 3.0±0.2 3.6±0.1 3.1±0.1 3.3±0.3 2.3±0.1 5.1±0.1 2.7±0.1 quercetin 6.0±0.7 10.9±0.1 6.8±0.1 8.5±0.6 5.7±0.6 2.3±0.1 6.2±0.1 6.5±0.0 3.5±0.2 1.8±0.1 4.4±0.0 3.8±0.2 kaempferol 0.2±0.1 0.3±0.0 0.1±0.0 0.1±0.0 0.1±0.0 0.1±0.0 0.1±0.0 0.1±0.0 0.1±0.0 0.1±0.0 0.1±0.0 0.1±0.0 isorhamnetin 0.3±0.1 2.3±0.0 1.1±0.0 0.7±0.1 1.4±0.3 0.1±0.0 0.2±0.0 1.1±0.0 0.6±0.0 0.1±0.0 0.8±0.0 0.6±0.1 § mean values ± sd, n=3 tab. 6: content§ of phenol compounds analyzed by high-performance liquid chromatography in 2011 phenols cdo1 cdo2 cdo3 cdo4 cdo5 cdo6 tgi1 tgi2 tgi3 tgi4 tgi5 tgi6 (mg/l) rutin 2.0±0.0 6.8±0.4 2.2±0.1 3.4±0.3 0.1±0.0 4.8±0.1 0.1±0.0 5.3±0.0 0.1±0.0 1.1±0.0 0.1±0.0 3.8±0.1 ethyl gallate 11.2±0.1 14.1±0.1 15.6±0.7 19.1±0.8 6.4±0.2 18.0±0.3 8.7±0.1 2.7±0.1 3.1±0.0 7.8±0.1 5.8±0.2 7.8±0.2 quercetin-3-β-d11.9±0.2 14.8±0.2 9.0±1.2 15.5±0.6 5.6±0.2 15.5±0.5 15.5±0.2 11.5±0.1 6.3±0.0 14.0±0.6 10.4±0.5 13.9±0.6 glucoside kaempferol-3-o0.4±0.0 0.7±0.0 1.4±0.1 0.6±0.0 1.2±0.2 0.8±0.0 0.1±0.0 0.2±0.0 0.5±0.0 0.3±0.0 1.0±0.0 0.2±0.0 glucoside isorhamnetin-3-o 1.5±0.1 3.0±0.1 2.5±0.0 0.7±0.0 1.2±0.0 2.0±0.0 1.3±0.0 0.1±0.0 0.1±0.0 0.1±0.0 2.4±0.1 2.4±0.3 glucoside myricetin 2.1±0.3 6.5±0.6 0.5±0.0 2.2±0.2 3.9±0.0 1.4±0.1 2.4±0.0 2.9±0.1 2.8±0.0 0.7±0.0 5.1±0.1 1.4±0.2 quercetin 2.6±0.0 7.2±0.3 0.4±0.0 1.7±0.3 4.1±0.0 0.7±0.0 4.3±0.1 3.6±0.1 2.6±0.0 0.3±0.0 4.4±0.0 0.5±0.0 kaempferol 0.1±0.0 0.1 ±0.0 0.1±0.0 0.1±0.0 0.1±0.0 0.1±0.0 0.1±0.0 0.1±0.0 0.1±0.0 0.1±0.0 0.1±0.0 0.1±0.0 isorhamnetin 0.1±0.0 1.7±0.2 1.4±0.1 0.1±0.0 1.1±0.0 0.1±0.0 0.1±0.0 0.6±0.0 1.3±0.5 0.1±0.0 0.8±0.0 0.1±0.0 § mean values ± sd, n=3 204 g. lombardi, l. cossignani, l. 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simonetti, p., pietta, p., testolin, g., 1997: polyphenol content and total antioxidant potential of selected italian wines. j. agric. food chem. 45, 1152-1155. doi: 10.1021/jf960705d singleton, v.l., rossi, j.a., 1965: colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagent. am. j. enol. vitic 16, 144-158. tourtoglou, c., nenadis, n., paraskevopoulou, a., 2014: phenolic composition and radical scavenging activity of commercial greek white wines from vitis vinifera l. cv. malagousia. j. food comp. anal. 33, 166-174. doi: 10.1016/j.jfca.2013.12.009 versari, a., parpinello, g.p., mattioli, a.u., 2007: characterisation of colour components and polymeric pigments of commercial red wines by using selected uv-vis spectrophotometric methods. s. afr. j. enol. vitic 28, 6-10. address of the corresponding author: university of perugia, department of pharmaceutical sciences, section of food science and nutrition, via san costanzo, 06126, perugia, italy e-mail: lina.cossignani@unipg.it © the author(s) 2017. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 91, 24 32 (2018), doi:10.5073/jabfq.2018.091.004 department of horticultural sciences, faculty of agriculture, university of hormozgan, iran effect of nitric oxide (no) on the induction of callus and antioxidant capacity of hyoscyamus niger under in vitro salt stress davood samsampour*, fatemeh sadeghi, mohammad asadi, atiye ebrahimzadeh (submitted: may 31, 2017; accepted: january 2, 2018) * corresponding author summary generation of reactive oxygen species (ros) under salt stress can cause oxidative damage. nitric oxide (no) is considered as a functional molecule in alleviating the oxidative damage of salinity to plants through modulating antioxidant metabolism. in the present study, effects of sodium nitroprusside (snp), a no donor, on induction of callus and seed germination, and antioxidant capacity of hyoscyamus niger seedlings were studied under 0, 50, 100 and 150 mm nacl stress. no stimulated the germination of nacltreated seeds. nacl treatment significantly induced accumulation of h2o2 and thiobarbituric acid-reactive substances (tbars) in hyoscyamus niger, and application of 100 μm snp stimulated rosscavenging enzymes and increased scavenging activity (dpph), hydroxyl radical (oh•) scavenging activity and chelating activity of ferrous ions, resulting in lower lipid peroxidation induced by nacl stress. therefore, it can be concluded that the increased antioxidant capacity by no might be responsible for its function in alleviating the inhibition of hyoscyamus niger growth and cell damage by salt stress. also the effect of snp on the induction of callus of hyoscyamus niger was investigated. callus fresh weight increased significantly in the combined effect of 50 μm snp with other plant growth regulators (pgr) compared to control. it is evident that snp has a direct effect on the induction of callus by interacting with cytokinins and auxins. key words: antioxidant capacity, callus, nitric oxide, hyoscyamus niger, salinity. introduction it is a well-known fact that excessive accumulation of salt ions in the soil is increasingly becoming a problem for agriculture production (zhu, 2003; baatour et al., 2010). the effects of salt stress on plant physiology have been well documented. salt stress can cause ion toxicity, osmotic stress and reactive oxygen species (ros) stress (mittler, 2002). in plant cells, mitochondria are major sites for the generation of reactive oxygen species (ros) in nonphotosynthetic cells (moller, 2001; fleury et al., 2002). as the results of oxidative stress, protein synthesis is inhibited, lipid is peroxided, enzyme is inactivated, and membrane systems are damaged (zhu et al., 2004; tanou et al., 2009). therefore, the balance between free radical generation and scavenging determines the survival of plants under salt stress. to counteract the oxidative stress, plants have evolved an antioxidant system which is composed of ros-scavenging enzymes such as catalase (cat), superoxide dismutase (sod), guaiacol peroxidase (gpx), ascorbate peroxidase (apx), dehydroascorbate reductase (dhar) and glutathione reductase (gr) (jung et al., 2000). many studies indicated that plant tolerance to salt stress was closely related with the expression and activities of these antioxidant enzymes (zushi and matsuzoe, 2009; seckin et al., 2010) as well as higher antioxidant capacity due to antioxidant metabolites (zushi and matsuzoe, 2009). enhancing the activity of antioxidant enzymes in plants organs is necessary for improving plant tolerance to salt stress. therefore, increasing free radical scavenging ability by exogenous substance application could be a practical measure in the detoxification of stress-induced excessive free radical production. recently, rapid and economically viable approaches have been proposed to alleviate the adverse effects of salt stress (ashraf and foolad, 2007). in a number of studies, it has been emphasized that exogenous application of elicitors, antioxidants, or plant growth regulators is a meaningful approach in inducing salt tolerance in crops (ashraf and foolad, 2007). nitric oxide (no) is a small, highly diffusible gas and a ubiquitous bioactive molecule. its chemical properties make nitric oxide a versatile signal molecule that functions through interactions with cellular targets via either redox or additive chemistry (lamattina et al., 2003). no produced by sodium nitroprusside (snp) has recently been considered a new phytohormones (leterrier et al., 2012) and reported to mediate the plants responses to both biotic and abiotic stresses (crawford and guo, 2005; delledonne, 2005); it also plays a crucial role in regulating plant growth and development, including germination, flowering, fruit ripening and organ senescence (arasimowicz and floryszak-wieczorek, 2007). moreover, no is believed to be involved in two respiratory electron transport pathways in mitochondria (yamasaki et al., 2001; zottini et al., 2002), where it mediates the modulation of ros and enhances antioxidant defense system in plants subjected to various abiotic stresses, such as heat (uchida et al., 2002), iron deficiency (sun et al., 2007), and salt and heavy metals (singh et al., 2008). in the past few years, research on function of no in salt stress tolerance has obtained much interest (yang et al., 2011; mostofa et al., 2015). however, the information available is sometimes contradictory, depending on the plant species, severity and duration of the salinity treatments (begara-morales et al., 2014; manai et al., 2014). in vitro culture of plant in the presence of high concentrations of salt provides a useful tool to study the adaptive mechanisms of plants living in adverse environments. mechanisms preventing oxidative burst in cells exposed to high salt concentrations are crucial for cell survival. although the antioxidant system is undoubtedly involved in the strategies used by the tissues to survive under high salt concentrations, the variation in the degree of responses has demonstrated that multiple mechanisms rather than a single mechanism which may be responsible for the adaptation of the tissues to resist salt stress. black henbane, hyoscyamus niger l., known as a medicinal herb belongs to the family solanaceae and is widely distributed in europe and asia (sawant, 2004). the plant contains tropane alkaloids (hyoscyamine, hyoscine and scopolamine) as well as flavonol glycosides (quercitin, rutin, kaempferol), which are amongst the oldest drugs used in medicine (el jaber-vazdekis, 2009). tropane alkaloids, are widely used in medicine for their mydriatic, anticholinergic, antispasmodic, analgesic and sedative properties. the seeds are used in traditional therapy for the treatment of a variety of health problems such as asthma, cough, colic, diarrhea and genitourinary complaints such as irritable bladder (gilani, 2008). in traditional chinese medicine the seeds of this plant are used as an antispasmodic, sedative, and analgesic agent, as well as for the treatment effect of nitric oxide on the induction of callus of hyoscyamus niger 25 of stomach cramps, heavy coughs, neuralgia, and manic psychosis (macy, 2002). hyoscyamus species is one of the four plants used in ayurveda for the treatment of parkinson’s disease. in previous research, sodium nitroprusside (snp) as a no donor was used to counteract the effect of salt stress. however not any data are available on the effect of no as a precursor in antioxidative responses of hyoscyamus niger against in vitro salt stress. the aim of this work is designed to study the effects of no on alleviation of oxidative damages induced by salt stress. comparing these responses can be useful for understanding the physiological and biochemical mechanisms of no in this plant that has to cope with salt stress. materials and methods experiment 1: seed germination and salt stress seeds of hyoscyamus niger were obtained from pakan bazr company, isfahan, iran. seeds surface were sterilized for 1 min with 70% ethanol and for 10 min with 10% sodium hypochlorite, then rinsed several times with sterile distilled water. sterile seeds then were placed on half-strength ms medium (murashige and skoog, 1962) containing treatment of 0, 50, 100 and 150 mm nacl and 0, 100 and 200 μm snp under aseptic condition. snp ([na2fe(cn)5] · no) was used as no donor. the medium was supplemented with 3% sucrose and solidified with 0.8% agar. the ph of the medium was adjusted to 5.8 before autoclaving. after four weeks some biochemical and physiological parameters were measured. experiment 2: induction of callus callus induction was obtained from hypocotyl explants of hyoscyamus niger obtained from seedling germinated on ms medium with a combination of snp (0 and 50 μm) and 2 concentrations (0.5 and 1 mg l-1) of α 6-benzyl aminopurine (bap), kinetin (kin), and 2,4-dichlorophenoxy acetic acid (2,4-d). before autoclaving at 121 °c for 15 min, ph of the media was adjusted to 5.8. seed germination percentage since all swollen seeds germinated, seed germination percentage was calculated by the following formula (thus differentiating swollen from unswollen seeds): seed germination percentage= number of seeds showing swelling of the embryo/total number of seeds determination of antioxidant enzyme activities three hundred mg fresh tissue were ground with 3 ml ice-cold 50 mm phosphate buffer (ph 7.8) containing 0.2 mm edta, 2 mm ascorbate and 2% pvp. the homogenates were centrifuged at 4 °c for 20 min at 12,000 × g and the resulting supernatants were used for determination of antioxidant enzyme activities. sod activity was assayed by measuring its ability to inhibit the photochemical reduction of nitroblue tetrazolium following the method of stewart and bewley (1980). cat activity was measured as the decline in absorbance at 240 nm due to the decrease of extinction of hydrogen peroxide (h2o2) according to the method of patra et al. (1978). pod activity was measured by the increase in absorbance at 470 nm due to guaiacol oxidation (nickel and cunningham, 1969). gr activity was measured according to foyer and halliwel (1976), which depends on the rate of decrease in the absorbance of nadph at 340 nm. determination of antioxidant activity for determination of antioxidant activities, 0.3 g sample was suspended in 3 ml of serine borate buffer (100 mm tris-hcl, 10 mm borate, 5 mm serine, and 1 mm diethylenetriaminepentacetic acid, ph 7.0). the slurry was centrifuged at 5,000 × g for 10 min at 4 °c and the supernatants were used for the in vitro antioxidant assays. all samples were placed on ice during the experiments. radical scavenging power (rsp) of tissue extracts were assessed by the method of manda et al. (2010). 100 μl of the extract was added to 2.9 ml of a dpph (1 × 10−4 m) solution, and kept for 30 min at room temperature under the darkness. the resulting color was measured at 520 nm against blanks. a decreasing intensity of purple color was related to a higher radical scavenging activity, which was calculated using the following formula: where a30 is absorbance of sample and b30 is absorbance of blank at 30 min reaction time. hydroxyl radical (•oh) scavenging activity was measured according to the method of manda et al. (2010). the reaction mixture (2.1 ml) contained 1 ml feso4 (1.5 mm), 0.7 ml h2o2 (6 mm), 0.3 ml sodium salicylate (20 mm) and 100 μl of extracts of hyos cyamus niger tissue. this mixture was incubated at 37 °c for 1 h, after which the absorbance of the solution was measured at 562 nm. the percentage scavenging effect was calculated as: where a0 was the absorbance of the control (without extract) and a1 was the absorbance in the presence of the extract, a2 was the absorbance without sodium salicylate. fe2+-chelating activity was measured according to the method of manda et al. (2010). the reaction mixture (2.0 ml) contained 100 μl of hyoscyamus niger tissue, 100 μl fecl2 (0.6 mm), and 1.7 ml deionised water. the mixture was shaken vigorously and left at room temperature for 5 min; 100 μl of ferrozine (5 mm in methanol) were then added, mixed, and left for another 5 min to complex was measured at 562 nm against a blank. disodium ethylenediaminetetraacetic acid (edta-na2) was used as the control. the chelating activity of the extract for fe2+ was calculated as: where a0 was the absorbance of the control (without extract) and a1 was the absorbance in the presence of the extract, a2 was the absorbance without ferrozine. determination of h2o2 concentration the h2o2 concentration was determined according to patterson et al. (1984). the assay was based on the absorbance change of the titanium peroxide complex at 415 nm. absorbance values were quantified using standard curve generated from known concentrations of h2o2. determination of lipid peroxidation lipid peroxidation was determined in terms of thiobarbituric acidreactive substances (tbars) concentration according to the method of cavalcanti et al. (2004). three hundred mg of fresh sample was homogenized in 3 ml 1.0% (w/v) tca at 4 °c. the homogenate was centrifuged at 12,000 × g for 20 min and 1 ml of the supernatant was 5 determination of antioxidant enzyme activities three hundred mg fresh tissue were ground with 3 ml ice-cold 50 mm phosphate buffer (ph 7.8) containing 0.2 mm edta, 2 mm ascorbate and 2% pvp. the homogenates were centrifuged at 4°c for 20 min at 12,000×g and the resulting supernatants were used for determination of antioxidant enzyme activities. sod activity was assayed by measuring its ability to inhibit the photochemical reduction of nitroblue tetrazolium following the method of stewart and bewley (1980). cat activity was measured as the decline in absorbance at 240 nm due to the decrease of extinction of hydrogen peroxide (h2o2) according to the method of patra et al. (1978). pod activity was measured by the increase in absorbance at 470 nm due to guaiacol oxidation (nickel and cunningham, 1969). gr activity was measured according to foyer and halliwel (1976), which depends on the rate of decrease in the absorbance of nadph at 340 nm. determination of antioxidant activity for determination of antioxidant activities, 0.3 g sample was suspended in 3 ml of serine borate buffer (100 mm tris–hcl, 10 mm borate, 5 mm serine, and 1 mm diethylenetriaminepentacetic acid, ph 7.0). the slurry was centrifuged at 5,000 × g for 10 min at 4°c and the supernatants were used for the in vitro antioxidant assays. all samples were placed on ice during the experiments. radical scavenging power (rsp) of tissue extracts were assessed by the method of manda et al. (2010). 100 µl of the extract was added to 2.9 ml of a dpph (1×10−4 m) solution, and kept for 30 min at room temperature under the darkness. the resulting color was measured at 520 nm against blanks. a decreasing intensity of purple color was related to a higher radical scavenging activity, which was calculated using the following formula: rsp(%) = �1 − ( 𝐴𝐴𝐴𝐴30 𝐵𝐵𝐵𝐵30 )� × 100 where a30 is absorbance of sample and b30 is absorbance of blank at 30 min reaction time. 6 hydroxyl radical (•oh) scavenging activity was measured according to the method of manda et al. (2010). the reaction mixture (2.1 ml) contained 1 ml feso4 (1.5 mm), 0.7 ml h2o2 (6 mm), 0.3 ml sodium salicylate (20 mm) and 100 µl of extracts of hyoscyamus niger tissue. this mixture was incubated at 37°c for 1 h, after which the absorbance of the solution was measured at 562 nm. the percentage scavenging effect was calculated as: scavenging effect(%) = �1 − ( 𝐴𝐴𝐴𝐴1 − a2 𝐴𝐴𝐴𝐴0 )� × 100 where a0 was the absorbance of the control (without extract) and a1 was the absorbance in the presence of the extract, a2 was the absorbance without sodium salicylate. fe2+-chelating activity was measured according to the method of manda et al. (2010). the reaction mixture (2.0 ml) contained 100 µl of hyoscyamus niger tissue, 100 µl fecl2 (0.6 mm), and 1.7 ml deionised water. the mixture was shaken vigorously and left at room temperature for 5 min; 100 µl of ferrozine (5 mm in methanol) were then added, mixed, and left for another 5 min to complex was measured at 562 nm against a blank. disodium ethylenediaminetetraacetic acid (edta-na2) was used as the control. the chelating activity of the extract for fe2+ was calculated as: chelating effect(%) = �1 − ( 𝐴𝐴𝐴𝐴1 − a2 𝐴𝐴𝐴𝐴0 )� × 100 where a0 was the absorbance of the control (without extract) and a1 was the absorbance in the presence of the extract, a2 was the absorbance without ferrozine. determination of h2o2 concentration the h2o2 concentration was determined according to patterson et al. (1984). the assay was based on the absorbance change of the titanium peroxide complex at 415 nm. absorbance values were quantified using standard curve generated from known concentrations of h2o2. determination of lipid peroxidation lipid peroxidation was determined in terms of thiobarbituric acid-reactive substances (tbars) concentration according to the method of cavalcanti et al. (2004). three hundred mg of fresh sample was homogenized in 3 ml 1.0% (w/v) tca at 4 ◦c. the homogenate was centrifuged at 6 hydroxyl radical (•oh) scavenging activity was measured according to the method of manda et al. (2010). the reaction mixture (2.1 ml) contained 1 ml feso4 (1.5 mm), 0.7 ml h2o2 (6 mm), 0.3 ml sodium salicylate (20 mm) and 100 µl of extracts of hyoscyamus niger tissue. this mixture was incubated at 37°c for 1 h, after which the absorbance of the solution was measured at 562 nm. the percentage scavenging effect was calculated as: scavenging effect(%) = �1 − ( 𝐴𝐴𝐴𝐴1 − a2 𝐴𝐴𝐴𝐴0 )� × 100 where a0 was the absorbance of the control (without extract) and a1 was the absorbance in the presence of the extract, a2 was the absorbance without sodium salicylate. fe2+-chelating activity was measured according to the method of manda et al. (2010). the reaction mixture (2.0 ml) contained 100 µl of hyoscyamus niger tissue, 100 µl fecl2 (0.6 mm), and 1.7 ml deionised water. the mixture was shaken vigorously and left at room temperature for 5 min; 100 µl of ferrozine (5 mm in methanol) were then added, mixed, and left for another 5 min to complex was measured at 562 nm against a blank. disodium ethylenediaminetetraacetic acid (edta-na2) was used as the control. the chelating activity of the extract for fe2+ was calculated as: chelating effect(%) = �1 − ( 𝐴𝐴𝐴𝐴1 − a2 𝐴𝐴𝐴𝐴0 )� × 100 where a0 was the absorbance of the control (without extract) and a1 was the absorbance in the presence of the extract, a2 was the absorbance without ferrozine. determination of h2o2 concentration the h2o2 concentration was determined according to patterson et al. (1984). the assay was based on the absorbance change of the titanium peroxide complex at 415 nm. absorbance values were quantified using standard curve generated from known concentrations of h2o2. determination of lipid peroxidation lipid peroxidation was determined in terms of thiobarbituric acid-reactive substances (tbars) concentration according to the method of cavalcanti et al. (2004). three hundred mg of fresh sample was homogenized in 3 ml 1.0% (w/v) tca at 4 ◦c. the homogenate was centrifuged at 26 d. samsampour, f. sadeghi, m. asadi, a. ebrahimzadeh added to 3 ml 20% tca containing 0.5% (w/v) thiobarbituric acid (tba). the mixture was incubated at 95 °c for 30 min and the reaction was stopped by quickly placing in an ice bath. the cooled mixture was centrifuged at 10,000 × g for 10 min, and the absorbance of the supernatant at 532 and 600 nm was read. after substracting the non-specific absorbance at 600 nm, the tbars concentration was determined by its extinction coefficient of 155 mm−1 cm−1. glycine betaine content the amount of glycine betaine was estimated according to the me thod of grieve and grattan (1983). the plants tissues were groun ded with 20 ml of deionised water for 24 h at 25 ºc. the samples were then filtered and filtrates were diluted to 1:1 with 2 n h2so4. aliquots were kept in centrifuge tubes and cooled in ice water for 1 h. cold ki-i2 reagent was added, and the reactants were gently stirred with a vortex mixture. the tubes were stored at 4 ºc for 16 h and then centrifuged at 10,000 × g for 15 min at 4 ºc. the supernatant was carefully aspirated with a new glass tube. the periodide crystals were dissolved in 9 ml of 1,2-dichloroethane. after 2 h, the absorbance was measured at 365 nm using glycine betaine as a standard and expressed in mg g−1 dw. total soluble protein content total soluble protein content was measured according to bradford (1976) using bovine serum albumin (bsa) as a protein standard. fresh leaf samples (1 g) were homogenized with 4 ml na-phosphate buffer (ph 7.2) and then centrifuged at 4 °c. supernatants and dye were pipetting in spectrophotometer cuvettes and absorbance was measured using a spectrophotometer at 595 nm. statistical analysis: the experiment was carried out as bi-factorial in a completely randomized design (4 salinity level × 3 snp level) with 3 replications. analysis of variance (anova) for different responses parameters were evaluated by sas 9.4 software package. least significance difference (lsd) test was used to determine the significant difference among the mean values at the 0.05 level. results in vitro seed germination: control of dormancy release and germination of plant seeds it was observed that the seeds started germination in the five days after placing on the medium. complete seedlings with two leaves appeared in the 2rd week. when salinity was applied singly, seed germination percentage was low compared to control. in all levels of salinity, medium supplemented with 100 μm snp significantly increased seed germination (fig. 1). medium supplemented with snp 100 μm in control resulted in 100% seed germination. interaction effect of salinity* snp showed that snp different levels in each level of salinity was significant (tab. 1). h2o2 concentrations and lipid peroxidation compared to control, salt stress significantly induced accumulation of h2o2 in hyoscyamus niger tissue (p < 0.05), and application of exogenous no dramatically reduced concentration of h2o2 (p < 0.05) (fig. 2), and effect of salt stress was blocked by addition no in medium. under normal conditions, exogenous no did not significantly affect h2o2 in hyoscyamus niger tissue. tbars was used to indicate lipid peroxidation, similar with the change of h2o2, salt stress significantly increased tbars concentration in hyoscyamus niger tissue (p < 0.05) (fig. 2). in 50, 100 and 150 mm nacl when seedlings were treated with 100 μm snp, tbars concentration was significantly lower than non-treated plant. in our experiment, it appears that no has protective effect on membrane damage hyoscyamus niger tissue. comparison of snp different levels in each level of nacl showed that snp effect was statistically significant for h2o2 and tbars concentration except in control (tab. 2). antioxidant enzyme activities as shown in fig. 3, salt stress significantly increased the activities of sod, cat, pod and gr in hyoscyamus niger than those of the control groups, which may be a reflection of the oxidative stress induced by salt stress. with increasing nacl in the medium, pod activities increased until 100 mm and then decreased (fig. 3). application of snp increased activities of all antioxidant enzymes under salt stress. under normal conditions, activities of pod and gr antioxidant enzymes were not significantly influenced by application of exogenous snp. treatment of plants with snp increased the activity of cat and apx enzymes in control, and stress conditions. interaction effect of salinity* snp showed that snp different levels in each level of salinity was significant except for pod and gr enzymes in the control (tab. 3). antioxidant capacity dpph scavenging capacity, •oh scavenging capacity and metal chelating capacity were usually used to express antioxidant capacity of plant tissues. compared to the control, salt stress dramatically decreased antioxidant capacity of hyoscyamus niger tissue, and reduced dpph scavenging capacity, •oh scavenging capacity and metal chelating capacity (fig. 4). while application of exogenous snp significantly alleviated the inhibition of antioxidant capacity by salt stress, and increased dpph scavenging capacity, •oh scavenging capacity and metal chelating capacity compared to the salt stress. under normal conditions, ap9 fig. 1. effects of snp on seed germination of hyoscyamus niger under nacl stress. means with different letters are significantly different (p<0.05). table 1. salt*snp effect separate by salt for germination. *, **, ***, significant at 0.05, 0.01, and 0.001 levels, respectively salt (mm) df mean square 0 2 234.10*** 50 2 143.44*** 100 2 74.08*** 150 2 115.96*** h2o2 concentrations and lipid peroxidation compared to control, salt stress significantly induced accumulation of h2o2 in hyoscyamus niger tissue (p < 0.05), and application of exogenous no dramatically reduced concentration of h2o2 (p < 0.05) (fig. 2), and effect of salt stress was blocked by addition no in medium. under normal conditions, exogenous no did not significantly affect h2o2 in hyoscyamus niger tissue. tbars was used to indicate lipid peroxidation, similar with the change of h2o2, salt stress significantly increased tbars concentration in hyoscyamus niger tissue (p < 0.05) (fig. 2). in d d e g a b d e bc c d f 0 20 40 60 80 100 120 0 50 100 150 % g er m in a9 o n salinity (mm) 0 snp 100 snp 200 snp tab. 1: salt*snp effect separate by salt for germination. *, **, ***, significant at 0.05, 0.01, and 0.001 levels, respectively. salt (mm) df mean square 0 2 234.10*** 50 2 143.44*** 100 2 74.08*** 150 2 115.96*** fig. 1: effects of snp on seed germination of hyoscyamus niger under nacl stress. means with different letters are significantly different (p<0.05). effect of nitric oxide on the induction of callus of hyoscyamus niger 27 fig. 2: effects of snp on the concentrations of h2o2 (a) and tbars (b) of hyoscyamus niger under nacl stress. means with different letters are significantly different (p<0.05). 10 50, 100 and 150 mm nacl when seedlings were treated with 100 µm snp, tbars concentration was significantly lower than non-treated plant. in our experiment, it appears that no has protective effect on membrane damage hyoscyamus niger tissue. comparison of snp different levels in each level of nacl showed that snp effect was statistically significant for h2o2 and tbars concentration except in control (table 2). fig. 2. effects of snp on the concentrations of h2o2 (a) and tbars (b) of hyoscyamus niger under nacl stress. means with different letters are significantly different (p<0.05) table 2. salt*snp effect by salt for h2o2 and tbars concentration. *, **, ***, significant at 0.05, 0.01, and 0.001 levels, respectively. ns, non-significant. mean square salt (mm) df h2o2 tbars 0 2 15.19ns 0.016ns 50 2 584.91*** 0.178* 100 2 587.32*** 1.50*** 150 2 155.39** 0.75*** g c b a g f e b g de cd b 0 20 40 60 80 100 120 140 160 0 50 100 150 h 2o 2 (n m o l g -1 f w ) salinity (mm) 0 snp 100 snp 200 snp h cd b a h f g de h ef bc b 0 0,5 1 1,5 2 2,5 3 3,5 4 0 50 100 150 tb a r s ( n m o l g -1 f w ) salinity (mm) 0 snp 100 snp 200 snp tab. 2: salt*snp effect by salt for h2o2 and tbars concentration. *, **, ***, significant at 0.05, 0.01, and 0.001 levels, respectively. ns, nonsignificant. mean square salt (mm) df h2o2 tbars 0 2 15.19ns 0.016ns 50 2 584.91*** 0.178* 100 2 587.32*** 1.50*** 150 2 155.39** 0.75*** 11 antioxidant enzyme activities as shown in fig. 3, salt stress significantly increased the activities of sod, cat, pod and gr in hyoscyamus niger than those of the control groups, which may be a reflection of the oxidative stress induced by salt stress. with increasing nacl in the medium, pod activities increased until 100 mm and then decreased (figure 3). application of snp increased activities of all antioxidant enzymes under salt stress. under normal conditions, activities of pod and gr antioxidant enzymes were not significantly influenced by application of exogenous snp. treatment of plants with snp increased the activity of cat and apx enzymes in control, and stress conditions. interaction effect of salinity* snp showed that snp different levels in each level of salinity was significant except for pod and gr enzymes in the control (table 3). g f de e g cd a bc g e b e 0 10 20 30 40 50 60 70 0 50 100 150 p o d (u .m g1 f w ) salinity (mm) 0 snp 100 snp 200 snp g f d b f c a a g e b b 0 10 20 30 40 50 60 70 0 50 100 150 c at (u .m g1 f w ) salinity(mm) 0 snp 100 snp 200 snp i gh e b h f c a h g d b 0 100 200 300 400 500 600 0 50 100 150 so d ( u m g1 fw ) salinity (mm) 0 snp 100 snp 200 snp g f d b g de c a g ef c b 0 0,5 1 1,5 2 2,5 0 50 100 150 g lu ta th io n e re d u ct as e ( u m g1 fw ) salinity (mm) 0 snp 100 snp 200 snp fig. 3: effects of snp on the activities of sod, cat, pod and gr of hyoscyamus niger under nacl stress. means with different letters are significantly different (p<0.05). tab. 3: salt*snp effect by salt for pod, cat, sod and gr. *, **, ***, significant at 0.05, 0.01, and 0.001 levels, respectively. ns, nonsignificant. salt (mm) df pod cat sod gr 0 2 8.38ns 73.50** 2918.11** 0.0032ns 50 2 129.53*** 273.79*** 661.44*** 0.036*** 100 2 245.29*** 346.77*** 2550.33*** 0.240*** 150 2 85.20** 43.11*** 868.11** 0.163*** plication of exogenous. no did not greatly change the antioxidant capacity of hyoscyamus niger tissue (fig. 4). comparison of snp different levels in each level of nacl showed that snp effect was not statistically significant in control for dpph scavenging and metal chelating and 150 mm nacl for dpph scavenging (tab. 4). glycine betaine concentration as shown in fig. 5, salt stress significantly induced accumulation of glycine betaine in hyoscyamus niger (p < 0.5), and application of snp significantly increased glycine betaine concentration under salt stress, like the change of other parameters, under normal conditions, glycine betaine concentrations in hyoscyamus niger tissue were not 28 d. samsampour, f. sadeghi, m. asadi, a. ebrahimzadeh significantly changed by application of exogenous snp. interaction effect of salinity* snp showed that snp different levels in each level of salinity was significant except in control (tab. 5). total soluble proteins addition of nacl caused a significant reduction in soluble proteins content. this reduction was more pronounced in 150 mm nacl. in the lower salt concentrations (control, 50 mm), when plants were treated with 100 μm snp, they showed higher amount of protein in comparison with their controls. 100 μm snp was found to be most effective (fig. 6). interaction effect of salinity*snp showed that snp different levels in each level of salinity was significant (tab. 6). callus induction: the results varied with respect to plant growth regulators (pgr) and snp (tab. 7). among eight callus induction media, media supplemented with 0.5 mg-1 2,4-d, 0.5 mg-1 bap and 50 μm snp was the most average of callus fresh weight (tab. 7). it was significantly higher than all other media combinations. the concentration of 14 salt(mm) df dpph scavenging oh scavenging metal chelating 0 2 10.023ns 66.33*** 0.22ns 50 2 142.69*** 57.33** 54.84*** 100 2 51.30* 44.77** 170.35*** 150 2 6.48ns 44.77** 94.63*** glycine betaine concentration as shown in figure 5, salt stress significantly induced accumulation of glycine betaine in hyoscyamus niger (p < 0.5), and application of snp significantly increased glycine betaine concentration under salt stress, like the change of other parameters, under normal conditions, glycine betaine concentrations in hyoscyamus niger tissue were not significantly changed by application of exogenous snp. interaction effect of salinity* snp showed that snp different levels in each level of salinity was significant except in control (table 5). fig. 5. effects of snp on glycine betaine of hyoscyamus niger under nacl stress. means with different letters are significantly different (p<0.05) h g e c i e d a hi f d b 0 5 10 15 20 25 0 50 100 150 g ly ci n b et ai n e (m g· g1 fw ) salinity 0 snp 100 snp 200 snp 13 comparison of snp different levels in each level of nacl showed that snp effect was not statistically significant in control for dpph scavenging and metal chelating and 150 mm nacl for dpph scavenging (table 4). fig. 4. effects of snp on dpph scavenging activity, hydroxyl radical (•oh) scavenging activity and fe2+ chelating activity of hyoscyamus niger under nacl stress. means with different letters are significantly different (p<0.05). table 4. salt*snp effect by salt for dpph scavenging, oh scavenging and oh scavenging. *, **, ***, significant at 0.05, 0.01, and 0.001 levels, respectively. ns, non-significant. salt(mm) df dpph scavenging oh scavenging metal chelating ab c d d a a c d a bc c d 0 10 20 30 40 50 60 0 50 100 150 d p p h s ca ve n ge r % salinity (mm) 0 snp 100 snp 200 snp a cd de ef b a b d cd bc d f 0 5 10 15 20 25 30 35 40 45 0 50 100 150 o h s ca ve n gi n g ca p ac it y % salinity (mm) 0 snp 100 snp 200 snp a d e g a bc c e a c d f 0 10 20 30 40 50 60 70 80 90 100 0 50 100 150 m et al c h el a9 n g ca p ac it y % salinity (mm) 0 snp 100 snp 200 snp fig. 4: effects of snp on dpph scavenging activity, hydroxyl radical (•oh) scavenging activity and fe2+ chelating activity of hyoscyamus niger under nacl stress. means with different letters are significantly different (p<0.05). tab. 4: salt*snp effect by salt for dpph scavenging, oh scavenging and oh scavenging. *, **, ***, significant at 0.05, 0.01, and 0.001 levels, respectively. ns, non-significant. salt (mm) df dpph oh metal scavenging scavenging chelating 0 2 10.023ns 66.33*** 0.22ns 50 2 142.69*** 57.33** 54.84*** 100 2 51.30* 44.77** 170.35*** 150 2 6.48ns 44.77** 94.63*** fig. 5: effects of snp on glycine betaine of hyoscyamus niger under nacl stress. means with different letters are significantly different (p<0.05). tab. 5: salt*snp effect by salt for glycine betaine. *, **, ***; significant at 0.05, 0.01, and 0.001 levels, respectively. ns, non-significant. salt (mm) df mean square 0 2 0.57ns 50 2 4.85*** 100 2 24.023*** 150 2 7.77*** higher bap and 2,4-d had a decrease in callus fresh weight. the minimum day until callus induction was recorded with media supplemented with 0.5 mg-1 2,4-d, 0.5 mg-1 kin and 50 μm snp (fig. 7). effect of nitric oxide on the induction of callus of hyoscyamus niger 29 tab. 7: effects of snp and plant growth regulators on callus induction of hyoscyamus niger hypocotyl. means with different letters are significantly different (p<0.05). pgr snp callus fw day until induction (μm) (g) of callus 0 0.25± 0.03 d 10± 0.57 b 0.5 2,4.d + 0.5 bap 50 0.54± 0.04 a 8± 0.57 c 0 0.19± 0.015 ef 11.66 ± 0.57 a 1 2,4-d + 1 bap 50 0.2± 0.01 ef 8 cd 0 0.35± 0.035 c 10.3± 0.57 b 0.5 2,4-d + 0.5 kin 50 0.43± 0.04 b 7.33± 0.57 d 0 0.16± 0.03 f 11.66 ± 0.57 a 1 2,4-d + 1 kin 50 0.23± 0.01 de 11.33± 0.57 a tab. 6: salt*snp effect by salt for total protein. *, **, ***, significant at 0.05, 0.01, and 0.001 levels, respectively. ns, non-significant. salt (mm) df mean square 0 2 366.093*** 50 2 271.37*** 100 2 18.78* 150 2 22.73* 15 table 5. salt*snp effect by salt for glycine betaine. *, **, ***; significant at 0.05, 0.01, and 0.001 levels, respectively. ns, non-significant. salt (mm) df mean square 0 2 0.57ns 50 2 4.85*** 100 2 24.023*** 150 2 7.77*** total soluble proteins addition of nacl caused a significant reduction in soluble proteins content. this reduction was more pronounced in 150 mm nacl. in the lower salt concentrations (control, 50mm), when plants were treated with 100 µm snp, they showed higher amount of protein in comparison with their controls. 100 µm snp was found to be most effective (fig. 6). interaction effect of salinity*snp showed that snp different levels in each level of salinity was significant (table 6). fig. 6. effects of snp on total soluble proteins of hyoscyamus niger under nacl stress. means with different letters are significantly different (p<0.05) c d ef g a b d f b c de g 0 10 20 30 40 50 60 70 80 0 50 100 150 p ro te in (m g· g1 fw ) salinity (mm) 0 snp 100 snp 200 snp fig. 6: effects of snp on total soluble proteins of hyoscyamus niger under nacl stress. means with different letters are significantly different (p<0.05). fig. 7: effects of snp and plant growth regulators on callus induction of hyoscyamus niger hypocotyl. a: 0.5 mg/l 2,4-d+ 0.5mg/l kinetin and 50 μm snp; b: 1 mg/l 2,4-d + 1 mg/l kinetin; c: 0.5 mg/l 2,4-d+ 0.5 mg/l bap; d: 0.5 mg/l 2,4-d+ 0.5 mg/l bap and 50 μm snp; e: 0.5 mg/l 2,4-d+ 0.5 mg/l kinetin and 50 μm snp; f: 0.5 mg/l 2,4-d+ 0.5 mg/l bap and 50 μm snp. discussion exogenous no has a strong stimulating effect on seed germination under stress or no-stress conditions (beligni and lamattina, 2000). some studies provided evidence that no could counteract the inhibitory effect of salinity in the process of germination (zheng et al., 2009). in agreement with these, we observed that exogenous no treatment significantly stimulated seed germination under severe salt stress in hyoscyamus niger (fig. 1). in this work, snp, as an exogenous donor, significantly reduced 30 d. samsampour, f. sadeghi, m. asadi, a. ebrahimzadeh oxidative stress in hyoscyamus niger imposed by salt stress, and reduced the accumulation of h2o2 and tbars accumulation. saltstressed hyoscyamus niger plant accumulated higher levels of h2o2 and tbars contents (fig. 2), which might be due to membrane destruction caused by ros-induced oxidative damage. exogenous application of no reduced levels of tbars and h2o2 in nacltreated hyoscyamus niger plants (fig. 2), which is in agreement with the observations of zheng et al. (2009) and khan et al. (2012). therefore, application of no could be an effective practice to protect plants against oxidative injury caused by salt stress. no alleviates abiotic stress through different metabolism, and antioxidant capacity modulation was reported to be one of important pathways in many investigations (hao et al., 2009; qiao and fan, 2008). the activities of sod, cat, pod and gr in the presence of snp under salt stress were also higher than those under salt stress alone (fig. 3). exogenous application of no increased activity of cat, sod, pod and apx in seashore mallow (guo et al., 2009), mustard (zeng et al., 2011), wheat (ruan et al., 2002) and protected plants from oxidative damage under salt stress. in tomato, exogenous application of no increased the activity of antioxidant enzymes sod, pod, cat, apx, non-enzymatic antioxidant ascorbate and reduced glutathione under salinity stress thus helping to alleviate salt-induced oxidative damage (wu et al., 2011). no can act (i) as a direct sca venger of ros, and (ii) antioxidant system inducer to enhance the expression of antioxidant enzyme-encoding genes (gross et al., 2013). no applied exogenously may also induce the synthesis of endogenous no (hao et al., 2008; zhao et al., 2009; fan and liu, 2012), which can function as signaling molecule or ros scavenger under prolonged stress conditions by regulating/enhancing the activities of antioxidant enzymes (hao et al., 2008; fan and liu, 2012). antioxidant capacity including dpph-radical scavenging activity, hydroxyl radical scavenging activity and the chelating activity of ferrous ions were measured for further investigation of no func tion in antioxidant capacity induction of hyoscyamus niger under salt stress. in the present experiment, exogenous no reversed the inhibition of dpph-radical scavenging activity, hydroxyl radical scavenging activity and the chelating activity of ferrous ions by salt stress in hyoscyamus niger (fig. 4), furthermore, compared to the change of antioxidant enzymes, their changing degrees were much higher related with lower accumulation of h2o2 and tbars, this was accordance with the observed results in cakile maritima (ksouri et al., 2007). h2o2 can also lead to the production of oh• and is very reactive on important biomolecules or membranes in plants (karuppanapandian et al., 2011). in this study, as suggested by results in lipid peroxidation in membranes of hyoscyamus niger plants, the oh• scavenging activity decreased at high-salt concentration. however, both snp concentrations under stress conditions improved the capacity of oh• scavenging. therefore, the enhanced scavenging ability of oh• inhibited ros accumulation in plant, and thus plants were protected from lipid peroxidation of membrane systems and oxidative damages. also jasid et al. (2008) reported that no acts as an antioxidant and ros scavenger increase of dpph radical scavenging activity, hydroxyl radical scavenging activity and the chelating activity of ferrous ions in plants usually depends on antioxidant metabolites (ksouri et al., 2007), there were reports indicated that exogenous no could induce syn thesis of these antioxidant metabolites (ksouri et al., 2007; wu et al., 2007; ferreira et al., 2010), therefore, the stimulation of antioxidant capacity by no might be attributed to induction of antioxidant metabolites synthesis and they might be greatly responsible for lower lipid peroxidation in hyoscyamus niger under salt stress. to make sure the hypothesis of metabolites function in increasing antioxidant capacity induced by no. osmolyte adjustment via glycine betaine accumulation has been reported to inhibit accumulation of ros, protect photosynthetic machinery and activate stress-related genes (chen and murata, 2008). in this study, nacl stress induced glycine betaine accumulation in hyoscyamus niger, and exogenous no increased glycine betaine level under salt stress (fig. 5). recently, many studies have indicated that no appears to be involved in the metabolism of glycine betaine in stress tolerance (lópez-carrión et al., 2008). in the present study, a similar accumulation trend of total soluble proteins was recorded in hyoscyamus niger under nacl stress (fig. 6). soluble proteins play a main role in osmotic adjustment under nacl stress and can provide storage form of nitrogen (singh et al., 1987). increase in soluble protein content under stress may be the result of enhanced synthesis of specific stress-related proteins (doganlar et al., 2010). thus, application of no to salt-stressed hyoscyamus niger provoked a remarkable increase in levels of total soluble proteins and glycine betaine perhaps to provide a better protection to plants exposed to stress. the protective nature of the osmolytes under no treatment corroborates with the findings of hayat et al. (2012), khan et al. (2012), kausar et al. (2013), and dong et al. (2014) in various plants. noticeable accumulation of glycine betaine, total soluble proteins due to no application might enhance salt tolerance of cells through osmotic regulation. also, the results indicated that addition of 50 μm snp led to increase in callus induction compared with the control (tab. 7). elicitations are considered to be an important strategy towards improved in vitro production of secondary metabolites. various biotic and abiotic factors added to the medium of callus production influence their production by activating genes for de novo synthesis or by stimulation the physiological processes leading to enhanced accumulation of such products. in a previous study conducted in cab-6p, gisela 6, and m.m 14 cherry rootstocks, the callus formation percentage was 100% in all snp (0-50 μm) + 17.6 μm bap + 2.68 mm naa treatments (sarropoulou et al., 2014). according to χu et al. 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biol. 6, 441-445. zhu, z., wei, g.q., li, j., qian, q.q., yu, j.q., 2004: silicon alleviates salt stress and increases antioxidant enzymes activity in leaves of saltstressed cucumber (cucumis sativus l.). plant sci. 167, 527-533. zottini, m., formentin, e., scattolin, m., carimi, f., schiavo, f.l., terzi, m., 2002: nitric oxide affects plant mitochondrial functionality in vivo. febs lett. 515, 75-78. zushi, k., matsuzoe, n., 2009: seasonal and cultivar differences in saltinduced changes in antioxidant system in tomato. sci. hort. 120, 181-187. address of the authors: davood samsampour, fatemeh sadeghi, mohammad asadi and atiye ebrahimzadeh, department of horticultural sciences, faculty of agriculture, university of hormozgan, iran e-mail: samsampoor@bmn.ir © the author(s) 2018. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 87, 279 285 (2014), doi:10.5073/jabfq.2014.087.039 1laboratoire d’ecologie et gestion des ecosystèmes naturels, faculté des sciences de la nature et de la vie, et des sciences de la terre et l’univers 2laboratoire des substances naturelles et bioactives (lasnabio) département de chimie, faculté des sciences, université de tlemcen, algérie 3université de corse, umr cnrs 6134, laboratoire chimie des produits naturels, campus grimaldi, corte, france control of fungal pathogens of citrus sinensis l. by essential oil and hydrosol of thymus capitatus l. leila tabti1, mohammed el amine dib2*, nassim djabou2, nassira gaouar benyelles1, julien paolini3, jean costa3, alain muselli3 (received september 11, 2014) * corresponding author summary essential oil, hydrosol extract and hydrosol of thymus capitatus l. from algeria were tested for antifungal activity against four phytopathogenic fungi (aspergillus niger, aspergillus oryza, penicillium italicum and fusarium solani) causing the deterioration of citrus sinensis fruits. essential oil and hydrosol extract showed strong in vitro antifungal activity based on the inhibition zone and minimal inhibitory concentration values against the pathogens. citrus sinensis fruits infected by penicillium italicum were treated in vivo with essential oil, hydrosol extract and hydrosol. 0.2 μg/ml of thymus hydrosol was needed for the absence of orange infection and causing 100 % mycelial growth inhibition. this activity can be correlated with chemical composition of extracts which are rich in carvacrol (more than 69 %). therefore, the preventive and curative effects of t. capitatus essential oil and hydrosol could be exploited as an ideal alternative to synthetic fungicides for using in the treatment of many fungal phytopathogens causing severe destruction to oranges. introduction citrus is an important crop with world production estimated at 115 million tons per year. during 2010-2011, 571 thousand tons were producer in algeria which is the 19th produced in the world and the 3rd in the arab maghreb union (lagha-benamrouche and madani, 2013). citrus are among the most popular fruits grown in algeria. furthermore, citrus production represents an important agricultural and economic activity in the country. oranges and mandarins are traditionally produced for local consumption and also for export. citrus is an important crop with world production estimated at 115 million tons per year. during 2010/2011, 571 thousand tons were produced in algeria which is the 19th producers in the world and the 3rd in the arab maghreb union (fao, 2012) (lagha-benamrouche and madani, 2013). oranges, lemons, grapefruits and mandarins represent approximately 98 % of industrial cultures. oranges are most pertinent with about 82 % of total (lagha-benamrouche and madani, 2013). fungal growth on fresh fruits and vegetables is responsible for food spoilage and numerous plant diseases, which lead to significant economic losses. penicillium and aspergillus were responsible for spoilage of many foods and causes decay on stored fruits damaged by insects, animals, early splits, and mechanical harvesting (tu et al., 2013; rojas et al., 2005). the industries of food products nowadays are using synthetic chemical preservatives to prevent the growth of pathogens, but these chemicals convert certain ingested materials into toxins and carcinogens (farag et al., 1989). alternative control methods are needed because of negative public perceptions about the use of pesticides, development of resistance to fungicides, and high cost for development of new chemicals preservatives (stojković et al., 2011). thus, there has been a growing interest on the research of the possible use of plant secondary metabolites for pest and disease control in agriculture (gikh et al., 2002). the plants have long been recognized to provide a potential source of different class of chemical compounds, known as phytochemicals, such as terpenoids, alkaloids, phenolics, glucosides, etc., which are effective products against pathogenic microorganisms (feng and zheng, 2007). essential oils are of growing interest both in the industry and scientific research because of their antibacterial and antifungal properties which make them useful as natural additives in foods (pattnaik et al., 1997). such antimicrobial activity is due to the presence of bioactive substances such as monoterpenes, sesquiterpenes and related alcohols, other hydrocarbons and phenols (griffin et al., 1999; kalemba and kunicka, 2003). thyme (thymus) is a genus containing about 350 species of aromatic perennial herbs and sub-shrubs to 40 cm tall, belong to the family lamiaceae. this family is distributed throughout the arid, temperate and cold regions including europe, north africa and asia. it is in leaf all year, flowering from july to september (gruenwald et al., 2004). several studies have assessed the ability of the thymus essential oils and their constituents as fumigants and repellents against a number of insect pests (clemente et al., 2003; lee et al., 2001; hori, 2003, salama et al., 2012). effective antifungal activity of t. propolis from regions of algeria was explained by their high content in thymol (49.3 %) and carvacrol (57.7 %) (melliou et al., 2007). molluscicidal activity of t. capitatus essential oils rich with carvacrol (32.98 %) and thymol (32.82 %) on adult and eggs of biomphalaria alexandrina as well as on different stages of culex pipiens was evaluated for their effectiveness on vector control (salama et al., 2012). askarne et al. (2012) showed that aqueous extract of thymus leptobotrys completely inhibited mycelial growth of penicillium italicum, a phytopathogenic fungi of citrus. on the other hand, there is no report on the hydrosol composition from this species. also, the antifungal activity of this hydrosol was reported for the first time against the development of fungi of citrus sinensis fruit. therefore, the present study was made (i) to examine the in vitro antifungal activity of the essential oil and hydrosol extract obtained from t. capitatus against four phytopathogenic fungi (aspergillus niger, aspergillus oryza, penicillium italicum and fusarium solani) and (ii) to test in vivo the essential oil, hydrosol extract and hydrosol against penicillium italicum responsible of rotting the oranges. materials and methods plant material the aerial parts of t. capitatus were collected from beni snous forests near tlemcen, algeria in may 2011. voucher specimens were deposited in the herbarium of the tlemcen university botanical laboratory (voucher number: utl 05.11). 280 l. tabti, m. el amine dib, n. djabou, n. gaouar benyelles, j. paolini, j. costa, a. muselli essential oil, hydrosol and hydrosol extract essential oil of fresh aerial parts (600 g) was isolated by hydrodistillation (hd) in a clevenger type apparatus for 5 h, giving clear yellow oil. the yield of the oils was 0.52 %. the obtained essential oil was stored at +4 °c until further tests. the first liter of hydrodistillate is recovered in order to obtain t. capitatus hydrosol. hydrosol was submitted to liquid liquid extraction (lle). half a liter of hydrosol was extracted three times with 200 ml of diethyl ether at room temperature. the organic layer dried over na2so4 and evaporated, so giving an oil yellowish, called hydrosol extract, with yield of 0.0016 %. (w/w). the 500 ml of hydrosol remaining were used to study the in vivo activity. gas chromatography analyses were carried out using a perkin elmer clarus 600 gc apparatus equipped with a dual flame ionization detection system and 2 fused-silica capillary columns (60 m x 0.22 mm i.d., film thickness 0.25 μm), rtx-1 (polydimethylsiloxane) and rtx-wax (polyethylene glycol). the oven temperature was programmed from 60 °c to 230 °c at 2 °c/min and then held isothermally at 230 °c for 35 min. injector and detector temperatures were maintained at 280 °c. essential oil and hydrosol extract were injected in the split mode (1/50), using helium as the carrier gas (1 ml/min); the injection volume was 0.2 μl. retention indices (ri) of the compounds were determined from perkin-elmer software. gas chromatography-mass spectrometry essential oil and hydrosol extract were analyzed with a perkin– elmer turbomass quadrupole analyzer, coupled to a perkin-elmer autosystem xl, equipped with 2 fused-silica capillary columns and operated with the same gc conditions described above, except for a split of 1/80. electronic impact (ei) mass spectra were acquired under the following conditions: ion source temperature 150 °c, energy ionization 70 ev, mass range 35-350 da (scan time: 1 s). component identification and quantification identification of the components of the essential oil obtained by hydrodistillation (hd) and the hydrosol extract obtained by lle was based (i) on the comparison of their gc retention indices (ri) on non-polar and polar columns, determined relative to the retention time of a series of n-alkanes with linear interpolation, with those of authentic compounds or literature data (jennings and shibamoto, 1980; könig et al., 2001; national institute of standards and technology, 2008) and (ii) on computer matching with commercial mass spectral libraries (mc lafferty and stauffer, 1994; mc lafferty and stauffer, 1988; national institute of standards and technology, 1999) and comparison of spectra with those of inhouse laboratory library. the quantification of essential oil components was carried out using peak normalization including response factors (rfs) with internal standard. the normalized % abundances were calculated, using the methodology reported by (bicchi et al., 2008) in order to perform the statistical analysis of the oil. tridecane was introduced in all sample oils at same concentration (0.7 g/100 g) as internal standard. pathogenic fungi four fungal isolates causing citrus rot. aspergillus niger, aspergillus oryza, penicillium italicum and fusarium solani were isolated directly from rotten c. sinensis fruits harvested from orchards of the el-fhoul cooperative in tlemcen (algeria). all isolated fungal species were transferred to sterilized three replicates 9 cm petri dishes containing fresh potato dextrose agar medium (pda) in the presence of a quantity of lactic acid (20 %) to stop the growth of bacteria. the plates were incubated at 25 ± 2 °c for 8 days and darkness. the developing fungal colonies were purified and identified up to the species level by microscopic examination through the help of the following references (barnett and hunter, 2006). in vitro antifungal assay the antifungal activity of t. capitatus essential oil and hydrosol extract was tested using radial growth technique (bajpai et al., 2007). appropriate volumes of the stock solutions of the natural mixtures (essential oil and hydrosol extract) in dimethyl sulfoxide (dmso) to 10 %, were added to pda medium immediately before it was poured into the petri dishes (9.0 cm diameter) at 40-45 °c to obtain a series of concentrations (0.01 to 0.5 μg/ml). each concentration was tested in triplicate. parallel controls were maintained with dmso mixed with pda. the discs of mycelial felt (0.5 cm diameter) of the plant pathogenic fungi, taken from 7-day-old cultures on pda plates, were transferred aseptically to the centre of petri’s dishes were incubated. amphotericin b (5.0 to 126 μg/ml) was used as a positive control for antifungal activity. the treatments were incubated at 27 °c in the dark. colony growth diameter was measured after the fungal growth in the control treatments had completely covered the petri dishes. percentage of mycelial growth inhibition was calculated from the formula (pandey et al., 1982): (i%) = [(dc-dt)/dc] x 100 (pandey et al., 1982); where dc and dt are average diameters of fungal colony of control and treatment, respectively. the measurements were used to deter-the measurements were used to determine the minimum inhibitory concentration (mic) (the minimum concentration causing 100 % mycelial growth inhibition). the fungistatic-fungicidal nature of essential oil and hydrosol extract was tested by observing resumption of growth of the inhibited mycelial disc following its transfer to non-treated pda. in order to determine fungistatic or fungicidal activity of volatile vapours of essential oils on mycelial growth, plates were further incubated at 26 °c for 7 days. fungi resuming mycelial growth were considered to be fungistatic. in vivo antifungal assay for the in vivo antifungal assay, we used method described and developed previously by dikbas et al. (2008). the selected orange fruits for the experiments were washed in water, dipped in ethanol (70 %) for 2 min, rinsed twice with double distilled sterile water (10 min each) and air-dried. surface-sterilized oranges were wounded with a flamesterilized nail to a uniform depth of 3 mm. the fungal inoculums containing 106 spores/ml was prepared by scraping spore material from the surfaces of the colonies with a wet cotton swab and resuspending the material in distilled water containing 0.5 % tween 80. for testing antifungal in vivo activity against p. italicum, the essential oil and hydrosol extract, were separately mixed vigorously with distilled water to obtain two concentrations 0.1 and 0.2 μg/ ml, respectively. however, the hydrosol was applied directly with the concentration of 20 μg/ml. the essential oil, hydrosol extract and hydrosol of t. capitatus and fungal inoculum were sprayed separately on wounded orange fruits. the antifungal activity was tested on healthy fresh oranges. these experiments were arranged as three different applications. fruits inoculated with only pathogen were used as positive control for each experiment. non-inoculated fruits with pathogen were used as negative control. the fruits were sealed in polyethylene-lined plastic boxes to retain 70 % humidity and incubated at 25 °c storage condition. the diameters of decay on fruits were measured at 3, 6, 9, 12 and 15th days after inoculation. antifungal activity of hydrosol against pathogens of citrus 281 tab. 1: chemical compositions of t. capitatus essential oil (eo) and hydrosol extract (hy). no.a components lria b ria c ripd eo hy identification e 1 α-thujene 932 924 1028 0.2 ri, ms 2 α-pinene 936 931 1028 0.9 ri, ms 3 camphene 950 945 1071 0.2 ri, ms 4 oct-1-en-3-ol 962 962 1441 0.5 0.2 ri, ms 5 β-pinene 978 972 1113 0.1 ri, ms 6 myrcene 987 982 1160 2.1 ri, ms 7 3-octanol 981 982 1366 tr 0.1 ri, ms 8 α-phellandrene 1002 999 1161 0.2 ri, ms 9 3-carene 1005 1006 1149 0.1 ri, ms 10 α-terpinene 1008 1011 1270 1.7 ri, ms 11 p-cymene 1015 1015 1270 12.4 ri, ms 12 (z)-β-ocimene 1029 1022 1234 0.6 ri, ms 13 γ-terpinene 1051 1050 1245 4.3 ri, ms 14 (e)-sabinene hydrate 1051 1054 1445 0.1 0.6 ri, ms 15 terpinolene 1082 1079 1281 0.2 ri, ms 16 linalool 1083 1085 1538 1.7 0.5 ri, ms 17 phenylacetaldehyde 1112 1108 1591 0.1 ri, ms 18 camphor 1123 1124 1506 0.1 ri, ms 19 isoborneol 1143 1144 1670 0.5 ri, ms 20 borneol 1148 1150 1688 0.3 ri, ms 21 terpinen-4-ol 1164 1162 1591 1.1 ri, ms 22 α-terpineol 1176 1176 1690 0.1 0.2 ri, ms 23 trans-dihydro carvone 1180 1182 1607 tr ri, ms 24 trans-myrtanol 1241 1242 1859 tr ri, ms 25 thymol 1266 1263 2181 0,6 0.1 ri, ms 26 carvacrol 1278 1286 2193 69.6 95.1 ri, ms 27 eugenol 1330 1329 2164 0.1 0.2 ri, ms 28 cis-carvyl acetate 1343 1345 1858 0.1 0.2 ri, ms 29 (e)-β-caryophyllene 1421 1416 1591 1.6 ri, ms 30 (e)-α-bergamotene 1434 1435 1573 tr ri, ms 31 α-humulene 1455 1448 1668 0.1 ri, ms 32 γ-humulene 1483 1480 1702 tr ri, ms 33 β-bisabolene 1503 1499 1721 0.1 ri, ms 34 δ-cadinene 1520 1511 1760 tr ri, ms 35 (e)-α-bisabolene 1530 1531 1755 0.1 ri, ms 36 spathulenol 1572 1560 2120 0,1 ri, ms 37 caryophyllene oxide 1578 1567 1969 0.1 1.1 ri, ms 38 humulene epoxide ii 1602 1599 2044 0.1 ri, ms total identification % 99.2 99.1 % hydrocarbon compounds 12.6 % monoterpene hydrocarbons 10.7 % sesquiterpene hydrocarbons 1.9 % oxygenated compounds 86.7 99.1 % oxygenated monoterpenes 3.5 2.2 % oxygenated sesquiterpenes 0.1 1.3 % non terpenic oxygenated compounds 0.5 0.4 % aromatic terpenes 82.6 95.2 a order of elution is given on apolar column (rtx-1), b retention indices on the apolar rtx-1 column (ria), c retention indices on the polar rtx-wax column (rip), d retention indices on the polar rtx-wax column (rip), e ri: retention indices; normalized % abundances; ms: mass spectrometry in electronic impact (ei) mode; eo: essential oil from aerial parts obtained by hd; hy: extract of hydrosol from aerial parts obtained by lle. 282 l. tabti, m. el amine dib, n. djabou, n. gaouar benyelles, j. paolini, j. costa, a. muselli tab. 2: antifungal activity of essential oil (eo) and hydrosol extract (hy) of t. capitatus against a. niger, a. oryzae, p. italicum and f. solani. extracts a. niger a. oryzae f. solani p. italicum mic a ec50 b mic a ec50 b mic a ec50 b mic a ec50 b eo 0.5 ± 0.06 d 0.121 ± 0.80 0.2 ± 0.01 d 0.143 ± 0.80 0.2 ± 0.01 d 0.132 ± 0.22 0.1 ± 0.01 e 0.022 ± 0.06 hy 0.5 ± 0.01 d 0.121 ± 0.81 0.2 ± 0.01 d 0.143 ± 0.56 0.2 ± 0.01 d 0.132 ± 0.32 0.1 ± 0.01 e 0.022 ± 0.06 am b c 46.2 ± 1.81 15.62 ± 1.06 46.2 ± 1.39 15.62 ± 1.50 126 ± 3.56 62.50 ± 1.79 61.2 ± 2.01 31.25 ± 1.11 a minimum concentration causing 100 % mycelial growth inhibition; b minimum concentration causing 50 % mycelial growth inhibition; c am b : amphotericin b (μg/ml) was used as reference antibiotic; d fungicidal effect; e fungicistatic effect; the results are expressed as mean ± standard deviation. (μg/ml) all treatments consisted of three replicates, and experiments were repeated three times and determined the averages of the repeated experimental results. taste panel sensory evaluation of oranges treated with hydrosol was assessed by a group of 20 untrained panellists. panellists were selected among students and staff of the laboratory of chemistry (lasnabio). orange samples were soaked into the hydrosol during 24 h, before the oranges were given to panellists to eat. the panellists were asked to evaluate flavor and odor of the orange samples on a scale from 5 to 1, where 1 = extremely dislike, 2 = dislike, 3 = neither like nor dislike, 4 = like; 5 = extremely like, according to a previous reports (stojković et al., 2011). a general taste score was calculated as the average of all grades. sensory evaluation was accomplished at 1st, 2nd and 3rd day. results were expressed as average grades given by 20 panellists. statistical analysis statistical analysis of variance (anova) was performed using the sas software and means were separated using the least significant difference (lsd) test at p ≤ 0.05. analysis of each test was performed in triplicate. results chemical analyses a total of 32 components accounting to 99.2 % of the essential oil (eo) composition of t. capitatus were identified by comparison of their ei-mass spectra and their retention indices (ri) with those of our own authentic compound library (tab. 1). the essential oil was highly dominated by oxygenated compounds (20 components: 74.4 %) with high amount of aromatic terpenic components (82.6 %). however, hydrocarbons appeared also in appreciable proportion (19 components: 25.0 %) with monoterpene hydrocarbons are well represented (23.1 %). indeed, the main constituents of essential oil were carvacrol (69.6 %), p-cymene (12.4 %) followed by γ-terpinene (4.3 %), myrcene (2.1 %), α-terpinene (1.7 %), linalool (1.7 %) and terpinen-4-ol (1.1 %). conversely, the analysis of hydrosol extract (hy) obtained by lle showed only 14 oxygenated compounds (7 monoterpenes, 3 sesquiterpenes, 3 non-terpenic components and 1 phenylpropanoid), no hydrocarbons were reported. in vitro antifungal activity of t. capitatus extracts against the development of fungi of c. sinensis the results obtained in assays of antifungal activity of t. capitatus essential oil and hydrosol extract by radial growth technique are reported in tab. 2. however, data analysis showed that the antifungal activity of the essential oil and hydrosol extract concentration against the four fungi tested exhibited a significant difference (p < 0.05). the results indicate that the inhibition of the mycelial growth of each strain was significantly influenced by the extract concentrations. essential oil and hydrosol extract inhibited completely all strains. the potent activity was observed against p. italicum with the ec50 of 0.022 μg/ ml followed by a. niger, f. solani and a. oryzae with the ec50 of 0.121, 0132 and 0.143 μg/ml, respectively. the minimum concentration causing 100 % mycelial growth inhibition against p. italicum, a. oryzae and f. solani phytopathogens were very effective at 0.1, 0.2 and 0.2 μg/ml, respectively. however, the minimum concentration causing 100% mycelial growth inhibition for a. niger strain was 0.5 μg/ml. the essential oil and hydrosol extract tested showed strong activity in comparison to the commercial drug amphotericin b. the most sensitive species were p. italicum, f. solani and a. oryzae with mic of 0.1, 0.2 and 0.2 μg/ml, respectively. a. niger (mic of 0.5 μg/ ml) was the most resistant species to the essential oil and hydrosol extract tested. while, mic of amphotericin b ranged to 46.2 to 126 μg/ ml for fungal species isolated. moreover, it is important to know the fungitoxic nature of the essential oils and hydrosol extract. indeed, the transfer of a mycelial disk of the plate containing a pda medium and samples on fresh pda (without oil and hydrosol) showed that no growth had developed after an incubation period of 7 days, suggesting a fungicidal effect of essential oils and hydrosol extract on f. solani, a. oryza, a. niger at 0.2, 0.2 and 0.5 μg/ml, respectively. on the other hand, essential oils and hydrosol extract was fungistatic on p. italicum (tab. 2). in vivo orange assay the results of in vivo p. italicum citrus rot treatment with essential oil, hydrosol extract and hydrosol are presented in tab. 3. according to the increase of concentration, a decrease of disease incidence was recorded. according to the results of lesion diameters on the fruits, both concentrations (0.1 and 0.2 μg/ml) of the essential oil and hydrosol extract showed strong antifungal activity even at the end of 15th day, there was no significant difference in lesion diameters among those treatments in comparison to the negative control. the concentration of 0.2 μg/ml of essential oil and hydrosol extract were needed for the absence of orange infection and low disease incidence. more, the hydrosol showed a complete absence of orange infection and no disease incidence with a concentration of 0.2 μg/ml. the result obtained from the hydrosol was showed on fig. 1. taste panel tab. 4 shows the acceptability scores of orange samples. results showed that there were no significant differences in the sensory properties between samples treated with hydrosol, essential oil and control (without hydrosol), since the sensory properties of oranges treated with hydrosol (0.2 μg/ml) and essential oil (0.2 μg/ml) antifungal activity of hydrosol against pathogens of citrus 283 tab. 3: means of decay diameters (mm) measured after 3, 6, 9, 12 and 15 days on orange fruits treated with 0.1 or 0.2 μg/ml of essential oil, hydrosol extract and hydrosol from t. capitatus. treatments means ± sd of decay diameters (mm) 3 days 6 days 9 days 12 days 15 days controls negative 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 controls positive 1.1 ± 0.2 2.8 ± 0.3 5.7 ± 0.5 8.1 ± 0.8 13.2 ± 0.2 essential oil 0.1 μg/ml 0.0 ± 0.0 0.0 ± 0.0 0.5 ± 0.01 1.5 ± 0.2 2.5 ± 0.5 0.2 μg/ml 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.5 ± 0.01 1.0 ± 0.2 hydrosol extract 0.1 μg/ml 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.5 ± 0.06 1.5 ± 0.2 0.2 μg/ml 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.5 ± 0.02 hydrosol 0.2 μg/ml 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 fig 1: decayed orange (left), inoculated with only pathogen p. italicum (positive control), and not decayed orange (right) onto which hydrosol (0.2 μg/ml) was applied 30 min before pathogen inoculation, kept at 25 °c for the period stated below the respective picture. 284 l. tabti, m. el amine dib, n. djabou, n. gaouar benyelles, j. paolini, j. costa, a. muselli were deemed acceptable by the panelists at the supplementation levels. more work on the acceptability of both extracts as oranges preservative will be necessary. discussion in the present investigation, a total of 14 and 38 compounds comprising 99.1 and 99.2 % of the hydrosol extract and essential oil were identified from t. capitatus respectively, carvacrol being the major component, comprising 95.1 and 69.6 % of the hydrosol and essential oil, respectively. this study agrees with the findings of ruberto et al. (1992), amarti et al. (2008) and tawaha et al. (2012), who reported carvacrol to be the major component of t. capitatus essential oil. in the present investigation, the reduction of the mycelial growth of colonies in presence of essential oil and hydrosol extract of t. capitatus showed that it effectively controlled all strains. this efficiency can be explained by the presence of active molecules that inhibited the growth of the phytopathogenic fungi. the antifungal properties of t. capitatus essential oil and hydrosol extract are probably associated with the high amount of phenolic terpenes, especially the main component carvacrol. indeed, carvacrol is used as a disinfectant, fungicide, and fragrance ingredient in cosmetic formulations. in addition, daferera et al. (2000) reported that the fungitoxic activity of essential oils may have been due to formation of hydrogen bonds between the hydroxyl group of oil phenolics and active sites of target enzymes. although the extracts or essential oils of t. capitatus have been screened for their antifungal activity under in vitro conditions (melliou et al., 2007; dikbas et al., 2008; nychas, 1996), there are no reports on the control of p. italicum under in vivo conditions by using the essential oil and hydrosol of t. capitatus. the essential oil and hydrosol of t. capitatus from algeria was characterized by high content of carvacrol (69.6 % and 95.1 %, respectively). little literature exists on the effect of carvacrol on these food pathogenic fungi (morcia et al., 2012), although carvacrol was effective on inhibiting spore germination of botrytis cinerea when applied to the potato dextrose agar (martinez-romero et al., 2007). in addition, markovic et al. (2011) demonstrated that carvacrol has a remarkably antifungal potential against aspergillus spp. and penicillium spp. in a study conducted by muller-riebau et al. (1995), carvacrol showed a remarkable antifungal activity by inhibition of the mycelial growth of fusarium spp. bouddine et al. (2012) revealed that aspergillus niger growth was completely inhibited by carvacrol at concentrations of 0.025 %. furthermore, this study confirmed the antifungal activity of this component against mycelial growth of p. italicum in vitro and in vivo assays. the world health organization (who) has stated that carvacrol residues in food are without danger to the consumer as long as they do not exceed 50 mg kg-1 (who, 2012). it is thus clear that treatment by hydrosol is an easy to prepare and the cost price of extraction is not expensive. however, the benefits of hydrosol (nontoxic at low doses, biodegradable, and no risk for resistance development) and disadvantages of chemical fungicides on health and on the environment make hydrolate of t. capitatus more interesting for citrus postharvest treatment. conclusion in conclusion, 38 compounds of t. capitatus essential oil and hydrosol extract were identified. this oil was characterized by high content of carvacrol. furthermore, this study confirmed the antifungal activity of the essential oil and hydrolate against mycelial growth and spore production of a. niger, a. oryzae, f. solani and p. italicum from in vitro assay. in vivo inhibitory properties of hydrosol on disease incidence of p. italicum-causal agent of penicillium rot on oranges were recorded. therefore, the preventive and curative effects of t. capitatus hydrolate could be exploited as an ideal alternative to synthetic fungicides for using in the treatment of many fungal phytopathogens causing severe destruction to oranges. acknowledgements the authors are indebted to the ministère des affaires etrangères et européennes through the research program france-algérie “partenariat hubert curien tassili”. references amarti, f., satrani, b., aafi, a., ghanmi, m., farah, a., aberchane, m., el ajjouri, m., el antry, s., chaouch, a., 2008: composition chimique et activité antimicrobienne des huiles essentielles de thymus capitatus et de thymus bleicherianus du maroc. phytotherapie 6, 342347. askarne, l., talibi, i., boubaker, h., boudyach, e.h., msanda, f., saadi, b., serghini, m.a., ait ben aoumar, a., 2012: in vitro and in vivo antifungal activity of several moroccan plants against penicillium italicum, the causal agent of citrus blue mold. crop prot. 40, 53-58. bajpai, v.k., rahman, a., kang, s.c., 2007: chemical composition and antifungal properties of the essential oil and crude extracts of metasequoia glyptostroboides miki ex hu, ind. crop prod. 26, 28-35. barnett, h.l., hunter, b.b., 2006: illustrated genera of imperfect fungi. 4th ed., the american phytopatological society, st. paul minnesota. bicchi, c., liberto, e., matteodo, m., sgorbini, b., mondello, l., zellner, b.a., costa, r., rubiolo, p., 2008: quantitative analysis of essential oils: a complex task. flav. fragr. j. 23, 382-391. bouddine, l., louaste, b., achahbar, s., chai, n., chamri, f., remmal, a., 2012: comparative study of the antifungal activity of some essential oils and their major phenolic components against aspergillus niger using three different methods. afr. j. biotechnol. 11, 14083-14087. clemente, s., mareggiani, g., broussalis, a., martino, v., ferraro, g., 2003: insecticidal effects of lamiaceae species against stored products insects. bol. san. veg. plagas. 29, 1-8. daferera, d.j., ziogas, b.n., polissiou, m.g., 2000: gc-ms analysis of essential oils from some greek aromatic plants and their fungitoxicity on penicillium digitatum. j. agric. food chem. 48, 2576-2581. dikbas, n., recep kotan, f., dadasoglu, f.s., 2008: control of aspergillus flavus with essential oil and methanol extract of satureja hortensis. int. j. food microbiol. 124, 179-182. farag, r.s., daw, z.y., hewedi, f.m., el-baroty, g.s.a., 1989: antimicrobial activity of some egyptian spice essential oils. j. food. protect. 52, 665-667. feng, w., zheng, x., 2007: essential oils to control alternaria alternata in vitro and in vivo. food control 18, 1126-1130. gikh, m., abdel rassoul, m.a., abdelgaleil, s.a.m., 2012: comparative antifungal activities and biochemical effects of monoterpenes on plant pathogenic fungi. pestic. biochem. phys. 103, 56-61. griffin, s.g., wyllie, s.g., markham, j.l., leach, d.n., 1999: the role of structure and molecular properties of terpenoids in determining their tab. 4: effect of essential oil (eo) and hydrosol (hy) on acceptability sensory scores of orange stored at 25 °c. treatment days of storage hy 1 2 3 acceptabilitya 4.5 4.6 4.2 eo 3.9 4.0 3.8 the results are expressed as the average of all grades. a 1 = extremely dislike, 2 = dislike, 3 = neither like nor dislike, 4 = like; 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204-211. stojković, d., soković, m., glamočlija, j., džamić, a., ćirić, a., ristić, m., grubišić, d., 2011: chemical composition and antimicrobial activity of vitex agnus-castus l. fruits and leaves essential oils. food chem. 128, 1017-1022. tawaha, k.a., hudaib, m.m., 2012: chemical composition of the essential oil from flowers, flower buds and leaves of thymus capitatus hoffmanns. & link from jordan. j. essent. oil bear pl. 15(6), 988-996. tu, q., chena, j., guoc, j., 2013: screening and identification of antagonistic bacteria with potential for biological control of penicillium italicum of citrus fruits. sci. hortic. 150, 125-129. who, 2012: http://apps.who.int/medicinedocs/en/d/js2200e/28. html2012 (accessed april). address of the corresponding author: e-mail: a_dibdz@yahoo.fr journal of applied botany and food quality 88, 145 151 (2015), doi:10.5073/jabfq.2015.088.021 1biotechnology division, taiwan agricultural research institute, taichung city, taiwan, roc 2hualien district agricultural improvement station, hualien county, taiwan, roc 3department of food science and technology, hungkuang university, taichung city, taiwan, roc comparative analysis of eating quality and yield of selected non-waxy red-pericarp aromatic rice mutants t.l. jeng1, c.s. chan2, s.y. lin1, d.p. shung2, c.z. pan2, y.j. shih1, j.m. sung3* (received july 31, 2014) * corresponding author summary red-pericarp rice variety kuanfu waxy aroma is highly valued for its grain quality in taiwan, but it has undesirable traits of awned rough grain and taller plant height. the present study compared the palatability of cooked rice grains and yields of kuanfu waxy aroma and its ten nan3-induced awnless non-waxy aromatic m6-generation mutants developed through single-seed-descent selection plus a nonwaxy aromatic rice variety tng71 (reference variety with good eating quality). the palatability of cooked rice grains was assessed by using a rice taste meter. results indicated that all the mutants exhibited awnless grain traits and reduced plant height. significant differences in the palatability of cooked rice were also observed among the mutants with am-425 (70.45) and am-430 (73.75) having higher palatability scores than tng71 (69.32). mutant am-425 also had higher aroma sensory score (1.33) than tng71 (1.17). two years yield trials indicated that am-425 and am-430 significantly outyielded kuanfu waxy aroma and can be recommended to rice growers. introduction rice (oryza sativa l.) is a tremendously variable species and has worldwide distribution. it is estimated that about 120,000 distinct rice varieties exist in the world (khush, 1997). among these, aromatic rice varieties that release a unique and pleasant aroma detectable through sensory test, chemical assessment or molecular marker identification (bradbury et al., 2005) constitute a small but important subgroup of rice varieties. aromatic rice varieties are popular throughout asia, and have also gained wider acceptance in europe, middle east, australia and the united states of america (sakthivel et al., 2009). however, most of the aromatic rice varieties have low yield compared to non-aromatic rice varieties, because the genes responsible for aroma also lead to a decreased ability to produce matured rice grains when under environmental stresses (fitzgerald et al., 2010). lack of high-yielding varieties is also associated with the low yield of aromatic rice that is mainly traditional and local landraces (abdul baset mia et al., 2012). therefore, various breeding efforts for increasing the yield of this type of rice varieties have been conducted in many countries. but in most cases, the outcomes are limited because the improvement of aromatic rice varieties is often challenged by the environment and genotype interactions for aromatic quality (gay et al., 2010). the rice variety kuanfu waxy aroma, which releases a pleasant flavor during cooking, is a red-pericarp waxy landrace indigenous in taiwan. it is primarily grown by the aboriginal peoples at kuanfu township of hualien county in the eastern part of taiwan. the produced kuanfu waxy aroma grains are sold at a premium price in local market because of their aroma and red-pericarp characteristics. moreover, the bran fraction of red-pericarp rice grains is endowed with many phytochemicals that are known to decrease oxidative stress in vivo and thus exert beneficial effects on human health (jeng et al., 2012). therefore, the produced kuanfu waxy aroma grains not only can be directly used for human consumption, but also can be used to produce high-value nutraceuticals for food and cosmetic uses (parrado et al., 2006, revilla et al., 2009; ryan, 2011). however, further expansion of kuanfu waxy aroma is limited by three drawbacks. first, the harvested kuanfu waxy aroma grains are mainly prepared and consumed in whole grain, as rice desserts or folk medicines, therefore, no rice brans milled off from kuanfu waxy aroma are currently available for producing high value by-products. secondly, the relative taller plant height (1.3-1.4 m) of kuanfu waxy aroma makes it susceptible to lodging. thirdly, the awned rough grains of kuanfu waxy aroma (fig. 1a) also cause problems during harvest and husking, even though this grain trait protects the developing rice grains from animal attack (minaei et al., 2007; hu et al., 2011). thus, if an awnless non-waxy aromatic rice variety with red-pericarp is readily available for farming, not only the polished rice grains can be consumed as a staple food, the resulted bran parts enriched with phytochemicals may also be collected and used to produce high-value functional substances. mutation has been used with distinct effects to bring about desirable changes in traits usually controlled by single gene or polygenes. previously, agriculture research institute in taiwan had used sodium azide to induce mutational changes in kuanfu waxy aroma, and several awnless red-pericarp mutants with non-waxy endosperm and reduced plant height were identified. this study examined the grain eating qualities of these awnless non-waxy mutants derived from kuanfu waxy aroma. the selected mutants were also grown in both spring and autumn crop seasons for grain yield comparisons. materials and methods plant materials and field planting ten m6 generation mutants namely am-419, am-420, am-422, am-423, am-424, am-425, am-426, am-427, am-430, am-433, derived from wild type variety kuanfu waxy aroma through nan3induced mutation, with reduced plant height, awnless rough grain and non-waxy endosperm were selected in the autumn of 2010. these m6 mutants with stabilized morphological traits were planted from spring 2011 to autumn 2012 for grain yield comparisons. all the selected mutants and wild type kuanfu waxy aroma were grown on the experimental farm of the agricultural research institute in the spring and autumn of 2011 and 2012. a non-waxy aromatic rice variety tng71 (a high eating quality aromatic variety with white-pericarp) was included to serve as a comparison. all the tested accessions were grown on the experimental farm of the agricultural research institute in the spring and autumn of 2011 and 2012. rice grains were sown in the nursery plots, where the seedlings grew up to the three-leaf stage before transplanting. the seedlings from the two spring crops were transplanted to the experimental plots on 5 february 2011 and 16 february 2012, respectively. the seedlings from the two autumn crops were transplanted to the experimental plots on 146 t.l. jeng, c.s. chan, s.y. lin, d.p. shung, c.z. pan, y.j. shih, j.m. sung 5 august 2011 and 30 july 2012, respectively. the experiment was laid out in a completely randomized block design with three replicates. one seedling per hill was planted with a spacing of 30 cm × 15 cm in each experimental plot of 3 m x 6 m. each plot received a basal application of fertilizer before transplanting (24 kg n ha-1, 36 kg p2o5 ha-1 and 24 kg k2o ha-1) and three top-dressings of fertilizer on the 20th day (6 kg n ha-1, 9 kg p2o5 ha-1 and 6 kg k2o ha-1), the 40th day (9 kg n ha-1, 13.5 kg p2o5 ha-1 and 9 kg k2o ha-1) and the 60th day (9 kg n ha-1, 13.5 kg p2o5 ha-1 and 9 kg k2o ha-1) after transplanting. diseases and pests were controlled using the standard practices in the study areas. protein and amylose contents determinations for grain protein and amylose contents analyses, the rice grains sampled from the spring crops of 2011 were used. sampled rough rice grains (50 g) were polished followed in an abrasive polisher (model satake pearler-tm05, satake corp., horoshima-ken, japan) with the degree of milling around 10 %. broken kernels were removed, and the polished rice samples were ground into fine powders in an rt08 mill (rong tsong, taichung, taiwan) and stored at -20 ℃ until analysis. the total nitrogen content (with three replicates) was de termined using the micro-kjeldahl procedure (zhang et al., 2005). the grain protein content was calculated as the nitrogen content multiplied by the factor of 5.95. for amylose content (with three re plicates) determination, milled rice grains were ground to pass through a 1.0 mm mesh screen by a udy cyclone mill (udy corp. boulder, co) to obtain rice flour. fifty mg of rice flour was used to determine the amylose content following the iodine colorimetry at 608 nm detailed by juliano (1971). aroma sensory and palatability tests for aroma sensory tests, rice grains sampled from the spring crops of 2011 were used by using the method of lestari et al. (2011) with some modifications. briefly, two g of polished rice grains were placed in a test tube of 15 mm × 150 mm previously filled with 10 ml of 1.7 % koh solution. the test tube was then covered with aluminum foil and heated in a 50 ℃ water bath for 10 minutes. after the sam-water bath for 10 minutes. after the samples were cool, the aluminum foil was removed, and samples were smelled and scored for aroma by six panelists. the scores were averaged and recorded with categories of non-aromatic (score 0), slightly aromatic (score < 1), moderately aromatic (score 1 ~ 2) and strongly aromatic (score 2 ~ 3). polished rice sample (30 g) were soaked in 40 ml of water for 30 min. the soaked rice samples were then cooked in an automatic electric cooker (sr-w180, panasonic, japan) for 25 min and held in the cooker covered for 10 min. afterwards, the cooked rice was cooled down at room temperature for 90 min. the palatability scores (with three replicates), based on the appearance, hardiness and stickiness of cooked rice grains, were determined by using a satake rice taste meter (sta1a, satake corp., hiroshima-ken, japan) according to the manufacturer’s instructions. grain yield and yield components determinations once the field-grown plants reached physiological maturity, 10 hills were marked in each experimental plot for the plant heights (from the plant base to the tip of the highest panicle) to be measured. since there was only one plant per hill, the number of panicles per hill was counted to determine the number of panicles per plant. for grain yield determinations, the plants in the middle four rows of 2 m length in each plot were hand-harvested. the harvested panicles were ovendried at 80 ℃ for 72 h to determine grain yields. a sub-sample of 20 panicles was taken at random and threshed to determine the number of spikelets per panicle, the percentages of fertility per panicle and the 1000-grain weights. the amount of developed and filled grains per panicle was recorded as the number of fully matured grains per panicle, while the sterile spikelets and grains with weight less than 10 mg were excluded from the calculation of fully mature grains. the percentage of fertility per panicle equaled the number of fully matured grains per panicle divided by the number of spikelets per panicle. the grain mass was weighed and the moisture content was measured. the grain yields were expressed at the 140 g kg-1 grain moisture content. statistical analysis variance analyses of the grain quality and agronomic traits data were performed with a statistical package for the social sciences (spss 10.0 for windows: spss inc., chicago, il, usa). differences among means were evaluated using the duncan’s multiple range test. the correlation coefficients between palatability scores of tested grains vs. grain quality traits and between yield components vs. grain yield were also calculated using the same spss statistical package. results agronomic traits the rough rice grains of kuanfu waxy aroma had long awn (fig. 1a), but some of nan3-induced mutants also produced awned grains (fig. 1b). nevertheless, all the selected mutants exhibited awnless grain trait (tab. 1). differentiation in other grain traits such as pericarp color (white-pericarp, red-pericarp and purple-pericarp grains) and grain shape (medium and short grains) also occurred among kuanfu waxy aroma and selected mutants (fig. 1b). based on our breeding goals, only 10 non-waxy mutants that exhibited awnless (tab. 1) and red-pericarp grain characteristics and wild type variety kuanfu waxy aroma plus a non-waxy white-pericarp high-yielding aromatic variety tng71 were selected and grown for agronomic traits comparisons. as shown in tab. 2 and 3, significant differences in plant height (p < 0.05) were observed among the tested mutants in both spring and autumn crop seasons. the plant heights of the selected mutants (ranged from 93 to 123 cm and from 83 to 112 cm for spring crop and autumn crop, respectively) were decreased considerably compared to kuanfu waxy aroma in both crop seasons (129 and 141 cm for the spring and autumn crops, respectively). significant differences in grain amylose and protein contents (p < 0.05) also existed among the selected non-waxy mutant (tab. 1). the contents of grain amylose ranged from 11.69 (mutant am-419) to 21.02 % (mutant am422). mutants am-422, am-424, am-427 and am-4337 showed higher grain amylose content than non-waxy aromatic variety tng71 (18.70 %). the protein contents of tested mutants ranged between 5.5 and 7.1 % (tab. 1). mutant am-430 had the lowest protein content of 5.5 %, which was slightly lower than the protein content of tng71 (5.8 %). grain eating qualities the aroma sensory test indicated that the milled grains of kuanfu waxy aroma showed a moderate level of aromatic score (1.83) (tab. 1). the aroma sensory scores of milled grains among the tested mutants ranged from 0.50 (am-433) to 1.50 (am-422) (tab. 1). mutants am-425 and am-422 had aroma sensory scores of 1.33 and 1.50, respectively, which were higher than the aroma sensory score of 1.17 recorded for non-waxy aromatic variety tng71 (tab. 1). the palatability scores of tested mutants ranged from 63.70 to 73.74 (tab. 1). two mutants am-425 and am-430 had the palatability scores of 70.45 and 73.75, respectively, which were higher than the eating quality of non-waxy aromatic mutants 147 palatability score of 69.32 recorded for reference variety tng71 (tab. 1). the correlation analyses (data not shown) between the palatability of cooked rice grains and related grain quality traits across all the mutants were conducted, but the results indicated only the grain protein content and palatability was negatively correlated (r = -0.9289, p < 0.01). grain yield the grain yields of selected aromatic mutants ranged from 6.26 to 8.09 ton ha-1 and from 4.72 to 6.63 ton ha-1 for spring and autumn crops, respectively (tab. 3 and 4). most of the mutants (except autumn-grown am-420 and am-426) produced higher grain yield than wild type kuanfu waxy aroma. the variance analyses demonstrated that there were significant differences in yield components of panicle number per plant, grain number per panicle, and 1,000-grain weight (p < 0.05) among the mutants (tab. 2 and 3). in both spring and autumn crop seasons, most of the mutants (except autumn-grown am-423) produced more panicles per plant than kuanfu waxy aroma (tab. 2 and 3). significant variations in the number of spikelets per panicle and the percentage of fertility per panicle (p < 0.05) were also obtained from the tested mutants (tab. 2 and 3). in the spring crop season, mutants am-423, am-424, am-425, am-430 and am433 produced more spikelets per panicle than kuanfu waxy aroma (tab. 2). in the autumn crop season, mutants am-423, am-424, am425, am-430 and am-433 produced more spikelets per panicle than kuanfu waxy aroma (tab. 3). some of mutants also exhibited higher percentages of fertility per panicle than kuanfu waxy aroma in both seasons (tab. 2 and 3). as for the 1000-grains weight, only mutant am-422 showed heavier grains weight than kuanfu waxy aroma in both crop seasons (tab. 2 and 3). as for the crop season effect on grain yield, all the mutants grown in the spring crop season delivered higher grain yields (p < 0.05) than the same mutants grown in the autumn crop season (tab. 2 and 3). to quantify the relationships among the examined yield components and grain yield, the correlation analysis was applied to the obtained data (tab. 4). correlation coefficients between the examined yields and yield ranged from 0.003 to 0.620. overall, significant correlation coefficients were found for % fertility per panicle vs. grain yield (r = 0.620, p < 0.01) and panicles per plant vs. grain yield (r = 0.340, p < 0.05). discussion the awn in cultivated rice is an undesirable trait that causing problems during harvesting and husking (minaei et al., 2007; hu et al., 2011). therefore, de-awning is the first objective in our kuanfu waxy aroma improvement program, and the results indicate that the nan3induced mutation is an effective approach to remove undesirable awn characteristic from kuanfu waxy aroma grain (tab. 1). another drawback of kuanfu waxy aroma is its relative taller plant height (about 1.3 m). nevertheless, all the selected mutants showed reduced plant height compared to kuanfu waxy aroma in both crop seasons (tab. 2 and 3). changing from waxy rice to non-waxy rice is a crucial criterion in determining whether the derived mutants are accepted and consumed as a daily staple by the general public in taiwan. therefore, selection of mutants with non-waxy endosperm is also one of our breeding objectives. as shown in tab. 1, all the selected mutants derived from kuanfu waxy aroma exhibited non-waxy type endosperm, each with different levels of grain amylose contents. however, breeding a rice variety with non-waxy endosperm does not guarantee that it will be accepted by the rice consumer. the consumers’ choice of a given rice variety is largely based on its eating quality, which covers various grain characteristics such as grain protein and amylose contents and sensory properties (bhonsle and sellappan, 2010; zhu et al., 2010). the milled rice with amylose content below 20 % is gene rally moist, soft and sticky once cooked, while the high amylose rice (25-30 %) tends to absorb more water and have a fluffy texture after cooking. on the other hand, the milled rice with high protein content tends to reduce water absorption and starch swelling, and therefore is less sticky when cooked. as shown in tab. 1, the high eating quality reference variety tng71 had the grain amylose and protein contents of 18.7 % and 5.83 %, respectively. several derived mutants including am-426, am-430 and am-433 had the grain amylose and protein contents comparable to that of reference variety tng71 (18.7 %) (tab. 1). aroma is a much valued quality factor for rice. various volatile aroma compounds, with 2-acetyl 1-pyrroline being the principal aromatic compound (sakthivel et al., 2009), have been identified in cooked rice grains of varieties originated from diverse regions (jezussek et al. 2002). a 2ap quantification using gas chromatography-mass spectrometer is available but the assay requires large tissue samples, and is expensive and time consuming (hien et al., 2006). thus, in this study, a simple and fast koh sensory test (sarhadi et al., 2008; lestari et al., 2011) was used to evaluate the aroma sensory scores fig. 1: the rough rice and brown rice grains of (a) wild type variety kuanfu waxy aroma and (b) some of its nan3-induced mutants. 148 t.l. jeng, c.s. chan, s.y. lin, d.p. shung, c.z. pan, y.j. shih, j.m. sung tab. 2: various agronomic characteristics and their respective results from variance analyses of results of the tested mutants, wild type variety kuanfu waxy aroma and non-waxy white-pericarp aromatic variety tng71 grown in spring crop season. mutant plant height panicles plant-1 % fertility spikelets 1000-grain weight yield (cm) panicle-1 panicle-1 (g) (ton ha-1) am-419 93.01g 14.63bc 88.91abc 87.06ef 27.86cde 6.95bcd am-420 93.17g 15.90ab 89.26abc 91.93de 28.34c 8.09ab am-422 97.17f 14.42bc 88.09abcd 78.16fg 30.21b 6.57cd am-423 102.17d 10.53def 85.37bcd 134.25ab 25.05f 6.92cd am-424 109.67c 10.07ef 89.79abc 128.44bc 27.04de 7.59bc am-425 120.11b 11.41de 92.32a 122.91c 28.16cd 7.99ab am-426 97.52ef 17.55a 90.04ab 69.04g 26.82e 6.36d am-427 94.17fg 16.53ab 87.72abcd 82.65ef 24.02gf 6.26d am-430 122.50b 11.26de 90.82ab 140.07ab 22.97±gh 7.31±bcd am-433 121.33b 12.65cd 87.35abcd 129.91bc 22.69h 7.15bcd tng71 103.17c 17.62a 82.01d 124.18c 23.07gh 8.97a kuanfu waxy aroma 140.83ª 8.21f 83.63d 98.75d 28.77c 4.18e analysis of variance mut ms 647.78 46.41 53.22 2917.16 49.84 6.57 error ms 5.09 3.45 24.98 58.26 0.87 0.85 f value 127.27** 13.44** 2.13** 50.07** 57.12** 7.71** **, significant at p = 0.01. values followed by different superscript letters in each column are statistically significantly different at p = 0.05. tab. 1: awn, amylose contents, protein contents, aroma sensory scores, and palatability scores as well as their respective results from variance analyses of the tested aromatic mutants, their wild type variety kuanfu waxy aroma and a non-waxy white-pericarp aromatice rice variety tng71. mutant awn pericarp color amylose content protein content aroma sensory palatability % % score score am-419 reddish-brown 11.9d 7.11a 1.00de 65.32±de am-420 reddish-brown 13.69cd 7.07a 1.17 cd 63.70e am-422 reddish-brown 21.02a 6.72±bc 1.50ab 66.08cd am-423 reddish-brown 18.85b 7.01a 0.67f 64.73e am-424 reddish-brown 19.37a 7.14a 1.17cd 65.71de am-425 reddish-brown 14.95c 6.33d 1.33bc 70.45±b am-426 reddish-brown 18.14b 6.67c 1.17cd 67.78bc am-427 reddish-brown 20.99a 6.97ab 1.00de 64.35e am-430 reddish-brown 18.59ab 5.52f 0.83ef 73.75a am-433 reddish-brown 19.30a 6.77bc 0.50f 66.01cd tng71 white 18.70b 5.83e 1.17cd 69.32b kuanfu waxy aroma + reddish-brown waxy nd 1.83a nd analysis of variance mut ms 16.41 0.81 0.69 22.49 error ms 2.15 0.02 0.11 0.72 f value 7.63** 40.50** 6.27** 31.24** **, significant at p = 0.01. nd, not determined. values followed by different superscript letters in each column are statistically significantly different at p = 0.05. eating quality of non-waxy aromatic mutants 149 of milled rice grains of selected mutants by a panel (tab. 1). the results indicate that significant differences in the sensory score (p < 0.05) existed among the tested aromatic mutants, with kuanfu waxy aroma, mutant am-422 (1.50) and am-425 (1.33) showing high sensory score than reference variety tng71 (1.17). the palatability of cooked rice, which is comprehensively determined by aroma, appearance, taste and texture, is a very important factor affecting the consumer acceptance. it is frequently evaluated by trained tasting panel (yoon et al., 2009; bhonsle and sellappan, 2010; lestari et al., 2011). however, several instrument-based methods for evaluating the palatability of cooked rice texture have also been developed with acceptable results (arai and itani, 2000; okadome, 2005; yoon et al., 2009). in taiwan, the instrument-based technique has been standardized and used to determine the palatability of newly developed rice variety before its nomenclature and release (chen et al., 2009). in this study, the palatability of cooked rice samples were judged by a commercially-produced tasting meter that evaluated the palatability based on the hardiness, stickiness and appearance, and subsequently provided an overall score of cooked rice samples. mutants am-425 and am-430 were found to have palatability scores of 70.45 and 73.75, respectively, which were higher than the palatability score of 69.32 recorded for non-waxy aromatic variety tng71 (tab. 1). the mutant am-430 also had grain amylose and protein contents very close to tng71. the contents of amylose and protein are two major components influencing the palatability of cooked rice (zhu et al., 2010). therefore, the correlation analyses between the tested palatabilities of cooked rice grains and related grain quality traits across all mutants were conducted, but the statistical analyses indicated that only the grain protein content was negatively correlated to palatability (r = 0.929, significant at p = 0.01) (data were not shown). similar finding was also reported by lestari et al. (2009). on the other hand, negative but insignificant correlation (r = -0.311) was found between grain amylose content and palatability (data not shown). this result is not unexpected because the existence of significant variation in grain amylose content (ranged from 11.69 to 21.02) among the tested mutants (tab. 1). moreover, selecting a proper water and grain ratio is important to obtain an optimum cooked rice texture for a given rice variety. the high amylose rice grains generally require more water for achieving optimum eating quality (srisawas and jindal, 2007). but, in the present study, a fixed amount of water and grain ratio (30 g rice grains/40 ml water) was given to all the rice mutants that differing in grain amylose content. this result may affect the palatability scores of cooked rice to some extent. as for the grain yield, aromatic rice varieties generally have fewer numbers of panicles and produce lower grain yield comparing to non-aromatic rice varieties. in the present study, all the selected aromatic mutants showed considerably higher grain yield than their wild tab. 3: various agronomic characteristics and their respective results from variance analyses of variance analyses of the tested mutants, wild type variety kuanfu waxy aroma and non-waxy white-pericarp aromatic variety tng71 grown in autumn crop season. mutant plant height panicles plant-1 % fertility panicle-1 spikelets panicle-1 1000-grain weight yield (cm) (g) (ton ha-1) am-419 82.21g 17.32b 76.37bc 83.66efg 25.37e 6.17bc am-420 90.53f 12.31cd 70.32ef 88.7ef 26.85d 4.72e am-422 93.17f 13.82c 87.58a 74.66fgh 30.01b 5.96bcd am-423 97.21e 9.45e 73.88±cde 180.28a 23.88fg 6.63ab am-424 102.67d 10.37de 67.13fg 154.31bc 24.25fg 6.23bc am-425 107.17c 10.31de 70.55def 142.97bcd 27.13d 6.12bc am-426 83.67g 21.22a 71.11def 58.83h 24.94fg 4.85de am-427 84.67g 21.93a 75.56bcd 65.98gh 23.36g 5.45cde am-430 111.52b 9.70e 63.85g 159.69b 21.81h 5.72bcd am-433 105.17cd 11.45de 79.16b 125.83d 22.07h 5.53bcd tng71 98.11e 14.27c 76.42bc 137.27cd 23.89fg 7.64a kuanfu waxy aroma 129.02a 9.53e 75.69abcd 95.72e 28.58c 4.93de analysis of variance mut ms 519.11 92.88 227.76 7760.24 43.64 3.63 error ms 3.60 3.55 15.47 197.35 0.77 0.78 f value 144.35** 26.17** 14.72** 39.32** 56.98** 4.64** **, significant at p = 0.01. values followed by different superscript letters in each column are statistically significantly different at p = 0.05. tab. 4: the correlation coefficients, across all non-waxy aromatic mutants and tng71, for the tested grain amylase contents, grain protein contents, aroma sensory scores and palatability scores. grain trait 1000-grain % fertility spikelets yield weight panicle-1 panicle-1 panicles plant-1 -0.203 0.085 -0.550** 0.340* 1000-grain weight 0.311 -0.444** 0.003 % fertility panicle-1 -0.264 0.620** spikelets panicle-1 0.144 *,**, significant at 0.05 and 0.01 levels, respectively. 150 t.l. jeng, c.s. chan, s.y. lin, d.p. shung, c.z. pan, y.j. shih, j.m. sung type variety kuanfu waxy aroma in both spring and autumn crop seasons. these results are mainly resulted from the improved % fertility per panicle (tab. 2 and 3). correlation analysis (r = 0.620, p < 0.01), across all the tested accessions in two crop seasons, confirms this finding (tab. 4). however, some of the mutants (e.g., am-420, am-426 and am-427) did show unstable yield responses when they were grown in autumn crop season (tab. 2 and 3). the autumn-grown am-420 exhibited significant declines in all the examined yield components comparing to spring-grown am-420, and resulted in a substantial yield decline. jeng et al. (2006) reported that the low air temperature tended to decrease the number of spikelets per panicle and the % fertility per panicle. the low air temperature and insufficient interception of solar radiation during autumn crop season would further decrease the post-heading accumulated dry matter, and therefore cause a significant yield decline. however, the increased panicles per plant (r = 0.340, p < 0.05) is also a factor for improved grain yield in the tested mutants (tab. 4). as shown in tab. 1-3, the mutants am425 and am-430, which scored relatively higher in aroma sensory and palatability tests, consistently produced significantly higher grain yield than kuanfu waxy aroma in both spring and autumn crop seasons. however, the grain yield of mutant am-430 was slightly lower than the reference variety non-waxy aromatic tng71, even though am-430 had greater palatability score (73.75) than tng71 (69.32). in conclusion, our results confirm the effectiveness of nan3-induced mutation on de-awning of the non-waxy red-pericarp mutants derived from awned rice variety kuanfu waxy aroma. nan3-induced mutation also reduced the plant heights of selected mutants. significant variations on aroma sensory and palatability scores existed among the tested mutants, with mutant am-425 having aroma sensory and palatability scores slightly higher than reference variety tng71. large variations in the number of panicles per plant, number of spikelets per panicle, percentages of fertility per panicle, 1000-grain weights and grain yields were also observed among the tested mutants grown in the spring and the autumn seasons. the red-pericarp aromatic mutants am-425 and am-430 with waxy endosperm produced slightly lower grain yield than tng71 in both spring and autumn crop seasons. these two mutants produced more yields than kuanfu waxy aroma. therefore, mutants am-425 and am-430 can be grown and the produced rice grains can be milled and used as a staple food for daily consumption. 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stud. 38, 21-41. wang, z.-y., zhang, f.-q., shen, g.-z., gao, j.-p., snustad, d.p., li, m.-g., zhang, j.-l., hong, m.-m., 1995: the amylose content in rice eating quality of non-waxy aromatic mutants 151 endosperm is related to the post-transcriptional regulation of the waxy gene. plant j. 7, 613-622. yoon, m.-r., koh, h.-j., kang, m.-y., 2009: pasting and amylose component characteristics of seven rice cultivars. j. korean soc. appl. biol. chem. 52, 63-69. zhang, l., hu, p., tang, s., zhao, h., wu, d., 2005: comparative studies on major nutritional components of rice with a giant embryo and a normal embryo. j. food biochem. 29, 653-661. zhu, b., li, b., zheng, x.d., xu, b.l., liang, s., kuang, x., ma, m.h., 2010: study on predictive models relating physicochemical properties to © the author(s) 2015. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). texture of cooked rice and the application in rice blends. j. texture stud. 41, 101-124. address of the corresponding author: e-mail: sungjm@sunrise.hk.edu.tw journal of applied botany and food quality 88, 322 (2015) buchbesprechung heilende gifte – toxische naturstoffe als arzneimittel dietrich mebs das buch beschreibt anwendungen giftiger naturstoffe in der medizin wie z.b. bei krebserkrankungen, bei der schmerztherapie sowie bei der behandlung von malaria, thrombose und diabetes. der autor liefert einblicke in die evolution, die im verlauf von millionen von jahren dazu geführt hat, dass tausende dieser hochwirksamen naturstoffe auf der erde hervorgebracht werden konnten. nach einem einführenden kapitel, das mit „die natur als dienstleister“ überschrieben ist, geht der autor auf die enorme vielfalt der von mikroorganismen, pflanzen und tieren synthetisierten giftstoffe ein. dabei weist er auch darauf hin, dass bisher nur eine vergleichsweise geringe anzahl pflanzen auf ihre inhaltsstoffliche zusammensetzung hin untersucht wurde und daher noch sehr umfangreiche aktivitäten notwendig sind, um die reichlich vorhandenen ressourcen weiter für die menschheit zu erschließen. in dem dritten kapitel wird der leser vom autor auf diverse sammelreisen giftiger pflanzen und tiere mitgenommen. es wird dabei u.a. erläutert, wie ethnobotaniker vorgehen, um die überlieferten erfahrungen und kenntnisse indigener naturvölker hinsichtlich der nutzung bestimmter pflanzen sowie deren zubereitung zu erforschen. im vierten kapitel wird schließlich näher auf anwendungen in der chemotherapie eingegangen, wobei auch nicht verschwiegen wird, dass die anzahl der heute in der krebstherapie eingesetzten naturstoffe eher überschaubar ist (z.b. vinblastin und vincristin aus madagaskar-immergrün (cataranthus roseus), taxol aus der rinde von eiben (taxus baccata), camptothecin aus dem glücksbaum (camptotheca acuminata) und podophyllotoxin aus dem fußblatt (podophyllum peltatum). ein weiteres kapitel ist der „medizin aus dem meer“ gewidmet. hier geht der autor insbesondere auf die artenvielfalt in den korallenriffen ein und weist darauf hin, dass von der pharmakologischen wirkungsweise in der marinen tier und pflanzenwelt bisher insgesamt nur wenig bekannt ist, obwohl bereits mehr als 10.000 naturstoffe aus den einzelnen organismen isoliert und identifiziert werden konnten. in weiteren kapiteln wird die jeweilige verwendung von froschtoxinen, schlangengiften sowie botulinum-toxin aufgeführt. das letzte kapitel beleuchtet schließlich die bedeutung pflanzlicher und tierischer giftstoffe in der diagnostik, insbesondere bei der erforschung des menschlichen nervensystems. das buch des renommierten toxikologen dietrich mebs vermittelt dem leser einblicke in die faszinierende welt der giftstoffe und liefert dabei eine fülle aktueller informationen hinsichtlich entdeckung, toxikologie, wirkungsweise und nutzung der angeführten giftstoffe. der text ist insgesamt gut verständlich und aufgrund der eingeflochtenen persönlichen reiseerfahrungen auch spannend zu lesen, weshalb auch toxikologische laien an dem buch gefallen finden dürften. das werk ist darüber hinaus als ein leidenschaftliches plädoyer für den weltweiten schutz der ökosysteme und den erhalt der biodiversität zu verstehen. dr. h. schulz email: hartwig.schulz@jki.bund.de bibliografie: dietrich mebs, heilende gifte – toxische naturstoffe als arznei mittel, wissenschaftliche verlagsgesellschaft mbh, stuttgart, 1. auflage, 174 seiten mit 162 meist vierfarbigen abbildungen, zahlreichen formelzeichnungen und 2 tabellen, preis: 39,80 €, isbn: 978-38047-2983-4 journal of applied botany and food quality 89, 258 263 (2016), doi:10.5073/jabfq.2016.089.033 süleyman demirel university, faculty of agriculture, field crops department, turkey caraway (carum carvi l.) seed treatments and storage temperature influences potato tuber quality during storage arif şanli (received june 13, 2016) summary the present research was conducted to determine the effects of chemical and natural sprout inhibitors on potato (solanum tuberosum ‘agria’) tuber quality at two storage temperatures (8 oc or 15 oc). four doses (0, 50, 100, 150 g/10 l) of ground and whole caraway (carum carvi l.), containing high levels of s-(+)-carvone, and chlorpropham (cipc) were applied. tubers were stored for a total of 135 days in plastic containers (10 l) and placed in cold storage rooms at 8 °c or 15 °c with 85 % relative humidity. sprout inhibitors were applied only once at the start of experiment. at the end of 135 days the tubers stored at 8 °c had high starch and vitamin c content with a higher degree of firmness as compared to the tubers stored at 15 °c. total soluble sugar content (tss) of tubers increased during the storage period and the increase was high at 15 °c than 8 °c of storage condition. low temperature storage and ground caraway treatments at high temperature caused accumulation of reducing sugars (rs). in general, ground caraway and cipc treatments were more effective in maintaining tuber quality than the control and whole caraway treatments during the storage period. key words: caraway seed, chlorpropham, potato, storage temperature, tuber quality introduction in most countries, potatoes are harvested once a year. adequate and steady supply of tubers depends on long term storage for domestic consumption, food-processing industries, starch production and planting material purposes. high moisture content and metabolic activity of potatoes lead to both losses of weight and quality. the primary causes of these losses are respiration, transpiration and sprouting (burton et al., 1992). respiration of tubers during storage and breakdown of dormancy result in sprouting, thereby causing the alterations in weight, texture, nutritional value, shrinkage and the formation of toxic alkaloids (suhag et al., 2006; delaplace et al., 2008). to increase the amount of marketable potatoes after storage and to avoid quality deterioration, good storage conditions and effective treatments are necessary. methods of storage have been designed to prolong the dormant period and to retard or inhibit undesirable quality changes. storage at low temperatures delays sprouting but also leads to sweetening of potatoes due to accumulation of rs. it is complicated to store potatoes intended for processing because relatively high storage temperatures are required (8-10 °c) to prevent rs accumulation. prolonged storage of potatoes at temperatures higher than 7-8 °c requires the use of sprout inhibitors. chlorporpham is the most effective sprout inhibitor registered for use in potato storage (saraiva and rodrigues, 2011). however, many countries have restricted the use of cipc due to problems associated with chemical residues (hartmans et al., 1995). an increasing public demand for the safety issues of chemicals raises to use the natural inhibition agents. the essential oils of aromatic plants are found to have this potentially. many studies have been conducted to investigate the sprout suppressing capacity of natural products with different active substances involving caraway (silva et al., 2007; şanli et al., 2010), clove (song et al., 2004; elsadr and waterer, 2005), dill (song et al., 2004; gomez et al., 2010), peppermint (frazier et al., 2004) and spearmint (frazier et al., 2004; song et al., 2004; elsadr and waterer, 2005). studies indicate that carvone, the major compound in caraway and dill seed oil, is an effective inhibitor of sprout growth in potatoes (song et al., 2004; şanli, 2012). suppressing effect by carvone is reversible (ooesterhaven et al., 1993), and thus carvone could be used as a sprout growth regulator of seed potatoes during storage (sorce et al., 1997). in addition to sprout suppression, several studies have shown that carvone can effectively inhibit the growth of certain fungi and bacteria (hartmans et al., 1995; ooesterhaven et al., 1995; frazi̇er et al., 2004). to date, (s)-(+)-carvone is commercially marketed (talenttm) in some european countries (gomez-castillo et al., 2013). however, its production is too costy and its application at storage rooms requires special arrangements. therefore, the use of pure chemical form of carvone is limited as compared to conventional sprout suppressants such as cipc (coleman et al., 2001). use of natural forms of these eos (i.e. seeds, leaves) may provide an alternative in order to manage sprouting during storage. even the active substances of essential oils are less effective than the pure active agents they will likely to be more readily available and cheaper. it is also expected that release of active substances from natural forms will be slower but will have more active substances present in the used parts of plants (elsadr and waterer, 2005). while the sprout suppression effects of many eos and their major compounds have been studied, the effects of these compounds on the quality of potato tubers have not been clearly defined. hence the objective of this study was to determine the effects of cipc and caraway seeds on the quality of potato tubers stored for a long time at low and high temperature conditions. materials and methods it was previously published a study on the effects of caraway seed applications on sprouting and weight loss of potato tubers (şanli et al., 2010). in the current study, it was aimed to investigate the effects of cipc and caraway seeds on the quality of potato tubers. this study is a continuation of the earlier study (şanli et al., 2010) with different test and analysis such as tuber quality after the applications. the two studies are linked and published in two parts. plant material and storage condition potatoes were grown at the research farms of suleyman demirel universty and tubers were harvested in october 2008. selected tubers (100 kg) were kept at 15 oc for one month for the curing period. at the end of the curing period, tubers were immediately analysed to determine initial contents for total starch, protein, ascorbic acid, tss, rs, and their firmness. potato tubers were stored under two different temperature regimes, 8 oc and 15 oc, respectively. the relative humidity of storage rooms were adjusted to 85 %. effects of caraway (carum carvi l.) seed on potato quality 259 caraway seed and cipc treatments caraway seeds were purchased from a local farmer in turkey and cipc (grostop 300 ec (300 g/l cipc) was commercially obtained from poland. eo content of caraway seeds (4.2 %) was determined by the clevenger hydrodistillation method (british pharmacopoeia, 1980). constituents of caraway eo were determined by gas chromatograph-mass spectrometry (gc-ms) and it was found that caraway oil contained approximately 56 % s-(+)-carvone. gc-ms analysis was performed on shimadzu 2010 plus gc-ms equipped with a quadrupole (qp-5050) detector. the analysis was performed under the following conditions: capillary column, cp-wax 52 cb (50 m × 0.32 mm, film thickness 0.25 μm); injector and detector temperature, 240 oc; stove heat program, from 60 oc (10 min. hold) to 90 oc rising at 4 oc/min., and increasing to 240 oc (11.5 min. hold) rising at 15 oc/min.; flow speed, 1 psi; detector: 70 ev; ionization type, ei; carrier gas, helium (20 ml/min.); sample injected 1 μl. identification of constituents was carried out with the help of retention times of standard substances by composition of mass spectra with the data given in the wiley, nist tutor library. the quantitative analysis was conducted using gas chromatography/ flame ionization detector (gc-fid), shimadzu model thermo ultra trace, operating at the same conditions of gc-ms. 50 μl of the volatile oil was solubilized in 5 ml of n-hexane and injected in to the split mode 1/100. treatment doses of caraway seeds were determined by taking into consideration ratio of commercial s-(+)-carvone application rate (600 ml per 1000 kg tuber) (hartmans et al., 1995) and were calculated according to eo and s-(+)-carvone content of caraway seeds. application rate of cipc was 20 g/ton tubers (application rate of gro-stop 300 ec was 60 ml/ton tubers), similar to that applied commercially (hartmans et al., 1995). twenty potato tubers (~ 4 kg) were placed in a plastic container (10 l) and one of four doses of whole or ground caraway seeds (0, 50, 100 and 150g/ 4 kg tuber) were applied to potato tubers. whole (w) and ground (g) caraway seeds were wrapped in cheese cloths and placed on the top of tubers within each container. chlorporpham was applied as a dry powder by dusting. caraway seed and cipc treatments were applied to each container once at the beginning of the experiment and the lids of containers closed tightly. in order to allow oxygen exchange for respiration of tubers, lids were opened every two days for 10 minutes. tubers were stored for a total of 135 days starting from november 2008 to march 2009. at the end of the storage period, firmness and quality changes of tubers were evaluated. tuber quality evaluation total starch content of potato was determined based on aoac (1984). firmness of the tubers was measured with a brand penetrometer (pce-ptr 200, pce instruments uk ltd, united kingdom, se=±0.1kg) equipped with a 0.8 cm wide conical probe (rezaee et al., 2011). for the determination of ascorbic acid content, 20 g of sliced potato sample was homogenized in the presence of 6 % hpo3 in a mixer for 5 min at 4000 rpm. the extract was filtered under vacuum with a whatman filter paper and volume was adjusted to 100 ml with 6 % hpo3. ascorbic acid content of the samples (mg/ 100 g fw) were determined by titration method and 2,6-dichlorophenolindolphenol solution used as colouring agent (aoac, 1984). for the determination of protein content, samples were ground and 2 g was used. the amount of n in each sample was determined using the kjeldahl method and the protein content (%) was calculated by multiplying the n amount of each sample by 6.25 (bradford, 1976). total soluble sugars and reducing sugar contents of the samples were determined with phenol sulphuric acid assay (dubois et al., 1956) and nelson-somogyi method (somogyi, 1952). results were expressed as mg/100 g fw. all analyses were performed in triplicate. data were subjected to analysis of variance (anova) procedure with sas (2009) statistical program (inc sas/stat user’s guide release 7.0, cary, nc, usa). means were compared using duncan’s multiple range test. results and discussion total starch content total starch content of tubers decreased significantly (p<0.01) during the storage and the decrease in starch content of tubers was higher at 15 oc storage. average total starch content of tubers was 13.7 % and 12.9 % for 8 oc and 15 oc storage conditions, respectively (tab. 2). ground caraway and cipc treatments yielded higher starch contents at both storage temperatures than the control treatment, and no significant difference was detected between the control and whole caraway treatments for starch content (tab. 1). ground caraway treatments yielded higher starch content at both storage temperatures than the whole caraway treatments, but the starch contents did not differ significantly between the doses for both ground and whole caraway treatments. the tuber starch content treated with ground caraway and stored at 8 oc was similar to the starch content of cipc treated tubers whereas the starch content of tubers stored at 15 oc was higher than that of cipc treated tubers. ground caraway treatments were effective in preserving starch content at both storage temperatures while starch content loss was more pronounced at 15 oc storage than that at 8 oc storage conditions (tab. 2). storage temperature is an important factor to minimize postharvest losses during the storage of potato tubers. sugars used as substrates for respiration are produced from the starch hydrolysis. the respiration rates of potato tubers have been reported to be minimum at 4 oc, and increase at higher temperatures (wustman and struic, 2007). lower storage temperatures (4 °c and 8 ºc) tended to be more effective in reduction of starch hydrolysis of the potato tubers as compared to higher temperatures (12 ºc and 25 ºc). higher temperatures increase metabolism, respiration and physiological aging of potato tubers, resulting in the observed earlier sprouting and higher carbohydrate consumption (wiltshire and cobb, 1996). loss tab. 1: analysis of variance (anova) results for the examined parameters in the experiment (f values). sources of variation ts (%) f (n) vit. c (mg/100 g) pc (%) tss (%) rs (mg/100 g fw) storage temperature (s) 91.3** 48.2** 384.9** 133.6** 91.6** 724.3** treatments (t) 24.8** 32.0** 179.7** 31.9** 41.3** 92.7** s × t interaction 4.2** 1.3 7.0** 3.1* 7.3** 28.3** coefficient of variation (%) 2.1 2.8 6.0 2.3 4.6 5.2 *: p≤ 0.05 ** p≤ 0.01 ts (%): total starch content, f (n): firmness, vit. c (mg/100 g fw): vitamin c content, pc (%): protein content, tss (%): total soluble sugar content, rs (mg/100 g fw): reducing sugar content. 260 a. şanli of dormancy results in sprouting which in turn causes tuber dehydration and weight losses by increased respiration and transpiration. sedlokova et al. (2003) reported that carvone concentration was higher in ground caraway than whole caraway seeds. caraway and cipc treated tubers stayed dormant for a period longer than the control tubers which explains the higher starch content observed in those treatments than the control. ground caraway treatments gave higher starch content than whole caraway seed treatments at both storage conditions which could be explained by the higher carvone concentrations released by grounded seeds. it was also observed that humidity caused by respiration of tubers during storage was absorbed by whole caraway seeds and caused seeds to deteriorate during storage, an effect detrimental to stored tubers. while, caraway seeds deteriorate was not observed in ground caraway applications. firmness firmness of tubers decreased significantly during the storage. overall the firmness of tubers stored at 8 oc and 15 oc was 81.3 n and 77.2 n; respectively (tab. 2). caraway and cipc applications reduced firmness loss as compared to the control at both storage conditions in the experiment. at 8 oc storage, no significant differences were detected for firmness between all doses of ground caraway, a dose of 150 g whole caraway and cipc treatments. at 15 oc storage, the ground caraway and cipc treatments gave better results for preserving firmness of tubers than the whole caraway treatments. at both storage conditions, the lowest firmness was observed in the control tubers (tab. 2). firmness of potatoes is related to turgidity, and the best way to preserve firmness is to restrict water loss of tubers during storage. firmness of tubers usually decrease steadily during storage and the loss of firmness is more pronounced over 12 oc storage conditions (kaur et al., 2008). high storage temperatures caused softening and irregularities of tuber surface due to water and solid matter loss, and these changes in turn reduced appearance, quality and marketability of tubers. nourian et al. (2003) reported that the tubers stored at low storage temperatures generally have better quality and marketability due to firmness and overall appeal of tubers. on the other hand afek et al. (2000) showed that tubers stored at 10 oc and high (94-98 %) and low (82-84 %) relative humidity had 74 n and 63 n firmness. in addition to water loss and infections during the storage cause softening of tubers during storage. in a pervious study (şanli et al., 2010), sprouting rate was found to be lower at both storage conditions for ground caraway treatments. consequently, firmness was higher for ground caraway treatments at both storage conditions. treatments that reduce water loss and preserve solid matter content will have higher firmness as evidenced in the present study. vitamin c content during the storage, vitamin c content of tubers decreased significantly (p<0.01). the reduction in vitamin c content of tubers was 16.3 % and 11.8 % at 8 oc and 15 oc storage conditions, respecitvely (tab. 1). at 8 oc significant differences were not observed between ground and whole caraway and cipc treatments. at 15 oc ground caraway and cipc treatments had higher vitamin c content as compared to whole caraway treatments (tab. 2). at both storage conditions, there was no difference between different doses of ground and whole caraway treatments (tab. 2). vitamin c is lost during the storage and processing due to oxidation emese and nagymate (2008). vitamin c loss depends on storage time and storage temperature, and it was reported to change between 35 and 60 % (mondy and munshi, 1993; nourian et al., 2003). vitamin c metabolism is closely related to carbohydrate metabolism in plants. storage temperatures and treatments that reduce sprouting and maintaining dry matter content could help reduce vitamin c losses during storage. different sprout inhibitors such as caraway and dill extracts and cipc reduce vitamin c loses during storage (şanli, 2012). protein content protein content of potatoes decreased significantly during the storage (p<0.01). storage temperature was also found to be an important factor for protein content of tubers during the storage (p<0.01). tubers stored at 8 oc had lower protein content (1.71 %) than those tubers (1.84 %) stored at 15 oc (tab. 2). control tubers had the lowest protein content at both 8 oc (1.59 %) and 15 oc (1.76 %) storage temperatures (tab. 2). at 8 oc storage, ground caraway and cipc treated tubers had higher protein content than control, however at the same storage temperature, there was no significant difference between whole caraway treated tubers and the control. significant differences were not detected between cipc and 50 g and 100 g ground caraway treatments for protein content, but the protein content of 150 g ground caraway treatment was found to be higher than tab. 2: comparison of means of total starch content (ts), firmness (f) and vitamin c of potato tubers with caraway seed and cipc applications at 8 oc and 15 oc temperature. ts (%) f (n) vit. c (mg/100 g fw) temperature 8 oc 15 oc 8 oc 15 oc 8 oc 15 oc initial 14.3±0.20a 86.3±1.5a 26.1±1.5a 50 13.9±0.33abc 13.5±0.20abcd 83.0±2.6abc 79.7±2.5def 16.1±0.7b 11.70.9±e 100 13.8±0.20abc 13.4±0.26bcd 83.7±2.5ab 81.3±1.5bcde 15.5±0.8bc 11.51.5±e 150 13.9±0.20ab 13.7±0.41abc 86.0±3.0a 82.0±1.7bcd 15.8±0.6bc 11.50.8±e 50 13.4±0.47bcd 12.3±0.13ef 78.0±1.7def 72.0±1.0g 14.6±1.0bcd 8.9±0.5f 100 13.4±0.40bcd 12.4±0.23ef 79.0±3.6cde 74.3±2.3g 14.6±0.9bcd 8.8±0.2f 150 13.4±0.37bcd 12.2±0.10ef 80.7±1.5abcd 75.3±1.5def 14.8±0.4bcd 8.9±0.2f cipc 13.8±0.28abc 12.9±0.29de 82.3±2.3abc 77.3±2.1f 15.8±0.7bc 10.4±0.4e control 13.1±0.38cd 11.8±0.16f 73.0±2.0h 66.0±3.0ı 13.4±0.7d 8.5±0.3f mean1 13.7a 12.9b 81.3a 77.2b 16.3a 11.8b mean values with different superscript letters in the same column differ significantly according to duncan’s test (p≤ 0.05). values are means ± sd (n = 3). 1: indicates the sum of storage temperature. ground caraway whole caraway treatments/ doses (g) effects of caraway (carum carvi l.) seed on potato quality 261 that of cipc treatment at 8 oc storage. the protein contents of cipc (1.88 %) and 150 g ground caraway (1.89 %) treated tubers were higher than those of whole caraway treated tubers at 15 oc storage (tab. 2). at the same storage conditions, ground caraway (50, 100 g) treatments and whole caraway treatments gave similar results for protein content. at both storage conditions, the protein contents of control and whole caraway applied tubers did not differ from each other significantly (tab. 2). similarly, pinto et al. (1993) reported a decrease in the protein content of potato tubers at high storage temperatures due to the increases in the activities of protease and amylase enzymes. during dormancy both proteinase activity and free amino acids levels are at the minimum level. however, after breaking of dormancy proteinase activity increases and the mobilizations of nitrogen reserves increase within tubers (nowak, 1977). dormancy was broken down at the earlier stages of storage in the control and whole caraway applied tubers which in turn leaded to an increase in proteinase acitivity but a decrease in protein content observed in those treatments. since the increase in sprout weight at higher temperature (şanli et al., 2010), indicated a higher available protein content at this temperature that was ultimately used during sprout growth. an increase in free amino acid content commonly occurred during the latter part of storage, caused by an upturn of proteinase activity on the break of dormancy. total soluble sugars total soluble sugar content of tubers increased depending on the storage temperature (p<0.01). the tubers stored at 8 oc had 1.53 % tss while the tubers stored at 15 oc had 1.72 tss (tab. 3). at both storage temperatures the highest tss value was observed in control tubers. the tss contents of ground caraway applied tubers at both temperatures did not differ significantly from each other however their tss contents were lower than those of the tubers treated with whole caraway at both storage conditions (tab. 3). while cipc treated tubers stored at 8 oc had similar tss content to ground caraway treated tubers, cipc treated tubers had higher tss content at 15 oc storage. the tss contents of the control and whole caraway apllied tubers had higher tss contents at 15 oc (tab. 3). respiration during the storage period causes breakdown of starch leading to increased tss content of the tubers (matsura-endo et al., 2004). storage temperature and duration affect tss content of potato tubers (karim et al., 2008). sprouting appeared earlier in the control and whole caraway treated tubers and consequently the control and whole caraway applied tubers had higher tss contents as compared to cipc and ground caraway applied tubers in the present study. energy requirement of sprouting is mainly supplied by the hydrolysis of glucose and sucrose which in turn increase tss content. razee et al. (2011) reported that radiation of tubers stored for 5 months delayed sprouting as compared to non radiated tubers and leaded to reduced tss content as compared to the control treatment. reducing sugars the effect of storage temperature on reducing sugars content was significant (p<0.01). initial reducing sugars content of tubers was 188 mg/100 g fw at the beginning of the experiment and at the end of the experiment final concentration of reducing sugars were 300 and 205 mg/100 g fw for 8 oc and 15 oc storage; respectively (tab. 3). while reducing sugars content increased in tubers stored at 8 oc during storage for all treatments, the changes in reducing sugars content of tubers were depended on treatments at 15 oc storage. at both storage temperatures the lowest amount of reducing sugars was observed from the control tubers. ground (50, 100, 150 g) and whole (150 g) caraway treatments did not differ from each other for reducing sugars content and they had the highest level of sugars observed in the experiment at 8 oc (tab. 3). ground caraway treatment had higher amount of reducing sugars than whole caraway treatment at 15 oc storage. chlorporpham treated tubers had lower reducing sugars than caraway treated tubers at 8 oc whereas cipc treatment did not affect reducing sugars content 15 oc storage (tab. 2). reducing sugars contents of tubers generally increase during the storage (matsuura-endo et al., 2004; rezaee et al., 2011). during storage, reserve starch accumulated in tubers are converted to sucrose, glucose and fructose to produce energy which in turn increases the level of reducing sugars content of tubers. under low storage temperatures, starch is converted to sucrose by udp-glucose pyrophosphorylase and sucrose phosphate synthase enzymes and sucrose is converted to reducing sugars by acid invertases (zrenner et al., 1996) and it is called low temperature tab. 3: comparison of means of protein content (pc), total soluble sugar (tss) and reducing sugar (rs) of potato tubers with caraway seed and cipc applications at 8 oc and 15 oc temprature. pc (%) tss (%) rs (mg/100 g fw) temperature 8 oc 15 oc 8 oc 15 oc 8 oc 15 oc initial 1.99±0.05a 1.38±0.08h 188±9.5f 50 1.75±0.05efg 1.85±0.03bcd 1.46±0.08gh 1.51±0.04efgh 347±20.6a 281±14.4d 100 1.73±0.02fgh 1.84±0.04bcd 1.46±0.07gh 1.53±0.07efgh 335±14.0abc 275±20.0d 150 1.78±0.03def 1.89±0.05b 1.45±0.09gh 1.52±0.06efgh 343±9.1ab 294±11.4d 50 1.62±0.02ıj 1.79±0.04def 1.61±0.08ef 1.91±0.06b 316±11.0c 166±7.6fg 100 1.63±0.06ıj 1.78±0.04def 1.63±0.06def 1.88±0.05b 323±12.9bc 162±5.3g 150 1.66±0.06hıj 1.81±0.05cde 1.54±0.13efg 1.84±0.03bc 329±13.0abc 169±11.4fg cipc 1.68±0.01ghı 1.88±0.08bc 1.49±0.08fgh 1.65±0.07de 274±19.4d 185±15.0fg control 1.59±0.02j 1.76±0.02ef 1.75±0.09cd 2.27±0.09a 243±8.1e 121±9.1h mean1 1.71b 1.84a 1.53a 1.72b 300a 205b cv 2.3 4.6 5.1 mean values with different superscript letters in the same column differ significantly according to duncan’s test (p≤ 0.05). values are means ± sd (n = 3). 1: indicates the sum of storage temperature. ground caraway whole caraway treatments/ doses (g) 262 a. şanli sweetening of tubers. at high storage temperatures, the increased level of respiration leads to the breakdown of fructose and glucose, which are used to produce necessary energy for sprout development, and reducing sugars levels are decreased (rezaee et al., 2011). at the present study, reducing sugar levels of tubers were lower at 15 oc than that of the tubers stored at 8 oc. dormancy breakdown and sprout development of tubers start earlier at 15 oc which explains lower levels of reducing sugars of tubers stored at higher temperature. conclusion in the present study, the effects of sprouting inhibitor cipc and caraway applications, a natural source of carvone, on the quality of tubers were studied using the tubers stored at different temperatures. at low storage temperature, the examined quality parameters were better with the exception of rs which negatively affects chips qaulity. ground caraway applications were more effective than the whole caraway applications overall, but the effects of caraway doses on the quality parameters were all similar. at low temperature condition, the effects of cipc and ground caraway applications on quality parameters were similar, but at the higher storage temperature ground caraway applications yielded better results as compared to cipc application. reducing sugar levels of ground caraway applications were high yet their levels were within acceptable levels for chips production (300-400 mg/100 g fw) (duplessis et al., 1996). at the end of the study, it was concluded that the ground caraway applications prevented quality losses of the tubers at high temperature storage conditions. there is no literature for the effects of caraway seed treatments on tuber taste and scent. in a study that we have done before, the tubers treated with caraway essential oil showed low carvone residue levels (1 ppm in peeled tubers and 4 ppm in peelings), as compared to those of s-(+)-carvone and cipc applied tubers (şanli, 2012). in addition, no carvone residues at 1520 days after storage were found at the same study. therefore, it is believed the tubers treated with caraway seed could not be showed a significant change in terms of flavor and taste. due to the fact that the carvone has a low human toxicity level (kerstholt et al., 1997) and has generally recognized as safe (gras) status (hall and oser, 1965), the use of ground caraway show promise as a replacement to cipc in prevent quality losses of tubers at high storage temperatures. acknowledgements we thank to suleyman demirel university faculty of agriculture and dr. muhammet tonguç for chemical analysis, and to dr. sulhattin yaşar and yaşar karakurt for editing the manuscript. references afek, u., orenstein, j., nuriel, e., 2000: using the tabor atomizer system to maintain weight and firmness in stored potato tubers. amer. j. potato res. 77, 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natural and applied sciences, field crops department, suleyman demirel university, turkey. wiltshire, j.j.j., cobb, a.h., 1996: a review of the physiology of potato tuber dormancy. ann. appl. biol. 129, 553-569. wustman, r., struic, p.c., 2007: the canon of potato science: seed and ware potato storage. potato. res. 50, 351-355. zrenner, r., schuler, k., sonnewald, u., 1996: soluble acid inver tase determines the hexose-to-sucrose ratio in cold-stored potato tubers. planta. 198, 246-252. address of the author: süleyman demirel university, faculty of agriculture, field crops department, turkey e-mail: arifsanli@sdu.edu.tr © the author(s) 2016. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 89, 191 201 (2016), doi:10.5073/jabfq.2016.089.024 1department of horticulture, mustafa kemal university, antakya, hatay, turkey 2 department of horticulture, erciyes university, melikgazi, kayseri, turkey 3alata horticultural research institute, erdemli, mersin, turkey 4agriculture county directorate, silifke, mersin, turkey effects of rootstocks on storage and shelf life of grafted watermelons ahmet erhan özdemir1, elif çandır1*, halit yetişir2, veysel aras3, ömer arslan4, özay baltaer1, durmuş üstün1, mustafa ünlü2 (received december 17, 2015) * corresponding author summary watermelon fruits from non-grafted or grafted ‘crimson tide’ (ct) and ‘crisby’ (cr) onto ferro, rs841, argentario and macis rootstocks were compared for their postharvest quality during storage at 7 °c for 21 days and additional 7 days at 21 °c. non-grafted and grafted ct and cr fruits did not exhibit chilling injury (ci) symptoms, but the 1-2 % of fungal decay occurred after shelf life period following storage. watermelons grafted on ferro and rs841 rootstocks had higher flesh firmness thicker rind, lower ripening rating, more intense (higher c*) brighter red (lower h° value) color and higher lycopene content after shelf life period following storage, compared to nongrafted fruits. all of the fruit tested by the panelists received high taste scores of >7.9 out of 8.5 at the beginning, but the scores decreased to >6.8 out of 7.7 at the end of shelf life period. watermelons could successfully be kept for 21 days at 7 °c and additional 7 days at 21 °c. watermelons grafted on ferro and rs841 rootstocks had higher postharvest quality, compared to the non-grafted fruits for both cultivars. introduction turkey is an important vegetable producing country with total of 27 billion metric tons vegetable production (faostat, 2014). the cucurbitaceae family comprises 28 % of total vegetable production of turkey. watermelon accounts for 14 % of total vegetable production. turkey is the second largest watermelon-producing country in the world after china, with production of 4 million tons per year. recently, grafted watermelon production has become widespread in turkey, because soil borne diseases are severe during early cultivation under plastic tunnels or later in the season in field production due to continuous and intensive cropping (kurt et al., 2002). soil borne diseases cause a decrease in yield and quality. there are different ways to prevent soil-borne diseases such as crop rotation, breeding programs, soil fumigant (yetisir and sari, 2004). however, the use of methyl bromide has been banned due to its effect on depletion of ozone layer. the use of seedlings grafted on cucurbita and lagenaria rootstocks, which have an acquired resistance to soil borne diseases, was suggested by several researchers as an environmentally safe alternative to methyl bromide (miguel et al., 2004). owing to environmental restrictions imposed on the use of chlorofluorocarbon-based soil fumigants, use of rootstocks resistant to soil borne pathogens has become an established practice in regions growing watermelon (davis and perkins-veazie, 2005). the most common rootstocks for watermelon are bottle gourd, interspecific hybrids between c. maxima and c. moschata, and wild watermelon (citrullus lanatus var. citroides) (davis et al., 2008). grafting of watermelon scions on squash, pumpkin, or bottle gourd rootstocks is practiced in all the major watermelon production regions of the world (lee, 1994; 2003). these rootstocks influenced resistance to soil borne diseases, plant growth, yield, and fruit quality. graft incompatibility and decrease in the fruit quality appeared depending on the scion-rootstock combination (lee and oda, 2003). although effects of rootstocks on yield have been reported, the information on fruit quality has been incidental and does not account for all correlative aspects of quality. abnormal fruit quality has been reported due to grafting for watermelon (yamasaki et al., 1994; lee and oda, 2003; liu et al., 2006; alexopoulos et al., 2007). however, there are other reports of positive effects of grafting on watermelon fruit quality (yetisir and sari, 2003; davis and perkins-veazie, 2005; karaca et al., 2012; çandir et al., 2013). the effect of the rootstocks on plant growth, fruit yield and quality of watermelon cv. crispy grafted onto tz-148 and rs841 of commercial hybrid of cucurbita maxima × cucurbita moschata and 64-18 of experimental bottle gourd rootstocks were studied. grafting resulted in higher yield by increasing in both fruit number and weight, however, no detrimental effect on fruit quality such as fruit index, rind thickness, and soluble solid contents on grafted plants was observed (alan et al., 2007). grafting on the local bottle gourd rootstocks improved plant growth parameters, yield (karaca et al., 2012) and the total soluble solid (tss), titratable acidity (ta), tss/ta ratio, sugar, organic acid, and carotenoid (β-carotene and lycopene) contents of crimson tide fruits (çandir et al., 2013). for short-term storage or transit to distant markets (>7 days), most recommendations use 7.2 °c (45 °f) and 85-90 % r.h. as the acceptable handling conditions for watermelons (suslow, 1997). watermelons are, however, prone to chilling injury at this temperature and extended holding at this temperature will induce chilling injury, rapidly evident after transfer to typical retail display temperatures (suslow, 1997). short-term storage at near ambient temperatures is the common practice for watermelon (chisholm and picha, 1986; perkins-veazie and collins, 2006; radulović et al., 2007). watermelons in most of the world are customarily handled postharvest under non-refrigerated conditions. shelf life for watermelon is 2-3 weeks at 10-15 °c depending on cultivar (rushing et al., 2001). there are few reports on the effects of grafting on storage and shelf life of watermelons. effects of grafting on postharvest quality of watermelon are not completely known and cause some speculation. previous studies have shown that grafting increased in flesh firmness of watermelon fruits in most scion-rootstock combinations (salam et al., 2002; yetisir et al., 2003; davis and perkins-veazie, 2005; roberts et al., 2007; cushman and huan, 2008; bruton et al., 2009; soteriou and kyriacou, 2015). firmer fruits are more likely to retain a desirable consistency and are expected to have a better shelf life than are softer fruit (roberts et al., 2007; king et al., 2010). therefore, effects of grafting on storage and shelf life performances of watermelon fruits have gained importance. the objective of this study was to determine quality changes of watermelon fruits cv. ‘crimson tide’ (ct) and ‘crisby’ (cr) grafted onto ferro, rs841, argentario, and macis rootstocks during storage at 7 °c for 21 days and additional 7 days at 21 °c and compare graf192 a.e. özdemir, e. çandır, h. yetişir, v. aras, ö. arslan, ö. baltaer, d. üstün, m. ünlü ted and non-grafted ‘crimson tide’ and ‘crisby’ fruits for postharvest quality. materials and methods the experiment was conducted at the alata horticultural research institute, erdemli, mersin, turkey. ‘crimson tide’ (ct) and crisby (cr) watermelon [citrullus lanatus (thunb.) matsum. and nakai] cultivars were grafted onto ferro and rs841 (c. maxima × c. moschata) and argentario and macis (lagenaria siceraria) rootstocks by using slunt cut grafting method (lee and oda, 2003). the grafted plants were supplied by the commercial seedling company of grow fide (antalya, turkey). the non-grafted ct and cr were used as control. fruits were harvested at full maturity when the 75 % of tendril and stipule on the same node with peduncle were desiccated. after harvest, fruits were stored at 7±0.5 ºc and 90±5 % relative humidity for 21 days in cold store and hold 21 days at 7 °c and subsequent 7 days at 21±0.5 ºc and 70±5 % relative humidity for shelf life. changes in fruit weight (g), diameter (mm) and length (mm), rind thickness (mm), weight loss (%), fruit flesh firmness (n), taste (1-9), total soluble solids (%), juice ph, titratable acidity (%), chilling injury and fungal decay (1-5), flesh color (l* c* hº) values, hallow heart (1-5), ripening (1-7), citric and malic acid (%), glucose (%), fructose (%), sucrose (%), total sugar (%), β-carotene (μg g-1), lycopene (μg g-1) were determined after shelf life period following storage at a weekly interval. fruit weight (g); 30 fruits were weighted by a laboratory balance with an accuracy of 0.01 g for grafted and non-grafted fruits of both cultivars; it was calculated by taking the arithmetic mean. diameter (mm) and length (mm); 5 fruits in each replicate were measured with a ruler. rind thickness was measured at two points on each fruit cross-section using an electronic caliper. weight loss (%); 30 fruits were numbered and they were weighted by a laboratory balance with an accuracy of 0.01 g for grafted and non-grafted fruits of both cultivars, the loss was calculated by subtracting the final weigh from the initial weigh in percent. fruit flesh firmness (n); flesh firmness of the middle of each fruit was determined with a penetrometer (now fhr-5 nippon optical works co. ltd. tokyo, japan). this involved measuring the force in kilograms required for a 12-mm conical probe to penetrate the cut surface to a depth of 5 mm at 3 locations in the mesocarp tissue; the results were then converted to newton (n). as described by motsenbocker and picha (1996), a 2.5-cm cross-sectional slice was taken from the middle of each fruit. the flesh of each slice was carefully removed from the outer rind and quartered. two opposite quarter pieces were combined and homogenized for one minute in a waring blender and filtered under suction through whatman #4 paper. portions of the homogenate were used to determine total soluble solid (tss) content, juice ph, titratable acidity (ta). tss content was measured using a digital refractometer (atago model atc-1e atago co. ltd., tokyo, japan) and juice ph with digital ph-meter (orion 5-star model thermo fisher scientific inc., ma, abd) at 20 °c. ta was measured by potentiometric method. the 5 ml of juice sample was diluted with distilled water to 100 ml and this sample was titrated with 0.1 n naoh until the ph value of 8.1 was read on digital ph-meter. the results were calculated as “g malic acid 100 ml-1 juice” in percent. the fruits were also scored at each evaluation for chilling injury (ci) and decay (1 = none, 2 = <10 % of surface area, 3 = 11 % to 25 %, 4 = 26 % to 50 %, and 5 = >50 %) (risse et al., 1990). incidence of ci and decay were determined after 7 days at 21 °c following each sto-rage period. for sensory evaluation, two opposite quarter pieces from the middle of each fruit were prepared as described above. ten trained panelists (non-smoker 7 male and 3 female, ages 20 to 45) evaluated taste (1-9) of fruits on hedonic scale of 1= disliked (lowest value) to 9 = liked (the best), hallow heart (1-5) of fruits on a scale of 1= none to 5 = very severe (50 % “more than hallow heart) and ripening (1-7) of fruits on a scale of 1= raw fruit and 3 = mature to 7= overripe extremely. fruit flesh color was measured using the cielab (l*a*b*) color space by a cr-300 minolta chroma meter (konica minolta, osaka, japan), calibrated using the manufacturer’s standard white plate. two readings were performed from the flesh of vertically cut fruits. l* represents lightness, ranging from 0 (black) to 100 (white). chroma (c*) represents color saturation, which varies from dull (low value) to vivid color (high value) and was calculated using the formula (a2 + b2)1/2. hue angle (h°) represents a color wheel with red-purple at an angle of 0°, yellow at 90°, bluish green at 180°, and blue at 270°, and it was calculated by h° = tan−1 (b/a) (mcguire, 1992). sugars and organic acids were extracted of the method described by çandir et al. (2013). briefly, frozen samples were homogenized using an ultra-turrax t25 model homogenizer (ikalabortechnik) at low speed with a 10-mm shaft. the resulting slurry was filtered through whatman no. 4 paper with a buchner funnel under vacuum. exactly 1 ml of sample was diluted with deionized distilled water to a total volume of 10 ml. after vortexing for 1 min, 20 μl of sample was injected directly into the hplc equipment after filtration through a millex-hv 0.45 μm filter (millipore). hplc analysis of sugars and organic acids was performed on lc-10a equipment consisting of lc-10ad pumps, an in-line degasser, a cto-10a column oven, an scl-10a system controller, an spd 10avp, a photo diode array (pda) detector, and a refractive index detector (rid), all operated by lc solution software (shimadzu). sugars were separated on an ec nucleosil carbohydrate 250 mm × 4 mm i.d. column (machereynagel) at 25 °c. the mobile phase was acetonitrile and water (80:20, v/v) at a flow rate of 2 ml min–1. organic acids were separated on a transgenomictm icsep ion300 300 mm × 7.8 mm i.d. column (transgenomic) at 65 °c. the mobile phase was 0.0085 n h2so4 at a flow rate of 0.4 ml min–1. sugars and organic acids were detected using the rid and pda detectors at 210 nm, respectively. the quantification was performed according to external standard solution calibrations. the results were expressed as g 100 g–1 fresh weight. carotenoids were extracted following a modified version of the method described by perkins-veazie and collins (2006). brefly, frozen samples were homogenized using the ultra-turrax homogenizer at low speed with a 10-mm shaft. three grams of puree were weighed into the centrifuge tube and extracted with hplc-grade solvents of 10 ml of hexane, 5 ml of ethanol, and 5 ml of acetone containing 0.05 % butylated hydroxytoluene (merck kgaa). samples were tightly sealed and placed on an orbital shaker (heidolph unimax 2010, heidolph instruments gmbh co. kg) for 15 min at 320 rpm, and then 3 ml of deionized distilled water was added and samples were shaken again for 10 min. afterwards, samples were put in a rack to allow solvent phase separation. the upper hexane layer was also filtered using a millex-hv 0.45-μm filter (millipore) and 20 μl of sample was injected directly into shimadzu hplc equipment (as previously described). carotenoids were separated on a ymc carotenoid column, c30 250 mm × 4.6 mm id, 5 μm particle size (ymc europe gmbh), operating at 30 °c with a flow rate of 1.5 ml min–1. the mobile phase was solvent a (methanol, methyl tertiary butyl ether, and deionized distilled water, 81:15:41) and solvent b (methanol and methyl tertiary butyl ether, 10:90) with elution with a linear gradient of 0-16 min with 100 % a and 16-60 min with 100 % b (liu et al., 2009). detection was carried out at 503 nm for lycopene and 452 nm for β-carotene using the pda detector. components were identified by comparison of their retention times to those of authentic standards under analysis conditions and were quantified by external standard method and expressed as μg g–1 fresh weight. the study was performed over a 2-year period, in 2009 and 2010. data are represented as the mean of 2 experimental years. in the watermelon production region where the experiment was conducted, storage and shelf life of grafted watermelons 193 farmers start the season with early season cultivar ‘crisby’ followed by mid-early/middle season cultivar ‘crimson tide’. crisby has a mealy texture while crimson tide is crisp. our objective in this experiment was to determine the commercial rootstock(s) with best storage and shelf life performance for each cultivar, not to determine the best scion/rootstock combination(s) for watermelon producing area. therefore, the experiment was conducted in completely randomized block design and the data were analyzed by one-way anova using sas software of sas institute, cary, n.c. (sas, 1999). the data were obtained from three replicates per scion/rootstock combination. each replicates contained 5 fruits. mean separation was performed by fisher’s least significance test at p<0.05 level. results and discussion watermelons grafted on rs841 and ferro rootstocks had the higher fruit weight for ct and cr cultivars (tab. 1). an increase in fruit weight due to grafting was reported in previous studies (yetisir et al., 2003; miguel et al., 2004; alexopoulos et al., 2007; alan et al., 2007; prioetti et al., 2008). grafting had no significant effect on fruit length and diameter for both cultivars (tab. 1). similarly, davis et al. (2008) reported that fruit shape, determined by length, circumference, and their ratio, did not change significantly from watermelon fruit harvested from grafted and not grafted plants. graf ting affected rind thickness dependent on the scion. consistent with these findings, soteriou and kyriacou (2015) reported that variability in rind thickness was derived mainly from the scion. at harvest, ct fruits from grafted plants had thicker rind than those from non-grafted plants while grafting did not affect rind thickness for cr watermelon cultivar (fig. 1). rind thickness of ‘crisby’ watermelon fruits grafted on tz-148, rs-841 and 64-18 of lagenaria rootstock was similar to non-grafted control fruits (alan et al., 2007). interspecific hybrid rootstocks (c. maxima × c. moschata) increased rind thickness of four watermelon cultivars (‘celebration’, ‘gallery’, ‘pegasus’ and ‘torpilla’) although their effect was very limited, indicating minimal rootstock effect on rind thickness compared with the effect of cultivar (kyriacou and soteriou, 2015). alexopoulos et al. (2007) reported that ‘crimson sweet’ fruits from grafted plants on rootstocks (long gourd, early max, max-2 and f-14 gourd), had a thicker rind than the fruits from non-grafted plants. davis et al. (2008) also found rind thickness increased for both seedless and seeded watermelon fruits when grafted to gourd rootstock ‘451’. in the study with ‘crimson tide’ watermelon fruits, all of the grafted plants on bottle gourd rootstocks produced fruit with a thicker rind than the control plants (karaca et al., 2012). rind thickness decreased in non-grafted and at a lesser extent in grafted fruits during storage at 7 °c for 21 days and additional 7 days at 21 °c in both cultivars (fig. 1). fruits grafted on rs841, argentario and ferro rootstocks had thicker rind compared to non-grafted fruits after shelf life period following storage for both cultivars (fig. 1). similarly kyriacou and soteriou (2015) reported that postharvest storage at 25 °c caused thinning of the rind after 7 and 14 days and all hybrid rootstocks resulted in thicker rind than the non-grafted control. thinning of the rind, known to characterize watermelon maturation (corey and schlimme, 1988), therefore may indicate an overripe fruit and one subjected to prolonged storage (kyriacou and soteriou, 2015). weight losses of grafted and non-grafted fruits were very low (<1 %) after shelf life period for 7 days at 21° following 21 days of storage at 7 °c for both cultivars. effect of rootstocks on weight loss was not significant (fig. 2). consistent with our results, perkins-veazie and collins (2006) reported the <1 % of weight loss in watermelon fruits at all temperatures (5 °c, 13 °c and 21 °c) after 14 days of storage. however, neto et al. (2000) determined higher weight loss (3.8 %) than our results. non-grafted ct and cr or ct and cr grafted onto different rootstocks did not exhibit ci symptoms, but the 1-2 % of fungal decay occurred during shelf life period after 21 days of storage (fig. 3). the decayed areas covered <10 % of rind surface of fruits for both cultivars. the graft combinations did not differ in the incidence of fungal decay for cr cultivar. with ct cultivar, fruits grafted on ferro tab. 1: effects of rootstocks on fruits weight, diameter and length of ‘crimson tide’ (ct) and ‘crisby’ (cr) watermelon fruits at harvest scion / rootstock fruit weight fruit diameter fruit length (g) (mm) (mm) cr control 4814.29c 21.07a 21.00a macis 5653.78ab 21.55a 21.80a argentario 5251.60bc 21.50a 21.92a rs841 6076.50a 22.01a 22.14a ferro 6058.54a 22.17a 22.95a ct control 6316.41c 21.26a 24.66a macis 6343.75c 22.26a 25.81a argentario 6668.41b 22.92a 26.37a rs841 6914.47a 21.60a 25.33a ferro 6814.06ab 21.99a 25.60a x mean separation was performed by fisher’s lsd test. means (n=3) followed by same letters within a column are not significantly different at p<0.05. fig. 1: effects of rootstocks on rind thickness of ‘crisby’ and ‘crimson tide’ watermelon fruits after shelf life period for 7 days at 21 °c following storage at 7 °c r in d th ic kn es s (m m ) r in d th ic kn es s (m m ) 194 a.e. özdemir, e. çandır, h. yetişir, v. aras, ö. arslan, ö. baltaer, d. üstün, m. ünlü (1.43 %) and rs841 (1.54 %) rootstocks had the lower fungal decay than those on argentario (1.87 %), macis (1.90 %) and control fruits (2.11 %) after shelf life period for 7 days at 21° following 21 days of storage at 7 °c. in this study, increased in storage time and subsequent shelf life period resulted in the surface decay caused by different fungi. botrytis cinerae, alternaria cucumerina, cladosporium cucumerinum, fusaium spp., penicilliım digitatum and p. italicum, the most encountered fungi identified followed by colletotrichum orbiculare and stemphylium spp. grafted and non-grafted watermelon fruits did not show differential susceptibility to the those pathogen. out of pathogen identified, b. cinerae, c. cucumerinum stemphylium spp. were first time described as additional pathogens to other previously known species causing postharvest decay of watermelon. postharvest rots caused by fusarium spp. and phytophthora capsici are of concern because control measures for these fungi in the field often are inadequate. with good disease control in the field, anthracnose (c. orbiculare) and black rot (didymella bryoniae) rarely develop on watermelon (rushing et al., 2001). flesh firmness decreased during storage at 7 °c for 21 days and additional 7 days at 21 °c for both cultivars and grafted fruits had firmer comparing to non-grafted fruits (tab. 2). our data suggest that effects of rootstocks on flesh firmness varied depending on the rootstock and the scion. watermelons grafted on ferro and rs841 rootstocks had the higher fruit flesh firmness for ct and cr cultivars. non-grafted fruits had the lowest fruit flesh firmness after shelf life period following storage for cr and ct cultivars. at harvest, an increase in flesh firmness due to grafting has been reported. (salam et al., 2002; yetisir et al., 2003; davis and perkins-veazie, 2005; roberts et al., 2007; cushman and huan, 2008; bruton et al., 2009; soteriou and kyriacou, 2015) while grafting on some rootstocks seems not affect watermelon firmness (karaca et al., 2012). the findings of kyriacou and soteriou (2015) indicated that c. maxima × c. moschata hybrids sustained higher postharvest flesh firmness compared with non-grafted controls, while rootstock effect was superior to that of cultivar and storage. watermelon fruit flesh firmness did not change or reduced during storage during 4 weeks of storage at 5°, 10°, 15° or 20 °c depending on storage temperature and cultivars (risse et al., 1990). depending on cultivar, seasonal variation and harvest maturity, postharvest decline in flesh firmness may compromise fruit quality within 14 days from harvest (kyriacou and soteriou, 2015). juice ph value slightly decreased during storage at 7 °c for 21 days and additional 7 days at 21 °c. similarly, lower ph values were reported in ‘charleston gray’ watermelons fruits after storage at 7 °c for 14-19 days (chisholm and picha, 1986) and ‘fantasy f1’ watermelons after storage at 20 °c for 14 days (radulović et al., 2007) compared to the ph values at harvest. in cr cultivar, non-grafted fruits had higher ph comparing to grafted fruits. in ct cultivar, fruits on rs841 rootstock resulted in lower ph than those on other rootstocks and non-grafted fruits after shelf life period following sto rage (tab. 3). grafting of mini-watermelons on commercial hybrid rootstock ps 1313 had no effect on juice ph (proietti et al., 2008) while grafted ‘crimson tide’ watermelons on some local bottle gourd rootstocks had lower juice ph, compared to control (çandir et al., 2013). ta content slightly increased in parallel with changes in juice ph during storage at 7 °c for 21 days and additional 7 days at 21 °c for both cultivars (tab. 3). in cr cultivar, fruits on rs841 and ferro rootstocks had higher ta than those on other rootstocks and nongrafted fruits after 7 days at 21 °c following 21 days storage at 7 °c. in ct cultivar, fruits on rs841 rootstock resulted in higher ta than those on other rootstocks and non-grafted fruits after shelf life period following storage (tab. 3). higher ta due to grafting was reported in watermelon fruits (proietti et al., 2008; çandir et al., 2013). the malic acid content varied from 0.23 % to 0.28 % for cr cultivar and 0.23 % to 0.31 % for ct cultivar and the citric acid content varied fig. 3: effects of rootstocks on fungal decay of ‘crisby’ and ‘crimson tide’ watermelon fruits after shelf life period for 7 days at 21 °c following storage at 7 °c fig. 2: effects of rootstocks on weight loss of ‘crisby’ and ‘crimson tide’ watermelon fruits after shelf life period for 7 days at 21 °c following storage at 7 °c storage and shelf life of grafted watermelons 195 tab. 2: effects of rootstocks on some quality attributes of ‘crisby’ (cr) and ‘crimson tide’ (ct) watermelon fruits after shelf life period for 7 days at 21 °c following storage at 7°c scion / rootstock days in storage at 7 °c +7 days at 21 °c mean 0+7 7+7 14+7 21+7 (rootstock) firmness (n) cr 6.16c 5.47c 5.25c 3.99c 5.22e control 6.31bc 6.24b 5.62c 5.13b 5.83d macis 6.86b 6.44b 6.33b 5.54b 6.29c argentario 7.65a 7.63a 7.40a 7.09a 7.44a rs841 7.47a 6.90b 7.31a 5.83a 6.88b ferro 6.16c 5.47c 5.25c 3.99c 5.22e ct control 5.74b 5.60c 4.38e 4.81c 5.13e macis 6.06b 6.46bc 5.16d 4.79c 5.62d argentario 7.03a 6.49bc 6.05c 5.72b 6.32c rs841 7.16a 6.88b 6.72b 6.75a 6.88b ferro 7.35a 8.29a 7.40a 7.24a 7.57a ripening (1-7) cr 4.0a 3.4a 3.6a 3.7ab 3.7ab control 3.8a 3.6a 3.8a 3.7ab 3.7ab macis 3.8a 3.7a 3.9a 4.0a 3.9a argentario 3.4a 3.5a 3.5a 3.6b 3.5bc rs841 3.5a 3.1a 3.6a 3.5b 3.4c ferro 4.0a 3.4a 3.6a 3.7ab 3.7ab ct control 4.6a 5.2a 5.0a 5.2a 5.0a macis 4.4a 4.2b 4.4b 5.1a 4.5b argentario 3.3b 4.0b 3.9bc 4.7b 4.0cd rs841 3.7b 4.1b 4.3b 4.6b 4.2c ferro 3.2b 3.5b 3.7c 4.5b 3.7d tss(%) cr control 10.6c 10.1c 10.4b 10.3c 10.4d macis 10.2bc 10.3c 10.6b 10.1c 10.3d argentario 10.8b 10.6b 10.6b 10.5bc 10.6c rs841 10.9ab 10.8ab 11.0a 10.8ab 10.9b ferro 11.4a 11.0a 11.1a 11.0a 11.1a ct control 10.9a 11.0a 10.8a 11.1a 10.9a macis 10.6a 10.4a 10.5a 10.7a 10.5b argentario 10.1a 10.6a 10.7a 10.7a 10.5b rs841 10.9a 10.9a 10.8a 11.2a 11.0a ferro 10.3a 10.7a 10.8a 11.1a 10.7ab taste (1-9) cr 8.1a 6.9a 6.2b 5.3a 6.6c control 8.0a 7.0a 6.3b 5.7a 6.8bc macis 8.1a 7.3a 6.5ab 5.9a 7.0ab argentario 8.2a 7.5a 6.9a 6.1a 7.2a rs841 8.4a 7.2a 6.9a 5.9a 7.1a ferro 8.1a 6.9a 6.2b 5.3a 6.6c ct control 7.6b 7.3a 6.7c 5.8c 6.8b macis 7.9b 7.3a 7.1bc 5.4bc 6.9b argentario 8.3a 7.9a 7.6b 6.3b 7.5a rs841 8.2a 7.8a 7.2bc 6.7bc 7.5a ferro 8.2a 7.6a 8.3a 6.5a 7.7a x mean separation was performed by fisher’s lsd test. means (n=3) followed by same letters within a column are not significantly different at p<0.05. 196 a.e. özdemir, e. çandır, h. yetişir, v. aras, ö. arslan, ö. baltaer, d. üstün, m. ünlü tab. 3: effects of rootstocks on juice ph, ta and organic acid content of ‘crisby’ (cr) and ‘crimson tide’ (ct) watermelon fruits after shelf life period for 7 days at 21 °c following storage at 7 °c scion / rootstock days in storage at 7 °c +7 days at 21 °c mean 0+7 7+7 14+7 21+7 (rootstock) juice ph cr control 5.80a 5.60a 5.55a 5.70a 5.66a macis 5.67b 5.48a 5.60a 5.61b 5.59bc argentario 5.68b 5.57a 5.56a 5.61b 5.60bc rs841 5.59c 5.52a 5.62a 5.48c 5.55c ferro 5.63bc 5.53a 5.56a 5.44c 5.54c ct control 5.69a 5.57a 5.62a 5.59a 5.62a macis 5.60a 5.57a 5.60ab 5.60a 5.59a argentario 5.62a 5.44b 5.53c 5.48a 5.52b rs841 5.59a 5.45b 5.55bc 5,39b 5.50b ferro 5.61a 5.47b 5.44d 5.50a 5.50b ta (%) cr control 0.16b 0.18a 0.17a 0.17b 0.17bc macis 0.16b 0.17a 0.15a 0.17b 0.16c argentario 0.16b 0.17a 0.16a 0.16b 0.16c rs841 0.18a 0.19a 0.16a 0.19a 0.18a ferro 0.17ab 0.18a 0.17a 0.19a 0.18ab ct control 0.17a 0.18b 0.17b 0.18b 0.18b macis 0.17a 0.16c 0.15c 0.16c 0.16c argentario 0.16a 0.18b 0.17b 0.17bc 0.17bc rs841 0.17a 0.20a 0.19a 0.20a 0.17a ferro 0.17a 0.19a 0.17b 0.17bc 0.18b citric acid (%) cr control 0.07a 0.08a 0.04ab 0.06a 0.06a macis 0.07a 0.06a 0.03b 0.06a 0.06a argentario 0.08a 0.07a 0.05a 0.07a 0.07a rs841 0.08a 0.09a 0.05a 0.07a 0.07a ferro 0.09a 0.07a 0.05a 0.07a 0.07a ct control 0.10a 0.12a 0.10a 0.06bc 0.09a macis 0.09a 0.07b 0.05b 0.04c 0.06c argentario 0.09a 0.11ab 0.04b 0.04c 0.07b rs841 0.10a 0.12a 0.06b 0.08a 0.09a ferro 0.07a 0.14a 0.08a 0.07ab 0.09a malic acid (%) cr control 0.22b 0.25bc 0.23bc 0.26bc 0.24b macis 0.22b 0.24c 0.22c 0.28c 0.24b argentario 0.21b 0.23c 0.23bc 0.24c 0.23b rs841 0.25a 0.29a 0.27a 0.33a 0.28a ferro 0.25a 0.27ab 0.26ab 0.35ab 0.28a ct control 0.24a 0.27c 0.25bc 0.24b 0.25bc macis 0.23a 0.25c 0.23c 0.21c 0.23c argentario 0.25a 0.38ab 0.25bc 0.23bc 0.28ab rs841 0.24a 0.43a 0.28ab 0.29a 0.31a ferro 0.19a 0.36b 0.31a 0.28a 0.29b ax mean separation was performed by fisher’s lsd test. means (n=3) followed by same letters within a column are not significantly different at p<0.05. storage and shelf life of grafted watermelons 197 from 0.06 % to 0.07 % for cr cultivar and 0.06 % to 0.09 % for ct cultivar after shelf life period following storage (tab. 3). the citric acid content was not affected by grafting for cr. in ct, fruits grafted on rs841 and ferro had higher citric acid content after shelf life period for 7 days at 21° following 21 days of storage at 7 °c, compared to other graft combination and control. malic acid was the predominant organic acid for both cultivars. compared to the control and fruits from other graft combination, watermelons grafted on rs841 rootstock had higher malic acid content for cr and ct cultivars during storage at 7 °c for 21 days and additional 7 days at 21 °c. we found a slight increase in ripening ratings during storage at 7 °c for 21 days and additional 7 days at 21 °c for both cultivars (tab. 2), indicating fruits became overripe toward the end of 21 days of storage and subsequent shelf life period. similar findings were reported by risse et al. (1990) for several watermelon cultivars during 4 weeks of storage at 5°, 10°, 15° or 20 °c. fruits grafted on rs841 and ferro rootstocks for cr cultivar and fruits grafted on rs841, argentario and ferro rootstocks for ct cultivar had the lowest ripening ratings after shelf life period following storage (tab. 2). ripening could be retarded by grafting in watermelon fruits at harvest. roberts et al. (2007) suggested that grafting may delay the harvest date by about 7 days. soteriou et al. (2014) found that as grafting retarded the ripening process, optimum harvest maturity in non-grafted plant was reached at 35-40 days post-anthesis (dpa) compared with 40-45 dpa in grafted plants. tss content remained above 10 % throughout during storage at 7 °c for 21 days and additional 7 days at 21 °c for both cultivars (tab. 2), rendering fruit acceptable for perceived sweetness as reported by (kyriacou and soteriou (2015). in, cr cultivar, fruits grafted on ferro rootstock had higher tss content after shelf life period for 7 days at 21° following 21 days of storage at 7 °c, compared to other graft combination and control (tab. 2). in case of ct cultivar, control and grafted fruits had similar tss content after shelf life period following storage (tab. 2). although some previous studies (miguel et al., 2004; proietti et al., 2008; bruton et al., 2009; balazs et al., 2011; bekhradi et al., 2011; soteriou and kyriacou, 2015) showed that grafting had no effect on tss, grafting on the bottle gourd rootstocks increased tss contents of watermelon fruits compared to the control (salam et al., 2002; karaca et al., 2012; çandir et al., 2013). unlike our findings, in other studies, grafted watermelon fruits had lower tss content compared to non-grafted controls (davis and perkins-veazie, 2005; roberts et al., 2007; kyriacou and sote riou, 2015). yetisir et al. (2003) reported that quality (brix, firmness, rind thickness, and fruit shape) of watermelon was greatly affected by grafting, but the results were dependent on the rootstock used. the differences in reported results may be due in part to different production environments, type of rootstock, interactions between specific rootstocks and scions, and harvest date (davis et al., 2008). the most abundant sugar was sucrose at harvest and during storage at 7 °c for 21 days and additional 7 days at 21 °c in both cultivars as reported previously (chisholm and picha, 1986; kyriacou and soteriou, 2015). in general, changes in on total soluble solid, total and individual sugar contents were not significant during storage and shelf life. in contrast to our findings, in previous studies, it was reported an accumulation of sucrose accompanied the decline in total soluble carbohydrates and soluble solids content in grafted and nongrafted watermelons during storage for 14 days at 25 °c (kyriacou and soteriou, 2015) and a significant decrease soluble solids and total sugar contents of watermelons during storage for 14 days at 20 °c (radulović et al., 2007). in another study showed that soluble solid content, sucrose, glucose, and fructose concentrations of watermelons mostly did not change during storage for 14 days at 0 °c plus 5 days at 23 °c, but all generally were reduced at higher storage temperatures (chisholm and picha, 1986). similarly, total soluble solid content of watermelon cultivars decreased with increased storage temperature (risse et al., 1990; perkins-veazie and collins, 2006). in our study, preservation of sugars at lower storage temperature may be attributed to a presumably lower rate of respiration. in cr cultivar, effect of grafting on total and individual sugar contents was not significant after shelf life period following storage (tab. 4). in ct cultivar, although sucrose and total sugar contents were not affected by grafting, fructose and glucose content were higher in fruits grafted on rs841, ferro and argentario rootstocks than those on macis and non-grafted fruits after shelf life period for 7 days at 21° following 7 days of storage at 7 °c (tab. 4). the differences in fructose and glucose contents between grafted and non-grafted fruits disappeared afterwards. in some studies, grafted watermelon fruits had lower (yetisir et al., 2003; davis and perkins-veazie, 2005; roberts et al., 2007) or similar (miguel et al., 2004; proietti et al., 2008; bruton et al., 2009; bekhradi et al., 2011) sugar content compared to non-grafted controls. similarly, kyriacou and soteriou (2015) reported that between the hybrid rootstocks, mean suc rose concentration was undifferentiated. in some studies, grafting on the local bottle gourd rootstocks increased fructose, glucose, and sucrose contents of ‘crimson tide’ watermelon fruits compared to the control and commercial rootstock grafts (çandir et al., 2013). on the other hand, the fruits of the non-grafted bonta watermelon plants had higher sucrose content than the fruits from the grafted plants on the interspecific hybrid rootstock rs 841 and the lagenaria rootstock fr strong, while the reducing sugar content (glucose and fructose) showed an opposite pattern (balazs et al. 2011). taste scores (1-9) declined to the lowest level after shelf life period for 7 days at 21° following 21 days of storage at 7 °c (tab. 2). the effect of rootstock on the taste of watermelon fruits was found to be significant. as the storage time extended, taste tented to decrease, all of the fruit tested by the panelists received high taste scores of >7.9 out of 8.5 at the beginning and decreased to scores of >6.8 out of 7.7 at the end of shelf life, with the exception of non-grafted fruits and fruits grafted on macis rootstock, which had lower taste scores than the other rootstocks after shelf life period following storage for cr and ct cultivars. lower taste score may be related to becoming of overripe of control fruits and grafted fruits on macis rootstock. furthermore, no off-flavors were detected in fruit from grafted plants. the similar results were obtained in another study conducted on the fruit from grafted watermelons (bruton et al., 2009). flesh color lightness (l* value) decreased during storage at 7 °c for 21 days and additional 7 days at 21 °c for both cultivars (tab. 5). similarly perkins-veazie and collins (2006) indicated darker (lower l* values) fruits after storage at 21 °c than in freshly harvested watermelons. grafting did not affect flesh color lightness during storage, but rs841and ferro rootstocks resulted in darker fruits after shelf life period for 7 days at 21° following 21 days of storage at 7 °c for both cultivars. kyriacou and soteriou (2015) reported that flesh color lightness (l*) of watermelon fruits was affected by rootstock and storage and compared to the non-grafted control, all hybrid rootstocks invariably maintained darker flesh color (lower lightness value) during storage. the overall intensity of flesh color (c* value), hue angle (h° value) and lycopene content were affected by storage time and rootstocks (tab. 5). watermelon flesh color changes from bright red (lower h°) to orange red (higher h°) as ripening level progresses (karaca et al., 2012). grafted and non-grafted fruits showed a progressive increase in h° value after shelf life period following storage, indicating a shift from red to orange-yellow. this changes in h° value, characteristic of over-ripening and senescence has been reported after prolonged postharvest storage of watermelons (kyriacou and soteriou, 2015). in both cultivars, c* value continuously decreased during shelf life period at 21 °c following storage at 7 °c. lycopene content peaked after shelf life period for 7 days at 21° following 7 days of storage at 7 °c for ct cultivars, but it tented to decrease for cr cultivars. fruits 198 a.e. özdemir, e. çandır, h. yetişir, v. aras, ö. arslan, ö. baltaer, d. üstün, m. ünlü tab. 4: effects of rootstocks on sugar contents of ‘crisby’ (cr) and ‘crimson tide’ (ct) watermelon fruits after shelf life period for 7 days at 21 °c following storage at 7 °c scion / rootstock days in storage at 7 °c +7 days at 21 °c mean 0+7 7+7 14+7 21+7 (rootstock) fructose (%) cr control 3.21a 3.10a 3.24a 3.46a 3.25a macis 3.22a 3.35a 3.08a 3.89a 3.39a argentario 2.94ab 3.43a 3.34a 3.43a 3.29a rs841 3.15a 3.31a 3.21a 4.23a 3.48a ferro 2.73b 3.57a 3.48a 3.90a 3.42a ct control 2.81ab 2.76b 2.67a 2.80a 2.76b macis 2.63b 2.83b 2.78a 2.70a 2.74b argentario 3.14a 3.42a 3.24a 3.00a 3.29a rs841 3.01a 3.79a 3.22a 3.01a 3.16a ferro 3.11a 3.74a 3.10a 3.34a 3.32a glucose (%) cr control 1.66a 1.64a 1.75a 1.75a 1.70a macis 1.66a 1.51a 1.63a 1.91a 1.68a argentario 1.55a 1.88a 1.81a 1.81a 1.76a rs841 1.54a 1.63a 1.67a 2.22a 1.76a ferro 1.32a 1.87a 1.85a 2.06a 1.77a ct control 1.79a 1.56c 1.62a 1.55a 1.63b macis 1.75a 1.63bc 1.68a 1.55a 1.65b argentario 1.73a 1.81bc 1.64a 1.61a 1.70b rs841 1.86a 1.86b 1.88a 1.78a 1.85a ferro 1.81a 2.16a 1.66a 1.80a 1.86a sucrose (%) cr control 4.47a 3.72a 3.89a 4.69a 4.19a macis 4.85a 4.31a 4.16a 4.65a 4.49a argentario 5.27a 4.33a 3.94a 5.26a 4.70a rs841 4.72a 4.41a 4.54a 3.57a 4.31a ferro 4.94a 4.06a 4.66a 4.12a 4.45a ct control 4.90a 5.39a 5.65a 4.81a 5.19a macis 4.47a 4.89a 4.77a 4.56a 4.67a argentario 4.35a 6.21a 4.76a 4.82a 5.04a rs841 4.86a 5.23a 4.39a 3.94a 4.61a ferro 4.27a 5.44a 4.51a 4.66a 4.72a total sugar (%) cr control 9.34a 8.45a 8.87a 9.89a 9.14a macis 9.73a 9.17a 8.87a 10.45a 9.55a argentario 9.76a 9.64a 9.08a 10.49a 9.74a rs841 9.40a 9.35a 9.42a 10.01a 9.54a ferro 8.97a 9.49a 9.99a 10.07a 9.63a ct control 9.50a 9.71b 9.93a 9.15a 9.57a macis 8.84a 9.34b 9.23a 8.81a 9.05a argentario 9.22a 11.85a 9.63a 9.42a 10.03a rs841 9.73a 10.45ab 9.49a 8.72a 9.60a ferro 9.18a 11.34a 9.27a 9.79a 9.90a x mean separation was performed by fisher’s lsd test. means (n=3) followed by same letters within a column are not significantly different at p<0.05. storage and shelf life of grafted watermelons 199 tab. 5: effects of rootstocks on color and total lycopene content of ‘crisby’ (cr) and ‘crimson tide’ (ct) watermelon fruits after shelf life period for 7 days at 21 °c following storage at 7 °c scion / rootstock days in storage at 7 °c +7 days at 21 °c mean 0+7 7+7 14+7 21+7 (rootstock) l* cr control 44.02a 45.50a 44.24a 44.04a 44.45ab macis 46.61a 46.77a 46.91a 42.29ab 45.65a argentario 44.84a 44.91a 43.48a 44.02a 44.31b rs841 44.99a 45.40a 43.29a 41.10b 43.70b ferro 43.08a 44.74a 43.60a 41.48b 43.23b ct control 44.87a 44.28a 43.70a 45.55a 44.60a macis 46.42a 43.96a 41.51a 44.36ab 44.06a argentario 44.87a 44.80a 44.72a 40.22c 43.65ab rs841 43.74a 43.02a 42.29a 41.52bc 42.64b ferro 44.76a 43.25a 41.73a 40.83c 42.64b c* cr control 32.75b 30.91b 26.95b 27.81b 29.61c macis 33.32b 30.86b 30.72a 31.40a 31.57b argentario 32.58b 34.26a 31.58a 31.29a 32.43ab rs841 35.76a 33.99a 31.74a 32.40a 33.47a ferro 35.86a 34.39a 31.37a 31.94a 33.39a ct control 35.27c 32.28a 15.49c 28.37c 27.85a macis 37.01bc 33.98a 16.15bc 30.16b 29.33a argentario 39.74a 36.33a 16.41bc 31.38ab 30.97a rs841 39.10ab 34.09a 16.94b 32.67a 30.70a ferro 37.96ab 35.27a 19.38a 31.68ab 31.07a h° cr control 44.58a 47.14a 47.54b 48.21a 46.87ab macis 44.27a 46.69ab 49.85a 47.62a 47.11a argentario 44.08a 46.68ab 46.25bc 47.36b 46.09b rs841 42.72b 44.12c 45.91c 45.04b 44.45c ferro 42.41b 45.44b 45.31c 44.34b 44.37c ct control 45.83a 46.33a 46.40a 45.62a 46.05a macis 44.38ab 44.09b 43.69b 47.56a 44.93ab argentario 42.72bc 43.72bc 42.95b 46.92a 44.08bc rs841 42.41bc 42.14b 43.15b 42.27b 42.50d ferro 42.34c 42.58c 42.30b 45.71a 43.23cd lycopene (μg g-1) cr control 32.06c 30.12b 25.03c 20.36c 26.89c macis 34.65bc 30.53b 22.34d 24.04d 27.89c argentario 40.47ab 31.80b 25.94a 26.06a 31.07b rs841 44.65a 38.04a 29.32b 33.18b 36.30a ferro 43.19a 36.36a 32.62a 29.75a 35.48a ct control 35.42a 39.95c 27.60d 26.81c 32.44b macis 36.91a 40.42bc 34.36c 27.61c 34.82b argentario 42.99a 43.83abc 40.23ab 33.55b 40.15a rs841 39.05a 44.55ab 35.78bc 40.09a 39.87a ferro 42.41a 47.35a 42.18a 36.86ab 42.20a x mean separation was performed by fisher’s lsd test. means (n=3) followed by same letters within a column are not significantly different at p<0.05. 200 a.e. özdemir, e. çandır, h. yetişir, v. aras, ö. arslan, ö. baltaer, d. üstün, m. ünlü grafted on rs841, argentario and ferro rootstocks had more intense (higher c*) brighter red (lower h° value) color and higher lycopene content after shelf life period following storage for both cultivars, compared to non-grafted fruits (tab. 5). in agreement with our results kyriacou and soteriou (2015) reported that h° value increased at 14 days of storage indicating yellowing of the flesh and non-grafted control watermelon fruits presented a greater postharvest transition to yellow than the hybrid rootstocks during storage at 25 °c. postharvest color changes and lycopene biosynthesis in watermelons can be affected by storage temperature and cultivar. perkins-veazie and collins (2006) reported that watermelons stored at 21 °c had increased c* value and lycopene content compared to fresh fruit whereas no or little change was observed in c* value and lycopene content of fruit held at 5 °c or 13 °c depending on cultivars. the c* value of grafted and non-grafted watermelon fruits peaked after 7 days at 25 °c was not affected by rootstock. however, the intensity of red color in particular, expressed by component a*, was higher in fruits on rootstocks “tz148” and “n101” than control fruits (kyriacou and soteriou, 2015). consistent with our results, previous studies have typically shown higher lycopene content in watermelon fruit from grafted plants at harvest (davis and perkins-veazie, 2005; davis et al., 2008; proietti et al., 2008; çandir et al., 2013) and during storage (kyriacou and soteriou, 2015). in the current study, changes in lycopene content supports changes in grafted and nongrafted cr and ct watermelon fruits flesh color after shelf life period following storage. the increase in lycopene content, the dominant pigment in watermelon, most likely contributed to the increased c* value as reported by (perkins-veazie and collins, 2006). degradation in lycopene during senescence of non-grafted watermelon fruits of both cultivar and grafted cr fruits after prolonged storage and consequent shelf life period led to decrease in c* and h° value. although grafted and non-grafted watermelons held for 14 days at 25 °c developed a yellowing of flesh (kyriacou and soteriou, 2015), we did not observed any yellowing of flesh in grafted or non-grafted fruit held for 7 days at 21° following 21 days of storage at 7 °c. flesh color changes was observed in non-grafted fruit, suggesting that fruit ripening occurs faster in non-grafted than in grafted fruit during shelf life period after storage. ripening ratings also confirmed these changes in non-grafted fruits which became overripe toward the end of storage and shelf life. β-carotene content did not significantly changed and was not affected by grafting during storage at 7 °c for 21 days and additional 7 days at 21 °c (data not presented). perkins-veazie and collins (2006) reported that watermelons stored for 14 days at 21 °c gained 50-139 % in β-carotene compared to fresh fruit, whereas fruit held at 5 and 13 °c changed little in β-carotene content. in our study, lower storage temperature may suppress increase in β-carotene content. in agreement with our results, a similar β-carotene content was reported between fruits grafted on some local bottle gourd rootstocks and nongrafted fruits (çandir et al., 2013). effects of grafting on hallow heart was not significant during storage at 7 °c for 21 days and additional 7 days at 21 °c for both cultivars (data not presented). cushman and huan (2008) reported a higher rate of hollow heart incidence in non-grafted watermelon plants than in those that had been grafted. this indicates that hollow heart is affected not only by rootstocks but also by other environmental and cultural conditions. many conflicting results have been reported on the changes in fruit quality resulting from grafting (salam et al., 2002; lee and oda, 2003; davis and perkins-veazie, 2005). bruton et al. (2009) observed a field and year effect due to soil and climatic conditions on watermelon quality parameters besides rootstock effect. the differences observed in previous studies may be explained by different production conditions, the type of rootstock/scion combinations used, or the harvest date. since flowering and harvest time are influenced by grafting, the duration of fruit harvest is prolonged, and the number of fruits per plant is increased in grafted plants (yetisir and sari, 2003), it is often difficult to harvest fully ripe fruit from grafted plants in large-scale watermelon production where all fruits are harvested in a single harvest. watermelons could successfully be kept for 21 days at 7 °c and additional 7 days at 21 °c. watermelons grafted on ferro and rs841 rootstocks had higher postharvest quality compared to the control for both cultivars. acknowledgments this research was supported by the scientific and technological research council of turkey (tubitak 108 o 391). the authors wish to thank the tubitak for their financial support. references alexopoulos, a.a, kondylis, a., passam, h., 2007: fruit yield and quality of watermelon in relation to grafting. j. food agric. environ. 5, 178-179. alan, ö., özdemir, n., günen, y., 2007: effect of grafting on watermelon plant growth, yield and quality. j. agron. 6, 362-365. balazs, g., kappel, n., fekete, d., 2011: the rootstock effect in the hungarian watermelon production. in: pospišil, m. 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(ed.), proceedings of the 2007 southeast regional vegetable conference, 33-36, savannah, georgia. rushing, j.w., fonseca, j.m., keinath, a.p., 2001: harvesting and postharvest handling. in: maynard, d.n. (ed.), watermelons − characteristics, production, and marketing, 156-164, alexandria, va, american society for horticultural science press. salam, m.a., masum, a.s.m.h., chowdhury, s.s., dhar, m., saddeque, a., islam, m.r., 2002: growth and yield of watermelon as influenced by grafting. online j. biol. sci. 2, 298-299. sas, 1999: sas/stat user’s guide, version 8. sas institute inc., cary, nc, usa. soteriou, g.a., kyriacou, m.c., 2015: rootstock mediated effects on watermelon field performance and fruit quality characteristics. int. j. veg. sci. 21, 344-362. soteriou, g.a., kyriacou, m.c., siomos, a.s., gerasopoulos, d., 2014: evolution of watermelon fruit physicochemical and phytochemical composition during ripening as affected by grafting. food chem. 165, 282-289. suslow, t.v., 1997: watermelon, recommendations for maintaining postharvest quality, http://rics.ucdavis.edu/postharvest2/produce/producefacts/ fruit/watermelon. shtml, accessed 14.10.2015. yamasaki, a., yamashita, m., furuya, s., 1994: mineral concentrations and cytokinin activity in the xylem exudate of grafted watermelons as affected by rootstocks and crop load. j. japan soc. hort. sci. 62, 817826. yetisir, h., sari, n., 2003: effect of different rootstock on plant growth, yield and quality of watermelon. aust. j. exp. agric. 43, 1269-1274. yetişir, h., sari, n., yücel, s., 2003: rootstock resistance to fusarium wilt and effect on watermelon fruit yield and quality. phytoparasitica 31, 163-169. yetisir, h., sari, n., 2004: effect of hypocotyl morphology on survival rate and growth of watermelon seedlings grafted on rootstocks with different emergence performance at various temperatures. turk. j. agric. for. 28, 231-237. address of the authors: assoc. prof. dr. ahmet erhan özdemir, prof. dr. elif çandır (corresponding author), özay baltaer, durmuş üstün, department of horticulture, mustafa kemal university, 31034 antakya, hatay, turkey. e-mail: ecandir@mku.edu.tr prof. dr. halit yetişir, department of horticulture, erciyes university, 38039 melikgazi, kayseri, turkey. veysel aras, mustafa ünlü, alata horticultural research institute, 33740 erdemli, mersin, turkey ömer arslan, agriculture county directorate, silifke, mersin, turkey © the author(s) 2016. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 89, 29 37 (2016), doi:10.5073/jabfq.2016.089.004 1croatian centre for agriculture, food and rural affairs, institute of pomology, zagreb, croatia 2department of food technology, faculty of food technology and biotechnology, zagreb, croatia 3department of pomology, faculty of agriculture, zagreb, croatia influence of four different dwarfing rootstocks on phenolic acids and anthocyanin composition of sweet cherry (prunus avium l.) cvs. ‘kordia’ and ‘regina’ bernardica milinović1, verica dragović-uzelac2, dunja halapija kazija1, tvrtko jelačić1, predrag vujević1, danijel čiček1, ante biško1, zlatko čmelik3 (received september 24, 2015) summary influence of four different dwarfing rootstocks: ‘gisela 5’, ‘gisela 6’, ‘phl-c’ and ‘piku1’ on the amount of polyphenols (total phenols, total and individual anthocyanins and individual hydroxy cinnamic acids) of sweet cherry cultivars ‘kordia’ and ‘regina’ was researched as well as correlations between polyphenols and colorimetrically measured fruit skin colour. total phenols (tp) and total anthocyanins (ta) were determined by spectroscopic methods, while individual anthocyanins and hydroxycinnamic were quantified and identified by high performance liquid chromatography (hplc). ‘gisela 6’ and ‘piku1’ had the strongest influence on the tp content of both cultivars. the main hydroxycinnamic acid identified was p-coumaric acid (p-ca) which content was influenced by cultivar, vegetation year and interaction of cultivar*rootstock*year. content of p-ca varied the most on rootstock ‘gisela 5’ for both cultivars, with its lowest amount obtained in 2013 and the highest in 2014 in comparison to other rootstocks. the largest difference in ta content in cultivar*rootstock interaction was identified in ‘gisela 6’. substantial difference in correlation pattern between cvs. ‘kordia’ and ‘regina’ lead to the conclusion that values l* and b* are better ta indicators rather than values a* and hue. introduction due to exceptional organoleptic qualities, market demand for cherries is growing worldwide, which has resulted with significant improvements in production and marketing (sansavini and lugli, 2008). similar to apple, modern sweet cherry production cannot be conceived without the use of dwarfing rootstocks. in selection of suitable sweet cherry rootstocks, the emphasis is given to their compatibility with majority of cultivars in use and adaptability to different agro-ecological conditions (miljković et al., 2002). the use of dwarfing rootstocks can be limited due to incompatibility issues connected with grafting of scion of different genetic origin (usenik et al., 2010). incompatibility is a complex, anatomical, physiological and biochemical process, not yet fully explained (guclu and koyuncu, 2012). with respect to grafted fruit trees incompatibility is defined as a phenomenon of premature senescence caused by physiological and biochemical processes (feucht and treutter, 1991). usenik and štampar (2000) found that low compatibility resulted in a pronounced accumulation of polyphenols, namely pcoumaric acid above the graft union of cv. ‘lapins’ grafted on different rootstocks (f 12/1, gisela 5, weiroot 158), as a stress response to grafting. higher content of p-coumaric acid above the graft point were found in apricot cultivars grafted on heterospecific rootstocks which led to an early graft incompatibility (usenik et al., 2006). mng’omba et al. (2008) have found that phenols, especially pcoumaric acids and anthocyanins caused poor callus formation at the union, and hence are implicated in graft incompatibility of wild loquat (uapaca kirkiana müell arg.). fruit quality depends mainly on the scion genotype, but could be influenced by the rootstock (scalzo et al., 2005; tavarini et al., 2011) and climatic conditions as well. different studies with prunus sp. have indicated that rootstock affects vegetative growth, fruit quality and yield efficiency of grafted cultivar (betrán et al., 1997; čmelik and družić-orlić, 2008). combined with fruit sweetness (soluble solids content), weight and firmness, fruit skin colour affects significantly consumer acceptance of sweet cherry cultivars as well (spinardi et al., 2005; fazzari et al., 2008). development of red colour in sweet cherry is often used as a ripeness indicator and is directly related with the content and concentration of plant pigments anthocyanins (usenik et al., 2015). major anthocyanins identified in sweet cherries responsible for their red colour are cyanidin-3-oglucoside and cyanidin-3-o-rutinoside, while peonidin-3-o-rutinoside and pelargonidin-3-o-rutinoside are present in smaller amounts (kelebek and selli, 2011). previous studies on anthocyanins and colour change in sweet cherries revealed that chromatic parameters l*, a*, b* and hue angle correlated negatively with total anthocyanin levels in sweet cherry (mozetič et al., 2004; gonçalves et al., 2007) and weakly correlated in plums (usenik et al., 2009). colorimetric ripeness estimation of sweet cherries is considered to be effective since strong correlation coefficient between a* and cyanidin-3-rutinoside was established (goncalves et al., 2007). research done by pedisić et al. (2009) on sour cherries indicated that correlation between fruit skin colour parameters and total anthocyanins are cultivar dependant as lower l* values were measured on cultivars with darker colored fruits. identification and quantification of polyphenols in a bark bellow and above graft point in different rootostock/cultivar combination is expensive and long procedure (usenik and štampar, 2000; usenik et al., 2006). it is assumed that higher content of polyphenols involved in incompatibility proces could be identified in fruits as well. higher content of hidroxycinnamic acids, namely neochlorogenic and chlorogenic acids, p-coumaric derivatives was determined in fruits of cv. ‘lapins’ grafted on ‘piku 1’ roostock than on ‘gisela 5’ (jakobek et al., 2009). the objectives of this research was to determine the influence of four different dwarfing rootstocks: ‘gisela 5’, ‘gisela 6’, ‘phl-c’ and ‘piku 1’ on the accumulation of polyphenols (total and individual hydroxycinnamic acids, total and individual anthocyanins) in fruits of sweet cherry cultivars ‘kordia’ and ‘regina’, and to find correlations between polyphenols and colorimetrically measured fruit skin colour. material and methods plant material the research was conducted in experimental orchard of the institute of pomology of the croatian centre for agriculture, food and rural affairs in donja zelina, near zagreb. experimental orchard is located on 180 m above mean sea level (amsl), 45° 55´ north 30 b. milinović, v. dragović-uzelac, d.h. kazija, t. jelačić, p. vujević, d. čiček, a. biško, z. čmelik latitude, 16° 14´ east longitude. the soil in orchard is described as albic stagnosol. the area is characterized by average annual temperature of 10.7 °c, and 855.1 mm of total rainfall. significant deviation was not recorded in average daily temperature during 2013, while total rainfall was slightely higher than average (970.6 mm). slightly higher average daily temperatures were recorded in april (13.3 °c), june (20.7 °c) and in july 2013 (24.0 °c). siginificantly higher precipitation than average were recorded in 2014 (up to 85 %), or precisely in february (279 mm) and june (136 mm). in the first quarter of 2014, significantly higher than average temperature were recorded as well (5.6 °c in january; 5.0 °c in february and 10.8 °c in march). the orchard is drip-irrigated with installed black anti-hail nets. conventional orchard management practices are implemented (pruning, fertilization, pest control). the trial was conducted on two sweet cherry cultivars ‘kordia’ and ‘regina’ grafted on four dwarfing rootstock: ‘gisela 5’, ‘gisela 6’, phl-c’ and ‘piku1’. genetic origin of tested rootstocks is shown in the tab. 1. trees were planted in a random block design: two trees in 3 repetitions (for each rootstock and cultivar combination). the two-year old trees were planted in 2006, at 4 × 2.5 m distance and trained as a spindle bush. absorbance of the solution was measured by the spectrophotometer unicam helios b (spectronic unicam, cambridge, uk) at 765 nm. the results were calculated according to the calibration curve for gallic acid (y = 0.0009x, y = absorbance at 765 nm, x = concentration of gallic acid in mg/l, r2 = 0.9986). the content of tp was expressed as mg of gallic acid equivalents (gae) per 100 g of fresh mass (fw) of edible part of fruits. the monomeric anthocyanin pigment content of the aqueous extracts was determined using the ph-differential method (giusti and wrolstad, 2001). a spectrophotometer unicam helios b (spectronic unicam, cambridge, uk) was used for spectral measurements at 520 and 700 nm. pigment content was calculated as milligrams cyanidin-3-glucoside/100 g fw using an extinction coefficient of 26900 l/cm/mol and molecular weight of 449.2 g/mol. hplc analysis of anthocyanins and hydroxycinnamic acids hplc analysis of anthocyanins and hydroxycinnamic was performed using agilent 1260 quaternary lc infinity system (agilent technologies, santa clara, ca, usa) equipped with uv/vis diode array detector (dad), an automatic injector and chemstation software. the separation of phenolic compounds (anthocyanins and hydroxycinnamic acid) was performed on a nucleosil 100-5c18, 5 μm (250 × 4.6 mm i.d.) column (macherey-nagel). the mobile phase composition and the used gradient conditions were described previously by mitić et al. (2012) and modified by zorić et al. (2014), instead of 5 % solvents contained 3 % of formic acid. for gradient elution, mobile phase a contained 3 % of formic acid in water, mobile phase b contained 3 % of formic acid in 80 % acetonitrile. the elution was as follows: from 0 to 28 min, 0 % b, from 28 to 35 min, 25 % b, from 35 to 40 min, 50 % b, from 40 to 45 min, 80 % b and finally for the last 10 min again 0 % b. operating conditions were: constant flow rate 0.8 ml/min, column temperature 22 °c, injection volume 5 μl and equilibration time 2 minutes. detection was performed with uv/vis-dad by scanning from 220 to 570 nm. identification of anthocyanins and hydroxycinnamic acids was carried out by comparing retention times and spectral data with those of the authentic standards (anthocyanins were identified at 520 nm, and hydroxycinnamic acids at 280 nm). the quantifications of anthocyanins, and hydroxycinnamic acids were made by the external standard method. anthocyanin standards, cyanidin-3-glucoside (cy-3-g), cyanidin-3-rutinoside (cy-3-r), were prepared as stock solutions in acidified methanol (1 % of formic acid (v/v) in methanol) at a concentration of 100 mg/l. standards solutions of anthocyanins were prepared by diluting the stock solution to yield five concentrations in a range from 16.67 to 100 mg/l. quantitative determination was carried out using the calibration curves of the standards cy-3-g: y = 21.952x, r2 = 0.99; cy-3-r: y = 14.969x, r2 = 0.99. standards of hydroxycinnamic acids were prepared as stock solutions in 80 % methanol (v/v) at following concentrations: chlorogenic (cha); p-coumaric acid (p-ca) 48 mg/l. standard solutions were prepared by diluting the stock solution to yield five concentrations in the range from 10.4 to 52 mg/l for cha and p-ca, from 9.6 to 48 mg/l for p-ca. quantitative determination was carried out using the calibration curves of the standards (cha: y = 14.571x, r2 = 0.99; p-ca: y = 10.729x, r2 = 0.99;). for neochlorogenic acid (ncha) lacking reference standard, identification was done according to cha, and p-coumaroylquinic acid according to p-ca. determination of colour parameters fruit skin colour measurements were carried out with portable konica minolta spectrophotometer cm-700d by using cie l*a*b* system. value l* represents lightness i.e. illumination from 0 (total tab. 1: tested rootstocks and their origin rootstock origin gisela 5, gisela 6 p. cerasus l. × p. canescens, germany piku 1 p. avium l. × (prunus canescens × prunus tomentosa), germany phl-c p. avium l. × p. cerasus l., czech republic at optimal picking maturity (determined by colour and organoleptically), samples were harvested from each tree in the trial. content of soluble solids in 2013 was in average for cv. kordia 16.17 °brix and for cv. regina 18.63 °brix, whereas in 2014 cv. kordia obtained 16.95 °brix and cv. regina 17.12 °brix. each sample for cultivar and rootstock combination was consisted of 360 fruits (60 fruits taken from each tree in the trial). fruits were taken from the middle part of the tree crown. harvesting of fruit samples for cv. ‘kordia’ in 2013 started on june 28, and in 2014 on june 16, and for cv. ‘regina’ on july 2 in 2013 and on june 23 in 2014. colour and polyphenol analysis o sweet cherry fruit samples was performed immediatelly after the harvest. analysis of total phenols and total anthocyanins phenolics were extracted from homogenized pitted fresh sweet cherry fruits. exactly 5 g of samples were weighed out and extracted using 20 ml of 80 % (by volume) aqueous methanol. the mixture was extracted for 20 min in ultrasonic bath at 50 °c, filtered through whatman no. 40 filter paper (whatman international ltd, kent, uk) using a büchner funnel. the filtrates were adjusted to 25 ml in a volumetric flask with 80 % aqueous methanol. the obtained extract was used for determination of total phenols (tp), total (ta) and individual anthocyanins and individual hydroxycinnamic acids (hca). for the determination of total phenols (tp), the adjusted method (ough and amerine, 1988; singleton and rossi, 1965) with folin-ciocalteu reagent was used. the content of tp was measured as follows: 0.25 ml of sample, 15 ml distilled water (dh2o), 1.25 ml folin-ciocalteu reagent (diluted with distilled water in 1:2 ratio) were added to a 25-ml volumetric flask containing and shaken. to the mixture 3.75 ml of saturated na2co3 solution (m/v) were added with mixing and the solution was immediately filled up to 25 ml with ddh2o. after incubation at 50 °c for 20 min, the dwarfing rootstock influence on sweet cherry polyphenols 31 dark or black) to 100 (total transparency or white). value a* measures redness/greenness (+a* represents the red, while -a* represents green). value b* measures yellowness/blueness (+b* represents yellow, while -b* represents blue) (hutchings, 1994). hue angle (°), hue = arctg (b*/a*), represents the hue of the colour (voss, 1992) which values are defined as follows: red pink: 0°, yellow: 90°, bluish green: 180° and blue: 270° (mcguire, 1992). chroma was calculated using the following formula: c = (a*2 + b*2)1/2, and it is a measure for chromaticity (c*), and represents the purity or colour saturation (voss, 1992). statistical analysis repeated measures analysis of variance (anova) in two replicates for cultivar * rootstock * year using the statistical software statistica 10.0 (stat soft, inc., usa) provided estimates of varietal, roostock and annual differences. standard error was determined using fisher lsd test with a 0.01 level of significance. correlation between fruit skin colour parameters and total and individual phenolic was determined by pearson’s r coefficient correlation. results and discussion fruit weight, trans cross-sectional area, yield efficiency index and phenolic content influence of rootstocks on the fruit quality of sweet cherry was studied intensively in past years due to large number of dwarfing rootstocks that can be found on the market. research was mainly done on one cultivar being grafted on several rootstocks (usenik et al., 2000; jakobek et al., 2009), or two or more cultivars grafted on one rootstock (spinardi et al., 2005). sweet cherries in our research were grafted on four different rootstocks which in our growing conditions influenced the vigour (measured as cross section of a tree trunk), yield efficiency and fruit weight of cvs. ‘kordia’ and ‘regina’ (tab. 2). both cultivars developed the smallest trees on gisela 5 rootstock, medium sized on piku1 and the highest on phl-c. rootstock influence on cultivars can be noticed at gisela 6, on which cv. ‘kordia’ produced trees of similar size as gisela 5 whereas cv. ‘re gina’ developed trees of similar size as phl-c. difference in maturity timing between rootstocks was not observed for neither of cultivars. fruit weight was the highest on phl-c for both cultivars but varied in other rootstocks. for cv. ‘kordia’ it decreased as follows: gisela 5 > gisela 6 > piku1, and for cv. ‘regina’ as follows: piku1 > gisela 6 > gisela 5 (tab. 2). special attention was made to harvest samples in optimal ripening stage for each cultivar to accurately evaluate influence of different rootstock on fruit quality. content of total phenols (tp) was influenced by rootstock, vegetation year and their mutual interaction as well as their interaction with tab. 2: effect of rootstocks on trans cross-sectional area (tcsa), yield efficiency index (yie) and fruit weight of cvs ‘kordia’ and ‘regina’ cultivar rootstock tcsa yei fruit weight (cm2) (%) (g) kordia gisela 5 57,75 ad 0,20 ab 9,13 a gisela 6 66,4 ab 0,21 ab 8,45 c phl-c 89,61 c 0,15 abc 9,72 b piku1 70,77 ab 0,24 b 7,84 d regina gisela 5 52,68 d 0,16 abc 8,42 c gisela 6 88,11 c 0,13 ac 9,48 ab phl-c 96,40 c 0,02 d 10,31 e piku1 72,49 b 0,09 cd 9,50 ab p 0,021 0,000 0,000 for each cultivar, means having the same letter in each column are not significantly different at p ≤ 0.01 by fisher lsd test tab. 3: phenolic content (mg/100 g fw) of sweet cherry cvs ‘kordia’ and ‘regina’ grafted on 4 different rootstocks*, average of 2 years cultivar rootstock cha ncha p-ca cy-3-g cy-3-r ta tp kordia gisela 5 1.90 ± 0.65 9.13 ± 1.46 10.53 ± 0.89 3.45 ± 1.04 24.80 ± 16.87 34.41 ± 6.07 175.61 ± 21.69 gisela 6 1.66 ± 0.27 9.49 ± 4,44 12.35 ± 5.65 3.89 ± 1.11 32.19 ± 5.38 36.78 ± 6.68 198.48 ± 47.67 phl-c 1.56 ± 0.29 8.88 ± 3.58 11.59 ± 4.78 3.46 ± 1.17 27.82 ± 7.49 39.85 ± 13.03 183.37 ± 41.14 piku1 1.76 ± 0.25 9.60 ± 4.65 10.67 ± 4.66 2.34 ± 0,.53 26.34 ± 3.63 33.18 ± 4.51 156.55 ± 25.13 regina gisela 5 1.48 ± 0.19 9.73 ± 3.16 10.21 ± 5.12 2.28 ± 1.51 23.33 ± 7.18 26.19 ± 12.98 149.45 ± 18.99 gisela 6 1.17 ± 0.52 8.18 ± 2.43 8.72 ± 2.63 1.17 ± 1.40 15.55 ± 6.21 17.53 ± 8.53 137.40 ± 35.92 phl-c 1.44 ± 0.39 9.77 ± 2,52 10.27 ± 3.27 1.76 ± 0.79 19.90 ± 4,.26 23.14 ± 9.66 198.54 ± 87.33 piku1 1.02 ± 0.57 7.71 ± 2,86 8.02 ± 1.82 1.42 ± 1.64 15.86 ± 7.50 20.74 ± 13.55 233.46 ± 156.73 p value cultivar 0.001 ns 0.006 0.000 0.000 0.000 ns rootstock ns ns ns 0.072 0.005 0.000 0.000 year ns 0.000 0.000 0.000 0.020 0.000 0.000 cultivar*rootstock ns 0.036 ns ns ns 0.000 0.000 cultivar*year 0.012 ns ns 0.001 0.000 0.000 0.000 rootstock*year 0.015 ns ns ns ns 0.000 0.000 cultivar*rootstock*year ns 0.024 0.009 ns 0.030 0.000 0.000 *values are expressed as the mean ± standard deviation; significant differences at p ≤ 0.05; ns = not significant (p > 0.05) cha – chlorogenic acid; ncha – neochlorogenic acid; p – coumaroylquinic acid; cy-3-g – cyanidin 3-glucoside; cy-3-r – cyanidin 3-rutinoside; ta – total anthocyanins, tp – total phenols 32 b. milinović, v. dragović-uzelac, d.h. kazija, t. jelačić, p. vujević, d. čiček, a. biško, z. čmelik cultivar (tab. 3) which is line with the results obtained by jakobek et al. (2009). also, previous studies reported that tp content in sweet cherries can range from 99.00 109.8 mg/100 gfw (kim et al., 2005; ferreti et al., 2010). our research resulted in higher tp content for both cultivars. rootstocks gisela 6 and piku1 had the strongest influence on the tp content of both cultivars, followed by gisela 5, and these differences where statistically significant (p ≤ 0.01). difference in tp content of both cultivars on phl-c was not significantly different. jakobek et al. (2009) also identified that cv. ‘lapins’ had higher total phenol content grafted on heterogenic rootstocks, with piku1 having the highest content of tp in comparison to weiroot 13, weiroot 158, gisela 5 and maxma 14. in the study conducted by gonçalves et al. (2005), tp content of cvs. ‘burlat’, ‘summit’ and ‘van’ was higher on vigorous rootstocks (maxma 14, cab 11e, prunus avium) as opposite to dwarfing and semi-dwarfing rootstocks (gisela 5, edabriz). in our research cv. ‘kordia’ shows less variation in tp content in combination with individual rootstocks (21 % difference between the lowest and the highest value), while this variation is much higher in cv. ‘regina’ (41.15 %). results indicate that this variation in tp content was not connected with either vigour, yield efficiency or fruit weight. further analysis of vegetation year influence on tp content shows significant variation during 2013 which could be attributed to higher temperatures during final stages of ripening (fig. 1). this is particularly visible at cv. ‘regina’ on phl-c and especially on piku1 rootstocks which in 2013 had the highest and in 2014 the lowest content of tp in comparison to all cultivars and rootstocks in trial, and this difference is statistically significant (p ≤ 0.01). during 2014, content of tp for both cultivars was significantly lower, however it followed the same pattern as in 2013 for cv. ‘kordia’. for cv. ‘regina’, the content of tp between years 2013 and 2014 varied the least in rootstock gisela 5 (21 %) and for cv. ‘kordia’ on gisela 5 (20 %) and piku1 (22 %) (fig. 1). as all four rootstocks in our research are of heterogenic origin (tab. 1), the level of variation in tp content has to be observed not only through genetic origin of rootstock but of both cultivars as well: cv. ‘kordia’ being a chance seedling and cv. ‘regina’ being a cross between cvs. ‘schneiders späte knorpelkirsche’ and ‘rube’. higher content of tp in sweet cherries grafted on heterogenic combinations can be explained by adjustment of genetically different metabolisms of rootstock and cultivar, which causes high stress levels (usenik et al., 2005). tp content in our research varied significantly less in cv. ‘kordia’ between two vegetation years which can lead to a conclusion that this cultivar has formed a more stable graft unions with all four rootstocks unlike cv. ‘regina’. hence, less variation in the inner quality parameters in sweet cherry fruits makes this cultivar more robust in struggling with adverse climatic conditions and therefore more suitable for intensive fruit production. hydroxycinnamic acids content variation in the content of hydroxycinnamic acids (hca) in fruit could be qualitative (presence or absence of certain hca) or quantitative (amount of present hca and their ratio), which is affected by numerous factors such as physiological stage of a plant, diseases and pests, and to a lesser degree by agro-ecological conditions (clifford, 1999). hca profile and the ratio between the major hcas, chlorogenic (cha), neochlorogenic (ncha) and p-coumaric acid (p-ca) are not maturation dependent, except in the very early stages (mozetič et al., 2004). out of researched individual hca, p-ca was the most abundant one followed by ncha and cha (tab. 3). how ever, this was not the case for cv ‘regina’ grafted on piku1 rootstock in 2013, and on gisela 5 in 2014 when higher content of ncha were identified in fruits (fig. 2 and 3). usenik et al. (2015) have identified higher ncha (62.9 64.8 %) and lower p-ca concentration (32.3 34.0 %) for cv. kordia/gisela 5 combination as well. serradilla et al. (2011) have obtained higher p-ca (62.0 %) to ncha (35.0 %) ratio in cv. ‘pico negro’ as well as in cv. ‘van’ (81.4 %; 18.3 %) respectively. hence, it can be concluded that genetic origin of cultivars can have a significant influence on the content and composition of individual hca. p-ca was influenced by cultivar, vegetation year and interaction of cultivar*rootstock*year. ncha was influenced by vegetation year and interaction of cultivar*rootstock and cultivar *rootstock*year. cha was influenced by cultivar and cultivar*year and rootstock*year interaction. these differences were statistically significant (p ≤ 0.01). rootstock did not have significant influence on the content of either of researched individual hca (tab. 3). content of p-ca of cv. kordia in combination with rootstocks was decreasing in following order: gisela 6 > phl-c > piku1 > gisela 5, whereas for cv ‘regina’ it followed different pattern: phl-c > gisela 5 > gisela 6 > piku1. these differences were not statistically significant. the content of ncha varied in both cultivars depending on rootstock, and these difference were statistically significant (p ≤ 0.01). this is especially visible at cv. ‘kordia’ which ncha content decreased in following order: piku1 > gisela 6 > gisela 5 > phl year 2013 year 2014cv kordia rootstock: gisela 5 gisela 6 phl-c piku1 50 100 150 200 250 300 350 400 450 t ot al p he no ls ( m g/ 10 0g f w ) cv regina rootstock: gisela 5 gisela 6 phl-c piku1 fig. 1 ncha p-cacv kordia rootstock gisela 5 gisela 6 phl-c piku -2 0 2 4 6 8 10 12 14 16 18 20 22 cv regina rootstock gisela 5 gisela 6 phl-c piku factors: levels year: 2013 fig. 2 pca a nd n ch a c on te nt (m g/ 10 0g f w ) fig. 1: total phenol content (mg/100 g fw) of cvs. ‘kordia’ and ‘regina’ grafted on 4 different rootstocks during 2013 and 2014 dwarfing rootstock influence on sweet cherry polyphenols 33 c. in cv. ‘regina’ content of ncha decreased in same manner as for p-ca (tab. 3). these differences in the individual hca variation in fruits of both sweet cherry cultivars depending on a graft union with individual rootstocks prompted further analysis to investigate the influence of vegetation year. content of both p-ca and ncha was higher in 2013 for all cultivar and rootstock combinations (fig. 2 and 3). cv. ‘kordia’ had higher content of both p-ca and ncha on all rootstocks except on gisela 5 during 2013. both cultivars had similar content of ncha on phl-c rootstock in 2013. cv. ‘kordia’ has shown more variation in the ratio of hca in 2013, which is especially visible in combinations with gisela 6 and phl-c rootstocks. rootstock piku1 and phl-c had similar influence on the content of p-ca of both cultivars in 2014 (fig. 3). the most significant influence on the content of p-ca was visible in rootstock gisela 5, on which cv. ‘regina’ obtained the lowest and cv. ‘kordia’ the highest amount of mentioned hca. weather conditions had influenced the ncha content of both cultivars in 2014. statistically significant differences in the content of ncha in 2014 were observed only in cv. ‘kordia’ in combination with gisela 5 and cv. ‘regina’ in combination with phl-c. cultivar*rootstock interaction in our research was significant only ncha p-cacv kordia rootstock gisela 5 gisela 6 phl-c piku -2 0 2 4 6 8 10 12 14 16 18 20 22 cv regina rootstock gisela 5 gisela 6 phl-c piku factors: levels year: 2014 fig. 3 year 2013 year 2014cv kordia rootstock: gisela 5 gisela 6 phl-c piku1 0 5 10 15 20 25 30 35 40 45 50 55 60 t ot al a nt ho cy an in s (m g/ 10 0 gf w ) cv regina rootstock: gisela 5 gisela 6 phl-c piku1 fig. 4 pca a nd n ch a c on te nt (m g/ 10 0g f w ) year 2013 year 2014cv kordia rootstock: gisela 5 gisela 6 phl-c piku1 50 100 150 200 250 300 350 400 450 t ot al p he no ls ( m g/ 10 0g f w ) cv regina rootstock: gisela 5 gisela 6 phl-c piku1 fig. 1 ncha p-cacv kordia rootstock gisela 5 gisela 6 phl-c piku -2 0 2 4 6 8 10 12 14 16 18 20 22 cv regina rootstock gisela 5 gisela 6 phl-c piku factors: levels year: 2013 fig. 2 pca a nd n ch a c on te nt (m g/ 10 0g f w ) fig. 2: p-coumaric acid (p-ca) and neochlorogenic acid (ncha) (mg/100 g fw) of cvs. ‘kordia’ and ‘regina’ grafted on 4 different rootstocks during 2013 fig. 3: p-coumaric acid (p-ca) and neochlorogenic acid (ncha) (mg/100 g fw) of cvs. ‘kordia’ and ‘regina’ grafted on 4 different rootstocks during 2014 34 b. milinović, v. dragović-uzelac, d.h. kazija, t. jelačić, p. vujević, d. čiček, a. biško, z. čmelik for the content of ncha. the influence of different rootstocks on the content of p-ca and their role in incompatibility process in sweet cherry with heterospecific rootstock combinations having higher p-ca content in phloem above the graft point was previously investigated (usenik and štampar, 2000). with regard to polyphenol content in specific cultivar and rootstock combinations it can be assumed that heterospecific graft combinations carry a low level of stress due to adjustment between two different genotypes. all of our combinations are of heterospecific genetic origin, therefore the presence of certain level of stress is expected and can be quantified through the content of individual hca. total and individual anthocyanins content in fruit content of total anthocyanins (ta) was influenced by cultivar, rootstock, vegetation year and their mutual interaction (tab. 3). difference in the ta content of cultivar and rootstock combinations can be better observed in their interaction with the vegetation year. ta was in average lower in 2014 than in 2013 for both cultivars on all rootstocks except for cv. kordia/gisela 5 combination which had 24 % higher content of ta in 2014. also, content of ta in cv. ‘kordia’ varied significantly on phl-c rootstock, and for cv. ‘regina’ varied the most on piku1 (fig. 4). cv. ‘regina’ showed similar distribution of ta content in both vegetation years forming in that sense more stable connection with all four rootstocks. cv. ‘kordia’, however reacted differently in combination with individual rootstocks when it comes to ta content in both vegetation years. distribution of ta content in 2013 and 2014 follows similar pattern as the distribution of p-ca (fig. 2, 3) except for kordia/gisela 5 combination in 2014. this can be explained by hca being a precursors in flavonoids biosynthesis via the phenylpropanoid pathway. the content of cyanidin 3-rutinoside (c-y3-r) for rootstock*cultivar combinations in trial followed ta pattern, except for cv. ‘kordia’, where the lowest c-y3-r content was determined in gisela 5, and highest in gisela 6. the amount of cy-3-r ranged between 84.75 % (cv. ‘kordia’) – 85.21 % (cv. ‘regina’), and cy-3-g ranged between 7.56 % (cv. ‘regina’) – 9.12 % (cv. ‘kordia’) which is in accordance with the ratio obtained by usenik et al. (2008; 2015). differences between mentioned individual anthocyanins for cultivar/rootstock combinations in trial were not statistically significant (tab. 3). colour parameters fruit skin colour is one of the most important fruit quality parameter and directly connected with consumer acceptance and purchase decisions (tijskens et al., 2011). it is renowned that cultivar and agroecological conditions affect cherry fruit colour which is linked to the ta content. our results revealed that not only cultivar and year, but rootstock and interaction of cultivar*year and cultivar*rootstock*year had significant influence on chromatic values l*, a*, b*, hue and chroma (tab. 4). cultivar * rootstock combinations in our research indicated that high chromatic value a* corresponds to higher values b* and chroma and to the lower hue value of the sweet cherry fruit skin. in contrast, chromatic value l* does not follow this pattern, nonetheless it is connected with the content of ta in cv. ‘regina’ (tab. 3, 4), as lower value l* of fruit skin corresponds to the higher ta content of cv. ‘regina’ on different rootstocks. for all cultivar and rootstock combinations, except for kordia/gisela 6, it was identified that lower value l* corresponds to higher ta, cy-3-g, cy-3-r and cha content. change in values a*, b*, chroma and hue is not linked with the change in phenolics content. fruits of cv. ‘regina’ were of lighter colour, with less red and yellow colour than cv. ‘kordia’s, hence the higher hue and chroma values. our results are in line with the research done by usenik et al. (2009) on cv. ‘kordia’ measured with chromameter konica minolta cr10 in slovenia. several authors have identified the rootstock influence on fruit skin colour, however their results differ depending on chromatic parameter which was affected by this interaction. cantin et al. (2010) have reported significant influence of different rootstocks on value l* of cvs. ‘van’ and ‘stark hardy giant’ sweet cherries. in our research, cultivar and rootstock interaction has affected the value b*, hue and chroma. gadže et al. (2010) have also identified significant influence of rootstocks to fruit colour of sweet cherry ‘lapins’. values l* and b* of cv. ‘lapins’ were higher and value a* was lower on gisela 5 the piku1, however these differences were not statistically different. correlation between phenols and colour parameters polyphenol content determination is time-consuming and expensive procedure. aiming to decrease these costs, different methods of fast fig. 4: total anthocyanin content (mg/100 g fw) of cvs. ‘kordia’ and ‘regina’ grafted on 4 different rootstocks during 2013 and 2014 ncha p-cacv kordia rootstock gisela 5 gisela 6 phl-c piku -2 0 2 4 6 8 10 12 14 16 18 20 22 cv regina rootstock gisela 5 gisela 6 phl-c piku factors: levels year: 2014 fig. 3 year 2013 year 2014cv kordia rootstock: gisela 5 gisela 6 phl-c piku1 0 5 10 15 20 25 30 35 40 45 50 55 60 t ot al a nt ho cy an in s (m g/ 10 0 gf w ) cv regina rootstock: gisela 5 gisela 6 phl-c piku1 fig. 4 pca a nd n ch a c on te nt (m g/ 10 0g f w ) dwarfing rootstock influence on sweet cherry polyphenols 35 tab. 4: colour characteristics of sweet cherry cvs ‘kordia’ and ‘regina’ grafted on 4 different rootstocks, average values for 2013 and 2014* cultivar rootstock/year l* a* b* hue chroma kordia gisela 5/2013 43.42 ± 0.45 4.52 ± 1.17 2.04 ± 0.39 24.69 ± 2.97 4.96 ± 1.21 gisela 5/2014 42,56 ± 0.67 3.70 ± 1.24 2.05 ± 0.39 29.99 ± 4.66 4.24 ± 1.26 gisela 6/2013 43.63 ± 0.45 4.33 ± 1.08 2.02 ± 0.36 25.30 ± 2.37 4.78 ± 1.12 gisela 6/2014 43.52 ± 0.48 5.78 ± 1.81 2.70 ± 0.57 25.55 ± 2.36 6.39 ± 1.88 phl-c/2013 43.34 ± 0.43 4.02 ± 0.84 1.86 ± 0.25 25.14 ± 2.41 4.43 ± 0.85 phl-c/2014 42.98 ± 0.59 3.59 ± 0.95 1.97 ± 0.30 29.48 ± 3.99 4.10 ± 0.96 piku1/2013 43.24 ± 0.35 4.35 ± 1.07 1.92 ± 0.38 24.13 ± 2.61 4.76 ± 1.12 piku1/2014 42.99 ± 0.34 4.29 ± 1.03 2.09 ± 0.28 26.53 ± 3.08 4.78 ± 1.04 regina gisela 5/2013 42.03 ± 0.36 3.46 ± 0.71 1.66 ± 0.18 26.07 ± 3.39 3.84 ± 0.70 gisela 5/2014 44.62 ± 0.47 6.33 ± 2.16 2.99 ± 0.74 25.91 ± 2.37 7.01 ± 2.26 gisela 6/2013 43.26 ± 0.42 3.68 ± 1.11 1.71 ± 0.34 25.46 ± 3.21 4.06 ± 1.13 gisela 6/2014 44.20 ± 0.27 4.63 ± 1.16 2.49 ± 0.35 28.84 ± 2.67 5.26 ± 1.18 phl-c/2013 42.76 ± 0.46 4.26± 0.96 1.75 ± 0.28 22.68 ± 2.71 4.61 ± 0.98 phl-c/2014 44.03 ± 0.31 4.56 ± 1.21 2.43 ± 0.39 28.61 ± 2.78 5.17 ± 1.25 piku1/2013 42.68 ± 0.56 4.06 ± 0.99 1.89 ± 0.30 22.55 ± 2.59 5.22 ± 1.72 piku1/2014 44.11± 0.34 4.85 ± 1.52 2.56 ± 0.53 28.47 ± 2.62 5.49 ± 1.59 p values cultivar 0.000 ns ns ns ns rootstock 0.000 ns 0.002 ns ns year 0.000 0.000 0.000 0.000 0.000 cultivar*rootstock ns 0.000 0.000 0.001 0.000 cultivar*year 0.000 0.000 0.000 ns 0.000 rootstock*year 0.006 0.004 ns 0.002 0.001 cultivar*rootstock*year 0.000 0.000 0.000 0.000 0.000 *values are expressed as the mean ± standard deviation; significant differences at p ≤ 0.01; ns = not significant (p > 0.01) and non-destructive identification of fruit inner quality parameters have been developed with higher or lesser accuracy. in this research we attempted to connect distribution of total and individual phenols, total and individual anthocyanins and individual hydroxycinnamic acids identified in cherry fruits (inner quality parameters) and colorimetric values of cherry fruit skin (outer fruit quality parameters) in order to determine whether these parameters are cultivar and rootstock dependant. following the statistical analysis of all above mentioned parameters, we have conducted further analysis concerning the average data for each cultivar and rootstock. overall, we have identified that colorimetrical values of cherry fruit skin (l*, a*, b*, chroma and hue) are negatively correlated with polyphenols identified in this research (tab. 5 and 6). mozetič et al. (2004) have also identified strong negative correlation between ta and value l* (r = -0.81), b* (r = -0.86), hue (r = -0.69) and chroma (r = -0.96) on sweet cherry cv. ‘petrovka’, which is darker coloured sweet cherry. in cv. ‘kordia’ we have not observed statistically significant correlation for value hue with any of the observed inner fruit quality parameter. correlation of medium strength was determined between value l* and a* and cha, value b* and ncha and ta. chroma was in medium negative correlation with both cha and ncha (tab. 5). in cv. ‘regina’, unlike cv. ‘kordia’ a* values and chroma did not have statistically significant correlation with any of the inner quality parameters (tab. 6). furthermore, the intensity of association between values l*, b* and hue and majority of polyphenols was much stronger than in cv. ‘kordia’ (tab. 6). substantial difference between cultivars when it comes to correlation coefficient between fruit skin colorimetric values and fruit phenolic content prompted further analysis on the rootstock level. obtained results showed that individual rootstocks have different correlation pattern. for gisela 5, value l* had strong association with p-ca (r = -0.76), cy-3-r (r = -0.83), cy-3-g (r = -0.87) and ta (r = -0.96). strong association was determined between values a*, b* and chroma and mentioned phenolic acids as well on gisela 5 (data not shown). in piku1 strong association was found as well between l* and cy-r-g (r = -0.83), cy-3-r (r = -0.84) and ta (r = -0.80). value b* correlated with ta (r = -0.93), cy-3-g (r = -0.76), cy-3-r (r = -0.74) and ncha (r = -0.75).gisela 6 had strong association only between value l* and ta (-0.74), and between hue and cha (r = -0.79). phl-c was the only rootstock where correlation between anthocyanins and chromatic values was not determined. the only association found for this rootstock was between tp and l* (r = -0.72) and hue (r = -0.71). mozetič et al. (2004) have determined that the content of anthocyanins rises during ripening of sweet cherries and leads to the development of new colour palette which results in decrease of the intensity of red colour. change in value l* and chroma during the ripening of sweet cherry ‘petrovka’ correlated strongly with the change in the ta content. gonçalves et al. (2005) obtained similar results on sweet cherry cultivars ‘burlat’, ‘summit’ and ‘van’. influence of the rootstock on sweet cherry fruit skin colour development and correlation association between outer and inner fruit quality parameters was 36 b. milinović, v. dragović-uzelac, d.h. kazija, t. jelačić, p. vujević, d. čiček, a. biško, z. čmelik not researched to this extent so far. our results instigate that when deciding on sweet cherry ripeness stage (harvest window), rootstock influence on individual cultivars must be taken into consideration as well. conclusions rootstock had strong influence on the quality parameters of cvs. ‘kordia’ and ‘regina’, namely on the content of tp, ta, cy-3-r and cy-3-g, as well as on the values l* and b*. ta content in fruits of both cultivars was influenced by vegetation year. cv. ‘regina’ showed similar distribution of ta content in both vegetation years forming in that sense more stable connection with all four rootstocks. cv. ‘kordia’, however reacted differently in combination with individual rootstocks when it comes to ta content in both vegetation years. with substantial difference in correlation pattern in our research between cvs. ‘kordia’ and ‘regina’ it can be concluded that values l* and b* are better ta indicators rather than values a* and hue as previous studies suggested. also, with ta content following the same distribution pattern as p-ca content, it can be assumed that mentioned chromatic values could be used as indicators of phenolics content for specific cultivar and rootstock combinations. having in mind that rootstock had strong influence on inner and outer fruit quality parameters which corresponds to existence of correlation association between researched phenolics and colour parameters, further analysis is needed to investigate influence of the cultivar*rootstock interaction on correlation pattern. tab. 5: correlation coefficients (r), p values and the probability levels for phenolic content as well as colour parameters of cv. ‘kordia’ l* a* b* hue chroma chlorogenic acid r -.5248 -.5101 -.4219 .4491 -.5037 p 0.037* 0.044* 0.104 ns 0.081 ns 0.047* neochlorogenic acid r .0414 -.4885 -.5744 .2103 -.5059 p 0.879 ns 0.055 ns 0.020* 0.434 ns 0.046* p-coumaroylquinic a. r .1373 -.3456 -.4499 .0712 -.3640 p 0.612 ns 0.190 ns 0.080 ns 0.793 ns 0.166 ns cyanidin 3 glucoside r .0352 .1360 .1366 -.1589 .1380 p 0.897 ns 0.616 ns 0.614 ns 0.557 ns 0.610 ns cyanidin 3 rutinoside r -.3013 .0271 .0599 -.0308 .0325 p 0.257 ns 0.921 ns 0.826 ns 0.910 ns 0.905 ns total anthocyanins r -.0355 -.3765 -.5068 .0867 -.3985 p .896 ns 0.151 ns 0.045* 0.749 ns 0,126 ns total phenols r .2288 -.1616 -.2353 .0136 -.1731 p 0.394 ns 0.550 ns 0.380 ns 0.960 ns 0.521 ns **, * = significant at p ≤ 0.01 and 0.05, respectively; ns = not significant (p > 0.05) tab. 6: correlation coefficients (r), p values and the probability levels for phenolic content and colour parameters of cv. ‘regina’ grafted on 4 different rootstocks l* a* b* hue chroma chlorogenic acid r -.3934 -.0823 -.3384 -.6451 -.1179 p 0.132 ns 0.762 ns 0.200 ns 0.007 ** 0.664 ns neochlorogenic acid r -.7711 -.4688 -.6844 -.5071 -.4749 p 0.000 ** 0.067 ns 0.003 ** 0.045 * 0.063 ns p-coumaroylquinic a. r -.7384 -.4365 -.6053 -.3509 -.4643 p 0.001 ** 0.091 ns 0.013 * 0,.183 ns 0,.070 ns cyanidin 3 glucoside r -.8341 -.4627 -.6688 -.5229 -.4500 p 0.000 ** 0.071 ns 0.005** 0.038 * 0.080 ns cyanidin 3 rutinoside r -.7898 -.4237 -.6516 -.5548 -.4314 p 0.000 ** 0.102 ns 0.006 ** 0.026 * 0.095 ns total anthocyanins r -.8683 -.4691 -.6887 -.5666 -.4564 p 0.000 ** 0.067 ns 0.003 ** 0.022 * 0.076 ns total phenols r -.5941 -.2933 -.4684 -.6079 -.2219 p 0.015 * 0.270 ns 0.067 ns 0.012 * 0.409 ns **, * = significant at p ≤ 0.01 and 0.05, respectively; ns = not significant (p > 0.05) dwarfing rootstock influence on sweet cherry polyphenols 37 references betrán, j.a., val, j., millán, l.m., monge, e., montañés, l., moreno, m.a., 1997: influence of rootstock on the mineral concentrations of flowers and leaves from sweet cherry. acta hortic. 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stampar, f., veberic, r., 2010: sweet cherry pomological and biochemical characteristics influenced by rootstock. j. agric. food chem. 58, 4928-4933. usenik, v., stampar, f., mikulic petkovsek, m., kastelec, d., 2015: the effect of fruit size and fruit colour on chemical composition in ‘kordia’ sweet cherry (prunus avium l.). j. food comp. anal. 38, 121-130. voss, d.h., 1992: relating colorimeter measurement of plant color to the royal horticultural society colour chart. hortscience 27, 1256-1260. zorić, z., dragović-uzelac, v., pedisić, s., kurtanjek, ź., garofulić, i.e., 2014: heat-induced degradation of antioxidants in marasca paste. food tech. biotech. 52, 101-108. address of the author: bernardica milinović, dunja halapija kazija, tvrtko jelačić, predrag vujević, danijel čiček, ante biško, croatian centre for agriculture, food and rural affairs, institute of pomology, rim 98, 10000 zagreb, croatia verica dragović-uzelac, department of food technology, faculty of food technology and biotechnology, pierottijeva 6, 10000 zagreb, croatia zlatko čmelik, department of pomology, faculty of agriculture, svetošimunska cesta 25, 10000 zagreb, croatia © the author(s) 2016. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 88, 222 227 (2015), doi:10.5073/jabfq.2015.088.032 1alata horticultural research station, erdemli, mersin-turkey 2 department of field crops, faculty of agriculture, namik kemal university, tekirdag-turkey 3department of horticulture, faculty of agriculture, ataturk university, erzurum-turkey genetic characterization of banana clones grown in turkey based on nuclear dna content and srap markers hasan pinar1, mustafa unlu1, mustafa bircan1, filiz baysal1, gulsemin savas tuna2, metin tuna2, sezai ercisli3* (received march 24, 2015) * corresponding author summary this study was conducted to investigate the genetic relationships among banana clones grown in turkey based on their nuclear dna contents and srap markers. four banana clones including ‘dwarf cavendish’, ‘grand nain’, ‘azman’ and local ‘erdemli’ were used as plant material. nuclear dna content of the banana cultivars was estimated by flow cytometer and varied between 1.766 pg (‘erdemli’) and 2.028 pg (‘grand nain’). ‘azman’ contained 1.973 pg and ‘dwarf cavendish’ 1.936 pg nuclear dna. genetic similarities of 4 banana clones were between 0.63 0.91 based on srap molecular markers. the local ‘erdemli’ banana clone was the most distinct from the others. in conclusion, there is a high level of genetic variation among erdemli and other banana clones grown in turkey and the local clone ‘erdemli’ is the most distinct one. this study showed that nuclear dna content analysis together with molecular markers could be useful to assess the relationships among banana clones. introduction fruits have long been valued as part of a nutritious and tasty diet. the flavours provided by different fruit species are among the most preferred in the world, and it is increasingly evident that fruits not only taste good, but also good for human health (bacvonkralj et al., 2014; rop et al., 2014). bananas and plantains belong to the genus musa and they are the most significant food and cash crops worldwide, especially in tropical and sub-tropical regions. world total annual banana produc tion is around 102 million tons (fao, 2013). the genus musa is divided into two sections: musa and callimusa; the musa section includes 33 species (hakkinen, 2013). the section contains triploid varieties evolved from two wild diploids (2n = 2x = 22) as of seminiferous species, m. acuminata colla (genomes aa) and m. balbisiana (genomes bb). in general, bananas consist of two major categories as of those consumed as dessert (constituting about 43 % of world production) and cooking bananas (constituting the remaining 57 % of world production) (jones, 2000). some banana clones can be cultivated in subtropical regions between 20° and 30° north and south of the equator. several subtropical regions protected from cold weather, such as the mediterranean coastline, are also suitable for growing bananas. banana has been grown in some subtropical parts of turkey since 1934 (gubbuk et al., 2004) and annual banana production is increasing each year and has reached 210.000 tons in turkey (fao, 2013). several direct or indirect methods have been used to identify the genomes in musa. these include molecular markers, isozymes, retrotransposons, in situ hybridization and flow cytometry. a number of techniques have been used to evaluate the level of genetic variability in musa via molecular markers, including random amplified polymorphic dna (rapd) (das et al., 2009), restriction fragment length polymorphism (rflp) (hippolyte et al., 2010), amplified fragment length polymorphism (aflp) (opara et al., 2010), inter simple sequence repeats (issr) (lu et al., 2011), sequence related amplified polymorphism (srap) (phothipan et al., 2005) and simple sequence repeats (ssr) markers (miller et al., 2010). sequence related amplified polymorphism (srap) was first used in brassicaceae for marker development and mapping (li and quiros, 2001). subsequently this system has been used in many fruit crops such as citrus (uzun et al., 2009), apricot (uzun et al., 2010) etc. for genetic diversity and fingerprinting studies because it is a simple and also efficient marker system that can be adapted for a variety of purposes in different crops, including map construction, gene tagging, genomic and cdna fingerprinting and map based cloning (li and quiros, 2001). flow cytometry is among the most common methods used to assess numerous cellular characteristics and to analyze cells individually (loureiro et al., 2006). the method was considered as the fastest and the most reliable technique (suda and travnicek, 2006). the method was initially developed for human cells but then was adapted for plant cells as a reliable tool for estimation of nuclear dna con tent and ploidy level constitutions in plants (dolezel et al., 1994; tuna et al., 2006; ozkan et al., 2010; tiryaki and tuna, 2012). dolezel et al. (1994) modified the technique for analysis of nuclear genomes of bananas and plantains. the method has been employed in finding out the dna nuclear contents of various banana varieties since the early 1980s (dolezel, 1991). d’hont et al. (1999) carried out a study with flow cytometry method to assess genetic differences between musa genomes and reported significant differences between the sizes of musa a, b, s and t genomes. he was able to indicate that s and t genomes were larger than a and b genomes and t genome was the largest one. the information provided by the researchers was then employed by the other researchers as a significant diagnostic tool to assess the differences between musa genomes and it was reported in previous studies that dna content of aa genome accessions varied between 1.22-1.27 pg (lysak, 1999) or between 1.20 -1.33 pg (kamate, 2001). arumuganathan and earle (1991) also reported the similar dna content estimations for triploid accessions with the estimations of lysak (1999) and kamate (2001). lysak et al. (1999) determined dna at a level of 1.81 pg in a triploid accession. in this study, nuclear dna contents of ‘dwarf cavendsish’, ‘grand nain’ and local clones ‘azman’ and ‘erdemli’, grown in turkey were determined for the first time through flow cytometry and also genetic relationships among these cultivars were assessed by srap marker. materials and methods plant material international ‘dwarf cavendish’ and ‘grand nain’, local ‘azman’ and ‘erdemli’ banana clones were used as the plant material of the determine of genetic relationships among banana clones by srap markers 223 present study. all plants were grown in protected areas of open fields and selected from a commercial orchard in erdemli, mersin. some yield and leaf characteristics such as finger length (cm), finger weight (g), plant height (cm), number of hands (bunch-1), number of fingers (hand-1), bunch weight (kg/plant), leaf petiole length (cm) and leaf petiole width (cm) were measured by using ten plants from each clone as shown in tab. 1. data were analyzed statistically via jmp 7.0 (copyright © 2007 sas institute inc.). dna isolation total genomic dna was extracted from fresh leaf tissues of four banana clones for molecular analysis by using the ctab procedure as described by doyle and doyle (1990). dna concentration was measured with a microplate spectrophotometer (biotek instruments, inc., vinooski, usa), and 10 ng/ml dna templates were made using te (10 mm tris-hcl, 0.1 mm edta, ph 8.0) buffer. srap analysis a total of 19 srap primer combinations were used for all banana clones (tab. 2). pcr reaction components and pcr cycling parameters were as described by uzun et al. (2009). pcr products were separated on 2 % agarose gels in 1x tbe buffer (89 mm tris, 89 mm boric acid, 2 mm edta) at 115 volts for 3 h. the fragment patterns were photographed under uv light for further analysis. a 100 bp standard dna ladder was used to confirm the appropriate markers for srap analysis. three replications were used to confirm markers. molecular analysis was carried out as follows: each band was scored as present (1) or absent (0) and data were analyzed with the numerical taxonomy multivariate analysis system (ntsys-pc) software (rohlf, 2000). a similarity matrix was constructed using srap data based on dice’s coefficient. the similarity matrix was used to construct a dendrogram using upgma (unweighted-pair group method with arithmetic average) to determine genetic relationships among the cultivars studied. the genetic similarity matrix and ultrametric distance matrix produced from the upgma-based dendrogram with coph module nested in the same software was compared using mantel’s matrix correspondence test (mantel, 1967). nuclear dna content nuclear dna content was carried out by a flow cytometer (cyflow® space, partec münster) equipped with a 30 mw 532 nm laser at the field crops department of namık kemal university. propidium iodide (pi) was used as a fluorochrome to stain the dna. suspensions of intact nuclei were prepared using a partec commercial kit (cystain pi absolute p). briefly, the procedure consists of chopping 40 mg of banana leaf tissue using a sharp razor blade together with 20 mg of rice leaf tissue as internal standard in a petri dish containing 0.5 ml extraction buffer. the crude nuclear suspension was then transferred to a labelled test tube through 50-micron nylon mesh. 1.5 ml of staining buffer was then added to the test tube and the samples were stored at 4 °c for approximately 60 min in dark. pi fluorescence emitted from nuclei was collected through a 560 nm long-pass filter and a 630 nm band-pass filter. for each sample, fluorescence data were acquired from at least 3000 nuclei. three independent replications were performed and histograms with coefficient of variation (cv) above 5.75 % were rejected. the formula used for converting fluorescence values to dna content was: nuclear dna content = (mean position of unknown peak)/(mean position of known) x dna content of known standard (rice, osmancik cultivar). results and discussion yield and leaf characteristics yield is the most important criterion for banana production. some yield and leaf characteristics of 4 banana clones grown in turkey are provided in tab. 1. ‘grand nain’ had the highest values with regard to finger length, finger weight, hand number, and bunch weight; ‘erdemli’ had the lowest value with regard to all these characteristics. ‘azman’ and ‘grand nain’ clones were similar to each other, but ‘dwarf cavendish’ clones had lower values when compared with ‘azman’ and ‘grand nain’ with regard to plant height and the other horticultural characteristics (tab. 1). srap analysis the number of morphological markers for identifying the clones of banana is limited and they do not often reflect genetic relationships because of interaction with the environment, epistasis and the largely unknown genetic control of traits (smith and smith, 1989). in contrast, dna markers are found in abundance and are not influenced by the environment or developmental stage of a plant, making them ideal for genetic relationship studies in plant species (reddy et al., 2002). in this study, genetic relationships among four banana clones grown in turkey since 1934 were evaluated by using srap molecular markers. nineteen srap markers gave polymorphisms ratio from 25 to 100 % (tab. 2). genetic similarities of 4 banana clones were between 0.63 0.91 (tab. 3). local ‘erdemli’ clone was found to be the most distant from the other clones (fig. 1). morphologically, this clone had short and thick fruits compared with the other clones (tab. 1). there is no information about the clone when it was brought to turkey. most probably the ‘erdemli’ clone had a different origin from the other bananas. ‘grand nain’ and ‘azman’ were genetically closer each other than ‘dwarf cavendish’ with ~ 0.91 similarity ratio. ‘azman’ has similar plant height (~5 m) to ‘grand nain’. also these two clones have the same fruit characteristics (tab. 1). genetic similarity tab. 1: some yield and leaf characteristics of four banana clones clones fl fw ph nh nf bw lpl lpw ‘grand nain’ 21.4a 151.7a 380.0a 16.3a 26.0a 49.0a 252.1a 115.3a ‘azman’ 21.2a 134.7ab 356.7a 15.2a 24.3a 44.7a 261.1a 107.1ab ‘dwarf cavendish’ 19.0b 116.7b 220.7b 14.7a 24.0a 38.3b 204.7b 100.1b ‘erdemli’ 14.1c 80.4c 382.0a 9.7b 17.3b 17.0c 192.4b 80.9c lsd 2.2 23.8 84 2.4 3.4 13.5 32.9 10.3 note: means with the same letters within columns are not significantly different at p <0.05. fl: finger length (cm), fg: finger weight (g), plant height (cm), nh: number of hands (bunch-1), nf: number of fingers (hand-1), bw: bunch weight (kg/ plant), lpl: leaf petiole length (cm), lpw: leaf petiole width (cm) 224 h. pinar, m. unlu, m. bircan, f. baysal, g. savas tuna, m. tuna, s. ercisli tab. 2: observed polymorphism with 19 srap primer combinations in 4 banana clones. primer total bands polimorphic % polimorphism combinations bands em14-me3 2 1 50.0 em2-me5 3 2 66.7 em1-me1 6 3 50.0 em2-me1 7 7 100.0 em3-me2 6 3 50.0 em6-me3 6 3 50.0 em3-me3 6 4 66.7 em4-me3 3 3 100.0 em6-me2 10 6 60.0 em6-me11 6 3 50.0 em7-me10 10 7 70.0 em8-me4 5 5 100.0 em10-me7 4 4 100.0 em11-me6 4 1 25.0 em12-me9 3 2 66.7 em14-me4 6 3 50.0 em16-me3 6 3 50.0 em3-me4 4 3 75.0 em8-me8 5 4 80.0 mean 5.37 3.53 66.32 total 102 67 fig. 1: dendrogram of 4 banana clones using upgma method obtained from srap markers, e: ‘erdemli’, a: ‘azman’, d: ‘dwarf cavendish’, g: ‘grand nain’. of ‘azman’ and ‘dwarf cavendish’ (0.88) was less than to ‘azman’ and ‘grand nain’ (0.91) (tab. 3). the lowest genetic similarity was observed between ‘grand nain’ and ‘erdemli’. brown et al. (2009) made molecular study on bana and reported that ‘grande nain’ (aaa), ‘williams’ (aaa), and ‘dwarf cavendish’ (aaa) were in the same cluster. loh et al. (2000) stated that the differences in phenotypic characters might not be due to euploidy differences but rather due to allelic differences in single or multiple genes in banana. they found a similar discrepancy among some of the banana clones. the cv. dwarf cavendish, grand nain, and raja udang, which were designated by simmond (1966) as having the aaa genomic con stitution based on morphological characterization, clustered separately. previously genetic similarities or differences of different banana clones have been determined by using different molecular marker techniques, but there are a few reports on srap molecular markers. srap markers were successfully used to analyze the genetic relationship among 29 accessions of banana in aa, aab and bb groups (phothipan et al., 2005). youssef et al. (2011) used srap and aflp to analyze the genetic variation and relationships among forty musa accessions (commercial cultivars and wild species of musa). srap had threefold more specific and unique bands than aflp. srap markers are demonstrated to be effective tools for discriminating amongst m. acuminate, m. balbisiana and m. schizocarpa in the musa section, as well as between plantains and cooking bananas within triploid cultivars. valdez-ojeda et al. (2014) aimed to determine the genetic variability within 71 accessions of musa (wild species and cultivars of different subgroups) using srap marker. the used accessions were consistently identified and separated by srap markers. a total of 330 polymorphic bands were detected using 12 primer combinations. the genetic similarity between accessions ranged between 0.44 and 0.97. they reported that srap marker system is useful to identify closely related accessions in the genus musa and facilitated the recognition of duplicates to be eliminated and clarified uncertainties or mislabeled banana accessions introduced to the collection. mattos et al. (2010) characterized banana accessions from brazil using agronomical, physical and physicochemical characteristics of fruit as well as simple sequence repeats (ssr) markers. thirteen microsatellite primers revealed an average of 7.23 alleles, which showed high variability. lu et al. (2011) identified genetic similarity of different cultivars and monitored somaclonal variations of banana during rapid mass micro propagation using inter simple sequence repeat (issr) primers and found about 85.1 % of total polymorphism. in another study, genetic similarities ranged from 0.3 to 1.0 with an average of 0.51 using random amplified polymorphic dna (rapd) markers with 27 accessions in the mauritian musa germplasm (brown et al., 2009). retnoningsih et al. (2011) used microsatellite markers to classify 116 banana accessions and placed 73 accessions in aa/aaa and aaa genomic tab. 3: similarity index of 4 banana clones obtained by upgama analysis using srap molecular marker data clones ‘grand nain’ ‘azman’ ‘dwarf ‘erdemli’ cavendish’ ‘grand nain’ 1.0000 ‘azman’ 0.9143 1.0000 ‘dwarf cavedish’ 0.8630 0.8876 1.0000 ‘erdemli’ 0.6102 0.6131 0.6575 1.0000 determine of genetic relationships among banana clones by srap markers 225 groups, 2 accessions in bb genomic group, 21 accessions in aab genomic group and 20 accessions in abb genomic group. the researchers also, reported 99 accessions as unique genotypes and the remaining 17 accessions as synonyms. in the same study, 116 accessions were separated into two main clusters with a similarity of 0.13 on upgma analysis. jain et al. (2007) investigated the genetic relationships among four banana clones (grand naine, red banana, nendran and rasthali) commonly cultivated in south india through random amplified polymorphic dnas (rapds). researchers indicated 43.47 % of the amplification products as monomorphic (common to all genotypes) and 30.43 % as unique; but only 26.08 % was able to reveal the genetic relationships among the investigated clones. the present findings about the genetic similarities between ‘dwarf cavendish’ and ‘grand nain’ clones comply with the results of grajel-martin et al. (1998), shoseyov et al. (1996) and gubbuk and pekmezci (2001). similar to current findings, grajel-martin et al. (1998) reported high similarity among lacatan, williams, petit nain, grand nain, robusta and valley, which belong to the cavendish subgroup (aaa). shoseyov et al. (1996) investigated genetic similarities among banana cultivars and indicated low polymorphism among grand nain, williams and natha banana clones and somaclonal variants of these cultivars. gubbuk and pekmezci (2001) used rapd markers and reported the genetic similarity ‘dwarf cavendish’ and ‘grand nain’ as 0.929. pinar et al. (2015) conducted a study on estimate genetic relationships and natural somatic mutations among selected banana genotypes from different region of turkey via srap molecular markers. ninety-six banana genotypes including 39 dwarf cavendish, 18 grand nain, 28 azman and 11 other bananas were evaluated. their study showed that there was high level of variation among the cultivars. in the present study, srap technique was demonstrated to be a useful tool in determining genetic diversity in 4 banana clones grown in turkey. evaluation of nuclear dna content of four banana clones using flow cytometry flow cytometry is the common method employed in estimation of nuclear dna content of bananas (novak, 1992). in this study, mean nuclear dna contents of four banana clones were evaluated by flow cytometer and are presented in fig. 2, 3, 4 and 5. rice (osmancik cultivar) used as an internal standard. results showed that ‘grand nain’ had the highest 2c nuclear dna content (2.028 pg) while ‘erdemli’ had the lowest (1.766 pg). ‘dwarf cavendish’ (1.936 pg) was close to ‘azman’ (1.976 pg) in nuclear dna content (tab. 4). ‘erdemli’ clones separated from the other clones by nuclear dna content as well as by molecular data (fig. 1, 2, 3, 4, 5). there is limited information in literature on nuclear dna content and genome sizes of musa cultivars (arumuganathan and earle, fig. 5: relative positions of the g1 peaks of ‘dwarf cavendish’ banana cultivar and rice (fırst peak) fig. 2: relative positions of the g1 peaks of ‘erdemli’ banana clone and rice (fırst peak) fig. 3: relative positions of the g1 peaks of ‘azman’ banana clone and rice (fırst peak) fig. 4: relative positions of the g1 peaks of ‘grand nain’ banana clone and rice (fırst peak) 226 h. pinar, m. unlu, m. bircan, f. baysal, g. savas tuna, m. tuna, s. ercisli 1991; kamate et al., 2001). of the available studies, two (lysak et al., 1999; kamate, 2001) of them included accessions with only a and b genomes and only one involved plants with s and t genomes (d’hont et al., 1999). arumuganathan and earle (1991) presented total 2c dna content (1.81 pg) and genome size (873 mbp) in musa for the first time. their estimations on dna content were similar to ones provided by kamate et al. (2001) for triploid accessions. lysak et al. (1999) in a previous study reported 2c dna content of a triploid accession as 1.81 pg. with regard to individual genomes of musa, b genome with 2c values of between 1.03 1.16 pg was reported to have the least nuclear dna content (lysak et al., 1999; kamate et al., 2001). the average 2c value of a genome was estimated to be 1.25 pg (kamate et al., 2001), 1.11 pg (d’hont et al., 1999) and 1.27 pg (kamate et al., 2001). such findings reveal significant differences (about 15 %) in dna contents of a and b genomes. a triploid accession with two b genomes (abb) was expected to have less dna than an accession with one b genome (aab). but the case was not as expected in lysak et al. (1999) and kamate et al. (2001). the genome size of simili radjah (abb) was identical to that of a tetraploid and 11% difference was identified in dna content of subspecies of the m. acuminata complex (kamate et al., 2001). kamate et al. (2001) also indicated wider range in dna contents of cultivars with similar genomes; while the dna contents of aab clones were between 1.61-1.79 pg and the dna contents of abb clones varied between 1.70 -2.23 pg. in the present study, the difference between the ‘erdemli’ clone and ‘dwarf cavendish’ clone was 9 %. the difference between ‘erdemli’ and ‘grand nain’ was 13 % and the difference between ‘erdemli’ and ‘azman’ was 10.5 %. such findings revealed that ‘grand nain’, ‘dwarf cavendish’ and ‘azman’ were placed in the same group (aaa) because of close 2c dna contents, ‘erdemli’ probably carried 1 b genome together with 2 a genomes (aab) or abb because of lower 2c dna content. similarly in previous studies, lysak et al. (1999) reported nuclear dna content of obino l’ewai (aab) as 1.777 pg, maritu (aab) as 1.847 pg and pelipita (abb) as is 1.751 pg. cizkova et al. (2013) reported nuclear dna content in aab as 1.786 pg and abb as 1.814 pg in banana. the differences in 2c dna contents of aaa and aab were between 6.5 8.3 % and the difference in 2c dna within this genome group was only 1.9 %. the difference in 2c dna within aab (5.5 %) was found to be significant. the largest difference in genome variation (8.9 %) was observed aa. variation in 2c nuclear dna content within both aa and bb genomes had significant contributions to existing differences within and between aaa and aab genomic groups (onyango et al., 2008). while the variation in 2c dna contents of triploid aab or abb genomic groups was found to be significant, the variation within aaa group was not significant. dolezel (2001) reported nuclear dna content of agbagba (aab) as 1.737 pg, pelipita (abb) as 1.751 pg, gross michel (aaa) as 1.881 pg and grand enano as 1.905 pg. bartos et al. (2005) reported relatively small but significant differences (3.4 %) between m. acuminata accessions. lysak et al. (1999) and dolezel et al. (1994) reported that a and b genomes of musa were different in size. lysak et al. (1999) indicated that b genome was generally significantly smaller (about 12 %) than a genome but the reserachers were not able to detect any variations among the m. balbisiana cultivars. in this study, ‘erdemli’ banana (er) clone was the most distinct genotype with regard to srap molecular data and nuclear dna content. this clone has different fruit and plant architecture than ‘grand nain’, ‘dwarf cavendish’ and ‘azman’ as well (tab. 1). ‘erdemli’ is also more tolerant to cold weather than the other clones. as previous studies suggested, this clone may have an aab or abb genome because ‘erdemli’ has a lower dna content than the other banana clones grown in turkey. ‘azman’ clone has dna content close to ‘grand nain’, and had similar fruit and plant characteristics, although ‘azman’ plant height is slightly greater than ‘grand nain’ (tab. 1). also ‘azman’ clone dna content is close to dwarf cavendish clone. but there were some differences between their fruit and plant characteristics. ‘grand nain’, ‘dwarf cavendish’, and ‘azman’ banana clones likely belong to the aaa genome group. conclusion in conclusion, bananas were brought to turkey as ornamentals in 1934 and subsequently put into commercial production in open fields. ‘dwarf cavendish’ was the first clone used in commercial production. after that, growers started to use ‘grand nain’ and ‘dwarf cavendish’. ‘azman’ and ‘erdemli’ are local clones but their names and origins are unknown. in this study, especially, ‘azman’ and ‘erdemli’ banana clones were determined to be different from ‘dwarf cavendish’ and ‘grand nain’ according to srap molecular markers, some yield and leaf characteristics and nuclear dna contents. references arumuganathan, k., earle, e.d., 1991: nuclear dna content of some important species, plant mol. biol. rep. 9, 208-218. bacvonkralj, m., jug, t., komel, e., fajt, n., jarni, k., zivkovic, j., mujici, i., trutic, n., 2014: effects of ripening degree and sample preparation on peach aroma profile 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teil 2: lebensmittel-wissenschaftliche aspekte; teil 3: grundlagen der angewandten humanernährung; teil 4: prävention und therapie ernährungsassoziierter erkrankungen. der interdisziplinäre anspruch des lehrbuches, die biochemie der ernährung, die angewandte humanernährung, die lebensmittelwissenschaft und die ernährungsmedizin dem leser in ihren komplexen zusammenhängen in verständlicher weise nahe zu bringen, ist den autoren insgesamt sehr gut gelungen. das werk kann daher allen mit ernährungsund gesundheitsfragen befassten personen als ständiger begleiter uneingeschränkt empfohlen werden. dr. h. schulz e-mail: hartwig.schulz@jki.bund.de bibliografie: a. hahn, a. ströhle und d. hahn, ernährung – physiologische grundlagen, prävention, therapie, wissenschaftliche verlagsgesellschaft mbh, stuttgart, 3. völlig neu bearbeitete und erweiterte auf lage, 1182 seiten mit 166 abbildungen und 380 tabellen, hard cover, preis: 79,80 €, isbn: 978-3-8047-2879-0 journal of applied botany and food quality 88, 249 254 (2015), doi:10.5073/jabfq.2015.088.036 1dipartimento di ingegneria civile ed ambientale, università degli studi di perugia, perugia, italy 2 dipartimento di scienze farmaceutiche, università degli studi di perugia, perugia, italy ethnobotanical knowledge and nutritional properties of two edible wild plants from central italy: tordylium apulum l. and urospermum dalechampii (l.) f.w. schmid aldo ranfa1, fabio orlandi1, angela maurizi2, mara bodesmo1* (received july 3, 2015) * corresponding author summary edible wild plants have provided an important source of food since time immemorial and have continued to do so until the present day. the study aimed to evaluate ethnobotanical uses and nutraceutical properties of tordylium apulum l. and urospermum dalechampii (l.) f.w. schmidt. the ethnobotanical data collected showed that knowledge of these two species was not limited to alimentary use, but also included folk medicinal properties. data obtained by nutraceutical analysis demonstrated how these species contain many of the so-called minor nutrients, such as carotenoids, tocopherol, and polyphenols. furthermore in a comparison with some cultivated species, these species showed higher calcium, iron, and phosphorus values. t. apulum also showed significant vitamin a, polyphenol and orac values. introduction in recent years many authors in europe (leonti et al., 2006; pardo de santayana et al., 2007; herrera et al., 2014), and in italy (ranfa 2005a; 2005b; nebel and heinrich 2009; łuczaj et al., 2013; caneva et al., 2013; vitalini et al., 2013; ranfa et al., 2014; maurizi et al., 2015), have shown renewed interest in edible wild plants (ewp), particularly for new cuisines, in folk medicine and in nutrition. however, one of the aspects having attracted recent interest is the nutraceutical properties of ewp and the health benefits that derive from their habitual consumption (romojaro et al., 2013). some studies have shown that these species have significant nutraceutical value, being rich in mineral elements and bioactive compounds, with proven benefits for human health (ranfa et al., 2011; guarrera and savo 2013; ranfa et al., 2014; maurizi et al., 2015). the nutraceutical components defined as natural ingredients are considered to be the source of health benefits over and above their nutritional contribution (food and culture encyclopedia, 2010). numerous phytochemicals (plant chemicals) that occur in fruits and vegetables are key issues of research, and evidence exists regarding their healthpromoting properties, in particular anti-oxidising properties which are fundamental in the prevention of cancer and ageing related diseases (ards) (yang et al., 2001). ewp play a fundamental role in the mediterranean diet, where anti-oxidising foods make up an important part of daily food intake (shad et al., 2013; ranfa et al., 2014) thanks to their high polyphenol and unsaturated fatty acid content (de lorgeril and salen, 2007). unesco recently (2010) declared the mediterranean diet to be part of the ‘intangible cultural heritage of humanity’, and in this diet ewp are habitually used in various recipes and dishes (simopoulos, 2001). this study analyses two of the most widely known ewps, t. apulum and u. dalechampii, which have been collected in umbria (ranfa 2005a; 2005b; ranfa et al., 2011; 2014). as research on these two species has been concentrated mainly on traditional uses in cooking and in folk medicine (local food nutraceuticals consortium, 2005; di tizio et al., 2012; dolina and łuczaj, 2014), this study shows that, apart from their many ethnobotanical uses, these species also possess important nutraceutical properties, even when com pared to some cultivated species. study area the study was conducted in umbria, a region of central italy, situated between the mediterranean and the continental biogeographical regions. umbria has a surface area of around 8,456 km² and a population of 895,259, and is rich in biodiversity, including many endemic species. such diversity is reflected in the 102 natura 2000 sites accounting for more than 15 % of the regional surface (orsomando et al., 2004; cagiotti et al., 2010) (see fig. 1 a). materials and methods in umbria the tradition of collecting ewps in many of its natural and semi-natural areas is still alive. various events such as fairs, themed exhibitions and local markets are held regularly to disseminate information and promote interest, and a wide range of publications is available on local wild vegetables and their uses (fig. 1 b-c-d-e). among such activities, the department of applied biology of the university of perugia has for many years now been collaborating with the perugia mycological and field naturalists’association in organizing exhibitions of wild species, general theory courses and field trips. ethnobotanical analysis tordylium apulum l. (umbelliferae family) and urospermum dalechampii (l.) f.w. schmidt (compositae family) were chosen because they belong to two different families and because they are among the most widely known and collected plant species in umbria (ranfa, 2005a; 2005b; ranfa et al., 2011; 2014). ethnobotanical data were collected using an ad hoc semi-structured interview during the many events centred on ewps which took place in umbria in 2013 and 2014, for example the ‘mostra delle erbe spontanee’ organized by the perugia mycological and field naturalists association. fifty questionnaires containing information on the two species were collected, 42 citations for t. apulum and 35 for u. dalechampii were obtained, with some interviewees completing the questionnaire for both species. the interviewees, 40 females and 10 males, average age 65 years, were asked to provide information on local names, alimentary use, folk medicine properties, parts used and traditions (see fig. 2). they were selected from among the elderly population in rural areas who still retain knowledge of traditional uses, having spent their lives as farm workers, thus acquiring survival skills and practical knowledge by gathering, preparing and eating wild plants throughout the year. some informants were able to provide citations 250 a. ranfa, f. orlandi, a. maurizi, m. bodesmo on numerous wild plants. various samples of the two species were collected, analysed with a stereomicroscope sx45 (vision engineering limited), determined and classified according to the checklist of italian vascular flora (conti et al., 2007). all the exsiccata of the aforementioned species are preserved in the erbario peru of the università degli studi di perugia. nutraceutical analysis eight samples for each of the two species were collected, each sample consisting of at least 500 g of the edible portion of the plants, i.e. basal leaves, young leaves and stems, taken from more than 15 randomly chosen individual plants, all with a healthy external appearance. the samples were collected in various rural and semirural areas near perugia, assisi and on the slopes of mt. subasio (assisi) on flat or undulating land among abandoned olive groves. the analyses were carried out in part on fresh material and in part on dry samples; in both cases the material was hand-ground. the moisture content was determined by drying the leaves in an oven at 100 °c until constant weight was obtained according to the association of official analytical chemists aoac methods (aoac, 1990). crude protein content was calculated using the conversion factor 6.25 (n× 6.25) from the nitrogen content determined by the kjeldahl method (aoac, 1990). total lipid content was evaluated using chloroform/methanol extraction based on the bligh and dyer procedure as described in the aoac method (aoac, 1990). ash content was determined according to the aoac method (aoac, 1990) by incineration in a muffle furnace at 500 °c for 6 h. total dietary fibre was determined by the enzymatic-gravimetric method according to the aoac method (aoac, 1995). carbohydrates were calculated by difference. according to the aoac method (aoac, 2006) iron, calcium and magnesium determination was conducted via a aa-6800 model flame (air-acetylene) atomic absorption spectrophotometer (aas) (shimadzu, kyoto, japan) on the ashes, obtained from 4 g of each lyophilised sample, dissolved in 10 ml of 6 n of hydrochloric acid transferring the solution into a 50 ml volumetric flask and adjusting the volume with distilled water. calcium, magnesium and iron certified stock standard solutions, 1000 ppm in hno3 (carlo erba, milan, italy) were used. for calcium analysis, lanthanum chloride (5 %) was added to each sample to avoid interference with the phosphate. standard working solutions were prepared according to the user manual in order to obtain the calibration curve. sodium and potassium determination was conducted using a pfp7 flame photometer (bibby scientific, jenway, techne inc., uk) on the same solution and certified stock industrial standard solutions were purchased from jenway (jenway, essex, uk). analysis accuracy was con firmed for all metals studied using certified standard reference material (nist-srm-1573rd, tomato leaves). phosphorus content was determined colorimetrically on 0.2 g of each lyophilised sample reading the absorbance at 650 nm using ammonium molybdate, hydroquinone and sodium sulphide solutions according to the aoac methods (aoac, 1990). polyphenols were determined by the folin-ciocalteu method with spectroscopic measurement at 760 nm (varian uv/vis 50 cary bio model, palo alto, ca, usa). the results were expressed as mg of gallic acid equivalent (gae) per 100 g fresh weight (singleton and rossi, 1965). the assay was carried out on 250 mg of each lyophilised sample. the values obtained were compared with the data relative to other ewps analysed in ranfa et al. (2014) and with some cultivated species (inran; ieo). principal components with antioxidant functions and total antioxidant capacity were assessed by oxygen radical absorbance capacity (orac) method (ou et al., 2001; usda). α-tocopherol (vitamin e) and β-carotene (provitamin a) were determined by a modified np-hplc method (redi, 1999) operating with a spectra physics sp8800 model ternary pump (mountain view, ca, usa), and a sample-injection valve rheodyne 7125 (cotati, ca, usa) with a 10 l injection loop. two detectors were joined in series in order to determine α-tocopherol and β-carotene with a single run. α-tocopherol was detected by a fluorometric detector (fld) (jasco fp-920 model, tokyo, japan) equipped with of a light source of 150 w xenon lamp, λex. = 290 nm and λem. = 330 nm while β-carotene was detected by a uv/vis detector (shimadzu spd-10 a vp model, kyoto, japan) set at 450 nm. a personal computer with appropriate software (varian star chromatograph ver. 6.00 walnut creek, ca, usa) for data acquisition and processing was used. isofig. 1: study area and some local events on edible wild plants in umbria, central italy. ethnobotanical knowledge and nutritional properties of edible wild plants in central italy 251 cratic separation was achieved on a merck hibar, lichrosorb si 60, 250 mm × 4 mm i.d., 5 m particle size column (merck, darmstadt, germany) using a mixture of n-hexane and 2-propanol (98:2, v/v) as eluent at a flow-rate of 1 ml/min. the standards stock solution of α-tocopherol containing 500 mg/ml was prepared by dissolving 50 mg of α-tocopherol (calbiochem a brand of emd biosciences, inc., an affiliate of merck kgaa, darmstadt, germany) in a 100 ml volumetric flask with absolute ethanol (j.t. baker, mallinckrodt baker, milan, italy). solutions containing 0.25, 0.50, 1.00, 2.00, 4.00 and 8.00 mg/ml of α-tocopherol used for the calibra tion curve were prepared by appropriate dilution with n-hexane (j.t. baker, mallinckrodt baker, milan, italy) the stock solution. the standard stock solution of β-carotene (calbiochem a brand of emd biosciences, inc., an affiliate of merck kgaa, darmstadt, germany) was prepared by dissolving 25.8 mg of β-carotene in 100 ml in a volumetric flask with n-hexane. solutions containing 0.10, 0.20, 0.40, 0.80, 1.60 and 3.20 mg/ml of β-carotene used for the calibration curve were prepared by appropriate dilution of the stock solution with n-hexane, stored at -20 °c and made freshly every week. the extraction of α-tocopherol and β-carotene from the samples was carried out by the addition 25 ml of n-hexane to 0.5 g of each fresh sample placed in a 50 ml polypropylene graduate conical tube falcon 2070 (falcon, boston dickinson lab ware, nj, usa). the mixture was homogenised with an ultra turrax (ika ti 25, milan, italy) to obtain a fine suspension and then was centrifuged (eppendorf 5810 r model, milan, italy) at 4000 rpm for 5 min. two ml of the supernatant solution were transferred to a 10 ml flask and the n-hexane was evaporated under a mild stream of nitrogen at 30 °c; dry residue was dissolved in 0.5 ml of mobile phase. the resulting solution was analysed by np-hplc. the data, collected by workstation software (varian star chromatograph software version 6.00, walnut creek, ca, usa), were processed to quantify α-tocopherol and β-carotene with the method of external standardisation. total antioxidant capacity determination was carried out by hydrophilic oxygen radical absorbance capacity (h-orac). the assay was conducted with a fluorescent microplate reader fluo star optima (bmg labth gmbh, germany), provided with a pump, set to λex. = 485 nm and λem. = 520 nm and interfaced with a com puter provided with a mars data analysis software ver. 2.00 (bmg labth gmbh, germany) for data acquisition and processing. costar 96 well black opaque plates (corning costar corporation, cambridge, ma, usa) were used. two hundred μl of 60 nm fluorescein (sigma-adrich, steinheim, germany) working solution was added to all experiments designated in the wells. in addition, blank wells received 20 μl of 75 mm of phosphate buffer (ph 7.2), while standard wells received 20 μl of each 10, 20, 40 and 80 mm trolox (a vitamin e analogue hydrosoluble, 6-hydroxy-2,5,7,8tetramethyl-chroman-2-carboxyl acid) (sigma aldrich, steinheim, germany) standard solution in phosphate buffer and each sample well received 20 μl of solution as prepared below. four wells for the blank, four wells for each standard solution of trolox and four wells for the three samples for a total of 32 wells were used. the plate was then allowed to equilibrate by incubating for 30 min at 37 °c and then the fluorescence in each well was read by plate reader (2 cycles). in the next cycle, the pump was programmed to inject 60 l of 160 mm of aaph of freshly-prepared solution into the respective wells to start the reactions. the data were collected every 2.3 min by monitoring the fluorescence. each sample extract was obtained adding 20 ml of a ch3coch3: h2o: ch3cooh (70:29.5:0.5 v/v/v) mixture (prior et al., 2003) to 0.5 g of each fresh leaf sample in a 50 ml polypropylene graduate conical tube (falcon 2070 blue max, boston dickinson labware, nj, usa). the mixture was subjected to homogenisation (ika ti 25 ultra turrex, milan, italy) to obtain a fine suspension. after centrifuging at 4000 rpm for 5 min (eppendorf centrifuge 5810 r model, eppendorf, milan, italy) the supernatant was diluted 1:50 with phosphate buffer and the resulting solution was used for orac evaluation. each analytical determination was replicated three times except for the h-orac assay, which was carried out in quadruple. mean values and standard deviations were reported. results and discussion ethnobotanical study from the ethnobotanical information collected, it was found that t. apulum, locally known as pimpinellone, zampa d’oca, saporitella, or ombrellini pugliesi is mainly used in salads or to flavour savoury pies or vegetables soups. in the past its seeds were threaded to make bracelets and necklaces. in folk medicine it was recommended to prevent hair-loss and ‘nervous illnesses’, as well as to bring on menstruation and as an expectorant. the leaves of u. dalechampii, also known as grugno, boccione maggiore, lattugaccio, cotecacchia, grugnole or radicchione selvatico, are cooked, mixed with other herbs (e.g. sonchus oleraceus, tragopogon pratensis, or apium nodiflorum) and used in the same way as spinach. the root, which resembles a potato, is also used. the floral buds are preserved in brine and used in the same way as capers. the plant is also used to produce an infusion to aid digestion (fig. 2). scientific names botanical family local italian names part(s) used preparation folk medicine properties citations tordylium apulum l. umbelliferae pimpinellone, pimpinella vellutata, zampa d'oca, saporitella, ombrellini pugliesi leaves, whole aerial parts raw in salads or boiled for hair loss, soothing, expectorant 42 urospermum dalechampii (l.) f.w.schmidt. compositae grugno, boccione maggiore, lattugaccio, cotecacchia, grugnole or radicchione selvatico leaves, floral buds, roots boiled, raw in salads, filling savoury pie, floral buds used in the same way as capers diuretic, digestive, tonic 35 fig. 2: the two edible wild plants analyzed. local use, common names, parts used, preparation, some folk medicine properties and number of ad hoc semistructured interview collected. 252 a. ranfa, f. orlandi, a. maurizi, m. bodesmo nutraceutical aspects it was found that u. dalechampii has high values of iron, calcium and potassium. t. apulum has higher β-carotene and vitamin e values than for example a. graveolens, b. vulgaris, c. intybus (see tab. 2). furthermore, when comparing the two species’ nutraceutical values with those of other ewps (ranfa et al., 2014) it was found that u. dalechampii contains significant protein values similar to sanguisorba minor and that both species contain higher phosphorus values than bellis perennis, bunias erucago and chondrilla juncea. particular attention needs to be drawn to antioxidant capacity calculated by means of the orac method. significant values were found particularly in s. minor whose antioxidant capacity was superior to cultivated species such as tomatoes, peppers and carrots (source usda). fiber content, which plays a role in improving nutrient bioavailability, was also considerable. (tab. 1). t. apulum showed significant total polyphenol and orac values. (tab. 2). conclusion the results obtained in this research demonstrate that the two species analyzed contain considerable nutraceutical properties to supplement a balanced daily diet, this being one of the principal reasons to preserve and divulge knowledge of ewps. their antioxidant capacity could certainly assist nutritionists and doctors in planning more balanced diets in an effort to prevent degenerative and chronic diseases. indeed ewps are being investigated as potential food supplements in various developing countries so as to increase the quality of daily food for rural populations. however, today we are witnessing a nutritional transition in the world’s poorest countries whereby traditional diets based on fruit and vegetables are being replaced by calorie-rich diets based on animal fats and sugars. another, no less important, aspect to consider is the environmental issue: our ethnobotanical heritage must be safeguarded as it represents an opportunity to preserve biodiversity, maintain the links between town and countryside, and encourage new forms of development such as environmental tourism (caneva et al., 2013). it would be important to retain and reinforce local knowledge of these species so as to develop new, relevant, and effective ways to revitalize languages, cultures, and ethnobotanical knowledge within contemporary contexts. references aoac, 1990: association of official analytical chemists. official methods of analysis, 15th ed. method number 930.04 (moisture), method number 978.04 (protein), method number 983.23 (lipid), method number 930.05 (ash), method number 931.01 (phosphorus), arlington, va. aoac, 1995: association of official analytical chemists. official methods of analysis, 16th ed. method number 985.29 (fiber), aoac international, gaithersburg, md. aoac, 2006: official methods of analyses of association of analytical chemistry, 18th ed. method number 975.03, aoac international, gaithersburg, md. cagiotti, m.r., landucci, f., marinangeli, f., bodesmo, m., ranfa, a., 2010: umbria. in: celesti-grapow, l., pretto, f., blasi, c. (ed.), flora alloctona d’italia, 95-100. palombi editore, roma. caneva, g., pieroni, a., guarrera, p.m., 2013: etnobotanica. conservazione di un patrimonio culturale come risorsa per uno sviluppo sostenibile. edipuglia srl, bari. conti, s., alessandrini, f., bacchetta, a., banfi, g., barberis, e., bartolucci, g., bernardo, f., bonacquisti, l., bouvet, d., bovio, m., brusa, g., del guacchio, e., foggi, b., frattini, s., galasso, g., gallo, l., gangale, c., gottschlich, g., grünanger, p., gubellini, l., iiriti, g., lucarini, d., marchetti, d., moraldo, b., peruzzi, l., poldini, l., prosser, f., raffaelli, m., santangelo, a., scassellati, e., scortegagna, s., selvi, f., soldano, a., tinti, d., ubaldi, d., uzunov, d., vidali, m., 2007: integrazioni alla checklist della flora vascolare italiana. nat. vicentina. 10, 5-74. dolina, k., łuczaj, ł., 2014: wild food plants used on the dubrovnik coast (south-eastern croatia). acta soc. bot. pol. 83(3), 175-181. tab. 1: comparison of chemical composition and mineral content (mg/100 g of edible parts) values of t. apulum and u. dalechampii with those of other cultivated species t. apulum u. dalechampii celery chard cultivated cichory arugula lettuce (apium (beta (cichorium (eruca sativa (lactuca sativa graveolens l.) vulgaris l.) intybus l.) hill.) l.) chemical composition water 85.5±0.91 86.1±1.03 88.3a 89.3a 95a 91a 94.3a protein 2.2±0.08 2.1±0.07 2.3a 1.3a 1.2a 2.6a 1.8a lipids 0.3±0.02 0.2±0.03 0.2a 0.1a 0.1a 0.3a 0.4a carbohydrates 5.4±0.87 5.0±0.74 2.4a 2.8a 1.7a 3.9a 2.2a dietary fiber 5.0±0.23 5.1±0.28 1.6a 1.2a 3.1b 0.9a 1.5a mineral content iron 2.5±0.74 3.0±0.91 0.5a 1a 1.5a 5.2a 0.8a calcium 153.7±12.51 174.9±15.73 31a 67a 150a 309a 45a phosphorus 24.2±4.21 29.5±6.48 45a 29a 26a 41a 31a sodium 42.0±3.21 68.4±2.47 140a 10a 7a 27b 9a potassium 344.5±28.16 287.5±32.35 280a 196a 180a 468a 240a magnesium 14.3±1.46 15.3±1.89 16a 38a a source: tabelle di composizione degli alimenti. inran istituto nazionale di ricerca per gli alimenti e la nutrizione. b source: banca dati di composizione degli alimenti per studi epidemiologici in italia (bda) – ieo istituto europeo di oncologia ethnobotanical knowledge and nutritional properties of edible wild plants in central italy 253 de lorgeril, m., salen, p., 2007: modified cretan mediterranean diet in the prevention of coronary heart disease and cancer. in: simopoulos, a.p., visioli, f. 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http://www.ars.usda.gov/main/site_main.htm?modecode=12-35-45-00 vitalini, s., iriti, m., puricelli, c., ciuchi, d., segale, a., fico, g., 2013: traditional knowledge on medicinal and food plants used in val tab. 2: β-carotene, vitamin e, total polyphenol content and orac values in 100 g of the edible parts of the two wild plants analyzed t. apulum u. dalechampii celery chard cultivated cichory arugula lettuce (apium (beta vulgaris (cichorium (eruca sativa (lactuca sativa graveolens l.) l.) intybus l.) hill.) l.) β-carotene 2,614±30 2,304±25 1242b 1578b 1315b 1422b 1374a (μg/100 g) vitamin a 436±6 384±5 207a 263a 267a 742a 229 (μgret. eq./100 g) α-tocopherol 2.8±0.04 2.1±0.08 0.48b 1.05b 2.26b 0.43b 0.66b (vit.e) (mg/100 g) total polyphenols 331±7.0 143±4.8 (mg gae/100 g) orac 493±34 437±41 (μmolte/100 g) asource: tabelle di composizione degli alimenti. inran istituto nazionale di ricerca per gli alimenti e la nutrizione. bsource: banca dati di composizione degli alimenti per studi epidemiologici in italia (bda) – ieo istituto europeo di oncologia 254 a. ranfa, f. orlandi, a. maurizi, m. bodesmo san giacomo (sondrio, italy) an alpine ethnobotanical study. j. ethnopharmacol. 145(2), 517-529. yang, t., devaraj, s., jialal, i., 2001: stress ossidativo e aterosclerosi. j. clin. ligand assay (ed. italiana) 24, 13-24. address of the corresponding author: mara bodesmo, dipartimento di ingegneria civile ed ambientale, università degli studi di perugia, borgo xx giugno, 06121 perugia, italy e-mail: bodesmo@hotmail.it © the author(s) 2015. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 89, 249 257 (2016), doi:10.5073/jabfq.2016.089.032 fraunhofer institute for process engineering and packaging, department of process engineering, freising, germany in vitro-study of antioxidant extracts from garcinia mangostana pericarp and riesling grape pomace – a contribution to by-products valorization as cosmetic ingredients judith wittenauer*, ute schweiggert-weisz, reinhold carle (received may 13, 2016) * corresponding author summary the objective of the present study was to compare extracts from two different plant sources regarding their suitability as antioxidant cosmetic ingredients. both, garcinia mangostana pericarp, in europe being widely considered as the non-edible part of a ‘superfruit’, and riesling grape pomace accruing from vinification, represent important by-products from food processing. mixtures of ethanol and water in different ratios were used for the preparation of polyphenol containing extracts. antioxidant properties of the extracts were determined using well-established in vitro assays (folin-ciocalteu, orac, dpph, and abts), and skin penetration was investigated by franz-type diffusion experiments with porcine skin. extraction of polyphenols was most effective using an equimolar ratio of ethanol and water for both raw materials. absolute polyphenol contents of mangosteen pericarp extract (65360.71 ± 1168.51 mg/kg dm) were higher than for grape pomace extract (18085.70 ± 411.50 mg/kg dm). however, folin-ciocalteu reducing capacities as well as in vitro antiradical activities did not adequately correspond to quantitative polyphenol estimations, as grape pomace extract showed relatively high antiradical capacities. when applied to pig skin, polyphenols from grape pomace extract were detected in low concentrations in the dermis as well as in the transdermal receptor fluid. in contrast, mangosteen pericarp xanthones were almost completely recovered with highest amounts detected in the dermis. in conclusion, both raw materials revealed potential as antioxidant ingredients for cosmetic formulations. keywords grape pomace; garcinia mangostana l.; antioxidants; polyphenols; franz-type diffusion introduction constituting the outer barrier of the body, the skin is perfectly designed for confining and protecting the inner organs from the outer environment. exposure to exogenous noxae such as uv radiation and toxins is a big challenge to this interface function. therefore, harmful reactive oxygen species (ros) and other free radicals are readily produced in the skin through diverse pathways (rittie and fisher, 2002; scharffetter-kochanek et al., 2000). like any other organ, the skin disposes of an antioxidant network that helps to cope with ros which are continuously formed intrinsically in the course of normal aerobic metabolism (shindo et al., 1994; thiele et al., 2002). however, overwhelming radical production by external prooxidant stimuli may disturb the sensitive balance between oxidants and antioxidants in the skin resulting in the harmful state of oxidative stress (thiele et al., 2002; thiele et al., 1998). consequently, inflammatory processes may occur, resulting in premature skin aging, and the formation of skin disorders such as photosensitivity and even cutaneous malignancy (bickers and athar, 2006). topically applied antioxidants are seen to be a potential measure to prevent such adverse effects at an early stage. some of the most commonly employed antioxidants in the cosmetic sector are synthetic vitamins, e.g. ascorbic acid, niacin and tocopherol, and also lipoic acid, buty lated hydroxytoluene, and butylated hydroxyanisole. in order to ensure stability and solubility of these substances in cosmetic emulsions, chemically modified derivatives thereof are applied (austria et al., 1997; kerscher and buntrock, 2011; thiele et al., 2005). since consumer acceptance of synthetic and chemically modified ingredients decreases, natural alternatives from plants are urgently demanded. due to their ubiquitous occurrence in nature, and because polyphenols act as potent hydrogen donators, they have been recognised as potent antioxidant bioactives (leopoldini et al., 2011; quideau et al., 2011; rice-evans et al., 1996). so far, only few plant extracts being rich in polyphenolics are applied in cosmetic products, and sound scientific data about their contribution to skin health are scarce (briganti and picardo, 2003; ribeiro et al., 2015). most studies have been performed with green tea, and, in particular, individual polyphenols thereof, e.g. epigallocatechin and epigallo catechin gallate, have been evaluated (dal belo et al., 2009; dvorakova et al., 1999; gianeti et al., 2013; wisuitiprot et al., 2011). due to their appealing image and with emphasis on marketing in the cosmetic sector, fruits, herbs and flowers are the most popular plant sources for cosmetic bioactives. the so-called ‘superfruits’ are considered to be most interesting for cosmetic purposes, since they are rich sources of antioxidants with alleged health benefits (gross, 2010). among this fruit category, mangosteen (garcinia mangostana l.), and, particularly, the purple coloured pericarp of the fruit has gained the attention of several research groups as they are rich sources of xanthones (obolskiy et al., 2009; pedraza-chaverri et al., 2008; wittenauer et al., 2012). the edible part of the fruit, constituting the aril, which is freshly eaten, dried, frozen or canned only amounts to about 30 % of its fresh weight, while the pericarp and seed are considered as waste (okonogi et al., 2007). thailand is the most important producer with an annual production of approximately 240,000 t. hence, sustainable production through waste valorization is an urgent challenge (diczbalis, 2009). xanthones recently identified in mangosteen pericarp seem to be suitable antioxidants in cosmetics for various reasons (wittenauer et al., 2012). firstly, due to the hydroxyl groups, attached to the unsaturated heterocyclic xanthone core, xanthones exert high antioxidant activity (martinez et al., 2011; sze lim et al., 2013). secondly, mangosteen extracts have been suggested as biopreservatives in food industry (sze lim et al., 2013). thirdly, the prenyl substituents might enhance skin penetration by increasing their lipophilicity, and, thus, the affinity to cell membranes (botta et al., 2005). by analogy to tropical fruit processing, large amounts of grape pomace accrue from vinification (balasundram et al., 2006), thus also representing polyphenol rich by-products which may be valorised for cosmetic purposes (balasundram et al., 2006; schieber et al., 2001). grape pomace has been reported to contain a complex spectrum of low molecular weight polyphenols such as phenolic acids and flavonoids as well as higher molecular procyanidins, and tannins (kammerer et al., 2004; katalinić et al., 2010; maier et al., 2009; schieber et al., 250 j. wittenauer, u. schweiggert-weisz, r. carle 2001). although many attempts have been made for the exploitation of grape pomace (maier et al., 2009; wittenauer et al., 2015), its valorisation has not been implemented in wine making industry up to now. as grape pomace extracts contain a variety of bioactive molecules, allowing intermolecular interactions of the individual compounds, such interdependencies may not only affect the overall antioxidant activity, but also their penetration behaviour to the targeted skin layer (abou samra et al., 2011; choueiri et al., 2012; seeram et al., 2004). the objective of the present study was the comparison of mangosteen pericarp and grape pomace extracts regarding their antiradical properties and their potential as antioxidative cosmetic ingredients. to allow their direct comparison, both plant extracts were made based on identical extraction conditions and by using the same aqueous ethanolic solvent, which can readily be used for their incorporation in cosmetic products due to their toxicological innocuousness. additionally, both plant materials were extracted with different concentrations of aqueous ethanol to obtain samples of different polyphenol pattern and polarities. the respective extracts were analysed by means of the commonly used in vitro assays orac, dpph, abts, and folin-ciocalteu. finally, franz-type diffusion experiments were executed with the most promising extracts of both raw materials enabling insight into the penetration depth of the polyphenols and, therefore, the presumed antioxidant performance of the polyphenolic molecules after topical application. materials and methods materials thai mangosteens (garcinia mangostana l.) were obtained from a local retailer. pericarp of the fruits was manually separated, minced in a bowl chopper, and immediately flash-frozen with liquid nitrogen prior to lyophilisation. the lyophilised mangosteen pericarp was kept at 5 °c until analysis. grape pomace from white wine grapes (vitis vinifera l. cv. ‘weisser riesling’) was used in this study. pomace samples were kindly provided by the baden-badener winzergenossenschaft (baden-baden-neuweier, germany) and directly collected after pressing the mash, lyophilised, and kept at 5 °c until further use. polyphenols used as standards for hplc-analysis were α-mangostin (97 %), trans-resveratrol (98 %) (abcr, karlsruhe, germany), gallic acid (≥97 %), epicatechin (≥99 %) (sigma, st. louis, mo, usa); caftaric acid (≥97 %), procyanidin b1 (≥90 %), procyanidin b2 (≥90 %), quercetin 3-o-glucoside (≥98 %), and quercetin 3-o-glucuronid (≥95 %) (fluka, buchs, switzerland). abts (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonicacid)), dpph (2,2-diphenyl-1-picrylhydrazyl), trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), sodium fluorescein, 2,2’-azobis(2-amidinopropane) dihydrochloride (aaph), folin-ciocalteu reagent, and gallic acid were used for the determination of the antiradical activity and the folin ciocalteu assay, respectively, and purchased from sigma (st. louis, mo, usa). preparation of polyphenol rich extracts lyophilised mangosteen pericarp and grape pomace, respectively, were ground using a zm 100 centrifugal mill with a 1 mm ring sieve (retsch, haan, germany). aliquots of 1 g of the powdered samples were extracted with 50 ml of different ethanol/water mixtures (30/70; 50/50; 70/30 (v/v)) at room temperature for 60 min under continuous stirring. the thus obtained plant extracts were filtered through a folded filter (munktell, bärenstein, germany), and directly used for antiradical activity assays. for hplc-analysis, the extracts were filtered through a 0.2 μm syringe filter (sartorius, göttingen, germany). hplc analyses identification and quantitation of individual polyphenols contained in the plant extracts were performed using an agilent hplc series 1200 system (agilent, waldbronn, germany) with chemstation software, a model g1379 degasser, a model g1312b binary gradient pump, a model g1367d thermo autosampler, a model g1316b column oven and a model g1315c diode array detector. for the separation of grape pomace polyphenols, a kinetex f5 column (150 × 3.0 mm i.d.; 4 μm particle size) was used. xanthones from mangosteen pericarp were separated on a kinetex c18 column (150 × 3.0 mm i.d.; 4 μm particle size) (phenomenex (torrance, ca, usa)) operated at 25 °c. the mobile phases were composed of 0.5 % acetic acid (eluent a) and acetonitrile (eluent b). analyses were performed in quadruplicate at 25 °c using gradient programs as follows: grape pomace extract 0-20 % b (80 min), 20-100 % b (20 min), 100 % b isocratic (3 min), 100-0 % b (10 min) at a flow rate of 0.4 ml/min; mangosteen pericarp extract 50-60 % b (20 min), 60-70 % b (35 min), 70-100 % b (5 min), 100 % b isocratic (3 min), 100-0 % b (2 min) at a flow rate of 0.6 ml/min. injection volumes varied between 8 and 40 μl. identification and quantitation of individual polyphenols was performed as described previously (wittenauer et al., 2012; wittenauer et al., 2015). total phenolic content (folin-ciocalteu) total phenolics were determined by the folin-ciocalteu method, according to singleton and rossi (1965) with slight modifications. aliquots of 20 μl diluted extract, 1 ml water and 0.1 ml folin reagent were mixed. after 3 min, 0.2 ml sodium carbonate solution (7.5 %, w/v) and 0.68 ml water were added. absorption was measured at 765 nm after 30 min using a specord 210 plus spectrophotometer (analytik jena, jena, germany). total polyphenols were quantitated by calibration using gallic acid (ga) as a reference. the reducing capacity was calculated as gallic acid equivalent (gae). all determinations were performed in triplicate. antiradical activity antiradical activity by abts+• was carried out following the method of re et al. (1999). abts+• stock solution was prepared dissolving 38.4 mg abts diammonium salt and 6.62 mg potassium persulfate in 10 ml water, and stored at 8 °c until needed. final radical solution was prepared by diluting the stock solution with water until an initial absorption value (734 nm) of 0.70 ± 0.02 was reached. for the determination of the antiradical activity, 10 μl of each extract was mixed with 9.99 ml abts radical solution, and the absorption was measured at 734 nm on a specord 210 plus spectrophotometer (analytik jena, jena, germany) after incubation at 30 °c for 15 min. ethanolic solutions of trolox in the range of 25-1500 μmol/l were used for calibration of the abts, dpph and orac assays. the dpph assay was performed according to the method of brandwilliams et al. (1995) with some modifications. dpph stock solution was prepared dissolving 24 mg dpph in 100 ml absolute ethanol (100 %), and stored at 8 °c until needed. the working solution was obtained by 10 fold dilution of the stock solution. aliquots of 10 μl of the extracts were mixed with 9.99 ml of the working solution. immediately after 15 min of incubation at 25 °c, the absorption was measured at 517 nm using a specord 210 plus spectrophotometer. the orac assay was carried out on an automated plate reader (synergy h1, biotek, winooski, vt, usa) with 96-well plates. the procedure was based on a previous report by huang et al. (2002). analyses were conducted at 37 °c in 75 mm phosphate buffer (ph 7.4) with 25 μl of extract. peroxyl radicals were generated using freshly prepared aaph (2,2’-azobis(2-amidino-propane) dihydro antioxidant extracts from garcinia mangostana pericarp and riesling grape pomace 251 chloride) solution for each run (153 mm). fluorescein was used as the substrate (4 nm). fluorescence conditions were as follows: excitation at 485 nm; emission at 520 nm, and fluorescence was recorded every minute after addition of aaph. when the absorbance value measured exceeded the linear range of the standard curve, samples were further diluted. orac values were calculated as described by cao and prior (1999) using a regression equation between the trolox concentration and the net area under the fluorescence decay curve. the area under the curve was calculated as where f0 is the initial fluorescence reading at 0 min and fi the fluorescence reading at time i. the net auc was obtained by subtracting the auc of the blank from that of the sample. all antiradical activity measurements were performed in triplicate, and results were expressed in μm te/ kg dry mass (dm). in vitro skin permeation studies preparation of pig ear skin pig ears were obtained from a local butcher immediately after slaughter. hairs were carefully removed by shaving. excision of skin segments from the underlying cartilage was carried out using a scalpel. the skin segments were carefully inspected to ensure the absence of perforations or other defects. skin disks of 25 mm were punched out with a hollow punch, wrapped in aluminium foil, and stored in polyethylene bags at -20 °c until use. franz-type skin permeation experiment in vitro skin permeation studies were performed according to zillich et al. (2013a) using static franz-type diffusion cells (permegear, hellertown, pa, usa) with effective diffusion area of 4.91 cm2 and inner diameters of 25 mm. permeation cells were heated by a thermostatic circulating water bath at 32 °c throughout the experiment. during the experiments, the receptor fluid (phosphate buffered saline, ph = 7.4) was continuously stirred by a magnetic stirrer. porcine skin disks were positioned between donor and receptor compartments, assuring their direct contact with the receptor fluid and the absence of air bubbles. after an equilibration time of 15 min, 500 μl of the plant extracts were pipetted on each skin sample. control cells were treated with 500 μl of ethanol/water (v/v). diffusion cells were covered with parafilm (brand, wertheim, germany), and protected from light by covering with aluminium foil. experiments were carried out 8 fold for a time period of 24 h. at the end of the permeation experiments, skin samples were dabbed carefully with a cellulose tissue to recover the remaining plant extract that did not penetrate the skin. subsequently, stratum corneum samples were obtained by 10 fold stripping of the permeation area each with adhesive tesa® tape (beiersdorf, hamburg, germany). the remaining skin was cut into small pieces using a fine scissor, and ground under cryogen conditions at -196 °c (cryomill, retsch, haan, germany), and lyophilized to obtain a powder. cellulose tissue, adhesive tape as well as the skin powder were extracted with 50 ml of methanol/water (90/10, v/v), evaporated to dryness at 40 °c in vacuo, dissolved in 1 ml of methanol and filtered through a 0.22 μm membrane filter (whatman, dassel, germany) for hplc analysis. the acceptor medium was concentrated from 21 to 1 ml by evaporation under nitrogen, membrane-filtered (0.22 μm), and used for hplc analysis. statistical analysis results are presented as mean values with their standard errors. statistical analysis was carried out using single-factor one-way anova. tukey’s post hoc test was performed to determine the significant difference between the groups. results were considered significantly different when p<0.05 was obtained. the relationship among hplc-, folin-ciocalteu and antiradical assay results was described by the pearson product-moment correlation coefficient (katalinić et al., 2010). results and discussion the polarity and type of solvent used for extraction of plant material had marked effects on the polyphenol composition of the antioxidant rich extracts. as ethanol is a usual solvent for the preparation of cosmetic ingredients, aqueous solutions of 30 %, 50 % and 70 % ethanol were applied for the extraction of grape pomace and mangosteen pericarp polyphenols. subsequently, polyphenol patterns, antioxidative capacities as well as the skin permeation behaviour of extract obtained from both plant sources were studied. identification and quantitation of polyphenolic constituents nine major polyphenols could be identified and quantitated in all grape pomace extracts by means of hplc analysis (fig. 1). in accordance with literature data (kammerer et al., 2004; wittenauer et al., 2015), the occurrence of gallic acid and caftaric acid, catechin and epicatechin, the procyanidins b1 and b2 as well as two quercetin-glycosides and resveratrol was confirmed. variation of ethanol contents of the extraction medium had impact on the total amounts as well as the proportion of the individual polyphenols in the grape pomace extracts. total amount of polyphenols, calculated as the sum of the nine individually quantified compounds, was highest using 50 % ethanol as solvent, resulting in 18,085.70 ± 411.50 mg/kg grape pomace (dm). in this case, highest extraction yields were obtained for catechin and epicatechin, but also flavanol-glycosides such as quercetin-3-o-glucuronide and quercetin-3-o-glucoside and resveratrol were extracted most efficiently. in contrast, yields of higher molecular procyanidins b1 and b2 reached a maximum, when applying 30 % of ethanol, probably due to their better water solubility (zanchi et al., 2009). application of 70 % ethanol as extraction solvent significantly lowered polyphenol contents of most compounds detected. in all three mangosteen pericarp extracts α-mangosteen was the major constituent followed by γ-mangosteen, as reported in our previous study (wittenauer et al., 2012) (fig. 2). variation of ethanol contents did not change the xanthones pattern. this might probably be due to their high structural similarity and polarities, in contrast to the complex composition of grape pomace extract comprising different phenol subclasses. efficient extraction of xanthones from mangosteen pericarp was obtained by ethanol concentrations of the extraction solvent exceeding 50 % (65,360.71 ± 1168.51 mg/kg dm), while applying 70 % of ethanol did only slightly raise the yield reaching 66,769.37 ± 1239.27 mg/kg dm. altogether, total amounts of individually quantified xanthones markedly exceeded that of grape pomace polyphenols when applying ethanol concentration of 50 % and 70 %. total polyphenol content (folin-ciocalteu) the folin-ciocalteu assay being a non-specific standardized method solely allows a first indication of the total reducing potential of all reductive compounds contained in a sample. as polyphenols are the main components present in the extracts under investigation, results of the folin-ciocalteu assay may represent the total phenolic content. however, additive contributions of ascorbic acid, thiols, aromatic amines, and reducing sugars must be considered (sanchez-rangel et al., 2013). immediately after 15 min of incubation at 25°c, the absorption was measured at 517 nm using a 204 specord 210 plus spectrophotometer. 205 the orac assay was carried out on an automated plate reader (synergy h1, biotek, winooski, vt, 206 usa) with 96-well plates. the procedure was based on a previous report by huang et al. (2002). 207 analyses were conducted at 37°c in 75 mm phosphate buffer (ph 7.4) with 25 µl of extract. peroxyl 208 radicals were generated using freshly prepared aaph (2,2’-azobis(2-amidino-propane) 209 dihydrochloride) solution for each run (153 mm). fluorescein was used as the substrate (4 nm). 210 fluorescence conditions were as follows: excitation at 485 nm; emission at 520 nm, and fluorescence 211 was recorded every minute after addition of aaph. when the absorbance value measured exceeded 212 the linear range of the standard curve, samples were further diluted. orac values were calculated as 213 described by cao and prior (1999) using a regression equation between the trolox concentration and 214 the net area under the fluorescence decay curve. the area under the curve was calculated as 215 𝐴𝐴𝐴𝐴𝐴𝐴 = 0.5 + � 𝑓𝑓1 𝑓𝑓0 � + � 𝑓𝑓2 𝑓𝑓0 � + � 𝑓𝑓3 𝑓𝑓0 � + ⋯ + ( 𝑓𝑓𝑓𝑓 𝑓𝑓0 ) where f0 is the initial fluorescence reading at 0 min and fi the fluorescence reading at time i. the net 216 auc was obtained by subtracting the auc of the blank from that of the sample. 217 all antiradical activity measurements were performed in triplicate, and results were expressed in µm 218 te/ kg dry mass (dm) 219 220 221 2.6 in vitro skin permeation studies 222 3.6.1 preparation of pig ear skin 223 pig ears were obtained from a local butcher immediately after slaughter. hairs were carefully 224 removed by shaving. excision of skin segments from the underlying cartilage was carried out using a 225 scalpel. the skin segments were carefully inspected to ensure the absence of perforations or other 226 defects. skin disks of 25 mm were punched out with a hollow punch, wrapped in aluminium foil, and 227 stored in polyethylene bags at -20°c until use. 228 229 2.6.2 franz-type skin permeation experiment 230 in vitro skin permeation studies were performed according to zillich et al. (2013a) using static franz-231 type diffusion cells (permegear, hellertown, pa, usa) with effective diffusion area of 4.91 cm2 and 232 inner diameters of 25 mm. permeation cells were heated by a thermostatic circulating water bath at 233 32°c throughout the experiment. during the experiments, the receptor fluid (phosphate buffered 234 saline, ph = 7.4) was continuously stirred by a magnetic stirrer. porcine skin disks were positioned 235 between donor and receptor compartments, assuring their direct contact with the receptor fluid and 236 the absence of air bubbles. after an equilibration time of 15 min, 500 µl of the plant extracts were 237 252 j. wittenauer, u. schweiggert-weisz, r. carle fig. 2: xanthone composition of mangosteen pericarp extracts obtained by extraction with different ethanol/water ratios (v/v) as determined by hplc-dad. *p-value < 0.05 fig. 1: polyphenolic composition of cv. ‘riesling’ grape pomace extracts obtained by extraction with different ethanol/water ratios (v/v) as determined by hplc-dad. * p-value < 0.05 concerning the three grape pomace extracts, maximum total polyphenol content was determined for the 70 % ethanol extract amounting to 50.94 g gae/kg dm (fig. 3). folin-ciocalteu values rose significantly with increasing ethanol content of the extraction medium from 30 % ethanol to 50 % ethanol. however, differences between folin-ciocalteu values of extracts prepared with 50 % ethanol and with 70 % ethanol were insignificant. results of the folin-ciocalteu assay for the grape pomace extracts are in contrast to the hplc results, where total polyphenol amounts were considerably lower for extracts produced with 70 % ethanol. poor correlation of folin-ciocalteu and hplc-dad values reflected this incongruity, which was supported by the moderate correlation coefficient of 0.717. it appears, that by extracting with 70 % ethanol further reducing compounds were dissolved, which were not determined by hplc; however, reacting with the folin-ciocalteu reagent. additionally, higher ethanol concentrations in the extract might increase the reducing capacities of the grape pomace polyphenols by a solvent induced enhancement of hydrogen-atom transfer, which has previously been reported for polar compounds such as the grape pomace polyphenols (nenadis and tsimidou, 2002). reducing capacities of mangosteen pericarp extracts ranged from 45.61 to 85.44 g gae/kg dm applying 30 % and 50 % ethanol, respectively. in contrast to the grape pomace extracts, folin-ciocalteu values of mangosteen pericarp extracts well correlated with the results of the hplc analysis (r = 0.997). despite their superior individual polyphenol contents as determined by hplc-analysis, reducing capacities of mangosteen pericarp extracts were only about 1.7 fold higher than those of grape pomace extracts. this may be due to the generally lower reducing power of individual xanthones, because, with the exception of γ-mangostin and garcinon e, potent antioxidant extracts from garcinia mangostana pericarp and riesling grape pomace 253 reductive ortho-diphenolic components such as gallic and caftaric acids, catechin and epicatechin, procyanidin b1 and b2 as well as quercetin-glycosides are typical of grape. in vitro antioxidant activity various in vitro assays mimicking the radical scavenging activity of isolated compounds and complex preparations are commonly carried out, each of them only partially reflecting their potential antioxidant power (lopez-alarcon and denicola, 2013; prior et al., 2005). to overcome specific limitations and reflect sample complexity as close as possible, analyses of the plant extracts were carried out based on multiple assays, namely the abts, dpph, and orac systems. the non-physiological radical molecules abts•+ and dpph• are useful to get first indications of the radial-scavenging ability of polyphenols by single electron transfer (set) reactions in general (jiménez et al., 2004). complementary to abts•+ and dpph•, inhibition of peroxyl radical induced oxidations, and, thus, to some extent, the classical radical chain-breaking antioxidant activity (prior et al., 2005) may be estimated by the orac assay. analyses of in vitro antioxidant capacities by the abts, dpph and orac assays resulted in higher values for the mangosteen pericarp extracts than for the grape pomace extracts (fig. 4). however, in accordance with the results of the folin-ciocalteu assay, the clearly higher contents of individual polyphenols contained in mangosteen pericarp extracts were not adequately reflected by the respective antioxidant activity values. all three grape pomace extracts showed similar antioxidant behaviour when applying the abts and dpph assays. with increasing ethanol contents of the extraction solvent, the antioxidant activity of the grape pomace extracts also increased. dpph values were generally lower than abts values, probably indicating less reactivity of grape pomace polyphenols towards this radical in contrast to abts radical. regarding the orac assay, increasing ethanol content of the extraction medium was paralleled by a significant rise of the antioxidant activity resulting in maximum values of 1131.12 mmol teq/kg (dm) for the highest ethanol concentration (70 %). investigation of the mangosteen pericarp extracts resulted in comparable trends for both the abts and orac assays. maximum abts (1063.69 mmol teq/kg (dm)) and orac (1543.09 mmol teq/ kg (dm)) values were found when using 50 % ethanol as extraction solvent. concordant with the results of grape pomace extracts, dpph values were generally lower. in addition, differences between the extracts resulting from three different ethanol concentrations were insignificant. to correlate the results obtained based on the in vitro antioxidant capacity assays with polyphenol concentrations in the extracts obtained by folin-ciocalteu and hplc measurements, pearson coefficients were calculated (tab. 1). results from the abts and dpph assays of grape pomace extracts fairly correlated with the amounts quantified by hplc yielding pearson coefficients of 0.81 and 0.62, respectively. by contrast, there was no correlation between the values attained by orac assay and the corresponding hplc data, while abts and dpph values correlate very well with the folin-ciocalteu values (r = 0.99). this may be due to similar reaction mechanisms of the latter assays, since the abts, dpph and folin-ciocalteu assay are all based on electron transfer reactions (huang et al., 2005). the considerably poorer correlation of the folin-ciocalteu values with the results of the orac assay (r = 0.74) confirmed this assumption, probably due to the underlying hydrogen atom transfer mechanism of the orac assay. the results indicate that the primary step of the antioxidant behaviour of the grape pomace polyphenols might be an electron transfer, as the detected compounds displayed stronger interaction with the abts and dpph radicals. in the case of man fig. 4: antioxidant activities of cv. ‘riesling’ grape pomace (a) and mangosteen pericarp (b) extracts as determined by the abts, dpph, and orac assays. * p-value < 0.05 fig. 3: reducing capacity of cv. ‘riesling’ grape pomace and mangosteen pericarp extracts as determined by folin-ciocalteu-assay. * p-value < 0.05 254 j. wittenauer, u. schweiggert-weisz, r. carle gosteen pericarp extracts, the abts and orac values showed remarkable correlation with the data of hplc and the folin-ciocalteu assay. it may be assumed that the primary antioxidative action of xanthones is based on both, an electron transfer and a hydrogen atom abstraction mechanism. in vitro skin permeation studies it was expected that the pattern and quantity of the polyphenols probably may change on their passage across porcine skin. there fore, information about the penetration of the applied polyphenols may be helpful to approximate to the resulting antioxidant activities in the different skin layers. according to literature, lipophilicity and molecular weight of the bioactives are key parameters influencing percutaneous absorption, apart from their release from the vehicle and the kind of vehicle used (abla and banga, 2013; alonso et al., 2014; dvorakova et al., 1999; suppasrivasuseth et al., 2006; zillich et al., 2013b). percutaneous absorption of polyphenols contained in the plant extracts was investigated by franz-type diffusion experiments. for this purpose, extracts from grape pomace and mangosteen pericarp prepared with 50 % ethanol were topically applied, as they showed maximum polyphenol contents and correspondingly high antioxidant activities. to minimalize interfering effects of the vehicle system regarding the penetration of bioactives, hydroalcoholic extracts were used for skin penetration experiments without further formulation. after their application on porcine ear skin, polyphenols from grape pomace extract were detected at low concentrations in the skin and in the transdermal receptor fluid after 24 h of expose (tab. 2). among the individual compounds recovered from the skin and receptor fluid, tab. 1: pearson correlation coefficients between the results of diverse antioxidant assays and total polyphenol contents as determined by hplc and folin-ciocalteu assay, respectively. grape pomace mangosteen pericarp hplc folinhplc folin ciocalteu ciocalteu abts 0.81 0.99 0.99 1.00 dpph 0.62 0.99 0.76 0.81 orac 0.07 0.74 0.98 1.00 antioxidant assay low molecular weight polyphenols such as gallic acid, and, in particular, caftaric acid were found to readily pass the stratum corneum and penetrate the skin, as only low amounts were contained in the extracts applied, and yet were detected in the skin and the receptor fluid. the octanol-water partition coefficients (log p) of gallic acid and caftaric acid were determined to be -0.5 and -0.6, respectively (lu et al., 2006; potts and guy, 1992). accordingly, penetration efficacy of these phenolic acids was expected to be rather low. presumably, low molecular weight of both substances may have had strong impact on their penetration. these findings are concordant with zillich et al. (2013b), who suggested superior penetration of low molecular weight polyphenols based on the release and penetration of some individual polyphenols. however, unexpectedly, significant amount of high molecular procyanidins b1 and b2 were also detected in the skin and the receptor fluid. this may be due to penetration enhancing effects of hydroalcoholic solvent systems directly applied to the skin. overall amounts of polyphenols recovered were low, in particular, for the catechins and glycosidically bound quercetin, possibly due to the high reactivity of these phenolic species. beside epimerization, oxidation and polymerization may occur upon exposure to light and temperature. additionally, polyphenol-proteininteractions might also be responsible for their poor recoveries from porcine tissues. this phenomenon has also been reported by other researchers (charlton et al., 2002; dall’acqua et al., 2012; zillich et al., 2013a). contrary to the grape pomace polyphenols, xanthones from mangosteen pericarp extract were almost completely recovered, indicating that these molecules are more stable without interacting with the skin proteins (tab. 3). consequently, highest xanthone concentrations were detected in the skin, while the polyphenol pattern of the pericarp extract remained completely identical. therefore, similar penetration behavior of the individual xanthones may be assumed, which is probably due to their similar structures, molecular weights, and polarities. in literature, octanol-water partition coefficient of α-mangostin is reported to be 6.4, confirming its strong hydrophobic nature, thus favoring skin penetration (koh et al., 2013). the higher concentrations of xanthones in the extracts may also contribute to improved penetration owing to their more pronounced concentration gradient. however, in our experiment, diffusion of lipophilic xanthones compounds may be restricted to the skin, as their poor water solubility prevents further penetration into the buffered aqueous receptor fluid. this might explain why xanthones were not detected in the receptor fluid. tab. 2: deposition of polyphenols in various tissues of porcine ear skin 24h after in vitro topical application of grape pomace extract as determined by hplc-dad. recovered amount [μg] polyphenol applied amount [μg] surface stratum corneum skin receiving chamber (crude extract) 1 gallic acid 1.74 ± 0.17 0.04 ± 0.01 0.01 ± 0.00 0.05 ± 0.01 0.66 ± 0.05 2 caftaric acid 5.99 ± 0.25 0.54 ± 0.01 n.d. 0.65 ± 0.04 4.58 ± 0.53 3 procyanidin b1 31.76 ± 0.82 2.64 ±0.83 n.d. 0.62 ± 0.21 14.41 ± 0.34 4 catechin 92.28 ± 11.09 1.09 ± 0.04 0.48 ± 0.06 1.98 ± 0.42 4.50 ± 0.64 5 procyanidin b2 51.24 ± 5.62 3.21 ±0.81 0.14 ± 0.11 4.41 ± 1.91 2.84 ± 1.52 6 epicatechin 115.02 ± 14.24 2.32 ± 0.47 0.89 ± 0.27 0.82 ± 0.04 3.10 ± 1.10 7 quercetin-3-glucuronide 26.87 ± 1.45 0.46 ± 0.01 n.d. 0.80 ± 0.00 0.82 ± 0.00 8 quercetin-3-glusoside 18.61 ± 0.48 0.56 ± 0.11 n.d. 0.93 ± 0.00 0.85 ± 0.00 9 resveratrol 5.41 ± 0.10 1.48 ± 0.01 n.d. 0.38 ± 0.12 0.62 ± 0.20 total 348.92 ± 34.22 12.34 ± 2.30 1.52 ± 0.44 10.64 ± 2.33 32.38 ± 4.38 antioxidant extracts from garcinia mangostana pericarp and riesling grape pomace 255 conclusions the results obtained in the present study demonstrate the antioxidant potential of ‘riesling’ grape pomace and mangosteen pericarp extracts in abts and orac systems, and hence, their potential as antioxidant ingredients in cosmetic formulations. deviating from the findings for mangosteen extracts, grape pomace extracts were de monstrated to contain further components in their matrix which seem to enhance their overall reducing capability. to evaluate the influence of such accompanying substances, further studies including fractionation and purification steps are required. their low reactivity towards the dpph radical was a common feature of phenolic extracts from both raw materials, thus confirming the necessity to apply multiple radical assays. when investigating the penetration behaviour of individual phenolic compounds, complete qualitative and quantitative recovery of xanthones from pig skin was achieved. in contrast, grape pomace polyphenols were only partially recovered after their topical application, indicating higher reactivity of these molecules and interactions with skin proteins. therefore, e.g. vehicle system may play a decisive role affecting skin permeability. finally, the results of this study confirmed a great potential of grape pomace as well as mangosteen pericarp extracts for their application as an antioxidant cosmetic ingredient. the high antioxidant acti vity of grape pomace constituents allows efficient topical application at low extract dosages in cosmetic products. in addition to its impressive antioxidant potential, our previous study demonstrated that grape pomace extract may also inhibit collagenase and elastase. the latter enzymes are responsible for the degradation of structural proteins of the dermis, thus confirming their suitability for antiaging cosmetic preparations (wittenauer et al., 2015). many attempts have been made to exploit grape pomace (balasundram et al., 2006; kammerer et al., 2014; maier et al., 2009; schieber et al., 2001); its value-added valorisation has not been implemented in industrial practice up to now. grape pomace utilisation was mainly considered for human nutrition but is limited due to its strong bitter and astringent taste (drewnowski and gomez-carneros, 2000). extracts from mangosteen pericarp also revealed their suitability for cosmetic purpose due to their rich content of antioxidant xanthones, which are readily able to penetrate the skin. the problems arising from mangosteen fresh waste disposals in the countries of production are recognized as a significant environmental concern, and only first steps are made for its exploitation (pathak et al., 2015; sze lim et al., 2013; takolpuckdee, 2014; wittenauer et al., 2012). the use of grape pomace and mangosteen pericarp extracts in cosmetics may be a more promising field of application for these underestimated by-products. acknowledgments this study was financially supported by the fraunhofer gesellschaft zur förderung der angewandten forschung (fhg). the authors are grateful to baden-badener winzergenossenschaft, baden-badenneuweier, germany, for providing the grape pomace. references abla, m.j., banga, a.k., 2013: quantification of skin penetration of antioxidants of varying lipophilicity. int. j. cosmet. sci. 35, 19-26. doi: 10.1111/j.1468-2494.2012.00728.x. abou samra, m., chedea, v.s., economou, a., calokerinos, a., kefalas, p., 2011: antioxidant/prooxidant properties of model phenolic compounds: part i. studies on equimolar mixtures by chemiluminescence and cyclic voltammetry. food chem. 125, 622-629. doi: http://dx.doi. org/10.1016/j.foodchem.2010.08.076. alonso, c., rubio, l., tourino, s., marti, m., barba, c., fernandezcampos, f., coderch, l., parra, j.l., 2014: antioxidative effects 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garbenstraße 25, d-70599 stuttgart, germany e-mail address: judith.wittenauer@fraunhofer.ivv.de ute schweiggert-weisz, fraunhofer institute for process engineering and packaging, department of process engineering, giggenhauser straße 35, d-85354 freising, germany reinhold carle, hohenheim university, institute of food science and biotechnology, chair plant foodstuff technology, garbenstraße 25, d-70599 stuttgart, germany king abdulaziz university, biological science department, faculty of science, p.o. box 80257, jeddah 21589, saudi arabia © the author(s) 2016. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 88, 322 326 (2015), doi:10.5073/jabfq.2015.088.046 1technische universität münchen, school of life sciences weihenstephan, associate professorship of fruit science, germany 2bayerisches obstzentrum, hallbergmoos, germany phenolic contents in fruit juices of plums with different skin colors katharina goldner1, sofia vio michaelis1, michael neumüller2, dieter treutter1* (received september 17, 2015) * corresponding author summary polyphenols in fruits are of increasing interest for consumers and for plant scientists because of their health beneficial potential and their role in plant physiology and disease resistance. anthocyanins contribute significantly to the attractive pigmentation of red and blue plums. mirabelles and several reineclaudes do usually not accumulate anthocyanins in the skin. is this linked to a general low phenolic level? both the health aspect and the pigmentation are interesting traits for the breeder. for this purpose, rapid analytical methods are necessary. one time consuming step is the extraction of polyphenols. however, fruit juices are easily produced and are anyhow used for estimation of quality traits such as sugars and acidity. here we show that hplc analysis of plum juices represent the phenolic profiles of the whole fruits. we analysed the phenolic patterns of juices from 43 plum varieties with yellow, blue and dark blue fruit skins. in most cases, a weak red pigmentation co-occurs with a low total phenol level. however, there are exceptions that may help the breeder to combine yellow fruit skin with a high level of health beneficial phenolic compounds by using the appropriate donor genotypes. the method described here offers a valuable tool for selection. introduction polyphenols are among the most important non-nutritive secondary compounds with antioxidant, anti-inflammatory and anticancerogenic activity in plant food (middleton et al., 2000). depending on ethnic origin and eating habit, the daily uptake of phenolic compounds ranges between 5 125 mg per person (ma nach et al., 2004). fruits contribute significantly to this uptake. the annual fruit consumption in germany is about 110 kg per person including an estimated plum percentage of 1 % (ludwig-ohm and dirksmeyer, 2013). since plums are a seasonal product, the consumption during summer and autumn may be above this mean value. furthermore, based on many studies it is assumed that polyphenols play a role in plant defense and resistance against biotic and abiotic stressors (treutter, 2005; lattanzio et al., 2008). the bioactivity of the diverse phenolic compounds depends on their respective structures. it is therefore of interest to know both the quantitative as well as the qualitative composition of phenolic constituents in fruits. european plum varieties exhibit a great diversity concerning their phenolic profiles both in the fruit skin (treutter et al., 2012) and the flesh (jaiswal et al., 2013). more than 40 individual phenolic compounds were identified; among these are 19 chlorogenic acids, 9 flavonols, 10 flavan-3-ols (jaiswal et al., 2013) as well as 5 anthocyanins (usenik et al., 2008; treutter et al., 2012). both the health aspect and the pigmentation are interesting traits for the breeder. because of their beneficial effects on human health, the content and profiles of phenolic compounds in fruits are of increasing consumer’s interest and fruit breeders are more and more asked for this new quality character of their products. thus, knowledge about the inheritance of the different phenolic classes is useful for the plum breeder. however, harvest time is a period of full workload since many hundreds of varieties have to be evaluated for fruit quality traits. thus, sampling of analytical probes cannot be extended too much. fruit juices are easily prepared and are used for the analyses of soluble solids, acid concentration and ph. the final analytical method for phenolic profiling is surely hplc. however, the time consuming extraction of fruits with organic solvents is a big deal and should be avoided. therefore, we attempted to use the fruit juice, which is routinely prepared, for phenolic profiling. material and methods sample preparation in 2013, fruits from trees grown in the experimental orchard of the school of life sciences weihenstephan at freising were harvested in a ripe stage as usual for the fresh market. the varieties of european plums analysed in this study belong to the species prunus domestica l. some myrobalanes (p. cerasifera l.) are also included. the fruits were grouped according to their skin colour. one group consists of mostly yellow plums with sometimes red dish skin spots including a mirabelle, reineclaudes and one myro balane-plum. these were ‘autumn compote’, ‘bellamira’ (mirabelle), ‘colora’, ‘early transparent’, ‘eibensbacher aprikosenpflaume’, ‘goldzwetsche’, ‘große grüne reneklode’ (green gage), ‘liegels gelbe’, ‘ontariopflaume’, p 34-56, ‘reine claude diane’, ‘tatjana’ (myrobalane plum). in a second group the varieties exhibiting blue or deep red skin color were combined: ‘angelina burdette’, ‘auerbacher’, ‘blaue wiener’ (myrobalane plum), ‘cacaks späte’, ‘carpatin’, ‘docera 5’ (myrobalane hybrid), ‘elena’, ‘felsina’, ‘gabrowska’, ‘haganta’, ‘isjumnaja’, ‘liablu’, ortenauer, p 63143-94, ‘topking’, ‘topper’, ‘toptaste’. the third group consists of varieties with dark blue skin: agri 2000 10/91, ‘bühler’ (clones doll and meier), ‘cacaks beste’, ‘cacaks julia’, ‘cacaks schöne’, ‘hanka’, ‘hauszwetsche’ (clones schüfer and wolff), ‘maria novella’, ‘oneida’, ‘topend plus’, ‘topgigant plus’, ‘tophit plus’, ‘topfive’. from 10 randomly selected fruits per variety the stones were removed and the flesh including the skin was crushed and the juice obtained using a household juicer. the juice was frozen and stored at -20 °c. before further analysis, the frozen juice was melted in a cooled ultrasonic water bath at 4 °c and then centrifuged twice for 30 min. with 10,000 g at 4 °c. 1 ml of the clear supernatant was subjected onto a spe cartridge prepacked with 1 ml c18 (bond elute, agilent chem). the sugars are not retained and elute immediately. further 0.5 ml water was added onto the cartridge. the corresponding fraction contained already about 10 to 15 % of the total neochlorogenic acid which was calculated from the neochlorogenic acid of all fractions. the remaining phenolic compounds were eluted with 1 ml methanol. the fractions containing the phenolic compounds were evaporated to dryness with a vacuum centrifuge (univapo, uniequipe) and redissolved in 250 μl methanol containing 0.05 mg/ml 3-methoxy flavone as internal standard. this solution was directly used for phenolic contents in fruit juices of plums with different skin colors 323 hplc analysis. in case of very high concentrations of neochlorogenic acid the sample had to be diluted and a second hplc run was necessary. the phenolic compounds were identified according to their uv-/ vis-absorbance spectra and their retention time in comparison to previously obtained information (treutter et al., 2012; jaiswal et al., 2013). the hydroxycinnamic acids include 3-caffeoylquinic acid (= neochlorogenic acid), 4-caffeoylquinic acid, 5-caffeoylquinic acid, 3-caffeoylshikimic acid, 3-p-coumaroylquinic acid, 3-feruloylquinic acid. four flavonols were found which are glycosides of quercetin (jaiswal et al., 2013) as well as the anthocyanins cyanidin-3-rutinoside, peonidin-3-rutinoside, cyanidin-3-glucoside, peonidin-3-glucoside. among the flavan-3-ols, catechin and epicatechin were identified. besides these two monomers, a number of 22 proanthocyanidins were detected. 13 of them are supposed to consist only of epicatechin units as stated earlier (jaiswal et al., 2013). the remaining oligomers are assumed to consist of catechin units or are mixed-type procyanidins consisting of catechin and epicatechin units; they are summated as groups. high performance liquid chromatography the hplc system and the gradient elution system were described by treutter et al. (2012). chlorogenic acids were quantified at 320 nm, flavonols at 350 nm and anthocyanins at 540 nm; for quantification they were calculated as chlorogenic acid, rutin and cyanidin3-glucoside, respectively. for the selective detection of the flavan3-ols a post-column derivatisation method was used with p-dimethylaminocinnamaldehyde as the reagent (treutter, 1989). the reaction products were detected at 640 nm. catechin and epicatechin were calculated as their respective standards; for quantification of procyanidins the response factor of procyanidin b2 was used. results and discussion comparison of phenolic profiles from fruit juices with fruit skin and flesh we prepared juices from the plum varieties ‘green gage’ (green skinned fruit), ‘tatjana’ (red skinned fruit), ‘ortenauer’ (blue skinned fruit) and ‘toptaste’ (dark blue skinned fruit) and estimated the concentrations of anthocyanins, flavonols, flavan-3-ols, and hydroxycinnamic acids. fig. 1a shows the differing profiles of the varieties’ juices. in order to check if these profiles may be representative for the whole fruit, which is essential for the interpretation by the breeder, we compared these profiles with the results of former studies. in those experiments fruit skin (treutter et al., 2012 ) or fruit flesh (jaiswal et al., 2013) were exhaustively extracted with methanol. the corresponding profiles are given in fig. 1b and 1c. for calcu fig. 1: phenolic profiles of plum fruit juices (a) in comparison to fruit skin (b) and fruit flesh (c). data of (b) and (c) are calculated from treutter et al (2012) and jaiswal et al. (2013), respectively. 324 k. goldner, s.v. michaelis, m. neumüller, d. treutter lation of the contents per 100 g fresh fruit the following estimation was used: skin 15 %, flesh 85 %, juice 46 %. when comparing the profiles of skin and flesh with that of the corresponding juice it is obvious that the juice’s profile combines those of both skin and flesh. even the anthocyanins and the flavonols that are restricted in the skin are found in the juice. hydroxycinnamic acids and flavan-3-ols occur in skin and flesh and the profile differences between the varieties are represented by the juices’ profiles. thus, the quantitative phenolic profiles of the plum fruit juices are a useful tool for the breeder to roughly estimate phenolic patterns of plum fruits for fruit quality purpose or for getting an idea about heredity and biosynthesis. the absolute amount of most phenolic compounds extracted by the simple juice-making is lower than the sum of the content in the respective tissues. the quantitative differences may also due to the year of harvesting the fruits, which were different. thus differing weather conditions have to be considered. for flavan-3-ols and flavonols this could also be explained by the exhaustive extraction of skin and flesh with methanol. the low anthocyanin levels in the juice might be due to the instability of these molecules in aqueous solution. only the chlorogenic acids represent the content of flesh plus skin. phenolic profiles of fruit juices of plums with different skin colours it is often stated that anthocyanin rich fruits and berries have a pronounced health beneficiary status, which is only partially related to the anthocyanin contents since non-visible phenolic compounds exhibit also health-promoting activities (aiyer et al., 2012; bradish et al., 2011; kaume et al., 2011; rodriguez-mateos et al., 2011). it is furthermore not known if a high anthocyanin content is generally linked to a high total phenol level. this may be true for some berry fruits which accumulate red pigments in all fruit cells. however, this may not be the case for plums which accumulate anthocyanins only in the skin. in order to clarify these questions, 43 plum varieties were divided into three groups according to their skin colour. 12 varieties are combined to a yellow group (y). the skin of the corresponding ripe fruits is green to yellow with some of them show reddish sprinkles. the blue-skinned group (b) include 16 varieties, and the third group shows dark blue skin (db). comparing the total phenolic contents (fig. 2) it can be seen that indeed most of the yellow varieties have lower levels than the blue and dark blue ones with now difference between blue and dark blue varieties. this difference is not due to the anthocyanins but to the hydroxycinnamic acids which in most juices represent more than 95 % of the phenolic content. there is no quantitative difference of flavan3-ols and flavonols between the three plum groups. the anthocyanin concentrations represent more or less the blue/dark blue grouping, which was visually made. the predominant anthocyanin in most juices is cyanidin 3-rutinoside (fig. 3) followed by peonidin 3-rutinoside according to usenik et al. (2008) and treutter et al. (2012). the juices of the dark blue varieties (db) show the highest values of these cyanidin-glycosides as compared to the blue-skinned group (b) wheres no such tendency was found for the peonidin-glycosides. trace amounts of anthocyanins were detected in the yellow varieties as expected. however, it has to be noted that even small amounts of red pigments from lightly red sprinkled fruit skins can be found in the respective juices. only peonidin-3-glucoside remained obviously below the detection limit in those juices. the level of total flavan-3-ols as indicated in fig. 2 is mainly due to the sum of procyanidins consisting of at least one catechin-type unit (c-/c-e-procyanidins) and the monomer catechin (fig. 4). epicatechin and its oligomers (e-procyanidins) play a minor role in most of the juices. this confirms former fruit flesh analyses (jaiswal et al., 2013). among the hydroxycinnamic acids, the neochlorogenic acid (3-caffeoylquuinic acid) is predominating in all juices (fig. 5). among the minor hydroxycinnamic acids, the 3-p-coumaroylquinic acid also exhibits lowest concentrations in the yellow group. there are no general group differences for 3-feruloylquinic acid, 3-caffeoylshikimic acid, 5-caffeoylquinic acid and 4-caffeoylquinic acid (fig. 5). conclusion red pigmentation is known to be dominantly inherited with an additive effect of genes (hartmann and neumüller, 2009; alehina, 1978). many of the blue skinned varieties seem to be heterozygote concerning this trait since crossings among them may produce descendants with yellow fruit skin which are homozygote related to this character. further chemical characterization is necessary to understand the relevant biosynthetic steps that regulate the pigmentation and the total phenol level. the results presented here seem to confirm fig. 2: box plots of total phenolic contents (mg/l), total hydroxycinnamic acids, total flavan-3-ols, total anthocyanins, and total flavonols in juices of plum varieties with yellow (y), blue (b) and dark blue (db) skin. quantification was performed by hplc and the individual components were summed up for getting the corresponding total values. respect the different scales. the box plots show the meridian (horizontal line), the interquartile range box between the first and the third quartile, and outliers (asterisk). the upper and lower whiskers extend to the highest and lowest data value, respectively. phenolic contents in fruit juices of plums with different skin colors 325 fig. 4: box plots of concentrations (mg/l) of the flavan-3-ols catechin and epicatechin and the corresponding proanthocyanidins consisting of epicatechintype units (e-procyanidins) or of catechin and mixed catechin-/epicatechin-type units (c-/c-e-procyanidins) in juices of plum varieties with yellow (y), blue (b) and dark blue (db) skin. fig. 5: box plots of concentrations (mg/l) of individual hydroxycinnamic acids in juices of plum varieties with yellow (y), blue (b) and dark blue (db) skin. fig. 3: box plots of concentrations (mg/l) of individual anthocyanins in juices of plum varieties with blue (b), dark blue (db) and yellow (y) skin. 326 k. goldner, s.v. michaelis, m. neumüller, d. treutter the tendency that weak red pigmentation of plum skin corresponds to low total phenol concentration in the juice. however, exceptions seem to be possible as indicated by an extreme value and by overlapping boxes in fig. 2. it may be a challenge for the breeder to combine the desired yellow fruit skin of a mirabelle or a reineclaude, for instance, with a high content of health-related chlorogenic acids. further studies are necessary to find the genetic donors of the respective traits. references aiyer, h.s., warri, a.m., woode, d.r., hilakivi-clarke, l., clarke, r., 2012: influence of berry polyphenols on receptor signaling and cell-death pathways: implications for breast cancer prevention. j. agric. food chem. 60, 5693-5708. alehina, e.m., 1978: inheritance of some signs in hybrid progenies got from the crossing prunus domestica varieties in the condition of north caucasus. acta hortic. 74, 79-81. bradish, c.m., perkins-veazie, p., fernandez, g.e., xie, g., jia, w., 2012: comparison of flavonoid composition of red raspberries (rubus idaeus l.) grown in the southern united states. j. agric. food chem. 60, 5779-5786. hartmann, w., neumüller, m., 2009: plum breeding. in: jain, s.m., priyadarshan, p.m. (eds.), breeding plantation tree crops: temperate species, 161-231. springer science + business media. kaume, l., howard, l.r., devareddy, l., 2012: the blackberry fruit: a review on its composition and chemistry, metabolism and bioavailability, and health benefits. j. agric. food chem. 60, 5716-5727. jaiswal, r., karaköse, h., rühmann, s., goldner, k., neumüller, m., treutter, d., kuhnert, n., 2013: identification of phenolic compounds in plum fruits (prunus salicina l. and prunus domestica l.) by highperformance liquid chromatography/tandem mass spectrometry and characterization of varieties by quantitative phenolic fingerprints. j. agric. food chem. 61, 12020-12031. lattanzio, v., kroon, p.a., quideau, s., treutter, d., 2008: introduction: plant phenolics – secondary metabolites with diverse functions. in: daayf, f., lattanzio, v. (eds.), recent advances in polyphenol research, 1-35. chichester, wiley-blackwell. ludwig-ohm, s., dirksmeyer, w., 2013: ausgewählte analysen zu den rahmenbedinungen und zur wettbewerbsfähigkeit des gartenbaus in deutschland. thünen working paper 6. manach, c., scalbert, a., morand, c., remesy, c., jimenez, i., 2004: polyphenols: food sources and bioavailability. am. j. clinical nutrition 6, 717-747. middleton, e. jr., kandaswami, c., theoharides, t.c., 2000: the effects of plant flavonoids on mammalian cells: implications for inflammation, heart desease and cancer. pharm. review 52, 673-751. rodriguez-mateos, a., cifuentes-gomez, t., tabatabaee, s., lecras, c., spencer, j.p.e., 2012: procyanidin, anthocyanin, and chlorogenic acid contents of highbush and lowbush blueberries. j. agric. food chem. 60 5772-5778. treutter, d., 1989: chemical reaction detection of catechins and proanthocyanidins with 4-dimethylaminocinnamoaldehyde. j. chromatogr. a, 467 185-193. treutter, d., 2005: significance of flavonoids in plant resistance and enhancement of their biosynthesis. plant biology 7, 581-591. treutter, d., wang, d., farag, m.a., argueta baires, g.d., rühmann, s., neumüller, m., 2012: diversity of phenolic profiles in the fruit skin of prunus domestica plums and related species. j. agric. food chem. 60, 12011-12019. usenik, v., štampar, f., veberič, r., 2008: anthocyanins and fruit colour in plums (prunus domestica l.) during ripening. food chem. 144, 529534. address of the corresponding author: dieter treutter, professur für obstbau, technische universität münchen, dürnast 2, 85354 freising, germany e-mail: dieter.treutter@wzw.tum.de © the author(s) 2015. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 90, 165 173 (2017), doi:10.5073/jabfq.2017.090.021 1agricultural botany department, faculty of agriculture, fayoum university, fayoum, egypt 2 king khalid university, faculty of science, biology department, saudi arabia 3 research center for advanced materials science (rcams) seed inoculation with azospirillum lipoferum alleviates the adverse effects of drought stress on wheat plants ramadan a. agami1*, hamed a. ghramh2, 3, mohamed hashem2, 3 (received november 12, 2016) * corresponding author summary drought is one of the major environmental stresses that adversely affects crop growth and productivity worldwide. the effect of inoculation with azospirillum lipoferum on growth, yield, water status, osmoprotectant, antioxidant system and grain anatomy of wheat plants under drought stress conditions was investigated. the plants exposed to the drought stress exhibited a significant reduction in growth, grains yield, relative water content and leaf photosynthetic pigments, as well as alterations in grain anatomy. however, the treatment with a. lipoferum alleviated the stress generated by drought and improved the above-mentioned parameters. drought stress increased proline, protein, soluble carbohydrates, relative membrane permeability and activities of antioxidant enzymes (sod and pox). the antioxidant enzymes, phenols and grain anatomy exhibited changes in response to a. lipoferum inoculation in the absence or presence of drought stress. our data suggest that inoculation with a. lipoferum could protect wheat plants from the harmful effects of drought stress through changes in the antioxidant defense system. keywords: triticum aestivum, drought stress, azospirillum lipoferum, antioxidant defense system, anatomy, compatible solutes introduction wheat (triticum aestivum l.) is one of the most important cereal crops in the world, however this crop is exposed to a variety of abiotic stresses, such as drought, salt loading and freezing that influence its development, growth and productivity. drought stress is among the most destructive abiotic stresses that increased in intensity over the past decades affecting world’s food security. it is expected to cause serious plant growth problems for more than 50% of the arable lands by 2050 (kasim et al., 2013). drought affects plant water potential and turgor, enough to interfere with normal functions (hsiao, 2000), and induces changes in physiological and morphological characters in the plants (rahdari and hoseini, 2012). common plant symptoms after water stress are: stunting, limiting in co2 diffusion to chloroplasts by stomatal closure, reduction in photosynthesis rate, and acceleration of leaf senescence. moreover, in wheat, a severe drought stress during the late growth stages (anthesis − post anthesis) induces chlorophyll degradation, cell solute leakage, and accelerated spike and grain maturation (beltrano et al., 1999). drought stress also causes severe alterations in cell membrane selective permeability (leakage of cell solutes), fluidity and microviscosity (navarri-izzo et al., 1993; beltrano et al., 1999). it induces free radicals affecting antioxidant defenses and reactive oxygen species (ros) such as superoxide radicals, hydrogen peroxide and hydroxyl radicals resulting in oxidative stress. high concentrations of ros can cause damage to various levels of organization (smirnoff, 1993), like initiate lipid peroxidation, membrane deterioration and degrade proteins, lipids and nucleic acids in plants (hendry, 2005; sgherri et al., 2000; nair et al., 2008). much of the injury in plants under abiotic stress is due to oxidative damage at the cellular level, which is the result of imbalance between the formation of reactive oxygen species (ros) and their detoxification. plant cells produce different antioxidant enzymes such as catalase (cat), peroxides (pox), superoxide dismutase (sod), glutathione peroxidase (gpx) and ascorbate peroxidase (apx) that scavenge the reactive free radicals (simova-stoilova et al., 2008). generally, drought negatively affects quantity and quality of the plant growth. therefore, to produce more food, the alleviation of drought stress is important to achieve the designated goals. globally, an extensive research is being carried out to develop strategies to cope with drought stress through development of drought tolerant varieties, shifting the crop calendars, resource management practices and others and other means (venkateswarlu and shanker, 2009) and most of these technologies are cost intensive. recent studies indicate that microorganisms can also help plants to cope with drought stress. bacteria of the genus azospirillum are among the best investigated plant growth promoting rhizobacteria (pgpr) detected in the rhizosphere of many crop plants. they are able to produce plants hormones such as auxin, and proteins like polyamines, fix n2, increase root growth and control pathogens. such abilities collectively result in the enhanced growth of plants under stress (ramos et al., 2002; bhaskara rao and charyulu, 2005; russo et al., 2008; cassan et al., 2009). many researchers have indicated that azospirillum spp. can mitigate the unfavorable effects of water stress on plant growth (arzanesh et al., 2009; pereyra et al., 2009). the present study was designed with the objective to examine changes in the antioxidant defense system of wheat plants under the effect of azospirillum lipoferum applied by seed inoculation and exposed to drought stress. the tested hypothesis is that a. lipoferum will positively modify the level of antioxidant system that will mitigate the injuries generated by drought stress. consequently, a. lipoferum will enhance the wheat performance under drought stress. materials and methods plant materials and bacterial strain seeds of wheat (triticum aestivum l. cv. giza 168) were obtained from the crop institute, agricultural research center, giza, egypt. bacterial strain azospirillum lipoferum n040 was obtained from agricultural microbiology department, faculty of agriculture, cairo university for research purpose. inoculum preparation and bacterial growth bacterial culture was prepared by growing the azospirillum lipoferum n040 in liquid nitrogen free biotin based (nfb) medium (piccoli et al., 1997) [5 g l-1 peptone and 3 g l-1 beef ph 7.0] at 25 ± 1 °c with shaking (150 rpm) for 48 h. the bacterial cells were 166 r.a. agami, h.a. ghramh, m. hashem pelletized by centrifugation (5000 rpm) for 10 min and re-suspended in sterilized tap water containing 0.025% (v/v) tween-20 to the desired concentration (108 cfu ml-1). experimental design and treatments two pot experiments were carried out at demo experimental farm, faculty of agriculture, fayoum university (southeast fayoum; 29° 17’n; 30° 53’e), during the two successive seasons of 2014 and 2015. wheat seeds were surface-sterilized with 70% ethanol (3 min), treated with 2% sodium hypochlorite (naclo) (5 min), and followed by repeated washing with sterile distilled water (3 times for 1 min). then surface-disinfected seeds were incubated in sterilized liquid nitrogen free biotin based medium (as control) or in bacterial suspension (108 cfu ml-1) for 2 h on a rotary shaker at 81 rpm. ten inoculated seeds (107 bacteria per seed) or non-inculcated seeds (control) were sown in each plastic pot (32 cm in diameter, 25 cm in deep) containing 15 kg of soil and were thinned to five plants, one week after germination. soil used in the pots had the following physic-chemical characteristics: sand, 2.7%; silt, 28.7%; clay, 68%; ph, 7.28 (1:2, w/v, soil and water solution); ec, 3.49 ds m-1 (1:2, w/v, soil and water solution); caco3 10.81% and organic matter 3.39%; total nitrogen, 39.6 (mg/kg dry soil); available phosphorus, 32.9 (mg/ kg dry soil); extractable potassium, 8.33 (mg/kg dry soil). the first experiment was conducted on 15 november, 2014 and the second one was conducted on 19 november, 2015 in an open greenhouse. the average day and night temperatures were 22 ± 3 oc and 11 ± 2 oc, respectively. the relative humidity ranged from 38.1 to 69.8%, and day-length from 10 to 11 h. pots were arranged in the greenhouse in a complete randomized block design with four replications for each treatment. recommended doses of n, p, and k fertilizers (150-10060 kg ha-1) were applied to each pot and equal amounts of tap water was added to the pots to maintain the optimal soil moisture depending on plant and soil conditions (up to 1000 ml). drought stress was applied after 30 days of planting to grain ripening. the two soil water conditions were either well-watered (100% of crop evapotranspiration (etc)) or dried up (60% of crop evapotranspiration). crop evapotranspiration was determined using gravimetrical method described by maoa et al. (2014). irrigation was applied twice a week during the experimental period. samples of wheat plants (36 per each treatment) were collected after 90 days from sowing to assess morphological data. length of shoots and spikes (cm) was measured by using a meter scale. numbers of fertile tillers per plant and numbers of spikelets per spike were coun ted. flag leaf area (cm2) was measured using a digital leaf meter (li3000 portable area meter produced by li-cor lincoln, ne, usa). samples of flag leaves were collected to estimate the concentration of total chlorophylls and total carotenoids, proline, total soluble protein, total soluble carbohydrates and total soluble phenols, relative membrane permeability, relative water content and activities of antioxidant enzymes. the experiment was terminated after 130 days from sowing after exposing the plants to water stress for 100 days. the 130-day-old plants from each treatment were collected for various measurements. the 130-day-old wheat plants were removed from the pots and moved smoothly to remove the adhering soil particles by dipped them in a bucket filled with water. roots and straw were weighed to record their fresh weight, then were placed in an oven at 70 °c to reach a constant dry weight (dw). grain yield per plant and 1000-kernel weight was also estimated. the powder of dried grains was used to determine the concentration of total soluble protein and total soluble carbohydrates. anatomical study for observation of grain anatomy, samples were taken from the main spike at the age of 110 days and fixed in faa solution (containing 50 cm3 of 95% (v/v) ethanol + 10 cm3 of formaldehyde + 5 cm3 of glacial acetic acid + 35 cm3 of distilled water) for 48 h. thereafter, the samples were washed in 50% ethanol, dehydrated and cleared in tertiary butanol series, and embedded in paraffin wax. cross sections, 25 μm thick, were cut by a rotary microtome (leitz, wetzlar, germany), adhered by a haupt’s adhesive, stained with a crystal violet-erythrosin combination (sass, 1961), cleared in carbol xylene, and mounted in canada balsam. the sections were observed and documented using an upright light microscope (axioplan, zeiss, jena, germany). measurements were done using a micrometer eyepiece and average of five readings was calculated. photosynthetic pigments determination total chlorophyll and carotenoids concentration (mg g-1 fw) were estimated according to the procedure given by arnon (1949). flag leaves discs (0.2 g) of 90-day-old plants were homogenized with 50 ml 80% acetone. the slurry was strained through a cheese cloth and the extract was centrifuged at 15,000 × g for 10 min. the optical density of the acetone extract was measured at 663, 645 and 470 nm using a uv-160a uv visible recording spectrometer, shimadzu, japan. free proline determination proline concentration in wheat flag leaves was measured following the rapid colorimetric method of bates et al. (1973). proline was extracted from 0.5 g of dry leaf samples by grinding in 10 ml of 3% sulpho-salicylic acid. the mixture was then centrifuged at 10,000 × g for 10 min. two ml of the supernatant was added into test tubes and 2 ml of freshly prepared acid-ninhydrin solution was added. tubes were incubated in a water bath at 90 °c for 30 min. the reaction was terminated in ice-bath. the reaction mixture was extracted with 5 ml of toluene and the vortex process was done for 15 s. the tubes were allowed to stand at least for 20 min in the dark at room temperature to allow the toluene and aqueous phases to be separated. the toluene phase was then carefully collected into test tubes and toluene fraction was read at 520 nm using a uv-160a uv visible recording spectrometer, shimadzu, japan. the proline concentration in the sample was determined from a standard curve using analytical grade proline. total soluble proteins determination the total soluble proteins concentration of the dry flag leaves and grains was determined according to the method described by bradford (1976) with bovine serum albumin as a standard. an amount of 0.2 g of samples was ground in a mortar with 5 ml of phosphate buffer (ph 7.6) and was then transformed to the centrifuge tubes. the homogenate was centrifuged at 8000 rpm for 20 min. the supernatant of different samples was put in separate tubes. the volume of the samples in tubes was then made equal by adding a phosphate buffer solution and the extraction were stored in the refrigerator at 4 °c for further analysis. after extraction, 30 μl of different samples were taken out in separate tubes and were mixed with 70 μl of distilled water. then, 2.9 ml of the coosmassic brilliant blue solution was added to each sample tube and mixed thoroughly. the total volume was 3 ml in each tube. all tubes were incubated for 5 min at room temperature and then, the absorbance was recorded at 600 nm against the blank. a standard curve of absorbance (600 nm) versus concentration (μg) of total soluble proteins was calculated. total soluble carbohydrates determination leaf and grains total soluble carbohydrates concentration were assessed by the method recommended by the association of of azospirillum lipoferum alleviates drought stress on wheat plants 167 ficial agricultural chemists (1990) using phenol sulphuric acid reagent method. total soluble phenols determination the soluble phenol concentration in wheat flag leaves was extracted as described by hsu et al. (2003). 0.2 g of dry leaves were homogenized in 80 ml methanol and kept overnight. the filtrates were diluted to 100 ml, and served as a stock solution. according to slinkard and singleton (1997), 200 μl of the stock solution was added to 1.4 ml distilled water, and 0.1 ml of 50% (1n) folin-ciocalteu phenol reagent. after three min., 0.3 ml of 20% (w/v) sodium carbonate was added. the mixture was allowed to stand for 2 h. after gentle vortex, the absorbance was determined at 765 nm. total soluble phenol concentration was standardized against tannic acid. relative water content determination relative water content (rwc) was determined in midrib excludingfresh flag leaf discs of 2 cm2 area. discs were weighed quickly and immediately floated on double distilled water in petri dishes to saturate them with water for the next 24 h, in dark. the adhering water of the discs was blotted and turgor mass was noted. dry mass of the discs was recorded after dehydrating them at 70 °c for 48 h. by placing the values in the following formula (hayat et al., 2007), rwc was calculated: rwc (%) = (fresh mass – dry mass) / (turgid mass – dry mass) × 100 relative membrane permeability determination for the rmp measurement, the flag leaves were cut into equal pieces and transferred to test tubes containing 20 ml of deionized distilled water. the test tubes were vortexed for 10 s and the solution was assayed for initial electrical conductivity (ec0). these tubes were kept at 4 °c for 24 h and then assayed for ec1. the same samples were autoclaved at 121 °c for 20 min to determine ec2. percent rmp was calculated as following the formula described by yang et al. (1996). rmp (%) = (ec1−ec0) / (ec2−ec0) × 100 enzyme assays flag leaves were excised from wheat plants and rapidly weighed. each 1.0 g sample was ground with a pestle in an ice-cold mortar containing 10 ml of 50 mm phosphate buffer, ph 7.0. the homogenate was centrifuged at 20,000 × g for 30 min at 4 °c. the supernatant was then filtered through two layers of cheese-cloth and used to measure various anti-oxidant enzyme activities. superoxide dismutase (sod) activity was determined according to the method of fridovich (1975). one unit of sod activity was defined as the amount of enzyme required to cause 50% inhibition of the rate of oxidation of nitroblue tetrazolium (nbt) at 560 nm. each 3 ml reaction mixture contained 50 mm phosphate buffer ph 7.0, 200 mm methionine, 1.125 mm nbt, 1.5 mm edta, 75 mm riboflavin, and 10-40 μl of crude enzyme extract. the riboflavin was added as the last component. the tubes were shaken and placed 30 cm below two 15 w fluorescent lights. the reaction was started by switching on the light, and allowed to run for 10 min. switching-off the light stopped the reaction. the tubes were then covered immediately with a black cloth and the absorbance was measured spectrophotometrically at 560 nm. a non-irradiated reaction mixture was set to zero absorbance as the blank. the volume of enzyme extracts that produced a 50% inhibition of the oxidation (color reaction) was read from the resulting graph. peroxidase (pox) activity in flag wheat leaves was measured using the method of thomas et al. (1981). pox was assayed using guaiacol as the substrate. the crude enzyme extract was prepared in a similar way to that used for sod. each reaction mixture consisted of 3 ml of 0.1 m phosphate buffer ph 7.0, 30 μl of 20 mm h2o2, 50 μl of crude enzyme extract, and 50 μl of 20 mm guaiacol. the reaction mixture was incubated for 10 min at room temperature in a cuvette. the absorbance was then measured at 436 nm. pox activity was expressed in a 436 units g -1fw leaf min-1. statistical analysis all the pots for two experiments (288) were arranged in a complete randomized design with nine pots per replicate and four replicates per treatment. analysis of variance was performed using the spss software package to determine the least significant difference (lsd) among treatments at p ≤ 0.05, and the duncan’s multiple range test was applied for comparing the means. results growth traits azospirillum lipoferum inoculation significantly improved the plant growth, straw and the grain yields of wheat in presence or absence of drought stress under open greenhouse conditions. however, drought stress significantly decreased the length of shoot, number of fertile tillers per plant, flag leaf area, length of spike, number of spiklets per spike, root fresh and dry weights (tab. 1). comparing to the control (100% etc), the above-mentioned parameters were decreased under 60% of etc by 27.17, 63.76, 64.85, 28.08, 19.95, 64.56 and 72.52%, respectively. after the inoculation of the wheat grains with tab. 1: effect of azospirillum lipoferum inoculation on the growth traits [length of shoot (cm), no of fertile tillers per plant, flag leaf area (cm2), length of spike (cm), no of spikelets per spike, root fresh mass (g) and root dry mass (g)] of wheat (triticum aestivum l., cv. giza-168) plants grown under non-stressed and drought-stressed condition. treatments parameters irrigation levels inoculation length of shoot no of fertile flag leaf area length of spike no of spikelets root fresh root dry weight tillers per plant per spike weight 100% etc no-inoculated 44.17±1.80b 3.67±0.58a 16.30±1.21b 9.83±0.20a 13.33±0.58b 0.790±0.06b 0.473±0.03b inoculated 50.00±0.58a 4.67±0.58a 27.60±2.52a 9.87±0.35a 15.33±0.58a 1.600±0.48a 1.090±0.06a 60% etc no-inoculated 32.17±0.58d 1.33±0.58c 5.73±0.66c 7.07±0.12c 10.67±0.58c 0.280±0.02c 0.130±0.02c inoculated 39.17±0.76c 1.67±0.58c 8.44±0.65c 8.20±0.75b 11.67±0.58c 0.280±0.03c 0.160±0.01c values are means ± sd (n=9) and differences between means were compared by the duncan’s multiple range test (lsd; p ≤0.05). mean pairs followed by different letters are significantly different. 168 r.a. agami, h.a. ghramh, m. hashem a. lipoferum n040 and in absence of the water stress, the growth in most cases was significantly higher than that of the control (p ≤ 0.05). inoculation with the bacteria also improved the growth of the plants grown under the water stress and the values in some cases were significantly higher than those of the plants grown under the stress alone. yield components data in tab. 2 show that wheat plants growing in the presence of water stress significantly decreased the yield components. however, the reductions were 52.45, 45.28, 71.97, 73.12, 38.49 and 19.27% for straw fresh weight per plant, straw dry weight per plant, spike fresh weight per plant, spike dry weight per plant, grain yield per plant and 1000-kernel weight respectively, compared to non-stressed and non-inoculated plants. inoculation of grains of triticum aestivum cv. giza 168 with a. lipoferum n040 alleviated these deleterious effects of drought stress. in the presence of water stress a. lipoferum treatment showed 24.79 and 11.31%, 54.33 and 58.67%, 79.35 and 3.32%, increases in straw fresh and dry weights per plant, spike fresh and dry weights per plant, grain yield per plant and 1000-kernel weight respectively, compared to non-inoculated control. however, incubation the wheat grains in a. lipoferum suspensions significantly increased the above-mentioned parameters especially in the absence of the water stress (100% etc) compared to the non-inoculated plants. anatomy of grain as concerns the grain anatomical structure, drought stress decreased height and width of the grain by 9.09 and 9.63%, height and width of the endosperm by 13.78 and 10.82% as well as pericarp and aleurone layer by 23.08, and 17.22%, respectively, in comparison to the control (tab. 3 and fig. 1). however, incubation the seeds in a. lipoferum suspension caused positive changes in the above-mentioned characteristics in absence or presence of the water stress. for example, the maximum increase was achieved in a. lipoferum pretreated plants in absence of the water stress which recorded increments of 16.49 and 14.66, 41.67, 17.66 and 15.53 and 24.49% for length and width of grain, pericarp, length and width of endosperm and aleurone layer, as compared to non-inoculated and water-stressed plants. photosynthetic pigments the water deficiency stress significantly decreased the concentration of total chlorophyll and carotenoids (tab. 4). comparing to non-in oculated control, the decrease reached 46.09 and 22.58% for total chlorophyll and carotenoids, respectively. after inoculation combined with drought stress the total chlorophyll and carotenoids content of wheat significantly increased compared to the non-inoculated plants (60% etc). in absence of the drought stress, a. lipoferum inoculation significantly promoted the increases in total chloro phyll and carotenoids by 23.48 and 16.13% for total chlorophyll and carotenoids, respectively compared to non-inoculated plants (100% etc). compatible solutes wheat plants subjected to drought stress exhibited a significant increase in the content of free proline, total soluble protein in leaves, total soluble carbohydrates in leaves and grains but showed a slight tab. 2: effect of azospirillum lipoferum inoculation on the yield component [straw fresh weight per plant (g), straw dry weight per plant (g), spike fresh weight per plant (g), spike dry weight per plant (g), grain yield per plant (g) and 1000-kernel weight (g)] of wheat (triticum aestivum l., cv. giza-168) plants grown under non-stressed and drought-stressed condition. treatments parameters irrigation levels inoculation straw fresh weight straw dry weight spike fresh weight spike dry weight grain yield 1000-kernel per plant per plant per plant per plant per plant weight 100% etc non-inoculated 9.60±0.40b 3.07±0.21b 11.95±1.00b 5.58±0.55b 2.52±0.03c 38.40±0.61b inoculated 18.32±0.21a 6.03±0.64a 15.95±0.80a 7.07±0.64a 6.51±0.11a 40.80±0.26a 60% etc non-inoculated 4.67±0.15d 1.68±0.03c 3.35±0.09d 1.50±0.10c 1.55±0.06d 31.00±2.00c inoculated 5.85±0.35c 1.87±0.06c 5.17±0.40c 2.38±0.18c 2.78±0.03b 32.03±0.84c values are means ± sd (n=9) and differences between means were compared by the duncan’s multiple range test (lsd; p ≤0.05). mean pairs followed by different letters are significantly different. tab. 3: effect of azospirillum lipoferum inoculation on the height and width of grain and endosperm, thickness of percarp and aleurone layer [μm] of wheat (triticum aestivum l., cv. giza-168) plants grown under non-stressed and drought-stressed condition. treatments parameters irrigation levels inoculation dimensions of grain dimensions of endosperm percarp aleurone layer thickness thickness height width height width 100% etc non-inoculated 2062.4±11.6b 3873±12.6b 1884.8±6.9b 3646.3±18.7b 52.0±2.0a 60.0±1.0a inoculated 2184.3±3.9a 4013±15.3a 1912.0±11.8a 3756.7±7.6a 56.7±3.1a 61.0±1.0a 60% etc non-inoculated 1875.0±10.0c 3500±20.0d 1625.0±7.0d 3251.7±12.6c 40.00±2.0c 49.7±1.5b inoculated 1875.2±13.9c 3752±17.6c 1711.8±11.5c 3619.0±16.8b 46.00±1.0b 57. 7±2.5a values are means ± sd (n=9) and differences between means were compared by the duncan’s multiple range test (lsd; p ≤0.05). mean pairs followed by different letters are significantly different. azospirillum lipoferum alleviates drought stress on wheat plants 169 decrease in total soluble protein in grains compared to the noninoculated and non-stressed plants (tab. 4). the increases were 31.86, 97.22, 35.82 and 18.61%, respectively. however, in presence of drought stress inoculation of grains with a. lipoferum alleviated these effects of drought stress on the previous compatible solutes and significantly decreased these parameters compared to non-inoculated and water-stressed plants. the reductions were 12.08, 16.20, 7.31 and 11.35%, for leaf free proline, leaf total soluble protein, total soluble carbohydrates in leaves and grains, respectively. total soluble phenols the stress generated by water deficit resulted in a slight decrease in total soluble phenols compared to non-water stressed plants (tab. 5). a b c d fig. 1: photographs of grain section of azospirillum lipoferum n040 inoculated triticum aestivum l. plants grown under water stress. a) non-inoculated + 100% etc; b) inoculated + 100% etc; c) non-inoculated + 60% etc; d) inoculated + 60% etc; al, aleurone layer; en, endosperm and pe, percarp, bars = 200 μm. tab. 4: effect of azospirillum lipoferum inoculation on total chlorophylls (mg g−1 fw), total carotenoids (mg g−1 fw), proline (mg g−1 dw), protein in leaves and grains (mg g−1dw) and total soluble carbohydrates in leaves and grains (mg g−1 dw) of wheat (triticum aestivum l., cv. giza-168) plants grown under non-stressed and drought-stressed condition. treatments parameters irrigation levels inoculation total total proline protein protein total soluble total soluble chlorophylls carotenoids in leaves in grains carbohydrates carbohydrates in leaves in grains 100% etc non-inoculated 1.15±0.03 0.31±0.02b 1.13±0.07c 2.16±0.05d 18.75±0.48b 112.8±0.67d 623.23±0.96d inoculated 1.42±0.10 0.36±0.01a 1.19±0.03c 3.05±0.13c 20.34±0.17a 131.7±0.47c 651.97±0.95c 60% etc non-inoculated 0.62±0.03 0.24±0.01d 1.49±0.07a 4.26±0.04a 17.85±0.87b 153.2±1.48a 739.20±0.61a inoculated 1.03±0.06 0.28±0.01c 1.31±0.03b 3.57±0.04b 18.10±0.21b 142.0±10.37b 655.27±1.01b values are means ± sd (n=6) and differences between means were compared by the duncan’s multiple range test (lsd; p ≤0.05). mean pairs followed by different letters are significantly different. 170 r.a. agami, h.a. ghramh, m. hashem however, this attribute was significantly improved by a. lipoferum inoculation in presence or absence of the drought stress. in presence of the water deficiency stress, a. lipoferum-inoculated grains had a 36.37% increase in the total soluble phenols over its non-inoculated control. relative water content (rwc %) and relative membrane permeability (rmp %) data shown in tab. 5 reveal that the rwc% of wheat plants was significantly reduced by 29.20% in presence of the water deficiency stress, but rmp% was significantly increased by 97.47% compared to the non-inoculated control (100% etc). in presence of the drought stress, a. lipoferum inoculation reduced the injurious effects of drought stress on wheat plants, and maintained their rmp% and rwc% values at the near levels as in control plants. antioxidant enzyme activities the activities of superoxide dismutase (sod) and peroxidase (pox) are shown in tab. 5. growing wheat plants in presence of the water deficiency stress significantly increased sod and pox activities by 19.22% and 51.58%, respectively, compared to the non-inoculated control (100% etc). in addition, a. lipoferum inoculation of grains further increased these enzyme activities in presence of the drought stress by 8.82% and 31.25%, respectively compared to the control (i.e. non-inoculated and drought-stressed plants). even in the absence of water deficiency stress a. lipoferum inoculation also significantly increased the activity of the two enzymes compared to the control (100% etc). discussion water stress is one of the most adverse factors affecting plants growth and productivity. in addition, water stress causes over-production of reactive oxygen species (ros) that can pose a threat to cells by causing oxidization of lipids, dna, rna and proteins, leading ultimately to cell death (smirnoff, 1995; mittler, 2002; cruz de carvalho, 2008; kar, 2011; sharma et al., 2012). a ba lance between the generation and degradation of ros is required to avoid oxidative injury and to maintain metabolic functions under stress conditions. in plant tissues, the level of ros is controlled by an antioxidant system that consists of antioxidant enzymes and nonenzymatic low molecular weight antioxidant molecules, including proline, ascorbic acid and carotenoids (schutzendubel and polle, 2002; semida and rady, 2014; agami, 2016). in this study, water deficiency stress significantly reduced the growth of wheat plants, in terms of reduced length of shoot, number of fertile tillers per plant, flag leaf area, length of spike, number of spikelets per spike, root fresh and dry weights per plant, straw fresh and dry weights per plant, spike fresh and dry weights per plant, 1000-kernel weight and grain yield per plant. the observed reduction in the above-mentioned growth parameters under drought stress condition in this study may be due to the disturbance in metabolic process of the plant including chlorophyll destruction and the cell division (tab. 3 and 4). water deficiency stress causes losses in tissue water content, which reduce turgor pressure in the cell, thereby inhibiting enlargement and division of the cells causing a reduction in plant growth (shao et al., 2007). moreover, water stress decreased the growth rate, stem elongation and leaf expansion (hale and orcutt, 1987). the decline in fresh weight may be due to the decrease in water content of the stressed plant cells and tissues which lose their turgor and thus shrink (boyer, 1988; soha e. khalil and el-noemani, 2012). the decrease in dry weights of the stressed plants could be attributed to the disturbances in metabolic processes, which lead to decreases in meristematic activity, thereby inhibiting division of cells causing a reduction in dry mass production. drought stress impairs mitosis, cell elongation and expansion result in reduced plant height, leaf area and crop growth (nonami, 1998; kaya et al., 2006; hussain et al., 2008). the deleterious effects of drought stress on growth were reported by several researcher i.e. beltrano and ronco (2008) they found that, dry weight per plant was decreased in wheat plants subjected to severe drought stress, arzanesh et al. (2011) they found that, straw yield and grain weight per ear were decreased under drought stress, agami (2013) who reported that, drought stressed plants showed a significant reduction in growth traits and yield of lettuce comparison to non-waterdeficiency stressed plants, naveeda et al. (2014) they found that, drought stress had drastic effects on growth of maize plants; wang et al. (2016) they found that severe stress had a negative impact on growth of heteropogon contortus plants. it has been shown from the results of this study that a. lipoferum inoculated grains in presence or absence of water deficit stress significantly improved plant growth characteristics and productivity of wheat plants. we have found that our bacterial strain has ameliorative effects on wheat growth grown in presence of water stress. this indicates that a. lipoferum is able to tolerate the drought stress and become active after the stress. this is very interesting regarding this strain, because there are so many situations in which the agricultural soils are subjected to moisture fluctuations. with such kind of ability of azospirillum strain can alleviate the drought stress on plant growth and yield through stabilizing the plant growth conditions including plant water characters. these observations are in accordance with previous reports on the potential of endophytic bacteria having multiple beneficial traits in improving plant productivity and to enhance drought tolerance in plants (sandhya et al., 2010; vardharajula tab. 5: effect of azospirillum lipoferum inoculation on relative water content (rwc %), relative membrane permeability (rmp %), total soluble phenols (mg g−1 dw) and the activities of superoxide dismutase (units g−1 fw leaf min−1) and peroxidase (units g−1 fw leaf min−1) of wheat (triticum aestivum l., cv. giza-168) plants grown under non-stressed and drought-stressed condition. treatments parameters irrigation levels inoculation relative water content relative membrane total soluble phenols sod activity pod activity permeability 100% etc non-inoculated 71.23±1.50a 6.33±0.25c 3.40±0.10b 14.93±0.61d 0.95±0.03d inoculated 73.50±0.61a 6.57±0.31c 4.57±0.21a 16.07±0.38c 1.61±0.03b 60% etc non-inoculated 50.43±1.80c 12.50±0.78a 3.30±0.10b 17.80±0.40b 1.44±0.10c inoculated 63.00±5.52b 8.03±0.32b 4.50±0.20a 19.37±0.42a 1.89±0.03a values are means ± sd (n=9) and differences between means were compared by the duncan’s multiple range test (lsd; p ≤0.05). mean pairs followed by different letters are significantly different. azospirillum lipoferum alleviates drought stress on wheat plants 171 et al., 2011). pgpr including azospirillum spp. affect plant growth through different activities including production of plant hormones such as iaa, n2-fixation and controlling pathogens (spaepen et al., 2008; jalili et al., 2009). production of plant hormones by azospirillum brasilense increased root growth through enhancing nutrient uptake (pereyra et al., 2009). in this study, the drought stress markedly reduced dimension of wheat grain. this was mainly due to the reduction in height and width of endosperm, pericarp and aleurone layer thickness (tab. 3 and fig. 1). tissues exposed to environments with low water availability have generally shown reduction in cell size, and increase in vascular tissue and cell wall thickness (guerfel et al., 2009). in contrast, a. lipoferum was found to be more efficient in mitigating the adverse effects of stress by inducing positive changes the grain anatomy. the beneficial effect of a. lipoferum on wheat grain structure may be due to the crucial role of a. lipoferum in improving n2-fixing potential, and plant growth regulators such as auxins, gibberellins and cytokinins which play a role in cell division and expansion. drought stress caused a significant reduction in the total chlorophylls and carotenoids concentration (tab. 4). this reduction may be attributed to the increase in activity of chlorophyll-degrading enzyme chlorophyllase under stress conditions (reddy and vora, 1986). a. lipoferum inoculation could alleviate the reduction in total chlorophylls and carotenoids concentration under water deficiency stress. a similar result was reported by heidari et al. (2011), who stated that inoculation of bacterial strain like pseudomonas sp., bacillus lentus, azospirillum brasilens, increased chlorophyll content in basil (ociumum basilicm l.) under drought stress. proline concentration in leaves of wheat plants were significantly increased under water deficiency stress (tab. 4) which may be due to up regulation of proline biosynthesis pathway to keep proline in high levels, which helps in maintaining cell water status, protects membranes and proteins from stress (yoshiba et al., 1997). the a. lipoferum inoculation in presence of drought stress showed lower values for the proline concentration than those in the water stress alone, suggesting that if proline is a stress indicator, wheat plants treated with a. lipoferum should have better drought tolerance. because of a. lipoferum in presence of drought stress supported the antioxidant system in wheat plants to enable them to tolerate drought stress. therefore, it could be expected that, the level of proline declined as result of recovery from stress. the reduced accumulation of proline may result in mitigating the stress effects of drought on wheat plants. this could be because proline is essential in proteins biosynthesis that necessary for cell division. soluble sugars are key osmolytes contributing towards osmotic adjustment. in our study, the concentration of total soluble carbohydrates significantly increased in leaves of wheat plants subjected to drought stress. the increase in sugar concentration may be a result from starch degradation (enebak et al., 1997). the increment in sugar concentration may be also a result of an interrupted starch metabolism to gain osmolytes for coping with the osmotic stress arising from water deficit. a. lipoferum inoculation decreased total soluble carbohydrates concentration in leaves of wheat plants under drought stress. this reduction in soluble carbohydrates concentration may be resulted from mitigating the stress generated by water stress. in this study inoculation also decreased total soluble proteins concentration in drought stress plants over than non-inoculated plants indicating a. lipoferum helps in alleviating the stress induced by water deficit. the relative water content is a good indicator of drought stress (fisher, 2000) and in this study, we noticed that drought stress caused a decrease in relative water content in both inoculated and non-inoculated plants compared to well watered plants, however, the inoculation of wheat stressed plants with the bacterium a. lipoferum n040 significantly increased the relative water content compared to the non-inoculated controls. this may be due to a reduction in the inhibitory effect of drought on roots and the development of a more effective root system in the inoculated plants (dodd et al., 2010). drought stress accelerated relative membrane permeability (rmp) in the inoculated and non-inoculated plants compared to wellwatered plants (100% etc). however, bacterial inoculation helped wheat plants to maintain the rmp and reduced leaf damage compared to non-inoculated plants under drought stress. a positive correlation between drought stress sensitivity and membrane damage were observed by vardharajula et al. (2011) and sandhya et al. (2010), and the bacterial inoculation reduced the membrane damage in plants stressed by drought stress. antioxidant enzymes are very good biochemical markers of stress and increasing their activities could be a potential to alleviate the oxidative stress-induced by water stress. in our study, drought induced activities of the two enzymes and this may be attributed to the generation of ros. consequently, the plant tries to force this by stimulation of antioxidants defense system (li et al., 2011). the inoculation showed further induction in activities of both sod and pod (tab. 5), increasing the antioxidant defense system efficiency against superoxide (o2-) radicals produced under water stress. it is most probable that a. lipoferum improved plant defense enzymes such as superoxide dismutase, peroxidase or phenolic compounds, to mitigate the oxidative damage elicited by drought stress. more work is necessary to find exactly the protecting roles of azospirillum sp. on antioxidant enzymes system under drought stress, to provide potential new mechanisms of a plant’s tolerance to drought stress, and to define the physiological roles of azospirillum sp. in relation to environmental stresses, including drought stress. conclusions the inoculation of wheat plants with the bacterium a. lipoferum n040 increased the activities of some key anti-oxidative enzymes (super-oxide dismutase and peroxidase) and the concentrations of non-enzymatic antioxidants such as total carotenoids and total phenols as well as the concentration of compatible solute such as proline, soluble carbohydrates and soluble proteins in wheat plants grown under drought stress. however, the antioxidant system exhibited considerable changes in response drought stress, suggesting that this increased antioxidant activity may be responsible, at least in part, for the greater tolerance of a. lipoferum treated wheat plants to drought stress, leading to improved plant growth, grain anatomy and productivity. 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rag01@fayoum.edu.eg © the author(s) 2017. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 88, 215 221 (2015), doi:10.5073/jabfq.2015.088.031 1department of citriculture, murcian institute of agriculture and food research and development (imida), la alberca, murcia, spain 2 department of economic and financial studies, miguel hernandez university, elche, alicante, spain 3 department of vegetable production and microbiology, miguel hernandez university, orihuela, alicante, spain 4 department of physics and computer architecture, miguel hernandez university, orihuela, alicante, spain quality and fruit colour change in verna lemon i. porras1, j.m. brotons2, a. conesa3, r. castañer4*, o. pérez-tornero1, f.j. manera4 (received may 15, 2015) * corresponding author summary while most lemon cultivars in the northern hemisphere are harvested in autumn-winter, verna, an autochthonous spanish cultivar, is harvested later (february to august), supplying the european market when lemons are in short supply, a market that is also served by imports from the southern hemisphere, mainly argentina and south africa. the aim of this study was to determine the temperature at which degreening begins naturally in verna lemon, noting the evolution of colorimetric parameter a and comparing the same with the equivalent measurements made in eureka lemon, the most widely cultivated lemon worldwide. the influence of solar radiation on the colorimetric parameters in verna was studied, and the influence of the minimum temperatures on the change from green to yellow, using the data collected over five growing seasons, was assessed. the results confirmed the relation between net solar radiation and degreening, a process that begins when net solar radiation reaches a value of between 2 and 4 mj/m2.day and when the mean temperature of the 14 days prior to sampling is 8.8 °c or when the daily mean temperature reaches 5.5 °c on two consecutive days. the information obtained will enable growers to predict the colour changes that will occur in the field and potential growers to ascertain whether a given geographical zone is suitable for the crop in question. introduction verna (berna or vernia) is an ancient cultivar (hodgson, 1967; ortiz et al., 1984; russo and spina, 1985), which blooms several times a year, but mainly in spring. the fruits in the first flowering period contain few seeds and are harvested later than most lemons (february to august) (garcía-lidon et al., 2003; ortiz et al., 1984; porras, 2014; russo and spina, 1985; saunt, 1990). suitable cultivation techniques can favour summer flowering, which produces fruit that can be harvested the following summer (known as (“rodrejos” in spanish and “verdelli” in italian). verna fruits keep well on the tree and in storage rooms; they are also resistant to handling and transport (porras, 2014). until the 1970s, verna was the most widely cultivated lemon in spain, but has since been superseded by fino. of the more than 39,000 ha dedicated to lemon cultivation in spain, 12 000 are dedicated to verna, with an annual production of 190 000 tons (magrama, 2014). this cultivar supplies the european market at a time (spring-early summer) when lemons are in scant supply (porras, 2014; saunt, 1990), since the harvesting of autumn-winter cultivars has finished and competition from the southern hemisphere does not begin until the end of may. eureka is perhaps the most widely grown lemon cultivar in the world, especially in california, south africa, chile and australia it is very productive and rapidly enters into production. however, the tendency of the fruit to appear at the end of branches makes it very susceptible to the cold and attack by aceria sheldoni ewing. it therefore needs special cultivation practices and, moreover, it is not a long-lasting tree (garcía-lidon et al., 2003). when night temperatures fall at the end of october-beginning of november, the chlorophylls present in the rind of verna begin to degrade (sinclair, 1984; soni and randhawa, 1969), and yellow carotenoids, which have already begun to be synthesised but were previously masked by the green colour, manifest themselves in the typical “lemon yellow” colour of the fruit. this colour indicates the stage of ripeness and stimulates the perception of freshness on the part of the potential customer (hutchings, 2003; joshi, 2001). the quantity of carotenoids in citrus fruit is very high (gross, 1977; lee and coates, 2003; meléndez-martínez et al., 2007a, b, 2010) but in lemon the total concentration of carotenoids is lower than in other citrus (yokoyama and vandercook, 1967; katoet al., 2004). the principal carotenoid is β-cryptoxanthin (katoet al., 2004). the decline in rind chlorophyll takes several months and the onset of carotenoid accumulation almost coincides with the disappearance of chlorophyll (spiegel-roy and goldschmidt, 1996). degreening with ethylene is practised when lemons have not yet degreened, especially in early cultivars (grierson et al., 1986; conesa et al., 2014). the lemon cultivars eureka, lisbon and fino are generally harvested green before undergoing degreening in an ethylene chamber. the colour change that they undergo on the tree is similar in all three cultivars (manera et al., 2012 a, b), while verna differs in this respect. this cultivar takes on its characteristic yellow colour later and can therefore be left longer on the tree, meaning that it does not need to be degreened artificially to reach an acceptable commercial colour. the aim of this study was to monitor the natural change in colour that takes place in verna and compare its behaviour with that of eureka, the most widely grown cultivar in the world (saunt, 1990). we also study whether verna needs lower temperatures than eureka for natural degreening to begin, and the influence that net solar radiation has on the colour change. verna fruit quality in spring, when no other cultivar in the northern hemisphere can supply the market, is analysed, but no comparison is made with the other cultivars (eureka, lisbon and fino) since they are not available at this time. the results obtained should be of interest for planning lemon plantations in new areas, based on natural degreening, with the aim of increasing the supply of lemons at a time of the year when the autumnwinter cultivars have ceased to produce. material and methods plant material the cultivar used in this study, verna clone 51 on citrus macrophylla rootstock, is designated as a spring-summer harvesting cultivar. its behaviour was compared with that of the widely cultivated cultivar, eureka on citrus macrophylla rootstock. although citrus 216 i. porras, j.m. brotons, a. conesa, r. castañer, o. pérez-tornero, f.j. manera macrophylla is an excellent rootstock and is well adapted to crop conditions of the mediterranean area, care must be taken when using nitrogenated fertilization in verna grafted onto this rootstock since, otherwise, the fruit has a tendency to grow too big and have a thick rind. verna grafted on citrus macrophylla is also more sensitive to injury than when grafted onto sour orange (riquelme et al., 2008). site description and experimental conditions the two cultivars were planted in april 1983 in a plot at imida (murcia institute of agriculture and food research and development), la alberca (murcia, se spain). trees were spaced 6 × 6 m. the soil is permeable and calcareous (17.1 % total calcium carbonate). all the trees used in the study were healthy and in full production. drip irrigation was used with five emitters per tree, with a flow rate of 4 l/h. water ph was 7.15, electrical conductivity 3.8 ds/m, cl8.63 mmol/l, na+ 12.28 mmol/l, ca2+ 10.70 mmol/l, k+ 0.79 mmol/l and mg2+ 10.65 mmol/l. potassium nitrate, ammonium nitrate (34 %), monoammonium phosphate and iron chelate were applied weekly from the first year of planting. zn and mn fertilizers were sprayed in spring. the trees were cultivated, pruned and sprayed with insecticides and fungicides, according to local practices. the annual average temperature is 18.4 °c, with a mean maximum temperature of 31.1 °c and mean minimum temperature of 4.5 °c. mean annual rainfall is 321 mm (siam, 2015). data analysis the external colour of the fruit was measured in the hunterlab colour space (hunter, 1967) with illuminant c as representative of daylight (macdougall, 2002), using a minolta c-300 reflection colorimeter: tristimulus with double light path in the visible light range, standard observer angle cie-2° and white calibration plate. three readings were taken in the equatorial zone of each fruit. in each reading, the colorimetric coordinates l, a and b were measured. the colour coordinate l measures lightness (100 for white and 0 for black). the colour coordinate a corresponds to the green-red axis, where the negative values correspond to green and the positive to red (-60 green, +60 red) (hutchings, 1994; macdougall, 2002), and the colour coordinate b measures variations from blue to yellow (-60 blue, +60 yellow). any decrease in the chlorophyll content of the fruit is associated with coordinate colorimetric a (kidsome et al., 2002; manera et al., 2012a). hue angle (hab) is defined as the angle between the hypotenuse and 0° on the a (bluish-green/red-purple) axis; hab is calculated from the arctangent of b/a (little, 1975; mcguire, 1992). colour differences expressed by δeab were calculated using l, a and b as geometric coordinates in the following formula: δeab = [(δl)2 + (δa)2 + (δb)2]1/2. values above 3 indicate that the colour per ceived by the human eye is different (manresa and vicente, 2007). the relation between net radiation, measured in mj/m2.day as described by allen et al. (1998), and the colorimetric coordinates was also studied. temperature and data for calculating net radiation, as described by allen et al. (1998), were provided by a weather station in the same plot, observatory mu62 of la alberca, murcia, which forms part of the network that imida maintains in the most important growing regions of the province of murcia (siam, 2015). net radiation (rn) is the difference between the incoming and outgoing radiation at long and short wavelengths. it is the balance between the energy absorbed, reflected and emitted by the earth’s surface (rb). rn is the radiation that remains in the crop and is calculated by deducting from the radiation that reaches the earth’s surface (rs) the reflected radiation (which depends on the albedo) and the losses of long wave length radiation (rb), which depend on the temperature, cloud cover and humidity level. rn is normally positive during the day and negative during the night (allen, 1998). rn = (1α) × rs rb, where rs = incident solar radiation. the radiation that reaches the earth’s surface (rs) can be calculated as a function of latitude, time of the year and cloud cover. the index α indicates the incident radiation that is reflected by the surface, and is known as “albedo”, with a value of between zero and one. for crops with more than 80 % soil cover, α = 0.23. a green vegetation cover has an albedo of about 0.20-0.25. for the green grass reference crop, α is assumed to have a value of 0.23 (allen et al., 1998). rb represents losses of long wave radiation from the surface. rn is measured in mj/m2.day. the relation between net radiation, measured in mj/m2.day, and the colorimetric coordinates was also studied. to smooth the radiation series and improve the correlation between net radiation and the colorimetric coordinates, the net radiation mean of the previous 14 days was used. from march to may, random samples of 15 fruits were harvested from each of the four verna trees analysed. the samples were weighed; fruit width (w) and peel thickness (t) were measured after cutting in half with a digital caliper and then the size; thickness indices (2 × t × 100 / w) indices were calculated. juice was extracted with an electric juicer. the resulting juice and the flesh were collected in a mesh bag (1 mm) and the remaining juice was extracted by squeezing the bag by hand until no juice remained. the juice content (%), total soluble solids (tss) and titratable citric acid (ta) were calculated. tss was determined by an atago digital refractometer (at 20 °c) and ta by titration with 0.1n naoh, using phenolphthalein indicator (anonymous, 1946). to quantify the organic acids present in the lemon juice (citric, malic, oxalic, tartaric, ascorbic and fumaric), their evolution was studied during maturation. the juice was centrifuged for 10 minutes at 7500 rpm at 4 °c, using a selecta model 540 centrifuge, and filtered through 0.45 μm filters before being introduced in the chromatograph for analysis. a heweltt packard 1100 (hplc) was used with a nucleosil column (12.5 × 0.4 cm; 3μm particle size). the mobile phase was a 0.01 m solution of kh2po4 taken to ph 2.25 with h3po4. the injection flow was 1 ml/min and the wavelength 206 nm. the data obtained were not compared with those of other cultivars because, at the time at which the verna samples were taken, no other cultivar bears fruit. experimental design at the beginning of the experiment, ten fruit (from inside the canopy, 5 north-facing and 5 south-facing) were labelled at random on four trees of each cultivar (40 fruits per cultivar − a total of 80 fruits between both cultivars per year). the colour of the fruit was measured by colorimeter every 7-15 days from september to january in eureka, and until april-may in verna, during the 2007/08, 2008/09, 2009/10, 2010/11 and 2011/12 growing seasons, providing approximately 13 measurements per year. to analyse the relation between the 14-day daily mean temperatures and the different colorimetric coordinates, the receiver operating characteristic curve (roc) was used since this shows the sensitivity (the probability that the colorimetic coordinate a will reflect the fact that degreening has begun when it really has begun) and the specificity (the probability that the coordinate will predict that degreening has not begun when it really has not). the roc curve is the plot that displays the full picture of trade-off between the sensitivity (true positive rate) and (1specificity) (false positive rate) across a series of cut-off points. as turning point temperature (responsible for initiating the process), we took the temperature at which the sum color in verna lemon 217 of both (sensitivity and specifity) was maximum. for this purpose, spss 22.0.0.0 was used. below, we determine the mean 14-day temperature at which this degreening process begins, taking the mean temperature of 14 days as test variable and another dichotomous variable (denominated a_01), which indicates whether the process has (a_01=1) or not (a_01=0) begun: (1) for each a*, a roc curve was constructed (fig. 1), and the a* curve for which the sum of the sensitivity and (1-specificity) is maximum was selected. this curve corresponds to the value of the mean 14-day temperature at which this degreening process begins. below, we study whether verna needs lower temperatures to reach a similar degreening level as eureka. if colour coordinate a remains above -2 in verna (fig. 2) the fruit can be kept on the tree another three months without losing its commercial quality, unlike eureka and other autumn-winter cultivars (e.g. lisbon and fino), which, once they reach a values above 0, begin to senesce and must be harvested before they fall to the ground. to ascertain whether differences in rind colour between eureka and verna can be appreciated visually, the value of δeab was calculated (fig. 3). results and discussion evolution of the colorimetric coordinate a the evolution of temperatures and coordinate a (eureka and verna cultivars) during the degreening process of 2012/13 are represented in fig. 2, where the differences in how the degreening process proceeds in both cultivars can be seen. in eureka degreening (mea sured as the increase in colour coordinate a) begins two weeks earlier and takes place much more rapidly than in verna. moreover, verna never reaches the values that are attained by eureka. fig. 1: roc curve constructed from sensitivity and 1-specifity fig. 2: evolution of coordinate a (eureka and verna cultivars) and mean minimum temperatures and standard deviation of the 14 days from september 2012 to may 2013. fig. 3: evolution of δeab in eureka and verna; mean values for the six harvests. when δeab is above 3, the fruit of both cultivars can be distinguished colorimetrically (manresa and vicente, 2007). the colour differences are higher in november and december, as is shown in fig. 3 and in accordance with fig. 2. influence of net solar radiation on degreening although temperature is considered the most important trigger of the degreening process (manera, 2012 a, b; sinclair, 1984), the influence of solar radiation on verna degreening was also studied. fig. 4 shows the evolution of mean net solar radiation (mj/m2.day) and colour coordinate a. at the beginning of autumn, net radiation reached values close to 12, falling as the year progressed. in all the years studied degreening (disappearance of chlorophyll) began at rn values of ≤ 4 mj/m2.day (during the first week of december). in the growing seasons of 2008 and 2009, the process began with rn values close to 2 mj/m2.day and in 2007 and 2012 at a slightly higher value, around 4 mj/m2.day. in 2007, 2008 and 2009, when rn ≥ 6 mj/m2.day (at the very end of winter, during the first week of march), the a value began to fall and the rind of verna turned from yellow to greenish. this agrees with the results of other studies, for example in the orange cultivar valencia, whose rind begins to turn green again, when the temperatures begin to rise (soler and soler, 2006; thomson et al., 1967; young et al., 1961). net solar radiation and degreening are related, the process beginning when nr falls to values of 2 and 4 mj/m2.day (fig. 3). relation between minimum temperature and colour coordinates fig. 5 shows the evolution of the mean values of colour coordinates l, a and b and of hue angle for verna and eureka, and the mean minimum temperatures for the 14 days preceding the measurements. although previous studies have taken the mean values for 21 days (manera et al., 2012 a, b), there is scant improvement in the correlations between variables with this increase in time, so we opted for a 14-day period. 218 i. porras, j.m. brotons, a. conesa, r. castañer, o. pérez-tornero, f.j. manera fig. 4: evolution of coordinate a and net radiation (mean of 14 days preceding the measurement, and standard deviation) for seasons 2007 to 2012. note that the series corresponding to eureka finish in mid-january, when the condition of fruit on the tree ceases to be commercially acceptable, while, at the same time, the fruit of the verna cultivar are still on the tree and can remain so for a further 3 months with no loss of quality. of note is the fact that all three colour coordinates (l, a, and b) and hue angle begin to change earlier and progress more rapidly in eureka than in verna. moreover, the final coordinate values reached are in general higher in eureka, while the hue angle is, by its very definition, lower. • coordinate l. in verna this coordinate does not begin to in crease until the second week of november, when mean minimum temperatures for the previous 14 days fall below 10 °c, whereas the same parameter begins to change one month earlier in eureka (mean minimum temperatures of around 16 °c), which agrees with the results of manera et al. (2012 a, b). however, the final values reached by l were similar in both verna and eureka (67-68). • coordinate a. in verna degreening begins with mean minimum temperatures for the previous 14 days of around 9 °c, and occurs later than in the case of eureka (around 16 °c), coinciding with the results of manera et al. (2012 a, b). however, a increases more slowly in verna than in eureka, in which the parameter reaches positive values (giving it a slightly reddish tone and the appearance of older fruit), while in verna the same parameter only reaches values of around -2 (greenish yellow and younger in appearance). • coordinate b. this begins with values that represent a yellowish colour (20-22), although masked by the green colour of the chlorophyll. the increase in yellow begins with when mean minimum temperatures 14 reach about 13 °c, while the final values are 35 and 38 in verna and eureka, respectively (slightly yellower). • lastly, hue angle in verna reaches 95 but does not exceed 90 in eureka, meaning that verna appears yellowish green and eureka yellow. color in verna lemon 219 determination of 14-day minimum temperature, below which degreening begins fig. 4, which depicts the evolution of coordinate a and net radiation for each of the six seasons studied, suggests that degreening begins when colorimetric coordinate a exceeds – 11. below, we determine the mean 14-day temperature at which this degreening process begins, taking the mean temperature of 14 days as test variable, and another dichotomous variable we denominate a_01, which indicates whether the process has (a_01=1) or not (a_01=0) begun: (2) using these two variables a roc curve was constructed for each temperature (test variable). different sensitivity and specificity values were obtained for each point of the roc curve. the highest sum was taken. this process was repeated for different values of the test variable (temperature), taking the highest sum of the sensitivity and specificity of each curve. finally, the maximum value of these sums was taken. the result corresponds to a mean minimum 14-day temperature of 8.8 °c. in other words, the value at which degreening in verna begins (9 °c in the above sections) (fig. 4) can now be corrected to 8.8 °c with the roc curve. therefore, it can be affirmed that the 14-day mean minimum temperature for degreening to begin is 8.8 °c. determination of daily minimum temperature below which degreening begins besides the relation observed between the mean minimum temperatures of 14 days and the colorimetric coordinates, we also studied how the degreening process is triggered by analysing the daily minimum temperatures and colorimetric coordinates. since coordinate a was seen to best explain the changes in yellowness (degreening), the relation is shown for the six years of the study. taking the daily temperature as test variable and the dichotomous variable we denominate a_01, which indicates whether the process has (a_01=1) or not (a_01= 0) begun, and repeating the same process as in the previous section, the daily minimum temperature for degreening to begin was found to be below 5.5 °c. physical-chemical characteristics of the fruit when the harvesting of autumn-winter cultivars of (eureka, lisbon and fino) finishes, the largest verna fruit can be picked, and the rest can be left on the tree for forthcoming months since they will maintain their condition (garcía-lidón et al., 2003; porras, 2014). however, care must be taken with irrigation and fertilisation to avoid excessively large fruit. in the time period studied in this work, the fruit reached their commercial size of 58-63 mm (tab. 1) and were suitable for marketing. if they are not harvested in april or may, many of the fruit grow too big for commercial purposes, although they remain in good condition on the tree. rind thickness is also excessive after april, fig. 5: evolution of colour coordinates l, a and b, hue angle and mean minimum temperatures (and standard deviation) for the 14 preceding days in eureka and verna cultivars. values are the means of all the growing seasons analysed. 220 i. porras, j.m. brotons, a. conesa, r. castañer, o. pérez-tornero, f.j. manera although the rind tends to be thicker in verna than in other cultivars at all times (ortiz et al., 1984; pérez-pérez et al., 2005). this characteristic and the low levels of essential oils in the rind make verna more resistant to damage from handling, storage and transportation (garcía-lidón et al., 2003; porras, 2014; riquelme et al., 2008). in general, fruits from trees on citrus macrophylla have higher ta and tss levels in march than in mid-may (54.9 g/l. and 6.7 °brix, compared with 45.3 g/l and 5.65 °brix, respectively). acidity is lower in verna than in eureka, lisbon and fino (ortíz et al., 1984). eureka grafted on citrus macrophylla shows higher acidity (67.8 g/l) in autumn (perez-perez et al., 2005), than was observed in our study. the percentage of juice, acidity and °brix of verna are lower than in other cultivars (ortiz et al., 1984; perez-perez et al., 2005). the organic acid with the highest concentration in verna was citric acid (92.16 %), followed by malic acid (4.45 %), which reflects the findings of sinclair (1984). other organic acid are present in very low proportions (tab. 2). conclusions 1. there was a clear relation between net solar radiation and the degreening of verna lemon in the growing seasons studied. degreening begins when net radiation reaches between 2 and 4 mj/ m2.day 2. in verna degreening begins when mean minimum temperatures of 14 days fall below 8.8 °c, as obtained by curve the roc calculation. 3. degreening in verna can also be said to begin when the daily minimum temperatures reach 5.5 °c. new plantations of verna will not be viable in any part of the world if the minimum temperature does not fall below 9 °c since fruit quality will not be sufficient for the fresh market. similarly, it must be borne in mind that both the beginning of degreening and its progress need rn values of ≤ 4 mj/m2.day. in the climatic conditions of the inland valleys of the province of murcia, verna would need 14 days with mean temperatures below 8.8 °c for the natural degreening process to begin on the tree. the fruit of verna remains on the tree during winter months and the beginning of spring, maintaining a stable yellow colour and remaining fresh in appearance. this permits the fruit to be harvested late (until the beginning of summer) with the fruit still in good condition. although overall quality of verna lemons is lower than that of autumn-winter cultivars, the fruit comply with quality norms and can supply the european market when no other cultivar is available and before the arrival of lemons grown in the southern hemisphere. acknowledgements the authors thank philip thomas for checking the english of the manuscript. this work was funded by the murcian institute of agriculture and food research and development and feder funds (feder-imida po-07-030). references allen, r.g., pereira, l.s., raes, d., smith, m., 1998: crop evapotranspiration – guidelines for computing crop water requirements. fao irrigation and drainage, paper 56. anonymous, 1946: official method for determining soluble solids to acid ratio for oranges and grapefruits. the bulletin department of agriculture, state of california, vol. xxxv, nº. 1, january-march 1946. conesa, a., brotons, j.m., manera, f.j., porras, i., 2014: the de greening of lemon and grapefruit in ethylene atmosphere: a cost analysis. scientia horticulturae 179, 140-145. dogv, 2006: orden de 4 de septiembre de 2006, de la conselleria de agricultura, pesca y alimentación por la que se especifican las condiciones mínimas de calidad para la comercialización de frutos cítricos en fresco. [2006/10409]. tab. 1: characteristics of verna lemon fruit in different dates 02/03 24/03 19/04 18/05 weight (g) 145.4±2.1 159.6±2.8 168.8±3.8 174.4±4.1 diameter (mm) 59.9±0.0 61.6±0.2 63.1±0.7 64.0±0.8 rind thickness 5.9±0.1 5.9±0.1 6.8±0.1 6.7±0.2 thickness index 19.8±0.4 19.2±0.3 21.7±0.3 20.9±0.6 % juice 28.6±1,0 34.4±0.7 33.4±0.7 32.5±0.7 acidity (g/l) 54.9±0.5 50.0±0.9 48.5±0.8 45.3±0.9 ºbrix 6.7±0.1 6.5±0.1 6.2±0.1 5.6±0.1 tab. 2: organic acid contents of verna lemon fruit in different dates organic acid 02/03 24/03 19/04 citric (mg/ml) 32.3±8.5 27.7±2.3 29.3±3.8 malic (mg/ml) 1.8±0.4 1.4±0.2 1.2±0.2 oxalic (mg/100 ml) 15.7±3.0 28±2.1 11.1±2.2 tartanic (mg/100 ml) 43.8±10.4 45.1±5.5 47.2±7.6 fumaric (mg/100 ml) 5.9±3.8 5.4±0.7 4.0±1.0 ascorbic (mg(100 ml) 47.2±9.4 39.1±3.6 40±5.5 the differences between total acidity expressed as anhydric citric acid and the sum of the organic acids can be attributed to the fact that aminoacids, polyphenolic substances and oxidryl radicals are evaluated in titratable acidity (rolle and vandercook, 1963, cited by sinclair, 1984). the ascorbic acid values were higher than those obtained in verna lemon by marín et al. (2002) (26.1 mg/100 ml). in general the values reflect those obtained by garcía lidón et al. (2003) and sinclair (1984) for numerous lemon cultivars throughout the world. in general the overall quality of verna is lower than that of autumnwinter cultivars (eureka, lisbon, fino), but it has the advantage of being able to supply the european market when the others have finished and before lemons arrive from the southern hemisphere (garcía-lidón et al., 2003; porras, 2014). they exceed the quality standards set for export to european markets (dogv, 2006). color in verna lemon 221 garcía lidón, a., del río, j.a., porras, i., fuster, m.d.y., ortuño, a., 2003: el limón y sus componentes bioactivos. serie técnica y de estudios nº 25. conserjería de agricultura, agua y medio ambiente. murcia. 127 pp. grierson, w., cohen, e., kitagawa, h., 1986: degreening. in: wardowsky, w.f., nagy, s., grierson, w. (ed.), fresh citrus fruits. avi publishing co, wesport. gross, j., 1977: carotenoid pigments in citrus. in: nagy et al. (ed.), citrus science and technology. s. avi. publishing co., wesport, connecticut. hodgson, r.w., 1967: horticultural cultivars of citrus. in: reuther, w., batchelor, l.d., webber, h.j. (eds.), the citrus industry. i (4), 431-591. univ. calif. hunter, r.s., 1967: development of the citrus colorimeter. food technol. 21, 100-105. hutchings, j.b., 1994: food colour and appearance. univ. press, cambridge, great britain. hutchings, j.b., 2003: expectations and the food industry. kluver academic/plenum publishers, new york. joshi, p., 2001: physical aspects of colour in foods. in: amesand, j.m., hoffman, t.f. (eds.), chemistry and physiology of selected foods colorants. acs symposium series 775. american chemical society. washington d.c. kato, m., ikoma, y., matsumoto, h., sugiura, m., hyodo, h., yano, m., 2004: accumulation of carotenoids and expression of carotenoid biosynthetic genes during maturation in citrus fruit. plant physiol. 134(2), 824-837. kidsome, u., edelenbos, m., nørbæk, r., christensen, l.p., 2002: color stability in vegetables. in: macdougall, d.b. (ed.), color in food. improving quality, 179-232. woodhead publishing, cambridge, england. lee, h.s., coates, g.a., 2003: effect of thermal pasteurization on valencia juice color and pigments. lebensm-wiss u technol. 36, 153-156. little, a.c., 1975: off on a tangent. j. food sci. 40, 410-411. macdougall, d.b., 2002: color measurement of food: principles and practice. in: douglas, b., macdougall, color in food. improving quality, 33-63. woodhead publishing limited, cambridge, england. magrama, 2014: anuario de estadística agraria 2012. www: magrama. gob.es/estadistica manera, j., brotons, j.m., conesa, a., porras, i., 2012a: relationship between air temperature and degreening of lemon (citrus lemon l. burm. f.) peel color during maturation. australian j. crop sci. 6(6), 1051-1058. manera, f.j., brotons, j.m., conesa, a., porras, i., 2012b: influence of temperature on the beginning of degreening in lemon peel. scientia horticulturae 145, 34-38. manresa, a., vicente, i., 2007: el color en la industria de los alimentos. ministerio de educación superior. ed. universitaria. la habana (cuba). mcguire, r.g., 1992: reporting of objective color measurements. hort. sci. 27(12), 1254-1255. marín, f.r., martínez, m., uribesalago, t., castilla, s., frutos, m.j., 2002: changes innutraceuticalcoposition of lemon juices according to different industrialextraction systems. food chem. 78, 319-324. meléndez-martínez, a.j., escudero-gilete, m.l., vicario, i.m., heredia, f.j., 2010: study of the influence of carotenoid structure and individual carotenoids in the qualitative and quantitative attributes of orange juice color. food res. int. 43, 1289-1296. meléndez-martínez, a.j., vicario, i.m., heredia, f.j., 2007a: review: analysis of carotenoids in orange juice. j. food compos. anal. 20(7), 638649. meléndez-martínez, a.j., vicario, i.m., heredia, f.j., 2007b: relationship between the color and the chemical structure of carotenoid pigments. food chem. 101, 1145-1150. ortíz, j.m., garcía-lidón, a., tadeo, j.l., fernández de córdova, l., 1984: rootstock effect on fruit and juice characteristics of lemons. proc. int. soc. citriculture i, 50-53. pérez-pérez, j.g., porras, i., garcía-lidón, a., botíal, p., garcíasánchez, f., 2005: fino lemon clones compared with two other lemon cultivars on two rootstocks in murcia (spain). scientia horticulturae 106, 530-538. porras, i., 2014: limonero, pomelo y lima. en: la fruticultura del siglo xxi en españa. coordinadores: juan josé hueso martín; julián cuevas gonzález. serie agricultura nº 10. 301-325. edita cajamar caja rural. isbn-13: 978-84-95531-64-3. riquelme, m.t., porras, i., riquelme, f., 2008: the rootstock and inter stock relations with the mecanical susceptibility of lemon 'verna'. acta hort. 773, 103-109. russo, f., spina, p., 1985: varietà coltivate. in: spina, p. (ed.), trattato di agrumicoltura, 117-156. edagricole. bolonia. saunt, j., 1990: citrus cultivars of the world. sinclair int. ltd., norwich, england. siam, 2014: cosult web page <htpp/www.imida.es> for further information. sinclair, w.b., 1984: the biochemistry and physiology of the lemon and other citrus fruits. univ. of california, u.s.a. soler, j., soler, g., 2006: cítricos. cultivares y técnicas de cultivo. mundi-prensa. madrid. soni, s.l., randhawa, g.s., 1969: changes in pigments and ash contents of lemon peel during growth. indian j. hortic. 26(1-2), 20-26. spiegel-roy, p., goldschmidt, e.e., 1996: biology of citrus. cambridge university press, great britain. thomson, w.w., lewis, l.n., coggins, c.w., 1967: the reversion of chromoplats to chloroplats in valencia oranges. cytologia 32, 117-124. yokoyama, h., vandercook, c.e., 1967: citrus carotenoids. i. comparison of carotenoids of mature-green and yellow lemons. j. food sci. 32, 42-48. young, l.b., erickson, l.c., 1961: influences of temperature on color change in valencia oranges. proc. am. soc. hortic. sci. 78, 197-200. address of the authors: i. porras, o. pérez-tornero, department of citriculture, murcian institute of agriculture and food research and development (imida), estación sericícola, calle mayor s/n. 30150, la alberca, murcia, spain. e-mail: ignacio.porras@carm.es j.m. brotons, department of economic and financial studies, miguel hernandez university, avda. de la universidad, s/n, 03202, elche, alicante, spain. e-mail: jm.brotons@umh.es a. conesa, department of vegetable production and microbiology, miguel hernandez university, ctra de beniel, km 3.2, 03312 orihuela, alicante, spain. e-mail: agustin.conesa@umh.es r. castañer, f.j. manera, department of physics and computer architecture, miguel hernandez university, ctra de beniel, km 3.2, 03312 orihuela, alicante, spain. e-mail: fco.manera@umh.es e-mail corresponding author: r.castaner@umh.es © the author(s) 2015. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 90, 359 367 (2017), doi:10.5073/jabfq.2017.090.045 1huzhou academy of agricultural sciences, huzhou, china 2institute of crop and nuclear technology utilization, zhejiang academy of agricultural sciences, hangzhou, china 3huzhou bureau of agriculture, huzhou, china seed weight and oil content response to branch position reveals the importance of carbohydrate and nitrogen allocation among branches in canola (brassica napus l.) genotypes yun ren1, jianfang zhu1, yichun wang3, shanlin ma1, genru ye1, shuijin hua2* (received march 24, 2017; accepted november 1, 2017) * corresponding author summary increasing seed weight and oil content are the main pathways to increase canola seed oil yield. the effect of branch position on seed weight and oil content has not been previously reported in canola. field experiments were conducted to explore the impact of branch position on seed weight and oil content. four canola genotypes, zheyou 50, zhongshuang 11, zheyou 18, and zheshuang 8, were used to evaluate seed weight, oil content, carbohydrate profile, and nitrogen content in the main inflorescence and branches from the top to the bottom of the main stem. seed weight and oil content decreased from the main inflorescence to the lower branches in the four genotypes. lower carbohydrate and nitrogen content in the seed and low transport efficiency of the two chemical compounds in the siliques and branches were responsible for the lower seed weight and oil content in zheshuang 8 and zheyou 18, respectively. however, the decreasing seed weight and oil content in the branches did not correspond with decreasing carbohydrate and nitrogen content in the branches from the top to the bottom. the result suggested complex carbohydrate and nitrogen metabolism in the canola seed in the different branches. introduction seed weight and oil content are two essential traits for canola (brassica napus l.) seed oil yield production. berry and spink (2006) estimated that canola seed weight should reach 5.0 mg or more per seed to achieve a yield of 6.5 t ha-1 of seed in the uk. canola germplasms with heavy seed exist in the natural accessions. kennedy et al. (2011) evaluated the performance of seed traits including seed weight in 1500 rapeseed accessions and the result revealed that the seed weight ranged from 1.3 to 7.0 mg per seed. consequently, there is a high potential for substantial improvement of canola seed weight in breeding programs. seed weight is one of the three yield components, namely, siliques per plant, seeds per silique, and seed weight in canola. in recent years, canola seed weight has been received more and more attentions by researchers and breeders. many researchers have investigated the genetic control of canola seed weight (cai et al., 2012; cai et al., 2014; li et al., 2014a; li et al., 2014b; li et al., 2015; liu et al., 2015). multiple qtls controlling canola seed weight have been identified. for example, more than 100 qtls affecting seed weight were found in genetically different canola populations and environments; while one qtl (qsw.a7-2) was detected in all environments (shi et al., 2009). these results indicate that canola seed weight is a complicated quantitative trait and governed not only by the genetic factors with different pathways but also the interactions with different environments. more recently, a gene encoding an auxin response factor, arf18, was found to regulate seed weight and silique length (liu et al., 2015). however, in addition to genetic and environmental factors, less attention is paid to the developmental issues modulating seed weight, for example, canola branch position. canola seed oil content is another important trait and is regulated by many factors as well. numerous reports revealed that canola seed oil content is controlled by many qtls (zhao et al., 2005; jiang et al., 2014; javed et al., 2016). although the pathway governing seed oil biosynthesis is well described, a key gene for seed oil content has not been isolated, possibly due to the lack of major effect qtls (baud and lepiniec, 2009). furthermore, seed oil content is heavily affected by crop management. nitrogen application has a positive effect on seed yield, yet, seed oil content can be negatively affected (zuo et al., 2016). planting date can also markedly affect seed oil content (robertson and green, 1981). however, there is no report on the relationship between canola seed oil content and branch position. after budding, branches establish and elongate rapidly in canola. the flowering sequence in the different branch positions is usually from the top to the bottom of the main stem, namely, from the main inflorescence, the first branch, second branch and so on. however, the flowering sequence on the branch begins from the bottom to the top on the main inflorescence and from the adaxial region on other branches. consequently, a gap of flowering time and seed and silique development among branches occurs in canola. however, regard less of this gap, canola is harvested at the same time and the whole plant is used as a harvesting unit. as a result, the differences of seed matter and oil accumulation potentially exist in different branches in canola. during silique wall and seed development, nitrogen-containing compounds and carbohydrates are relocated from the main stem and are also produced through silique wall photosynthesis (bennett et al., 2011). furthermore, the canopy of canola consists of siliques with different branches; levels of irradiation will differ depending on branch location. the resultant photosynthetic activity of siliques on different branches would lead to varying seed weight and oil content since carbohydrates are essential for seed matter accumulation and oil biosynthesis (hua et al., 2014). the aim of this study was to evaluate seed weight and oil content at different branch locations in four canola cultivars. moreover, carbohydrate profiling and nitrogen content were determined in the seed, silique, and the branch stem to investigate the effect of the distribution of the compounds on seed weight and oil content. materials and methods plant materials and crop management the field experiments were carried out over two growth seasons in 2012-2013 and 2013-2014 at huzhou academy of agricultural sciences experimental station. four canola (brassica napus l.) cultivars, zheyou 50, zhongshuang 11, zheyou 18, and zheshuang 8, were selected as plant materials. zheyou 50 and zhongshuang 11 are two high seed oil content cultivars in china whose oil content can reach 49.00% and 49.04%, respectively. the two cultivars were bred by zhejiang academy of agricultural sciences and institute of oil crops, chinese academy of agricultural sciences, respectively. 360 y. ren, j. zhu, y. wang, s. ma, g. ye, s. hua zheyou 18 and zheshuang 8 were released by zhejiang province, china. the cultivars were bred by zhejiang academy of agricultural sciences. zheyou 18 was the first cultivar successfully bred for mechanical harvesting in zhejiang province. three to five seeds of the four cultivars were directly sown in a shallow hole (about 3 cm) and covered by a layer of soil on 1 october 2012-2013 and 2013-2014. at the five-true-leaf stage, seedlings were thinned to one plant in each hole. disease and pest control were performed to maximize the canola seed yield and quality. urea was used as a nitrogen fertilizer and 150 kg n ha-1 of urea was applied into the soil before sowing. a second nitrogen application was performed as a top dressing with 75 kg n ha-1 nitrogen fertilizer (urea) at the end of january in each growth season. calcium superphosphate, potassium oxide, and borax were applied at a rate of 375, 120, and 15 kg ha-1, respectively, as a basal fertilizer. no irrigation was applied during two growing seasons. experimental design and sampling the experiment used a randomized complete block design with four canola cultivars as experimental treatments. three replications were conducted in the experiment. the plot was 20 m in length and 3.2 m wide. canola plants were planted in eight rows with a 0.2 m space between plants and 0.35 m space between rows. during harvesting, ten plants were randomly selected in each plot excluding boarder plants. the ten plants were dug with root and soil and rinsed by tapped water to carefully remove soil from the root. the whole plants were quickly transported to the lab and separated into main inflorescence, the first branch (from the top to the bottom regarding the sequential number of branch) until the tenth branch. the branches below the tenth were mixed. the main stem and root were separately reserved. each branch was further divided into branch stem, silique wall, and seed. the seeds were dried in the sunlight until the seed water content reached 8 g 100 g-1. silique wall, branch stem, main stem, and root were dried in an oven at 80 °c until the dry weight became constant. seed weight, oil content, carbohydrate profiling, and nitrogen (n) determination the one-thousand seed weight was recorded using a balance. seed oil content was determined by near-infrared (anataris ii, ftnir analyzer, thermo fisher scientific inc., madison, wi, usa). carbohydrates including total soluble sugar, sucrose, and starch content were determined. the dried sample was ground to a powder. briefly for carbohydrate profiling, about 50 mg of powder was boiled twice in a water bath using 10 ml of 800 ml l-1 ethanol for 30 min and then cooled to room temperature. the extract was centrifuged at 10000 × g for 10 min. one hundred milligrams of activated charcoal was added to the supernatant and kept at 80 °c in a water bath to remove the chlorophyll. the chlorophyll-free supernatant was then used for the determination of total soluble sugar and sucrose content according to hendrix (1993). the starch content in the pellets was digested with amyloglucosidase for 100 min at 55 °c and then determined according to hendrix (1993). n content was measured using the standard kjeldahl method. statistical analysis mean data of one thousand seed weight, seed oil content, total soluble sugar, sucrose, starch, and nitrogen content in branch stem, silique wall, and seed with different branches and genotypes were analyzed using the mixed procedure of sas (sas institute, 2004). branch position, genotype, and the interaction between branch position and genotype were considered as fixed effects. year, block, and all interactions among these effects were considered as random effects. when the main effect of branch position was significant, mean comparisons were further conducted using duncan’s method (α = 0.05). results seed weight in different branch and genotype seed weight was significantly affected by branch position, genotype, and year with a strong interaction between these factors (tab. 1). genotypic variation of seed weight revealed that zhongshuang 11 had heavier seed weight than other genotypes. on average, the seed weight in zhongshuang 11 was 7.9%. 23.4%, 20.9% in 2012 and 6.2%, 16.2%, 16.3% in 2013 higher than zheyou 50, zheyou 18, and zheshuang 8, respectively (fig. 1a and b). the upper position of branches had the greatest seed weight in all genotypes. seed weight of the main inflorescence and the first branch were on average 24.3% and 21.4% higher, respectively, than that of the tenth and below the tenth branches in zhongshuang 11 and zheyou 50 (fig. 1a and b). generally, variations of seed weight in branches located in the middle position of the stalk were less because of the close seed weight among these branches (fig. 1a and b). seed oil content in different branch and genotype seed oil content was also significantly influenced by genotype, tab. 1: analysis of variance (branch position, genotype, and year) of seed weight (sw), seed oil content (soc), silique total soluble sugar content (si-tssc), seed soluble sugar content (se-tssc), branch soluble sugar content (b-tssc), silique sucrose content (si-su), seed sucrose content (se-su), branch sucrose content (b-su), silique starch content (si-st), seed starch content (se-st), branch starch content (b-st), silique n content (si-n), seed n content (se-n), and branch n content (b-n) sw soc si-tssc se-tssc b-tssc si-su se-su b-su si-st se-st b-st si-n se-n b-n source p value <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 year (y) <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.032 <0.001 0.265 <0.001 0.906 0.214 bp*g <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 bp*y <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.034 0.214 g*y 0.018 <0.001 <0.001 <0.001 0.005 <0.001 <0.001 0.099 0.003 <0.001 0.003 <0.001 0.020 0.776 bp*g*y <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.002 <0.001 branch position (bp) genotype (g) effect of branch position on seed weight and oil content in canola 361 branch position, growth season, and their interactions among these factors (tab. 1). seed oil content of the four genotypes in 2012 was significantly lower than that in 2013 and the gap of the seed oil content between two years averagely ranged from 13.8 to 28.0 mg g-1 with different genotypes (fig. 2a and b). furthermore, seed oil content among the four genotypes differed significantly. zheyou 50 contained far more oils than other genotypes. on average, it was 4.2%, 9.6%, and 6.8% more oils than that in zhongshuang 11, zheshuang 8, and zheyou 18, respectively (fig. 2a and b). as for the branch position, seed oil content decreased significantly from top to bottom in all genotypes. in general, the gap of seed oil content between the main inflorescence and the lowest branch ranged from 24.4 (zheyou 50 in 2013) to 38.8 (zheshuang 8 in 2012) mg g-1 (fig. 2a and b). the difference in seed oil content was much smaller on the upper branches yet dropped dramatically below the fifth branch in the four genotypes (fig. 2a and b). total soluble sugar content (tssc) in seed, silique, and branch stem in different branch and genotype generally, tssc in each branch stem was below 2 mg g-1 in all genotypes. silique wall tssc was much higher as compared with that in branch stem and ranged from 1.5 to 5 mg g-1. the highest tssc was found in the seed which was greater than 14 mg g-1 (fig. 3a to f). in the branch stem, zheyou 18 had the highest tssc while zheshu ang 8 possessed the lowest content. the mean tssc of each branch in zheyou 18 was 43.75% and 43.94% higher than that in zheshu ang 8 in two years, respectively (fig. 3a and b). close tssc values were observed in zhongshuang 11 and zheyou 50 at each branch. regarding branch position, tssc at the higher stalk position was generally less than at the lower position. in the seed, zhongshuang 11 and zheyou 50 had similar tssc, which were on average 19.30 mg g-1 and 19.38 mg g-1, respectively (fig. 3 c and d). seed tssc in zheshuang 8 was significantly higher than that in zheyou 18 below the second branch. the mean seed tssc in the branches below the second branch in zheshuang 8 was 8.36% and 9.96% higher than that in zheyou 18 over the two growth seasons (fig. 3c and d). zhongshuang 11 had significantly lower tssc in the main inflorescence than other branches except the branch below the tenth. a stable tssc was observed from the first to the sixth and the first to the ninth branches in the two growth seasons, respectively, and then decreased rapidly in zhongshuang 11. seed tssc from the second branch onwards remained relatively stable in zheshuang 8. conversely, zheyou 18 showed decreasing seed tssc from the main inflorescence. however, the tssc from the sixth to the ninth branch showed very few variations in the 2012-2013 growth season in zheyou 18 (fig. 3c and d). like branch stem, zheyou 18 had the highest amount of tssc in the silique of each branch. zhongshuang 11 exhibited the lowest tssc above the sixth branch, however, it showed much higher tssc below the sixth branch as compared with zheshuang 8. zheyou 50 had significantly higher tssc in comparison with zheshuang 8. on average, silique tssc of each branch in zheyou 18 was 15.23%, 31.73%, and 35.09% higher than that in zheyou 50, zheshuang 8, and zhongshuang 11, respectively (fig. 4e and f). branches in the middle position presented higher tssc than other ones in general (fig. 3e and f). for example, silique tssc peaked at the fourth branch in zheyou 18 in both years while, zheyou 50 and zheshuang 8 obtained the maximum content moving to the second or lower branch. 2,5 3 3,5 4 4,5 5 5,5 0 1 2 3 4 5 6 7 8 9 10 >10 zhongshuang 11 zheshuang 8 zheyou 18 zheyou 50 3,5 4 4,5 5 5,5 6 6,5 0 1 2 3 4 5 6 7 8 9 10 >10 a: 2012-2013 b: 2013-2014 branch position s ee d w ei g h t (m g 1 0 0 0 -1 ) s ee d w ei g h t (m g 1 0 0 0 -1 ) fig. 1: seed weight in the main inflorescence and different branches from the top to the bottom of the main stem in the four cultivars, zheyou 50, zhongshuang 11, zheyou 18, and zheshuang 8 in (a) 2012-2013 and (b) 2013-2014 growth seasons. fig. 2: seed oil content in the main inflorescence and different branches from the top to the bottom of the main stem in the four cultivars, zheyou 50, zhongshuang 11, zheyou 18, and zheshuang 8 in (a) 2012-2013 and (b) 2013-2014 growth seasons. 420 430 440 450 460 470 480 490 500 510 0 1 2 3 4 5 6 7 8 9 10 >10 380 390 400 410 420 430 440 450 460 470 480 490 0 1 2 3 4 5 6 7 8 9 10 >10 zhongshuang 11 zheshuang 8 zheyou 18 zheyou 50 a:2012-2013 b: 2013-2014 branch position s ee d o il co n te n t (m g 1 0 0 0 -1 ) s ee d w ei g h t (m g 1 0 0 0 -1 ) 362 y. ren, j. zhu, y. wang, s. ma, g. ye, s. hua sucrose content in branch stem, seed, and silique wall in different branch and genotype in the branch stem, two genotypes, zheshuang 8 and zhongshu ang 11, showed higher sucrose content than zheyou 18 and zheyou 50 except at the tenth or below tenth branches in both growth seasons. average sucrose content of each branch in zhongshuang 11 and zheshuang 8 was 7.2 mg g-1 and 7.8 mg g-1, respectively, while zheyou 18 and zheyou 50 was 5.4 mg g-1 and 4.4 mg g-1 (fig. 4a and b). sucrose content peaked at the second to the fourth branch in zhongshuang 11 and zheshuang 8 in the two growth seasons, respectively (fig. 4a and b). for zheyou 18, branch sucrose content increased and peaked at the sixth and fourth branch in the two growth seasons, respectively (fig. 4a and b). for zheyou 50, suc rose content remained relatively stable from the main inflorescence to the eighth branch and then increased quickly in 2012-2013 while, few fluctuations were observed from the second to the branch below the tenth in 2013-2014 (fig. 4a and b). significant genotypic variation of sucrose content in the seed was observed (fig. 4c and d). zheyou 18 had the lowest seed sucrose content in each branch. zheyou 50 and zhongshuang 11 had the highest seed sucrose content. the mean seed sucrose content of each branch in zheyou 18 was 9.69%, 16.75%, and 17.78% lower than that in zheshuang 8, zheyou 50, and zhongshuang 11, respectively (fig. 4c and d). sucrose content was relatively stable above the ninth and eighth branch in the two growth seasons, respectively, and then decreased rapidly in zheyou 18. the sucrose content below the tenth branch was on average 12.70% less than that in the ninth or eighth branch in zheyou 18 (fig. 4c and d). a similar trend of sucrose content was found in zheshuang 8 as compared with zheyou 18 in 2012-2013 in each branch, however, the content was almost evenly distributed in the 2013-2014 growth season (fig. 4c and d). for zhongshuang 11, seed sucrose content slightly decreased from the main inflorescence to the ninth branch, and then decreased dramatically in the 2012-2013 growth season. sucrose content increased from the main inflorescence to the second branch until the ninth branch, then decreased rapidly again in the 2013-2014 growth season (fig. 4c and d). changes of seed sucrose content in zheyou 50 were similar to zhongshuang 11 (fig. 4c and d). silique sucrose content in zheshuang 8 was the highest while that in zheyou 50 was the lowest (fig. 4e and f). zheyou 18 had significantly higher silique sucrose content than that of zhongshuang 11 at most branches. on average, silique sucrose content at each branch of zheshuang 8 was 15.95%, 28.32%, and 26.59% higher than that in zheyou 18, zhongshuang 11, and zheyou 50, respectively (fig. 4e and f). silique sucrose content increased from the main inflorescence and peaked at the third branch and then decreased in zheshuang 8 in both growth seasons. however, the content increased again from the ninth branch in 2012-2013 yet, decreased drastically from the eighth branch in 2013-2014 in zheshuang 8 (fig. 4e and f). for zheyou 18, silique sucrose content showed few variations except at the first branch in 2012-2013 and the main inflorescence in 20132014. for zhongshuang 11 and zheyou 50, silique sucrose content 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 1,8 2,0 0 1 2 3 4 5 6 7 8 9 10 >10 zhongshuang 11 zheshuang 8 zheyou 18 zheyou 50 0,0 0,5 1,0 1,5 2,0 2,5 0 1 2 3 4 5 6 7 8 9 10 >10 15 16 17 18 19 20 21 22 0 1 2 3 4 5 6 7 8 9 10 >10 14 15 16 17 18 19 20 21 0 1 2 3 4 5 6 7 8 9 10 >10 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0 0 1 2 3 4 5 6 7 8 9 10 >10 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0 0 1 2 3 4 5 6 7 8 9 10 >10 a branch stem 2012-2013 b branch stem 2013-2014 c seed 2012-2013 d seed 2013-2014 e siliquewall 2012-2013 f siliquewall 2013-2014 branch position branch position t o ta l so lu b le su g ar co n te n t (m g g 1 ) fig. 3: total soluble sugar content in the main inflorescence and different branches from the top to the bottom of the main stem in the branch stem in (a) 2012-2013 and (b) 2013-2014, seed in (c) 2012-2013 and (d) 2013-2014, and silique wall in (e) 2012-2013 and (f) 2013-2014 growth seasons in the four cultivars, zheyou 50, zhongshuang 11, zheyou 18, and zheshuang 8. effect of branch position on seed weight and oil content in canola 363 was much more stable than other genotypes at each branch (fig. 4e and f). starch content in branch stem, seed, and silique wall in different branch and genotype starch content in different organs showed that seed starch content was significantly lower than branch stem (fig. 5a to f) and silique. for branch stem, zheyou 18 had the lowest starch content in each branch. zheyou 50 had the highest starch content at the main inflorescence and the first branch and then decreased quickly, which was significantly lower than zheshuang 8 and zhongshuang 11 (fig. 5 a and b). compared with zheshuang 8, zhongshuang 11 had higher starch content from the main inflorescence to the fourth branch while, the opposite trend was found at lower branch position (fig. 5a and b). zhongshuang 11 peaked at the second and fourth branch in the two growth seasons, respectively. for zheshuang 8, starch content increased from the main inflorescence to the seventh or eighth then declined sharply in the two growth seasons (fig. 5a and b). for zheyou 50, starch content decreased with decreasing branch height. unlike the other three genotypes, starch content in zheyou 18 was relatively stable at each branch position (fig. 5a and b). like branch stem starch content, seed starch content in zheyou 18 was the lowest (fig. 5c and d). for zhongshuang 11, seed starch content was significantly lower than zheshuang 8 and zheyou 50 above the sixth branch and then sharply increased below the sixth branch, which exceeded the two genotypes (fig. 5c and d). the maximum seed starch content was observed at the main inflorescence and the first branch in zheyou 50, which was higher than other genotypes. however, the seed sucrose content from the second to the sixth branch ranked the second, which was lower than zheshuang 8. the seed sucrose content below the sixth branch was ranked third in zheyou 50 (fig. 5c and d). seed starch was relatively stable in each branch in zheyou 18 in both growth seasons as compared with other genotypes (fig. 5c and d). for zhongshuang 11, branches at the middle positions, which was from the second to the sixth branch in 2012-2013 growth season and from fourth to the sixth branch position in 20132014 growth season, showed the lowest content compared with other branches (fig. 5c and d). the seed starch content increased from the sixth branch and peaked at the ninth and the seventh branch in the two growth seasons, respectively in zhongshuang 11 (fig. 5c and d). for zheshuang 8, the seed starch content increased from the main inflorescence to the second branch and then decreased yet, increased at the sixth and seventh branch in 2012-2013 growth season (fig. 5c and d). for zheyou 50, seed starch content continuously decreased from the main inflorescence (fig. 5c and d). silique starch content of zheyou 18 was the lowest while zhong shuang 11 was the highest in both growth seasons (fig. 5e and f). silique starch content in zheyou 50 ranked second, however, the content was similar to zhongshuang 11 (fig. 5e and f). silique starch content decreased from the main inflorescence to the second and the third branch and then remained relatively stable in zheyou 18 3 4 5 6 7 8 9 10 11 0 1 2 3 4 5 6 7 8 9 10 >10 6,0 6,5 7,0 7,5 8,0 8,5 9,0 9,5 0 1 2 3 4 5 6 7 8 9 10 >10 6,0 6,5 7,0 7,5 8,0 8,5 9,0 9,5 10,0 10,5 0 1 2 3 4 5 6 7 8 9 10 >10 0 2 4 6 8 10 12 0 1 2 3 4 5 6 7 8 9 10 >10 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10 >10 3 4 5 6 7 8 9 10 11 0 1 2 3 4 5 6 7 8 9 10 >10 zhongshuang 11 zheshuang 8 zheyou 18 zheyou 50 a branch stem 2012-2013 b branch stem 2013-2014 c seed 2012-2013 d seed 2013-2014 e siliquewall 2012-2013 f siliquewall 2013-2014 s u cr o se co n te n t (m g g 1 ) branch position branch position fig. 4: sucrose content in the main inflorescence and different branches from the top to the bottom of the main stem in the branch stem in (a) 2012-2013 and (b) 2013-2014, seed in (c) 2012-2013 and (d) 2013-2014, and silique wall in (e) 2012-2013 and (f) 2013-2014 growth seasons in the four cultivars, zheyou 50, zhongshuang 11, zheyou 18, and zheshuang 8. 364 y. ren, j. zhu, y. wang, s. ma, g. ye, s. hua in both growth seasons (fig. 5d and e). for zheshuang 8, silique starch content above the eighth or seventh showed small variations in two growth seasons, respectively. however, the content from the eighth branch decreased in 2012-2013 growth season while, the opposite trend was observed in 2013-2014 in zheshuang 8 (fig. 5d and e). for zheyou 50, the middle position branches had much higher silique starch content. for example, silique starch content was higher than other branches from the fifth to eighth branch in the 2012-2013 growth season and that from the sixth to the eighth branch in the 2013-2014 growth season (fig. 5d and e). for zhongshuang 11, large variation of silique starch content in different branches was found in 2012-2013 especially below the fourth branch. the starch content increased from the fourth branch and then decreased quickly in zhongshuang 11 (fig. 5d and e). total n content in branch stem, seed, and silique wall in different branch position with genotypes in general, n content in the branch stem was the lowest while that was the highest in the seed (fig. 6 a, b, c, d, e, and f). the result indicated that a majority of n in the stem and silique shifted to the seed for its development. significantly genotypic variation of n content was found in branch stems (fig. 6a and b). branch stem n content in zheyou 18 was significantly higher than other genotypes from the main inflorescence to the sixth branch. however, the content below the sixth branch in zheyou 18 was lower than that in zhongshuang 11 (fig. 6a and b). branch stem n content in zheshuang 8 and zheyou 50 showed far lower amount as a comparison with zhongshuang 11 and zheyou 18 (fig. 6a and b). the upper position branches in zheyou 50 had lower n content as compared with zheshuang 8. however, the reverse trend was found after the seventh branch (fig. 6a and b). branch stem n content in zheshuang 8 was very stable in each branch except the lowest branch in the 2013-2014 growth season (fig. 6a and b). for zheyou 50, higher n content was found as the branch position lowered (fig. 6a and b). branch stem n content in zhongshuang 11 increased from the main inflorescence to the ninth and sixth branch in the two growth seasons, respectively (fig. 6a and b). a similar trend in n content was observed in zheyou 18 (fig. 6a and b). however, the peak n value occurred earlier, at the fourth and the fifth branch in the two growth seasons, respectively (fig. 6a and b). as for seed n content, zhongshuang 11 exhibited the highest n amount in each branch position. zheyou 18 showed the lowest seed n content. furthermore, seed n content in the four genotypes showed relatively stable in each branch (fig. 6c and d). silique n content in zhongshuang 11 and zheshuang 8 was significantly higher than that in zheyou 50 and zheyou 18 (fig. 6e and f). small fluctuations of silique n content were observed in each branch in the four genotypes in general. however, a drastic increase of silique n content from the eighth branch in zheshuang 8 was found in the 2012-2013 growth season (fig. 6e and f). 20 25 30 35 40 45 50 55 60 0 1 2 3 4 5 6 7 8 9 10 >10 zhongshuang 11 zheshuang 8 zheyou 18 zheyou 50 20 25 30 35 40 45 50 55 0 1 2 3 4 5 6 7 8 9 10 >10 5 6 7 8 9 10 11 12 13 14 15 0 1 2 3 4 5 6 7 8 9 10 >10 6 7 8 9 10 11 12 13 14 15 16 0 1 2 3 4 5 6 7 8 9 10 >10 20 25 30 35 40 45 0 1 2 3 4 5 6 7 8 9 10 >10 20 25 30 35 40 45 0 1 2 3 4 5 6 7 8 9 10 >10 a branch stem 2012-2013 b branch stem 2013-2014 c seed 2012-2013 d seed 2013-2014 e siliquewall 2012-2013 f siliquewall 2013-2014 branch position branch position s ta rc h co n te n t (m g g 1 ) fig. 5: starch content in the main inflorescence and different branches from the top to the bottom of the main stem in the branch stem in (a) 2012-2013 and (b) 2013-2014, seed in (c) 2012-2013 and (d) 2013-2014, and silique wall in (e) 2012-2013 and (f) 2013-2014 growth seasons in the four cultivars, zheyou 50, zhongshuang 11, zheyou 18, and zheshuang 8. effect of branch position on seed weight and oil content in canola 365 discussion seed traits including weight and oil content are important for canola oil production. although there are numerous studies on seed weight and oil content of canola (fafaji, 2014; rad et al., 2014; li et al., 2015), few have investigated the effect of canola developmental issues on seed weight and oil content such as branch position. therefore, the objective of the current study was to understand the effect of branch position on seed weight and oil content. in this study, we found significant differences in canola seed weight and oil content with different branches. both the seed weight and oil content decreased from the top to the bottom branch suggesting a difference in the vertical allocation of carbohydrate and nitrogen for dry matter accumulation and lipid biosynthesis in different branches. in wheat, seeds on the head of the main tiller were heavier than any other position (nik et al., 2012). in rice, the phenomenon exists as well and leads to the appearance of superior and inferior grains with different positions in the same spikelet. consequently, the development of grains with different position results in a large difference of seed mass accumulation and quality formation between the two types of grain (tan et al., 2009; fu et al., 2011; dong et al., 2012; das et al., 2016). in canola, the sequence of branch establishment initiates from the main inflorescence and then from the top to bottom of the stalk. the flowering sequence initiates at the bottom of the main inflorescence and the proximal upper position branch as the middle and lower position branches develop. as a result, temporal and spatial differences in seed development follow. consequently, the hypotheses for the difference in seed weight and oil content are (1) the photosynthetic product and nitrogen in the elongating stem is preferentially supplied to the main inflorescence and then from the upper to the lower branches; and (2) photosynthetic products in the silique decrease from the main inflorescence and the upper to the lower branches. as flowering initiates, several important issues occur simultaneously including old leaf senescence, new leaf development, stem elongation, flowering, and silique development. each issue, except old leaf senescing, demands considerable carbohydrate and nitrogen supplies both for the organ volume expansion and elongation. during the initial flowering, the early opened flowers commence seed (embryo) development after fertilization and the young bud is still in the state of floral organ development. the process continues to the end of flowering and this is the first gap for seed development among different branch positions. after the end of flowering, the canola plant enters silique wall and seed development. normally, this stage ranges from 30 to 50 days (hua et al. 2014). there are two possible pathways leading to the difference in the seed weight and oil content. firstly, the photosynthetic ability of the silique wall in different branches since the canopy consists of siliques (gammelvind et al., 1996; king et al., 1997). secondly, the difference fig. 6: nitrogen content in the main inflorescence and different branches from the top to the bottom of the main stem in the branch stem in (a) 2012-2013 and (b) 2013-2014, seed in (c) 2012-2013 and (d) 2013-2014, and silique wall in (e) 2012-2013 and (f) 2013-2014 growth seasons in the four cultivars, zheyou 50, zhongshuang 11, zheyou 18, and zheshuang 8. 0,00 0,50 1,00 1,50 2,00 2,50 3,00 3,50 4,00 4,50 0 1 2 3 4 5 6 7 8 9 10 >10 0,00 0,50 1,00 1,50 2,00 2,50 3,00 3,50 4,00 4,50 0 1 2 3 4 5 6 7 8 9 10 >10 0,00 1,00 2,00 3,00 4,00 5,00 6,00 0 1 2 3 4 5 6 7 8 9 10 >10 0,00 1,00 2,00 3,00 4,00 5,00 6,00 0 1 2 3 4 5 6 7 8 9 10 >10 6,00 6,50 7,00 7,50 8,00 8,50 9,00 9,50 0 1 2 3 4 5 6 7 8 9 10 >10 6,00 6,50 7,00 7,50 8,00 8,50 0 1 2 3 4 5 6 7 8 9 10 >10 a branch stem 2012-2013 b branch stem 2013-2014 c seed 2012-2013 d seed 2013-2014 e siliquewall 2012-2013 f siliquewall 2013-2014 branch position branch position n it ro g en co n te n t (m g g 1 ) zheyou 18 zheyou 50 zhongshuang 11 zheshuang 8 366 y. ren, j. zhu, y. wang, s. ma, g. ye, s. hua in carbohydrate reallocation from the old leaves and stem. however, it is difficult to estimate precisely which pathway contributes more to seed mass accumulation and seed oil content presently. however, regardless of the pathways, both aim to maintain seed filling and volume expansion at seed developmental stage. in the present study, the soluble sugar content in each branch and silique wall were very low. there were two possible destinations for those sugars in branch and silique wall. first, the sugars were transformed into structural compounds such as lignin and cellulose in branch and silique wall (günl and pauly, 2011; anderson et al., 2015). second, sugars were transported to the seed since the seed is a strong sink during development (andrianasolo et al., 2017; asseng et al., 2017). furthermore, the considerable carbohydrate reserves in the seed suggest the large amount of carbohydrate was deprived from those two tissues. the genotypic variation of soluble sugar content revealed that the difference of carbohydrate utilization can lead to the different seed weight and oil content. for example, zheyou 18 had the highest branch stem and silique wall soluble sugar content while, the lowest content in the seed suggesting lower sugar transportation from these two tissues to the seed. although zheshuang 8 had similar seed weight to zheyou 18 and lower seed oil content, its seed soluble sugar content was much higher than zheshuang 8 and branch stem had the lowest soluble sugar content. the result implied that most of the soluble sugars in the branch stem and silique wall were transported into the seed in zheshuang 8. however, the sugar was possibly used for seed mass accumulation but not for seed oil biosynthesis in zheshuang 8. surprisingly, zhongshuang 11 had very low silique soluble sugar content possibly suggesting significant contribution of silique wall on seed soluble sugar content. seed sucrose and starch content were the lowest in zheyou 18 suggesting the lighter seed weight and lower seed oil content is due to the insufficient carbohydrate supply during seed mass accumulation and lipid biosynthesis. zheyou 50 ranked second in seed weight and first in seed oil content in each branch. a medium amount of total soluble sugar and starch content, low sucrose content in branch and silique, and higher sucrose content in seed revealing the intensive input of sugars to the seed should be a strong indicator for seed weight and oil content accumulation. however, the sugars may flow into the seed lipid biosynthetic pathway due to the higher seed oil content. unlike carbohydrate, seed nitrogen-derivatives occupy the cellular content following carbon-derivatives and come from silique wall and other tissues. zhongshuang 11 had higher seed nitrogen content which might be beneficial for seed weight while, zheyou 50 had relatively lower seed nitrogen content in comparison with zhongshuang 11, which is helpful for increasing seed oil content as there are negative correlations between seed oil and protein content (the main nitrogenderivative) (li et al., 2014c; zuo et al., 2016). the different responses of the carbohydrate type and organs to the branch position were found in present investigation. for total soluble sugar content, higher content in lower branches may suggest that soluble sugars in the lower position branch had lower transformation efficiency to secondary metabolites due to late development. alternatively, seeds on the upper branches develop earlier than that on the lower branches; therefore, branch stem soluble sugar content was almost fully transported into the seed leading to a lower soluble sugar content. unlike branch stem, soluble sugar content in the siliques was higher in the middle position. from canola plant morphology, siliques on the upper branches usually receive the highest irradiation due to space and earlier development. however, the middle and lower position branches rapidly develop due to higher temperatures after flowering (zhang et al., 2016); the soluble sugars may remain in the silique wall. furthermore, the additive effect of the transportation of soluble sugars in the branch stem to silique wall might simultaneously lead to the higher soluble sugar content since the silique wall is an intermediate tissue between branch stem and seed (bennett et al., 2011). in seed, total soluble sugar in the upper and middle positions were similar but decreased in the lower position. seed development demands large amounts of carbohydrates, however, lower soluble sugar content in the lower branch position may not meet the requirements of development. for sucrose and starch content, both are cleaved into smaller molecules such as fructose and glucose (weber and roitsch, 2000; fallahi et al., 2008). two genotypes, zheshuang 8 and zhongshuang 11, had higher sucrose content in the upper branches. however, in the seed and silique wall, fewer fluctuations and lower sucrose content were observed in the lower branches. this is in accordance with total soluble sugar content. a similar trend was observed in nitrogen content. therefore, sugar content including total soluble sugar, sucrose and starch content, and nitrogen content in the different branch positions does not precisely reflect the trend of seed weight and oil content. conclusions in conclusion, both seed weight and oil content decreased in the four canola genotypes from the main inflorescence to the lower branches in the main stem. the results suggest that the seed developmental schedule in the different branches affected seed dry matter accumulation and lipid biosynthesis. the mechanism of lower seed weight and oil content in zheyou 18 and zheshuang 8 was different. for zheyou 18, low output efficiency of sugars from branch and silique wall to the seed was the main reason for dry matter and oil accumulation. however, for zheshuang 8, lower sugar content in the branch and photosynthetic efficiency in the silique wall resulted in the lower seed weight and oil content. although zhongshuang 11 and zheyou 50 had moderate sugar content in different organs, the destinations of the sugars were different. for zhongshuang 11, the sugars mainly flowed into the dry matter pathway yet, into the lipid biosynthesis in zheyou 50. although seed weight and oil content decreased with decreasing branch height, there was no obvious correlation between seed weight (or oil content) 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nitrogen rates and genotype on seed protein and amino acid content in canola. j. agric. sci. 154, 438-455. doi: 10.1017/s0021859615000210 address of the corresponding author: e-mail: sjhua1@163.com © the author(s) 2017. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 91, 103 108 (2018), doi:10.5073/jabfq.2018.091.014 department of food technology, warsaw university of life sciences – sggw (wuls-sggw), warsaw, poland the study of palm and rapeseed oil stability during frying magdalena maszewska, anna florowska*, katarzyna matysiak, katarzyna marciniak-łukasiak, elżbieta dłużewska (submitted: december 12, 2017 accepted: march 20, 2018) * corresponding author summary palm oil is characterized by high oxidation stability, high smoke point, low foam making properties, limited penetration into the product, what makes it ideal for processes requiring thermal treatment such as frying. the aim of the study was to investigate the chemical composition and thermooxidative stability of red palm olein, rapeseed oil and their mixtures during deep-frying of french fries. analysis of fatty acids composition and basic parameters of fresh oils (acid number, peroxide value, polar compounds content, induction time) were performed. during frying, changes in acid number, polar compounds in oils as well as consumers’ acceptance of the fries fried in these oils were investigated. during the 32-hour of frying, the lowest chemical changes occurred in palm olein, what was confirmed by low acid values (0.99 mg koh/g) and low polar content (14.4%). at the end of the experiment, the oil mixture had the highest polar fraction value of 25.0%. in the opinion of consumers, fries fried in rapeseed oil were “the best”, while french fries fried on palm oil were considered “artificial”, “chemical” and “disgusting”. the reason for this opinion was the addition of β-carotene to this oil. on the other hand β-carotene from palm olein had a great positive effect on the colour of the fries, but at the same time had a negative effect on the taste. introduction the main components of edible oil are triacylglycerols (tag) which are about 94-99%. they determine the physical properties of oil. in composition of palm oil there is almost equal the quantity of saturated (49.9%) and unsaturated (49.7%) fatty acids. most of unsaturated fatty acids occur in 2nd position in a molecule of triacylglycerol (nagendran et al., 2000; sambanthamurthi et al., 2000). raw palm oil is a rich source of carotenoids which impart a deep orangered colour (nagendran et al., 2000; ong and goh, 2002; mba et al., 2017). their contents vary between 700-800 mg/kg (of which 37% is alfaand 50% is beta-carotene). this oil is also a rich source of tocopherols and tocotrienols (vitamin e). its contents vary between 600-1000 mg/kg (nagendran et al., 2000; sambanthamurthi et al., 2000; ong and goh, 2002; mba et al., 2017). carotenoids and vitamin e are natural antioxidants, however, because of the processing they are partly lost. they modify highly reactive radicals, mainly hydroxyl radical and peroxy radical, into less active forms. thus they protect the oil from oxidation (nagendran et al., 2000; sambanthamurthi et al., 2000; ong and goh, 2002; edem, 2002; berger, 2005). palm oil is recommended for frying because of its high stability due to relatively high contents of oleic acid, high contents of saturated acids and large amounts of natural antioxidants. it slightly penetrates the fried product and it can be used repea tedly (ong and goh, 2002). it gives the products the colour of gold. besides, there is no loss of nutritional value during frying in this fat (berger, 2005). however, according to norizzah et al. (2004) the use of olein is limited, because of the excessive foaming during heating caused by the presence of short chain fatty acids. in the rapeseed oil 25% of the tag creates triolejan. the saturated acids are mainly present in the molecule of tag in 1st or 3rd positions, while unsaturated acids are present in 2nd position (przybylski, 1999; richard and o’brien, 2004). the rapeseed oil as opposed to palm oil contains small quantities of saturated acids (10 times). the dominant fatty acid is mono-unsaturated oleic acid (> 60%), and contains linoleic to α-linolenic essential fatty acids ratio of 2:1, making it nutritious. moreover, rapeseed oil is rich source of natural antioxidants, including tocopherols, polyphenols and phytosterols (scarth and mcvetty, 1999). traditional rapeseed oil used for frying is characterized by the following properties: it does not transfer taste, it has a high smoke point of 204-230 °c, it almost completely filters, if it is not well hot it quickly absorbs into the product (przybylski, 1999; matthaüs, 2006). the smoke point for palm olein is 195 °c (matthaüs, 2006). the aim of the study was to investigate the chemical and thermooxidative stability of palm oil, rapeseed oil and their mixtures during frying. materials and methods materials the research material were: fractionated palm olein (poland) (betacarotene – 375 mg/kg of oil), refining rapeseed oil (poland), palm olein and rapeseed oil mixture composed in the ratio of 1 to 1, prefried and frozen french fries (poland). fresh oils, oils during frying and oils after frying were tested. frying of potato fries the oil was heated to a temperature of 175 °c. french fries were fried in 1 liter oil everyone hour for about 6.5 min – starting from 100 g portions in fryer tefal type fa 700 te. in orderto maintain the same share of the product in the ratio to oil, that is in the ratio of 1 to 10, the portions of fries were reduced every 2 h by about 5 g (in proportion to the loss of oil during frying). the study lasted 32 h, 8 h per day for 4 days. the samples for oil analysis were collected after 3, 12, 24 and 32 h of frying. in the samples taken, acid value and the contents of polar fraction were measured. organoleptic characteristics of fries fried in fresh oils were also rated. determination of fatty acid composition fatty acid methyl esters (1 μl), prepared by iso 5509:2000 standard method, were separated on a gc-fid system (trace™ 1300, thermo scientific, waltham, ma, usa) equipped with a bpx 70 capillary column (60 m length, 0.22 mm i.d., 0.25 mm film thickness). helium was used as a carrier gas at a flow rate of 1.5 ml/min. a split/splitless injector was operated at a temperature of 230 °c with a split rate set to 100:1, and the detector was the gc-fid interface temperature was 220 °c, the ion source temperature was 200 °c. the gc’s oven temperature was programmed as follows: 80 °c hold for 2 min, ramped to 230 °c at a rate of 2.5 °c/min, hold for 5 min. fatty 104 m. maszewska, a. florowska, k. matysiak, k. marciniak-łukasiak, e. dłużewska acids were identified by comparing their retention times with authentic standards, and the results were reported as weight percentages. oil quality analysis acid value was determined according to iso 660:2005. the results were shown in mg koh per gram (mg koh/g). peroxide value was determined according to iso 3960:1996. the results were shown in miliequivalent o2 per kg (m eq o2/kg). oxidative stability determined via accelerated stability test (rancimat) the rancimat test − oxidative stability of oils was determined according to iso 6886:2006. the research was carried out by using rancimat type 679 (a temperature of 120 °c, air circulation 20 l/h) produced by “metrohm” company (switzerland). the contents of polar compounds polar compound was determined according to iso 8420:2002. the contents of polar compounds (w) were calculated from the difference between the mass of oil sample and the mass of eluted non-polar compounds, according to the formula (eq. 1): ω = 100 − m1 − m2 × 100 m (eq. 1) where: m1: the mass of the flask and the mass of the non-polar compounds (g), m2: the mass of empty flask (g); m: the mass of oil sample (g). sensory analysis organoleptic assessment of french fries was determined according to iso 5492:2008 and baryłko-pikielna and matuszewska (2009) using a 0-10-point scale, where “0” implies the absence of a feature and “10” implies the greatest severity of characteristics. sensory evaluation was performed in triplicate with a selected and trained panel consisting of 10 persons. flavour attributes – colour, oil odour, off-odour, fatness, gumminess, tenderness, oil taste, potato taste, off-flavour, a general quality – were investigated. a quantitative sensory description was conducted using a graded 10-point scale to measure the intensity of attributes, leading from zero (“not detectable”) to ten (“intense”). statistical analysis all experiments were carried out in triplicate. statistical analysis was performed using statistica 10 software. data were expressed as mean ± sd or as percentage. variables were compared by t test, oneway anova; post hoc tukey test and the significance of differences among means were determined at p<0.05. results and discussion the characteristics of the material the oils used in the research have been chemically analysed. the acid value, peroxide value, the contents of polar fraction and induction time in the rancimat test were determined (tab. 1). refined oils, according to codex stan 210-1999 should not have an acid value more than 0.6 mg koh/g and peroxide value more than 10 m eq o2/ kg. all the oils and the mixtures prepared of them and used for the test fulfill the conditions of codex stan 210-1999. fatty acids composition (tab. 1) in palm olein was as follows: palmitic acid – 43.3%, oleic acid – 38.9%, linoleic acid – 10.2%, and it followed observations of other authors [sambanthamurthi et al., 2000; mba et al., 2017; aniołowska and kita, 2016; li et al., 2017]. rapeseed oil contained (tab. 1): palmitic acid – 4.9%, oleic acid – 51.1%, linoleic acid – 24.0%, linolenoic – 12.7% and it followed observations of codex stan 210-1999, scarth and mcvetty, 1999 and mba et al., 2017. determination of oxidative stability of oils – the rancimat test the rancimat test is one of the dynamic, fast methods used to determine oxidative stability of fats. changes taking place in fats which are the cause of the formation of secondary products of its decomposition are the basis for the designation of kinetic curves based on which the induction time is determined. the induction time is the conventional measure of oxidative stability of fats, which is a measure of the rate of oxidation (litwinienko, 2001). the shorter the tab. 1: characteristics of used material palm olein rapeseed oil mixed oil x ± sd x ± sd x ± sd acid value [mg koh/g] 0.22 ± 0.000 0.17 ± 0.064 0.30 ± 0.000 peroxide value [m eq/kg] 0.76 ± 0.005 2.01 ± 0.047 3.33 ± 0.000 polar compounds [%] 6.200 ± 0.378 3.024 ± 0.382 5.610 ± 1.338 induction time [h] 5.17a ± 0.154 4.32b ± 0.066 4.45b ± 0.280 fatty acid composition: c16:0 43.3 4.9 24.3 c16:1 0.3 0.05 0.16 c18:0 4.6 2.45 3.55 c18:1 38.9 51.1 49.7 c18:2 10.2 24.0 17.0 c18:3 0.3 12.7 6.4 c20:0 0.1 0.6 0.31 c20:1 nd 2.8 1.4 values x represent mean ± standard deviation sd (n = 3). different small letters in superscript in a row indicate statistically significant differences at the level p < 0.05. stability of palm and rapeseed oil during frying 105 induction times the more unstable oil. the induction time (in hours) of fats used in the study is shown in the table (tab. 1). the shortest induction time was for liquid rapeseed oil (4.32 h). therefore this oil is the most unstable. this is due to its fatty acids composition, that is the presence of large quantities (10 times more in comparison to palm olein) unsaturated fatty acids which are easily oxidized due to the double chemical bonds. the longest induction time was for palm olein 5.17 h. acid value acid value which is a measure of free fatty acids which arise because of hydrolytic degradation of fat during frying. according to eu le gislation the acid value must not exceed 2.5 or 5 mg koh/g and it depends on a country. in poland it must not exceed 2.5 mg koh/g. changes in acid value and the time of frying for each test of fresh oil, and after 3, 12, 24, 32 h of frying were shown in the fig. 1. after statistical analysis on the significance level equal to 0.05 (p=0.05) it was found that frying time has statistically significant impact on the average value of acid value. while analyzing the changes in acid value during frying its growth in the case of any oil used for frying was observed (fig. 1). however, acid value in any of the test fats did not exceed 2.5 mg koh/g after the last hour, that is after 32nd h of frying, which otherwise would mean the withdrawal of oil from further use. therefore, these oils can be used for frying for longer than 32 h. this is also confirmed by other authors (aniołowska and kita, 2016). pufa contents in refined palm olein after about 30 h of frying in 180 °c were labeled at the level of around 0.52% by jaswir et al. (2000), in rapeseed oil after 7 days of frying in temperature of 160 °c were labeled at the level of around 5.2% by paul and mittal (1996), while acid value in rapeseed oil after 24 h of frying in temperature of 180 °c was labeled at the level of around 0.33 mg koh/g by danowska-oziewicz and karpińska-tymoszczuk (2005). basing on excel’s reglinx function [excel 2007], which predicts future value based on available values, the time after which the examined oils will be discarded when they reach av = 2.5 mg koh/g) were calculated. this was as follow: • palm olein – after 94 h • rapeseed oil – 47.4 h • mixed oil – 95.5 h graphical presentation of the increase in acid number with frying time up to av = 2.5 is shown in the graph below (fig. 2). one-way anova was perfomed in order to verify whether the type of oil affects the changes of acid value during frying. based on that a statistically significant effect of the type of oil on acid value was found. on the basis of this analysis it is concluded that between palm olein and mixture there are no statistically significant differences which means that these two oils behave similarly during frying. a separate homogenous group constitutes rapeseed oil which is characterized by the lowest resistance to hydrolytic changes which meansthat the most free fatty acids arise from it during the frying. it may be associated with a more unsaturated fatty acid composition in comparison to palm olein. the contents of polar compounds the contents of polar compounds is currently considered to be the best indicator of the quality of frying fat (berger, 2005; anio łowska and kita, 2016; hein et al., 1998; gertz, 2000), because it determines the degree of degradation of fat induced by thermooxidative changes. with the contents of polar fraction equal to 24% its further use is considered and limited and at 27% it is prohibited (berger, 2005; hein et al., 1998; gertz, 2000; gill et al., 2004). however, in majority of countries the contents of polar compounds of 25% mean that the frying fats are not allowed be used (berger, 2005; li et al., 2017; hein et al., 1998; gertz, 2000). the changes in the contents of polar fraction and time of frying for different oils are presented in the fig. 3. after statistical analysis on significance level equal to p = 0.05 it was concluded that frying time has statistically significant influence on the content of polar compounds. for each oil, an increase of contents of polar compounds with frying time was observed. this is also confirmed by other authors (matthaüs, 2006; li et al., 2017; paul and mittal, 1996; gill et al., 2004; ledóchowska and hazuka, 2006). after the final determination, that is after 32 h, the highest contents of this fraction was reached by mixed oil at 25.6% which means the withdrawal of the oil from further use. the lowest content of polar compounds was determined in rapeseed oil (11%). each oil had its own characteristic initial contents of polar compounds. the largest quantity of fig. 2: the increase in acid number with frying time up to av = 2.5 fig. 1: av changes in oil during frying. fig. 3: polar compounds changes in oil during frying. 106 m. maszewska, a. florowska, k. matysiak, k. marciniak-łukasiak, e. dłużewska this fraction containedpalm olein, which is related to its chemical composition as it naturally contains 6-10% diacyloglycerol, which in the chemical determination are the part of polar fraction. referring the results of the determinations to the initial contents of polar compounds and comparing them to the final results it was noticed that after 32 h of frying its contents increase 2.3 times in palm olein, 3.6 times in the rapeseed oil, and 4.6 times in mixed oil. similar dependancies between palm and rapeseed oil was observed by matthaüs (2006) after 72 h of frying. as a result, it can be stated that the contents of polar fraction increased in the slightest degree in palm olein, which is tantamount to the fact that thermooxidative changes in it occur the slowest. paul and mittal (1996) marked the contents of polar compounds in rapeseed oil at 15% after 7 days of frying at 160 °c, while gill et al. (2004) labeled in a double frac tionated palm oil at 19% after frying about 32 h at 145 °c. using excel›s reglinx function a frying time after which palm olein and rapeseed oil exceed the illegal content of the polar fraction (polar fraction more than 25%) were provided, (fig. 4). this is as follows: • palm olein – after 70 h • rapeseed oil – after 87 h the mixed oils were not considering because they reach 25% of polar fraction at 31 h. analyzing the results of one-way anova for frying time of palm olein, no statistically significant differences were found in the contents of polar compounds between the fresh oil and after 3 h of use, between 3 and 12 h, and 24 and 32 h. in the case of rapeseed oil and mixed one, no statistically significant differences were found only between the contents of polar compounds in output oil and after 3 h of frying. one-way anova was perfomed to check whether the type of oil is changing the contents of polar compounds with the time of frying. it has been found that the type of oil affects statistically significant changes in the contents of polar compounds during frying. it was also noticed that there were important differences between tested oils and, therefore, in each of these oils thermooxidative changes occur in varying degrees. to examine whether there is a correlation between the value of acid value and the contents of polar compounds, simple correlation analysis was performed at the significance level p=0.05. it was found that for all tested oils, there is a statistically significant correlation between the contents of polar compounds and the acid value. interdependence is very high for each of the oils (oil mixed r=95.1%, palm olein r=98.2%, rapeseed oil r=99.6%) and directly proportional, which means that the increase of acid value increases the contents of polar fraction and vice versa. and thus, with the increase in free fatty acids contents, the contents of polar compounds also increase. this is since free fatty acids belong to the polar fractions. organoleptic characteristic of french fries − profile method in the organoleptic evaluation in consumers opinion the best fries were fried in rapeseed oil (9.4), similar results were obtained by rutkowska and jaworska (2006) but other results have been obtained by matthaüs (2006). the worst fries were fried in palm olein (4.6). consumers issued an opinion that french fries in palm olein looked artificial and had off-flavour. the reason for that consumer sensation was probably the content of carotenoids, which gave an unpleasant aftertaste and a yellow colour of french fries. based on these opinions we should not fry on the palm olein with of beta-carotene, because it might gives, after termal treatment, negative sensory characteristics of french fries (fratianni et al., 2010; fratianni et al., 2017; xiao et al., 2018). it can be assumed that it would also have similar influence on other products. graphic presentation of the organoleptic evaluation is illustrated in fig. 5. fig. 4: provides a frying time after which oils exceed the illegal content of the polar fraction 25%. fig. 5: organoleptic characteristics of french fries fried in palm oil, rapeseed oil and mixed oil. stability of palm and rapeseed oil during frying 107 generally, fries rating were determined by the type of oil, which was also proved by matthaüs (2006) and rutkowska and jaworska (2006). fries fried in mixed oil and rapeseed oil did not show significant differences for such parameters as the smell of oil, strange smell, strange taste and general quality. between the french fries in palm olein and mixed no significant differences were shown for the assessment of tenderness, taste of potato or oil. among all the oils used, significant differences appeared in colour of the french fries, which means that each oil gave a characteristic colour of the fries depending on the content of carotenoids. correlation analysis was performed at the significance level p=0.05 to check whether there is a correlation between the assessed attributes and general rating. on that basis, it was found that there is a very strong and directly proportional correlation between colour and general assessment of french fries in palm olein (r=91.9%) and mixed oil (r=91.3%). this is due to the presence of carotenoids, which gives intense yellow colour of the fries, a bit softer in fries fried in mixed oil. also, the existence of a very strong, directly proportional correlation was proved for the fragility – overall assessment (r=91%) and taste of the potato – overall assessment (r=91.3%) for the french fries in rapeseed oil. very strong, but inversely proportional correlation emerged between the sensation of rubbery and the overall assessment (r=-91.3%) for the french fries in mixed oil. this means that the more chips will be rubbery, the less desirable by consumers. in fries fried in palm olein the existence of very strong, direct proportional correlation was found between the strange taste and general assessment (r=91.7%). conclusion the tested oils and mixtures made of them in a 1:1 ratio were of good quality which was confirmed by chemical determinations. because of a 32-hour frying the smallest thermooxidative changes occurred in palm olein, as evidenced by the value of acid value and the contents of polar compounds. at the end of the experiment mixed oil showed the highest contents of polar fraction more than 25.0%, which means the prohibition of further use of this oil. in the consumer rating french fries fried in rapeseed oil were the best assessed. french fries in palm olein have been recognized by consumers as “artificial”, “chemical”, and “distasteful”. the reason for such opinions was the content of beta-carotene to this oil. beta-carotene had a significant influence on the colour of french fries. however, due to the rapid disintegration of beta-carotene such oil would not find application in industrial frying. palm olein compared to rapeseed 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jaworska, d., 2006: stabilność przeciwutleniająca i jakość sensoryczna jako kryteria przydatności olejów rafinowanych do smażenia. żywność. nauka. technologia. jakość. 46, 136-142. in polish. sambanthamurthi, r., sundram, k., yew-ai, t., 2000: chemistry and biochemistry of palm oil. prog. lipid res. 39, 507-558. doi: 10.1016/s0163-7827(00)00015-1 scarth, a., mcvetty, p.b.e., 1999: designer oil canola – a review of new food-grade brassica oils with focus on high oleic, low linolenic types. new horizons for an old crop. proceedings 10th intl. rapeseed congress. canberra. australia. 57. http://www.regional.org.au/au/gcirc/4/57.htm#topofpage xiao, y.-d., huang, w.-y., li, d.-j., song, j.-f., liu c.-q., wei, q.-y., zhang, m., yang, q.-m., 2018: thermal degradation kinetics of alltrans and cis-carotenoids in a light-induced model system. food chem. 239, 360-368. doi: 10.1016/j.foodchem.2017.06.107 address of the authors: department of food technology, warsaw university of life sciences – sggw (wuls-sggw), ul.nowoursynowska 159c, 02-787 warsaw, poland © the author(s) 2018. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 89, 89 97 (2016), doi:10.5073/jabfq.2016.089.011 1department of horticulture, faculty of agriculture and natural sciences, bozok university, yozgat, turkey 2department of horticulture, faculty of agriculture and natural sciences, recep tayyip erdogan university, rize, turkey 3department of horticulture, faculty of agriculture, ataturk university, erzurum, turkey influence of arbuscular mycorrhizae and plant growth promoting rhizobacteria on proline content, membrane permeability and growth of strawberry (fragaria × ananassa duch.) under salt stress aysen koc1, gulden balci1, yasar erturk1, hakan keles1, nalan bakoglu2, sezai ercisli3* (received november 30, 2015) * corresponding author summary salinity is one of the most important factors negatively effecting the yield in crop species. in this study the effect of plant growth promoting rhizobacteria (pgpr) and arbuscular mycorrhizae on proline content, membrane permeability and growth of strawberry cv. ‘san andreas’ were studied under different salt treatments (0, 30 and 60 mm/l nacl). the leaf area was measured 0, 15, 30, 45 and 60 days after saline solution applications on the plants. the results showed that increasing concentrations of nacl decreased all growth parameters. increased salt concentration led to increased proline level compared to the control. bacterial application at 60 mm/l nacl concentration provided the highest ameliorative effect and therefore determined the most effective protection of the plant against salt stress. it was observed that the anthocyanin content increased in line with the increasing salt concentration. in general, the salt applied on the plants causes an increase in membrane permeability and thus disrupts membrane stability and becomes a significant factor damaging the plant. membrane permeability increased at applications with 30 mm/l and 60 mm/l nacl. our results revealed that bacteria application can have an ameliorative effect that helps the plant to tolerate the negative effects of salt stress by increasing proline and anthocyanin levels. introduction strawberries (fragaria × ananassa duch.) dominate the world berry production and are cultivated in europe, asia, north and south america with a big commercial importance (alkan torun et al., 2014). the total strawberry production of the world is more than 4.516.000 tons with usa taking the first places as highest strawberry producing country (1.367.000 tons), followed by mexico (360.426 tons), and turkey (353.173 tons), respectively) (fao, 2012). strawberry cultivation is suitable for both greenhouse and open field condition and it has high capability to adapt to diverse ecologic conditions. strawberries are relatively low salt tolerant plants and one of the main problem for their cultivation is salinity that restrict plant growth (keutgen and pawelzik, 2009). in particular in greenhouse conditions, salinization is serious problem due to the fact that a certain area of space is used continuously and intensively with intense use of salt included fertilizers. it is reported that soil and water salinity decrease the usefulness of ground water and thus affect plant growth negatively (tanji, 1990). one of the major effects of salinity on plants is the ethylene accumulation in their roots, which decrease root growth and finally reduce the yield of crops. pgprs are able to produce acc-deaminase in plants rhizosphere and they can consume pre-produced ethy lene (acc) and convert it to α-ketobutyrate and ammonium, so they are able to reduce ethylene level in plants and hence, increase their growth (penrose and glick, 2003). arbuscular mycorrhizal (am) fungi were reported by several researchers as enhancer of root systems and they are known to support stronger, healthier, higheryielding plants through increased nutrient acquisition (miller et al., 2010), reduce levels of water stress (auge, 2001), and increase phytohormone production (shaul-keinan et al., 2002). hence, mycorrhizal plants are able to increase their tolerance to salinity stress due to their high soil exploration conferred by their hypha structure and growth (miransari et al., 2008). anthocyanins are a large class of water soluble pigments in the flavonoid group found in all plant tissues, are largely responsible for coloration in higher plants (iwashina, 2000). these substances accumulate in different plant tissues under the influence of various environmental stimuli (gould et al., 2000). previously, it is reported that anthocyanins in plant tissue is increased with elevated salt stress (chalker-scott, 1999). it is known that under salt stress conditions, plants increase cellular osmotic pressure by producing secondary metabolites, various chemicals and particularly stress proteins (such as proline, etc.), and thus they sustain their existence by balancing the high osmotic pressure that emerges in the nutrient medium. physiological reactions of plant species cultivated in media with increasing salinity vary and to avoid negative effects of the osmotic pressure due to increasing salinity, plants increase their proline content. plants and species capable of generating more proline are more likely to resist the stress and grow healthily (edreva, 1998). in this study, the ameliorative effects of some rhizobacteria and mychorriza applications on growth and the biochemical changes of strawberry plants under salt stress were investigated. material and methods plant material and site description the day-neutral strawberry cultivar ‘san andreas’ was used in this study. first class health cold-stored frigo plants (seedlings) were planted in pots (25 × 18 × 20 cm) filled with perlite: turf media (1:1) (sahin et al., 2002) at may 6th 2013. the experiment was established with 1 cultivar × 3 salt concentrations (0, 30, 60 mm/l nacl) × 4 treatments (control, mychorriza, bacteria and mychorizza + bacteria) × 3 repetitions × 20 seedlings in each repetition = 720 seedlings in factorial randomized block design. the experiment was conducted at the bozok university gedikhasan experimental farm. bacterial application the bacteria used in the study were isolated from rhizosphere soils of a total 56 tea growing orchards located different agroclimatic regions in rize and trabzon provinces in black sea region in turkey and were identificated according to their fatty acid methyl esters (fames) analysis conducted in sherlock microbial identification system and confirmed with biolog system. among bacteria 90 a. koc, g. balci, y. erturk, h. keles, n. bakoglu, s. ercisli isolated two bacteria of them (bacillus cereus rcp 3/1 + rhizobium radiobacter rcr 11/2) were selected for their ability to grow in a saline culture medium (10 % sodium chloride (nacl). the phosphate dissolving and nitrogen fixing capacities of bacteria were determined previously (tab. 1) (cakmakci et al., 2010). for this experiment, the bacterial strains were grown on nutrient agar. a single colony was transferred to 250-ml flasks containing nutrient broth and grown aerobically in flasks on a rotating shaker (95 rpm) for 24 h at 27 °c. inoculation of bacterial treatments was performed using a dipping method in which plant roots in celled trays were inoculated with the bacterial suspensions of the concentration of 108 colony-forming units/ml in sterile water for 30 min before planting. control plants were dipped into sterile water. mychorriza application in mychorriza application, preparates in commercial powder form containing 9 different glomus fungi were used. in the preparation fungi types and ratios were; glomus intraradices (21 %), glomus aggregatum (20 %), glomus mosseage (20 %), glomus clarum (1 %), glomus monosporus (1 %), glomus deserticola (1 %), glomus brasilianum (1 %), glomus etunicatum (1 %), and gigaspora margarita (1 %). mychorrizal applications were performed by inoculating the plant roots by fungi in the solution prepared by mixing 10-liter water and 250 gr powder in packages before planting. salt application 40 days after planting (when 3-4 leaves were formed, 17.06.2013) salt solution including 0, 30 and 60 mm/l nacl were applied with an amount of 100 ml, 2 times a week. also, in order to ensure plants are nourished, 100 gram of 12-2-14 fertilizer solution (11.7 % nitrate, 0.3 % ammonium, 2 % phosphoric acid, 14 % potassium, 6 % calcium, 3 % magnesium and trace elements) per pot was applied three times a week. growth parameters growth parameters were measured after plants rooted out on 0, 15th, 30th, 45th and 60th days after salt application on the plants. leaves surface area (lsa) were measured in cm2 by adc bioscientific area meter am300. the roots and shoot dry weight (rdw and sdw) were determined in a drying cabin immediately after their fresh weights were determined. they were kept in drying cabin until their weights did not change at 65 °c and then, their weights were measured. weighing operations were performed by balances sensitive to 0.001 g. proline analysis proline content was determined according to bates et al. (1973). leaves were ground and homogenized in sulfosalicylic acid. the homogenate was filtered through filter paper. after the addition of ninhydrin reagent, the mixture was heated to 100 °c for one hour. the reaction was then stopped in ice. the mixture was extracted with 4 ml toluene, and the sample was vigorously shaken for 15 s. sample absorbance of the toluene layer was read at 520 nm. proline concentration was determined by using a calibration curve and expressed as μm per 100 mg fresh weight (fw). anthocyanin content the acm-200 plus anthocyanin content meter provides a fast estimate of anthocyanin content on the intact leaves of plants. the nondestructive technique allows researchers to monitor anthocyanin without the costly and time consuming extraction. for each plant, measurements were taken at four locations on each leaf; two on each side of the mid rib on all fully expanded leaves and then averaged (khan et al., 2003). electrolyte leakage (membrane permeability) for measurement of electrolyte leakage, 10 leaf discs (10 mm in diameter) from the young fully expanded leaves from two plants per replicate were placed in 50 ml glass vials, rinsed with distilled water to remove electrolytes released during leaf disc excision. vials were then filled with 30 ml of distilled water and allowed to stand in the dark for 24 h at room temperature. electrical conductivity (ec1) of the bathing solution was determined at the end of the incubation period. vials were heated in a temperature-controlled water bath at 95 ºc for 20 min and then cooled to room temperature and the electrical conductivity (ec2) was again measured. electrolyte leakage (membrane permeability – mp) was calculated as a percentage of ec1/ec2 (shi et al., 2006). data analysis the experiment was arranged in a randomized blocks in factorial design with three replications. data were tested by spss 20.0 for windows program. the differences between the means were compared using the duncan test (p < 5 %). results and discussion salt stress is complex and has toxic effects on plants and lead to metabolic changes (latrach et al., 2014). strawberry is considered as a nacl salinity sensitive species and it has been shown to reduce number of runners, runner length, leaf number, fresh and dry root weight (saied et al., 2005). within the scope of our study, it was determined that effects of salt concentrations, applications, and uprooting days on leaf area are statistically significant (p < 0.05, fig. 1). leaf areas were observed to decrease with the increasing concentrations of salt. while the leaf area with 60 mm/l nacl concentration was determined to be 33.37 cm2, with 0 mm/l nacl concentration leaf area was measured as 38.43 cm2. in terms of applications, it was observed that mycorrhiza and bacteria applications have positive impact on leaf area. tab. 1: laboratory test results on the used isolates mis results oxidase test catalase test n-free media sucrose test nbrip-bpb amylase test development media development bacillus cereus rcp3/1 rhizobium radiobacter 11/2 s+ = strong positive; w+ = weak positive w+ s+ s+ w+ s+ + s+ + arbuscular mycorrhizae and plant growth promoting rhizobacteria affects strawberry (fragaria × ananassa duch.) growth under salt stress 91 while leaf area increased up to 40.50 cm2 in plants that were subjected to bacteria application, the plants of the control group, had an average leaf area of 32.76 cm2. in terms of uprooting days, while the largest leaf area was determined at the plants uprooted on the 45th day, it was observed that leaf areas decrease at the uprootings of the 60th day (fig. 1). in salt × application interaction while the largest leaf area was measured at plants with 30 mm/l nacl concentration and bacteria application, the smallest areas were determined at the plants subjected to mycorrhiza+bacteria combined application (tab. 2). bacteria application at 0 mm/l nacl concentration on the plants uprooted on the 45th day gave the highest average leaf area value (fig. 1). salinity stress is reduction in the rate of leaf surface expansion leading to cessation of expansion as salt concentration increases (wang and nil, 2000). salt stress also results in a considerable decrease in the dry weights of leaves, stems, and roots (chartzoulakis and klapaki, 2000). while the effects of salt concentration, applications and salt × application interaction were determined to be statistically insignificant on offshoot dry weight, the effects of uprooting dates and day × salt × application interaction on the same were determined to be significant. examining the uprooting dates showed that the average shoot dry weight reached its highest value on the plants uprooted on the 45th day, while the weight started to decrease on the uprootings of the 60th day (tab. 3). as for the day × salt × application interaction, mycorrhiza + bacteria application at 0 mm/l nacl concentration on the plants uprooted on the 45th day gave the highest average value (tab. 3). it was further determined that the applications, uprooting dates and the day × salt × application interaction had significant effects in terms of root dry weight. while the control and bacteria application caused an increase in root dry weight, mycorrhiza and myc+bac application caused a decrease. in terms of uprooting days, the uprootings of the 30th and the 45th days gave the highest root dry weight (fig. 3). as for the day × salt × application interaction, control application at 30 mm/l nacl concentration on the plants uprooted on the 30th day gave the highest average dry root weight (tab. 3). shoot dry weight, and root dry weight of strawberry plants were lower at salt stress treatment as compared to non-saline conditions (p < 0.05). similar result has been shown (saied et al., 2005). previous study indicating that the use of mycorrhiza and pgpr that produce acc (1-aminocyclopropane-1-carboxylate) deaminase (by glomus intraradices) enhanced phytoremediation in saline soils, and the study also showed that the use of pgpr and/or mycorrhiza increase the volume of plants (chang, 2007). in another study pseudomonas mendocina palleroni alone or in combination with either glomus intraradices (schenk & smith) or glomus mosseae (nicol & gerd) applied on lettuce and revealed that the plants inoculated with p. mendocina had higher offshot volume than the control plants at both salinity levels, and that mycorrhiza applications increased offshoot volume only at fig. 1: surface areas of leaves of strawberry ‘san andreas’ plants under salt (0, 30 and 60 mm/l nacl) stress and applications (control, mycorrhiza, bacteria and myc+bac) for the periods of 0, 15, 30, 45 and 60 days. fig. 2: shoot dry weights of strawberry ‘san andreas’ plants under salt (0, 30 and 60 mm/l nacl) stress and applications (control, mycorrhiza, bacteria and myc+bac) for the periods of 0, 15, 30, 45 and 60 days. 92 a. koc, g. balci, y. erturk, h. keles, n. bakoglu, s. ercisli tab. 2: surface areas of leaves, shoot and root dry weights, proline, anthocyanins and membrane permeability in the leaf tissues of strawberry ‘san andreas’ plants under salt (0, 30 and 60 mm/l nacl) stress and applications (control, mycorrhiza, bacteria and myc+bac). salt applications control mycorrhiza bacteria myc+bac means surface areas of leaves (cm2) 0 mm/l nacl 34,35 ac 40,55 ac 42,62 ab 36,18 ac 38,43 a 30 mm/l nacl 31,52 bc 39,83 ac 44,71 a 30,95 c 36,75 ab 60 mm/l nacl 32,40 bc 32,83 bc 34,16 ac 34,09 ac 33,37 b means 32,76 b 37,74 ab 40,50 a 33,74 b shoot dry weights (g) 0 mm/l nacl 5,15 ns 5,12 5,08 5,48 5,21 30 mm/l nacl 4,64 4,37 5,44 5,13 4,90 60 mm/l nacl 4,30 4,54 4,50 5,11 4,61 means 4,70 4,68 5,01 5,24 root dry weights (g) 0 mm/l nacl 3,37 ns 3,26 3,57 2,86 3,27 30 mm/l nacl 3,89 2,69 3,29 3,09 3,24 60 mm/l nacl 3,42 2,82 2,76 2,78 2,95 means 3,56 2,92 3,21 2,91 proline (μmol/100 mg) 0 mm/l nacl 0,582 df 0,558 ef 0,627 df 0,541 f 0,577 c 30 mm/l nacl 0,835 b-f 0,776 c-f 1,139 b 0,685 c-f 0,858 b 60 mm/l nacl 1,023 bc 0,933 b-d 1,459 a 0,912 b-e 1,082 a means 0,813 b 0,756 b 1,075 a 0,713 b anthocyanins 0 mm/l nacl 8,364 cd 7,597 d 8,735 b-d 7,373 d 8,017 c 30 mm/l nacl 10,013 a-d 9,653 b-d 10,454 a-d 8,871 b-d 9,748 b 60 mm/l nacl 12,297 ab 11,746 a-c 13,543 a 9,336 b-d 11,731 a means 10,224 b 9,665 b 10,911 a 8,527 c membrane permeability 0 mm/l nacl 9,217 bc 8,886 cd 8,538 d 8,496 d 8,784 b 30 mm/l nacl 9,563 ab 9,349 b 9,438 b 9,262 bc 9,403 a 60 mm/l nacl 9,914 a 9,404 9,372 b 9,419 b 9,527 a means 9,565 a 9,213 b 9,116 b 9,059 b *values within by the same letter are not significantly different at p < 0.05 by duncan ns not significant medium level of salinity (kohler et al., 2009). it is known that under salt stress conditions plants increase cellular osmotic pressure by producing secondary metabolites, various chemicals and particularly stress proteins (such as proline, etc.), and thus they sustain their existence by balancing the high osmotic pressure that emerges in the nutrient medium (edreva, 1998). in present study, the effects of salt concentrations, applications, uprooting days, the interaction between salt and application, and the interaction between day, salt and application were determined to be statistically significant (p < 0.05) on proline content. salt application, in general, caused an increase in the proline content of plants. while the proline content was 1.082 μmol/100 mg at 60 mm/l nacl concentration, it was determined to be 0.577 μmol/100 mg at 0 mm/l nacl. examining the effects of the applications showed that the bacteria application caused the proline content to reach its maximum value. in terms of uprooting days, on the other hand, the highest proline content was determined to be in the first uprooting day, and it decreased on the following uprooting days (fig. 4). in salt × ap plication interaction, bacteria application at 60 mm/l nacl con centration provided the highest proline content as for the day × salt × application interaction, bacteria application at 60 mm/l nacl concentration on the plants uprooted on the 45th day gave the highest average value (tab. 2, 4). similarly, a study conducted on tomato (aziz et al., 1999) reported that introducing salt concentrations increases plants’ proline content. in another study were used six nacl levels (0, 25, 50, 75, 100 and 150 mm) and two strawberry cultivars, ‘camarosa’ and ‘albino’. elevated salinity level significantly increased leaf proline content of both cultivars. ‘albino’ leaves accumulated higher proline compared with ‘camarosa’ at salinized and non-salinized treatments (al-shorafa et al., 2014). keutgen arbuscular mycorrhizae and plant growth promoting rhizobacteria affects strawberry (fragaria × ananassa duch.) growth under salt stress 93 tab. 3: surface areas of leaves, shoot and root dry weights in the leaf tissues of strawberry ‘san andreas’ plants under salt (0, 30 and 60 mm/l nacl) stress and applications (control, mycorrhiza, bacteria and myc+bac) for the periods of 0, 15, 30, 45 and 60 days. salt applications 0 15 30 45 60 surface areas of leaves (cm2) 0 mm/lt nacl control 25,340 f-j 29,510 d-j 29,730 d-j 45,067 a-h 42,097 a-j mycorrhiza 29,160 d-j 44,253 a-i 44,180 a-i 53,780 a-c 31,353 c-j bacteria 31,497 c-j 30,613 c-j 44,873 a-h 60,527 a 45,603 a-h myc+bac 24,743 g-j 33,230 c-j 36,737 b-j 48,980 a-f 37,230 b-j 30 mm/lt nacl control 30,693 c-j 20,933 ij 33,873 c-j 46,507 a-h 25,580 f-j mycorrhiza 36,733 b-j 35,127 b-j 32,297 c-j 57,847 ab 37,123 b-j bacteria 31,500 c-j 50,647 a-e 49,973 a-e 52,543 a-d 38,890 a-j myc+bac 23,103 h-j 25,980 f-j 30,203 c-j 46,737 a-h 28,747 e-j 60 mm/lt nacl control 37,043 b-j 26,237 f-j 36,620 b-j 42,360 a-j 19,757 j mycorrhiza 29,563 d-j 31,437 c-j 33,673 c-j 49,920 a-e 19,533 j bacteria 32,057 c-j 30,117 d-j 24,793 g-j 48,327 a-g 35,513 b-j myc+bac 20,643 ij 35,900 b-j 28,903 d-j 51,730 a-e 33,273 c-j shoot dry weights (g) 0 mm/lt nacl control 1,817 kl 3,237 e-l 6,577 b-l 8,283 b-e 5,850 b-l mycorrhiza 2,283 h-l 3,810 c-l 6,907 b-k 8,353 b-d 4,243 b-l bacteria 2,793 g-l 4,263 b-l 5,540 b-l 5,860 b-l 6,920 b-k myc+bac 3,350 d-l 2,840 g-l 4,440 b-l 13,330 a 3,460 d-l 30 mm/lt nacl control 2,947 g-l 3,557 d-l 6,300 b-l 5,763 b-l 4,613 b-l mycorrhiza 2,470 h-l 2,300 h-l 3,810 c-l 8,847 bc 4,443 b-l bacteria 3,207 e-l 2,730 g-l 7,063 b-j 7,693 b-g 6,520 b-l myc+bac 1,720 l 3,053 g-l 4,237 b-l 8,177 b-f 8,437 b-d 60 mm/lt nacl control 2,800 g-l 2,717 g-l 4,767 b-l 6,570 b-l 4,623 b-l mycorrhiza 2,077 j-l 3,923 c-l 5,983 b-l 7,257 b-i 3,437 d-l bacteria 2,750 g-l 1,920 kl 7,337 b-h 7,343 b-h 3,140 f-l myc+bac 2,143 i-l 3,110 f-l 3,910 c-l 9,033 b 7,360 b-h root dry weights (g) 0 mm/lt nacl control 1,510 g 3,353 b-g 4,220 b-g 4,290 b-g 3,477 b-g mycorrhiza 1,627 fg 5,683 ab 3,970 b-g 2,547 c-g 2,493 d-g bacteria 2,070 d-g 4,647 a-e 3,983 b-g 2,853 b-g 4,283 b-g myc+bac 2,583 c-g 2,113 d-g 2,480 d-g 5,450 a-c 1,650 fg 30 mm/lt nacl control 1,573 g 4,037 b-g 7,130 a 2,777 b-g 3,950 b-g mycorrhiza 2,520 c-g 1,693 e-g 2,463 d-g 3,730 b-g 3,023 b-g bacteria 2,897 b-g 2,983 b-g 3,927 b-g 3,730 b-g 2,930 b-g myc+bac 1,627 fg 2,823 b-g 3,263 b-g 4,077 b-g 3,667 b-g 60 mm/lt nacl control 2,643 c-g 3,937 b-g 4,540 a-f 3,187 b-g 2,773 b-g mycorrhiza 1,827 e-g 2,203 d-g 4,160 b-g 2,957 b-g 2,940 b-g bacteria 2,007 d-g 2,513 c-g 4,250 b-g 3,117 b-g 1,917 e-g myc+bac 1,797 e-g 1,673 fg 2,547 c-g 4,880 a-d 3,013 b-g *values within by the same letter are not significantly different at p < 0.05 by duncan and pawelzi̇k (2009) reported that free amino acids and proline accumulated in salt conditions in cultivars, ‘elsanta’ and ‘korona’, but the latter revealed higher free amino acid content. however, higher accumulation of proline has been linked with better osmotic adjustment and, consequently, higher adaptation to salt conditions (saied et al., 2005). in strawberry cultivars, a dramatic accumulation of proline following salt stress was observed (keutgen and pawelzik, 2009). the effects of salt concentrations, applications, uprooting days, the interaction between salt and application, and the interaction between day, salt and application were determined to be statistically significant (p > 0.05) on anthocyanin level. it was observed that the anthocya94 a. koc, g. balci, y. erturk, h. keles, n. bakoglu, s. ercisli fig. 3: root dry weights of strawberry ‘san andreas’ plants under salt (0, 30 and 60 mm/l nacl) stress and applications (control, mycorrhiza, bacteria and myc+bac) for the periods of 0, 15, 30, 45 and 60 days. fig. 4: proline in the leaf tissues of strawberry cv. ‘san andreas’ plants under salt (0, 30 and 60 mm/l nacl) stress and applications (control, mycorrhiza, bacteria and myc+bac) for the periods of 0, 15, 30, 45 and 60 days. nin content increased in line with the increasing salt concentration. anthocyanin content increased in plants subjected to bacterial application. on the other hand, the 60th day uprooting was determined to be the uprooting with the highest content of anthocyanin (fig. 5). in terms of salt × application interaction, it was determined that the anthocyanin content was high at 30 mm/l nacl concentration with control and bacteria applications and at 60 mm/l nacl concentration with control, mycorrhiza and bacteria applications. anthocyanin content was determined to be rather low at both salt concentrations with the application of mycorrhiza and bacteria (tab. 2). concerning the interaction between uprooting days, salt concentrations and implemented applications, high levels of anthocyanin were determined in plants subjected to bacteria application made on the plants uprooted on the 60th day with 30 mm/l nacl concentration, and on those that were subjected to control, mycorrhiza and bacteria applications made on the plants uprooted on the 60th day with 60 mm/l nacl concentration (tab. 4). keutgen and pawelzik (2009) reported that salt stress increased the contents of anthocyanins in fruits of both cvs and the highest increase of 94 % occurred in cv. ‘elsanta’ at 40 mmol nacl/l. membrane permeability is an important indicator that points out to membrane stability. a low level of membrane permeability is important for the robustness of the membrane. in general terms, the salt applied on the plants causes an increase in membrane permeability thus disrupts membrane stability and becomes a significant factor damaging the plant. in studies conducted on strawberry, it was determined that application of salt increases membrane permeability (kaya et al., 2003). in addition, subjecting plants under salt stress to bacteria application caused a decrease in membrane permeability and thus helped in the maintenance of membrane stability. in our study, the effects of salt concentrations, applications, uprooting days, the interaction between salt and application, and the interaction between day, salt and application were determined to be statistically significant (p < 0.05) on membrane permeability. membrane permeability increased at applications with 30 mm/l and 60 mm/l nacl concentrations. among the applications, it was determined that the membrane permeability of the control application increased and in other mycorrhiza, bacteria and myc+bac applications it was lower. it was observed that membrane permeability increased in the uprootings of the 15th, 45th and 60th days (fig. 6). in salt × application interaction, membrane permeability was determined to be high in plants subjected to control application at 30 mm/l and 60 mm/l nacl concentrations. the lowest interaction value was measured with bacteria and myc+bac applications at 0 mm/l nacl concentration (tab. 2). concerning the interaction between uprooting days, salt concentrations and applications, it was determined that the control application arbuscular mycorrhizae and plant growth promoting rhizobacteria affects strawberry (fragaria × ananassa duch.) growth under salt stress 95 was statistically different (p < 0.05) at 60 mm/l nacl concentration on the plants uprooted on the 45th day (tab. 4). conclusions our study demonstrates that under saline conditions, proline, anthocyanins and membrane permeability increase in strawberry leaves, while lsa decreases. the mycorrhiza and bacteria applications carried out during planting caused an increase in the leaf area of plants under salt stress. bacteria application was determined to be the prominent application that helps the plant to tolerate the negative effects of salt stress by increasing proline and anthocyanin levels. acknowledgements we would like to thank to dr. ramazan çakmakçi for providing the bacteria and to bioglobal company for providing mychorriza, and to bozok university scientific reseach projects division for providing financial support to my project. references al-shorafa, w., mahadeen, a., al-absi, k., 2014: evaluation for salt stress tolerance in two strawberry cultivars. am. j. agric. biol. sci. 9, 334-341. alkan torun, a., aka kaçar, y., bicen, b., erdem, n., serce, s., 2014: in vitro screening of octoploid fragaria chiloensis and fragaria virginiana genotypes against iron deficiency. turk. j. agric. for. 38, 169-174. augé, r.m., 2001: water relations, drought and va mycorrhizal symbiosis. mycorrhiza 11, 3-42. aziz, a., martin-tonguy, j., larher, f., 1999: salt stres, induced proline accumulation and changes in tyramine and polyamine levels are linked to ionic adjustment in tomato leaf discs. plant sci. 145, 83-91. bates, l., waldren, r.p., teare, i.d., 1973: rapid determination of free proline for water-stress studies. plant and soil. 39, 205-207. cakmakci, r., donmez, m.f., erturk, y., erat, m., haznedar, a., sekban, r., 2010: diversity and metabolic potential of culturable bacteria from the rhizosphere of turkish tea grown in acidic soils. plant soil. 332, 299-318. fig. 6: membrane permeability in the leaf tissues of strawberry cv. ‘san andreas’ plants under salt (0, 30 and 60 mm/l nacl) stress and applications (control, mycorrhiza, bacteria and myc+bac) for the periods of 0, 15, 30, 45 and 60 days. fig. 5: anthocyanins in the leaf tissues of strawberry cv. ‘san andreas’ plants under salt (0, 30 and 60 mm/l nacl) stress and applications (control, mycorrhiza, bacteria and myc+bac) for the periods of 0, 15, 30, 45 and 60 days. 96 a. koc, g. balci, y. erturk, h. keles, n. bakoglu, s. ercisli tab. 4: proline, anthocyanins and membrane permeability in the leaf tissues of strawberry cv. ‘san andreas’ plants under salt (0, 30 and 60 mm/l nacl) stress and applications (control, mycorrhiza, bacteria and myc+bac) for the periods of 0, 15, 30, 45 and 60 days. salt applications 0 15 30 45 60 proline (μmol/100 mg) 0 mm/lt nacl control 1,536 b-e 0,746 m-t 0,315 w-z 0,157 z 0,156 z mycorrhiza 1,209 f-i 0,725 n-t 0,188 yz 0,323 v-z 0,345 u-z bacteria 1,105 h-k 0,775 l-s 0,538 r-x 0,276 x-z 0,442 t-z myc+bac 1,165 g-j 0,696 n-t 0,160 z 0,355 u-z 0,327 v-z 30 mm/lt nacl control 1,534 b-e 0,897 j-p 0,462 t-z 0,842 k-r 0,439 t-z mycorrhiza 1,252 e-h 1,024 h-m 0,523 s-x 0,563 r-x 0,515 s-x bacteria 1,232 f-h 1,442 d-g 0,634 o-v 1,636 b-d 0,748 m-t myc+bac 1,159 g-j 0,969 h-n 0,341 u-z 0,492 s-y 0,462 t-z 60 mm/lt nacl control 1,920 a 1,045 h-l 0,598 p-w 0,873 j-q 0,680 n-t mycorrhiza 1,450 d-g 1,418 d-g 0,484 s-y 0,646 o-u 0,668 n-t bacteria 1,494 c-f 1,814 ab 0,915 i-o 1,951 a 1,124 h-k myc+bac 1,768 a-c 1,062 j-l 0,605 p-w 0,555 r-x 0,573 q-x anthocyanins 0 mm/lt nacl control 11,470 e-k 7,643 o-u 3,557 yβ 8,627 l-s 10,523 g-m mycorrhiza 7,457 o-v 4,970 v-β 2,487 β 9,970 i-o 13,100 c-g bacteria 8,627 l-s 5,753 t-y 2,873 z β 11,003 e-m 15,417 bc myc+bac 7,890 n-t 5,257 u-z 2,633 β 8,547 l-s 12,537 d-h 30 mm/lt nacl control 12,657 d-h 8,437 m-s 3,217 yβ 12,587 d-h 13,167 c-f mycorrhiza 10,720 f-m 7,147 p-v 3,577 yβ 13,427 c-e 13,397 c-e bacteria 10,457 h-n 6,970 q-w 3,487 yβ 13,530 c-e 17,827 a myc+bac 9,327 k-q 6,217 s-x 3,110 z β 11,097 e-l 14,607 cd 60 mm/lt nacl control 14,610 cd 9,743 j-p 3,870 xβ 14,400 cd 18,860 a mycorrhiza 13,460 c-e 8,970 k-r 4,487 wβ 14,183 cd 17,630 ab bacteria 12,110 d-j 14,740 cd 5,370 t-z 15,533 bc 19,963 a myc+bac 9,360 k-q 6,577 r-w 3,120 z β 12,347 d-i 15,277 bc membrane permeability 0 mm/lt nacl control 8,918 b-g 9,265 b-f 9,150 b-f 9,378 b-f 9,375 b-f mycorrhiza 7,913 hi 9,113 b-g 8,633 d-h 9,222 b-f 9,548 b-d bacteria 8,808 c-h 9,070 b-g 8,396 f-h 7,964 hi 8,455 e-h myc+bac 8,819 c-h 9,088 b-g 7,244 i 8,146 gh 9,184 b-f 30 mm/lt nacl control 9,369 b-f 9,813 bc 9,421 b-e 9,626 b-d 9,584 b-d mycorrhiza 9,081 b-g 9,679 bc 9,114 b-g 9,397 b-f 9,476 b-e bacteria 9,202 b-f 9,436 b-e 9,575 b-d 9,467 b-e 9,509 b-d myc+bac 8,971 b-g 9,272 b-f 9,129 b-g 9,455 b-e 9,484 b-d 60 mm/lt nacl control 9,295 b-f 9,898 b 9,556 b-d 11,012 a 9,811 bc mycorrhiza 9,062 b-g 9,347 b-f 9,481 b-d 9,457 b-e 9,674 bc bacteria 9,136 b-f 9,280 b-f 9,442 b-e 9,456 b-e 9,544 b-d myc+bac 9,004 b-g 9,551 b-d 9,608 b-d 9,279 b-f 9,655 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agricultural salinity assessment and management. asce manual reports on engineering practices 71, 1-41. wang, y., nil, n., 2000: changes in chlorophyll, ribulose biphosphate carboxylase-oxygenase, glycine betaine content, photosynthesis and transpiration in amaranthus tricolor leaves during salt stress. j. hortic. sci. biotechnol. 75, 623-627. address of the corresponding author: e-mail: sercisli@gmail.com © the author(s) 2016. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 90, 339 345 (2017), doi:10.5073/jabfq.2017.090.042 1institute of horticultural sciences, university of agriculture, faisalabad, pakistan 2department of horticultural sciences, university college of agriculture and environmental sciences, the islamia university of bahawalpur, punjab, pakistan 3nuclear institute of agriculture and biology, faisalabad, pakistan 4department of soil sciences, bahauddin zakariya university, multan, pakistan 5department of agronomy, university college of agriculture and environmental sciences, the islamia university of bahawalpur, pakistan 6departments of environmental sciences, bahauddin zakariya university, multan, pakistan exploring the better genetic options from indigenous material to cultivate tomato under high temperature regime araiz nazir1, muhammad rashid shaheen2, choudhary muhammad ayyub1, rashid hussain2, nadeem sarwer3, muhammad imran4, muhammad aurangzaib5, muhammad nawaz6*, muhammad faizan ali khan6, yussra jawad6, munawar iqbal6 (received may 5, 2017; accepted september 19, 2017) * corresponding author summary screening test was conducted on 54 genotypes of tomato to analyze the effect of heat stress and categorize them as heat tolerant or heat susceptible ones. seedlings were grown at temperatures of 28/22 oc day/night. four weeks after sowing, plants were exposed to high temperatures of 40/32 oc day/night for one week. data for various morphological (root and shoot length, root and shoot fresh and dry weight, number of leaves) and physiological parameters (chlorophyll contents, sub-stomatal co2, transpiration rate, stomatal conductance, photosynthetic rate, water use efficiency and leaf temperature) were recorded. heat stress had a negative effect on all physiological and morphological processes of the genotypes. the results of this study revealed that “parter improved” and “legend” were more heat tolerant genotypes whereas “grus chovka” and “nepoli” were more heat sensitive among the genotypes under consideration. keywords: tomato, genotypes, screening, heat stress introduction over 20th century, the global average surface temperature has risen about 0.6 ± 2 oc and is expected to further increase up to 1.4 5.8 oc during this century (houghton et al., 2001). the extreme climates including high temperature stress might result in loss of crop productivity which in turn would lead to famine (bita and gerats, 2013). a manifestation of the ever-increasing and unexpected climate changes in plants is through appearance of stress symptoms. stress is defined as “the negative effect that an organism may suffer” and may be internal or external (madlung and comai, 2004) and has been reported to have reduced agricultural productivity to as much as 50%. it has been estimated that each degree celsius rise in temperature in average growing season results in 17% decrease in yield (lobell and asner, 2003). bray et al. (2000) estimates that 51 82% of the potential yield of annual crops is lost due to abiotic stresses. among the abiotic stresses, heat stress has created the most alarming situation for pakistan’s agriculture, causing several physical, physiological, biochemical and anatomical distortions in crop plants. rise in temperature at vegetative stage of plants may have direct or indirect effects. direct ones include protein aggregation and denaturation (wahid et al., 2007; golam et al., 2012) while indirect ones are the limited protein production, inactivation of enzymes in chloroplast and mitochondria (wahid et al., 2007). visually, high temperature manifests itself through burning of twigs, sunburn, hindered development of root (batts et al., 1998; porter and gawith, 1999) and shoot (golam et al., 2012), scorching of leaves, leaf senescence and abscission, inhibition of growth and ultimately decreased productivity (guilini et al., 1997; ismail and hall, 1999; vollenweider and gunthardt-goerg, 2005). heat stress adversely affects radical growth in eggplant seedlings (sekara et al., 2012) and has been reported to have markedly reduced plant height, stem fresh and dry weight, leaf fresh and dry weight and leaf area of tomatoes (abdelmageed et al., 2003). abdelmageed (2009) found poor and stunted growth in tomatoes and negative impacts on leaf area, leaf fresh weight, leaf dry weight, stem fresh weight and stem dry weight, leaf area ratio and leaf weight ratio of the vegetable when exposed to high temperatures. prasad et al. (2006) stated after experimentation on easter lilies that se vere heat stress resulted in decreased plant height and that day and night temperatures affect stem length in the flower (erwin et al., 1989). after an experiment on rose with temperature ranges of 0, 6 and 10 °c for 2 and 14 days, it was concluded that increasing bandwidths reduced shoot lengths as well as their fresh weight at harvestable stage, irrespective of the days for which the temperature was applied (dieleman et al., 2005). porter and gawith (1999) indicated that root growth is comparatively more sensitive to heat stress than other organs, and decreases with heat stress. high temperature stress decreases root length as well as diameter. during reproductive phase, decreased carbon partitioning in roots causes the reduction in number of roots as well as their length (batts et al., 1998). heat stress increases rate of evaporation and transpiration by influencing soil temperature and increasing water vapor deficit, respectively (prasad et al., 2008), and has been said to be associated with lack of water availability (simoes-araujo et al., 2003). these alterations affect water relations, and tend to be severer during day time than night time (wahid et al., 2007). although drought has been known to be the major cause of water loss from plants, the severity of temperature tends to aggravate the situation (machado and paulson, 2001). especially during the day, water tends to decrease in plants due to transpiration which leads to decreased water potential and thus disturbance of many plant processes (tsukaguchi et al., 2003). morales et al. (2003) reported heat stress to damage water relations and root hydraulic activity in tomato plants. in sugarcane, high temperature tends to change leaf water potential, even though the soil water content and relative humidity are optimal (wahid and close, 2007). todorov et al. (2003) and sharkey and zhang (2010) indicated that heat stress not only adversely affects photosynthesis but also 340 a. nazir, m.r. shaheen, c.m. ayyub, r. hussain, n. sarwer, m. imran, m. aurangzaib, m. nawaz, m.f.a. khan, y. jawad, m. iqbal respiration. the rise in chlorophyllase activity and decreased photo synthetic pigments leads to reduced photosynthetic and respirato ry processes of plants. with increasing temperatures, respiration initially increases. however, at temperatures above 50 °c, damage to the respiratory mechanism causes decrease in the process (prasad et al., 1998). in a study conducted by sato et al. (2000), effect of heat stress on respiration, photosynthesis, pollen production and release and dehiscence was examined and it was found that all plants kept under high temperature showed reduced photosynthesis and increased respiration. photosynthesis is said to be the physiological process most sensitive to heat stress (wahid et al., 2007). an increase in temperature (>40 °c) (prasad et al., 2008) damages the structural organization of thylakoid and disturbance in grana stacking, leading to reduced photosynthesis (wahid et al., 2007). the rate of the energy con suming photorespiration increases (lea and leegood, 1999; nakamoto and hiyama, 1999) and photosynthesis decreases (schuster and monson, 1990). wahid et al. (2007) reported several changes in grapes when exposed to high temperature stress whereby chloroplast in the mesophyll cells of grape plants assumed a rounded shape, the stroma lamellae swelled, whilst the cristae were disturbed and mitochondria got to be vacant. such changes bring about the structuring of antenna-depleted psii and henceforth diminish photosynthetic and respiratory processes. damage to photosystem ii has also been reported in several studies (santarius, 1975; santarius and müller, 1979; berry and björkman, 1980; enami et al., 1994). prasad et al. (1998) have indicated psii to be most sensitive to heat stress which occurs at temperatures as high as 35 40 °c (terzaghi et al., 1989; thompson et al., 1989; gombos et al., 1994; çjánek et al., 1998; yamane et al., 1998). however, moderate heat stress does not damage psii but reduces photosynthetic activity (sharkey, 2005). heat stress is also reported to be a cause of formation reactive oxygen species (ros) (zinnet al., 2010). these ros are formed due to alteration in protein aggregation (wahid et al., 2007; golam et al., 2012), limited protein production, and inactivation of enzymes in chloroplast and mitochondria (wahid et al., 2007). in spite of being a summer vegetable, tomatoes are also affected by rise in temperature beyond their threshold level. kuo et al. (1993) has categorized tomato as a heat sensitive vegetable with more than 75% high temperature injury. germination, seedling, flowering and fruit setting and ripening is adversely affected at temperatures above 35 oc (miller et al., 2001). the present study was aimed at screening of indigenous tomato genotypes to estimate its heat tolerance potential and categorizing them as heat tolerant and heat susceptible ones. materials and methods the present study was conducted in growth room in controlled conditions of photoperiod, temperature and humidity. fifty-four tomato genotypes were screened against heat stress. each treatment consisted of four replications. plants were grown in plastic pots of 8-inch diameter and sterilized sand was used as growth medium. each pot was filled with 850 g sand and 160 ml water and 10 ml hoagland’s solution was applied prior to sowing. hoagland’s solution was later applied periodically as the nutrient medium. optimum temperature of 28/22 oc (day/night) was provided for four weeks during germination and growth. heat stress was applied four weeks after seedling growth by gradually increasing 2 oc temperature per day, to avoid osmotic shock until desired high temperature of 40/32 oc day/night temperature was achieved. plants were kept at this temperature for one week. after one week of stress, data was recorded. morphological attributes such as number of leaves, shoot length (cm), root length (cm) were measured with meter rod, shoot fresh weight (g), root fresh weight (g), shoot dry weight (g) and root dry weight (g) was recorded with digital weighing balance. physiological parameters such as transpiration rate (mmol/m2/s), photosynthetic rate (μmol/m2/s), sub-stomatal co2 (vpm) and sub-stomatal water and leaf surface temperature (ºc) were all recorded using portable photosynthetic meter (model lci-sd adc bioscientific, uk). water use efficiency was calculated using the formula: rate of photosynthesis (pn) water use efficiency (pn/e) = rate of transpiration (e) chlorophyll contents (spad value) were measured using a chlorophyll meter (cm 200plus, bio-scientific usa). completely randomized design (crd) was applied to the experiment. collected data was analyzed statistically by employing fisher’s analysis of variance technique and significance of treatments were tested (steel et al., 1997). statistical analysis and correlations between variables were also estimated by using r. principal component analysis (pca) was employed to identify the patterns in data and to graphically express the data in such a way as to emphasize their similarities and differences. results and discussion as stated by vollenweider and gunthardt-goerg (2005), heat stress can cause marked reduction in shoot and root growth. ex periments conducted by rahmani et al. (2013) on impact of day and night temperatures on cauliflower showed that greater curd length, diameter, fresh weight and dry weight was observed at warmer night temperatures than day temperatures whereas greater leaf growth, leaf area, stem length, stem fresh and dry weight was observed at warmer day temperatures. screening results (tab. 1) demonstrated that “parter imported” exhibited greatest tolerance against heat stress, with 185 cm shoot length. “grus chovka” was most susceptible to heat stress with 4.9 cm shoot length. the genotype “alaskan fancy” had maximum root length under heat stress with 13.18 cm (tab. 1). “kaldera” was most sensitive to heat stress with root length of 4.12 cm. maximum shoot fresh weight was recorded for “roma” (4.06 g) (tab. 1) while “grus chovka” (0.7 g) had least shoot fresh weight. these results were similar to the results obtained by rahmani et al. (2013) who conducted experiments on impact of day and night temperatures on cauliflower. temperatures of 24/12 °c, 12/24 °c, 20/16 °c, 16/20 °c and 20/20 °c in the first run and 24/20 °c, 20/ 12 °c and 20/16 °c in the second run revealed that greater stem fresh and dry weight was observed at warmer day temperatures. genotype “bush beef steak” had maximum root fresh weight of 2.02 g (tab. 1). minimum root fresh weight was recordrd for “kaldera”, having 0.21 g. “roma” showed greatest shoot dry weight of 0.31 g (tab. 1). this genotype also had the highest shoot fresh weight. similarly, “grus chovka” had least shoot dry weight of 0.07 g and also had least shoot fresh weight. highest root dry weight was recorded to be 0.52 g (tab. 1) which was for “roma” and least was 0.06 g which was for “pakit”. highest number of leaves (33) were recorded for “parter improved” (tab. 1). “uc-134” had 14 leaves which were recorded as the least number of leaves. analysis of variance for individual traits i.e. morphological traits is given in (tab. 3) which showed that root length, shoot length, root and shoot fresh and dry weight, and number of leaves were highly significant among the genotypes under study. similarly analysis of variance for physiological traits is given in (tab. 4) which showed that chlorophyll content, sub-stomatal co2, stomatal conductance to water, photosynthetic rate, transpiration rate, water use efficiency and leaf temperature were highly significant in genotypes under observation. screening of local germplasm for cultivation under heat stress conditions 341 v1 v2 v3 v4 v5 v6 v7 v8 v9 v10 v11 v12 v13 v14 v15 v16 v17 v18 v19 v20 v21 v22 v23 v24 v25 v26 v27 v28 v29 v30 v31 v32 v33 v34 v35 v36 v37 v38 v39 v40 v41 v42 v43 v44 v45 v46 v47 v48 v49 v50 v51 v52 v53 v54 tab. 1: shoot length, root length, shoot fresh weight, root fresh weight, shoot dry weight, root dry weight and number of leaves of tomato genotypes under high temperature stress genotype genotype shoot length root length shoot fresh root fresh shoot dry root dry no. of leaves number (cm) (cm) weight (g) weight (g) weight (g) weight (g) cln-2366 a la-2662 la-3120 early annie sasha altai kht-15 subartic way ahead jagour iles yellow latvian zarnitza pakit uc-134 brdley subarctic lomg keeper parter improved roma cchaus legend alaskan fancy raad red early wonder polar beauty zhezha camp bells bonita rio grande new yarker beef steak leeper la-2010 grus chovka nepoli dona pres cott tai-1042 bush beef steak cold set naqeeb kaldera manatoba caro rich tomato forme de coeur nth-671 spekled sibrian northern delight anahu taxi nagina rio grand quantum tima france tomato 3383 f1 cm selection 10.90±1.09 f-p 13.48±0.85 a-j 16.60±0.38 a-d 10.28±0.87 h-p 16.80±0.85 abc 8.88±1.71 i-q 12.60±0.85 b-l 7.23±0.70 m-q 11.50±1.02 d-n 10.90±1.39 f-p 8.68±1.06 j-q 7.10±0.81 m-q 10.65±0.53 g-p 14.48±0.80 a-h 12.53±0.92 b-l 12.73±0.98 b-l 18.50±0.54 a 14.75±0.75 a-h 11.50±0.74 d-n 16.38±1.13 a-e 15.60±0.58 a-g 11.45±0.55 e-n 12.10±0.50 c-m 17.33±1.22 ab 13.00±0.88 b-k 11.25±1.21 e-o 12.60±0.73 b-l 14.70±1.17 a-h 10.23±1.18 h-p 9.90±1.11 h-q 9.83±0.88 h-q 11.65±0.41 d-n 4.90±0.41 q 10.23±0.56 h-p 9.10±0.63 i-q 13.25±0.73 b-k 9.05±1.23 i-q 8.23±0.35 k-q 13.88±1.61 a-i 13.50±0.96 a-j 15.88±1.84 a-f 6.18±0.12 opq 10.50±0.61 g-p 5.00±0.74 q 10.83±1.01 f-p 13.50±1.02 a-j 11.10±0.46 f-p 10.63±0.77 g-p 6.75±0.25 n-q 8.73±0.63 j-q 11.00±0.35 f-p 6.08±0.88 pq 10.38±0.80 h-p 7.75±0.60 l-q 9.30±1.19 a-f 8.25±1.59 a-f 8.93±0.62 a-f 6.95±1.22 c-f 8.38±0.90 a-f 8.45±0.92 a-f 12.88±1.13 ab 6.18±0.62 c-f 9.75±0.41 a-e 9.38±1.83 a-f 7.83±1.34 b-f 5.90±0.46 def 7.38±0.31 c-f 7.68±0.84 b-f 8.30±0.45 a-f 9.35±1.43 a-f 8.90±1.33 a-f 9.08±0.79 a-f 6.95±0.55 c-f 7.98±0.71 a-f 13.18±1.37 a 8.23±1.17 a-f 11.43±1.17 abc 8.88±0.88 a-f 9.13±0.83 a-f 7.00±0.51 c-f 8.93±0.72 a-f 7.98±0.86 a-f 8.00±0.95 a-f 8.60±0.76 a-f 6.80±1.20 c-f 4.50±0.84 ef 5.88±1.09 def 7.00±0.71 c-f 7.15±1.41 c-f 5.88±0.59 def 6.88±1.68 c-f 5.38±0.38 def 4.13±0.13 f 7.75±0.92 b-f 4.13±0.63 f 7.05±0.57 c-f 10.65±0.83 a-d 7.48±0.93 c-f 6.00±0.29 def 5.25±1.03 ef 5.45±1.28 def 5.38±0.69 def 7.10±0.58 c-f 5.93±0.40 def 7.40±0.36 c-f 5.88±0.43 def 8.38±0.92 a-f 7.55±0.73 b-f 2.38±0.27 b-l 3.26±0.31 a-f 3.35±0.16 a-e 2.10±0.36 b-m 3.08±0.36 a-h 1.73±0.44 f-m 2.87±0.27 a-j 1.38±0.32 i-m 2.24±0.12 b-m 3.25±0.38 a-f 1.64±0.27 g-m 1.01±0.09 lm 1.36±0.18 i-m 2.68±0.42 a-k 2.09±0.14 b-m 2.23±0.40 b-m 3.69±0.33 ab 4.06±0.41 a 2.08±0.35 c-m 3.51±0.31 abc 3.11±0.27 a-g 2.34±0.42 b-l 3.43±0.36 a-d 2.43±0.33 b-l 2.57±0.33 a-l 2.02±0.34 c-m 2.93±0.37 a-i 2.28±0.43 b-m 1.51±0.31 g-m 2.07±0.28 c-m 1.10±0.20 klm 1.36±0.32 i-m 0.70±0.13 m 1.19±0.14 klm 1.51±0.17 g-m 1.89±0.20 d-m 1.47±0.30 i-m 1.73±0.08 f-m 1.78±0.38 e-m 1.66±0.31 f-m 1.40±0.25 i-m 1.49±0.04 h-m 2.12±0.18 b-m 1.31±0.18 j-m 1.99±0.18 c-m 1.95±0.30 c-m 1.98±0.25 c-m 2.01±0.14 c-m 1.12±0.07 klm 1.64±0.08 g-m 1.62±0.11 g-m 1.06±0.11 lm 2.12±0.23 b-m 1.68±0.34 f-m 0.94±0.26 b-j 0.76±0.15 c-j 1.20±0.31 a-h 0.97±0.27 b-j 0.52±0.10 e-j 0.40±0.09 g-j 1.20±0.24 a-h 0.63±0.15 d-j 0.75±0.04 c-j 0.88±0.17 c-j 0.44±0.05 g-j 0.45±0.07 g-j 0.52±0.04 e-j 0.58±0.12 d-j 0.75±0.15 c-j 0.49±0.16 f-j 1.08±0.26 b-i 1.37±0.28 a-e 0.45±0.08 g-j 0.66±0.13 c-j 1.01±0.18 b-j 0.70±0.17 c-j 0.89±0.20 b-j 0.48±0.08 g-j 0.48±0.11 g-j 0.45±0.07 g-j 0.78±0.18 c-j 0.45±0.09 g-j 0.46±0.09 g-j 0.56±0.05 d-j 0.31±0.06 ij 0.69±0.11 c-j 1.13±0.12 b-i 1.40±0.14 a-d 1.74±0.31 ab 1.34±0.27 a-f 1.51±0.22 abc 2.02±0.09 a 1.25±0.32 a-g 0.83±0.01 c-j 0.21±0.04 j 0.38±0.05 hij 0.35±0.08 hij 0.30±0.03 ij 0.35±0.06 hij 0.32±0.06 ij 0.50±0.12 f-j 0.32±0.02 ij 0.32±0.03 ij 0.35±0.05 hij 0.41±0.05 g-j 0.27±0.02 ij 0.37±0.04 hij 0.40±0.03 g-j 0.20±0.04 a-h 0.26±0.03 a-e 0.27±0.02 a-d 0.15±0.03 b-h 0.23±0.03 a-f 0.14±0.02 c-h 0.26±0.03 a-e 0.10±0.03 fgh 0.20±0.02 a-h 0.23±0.03 a-f 0.13±0.03 c-h 0.10±0.01 fgh 0.14±0.02 c-h 0.17±0.03 a-h 0.19±0.02 a-h 0.16±0.03 a-h 0.30±0.05 ab 0.31±0.03 a 0.17±0.04 a-h 0.28±0.03 abc 0.23±0.03 a-g 0.20±0.04 a-h 0.27±0.03 a-d 0.21±0.02 a-h 0.20±0.03 a-h 0.18±0.02 a-h 0.25±0.04 a-f 0.22±0.03 a-h 0.13±0.03 c-h 0.20±0.03 a-h 0.08±0.02 gh 0.15±0.01 b-h 0.07±0.02 h 0.11±0.01 e-h 0.13±0.02 d-h 0.16±0.02 b-h 0.15±0.03 b-h 0.18±0.03 a-h 0.16±0.04 b-h 0.19±0.04 a-h 0.14±0.02 c-h 0.12±0.01 d-h 0.20±0.02 a-h 0.13±0.03 c-h 0.20±0.00 a-h 0.18±0.03 a-h 0.17±0.03 a-h 0.19±0.02 a-h 0.10±0.00 fgh 0.15±0.02 c-h 0.16±0.01 b-h 0.13±0.00 d-h 0.21±0.02 a-h 0.16±0.02 b-h 0.13±0.03 k-s 0.29±0.01 c-l 0.29±0.04 c-m 0.31±0.04 b-k 0.12±0.03 l-s 0.08±0.02 qrs 0.32±0.05 b-j 0.21±0.03 f-s 0.22±0.02 e-s 0.37±0.06 a-g 0.08±0.02 rs 0.06±0.01 s 0.12±0.02 l-s 0.08±0.02 rs 0.20±0.03 g-s 0.11±0.02 m-s 0.43±0.03 abc 0.52±0.05 a 0.11±0.02 n-s 0.25±0.02 d-q 0.23±0.02 d-s 0.21±0.05 f-s 0.27±0.05 c-o 0.14±0.02 k-s 0.12±0.02 l-s 0.13±0.02 l-s 0.28±0.05 c-n 0.10±0.03 o-s 0.17±0.03 i-s 0.19±0.03 h-s 0.07±0.02 rs 0.27±0.02 c-o 0.38±0.05 a-f 0.36±0.03 a-h 0.39±0.04 a-e 0.39±0.04 a-e 0.34±0.03 b-i 0.47±0.05 ab 0.24±0.02 d-r 0.40±0.03 a-d 0.06±0.03 s 0.11±0.03 m-s 0.09±0.04 p-s 0.08±0.01 rs 0.07±0.03 rs 0.09±0.03 p-s 0.15±0.03 j-s 0.07±0.01 rs 0.26±0.02 c-p 0.09±0.02 p-s 0.08±0.02 qrs 0.08±0.01 rs 0.07±0.02 rs 0.09±0.02 p-s 27.25±2.29 a-h 30.50±1.04 abc 28.50±2.50 a-g 21.25±1.65 e-o 16.50±1.76 k-o 18.75±1.70 h-o 32.75±2.29 ab 22.00±2.35 c-o 22.75±0.95 c-n 26.00±2.04 a-j 21.50±1.55 d-o 16.00±0.91 mno 14.00±1.35 o 24.75±1.80 a-l 30.00±1.96 a-d 23.50±1.55 c-n 33.25±2.21 a 26.25±1.75 a-i 25.00±1.41 a-k 29.25±1.80 a-e 29.00±1.47 a-f 27.25±2.56 a-h 30.00±1.08 a-d 25.00±0.41 a-k 24.25±0.63 b-m 26.00±1.47 a-j 23.75±1.31 c-n 21.75±1.03 d-o 22.50±1.66 c-o 20.50±1.85 f-o 17.75±1.11 i-o 19.50±0.96 h-o 15.50±1.19 no 15.25±1.55 no 23.25±1.49 c-n 18.75±1.38 h-o 18.00±1.08 i-o 18.00±1.35 i-o 18.75±0.75 h-o 17.25±2.02 k-o 21.25±1.65 e-o 27.00±2.12 a-h 20.75±1.31 e-o 17.75±1.03 i-o 20.00±1.22 g-o 23.25±0.95 c-n 19.25±1.31 h-o 16.75±1.38 k-o 15.75±1.49 mno 15.25±0.48 no 15.50±0.50 no 15.75±1.44 mno 16.25±1.31 l-o 17.50±0.87 j-o means sharing similar letter in a column are statistically non-significant (p>0.05) 342 a. nazir, m.r. shaheen, c.m. ayyub, r. hussain, n. sarwer, m. imran, m. aurangzaib, m. nawaz, m.f.a. khan, y. jawad, m. iqbal tab. 2: chlorophyll contents, sub-stomatal co2, transpiration rate, stomatal conductance, photosynthetic rate, water use efficiency and leaf temperature of tomato genotypes under high temperature stress genotypes chlorophyll sub-stomatal transpiration stomatal cond. photosynthetic water use leaf content co2 rate water rate efficiency temperature (spad value) (vpm) (mmol/m2/s) (ºc) (μmol/m2/s) (pn/e) (ºc) cln-2366 a la-2662 la-3120 early annie sasha altai kht-15 subartic way ahead jagour iles yellow latvian zarnitza pakit uc-134 brdley subarctic lomg keeper parter improved roma cchaus legend alaskan fancy raad red early wonder polar beauty zhezha camp bells bonita rio grande new yarker beef steak leeper la-2010 grus chovka nepoli dona pres cott tai-1042 bush beef steak cold set naqeeb kaldera manatoba caro rich tomato forme de coeur nth-671 spekled sibrian northern delight anahu taxi nagina rio grand quantum tima france tomato 3383 f1 cm selection 21.53±1.14 b-f 20.78±2.59 b-f 21.70±1.55 b-f 17.43±1.88 ef 25.28±2.68 b-f 20.53±1.86 b-f 28.25±3.32 b-f 18.85±2.09 c-f 31.38±1.97 b-e 23.50±1.84 b-f 19.95±2.90 b-f 49.03±3.26 a 22.45±3.34 b-f 20.78±4.02 b-f 22.10±1.03 b-f 27.55±2.30 b-f 21.45±4.80 b-f 18.68±0.79 def 30.30±3.19 b-f 17.28±2.51 ef 26.30±2.94 b-f 26.65±2.49 b-f 16.38±1.09 ef 22.95±2.57 b-f 34.28±3.18 a-d 16.15±2.80 ef 16.83±2.76 ef 23.50±2.79 b-f 23.43±2.70 b-f 21.50±1.44 b-f 25.60±4.82 b-f 20.33±1.45 b-f 31.13±2.95 b-e 18.65±1.44 def 34.53±2.43 abc 23.95±2.55 b-f 24.73±2.11 b-f 22.35±3.23 b-f 21.90±3.15 b-f 23.50±3.21 b-f 25.13±4.27 b-f 21.70±4.04 b-f 20.73±2.56 b-f 34.98±2.53 ab 25.68±3.26 b-f 14.85±3.48 f 17.05±2.42 ef 21.33±3.11 b-f 22.40±3.63 b-f 19.95±3.11 b-f 27.13±2.91 b-f 26.25±1.93 b-f 23.43±2.39 b-f 18.85±0.63 c-f 802.5±102.47 h-u 753.5±34.13 j-v 709.8±29.55 k-v 1113.8±24.37 c-h 1096.8±66.39 d-j 1394.0±37.52 a-d 766.3±55.43 i-v 1150.3±39.80 b-g 1103.0±57.68 d-i 1452.5±40.49 abc 1334.5±20.41 a-e 1487.5±15.95 ab 1555.0±67.57 a 989.0±91.27 f-m 883.3±127.85 f-s 549.0±6.86 r-v 467.8±8.68 uv 454.5±15.54 v 1153.8±126.24 b-f 1001.5±112.04 e-m 1036.0±142.82 e-l 944.8±85.87 f-o 1042.5±100.01 e-k 804.5±25.09 h-u 734.3±72.79 k-v 720.0±13.56 k-v 696.0±7.74 l-v 704.0±12.40 k-v 690.3±24.64 m-v 704.8±80.17 k-v 593.3±82.27 p-v 700.5±160.61 k-v 587.5±25.93 q-v 665.3±14.23 m-v 603.5±38.82 o-v 619.3±25.99 n-v 539.3±16.56 s-v 529.8±71.26 tuv 741.5±28.69 k-v 943.5±69.80 f-o 824.8±9.66 f-t 880.0±12.32 f-s 821.3±42.25 f-t 806.3±9.81 g-u 842.5±19.32 f-t 821.0±21.89 f-t 831.5±30.99 f-t 889.3±40.43 f-r 955.5±58.54 f-n 906.8±41.67 f-q 966.8±26.16 f-m 936.0±6.87 f-p 862.0±44.01 f-t 932.3±16.63 f-q 0.92±0.19 gh 1.63±0.17 a-h 2.30±0.27 a-h 2.10±0.11 a-h 2.82±0.63 a-d 1.99±0.28 a-h 2.49±0.13 a-g 2.21±0.19 a-h 2.61±0.24 a-f 3.18±0.35 ab 2.68±0.46 a-e 1.54±0.14 b-h 3.22±0.45 a 1.13±0.24 e-h 2.27±0.30 a-h 2.14±0.09 a-h 1.64±0.28 a-h 2.43±0.27 a-g 0.95±0.18 fgh 0.95±0.21 fgh 1.44±0.08 c-h 2.01±0.19 a-h 1.64±0.20 a-h 1.56±0.16 a-h 2.17±0.38 a-h 2.05±0.21 a-h 1.71±0.43 a-h 1.42±0.34 c-h 1.89±0.32 a-h 1.32±0.33 d-h 1.46±0.22 c-h 1.04±0.23 e-h 2.29±0.26 a-h 1.90±0.33 a-h 1.75±0.20 a-h 1.96±0.33 a-h 1.69±0.18 a-h 0.66±0.28 h 1.00±0.26 fgh 1.95±0.48 a-h 2.14±0.33 a-h 1.99±0.61 a-h 0.73±0.16 h 1.16±0.20 d-h 1.27±0.16 d-h 1.47±0.15 c-h 1.51±0.25 b-h 1.97±0.21 a-h 2.28±0.43 a-h 1.51±0.38 b-h 3.05±0.14 abc 2.32±0.23 a-h 1.38±0.41 c-h 2.46±0.27 a-g 0.055±0.017 a-g 0.085±0.010 a-f 0.115±0.016 ab 0.078±0.006 a-g 0.120±0.032 a 0.060±0.012 a-g 0.078±0.005 a-g 0.070±0.007 a-g 0.083±0.010 a-g 0.100±0.015 a-d 0.085±0.018 a-f 0.040±0.004 c-g 0.110±0.024 abc 0.048±0.013 a-g 0.093±0.016 a-e 0.080±0.004 a-g 0.055±0.012 a-g 0.085±0.012 a-f 0.073±0.017 a-g 0.060±0.020 a-g 0.068±0.005 a-g 0.078±0.012 a-g 0.050±0.008 a-g 0.043±0.005 b-g 0.068±0.015 a-g 0.060±0.008 a-g 0.050±0.017 a-g 0.038±0.011 c-g 0.055±0.013 a-g 0.035±0.012 d-g 0.043±0.008 b-g 0.025±0.006 efg 0.070±0.009 a-g 0.055±0.012 a-g 0.048±0.006 a-g 0.055±0.012 a-g 0.045±0.006 b-g 0.010±0.007 g 0.020±0.007 efg 0.053±0.017 a-g 0.055±0.012 a-g 0.055±0.022 a-g 0.015±0.005 fg 0.073±0.019 a-g 0.060±0.011 a-g 0.055±0.006 a-g 0.053±0.011 a-g 0.065±0.010 a-g 0.078±0.021 a-g 0.040±0.013 c-g 0.103±0.008 a-d 0.068±0.006 a-g 0.035±0.013 d-g 0.070±0.011 a-g 5.56±1.35 a-e 6.37±0.66 a-e 7.42±0.53 a-e 5.12±0.90 b-e 3.56±1.15 cde 5.52±0.75 a-e 6.36±2.39 a-e 5.14±0.53 b-e 4.26±1.56 cde 4.38±1.23 cde 3.73±0.92 cde 3.92±0.34 cde 4.62±1.09 b-e 4.77±0.50 b-e 7.37±1.50 a-e 2.40±0.18 de 4.91±0.84 b-e 3.15±1.05 cde 12.15±1.69 a 9.21±1.10 abc 9.03±2.00 a-d 11.12±2.35 ab 7.45±1.52 a-e 2.00±0.75 e 6.11±1.94 a-e 3.54±1.29 cde 2.34±0.79 de 1.58±0.66 e 3.60±0.45 cde 2.40±0.91 de 3.85±1.59 cde 1.62±0.82 e 2.94±1.13 cde 0.95±0.33 e 3.12±0.95 cde 5.18±2.88 b-e 1.02±0.33 e 1.12±0.42 e 1.04±0.17 e 2.62±1.25 cde 1.84±0.81 e 2.09±0.50 e 1.05±0.38 e 2.74±1.41 cde 1.60±0.83 e 2.44±0.94 de 1.29±0.63 e 2.76±1.06 cde 1.32±2.37 e 2.16±0.76 e 3.23±0.63 cde 2.69±1.10 cde 2.17±0.20 e 2.73±0.67 cde 6.97±1.79 bc 4.10±0.71 c-f 3.49±0.78 c-f 2.46±0.44 c-f 1.74±0.87 c-f 3.15±0.99 c-f 2.60±1.07 c-f 2.41±0.39 c-f 1.85±0.81 c-f 1.46±0.48 def 1.71±0.74 c-f 2.58±0.20 c-f 1.42±0.29 def 4.66±0.77 c-f 3.38±0.81 c-f 1.13±0.08 def 3.03±0.16 c-f 1.34±0.53 def 13.87±2.72 a 10.66±1.83 ab 6.38±1.54 bcd 5.99±1.61 b-e 4.82±1.37 c-f 1.25±0.44 def 3.38±1.36 c-f 1.80±0.66 c-f 1.31±0.16 def 0.99±0.24 def 2.18±0.55 c-f 2.32±1.06 c-f 2.70±0.89 c-f 2.93±2.22 c-f 1.19±0.40 def 0.53±0.16 f 1.71±0.41 c-f 2.78±1.82 c-f 0.59±0.17 ef 2.51±1.22 c-f 1.47±0.57 def 1.59±0.62 c-f 1.05±0.52 def 1.15±0.13 def 1.57±0.67 def 2.02±0.66 c-f 1.22±0.58 def 1.70±0.60 c-f 1.10±0.66 def 1.63±0.82 c-f 0.87±0.88 ef 1.78±0.69 c-f 1.08±0.22 def 1.11±0.36 def 2.10±0.70 c-f 1.15±0.29 def 27.30±0.45 s 30.83±0.19 q 32.38±0.19 mno 34.20±0.11 e-j 34.78±0.09 b-g 35.30±0.04 a-d 35.48±0.02 abc 35.63±0.02 ab 35.63±0.03 ab 35.15±0.03 a-d 34.65±0.06 c-g 34.60±0.00 c-g 34.40±0.07 d-i 29.23±0.29 r 30.83±0.18 q 31.68±0.11 opq 33.20±0.07 klm 33.45±0.03 jkl 26.68±0.49 s 29.25±0.37 r 31.93±0.30 op 33.63±0.31 i-l 35.23±0.19 a-d 34.60±0.04 c-g 34.68±0.08 c-g 34.75±0.03 b-g 34.70±0.00 b-g 34.68±0.08 c-g 34.78±0.09 b-g 34.98±0.08 a-g 34.05±0.09 g-k 34.08±0.03 g-k 34.10±0.04 f-k 34.18±0.05 e-j 34.18±0.02 e-j 34.48±0.25 d-i 34.48±0.06 d-i 34.58±0.06 c-h 35.05±0.06 a-e 35.80±0.04 a 35.45±0.06 abc 35.45±0.03 abc 34.75±0.12 b-g 26.93±0.38 s 29.40±0.25 r 31.05±0.13 pq 32.23±0.15 no 33.10±0.08 lmn 33.65±0.06 h-l 34.05±0.05 g-k 34.45±0.06 d-i 34.80±0.04 b-g 35.03±0.05 a-f 35.08±0.05 a-e means sharing similar letter in a column are statistically non-significant (p>0.05) screening of local germplasm for cultivation under heat stress conditions 343 in this investigation, pca was used in order to investigate how the different genotypes perform different under high temperature stress condition. the biplot generated for different traits like shoot length, root length, shoot fresh weight, root fresh weight, shoot dry weight, root dry weight, number of leaves, chlorophyll contents, sub-stomatal co2, transpiration rate, stomatal conductance, photosynthetic rate, water use efficiency and leaf temperature is given in fig. 1. the biplot generated for 54 genotypes is given in fig. 2. according to fig. 1, on the basis of leaf temperature (l tmp) genotypes v41, v50, v54, v48, v53, v43, v45, v26, v28, v30 and v42 are the most tolerant for high temperature as they lay in the same region in the biplot generated for the genotypes in fig. 2. on the other hand, genotypes v19, v22, v7, v21, v3 v18, v25 and v9 are most sensitive for leaf temperature as these genotypes lay in the opposite to the leaf temperature tolerant genotypes in the biplot in fig. 2. in the same way on the behalf on fig. 1, for photosynthesis (photo), water use efficiency (wue), root length (rl), stomatal conductance to water (st conductance) genotypes v19, v22, v7, v21, v3 v18, v25 and v9 are most heat tolerant on the basis of the traits because these fall in the same region in biplot for genotypes given in fig. 2. genotypes v41, v50, v54, v48, v53, v43, v45, v26, v28, v30 and v42 are most sensitive for these traits as these genotypes lay in the opposite to the photosynthesis (photo), water use efficiency (wue), root length (rl), stomatal conductance to water (st conductance) tolerant genotypes in the biplot in fig. 2. according to fig. 1, on the basis of shoot fresh weight (sfd), shoot dry weight (sdw), shoot length (sl), root fresh weight (rfw), root dry weight (rdw) genotypes v17, v23, v24, v27, v5 and v14 are most heat tolerant on the basis of the traits because these fall in the same region in biplot for genotypes given in the fig. 2. while genotypes v33, v35, v12, v38, v37, v44, v34, v8, v48, v52, and v13 are most sensitive for these traits as these genotypes lay in the opposite to these traits in the biplot for genotypes in fig. 2. in the similar way in fig. 1, for sub-stomatal conductance to co2, transpiration rate and chlorophyll content genotypes v33, v35, v12, v38, v37, v44, v34, v8, v48, v52, and v13 are most heat tolerant on the basis of the traits because these fall in the same region in biplot for genotypes given in the fig. 2. while genotypes v17, v23, v24, v27, v5 and v14 most sensitive for these traits as these genotypes lay in the opposite of these traits in the biplot for genotypes in fig. 2. sub-stomatal co2 was highest (tab. 2) at 1555.6 vpm for “uc134” and lowest for “roma”at 454.5 vpm. as revealed by studies conducted on co2 concentration and its relation with plant growth rate, the results have varied. some studies revealed co2 to have a positive effect on photosynthetic rates and plant tolerances to heat stress (faria et al., 1996; ferris et al., 1998; huxman et al., 1998; taub et al., 2000) whereas others reported the effect to be tab. 3: analysis of variance for individual trait (morphological traits) serial no trait significant level 01 shoot length ** 02 root length ** 03 shoot fresh weight ** 04 root fresh weight ** 05 shoot dry weight ** 06 root dry weight ** 07 no. of leaves ** ns = non-significant (p>0.05); * = significant (p<0.05); ** = highly significant (p<0.01) tab. 4: analysis of variance for individual trait (physiological traits) serial no trait significant level 01 chlorophyll content ** 02 sub-stomatal co2 ** 03 stomatal conductance to water ** 04 photosynthetic rate ** 05 transpiration rate ** 06 water use efficiency ** 07 leaf temperature ** ns = non-significant (p>0.05); * = significant (p<0.05); ** = highly significant (p<0.01) fig. 2: biplot graph of 54 genotypes under high temperature stress on the basis of various traits. fig. 1: biplot graph of 54 genotypes under high temperature stress for various traits. 344 a. nazir, m.r. shaheen, c.m. ayyub, r. hussain, n. sarwer, m. imran, m. aurangzaib, m. nawaz, m.f.a. khan, y. jawad, m. iqbal negative (roden and ball, 1996; huxman et al., 1998; taub et al., 2000). some studies suggested there to be no effect of co2 on photosynthetic rates. “uc-134” showing highest sub-stomatal co2 also had highest transpiration rate of 3.22 mmol/m2/s (tab. 2). “bush beef steak” with 0.66 mmol/m2/s had the lowest transpiration rates. greatest amount of sub-stomatal water was recorded for “sasha altai” with 0.120 whereas “bush beef steak” which had the lowest transpiration rate also had the lowest sub-stomatal water of 0.010. the highest photosynthetic activity was of “cohaus” with 12.15 μmol/ m2/s, whereas “nepoli” had the least photosynthetic rate of 0.95 μmol/m2/s (tab. 2). data collected for water use efficiency showed that “cohaus” had highest water use efficiency (13.87). least water use efficiency was recorded for “nepoli” (0.53).“naqeeb” showed highest leaf surface temperature of 35.08 °c, while the least leaf surface temperature was recorded for “cohaus” at 26.68 °c. after experimentation it was revealed that tolerant genotypes of tomato and sugarcane exhibited the tendency of increasing their chlorophyll a:b and decreasing chlorophyll carotenoid content (camejo et al., 2005). screening results showed that highest chlorophyll contents were recorded for “pakit” with a spad value of 49.03. “spekled sibrian” had the least chlorophyll contents of 14.85 followed by “campbells” with 16.15 spad value. conclusion it may be concluded from the present study that genotypes varied significantly for their heat tolerance potential and this variation can successfully be implied in breeding programs. the screening tests for 54 genotypes revealed that “parter improved” and “legend” were the most tolerant genotypes whereas “grus chovka” and “nepoli were susceptible to heat stress. acknowledgements we are very thankful to ayub agriculture research institute faisalabad (aari) and 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article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 88, 234 240 (2015), doi:10.5073/jabfq.2015.088.034 1 department of environment, ecology and plant sciences, stockholm university, stockholm, sweden 2 department of land and water resources engineering, royal institute of technology, stockholm, sweden influence of silicon on arsenic uptake and toxicity in lettuce maria greger1*, claes bergqvist1, arifin sandhi1, 2, tommy landberg1 (received november 20, 2014) * corresponding author summary lettuce grown in soil is found to contain high concentrations of arsenic (as). this paper investigates the uptake and speciation of as in lettuce as well as the influence of silicon (si) on as uptake, since si may decrease it. lettuce plants were cultivated in nutrient solution containing arsenite or arsenate with or without silicate. the uptake and distribution of as between roots and shoots, as accumulation in cell walls, as speciation, and toxic effects on growth were analysed. results indicate that arsenite was more toxic to lettuce than was arsenate. silicate decreased arsenate toxicity but had little effect on arsenite toxicity. in contrast, si decreased arsenite uptake more than arsenate uptake. the concentration of arsenate was higher than that of arsenite in the plants independent of the as species added. when arsenate was added, the as concentration in shoots was half of that in the roots and this distribution did not change with si addition. when arsenite was added, approximately 10 % of as was found in the shoots and 90 % in the roots; this pattern changed in the presence of si, and as became evenly distributed in the plant. in both roots and shoots, approximately 40 % of the as was found in the cell wall fraction; when arsenite was added, the presence of si increased this fraction to 47 %, but only in the shoots. the extraction efficiency when analysing the as species was lower in shoots than in roots, especially in the presence of arsenite and si. the opposite was found for as concentration in pellets after extraction. this indicated variation in the binding strength of arsenite and arsenate between roots and shoots and between siand non-sitreated plants. introduction arsenic (as) is ubiquitous in nature due to natural causes and anthropogenic activity. arsenic is toxic to humans and may be taken into the human body via polluted waters and food (bundschuh et al., 2012). crop plants accumulate as and lettuce (lactuca sativa) accumulates high as levels in the edible parts compared with other crops (greger, 2006; bergqvist et al., 2014). arsenic exists in various species in nature, the inorganic species arsenite and arsenate being predominant (sadiq, 1997). in water or waterlogged systems, arsenite is the predominant as species, while arsenate is more common in terrestrial systems with higher redox potential (sadiq, 1997). small amounts of methylated arsenic species such as methylarsonic acid (mma) and dimethylarsinic acid (dma) may also be found due to microbiological activity (wood, 1974). plants mainly contain the inorganic as species arsenate and arsenite (smith et al., 2008). several plant species also contain methylated as species (bergqvist and greger, 2012). the levels of methylated as are usually low (raab et al., 2007), and some argue that methylated as is not formed in plants but is taken up from the surroundings (lomax et al., 2012). arsenic is generally considered toxic to plants, such as lettuce (sturchio et al., 2011). the toxicity, however, differs between as species. to most organisms, inorganic as species are regarded as more toxic than organic as species, and of the inorganic as species, arsenite is considered more toxic than arsenate (meharg and hartley-whitaker, 2002). plants generally contain small amounts of as, especially in their aboveground parts, possibly due to restricted uptake and trans location from roots to shoots (wang et al., 2002). however, some plants, such as the radish (raphanus sativus), when cultivated hydroponically, accumulate higher concentrations of as in the shoots than the roots (smith et al., 2008). arsenite-sulphur compounds localized in the phloem could account for the high distribution to shoots in the radish (smith et al., 2008). much of the as in plants is bound in the apoplasmic cell-wall fraction, and 30-60 % was found in the apoplasm of rice (oryza sativa) and cretan brake (pteris cretica) (bravin et al., 2008; feng et al., 2011). cellular uptake of arsenate is thought to occur through the high-affinity phosphate transporters in terrestrial plants (moreno-jiménez et al., 2012), while cellular uptake of arsenite occurs through aquaporins used for glycerol and silicon (bienert et al., 2008). the as accumulated in the cytoplasm is generally thought to be reduced to arsenite, bound to phytochelatins, and transported further into the vacuole (moreno-jiménez et al., 2012). some plants additionally display arsenite cellular efflux ability when growing in an as-containing medium (bienert et al., 2008). silicon is one of the most abundant elements on earth, but most of it has low availability to plants. although si is beneficial for some plant species, such as rice, bamboo, and sugar cane, its essentiality to other plants has been questioned (epstein, 2009). silicon was found to prevent the toxicity and decrease the uptake of toxic elements in plants (treder and cieslinski, 2005). silicon may also alleviate the negative effects of as on plants; for example, in rice, si inhibits the uptake of as, possibly due to the competition between silicic acid and arsenite (bogdan and schenk, 2008). such effects, however, are not seen in the as hyperaccumulator chinese brake (pteris vittata), whose uptake mechanisms and routes differ from those of non-accumulators (wang et al., 2010). lettuce is not a si-accumulator and not an as hyperaccumulator. the influence of si on as uptake and toxicity to lettuce is therefore difficult to predict from data on si-accumulators such as rice and as accumulators such as pteris vittata. crops are cultivated on as rich soils, such as fertile alum shale soils, and lettuce is a popular vegetable to be cultivated. it is thus im portant to elucidate the influence of si as an agent to decrease as uptake in lettuce. the aim of the study was therefore to investigate the effect of si on the toxicity, accumulation, and speciation of as in lettuce (lactuca sativa) when as was added as arsenate and arsenite. the hypothesis was that si would decrease the arsenic uptake by, accumulation in, and toxicity to lettuce. the effect would likely be greatest when arsenite was added. arsenite and silicate are thought to have the same uptake site on cell membranes; because si decreases the cellular entrance of arsenite, a higher as level would be found in the apoplasm. more as would therefore accumulate in roots in relation to shoots with the addition of si. by means of these effects, as toxicity will likely decrease with si addition. silicon effect on arsenic in lettuce 235 material and methods plant material seeds of lettuce (lactuca sativa l. cv amerikanischer brauner) were germinated in vermiculite for approximately 10 days until the plants were 1 cm tall. the plants were then transferred to 25 %-strength nutrient medium; 100 % medium contained the following concentrations of nutrients: 10 mm kno3, 1.5 mm kh2po4, 3.0 mm ca(no3)2, 2.0 mm mgso4, 0.5 mm (nh4)2 hpo4, 25 μm fecl3, 18.8 μm naedta, 10 μm h3bo3, 16 μm mncl2, 0.16 μm cuso4, 0.35 μm znso4, and 0.21 μm na2moo4. the ph was 6.4. when the plants were 10 cm tall, they were transferred to 50 %-strength nutrient medium, three plants per 1-l black pot. the nutrient solution was changed every week. two weeks later, the plants were used in the toxicity tests and uptake experiments. plants were grown in a climate chamber with a 16 h light / 8 h dark regime at 23 °c / 19 °c and a photon flux density of 270 μmol m–2 s–1 during the light period. the humidity was 80 %. toxicity test the plants were weighed and two plants were transferred to each 1-l pot containing 50 %-strength nutrient solution. the solution was supplemented with various concentrations (i.e., 0, 0.1, 1, 10, 50, 100, 500, 1000, or 5000 μm) of arsenite (naaso2) or arsenate (na2haso4 × 7h2o), with or without 1 mm k2sio3 added. the silicate concentration was chosen from a pilot study were 0-5 mm k2sio3 and 0-10000 μm arsenate or arsenite was used under the same condition as described above. after five days treatment plants were divided into roots and shoots and weighed, dried at 80 °c for 48 hrs, and weighed again. the ph of the solution at the end of the five days of treatment was measured to be approximately 6.0 without and 6.5 with si; four replicates were used. arsenic uptake two plants were transferred to each 1-l pot containing 50 %-strength nutrient solution. after 24 h, the solution was replaced with a new nutrient medium containing 10 μm arsenite or arsenate in combination with 0 or 1 mm k2sio3 added and treated during four days. the plants were then harvested and total as was measured in the whole tissue and in the cell wall fraction of the roots and shoots. in addition, the as speciation was analysed in the tissue fraction. this experiment was performed in four replicates. cell walls were prepared according to lozano-rodriguez et al. (1997) as follows. plant material was first homogenized in liquid nitrogen using a mortar, and then ultra-mixed using a polytron pt2000 homogenizer (kinematica ag, luzern, switzerland) in extraction buffer containing 500 mm sucrose, 50 mm hepes, 1 mm sodium dithionite, 5 mm ascorbic acid, and adjusted to ph 7.5. the sample was centrifuged at 500 g for 10 min. the pellet was used for as analysis of cell walls. short term arsenic uptake two times 1 ml samples were taken from 1-l pots containing 50 %-strength nutrient solution in a combination of 1 μm arsenate or arsenite with or without 1 mm k2sio3 added. one plant was placed in each1-l pot and 2 times 1 ml samples were taken after 10, 20, 30, 60 min, 2, 4, 6 and 24 hours. the total content of as in the samples was analysed in the samples using an atomic absorption spectrophotometer (spectraa 55b, varian inc., palo alto, ca) with the vapour generation technique (vga-77, varian inc.). sodium borohydride (3 %; merck, whitehouse station, nj), sodium hydroxide (2.5 %; eka chemicals), and hydrochloric acid (6m; vwr international, radnor, pa) were used for hydride generation. standards of as were added to the samples to eliminate matrix interaction effects. the detection limit was 7 μg as l–1. the total as uptake was calculated based on plant fresh weight and the as content in the solution samples. five replicates were used. analysis of arsenic cell walls and whole tissue of roots and shoots were dried at 80 °c for 48 hrs. the material was then wet digested in concentrated hno3:hclo4, (7:3 v:v). the total content of as in the wet digested plant material, and solution from the short term uptake study, was analysed using an atomic absorption spectrophotometer. arsenic was extracted from plant material to analyse as speciation according to a modified mir et al. (2007) method. approximately 0.5 g dw of air-dried plant material was put in 50-ml falcon tubes and then ultra-mixed using a polytron pt2000 homogenizer (kinematica ag) at maximum speed for approximately 10 s in 10 ml of meoh:h2o (1:1). the tubes were then placed in a sonicator (transenic digital s; elma, singen, germany) for 10 min followed by centrifugation at 3000 × g for 10 min. the supernatant was extracted into a new 50-ml falcon tube. to each 50-ml falcon tube, 10 ml of meoh:h2o (1:1) was added to the pellets for resuspension and the extraction procedure was repeated. in total, this extraction procedure was performed twice, rendering 20 ml of meoh:h2o (1:1) solution. the same procedure was then repeated two additional times with 0.1% hcl, rendering 20 ml of hcl solution. the volume of the extracted meoh:h2o (1:1) solution was reduced at 60 °c and resuspended in deionized h2o to 3 ml to remove meoh and to concentrate the low levels of organic as before analysis. in the case of hcl, the extracted solution was also concentrated to 3 ml. arsenic species in samples were separated using ion chromatography (ic) with a prp x-100 (250 × 4.6 mm) anion exchange column (hamilton, reno, nv). before injection of 100 μl of solution, samples were filtered through a 0.22-μm filter. the eluent was ammonium-phosphate buffer (ph 5.8) with a flow rate of 1 ml min–1. the atomic absorption spectrophotometer (spectaa 55b, varian inc.) vapour generation technique (vga-77, varian inc.) was used for peak detection. sodium borohydride (3 %; merck), sodium hydroxide (2.5 %; eka chemicals), and hydrochloric acid (6m; vwr international) were used for hydride generation. peak amounts of as were quantified by means of external calibration with standard solutions of arsenate, arsenite, mma, and dma using the following chemicals: sodium arsenate dibasic heptahydrate (sigma-aldrich, st. louis, mo) for arsenate, sodium-meta-arsenite (merck) for arsenite, sodium methylarsonate (sigma-aldrich) for mma, and cacodylic acid (sigma aldrich) for dma. spectraa worksheet oriented aa software, version 5.1 (varian inc.) was used to detect the peaks, and a formula devised by burriel-marti et al. (1968) was used to calculate peak area and implemented in microsoft office excel. analysis of silicon roots and shoots of plants treated five days in nutrient medium added 0 and 1 mm k2sio3 combined with 0 and 10 μm arsenite or arsenate, originating from the toxicity experiment, were dried at 80 °c for 48 hrs. the material was then wet digested in concentrated hno3:hclo4, (7:3 v:v). the total content of si in the wet digested plant material was analysed using atomic absorption spectrophotometty (varian spectraa 55b) with furnace. calculations and statistics the dose-response and sensitivity of various as and silicon combinations in the external medium were evaluated using the modified weibull function (1) according to taylor et al. (1991). this was 236 m. greger, c. bergqvist, a. sandhi, t. landberg done for the increase in the fresh weight of the whole plants over five days. y = a + b exp[– (x/c) d ] (1) where y is the dependent variable, yield; x is the independent variable, the concentration of metal in the growth solution; a is the absolute minimum growth; b is the magnitude of the uninhibited growth response above the absolute minimum growth; and c and d indicate the shape of the curve, with parameter c altering the scaling on the x-axis and parameter d affecting the skewness of the dose-response (taylor et al., 1991, 1992). the modified weibull function provides direct estimates of some biological parameters of toxicity. these parameters are maximum unit toxicity (utmax), defined as the maximum reduction in growth per unit of concentration of arsenic (2), and the empirical toxicity threshold (tt95b), which is the concentration of arsenic that results in a yield reduction of 5% (3) (taylor et al., 1991). when d was <1, the utmax was calculated as dy/dx at tt95b (taylor et al., 1991): utmax = bd/cd exp[–(d –1)/d] c(d –1) [(d –1)/d][(d –1)/d] (2) tt95b = c[(– ln 0.95)(1/d)] (3) when comparing two values, a higher tt95b and a lower utmax indicate that a plant is less sensitive to arsenic than is a plant with a lower tt95b and a higher utmax. the concentration of as that induced a 50 % reduction in growth increase was also calculated (i.e., ec50 values). the student t-test and anova were used to detect differences between the treatments and linear regression was used to find significant differences between lines. significance levels at p ≤ 0.05 were calculated using the statistical program jmp (version 10.0, 2012; sas institute inc., cary, nc). results arsenic affected the growth of lettuce to different extents depending on the as species added. arsenite produced a more severe effect and inhibited the plant-growth increase at lower concentrations than did arsenate (fig. 1, tab. 1). the lower tt95b and ec50 and higher utmax values for arsenite than for arsenate indicate this. plants died when treated with concentrations higher than 50 μm arsenite but survived arsenate at concentrations as high as 1000 μm (not shown). the dry weight:fresh weight (dw:fw) ratio increased with increasing as addition, as fresh weight was more affected than was dry weight (tab. 2). the shoot:root ratio (based on fresh weight) did not change with arsenate addition, but decreased slightly with high arsenite additions (tab. 2). addition of si decreased the effect of arsenate on growth more than that of arsenite, and the presence of si increased tt95b and ec50 but decreased utmax (tab. 1). the si effect was especially pronounced in the as concentration range of 10 -100 μm (fig. 1, tab. 2). although si influenced the effect of as on fresh and dry weight, it did not influence the change in dw:fw ratio or shoot:root ratio caused by as (tab. 2). lettuce accumulated more as in roots than in shoots (tab. 3), independent of whether as was added as arsenite or arsenate. the [as] shoot:[as]root ratio was lower when as was added as arsenite than as arsenate. when adding si to the arsenite treatment, however, the as concentration increased in shoots and decreased in roots compared with the treatment without si. no such si effect was found in the arsenate case. the same concentrations found in plant tissue were also found in the cell wall fraction. approximately 40 % of as in the plant tissue was found in the cell walls in both roots and shoots. however, when si was added to the arsenite treatment, a larger percentage, 47 %, of as was bound to the cell walls in shoots. the si concentration in lettuce roots increased about 30 % when arsenite or arsenate was added but not si (fig. 2). similar effect was found in shoots, but only when arsenite was added. in the presence of 1 mm si, however, the si concentration in both roots and shoots decreased upon addition of arsenite or arsenate. this decrease was larger in shoots than in roots and as species added did not influence the magnitude. the short-term uptake of as is shown in fig. 3. in the first 30 min, when apoplasmic uptake occurs, treatment with arsenite + si resulted in a lower uptake rate and the curve was saturated at a lower internal as concentration than in the other treatments. in the later part of the curve, showing symplasmic uptake, less as was taken up from the arsenite solution than the arsenate solution; furthermore, when si was added, less as was taken up from either the arsenate or arsenite solution. the effects of various treatments on as speciation in plant tissue are presented in tab. 4. in all cases, arsenite and arsenate were found in tab. 1: assessing response of fresh weight increase of lettuce to various combinations of arsenite or arsenate with potassium silicate using the modified weibull function (see calculations and statistics). treatment weibull parameter tt95b ec50 utmax a b c d r2 (μm as g-1 fw) (μm as g-1 fw) (%) arsenate 0.505 1.095 67 1.01 0.455 3.53 46.5 0.016 arsenate + si 0.170 1.095 615 1.01 0.685 32.50 428.0 0.002 arsenite – 0.406 1.953 42 1.01 0.585 2.25 29.5 0.044 arsenite + si – 0.368 1.743 66 1.01 0.684 3.47 45.8 0.025 fig. 1: fresh weight decrease in relation to control of lettuce over five days of treatment with arsenite or arsenate in the presence or absence of 1 mm potassium silicate using modified weibull function; ±se is included in the figure, n = 4. silicon effect on arsenic in lettuce 237 ta b. 2 : d ry w ei gh t ( d w ), dr y w ei gh t:f re sh w ei gh t r at io (d w :f w ) o f w ho le p la nt s, a nd s ho ot :w ho le p la nt ra tio b as ed o n fr es h w ei gh t o f l et tu ce a ft er tr ea tm en t f or fi ve d ay s w ith v ar io us c on ce nt ra tio ns o f a rs en ite or a rs en at e w ith o r w ith ou t 1 m m p ot as si um s ili ca te ; n = 4 , ± se . c ha ng e in w ei gh t w ith a s ad di tio n, % d w :f w r oo t:s ho ot a s, a rs en at e a rs en at e a rs en ite a rs en ite a rs en at e a rs en at e a rs en ite a rs en ite a rs en at e a rs en at e a rs en ite a rs en ite µm + si + si + s i + si + si + si d w fw d w fw d w fw d w fw 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 5. 58 ± 0 .6 9 5. 89 ± 0 .4 6 5. 58 ± 0 .6 9 5. 89 ± 0 .4 6 82 .9 ± 1 .2 4 82 .0 ± 1 .0 1 82 .9 ± 1 .2 4 82 .0 ± 1 .0 1 0. 1 93 80 10 4 90 95 76 99 91 5. 15 ± 0 .3 2 7. 15 ± 0 .8 2 5. 98 ± 0 .3 3 5. 38 ± 0 .1 2 79 .9 ± 0 .3 5 79 .2 ± 1 .7 0 79 .1 ± 1 .9 7 82 .6 ± 0 .2 0 1 95 83 98 82 94 75 10 8 11 8 6. 26 ± 0 .8 3 6. 71 ± 0 .8 3 5. 82 ± 0 .3 4 5. 91 ± 0 .0 6 77 .9 ± 1 .8 6 81 .0 ± 1 .3 5 82 .5 ± 0 .7 9 83 .5 ± 0 .8 6 10 88 54 10 3 10 7 98 77 96 57 6. 26 ± 0 .3 2 5. 82 ± 0 .7 3 6. 57 ± 0 .4 9 8. 34 ± 0 .1 6 77 .9 ± 1 .8 4 80 .5 ± 1 .2 7 82 .2 ± 1 .3 7 85 .6 ± 0 .9 6 50 90 57 10 5 91 88 43 91 42 6. 77 ± 0 .4 5 7. 32 ± 0 .2 4 8. 11 ± 0 .3 2 8. 47 ± 0 .5 8 82 .9 ± 1 .2 2 78 .1 ± 0 .9 2 81 .9 ± 1 .7 2 84 .0 ± 0 .2 9 10 0 92 58 98 62 81 24 91 45 6. 85 ± 0 .3 2 8. 46 ± 0 .5 6 8. 40 ± 0 .2 2 8. 32 ± 1 .3 8 78 .9 ± 1 .5 9 79 .1 ± 1 .2 7 81 .9 ± 1 .4 6 83 .2 ± 1 .3 1 50 0 91 51 10 3 80 85 26 82 20 7. 74 ± 0 .9 0 8. 08 ± 0 .4 7 11 .1 1 ± 0. 71 8. 02 ± 0 .4 0 83 .4 ± 0 .4 8 82 .7 ± 1 .8 6 78 .7 ± 1 .2 5 81 .0 ± 1 .1 3 10 00 95 52 93 47 84 27 90 31 9. 02 ± 1 .1 8 8. 33 ± 0 .6 4 10 .2 2 ± 0. 49 10 .8 8 ± 1. 32 79 .3 ± 2 .4 1 79 .9 ± 1 .1 6 76 .0 ± 0 .7 6 77 .8 ± 0 .6 9 50 00 95 59 89 38 79 17 80 12 8. 15 ± 0 .6 3 8. 23 ± 0 .0 2 9. 59 ± 1 .1 8 9. 85 ± 2 .4 6 82 .1 ± 1 .4 3 76 .1 ± 2 .3 9 73 .5 ± 3 .6 9 74 .0 ± 2 .9 8 both plant parts. independent of the as species added, arsenate predominated in both roots and shoots. the arsenite:arsenate concentration ratio was higher when arsenite was added, and this ratio was higher in shoots than in roots. the addition of silicon decreased the arsenate and arsenite concentrations in both roots and shoots, either significantly or as a tendency. adding silicon did not change the ratio between arsenite and arsenate in either roots or shoots. monomethylarsonic acid was detected in both the absence and presence of si, but only when arsenate was added (tab. 4). silicon addition decreased the mma concentration in both plant parts. to determine whether the mma concentration in plants was due to bacterial activity in nutrient medium containing arsenate, sterilized and unsterilized media without plants were analysed after four days. the results indicated that the unsterilized solution contained 0.03 ± 0.006 (se) μm mma, while the sterilized solution contained no mma (not shown). arsenite or arsenate concentrations did not differ between the sterilized and unsterilized solutions and no dma was detected. no dma was found in the plants after the various treatments (tab. 4). the extraction efficiency for the as species analysis of lettuce tissue was higher in roots (≥ 60 %) than in shoots (3145 %; tab. 4). the addition of si reduced the extraction efficiency in shoots but significantly only in the shoots of arsenite-treated plants, where the efficiency decreased from 45 % to 13 %. during extraction for as species analysis, a pellet fraction was formed as a by-product. when si was added, a higher percentage of as was found in this pellet fraction for the shoots of arsenite-treated plants. discussion this study demonstrated that silicon influences the toxicity, accumulation, and speciation of as in lettuce (lactuca sativa). as suggested by the hypothesis, the uptake of as, as both arsenite and arsenate, decreased when si was present (fig. 3). arsenite, arsenate, and silicate may interact with each other at uptake into the root tissue. the interaction between arsenite and silicate was likely strongly antagonistic in the cell wall sites, since the apoplasmic uptake of as, as shown in the first 30 min in fig. 3, was significantly affected by si only when as was added as arsenite. the ionic interactions in the cell walls resulting in the decreased arsenite accumulation in the apoplasmic compartments could have been due to si-induced secondary cell wall modifications (yamamoto et al., 2012), for example, of as binding functional groups (vithanage et al., 2012), reducing the binding affinity for arsenite. low cell wall binding may increase the efflux of arsenite from roots, as suggested by bienert et al. (2008). in addition, lack of binding in root apoplasm may increase as translocation to shoots, increasing the as concentration in the shoots and reducing it in roots (tab. 3). at the same time as addition decrease the translocation of si to the shoot; the decrease of si concentration of shoots more than that of roots (fig. 2), showing that interaction between si and as occurs in both directions. the effect of si on the cellular uptake of as (shown after 30 min in fig. 3) was, on the other hand, similar for arsenite and arsenate. silicate might therefore interact with arsenite at aquaglyceroporins, i.e., the site of both arsenite and silicon uptake (meharg and jardine, 2003; bienert et al., 2008), and with arsenate at the high-affinity phosphate transporters (guo et al., 2007; moreno-jiménez et al., 2012). after being taken up, as distribution in the plants depended on the as species added and on whether or not si was present. most as was found in roots, especially when added as arsenite (tab. 3). it is known that arsenite is taken up by the root cells, where it is detoxified by being bound to phytochelatins, while arsenate may be translocated further to the shoots (meharg and hartley-whitaker, 2002). for this reason, more as may be found in the shoots when 238 m. greger, c. bergqvist, a. sandhi, t. landberg in the arsenate concentration, nor in the arsenite concentration, in shoots in the presence of si (tab. 4). adding si did not result in any changes in the ratio between arsenite and arsenate in either roots or shoots compared with the non-silicon treatments. this suggests that enzymes responsible for arsenate/arsenite metabolism (morenojiménez et al., 2012) were unaffected by si. the change in as concentration in shoots, but no change in arsenite or arsenate, might therefore depend on the increase of some other form of as in the shoots, for example, mma or dma. dimethylarsinic acid (dma) was, however, not found at all in lettuce (tab. 4). methylarsonic acid (mma) was only found in roots and shoots when as was added as arsenate but not as arsenite, and the mma concentration decreased in the presence of si (tab. 4). the mma originated from the unsterilized nutrient solution (not shown) and was unlikely produced in the plant itself. therefore, the formation of dma or mma could not contribute to the increased as concentration in shoots after si addition. one possibility of si increases as concentration in shoot at arsenite addition, but no change in arsenite and arsenate, is that the addition of si may decrease the ability of as to be extracted from the plant material. this is seen by the lower extraction efficiency in the arsenite + si-treated lettuce (12 %) than in the non-arsenite + si-treated lettuce (44 %; tab. 4). thus, as would be more bound to the shoot material in the presence than in the absence of si. when analysing the different as species, the extraction efficiency was lower in shoots than in roots, meaning that the binding of as to the tissue likely differs between roots and shoots. pellets remained from the extraction fig. 2: silicon concentration in root and shoot of lettuce after cultivation for five days in 0 and 1 mm potassium silicate with or without 10 μm arsenite (asiii) or arsenate (asv); n = 9, ±se. * indicate significant difference from control. fig. 3: uptake of as by lettuce over 24 h from nutrient medium containing 1 μm arsenite or arsenate with or without 1 mm potassium silicate; n = 5, ±se. tab. 3: arsenic concentration in whole tissue and cell walls of roots and shoots of lettuce after treatment with 10 μm arsenite or arsenate with or without 1 mm potassium silicate for four days; n = 4, ±se. treatment as concentration, μg gdw-1 as in cell wall fraction, % whole tissue cell wall root shoot root shoot root shoot arsenate 1260 ± 106a 508 ± 54c 1255 ± 53s 511 ± 37u 40.7 ± 6.04 35.6 ± 2.06 arsenate + si 1082 ± 220a 404 ± 28c 1089 ± 199s 456 ± 47u 37.0 ± 2.76 38.9 ± 1.45 arsenite 1131 ± 250a 179 ± 38d 1017 ± 211s 196 ± 35v 34.4 ± 1.41 38.8 ± 3.87 arsenite + si 560 ± 51b 488 ± 43c 547 ± 66t 581 ± 32u 38.2 ± 2.36 47.2 ± 3.57 different letters indicate significant differences between values in each column. added as arsenate than as arsenite, which was the case in this study (tab. 3). silicon affected the distribution of as between roots and shoots in lettuce when added as arsenite, but not as arsenate (tab. 3). much more as was translocated to the shoots in the presence than in the absence of si, resulting in similar concentrations in shoots and roots in the case of arsenite (tab. 3). silicate interaction might have inhibited the cellular uptake via aquaporines of arsenite consigning arsenite to instead be translocated to the shoot. silicate might have promoted the transformation of arsenite to arsenate in roots, which was thereafter translocated to shoots. however, there was no change silicon effect on arsenic in lettuce 239 tab. 4: concentration of various as species (μg g–1 dw) in roots and shoots of lettuce after treatment with 10 μm arsenite or arsenate with or without 1 mm potassium silicate for four days. indicated here is as in pellets left after extraction for as species in relation to total as in tissue as well as the extraction efficiency of arsenite species; n = 4, ±se, nd = not detected. treatment arsenate arsenite mma dma extraction as in pellet efficiency, % in relation to total as in tissue, % roots arsenate 1226 ± 50a 40.9 ± 1.34a 50.8 ± 2.38a nd 58.0 ± 4.5a 6.9 ± 1.5ab arsenate + si 1109 ± 21a 29.9 ± 3.01b 43.7 ± 2.05a nd 65.7 ± 10.4ab 22.1 ± 10.2abc arsenite 1145 ± 74a 56.8 ± 0.43c nd nd 70.3 ± 19.5ab 8.0 ± 1.4ab arsenite + si 928 ± 61b 42.9 ± 4.98a nd nd 96.2 ± 6.7b 4.8 ± 1.7b shoots arsenate 180 ± 15a 43.0 ± 4.09a 49.7 ± 1.38a nd 31.6 ± 4.6ab 45.6 ± 8.0c arsenate + si 92 ± 11b 21.3 ± 2.73b 27.9 ± 4.13b nd 20.1 ± 2.9bc 31.4 ± 7.6c arsenite 70 ± 3b 44.1 ± 0.28a nd nd 44.6 ± 10.4a 29.8 ± 10.7cb arsenite + si 61 ± 8b 40.0 ± 3.30a nd nd 12.6 ± 1.2c 83.9 ± 12.1d different letters indicate significant differences between values in the same column. procedures before the analysis of arsenite and arsenate, and the as concentration in the pellets in relation to the total concentration in the tissue was higher in shoots than in roots (tab. 4). in addition, in the shoots of arsenite-treated plants, the share of as in the pellet fraction in relation to the as in the tissue was even higher when si was present (tab. 4). the same trend was found for as in the cell wall fraction (tab. 3). one suggested explanation of this is that the pellet material originated largely from the cell walls and that si promoted a tighter binding of as to the cell walls, possibly by modifying functional groups in the walls responsible for as binding (vithanage et al., 2012), thereby increasing the as concentration in the pellets. in this study, we demonstrated that as was toxic to lettuce at elevated concentrations, and that arsenite was more toxic than arsenate (fig. 1, tab. 1 and 2). the latter has been demonstrated for other plant species as well (meharg and hartley-whitaker, 2002). less arsenite than arsenate was distributed to the shoots, likely to prevent the toxic effects of arsenite on the photosynthetic apparatus, which has also been seen in cucumber (cucumis sativus; uroic et al., 2012). one would think from the uptake data, indicating that si influenced the uptake of arsenite more than that of arsenate (fig. 3), that si would have influenced the toxicity of arsenite more than that of arsenate. however, the opposite was found. silicate decreased the toxic effects of both arsenite and arsenate, but most efficiently decreased the toxicity caused by arsenate (fig. 1, tab. 1 and 2). one reason why si generally decreased the arsenate and arsenite effect on growth was that si decreased the net as uptake (fig. 3), as also demonstrated by bogdan and schenk (2008). in addition, si may increase antioxidant activities in plants, alleviating the negative effects of reac tive oxygen species (liu et al., 2009). silicon would decrease the toxicity of arsenate more than that of arsenite because, when arsenate was added, both arsenite and arsenate concentrations decreased in both roots and shoots, while with arsenite addition, the concentration of both species decreased only in roots in the presence of si (tab. 3). in the latter case, therefore, the effect on photosynthesis would be the same as without added si. in addition, si might decrease the reactivity of the arsenate molecule. we conclude that silicon can be used to decrease as uptake by lettuce, and thus, reducing toxic effects on the plant and, thus, in lettuce-crops. lowered uptake also reduces the arsenic health-risk in lettuce as human food. during lettuce production, arsenate will be the major problem, while in solution culture, arsenite could be an additional problem due to the lower redox potential than in terrestrial conditions. in addition, cultivation in soil might include si effects on the binding of as to soil colloids, which would in turn influence the as uptake (seyfferth and fendorf, 2012). lettuce has a relative high uptake of arsenic compared to many other crop plants (mcbride, 2013) but there are no international accepted limit for arsenic in food. there are suggestions eg. 200 mg/kg and 15 μg/ kg b.w (efsa, 2009). in field, lettuce can reach these levels and silicon would therefore be a useful tool to reduce as in food crops. acknowledgements this work was funded by the knut and alice wallenberg foundation and the swedish research council. we would like to acknowledge lillvor wikander for assisting with the experiments. references bergqvist, c., greger, m., 2012: arsenic accumulation and speciation in plants from different habitats. appl. geochem. 27, 615-622. bergqvist, c., herbert, r., persson, i., greger, m., 2014: plants influence on arsenic availability and speciation in the rhizosphere, roots and shoots of three different vegetables. environ. pollut. 184, 540-546. bienert, g.p., thorsen, m., schüssler, m.d., nilsson, h.r., wagner, a., tamás, m.j., jahn, t.p., 2008: a subgroup of plant aquaporins facilitate the bi-directional diffusion of as(oh)3 and sb(oh)3 across membranes. bmc biol. 6, article no. 26. bogdan, k., schenk, m.k., 2008: arsenic in rice (oryza sativa l.) related to dynamics of arsenic and silicic acid in paddy soils. environ. sci. technol. 42, 7885-7890. bravin, m.n., travassac, f., le floch, m., hinsinger, p., garnier, j.m., 2008: oxygen input controls the spatial and temporal dynamics of arsenic at the surface of a flooded paddy soil and in the rhizosphere of lowland rice (oryza sativa l.): a microcosm study. plant soil 312, 207-218. bundschuh, j., nath, b., bhattacharya, p., liu, c.w., armienta, m.a., moreno lópez, m.v., lopez, d.l., jean, j.s., cornejo, l., lauer macedo, l.f., filho, a.t., 2012: arsenic in the human food chain: the latin american perspective. sci. total environ. 429, 92-106. 240 m. greger, c. bergqvist, a. sandhi, t. landberg burriel-marti, f., condal-bosch, l., gassiot-matas, m., 1968: chronometrical method for calculation of chromatographic peak areas. chromatographia 1, 507-509. efsa, 2009: scientific opinion on arsenic in food. efsa panel on contaminants in the food chain (contam). efsa j. 7(10), 1351. epstein, e., 2009. silicon: its manifold roles in plants. ann. appl. biol. 155, 155-160. feng, r., wei, c., tu, s., tang., s., wu, f., 2011: simultaneous hyperaccumulation of arsenic and antimony in cretan brake fern: evidence of plant uptake and subcellular distributions. microchem. j. 97, 38-43. greger, m., 2006: metallupptag i växter odlade i rödfyroch alunskifferjord. 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environ. exp. bot. 68, 222-229. wood, j.m., 1974: biological cycles for toxic elements in the environment. science 183, 1049-1052. yamamoto, t., nakamura, a., iwai, h., ishii, t., ma, j.f., yokoyama, r., nishitani, k., satoh, s., furukawa, j., 2012: effect of silicon deficiency on secondary cell wall synthesis in rice leaf. j. plant res. 125, 771-779. address of the corresponding author: e-mail: maria.greger@su.se © the author(s) 2015. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 91, 8 13 (2018), doi:10.5073/jabfq.2018.091.002 1forestry, wildlife and fisheries department, government of the punjab, lahore, pakistan 2institute of geology, university of the punjab, lahore, pakistan 3department of botany, university of the punjab, lahore, pakistan 4department of zoology, university of gujrat, gujrat, pakistan 5department of agricultural sciences, university of haripur, haripur, pakistan phytoremediation of arsenic-contaminated soils by eucalyptus camaldulensis, terminalia arjuna and salix tetrasperma firoz ud din ahmad1, nasir ahmad2, khan rass masood3, mubashar hussain4*, muhammad faheem malik4, abdul qayyum5 (submitted september 23, 2017, accepted january 2, 2018) * corresponding author summary rising levels of arsenic in ground water posing threats to the lives of millions of people residing in the indus plains, whereas the magnitude of the risk is alarming that needs to control arsenic from leaching down into the ground water becomes essential. the study was designed to assess the potential of three tree seedlings to reclaim the arsenic-affected soils in pakistan and determining the impact of arsenic on growth parameters and its accumulation in tree seedlings. the experiment conducted at the botanical garden, university of the punjab, lahore, revealed that eucalyptus camaldulensis, termina lia arjuna and salix tetrasperma showed varying adaptability to survive under the arsenic stress environment, suggesting them as strong candidates to be exploited for arsenic remediation. arsenictreated tree seedlings showed reduced growth in terms of stem height (21.76%), stem diameter (25%), number of branches (22.3%), number of leaves (21%), root length (24.8%), total plant length (25.6%), fresh biomass production (40%) and dry biomass production (49%) as compared to plants grown without arsenic treatment. arsenic accumulated in all vegetative parts of the seedlings, however maximum arsenic accumulation was recorded in roots of e. camaldulensis (37.25 mg kg-1) followed by s. tetrasperma (35.76 mg kg-1) and t. arjuna (24.13 mg kg-1) when arsenic was applied at a concentration of 4.0 mg l-1. the study has shown that these tree species can be grown on arsenic-contaminated fields to reclaim the soil from arsenic content resulting in its substantial reduction in leaching to the groundwater. the results suggest that e. camaldulensis plants accumulated maximum arsenic in its vegetative parts and confirmed it as a tree species with a potential of arsenic phyto-extraction and a good candidate for phytoremediation. key words: arsenic accumulation, metalloid, phytoremediation, reclamation. introduction arsenic-contaminated soils and water cause environmental and human health problems and the situation is becoming grave with rapid industrialization and disturbance in natural geochemical cycle (mieke and rengel, 2005; madhumita et al., 2014; paul et al., 2015; singh et al., 2015). many areas of the world are affected by arsenic contamination of the underground water (mukherjee et al., 2006; singh et al., 2015). arsenic-contaminated soils and water have been attempted to remove arsenic using a wide range of remediation technologies (uddin et al., 2001; manning et al., 2002; gavrilescu, 2006; tonni et al., 2006; pavel and gavrilescu, 2008; singh et al., 2015). generally all these methods are costly, require high technology and by-products from these technologies can be harmful (ali et al., 2013). phytoremediation of toxic substances has emerged as a promising, cost-effective, eco-friendly and sustainable approach by using green plants (trees, shrubs, grasses and aquatic plants) to remove and uptake, degrade or isolate toxic substances (heavy metals, metalloids, trace elements, organic and inorganic compounds, and radioactive compounds) from the soils and water (ensley, 2000; mendez and maier, 2008; dickinson et al., 2009; luqman et al., 2013). in this study, tree seedlings of e. camaldulensis, t. arjuna and s. tetrasperma were used to reclaim arsenic-contaminated soils. in this study, an attempt has been made to determine the potential of three tree species to uptake and accumulate arsenic from arseniccontaminated soil in their vegetative parts and to demonstrate their tolerance against arsenic toxicity, so that these trees can be planted on arsenic-contaminated land to remediate the soil through phytoremediation. materials and methods raising of seedlings: six month old seedlings of eucalyptus camaldulensis dehnh., myrtaceae, terminalia arjuna (roxb.) wight & arn., combretaceae, and s. tetrasperma, salicaceae, were obtained from a nursery of the forest department, government of the punjab, and were planted in plastic pots (40 cm height × 35 cm internal diameter) containing loamy soil at the botanical garden, university of the punjab, lahore. application of arsenic: plants were given different doses of arsenic (0.5, 1.0, 2.0, 4.0 mg l-1) for 18 months. these doses were given to plants once a week in the winter season (october march) and on alternate days during summer (april june) and plants were dosed twice a week during the monsoon season (july september). sodium arsenite salt (naaso2) was used by making stock solutions and given through irrigation water. the level of arsenic in the tap water given to control plants was 5.5 μg l-1. no fertilizer was used in this experiment. determination of plant growth and biomass: the shoot length and diameter (of collar) of arsenic treated and untreated (control) plants were measured monthly during the experiment. at the end of experiment, seedlings were uprooted and thoroughly washed with tap water and rinsed with distilled water. the leaves and number of branches were counted and weighed separately. dry weight of leaves, shoots and roots was determined after oven drying at 70 ºc for 48 hrs. phytoremediation of arsenic-contaminated soils by different plant species 9 arsenic analysis: oven dried plant parts (roots, shoots and leaves) were ground separately using willey mill (2000 sm, retsch, germany) and sieved with mesh (2 mm). one g of plant material was transferred into conical flasks and digested in a solution of hno3 : hclo4 with 2:1 ratio (v/v) by placing it on a hot plate for about 2 hrs until the plant material became transparent. the concentrations of arsenic were determined by using hydride generation atomic absorption spectrophotometer (aa-7000, shimadzu, japan). statistical analysis: the data recorded for each treatment was subjected to the analysis of variance (anova) and means of various treatments were compared using tukey’s hsd test at 5% probability level (steel and torrie, 1980). results effects of arsenic on plant growth effects of various doses of arsenic on the growth of e. camaldulensis, s. tetrasperma and t. arjuna plants are shown in tab. 1. stem height (cm) stem height varied significantly with increase in arsenic concentration. a reduced value of 272 cm in stem height of e. camaldulensis was recorded in t4 as compared with 331.3 cm in control (t0) while in the case of s. tetrasperma, it was reduced to 92 cm in t4 compared with 141.3 cm in t0. in the case of t. arjuna, however it was reduced to 166.7 cm in t4 from 221 cm in t0. a gradual smooth decreasing trend was observed in stem height (average reduction of 25.8% as compared to control) in all three plant species with the increase in the concentration of arsenic. stem diameter (cm) stem diameter significantly decreased with the increased concentration of arsenic. a maximum reduction in stem diameter of e. camaldulensis was found to be 2.63 cm that was recorded in t4 compared with 3.8 cm recorded in control (t0) while in the case of s. tetrasperma, it was reduced to 1.4 cm in t4 down from 1.72 cm in t0. in case of t. arjuna plant, however it was recorded to be 2.65 cm at t4 down from 3.6 cm at t0. a gradual smooth decreasing trend in stem diameter was observed with an average reduction of 25% for all three plants species when the concentrations of arsenic were increased. number of branches similarly, reductions in number of branches were observed with an increase in arsenic concentration. a maximum reduction to the level of 163 (nos.) in t4 in number of branches of e. camaldulensis was recorded as compared to average 217.3 branches per plant in control (t0) while in the case of s. tetrasperma, it was recorded as 79.8 in t4 down from 103.7 in t0. in the case of t. arjuna plant, however, it was found to be reduced to 64.7 in t4 compared with 79.7 in control (t0). a gradual smooth decreasing trend was observed with an average reduction of 22.27% for all three plants species when the concentration of arsenic was increased. number of leaves number of leaves was also significantly varied with an increase in arsenic concentration in all species. a maximum reduction in total number of leaves of e. camaldulensis plant was recorded to be 449.7 in t4 as compared with 610.7 in control (t0) while in the case of s. tetrasperma, the number was reduced to 348 in t4 as compared with 430 in t0. in the case of t. arjuna, however it was reduced to 408.7 in t4 down from 510.7 in t0. a gradual smooth decreasing trend was observed with an average reduction of 21.76% for all three plants species as the doses of arsenic were increased. tab. 1: effects of arsenic application on plant growth parameters. treatments species stem height stem diameter no. of branches no. of leaves root length total length (cm) (cm) (cm) (cm) e. camaldulensis 331.3±3.41 a 3.8±0.09 a 217.3±2.22 a 610.7±3.5 a 165.3±1.45 a 496.7±4.77 a t0 s. tetrasperma 141.3±2.82 def 1.7±0.33 d 103.7±1.15 d 430.0±8.71 fgh 149.2±3.33 ab 290.5±3.21 def t. arjuna 221.0±4.50 bc 3.6±0.33 ab 79.7±0.88 ef 510.7±2.19 bc 134.0±2.50 ab 355.3±4.07 bcde e. camaldulensis 292.0±4.51 a 3.3±0.33 abc 193.7±2.70 b 564.0±15.10 ab 155.0±3.04 ab 447.0±2.52 ab t1 s. tetrasperma 130.3±2.71 def 1.6±0.08 d 90.0±1.48 de 400.3±1.04 ghij 132.3±1.86 ab 262.6±2.70 ef t. arjuna 189.5±2.88 cd 3.0±0.10 abc 78.0±1.51 ef 489.7±2.19 cde 117.0±1.76 ab 306.5±2.15 cdef e. camaldulensis 283.0±3.04 ab 3.0±0.16 abc 182.0 ±2.86 bc 502.0±7.51 cd 147.1±1.50 b 380.1±4.02 bcd t2 s. tetrasperma 120.2±1.00 ef 1.5±0.33 d 86.0±1.67 de 381.0±2.08 hij 121.0±1.15 ab 241.2±2.33 ff t. arjuna 178.0±1.53 cde 2.8±0.11 bc 72.3±0.23 ef 460.0±3.51 cdef 110.2±1.00 ab 288.2±1.88 def e. camaldulensis 276.0±1.86 ab 2.9±0.12 bc 171.0±1.15 c 485.0±10.54 cde 134.0±1.15 ab 410.0±2.85 abc t3 s. tetrasperma 95.3±1.86 f 1.5±0.10 bc 79.8±0.58 ef 362.0±0.44 ij 116.0±3.86 ab 211.3±2.69 f t. arjuna 170.0±2.61 cde 2.7±0.09 c 64.7±0.67 f 437.7±1.45 efg 102.0±0.00 ab 272.0±3.93 ef e. camaldulensis 272.0±3.30 ab 2.6±0.33 c 163.0±2.52 c 449.7±1.76 defg 130.3±0.88 ab 402.2±2.89 abc t4 s. tetrasperma 92.0±0.88 f 1.4±0.07 d 79.8±1.76 ef 348.0±0.20 j 111.1±1.00 ab 203.1±3.17 f t. arjuna 166.7±1.67 cde 2.7±0.10 c 64.7±0.58 f 408.7±1.76 fghi 97.0±0.58 b 263.7±2.37 f t0 = tap water (control); t1 = 0.5 mg l-1 arsenic; t2 = 1.0 mg l-1 arsenic; t3 = 2.0 mg l-1 arsenic; t4 = 4.0 mg l-1 arsenic. values with different letters are significantly different from each other at p ≤ 0.05. 10 f.u.d. ahmad, n. ahmad, k.r. masood, m. hussain, m.f. malik, a. qayyum root length (cm) variation in root length was observed with change in arsenic doses in all three species. a maximum reduction in root length of e. camaldulensis was recorded to be 130.3 cm in t4 as compared with 165.3 cm in t0, while in the case of s. tetrasperma it was reduced to 111.1 cm in t4 down from 149.2 cm in t0. in the case of t. arjuna, however, the root length was observed as reduced to 97 cm in t4 in comparison to 134 in control (t0). a gradual smooth decreasing trend was observed for all three plant species with increasing concentration of arsenic having an average reduction of 24.8%. total plant length (cm) total plant length varied significantly with an increase in arsenic doses. a maximum reduction in total plant length of e. camaldulensis was observed to be in t4 due to the highest dose of arsenic whereas it was recorded to be 402.2 cm as compared with 496.7 cm at control (t0) while in the case of s. tetrasperma it reduced to 203.1 cm in t4 as compared with 290.5 cm in t0. in the case of t. arjuna, however, it was reduced to 263.7 cm down from 355.3 cm in control (t0). a gradual smooth decreasing trend was observed for all three plants species with increasing concentration of arsenic having an average reduction of 25.6%. effect of arsenic doses on biomass production of plants the results of the study regarding effects of varying concentrations of arsenic application on biomass production of e. camaldulensis, t. arjuna and s. tetrasperma are shown in tab. 2. leaf fresh biomass (g) increase in arsenic contents resulted in reduction of leaf fresh biomass. with an increase in arsenic doses, a reduced value of 201 g in leaf fresh biomass of e. camaldulensis was recorded in t4 as compared with 300.8 g in control (t0) while in the case of s. tetrasperma, it reduced to 28.3 g in t4 compared with 49.5 g in t0. in the case of t. arjuna, however, it was reduced to 61 g in t4 from 125.3 g at t0. stem fresh biomass (g) increase in arsenic contents resulted in reduction of stem fresh biomass. a 955.7 g in stem fresh biomass of e. camaldulensis was recorded in t4 as compared with 1221.7 g in control (t0) while in the case of s. tetrasperma, it reduced to 55 g in t4 compared with 195 g in t0. in the case of t. arjuna, however, it was reduced to 225 g in t4 from 372 g at t0. root fresh biomass (g) similarly, root fresh biomass also varied with arsenic increase. in response to arsenic doses, a reduced value of 457.8 g in root fresh biomass of e. camaldulensis was recorded in t4 as compared with 555.6 g in control (t0) while in the case of s. tetrasperma, it reduced to 65 g in t4 compared with 164.2 g in t0. in the case of t. arjuna, however, it was reduced to 146 g in t4 from 256.6 g at t0. leaf dry biomass (g) increase in arsenic contents resulted in reduction of leaf dry biomass. in response to arsenic doses, a reduced value of 105 g in leaf dry biomass of e. camaldulensis was recorded in t4 as compared with 160.5 g in control (t0) while in the case of s. tetrasperma, it reduced to 14 g at t4 compared with 29 g in t0. in the case of t. arjuna, however, it was reduced to 27 g in t4 from 62.5 g in t0. stem dry biomass (g) increase in arsenic contents resulted in reduction of stem dry biomass. a reduced value of 506.2 g in stem dry biomass of e. camaldulensis tab. 2: effects of arsenic doses on biomass production of tree seedlings of e. camaldulensis, s. tetrasperma and t. arjuna. treatments species fresh weight (g) oven dry weight (g) leaf stem root leaf stem root e. camaldulensis 300.8 ± 3.23 a 1221.7 ± 9.99 ab 555.6 ± 2.67 b 160.5 ± 1.49 b 705.2 ± 8.45 a 216.0 ± 2.66 d t0 s. tetrasperma 49.5 ± 1.04 fgh 195.0 ± 2.00 b 164.2 ± 2.40 b 29.0 ± 1.76 b 92.3 ± 1.67 fg 80.0 ± 1.36 a t. arjuna 125.3 ± 2.14 d 372.0 ± 3.00 b 256.6 ± 3.12 b 62.5 ± 1.51 b 182.5 ± 3.49 c 141.7 ± 1.68 d e. camaldulensis 255.8 ± 2.73 b 1096.0 ± 8.53 ab 495.8 ± 1.53 b 128.9 ± 5.85 a 604.2 ± 2.19 b 192.5 ± 2.81 b t1 s. tetrasperma 41.0 ± 1.53 gh 125.3 ± 1.27 b 104.0 ± 3.22 b 22.0 ± 2.08 b 68.0 ± 1.16 efg 55.0 ± 1.16 ght t. arjuna 100.8 ± 1.82 de 297.0 ± 2.06 b 199.0 ± 3.53 b 47.5 ± 2.90 b 151.0 ± 2.08 cd 95.3 ± 1.76 e e. camaldulensis 235.8 ± 1.15 bc 1076.7 ± 5.33 ab 475.8 ± 2.00 b 119.5 ± 1.20 b 584.2 ± 4.70 b 170.8 ± 2.22 c t2 s. tetrasperma 35.0 ± 1.33 gh 101.7 ± 1.76 a 99.0 ± 1.56 b 20.8 ± 0.67 b 55.0 ± 1.53 fg 51.3 ± 1.33 hi t. arjuna 81.0 ± 0.22 ef 278.0 ± 3.35 b 177.7 ± 2.40 b 40.0 ± 2.52 b 137.0 ± 2.40 cd 87.0 ± 1.33 ef e. camaldulensis 222.8± 1.73 bc 1037.7 ± 6.67 a 466.0 ± 3.93 a 112.0 ± 2.00 b 561.5 ± 2.60 b 151.2 ± 2.69 cd t3 s. tetrasperma 27.0 ± 0.67 h 75.0 ± 3.51 b 88.0 ± 1.11 b 16.0 ± 1.67 b 45.0 ± 1.53 fg 46.0 ± 0.16 i t. arjuna 70.0 ± 1.16 efg 250.0 ± 2.67 b 161.0 ± 2.76 b 35.0 ± 1.76 b 126.0 ± 2.85 cde 75.0 ± 1.40 efgh e. camaldulensis 201.0 ± 1.00 c 955.7 ± 5.33 ab 457.8 ± 1.16 b 105.0 ± 1.00 b 506.2 ± 1.16 b 132.8 ± 1.20 cd t4 s. tetrasperma 28.3 ± 1.76 h 55.0 ± 3.06 b 65.0 ± 2.19 b 14.0 ± 2.03 b 40.0 ± 1.53 g 30.0 ± 0.73 i t. arjuna 61.0 ± 1.76 fgh 255.0 ± 3.06 b 146.0 ± 2.0 b 27.0 ± 2.00 b 115.0 ± 2.52 def 63.0 ± 1.73 efg t0 = tap water (control); t1 = 0.5 mg l-1 arsenic; t2 = 1.0 mg l-1 arsenic; t3 = 2.0 mg l-1 arsenic; t4 = 4.0 mg l-1 arsenic. values with different letters are significantly different from each other at p ≤ 0.05. phytoremediation of arsenic-contaminated soils by different plant species 11 was recorded in t4 as compared with 705.2 g in control (t0) while in the case of s. tetrasperma, it reduced to 40 g in t4 compared with 92.3 g in t0. in the case of t. arjuna, however, it was reduced to 115 g in t4 from 182.5 g at t0. root dry biomass (g) increase in arsenic contents resulted in reduction of root dry biomass. in response to arsenic doses, a reduced value of 132.8 g in root dry biomass of e. camaldulensis was recorded in t4 as compared with 216 g in control (t0) while in the case of s. tetrasperma, it reduced to 30 g in t4 compared with 80 g in t0. in the case of t. arjuna, however, it was reduced to 63 g in t4 from 141.7 g in t0. accumulation of arsenic by tree seedlings effects of arsenic doses on the uptake and accumulation of arsenic in roots, shoots, leaves is shown in tab. 3. accumulation of arsenic in roots arsenic accumulation tends to increase in roots with the higher dose of arsenic in all tested plant species. an increased value of 37.25 mg kg-1 of as in roots of e. camaldulensis was recorded in t4 as compared with 1.83 mg kg-1 in control (t0) while in the case of s. tetrasperma, it increased to 35.76 mg kg-1 in t4 compared with 1.70 mg kg-1 in t0. in the case of t. arjuna, however, it was increased to 24.13 mg kg-1 in t4 from 1.98 mg kg-1 at t0. accumulation of arsenic in shoots arsenic accumulation tends to increase in shoots with the higher dose of arsenic in all tested plant species. in response to arsenic doses, an increased value of 25.69 mg kg-1 of arsenic in shoots of e. camaldulensis was recorded in t4 as compared with 1.08 mg kg-1 in control (t0) while in the case of s. tetrasperma, it increased to 13.76 mg kg-1 in t4 compared with 1.03 g in t0. in the case of t. arjuna, however, it was increased to 12.52 mg kg-1 in t4 from 0.94 mg kg-1 in t0. accumulation of arsenic in leaves arsenic accumulation tends to increase in leaves with the higher dose of arsenic in all tested plant species. in response to arsenic doses, an increased value of 6.55 mg kg-1 of arsenic in leaves of e. camaldulensis was recorded in t4 as compared with 0.57 mg kg-1 in control (t0) while in the case of s. tetrasperma, it increased to 6.03 mg kg-1 in t4 compared with 0.57 g in t0. in the case of t. arjuna, however, it was increased to 4.22 mg kg-1 in t4 from 0.67 mg kg-1 in t0. discussion an inhibition of plant growth is directly related to the enhanced uptake and accumulation of heavy metals in plants because metal elements adversely affect the plant growth through the generation of reactive oxygen species (ros) causing constant damage to cellular structures, interfere metabolic process in plants (marin et al., 1993; mallick et al., 2011). upon translocation to the shoot, arsenic severely inhibits plant growth by slowing biomass accumulation (finnegan and chen, 2012). results of this study shows that stem height of all three plants species gradually reduced with increasing doses of arsenic and averagely it reduced to 25.8% at 4 mg l-1 arsenic dose. reduction in shoot growth could be attributed to the reduction in chlorophyll contents and activity of photosystem i (psi) induced by heavy metal stress (skorzynska-polit and baszynski, 1997). moreover, accumulation of metal elements to above ground plant part can reduce plant height because of disturbance in cellular tab. 3: arsenic accumulation in leaves, shoots and roots of tree seedlings of e. camaldulensis, s. tetrasperma and t. arjuna. treatments species arsenic content arsenic content arsenic content accumulated in leaves accumulated in shoot accumulated in root (mg kg-1) (mg kg-1) (mg kg-1) e. camaldulensis 0.57 ± 0.33 i 1.08 ± 0.06 j 1.83 ± 0.07 i t0 s. tetrasperma 0.57 ± 0.17 i 1.03 ± 0.04 j 1.70 ± 0.09 i t. arjuna 0.67 ± 0.06 i 0.94 ± 0.03 j 1.98 ± 0.11 i e. camaldulensis 2.73 ± 0.33 d 9.65 ± 0.38 f 14.84 ± 0.39 f t1 s. tetrasperma 1.87 ± 0.08 h 5.41 ± 0.33 hi 12.82 ± 0.46 g t. arjuna 1.73 ± 0.33 h 4.50 ± 0.33 i 10.45 ± 0.33 h e. camaldulensis 3.31 ± 0.33 c 15.61 ± 0.45 c 20.56 ± 0.67 e t2 s. tetrasperma 2.85 ± 0.39 fg 9.78 ± 0.30 g 19.98 ± 0.43 e t. arjuna 2.22 ± 0.17 gh 6.85 ± 0.58 h 13.01 ± 0.31 g e. camaldulensis 5.27 ± 0.33 b 20.94 ± 0.50 b 29.16 ± 0.58 b t3 s. tetrasperma 4.50 ± 0.37 e 10.91 ± 0.31 e 27.01± 0.58 c t. arjuna 3.22 ± 0.31 f 9.26 ± 0.33 g 19.92 ± 0.46 e e. camaldulensis 6.55 ± 0.67 a 25.69 ± 0.88 a 37.25 ± 0.61 a t4 s. tetrasperma 6.03 ± 0.33 d 13.76 ± 0.30 d 35.76 ± 1.44 a t. arjuna 4.22 ± 0.18 e 12.52 ± 0.33 de 24.13 ± 0.33 d t0 = tap water (control); t1 = 0.5 mg l-1 arsenic; t2 = 1.0 mg l-1 arsenic; t3 = 2.0 mg l-1 arsenic; t4 = 4.0 mg l-1 arsenic. values with different letters are significantly different from each other at p ≤ 0.05. 12 f.u.d. ahmad, n. ahmad, k.r. masood, m. hussain, m.f. malik, a. qayyum metabolism of shoots (shanker et al., 2005). a significant reduction in stem growth of pea (pisum sativum) seedlings was also recorded when arsenic was applied at the rate of 5 mg l-1 (castillo-michel et al., 2007) which is almost similar to this study. similarly, stem diameter gradually reduced with increasing doses of arsenic and on average it reduced to 25% at 4 mg l-1 arsenic dose in case of all three plants species. leaf growth and total number of leaves are considered a suitable indicator of heavy metal toxicity. reza et al. (2009) found a decrease in number of leaves per plant with gra dual increase of as application to amaranthus retroflexus plants. similarly, a decrease in leaf number was observed in this study, with an average reduction of 22.27% for all three plants species as the doses of arsenic were increased. root growth is also an important indicator related to the enhanced uptake and accumulation of heavy metals in plants. high concentration of heavy metals retard root respiration leading to reduction in root growth (menon et al., 2005). moreover, due to high solute potential in the rhizosphere, nutrient uptake by plants is disturbed causing a decrease in mitotic division of meristematic cells (odjegba and fasidi, 2004). root growth of all three plants species decreased with increasing doses of arsenic with an average reduction of 24.8% at 4 mg l-1 dose. shri et al. (2009) also observed a decrease in root growth of rice seedlings. the reduction in biomass at high doses of as might be due to the high lipid peroxidation which might have damaged chloroplast, and inhibited chlorophyll and protein contents (singh et al., 2006; garg and singla, 2011). upon translocation to the shoot, arsenic severely inhibits plant growth by slowing biomass accumulation (finnegan and chen, 2012) resulting in biomass reduction. aydin and aksoy (2009) applied arsenic on chickpea (cicer arietinum l.) plants and found reduction in fresh and dry biomass production of chickpea plants with increasing arsenic levels. arsenic is non-essential for plant growth; therefore, plants have no specific mechanism for its uptake and translocation in plant system. it enters by means of the same uptake transporter as essential nutrients. higher levels of arsenic in soil interfere with nutrient uptake by plants and their distribution leading to a deficiency of essential nutrients in plants (mahimairaja et al., 1994). since roots are the first plant tissue to come in contact with soil and therefore, metal accumulation was more pronounced in roots compared to shoots and leaves (kuzovkina et al., 2004). higher accumulation of heavy metals in roots might be due to poor translocation to above ground parts and sequestration in the vacuoles of root cells (shanker et al., 2005). metal accumulation in plant tissues was in order of roots>shoot>leaves and the increase was proportional to the external application of metal elements. the variability of metals in various plant tissues may be due to compartmentalization and translocation in the vascular system (kim et al., 2003). arsenic in high concentration may hamper with metabolism of plants and adversely affected the uptake of nutrients by competing with plant nutrients for transporters (meharg and macnair, 1990; mokgalaka-matlala et al., 2008). cell membranes are vulnerable targets of arsenic stress and damage of membrane is used as an indicator for tolerance to arsenic stress. arsenic adversely affects the membrane leading to unbalanced nutrient uptake and water content in plant cells (gadallah, 1999; garg and singla, 2011). conclusion in this study the growth of tree seedlings was determined and shown to be affected by the uptake of arsenic as it accumulated in the vegetative parts of the plants grown under arsenic treated media. e. camaldulensis, t. arjuna and s. tetrasperma have demonstrated adaptability to recover the soil from arsenic with varying magnitude. although arsenic-treated plants were affected and growth parameters demonstrated reduced number of branches, leaves, reduced root length and low biomass production as compared to plants grown without arsenic treatment. they significantly reduced arsenic from the media. among the three tree species investigated, e. camaldulensis showed the highest tolerance against arsenic toxicity and maxi mum arsenic accumulation in its vegetative parts. thus, the 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a biometric approach of statistics. 2nd ed. new york, usa: mcgraw hill press. tonni, a.k., chana, g.y.s., lo, w., sandhya, b., 2006: physico-chemical treatment techniques for wastewater laden with heavy metals. adv. biochem. eng. 118, 83-98. uddin, m.t., majumder, m.s.i., figoli, a., islam, m.a., drioli, e., 2001: arsenic removal by conventional and membrane technology: an overview. indian j. chem. technol. 14, 441-45. address of the corresponding author: e-mail: dr.mubashar@uog.edu.pk journal of applied botany and food quality 89, 290 298 (2016), doi:10.5073/jabfq.2016.089.038 1 department of plant physiology, faculty of sciences, university of granada, granada, spain 2 phytochemistry lab, department of food science and technology, cebas-csic, campus universitario espinardo, espinardo, murcia, spain evaluation of hydrogen sulfide supply to biostimulate the nutritive and phytochemical quality and the antioxidant capacity of cabbage (brassica oleracea l. ‘bronco’) david montesinos-pereira1*, yurena barrameda-medina1, nieves baenas 2, diego a. moreno 2, eva sánchez-rodríguez1, begoña blasco1, juan m. ruiz1 (received january 25, 2016) * corresponding author summary the potential effects of the hydrogen sulfide on shoot biomass, nutritional quality and antioxidant capacity of brassica oleracea, were investigated through the application of increasing doses of nahs (h2s donor nahs; 0.5, 1, 2.5, and 5 mm). the results showed that the 0.5 and 1 mm nahs treatments increased biomass and the quality composition of ‘bronco’ cabbage (i.e. chlorophylls, carotenoids, anthocyanins, flavonols, total phenolics and sinigrin). on the other hand, there was an increase in lipid peroxidation and hydrogen peroxide content with the application of doses higher than 2.5 mm nahs. therefore, we selected the 0.5 and 1 mm nahs dosages as optimal for cabbage. the 2.5 and 5 mm nahs produced an excessive lipid peroxidation, decreases in plant biomass and losses of chlorophylls, being all considered negative effects, and clear evidences of stressful situation for the plants. for practical purposes, this study suggested that exogenous application of h2s donor nahs at 0.5 and 1 mm may be useful as bio-stimulant to boost the yield and the health-promoting composition of ‘bronco’ cabbage (brassica oleracea l.). keywords hydrogen sulfide, health-promoting compounds, brassica oleracea, antioxidant capacity. introduction sulfur is an essential mineral nutrient element, crucial for sulfuramino acids (cysteine and methionine), natural antioxidants (reduced glutathione; gsh), co-enzymes, prosthetic groups, vitamins, secondary metabolites, phytochelatins (pcs) and lipids (khan et al., 2014 a; khan et al., 2014 b). with respect to the different forms of appli cation to plants of this macronutrient in last few years there has been an increased interest in the study of hydrogen sulfide (h2s) on plant physiology (lisjak et al., 2013). high doses of h2s in greenhouses caused leaf lesions, defoliation, reduced growth, and death of sensitive species such as alfalfa (medicago sativa l.), lettuce (lactuca sativa), sugar beet (beta vulgaris) (thompson and kats, 1978). although several works showed that this compound can adversely affects the growth and physiology of crops (koch and erskine, 2001), recent works suggests that h2s is a more fundamental mo lecule which is produced by plants and used to control plant func tion (zhang et al., 2010). so, h2s has shown its action as a signal molecule and it has been used to promote the antioxidant enzyme activities and uptake of elements against abiotic stress in barley (gadalla and snyder, 2010; dawood et al., 2012). moreover, it was observed that h2s involved in the antioxidant response against osmotic stresses (i.e. excessive boron and drought) in cucumber and sweet potato respectively (zhang et al., 2009; wan et al., 2010). nonetheless, the application of h2s increased the antioxidant capacity and quality parameters in fruits and berries (i.e., mulberry, kiwi, and strawberry) (hu et al., 2012; hu et al., 2014; zhu et al., 2014). brassicaceae vegetables are economically relevant crops and important human foods worldwide, highly used in china, japan, india, and european countries (cartea et al., 2010). the popularity and consumption of brassica is growing because of their renewed relevance on human health through the prevention of certain degenerative diseases (i.e. cardiovascular, cognitive, alzheimer’s and parkinson’s, etc.) and reduction in the risks of suffering from cancer (i.e., lung, breast, colon, prostate) (cartea et al., 2010; podsedek, 2007; bazzano et al., 2002; smith-warner et al., 2003; cho et al., 2004). these health-promoting properties are attributed to their composition very rich in intrinsic and indirect antioxidants (cartea et al., 2010). natural antioxidants in brassica, includes ascorbate, anthocyanins, phenolic compounds and carotenoids (galati and o’brien, 2004). of particular relevance, ascorbate or vitamin c and phenolic com pounds are highlighted, as well as the phenolic compounds, which has been shown to exert antioxidant, anticarcinogenic, antimicro bial, antiallergic, antimutagenic, and anti-inflammatory activities (martínez-valverde et al., 2002). moreover, essentially unique to cruciferous crops and foods, and more relevant in terms of health-promoting effects are the gluco sinolates (glss). the glss are a heterogeneous family of molecules characterized by a similar basic structure containing a sulphurlinked β-d-glucopyranoside, a sulphonated oxime, and a side chain derived from different amino acids, which allow their classification in: aliphatic, aromatic, and indolic glss (fahey et al., 2001). glss have gained growing attention for their potential health-promoting properties, as their hydrolysis products (isothiocyanates, itcs) are able to induce phase 2 detoxification enzymes and protect mammals against chemically induced cancer (zhang et al., 1992). so, the consumption of vegetables containing glucosinolates, such as brassica oleracea varieties, may confer protection against different types of cancer (cartea et al., 2010; zhang et al., 1992 ). one strategy to improve the nutritional characteristics of crops is the optimal application of different forms of s. the most widely used form of s for this purpose has been so4-, favoring yield in number of fruits and the vitamin c content in strawberries (eshghi and jamali, 2014). others authors also reported increased total phenolics and antioxidant capacity in mango fruits upon application of sodium bisulfate (siddiq et al., 2013). in addition to so4-, in the last few years it has been observed that the application of s in a more reduced form than so4such as h2s could have a positive effect on the nutritional quality, but such treatments were performed during postharvest, for example in mulberry, after applying 0.8 mm of the h2s donor nahs, increased contents of ascorbate and soluble proteins, were found, maintaining the postharvest quality (hu et al., 2014). using the same can nahs biostimulate nutritive quality and antioxidant capacity of brassica oleracea l.? 291 fumigation (0.8 mm of the h2s donor nahs) resulted in increased firmness and colour, and delayed respiratory damage in strawberries, prolonging postharvest shelf life (hu et al., 2012). nevertheless, there are very limited studies to examine the potential beneficial effects of h2s on the nutritional quality, yield and phytochemical quality of leafy vegetables not affected by any abiotic or biotic stress, and therefore, the aim of this study was to determine the influence of h2s application (as sodium hydrosulfide hydrate, nahs + h2o) on the biomass production and antioxidant capacity response and the nutritional and phytochemical quality of ‘bronco’ cabbage (brassica oleracea l.). material and methods plant material and treatments seeds of b. oleracea cv. bronco (saliplant s.l., spain) were germinated and grown for 35 days in cell flats of 3 cm × 3 cm × 10 cm filled with a perlite mixture substratum. the flats were placed on benches in an experimental greenhouse located in southern spain (saliplant s.l., motril, granada). after 35 days, the seedlings were transferred to a growth chamber under the following controlled environmental conditions: relative humidity 50 %; day/night temperatures 25/18 °c; 16/8 h photoperiod at a photosynthetic photon flux density (ppfd) of 350 μmol m -2s -1 (measured at the top of the seedlings with a 190 sb quantum sensor, li-corinc., lincoln, nebraska, usa). under these conditions the plants were grown in hydroponic culture in lightweight polypropylene trays (60 cm diameter top, bottom diameter 60 cm and 7 cm in height) of 3 l volume. throughout the experiment the plants were treated with a growth solution made up of 4 mm kno3, 3 mm ca(no3)2·4h2o, 2 mm mgso4·7h2o, 6 mm kh2po4, 1 mm nah2po4·2h2o, 2 μm mncl2·4h2o, 10 μm znso4·7h2o, 0.25 μm cuso4·5h2o, 0.1 μm na2moo4·2h2o, 5 mg l-1 fe-chelate (sequestrene; 138 feg100) and 10 μm h3bo3. this solution, with a ph of 5.5-6.0, was changed every three days. experimental design the nahs treatments were initiated 43 days after germination and were maintained for 30 days. to determine the concentrations of nahs to apply, it was carried out a previous culture in which it was observed the response of brassica oleracea l. ̔bronco’ to a wide range of concentrations of nahs, ranging from 0.01 mm to 6 mm in which was observed as the most appropriate doses to study the effects of fortification and toxicity in this species were: 0.5, 1, 2.5 and 5 mm of nahs. for that reason the experiment consisted in a randomized complete block design with five treatments that were supplied with the irrigation solution (complete nutrient solution amended with different nahs levels: 0 mm nahs; and 0.5 mm, 1 mm, 2.5 mm and 5 mm nahs), arranged in 8 plants per tray (in expanded polystyrene support) and three replicates per treatment. plant sampling plants of each treatment (73 days after germination) were divided into roots and leaves, washed with distilled water, dried on filter paper and weighed, thereby obtaining fresh weight (fw). half of leaves from each treatment were frozen at -30 °c for further work and biochemical assays and the other half of the plant material was lyophilised for 48 h to obtain the dry weight (dw) and the subsequent analysis of phenolics and glss. determination of h2s content 0.1 g of brassica oleracea leaves were ground under liquid nitrogen and extracted by 1 ml phosphate buffered saline (50 mm, ph 6.8) containing 0.1 m edta and 0.2 m ascorbic acid. after centrifugation at 12000 rpm for 15 min at 4 ºc, 400 ml of the supernatant was injected to 200 ml 1 % zinc acetate and 200 ml 1 n hcl. after 30 min reaction, 100 ml 5 mm dimethyl-p-phenylene-diamine dissolved in 7 mm hcl was added to the trap followed by the injection of 100 ml 50 mm ferric ammonium sulfate in 200 mm hcl. after 15 min incubation at room temperature, the amount of h2s was determined at 667 nm. solutions with different concentrations of na2s were used in a calibration curve (sekiya et al., 1982). chlorophyll concentration and lipid peroxidation for the extraction of chlorophylls, chlorophyll a (chla) and b (chlb), 0.1 g of leaves were ground in semidarkness and resuspended in 10 ml of cold acetone at 80 %. immediately afterwards, the samples were centrifuged at 800 rpm and the absorbance of the supernatant was measured at 653 and 666. the concentrations of chl a and chl b were calculated (wellburn, 1994). for the mda assay, leaves were homogenized with 3 ml of 50 mm solution containing 0.07 % nah2po4·2h2o and 1.6 % na2hpo4· 12 h2o and centrifuged at 15.000 rpm for 25 min in a refrigerated centrifuge. for measurement of mda concentration 3 ml of 20 % trichloroacetic acid (tca) containing 0.5 % thiobarbituric acid (tba) was added to a 1 ml aliquot of the supernatant. the mixture was heated at 95 ºc for 30 min, quickly cooled in an ice bath and then centrifuged at 10 400 rpm for 10 min. the absorbance of the supernatant was read at a532 and a600 nm (heath and packer, 1968). the result of mda was expressed as abs g-1 fw. the h2o2 content of leaf samples was colorimetrically measured (mukherjee and choudhuri, 1983). leaf samples were extracted with cold acetone to determine the h2o2 levels. an aliquot (1 ml) of the extracted solution was mixed with 200 ml of 0.1 % titanium dioxide at 20 % (v/v) h2so4 and the mixture was then centrifuged at 8 000 rpm for 15 min. the intensity of yellow colour of the supernatant was measured at 415 nm. the result of h2o2 concentration was expressed as mg g-1 fw. antioxidant activity test in the free radical scavenging effect (dpph) assay, antioxidants reduce the free radical 2, 2-diphenyl-1-picrylhydrazyl, which has an absorption maximum at a515. to measure the dpph test, the absorbance of the reaction mixture at a517 was read with a spectrophotometer. methanol (0.5 ml), replacing the extract, was used as the blank. the percentage of free-radical scavenging effect was calculated as follows: scavenging effect (% g-1) = [1 − (a517 sample/ a517 blank)] × 100 [29]. to measure the reducing power test, 300 μl of leaves extract (0.1 g per ml methanol 80 %), phosphate buffer (0.2 m, ph 6.6, 0.5 ml) and k3fe (cn)6 (1 % v/w, 2.5 ml, fluka, steinheim, germany) was placed in a ependrof tube and allowed to react for 20 min at 50 ºc. the tube was immediately cooled over crushed ice, and then 150 μl cl3ccooh (10 %, fluka) was added. after centrifugation at 5 000 rpm for 10 min, an aliquot of 300 μl supernatant was mixed with 300 μl distilled water and 40 μl fecl3 (0.1 %).then, the absorbance at 700 nm was measured with a spectrophotometer. increased ab sorbance of the reaction mixture indicated greater reducing power (hsu et al., 2003). antioxidant compounds for the extraction of total carotenoids, 0.1 g of leaves were ground in semidarkness and resuspended in 1 ml of cold acetone at 80 %. immediately afterwards, the samples were centrifuged at 6.000 rpm and the absorbance of the supernatant was measured at 470 nm. to calculate the concentrations of total carotenoids and anthocyanins, 292 d. montesinos-pereira, y. barrameda-medina, n. baenas, d.a. moreno, e. sánchez-rodríguez, b. blasco, j.m. ruiz the edible leaves were homogenized in propanol: hcl:h2o (18:1:81) and further extracted in boiling water for 3 min. after centrifugation at 7.400 rpm for 40 min at 4 ºc, the absorbance of the supernatant was measured at 535 and 650 nm (wellburn, 1982). the absorbance due to anthocyanins was calculated as a = a535-a650 (lange, shropshire and mohr, 1971). finally the determination of reduced ascorbate (asa) in leaf extracts was following the method based on the reduction of fe3+ to fe2+ by asa in acid solution. leaves material were homogenized in liquid n2 with metaphosphoric acid at 5 % (w/v) and centrifuged at 4 ºc for 15 min. absorbance was measured at a525 nm against a standard asa curve that followed the same procedure as above. the results of reduced asa were expressed as μg g-1 fw (law et al., 1983). extraction and determination of phenolic compounds and glucosinolates lyophilised samples (50 mg) were extracted with 1 ml of methanol 70 % v/v in a vortex for 1min, then heated at 70 ºc for 30 min in a heating bath, with shaking every 5 min using a vortex stirrer, and centrifuged (12 000 × g, 10 min, 4 ºc). the supernatants were collected and methanol was completely removed using a rotary evaporator. the dry material obtained was re-dissolved in 1 ml of ultrapure water and filtered through a 0.22 μm millex-hv13 filter (millipore, billerica, ma, usa). hplc-dad-esi-msn qualitative and quantitative analysis glucosinolates and phenolic compounds were determined using a lc-ms multipurpose method that simultaneously separates intact glucosinolates and phenolics (francisco et al., 2009), with slight modifications. firstly, the glss were identified from the extracted samples following their ms2 [m-h] fragmentations in hplc-dadesi-msn, carried out on a luna c18 100a column (250 × 4.6 mm, 5 μm particle size; phenomenex, macclesfield, uk). water:formic acid (99:1, v/v) and acetonitrile were used as mobile phases a and b, respectively, with a flow rate of 800 μl/min. the linear gradient started with 1 % of solvent b, reaching 17 % solvent b at 15 min up to 17 min, 25 % at 22, 35 % at 30, 50 % at 35, which was maintained up to 45 min. the injection volume was 5 μl. the hplc-dad-esi/ msn analyses were carried out in an agilent hplc 1200 (agilent technologies, waldbronn, germany) and coupled to a mass detector in series. the hplc system consisted of a binary capillary pump (model g1376a), an autosampler (model g1377a), a degasser (model g1379b), a sample cooler (model g1330b), and a photodiode array detector (model g1315d), and controlled by chem station software (v.b.0103-sr2). the mass detector was a bruker, model ultra hct (bremen, germany) ion trap spectrometer equipped with an electrospray ionization interface (esi) and controlled by bruker daltonic esquire software (v.6.1). the ionization conditions were adjusted at 350 °c and 4 kv for capillary temperature and voltage, respectively. the nebulizer pressure and flow rate of nitrogen were 60.0 psi and 11 l/min, respectively. the full-scan mass covered the range from m/z 50 up to m/z 1000. collision induced fragmentation experiments were performed in the ion trap using helium as the collision gas, with voltage ramping cycles from 0.3 up to 2 v. mass spectrometry data were acquired in the negative ionization mode for glucosinolates. msn was carried out in the automatic mode on the more abundant fragment ion in ms (n-1). chromatograms were recorded at 227 nm for glucosinolates and 330 nm for phenolic compounds. sinigrin was used as aliphatic glucosinolate and glucobrassicin as indolicglucosinolate external standards (phytoplan, heidelberg, germany). caffeoylquinic acid derivatives were quantified as chlorogenic acid (5-caffeolylquinic acid, sigma-aldrichchemie gmbh, steinheim, germany), flavonols (mainly quercetin and kaempferol derivatives) as quercetin-3-rutinoside (merck, darmstadt, germany), and sinapic acid derivatives as sinapinic acid (sigma). statistical analysis for statistical analysis, data compiled were submitted to anova, and differences between the means were compared with fisher’s least significant difference (lsd; p < 0.05). results effects of nahs on shoot biomass and h2s foliar concentration the greatest increase in shoot biomass was observed after the application of the dose 0.5 and 1 mm of nahs, while higher doses 5 mm resulted in a significant decrease of biomass respect to the control (fig. 1). the content of h2s was found at a moderate increase at 0.5 and 1 mm nahs, and a higher increase was observed at 2.5 and 5 mm nahs (fig. 2). fig. 2: effects of different doses of nahs on h2s shoot concentration. columns are mean ± s.e. (n = 9). different letters indicate significant difference between values. fig. 1: effects of different doses of nahs on shoot biomass. columns are mean ± s.e. (n = 9). different letters indicate significant difference between values. effects of nahs on chlorophylls concentration and lipid peroxidation tab. 1 shown chlorophylls (chl a and chl b), malonyldialdehyde (mda) and h2o2 contents in ‘bronco’ cabbage plants treated with nahs. the chl a concentration increased slightly and significantly at 0.5 mm nahs, but decreased at 5 mm nahs respect to control plants (tab. 1). the treatments of 1 and 2.5 mm nahs did not show any significant difference respect to the untreated control (tab. 1). the concentration of chl b only showed a significant increase in can nahs biostimulate nutritive quality and antioxidant capacity of brassica oleracea l.? 293 tab. 1: effects of different doses of nahs on chlorophylls, lipid peroxidation and hydrogen peroxide concentration. treatments chlorophyll a chlorophyll b mda h2o2 (mg·g-1fw) (mg·g-1fw) (abs·g-1fw) (mg·g-1fw) control 0.149 ± 0.003 b 0.069 ± 0.002 b 0.608 ± 0.004 d 0.429 ± 0.008 c 0.5 mm nahs 0.162 ± 0.004 a 0.073 ± 0.001 a 0.656 ± 0.009 c 0.471 ± 0.007 b 1.0 mm nahs 0.145 ± 0.002 b 0.071 ± 0.000 ab 0.733 ± 0.011 b 0.466 ± 0.010 b 2.5 mm nahs 0.152 ± 0.002 b 0.072 ± 0.001 ab 0.715 ± 0.013 b 0.448 ± 0.002 ab 5.0 mm nahs 0.119 ± 0.001 c 0.060 ± 0.001 ab 1.017 ± 0.015 a 0.533 ± 0.008 a p-value *** * *** *** lsd 0.05 0.008 0.004 0.033 0.028 values are mean ± s.e. (n=9). lsd, least significant difference; ns, not significant * p<0.05; ** p<0.01; *** p<0.001 0.5 mm dose, whereas there were no significant differences in other treatments respect to the control plants (tab. 1). a higher mda concentration was observed at 1, 2.5 and 5 mm of nahs (tab. 1). finally, the concentration of h2o2 increased moderately at 0.5, 1, and 2.5 mm nahs and showed an acute increase at 5 mm (tab. 1). antioxidant capacity tests dpph and the reducing power antioxidant capacity tests are presen ted in fig. 3. the dpph assay results showed a significant increase only at the 5 mm nahs dose. the reducing power test showed a significant increase at the 2.5 and 5 mm nahs treatments, compared to the control. natural antioxidants with respect to the carotenoids there was a significant increase with the application of all doses of nahs respect to the control plants (0 mm nahs) (tab. 2). the concentration of anthocyanins significantly increased with the increasing nahs dosages (tab. 2). vitamin c (reduced asa) concentration increased at 2.5 and 5 mm nahs treatment and of the levels at 0.5 and 1 mm of nahs were in the same range of the control (tab. 2). phenolic compounds and glucosinolates tab. 3 shows the different types of phenolics quantified in the samples. the application of the 0.5 mm nahs dose exerted a significant effect on the content, while at 5 mm nahs there was a significant decrease respect to the untreated control (tab. 3). on the other hand, we did not observe any statistically significant difference in the contents of caffeic and sinapic acid derivatives upon the treatments, only and slight increase in sinapic acid derivatives at 0.5 mm nahs not statistically significant (tab. 3). then the addition of total phe nolics, following response of the major components in the samples (flavonols and sinapic acid derivatives), showed increased content at 0.5 mm nahs, and a significantly reduced content at 5 mm res pect to the control (tab. 3). the analysis of intact glucosinolates in ‘bronco’ cabbage (tab. 4), revealed only slight and significant in creases at 1 mm for sinigrin, and for 4-hydroxyglucobrassicin at 2.5 and 5 mm. the concentrations of glucoberin and glucobrassicin, as well as the addition of aliphatic or indolic glss varied only slightly according to the treatments, and at the end, the total glss remained unchanged (tab. 4). it is worth mentioning that the glucoiverberin, a common glucosinolate in this species was absent in the studied samples (data not shown). fig. 3: effects of different doses of nahs on dpph (a) and reducing power (b) antioxidant tests. columns are mean ± s.e. (n = 9). different letters indicate significant difference between values. 294 d. montesinos-pereira, y. barrameda-medina, n. baenas, d.a. moreno, e. sánchez-rodríguez, b. blasco, j.m. ruiz discussion previous studies found that applications of low concentrations of h2s at 100 mg/l, could significantly increase the growth of alfalfa, lettuce and sugar beets (thompson et al., 1979). under the 0.5 and 1 mm nahs treatments the cabbage (brassica oleracea l. ‘bronco’) shoots biomass improved in final result by 64 and 65 % respectively if compared to the untreated plants (fig. 1). in leaves of brassica tab. 2: effects of different doses of nahs on carotenoids, anthocyanin and reduced asa concentration. treatments carotenoids anthocyanin reduced asa (mg · g-1fw) (a535· g-1 fw) (μg g-1 fw) control 0.023 ± 0.0003 c 0.780 ± 0.005 d 674.62 ± 36.57 c 0.5 mm nahs 0.028 ± 0.0008 a 0.875 ± 0.008 c 713.63 ± 12.13 c 1.0 mm nahs 0.025 ± 0.0004 b 0.884 ± 0.007 c 674.09 ± 09.56 c 2.5 mm nahs 0.026 ± 0.0002 ab 1.137 ± 0.056 b 818.59 ± 12.72 b 5.0 mm nahs 0.025 ± 0.0002 b 1.270 ± 0.011 a 880.90 ± 10.84 a p-value *** *** *** lsd 0.05 0.001 0.074 55.06 values are mean ± s.e. (n=9). lsd, least significant difference; ns, not significant * p<0.05; ** p<0.01; *** p<0.001 different letters indicate significant difference between values. tab. 3: effects of different doses of nahs on phenolic compounds. phenolic compounds (mg g-1dw) flavonols caffeic acid derivatives sinapic acid derivatives total phenolics control 7.88 ± 0.05 b 1.36 ± 0.04 a 39.93 ± 1.28 a 49.27 ± 1.63 b 0.5 mm nahs 10.53 ± 0.21 a 1.44 ± 0.17 a 47.68 ± 4.70 a 64.40 ± 1.44 a 1.0 mm nahs 7.95 ± 0.20 b 1.20 ± 0.28 a 39.07 ± 0.36 a 49.56 ± 1.36 b 2.5 mm nahs 6.51 ± 0.09 b 1.41 ± 0.18 a 38.07 ± 3.95 a 46.01 ± 4.59 bc 5.0 mm nahs 4.47 ± 0.01 c 1.01 ± 0.03 a 34.98 ± 1.79 a 40.56 ± 2.56 c p-value *** n.s n.s *** lsd 0.05 1.46 0.54 9.22 8.25 values are mean ± s.e. (n=9). lsd, least significant difference; ns, not significant * p<0.05; ** p<0.01; *** p<0.001 different letters indicate significant difference between values. tab. 4: effects of different doses of nahs on glucosinolate concentrations. glucosinolates (mg g-1dw) glucoiberin sinigrin 4-hydroxyglucobrassicin total aliphatics total indolics total glss glucobrassicin control 1.84 ± 0.05 a 0.74 ± 0.04 b 0.02 ± 0.01 b 0.76 ± 0.05 a 2.59 ± 0.08 a 0.78 ± 0.02 a 3.70 ± 0.05 a 0.5 mm nahs 1.49 ± 0.21 a 0.63 ± 0.09 b 0.01 ± 0.00 b 0.47 ± 0.05 a 2.12 ± 0.30 a 0.49 ± 0.08 b 2.61 ± 0.05 a 1.0 mm nahs 1.47 ± 0.20 a 1.01 ± 0.06 a 0.02 ± 0.00 b 0.50 ± 0.05 a 2.49 ± 0.27 a 0.53 ± 0.01 b 3.02 ± 0.05 a 2.5 mm nahs 0.80 ± 0.09 b 0.39 ± 0.02 c 0.03 ± 0.00 ab n.d 1.18 ± 0.07 b 0.03 ± 0.00 c 1.21 ± 0.05 a 5.0 mm nahs 0.31 ± 0.01 c 0.22 ± 0.01 c 0.04 ± 0.03 a n.d 0.53 ± 0.53 c 0.04 ± 0.00 c 0.57 ± 0.05 a p-value *** *** * n.s *** *** n.s lsd 0.05 0.44 0.17 0.016 0.11 0.6 0.7 8.25 values are mean ± s.e. (n=9). lsd, least significant difference; ns, not significant * p<0.05; ** p<0.01; *** p<0.001 different letters indicate significant difference between values. can nahs biostimulate nutritive quality and antioxidant capacity of brassica oleracea l.? 295 napus the application of h2s at 0.2 mm increased the growth in terms of plant height by 5 % (basharat ali, 2014). on the other hand in the 5 mm nahs treatment a significant decrease in shoots biomass was found (fig. 1). the application of nahs at elevated concentrations may exert a toxic effect to the b. oleraceaa indicated by the decreased biomass (koch and erskine, 2001; lamers et al., 2013). supported by the results, the effect of nahs on the shoots biomass is positive, at low dosages (0.5 and 1 mm) while the high levels (5 mm) applied are negative for the plant growth and biomass with significant losses of 55 % (fig. 1). although, the physiological mechanisms involved in the effects of the h2s are unknown, it is clear that the intervention of secondary metabolites and proteins (i.e. enzymes) and alterations in cell membranes be involved and probably not as simple disturbance in the sulphur balance in the plant (kuramata et al., 2009). on the other hand the phytotoxicity of h2s appears to result from its high affinity to the metallo-groups of proteins (beauchamp et al., 1984). both, damage to cell membranes and the interaction of h2s with cell enzymes, could explain the reduction of biomass that occurs at 2.5 and 5 mm of nahs. these results are correlated with the foliar levels of h2s found in the samples (fig. 2). these data are consistent with previous works where an increased concentration of h2s after the supply of 200 mg h2s kg-1 to wheat plants (khan et al., 2015). others reports also showed increased h2s concentrations in mulberry after application of 0.8 mm of nahs (hu et al., 2014). the main indicators of the presence or absence of stress in plants, in addition to the production of biomass, are chls levels and lipid peroxidation (van kooten and snel, 1990; ashrafi et al., 2015). in our study chl a and chl b significantly increased at 0.5 mm nahs (tab. 1). these results agreed with those obtained in spinacia oleracea after applying 0.1 mm nahs resulting in a significant increase in plant biomass, chlorophyll content and the number of grana lamel lae in chloroplasts (chen et al., 2011), and with the observation of an increased chlorophyll content when elemental sulfur was applied to wheat (triticum aestivum) (khan et al., 2015). on the contrary, in our study we observed a decrease in chl when applying the 5 mm treatments, and it is well known that the reduction in leaf pigments is a typical oxidative stress indicator, which might be attributed to pigment photo-oxidation, chlorophyll degradation and/or chlorophyll synthesis deficiency (ahmed et al., 2009). with regard to the h2o2, the higher dose of nahs (5 mm) gave rise to the highest levels (tab. 1), and could be related to a decline in biomass (fig. 1). so in this case may the overproduction of radicals, result in severe cellular damages or trigger a genetically controlled cell death program (van breusegem et al., 2001). on the other hand mda content presents a very charged increase in treatment 5 mm (67 %) relative to the control (tab. 1), which confirms that a type oxidative stress is occurring. the increases in mda and h2o2 levels and the degradation of chlorophyll were observed in brassica oleracea exposed to the highest dose (5 mm) (tab. 1), must be, direct ly or indirectly, attributable to the high dose of nahs-induced oxidative damage. however we observed a small increase in 0.5 and 1 mm treatments, which does not correspond to a decrease in biomass (tab. 1 and fig. 1). this could be explained because hydrogen peroxide production could act as a signal to trigger mechanisms involved in the antioxidant response in plants (wimalasekera et al., 2011). the toxic or beneficial effects a priori produced by nahs treat ments could affect the nutritional characteristics of plants (hu et al., 2014). therefore it is appropriate to measure the antioxidant capacity of fruits and vegetables to get an index of its health benefits (molyneux et al., 2004). there are a number of tests that can be used to measure the antioxidant capacity, among them are reducing power test, significant indicator of potential antioxidant activity and dpph free-radical scavenging test, based on the removal of 1,1-diphenyl-2picryl-hydrazyl radical. in our study we observed an increase in both test with the doses 2.5 and 5 mm (fig. 3). these results corroborate the obtained in others works that found an increase in the dpph and reducing test after application of h2s in cucumber and lotus roots (yu et al., 2013; sun et al., 2015). correlating the results obtained in the antioxidant test, exogenous toxic nahs application could be enhancing the antioxidant response through the increase in the synthesis of antioxidant compounds in treatments 2.5 and 5 mm with respect to control plant (fig. 3). one of these group of compounds are carotenoids that in our study showed a significant increase (by 22 %) when plants were subjected to 1 mm of nahs as compared with the control (tab. 2). these results are consistent with those obtained by other researchers after applying h2s to broccoli plants, and finding an increased content of carotenoids (li et al., 2014). with respect to the anthocyanins concentration, we observed a concomitant increase in the content of anthocyanins to the applied dose of nahs, presenting the highest concentrations at 5mm (tab. 2). finally, we found an increase in vitamin c content at the 2.5 and 5 mm of nahs, by 21 and 30 % respectively, and non-significant differences in the other treatments, with respect to the control (tab. 2). this increase may be resulting from the promoted ascorbate homeostasis in response to high dose of nahs. according to this, increased contents of asa were observed with the application of nahs to mulberry fruits and broccoli plants (hu et al., 2014). the highest dosages of nahs (2.5 and 5 mm) in our study promoted higher contents of natural antioxidants, which could be part of a stress response mechanism. however the application of beneficial dosages in our work as 0.5 and 1 mm of nahs potentiate the increase in concentrations of antioxidant compounds like carotenoids, several of them are precursors of vitamin a (i.e. β-carotene, γ-carotene, and β -cryptoxanthin), and due to conjugated double bonds they are both radical scavengers and quenchers of singlet oxygen. lower serum β-carotene levels have been linked to higher rates of cancer and cardiovascular diseases, as well as to increased risk of myocardial infarction among smokers (rice-evans et al., 1997). another im portant antioxidant compound which are increased in our study after application of nahs are anthocyanins. these compounds are present in various fruits and vegetables provide the natural pigmentation and exhibit a wide range of antioxidant protection and therapeutic benefits including the integrity of genomic dna, potent cardioprotective, neuroprotective, antiinflammatory, and anticarcinogenic propertie (juranić and zizak, 2005). finally, more than 85 % of vitamin c in human diets is supplied by fruits and vegetables (lee and kader, 2001). in fact vitamin c has many biological activities in human body and has been found that reduce levels of c-reactive protein (crp), a marker of inflamma tion and possibly a predictor of heart disease (block et al., 2004). therefore the increase in concentration of these compounds in leafly vegetables like brassica oleracea by the application of adequate doses of h2s as 0.5 and 1 mm can increase the production of biomass and its nutritional quality making their consumption may be beneficial to human health. in addition to the antioxidant compounds that we have just described, in the present study we observed an increase in the concentration of flavonols and total phenolics with application of 0.5 mm of nahs (tab. 3). recent works showed an increase in phenol content after the application of 15 μl l-1 of h2s in lotus roots (sun et al., 2015). the health benefits of phenolic compounds have been widely studied in recent years, including cardioprotective, anti-inflammatory, anticarcinogenic, and antimicrobial activities, mainly attributed to their antioxidant and radical scavenging properties, and their metal chelation ability (del rio et al., 2013). so the increase in flavonols concentration and total phenols with the application of 0.5 mm of nahs increases the nutritional quality of brassica oleracea. on the 296 d. montesinos-pereira, y. barrameda-medina, n. baenas, d.a. moreno, e. sánchez-rodríguez, b. blasco, j.m. ruiz other hand the flavonols and total phenols concentration decreased in 5 mm nahs treatments since this high dose could be causing a stress in the plants of brassica oleracea that inhibits the formation of these compounds (tab. 3), similar results obtain others authors when after applying 2.4 mm h2s donor nahs in broccoli plants produced a decrease in the content of total phenols (li et al., 2014). in terms of the caffeic acid derivatives and sinapic acid derivatives content not showed significant difference in any of the treatments with respect to the control. in addition to antioxidant vitamins, carotenoids, and polyphenols, brassica vegetables provide a large group of glss (plumb et al., 1996). these compounds possess rather low antioxidant activity, but the products of their hydrolysis can protect against cancer (keum et al., 2004). some authors observed in their studies that the glss content was not affected by h2s and so2 exposure, demonstrating that these sulfur compounds did not form a sink for excessive atmospheric sulfur supplied (westerman et al., 2001; aghajanzadeh et al., 2014). however in our study only the aliphatic sinigrin increased at 1 mm nahs but the 4-hidroxiglucobrasicin was slightly higher at 2.5 and 5 mm dose (tab. 4). this increase in sinigrin with the treatment 1 mm of nahs is interesting in terms of improving the nutritional quality because hydrolysis products from sinigrin (i.e. allyl isothiocyanate), is biologically active against cancer (park et al., 2013). it is also interesting that indolic glucosinolates (i.e. glucobrassicin) responded to the highest dosages, selected as toxic or dangerous for these plants, and could be also a sign of stress in the plant, since the indolic glucosinolates always respond to the stress conditions and the aliphatic glucosinolates are more influenced by the variety than to the environment (fahey et al., 2001; charron et al., 2005). besides, the higher sinigrin content can have a significant impact on the organoleptic parameters (aroma and taste) of the cabbage (banerjee et al., 2014). a decrease in the content of indolic and aliphatic glucosinolates witch 2.5 and 5 mm treatments was observed in our study and this decrease in the content of these bioactive compounds could have a negative effect on the nutritional quality of brassica oleracea. finally the application of certain doses of h2s as we have seen is an effective strategy for modulate glucosinolates profile and thus improve the nutraceutical value of brassica vegetables (banerjee et al., 2014). in view of the results obtained, we can conclude that the effect of the application of nahs in plants of brassica oleracea l. ‘bronco’ is dose-dependent (scheme 1). in the present study the application in the range of 0.5 to 1 mm of nahs to a crop of leafy vegetables like brassica oleracea seems to increase the production of antioxidant compounds such, carotenoids, anthocyanins, total phenols, flavonols, and the glucosinolate. together with an increase in the biomass doses of 0.5 to 1 mm of nahs can be defined as ‘optimal’ to improve the yield and nutritional quality of brassica oleracea var. ‘bronco’. 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protective enzymes from broccoli: isolation and elucidation of structure. proc. natl. acad. sci. u.s.a. 89, 2399-2403. zhu, l., wang, w., shi, j., zhang, w., shen, y., du, h., wu, s., 2014: hydrogen sulfide extends the postharvest life and enhances antioxidant activity of kiwi fruit during storage. j. sci. food agric. 94, 2699-2704. address of the corresponding author: david montesinos pereira e-mail: dmontesinos@ugr.es journal of applied botany and food quality 89, 299 306 (2016), doi:10.5073/jabfq.2016.089.039 1 universidade de trás-os-montes e alto douro, utad, quinta de prados, vila real, portugal 2 centre for the research and technology of agro-environmental and biological sciences citab, universidade de trás-os-montes e alto douro, utad, quinta de prados, vila real, portugal leaf age, seasonal and annual variations in salvia officinalis l. var. purpurascens biochemical characteristics f. martins1, i. oliveira2*, a. barros2, c. amaral2, s. afonso2, h. ferreira2, b. gonçalves2 (received september 15, 2016) * corresponding author summary medicinal and aromatic plants (map’s) have gain new attention in the past years due to their content in bioactive compounds and recognized health-promoting effects. one of the most important species of map’s is salvia officinalis l., rich in several phytoche micals (essential oil, phenolic compounds) and vitamins. besides, it has various uses and pharmacological effects (including antibacte rial, antiviral, antioxidative, anti-inflammatory, antidiabetic and antitumour activities). salvia officinalis l. has many cultivars, including salvia officinalis l. var. purpurascens, which is currently understudied. as few is known about this specific cultivar, characterization of this plant, as well as the study of biochemical variations occurring during its development, is of great significance. hence, in this work, young and adult leaves of salvia officinalis l. var. purpurascens, were collected in two different seasons (june and september) and in two different years (2011 and 2013). several biochemical traits were analyzed, namely carbohydrate content, photosynthetic pigments concentration, total phenolics, soluble proteins, as well as oxidation parameters (thiobarbituric acid reactive substances, thiols and electrolyte leakage). the year factor significantly influenced carbohydrate content (higher in 2013 for non-structural carbohydrates and soluble sugars, but lower for starch), but also chlorophyll and carotenoid content (higher in 2011), with a similar influence recorded for the season of harvest (higher values for starch, chlorophyll and carotenoids in september, but lower for soluble sugars). the developmental stage of leaves showed significant influence mainly in the content of photosynthetic pigments, with higher values of chlorophyll and carotenoids recorded in young leaves. the results show the biochemical variations occurring in plants of salvia officinalis l. var. purpurascens, during developmental stages, and others associated to season of harvest and year, and their relation to climatic factors. the gathered data, besides useful for the characterization of this plant, is also valuable when aiming for the optimization of sage cultivation. keywords salvia officinalis l. var. purpurascens; carbohydrates; chlorophyll, carotenoids; total phenolics introduction in recent years, there has been an increased interest in herbs and spices, due to their documented and studied beneficial health effects. these plants have in their composition a large number of bioactive and health-promoting compounds, including vitamins, terpenoids and polyphenols, with several studies showing their beneficial effects in cancer, cardiovascular disease and neurodegenerative disorders (halliwell, 1996). one of the most studied aromatic plants is salvia officinalis l. – common sage – native to the mediterranean region and asia minor, which has been used in folk medicine for centuries, it is known to have pharmacological effect, which resulted in its inclusion in pharmacopoeias throughout the world (tepe, 2008). together with the increased demand of new sources of bio active compounds, based in a global trade of herb-based products that reached an estimated amount s$ 60 000 million in 2000 (world health organization, 2003), renovated interest in sage has been triggered. due to its economic value, this plant has been included in cultivation systems, which can help to achieve higher plant quality (schippmann et al., 2006). however, sage is a droughtsusceptible species (tounekti et al., 2011), and in the mediterranean areas, this plant in under summer drought stress, together with high temperatures, which can lead to variations on several characteristics. some available works regarding salvia officinalis l. focused on the variations of growth parameters, volatile composition, essential oils and phenolics, caused by the type of cultivation (field or greenhouse) (yi and wetzstein, 2010), saline stress (taârit et al., 2012), low light conditions (mapes and xu, 2014) or water deficit (bettaieb et al., 2011) rather than in biochemical parameters of this plant. furthermore, salvia officinalis l. has many known varieties, being salvia officinalis var. purpurascens one of the less studied ones. hence, it is of essential interest to study the biochemical variations occurring in sage leaves that can be of great importance to achieve maximum quality, from the producers’ standpoint, but also from consumer’s point of view. therefore, in this work we present results from biochemical characteristics, and their variation caused by leaf age, season or year in salvia officinalis var. purpurascens. materials and methods plant material salvia officinalis l. var. purpurascens plants were collected from the botanical garden of the university of trás-os-montes e alto douro (utad), vila real (41º19’ n, 7º44’ w, 450 m above sea level), having the herbarium specimen number “hvr13737, gerês, 05-11-2007, j.m. neves”. plants are on a dystric cambisol (nonhumic litholic) derived from shale. is presents a medium texture (fine-sandy) with acidic ph (5.4), a percentage of organic matter of 1.45 and average phosphorus (63 ppm) and very high potassium (348 ppm) contents. no fertilization is applied, and watering of plants is performed regularly. climatic data was recorded by a standard weather station located near the experimental site. the study was carried out in 2011, 2012 and 2013, but, considering the differences regarding several climatic conditions (namely precipitation, temperature and total radiation) recorded for 2011 and 2013 (fig. 1) (while conditions were similar between 2012 and 2013), we choose to present result concerning these two years. healthy leaves, presenting two developmental stages (young, collected from the upper part of the plant and adult, collected from the middle third), were collected at two different harvest dates (june and september) and 300 f. martins, i. oliveira, a. barros, c. amaral, s. afonso, h. ferreira, b. gonçalves years (2011 and 2013). samples were obtained from twelve 4 yearsold plants and eight repetitions of all methodologies were performed in randomly selected leaves. leaf discs were prepared in the field, deep-frozen in liquid nitrogen and stored at -80 ºc until analysis. carbohydrate content carbohydrate content was measured using the methodology of irigoyen et al. (1992), by heating (80 °c) one leaf disc in 80 % (v/v) ethanol/distilled water solution, for 1 hour. afterwards, 0.2 ml of the previous extract and 3 ml of anthrone were mixed and placed in a water bath at 100 ºc, during 10 minutes. the liquid fraction was used for soluble sugar quantifications and the solid fraction was used for starch analysis. the solid fraction was extracted with 30 % perchloric acid and quantified according to osaki et al. (1991), following the anthrone procedure described in the soluble sugars methodology, using glucose as standard in both methodologies. photosynthetic pigments the quantification of chlorophyll (cla and clb) and total carotenoids was performed using the methods of sesták et al. (1971) and lichtenthaler (1987), respectively, by spectrophotometry in extracts of 80 % acetone with distilled water (v/v). total phenolics a modified procedure of the folin-ciocalteu method (tsao et al., 2003) was used for accessing the concentration of phenolic compounds, using the same extracts obtained for the quantification of photosynthetic pigments. results are expressed as mg of gallic acid equivalents, using a calibration curve obtained by preparing different known concentrations of gallic acid, measured using the same procedure. soluble proteins soluble proteins were quantified by homogenising samples in a grinding medium containing 50 mm phosphate buffer (ph 7.5), 0.1 mm ethylenediaminetetraacetic acid (edta), 100 mm phenyl methylsulfonyl fluoride (pmsf) and 2 % (w/v) polyvinylpyrolli done (pvp), followed by centrifugation at 22 000 g for 30 minutes, at 4 ºc. absorbance was read at 595 nm, and bovine serum albumin (bsa) was used as a standard (bradford, 1976). thiobarbituric acid reactive substances the concentration of total thiobarbituric acid reactive substances (tbars) was calculated to evaluate membrane integrity. lipid peroxidation in salvia officinalis var. purpurascens leaves was esti mated following the method described in bacelar et al. (2006). briefly, samples previously frozen with liquid nitrogen were ground in 3 ml of 20 % (w/v) trichloroacetic acid (tca) using mortar and pestle, homogenized and centrifuged at 3500 g for 20 min. afterwards, 1 ml of the supernatant was added to 1 ml of 20 % tca containing 0.5 % (w/v) thiobarbituric acid and to 100 μl 4 % (w/v) butylated hydroxytoluene (bht). this mixture was heated at 95 ºc for 30 min, quickly cooled in an ice bath and centrifuged at 10,000 g for 20 min and the absorbance of the supernatant was measured at 532 nm. the value for the non-specific absorption at 600 nm was subtracted. the tbars concentration was expressed in terms of nmol/g and nmol/cm2, using an extinction coefficient of 155,000 m-1 cm-1. thiols total thiol content (–sh) of soluble protein extract was assayed as described in bacelar et al. (2006), using 5,5-dithiobis (2-nitrobenzoic acid) (dtnb). electrolyte leakage electrolyte leakage was measured following the methodology of lutts et al. (1996). leaf discs were washed with deionised water to remove surface-adhered electrolytes, placed in closed vials containing 10 ml of deionised water and incubated at 25 ºc on a rotary shaker for 24 h. afterwards, electrical conductivity (ec1) of the solution was determined. samples were then autoclaved at 120 ºc for 20 min and the electrical conductivity (ec2) was obtained after equilibration at 25 ºc. the electrolyte leakage was calculated as follows: electrolyte leakage = × 100. statistical analysis data are presented as mean ± standard deviation, and the results presented by weight refer to dry weight of sage leaves. differences among means were determined by analysis of variance (anova), using spss (statistical package for social sciences) software, version 19.0 (ibm corporation, new york, u.s.a.) software. the fulfilment of the anova requirements, namely the normal distribution of the residuals and the homogeneity of variance, were evaluated by means of the kolmogorov-smirnov with lilliefors correction (if n > 50) or the shapiro-wilk’s test (if n < 50), and the levene’s tests, respectively. fig. 1: average monthly temperature (°c), total monthly precipitation (mm) and global solar radiation (kj/m2) for 2011 and 2013. ec1 ec2 salvia officinalis l. biochemical dynamics 301 results and discussion there are few previous works regarding the analyzed biochemical parameters of salvia officinalis var. purpurascens, thus, most of the comparisons here presented are made with salvia officinalis related works (rather than with var. purpurascens), unless stated otherwise. the sampling year proved to be the factor that significantly influenced a higher number of parameters (tab. 1, 2 and 3). considering all the results, sixteen of the twenty-five analyzed biochemical parameters were affected by the year of study. of those, non-structural carbohydrates, soluble sugars, soluble sugars/starch ratio, total chlorophyll/total carotenoids ratio, total phenolics and electrolytes leakage presented higher values in 2013, while starch, chlorophyll a (expressed as mg/g), total chlorophyll (expressed as mg/g), total carotenoids, and tbars (expressed as nmol/g) showed higher values in 2011. seasonal variations of the studied biochemical parameters were also detected (tab. 1, 2 and 3). of those significantly affected by the sampling date, starch, chlorophyll a, total chlorophyll (expressed as mg/g) and total carotenoids were present in higher amounts in leaves collected in september, while soluble sugars, soluble sugars/starch, total chlorophyll (expressed as mg/ dm2) and thiols were detected in higher concentrations when leaves were collected in june. finally, the leaf developmental stage caused fewer significant variations in biochemical parameters of sage. the leaf age (young or adult) only significantly affected ten out of 25 parameters. of those, only sugars/starch ratio was higher in adult leaves, while content of chlorophyll a, chlorophyll b (expressed as mg/dm2), total chlorophyll, total carotenoids and tbars was higher in young leaves. few works are available regarding carbohydrate (soluble sugars and/ or starch) content of salvia officinalis. sahar and colleagues (2011) recorded contents for soluble sugars of about 9 mg/g of fresh leaf, while castrillo et al. (2005) refer about 1.25 g/m2. when reporting our data in fresh weight (the leaves tested in this work had an average of 73 % of water content – data not shown), a content of about 50 mg/g of dry weight will correspond to about 13.5 mg/g fresh weight, similar to the reported 9 mg/g of sahar et al. (2011). the values of 151.34±8.67 mg/g will represent over 40 mg/g expressed in fresh weight, considerably higher than those previous reports indicate. on the other hand, the values reported by castrillo et al. (2005) (1.25 g/m2, corresponding to 12.5 mg/dm2) can also be considered similar to the ones detected in the present work (ranging from 4.57±0.31 mg/dm2 in leaves from 2011 to 15.36±0.76 in leaves from 2013). the concentration of soluble sugars has been correlated to specific leaf mass, as well as with irradiance, but also to water stress, as they are known osmoprotectants and carbon sources (castrillo et al., 2005). total carbohydrate content of sage has been reported to be 15 μg/mg fresh weight (pellegrini et al., 2015), well below the content detected by us, of around 54 μg/mg fresh weight (again, if considering 73 % of water content). no reports concerning the starch content of sage are known. however, starch content in other aro matic and medicinal plants has been reported to present seasonal changes, increasing through spring until autumn (kofidis et al., 2007). the lower value of starch in june, combined with higher values of soluble sugars, with an inverse behaviour in september, can be related to the mobilization of starch and its conversion in soluble sugars, as detected in other species. this will take place in order to support the increased metabolism during the flowering period of sage, which occurs between april and july, but always depending of climatic conditions. as sage is an important aromatic plant, effects of the composition on processing parameters (including cutting, drying or freezing) is of interest. no data regarding how carbohydrate content of sage can influence or be influenced by processing parameters is available. nonetheless, reports concerning the effects of drying methodologies in the sugar content of several plants show considerable variations, depending on the selected process (e.g. gao et al., 2012). concerning the chlorophyll content found in leaves of sage in the present work, similar amounts have been previously reported (castrillo et al., 2005; bettaieb et al., 2011; tounekti et al., 2012). however, many reports also indicate higher (mapes and xu, 2014; pellegrini et al., 2015) or lower values (nasta et al., 2014). several factors affecting the content of chlorophyll in sage, including ozone stress, light conditions, salinity, drought and temperature, can help to explain some of the variations detected between 2011 and 2013. higher values for chlorophyll june, with a decrease in september can be related to the flowering period of sage. in fact, the work of coisin et al. (2010), with salvia nemorosa, recorded an increase in chlorophyll content when plants are in the anthesis stage, probably due to increased sugar synthesis, to support the metabolism of the plant. furthermore, values of precipitation show a considerable difference between the amount of water that will be available for plants (average of both years for june is 5.6 mm, and is september of 67.8 mm). considering that salvia officinalis is a drought-susceptible species (tounekti et al., 2011) this factor can also help to explain the recorded variations on chlorophyll content between june and september. the beginning of leaf senescence can be the cause for the decrease of chlorophyll content in adult leaves. in fact, during the senescence process, were reserves are mobilized for younger tissues, like developing leaves or flowers (abreu and munné-bosch, 2007), chloroplasts are one of the first organelles to be subjected to senescence (dangl et al., 2000). significant variations were also detected in total carotenoids, correlated to all the factors under study (age, season and year). although similar values have been previously published (tounekti et al., 2012), other authors point out lower (tounekti et al., 2011) or higher content (mapes and xu, 2014). several factors are known to influence carotenoid content in sage, like ozone (pellegrini et al., 2015), salinity (tounekti et al., 2012) or light levels (mapes and xu, 2014). our results show significant differences between young and adult leaves in the total carotenoid content, as detected for the chlorophyll content, which may be due to the onset of senescence that leads to losses of the compounds (abreu and munné-bosch, 2007). furthermore, differences in total carotenoid content between seasons and years are likely related to climatic factors, known to influence carotenoid content (munné-bosch and alegre 2002), as they protect cellular structures by dissipating excess energy reaching the chloroplast, and by preventing the formation and/or scavenge any singlet oxygen that can result in lipid peroxidation in photosynthetic membranes. in fact, it was in 2011 that higher carotenoids and total radiation were recorded, results that may indicate a response to avoid oxida tive damage caused by excess radiation. however, this same rationale cannot be used for results regarding seasonal variation. in fact, total radiation was lower in september; contrarily to what was found to carotenoid content (higher in september), indicating that other stress-causing factors, namely low rainfall, that occurred in june, may be affecting carotenoids content. no data was found regarding the correlation between processing of sage and its content in chlorophylls and carotenoids. however, data concerning other aromatic plants indicate a considerable reduction of the content of those compounds with processing (divya et al., 2012). it can be expected that, during processing, leaves containing higher levels of carotenoids are less prone to suffer oxidative processes, as these compounds are known to have strong antioxidant activity (e. g. krinsky, 1989). for total phenolic content (tab. 3), the recorded values can be considered similar to those found in previous works (e.g. taârit et al., 2012; yi and wetzstein, 2012). however, other reports are avail able (roby et al., 2013) that obtained different values of phenolic content in sage. these may be linked to several factors, such as growing conditions, but also to different approaches in the quantification of these compounds. for phenolics, interaction between 302 f. martins, i. oliveira, a. barros, c. amaral, s. afonso, h. ferreira, b. gonçalves tab. 1: values (mean ± standard deviation) for carbohydrate content of salvia officinalis leaves and probability levels of the effects of age, season and year, as determined by anova. ns: not significant. in bold, results that showed to be affected by the studied factors and/or their interaction. non-structural non-structural starch starch soluble sugars soluble sugars soluble carbohydrates carbohydrates sugars/ (mg/dm2) (mg/g) (mg/dm2) (mg/g) (mg/dm2) (mg/g) starch age (a) young 194.91±68.16 196.92±68.15 94.96±63.60 99.64±75.04 99.95±55.63 97.28±47.62 3.06±4.33 old 202.30±80.35 213.69±80.35 102.95±80.93 109.27±83.19 99.35±71.19 104.43±75.93 6.02±10.03 season (s) june 189.97±75.64 193.98±58.36 72.26±78.50 76.19±85.11 117.61±72.23 117.79±74.41 8.36±9.67 september 207.34±86.99 216.63±86.99 125.65±54.71 132.73±60.89 81.68±47.74 83.91±43.92 0.71±0.49 year (y) 2011 163.45±67.14 184.92±45.59 117.72±60.97 134.55±78.41 45.73±17.56 50.37±19.82 0.48±0.29 2013 233.76±63.79 225.70±67.34 80.19±78.64 74.36±67.69 153.57±43.03 151.34±49.04 8.59±9.49 a*s young*june 164.71±46.24 149.49±46.24 47.02±36.94 41.47±28.61 117.68±64.91 108.02±60.65 5.52±5.08 young*september 225.12±74.27 244.35±74.27 142.90±45.75 157.82±60.00 82.21±38.88 86.53±27.62 0.59±0.26 old*june 215.04±56.57 238.48±59.57 97.49±100.07 110.90±107.62 117.55±81.05 127.58±86.97 11.21±12.26 old*september 189.56±58.36 188.92±97.19 108.41±58.81 107.64±52.13 81.15±56.56 81.28±56.64 0.82±0.63 a*y young*2011 157.49±45.59 180.55±45.59 103.59±42.87 121.29±78.88 53.89±15.75 59.26±18.83 0.62±0.30 young*2013 232.33±67.34 213.29±67.34 86.33±79.77 77.99±66.46 146.00±40.29 135.29±35.35 5.49±5.09 old*2011 169.39±84.63 189.29±84.63 131.85±73.61 147.82±78.16 37.55±15.71 41.47±16.99 0.35±0.21 old*2013 235.20±80.35 238.11±62.21 74.06±79.61 70.72±70.90 161.14±45.62 167.39±56.31 11.68±11.82 s*y june*2011 182.32±75.64 190.03±75.54 130.87±73.33 139.06±80.72 51.45±18.15 50.97±9.09 0.54±0.36 june*2013 197.42±35.56 197.93±34.56 13.65±5.49 13.31±4.63 183.77±33.39 184.63±4.85 16.19±7.91 september*2011 144.56±53.28 179.80±53.24 104.57±43.98 130.04±78.40 39.99±15.42 49.76±26.99 0.43±0.21 september*2013 270.11±66.31 253.46±66.31 146.65±57.47 135.41±38.72 123.37±27.68 118.06±27.74 0.98±0.53 a*s*y young*june*2011 134.88±42.63 118.26±42.62 76.24±31.01 66.42±17.94 58.64±19.81 51.83±10.56 0.83±0.27 young*june*2013 194.52±27.06 180.73±27.06 17.80±3.16 16.52±2.93 176.72±25.85 164.21±23.52 10.20±2.25 young*september*2011 180.10±38.33 242.85±38.33 130.94±35.59 176.16±78.29 49.15±9.37 66.69±22.84 0.40±0.14 young*september*2013 270.14±75.62 245.74±75.62 154.86±53.77 139.47±28.58 115.28±25.55 106.37±14.59 0.79±0.19 old*june*2011 229.76±72.80 261.81±72.80 185.50±61.16 211.70±39.75 44.26±13.98 50.11±7.92 0.24±0.05 old*june*2013 200.32±42.52 215.14±42.52 9.49±3.88 10.10±3.72 190.83±40.19 205.04±49.29 22.18±6.86 old*september*2011 109.03±41.45 116.76±41.45 78.19±35.89 83.93±46.71 30.84±15.18 32.83±19.62 0.46±0.26 old*september*2013 270.11±66.31 261.08±60.86 138.62±63.53 131.34±48.56 131.46±28.96 129.74±33.53 1.18±0.69 f values and probability levels age (a) 0.314n.s. 1.762n.s. 0.575n.s. 0.884n.s. 0.010n.s. 1.212n.s. 21.232*** season (s) 1.753n.s. 3.211n.s. 25.678*** 30.514*** 35.357*** 27.222*** 142.081*** year (y) 28.428*** 10.411** 12.685** 34.584*** 318.585*** 241.630*** 159.238*** a*s 10.604** 32.637*** 16.258*** 34.138*** 0.006n.s. 3.643n.s. 18.146*** a*y 0.117n.s. 0.405n.s. 3.689n.s. 2.725n.s 6.790* 14.742*** 25.235*** s*y 17.529*** 6.767* 57.211*** 41.022*** 16.408*** 25.310*** 138.255*** a*s*y 9.208** 24.547*** 13.367*** 33.169*** 0.062n.s. 0.319n.s. 22.700*** salvia officinalis l. biochemical dynamics 303 ta b. 2 : v al ue s (m ea n ± st an da rd d ev ia tio n) fo r p ho to sy nt he tic p ig m en ts o f s al vi a of fic in al is le av es a nd p ro ba bi lit y le ve ls o f t he e ff ec ts o f a ge , s ea so n an d y ea r, as d et er m in ed b y a n o va . n s: n ot s ig ni fic an t. in b ol d, re su lts th at s ho w ed to b e af fe ct ed b y th e st ud ie d fa ct or s an d/ or th ei r i nt er ac tio n. c hl or op hy ll a c hl or op hy ll a c hl or op hy ll b c hl or op hy ll b to ta l to ta l c la /c lb to ta l to ta l to ta l ch lo ro ph yl l ch lo ro ph yl l ca ro te no id s ca ro te no id s ch lo ro ph yl l (m g/ dm 2 ) (m g/ g) (m g/ dm 2 ) (m g/ g) (m g/ dm 2 ) (m g/ g) (m g/ dm 2 ) (m g/ g) /c ar ot en oi ds a ge (a ) y ou ng 3. 54 ±0 .6 3 3. 73 ±1 .5 3 1. 46 ±0 .5 9 1. 55 ±0 .8 5 4. 99 ±1 .1 8 5. 29 ±2 .2 9 2. 56 ±0 .4 2 1. 07 ±0 .2 0 1. 12 ±0 .4 3 4. 72 ±1 .0 5 o ld 2. 89 ±0 .6 3 3. 10 ±0 .7 9 1. 17 ±0 .4 4 1. 26 ±0 .5 1 4. 06 ±0 .9 6 4. 36 ±1 .1 9 2. 62 ±0 .5 1 0. 87 ±0 .1 2 0. 94 ±0 .1 9 4. 66 ±0 .8 3 se as on (s ) ju ne 2. 95 ±0 .6 6 3. 00 ±0 .7 1 1. 25 ±0 .5 6 1. 28 ±0 .6 1 4. 19 ±1 .1 2 4. 28 ±1 .2 2 2. 53 ±0 .5 1 0. 94 ±0 .1 6 0. 95 ±0 .1 5 4. 53 ±1 .1 2 se pt em be r 3. 48 ±0 .6 8 3. 83 ±1 .5 2 1. 38 ±0 .5 0 1. 54 ±0 .7 8 4. 86 ±1 .1 3 5. 37 ±2 .2 4 2. 64 ±0 .4 2 1. 01 ±0 .2 2 1. 11 ±0 .4 5 4. 84 ±0 .6 9 y ea r (y ) 20 11 3. 33 ±0 .7 5 3. 82 ±1 .5 5 1. 32 ±0 .5 2 1. 51 ±0 .7 8 4. 65 ±1 .2 4 5. 33 ±2 .2 7 2. 66 ±0 .3 9 1. 05 ±0 .2 1 1. 18 ±0 .4 2 4. 44 ±0 .8 2 20 13 3. 09 ±0 .6 7 3. 02 ±0 .6 7 1. 31 ±0 .5 5 1. 30 ±0 .6 2 4. 41 ±1 .0 9 4. 32 ±1 .1 9 2. 51 ±0 .5 2 0. 89 ±0 .1 4 0. 87 ±0 .1 3 4. 92 ±0 .9 9 a *s y ou ng *j un e 3. 33 ±0 .6 4 3. 10 ±0 .7 9 1. 39 ±0 .6 9 1. 31 ±0 .7 5 4. 72 ±1 .2 8 4. 41 ±1 .4 9 2. 55 ±0 .4 3 1. 04 ±0 .1 4 0. 96 ±0 .1 4 4. 60 ±1 .3 5 y ou ng *s ep te m be r 3. 74 ±0 .6 3 4. 37 ±1 .8 4 1. 53 ±0 .4 8 1. 79 ±0 .8 9 5. 27 ±1 .0 4 6. 16 ±2 .6 4 2. 56 ±0 .4 4 1. 10 ±0 .2 5 1. 28 ±0 .5 6 4. 83 ±0 .6 3 o ld *j un e 2. 57 ±0 .4 2 2. 90 ±0 .6 3 1. 09 ±0 .3 7 1. 24 ±0 .4 6 3. 67 ±0 .5 9 4. 14 ±0 .8 9 2. 52 ±0 .5 9 0. 83 ±0 .0 9 0. 94 ±0 .1 7 4. 46 ±0 .8 6 o ld *s ep te m be r 3. 21 ±0 .6 5 3. 29 ±0 .9 1 1. 24 ±0 .4 9 1. 28 ±0 .5 6 4. 46 ±1 .1 1 4. 57 ±1 .4 2 2. 72 ±0 .4 0 0. 92 ±0 .1 4 0. 94 ±0 .2 2 4. 84 ±0 .7 8 a *y y ou ng *2 01 1 3. 63 ±0 .7 3 4. 25 ±1 .9 5 1. 48 ±0 .5 0 1. 73 ±0 .9 2 5. 11 ±1 .1 7 5. 98 ±2 .7 9 2. 55 ±0 .3 7 1. 17 ±0 .2 1 1. 34 ±0 .5 2 4. 37 ±0 .7 4 y ou ng *2 01 3 3. 44 ±0 .5 8 3. 22 ±0 .6 9 1. 45 ±0 .6 8 1. 38 ±0 .7 5 4. 88 ±1 .2 2 4. 59 ±1 .4 3 2. 56 ±0 .4 9 0. 97 ±0 .1 4 0. 90 ±0 .1 0 5. 06 ±1 .2 1 o ld *2 01 1 3. 03 ±0 .6 7 3. 39 ±0 .8 8 1. 16 ±0 .5 2 1. 29 ±0 .5 6 4. 19 ±1 .6 5 4. 68 ±1 .4 0 2. 76 ±0 .4 1 0. 92 ±0 .1 3 1. 03 ±0 .1 9 4. 52 ±0 .9 2 o ld *2 01 3 2. 75 ±0 .5 7 2. 81 ±0 .5 9 1. 17 ±0 .3 5 1. 22 ±0 .4 6 3. 93 ±0 .7 2 4. 04 ±0 .8 6 2. 47 ±0 .5 7 0. 82 ±0 .0 9 0. 84 ±0 .1 4 4. 79 ±0 .7 4 s* y ju ne *2 01 1 2. 79 ±0 .3 9 2. 94 ±0 .6 9 1. 01 ±0 .1 9 1. 06 ±0 .2 8 3. 81 ±0 .5 8 4. 00 ±0 .9 7 2. 79 ±0 .2 0 0. 97 ±0 .1 7 1. 00 ±0 .1 7 3. 97 ±0 .3 9 ju ne *2 01 3 3. 11 ±0 .8 3 3. 06 ±0 .7 4 1. 48 ±0 .7 1 1. 49 ±0 .7 7 4. 59 ±1 .3 9 4. 55 ±1 .4 0 2. 28 ±0 .6 0 0. 90 ±0 .1 4 0. 89 ±0 .1 1 5. 09 ±1 .3 2 se pt em be r* 20 11 3. 87 ±0 .6 4 4. 69 ±1 .6 9 1. 63 ±0 .5 8 1. 96 ±0 .8 7 5. 49 ±1 .1 5 6. 65 ±2 .4 4 2. 53 ±0 .5 0 1. 13 ±0 .2 2 1. 36 ±0 .5 1 4. 92 ±0 .8 7 se pt em be r* 20 13 3. 09 ±0 .6 7 2. 97 ±0 .6 1 1. 14 ±0 .2 4 1. 11 ±0 .3 4 4. 22 ±0 .6 9 4. 08 ±0 .9 3 2. 75 ±0 .5 9 0. 89 ±0 .1 4 0. 85 ±0 .1 4 4. 75 ±0 .4 8 a *s *y y ou ng *j un e* 20 11 3. 04 ±0 .3 3 2. 81 ±0 .5 9 1. 15 ±0 .1 3 1. 06 ±0 .2 3 4. 18 ±0 .4 5 3. 87 ±0 .8 2 2. 65 ±0 .1 0 1. 08 ±0 .1 6 0. 99 ±0 .1 8 3. 91 ±0 .3 1 y ou ng *j un e* 20 13 3. 62 ±0 .7 5 3. 39 ±0 .8 9 1. 65 ±0 .9 2 1. 56 ±1 .0 0 5. 27 ±1 .6 3 4. 96 ±1 .8 6 2. 46 ±0 .5 9 1. 00 ±0 .1 2 0. 93 ±0 .0 9 5. 29 ±1 .6 5 y ou ng *s ep te m be r* 20 11 4. 23 ±0 .4 7 5. 68 ±1 .7 5 1. 81 ±0 .5 2 2. 40 ±0 .8 6 6. 03 ±0 .8 8 8. 08 ±2 .4 4 2. 46 ±0 .5 1 1. 26 ±0 .2 3 1. 69 ±0 .5 3 4. 84 ±0 .7 6 y ou ng *s ep te m be r* 20 13 3. 26 ±0 .2 9 3. 05 ±0 .4 4 1. 25 ±0 .2 2 1. 19 ±0 .3 7 4. 50 ±0 .4 2 4. 24 ±0 .7 8 2. 66 ±0 .3 6 0. 94 ±0 .1 5 0. 88 ±0 .1 1 4. 82 ±0 .5 3 o ld *j un e* 20 11 2. 55 ±0 .3 0 3. 07 ±0 .8 4 0. 88 ±0 .1 4 1. 06 ±0 .3 4 3. 43 ±0 .4 4 4. 13 ±1 .1 5 2. 93 ±0 .1 9 0. 86 ±0 .0 9 1. 01 ±0 .1 8 4. 03 ±0 .4 7 o ld *j un e* 20 13 2. 59 ±0 .5 4 2. 73 ±0 .3 9 1. 32 ±0 .4 0 1. 41 ±0 .5 1 3. 91 ±0 .6 6 4. 15 ±0 .6 3 2. 11 ±0 .5 9 0. 81 ±0 .0 8 0. 86 ±0 .1 2 4. 89 ±0 .9 7 o ld *s ep te m be r* 20 11 3. 51 ±0 .5 9 3. 70 ±0 .8 9 1. 45 ±0 .6 1 1. 52 ±0 .6 7 4. 96 ±1 .1 7 5. 22 ±1 .4 9 2. 60 ±0 .5 2 0. 99 ±0 .1 2 1. 04 ±0 .2 1 5. 01 ±1 .0 2 o ld *s ep te m be r* 20 13 2. 92 ±0 .5 9 2. 89 ±0 .7 8 1. 03 ±0 .2 3 1. 03 ±0 .3 2 3. 95 ±0 .8 1 3. 92 ±1 .0 8 2. 84 ±0 .2 1 0. 84 ±0 .1 2 0. 83 ±0 .1 8 4. 69 ±0 .4 7 f va lu es a nd p ro ba bi lit y le ve ls a ge (a ) 25 .3 24 ** * 7. 79 9* * 6. 09 1* 3. 92 8n .s . 17 .1 83 ** * 7. 02 3* 0. 34 8n .s . 31 .3 35 ** * 9. 40 4* * 0. 07 4n .s . se as on (s ) 17 .1 54 ** * 13 .4 85 ** 1. 32 6n .s . 2. 98 9n .s . 8. 69 6* * 9. 67 9* * 0. 31 6n .s . 4. 43 7* 7. 35 2* * 1. 96 1n .s . y ea r ( y ) 3. 44 3n .s . 12 .3 77 ** 0. 00 7n .s . 2. 02 1n .s . 1. 19 7n .s . 8. 29 8* * 0. 18 0n .s . 17 .9 10 ** * 26 .7 66 ** * 4. 83 3* a *s 0. 80 7n .s . 3. 67 8n .s . 0. 00 4n .s . 2. 23 5n .s . 0. 29 4n .s . 3. 52 3n .s . 0. 36 0n .s . 0. 07 4n .s . 7. 37 0* * 0. 13 0n .s . a *y 0. 11 5n .s . 0. 99 5n .s . 0. 02 8n .s . 0. 88 6n .s . 0. 01 1n .s . 1. 09 4n .s . 0. 17 4n .s . 1. 88 4n .s . 4. 41 6* 0. 89 8n .s . s* y 18 .4 80 ** * 16 .4 86 ** * 16 .3 37 ** * 18 .3 29 ** * 20 .7 78 ** * 19 .7 96 ** * 0. 00 1n .s . 5. 85 8* 11 .5 54 ** 8. 85 4n .s . a *s *y 3. 27 1n .s . 9. 13 8* * 0. 17 8n .s . 2. 11 2n .s . 1. 55 0n .s . 6. 62 5* 0. 11 9n .s . 0. 90 5n .s . 8. 23 0* * 0. 06 3n .s . 304 f. martins, i. oliveira, a. barros, c. amaral, s. afonso, h. ferreira, b. gonçalves ta b. 3 : v al ue s (m ea n ± st an da rd d ev ia tio n) f or p he no lic , t b a r s, p ro te in a nd th io ls c on te nt , a nd e le ct ro ly te le ak ag e, o f sa lv ia o ffi ci na lis le av es a nd p ro ba bi lit y le ve ls o f th e ef fe ct s of a ge , s ea so n an d y ea r, as de te rm in ed b y a n o va . n s: n ot s ig ni fic an t. in b ol d, re su lts th at s ho w ed to b e af fe ct ed b y th e st ud ie d fa ct or s an d/ or th ei r i nt er ac tio n. to ta l p he no lic s to ta l p he no lic s t b a r s t b a r s p ro te in p ro te in t hi ol s e le ct ro ly te s (m g g a /d m 2 ) (m g g a /g ) (n m ol /c m 2 ) (n m ol /g ) (m g/ dm 2 ) (m g/ g) (n m ol /m g pr ot ei n) le ak ag e (% ) a ge (a ) y ou ng 39 .4 4± 12 .6 7 37 .4 9± 12 .5 5 3. 02 ±1 .7 8 31 3. 89 ±1 91 .8 4 13 6. 45 ±1 51 .0 8 14 6. 26 ±1 76 .9 2 52 .5 9± 60 .4 6 14 .8 7± 2. 48 o ld 36 .3 1± 11 .2 0 40 .1 2± 10 .3 4 1. 43 ±0 .4 9 15 3. 19 ±5 2. 29 12 8. 72 ±9 3. 58 13 6. 33 ±9 7. 18 41 .6 9± 34 .7 6 15 .1 2± 4. 90 se as on (s ) ju ne 38 .4 9± 9. 86 38 .9 9± 10 .4 8 2. 18 ±1 .7 8 21 5. 25 ±1 61 .9 2 14 5. 72 ±1 57 .1 2 15 0. 33 ±1 63 .2 4 76 .3 6± 54 .9 2 14 .7 0± 2. 31 se pt em be r 37 .2 5± 13 .8 9 38 .6 2± 12 .5 7 2. 27 ±1 .2 3 25 1. 83 ±1 61 .0 5 11 9. 44 ±8 1. 07 13 2. 26 ±1 18 .2 3 17 .9 3± 12 .1 3 15 .1 8± 4. 53 y ea r (y ) 20 11 32 .6 7± 12 .3 8 35 .2 9± 11 .5 2 2. 96 ±1 .7 5 31 2. 12 ±1 79 .5 5 11 8. 98 ±1 03 .6 1 13 6. 88 ±1 33 .2 9 52 .5 3± 62 .7 0 12 .8 8± 2. 72 20 13 43 .0 8± 9. 05 42 .3 1± 10 .4 8 1. 49 ±0 .7 3 14 5. 96 ±6 9. 23 14 6. 19 ±1 43 .1 7 14 5. 71 ±1 51 .6 2 41 .7 6± 30 .5 5 15 .6 3± 3. 94 a *s y ou ng *j un e 39 .6 6± 9. 91 36 .3 2± 9. 11 2. 99 ±2 .1 9 27 6. 02 ±2 05 .4 3 15 2. 13 ±1 96 .4 1 14 9. 44 ±2 07 .6 9 88 .1 2± 68 .7 5 15 .4 2± 2. 54 y ou ng *s ep te m be r 39 .2 1± 15 .2 8 38 .6 6± 15 .4 8 3. 04 ±1 .3 1 35 1. 76 ±1 75 .5 0 12 0. 77 ±8 9. 85 14 3. 07 ±1 46 .7 2 17 .0 7± 11 .6 1 14 .5 3± 2. 47 o ld *j un e 37 .3 4± 9. 98 41 .6 6± 11 .3 5 1. 36 ±0 .5 8 15 4. 48 ±6 4. 40 13 9. 32 ±1 11 .1 5 15 1. 21 ±1 09 .2 3 64 .6 0± 34 .7 9 13 .9 9± 2. 31 o ld *s ep te m be r 35 .2 9± 12 .5 5 38 .5 7± 9. 32 1. 51 ±0 .3 9 15 1. 90 ±3 8. 72 11 8. 11 ±7 4. 19 12 1. 45 ±8 4. 35 18 .7 9± 12 .9 5 15 .8 3± 5. 95 a *y y ou ng *2 01 1 36 .3 1± 14 .5 7 35 .1 3± 13 .9 6 4. 34 ±1 .4 0 46 9. 21 ±1 31 .1 2 11 1. 28 ±9 9. 56 13 4. 63 ±1 53 .4 8 58 .9 3± 77 .5 3 14 .9 7± 2. 03 y ou ng *2 01 3 42 .5 6± 9. 93 39 .8 5± 10 .8 9 1. 69 ±0 .9 0 15 8. 67 ±8 6. 05 16 1. 63 ±1 89 .5 0 15 7. 88 ±2 02 .0 9 46 .2 6± 38 .1 9 14 .8 4± 2. 65 o ld *2 01 1 29 .0 4± 8. 75 35 .4 5± 8. 90 1. 57 ±0 .5 0 17 3. 04 ±5 1. 46 12 6. 67 ±1 10 .2 3 13 9. 13 ±1 14 .6 8 46 .1 3± 45 .0 3 10 .8 0± 1. 29 o ld *2 01 3 43 .5 9± 8. 36 44 .7 8± 9. 76 1. 29 ±0 .4 5 13 3. 35 ±4 6. 49 13 0. 75 ±7 7. 08 13 3. 54 ±7 9. 69 37 .2 6± 20 .6 8 16 .4 2± 4. 85 s* y ju ne *2 01 1 31 .7 1± 8. 86 32 .5 2± 8. 25 3. 27 ±1 .9 3 31 9. 55 ±1 66 .0 8 10 8. 56 ±9 8. 76 11 4. 43 ±9 8. 16 96 .0 5± 63 .5 1 n. a. ju ne *2 01 3 45 .2 9± 4. 89 45 .4 6± 8. 35 1. 09 ±0 .5 5 11 0. 95 ±5 8. 59 18 2. 90 ±1 95 .7 5 18 6. 23 ±2 06 .6 1 56 .6 8± 37 .0 4 14 .7 0± 2. 31 se pt em be r* 20 11 33 .6 4± 15 .3 8 38 .0 7± 13 .7 7 2. 65 ±1 .5 4 32 2. 69 ±1 97 .5 7 12 9. 40 ±1 10 .4 6 15 9. 33 ±1 61 .2 7 9. 01 ±7 .2 1 12 .8 8± 2. 72 se pt em be r* 20 13 40 .8 7± 11 .6 1 39 .1 6± 11 .6 7 1. 90 ±0 .6 7 18 0. 97 ±6 2. 10 10 9. 48 ±3 4. 19 10 5. 19 ±3 6. 32 26 .8 5± 9. 08 16 .5 6± 4. 89 a *s *y y ou ng *j un e* 20 11 33 .9 3± 10 .9 9 30 .2 1± 7. 26 4. 84 ±1 .4 4 44 3. 94 ±1 48 .0 6 98 .9 0± 69 .4 9 94 .7 3± 68 .4 7 10 9. 71 ±8 3. 25 n. a. y ou ng *j un e* 20 13 45 .3 8± 3. 84 42 .4 3± 6. 32 1. 14 ±0 .6 4 10 8. 09 ±6 3. 68 20 5. 37 ±2 67 .1 3 20 4. 16 ±2 84 .4 4 66 .5 3± 46 .1 9 15 .4 2± 2. 55 y ou ng *s ep te m be r* 20 11 38 .6 8± 17 .9 2 40 .0 6± 17 .6 0 3. 84 ±1 .2 5 49 4. 47 ±1 16 .0 3 12 3. 66 ±1 26 .7 2 17 4. 53 ±2 05 .3 1 8. 14 ±7 .3 7 14 .9 7± 2. 04 y ou ng *s ep te m be r* 20 13 39 .7 5± 13 .3 6 37 .2 7± 14 .1 2 2. 24 ±0 .8 0 20 9. 04 ±7 7. 38 11 7. 89 ±3 4. 97 11 1. 61 ±4 1. 38 25 .9 9± 7. 23 14 .2 6± 2. 76 o ld *j un e* 20 11 29 .4 8± 6. 02 34 .8 3± 9. 01 1. 69 ±0 .4 9 19 5. 16 ±4 2. 70 11 8. 21 ±1 25 .9 3 13 4. 13 ±1 14 .2 4 82 .3 8± 35 .8 8 n. a. o ld *j un e* 20 13 45 .1 9± 6. 04 48 .4 9± 9. 39 1. 03 ±0 .4 7 11 3. 81 ±5 7. 29 16 0. 43 ±9 7. 96 16 8. 29 ±9 9. 13 46 .8 2± 24 .1 9 13 .9 9± 2. 31 o ld *s ep te m be r* 20 11 28 .5 9± 11 .2 8 36 .0 8± 9. 36 1. 45 ±0 .5 2 15 0. 91 ±5 2. 28 13 5. 14 ±1 00 .0 6 14 4. 12 ±1 14 .2 4 9. 89 ±7 .4 4 10 .8 0± 1. 29 o ld *s ep te m be r* 20 13 41 .9 9± 10 .3 7 41 .0 6± 9. 17 1. 56 ±0 .2 3 15 2. 89 ±2 1. 84 10 1. 08 ±3 3. 47 98 .7 8± 31 .9 4 27 .7 0± 11 .0 7 18 .8 5± 5. 58 f va lu es a nd p ro ba bi lit y le ve ls a ge (a ) 1. 32 2n .s . 0. 93 4n .s . 58 .4 65 ** * 61 .5 02 ** * 0. 07 5n .s . 0. 05 9n .s . 1. 35 4n .s . 1. 78 4n .s . se as on (s ) 0. 21 0n .s . 0. 01 9n .s . 0. 22 4 3. 18 7n .s . 0. 24 8n .s . 0. 68 0n .s . 38 .9 46 ** * 3. 26 4n .s . y ea r ( y ) 14 .6 94 ** * 6. 68 7* 49 .9 75 ** * 73 .0 76 ** * 0. 05 9n .s . 0. 72 9n .s . 1. 32 3n .s . 9. 59 0* * a *s 0. 08 7n .s . 1. 00 4n .s . 0. 05 2 0. 36 5n .s . 0. 10 4n .s . 0. 02 5n .s . 1. 81 8n .s . 8. 60 3* * a *y 2. 33 3n .s . 0. 72 0n .s . 32 .7 85 ** * 43 .7 16 ** * 0. 15 8n .s . 0. 52 7n .s . 0. 04 1n .s . 13 .6 65 ** s* y 1. 36 7n .s . 4. 76 1* 11 .9 63 ** * 2. 66 3n .s . 3. 01 3n .s . 2. 18 6n .s . 9. 33 4* * n. a. a *s *y 0. 55 1n .s . 0. 34 0n .s . 2. 53 0 0. 16 1n .s . 0. 40 9n .s . 0. 08 0n .s . 0. 04 2n .s . n. a. salvia officinalis l. biochemical dynamics 305 season and year factors showed influence in the results reported by leaf weight (tab. 3). considerable variation of the content on phenolics present in aromatic and medicinal plants has been reported by other authors (kofidis et al., 2007). the relationship to harvest season, with increase in summer months, has been linked to their protective properties against excessive uv-b radiation that might reach mesophyll chloroplasts and nuclei, therefore shielding leaf cells from structural and functional damages (manukyan, 2013). however, our results do not show this behaviour, and the detected influence caused by the season*year interaction appears to be more likely caused by the year factor. as referred before, total phenolic compounds present in the leaves of sage were influenced by the year of study, with higher values recorded in 2013, with the same pattern observed for electrolyte leakage. these results appear to indicate that in 2013, the plants were under increased stress conditions, as it is well established that phenolics act as defense mechanisms against stress conditions (e.g. manukyan, 2013), while electrolyte leakage is used to estimate membrane integrity (rolny et al., 2011). climatic data concerning precipitation and temperature show that 2011 was one of the hottest years in portugal since 1930, and that precipitation was also considerably lower than common records (ipma, 2015), while 2013 was considered an average year. this information about climatic data gives more consistency with the results observed for tbars. this methodology allows an overview regarding peroxidation of membrane, and usually, higher values indicate a higher exposure to stress, which apparently, and as stated above, occurred in 2011. therefore, some other factors would have been implicated in this higher amount of phenolics and of electrolyte leakage. by one hand, the higher values of total phenolics in 2013, quantified using the folin-ciocalteu method can be due to the higher amount of sugars found is this year, compounds, that among others, are known to interfere with this methodology (prior et al., 2005). sugars may also be associated to the values of electrolyte leakage, as they have been correlated to an increase in this parameter (shi et al., 2012), although they are known as key osmolytes, improving stress tolerance by protecting and stabilizing membranes. the age factor also significantly influenced the tbars content (expressed as nmol/g), with higher values recorded in young leaves. although several reports indicate that the amount of these compounds may be higher in adult leaves of several species (lepeduš et al., 2011), higher lipid peroxidation measured by the tbars assay in young leaves has been reported (carvalho et al., 2015). interestingly, the total carotenoid content in young leaves was also higher (tab. 2), and it can be argued that their accumulation in young leaves can be related not to their photosynthetic role, but instead to their known protective antioxidant characteristics (krinsky, 1989). there is a lack of information about how the presence of phenolics can influence the processing of aromatic plants. it can be expected that leaves with higher content of phenolics can be less susceptible to oxidation, as those compounds are proved to be antioxidants (e.g. gião et al., 2013). the presence of these compounds can also influence leaf sensorial attributes, as they have associated two main descriptors, namely bitterness and astringency, which are linked to negative consumer reactions (lesschaeve and noble, 2005). for thiol content (tab. 3), a strong influence of season was found, with samples collected in june presenting higher values than those recorded in september. these compounds act like intracellular antioxidants, either through scavenging free radicals or by enzymatic reactions, and some of them, although being water-soluble, are able to protect biological membranes against lipid peroxidation (mascio et al., 1991). interestingly, none of the other studied parameters that evaluate oxidation processes show this behavior pattern of higher values in june. in this month, although temperatures were similar to those recorded in september (average of 18.2 °c in june and 19.6 °c in september), rain was considerably lower (average of 5.6 mm in june and 67.8 mm in september), and, furthermore, total radiation was significantly higher (average of 27.6 kj/m2 in june and 16.9 kj/m2 in september). hence, this increase in thiols concentration can be a response to those stresses, in order to reduce oxidation (mascio et al., 1991) and cell damage. conclusions this work allowed a detailed characterization of several biochemical parameters of salvia officinalis l. var. purpurascens, as well as an evaluation of their dynamics, considering developmental stage, season and year factors. this latter factor was the one that exerted a significant influence in a higher number of parameters, situation that is likely linked to differences in climatic conditions (considerable difference of precipitation between the years of study, but also of temperature values), while, on the other hand, the developmental stage of leaves influenced almost only the content on photosynthetic pigments. these results are key to understand biochemical variations occurring in salvia officinalis l. var. purpurascens, and helpful when designing cultivation strategies for this specific plant. acknowledgements this work is supported by: european investment funds by feder/ compete/poci – operacional competitiveness and internacio nalization programme, under project poci-01-0145-feder-006958 and national funds by fct – portuguese foundation for science and technology, under the project uid/agr/04033.” references abreu, m., munné-bosch, s., 2007: photoand antioxidant protection and salicylic acid accumulation during post-anthesis leaf senescence in salvia lanigera grown under mediterranean climate. physiol. plant. 131, 590-598. bettaieb, i., hamrouni-sellami, i., bourgou, s., limam, 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(eds.), medicinal and aromatic plants springer, 75-95. the netherlands. sesták, z., castky, j., jarvis, p., 1971: plant photosynthetic production. manual of methods. dr. w. junk publishers, hague, netherlands. shi, h., wang, y., cheng, z., ye, t., chan, z., 2012: analysis of natural variation in bermudagrass (cynodon dactylon) reveals physiological responses underlying drought tolerance. plos one. 7, e53422. taârit, m., msaada, k., hosni, k., marzouk, b., 2012: physiological changes, phenolic content and antioxidant activity of salvia officinalis l. grown under saline conditions. j. sci. food. agric. 92, 1614-1619. tepe, b., 2008: antioxidant potentials and rosmarinic acid levels of the methanolic extracts of salvia virgata (jacq), salvia staminea (montbret & aucher ex bentham) and salvia verbenaca (l.) from turkey. bioresour. technol. 99, 1584-1588. tounekti, t., abreu, m., khemira, h., munné-bosch, s., 2012: canopy position determines the photoprotective demand and antioxidant protection of leaves in salt-stressed salvia officinalis l. plants. environ. exp. bot. 78, 146-156. tounekti, t., hernández, i., müller, m., khemira, h., munnébosch, s., 2011: kinetin applications alleviate salt stress and improve the antioxidant composition of leaf extracts in salvia officinalis. plant physiol. biochem. 49, 1165-1176. tsao, r., yang, r., young, j., zhu, h., 2003: polyphenolic profiles in eight apple cultivars using high-performance liquid chromatography (hplc). j. agric. food chem. 51, 6347-6353. world health organization, 2003: who guidelines on good agricultural and collection practices (gacp) for medicinal plants. geneva: world health organization. yi, w., wetzstein, h., 2010: biochemical, biological and histological evaluation of some culinary and medicinal herbs grown under greenhouse and field conditions. j. sci. food agr. 90, 1063-1070. address of the authors: f. martins, universidade de trás-os-montes e alto douro, utad, quinta de prados, 5000-801 vila real, portugal i. oliveira, a. barros, c. amaral, s. afonso, h. ferreira, b. gonçalves, centre for the research and technology of agro-environmental and biological sciences – citab, universidade de trás-os-montes e alto douro, utad, quinta de prados, 5000-801 vila real, portugal e-mail of the corresponding author: ivo.vaz.oliveira@utad.pt © the author(s) 2016. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 91, 187 193 (2018), doi:10.5073/jabfq.2018.091.025 1 fujian provincial key laboratory of eco-industrial green technology (wuyi university), wuyishan, china 2 college of life sciences, longyan university, longyan, china 3 key laboratory of agroecological processing and safety monitoring, fujian agriculture and forestry university, fuzhou, china characteristic amino acids in tea leaves as quality indicator for the evaluation of wuyi rock tea in different culturing regions xiaoli jia1,3, jianghua ye1,3, haibin wang2,3, li li3, feiquan wang1, qi zhang3, jiebo chen3, xinyu zheng3, haibin he3* (submitted: february 28, 2018; accepted: june 20, 2018) * corresponding author summary free amino acid compositions in wuyi rock tea leaves from yu (authentic rock region), guiyan (semi-authentic rock region) and qishan (ordinary region) tea plantations were analyzed. results showed that contents of 18 free amino acids were 1.6-2.0 times higher in yu and guiyan than that in qishan. the theanine contents reached to 17-20 mg g-1 in yu and guiyan, while it was less than 10 mg g-1 in qishan. hierarchical cluster analysis and principal component analysis were effective in distinguishing rock tea from different regions. the ratios of theanine, sweet and umami amino acids were 8%, 5% and 6% higher, respectively in yu than that in qishan. sensory evaluation score were positively correlated with the ratios of theanine, sweet and umami amino acids (p < 0.05 or p < 0.01). our results highlight that the favourite characteristic amino acids are dominant contributors to sweet aftertaste of rock tea. keywords: authentic rock region; sweet amino acids; umami amino acids; theanine; bitter amino acids; sweet aftertaste introduction wuyi rock tea is one of china’s top ten teas, named specially after its origin from the rocky mountain region of wuyishan county, fujian province of china. the authentic wuyi rock tea has a unique flavour termed ‘rock flavour’ (yanyun in chinese) (gb/t 144872008) – a rock characteristic aroma and lasting sweet aftertaste, which should be significantly different from other teas. rock flavour is also approved by government as a geographical indication of wuyi rock tea. traditionally, local farmers use the valley, coves, pits, curve and flat slopes, making staircases with stone, masonry stone blocks, embankments, and constructing “potted style” tea plantations along mountains. these tea plantations are known as ‘the authentic rock regions’ because the tea products from these regions are the high-grade rock tea. however, rock tea produced from the authentic rock regions could far from satisfy consumer demand, and so farmers reclaimed a large number of tea plantations in the non-rocky regions, and even planted tea in the plains farmland. to distinguish different quality of rock tea, in the folk custom of wuyi county, the tea producing from the core rock regions with high-grade quality is called as ‘authentic rock tea’ (zhengyan cha in chinese) (fig. 1), those from the regions located around the core rock regions with middle-grade quality as ‘semi-authentic rock tea’ (banyan cha in chinese), and those from the ordinary regions as ‘continent tea’ (zhou cha in chinese) (ye et al., 2004; chen et al., 2016). the fresh leaves of ‘authentic rock tea’ sells for > 400 yuan (rmb) kg-1, while of ‘continent tea’ sells only for < 20 yuan (rmb) kg-1, less than 5% of the authentic rock tea. amino acids, as an most important contributor to tea infusion taste, are usually used as evaluation index for tea quality (nakagawa, 1975; nagata et al., 1986; nishimuraa et al., 1988; harbowy et al., 1997; hayashi et al., 2008; akitomi et al., 2013; chen et al., 2016), characters of tea cultivars and types (horanni et al., 2013; huo et al., 2014), quality control of tea processes and growth conditions (tan et al., 2016; zhu et al., 2016), levels of tea leaves with age, plucking seasons and positions (nakagawa, 1970; xu et al., 2012; liu et al., 2016), as well as identification of tea geographical origins (he et al., 2009; song et al., 2012; lee et al., 2015). alcazar et al. (2007) used the differentiation of free amino acids content as chemometric descriptor for classification of green, white, black, oolong, and pu-erh teas. miyauchi et al. (2014) reported that in high-quality japanese green tea (tencha), glutamine, arginine, and theanine are the main constituent amino acids, and could be as an indicator to evaluate the quality of indoor-planted tea. feng et al. (2014) showed that in albino tea leaves, an increase in total amino acid content combined with a decrease in catechins and caffeine reduced its astringency and bitterness, thus enhancing the umami taste, which is one of the indicators of high quality in tea. the amino acid content has a positive correlation with the quality of green tea, oolong tea, and pu-erh tea (nakagawa, 1970; liang fig. 1: classification of rock tea region and the selected tea plantations. a – authentic rock region; b – semi-authentic rock region; c – ordinary region; y –yu, an authentic rock tea plantation located in latitude 27°38′42″–45″ and longitude 117°56′38″–44″; g – guiyan, a semiauthentic rock tea plantation located in latitude 27°36′26″–34″ and longitude 117°57′52″–58′1″; q – qishan, an ordinary tea plantation, located in latitude 27°17′32″–39″ and longitude 117°54′41″–56″. 188 x.l. jia, j.h. ye, h.b. wang, l. li, f.q. wang, q. zhang, j.b. chen, x.y. zheng, h.b. he et al., 2005; wang et al., 2009, 2010; xu et al., 2012). but so far, few study refers to the differentiation of free amino acids content for wuyi rock tea cultured in different regions. no report refers to the relationship between sweet aftertaste and characteristic amino acids in wuyi rock tea. in this paper, we selected three representative tea cultivars of wuyi rock tea which planted in the authentic rock region, semi-authentic rock region, and ordinary region, respectively. contents of free amino acids in fresh tea leaves were analyzed, and the contribution of some characteristic amino acids to taste feature of wuyi rock tea were further revealed. materials and methods sampling sites and processing three plantations qualifications of which have been recognized by the government were chosen as sampling sites, they are yu plantation as a representative of authentic rock regions, guiyan plantation as a representative of semi-authentic rock regions, and qishan plan tation as a representative of ordinary regions (fig. 1). three most representative cultivars of wuyi rock tea – dahongpao, shuixian and rougui – grown in these plantations as the study material. the sampling time was the tea plucking season in may 2014. the tea plants were chosen from each tea cultivar in each plantation using an equidistant sampling method, in which the length of the tea plant rows were divided into 5 equal sections, and then leaf buds with 3 leaves were randomly picked from each section. the fresh tea leaves were over-dried at 105 °c for 30 min to inactivate quickly enzymes, and then dried at 80 °c for 48 h, grounded and sieved by a 60-mesh. the tea powder was used to determine contents of free amino acids subsequently. the tea products of the three tea cultivars from these plantations were used for sensory evaluation. determination of the free amino acids composition in tea leaves the composition of free amino acids was measured according to the national standards of the people’s republic of china (gb/t 5009. 124-2003), using a hitachi l-8900 automatic amino acid analyzer and using external standards of mixed amino acids as calibrators. in brief, 3 g tea powder was placed in a 500-ml conical flask, 450 ml boiling distilled water was added, and the flask was placed in a boiling water bath for extraction for 45 min (shaking once every 5 min). after the completion of extraction, while still hot, the tea was filtered under reduced pressure, the residue was washed with a small amount of hot distilled water 3 times, and after cooling the filtrate was transferred into a 500-ml volumetric flask marked, and shaken to homogenize. there were three replicates for all the tea samples. theanine was assayed according to the national standards of the people’s republic of china (gb/t 23193, 2008) using a sykam s-433d automatic amino acid analyzer, and calibrating with a theanine external standard. samples were prepared by taking 100 mg of tea powder, adding 10 ml deionized water, placing in a 90 °c water bath for 15 min, centrifuging at 10,000 rpm for 10 min, mixing 1:4 with a ph 2.2 dilution solution, filtering, and then loading the sample. instrument conditions were as follows: flow rate 0.25 ml min-1, pressure 35 bar, reactor temperature is 130 °c, injection volume 10 μl, and detection wavelength 338 nm. there were three replicates for all the tea samples. sensory evaluation the quality of tea products from the above plantations were assessed by a tea tasting panel consisting of five professional panelists, according to standardised procedure of ‘methodology of sensory evaluation of tea’, the national standards of the people’s republic of china (gb/t 23776-2009). the total score of 100 was summed of five quality features for oolong tea, of which 20% for the appearance of dry tea, 30% for the tea aroma, 35% for the taste, 10% for the infused leaves, and 5% for the liquor colour (gb/t 23776-2009). the evaluation procedure was as follows. five gram tea was infused in a 110 ml tea tasting porcelain cup with freshly boiled water, covered for 1 min, smelling the flavour of cup cover for tea aroma assessment, for 2 min the liquor was poured into a tea bowl for assessment of liquor colour, taste and infused leaf aroma; secondly, refilled freshly boiled water to cup, covered for 2 min for tea aroma assessment, and for 3 min the liquor was poured into a tea bowl for assessment of liquor colour, taste and infused leaf aroma again; thirdly, refilled freshly boiled water to cup, covered for 3 min for tea aroma assessment, and for 5 min the liquor was poured into a tea bowl for assessment of liquor colour, taste and infused leaf aroma. finally, the tea leaves were poured out for assessment of infused leaf. the each tea feature was examined and scored by each of the five panelists independently. the sum total of the five quality features was expressed as the total quality score (tqs). data analysis all experimental data were presented as mean ± standard error (se). they were analysed using a one-way analysis of variance (anova), and followed by the least significant difference (lsd). hierarchical cluster analysis (hca), principal component analysis (pca) and correlative analysis were all conducted with spss 20.0 software. results and discussion the free amino acids composition in fresh tea leaves the contents of 18 free amino acid compositions were showed in tab. 1. there were significant differences among tea leaves from the 3 tea plantations in each of the 18 amino acid content and the total content (p < 0.05), except valine of dahongpao and glycine of rougui between yu and qishan. the total free amino acids content of three tea cultivars was about 20 mg g-1 in qishan, while they were about 30-40 mg g-1 in guiyan and yuchayuan. it was 1.6-1.7 times higher in yu and 1.9-2.0 times higher in guiyan than that in qishan. the theanine contents of three tea cultivars reached to 17-20 mg g-1 in guiyan and yu tea plantations, while it was less than 10 mg g-1 in qishan, only about half of that in yu and guiyan (tab. 1). high level of amino acids is essential for high quality of tea products (harbowy et al., 1997; miyauchi et al., 2014). nakagawa (1970, 1975) found that about 70% of brothy taste and sweetness in green tea was due to amino acids, and suggested that the higher the con tent of amino acids the better is the taste. the content of amino acids has been widely used as quality index in various teas (harbowy et al., 1997; alcazar et al., 2007; hayashi et al., 2008; chen et al., 2011; xu et al., 2012; feng et al., 2014; miyauchi et al., 2014; chen et al., 2016; zhu et al., 2016). the results indicated the quality of tea leaves was higher in yu and guiyan than in qishan, based on the contents of total free amino acids and theanine. hierarchical cluster analysis (hca) hca was applied on the 18 free amino acids compositions to group the cultured regions. the results showed that the 9 tea samples could be classified 3 types correctly according to their cultured regions (fig. 2). the tea samples between guiyan and yu tea plantations had similar characteristic based on the 18 free amino acids compositions. the results indicated the free amino acids compositions combined with hca could distinguish clearly wuyi rock tea from different cultured regions. characteristic amino acids in wuyi rock tea 189 principal component analysis (pca) the contents of 18 amino acids components were imported to soft ware for pca analysis (fig. 3). the results showed that three sampling plantations dispersed in different quadrants and each group represented independently one type of the 3 tea plantations. the first two principal components, pc1 (94.595%) and pc2 (3.850%), explained 98.445% of the total system variance, which means that the first two principal components can presents most of discrimination of wuyi rock tea in the three plantations. the results indicated the amino acids components could be used to distinguish from tea leaves quality of different tea plantations. characteristic amino acids profile in tea fresh leaves each amino acid has a different taste. for example, glycine and alanine, because of a small molecular chain, can bind taste receptors and produce considerably strong sweetness, while amino acids with a branched chain, such as leucine, isoleucine, and valine, are particularly bitter taste (nishimuraa et al., 1988; scharbert et al., amino acid tab. 1: contents of free amino acids in tea leaves of the different plantations (mg g-1) dahongpao shuixian rougui qishan guiyan yu qishan guiyan yu qishan guiyan yu threonine 0.51±0.01c 0.88±0.02a 0.63±0.02b 0.42±0.01c 0.87±0.03a 0.58±0.02b 0.47±0.02c 0.94±0.03a 0.64±0.03b valine 0.80±0.01b 1.29±0.02a 0.73±0.02b 0.69±0.02c 1.25±0.04a 0.95±0.02b 0.77±0.04c 1.28±0.07a 0.99±0.05b methionine 0.29±0.01c 0.70±0.02a 0.45±0.03b 0.22±0.01c 0.68±0.04a 0.40±0.03b 0.25±0.03c 0.73±0.05a 0.44±0.02b isoleucine 0.72±0.02c 1.21±0.03a 0.95±0.08b 0.62±0.02c 1.20±0.05a 0.89±0.02b 0.69±0.07c 1.26±0.04a 1.00±0.03b leucine 0.63±0.02c 1.09±0.02a 0.86±0.01b 0.50±0.02c 1.03±0.05a 0.69±0.09b 0.60±0.03c 1.08±0.05a 0.82±0.02b phenylalanine 0.72±0.02c 1.22±0.01a 0.94±0.01b 0.64±0.03c 1.14±0.04a 0.82±0.02b 0.65±0.07c 1.19±0.06a 0.87±0.04b lysine 0.53±0.01c 1.01±0.01a 0.74±0.02b 0.49±0.02c 0.98±0.04a 0.68±0.03b 0.54±0.02c 1.05±0.04a 0.74±0.04b asparagine 0.89±0.02c 1.39±0.02a 1.12±0.02b 0.84±0.02c 1.35±0.09a 1.08±0.03b 0.90±0.04c 1.34±0.12a 1.09±0.05b serine 0.75±0.03c 1.27±0.03a 1.03±0.02b 0.69±0.02c 1.21±0.04a 0.92±0.01b 0.75±0.04c 1.28±0.10a 0.98±0.06b glutamic acid 1.16±0.04c 1.72±0.01a 1.42±0.03b 1.04±0.03c 1.58±0.06a 1.27±0.04b 1.08±0.06c 1.63±0.10a 1.38±0.04b glycine 0.61±0.03c 1.03±0.02a 0.75±0.01b 0.50±0.03c 0.94±0.08a 0.66±0.02b 0.61±0.07b 1.04±0.05a 0.74±0.03b alanine 0.63±0.03c 1.26±0.03a 0.98±0.01b 0.65±0.04c 1.19±0.03a 0.89±0.02b 0.71±0.04c 1.24±0.06a 0.93±0.05b cysteine 0.44±0.03c 0.89±0.02a 0.60±0.01b 0.36±0.04c 0.85±0.03a 0.54±0.02b 0.43±0.03c 0.90±0.04a 0.60±0.04b tyrosine 0.65±0.03c 1.21±0.02a 0.93±0.02b 0.57±0.04c 1.18±0.03a 0.88±0.01b 0.63±0.04c 1.20±0.05a 0.90±0.04b histidine 0.26±0.02c 0.82±0.01a 0.55±0.02b 0.22±0.02c 0.73±0.03a 0.43±0.02b 0.27±0.02c 0.80±0.05a 0.51±0.03b arginine 0.82±0.03c 1.39±0.02a 1.09±0.02b 0.79±0.01c 1.32±0.04a 1.01±0.02b 0.81±0.03c 1.37±0.06a 1.05±0.04b proline 0.33±0.01c 0.81±0.02a 0.55±0.02b 0.28±0.03c 0.78±0.03a 0.46±0.01b 0.32±0.04c 0.84±0.06a 0.53±0.05b theanine 9.19±0.20c 17.85±0.46a 16.84±0.17b 9.62±0.71c 20.01±0.11a 18.60±0.34b 9.95±0.32c 20.57±0.33a 18.26±0.35b total 19.92±0.47c 37.03±0.73a 31.16±0.44b 19.13±1.07c 38.29±0.77a 31.74±0.67b 20.41±0.88c 39.74±1.32a 32.46±0.95b means standard error (±se) from three replications for each determination is shown. the lowercase letters represent a significant level at p < 0.05. fig. 2: dendrogram of the 9 tea samples in different tea plantations by hierarchical cluster analysis (hca) according to the 18 free amino acids compositions. the 1, 2 and 3 represent dahongpao, shuixian, and rougui cultivars, respectively in qishan plantations, 4, 5 and 6 represent dahongpao, shuixian, and rougui cultivars, respectively in guiyan plantations, and 7, 8 and 9 represent dahongpao, shuixian, and rougui cultivars, respectively in yu plantations. fig. 3: loadings for principal component analysis (pca) derived from the 18 free amino acid contents of tea leaves from the different plantations. a: yu. b: qishan. c: guiyan. ∆: dahongpao. □: shui xiang. ○: rougui. 190 x.l. jia, j.h. ye, h.b. wang, l. li, f.q. wang, q. zhang, j.b. chen, x.y. zheng, h.b. he 2005; akitomi et al., 2013). matoba et al. (1972) study indicated that the more hydrophobic amino acids, the stronger the bitterness. the dissolution of hydrophilic amino acids in tea infusion is much faster than that of hydrophobic amino acids, and within 15 min theanine comprises about 30% of the free amino acids in tea infu sion (kocadağlı et al., 2012). it was suggested that theanine could be synthesized from glutamic acid and ethylamine, and theanine and glutamate are the major contributors to the umami taste of green tea (feldheim et al., 1986; scharbert et al., 2005; kaneko et al., 2006; deng et al., 2008). on in-depth analysis of these special characteristics of amino acids, we found that the percentage of hydrophilic amino acids (threonine, lysine, serine, glycine, cysteine, tyrosine, histidine, arginine, as paragine, glutamic acid, and theanine) to the total free amino acids in tea leaves were 82-84% in yu tea plantation, and 79-81% in guiyan and qishan, respectively, while the percentage of hydropholic amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine) were 16.0-17.5% in yu tea plantation, and 19.0-20.7% in guiyan and qishan, respectively (fig. 4). further, the percentage of sweet amino acids (glutamic acid, alanine, glycine, and theanine) were 64.2-67.5% in yu tea plantation, 58.2-62.0% in guiyan and qishan, respectively, was about 5% higher in the authentic rock region (yuchayuan) than that in the ordinary region (qishan). while the percentage of bitter amino acids (valine, leucine, and isoleucine) were 8.0-8.7% in yu tea plantation, 9.1-9.7% in guiyan, and 9.410.8% in qishan, respectively, was about 2% lower in the authentic rock region (yuchayuan) than that in the ordinary region (qishan) (fig. 4). and more, the proportion of umami amino acids (glutamic acid and theanine) were 58.6-62.6% in yu tea plantation, 52.8-56.4% in guiyan, and 51.9-55.7% in qishan, respectively, was about 6% higher in the authentic rock region (yu) than that in the ordinary region (qishan) (fig. 4). all proportions of those favourite amino acids in tea leaves was with the order of yu > guiyan ≈ qishan. these data further support the sweet aftertaste is the characteristic of high-grade rock tea. theanine is a unique amino acid in tea plants, which is a key dedicator to the umami and sweet tastes of green tea (nakagawa, 1975; hayashi et al., 2008; kurihara, 2015). the umami taste and sweetness in green tea was highly positively correlated with theanine concentration (yin et al., 2014). theanine can reduce the bitterness and astringency in tea (hayashi et al., 2013; yin et al., 2014). as a predominant amino acid present in high-grade tea and usually accounts for more than a half of the total amino acids content, theanine has the greatest impact of all the amino acids on fig. 4: the percentage of characteristic amino acids in tea leaves of the different plantations. hydrophilic amino acids including threonine, lysine, serine, glycine, cysteine, tyrosine, histidine, arginine, asparagine, glutamic acid, and theanine. hydropholic amino acids including alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine. sweet amino acids including glutamic acid, alanine, glycine, and theanine. bitter amino acids including valine, leucine, and isoleucine. umami amino acids including glutamic acid and theanine. characteristic amino acids in wuyi rock tea 191 tab. 2: t he score of five quality features and the total quality score (tqs) by sensory evaluation cultivar & appearance aroma taste infused leaves liquor color tqs plantation (20%) (30%) (35%) (10%) (5%) (100%) dahongpao qishan 18.20±0.84a 21.20±2.17b 25.40±0.55c 7.80±0.84a 4.20±0.84a 76.80±3.19b guiyan 15.60±0.55a 23.80±2.28b 27.80±0.84b 8.80±0.84a 4.40±0.55a 80.40±2.70b yu 18.00±0.71a 28.20±1.48a 34.40±1.14a 8.20±0.84a 4.60±0.55a 93.40±1.82a shuixian qishan 18.20±0.84a 22.20±1.79b 23.60±0.55c 8.20±0.84a 4.20±0.45a 76.40±2.07c guiyan 16.80±1.48a 25.40±1.67b 26.00±0.71b 9.60±0.55a 4.80±0.45a 82.60±2.61b yu 18.20±0.84a 30.40±0.55a 32.20±2.17a 8.80±0.45a 4.40±0.89a 94.00±3.74a rougui qishan 16.40±1.14a 23.20±0.84c 25.40±0.55c 9.00±1.00a 4.00±0.71a 78.00±2.65c guiyan 17.60±1.67a 26.20±0.84b 28.00±1.00b 8.40±0.55a 4.80±0.45a 85.00±2.55b yu 17.60±1.14a 29.40±0.55a 33.60±1.52a 9.00±0.71a 4.80±0.45a 94.40±1.14a means standard error (±se) from the five panelists independently for each sample is shown. the lowercase letters represent a significant level at p < 0.05. the tea’s natural quality (horanni et al., 2013; miyauchi et al., 2014; kurihara, 2015). our results showed that the percentage of theanine to the total amount of the 18 free amino acids in tea leaves were 54.0-58.6% in yuchayuan, 48.2-52.3% in guiyan, and 46.1-50.3% in qishan (fig. 4). the ratio of theanine to the 18 free amino acids contents in tea leaves was 8% higher in the authentic rock region (yu) than that in the ordinary region (qishan). this data reflects the real differential of quality between ‘authentic rock tea’ and ‘continent tea’. we suggested that rather than net contents, the ratio of those favourite amino acids such as sweet amino acids and umami amino acids, as well as theanine, may a solid indicator to evaluate quality of wuyi rock tea. sensory evaluation in china, the taste is the most important quality index in oolong tea, account for 35% of the five quality features (gb/t 23776-2009). the sensory assessment results shown that the total quality scores (tqs) were yu > guiyan > qishan in all the three tea cultivars (tab. 2), and had significant different in the three different plantations (p < 0.05), except the dahongpao between guiyan and qishan. the highest tqs for three tea cultivars from yu tea plantation was in good agreement with its reputation and high quality of genuine rock tea. further, no significant differences were observed in the three different plantations in the appearance of dry tea, the infused leaves, and the liquor colour. however, the scores for the taste were yu > guiyan > qishan in all the three tea cultivars, and had significant different in the three different plantations (p < 0.05). with regard to the attributes of the taste in tqs, we suggested that the taste is the key factor for wuyi rock tea and is a distinct index of genuine rock tea plantation distinguishing from other plantations. correlation analysis between characteristic amino acids and sensory evaluation correlation analysis was conducted to investigate the relationship between the each of five quality features, tqs and the characteristic amino acids. as shown in tab. 3, most of the characteristic amino acids in the three tea leaves have no significant correlation with the appearance of dry tea, infused leaves, and liquor colour (except liquor colour of dahongpao cultivar). however, tqs of all the three cultivars were significantly positively correlated to theanine, sweet amino acids, and umami amino acids, and were significantly negatively correlated to bitter amino acids (p < 0.01 or p < 0.05). these results revealed that the characteristic amino acids are more responsible for wuyi rock tea quality than the appearance, infused leave and liquor colour. correspondingly, the taste feature of three cultivars were significantly positively correlated to hydrophilicity amino acids, theanine, sweet amino acids, and umami amino acids, and were significantly negatively correlated to hydrophobicity amino and bitter amino acids (p < 0.01 or p < 0.05). not coincidentally, the perceived taste score and tqs positively correlated with the concentration of amino acids and theanine in longjing tea (p < 0.05), oolong tea (p < 0.01), and pu-erh tea (p < 0.01) (liang et al., 2005; wang et al., 2009, 2010; gao et al., 2016). our results further revealed that those favourite amino acids such as theanine, sweet and umami amino acids were all positively responsible for tqs and taste score, while unfavourite amino acids such as bitter amino acids were negatively responsible. as sweet aftertaste is the most notable character of wuyi rock tea and is a constituent of rock flavour, the characteristic amino acids are dominant contributors to sweet aftertaste of wuyi rock tea. it must be pointed out that although amino acids is an important contributor for tea taste in all tea types, other chemical components such tea polyphenols, caffeines, catechins, and other soluble substan ces in tea infusion also contribute to perceived taste (nakagawa, 1970; nagata et al., 1986; harbowy et al., 1997; liang et al., 2005; wang et al., 2009, 2010; xu et al., 2012; hayashi et al., 2013; feng et al., 2014; gao et al., 2016). as the multicomponent composition resulting in tea taste, the particular feature and the coeffect of tea secondary metabolites need to further uncover the influence on the rock flavour of wuyi rock tea. metabonomic strategies, as an effective and powerful approach to gathering integrated information of tea metabolites (lee et al., 2015; liu et al., 2016; tan et al., 2016; you et al., 2017), may be a helpful method for unveiling the fascinating rock flavour of wuyi rock tea. conclusion by the detailed analysis on the content of characteristic amino acids in the three cultivars of wuyi rock tea sampled from the different culture regions, and their correlation with the sensory evaluation, we found that the tea taste score and tqs significantly positively correlated with the concentration of theanine, sweet amino acids, 192 x.l. jia, j.h. ye, h.b. wang, l. li, f.q. wang, q. zhang, j.b. chen, x.y. zheng, h.b. he tab. 3: correlation between the score of sensory evaluation and the proportion of characteristic amino acids quality feature theanine hydrophobicity hydrophilicity sweet bitter umami amino acids amino acids amino acids amino acids amino acids dahongpao appearance 0.27 -0.45 0.45 0.38 -0.1 0.39 aroma 0.98* -0.93 0.93 0.95* -1.00** 0.95 taste 1.00** -0.96* 0.96* 0.98* -1.00** 0.98* infused leaves -0.27 0.45 -0.45 -0.38 0.1 -0.39 liquor color 0.97* -1.00** 1.00** 0.99** -0.91 0.99** tqs 1.00** -0.97* 0.97* 0.99* -0.99** 0.99* shuixian appearance 0.29 -0.55 0.55 0.47 -0.28 0.42 aroma 0.99* -0.91 0.91 0.94 -0.99** 0.96* taste 1.00** -0.96* 0.96* 0.98* -1.00** 0.99* infused leaves 0.69 -0.45 0.45 0.53 -0.69 0.58 liquor color -0.29 0.55 -0.55 -0.47 0.28 -0.42 tqs 0.99** -0.92 0.92 0.95* -0.99** 0.97* rougui appearance 0.81 -0.62 0.62 0.67 -0.95* 0.72 aroma 0.99** -0.93 0.93 0.95* -0.98* 0.97* taste 1.00** -0.98* 0.98* 0.99** -0.92* 1.00** infused leaves 0.11 -0.37 0.37 0.31 0.21 0.24 liquor color 0.81 -0.62 0.62 0.67 -0.95* 0.72 tqs 1.00** -0.96* 0.96* 0.98* -0.95* 0.99** tqs – total quality score. * and ** represent significant correlation at p < 0.05 and p < 0.01. and umami amino acids, and significantly negatively correlated with the concentration of bitter amino acids (p < 0.05 or p < 0.01). and the ratios of these favourite amino acids (sweet and umami amino acids, as well as theanine) were higher, while the ratios of bitter amino acids were lower in tea leaves from the authentic rock region (yu) than that from the ordinary region (qishan). we suggested that the ratios of those favourite amino acids are dominant contributors to sweet aftertaste of wuyi rock tea and could be as quality indicators to evaluate quality of wuyi rock tea. acknowledgments this study was supported by the national 948 project of china (2014z36), the fujian-taiwan joint innovative center for germplasm resources and cultivation of crop (fj 2011 program no. 201575), the project of the collaborative innovation center of chinese oolong tea industry (201310), the project of scientific research of young and middle-aged teachers, fujian province (jat160504) and the foundation of wuyi university (xd201502). we thank hexiang tea industry co., ltd (wuyishan), shanming chen, chairman of shanming chen ecological tea co., ltd (wuyishan), shixian cao, tea technician of yuchayuan plantation, 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(nat. sci. edi.) 33, 459-462. yin, j.f., zhang, y.n., du, q.z., chen, j.x., yuan, h.b., xu, y.q., 2014: effect of ca2+ concentration on the tastes from the main chemicals in green tea infusions. food res. int. 62, 941-946. doi: 10.1016/j.foodres.2014.05.016 you, r., guan, y., li, l., 2017: chinese herbal teas as a complementary therapy metabonomics: a developing platform for better understanding. int. j. food sci. techn. 52, 13-21. zhu, y., luo, y., wang, p., zhao, m., li, l., hu, x., chen, f., 2016: simultaneous determination of free amino acids in pu-erh tea and their changes during fermentation. food chem. 194, 643-64. doi: 10.1016/j.foodchem.2015.08.054 address of the corresponding author: haibin he, key laboratory of agroecological processing and safety monitoring, fujian agriculture and forestry university, fuzhou, china © the author(s) 2018. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 91, 321 331 (2018), doi:10.5073/jabfq.2018.091.041 universidad autónoma agraria antonio narro, saltillo, coahuila, méxico 1departamento de horticultura, 2doctorado en agricultura protegida, 3departamento de nutrición animal y 4departamento de maquinaria agrícola effect of selenium application on mineral macroand micronutrients and antioxidant status in strawberries willian alfredo narváez-ortiz1, mariano martínez-hernández1, laura o. fuentes-lara3, adalberto benavides-mendoza1, 2, jesús rodolfo valenzuela-garcía4, josé a. gonzález-fuentes1, 2* (submitted: june 4, 2018; accepted: september 21, 2018) * corresponding author summary the application of selenium (se) as sodium selenite (na2seo3) at 0, 2, and 4 mg l-1 concentrations in nutrient solution to strawberry plants was evaluated. selenium did not modify the dry weights of the roots, stems, leaves, and fruits, or the fresh weights of the stems and fruits. the 4 mg l-1 concentration caused decreases in the fresh weights of the roots and leaves and in the yield. the mineral content of different plant organs changed but was not adversely affected by se applications, with the 2 mg l-1 treatment having a lower impact on mineral concentration variation, as well as temporary positive effects on the fruits’ antioxidant status. the fruit ph was not adversely affected by application of se. se application in nutrient solution proved to be an adequate technique to increase the se content in all plant organs. se concentration exhibited a differential distribution, with the highest levels in the roots, followed by the leaves and crowns; the fruits had the lowest levels, reaching an average concentration of 31.2 mg kg-1 of dry weight. by contrast, fruits from the untreated plants obtained an average concentration of only 6.35 mg kg-1, with no decreases in the concentrations of other mineral elements in treated plants. keywords: fragaria, fruit quality, sodium selenite, biofortification. introduction selenium (se) is an essential trace element for humans and other animals its biological importance lies in the fact that it forms a structural part of more than 30 selenoenzymes that regulate oxidative metabolism by mitigating cell damage caused by free radicals (mangiapane et al., 2014). low se intake in humans is associated with health disorders, reduced fertility and immune function, and an increased risk of cancer (broadley et al., 2006). the primary source of se is the soil, from which it is absorbed by plants and reaches humans directly or indirectly via the food chain (steinnes, 2009). therefore, soils with low levels of se are associated with human populations with a higher risk of deficiency of this element (hartikainen, 2005). although se is not essential for plants, it is a beneficial nutrient (pilon-smits et al., 2009) that enhances plant growth and antioxidant activity (benavides-mendoza et al., 2012). however, at high concentrations, it causes toxicity in plants (lyons et al., 2005). in current practice, the objective is to increase se intake in humans through biofortification of crops, which is achieved through mineral fertilization or genetic improvement (gonzález-morales et al., 2017). biofortification using fertilizer enriched with se has been successfully tested in the field and greenhouse (bañuelos et al., 2015). in extensive crops such as winter wheat, se application did not cause changes in yield or harvest index but did have a positive effect on se concentration in grains, with increases ranging from 16 to 26 ng g-1 se of fresh weight per each gram of se applied per hectare (broadley et al., 2010). similar results were observed in rice, in which the average content of se was 0.025 ± 0.011 μg g-1; when foliar fertilizers enriched with se were applied, the average se content increased until it reached 0.471 to 0.640 μg g-1 (chen et al., 2002). in horticultural species such as carrots, the foliar application of selenite or selenate solutions at a se concentration of 100 μg ml-1 resulted in se levels of up to 2 μg g-1 in dry matter of edible parts (kápolna et al., 2009). in potatoes, application of this mineral resulted in higher concentrations of starch in the leaves, as well as an improved yield of tubers (turakainen et al., 2004). supplying se through a nutrient solution, soil fertilization, or a foliar spray has been demonstrated to increase se levels in tomato fruits (becvortazcurra et al., 2012; castillo-godina et al., 2016), rice grains (lidon et al., 2018), carrots (oliveira et al., 2018), turnips (li et al., 2018), and lettuce (hawrylak-nowak et al., 2018). studies have also been reported regarding se application and its interaction with other beneficial elements such as iodine; however, the literature is very scarce concerning simultaneous biofortification with iodine and se and possible interactions in the uptake processes (jerše et al., 2018). in a recent study of strawberry plants, mimmo et al. (2017) used sodium selenate as a se source in concentrations of approximately 0.79 and 7.9 mg l-1; supplementation with se did not negatively affect growth, fruit yield, or mineral accumulation in the different plant organs, and fruits were obtained with se concentrations of up to 46.04 mg kg-1. on the other hand, selenate application at concentrations of up to 2.4 mg l-1 favored se accumulation in strawberry plants and increased the fresh weight of the fruits (melo santiago et al., 2018). however, at present there are few studies of biofortifi cation with se – more specifically, as selenite – and its impact on the mineral composition of strawberries, which contain a wide range of nutrients and phytochemicals and are considered one of the most relevant commercial berry crops in many parts of the world (sandhu et al., 2018). for these reasons, this study aimed to determine the impact of fertilization with sodium selenite on growth, yield, and se concentration, as well as the concentration of macroand micronutrients in the roots, crowns, leaves, and fruits of strawberry plants. in addition, this study sought to verify the effect of se on the fruits’ antioxidant status. materiels and methods the study was conducted at universidad autónoma agraria antonio narro, located in saltillo, coahuila, méxico. fragaria x ananassa var. ‘festival’ strawberry plants were used as plant material. the plants were grown under greenhouse conditions, with a temperature 322 w.a. narváez-ortiz, m. martínez-hernández, l.o. fuentes-lara, a. benavides-mendoza, j.r. valenzuela-garcía, j.a. gonzález-fuentes range of 12 °c to 32 °c. the plants were placed in polyethylene bags with a capacity of 5 l that contained a mixture of peat moss and perlite at a ratio of 1:3 (v:v). the bags were placed in three 0.8 × 10 m beds with black plastic mulch. for the first 15 days, the plants were irrigated with previously characterized water (tab. 1). crop nutrition was carried out through the application of a steiner nutrient solution (steiner, 1961), beginning 16 days after transplant (dat). the nutrient solution concentration was increased according to the growth of the crop: 15% up to 30 dat, 50% at 31-50 dat, and 100% from 51 dat until the end of the experiment. the ph of the solution was maintained at approximately 6.0, using sulfuric acid and a maximum electrical conductivity of approximately 2 ds m-1. the treatments consisted of a control group (without se application) and se addition in nutrient solution at concentrations of 2 and 4 mg l-1 of se as sodium selenite (na2seo3, merck), initiated at 26 dat. according to the analyses conducted before the start of the research, the basal se concentration in the mentioned substrates was 0.175 mg kg-1 (perlite) and 0.016 mg kg-1 (peat moss), while in the irrigation water, it was 0.018 mg l-1. during plant development, two samplings were carried out. the first one, at 60 dat, was conducted to determine the fresh weights (fw), dry weights (dw), se content, and other minerals in the roots, crowns, and leaves of the plants. the second sampling, was carried out at 98 dat, again to determine the variables above and also to analyze additional samples of mature fruits for concentrations of se and other minerals, oxidation-reduction potential (orp) as a measure of antioxidant status, and ph. harvesting of the fruits began at 98 dat and ended at 129 dat (14 sampling dates). determination of biomass and yield, chemical variables, and mineral concentrations for each sampling, five strawberry plants from each treatment con dition were dissected into their various organs (roots, crowns, leaves, and fruits). the fresh weights of each fresh plant part were weighed using a sartorius cp224s analytical balance. the samples were then placed in an arsa drying stove at a temperature of 80 °c for 72 hours, and the dry weights were subsequently measured. the ripe strawberry fruits were collected and weighed to obtain the yield per plant. this determination began at 98 dat and ended at 129 dat (14 sampling dates). the p content was obtained using a spectrophotometric method (aoac, 1990). k, ca, mg, na, fe, mn, cu, and zn levels were determined in samples submitted to acid digestion (jones and case, 1990) using a varian aa-1275 atomic absorption spectrophotometer. the se concentration was determined in samples submitted to acid digestion (aoac, 2000) using a varian 725-es inductively coupled plasma optical emission spectrometer (icp-oes). the ph and orp in the fruits were determined by a hanna hi 98121 potentiometer using the technique described by benavidesmendoza et al. (2002). for this determination, five fully mature fruits were collected (one replicate). the fruits were washed with distilled water and subsequently macerated. electrodes were placed in the macerate obtained from the five fruits, and readings were taken. between readings, the electrodes were washed with distilled water. this procedure was performed three times (three replicates) for each treatment and for each of the 14 sampling dates (98 dat to 129 dat). experimental design the experiment was carried out using a completely randomized design with 40 replicates (40 plants) per treatment; each se concentration was considered a treatment and each potted plant an experimental unit. statistical analyses the wilcoxon signed rank test was performed to determine the differences between treatments for the ph and orp variables in fruits over time. the spearman correlation coefficient was calculated to determine the degrees of correlation for the different variables evaluated in the plants and the se concentrations in the different plant parts, using the statistical package r, version 3.1.1 (r develop ment core team, 2014). data were analyzed by one-way anova, also using r, version 3.1.1 (r development core team, 2014). the lsd simultaneous test (p ≤ 0.05) was used for means separation. results biomass and yield there were few differences in plant biomass between the different treatments. at 60 dat (tab. 2), increases in fresh and dry biomass of the crowns were observed due to the effect of the se treatments. in the second sampling, for plants treated with se applied at a concentration of 4 mg l-1, adverse effects were observed for the fresh weight of the roots and dry weight of the leaves, while all other evaluated variables demonstrated no significant effects (p < 0.05). when the overall weight of plants (roots + crowns + leaves) and the yield were obtained (fig. 1), a negative effect of treatment with se applied at 4 mg l-1 was observed in comparison with the control plants (98 dat), which was assumed to be the result of se accumulation. selenium accumulation and its effect on the content of other minerals selenium distribution in plants when the se nutrient solution was applied, increases in the concentrations of se were found in strawberry plants at 60 and 98 dat (fig. 2). for both samplings of plants treated with 2 mg l-1 se and for the second sampling of plants treated with 4 mg l-1 se, the highest se concentration was detected in the roots, followed by the leaves, crowns, and fruits. the first sampling of plants treated with 4 mg tab. 1: general characteristics (salinity/sodicity, cations, anions, and micronutrient determinations) of irrigation water. salinity / sodicity cations anions micronutrients (mg l-1) (mg l-1) (mg l-1) ph 7.79 ca 95.2 so4 38.9 b 0.17 e.c (ds m-1) 0.85 mg 23.4 hco3 329 fe 0.0091 sar 0.96 na 40.3 cl 54.3 mn 0.0017 saraj 1.23 k 3.12 co3 35.4 cu 0.0003 n-no3 4.90 zn 0.2018 e.c: electrical conductivity; ars: absorption ratio of sodium; arsaj: adjusted sodium adsorption ratio; nd: not detected. effects of selenium application in strawberry 323 l-1 of se exhibited a different trend, obtaining the highest se concentration in the roots, followed by the crowns and then the leaves. the addition of se at 2 and 4 mg l-1 increased the concentration of this element in fruits, reaching values fivefold higher than the control treatment but without differing from each other (fig. 2). macroand micronutrient concentrations in plants the concentrations of mineral nutrients in the different organs evaluated in strawberry plants is illustrated in tab. 3. roots: changes in concentrations of k, ca, mg, and zn were observed. at a concentration of 4 mg l-1 se, there were no significant effects on the first sampling date, while for the second sampling, a reduction of these elements was observed. when 2 mg l-1 of se was applied, no changes in the concentrations of these minerals were detected, except for that of ca, which increased. na, cu, and mn concentrations for the first sampling were increased by treatment with 4 mg l-1 se; a similar response was observed for the second sampling in plants treated with sodium selenite at both 2 and 4 mg l-1 se. fe demonstrated the same behavior in the two sampling dates, with treatment at 4 mg l-1 se resulting in a higher concentration of this mineral element. p had no differences associated with treatments for either sampling date. crowns: for the first sampling, the treatments had no significant effect on k, na, cu, mn, and fe concentrations, while for the second sampling, treatment with 2 mg l-1 se resulted in an increase in fe but no changes in the concentrations of k and cu compared to the control plants. na was increased by treatment with sodium selenite; mn also increased, but only in plants treated with 4 mg l-1 se. the concentrations of p, mg, and zn were higher at 2 mg l-1 se treatment for the first sampling; for the second sampling, this effect disappeared for p and mg, which demonstrated no significant differences, while zn decreased at this low concentration of 2 mg l-1 compared to control plants. when se was applied at a concentration of 4 mg l-1, ca concentration in the crowns was higher for both sampling dates. leaves: for the first sampling, no significant differences were detec ted in the concentrations of p, ca, zn, and fe between the treatments; however, for the second sampling, modifications in concentrations of these minerals were observed. zn decreased due to the se treatments, while fe increased when 2 mg l-1 se was applied. treatment with 2 mg l-1 se resulted in no changes to the concentrations of p and ca compared to the control plants. treatment with 4 mg l-1 se increased the concentrations of k and cu for the first sampling, while for the second sampling, it had an opposite effect, causing the concentrations of k and cu to decrease in comparison to the untreated plants. neither the control plants nor those treated with 2 mg l-1 se demonstrated changes in na concentration in the first sampling, tab. 2: fresh and dry weight averages for different organs of strawberry plants treated with different concentrations of se in nutrient solution. treatment first sampling second sampling treatment first sampling second sampling (60 dat) (98 dat) (60 dat) (98 dat) 0 mg l-1 se 6.91 a¥ 24.15 a 0 mg l-1 se 7.56 a 26.60 a 2 mg l-1 se 4.96 a 21.74 ab 2 mg l-1 se 7.88 a 23.67 a 4 mg l-1 se 5.92 a 18.67 b 4 mg l-1 se 7.22 a 16.26 a 0 mg l-1 se 1.90 a 2.89 a 0 mg l-1 se 1.81 a 6.65 a 2 mg l-1 se 1.15 a 2.80 a 2 mg l-1 se 1.94 a 6.13 a 4 mg l-1 se 1.42 a 2.76 a 4 mg l-1 se 1.86 a 4.24 b 0 mg l-1 se 0.78 b 5.79 a 0 mg l-1 se ------6.55 a 2 mg l-1 se 2.07 ab 5.39 a 2 mg l-1 se ------7.12 a 4 mg l-1 se 4.70 a 5.43 a 4 mg l-1 se ------7.73 a 0 mg l-1 se 0.17 b 1.34 a 0 mg l-1 se ------1.04 a 2 mg l-1 se 0.61 ab 1.31 a 2 mg l-1 se ------1.22 a 4 mg l-1 se 1.54 a 1.35 a 4 mg l-1 se ------1.08 a dat = days after transplanting. rfw = root fresh weight, rdw = root dry weight, cfw = crown fresh weight, cdw = crown dry weight, fwl = fresh weight of leaves, dwl = dry weight of leaves, ffw = fruit fresh weight, fdw = fruit dry weight. ¥averages with different literal were statistically different according to lsd (p ≤ 0.05). rfw (g) rdw (g) cfw (g) cdw (g) fwl (g) dwl (g) ffw (g) fdw (g) fresh weight g pl an t-1 0 20 40 60 80 100 120 first sampling (60 dat) second sampling (98 dat) dry weight a a a a a a a a bab a b 0 m g s e l1 2 m g s e l1 4 m g s e l1 0 m g s e l1 2 m g s e l1 4 m g s e l1 0 m g s e l1 2 m g s e l1 4 m g s e l1 a b ab 0 m g s e l1 2 m g s e l1 4 m g s e l1 0 m g s e l1 2 m g s e l1 4 m g s e l1 yield fig. 1: total fresh and dry weights (roots + crowns + leaves) and yields of strawberry plants treated with sodium selenite at concentrations of 0, 2, and 4 mg l-1 in irrigation water. results of the two samplings are included. the bar in each column represents the standard error. averages with different letters were statistically different according to lsd (p ≤ 0.05). 324 w.a. narváez-ortiz, m. martínez-hernández, l.o. fuentes-lara, a. benavides-mendoza, j.r. valenzuela-garcía, j.a. gonzález-fuentes 0 50 100 150 200 250 300 0 20 40 60 80 0 20 40 60 80 100 0 10 20 30 40 50 a b a c a a b c b a a b root crown leaves fruit second sampling (98 dat) 0 50 100 150 200 250 300 0 20 40 60 80 0 20 40 60 80 100 first sampling (60 dat) a b b a b ab a b c root crown leaves 0 mg se l-1 2 mg se l-1 4 mg se l-1 m g se k g1 d w m g se k g1 d w m g se k g1 d w 0 mg se l-1 2 mg se l-1 4 mg se l-1 0 mg se l-1 2 mg se l-1 4 mg se l-1 0 mg se l-1 2 mg se l-1 4 mg se l-1 m g se k g1 d w 0 mg se l-1 2 mg se l-1 4 mg se l-1 m g se k g1 d w m g se k g1 d w 0 mg se l-1 2 mg se l-1 4 mg se l-1 m g se k g1 d w 0 mg se l-1 2 mg se l-1 4 mg se l-1 fig. 2: effect of se application as sodium se in nutrient solution on accumulation of elements in different organs of strawberry plants for samplings made at 60 and 98 dat. the bar in each column represents the standard error. averages with different letters were statistically different according to lsd (α ≤ 0.05). while for the second sampling, se treatment produced an increase in the concentration of na. for the first sampling, mn decreased in the presence of se, while for the second sampling, it increased in plants treated with the higher concentration (4 mg l-1) of se. no differences in mg concentration were detected for either sampling. fruits: applying se in nutrient solution did not have significant effects on the concentrations of ca, na, and fe. when treated with 2 mg l-1 se, the concentrations of k, zn, and mn in the fruits did not change compared to the control plants; similar behavior was observed for mg concentration in plants treated with 4 mg l-1 se. however, at this latter concentration, p increased, whereas cu decreased. for se application in nutrient solution at concentrations of both 2 and 4 mg l-1, changes in the concentrations of elements in the different organs were greater for the second sampling (fig. 3). correlation analysis tab. 4 indicates the correlations between se concentration in different plant organs (roots, crowns, leaves, and fruits) and mineral content and biomass for the second sampling at 98 dat (fruiting stage). for purposes of presenting and discussing these results, only the spearman correlation coefficients (ρ) that are statistically significant with absolute value equal to or greater than 0.70 were considered. a positive correlation was observed between se concentration in the fruits and se concentrations in the roots and crowns. in addition, se concentration in the crowns correlated positively with se concentration in the leaves. regarding se’s effects on minerals, it was observed that se in different plant organs was associated with an increase of na in the crowns, leaves, and fruits. on the other hand, increased se in the crowns, leaves, and fruits was associated with decreased zn in all plant organs. it was also found that higher se concentration in the crowns was correlated with decreased k in the roots, leaves, and fruits, while se concentration in the leaves was negatively correlated with k in the roots and crowns. k levels decreased in the roots and leaves when there was greater se accumulation in the fruits. for other minerals, no clear pattern due to the effect of se was observed. in the roots, se concentration correlated negatively with the concentration of cu in the leaves and fruits. se was also associated positively with the concentrations of p and mg in the fruits. increased se in the crowns was associated with higher concentrations of cu and mn in the roots, as well as with ca in the crowns and p in the effects of selenium application in strawberry 325 fruits. on the other hand, se in the crowns was negatively correlated with p concentration in the leaves and mn in the fruits. the highest level of se in the leaves was accompanied by increases in fe, cu, and mn in the roots, ca in the crowns, and p in the fruits. a negative correlation was also found between se concentration in the leaves and p in the leaves. se concentration in the fruits was positively associated with p concentration and negatively associated with mg concentration in the fruits. the increase in se concentration in the crowns and leaves was accompanied by a decrease in the fresh and dry weights of the leaves, as well as the fresh weight of the roots; this latter variable was also affected negatively by increased se in the fruits. ph and orp dynamics in fruits the wilcoxon and lsd tests indicated no differences (p > 0.05) in the dynamic behavior of fruit ph among different treatments, as well as for the different samplings performed (fig. 4). the above suggests that the higher se concentration did not modify the metabolic processes that maintain the ph at between 3 and 4, which is considered suitable for the pulp of strawberry fruits. on the other hand, for the orp of fruits, the wilcoxon test indicated that differences exist (p ≤ 0.05) between treatment with se applied at 4 mg l-1 and the other treatments: control and 2 mg l-1 se (fig. 5). in consideration of its dynamics, the orp demonstrated a sigmoidal behavior, observing a temporal progression from 108 to 122 dat, with subsequent stability oscillating between approximately 20 and 60 mv. the wilcoxon test indicated that the control condition and treatment with 2 mg l-1 se are statistically equal. univariate analysis using the lsd test revealed significant differences in specific evaluation dates (fig. 5) associated with the control condition and treatment with 2 mg l-1 se. discussion biomass similar results to those obtained for the first sampling have already been documented in other crops (lópez-gutiérrez et al., 2015; castillo-godina et al., 2016). in brassica juncea l., it has been reported that the application of 0.5 mg kg-1 se-selenite stimulated growth and yield of dry matter (singh et al., 1980). the same effect tab. 3: mineral content in roots, crowns, leaves, and fruits of strawberries plants treated with different concentrations of se in nutrient solution. treatments minerals sampling 1 sampling 2 root crown leaves root crown leaves fruit 0 mg l-1 se 0.9 a¥ 1.5 b 3.1 a 1.6 a 2.3 ab 4.3 a 1.7 b 2 mg l-1se p (g kg-1) 1.1 a 2.9 a 2.1 a 1.7 a 2.4 a 3.7 a 3.0 ab 4 mg l-1se 2.5 a 1.7 ab 2.7 a 1.5 a 1.6 b 2.7 b 4.5 a 0 mg l-1 se 5.4 a 7.6 a 6.7 b 16.2 a 12.1 a 22.1 a 18.3 a 2 mg l-1 se k (g kg-1) 6.2 a 13.1 a 14.5 ab 12.9 a 10.9 ab 19.8 b 17.4 ab 4 mg l-1se 12.8 a 7.7 a 18.0 a 8.6 b 9.4 b 18.3 c 16.4 b 0 mg l-1 se 4.7 a 6.5 b 6.1 a 8.1 b 7.9 b 7.7 a 4.3 a 2 mg l-1 se ca (g kg-1) 9.1 a 6.6 b 4.9 a 10.0 a 8.4 ab 9.1 a 5.5 a 4 mg l-1 se 12.8 a 12.7 a 4.9 a 8.2 b 10.5 a 5.7 b 4.9 a 0 mg l-1 se 2.8 a 2.3 b 3.5 a 4.2 a 2.7 a 3.4 a 2.5 a 2 mg l-1 se mg (g kg-1) 3.5 a 3.8 a 2.7 a 4.5 a 2.4 a 3.4 a 2.2 b 4 mg l-1 se 8.4 a 2.4 ab 3.2 a 3.5 b 2.6 a 3.3 a 2.3 ab 0 mg l-1 se 1.7 b 2.7 a 1.5 ab 1.9 c 2.3 b 0.7 c 1.9 a 2 mg l-1 se na (g kg-1) 2.4 b 5.2 a 2.0 a 3.9 a 3.1 a 2.0 a 2.9 a 4 mg l-1 se 6.9 a 2.6 a 1.4 b 3.0 b 3.2 a 1.5 b 3.0 a 0 mg l-1 se 32.6 a 58.3 b 47.6 a 77.6 a 139.3 a 54.3 a 66.0 a 2 mg l-1 se zn (mg kg-1) 59.3 a 110.0 a 41.6 a 74.0 a 112.0 b 44.3 b 74.2 a 4 mg l-1 se 69.3 a 74.0 ab 45.0 a 62.3 b 69.0 c 37.6 c 42.3 b 0 mg l-1 se 32.3 ab 45.3 a 8.0 b 11.3 b 27.3 ab 8.0 a 19.0 a 2 mg l-1 se cu (mg kg-1) 27.6 b 36 .0 a 9.2 ab 26.3 a 28.3 a 5.6 c 5.8 c 4 mg l-1 se 67.0 a 42.0 a 11.6 a 31.3 a 23.3 b 7.0 b 9.0 b 0 mg l-1 se 35.0 b 59.3 a 63.6 a 65.0 b 55.0 a 29.0 b 30.0 a 2 mg l-1 se mn (mg kg-1) 61.3 b 73.3 a 37.0 b 93.3 a 27.6 b 18.6 c 20.8 ab 4 mg l-1 se 313.3 a 116.6 a 41.3 b 110.3 a 50.3 a 42.3 a 10.0 b 0 mg l-1 se 420.0 b 460.0 a 197.6 a 236.6 b 250.0 b 133.3 b 420.0 a 2 mg l-1 se fe (mg kg-1) 600.0 b 646.6 a 213.3 a 253.3 b 456.6 a 213.33 a 563.3 a 4 mg l-1 se 1646.6 a 683.3 a 253.3 a 550.0 a 270.0 b 140.00 b 350.0 a ¥ in each column the averages per element with different literal were statistically different according to lsd (α ≤ 0.05). 326 w.a. narváez-ortiz, m. martínez-hernández, l.o. fuentes-lara, a. benavides-mendoza, j.r. valenzuela-garcía, j.a. gonzález-fuentes fig. 3: effect of se application (2 and 4 mg l-1) in nutrient solution on mineral concentration in roots, crowns, leaves, and fruits of strawberry plants at 98 dat. to illustrate the impact of se on mineral composition in a particular plant organ, each organ was analyzed separately for p, k, ca, mg, na, zn, cu, mn, and fe. for elements not included in the figure, no significant differences were found between the treatments. signs to the right of each element’s symbol indicate increase (+), decrease (-), or equality (=) in comparison with the control. was obtained in soybean plants with foliar application of 50 mg l-1 selenate (djanaguiraman et al., 2005). on the other hand, da silva et al. (2017) found no effect associated with the application of seselenite in concentrations of up to 3.2 mg l-1 in lettuce plants. in this study, the fresh weights obtained for roots (60 dat) and for crowns and leaves (98 dat) are consistent with those reported by mimmo et al. (2017), who did not find significant differences between treatment with se-selenate at approximately 0.79 mg l-1 but observed an increase in fresh shoot weight in strawberries at a concentration 10 times higher (approximately 7.9 mg l-1 se). the decreased biomass found for the second sampling was associated with the highest concentration of se applied and was reflected in the weights of the roots and leaves, the organs where se accumulation was highest; therefore, the results can be interpreted as a sign of toxicity. it is known that the impact of se on plants depends mainly on its concentration (hamilton, 2004); low se levels will promote growth, but high levels can inhibit it (boldrin et al., 2016). ahmed (2010) observed a decrease in the fresh weight of tomato plants after applying se in the forms of sodium selenite and organic se in doses of 2 and 30 mg kg-1 of soil, respectively. ramos et al. (2010) reported a decrease in the dry weight of lettuce plants grown in hydroponics when se was applied in concentrations greater than 0.6 mg l-1; both the selenite and sodium selenate forms were used, with selenite having a more significant effect. it has been suggested that se’s negative effect on growth is partly due to the substitution of sulfur by se in proteins, which could modify the functionality of those proteins and sulfur metabolism in the plant (boldrin et al., 2016). perhaps this effect is different from one plant organ to another, which could explain the positive results in some organs and either the absence of response or undesirable effects in others. selenium distribution in plants the differential distribution of se (roots > crowns > leaves) in strawberry plants was also reported for tomatoes (becvort-azcurra et al., 2012), strawberries (mimmo et al., 2017), and beans (arvy, 1982). for the first sampling, se concentrations decreased as the distance of different organs from the root increased. the roots absorb the selenite through phosphate transporters dependent on the co-transport of h+, and once in the root tissue, it is rapidly transformed into organic forms of se that have less mobility in the xylem than ionic forms (sánchez-rodas et al., 2016). the data from the first sampling seem to indicate that the distribution of se resulted from the mobility of selenite and its organic forms. for the second sampling, however, the concentration in the leaves was higher than in the crowns, possibly indicating a type of foliar storage dependent on exposure time. the average se concentration in the fruits obtained in this study was 31.2 mg kg-1, which is lower than that reported in strawberries treated with selenate at a concentration of 7.9 mg l-1 (46 mg kg-1) (mimmo et al., 2017). similarly, se concentrations were higher in other types of fruits and vegetables fertilized with se, measuring 46.7 mg kg-1 for opuntia (bañuelos et al., 2011), 35.8 mg kg-1 for tomatoes (castillo-godina et al., 2016), and 84.32 mg kg-1 for radishes (schiavon et al., 2016). however, the concentration obtained in this study is high enough to meet the recommended daily intake in the united states (55 μg d-1 se) and in the united kingdom (60-75 μg d-1 se) (white and broadley, 2005), assuming that 16 to 23 g fw of biofortified strawberries (2-3 fruits) are consumed and that 92%-95% of the weight of these fruits is water. on the other hand, eating 8.2 g d-1 − the per capita consumption (lucier et al., 2006) − of strawberries with se concentrations found in this study would contribute 0.026 mg se, approximately half of the daily intake recommended in the united states. the se concentration found in the vegetative organs of strawberries corroborate other results indica ting that as the se concentration in the medium increases, the greater the concentration in plant tissues (schiavon et al., 2016; da silva et al., 2017). concentrations of other minerals in plants changes in the concentrations of some elements are probably one of the first observable signs of se presence in plants (pazurkiewiczkocot et al., 2003). modifications in the absorption and accumulation of different elements in strawberry plants could be due to an alteration in the absorption path or a change in the permeability coefficient of plasma membranes (pazurkiewicz-kocot et al., 2003). the result can be damage to the membrane, triggering oxidative stress and production of reactive oxygen species (ros) (hartikainen et al., 2000), caused either by excess se or by se’s inhibitory effect on zn concentration (fargašová et al., 2006). it is known that ros production induces membrane depolarization, which modifies the flow of ions, including chloride and potassium efflux and calcium influx (demidchik, 2014). the slight increases in ca and na, as well as the decreases in k and zn, observed in this study could possibly be due to these ions’ involvement in the regulation of cell membrane potential and turgor (pazurkiewicz-kocot et al., 2003). on the other hand, the increase in na can also be attributed to the application of 2 and 4 ml l-1 se as sodium selenite, which contributed 1.16 and 2.32 mg l-1 of na, respectively. however, these results differ from those found in lettuce plants, in which na concentration decreased in plants treated with up to approximately 3.15 mg l-1 se as sodium selenite (da silva et al., 2017). for the second sampling, k was possibly replaced by na, resulting in reduced k concentrations in the roots, crowns, and leaves (benton, 2012). similar results have been documented in corn plants treated with approximately 4 mg l-1 se in nutrient solution, in which k content effects of selenium application in strawberry 327 decreased and ca increased in both the shoots (hawrylak-nowak, 2008) and roots; additionally, na increased in the leaves, in which turgor was also observed (pazurkiewicz-kocot et al., 2003). this study’s results differ from those reported in lettuce plants treated with 3 mg l-1 se-selenite; in that study, ca concentration decreased (rios et al., 2013), whereas in plants treated with approximately 9.4 mg l-1 se-selenite, k concentration increased (rios et al., 2013). in the same way, increases in k in tomato plants treated with 2 mg l-1 se-selenite have also been recorded (castillo-godina et al., 2016). mg also plays a protective role in maintaining the integrity of plant tissue (wilkinson et al., 1990) as a se tolerance mechanism (feng et al., 2009). therefore, an increase of this ion would be expected; however, in this study, mg decreased in the roots. in other studies, conflicting results were found in strawberry roots (mimmo et al., 2017) and in tomato roots and leaves (schiavon et al., 2013; castillo-godina et al., 2016), whereas in corn plants (hawrylaknowak, 2008) and tomato leaves (schiavon et al., 2013), no significant effects associated with se treatments were found. se has also been found to modify the transport or absorption of other elements (djanaguiraman et al., 2005; pilon-smits et al., 2009). depending on the se concentration, se will cause synergistic or antagonistic effects, such as those observed in this study for p concentration in different organs. the antagonistic effect of se-selenite on p observed in the leaves is probably the result of competition between these ions (hopper and parker, 1999), since both are absorbed through phosphate transporters (zhao et al., 2010). this antagonistic effect has also been reported in lettuce plants treated with se levels ranging from approximately 1.58 to 9.5 mg l-1 (rios et al., 2013). the synergistic effects observed in fruits between p and se have also been reported in lettuce plants treated with se-selenite at concentrations of approximately 1.97 mg l-1 (da silva et al., 2017) and approximately 0.79 mg l1 (rios et al., 2013), and in corn plants when se increases from approximately 0.4 to 4 mg l-1 (hawrylaknowak, 2008). the decrease of p in leaves and its increase in fruits is hard to explain but probably was due to some translocation process of this element associated with fruit growth or maturation. another metabolic pathway modified by se is cellular redox balance variation (djanaguiraman et al., 2005; pilon-smits et al., 2009), which involves antioxidant synthesis associated with the reduction of ros levels (feng et al., 2013). the change probably occurs due to modifications in the concentrations of some microelements used as cofactors for superoxide dismutase enzymes (fe, mn, cu, and zn), the peroxidase enzyme (fe), the catalase enzyme (fe), and enzymes involved in the biosynthesis pathway for chlorophyll (fe) (feng et al., 2013). depending on se concentration, a pro-oxidant or antioxidant response can be induced, modifying gene expression (flohé et al., 2000) and changing the transcripts’ abundance and post-transcriptional regulation of different proteins (schiavon et al., 2015), among which some transport proteins can be found. this could partially explain the variability in the concentration of some elements assessed in plants. it has been reported that high se concentrations induced the highest fe absorption, while low se concentrations reduced fe absorption (feng and wei, 2012). however, this effect on fe is not a constant, since fargašová et al. (2006) found that the absorption tab. 4: spearman correlation coefficients obtained from the relationships between growth and mineral variables and concentrations of se present in different plant organs, evaluated at 98 dat. [se] [se] root crown leaves fruit root crown leaves fruit p -0.14 -0.27 -0.30 -0.18 p -0.18 -0.55 -0.50 -0.35 ca 0.43 -0.01 0.03 0.29 ca 0.18 0.88 * 0.70 * 0.42 k -0.67 -0.88 * -0.80 * -0.82 * k -0.31 -0.68 -0.73 * -0.49 mg -0.04 -0.49 -0.47 -0.16 mg -0.41 0.01 0.03 -0.21 na 0.63 0.48 0.45 0.64 na 0.42 0.79 * 0.78 * 0.54 root fe -0.10 0.66 0.78 * 0.08 crown fe 0.59 -0.05 0.06 0.34 zn -0.43 -0.79 * -0.82 ** -0.48 zn -0.68 -0.83 * -0.93 ** -0.73 * cu 0.57 0.70 * 0.85 ** 0.44 cu -0.45 0.16 0.22 -0.13 mn 0.57 0.92 ** 0.92 ** 0.62 mn -0.64 -0.28 -0.43 -0.48 se 1 0.52 0.52 0.82 * se 0.52 1 0.90 * 0.73 * rdw 0.20 -0.32 -0.22 -0.18 psc 0.32 -0.23 -0.18 -0.03 rfw -0.59 -0.81 ** -0.79 * -0.79 * pfc 0.28 -0.23 -0.27 -0.03 p -0.67 -0.79 * -0.83 ** -0.69 p 0.72 * 0.84 ** 0.73 * 0.89 ** ca 0.16 -0.46 -0.46 0.04 ca 0.24 -0.19 0.19 -0.11 k -0.59 -0.97 ** -0.90 -0.76 * k -0.65 -0.72 * -0.67 -0.58 mg -0.28 -0.61 -0.63 -0.35 mg -0.91 ** -0.44 -0.29 -0.77 * na 0.81 ** 0.47 0.42 0.71 * na 0.83 ** 0.49 0.54 0.68 leaves fe 0.53 0.01 0.08 0.25 fruit fe 0.30 -0.25 0.00 0.08 zn -0.65 -0.90 ** -0.88 ** -0.84 ** zn -0.27 -0.55 -0.50 -0.35 cu -0.81 ** -0.47 -0.47 -0.69 cu -0.72 * -0.50 -0.43 -0.62 mn -0.14 0.49 0.39 0.16 mn -0.53 -0.81 ** -0.66 -0.64 se 0.52 0.90 * 1 0.60 se 0.82 * 0.73 * 0.60 1 dwl 0.02 -0.77 * -0.78 * -0.27 fdw 0.23 0.15 -0.05 0.22 fwl -0.60 -0.97 ** -0.85 ** -0.60 ffw 0.63 0.45 0.20 0.65 * = significant (p <0.05); ** significant (p <0.01). rfw= root fresh weight, rdw= root dry weight, cfw= crown fresh weight, cdw= crown dry weight, fwl= fresh weight of leaves, dwl= dry weight of leaves, ffw= fruit fresh weight, fdw= fruit dry weight. 328 w.a. narváez-ortiz, m. martínez-hernández, l.o. fuentes-lara, a. benavides-mendoza, j.r. valenzuela-garcía, j.a. gonzález-fuentes of elements such as zn and fe is inhibited by increasing se levels. furthermore, longchamp et al. (2016) detected a 30% decrease in zn content in the leaves and stems of corn plants submitted to 1 mg l-1 se-selenite, while at 0.01 mg l-1 concentrations, fe concentration in the roots increased; no significant effects were found for zn, cu, and mn. da silva et al. (2017) also demonstrated that treatment with selenite decreased the concentrations of cu and fe. for the second sampling, additional changes in the concentrations of elements were evident in different plant organs (fig. 1). the changes undoubtedly occurred because the longer exposure time to the se solution (38 days elapsed between the first and second sampling dates) increased the concentration of se, since it is known that the concentration of this element in plant tissues is a function of its availability in the growing medium (becvort-azcurra et al., 2012). the results of foliar analyses suggest that se application in nutrient solution at concentrations of 2 and 4 mg l-1 was not associated with adverse effects on macronutrient concentrations, which remained above the sufficiency ranges (benton, 2012). the same pattern was repeated for micronutrients, except for mn, which decreased to concentrations below the sufficiency ranges in treated and untreated plants. in a study of strawberry plants, se-selenate application in concentrations of 10 and 100 μm (approximately 0.79 and 7.90 mg l-1) did not significantly modify the concentrations of p, k, ca, mg, s, fe, and mn in the roots, shoots, and fruits (mimmo et al., 2017). moreover, in cucumber plants, selenite and selenate application in lower concentrations than those used in this study (<10 μm, or approximately 0.79 mg l-1) did not substantially modify the concentrations of n, p, k, mg, ca, and s (hawrylak-nowak et al., 2015). in tomato plants, treatment with se-selenite at concentrations of 2 and 5 mg l-1 did not interfere negatively with the accumulation of n, p, k, ca, and mg in the stems, leaves, and fruits (castillo-godina et al., 2016). in lettuce plants treated with se-selenite at concentrations of 5 and 10 mg l-1, the concentrations of n, p, k, ca, mg, na, zn, mn, and cu were not substantially modified (lópez-gutiérrez et al., 2015). ph and orp in fruit dynamics for the duration of the experiment, the ph of the fruit pulp was maintained at an average value of 3.32 for the three treatments, a value considered as acceptable in strawberry fruits (roudeillac and trajkovski, 2004; pérez de camacaro et al., 2005). low orp values obtained in the first 12 days of harvest indicated that se applied at 2 mg l-1 had an impact on the reducing capacity of the fruit pulp. the orp values mentioned indicate the antioxidant capacity − that is, the capacity of the system under analysis to give electrons in comparison with a hydrogen electrode (benavidesmendoza et al., 2002). the lower the orp value, the higher the capacity to release electrons to function as an antioxidant. this transient response to se application could be caused by the capacity of this metalloid to induce oxidative stress, the magnitude of which depends on se concentration. at low concentrations, it can be an activator of the plants’ antioxidant system (kong et al., 2005), as seemed to occur with treatment at 2 mg l-1 during the first 122 days. in contrast, at high concentrations, it functions as a prooxidant agent (hartikainen et al., 2000), a situation that undoubtedly occurred when se was applied at a concentration of 4 mg l-1, as well as after 122 days when applied at a concentration of 2 mg l-1. these results indicate that if the purpose of se application is to increase the antioxidant state of the fruits, the applied concentration should be less than 2 mg l-1; otherwise, se should be applied intermittently (for example, once a week) instead of continuously, as was done in this study. se application as sodium selenite in nutrient solution at a concentration of 2 mg l-1 was determined to be a proper enrichment technique for strawberry fruits. plant biomass decreased when se was applied at concentrations of 2 and 4 mg l-1, but fruit production was not diminished. mineral concentrations in different plant organs were both positively and negatively associated with se application, but the concentrations did not decrease below the recommended ranges. neither ph nor orp in fruit pulp were modified by se treatments, and a positive effect was observed on the antioxidant status of the fruits in the first days of harvest when treated at 2 mg l-1. references ahmed, h.k., 2010: differences between some plants in selenium accumulation from supplementation soils with selenium. agric. biol. j. n. am., 1, 1050-1056. doi: 10.5251/abjna.2010.1.5.1050.1056 aoac, 1990: official methods of analysis of the association of official analytical chemists. official methods of analysis of the association of official analytical chemists., 15th edn. washington, d.c. usa. ph 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 0 mg l-1 se 2 mg l-1 se 4 mg l-1 se days after transplant 98 10 1 10 2 10 3 11 2 11 4 11 5 11 6 11 7 12 3 12 4 12 5 12 8 12 9 a a a a a a a a a a a a a b b a a a a a a a a a a a a a a a a a a a a a a a a a a a days after transplant o r p (m v ) -80 -60 -40 -20 0 20 40 60 80 0 mg l-1 se 2 mg l-1 se 4 mg l-1 se a a a a a b a b c a b b a b b a a b a a a b a ab a c a b c a a a a a a a b b a a b 98 10 1 10 2 10 3 11 2 11 4 11 5 11 6 11 7 12 3 12 4 12 5 12 8 12 9 b fig. 4: ph values over time of strawberry fruits harvested from plants trea ted with different concentrations of se in nutrient solution. averages with different letters were statistically different according to lsd (α ≤ 0.05). fig. 5: oxidation-reduction potential (orp) values over time of strawberry fruits harvested from plants treated with different concentrations of se in nutrient solution. averages with different letters were statistically different according to lsd (α ≤ 0.05). effects of selenium application in strawberry 329 aoac, 2000: official methods of analysis of the association of official analytical chemists. official methods of analysis of the association of official analytical chemists., 17th edn. gaithersburg, md. usa. arvy, m.-p., 1982: translocation of selenium in the bean plant (phaseolus vulgaris) and the field bean (vicia faba). physiol. plant. 56, 299-302. doi: 10.1111/j.1399-3054.1982.tb00342.x bañuelos, g.s., arroyo, i., pickering, i.j., yang, s.i., freeman, j.l., 2015: selenium biofortification of broccoli and carrots grown in soil amended with se-enriched hyperaccumulator stanleya pinnata. food chem. 166, 603-608. doi: 10.1016/j.foodchem.2014.06.071 bañuelos, g.s., fakra, s.c., walse, s.s., marcus, m.a., yang, s.i., pickering, i.j., pilon-smits, e.a.h., freeman, j.l., 2011: selenium accumulation, distribution, and speciation in spineless prickly pear cactus: a drought-and salt-tolerant, selenium-enriched nutraceutical fruit crop for biofortified foods. plant physiol. 155, 315-327. doi: 10.1104/pp.110.162867 becvort-azcurra, a., fuentes-lara, l.o., benavides-mendoza, a., ramírez, h., robledo-torres, v., rodríguez-mendoza, m. de las n., 2012: application of selenium in tomato: effects on plant growth, productivity and fruit antioxidant status. terra latinoam. 30, 291-301. benavides-mendoza, a., ramírez, h., robledo-torres, v., cornejooviedo, e., maiti, r.k., 2002: productivity, co2 assimilation, and mineral tissue concentrations in onion plants under colored plastic films. crop res. 24, 26-39. benavides-mendoza, a., ramírez, h., robledo-torres, v., fuenteslara, l.o., sandoval-rangel, a., 2012: trace elements and nutritional quality, cases of iodine, zinc, and selenium. in: rocha-estrada, a., moreno-limón, s., gonzález-de la rosa, m. del c. 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accepted: june 6, 2018) * corresponding author summary hplc-pda-esi-msn analysis of different parts such as stem bark, shoot, flower, fruit and root of capparis spinosa (c. spinosa) and capparis decidua (c. decidua), collected in rainy and dry seasons from the cholistan desert of pakistan, depicted the occurrence of a wide array of phenolics with quercetin, apigenin and kaempferol derivatives along with dicaffeoylquinic acid, caffeoylquinic acid and feruloylquinic acid as the main compounds. kaempferol-3-glucoside (28.02-167.21 µg g-1dw) was found to be the principal component in all tested parts of both species while dicaffeoylquinic acid was detected only in the flowers and roots. the roots exhibited maximum contents of flavonoids and hydroxycinnamic acid derivatives. the harvesting period significantly (p<0.05) affected the concentration of phenolics wherein the samples collected in rainy season offered greater levels of phenolics than their counterpart. the roots and fruits of both species were found to be rich sources of phenolics. the findings of this research suggest the harvesting of the selected wild capparis species in rainy season to maximize their antioxidant and nutraceutical benefits. keywords: capparaceae; flavonoids; hydroxycinamic acids; polyphenols introduction plants are valued as a rich source of a wide array of secondary metabolites. among secondary metabolites, phenolics are one of those bioactive compounds which are widely distributed in the plant kingdom (muhammad et al., 2015; sahib et al., 2013; shahidi, 1997). it is widely accepted that nutraceutical and antioxidant attributes of plant foods are mainly associated with their phenolics (shahidi, 1997). in this regard, consumption of selected vegetables and fruits as well as several other plant foods, is strongly linked with health benefits and/or reduced incidence of degenerative diseases such as aging, inflammation and certain cancers (lodovici et al., 2001; oomah, 2001; robbins, 2003). capparis is one of the important genera with known 250 species distributed world wide. of the capparis species, capparis spinosa and capparis decidua are native to pakistan (gull et al., 2015a). these plants have been investigated for their anti-atherosclerosis, anti hypertensive, anti-inflammatory, analgesic, anti-asthmatic, anti-hyper lipidemic, hepatoprotective, antibacterial, and antifungal activities (chahlia, 2009; duman et al., 2013; hundiwale et al., 2005; mali et al., 2005). besides, stem bark, shoot, root, flower and fruits of both of these species have been reported as good sources of valuable nutrients such as minerals, crude fiber and protein (gull et al., 2015b; gull et al., 2015c; gull et al., 2015d). different parts of these species are also being employed in the traditional medicine systems for the treatment of several diseases (gull et al., 2015a). for example, the root extract of c. spinosa has been reportedly used to prepare liver protecting drugs named liv-52 and cm-52 which are used to cure hepatitis b and liver cirrhosis (eddouks et al., 2005; rajesh et al., 2009). the hepatoprotective efficacy of c. spinosa might be attributed to the potential antioxidant activities of its bio actives following the restoration of liver cell membrane and its permeability, while the exact mechanism of action needs to be explored. moreover, c. decidua extracts have also been reported for hepatoprotective effects via decreasing the levels of the enzymes sgpt (serum glutamate pyruvate transaminase), sgot (serum glutamic oxaloacetic transaminase), and alp (alkaline phosphatase) (jhajharia et al., 2010). different parts of capparis species are also used to cure/prevent diabetes, cardiac diseases, toothache and intermittent fever (marwat et al., 2011; özcan, 2005; sharma and kumar, 2008). it has been reported in various studies that plant phenolics composition is affected by geographical zones, climatic conditions and seasonal variability (nouman et al., 2016; olson et al., 2016). researchers are focusing on profiling of the phenolics in vascular plants but there are few studies available revealing the variation in these compounds under seasonal fluctuations (sampaio et al., 2016) while no study is available explaining the variations in concentration of phenolic acids in different parts of c. spinosa and c. decidua as affected by harvesting seasons. the concentration and availability of phenolic bioactive compounds vary within plants of the same species and their parts. variation in expression and profiling of these bioactive compounds also vary with respect to cultivar, geogra phical zones, climatic conditions and seasonal fluctuations (cartea et al., 2008; ciska et al., 2000; garcía-salas et al., 2014; ray et al., 2013). for example, variation in biological activities of different brassica vegetables under seasonal fluctuations such as temperature and precipitation might affect the composition and concentration of bioactive compounds resulting in affecting antioxidant activities of brassica vegetables (aires et al., 2011). likewise, variation in bioactive compounds, enzymatic antioxidants and antiradical activities of moringa oleifera leaves as affected by tree age, climatic factors and soil condition has been reported (nouman et al., 2016; vázquez-león et al., 2017). keeping in view the possible variations among bioactive compounds of various plant species and genera, it can be hypothesized that different parts of c. spinosa and c. decidua can yield varying concentration and profile of bioactive compounds under seasonal effects. the present study was aimed to investigate whether or not the two 74 t. gull, b. sultana, f. anwar, w. nouman, e. rosa, r. domínguez-perles different seasons such as rainy and dry affect the profile of phenolics in different parts of wild c. spinosa and c. decidua harvested in their natural habitat. materials and methods reagents all lc-ms grade solvents were obtained from j.t. baker (phillipsburg, nj). formic acid was purchased from panreac (barcelona, spain). the standards (-)-epigallocatechin, quercetin-3-o-glucoside, 5-o-caffeoylquinic acid were from sigma aldrich (steinheim, germany). ultrapure water was produced using a millipore water purification system. collection of plant samples and pretreatment as a representative of dry and rainy seasons, two months including april and september were selected for plant sampling based on low/high temperature and rainfall intensities, respectively. rainfall and temperature data were obtained from pakistan council of research in water resources (pcrwr), bahawalpur, pakistan. fig. 1 depicts an average weather data for the sampling site while in particular sampling months (april and september), mean temperature (27.9 and 31.1 °c) and average rainfall (7 and 41 mm) was noted, respectively. different parts including stem bark, shoot, fruits, flowers and roots of c. spinosa and c. decidua were collected from cholistan desert area of bahawalpur, punjab, pakistan (desert region, latitude 28-15° n; longitude 70-45° ' e and altitude 89 m above mean sea level) in april and september 2013. the samples of selected parts were harvested from ten different plants of each of the mentioned species and then these were pooled in three replications for further analyses. the specimen were further identified and authenticated by dr. mansoor hameed, taxonomist, department of botany, university of agriculture faisalabad, pakistan. the collected samples were dried at room temperature for 24 hours followed by oven drying at 45 °c for further studies (nouman et al., 2016). sample preparation and analysis samples of the selected parts including stem bark, shoot, flower, fruit and root of c. spinosa and c. decidua were prepared for the analysis of phenolic compounds using hplc-pda-esi-msn and hplc-pad following the protocol as described by our research group (nouman et al., 2016). briefly, samples were air-dried in an oven at 45 °c for 72 h and were ground to a fine powder and stored at -20 ºc for further analysis. each sample (40 mg) was extracted with 1.5 ml of 70% methanol for 30 min at 70 ºc, vortexed every 5 min to improve extraction efficacy and then centrifuged (10000×g for 20 min at 4 ºc) (model sigma 1-13, b braun biotech international, osterode, germany). supernatants were collected and methanol was removed under reduced pressure using a rotary evaporator (eyela, n-n series, rikakikai co., tokyo, japan). the dried residue was reconstituted in ultrapure water (1 ml) and filtered through a 0.22 μm polypropy lene membrane filter (anotop 10 plus; whatman, maidstone, uk). each sample (20 μl) was analyzed in a lc-pad-esi/msn (thermoscientific, loughborough, uk) and hplc-pad (gilson inc., mettmenstetten, switzerland) for phenolics identification/authentication and quantification, respectively. the method described by nouman et al. (2016) was used for these analyses as mentioned below. analysis of phenolic compounds by hplc-pda-esi-msn and hplc-pda the phenolics profiling, using hplc-pda-esi-msn chromatographic analysis, was conducted on a luna c18 column (150 × 2.1 mm, 2.6 μm particle size; thermo-scientific, loughborough, uk). the mobile phase was a mixture of distilled water/formic acid (99:1, v/v) (solvent a) and acetonitrile/formic acid (99:1, v/v) (solvent b). the flow rate was of 0.6 ml/min in a linear gradient following the scheme (t in min; %b): (0; 0%), (5; 20%), (30; 50%), (45, 100%), and (55; 0%). the chromatograms were recorded at 320 and 520 nm. the equipment consisted of a lc pump (srvyr-lpump), an autosampler (srvyr-as), and a photodiode array detector (srvyr-pda5). the hplc-pda-esi-msn analysis was performed in a mass detector in series, which was an ion trap spectrometer (model lcq-advantagemax) equipped with an electrospray ionization interface controlled by tune plus 1.3 sr1 software (fisher scientific, lisboa, portugal) and operated in negative mode. the ionization conditions were adjusted at 250 ºc and 4.0 kv for capillary temperature and voltage, respectively. the nebulizer pressure and flow rate of nitrogen were 2.0 bar and 8.0 l/min, respectively. the full scan mass covered the range from m/z 100 up to m/z 1500. collision-induced fragmentation experiments were performed in the ion trap using helium as the collision gas, with voltage ramping cycles from 0.3 up to 2.0 v. 16 360 361 fig. 1: mean annual temperature and rainfall data of cholistan desert, district bahawalpur 362 363 0 5 10 15 20 25 30 35 40 45 50 jan feb mar apr may jun jul aug sep oct nov dec average rainfall (mm) mean temerature (°c) fig. 1: mean annual temperature and rainfall data of cholistan desert, district bahawalpur profiling of polyphenolics in capparis species 75 quantitative analysis was carried out by a hplc-pda system (gilson inc., mettmenstetten, switzerland) using the same chromatographic conditions as described for identification. the equipment consisted of a lc pump (gilson, 305/306-pump), a dynamic mixer chamber (gilson, 811c), an autosampler (gilson, autoinjector-234), and a photodiode array detector (pda-plus detector finnigan suriveyor, thermo-scientific). cinnamic acids derivatives were quantified as 5-o-caffeoylquinic acid at 320 nm and flavonols as quercetin3-o-glucoside at 360 nm. statistical analysis data were processed using mstat-c statistic software package (michigan state university, michigan, us). a multi factorial analysis of variance (anova) and multiple range test (tukey’s test) were carried out to evaluate statistical differences. the level of significance was set at p < 0.05. results in the present study, on the basis of retention time (rt), molecular masses, and fragmentation patterns, twelve flavonols and five hydroxycinnamic acids were detected and reported for the first time in the selected capparis species from pakistan. a significant (p < 0.05) variation in the qualitative and quantitative composition of flavonoids and hydroxycinnamic acids was recorded among parts of both species as well as function of two seasons. overall, maximum concentration of total flavonoids was observed in c. spinosa in rainy season (554.53 μg g-1 dw) in comparison with dry months (488.07 μg g-1 dw) which were 11% lower (tab. 1-5). a similar trend was recorded for hydroxycinnamic acid derivatives as well. over all, in comparison with c. spinose, c. decidua offered 19.23 and 21.97% higher flavonoids and hydroxycinnamic acids, respectively (tab. 1-5). stem bark of both species showed different compounds with varying concentration in both seasons. the stem bark of c. spinosa con tained lower amount of kaempferol-3-glucoside (114.80 μg g-1 dw, on average) than that of c. decidua (144.78 μg g-1 dw, on average). the stem bark of c. decidua exhibited a reasonable level ofapige nin-7-c-glucoside (isorhoifolin) and quercetin-3-glucoside; however, these compounds were present only in traces in the stem bark of c. spinosa. c. decidua stem bark showed higher contents of the above mentioned phenolics in rainy season while c. spinosa stem bark presented more concentration in dry season (tab. 1). beside, 3-p coumaroylquinic acid and feruloylquinic acid were also detected in c. spinosa stem bark while c. decidua stem bark showed the pre sence of only feruloylquinic acid (tab. 1). shoot of c. spinosa and c. decidua showed lower concentration of these phenolics in comparison with other parts. apigenin-8-c-glucoside (isovitexin) (3.15 μg g-1 dw, on average), quercetin-3-glucoside (5.23 μg g-1 dw, on average) and kaempferol-3-glucoside (45.93 μg g-1 dw, on average) were recorded in c. spinosa shoot while quercetin3-rhamanoside was recorded in lower quantity. on the other hand, c. decidua shoot contained quercetin-3-glucoside (2.87 μg g-1 dw, on average), apigenin-7-c-glucoside (isorhoifolin) (5.48 μg g-1 dw, on average), kaempferol-3,7-diglucoside (4.34 μg g-1 dw, on average) and kaempferol-3-glucoside (51.71 μg g-1 dw, on average) in both seasons. in case of hydroxycinnamic acids, only feruloylquinic acid was recorded in both species with its concentration higher in c. spinosa shoot samples collected in rainy season than c. decidua shoot samples of both seasons (tab. 2). the other flavonoid and hydroxycinna mic acids were detected in traces. fruits of both species were noted to be a good source of flavonoids and hydroxycinnamic acids compared to other parts with c. decidua tab. 1: identification and quantification of phenolic acids and flavonoids (μg g-1 dw) in stem bark of c. spinosa and capparis decidua compounds capparis spinosa capparis decidua dry season rainy season dry season rainy season flavonoids quercetin-3,7-diglucoside 0.08±0.01 e 0.06±0.10 e traces traces quercetin-3,7-diglucoside (isomer) traces traces traces traces apigenin-8-c-glucoside (isovitexin) traces traces traces traces quercetin-3-glucoside traces traces 22±1.27 cd 13±0.43 d apigenin-7-c-glucoside (isorhoifolin) traces traces 39±0.94 c 44±1.41 c kaempferol-3,7-diglucoside traces traces 8±1.03 c 8±1.13 c apigenin-7-rutinoside traces traces 6±0.87-d 8±0.54 d quercetin-3-rhamanoside 0.46±0.09 e 0.42±0.07 e 0.34±0.02 0.39±0.05 quercetin-3-sophoroside 0.24±0.05 e 0.12±0.02 e traces 0.49±0.08 quercetin-3-acetyl-glucoside traces traces 0.11±0.03 traces kaempferol-3-glucoside 122±3.59 a 107±2.98 ab 122±1.80 b 167±5.96 a kaempferol-7-glucoside traces traces traces traces total 123 a 108 b 197 b 242 a hydroxycinamic acids dicaffeoylquinic acid traces traces traces traces 5-caffeoylquinic acid traces traces traces traces 3-caffeoylquinic acid traces traces 0.07±0.01 traces 3-p-coumaroylquinic acid 31±1.11 c 52±1.73 b traces traces feruloylquinic acid 16±1.38 d 35±1.12 c 22±1.27 cd 35±1.18 c total 48 b 87 a 22 bc 35 b values (means ±sd) are average of three samples of each part, analyzed individually in triplicate (p< 0.05). 76 t. gull, b. sultana, f. anwar, w. nouman, e. rosa, r. domínguez-perles tab. 2: identification and quantification of phenolic acids and flavonoids (μg g-1 dw) in shoot of c. spinosa and capparis decidua compounds capparis spinosa capparis decidua dry season rainy season dry season rainy season flavonoids quercetin-3,7-diglucoside traces traces traces traces quercetin-3,7-diglucoside (isomer) traces traces traces traces apigenin-8-c-glucoside (isovitexin) 2.3±0.07 c 4.0±0.05 bc traces traces quercetin-3-glucoside 4.4±0.83 bc 6.0±0.19 b 2.4±0.22 d 3.4±0.32 d apigenin-7-c-glucoside (isorhoifolin) traces traces 7.75±1.23 c 3.22±0.71 d kaempferol-3,7-diglucoside traces traces 4.0±0.47 d 4.7±0.38 d apigenin-7-rutinoside traces traces traces traces quercetin-3-rhamanoside 0.73±0.11 d 0.29±0.08 d traces traces quercetin-3-sophoroside traces traces traces traces quercetin-3-acetyl-glucoside traces traces traces traces kaempferol-3-glucoside 43±0.57 a 48±1.89 a 42±1.40 b 61±2.08 a kaempferol-7-glucoside traces traces traces traces total 51 b 59 b 57 b 72 a hydroxycinamic acids dicaffeoylquinic acid traces traces traces traces 5-caffeoylquinic acid traces traces traces traces 3-caffeoylquinic acid traces traces traces traces 3-p-coumaroylquinic acid traces traces traces traces feruloylquinic acid 7.5±0.96 b 17±0.99 b 2.4±0.22 d 3.9±0.47 d total 7.5 b 17 a 2.4 a 3.9 a values (means ±sd) are average of three samples of each part, analyzed individually in triplicate (p< 0.05). tab. 3: identification and quantification of phenolic acids and flavonoids (μg g-1 dw) in fruit of c. spinosa and capparis decidua compounds capparis spinosa capparis decidua dry season rainy season dry season rainy season flavonoids quercetin-3,7-diglucoside traces traces traces traces quercetin-3,7-diglucoside (isomer) traces traces traces traces apigenin-8-c-glucoside (isovitexin) 17±1.03 c 20±0.1 c 2.6±0.42 e 2.9±0.31 e quercetin-3-glucoside 0.08±0.009 0.12±0.009 25±1.30 c 32±1.08 c apigenin-7-c-glucoside (isorhoifolin) traces traces 0.42±0.15 f traces kaempferol-3,7-diglucoside traces traces 0.21±0.02 f 1.2±0.14 ef apigenin-7-rutinoside traces traces 7.2±0.49 d 11±0.48 d quercetin-3-rhamanoside 0.47±0.08 e 0.89±0.12 e 0.41±0.08 1.11±0.18 quercetin-3-sophoroside 0.12±0.15 e 0.78±0.23 e traces traces quercetin-3-acetyl-glucoside 0.11±0.05 e 0.43±0.09 e 0.19±0.009 f 0.97±0.10 f kaempferol-3-glucoside 123±1.66 b 147±1.34 a 116±1.54 b 131±5.38 a kaempferol-7-glucoside traces traces traces traces total 140 b 170 a 152 b 180 a hydroxycinamic acids dicaffeoylquinic acid traces traces traces traces 5-caffeoylquinic acid traces traces traces 0.49±0.08 3-caffeoylquinic acid traces traces 0.09±0.012 f 1.1±0.17 ef 3-p-coumaroylquinic acid 4.1±0.59 de 5.0±0.47 de 2.6±0.42 e 5.0±0.97de feruloylquinic acid 7.6±0.85 d 8.2±0.37 d 24.9±1.30 c 29.0±1.03 c total 12 a 13 a 28 b 36 a values (means ±sd) are average of three samples of each part, analyzed individually in triplicate (p< 0.05). profiling of polyphenolics in capparis species 77 tab. 4: identification and quantification of phenolic acids and flavonoids (μg g-1 dw) in flower of c. spinosa and capparis decidua compounds capparis spinosa capparis decidua dry season rainy season dry season rainy season flavonoids quercetin-3,7-diglucoside traces traces 1.18±0.56 b 1.16±0.32 b quercetin-3,7-diglucoside (isomer) 3.6±0.12 de 2.7±0.09 traces traces apigenin-8-c-glucoside (isovitexin) 2.6±0.04 de 5.0±0.20 d traces traces quercetin-3-glucoside 2.8±0.40 de 3.5±0.13 de 1.8±0.09 b 2.2±0.11 b apigenin-7-c-glucoside (isorhoifolin) traces traces 2.2±0.31 b 2.1±0.27 b kaempferol-3,7-diglucoside traces traces traces traces apigenin-7-rutinoside traces traces traces 0.61±0.19 quercetin-3-rhamanoside 0.82±0.05 e 1.3±0.13 e 0.9±0.03 1.4±0.09 quercetin-3-sophoroside traces traces traces traces quercetin-3-acetyl-glucoside traces traces traces traces kaempferol-3-glucoside 41±0.78 b 52±1.77 a 40±1.07 ab 48±1.97 a kaempferol-7-glucoside traces traces 2.1±0.85 3.0±0.78 total 51 b 65 a 48 b 59 a hydroxycinamic acids dicaffeoylquinic acid 17±0.52 c 24±0.88 c traces traces 5-caffeoylquinic acid traces traces traces traces 3-caffeoylquinic acid traces traces traces traces 3-p-coumaroylquinic acid traces traces traces traces feruloylquinic acid traces traces 1.8±0.59 b 2.3±0.12 b total 17 b 24 a 1.8 a 2.3 a values (means ±sd) are average of three samples of each part, analyzed individually in triplicate (p< 0.05). tab. 5: identification and quantification of phenolic acids and flavonoids (μg g-1 dw) in root of c. spinosa and capparis decidua compounds capparis spinosa capparis decidua dry season rainy season dry season rainy season flavonoids quercetin-3,7-diglucoside 0.10±0.01 e 0.32±0.01 e traces traces quercetin-3,7-diglucoside (isomer) traces traces traces traces apigenin-8-c-glucoside (isovitexin) 14.±1.5 cd 19.±0.65 c 18±1.25 f 19±0.62 f quercetin-3-glucoside traces traces 55±0.82 c 69±2.21 bc apigenin-7-c-glucoside (isorhoifolin) traces traces 48±0.45 d 59±2.15 kaempferol-3,7-diglucoside traces traces 6.0±0.35 g 8.0±0.37 g apigenin-7-rutinoside traces traces 21±0.77 f 18±1.02 quercetin-3-rhamanoside 0.48±0.08 e 0.97±0.09 e 51±1.20 d 69±2.07 bc quercetin-3-sophoroside traces traces traces traces quercetin-3-acetyl-glucoside 0.51±0.008 e 1.19±0.10 e 44±0.66 de 57±2.17 c kaempferol-3-glucoside 107±1.76 b 132±4.34 a 28±1.87 e 44±1.71 de kaempferol-7-glucoside traces traces 20±1.05 25±1.13 ef total 123 b 154 a 290 b 368 a hydroxycinamic acids dicaffeoylquinic acid traces traces 29±1.29 e 34±1.19 e 5-caffeoylquinic acid traces traces 3±0.16 g 4±0.19 g 3-caffeoylquinic acid traces traces 78±1.03 b 97±3.21 a 3-p-coumaroylquinic acid 8±1.00 d 11±0.85 cd 18±1.25 f 30±1.13 e feruloylquinic acid 7.0±0.55 d 22±1.10 c 55±0.82 c 61±2.47 bc total 15 b 33 a 183 b 226 a values (means ±sd) are average of three samples of each part, analyzed individually in triplicate (p< 0.05). 78 t. gull, b. sultana, f. anwar, w. nouman, e. rosa, r. domínguez-perles fruits offering higher concentration of these compounds (tab. 3). the fruits from c. spinosa exhibited maximum concentration of kaempferol-3-glucoside (134.80 μg g-1 dw, on average) followed by apigenin-8-c-glucoside (isovitexin) (18.62 μg g-1 dw, on average) while c. decidua fruit contained quercetin-3-glucoside (28.21 μg g-1 dw, on average) and kaempferol-3-glucoside (123.50 μg g-1 dw, on average). moreover, 3-p-coumaroylquinic acid and feruloylquinic acid were recorded in fruits of both species in both seasons while 3-caffeoylquinic acid was recorded only in c. decidua fruit (tab. 3). likewise, shoot and flowers of both species exhibited lower quantities of identified flavonoids and phenolic acids. among flavonoids, only kaempferol-3-glucoside was detected in both species while the other compounds were present in traces. dicaffeoylquinic acid (20.56 μg g-1 dw, on average) was quantified in flowers of c. spinosa while feruloylquinic acid was present in c. decidua flowers (tab. 4). it can be guessed from the data of tab. 5 that roots of c. decidua contained higher amount of flavonoids and hydroxycinnamic acids than its counterpart. meanwhile, the roots from c. spinosa were found to be a good source of kaempferol-3-glucoside (119.66 μg g-1 dw, on average) while the other flavonoids were found in traces. regarding hydroxycinnamic acids, dicaffeoylquinic acid, 5-caffeoyl quinic acid and 3-caffeoylquinic acid were identified in traces in c. spinosa roots while 3-p-coumaroylquinic and feruloylquinic acids were found in reasonable concentration. on the other hand, c. decidua roots exhibited maximum amount of 3-caffeoylquinic acid (87.58 μg g-1 dw, on average) followed by feruloylquinic acid (58.28 μg g-1 dw, on average), dicaffeoylquinic acid (31.40 μg g-1 dw, on average), 3-p-coumaroylquinic acid (23.69 μg g-1 dw, on average) and 5-caffeoylquinic acid (3.28 μg g-1 dw, on average) (tab. 5). discussion hplc-pda-esi-msn analysis of aqueous methanolic extracts of different parts of c. spinosa and c. decidua demonstrated a variable composition of phenolic compounds in dry and rainy seasons. among these compounds, flavonols were found as the main consti tuents as shown in tab. 1-5. beside these, hydroxycinnamic acids were also found in the tested parts of these species with varying concentrations. previously, few studies have shown the presence of rutin, kaempferol, kaempferol rutinoside and quercetin rutinoside (argentieri et al., 2012; inocencio et al., 2000; rodrigo et al., 1992) while the others were detected first time in the present study. the fruits of c. spinosa presented highest amount of total flavonoids in rainy seasons (169.50 μg g-1 dw) followed by roots (153.61 μg g-1 dw) and fruits of dry season (140.35 μg g-1 dw). total flavonoids, expressed in dry season by stem bark and roots of c. spinosa, were 27% lower than fruits (tab. 1-5). kaempferol-3-glucoside was noted as main flavonoid in c. spinosa fruits (134.80 μg g-1 dw, on average) followed by apigenin-7-c-glucoside (isorhoifolin) (tab. 3). apigenin and kaempferol have previously been reported in c. spinosa fruits collected from xinjiang province of china (yu et al., 2006; zhou et al., 2010; zhou et al., 2011) but these compounds were identified for the first time in capparis fruit samples collected from pakistan. beside this, a few other derivatives of kaempferol were reported in c. spinosa shoots which were confirmed in the present investigation (siracusa et al., 2011). it was noted that c. spinosa shoot is a rich source of kaempferol derivative. such variation in flavonoids distribution within plants of different origins may be linked to varying mode of extraction employed, and genetic and agro-climatic factors prevailing in different sites (hashempour et al., 2010; islam et al., 2003). the flavonoid compounds detected in the present analysis of capparis species were consisted of twelve compounds including quercetin-3,7-diglucoside, quercetin-3,7-diglucoside (isomer), apigenin 8-c-glucoside, quercetin-3-glucoside, apigenin-7-c-glucoside (iso rhoifolin), kaempferol-3,7-diglucoside, apigenin-7-rutinoside, quercetin-3-rhamnoside, quercetin-3-o-sophoroside, quercetin-3-acetyl glucoside, kaempferol-3-glucoside, and kaempferol-7-glucoside with their composition significantly differed among different parts of both species in either of the seasons. as stated earlier, fruits and roots of c. spinosa presented maximum content of flavonoids while a few such as apigenin-7-c-glucoside (isorhoifolin), kaempferol-3,7diglucoside, apigenin-7-rutinoside and kaempferol-7-glucoside were detected only in traces amount in c. spinosa. in case on hydroxycinnamic acids, stem bark and fruits of c. spinosa exhibited maximum amount of these acids in either of the dry and rainy seasons, respectively while 5-caffeoylquinic acid and 3-caffeoylquinic acid were recorded in traces (tab. 1-5). kaempferol-3-glucoside was the most abundant constituent (437.0 and 486.9 μg g-1 dw) and represented 89.0 and 87.8% of flavonoids detected in in dry and rainy seasons, respectively. apigenin-8-c-glucoside (isovitexin) was found as the second most abundant flavonoid in c. spinosa with contribution 36.09 and 48.49 μg g-1 dw in dry and rainy seasons, respectively. apigenin-8-c-glucoside (isovitexin) was mainly present in the fruits representing 43.3% of total apigenin-8-c-glucoside (isovitexin) contents of c. spinosa while in stem bark, it was not detected. the least amounts of flavonoids were quantified in c. spinosa shoot. dicaffeoylquinic acid, 5-caffeoylquinic acid, 3-caffeoylquinic acid, 3-p-coumaroylquinic acid, and feruloylquinic acid were identified in c. spinosa (tab. 1-5). as a whole, c. spinosa presented 99.10 and 173.80 μg g-1 dw of hydroxycinnamic acids in dry and rainy months, respectively (tab. 1-5). stem bark and roots exhibited maximum amount of these acids (47.85 and 33.06 μg g-1 dw, respectively) while the least count was recorded in shoots. of the studied hydroxycinnamic acids, 3-p-coumaroylquinic acid presented maximum amount in dry month (43.33 μg g-1 dw) while in rainy month feruloylquinic acid gave maximum quantity (81.44 μg g-1 dw). both caffeoylquinic acid derivatives were found in traces while dicaffeoylquinic acid was as high as 17.38 and 23.74 μg g-1 dw (tab. 1-5). the availability of these phenolic acids supports the traditional health benefits of capparis. the researchers reported that roots, fruits and flowers of these species are a good for curing infectious diseases (iwu et al., 1999). such functions of capparis tissues might be attributed to the phenolic acids (sharma and kumar, 2008). the researchers extracted kaempferol, apigenin and cinnamic acids from fruit extracts of c. spinosa and identified these as anti-inflammatory agents against induced rat paw edema (zhou et al., 2010). so, the findings of the pre sent investigation support that capparis tissues, especially roots can be used in pharmaceutical industries. c. decidua presented more amounts of flavonoids and hydroxycinnamic acids. in dry and rainy months, c. decidua exhibited 65.5 and 52.9% more flavonoids than c. spinosa, respectively. these data also showed that c. decidua provided more flavonoids in rainy months rather than dry ones (920.38 and 734.49 μg g-1 dw, respectively) as was recorded in case of c. spinosa. among different parts of c. decidua, roots ranked higher in flavonoids followed by stem bark and fruits. the least amount of flavonoids was recorded in shoots (64.42 μg g-1 dw, on average) which was 80.6% lower than roots and 70.7% lower than stem bark. among all studied flavonoids, quercetin-3,7-diglucoside (isomer) was found in traces while all others were also detected except quercetin-3-sophoroside and quercetin-3,7-diglucoside (0.49 and 1.17 μg g-1 dw on average, respectively). kaempferol-3-glucoside ranked highest among all detected flavonoids (400.10 μg g-1 dw, on average) followed by quercetin-3-glucoside and apigenin-7-c-glucoside (isorhoifolin). these three compounds re presented 74.00% of total flavonoids. interestingly, a few flavonoids like apigenin-7-rutinoside and quercetin-3-sophoroside were not detected in dry months but these were found in the samples (flowers and stem bark) harvested/plucked during rainy month. previously, various compounds like apigenin, kaempferol, kaempferol-3-o profiling of polyphenolics in capparis species 79 rutinoside and quercetin-3-o-rutinoside have been isolated from c. spinosa fruits (yu et al., 2006; zhou et al., 2010; zhou et al., 2011). beside these, capparis leaves and shoots have also been reported as a good source of kaempferol and quercetin. rutin, kaempferol, 3-orutinoside, and isorhamnetin 3-o-rutinoside have also been reported as major flavonoids in c. spinosa (siracusa et al., 2011). hydroxycinnamic acids such as dicaffeoylquinic acid, 5-caffeoylquinic acid, 3-caffeoylquinic acid, 3-p-coumaroylquinic acid, and feruloylquinic acid were also detected first time in c. decidua. these were found in all parts of c. decidua in appreciable amount with maximum levels recorded in roots of this species followed by fruits and stem bark. roots represented 77.00 and 74.72% of total hydrocinnamic acids in dry and rainy months, respectively. among all hydroxycinnamic acids, feruloylquinic acid ranked highest followed by 3-caffeoylqui nic acid > 3-p-coumaroylquinic acid > dicaffeoylquinic acid > 5 caffeoylquinic acid. the comparative profile of flavonoids and hydroxycinnamic acids of c. spinosa and c. decidua is first time investigated and reported in the present investigation. it can be noted that c. decidua is an important source of studied phenolics as it possesses 59.2 and 50.7% more flavonoids and hydroxycinnamic acids than c. spinosa on average, respectively. both species exhibited higher contents of phenolics in rainy season than dry ones. the reasons for higher concentration of phenolic acids might be rainfall as researchers reported a positive correlation of rainfall with phenolics and flavonoid contents in seve ral studies (sarrazin et al., 2015; sousa et al., 2010). yang et al., (2006) studied 120 tropical and sub-tropical edible plants for their antioxidant potential and reported a higher antioxidant activity of these plants in hot wet season. higher antioxidant activity is an indicator for the presence of higher phenolic acids (siddhuraju and becker, 2003). the varying concentration of bioactive compounds such as rutin has been reported to be linked with varying daylight in different capparis parts. for example, maximum rutin contents in flowers and leaf samples of c. spinosa were detected at morning and night times while floral buds of c. spinosa gave maximum rutin in the morning. stem and fruits of this species provided maximum rutin contents in morning at 11:00 and afternoon at 16:00 h (behnaz et al., 2013). in the present work, variations in the phenolics profile can be mainly linked to varying climatic factors such as temperature, humidity and rainfall fluctuation in both seasons. conclusion in the present comprehensive study, we for the first time reported that how different parts of c. spinosa and c. decidua exhibit varying le vels of polyphenolics in dry and rainy seasons. a notable variation in the profile of phenolics was recorded among selected species parts as function of both harvesting seasons. different parts of both species exhibited impressive range of phenolics but the roots possessed maxi mum amount of these compounds which were mostly derivatives of quercetin such as kaempferol, apigenin, caffeoylquinic acid and feruloylquinic acid. these findings support the nutra-pharmaceutical potential of c. spinosa and c. decidua due to offering a wide array of polyphenols. moreover, from the findings of this study, it may be concluded that the selected species exhibited more polyphenols in 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fruit aqueous extract and the isolation of main phytochemicals. j. agric. food chem. 58, 12717-12721. doi: 10.1021/jf1034114 zhou, h.f., xie, c., jian, r., kang, j., li, y., zhuang, c.-l., yang, f., zhang, l.-l., lai, l., wu, t., 2011: biflavonoids from caper (capparis spinosa l.) fruits and their effects in inhibiting nf-kappa b activation. j. agric. food chem. 59, 3060-3065. doi: 10.1021/jf105017j addresses of the authors: tehseen gull1, 2, bushra sultana2, farooq anwar3*, wasif nouman4*, eduardo rosa5, raúl domínguez-perles5, 6 1 department of chemistry, university of central punjab, lahore, pakistan 2 department of chemistry, university of agriculture faisalabad, pakistan 3 department of chemistry, university of sargodha, sargodha, pakistan 4 department of forestry & range management, bahauddin zakariya university, multan, pakistan 5 centre for the research and technology for agro-environment and biological sciences, university of trás-os-montes e alto douro (citab/ utad), 5001-801, vila real, portugal 6 centro de edafología y biología aplicada del segura, national council for scientific research (cebas-csic) univeristy campus of espinardo, edif. 25. 30100 espinardo, spain * corresponding author’s e-mail: wnouman@gmail.com; fqanwar@yahoo.com © the author(s) 2019. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). mailto:wnouman@gmail.com mailto:fqanwar@yahoo.com journal of applied botany and food quality 89, 264 269 (2016), doi:10.5073/jabfq.2016.089.034 1department of chemistry of natural compounds, faculty of food and biochemical technology, university of chemistry and technology prague, prague, czech republic 2 forensic laboratory of biologically active substances, university of chemistry and technology prague, prague, czech republic 3 department of quality of agricultural products, faculty of agrobiology, food and natural resources, czech university of life sciences prague, prague, czech republic 4department of soil science and soil protection, faculty of agrobiology, food and natural resources, czech university of life sciences prague, prague, czech republic 5department of food analysis and nutrition, faculty of food and biochemical technology, university of chemistry and technology prague, prague, czech republic 6school of agriculture and food technology, faculty of business and economy, the university of the south pacific, independent state of samoa 7department of nutritive substances, food research institute prague, prague, czech republic 8 department of crop sciences and agroforestry, faculty of tropical agrisciences, czech university of life sciences prague, prague, czech republic fatty acids, minerals, phenolics and vitamins in the seeds of inocarpus fagifer, a pacific island underutilized legume lukas huml1, petra miksatkova1,2, pavel novy3, ondrej drabek4, monika sabolova5, mohammed umar6, vaclav tejnecky4, barbora pohorela5, lenka kourimska3, eva maskova7, anabella tulin6, oldrich lapcik1, ladislav kokoska8,* (received august 16, 2016) * corresponding author summary recently, pacific nations have faced to alarming increase in pre valence of noncommunicable diseases connected with consumption of non-traditional processed food. it is believed that re-introduction of native diet may mitigate these negative trends. one of the traditional staple food of pacific region are seeds of underutilized leguminous tree inocarpus fagifer. nevertheless, information on their chemical composition and nutritional properties are missing. therefore we decided to analyze this crop for the presence of fatty acids, minerals, phenolics and vitamins. performed analyses revealed a slightly predominating portion of unsaturated (e.g. 18:2 n-6; 18:1 n-9 and 18:3α n-3) over saturated (e.g. c18 and c16) fatty acids. consi dering minerals, the substantial concentrations of copper, magnesium, manganese, potassium and zinc (19.32; 1823.21; 8.44; 23308.41 and 77.99 mg kg-1 of dry matter respectively) were recorded. ferulic and coumaric acids were the most abundant phenolics (3.23 and 1.48 mg kg-1 of dry matter respectively), whereas flavonoids, isoflavonoids and coumestrol were also present. regarding vitamins, niacin and riboflavin were found in respective concentrations 131.80 and 4.47 mg kg-1 of dry matter. our findings suggest i. fagifer seeds as a prospective food source of several health-beneficial constituents which might contribute to the well-being of pacific islanders. introduction globalization of processed, carbohydrate rich diets with lack of micronutrients leads to a higher vulnerability to overweight and non-communicable diseases (ncds) not only in the developed world but also in lowand middleincome countries where almost three quarters of the world‘s ncd deaths currently occur. despite the remote location, pacific islanders have also been facing severe changes in dietary habits, whereas the current prevalence of raised blood glucose and obesity is dramatically increasing in certain re gions. for example in samoa, the levels of both above mentioned factors are more than two-fold higher than the global average (curtis, 2014; who, 2014; who, 2002; win tin et al., 2015). since many underutilized crops are being recognized for their dietetic value due to the presence of specific health-beneficial constituents, their re-introduction to the diet has a potential to reverse the negative impact of recently occurring trends in the nutrition and health of the local populations (ebert, 2014; gibson et al., 2015; thies, 2000). nevertheless, before any recommendations can be made on consumption of novel plant foods, it is necessary to assess their dietetic and toxicological properties by detailed analysis (nasi et al., 2009). edible seeds of leguminous plants are considered as a valuable source of nutrition and functional compounds. besides the basic nutritional proteins and oligosaccharides they also provide other essential factors such as unsaturated omega-3 and omega-6 fatty acids, minerals and vitamins, which play an irreplaceable role in the human metabolism and health (combs, 2001; schieber et al., 2001; yildiz, 2010). characteristic secondary metabolites of legumes are isoflavonoids (veitch, 2013), which exert various biological effects. some of them possess an ability to bind to the estrogen receptor (andres et al., 2015), while it might lead to e.g. relief of menopausal symptoms, inhibition of intestinal glucose-uptake (vedavanam et al., 1999) or prevention of steroid hormone-dependent cancers (perabo et al., 2008). other biologically active phenolic derivatives found in leguminous plants are flavonoids and phenolic acids (dewick, 2009). their consumption is connected with a lower risk of oxidative-stress related diseases due to their antioxidant potential (gordon, 1996). inocarpus fagifer (parkinson) fosberg, known as tahitian or polynesian chestnut, is a medium-sized tree belonging to the leguminosae family (lewis et al., 2005) and naturalized in the lowland tropical forests of melanesia, micronesia and polynesia. its indehiscent pod contains a kidney-shaped seed with an edible kernel which when prepared raw or cooked is considered to be among the most important cash crops and culinary nut species of the pacific (lim, 2012), however, as a food, it is practically unknown in the global context. in samoa, this multipurpose tree called “ifi” has played an important role in the livelihood of local inhabitants and even nowadays its seed is considered a staple food. it has been also consumed in other pacific regions, where it is traditionally prepared in many different ways, including grilling, roasting, baking, boiling or mashed in pudding. moreover, significance of this tree also relies pioneering study on nutritional components of inocarpus fagifer 265 on the medicinal use of various tree parts and its timber is suitable for light constructions, tool handles, furniture and canoes manufacturing (pauku, 2006; whistler, 2002). for above mentioned reasons, i. fagifer was designated as one of the priority crops for the pacific region (apaari, 2011). despite the well recorded traditional food use of i. fagifer in many pacific island nations, the plant has received little attention from research and extension services and the data on chemical composition and nutritional properties of this species are totally missing. therefore we decided to determine the presence of certain basic nutritional components of its seeds collected in samoa. materials and methods plant material the fruits of inocarpus fagifer were collected from a wild population of trees (13°30.743´ s, 172°47.828´ w) growing in the coastal area near to the village of falealupo (vaisigano district) located at savai’i island of the independent state of samoa in august 2014. during randomized collection, 30 fruits were gathered from three different trees (ten fruits from each tree). the plant was authenticated by prof. ladislav kokoska and voucher specimen (no. 03015kbfr) is deposited in the herbarium of the department of botany and plant physiology of the faculty of agrobiology, food and natural resources of the czech university of life sciences prague (czech republic). sample preparation and dry matter determination a fleshy mesocarp and testa, which are inedible for humans, were removed and hulled seeds were lyophilized in the free-zone 1 freeze dry system (labconco, kansas city, mo, usa). immediately after lyophilization the seeds were pulverized using an ika a11 analy tical mill (ika werke gmbh & co. kg, staufen, germany). homogenized material was stored under -20 °c until analysis. the residual moisture of lyophilized samples was determined gravimetrically at 130 °c for 60 min by the scaltec smo 01 analyzer (scaltec instruments, gottingen, germany) according to the official methods of analysis of aoac international (2012). chemicals and reagents bacto niacin assay medium was purchased from becton, dickson and company (franklin lakes, nj, usa), coumestrol, daidzein, daidzin, 7,4-dimethoxyisoflavone, formononetin, genistein, genistin, glycitein, glycitin, kaempferol, liquiritigenin, luteolin, naringenin, naringenin-7-o-glucoside, prunetin, rutin, sissotrin from indofine (hillsborough, nj, usa), chloroform, nitric acid, sodium carbonate, sodium hydroxide, sodium sulfate from lach-ner (nerato vice, czech republic), ethanol, folin-ciocalteu reagent, formic acid, gallic acid, methanol from merck (darmstadt, germany), acetic acid, dichlormethan, hydrogen peroxide, n-hexane, sodium acetate, sulfuric acid, potassium permanganate from penta (prague, czech republic), tectoridin from phytolab (vestenbergsgreut, germany), apigenin-7-o-glucoside, boron trifluoride-methanol solution, bromothymol blue indicator, caffeic acid, p-coumaric acid, (-)-epicatechin, ferulic acid, gallic acid, isoquercitrin, nicotinic acid, salicylic acid, sinapic acid and takadiastase (ec 3.2.1.1) from aspergillus oryzae from sigma-aldrich (saint luis, mo, usa), standard 37 component fame mixture was obtained from supelco (bellefonte, pa, usa). minerals analysis determination of minerals was carried out after acid digestion of samples performed according to the method previously described by vanek et al. (2010). initially, a 0.5 g of sample was kept overnight at laboratory temperature in a closed, but not sealed, teflon vessel (savillex, eden prairie, mn, usa) with 10 ml of 65 % nitric acid. then the teflon vessel was sealed and the mixture was heated at 120 °c on a hot plate for 2 hours. the digested solution was then quantitatively transferred to the 50 ml volumetric flask and filled up to the mark with deionized water. the solution was filtered through 0.45 μm nylon disk filters (cronus, gloucester, united kingdom) prior to analysis. an inductively coupled plasma optical emission spectrometer duo icap 7000 (thermo scientific, waltham, ma, usa) operating at 61.15 kw with respective nebulizer and auxiliary gas flow rates of 0.5 and 1 l/min was used for contents determination of selected elements, which were monitored at the following spectral lines (wavelength in nm): boron (b, 208.959), calcium (ca, 317.933), copper (cu, 224.700), iron (fe, 238.204), magnesium (mg, 279.079), manganese (mn, 257.610), phosphorus (p, 177.495), potassium (k, 766.490), sodium (na, 589.592), sulfur (s, 180.731), and zinc (zn, 202.548). quality of digestion and analysis were controlled using blanks and the standard reference materials (nist srm 1575a pine needles and ncs dc 73351 tea). fatty acids analysis the oil was obtained from the sample (about 3 g) by multiple extraction with 180 ml n-hexane:dichlormethan 80:20 v/v in a soxhlet apparatus for 6 h. the fat was recovered under vacuum and dried over anhydrous sodium sulfate and transferred to a capped bottle and stored at 4 °c until used for analysis. derivatization of fatty acids was based on the base-catalyzed reaction using sodium hydroxide-methanol as a reagent with boron trifluoride as a catalyst. fatty acid methyl esters (fames) were then extracted to hexane. fames were analyzed by gas-liquid chromatography using a sp-2560 fused silica capillary column (100 m × 0.25 mm i.d., 0.2 μm film thickness) from supelco (bellefonte, pa, usa) in agilent 6890 gas chromatograph (agilent, palo alto, ca, usa) equipped with a flame ionization detector. the oven temperature was 175 °c for 30 min, then it was increased by 1 °c min-1 to 210 °c and this temperature was maintained for 140 min. detector and injection port temperatures were 220 °c and the nitrogen carrier gas flow was 1 ml min-1. for the identification of fames, standard fame mixtures were analyzed. to confirm the identi fication of some fames, gc/ms analysis was carried out in the gc/msd system agilent 5975 (agilent, palo alto, ca) with the same column and temperature conditions as above, except for the helium flow, which was 0.6 ml/min and the detector temperature was 250 °c. gc-fid chromatogram of fatty acids composition in i. fagifer seed extract is available in supplementary information (fig. s1). uhplc-ms/ms analysis of phenolic compounds prior to analysis, the plant material (2 g) was extracted in a soxhlet like fex ika 50 extractor (ika werke gmbh & co. kg, staufen, germany) in 70 % ethanol in 1/20 (w/v) proportion during three 7-min cycles at 130 °c followed by cooling to 50 °c. the extracts were evaporated on a rotary evaporator, dissolved in 5 ml of 40 % methanol and filtered through a teflon (ptfe) syringe filter (0.45 μm) (labicom, olomouc, czech republic). standard stock solutions of coumestrol, daidzein, daidzin, 7,4-dimethoxyisoflavone, formononetin, genistein, genistin, glycitein, glycitin, kaempferol, liquiritigenin, luteolin, naringenin, naringenin-7-o-glucoside, prunetin, rutin, sissotrin, tectoridin, apigenin-7-o-glucoside, caffeic acid, p-coumaric acid, (-)-epicatechin, ferulic acid, gallic acid, isoquercitrin, salicylic acid and sinapic acid were prepared in methanol at a concentration of 1 mg ml-1 and were subsequently diluted in 40 % (v/v) methanol-water solution at concentrations ranging from 0.1 to 1000 ng ml-1. 266 l. huml, p. miksatkova, p. novy, o. drabek, m. sabolova, m. umar, v. tejnecky, b. pohorela, l. kourimska, e. maskova, a. tulin, o. lapcik, l. kokoska uhplc-ms/ms analysis of polyphenolic compounds was carried out using an agilent 1290 infinity instrument (agilent, santa clara, ca, usa) equipped with a binary pump (g4220b), an autosampler (g4226a), an autosampler thermostat (g1330b) and a column compartment thermostat (g1316c), coupled to an agilent triple quadrupole mass spectrometer (6460a) with a jet stream esi ion source. a kinetex pfp column (2.6 μm, 100a, 150 × 3.00 mm) from phenomenex (torrance, ca, usa) was used for the chromatographic separation of the extracts. column temperature was set at 35 °c and the injection volume was 3 μl. 10 mm formic acid (eluent a) and methanol (eluent b) were used as mobile phases for a gradient elution. the linear gradient solvent system started in 40 % of eluent b and increased to 100 % of eluent b in 10 min. this was maintained for 4 min and in 1 min the conditions returned to the initial ratio, which was maintained for a further 4 min. the flow rate was set at 0.3 ml min-1. the ms/ms apparatus was operating in positive and negative mode in the same analysis. the applied conditions of jet stream ion source were: drying gas temperature 290 °c; drying gas flow 4 l min-1; sheath gas temperature 380 °c; sheath gas flow 10 l min-1; nebulizer pressure 35 psi; capillary voltage was set at 3500 and 5000 v in positive and negative acquisitions, respectively. multiple reaction monitoring (mrm) mode was used for the detection. except for p-coumaric acid, the two most abundant transitions per analyte were used. the transitions and specific ms/ms parameters used for each compound are available in supplementary information (tab. s1). an agilent mass hunter (agilent, santa clara, ca, usa) was used for data acquisition and quantification of samples. extracted ion chromatograms of phenolic compounds detected in i. fagifer seed extract in mrm mode can be found also in supplementary information (fig. s2). total phenolic content total phenolic content was measured using slightly modified method previously developed by singleton et al. (1998). firstly, each sample in volume of 100 μl was added to a 96-well microtiter plate. thereafter, 25 μl of pure folin-ciocalteu reagent was added. the plate was inserted in an orbital shaker at 400 rpm for 5 min. reaction was started by adding 75 μl of 12 % sodium carbonate. the mixture was kept in dark at 37 °c for 1 h. absorbance was measured at 760 nm by infinite m200 pro (tecan, grödig, austria). results were expressed as gallic acid equivalents. vitamin b2 and b3 content the content of vitamin b2 (riboflavin) was evaluated by fluorimetric method after its conversion to lumiflavin according to the czech technical standard method csn 560054 (1972). the riboflavin was extracted after hydrolysis with 0.05m sulfuric acid at 100 °c for 30 min followed by dephosphorylation using an enzymatic treatment (takadiastase from aspergillus oryzae) at 37 °c for 16 h after ph adjustment to 4.5 by 2.5m sodium acetate solution. the sample was filtrated and the aliquot of the filtrate was shaken with chloroform. concentrated sodium hydroxide solution (7m) was added to the clear aqueous layer; the solution was then illuminated with a 40 w fluorescent tube and riboflavin in the sample converted to lumiflavin. the solution was acidified with a few drops of acetic acid and interfering substances of reaction were inactivated by oxidation with 4 % potassium permanganate solution. after 1 minute of exposure the decolorization of the solution by 3 % of hydrogen peroxide was performed. lumiflavin was then extracted by chloroform and measured fluorimetrically (excitation 435 nm, emission 525 nm) at spekol 11 (carl zeiss, oberkochen, germany). the microbiological determinations of vitamins b3 (niacin) was performed according to the czech technical standards csn 56 0051 (1987). the sample was hydrolyzed with 0.5m sulfuric acid at 121 °c for 60 min and then cooled to room temperature. the ph of the sample was adjusted to 4.5, filtered and diluted to a concentration suitable for the assay calibration range. for determination of vitamin b3, ph was adjusted by 1m sodium hydroxide solution to 6.8. the diluted filtrate, appropriate medium (bacto niacin assay medium), standard solution of nicotinic acid and deionized water were pipetted into assay tubes. tubes were sterilized by autoclaving (116 °c, 15 min) and after cooling to room temperature inoculated with lactobacillus plantarum atcc 8014 (the czech national collection of type cultures, the national institute of public health, prague, czech republic). after 72 hours of incubation at 30 °c, the content of b3 was determined by titration with 0.1m sodium hydroxide and bromothymol blue indicator. the growth response of the tested microorganism was compared quantitatively to that of known standard solutions. statistical analysis and calculations all analytical experiments were performed in triplicate and results are expressed as mean and standard deviation. for comparison of our results with contents of minerals and vitamins reported by the usda (2014), the conversion of nutrient values (nv) from fresh weight (fw) to dry matter (dm) basis was performed according to the formula nv in g kg-1 of dm = (100 × nv in g kg-1 of fw)/(100 – water content in g kg-1 of fw) as recommended in fao/infoods guidelines (2012). results and discussion a complex set of analysis has revealed substantial contents of several minerals together with the presence of various fatty acids, phenolic compounds and b vitamins. the complete results are shown in the tab. 1-3. the determined oil content of i. fagifer seed (2.2 ± 0.2 % w/w) correlates with oil content of other legumes such as peas, lentils and beans (usda, 2015). within the oil fraction, overall 14 unsaturated fatty acids (ufas) and 21 saturated fatty acids (sfas) were detected while the quantity of each compound was expressed as a relative percentage (tab. 1). the most abundant ufas determined in our study were linoleic (18:2 n-6; represented by 26.72 %), followed by oleic (18:1 n-9; 18.49 %); α-linolenic (18:3α n-3; 4.42 %) and vaccenic acid (18:1 n-7; 1.27 %). the rest of ufas were represented by less than 1 %. among sfas, palmitic (c16; 22.73 %), stearic (c18; 7.35 %), myristic (c14; 4.41 %) and lignoceric (c24; 4.20 %) acids were the most predominant. behenic (c22), arachidonic (c20) and cerotic acid (c26) were detected in respective amounts of 1.70, 1.33 and 1.17 %. remaining sfas were found in lower concentration than 1 %. the complete results of fatty acids (fas) analysis showed that ufas were slightly predominating (53.57 %) over sfas (46.22 %). the overall profile also demonstrated a greater portion of polyunsaturated fas (31.14 %) over monounsaturated fas (22.06 %). recent nutritional studies are focusing on the omega-6/omega-3 polyunsaturated fas proportion as one of the keystones to prevent health complications caused by an unhealthy diet. it is proposed that their high ratio (1:15-25) occurring in the western diet is connected with the development of ncds (simopoulos, 2012). in the case of i. fagifer seed, the results showed a relatively desirable proportion of omega-6/omega-3 fas (1:6.05) whereas this ratio is comparable to those of other legumes such as soybean (rusnikova et al., 2013). these findings may be interpreted as indicating the potential of this traditional crop to be an alternative to current diets with an undesirable ufas ratio. the results of mineral elements analysis (tab. 2) showed that calcium (ca), potassium (k), magnesium (mg), phosphorus (p) and pioneering study on nutritional components of inocarpus fagifer 267 tab. 2: minerals and vitamins content of inocarpus fagifer seed minerals content (mg kg-1 dry matter) sd a boron 37.77 19.45 calcium 1536.39 151.27 copper 19.32 10.11 iron 44.84 42.30 magnesium 1823.21 86.32 manganese 8.44 0.69 phosphorus 3904.02 34.48 potassium 23308.41 570.80 sodium 348.35 98.01 sulfur 2179.05 41.31 zinc 77.99 7.01 vitamins niacin 131.80 2.67 riboflavin 4.47 1.62 a standard deviation (n=3) sulfur (s) were the most abundant entities detected in the i. fagifer seeds; while boron (b), copper (cu), iron (fe), manganese (mn), sodium (na) and zinc (zn) were found in lower concentrations. k was present in the highest amount of 23.31 g kg-1, exceeding those of soybean and peanut (19.65 and 7.54 g kg-1 respectively) (usda, 2015). according to the minimum recommended dietary allowances (rda; 1.6-2 g day-1 per healthy adult) this study indicates i. fagifer as a valuable dietary source of this element (nrc, 1989a). considering the importance of k in the human diet, this finding could boost its availability and intake for people from pacific island nations. relatively high na/k ratio (1:67) with low na concentration (348.35 mg kg-1) also suggests a possible beneficial effect of seed consumption in persons suffering from hypertension (nrc, 1989b; perez and chang, 2014). taking into the account the rda of other micronutrients, the notable concentrations of cu (19.32 mg kg-1), mg (1823.21 mg kg-1), mn (8.44 mg kg-1) and zn (77.99 mg kg-1) were detected. these results indicate that 100 g of the dry matter edible portion of i. fagifer seed contains approximately half of the rda of mentioned micro-elements (cu = 1.5-3 mg day-1; mg = 200 350 mg day-1; mn = 2-5 mg day-1; zn = 11-15 mg day-1 per healthy adult) (nrc, 1989a) whereas analyzed material has higher content of zn than soybean (53.47 mg kg-1) (usda, 2015). in order to evaluate the presence of phenolic compounds in i. fagifer seed, the total phenolic content (tpc) was determined (63.75 ± 4.62 mg of gallic acid equivalents kg-1 of dry matter). furthermore, 27 phenolic entities with a total sum 6.81 mg kg-1 were detected by detailed analysis (tab. 3). the most abundant phenolic compounds found in the seed of i. fagifer were hydroxycinnamic acids, namely ferulic and coumaric, in respective amounts of 3.23 and 1.48 mg kg-1 while other phenolic acids were determined in much lower concentrations. the analysis also revealed the presence of isoflavonoids. from this class of compounds, the most abundant were glycitein, its glycoside glycitin and formononetin (568.64; 71.28 and 179.73 μg kg-1 respectively). daidzein and genistein were identified along with their glycosides daidzin and genistin in concentrations ranging from 2.46 to 14.48 μg kg-1. it is known that quantities of isoflavonoids may vary in underutilized legumes significantly (leuner et al., 2013). although the amounts of isoflavonoids detected in i. fagifer seeds are rather low, the presence of this metabolic pathway within this species is in agreement with authors studying other members of the dalbergieae tribe (hegnauer and hegnauer, 2001). coumestrol was the only coumestan found (530.06 μg kg-1). among flavonoids, the highest concentration was recorded in the case of liquiritigenin and rutin (152.92 and 123.68 μg kg-1respectively). the determined tpc of i. fagifer seed was by several orders of magnitude lower in the comparison with other pulses e.g. soybean and peanut (djordjevic et al., 2011). although it is obvious that tab. 1: fatty acids composition of inocarpus fagifer seed oil fatty acid shorthand designation content (%) sda saturated fatty acids (sfa) c 6:0 0.15 0.02 c 8:0 0.15 0.02 c 10:0 0.10 0.02 c 12:0 0.91 0.07 c 14:0 4.41 0.27 c 15:0 0.22 0.02 c 16:0 22.73 0.57 c 17:0 0.23 0.02 c 18:0 7.35 0.26 c 19:0 0.16 0.02 c 20:0 1.33 0.11 c 21:0 0.07 0.02 c 22:0 1.70 0.14 c 23:0 0.49 0.06 c 24:0 4.20 0.38 c 25:0 0.43 0.06 c 26:0 1.17 0.12 c 27:0 0.09 0.02 c 28:0 0.20 0.04 c 29:0 0.05 0.02 c 30:0 0.08 0.02 total sfa 43.22 unsaturated acid (ufa) c14:1 (cis 9) tr.b c16:1 (cis 7) 0.29 0.03 c16:1 (cis 9) 0.88 0.07 c16:1 (cis 11) 0.60 0.05 c17:1 (cis 9) 0.03 n.a.c c18:1 (trans isomers) tr. c18:1 (cis 9) 18.49 0.46 c18:1 (cis 11) 1.27 0.10 c18:1 (cis isomers) tr. c18:2 (cis trans isomers) 0.19 n.a. c18:2 (cis 9, cis 12, n-6) 26.72 0.67 c18:3 (cis trans isomers) 0.18 n.a. c18:3 (cis 9, cis12, cis15, n-3) 4.42 0.27 c20:1 (cis 11) 0.50 0.05 total ufa 53.57 astandard deviation (n=3), btraces (below 0.01 %), cnot applicable 268 l. huml, p. miksatkova, p. novy, o. drabek, m. sabolova, m. umar, v. tejnecky, b. pohorela, l. kourimska, e. maskova, a. tulin, o. lapcik, l. kokoska this crop represents a rather weak source of phenolics, as long as it serves as a staple food in certain regions, it would appear that the cumulative amount consumed on a daily bases might contribute to the prevention of oxidative stress-related diseases. because the determined value of tpc and the sum of quantified phenolics do not match, it seems that a considerable amount of other phenolic entities might be present within the examined material. possible candidates include condensed tannins which are found in numerous legumes (coda et al., 2015; curiel et al., 2015). considering the vitamins content (tab. 2), representatives of group b, niacin and riboflavin were found in respective concentrations of 131.80 and 4.47 mg kg-1. niacin was present in the seed of i. fagifer in a higher concentration than in soybean or peanut (usda, 2015), while a 100 g dry matter edible portion along with sufficient intake of tryptophan (200 mg) should cover daily recommended minimum consumption (nrc, 1989a). administration of niacin increases adiponectin secretion and may therefore contribute to the reduction of obesity via lipid-lowering effect (westphal et al., 2010). in the case of riboflavin, a 100 g dry matter edible portion represents ca. 1/3 of re-commended minimum intake (1.2 mg day-1 per healthy adult) (nrc, 1989a). conclusion this study, to our best knowledge, is a pioneering investigation on the chemical composition of i. fagifer seed highlighting the presence of minerals, fatty acids, phenolics and b vitamins. of great significance is the presence of micronutrients such as cu, mg, mn and zn in substantial amounts. these micronutrients together with k are essential to the human metabolism. appropriate to the human diet seem to be also concentrations of niacin and riboflavin together with determined ratios of sodium/potassium and omega-6/omega-3 ufas, indicating its potential as a valuable dietary source. considering the obtained results, it seems that this crop possesses several health-beneficial attributes and therefore might potentially play a positive role in the lowering of prevalence of obesity and ncds mainly in pacific island nations where it is traditionally consumed as a staple food. however, further studies on chemical composition, biological activities and toxicology of this underutilized crop which has received little attention from researches in the past, are necessary to verify its potential as a healthy food. acknowledgements this research was supported by the internal grant agency of the czech university of life sciences prague (project no. iga 20155012) and by specific university research financial support (msmt no 20/2015). references 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food biochemistry, 250-267. boca raton, usa: crc press. addresses of the authors: 1department of chemistry of natural compounds, faculty of food and biochemical technology, university of chemistry and technology prague, technicka 5, 166 28, praha 6 dejvice, czech republic 2forensic laboratory of biologically active substances, university of chemistry and technology prague, technicka 5, 166 28, praha 6 dejvice, czech republic 3 department of quality of agricultural products, faculty of agrobiology, food and natural resources, czech university of life sciences prague, kamycka 129, 165 21, prague 6 suchdol, czech republic 4department of soil science and soil protection, faculty of agrobiology, food and natural resources, czech university of life sciences prague, kamycka 129, 165 21, prague 6 suchdol, czech republic 5department of food analysis and nutrition, faculty of food and biochemical technology, university of chemistry and technology prague, technicka 5, 166 28, praha 6 dejvice, czech republic 6school of agriculture and food technology, faculty of business and economy, the university of the south pacific, independent state of samoa 7department of nutritive substances, food research institute prague, radiova 7, 102 31 prague 10 hostivar, czech republic 8department of crop sciences and agroforestry, faculty of tropical agrisciences, czech university of life sciences prague, kamycka 129, 165 21, prague 6 suchdol, czech republic e-mail: kokoska@ftz.czu.cz © the author(s) 2016. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). i supplementary material fig. s1: gc-fid chromatogram of inocarpus fagifer seed extract. peak identification: 1 = caprylic acid, 2 = capric acid, 3 = lauric acid, 4 = myristic, 5 = pentadecylic acid, 6 = palmitic acid, 7 = hexadecenoic acid (cis 7), 8 = hexadecenoic acid (cis 9), 9 = hexadecenoic acid (cis 11), 10 = heptadecanoic acid, 11 = heptadecenoic acid (cis 9), 12 = stearic acid, 13 = oleic acid (cis 9), 14 = octadecenoic acid (cis 11), 15 = nonadecanoic acid, 16 + 17 = octadecadienoic acid (cis trans isomers), 18 = linoleic acid, 19 = arachidic acid, 20 = eicosenoic acid (cis 11), 21 = α-linolenic, 22 = heneicosanoic acid, 23 = unidentified fatty acid, 24 = behenic acid, 25 = tricosanoic acid, 26 = lignoceric acid, 27 = pentacosanoic acid, 28 = cerotic acid, 29 = heptacosanoic acid, 30 = octacosanoic acid, 31 = nonacosanoic acid, 32 = triacontanoic acid. fig. s2: extracted ion chromatograms of phenolic compounds detected in inocarpus fagifer seed extract. supplementary material ii2    t ab le s 1. t h e tr an si ti o n s an d s p ec if ic m s /m s p ar am et er s u se d f o r an al y si s o f p h en o li c co m p o u n d s 13   io n is at io n m o d e re te n ti o n ti m e (m in )a fr ag m en to r (v ) p re cu rs o r io n ( m /z ) p ro d u ct i o n (m /z ) l o d (n g /m l )c l o q (n g /m l )d q u an ti fi ca ti o n tr an si ti o n c e (e v )b co n fi rm at io n tr an si ti o n c e (e v )b 7 .4 ´d im et h o x y is o fl av o n e e s i + 9 .0 5 ( 0 .5 ) 1 1 7 2 8 3 .1 0 1 7 1 .3 4 2 1 7 6 .0 1 8 0 .4 1 .4 ap ig en in -7 -o -g lu co si d e e s i + 5 .5 4 ( 0 .5 ) 1 0 9 4 3 3 .1 2 2 7 1 .0 1 3 1 5 3 .0 6 0 0 .2 0 .8 ca ff ei c ac id e s i 3 .6 0 ( 0 .4 ) 8 1 1 7 9 .0 0 8 9 .0 3 0 1 3 5 .1 1 3 2 .3 7 .7 ch lo ro g en ic a ci d e s i 3 .0 1 ( 1 .0 ) 8 1 3 5 3 .0 9 1 9 1 .1 9 0 .6 1 .9 pco u m ar ic a ci d e s i + 4 .3 7 ( 0 .5 ) 6 0 1 6 5 .0 5 1 4 7 .1 9 0 .9 3 .0 co u m es tr o l e s i + 7 .8 8 ( 0 .5 ) 1 2 1 2 6 9 .0 5 1 1 5 .0 5 3 1 5 7 .0 3 3 0 .5 1 .6 d ai d ze in e s i 6 .0 1 ( 0 .5 ) 1 2 6 2 5 3 .0 5 1 3 2 .1 4 2 9 1 .0 3 4 0 .1 0 .3 d ai d zi n e s i + 3 .6 6 ( 0 .5 ) 8 5 4 1 7 .1 2 2 5 5 1 4 1 5 2 .0 7 8 0 .1 0 .2 ep ic at ec h in e s i 3 .1 0 ( 0 .5 ) 1 1 1 2 8 9 .0 7 1 0 9 1 3 2 4 5 .1 5 0 .7 2 .4 fe ru li c ac id e s i + 4 .6 5 ( 0 .5 ) 6 3 1 9 5 .0 7 1 4 5 .0 1 3 1 7 7 .0 5 1 .0 3 .2 g al li c ac id e s i 2 .4 3 ( 0 .6 ) 7 5 1 6 9 .0 1 1 2 4 .9 9 1 6 9 .0 5 0 .7 2 .2 g en is te in e s i 7 .2 1 ( 0 .5 ) 1 2 3 2 6 9 .0 4 1 3 3 .0 3 0 6 3 .1 3 4 0 .1 0 .4 g en is ti n e s i 4 .6 2 ( 0 .5 ) 1 6 2 4 3 1 .1 0 2 6 9 .0 1 0 2 3 9 .1 4 6 0 .1 0 .4 g ly ci te in e s i 6 .3 2 ( 0 .5 ) 9 9 2 8 3 .0 6 2 4 0 .0 1 8 2 6 8 .0 1 0 0 .1 0 .2 g ly ci ti n e s i + 3 .9 6 ( 0 .5 ) 8 2 4 4 7 .1 3 2 8 5 .0 1 4 2 7 0 .0 4 6 0 .1 0 .1 is o q u er ci tr in e s i 4 .8 7 ( 0 .5 ) 1 5 0 4 6 3 .0 9 3 0 0 .3 1 8 2 7 1 .0 4 2 0 .4 1 .2 k ae m p fe ro l e is + 7 .5 5 ( 0 .6 ) 1 6 1 2 8 7 .0 6 1 5 3 .0 4 1 6 9 .1 5 3 1 .1 3 .6 li q u ir it ig en in e s i 6 .0 6 ( 0 .5 ) 9 0 2 5 5 .0 6 1 3 5 .0 9 1 1 9 .1 2 1 0 .1 0 .3 lu te o li n e s i 7 .2 3 ( 0 .8 ) 1 2 8 2 8 5 .0 4 1 3 3 .0 3 3 1 5 1 .0 2 0 0 .4 1 .4 n ar in g en in e s i 7 .0 5 ( 0 .5 ) 9 3 2 7 1 .0 6 1 1 9 .0 2 1 1 5 1 .0 9 0 .1 0 .1 n ar in g en in -7 -o -g lu co si d e e s i 4 .7 6 ( 0 .5 ) 1 1 7 4 3 3 .1 1 2 7 1 .1 1 0 1 1 9 .0 5 0 0 .1 0 .4 p ru n et in e s i 8 .8 2 ( 0 .3 ) 1 1 1 2 8 3 .0 6 2 6 8 .0 1 3 2 3 9 .0 2 5 0 .3 0 .9 ru ti n e s i 4 .6 9 ( 0 .5 ) 1 6 3 6 0 9 .1 4 2 7 1 .0 6 1 3 0 0 .0 6 5 0 .3 0 .9 sa li cy li c ac id e s i 5 .2 2 ( 0 .6 ) 7 2 1 3 7 .0 2 9 3 .1 1 3 6 5 .1 2 9 0 .5 1 .8 si n ap ic a ci d e s i 4 .8 0 ( 0 .4 ) 8 1 2 2 3 .0 6 2 0 8 .1 9 1 4 9 .0 1 7 0 .1 0 .2 si ss o tr in e s i 6 .5 5 ( 0 .5 ) 1 6 2 4 4 5 .1 1 2 8 3 .1 1 0 2 6 8 .0 3 0 0 .1 0 .3 te ct o ri d in e s i 4 .7 4 ( 0 .5 ) 1 3 8 4 6 1 .1 1 2 9 9 .0 1 3 2 8 3 .0 2 9 0 .1 0 .4 ta b. s 1: t he tr an si tio ns a nd s pe ci fic m s/ m s pa ra m et er s us ed fo r a na ly si s of p he no lic c om po un ds a r et en tio n tim e w in do w (m in ) i s gi ve n in b ra ck et s, b c ol lis io n en er gy , c l im its o f d et ec tio n (s ig na lto -n oi se ra tio o f 3 ), d l im its o f q ua nt ifi ca tio n (s ig na lto -n oi se ra tio o f 1 0) journal of applied botany and food quality 91, 194 201 (2018), doi:10.5073/jabfq.2018.091.026 1department of botany, kohat university of science and technology (kust), kohat, pakistan 2national institute for food and agriculture (nifa) peshawar, pakistan 3department of microbiology, abdul wali khan university mardan, pakistan 4department of biotechnology and genetic engineering, kohat university of science and technology (kust), kohat, pakistan 5department of botany, abdul wali khan university mardan, pakistan effects of neem (azadirachta indica) seed and turmeric (curcuma longa) rhizome extracts on aphids control, plant growth and yield in okra uzair muhammad1, tariq nawaz khattak2, hazir rahman3, m.k. daud4, waheed murad5, azizullah azizullah1* (submitted: june 3, 2017; accepted: march 12, 2018) * corresponding author summary the use of synthetic pesticides to control pests and increase crop yield is a common practice, but they cause several environmental and health problems. therefore, there is a need to explore alternative approaches to reduce the sole dependence on synthetic pesticides. the present study was conducted to screen the extracts of neem seed and turmeric rhizome for pesticidal activities against okra pests (aphids: aphis gossypii). experiments were conducted in field with four plots. one plot was kept as a control (unsprayed) and one was sprayed with synthetic pesticides, one with neem seeds extract and one with turmeric rhizome extract. the effect on number of pests, plant growth and yield was observed at regular intervals. a significant reduction in pests was recorded in all treatments as compared to the control. neem seed extract was more effective than turmeric rhizome extract as revealed by 73% decrease in aphids by neem extract in comparison to 54% by turmeric extract after last application. both the extracts were found to be more effective than the synthetic pesticides in controlling okra pests. both the extracts had stimulatory effects on okra growth and yield. for example, the total yield of plots sprayed with neem (53.3 kg plot-1) and turmeric extract (47.7 kg plot-1) was higher than the yield of control plot (33.8 kg plot-1) and plot sprayed with synthetic pesticides (39 kg plot-1). it is concluded that neem and turmeric extracts can be used as alternative of synthetic pesticides for controlling pests’ attacks in okra. key words: neem, turmeric, aphids, okra, pesticidal properties, growth and yield introduction food crops are generally attacked by a number of pests like insects, weeds, and other pathogens which cause substantial losses in agriculture. crop losses due to different factors like diseases, animal pests and weeds range between 20 and 40% of the total yield (savary et al., 2006). according to some estimations, the total loss (without crop protection practices) due to pests varied from about 50% in wheat to more than 80% in cotton (dhaliwal et al., 2002). generally, crops are attacked by more than 10,000 species of insects, 30,000 species of weeds and by a thousand species of nematodes (dhaliwal et al., 2010). in addition, about 100,000 diseases in plants are caused by fungi and other microorganisms (dhaliwal et al., 2010). it has been observed during the last 40 years that despite a continuous increase in pesticide application, pest attacks have not been significantly controlled and crops losses continue (pimentel, 2007). approximately 5.2 billion pounds of total pesticides were used worldwide during 2006 and 2007, with 40% herbicides, 17% insecticides and 10% fungicides (kiely et al., 2004). only in the u.s, approximately 1.1 billion pounds of pesticides were used during 200607, which was 22% of the total pesticides used worldwide (kiely et al., 2004). only to some extent, pesticide use has enabled farmers to minimize crop losses caused by pest attacks (hamill et al., 2004). despite their useful effect of protecting crops, synthetic pesticides cause hazardous impacts on the surrounding environment by affec ting non-target organisms. the use of pesticides kill targeted orga nism but as well as kill some beneficial organisms also (gilden et al., 2010). synthetic pesticides have serious effects on human health and cause various disorders like breast cancer, the suppression of immune system, disorder of nervous system, respiratory disorder and hormonal damages (benbrook, 2004). some chemicals of the commercial pesticides have been investigated for the disruption of male reproductive hormones, low sperm count in men and birth defects in babies (deepa et al., 2011). it has been estimated that about $1.1 billion dollar per year are spent worldwide on public health problems associated with pesticide-related acute poisonings and cancer (benbrook, 2004). in addition to their adverse effects on animals and humans, pesticides also adversely affect non-target plants. they reduce metabolite synthesis and translocation and inhibit the processes like photosynthesis, respiration and sterol syn thesis in non-targeted crops like tomato, maize, pea, grapes, bean and barley (magné et al., 2006). pesticides have some direct harmful effect on non-target plant growth such as shoot yellowing, poor root hair development and reduced plant growth (walley et al., 2006). pesticides also affect size, number and morphology of fruits and seeds which can lead to reduce yield and low quality (magné et al., 2006). in comparison, plants derived pesticides or botanical extracts are non-toxic to the user, easily biodegradable and specific in their action and can serve as models for the development of new synthetic analogues with favorable biological and physicochemical properties (akbar et al., 2010). neem (azadirachta indica) is a large tree with semi-straight to straight trunk and spreading branches. it has been widely regarded as a natural source of pesticides and other agrochemicals. neem was reported to be the most effective among the 2,400 plant species that have shown pesticidal activities (girish and shankara, 2008). the extracts of different parts of neem (leaves, seeds and bark) were found to control a variety of arthropods, nematodes, fungi, viruses, snails and crustacean species (girish and shankara, 2008). the extract of its seeds was effective larvicidal and indicated 70-90% mortality against culex larvae (hashmat et al., 2012). neem extract was recommended as the most promising pesticide for effective control of tomato fruit worm, helicoverpa armigera (shah et al., 2013). turmeric (curcuma longa) is the rhizomatous perennial plant of the family zingiberaceae (ginger family). it possesses a variety of bioactive ingredients that act as repellent and insecticidal agents (damalas, 2011). rhizomes powder of turmeric showed repellency against different insects of stored products like sitophilus granaries, tribolium castaneum, and rhyzopertha dominica (damalas, 2011). effects of neem seed and turmeric rhizome extracts on aphids control in okra 195 its extract was also effective against rice insect sitophilus oryzae (damalas, 2011). turmeric powder with ash showed pesticidal acti vity against different pests of rice (oxya nitidula and cnaphalocrocis medinalis) and eggplant (epilachna vigintioctopunctata) (sankari and narayanasamy, 2007). similarly, its rhizomes powder was shown to be effective against store-grain pests like lesser grain borer (rhyzopertha dominica) in rice (chander et al., 2003). okra (abelmoschus esculentus) is one of the hot summer and annual vegetable grown in plain areas of the world. in pakistan, okra is cultivated on an area of about 2.21 × 105 hectares (kashif et al., 2008). it is attacked by a number of insects which reduces its growth and yield. some of the major destructive insect pests of okra are aphids (e.g. aphis gossypii), whitefly (e.g. bemisia tabaci), jassid (e.g. amrasca devastans), thrip (e.g. thrips tabaci) and spotted bollworm (e.g. helicoverpa armigera) (aziz et al., 2012). among these insects, aphids are the major pests of okra in pakistan (gardiner et al., 2009). aphid infestation causes heavy losses in different crops. for example, aphids attack causes a reduction of up to 4.57% in wheat yield in pakistan (khan et al., 2012). aphids damage maize crops and cause up to 60% loss in yield (hafeez and zia, 2009). aphids also act as a vector and transfer several viruses in potato crops and cause yield losses up to 90% depending on cultivar, infestation and environmental conditions (saljoqi, 2009). according to an estimate, aphis gossypii causes about 20-40% yield losses in cotton crops in pakistan (aslam et al., 2004). keeping in view the economic importance of okra and losses in its yield due to pest attacks, the present study was conducted to evaluate the extract of neem seeds and turmeric rhizome for pesticidal activities against aphids in okra to provide an easy and cost effective way for the control of aphid attack on okra in pakistan. materials and methods experimental field the present experiments were conducted at nuclear institute for food and agriculture (nifa) peshawar, pakistan. okra was grown in plots with a size of 88 × 17 feet. the experimental field was consisted of four main plots: one each for the control (no treatment), synthetic pesticides (cypermethrin and bifenethrin), turmeric rhizome extract and neem seeds extract (fig. 1). each plot was further divided into three equal blocks. each plot was sprayed with the respective solution at specific intervals as shown in tab. 1. normal agronomic practices like soil preparation, irrigation and fertilization were done. fig. 1: images of experimental plots. (a) control plot, (b) pesticides treatment plot (c) turmeric treatment plot, (d) neem treatment plot. tab. 1: dates of extracts application and pests observations. application of extracts / application date pests observation date pesticides 1st 15.05.2014 21.05.2014 2nd 26.05.2014 01.06.2014 3rd 03.06.2014 11.06.2014 4th 16.06.2014 25.06.2014 5th 30.06.2014 06.07.2014 6th 10.07.2014 18.07.2014 preparation of plant extracts and pesticides solution neem seeds and turmeric rhizome were purchased from qissakhwani bazar in peshawar. dried seeds of neem and rhizome of turmeric were crushed with electronic blender separately. the ob196 u. muhammad, t.n. khattak, h. rahman, m.k. daud, w. murad, a. azizullah tained powder was mixed with petroleum ether at the rate of 125 g per liter following the method of soxlet. the mixture was stirred by orbital shaker for five days and then was filtered through fine gauze to remove bigger pieces. the extract was then mixed with 5 liters of water to prepare the final solution for application (aziz et al., 2012). for comparison purpose, two synthetic pesticides, i.e. cypermethrin and bifenethrin, were applied in a mixture form by dissolving 10 ml of each pesticide in 5 liters of water. application/spray of extracts the prepared extracts and synthetic pesticides solutions were applied on their respective plots at different time intervals as shown in tab. 1, while the control plot was left unsprayed. the first spray was applied after the appearance of pests. all solutions were applied on the same day using a knapsack sprayer. the sprayer was rinsed with clean water before using for each solution. a total of six applications were made for each treatment at different times as shown in tab. 1. pest monitoring for the investigation of extracts effect on pests, the number of pests per plant and percentage of infected plants were determined after each application. a total of 20 plants were randomly selected in each block of every plot. to collect aphids, a piece of paper was laid under one side of plant, shaken the plant gently on the paper and collected the aphids. the average number of aphids per plant was calculated and the percentage of infected plants in each block was determined. a total of six observations were made at specific interval during the course of experiment as shown in tab. 1. determination of plant growth and yield parameters determination of shoot length after sixty days of plant growth, three plants were randomly collected from each block of every plot. the shoot length in centimeter (cm) was measured with a measuring tape. the average shoot length was calculated as follow: average shoot length (cm) = sum of length of all plants/ total number of plants determination of leaf area three leaves per plant were collected from randomly selected 20 plants in each block of every plot. the leaf area (cm2) was calculated from the length and width of a leaf. determination of fruits number for the determination of fruit number, three plants were randomly selected in each block of every plot and the number of fruits per plant was calculated as: average number of fruits per plant = sum of total number of fruits / total number of plants determination of fruit length three fruits per plant were collected from 20 plants in each block of every plot and the fruit length was determined in cm using a measuring tape. average fruit length (cm) = sum of total length of fruits / total number of fruits determination of okra yield for measuring the okra yield, the fruits were plucked after every four to five days and the yield was measured by an electronic balance in kilogram (kg). data analysis mean and standard deviation of the three replicates for each parameter were calculated using microsoft excel. one-way analysis of variance (anova) was applied to measure the significance of differences among different treatments and the control. the difference was considered to be significant if p value was smaller than 0.05 (p < 0.05). results effect of neem and turmeric extracts on aphids effect on percentage of infected plants the percentages of infected plants in different plots are shown in tab. 2. all the applied solutions, i.e., the neem and turmeric extracts and the synthetic pesticides significantly protected okra plants from aphid attacks. in the first observation (after first application), there were 53.33%, 81.66% and 70% infected plants in neem extract, turmeric extract and pesticides treated plots, respectively, as compared to 90% in the control. in the second observation (after second application) 50%, 78.33% and 70% plants were attacked by aphids in neem, turmeric and pesticides treated plots, respectively, as compared to 85% in the control. the reduction in aphids attacks by turmeric extract was statistically not significant as compared to the control, but the neem extract caused a significant reduction in aphids attacks as compared to the control and pesticides. after third spray, 45%, 60% and 63.33% plants were infected by aphids in neem, turmeric and pesticides treated plots, respectively, where 80% plants in the control were attacked. in this case both neem and turmeric extracts significantly controlled aphids attacks in comparison to the control, but the difference was statistically significant only in the case of neem extract when compared to the plot sprayed with synthetic pesticides. both the extracts were shown to be more effective with the passage of time as revealed from third spray and observation onward, where both the neem and turmeric extracts caused a more prominent and significant reduction in aphids attacks as compared to the control. although a natural decrease in pest attacks was observed with time as revealed by the number of infected plants in the control plot at different observation, a prominent effect of neem and turmeric extracts can be observed in comparison to the control. the extracts, especially neem extract was found to be more effective even than the synthetic pesticides (tab. 2). tab. 2: percentage of infected okra plants observed at six different intervals. 1st 2nd 3rd 4th 5th 6th control plot 90±00a 85±05a 80±00a 68.33±2.88a 60±00a 60±00a pesticides plot 70±00b 70±00b 63.33±2.88b 63.33±2.88b 36.66±5.77b 36.66±5.77b turmeric plot 81.66±5.77a 78.33±7.63a 60±00b 46.66±5.77b 33.33±5.77b 33.33±5.77b neem plot 53.33±2.88c 50±00c 45±00c 33.33±7.63b 23.33±5.77c 23.33±5.77c values given are mean ± standard deviations of three replicates. different letters on each two values indicate significant difference from each other. effects of neem seed and turmeric rhizome extracts on aphids control in okra 197 effects on number of aphids per plant the effect of neem and turmeric extracts on number of aphids per plant is shown in fig. 2. the obtained results indicate that after first two applications, no treatment caused any significant decrease in aphids density per plant in comparison to the control. however, thereafter both the neem and turmeric extracts were shown to cause a decrease in the number of aphids per plant in comparison to the control as well as pesticides treated plot. for example, after third observation, there were 18.66 and 20.66 (mean values) of aphids per plant in neem and turmeric treated plots, as compared to 25.66 and 27.66 in the control and pesticides treated plot, respectively. the same trend continued and both the extracts were observed to decrease the number of aphids per plant in all the remaining observations. both the extracts were found to show enhanced protection with the passage of time and repeated application. for example, after sixth and last application, there were only 5.66 and 9.66 (mean) aphids per plant in neem and turmeric treated plots in comparison to 17 and 21 aphids in pesticides and control plots, respectively. after last application, neem and turmeric caused a 73 and 54% reduction in the number of aphids per plant, respectively. it was also observed that both the extracts were more effective than the synthetic pesticides as revealed by the obtained results (fig. 2). although after fourth application, the synthetic pesticides significantly controlled aphids in comparison to the control plot but this effect was much smaller than the neem and turmeric extracts. a comparison of both the extracts revealed that neem seeds extract was more effective than the turmeric rhizome extract as can be seen by a 73% decrease in aphids by neem extract in comparison to 54% by turmeric extract after last application. it is noteworthy to mention that like the natural decrease in the per centage of attacked plants, a natural decrease in the number of aphids per plant was observed as is evident from the data of control plot at different intervals (fig. 2). ticides treated and control plots, respectively. both the extracts were shown to cause a slight but statistically significant increase in the leaf area of okra (fig. 4), and the average leaf area in in plants sprayed with neem and turmeric extracts reached 193.26 and 192.66 cm2 in comparison to 187.66 and 183.9 cm2 in pesticides treated and control plots, respectively. effect on yield parameters effect on fruit yield the effect of neem and turmeric extracts on fruit yield in term of weight is shown in fig. 5. all the applied solutions (neem, turmeric extracts and synthetic pesticides) significantly protected the okra plants from aphid attacks and resulted in high yield. in first two plucking periods, there was no increase in fruit yield in neem, turmeric and pesticides treated plots in comparison to the control. however, thereafter both neem and turmeric extracts increased its yield in comparison to the control as well as pesticides treated plots. for example, in third plucking period there were 8.6 kg, 8.5 kg and 8.0 kg fruit yields in neem, turmeric and pesticides treated plots, respectively, as compared to 7.0 kg in the control. the same trend continued and both the extracts were observed to increase fruits yield in all the remaining observations. both the extracts were found to give higher yield with the passage of time and repeated application as compared to the control. for example, in ninth plucking period there fig. 2: number of aphids per okra plant noted at different observations. the chart bars show the mean value of three replicates in each set of experiment while the standard deviation is shown by error bars. different letters on different chart bars indicate significant difference from each other. fig. 3: shoot length of okra in four experimental plots measured after three months of growth. the chart bars show the mean value of three replicates in each set of experiment while the standard deviation is shown by error bars (cont. means control plot). fig. 4: area of okra leaf in the four experimental plots measured after three months of growth. the chart bars show the mean value of three replicates in each set of experiment while the standard deviation is shown by error bars. different letters on different chart bars indicate significant difference from each other (cont. means control plot). effects of plant extracts on growth parameters of okra in order to evaluate whether the application of neem and turmeric extracts for pest control will have any adverse effect on plant growth, the effect of both extracts on two growth parameters (shoot length and leaf area) was investigated (figs. 3 and 4). both the extracts did not cause any adverse effect on shoot length of okra, but rather caused a slight and non-significant increase in shoot length. plots sprayed with neem and turmeric extracts, the average shoot length was 148.83 and 146.10 cm in comparison to143.90 and 142.20 cm in pes198 u. muhammad, t.n. khattak, h. rahman, m.k. daud, w. murad, a. azizullah were 5.0 kg and 4.0 kg fruits yield in neem and turmeric extracts treated plots as compared to 1.2 kg and 1.8 kg in the control and pesticides treated plots, respectively. in tenth plucking period there were 6.0 kg and 4.5 kg fruits yield in neem and turmeric treated plots in comparison to 0.5 kg and 1.2 kg in the control and pesticides treated plots, respectively. it was also shown that both the extracts were more effective than the synthetic pesticides as revealed by the obtained results (fig. 5). although after fourth application, synthetic pesticides increased fruits yield in comparison to the control plot but this effect was much smaller than the neem and turmeric extracts. a comparison of both the extracts revealed that neem seeds extract was more effective than the turmeric rhizome extract as can be seen by 6.0 kg fruits yield by neem extract in comparison to 4.5 kg by turmeric extract after last application. the obtained results (figs. 5 and 6) revealed that a natural decrease in fruits yield was observed with the passage of time as is evident from the data of control plot at different intervals. the effect of neem and turmeric extracts on total fruits yields of okra in term of weight is shown in fig. 6. the mean data indicated that both the neem and turmeric extracts caused an increase in total yield (kg) in comparison to the control and synthetic pesticides trea ted plots. for example, there were total 53.3 kg and 47.7 kg of fruit in neem and turmeric extracts treated plots in comparison to 33.8 kg and 39.0 kg in the control and pesticides treated plots, respectively. effect on fruit length the average fruit length of okra in different plots is shown in fig. 8. there was no prominent effect of any extract on fruit length in okra except a slight but significant increase by the neem extract. fig. 5: yield of okra measured at different intervals. fig. 6: total yield of okra (sum of yield of all observations) in four different plots. effect on fruit number per plant the effect of different treatments on fruit number per okra plant is shown in fig. 7. the obtained results indicate that the extracts of both plants caused a significant increase in fruit number per plant. the average number of fruits on a plant in the control plot was 9.33 which reached 19.66 and 16.66 in plots sprayed with neem and turmeric extracts, respectively. fig. 8: average length of fruit in different plots. the chart bars show the mean value of three replicates in each set of experiment while the standard deviation is shown by error bars. different letters on different bars indicate significant difference from each other (cont. means control plot). fig. 7: number of fruits per okra plant in the four experimental plots. the chart bars show the mean value of three replicates in each set of experiment while the standard deviation is shown by error bars. different letters on different chart bars indicate significant difference from each other (cont. means control plot). discussion crop losses due to pest attacks is one of the major global problems. application of chemical pesticides is the most common and successful methods for pests’ control, but synthetic pesticides often have undesirable side effects on the environment such as damaging of non-targeted organisms and accumulation as residue in the environment and food. therefore, researchers have always been in search of pesticides that are more environmentally friendly and safer from health point of view. this search has led to screening of plants for their pesticidal activities against common pests which has resulted in reporting a number of plant species as promising candidates for use as a source of new plants derived pesticides. the present study was conducted to screen the extracts of neem seeds and turmeric rhizome against aphids in okra, which causes 1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th 11th yield yield yield yield yield yield yield yield yield yield yield effects of neem seed and turmeric rhizome extracts on aphids control in okra 199 huge losses of okra every year. it was observed that the extracts of both plants effectively reduced plant infestation by aphid as revealed by the percentage of infected plants and number of aphids per plant. the present observations are comparable to previous reports that extracts of plants like tobacco and neem effectively reduced the population of bollworm in okra where 11-13% plants were damaged in treated plot as compared to 25-29% in untreated control (shabozoi et al., 2011). similarly, the extracts of neem, turmeric, garlic and henge significantly reduced jassids population in okra (sohail et al., 2015). another study reported that the extract of turmeric caused a noticeable reduction (36%) in the population of insect sitophilus oryzae in rice crop (damalas, 2011). turmeric rhizomes was found to significantly reduce insects of eggplant (epilachna vigintioctopunctata) and pests of okra including amrasca devastans, anomis flava, dysdercus cingulatus, earias vittella, oxycarenus hyalinipennis, tetranychus neocaledonicus, and spodoptera litura (damalas, 2011). furthermore, the extracts of plants in the present study, particularly of neem, were found to be more effective than the synthetic pesticides. this observation is supported by a previous report that botanical pesticides, like neem and tobacco extracts, had similar or even higher inhibitory effect against pests as compared to the conventional pesticides (shabozoi et al., 2011). another study found that neem extract showed the highest activity against the insect pest, red flour beetle (caused 68.69% reduction) followed by sweet flag, harmal and turmeric extracts (iqbal et al., 2010). similarly, hossain et al. (2013) reported that neem extract was more effective than synthetic pesticides like carbamate butocarboxin against pests in tomato, onion and okra. a comparison of the effectiveness of the extracts of the two plants used in this study revealed that neem seeds extract was more effective than the turmeric rhizome. it is supported by a previous study of schmutterer (1990) who found that neem was the most effective among neem, turmeric and garlic extracts when tested against oxycaremus loetus. the pesticidal activities of neem and turmeric have been attributed to the presence of a number of active compounds in them (tab. 3). extracts of these plants might have controlled aphids by acting as repellent, antifeedant and/or as growth and reproductive inhibitors. a previous report suggested that neem can act as repellent and larvicidal as well as affects egg hatching and reduces fertility in insect pest which results in their progeny inhibition. it can also disturb chemoreceptors of insects and impair their feeding (iqbal et al., 2006). turmeric had also been found to possess a variety of bioactive constituents which acts as a repellent and interfere insect behavior and growth (damalas, 2011). according to previous reports extracts of plants like neem, turmeric, harmal and sweet flag showed repellent effect against red flour beetle in wheat (matter et al., 2008). compounds like azadirachtin a and b, salannin and meliantriol present in neem have found to protect from insects due to their repellent effects (james et al., 2014). the passage of time and repeated applications of extracts in the present study were found to enhance the effectiveness of extracts. it is similar to a previous reports where neem extract was found to cause a higher mortality of bollworm in okra as exposure time increased (ahmed, 2000). similarly, adesina and afolabi (2014) observed that plant extracts were less effective after initial applications but killed 89% of flea beetles with the passage of time. this enhancement could be due to richness of pesticidal activities of neem seed and turmeric rhizome extracts. the insects continued to feed on the treated plants for some time which caused increase in its efficiency (schmutterer, 1990). in order to evaluate whether the application of neem and turmeric extracts for pest control would have any adverse effect on okra growth and yield, the effect on plant growth and fruit yield was obtab. 3: some bioactive compounds from neem seeds and turmeric rhizomes. neem compounds biopesticidal activities against aphids references azadirachtin a, b nimbolide nimbin sodium nimbidate mahmoodin salannin tetranotriterpenoid (limonoid) salannol azadirachtin c, g salannolacetate methoxyazadirachtin vilasinin nonterpenoidal 2,3-dehydrosalannol turmeric compounds biopesticidal activities against aphids curcuminoids curcumin i curcumin ii curcumin iii sesquiterpenes turmerone demethoxycurcumin turmeronol a, b curcumenone curcumanolide a tetrahydroxycurcumin cyclocurcumin monoterpen curculonone a, b, c, d antifeedancy, oviposition repellency, reduced in life span, mortality, larval growth inhibition, interruption of aphid reproduction, blocking the development of vector-borne pathogen, growth regulation, fecundity suppression, sterilization (su, 1999; iqbal et al., 2006; james et al., 2014 and lokanadhan et al., 2012) mortality, adult repellency, changes in biological fitness, fecundity suppression and sterilization, antifeedant, growth-inhibiting effect, fumigant toxicity, reduced oviposition and eggs hatching (li et al., 2011; roy et al., 2015; siddiqi et al., 2011 and abida et al., 2009) 200 u. muhammad, t.n. khattak, h. rahman, m.k. daud, w. murad, a. azizullah served. we found that these extracts had a positive impact on okra growth and yield. ostermann (1992) also found that in neem seed extract caused an increased growth of okra plants. the higher growth and yield by these extracts can be attributed to the low incidence of pest attacks as revealed by hossain et al. (2013). the lower growth and yield in the control can be probably due to high occurrence of aphids. untreated plants possess high numbers of aphids which have caused leaf damages, reduced photosynthesis and consequently reduced growth and yield. the reduction in the number of aphids on okra plants sprayed with plant extracts resulted in superior vegetative growth which in turn produced higher okra fruit yields. neem extract when applied as pesticide was found to give better yield than conventional pesticides (satti and nasr, 2008). extracts of other plants like garlic and henge were also found to effectively reduce the population of pests in okra and increased its yield (mudathir and basedow, 2004). similarly, neem and tobacco extracts increased cotton yield to 1450 kg/ha as compared to the 1000 kg/ha in the control (rajaram et al., 2006). conclusions it is concluded that neem seeds and turmeric rhizome extracts effectively controlled aphid’s attacks on okra and enhanced it growth and yield. both extracts were found to be more effective than the synthetic pesticides. based on the present results and previous literature reviewed here, the extracts of neem seeds and turmeric rhizome are recommended as effective, ecofriendly, cheap, and easily available remedy for aphids attack in okra. acknowledgement we are thankful to nifa, peshawar and kust, kohat for supporting this study. conflict of interest all authors have participated in this work and all have read and approved the final version of the manuscript. the authors declare no conflict of interest. the paper was a part of m.phil thesis of mr. uzair muhammad submitted to kust, pakistan under the title “screening of neem seeds and turmeric rhizome extracts for biopesticidal activities against aphids in okra”. authors contributions: um and tnk: conducted experiments hr, mkd and wm: data analysis; initial drafting of publication aa: designed experiments; overall supervision of experiments; final drafting and correction 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technology proceedings, 121123. address of the corresponding author: dr. azizullah, assistant professor, department of botany, kohat university of science and technology, 26000 kohat, pakistan e-mail: azizswabi@hotmail.com © the author(s) 2018. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 90, 68 75 (2017), doi:10.5073/jabfq.2017.090.010 1 department of agronomy and plant breeding, college of agriculture, isfahan (khorasgan) branch, islamic azad university, isfahan, iran 2 ataturk university, agricultural faculty, department of horticulture, erzurum, turkey embryo and callus induction by different factors in ovary culture of cucumber maryam golabadi1*, samaneh ghanbari1, kourosh keighobadi1, sezai ercisli2 (received june 23, 2016) * corresponding author summary haploid and doubled haploid lines can be obtained in a short time using in vitro methods. in this study, unfertilized ovaries of cucumber were harvested and placed on semi-solid ms media as explants in petri dishes. the thermal shock pretreatment, cucumber genotypes, hormonal combinations and agno3 were evaluated as experimental factors in three consecutive experiments. the results of first experiment revealed that the thermal shock pretreatment had a significant influence on embryo induction, and the highest rate of embryogenesis was produced in presence of thermal shock pretreatment and the naa+2,4-d+kin+bap (0.5+0.7+1+1.8) mg/l hormonal combination. the highest rate of callus induction was recorded in combination of absence thermal shock and the naa+2,4-d+kin+bap (1.5+0.7+1+1.8) mg/l hormonal combination. according to the second experiment results, genotypes and the other hormonal combinations in media culture had highly significant effects on embryo and callus induction. the nbdc6*6/32441 genotype had the highest effects on these traits. a combination of bap (4 mg/l) and 2,4d (1.5 mg/l) was found to be optimal for embryogenesis. in third experiment, the highest level of embryogenesis (24.93 %) and callus (24.19 %) induction were found in the local iranian cultivar in comparison to nbdc6*6/32441 genotype. silver nitrate treatment had significant effects on the embryo and callus induction. the highest rate of embryo induction was recorded in presence of silver nitrate; however the absence of agno3 had a positive effect on the callus induction. in conclusion, the thermal shock pretreatment, silver nitrate, genotype and hormonal combination factors could play key roles in embryo and callus production, independently and simultaneously. keywords: ovary culture, hormone, agno3, genotype, cucumber introduction cucumber (cucumis sativus l.) is one of the world’s most important vegetable crops belonging to the family cucurbitaceae and the fruit is rich in phosphorus, potassium and oxalic acid. it is also called “khiar” and has been used for 3,000 years in particular asia and africa (khan et al., 2015). one of important genetic materials in vegetable breeding programs is doubled haploid (dh) line. the use of completely homozygous dh lines would be considerably shortened breeding program. one of the most significant biotechnological advances in plant breeding has been the development of in vitro techniques to produce haploid plants that can be used to generate homozygous lines (evans et al., 2003). there are several processes leading to haploid and dh cucurbits. these include primarily haploid parthenogenesis (induced primarily by pollination with irradiated pollen), in vitro gynogenesis (during in vitro culture of ovules and ovaries) and in vitro androgenesis (during in vitro culture of microspores and anthers) (galazka and niemirowicz-szczytt, 2013). dh lines have been used successfully in plant breeding programs for many crops such as barley, wheat, maize, pepper, rice, and tobacco (thomas et al., 2003). the main advantage of double haploid lines is their complete homozygosis. this facilitates the phenotypic selection for qualitative and quantitative characters much easier. thus, dhs can improve the efficiency and it is well known as time-consuming and labor-intensive. dh systems are widely applied in breeding (thomas et al., 2003) and genetic mapping (forster and thomas, 2005). ovary culture has been one of the approaches used to generate homozygous lines for commercial production of f1 hybrid varieties and genetic studies (diao et al., 2009). it has been pointed out in many experiments. on the other hand, successful regeneration of haploids and doubled haploids in ovary or ovule culture depend on several factors, i.e., pretreatment, genotype, female gametophyte development stage, and culture media composition (diao et al., 2009). diao et al. (2009) examined the effect of several factors in detail, i.e. the duration of thermal shock pretreatment at 35 ºc, the concentrations of thidiazuron (tdz) and the presence of silver nitrate and investigated their effects on embryo formation in ovary culture of cucumber (cucumis sativus l.). their results showed that a thermal shock for 3 days at 35 °c at the start of the culture in contrast to 2 or 4 days would result in higher frequency of embryo formation. thus the tdz has a positive effect on embryo formation. the highest embryo formation frequency (72.7 %) was recorded when 0.04 mg/l tdz into the induction medium. the results also revealed that was added to agno3 to the induction medium had no significant effect on frequency of embryo formation, however embryo germination period was shortened and the number of embryos increased in each ovary slice. malik et al. (2011) observed that thermal pre-treatment (4 °c) for 4 days increased embryo formation frequency (63.3 %) significantly in melon (cucumis melo l.). their experiment showed that tdz (0.04 and 0.02) mg/l to induction medium significantly increased the number of responding ovaries (46.6 % and 65.83 %, respectively). the maximum number of plantlet regeneration (22.5 %) was achieved by culturing the ovary derived embryos on murashige and skoog medium (ms medium) supplemented with 0.6 mg/l 6-benzylaminopurine. the objectives of this study were 1) to evaluate the effect of different concentrations of hormones and also hormone combinations on callus and embryo induction, 2) the influences of low and high temperature pre-treatment on callus and embryo induction, 3) the effect of silver nitrate on embryo and callus formation and 4) the influence of genotypes on callus and embryo induction, after all, as a local variety of cucumber in iran in terms of taste and aroma, it is highly regarded and highly marketable for the iranian consumers. material and methods plant material the present experiment was carried out at the plant improvement and seed production research center of the islamic azad university, isfahan (khorasgan) branch, iran, (51° 36´ longitude and 32° ovary culture in cucumber 69 63´ latitude) during 2013-2014. in this experiment one local variety of open field cucumber and two breeding lines (nbdc20*15/53211 and nbdc6*6/32441) of greenhouse cucumbers, that have been released in this center, were used. all three genotypes were planted in a greenhouse and managed using standard agronomic practices. during the growing period, the diurnal greenhouse air temperature was kept within 25-30 °c and the nocturnal one within 19-21 °c with a relative humidity of about 60 %. ovary culture the female flowers were picked one day before opening and transferred to laboratory in the ice box. most of the flowers were selected from first nodes (shalabi, 2007). unfertilized ovaries were isolated and surface sterilized by soaking in water for 3 min, followed by a 3 min washing in a water and detergent solution, and then rinsed three times in sterile distilled water. ovaries were then placed in 10 % sodium hypochlorite for 10 min, rinsed three or four times in sterile distilled water and finally, rinsed in 70 % ethanol for 5 min. the ovaries were sliced into 1 mm cross section under sterile con ditions and placed carefully in a semi-solid medium in a petri dish. ms medium (moqbeli et al., 2013) was used as basal medium in all experiments. all media were supplemented with 3 % (w/v) sucrose and solidified with 0.8 % (w/v) agar. the ph of the medium was adjusted to 5.8 before autoclaving at 121 °c, 1.1 kg/cm2 for 20 min. all cultures were incubated at 24±2 °c under a 16/8 h (light/dark) photoperiod with light provided using cool-white fluorescent lamps with 3000 lux. experimental factors in this study, consecutive experiments were conducted with the individual results used for the preceding ones. experiment 1: this experiment was performed with respect to the two factors; factor i consisted of thermal shock pretreatment (35 °c pretreatment for 4 days and without pretreatment), and factor ii consisted of various concentrations of cytokinins and auxins (2, 4-d, kin, iba, naa, bap, iaa from 0.1 to 3 mg/l). immediately after placing the ovary slices on induction medium, cultures were exposed to thermal shock pretreatment at 35 °c for 4 days. after the thermal pretreatment, all cultures were transferred to the 25 °c growth chamber. control petri dishes (without pretreatment) had been transferred to the 25 °c growth chamber from the beginning. experiment 2: according to the results of thermal shock pretreatment, the effect of genotype and different concentrations of hormones were investigated in the second experiment. the first factor consisted of two greenhouse inbred lines (nbdc515*20/3211 and nbdc6*6/32441) and the second factor consisted of 20 treatments of different concentrations and combinations of cytokinin and auxin plant growth regulators (2, 4-d, kin, iba, naa, bap, iaa from 0.1 to 3 mg/l). experiment 3: the most suitable greenhouse inbred line (nbdc6*6/ 32441) was selected and compared with the cucumber local cultivar as the first factor. the experiment carried out in presence and absence of silver nitrate in the culture medium as the second factor. finally, seven selected concentrations and combinations of different hormones (2, 4-d, kin, iba, naa, bap, tdz from 0.1 to 0.8 mg/l) were considered as the third factor. after thermal shock pretreatment, all treatments were transferred to the 25 °c growth chamber. statistical analysis all cultures were observed when one month had elapsed from the first day of ovary slices incubation. the frequency of embryo and callus formation was recorded based on the ratio of the number of ovaries responding to the total number of treated ovaries. the results of all three experiments were analyzed based on the factorial experiment in a completely randomized design in three replications and three petri dishes in every replicate. analysis of variance was performed by sas (ver. 9) software, and mean comparisons were assayed by an lsd (least significant differences) test at p≤0.05 proportion values were transformed with arcsine transformation before they were used in the statistical analysis. interaction mean comparisons were carried out by mstatc software. results and discussion effect of thermal pretreatment and growth regulators on callus and embryo induction (first experiment) in the first experiment, analysis of variance indicated that thermal shock pretreatment (2 levels) and various concentrations of hormones (6 levels) had significant effects on callus induction and embryogenesis. data presented in tab. 1 showed that the most embryogenesis (38.98 %) occurred during the thermal shock pretreatment. dubas et al. (2014) revealed that the distribution of auxin changes during embryo development and this innovation depends on the temperature-inducible in vitro culture conditions. many factors effect on successful ovary culture. for example, thermal treatment, (i.e. high or low temperature) applied to ovary or during in vitro culture, may have a strong influence on embryo formation (yang, 1982). in cucumber, 35 ºc thermal shock pretreatment was effective on ovary culture (gemes et al., 2002). specific changes that occur in cellular architecture during the switching from gametophytic to embryogenic pathway period following a heat shock treatment in brassica napus had been reported earlier (satpute et al., 2005). the diao et al. (2009) results showed that with high temperature (35 °c) treatments, the amount of embryogenesis increased and the most embryogenesis observed in 3-day treatments. the main objective of the koleva-gudeva et al. (2007) study was to evaluate the response of anthers from different genotypes to various media and heat and cold-shock pre-treatments regarding the direct somatic embryogenesis. their findings indicated that the cold and heat shocks (at 7 °c and 35 °c respectively, both in darkness) as a precondition will push the anther cultures towards direct somatic embryogenesis. shalaby (2007) found that ovules exposed to 32 °c for 4 days produced the greatest number of gynogenic ovules in squash. kurt and evans (1998) revealed that the pretreatment of flower buds (at 4 °c for three days) significantly lowered the number of calli induced in solid medium but, it could not have that statistically significant effect in the liquid medium. the fact that temperature can control the development of sexual explants such as isolated microspores has been well documented in brassica napus. in this plant lower temperatures favor asymmetric divisions, while heat treatment favors symmetric divisions (segulsimarro et al., 2003). the bueno et al. (1997) results were revealed that stress, particularly heat shock, sucrose starvation or a combination of both treatments could be the major and general sign of the inhibition of the normal gametophytic development of microspores and for the induction of the alternative embryogenic pathway. tab. 1: effects of thermal pretreatment on embryo and callus induction in experiment 1. traits thermal shock control pretreatment embryogenesis (%) 38.98 a 7.20 b callus induction (%) 28.60 b 35.73 a means followed by a common letter are not significantly different at 5 % level according to lsd test 70 m. golabadi, y. ghanbari, k. keighobadi, s. ercisli plant growth regulators such as cytokinin and auxin are the principal hormones in the plants involved in cell division and regulation of the differentiation (feher et al., 2003). hormone combinations had a significant effect on callus induction and embryogenesis. according to fig. 1, combination of 2,4-d+kin (1.5+1) mg/l showed the highest level of embryogenesis (52.76 %). however, this combination had no significant difference with that of naa+2,4-d+kin+bap (0.8+0.8+0.8+2.5) mg/l. among the similar somatic embryogenesis production factors, the 2,4-d is one of the highest efficiency, and thus it is used in the majority of embryonic cell and tissue culture procedures (feher et al., 2003). mean comparison results showed that the highest rate of callus in duction (47.76 %) is observed in combination of 2,4-d+naa+kin+ bap (0.5+0.5+0.5+1.5) mg/l, however it didn’t have any significant difference with naa+2,4-d+kin+bap (1.5+0.7+1+1.8) mg/l combination (fig. 1). the least embryogenesis and callus induction was observed in iba+naa+kin+bap (0.4+0.1+1+0.5) mg/l. in general, the medium containing 2,4-d and kin had the highest rate of embryogenesis, and the medium containing naa had the highest rate of callus induction. however, with increased bap, the amount of callus induction decreases. both auxins and cytokinins are essential for cell division and play a significant role in the meristem’ establishment and activity, however fluctuation of cytokinin and auxin ratios favor development of either root or shoot meristems (sugimoto et al., 2011). in general, increasing the ratio of cytokinin to auxin results in a shift from root to shoot organogenesis (hill and schaller, 2013). the koli and murthy (2013) results showed that the maximum callus induction observed in naa+bap (2+1) mg/l, 2,4-d+tdz (2+2) mg/l and naa+kin (5+2) mg/l combinations and also reported that the more cytokinins and the less auxins the more callus induction occurs. the analysis of variance indicated that there was a significant inter action between the thermal shock pretreatment and the hormonal combinations of the culture medium. the highest rate of embryogenesis was obtained in the t4 hormone combination with thermal shock pretreatment, which is indicative of the fact that there is a significant difference between this treatment and other treatments (tab. 2). the lowest rate of embryogenesis was observed in hormonal combinations iba+naa+kin+bap (0.4+0.1+1+0.5) mg/l and iaa+kin+bap (0.7+1.5+3) mg/l without thermal shock pretreatment. hormonal combination of naa+2,4-d+kin+bap (1.5+0.7+1+1.8) mg/l without thermal shock pretreatment showed the maximum callus production induction, although it did not have any significant difference with 2,4-d+naa+kin+bap (0.5+0.5+0.5+1.5) mg/l hormonal combination (tab. 2). effect of genotypes and growth regulators on callus and embryo induction (second experiment) according to the results of the first experiment, the thermal shock pretreatment compared to no heat treatment had a better effect on the traits in question. therefore, thermal shock pretreatment was applied to all ovary cultures in this experiment. on the other hand, the second experimental hormonal compounds were designed on the basis of the best combination and concentration of pgrs in the first experiment. as the results of anova showed, genotypes in terms of embryogenesis and callus induction were significantly different. a comparison of means between genotypes (tab. 3) showed that genotype 6*6/32441 resulted in the highest rate of embryogenesis and callus production induction. in a similar study rakhi et al. (2010) cultured cotyledon explants from seven varieties of pumpkin. according to their results, percentage of callus production induction was different among various genotypes. the separate analyses of the kurt and evans (1998) demonstrated that cultivar significantly influenced callus production induction in both solid and liquid media. mean comparison for hormonal composition showed that combi nation of 2,4-d+bap (1.5+4) mg/l had the highest rate of embryo genesis (i.e. 37.5 %), although the difference among the four hormonal combinations naa+2,4-d+bap (0.7+0.1+2.5) mg/l, naa+ fig. 1: effects of different hormonal combinations on embryogenesis and callus induction in experiment 1. for every trait, letters compared separately. means followed by a common letter are not significantly different at 5 % level according to lsd test t1: 2,4-d+kin (1.5+1 mg·l-1); t2: iba+naa+kin+bap (0.4+0.1+1+0.5 mg·l-1); t3: iaa+kin+bap (0.7+1.5+3 mg·l-1); t4: naa+2,4-d+kin+bap (0.5+0.5+0.5+1.5 mg·l-1); t5: naa+2,4-d+kin+bap (0.8+0.8+0.8+2.5 mg·l-1); t6: naa+2,4-d+kin+bap (1.5+0.7+1+1.8 mg·l-1) tab. 2: effects of thermal pretreatment and different hormonal combinations on embryogenesis and callus induction in experiment 1. growth regulators embryogenesis (%) callus induction (%) treatment concentration thermal shock control thermal shock control (mg/l) pretreatment pretreatment 2,4-d+kin 1.5+1 57.20 b 48.33 b 0.00 e 26.66 bc iba+naa+kin+bap 0.4+0.1+1+0.5 25.00 c 0.00 d 0.00 e 0.00 e iaa+kin+bap 0.7+1.5+3 51.66 b 0.00 d 0.00 e 50.00 b naa+2,4-d+kin+bap 0.5+0.5+0.5+1.5 70.53 a 23.33 c 43.33 bc 52.20 ab naa+2,4-d+kin+bap 0.8+0.8+0.8+2.5 23.33 c 25.00 c 0.00 e 21.66 de naa+2,4-d+kin+bap 1.5+0.7+1+1.8 51.66 b 29.43 c 0.00 e 63.86 a for every trait, letters compared separately. means followed by a common letter are not significantly different at 5 % level according to lsd test ovary culture in cucumber 71 tab. 3: effects of genotypes on callus and embryo induction in experiment 2. traits genotype nbdc6*6 / 32441 nbdc20*15 / 53211 embryogenesis (%) 17.44 a 12.30 b callus induction (%) 70.91a 38.91b means followed by a common letter are not significantly different at 5 % level according to lsd test bap+kin (1+1.5+2.5) mg/l, iba+bap+kin (1+1.5+2.5) mg/l and naa+2,4-d+bap (1+2+1.75) mg/l was not significant (fig. 2). the most callus induction was observed in hormone combinations of iaa+kin (1+1.5) mg/l (t2) and 2,4-d+bap (0.5+2) mg/l (t3), although their difference with kin+bap+iaa+iba (0.5 mg/l) (t5) and 2,4-d (3 mg/l) (t4) hormonal combinations was not significant (fig. 2). these results show that the presence of auxin in the culture medium increased the callus formation rate. auxins have several effects including cell enlargement, cell division, root initiation and apical dominance. in the culture medium high concentration of auxin inhibits root formation and leads to callus formation. culture medium is a principal factor controlling the induction and development of intact plants (mukhambetzhanov, 1997). in some culture media, the presence of low levels of auxin is essential for stimulating explants to the embryos formation. this result is consistent with those of ugandhar et al. (2011), on the culturing of cucumber cotyledons. they showed that the presence of 0.5 mg/l iaa and 3 mg/l bap or 3 mg/l kin, helped to increase accountability and induce root formation from explants. effect of genotype, absence or presence of agno3 and growth regulators on callus and embryo induction (third experiment) based on the results of anova and comparison of the hormones and genotypes in the second experiment, seven hormonal combinations and genotype nbdc6*6.32441 were selected, optimized and prepared for the third experiment. in the third experiment, one local and one greenhouse genotypes of cucumber (nbdc6*6/32441) were compared. the results showed that the genotypes used in this experiment (the local open field and greenhouse cucumber) made a significant difference in the percentage of embryogenesis and callus induction (tab. 4). according to tab. 4, the local genotype with the 24.93 % had the highest rate of embryogenesis. it also had the highest callus induction rate with 24.19 %. therefore, the iranian local variety was more successful in producing embryos and plantlets compared to the other genotype. shalaby (2007) stated that differences among genotypes indicated that the genotype-specific results can be obtained depending on the genetic structure of the individually used hybrids. this agreed with the results obtained by dumas-de-valux and chambonnet (1986) in squash, lux et al. (1990) in sugar beet, kobayashi et al. (1993) in sweet potato and alan et al. (2004) in onion. therefore, if the aim of tissue culture is to produce pure lines of local varieties, the results of this experiment will be applicable. the inbred line production in indigenous varieties could be used in order to transfer desirable traits such as fruit taste and quality of the commercial cultivars. fig. 2: effects of different hormonal combinations on embryogenesis and callus induction in experiment 2. for every trait, letters compared separately. means followed by a common letter are not significantly different at 5 % level according to lsd test t1: kin+2,4-d (1.25+0.5 mg·l-1); t2: kin+iaa (1.5+1 mg·l-1); t3: bap+2,4-d (2+0.5 mg·l-1); t4: 2,4-d (3 mg·l-1); t5: kin+bap+iaa+iba (0.5+0.5+ 0.5+0.5 mg·l-1); t6: bap+2,4-d+naa (1.75+2+1 mg·l-1); t7: bap+naa (3+1.5 mg·l-1); t8: kin+bap+iaa (2.5+1.5+1 mg·l-1); t9: kin+bap+2,4-d (2.5+ 1.5+1 mg·l-1); t10: kin+bap+naa (2.5+1.5+1 mg·l-1); t11: kin+bap+iba (2.5+1.5+1 mg·l-1); t12: bap+2,4-d (4+1.5 mg·l-1); t13: bap+iba (1+1.5 mg·l-1); t14: kin+bap+2,4-d (0.75+1.5+0.5 mg·l-1); t15:kin+bap+iba (1.3+0.6+0.5 mg·l-1); t16: kin+bap+2,4-d (0.5+2+1.8 mg·l-1); t17: kin+bap+2,4-d (0.5+3.5+1.8 mg·l-1); t18: bap+2,4-d+naa (2.5+0.1+0.7 mg·l-1); t19: kin+2,4-d+iaa+iba (0.3+1.1+0.7+0.5 mg·l-1); t20: kin+ bap+iaa (3+0.75+0.5 mg·l-1) tab. 4: effects of genotypes on embryogenesis and callus induction in experiment 3. traits genotype nbdc6*6 / 32441 local cultivar embryogenesis (%) 11.93 b 24.93 a callus induction (%) 3.57 b 24.19 a means followed by a common letter are not significantly different at 5 % level according to lsd test 72 m. golabadi, y. ghanbari, k. keighobadi, s. ercisli few characteristics of agno3 such as stability, availability, watersolubility and specificity make it very useful for several applications in regulation used in the plant growth and morphogenesis in vivo and in vitro (kumar et al., 2009). in this research, silver nitrate had the significant effect on embryogenesis and induction of callus production. the highest rate of embryogenesis (15.71 %) was observed in the medium with silver nitrate, but the highest rate of the callus production induction (18.34 %) was obtained in the medium without silver nitrate (tab. 5). thus, the application of silver nitrate had a positive effect on the regeneration of ovary culture. silver ions in the form of nitrate, such as agno3, play a major role in somatic embryogenesis efficient shoot and root formation (bais et al., 2001). agno3 is used in plant tissue culture as an ethylene antagonist. (beyer, 1976a). the results of the kumar et al. (2009) were revealed that agno3 promotes embryogenesis by blocking the inhibitory effect of endogenous ethylene on embryo formation (kumar et al., 2009). embryo production were significantly increased in the majority of the morphotypes by addition of silver nitrate to the medium, and agno3 treatment produced embryos in what would otherwise have been unresponsive morphotypes (dias and martins, 1999). li et al. (2013) conducted a study on the culturing of cucumber ovaries in two inbred lines cucumbers, four tdz and three silver nitrate concentrations (0.5 and 10 mg/l). their results showed that the biggest number of the regenerated plants produced in the presence of silver nitrate at a concentration of 10 mg/l. their results also showed that the presence of silver nitrate in the culture media prolongs the time needed for the formation of the embryo. they stated that the presence of silver nitrate increased the number of the embryos, though it did not make a significance difference with culture without silver nitrate. there are various opinions and experimental documents on the silver subject. according to this hypothesis, the ethylene role in plants is inhibited by weak antagonists like co2 and strong antagonists such as ag compounds. these procedures are possibly due to oxidation of ethylene by a metal-ion enzyme system. another theory is that silver nitrate inhibits the ethylene role by means of silver ions by decreasing the receptor capacity to bind ethylene, thus inhibiting the earlier steps of its own pathway (kumar et al., 2009). the culture media with different hormone concentrations and combinations used in this experiment made a significant difference with respect to the measured traits. the highest rate of embryogenesis was observed in 2,4-d+kin+bap (1.5+1+1.8) mg/l (i.e. 20.83 %), and the difference between this treatment and other hormonal compounds like iba+2,4-d+tdz+kin+bap (0.8+1+1+0.5+1.5) mg/l and 2,4-d+tdz+kin (1.5+0.5+1.5 mg/l) was significant (tab. 6). the highest rate of callus induction (i.e. 20.83 %) was detected in the hormonal compound naa+2,4-d+tdz+bap (0.5+0.5+0.5+1.5) mg/l. however, the difference with hormonal compounds iba+2,4d+tdz+kin+bap (0.8+1+1+0.5+1.5) mg/l, naa+kin+bap (0.1+ 1.5+1.5) mg/l and iba+2,4-d+bap (0.8+1+0.8) mg/l was not significant (tab. 9). the least rate of callus induction (i.e. 3.33 %) was observed in the hormonal compound 2,4-d+tdz+kin (1.5+0.5+1.5) mg/l. the interaction between genotype and silver nitrate was not significant for embryogenesis and callus induction traits. this means that regardless of what genotype has been used (local or commercial), silver nitrate is able to increase the embryogenesis. the interactive effect of genotype and hormonal combinations for these traits was significant. the local cultivar with combination of naa+2,4-d+tdz+bap (0.1+0.3+1+2) mg/l, had the highest rate of embryogenesis, even though its difference with some other hormonal combinations and local and inbred line genotypes was not statistically significant (tab. 7). tab. 5: effects of presence or absence of silver nitrate on embryogenesis and callus induction in experiment 3. traits silver nitrate presence absence embryogenesis (%) 15.71a 12.24 b callus induction (%) 11.19 b 18.34 a means followed by a common letter are not significantly different at 5 % level according to lsd test tab. 6: effects of different hormonal combinations on embryogenesis and callus induction in experiment 3. growth regulators embryogenesis (%) callus induction (%) t1 4.17 c 20.83 a t2 15.00 ab 12.50 c t3 12.50 b 3.33 d t4 16.66 ab 20.83 a t5 20.83 a 10.83 cd t6 15.16 ab 20.16 ab t7 13.63 ab 14.54 ab means followed by a common letter are not significantly different at 5 % level according to lsd test t1: iba+2,4-d+tdz+kin+bap (0.8+1+1+0.5+1.5 mg·l-1); t2: naa+2,4d+tdz+bap (0.1+0.3+1+2 mg·l-1); t3: 2,4-d+tdz+kin (1.5+0.5+1.5 mg·l-1); t4: naa+2,4-d+tdz+bap (0.5+0.5+0.5+1.5 mg·l-1); t5: 2,4-d+ kin+bap (1.5+1+1.8 mg·l-1); t6: naa+kin+bap (0.1+1.5+1.5 mg·l-1), t7: iba+2,4-d+bap (0.8+1+0.8 mg·l-1) the inbred line genotype showed the lowest embryogenesis rate with 3.33 % in hormonal treatment of iba+2,4-d+tdz+kin+bap (0.8+1+1+0.5+1.5) mg/l, the highest rate of callus induction was observed in local cultivar treated with naa+2,4-d+tdz+bap (0.5+0.5+0.5+1.5) mg/l, although their difference from the two different hormone and genotype treatments was not significant (tab. 7). the interactive effect of silver nitrate and hormone combinations was significant for embryogenesis and callus induction. the biggest percentage of embryogenesis (i.e. 25.88 %) was observed in the presence of silver nitrate and hormonal combination naa+2,4-d+tdz+bap (0.5+0.5+0.5+1.5) mg/l (fig. 3). this treatment made a significant difference with some other treatments. silver nitrate has been used in several studies for its similar hormonal activity and transgender cucumber, which indicates the key role of this material in plant physiological changes in the vegetative and reproductive phases (stankovic and prodanovic, 2002; nagar et al., 2014; law et al., 2002). the lowest rate of embryogenesis (i.e. 1.65 %) was observed in the absence of silver nitrate and hormonal combination iba+2,4d+tdz+kin+bap (0.8+1+1+0.5+1.5) mg/l. the highest rate of callus induction was observed in the hormonal combination iba+2,4d+tdz+kin+bap (0.8+1+1+0.5+1.5) mg/l without silver nitrate, although it did not make any statistically significant difference with a1t6, a2t4 and a2t7 treatments (fig. 3). in many crop species, tdz stimulated embryogenesis with a higher efficiency and frequency than other cytokinins or the combined treatments of auxins and cytokinins (visser et al., 1992; zhang et al., 2001). according to the analysis of variance results, interaction of genotype×silver nitrate×hormonal combinations was also significant for the rate of embryogenesis and callus induction. the highest rate of embryogenesis was observed in the treatment of local cultivar ovary culture in cucumber 73 with 2,4-d+tdz+kin (1.5+0.5+1.5) mg/l in the presence of silver nitrate. however, the difference among treatments with c1a1t2, c1a1t4, c1a2t5, c1a2t6, c2a1t2, c2a1t4, c2a1t5, c2a1t7, c2a2t2, c2a2t5 and c2a2t7 was not statistically significant (tab. 8). callus production induction in the local cultivar with hormonal combination of iba+2,4-d+tdz+kin+bap (0.8+1+1+0.5+1.5) mg/l in the absence of silver nitrate (c2a2t1) resulted in the highest rate (i.e. 13.56 %) of callus production which is significantly different from all of the other treatments with the exception of c2a2t5 and c2a1t6. conclusion haploids can be obtained using ovary as well as other sexual explants. the current study reveals different factors for the efficient ovary embryogenesis induction in cucumber using various treatments. our findings demonstrated that thermal shock (at 35 °c) as a pretreatment favors an embryogenesis pathway. other experiments in the present study showed that rate of embryo production induction was different among genotypes, and that silver nitrate had a significant effect on the embryogenesis and callus production induction. in a nutshell, the results of the present assessment can be described and summarized as follows: a the thermal shock pretreatment compared to no heat treatment produced a better effect on embryo and callus production induction. therefore, thermal shock pretreatment was applied to all ovary cultures. b the results showed that the genotype 6*6/32441 produced the highest rate of embryogenesis and callus production induction, and combination of 2,4-d+bap (1.5+4 mg · l-1) produced the highest rate of embryogenesis. c the application of silver nitrate had a positive effect on the regeneration of ovary culture. references alan, a.r., brants, a., cobb, e., goldschnied, p.a., 2004: fecund gynogenic lines from onion (allium cepa l.) breeding materials. plant sci. 167, 1055-1066. doi: 10.1016/j.plantsci.2004.06.007. bais, h.p., sudha, g.s., ravishankar, g.a., 2001: influence of putrescine agno3 and polyamine inhibitors on the morphogenetic response in untransformed and transformed tissues of chichorium intybus and their regenerants. plant cell rep. 20, 547-555. doi:10.1007/s002990100367. beyer, j.r.e., 1976a: silver ion: a potent antiethylene agent in cucumber and tomato. hort sci. 11, 195-196. bueno, m.a., gomez, a., boscaiu, m., jose, j.a., vicente, o., 1997: stress-induced formation of haploid plants through anther culture in cork oak (quercus suber). physiol plantarum 99, 335-341. doi:10.1111/j.1399-3054.1997.tb05421.x. diao, w.p., chen, j.f., jia, y.y., song, h., zhang, x.q., lou, q.f., 2009: effcient embryo induction in cucumber ovary culture and homozygous identification of the regenetants using ssr markers. scientia hortic. 119, 246-251. doi:10.1016/j.scienta.2008.08.016. dias, j.s., martins, m.g., 1999: effect of silver nitrate on anther culture embryo production of different brassica oleracea morphotypes. scientia hortic. 82, 299-307. doi: 10.1.1.469.9966&rep=rep1. dumas de vaulx, r., chambonnet, d., 1986: obtention of embryos and plants from in vitro culture of unfertilized ovules of cucurbita pepo l. in: horn, w., jensen, c.j., odenbach, w., schieder, o. (eds.), genetic manipulation in plant breeding. proceedings of the international symposium organised by eucarpia, september 8-12, 1985. walter de gruyter & co., berlin, 295-297. evans, d.c., coleman, j.d., kearns, a., 2003: plant cell culture. bios scientific publications, london, 192 pp., isbn 185 996320x. feher, a., pasternak, t.p., dudits, d., 2003: transition of somatic plant cells to an embryogenic state. plant cell tiss. org. 74, 201-228. doi: 10.1023/a:1024033216561. fig. 3: interaction effects of silver nitrate and different hormonal combinations on embryogenesis. means followed by a common letter are not significantly different at 5 % level according to lsd test t1: iba+2,4-d+tdz+kin+bap (0.8+1+1+0.5+1.5 mg·l-1); t2: naa+2,4d+tdz+bap (0.1+0.3+1+2 mg·l-1), t3: 2,4-d+tdz+kin (1.5+0.5+1.5 mg·l-1); t4: naa+2,4-d+tdz+bap (0.5+0.5+0.5+1.5 mg·l-1), t5: 2,4-d+ kin+bap (1.5+1+1.8 mg·l-1); t6: naa+kin+bap (0.1+1.5+1.5 mg·l-1), t7: iba+2,4-d+bap (0.8+1+0.8 mg·l-1) tab. 7: interaction effects of different hormonal combinations and genotype on embryogenesis and callus induction in experiment 3. growth regulators embryogenesis (%) callus induction (%) treatment concentration (mg/l) greenhouse line local cultivar greenhouse line local cultivar ba+2,4-d+tdz+kin+bap 0.8+1+1+0.5+1.5 3.33 f 6.16 ef 1.65 d 38.33 a naa+2,4-d+tdz+bap 0.1+0.3+1+2 11.21 b-f 23.68 a 0.00 d 25.55 b 2,4-d+tdz+kin 1.5+0.5+1.5 6.10 ef 19.28 abc 0.00 d 6.60 cd naa+2,4-d+tdz+bap 0.5+0.5+0.5+1.5 15.71 a-e 18.01 a-d 0.00 d 42.66 a 2,4-d+kin+bap 1.5+1+1.8 20.08 ab 18.18 a-d 8.46 cd 11.53 cd naa+kin+bap 0.1+1.5+1.5 21.48 a 9.81 c-f 1.66 d 39.25 a iba+2,4-d+bap 0.8+1+0.8 8.15 d-f 20.10 ab 8.85 cd 17.60 bc for every trait, letters compared separately. means followed by a common letter are not significantly different at 5 % level according to lsd test 74 m. golabadi, y. ghanbari, k. keighobadi, s. ercisli forster, b.p., thomas, w.t.b., 2005: doubled haploids in genetics and plant breeding. in: janick, j. 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8: triple interaction effects of different hormonal combinations, genotypes and absence or presence of agno3 on embryogenesis and callus induction in experiment 3. embryogenesis (%) callus induction (%) greenhouse line (c1) local cultivar (c2) greenhouse line (c1) local cultivar (c2) presence absence presence absence presence absence presence absence ag (a1) ag (a2) ag (a1) ag (a2) ag (a1) ag (a2) ag (a1) ag (a2) t1 6.66 e-g 0.00 g 9.03 d-g 3.30 fg 0.00 h 3.30 gh 23.43 c-f 56.13 a t2 22.43 a-d 0.00 g 19.43 a-c 27.93 ab 0.00 h 0.00 h 22.13 c-g 28.96 cd t3 3.16 fg 9.03 d-g 31.43 a 7.13 e-g 0.00 h 0.00 h 3.13 gh 9.80 e-h t4 28.10 ab 3.33 fg 23.66 a-c 12.36 c-g 0.00 h 0.00 h 34.56 bc 50.93 a t5 12.23 c-g 27.93 ab 19.03 a-e 17.33 a-f 0.00 h 16.93 c-h 5.53 f-h 17.53 ch t6 14.53 b-f 28.43 ab 10.13 c-g 9.50 d-g 0.00 h 3.16 gh 50.10 ab 28.40 c-e t7 6.46 e-g 9.83 cg 20.16 a-e 20.00 a-e 0.00 h 17.70 c-h 15.58 d-h 20.20 c-e for every trait, letters compared separately. means followed by a common letter are not significantly different at 5 % level according to lsd test. t1: iba+2,4-d+tdz+kin+bap (0.8+1+1+0.5+1.5 mg·l-1); t2: naa+2,4-d+tdz+bap (0.1+0.3+1+2 mg·l-1); t3: 2,4-d+tdz+kin (1.5+0.5+1.5 mg·l-1); t4: naa+2,4-d+tdz+bap (0.5+0.5+0.5+1.5 mg·l-1); t5: 2,4-d+kin+bap (1.5+1+1.8 mg·l-1); t6: naa+kin+bap (0.1+1.5+1.5 mg·l-1); t7: iba+2,4-d+bap (0.8+1+0.8 mg·l-1) growth regulators ovary culture in cucumber 75 address of the corresponding author: maryam golabadi, department of agronomy and plant breeding, college of agriculture, isfahan (khorasgan) branch, islamic azad university, isfahan, iran e-mail: golabadim@gmail.com, m.golabadi@khuisf.ac.ir © the author(s) 2017. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 89, 142 149 (2016), doi:10.5073/jabfq.2016.089.017 1department of food technology, pir mehr ali shah, arid agriculture university rawalpindi, pakistan 2 department of agricultural sciences, university of haripur, pakistan 3 division of plant and soil sciences, west virginia university, morgantown, usa transition in tuber quality attributes of potato (solanum tuberosum l.) under different packaging systems during storage kashif sarfraz abbasi1, tariq masud1, abdul qayyum2*, sami ullah khan2, asif ahmad1, ayaz mehmood2, abid farid2, matthew a. jenks3 (received december 19, 2015) * corresponding author summary the suitability of different packaging materials i.e. jute, nylon, polypropylene, cotton, low density polyethylene, medium density polyethylene, and high density polyethylene were studied for tubers of the premium potato (solanum tuberosum l.) variety “lady rosetta”. after harvest, potato tubers were washed, sorted, graded, cured, and subsequently stored in different packaging materials at ambient temperature (25 ± 2 °c). changes in quality attributes of potato tubers under different packaging materials were studied on the basis of their physico-chemical and functional parameters. overall results revealed that packaging materials had a significant (p ≤ 0.05) effect on many important quality attributes. generally, weight loss, glucose and glycoalkaloid amounts, and polyphenol oxidase and peroxidase activities increased, while ascorbic acid contents decreased with increasing storage time. total phenolic contents and radical scavenging activity showed a nearly parabolic trend during the storage period. amongst the different packaging materials employed, potatoes stored in polypropylene and low-density polyethylene pre sented the best overall retention of vital quality attributes during 63 days storage. however, the higher tensile strength of polypropylene packaging made it a more durable and thus more effective material for prolonged potato tuber storage, a characteristic having clear advantages when used in typical marketing supply chains. introduction potato is the most highly produced non-grain staple crop in the world, with one third of total production harvested in densely-populated developing countries, like china and india (pic, 2008). as such, it is critically important for world food security. moreover, it is considered the most important crop in south america, the second most important crop in europe, and fourth on a global scale, after wheat, rice, and maize (messer, 2000). one of the greatest challenges to potato tuber production is storage after harvest and greatest quantity of potatoes is lost each year due to inadequate storage conditions after harvest and during marketing. once the potato tuber is harvested, it is washed and then cured for storage. the curing process leads to the potato tuber entering physiological dormancy (pande et al., 2007), and modern commercial potato cultivars grown for the processing industry often have shorter dormancy periods and more susceptibility to storage disorders like sweetening, greening, and others. there is still much work needed to establish appropriate postharvest interventions to ensure optimum tuber storage (quality and duration) to facilitate continuous supply (suttle, 2007). use of suitable packaging systems extends the storage life of potato tubers during transit, in part by slowing down the ripening process during postharvest storage and marketing (sanchez et al., 2003). in developing countries, the development of effective packaging materials for perishable commodities like potatoes can have tremendous advantages over more expensive technologies like controlled atmosphere storage facilities, and/or irradiation treatments, as a means to prolonged postharvest storage life. besides improving food security within country, improved packing strategies that extend commodity shelf life create new options for long-distance shipping of produce to more distant markets. in many cases today, various packaging systems can be supplemented with low temperature storage (kyriacou et al., 2009), sprout inhibitors/suppressants (frazier et al., 2004), ethylene scavengers (abbasi et al., 2004), and irradiation (blessington et al., 2007) as part of an integrated strategy to minimize postharvest losses. the packaging materials themselves are often processed as sheets, wraps, pouches, or more solid containers having diverse barrier proper ties that modulate gas exchange between the internal and external package atmosphere (hong et al., 2003). potato being an important cash crop with substantial export potential could have a major impact on farm incomes and foreign exchange earnings for the exporting country. in developing countries such as pakistan with a growing potato industry, large bags made of jute (a natural plant fiber, corchorus olitorius l.) with typical carrying capacity of 100 kg are commonly used to store potato tubers after harvest. however, more highly engineered packaging systems that utilize polyethylene, polypropylene, polystyrene, various biodegradable plastics, and wound preventing corrugated cartons and other cushioning materials, are a necessity in many marketing systems to maintain compliance with food safety standards and to compete on the basis of quality in the international market. the quality of horticultural commodities is a critical consideration in decisions made by producers, exporters, and consumers, and the packaging system utilized plays a principle role in determining quality attributes. the present study was designed to identify an optimized packaging system for postharvest storage of the potato variety “lady rosetta” at ambient temperature. materials and methods tubers of potato (solanum tuberosum l.) variety “lady rosetta” were harvested from the potato research institute, sahiwal (punjab, pakistan). tubers were transported the same day to the postharvest technology laboratory, department of food technology pmasarid agriculture university rawalpindi, pakistan. on the same day, the tubers were washed, sorted and graded by size and then cured for one week at temperatures between 15-20 °c before being sampled for analysis here. potato tubers were packed in different packaging materials (30 cm × 40 cm bags) procured from ms. multi packages ltd. (lahore, pakistan). the set of treatments included control, jute packaging, nylon packaging, polypropylene packaging, cotton packaging, low density polyethylene packaging (ldpe), medium density polyethylene packaging (mdpe) and high density polyethylene packaging (hdpe). one kg of potato tubers were used for each replication, sampling from a total of 30 kg of potato tubers subjected to different physico-chemical assessments each week. all the changes in potato tuber quality under different packaging systems 143 packaged potatoes were stored at approximately 25 ± 2 oc and 70 ± 5 % relative humidity. weight loss the weight loss (%) in different experiments at specified storage intervals was determined by weighing the samples with digital balance (ohaus, model ts4kd florham park, nj, usa) and reported as percent loss in sample weight based on initial weight. weight loss (%) = (initial weight final weight / initial weight) × 100 glucose content glucose quantity was determined using glucose test strips imported from snack food association, (arlington virginia usa). the color change was correlated with a standard color chart, and the values expressed in mg glucose 100 gm-1 sample. the tests were conducted in triplicate per treatment. ascorbic acid content ascorbic acid (aa) quantity was determined using titrametric method by employing 2, 6, di-chlorophenol indophenol dye (redox dye) as explained in (aoac, 1990) method no. 967.21. a 10 g representative sample was placed in a beaker and brought up to volume with 100 ml 3 % phosphoric acid and filtered. 10 ml of filtrate was titrated with the standard dye solution. the ascorbic acid contents (mg 100 g-1 dry matter) were quantified as: ascorbic acid = (dye factor × titration × volume made up) / (weight of sample × volume of filtrate) × 100 dye standardization five milliliters of standard ascorbic acid solution was diluted with five milliliter of metaphosphoric acid (3 %) and titrated with dye solution until a pink coloration could be observed to endure for ten sec. dye factor was determined (mg ascorbic acid ml-1 of dye) as: dye factor (d.f) = 0.5 / titration glycoalkaloid content the total glycoalkaloids (tga) were determined by the method described by grunenfelder et al. (2006). ground lyophilized potato tissue (500 mg) was extracted in 10 ml of 80 % ethanol at 85-90 °c for 25 min. the extract were filtered and reduced to 3-5 ml on rotary evaporator at 50 °c. each extract was rinsed twice with 3 ml of 10 % (v/v) acetic acid and then centrifuged at 10000 g for 30 min at 10 °c. the ph of the supernatants was adjusted at 9.0 with nh4oh. the extract was refluxed at 70 °c for 25 min followed by overnight storage at 4 °c temperature. the extracts were centrifuged, and after discarding the supernatants the resulting pellets were dissolved in 0.5 ml of 7 % (v/v) phosphoric acid and stored at -20 °c. the total glycoalkaloids were estimated by adding 200 μl of extract in 1 ml of 0.03 % (w/v) in concentrated phosphoric acid. the contents were allowed to settle for 20 min and absorbance was measured at 600 nm. tga concentrations were quantified based on α-solanine (sigma-aldrich) standard curve using a ce-2021, spectrophotometer (cecil instruments cambridge, england) and expressed as mg tga 100 g-1 dry weight. phenolic content total phenolic content (tpc) in terms of gallic acid equivalent (gae) were carried out by folin-ciocalteu (fc) assay (lachmann et al., 2008) with few modifications. tubers randomly selected were freeze dried and then extracted with 80 % ethanol. a total of 2 gm extract was quantitatively converted into 100 ml volumetric flask and adjusted with 80 % ethanol. in 5 ml of the sample slightly diluted with distilled water, 2.5 ml of fc and 7.5 ml of 20 % solution of sodium carbonate were added. the contents were allowed to settle for 2 hrs and absorbance was measured at 765 nm using a ce-2021, spectrophotometer (cecil instruments cambridge, england). total phenolic contents were quantified by standard calibration curve de rived from the absorbance of known gallic acid concentration (10100 ppm). results were articulated as mg gallic acid equivalents (gae) 100 g-1 dry weight. radical scavenging activity (rsa) antioxidant activity was measured as radical scavenging activity (rsa) using methods described by singh and rajini (2004) that involved electron transfer reaction based assay by employing free radical 2, 2-diphenyl-1-picrylhydrazyl (dpph). five mg of freeze dried potato extract was incubated with 1.5 ml of dpph solution (0.1 mm in 95 % ethanol). the reaction mixture was shaken and allowed to stand for 20 min under ambient temperature. absorbance of the resultant mixture was determined at 517 nm against a blank. the radical scavenging activity was determined as a decrease in the absorbance of dpph using the following equation: scavenging (%) = 1 (absorbance of sample at 517 nm / absorbance of control at 517 nm) × 100 enzyme determination enzyme extraction was carried out by the method described by yemenicioglu (2002), with some modifications. washed, peeled, and diced potato tubers were placed under -20 °c before homogenate preparation. 200 gm frozen potato tuber was homogenized with 300 ml acetone and 1 gm of polyvinylpolypyrrolidone (pvpp) in waring blender. the resultant mixture was homogenized for 2 min and then filtered through whatman no.1 filter paper. acetone powder preparation was carried out by repeated extraction and evaporation. the extraction mixture was prepared by mixing 0.4 g pvpp, 2 g acetone powder, and 50 ml of cold 8.8 % sodium chloride solution. extraction was completed at 4 °c temperature 3 hrs under magnetic stirrer. the extract was filtered, centrifuged at 11000 g for 20 min, and then stored at -20 °c prior to quantification of enzyme activity. protein extraction was carried out with the lowry et al., (1951) method using bovine serum albumin (bsa) as standard. protein standards of crude and partially purified extracts were prepared in 8.8 % nacl solution and deionized water respectively. all the assays were carried out in triplicate. the enzyme activity was calculated as u 100 g-1 fresh weight, in spectrophotometric assay 1 u was defined as 0.001 change in absorbance min-1 ml-1 of enzyme extract. polyphenol oxidase assay polyphenol oxidase (ppo) activity was determined as described by yemenicioglu (2002). the reaction mixture contained 2 ml 0.01 m sodium phosphate buffer (ph 7.0), 0.2 ml of 0.25 m catechol, and 0.3 ml enzyme extract to the total volume of 2.5 ml. the optical density (od) of the reaction mixture was determined spectrophotometerically at 420 nm. polyphenol oxidase activity was calculated by the change in od over a period of thirty sec and expressed as u 100 g-1 fresh weight. peroxidase assay peroxidase (pod) estimation was carried out as reported by abbasi et al. (1998). reaction mixture consisted of 2.1 ml, 15 mm nakpo4 144 k.s. abbasi, t. masud, a. qayyum, s.u. khan, a. ahmad, a. mehmood, a. farid, m.a. jenks buffer (ph 6.0), 0.3 ml 1 mm h2o2, 0.3 ml 0.1 mm guaiacol and 0.3 ml enzyme extract to the total volume of 3 ml. the optical density (od) of the reaction mixture was determined spectrophotometerically at 470 nm. pod activity was estimated by the change in od due to guaiacol oxidation over thirty sec time and expressed as u 100 g-1 fresh weight. statistical analysis data obtained as a mean of three replications were statistically analyzed by a two-factor factorial in completely randomized design (crd) and treatments and storage interval means were compared using a duncan multiple range test using m-stat-c statistical software as described by steel et al. (1997). results weight loss tuber weight loss (%) occurred in all treatments over time; however, the rate of weight loss was slower in packaged tuber than nonpackaged controls during the storage period. treatment means of packaged potato tubers showed non-significant differences between jute and hdpe, polypropylene and ldpe, while all other differed significantly. data on weight loss revealed significant differences between all the storage interval means. the interaction between treatment means and storage intervals showed maximum weight loss (%) in control and minimum in ldpe at the end of storage. in general potato packed in different polyethylene packaging materials (ldpe, mdpe and hdpe) showed lesser weight loss as compared to jute, nylon and cotton packagings (fig. 1). creased up to 333.5 mg 100 g-1 (0.333 %) by the end of storage. nonsignificant differences were recorded between ldpe and mdpe packaging at the end of their respective storage periods, and attained 220 mg 100 g-1 and 215 mg 100 g-1 respectively. the least glucose contents were estimated in polypropylene packaged potato with less than 200 mg 100 g-1 (0.2 %) glucose contents throughout the storage period (fig. 2). fig. 1: weight loss in potato tubers stored using different packaging materials was highest throughout the storage period when stored in polypropylene and ldpe packages. vertical bars show ±se of means (n-3). interaction between storage intervals and packaging materials was significantly different at p ≤ 0.05. glucose content glucose contents showed steady increase throughout the storage period, with an observed impact of applied packaging materials. treatment means showed significant difference in stored potato with control having maximum while ldpe having minimum glucose over time. maximum glucose contents were recorded in the last week while minimum during the first week storage. the interaction between storage intervals and treatments was less significant during the early storage, but differed significantly with the progression in the storage period. the prominent increase in glucose contents in the non-packaged control started on the 28th day which gradually inascorbic acid content in response to different packaging systems, ascorbic acid (aa) was amongst the parameters that decreased significantly during the storage period. treatment means revealed that the maximum aa retention was observed in ldpe and polypropylene packagings were also found statistically similar at the 5 % level of significance. potatoes packaged in jute, mdpe and ldpe also maintained higher aa contents than the control. storage intervals showed significant impact in their aa contents with maximum and minimum values estimated during the first and last weeks, respectively. the interaction between storage intervals and treatments showed substantial aa retention in packaged potato tubers as compared to controls. amongst different treatments, potato packed in polypropylene packaging and ldpe packaging retained maximum aa contents by the end of storage period. minimum retention in aa contents was observed in control (15.6 mg 100 g-1) while maximum in polypropylene (19.97 mg 100 g-1) packaging and lpde 19.2 mg 100 g-1) packaging by the end of storage period (fig. 3). glycoalkaloid content total glycoalkaloid (tga) accumulation in terms of solanine equivalent increased during storage in all the treatments. treatment means revealed significant difference between the tubers in their tga contents for the various packaging materials. maximum and minimum tga contents during the storage period were identified in control and hdpe packaging, respectively. storage interval means showed significant differences in their tga contents, and these were found to be maximal at the end of the storage period. results expressed on dry weight basis showed increase in tga content in potato tubers during storage. however, in all treatments except in control, the tga levels remained under a safe limit i.e. 20 mg 100 g-1, as suggested by nema et al. (2008). in general, irrespective of packaging types, considerable increase in tga content started on fig. 2: glucose content in stored potato tubers stored using different packaging materials was lowest by the end of the storage period when stored in polypropylene packages. vertical bars show ±se of means (n-3). interaction between storage intervals and packaging materials was significantly different at p ≤ 0.05. changes in potato tuber quality under different packaging systems 145 the 28th day, which continued till the end of storage. maximum increase in tga was about eight folds (7.50 mg 100 g-1 to 63.80 mg 100 g-1) in control at the end of storage as compared to six folds (7.50 mg 100 g-1 to 47.20 mg 100 g-1) increase in tubers in the polypropylene packaging (fig. 4). gae 100 g-1, 124.07 mg gae 100 g-1 and 120.5 mg gae 100 g-1 tpc, respectively, in contrast to lowest tpc i.e. 88.77 mg gae 100 g-1 estimated in control (fig. 5). fig. 3: ascorbic acid content in potato tubers stored using different packaging materials was highest by the end of the storage period in polypropylene packages. vertical bars show ±se of means (n-3). interaction between storage intervals and packaging materials was significantly different at p ≤ 0.05. fig. 4: total glycoalkaloids (tga) in potato tubers stored using different packaging materials was highest in non-packaged controls by the end of the storage period. vertical bars show ±se of means (n-3). interaction between storage intervals and packaging materials was significantly different at p ≤ 0.05. phenolic content total phenolic content (tpc) initially showed a trend toward increasing, followed by a decline till the end of storage period. treatment means showed significant difference in tpc in response to different packagings. polypropylene and mdpe packagings maintained maximum tpc while control had minimum phenolic content. the interaction between treatments and storage intervals showed maximum tpc in control and polypropylene packaging on 28th and 49th day of storage, respectively. in general, considerable increase in tpc was observed in all treatments up to 42nd day, which subsequently declined by the end of storage. this decline in tpc was more significant in control, nylon, hdpe, and cotton packaged potatoes. after 63rd days storage, potato tubers stored in polypropylene, ldpe, jute, and mdpe packaging retained 137.23 mg gae 100 g-1, 130.23 mg radical scavenging activity radical scavenging activity (rsa) determined in terms of % inhibition of dpph showed slight increase initially, followed by a gradual decrease during potato tuber storage. treatment means showed ldpe packaging maintained maximum activity followed by mdpe, polypropylene and hdpe packaging, while control demonstrated minimum activity. potato storage in jute, nylon and cotton packaging was found statistically similar, and retained moderate activities throughout the storage period. radical scavenging activity increased during the first week and attained maximum levels by the second week in all the treatments. in general, maximum rsa activity in tubers occurred between 14-21 days storage, and then progressively decreased after. a considerable reduction in activity was observed in all treatments on the 28th day storage. potato tubers packed in polypropylene and polyethylene packaging exhibited substantial rsa that retained substantial higher after the fourth week till the end of storage period, with no significant difference between them. the loss in rsa after 14th day till the end of storage in polypropylene was 45.8 % 23.8 % as compared to 48.0 % 17.8 % in control (fig. 6). polyphenol oxidase activity in response to different packaging materials, polyphenol oxidase (ppo) activity in potato tubers generally increased with time. treatment means demonstrated maximum activity in control, while minimum was recorded in polypropylene and ldpe packaging (statistically similar at 5 % level of significance). moderate ppo activity was observed in jute and hdpe packaging (statistically similar at 5 % level of significance). interaction between storage intervals and treatments was significant with maximum activity estimated in control (68.5 u 100 g-1 fresh weight) at the end of the storage period. in general, steady increase with no significant differences in ppo activity were observed in all the treatments till 28th day, afterward prominent increase was estimated in control till the end. nevertheless, irrespective of different treatments the ppo activity increased after the fourth week storage. lowest ppo activity was observed in polypropylene, ldpe and mdpe packaged potato tubers and were found statistically similar during most of the storage period (fig. 7). fig. 5: total phenolic contents in potato tubers stored using different packaging materials were highest at the end of the storage period when stored in polypropylene packages. vertical bars show ±se of means (n-3). interaction between storage intervals and packaging materials was significantly different at p ≤ 0.05. 146 k.s. abbasi, t. masud, a. qayyum, s.u. khan, a. ahmad, a. mehmood, a. farid, m.a. jenks peroxidase activity peroxidase (pod) activity in potato tubers showed a steady increase in all the treatments during storage. however the increase was most prominent in the control. increased pod activity in potato tuber was comparable to their ppo activity but observed at slower pace. treatments means revealed maximum pod activity in control and minimum in ldpe packaging during the complete storage. nonsignificant difference was observed in jute and nylon packaging, whereas, cotton and hdpe were also found to be statistically similar during their storage period. in general, pod activity was maximum and minimum during the last and first weeks, respectively. maximum activity pod activity (32.40 u 100 g-1) was estimated in control and minimum (23.20 u 100 g-1) in ldpe packaging at the end of storage. polypropylene and mdpe packaging retained comparatively lower pod activity i.e. 23.43 u 100 g-1 and 26.47 u 100 g-1, respectively, during the same storage periods (fig. 8). discussion as the potato tuber is one of the most important crops supporting worldwide food security, its extended storage life at high quality is the focus of much interest by growers, exporters, consumers, and researchers. weight loss by potato tubers during storage results in significant post harvest losses. as an edible plant stem typically forming underground, weight loss is primarily attributed to the water loss that occurs through the outermost skin tissues during the processes of respiration and sprouting (tester et al., 2005). the phenomenon is thus considered as an important stability index for the storage life assessment. packaging systems confer barrier properties to the physiological gaseous exchange of stored tubers and decrease the rate of weight loss during storage (hong et al., 2003). different types of packaging material assessed in the present study had a significant impact on potato tuber weight loss during storage. potato tubers stored in different polyethylene packaging (ldpe, mdpe and hdpe) showed increased percentage weight loss with the increase in the thickness of polyethylene packaging, which might be the reason behind increased weight loss in high density polyethylene packaging as compared to those packed in low density polyethylene. similar impacts due to thickness and permeability of packaging materials have also been reported by rakotonirainy et al. (2001). application of polyethylene packaging in horticultural products like potato tuber (rosenfeld et al., 1995) and tomato (solanum lycopersicum l.) fruit (sammi and masud, 2007) have been shown to reduce weight loss during the postharvest storage. use of polypropylene packaging material has also been shown to effectively prolong storage stability in sweet cherry (conte et al., 2009) and fresh cut pineapple (calderon et al., 2008). the hydrolysis of sucrose by the invertase enzyme leads to the formation of glucose and fructose monomers within potato tubers (kumar et al., 2004).the presence of either kind of sugars is highly undesirable for tuber processing, as high glucose and fructose are reducing sugars that have a negative effect on potato chip fry color. in addition, high levels of these sugars are a safety concern due to their active participation in toxic acrylamide formation at elevated processing temperatures (mottram, 2002). the significant increase in glucose contents we report here later in the storage period in treatments like control, jute, nylon, cotton, and hdpe are likely due to earlier sprouting and the associated depletion of carbohydrate reserves in the tuber (blenkinsop et al., 2002). by comparison, potato tubers stored in polypropylene, ldpe, and mdpe packaging were observed to delay the dormancy break relative to other materials, and thus retained lower glucose contents at the end of the storage period. the maximum glucose content was reported at the end of storage period for the non-packaged control treatments was associated to fig. 6: radical scavenging activity (rsa) in potato tubers stored using different packaging materials was highest by the end of the storage period when stored in polypropylene. vertical bars show ±se of means (n-3). interaction between storage intervals and packaging materials was significantly different at p ≤ 0.05. fig. 7: polyphenol oxidase (ppo) in potato tuber stored in different packaging materials was lowest by the end of the storage period when stored in polypropylene and ldpe. vertical bars show ±se of means (n-3). interaction between storage intervals and packaging materials was significantly different at p ≤ 0.05. fig. 8: peroxidase (pod) in potato tubers stored in different packaging materials was lowest by the end of the storage period when stored in polypropylene and ldpe. vertical bars show ±se of means (n-3). interaction between storage intervals and packaging materials was significantly different at p ≤ 0.05. changes in potato tuber quality under different packaging systems 147 more rapid progress toward dormancy break at the end of the storage period, similar to results of fauconnier et al. (2002). ascorbic acid is the predominant vitamin in potato tuber and of significant importance in the human diet. depletion of ascorbic acid has been implicated with reduced nutritional quality; therefore their assured stability during storage has been a major concern of the postharvest technologists. hagg et al. (1998) reported that aa content significantly decreases during storage of potato tuber. the reduction is ascribed to the oxidation of ascorbic acid into dehydro ascorbic acid and afterward to diketo-gluconic acid. being a water-soluble vitamin and susceptible to oxidation, aa contents rapidly decrease with increasing rates of respiration and water loss in storage. the present study revealed continuous reduction in aa content in all treatments, but especially then on-packaged controls. our application of certain packaging materials such as polypropylene and polyethylene could be shown to significantly reduce the rate of water loss from potato, and this was apparently associated with less oxidation of ascorbic acid compared to the controls and other treatments. the efficacy of modified atmosphere packaging in retention of high aa contents has likewise been described in tomato by sammi and masud, (2007). glycoalkaloids contents in potato tuber are attributed to their medicinal and toxicological properties. low quantities (below 15 mg 100 g-1 fresh weight) impart flavor and functional value, while high quantities (above 20 mg 100 g-1 fresh weight) can impart bitter taste and even may even cause death at excessive intake (28 mg 100 g-1 fresh weight) (mensinga et al., 2005). nema et al. (2008) reported that total glycoalkaloids (tga) contents increased during the storage under different packaging systems. he proposed that color, type, and permeability of packaging material effect tga formation during storage. similar observations regarding the effect of different packaging materials on tga contents have been documented by rosenfeld et al. (1995). in addition, potato tubers produced high level of tga in the tubers close to sprouting stage, which confirmed the previous findings of sengul et al. (2004). large amounts of phenolic compounds can cause tuber discoloration, as phenolics act as a substrate in potato browning mediated through the activities of polyphenol oxidase (ppo) in melanin formation (anthon and barrett, 2002). however, a high amount of phenolics during storage is attributed to low ppo and high antioxidant activity in potatoes (lachman et al., 2008). previous studies have revealed that the phenolics content in potato continued to increase during storage period until the onset of ppo activity (madiwale et al., 2011). the presence of ample molecular oxygen in control likely caused a significant decline in total phenolics (tpc) as compared to other potatoes stored in different packaging. our results indicated that packaging materials in general and ldpe and polypropylene packaging in particular curtailed the decline in tpc relative to control, similar as reported by gonzalez et al. (2004). fruits and vegetables, owing to their rich vitamins and poly phenolic contents, possess significant radical scavenging activity that corresponds to their antioxidant potential (kondo et al., 2004). these antioxidants have the capacity to quench free radicals (peroxides, super oxides, hydroxyl radicals) thus, protect the cellular structures and proteins from membrane peroxidation and degeneration (ding et al., 2002). in the present investigation, our observation of an initial increase in radical scavenging activity (rsa) might be due to the increase in total phenolic contents observed early in the storage period, as was also reported by (padda and picha, 2008). moreover, minimum loss in rsa during the last three weeks of storage and was associated with all packaged potatoes. potentially, this is due to the regeneration of antioxidant compounds like ascorbic acids in potato to counter balance the increased free radicals produced during senescence. packaged potato expressed higher antioxidant activity as compare to the non-packaged controls, which might be explained by greater retention of ascorbic acids, phenolics and other functional compounds. this improved response at later stages of storage in packaging was similar to previous reports of horticultural commodity packaging reported by sonia and chavez (2006) and ding et al., (2002). polyphenol oxidase activity increases in potato due to the availability of substrate and its subsequent oxidation during storage. the non-significant changes during the 1st month storage in most of the treatments might be due to absence of physical damages and appropriate curing which was also observed by nourian et al. (2003). prominent increase in the ppo activity in different treatments after the 1st month till the end of the storage period might be associated with an increased substrate (polyphenol) oxidation. however, packaging materials conferred barrier properties to substrate oxidation resulted in low eventual activity as compare to control. in addition, increased ppo activity in potato during postharvest storage has also been implicated with increased sprouting percentage and accelerated senescence (abbasi et al., 2015). in the present study, all the packaging materials effectively maintained modified atmospheric conditions around potato better than the non-packaged control. this resulted in lower moisture loss and limiting oxygen availability for the polyphenol oxidations. the variation in ppo activity within different packaging materials might be attributed to the difference in their oxygen permeability, which was also observed by rakotonirainy et al. (2001). kader (2002) also reported substrate inhibition for ppo enzymes under modified atmosphere packaging, which led to low ppo activity during storage just as we observed in the present investigation. enzymatic browning in potato may cause substantial loss by deteriorating nutritional and sensorial attributes, and this is primarily associated with the activities of peroxidase and polyphenol oxidase enzymes during storage (loaiza and saltveit, 2001). peroxidase (pod), being thermally stable and omnipresent in all part of the plant, has a wider range of substrate-based activity than ppo (anthon and barrett, 2002). increased pod activity is associated with oxidation of phenolic compounds under physiological stress leading to decay and loss of quality during storage (ding et al., 2002). in addition, pod can also degrade natural antioxidants and liberate damaging free radicals (rojas et al., 2008). both these processes, mediated through peroxidase activity, accelerate potato browning and affect the ultimate postharvest storage life. aydin and kadioglu (2001) reported increased pod activity in fruits and vegetables under stress conditions, and with progression in the physiological stages from ripening through senescence, just as we observe in the present study as affected by packaging material by the end of storage period. our results showed that different packaging materials could maintain low pod activity in potato tuber as compare with non-packaged controls at ambient temperature storage, presumably due to lower available oxygen required for the oxidation of phenols and peroxides. the increased pod activity by the end of the storage period, particularly in the non-packaged controls, might be attributed to the physiological stress associated with senescence and sprouting in these tubers, as previously reported by afify et al. (2012) and abbasi et al. (2015). conclusion this study revealed that storage life in potato tubers was significantly affected by different packaging materials. in general, weight loss, total soluble solids, glucose, total sugars, glycoalkaloids, polyphenol oxidase, and peroxidase increased during the storage period. ascorbic acids decreased with the increase in storage time. total phenolic contents and radical scavenging activity of the stored tubers increased initially and then declined later in the storage period. amongst the different packaging materials studied here, the potato tubers 148 k.s. abbasi, t. masud, a. qayyum, s.u. khan, a. ahmad, a. mehmood, a. farid, m.a. jenks stored in polypropylene and low density polyethylene packaging showed best overall retention of vital quality attributes during 63rd day’s storage. however, tensile strength of polypropylene packaging made it advantageous for prolonged potato tuber storage, which helps prevent potential losses during transit operations and shipping 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(eds.), potato biology and biotechnology. amsterdam: elsevier science b.v. tester, r.f., ansell, r., snape, c.e., yusuph, m., 2005: effect of storage temperatures and annealing conditions on the structure and properties of potato (solanum tuberosum) starch. int. j. biol. macromol. 36, 1-8. yemenicioglu, a., 2002: control of polyphenol oxidase in whole potatoes by low temperature blanching. eur. food res. technol. 214, 313-319. address of the corresponding author: e-mail: aqayyum@uoh.edu.pk © the author(s) 2016. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 90, 306 314 (2017), doi:10.5073/jabfq.2017.090.038 1faculty of engineering, chemical and bioprocess engineering department 2faculty of agriculture and forestry, plant science department 3faculty of chemistry, inorganic chemistry department, pontificia universidad católica de chile, macul, chile relation between composition, antioxidant and antibacterial activities and botanical origin of multi-floral bee pollen p. velásquez1, k. rodríguez2, m. retamal3, a. giordano3, l.m. valenzuela1, g. montenegro2* (received february 9, 2017; accepted march 24, 2017) * corresponding author summary harvested bee pollen is valuable for its nutritional value and healthy properties. this work relates the botanical origin of sixteen bee pollens from chile with their phenolic, protein and carotenoid content, and antioxidant/antibacterial activities. our results showed that the chemical properties of different bee pollens are associated with the plant’ species from which each one was derived from. some correlations between chemical properties and botanical origin were observed. bee pollen showed between 20.0-30.4% protein, 2.8-50.2 mg/kg carotenoids, 22.8-918.4 mg/kg phenolics, and 4.51-91.19 mmol fe+2/kg pollen. antibacterial activity was observed against all bacteria assayed even surpassing the activity of traditional antibiotics. brassica sp. and galega officinalis are an abundant source of antioxidants and antibacterial compounds. other species such as those derived from fruit and endemic plants from chile, although they occur less frequently, are also good source of these compounds. some correlations between botanical origin and chemical, antioxidant and antibacterial properties were observed. knowing the influence of plant species over the antioxidant or antibacterial properties of bee pollen, will allow selecting the best location for honeycombs and will allow beekeepers to differentiate and add value to their products. keywords: brassica sp., galega officinalis, trevoa quinquenervia, prunus sp., medicago sp., frap, diameter of inhibition, hplcdad, protein, carotenoid. introduction bee pollen corresponds to microspores of spermatophytes and entomophilous plants with flowers collected and transported by bees in their last pair of legs as granules or pollen-loads. once in the hive, bee adds salivary enzymes (e.g., amylase, catalase) to the pollenloads and reserved them as nutrient resource for honeycomb. bee pollen is valuable for its nutritional value and healthful properties and is considered by many beekeepers as a mean of diversifying and increasing their income. bee pollen products are valuable for their nutritional value and healthful properties. as a nutrient, bee pollen provides to the human diet protein, fat and other components in lesser amount. bee pollen presents all essential amino acids to the human diet and its content varies between 10 and 40% (bogdanov, 2014). dry bee pollen presents an average protein content about 23.8% (almeida-muradian et al., 2005). fatty acids are 3%, from which about half of them are oleic (omega-3), linoleic (omega-6) and linolenic acids (omega-3) (bogdanov, 2014). carbohydrates in bee pollen are mainly polysaccharides such as starch and sugars, and represent between 13 and 55 g/100 g of sample. with respect to healthful characteristics, bee pollen has been described as anti-anemic, tonic and restorative, hormone regulator, intestinal regulator, vasoprotector, hepatoprotective, anti-atheroscleorotic agent, antiallergic, anticarcinogenic, antioxidant, antibacterial and as antifungal (denisow and denisowpietrzyk, 2016; graikou et al., 2011). phenolic acids, flavonoids and pigments as β-carotene, are mainly related to the healthy properties exhibited by bee pollen such as antioxidant and antibacterial (aloisi and ruppel, 2014; alicic et al., 2014). phenolic acids and flavonoid glycosides are present in the nectar of flowers visited by bees, which are hydrolyzed and transferred to the bee pollen. the number and variety of phenolic acids and flavonoids are highly variable, since beekeepers mix bee pollen from different botanical origins (morais et al., 2011; leja et al., 2007). the main group of pigments that compose bee pollen corresponds to carotenoids, especially β-carotene (17% of all carotenoids), whose concentration also depends on the botanical origin of the pollen (almeida-muradian et al., 2005). the type and concentration of the polyphenolic compounds influence the antibacterial and antioxidant activity exhibited by bee pollen. the most important polyphenolic compounds related with these activities are vanillic acid, protocatechuic acid, gallic acid, p-coumaric acid, hesperidin, rutin, kaempferol, apigenin, luteolin, quercetin, and isorhamnetin (alicic et al., 2014). these compounds also serve as biochemical markers of bee pollen (tomás-barberán et al., 1989) related with the botanical origin. bee pollen rich in these compounds has shown activity against specific pathogens such as staphylococcus aureus (cabrera and montenegro, 2013), escherichia coli (libonatti et al., 2014; cabrera and monte negro, 2013), streptococcus viridians (campos et al., 2010; bisno and stevens, 1996), and pseudomonas aeruginosa (abouda et al., 2011; carpes et al., 2007). in order to establish relatioships between the botanical origin of multifloral chilean bee pollen and their phenolic, protein and carotenoid content, and antioxidant/antibacterial activities, we present a characterization by hplc-dad of the phenolic compounds present in their extracts, their botanical origin, and a quantification of their total carotenoid and protein content. in vitro antioxidant and anti bacterial activities were evaluated by ferric reducing antioxidant power (frap) and determining the zone of inhibition against e. coli, s. aureus, p. aeruginosa, and s. pyogenes, respectively. cha racterization on antioxidant and antibacterial properties present in samples of multiflora bee pollen will allow the beekeeping sector to add value to this product. materials and methods bee pollen sixteen samples of commercial bee pollen were purchased from local beekeepers of central chile between december 2013 and february 2014. samples were lyophilized and stored at -20 ºc. the determination of botanical origin was performed using palynological analysis method described at chilean regulation (nch3255, 2011). multi-floral bee pollen properties 307 five grams of each type of bee pollen corbiculae were separated by color and each fraction was weighed. then one corbiculae of each kind of bee pollen sample was crushed with alcohol to disperse the pollen grains. several drops of red calberla were used to stain the grains allowing their observation under light microscope. to determine the botanical origin specific literature (marticorena and quezada, 1985; heusser, 1971) and the botanical palinoteca of botanical laboratory at pontificia universidad católica de chile were consulted. total protein content protein determination was performed by kjeldahl method based on standard aoac (1984). one gram of sample was weighted and homogenized. in the digestion step organic nitrogen in the sample was decomposed by a solution of concentrated sulfuric acid, sodium sulfate, cuprum dioxide and applying a temperature cycle: 120 ºc for 15 min, 200 ºc for 2 min, 300 ºc for 2 min, and 402 ºc for 40 min, on a dk 6 kjeldahl digestion unit (velp scientifica). sodium hydro xide was added and distillation on 3% boric acid was performed using a udk 129 kjeldahl distillation unit (velp scientifica). titration was performed with 0.1 m hydrochloric acid. conversion factor used was 6.25. total carotenoid content carotenoid extraction was performed weighting 4 g of bee pollen, milled and sonicated in an ultrasonic bath for 15 minutes with 20 ml of petroleum ether-acetone mixture (1:1 v/v). the extract was transferred to a separator funnel and washed with 60 ml of distilled water. the aqueous phase was discarded and the organic portion was passed through 2 g of anhydrous sodium sulfate. the whole process was repeated until the sample showed no coloration. finally, the extract was evaporated to dryness under a stream of nitrogen, reconstituted in 2 ml of butanol and quantified by hplc-dad at 440 and 480 nm. bee pollen extracts bee pollen extraction process was based and adapted from leblanc et al. (2009). ten grams of multiflora bee pollen were mixed with 10 ml of distilled water and ultrasonicated for one hour. the mixture was centrifuged at 6000 rpm for 20 minutes and the supernatant was stored at 4 °c in darkness. this process was repeated 5 times. the collected supernatants were combined and filtered using qualitative paper (whatman no. 2). finally they were evaporated (rotary evaporator buchi r-210) and the dry extract was reconstituted with 10 ml of ultrapure water, filtered (edlab ca syringe filter 0.45 mm) and stored at -20 ºc. total phenolic compounds colorimetric determination of the phenolic content was evaluated by the folin-ciocalteu reaction (fc). assays were performed on bee pollen extracts. the absorbance at 765 nm of the mixture of 200 μl extract, 50 μl folin-ciocalteu reagent, 150 ml of 20% w/v sodium carbonate solution (na2co3) and 600 ml of ultrapure water was measured in triplicate after 30 minutes of reaction. a calibration curve was constructed with gallic acid concentrations between 10 and 50 mg/ml. antioxidant activity the antioxidant activity was determined by the ferric reducing antioxidant power (frap) assay. 200 μl of pollen extract w mixed with 1.8 ml of frap reagent, after 15 minutes in the dark the absorbance was measured at 593 nm. frap reagent was prepared as follow: 25 ml of acetate buffer, 2.5 ml tptz solution (10 mmol/l of tptz in hcl 40 mmol/l) and 2.5 ml of 20 mm fecl3 · 6h2o). a calibration curve was calculated with known solutions of feso4 · 7h2o in a concentration range of 20 and 100 μg/ml. in order to compare the antioxidant power of these samples, this test was performed to a blueberry sample, which is a recognized natural source of antioxidant compounds. identification and quantification of flavonoids and phenolic acids the identification and quantification of flavonoids and phenolic acids on bee pollen extracts were performed by high performance liquid chromatography with a diode array detector based on benzie and strain (1996). elite merck lachrom hplc hitachi was used in a reverse phase column (lichrocart rp-18) with a mobile phase of aqueous formic acid 5% (v/v) and methanol at constant solvent flow of 1 ml/min at 30 °c. samples were injected manually. chromatograms were monitored at 290 and 340 nm. a calibration curve was made with high purity standards and area of peaks found with the ezchrom elite v.3.3.1 (scientific software inc. 1988-2005; agilent 2005-2008) program. antibacterial activity the antibacterial activity of bee pollen extracts was evaluated by diameter of inhibition against escherichia coli atcc-25922, staphylococcus aureus atcc-25923, pseudomonas aeruginosa atcc 27853 and streptococcus pyogenes i.s.p. 364-00 (supplied by chilean public health institute). diameter of inhibition was determined using the standard reported by clsi (2006): bacterial strains were inoculated on mueller hinton agar for 24 hours at 37 ºc. after that time, colonies were selected and diluted in saline solution to a concentration of 10-3 ufc by visual comparison with a standard of 0.5 mcfarland (1.5×108, becton & dickinsson company, usa). once strains were swab on the agar, 6 mm diameter holes were made, and 100 μl of each extract were deposited in each hole. petri dishes were incubated between 18 to 24 hours at 37 °c until measurements. the inhibition diameter that appeared around each hole was measured. tetracycline, ampicillin and chloramphenicol were used as controls. statistical analysis statistical analysis of the results was performed using a one-way variance analysis (anova) followed by tukey hsd method with 95% (p<0.05) level of confidence and computed by statgraphics centurion xv software 15.02.05. samples were analyzed in triplicate. correlations between results were made using the pearson’s correlation coefficient (r) (p<0.05). results and discussion the botanical origin described the presence of different plant sources used by bees to produce the bee pollen. this description permitted to classify them as native/non-native/mixed and unifloral/bifloral/ multiflora bee pollen (nch 3255, 2011) (tab. 1). floral species found in the samples are closely related with the geographic location of hives. the analyzed samples of bee pollen were predominantly derived from non-native floral species and frequently from only one of them. the majority of samples analyzed corresponded to non-native unifloral (nine), followed by non-native multifloral, mixed multifloral, and non-native bifloral (two samples of each), and native unifloral (one). among the samples analyzed, galega officinalis predominated in thirteen samples and brassica sp. was present in eleven samples. brassica sp. accounted for 34% of the average weight of each sample, 308 p. velásquez, k. rodríguez, m. retamal, a. giordano, l.m. valenzuela, g. montenegro while galega officinalis accounted for 40%. this indicates that brassica sp. and galega officinalis are important sources of pollen collection. the asteraceae family was the least frequently detected in the samples analyzed, comprising only 2% of the average sample weight when present. total protein content total protein of the samples ranged between 20.0 and 30.4%, with an average of 25.4% (fig. 1). the values observed are similar to the amounts reported in literature (almeida-muradian et al., 2005; bogdanov, 2014; balkanska and ignatova, 2012). this result tab. 1: botanical origin and classification of samples. data represent analyses of 300 pollen grains counted in 5 distinct optical areas in three different samples. sample predominant pollen secondary pollen important minor pollen minor pollen classification (>45%) (16 45%) (3 15%) (<3%) specie % specie % specie % specie % 1 brassica sp. 57.8 galega officinalis 42.2 non-native unifloral 2 eschscholzia cali34.8 medicago sativa 12.2 apiaceae 2.0 non-native fornica schinus sp. 10.2 fungal spores 2.0 bifloral brassica sp. 30.6 olea europaea 8.2 3 schinus sp. 37.5 mutisia sp. 2.5 mixed brassica sp. 22.9 medicago poly0.4 multifloral morpha medicago sativa 0.4 4 galega officinalis 50.0 asteraceae 26.0 non-native raphanus sp. 24.0 unifloral 5 medicago sativa 58.0 galega officinalis 39.6 hypochaeris/ 2.4 non-native taraxacum unifloral 6 brassica sp. 36.5 galega officinalis 15.6 non-native medicago poly27.1 multiflora morpha convolvulus sp. 20.8 7 brassica sp. 51.0 adesmia sp. 2.0 non-native galega officinalis 46.9 unifloral 8 prunus sp. 52.2 maytenus boaria 2.2 native trevoa 45.7 unifloral quinquenervia 9 brassica sp. 58.3 sonchus sp. 22.9 oxalis sp. 12.5 asteraceae 2.1 non-native papilonaceae 4.2 unifloral 10 brassica sp. 32.7 medicago sativa 8.2 trifolium sp. 2.0 non-native galega officinalis 26.5 hypochaeris/ 8.2 asteraceae 2.0 multifloral dysopsis sp. 20.4 taraxacum 11 brassica sp. 72.9 actinidia deliciosa 12.5 galega officinalis 2.1 non-native fabaceae 4.2 asteraceae 2.1 unifloral hypochaeris/ 2.1 taraxacum quillaja saponaria 2.1 vicia sp. 2.1 12 galega officinalis 74.5 brassica sp. 19.6 fern spores 2.0 non-native trifolium repens 2.0 unifloral fabaceae 2.0 13 convolvulus sp. 43.2 chenopodiaceae 2.3 non-native convolvulus sp. 38.6 bifloral brassica sp. 16.0 14 chenopodiaceae 58.0 convolvulus sp. 24.0 brassica sp. 12.0 chenopodiaceae 2.0 non-native clarkia tenella 4.0 unifloral 15 cactaceae 51.2 galega officinalis 34.1 amaranthaceae 12.2 tecophilaceae 2.4 mixed multifloral 16 medicago sp. 62.5 asteraceae 10.4 non-native mirtaceae 10.4 unifloral galega officinalis 8.3 malvaceae 8.3 multi-floral bee pollen properties 309 fig. 1: total protein and carotenoid content of samples (mean ± sd; n = 3). in each column different letters imply significant differences (p<0.05). confirms that bee pollen could be a good source of vegetable protein replacing dietary animal sources such as meat (20% protein content, schmidt et al., 1985), that currently are highly criticized for causing or increasing the likelihood of developing diseases (who, 2015; bernstein et al., 2010). bee pollen is even a better vegetable protein than quinoa (12-23% protein content, james, 2009). the samples composed by prunus sp. 52.2% / trevoa quinquenervia 45.7% (sample 8) and by medicago sativa 58.0% / galega officinalis 39.6% (sample 5) have the highest protein contents. these results are in agreement with vanderplanck et al. (2014), whom reported 25.8% of protein in prunus sp. bee pollen. there is not reported protein content of bee pollen from trevoa quinquenervia. andrada and tellería (2005) reported that medicago sativa has 22% of protein content and according to peiretti and gai (2006) galega officinalis has 20%. moreover, samples such as eschscholzia californica 34.8% / brassica sp. 30.6% / medicago sativa 12.2% / schinus sp. 10.2% / olea europaea 8.2% (sample 2), and brassica sp. 58.3% / sonchus sp. 22.9% / oxalis sp. 12.5% (sample 9) samples have poorest content. these results are also expected since forcone et al. (2013) reported 21.1% of protein content in bee pollen from eschscholzia californica. regarding the correlation of the protein content with the botanical origin, no relation was found with a confidence level of 95% (p <0.05). this indicates that the protein content is not dependent on any particular species. it is also observed that the bee pollen samples did not present significant difference in protein content between them (p <0.05). total carotenoid content carotenoids were observed in nine bee pollen samples, which ranged between 2.8 and 50.2 mg/kg of pollen with 12.0 mg/kg of pollen in average (fig. 1). the values obtained in our samples were lowers than those ranged between 10 200 mg/kg reported in the literature from other multiflora bee pollen samples (almeida-muradian, 2005; mărgăoan et al., 2010). this difference can be explained by the wide difference in carotenoid content between genus, families and species. significant differences (p<0.05) were observed in total carotenoid content, where the samples of galega officinalis 50% / asteraceae 26.0% / raphanus sp. 24.0% (sample 4) and brassica sp. 57.8% / galega officinalis 42.2% (sample 1) presented the highest carotenoid content. meanwhile the sample composed by cactaceae 51.2% / galega officinalis 34.1% / amaranthaceae 12.2% / tecophilaceae 2.4% (sample 15) showed the lowest content that indicates that bee pollen from these species are poor as carotenoid sources. it is possible that the high content present in sample 4 was due to the presence of bee pollen from galega officinalis since bee pollen from brassica sp has been reported as very poor in carotenoids (stanciu et al., 2016) but also exists a high variation in carotenoid content inside species that compose this genus (jahangir et al., 2009). oliveira et al. (2009) and bobis (2014), has been reported that asteraceae and raphanus sp. are sources of high carotenoid content. there is no information about carotenoid contents of bee pollen from cactaceae, amaranthaceae and tecophilaeaceae families but probably have lower contents. a positive correlation was found between carotenoid content and asteraceae (r=0.92; n=5; samples 4, 9, 10, 11, 16) and raphanus sp. (r=0.95; n=1; sample 4). a moderate interdependence between carotenoid content and galega officinalis was observed (r=0.45; n=13; samples 1, 3, 4, 5, 6, 7, 10, 11, 12, 13, 14, 15, and 16). however, it is not possible to affirm that the presence of these species really correlated with the carotenoid content since there is no information about these parameters in samples of bee pollen from these species. identification and quantification of flavonoids and phenolic acids six phenolic acids (p-coumaric, chlorogenic/caffeic, ferulic, sinapic, and cinnamic acids) and two flavonoids (kaempferol, luteolin) were identified by liquid chromatography with diode array (tab. 2). p-coumaric acid was presented in all samples and chlorogenic/ caffeic and ferulic acids were presented in ten samples. the most frequent flavonoid was kaempferol and the least frequent was luteolin. kaempferol was presented in six samples and luteolin in only one. samples with the highest polyphenolics concentrations (i.e., phenolic acid +flavonoids) were medicago sp. 62.5% (sample 16, 918.42 mg/kg) and brassica sp. 51.0% / galega officinalis 46.9% (sample 7, 638.63 mg/kg). these results differ from phenolic acids and flavonoids concentration previously reported for similar botanical origin bee pollen. the presence of kaempferol in bee pollen derived from brassica sp. has been previously reported (fatrcová-šramková et al., 2013). it has been previously observed that bee pollen from brassica napus subsp. napus l. contains luteolin (fatrcová-šramková et al., 2013). however, only one of the samples in this study that contain this botanical origin (brassica sp. 51.0% / galega officinalis 46.9% / adesmia sp. 2%) presents luteolin, with a concentration 10 times higher than that reported by fatrcová-šramková et al. (2013). apigenin, a common flavonoid present in bee pollen with a biological activity was not found in any sample. this variability in bee pollen 310 p. velásquez, k. rodríguez, m. retamal, a. giordano, l.m. valenzuela, g. montenegro is derived by the variability of phenolic compounds produced by plants, which depends on the stress conditions, geographic location and vegetation around the apiaries, which conditioned the flowering (morais et al., 2011; leja et al., 2007). regarding the correlation between phenolic acids/flavonoids and botanical origin several dependences were observed. medicago showed a positive correlation with p-coumaric acid (r=0.67; n=16) that indicates high concentrations of this compound in samples that include it (i.e., samples 2, 3, 5, 6, 10, and 16). bee pollen from eschscholzia californica and olea europaea has a positive correlation with cinnamic acid content (r= 0.54, 0.54, respectively) that is in agreement with high content of this compound at samples with these kind of bee pollen (i.e., sample 2). samples with bee pollen from malvaceae has a high correlation with chlorogenic/caffeic acid and p-coumaric acid (r=0.67, 0.87, respectively) that indicates a high content of this compounds at samples that have bee pollen from these species (i.e., sample 16). samples with mirtaceae bee pollen also have a high correlation with chlorogenic/caffeic acid and p-coumaric acid (r=0.67, 0.87, respectively) that is present in sample 16. bee pollen from sonchus sp., oxalis sp and papilonaceae have a high correlation with kaempferol (r=0.97, 0.97, 0.97, respectively; n=16) that is in agreement with the high content of kaempferol at sample 9. sample 10, composed by bee pollen from dysopsis sp. has a positive correlation with ferulic and sinapic acid (r=0.57, 0.52) that indicates a high content of this compound. finally, bee pollen from actinidia deliciosa has a positive correlation with sinapic acid (r=0.68) that is in agree with the higher content at sample 11. determination of total phenolic content and ferric reducing antioxidant power (frap) total phenolic content and ferric reducing antioxidant power (frap) for bee pollen samples are showed in fig. 2. the total phenolic con tent of samples ranged between 6.86 and 52.99 g gae/kg of pollen, with an average value of 12.64 g gae/kg of pollen. these contents are higher than other reports such as values published by pascoal et al. (2014) and feás et al. (2012) which showed ranges between 4.96 and 19.80 g gae/kg, respectively. the sample of prunus sp. 52.2% /trevoa quinquenervia 45.7% / maytenus boaria 2.2% (sample 8) and brassica sp. 58.3% / sonchus sp. 22.9% / oxalis sp. 12.5% (sample 9) have the highest values (33.34 and 52.99 g gae/ kg, respectively). mărgăoan et al. (2013) and stanciu et al. (2016) reported a content of 8.87g gae/kg and 7.57g gae/kg on average respectively for bee pollen from prunus sp. in addition, stanciu et al. (2016) reports that the content of bee pollen from brassica sp. is 11.62 g gae/kg and 5.46 g gae/kg for oxalis sp. bee pollen. all values being higher than the bee pollen of another species. as for the phenolic content of bee pollen from sonchus sp., trevoa quinquenervia and maytenus boaria, there are no reports, however they may have a high content considering the total content of the samples. the high phenolic value of brassica sp. 58.3% / sonchus sp. 22.9% / oxalis sp. 12.5% / papilonaceae 4.2% / asteraceae 2.1% (sample 9) may be due to the high content reported at tab. 2. brassica sp. has been reported with a high content of kaempferol associated with its antioxidant activity (fatrcová-šramková et al., 2013; li et al., 2016). there are no reports of the presence of kaempferol in bee pollen of the other species present in the sample 8 and 9. frap values ranged between 4.51 and 91.19 mmol fe+2/kg pollen, with an average of 35.95 mmol fe+2/kg pollen (fig. 3). the sample of prunus sp. 52.2% / trevoa quinquenervia 45.7% / maytenus boaria 2.2% (sample 8) has the highest frap value with 91.19 mmol fe+2/ kg pollen. this value was higher than the frap value of blueberry, a very well known natural antioxidant (between 58.99 and 63.41 fe+2 mmol/kg). however, compared to the frap values found in literature (5.36 mm fe+2/g, marghitas et al., 2009 and 21 mm eq. fe+2/g, montenegro et al., 2013), our results are much smaller (less tab. 2: hplc-dad profile of bee pollen samples evaluated (mean ± sd; n = 3). in each column different letters imply significant differences (p<0.05). nd: non-detected (under detection threshold). sample chlorogenic + ferulic acid sinapic acid p-coumaric acid cinnamic acid kaempferol luteolin caffeic acid (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) tr (min) 6.90 + 7.06 11.74 9.09 8.42 11.74 12.67 11.96 λ(nm) 340 290 340 290 340 340 340 1 18.16 ± 0.73ab nd nd 4.02 ± 0.16a nd nd nd 2 18.07 ± 0.72ab 5.66 ± 0.23b nd 92.01 ± 3.68bc 8.93 ± 0.36d nd nd 3 nd nd nd 287.37±11.50g nd nd nd 4 nd nd nd 109.91 ± 4.40c nd nd nd 5 nd nd nd 458.52 ±18.34i nd nd nd 6 nd nd nd 337.05±13.48h nd nd nd 7 nd 9.97 ± 0.40d nd 255.17±10.21ef nd 57.49 ± 2.29c 316.00±2.64a 8 26.94 ± 1.08bc nd nd 71.55 ± 2.86b nd nd nd 9 nd 13.35±0.53e nd 4.02 ± 0.16a nd 344.20±13.76d nd 10 29.79 ± 1.19c 26.33 ±1.05h 72.95±2.92a 286.21±11.45fg nd 17.11 ± 0.68b nd 11 111.60± 4.46f 22.61±0.90g 89.67±3.59b 169.60 ± 6.78d nd 63.68 ± 2.55c nd 12 43.37 ± 1.73d 4.09 ± 0.16a 22.28±0.89c 82.19 ± 3.29bc nd nd nd 13 86.11 ± 3.44e 5.48 ± 0.22b nd 73.28 ± 2.93b 7.30 ± 0.29b 5.33 ± 0.21a nd 14 45.16 ± 1.81d 7.50 ± 0.30c 9.12 ± 0.36d 191.91 ± 7.68d 6.49 ± 0.26a nd nd 15 11.29 ± 0.45a 6.72 ±0.27bc 51.84±2.07e 243.21 ± 9.73e nd 19.23 ± 0.77b nd 16 258.92±10.36g 20.58±0.82f nd 630.92 ±25.24j 8.00 ± 0.32c nd nd multi-floral bee pollen properties 311 fig. 3: antibacterial activity showed by bee pollen extracts against streptococcus pyogenes, staphylococcus aureus, pseudomonas aeruginosa and escherichia coli. dashed line indicate diameter of hole with bee pollen extracts (mean ± sd; n = 4). fig. 2: total phenolic content and antioxidant activity of samples (mean ± sd; n = 3). than 0.1 mmol fe+2/g). this may be due to the fact that our extracts were obtained using water instead of ethanol or methanol (do et al., 2014). as expected from similar studies (ulusoy and kolayli, 2014; borycka et al., 2016), there is a positive correlation coefficient between total phenolic content and frap: pearson’s correlation coefficient of 0.59 (fig. 3). thus, the antioxidant power showed by bee pollen samples can be attributed to their phenolic content. a positive correlation was also found between total phenolic content and prunus sp. (n=16; r=0.50), trevoa quinquenervia (n=16; r=0.50), oxalis sp. (n=16; r=0.79), papilonaceae (n=16; r=0.79) and sonchus sp. (n=16; r=0.79), and between frap with prunus sp. (n=16; r=0.76) and trevoa quinquenervia (n=16; r=0.76). furthermore a high dependence between total phenolic content and kaempferol (n=16; r=0.78) was observed. these results indicate that phenolic content of samples that includes bee pollen from prunus sp., trevoa quinquenervia or sonchus sp. are result of kaempferol content. antibacterial activity antibacterial assays showed that bee pollen is more active against gram-positive (i.e., s. aureus and s. pyogenes) than gram-negative bacteria (i.e., p. aeruginosa and e. coli) (fig. 3). fifteen samples inhibited s. pyogenes, fifteen samples inhibited s. aureus, five samples inhibited p. aeruginosa and only one showed inhibition against e. coli. these results show that gram-positive bacteria are better controlled by the bee pollen than gram-negative bacteria, which showed more resistance. this tendency was also reported in another study from our research group (cabrera and montenegro, 2013). gram-negative bacteria have a double cell wall, composed by lipopolysaccharides and proteins, which hinders the antibacterial action of bee pollen thus e. coli and p. aeruginosa because are more resistant therefore were less controlled (tafur et al., 2008). fifteen out of sixteen samples showed control against streptococcus pyogenes. the range of inhibition was observed between 9.3 and 31.3 mm, similar to the range of between 9 and 28 mm reported in the literature (abouda et al., 2011; cabrera and montenegro, 2013). the highest diameter of inhibition was observed in sample 13 (31.3 mm), higher than tetracycline and closer to ampicillin. this sample was composed by convolvulus arvensis 43.2% / galega officinalis 38.6% / brassica sp. 16%. there are no reports indica ting that galega officinalis and convolvulus arvensis bee pollen have antibacterial activity against s. pyogenes. cabrera and montenegro (2013) report one sample that contains 30% of brassica sp. bee pollen which controlled s. pyogenes. there is a positive correlation between chenopodiaceae (r=0.65; 312 p. velásquez, k. rodríguez, m. retamal, a. giordano, l.m. valenzuela, g. montenegro n=16) and convolvulus sp. (r=0.58; n=16) with the inhibition activity against s. pyogenes, and a negative correlation with eschscholzia californica (r=-0.58; n=16) and olea europaea (r=-0.57; n=16). these correlations would indicate that some species enhance the antibacterial activity of bee pollen while others decrease it when are present. these species could contain phenolic/flavonoid compounds that would not necessarily be poor antibacterials since they could compete for sites of action with phenolic/flavonoid compounds from other species or results in antagonistic effects (kumar and pandey, 2013; mandalari et al., 2010; palafox-carlos et al., 2012). it is also possible that samples containing bee pollen from chenopodiaceae and convolvulus sp. contain compounds that inhibit the bacterial growth of s. pyogenes, however do not exist in the literature reports on this. since eschscholzia californica, olea europaea and chenopodiaceae correlates with cinnamic acid (r=0.54, 0.54 and 0.56, respectively) it suggest a relation with antibacterial exerted against s. pyogenes. however 15 samples inhibited s. pyogenes and only 4 samples have cinnamic acid. therefore no correlation between botanical origin and antibacterial activity against s. pyogenes. fifteen out of sixteen samples showed growth inhibition against staphylococcus aureus. sample 5 showed the highest inhibition diameter with 18.7 mm, similar to half of the ampicillin and tetracycline diameters. medicago sativa (58.0%) and galega offici nalis (39.6%) predominate in sample 5. there is no data indicating that the bee pollen obtained from these species has antibacterial activity. however, there are many studies indicating that some of these plants have antibacterial activity against s. aureus, which has been attributed to certain flavonoids, saponins and peptides (rodrigues et al., 2013; karakas et al., 2012; erturk, 2010). there is a positive correlation between antibacterial activity of bee pollen samples against s. aureus and medicago sativa (n=16; r=0.50) and a negative correlation with malvaceae (n=16; r=-0.72), medicago sp. (n=16; r=-0.81) and mirtaceae (n=16; r=-0.81). the antibacterial activity in this case is mainly a result of chlorogenic/ caffeic and p-coumaric acids since correlation were found between these compound and bee pollen from these species (see section above: identification and quantification of flavonoids and phenolic acids). the samples with a higher concentration of p-coumaric acid than chlorogenic/caffeic acids (i.e., samples 2, 3, 5, 10, 16) showed a higher antibacterial activity. the bacterial growth inhibition exerted by bee pollen extracts against p. aeruginosa was less effective than the other bacteria assayed. only 5 out of sixteen samples showed positive results. the sample composed by medicago sativa 58.0% / galega officinalis 39.6% / hypochaeris-taraxacum 2.4% (sample 5) showed the highest diameter of inhibition (11.3 mm). this result was similar to tetracycline and higher than ampicillin that did not have inhibition against p. aeruginosa. the inhibition diameters observed were found to be similar to that reported by abouda et al. (2011). there is a positive correlation between antibacterial inhibition showed against p. aeruginosa and hypochaeris-taraxacum sp. (r=0.52; n=16). since a moderate dependence were found between hypochaeristaraxacum sp. and ferulic and sinapic acids, it suggests a relation of these compounds with antibacterial effect exerted against s. pyogenes. however, only two out of five samples that inhibited s. pyogenes have ferulic and sinapic acids in their composition. therefore no correlation between botanical origin and antibacterial activity against p. aeruginosa. escherichia coli was controlled only by one sample (sample 12) that formed an inhibitory diameter of 10.0 mm. however, zones of inhibition were reported in the literature ranging from 15 to 40 mm (khider et al., 2013). this difference could be explained by the different botanical origin of bee pollen sample compared with khider et al. (2013) and the extractant used (i.e., methanol/ hexane vs. water). the botanical origin of sample 12 was composed mainly by galega officinalis 74.5% / brassica sp. 19.6%, which have shown inhibition against gram-negative bacteria, so they should be further investigated for the responsible compounds of their activity (erturk, 2010). inhibition activity against e. coli showed a positive and moderate dependence the presence of bee pollen from galega officinalis (r=0.52; n=16). however, the antibacterial activity observed cannot be attributed to galega officinalis since is also presented in other samples that no showed this control against e. coli. conclusion s we reported for the first time the relationship bet ween botanical origins of bee-pollen from chile and their phenolic, protein and carotenoid content, and antioxidant/antibacterial activities. it was demonstrated that several plant species contribute these parameters, mainly brassica sp. and galega officinalis. less frequent species such as fruit and endemic species as medicago sativa, prunus sp., trevoa quinquenervia, prunus sp., and convolvulus arvensis contribute to differences between composition and antioxidant/antibacterial activities of bee pollen samples. in addition species present in lower concentrations also help to accentuate these differences. samples with high protein content are composed by bee pollen from prunus sp., trevoa quinquenervia, medicago sativa and galega officinalis. samples with high carotenoid content are composed by bee pollen from galega officinalis, asteraceae, raphanus sp. the samples with higher content of polyphenols are composed by medicago, brassica sp. and galega officinalis bee pollen. six phenolic acids (p-coumaric, chlorogenic / caffeic, ferulic, synapic and cinnamic acids) and two flavonoids (kaempferol and luteolin) were identified in the samples. the highest content of phenolics was presented in samples composed by prunus sp. and brassica sp. bee pollen. while those with higher antioxidant power (frap) presented prunus sp. and trevoa quinquenervia bee pollen. samples composed by convolvulus arvensis, galega officinalis and brassica sp. showed inhibitory activity against s. pyogenes; those which contain medicago sativa and galega officinalis bee pollen inhibited s. aureus and p. aeruginosa; e. coli was controlled by samples with galega officinalis and brassica sp. bee pollen. botanical origin analysis of bee pollen permits correlation with some chemical, antioxidant and antibacterial properties, suggesting new resources of bioactive compounds. further studies with monofloral bee pollen loads are needed in order to provide more accurate correlations between botanical origin and composition and antioxidant/antibacterial activities. likewise, a more accurate determination of the phenolic/ flavonoid and other active compounds that make up the extracts is needed since they play a crucial role in the bioactivity of bee pollen. acknowledgements we 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gram negative bacteria. infectio 12, 227-232. accessed on august 05, 2015. available online at: http://www.scielo.org.co/scielo. php?script=sci_arttext&pid=s0123-93922008000300007 tomás barberán, f.a., tomás lorente, f., ferreres, f., garcia viguera, c., 1989: flavonoids as biochemical markers of the plant origin of bee pollen. j. sci. food agr. 47, 337-340. doi: 10.1002/jsfa.2740470308 ulusoy, e., kolayli, s., 2014: phenolic composition and antioxidant properties of anzer bee pollen. j. food biochem. 38, 73-82. doi: 10.1111/jfbc.12027 vanderplanck, m., leroy, b., wathelet, b., wattiez, r., michez, d., 2014: standardized protocol to evaluate pollen polypeptides as bee food source. apidologie 45, 192-204. doi: 10.1007/s13592-013-0239-0 who (world health organization). international agency for research on cancer (iarc). press release n°240: iarc monographs evaluate consumption of red meat and processed meat, accessed on october 24, 2015. available online at: http://www.iarc.fr/en/media-centre/pr/2015/ pdfs/pr240_e.pdf address of the corresponding author: e-mail: gmonten@uc.cl © the author(s) 2017. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 91, 237 248 (2018), doi:10.5073/jabfq.2018.091.032 1jimma university, college of agriculture and veterinary medicine, jimma, ethiopia 2universität kassel − fg agrartechnik, witzenhausen, germany actors’ post-harvest maize handling practices and allied mycoflora epidemiology in southwestern ethiopia: potential for mycotoxin-producing fungi management chemeda abedeta garbaba1, 2, *, lemlem gurmu denboba1, esayas mendesil1, fikre lemessa ocho1, oliver hensel2 (submitted: april 4, 2018; accepted: september 5, 2018) * corresponding author summary maize plays a key role in household food security in ethiopia, but its benefit has been limited with high post-harvest losses. this study was initiated to assess post-harvest practices and associated fungi pathogen epidemiology along the maize supply chain in southwestern ethiopia. the study was conducted in five purposively selected districts and a three-stage sampling procedure was employed for selection of the target groups. in total, 342 participants from different actors were interviewed using a semi-structured questionnaire. maize samples were collected every month from 63 randomly selected actors for mycological analysis during six months storage period. post-harvest loss was estimated to be 31% and loss during storage was identified as a critical loss point. com paring all biological agents, loss due to fungal pathogens in the store ranked on top. moisture content at loading stage could not increase the shelf life of the commodity. germination tests showed a significant (p < 0.01) decrease as storage duration increased, while mould incidence on cobs and kernels significantly (p < 0.05) increased. in total, seven fungal genera were isolated, characterized and identified, with fusarium, penicillium and aspergillus being predominant. most of the post-harvest practices are not effective in reducing post-harvest losses. especially, farmers’ traditional storage structures can be influenced by external climatic conditions and make the grains liable to develop mould during the rainy season. this research, therefore, highlights the need to design, develop or modify existing storage technologies that reduce post-harvest loss due to mycotoxin-producing fungal pathogens. furthermore, postharvest drying to obtain optimum moisture content is also crucial to reduce losses. keywords: mould, post-harvest management, post-harvest loss, storage fungi, stored maize introduction food security is a major challenge in sub-saharan african countries. whilst increasing production through crop intensification has been suggested, the reduction of post-harvest losses (phls) has received little attention. globally, phls has been estimated at one-third of the production but it could be even higher in sub-saharan africa (fao, 2011). the magnitude of phls due to deterioration of quality seems to be at a level similar to quantity losses. for instance, the caloric loss is estimated at 24% of all food produced (lipinski et al., 2013). this suggests that phl reduction can play a key role to improve food, nutritional security and household income. it is also considered as the easiest and cheapest option for resource conservation (yahia, 2008). the food security and economic wellbeing of ethiopia, in general, depends on agriculture. maize is considered amongst the top commodities contributing to food security due to its wide adaptability, high production, productivity and relatively cheap calories compared to other cereals. as a result, it has been included in the national food security strategy via intensive agriculture systems (abate et al., 2015). maize production and productivity in ethiopia has doubled in less than two decades and is the second in sub-saharan africa in yield/ha (abate et al., 2015). however, this boost in production and productivity threatens to be negated by high phls, further affecting food security. for instance in africa, maize phls were estimated at 14% to 36% (tefera, 2012). losses in weight of the same commodity in eastern and southern africa was estimated at 17.5% (rembold et al., 2011) and 41% to 80% for maize stored for six months using farmers traditional storage in ethiopia (sori and ayana, 2012). in ghana, up to 15% weevil attack was reported for five weeks stored maize (baidoo et al., 2010). maize phls occur along the whole activity chain including harvesting, drying, shelling, transport to store, storage, transport to market and processing for consumption (rembold et al., 2011). as a result, different research recommended commodity handling system analysis as the rational step in identifying suitable tactics for reducing phls along the activity chain (kitinoja and gorny, 1999; kader, 2005). however, several authors have reported maize phls in ethiopia in general and in southwestern ethiopia in particular without considering those chain of activities and analysis handling systems (ashagari, 2000; dubale et al., 2012; sori and ayana, 2012; befikadu, 2014). consequently, issues leading to high phls were not fully identified and characterized along the activity chain in order to reduce losses. identifying locally available post-harvest (ph) technologies and practices along the maize ph activity chain should be the first step in designing loss reduction strategies. an efficient phl reduction strategy for maize basically depends on the ecological conditions of storage which includes, physical, chemical and biological characteristics of the maize grain; the storage period and type; and functional characteristics of the facility (golob et al., 2002; ifpri, 2010; dubale et al., 2012; befikadu, 2014). despite the realization of the importance of storage, the potential impact of destructive storage pests (especially fungi) that cause quality deterioration leading to quantity, nutritional and financial losses has not been well researched (fourar-belaifa et al., 2011). furthermore, maize phl by fungal pathogens is not only of economic importance, but is also a public health concern due to the possible production of mycotoxins (golob, 2009). aspergillus, fusarium and penicillium spp. are the top three mycotoxins producing fungi in food and feed in the tropics and sub-tropics along production chains. generally, contaminated kernels by consuming those mycotoxins, particularly aflatoxin, resulted in illness, death, immunological suppression, liver cancers and nutritional interference (rundbergeta et al., 2002; jonathan et al., 2004; stumpf et al., 2013; kiarie et al., 2016). study findings reported that on-farm storage practices and structures in southwestern ethiopia, such as gombisa, can make maize susceptible to different types of damage, including storage pests and mould development (ifpri, 2010; dubale et al., 2012; befikadu, 2014). 238 c.a. garbaba, l.g. denboba, e. mendesil, f.l. ocho, o. hensel furthermore, research findings reported maize phl based on point data taken once or twice, and sample collection for loss assessment without considering the full storage duration until the product was depleted. at the same time, previous researches only focused at the farmer level without considering other actors along the maize supply chain that play a key role in maize transactions. therefore, the current research was designed to assess maize post-harvest handling practices and the fungal pathogens dynamic associated with maize stored by producers, collectors, and wholesalers in selected districts of the jimma zone in southwestern ethiopia. materials and methods study site the study was conducted in jimma zone, which is situated in southwest ethiopia at 7°15´ and 8°56´ n latitude and 36°00´ and 38°38´ e longitude. the elevation of jimma zone ranges from 800 to 3360 m.a.s.l. the agro-ecological setting includes highlands (15%), midlands (67%) and lowlands (18%) (csa, 2009; zofedo, 2013). for the current study, five districts namely dedo from the highlands; kersa, omonada and mana from the midland; and sokoru from the lowlands’ agro-ecology were purposely selected based on their high maize production potential and varied agro-ecological conditions (fig. 1). participant selection a three-stage sampling procedure was used to select participants. jimma zone was purposively selected from the southwest part of the country due to its high maize producing potential. five districts ranking among top maize producers and representing variable agroecology from the aforementioned zone were selected purposely based on secondary data from jimma zonal agricultural office. after discussion with agricultural office experts and extension agents from each district, three kebeles (the lowest administrative region, peasant associations, pas) were selected based on agro-ecological settings and the potential for maize production. considering the total population of each pa, sample size (the number of participants) selected from the households was determined using sampling formula with a 95% confidence level (yamane, 1967 as cited by ajay and micah, 2014) as indicated below. n = (e) 2 where n is the sample size; e is the level of precision at 5% and n is the total number of maize producing household in selected pa. after determination of sample size, a list of all household was collected from each pa and respondents were selected randomly using minitab randomization software. similarly, a number of collectors from each district and wholesalers from jimma town were randomly selected and used for data collection. for triangulation and validation of the information collected, key informants from farmers, developmental agents, experts at districts and zonal level were also interviewed. in order to acquire the desired depth of information, a total of 342 participants were involved in the study. data collection techniques the study was conducted from january 2014 to june 2015. both quantitative and qualitative data were collected from secondary and primary sources in the selected districts and communities. secondary data were collected from the archives of various organizations. semistructured questionnaires covering socio-economic, demographic data, maize post-harvest handling practices and associated issues were collected during field surveys via village meetings, key informants, and individual interviews. house to house interviews were carried out with the help of developmental agents from each pa. semi-structured questionnaires were prepared accordingly for each participant (farmers, key informants, traders and experts) to generate reliable data. pre-test interviews were conducted before actual data collection at each study site and amendments were made before the final interview. fig. 1: study area map with different agro-ecology of the study districts of jimma zone, southwestern ethiopia. n 1 + n actors’ post-harvest maize handling practices and allied mycoflora epidemiology 239 from each pa, three farmers were randomly selected from those growing the dominant bh-660 maize variety and storing their maize in a local storage structure, called gombisa, of uniform structure. in total, 45 farmers were included for the mycological study of the maize samples collection. similarly, three local collectors from each district (15 in total) and three wholesalers from jimma town were also included in the fungal pathogens assessment of maize kernels sample. disease assessment and sample collection started at harvest and loading stage (farmers) then continued with monthly intervals up to six months, at which time most of the participants’ stored product depleted from all actors store. experimental designs a 3 × 6 factorial design was used for the determination of the germination test, mould incidence on cobs and maize kernels stored in the farmers’ traditional storage structures. three agroecological levels (highland, midland, and lowland) and six-months storage duration with monthly interval data collection were used at the farmer level. three farmers from highland and lowland agroecological setting were used as replicates. however, the average of three districts used as replication for midland agro-ecology as more dominantly maize is produced in this agro-ecology setting. all factors including storage structures, maize variety, and management practices were kept uniform to minimize experimental error. similarly, for the collectors 3 × 6 factorial design was used that included the three agro-ecological levels of the respective districts and six-month storage duration with a monthly interval for the determination of germination test and mould incidence of maize kernels. for all collectors, the same maize variety, open-weave sacks and management practices used were uniforms. in a similar manner, three collectors from each district were used as replicates. for the wholesalers, three actors were included in the study with six level of storage duration. a completely randomized design was used for determination of germination test, mould incidence on kernels, cobs and maize as samples were collected from jimma town alone for wholesalers condition. germination test and moisture content the germination test was undertaken by randomly selecting 150 maize kernels from each sample lot. the test was done in triplicate: 50 kernels per replication. maize kernels were sown in 9 cm petridishes lined with filter paper (whatman no. 1), moistened with distilled water and then placed on a clean laboratory bench at room temperature (25 °c) for 7 days. the germinated seeds were visually examined for the appearance of radical and/or plumule and the germination percentage was computed following developed method (ogendo et al., 2004). germination (%) = × 100 where a stands for a number of germinated kernels, and b stands for the total number of plated kernels. the moisture content of the sample maize was determined using a digitally calibrated moisture tester (wile55 tr serial number 554601 by farm comp agro-electronics, france) on the spot. mycological analysis six cobs from each farmer’s store were brought for laboratory analysis. similarly, two kilograms of maize kernels from every trader’s store were sampled by deep probing at three layers of each sack, then mixed together and brought for mycological analysis under laboratory conditions. mould incidence on-cobs-maize sixty cobs were picked from a different level of each farm storage structure (top, centre, and bottom) and fed through pvc pipe fitted at the middle and bottom of the gombisa which allowed removal of sample cobs from the store. twenty cobs sampled from each layer were used as replication (atukwase et al., 2012). a visual inspection and scrutiny for kernel infections on each cob was made to record mould in order to calculate the incidence following (meer et al., 2013). mould incidence (mi) on-cobs-maize = × 100 where a stands for a number of infected cobs while b stands for a total number of cobs assessed. mould incidence on kernels a blotter test was used to determine mould incidence on maize kernels and the test was carried out following developed procedures (fandohan et al., 2003; hajihasani et al., 2012). mould incidence on kernel (%) = × 100 where a stands for infected kernels, while b total number of kernels plated. isolation and identification of fungal genera fungal pathogens on the maize kernels were grown, isolated and identified to the genus level on a monthly basis until the six month of storage period following standard techniques and procedures (narayanasamy, 2006; pitt and hocking, 2009; atukwase et al., 2012). the isolated fungal genera frequency of occurrence and relative density were calculated (mostafa and kazem, 2011; meer et al., 2013). fungal frequency (ff) = × 100 where nf number of particular fungal genera and nt a total number of samples/kernels. relative density (rd) = × 100 where ng stands for a number of the specific isolated genus, while tg for a total number of the fungal genus. data analysis statistical package for social sciences (spss) version 20.0 software was used for descriptive analysis of data (socioeconomics, maize phm practices, the frequency of occurrence and relative density of fungal genera recorded). whereas germination tests, mould inci dence on kernels and on-cobs-maize were analyzed using sas software version 9.0 after checking anova assumptions. analysis of variance was carried out using general linear model (glm). wherever significant difference was observed, mean separation was carried out using tukey’s honestly significant difference (hsd) test at the 5% probably level. results socio-economic characteristics most of the respondent farmers and traders were between 30 and 59 years old. but, about 78% of the experts participating were less than 30 years old. the majority of the respondent farmers and traders had primary education. more than 50% of the farmers had up to two decades’ experience in maize production but only a few traders more than that. however, none of the participant experts had more than five years’ experience in maize post-harvest management. most of the a b nf nt ng tg a b a b 240 c.a. garbaba, l.g. denboba, e. mendesil, f.l. ocho, o. hensel tab. 1: socio-economic characteristics of the participants actor agro-ecology statistical test lowland midland highland mean χ²-value p-value farmer sex (%) 0.278 0.870ns • male 95.5 94.6 92.7 94.3 • female 4.5 5.4 7.3 5.7 educational level 3.921 0.687ns • none 4.5 20.8 17.1 14.1 • basic 18.2 15.4 14.6 16.1 • 1 to 6 59.1 46.2 53.7 53.0 • 7 and above 18.2 17.7 14.6 16.8 age (years) 12.896 0.012* • less than 30 9.1 5.4 24.4 13.0 • 30 to 59 81.8 86.9 70.7 79.8 • more than 60 9.1 7.7 7.3 8.0 experience (years) 7.834 0.098ns • less than 10 22.7 7.7 17.1 15.83 • 10 to 20 31.8 24.6 29.3 28.57 • more than 20 45.5 67.7 53.7 55.63 traders sex 2.974 0.245ns • male 100.0 100.0 83.3 • female 0.0 0.0 16.7 educational level 8.922 0.063ns • 16 33.3 60.0 0.0 31.1 • 710 50.0 30.0 33.3 37.8 • 1112 16.7 10.0 66.7 31.1 age (years) 7.145 0.128ns • less than 30 0.0 50.0 16.7 22.2 • 30 to 59 66.7 50.0 66.7 61.1 • 60 and above 33.3 0 16.7 16.7 experience (years) 7.559 0.109ns • less than 10 16.7 70.0 83.3 56.7 • 10 to 20 66.7 30.0 16.7 37.8 • more than 20 16.7 0.0 0.0 5.6 experts sex 0.297 0.862ns • male 90.0 87.5 80.0 85.8 • female 10.0 12.5 20.0 14.2 age (years) 2.385 0.304ns • less than 30 70.0 62.5 100.0 77.50 • 30 to 59 30.0 37.5 0.0 22.50 experience (years) 1.179 0.555ns • less than 3 20.0 12.5 0.0 10.8 • 3 and 4 80.0 87.5 100.0 89.2 statistically significant at, *p < 0.05; ns = not significant. socio-economic characteristics did not show significant differences (p >0.05) between surveyed agro-ecological settings (tab. 1). survey results showed a majority of the participants (74%) had a family size of 5-10 and 58% of the farmers had less than five workforces in the household. on average, most of the participant farmers (68.4%) produced maize on less than 1 ha of land. similarly, most of the farmers (51.3%) allotted up to 50% of their land for maize, while 34.7% of the farmers allocated up 75% out of their total land, the rest allotted one-quarter of their land for maize production. in the study areas, participant farmers mainly produce maize for household consumption (67.9%) while 32.1% of the producers used it both for consumption and for selling (as an income source). most of the collectors collected maize from nearby pas in their district. about 50% of collectors sold their maize to individual consumers. wholesalers also sold to both local traders and consumers (33.3%) and addis ababa traders (50%). low quality maize especially discoloration and irregularity of maize supply was the major trading problem mentioned by both collectors and wholesalers. maize post-harvest practices in the current study, 10 post-harvest handling practices have been identified at producer level but harvesting, transportation, drying, storage and shelling are amongst the key activities carried out by producers (fig. 2). some of handling practices to maintain ph quality also carried out by traders too. harvesting maize harvesting in the study area started in september and lasted until december. farmers used visual observation, crop calendar method, shelling and observing kernel dryness; and checking seed socio-economic characteristics actors’ post-harvest maize handling practices and allied mycoflora epidemiology 241 hardness with the proportion of 61.1%, 28.8%, 8.8%, and 2.1%, respectively to determine the maturity of the crop for harvesting. however, none of the respondent farmers used the moisture testing method to harvest maize for safe storage. the moisture content at harvest and loading stage ranged between 16% to 28% which is far more than the optimum moisture content recommended for long term storage (fig. 3). drying the majority of the farmers (75.1%) practiced on-farm drying with the cobs still attached to the stalk (fig. 2), creating favorable conditions for fungal infection. the drying process was usually done by heaping up or spreading out the cobs on bare ground for a couple of days which inadvertently creates favorable conditions for a mycotoxin-producing fungal contamination. transportation about 55.0% of the respondent used animals as transportation means, while the remaining 45.0% used human labor (fig. 2). however, spillage loss of the harvested product was high during transportation storage the most common maize storage structure used by farmers across all agro-ecological settings was gombisa to store maize-on-cobs dominantly without sheaths. gombisa is the type of circular granary and is made by interweaving locally available materials; mostly bamboo split by local artisans. the roof is covered with natural grass or thatch. in rare cases, the corrugated sheet is used. the most critical problem observed in gombisa was not climatically controlled structure, resulting in high moisture leakage during the rainy season and the common formation of mould on stored maize. newly purpose of the practices limitations or associated constraints phpractices and practitioners (%) • tominimizemould development in the store • to collect the product at right maturity stage • less cost and easy for transportation • to dry and sort mouldy cobs and maintain quality "• sanitation purpose and easy for management • " • tomaintain the quality and reduce pest infestations • " • it is assumed as it facilitate drying and reduce temperature" • weevil problemdue to over dried and not reliable for safe storage • donemanually • coincidewith rain and increase phls • high spillage loss " • loss due to animals • no drying facilities • nomoisture test • assumed increase weevil infestation • costly, dosage problem and treatedmaize not consumed immediately • less efficient • not protective from external environment • highphls in store • not efficient to maintain quality • to control storage pests and prolong shelf life • shelledmaize easier for insecticides application on farmdrying 75.10% harvesting 100% transportationwith animals 55% temporary storage 24.9% storingmaize cobs without sheath 75.6% sanitation of the store at loading 99.5% storage using gombisa 94.6% use of insecticides 92.8% " maize shelling using sticks hitting 82.4% • physical damage • not efficient • time consuming maize selling 65.3% • cheap price of the commodity • source of income fig. 2: flow chart for maize post-harvest activity chain, its function, and associated constraints purposeofthe practices limitationsor associatedconstraints phpracticesand practitioners(%) • tominimizemould developmentinthe store • tocollectthe productatright maturitystage • lesscostandeasy fortransportation • todryandsort mouldycobsand maintainquality "• sanitationpurpose andeasyfor management • " • tomaintainthe qualityandreduce pestinfestations • " • itisassumedasit facilitatedryingand reducetemperature" • weevilproblemdueto overdriedandnot reliableforsafestorage • donemanually • coincidewithrain andincreasephls • highspillageloss" • lossduetoanimals • nodryingfacilities • nomoisturetest • assumedincrease weevilinfestation • costly,dosageproblem andtreatedmaizenot consumedimmediately • lessefficient • notprotectivefrom externalenvironment • highphlsinstore • notefficientto maintainquality • tocontrolstorage pestsandprolong shelflife • shelledmaizeeasier forinsecticides application onfarmdrying 75.10% harvesting 100% transportationwith animals55% temporarystorage 24.9% storingmaizecobs withoutsheath75.6% sanitationofthe storeatloading99.5% storageusing gombisa94.6% useofinsecticides 92.8% " maizeshellingusing stickshitting82.4% • physicaldamage • notefficient • timeconsuming maizeselling 65.3% • cheappriceofthe commodity • sourceofincome purposeofthe practices limitationsor associatedconstraints phpracticesand practitioners(%) • tominimizemould developmentinthe store • tocollectthe productatright maturitystage • lesscostandeasy fortransportation • todryandsort mouldycobsand maintainquality "• sanitationpurpose andeasyfor management • " • tomaintainthe qualityandreduce pestinfestations • " • itisassumedasit facilitatedryingand reducetemperature" • weevilproblemdueto overdriedandnot reliableforsafestorage • donemanually • coincidewithrain andincreasephls • highspillageloss" • lossduetoanimals • nodryingfacilities • nomoisturetest • assumedincrease weevilinfestation • costly,dosageproblem andtreatedmaizenot consumedimmediately • lessefficient • notprotectivefrom externalenvironment • highphlsinstore • notefficientto maintainquality • tocontrolstorage pestsandprolong shelflife • shelledmaizeeasier forinsecticides application onfarmdrying 75.10% harvesting 100% transportationwith animals55% temporarystorage 24.9% storingmaizecobs withoutsheath75.6% sanitationofthe storeatloading99.5% storageusing gombisa94.6% useofinsecticides 92.8% " maizeshellingusing stickshitting82.4% • physicaldamage • notefficient • timeconsuming maizeselling 65.3% • cheappriceofthe commodity • sourceofincome • less efficient 242 c.a. garbaba, l.g. denboba, e. mendesil, f.l. ocho, o. hensel constructed gombisa can be used for about 10 years and farmers use the same structure every year which serves as an inoculation source to enhance damage by insect pests and stored fungal pathogens. about 94.8% of farmers stored their new maize separate from the old maize (if available) while, 5.2% of the farmers mixed it with the old maize. participant farmers stored maize separately from other cereals to prevent from insect damage (73.6%), to avoid difficulty during storage management (11.9%) and to control mould problems (8.3%). the remaining farmers mixed maize with other cereals such as sorghum or teff. maize was stored in different forms by farmers. de-husked cob was most common (75.6%), both as cob and shelled kernels (21.2%) and sheath kernels alone (2.6%). maize cobs were stored on average for six months in gombisa then shelled and stored inside the house with sacks for a few months as it depleted mostly at six months. both collectors and wholesalers stored shelled maize with sacks inside a house-like structure made up of wood from different trees and roofed by a corrugated iron sheet. in most cases, the inside and outside wall were sealed by mud (81.5%) or cement (18.8%) and the floor was either cemented (56.3%), mud (25%) or mud covered with plastic (18.8%). only 18.8% of the participant maize collectors store had windows and most of the stores had no ventilation system. however, half of the wholesalers’ stores possessed windows and a ventilation system. still the moisture content was not optimal (fig. 3). in general, the storage structure of both collectors and wholesalers were not protected from insects, fungal pathogens, and other pests which resulted in high phls. in addition, sanitation was the main problem observed in stores belonging to traders. shelling farmers in the study area shelled maize kernels from cobs manually. farmers were hitting cob by stick inside the sack, finger palm shelling and hitting cob inside the house which physically damaged and made the kernels prone to fungal damage. post-harvest loss estimation and causes actors estimated 31% of maize phl along the activity chain (harvesting, post-harvest drying, shelling, storage, transportation and selling) out of this, 14% happened during storage (fig. 4). the proportion of phls due to biological agents is the most significant factor which starts as the crop reaches physiological maturity. respondent estimated that the highest proportion of phl (18%) was due to mould development followed by insect pest, rodents, wild animals and domestic animals in decreasing order. germination test a significant (p = 0.01) difference was observed due to the interaction of storage duration and variation in agro-ecology which affected germination percent of maize stored in farmers’ storage systems (tab. 2). similarly, highly significant (p < 0.0001) effect on the germination percentage of maize kernels stored under collector condition was observed without interaction between storage duration and agro-ecological variations (fig. 5). maize kernels stored under wholesaler stores were significantly (p = 0.0012) affected by storage duration (tab. 2). the maximum and minimum kernels germina tion was 93.1 and 65.2% at lowland and highland agro-ecology, respectively. mycological analysis mould incidence both storage duration and variation in agro-ecology exhibited highly significant (p < 0.0001) effects on the mi of on-cobs-maize stored fig. 3: the moisture content of maize measured with monthly interval from loading stage to six month of storage periods. fig. 4: actors’ maize phl estimation for major activities. t & m = transportation and marketing activities. actors agro ecology tab. 2: mean separation for germination (%) test of stored maize kernels under farm and wholesaler conditions. storage duration (months) 1 2 3 4 5 6 p-value farmer lowland 93.1±1.7a 82.2±1.7b-d 83.3±1.7bc 78.5±1.7c-f 77.1±1.7c-f 71.2±1.7fg 0.01 midland 86.9±1.7b 86.2±1.7b 83.5±1.7bc 80.7±1.7cd 79.2±1.7cd 72.6±1.7fg highland 83.6±1.7bc 84.1±1.7bc 78.1±1.7c-f 74.6±1.7d-f 72.9±1.7e-g 65.2±1.7g wholesaler 91.9±3.9a 83.2±3.9ab 81.3±3.9ab 76.7±3.9a-c 64.7±3.9bc 61.9±3.9c 0.0012 values are mean ± se of triplicate samples. means followed by the same letter among columns and/or rows are not significantly different from each other at p < 0.05 for the farmer and along row for the wholesalers. major post-harvest practices actors’ post-harvest maize handling practices and allied mycoflora epidemiology 243 under farm conditions (fig. 6). significant interaction between agro ecology and differences in storage duration were observed in mi of maize kernels sampled from both farmer and collector stores (tab. 3 and fig. 7), respectively. for wholesaler storage systems, the mi on kernels differs significantly (p = 0.0017) with storage duration (fig. 8). fungal genera a total of seven fungi genera were isolated, characterized and fig. 5: box plots for germination test of maize stored in collector stores a) varying storage duration and b) different agro-ecology. p < 0.0001 for both storage duration and agro-ecology. box plots with the same letter(s) for each figure do not significantly different from each other at p < 0.05. error bars are range values. tab. 3: mean separation for mould incidence (%) of maize kernels stored under farm condition storage duration (months) 1 2 3 4 5 6 p-value lowland 10.5±2.7k 24.7±2.7hi 30.9±2.7g-i 37.8±2.7e-h 45.2±2.7d-f 51.8±2.7b-d 0.001 midland 17.1±1.6jk 27.6±1.6hi 34.2±1.6f-h 41.7±1.6d-g 49.6±1.6cd 61.6±1.6b highland 21.8±2.7i-k 28.4±2.7g-i 41.9±2.7d-g 49.2±2.7c-e 59.9±2.7bc 78.9±2.7a values are mean ± se of triplicate samples. means with the same letter(s) are no significantly different from each other along the columns and/or rows at p < 0.05. agro-ecology fig. 6: box plots for mould incidence of on-cobs-maize stored under farm conditions a) storage duration b) agro-ecology. p < 0.0001 for both storage duration and change in agro-ecology. box plots with the same letter(s) for each figure do not significantly different from each other at p < 0.05. error bars are range values. identified in maize kernels from each actor’s store, except for wholesalers; where six fungi genera were recovered. genus fusarium was the most common fungi based on the frequency of occurrence and relative density followed by penicillium and aspergillus throughout the study period and location (tab. 4). in the current study, fusarium had the highest frequency of occurrence and relative density during the first two months of storage then slightly decreased as storage duration increased. comparison of the first month’s data with the last month’s exhibited a negative increment for fusarium but a positive one for both penicillium and aspergillus (tab. 5-7). 244 c.a. garbaba, l.g. denboba, e. mendesil, f.l. ocho, o. hensel discussion the present study identified 10 post-harvest handling activities in the maize ph supply chain. generally, the moisture content at harvest and loading stage was not optimal to increase the shelf life of the stored product; it actually favored the development of mould in the store. high kernel moisture levels of above 12% increase the chances of fungal growth in the store (dubale et al., 2012). it was also suggested that for either mechanical or manual harvesting, the maize grain must be dried to safe moisture levels (fao, 2011). furthermore, most farmers in the study area did not use post-harvest drying but wait until the crop dries on the field. unfortunately, this mostly coincides with rainfall which will also facilitate mould development when the maize is stored. delayed harvesting causes maize ear rot and fusarium spp. which are the principal pathogenic fungi responsible for causing rotting of maize ears (pitt and kocking, 2009). the report also indicated, aspergillus spp. often encountered on maize kernels that were allowed to dry in the field before harvesting (owolade et al., 2005). producers commonly used domestic animals, both for transportation of harvested maize to stores and to market; loss due to spillage was the most common fig. 8: box plots for mould incidence on maize kernels collected from wholesaler warehouse. p-value = 0.0017. box plots with the same letter(s) are do not significantly different from each other at p < 0.05. error bars are range values. fig. 7: mould incidence of stored maize kernels in collector store-house for six months. p-value = 0.005; values are mean ± se of triplicate samples. means with the same letter(s) do not differ significantly from one another at p < 0.05, both for storage duration and agro-ecology. tab. 4: frequency of occurrence and relative density of fungal genera associated to maize under different actors store fungi genera (%) actors penicillium aspergillus fusarium phoma geotrichum cloudosporium drechslera fr rd fr rd fr rd fr rd fr rd fr rd fr rd farmer 19.10 17.48 7.80 7.45 48.05 67.33 0.58 1.05 1.57 1.00 3.02 2.68 0.60 0.65 collector 28.02 23.60 5.95 8.33 61.13 59.18 0.00 0.00 2.67 2.50 1.70 1.38 0.52 0.58 wholesaler 35.70 20.22 31.02 16.68 36.38 44.95 11.12 1.85 0.00 2.37 6.67 2.53 0.00 0.00 rd = relative density, fr = frequency of occurrence tab. 5: frequency of occurrence and relative density of major fungal genera associated to stored maize under farmers condition storage duration (months) 1 2 3 4 5 6 mean sd increment (%)* penicillium frequency 2.20 3.40 12.40 34.10 26.20 36.30 19.10 15.15 93.94 relative density 5.80 3.40 26.50 24.40 18.70 26.10 17.48 10.39 77.78 aspergillus frequency 1.10 1.50 2.70 11.20 12.70 17.60 7.80 6.96 93.75 relative density 2.70 4.70 3.80 7.80 9.30 16.40 7.45 5.04 83.54 fusarium frequency 67.40 29.10 27.00 66.60 72.60 36.60 48.05 23.00 -84.15 relative density 83.60 86.10 64.40 53.90 66.40 49.60 67.33 14.97 -68.55 where sd = standard deviation of mean, * = % increment calculated by subtracting last month data from first-month data, then divided the value by last month data and multiply by 100 for each fungal genera frequency of occurrence and relative density. fungal genera parameters actors’ post-harvest maize handling practices and allied mycoflora epidemiology 245 problem observed when using domestic animals. pack animals especially donkey and mules are used for transportation of goods in most parts of africa and ethiopia (fernando and starkey, 2004; tolera and abebe, 2007). gombisa is the most dominant traditional storage structure used to store maize in cob form. studies conducted in various areas of ethiopia showed that most farmers across the country used gotera, gombisa and sacks to store maize (tadesse and basedow, 2004; dubale et al., 2012). traditional granaries (cribs), usually made up of locally available materials such as timber, bamboo, etc. are used in humid countries both for drying and for the storage of maize (nukenine, 2010). newly constructed gombisa can be used for up to ten years for the same purpose in the study area. hell et al. (2000) reported most storage structures for maize in benin were used for up to 5 years. this accelerated the risk of contamination with the increasing age of the storage structure. maize storage with sheath was only common in lowland maize producing districts and farmers in the study area assumed it reduced weevil damage. a study conducted at bako research centre, in western ethiopia, showed good sheath cover is considered as protecting the ear from insect and fungi damage (demissie et al., 2008). in general, the traditional storage structure, gombisa provides less protection from pests and not effective in protecting the stored products from external climatic conditions (like rainfall and high temperatures) which facilitate the development of fungi in the store (narayanasamy, 2006). on an average, after six months of storage, shelling is another main activity that is carried out to store the remaining kernels in the sack. however, shelling is totally carried out manually using stick hitting. this action causes physical damage or breakage, splitting or cracking of kernels which make them prone to fungal damage by other pests resulting in high phl (tadesse and basedow, 2004; ifpri, 2010). it has been reported that physical damage during shelling favors fusarium infections and using mechanical shelling can reduce fumonisin levels by 57% 65% in maize (fandohan et al., 2003). current survey results revealed that maize phl estimated at 31%. in ethiopia, the major maize production challenge for farmers is high phl, ranging from 15 to 30% (ifpri, 2010) and up to 19% (ashagari, 2000). in sub-saharan africa, about 37% of losses occur during storage and handling (lipinski et al., 2013). it was also stated that poor ph management results in large amounts of maize loss after harvest (kaaya et al., 2006). declining of germination percentage along the storage duration was observed for all actors’ storage systems; mainly due to the increment of mould which resulted in kernels quality loss. therefore, the current study shows that high fungi damage resulted in reduction of maize kernel germination. this result is in accordance with befikadu (2014) who reported that germination reduction of maize grain stored for six months in traditional farmers’ storage was 98% to 68.5% under intermediate and 97.5% to 70.17% in lowland agro-ecology. de crease in the germination percentage of stored maize to 28% after 180 days of storage at 35 oc with 14% moisture content has been re ported (tabatabaei and naghibalghora, 2013). somda et al. (2008) and govender et al. (2008) also reported that fungal infection caused a reduction in germination rates of maize kernels. for all storage systems studied, the trend in mould development rose along with the storage duration and agro-ecology. both mould incidence on cobs and kernels were more significant in the highland region of the area studied and were also related to an increase of storage duration which coincided with the region’s next rainy season. this may be because of higher relative humidity that favors fungal growth in less protected traditional storage structures. insufficient drying and humid conditions favor the development of fungi in tropics (suleiman et al., 2013). groot (2004) also stated that humidity is crucial for the development of fungi; even at low temperatures, some mould development may occur if the relative humidity of the air is high. similarly, garuba et al. (2011) reported that occurrence and frequency of mould were higher on maize stored with a higher moisture content of 19.0%. furthermore, damage caused by weevils can allow fungi to enter more easily and at the same time serve as an agent transferring fungi spores from infected to healthy grain (kankolongo et al., 2009; suleiman et al., 2013). moreover, even if maize can be harvested during the dry season, the rainy season fungal genera parameters tab. 6: frequency of occurrence and relative density of major fungal genera associated to stored maize under collector condition storage duration (months) 1 2 3 4 5 6 mean sd increment (%)* penicillium frequency 6.90 12.50 23.80 41.80 24.90 58.20 28.02 17.39 88.14 relative density 8.50 9.70 20.80 39.00 21.20 42.40 23.60 13.07 79.95 aspergillus frequency 1.80 0.40 3.30 4.00 12.00 14.20 5.95 5.22 87.32 relative density 0.40 0.40 10.60 5.50 17.30 15.80 8.33 6.77 97.47 fusarium frequency 81.40 70.30 49.80 46.70 70.20 48.40 61.13 13.39 -68.18 relative density 91.10 83.20 47.50 39.60 52.10 41.60 59.18 20.31 -118.99 tab. 7: frequency of occurrence and relative density of major fungal genera associated to stored maize under wholesaler condition storage duration (months) 1 2 3 4 5 6 mean sd increment (%)* penicillium frequency 28.80 32.00 2.10 68.90 48.90 35.60 36.05 22.21 19.10 relative density 9.20 9.00 1.88 44.40 34.40 24.30 20.53 16.65 62.14 aspergillus frequency 1.60 6.70 44.40 66.70 17.80 48.90 31.02 26.09 96.73 relative density 5.60 11.30 8.90 27.80 16.60 29.90 16.68 10.11 81.27 fusarium frequency 60.90 60.90 8.10 12.11 57.80 30.60 38.40 24.73 -99.02 relative density 78.00 72.50 24.40 9.08 49.00 45.80 46.46 26.70 -70.31 where sd = standard deviation, * = % increment calculated by subtracting last month data from first-month data, then divided the value by last month data and multiply by 100 for each fungal genera frequency of occurrence and relative density. fungal genera parameters 246 c.a. garbaba, l.g. denboba, e. mendesil, f.l. ocho, o. hensel that follows will facilitate mould development in traditional storage structures which are not climatically controlled and allow entry of moisture from outside. the present findings show that there were seven fungal genera associated with stored maize but fusarium, penicillium and aspergillus are the most dominant ones. tesfaye and abate (2000) also reported that fusarium, penicillium and aspergillus spp. were the most significant toxigenic fungal pathogens in ethiopian maize. fusarium was the most common of the above three. in a study conducted in the jimma zone, fusarium, penicillium and aspergillus spp. were identified from farmers’ storage systems; in both gombisa and in sacks (befikadu, 2014). however, this study confirmed that these fungi genera did not only dominate at producer level but also at collector and wholesaler level in the maize supply chain, including different agro-ecologies, storage periods and types. kaaya et al. (2006) reported that predominantly fusarium, penicillium, and aspergillus were identified from traders samples collected from different ugandan agro-ecologies. however, a report from cameron showed that the infection levels of stored maize were: aspergillus (up to 96%), penicillium (up to 63%) and lastly by fusarium (up to 32%) (tagne et al., 2003). similarly, several studies have reported that these three fungi genera were the most significant in stored maize (mostafa and kazem, 2011; bosah and omorusi, 2014). however, most studies did not cover the whole commodity supply chain but focused on the producer level. the findings showed that as the storage duration increased, the occurrence of most toxinogenic fungi, aspergillus spp. and peni cillium spp. increased along the maize supply chain. aspergillus species (particularly aspergillus flavous and a. parasticus) are the major aflatoxin producing fungi species in food and feed in the tropics and sub-tropics along production chains (kiarie et al., 2016), but are particularly significant in the storage phase (jonathan et al., 2004). depending on dose and duration of the exposure, aflatoxin can cause acute illness and death, immunological suppression, liver cancers and nutritional interference (jonathan et al., 2004). several species of penicillium are able to produce mycotoxins in the storage phase (rundbergeta et al., 2002). types of fusarium are also among the main fungal diseases that contribute to a loss in quality and the contamination of maize kernels with mycotoxins (stumpf et al., 2013). conclusion the present study clearly demonstrated that significant amount of losses occurred during post-harvest activities along the maize supply chains. traditional storage structures in the study area did not protect the stored maize from external environmental conditions and pest problems. thus, losses during storage were particularly significant and identified as a critical intervention point. the study also showed that, among the seven fungi genera identified, fusarium, penicillium and aspergillus spp. were the predominant fungi occurring in all the maize sampled along the supply chain. at the same time, these were the top three fungi able to produce mycotoxins and cause health hazards both to humans and animals that feed on it. however, the trend showed that fusarium spp. were slightly decreased over time but aspergillus and penicillium spp. increased rapidly with storage duration throughout the maize storage duration. in general, mould incidence both on-cobs-maize and kernels increased with storage duration for all storage situations studied. in order to reduce phls it is important to implement good postharvest practices such as selecting the optimal harvesting time, drying techniques and maize shelling method. in addition, traditional storage structures should be improved and disseminated to producers. also, current research findings highlight the need to investigate quality loss that occurs 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(eds.), using food science and technology to improve nutrition and promote national development, 10-16. international union of food science & technology. rome, italy. yamane, t., 1967: statistics: an introductory analysis. 2nd edition. harper and row, new york, usa. zofedo (zonal office of finance and economic development office of jimma), 2013: physical and socio-economic profile of jimma zone. jimma. unpublished report. 248 c.a. garbaba, l.g. denboba, e. mendesil, f.l. ocho, o. hensel address of the corresponding author: chemeda abedeta garbaba, jimma university, college of agriculture and veterinary medicine, p. o. box 307, jimma, ethiopia. universität kassel − fg agrartechnik, nordbahnhofstr. 1a, 37213 witzen hausen, germany e-mail: chemedaa@yahoo.com © the author(s) 2018. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 88, 16 21 (2015), doi:10.5073/jabfq.2015.088.004 1department of plant breeding and molecular genetics, university of poonch, rawalakot azad kashmir, pakistan 2department of chemistry, university of poonch, rawalakot, azad kashmir, pakistan 3university of azad jammu and kashmir, muzaffarabad, azad kashmir, pakistan 4phytochemical research laboratory, department of industrial pharmacy, federal university of santa maria, santa maria, brazil 5department of biochemistry, university of baluchistan, pakistan 6pakistan agricultural research centre, islamabad, pakistan 7department of eastern medicine, university of poonch, rawalakot, azad kashmir, pakistan antioxidant activities and phenolic composition of olive (olea europaea) leaves abdul khaliq1, syed mubashar sabir2*, syed dilnawaz ahmad3, aline augusti boligon4, margareth linde athayde4, abdul jabbar5, imtiaz qamar6, asmatullah khan7 (received october 25, 2014) * corresponding author summary the present study compares the antioxidant activities of leaves of eight cultivars of olive. the aqueous extracts of leaves showed inhibition against thiobarbituric acid reactive species (tbars) induced by pro-oxidant (10 μm feso4) in mice liver. the order of the antioxidant activity among cultivars on lipid peroxidation assay is gemlik > frantio > doleca-agogia > moriolo > mission > uslu > leccino > carotina. different varieties of olive showed good antioxidant properties, ic50 values ranged between 22.46 to 198 μg/ml on 2,2-diphenyl-1-picrylhydrazyl (dpph) assay. the major phenolic acids, some flavonoid aglycone and glycosides were identified in leaves by high performance liquid chromatography. ellagic acid (29.80 ± 0.02 mg/g), caffeic acid (15.73 ± 0.01mg/g), gallic acid (15.69 ± 0.01 mg/g), rutin (34.56 ± 0.03 mg/g), quercetin (16.41 ± 0.01 mg/g), epicatechin (11.04 ± 0.01 mg/g) and quercitrin (15.32 ± 0.03 mg/g) were predominant in infusion of olive. introduction during metabolism, reactive oxygen species (ros) are generated spontaneously in cells and are implicated in the aetiology of different degenerative diseases, like heart diseases, stroke, rheumatoid arthritis, diabetes and cancer (halliwell et al., 1992). a number of studies have shown that the use of polyphenolic compounds found in tea, fruits and vegetables is associated with low risk of these diseases (hertog et al., 1993). consequently, there is a great deal of interest in edible plants that contain antioxidants and healthpromoting phytochemicals as potential therapeutic agents. one of such plant is olive (olea europaea l.) which belongs to the family oleaceae and is native to tropical and warm temperate regions of the world. olive is also considered as multipurpose crop with great yield potential. the tree is famous for its fruit and is also called the olive, is commercially important in the mediterranean region as an important source of olive oil. the olive is typically distributed in the coastal areas of the eastern mediterranean basin, the adjoining coastal areas of southeastern europe, western asia and northern africa as well as northern iran at the south end of the caspian sea. the olive tree possesses medicinal and nutritional values. over the centuries, extracts obtained from leaves of olive have been used for promoting health and used in preservation. similarly, olive is a famous folk remedy to treat fever and some tropical diseases such as malaria (soler-rivas et al., 2000). a study has indicated that extract of olive leaves had a capacity to lower blood pressure in animals and increase blood flow in coronary arteries, relieved arrhythmia and prevented intestinal muscle spasms (benavente-garcia et al., 2000), diarrhea, to treat respiratory and urinary tract infections; olive oil and olive leaf extracts are some of these foodstuffs with recognized medicinal benefits and food preservation properties dating back to the egyptian empire (medina et al., 2007). antioxidant activity is used to measure a compound to reduce the pro-oxidants or reactive species of pathologic significance (somogyi et al., 2004). much attention has been focused on natural antioxidants capable of inhibiting lipid peroxidation which is mediated in several pathological conditions such as atherosclerosis, cancer and aging (frei, 1994). ferric reducing antioxidant power (frap), total phenolic assay by using the folin-ciocalteu reagent, total flavonoid content and dpph radical scavenging activity are the common methods used to evaluate the antioxidant properties. the demands of natural antioxidants are high for application as nutraceuticals and as food additives because of consumer preferences (kumar and chatoopadhyay, 2007). since antioxidants from plant source are safe and easily available, olive leaves was subjected to determine and quantify various antioxidant activities. there is limited information available on the antioxidant activity and phenolic profile of these olive cultivars. in view of the potential role of olive as dietary source of flavonoids as well as its possible use as functional food, this study was aimed to evaluate the antioxidant and inhibitory effect of eight important cultivars of olive on fe(ii) induced lipid peroxidation and to find out the phenolic profile of olive for their possible use in food and phytotherapy. the dpph radical activity, total antioxidant activity, phenolic and flavonoid contents of these cultivars were also determined. materials and methods chemical and reagents methanol, formic acid, gallic acid, chlorogenic acid, caffeic acid and ellagic acids purchased from merck (darmstadt, germany). epicatechin, quercetin, quercitrin, rutin and kaempferol were acquired from sigma chemical co. (st. louis, mo, usa). thiobarbituric acid (tba), malonaldehyde-bis-dimethyl acetal (mda), 2,2-diphenyl-1picrylhydrazyl (dpph), and ammonium molybdate was purchased from sigma aldrich (st. louis, mo, usa). ferrous sulphate was obtained from biochemicals (lahore). all chemical were of analytical grade. preparation of the leaves extracts the leaves of different cultivars of olive which include doleceagogia, mission, moriolo, maurino, carotina, leccino, gemlik, uslu were collected from three locations, quetta, zhoab and lorallia research stations of pakistan at the same harvesting time. leaves were washed and dried in hot air at 40 oc and ground to a fine powder in mill. ground material (5 g) was extracted with hot water (250 ml) for 30 minutes followed by filtration through whatman no.1 filter paper. the obtained residues were re-extracted under the antioxidant activities of olive 17 same conditions. the combined filtrates were evaporated in rotary evaporator below 40 oc. the extracts obtained after evaporation of solvent was weighed to determine the yield and stored at -20 oc. the extract at a final concentration (1 mg/ml) was then serially diluted to obtain the desired concentration of plant for the experiment. test animals all animal studies were in strict accordance with the nih guide for the care and use of laboratory animals. male balb/c mice (2.0 2.5 months and 22-30 g), were purchased from national institute of health islamabad, were used for in vitro studies. the animals were kept in separate cages with continuous access to food and water in a room with controlled temperature (22 ± 3 oc) and on a 12 h light/ dark cycle with lights turned on at 7:00 a.m. production of tbars from liver tissues production of tbars was determined using a modified method (ohkawa et al., 1997). the mice were anaesthetised with chloroform, sacrificed by decapitation and the liver was quickly removed and placed on ice. one gram of liver tissues were homogenised in cold 100 mm tris buffer ph 7.4 (1:10 w/v) and centrifuged. the homogenates (100 μl) were incubated with or without 50 μl of the various freshly prepared oxidant (iron) and different concentrations of the leaves extracts together with an appropriate volume of deionised water to give a total volume of 300 μl at 37 oc for 1 h. the colour reaction was carried out by adding 200, 500 μl each of the 8.1 % sodium dodecyl sulphate (sds), acetic acid (ph 3.4) and 0.6 % tba respectively. the reaction mixtures, including those of serial dilutions of 0.03 mm standard mda were incubated at 97 oc for 1 h. the absorbance was read after cooling the tubes at 532 nm in a spectrophotometer. antioxidant activity by dpph radical scavenging the antioxidant activities of the plant extracts were measured by scavenging of stable dpph radical according to the method of hatano et al. (1998). briefly 0.25 mm solution of dpph radical (0.5 ml) was added to the sample solution in ethanol (1 ml) at different concentrations (25 200 μg/ml) of aqueous extracts. the mix ture was shaken vigorously and left to stand for 30 minutes in the dark, and the absorbance was measured at 517 nm. the capacity to scavenge the dpph radical was calculated using the following equation: (%) scavenging = [(ao a1)/ao)] x 100, where, ao is the absorbance of the control reaction and a1 is the absorbance of the sample itself. the ic50 values (extract concentration that cause 50 % scavenging) were determined from the graph of scavenging effect percentage against the extract concentration. all determinations were carried out in triplicate. total antioxidant assay the phosphomolybdenum assay was based on the reduction of molybdenum, mo (vi)-mo (v) by the extracts and subsequent formation of a green phosphate/mo (v) complex at acidic ph (prieto et al., 1998). the extracts at concentration (100 μg/ml) were mixed with 3 ml of the reagent solution (0.6 m h2so4, 28 mm sodium phosphate and 4 mm amonium molybdate). the tubes were incubated at 95 oc for 90 mins. the mixture was cooled to room temperature and the absorbance of the solution was measured at 695 nm. determination of phenolics the total phenolic content as gallic acid equivalent was determined by the method of singleton et al. (1999). this method quantifies various phenolic acids and flavonoids in the extract. the aqueous extract (0.5 ml) was added to 2.5 ml, 10 % folin-ciocalteau’s reagent (v/v) and 2 ml of 7.5 % sodium carbonate. the reaction mixture was incubated at 45 oc for 40 min and the absorbance was measured at 765 nm in the spectrophotometer. the total phenol content was expressed as milligrams of gallic acid equivalents/g extract was calculated using the following linear equation based on calibration curve: y = 0.0063 x + 0.0396 (r2 = 0.99) where, y is the absorbance at 765 nm and x is the total phenolic content of different extracts of olive. determination of flavonoids the total flavonoid content was expressed as quercetin equivalents. a standard curve was prepared from quercetin [0.04, 0.02, 0.0025 and 0.00125 mg/ml in 80 % ethanol (v/v)]. the standard solutions or extracts (0.5 ml) were mixed with 1.5 ml of 95 % ethanol (v/v), 0.1 ml of 10 % aluminium chloride (w/v), 0.1 ml of 1 mol/l sodium acetate and 2.8 ml of water. the volume of 10 % aluminium chloride was substituted by the same volume of distilled water in the blank. after incubation at room temperature for 30 min, the absorbance of the reaction mixture was measured at 415 nm. the mean of three readings was used and the total flavonoid content was expressed as milligrams of quercetin equivalents/g of extract by the method of kosalec et al. (2004). the total flavonoid content was calculated using the following linear equation based on calibration curve: y = 0.0026x + 0.0114 (r2 = 0.996) where, y is the absorbance at 765 nm and x is the total flavonoid content of different extracts of olive. quantification of phenolic compounds by hplc-dad reverse phase chromatographic analyses were carried out under gradient conditions using c18 column (4.6 mm x 150 mm) packed with 5 μm diameter particles; the mobile phase was water contain ing 1 % formic acid (a) and methanol (b), and the composition gradient was: 13 % of b until 10 min and changed to obtain 20 %, 30 %, 50 %, 60 %, 70 %, 20 % and 10 % b at 20, 30, 40, 50, 60, 70 and 80 min, respectively, following the method described by pereira et al., (2014) with slight modifications. olea europaea extract and mobile phase were filtered through 0.45 μm membrane filter (millipore) and then degassed by ultrasonic bath prior to use, the olea europaea extract was analyzed at a concentration of 15 mg/ml. the presence of nine phenolic compounds, namely, gallic acid, chlorogenic acid, caffeic acid, ellagic acid, epicatechin, rutin, quercitrin, quercetin and kaempferol, was investigated. identification of these compounds was performed by comparing their retention times and uv absorption spectra with those of external standards. the flow rate was 0.7 ml/min, injection volume 50 μl and the wavelength were 254 for gallic acid, 280 for epicatechin, 327 nm for chlorogenic, caffeic and ellagic acids, and 365 nm for quercetin, quercitrin, rutin and kaempferol. stock solutions of standards references were prepared in the hplc mobile phase at a concentration range of 0.025 0.300 mg/ml for kaempferol, quercetin, quercitrin, rutin and epicatechin; and 0.050 0.450 mg/ml for ellagic, gallic, caffeic and chlorogenic acids. chromatography peaks were confirmed by comparing its retention time with those of reference standards and by dad spectra (200 to 500 nm). calibration curve for gallic acid: y = 12754x + 1197.3 (r = 0.9998); chlorogenic acid: y = 11864x + 1267.5 (r = 0.9999); caffeic acid: y = 12693x + 1308.1 (r = 0.9995); ellagic acid: y = 13172x + 1246.9 (r = 0.9998); epicatechin: y = 11849x + 1287.6 (r = 0.9993); quercitrin: y = 12581x + 1317.4 18 a. khaliq, s.m. sabir, s.d. ahmad, a.a. boligon, m. linde athayde, a. jabbar, i. qamar, a. khan (r = 0.9999); rutin: y = 13028x + 1267.7 (r = 0.9998); quercetin: y = 11896x + 1197.5 (r = 0.9994) and kaempferol: y = 11679x + 1265.8 (r = 0.9999). all chromatography operations were carried out at ambient temperature and in triplicate. the limit of detection (lod) and limit of quantification (loq) were calculated based on the standard deviation of the responses and the slope using three independent analytical curves. lod and loq were calculated as 3.3 and 10 σ/s, respectively, where σ is the standard deviation of the response and s is the slope of the calibration curve (boligon et al., 2013). statistical analysis the results were expressed as means ± standard deviation. the data was analyzed by one way anova and different group means were compared by duncan’s multiple range (dmr) and turkey test where necessary. p < 0.05 was considered significant in all cases. the software package statistica (version, 4.5) was used for analysis of data. results inhibition of lipid peroxidation using iron as pro-oxidant in mice liver lipid peroxidation (an index of oxidative stress) in mice liver homogenate was induced with iron and the potential antioxidant effect of aqueous extract of different cultivars of olive was determined. fig. 1 shows the antioxidant effect of different cultivars of olive in mice liver. here, the tbars was induced with 10 μm iron. the results revealed that treatment with fe(ii) caused a significant (p < 0.05) increase in thiobarbituric acid reactive substances (tbars) compared to the basal. separate controls were used for different cultivars of olive (fig. 1). treatment with different concentrations of olives caused a marked decrease in lipid peroxidation (fig. 1). all the cultivars were found to be capable of reducing the lipid peroxidation almost comparable to the basal level. in order to compare the antioxidant activity among different cultivars ic50 values (concentration that cause 50 % inhibition) were calculated (tab. 1). thus the order of antioxidant activity is gemlik > frantio > doleca-agogia > moriolo > mission > uslu > leccino > carotina (tab. 1). dpph radical scavenging activity the free radical scavenging activities of extracts was measured by the ability to scavenge dpph radical. now dpph radical has been widely used in assessment of radical scavenging activity because of its ease and convenience. five different concentrations (25, 50, 100, 150 and 200 μg/ml) of extracts were used to analyze the free radical scavenging activity. all the cultivars showed their abilities to scavenge the dpph radical which were evaluated in terms of their ic50 values (fig. 2a). all the cultivars showed their ability to reduce the dpph. however, their order of reactivity is (ic50, 22.46 μg/ml (leccino), 23.5 μg/ml (gemlik), 29.64 μg/ml (dolecaagogia), 46.34 μg/ml (moriolo), 82.1 μg/ml (mission) and 115.5 μg/ ml (uslu), 196 μg/ml (frantio) and 198 μg/ml (carotina) (tab. 1). total antioxidant activity by phosphomolybdenum assay the total antioxidant activity of different cultivars was expressed as ascorbic acid equivalent is shown in fig. 3. there was a statistically significant (p < 0.05) difference among the antioxidant activity of all cultivars. however, the order of their reactivity was dolece-agogia > moriolo > leccino > gemlik > mission > picholino > frantio > carotina (fig. 2b). total phenolics and flavonoid contents the total phenolic content was expressed as gallic acid equivalent, while, flavonoid content as quercetin equivalent (fig. 3). there was a statistically significant difference (p < 0.05) among different cultivars. the phenolic content was found in the range of 109.6 ± fig. 1: the inhibitory effect of aqueous extract of eight cultivars of olive on iron sulphate induced lipid peroxidation in mice liver. the results are means of three separate experiments ± sd. p < 0.05 is significantly different from control by dmr test. all the values are significantly different (p < 0.05) from control by dmr test. tab. 1: ic50 values of different genotypes of olive genotypes ic50 (μg/ml) ic50 (μg/ml) for dpph assay for tbars assay doleca-agoga 29.64 28.4 mission 82.1 44.71 moriolo 46.34 32.05 carotina 198 64 leccino 22.46 59.32 gemlik 23.5 21.18 uslu 115.5 57.42 frantio 196 26 antioxidant activities of olive 19 3.68 to 161 ± 1.9 mg/g. gemlik cultivar which showed the highest antioxidant activity showed the highest phenolic content. while, leccino contained the lowest amount of phenolics (fig. 3). the flavonoid content was found between 42 ± 1.97 to 57 mg/g. gemlik contained the highest amount of flavonoid. whereas, uslu contained the least amount of flavonoid (fig. 3). fig. 2: antioxidant activities of olive cultivars (a). dpph radical scavenging activity of different cultivars of olive. values are means ± sd (n=3). (b). total antioxidant activity of different cultivars of olive at 100 µg/ ml measured by phosphomolybdenum reduction assay. results are means ± sd (n=3).values in figure followed by different letter are significantly (p < 0.05) different from control by dmr test. fig. 3: total phenolic and flavonoid contents among different cultivars of olive. values in figure followed by different letter are significantly (p < 0.05) different from control by dmr test. results are means ± sd (n=3). fig. 4: representative high performance liquid chromatography profile of olea europaea extract, detection uv was at 327 nm. gallic acid (peak 1), chlorogenic acid (peak 2), caffeic acid (peak 3), ellagic acid (peak 4), epicatechin (peak 5), rutin (peak 6), quercitrin (peak 7), quercetin (peak 8) and kaempferol (peak 9). " tab. 2: phenolics and flavonoids composition of olea europaea extract compounds olea europaea lod loq mg/g % mg/ml mg/ml gallic acid 15.69 ± 0.01 a 1.56 0.017 0.058 chlorogenic acid 3.17 ± 0.03 b 0.31 0.025 0.083 caffeic acid 15.73 ± 0.01 a 1.57 0.031 0.107 ellagic acid 29.80 ± 0.02 c 2.98 0.029 0.096 epicatechin 11.04 ± 0.01 d 1.10 0.016 0.054 rutin 34.56 ± 0.03 e 3.45 0.035 0.115 quercitrin 15.32 ± 0.03 a 1.53 0.007 0.025 quercetin 16.41 ± 0.01 a 1.64 0.012 0.039 kaempferol 7.38 ± 0.02 f 0.73 0.009 0.028 results are expressed as mean ± standard deviations (sd) of three determinations. averages followed by different letters differ by tukey test at p < 0.05. content of phenolics, flavonoids and hplc characterization of phenolic compounds hplc fingerprinting of olea europaea was also acquired (fig. 4). the samples of olea europaea (gemlik) displayed the highest antioxidant activity, therefore, its phenolic profile was determined. the compounds were identified based on the spectral characteristics and retention times compared with authentic standards. olea europaea extract revealed the presence of gallic acid (tr = 11.73 min; peak 1), chlorogenic acid (tr = 21.09 min; peak 2), caffeic acid (tr = 25.01 min; peak 3), ellagic acid (tr = 31.65; peak 4), epicatechin (tr = 34.81 min; peak 5), rutin (tr = 40.25 min; peak 6), quercitrin (tr = 45.19 min; peak 7), quercitrin (tr = 50.97 min; peak 8) and kaempferol (tr = 58.36 min; peak 9) (fig. 4, tab. 2). discussion oxidative stress is now recognised to be associated with more than 200 diseases, as well as with the normal aging process (ghasanfari et al., 2006). there is a strong correlation between thiobarbituric acid-reactive substances (tbars) as a maker of oxidative stress and products that reflect oxidative damage to dna (chen et al., 2005). it is known that metal-catalysed generation of ros results in an attack not only on dna and proteins, but also on other cellular components involving polyunsaturated fatty acid residues of phospholipids, which are extremely sensitive to oxidation.17 increases in the formation of tbars in iron(ii) sulphate (10 μm)-induced oxidative stress, as compared to the normal, suggest possible damage of tissues with an overload of iron. free iron in the cytosol and mitochondria can cause considerable oxidative damage by increasing superoxide production, 20 a. khaliq, s.m. sabir, s.d. ahmad, a.a. boligon, m. linde athayde, a. jabbar, i. qamar, a. khan which can react with fe(iii) to regenerate fe(ii) that participates in the fenton reaction (fraga and oteiza, 2002). iron overload results in the formation of lipid peroxidation products, which have been demonstrated in a number of tissues, including the liver and kidneys (houglum et al., 1990). the decrease in the fe(ii) induced lipid peroxidation in the mice liver homogenates in the presence of the extracts could be as result of the ability of the extracts to chelate fe(ii) and/or scavenge free radicals produced by the fe(ii) catalyzed production of reactive oxygen species (ros) in the mice liver. the cultivars, gemlik, frantio and dolece-agogiab-mission possessed strong antioxidant activity and showed comparatively higher ability to reduce the tbars and thus can be utilized against potential overload of iron in liver disease. dpph radical scavenging activity is usually performed as a measure of electron denoting capacity of antioxidant (baumann et al., 1997). antioxidants are substances that neutralize free radicals and their negative effects. they act at different stages (prevention, interception and repair) and by different mechanisms: reducing agents by donating hydrogen, quenching singlet oxygen, acting as chelators and trapping free radicals (devasagayam et al., 2004). dpph• is considered to be a model of a stable lipophilic radical. a chain reaction of lipophilic radicals is initiated by lipid auto-oxidation. antioxidants react with dpph•, reducing the number of dpph free radicals to the number of their available hydroxyl groups. there fore, the absorption at 517 nm is proportional to the amount of residual dpph•. it is visually noticeable as a discoloration from purple to yellow. the high dpph radical scavenging activity of these cultivars suggests their use in diseases arising from free radical attack. the phosphomolybdenum method is quantitative since the total antioxidant activity is expressed as the number of equivalents of ascorbic acid (prieto et al., 1999). the phosphomolybdenum me thod usually detects antioxidants such as ascorbic acid, some phenolics, tocopherols and carotenoids. dolece-agogia showed the highest reducing activity on phosphomolybdenum assay whereas, carotina showed the least. plant-derived polyphenolic flavonoids are well known to exhibit antioxidant activity through a variety of mechanisms including scavenging of ros, inhibiting lipid peroxidation and chelating metal ions (shahidi, 1997). in vitro experimental systems have also shown that flavonoids possess anti-inflammatory, antiallergic, antiviral and anticarcinogenic properties (middleton, 1998). the hot water extracts of olive showed high content of phenolics and flavonoids. ellagic acid, caffeic acid, gallic acid, rutin, quercetin, epicatechin and quercitrin were predominant in infusion of olive. this is a detailed phytochemical study which reports different phenolics and flavonoids in olive leaves. earlier studies have shown that the main phenolic compounds in olive leaves are oleuropein, verbascoside, ligstroside, tyrosol and hydroxytyrosol (caturla et al., 2005). the observed antioxidant activity and protecting ability of olive against lipid peroxidation is due to the phenolic acids (ellagic acid, gallic acid, chlorogenic acid and caffeic acid) and flavonoids (rutin, quercitrin, epicatechin, and quercetin and kaempferol). the exogenous antioxidants from olive extracts may act directly or indirectly with the internal antioxidant system for synergetic effects to protect several diseases linked to free radicals such as heart diseases, neurodisorders and other stress related disorders. from the results of this study, we conclude a high efficacy of the crude aqueous extract of eight cultivars of olive, in free radical scavenging, inhibition of reactive oxygen species and lipid peroxidation which may be associated with its high medicinal use as a functional food and effectiveness in treatment of different diseases amongst which the liver disease is the most important. however, more detailed in vivo studies are required to evaluate the antioxidant activity and bioavailabity of olive. conflict of interest none declared. references baumann, j., wurn, g., bruchlausen, f.v., 1979: prostaglandin synthetase inhibiting o2-radical scavenging properties of some flavonoids and related phenolic compounds. arch. pharmacol. 313, 330-337. benavente-garcia, o., castillo, j., lorente, j., ortuno, a., del rio, j.a., 2000: antioxidant activity of phenolics extracted 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(eds.). academic press, london, uk. ghasanfari, g., minaie, b., yasa, n., leilu, a.n., azadeh, m., 2006: biochemical and histopathological evidences for beneficial effects of satureja khuzestanica jamzad essential oil on the mouse model of inflammatory bowel diseases. toxicol. mech. methods 16, 365-372. halliwell, b., gutteridge, j.m.c., cross, c.e., 1992: free radicals, antioxidants and human disease: where are we now? j. lab. clin. med. 119, 598-620. hatano, t., kagawa, h., yasuhara, t., okuda, t., 1998: two new flavonoids and other constitnduents in licorice root; their relative astringency and radical scavenging effects. chem. pharm. bull. 36, 20902097. hertog, m.g.l, hollman p.c.h., van, d.p.b., 1993: content of potentially anticarcinogenic flavonoids of tea infusions, wines and fruit juice. j. agri. food chem. 41, 1242-1246. houglum, k., filip, m., witztum, j.l., chojkier, m., 1990: malondialdehyde and 4-hydroxynonenal protein adducts in plasma and liver of rats with iron overload. j. clin. inves. 86, 1991-1998. karadag, a., ozcekik, b., saner, s., 2009: review of methods to determine antioxidant capacities. food anal. methods 2, 41-60. kosalec, i., bakmaz, m., pepeliniak, s., vladimir-knezevic, s., 2004: quantitative analysis of the flavonoids in raw propolis from northern croatia. acta pharm. 54, 65-72. kumar, a., chattopadhyay, s., 2007: dna damage protecting activity and antioxidant potential of pudina extract. food chem. 100, 1377-1384. medina, e., romero, c., brenes, m., garcia, p., de castro, a., garcia, a., 2008: profile of anti-lactic acid bacteria compounds during the storage of olive which are not treated with alkali. eur. food res. tech. 228, 133-138. antioxidant activities of olive 21 middleton, e.j., 1998: effect of plant flavonoids on immune and inflammatory cell function. adv. exp. med. biol. 439, 175-82. myriam, b.s., hafedh, a., manef, a., 2012: study of phenolic composition and biological activities assessment of olive leaves from different varieties grown in tunisia. med. chem. 2, 107-111. obeid, h.k., bedgood, jr. d.r., prenzler, p.d., robards, k., 2007: bio screening of australian olive mill waste extracts: biophenol content, antioxidant, antimicrobial and molluscicidal activities. food chem. toxicol. 45, 1238-1248. ohkawa, h., ohishi, n., yagi, k., 1997: assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. anal. biochem. 95, 351-358. pereira, r.p., boligon, a.a., appel, a.s., fachinetto, r., ceron, c.s., tanus-santos, j.e., athayde, m.l., rocha, j.b.t., 2014: chemical composition, antioxidant and anticholinesterase activity of melissa officinalis. ind. crop. prod. 53, 34-45. prieto, p., pineda, m., aiguel, m., 1999: spectrophotometer quantization of antioxidant capacity through the formation of phosphomolybdenum complex: specific application to the determination of vitamin e. anal. biochem. 269, 337-341. shacter, e., 2000: protein oxidative damage. methods enzymol. 319, 428436. shahidi, f., 1997: natural antioxidants: an overview. in: shahidi, f. (ed.), natural antioxidants: chemistry, health effects and applications, 1-7. champaign, il: aocs press. singleton, v.l., orthofer, r., lamuela-raventos, r.m., 1999: analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu’s reagent. methods enzymol. 299, 152-178. somogyi, a., rosta, k., pusztai, p., tulassay, z., nagy, g., 2007: antioxidant measurements. physiol. meas. 28, 41-55. soler-rivas, c., espin, j.c., wichers, h.j., 2000: oleuropein and related compounds. j. sci. food agri. 80, 1013-1023. address of the corresponding author: e-mail: mubashersabir@yahoo.com journal of applied botany and food quality 90, 259 265 (2017), doi:10.5073/jabfq.2017.090.032 1institute of food technology, 2department of agrotechnology, faculty of agriculture and biotechnology, university of science and technology, poland influence of mechanical damage and storage on various quality aspects of potatoes jarosław pobereżny1*, katarzyna gościnna1, elżbieta wszelaczyńska1, małgorzata szczepanek2 (received may 5, 2017; accepted may 18, 2017) * corresponding author summary the aim of this study was to determine the effects of mechanical damage on both the contents of dry matter and chlorogenic acid and the degree of blackspot for five cultivars of potatoes of various earliness groups. the study was conducted immediately after the harvest as well as after two, four and six months of storage under constant conditions (air temperature +4 °c and rh 95%). mechanical damage leads to a greater accumulation of chlorogenic acid and increases the tubers’ susceptibility to blackening, irrespective of the earliness group. the duration of storage significantly determines the dry matter content of chlorogenic acid and the susceptibility to blackening of raw tuber flesh to the greatest extent for cultivars of the medium-early group. a significant (p < 0.01) correlation was demonstrated between the dry matter and chlorogenic acid contents and the degree of blackspot, which was higher on damaged tubers. keywords blackening, chlorogenic acid, early, mechanical damage, potato introduction potato (solanum tuberosum spp.), along with wheat, maize and rice, is one of the main crops for feeding the world’s population (devaux et al., 2014; gancarz, 2016). in many countries of the world, an increase in the popularity of potatoes as been noted, which is due to its nutritional value and the high energy value in relation to the area under cultivation. the main trends in the use of the potato in the food processing sector include the production of table potatoes, frozen products (salads, pancakes, dumplings, etc.), wet products (salads, purees, etc.), fried products (crisps, chips) and dried products, as well as the production of starch and ethanol (ciesarova et al., 2006; erturk and picha, 2007). potatoes are delivered to stores in a packaged and customised form, i.e. cleaned with a brush, washed and pre-packaged (erturk and picha, 2007). one of the major problems in the production of potatoes is mechanical damage, which increases the tendency to blacken the tubers and reduces the raw material quality (opara and pathere, 2014). the process of raw tuber flesh blackening is due to the enzymatic oxidation of phenols (mainly tyrosine and phenolic acids: chlorogenic and caffeic) in the presence of an enzyme called phenolase, which oxidises these compounds to dark coloured products, namely melanins (stevens and davelaar, 1996; delago et al., 2001a). mechanical damage (bruising and abrasions) affects the cell structure and results in an increase in the polyphenolic compound content, primarily the content of chlorogenic acid, which accounts for approx. 90% of all polyphenolic compounds of the potato (keutgen et al., 2014). damage also results in an increase in polyphenol oxidase activity and, consequently, in intensification of the enzymatic oxidation reaction of polyphenols. chlorogenic acid content and the susceptibility of tubers to damage and blackening are, to a great extent, determined by the cultivar and its earliness group (finotti et al., 2011). high susceptibility to dark spotting after being hit is also a characteristic of starch cultivars with a higher content of dry matter and roughage. during storage, the tendency toward dark spotting increases, which is due to the intensity of life processes. an increased tendency to flesh blackening occurs at lower temperatures. many authors have observed a more intense blackening of the tuber flesh during storage at a temperature of 2-4 °c, compared to tubers stored at a temperature of 8 °c. in turn, a reduced relative air humidity and the duration of potato storage increases their susceptibility to blackspot (delago et al., 2001a, b; keutgen et al., 2014). a study was undertaken to examine the relationship between the degree of tuber damage and the susceptibility to tuber blackening as determined by the contents of total dry matter and chlorogenic acid of various potato cultivars and storage durations. materials and methods materials the raw material used in the study included five potato cultivars (‘denar’ – a very early cv., ‘bila’ and ‘rosalind’ – early cvs., ‘satina’ – a medium-early cv., and ‘tajfun’ – a medium early cv.). the different cultivars were studied each year over a three year period. ‘denar’, ‘bila’ and ‘tajfun’ are polish cultivars., while ‘rosalind’ and ‘satina’ are german cultivars. all of them are characterised by very large round and oval tubers with shallow eyes and light-yellow flesh. ‘rosalind’ has a characteristic, red-coloured skin. ‘denar’ is a table ab and salad cultivar, which can also be used for canned and frozen products. ‘bila’ and ‘satina’ are table b cultivars of general use, while ‘tajfun’ is a b-bc table cultivar. ‘tajfun’ is characterised by the highest starch content (16.9%) followed by ‘rosalind’ (13.5%), ‘bila’ (12.8%), ‘satina’ (12.3%) and ‘denar’ (11.6%). all cultivars are characterised by a low discoloration potential, determined at a level of 8-8.5 points in the opposite danish scale 9 (9, non-darkening; 1, black), with chotkowski and stypa (2010). field experiments were carried out at the research station of the department of agriculture and technology, technical and natural science university, in mochełek (53°13’ n, 17°51’ e; 100 m a.s.l.). the harvested tubers were stored as 10 kg samples in a storage chamber for 6 months under constant conditions (temperature +4 °c and air relative humidity 95%). this experimental storage chamber was 2 m high, 2 m wide and 3.8 m in depth with milky white translucent polypropylene plate as tank shell material, which was flame retardant, thermally insulating, moisture proof and lightfast. moreover, to simulate the temperature environment and to reduce heat loss, the experimental storage chamber was covered with foam insulation material of 20 mm thickness. the contents of total dry matter (tdm) and chlorogenic acid (ca) as well as the susceptibility to tuber blackening (bs) were determined in both damaged and undamaged tubers immediately after the harvest as well as after 2, 4 and 6 months of storage. in order to determine the potatoes’ susceptibility to mechanical damage, a rotary tuber damage simulator was used; the devise included a drum with metal rods placed inside (a modified concrete 260 j. pobereżny, k. gościnna, e. wszelaczyńska, m. szczepanek mixer operating at a speed of 30 revolutions per minute). during the tests, the potato samples were subjected to damage in the drum for 1 minute. mechanical damage was inflicted to tubers 24 hours prior to the qualitative assessment. the chemicals urea, acetic acid, sodium nitrate, sodium hydroxide and phosphate buffer were purchased from merck kgaa, darmstadt, germany, and ca from sigma-aldrich co., llc, usa. determination of total dry matter the tdm content of potato tubers was determined according to the aoac method 950.01 (aoac, 1990). five tubers were washed, dried, and cut into cubes. the cubes were homogenized in a laboratory mixer (bosch, model msm67170, bsh gmbh germany) until a homogeneous pulp was obtained. about 10 g of the pulp was poured into a petri dish and then heated at 60 °c for 15 h. studies in the dryer were performed using a wamed, model sup – 100 dryer (poland). afterwards, the oven temperature was raised to 105 °c. after three hours at 105 °c, the petri dish with the dry potato was cooled to room temperature in the desiccators and weighed. the total tdm content of the potato tubers was then calculated. measurement of chlorogenic acid the ca content was determined colorimetrically by the method of griffiths et al. (1992). briefly, the diluted extract was vortexed with 2 ml of urea (0.17 m) and acetic acid (0.10 m). to this, 1 ml of sodium nitrite (0.14 m) was added, followed by 1 ml of sodium hydroxide (0.5 m) after incubation at room temperature for 2 min. the suspension was then centrifuged (hettina zentrifugen, rotina 420 r, germany) at 2250 g for 10 min. an aliquot of the supernatant was taken and the absorbance of the cherry red complex formed was read at 510 nm (uv-1800, uv spectrophotometer system, japan). a standard curve was prepared using different concentrations of ca and the results were expressed as mg of ca/1 kg of fresh potato tubers. measurement of blackspot discoloration potential analysis was carried out using the colorimetric method. for the homogenization method, equal portions of 25 g of each of the apical and basal ends of six tubers were homogenized in a laboratory mixer (bosch, model msm67170, bsh gmbh germany) for 30 s in a 25 ml 0.02 m phosphate buffer as described by delgado et al. (2001a, 2001b). the homogenate was left to oxidize for 24 h. the absorbance was measured at 475 nm with an shimadzu uv-1800, uv-vis spectral photometer system (japan). the samples were diluted at a 1:3 ratio before the photometric measurements. the results are the mean of three measurements and are presented as absorbance units (au475) at 475 nm. statistical analysis each analysed variant of experimental factors was replicated four times independently with different potato cultivars in three laboratory replications. the significance of the effects of the tested factors on the analysed characteristics was determined using the two-factor variance analysis (anova) with a statistica 12.5 software. for the testing of differences between average values at a significance level p < 0.05, fisher’s lsd test was used. in addition, coefficients of linear correlation between the tested quality characteristics of potatoes were calculated. results and discussion the results of the determination of tdm content of tubers of five cultivars of undamaged and damaged potatoes, performed immediately after the harvest and after storage, are provided in tab. 1. the results revealed that the tdm content of table potatoes was genetically conditioned and ranged from 190 to 227 g · kg-1 (tab. 1). such a result was confirmed by other authors who found that the tdm content is a varietal characteristic, and for table cultivars, it ranges from 201-230 g · kg-1 (haase, 2003; murnice et al., 2011). as for undamaged tubers, the earlycultivar ‘bila’ was characterised by the lowest tdm content (201 g · kg-1) while the highest tdm (227 g · kg-1) was noted for the medium-early cultivar ‘tajfun’. similar results were obtained by krzysztofik and skonieczny (2010) who noted that early cultivars were characterised by a lower content of tdm compared to the medium-early cultivars. however, other authors suggest that the tdm content does not depend on a cultivar’s earliness, provided that the harvest is performed in full processing maturity (murnice et al., 2011; zgórska and grudzińska, 2012) as for damaged tubers, the tdm content results were similar. ‘bila’ and ‘rosalind’ were characterised by the lowest tdm content, while ‘tajfun’ was characterised by the highest tdm content. the greatest changes to the tdm content were noted for damaged tubers after a 2-month storage period for ‘tajfun’ (4.7%), and after a 4-month storage period for ‘bila’ (3.2%) (tab. 1). the storage of potato tubers for a six-month period resulted in the significantly (p < 0.05) greatest drop in the tdm content (fig. 1). the losses ranged from 4.2% for ‘bila’ to 8.1% for ‘tajfun’ tubers, the greatest drop in the tdm content (5.6%) was noted as early as after 2 months of storage. further storage led to small, statistically tab. 1: the total dry matter (g · kg-1) of potato tubers as affected by mechanical damage and length of storage cultivar date of investigation immediate after harvest after storage 2 months 4 months 6 months ud* d* ud d ud d ud d denar 207 a 213 a 206 a 210 b 202 bc 204 a 198 bc 200 bc bila 201 a 208 a 196 c 201 a 192 a 195 a 190 a 194 ab rosalind 206 a 207 a 202 a 203 a 197 ab 203 c 195 ab 199 a satina 215 b 218 b 212 b 213 bc 207 cd 211 b 202 c 203 c tajfun 227 c 228 c 215 b 217 c 210 d 215 b 209 d 213 d *ud – undamaged, d – damaged means sharing the same letter in column are not significantly different from each other (fischer’s significant difference test, p < 0.05). data are the averages (n = 12). mechanical damage and storage on potato quality 261 insignificant changes to the tdm content for the cultivar concerned. on the other hand, for ‘bila’, the greatest drop in the tdm content was noted after 4 months and amounted to 6.6%; the extension of the period to 6 months had no significant effect. for ‘satina’ and ‘denar’, a significant drop in the tdm content was noted after a longer duration of storage, namely, after 4 and 6 months (tab. 1). similar relationships for table potatoes stored for 3 and 6 months were obtained by pobereżny and wszelaczyńska (2011) in a study in which tdm losses were at an average level of 7.1%. according to zgórska and grudzińska (2012), storage at higher temperatures of 8-10 °c results in an increase in the tdm content, which is false and associated with the loss of water. it results from the more intense life processes (i.e. transpiration and respiration) at higher temperatures. in their study, the authors obtained an increase in the tdm content from 10.5% to 18.5% when storing tubers of various cultivars at 8 °c. the damage and earliness group of a potato is also of significance to the tdm content of tubers after the storage (fig. 2). irrespective of the time of a study, damaged tubers of the medium-early group exhibited the greatest increase in the tdm content. this is associated with an increase in transpiration rate due to life processes and the sprouting of tubers during storage (casanas et al., 2003). during the study, the smallest increase in the tdm content was noted for damaged potatoes of the early cultivars immediately after the harvest and two months of storage. on the other hand, after four and six months of storage, the smallest increase in the tdm content was noted for the very early potato cultivars. obtaining such a result may be due to the more rapid completion of the natural dormancy period during the storage of tubers, resulting from genetic predispositions in relation to other earliness groups (murnice et al., 2011; casanas et al., 2003). as for damaged tubers, for all cultivars except ‘tajfun’ (tab. 1), greater losses in the tdm content were noted during storage than forundamaged tubers stored under the same conditions. the average tdm content of damaged tubers analysed after 6 months of storage dropped considerably, which was confirmed by the analysis of variance, and ranged from 19.4% for ‘bila’ to 21.3% for ‘tajfun’. a change to the tdm concentration is at the same time associated with the change to starch content (pobereżny and wszelaczyńska, 2011). in addition, mechanical damage resulted in an increase in starch losses at a level of 2%-3%, which at the same time leads to a decrease in the tdm content during the storage. according to wang et al. (2015) and olsen et al. (2003), healing of wounds during storage is an energy intensive process. the intensity of respiration increases threeto fourfold during the initial three days after dama ging the tubers. however, after a few more days it decreases but remains 1.5-2 times higher than the intensity of respiration for undamaged tubers. as reported by pagan et al. (2010), structural carbohydrates found in undamaged tubers are responsible for the intact structure and the rigidity of the potato tuber skin. even though the percentage content of structural carbohydrates (cellulose, hemicellulose) in the skin is lower than the starch content, damage leads to an intensified activation of enzymes (cellulase, xylanase), which results in the loosening or breaking of the bonds of the complex of these polymeric units. consequently, processes of exoand endocorrosion of starch granules occur due to the intensified activity of enzymes (e.g. amylase), which results in losses (bishai et al., 2015; collins et al., 2005; sujka and jamroz, 2007), but fructose, sucrose and total sugar content increased (erturk and picha, 2007). the ca content in undamaged tubers immediately after the harvest ranged from 174 mg · kg-1 f.w. for the medium-early cultivar ‘satina’ to 232 mg · kg-1 f.w for the very early cultivar ‘denar’ (tab. 2). as reported by finotti et al. (2011), andre et al. (2007), navarre et al. (2010), shakya and navarre (2006), the ca content in potato tubers varies widely from 600 to 2,920; 1,000 to 2,220; 174.0 to 12,746 mg · kg-1 d.m. and from 33 to 6,370 mg · kg-1 f.w., respectively. in the authors’ own study, storage resulted in a significant increase in the ca content of undamaged tubers, and the highest values were noted for tubers stored for a period of 6 months. an increase in the content in relation to tubers after the harvest ranged from 39.5% for fig. 1: the significant relationship between the date of investigation and total dry matter content in the potato tubers. a) undamaged, b) damaged. r – indicates that the correlation is significant at the 0.05 probability level. a) undamaged b) damaged fig. 2: the effect of damage on the total dry matter content of early potato tubers. vertical bars show ±se of means (n = 12). the interaction between storage duration and damage was significantly different at p < 0.05. 262 j. pobereżny, k. gościnna, e. wszelaczyńska, m. szczepanek ‘tajfun’ to 67.7% for ‘rosalind’. in relation to the content after the harvest, each of the storage periods resulted in a significant (p < 0.05) increase in ca content in all tested cultivars (fig. 3). only for ‘denar’ tubers, no significant increase was obtained after two months of storage. for ‘rosalind’ and ‘tajfun’, the extension of the storage period from two to four and six months had no significant effect on the content of the tested compound (tab. 2). this is consistent with the results obtained by stushnoff et al. (2008). on the other hand, grudzińska and zgórska (2011) demonstrated that the duration of storage had no significant effect on the changes to the content of polyphenolic compounds, the main component of which is ca. in turn, keutgen et al. (2014) noted a drop in the total polyphenolic compound content after a 6-month period of storage of tubers of various cultivars by as much as 81%. however, as an indicator of antioxidant activity, total polyphenol content was determined in industrially dehydrated potatoes (lyophilized material) instead of fresh materials. as expected, total polyphenol content was variable according to the vegetable composition; the lowest phenolic content was found in potatoes (gamboa-santos et al., 2012). for tubers subjected to mechanical damage, the ca content was significantly higher than in undamaged tubers and ranged from 216 mg · kg-1 f.w. for ‘satina’ to 311 mg · kg-1 f.w. for ‘rosalind’ (tab. 2). an increase in ca content due to mechanical damage was also found in studies by cantos et al. (2002) and torrescontreras et al. (2014). as shown by wang et al. (2015) and faller and fialho (2009), this is associated with the physiological response of potato tubers to damage, since under conditions stressful to the plant an intensified attack of pathogens occurs, which results in an intensified synthesis of polyphenolic compounds. for damaged tubers, the duration of storage was of less significance. the increase in ca content due to the extension of storage duration was smaller. it should be noted that, similar to undamaged tubers, the highest acid content was reported for damaged tubers stored for 6 months. the increase in the content of this compound may also result from the release of polyphenols from damaged potato flesh cells, as it was demonstrated by torres-contreras et al. (2014) in a study in which the content of ca increased with an increase in the extent of mechanical damage. similar to the tdm, damaged tubers of the medium-early group exhibited the greatest increase in ca content (fig. 4). the smallest increase in ca content was noted for. tubers of early cultivars after two months and for tubers of very early cultivars. after six months. it should be noted that mechanical damage resulted in an increase in ca content by as much as 84% in tubers of the medium-early group. this confirms that mechanical damage has a greater effect on the tab. 2: the chlorogenic acid (g · kg-1 f.w.) of potato tubers as affected by mechanical damage and length of storage cultivar date of investigation immediate after harvest after storage 2 months 4 months 6 months ud* d* ud d ud d ud d denar 232 c 294 bc 243 ab 329 b 282 a 390 a 353 b 399 d bila 187 ab 271 b 248 ab 282 ab 251 c 362 a 285 a 367 c rosalind 224 bc 311 c 319 c 316 b 352 d 360 d 376 b 430 e satina 174 a 216 a 212 a 238 a 234 b 313 b 251 a 324 a tajfun 204 abc 235 a 268 b 250 ab 278 a 277 c 285 a 282 b *ud – undamaged, d – damaged means sharing the same letter in column are not significantly different from each other (fischer’s significant difference test, p < 0.05). data are the averages (n = 12). fig. 3: the significant relationship between the date of investigation and chlorogenic acid contentin the potato tubers. a) undamaged, b) damaged. r – indicates that the correlation is significant at the 0.05 probability level. a) undamaged b) damaged mechanical damage and storage on potato quality 263 synthesis of phenolic compounds than the duration of storage. this study demonstrated that the cultivars differed significantly in the tendency to blacken the flesh of both undamaged and mechanically damaged tubers (tab. 3). as for the tested cultivars, ‘rosalind’ was characterised by the greatest tendency for bs, which was followed by ‘denar’, ‘satina’, ‘bila’, and ‘tajfun’. susceptibility of potato tubers to the bs processes is a varietal characteristic, which is also proven by results of studies by other authors (keutgen et al., 2014; zgórska and grudzińska, 2012; praeger et al., 2012). the flesh of undamaged and mechanically damaged tubers of the tested cultivars exhibited a significant (p < 0.05) tendency toward intensified bs after each storage period and reached the highest absorbance values (au475) after six months (fig. 5). dean et al. (1993) and laerke (2002) have a different opinion, as they obtained changes in the tendency to blacken tuber flesh during storage. these changes did not take place consistently throughout the entire period. during the initial months of the storage they noted an increase in the tendency to blacken, and a drop at the end of the storage period. according to wszelaczyńska (2004), an increase in the susceptibility to bs of raw tuber flesh after the storage is not due to the duration of the storage period but is associated with changes in its chemical composition. these changes relate primarily to the decrease in the concentration of both ascorbic and citric acid and to the increase in ca content. the bs of the flesh in mechanically damaged tubers immediately after the harvest was greater than for the flesh of undamaged tubers by an average of 10% (tab. 3). this is associated with the damage to plant cells, which results in an increase in enzyme activity. this process results in an increase in the content of phenolic compounds, fig. 4: the effect of damage on the chlorogenic acid content of early potato tubers. vertical bars show ±se of means (n = 12). the interaction between storage duration and damage was significantly different at p < 0.05. tab. 3: the discoloration potential (au475*1000) of potato tubers as affected by mechanical damage and length of storage cultivar date of investigation immediate after harvest after storage 2 months 4 months 6 months ud* d* ud d ud d ud d denar 229 c 231 b 280 c 332 a 324 c 337 a 378 c 419 c bila 173 a 196 a 190 a 220 a 213 ab 238 a 240 ab 309 a rosalind 268 d 310 c 346 d 396 c 388 d 415 c 429 d 434 b satina 180 a 197 a 227 b 224 a 232 b 232 a 262 b 288 a tajfun 152 b 179 a 178 a 211 a 19 a 216 a 224 a 236 c *ud – undamaged, d – damaged means sharing the same letter in column are not significantly different from each other (fischer’s significant difference test, p < 0.05). data are the averages (n = 12). fig. 5: the significant relationship between the date of investigation and discoloration potential of potato tubers. a) undamaged, b) damaged. r – indicates that the correlation is significant at the 0.05 probability level. a) undamaged b) damaged 264 j. pobereżny, k. gościnna, e. wszelaczyńska, m. szczepanek which are substrates for oxidising enzymes responsible for the formation of melanin compounds. melanin pigments are reactive quinone compounds, which lead to the formation of brown, black and red pigments in plant products. for this reason, the appearance of the products is less attractive to the consumer, and some nutritional quality is lost (cantos et al., 2002; gonzalez-santoyo and cordoba-aguilar, 2012). the smallest average increase in the tendency to blacken after a mechanical damage was noted for tubers of cultivars of the very early group, and the highest average increase was for tubers of cultivars of the medium-early group (fig. 6) associated with the highest average ca content (tab. 3). the results of the study indicate a high positive correlation between the degree of bs and the ca content (p < 0.01) and a high negative correlation between the degree of bs and the tdm content (p < 0.01) in both undamaged and mechanically damaged tubers (tab. 4). more significant relationships between these parameters were noted for damaged tubers. this indicates a significant participation of these compounds in the course of the bs reaction and a great effect of mechanical damage on the relationships concerned (wang et al., 2015; cantos et al., 2002; torres-contreras et al., 2014). relationship was also demonstrated between the tdm and ca con tents and the degree of bs. these relationships were more significant for damaged tubers. potatoes that were not mechanically damaged and were stored for a period of six months under cooling conditions retained a higher quality than tubers subjected to mechanical damage. references aoac official method 950.01. determination of dry matter. in: helrich, k. 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[in polish] tomás-barberán, f.a., espin, j.c., 2001: phenolic compounds and related enzymes as determinants of quality in fruits and vegetables. j. sci. food agric. 81, 853-876. doi: 10.1002/jsfa.885. zgórska, k., grudzińska, m., 2012: changes in selected quality parameters of potato tubers during storage. acta agroph. 19. e-issn: 2300-6730. [in polish] address of the corresponding author: e-mail: poberezny@utp.edu.pl © the author(s) 2017. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 91, 109 113 (2018), doi:10.5073/jabfq.2018.091.015 1 college of food and bioengineering, zhengzhou university of light industry, zhengzhou, henan, china 2 department of horticulture, oregon state university, mid-columbia agricultural research and extension center, hood river, oregon, usa effect of hydrogen sulfide on surface pitting and related cell wall metabolism in sweet cherry during cold storage huanhuan zhi1, yu dong2* (submitted: december 25, 2017 accepted: march 28, 2018) * corresponding author summary this study was to determine whether postharvest hydrogen sulfide (h2s) fumigation effected surface pitting development of ‘lapins’ and ‘regina’ cherries after cold storage, and if so, how it minimizes surface pitting injury and its relation to cell wall metabolism. fruit were exposed to h2s gas released from an aqueous solution of sodium hydrosulfide (nahs) at rates of 0.5, 1, 2, or 3 mm, then stored at 0 °c for 4 weeks. fruit of both cultivars treated with 1 or 2 mm nahs had a greater firmness and a lower respiration rate compared to control fruit. however, no treatments retarded losses in soluble solids content (ssc) and titratable acidity (ta). fruit fumigated with h2s displayed significantly suppressed stem browning, decay, and pitting incidences, but not weight loss. the positive effect of h2s on reduced pitting might be due to the lower yields of water-soluble polysaccharides (wsp) and cdta-soluble polysaccharides (csp) or/ and due to the lower activities of polygalacturonase (pg), pectate lyase (pl), and β-d-galactosidase (β-gal). overall, results demonstrated that h2s applied to sweet cherry as a postharvest vapor has ability to control surface pitting development by stabilizing cell wall structure and regulating cell wall catabolism. keywords: sweet cherry; hydrogen sulfide; surface pitting; storage quality; cell wall metabolism abbreviations: h2s, hydrogen sulfide; nahs, sodium hydrosulfide; ssc, soluble solids content; ta, titratable acidity; wsp, water soluble polysaccharides; cdta, cyclohexanetrans-1, 2-diamine tetra acetate; csp, cdta-soluble polysaccharides; nsp, na2co3-soluble polysaccharides; air, alcohol insoluble residue; pg, polygalacturonase; pme, petin methylesterase; pl, pectate lyase; β-gal, β-dgalactosidase. introduction fresh sweet cherries (prunus avium l.) gain popularity among consumers worldwide due to their attractive dark mahogany color, pleasing flavor, desirable taste and high nutrients. however, they are highly perishable, non-climacteric, and available only a few weeks after storage under the optimal conditions (serrano et al., 2005; wani et al., 2014). changes in fruit appearance (i.e. color, surface pitting), flavor, softening, stem quality, and dehydration are major symptoms of quality deterioration. under current marketing conditions, pitting greatly influences consumer perception of fruit quality (kappel et al., 2006). fruit pulp temperature had been identified as an important factor implicated in the development of pitting during handling, storage, and shipping of sweet cherry (lidster and tung, 1980; zoffoli and rodriguez, 2009). rapid cooling of fruit to almost 0 °c immediately after harvest is an extremely important step in postharvest handing, known to reduce pitting (drake et al., 1988; wade and bain, 1980; young and kupferman, 1994). during storage or shipping, relatively stable low-temperature (0 °c) maintain fruit quality and low respiratory activity that retard skin darkening and loss of flavor and acidity; these quality changes, when not restricted, increase cherry susceptibility to pitting (wang and long, 2014). however, cherry stored at higher temperatures and lower levels of humidity developed increased pitting, likely due to rapid water loss from the fruit (patterson, 1987; schick and toivonen, 2002). additionally, pitting development may be related to the length of storage (lidster and tung, 1980; drake and elfving, 2002). it is clear that surface pitting occurs either during handling or storage and shipping; therefore improved strategies discourage development of pitting during the postharvest period will have economic benefits for growers. pre-harvest sprays of plant growth regulators such as cacl2, gibberellic acid, or benzyladenine increase fruit firmness and improve resistance to pitting disorder (canli et al., 2015; einhorn et al., 2013; wang et al., 2015). however, sweet cherries’ response to these applications is known to delay on-tree fruit maturation and fruit with lower sugar and acid levels that are likely subject to quality deterioration or mold (kappel and macdonald, 2002). recently, hydrogen sulfide (h2s), an endogenous signaling molecule similar to carbon monoxide and nitric oxide, has been reported to activate defense responses and prolong fruit life in studies on kiwi (zhu et al., 2014), banana (li et al., 2016), strawberry (hu et al., 2012), and pear (hu et al., 2014) fruit. it has been hypothesized that h2s could up-regulate antioxidant enzymes while down regulating the expression of chlorophyll degradation-related genes and reduce membrane oxidative damage caused by reactive oxygen species (fotopoulos et al., 2015). however, whether h2s plays a functional role in reducing susceptibility to surface pitting injury remains unclear. the objective of this work was to investigate (1) the effect of h2s on fruit quality and pitting development of ‘lapins’ and ‘regina’ sweet cherries during cold storage and (2) to evaluate the possible mechanism of h2s in reducing development of pitting. materials and methods plant materials ‘lapins’ and ‘regina’ cherries were harvested from the orchard of the mid-columbia agriculture research and extension center in hood river, or, usa (latitude 45.7°n, longitude 121.545.7°w, elevation 150 m, average annual rainfall ~800 mm). for both cultivars, trees were 15-years old and on mazzard rootstock. they were maintained with standard cultural, fertilizer, herbicide and pesticide practices. twenty-five boxes (~8 kg/box) of each cultivar, selected to be free of any visible damage or fungal infection and of uniformity size were picked at commercial maturity. commercial maturity was determined as color grade 5 according to the pacific northwest dark sweet cherry development index chart developed by oregon state university, in which 1 = blush, 2 = rose, 3 = ruby, 4 = crimson, 5 = currant, 6 = merlot, and 7 = mahogany. fruit of each cultivars with pedicels intact were divided into 5 treatments × 3 replications × 1 lot per replication = 15 lots of 10 kg/lot. each lot was loaded 110 h. zhi, y. dong into a 70-l latching box (650 × 430 × 340 mm) and fumigated with 0.5, 1.0, 1.5, or 3.0 mm of sodium hydrosulfide (nahs) dissolved in 500 ml distilled water. control fruit were fumigated with the same volume of distilled water. after fumigating, h2s gas concentration in each lot was monitored using an h2s gas detector (ventis mx4, industrial scientific, pasadena, tx, usa). the h2s concentration reached approximately 0.1, 0.6 2.4, and 4.8 × 10-4 μm after 30 min when the fruit were exposed to 0.5, 1.0, 1.5, or 3.0 mm nahs, res pectively, then maintained at a constant concentration for 24 h during the fumigation treatment. all latching boxes were held at 0 °c for 24 h. after treatment, fruit were packed into commercial poly ethylene bags (~1 kg), then stored at 0 °c and >90% relative humi dity for 4 weeks. respiration rate, storage quality, weight loss, stem browning, decay and pitting incidence, cell wall compositions, and related wall-modifying enzymes were evaluated. methods respiration rate thirty fruit per replicate from each treatment were equilibrated in air at 0 °c for 5 h before being place into hermetically sealed glass containers (960 ml). after 1 h incubation, the headspace co2 were determined using co2 analyzer (900161, bridge analyzers inc., ala meda, ca, usa). respiration rate was expressed as μg co2 kg-1 s-1. fruit firmness, ssc, and ta before quality evaluations, fifty fruit from each treatment per re plicate were randomly selected and placed in the laboratory at 20 °c for 4 h, until condensation on the surface of the fruit was dried. fruit firmness was measured on opposite sides of each fruit, midway between the pedicel and calyx using a firmtech 2 fruit firmness instrument (bioworks inc., stillwater, ok, usa) and expressed as g mm-1. juice was prepared for ssc and ta measurements using a juicer (acme model 6001, acme juicer manufacturing co., sierra madre, ca, usa) equipped with a uniform milk filter strip (schwartz manufacturing co., two rivers, wi). ssc was determined using a refractometer (pal-1, atago, tokyo, japan). ta was determined by titrating 10 ml of cherry juice, diluted with 40 ml distilled water to ph 8.1 with 0.1 n naoh using a titrator (dl-15, mettler-toledo, zurich, switzerland) and expressed as the equivalent percentage of malic acid. weight loss, stem browning, decay, and surface pitting incidence the bags of fruit were weighed initially and after 4 weeks of sto rage. weight loss was expressed as percentage loss of original weight. stem browning was recorded for those pedicels exhibiting >30% of the entire surface browned. fruit decay was assessed as a percent of fruit from one hundred fruit samples showing any type of decay, however, decay organisms were not identified. surface pitting was evaluated and recorded as a percentage of one hundred fruit samples showing visible pitting. one hundred fruit from each treatment of each replicate were randomly selected and were scored for stem browning and surface pitting. isolation and measurement of cell wall polysaccharides cell wall polysaccharides were isolated and measured as described by salato et al. (2013). flesh tissue samples (10 g) were homogenized in 20 ml of 80% ethanol, then boiled for 30 min, and centrifuged at 9,000 g for 10 min at 20 °c. the supernatant was decanted and the residue twice suspended with ethanol, then twice with acetone, and dried at 75 °c for 6 h. the samples were air. air (100 mg) was treated with distilled water twice, centrifuged, and two supernatants were collected as wsp. next, the residue was treated with 50 mm sodium acetate buffer (ph 6.5) containing 50 mm cdta twice, centrifuged, and two supernatants were collected as csp. finally, the residue was treated with 50 mm na2co3 containing 10 mm nabh4 twice, centrifuged, and two supernatants were collected as nsp. extract polysaccharides solution (0.2 ml) was added to 1 ml of sulfuric acid containing 75 mm sodium borate. after boiling for 10 min, 20 μl of 0.5% naoh containing 0.15% m-phenylphenol was added to the mixture and loaded in a 96-well plate. absorbance at 520 nm was monitored in a plate reader (elx800, bio-tek instruments co., winooski, vt, usa). a standard calibration curve was constructed using galacturonic acid and the data were expressed as air mg g-1. cell wall-modifying enzymes pg activity was performed as described by gross (1982). flesh tissue samples (0.25 g) were homogenized in 5 ml of 0.3 m nacl. after centrifugation at 12,000 g for 15 min at 4 °c, the supernatant (50 μl) was added to 0.6 ml of 0.1% polygalacturonic acid, 0.6 ml of 50 mm sodium acetate buffer (ph 4.5), and 0.15 ml of 0.3 m nacl, then incubated at 37 °c for 2 h. after adding 4 ml of 0.1 m borate buffer (ph 9.0) and 0.6 ml of 1% 2-cyanoacetamide, the mixture was boiled in a water bath for 10 min. one unit of pg activity was defined as the release of 1 μg of galacturonic acid at 276 nm absorbance per min. pme activity was performed as described by basak and ramas wamy (1996). all solutions, including distilled water, nacl, pectin, and supernatant were adjusted to ph 7.5 with 0.1 m naoh. flesh tissue samples (4 g) were homogenized in 4 ml of 0.3 m nacl. after centrifugation, the supernatant (2 ml) was added to 25 ml of 0.5% pectin and then incubated for 1 h at 37 °c. the mixture was titrated to ph 8.1 with 0.1 m naoh and the volume of naoh solution was recorded. one unit of pme activity was defined as the release of 1 mm of ester hydrolyzed per min. pl activity was performed as described by belge et al. (2015). flesh tissue samples (0.25 g) were homogenized in 5 ml of 50 mm trishcl (ph 8.5) containing 0.6 mm cacl2, 5 mm edta, and 0.05% triton x-100. after centrifugation, the supernatant (0.4 ml) was added to 3.1 ml of 50 mm tris-hcl (ph 8.5) containing 0.6 mm cacl2 and 0.24% polygalacturonic acid, then incubated at 37 °c for 30 min. after boiling in a water bath for 10 min, one unit of pl activity was defined as the formation of 1 μm of 4,5-unsaturated product at 232 nm absorbance per min. β-gal was performed as described by belge et al. (2015). flesh tissue samples (0.5 g) were homogenized in 5 ml of 0.3 m nacl. after centrifugation, the supernatant (0.1 ml) was added to 2 ml of 0.04% p-nitrophenyl-β-d-galactoside and 0.3 ml of 0.3 m nacl, then incubated at 37 °c for 30 min. after adding 2 ml of 0.2 m na2co3, one unit of β-gal activity was defined as the release of 1 mg of p-nitrophenol at 400 nm absorbance per min. statistical analysis the experiment design was completely randomized with three replicates for each treatment since the trees were uniform in the orchard. homogeneity tests were performed for two continuous years and the data were homogeneous for each parameter tested. the data were combined and subjected to analysis of variance using spss software (spss inc., chicago, il, usa). when appropriate, means were separated by fisher’s protected least significant difference test at p < 0.05. results and discussion effect of h2s on fruit respiration postharvest application of h2s has been shown to reduce fruit qua lity deterioration, including appearance and texture, to resist patho effect of h2s on pitting in sweet cherry 111 gen infection, and to extend fruit storability (hu et al., 2012; hu et al., 2014; li et al., 2016; zhu et al., 2014). respiration rate is a major indicator of physiological and biochemical activity, and it increases as fruit deterioration. in this study, respiration rates were inhibited by h2s in both cultivars, especially in 1 and 2 mm nahs treatments, compared to the control (tab. 1). this demonstrated that h2s may lower respiration and lower the loss of quality for stored fruit. decay, and surface pitting (tab. 2). hu et al. (2002) reported reduced physiological disorders as a response to greater antioxidant enzymes (i.e. catalase, guaiacol peroxidase, ascorbate peroxidase, and glutathione reductase) activity which might be activated by h2s. other reports indicated that the effect of h2s on quality deterioration disorders was probably due to fruit higher energy status (li et al., 2016). however, susceptibility to pitting is an intrinsic characteristic of cherry skin. therefore, cell wall metabolism seems to be implicated in the development of pitting. tab. 1: effect of h2s on respiration rate, fruit firmness, soluble solids content (ssc), and titratable acidity (ta) of ‘lapins’ and ‘regina’ cherries at harvest and after 4 weeks of storage at 0 °c. treatments respiration rate fruit firmness ssc (%) ta (%) (μg co2 kg-1 s-1) (g mm-1) ‘lapins’ at harvest 3.21 da 315.01 e 17.01 a 0.59 a control 4.55 a 335.12 c 16.00 b 0.47 b 0.5 mm nahs 4.39 a 328.49 d 16.01 b 0.45 b 1 mm nahs 3.51 c 347.30 a 15.93 b 0.46 b 2 mm nahs 3.44 c 346.07 a 15.87 b 0.46 b 3 mm nahs 3.87 b 342.10 b 15.64 b 0.47 b ‘regina’ at harvest 2.98 d 361.14 d 18.10 a 0.45 a control 3.98 a 370.11 c 18.01 a 0.38 b 0.5 mm nahs 3.85 ab 371.08 c 17.83 a 0.41 b 1 mm nahs 3.34 c 391.10 a 17.93 a 0.41 b 2 mm nahs 3.31 c 390.08 a 18.06 a 0.41 b 3 mm nahs 3.76 b 379.33 b 18.03 a 0.38 b ameans within a column in each cultivar followed by different letters indicate significant differences according to the fisher’s protected least significant difference test p < 0.05. effect of h2s on fruit quality ‘lapins’ and ‘regina’ cherries were picked at typical commercial harvest maturity; fruit firmness was 315.01 and 361.14 g mm-1, ssc was 17.01% and 18.10%, and ta was 0.59% and 0.45%, respectively. after 4 weeks of storage at 0 °c, firmness increased in all treatments. this increased firmness may be related to moisture loss (drake and elfving, 2002), although no significant weight losses were detected between the control and h2s-treated fruit after 4 weeks (tab. 2). in both cultivars, h2s significantly increased fruit firmness, while losses in ssc and ta were not delayed by the treatment. both 1 and 2 mm nahs treatments in both cultivars led to significantly higher firmness than control and other nahs treatments. the 0.5 mm nahs treatment did not affect storage quality. the effect of the 3 mm nahs treatment were similar to the 1 or 2 mm nahs treatment for firmness, but fruit firmness was lower than that in 1 or 2 mm nahs treatment (tab. 1), indicating that high h2s concentration might increase the levels of malondialdahyde, and oxidative damage to cell membrane systems (zhang et al., 2011). these results indicated that the effect of h2s in both cherry cultivars was dosedependent, the greatest positive effect exerted by the 1 to 2 mm dose. facteau (1982) reported that fruit firmness was negatively related to pitting development in ‘bing’ cherries; our finding in h2s-treated fruit confirmed his observations. as expected, consumers favor sweet cherries with less surface pitting (chauvin et al., 2009). in both cultivars, fruit treated with h2s expressed delayed deterioration of fruit indicators stem browning, effects of h2s on cell wall composition and metabolism pitting appears as one or more irregular depressions around the fruit surface and can occur anywhere without particular location (porritt et al., 1971). examinations of the ultrastructure of fruit skin tissue has shown that a single layer of epidermal cells covers the surface of fruit, 4-5 layers of hypodermal cells with thicker walls are found below the epidermal layer, underneath the hypodermal layers are composed of radially elongated and rounded parenchy matic mesocarp cells (lane et al., 2000; param and zoffoli, 2016). pitting originating from mechanical damage is caused by the collaps of injured cells in the epidermis and hypodermis or/and paren chymatic mesocarp. anatomical differences among epidermal, hypodermal and mesocarp cells across cultivars affects cell layout, cell number, cell size, and susceptibility to pitting (porritt et al., 1971; param and zoffoli, 2016). ‘regina’ had a great uniformity in cell size and arrangement with less susceptibility to pitting, while ‘lapins’ had smaller and less-uniform cells and greater susceptible to pitting (param and zoffoli, 2016). in this study, even though h2s did not affect the initiation of mechanical damage in either cultivars, it postponed the development of macroscopically visible dents that might alter the structural composition of the cell wall in fruit skin tissue. compared to the control, h2s-treated cherries of both cultivars had lower wsp and csp and higher nsp (tab. 3), especially in 1 mm nahs treatments. previous work failed to show a relationship between the depolymerization of cell wall polysaccharides and pitting development (facteau et al., 1982; kappel et al., 2006; salato et al., 2013). our results indicated that h2s has the ability to rein force the connection between cell wall polysaccharides and the cell tab. 2: effect of h2s on weight loss, stem browning, decay, and surface pitting of ‘lapins’ and ‘regina’ cherries after 4 weeks of storage at 0 °c. treatments weight loss stem browning decay surface pitting (%) (%) (%) (%) ‘lapins’ control 0.87 aa 34.51 a 6.71 a 13.34 a 0.5 mm nahs 0.85 a 33.93 a 5.11 b 12.19 a 1 mm nahs 0.87 a 23.66 c 3.52 d 8.76 c 2 mm nahs 0.86 a 23.71 c 3.30 e 8.97 c 3 mm nahs 0.86 a 26.87 b 4.01 b 10.34 b ‘regina’ control 0.84 a 23.51 a 5.87 a 9.97 a 0.5 mm nahs 0.83 a 23.32 a 5.57 ab 9.35 ab 1 mm nahs 0.83 a 13.21 b 3.41 b 6.13 d 2 mm nahs 0.84 a 13.17 b 3.67 b 7.07 c 3 mm nahs 0.83 a 23.50 a 5.14 b 8.64 b ameans within a column in each cultivar followed by different letters indicate significant differences according to the fisher’s protected least significant difference test p < 0.05. 112 h. zhi, y. dong wall. firmer fruit was more resistant to mechanical damage, and displayed reduced susceptibility to surface pitting (toivonen et al., 2004; kappel et al., 2006). andrews and li (1995) reported that the cell wall-modifying enzymes including pg, pme, and carboxymethylcellulase increased in sweet cherry during development and reached to maximum acti vity when the fruit skin color turned to dark-red. after fruit were harvested and subjected to cold storage, the activity of pg and β-gal continued to increase, but pme activity was not affected by cold temperatures (belge et al., 2015). in this study, the activity of cell wall-modifying enzymes in ‘lapins’ and ‘regina’ cherries at harvest were pg of 118.96 and 84.79 u g-1, pme of 0.44 and 0.35 u g-1, pl of 229.20 and 187.63 u g-1, and β-gal of 437.64 and 227.67 u g-1, respectively. after 4 weeks of storage, the activity of pg and β-gal in control fruit of both cultivars increased, while no significant differences were observed in pme activity (tab. 4). fruit treated with 1 mm nahs in both cultivars displayed a significant reduction in pg activity over the control as reported in strawberry (hu et al., 2002). furthermore, pg and β-gal activity were delayed by the 1 mm nahs treatment, while pme activity was not effected by h2s (tab. 4). these results indicate that pg and β-gal may play important roles in the development of surface pitting, and that h2s could inhibit their activity, and result in reduced cell wall disassembly and improved resistance to pitting injury. belge et al. (2015) reported that pl did not show any distinct changes in ‘celeste’ or ‘somerset’ cherries when fruit were held at 0 °c for 14 days. by contrast, pl in the control of both varieties studies here increased after 4 weeks of storage; the h2s-treated fruit displayed significantly inhibited pl activity. further studies should be conducted to see if the low acti vity of β-gal in cherries is protects fruit against the development of surface pitting. conclusion this study demonstrated that postharvest application of h2s gas (1-2 mm nahs) increased fruit firmness and reduced respiration rate, stem browning, decay, and surface pitting in ‘lapins’ and ‘regina’ cherries after 4 weeks of cold storage. the potential benefit of h2s in minimizing pitting development was associated closely with reduction of the degradation of cell wall polysaccharides and suppression of cell wall-modifying enzymes (pg, pl, and β-gal) activities. these results are the first report on the effect of h2s on surface pitting development in ‘lapins’ and ‘regina’ cherries. although approval of the use of h2s gas on food has not yet been granted, an alternative reducing agent gas based on h2s tended to be effective in controlling physiological disorders and improving fruit quality in sweet cherry. acknowledgement we are grateful to the columbia gorge fruit growers association and the nw fresh pear research committees for financial support. references andrews, p.k., li, s., 1995: cell wall hydrolytic enzyme activity during development of nonclimacteric sweet cherry (prunus avium l.) fruit. j. hortic. sci. 70, 561-567. doi: 10.1080/14620316.1995.11515327 basak, s., ramaswamy, h.s., 1996: ultra high pressure treatment of orange juice: a kinetic study on inactivation of pectin methyl esterase. food res. int. 29, 601-607. doi: 10.1016/s0963-9969(96)00068-3 belge, b., comabella, e., graell, j., lara, i., 2015: post-storage cell wall metabolism in two sweet cherry (prunus avium l.) cultivars displaying different postharvest performance. food sci. technol. int. 21, 416-427. doi: 10.1177/1082013214541863 canli, f.a., sahin, m., ercisli, s., yilmaz, o., temurtas, n., pektas, m., 2015: harvest and postharvest quality of sweet cherry are improved by pre-harvest benzyladenine and benzyladenine plus gibberellin applications. j. appl. bot. food qual. 88, 255-258. doi: 10.5073/jabfq.2015.088.037 chauvin, m.a., whiting, m., ross, c.f., 2009: the influence of harvest tab. 3: effect of h2s on water-soluble polysaccharides (wsp), cdtasoluble polysaccharides (csp), and na2co3-soluble polysaccharides (nsp) of ‘lapins’ and ‘regina’ cherries after 4 weeks of storage at 0 °c. treatments wsp csp nsp (aira mg g-1) (air mg g-1) (air mg g-1) ‘lapins’ control 11.72 ab 8.96 a 13.44 c 0.5 mm nahs 10.86 b 8.89 a 14.47 b 1 mm nahs 7.00 e 6.17 d 17.71 a 2 mm nahs 7.72 d 6.64 c 17.33 a 3 mm nahs 8.53 c 7.16 b 17.04 a ‘regina’ control 8.89 a 6.44 a 15.41 c 0.5 mm nahs 7.96 b 5.98 a 15.39 c 1 mm nahs 5.43 c 3.98 b 18.46 a 2 mm nahs 5.19 c 4.17 b 17.41 b 3 mm nahs 7.79 b 6.35 a 14.97 c aair = alcohol insoluble residue bmeans within a column in each cultivar followed by different letters indicate significant differences according to the fisher’s protected least significant difference test p < 0.05. tab. 4: effect of h2s on activities of polygalacturonase (pg), petin methylesterase (pme), pectin lyase (pl), and β-d-galactosidase (β-gal) at harvest and after 4 weeks of storage at 0 °c. treatments pg pme pl β-gal (u g-1) (u g-1) (u g-1) (u g-1) ‘lapins’ at harvest 118.96 ba 0.44 a 229.20 e 437.64 e control 125.69 a 0.43 a 420.59 a 950.22 a 0.5 mm nahs 123.34 a 0.44 a 367.33 c 828.87 b 1 mm nahs 117.50 b 0.45 a 289.76 d 685.89 d 2 mm nahs 121.36 a 0.43 a 294.20 d 722.88 c 3 mm nahs 127.62 a 0.44 a 409.60 b 841.30 b ‘regina’ at harvest 84.79 c 0.35 a 187.63 e 227.67 e control 99.87 a 0.36 a 338.46 a 556.34 a 0.5 mm nahs 102.23 a 0.34 a 348.54 a 413.60 b 1 mm nahs 88.46 b 0.35 a 223.39 d 399.85 b 2 mm nahs 85.35 bc 0.36 a 249.98 c 427.66 b 3 mm nahs 96.98 a 0.38 a 290.30 b 405.57 b ameans within a column in each cultivar followed by different letters indicate significant differences according to the fisher’s protected least significant difference test p < 0.05. effect of h2s on pitting in sweet cherry 113 time on sensory properties and consumer acceptance of sweet cherries. horttechnol. 19, 748-754. drake, s.r., kupferman, e.m., fellman, j.k., 1988. bingrsquo; sweet cherry (prunus avium l.) quality as influenced by wax coatings and storage temperature. j. food sci. 53, 124-126. doi: 10.1111/j.1365-2621.1988.tb10191.x drake, s.r., elfving, d.c., 2002: indicators of maturity and storage quality of ‘lapins’ sweet cherry. horttechnol. 12, 687-690. einhorn, t.c., wang, y., turner, j., 2013: sweet cherry fruit firmness and postharvest quality of late-maturing cultivars are improved with lowrate, single applications of gibberellic acid. hortsci. 48, 1010-1017. facteau, t.j., 1982: 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w., shi, j., zhang, w., shen, y., du, h., wu, s., 2014: hydrogen sulfide extends the postharvest life and enhances antioxidant activity of kiwifruit during storage. j. sci. food agric. 94, 2699-2704. doi: 10.1002/jsfa.6613 zoffoli, j.p., rodriguez, j., 2009: fruit temperature affects physical injury sensitivity of sweet cherry during postharvest handling. acta hortic. 1020, 111-114. doi: 10.17660/actahortic.2014.1020.13 address of the author: huanhuan zhi, college of food and bioengineering, zhengzhou university of light industry, zhengzhou, henan 450002, china yu dong, department of horticulture, oregon state university, midcolumbia agricultural research and extension center, 3005 experiment station drive, hood river, or 97031, usa e-mail: dongyu@oregonstate.edu; dongyu8306@hotmail.com © the author(s) 2018. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 90, 323 329 (2017), doi:10.5073/jabfq.2017.090.040 1institute of plant molecular and cellular biology (ibmcp, upv-csic), universitat politècnica de valència, valencia, spain 2faculty of biology, ioan cuza university, iasi, romania 3dipartimento di scienze agrarie e forestali, università degli studi di palermo, palermo, italy 4agroforest mediterranean institute (iam, upv), universitat politècnica de valència, valencia, spain 5institute of plant biology and biotechnology, university of agriculture, krakow, poland (permanent address) 6the new zealand institute for plant & food research limited (pfr), mt albert research centre, auckland, new zealand (present address) 7department of botany, university of delhi, india (permanent address) characterizing the effects of salt stress in calendula officinalis l. aleksandra kozminska1,5, mohamad al hassan1,6, dinesh kumar1,7, lacramioara oprica2, federico martinelli3, marius nicusor grigore2, oscar vicente1, monica boscaiu4* (received march 3, 2017; accepted october 17, 2017) * corresponding author summary in this study the effects of salt stress on growth and several stress markers were investigated in the ornamental and medicinal plant calendula officinalis. one-month-old plants were submitted to increasing salt concentrations, up to 150 mm nacl, for a period of 30 days. salinity affected growth in terms of relative reduction of stem length and fresh weight of the plants, but water content remained unchanged indicating a certain tolerance to low and mild nacl concentrations. although na+ and clincreased in parallel to increasing salinity, the levels of k+ and ca2+ showed no significant change, while mg2+ levels recorded a twofold increase upon the ap plication of the highest salt concentration. other measured para meters showed a more significant change, notably proline levels, which registered a nine-fold increase in the presence of 150 mm nacl. in conclusion, although plants suffered from salt stress, as shown by the degradation of photosynthetic pigments and induction of oxidative stress (increased mda levels), they continued their ve getative growth under low concentrations of salt. the main mechanisms of response to salt stress in this species appear to be based on the maintenance of k+ and ca2+ homeostasis and the accumulation of proline as a functional osmolyte. keywords: calendula; ions; osmolytes; proline; salt stress introduction given the ever rising human population and the spreading of soil salinization in the context of global climate change, stress tole rance studies are becoming increasingly important. salinity can affect growth and yield of most plants (munns, 1993), by inducing reduced mitosis in roots (sharp et al., 1988) and leaves (durand et al., 1995), increase in reactive oxygen species (ros) levels (apel and hirt, 2004) and disrupted ability to detoxify them (munns and tester, 2008), chlorophyll degradation (kato and shimizu, 1987), and tissue injury (leaf senescence), among other effects (allu et al., 2014), leading to the plants’ death in case of severe or prolonged exposure to salinity. salinity induces significant changes in plants due to ionic toxicity and osmotic stress, both of which generate oxidative stress (grattan and grieve, 1999; parida and das, 2005). salinity modifies the nutritional balance of plants, notably on the ionic level, resulting in higher ratios of na+/ca2+, na+/k+, na+/mg2+, cl-/no3and cl-/h2po4-, thus causing growth retardation (grattan and grieve, 1999). growing more salt-tolerant ornamentals and non-crop species with economic importance (such as medicinal plants), can open up salt affected croplands – amounting to nearly 400 million hectares worldwide (al-sadi et al., 2010) – for re-use, and thus make more cultivable land available for crops that could be more affected by salinity in terms of yield. for example, saline soils that cannot support standard crops might be used to grow medicinal plants as novel crops (muhammad and hussain, 2010). one medicinal plant – which is also used as ornamental – with unknown salt tolerance potential is calendula officinalis l., commonly known as marigold (not to be confused with tagetes species, commonly known as french and african marigolds). c. officinalis has a very long history of medi cinal use, especially for its anti-inflammatory properties. calendula extract-containing creams and gels are commonly used to treat skin irritation, inflammation and burns, especially after radiotherapy, and to aid wound healing (edwards et al., 2015). the species is a short perennial that rarely survives hot summers or hard winters. it does however tolerate a wide range of soil types, and as such is considered among the most versatile flowers for ornamental and domestic use. c. officinalis is native to southern europe, but it has been naturalized in northern europe, and many temperate regions of north africa, china, and the united states. despite the large body of literature on salt stress, little is known about the effects of salinity on this plant. several studies have focused on the effects of salinity on seed germination or on seedling growth in c. officinalis (antonello and espindola, 2008; torbaghan, 2012, gharineh et al., 2013), whereas some others discussed water relations and ionic balance (chaparzadeh et al., 2003), or some biochemical responses (chaparzadeh et al., 2004; oprica et al., 2015). taking into account the economic potential of this species, further studies regarding the effects of salt stress on c. officinalis are deemed necessary. the aim of this study was to analyze the growth of c. officinalis at low and moderate concentrations of nacl and to assess which are the most relevant biochemical responses to salinity in this species. materials and methods plant material and growth conditions seeds of c. officinalis were obtained from the agricultural research and development station, secuieni, neamt county, romania, and were sterilized with 5% sodium hypochlorite solution before being thoroughly rinsed with milli-q water, prior to sowing on a mixture of commercial peat and vermiculite (3:1). after germination, seedlings were transplanted into individual polyethylene pots (ø = 11 cm) with the same substrate. during the growth period, plants were irrigated with hoagland’s nutrient solution (hoagland and arnon, 1950). both the growth (4 weeks) and the following treatment (4 weeks) periods were carried out in the greenhouse of the institute of plant molecular and cellular biology, polytechnic university of valencia, 324 a. kozminska, m. al hassan, d. kumar, l. oprica, f. martinelli, m.n. grigore, o. vicente, m. boscaiu spain. the greenhouse maintained a controlled environment, with regulated temperatures ranging between 17 and 23 °c, under a longday photoperiod (16 h light / 8 h dark), with light intensity of 130 μe m-2 s-1. four-week-old plants were treated for a period of additional four weeks, being watered twice per week with 2 l of hoagland’s nutrient solution per tray (each tray with 10 individual pots), containing nacl to the final concentrations of 0 (for the control treatment), 50, 100, or 150 mm; therefore, 10 replicates (individual plants) were used for each treatment. plant growth parameters after four weeks of treatments, the aerial part of each individual plant was harvested and the following growth parameters were determined: stem length, leaf number, fresh weight (fw), and water content percentage. fresh material was stored at -20 °c for further studies. fresh weight was expressed in percentage of the values corresponding to the non-stressed controls. to determine water content, part of each sample was weighed (fw), dried at 65 °c until constant weight (96 h), and then weighed again (dw); water content of each sample (%) was calculated as described in gil et al. (2014). photosynthetic pigments total carotenoids (caro), chlorophyll a (chl a) and chlorophyll (chl b) were measured following lichtenthaler and wellburn (1983): 0.1 g of fresh leaf material was ground in the presence of 20 ml of ice-cold 80% acetone, mixed by vortexing and centrifuged. the supernatant was collected and its absorbance was measured at 663, 646, and 470 nm. the concentrations of chl a, chl b, and caro were calculated according to the equations described by lichtenthaler and wellburn (1983). the final values were expressed in mg g−1 dw. monovalent and divalent ions ion measurements were performed according to weimberg (1987) in aqueous extracts obtained by heating the samples (0.05-0.1 g of dried, ground plant material in 25 ml of water) in a water bath, for 15 min at 99 °c, followed by filtration through a 0.45 μm filter (gelman laboratory, pall corporation). sodium and potassium ions were quantified with a pfp7 flame photometer (jenway inc., burlington, usa), and chlorides were measured using a merck spectroquant nova 60® spectrophotometer and its associated test kit (merck, darmstadt, germany), while calcium and magnesium ions were measured with an atomic absorption spectrometer spectraa 220 (varian, inc., ca, usa). proline and total soluble sugars proline and total soluble sugars (some of the most common osmolytes in plants) were determined in the stressed and control plants. proline (pro) was extracted with 3% (w/v) sulfosalicylic acid, from 0.05 g of dry plant material, and was quantified according to the acidninhydrin method described by bates et al. (1973). pro concentration was expressed in terms of μmol g-1 dw. total soluble sugars (tss) were measured in 0.05 g dry plant material suspended in 3 ml 80% (v/v) methanol, following the phenol / sulfuric acid method, according to dubois et al. (1956). tss contents were expressed as ‘mg equivalent of glucose’ per g dw. mda and non-enzymatic antioxidants malondialdehyde (mda) content was determined according to the method described by hodges et al. (1999) in the same extracts as those used to measure tss. the samples were mixed with 0.5% thiobarbituric acid (tba) prepared in 20% tca (or with 20% tca without tba for the controls), and then incubated at 99 °c for 15 min. after stopping the reaction on ice, the absorbance of the supernatants was measured at 532 nm. total phenolic compounds (tpc) were quantified according to blainski et al. (2013), by reaction with the folin-ciocalteu reagent. absorbance was measured at 765 nm, and the results expressed in equivalents of gallic acid (mg · eq · ga g-1 dw). total flavonoids (tf) were measured following the method described by zhishen et al. (1999), based on the reaction of catechol groups with alcl3; the absorbance was measured at 510 nm, and the amount of flavonoids was expressed in equivalents of catechin (mg · eq · c g-1 dw). statistical analysis statistical analysis was performed using the program statgraphics centurion v. xvi (statpoint technologies, warrenton, virginia, usa). before the analysis of variance, the shapiro-wilk test was used to check for validity of normality assumption and levene test for the homogeneity of variance. if anova requirements were accomplished, the significance of the differences among treatments was tested by one-way anova at a 95% confidence level and posthoc comparisons were made using the tukey hsd test. results plant growth parameters an inhibition of vegetative growth due to salt stress was observed in terms of stem length and fresh weight percentage (fig. 1). stem length decreased, as compared to the non-stressed plants, with those from the 150 mm nacl treatment recording a reduction of 40% of the control (fig. 1a). concerning fresh weight, a decrease of nearly 40% was also observed in plants treated with the highest salt concentration, 150 mm nacl (fig. 1b). on the other hand, water content (wc%) did not change significantly in response to the nacl treatments (fig. 1c). photosynthetic pigments sodium chloride affected photosynthetic pigments levels in the leaves of c. officinalis in all treatments (fig. 2). chl a contents decreased by nearly 50% under the strongest salt treatment applied (fig. 2a), while chl b and caro recorded reductions of about 40% in the presence of 150 mm nacl (fig. 2b, 2c, respectively). monovalent and divalent ions na+ increased in parallel to increasing external salinity, from approx. 350 μmol g-1 dw in the leaves of control plants to 1500 μmol g-1 dw in the presence of 150 mm nacl (fig. 3a), and clfrom 450 μmol g-1 dw to 2300 μmol g-1 dw under the same conditions (fig. 3b). k+ levels did not show any statistically significant change in response to the application of nacl (fig. 3c). average leaf levels of divalent cations increased with increasing nacl concentrations in the nutrient solution; however, the observed changes in ca2+ contents were not statistically significant (fig. 3d), while a significant increase of nearly twofold over the control, non-stressed plants was measured in the case of mg2+ (fig. 3e). osmolytes (proline and total soluble sugars) in order to maintain osmotic pressure under different abiotic stress conditions causing cell dehydration, plants accumulate certain sol effects of salinity in calendula 325 fig. 1: growth parameters in c. officinalis after four weeks of salt treatments. (a) stem length (cm), (b) fresh weight of the aerial part (% of the control), and (c) water content percentage (wc %). means with sd (n = 10). different lowercase letters above the bars indicate significant differences between nacl treatments, according to the tukey test (α = 0.05). fig. 2: photosynthetic pigments in c. officinalis after four weeks of salt treatments. (a) chlorophyll a (chl a), (b) chlorophyll b (chl b), and (c) total carotenoids (caro). means with sd (n = 10). different lowercase letters above the bars indicate significant differences between nacl treatments according to the tukey test (α = 0.05). uble inert compounds – compatible solutes or osmolytes – such as proline (pro) and soluble sugars (tss, e.g. glucose, fructose and sucrose, among others). the application of nacl induced an increase in pro levels in a concentration-dependent manner, reaching a 9-fold increment over the control in plants submitted to the 150 mm nacl treatment (fig. 4a). on the other hand, the changes observed in tss levels did not correlate with the applied salt concentrations. mda and non-enzymatic antioxidants leaf contents of malondialdehyde (mda), an oxidative stress biomarker that usually peaks under abiotic stress conditions, showed an increase in response to the salt treatments, up to twofold over the control in the presence of 150 mm nacl (fig. 5a). antioxidants such as phenolic compounds and flavonoids tend to increase under stress in many species, to alleviate the secondary oxidative stress accompanying salt and other abiotic stresses. however, this does not seem to be the case in c. officinalis, as we did not observe such an increase in the leaf contents of total phenolic compounds (tpc, fig. 5b) or total flavonoids (tf, fig. 5c) upon the application of nacl; in fact, a slight reduction in the levels of these compounds was detected in the presence of salt (figs. 5b, 5c). discussion salt stress reduces plant growth and productivity by affecting morphological, anatomical, biochemical and physiological characteristics, processes and functions. disturbed water and nutritional balance of plants may cause reduced crop yields in saline soils. reduction in plant height and other growth parameters are the most distinct and obvious effects of salt stress, since inhibition of growth is probably the most general response of plants to stress (munns, 2002; munns and tester, 2008). depressed growth due to high salinity is attributed to several factors such as osmotic stress, specific ion toxicity and ion imbalance, and induced nutritional deficiency. in the present study, plant height decreased under salt stress, as found in all other studies performed on this species (chaparzadeh et al., 2003; bayat et al., 2012; mirlotfi et al., 2015; nofal et al., 2015). although salinity affects growth in calendula, low concentrations of 50 and 100 mm nacl did not reduce drastically the stem length and fresh weight of the aerial part. plants from the 50 and 100 mm treatments had only a 20% lower fw in comparison to the control, which is similar to the reduction of only 10 and 27% at 50 and 100 mm nacl, respectively, reported by chaparzadeh (2003); this degree of saltinduced growth inhibition is much lower than that reported under similar conditions for stress sensitive cultivars of different crops (see, for example, bartha et al., 2015; al hassan et al., 2016a, among 326 a. kozminska, m. al hassan, d. kumar, l. oprica, f. martinelli, m.n. grigore, o. vicente, m. boscaiu many other references). moreover, salt treatments did not produce a significant change in the water content of the plants leaves, indica ting as well a relatively high resistance to dehydration, which will certainly contribute to some degree of salt tolerance in this species. although clearly a glycophyte, c. officinalis is able to maintain vegetative growth at low concentrations of salt. therefore, it may be fig. 3: ion concentrations in c. officinalis after four weeks of treatments. (a) sodium (na+), (b) chloride (cl-), (c) potassium (k+), (d) calcium (ca2+), and (e) magnesium (mg2+). means with sd (n = 10). different lowercase letters above the bars indicate significant differences between nacl treatments according to the tukey test (α = 0.05). fig. 4: osmolytes levels in c. officinalis after four weeks of treatments. (a) proline (pro) and (b) total soluble sugars (tss). means with sd (n = 10). different lowercase letters above the bars indicate significant differences between nacl treatments according to the tukey test (α = 0.05). considered for cultivation on soils that are not optimal for more salt sensitive crops. photosynthetic pigment concentrations in the leaves of salt stressed c. officinalis recorded a significant decrease in comparison with those measured in unstressed controls, which is likely due to chlorophyll degradation induced by toxic levels of nacl. these results are consistent with those reported by bayat et al. (2012), who indicated that chlorophyll content significantly decreased in the leaves of c. officinalis with increasing nacl concentration. reduction of chlorophyll levels in salt-treated plants is due to the inhibition of chlorophyll synthesis, together with the activation of its degradation by the enzyme chlorophyllase (santos, 2004). yet, this is not the only reason for the inhibition of photosynthesis in the presence of salt, since nacl also inhibits key enzymes involved in this process, such as rubisco and pep carboxylase (soussi et al., 1998). there are, however, some reports suggesting that chlorophyll and caro tenoid contents in c. officinalis are not affected by salinity (mirlotfi et al., 2015) or can even increase in the presence of salt (oprica et al., 2015). this apparent contradiction may be due to the use of different experimental growth conditions in these reports. in any case, a reduction of photosynthetic pigment levels appear to be a general response to salt stress (parihar et al., 2015), as we have shown in previous work with other species (al hassan et al., 2015, 2016a, b; kumar et al., 2017). an interesting pattern of variation was found in the ion content of foliar tissue. as the applied salt concentration increased, na+ and cl levels also increased, in a concentration-dependent manner, reaching more than four-fold and five-fold, respectively, over the corresponding controls in the presence of 150 mm nacl. sodium accumula tion in plants is usually accompanied by the inhibition of many enzymatic activities and physiological processes, and by a reduction in the concentrations of potassium, as both ions compete for the same membrane transporters (rodríguez-navarro, 2000). moreover, k+ deficiency has negative effects on photosynthesis, osmoregulation, protein biosynthesis and turgor driven movements (gierth and mäser, 2007). intracellular k+ and na+ homeostasis is important for effects of salinity in calendula 327 the activities of many cytosolic enzymes, and for maintaining membrane potential and an appropriate osmoticum for cell volume regulation. therefore mechanisms which allow maintaining relatively low na+/k+ ratios are essential for salt tolerance of plants (niu et al., 1995; rodríguez-navarro, 2000). contrary to this general be havior, this study showed that the accumulation of na+ in the leaves of c. officinalis did not lead to a significant reduction of k+, indi cating that a possible defense mechanism against salt stress is the activation of k+ transport from roots to the aerial parts of plants, avoiding excessively high na+/k+ ratios in the leaves, as we have also reported for some other species (schiop et al., 2015; al hassan et al., 2016b). salt tolerance is also depending on the plant’s capacity to accumulate na+ and clin the vacuole, to avoid reaching toxic concentrations in the cytoplasm, a mechanism that is especially efficient in some succulent, highly tolerant dicotyledonous halophytes. this ‘ion compartmentalisation hypothesis’ (flowers et al. 1977; wyn jones et al., 1977; glenn et al., 1999) is generally accepted, although it is supported by few experimental data, given the technical difficulties to determine ion concentrations in the vacuole and the cytoplasm separately. since we also measured ion contents in the whole leaf material, without any subcellular discrimination, we cannot estimate the possible contribution of this mechanisms to the salt tolerance of c. officinalis. regarding changes in divalent cations, their average leaf concentrations increased under salt treatments, but this variation was significant only for mg2+ (not for ca2+). the divalent cation ca2+, an essential mineral nutrient, has an important and well-known role in the mechanisms of plant responses to salinity, reducing the harmful effects of na+ (bressan et al., 1998; hepler, 2005; gul and khan, 2006). the possible role of mg2+ in those mechanisms is not so well established, but it has been shown that inhibition by salt of some enzymes that use mg2+ as cofactor is due to its displacement from the active centre by na+ (albert et al., 2000). therefore, increased concentrations of intracellular mg2+ (without reaching toxic levels) may partially counteract the inhibitory effect of na+ and contribute to salt stress tolerance in plants (boscaiu et al., 2011; grigore et al., 2012). osmolyte accumulation in the cytosol is a general response to abiotic stress in all plants, both halophytes and glycophytes. besides their main function in osmotic adjustment, osmolytes play many other important roles in the mechanisms of stress defense, as low-molecular weight chaperones, reactive oxygen species (ros) scavengers or signalling molecules (ashraf and foolad, 2007; szabados and savouré, 2010; gil et al., 2013). proline is one of the most studied compatible solutes of plants, and is considered to play the primary role in protecting plants against osmotic stress in many different species (ashraf and foolad, 2007; verbruggen and hermans, 2008; al hassan et al., 2016b). in this study, we found a clear positive correlation between pro accumulation and the intensity of the applied stress treatments, suggesting its participation in the mechanisms of salt resistance in c. officinalis, as osmoprotectant and contributing to cellular osmotic adjustment under stress conditions. the role of soluble sugars and polyalcohols in salt tolerance is also well documented (gil et al., 2011, 2013). total soluble sugars accumulate depending on the intensity of salt stress, time exposure and organs of plants where it has been measured (nemati et al., 2011). in our experiments we did not find a uniform pattern regarding the accumulation of total sugars in response to the exposure to salt, as changes in leaf sugar contents did not correlate with increasing external nacl concentrations. therefore, with the present data it is difficult to speculate about their possible role in salt stress tolerance in c. officinalis. it cannot be ruled out that, in addition to pro, some specific sugar may act as a functional osmolyte in c. officinalis, accumulating specifically in the leaves of salt-treated plants, this being masked by changes, not related to salt stress responses, in the concentrations of other sugars. fractionation of the extracts, for example by hplc, identification and quantification of individual sugars would be required to check this hypothesis. a clear symptom of oxidative damage is cell membrane degradation; therefore, mda – a product of membrane lipid peroxidation – is an excellent marker of oxidative stress (del rio et al., 2005). in this study a significant increase of mda levels in c. officinalis upon salt stress treatments of the plants was observed, indicating that plants suffered from secondary oxidative stress associated with salinity. c. officinalis is rich in several compounds considered as ‘health-promoting’, such as carotenoids, flavonoids and other phenolics (raal and kirsipuu, 2011). these secondary metabolites play multiple roles in plants, including scavenging of ros induced under different stress conditions and causing oxidative stress. in our study, leaf levels of carotenoids, total phenolics and flavonoids, all showed a significant, concentration-dependent decrease in c. officinalis salt-treated plants; this suggests that these compounds do not play any major role in the mechanisms of tolerance to salinity in this species. in conclusion, although c. officinalis is affected by salt stress at the vegetative growth stage, it shows nonetheless a certain tolerance to low and moderate concentrations of nacl (50 100 mm). the responses to increasing salt concentrations contributing to this tole fig. 5: oxidative stress marker mda and chemical antioxidants in c. officinalis after 4 weeks of treatments. (a) malondialdehyde (mda), (b) total phenolic compounds (tpc), and (c) total flavonoids (tf). means with sd (n = 10). different lowercase letters above the bars indicate significant differences between nacl treatments according to the tukey test (α = 0.05). 328 a. kozminska, m. al hassan, d. kumar, l. oprica, f. martinelli, m.n. grigore, o. vicente, m. boscaiu rance appear to be related to the maintenance of k+ and ca2+ leaf levels and a slight increase in mg2+, to counterbalance the negative effects of na+, and to the synthesis and accumulation of proline as a physiological osmolyte. therefore, c. officinalis might be considered as a possible candidate for cultivation on soils regarded unfit for more sensitive crops, as it would tolerate low quality, slightly saline water for irrigation. however, if grown as a medicinal plant, it should be taken into account that the contents of some interesting secondary metabolites, such as antioxidant carotenoids and phenolics, will decrease in c. 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(eds.), regulation of cell membrane activities in plants, 121-136. elsevier, amsterdam. zhishen, j., mengcheng, t., jianming, w., 1999: the determination of flavonoid contents in mulberry and their scavenging effects on super oxide radicals. food chem. 64, 555-559. address of the corresponding author: agroforest mediterranean institute (iam, upv), universitat politècnica de valència, spain, camino de vera s/n, 46022, valencia, spain e-mail: mobosnea@eaf.upv.es © the author(s) 2017. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 89, 73 81 (2016), doi:10.5073/jabfq.2016.089.009 1pear research station, national institute of horticultural and herbal science, naju, korea 2 functional food research center, bk21 plus program, graduate school of chonnam national university, gwangju, korea physiochemical, nutritional and functional characterization of 10 different pear cultivars (pyrus spp.) sun-hee yim1, seung-hee nam2* (received september 29, 2015) * corresponding author summary this study was performed to compare the physiochemical properties and nutritional components including sugars, amino acids, and minerals of 10 common pear cultivars cultivated in korea (four pyrus spp.). furthermore, the pear cultivars were characterized for functional properties with respect to phenolic compounds by hplc/ dad analysis and antioxidant activities using dpph and abts assays. among the 10 pear cultivars that were tested, niitaka and hanareum pears show the best physiochemical properties such as higher sugar/acid ratio and proper firmness. they also showed relatively enriched soluble sugar (12.6 ~ 13.0 g/100 g fw), amino acid (4.5 ~ 7.3 g/100 g dw) or mineral contents with high k/na ratio. for functional properties, niitaka and hanareum pears have significantly higher contents of total phenolics (240 mg/100 g dw), arbutin (103 ~ 124 mg/100 g dw), and chlorogenic acid (11 mg/ 100 g dw) as well as strong antioxidant activities (49 % or 86 %) among cultivars. these results indicate that niitaka, and hanareum cultivars, could be best for consumption or favorable processing due to excellent product quality and high concentrations of nutritional and functional compounds. introduction pear fruit (pyrus spp.) is one of the most widely consumed fruits in the world, and the fruit type is comprised of oriental and occidental varieties due to different geographical developments. oriental pears are cultivated mainly throughout eastern asia including china, the koreas, and japan. the major species of oriental pears include pyrus bretschnrideri, pyrus ussuriensis, pyrus pyrifolia, and pyrus sinkian, while most occidental pears belong to the pyrus communis species (cui et al., 2005). as pear is a seasonal fruit, it is typical ly eaten fresh and is often found in processed foods such as juice, puree, jellies, and jams. for many years, however, pear has also been used as a herbal medicine for the relief of coughing, and for its antitussive, anti-inflammatory, and diuretic activities (li et al., 2014). recently, researchers have focused on analyses and comparisons involving the nutritional components of pear such as sugars, vitamins, amino acids, minerals, and fatty acids (tanrioven and eksi, 2005). these nutrient components play important roles in a qualitative evaluation of pear fruits, wherein attributes such as color, taste, and natural value are assessed (ashoor and kanox, 1982). the sugar, amino acid, and fatty acid compounds of eight commercial pear cultivars from china were identified and quantified using hplc and gas-chromatography (gc) (chen et al., 2007). during storage, yali pear from pyrus bretschneideri was also studied for changes of the volatile aroma components, sugars, organic acids, minerals, and amino acids (wang et al., 2002; chen et al., 2006). certain chemicals of pear fruits such as total sugars, titratable acidity (ta), and soluble solids content (ssc) have been foremost quanti fication parameters because of their influence on the fruit’s organoleptic properties (teng and liu, 1999). additionally, occidental pear (pyrus communis) and prickly-pear cultivars were studied for soluble solids, total ta, and ph during the ripening process (hemandez-perez et al., 2005; barroca et al., 2006). apart from the above nutritional and chemical components, a variety of phenolic compounds such asarbutin, chlorogenic acid, catechin, and epicatechin have also been identified as primary active components in pears (li et al., 2012; chaalal et al., 2013; garcia-cruz et al., 2013). the phenolic compounds in pear cultivars have also been further evaluated for beneficial health functions such as antioxidant, antiinflammatory, and antimicrobial activities. the phenolic compounds of some pear cultivars distributed in ankara and bursa, turkey, and southwestern germany including arbutin, chlorogenic acid, and epicatechinin were determined by hplc (schieber et al., 2001; tanrioven and eksi, 2005). the peels and flesh of 10 different pear varieties (pyrus spp.) from china were analyzed for their totalphenolic, total-flavonoid, and total-triterpene contents, and were also measured for antioxidant and anti-inflammatory activities (cui et al., 2005; li et al., 2014). the whole and ground seeds of three pricklypear cultivars were studied for phenolic compounds, in vitro antioxidant capacity, and antiradical potential (chaalal et al., 2013). in another study, the peel, flesh, and cores of three pear cultivars were analyzed for phenolic compounds and antioxidant activity during the ripening process (zhang et al., 2006). in our previous work, five unripe pear cultivars (pyrus pyrifolia) were measured for total-phenolic and arbutin contents, characterized with respect to antioxidant activities, and their whitening functions were examined according to the inhibition of tyrosinase and cellular melanin formation (yim and nam, 2015). so far, most studies have focused on the physiochemical and/or nutritional properties or the functional characterization of some pear cultivars, but a simultaneous study of all of these has never been conducted for 10 different pear cultivars. this research focused on an analysis and comparison of the physiochemical properties and nutritional components such as minerals, sugars, and amino acids among 10 common pear cultivars that are grown in korea (four pyrus spp.). furthermore, the pear cultivars were compared for functional properties with respect to phenolic compounds and antioxidant activity by using hplc analysis, dpph assay, and abts assay. a more detailed knowledge of the variability of the physiochemical and functional properties of pear cultivars will be helpful in the selection of pear cultivars with more beneficial nutritional and functional properties and favorable processing characteristics. materials and methods plant materials ten pear cultivars from four species were used in this study. to compare the whole-fruit contents of different species and cultivars, the following 8 cultivars of 3 species of oriental pear (pyrus bretschneideri, pyrus ussuriensis, and pyrus pyrifolia) and 2 cul74 s.-h. yim, s.-h. nam tivars of occidental pear (pyrus communis) were sampled. there are jules d’airolles, abate fetal (p. communis), laiyangchili, yali (p. bretschneideri), ingyebae, cheongbae (p. ussuriensis), wonwhang, niitaka, hanareum, and chuwhang (p. pyrifolia). all of the cultivars were picked from 15 year olds trees that were located at an orchard of the naju national pear experimental station in the chonnam province of korea, and were harvested at the mature fruit stage only. seven to ten fruits from each of the selected standard pear trees were used for the experiments. all of the samples were of a uniform size and consisted of no defects. each of the whole pear fruits was washed, rapidly cut into thin slices, and lyophilized by freeze-dryer (fd8512, ilshin, korea). the samples were then grinded by a pulverizer (fm-681c, hanil electric, korea) and stored at -20 °c in polyethylene bags until further analysis. the methanol extracts of the pear cultivars were prepared for total flavonoid contents, antioxidant capacities, and hplc analysis of phenolic compounds. the pear powder of 10 cultivars (10 g) had been previously homogenized in a moulinex stirrer, and was later extracted with 80 % ethanol using the soxhlet extraction method at 60 °c for 6 h. the extracts were then filtered through whatman no 1 filter paper. the residues were extracted again with 80 % ethanol using the previously mentioned method, and the extracts were then combined and evaporated to dryness under a vacuum. the dried extract was then prepared at a concentration that consisted of 0.1 g/ml of the freeze-dried pear in methanol. otherwise, fresh pear fruits from the 10 cultivars were used to determine physical properties or sugar content. reagents arbutin, gallic acid, catechin, chlorogenic acid, caffeic acid, and pcoumaric acid (pheolic-compound standards) were purchased from wako pure-chemical industries (japan). folin-ciocalteu’s phenol reagent, dpph (1, 1-diphenyl-2-picrylhydrazyl), abts (2, 2-azinobis3-ethylbenzothiazoline-6-sulfonic acid), trolox (6-hydroxy-2, 5, 7, 8-tetramethyl chromane 2 carboxylic acid), citric acid, and quercetin were purchased from sigma chemical co. (st. louis, mo, u.s.a.). hplc-grade acetonitrile and formic acid (98 % purity) were also purchased from sigma aldrich-fluka (st. louis, mo, u.s.a.). all of the other solvents and chemicals were of analytical grade. physiochemical properties weight, ssc, ta, and firmness were analyzed using fresh pears from the 10 cultivars. the fresh weight was measured by weighing 20 randomly chosen pear fruits using an analytical balance. the ssc was determined by a digital refractometer (model ra-250he, kyoto electronics co. ltd., japan) at 22 °c. the ta was determined by an automatic titrator with sodium hydroxide, and it is expressed as the percentage of citric acid (valente et al., 2013). firmness is measured after peel-removal at the two opposite sides on the equator of each pear using a penetrometer with an 8 mm diameter tip. puncture tests were performed using a ta-xt2 texture analyzer (stable micro systems, surrey, uk). the work that was required to penetrate the flesh to the maximum penetration depth was retained as the penetrometer-firmness parameter (valente et al., 2013). this parameter, expressed in n mm, was obtained by calculating the area under the force displacement curve. ssc, ta, and firmness analyses were carried out in quintuplicate on the 10 pear cultivars. nutritional compositions: soluble sugars, amino acids, and minerals soluble sugar, amino acids, and minerals were determined among the 10 pear cultivars. seven to ten fruits from each standard pear trees were selected for the experiments with uniformity in size and no defects. pooled pears from each cultivar were washed, rapidly cut into thin slices, and grinded with homogenizer for fruit juice or lyophilized by freezedryer for dry powder. soluble sugar composition was determined according to the previous study (chen et al., 2007), the pear fruit juice (10 g) was milled and diluted to 100 ml with redistilled water, and was then filtered through a 0.45 μm millipore filter. an aliquot of 20 μl was injected into the hplc system. a waters hplc system (waters 717 plus auto sampler, milford, ma, u.s.a.) with a refractive index detector was employed in a monosaccharide analysis wherein a 5 μm aminopropyl column was utilized (phenomenex, torrance, ca, u.s.a.). the mobile phase consisted of acetonitrilewater (85:15, v/v) at a flow rate of 1.5 ml/min, at 30 °c for 20 min. sucrose, glucose, fructose, and sorbitol (sigma chemical co, st. louis, mo, u.s.a.) were used as standards. chromatographic data were analyzed using millennium software (pharmaceutics international, hunt valley, md, u.s.a.). sugar contents were expressed in g/100 g of fresh weight (fw). the amino acid contents of the 10 pear cultivars were determined according to the previous study using an amino-acid analyzer (hewlettpackard amino quant series ii, hp 1090) (bora et al., 2003). pear powder (0.1 g) was added to an ampoule and hydrolysis was carried out using 5 ml of 6 n hcl containing 0.05 % mercaptoethanol, at 105 °c for 24 h. filtered hydrolyzate was dried in a vacuum desiccator and redissolved in 0.1 n hcl. sample solution (10 μl) was injected directly into the amino-acid analyzer with a reverse-phase column using a buffer (20 mm sodium-acetate, ph 7.2, 0.018 % trimethylamine, 0.3 % tetrahydrofuron) and b buffer (100 mm sodium-acetate, ph 7.2, 40 % acetonitrile, 40 % methanol). double pre-derivatization of the amino acids was achieved by reacting with orthophthalide, which was derivatized with 9-fluorenylmethyl chloroformate. the carrier gas was maintained at a flow rate of 0.45 ml/ min, and the gradient was 0 % to 60 % of channel b in 17 min. an amino-acid standard solution (sigma chemical co, st. louis, mo, u.s.a.) was used for obtaining qualitative and quantitative information. free amino-acid contents were expressed in mg/100 g of dry weight (dw). minerals were quantified by using flame atomic-absorption spectrometry (icpms) (hewlett-packard 4500) according to previous study (chen et al., 2007). dried fruit samples (0.5 g) were stored overnight in 10 ml of concentrated hno3. then, 1.5 ml of concentrated hclo4 and 2 ml of concentrated h2so4 were added. the temperature was gradually raised to the range of 200 ~ 250 °c and was maintained until complete charring was achieved. the oven temperature was then raised to 550 °c and maintained for 6 h to 8 h until a white ash remained. the concentrations of each element (k, ca, na, mg, zn, fe, or cu) in the pear fruits was determined using inductivelycoupled plasma-flame emission spectrometry (icp-fes) (shimadzu aa6701 equipped with shimadzu hvg-1 hydride vapor generator). a calibration curve to quantitate each mineral was established and the correlation coefficient is higher than 0.995. mineral contents were expressed in mg/100 g of dw. total phenolic and flavonoid contents the total phenolic content in the extracts of the 10 pear cultivars (0.1 g/ml) was determined using folin-ciocalteu’s reagent as described in previous study (cui et al., 2005). the absorbance of each sample was measured at 765 nm with the uv/visible spectrophotometer (jp/u-3900, hitachi, japan.) after incubation at 25 °c for 2 h. total phenolic content was calculated from a calibration curve where gallic acid was used as the standard (160 ~ 960 mg/100 g dw). the flavonoid content was measured according to the aluminum-chloride colorimetric method (boo et al., 2009) and quercetin was used as the standard. the flavonoid content was determined at 506 nm with a spectrophotometer. the data were expressed as mg /100 g dw, and the calibration curve ranged from 40 mg to 840 mg. characterization of 10 pear cultivars 75 phenolic compounds by hplc-dad phenolic compounds from the pear extracts were determined using a shimadzu prominance lc-20a hplc-pad (photodiode array detector) system using ace 15 c18 hplc column (250 × 4.6 mm, advanced chromatography technologies ltd., scotland, uk). pear methanol extract (0.1 g/ml) was filtered through a 0.22 μm filter (agilent technologies, seoul, korea), and 10 μl of each of the standard and sample solutions were injected into the hplc system. the mobile phases were 2 % (v/v) aqueous acetic acid (solvent a) and 0.5 % (v/v) acetic acid in 50 % acetonitrile (solvent b). the following binary elution system was applied: 2 % b at initial 5 min to wash the column, and a linear gradient of 55 % b (50 min), 100 % b (10 min), and 10 % b (5 min). after 70 min, the organic-phase concentration was brought back to 2 % (b) and lasted for 6 min for column equilibration. the flow rate was set at 1 ml/min at 25 °c, and simultaneous monitoring was performed at 280 nm for arbutin, gallic acid, and catechin, and 320 nm for chlorogenic acid, caffeic acid, and p-coumaric acid. the phenolics in the hplc chromatogram were identified by the time of retention. retention times were 3 min (arbutin), 6 min (gallic acid), 15.4 min (chlorogenic acid), 6.9 min (caffeic acid), 20 min (catechin), and 27 min for p-coumaric acid under the conditions of the analysis. dpph and abts radical-scavenging activity the dpph radical scavenging activities of the pear extracts were determined as described previously (blios, 1958). a 0.25 ml of sample (0.1 g/ml) and 0.8 ml of 0.15 mm dpph· (methanol) were added and shaken vigorously, followed by incubation at room temperature for 30 min in the dark. the control sample was prepared with 80 % ethanol instead of pear extract. the decrease of absorbance in the presence of dpph· was measured at 517 nm using a spectrophotometer (jp/u-3900, hitachi, tokyo, japan) wherein trolox was used as a reference compound. the radical-scavenging activity was expressed as a % of the inhibition of dpph· radicals using the following equation: dpph radical scavenging activity (%) = 1 − (sample absorbance/ control absorbance) × 100 the abts radical scavenging activity of the pear extracts was measured by the abts cation decolorization assay with some modifications (re et al., 1999). the abts radical cation (abts+·) was produced by a reaction of 7 mm abts stock solution with 2.45 mm potassium persulfate, which was allowed to stand in the dark at room temperature for 12 h to 16 h before use. the abts+· solution was diluted with methanol to give an absorbance of 0.7 ± 0.01 at 734 nm. the pear extracts (0.1 g/ml) were allowed to react with 2 ml of the abts+· solution for 1 min, after which time the absorbances were measured at 734 nm. trolox was used as the reference compound. the radical-scavenging activity was expressed as the % of the inhibition of abts radicals using the following equation: abts radical scavenging activity (%) = (1 − sample absorbance/ control absorbance) × 100 statistical analysis all of the experiments were carried out with three replicates and the results were expressed as mean ± standard deviation. the data were analyzed using a one-way anova, and the means of different groups were compared to the duncan’s multiple-range test (dmrt) using the spss version 17.0 statistical-software package. values of p < 0.05 are considered significant in all cases. results and discussion physiochemical properties the physiochemical properties of the 10 pear cultivars are presented in tab. 1. the pears that were examined include the following 10 major cultivars of korea from 4 species: jules d’airolles, abate fetal (p. communis), laiyangchili, yali (p. bretschneideri), ingyebae, cheongbae (p. ussuriensis), wonwhang, niitaka, hanareum, and chuwhang (p. pyrifolia). the level of fresh weight, the ssc, the ta, the ratio of sugar to acid, and the firmness differed considerably across the 10 cultivars. a high sugar/acid ratio and appropriate firmness are considered favorable intrinsic characteristics for the selection of appropriate cultivars to eat fresh due to the beneficial taste effect (chen et al., 2007). in terms of fruit weight, niitaka, wonwhang, and chwhang pears showed a 2.5 times higher than yali and cheongbae pears which have relatively lower weights among pear cultivars. it has been suggested that sugar/acid ratio and hardness can be used as criteria for the ripeness of fresh fruits (pal and kumar, 1995). the jules d’airolles and abate fetal pears in our study show relatively high levels of ssc and tab. 1: physiochemical properties of 10 different pear cultivars. species cultivars weight ssc1 ta2 sugar/acid firmness (g) (%) (%) (n) jules d’airolles 454 ± 26c 15.4 ± 0.2a 0.37 ± 0.01a 42.0 ± 1.0f 14.7 ± 1.1a abate fetal 349 ± 20d 14.9 ± 0.1b 0.21 ± 0.00b 70.1 ± 1.4cde 13.3 ± 0.5b laiyangchili 289 ± 24e 12.3 ± 0.1f 0.14 ± 0.01e 84.0 ± 1.9c 9.4 ± 0.5d yali 238 ± 34f 10.9 ± 0. 1g 0.21 ± 0.01bc 52.0 ± 1.1ef 7.4 ± 0.1e ingyebae 342 ±37d 10.7 ± 0.2h 0.19 ± 0.00c 56.8 ± 1.7def 8.6 ± 0.3d cheongbae 233 ± 11f 12.2 ± 0.2f 0.19 ± 0.00b 64.4 ± 1.1cde 6.7 ± 0.2e wonwhang 532 ± 9b 12.3 ± 0.1f 0.16 ± 0.00d 76.4 ± 1.4cd 14.1 ± 0.6ab niitaka 635 ± 49a 12.5 ± 0.1e 0.09 ± 0.02f 138.9 ± 31.2b 11.0 ± 0.4c hanareum 361 ± 5d 14.3 ± 0.1c 0.07 ± 0.01g 192.5 ± 11.7a 11.5 ± 0.8c chuwhang 525 ± 13b 14.0 ± 0. 1d 0.21 ± 0.01bc 66.9 ± 2.2cde 11.1 ± 0.3c 1 ssc (soluble solids content) 2 ta (titratable acidity) 3 nd (not detected) values are means ± sd and means with the same letter within columns are not significantly different (dmrt, p < 0.05). pyrus communis pyrus bretschneideri pyrus ussuriensis pyrus pyrifolia 76 s.-h. yim, s.-h. nam ta, while ingyebae and cheongbae pears have lower ssc and ta levels. the sugar/acid ratios of jules d’airolles, abate fetal, ingyebae, and cheongbae pears are the lowest with ratios of 42 ~ 70 %. however, niitaka and hanareum pears have the highest soluble solids but the lowest acid contents. the sugar/acid ratios of niitaka and hanareum pears are 2 to 3 times higher than those of other pears with ranging from 138 % to 192 %. the fruit firmness is relatively very high for the jules d’airolles and abate fetal pears with 13.3 ~ 14.7 n, whereas it is low for the ingyebae and cheongbae pears with 6.7 ~ 8.6 n. the fruit firmness of niitaka and hanareum pears are moderate with 11 n. the sugar/acid ratios of our pear cultivars (42 ~ 192 %) are similar or slightly higher than those of major pear cultivars from china (23 ~ 125 %) (chen et al., 2007). some studies report that the sugar/acid ratio of pear fruit is not only affected by the ripeness but also the pear cultivar or cultivation conditions (hemandez-perez et al., 2005; barroca et al., 2006). among the 10 tested pear cultivars, the niitaka and hanareum pears show the best physiochemical properties such as higher sugar/acid-ratio levels and proper firmness. nutritional compositions: soluble sugars, amino acids, and minerals soluble sugars sugars are one of the biochemical components of fruit quality, and their kinds and amount directly influence fruit-flavor components such as sweetness (moriguchi et al., 1992). fructose, glucose, sucrose, and sorbitol were identified in this study as the principal saccharides of each pear fruit (tab. 2). regarding the total sugar contents, the jules d’airolles, abate fetal, hanareum, and chuwhang pears showed relatively high contents while laiyangchili pear had low. for individual sugars, fructose is the predominant sugar, accounting for 35 % to 56 % of the total sugars. the fructose contents in the pears are approximately 2 times higher than glucose or sorbitol contents. interestingly, laiyangchili pear contains remarkably higher sorbitol content than the other pears. as the sweetness characteristic of the fruit, a large variety of sucrose levels was observed among the pear cultivars, ranging from 0.2 to 3.0 g per 100 g fw. the determined values of the sucrose in pears were much lower by approximately 5 ~ 10 times than fructose contents. the trends of the fructose and glucose contents across the 10 cul tivars are similar to the trend of the total soluble sugar contents. however, sugar contents of the pears show different patterns from those of fructose or glucose. since sugar contents of the jules d’airolles, wonwhang, and niitaka cultivars are clearly high but those of laiyangchili is relatively low among cultivars. among four pear species, p. communis (jules d’airolles, abate fetal) and p. pyrifolia (hanareum, chuwhang) pears possess relatively higher soluble sugar contents, whereas p. bretschneideri (laiyangchili, yali) and p. ussuriensis (ingyebae, cheongbae) pears have relatively lower soluble sugar contents (tab. 1 and 2). the quantification of the glucose and fructose contents in this study agrees with the previously reported ranges for pear (moriguchi et al., 1992; chen et al., 2007). in this study, the species and geographical origin influence the sugar and phenolic contents more than the other factors (chen et al., 2007; li et al., 2012). amino acids six amino acids out of the 15 kinds of free amino acid standards were determined from the 10 pear cultivars and are presented in tab. 3. the amino acids of the 10 pear cultivars are aspartic acid, glutamic acid, proline, threonine, valine, and phenylalanine in descending order of abundance. the most abundant amino acids of the 10 pears in this study are aspartic acid and glutamic acid. these results agree with the existing research that reports glutamic acid as the major amino acid among 15 free amino acids with respect to the major pear cultivars (chen et al., 2007). the role of gluta mic acid has been reported as the provision of the characteristic ‘‘umami taste’’ to foods with high, free glutamate content such as cheese, tomato, and mushrooms, which are also major ingredients in cooking (bellisle, 1999). regarding the total free amino acid contents, niitaka, and chuwhang pears show relatively high contents in our study, with 6.8 ~ 7.3 g/100 g dw, whereas the jules d’airolles and abate fetal pears contain low contents, with 1.8 ~ 2.1 g/100 g dw. large variations of proline values are shown among the pear cultivars, ranging from 3 ~ 190 g/100 g dw (tab. 3). this pattern agreed with a previous report concerning proline content among pear cultivars (chen et al., 2007). among the pears tested, cheongbae pear had significantly higher proline content and was one of disease resistant cultivars. since proline plays an important role in plant growth and defense as a key determinant of many cell wall proteins. it accumulates both under stress and non-stress conditions as a beneficial solute in plants (stintzing et al., 2001). this may be the reason why cheongbae pear results in significantly high proline content (stintzing et al., 2001). pyrus communis pyrus bretschneideri pyrus ussuriensis pyrus pyrifolia tab. 2: soluble sugar compositions of 10 different pear cultivars (g/100 g fw). species cultivars sucrose glucose fructose sorbitol total1 jules d’airolles 2.72 ± 0.01c 2.46 ± 0.01f 6.29 ± 0.01 d 2.13 ± 0.01 h 13.6 ± 0.4c abate fetal 1.58 ± 0.10 e 3.36 ± 0.01b 6.61 ± 0.02 b 2.98 ± 0.01 d 14.5 ± 0.1a laiyangchili 0.26 ± 0.01i 2.05 ± 0.01i 5.04 ± 0.01 g 3.69 ± 0.01 a 11.1 ± 0.3h yali 0.53 ± 0.05g 2.30 ± 0.02 g 4.83 ± 0.02 h 3.56 ± 0.01 b 11.2 ± 0.1g ingyebae 0.84 ± 0.01f 2.26 ± 0.02h 6.21 ± 0.01e 1.73 ± 0.01i 11.0 ± 0.1h cheongbae 0.39 ± 0.01h 3.90 ± 0.01 a 5.12 ± 0.02f 3.01 ± 0.02c 12.4 ± 0.1f wonwhang 3.06 ± 0.01a 2.82 ± 0.01d 4.47 ± 0.02 i 2.18 ± 0.01 g 12.5 ± 0.2e niitaka 2.78 ± 0.01b 2.88 ± 0.02c 4.21 ± 0.02 j 2.71 ± 0.01 e 12.6 ± 0.1e hanareum 1.56 ± 0.01g 2.40 ± 0.03f 6.36 ± 0.01 c 2.71 ± 0.01 e 13.0 ± 0.1d chuwhang 1.92 ± 0.02d 2.79 ± 0.01 e 6.80 ± 0.01 a 2.56 ± 0.01 f 14.1 ± 0.3b 1 total sugar is the sum of individual sugars. value are means ± sd (n = 3), and means with the same letter within columns are not significantly different (dmrt, p < 0.05). characterization of 10 pear cultivars 77 minerals minerals are dietary requirements for humans and exert various physiological effects. the mineral compositions of the 10 pear cultivars are listed in tab. 4. k and ca are minerals that are essential for controlling the salt balance, bone structure, and functions of the human body. mg is also useful to the body as a minor component of bones and plays a catalytic role in respiration. with respect to macrominerals, k is the most abundant mineral in pears, followed by na, ca, and mg. the k/na ratio may be useful for compensating for high na levels in a typical human diet. the k/na ratio is relatively higher at cheongbae and chuwhang pear but lower at laiyangchili among 10 cultivars since cheongbae and chuwhang pears contained relatively high contents of potassium among cultivars (tab. 4). the 10 cultivars show similar calcium (20 ~ 33 mg/100 g dw) and magnesium levels (10 ~ 26 mg/100 g dw). for microminerals, zn is especially important for the normal functioning of the immune system, and fe is the major component of essential biological compounds such as transferrin, ferritin, and haemoglobin (brody, 1994). large variations of zn values are shown among the pear cultivars, ranging from 0.8 mg to 3.0 mg/100 g dw (tab. 4). meanwhile, other microminerals such as fe, cu, and mn show similar values among the pear cultivars. cheongbae pear has higher amounts of microminerals than the other pears. micromineral levels obtained in this work are similar to those obtained from other studies. however, the contents of macrominerals in the pear cultivars are much lower than those of other cultivars (tab. 4), and this may be due to the differences between pear cultivars rather than the individual method that we used (chen et al., 2007; salvador et al., 2010). soluble sugars, amino acids, and mineral compositions are important factors for qualitatively evaluating the nutritional value of fruits and their potential use for different products. among the 10 pear cultivars that we investigated, niitaka, hanareum and chuwhang pears show the highest levels of biologically-active chemical compounds. functional characterization: total phenolics and flavonoids, phenolic compounds by hplc, and antioxidant activity total phenolic and flavonoid contents the total phenolic and flavonoid contents of the 10 cultivars (pyrus spp.) are presented in fig. 1. the 10 pear cultivars that were tested have total phenolic contents ranging from 109 ~ 261 mg/100 g dw, while the flavonoid contents were between 68 mg and 177 mg/100 g tab. 3: major amino-acid compositions of 10 different pear cultivars (mg/100 g dw). species cultivars proline valine threonine aspartic acid glutamic acid phenylalanine total1 jules d’airolles 73.0 ± 1.6c 2.9 ± 0.1gh 16.6 ± 2.5d 1582 ± 102f 491 ± 19de 1.17 ± 0.14h 2167 ± 113ef abate fetal 129.9 ± 9.4 b nd2 nd 1151 ± 141g 583 ± 52c 2.56 ± 0.15g 1867 ± 192f laiyangchili 12.7 ± 1.1e 6.8 ± 0.9def 16.1 ± 1.5d 1833 ± 68 f 533 ± 35 cd 6.71 ± 0.31c 2408 ± 47e yali 9.1 ± 0.4e 8.4 ± 1.1cd 15.3 ± 0.6d 3061 ± 164 e 1009 ± 45 a 5.86 ± 0.21 d 4109 ± 199d ingyebae 18.8 ± 2.4 e 4.1 ± 0.5 fg nd 5951 ± 195 b 478 ± 19 de 4.13 ± 0.15 f 6457 ± 207b cheongbae 190.7 ± 11.9a 37.4 ± 4.6a 58.2 ± 8.5a 4475 ± 311 c 402 ± 31 e 3.80 ± 0.34f 5168 ± 368c wonwhang 38.4 ± 3.0d 10.7 ± 0.5bc 16.2 ± 0.8d 1906 ± 55f 406 ± 47 e 8.58 ± 0.39b 2387 ± 100e niitaka 17.5 ± 2.0e 7.6 ± 1.1 cde 24.2 ± 2.4c 6785 ± 418a 479 ± 88 de 9.66 ± 0.41 a 7324 ± 508a hanareum 4.2 ± 0.5e 13.6 ± 2.9b 28.0 ± 2.3c 4034 ± 244d 450 ± 55de 3.81 ± 0.26f 4535 ± 291d chuwhang 3.1 ± 0.7e 4.9 ± 0.7efg 36.0 ± 2.1b 5894 ±161b 908 ± 49 b 5.18 ± 0.08e 6852 ± 203b 1 total means the sum of individual amino acids 2 nd (not detected). value are means ± sd (n = 3), and means with the same letter within columns are not significantly different (dmrt, p < 0.05). pyrus communis pyrus bretschneideri pyrus ussuriensis pyrus pyrifolia pyrus communis pyrus bretschneideri pyrus ussuriensis pyrus pyrifolia tab. 4: mineral compositions of 10 different pear cultivars (mg/100 g dw). species cultivars macro minerals micro minerals k na ca mg zn fe cu mn jules d’airolles 170 ± 36ab 70 ± 9ab 23 ± 5ab 16 ± 6abc 3.01 ± 0.51 a 4.32 ± 0.31de 1.20 ± 0.20c 1.11 ± 0.10c abate fetal 170 ± 10ab 57 ± 6ab 20 ± 6b 10 ± 1c 2.21 ± 0.61b 6.00 ± 0.32 b 0.72 ± 0.10d 0.51 ± 0.09e laiyangchili 157 ± 47bcd 83 ± 4 a 30 ± 4ab 23 ± 1ab 1.32 ± 0.22c 4.32 ± 0.71de 1.61 ± 0.44ab 0.55 ± 0.08e yali 140 ± 10bcd 63 ± 5 ab 26 ± 5ab 17 ± 1abc 2.32 ± 0.50ab 2.15 ± 0.80f 0.92 ± 0.11cd 2.32 ± 0.21 a ingyebae 150 ± 10bcd 57 ± 5 ab 23 ± 5ab 20 ± 1abc 2.81 ± 0.51ab 4.42 ± 0.32de 1.61 ± 0.31b 0.71 ± 0.04 d cheongbae 210 ± 10a 60 ± 4ab 33 ± 5a 26 ± 5a 2.72 ± 0.31 ab 5.52 ± 0.40bc 2.00 ± 0.11 a 1.41 ± 0.11 b wonwhang 123 ± 6cd 50 ± 7b 20 ± 2b 17 ± 5 abc 2.62 ± 0.11ab 12.8 ± 0.22a 0.81 ± 0.11d 1.12 ± 0.10c niitaka 140 ± 10bcd 60 ± 6ab 23 ± 5ab 13 ± 5bc 1.35 ± 0.40c 4.12 ± 0.50de nd 0.52 ± 0.05e hanareum 120 ±11d 57 ± 6ab 20 ± 1b 20 ± 3abc 1.10 ± 0.10c 3.50 ± 0.40e 0.91 ± 0.09cd 0.40 ± 0.04e chuwhang 167 ± 15bc 60 ± 4ab 23 ± 5ab 20 ± 3abc 0.80 ± 0.21c 4.90 ± 1.10cd 0.60 ± 0.04d 0.42 ± 0.03e value are means ± sd (n = 3), and means with the same letter within columns are not significantly different (dmrt, p < 0.05). 78 s.-h. yim, s.-h. nam dw. among the species tested, the cheongbae, niitaka, and hanareum pears have significantly higher total phenolic contents with 230 ~ 261 mg/100 g dw, while jules d’airolles and abate fetal show lower value of 109 ~ 120 mg/100 g dw. the flavonoid content followed a similar trend to that of the total phenolic content, with higher values from 115.7 mg ~ 129.5 mg/100 g dw in cheongbae, niitaka, and hanareum. here, niitaka, the representative cultivar with above 70 % domestic production, has higher values for total phenolics and flavonoids. in general, total phenolic contents in pear cultivars are higher by about 1.2 ~ 3.0 times than those of flavonoid contents. this agrees with the research that reports the concentrations of phenolics of both oriental and occidental pears as much greater than those of flavonoids (galvis-sanchez et al., 2003; valente et al., 2013; li et al., 2014). the total phenolic contents of the 10 pears (261 mg/100 g dw) were similar or less than those of other studies (263 ~ 823 mg/100 g dw) (cui et al., 2005; li et al., 2012; 2014). it could be reasoned from different cultivated environments, cultivars or detection methods. with respect to total phenolic content (fig. 1), our values are comparable with the contents in other kinds of fruit such as guava (179 mg/100 g fw), banana (51 mg/100 g fw), and peach (112 ~ 126 mg/100 g fw) (veberic et al., 2008; arranz et al., 2009). phenolic compounds by hplc-dad the different phenolic compounds in the ethanol extracts of the 10 pears were determined by hplc/dad. the phenolic contents of the 10 pears were comprised of one phenolic glucoside (arbutin), one flavanol (catechin), and three phenolic acids (chorogenic acid, caffeic acid, and gallic acid) and are given in tab. 5. some variations (p < 0.05) of the phenolic concentrations were noted among the 10 pear samples. the phenolic content varies greatly among the pear cultivars according to tab. 5. in terms of total phenolic content, cheongbae, niitaka, and hana reum show higher levels of 135.2 ~ 161.2 mg/100 g dw, but jules d’airolles and abate fetal display lower levels of 60.6 ~ 81.0 mg/100 g dw. the 10 pears show similar trends in the contents of the four phenolic constituents with the exception of catechin (tab. 5). the highest amounts were exhibited in the case of arbutin, followed by chlorogenic acid, caffeic acid, and gallic acid, with a trace of catechin, over 10 tests. arbutin is the predominant compound across all of the cultivars, accounting for 43 % to 72 % of the total phenolic compounds. the chlorogenic acid contents of the pears are approximately 20 % to 50 % of the arbutin content, but are slightly higher than the caffeic acid content or gallic acid content. the amounts of catechin in pears are also quite low with large variations (1.0 ~ 11.9 mg/100 g dw). arbutin (4-hydroxyphenyl β-d glucopyranoside) is the main phenolic constituent in pear fruit and attracted attention for its wide use as an antioxidant or human skin whitening agent for the prevention of unnecessary spots and freckles (maeda and fukuda, 1996). chlorogenic acid is a potential chemopreventive agent, and possesses important bioactivities including antioxidant, antitumor, and immune system enhancement activities (krakauer, 2002). in particular, the contents of arbutin and chlorogenic acid among the cultivars seem to be positively correlated with the trend of the total phenolic contents (tab. 5 and fig. 1). the arbutin and chlorogenic acid contents obtained from the cheongbae, niitaka, and hanareum cultivars are obviously higher than those of the other cultivars, ranging from 103.7 ~ 124.4 mg and 11.0 ~ 20.7 mg per 100 g dw, respectively. this agrees with the report that niitaka contains greater arbutin and chlorogenic acid concentrations than the other cultivars, like chuwhang or hosui (zhang et al., 2006). interestingly, the occidental pears, jules d’airolles and abate fetal displayed total phenolic or arbutin amounts that are half those of the other 8 cultivars from oriental pears (tab. 5 and fig. 1). it shows similar result with other fig. 1: total phenolic and flavonoid contents of 10 different pear cultivars. value are means ± sd (n = 3). different letters (a to g) of each indicate significant difference at p < 0.05 according to dmr test. the cultivars used are jules d’airolles (jd), abate fetal (af), laiyangchili (ly), yali (yl), ingyebae (ig), cheongbae (ch), wonwhang (ww), niitaka (nk), hanareum (ha), and chuwhang (cw). tab. 5: phenolic compounds of 10 different pear cultivars (mg/100 g dw). species cultivars arbutin gallic acid catechin chlorogenic acid caffeic acid total1 jules d’airolles 26.21 ± 2.21h 7.85 ± 0.11 d 5.09 ± 0.18d 11.59 ± 0.11c 9.89 ± 0.21bc 60.6 ± 2.1i abate fetal 41.15 ± 1.22 g 7.95 ± 0.11d 10.46 ± 0.86b 11.64 ± 0.17 c 9.88 ± 0.11bc 81.0 ± 2.2h laiyangchili 79.85 ± 1.33 ef 9.12 ± 0.23 ab 3.17 ± 0.13 f 11.86 ± 0.24c 9.42 ± 0.80c 113.3 ± 3.2e yali 84.94 ± 2.57 d 8.76 ± 0.20abc 1.06 ± 0.17h 16.86 ± 0.50b 10.55 ± 0.41 a 122.1 ± 2.6cd ingyebae 78.15 ± 1.35f 9.40 ± 0.30a 11.98 ± 0.84a 11.30 ± 0.22cd 9.90 ± 0.13bc 120.6 ± 4.0d cheongbae 116.01 ± 2.55b 8.36 ± 0.72bcd 5.91 ± 0.22c 20.78 ± 0.12a 10.16 ± 0.15ab 161.2 ± 2.5a wonwhang 79.71 ± 1.45 ef 8.46 ± 0.10bcd 1.25 ± 0.04h 11.16 ± 0.25 cd nd2 100.5 ± 4.2g niitaka 124.45 ± 2.67a 8.36 ± 0.15bcd 1.20 ± 0.09 h 11.21 ± 0.14cd nd 145.2 ± 7.3b hanareum 103.75 ± 5.62c 8.27 ± 0.08cd 2.47 ± 0.05g 11.08 ± 1.02d 9.69 ± 0.73bc 135.2 ± 5.6c chuwhang 82.82 ± 2.21de 8.39 ± 0.05bcd 3.44 ± 0.04 e 5.51 ± 1.00e 9.85 ± 0.12bc 110.0 ± 2.4 f 1 total means the sum of individual phenolic compounds 2 nd (not detected). value are means ± sd (n = 3), and means with the same letter within columns are not significantly different (dmrt, p < 0.05). pyrus communis pyrus bretschneideri pyrus ussuriensis pyrus pyrifolia characterization of 10 pear cultivars 79 study in that the mean concentration of arbutin in oriental pear cultivars is twice as high as that of occidental pear cultivars (cui et al., 2005). however, differences exist between the findings of our study and previously research that reports much higher concentrations of chlorogenic acid in occidental pears than oriental pears (cui et al., 2005). our results show that the concentrations of chlorogenic acid in the oriental pear cultivars are similar to or slightly higher than those of the occidental pear cultivars. the values of chlorogenic acid in pears could be attributed to the different cultivars rather than their geographical origin (li et al., 2014; chen et al., 2006). it is worth mentioning that the yali pear contains remarkably higher amounts of chlorogenic acid or caffeic acid than the other pears, with values of 16.8 mg or 10.5 mg/100 per g dw, respectively. besides arbutin and chlorogenic acid, caffeic acid, gallic acid, and catechin are also important biologically active constituents of pear and variations of their levels are similar among the 10 pear cultivars. no detectable amounts of caffeic acid were found in the wonwhang or niitaka pears. this result agrees with the study that reported that caffeic acid was not detectable in the niitaka cultivar (zhang et al., 2006). abate fetal and ingyebae cultivars display the highest amounts of catechin, from 10.5 ~ 11.9 mg/100 g dw. the 10 pears do not have measurable amounts of p-coumaric acid, with the exception of laiyangchili (5.7 mg/100 g dw). dpph and abts radical-scavenging activity the antioxidant activities of the 10 pear extracts were evaluated by dpph· or abts+· radical scavenging assays (fig. 2). dpph· can only be dissolved in organic media (especially in alcohols), not in aqueous media, and this limits the ability to interpret the role of hydrophilic antioxidants. in contrast, abts+· can be solubilized in either aqueous or organic media, allowing for the interpretation of the roles of hydrophilic and lipophilic antioxidants. with regard to the dpph· scavenging activity, the 10 pear cultivars show significant differences (p > 0.05) with dpph bleaching abilities from 20.5 ~ 58.8 %. the free radical scavenging activity of the pear cultivars increased linearly with increases of the sample concentration (data not shown). it was reported that the antioxidant capacities of the pear extracts according to a dpph assay were significantly different among the pear cultivars, ranging from 10 ~ 85 % (li et al., 2014; 2012). among the cultivars examined in this study, the cheongbae, niitaka, and hanareum cultivars have the highest antioxidant activities, whereas occidental pears, jules d’airolles and abate fetal cultivars, displayed relatively lower activities. this result was similar to that of other study (li et al., 2012), where by the antioxidant activity of occidental pear (p. communis) was lower than those of oriental pears (p. bretschneideri or p. ussuriensis). pear cultivars showed similar activity values when the antioxidant capacities per serving (100 g) were compared in other study (galvis-sanchez et al., 2003). in comparison to other fruits, the pear fruits show slightly lower antioxidant activities with 59 %. chinese plum and peach are reported to have antioxidant activities of 80.9 % and 76.8 %, respectively, at the same sample concentrations of 100 mg/ml (lee et al., 2008; kim et al., 2009). in terms of abts+· radical scavenging activity, 10 pear cultivars show similar values ranging from 31.7 ~ 98.3 % of antioxidant activity, although some differences were observed among the cultivars (p < 0.05). for the 10 pear cultivars, the antioxidant capacities by the two methods show sound agreement. cheongbae, niitaka, and hanareum presented the strongest abts+· bleaching activities (84 ~ 98 %), while jules d’airolles and abate fetal showed the lowest dpph scavenging capacity (31.7 ~ 60.5 %) (fig. 2). cheong bae, niitaka, and hanareum, with high total phenolic, flavonoid, and arbutin contents, exhibited significantly higher antioxidant abilities than the other cultivars (fig. 1 and 2). therefore, it can be deduced that total-phenolic and flavonoid contents provide major contributions to the antioxidant capacities of pears. a number of studies have reported that phenolic compounds including arbutin and chlorogenic acid are the main phytochemicals responsible for the antioxidant capacities of vegetables and fruits (du et al., 2009; salta et al., 2010). the phenolic compounds of pears provide a much greater contribution to antioxidant capacity than vitamin c (galvis-sanchez et al., 2003). the sugar/acid ratio of pear was commonly used to determine the flavor quality of fruit, which is consistent with the preference of consumers for pear fruit. it was noteworthy that sugar/acid ratios of niitaka, and hanareum pears had the highest among cultivars due to their highest soluble solids but the lowest acid content. sugar/ acid ratios in niitaka, and hanareum pears were 139 or 192, which are greater values than found in the other study, ranged from 23 ~ 125 (chen et al., 2007). niitaka, and hanareum pears considered as appropriate cultivars for eating fresh at home or commercial pear juice concentrates. besides high sugar/acid ratios, niitaka, and hanareum pears possess enriched nutritional or functional compounds as well as strong antioxidant activities. niitaka is the representative cultivar with above 70 % domestic production in korea due to high yield. however, niitaka pear showed relatively lower sweetness but chuwhang pear had high sweetness and late maturing of flower. thus, hanareum pear is hybrid progeny of niitaka × chuwhang to increase the sweetness of niitaka. according to tab. 2 and 3, it seems to be successful to produce optimum pear, hanareum with high yield and sweetness. further research should be conducted to produce excellent pear cultivars with enhanced stress or disease resistant property by hybridization of niitaka or hanareum × cheongbae. pear quality, nutrition, and function are affected by many factors such as fruit cultivar, region diversity, and cultivation condition. here, our research can provide ten pears chemical and functional composition characteristics. this work will provide valuable information for further research on such fruits, and will also provide insights into the potential health benefits of the pear fruit, thereby supporting its nutritional and functional applications. conclusions current study is a first evaluation report on physiochemical and nutritional properties as well as functional characterization of 10 pear cultivars in korea (four pyrus spp.). among pear cultivars investigated, niitaka, and hanareum pears showed optimum physiochemical properties like high sugar/acid ratio and high nutritional compounds fig. 2: antioxidant activities of 10 different pear cultivars according to dpph and abts assays. value are means ± sd (n = 3). different letters (a to g) of each indicate significant difference at p < 0.05 according to dmr test. the cultivars used are jules d’airolles (jd), abate fetal (af), laiyangchili (ly), yali (yl), ingyebae (ig), cheongbae (ch), wonwhang (ww), niitaka (nk), hanareum (ha), and chuwhang (cw). 80 s.-h. yim, s.-h. nam such as sugars, amino acids, and minerals. in addition, niitaka, and hanareum pears possessed higher phenolic and flavonoid contents, enriched arbutin and chlorogenic acid, and strong antioxidant activity. those results indicate that niitaka, and hanareum cultivars, could be best for consumption or favorable processing due to excellent product quality and high concentrations of nutritional and functional compounds. acknowledgements this work was carried out with the financial support of the cooperative research program for agriculture science & technology development (project no. pj01002401) at the rural development administration, republic of korea. references arranz, s., saura-calixto, f., shaha, s., kroon, p.a., 2009: high contents of 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ultrasound for a better mango fruit quality characterization. j. food eng. 116, 57-64. veberic, r., colaric, m., stampar, f., 2008: phenolic acids and flavonoids of fig fruit (ficuscarica l.) in the northern mediterranean region. food chem. 106, 153-157. wang, j., xu, j.z., chen, h.j., zhang, h.j., li, s.l., 2002: the determination of volatile matters and amino acids content in the fruit of ya pear. food technol. 9, 71-73. yim, s.h., nam, s.h., 2015: antioxidant and whitening activities of five unripe pear cultivars. j. appl. bot. food qual. 88, 186-191. zhang, x., koo, j., eun, j.b., 2006: antioxidant activities of methanol extracts and phenolic compounds in asian pear at different stages of maturity. food sci. biotechnol. 15, 44-50. characterization of 10 pear cultivars 81 address of the corresponding author: seung-hee nam, functional food research center, bk21 plus program, graduate school of chonnam national university, gwangju 61186, korea e-mail: namsh1000@ hanmail.net © the author(s) 2016. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 90, 83 105 (2017), doi:10.5073/jabfq.2017.090.012 thünen institute of biodiversity, braunschweig, germany impact of tropospheric ozone on terrestrial biodiversity: a literature analysis to identify ozone sensitive taxa e. bergmann, j. bender*, h.j. weigel (received december 19, 2016) * corresponding author summary tropospheric ozone has long been known as highly phytotoxic. however, currently hardly anything is known whether this air pollutant can also pose a threat to the overall biodiversity in terrestrial ecosystems. identifying the relative ozone sensitivities of relevant taxa or species can be a first step in an assessment if biodiversity is at risk from ozone. a literature survey was conducted describing ex perimental and observational results of exposure of organisms and particularly plant species to ozone at environmentally relevant concentrations. for plants ozone effects considered were vegetative growth (e.g. biomass of shoots, foliage, single leaves, stems, and roots), reproduction (number and biomass of seeds and flowers), species development, and symptoms of visible foliar injury. a total of 474 literature references were evaluated which described such effects. for crop plants 54 species with 350 varieties could be considered, while (semi)natural vegetation was represented by 465 vascular plant species comprising 298 herbaceous and 165 woody plant species. overall, these ozone studies cover only a small fraction of the entire global flora. about two third of woody and about one half of native herbaceous plant species investigated so far have been described as ozone sensitive in at least one study. ozone sensitivity is slightly higher with respect to visible leaf injury as compared to growth effects, and herbs and deciduous tree species are more responsive than grasses and coniferous trees. observational results from field surveys conducted along ozone gradients to assess eco system effects of ozone in north america and europe revealed visible macroscopic leaf injuries for 258 herbaceous species. however, these findings often have not been verified under experimental ozone exposure. albeit the numbers of ozone studies related to a particular plant family varied considerably, high proportions of ozone sensitive species were found e.g. for the families myrtaceae, salicaceae and onograceae, while low proportions of ozone sensitive species were found e.g. for the families brassicaceae, boraginaceae and plantaginaceae. intra-specific variations of ozone sensitivity of vascular plants were primarily detected in crop species (e.g. wheat, soybean, snap bean, clover, rice), most often derived from screening studies of cultivars for their relative ozone sensitivity / tolerance to ozone. in some cases intra-specific variation of ozone sensitivity is also true for different populations of woody and herbaceous plant species, which often resulted from temporal or spatial differentiation of the relative ozone susceptibility. therefore, there is some evidence that ozone pollution in the past has already affected plant selection and modified the genetic pool of ozone sensitive genotypes. information on direct ozone effects on species other than vascular plants (e.g. ferns, mosses, fungi, algae, vertebrates) is very poor or irrelevant, i.e. ozone sensitivities for these taxa could not be described. this is also true for organisms like microbes, arthropods or insects which have not been tested so far for their responses to direct ambient ozone exposure. however, these organisms may be indirectly impaired by ozone via loss of vitality of the plant system to which they are associated. 1. introduction the loss of biological diversity (biodiversity) is one of the most prominent examples of global change. according to the definition of the convention of biological diversity (cbd) biodiversity is defined as “the variability among living organisms from all sources including, inter alia, terrestrial, marine and other aquatic ecosystems and the ecological complexes of which they are part; this includes diversity within species, between species and of ecosystems”. significant dri vers of the past and current loss of biodiversity are land use changes, changes of climate and atmospheric chemistry, invasive species, as well as soil and air pollution (sala et al., 2000). while there is already scientific evidence and public awareness, respectively, that climate change or excessive deposition of nitrogen from the atmosphere must be regarded as major threats to biodiversity (cbd; stevens et al., 2010a; stevens et al., 2010b), there is currently little infor mation if and to what extent biodiversity is at risk from tropospheric or ground level ozone pollution. this lack of information is all the more serious as during the last 60 years there has been undeniable evidence that tropospheric ozone has significant adverse effects on plant growth, crop yields and forest health and that this air pollutant has emerged as a problem of global dimension (royal society, 2008). typical effects of ozone on sensitive species include alterations of carbon allocation patterns, symptoms of visible injury, enhanced senescence, reduced growth and yield, or reduced flowering and seed production. each of these effects can impact on the vitality of component species in plant communities, which may have implications for biodiversity. therefore, identifying and understanding the relative sensitivity of individual species and genotypes to ozone is a central prerequisite for estimating effects at the community and ecosystem level and for the development of critical ozone levels to protect vegetation. however, there are hardly any systematic assessments which taxa are particularly affected by ozone. in the present study we summarize the existing information of the relative susceptibility of different vascular plant species and genotypes, respectively (native herbaceous plants, woody plant, agricultural plants) to near-ambient ozone concentrations and – based on this information – identify ozone sensitive taxa. we restrict our analysis to assessments of ozone effects on ecologically relevant parameters, i.e. biomass growth, productivity, reproduction, and easily accessible visible symptoms of the respective organisms and do not consider studies with a focus on physiological and biochemical ozone effects. while ozone effects on vascular plants provide the overwhelming majority of information of the ozone susceptibility of organisms in terrestrial ecosystems, we also reviewed existing information of ozone effects on non-vascular plant species and on other non-plant organisms. in the following text we first provide short summaries of ozone pollution trends, methods to study its effects and its mechanism of effects on plant organisms. we then provide detailed information on the relative ozone susceptibilities of the different taxa for which ozone effects have been described. 84 e. bergmann, j. bender, h.j. weigel 2 ozone pollution trends as a secondary air pollutant ozone is formed in the troposphere through a number of sun-light driven photochemical reactions involving the main precursor substances nitrogen monoand dioxide (no/ no2), volatile organic compounds (voc), methane (ch4) and carbon monoxide (co) (staehelin, 2003). these precursors are of natural or anthropogenic origin such as vehicles, power plants, biomass burning and all other forms of combustion. naturally occurring global ground-level background ozone concentrations in the pre-industrial era ranged between approx. 5 20 parts per billion (ppb) (marenco et al., 1994). since that time annual mean background ozone concentrations have increased to values between approx. > 20 45 ppb depending on the geographical location (vingarzan, 2004) with a rate of increase of annual mean values ranging between 0.1 1.0 ppb per year. this increase has been observed over large areas of europe and north america, and more recently in many countries in asia (e.g. china, india, pakistan), south america (e.g. brazil) and africa with rapidly emerging industrialization and hence, increasing emissions of precursors of ozone. very high concentrations episodically occur, but for large parts of western europe there has been a noticeable lack of the short-term high ozone concentrations that had previously been experienced. future ozone levels will be determined by the trends of the emissions of the precursors and of temperature and solar radiation. predictive models, e.g. based on ipcc-sres global emission scenarios indicate that background ozone concentrations will continue to increase at a rate of 0.5 % 2 % per year in the northern hemisphere during the next 100 years and will be in the range of ca. 42 84 ppb by 2100 (jacob and winner, 2009; prather et al., 2003; vingarzan, 2004). on the other hand, a recently published model study predicted more moderate increases of ozone levels until 2050 (wild et al., 2012). in germany there was also a slight increase in the annual mean ozone concentration between 1990 and 2011, but in recent years it is not possible to identify such statistically significant trend (uba, 2016). according to andersson and engardt (2010) and percy et al. (2003) about 50 % of forests worldwide are expected to be exposed to ozone concentrations above 60 ppb by 2100. groundlevel ozone concentrations influenced by human activities vary sig nificantly with time (diurnally, seasonally, inter-annually) and with geographic location. as ozone formation is dependent on sunlight and as some of the chemical reactions involved in the ozone formation in the troposphere are temperature-dependent, its concentrations are particularly high at warm sunny days (royal society, 2008). while at low elevation sites ozone concentrations show diurnal cycles with low concentrations during the night and in the morning and high and peak concentration during the afternoon, high elevation sites mostly do not show such distinct diurnal variation (stockwell et al., 1997). in general, at a particular location formation of high ozone concentrations depends on the local meteorology, the topography and the regional sources of ozone precursors. in europe, the highest average ozone levels occur in central and southern europe (royal society, 2008). 3 methods to study ozone effects on plants and terrestrial eco-systems experimental techniques to expose single plants, plant communities and segments of ecosystems to ozone range from controlled-environ ment growth chambers, greenhouses, field chambers to open-air ozone exposure systems (weigel et al., 2015). in these systems targeted ozone concentrations are supplied to the test organisms by adding ozone to either ambient or charcoal-filtered air. most of the information of ozone effects on plants is derived from the use of various types of indoor and outdoor chambers. for example, laboratory fumigation chambers of various designs which provide highly reproducible environmental and ozone exposure conditions have widely been used for assessing visible injury or physiological and biochemical ozone effects (heck et al., 1978). however, due to different microclimatic conditions in these chambers compared to ambient air (“chamber effect”) plants often show morphological or physiological differences compared to field-grown plants which modify the response to ozone. open-top field chambers (otc, heagle et al., 1973) have been the most widely used ozone exposure system up to now (heagle et al., 1988; jäger et al., 1999; oksanen et al., 2013; zheng et al., 2013). open-top chambers offer the opportunity to expose individual plants, model ecosystems and canopies of field plots for one to several growing seasons to either ambient and filtered air or to elevated levels of ozone induced by ozone addition. open-top chambers are best suited for in situ studies with low stature vegetation, e.g. like most crop or grassland species. to allow studies with taller trees large versions of otcs have been constructed (musselman and hale, 1997). hemispherical greenhouses (“solardomes”) represent another type of closed outdoor fumigation chambers that are used in the uk (lucas et al., 1987). the necessity to avoid chamber effects and space limitations and to investigate ecosystems under in situ conditions in an undisturbed environment led to the development and utilisation of free-air ozone exposure systems (percy et al., 2010) which have been used in a very limited number of experiments. one type of a chamberless expo sure system for ozone effect studies is a modification of the circular free air carbon dioxide enrichment (face) system (hendrey et al., 1999; miglietta et al., 2001) which was modified to dispense ozone into plant canopies. this type of exposure systems has been used for ozone effects studies with soybean (morgan et al., 2004) and with young tree species (karnosky et al., 1999; watanabe et al., 2013). a similar custom-designed circular free-air ozone exposure system was used by volk et al. (2003) in a swiss grassland system. a free-air ozone fumigation system in mature tree crowns of beech and spruce in germany was developed by werner and fabian (2002) and tested and used by matyssek et al. (2010b, 2013). in free-air ozone exposure systems the coupling between the atmosphere and the plant canopy as well as between the canopy and the respective soil volume largely remains unchanged. thus, in situ water and nutrient fluxes at the ecosystem level as well as biotic interactions (pollination, herbivory) can be investigated. a second chamberless method to assess effects of ambient ozone levels on plants is the use of protecting chemicals against ozone stress (manning et al., 2011). while this approach has long been known but rarely been applied, it has recently been utilised again with crop species in europe (mills and harmens, 2011) and asia (oksanen et al., 2013; rai and agrawal, 2012) or with tree species (paoletti, 2007; paoletti et al. 2011). methods of ozone exposure where there is no manipulation of the ozone concentration surrounding the plants are field observations and ambient ozone gradient studies where impacts of current or past ozone exposure scenarios in complex ecosystems are monitored and assessed. for example, the survey of ozone-specific leaf injury symptoms is a common worldwide established tool to assess significant ozone -response relationships. prominent examples, where forest tree species and ecosystem responses to ozone have been assessed using ozone gradient approaches are studies in the usa (mclaughlin et al., 2007; miller and mcbride, 1999), in the carpathian mountains (bytnerowicz et al., 2003) and in italy (ferretti et al., 2005). 4 mechanisms of ozone uptake and effects on biota terrestrial ecosystems are the major sink for tropospheric ozone and consequently, vegetation is at particular risk from this pollutant. with respect to vegetation the mechanisms of ozone uptake and its effects have predominantly been investigated for vascular plants and ozone sensitive taxa 85 will be briefly described here. the uptake of ozone by vegetation is attributed to both non-stomatal and stomatal deposition. non-stomatal deposition includes deposition to soil, stems, cuticles and other external surfaces. field measurements of ozone deposition (flux) in various ecosystems indicate that total dry deposition is largely dominated by stomatal uptake during the most active parts of the growing season, but, at other times of the year and depending on vegetation type and weather conditions, nonstomatal deposition can be larger than stomatal uptake (cape et al., 2009; cieslik, 2004). it has long been known that penetration of ozone through the plants cuticle is of minor importance in comparison to the route of uptake through the stomata (kerstiens and lendzian, 1989; massman and grantz, 1995). this transfer of the gas through the atmosphere by turbulent diffusion, which is governed by micro-meteorological conditions (radiation, temperature, wind, etc.) and the roughness of the vegetation, into the plant via molecular diffusion through the stomata is currently considered the key process in relating ozone exposure to plant responses (fowler et al., 2009). consequently, all environmental factors that modify the stomatal aperture (e.g. temperature, light and soil water conditions, other pollutants, atmospheric co2 concentration) and which thus affect leaf gas exchange have an influence on the uptake of ozone into the plant interior (fiscus et al., 2005; fuhrer, 2009) and consequently on its effects. once ozone has passed the stomatal pore the ozone molecule as a strong oxidant reacts with the apoplastic fluid and this results in the generation of reactive oxygen species (ros) like the hydroxyl radical (·oh) and the superoxide radical (·o2−). these breakdown products of ozone impact on the cell membrane structure and function, change the cell metabolism and cellular events, which results in reduced photosynthetic rates and finally in the generation of observable plant responses like visible chlorotic or necrotic tissue damage, reduced photosynthesis, temporal shifts in the plant‘s development, and losses in productivity (cho et al., 2011; dizengremel et al., 2013). the accumulation of ros induces defence reactions by the plant that are similar to other oxidative stress responses or pathogen attack and may result in a programmed cell death (“hypersensitive response”), a process which is thought to have the biological significance of limiting the spreading of the oxidative burst (bartoli et al., 2013; kangasjärvi et al., 2005; langebartels et al., 2002; wohlgemuth et al., 2002). defence mechanisms involved in detoxifying ros, directly or indirectly derived from ozone exposure, may consist in enzymatic and non-enzymatic reactions among which the apoplastic ascorbate pool seems to be particular important (fuhrer, 2009; iriti and faoro, 2008). defence reactions require energy for regeneration of antioxidants, i.e. particularly at prolonged ozone exposure the detoxification capacity may decline due to decreased rates of carbon assimilation and limited available energy (wieser and matyssek, 2007). visible injury resulting from cellular ozone impacts has been observed on a wide range of plant species including trees, crops, and species of semi-natural vegetation, e.g. in north-america and in europe (flagler, 1998; innes et al., 2001; mills et al., 2011). while on broad-leaved plants visible injuries include stippling, flecking, surface bleaching, bifacial necrosis, pigmentation (e.g. bronzing) and chlorosis, for conifers visible injury has been described as chlorotic banding, tip burn, flecking and chlorotic mottling. for both plant types ozone induced symptoms of premature senescence of leaves and needles, respectively, can be observed. these foliar lesions can vary between and within taxonomic groups and the degree and extent of visible foliar injury development may vary from year to year and site to site. under prolonged, i.e. chronic ozone exposure, visible injury is often not observed, but decreased rates of photosynthesis indicate adverse effects of ozone. the response of photosynthesis to ozone has received much attention in order to explain ozone induced losses of plant productivity. it may be assumed that plant growth retardation under longer-term ozone exposure at moderately enhanced concentrations is mostly the result of reduced rates of co2 assimilation at the leaf level. however, in trees within-tree alterations of carbon allocation due to disturbed phytohormonal regulation have also been shown to affect growth (kitao et al., 2012; winwood et al., 2007). although many different changes are observed in the photosynthetic apparatus, decreased activity and amount of the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) appear to be the prevailing causes of loss of photosynthetic capacity. reduced photosynthesis due to ozone exposure may finally result in decreased growth rates and reduced overall plant productivity. along with these effects impaired translocation of assimilates from source (e.g. leaves) to sink (e.g. roots; seeds) organs and early senescence likely contribute to ozone effects on plant growth and reproduction. in the past four decades ozone effects on crops (reviewed by e.g. booker et al., 2009; fiscus et al., 2005; heagle, 1989; heagle et al., 1989; mills and harmens, 2011) and particularly on decidu ous and coniferous trees (reviewed e.g. matyssek et al., 2013; 2010a; 2010b; percy et al., 2003; sandermann et al., 1997) have been investigated. other types of natural or semi-natural vegetation have only recently received attention (reviewed by e.g. ashmore, 2005; davison and barnes, 1998; fuhrer, 1997; weigel et al., 2015). very few studies have addressed ozone effects on other organisms than vascular plants (mosses, ferns, algae) in terrestrial ecosystems. some studies investigated the respiratory and thermoregulatory behaviour of ozone-exposed vertebrates, namely of amphibians and reptiles. other vertebrate species have only been investigated as test objects in medical research studies and are not considered here. 5 the literature database of the present study a literature search was performed using web of sciencetm (core collection, biological abstracts, and cab abstracts) encompassing reviewed papers, book chapters, research reports, or conference proceedings starting with the year 1980 and describing results of controlled exposure of organisms to ozone with at least two different levels of ozone. for vascular plants, which represent the predominant majority of studies, exclusively studies with single plants or monocultures which had been performed in either outdoor ozone exposure facilities (free-air fumigation systems; open-top chambers) or in greenhouses or solardomes were included. controlled environment studies in growth chambers were considered only, if hourly ozone exposure concentration did not exceed 100 ppb (exceptions are indicated). ozone effects considered were yield effects (crops only) or general growth effects (i.e. reduction or increase in shoot, foliage, single leaf biomass, stem, root, seed and flower biomass, no. of flowers), change of root/shoot ratio, enhancement and delay of flowering, reduction in germination rate of produced seeds or symptoms of visible leaf/needle injury (unspecific symptoms, senescence, colouring, and ozone specific symptoms). a total of 418 literature references were evaluated which met these requirements. in addition, further 56 publications from forest health monitoring programs were evaluated to identify native plant species, which have been recog nised as ozone-sensitive in terms of the expression of ozone-specific symptoms in the field. all data were compiled into a database separated into seven parts. these source data can be downloaded at https://www.thuenen.de/en/bd/fields-of-activity/biodiversity-andclimate-change/phytotoxicology-of-air-pollutants/. unless otherwise stated, ozone effects were considered as either “none”, “statistically significant” or “not significant” as taken from the original publication. for plants native to germany, further taxonomical information and spatial distribution is given according to bfn (bundesamt für naturschutz; http://www.floraweb.de/index.html). 86 e. bergmann, j. bender, h.j. weigel 6 identifying ozone sensitive taxa from experimental studies in the following text the description of the ozone sensitivity of organisms is grouped according to their taxonomy (vascular plants, bryophytes/pteridophytes, algae, lichens, fungi and vertebrates). ozone effects on microorganisms and invertebrates mostly result from ozone effects on the plant or plant community, respectively, interacting with these organisms. for this reason, these groups of organisms are disregarded here. the large number of studies with vascular plants was further grouped into natural/semi-natural vegetation and crop plants. (semi)natural vegetation comprises herbaceous and woody plant species including both native wild plants, extensively managed pasture plants and forest trees. 6.1 native herbaceous and pasture plants results from the database during the last decades an increasing number of experimental studies have been performed to assess the relative sensitivity of natural or semi-natural herbaceous plant species to ozone. in tab. 1 and tab. 2 summaries of assessments of ozone responses as indicated by visible symptoms and growth effects of native herbaceous and pasture plant species are shown. overall, 62 publications were reviewed reporting on either only one or on up to 44 different species. from these publications a number of 554 indications (i.e. entries in the database) were collected which provide information about 298 species belonging to 47 plant families. for 56 species more than two studies are available and 169 species were tested only once. in the majority of studies (documented by 37 publications) open-top chambers or solardomes were used as ozone exposure facilities, however, for 74 species the only available data result from experiments using controlled environment fumigation chambers. about 60 % of the species tested were perennials and 20 % of all species were grasses. in total, for 188 species out of the 298 species investigated a response to ozone has been documented. across the whole data set of herba ceous non-crop plants 53 % and 47.5 % of all species tested were found to express visible injury symptoms and changes in biomass production, respectively, in at least one experiment. in terms of growth effects (tab. 2) the proportion of ozone sensitive species is higher for herbs than for grasses, however, the number of herb species which were tested for their ozone responses is 3.3 times higher than the number of grasses. a noticeable difference becomes obvious with respect to life history: regarding the parameter visible leaf injury (tab. 1), the proportion of ozone sensitive species is lower for annuals and biennials as com pared to perennials, but the proportion of species responding with ozone impacts on growth (tab. 2) is higher for annuals and biennials than for perennials. the observation that about one half of the species tested responded to ozone in a sensitive way is also true for species native to germany including neophytes and archaeophytes (tab. 1). in tab. 3 recorded ozone responses of species are grouped according to the respective plant families. there were six families which were represented by at least ten different species. in terms of visible leaf injuries, six frequently studied families covered more than 50 % of species classified to be ozone sensitive for which the following order of decreasing sensitivity could be derived from the dataset: onagraceae > fabaceae > cyperaceae > lamiaceae > asteraceae > poaceae. considering growth effects, 70 % of the species of the fabaceae tested for their ozone sensitivity were impaired by ozone and about 40 % of all species tested to be sensitive towards ozone belong to the families polygonaceae, poaceae, asteraceae, lamiaceae, and plantaginaceae. these results for both parameters point to the fact that fabaceae (legumes) seem to be highly ozone sensitive and that a high proportion of members of poaceae and asteraceae families are also sensitive to ozone. on the other hand, for brassicaceae (crucifers) eight of nine species tested so far proved to be insensitive to ozone exposures. about one half of all native herbaceous species listed in the present database are also native to germany, 15 of these species are considered to be “endangered” and additional 12 species are classified as “near-threatened”. out of these, 16 species (eight within each classification group) are responsive to ozone (tab. 4). special attention should be paid to the species comarum palustre, medicago minima, nardus stricta and trifolium striatum because for these species ozone impacts have been shown for both, growth and leaf injury. tab. 1: summary of assessments of ozone effects on native herbaceous or pasture plant species as indicated by visible leaf injury symptoms. results are classified into different descriptive groups and given as the numbers of total species tested and the numbers of species showing a particular ozone response: spec. = specific ozone symptoms; col. = non-specific discolouration; sen. = symptoms of senescence; not spec. = symptoms not characterized; none = no visible symptoms observed. the percentage of species which were injured to ozone at least in one study is also shown. no. of species kind of visible symptoms descriptive group tested spec. col. sen. not spec. none % injured total 276 28 4 28 112 164 52.9 herbs 211 23 4 16 86 122 51.7 grasses 58 3 0 10 25 40 55.2 sedges 11 2 0 2 1 2 45.5 annuals or biennials 100 4 0 8 34 65 42.0 perennials 175 23 4 20 77 99 58.9 species native to germany1 195 27 4 28 79 120 56.9 endangered1 13 0 0 0 3 10 23.1 near threatened1 12 5 1 1 2 8 58.3 1according to bundesamt für naturschutz. floraweb: http://www.floraweb.de/index.html. sum of responsive and non-responsive species numbers is not equivalent to the total number of species assessed because of divergent results within different publications. ozone sensitive taxa 87 tab. 3: summary of assessments of ozone effects on native herbaceous or pasture plant species categorized according to the respective plant families. the total number of species tested (total) and the number of species showing a response to ozone indicated by visible symptoms and growth effects (see tab. 1 and tab. 2) are shown. statistically significant effects (see chap. 5) are shown in parentheses. only those families are considered which are represented by at least three different species. visible leaf injuries growth effects reduction increase not family total tested injured not injured tested total (sign.) total (sign.) responsive apiaceae 4 3 3 0 3 2 (2) 0 (0) 1 apocynaceae 4 4 4 4 1 1 (1) 0 (0) 0 asteraceae 59 54 30 26 41 22 (14) 4 (2) 25 boraginaceae 12 12 4 8 2 2 (1) 0 (0) 0 brassicaceae 9 9 1 8 2 2 (2) 0 (0) 1 caryophyllaceae 10 8 1 8 10 5 (4) 2 (2) 8 cyperaceae 10 9 7 2 10 3 (3) 1 (0) 6 fabaceae 29 28 22 7 25 20 (16) 1 (0) 6 geraniaceae 3 3 2 2 1 0 0 2 hyperaceae 3 3 2 2 2 1 (0) 0 1 lamiaceae 9 9 5 5 5 2 (2) 2 (1) 3 malvaceae 3 3 2 1 3 2(1) 0 1 onagraceae 8 8 8 4 2 2 (2) 0 1 papaveraceae 5 5 2 3 3 2 (2) 0 2 plantaginaceae 7 7 2 7 6 3(1) 1 (0) 5 poaceae 64 55 30 39 58 29 (23) 7 (4) 42 polemoniaceae 3 3 0 3 0 n.d. n.d. n.d. polygonaceae 9 8 5 5 6 5 (5) 1 (1) 3 ranunculaceae 4 4 2 3 3 1 (1) 0 3 rosaceae 7 6 3 5 7 4 (4) 2 (0) 3 saxifragaceae 3 3 0 3 3 0 0 3 scrophulariaceae 4 4 1 3 3 3 (3) 0 2 violaceae 3 3 1 2 3 1 (1) 0 2 tab. 2: summary of assessments of ozone effects on native herbaceous or pasture plant species as indicated by growth effects (change in biomass of shoot, foliage, stem, root, seed or flowers, root/shoot ratio or change in germination rate of produced seeds = seed quality). results are classified into different descriptive groups and given as the numbers of total species tested and the numbers of species showing a particular ozone response. the percentage of species which were responsive to ozone at least in one study is also shown. significant changes (see chapt. 5) are given in parentheses. no. of species kind of growth response reduced growth descriptive group tested or seed quality increased growth none % responsive total 223 119 (95) 24 (11) 135 61.0 (47.5) herbs 155 89 (69) 15 (7) 87 63.9 (47.7) grasses 61 30 (24) 7 (4) 44 55.7 (45.9) sedges 7 2 (2) 1 (0) 4 42.9 (28.6) annuals or biennials 55 32 (25) 4 (2) 30 63.6 (49.1) perennials 166 83 (67) 20 (9) 105 59.0 (44.6) species native to germany1 187 97 (76) 22 (10) 124 59.9 (44.9) endangered1 12 5 (5) 2 (1) 6 58.3 (50.0) near threatened 12 3 (2) 0 (0) 12 25.0 (16.7) 1according to bundesamt für naturschutz. floraweb: http://www.floraweb.de/index.html. sum of responsive and non-responsive species numbers is not equivalent to the total number of species assessed because of divergent results within different publications. 88 e. bergmann, j. bender, h.j. weigel tab. 4: vascular plant species at risk (species red list) which have been tested for their response to ozone with respect to visible injuries and growth effects. if distinct effects have been published, the most sensitive one is listed. red. = reduction, inc. = increase, n.s. = not significant, -= not determined. risk status according to “bundesamt für naturschutz”: nt – near threatened, 3: vulnerable, 2: endangered, 1: critically endangered, 0: extinct in the wild, r: extremely rare species status visible injury growth effect reference1 antennaria dioica 3+ -shoot red. mortensen, 1993 avenula pratensis nt no no ashmore et al., 1996 briza media nt no no ashmore et al., 1996; 1995 carex laevigata 3 no no hayes et al., 2006 carex panicea nt specific no hayes et al., 2006 carum carvi nt specific no bungener et al., 1999b cirsium dissectum 2 no shoot red., n.s. franzaring et al., 2003; 2000 coramrum palustre nt yes growth red. mortensen, 1994 eriophorum vaginatum nt -no morsky et al., 2011 gentiana asclepiadea 3 yes -manning and godzik, 2004 juncus squarrosus nt no inc. in growth hayes et al., 2006 lychnis flos-cuculi nt coloured/ bungener et al.; 1999b; batty et al., 2001; specific no franzaring et al., 2000; tonneijck et al., 2004 lychnis viscaria nt no no batty et al., 2001 medicago minima 3 yes growth red. gimeno et al., 2004 micropyrum tenellum 0 no -bermejo et al., 2003 nardus stricta nt specific shoot red. ashmore et al., 1996; hayes et al., 2006 narthecium ossifragum 3 no no hayes et al., 2006 primula farinosa 3+ no no batty et al., 2001 rhodiola rosea r -no batty et al., 2001 rubus chamaemorus 1 no -mortensen and nilsen, 1992 salvia pratensis nt senescent no bungener et al., 1999a; 1999b saussurea alpina r -inc. in growth. mortensen, 1993 scrophularia auriculata 3 no growth red. batty et al., 2001 senecio sarracenicus 3 no inc. in shoot hayes et al., 2006 silene noctiflora nt no no batty et al., 2001 succisa pratensis nt -no franzaring et al., 2000 tragopogon orientalis nt specific no bungener et al., 1999a; 1999b trifolium striatum 3 yes shoot / seed red. bermejo et al., 2003; gimeno et al., 2004; sanz et al., 2007 1) ashmore, m.r., power, s.a., cousins, d.a., ainsworth, n., 1996: in: l. kärenlampi, l. skärby (eds.), critical levels for ozone: un-ece workshop report, kuopio, 193-197. 2) ashmore, m.r., thwaites, r.h., ainsworth, n., 1995: water air soil pollut 85, 1527-1532; batty, k., ashmore, m., power, s.a., 2001: in: d. fowler et al. (eds.), the ozone umbrella project ceh project: c00970 – final report, centre for ecology and hydrology, edinburgh; bermejo, v., gimeno, b.s., sanz, j., de la torre, d., gil, j.m., 2003: atmos. environ. 37, 4667-4677; bungener, p., balls, g.r., nussbaum, s., geissmann, m., grub, a., fuhrer, j., 1999a: new phytol. 142, 271-282; bungener, p., nussbaum, s., grub, a., fuhrer, j., 1999b: new phytol. 142, 283-293; franzaring, j., dueck, t.a., tonneijck, a.e.g., 2003: in: p.e. karlsson, g. selldén, h. pleijel (eds.), establishing ozone critical levels ii, unece workshop report b 1523. ivl swedish environmental research institute, gothenburg, sweden, 224-229; franzaring, j., tonneijck, a.e.g., kooijman, a.w.n., dueck, t.a., 2000: environ. exp. bot. 44, 39-48; gimeno, b.s., bermejo, v., sanz, j., de la torre d., elvira, s., 2004: environ. pollut. 132, 297-306; hayes, f., mills, g., williams, p., harmens, h., buker p., 2006: atmos. environ. 40, 4088-4097; manning, w.j., godzik, b., 2004: environ. pollut. 130, 33-39; morsky, s.k., haapala, j.k., rinnan, r., saarnio, s., silvola, j. et al., 2011: environ. exp. bot. 72, 455-463; mortensen, l.m., 1993: norweg. j. agric. sci. 7, 129-138; mortensen, l.m., 1994: norweg. j. agric. sci. 8, 91-97; mortensen, l.m., nilsen, j., 1992: norweg. j. agric. sci. 6, 195-204; sanz, j., bermejo, v., gimeno, b.s., elvira, s., alonso, r., 2007: atmos. environ. 41, 8952-8962; tonneijck, a.e.g., franzaring, j., brouwer, g., metselaar, k., dueck, t.a., 2004: environ. pollut. 131, 205-213. literature context: approaches to describe functional patterns of plant responses to ozone during the last decades an increasing number of experimental studies have been performed to assess the relative sensitivity of natural or semi-natural herbaceous plant species to ozone. the majority of these studies focused on highland or alpine (bungener et al., 1999a; bungener et al., 1999b), dehesa (gimeno et al., 2004), wetland (batty et al., 2001; franzaring et al., 2003; franzaring et al., 2000) or ruderal plant species (bender et al., 2006; bergmann et al., 1999), and in most of the studies rare species were included ozone sensitive taxa 89 as well. beyond the repeated documentation of widespread ozone sensitivity of native herbaceous species it is impossible to assess the ozone risk of the total flora only by doing standardised screening experiments. therefore, several functional approaches have been described in order to identify ecological characteristics which might be associated with the sensitivity of the native herbaceous plants to ozone. relating eco-physiological characteristics to ozone sensitivity of different species, harkov and brennan (1982) concluded that herbaceous species are generally more sensitive than woody plants. in his unifying theory reich (1987) related the strong dependence of phytotoxic ozone effects to the gas exchange properties of the target plant, thus indicating that water status and transpiration rate might be the critical factor for determining the ozone responsibility of a plant. franzaring et al. (1997) tested the hypothesis that hygroand mesomorphous species from moist sites are more affected by ozone than scleromorphous species adapted to dry sites. however, the authors failed to evidence a relationship between ozone sensitivity ranking and ellenberg-moisture values, especially when the assessment was based on growth parameters. in open-top chamber fumigation experiments franzaring et al. (2000) and batty et al. (2001) chose wet grassland species which are thought to suffer less water stress in ozone episodes and are therefore considered to be particularly sensitive because of a higher stomatal conductance. in both studies, 36 % of the species responded with growth changes and 56 % expressed symptoms of leaf injury. this reflects a ratio of sensitivity below or similar to that calculated for the whole data set over all habitats as presented in the present study. moreover, there was no association between ecological indicator values for either moisture, light, ph and fertility or ozone sensitivity in the short-term experiments of batty et al. (2001). however, the most sensitive species in the experiments were characterised by a high stomatal conductance. from a meta-analysis hayes et al. (2007) identified three significant relationships between relative sensitivity to ozone and ecological habitat requirements, i.e. light, moisture and salt content of soils. these relationships provided an opportunity to model the relative sensi tivity to ozone of species (jones et al., 2010). in a review of ozone impacts on european grasslands bassin et al. (2007) pointed out that stomatal conductance, specific leaf area (sla) and defense capacity are three main plant traits that determine ozone sensitivity. low relative growth rate (rgr) and sla are characteristic for stress tolerating species (s-strategy sensu the c-s-r strategy model of grime (1979), but there is limited evidence that these species are less sensitive towards ozone than species with the highest ranking of c (competitive)or r (ruderal)-strategy (bassin et al., 2007; bungener et al., 1999; franzaring et al., 1997; hayes et al., 2007). however, a basic requirement for deriving any kind of relationship between a response to ozone and ecological properties is to embrace a critical number of species covering the broad part of the total ecological amplitude of plants e.g. families, life forms, habitat types, plant traits etc. at present, most of these characteristics are not sufficiently represented in the databases of experiments under realistic conditions (bassin et al., 2007; hayes et al., 2007) narrowing the success in grouping the plant kingdom into broad classes of ozone sensitivity. hayes et al. (2007) determined an index to describe the relative sensitivity to ozone for 83 native plant species from existing publications. approximately one-third of the species in this study showed above-ground biomass reductions of about > 10 % under ozone exposure. the authors concluded that plants of the fabaceae family and species with a therophyte life form have to be regarded as particularly sensitive to ozone. the ranking of species in this study derived from ozone dose-response relationships and was related to a standardized ozone exposure and is thus of high confidence. nevertheless, the main observations of this study comply with our findings when the data set for the 298 species shown tab. 2 and tab. 3 is regarded. using the database of hayes et al. (2007) 54 eunis (european nature information system) level 4 communities were identified as potentially ozone-sensitive with the largest number of species associated with grasslands (mills et al., 2007). within the grasslands classification, the communities e4 (alpine and sub-alpine grasslands), e5 (woodland fringes and clearings) and e1 (dry grasslands) have been found to be the most sensitive. in contrast, bassin et al. (2007) concluded that species grown in less productive habitats (eunis e4 and e1) are thought to be less sensitive than species grown under favorable growth conditions or in productive habitats as mesotrophic pastures. recently, van goethem et al. (2013) presented an approach to consider a cumulative stressor-response distribution for ozone exposure on natural vegetation named ‘species sensitivity distributions’, ssd. their findings indicate that annual grassland species, as a species assemblage, are more sensitive to ozone than perennial grassland species. with respect to the present study these results can only be confirmed for the occurrence of growth effects but not for visible ozone injuries. 6.2 woody plants results from the data base this chapter summarizes existing information on the ozone sensitivity of woody plants species based on their responses with respect to visible symptoms (tab. 5) or growth effects (tab. 6) as well as their classification to plant families (tab. 7). the data set contains results taken out of a total of 142 references, with 56 originating from america, 16 from asia and 67 from europe. in summary, a total of 360 entries covering 165 species, 69 genera and 39 families have been found within the references selected. about two third of the species listed are categorised as trees, the rest are shrubs or climbers and an equal proportion as deciduous in contrast to evergreen species. with respect to the german situation 39 species that were described in ozone exposure studies are native to germany, and 28 species are neophytes. populus nigra was the only species classified as endangered in germany. the majority of results (92 studies) was achieved using open top chambers as an ozone exposure system while only 13 studies used free-air fumigation facilities. only six studies presented here investigated mature trees of the species european larch (havranek and wieser, 1993), red oak (kelting et al., 1995; samuelson et al., 1996), scots pine (manninen et al., 2003), beech (nunn et al., 2005), and apple (wiltshire et al., 1993) whereas all other investigations are based on experiments with cuttings or seedlings (one to eight years old). however, while more recently there is a number of ozone exposure studies working with mature trees, these studies focussed on eco-physiological issues, which are not considered here. about 64 % of all woody species (105 species) were investigated in only one study and 18 % and 20 %, respectively, in two or more studies. the most frequently studied species were liriodendron tulipifera, viburnum lantana, acer saccharum, pinus sylvestris, populus deltoides × nigra, prunus serotina, quercus rubra, and betula pendula (6 to 10 times) and pinus taeda, fraxinus excelsior, fagus sylvatica, and picea abies (> 10 times). a total of 148 species was shown to be responsive to ozone. in terms of visible leaf injuries more than 80 % of 135 species tested expressed symptoms when exposed to ozone. this percentage is slightly higher for deciduous and broadleaved species than for evergreen or coniferous species. about one half of all observations, which represent 62.6 % of the species, revealed significant growth responses to ozone. differences in the ozone sensitivity between coniferous and broadleaved species are minimal with a slight tendency to more sensitive responses of deciduous versus evergreen species. in general, there is no evidence of a significant increase in growth due to an ozone exposure. similar to native herbaceous species (chap. 6.1), a higher percentage of responsive species was detected when assessing visible 90 e. bergmann, j. bender, h.j. weigel leaf injury rather than growth measurements. however, the averaged percentage of ozone responsive woody species is higher than that of herbaceous species (see tab. 1 and tab. 2). for 64 species both parameters, visible injury and growth, have been investigated simultaneously. for a majority of more than 70 % (46 species) concordant results were recorded i.e. an impact of ozone was evidenced by both, visible injury and growth effects. tab. 7 summarizes the ozone responses of species according to the respective plant families. the family of the pinaceae was represen ted by the highest number of species (28) followed by the families of salicaceae, rosaceae, fagaceae, and caprifoliaceae. these 5 fre quently studied families cover 54 % of all species tested and involve about one third of species shown to be responsive to ozone. irrespective of the kind of ozone response (i.e. visible injuries or growth effects) the families myrtaceae, oleaceae, salicaceae, and betulaceae comprise the highest proportion of responsive species, followed by fagaceae, sapindaceae, rosaceae, and pinaceae. however, because of the general high proportion of responsive species the differences between the plant families are quite small. for example, out of the most frequently investigated family of the pinaceae, which are classified here to be less sensitive to ozone, 72 % of the species were found to express visible symptoms and 52 % showed significant growth effects at least in one investigation. literature context: approaches to evaluate the sensitivities of species to ozone based on ecophysiological parameters the data listed in the present study clearly reflect the fact that our available knowledge about the ozone sensitivity of trees and shrubs derives predominantly from potted juvenile individuals. accordingly, karnosky et al. (2007) pointed out that during more than 50 years of research negative growth effects on forest trees have been demonstrated mainly for seedlings, while ozone response of adult forest trees has rarely been examined experimentally. in order to determine whether seedlings and mature trees responded similarly to ozone, samuelson and edwards (1993) exposed 2-yr-old seedlings and 30-yr-old trees of quercus rubra to ozone. their results indicated that the ozone sensitivity of larger and more physiologically mature trees will be underestimated when represented by young seedlings within exposure experiments. a first step of a move towards field conditions was made by the use of a free-air exposure facility in a northern temperate forest (aspenface, karnosky et al., 2003b). for mature trees some attempts to investigate ozone effects were made by the use of branch cuvettes (wieser et al., 2012), while the free-air ozone exposure system established in germany in a mixed stand of about 60-year-old fagus sylvatica and picea abies trees (nunn et al., 2002) remains unique. nevertheless, research activities on ozone effects on woody plant species performed within the last decades revealed a broad knowledge about visible symptoms, effects on growth, carbohydrate allocation, ozone detoxification and to a high extent about effects of tab. 5: summary of assessments of ozone effects on woody plant species indicated by visible leaf injury symptoms. results are classified into different descriptive groups and given as the numbers of species tested and the number of species showing a particular ozone response. the percentage of species which were responsive to ozone at least in one study is also shown. no. of species descriptive group tested injured not injured % injured total 135 114 39 84.4 shrubs 33 28 8 84.8 trees 84 70 23 83.3 climbers 3 2 1 -deciduous 90 78 23 86.7 evergreen 41 31 16 75.6 conifers 18 13 9 72.2 broadleaved 118 101 30 85.6 native to germany1 63 59 17 93.7 1 according to bundesamt für naturschutz. floraweb: http://www.floraweb.de/ index.html tab. 6: summary of assessments of ozone effects on woody plant species indicated by growth effects (change of total plant or organ biomass). results are classified into different descriptive groups and given as the numbers of species tested and the number of species showing a particular ozone response. statistically significant effects (see chap. 5) are shown in parentheses. the percentage of species which were responsive to ozone at least in one study is also shown; none = no ozone effects observed. no. of species decreased growth increased growth % responsive descriptive group tested total (sign.) total (sign.) none total (sign.) total 99 76 (62) 2 (0) 54 76.8 (62.6) shrubs 3 2 (2) 0 2 -trees 89 71 (56) 2 (0) 37 79.8 (62.9) climbers 1 1 (1) 0 0 -deciduous 51 41 (33) 2 (0) 19 80.4 (64.7) evergreen 48 34 (28) 0 23 70.8 (58.3) conifers 30 19 (16) 0 17 63.3 (53.3) broadleaved 69 57 (46) 2 (0) 25 82.6 (66.7) native to germany1 36 28 (22) 2 (0) 20 77.8 (61.1) sum of responsive and non-responsive species numbers is not equivalent to the total number of species assessed because of different results within different publications. 1 according to bundesamt für naturschutz. floraweb: http://www.floraweb.de/index.html ozone sensitive taxa 91 tab. 7: summary of assessments of ozone effects on woody plant species categorized according to the respective plant families. the total number of species tested (total) and the number of species showing a response to ozone indicated by visible symptoms and growth effects (tab. 5 and tab. 6) are shown. statistically significant effects (see chap. 5) are shown in parentheses. only those families are considered which are represented by at least three different species. visible leaf injuries growth effects responsive not family total tested injured not injured tested total (sign.) responsive anacardiaceae 3 3 3 1 0 0 (0) 0 betulaceae 8 6 5 2 6 5 (5) 2 caprifoliaceae 11 11 8 5 0 0 (0) 0 cornaceae 3 3 3 1 0 0 (0) 0 cupressaceae 3 0 0 0 3 2 (2) 1 fabaceae 3 2 1 2 2 2 (1) 0 fagaceae 14 9 8 3 10 8 (7) 5 lauraceae 3 2 1 1 2 2 (2) 1 myrtaceae 9 9 8 1 8 8 (8) 1 oleaceae 5 5 5 1 4 4 (2) 1 pinaceae 28 18 13 9 27 17 (14) 16 rosaceae 16 14 12 4 11 9 (5) 4 salicaceae 20 16 15 2 9 9 (7) 3 sapindaceae 8 7 6 3 4 3 (2) 0 tiliaceae 3 3 2 1 0 0 (0) 0 ozone on photosynthesis and stomatal functioning (e.g. reviewed by gomez-garay et al., 2013; paoletti, 2007). these measurements suggested that ozone reduces stomatal conductance and may impair stomatal control and predispose trees to drought stress under dynamic conditions. paoletti (2007) concluded that the most significant ozone impact is on the regulatory capacity of resource allocation rather than on productivity. as already pointed out for (semi-)natural herbaceous plants speciesspecific and individual-specific responses to ozone may affect forest competition and biodiversity (paoletti, 2007). thus, in terms of biodiversity issues the knowledge about different ozone sensitivity of species is important. for example, based on the 38 experiments reviewed by huttunen and manninen (2013), pinus sylvestris may be considered as an ozone sensitive conifer species, with mature pines being more sensitive than younger trees. for field-grown mature coniferous trees the higher ozone sensitivity of the deciduous species larix decidua was associated with a higher ozone uptake when compared to the evergreen picea abies or pinus cembra (wieser et al., 2013). in contrast, schaub et al. (2003) reported on similar ozone uptake rates of two deciduous species (prunus serotina and fraxinus americana) differing in ozone sensitivity as shown by means of visible injury. also, zhang et al. (2001) found that there was no correlation between foliar injury and stomatal conductance when comparing 11 deciduous broad leaved trees. the authors suggested that species-specific leaf biochemical processes and environmental interactions must be considered in determining species’ sensitivity to ozone. there is evidence that differences in ozone sensitivity can be attributed either to anatomical characteristics (deciduous trees, bennett et al., 1992) or foliage type specific differences in specific leaf area (evergreen and deciduous conifers, wieser et al., 2013). for example, sclerophyllous mediterranean species are known to have a high biochemical capacity to cope with oxidative stress and thus are expected to be less sensitive to ozone (bussotti and gerosa, 2002). similarly, the present analysis revealed a slight trend of higher ozone tolerance for evergreen or coniferous species (tab. 5 and tab. 6). 6.3 agricultural and horticultural crops results from the data base in total 478 entries were found in 195 literature references that meet the above mentioned requirements for the experimental setup and comprise information on the response to ozone of 54 crop species. with very few exceptions, the cultivars of these species tested are exactly specified, so that in total data are available for 350 different genotypes (tab. 8 ). more than half of the studies were performed using open-top chambers as exposure systems. recently data for glycine max, oryza sativa, phaseolus vulgaris, solanum tuberosum, and triticum aestivum are also available from free-air fumi gation systems. the majority of data contained in the database derived from european studies but due to a growing awareness of air pollution impacts in china and india for example, a high number of data derived from more recently published asian studies. different from the other plant groups, testing of agricultural and horticultural crop plants for their ozone sensitivity has started already in the 1950s in the usa. moreover, as these studies were always related to the commercial use of agricultural and horticultural crop species they nearly always comprised one or several clearly defined cultivars of a species. implicitly, the use of such cultivars also resembled the result of breeding activities which intended to optimize a cultivar to its particular environment including ozone pollution (see below). thus the studies referred to in the present investigation (not before 1980) have been performed when the sensitivity of many crop species and cultivars, respectively, was already known. apart from some new screening studies in africa or asia after 1980 mainly sensitive species have been included into experimental studies. as a result, about 90 % of species and 83 % of all cultivars showed a negative growth effect (tab. 8). out of the 55 species considered here 17 have only been investigated in one study which for example comprises six ancestors of modern wheat cultivars. generally, wheat was the most frequently studied species, followed by bean, rice and soybean (tab. 9), each represented by 29 to 49 different cultivars. according to the present data 75 % 92 e. bergmann, j. bender, h.j. weigel of all cultivars were studied only once, 15.2 % (52 cvs) twice and only 6.4 % (22 cvs) three times. cultivars investigated repeatedly (more than three experimental studies) were lactuca sativa cv. paris island (4×), triticum aestivum cv. turbo, oryza sativa cv. koshihikari, and solanum tuberosum cv. bintje (5×), glycine max cv. essex (7×) and phaseolus vulgaris cv. lit (8×). although about 90 % of all experiments proved an ozone sensitive response of the considered cultivar, for the following cultivars divergent responses to ozone were observed in different studies: brassica campestris cv. wisconsin fast plants, fragaria × ananassa cv. elsanta, glycine max cv. essex, lactuca sativa cv. paris island, lycopersicon esculentum cv. pusa ruby, oryza sativa cvs ir 64, kasalath, koshihikari, nipponbare, yangdao 6, phaseolus vulgaris cvs r123, r331, s156, raphanus sativus cv. cherry belle, solanum tuberosum cv. bintje, triticum aestivum cvs yangfumai 2, yangmai 15 (growth), solanum lycopersicum cv. pusa ruby, phaseolus vultab. 8: summary of information on growth and visible injury effects of ozone on agricultural and horticultural crops. numbers of records (entries in the database), species, and cultivars or genotypes investigated described in 195 relevant publications including the relative share of responses to ozone. tested (no.) responsive (%) visible injury growth both visible injury growth both records 214 413 150 88.3 80.9 80.0 species# 48 47 35 89.6 91.5 91.4 cultivars/genotypes* 179 308 127 88.3 82.8 80.3 # irrespective of ssp. or var., * except for those, whose response was given as an average exclusively tab. 9: summary of the ozone sensitivity of 55 agricultural and horticultural crop species. data refer to the number of cultivars of each species and are shown as the number of responsive cultivars in relation to the total number of cultivars investigated. species responsive total species responsive total aegilops tauschii 1 1 medicago sativa 5 5 allium ampeloprasum 0 1 nicotiana tabacum 7 9 allium cepa 5 5 oryza sativa 31 47 arachis hypogaea 1 1 phaseolus vulgaris 39 42 avena sativa 0 1 pisum sativum 1 1 beta vulgaris 2 2 raphanus sativus 3 4 brassica campestris 10 10 saccharum spp. 1 1 brassica juncea 2 2 solanum lycopersicum 10 11 brassica napus 2 2 solanum tuberosum 7 7 brassica napus ssp. oleifera 1 5 spinacia oleracea 1 ? brassica oleracea 2 5 trifolium alexandrinum 6 6 brassica rapa 4 4 trifolium repens 5 5 cicer arietinum 2 2 trigonella foenum-graecum 1 1 citrullus lanatus 7 7 triticosecale wittmack 1 1 corchorus olitorius 1 1 triticum aestivum 50 50 coriandrum sativum 1 1 triticum boeoticum 1 1 cucumis melo 2 2 triticum dicoccum 1 1 cucurbita pepo 1 3 triticum durum 11 11 daucus carota 2 2 triticum monococcum 1 1 eruca sativa 2 2 triticum polonicum 1 1 fragaria × ananassa 4 4 triticum timopheevii 1 1 glycine max 27 28 valerianella locusta 1 1 gossypium barbadense 1 4 vicia faba 0 1 gossypium hirsutum 8 8 vigna mungo 1 1 hordeum vulgare 3 8 vigna radiata 7 7 lactuca sativa 9 11 vigna unguiculata 2 2 linum usitatissimum 2 2 zea mays 6 6 lycopersicon pimpinellifolium 1 1 ozone sensitive taxa 93 garis cv. bush blue lake 274, and triticum aestivum cv. riband (injury). in order to define sensitivity rankings for cultivars an experimental design is required, which allows to compare the response of different cultivars or genotypes directly. the complete database comprises 465 entries and put emphasis on cultivar comparisons. referred to the literature considered, 82 studies met these requirements. a total for 29 species have been investigated comparatively within one experiment, in which the number of cultivars per study ranged from 2 to 20 (oryza sativa). the species, which most frequently were subjected to cultivar comparisons or screenings were glycine max, nicotiana tabacum, oryza sativa, phaseolus vulgaris, and triticum aestivum. for the following 22 species no comparative studies were found: aegilops tauschii, allium ampeloprasum, arachis hypogaea, avena sativa, corchorus olitorius, coriandrum sativum, cucurbita pepo, daucus carota, eruca sativa, pisum sativum, raphanus sativus, saccharum spp, solanum tuberosum, spinacia oleracea, trigonella foenum-graecum, triticosecale wittmack, triticum monococcum, triticum polonicum, triticum timopheevii, and valerianella locusta literature context: importance of breeding activities in order to maintain the productivity of agricultural and horticultural crops in a high ozone environment the development and use of ozone insensitive/tolerant cultivars is suggested to be the most economical and practical solution (avnery et al., 2013; biswas et al., 2009; burkey and carter, 2009; foy et al., 1995) and the knowledge about the genetic background of ozone sensitivity/tolerance is fundamental for breeding ozone tolerant cultivars (biswas et al., 2008a). due to its importance for human nutrition wheat (triticum aestivum) has repeatedly been tested for its ozone sensitivity (83 records, 50 cultivars; tab. 9). intraspecific variations of ozone sensitivity of wheat has been found for some modern cultivars (kou et al., 2012; kou et al., 2013; reichenauer et al., 1998; sarkar and agrawal, 2010; tiwari et al., 2005), while in other studies different cultivars responded similarly to ozone (akhtar et al., 2010; barnes et al., 1995; zhu et al., 2011). a screening study of yields of old and modern cultivars clearly showed that the more modern cultivars exhibited a greater sensitivity to ozone than older cultivars (barnes et al., 1990; biswas et al., 2008a; biswas et al., 2008b; velissariou et al., 1992). it was assumed that selection by plant breeders for higher stomatal conductance and hence, higher co2 assimilation, inadvertently led to higher ozone uptake rates in modern cultivars resulting in higher sensitivity to ozone than their predecessors (barnes et al., 1990; velissariou et al., 1992). while for wheat this has also been questioned (biswas et al., 2009; feng et al., 2011; inada et al., 2012), for soybean, however, the same mechanism, i.e. an increasing ozone sensitivity of more modern cultivars, was suggested by osborne et al. (2016). the latter authors analysed 28 experimental studies reporting on 49 soybean cultivars in total and found an increase in ozone sensitivity of soybean by an average of 32.5 % between 1960 and 2000 based on the year of cultivar release. although the underlying mechanisms remain unclear, the fact that ozone sensitivity has been changed due to selection by plant breeders seems to be obvious. 6.4 mosses and ferns there were only 9 references which were selected to be appropriate to describe effects of ozone on mosses and ferns. this resulted in 21 records of 14 species (tab. 10). in these studies exposure to ozone included controlled environment and open-top chamber studies and comprised two fern species (atyrium felix-femina and onoclea sensibilis), 8 species of the sphagnum genus and the two other moss species (dicranum polysetum and pleurozium schreberi). visible injury was either absent or not determined and reductions in growth or spore germination were observed on two species each (tab. 10). on the other hand, the species regarded here proved to be highly ozone sensitive when physiological parameters, i.e. membrane permeability or ultrastructure of organelles were considered. however, overall the data basis for these taxa is very small and it remains difficult to assess the sensitivity of mosses and especially of ferns towards ozone in established ecosystems. as outlined by niemi (2003) due to their morphological structure, stomatal control does not play a role for ozone uptake into the mosses and the permanent water film covering the leaf surfaces of mosses is thought to act as an ozone scavenger. this argument does not point to a particular ozone risk for these species. 6.5 algae, lichens, and phyllosphere fungi most experimental studies with algae species have been carried out either with chlorella sorokiniana (heath, 1984; heath et al., 1982; swanson et al., 1982) or euglena gracilis (bilodeau and chevrier, 1998; chevrier et al., 1990; chevrier and sarhan, 1992; chevrier et al., 1988). in these studies the algae were grown in cell cultures and ozone was injected into the liquid growth medium. all relevant studies refer to investigations on the mechanisms of ozone-induced oxidative damages and repair processes at the cellular level using the algae as model cells. lichens represent a symbiotic system of fungi and algae. similar to mosses lichens lack a waterproofing cuticle and stomata, and are thus exposed to ozone directly at their surface cell layers. lichens are known to be highly responsive to so2 (ahn et al., 2011; lackovicova et al., 2013) and hno3 (riddell et al., 2012). studies with lichens using experimental ozone exposures are listed in tab. 11 and comprise mainly controlled environment conditions. in total 31 lichen species have been tested for their ozone sensitivity. with the exception of the observation of visible injuries described by ruoss and vonarburg (1995) and scheidegger and schroeter (1995) physiological and structural impairments have frequently been observed as a result of controlled exposure to high ozone levels. from a study with three lichen species using open-top chambers bertuzzi et al. (2013) concluded, that lichens may be considered as rather ozone-tolerant organisms. lichens were frequently used as bioindicators for photochemical oxidant air pollution e.g. in the usa (mccune, 1988; will-wolf et al., 1996), scandinavia (oksanen et al., 1990; olsson, 1995), switzerland (ruoss and vonarburg, 1995), italy (lorenzini et al., 2003; nali et al., 2007), slovenia (batic and kralj, 1996), and korea (ahn et al., 2011; hur and kim, 2000). with respect to ozone their suitability as bioindicators for this pollutant was deduced from field observation in the san bernardino mountains e.g. (nash and sigal, 1999). however, the lack of a correlation with other ozone response data (batic and kralj, 1996; lorenzini et al., 2003) or ozone exposures indices (ahn et al., 2011; nali et al., 2007; ruoss and vonarburg, 1995) led to the conclusion that lichens are not suitable for monitoring ozone episodes. few studies have addressed the question if and to what extent fungi respond to an ozone exposure and these studies focused on phyllosphere species. magan et al. (1995) analysed the phyllosphere microbial populations inhabiting the needles surfaces of conifer species in an open-air fumigation experiment. after three years of exposure to ozone they found an increase in the occurrence of sclerophoma pythiophila on picea sitchensis but a decrease of epicoccum nigrum and cladosporium spp. on pinus sylvestris, while the total fungal populations or the fungal biomass was increased on the needles of this species. fenn et al. (1989) examined populations of phyllosphere fungi from leaves of the tree species sequoiadendron giganteum and quercus kelloggii which were exposed to 1.5× of the ambient 94 e. bergmann, j. bender, h.j. weigel ozone concentration within otc’s for 9 to 11 weeks. for none of the tree species total numbers of fungi isolated or the frequency of occurrence of dominant fungi was affected by ozone. however, there was a significant chamber effect since five fungal species had significantly higher isolation frequencies in the open-air treatment compared with those in the treatments within chambers. similarly, von tiedemann et al. (1991) in an otc study reported on a chamber effect on the saprobial colonization of the phyllosphere of triticum aestivum, while there was no ozone effect. 6.6 vertebrates information on responses of vertebrates to controlled ozone exposures found in the literature mainly refer to ozone exposure studies with laboratory test animal associated with medical (pulmonary) research and are not relevant in the present context. the few existing studies that might be relevant for terrestrial ecology exclusively used acute exposure to high levels of ozone (up to 800 ppb) and are thus also not further described here. nevertheless, the species tested in these studies included guinea pig (cavia porcellus, su and gordon, 1997), toad (bufo marinus, dohm and mautz, 2001; dohm et al., 2008; dohm et al., 2001; johnson et al., 2009), frog and lizard (pseudacris cadaverina and sceloporus occidentalis, mautz and dohm, 2004). for these vertebrates species adverse ozone effects on immune function, respiration, and feeding behaviour as well as induced hypothermia are described. no classification of an ozone sensitivity can be derived from these studies. 7 identifying sensitive taxa from field observations and case studies in contrast to the previously described experimental studies in the following chapter observations are compiled that describe actual impacts of the prevailing ambient ozone exposure scenario at a particular site or region on constituents of the ecosystem. with the raising awareness of symptoms of forest damage several national and international monitoring and research programs have been initiated to assess both the extent of ecosystem impacts and their causal agents including ozone. examples at a national, international and regional scale are the us ‘forest health monitoring (fhm)’ or the ‘international cooperative program on assessment and monitoring of air pollution effects on forests (icp forests)’ of the unece, the us ‘san bernardino mountains network study (sbm)’, the us ‘the vital signs program of the u.s. national park tab. 10: effects of ozone exposure on moss and fern species. detailed list of all experimental studies considered. ozone concentrations (ppb) are given in parentheses. controlled = controlled environment studies; otc = open top chambers studies species exposure effect reference athyrium felix-femina controlled (50, 100, 150) reduced spore germination bosley et al. (1998) dicranum polysetum otc (80) no injury nygaard (1994) hylocomium splendens otc (80) no injury nygaard (1994) onoclea sensibilis controlled (50, 100, 150) reduced spore germination bosley et al. (1998) pleurozium schreberi otc (80) no injury nygaard (1994) polytrichum commune controlled (50, 100, 150) none bosley et al. (1998) controlled (50, 100, 150) no effect on protonematal growth petersen et al. (1999) or gametophore production otc (70-80) (reduced growth) potter et al. (1996a) sphagnum angustifolium controlled (50, 100, 150) decreased cell cross-sectional area rinnan and holopainen (2004) occupied by chloroplasts controlled (50, 100, 150) increase in membrane perameability niemi et al. (2002) sphagnum capillifolium controlled(50, 100, 1150) none potter et al. (1996b) sphagnum cuspidatum controlled(50, 100, 1150) none potter et al. (1996b) sphagnum flexuosum otc (80) none gagnon and karnosky (1992) sphagnum magellanicum controlled (50, 100, 150) ultrastructural changes rinnan and holopainen (2004) otc (80) reduced chlorophyll concentration gagnon and karnosky (1992) sphagnum papillosum controlled (150) none potter et al. (1996b) controlled (50, 100, 150) cellwall became thinner, rinnan and holopainen (2004) decreased chloroplast size aa no growth effects morsky et al. (2011) sphagnum recurvum otc (70-80) reduced growth potter et al. (1996a) controlled (150) reduction in photosynthesis, potter et al. (1996b) increased membrane leakage sphagnum rubellum otc (80) reduced chlorophyll concentration gagnon and karnosky (1992) bosley, a., petersen, r., rebbeck, j., 1998: bryologist 101, 512-518; gagnon, z.e., karnosky, d.f., 1992: j. bryol. 17, 81-91; morsky, s.k., haapala, j.k., rinnan, r., et al., 2011: environ. exp. bot. 72, 455-463; niemi, r., martikainen, p.j., silvola, j., holopainen, t., 2002: sci. total environ. 289, 1-12; nygaard, p.h., 1994: rapport fra skogforsk, 1-17; petersen, r.l., bosley, a., rebbeck, j., 1999: bryologist 102, 398-403; potter, l., foot, j.p., caporn, s.j.m., lee, j.a., 1996a: new phytol. 134, 649-656; potter, l., foot, j.p., caporn, s.j.m., lee j.a., 1996b: j. bryol. 19, 19-32; rinnan, r., holopainen, t., 2004: ann. bot.-london 94, 623-634. ozone sensitive taxa 95 tab. 11: effects of ozone exposure on lichens. detailed list of all experimental studies considered. ozone concentrations (ppb) are given in parentheses. controlled = controlled environment studies; otc = open top chambers studies species exposure effect references anaptychia ciliaris controlled (150 ) discolored parts ruoss and vonarburg (1995) field fumigation (90 ) decreased chlorophyll fluorescence, scheidegger and schroeter (1995) decreased photosynthesis controlled (300 ) none calatayud et al. (2000) bryoria capillaris controlled (300 ) ultrastructural changes, tarhanen et al. (1997) increased membrane permeability bryoria fuscescens controlled (150 ) none ruoss and vonarburg (1995) controlled (300 ) increased membrane permeability tarhanen et al. (1997) collema nigrescens field fumigation (90 ) decreased chlorophyll fluorescence scheidegger and schroeter (1995) evernia prunastri field fumigation (90 ) decreased chlorophyll fluorescence scheidegger and schroeter (1995) controlled (300 ) none calatayud et al. (2000) controlled (150 ) discolored parts ruoss and vonarburg (1995) flavoparmelia caperata otc none bertuzzi et al. (2013) flavopunctelia flaventior controlled (80 ) red photosynthesis riddell et al. (2012) hypogymnia bitteri field fumigation (90 ) decreased chlorophyll fluorescence scheidegger and schroeter (1995) hypogymnia imshaugii controlled (80 ) none riddell et al. (2012) hypogymnia physodes controlled (300 ) ultrastructural changes tarhanen et al. (1997) controlled (150 ) discoloured parts ruoss and vonarburg (1995) controlled (300 ) none calatayud et al. (2000) lobaria pulmonaria controlled (60-175 ) no effect on photosynthesis sigal and johnston (1986) field fumigation (90 ) visible symptoms, decreased scheidegger & schroeter (1995) chlorophyll fluorescence parmelia glabra controlled (150 ) marginal blackening ruoss and vonarburg (1995) parmelia quercina controlled (300 ) none calatayud et al. (2000) parmelia sulcata controlled (150 ) no morphological changes, ruoss and vonarburg (1995) discoloured parts, blackening controlled (300 ) none calatayud et al. (2000) parmelia tialicea controlled (150 ) discoloured parts ruoss and vonarburg (1995) parmotrema perlatum otc none bertuzzi et al. (2013) physconia enteroxantha + controlled (80 ) none riddell et al. (2012) physconia isidiigera physconia perisidiosa controlled (150 ) none ruoss and vonarburg (1995) platismatia glauca controlled (150 ) discoloured parts ruoss and vonarburg (1995) pseudevernia furfuracea controlled (150 ) marginal blackening ruoss and vonarburg (1995) field fumigation (90 ) collapsed photobiont cells scheidegger and schroeter (1995) pseudocyphellaria anthraspis controlled (80 ) none riddell et al. (2012) pseudoparmelia caperata controlled (100 ) decreased photosynthesis ross and nash iii (1983) pseudovernia furfuracea controlled (150 ) discoloured parts ruoss and vonarburg (1995) ramalina farinacee controlled (150 ) marginal blackening, discoloured parts ruoss and vonarburg (1995) ramalina fraxinea controlled (150 ) marginal blackening, discoloured parts ruoss and vonarburg (1995) ramalina menziesii controlled (100 ) no reduction in photosynthesis ross and nash iii (1983) controlled (80 ) none riddell et al. (2012) usnea hirta controlled (80 ) none riddell et al. (2012) controlled (300 ) ultrastructural changes tarhanen et al. (1997) usnea lapponica controlled (150 ) none ruoss and vonarburg (1995) usnea rigida field fumigation (90 ) visible symptoms, scheidegger and schroeter (1995) decreased photosynthesis xanthoria parietina otc none bertuzzi et al. (2013) bertuzzi, s., davies, l., power, s.a., tretiach, m., 2013: ecol. indic. 34, 391-397; calatayud, a., temple, p.j., barreno, e., 2000: photosynthetica 38, 281-286; riddell, j., padgett, p.e., nash, t.h., 2012: environ. pollut. 170, 202-210; ross, l.j., nash, iii t.h., 1983: environ. exp. bot. 23, 71-77; ruoss, e., vonarburg, c., 1995: cryptogam. bot. 5, 252-263; scheidegger, c., schroeter, b., 1995: environ. pollut. 88, 345-354; sigal, l.l., johnston, j.w., jr., 1986: environ. exp. bot. 26, 59-64; tarhanen, s., holopainen, t., oksanen, j., 1997: ann. bot.-london 80, 611-621. 96 e. bergmann, j. bender, h.j. weigel service‘, the ‘conecofor (controlli ecosistemi forestali)’ program in italy and the ‘international long-term ecological studies in the carpathian mountains‘ in europe. overall, the parameter “visible leaf injury” is of particular relevance for a first assessment of the relative ozone sensitivity of a species under field conditions as it indicates an overall impairment of the respective organism. experimental verification of these ozone symptoms observed in the field (bussotti et al., 2003; gravano et al., 2004; kline et al., 2008; skelly et al., 1999; vollenweider et al., 2003) and pictorial guides of ozone effects (flagler, 1998; innes et al., 2001) helped to confirm these assessments. in total, 49 out of 349 relevant publications provide information on the expression of foliar symptoms in the field that were attributed to ambient ozone levels. overall, there were 528 indications for 245 native woody and herbaceous plant species and additionally for 28 genera without a species identification for which ozone-specific or ozone-like foliar symptoms have been observed in the field. tab. 13 provides a summary of the whole data set. for herbaceous species the information is dominated by perennials, for trees and shrubs by deciduous species. for the majority of species (60 %) visible foliar symptoms due to ozone were documented in one single study only, while for 55 species symptoms were reported by at least three studies. the species with the highest number of symptom records were prunus serotina (10×), corylus avellana (9×), fraxinus excelsior (9×), ailanthus sylvatica (8×), and fagus sylvatica (8×), which may point to a particular ozone sensitivity of these species. however, only ca. 30 % of the species (23 herbaceous and 48 woody species) described to be responsive to ozone in these field observation studies are also subjected to controlled exposure studies shown in chapters 6.1 and 6.2, i. e. for only 71 species out of 245 the ozone response was actually verified by experimental studies. however, it points to the necessity to use both types of information for a detailed ozone cause-effect analysis for vegetation. nevertheless, an assessment of such species-specific responses and their temporal and spatial variation based on long-term observations may be regarded as a first valuable information on a risk for biodiversity. 8 evidence for natural selection for ozone tolerance in (semi) natural the vegetation 8.1 spatial and temporal variations in ozone sensitivity/tolerance the inheritance of ozone tolerance as known from crops (burkey and carter, 2009; heggestad, 1991; mebrahtu et al., 1990) has also been documented for wild plant species (lee et al., 2002; whitfield et al., 1997). intraspecific variation in ozone tolerance have been evidenced for woody and herbaceous wild plant species and demonstrated that wild genotypes may differ greatly in sensititab. 12: effects of ozone exposure on vertebrates. detailed list of all experimental studies. species exposure effect reference bufo marinus acute no effect on the preferred body temperatures, dohm et al. (2001) higher evaporative water loss rates acute no effect on metabolism associated with food processing johnson et al. (2009) acute depressed feeding behaviour dohm et al. (2008) acute no differences in macrophage functions dohm et al. (2005) acute adverse effect on immune function dohm and mautz (2001) cavia porcellus acute induction of heat shock proteins su and gordon (1997) pseudacris cadaverina acute alteration of respiration mautz and dohm (2004) sceloporus occidentalis acute hypothermia mautz and dohm (2004) dohm, m.r., mautz, w.j., 2001: am. zool. 41, 1429-1429; dohm, m.r., mautz, w.j., andrade, j.a., et al., 2005: environ. toxicol. chem. 24, 205-210; dohm, m.r., mautz, w.j., doratt, r.e., stevens, j.r., 2008: environ. toxicol. chem. 27, 1209-1216; dohm, m.r., mautz, w.j., looby, p.g., gellert, k.s., andrade, j.a., 2001: environ. res. 86, 274-286; johnson, s.r., mautz, w.j., dohm, m.r., 2009: integr. compar. biol. 49, e250-e250; mautz, w.j., dohm, m.r., 2004: comp. biochem. phys. a 139, 371-377; su, w.y., gordon, t., 1997: j. appl. physiol. 83, 707-711. tab. 13: number of native species for which ozone-specific foliar symptoms or ozone-like injuries have been observed in field monitoring surveys across different continents. the data are split into different descriptive groups and sites of observation. continent of observation europe north-america central america asia total total species 157 49 6 33 245 genera without species identification 19 6 3 0 28 herbaceous species 69 24 1 3 97 annual/biennial 5 8 0 3 16 perennial 64 16 1 0 81 woody species 88 25 5 30 148 deciduous 77 18 1 28 124 evergreen 11 7 4 2 24 ozone sensitive taxa 97 vity. species for which this has been shown are populus tremuloides (karnosky et al., 1992; karnosky et al., 2003a); p. tremuloides × p. tremula l. (oksanen et al., 2001); prunus serotina (lee et al., 1999); betula pendula (oksanen, 2003; pääkkönen et al., 1993; pääkkönen et al., 1996); pinus densiflora (lee et al., 2006); pinus taeda (taylor, 1994); quercus coccifera (elvira et al., 2003); anthoxanthum odoratum (dawnay and mills, 2009); arabidopsis thaliana (brosche et al., 2010); phleum pratense and phleum alpinum (danielsson et al., 1999). with respect of steadily increasing ozone exposure levels over the last decades it has been hypothesised that this could also have resulted in an increase of the ozone tolerance of native (plant) species. there are several studies reporting on spatial approaches to test whether geographically separated plant populations, which were exposed to different levels of ozone over many years exhibit differences in their ozone sensitivity/tolerance corresponding to the prevailing ozone exposure regime at the respective site. the degree of the sensitivity/tolerance of a population was tested experimentally under controlled exposure conditions. in tab. 14 we have compiled studies where associations between the ozone sensitivity/tolerance of a plant population and the “ozone climate” at different location have been demonstrated. the most prominent example of spatial variation in ozone tolerance is the herbaceous wild plant species plantago major (tab. 14). testing the relative ozone sensitivity/tolerance of 28 geographically separated populations, which had been collected across uk, it was demonstrated that the populations differed in their ozone sensitivity/ tolerance and that these differences were statistically related primarily to the “ozone climate” of its site of origin (lyons et al., 1997; pearson et al., 1996; reiling and davison, 1992). however, as also shown in tab. 14, in some studies intraspecific differences in ozone sensitivity/tolerance between populations originating from different proveniences were also shown, but a correlation with the “ozone climate” could not statistically be verified (tab. 14). this was partly be explained by a high variability in the ozone sensitivity/tolerance of the individuals within the populations (kline et al., 2009). in addition to spatial variations for plantago major also a temporal change in ozone resistance/tolerance over a short period of time was reported. for two populations in uk an increase in ozone tolerance has been shown in experiments for plants grown from seed material collected after summers when ozone concentrations were high (davison and reiling, 1995; whitfield et al., 1997). heagle et al. (1991) exposed trifolium repens plants under field conditions to dif ferent levels of ozone under conditions of intraand interspecific competition with festuca arundinacea and propagated those individuals by cloning that survived the ozone treatment. only after two years clones sampled from the high ozone plots exhibited a higher percentage of ozone tolerant clones than those treated with low ozone. in contrast, repeated exposure of betula pendula trees to ozone for six years led to an increase in the ozone sensitivity of the trees, which was partly explained by an ozone induced change in the growth form and deleterious carry-over effects (oksanen, 2003). tab. 14: plant species for which spatial variation in the ozone sensitivity/tolerance was indicated by differential responses of populations from different sites; no. = number of populations or sites considered; exposure conditions for controlled sensitivity tests include exposure facility (aa, aa+ ambient air, ambient air plus ozone addition; gh greenhouse; gc growth chamber; nf non-filtered; otc open-top chamber), ozone concentration (ppb) and duration; stat. sign. = statistically significant. species study area no. exposure conditions stat. sign. reference plantago major uk 28 gc 70 ppb, 2wk yes reiling and davison (1992) plantago major different countries in europe 20 gc 70 ppb, 2wk yes lyons et al. (1997) trifolium campestre switzerland, north / south 2 otc aa, aa+ yes fuhrer et al. (1998) populus tremuloides usa, eastern national parks 5 gh 180 ppb, 6h yes berrang et al. (1986) usa, national parks 15 gh 150 ppb, 6h yes berrang et al. (1991) usa, new york, michigan 5 aa yes berrang et al. (1989) usa, great lakes region 6 aa yes karnosky et al. (2003) trifolium repens switzerland 100 ppb, 6 d 150 ppb, 3 d no nebel and fuhrer (1994) trifolium pratense phleum pratense scandinavia 9 otc cf+70 ppb, nf+50 ppb no danielsson et al. (1999) epilobium hirsutum uk 18 no davison and haley (2001) asclepias syriaca midwestern usa 9 gh 40 to 80 ppb no kline et al. (2009) apocynum cannabinum midwestern usa 16 gh 40 to 80 ppb no kline et al. (2009) prunus serotina usa, pennsylvania, west virginia 15 aa no lee et al. (1999) betula pubescens scandinavia 5 gh 20 to 116 ppb no mortensen (1998) berrang, p., karnosky, d.f., bennett, j.p., 1989: can. j. for. res. 19, 519-522; berrang, p., karnosky, d.f., bennett, j.p., 1991: can. j. for. res. 21, 1091-1097; berrang, p., karnosky, d.f., mickler, r.a., bennett, j.p., 1986: can. j. for. res. 16, 1214-1216; danielsson, h., gelang, j., pleijel, h., 1999: environ. exp. bot. 42, 41-49; davison, a.w., haley, h., 2001: in: d. fowler, m. coyle, r. storeton-west, h. lewis, t. mansfield, n. paul, p.s. de silva, m. ashmore, k. batty, a. davison, h. haley (eds.), the ozone umbrella project ceh project: c00970 final report, centre for ecology and hydrology, edinburgh; fuhrer, j., endtner, v., bungener, p., nussbaum, s., grub, a., 1998: in: b. boller, f.j. stadelmann (eds.), breeding for a multifunctional agriculture. proceedings of the 21st meeting of the fodder crops and amenity grasses section of eucarpia, kartause ittingen, switzerland, 9-12 september 1997, 191-194; karnosky, d.f., percy, k.e., mankovska, b., prichard, t., noormets, a., dickson, r.e., jepsen, e., isebrands, j.g., 2003: in: d.f. karnosky, k.e. percy, a.h. chappelka, c. simpson, j. pikkarainen (eds.), air pollution, global change and forests in the new millennium. elsevier, 199-209; kline, l.j., davis, d.d., skelly, j.m., decoteau, d.r., 2009: north east. nat. 16, 307-313; lee, j.c., skelly, j.m., steiner, k.c., zhang, j.w., savage, j.e., 1999: environ. pollut. 105, 325-331; lyons, t.m., barnes, j.d., davison, a.w., 1997: new phytol. 136, 503-510; mortensen, l.m., 1998: scand. j. forest res. 13, 189-196; nebel, b., fuhrer, j., 1994: angew. bot. 68, 116-121; reiling, k., davison, a.w., 1992: new phytol. 122, 699-708. 98 e. bergmann, j. bender, h.j. weigel 8.2 implication of variation in ozone tolerance for the genetic structure of plant populations any indication of a loss of ozone sensitive genotypes in areas with high ozone pollution carries the risk for a loss of useful rare alleles, particularly, if some genes are found exclusively in the sensitive genotypes as this has been suggested by isozyme studies of picea abies clones (karnosky et al., 1989a). as a consequence, ozone pollution would have the potential to affect the genetic structure of plant populations. for example, the finding that ozone and/or pollutant sensitive genotypes are under-represented within populations of plant species in eastern north america was interpreted as a first stage of natural selection, i.e. the elimination of sensitive genotypes attributed to the impact of ozone and/or other regional air pollutants (berrang et al., 1991; karnosky et al., 1989a; b). there is evidence that micro-evolutionary processes could take place in response to long-term elevated ozone exposure and at some regions even the prevailing ambient ozone levels are sufficiently high to promote this evolution (berrang et al., 1991; 1986; kolliker et al., 2008; lyons et al., 1997). there are some indications for this assumption in the literature for both trees and herbaceous plants. staszak et al. (2004) reported on a relationship between ozone injury of pinus ponderosa needles and heterozygosity, as ozone tolerant trees were more heterozygous than ozone sensitive individuals. moreover, the signatures of the genetic structure between saplings and mature pine trees suggest that the increase in air pollution over the last 50 years together with episodic drought stress may have affected the genetic structure of two pine species in the us sequoia national park (staszak et al., 2007). wolff et al. (2000) analysed genetic markers associated with ozone tolerance in plantago major at 27 continental european sites. they found that gaining tolerance to ozone was associated with a decrease in genetic variation over time. because the genetic composition showed no drastic changes, it was assumed that the change in tolerance to ozone was probably the result of a selection of genotypes already present in local populations (selection in situ). in addition, their finding revealed that selection for ozone tolerance may involve a number of genetically determined traits and thus the authors concluded that plants with similar degrees of ozone tolerance are not closely related (davison and reiling, 1995; whitfield et al., 1997). kolliker et al. (2008) recently demonstrated that differences in the genetic composition and diversity were only detectable in populations of the species plantago lanceolata after exposure of an old semi-natural grassland to elevated ozone for five years. bassin et al. (2004) examined the genetic distinctiveness of five centaurea jacea populations originating from different european countries (norway, hungary, switzerland, italy, slovenia) showing a high degree of intraspecific variability in ozone sensitivity. their results indicate a qualitative relationship between population genetic divergence and variability in ozone sensitivity. as shown by dna fingerprinting assays populations of rudbeckia laciniata sampled at different sites in the great smoky mountains national park, usa, differ in genetic diversity (davison et al., 2003). according to karnosky et al. (1989a) germplasm loss and the subsequent decrease in genetic diversity could be a more important air pollution impact in the long run than short-term economic losses. conclusion there is currently insufficient information available, if and to what extent tropospheric ozone might contribute to biodiversity changes in terrestrial ecosystems. as a first step into such an assessment the relative ozone sensitivity of the respective organisms should be known. therefore, the aim of the present study was to systematically analyse possible ozone impacts on different taxa and species through a literature research. worldwide information was collected about 350 varieties across 54 crop plant species and 465 vascular and fern plant species belonging to (semi)-natural vegetation types and used as a database. overall, the available ozone studies covered only a small fraction of the entire global flora, e.g. for germany only about 6.1 % of all known plant species. fig. 1: ranking of ozone sensitivity of native herbaceous or woody plant species based on their taxonomic classification. percentage of the number of ozone responsive and non -responsive species within a family are shown (according to chap. 6, tab. 3 and tab. 7). only those families are considered which are represented by at least five different species. about two third of woody and about one half of herbaceous vascular species listed in the database were described as ozone sensitive in at least one study. these proportions are slightly higher for visible leaf injury than for growth effects and herbaceous and deciduous woody plants are more responsive to ozone than grasses and coniferous tress. based on this information we make an attempt to identify and rank plant families according to the proportion of ozone sensitive species within a particular family as shown in fig. 1. for example, while many species in the family of myrtaceae and salicaceae seem to be rather sensitive towards ozone this is not true for the boraginaceae and the brassicaceae. with respect to the german situation there is evidence from the database that a number of plant species that are already categorised as endangered or near-threatened (tab. 4) seem to be sensitive towards an ozone exposure. probably, these plant species should be more thoroughly observed under aspects of air pollution impacts. in addition the present database has shown that there is some evidence that ozone pollution in the past has affected plant selection and modified the genetic pool of genotypes. although there is still 13        fig.  1:   ranking   of   ozone   sensitivity   of   native   herbaceous   or   woody   plant   species   based   on   their   taxonomic  classification.  percentage  of   the  number  of  ozone  responsive  and  non   -­‐responsive  species   within  a  family  are  shown  (according  to  chap.  6,     tab.  3  and     tab.  7).  only  those  families  are  considered  which  are  represented  by  at  least  five  different  species.   brassicaceae   boraginaceae   plantaginaceae   caryophyllaceae   papaveraceae   poaceae   asteraceae   polygonaceae   caprifoliaceae   pinaceae   adoxaceae   rosaceae   fagaceae   sapindaceae   fabaceae   cyperaceae   scrophulariaceae   betulaceae   oleaceae   onagraceae   salicaceae   myrtaceae   -­‐150   -­‐100   -­‐50   0   50   100              %  species   not  responsive  ←      →  responsive       ozone sensitive taxa 99 unsufficient evidence whether rapid changes in plant tolerance to ozone impacts is an overall phenomenon in non-managed ecosystems. however, due to a broad intraspecific variation of ozone sensitivity of plant species it has to be kept in mind that classifying a species as either ozone sensitive or tolerant might be an oversimplification because the ozone response of a particular genotype seems to reflect its ozone exposure history rather than a generic species specific trait. in the context of ozone risk assessments for biodiversity this must be taken into account. information on direct effects of ozone on species other than vascular plants (mosses, ferns, lichens, algae, fungi, vertebrates) is very poor and does not allow a sensitivity assessment. other organisms like microorganisms, arthropods or insects are known to not respond directly to ambient ozone, but to be affected indirectly via impairment of the vitality of plants they are associated with (weigel et al., 2015). these organisms were not considered here. also, we did not consider if and to what extent other environmental factors (e.g. drought, nutrient limitation, climate change, elevated co2) modify the response of a particular species or genotype to ozone. nevertheless, the information summarised provided in the present study provides sufficient evidence that current tropospheric ozone levels interact with many elements of the terrestrial biodiversity. acknowledgement this work was partly supported by the german umweltbundesamt (project no. 3711 63 235). 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de botánica, 3 departmento de horticultura, universidad autónoma agraria antonio narro, saltillo, coahuila, méxico 2 centro de investigación en química aplicada, saltillo, coahuila, méxico cu nanoparticles absorbed on chitosan hydrogels positively alter morphological, production, and quality characteristics of tomato antonio juárez-maldonado1, hortensia ortega-ortíz2, fabián pérez-labrada3, gregorio cadenas-pliego2, adalberto benavides-mendoza3* (received january 26, 2016) * corresponding author summary use of nanoparticles as nano copper (ncu) may be useful in agriculture. the objective of the present study was to evaluate responses in plant growth and antioxidants in tomato fruits upon application of ncu absorbed on chitosan hydrogels. the study was performed in two stages. the first stage, with tomato seedlings, was done to determine the most appropriate ncu concentration. ncu was absorbed on a chitosan hydrogel at 100 mg ncu kg-1 of hydrogel, and five different treatments of hydrogel were applied to the substrate prior to transplantation: 0.3, 0.15, 0.06, 0.03 and 0.015 g l-1, plus a control. the second stage evaluated the best treatment results from the previous stage, a chitosan treatment without ncu and a control. effects of the treatments on antioxidants in the leaves and fruit were evaluated, along with fruit quality. the results from the first stage demonstrated that 0.06 g l-1 ncu-chitosan hydrogel treatments had better results. outcomes from the second stage demonstrated that treatment with ncu had the best results for most of the plant growth variables, with differences in catalase activity in the leaves and lycopene concentration in the fruit. application of chitosan hydrogels with ncu was favorable to tomato growth and quality. introduction the use of nanoparticles (nps) on a scale from 1 to 100 nm is increasingly common for a variety of clinical and commercial purposes (somasundaran et al., 2010). nps are also used in agriculture and the food industry. compared to bulk materials, nps exert effects on organisms at very low concentration thresholds, interact with membrane transport proteins and, because of their small size, are able to enter the cell cytoplasm directly through the membrane (adhikari et al., 2013). despite these potentially beneficial features, the use of nanotechnology in agriculture is still limited due to the lack of information on nanomaterial or nanoparticle toxicity (narayanan et al., 2013). plants need metals like fe, zn, mn, and cu, to develop biochemical redox reactions for the activation of enzymes and proteins and their cofactors and gas transport, such as co2 and o2 (epstein and bloom, 2005). because the availability of transition metals in soils is highly variable, plants have developed different mechanisms for their capture, transport and assimilation. the uptake of these metals usually occurs in the roots, where they are then transported to the rest of the plant. some of these elements can be found stored in ionic form in the cell vacuole but are usually complexed with organic acids, in non-charged metallic nanostructured particles or in specialized proteins for storage, such as ferritin (lobréaux et al., 1992). the concentrations of transition metals at sites of absorption, transport and storage are under very fine control, as even in small concentrations, these metal ions cause oxidative damage through fenton reactions in membranes, proteins, and nucleic acids (pastori and foyer, 2002). many studies have been reported where toxic effects of nps of different elements (musante and white, 2012; song et al., 2013) are verified. however it is known that the toxic effect depends on the concentration; the nps in low concentration exert stimulatory effects of plant growth (iavicoli et al., 2014). sio2 nps promote the activities of superoxide dismutase and indoleacetic acid in cotton plants, being present in the root and transported to the rest of the plant by the xylem (nhan et al., 2014). in rice, the si and mo nps performed best at concentrations of 40 and 5 mg l-1, respectively (adhikari et al., 2013); in soybeans and chickpeas, it was observed that the optimum concentration of ncu to promote growth was between 60 and 100 mg l-1 (adhikari et al., 2012). however, growth was inhibited beyond these concentrations, attributed to the excessive accumulation of nps in the roots. recently, several studies have demonstrated that cu nps (ncu) are absorbed by plants and accumulate in the cells of roots, leaves and other plant tissues (shi et al., 2014). during this process, the activity of some enzymes can increase, such as catalase, or decrease, such as ascorbate peroxidase (trujillo-reyes et al., 2014). it is believed that the stimulatory effects of cu nps relate to the induction of antioxidant activity (fu et al., 2014). thus, it would be interesting verify the effect of ncu in the tomato fruit which is consumed by its nutraceutical value (shalaby and el-banna, 2013). however, this requires a system that controls the ncu concentration in the medium. hydrogel is a gel-type polymer network that is water-insoluble due to its extensive cross-linking; it has significant water retention capacity, which is attributed to the presence of several functional groups (e.g., amino, carboxyl, amide, hydroxyl and sulfonic acid) in the polymers of the hydrogel network (peppas and khare, 1993). these hydrogels are used in pharmaceutical products (singh et al., 2010; kashyap et al., 2005), agriculture (rudzinski et al., 2002), biomedical applications (kashyap et al., 2005), the separation of biomolecules or cells (wang et al., 2010), and biosensors (krsko et al., 2009). chitosan is a linear polysaccharide with good chemical functionality in hydrogel synthesis due to the greater crosslinking capacity and presence of a large number of amino groups (-nh2) (ravi-kumar, 2000). chitosan is a natural biodegradable polymer that is extracted from the shells of crustaceans, such as crabs and shrimp (bautista et al., 2006). several studies have shown that chitosan has antimicrobial properties (el-hadrami et al., 2010), promotes changes in gene expression and increases flower production of orchids (limpanavech et al., 2008), and operates as a resistance inducer (khan et al., 2003; el-hadrami et al., 2010). chitosan hydrogels are prepared with the aim of encapsulating substances to produce dosing systems with applications in industry, agriculture, medicine, pharmacy and biotechnology. in the case of nps, the use of hydrogels allows a greater control of the release of the materials in soils and substrates, with relatively little restriction for root exploration. to date, there are no reports on the agricultural use of ncu encapsulated in chitosan hydrogels, which could be advantageous from the standpoint of controlling release of nps to the soil. 184 a. juárez-maldonado, h. ortega-ortíz, f. pérez-labrada, g. cadenas-pliego, a. benavides-mendoza the objective of the present study was to evaluate different concentrations of ncu absorbed in chitosan hydrogels on tomato plants to known their effects on plant growth characteristics and antioxidant compound induction in the fruits. materials and methods this study was conducted in a hood-type greenhouse with a polycarbonate cover located in the universidad autónoma agraria antonio narro, located in saltillo, mexico (25°21’5” latitude north and 101°1’47” longitude west, elevation 1742 masl). the study was conducted in two stages that are described below. the ncu used in this study were synthesized at the centro de investigación en química aplicada (ciqa) using the methodology of cadenaspliego et al. (2013). the chitosan hydrogels were also synthesized at the ciqa from marine chemicals brand chitosan (mv=200,000) using cross-linked glutaraldehyde to obtain a hydrogel with a water absorptive capacity of 300 % based on its weight. stage 1. preliminary study to determine the range of concentrations of ncu suitable for application to the chitosan hydrogel substrate in this first stage, seeds of the saladette tomato hybrid variety “toro” with determinate growth were germinated in polystyrene trays. this variety was selected by its precocity and uniformity, also, because in the first stage it was not necessary the production of fruits. the seedlings developed for 30 days before being transplanted into 2 l pots with a mixture of peat moss and perlite 50:50 (v/v). steiner nutrient solution was used (steiner, 1961) with a targeted irrigation system to provide the nutrients required for growth. microele ments of the nutrient solution were applied in the form of chelates. nutrient applications were adjusted by managing the concentration of steiner solution according to the growth stage of the crop and its capacity of nutrient uptake: 25 % of concentration at two weeks after transplantation, 50 % of concentration at weeks 3 and 4, 75 % of concentration at weeks 5 and 6, and 100 % of concentration from week 7. this management was the same for the two experimental stages. in the first stage, an exploratory study was conducted to determine the most suitable ncu concentration for plant growth, starting with a chitosan hydrogel with ncu absorbed at a concentration of 100 mg kg-1. the five different treatments consisted of the application of the ncu-chitosan hydrogel substrate prior to transplantation: 0.3, 0.15, 0.06, 0.03 and 0.015 g l-1, plus a control without application of ncu-chitosan hydrogel, which corresponded to 0.03, 0.015, 0.006, 0.003, 0.0015, and 0 mg ncu l-1 of substrate. the treatments were prepared by placing the first 1 l of substrate followed with the ncuchitosan hydrogel in the middle of each pot, and finally, the remaining substrate was added to complete the 2 l. at 40 days after transplantation (dat), plant growth was measured using indices such as plant height, number of leaves, stem diameter and number of floral clusters. in addition, the abaxial stomatal conductance was measured in the third leaf using a model sc-1 porometer (decagon devices, inc.). subsequently, the plants were collected, weighed and dried in a drying oven at 80 °c for 72 h to determine the biomass of the dry shoot (leaves and stems). these results were used to determine the range of the most suitable ncu concentration. the treatment with the best results was selected for evaluation in the second stage. stage 2. study of chitosan hydrogels with ncu applied to the substrate and their inductive capacity of antioxidants once the most suitable concentration of absorbed ncu applied to the chitosan hydrogel was determined in stage 1, the treatment was evaluated in terms of its capacity to promote growth in adult plants and to induce antioxidant compounds in the fruits. for this assessment, a comparison was conducted with the same concentration of chitosan hydrogel but without any ncu content and with an absolute control without application of either chitosan hydrogel or ncu. the same application system was used for the ncu absorbed on chitosan hydrogel as described for stage 1, with the difference being that in this stage, 12 l pots were used for crop development. hydrogels were applied in three parts, first by adding 3 l of substrate to the pot and then 0.33 g of hydrogel, repeating this process until the required 1 g of hydrogel and substrate was obtained. for the second stage, seeds from the ball-type tomato hybrid variety “imperial” of indeterminate growth were used. this variety was selected by its uniformity and great production fruit capacity, also is commonly used under greenhouse conditions. the seeds were germinated in polystyrene trays and left to develop for 30 days before transplantation into 12 l pots. for evaluating the effects of treatments, at 90 dat, the abaxial stomatal conductance and spad units were measured with a model sc-1 porometer (decagon devices, inc.) and a spad minolta 502, respectively. at 120 dat, growth was measured using plant height, number of leaves, stem diameter, number of floral clusters, and fruit number and weight as indices. subsequently, the plants were collected, weighed and dried in a drying oven at 80 °c for 72 h to determine the dry shoot biomass (leaves and stems). in addition, the mineral contents (k, ca, mg, fe, cu and zn) were measured in the different organs, including roots, leaves and fruits, using a plasma emission spectrophotometer (icp; model thermo jarrel ash). the plants were collected, dried in a drying oven at 80 °c for 72 h, ground and finally subjected to acid digestion according to the standard techniques of the aoac (1990). quantification of total proteins and enzyme activity was conducted using the fresh tissue of leaves and fruits collected at 60 dat corresponding to first harvest of fruits. the samples were frozen at -20 °c and then lyophilized. next, 200 mg of lyophilized tissue was ground in a mortar and placed in a microcentrifuge tube, to which 20 mg of polyvinylpyrrolidone and 1.5 ml of phosphate buffer ph 7-7.2 (0.1 m) were added. the extract obtained was centrifuged at 12,000 rpm for 10 minutes at 4 °c in a microcentrifuge (labnet int. inc., model prismtm r), and the supernatant was collected and filtered with a nylon membrane (ramos et al., 2010) and then diluted at a ratio of 1:20 with phosphate buffer for analysis according to the following methodologies. quantification of total protein: plant tissue protein concentrations were determined using the bradford (1976) colorimetric technique with bovine serum albumin (bsa) as a standard at a wavelength of 595 nm in a uv-vis spectrophotometer (thermo scientific model g10s). superoxide dismutase (sod) (eq 1.15.1.1): measurement of sod enzymatic activity was conducted using the sigma 19160 determination kit. the reaction mixture (made in white eppendorf microplates) contained 20 μl of protein extract, 200 μl of water soluble tetrazolium salt (wst) and 20 μl of enzyme working solution (xo/x). the plate was then incubated at 37 °c for 20 minutes and read using a plate reader (biotek el×808) at an absorbance of 450 nm. the sod activity was expressed as a rate of inhibition assuming that wst inhibition is attributed to the neutralization of superoxide radicals by sod. measurement of catalase enzymatic activity (eq 1.11.1.6): catalase activity was quantified by measuring 2 reaction times using the spectrophotometric method of dhindsa et al. (1981). the reaction mixture contained 100 μl of protein extract, 1 ml of h2o2 (100 mm) and 400 μl of 5 % h2so4 (added to stop the reaction). the reaction was performed in microcentrifuge tubes at a temperature of 20 °c under constant agitation. h2o2 consumption was monitored using a previously traced peroxide calibration curve at 270 nm, and en cu nanoparticles and chitosan hydrogels in tomato 185 tab. 1: morphological variables of tomato plants treated with ncu-chitosan hydrogel. treat height nl sd nfc fws dws sc (cm) (mm) (g) (g) (mmol m-2 s-1) 0.03 mg l-1 84.7 a 15.5 a 7.85 bcd 3.75 a 274 a 31.5 c 889 a 0.015 mg l-1 78.9 b 14.7 a 9.01 a 3.60 a 275 a 38.0 a 806 b 0.006 mg l-1 85.1 a 14.2 a 8.42 ab 3.60 a 283 a 36.0 ab 877 a 0.003 mg l-1 87.1 a 15.4 a 7.30 d 3.95 a 269 a 33.9 bc 815 b 0.0015 mg l-1 87.3 a 14.5 a 7.70 cd 4.00 a 267 a 33.1 bc 731 c t0 82.8 ab 15.2 a 7.90 bc 3.45 a 262 a 32.3 c 664 d treat: treatments on base to ncu concentration. t0: absolute control without application of either chitosan hydrogel or ncu. nl: number of leaves. sd: stem diameter. nfc: number of floral clusters. fws: fresh weight of shoot. dws: dry weight of shoot. sc: stomatal conductance. data is the mean of 20 samples. different letters by column indicate significant differences among treatments, at p ≤ 0.05, according to the lsd test. zymatic activity was expressed in mm of h2o2·min-1·total proteins (mg g-1). glutathione peroxidase (eq 1.11.1.9): to measure glutathione peroxidase activity, the method modified by flohé and günzler (1984) using h2o2 as a substrate was used. for the assay, 0.4 ml of reduced glutathione (0.1 m) and 0.2 ml of na2hpo4 (0.067 m) were added to 0.2 ml of protein extract. this mixture was then pre-warmed in a water bath at 25 °c for 5 minutes, and 0.2 ml of h2o2 (1.3 mm) was subsequently added to initiate the catalytic reaction. this mixture was allowed to react for 10 min and was then stopped by the addition of 1 ml of 1 % trichloroacetic acid, followed by incubation in an ice bath for 30 min. the sample was then centrifuged at 3000 rpm for 10 min. finally, 0.48 ml of the supernatant was placed in a cell to which 2.2 ml of na2hpo4 (0.32 m) and 0.32 ml (1 mm) of the stain 5,5’ dithiobis(2-nitrobenzoic acid) (dtnb, sigma) were added. finally, the mixture was read in a uv-vis spectrophotometer at 412 nm (xue et al., 2001). glutathione: glutathione assessment was performed colorimetrically through reaction with 5,5’ dithiobis(2-nitrobenzoic acid) (dtnb, sigma). for the assay, 0.48 ml of the protein extract was added to 2.2 ml of na2hpo4 (0.32 m) and 0.32 ml of stain dtnb (1 mm) to a test tube. the reaction was mixed completely and then read using a uv-vis spectrophotometer at 412 nm (xue et al., 2001), and the absorbance obtained was interpolated using a calibration curve previously standardized with reduced glutathione. ascorbate peroxidase (eq. 1.11.1.1): quantification of the ascorbate peroxidase activity was performed according to the protocol established by nakano and asada (1987). for the assay, 0.1 ml of enzymatic extract, 0.5 ml of ascorbate (10 mg l-1) and 1 ml of h2o2 (100 mm) were added for a final volume of 2 ml and were incubated at a temperature of 22 °c. the reaction was stopped after one minute by adding 0.4 ml of 5 % h2so4. the rate of ascorbate oxidation was quantified by the decrease in absorbance at 266 nm in a uv-vis spectrophotometer. total phenols: one milliliter of a water:acetone solution (1:1) was added to 200 mg of lyophilized tissue to extract the total phenolic compounds (yu and dahlgren, 2001). the sample was mixed in a vortex for 30 seconds and later using a bransonic brand ultrasonic cleaner (model 1510r-dth) for 5 minutes, after which the sample was centrifuged at 4 °c at 12,500 rpm for 10 min, and the supernatant (phenolic extract) was collected. the quantification was performed colorimetrically using 50 μl of phenolic extract, 200 μl of the reagent folin-ciocalteu, 500 μl of na2co3 (at 20 %) and 5 ml of distilled water, which were mixed with a vortex. the mixture was then incubated at 45 °c for 30 min to allow the reaction to occur (sultana et al., 2009; nsor-atindana et al., 2012). finally, readings were taken using a uv-vis spectrophotometer at 750 nm. the results are expressed in mg of gallic acid g-1 of fresh tissue. the calibration curve was performed with gallic acid. the variables for fruit quality were determined in fully ripe tomato fruits. a maturity rating scale was used when collecting the fruits, based on us standards for fresh tomato coloration, selecting those called “red” (those with over 90 % of the surface red in color) (united states department of agriculture [usda], 1997). firmness was measured at the time of harvest in five fruits, with a second measurement taken 15 days after maintaining the fruit at room temperature (t max 25 °c and t min 18 °c), for this a fruit test penetrometer (ft20 wagner instruments) was used. the refractive index (% soluble solids) was determined with the pulp of the five fruits using a manual refractometer from 0 to 32 % (atago model atc1e), along with electrical conductivity and ph using an hi 98130 potentiometer (hanna instruments), while redox potential was determined using an hi 2211 ph/oxidation reduction potential (orp) potentiometer hi 2211 (hanna instruments). the titratable acidity (% citric acid) was calculated using 10 ml of pulp from each fruit, to which 2 drops of phenolphthalein (1 %) were added and titrated with 0.1 n naoh (aoac, 1990). finally, the fruit lycopene content was quantified using the methodology cited by fish et al. (2002) with a mixture of completely ripe fruit for the sample evaluated. statistical analysis the experimental unit was a plant in a pot. for the two stages of the study, the growth and physiological variables were measured using 20 replicates for each treatment. for the minerals, biochemical and fruit quality variables five replications per treatment were performed. a completely randomized design was used for the experimental development. a factorial analysis considering two factors was performed to evaluate minerals only in the second stage. one factor was the organ evaluated (root, leaf and fruit), and the other factor was treatments applied (chitosan hydrogel only, ncu-chitosan hydrogel, and a control). significant differences among treatments were detected using an analysis of variance (anova) and least significant difference (lsd) means separation test (p ≤ 0.05). all of the statistical analyses were performed using the statistical software sas v9.1. results and discussion stage 1. preliminary study to determine the range of ncu concentrations suitable for application to the chitosan hydrogel substrate the results of the evaluation of the agronomic variables of tomato plants are shown in tab. 1. significant differences were observed (lsd, p ≤ 0.05) in plant height, stem diameter, dry weight of the shoots and stomatal conductance; however, the variables number of 186 a. juárez-maldonado, h. ortega-ortíz, f. pérez-labrada, g. cadenas-pliego, a. benavides-mendoza tab. 2: morphological, fruit yield and physiological variables of tomato plants treated with ncu-chitosan hydrogel at a concentration of 0.006 mg ncu l-1. treat: treatments. ncu: ncu-chitosan hydrogel. chitosan: chitosan hydrogel without ncu. t0: absolute control without application of either chitosan hydrogel or ncu. nl: number of leaves. sd: stem diameter. nfc: number of floral clusters. nf: number of fruits. fwf: fresh weight of fruits. fws: fresh weight of shoots. dws: dry weight of shoot. sc: stomatal conductance. data is the mean of 20 samples. different letters by column indicate significant differences among treatments, at p ≤ 0.05, according to the lsd test. leaves, number of clusters, shoot fresh weight, fresh weight of leaves and fresh weight of stems did not differ. for stem diameter and shoot dry weight, the best treatments were ncu at 0.015 and 0.006 mg l-1, which were greater than the control by 6.5-14 % and 11-17 % for each variable, respectively. this result is consistent with adhikari et al. (2012), who report more biomass in the root and shoots of soybeans and chickpeas after seed treatment with cuo nps at 60 mg l-1 and 100 mg l-1, respectively. the best treatments for stomatal conductance were 0.03 and 0.006 mg ncu l-1, which were greater than the control by approximately 32 % (lsd, p ≤ 0.05). for plant height, the 0.015 mg ncu l-1 treatment was the lowest, with the remaining treatments with ncu being greater than or equal to that treatment. umber et al. (2015), report that the application of 100 mg kg-1 tio2 nps in soil significantly increased plant dry weight and height in lettuce. based on these results, treatment with 0.006 mg l-1 of ncu was selected for evaluation in the second stage. stage 2. study of chitosan hydrogels with ncu applied to the substrate and their inductive capacity of antioxidants the results corresponding to the growth and physiological variables of the second experimental stage are shown in tab. 2. differences were detected among treatments in the number of floral clusters, number of fruits, weight of fruits, shoot fresh weight, shoot dry weight, stomatal conductance, and spad units. there were no differences in height, number of leaves, or stem diameter (lsd, p ≤ 0.05). compared to the control, treatment of chitosan hydrogel with ncu showed the following significant increases: 11 % more clusters, 29 % more fruits, 25 % higher fruit weight, 20 % higher shoot fresh weight, 29 % higher shoot dry weight, 7 % more stomatal conductance and values of of spad units 9 % highest. applications using chitosan hydrogels without ncu were never greater than the control (tab. 2). this response is possibly due to the capacity of the ncu to induce physiological or biochemical adjustment responses due to their size, surface area and capacity to easily traverse cellular barriers to interact with intracellular structures (shobha et al., 2014). the exact mechanisms for response induction for ncu have not been understood, but the formation of reactive oxygen species (ros) and release of cu2+ are mentioned in the literature (phogat et al., 2016). it has been reported that the application of solution of 60 mg l-1 cuo nps at the seeds significantly increased the root and shoot biomasses in soybeans, while application of 100 mg l-1 in chickpeas obtained similar results (adhikari et al., 2012). the application of tio2 nps significantly increased shoot dry weight and height in lettuce; however, the authors used soil concentrations of 100 mg kg-1 (umber et al., 2015). in contrast, trujillo-reyes et al. (2014) reported that the application of ncu decreased water content, root length and dry biomass in lettuce plants grown in solution with the nps. additionally, nhan et al. (2014) reported negative effects for growth, root biomass and shoot biomass in cotton grown in solution after application of sio2 nps at concentrations of 10 to 2000 mg l-1. these findings indicate that nps have different effects depending on their concentration, the type of nps and even the vegetable species to which they are applied. in the case of spad units, a significant increase (lsd, p ≤ 0.05) of approximately four spad (9 %) units compared to the control was observed (tab. 2). however, the opposite was reported for lettuce after application of ncu at concentrations of 10 and 20 mg l-1, decreasing the quantity of spad to 17 % (trujillo-reyes et al., 2014). the results obtained in the present study can be explained by the low dose of ncu used (0.006 mg ncul-1 of substrate) compared to the dose used by trujillo-reyes et al. (2014), as it is known that cu is very toxic, even in its ionic form, and that its toxicity increases in the form of nps (shobha et al., 2014). the mineral content results in the different tomato plant organs are shown in tab. 3. significant differences between the plant organs were observed for all of the minerals evaluated (lsd, p ≤ 0.05). the cu, fe and zn contents were highest in the root compared to the leaf and fruit. the ca and mg contents were highest in the leaf, and finally, the k content was highest in the fruit. these results are within the expected range, as has been reported by different authors the concentrations of the tomato plant organs are different even among growth stages (betancourt and pierre, 2013; quesada-roldán and bertsch-hernández, 2013). regarding the effects of the different chitosan hydrogel treatments, significant differences were observed only regarding cu concentration. there were no significant differences between the three treatments evaluated for k, ca, mg, and fe (lsd, p ≤ 0.05) (tab. 3). the treat height nl sd nfc nf fwf fws dws sc spad (cm) (mm) (kg) (kg) (g) (mmol units m-2 s-1) ncu 267 a 30.4 a 11.4 a 9.75 a 25.4 a 3.56 a 1.45 a 172.8 a 490.5 a 45.0 a chitosan 276 a 29.9 a 11.2 a 9.35 ab 20.9 b 2.90 b 1.23 b 127.6 b 459.2 b 40.3 b t0 257 a 28.6 a 11.0 a 8.75 b 19.6 b 2.84 b 1.20 b 133.6 b 458.2 b 41.1 b factor treatk ca mg fe cu zn ment root 18147 b 5323 b 1922 b 303 a 6.19 a 73.23 a leave 17707 b 13381 a 2877 a 144 b 3.71 b 27.67 b fruit 24532 a 755 c 1873 b 115 b 2.47 c 19.88 b ncu 21202 a 6828 a 2324 a 171 a 3.59 b 32.95 a chitosan 19044 a 6175 a 1942 a 165 a 3.97 b 46.41 a t0 20140 a 6456 a 2407 a 226 a 4.81 a 41.43 a o rg an h yd ro ge l tab. 3: mineral concentration (μg g-1 [dry weight]) in tomato plants treated with ncu-chitosan hydrogel at a concentration of 0.006 mg ncu l-1. ncu: ncu-chitosan hydrogel at a concentration of 0.006 mg ncu l-1. chitosan: chitosan hydrogel without ncu. t0: absolute control without application of either chitosan hydrogel or ncu. data is the mean of five samples. the data of hydrogel factor are the mean of roots, leaves and fruits. different letters by column indicate significant differences among treatments, at p ≤ 0.05, according to the lsd test. cu nanoparticles and chitosan hydrogels in tomato 187 cu content was highest in the control and lowest in both treatments with chitosan hydrogels, regardless of the presence of ncu. these results suggest that chitosan could negatively influence cu absorption and translocation. however, trujillo-reyes et al. (2014) reported that the application of 20 mg l-1 of cu/cuo nps increased the quantity of cu in the roots and leaves of lettuce. this difference between the two studies may be due to the difference in ncu concentrations used and for the presence of chitosan hydrogels. as explained by trujillo-reyes et al. (2014), ncu can be absorbed by the root due to its tiny size and then later translocated to the leaves of lettuce plants, causing increases in the cu concentrations in the roots and leaves. the same authors also reported effects on the concentrations of different minerals in lettuce tissues (al, zn, mn, mg, p, s and ca); however, in the present study, differences were only observed in the cu concentration. this finding confirms the feasibility of using ncu, provided it is applied at appropriate concentrations. of note, nhan et al. (2014) reported that the application of sio2 nps had significant effects on the concentrations of na, mg and cu in the shoots of cotton plants. these results indicate that the effects on mineral concentration of the different plant tissues can vary depending on np type and concentration. the results of the content of antioxidant compounds in the leaves and fruits of tomato plants are shown in tab. 4. catalase activity measured in the leaves had significant differences among treatments (lsd, p ≤ 0.05). for this variable, the ncu-chitosan hydrogel treatment was more than five times higher than the control and more than two times higher than chitosan hydrogel without ncu. this finding agrees with that reported by trujillo-reyes et al. (2014), as they mention that when applying 10 mg l-1 of ncu, catalase activity increased but ascorbate peroxidase activity decreased in the root. this result may be due to the oxidative stress caused by the ncu, as suggested by the same authors. in terms of the variables ascorbate peroxidase, total phenols, proteins, glutathione peroxidase, glutathione and superoxide dismutase evaluated in the leaves, there were no significant differences among treatments. although trujillo-reyes et al. (2014) observed a decrease in the activity of ascorbate peroxidase in lettuce leaves after applying 20 mg l-1 of ncu and nhan et al. (2014) reported an increase in the activity of superoxide dismutase in cotton by applying 10 and 500 mg l-1 sio2nps, these differences could be due to the high concentrations of nps used in the studies mentioned compared to the present study. there were also no differences in the variables evaluated in the fruit (lsd, p ≤ 0.05) (tab. 4). the results of the evaluation of variables for tomato fruit quality are shown in tab. 5. there were differences (lsd, p ≤ 0.05) in fruit firmness, ph, titratable acidity and lycopene at harvest; however, there were no differences observed in firmness and soluble solids measured 15 days after harvest. compared to the control, there was an increase in lycopene of approximately 12 % after applying the ncu-chitosan hydrogel. however, treatment of the chitosan hydrogel without ncu had a positive effect on fruit firmness at the time of the harvest, being 16 % greater than the control. nevertheless, at 15 days after harvest, firmness was equal between the three treatments (lsd, p ≤ 0.05). the treatment of the chitosan hydrogel without ncu also increased the fruit titratable acidity, resulting in a value 25 % greater than the control. it was also observed that the chitosan hydrogel and the ncu-chitosan hydrogel treatments decreased the orp compared to the control. this result is positive because, according to benavides et al. (1999), a reduction in the potential redox values denotes a greater antioxidant potential, indicating a higher nutritive quality in fruit. conclusions application of ncu in chitosan hydrogels had positive effects on almost all of the variables related to the vigor of tomato plants, as organ treat protein (mg l-1) cea sod gpx glu apx tf ncu 316.2 a 5.06 a 66.1 a 10085 a 267.7 a 55.3 a 921 a chitosan 263.6 a 2.33 b 51.9 a 9471 a 239.7 a 56.9 a 1007 a t0 246.4 a 0.90 b 52.3 a 10224 a 204.3 a 55.4 a 869 a ncu 102.4 a 1.46 a 35.2 a 9587 a 207.0 a 53.5 a 580.4 a chitosan 79.2 a 2.23 a 37.1 a 9676 a 135.0 a 50.8 a 410.2 a t0 100.3 a 0.79 a 39.4 a 9586 a 141.7 a 50.5 a 483.8 a l ea ve s fr ui t tab. 4: antioxidant compounds in tomato leave and fruits treated with ncu-chitosan hydrogel at a concentration of 0.006 mg ncu l-1. treat: treatment. ncu: ncu-chitosan hydrogel at a concentration of 0.006 mg ncu l-1. chitosan: chitosan hydrogel without ncu. t0: absolute control without application of either chitosan hydrogel or ncu. cea: catalase enzymatic activity (mm of h2o2 min-1 total proteins [mg g-1]). sod: superoxide dismutase (inhibition rate [%] of water soluble tetrazolium salt). gpx: glutathione peroxidase (iu mg protein-1). glu: glutathione (iu mg protein-1). apx: ascorbate peroxidase (μmol oxidized ascorbate min-1 mg-1 protein-1). tf: total phenols (mg gallic ac g-1 fresh tissue). data is the mean of five samples. different letters by column indicate significant differences among treatments, at p ≤ 0.05, according to the lsd test. treatment firmness firmness 15 dah soluble solids ph orp ta lycopene (kg cm-2) (kg cm-2) (°brix) (mv) (% ca) (μg g-1) ncu 4.36 ab 3.47 a 5.11 a 4.79 a 61.7 b 0.38 b 0.0419 a chitosan 4.64 a 3.56 a 5.04 a 4.70 b 65.8 b 0.45 a 0.0370 b t0 4.00 b 3.34 a 5.03 a 4.66 b 102.1 a 0.36 b 0.0372 b tab. 5: fruit quality variables of tomato treated with ncu-chitosan hydrogel at a concentration of 0.006 mg ncu l-1. ncu: ncu-chitosan hydrogel at a concentration of 0.006 mg ncu l-1. chitosan: chitosan hydrogel without ncu. t0: absolute control without application of either chitosan hydrogel or ncu. dah: days after harvest. orp: oxidation reduction potential. ta: titratable acidity. ca: citric acid. data is the mean of five samples. different letters by column indicate significant differences among treatments, at p ≤ 0.05, according to the lsd test. 188 a. juárez-maldonado, h. ortega-ortíz, f. pérez-labrada, g. cadenas-pliego, a. benavides-mendoza the floral cluster numbers, fruit numbers, fruit weight, fresh and dry shoots, stomatal conductance and spad units all significantly increased. additionally, the application of ncu-chitosan hydrogels increased the catalase activity in the leaves and the lycopene content in the fruit, along with the ph in the pulp of the tomato fruits. treatment with chitosan increased the firmness of fruit at the time of harvest, but after 15 days, there were no differences in this variable. application of chitosan only and chitosan hydrogels with ncu decreased the orp of the fruit extract, indicating greater antioxidant capacity. with respect to titratable acidity, treatment with chitosan increased the value of this variable compared to the control; however, in contrast, ncu treatment was not different from the control. the lycopene concentration in the fruit was higher in the chitosan treatment with ncu. application of the chitosan only and ncu-chitosan hydrogels also affected the cu concentration in the tissues of the tomato plants, reducing the content of this mineral. however, there were no differences in the concentrations of other minerals. the application of cu nanoparticles in chitosan hydrogels at a concentration of 0.06 g l-1 had positive effects on tomato growth, yield and nutritional characteristics. these results indicate that application of cu nanoparticles in chitosan hydrogels can be used as a tool to increase the yield of tomato crop as well as the beneficial 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response to soil applied tio2 nanoparticles. pakistan j. agr. sci. 52, 177-182. usda, 1997: united states department of agriculture. agricultural marketing service. united states standards for grades of fresh tomatoes. retrieved from: http://www.ams.usda.gov/amsv1.0/getfile?ddocname= stelprdc5050331. wang, f., zhenqing, l., mahmood, k., kenichi, t., periannan, k., 2010: injectable, rapid gelling and highly flexible hydrogel composites as growth factor and cell carriers. acta biomater. 6, 1978-91. xue, t., hartikainen, h., piironen, v., 2001: antioxidative and growthpromoting effect of selenium on senescing lettuce. plant soil. 237, 5561. yu, z., dahlgren, r.a., 2000: evaluation of methods for measuring polyphenols in conifer foliage. j. chem. ecol. 26, 2119-2140. address of the authors: antonio juárez-maldonado, departamento de botánica, universidad autónoma agraria antonio narro, calzada antonio narro 1923, cp 25315, saltillo, coahuila, méxico e-mail: juma841025@hotmail.com hortensia ortega-ortíz and gregorio cadenas-pliego, centro de investigación en química aplicada, blvd. enrique reyna no. 140, col. san josé de los cerritos, cp 25294, saltillo, coahuila, méxico e-mail: hortensia.ortega@ciqa.edu.mx; gregorio.cadenas@ciqa.edu.mx fabián pérez-labrada and adalberto benavides-mendoza, departamento de horticultura, universidad autónoma agraria antonio narro, calzada antonio narro 1923, cp 25315, saltillo, coahuila, méxico e-mail: fabperlab@outlook.com; abenmen@gmail.com © the author(s) 2016. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 91, 296 303 (2018), doi:10.5073/jabfq.2018.091.038 1fruit research institute cacak, department for technology of fruit growing, cacak, serbia 2university of belgrade, faculty of agriculture, belgrade-zemun, serbia application of 1-methylcyclopropene in fruit of five apple cultivars grown in serbia m. milinkovic1*, b. lalevic2, v. raicević2, s.m. paunovic1 (submitted: march 15, 2018; accepted: october 4, 2018) * corresponding author summary fruits of five apple cultivars were treated using 1-methylcyclopropene (1-mcp or smartfreshtm) after cropping and were stored at normal atmosphere 2 ± 0.5 °c, 90 ± 5% relative humidity (rh) and 20.9 kpa o2 + <0.5 kpa co2. fruit firmness was assessed at three periods: 7 d after storing, 120 d after storing and 30 d after the second assessment and storing at room temperature. contents of k in all of the cultivars and in all years of study varied within the average values between 1390.5 and 2028.0 mg kg-1, while the ca content varied between 21.7 and 59.5 mg kg-1. the k:ca ratio was the lowest in cultivar ‘granny smith’ (24.0) and the highest in ‘redchief’ (99.1). application of 1-mcp made the strongest impact on fruit firmness of the cultivars ‘granny smith’ and ‘idared’ in all measuring periods. cultivars ‘redchief’, ‘čadel’ and ‘morrens ionagored’ responded well to the application of 1-mcp in the storage conditions, whereas the effect of its application influenced conservability of the fruits stored at room temperature except in fruits of the cultivar ‘morens jonagored’. application of 1-mcp made an important effect on the preservation of fruit firmness, all in accordance with the degree of ripeness of the fruits subjected to the treatment and the contents of k, ca and k:ca ratio. this study indicates that the use of 1-mcp treatment in post harvest handling of apples is promising for maintaining the freshness and quality of fruits. key words: apple, degree of ripeness, firmness of fruits, storage quality, 1-methylcyclopropene. introduction as one of the world’s most commonly cultivated crops, apple is undoubtedly a top global fruit (sheffield et al., 2016). apple is nowadays the most commonly grown and the most significant cultivar in the world’s temperate areas (zohary and hopf, 2000; cornille et al., 2012). as the most important pome fruit species, it covers the surface of 23,737 ha in serbia coming right after plum in the respective area (keserovic and magazin, 2014). different assortments and varied fruits storing conditions are an increasingly important topic for the fruit growers, especially from the aspect of preserving the quality of the fruits intended for sale in the market several months after the harvest, when they reach a higher price. for fresh fruit consumption, harvest maturity is determined differently depending on species, cultivar, storage conditions, remoteness of consumers, etc. choosing apple cultivars – with long-term preservation characteristics and resistance to transportation – is as important as any other work or process carried out in the orchard before harvesting or du ring handling and storing of fruits after harvest (molina et al., 2006; pineiro and luz, 2007). along the fruit supply chain from farm to fork, the intrinsic quality of fruits is becoming increasingly important, with external colour, fruit size and fruit firmness being the dominant factors in consumer acceptance of apple. the storability of apples depends, to a great extent, on the harvest date (szalay et al., 2013). starch hydrolysis begins at the end of the fruit development process, around 2 to 3 weeks before the start of ethylene production lau (1985). a close correlation has been found between the rate of starch degradation and the ethylene production (tomala and piestrzeniewicz, 1998). harvest date in different maturity periods is determined using iodine-starch test index (pashazadeh et al., 2017). quality of apple changes during storage and thus, substantially affects consumer acceptability (vieira et al., 2009). at first instance, it is judged by appearance comprising colour, gloss, size and secondly by texture, total soluble solids (tss) content and/or titratable acidity. dehydration reduces the weight and quality of fruits affecting both organoleptic characteristics and appearance of fruits as it comes to skin traction. the loss of fruit weight occurs as a result of respiration and combustion of organic materials such as sugars (hajnajari et al., 2010). fruits harvested later than at optimum harvesting stage contain heightened values of tss (kvikliene and valiuškaite, 2009). tss value points to the amount of converted starch into sugars, which may serve as ripe index (tromp et al., 2005). optimum nutrition of fruits provides balance in the composition of mineral substances in fruits. calcium is believed to be the most important element to determine the conservability of apple fruits as it affects the reduction of physiological disorders: fruit firmness and water content, skin traction, ethylene production and other physiological disorders (conway et al., 2002). content of calcium (ca) and its relation with potassium is also important for the conservability of apple (lanauskas and kvikliene, 2006). storage of apples is mainly carried out in the conditions of cooling and additionally under controlled atmosphere (ca) to delay ripening and provide longer shelf-life (bekele et al., 2015). such system of storing apples requires cooling in a controlled atmosphere with partial pressure and an increased concentration of carbon dioxide. application of cooling systems with controlled atmosphere (ca) and the treatment of apple fruits with 1-methylcyclopropene (1-mcp) contributes to preserving the fruit quality after harvest. preservation of fruit firmness after storage is one of the most important quality parameters. valero and serrano (2010) have stated that fruit softening is closely related to ethylene. the application of 1-mcp inhibits the maturation of fruits by blocking ethylene receptors (gwanpua et al., 2017). it has been found that the inhibition of ethy lene production, with 1-mcp causes changes in the quality para meters such as colour, strength, and weight loss (poyesh et al., 2017). incorrect storage conditions can lead to storage disorders and loss of quality, which can make entire batches unsuitable for consumption (thompson, 1998). considering that in our country the predominant method for storing apple are normal atmosphere conditions with cooling, the aim of this study is to determine the impact made by application of 1-mcp on fruit firmness from different assortment in the stated storing conditions. materials and methods plant material apple fruits were collected from the plantations established in 2007 plantations, grafted on m-9 rootstock at the experimental plantation application of 1-methylcyclopropene in fruit of five apple cultivars 297 of the fruit research institute in cacak in the village donja trepča (serbia), (n 43° 53’’63.4’’, e 20° 26’ 07.6’’, 232 m), under conditions of temperate continental climate, during 2015 and 2016. the study included the following cultivars: ‘red chief’, ‘morrens jonagored’, ‘čadel’, ‘idared’ and ‘granny smith’. the fruits of the assessed cultivars were harvested at two dates: the first was in september 16th and the second was in october 22nd, in both of the trial years. while the fruits harvested during the first date were subjected to iodine-starch test (szalay, 2013) and measurement of total soluble solids, the fruits collected in the second harvest period were used for chemical analyses, measuring of the ethylene levels, determination of fruit firmness and treatment with 1-methylcyclopropene (1-mcp). climatic factors perennial average air temperature for the period 1997-2016 in the experimental field was 12.5 °c. in the first year of study in 2015, the annual average temperature was 12.8 °c, with the average highest mean temperatures in july, 23.6 °c and 22.4 °c in august. in 2016, the average annual air temperature was 11.3 °c, with the average high temperature of air in june (20.7 °c), compared to the same month of 2015 (19.3 °c), while in other months the average temperature was lower compared to 2015. fig. 1 shows average monthly air temperatures for several years, average score and period of investigation. the average amount of rainfall (1997-2016) was 672.77 mm m-2, the highest values of precipitations in the months of june or july (76.9679.03 mm m-2). the total rainfall in 2015 amounted to 432.3 mm m-2, with the highest rainfall in june, 86.0 mm m-2. in the second year of study (2016), > 100 mm m-2, it was noted in march, may, august and october, which contributed to the amount of rainfall to be 789.6 mm m-2 (fig. 2). 1-methylcyclopropene (1-mcp) treatment apple fruits of all the examined cultivars of even shape (of 70-85 mm diameter depending on the cultivar) and with no visible mechanical damage were selected for the purposes of the experiment, to include 30 fruits in three replications for each cultivar, whereas an equal number of samples were set aside in the cold storage, to serve as the control variant. after selecting the samples for the analysis, the fruits were placed inside the cold storage with normal atmosphere (na) for 2 days, at the temperature of 10 °c. fruits selected for treatment were arranged into crates, which were then placed into plastic bags of 1 m3 volume. treating the fruits with the agent acting as an ethylene blocker was performed by dissolving three tablets of 1-mcp (pink smartfreshtm research tablets) with a single activator tablet (blue activator tablets) in 20±0.5 ml of citric acid (20 °c). after diluting 1-mcp (smartfreshtm protab), the plastic bag was sealed airtight for the next 24 hours, until the gas concentration of 937 ppb was achieved. a small battery-drive air fan was attached to the jar, for securing a better distribution of 1-мcp gas inside the plastic bag. after this, the bag was removed and the fruits were kept in a cold storage for the next 120 days. fruit storage and sampling all of the replications of the fruits selected for treatment, as well as the ones that were not subjected to the 1-mcp treatment, were placed in wooden crates and kept in vertical tiers. the fruits were kept in cold atmosphere: (2 ± 0.5) °c, (90 ± 5) % relative humidity (20.9kpa 02 + <0.5kpa co2), over a period of 120 days. in order to determine fruit firmness, the sampling of both treated and nontreated apple fruits was conducted at three periods: 7 days and 120 days following the application of 1-mcp on fruits kept in the cold storage and 30 days following the 2nd measurement. after taking the samples from the cold storage, the fruits were left in conditions of normal atmosphere: 21 ± 1 °c and 60 ± 5% rh, for 2 hours. fruits for the 3rd measurement were kept in the same conditions of at room temperature, over a period of 30 days. the fruit measuring was performed using the penetro-meter fruit pressure tester ft 327 (winopal forschungsbedarf gmbh, germany) with a 11-mm probe. two measurements were made fig. 2: average rainfall for the period 1997-2016, 2015 and in 2016 (mm m-2) fig. 1: average monthly air temperature for the period 1997-2016, 2015 and 2016 (°c) 298 m. milinkovic, b. lalevic, v. raicević, s.m. paunovic in six fruits per each replication on both sides expressed in newton (n/cm2) = fruit firmness value (kg/cm2) × 9.81. assessments the starch index (si) was evaluated using the starch iodine test using the solution of 1% iodine flakes and 4% potassium iodide. five fruits each were picked from five trees of each cultivar (25 fruits/ per cultivar). the fruits were cut into equatorial halves and left for 10 seconds to soak in the solution after which dried for at least 5-10 min with surface up in the air or face down. determining, reading the starch-iodine test and calculating the index was performed according to the (code amidon, ctifl, 2002). total soluble solids (tss) were determined using a hand refractometer (0 to 32%), carl zeiss 3828, germany. chemical analysis of the fruits included: the analysis of ethylene content μl l-1 and determination of calcium (ca), potassium (k) content and the k:ca ratio. sampling of fruits for the ethylene content analysis was performed before the smartfreshtm treatment and 24 h after treating the fruits, which included six apple fruits per cultivar. both treated and non-treated (control) fruits were kept for 7 days at room temperature before individual samples were laid for 6 hours in the airtight vessel of 3.7 l volume, in order to measure the ethylene production using the ica 56 device. the content of macro-elements was determined before the experiment setup, by sampling 2 kg of average samples in three replications, for each cultivar. the macro-elements content and the k:ca ratio were determined using the following methods: k (by atomic absorption spectrophotometry-emission) and ca (by atomic absorption spectrophotometry-absorbance), whereas k:ca ratio was determined using calculation. in addition to measuring apple fruit firmness, the occurrence of scald and fruit rot was also monitored, after each sampling for fruit firmness and storing at room temperature. statistical analysis the data was subjected to analysis of variance (anova) using statistical package mstat-c (michigan state university, usa). the least significance difference (lsd) was used to compare treatment means and treatments declared different at p = 0.05 level of significance. results and discussion fruit ripeness degree determination of the ripeness degree in apple fruits using the iodine starch method is based on the hydrolysis of starch into sugar, which takes place with the gradual ripening of the fruits. the ripening process starts from the core (inner parts) of the fruits and progresses towards the perimeter. the process may vary in length, depending on the cultivar-specific characteristics. there are different optimum maturity levels for the same cultivars, depending on the intended use and storage life desired. the maturity level of fruits in the first and second measurement time period was substantially greater in 2016 than in 2015, which was determined by the comparative analysis (axb) of measurement date and the study year on five tested apple cultivars. ripeness degree was a major factor in the impact of 1-mcp application, as a cultivar-specific characteristic in combination with climatic conditions (tab. 1). if the fruits were harvested when overmature, problems with fruit drop would appear and apples could no longer be used for long-term storage as this could lead to a soft mealy texture, off flavours and greasy skin (little and holmes, 2000). although on-tree softening was slow, different harvest dates resulted in different initial firmness values affecting fruit quality throughout the whole postharvest life. the contents of total soluble solids tab. 2 showed significant differences between two different measurements in all of the tested cultivars, whereas the impact of the year of trial was of a statistical importance only in the cultivar ‘idared’. the established tss values were slightly higher, compared to the research (jha et al., 2012). according to the results shown in soska and tomala (2006), in addition to the impact made by the cultivar-specific characteristics, the tss variations were also under the influence of the climate, which was established by our research in the cultivar ‘idared’ (fig. 1, 2). in their investigations, skic et al. (2016) have indicated that the choice of optimum fruit maturity is determined by several parameters: total soluble solid content (tssc), starch index (si) firmness (f) and others. this methodology has an indestructible character in comparison with chemical analysis and allows farmers to determine their harvest time and storage using alternative methods. it has been found that there is a correlation between the examined parameters of fruit maturity. juhņeviča-radenkova and radenkovs (2016) have found that harvest time impacts physical and chemical properties of apples stored after harvest. furthermore, it has been found that after 6 months of storage, apples are sweeter and more aromatic preserving the juiciness and acidity. the plant hormone, ethylene, produced during metabolism of fruits is mainly responsible for intensifying ripening and consequently, reducing the shelf life of fruits (watkins, 2006). based on the results shown in tab. 3, there is a statistically significant difference in the ethylene content between the apple fruits treated with 1-mcp and those not treated, in all of the examined cultivars. statistically, differences in the ethylene content in different years of trial were tab. 1: values the starch index (si) on a different assortment of apples in periods. factor measurements ‘red chief’ ‘morrens jonagored’ ‘čadel’ ‘idared’ ‘granny smith’ a 1st measurement 2.10±0.19b 4.95±0.49b 5.55±0.37b 2.48±0.26b 2.48±0.22b 2nd measurement 6.55±0.35a 6.75±0.32a 7.05±0.25a 5.00±0.19a 5.15±0.43a b 2015 3.30±0.44b 4.20±0.35b 5.05±0.29b 3.10±0.35b 2.50±0.22b 2016 5.35±0.61a 7.50±0.14a 7.56±0.11a 4.38±0.32a 5.13±0.44a a × b 1st measurement 2015 1.50±0.22d 2.90±0.23d 4.00±0.21d 1.70±0.21d 1.70±0.21c 1st measurement 2016 2.70±0.15c 7.00±0.15b 7.10±0.10b 3.25±0.31c 3.25±0.13b 2nd measurement 2015 5.10±0.23b 5.50±0.31c 6.10±0.23c 4.50±0.22b 3.30±0.15b 2nd measurement 2016 8.00±0.00a 8.00±0.00a 8.00±0.00a 5.50±0.22a 7.00±0.00a anova a * * * * * b * * * * * a × b * * * * * note: means with different letters are significantly different (fisher’s lsd, p = 0.05); * – statistically significant. application of 1-methylcyclopropene in fruit of five apple cultivars 299 significantly different in cultivars ‘red chief’, ‘morrens jonagored’ and ‘granny smith’. different correlations between the optimal time of harvest and the ethylene production rate can be found depending on the cultivar (watkins, 1989). a lot of effort has been invested in profiling the changes in ethylene biosynthesis and quality in apple dependent on: harvesting (lin and walsh, 2008), harvest maturity (johnston, 2002), application of chilling conditions and re-warming (lara and vendrell, 2003; vilaplana et al., 2007), or shelf-life conditions (xuan and streif, 2005; dal, 2007). by treating apple fruits with 1-mcp, ethylene biosynthesis is almost completely suppressed during the storage period, except when fruits are harvested beyond optimal deadline, i.e. at late harvest, because such fruits get the ability to form ethylene in two weeks (bulens at al., 2012). in their study, valero and serrano (2010) found a decrease in the production of ethylene in a large number of cultivars. the results obtained correspond to our research results. content of k, ca and k:ca ratio chemical composition of fruits is determined by a number of both ecological and genetic factors. fruits with low ca and high k content, and consequently, a high k:ca ratio, are susceptible to bitter pit occurrence. moreover, development of this disorder is also influenced by storage conditions and the activity of the enzymes involved in fruit respiration (wińska-krysiak and łata, 2010). potassium (k) is often considered to be detrimental to fruit quality through interference with the uptake and utilization of ca. however, over-emphasis on this issue can sometimes lead to k deficiency, particularly in high-density production systems on coarse-textured soils, or wherever soils are naturally low in k, due to the parent material or presence of k-fixing clay minerals (neilsen and neilsen, 2009). k content was manifested depending on the cultivar, whereas the differences in the content relative to the year of study were significant in all of the tested cultivars except cultivar ‘granny smith’. on average, the lowest k content (1390.5 mg kg-1) was observed in the ‘granny smith’, whereas the highest k content (2028.0 mg kg-1) was observed in ‘morrens jonagored’ (tab. 4). calcium (ca) is a mineral nutrient, which has been most highly implicated in the quality of fruits, particularly with respect to disorders, which affect storage. unlike the content of k, presence of ca in the trial years revealed differences in cultivars ‘red chief’, ‘čadel’ and ‘idared’. the highest average value of ca was recorded in the cultivar ‘granny smith’, whereas the lowest average ca value was observed in ‘red chief’, with all the other cultivars recording similar values of this parameter, in the range 39.4 to 43.4 mg kg-1. in their manuscript, lu et al. (2015) have found that application of larger quantities of k after flowering stage leads to decreased quantities of ca in fruits with negative influence on the fruit quality. tab. 2: content of total soluble solids (tss %) at different measuring periods on a different assortment of apples in two measurement periods. factor measurements ‘red chief’ ‘morrens jonagored’ ‘čadel’ ‘idared’ ‘granny smith’ a 1st measurement 12.59±0.17b 12.81±0.26b 12.86±0.14b 10.99±0.12b 12.18±0.36b 2nd measurement 15.21±0.15a 13.96±0.20a 14.10±0.21a 12.94±0.26a 14.53±0.12a b 2015 14.10±0.29a 13.18±0.34a 13.66±0.27a 12.30±0.37a 13.30±0.35a 2016 13.70±0.38a 13.59±0.15a 13.30±0.16a 11.63±0.19b 13.41±0.41a a × b 1st measurement 2015 12.99±0.21b 11.86±0.21c 12.68±0.20c 10.94±0.19c 11.97±0.28b 1st measurement 2016 12.18±0.22c 13.76±0.23b 13.04±0.18bc 11.03±0.15c 12.38±0.68b 2nd measurement 2015 15.21±0.21a 14.50±0.25a 14.63±0.22a 13.66±0.36a 14.62±0.19a 2nd measurement 2016 15.21±0.21a 13.42±0.19b 13.56±0.26b 12.22±0.21b 14.43±0.16a anova a * * * * * b ns ns ns * ns a × b * * * * ns note: means with different letters are significantly different (fisher’s lsd, p = 0.05); ns – not significant, * – statistically significant. tab. 3: content (μl l-1) of ethylene in apple fruits prior to and following 1-methylcyclopropene (1-mcp) application. factor treatments ‘red chief’ ‘morrens jonagored’ ‘čadel’ ‘idared’ ‘granny smith’ a control 46.45±0.10a 58.95±2.77a 14.20±0.98a 43.00±3.31a 33.60±3.41a 1-mcp 12.15±4.58b 12.25±4.89b 2.30±0.38b 16.95±2.44b 8.90±3.66b b 2015 24.80±10.08b 30.80±13.35b 8.40±2.95a 31.20±8.15a 13.85±5.84b 2016 33.80±5.47a 40.40±7.89a 8.10±2.56a 28.75±4.22a 28.65±5.48a a × b control 2015 47.30±1.45a 60.20±5.16a 14.80±1.48a 49.20±2.57a 26.80±1.66b control 2016 45.60±1.46a 57.70±3.20a 13.60±1.51a 36.80±3.12b 40.40±3.02a 1-mcp 2015 2.30±0.21c 1.40±0.26c 2.00±0.67b 13.20±1.14c 0.90±0.15d 1-mcp 2016 22.00±2.83b 23.10±1.27b 2.60±0.44b 20.70±3.79c 16.90±1.76c anova a * * * * * b * * ns ns * a × b * * ns * * note: means with different letters are significantly different (fisher’s lsd, p = 0.05); ns – not significant, * – statistically significant. 300 m. milinkovic, b. lalevic, v. raicević, s.m. paunovic the k:ca ratio did not reveal significant differences in the tested cultivars in different years of the study. the average values of the k:ca ratio were in the range from 24.0 in ‘granny smith’ to 99.1 in ‘red chief’(tab. 6). wińska-krysiak and łata (2010) found that the content of ca in fruits differed depending on the cultivar, whereas the content of k was identical in both sampling periods. the same authors established that the mean of the k:ca ratio was 33.2, which was a lower value than the one observed in our study (tab. 6). comparing the research results with other authors, in their work, demuth and sundrud (2012) have reported that if the calcium content in apple is greater than 30 mg kg-1 then it is unlikely for physiological disorders to be developed, and if the content is lower than 19 mg kg-1, the greater the chance of the incidence of bitter pit and other physiological disorders. for values between 19 to 30 index mg kg-1, the content of mg and k is important as well as their relationship with ca. the aforementioned authors have also found the most common concentrations of cations in the fruits of apple, for calcium 10 to 50 mg kg-1 and potassium 500 to 1500 mg kg-1. in their study, they have found that in different apple cultivars the content of calcium ranges 28-110 mg kg-1, whereas potassium content is 690 to 1200 mg kg-1. the most common values of ca content are 10-50 mg kg-1. in our studies, the average content of k is 1390.5 to 2028.0 mg kg-1, ca 21.7 to 59.5 mg kg-1, and k: ca 24.0 to 99.1. most of the tested cultivars have increased values of k and average values of ca in accordance with aforementioned authors. the difference exists only in the cultivar ‘granny smith’. in the investigations by piestrzenievicz and tomala (2001), it has been confirmed that when ratio k:ca in ripe ‘jonagold’ fruits does not exceed 28. values of the content of certain ions in apple fruits depend on the cultivar and genetic resources. firmness of apple fruits firmness of fruits of different apple cultivars was significantly different at all different measurement periods (tab. 7). the highest value of the initial fruit firmness was recorded in ‘granny smith’, whereas the cultivars ‘red chief’, ‘čadel’ and ‘idared’showed no statistically significant difference of this parameter. during the different measuring periods, including the final measurement, the largest firmness of fruits was established in the cultivar ‘granny smith’, compared to the initial measurement. in south tyrol (italy), a region that grows approximately 1/10 of european apples, these methods are therefore more commonly used on cultivars susceptible to superficial scald approx. 30% of the whole production – respectively distributed among the cultivars ‘red delicious’, ‘granny smith’, ‘rome beauty’, ‘fuji’, ‘winesape’ and ‘cripps pink’, than on those resistant to this post-harvest disorder (zanella and stürz, 2013), where the advantage could be the improved firmness. while ripening on-tree, ‘jonagold’ apples softened slowly (0.5 n/day), but during shelf-life after extended storage the rate of firmness loss was much higher (1.07 n/day). application of 1-mcp resulted in increased fruit firmness, compared to non-treated fruits. the fruit firmness showed significant differences in the different years of the trial, with the exception of the non-treated fruits from the 1st measurement period, and the 3rd measurement period of all the examined cultivars. applying the 1-mcp treatment, fruit firmness losses during storage can be reduced (deell et al., 2007; hoehn et al., 2008). the extent to which these losses may be reduced varies in relation to several factors, such as cultivar, ripening stage, storage conditions, temperatures at application, timing of application, and duration of application (deell et al., 2002; blankenship and dole, 2003). different harvest dates resulted in tab. 4: content (mg kg-1) of potassium (k) in fruits of different apple cultivars. factor measurements ‘red chief’ ‘morrens jonagored’ ‘čadel’ ‘idared’ ‘granny smith’ a 2015 1783.0±19.08a 2082.0±7.55a 1860.0±14.29a 1659.0±18.88a 1450.0±52.54a 2016 1442.3±109.44b 1974.0±25.38b 1741.7±33.46b 1523.3±20.28b 1331.0±49.34a average 1612.5 2028.0 1800.9 1591.2 1390.5 anova * * * * ns note: means with different letters are significantly different (fisher’s lsd, p = 0.05); ns – not significant, * – statistically significant. tab. 5: content (mg kg-1) of calcium (ca) in apple fruits. factor measurements ‘red chief’ ‘morrens jonagored’ ‘čadel’ ‘idared’ ‘granny smith’ a 2015 28.0±8.74a 49.0±4.36a 48.0±3.46a 47.0±2.31a 57.0±6.24a 2016 15.3±5.04b 37.7±2.40a 30.7±2.96b 34.3±2.96b 62.0±5.51a average 21.7 43.4 39.4 40.7 59.5 anova * ns * * ns note: means with different letters are significantly different (fisher’s lsd, p = 0.05); ns – not significant, * – statistically significant. tab. 6: potassium and calcium (k:ca) ratio in apple fruits. factor measurements ‘red chief’ ‘morrens jonagored’ ‘čadel’ ‘idared’ ‘granny smith’ a 2015 87.3±38.47a 43.2±3.76a 39.2±2.96a 35.5±2.06a 26.0±2.85a 2016 110.9±26.19a 52.9±3.67a 58.2±7.09a 45.1±4.04a 22.0±2.86a average 99.1 48.1 48.7 40.3 24.0 anova ns ns ns ns ns note: means with different letters are significantly different (fisher’s lsd, p = 0.05); ns – not significant, * – statistically significant. application of 1-methylcyclopropene in fruit of five apple cultivars 301 different initial firmness values affecting fruit quality throughout the whole postharvest life (bulens et al., 2012). monitoring the impact, made by 1-mcp on fruit firmness in the 2nd measurement (120 days), it is possible to observe a significant difference between the treated and non-treated fruits. in cultivars ‘red chief’ and ‘morrens jonagored’, the treated fruits had considerably higher firmness compared to non-treated fruits, in a situation when the initial firmness was higher, and vice versa. in both the cultivars ‘čadel’ and ‘idared’, it is possible to observe the effect made by the ethylene blockers in both trial years, whereas this effect was the most prominent in the cultivar ‘red chief’, and, in ‘granny smith’, a substantial fruit firmness in all measuring periods has been preserved. magazin et al., (2010) have found a positive effect of 1-mcp on the quality of cultivar ‘granny smith’ fruits was established. in the cultivars ‘morrens jonagored’ and ‘čadel’, no significant difference was established in the 3rd measurement regarding the fruit firmness in relation to the year of study, despite a significant difference in the initial firmness of fruits. this finding combined with the difference between treated and non-treated fruits confirmed that the application of 1-mcp had no impact on preservation of fruit firmness in fruits of this cultivar kept at room temperature for a prolonged period of time (tab. 7). the effect of treatment with 1-mcp on the ethylene production rate was much more profound than the effect of the different harvest dates. in 1-mcp treated apples respiration also increased but to a lower level compared to the untreated apples (bulens et al., 2012). pashazadeh et al., (2017) have found that by application of 1-mcp the loss of weight and firmness of fruit flesh is reduced compared to control. it is notable also that the survey results differ between the cultivars studied. according to the available results (jeziorek et al., 2010; sardabi et al., 2014), mcp-1 treatment promotes the preservation of fruit firmness of the cultivar ‘golden delicious’ whereas the same effect is noticed in other treated apple cultivars. similar results have been found by zanella and rossi (2015) in the cultivar ‘red delicious’ by storing fruits in different storing conditions compared to normal cold storage, where application of 1-mpc contributes to preservation of fruit firmness. positive effects of the 1-mcp application are shown in studies (akbudak et al., 2009; poyesh et al., 2017). the act of harvesting did not affect fruit softening on the short term except in the case of late harvested apples, which showed an increased softening during the shelf life directly after harvest. these results are in accordance with studies described in lin and walsh (2008). in accordance with our research results, other authors have found similar results indicating that fruit firmness after storage and application of 1-mcp depends on the cultivar. conclusions the impact made by application of 1-mcp on the quality and firmness of fruits depends on the degree of fruit ripeness and cultivar-specific characteristics, which may be under the influence of agro-ecological conditions in the year of the trial. each of the examined cultivars has its own optimum harvesting period, enabling optimum storage and application of ethylene blocker, and these periods tend not to coincide in different years of study, which was additionally confirmed by this experiment in red chief, morrens jonagored and čadel cultivars. for this reason, it is necessary to examine maturity in several periods to select the appropriate one for storing with cultivars of the same ripening period. in cultivars, tab. 7: firmness (n/cm-2) of apple fruits at different measurement periods: 7 and 120 days following the application of 1-mcp on fruits kept in the cold storage and 30 days following the 2nd measurement in terms of room temperature. firmness factor parameter control ø 1st measurement 2nd measurement 3rd measurement – 7 days – 120 days – 120 + 30 days control 1-mcp control 1-mcp control 1-mcp a ‘red chief’ 71.12±0.47b 53.37±0.16d 71.52±0.35b 45.22±0.22d 59.74±0.38c 35.02±0.16d 51.80±0.35cd ‘morrens jonagored’ 64.35±0.23c 51.99±0.17d 61.80±0.18c 45.71±0.08d 56.11±0.19c 47.09±0.16c 47.58±0.09d ‘čadel’ 70.14±0.39b 58.57±0.14c 62.69±0.23c 51.50±0.14c 56.21±0.11c 49.83±0.12c 52.39±0.11c ‘idared’ 67.10±0.15bc 64.45±0.09b 72.89±0.14b 56.60±0.16b 65.83±0.17b 55.03±0.28b 65.33±0.27b ‘granny smith’ 92.41±0.25a 92.02±0.17a 93.59±0.23a 84.37±0.27a 92.12±0.21a 76.22±0.26a 88.39±0.22a b 2015 82.70±0.23a 64.55±0.26a 77.98±0.23a 52.19±0.19b 71.71±0.23a 46.50±0.18b 62.20±0.25a 2016 63.37±0.18b 63.57±0.20a 67.00±0.19b 60.72±0.27a 60.33±0.23b 58.76±0.25a 59.94±0.27a a × b ‘red chief’ 2015 88.58±0.42b 57.09±0.26e 82.60±0.43b 36.69±0.15h 74.75±0.23c 30.71±0.19g 63.08±0.38d ‘red chief’ 2016 60.53±0.23d 53.66±0.11d 53.76±0.26e 49.74±0.13fd 44.73±0.22g 39.34±0.18d 40.61±0.27g ‘morrens jonagored’ 2015 69.36±0.38c 45.81±0.11f 62.20±0.34d 45.03±0.10fg 61.90±0.25e 41.79±0.18d 46.21±0.11fg ‘morrens jonagored’ 2016 59.25±0.15de 58.27±0.15e 59.06±0.13d 52.48±0.10d 50.33±0.13f 44.54±0.12d 49.05±0.12ef ‘čadel’ 2015 85.45±0.22b 60.23±0.25de 72.10±0.14c 54.35±0.20d 59.45±0.14e 50.13±0.19c 55.13±0.16e ‘čadel’ 2016 56.99±0.14e 54.94±0.21e 53.37±0.10e 48.66±0.14ef 53.07±0.07f 49.54±0.17c 49.74±0.11ef ‘idared’ 2015 72.40±0.09c 63.37±0.09cd 68.47±0.28c 50.62±0.12de 62.88±0.15e 43.46±0.09d 55.13±0.19e ‘idared’ 2016 73.48±0.28c 65.53±0.14c 65.83±0.13cd 66.61±0.10c 68.87±0.27d 62.49±0.12b 75.54±0.17c ‘granny smith’ 2015 101.93±0.18a 96.53±0.20a 100.94±0.28a 74.36±0.18b 99.47±0.22a 66.32±0.14b 91.63±0.27a ‘granny smith’ 2016 94.47±0.19a 87.60±0.18b 86.33±0.15b 86.23±0.021a 84.76±0.12b 82.99±0.15a 85.05±0.32b anova a * * * * * * * b * ns * * * * ns a × b * * * * * * * note: means with different letters are significantly different (fisher’s lsd, p = 0.05); ns – not significant, * – statistically significant. 302 m. milinkovic, b. lalevic, v. raicević, s.m. paunovic characterized by later technological maturity, there was a smaller difference in the initial hardness, but the impact made by 1-mcp produced different effects in different periods when the hardness of fruits was measured, occurring as a result of the interaction of other parameters of ripeness and contents of macro-elements, as shown in the results of our research. the strongest impact of 1-mcp application was observed in the granny smith cultivar where the fruits retained firmness comparable to the one existing during the harvest, after storing the fruits for 30 days at room temperature. a strong effect of the 1-mcp application was observed in the idared cultivar and ‘red chief’, whereas the fruit firmness of other cultivars (‘morrens jonagored’ i ‘čadel’) preserved in the cold storage cannot be sustained at room temperature over a prolonged period. acknowledgments this research was partly supported by the ministry of education, science and technological development of the republic of serbia (grant number tr 31080) and partly by pro fruit in serbia, whom we thank for their collaboration. references akbudak, b., ozer, m.h., erturk, u., cavusoglu, s., 2009: response of 1-methylcyclopropene treated ‘granny smith’ apple fruit to air and controlled atmosphere storage conditions. j. food qual. 32,18-33. doi: 10.1111/j.1745-4557.2008.00233.x bekele, e.a., beshir, w.f., hertog maarten, l.a.t.m., nicolai, b.m., geeraerd, a.h., 2015: metabolic profiling reveals ethylene mediated metabolic changes and a coordinated adaptive mechanism of ‘jonagold’ apple to low oxygen stress. physiol. plant. 155, 232-47. doi: 10.1111/ppl.12351 blankenship, s.m., dole, j.m., 2003: 1-methylcyclopropene: a review. postharv. biol. technol. 28, 1-25. doi: 10.1016/s0925-5214(02)00246-6 bulens, i., van de poela, b., hertoga, m.l.a.t.m., de proftb, m.p., geeraerda, a.h., nicolai, b.m., 2012: influence of harvest time and 1-mcp application on postharvest ripening and ethylene biosynthesis of ‘jonagold’ apple. postharv. biol. technol. 72, 11-19. doi: 10.1021/jf0525075 conway, w.s., sams, c.e., hickey, k.d., 2002: preand postharvest calcium treatment of apple fruit and its effect on quality. in: tagliavini, m. et al. 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(formerly called pennisetum pedicellatum trin., “kyasuwa”), belongs to the poaceae family and is a c4 grass (schmelzer, 1996). it is native to west africa and was introduced to india from where it has since spread to south east asia and northern australia (schmelzer, 1996) where it is invasive and regarded as an environmental weed (quddus et al., 2014). kyasuwa is tolerant of disturbance with broad climatic amplitude, produces large quantities of seeds with an efficient dispersal mechanism (schmelzer, 1996). it occurs along road edges, disturbed and abandoned lands and thrives on soils of a wide ph scale in rainfall regimes ranging between 500-1500 mm with severe drought lasting 4-6 months (fao, 2010; schmelzer, 1996). kyasuwa provides succulent, palatable and nutritious forage over a long growing season including the dry periods of october-november and contributes to meeting cattle fodder requirements in rural areas (schmelzer, 1996; fao 2010). it is also used as a soil stabilizer, mulch and in soil erosion control (schmelzer, 1996; fao, 2010). climate change is one of the most severe challenges of our time, with predicted increases in global mean temperature, length and severity of drought events and atmospheric co2 concentration, due to human activities (ipcc, 2014). many plants respond to elevated atmospheric carbon dioxide (eco2) concentrations by increased growth, biomass and productivity, with c3 plants generally benefitting more than c4 plants (santos et al., 2014; ainsworth and rogers, 2007). moreover, there are also reports of significant changes in the chemical composition of plants under eco2 (myers et al. 2014). nonetheless, plant responses to eco2 are not easily predictable because they depend on multiple environmental factors which are not necessarily additive (ackerly et al., 1992). water and nitrogen (n) availability are two of the most limiting plant resources and have been repor ted to interact with eco2 (erbs et al., 2015). several studies have shown that the photosynthetic capacity of plants grown at eco2 will be acclimated and even down-regulated due to feedback repression of accumulated carbohydrates (paul and driscoll, 1997). this has especially been observed when the plant’s c/n status is high due to deficient n supply, leading to a decrease in rubisco activity and thus, lower photosynthetic rates (leakey et al., 2012). biomass enhancement of eco2 may therefore be dependent on sufficient n supply in some species (dong et al., 2016). in contrast, some species may show enhanced c gain per leaf n due to a suppression of photorespiration under eco2 (leakey et al., 2012). there is a general lack of data on responses of tropical and sub tropical plants to future climate changes and more research on plant responses of c3 than c4 species (leakey, 2009). the purpose of this work was to determine the effects of future elevated atmospheric co2 concentration on biomass development which aids invasive potential and nutritive value of kyasuwa grass. the objectives of the study were, (1) to assess the effects of eco2 on kyasuwa growth, biomass allocation and nutritive value, (2) to evaluate how different water and fertilization regimes affect kyasuwa growth, biomass allocation and nutritive value and, (3) to estimate interactive effects of the three resource factors on these traits of kyasuwa. we hypothesize future increases in atmospheric co2 will increase kyasuwa growth and biomass thereby enhancing competitiveness. we also hypothe size that nutritive value of kyasuwa will increase with eco2 especially at high fertilization regimes. materials and methods plant preparation and growth conditions seeds for the study were harvested in january 2016 from 150 plants located in tolon in the guinea savanna zone of ghana (09° 25’n, 00° 58’w). seeds were transported within 3 weeks of picking to the greenhouse of universität hamburg, germany (53° 30’n, 10° 12’e). germination and pre-treatment growth were carried out as follows in the greenhouse. after a germination period of 3-6 days in germination trays, two plants were transplanted into a 3 l plastic pots of 15 cm height, filled with a mixture of 4:1:0.5 (v/v) sand (0.130.36 mm), clay and standard organic substrate (tks1, floragard vertriebs-gmbh, oldenburg). the bottom of the pots was secured with a weed mat to prevent loss of substrate. after pre-treatment growth for 20 days, the pots were randomly assigned to two growth chambers (each 28.5 m2) of different co2 concentrations in a green kyasuwa biomass and nutritive value under elevated co2 89 house with controlled growth conditions (day/night air temperature 25 °c/22 °c; 70%-80% relative air humidity; length of photo period same as hamburg area). we stimulated the atmospheric co2 concentration of 950 ppm based on the representative concentration pathway (rcp) 8.5 scenario by 2100 (ipcc, 2014). in the first growth chamber, the co2 concentration was the same as in ambient atmospheric air (aco2; 400 ppm) while in the other chamber, the co2 was elevated to 950 ppm (eco2). three fertilization treatments were applied using commercial li quid fertilizer with npk values of 8-8-6 (wuxal super, aglukon spezialdünger gmbh, düsseldorf, germany). the treatments corresponded to the equivalent of 75 kg n ha-1 (n-1), 100 kg n ha-1 (n-2) and 125 kg n ha-1 (n-3) and were applied at planting, 20 days after planting and 40 d after planting. the wuxal super fertilizer has the full complement of macro and micro nutrients. during the ex perimental period of 68 days two watering regimes of continuous watering (wet), and a stimulated drought period of no watering (dry) for 2 weeks were implemented. the complete design of the study comprised of two levels of co2, two watering regimes, three fertili zation regimes and six replicates, resulting in a total of 72 pots for the entire experiment. the pots were equally distributed among the two growth chambers which resulted in 36 pots in each chamber. to avoid edge and chamber effects, the positions of individual potted plants were rotated on the tables on a weekly basis but, it was practically impossible to switch co2 concentrations between the two chambers however, the growth conditions were similar. during the experiments, the co2 level, temperature and air humidity were monitored by the computer climate model cc 600 (ram co. measurement and control, germany) every 12 minutes. the light conditions in the two chambers were measured for a month with quantum sensors (li190r, li-cor, lincoln, ne, usa) connected to data loggers (cr 1000, campbell scientific, logan, ut, usa). the grasses were exposed to the experimental treatments for a total of 68 days during the growth period (may-july 2016). plant growth and physiological measurements to assess the effects of eco2, watering and fertilization regimes, the growth characteristics were recorded before harvesting the grass 80-90 days after sowing. shoot height was measured from ground level to the base of the top-most, fully developed leaf or to the base of the panicle depending upon the stage of the particular plant. the stomatal conductance was measured on two leaves for three replicates within all treatments using a leaf porometer (sc-1, decagon devices, pullman, wa, usa) on young but fully developed leaves. at harvest, the area of the individual grass leaves was determined using an area meter (li 3100, li-cor, lincoln, ne, usa) while the fresh weight of leaves and stems were measured separately and the leaf to stem ratio (l:s) estimated from these measurements. roots were thoroughly washed from the soil over a sieve with 1 mm mesh size. the biomass fractions (leaves, stems and roots) were oven dried for 48 hours at 65°c. roots were denominated below-ground biomass (bgb). above-ground biomass (agb) was estimated by adding dry weights of leaves and stems, while total biomass (tb) was the sum of agb and bgb. the root to shoot ratio (r:s) was calculated by dividing bgb by agb. forage nutrient analysis to evaluate the effects of eco2, watering and fertilization regimes on nutritive value of kyasuwa, oven-dried leaves and stems were ground separately with a micro hammer mill (culatti ag, zürich, switzerland) fitted with a 1 mm sieve. neutral detergent fibre (ndf) and acid detergent fibre (adf) were measured sequentially with the ankom filter bag method according to the manufacturer, using a fibre analyzer (ankom-200 fiber analyzer, ankom technology, macedon, ny, usa). ground leaf and stem material (500 mg) was placed in ankom f57 filter bags and sealed with heat. all 72 samples were first extracted with neutral detergent, and the residue was weighed to determine percentage of ndf. the ndf residue was then extracted with acid detergent solution, followed by extraction with 72% h2so4 and ashing to determine the percentage of acid deter gent lignin (adl) (van soest, 1994; ryan et al., 1990). the following nutrient parameters were estimated: % ndf, % adf, and % adl. total nitrogen and carbon concentration of leaves was measured in aliquots of oven dried samples by an elemental analyzer following pyrolysis (euro-ea 3000, euro vector, italy). mass calibration was conducted by the use of the certified standard 2,5-bis (5-tertbutyl-2-benzoxazol-2-yl) thiophene (6.51% n; 72.52% c; hekatech, germany). the percentage of proteins in leaves was measured according to bradford (1976): dried leaves of all treatments were finely ground using a retsch mixer mill (mm-400, fischer scientific, suwanee, usa) and thereafter digested in 0.1 m naoh for 30 minutes (jones et al., 1989). a volume of 100 μl aliquots of centrifuged supernatant were assayed with 50 μl bio-rad bradford dye (coomasie brilliant blue). absorbance was measured at 595 nm after 15 minutes using a multi-mode microplate reader (synergy ht, biotek instruments, winooski, usa). data analysis all statistical analyses were carried out using the software package statistica 13 (stat-soft inc., tulsa, ok, usa). growth, biomass and nutritive value parameters were analyzed by three-way analysis of variance (anova) to test for significant main effects of the factors co2 concentration, fertilization levels and watering regime as well as all factor interactions, followed by the tukey hsd post hoc test of significant differences. for analysis of nutritive value, data of stems and leaves were averaged after chemical assays. prior to the 3-way anova, all data were analyzed for homoscedasticity using levene’s test, and data were transformed appropriately where necessary. in addition, residual plots and normal probability plots were inspected to ensure that the assumptions of anova were met. results environmental conditions during the study throughout the experiment, the monitored average daily temperature in both the ambient and elevated co2 chambers was 26 °c ± 1 °c. the relative humidity of the ambient co2 chamber was 79% ± 7% while that of the elevated co2 chamber was similarly 73% ± 8%. the light conditions of the two chambers were similar. overall, the environmental conditions in two chambers were stable and similar throughout the duration of the study except the ambient co2 (400 ppm) and the elevated co2 (950 ppm) in the elevated chamber. effects of elevated co2 on kyasuwa kyasuwa grass grew well in the two co2 chambers and showed no signs of nutrient deficiencies or pest attacks. elevated co2 significantly reduced stomatal conductance (p < 0.001), leaf to stem ratio (p = 0.04) and root to shoot ratio (p = 0.01) by 40%, 3% and 5%, respectively (tab. 1, fig. 1). however, leaf area (p = 0.014) was significantly increased by 2% with increased carbon dioxide concentration (fig. 1, tab. 1). above-ground and total biomass was not significantly affected by atmospheric co2 concentration (tab. 1). growth under eco2 significantly increased structural carbohydrates (adf) by 2% (p < 0.001). however, adl and percentage protein were reduced by 23% (p < 0.001) and 14% (p < 0.001), respectively (fig. 1, tab. 1). the c/n ratio was not significantly affected by co2 concentration. 90 d. tom-dery, f. eller, k. jensen, c. reisdorff water and fertilization regime effects on kyasuwa growth and nutritive value the high-watering level significantly increased by 1% (p < 0.001) the adf content (fig. 1). increased nutrient availability reduced the leaf to stem ratio by 6% between n-3 and n-1 (p = 0.002), and by 4% (p = 0. 04) between n-2 to n-1 (tab. 1, fig. 1). we found significant interactions between watering and fertilization regimes for agb, bgb and, tb (p = 0.02; 0.01; and 0.005; respectively) (fig. 2a). increasing fertilization caused significant increases in these three biomass parameters but only with concurrently high water availability. at low water availability, no biomass responses to fertilization regime could be detected. for nutritive components, we observed two way interactions of watering and fertilization regimes for the c/n ratio (p = 0.005) and adl (p = 0.046) (fig. 2b, c). the c/n ratio was lowered with higher levels of fertilization, but only in drought-exposed plants. the adl concentration was higher at the highest than the two lower fertilization levels, but only in well-watered plants. interactive effects of elevated co2 with fertilization and/or watering regimes there was a two-way interaction between carbon dioxide enrichment and fertilization regime for ndf (p = 0.008; tab. 1; fig. 3a). here, eco2 had no effect on ndf at the low fertilization level, while it resulted in increased ndf at the higher nutrient availability n-3 (fig. 3a). a significant three-way interaction (p= 0.02) of co2 concentration, water and fertilization regimes was observed for shoot height (fig. 3b). under aco2, the higher fertilization regimes (n-2 and n-3) increased shoot height in well-watered plants but decreased shoot height in drought-treated plants. however, under eco2, higher fertilization regimes (n-2) resulted in increased shoot height at lower water regimes, but well-watered plants had taller shoots than drought-treated plants though no fertilization effects were recorded. discussion growth and biomass most plants, both c3 and c4, respond to eco2 by reducing stomatal conductance (ainsworth and rogers, 2007; ainsworth and long, 2005). in the present study, kyasuwa responded to eco2 by reducing its stomatal conductance (gs), which usually translates into reduced water loss through lower transpiration rates and higher water use efficiency at the leaf level (eamus et al., 2008). a review of the effect of eco2 on both c3 and c4 vegetables pointed to downregulating of stomatal conductance and lowering of transpiration, the combination of which leads to higher water use efficiency (bisbis et al., 2018). there are several studies showing that both tropical and temperate c4 grasses benefit from eco2 by down-regulating their stomatal conductance and thus, water loss, rather than increasing their above ground and or total biomass (xu et al., 2014; kakani and reddy, 2007). except shoot height, we did not find any interaction of wa tering regime and atmospheric co2 concentration on any other investigated parameters, so it is unlikely that eco2 had any effects on the integrated water-use efficiency of kyasuwa, although gs was lower under eco2, which at first sight might be indicative of an increased instantaneous water-use efficiency. in the case of shoot height there was a three-way interaction with differences recorded in the low water treatments of aco2 and eco2 in higher fertilization regimes. since consequently the net co2 flux into the leaves might have been the same at both high and ambient co2 concentrations, irrespective of the water availability, the lower stomatal conductivity in eco2 was the result of an adjustment of the co2 influx to an apparently unchanged co2 demand reflecting a similar assimilation potential under both atmospheric co2 conditions (ghannoum et al., 2000). this suggestion is further supported by similar c/n ratios at aco2 and eco2. the effects of eco2 on kyasuwa growth and biomass seemed to be confined to changes in biomass allocation patterns in the present tab. 1: f-values of three-way anova of all measured parameters of kyasuwa grass grown under elevated and ambient co2, three fertilization and two watering regimes. n=6 sources of variation parameters co2 (c) water (w) fertilization c*w c*n w*n c*w*n (n) growth & biomass height (cm) leaf area (cm2) stomatal conduc-tance (mmol mol-1) leaf to stem ratio root to shoot ratio above ground biomass (g) below ground biomass (g) total biomass(g) nutritive value c/n ratio % protein % neutral detergent fibre % acid detergent fibre % acid detergent lignin 10,63** 6,37** 7,30** 4,62* 7,09** 0,00 9,30** 0,71 0,59 30,38*** 23,45*** 111,34*** 14,38*** 47,87*** 0,03 0,73 2,49 2,41 63,52*** 36,69*** 54,04*** 53,98*** 1,17 1,42 16,61*** 2,06 0,77 0,44 0,13 7,04** 0,46 9,67*** 9,95*** 10,10*** 11,60*** 0,46 2,36 1,79 0,78 0,58 0,00 2,54 0,10 0,09 1,32 3,11 1,26 1,64 0,44 1,03 0,00 3,87 0,07 0,18 0,23 2,06 0,17 2,59 2,12 2,26 0,39 0,44 5,31** 1,44 1,64 1,42 0,64 0,73 0,06 0,23 7,02*** 4,90** 5,68** 5,88** 0,72 2,34 0,82 3,23* 4,15* 0,33 1,03 0,60 0,09 2,56 2,81 2,75 1,22 1,31 0,55 0,23 0,30 statistically significant values in bold; * <0,05, **<0,01, ***<0,001 probability levels kyasuwa biomass and nutritive value under elevated co2 91 study. the decrease in the root to shoot ratio and the general reduction in below-ground biomass with eco2 has previously been observed (kakani and reddy, 2007). the effect of eco2 on r:s is mechanistically not well understood, but it varies with plant types and resource supply (rogers et al., 1996). other studies have both found an increase (arnone et al., 2000) and no change in r:s (körner et al., 1997). as a general principle of allocation, under changing resource availability (light, water, nutrients, co2), plants tend to enhance organs that can increase the capture of the resource that is becoming limiting, partly to the detriment of other organs. in our study, as in others, increased co2 supply caused lower stomatal conductivity leading to reduced water demand. consistent with the above-mentioned principle, the allocation to root growth has been reduced, whilst a significant increase in leaf area as a response to eco2 has been observed which is also reported in other studies (ackerly et al., 1992). by lowering investment in below-ground biomass, the plant’s rooted soil volume is reduced and, thus, not only less nutrients but also less soil water is available to the plant. consequently under eco2 shorter dry spells could be withstood devoid of severe distress. however, kyasuwa will be more susceptible to extended drought because less soil volume will be used for water uptake and thus less competitive in the drier savanna. the observed decrease in leaf to stem ratio (l:s) signifies that eco2 resulted in higher allocation of biomass to stems, which has been previously reported in other c4 grasses (santos et al., 2014). on one hand, if the lower l:s ratio is combined with the increased leaf area, plants grown at eco2 developed overall broader leaves and denser shoots than those grown at aco2, which is beneficial for future use of the species, as livestock prefer leaves with broader blades (batistoti et al., 2012). on the other hand, a high leaf to stem ratio is generally preferred as an important factor in diet selection, quality and forage intake (smart et al., 2004). however, l:s was significantly reduced with higher fertilization regimes because of higher accumulation of stems as reported by other studies (salvador et al., 2016). water availability is one of the main biophysical limitations of grass growth in savannas (del grosso et al., 2008). water in general is one of the most important limiting resources to plant growth, and in the present study, low soil water content decreased all the biomass parameters as well as grass shoot height. water deficiency decreases transpiration rate via decreased stomatal conductance, which results in a decline of net photosynthetic rates (flexas and medrano, 2002). although c4 species have inherently lower stomatal conductance than c3 and thus have higher water use efficiency, they can still % n eu tr al d et er ge nt fi be r 10 30 50 70 % a ci d de te rg en t l ig ni n 1 3 5 7 le af to s ho ot r at io ( l: s ) 0,2 0,4 0,6 n-1 n-2 n-3 % p ro te in 0,5 1,5 2,5 % a ci d de te rg en t f ib er 0,8 10 20 30 40 a ci d de te rg en t f ib er 10 20 30 40 lw hw a b a b a b a bcb a b a s to m at al c on du ct an ce (m m ol m ol -1 ) 0,8 50 100 150 200 aco2 eco2 le af to s ho ot r at io ( l: s ) 0,2 0,4 0,6 r oo t t o sh oo t r at io n (r :s ) 0,1 0,3 le af a re a (c m ) 20 60 100 t ot al b io m as s (g d ry m as s) 0,5 5 15 25 b el ow -g ro un d bi om as s (g d ry m as s) 2 4 6 8a b a a a b a b b a a b fig. 1: main effects of co2 concentrations, watering and fertilization regimes on growth, biomass parameters and nutritive values of kyasuwa. different lowercase letters represent significant differences among treatments. lsm ± se. eco2, elevated co2 concentration; aco2, ambient co2 concentration; lw, low water treatments; hw, high water treatment; n-3, high fertilization regime; n-2, medium fertilization regime; n-1, low fertilization regime. 92 d. tom-dery, f. eller, k. jensen, c. reisdorff be threatened by drought. in the present study, it is more likely that the effects of water availability on growth were displayed through indirect effects for example lower mobility of nutrients in dry soil rather than a direct effect via stomatal conductance, which did not respond to watering regime. the water by fertilization regime interactions for kyasuwa showed that water supply was the main limiting factor for biomass accumulation and height growth. water avail ability is pivotal for increasing nutrient availability for kyasuwa growth and biomass production, and elevated co2 did not help to improve the water use efficiency of this species. however, some plants do not reduce water loss through stomata closure but, react to water shortage by investing assimilates into protective substances (i.e. synthesis of lea-proteins and other osmoprotectants, compatible so lutes etc) and consequently they have a lower biomass under drought treatment (schulze et al., 2005). kyasuwa tissue nutritive value the significant reduction of the protein content under eco2 has been attributed more to a dilution-effect due to an overall increase in total non-structural carbohydrates, rather than an absolute decrease of the protein content in leaves (dumont et al., 2015). this is unlikely to be the case in the present study, since the c/n ratio was unaffected by co2 treatment. other studies implicate the decrease in rubisco concentration (ainsworth and long, 2005) due probably to carbohydrate-dependent decrease in expression of photosynthetic genes (moore et al., 1999) and decreased transpiration and stomatal conductance (del pozo et al., 2007). a meta-analysis on proteins in food crops also indicates a reduction of proteins at elevated co2 (taub et al., 2008). we suggest that a re-allocation of n to resources other than proteins occurred within kyasuwa, for example to structural n-compounds or free amino acids or even to vacuolar nitrates, leaving a similar c/n ratio in the aco2 and eco2 treatment. the lack of effect of eco2 on the c/n ratio could be a result of c4 plants photosynthesis and biomass accumulation being less affected by eco2 (wang et al., 2012). c4 plants are less affected by eco2 because of their c-accumulation strategy, which minimizes photorespiration through anatomical and biochemical specializations that concentrate co2 at the active site of rubisco (sage, 2004) and are virtually co2 saturated already at aco2. low soil water potential generally impairs plant metabolism and there are suggestions that water availability plays a key role in nutrient limitations to grasses of semi-arid areas (lü et al., 2012). this is because of the vital role water plays in nutrient transport and availability for nutrient uptake in plant roots. the interactive effect of watering and fertilization regimes on c/n ratios showed higher fertilization regimes reducing c/n ratios when water availability was limiting. several studies support the finding of an interaction between water availability and nitrogen nutrition for c/n ratio (lü et al., 2012). in nature, soil water availability influences soil n availability via many microbial aided pathways like litter decomposition (liu et al., 2006) and n mineralization (wang et al., 2006). in the present study, the high water availability probably had a direct effect on nutrient availability to plant roots by increasing their diffusion and mass flow in the soil. the lower c/n ratio may therefore be an expression for a relative decrease in c-assimilation rates under dry conditions when nutrient supply was high. ndf concentration was increased at high fertilization regimes only under eco2, which is in concert with previous observations of soil n supply limiting the ability of plants to respond to eco2 (dong et al., 2016). plants with low n-demand, however, may respond to eco2 even in n-poor soils (norby et al., 1992). the present study fig. 2: two-way interactions between watering and fertilization regimes on biomass of kyasuwa (a). interactive effects of fertilization and watering regimes on nutritive value parameters of kyasuwa (b, c). different lowercase letters represent significant differences among treatments. lsm ± se. c /n r at io 20 60 100 lw hw n-1 n-2 n-3 % a ci d de te rg en t f ib re 2 4 6 8 b io m as s (g d ry m as s) 5 15 25 agb bgb agb bgb a b a b a b a b a b a b a a a a a b a a a b a b (a) (b) (c) kyasuwa biomass and nutritive value under elevated co2 93 showed that the main effects of eco2 on ndf were significant but rather small, as reported by other studies (fritschii et al., 1999; akin et al. 1995), and therefore future atmospheric co2 concentration can be expected to increase fiber content of kyasuwa to a minimal extent, and only if a high fertilization load is provided. adf concentration was significantly reduced by drought, which may have been caused by delayed plant maturity due to stressful arid growth conditions (küchenmeister et al., 2013). plant fibre contents turn to increase with age since the stem to leaf ratio increases with age, and fiber content of stems is considerably higher than leaves (bruinenberg et al., 2002). the increases in structural carbohydrates with eco2 recorded in this study have previously been observed and seem to be species-dependent (dumont et al., 2015; milchunas et al., 2005; fritschi et al., 1999; akin et al., 1995). in wheat for instance, akin et al. (1995) recorded increased contents in fibre fractions with eco2, while fritschi et al. (1999) reported increases in structural carbohydrates of arachis glabrata leaves. milchunas et al. (2005) likewise recorded increases in combined cellulose and hemicellulose in bouteloua gracilis with eco2. however, a recent meta-analysis of forage quality of mediterranean grasslands pinpointed no change in structural carbohydrates with eco2 (dumont et al., 2015). adl concentration was increased at the highest fertilization level, but only in well-watered plants, which could be the result of lower l:s ratio with higher fertilization regimes. moreover, adl was lowered by eco2, which means that the tissue quality as forage can be expected to be higher under future eco2 concentrations. the reducing effect of eco2 on lignin components of forages has been reported in previous studies (milchunas et al., 2005; akin et al., 1995) which may be caused by lignin being connected chemically to proteins and carbohydrates in the cell wall to form large macromolecules (moore and jung, 2001). a higher ndf concentration reduces animal intake while a higher adf concentration decreases digestibility (saha et al., 2013). however, decreases in adl increa ses digestibility because lignin limits digestion (moore and jung, 2001). we therefore propose that the overall quality of kyasuwa as forage will be lower under eco2. vegetables are reported to increase in sugars, vitamin c, phenols, flavonoids and antioxidant capacity as a result of eco2, however the macro and micronutrients are reduced (bisbis et al., 2018). conclusion elevated co2 increased individual leaf area of kyasuwa which would make the grass attractive as forage. however, eco2 resulted in a change in biomass allocation towards a lower r:s ratio, that ultimately may be harmful for the species especially under dry conditions and low nutrient availability making it less competitive. moreover, eco2 will result in changes in the chemical composition of kyasuwa with increases in structural carbohydrates (ndf and adf) and reduction in adl and protein which will reduce the nutritive value of kyasuwa overall. water and fertilization were the two most limiting resources for kyasuwa compared to co2 and did not interact with co2 except in shoot height and ndf. as a compromise for future tissue quality, we suggest to avoid over-fertilization of kyasuwa to avoid an undesirable increase in fibre content. acknowledgement the authors are grateful to prof. dr. jörg fromm and the institute of wood science/vti hamburg for allowing us to use their greenhouse. we also wish to thank prof. dr. jörg ganzhorn of animal ecology fig. 3: two-way interaction of co2 concentrations and fertilization regimes on neutral detergent fibre of kyasuwa (a) and three-way interaction between co2 concentrations, watering and fertilization regimes on kyasuwa shoot height (b). different lowercase letters represent significant differences among treatments. lsm ± se. lw hw lw hw s ho ot h ei gh t ( cm ) 20 60 100 140 n-1 n-2 n-3 % n eu tr al d et er ge nt fi br e 10 30 50 70 n-1 n-2 n-3 a b b a ca a b b a c c a abb c c c aco2 eco2 aco2 eco2 (a) (b) 94 d. tom-dery, f. eller, k. jensen, c. reisdorff and conservation, university of hamburg for allowing us to use their fiber analyzer for nutrient analysis. references ackerly, d.d., coleman, j.s., morse, s.r., bazzaz, f.a., 1992: co2 and temperature effects on leaf area production in two annual plant species. ecology 73, 1260-1269. doi: 10.2307/1940674 ainsworth, e.a., long, s.p., 2005: what have we learned from 15 years of free-air co2 enrichment (face)? 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applied botany and food quality 90, 126 131 (2017), doi:10.5073/jabfq.2017.090.015 1laboratoire d’etudes et développement des techniques d’epuration et de traitement des eaux et gestion environnementale, département de chimie, ecole normale supérieure de kouba, algiers, algeria 2 département de biologie, ecole normale supérieure de kouba, algiers, algeria 3 département de physique, ecole normale supérieure de laghouat, algeria 4 département des sciences et techniques, faculté de technologie, université amar télidji laghouat, algeria starch digestion in pearl millet (pennisetum glaucum (l.) r. br.) flour from arid area of algeria mohamed lemgharbi 1, 2, rachid souilah 1, 3, badreddine belhadi 1, 4, ladjel terbag 1, 3, djaffar djabali 1, boubekeur nadjemi 1 (received august 28, 2016) * corresponding author summary to assess the nutritive value of minor cereals cultivated in arid areas of algeria, nine pearl millet landraces were sampled from two regions: tidikelt and hoggar. some qualitative and quantitative characters of the panicle and grain were measured, as well as in vitro starch digestion of the grain flour. considerable variation was recorded in seed color, endosperm texture and nutritional value of starch and protein content. in vitro starch digestion displayed a first-order kinetic model. for all pearl millet landraces, starch was digested to a different extent; the hydrolysis index (hi) ranged from 22.29 % to 35.52 % and the expected glycemic index (egi) ranged from 27.41 to 38.82. the results show that there is diversity in the physical and chemical properties of pearl millet accessions from the arid areas of algeria: tidikelt and hoggar. this study confirms that pearl millet has an acceptable nutritional value with a low glycemic index suitable for human health and nutrition. keywords: pearl millet, starch digestion, first-order kinetics, glycemic index, nutrition. introduction pearl millet [pennisetum glaucum (l.) r. br.] is a small-seeded grass belonging to the poaceae family and the panicoideae subfamily. as it is a drought tolerant crop and has the ability to grow on low fertility soil and moisture (fao and icrisat, 1996), it is mostly cultivated in arid and semi-arid areas of the sahel in africa and in asia, where it is a major food source. millet is the 6th largest cereal crop in terms of world agriculture production. furthermore, millets have resistance to pests and diseases, a short growing season, and are productive under drought conditions, compared to major cereals (devi et al., 2014). millets are unique among the cereals because of their richness in calcium, dietary fiber, polyphenols and protein. furthermore, as they do not contain gluten, they can be recommended for use by celiac patients (devi et al., 2014). in addition to their nutritive value, several potential health benefits have been reported such as lowering the risk of cancer and cardiovascular disease, lowering blood pressure and risk of heart disease, lowering cholesterol and rate of fat absorption, and delaying gastric emptying by supplying gastrointestinal bulk (saleh et al., 2013). there are several plant characteristics that define grain quality, such as their structural and biochemical characteristics, digestibility, and bioavailability of nutrients. the structure of the grain kernels varies significantly because of environmental and genetic factors. shape, size, proportion and nature of the endosperm, germ, and pericarp, the presence and absence of a subcoat, and color of the pericarp are all genetically determined (rooney and murty, 1982). grain digestibility also is important. several works have been conducted to study the kinetics of starch digestion of different grains by alpha-amylase (ezeogu et al., 2005; frei et al., 2003; goni et al., 1997). the glycemic index (gi) is an in vitro measurement based on glycemic response to carbohydrate-containing foods, and allows ranking of food on the basis of the rate of digestion and absorption of carbohydrates that they contain (englyst et al., 1992; jenkins et al., 1981). in vitro methods have also been used to classify foods based on their digestion characteristics similar to the in vivo situation, and to identify slow release of carbohydrate in foods (schweizer et al., 1988; jenkins et al., 1984). food materials with gi values more than 70 %, between 56 and 69 % and lower than 55 % are classified as high, medium and low gi foods, respectively (brand-miller et al., 2003). in the sahara of algeria, the tidikelt and hoggar regions are characterized by a typical desert climate; they are very hot and very dry. moreover, rains have been rare during the last ten years. aridity of the climate is extreme and the ambient temperature is very high in tidikelt (in salah), which is known to have temperatures ranging from 7.8 to 45.2 °c, a very low annual rainfall (16.9 mm), and irrigation is done with saline water. the hoggar region is known to have temperatures ranging from 10 to 38 °c, with daily temperatures ranging over 23 °c, annual rainfall rate ranging from 7 to 160 mm, and irrigation is done with ground water. despite these local hard climatic conditions, indigenous millet has maintained its original morphological diversity for centuries, and it has been able to accumulate significant genetic diversity between populations. however, these environmental factors have affected the starch properties in different sorghum genotypes (belhadi et al., 2012; boudries et al., 2009; mastsuki et al., 2003). for example, pearl millet; (p. glaucum), is a cereal that also is called mil, mil à chandelle (french), and dokhen (arabic); the local appellation is “bechnna” and “inélé”, originally from west africa, particularly, in the area north-east of the senegal river. millet was probably introduced during the eighth century into north africa; its culture was intended to produce seed and fodder (tostain, 1998). actually, pearl millet production in these marginalized areas depends on traditional harvesting and processing. most of the harvest is used as animal feed and rarely for human consumption. in the past, a wide range of traditional food products has been made from millet including kisra, porridge, and beverage. one of the objectives of our laboratory research is to study the nutritional and quality traits of sorghum and pearl millet grains as well as the isolation of starch and protein fractions and their beneficial characteristics for food and non-food uses (souilah et al., 2014; belhadi et al., 2012; hadbaoui et al., 2010; mokrane et al., 2010; boudries et al., 2009; mokrane et al., 2009). in a previous work, the protein nutritional quality of seven sorghum cultivars cultivated in the sahara of algeria was assessed (mokrane et al., 2010). high percentages of protein, up to 16 % db, were found in these cultivars with a favorable amino acid composition. the measure of in vitro starch digestion in pearl millet flours 127 pepsin digestibility shows that some cultivars exhibit high digestibility, whereas, other cultivars are characterized by their low digestibility (mokrane et al., 2010). the measurement of in vitro starch digestibility shows that the nine local sorghum cultivars exhibit high digestibility of up to 90 % (souilah et al., 2014). the aim of the present study is to evaluate the digestibility of starch in algerian arid area pearl millet grain cultivars by investigating starch in vitro digestion in pearl millet grain flour, assessing the in vitro digestion kinetic data, and evaluating the effect of landrace differences on kinetic parameters. materials and methods materials nine pearl millet [pennisetum glaucum (l.) r. br.] grains from local landraces were sampled from the arid sahara areas of south algeria: i.e., within tidikelt and hoggar. landraces labeled as mlt.p, mlt.p.p, mlt.saf, mdt.smix, mlt.ham, mdt.sepl, mlh.z, mlh.epc and mdh.saf.t were harvested from different localities within tidikelt and hoggar; djafou, foggaret ezzoua, el malah, in amghel, tamanrasset and abalessa. the samples were characterized by their kind of use (eg., cultivated millet, and domestic) and by different harvest years (2008, 2010 and 2011). tab. 1 lists pearl millet landraces from arid areas of algeria. millet grains were ground to flour in ika labotechik using an a10 sample mill. the flour was manually sieved using a 500 μm sieve. all reagents were analytical grade. methods pearl millet grain analysis some qualitative and quantitative characters of pearl millet grains (seed envelop, seed color, seed form, thousand seed weight and bulk density) were categorized (ibpgr and icrisat, 1993). grain color was assessed by the royal horticultural society (rhs) color codes (icrisat, 1993). endosperm texture was defined as the proportion of corneous relative to floury endosperm in the grain, which was determined subjectively by viewing sectioned kernels using a stereomicroscope, and comparing them to sorghum standards (taylor and taylor, 2008). the kernels were classified as corneous, intermediate or floury (icc, 2008). moisture content was determined according to aacc methods 44-15a. the crude protein content was determined according to micro kjeldahl method using a nitrogen conversion factor of 5.83, based on an adaptation of the aacc 46-13a method (aacc, 2000). total starch (ts) was determined by the enzymatic method of goni et al. (1997). in vitro starch digestion in vitro starch digestion was determined according to the modified method of goni et al. (1997). around 600 mg of pearl millet flour was prepared in large tubes containing 25 ml of phosphate buffer (ph 6.9). to start starch hydrolysis, 5 ml of α-amylase (2 × 10-4 mg/ ml), type vi.b from porcine pancreas (a3172, sigma-aldrich) was added. the prepared mixture was incubated at 37 °c for 2 h with constant shaking. aliquots of 0.2 ml were withdrawn at 5, 10, 15, 20, 30, 60, 90 and 120 min. α-amylase was inactivated immediately by placing the tubes in a boiling water bath for 5 min. then, 0.6 ml of a 0.4 m sodium-acetate buffer solution (ph 4.75), and 0.2 ml of an enzyme solution containing 0.833 μl of amyloglucosidase from aspergillus niger (300 u/ml, sigma, a-7095) were added. in order to hydrolyze digested starch into glucose, the sample was incubated at 60 °c for 45 min. finally, the volume was adjusted to 1-5 ml with distilled water and glucose concentration in the digesta was measured within the range (0.1-0.5 g/l) using an oxidase-peroxidase kit (biomaghreb, tunisia). a concentration of free glucose (0.459 g/l) was used to correct the starch digestion values. starch digestion was expressed as percentage on the amount of starch present at the start of the reaction. modelling of starch digestograms first-order exponential kinetics were used to estimate starch hydrolysis and glycemic indices in the food and feed studies (ezogu et al., 2005; frei et al., 2003; goni et al., 1997). starch amylolysis data was fitted to a first-order equation (eq. (1)): ct = c∞ (1-exp [-kt]) (1) where ct corresponds to the percentage of starch hydrolysis at time t, c∞ is the equilibrium percentage of starch hydrolyzed after 120 min, k is the kinetic constant and t is the time (min). glycemic and hydrolysis indices were determined from the area under the hydrolysis curve (aucexp), which was obtained by integrating eq. (1) between times t0 = 0 min and tf = 120 min getting eq. (2) aucexp = c∞ tf c∞ / k (1-exp [-k tf ]) (2) the hydrolysis index (hi) expressed the ratio of the aucexp of the sample from 0 to 120 min relative to the area under the hydrolysis curve of white bread (~ 7444 % min) (goni et al., 1997). the expected glycemic index egi was calculated by the equation egi=8.198 + 0.862 hi as described by granfeldt et al. (1992). statistical analysis all the parameters of pearl millet panicle and grain quality were measured in three replicates, and expressed as mean±sd. data analyses were performed using sigmaplot v.10.0 (systat software inc, chicago, illinois, usa) for windows. tab. 1: pearl millet [p. glaucum (l.) r. br.] landraces from the hyper area of algeria: tidikelt and hoggar. no. landraces codes locality region status harvest date 01 mlt.p djafou tidikelt cultivated millet 2008 02 mlt.p.p foggarat ezzoua tidikelt cultivated millet 2008 03 mlt.saf foggarat ezzoua tidikelt cultivated millet 2008 04 mdt.smix el malah tidikelt domestic* 2011 05 mlt.ham djafou tidikelt cultivated millet 2010 06 mdt.sepl el malah tidikelt domestic* 2011 07 mlh.z in amgheul hoggar cultivated millet 2008 08 mlh.epc tamanrasset hoggar cultivated millet 2011 09 mdh.saf.t abalessa hoggar domestic* 2011 domestic*: introduced from neighboring countries; mali, niger, sénégal… (local appellation is sudan) 128 m. lemgharbi, r. souilah, b. belhadi, l. terbag, d. djabali, b. nadjemi tab. 2: qualitative and quantitative characters of pearl millet grains. n° landraces codes se sc sf 01 mlt.p exposed(3) grey (201) obevate 02 mlt.p.p intermediate(5) grey (201) oblanceolate 03 mlt.saf exposed (3) yellow (8c) oblanceolate 04 mdt.smix intermediate grey brown (199) hexagonal 05 mlt.ham intermediate (5) brown (200) oblanceolate 06 mdt.sepl enclosed (7) ivory (158a) obevate 07 mlh.z exposed (3) deep grey (202b) oblanceolate 08 mlh.epc exposed(3) deep grey (202b) obevate 09 mdh.saf.t exposed (3) yellow (8c) globular se: seed envelop, sc: seed color, sf:seed form, tsw: thousand seed weight, g, bd: bulk density, g/l, h: moisture, %, ts: total starch, %, p: protein, %. n° landraces codes tsw (g) bd (g/l) h (%) ts (%) p (%) endosperm texture (%) corneous intermediate starchy 01 mlt.p 9.50 ±0.15 782.60 ± 3.15 09.61 57.02 ± 1.92 17.18 ± 0.58 95 5 0 02 mlt.p.p 9.10 ±0.10 780.40 ± 6.32 11.35 58.82 ± 5.56 15.18 ± 0.71 5 85 10 03 mlt.saf 9.20 ± 0.70 782.50 ± 4.56 10.95 65.29 ± 7.19 11.41 ± 0.20 0 5 95 04 mdt.smix 6.86 ± 0.22 778.00 ± 3.66 10.42 58.44 ± 7.97 14.15± 0.12 90 10 0 05 mlt.ham 9.50 ± 0.08 782.60 ± 7.15 11.75 65.81 ± 2.12 16.89 ± 0.76 90 10 0 06 mdt.sepl 6.20 ± 0.04 782.60 ± 3.55 10.27 65.87 ± 2.48 13.27 ± 1.83 80 20 0 07 mlh.z 9.40 ±0.14 782.60 ± 2.81 13.11 69.07 ± 3.09 14.87 ± 0.50 100 0 0 08 mlh.epc 9.20 ±0.05 782.60 ± 5.16 11.55 63.06 ± 4.19 13.30± 0.89 0 5 95 09 mdh.saf.t 9.80±0.12 782.60 ± 4.46 10.00 59.53 ± 9.69 14.24± 1.55 0 5 95 mlt.p : pearl millet local fromtidikelt, mlt.p.p : pearl poilue (hairy), mlt.saf: safra(yellow), mdt.smix: domesticated from soudan mix, mlt.ham: hamra(red), mdt.sepl: soudan epi log (tall panicle), mlh.z: millet local from hoggar. zarga (blue), mlh.epc : local hoggar epi court (short panicle), mdh.saf.t : domesticated hoggar safra (yellow) from tidikelt. results and discussion pearl millet grain analysis some qualitative and quantitative characters of pearl millet grain were determined. as shown in tab. 2, qualitative characters assessed were seed envelop (se), seed color (sc) and seed form (sf). seed envelop was assessed as exposed, intermediate or enclosed; seed color was grey, yellow, grey brown, brown, ivory or deep grey, and seed form was obevate, oblanceolate, hexagonal or globular. in general, pearl millet landraces showed variation in phenotypic characters, indicating that algeria has a high diversity in millet traits. the quantitative characters for all pearl millet landraces were thousand seed weight, bulk density, moisture, total starch and protein. thousand seed weight varied from 6.20 ± 0.04 to 9.80 ± 0.12 g with a mean value of 8.75 g, which was in the range of the majority of millets varieties from the data collected by dendy (1995), which varied between 2.5 and 14.7 g. the bulk density varied from 778.00 ± 3.66 to 782.60 ± 7.15 g/l with a mean value of 781.83 g/l; these mean values were lower than those reported for three pearl millets (850 g/l) looked at by jain (1997). moisture content in pearl millet grains ranged from 09.61 % to 13.32 %. visual examination of endosperm texture varied in percentage of corneous (0 to 100 %), intermediate (0 to 85 %), and starchy (0 to 95 %) fractions (tab. 2). this variation in endosperm texture indicated that the grains should be classified as corneous, floury, mixed and intermediate endosperm type as described by the international association for cereal science and technology (2008). total starch (ts) content of pearl millet flour ranged from 51.35 ± 5.35 to 69.07 ± 3.09 % db with a mean value of 61.43 % (tab. 3). the grain chemical composition of pearl millet genotypes from the world collection at icrisat showed that starch composition was between 62.8 % and 70.5 % with a mean value of 66.7 % (fao, 1995). when compared to our results, the total starch content in the algerian pearl millet samples was lower than the mean value. moreover, the grain starch contents in the ten studied pearl millet landraces were lower than those in wheat (65 %) and higher than those observed in rye (60 %) and barley (55 %) (choct and hughes, 2000). however, our samples exhibited a lower total starch content than maize (75 %) and rice (80 %) (choct and hughes, 2000). the protein (p) content of pearl millet flour ranged from 09.62 ± 0.01 to 17.18 ± 0.58 % db with a mean value of 14.01 % (tab. 3). the grain chemical composition of pearl millet genotypes from the world collection at icrisat showed that protein content was between 5.8 % and 20.9 % with a mean value of 10.6 % (fao, 1995). the grain protein contents in the ten pearl millet landraces studied were higher than the mean value. a large variation for grain qualitative and quantitative traits was observed in algerian pearl millet landraces. based on this variation, probably due to environmental conditions, high genotype diversity is found among landraces (rooney and miller, 1982). in vitro kinetic starch digestion and modelling experimental data and computed digestibility curves were shown for all pearl millet flours in (fig. 1). the kinetic curves show that the starches in pearl millet flours from algeria landraces were hydro starch digestion in pearl millet flours 129 tab. 3: starch digestibility and expected glycemic index parameters of the first-order model for the pearl millet flours a. n° landraces codes k(min-1) r2 see (%) c ∞(%) hi(%) egi 01 mlt.p 0.24 0.97 4.40 22.83 35.52 38.82 02 mlt.p.p 0.13 0.99 0.86 19.42 29.30 33.45 03 mlt.saf 0.23 0.99 1.44 15.54 24.14 29.01 04 mdt.smix 0.22 0.99 0.78 19.54 30.31 34.32 05 mlt.ham 0.21 0.96 4.50 21.25 32.90 36.55 06 mdt.sepl 0.17 0.98 2.18 22.23 34.08 37.57 07 mlh.z 0.29 0.91 9.23 14.24 22.29 27.41 08 mlh.epc 0.15 0.99 0.94 22.22 33.83 37.36 09 mdh.saf.t 0.20 0.90 6.64 18.15 28.04 32.37 a values are estimated from fit to experimental data, with r2> 0,9 and standard error of estimate (see) < 6% for most landraces. fig. 1: digestibility curves obtained for pearl millet flours. lyzed by amylases. the extent of the reaction indicates that these flours have a low susceptibility to digestion. predicted values for variables affecting starch amylolysis (c∞ and k), that were obtained from the first-order model, fit to the experimental data. overall, computed digestibility curves provided a very good fit to all experimental data, with r2 > 0.9 and standard error of estimate (see) < 6 % for most landraces. predicted values (c∞, and k) were obtained from the first-model fit 130 m. lemgharbi, r. souilah, b. belhadi, l. terbag, d. djabali, b. nadjemi to the experimental data, reported in tab. 3. the constant k ranged between 0.13 and 0.29 min-1; starch hydrolysis at infinite time c∞ varied from 14.24 to 26.38 %, and model-fit analysis of digestibility data was particularly well-suited to these studies. first-order model properties have been demonstrated in in vitro starch digestion of raw and processed food and feed (ezogu et al., 2005; frei et al., 2003; goni et al., 1997). the kinetic constant k of amylolysis has been proposed as a reliable index of the inherent susceptibility of flour starches to amylase hydrolysis (frei et al., 2003; goni et al., 1997). modeling of starch digestion kinetics is required to derive more quantitative information on digestibility properties. the hydrolysis index hi and expected glycemic index egi are reported in tab. 3. the hydrolysis index (hi) obtained by use of a first-order model fit to the experimental data ranged from the lowest in mlh.z (22.29 %) to the highest mlt.p (35.52 %). this variation in starch digestibility of pearl millet flours was due to grain quality differences in the sampled pearl millet landraces (rooney and pflugfelder, 1986). the expected glycemic index egi varied from 27.41 to 38.82. relative to the glycemic index suggested by brand-miller et al. (2003), for algerian pearl millet landraces (egi < 55), the results of the gi for the eight local cultivars of sorghum were high and ranged from 74.02 to 94.14 (souilah et al., 2014). in the 2002 edition of the international table of glycemic index and glycemic load reported by foster-powell et al. (2002), the glycemic index of boiled millet (canada) was found to be 101, while that of millet flour porridge (kenya) was 153 ± 14. mani et al. (1993) also found that gi ranged from 55 ± 13 to 104 ± 13 after testing six commonly consumed sorghum foods of india. the results indicate that starches from pearl millet samples in our work can be classified as having a low gi, although the gi values of sorghum flours grown in algeria are high with a gi value of up to 70 (souilah et al., 2014). the hi and egi values of pearl millet flours grown in algeria were much lower, possibly due to differences in genetic source, growing conditions, and the employed methods used to determine hi and egi. conclusions the present study points out that differences in some morphological characteristics and biochemical components of the panicle and grains demonstrate diversity in the phenotype of pearl millet landraces in algeria. moreover, measure of in vitro starch digestibility shows that the nine local landraces examined exhibited low digestibility (< 40 %) and a low glycemic index (< 55). first-order kinetic analysis was used to model starch 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chem. 86, 487-491. doi: 10.1094/cchem-86-5-0487 mokrane, h., amoura, h., belhaneche-bensemra, n., courtin, c.m., delcour, j.a., nadjemi, b., 2010: assessment of algerian sorghum protein quality [ sorghum bicolor (l.) moench] using amino acid analysis and in vitro pepsin digestibility. food chem. 121, 719-723. doi: 10.1016/j.foodchem.2010.01.020 rooney, l.w., miller, f.r., 1982: variation in the structure and kernel characteristics of sorghum. proccedings of the international symposium on sorghum grain quality, 143-162, icrisat, patancheru, india, rooney, l.w., murty, d.s., 1982: evaluation of sorghum food quality. proccedings of the international symposium on sorghum grain quality, 571588. icrisat, patancheru, india. rooney, l.w., pflugfelder, r.l., 1986: factors affecting starch digestibility with special emphasis on sorghum and corn. j. animal sci. 63, 1607-1623. doi:10.2527/jas1986.6351607x saleh, a.s.m., zhang, q., chen, j., shen, q., 2013: millet grains: nutritional quality, processing, and potential health benefits. comp. rev. food sci. food safety. 12, 281-295. doi/10.1111/1541-4337.12012 schweizer, t.f., reimann, s., wursch, p., 1988: definition and measurement of a starch digestion index and a study of factors determining starch digestion rates in foods. food sci. technol. 21, 352-357. souilah, r., djabali, d., belhadi, b., mokrane, h., boudries, n., nadjemi, b., 2014: in vitro starch digestion in sorghum flour from algerian cultivars. food sci. nutrition. 2, 251-259. doi: 10.1002/fsn3.104 tatlor, j.r.n., taylor, j., 2008: five simple methods for the determination of sorghum grain end-use quality. intsormil scientific publications, 9-11. address of the corresponding author: mohamed lemgharbi, laboratoire de biochimie, département de biologie, ecole normale supérieure, bp 92 kouba, algiers, algeria. e-mail address: lemgharbim@yahoo.com or md.lemgharbi@hotmail.fr. © the author(s) 2017. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 92, 64 72 (2019), doi:10.5073/jabfq.2019.092.009 1department of entomology, phytopathology and molecular diagnostics, faculty of environmental management and agriculture, university of warmia and mazury in olsztyn, poland 2chair of plant food chemistry and processing, faculty of food sciences, university of warmia and mazury in olsztyn, poland 3department of plant breeding and seed production, faculty of environmental management and agriculture, university of warmia and mazury in olsztyn, poland phenolic and lipophilic compounds of wheat grain as factors affecting susceptibility to infestation by granary weevil (sitophilus granarius l.) bożena kordan1, marta skrajda-brdak2, małgorzata tańska2, iwona konopka2, robert cabaj1, dariusz załuski3 (submitted: june 13, 2018; accepted: november 30, 2018) summary the impact of certain groups of polyphenolic (phenolic acids and alkylresorcinols) and lipophilic compounds (total lipids, fatty acids, sterols, tocols and carotenoids) on susceptibility of bread wheat (triticum aestivum l.) kernels to sitophilus granarius infestation was studied. in the experiments, six cultivars of spring wheat with comparable protein content, endosperm hardness and overall technological quality were used. twenty grams of grain were infested by 10 pairs of beetles and stored for one week or eight weeks at 28 ± 2 oc and relative humidity of 60%. the intensity of growth and feeding of s. granarius varied significantly in the used cultivars. the antixenosis effect of the studied grain chemicals, observed after one week of infestation, was the lowest for łagwa cv., which was characterized by the highest total lipid and sterol contents. other cultivars showed a similar antixenosis effect. for antibiosis effect, the most attractive for s. granarius infestation was ostka smolicka cv., which was characterized by the lowest content of total phenolic acids. in contrast, the highest antibiosis effect was found for arabella and izera cvs. with the lowest values of sterol content and average values of other determined phytochemicals. keywords: alkylresorcinols, infestation by pests, lipophilic compounds, phenolic acids, wheat grain introduction post-harvest losses of cereal grain during storage may be as high as 83% of mass, especially in tropical and subtropical areas (pingale, 1964). in temperate zones, the losses to the stored grains and pro ducts range from 5% to 15% (rajendran and sriranjini, 2008). the main reason for these losses is an attack of insect pests (fornal et al., 2007). according to fao estimates, grain losses from pests are about 96 million tonnes annually (nietupski et al., 2006). chemical pesticides can reduce these losses, but their use is detrimental to the environment and to the health of the warehouse staff. additionally, there is a possibility of residual toxicity in the final products used in the feeding of people and animals (singh and kaur, 2018). an additional problem with the use of chemical control is the increment of the pest resistance to almost all groups of pesticides (georghiou, 1990; singh, 2017). although the use of plant natural repellents (powders, essential oils and extracts) as an alternative to synthetic compounds is possible (singh, 2017), the basic strategy is still limited to creating the least favourable conditions (temperature, humi dity) for their development (niewiada et al., 2005). the most dangerous pest of stored grain in temperate climates is the granary weevil (sitophilus granarius l.) (campbell and sinha, 1976). it is considered as a cosmopolitan species (padin et al., 2002). grain weight losses caused by s. granarius range from 2% to 5% in temperate countries (ignatowicz, 1999; rajendran and sriranjini, 2008). adult beetles eat from 0.5 to 1 mg per day (nawrot, 2001) and produce from 0.1 to 0.3 mg of dust (piaseckakwiatkowska, 1999). larvae of s. granarius develop inside the grains and destroy more than half of the grain volume (hansen and steenberg, 2007). in the interior of the infested kernels, there are moults or dead individuals. however, the number/mass of tole rable insect fragments is not regulated by european regulations, milling products made from grains heavily infested by s. granarius are harmful to humans and animals (perez-mendoza et al., 2005) and may cause atopic bronchial asthma in employees of cereal-mills (skjold et al., 2007). additionally, the infestation by s. granarius results in an increase in kernel moisture content and temperature, which contributes to an increase in enzyme activity (grain sprou ting), facilitates the invasion of secondary insect pests, bacteria, mites and fungi (nawrot et al., 2006; grineva et al., 2014) and favours the infection of the grain with mycotoxins produced by aspergillus and fusarium species (ragunathan et al., 1974). susceptibility to postharvest infestation by granary weevils is highly variable between wheat cultivars. for example, the most and the least susceptible cultivars can differ approx. 8-fold in insect fertili ty and approx. 3-fold in insect growth index and grain weight loss after three months of storage (mebarkia et al., 2010). in a similar study conducted by khan et al. (2014) on the resistance against rice weevil (s. oryzae), the extreme wheat genotypes differed by at least 2-3-fold in the number of damaged grains, percent weight loss and adult population during an experiment conducted over five months. in turn, fourar-belaifa et al. (2011) found a 5-fold difference in the number of s. oryzae after a 160-day storage of three wheat cultivar grains. similarly, nawrot et al. (2006) determined significant differences of the number of offspring and the mass of produced dust between two bread wheats (cvs. begra and korweta) and du rum wheat (cv. lgr896/64a). the phenomenon of various wheat cultivars susceptibility to pest damage is related to many chemical compounds, such as the content of total protein or gluten, total lipids and cuticular lipids (nawrot, 1983; niewiada et al., 2005; mebarkia et al., 2010; nawrot et al., 2010) and physical kernel features, such as endosperm hardness or vitreosity and thickness of seed coat (nawrot et al., 2006; fourar-belaifa et al., 2011), but it is still not fully explained. these relationships are much better understood for maize. according to lópez-castillo et al. (2018), maize kernel resistance against maize weevil (sitophilus zeamais) and large grain borer (prostephanus truncates) is manifested by antibiosis and antixenosis mechanisms. the cited authors concluded that kernelpest interactions are determined by biophysical factors (pericarp thickness/toughness, kernel hardness and endosperm vitreosity) and biochemical factors (hydroxycinnamic acids, hydroxyproline-rich glycoproteins, extensins, zeins, arabinoxylans, peroxidises and phenolic acid amides) under the control of genetic factors. the result of these interactions is kernel modification, which leads to limited factors affecting infestation of wheat grain by granary weevil 65 accessibility or toxicity to invading pests (lópez-castillo et al., 2018). the objective of the study was to determine if the susceptibility of grain of bread wheat cultivars to s. granarius infestation is influenced by certain groups of grain polyphenolic and lipophilic compounds, since these secondary metabolites are highly varied between various wheat grain samples. to avoid the impact of grain protein content and endosperm hardness, only grain samples of similar values of these features were used. based on a one week and eight-week infestation experiment, the antixenosis or antibiosis effect of the determined chemical compounds was determined. material and methods biological material wheat kernel s. granarius interaction was determined for six spring cultivars of bread wheat: arabella, izera, kws torridon, łagwa, ostka smolicka and tybalt obtained from the department of experimental evaluation of varieties in radostow, pomeranian voivodeship, poland, from the harvest in 2014 (fig. 1). the used grain samples were similar in moisture content (13.7 13.9%), protein content (13.9 14.2%), endosperm hardness (14.4 14.8 n) and grain overall quality (results of preliminary analyses) and represented the same technological class (quality a – bread wheat) according to the guidelines and criteria of the research centre for cultivar testing in poland (coboru, 2018). granary weevils originated from the mass breeding conducted in the department of entomology, phytopathology and molecular diagnostics of the university of warmia and mazury in olsztyn on wheat kernels of the korweta cv. (triticum aestivum). experimental setup for infestation the experiment was performed in 10 replicates for each cultivar, using 80 mm vinyl vials and 35 mm height with a single-centimetre ventilation opening. each replicate containing 20 g of whole kernels was infested with 20 five-day adults, 10 males and 10 females, sexed by examining the rostrum and abdominal shape, as reported by halstead (1963) and dinuta et al. (2008). infested kernels were placed for seven days in a sanyo mlr-350h air-conditioned chamber for incubation in 28 ± 2 °c and 60% relative humidity. after this time, the adults were removed and the percentage of damaged kernels (dk), the grain weight loss (gwl), the number of eggs laid (ne) and the mortality of the parents (mp) were determined. the kernels were further incubated for insect development during eight weeks. after that time, the selected parameters were recorded: the number of the adult progeny (ap), the total grain weight loss (tgwl), the weight of the dust/flour production (fp). the dobie susceptibility index (di) was calculated according to the formula given by dobie and kilminster (1978). based on the di, used wheat cultivars were assigned as moderately resistant/susceptible to pest infestation with values in the 7-8 range (dobie, 1974). determination of chemical composition in this part of experiment intact grain samples, manually cleaned from broken and damaged kernels, were utilized. before subsequent extraction of chemicals, grains were milled to obtain particles smaller than 400 μm. phenolic acids chromatography analysis was performed by the rp-hplc technique according to the method described by bojarska et al. (2011). total phenolic acids were extracted by triplicate with diethyl ether from alkaline hydrolysate of milled grains, while free phenolic acids from untreated samples were extracted according to konopka et al. (2012). an agilent technologies (santa clara, usa) 1200 series system fitted out with a photodiode detector and with an agilent technologies eclipse xdb-c18 column (150 mm × 4.6 mm, 5 μm) at the temperature of 30 °c was used. the mobile phase consisted of water : acetonitrile : formic acid (88:10:2, v/v/v). the isocratic flow rate was equal to 0.8 ml/min. detection was performed at the wavelength of 260 and 320 nm. identification and quantification of phenolic acids was performed by comparison of the retention times and absorption spectra of the sample with reference standards (sigmaaldrich, st. louis, mo, usa). the content of phenolic acids was determined from calibration curves of reference phenolic acids (sigmaaldrich, st. louis, mo, usa) and expressed as μg per g of a sample dry mass (dm). the repeatability for phenolic acid determination (expressed as coefficient of variation) was at least 2.1 and 3.5% (for free and total form, respectively). limit of quantification was at least 0.05 μg/g of sample dm, while linearity of calibration curves was confirmed in range of 1-150 μg/ml. free phenolic acids content is presented as a sum of identified compounds. alkylresorcinols extraction of alkylresorcinols was done with the use of acetone (10 ml/g of a sample) according to the method described by sampietro et al. (2009) with small modifications. firstly, samples were sonicated for 15 minutes in an ultrasonic bath (intersonic, olsztyn, poland) and subsequently put aside in a dark place at room temperature for 48 hours. after that time, the 15-minute ultrasonic treatment was repeated. then, the extract was centrifuged in an eppendorf centrifuge (eppendorf, type 5810 r, hamburg, germany) fig. 1: images of kernels of the studied spring wheat cultivars: ostka smolnicka (a), arabella (b), izera (c), tybalt (d), kws torridon (e), łagwa (f). 66 b. kordan, m. skrajda-brdak, m. tańska, i. konopka, r. cabaj, d. załuski at 1,000 rpm for 10 minutes, at 22 °c. the supernatants were collec ted in clean flasks and evaporated to dryness on a vacuum evaporator (büchi, type r-210; büchi labortechnik). the residue was dissolved in 1 ml of methanol and centrifuged on a centrifuge (eppendorf, type 5417 r) at 1,600 rpm for 10 minutes. colour reaction was performed by adding 2 ml of 0.05% fast blue rr reagent (4-benzoylamino-2,5-dimethoxybenzenediazonium chloride hemi (zinc chloride) salt) (sigma-aldrich, st. louis, mo, usa) diluted with methanol (1:5) and 10 ml of 10% k2co3 solution to each extract. absorbance measurements were carried out after 20 min at 480 nm using a unicam uv/vis uv2 spectrophotometer (ati unicam, cambridge, united kingdom). the content of alkylresorcinols was calculated using a standard curve prepared for olivetol and expressed in μg/g of grain dm. the repeatability for olivetol determination (expressed as coefficient of variation) was 2.5%. limit of quantification was 0.05 μg/g of sample dm, while linearity of calibration curve was confirmed in range of 1-15 μg/ml. the composition of alkylresorcinols was determined with the use of the gc-ms qp2010 plus, manufactured by shimadzu (kyoto, japan). for this purpose extract re-dissolved in methanol and centrifuged was transferred into vials, and evaporated under a nitrogen stream. to the residue pyridine and n,o-bis (trimethylsilyl) trifluoroacetamide (bstfa) with 1% trimethylchlorosilane (tmcs) (sigma-aldrich, st. louis, mo, usa) were added, and the mixture was heated at 60 °c for 60 min. after complete derivatization heptane was added, and the mixture was analysed using the gc-ms qp2010 plus manufactured by shimadzu (kyoto, japan). alkylresorcinols were separated on a zb5ms capillary column (30 m × 0.25 mm × 0.25 μm) (phenomenex, torrance, usa), and with helium as a carrier gas with a 0.9 ml/ min flow rate. the temperatures were as follows: injector – 230 °c, column – 70 °c increased to 230 °c at 15 °c/min, and to 310 °c at 3 °c/min, and maintained for 10 min, gc-ms interface – 240 °c, ion source – 220 °c. electron energy was set to 70 ev. the total ion current (tic) mode was used for quantification (100-600 m/z range). alkylresorcinols were identified using retention times and mass spectra of peaks. lipids the content of lipids was determined by soxhlet method with hexane according to polish standard pn-en iso 659:2010p. fatty acids the fatty acids methylation was carried out by heating the vials at 70 °c for 2 hours according to the method described by zader nowski and sosulski (1978). obtained methyl esters were analysed by gas chromatography with a gc-ms qp2010 plus (shimadzu, japan) system according to the parameters described by czaplicki et al. (2016). separation of fatty acids methyl esters was performed on a bpx70 (25 m × 0.22 mm × 0.25 μm) capillary column (sge analytical science, victoria, australia) with helium as a carrier gas at a flow rate of 0.9 ml/min. the column temperature was programmed as follows: a subsequent increase from 150 °c to 180 °c at the rate of 10 °c/min, to 185 °c at the rate of 1.5 °c/min, to 250 °c at the rate of 30 °c/min, and then 10 min hold. the interface temperature of gc-ms was set at 240 °c. the temperature of the ion source was 240 °c and the electron energy 70 ev. the total ion current (tic) mode was used in 50-500 m/z range. the results were expressed as percentages of total fatty acids. sterols the content of sterols was determined according to the gc/ms method described by roszkowska et al. (2015), with modifications. extraction of sterols from finely milled grain was carried out using acetone (10 ml/g of a sample). the collected extracts were evaporated to dryness at temperatures below 50 °c in a vacuum evaporator (büchi, type r-210; büchi labortechnik, flawil, switzerland). the residue was dissolved in ethanol, and 5α-cholestane (sigma-aldrich, st. louis, mo, usa) solution was added as an internal standard. the mixture was saponified by adding 0.5 ml 10 m koh solution in ethanol at a temperature of 70 oc for 30 min. the mixtures were transferred to separatory funnels containing deionized water, and non-saponifiable components were extracted twice with diethylether. the collected ether layers were washed twice with 0.5 m koh and four times with deionized water. the ether fractions were filtered through a filter with anhydrous sodium sulphate and evaporated in a vacuum evaporator at 45 °c. the dry extract was re-dissolved in hexane, centrifuged on a centrifuge (eppendorf, hamburg, germany), transferred into vials, and evaporated under a nitrogen stream. to the residue pyridine and n,o-bis (trimethylsilyl) trifluoroacetamide (bstfa) with 1% trimethylchlorosilane (tmcs) (sigma-aldrich, st. louis, mo, usa) were added and the conditions of derivatization, separation and identification were identical to those in alkylresorcinol analysis. sterols were identified using retention times and mass spectra of peaks, and their content was determined based on the concentration of the internal standard, and was expressed as μg of 5α-cholestane per g of grain dm. the repeatability for 5α-cholestane determination (expressed as coefficient of variation) was 2.5%. limit of quantification was 0.05 μg/g of sample dm. tocols the content of tocols was determined according to the method described by konopka et al. (2012). each sample of finely milled grain was extracted using n-hexane (10 ml/0.5 g of sample). the collected extracts were evaporated to dryness at temperatures below 50 °c in a vacuum evaporator (büchi, type r-210; büchi labortechnik) and then rediluted in n-hexane and subsequently centrifuged (10 min, 25,000 g) in a 5417r-type eppendorf centrifuge (eppendorf ag, hamburg, germany). chromatography analysis was performed by the rp-hplc technique according to method described by pheno menex (2015). the analysis was carried out using an agilent tech nologies 1200 rp-hplc apparatus, equipped with a fluorescence detector from the same manufacturer. separation was performed on a phenomenex kinetex f5 column (100 mm × 2.1 mm × 2.6 μm). a methanol (a)-water (b) gradient was used for elution of the tocols. the gradient was programmed: 0-1 min 85% of a, until 15 min increased to 100% of a and persisted until 17 min. then it decreased to 85% of a and was stable until 23 min. the flow rate was equal to 1.2 ml/min. the fluorescence detector was set at 296 nm of excitation and 330 nm of emission. tocols were quantified using standards of α-, β-, γ-, and δ-tocopherols (sigma-aldrich, st. louis, mo, usa). their content was calculated using external calibration curves, and was expressed as μg/g of grain dm. the repeatability for α-, β-, γ-, and δ-tocopherol determination (expressed as coefficient of variation) was 2.5%. limit of quantification was respectively 0.45, 0.4, 0.4 and 0.2 μg/g of sample dm, while linearity of calibration curve was confirmed in range of 0.02-16 μg/ml. carotenoids the content of carotenoids was determined according to the method described by czaplicki et al. (2016). to each 10 g of fine milled grain sample, placed in brown glass flask, were added 20 ml mixture of hexane, acetone, absolute ethanol and toluene (10:7:6:7, v/v/v/v), 5 ml of 40% koh in methanol, 1 ml of 0.1% bht in ethanol and β-apo-8’-carotenal (sigma-aldrich, st. louis, mo, usa) as an internal standard. the solutions were vigorously shaken in multi rotator rs-60 (biosan, riga, latvia) in the dark at room temperature for factors affecting infestation of wheat grain by granary weevil 67 tab. 1: parameters of development of s. granarius on grain of the studied spring wheat cultivars after 1 week and 8 weeks of storage. parameter ostka smolicka arabella izera tybalt kws torridon łagwa dk [%] 69.0±2.5b 71.2±2.5b 67.7±4.9b 69.1±8.0b 69.9±6.5b 82.6±2.4a ne [pieces] 322±12ab 330±14a 291±19b 291±32b 299±27b 349±13a gwl [%] 2.80±0.33a 1.71±0.47b 2.31±0.68ab 1.71±0.39b 1.85±0.54b 2.11±0.22ab mr [%] 3.75±3.54ab 0.63±1.77ab 1.25±2.31ab 3.75±3.54ab 4.38±3.20a 0.00±0.00b ap [pieces] 266±31a 160±16c 160±18c 189±16b 186±11b 189±16b tgwl [%] 45.8±7.5a 28.7±3.8b 28.2±3.9b 36.2±7.4b 34.7±9.2b 36.2±7.4b di 7.97±0.18a 7.25±0.14c 7.25±0.16c 7.48±0.12b 7.46±0.08b 7.48±0.12b resistant category mr/s mr mr mr mr mr abbreviations: dk – percentage of damaged kernels, ne – number of eggs, gwl – percentage grain weight loss, mp – parent mortality, ap – adult progeny, tgwl – percentage grain weight loss, di – dobie index, s – susceptible cultivar based on di, mr – moderately resistant cultivar based on di the results are presented as mean values ± standard deviation. different letters in the same raw indicate significant differences (p≤0.05). 16 h saponification. after saponification 10% na2so4 was added to each sample and extraction of carotenoids were performed fourfold with hexane. collected extracts were evaporated to dryness using vacuum evaporator (büchi̇ r-200 type). finally, the dry extracts were re-dissolved in methanol and dichloromethane mixture (45:55, v/v) and subsequently centrifuged (10 min, 25,000 g) in a 5417r-type eppendorf centrifuge. the chromatographic analysis was carried out using a 1200 series agilent technologies chromatograph equipped with a diode array detector (dad). separations were performed at 30 °c on ymc-c30 column (250 mm × 4.6 mm × 5 μm; komatsu, japan) and ymc-c30 pre-column (10 mm × 4.6 mm × 3 μm). a methanol (a) vs. methyl tert-butyl ether (mtbe) (b) gradient was used for elution. the gradient was programmed: 0-5 min 95% of a, until 25 min decreased to 72% of a and kept on decreasing until 33 min to 5% of a. then it increased to 95% of a and was stable until 60 min. absorbance was measured at 450 nm. carotenoids were identified based on the retention times of standards and their spectral properties, and their content was determined based on the concentration of the internal standard, and was expressed as μg of β-apo-8’carotenal equivalent per g of grain dm. the repeatability for β-apo8’-carotenal determination (expressed as coefficient of variation) was 2.5%. limit of quantification was 0.05 μg/g of sample dm, while line arity of calibration curve was confirmed in range of 1-150 μg/ml. statistical analysis all chemical analyses were done in triplicate and analysed using statistica 12.5 pl software (statsoft, kraków, poland) at p ≤ 0.05 significance level. the differences of grain samples composition were determined using anova with duncan’s test. the relationships between biological susceptibility to pest infestation and chemical composition of used grain were examined using principle component analysis (pca). results and discussion wheat cultivar differentiation based on susceptibility to granary weevil infestation in the first stage of the study, selected indices related to the intensity of growth and feeding of s. granarius for six spring wheat cultivars were determined (tab. 1). host plant attractiveness for insects to laying eggs and feeding (antixenosis mechanism) was assayed 7 days after grain infestation. it was found that the percentage of damaged kernels ranged from 67.7% (izera cv.) to 82.6% (łagwa cv.), while the percentage of grain weight loss was from 1.71% to 2.80%. at the same time, the number of folded eggs varied from 291 (tybalt and izera cvs.) to 349 (łagwa cv.). the mortality of the beetles was relatively low and ranged from 0 (łagwa cv.) to 4.38% (kws toridon cv.). considering the results of all these infestation indices, łagwa cv. was the most attractive for s. granarius to laying eggs and feeding (the low visible antixenosis mechanism). after 8 weeks of infestation, the antibiosis mechanism was determined, which refers to adverse biologically consequences on the life cycle of pests as a result of feeding activity on the resistant host plant. symptoms of the antibiosis mechanism can include: larval mortality, increased mortality of pupae, failure of the adult out of the pupae, low adult fertility, short insect life cycle and other forms of abnormality (sulistyo and inayati, 2016). for the used wheat cultivars, the contrast antibiosis effect was stated for arabella and izera cvs. (the highest effect) and ostka smolicka cv. (the lowest effect) with the values of an adult progeny 160 and 266, respectively. it was also confirmed by a final percentage grain weight loss close to 28% and 45.8%, respectively. wheat cultivar differentiation based on the profile of phenolic and lipophilic compounds in grain phenolic compounds act as natural plant pesticides and protect plants against external aggression and predators (drewnowski and gomez-carneros, 2000). in cereal grain, the main representatives of these compounds are phenolic acids and alkylresorcinols (konopka et al., 2017). most of the phenolic acids are deposited in pericarp cell walls and cells of the aleurone layer (hansen et al., 2002; lopez-castillo et al., 2018), while alkylresorcinols (ar) as a phenolic lipids (ross et al., 2003) are mainly (99% of them) deposited in an intermediate layer of the caryopsis, including the hyaline layer, testa and inner pericarp surrounding the cereal kernel (landberg et al., 2008). phenolic compounds content in wheat grain is highly affected by genetic factors (species/cultivar) and by environmental conditions (mainly biotic stress). the phenolic compound contents and compositions of the used wheat cultivars are presented in tab. 2. grain contained from 332 μg/g (ostka smolicka cv.) to 482 μg/g (kws torridon cv.) of total phenolic acids (tpa). among them, ferulic acid prevailed with a share from 79.6% (ostka smolicka cv.) to 86.0% (tybalt cv.). the second in quantity was sinapic acid and its share varied from 8.9% (kws torridon cv.) to 15.6% (ostka smolicka cv.). other identified phenolic acids (p-coumaric, protocatechuic, vanillic, p-oh benzoic and caffeic) were less frequent. only up to 1.8% of these acids existed in an unbound state. the highest content of unbound phenolic acids was determined in grain of the ostka smolicka cv. (5.89 μg/g). in pa ra m et er s at 1 w ee k pa ra m et er s at 8 w ee ks 68 b. kordan, m. skrajda-brdak, m. tańska, i. konopka, r. cabaj, d. załuski tab. 2: quantification of phenolic and lipophilic compounds in grain of the studied spring wheat cultivars. parameter ostka smolicka arabella izera tybalt kws torridon łagwa total phenolic acids 332±16a 441±13c 342±10a 472±4d 482±17d 404±2b (mg/g of dm) ferulic 265±14a 373±10d 284±8b 406±3e 414±11e 341±0c sinapic 51.8±1.4b 49.2±2.4ab 43.4±2.2ab 43.3±6.5ab 42.7±4.5ab 42.3±1.7a p-coumaric 5.92±0.22a 5.82±0.21a 5.36±0.23a 7.69±0.09b 8.56±0.62c 8.23±0.10bc protocatechuic 2.13±0.01a 5.09±0.32c 3.26±0.02b 5.81±0.22d 5.53±0.24cd 5.20±0.30c vanillic 2.85±0.06ab 2.64±0.17a 2.37±0.13a 3.77±0.20c 4.61±0.31d 3.31±0.20bc p-oh benzoic 2.42±0.07a 3.21±0.16c 2.47±0.00a 2.91±0.17bc 3.61±0.22d 2.77±0.17ab caffeic 2.04±0.07bc 1.96±0.19b 0.71±0.07a 2.35±0.16c 2.32±0.16c 1.80±0.10b free phenolic acids 5.89±0.24b 3.68±0.10a 3.46±0.13a 5.76±0.15b 5.50±0.17b 5.82±0.02b (mg/g of dm) total alkylresorcinols 291±ab 261±8a 344±8bc 356±4c 348±18bc 269a (mg/g dm) ar 17:0 14.0abc 13.0±0.5ab 13.8±0.1ab 16.3±0.4bc 17.5±1.1c 11.1a ar 19:0 105ab 94.0±2.1ab 107±2bc 123±2c 124±5c 88.7a ar 21:0 137ab 129±3a 178±2c 179±4c 168±7bc 139ab ar 23:0 22.8ab 15.4±0.9a 27.7±2.0b 23.6±1.4ab 22.2±0.7ab 19.2a ar 25:0 7.9a 5.8±0.13a 13.8±0.85c 8.96±0.43ab 12.3±0.71bc 8.6a others 3.97ab 3.21±0.11ab 4.32±0.59b 4.70±0.60b 3.97±0.20ab 2.36a total lipids (% of dm) 2.04±0.20bc 1.78±0.01ab 1.47±0.04a 2.37±0.16d 2.15±0.13cd 2.39±0.12d fatty acids composition (%): c16:0 17.97±0.13d 17.68±0.19cd 17.64±0.11cd 16.96±0.15ab 16.75±0.06a 17.25±0.18bc c18:0 0.81±0.01ab 0.94±0.00b 0.83±0.13ab 0.72±0.02a 0.86±0.02ab 0.85±0.06ab c18:1 13.58±0.02a 15.77±0.01de 15.42±0.04c 15.65±0.06cd 15.03±0.25b 16.03±0.02e c18:2 63.17±0.06c 61.82±0.00a 62.03±0.04ab 63.21±0.33c 63.79±0.33d 62.49±0.09b c18:3 3.90±0.13d 3.19±0.01b 3.60±0.04c 3.00±0.10b 3.03±0.08b 2.72±0.06a c20:1 0.58±0.05abc 0.61±0.00bc 0.49±0.04a 0.47±0.04a 0.56±0.04ab 0.68±0.06c total sterols 675±7b 522±20a 500±7a 675±30b 638±30b 729±28b (mg/g of dm) campesterol 191±2c 132±4ab 115±2a 132±5ab 134±1.3ab 157±8bc β-sitosterol 455±17b 367±16a 358±5a 509±20bc 467±21bc 538±13c stigmasterol 28.7±0.7a 23.3±0.6a 26.0±0.4a 34.4±2a 37.3±0.2a 33.9±0.5a total tocols 49.9±1.2a 45.3±1.3a 49.2±4.1a 50.2±3.1a 45.7±0.6a 44.0±4.5a (mg/g of dm) α-t 19.6±0.4b 16.8±0.3a 17.5±0.9ab 17.2±0.2ab 15.9±0.1a 15.7±2.3a α-t3 8.34±0.5b 6.97±0.68ab 7.37±0.45ab 6.93±0.72ab 7.19±0.22ab 6.03±0.52a β-t 5.15±0.21bc 4.85±0.07b 5.54±0.17c 4.75±0.42b 3.99±0.04a 4.70±0.42b β-t3 15.9±0.2a 15.7±0.8a 17.8±1.25ab 20.2±1.4b 17.6±0.3ab 16.6±1.2ab δ-t 0.95±0.0a 0.95±0.02a 0.99±0.05ab 1.04±0.03b 0.98±0.01ab 0.96±0.04ab total carotenoids 3.51±0.18d 2.22±0.13bc 2.80±0.11cd 1.87±0.15b 1.24±0.10a 2.52±0.05c (mg/g of dm) lutein 2.78±0.13e 1.56±0.11bc 2.15±0.08d 1.27±0.09b 0.78±0.06a 1.88±0.01c zeaxanthin 0.53±0.03b 0.33±0.01a 0.48±0.02b 0.38±0.04a 0.28±0.02a 0.49±0.04b β-carotene 0.09±0.01b 0.20±0.00c 0.05±0.00a 0.12±0.01b 0.09±0.01b 0.04±0.00a 9-cis β-carotene 0.02±0.00a 0.08±0.00b 0.01±0.00a 0.04±0.00a 0.04±0.00a 0.01±0.00a others 0.11±0.01a 0.06±0.00a 0.10±0.00a 0.06±0.01a 0.04±0.00a 0.10±0.01a the results are presented as mean values ± standard deviation. different letters in the same raw indicate significant differences (p≤0.05). factors affecting infestation of wheat grain by granary weevil 69 contrast, this fraction was the lowest (3.46-3.68 μg/g) in grain of arabella and izera cvs. the content of alkylresorcinols varied from 261 (arabella cv.) to 356 μg/g (tybalt cv.). among them, homologues with an alkyl chain length of ar19:0 and ar21:0 prevailed, reaching 34% and 50% on average, respectively. the determined contents of phenolic acids and alkylresorcinols were relatively low, but still within the ranges given by other authors (shewry and ward, 2012; ziegler et al., 2015; shewry and hey, 2015). the composition of grain lipids in tested samples is presented in tab. 2. lipids usually account for approx. 2-3% of wheat grain mass (konopka et al., 2017). most of them are triacylglycerols deposited in endosperm and germ in the form of storage oil bodies. they are accompanied by sterols, tocols and carotenoids with various protectant activities against plant stresses during growth. usually, the overall content of low molecular lipophilic compounds does not exceed 1,000 μg/g of grain (konopka et al. 2017). the content of lipids varied from 1.47% in izera cv. to 2.39% in łagwa cv. the used wheat cultivars were only slightly differentiated by the shares of fatty acids in grain, with three main compounds: c18:2 (ca. 62%), c16:0 (ca. 17%) and c18:1 (ca. 15%). more variable was the content of sterols, which ranged from 500 μg/g (izera cv.) to 729 μg/g (łagwa cv.). only three homologues were found among this group: β-sitosterol, campesterol and stigmasterol, with average shares of 72%, 23% and 5%, respectively. tocols content was the least variable feature among the tested cultivars (with a coefficient of variation 5.7%) and varied from 44.0 μg/g (łagwa cv.) to 50.2 μg/g (tybalt cv.). in all samples, five homologues were determined in order of participation: β-tocotrienol and α-tocopherol (ca. 36%), α-tocotrienol (ca. 15%), β-tocopherol (ca. 10%) and δ-tocopherol (ca. 2%). the smallest part of lipophilic compounds constituted carote noids, with an average amount of 2.36 μg/g and high variation from 1.24 μg/g (kws torridon cv.) to 3.51 μg/g (ostka smolicka cv.). the main homologues of this group were lutein (ca. 72%), zeaxanthin (ca. 18%), and β-carotene (ca. 5%). shares of minor compounds such as β-carotene and 9-cis-β-carotene were highly variable between the used cultivars. the determined profile of low molecular lipophilic compounds was within the limits given by other authors since bread wheat grain usually contains from 225 μg/g to 959 μg/g of sterols, from 23.3 μg/g to 79.7 μg/g of tocols and from 1.40 μg/g to 4.90 μg/g of carotenoids (shewry and ward, 2012; konopka et al., 2017). correlations between biological indices of wheat grain susceptibility to granary weevil infestation and phenolic and lipophilic grain compounds in summary, a total of 6 biological indices and 37 chemical compounds were analysed. the correlations between biological and che mical properties of the used wheat cultivars are presented in tab. 3. it has been found, that the percentage of damaged kernels after one-week of s. granarius infestation was only positively correlated (r=0.82) with the share of c20:1 fatty acid, number of laid eggs with content of ar 19:0 (r=-0.86) and ar 21:0 (r=-0.90) and with the share of c20:1 fatty acid (r=0.96), while grain weight loss negatively (r in range 0.90 0.94) with total phenolic acids and some individuals (ferulic acid and protocatechuic acid) and positively with total carotenoids and lutein and zeaxanthin content (r in range 0.83-0.88). parent mortality after one-week infestation was correlated with ar 17:0 (r=0.88) and ar 19:0 (r=0.87) and the share of c18:2 fatty acid (r=0.88). in contrast, adult progeny after eight-week of s. granarius infestation was mostly correlated with campesterol content (r=0.93) and with the share of c18:1 fatty acid (r=-0.86). in the case of impact of composition of low molecular phytochemicals on the final grain weight loss (after 8-weeks) the significant relationships were determined for the content of free phenolic acids (r=0.83) and campesterol (r=0.91). as mentioned in the introduction part, the susceptibility of a cultivar to infestation by pests is a result of the cumulative effect of kernel physical features and various chemical compounds. the data about the impact of grain phenolic compounds and grain lipids on resistance or attractiveness of wheat grain to s. granarius infestation are relatively scarce. it was previously found, that the grain lipid fraction is attractive for the egg-laying process. triacylglycerols were recognized as strong attractants of the granary weevil (nawrot, 1983). cuticular lipids were determined as crucial for the feeding and oviposition behaviour of this beetle (niewiada et al., 2005). in gene ral, beetles produced 64-95% less dust and laid 7-16% fewer eggs in kernels from which cuticular lipids had been removed. these cuticular lipids consisted mainly of alkanes: n-heptacosane (c27), n-nonacosane (c29) and n-hentriacontane (c31), wax esters, triacyl glycerols, c16:0 and c18:1 free fatty acids, triterpene alcohol amyrine, and sterols (niewiada et al., 2005). this implies that these compounds may have a major role in food selection and the search for an oviposition site prior to grain infestation (nawrot et al., 2010). fungal volatile metabolites such as 1-octanol and 2-methyl1-butanol can also increase egg lying on wheat kernels (niewiada et al., 2005). in the case of infestation of rice grain by s. zeamais maeshima et al. (1985) concluded that oviposition was stimulated by a synergistic action – a mixture of ferulates, diglycerides and free sterols. in the current study, a strong positive correlation was found between indices of long-term infestation and campesterol content. insects cannot synthesize sterol structures and need cholesterol or phytosterols as precursors from food for the biosynthesis of ecdy steroids, which are insect moulting and sex hormones (singh and kaur, 2018). campesterol is preferentially deposited in some insect tissues (behmer and nes, 2003), but specific data for s. granarius are not available. nwosu (2016) discovered that mechanisms of maize resistance to s. zeamais were antibiosis, antixenosis and preference, as the increases in maize varietal crude fibre, phenolic acids and trypsin inhibitor significantly increased the mortality of the maize weevil adults and significantly reduced their survival rate and oviposition by the adult females. at the same time, the percent of grain damage, percent of weight loss and weight of grain flour was also reduced. the significant increase of phenolic acid and trypsin inhibitor in grains resulted in low infestation (nwosu, 2016). these results were in accordance with earlier findings by serratos et al. (1993), that there was a significant negative correlation between phenolic content (i.e., hydroxycinnamic acid accumulation in maize grain) and susceptibility of the grain to weevil infestation. likewise, arnason et al. (1994) found that the variation in resistance of mexican landraces of maize to maize weevil was negatively correlated to phenolic and protein content of the variety tested. a detailed analysis of the phenolics revealed the presence of diferulate which may contribute to the mechanical resistance of the kernel by the cross-linking of cell wall hemicelluloses (arnason et al., 1994). apart from the impact on the mechanical resistance of the kernel, phenolic compounds are also able to precipitate of proteins, and this effect can affect pest digestive enzymes. our study confirmed the only protectant impact of grain phenolic acids on the dietary habit of s. granarius manifesting as reduced grain weight loss after one-week infestation. in contrast, these compounds did not affect other indices of infestation. it can be explained by relatively low concentration of these compounds in used grain since wheat grain samples can contain 2-3-fold highest content of phenolic acids (shewry and ward, 2012; ziegler et al., 2015). positive correlation between free phenolic acids and loss of grain mass after an eight-week infestation indicates the attractiveness of grain with slightly higher degradation of cell walls, which is hypothetically indicated by the increased content of free phenolic compounds. 70 b. kordan, m. skrajda-brdak, m. tańska, i. konopka, r. cabaj, d. załuski conclusions this study showed that the susceptibility of six polish spring wheat cultivar grains to s. granarius infestation varied significantly. this phenomenon cannot be explained by variation in grain protein and hardness since the used cultivars were similar in these factors and had similar overall quality. however, these cultivars differed in the content and composition of specific phenolic and lipophilic phytochemicals. some of these compounds may act as pest attractants (potential candidate is c20:1 fatty acid) or favour the growth during tab. 3: correlations between parameters of development of s. granarius and chemical composition. parameter at 1 week at 8 weeks dk ne gwl mp ap tgwl total phenolic acids 0.06 -0.17 -0.90 * 0.26 -0.41 -0.26 ferulic acid 0.07 -0.18 -0.91 * 0.24 -0.45 -0.29 sinapic acid -0.31 0.29 0.48 0.09 0.57 0.41 p-cumaric acid 0.49 0.08 -0.44 0.27 -0.02 0.20 protocatechuic acid 0.33 -0.01 -0.94 * -0.05 -0.58 -0.40 vanilic acid 0.08 -0.23 -0.44 0.62 0.05 0.22 p-hydroxybenzoic acid 0.01 -0.08 -0.76 0.24 -0.40 -0.31 caffeic acid 0.08 0.13 -0.36 0.55 0.37 0.49 free phenolic acids 0.34 0.19 0.17 0.50 0.68 0.83 * total alkylresorcinols -0.42 -0.80 0.07 0.67 0.11 0.17 ar 17:0 -0.67 -0.80 -0.30 0.88 * 0.04 0.08 ar 19:0 -0.65 -0.86 * -0.23 0.87 * 0.06 0.11 ar 21:0 -0.47 -0.90 * -0.23 0.43 -0.31 -0.26 ar 23:0 -0.49 -0.74 0.41 0.39 0.11 0.08 ar 25:0 0.63 0.64 0.69 -0.19 0.64 0.67 total lipids 0.52 0.28 -0.23 0.28 0.34 0.55 fatty acid c16:0 -0.15 0.33 0.68 -0.35 0.34 0.15 fatty acid c18:0 0.23 0.51 -0.14 -0.53 -0.35 -0.45 fatty acid c18:1 0.47 0.12 -0.76 -0.62 -0.86 * -0.74 fatty acid c18:2 -0.17 -0.33 0.04 0.88 * 0.52 0.62 fatty acid c18:3 -0.66 -0.26 0.76 0.26 0.50 0.30 fatty acid c20:1 0.82 * 0.96 * 0.10 -0.53 0.13 0.17 total sterols 0.55 0.36 0.14 0.27 0.58 0.75 campesterol 0.25 0.54 0.68 0.20 0.93 * 0.91 * ß-sitosterol 0.58 0.26 -0.06 0.23 0.38 0.58 stigmasterol 0.27 -0.15 -0.22 0.51 0.17 0.36 total tocols -0.73 -0.67 0.40 0.54 0.40 0.33 α-t -0.56 -0.17 0.73 0.36 0.71 0.56 α-t3 -0.76 -0.37 0.64 0.59 0.62 0.45 ß-t -0.24 -0.06 0.55 -0.39 0.06 -0.09 ß-t3 -0.30 -0.74 -0.42 0.42 -0.25 -0.12 δ-t -0.34 -0.74 -0.43 0.40 -0.26 -0.14 total carotenoids 0.01 0.33 0.86 * -0.27 0.55 0.41 lutein 0.01 0.31 0.88 * -0.25 0.56 0.42 zeaxantin 0.23 0.30 0.83 * -0.28 0.51 0.45 ß-caroten -0.32 0.04 -0.51 0.03 -0.19 -0.24 9-cis ß-caroten -0.24 0.03 -0.66 0.02 -0.33 -0.35 * correlation coefficient statistically significant (p≤0.05). abbreviations: dk – percentage of damaged kernels, ne – number of eggs, gwl – percentage grain weight loss, mp – parent mortality, ap – adult progeny, tgwl – total percentage grain weight loss. factors affecting infestation of wheat grain by granary weevil 71 long-term infestation (campesterol). the final results of s. granarius infestation may have also been affected by phenolic acids, which are responsible for compactness of the pericarpand aleuronic-layer cell wall structures and can affect dietary habit of this beetle. further studies are needed using grains from highly differentiated cultivars in terms of the cited compounds. references arnason, j.t., baum, b., gale, lambert, j.d.h., bergvinson, d., philogene, b.j.r., serratos, j.a., mihm, j., jewell, d.c., 1994: variation in resistance of mexican landraces of maize to maize weevil sitophilus zeamais, in relation to taxonomic and bichemical parameters. euphytica 74, 227-236. doi: 10.1007/bf00040405 behmer, s.t., nes, w.d., 2003: insect sterol nutrition and physiology: a global overview. adv. insect physiol. 31, 1-72. doi: 10.1016/s0065-2806(03)31001-x bojarska, j.e., zadernowski, r., czaplicki, s., 2011: ellagic acid content in fruits of selected strawberry cultivars. pol. j. natur. sci. 26, 171-177. campbell, a., sinha, r.n., 1976: damage oh wheat by feeding of some stored beetle. j. econ. entomol. 69, 11-3. coboru, 2018: polish national list of agricultural plants, issn 1231-8299, www.coboru.pl/publikacje_coboru/listy_odmian/lo_ rolnicze_2018.pdf czaplicki, s., tańska, m., konopka, i., 2016: sea-buckthorn oil in vegetable oils stabilisation. ital. j. food sci. 28, 412-25. doi: 10.14674/1120-1770/ijfs.v252 dinuta, a., bunescu, h., proorocu, m., bodis, i., oros, s., 2008: researches concerning the external morphology of granary weevil’s adult (sitophilus granarius l.), a major pest of the stored cereals. bull. univ. agric. sci. agriculture 65, 72-7. dobie, p., 1974: the laboratory assessment of the inherent susceptibili ty of maize varieties to post-harvest infestation by sitophilus zeamais motshh. 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sulistyo, a., inayati, a., 2016: mechanisms of antixenosis, antibiosis, and tolerance of fourteen soybean genotypes in response to whiteflies (bemisia tabaci). biodiversitas 17, 447-453. doi: 10.13057/biodiv/d170207 zadernowski, r., sosulski, f., 1978: composition of total lipids in rapeseed. j. am. oil chem. soc. 55, 870-2. doi: 10.1007/bf02671409 ziegler, j.u., steingass, c.b., longin, c.f.h., würschum, t., carle, r., schweiggert r.m., 2015: alkylresorcinol composition allows the differentiation of triticum spp. having different degrees of ploidy. j. cereal sci. 65, 244-251. doi: 10.1016/j.jcs.2015.07.013 address of the authors: bożena kordan, robert cabaj, department of entomology, phytopathology and molecular diagnostics, faculty of environmental management and agriculture, university of warmia and mazury in olsztyn, poland marta skrajda-brdak, małgorzata tańska, iwona konopka, chair of plant food chemistry and processing, faculty of food sciences, university of warmia and mazury in olsztyn, poland dariusz załuski, department of plant breeding and seed production, faculty of environmental management and agriculture, university of warmia and mazury in olsztyn, poland © the author(s) 2019. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). journal of applied botany and food quality 91, 96 102 (2018), doi:10.5073/jabfq.2018.091.013 1norwegian institute of bioeconomy research, ås, norway 2arctic university of norway, uit, tromsø, norway seasonal and yearly variation of total polyphenols, total anthocyanins and ellagic acid in different clones of cloudberry (rubus chamaemorus l.) anne linn hykkerud1, eivind uleberg1, espen hansen2, marieke vervoort1, jørgen mølmann1, inger martinussen1 (submitted: june 30, 2017; accepted: march 1, 2018) * corresponding author summary cloudberry (rubus chamaemorus l.) is a wild perennial shrub growing on peatland with a circumpolar distribution. the combined berries have a high polyphenol content comprised primarily of ellagitannins. a few commercial cultivars are available, and prebreeding trials on clonal material from different geographical origins are in progress. the objective of this study was to investigate how the content of polyphenols of four different cloudberry cultivars were affected by harvesting time and climatic variations during a 3-yearperiod. plants were grown outside in plots and berries were harvested when mature. berries were analyzed for total polyphenols and total anthocyanins by spectrophotometer. total ellagic acid was identified and quantified using hplc-ms after hydrolysis of the extracts. results showed that all measured parameters; total anthocyanins, total polyphenols and ellagic acid are strongly influenced by the genetic background. although low anthocyanin contents were present in all genotypes, they were highly affected by climatic conditions, being highest at low temperatures. however, the content of ellagic acid was less affected by environmental conditions and showed little response to changing temperatures. in conclusion, ellagitannin content was the most dominating polyphenol group observed in this study and was affected by genetics and is therefore a good breeding criterion for increased health benefit of cloudberry. keywords: climatic effects, cloudberry, ellagic acid, gene × environment interaction, polyphenols, rubus chamaemorus abbreviations ea ellagic acid, dw dryweight, ga gallic acid, gdd growing degree days, ta total anthocyanins, tp total polyphenols introduction cloudberry (rubus chamaemorus l.) is a dioecious perennial rhizomatous plant, native to the subarctic regions. berries have a distinct aromatic flavor and are highly valued in the nordic countries (thiem, 2003). in addition, the market and interest in wild berries is increasing, especially in the east asian region (paassilta et al., 2009; turtiainen and nuutinen, 2012). although most of the commercial harvesting comes from wild stands, four commercial varie ties have been released (rapp and martinussen, 2002). additional ly, there is ongoing testing of new cultivars (uleberg et al., 2011). previous breeding strategies used the number of pistils, flowers and shoots as selection criteria (rapp, 1989). berries are known to be rich in a variety of bioactive compounds including phytochemicals related to human health benefits (duthie, 2007). cloudberries contain a range of bioactive compounds and are especially rich in polyphenols (martinussen et al., 2010; jaakkola et al., 2012). the nutritional quality has become a priority for breeding and biotechnological strategies, in order to control or to increase the content of specific health-related compounds in fruits and berries (mazzoni et al., 2015). polyphenols have unique physical, chemical, and biological properties (manach, 2004) and polyphenol content has been shown to be highly correlated with antioxidant capacity (dobson et al., 2012). total polyphenols in cloudberry has been observed to be affected by genotype (uleberg et al., 2011). ellagitannin occurrence in common food is limited to a few fruit and nut species, and cloudberry is among the fruits with the highest reported levels (koponen et al., 2007) where they are the dominating polyphenol group (häkkinen, 1999; koponen et al., 2007; kylli, 2011). ellagitannins contain ester bonds, which upon hydrolysis give ellagic acid. the gut derivate of ellagic acid have been linked to inhibition of prostate cancer cell growth (seeram et al., 2007) and have vascular health benefits (larrosa et al., 2010). in cloudberry, only a minor part of the total ellagic acid content is found as free ellagic acid. most ellagitannins are bound and are released upon acid hydrolysis (kylli, 2011). it has previously been reported that different growing locations and seasons affected the ellagic acid content in cloudberry (li et al., 2016). anthocyanins, another group of polyphenols, are sparse in cloud berries, strongly correlated with berry color, and have higher levels at low temperatures (martinussen et al., 2010). climatic conditions of high latitude areas, where cloudberries grow, differ significantly between years, as well as within season, affecting the phenology and quality of the berries. light conditions vary greatly during the growing season, with midnight sun until late july and a minimum of 12 h daylight until the end of september (bliss, 1962). effects of climatic conditions on various quality parameters, including the content of bioactive compounds, have been evaluated in previous cloudberry studies (martinussen et al., 2010; jaakkola et al., 2012; li et al., 2016), as well as other berries including bilberry (uleberg et al., 2012; zoratti et al., 2014), blueberry (connor et al., 2002) and raspberry (mazur et al., 2014). however, phytochemical content varies greatly among genotypes (antonnen et al., 2005; scalzo et al., 2005; uleberg et al., 2011). by investigating the genetic and environmental effects and their interactions on the polyphenol levels, we will have a better insight into the regulation and variety consistency. the objective of this paper is to investigate how the content of total phenols, total anthocyanins and levels of ellagic acid of four different clones of cloudberry are affected through season and between years. materials and methods plant material in 2012, 2013 and 2014, cloudberries from four different clones (‘fjordgull’, ‘fjellgull’, ‘102’ and ‘306’) were harvested at holt in tromsø 69°39’n 18°57’e. the different clones were grown in two open outdoor benches in separated squares (2 × 2 m), and every clone was represented at random positions in the bench. the two replicated benches consisted of 6 plots, including the female clones ‘fjordgull’, seasonal and yearly variation of secondary metabolites in cloudberry 97 ‘fjellgull’, ‘102’ and ‘306’, while the last two plots in each bench consisted of a mixture of three different male clones (‘apollen’, ‘apolto’ and ‘h510’), to ensure sufficient pollination (see tab. 1 for the geographical origin of the clones). the clones ‘102’ and ‘306’ were included in this study based on their production performances in earlier experiments (uleberg et al., 2011; trost et al., 2013), in addition to the released cultivars ‘fjellgull’ and ‘fjordgull’. the plots with male plants were located between the plots with female plants so every plot with a female clone was next to one of the plots with male plants. data for flowering and harvest were registered per each 2 × 2 m plot. individual berries were harvested when ripen, weighed and frozen at storage -80 °c and freeze dried before extraction. depending on the year, the length of the harvest season was approximately one month (tab. 2). for each year, an early, middle and late harvest date were selected and berries from these dates were analyzed (tab. 2). the selection criteria for the selected harvesting dates was the earliest and latest date where all clones had mature berries, the middle time was selected as close as possible to the mean of these two dates. experimental conditions weather data information was downloaded from the weather station at nibio holt, tromsø collected data included average daily temperature as well as daily precipitation. from these data, monthly and total precipitation and monthly and seasonal mean temperatures were extracted. daily, monthly and aggregated growing day degrees above 5 °c were calculated and used for analysis of weather impacts on cloudberry quality (tab. 3). the berry samples consisted of the pooled berries sampled from a cultivar a given day, never less than two berries. the samples were freeze-dried for minimum 48 h. extraction freeze-dried powder (100 mg) from ripe harvested cloudberries were extracted according to trošt et al. (2013) with some modifications in 5 ml 80% methanol and vortexed in 1 min and then centrifuged. the supernatant was decanted, and then added 5 ml 80% methanol and vortexed in 1 min before centrifugation. total polyphenols total phenolic content was determined by the folin-ciocalteu method (singelton and rossi, 1965) with minor modifications as described by uleberg et al. (2011), in which gallic acid was used as a calibration standard in spectrophotometric measurements. samples (20 μl) was combined with 1.58 ml of dh2o and 100 μl folin-ciocalteu reagent. the mixture was incubated for 5 min at room temperature before adding 300 μl na2co3 (saturated sodium) carbonate solution. after a 2 h incubation at room temperature, the absorbance of the mixture was measured at 765 nm in a spectrophotometer (smartspec plus, bio-rad, hercules, usa). a gallic acid (ga) reference absorbance curve was subsequently used to calculate the polyphenol content in mg of ga per gram dry weight (dw) sample. hydrolysis methanolic extracts (2 ml) were transferred to glass tubes and subject to acid hydrolysis by addition of methanol and 37% hcl to 2.5 m at 85 °c for 6 h. hydrolyzed extracts (1 ml) were diluted with methanol (2 ml), before uhplc-injections (5 μl). ellagic acid ellagic acid was identified and quantified using uhplc-pda-hrms on a waters acquity uplc (milford, ma, usa), waters 2996 uv-detector and waters lct premiere time-of-flight ms with electab. 1: geographical origin of the different cloudberry (rubus chamaemorus l.) clones and varieties used in the study. clone origin (county) sex ‘102’ 58°30‘n, aust-agder female ‘306’ 66°30‘n, nordland female ‘fjordgull’ 69°06‘n, andøya, nordland female ‘fjellgull’ 70°24‘n, ifjordfjellet, finnmark female ‘apollen’ 69°06‘n, andøya, nordland male ‘apolto’ 69°06‘n, andøya, nordland male ‘h510’ 69°06‘n, andøya, nordland male tab. 2: harvesting season during 2012-2014. first flower last flower flower season first harvest last harvest harvest period early middle late 2012 15.06 28.06 13 01.08 06.09 36 15.08 20.08 28.08 2013 28.05 13.06 16 08.07 16.08 39 15.07 29.07 06.08 2014 04.06 03.07 29 23.07 14.08 22 30.07 04.08 11.08 tab. 3: mean monthly and growing season: air temperature, precipitation and growing degree-days (gdd) in tromsø during 2012-2014. month mean temperature (°c) precipitation (mm) growing degree days (gdd) 2012 2013 2014 2012 2013 2014 2012 2013 2014 may 4.5 8.7 5.1 107.4 12.0 42.4 30.2 127.1 41.1 june 9.1 11.7 9.1 35.6 52.1 38.4 115.2 201.4 125.0 july 10.9 11.9 15.2 90.1 130.3 30.0 184.0 214.6 314.9 august 9.9 11.9 11.8 29.1 95.0 71.0 151.7 214.5 209.5 september 7.6 10.2 7.9 83.9 40.0 97.4 84.3 157.0 96.8 total growing 8.4 10.9 9.8 346.1 329.4 279.2 565.4 914.6 787.3 98 a.l. hykkerud, e. uleberg, e. hansen, m. vervoort, j. mølmann, i. martinussen trospray ionisation. masslynx version 4.1 (waters) was used for instrument control and data processing. the extracts were injected on a waters acquity ethylene bridged hybrid (beh) c18 column (2.1 × 100 mm, 1.7 μm) and ellagic acid was separated using a gradient of 5-30% acetonitrile in water (both containing 0.1% formic acid) over 6 min at a flow rate of 0.5 ml/min. the column was kept at 40 °c, and 5 μl of the extracts was injected. the samples were ionized with negative electrospray (esi-), and data from 150 to 1500 da were acquired at a scan time of 0.25 s. capillary and cone voltages were set to -2.8 kv and -50 v, respectively, whereas source and desolvation temperatures were set to 120 °c and 350 °c, respectively. nitrogen was used as desolvation gas at 650 l/min. the ms was tuned to a resolution of 10,000 (fwhm) and leucine-enkapheline was infused through the reference probe for internal calibration during data acquisition. for quantification of ellagic acid, uvabsorption at 258.2 nm was used. a calibration curve for ellagic acid was made using a commercial standard (sigma-aldrich, darmstadt, germany). ellagic acid eluted at 3.55 min and was identified as a deprotonated species ([m-h]m/z 300.9983 calculated 300.9990), a deprotonated dimer ([2m-h]m/z 603.0041 calculated 603.0053) and a deproto nated trimer ([3m-h]m/z 905.0095 calculated 905.0116). the formation of deprotonated multimers of ellagic acid in the ion source of the ms is concentration dependent; it is therefore difficult to quantify the compounds using ms data. ellagic acid absorbs very well in the uv-spectrum, and as no other compounds eluting close to ellagic acid (as detected with uv and esi-), we quantified ellagic acid by integrating the peak at 258.2 nm (± 0.5 nm). total anthocyanins total anthocyanins were estimated by a ph differential as described by (giusti and wrolstad, 2001; lee et al., 2005). each sample was diluted in a 0.025 m potassium chloride buffer, ph 1.0 and a 0.4 m sodium acetate buffer, ph 4.5. of these mixtures the λvis-max (510 nm) and 700 nm (haze) absorbance was measured. the absorbance of the diluted sample then equals (a510 nm – a700 nm) ph 1.0 – (a510 nm – a700 nm) ph 4.5 and the monomeric anthocyanin pigment in mg/l was calculated using (a × mw × df × 1000)/ (ε × 1) with mw = 449.2 and ε = 26,900 defining the major pigment content as cyanidin-3-glucoside. the anthocyanin content for all samples was expressed in mg cyanidin-3-glucoside per gram dw. statistical analyses data for total polyphenols, total anthocyanins and ellagic acid were analyzed by the glm procedure of r (r-project) in a model that included the main effects clone/genotype, harvesting time and year and their interactions. pearson correlation analysis (minitab 17) was used to indicate potential impact of climate factors (temperature and precipitation) on berry quality. furthermore, pearson correlation analysis was carried out to show relationships between the quality parameters total polyphenols, total anthocyanins and ellagic acid. results plant development weather data for the growing seasons of 2012, 2013 and 2014 are summarized (tab. 3). heat unit accumulation evident with growing degree-days (gdd) is presented (fig. 1). results illustrated that the time for early-, middleand late-harvest varied considerably between the three years. the coldest summer, 2012, had a mean temperature of 8.4 °c and 565.4 gdd. the warmest summer was in 2013, the second year of the experiment, with a mean temperature of 10.9 °c and 914.6 gdd. in 2014 the mean temperature was 9.8 °c and accumulated gdd was 787.3 (fig. 1, tab. 3). mean temperature during berry ripening, june, july and august, differed from 10.0 °c in 2012 to 11.8 °c in 2013 and 12.0 °c in 2014. thus, the highest temperature during berry ripening occurred in 2014 while 2013 was the overall warmest summer. time for first flowering, first and last harvesting and time points for early, middle and late harvest are presented (tab. 2). berries were harvested 2-3 times per week during the harvesting seasons. as ‘306’ and ‘fjellgull’ are earlier than ‘fjordgull’ and particularly ‘102’, this implies that ‘306’ and ‘fjellgull’ produced ripe berries before early harvest and ‘102’ produced ripe berries after late harvest time point. generally, late-season harvesting involves lower light intensity, shorter day length and lower temperature; compared with early and mid-season especially in 2013 (fig. 2, tab. 2). total polyphenols total polyphenol content was significantly affected by genotype, year and harvest time (tab. 4 and 5). in the statistical model, 17% of the variation of total polyphenols were assigned to genotype (tab. 4). ‘fjellgull’ (22.99 mg/ga/g dw) was the cultivar with the highest level of total polyphenols while ‘102’ (21.67 mg/ga/g dw) had the second highest level (fig. 2, tab. 5). berries from cultivar ‘306’ (19.18 mg/ga/g dw) had the lowest levels of total polyphenols all years, but not statistically different from ‘fjordgull’. additional ly, approximately 17% of the variation was associated with yearly variation in weather conditions (tab. 4). year 2012, with the lowest mean temperature, had significantly higher levels of total polyphe nols than 2013 and 2014 (fig. 1, tab. 5). all cultivars showed a reduction in levels of total phenols during the three years period of experiment, but the reduction from 2013 to 2014 was not significant (fig. 2). the yield and mean berry sizes of the plots were higher in 2013 compared to 2012 and 2014 (data not shown). the berries that matured early had the highest levels of total polyphenols (fig. 2, tab. 5) and berries from early harvest had significant higher levels of polyphenols than berries harvested late in the harvesting season. berries harvested in the mid-season were not statistically different from early and late harvested berries. there were no significant interactions between the main effects for total polyphenols. the contents of total polyphenols were negatively correlated with temperature (tab. 6). fig. 1: development of growing degree days (gdd) through the growing season. time points for early harvest, middle harvest and late harvest for the harvesting years 2012, 2013 and 2014 are shown. seasonal and yearly variation of secondary metabolites in cloudberry 99 ellagic acid ellagic acid content after hydrolysis varied from 8.05 mg/g dw in ‘fjellgull’ to 5.44 mg/g dw in ‘fjordgull’ (fig. 2, tab. 4). ellagic acid content was significantly affected by genotype (32% of the variation) (tab. 5). ‘fjordgull’, which contained the highest levels of total anthocyanins, had the lowest levels of ellagic acid at all harvest points for all three years. there were also significant effects of year and harvest time and interactions between harvesting time and year as well as between cultivar and harvesting time (tab. 4). ellagic acid levels were significantly higher in berries collected in early and middle season compared to late season (fig. 2, tab. 4). the cultivars varied in response to ellagic acid content throughout the harvest season during the study, indicating that other factors than harvest time may have influenced the seasonal variations. the levels were significantly higher in the coolest year, 2012, than in the warmest year, 2014, while 2013 was not statistically different from the other years (fig. 2, tab. 4). still, ellagic acid content showed a small negative correlation with temperature (tab. 5). total anthocyanins the highest content of total anthocyanins was observed in berries harvested in 2012, the year with lowest gdd, additionally, berries harvested early in the 2012 season had the highest level of total anthocyanins. as much as 45% of the variation was linked to the year and harvest time (tab. 5). the contents of anthocyanins were clearly negative correlated with temperature (tab. 6). levels of anthocyanins in 2012 were 70% higher than 2013 and 63% higher than in 2014 (fig. 2, tab. 4). all clones had the highest levels in 2012, but the lowest levels for ‘102’ were measured in 2014 while the other clones had the lowest amounts of total anthocyanins in 2013 (fig. 2, tab. 4). additionally, there was an interaction between year and harvesting time (tab. 5). in 2013 the anthocyanin levels decreased through the harvesting season from early to middle and late. in 2013, levels were highest in the early harvest and in alignment middle and late harvest, while in 2014 the levels were high and leveled in early and middle harvest and lowest in late harvest. thus, the middle harvest levels differed while the high contents in early season harvesting and low contents in late harvesting were evident all three years. in addition to the environmental conditions, there was also a significant effect of genotype and an interaction between genotype and year. ‘fjordgull’ and ‘fjellgull’ had significant higher contents than ‘102’ and ‘306’. discussion influence of genotype the present study confirms that genotype significantly influences the content of total polyphenols, ellagitannins and total anthocyanins in cloudberries. this is in alignment with previous studies on fig. 2: yearly (from 2012-2014) and harvesting time variation in the content of a) total anthocyanins (ta) mg/g dry weight (dw) sample; b) total phenols (tp) expressed as mg of ga (gallic acid) equivalents per gram dry weight (dw) of sample and c) ellagic acid (ea) mg per gram dw of sample in cloudberry (rubus chamaemorus l.) clones ‘102’, ‘306’ and the commercial varieties ‘fjellgull’ and ‘fjordgull’. the growth harvesting season are divided into ‘early’, ‘middle’ and ‘late’. tab. 4: effects of genotype, year and harvesting time and their interactions on the quality parameters total anthocyanin (ta), total polyphenol (tp) and ellagic acid (ea) determined in cloudberry berries. the levels are expressed as mg per g dry weight (dw) of sample, total polyphenols were expressed as mg of ga (gallic acid) equivalents per gram dw of sample. clone tp ea ta mg/ga/g dw mg/g dw mg/g dw genotype ‘102’ 21.67 7.28 0.14 ‘306’ 19.18 6.83 0.14 ‘fjellgull’ 22.99 8.05 0.21 ‘fjordgull’ 20.01 5.44 0.24 year 2012 23.19 7.22 0.32 2013 20.51 7.11 0.10 2014 19.57 6.54 0.12 harvest time early 22.12 7.41 0.24 middle 20.97 7.03 0.19 late 20.09 6.35 0.13 100 a.l. hykkerud, e. uleberg, e. hansen, m. vervoort, j. mølmann, i. martinussen 20 different cloudberry clones, including the four tested here, which revealed large variation on total polyphenol levels and total anthocyanins (uleberg et al., 2011). in raspberry (rubus idaeus) stu dies, the content of total phenolics, ellagic acid, and total anthocyanins also varied greatly between cultivars (anttonen et al., 2005; bobinaite et al., 2012; mazur et al., 2014). additionally, our results showed that genotype had greatest influence on total polyphenols and ellagic acid and less for anthocyanin content, which was more affected by environment. this is in line with findings in black currant (ribes nigrum) (vagiri et al., 2013) where the large variation in total anthocyanins was mainly affected by yearly variations and less by genotype while polyphenolics to be more affected by genotype. likewise, the polyphenol content in blackberry was highly affected by cultivar and less by yearly variations (milošević et al., 2012). on the contrary, another study on raspberry (mazur et al., 2014), found small yearly variations on the anthocyanin content, however, the temperature difference between the two selected years was smaller than the yearly variations in our study. cultivar effect on anthocyanins have also been detected in other berry species such as cranberry (vaccinium oxycoccos) (borowska et al., 2009) and lowbush blueberry (vaccinium angustifolium) (kalt et al., 1996). these results indicate that total polyphenol content is most affected by genotype while total anthocyanins are more controlled by environmental conditions. ellagitannins are the major phenolic constituent in cloudberry (kähkönen et al., 2001; määttä-riihinen et al., 2004; kylli, 2011) and given this would be an interesting qualitative trait in future plant breeding activities. in this study, there were significant effects of year and harvesting time during the season, yet genotype was the factor with the greatest impact on ellagic acid content. in a finnish study, ellagic acid content in cloudberry was found to be affected by the location, which could be due both to genetic and environmental differences (häkkinen et al., 1999), while li et al. (2016) did not find a location effect in four different locations in atlantic canada. our results are consistent with those of anttonen and karjalainen (2005) and bobinaite et al. (2012) that described large variations in ellagic acid content between cultivars of raspberries and atkinson et al. (2006) that reported a strong seasonal effect on ellagic acid content in strawberry. mazur et al. (2014) reported that genotype was more important for ellagitannin content in raspberry than seasonal variation. additionally, genotype effect was found to be smaller in red raspberry as compared to blackberry (vrhovsek et al., 2008), indicating differences between species. the correlation between total phenolics and ellagic acid suggest that selection for a high total phenolic content will be a good indirect selection criterion for ellagic acid content. in the present study, the differences between the genotypes were less profound than findings from raspberries but still significant for all investigated compounds. clones ‘102’ and ‘306’ were included in this study based on their performances in earlier experiments (uleberg et al., 2011; trost et al., 2013), in addition to the released cultivars ‘fjellgull’ and ‘fjordgull’. thus, the studied clones are already selected and we expect less variation here than we would in a study with wild material. influence of climatic conditions total polyphenolic and ellagic acid content were significantly affected by year and harvest time. li et al. (2016) also found a yearly ef fect on the total polyphenol content as well as the ellagic acid content in cloudberries collected at four different locations during a two-year period in atlantic canada. total phenols showed a negative correlation with gdd and the highest polyphenol content was observed in the year with the lowest growing temperature, while the levels were reduced the warmest summers (2013 and 2014). conversely, ellagic acid content was less correlated to temperature, but behaved similar as total phenols through the harvesting season and between years. on the contrary, remberg et al. (2010) reported significant higher levels of the analyzed two prevalent ellagitannins, lambertianin c and sanguiin hg, in raspberries at increasing temperature. our results indicate that although ellagitannins are dominating, there are other polyphenols more affected by temperature. in cloudberries, a previous study suggests that in addition to temperature, sunlight is the main factor affecting the chemical composition of cloudberry (jaakkola et al., 2012). total anthocyanin levels in the four studied cloudberry clones were strongly affected by year, time of harvesting and climatic conditions as described previously by trôst et al. (2013), uleberg et al. (2011) and martinussen et al. (2010). in the present study, anthocyanin levels were negatively correlated with gdd. the anthocyanin levels were highest in 2012, the coldest summer, while the lowest contents were found in 2013, which had the warmest summer. findings in the present study thus confirm the results described by martinussen et al. (2010) that found enhanced levels of anthocyanins in the cultivar ‘fjellgull’ at 9 °c or 12 °c compared to 15 °c and 18 °c jaakkola et al. (2012) found similar results in cloudberry from different locations over several years; in a cold and rainy summer, the tab. 5: percentage of total variation (sum of squares) ascribed to clone, harvesting time, and year and their interactions on the content of total polyphenols, ellagic acid and total anthocyanins in cloudberry grown during three different years. total polyphenol total anthocyanin ellagic acid % of variations p-value % of variations p-value % of variations p-value genotype (g) 17.1 8.3 × 10-11 10.2 6.0 × 10-9 32.3 2.0 × 10-16 year (y) 17.2 2.0 × 10-12 34.4 2.0 × 10-16 3.4 1.1 × 10-3 harvest time (h) 5.7 1.2 × 10-4 10.1 1.7 × 10-9 5.7 1.3 × 10-4 g × y 1.3 0.24 3.2 3.2 × 10-3 0.12 0.94 g × h 3.6 0.07 1.6 0.31 5.2 0.01 y × h 0.6 0.35 1.9 0.01 0.0 0.93 tab. 6: correlations (pearson’s) between total anthocyanins, total phenols, ellagic acid, mean temperature and total precipitation during the ripening period, and berry weight. ta tp ea temp precipitation tp 0.548 ea 0.131 0.629 temp -0.502 -0.434 -0.214 precipitation -0.24 -0.003 0.161 0.062 berry weight 0.158 0.363 0.284 -0.141 0.301 seasonal and yearly variation of secondary metabolites in cloudberry 101 content of anthocyanins were significantly higher than in a warm and dry summer. higher anthocyanin levels at low temperatures have been described in various species (chalker-scott, 1999), and at high temperatures for others berry species (uleberg et al., 2012; josuttis et al., 2012). the regulation of anthocyanins is found to be a stress response in plants. depending on the stress conditions production of specific anthocyanins is induced (kovinich et al., 2014). in addition to the year and temperature effects, there were also interactions; year by genotype and year by harvesting time (tab. 3). the sensitivity of anthocyanin accumulation at temperature changes related to different genotypes have also been found to differ significantly in flowers of plantago lanceolata from different genotypes (stiles et al., 2007). in our study, anthocyanin levels decreased from early to late harvesting time. the accumulated gdd (tab. 2) could explain the reduction in anthocyanin contents that was observed from early to late harvest. in a recent study barnuud et al. (2014), using climate-variable-based empirical models to predict quality changes of different cultivars of grapes (vitis vinifera), found that in a warmer climate berry anthocyanins would decline. dark inhibition of the anthocyanin biosynthesis has been described by zhang et al. (2015) who found that light was necessary for anthocyanin production in begonia leaves under low temperature. effect of other environmental factors the variation between years was significant and all genotypes showed a total polyphenolic reduction during the three years period of experiment, even if the reduction from 2013 to 2014 was not significant. the explanation could be weather conditions, as discussed above or other factors as age of the plantlets, total yield or nutritional status in the plots. cloudberry spread vegetatively so the restricted area of each plot gradually gets more and more populated, increasing the competition for nutrients. nevertheless, the yields in the plots were highest in 2013 (2421 g) while similar in 2012 and 2014 (1807 g and 1648 g, respectively). berry yields in cloudberry are highly correlated to the weather conditions during pollination, since insect pollination is necessary for fruit development in the dioecious cloudberry (rapp and martinussen, 2002). the high mean temperature in june 2013 (11.7 °c) compared to 2012 and 2014 (9.1 °c) indicate that the conditions for pollination was better this year, which could explain the observed yield differences. the mean berry weights were similar in 2012 (1.69 g) and 2013 (1.89 g) while significantly lower in 2014 (1.18 g). even if the berry size was reduced, good berry yields were obtained in 2014, indicating that nutrients were available for fruit growth and development. conclusions to investigate the breeding potential of cloudberry clones with high content of health beneficial compounds total anthocyanins, total polyphenols and ellagic acid content were analyzed in berries harvested from four clones of cloudberry from a three years period. the results illustrated that although climatic effects have strong impact on total anthocyanins, the levels are highly genotype dependent. although genotype affects the polyphenolic levels in cloudberry, final content was also dependent upon environmental parameters. in addition, results indicated that the different clones respond differently to environmental factors. the presented study expands our know ledge about variation in the content of polyphenols in cloudberry clones and how they are affected by environmental conditions. this knowledge may help for the selection and validation of the clones with the highest health benefit. the strong correlation between total polyphenols and ellagic acid content of cloudberry suggest that selection for a high total phenolic content will be a good indirect selection criterion for ellagic acid content. acknowledgements the work has been funded by the czech-norwegian research program cz09, project id 7f14122. thanks to kirsten jakobsen for valuable technical assistance. references anttonen, m.j., karjalainen, r.o., 2005: environmental and genetic variation of phenolic compounds in red raspberry. j. food. compos. anal. 18, 759-769. doi: 10.1016/j.jfca.2004.11.003 atkinson, c.j., dodds, p.a.a., ford, y.y., le miere, j., taylor, j.m., blake, p.s., paul, n., 2006: effects of cultivar, fruit number and reflected photosynthetically active radiation on fragaria × ananassa productivity and fruit ellagic acid and ascorbic acid concentrations. ann. bot. 97, 429-441. doi: 10.1093/aob/mcj046 barnuud, n.n., zerihun, a., gibberd, m., bates, b., 2014: berry composition and climate: responses and empirical models. i j. biometeorol. 58, 1207-1223. doi: 10.1007/s00484-013-0715-2 bliss, l.c., 1962: adaptations of arctic and alpine plants to environmental conditions. arctic 15, 117-144. bobinaitė, r., viškelis, p., venskutonis, p.r., 2012: variation of total phenolics, anthocyanins, ellagic acid and radical scavenging capacity in various raspberry (rubus spp.) cultivars. food chem. 132, 1495-1501. doi: 10.1016/j.foodchem.2011.11.137 chalker-scott, l., 1999: environmental significance of anthocyanins in plant stress. responses. photochem. photobiol. 70, 1-9. doi: 10.1111/j.1751-1097.1999.tb01944.x connor, a.m., luby, j.j., tong, c.b.s., finn, c.e., hancock, j.f., 2002: genotypic and environmental variation in antioxidant activity, total phenolic content, and anthocyanin content among blueberry cultivars. j. amer. soc. hort. sci. 127, 89-97. dobson, p., graham, j., stewart, d., brennan, r., hackett, c.a., mcdougall, g.j., 2012: over-seasons analysis of quantitative trait loci affecting phenolic content and antioxidant capacity in raspberry. j. agric. food chem. 53, 625-634. doi: 10.1021/jf3005178 duthie, s.j., 2007: berry phytochemicals, genomic stability and cancer: evidence for chemoprotection at several stages in the carcinogenic process. mol. nutr. food res. 51, 665-674. doi: 10.1002/mnfr.200600257 giusti, m.m., wrolstad, r.e., 2001: characterization and measurement of anthocyanins by uv-visible spectroscopy. in: wrolstad, r.e. 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a., oberg, e., johansson, e., andersson, s.c., rumpunen, k., 2013: phenols and ascorbic acid in black currants (ribes nigrum l.): variation due to genotype, location, and year. j. agric. food chem. 61, 9298-9306. doi: 10.1021/jf402891s vrhovsek, u., giongo, l., mattivi, f., viola, r., 2008: a survey of ellagitannin content raspberry and blackberry cultivars grown in trentino (italy). eur. food res. technol. 226, 817-824. doi: 10.1007/s00217-007-0601-4 zhang, k.m., guo, m.l., he, d., wu, r.h., li, y.h., 2016: the inhibition effect and excessive carbon flux resulting from blocking anthocyanin biosynthesis under darkness in begonia semperflorens. j. plant growth reg. 35, 22-30. doi: 10.1007/s00344-015-9503-z zoratti, l., sarala, m., carvalho, e., karppinen, k., martens, s., giongo, l., hägman, h., jaakola, l., 2014: monochromatic light increases anthocyanin content during fruit development in bilberry. bmc plant biol. 14, 377-387. doi: 10.1186/s12870-014-0377-1 address of the authors: anne linn hykkerud, eivind uleberg, marieke vervoort, jørgen mølmann, inger martinussen, norwegian institute of bioeconomy research, p.o. box 115, no-1431 ås, norway e-mail: anne.linn.hykkerud@nibio.no espen hansen, arctic university of norway, uit, p.o. box 6050 langnes, n-9037 tromsø, norway © the author(s) 2018. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 90, 238 245 (2017), doi:10.5073/jabfq.2017.090.030 department of medicinal and aromatic plants, szent istván university, budapest, hungary morphological, phytochemical and molecular characterization of intraspecific variability of wormwood (artemisia absinthium l.) huong thi nguyen, katalin inotai, péter radácsi, szilvia tavaszi-sárosi, márta ladányi, éva zámboriné-németh* (received february 10, 2017; accepted march 24, 2017) * corresponding author summary a trial with nine wormwood accessions was installed to carry out a systematic evaluation of intraspecific diversity. six morphological features, essential oil (eo) yield and thujone content were measured. besides, 11 rapd and 15 issr molecular markers were tested to determine the genetic diversity of the accessions. the experiment was carried out in open field in 2016. accession “pákozd” exhibited largest growth (64.9 cm) and genotype “norwegen” was the smallest (29.9 cm). this latter accession had also the smallest but thickest leaves. concerning morphological features, the norwegian population was the most homogenous one (cv%: 10.6 -20.1) while “belgin” brought about largest variability (cv%: 18.4-45.3). based on eo yield, the studied accessions were divided into three significantly diverse groups. the highest yield was produced by “spanish” accession (3.215 ml/100 g), “norwegen” and “belgien” produced medium values (1.569-1.892 ml/100 g) and six accessions showed eo yields below 1% (0.349-0.832 ml/100 g). three accessions (“leipzig”, “belgien” and “norwegen”) had high amount of thujone in the oil (50-89%) while in all other accessions thujones were absent or present only below 1%. “belgien” accession had balanced ratio of αand β-thujones while in the other ones β-thujone was the absolute main component. high polymorphism was found among the wormwood accessions also by molecular markers: 81.15% for rapd and 73.10% for issr primers. based on the nei’s genetic distances the three groups of genotypes were identical to those in the case of eo yield. the study confirmed the large intraspecific variability of wormwood but revealed that it is not definitely connected to geographical origin of the populations. abbreviations: ema-hmpc: european medicines agency – committee on herbal medicinal products; cv: coefficient of variation; dna: deoxyribonucleic acid; issr: inter simple sequence repeat; rapd: random amplified polymorphic dna; eo: essential oil keywords: essential oil, leaf shape, thujone; radp; issr; dna; morphology. introduction artemisia absinthium (compositae) is a perennial herb, growing to 40-150 cm in height (maw, thomas and stahevitch, 1985) and developing abundantly branching shoots. wormwood oil has been used for centuries as anthelmintic, anticold, anti-inflammatoy, antimicrobial, antidepressant, digestive, carminative, choleretic drug, curing insect and spider bites, herpes and parasitic worm infections (guarrera, 2005; goud and swamy, 2015). the indications of ema-hmpc (2009) monograph include temporary loss of appetite and gastro-intestinal disorders. the species exhibit a very large intraspecific variability concerning its morphological traits and active ingredients. systematic research is, however, rather scarce on this aspect, except references on essential oil composition. studies on morphological and anatomical features of the genus detected significant marker characteristics of selected species within the genus. thus, haghighi and co-workers (2014) used seven morphological characters such as leaf type, leaf colour on adaxial and abaxial surfaces, attachment of capitalism, etc. to differenciate 15 species including a. absinthium. other in vestigations focused on leaf anatomy as important qualitative feature in classification of this species (mehrotra et al., 1990; nazar and mahmood, 2011). noorbakhsh and co-workers (2008) described 13 leaf characteristics in 28 species of artemisia but they had no stable relationship with taxonomy. the most important active constituents of the herb are volatile compounds and bitter substances. the major volatile components of wormwood are thujone, myrcene, sabinene, transand cis-epoxyocimene, trans-verbenol, carvone, sabinyl acetate and chrysanthenyl acetate. however, numerous studies have shown that artemisia absinthium displays significant intraspecific variation in the terpenic constituents and the spectrum is varying from region to region (basta et al 2007; mohammadi et al., 2015). quantitative evaluation of phytochemical diversity of wormwood populations from different natural geographic areas supports the existence of distinct natural chemotypes within the species. wormwood is usually known and reported to be rich in bicyclic monoterpene thujone (juteau et al., 2003; meschler and howlett, 1999). the presence and concentration of both isomeric forms αand β-thujones in the oil were described by several authors. nin et al. (1995) and juteau et al. (2003) defined characte ristic “pure” thujone chemotype while on the other side, absence of thujones in wormwood oil was reported also several times by orav and co-workers (2006), carnat et al. (1992), ariño et al. (1999c) etc. because of restricted concentration of thujones in food products (eu council directive, 1988), these thujone-free chemotypes might raise considerable attention. molecular markers have only exceptionally been used for studying intraspecific variability and relationship of accessions of this genus and especially of this species. rapd investigations in artemisia annua were performed in india already 17 years before (sangwan et al., 1999). nazar and mahmood (2011) established the genetic diversity of some collected artemisia species including a. absinthium in pakistan, using rapd markers as well. application of issr analysis has been used for a. herba-alba in tunisia to detect genetic polymorphism within this species. according to our knowledge, the combination of both rapd and issr markers for revealing genetic variability of a. absinthium has not been published yet. wild growing plants assure rich resources for optimising the botanical raw material for diverse utilization areas. optimisation of the raw material, however, presumes the thorough knowledge on the genotypes used in breeding programs and production. the objective of our study was the comparison and better recognition of 9 genetic stock accessions originating from different european regions. the populations were grown under uniform environmental conditions in hungary and evaluated by morphological, chemical and genetic tools. characterisation of wormwood accessions 239 materials and methods plant material and cultivation plant material for the study included gene bank collections, market items and seeds collected from wild habitats (tab. 1). propagation was carried out by seed sowing in march 2016 and seedling raising in greenhouse for 2 months. the seedling with 5-6 leaves were planted into open field plots in june, installing three replicates at the experimental station of szent istván university in budapest. the soil is sandy-loam, ph 7.8, humus content is low. the plants were grown without additional fertilization; mechanical weed control and irrigation were assured in dry periods. measurements and sampling were carried out at the beginning of august at vegetative stage. 10 in dividuals from each accession were randomly chosen to evaluate harvest. details of the measurements are described below. the oils were collected, stored in an airtight vial and kept in a refrigerator at 4 °c until the analysis took place. gas chromatography the gc analyses were carried out using an agilent technologies 6890n instrument equipped with hp-5 and hp-5ms capillary columns (5% phenyl, 95% dimethyl polysiloxane, length: 30 m, film thickness: 0.25 mm, id. 0.25 mm), programed as follows: initial temperature 60 °c, then by a rate of 3 °c/min up to 240 °c; the final temperature was kept for 5 min; injector and detector temperature: 250 °c; carrier gas: helium (constant flow rate: 1 ml/min); split ratio: 30:1. mass spectrometric analysis gc-ms analyses were carried out using an agilent technologies 6890n gc equipped with an agilent technologies ms 5975 inert mass selective detector and an ionization energy of 70 ev. the ms were recorded in full scan mode that revealed the total ion current (tic) chromatograms. αand β-thujone content were identified by linear retention indices that were calculated using the generalized equation of van den dool and dec. kratz (1963) by using nist ms search 2.0 library and adams mass spectra library (p. adams, 2007). dna isolation young leaves from 10-15 plants of each accession were collected and ground in liquid nitrogen to a fine powder. genomic dna was extracted from fresh leaves by a dn easy plant mini kit (qiagen, bioscience, hungary). the concentration and quality of extracted dna from each accession were assessed using nanodrop (bioscience, hungary) and visually checked on 1.5% agarose gel. out of the primarily screened 13 rapd and 15 issr primers only 11 rapd and all the 15 issr primers produced clear, reproducible and scorable bands, thus the investigations have been carried out by these ones. their sequences are presented in tab. 2. rapd and issr amplification the pcr reaction was performed in a 12 ml volume containing the reaction buffer (approximately 15-25 ng genomic dna, 1 mm primer (for rapd) or 2mm primer (for issr), 6 ml of 2× gotaq hot start green master mix (promega), 3 mm mgcl2 and nuclease free tab. 1: list of the investigated a. absinthium accessions accession origin of population no. sign 1 belgien accession of gatersleben genebank (collected in belgium) 2 csór wild collected population: csór, hungary 3 english market item from england (‘premier seeds direct’ company) 4 hungarian market item from hungary (‘herbaria’ company) 5 leipzig accession of gatersleben genebank (collected in germany) 6 norwegen accession of gatersleben genebank (collected in norway) 7 pákozd wild collected population: pákozd, hungary 8 spanish wild collected population: teruel, spain 9 wild soroksár wild collected population: soroksár, hungary morphological measurements six randomly selected individuals were chosen to collect seven leaves at the central part of the shoots from each of them. in total, 378 leaves of nine accessions were investigated at leaf rosette period. after collecting, the leaves were pressed and a herbarium was prepared following the techniques of dewolf (1968). the thickness (as mean of five target points on midway between the margins and midrib at the widest part of the leaf) was measured using a digimatic thickness gauge equipment. the total length of the leaf and that of the blade were measured by vernier caliper at the herbarium specimens and the ratio between them has been calculated (fig. 1). plant height (from ground level to tallest shoot) and width (mean of the widest diameters) were measured immediately before start of harvesting. essential oil extraction after the morphological measurements, the shoots of each individual were cut at about five cm height above the soil surface. the plant material was dried at room temperature (20-25 °c) in shade for two weeks. leaves were separated from stem parts and only the former ones were used for essential oil distillation. plant samples (g) were distilled in a clevenger-type apparatus according to the method recommended by the vii. hungarian pharmacopoeia. essential oils were extracted from 50 g dried leaves of each accession by hydro-distillation (500 ml water) for 2.5 hours. ! fig. 1: herbarium leaf of a. absinthium and devices for leaf measurements 240 h.t. nguyen, k. inotai, p. radácsi, s. tavaszi-sárosi, m. ladányi, é. zámboriné-németh water). pcr amplification was conducted in a supercycler sc-200 thermocycler (kyratec). the thermal cycle used for rapd primers was 2 min at 95 °c followed by 35 cycles at 94 °c for 30 s; 1 min at specific annealing temperature (the optimal annealing temperature was determined for each individual primer); 1 min at 72 °c and final extension at 72 °c for 5 min. the program used for issr primers was 3 min at 95 °c followed by 35 cycles at 94 °c for 30 s; 45 s at specific annealing temperature; 45 s at 72 °c and final extension at 72 °c for 5 min. amplified dna fragments were separated in a 1.5% agarose gel (seakem le agarose, lonza) at 100 v for 90-120 min in 1× trisacetate edta (tae) buffer (ph 8.0) and stained by 1% (w/v) ethidium bromide. the pcr products were visualized under uv light by alphaimager ep imaging system (cell bioscience). the 100 bp ladder (promega) was used as a molecular weight size marker. data analysis spss version 22 was used to analyze the data. manova test has been conducted for evaluating the measured morphological characters of tab. 2: the evaluated rapd and issr primers rapd primer name sequence size of fragment number of fragments (bp) a10 5’-gtgatcgcag-3’ 1450-250 10 b10 5’-ctgctgggac-3’ 1500-300 16 e3 5’-ccagatgcac-3’ 1000-250 11 m2 5’-acaacgcctc-3’ 1300-300 15 m3 5’-gggggatgag-3’ 1300-300 14 opa-02 5’-tgccgagctg-3’ 1100-300 11 op a20 5’-gttgcgatcc-3’ 1500-400 6 opb-11 5’-gtagacccgt-3’ 1200-300 10 op g18 5’-ggctcatgtg-3’ 1300-350 10 op g13 5’-ctctccgcca-3’ 1300-300 11 seq-2 5’-gggtttaggg-3’ 1300-400 8 total no. of bands (radp) 122 number of polymorphic loci 99 percentage of polymorphic loci 81.15% issr primer name sequence size of fragment number of fragments (bp) 443 5’-acacacacacacacacact-3’ 1200-200 14 818 5’-cacacacacacacag-3’ 1100-200 13 825 5’-acacacacacacacact-3’ 1300-200 16 849 5’-gtgtgtgtgtgtgtgtcg-3’ 1100-240 13 a7 5’-agagagagagagagagagagt-3’ 950-200 14 caa5 5’caacaacaacaacaa-3’ 1100-200 11 cag5 5’cagcagcagcagcag-3’ 1000-180 17 ctc4rc 5’-ctcctcctcctcrc-3’ 1000-230 16 issr1 5’-cacacacacacacacagt-3’ 1500-150 14 issr2 5’-gagagagagagagagag-3’ 900-220 12 issr3 5’-gtgtgtgtgtgtgtgtc-3’ 850-300 12 issr4 5’-acacacacacacacactg-3’ 1500-250 12 issr5 5’-agtgagtgagtgagtg-3’ 1300-120 17 issr6 5’-gatagatagatagatagata-3’ 1100-250 8 issr7 5’-tcttcttcttcttcttct-3’ 1500-530 7 total no. of bands (issr) 196 number of polymorphic loci 144 percentage of polymorphic loci 73.10% total no. of bands (radp+issr) 318 number of polymorphic loci 243 percentage of polymorphic loci 76.18% characterisation of wormwood accessions 241 the leaves. one-way analysis of variance (anova) and tukey’s hsd post hoc test were carried out to analyze the significant differences of eo content of the nine accessions. homogeneity of variances was checked by levene’s test and the normality of variances was checked by kolmogorov-smirnov method. in case of violated homogeneity of variances, games-howell was used in post hoc test to determine the significant difference between accessions. amplified dna fragments with reproducible bands of each locus were scrored as binary present (1) or absent (0) and data matrices of radp and issr loci were assembled for further analysis. the results were summarized in microsoft excel table. popgene version 1.32 (yeh et al., 1997) was used to estimate number of polymorphic bands, percentage of polymorphic bands, nei’s (1972) gene diversity (h) and shannon’s information index (i) (lewontin, 1972) for dominant marker data or all loci and also for each population separately. genetic relationship among genotypes was studied by upgma (unweighted pair group method with arithmetic averages) cluster analysis and principal component (pca) analysis using past software (hammer et al., 2001). results and discussion morphological measurements according to manova tests of between-subjects effects, there have been significant differences detected in the case of each of the four leaf morphological characters: f. thickness(8;363)=13.77 (p<0.001); f. lenght leaf(8;363)=18.17 (p<0.001); f. length petiole(8;363)=15.03 (p<0.001); f. ratio blade petiole(8;363)=9.84 (p<0.001). the results are summarized in tab. 3. the thickness of blades of the investigated accessions ranged from 0.31 mm to 0.49 mm. the games-howell test distinguished 4 subsets at p = 0.05 significance level. accessions from “csór” and “norwegen” have the smallest thickness of leaf while “belgien” was characterised by the thickest leaves. the length of leaf and the length of petiole seem to be in tight connection, as highest values for both of them were found in accessions “hungarian” and “leipzig” while the length of leaf and petiole were shortest in the accession “norwegen”. the marginal values for the leaf length were 209 mm (“leipzig”) and 124 mm (“norwegen”). the ratio between blade and petiole was influenced by the origin, too. it varied from 0.76 (“spanish”) to 1.12 (“belgien”). these accessions proved to be significantly different from all the other ones and also from each other. for these values, the investigated four accessions of hungarian origin revealed similar ratios between 0.8-0.9. concerning the evaluated leaf morphological traits, it was established that the norwegian population can be considered as the most homogenous one (cv%: 10.6 -20.1) while the greatest variability was shown by the accession “belgien” (cv%: 18.4-45.3). the four hungarian accessions had taller plants than all the accessions of other origin except the accession “english” (tab. 4.). in the whole population, genotype “pákozd” exhibited the highest growth (46.2 cm) and genotype “norwegen” had the shortest shoots (18.7 cm). this latter accession was determined as the most homogenous one (cv%=11%). on the other side, the largest variability was detected in population “english” (cv%=31.17%). similarly to the discussed other characteristics, plant width brought about considerable differences among accessions (tab. 4). “english” and “pákozd” were the biggest with largest values for plant width (59.4-64.9 cm) while “norwegen” was separated from all the others presenting the smallest bushes (29.9 cm). it should be mentioned, that the thinnest leaf blade and smallest leaves were also detected in this latter one. tab. 3: leaf characteristics of nine wormwood accessions accession thickness of blade (mm) length of leaf (mm) length of petiole (mm) ratio of blade/petiole mean st. dev. mean st. dev. mean st. dev. mean st. dev. belgien 0.49d 0.089 167b,c,d 50.5 83b,c 37.8 1.12e 0.306 csór 0.31a 0.075 181c,d,e 32.1 97c,d 21.9 0.89b,c 0.164 english 0.37a,b 0.098 185d,e 42.9 100c,d 33.1 0.91a,b,c,d 0.278 hungarian 0.40b 0.095 206e 55.2 115d 35.3 0.81a,b 0.218 leipzig 0.42b,c 0.075 209e 56.2 111d 33.2 0.91b,c,d 0.181 norwegen 0.32a 0.056 124a 13.0 61a 8.6 1.03d,e 0.207 pákozd 0.37a,b 0.161 148b 42.0 78b 27.4 0.93b,c,d 0.220 spanish 0.47c,d 0.097 155b 36.5 90b,c 24.8 0.76a 0.172 wild soroksár 0.41b,c,d 0.131 151b 34.7 79b 21.3 0.94b,c,d 0.195 mean 0.39 170 91 0.92 different letters at column represent statistical significant difference between accessions according to games-howell test at p=0,05 tab. 4: height and width of different wormwood accessions accession height (cm) width (cm) mean st. dev. mean st. dev. belgien 41.1b,c 9.11 42.9b,c 10.16 csor 42.0b,c 9.16 54.0b,c,d 11.08 english 47.5c 14.80 59.4c,d 11.78 hungarian 40.2b,c 9.37 50.3b,c,d 4.55 leipzig 40.8b,c 13.46 50.8b,c,d 9.85 norwegen 18.7a 2.06 29.9a 4.25 pakozd 46.2c 8.84 64.9d 16.74 spanish 32.7b 7.53 47.4b,c,d 5.72 wild soroksar 42.7b,c 12.60 56.3d 8.25 mean 39.1 50.7 different letters at column represent statistical significant difference between accessions according to games-howell test at p=0,05 242 h.t. nguyen, k. inotai, p. radácsi, s. tavaszi-sárosi, m. ladányi, é. zámboriné-németh characteristics of the essential oil essential oil yield the essential oil yield of the investigated accessions was 1.107 ml/ 100 g as a mean, however, it varied on a large scale: between 0.349 ml/100 g (“wild soroksár”) and 3.215 ml/100 g (“spanish”). according to anova test of between-subjects effects for this trait, significant differences could be detected among accessions: f(8;81)=58.707 (p<0.001). the tukey test provided 3 subsets at p=0.05 significance level (tab. 5). based on this, all accessions from hungary together with “english” and “leipzig” are statistically equal, practically below 1%. among them, highest mean value was reached by “leipzig” and lowest level by “wild soroksár”. in general, these values correspond to former data of orav and co-workers (2006) who obtained 0.1-1.1% essential oil from plant material coming from different european regions or to the reference of basta et al. (2007) about greek wild plants (0.31%). relatively high concentrations of 1.569 ml/100 g and 1.892 ml/ 100 g were determined from accessions “norwegen” and “belgien”, respectively. these results are similar to the findings of msaada and co-workers (2015) in tunisia who described an essential oil content of 1.1-1.46% in wild growing plants. also plant material from cuba produced similar yield (1.25%), (pino et al., 1997). the “spanish” accession in our study presented an exceptionally high level of volatile compounds which is significantly different from each of the other ones (3.215 ml/100 g). this high level could hardly be found in the literature. the “spanish” accession was the most homogenous one in term of essential oil yield (cv%=12%) while the largest variability was determined in the population “leipzig” from germany (cv%=64.4%). our results ascertain that the yield of essential oil of wormwood may differ considerably depending on the investigated plant material (nguyen and németh, 2016). at the same time, it can be concluded that the differences are based on genetic variability instead of being the result of diverse ecological conditions as they have manifested themselves in the same environment at our experimental site. thujone content both isomeric forms of thujone, αand β-thujone were determined and quantified in all studied wormwood oils (tab. 6). there are well established differences among the oils. three accessions (“leipzig”, “belgien” and “norwegen”) have high amounts of thujonesin the oil while in the case of all other accessions thujones were absent or present only in trace amounts (below than 1%). thujone content has been shown the variable distribution within individuals of these accessions (fig. 2). the highest proportions (up to 91.55%) were found in an individual of “belgien”. it seems to be a unique chemical character which may be detected only by individual sampling like in our study. tab. 5: essential oil yield (ml/100 g dm) of dried leaves of the studied artemisia absinthium accessions grouped according to the tukey test n subset 1 2 3 wild soroksar 10 0.3490 hungarian 10 0.3620 csor 10 0.5210 pakozd 10 0.5530 english 10 0.6750 leipzig 10 0.8320 norwegen 10 1.5690 belgien 10 1.8920 spanish 10 3.2150 sig. .152 .662 1.000 means for groups in homogeneous subsets are displayed. the error term is: mean square(error) = 0.156. a. uses harmonic mean sample size = 10.000. b. alpha = 0.05 wormwood accessions tab. 6: thujone contents of the studied a. absinthiumaccessions (gc area %) accession α-thujone β-thujone min. max. st.dev. min. max. st.dev. belgien 0.00 51.68 33.24 0.00 89.89 30.09 csor 0.00 0.27 0.18 0.00 0.30 0.12 english 0.00 0.25 0.63 0.00 1.92 0.63 hungarian 0.00 0.27 0.10 0.00 0.70 0.24 leipzig 0.00 0.75 0.72 0.00 84.44 32.51 norwegen 0.00 24.83 10.60 0.00 26.77 9.72 pakozd 0.00 0.12 0.04 0.00 0.19 0.08 spanish 0.00 0.00 0.00 0.00 0.06 0.02 wild soroksar 0.00 0.13 0.05 0.00 2.12 0.67 mean 0.00 3.33 0.00 14.56 spanish   0.44   0.39   0.37   0.46   0.40   0.40   0.43   0.38   1  132 133  134 fig.  1:  herbarium  leaf  of  a.absinthium  and  devices  for  leaf  measurements  135 136  137 figure  2:    distribution  of  individual  values  in  the  thujon  containing  accessions  (gc  area  %)  138  139 0   20   40   60   80   100   1   2   3   4   5   6   7   8   leipzig   norwegen   belgien   xxx xxx 31.5.17 16:09 gelöscht: accession ... [6] fig. 2: distribution of individual values in the thujon containing accessions (gc area %) among the thujone chemotypes, in the oils of accessions “leipzig” and “belgien” β-thujone content was much higher than that of the a isomer. in these spectra β-thujone varied from 50% to 89% of total oil while α-thujone was observed in less than 2% of total area percentage of the investigated oils. the majority of the references on wormwood indicate similar tendency. blagojević et al. (2006) investigated serbian natural populations and brought about 63.4% concentration for β-thujone with 0.4% for α-thujone. “belgien” accession has balanced percentages of both forms of thujone which may be compared to the data of rezaeinodehi and khangholi (2008) on wild collected iranian wormwood: 18.6% and 23.8%, for αand β-thujones, respectively. the remaining genotypes of our study (all accessions from hungary, “english” and “spanish”), however, can be declared as “thujonless” ones because thujones could not be demonstrated among the main components of their oils. several characterisation of wormwood accessions 243 authors reported oil composition without or with trace amounts of thujones. these samples included raw material from different regions of the world, e.g. estonia (orav et al., 2006), france (carnat et al., 1992), cuba (pino et al., 1997), etc. molecular aspect using 11 rapd primers, we detected 122 bands which means 11 bands per primer on average. the bands were in the range of 250-1500 bp. the primer b10 gave the highest number of bands (16 bands) while the primer op-a20 gave the lowest number of bands (6 bands). tab. 2 shows the number and the size of dna fragments produced by each of the rapd and issr primers. a total of 196 scorable bands were generated from 15 issr primers. the average number of amplified bands per primer was 13. the primer issr5 and cag5 composed of trinucleotide repetitions produced as many as 17 bands while primer issr7 amplified the lowest number of bands (7 ones). the size of the bands amplified by the issr primers ranged from 120 bp (by primer issr5) to 1500 bp (by issr1, issr4 and issr7) (tab. 2). the proportions of polymorphic bands among wormwood accessions were high, with 81.15% for rapd and 73.10% for issr. when comparing 15 samples of three artemisia species (a. vulgaris, a. absinthium, a. roxburghiana), the proportion of polymorphic bands was 68% (nazar and mahmood, 2011). sangwan et al. (1999) indicated 63.6% polymorphism for rapd primers during the investigations on 8 plants of artemisia annua l. in india. however, these and related studies are hardly comparable to the present one as they evaluated interspecific variability or intraspecific diversity of other artemisia species. the largest genetic distance coefficients were determined for accessions “belgien” and “spanish” (>0.4 value detected in 4 cases out of 8), (tab. 7). nei’s genetic distances among the accessions from hungary are relatively low, between 0.26 and 0.34. the closest similarity was observed between samples collected from wild growing regions in hungary: “pákozd” and “wild soroksar” (0.26) while the greatest dissimilarity was observed between the accessions “belgien” and “csór” (0.47). based on the genetic distance matrix of the 9 accessions, the upgma dendogram demonstrates the grouping of the investigated accessions in three main clusters (fig. 3). “spanish” accession seems to form alone a distinct group (group 1), accessions “norwegen” and “belgien” were classified into group 2 while all the hungarian accessions were located in the same group 3 with two further accessions, one from market in england and the other from german seed bank exchange (“leipzig”). conclusions our results show that the data obtained by different methods such as morphological, chemical and molecular analysis may effectively demonstrate the wide intraspecific variability of wormwood. at the same growing habitat, under the conditions of our experiment, characteristic differences in growth, width of the bushes, size and form of the leaves were registered. besides the actual mean values, the internal homogeneity of the studied accessions proved to be different. the coefficient of variation for the morphological traits ranged from 10,6% till 45.3% and was the highest in the case of the length of petiole. differences in leaf shape of the same individual contribute to the heterogeneity of populations and present additional difficulties in evaluation. it could be established that the accession “norwegen” presented the smallest bushes, thinnest leaf blade and smallest leaves. however, on the other side “english” demonstrated the highest growth, “spanish” developed the thickest leaves while accessions “leipzig” and “hungarian” provided the largest leaves. thus, unfortunately, a well established connection among these morphological traits can be excluded. the yield of the eo demonstrated similarly large differences among genotypes, it was determined between 0.349 and 3.215 ml/100 g. besides, intra-accession differences were also considerable, even higher than in the case of the morphological traits: cv for eo yield ranged between 18.4% and 64.4%. the three groups representing significantly different levels of this yield do not seem to be in any tab. 7: genetic distance matrix of the investigated wormwood accessions based on rapd and issr data accession csór wild sorok. pákozd hungarian english norwegen belgien leipzig spanish csór 1 wild soroksár 0.30 1 pákozd 0.34 0.26 1 hungarian 0.32 0.35 0.3 1 english 0.33 0.27 0.34 0.27 1 norwegen 0.30 0.28 0.35 0.33 0.29 1 belgien 0.47 0.40 0.37 0.31 0.44 0.42 1 leipzig 0.43 0.34 0.34 0.34 0.39 0.35 0.38 1 spanish 0.44 0.39 0.37 0.46 0.40 0.40 0.43 0.38 1 fig. 3: dendrogram showing the relationship among the studied a. absinthium accessions (generated by combination of rapd and issr primers using upgma method) 244 h.t. nguyen, k. inotai, p. radácsi, s. tavaszi-sárosi, m. ladányi, é. zámboriné-németh connection with the origin of the accession. the genotypes showing significantly similar values such as “norwegen” and “belgien” are both genebank accessions of presumably wild origin, thus they have no obvious relationship with each other. in the largest group of relatively low eo yield can be found all the hungarian wild growing populations. however, they are grouped together with geographically very distinct origins such as the english and the german one. unfortunately, the former accession has been obtained from a market item, thus may not represent a natural habitat. further systematic study would be necessary to establish any tendency for the yield of eo in connection with geographical directions. especially interesting is the variation concerning thujone content of the eo. our results demonstrate that thujone is not necessarily the main compound of the essential oil of wormwood as it is frequently interpreted by lachenmeier et al. (2006). among the 9 investigated accessions only three accumulated considerable concentrations of thujones in their essential oil. the variability for thujone content exceeded both that of the morphological traits and that of the accumulation level of volatile compounds: e.g. in accession “belgien” no individuals were found without thujones while in accession “leipzig” the individual deviations ranged from 0.0% to 86.2%. as for the isomers, β-thujone proved to be the major one, in some cases α-thujone reached comparable percentages, however, no sample was found where this latter one would have been the only isomer. based on the accumulation level of thujone and its two isomers, it seems to be relevant defining the following chemotaxonomic groups of the species: • high content of β-thujone (above 50%) + low content of α thujone (below 10%), • medium content of β-thujone (from 10 to 50%) + medium content of α-thujone (from 10 to 50%), • low content of β-thujone (below 10%) + low content of αthujone (below 10%). as the minimum values depend to some extent on the sensitivity of the equipment and investigation parameters, it does not seem reasonable to identify an absolute zero variant in this respect. based on the study, it may be concluded that large variability of morphological traits such as growth and leaf shape cannot be necessarily connected to the essential oil accumulation potential of the accessions. similarly, no characteristic marker trait has been found for essential oil composition and chemotype although it would be an advantageous phenomenon for breeding and cultivation. at the same time, the grouping of the accessions based on the eo yield coincides with the groups based on the applied rapd and issr molecular markers. acknowledgement the work was supported by the stipendium hungaricum scolarship. special thanks for prof. antonio llorens-molina (valencia) for providing us the spanish accession. conflict of interest the authors declare that they have no conflict of interest. references adams, r.p. phd., 2007: identification of essential oil components by gas chromatography/mass spectrometry, 4th edition. carol stream, ill: allured pub corp. ariño, a., arberas, i., renobales, g., arriaga, s., dominguez, j.b., 1999c: essential oil of artemisia absinthium l. from the spanish pyrenees. j. essent. oil res. 11, 182-184. doi: 10.1080/10412905.1999.9701105 basta, a., tzakou, o., couladis, m., pavlović, m., 2007: chemical composition of artemisia absinthium l. from greece. j. essent. oil res. 19, 316-318. doi: 10.1080/10412905.2007.9699291 blagojević, p., radulović, n., palić, r., stojanović, g., 2006: chemical composition of the essential oils of serbian wild-growing artemisia absinthium and artemisia vulgaris. j. agr. food. chem. 54, 4780-4789. doi: 10.1021/jf060123o carnat, a.-p., madesclaire, m., chavignon, o., lamaison, j.-l., 1992: cis-chrysanthenol, a main component in essential oil of artemisia absinthium l. growing in auvergne (massif central), france. j. essent. oil res. 4, 487-490. doi: 10.1080/10412905.1992.9698115 dewolf, g.p., 1968: notes on making an herbarium. arnoldia 28, 69-111. ema-hmpc, 2009: community herbal monograph on artemisia absinthium l., herba. committee on herbal medicinal products, european medicines agency. eu council directive, 1988: council directive 88/388/eec of 22 june 1988 on the approximation of the laws of the member states relating to flavourings for use in foodstuffs and to source materials for their production. o.j.l. 184, 61-66. goud, b.j., swamy, b.c., 2015: a review on history, controversy, traditional use, ethnobotany, phytochemistry and pharmacology of artemisia absinthium linn. int. j. adv. res. eng. appl. sci. 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54-69. orav, a., raal, a., arak, e., muurisepp, m., kailas, t., 2006: composition of the essential oil of artemisia absinthium l. of different geographi characterisation of wormwood accessions 245 cal origin. proc. estonian. acad. sci. 55, 155. pino, j.a., rosado, a., fuentes, v., 1997: chemical composition of the essential oil of artemisia absinthium l. from cuba. j. essent. oil res. 9, 87-89. doi: 10.1080/10412905.1997.9700720 rezaeinodehi, a., khangholi, s., 2008: chemical composition of the essential oil of artemisia absinthium growing wild in iran. pak. j. biol. sci. 11, 946-949. doi: 10.3923/pjbs.2008.946.949 sangwan, r.s., sangwan, n.s., jain, d.c., kumar, s., ranade, s.a., 1999: rapd profile based genetic characterization of chemotypic variants of artemisia annua l. iubmb life, 47, 935-944. van den dool, h., dec. kratz, p., 1963: a generalization of the retention index system including linear temperature programmed gas-liquid partition chromatography. j. chromatogr. a, 11, 463-471. doi: 10.1016/s0021-9673(01)80947-x yeh, f.c., yang, r.-c., boyle, t., ye, z.-h., mao, j.x., 1997: popgene: the user-friendly shareware for population genetic analysis. molecular biology and biotechnology centre. address of the authors: dr. éva németh-zámbori, department of medicinal and aromatic plants, szent istván university, 1518 budapest, p.o. box 53, hungary e-mail: zamborine.nemeth.eva@kertk.szie.hu huong nguyen thi, szilviatavaszi-sárosi, péter radácsi, katalin inotai, department of medicinal and aromatic plants, szent istván university of budapest, h-1118 budapest, villányi str. 29-35., hungary márta ladányi, department of biometrics and agricultural informatics, szent istván university, h-1118 budapest, villányi str. 29-35., hungary ladanyi.marta@kertk.szie.hu © the author(s) 2017. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 88, 5 9 (2015), doi:10.5073/jabfq.2015.088.002 1igdir university, agricultural faculty, department of horticulture, igdir-turkey 2oregon state university, agricultural faculty, department of horticulture, corvallis, oregon-usa *3atatürk university, agricultural faculty, department of horticulture, erzurum-turkey 4igdir university, agricultural faculty, biometry genetics unit, igdir-turkey phytochemical profiles and antioxidant activity of some grape accessions (vitis spp.) native to eastern anatolia of turkey sadiye peral eyduran, meleksen akin2, sezai ercisli3*, ecevit eyduran4 (received november 3, 2014) * corresponding author summary phytochemical profiles and antioxidant activity of four historical grape accessions (‘kuzu kuyrugu’, ‘miskali’, ‘erkek miskali’, and ‘kirmizi kismis’) grown in igdir province located in eastern anatolia region of turkey were examined. the amount of vitamin c, organic acids (citric acid, tartaric acid, malic acid, succinic acid and fumaric acid), sugars (fructose and glucose), phenolic acids (catechin, rutin, quercetin, chlorogenic acid, ferulic acid, o-coumaric acid, p-coumaric acid, caffeic acid, syringic acid, vanillic acid, and gallic acid), and antioxidant capacity (trolox equivalent antioxidant capacity, teac assay) were determined. accession was found to be important source of variation for all the parameters identified above (p<0.01). among the grape accessions analyzed, ‘kuzu kuyrugu’ had the predominant vitamin c (47.19 mg/100 g), chlorogenic acid (2.687 mg/l), ferulic acid (1.303 mg/l), o-coumaric acid (1.317 mg/l), and syringic acid content (1.687 mg/l). the highest citric acid (0.873 g/l), fructose (10.36 g/100g), glucose (11.51 g/100g), and catechin (1.353 mg/l) were recorded in ‘miskali’ accession. ‘kirmizi kismis’ was determined to be the accession with the highest tartaric acid (10.80 g/l), succinic acid (0.94 g/l), and caffeic acid (2.137 mg/l) levels. ‘erkek miskali’ accession produced the paramount contents for fumaric acid (0.0042 g/l), rutin (2.477 mg/l), quercetin (0.447 mg/l), and vanillic acid (0.313 mg/l). the investigated grape accessions showed notable levels of sugars, organic acids and phenolic compounds. these accessions could be valuable in breeding programs for improving grape quality and nutrition, as well as enhancing commercial worth and production of the grapes in igdir province of turkey. introduction horticultural plants including fruits, vegetables and grapes have long been valued as part of a nutritious and tasty diet. the flavours provided by different horticultural species are among the most preferred in the world, and it is increasingly evident that horticultural plants not only taste good, but are also good for human health (bacvonkralj et al., 2014; rop et al., 2014). it is well established that horticultural plants and products are a rich source of vitamins, organic acids, sugars, phenolics, minerals and dietary fibre (nonstarch polysaccharides) that are essential for normal growth and development and overall nutritional well-being (ercisli and esitken, 2004; hegedus et al., 2010). grapes are among the most economically important horticultural crops with a total worldwide production of 67 000 000 mt. china is the leading producer followed by usa. turkey ranks 6th in the world in total grape production and has the 4th largest vineyard area in the world after spain, france and italy (fao, 2012). grapes are classified as table grapes, wine grapes, raisin grapes and juice grapes (patil et al., 1995). especially in the fresh market, flavor is the most important quality criterion, mainly determined from the ratios of organic acids and sugars, which are also responsible for the taste of the fruit. tartaric, malic, citric, and succunic acid are among the most important organic acids, not exceeding the total juice weight more than 1 %. differences in organic acids content in grapes are generally associated with genotype, maturity, environmental conditions and other factors (soyer et al., 2003; robredo et al., 2011). other important chemical components of the grape are the polyphenols, well-known for their beneficial effects on human health due to their antioxidant activity (xia et al., 2010). anthocyanins, flavonoids and phenolic acids are among the main phenolic compounds (spacil et al., 2008). anthocyanins are pigments corresponding for the color of the fruit. flavonoids are mainly formed as catechin, epicatechin and procyanidin polymers. gallic, caffeic, chlorogenic, catechin, epicathechin are among the most important phenolic acids (xia et al., 2010; santos et al., 2011). many reports are available about the beneficial effects of phenolic compounds on human health as inhibiting cardiovascular diseases and cancer, as well as slowing aging (tsanga et al., 2005; god et al., 2007; shanmuganayagam et al., 2007). grapes are also very rich source in vitamins, minerals, carbohydrates and fibers, which are key components in human nutrition (xia et al., 2010). factors like genotype and environmental conditions have influence on the chemical composition and antioxidant activity of grapes (lima et al., 2014). in literature, there were several reports about the extraction and detection of sugars, organic and phenolic acids of the divergent grape cultivars grown under different ecological conditions in turkey (orak et al., 2007; sabir et al., 2010), but no published knowledge is currently available for historical grape accessions cultivated in the igdir province of turkey. therefore, the current study was conducted with the objective to determine the organic acid, sugar and phenolic acids and the anti oxidant capacities of vitis vinifera grape accessions grown in kadikislak village of igdir province of turkey. knowledge of phytochemical content and antioxidant activity of the historical accessions will be useful for the progress of the food industry related to grape production in igdir province of turkey neighboring on armenia, nackhcivan, and iran, and more specifically will provide a precious reference for further researches who are interested in the related topics. materials and methods plant material accessions ‘kuzu kuyrugu’, ‘miskali’, ‘erkek miskali’, and ‘kirmizi kismis’ were used in this study. the berry colour of ‘kuzu kuyrugu’ and ‘erkek miskali’ was white, the other accessions had red berry colour. the collection site is located in the village of kadikislak village. the village has continental climate with latitude 40° 2’ 60 n, longitude 44° 4’ 60 e, and altitude 858 meters. fruits (berries) for analyses were collected from one bunch with 6 s.p. eyduran, m. akin, s. ercisli, e. eyduran 8 grapevine plants ensuring representative samples per accessions. berries of those grape accessions were taken during commercial ripening stage varying between 1 to 20 august of the year 2013. thus, the sampling was structured according to different ripening stages across the individual accessions. the plants were established with a planting scheme of 2.5 m x 2.5 m in distance. during winter months, the plants were covered by soil to avoid winter injury. pruning was applied in feb-ruary and 3-4 buds were left on shoots. analysis of organic acids the juice of the grapes was extracted by hand mashing using cheesecloth, and stored at (-20 °c) until analyzed for the following organic acids of succinic, citric, malic, fumaric and tartaric. aforementioned organic acids were extracted according to the modified method of bevilacqua and califano (1989). each sample of 5 ml was blended with 20 ml 0.009 n h2so4 (heidolph silent crusher m, germany). for homogenization, the mixtures were stirred with a shaker (heidolph unimax 1010, germany) for 1 hour, afterwards centrifuged for 15 min at 15000 g. following initial filtration with filter paper and twice with membrane filter of 0.45 μm (millipore millex-hv hydrophilic pvdf, millipore, usa), the supernatants were run through sep-pak c18 cartridge. using aminex column (hpx 87 h, 300 mm x 7.8 mm, bio-rad laboratories, richmond, ca, usa), hplc was commanded by agilent package program with a dad detector (agilent, usa) adjusted to 214 and 280 nm wavelengths. analysis of phenolic compounds the following phenolic compounds of gallic acid, catechin, chlorogenic acid, caffeic acid, p-coumaric acid, o-coumaric acid, ferulic acid, vanillic acid, rutin, syringic acid and quercetin were extracted and determined according to the modified method of rodriguezdelgado et al. (2001). the samples were diluted with distilled water in a ratio of 1:1, and centrifuged for 15 min at 15000 g. following initial filtration with filter paper and twice with 0.45 μm membrane filter (millipore millex-hv hydrophilic pvdf, millipore, usa), the supernatants were passed through hplc. chromatographic analysis was performed by agilent 1100 (agilent) hplc system using dad dedector (agilent. usa) with 250 x 4.6 mm, 4μm ods column (hichrom, usa). as a mobile phase, solvent a methanol:acetic acid:water (10:2:28) and solvent b methanol:acetic acid:water (90:2:8) were used, with a flow rate of 1ml/min and 20 μl injection volume for spectral measurements at 254 and 280 nm. sugar analysis the sugar analysis was performed using the modified method of melgarejo et al. (2000). standards of fructose and glucose were used. homogenized samples of 5 ml were centrifuged for 2 min at 12000 g for 2 min and filtered, afterwhich run through sep-pak c18 column. μbondapak-nh2 column with 85 % acetonitrile liquid phase was used in the hplc device having a refractive index detector. calculation of the sugar concentrations was done based on fruit juice standards. analysis of ascorbic acid 5 ml juice of each sample from the fruit was transferred to test tubes and mixed with 2.5 % m-phosphoric acid solution. the mixture was centrifuged for 10 min at 6500 rpm and 4 °c. 0.5 ml from the clear part of the blend was complemented to 10 ml using % 2.5 m-phosphoric solution. afterwards filtered by 0.45 μm teflon filter and injected to hplc. c18 column (phenomenex luna c18, 250 x 4.60 mm, 5 μ) was used in the hplc device, and temperature was adjusted to 25 °c. ultra distilled water with 1 ml/min flow rate and ph of 2.2 adjusted with h2so4 was used as a mobile phase. the readings were made with dad detector at 254 nm wavelength. for determination of ascorbic acid, different concentration levels of l-ascorbic acid (sigmaa5960) (50, 100, 500, 1000, and 2000 ppm) were used (cemeroglu, 2007). determination of trolox equivalent antioxidant capacity (teac) for a standard teac measurement, abts acetate was prepared with potassium persulphate by dissolving in a buffer (ozgen et al., 2006). to keep the stability of the mixture for a long time, abts+ was diluted with 20 mm sodium acetate buffer at ph of 4.5, with absorbance of 734 nm, 0.700 ± 0.01. for spectrophotometric measurements, 3 ml abts+ solution was mixed with 20 μl fruit extract, and incubated for 10 min. absorbance values were obtained at 734 nm. statistical analysis eight grapevine plants for each accession were used for sampling. fruits (berries) were collected from different bunch with 8 plants per accession. the descriptive statistics for all quantitative characteristics under investigation were expressed as mean ± se. the data were analyzed through one-way anova with four replications and mean separation was done by using ducan multiple comparison tests. in the present paper, a significance level of 1 % was considered. all statistical computations were performed using a spss 20 programme. results and discussion the current study is a preliminary evaluation report on identification of some organic acids, sugars, and phenolic acids and antioxidant activity of native historical four grape accessions grown in kadikislak village of igdir province of turkey. results of statistical analysis in terms of organic acids from the local grape accessions in kadikislak village of igdir province of turkey are summarized in tab. 1. results of analysis of variance revealed apparently in tab. 1 that the accession was a prominent factor for organic acids in the current study (p<0.01). the current finding on organic acids was compatible with those reported in pretab. 1: organic acids content in berries of grape accessions. citric acid g/l tartaric acid g/l malic acid g/l succinic acid g/l fumaric acid g/l ‘kuzu kuyrugu’ 0.780±0.02b 8.63±0.26b 3.02±0.23a 0.44±0.01c 0.0036±0.01b ‘miskali’ 0.873±0.01a 4.30±0.20d 2.17±0.11b 0.63±0.02b 0.0013±0.01c ‘erkek miskali’ 0.298±0.01d 5.63±0.16c 2.32±0.17b 0.44±0.01c 0.0042±0.02a ‘kirmizi kismis’ 0.345±0.02c 10.80±0.19a 3.59±0.19a 0.94±0.02a 0.0036±0.01b the difference between the means with different letters in same column was significant (p<0.01) antioxidant and chemical characteristics of local grapes 7 vious published studies (soyer et al., 2003; robredo et al., 2011; liang et al., 2014). at harvest, the differences in terms of the organic acids may be attributed to cultivars, environmental conditions, and storage time (robredo et al., 2011). robredo et al. (2011) mentioned that the sugar and organic acids composition determine the flavor of the table grapes, which is a very important quality criterion in fresh market. also, they informed that organic acids provide contribution to the sourness of the fruits. the organic acids were also reported to be a significant indicator on maturity of the grapes that is an important factor for identification of the proper harvesting time (soyer et al., 2003). the order of the present accessions for citric acid was ‘miskali’ > ‘kuzu kuyrugu’ > ‘kirmizi kismis’ > ‘erkek miskali’. according to the current results, ‘kirmizi kismis’ was determined to be the prominent accession in tartaric acid. the order of the statistically significant differences for tartaric acid was ‘kirmizi kismis’ > ‘kuzu kuyrugu’ > ‘erkek miskali’ > ‘miskali’. for malic acid, the statistical order with respect to statistical test was found as ‘kirmizi kismis’ = ‘kuzu kuyruğu’ > ‘erkek miskali’ = ‘miskali’. as understood from tab. 1, corresponding accession orders for succinic and fumaric acids were ‘kirmizi kismis’ > ‘miskali’ > ‘kuzu kuyrugu’ = ‘erkek miskali’, and ‘erkek miskali’ > ‘kuzu kuyrugu’ = ‘kirmizi kismis’ > ‘miskali’, respectively. compo sition of organic acids was very important in the grape flavor, and the composition influenced by cultivars should be identified (robredo et al., 2011; soyer et al., 2003). sabir et al (2010) reported varied ranges of tartaric acid, malic acid and citric acid of five grape cultivars as 3.8-5.2 g/l, 2.8-3.6 g/l and 0.2-0.4 g/l, respectively. for red globe, thompson seedless, and crimson seedless table grapes usually grown in inner western anatolia, robredo et al. (2011) recorded 1.28, 2.05 and 1.80 g/l for tartaric acid content; 0.39, 1.80, and 0.51 g/l for malic acid content, respectively. burin et al. (2010) notified to be the ranges of 11.64-17.15 g/l for tartaric acid levels of commercial grape juices. the differences may be ascribed to the common effect of accession because all accessions were grown at a single location applying similar cultivation practices. results of statistical assessments made for antioxidant activity (teac assay), vitamin c, fructose and glucose are presented in tab. 2. as depicted in tab. 2, the analysis results of variance illustrated that the accession is of big importance in characterizing teac, vitamin c, fructose, and glucose levels (p<0.01). the highest contents for teac were statistically enabled by ‘kuzu kuyrugu’ = ‘kirmizi kismis’, followed by ‘erkek miskali’ > ‘miskali’. burin et al. (2010) notified that there were 2.51-11.05 mm for teac levels of commercial and grape juices. however, the pre dominant content for vitamin c was yielded with ‘kuzu kuyrugu’ accession compared to other accessions used in this study. the statistical significance order of the grape accessions for vitamin c was in the form of ‘kuzu kuyrugu’ > ‘erkek miskali’ > ‘miskali’ > ‘kirmizi kismis’. the accession significance order was obtained for fructose, ‘miskali’ > ‘erkek miskali’> ‘kirmizi kismis’ = ‘kuzu kuyrugu’ (tab. 2). significance order of the accessions in glucose content was noted as ‘miskali’ > ‘erkek miskali’> ‘kuzu kuyrugu’ > ‘kirmizi kismis’. for red globe, thompson seedless, and crimson seedless table grapes, robredo et al. (2011) reported, at harvest time, 8.74, 8.05, and 7.74 g/100 g for fructose content, 8.06, 8.71, and 8.03 g/100 g for glucose content, respectively. the previous results of robredo et al. (2011) were higher markedly than the glucose and fructose contents of ‘kuzu kuyrugu’, ‘erkek miskali’, and ‘kirmizi kismis’ accessions evaluated in the current study, but lower than corresponding contents of ‘miskali’ accession. additionally, several authors have reported previously that glucose and fructose contents were available in similar amounts (sabir et al., 2010; robredo et al., 2011). the mean vitamin c contents from other accessions with the exception of ‘kirmizi kismis’ accession were not in agreement with the content reported by sousa et al. (2014). the differences could be related to the individual genetical background or environmental influences. in the study of sabir et al. (2010), it was obtained that the discriminating ranges were 73.1-94.1 g/l and 86.4-107.0 g/l for fructose and glucose sugars, respectively, which was very higher when made comparison with the corresponding ranges (54.8-83.9 g/l and 50.989.9 g/l and) of rusjan and korosec koruza (2007) reported for 15 red wine cultivars. results of statistical analysis for 11 phenolic compounds are summarized in tab. 3 and 4, respectively. anova results represented that accession had a significant influence on the studied compounds notified in tab. 3 and 4 (p<0.01). in terms of catechin content, the highest content was obtained by ‘miskali’ accession (1.353 mg/l), but the lowest content by ‘kirmizi kismis’ accession. the accession significance order for catechin content was ‘miskali’ > ‘erkek miskali’ > ‘kuzu kuyrugu’ > ‘kirmizi kismis’. rutin content of the grapes in the current study ranged from 0.950 to 2.477 mg/l. with regard to duncan results, the statistical significance order of tab. 2: content of vitamin c, fructose and glucose and trolox equivalent antioxidant capacity (teac) in berries of grape accessions. teac (μmol te g-1 ) vitamin c mg/100 g fructose g/100 g glucose g/100 g ‘kuzu kuyrugu’ 8.15±0.392ay 47.19±0.30a 5.37±0.15c 6.55±0.25c ‘miskali’ 3.35±0.159c 16.17±0.21c 10.36±0.17a 11.51±0.25a ‘erkek miskali’ 6.29±0.332b 23.62±0.38b 6.59±0.22b 7.34±0.17b ‘kirmizi kismis’ 7.29±0.190a 9.85±0.31d 5.43±0.16c 5.86±0.06d the difference between the means with different letters in same column was significant (p<0.01) tab. 3: phenolic compounds in berries of the grape accessions. catechin mg/l rutin mg/l quercetin mg/l chlorogenic acid mg/l ferulic acid mg/l ‘kuzu kuyrugu’ 0.680±0.006cy 0.950±0.012c 0.337±0.018b 2.687±0.015a 1.303±0.043a ‘miskali’ 1.353±0.012a 2.270±0.090b 0.193±0.009c 2.307±0.049b 0.057±0.003d ‘erkek miskali’ 1.073±0.026b 2.477±0.041a 0.447±0.020a 0.947±0.018c 0.617±0.019b ‘kirmizi kismis’ 0.347±0.012d 1.097±0.041c 0.220±0.012b 0.743±0.019d 0.327±0.009c the difference between the means with different letters in same column was significant (p<0.01) 8 s.p. eyduran, m. akin, s. ercisli, e. eyduran the accessions for rutin content was assigned as ‘erkek miskali’ > ‘miskali’ > ‘kirmizi kismis’ = ‘kuzu kuyrugu’. as perceived expressly from tab. 3, ‘erkek miskali’ was the grape accession containing the maximum quercetin content. as to tab. 3, it was clear that the accession with the maximum content in chlorogenic acid was found to be ‘kuzu kuyrugu’, followed statistically by ‘miskali’ > ‘erkek miskali’ > ‘kirmizi kismis’ with the range of 0.743 to 2.687 mg/l, respectively. for ferulic acid content with the range of 0.057 to 1.303 mg/l, the order of the grape accessions was ‘kuzu kuyrugu’ > ‘erkek miskali’ > ‘kirmizi kismis’ > ‘miskali’ (tab. 3). the pre sent results from tab. 3 were quite lower compared to those of lima et al. (2014), who obtained the ranges in catechin (4.7 21.0 mg/l), and chlorogenic acid (2.1-21.3 mg/l), for new brazilian grape cultivars, respectively. but, the findings were in disagreement with the ranges of rutin (0.9-5.9 mg/l), and quercetin (0.5 0.8 mg/l) reported by lima et al. (2014). for emir, kalecik karası, and narince grape cultivars, baydar (2006) confirmed 0.28, 18.95, and 1.08 μg/g for rutin content; 0.35, 0.87, and 0.60 μg/g for quercetin content; 0.20, 0.21, and nd for ferulic acid content, and 0.22, nd, and 2.26 for chlorogenic acid content, respectively (nd=not detected). the highest content of o-coumaric acid in the current investigation was recorded for ‘kuzu kuyrugu’ accession and the significance order for o-coumaric acid of the accessions assessed herein was ‘kuzu kuyrugu’ > ‘miskali’ > ‘kirmizi kismis’ > ‘erkek miskali’ as also seen in tab. 4. the means of o-coumaric acid content obtained for the inspected accessions were lower noticeably than those of the p-coumaric acid contents obtained for the accessions (tab. 4). for the p-coumaric acid content, the accession significance order verified statistically in tab. 4 was indicated as ‘kirmizi kismis’ = ‘erkek miskali’ > ‘miskali’ = ‘kuzu kuyrugu’. as also depicted in tab. 4, within the accessions, ‘kirmizi kismis’ with the mean of 2.137 mg/l had the uppermost level for caffeic acid content with the statistical accession order, ‘kirmizi kismis’ > ‘erkek miskali’= ‘kuzu kuyrugu’ > ‘miskali’. ‘kuzu kuyrugu’ accession produced the prominent syringic acid content with 1.687 mg/l, but the lowest content of 0.317 mg/l was extracted from ‘kirmizi kismis’ grape accession. from tab. 4, the accession order in syringic acid was statistically detected as ‘kuzu kuyrugu’> ‘erkek miskali’> ‘miskali’ > ‘kirmizi kismis’. the vanillic acid content of ‘miskali’ amongst accessions was not quantifiable, and the statistically significance order was given as, ‘erkek miskali’ (0.313 mg/l) > ‘kirmizi kismis’ (0.143 mg/l) > ‘kuzu kuyrugu’ (0.087 mg/l) in tab. 4. the order was formed for gallic acid content, ‘erkek miskali’= ‘kuzu kuyrugu’> ‘miskali’ > ‘kirmizi kismis’. the present results from tab. 4 were higher than those of lima et al. (2014), who detected the very wide range for p-coumaric acid (2.1-9.0 mg/l) extracted from new brazilian grape cultivars, respectively. for three grape cultivars, viz. emir, kalecik karası, and narince, baydar (2006) reported 0.30, 0.46 and 1.22 μg/g for o-coumaric content; 0.13, 0.15, and 0.46 μg/g for p-coumaric content; 0.18, nd, and 0.33 μg/g for caffeic acid content; nd, 0.55, and nd μg/g for syringic acid content; 0.62, 5.20, and 1.30 μg/g for gallic acid content respectively (nd=not detected). the difference may be due to cultivars and environmental variations (baydar, 2006) in terms of phenolic compounds. conclusion the current study is a first evaluation report on the organic acids, sugars, phenolic compounds and antioxidant capacity of the four grape accessions grown in kadikislak village of igdir province, turkey. ‘kuzu kuyrugu’ accessions were found to contain very high content of ascorbic acid, chlorogenic acid and ferulic acid and show high antioxidant activity. ‘miskali’ and ‘erkek miskali’ were found rich in terms of rutin and catechin. ‘kirmizi kismis’ contained high content of tartaric acid. therefore, the present results are expected to provide some useful information for further use of promising accessions in grape breeding to obtain high levels of phytochemicals in berries for human health and nutrition. references bacvonkralj, m., jug, t., komel, e., fajt, n., jarni, k., zivkovic, j., mujici, i., trutic, n., 2014: effects of ripening degree and sample preparation on peach aroma profile characterization by headspace solidphase microextraction. turk. j. agric. for. 38, 676-687. baydar, n.g., 2006: organic acid, tocopherol, and phenolic compositions of some turkish grape cultivars. chem. nat. compd. 4 (2), 156-159. bevilacqua, a.e., califano, a.n., 1989: determination of organic acids in dairy products by high performance liquid chromatography. j. food sci. 54, 1076-1079. burin, v.m., falcao, l.d., gonzaga, l.v., fett, r., rosier, j.p., bordignon-luiz, m.t., 2010: colour, phenolic content and antioxidant activity of grape juice. ciénc. tecnol. aliment., campinas. 30, 10271032. cemeroglu, b., 2007: gıda analizleri. gıda teknolojisi derneği yayınları. 34, 168-171. ankara, turkey. ercisli, s., esitken, a., 2004: fruit characteristics of native rose hip (rosa spp.) selections from the erzurum province of turkey. new zeal. j. crop hort. 32, 51-53. fao, 2012. agricultural data. <http:// faostat.fao.org/ >. 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(eds.), handbook of fruit science and technology, 7-38. new york, marcel dekker. robredo, p.m., robledo, p., manriquez, d., molina, r., defilippi, g., 2011: characterization of sugars and organic acids in commercial varieties of table grapes. chil. j. agr. res. 71, 452-458. rodriguez-delgado, m.a., malovana, s., perez, j.p., borges, t., garcia-montelongo, f.j., 2001: separation of phenolic compounds by high-performance liquid chromatography with absorbance and fluorimetric detection. j. chroma. 912, 249-257. rop, o., ercisli, s., mlcek, j., jurikova, t., hoza, i., 2014: antioxidant and radical scavenging activities in fruits of 6 sea buckthorn (hippophae rhamnoides l.) cultivars. turk. j. agric. for. 38, 224-232. rusjan, d., korosec-koruza, z., 2007: morphometrical and biochemical characteristics of red grape varieties (vitis vinifera l.) from collection vineyard. acta agric. slovenica 89 (1), 245-257. sabir, a., kafkas, e., tangolar, s., 2010: distribution of major sugars, acids and total phenols in juice of five grapevine (vitis spp.) cultivars at different stages of berry development. spanish j. agric. res. 8 (2), 425-433. santos, l.p., morais, d.r., souza, n.e., cottica, s.m., boroski, m., visentainer, j.v., 2011: phenolic compounds and fatty acids in different parts of vitis labrusca and v. vinifera grapes. food res. int. 44, 1414-1418. shanmuganayagam, d., warner, t.f., krueger, c.g., reed, j.d., folts, j.d., 2007: concord grape juice attenuates platelet aggregation, serum cholesterol and development of atheroma in hypercholesterolemic rabbits. atherosclerosis 190, 135-142. sousa, e.c., uchôa-thomaz, a.m.a., carioca, j.o.b., maia de morais, s.d.e., lima, a., martins, c.g., alexandrino, c.d., ferreira, p.a.t., rodrigues, a.l.m., rodrigues, s.p., do nascimento silva, j., rodrigues, l.l., 2014: chemical composition and bioactive compounds of grape pomace (vitis vinifera l.), benitaka variety, grown in the semiarid region of northeast brazil. food sci. tech. 34 (1), 135142. soyer, y., koca, n., karadeniz, f., 2003: organic acid profile of turkish white grapes and grape juices. j. food comp. anal. 16, 629-636. spacil, z., novakova, l., solich, p., 2008: analysis of phenolic compounds by high performance liquidchromatography and ultra performance liquid chromatography. talanta. 76, 189-199. tsanga, c., higginsa, s., duthiea, g.g., duthiea, s.j., howiea, m., mullena, w., leana, m.e.j., crozier, a., 2005: the influence of moderate red wine consumption on antioxidant status and indices of oxidative stress associated with chd in healthy volunteers. br. j. nutr. 93, 233-240. xia, e., deng, g., guo, y., huabin, l., 2010: biological activities of polyphenols from grapes. int. j. mol. sci. 11, 622-646. address of the corresponding author: e-mail: sercisli@gmail.com journal of applied botany and food quality 90, 115 125 (2017), doi:10.5073/jabfq.2017.090.014 1 university of bandırma onyedi eylül, bandırma vocational school, department of food processing, balıkesir, turkey 2 university of uludag, agricultural faculty, department of food engineering, bursa, turkey 3 kurtsan pharmaceuticals inc., bandırma, balikesir, turkey 4 university of balikesir, engineering and architechture faculty, department food engineering, balikesir, turkey influence of hot air drying on phenolic compounds and antioxidant capacity of blueberry (vaccinium myrtillus) fruit and leaf nurcan değirmencioğlu1*, ozan gürbüz2, gözde erdem karatepe3, reyhan irkin4 (received october 20, 2016) * corresponding author summary the present study was undertaken to assess the effects of hot air drying on phenolic compositions, total phenolic (tp) content, total anthocyanin (ta) content, as well as antioxidant capacities of methanol extracts from blueberry (vaccinium myrtillus) fruit and leaf introduced in kapıdağ region of turkey climate conditions. a total of twenty-two phenolic standards were screened by hplc, total phenols were measured by spectrophotometric methods, antioxidant capacity was determined using dpph, cuprac, abts, and frap assays in the blueberry fruit and leaf extracts. analysis by hplc revealed that fruit extracts have different phenolic profiles due to drying process and contain syringic acid, myricetin, naringin, (-)-epicatechin, and malvidine-3-o-glucoside chloride as the main compounds. leaf extracts had higher resveratrol concentrations than fruit extracts. the tp and ta contents gradually increased when the blueberry fruits were dried under hot air condition. the fresh and dried blueberry fruit and leaf extracts showed similar antioxidant capacity values. significant relationships between antioxidant capacity and tp were found. keywords vac. myrtillus; blueberry; hot ait drying; fruit; leaf; phenolics; antioxidant capacity introduction blueberry, a perennial shrub of the genus vaccinium, family ericaceae, became well known around the world due to high levels of phenolic compounds (ehlenfeldt and prıor, 2001; prıor et al., 2001; kım et al., 2010; routray et al., 2014). these compounds have been reported to have numerous valuable health benefits including superb antioxidant, anti-hypertensive, anti-diabetic, anti-leukemia, anti-obesity, anti-inflammatory, and anti-microbial activity, as well as neuroactive properties, to protect against cancer and stroke (ehlenfeldt and prıor, 2001; deng et al., 2014; lı et al., 2013). blueberries are considered to be one of the richest sources of phenolic compounds and antioxidant phytochemicals among fruits and vegetables, and they contain significant levels of anthocyanins, flavonols, flavonons, proanthocyanidins, and phenolic acids (castrejón et al., 2008; wang et al., 2012). factors that have an impact on the total phenolic content, total anthocyanins, and the antioxidant capacity of blueberries fruit and leaves, include genetic differences, the cultivar type, growing location and season, agronomic factors, the degree of maturity at harvest, and postharvest storage conditions (ehlenfeldt and prıor, 2001; deng et al., 2014). drying, as a preservation method, is a very important aspect of food processing. the main functions of drying are lowering the water activity, inhibiting the growth of microorganisms, decreasing chemical reactions, extending shelf life, allowing for room temperature storage, reducing transportation costs with regard to refrigeration, and also enhancing visual and taste of cereals, confections, and baked goods. several drying techniques such as sun drying, convection oven drying, freeze drying, microwave drying etc., have been employed in an effort to achieve high quality dried blueberries (mejıa-meza et al., 2008; hamrounı-sellamı et al., 2013). the selection of drying methods to be used is dependent on the use of the end product, economic viability, availability of resources, and composition of the biomaterial (routray et al., 2014). convective hot air drying is a traditional, low cost technique that is widely used to lower the water content of fresh products at present, nevertheless it requires relatively long times and high temperatures, causes degradation of important nutrients, has shrunken and toughened dried products with noticeable browning, and allows for little rehydration ability (sellappan et al., 2002). the aim of this study was to determine the influence of location, part of plant (fruit or leaf), and oven drying on the phenolic compounds, total phenol and anthocyanin contents, and antioxidant capacities of blueberries (vac. myrtillus). material and methods sample collection and preparation blueberry leaves and fruit grown at three different locations of erdek (sea level, balikesir, turkey) and kapıdağ (altitudes of 650 m, balıkesir, turkey) regions were randomly hand-picked during october and november, 2014, and transported to the laboratory within the same day. upon arrival, the fruit and leaves were hand selected and separated into two lots of equal weight. one lot of fruit and leaves were stored separately fresh, and the other lots were firstly dried at 50 °c for 2 h in an air oven type fn 055 (nuve, i̇stanbul, turkey) then cooled. the two lots were then separately vacuum packaged (vc 999/k12na packing machine, verpackungssysteme ag, herisau, switzerland) in fmxbk polyamide-polyethylene film (po2=15 cm3/ m2/24 h at 23 °c and 75 % relative humidity; flexopack s.a. plastics industry, koropi, greece) and stored at -20 °c (sf 312, dairei, tokyo, japan) until further analysis. extracts of vac. myrtillus fruit and leaf were prepared according to the method of described by ehlenfeldt and prıor (2001) and prıor et al. (2001), with some modifications. fresh and dried fruit and leave samples (2 g) were separately extracted twice with 20 ml of methanol:formic acid (99.5/0.5, v/v, for fruit), and acetone:formic acid (99.5/0.5, v/v, for leaf) mixture in an ultrasonic bath at room temperature (20 °c) for 15 min. extracts then were separately centrifuged at 3500 rpm for 10 min at 4 °c in a centrifuge (sigma 3k30, uk). the supernatants were combined, after removal of methanol and acetone with a rotary evaporator (heidolph laborota 4001, germany) under vacuum conditions at 40 °c, the residual extracts were subjected to a liquid-liquid partition with methanol:formic acid (99.5/0.5, v/v, for fruit) and acetone:formic acid (99.5/0.5, v/v, for leaves), respectively, filtered through a nylon filter membrane (sigma z290793, pore size 0.45 μm, diam. 47 mm), transferred to vials, and stored -20 °c until further analysis. 116 n. değirmencioğlu, o. gürbüz, g.e. karatepe, r. irkin chemicals phenolic standards were obtained from fluka (st. louis, mo, usa) (gallic acid; cas:149-91-7, ferulic acid; cas:537-98-4, resveratrol; cas:501-36-0, (+)-catechin; cas:154-23-4, (-)-epicatechin; cas:490-46-0, myricetin; cas:529-44-2, kaempferol; cas:52018-3, (-)-epigallocatechin; cas:970-84-1, sigma (st. louis, mo, usa) (quercetin; cas:117-39-5, caffeic acid; cas:331-39-5, syringic acid; cas:530-87-4, p-coumaric acid; cas:501-98-4, naringin; cas:10236-47-2, hesperidin; cas:520-26-3, neohesperidin; cas:13241-33-3, rutin hydrate; cas:207671-50-9, cyanidin-3-oglycoside chloride; cas:7084-24-4, malvidine-3-o-glycoside chloride; cas:7228-78-6), aldrich (st. louis, mo, usa) (vanillic acid; cas:121-34-6, trans ferulic acid; cas:537-98-4, 3-hydroxy-4 metoxy-cinnamic acid; cas:637-73-5), hwi analytik gmbh (ruelzheim, germany) (chlorogenic acid; cas:327-97-9). calibration curves were made by diluting stock standards in methanol. determination of phenolic composition using hplc phenolic compositions were analysed according to a previously reported method with modifications in hplc elution conditions (sellappan et al., 2002). the phenolic extracts, phenolic standards and also all solvents were filtered through a nylon filter membrane (sigma z290793, pore size 0.45 μm, diam. 47 mm) prior to hplc analysis and then analysed in a hplc chromatography system (shimadzu class vp v.6.14 sp1, usa) equipped with shimadzu diode array detector (spd-m 10a), vp and reversed-phase c18 column (zorbax eclipse xdb, agilent, 4.6 mm, 150 mm, 5 μm). the temperature of the column oven was set at 40 °c. the wavelengths used for the quantification of phenolic compounds by the detector were: 280 nm for syringic acid, gallic acid, (+)-catechin, neohesperidin, caffeic acid, hesperidin, (-)-epigallocatechin, (-)-epicatechin, naringin, vanillic acid; 320 nm for trans-ferulic acid, chlorogenic acid, 3-hydroxy-4-metoxy-cinnamic acid, resveratrol, p-coumaric acid, ferulic acid; 360 nm for myricetin, rutin hydrate, kaempferol, quercetin; and 520 nm for cyanidin-3-o-glycoside chloride, malvidine-3-o glycoside chloride. a gradient elution was employed with mobil phase consisting of methanol:water:formic acid (3.5/96.4/0.1, v/v/v, solvent a) and acetonitril:formic acid (98/2, v/v, solvent b) as follows: the composition of b was increased from 0.5 % to 7.5 % after 31 min, increased to 10 % for 9 min, and increased to 14 % for 5 min, increased to 18 % for 5 min, increased to 30 % for 10 min, increased to 45 % for 5 min, and increased to 60 % for 5 min. the composition was decreased to 40 % for 5 min. the injection volume was 20 μl, the flow rate was 0.7 ml/min at room temperature, the duration of a single run was 75 min. all phenolic acids were quantified using an external standard. the total phenolic extracts and standard compounds were analyzed under the same analysis conditions and a 10 min equilibrium time was allowed between injections. all standard and sample solutions were injected in triplicate. determination of total phenolic (tp) content the total phenolic (tp) contents of fresh and dried blueberry fruit and leave extracts were measured by the folin-ciocalteu method described by sıngleton et al. (1999), with some modifications. briefly, an aliquot (0.5 ml) of appropriately diluted extracts, or standard solutions of gallic acid, 1.5 ml of double distilled water and 2.5 ml folin-ciocalteu reagent, were mixed within volumetric flasks at room temperature. after 10 min, 0.25 ml of 7.5 % sodium carbonate (1:3 diluted with double distilled water) solution (m/v) was added and mixed thoroughly. the absorbance of the solution was measured using a spectrophotometer (uvmecasys optizen 3220) at 750 nm after 30 min in the dark at room temperature. methanol was used as the blank and gallic acid was used for calibration of the standard curve (0-500 mg/l). the results were expressed as mg of gallic acid equivalents (gae) per kg. each extract was measured in triplicate. determination of total anthocyanin (ta) content the total anthocyanin content of extracts obtained from blueberry fruit and leaves were determined by means of the ph-differential method as described by sellappan et al. (2002). the absorbance was measured using a spectrophotometer (uvmecasys optizen 3220) at 700 nm and at the wavelength of maximum absorption (520 nm) against a blank and calculated as: a = (a520 – a700) ph1.0 (a520 – a700) ph4.5 monomeric anthocyanin pigment concentration of extracts was calculated as cyanidin-3-glucoside equivalent and each extract was measured in triplicate. monomeric anthocyanin pigment (mg/l) = a × mw × df × 1000 (ε × 1) where a = absorbance, mw = molecular weight (449.2), df = dilution factor, ε = molar absortivity (26900). the final concentration of total anthocyanins (mg/kg) was calculated based on total volume of extract and weight of sample. determination of antioxidant capacity by cupric ion reducing antioxidant capacity (cuprac) assay determination of cuprac was conducted according to the method by apak et al. (2007). one ml 10 mmol/l cucl2, 1 ml 7.5 mmol/l neocuproine, 1 ml 1 m nh4ac, × ml extract, and (4-×) ml h2o were mixed. the tubes were stopped and after 30 min the final absorbance was recorded using a spectrophotometer (uvmecasys optizen 3220) at 450 nm against a reagent blank. a standard curve was prepared using different concentrations of trolox. the calculations of the antioxidant capacity of phenolic antioxidants were expressed as μmol of trolox equivalent (te) per gram. each extract was measured in triplicate. determination of antioxidant capacity by dpph (2,2-diphenyl-2picrylhydrazyl) free radical assay the free radical scavenging capacity of the blueberry fruit and leave extracts were determined by colorimetric method described by brand-wıllıams et al. (1995). in brief, the appropriately diluted extracts (× ml), methanol (4-× ml), and dpph solution (3.9 ml, 50 μm) in methanol were incubated in a water bath at 37 °c for 30 min. after incubation, the absorbance was measured at 515 nm with a spectrophotometer (uvmecasys optizen 3220). the results were calculated against methanol without dpph and compared to a different concentration of trolox standard curve. each extract was measured in triplicate. dpph values, derived triplicate analyses, were expressed as μmol of trolox equivalent (te) per gram and were calculated as follows: dpph radical scavenging capacity (%) = (1-[asample/acontrol]) × 100 determination of antioxidant capacity by abts [2,2-azinobis(3ethylbenzothiazoline-6-sulphonic acid)] assay the abts method is based on the deactivation of the antioxidant radical cation abts·+. the abts method was performed as described by re et al. (1999). abts radical cation (abts+) was produced by reacting 7 mm abts solution with 2.45 mm potassium persulfate (k2s2o8) aqueous solution and allowing the mixture to stand in the dark at room temperature for 12-16 h before use. different concentrations of fruit and leaf extracts were mixed with 1 ml of diluted abts·+ solution and the reduction of abts·+ radical was measured by the decrease in absorbance at 734 nm after 6 min by bioactive compounds in fruits and leaves of blueberry 117 using the spectrophotometer uvmecasys optizen 3220. to develop a standard curve, a standard trolox solution was diluted with ethanol and added to 1 ml of the diluted abts·+ solution. the controls contained the extraction solvent instead of the test samples. each extract was measured in triplicate. the scavenging capacity of abts free radical was calculated as: abts radical scavenging capacity (%) = (1-[asample/acontrol]) × 100 determination of antioxidant capacity by frap assay the frap assay was conducted according to benzıe and straın (1996). this method is based on an increase of the absorbance at 593 nm due to the formation of tripyridyl-s-triazine complexes with fe2+ [tptz-fe(ii)] in the presence of a reductive agent. the frap reagent was prepared by mixing tptz solution (10 mmol/l) in hydrochloric acid (40 mmol/l) and fecl3 solution (20 mmol/l) mixed with 25 ml of acetate buffer (0.3 mol/l, ph=3.6). an appropriately diluted sample extract (× μl) and frap reagent (1-× ml) were added and, the mixture and extraction or solvent for the reagent blank were incubated at 37 °c for 30 min. at the end of incubation, absorbance was immediately measured using a spectrophotometer (perkin elmer uv/vis lambda35) at 595 nm. solutions of trolox dissolved in extraction solvent, ranging from 10-100 μmol/l were used for preparation of a calibration curve. frap values, derived from triplicate analyses, and were expressed as μmol of trolox equivalent (te) per gram. each extract was measured in triplicate. statistical analysis statistical differences between the data sets were determined by two-way analysis of variance (anova) using the spss statistical package (spss 16.0, chicago, il). differences between treatments that are described subsequently as being significant, were determined at least p<0.05. the least significant difference (lsd) test was used to determine differences between means. results and discussion phenolic compositions the data set for the contents of phenolic acids, flavonols, flavanones, monomeric of flavan-3-ol derivatives, anthocyanins, and the stilbene in the vac. myrtillus fresh and dried fruit and leaf extracts are given in tab. 1-4. these compounds can act as antioxidants and may be important components of functional foods. the dominant phenolic acid was syringic acid in fresh fruit extracts grown in erdek and kapıdağ regions (28.79-637.43 mg/kg fw, 339.13-995.15 mg/kg fw, respectively), and their content was especially high (p<0.05) in dried fruits. in fresh and dried vac. myrtillus leaf extracts, syringic, p-coumaric, gallic and vanillic acids were the most abundant phenolic acids. some research has reported that chlorogenic acid, referred to as 5-o-caffeoylquinic acid (5-cqa), is considered a major colourless phenolic acid in blueberry fruit and leaf (harrıs et al., 2007; kım et al., 2010), and a more readily available sources of 5-cqa, even compared to green coffee beans (kım et al., 2010). prıor et al. (2001) found the level of chlorogenic acid in blueberries to be 60100 mg/g of fresh fruit, while harrıs et al. (2007) detected this compound 30 times more concentrated in the leaf extract than in fruit. nevertheless, in this study high chlorogenic acid levels in the fruit and leaf extracts were not determined. on the other side, vanillic acid, 3-hydroxy-4-metoxy cinnamic acid, and ferulic acid were obtained in fresh fruit extracts, whereas in dried fruit extracts, higher levels of vanillic acid, gallic acid, p-coumaric acid, ferulic acid, and 3-hydroxy-4-metoxy cinnamic acid were determined. apart from chlorogenic acid, the phenolic acids have been found to be present in smaller concentrations, such as caffeic, p-coumaric and ferulic acid (sellappan et al., 2002). in addition, other phenols that may be found include gallic, p-hydroxybenzoic, m-hydroxybenzoic, ellagic, vanillic, protocatechuic, gentisic, syringic, sinapic and salicylic acids, and catechin, epicatechin, myricetin, and kaempferol (sellappan et al., 2002; harrıs et al., 2007). in our samples obtained from erdek and kapıdağ, we found some of these components, and most phenolic compounds detected in this study were consistent with previous reports on blueberry fruit and leaves from different locations in the world. it is believed that the significant qualitative and quantitative differences (p<0.05) of phenolic compounds profiles which have been confirmed in blueberries, are due to variations in genotypes, locations, cultivation conditions, increased maturity, different parts of plants examined, stresses, organically grown, extraction methods, and all can have varying effects on the level of total anthocyanins, total phenolics and antioxidant capacity (xıaoyong and lumıng, 2014). also drying conditions can cause differences. some recent studies have been accomplished on the content of blueberry and bilberry native flavonols (sellappan et al., 2002; harrıs et al., 2007; moze et al., 2011; vrhovsek et al., 2012). in this study, fresh vac. myrtillus fruit extracts contained low flavonols contents (0.79-91.98 mg/kg fw), whereas dried fruit and leaf extracts contained relatively high flavonols. while myricetin was found to be the main flavonol compound in the leaves, kaempferol and quercetin were also detected, in agreement with other studies. previous research also determined that the green leaves of blueberry contained a much larger amount of flavonoids (quercetin and kaempferol) and hydroxycinnamic acid (p-coumaric and caffeic acid) than fruits (rııhınen et al., 2008). nevertheless, in this study the concentrations of myricetin were higher in the fresh leaves extracts than fresh fruit extracts (p<0.05). also in the dried fruit extracts, the concentrations of flavonols were considerably higher than in the dried leaf extracts. as mentioned by jaakola et al. (2004) and oszmiański et al. (2011), p-coumaric acid is the precursor of flavonoids, and the increase in p-coumaric acid concentration in the leaves, growing under high solar radiation, can also reflect the overall activation of flavonoid biosynthesis. tab. 1-4 shows that all the flavonol compounds had some significant (p<0.05) level of variability due to altitude, and due to the drying process. rutin hydrate, which has been reported in high amounts in buckwheat (moze et al., 2011), was found in both regions of vac. myrtillus fruits under investigation, the dried fruit extracts containing more rutin hydrate than fresh fruit extracts and also compared to the fresh and dried leaf extracts. moze et al. (2011) first detected rutin in bilberry and blueberry samples (0.2 and 3.1 mg/100g fw, respectively), and also rutin (quercetin-3-o-rutinoside) was the major phenolic compounds in leaves of rabbiteye blueberry cultivated in japan (lı et al., 2013). harrıs et al. (2007) determined this compound as 3.10 mg/100 g in vac. corybocum l. fruits, whereas our results were higher than their results. bioflavonoids like rutin and naringin have been proven to be efficacious antioxidants and are widely distributed in fruits and vegetables. rutin belongs to the class of flavonols and naringin belongs to flavanones. it is well noted and proven in several studies, that flavonols are very active in conveying therapeutic benefit compared to flavonones (akondı et al., 2011). the flavonones (naringin, hesperidin, and neohesperidin) were detected in studied vac. myrtillus extracts. naringin is one of the most abundant flavanone in fresh and dried fruit and leaf extracts in this study. it was also observed that naringin levels were greater in samples harvested from high altitude compared to those samples originating from areas at sea level. some reports have indicated that drying methods can affect phenolic contents and the antioxidant capacity of plant materials due to drying time/temperature, light intensity, packaging, and storage time etc. (lu and luthrıa, 2014). the three flavanols were found in both regions of vac. myrtillus, the dried fruit extracts being a better source of (+)-catechin, (-)-epicate 118 n. değirmencioğlu, o. gürbüz, g.e. karatepe, r. irkin tab. 1: phenolic compounds concentrations of fresh vac. myrtillus fruits grown in erdek ve kapıdağ regions (mg/kg) phenolic compounds e1frf* e2frf e3frf k1frf k2frf k3frf gallic acid 19.19 ± 2.12cb** 36.82 ± 4.58aa 30.29 ± 3.14ba 10.00 ± 1.13dk 8.10 ± 1.10dl 0.87 ± 0.12em vanillic acid 532.97 ± 25.16aa 52.36 ± 3.25cc 289.61 ± 5.12bb 20.91 ± 1.95el 9.25 ± 1.63fm 29.75 ± 1.98dk caffeic acid 5.46 ± 1.23aa 4.84 ± 1.10bc 5.35 ± 1.00ab 1.87 ±0.12ck 1.86 ± 0.24ck 1.73 ± 0.23dl chlorogenic acid 1.25 ± 0.25ec 4.37 ± 0.85aa 1.71 ± 0.26cb 1.42 ± 0.10dl 1.50 ± 0.27cdl 2.71 ± 0.45bk syringic acid 637.43 ± 3.45ba 28.79 ± 4.85fc 32.30 ± 1.26eb 529.53 ± 6.52cl 995.15±6.54ak 339.13± 3.21dm p-coumaric acid 7.63 ± 1.48ca 1.81 ± 0.12ec 4.04 ± 1.85db 11.18 ± 2.13bl 20.05 ± 2.31ak 8.68 ± 1.00cm ferulic acid 10.60 ± 2.69da 6.06 ± 0.95eb 4.13 ± 0.74fc 20.54 ± 2.31bl 24.87 ± 1.98ak 14.77 ± 1.14cm trans-ferulic acid 5.99 ± 1.78da 1.89 ± 0.14eb 5.45 ± 1.23da 8.49 ± 2.85cm 11.50 ± 2.14bl 19.40 ± 1.56ak 3-hydroxy-4-methoxy 50.63 ± 4.65db 75.95 ± 4.65ca 26.29 ± 2.32ec 78.11 ± 3.12bcm 103.39 ± 6.54ak 80.61 ± 3.24bl cinnamic acid myricetin 51.64 ± 2.98db 55.12 ± 3.12da 43.06 ± 2.96ec 79.78 ± 2.96cm 91.98 ± 3.45ak 83.39 ± 3.59bl quercetin 1.62 ± 0.12ec 4.27 ± 1.00bb 6.93 ± 1.85aa 2.48 ± 0.62dl 3.71 ± 0.56ck 2.24 ± 0.46dl kaempferol 1.72 ± 0.45aba 0.94 ± 0.16db 1.82 ± 0.15aa 0.79 ± 0.10em 1.01 ± 0.15dl 1.67 ± 0.37bk rutin hydrate 3.84 ± 1.10da 1.31 ± 0.25fc 2.07 ± 0.23eb 13.30 ± 1.10bl 24.01 ± 1.47ak 7.64 ± 0.96cm naringin 5.50 ± 1.14dc 33.60 ± 3.74bb 79.37 ± 3.41aa 4.42 ± 0.65em 6.97 ± 0.84ck 6.13 ± 0.45dl hesperidin 7.07 ± 1.56cc 27.45 ± 2.85bb 31.59 ± 2.16aa 2.40 ± 0.13el 4.93 ± 0.16dk 2.35 ± 0.78el neohesperidin 2.50 ± 0.45bb 8.38 ± 2.14aa 8.80 ± 1.62aa 1.83 ± 0.14cdl 1.92 ± 0.11ck 1.73 ± 0.15cbm (+)-catechin 2.72 ± 0.41dc 29.57 ± 2.48aa 9.45 ± 1.64bb 6.41 ± 0.64ck 1.36 ± 0.12em 2.63 ± 0.43dl (-)-epicatechin 28.54 ± 3.41da 10.43 ± 1.96eb 8.63 ± 1.18fc 50.63 ± 2.63bl 80.23 ± 4.85ak 46.04 ± 2.45cm (-)-epigallocatechin 6.66 ± 1.58dc 24.99 ± 3.11bb 82.62 ± 3.98aa 17.88 ± 1.84cl 22.79 ± 2.41bk 16.32 ± 1.47cm cyanidin-3-o-glucoside 373.38 ± 15.69ca 70.94 ± 4.23eb 384.06 ± 4.85ca 438.33 ± 5.23bl 781.26 ± 6.52ak 335.60 ± 6.21dm chloride malvidine-3-o-glucoside 3231.38 ± 25.89ca 212.93 ± 6.52fc 329.94 ± 6.12eb 3644.61 ± 8.95bl 4433.19 ± 9.96ak 2433.10 ± 5.27dm chloride resveratrol 1.01 ± 0.05aa 0.87 ± 0.05bb 0.78 ± 0.05bb 0.70 ± 0.06bm 0.84 ± 0.06bl 1.00 ± 0.00ak * e: erdek, k: kapıdağ, fr: fresh, f: fruit, 1-3: codes of samples collected from different regions, ** mean values (mg/kg)±standard deviation (n=3×2) with different capital letters (a-f) in the same row are significantly different (p<0.05) according to collected from different region at fresh fruit. mean values±standard deviation (n=3×2) with different lowercase (a-c. k-m) in the same row are significantly different (p<0.05) according to collected from the same region at fresh fruit. tab. 2: phenolic compounds concentrations of dried vac. myrtillus fruits grown in erdek ve kapıdağ regions (mg/kg (mg/kg) phenolic compounds e1drf* e2drf e3drf k1drf k2drf k3drf gallic acid 43.53 ± 4.78cb** 28.11 ± 1.78ec 57.63 ± 5.41ba 52.29 ± 2.18bl 10.43 ± 1.10dm 72.59 ± 3.25ak vanillic acid 244.73 ± 3.65bb 352.64 ± 3.65aa 123.76 ± 3.29cc 42.04 ± 2.87em 117.16 ± 2.85ck 71.00 ± 4.58dl caffeic acid 3.92 ± 1.85db 2.19 ± 0.18ec 6.99 ± 1.23ca 3.80 ± 0.51dl 35.14 ± 1.47ak 31.88± 1.95bm chlorogenic acid 1.19 ± 0.14eb 0.94 ± 0.02fb 1.66 ± 0.45da 2.11 ± 0.11cm 8.04 ± 1.45ak 2.86 ± 0.10bl syringic acid 1338. 96 ± 16.84db 245.49 ± 1.85fc 2971.61 ± 25.56ca 3344.54 ± 28.62bl 5627.47 ± 32.14ak 304.33 ± 3.85em p-coumaric acid 22.25 ± 2.14eb 9.09 ± 1.63fc 73.98 ± 3.45da 390.96 ± 3.61bl 557.22 ± 3.25ak 137.30 ± 4.45cm ferulic acid 11.92 ± 3.16db 7.49 ± 1.74ec 24.32 ± 2.89ca 94.46 ± 3.54ak 77.55 ± 1.85bl 71.48 ± 6.18bl trans-ferulic acid 1.54 ± 0.14eb 1.06 ± 0.16ec 2.52 ± 0.85da 20.17 ± 4.52ak 15.59 ± 3.85cm 18.13 ± 2.85bl 3-hydroxy-4-methoxy 89.40 ± 2.96cb 30.19 ± 2.65ec 144.22 ± 2.84aa 98.70 ± 3.21bl 145.96 ± 3.84ak 71.86 ± 4.78dm cinnamic acid myricetin 122.64 ± 4.32db 94.79 ± 2.85ec 311.82 ± 9.12aa 310.39 ± 9.85ak 241.37 ± 5.45bl 194.10 ± 6.59cm quercetin 2.94 ± 0.56db 1.13 ± 0.13ec 4.67 ± 1.12ca 9.67 ± 1.25bl 11.35 ± 1.84ak 4.36 ± 0.19cm kaempferol 8.87 ± 1.12db 3.82 ± 0.41ec 21.49 ± 1.45ca 70.19 ± 7.84ak 21.42 ± 2.14cm 28.07 ± 1.18bl rutin hydrate 23.93 ± 1.45eb 8.53 ± 0.45fc 83.39 ± 3.12ca 236.59 ± 6.10ak 226.05 ± 6.95bl 41.86 ± 2.14dm naringin 119.57 ± 2.34db 117.91 ± 2.95db 288.42 ± 4.59ca 343.55 ± 6.25ak 326.60 ± 6.54abkl 313.44 ± 5.61bl hesperidin 2.69 ± 0.16db 2.30 ± 0.16db 3.55 ± 0.48ca 12.66 ± 1.42ak 12.27 ± 2.14ak 8.46 ± 1.26bl neohesperidin 1.23 ± 0.14deb 0.94 ± 0.05ec 1.66 ± 0.12da 3.58 ± 0.95bl 2.97 ± 0.23cm 6.14 ± 1.14ak (+)-catechin 1.70 ± 0.19fc 5.63 ± 0.96eb 32.39 ± 0.41ba 7.21 ± 1.58dm 23.91 ± 1.45cl 47.75 ± 4.40ak (-)-epicatechin 262.33 ± 2.64db 61.12 ± 2.46ec 661.53 ± 9.65ca 2599.73 ± 29.41bl 3675.69 ± 42.41ak 622.78 ± 9.98cm (-)-epigallocatechin 35.34 ± 1.97eb 28.40 ± 1.85fc 89.71 ± 5.12da 195.44 ± 2.52bl 254.97 ± 6.95ak 138.53 ± 3.48cm cyanidin-3-o-glucoside 2079.27 ± 6.45db 444.10 ± 3.47ec 5297.84 ± 23.14ba 3649.42 ± 10.85cl 6154.05 ± 58.42ak 153.29 ± 3.87fm chloride malvidine-3-o-glucoside 3891.21 ± 5.23db 1566.54 ± 5.42ec 12933.81 ± 45.87aa 6095.81 ± 19.84cl 7985.69 ± 23.87bk 1520.40 ± 27.95em chloride resveratrol 1.07 ± 0.12deab 0.97 ± 0.04eb 1.12 ± 0.14da 1.37 ± 0.10cm 2.18 ± 0.12bl 2.58 ± 0.13ak * e: erdek, k. kapıdağ, dr: dried, f: fruit, 1-3: codes of samples collected from different regions, ** mean values (mg/kg)±standard deviation (n=3×2) with different capital letters (a-f) in the same row are significantly different (p<0.05) according to collected from different region at dried fruit. mean values±standard deviation (n=3×2) with different lowercase (a-c. k-m) in the same row are significantly different (p<0.05) according to collected from the same region at dried fruit. bioactive compounds in fruits and leaves of blueberry 119 tab. 3: phenolic compounds concentrations of fresh vac. myrtillus leaves grown in erdek ve kapıdağ regions (mg/kg) phenolic compounds e1frl* e2frl e3frl k1frl k2frl k3frl gallic acid 12.09 ± 1.65cb** 33.33 ± 2.14ba 6.53 ± 0.96ec 46.64 ± 3.58ak 12.53 ± 1.26cl 7.17 ± 1.12dm vanillic acid 159.69 ± 6.85ba 92.79 ± 6.51cb 48.28 ± 3.48ec 18.00 ± 2.14fm 167.59 ± 15.20ak 62.69 ± 9.87dl caffeic acid 248.76 ± 9.78aa 197.65 ± 12.85bb 28.66 ± 2.14dc 51.85 ± 4.59ck 27.82 ± 3.85dl 14.49 ± 2.15em chlorogenic acid 9.07 ± 1.23aa 3.16 ± 0.29bc 3.41 ± 0.27bb 2.27 ± 0.52cdl 2.20 ± 0.84dl 2.49 ± 0.48ck syringic acid 31.93 ± 2.14cb 960.56 ± 15.26aa 26.05 ± 3.56dc 24.18 ± 3.65el 130.15 ±10.84bk 24.09 ± 2.47el p-coumaric acid 123.44 ± 6.54aa 69.26 ± 6.58cb 50.35 ± 5.84dc 92.83 ± 9.85bl 129.87 ± 15.20ak 36.23 ± 3.65em ferulic acid 5.51 ± 1.10bca 5.23 ± 0.95db 5.33 ± 1.10cdab 5.59 ± 1.05bcl 5.68 ± 0.95bl 6.50 ± 0.98ak trans-ferulic acid 9.98 ± 1.18bb 11.68 ± 1.84aa 5.19 ± 0.56cc 4.48 ± 0.96dl 4.38 ± 0.28dl 5.27 ± 0.82ck 3-hydroxy-4-methoxy 68.30 ± 3.87ba 35.45 ± 3.58fc 59.22 ± 3.98cb 49.34 ± 4.12dl 86.71 ± 6.54ak 41.80 ± 6.14em cinnamic acid myricetin 118.02 ± 6.41ba 57.06 ± 4.50dc 87.53 ± 6.52cb 84.26 ± 9.23cl 141.45 ± 8.59ak 56.33 ± 10.20dm quercetin 2.26 ± 0.25db 0.99 ± 0.19ec 9.32 ± 1.12ca 9.97 ± 1.10bcm 11.83 ± 1.65ak 10.84 ± 3.45abl kaempferol 2.56 ± 0.39cb 1.28 ± 0.25fc 9.91 ± 1.48aa 1.79 ± 0.27dl 1.38 ± 0.29em 8.78 ± 2.15bk rutin hydrate 5.74 ± 0.48dc 24.18 ± 1.17aa 9.81 ± 2.00bb 5.53 ± 0.52dl 2.46 ± 0.54em 8.95 ± 1.84ck naringin 21.66 ± 1.85aa 4.45 ± 0.98dc 14.39 ± 2.58bb 5.10 ± 0.29dl 13.40 ± 2.87ck 14.04 ± 2.14bck hesperidin 13.48 ± 1.74aa 7.50 ± 1.25cc 9.56 ± 2.05bb 6.31 ± 0.84dl 5.89 ± 0.68em 7.73 ± 0.96ck neohesperidin 2.81 ± 0.69bcb 2.68 ± 0.62cb 7.73 ± 2.14aa 2.36 ± 0.26dl 2.19 ± 0.39em 3.12 ± 0.25bk (+)-catechin 33.20 ± 2.98ecc 47.15 ± 3.48cb 61.71 ± 9.51ba 78.83 ± 8.63ak 44.65 ± 5.62dl 22.51 ± 2.15fm (-)-epicatechin 18.72 ± 2.45ba 7.06 ± 1.18db 5.60 ± 1.10ec 2.55 ± 0.35fm 14.00 ± 2.10cl 20.55 ± 2.46ak (-)-epigallocatechin 9.84 ± 2.12cc 26.79 ± 3.15bb 61.26 ± 8.56aa 8.70 ± 0.95dk 7.23 ± 1.98el 9.12 ± 1.10cdk cyanidin-3-o-glucoside 1.51 ± 0.23ba 1.02 ± 0.20cb 0.96 ± 0.18db 0.98 ± 0.27cdl 0.97 ± 0.19cdl 23.61 ± 2.58ak chloride malvidine-3-o-glucoside 1.57 ± 0.15ba 1.10 ± 0.14cb 0.96 ± 0.12db 1.05 ± 0.16cdl 0.00 ± 0.00em 42.66 ± 5.00ak chloride resveratrol 1.57 ± 0.10ec 4.11 ± 0.58cb 6.29 ± 1.10aa 3.91 ± 0.56dm 5.89 ± 0.84abk 5.31 ± 0.85bl * e: erdek, k: kapıdağ, fr: fresh, l: leaf, 1-3: codes of samples collected from different regions, ** mean values (mg/kg)±standard deviation (n=3×2) with different capital letters (a-f) in the same row are significantly different (p<0.05) according to collected from different region at fresh leaf. mean values±standard deviation (n=3×2) with different lowercase (a-c. k-m) in the same row are significantly different (p<0.05) according to collected from the same region at fresh leaf. tab. 4: phenolic compounds concentrations of dried vac. myrtillus leaves grown in erdek ve kapıdağ regions (mg/kg) phenolic compounds e1drl* e2drl e3drl k1drl k2drl k3drl gallic acid 176.95 ± 6.98cc** 352.30 ± 3.59aa 201.37 ± 3.29bb 203.48 ± 4.36bk 67.43 ± 3.69em 110.28 ± 9.62dl vanillic acid 271.17 ± 5.89bb 1156.80 ± 18.29aa 249.72 ± 4.62cc 157.27 ± 2.48dl 230.63 ± 13.52ck 20.78 ± 2.98em caffeic acid 27.05 ± 3.21aa 5.28 ± 1.14cb 28.24 ± 2.12aa 8.61 ± 1.10bk 4.65 ± 0.85dl 3.28 ± 0.68em chlorogenic acid 3.31 ± 0.45ba 3.23 ± 0.58ca 1.25 ± 0.26eb 4.14 ± 0.87ak 1.94 ± 0.34dm 3.25 ± 0.52bcl syringic acid 202.99 ± 5.47aa 84.36 ± 6.47dc 113.56 ± 3.29bb 95.71 ± 6.54ck 47.56 ± 3.85el 32.46 ± 5.20fm p-coumaric acid 15.93 ± 2.85ba 11.71 ± 2.19dc 13.18 ± 1.23cb 8.51 ± 1.98em 15.24 ± 2.18bl 17.58 ± 2.16ak ferulic acid 3.13 ± 0.95eb 3.93 ± 0.48ca 3.80 ± 0.48cda 9.99 ± 2.57ak 3.50 ± 0.84dem 6.56 ± 1.05bl trans-ferulic acid 12.29 ± 2.14db 11.90 ± 2.43deb 24.93 ± 1.15aa 16.03 ± 2.18bk 15.89 ± 2.17cl 10.95 ± 2.84em 3-hydroxy-4-methoxy 111.93 ± 4.58cb 254.11 ± 4.56aa 88.46 ± 3.58ec 141.86 ± 9.84bk 96.34 ± 9.27dl 51.75 ± 9.13fm cinnamic acid myricetin 101.45 ± 4.32db 237.56 ± 5.42aa 94.76 ± 4.51dc 152.97 ± 11.18bk 140.59 ±12.17cl 49.44 ± 7.68em quercetin 4.63 ± 0.47cb 2.07 ± 0.23dc 8.10 ± 1.18ba 4.27 ± 0.87cl 2.75 ± 0.96dm 11.38 ± 2.34ak kaempferol 1.63 ± 0.28dc 3.37 ± 0.43aa 2.17 ± 0.18bb 2.20 ± 0.59bl 3.30 ± 0.95ak 2.07 ± 0.56bl rutin hydrate 12.27 ± 1.15dc 79.69 ± 6.58aa 16.63 ± 2.15cb 21.79 ± 2.51bk 11.98 ± 3.25el 8.08 ± 1.02fm naringin 95.12 ± 5.21bb 261.38 ± 5.53aa 83.45 ± 6.41cc 34.66 ± 3.26el 43.08 ± 8.26dk 1.14 ± 0.38fm hesperidin 82.06 ± 5.10bb 134.01 ± 4.87aa 60.14 ± 4.28cc 36.93 ± 3.48dk 7.45 ± 2.14fm 9.74 ± 1.36el neohesperidin 6.52 ± 0.75ba 3.18 ± 0.59cb 6.06 ± 0.58ba 17.24± 2.47ak 2.63 ± 0.51cm 16.20 ± 2.01al (+)-catechin 17.50 ± 1.48db 95.59 ± 3.57aa 21.94 ± 2.43bb 19.73 ± 2.52cl 7.31 ± 2.18em 22.71 ± 3.20bk (-)-epicatechin 49.08 ± 3.47ba 12.78 ± 2.15db 10.06 ± 1.64dc 84.06 ± 9.52ak 37.31 ± 6.20cm 49.38 ± 4.02bl (-)-epigallocatechin 52.07 ± 3.65ca 18.20 ± 2.39ec 28.78 ± 3.28db 67.26 ± 6.53bl 29.84 ± 3.27dm 197.80 ± 12.10ak cyanidin-3-o-glucoside 0.00 ± 0.00cb 1.00 ± 0.06aba 1.01 ± 0.17aba 1.06 ± 0.26ak 1.01 ± 0.18abkl 0.95 ± 0.09bl chloride malvidine-3-o-glucoside 1.09 ± 0.18aba 0.00 ± 0.00cb 1.14 ± 0.16aba 1.20 ± 0.19ak 1.11 ± 0.24abkl 0.95 ± 0.12bl chloride resveratrol 3.32 ± 0.42ba 1.54 ± 0.28cb 1.50 ± 0.45cb 8.89 ± 2.21ak 8.55 ± 1.00ak 3.85 ± 0.45bl * e: erdek, k. kapıdağ, dr: dried. l: leaf, 1-3: codes of samples collected from different regions, ** mean values (mg/kg)±standard deviation (n=3×2) with different capital letters (a-f) in the same row are significantly different (p<0.05) according to collected from different region at dried leaf. mean values±standard deviation (n=3×2) with different lowercase (a-c. k-m) in the same row are significantly different (p<0.05) according to collected from the same region at dried leaf. 120 n. değirmencioğlu, o. gürbüz, g.e. karatepe, r. irkin fig. 1: total phenolic (tp) contents (mg gae/kg) of fresh and dried vac. myrtillus fruit and leaf extracts (e: erdek, k. kapıdağ, fr: fresh, dr: dried, f: fruit, l: leaf, 1-3: codes of samples collected from different regions, different letter(s) on bar indicate statistically significant differences, p<0.05) 0,00 5000,00 10000,00 15000,00 20000,00 25000,00 30000,00 e1frf e2frf e3frf k1frf k2frf k3frf m g g a e /k g fresh vac.myrtillus fruit extracts total phenol d d d d b a c 0,00 20000,00 40000,00 60000,00 80000,00 100000,00 120000,00 e1drf e2drf e3drf k1drf k2drf k3drf m g g a e /k g dried vac.myrtillus fruit extracts total phenol de d d c d b d a d de 0,00 5000,00 10000,00 15000,00 20000,00 25000,00 e1frl e2frl e3frl k1frl k2frl k3frl m g g a e /k g fresh vac.myrtillus leaf extracts total phenol c c e a d b 0,00 20000,00 40000,00 60000,00 80000,00 100000,00 120000,00 140000,00 e1drl e2drl e3drl k1drl k2drl k3drl m g g a e /k g dried vac.myrtillus leaf extracts total phenol d c b b a a chin, and (-)-epigallocatechin compared to fresh fruit and leaf and also dried leaf extracts. the flavanol contents in fruit and leaf extracts were determined to be increasing or decreasing depending on the altitude, the area, and the harvesting time where the samples were collected as well as the drying process. drying and the drying conditions (oxygen, high temperature, length of time, without vacuum, etc.) may cause polymerization reactions, reduce some polyphenols and their antioxidant capacity, and induce the formation of new compounds (kendarı et al., 2012). as a stated by kendarı et al. (2012) catechin oxidation mechanism initially constructs a semiquinon, which then converts into a quinon. the quinon compound can react with amino acid, protein, or other polymers to produce proanthocyanidin. and then proanthocyanidin can degrade into catechin because proanthocyanidin represents an oligomer or polymer from flavan-3-ol (catechin/epicatechin). these flavanols have already been detected in some berry fruit and leaf (sellappan et al., 2002; harrıs et al., 2007; vrhovsek et al., 2012; deng et al., 2014). in blueberry skin and flesh, delphinidin, cyanidin, petunidin, peonidin, and malvidin monoglycosides are the main anthocyanins. because of the diversity in the glycosylation and acylation pattern, more than 25 anthocyanins have been identified in blueberries (harrıs et al., 2007; moze et al., 2011). the amounts and distribution of anthocyanins in the berries differ depending on their plant species, cultivation conditions in which the fruit have been grown or stored (e.g. light, temperature), and producing districts, due to genetic differences among wild and cultivated varieties (ehlenfeldt and prıor, 2001). blueberry leaves are by-products of the blueberry industry. the leaves contain in an excessively amount of polyphenols, which creates an opportunity for their use in the neutraceutical industry, more so than the fruits (ehlenfeldt and prıor, 2001; kım and um, 2011; lı et al., 2013; deng et al., 2014). but no anthocyanins have been found in fresh green leaves (harrıs et al., 2007; lı et al., 2013). we found fresh and dried vac. myrtillus fruit extracts with high levels of malvidin-3-o-glucoside chloride (212.93-4433.19 mg/kg fw and 1520.40-12933.81 mg/kg dw) and cyanidin-3-o-glucoside chloride (70.94-781.26 mg/kg fw and 153.29-6154.05 mg/kg dw) collected from turkey, respectively. as mentioned earlier, fresh and dried blueberry leaf extracts have significantly less (p<0.05) anthocyanins than fruit extracts. nevertheless, there were numerous reports that anthocyanins were detected in blueberry leaves (harrıs et al., 2007; kım and um, 2011). these differences are possibly related to the harvesting season of leaves. within the anthocyanidin reductase or leucoanthocyanidin reductase activity, the transcriptional control favors the expression of the anthocyanidin synthase in sun-exposed leaves. the decrease in proanthocyanidin content in favor of anthocyanin production has also been observed during fruit development at the point when leaves turned red, activating the biosynthesis of cyanidin glycosides (jaakola et al., 2004; lı et al., 2013). in a previous study reported by lı et al. (2013) and rııhınen et al. (2008) anthocyanins were not detected in green leaves of blueberry and bilberry, but a low amount was detected in red leaves. resveratrol is a type of natural phenol and a phytoalexin produced naturally by several plants in response to injury or when the plant is under attack by pathogens such as bacteria or fungi (frémont, 2000). the occurrence of resveratrol in vaccinium berries should not be sur prising, as its occurrence in the plant kingdom appears to be widespread (rımando et al., 2004). food sources of resveratrol include the skin of grapes, blueberries, raspberries, and mulberries (jasıńskı et al., 2013) and the variability in the resveratrol content in fruit such as blueberries changes due to food processing or preparation. recently, resveratrol was reported by lyons et al. (2003) at levels of approximately 0.0002-0.0006 ng/g sample (rımando et al., 2004). the resveratrol contents of blueberries and the related bilberry, vac. myrtillus l., were cultivated in several different geographical regions (lyons et al., 2003). in our study, resveratrol was found in fresh fruit and leaf extracts (0.70-1.01 mg/kg fw and 1.57-6.29 mg/kg fw, respectively) from both regions. the previous data for trans-resveratrol content (0.4 mg/100 g fw) in blueberry (moze et al., 2011) agreed with ours, but it has also been determined to be lower for blueberries (wang et al., 2008). also, as stated by lyons et al. (2003), blue berries and bilberries were found to contain resveratrol and the level of this chemoprotective compound in these fruits was <10 % of that reported for grapes. in grapes, the levels of resveratrol were found to vary with the time of harvest, environmental and climatic conditions, fig. 1: total phenolic (tp) contents (mg gae/kg) of fresh and dried vac. myrtillus fruit and leaf extracts (e: erdek, k. kapıdağ, fr: fresh, dr: dried, f: fruit, l: leaf, 1-3: codes of samples collected from different regions, different letter(s) on bar indicate statistically significant differences, p<0.05) bioactive compounds in fruits and leaves of blueberry 121 and plant developmental stage (rımando et al., 2004). these factors possibility affected the differences in resveratrol contents of the vac. myrtillus extracts in this study. total phenol and total anthocyanin contents the tp contents were significantly different among the fruit and leaf vac. myrtillus extracts (fig. 1). the tp was 6152.05-25688.90 mg gae/kg in fresh fruit extracts, whereas in dried fruit were determined to be 14512.49-97214.25 mg gae/kg. for the fresh and dried blueberry leaf extracts, their tp contents were significantly (p<0.05) higher than those in fresh and dried fruits. in the leaf tissues of 87 highbush blueberries, the mean values of polyphenol contents were ~ 30 times higher than observed in fruits on a fw basis (ehlenfeldt and prıor, 2001). skupıeń et al. (2006) reported that the tp contents in vac. corymbosum l. leaf extracts was 111.5 mg/100 g dw dried leaves. in addition, the tp of the leaf extracts of vac. myrtillus in erdek and kapıdağ regions were significatly higher (p<0.05) than those of blackberry leaves (82.8-91.6 mg of gae/g of dw) and strawberry leaves (55.2 mg of gae/g of dw) reported by wang and lın (2000). lı et al. (2013) and oszmıańskı et al. (2011) indicated that the polyphenol content of the blueberry leaves was much higher than those of any other leaves of tested berries (blackberry, raspberry, honeyberry, and strawberry). the differences in tp contents between fruit and leaf extracts were statistically significant (p<0.05). gene rally, the tp of plant extracts can be affected by solvent, its pola rity, its concentration, and/or extraction method method (deng et al., 2014; xıaoyong and lumıng, 2014). it is also well known that genetic, agronomic or environmental factors play important roles in phenolic composition and nutritional quality of crops (yang et al., 2009). when the tp contents of these extracts are compared with white wines, these plants could contribute the same health benefit as those wines in terms of polyphenols. a large variation was observed among fruit and leaf extracts for ta content (fig. 2), ranging from 2805.08 to 5973.69 mg/kg (in fresh fruit), from 2094.56 to 5975.14 mg/kg (in dried fruit), from 12.89 to 262.02 mg/kg (in fresh leaf), and from 51.91 to 318.30 mg/kg (in dried leaf). fresh and dried fruit samples are a good source of anthocyanin (tab. 1-4), however, fresh fruit samples recorded decreases/increases in ta content. the ta content in blueberry fruit and leaf extracts in this study was comparable to the quantity reported by ehlenfeldt and prıor (2001), sellappan et al. (2002), lohachoompol et al. (2008), and wang et al. (2015). results obtained from several studies suggest that the tp and ta contents in blueberry fruit are influenced by the cultivar, harvest time, the growing season, fruit mass, maturity, environmental growing conditions, growing location, postharvest storage conditions, drying process, different extraction methods, irridation, temperature, and pathogen attacks (routray et al., 2014). antioxidant capacity blueberry fruit and leaf show high antioxidant capacity, correlated especially with their anthocyanin and other phenolic compounds content, and may be considered as one of the highest antioxidant sources among fruits and vegetables (vrhovsek et al., 2012). the values found in the fresh vac. myrtillus fruit extracts for antioxidant capa city (fig. 3) were 8.37-23.26 μmol te/g fw by cuprac, 8.5619.23 μmol te/g fw by dpph, 4.26-9.56 μmol te/g fw by abts, and 0.97-1.73 μmol te/g fw by frap method, values lower/higher than or close to those found in fresh leaf extracts (fig. 4). also in blueberry fruits and leaves studied by wang and lın (2000), ehlenfeldt and prıor (2001), sellappan et al. (2002), lı et al. (2013), and wang et al. (2015) using the same methods, the vac. myrtillus fruit and leaf extract results obtained in this study were similar to those studies. therefore, as a reported by wang and lın (2000) and park et al. (2012), because of their high antioxidant content, blueberry leaves can also be added to tea mixes to increase the antioxidant capacity level in the beverages for greater benefits to human health. previous studies found a direct relationship between the antioxidant capacity, and the tp and tas contents in blueberry fruits and leaves (ehlenfeldt and prıor, 2001; lı et al., 2013). in this study, there was a strong correlation between antioxidant capacity and tp content fig. 2: total anthocyanin (ta) contents (mg/kg) of fresh and dried vac. myrtillus fruit and leaf extracts (e: erdek, k. kapıdağ, fr: fresh, dr: dried, f: fruit, l: leaf, 1-3: codes of samples collected from different regions, total anthocyanins were expressed as cyanidin-3-glucoside equivalents, different letter(s) on bar indicate statistically significant differences, p<0.05) fig. 2: total anthocyanin (ta) contents (mg/kg) of fresh and dried vac. myrtillus fruit and leaf extracts (e: erdek, k. kapıdağ, fr: fresh, dr: dried, f: fruit, l: leaf, 1-3: codes of samples collected from different regions, total anthocyanins were expressed as cyanidin-3glucoside equivalents, different letter(s) on bar indicate statistically significant differences, p<0.05) 0,00 1000,00 2000,00 3000,00 4000,00 5000,00 6000,00 7000,00 e1frf e2frf e3frf k1frf k2frf k3frf m g/ kg fresh vac.myrtillus fruit extracts total anthocyanin c a b c d a 0,00 1000,00 2000,00 3000,00 4000,00 5000,00 6000,00 7000,00 e1drf e2drf e3drf k1drf k2drf k3drf m g/ kg dried vac.myrtillus fruit extracts total anthocyanin b a a bc c b 0,00 50,00 100,00 150,00 200,00 250,00 300,00 350,00 e1frl e2frl e3frl k1frl k2frl k3frl m g/ kg fresh vac.myrtillus leaf extracts total anthocyanin bc cd a d b cd 0,00 50,00 100,00 150,00 200,00 250,00 300,00 350,00 e1drl e2drl e3drl k1drl k2drl k3drl m g/ kg dried vac.myrtillus leaf extracts total anthocyanin a a a a a a 122 n. değirmencioğlu, o. gürbüz, g.e. karatepe, r. irkin fig. 3: antioxidant capacities (μmol te/g) of fresh and dried vac. myrtillus fruit extracts (e: erdek, k. kapıdağ, fr: fresh, dr: dried, f: fruit, 1-3: codes of samples collected from different regions, different letter(s) on bar indicate statistically significant differences, p<0.05) in the dried fruit and fresh leaf extracts, while weak correlation ta content in fresh fruit extracts (tab. 5, is available in supplementary material). these results indicated that the phenolics, rather than the anthocyanins alone, play an important role in contributing to the whole antioxidant capacity. the reason may be that anthocyanins in fruit and leaves could transform into other types of phenolics, which have higher levels of antioxidant capacity, via the drying and sample preparation processes. as a reported by previously study, in the dry heating experiment, all phenolics including anthocyanins in the blueberry pomace were completely degraded to small fragments that had no antioxidant capacity. thus, the loss of the anthocyanins and other phenolics straightforwardly linked to the decrease of antioxidant capacity of the dry-heated pomace (bener et al., 2013). conclusion the drying method affects the quality of the end products such as color, texture, aroma, along with its chemical constituents (routray et al., 2014). therefore, the control method chosen during this study was fresh storage after vacuum packaging. oven-dried fruit and leaf samples retained higher/lower amounts of phenolic content, tp and ta according to the degree of resistance to the drying process of the phenolic compounds and the growing season, suggesting that enviromental growing conditions, harvesting altitude, and length and type of drying time used for the dried samples when compared to the amount obtained from fresh samples. also, anthocyanins, flavonols, and proanthocyanidins are located mainly in the peel while hydroxycinnamates are found in the flesh (goldıng et al., 2001), therefore, it is believe that they are affected by different degrees fig. 3: antioxidant capacities (µmol te/g) of fresh and dried vac. myrtillus fruit extracts (e: erdek, k: kapıdağ, fr: fresh, dr: dried, f: fruit, 1-3: codes of samples collected from different regions, different letter(s) on bar indicate statistically significant differences, p<0.05) 0,00 10,00 20,00 30,00 40,00 50,00 60,00 e1frf e2frf e3frf k1frf k2frf k3frf µm ol t e /g fresh vac.myrtillus fruit extracts cuprac c c d b a c 0,00 10,00 20,00 30,00 40,00 50,00 60,00 e1drf e2drf e3drf k1drf k2drf k3drf µm ol t e /g dried vac.myrtillus fruit extracts cuprac b ab a d cd c 0,00 5,00 10,00 15,00 20,00 25,00 30,00 35,00 40,00 e1frf e2frf e3frf k1frf k2frf k3frf µm ol t e /g fresh vac.myrtillus fruit extracts dpph b c d cd a cd 0,00 5,00 10,00 15,00 20,00 25,00 30,00 35,00 40,00 e1drf e2drf e3drf k1drf k2drf k3drf µm ol t e /g dried vac.myrtillus fruit extracts dpph a a b d d c 0,00 2,00 4,00 6,00 8,00 10,00 12,00 14,00 16,00 18,00 20,00 e1frf e2frf e3frf k1frf k2frf k3frf µm ol t e /g fresh vac.myrtillus fruit extracts abts b b b a a b 0,00 2,00 4,00 6,00 8,00 10,00 12,00 14,00 16,00 18,00 20,00 e1drf e2drf e3drf k1drf k2drf k3drf µm ol t e /g dried vac.myrtillus fruit extracts abts c ab a b d c 0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00 e1frf e2frf e3frf k1frf k2frf k3frf µm ol t e /g fresh vac.myrtillus fruit extracts frap c c b a ab c 0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00 e1drf e2drf e3drf k1drf k2drf k3drf µm ol t e /g dried vac.myrtillus fruit extracts frap c ab b a d d bioactive compounds in fruits and leaves of blueberry 123 fig. 4: antioxidant capacities (μmol te/g) of fresh and dried vac. myrtillus leaf extracts (e: erdek, k. kapıdağ, fr: fresh, dr: dried, l: leaf, 1-3: codes of samples collected from different regions, different letter(s) on bar indicate statistically significant differences, p<0.05) from an applied drying method, while the antioxidant capacities of the samples increase under the same conditions. if combined with the other protective methods, oven-drying proved to be a suitable method for vac. myrtillus samples preservation because the phenolic compounds and their functional properties were either increased or at least decreased. consequently, vac.myrtillus fruit and leaf can be recommended as an addition to food composition, to increase the antioxidant capacity, because of their high antioxidant properties. acknowledgements the authors are grateful to balikesir university scientific research projects unit (project no: 2012-117) for the financial support of this research. references akondı, r.b., kumar, p., annapurna, a., pujarı, m., 2011: protective effect of rutin and naringin o sperme quality in streptozotocin (stz) induced type 1 diabetic rats. iran j. pharm. res. 10, 585-596. doi:10.1211/jpp.58.10.0011. apak, r., guclu, k., demırata, b., ozyurek, m., celık, s.e., bektasoglu, b., berker, k.ı., ozyurt, d., 2007: comparative evaluation of various total antioxidant capacity assays applied to phenolic compounds with the cuprac assay. molecules. 12, 1496-1547. doi:10.3390/12071496. bener, m., shen, y., apak, r., fınley, j.w., xu, z., 2013: release and degradation of anthocyanins and phenolics from blueberry pomace during thermal acid hydrolysis and dry heating. j. agric. food chem. 61, 66436649. doi:10.1021/jf401983c. benzıe, ı.f.f., straın, j.j., 1996: the ferric reducing ability of plasma fig. 4: antioxidant capacities (µmol te/g) of fresh and dried vac. myrtillus leaf extracts (e: erdek, k: kapıdağ, fr: fresh, dr: dried, l: leaf, 1-3: codes of samples collected from different regions, different letter(s) on bar indicate statistically significant differences, p<0.05) 0,00 20,00 40,00 60,00 80,00 100,00 120,00 e1frl e2frl e3frl k1frl k2frl k3frl µm ol t e /g fresh vac.myrtillus leaf extracts cuprac c b b a d c 0,00 20,00 40,00 60,00 80,00 100,00 120,00 e1drl e2drl e3drl k1drl k2drl k3drl µm ol t e /g dried vac.myrtillus leaf extracts cuprac c b b bc a b 0,00 5,00 10,00 15,00 20,00 25,00 30,00 35,00 40,00 e1frl e2frl e3frl k1frl k2frl k3frl µm ol t e /g fresh vac.myrtillus leaf extracts dpph c b b a c d 0,00 5,00 10,00 15,00 20,00 25,00 30,00 35,00 40,00 e1drl e2drl e3drl k1drl k2drl k3drl µm ol t e /g dried vac.myrtillus leaf extracts dpph ab c a b ab ab 0,00 5,00 10,00 15,00 20,00 25,00 30,00 e1frl e2frl e3frl k1frl k2frl k3frl µm ol t e /g fresh vac.myrtillus leaf extracts abts bc b a d bc cd 0,00 5,00 10,00 15,00 20,00 25,00 30,00 e1drl e2drl e3drl k1drl k2drl k3drl µm ol t e /g dried vac.myrtillus leaf extracts abts b b a a a a 0,00 2,00 4,00 6,00 8,00 10,00 12,00 14,00 e1frl e2frl e3frl k1frl k2frl k3frl µm ol t e /g fresh vac.myrtillus leaf extracts frap b a b a b b 0,00 2,00 4,00 6,00 8,00 10,00 12,00 14,00 e1drl e2drl e3drl k1drl k2drl k3drl µm ol t e /g dried vac.myrtillus leaf extracts frap d a d b c bc 124 n. değirmencioğlu, o. gürbüz, g.e. karatepe, r. irkin (frap) as a measure of “antioxidant power”: the frap assay. anal. biochem. 23, 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myrtillus fruit and leaf extracts table 5. correlation coefficient of total phenolic content. total anthocyanin content and antioxidant capacity of fresh and dried vac. myrtillus fruit and leaf extracts total phenol total anthocyanin cuprac dpph abts frap total phenol 1 -0.406 0.935** 0.323 0.984** 0.704 total anthocyanin -0.406 1 -0.679 -0.774 -0.638 -0.632 cuprac 0.935** -0.679 1 0.545 0.987** 0.745 dpph 0.323 -0.774 0.545 1 0.420 0.187 abts 0.948** -0.638 0.987** 0.420 1 0.811 f re sh fr ui t e xt ra ct frap 0.704 -0.632 0.745 0.187 0.811 1 total phenol total anthocyanin cuprac dpph abts frap total phenol 1 0.572 0.997** 0.869* 0.949** 0.981** total anthocyanin 0.572 1 0.630 0.835* 0.777 0.642 cuprac 0.997** 0.630 1 0.900* 0.970** 0.984** dpph 0.869* 0.835* 0.900* 1 0.955** 0.919** abts 0.949** 0.777 0.970** 0.955** 1 0.960** d ri ed fr ui t e xt ra ct frap 0.981** 0.642 0.984** 0.919** 0.960** 1 total phenol total anthocyanin cuprac dpph abts frap total phenol 1 0.861* 0.981** 0.738 0.957** 0.918** total anthocyanin 0.861* 1 0.932** 0.659 0.925** 0.930** cuprac 0.981** 0.932** 1 0.782 0.978** 0.958** dpph 0.738 0.659 0.782 1 0.814* 0.814* abts 0.957** 0.925** 0.978** 0.814* 1 0.992** f re sh le af e xt ra ct frap 0.918** 0.930** 0.958** 0.814* 0.992** 1 total phenol total anthocyanin cuprac dpph abts frap total phenol 1 0.474 0.793 -0.020 0.420 0.238 total anthocyanin 0.474 1 0.607 0.576 0.971** -0.131 cuprac 0.793 0.607 1 0.048 0.444 0.231 dpph -0.020 0.576 0.048 1 0.653 -0.863* abts 0.420 0.971** 0.444 0.653 1 -0.250 d ri ed le af e xt ra ct frap 0.238 -0.131 0.231 -0.863* -0.250 1 **. correlation is significant at the 0.01 level (2-tailed). *. correlation is significant at the 0.05 level (2-tailed). journal of applied botany and food quality 91, 14 23 (2018), doi:10.5073/jabfq.2018.091.003 1lset, cadi ayyad university, marrakech, morocco 2 biotechnologie de la valorisation et la protection des agro-ressources chimie bio-organique et macromoléculaire, cadi ayyad university, marrakech, morocco solar drying, hygroscopic equilibrium and biochemical quality of punica granatum legrelliae’s flowers hicham el ferouali1, ahmed zoukit1, naima zehhar2, fatiha benkhalti2, hafida bouamama2, said doubabi1, naji abdenouri1* (submitted: september 24, 2017; accepted: january 3, 2018) * corresponding author summary punica granatum legrelliae is a valuable medicinal plant that is widely planted in morocco. the equilibrium moisture content was investigated. peleg model was found the most suitable to describe the sorption phenomenon. the drying kinetic of punica granatum legrelliae’s flowers was investigated by using a convection solar dryer. midilli-kucuk model described well the drying curves’ trend. the effective moisture diffusivity values were obtained. the arrhenius relation, with an activation energy value of 92.91±2.51 kj mol-1 expressed the temperature effect on the diffusion coefficient. finally, the effect of drying these flowers at different temperatures on their quality was investigated. to assess the quality of the product after solar drying, the color, polyphenols content, antioxidant activity, and polyphenoloxidase and peroxidase activities were considered. the best drying temperature for the preservation of color and bioactive molecules with antioxidant property was 40 °c. keywords: biochemical characterization; punica granatum le grelliae; quality; solar drying process; sorption; storage. introduction medicinal and aromatic plants have a crucial value in the pharmaceutical industry and traditional medicine. but, long-term storage of these products requires specific treatments to dehydrate, inactivate tissue enzymes and microorganisms and protect against further contaminations. drying is the most used preservation operation in the storage of medicinal and aromatic plants and it is a part of the extraction process of high value substances. it aims to reduce the water content and minimizes there biochemical, chemical and microbiological deterioration. direct sunlight drying is considered as one of the most common drying methods of aromatic and medicinal plants in developing countries. however, this drying technique highly affects the quality of dried product and exposes it to dust and other environmental contaminations. in industry, other techniques are usually used such as freeze-drying and microwave drying but they have always been recognized as expensive processes in terms of energy consumption (karam et al., 2016). therefore, the indirect solar drying is more adapted to developing countries; it is economic, fast and leads to a homogeneous and edible product. thus, it can be strongly considered for the industrial exploitation. punica granatum legrelliae (punicaceae) is a flowering shrub without fruiting, native to the mediterranean and asia region and has been used extensively in the folk medicine. the flowers are a traditional antidiabetic medicine (li et al., 2007), and are strongly astringent (huang et al., 2005). the flowers are also used to treat chronic diarrhea (zhang et al., 2011), and the passive bleeding. they contain polyphenols including gallic, ellagic acids and triterpenes (zhang et al., 2011). they also contain a variety of secondary metabolites. the bright color of the flowers is due to anthocyanins. many studies have been conducted on the solar drying processes and water activity of various medicinal plants (argyropoulos et al., 2011; desmorieux and decaen, 2005). however, few works have studied the drying process effect on the quality of the dried product. furthermore, no studies have so far been reported on the physical and the hygroscopic behavior of punica granatum legrelliae’s flowers. the sorption isotherms are a good way to describe how active water is bound to a wet product (abdenouri et al., 2010). they are also an extremely valuable tool because it provides precious information about the hygroscopic equilibrium of the product. in other words, it is necessary to determine the optimum moisture content and the water activity that must be achieved during drying for better preservation of the dried product (chirife and iglesias, 2007). only experimental studies allow determining the drying kinetics of products. therefore, it is interesting to study variations of moisture content for different controllable aerothermal parameters. the drying kinetics depends on the heat and mass transfers that occur within products. the mechanisms of these transfers are very complex and they lead to large physical, chemical and biological changes. hence, the evolution of these parameters must be controlled in order to achieve the best quality (değirmencioğlu et al., 2017; el ferouali et al., 2017a; el ferouali et al., 2017b). in addition, the products’ color evolution has to be examined during drying insofar color is one of the determinant parameters of dried products’ quality and it sorely influences their commercial value. in fact, color change is a key parameter in the quality of several dried products such as kiwi (orikasa et al., 2014) and tomato (santossánchez et al., 2012). the main objectives of this study are: • to determine the effect of temperature on the moisture desorption and adsorption isotherms of punica granatum legrelliae’s flowers, and to find the appropriate model that describes its sorption curves. • to determine the optimal storage condition of the product and the isosteric heat of sorption. • to study the drying kinetics of punica granatum legrelliae’s flowers for different controllable aerothermal parameters by using a convective solar dryer. • to determine the effective moisture diffusivity for different temperatures and the corresponding activation energy. • to study the effect of three drying temperatures (40 °c, 50 °c and 60 °c) on the color change and the biochemical parameters of punica granatum legrelliae’s flowers. the parameters taken into account are the total polyphenol content, polyphenoloxidase and peroxidase activities and antioxidant activity. hygroscopic and biochemical characterization of dried punica granatum legrelliae 15 nomenclature: ∆hd isosteric heat of sorption (kj mol-1) ∆hd net isosteric heat of sorption (kj mol-1) ∆hvap heat of vaporization of pure water at 35 °c (43.53 kj mol-1) ads. adsorption aw water activity deff effective diffusivity (m2 s-1) des. desorption dm dry matter dv drying air flow rate (m3 s-1) ea activation energy (kj mol-1) f dimensionless drying rate h half thickness of the dried samples (m) l*, a*, b* color parameters mbe mean bias error md mass of dry matter (kg) meq mass at the hygroscopic equilibrium (kg) mre mean relative error (%) mw mass of wet matter (kg) n number of experimental points r correlation coefficient r universal gas constant (8.3145 j mol-1 k-1) rh relative humidity (%) sd standard deviation sem standard error of moisture t absolute temperature (k) x moisture content at any time during drying (% d.b.) x* moisture ratio x0 initial moisture content (% d.b.) xeq equilibrium moisture content (% d.b.) xf final moisture content (% d.b.) θ temperature (°c) χ² reduced chi-square materials and methods sorption isotherms of punica granatum legrelliae’s flowers the hygroscopic equilibrium was achieved by the static gravimetric technique, it consists on using six saturated salt solutions (koh, mgcl2 6h2o, k2co3, nano3, kcl, bacl2 2h2o). these salts provide a range of relative humidity of 5-90 % (greenspan, 1977). the salts were prepared in six glass jars of 1 l each with an insulated lid. for each solution, the samples were suspended in the jars above salts and remained in a stabilized temperature and humidity environment. the samples were weighed every two days in order to determine their equilibrium moisture content xeq. as soon as the masses had become stationary, the samples were weighed and placed in a drying oven whose temperature is fixed at 105 °c for 24 h. the difference of mass before and after drying at 105 °c (respectively meq and md) allows determining the product’s moisture content xeq at the hygroscopic equilibrium by eq. (1). (1) the samples that were used for desorption were fresh and weighed between 0.093 and 0.102 g. the ones that were used for adsorption had been dried for 24 h in an oven at 105 °c before putting them on the glass jars; their weight were between 0.030 and 0.035 g. this process was performed at three different temperatures (30, 40 and 50 °c). description of the solar drying the experimental apparatus consists of an indirect forced convection solar dryer, with a solar air collector, an electric auxiliary heater, a circulation fan and a drying chamber. the solar dryer was described in detail by el ferouali et al. (2016). the same mass of fresh punica granatum legrelliae’s flowers (8.50 ± 0.01 g) was used for each drying experiment. samples were uniformly spread forming a thin layer on the drying rack. in solar drying processes, the air temperature can vary with the magnitude of the solar radiation. the auxiliary heater was so used to control the air temperature. the variation of the weight of the product versus time was determined by a digital weighing apparatus (±0.01 g). drying experiments were performed for three drying air temperatures (40, 50 and 60 ± 0.5 °c) with the air flow rate of 0.083 ± 0.001 m3 s-1. biochemical characterization protocol total polyphenols determination 100 mg of samples were ground in 2 ml of methanol at 80%. then, the mixture was sonicated for 10 min in an ultrasonic bath containing distilled water. the homogenate was centrifuged at 15,000 × g during 10 min. supernatant was tested by a differential assay in the presence and absence of polyvinylpolypyrrolidone (pvpp) according to bridi et al. (2014). samples were passed through three cycles of pretreatment in order to improve the ability of pvpp to absorb polyphenols. at first, 100 mg of pvpp was added to 1 ml of the supernatant, and the solution was stirred for 30 s using a vortex and then centrifuged at 10,000 × g for 5 min (one-cycle). after thus, 500 μl of the supernatant, obtained from the one-cycle procedure, was exposed to 50 mg of pvpp, and the obtained mixture was stirred and centrifuged in the same experimental conditions as mentioned above (second-cycle). finally, 250 μl of the supernatant (obtained from the second cycle process) was treated by 25 mg of pvpp, and the obtained mixture was stirred and centrifuged again in the same experimental conditions as mentioned above (third-cycle). the total polyphenol concentration was determined using the folin-ciocalteu reagent according to the method adapted by singleton and rossi (1965). 1 ml of reagent was added to 75 μl of phenol extract, followed by the addition of 200 μl na2co3 (20%) and 225 μl of distilled water. the mixture was incubated in the dark for 30 min. the absorbance was determined at 765 nm. the obtained values were corrected by subtracting the value related to the absorbance with pvpp. the total polyphenol content was then calculated based on a calibration curve established from a standard range with known concentrations of gallic acid. measurement of polyphenoloxidase and peroxidase activities 50 mg of samples were ground in 1 ml of phosphate buffer (0.1 m, ph 6) containing the insoluble pvp (5%), and then centrifuged for 30 min at 12,000 × g. for the polyphenoloxidase activity, 100 μl of the obtained supernatant were added to 2 ml of catechol (10 mm), and then the absorbance was determined at 410 nm after 3 min. for the peroxidase activity, 100 μl of the supernatant were added to 1 ml of guaiacol (20 mm). the reaction was initiated by the addition of 0.5 ml of hydrogen peroxide (0.1%), and the absorbance was deter mined at 470 nm after 3 min. antioxidant activity the determination of antioxidant activity was based on 1,1-diphenyl2-picrylhydrazyl (dpph) free radical scavenging. thus, 100 μl of different concentrations of each phenolic extract were mixed to 0.9 ml of a 0.004% methanol solution of dpph. the negative control was prepared by mixing 100 μl methanol and 0.9 ml of methanol solution of ddph. after 30 min of incubation in the dark, the absorbance was measured at 517 nm by using a spectrophotometer (chen et al., 2013). 5 x0 initial moisture content (% d.b.) xeq equilibrium moisture content (% d.b.) xf final moisture content (% d.b.) θ temperature (°c) χ² reduced chi-square 2. materials and methods 2.1. sorption isotherms of punica granatum legrelliae’s flowers the hygroscopic equilibrium was achieved by the static gravimetric technique, it consists on using six saturated salt solutions (koh, mgcl2 6h2o, k2co3, nano3, kcl, bacl2 2h2o). these salts provide a range of relative humidity of 5– 90 % (greenspan, 1977). the salts were prepared in six glass jars of 1l each with an insulated lid. for each solution, the samples were suspended in the jars above salts and remained in a stabilized temperature and humidity environment. the samples were weighed every two days in order to determine their equilibrium moisture content xeq. as soon as the masses had become stationary, the samples were weighed and placed in a drying oven whose temperature is fixed at 105 °c for 24 h. the difference of mass before and after drying at 105 °c (respectively meq and md) allows determining the product’ s moisture content xeq at the hygroscopic equilibrium by eq. (1). (1) the samples that were used for desorption were fresh and weighed between 0.093 and 0.102 g. the ones that were used for adsorption had been dried for 24 h in an oven at 105 °c before putting them on the glass jars; their weight were between 0.030 and 0.035 g. this process was performed at three different temperatures (30, 40 and 50 °c). 2.2. description of the solar drying the experimental apparatus consists of an indirect forced convection solar dryer, with a solar air collector, an electric auxiliary heater, a circulation fan and a drying chamber. the solar dryer was described in detail by el ferouali et al. (2016). the same mass of fresh punica granatum legrelliae’ s flowers (8.50 ±0.01 g) was used for each drying experiment. samples were uniformly spread forming a thin layer on the drying rack. in solar drying processes, the air temperature can vary with the magnitude of the solar radiation. the 16 h. el ferouali, a. zoukit, n. zehhar, f. benkhalti, h. bouamama, s. doubabi, n. abdenouri the results were expressed in ic 50 that is the amount of antioxidant necessary to decrease the initial concentration of dpph by 50%. radical dpph was graphically determined by the linear regressions of the plotted graphs (inhibition percentages as a function of different concentrations of samples). the lower ic 50 values indicate a higher antioxidant activity. statistical analysis all measurements were performed in triplicate and were reported as mean (± standard deviation). all data were analyzed using the spss 21.0 statistical package. an analysis of variance (anova) followed by the student newman-keuls post hoc test was used to compare differences in the means. the level of significance was defined as p<0.05. results and discussion desorption and adsorption isotherms the hygroscopic equilibrium of punica granatum legrelliae’s flowers was reached in 11 days for desorption and 10 days for adsorption. figs. 1 and 2 show the effect of temperature on desorption and adsorption of punica granatum legrelliae’s flowers. xeq increases by decreasing temperature at constant water activity. this result may be explained by the higher excitation state of water molecules at higher temperature thus decreasing the attractive forces between them. the sorption isotherms are type ii of the iupac classification and exhibit a sigmoidal shape; this is consistent with the behavior of other medicinal and aromatic plants (mujumdar, 2006). the hysteresis appears in fig. 3. this phenomenon is explained by thermodynamical irreversible processes that occur during desorption or adsorption or both (chirife and iglesias, 2007). 8 are type ii of the iupac classification and exhibit a sigmoidal shape; this is consistent with the behavior of other medicinal and aromatic plants (mujumdar, 2006). the hysteresis appears in fig. 3. this phenomenon is explained by thermodynamical irreversible processes that occur during desorption or adsorption or both (chirife and iglesias, 2007). 3.2. modeling of sorption isotherms in the present study, the relationship between xeq, aw at 30, 40 and 50 °c was predicted by applying mathematical models. the used models’ equations (gab, modified henderson, modified halsey, modified oswin and peleg) are given by basu et al. (2006), and the enderby model is given by popovski and mitrevski (2004). levenberg– marquardt non linear optimization method was used to calculate the models’ coefficients that describe the equilibrium curves and their statistical parameters. the best model, describing sorption isotherms of the product, has the highest value of the correlation coefficient r and smallest values of standard error of moisture (sem) and mean relative error (mre). these statistical parameters are defined respectively by eqs. (2), (3) and (4): (2) where, the experimental average moisture content is defined by: (3) (4) where xeqi,exp is the ith experimental moisture content, xeqi,pre is the ith predicted moisture content, and df is the freedom degree of the regression model. it can be seen from tab. 1 that the peleg model gives the best fitting to the experimental data with a correlation coefficient r of 0.9847 and 0.9916, sem of 1.9771 and 1.1692, and 8 are type ii of the iupac classification and exhibit a sigmoidal shape; this is consistent with the behavior of other medicinal and aromatic plants (mujumdar, 2006). the hysteresis appears in fig. 3. this phenomenon is explained by thermodynamical irreversible processes that occur during desorption or adsorption or both (chirife and iglesias, 2007). 3.2. modeling of sorption isotherms in the present study, the relationship between xeq, aw at 30, 40 and 50 °c was predicted by applying mathematical models. the used models’ equations (gab, modified henderson, modified halsey, modified oswin and peleg) are given by basu et al. (2006), and the enderby model is given by popovski and mitrevski (2004). levenberg– marquardt non linear optimization method was used to calculate the models’ coefficients that describe the equilibrium curves and their statistical parameters. the best model, describing sorption isotherms of the product, has the highest value of the correlation coefficient r and smallest values of standard error of moisture (sem) and mean relative error (mre). these statistical parameters are defined respectively by eqs. (2), (3) and (4): (2) where, the experimental average moisture content is defined by: (3) (4) where xeqi,exp is the ith experimental moisture content, xeqi,pre is the ith predicted moisture content, and df is the freedom degree of the regression model. it can be seen from tab. 1 that the peleg model gives the best fitting to the experimental data with a correlation coefficient r of 0.9847 and 0.9916, sem of 1.9771 and 1.1692, and 8 are type ii of the iupac classification and exhibit a sigmoidal shape; this is consistent with the behavior of other medicinal and aromatic plants (mujumdar, 2006). the hysteresis appears in fig. 3. this phenomenon is explained by thermodynamical irreversible processes that occur during desorption or adsorption or both (chirife and iglesias, 2007). 3.2. modeling of sorption isotherms in the present study, the relationship between xeq, aw at 30, 40 and 50 °c was predicted by applying mathematical models. the used models’ equations (gab, modified henderson, modified halsey, modified oswin and peleg) are given by basu et al. (2006), and the enderby model is given by popovski and mitrevski (2004). levenberg– marquardt non linear optimization method was used to calculate the models’ coefficients that describe the equilibrium curves and their statistical parameters. the best model, describing sorption isotherms of the product, has the highest value of the correlation coefficient r and smallest values of standard error of moisture (sem) and mean relative error (mre). these statistical parameters are defined respectively by eqs. (2), (3) and (4): (2) where, the experimental average moisture content is defined by: (3) (4) where xeqi,exp is the ith experimental moisture content, xeqi,pre is the ith predicted moisture content, and df is the freedom degree of the regression model. it can be seen from tab. 1 that the peleg model gives the best fitting to the experimental data with a correlation coefficient r of 0.9847 and 0.9916, sem of 1.9771 and 1.1692, and fig. 2: adsorption isotherms of punica granatum legrelliae’s flowers at 30, 40 and 50 °c, sdmax=1.08. fig. 1: desorption isotherms of punica granatum legrelliae’s flowers at 30, 40 and 50 °c, sdmax=1.07. modeling of sorption isotherms in the present study, the relationship between xeq, aw at 30, 40 and 50 °c was predicted by applying mathematical models. the used models’ equations (gab, modified henderson, modified halsey, modified oswin and peleg) are given by basu et al. (2006), and the enderby model is given by popovski and mitrevski (2004). levenberg-marquardt non linear optimization method was used to calculate the models’ coefficients that describe the equilibrium curves and their statistical parameters. the best model, describing sorption isotherms of the product, has the highest value of the correlation coefficient r and smallest values of standard error of moisture (sem ) and mean relative error (mre). these statistical parameters are defined respectively by eqs. (2), (3) and (4): (2) where, the experimental average moisture content is defined by: (3) (4) fig. 3: desorption and adsorption isotherms of punica granatum legrelliae’s flowers at 40°c, sdmax=0.64. hygroscopic and biochemical characterization of dried punica granatum legrelliae 17 where xeqi,exp is the ith experimental moisture content, xeqi,pre is the ith predicted moisture content, and df is the freedom degree of the regression model. it can be seen from tab. 1 that the peleg model gives the best fitting to the experimental data with a correlation coefficient r of 0.9847 and 0.9916, sem of 1.9771 and 1.1692, and mre of 10.5050% and 12.1983% respectively for desorption and adsorption isotherms. the peleg coefficients (a, b, c, d) are dependent on temperature and they are presented in tab. 2. determination of the optimum conditions for the storage all experimental data of desorption and adsorption at 30, 40 and 50 °c were gathered on the same graph (fig. 4). the sorption isotherm may particularly be described by a 3rd degree polynomial. the polynomial fitting is presented in the same figure and its relative equation is expressed by eq. (5). (5) this curve allowed to determine the value at which the second derivative of xeq equals to zero (inflection point) and consequently the optimum value of water activity for storage. the found value for punica granatum legrelliae’s flowers (awop=0.35±0.005) is in agreement with the general stability domain of biological products that is between 0.2 and 0.4 (le meste et al., 2001). this result reveals that relative humidity for storage and conservation should be less than 35±0.5% in order to ensure better stability of the product. net isosteric heat the net isosteric heat of sorption (∆hd) or differential enthalpy stands for the binding energy of the water to the substrate. this energy must be added to the heat vaporization energy (∆hvap) for dehydratation (eq. (6)). the net isosteric heat of sorption was calculated from the experimental data using the clausius clapeyron equation given by eq. (7) (beristain et al., 1996): (6) (7) this equation involves determining the isotherms at different temperatures in order to calculate the logarithmic variation of the water activity as a function of inverse temperature for fixed equilibrium moisture content. it is assumed that isosteric heat does not vary with the temperature. hence, ∆hd is determined from the slope -∆hd/r as it is given in eq. (8). tab. 1: statistical parameters: r, sem, and mre of the six models fitted to the sorption isotherms. gab modified peleg modified modified enderby henderson halsey oswin des. r 0.9819 0.9661 0.9847 0.9745 0.9761 0.9629 sem 1.7945 0.1026 1.9771 0.0787 2.3731 2.8614 mre (%) 12.0542 22.2729 10.5050 13.2845 16.3544 22.0315 ads. r 0.9958 0.9729 0.9916 0.9766 0.9859 0.9960 sem 1.0429 0.0873 1.1692 0.0803 1.4105 1.0122 mre (%) 17.8972 29.1219 12.1983 13.1607 12.9449 18.0610 tab. 2: peleg’s coefficients for sorption isotherms of punica granatum legrelliae’s flowers. model’s expression temperatures 30 °c 40 °c 50 °c des. ads. des. ads. des. ads. a 15.6804 25.3289 16.9795 39.2313 41.5141 41.5141 b 45.8227 9.6476 24.8467 8.0212 10.9518 10.9518 c 0.6162 2.4406 0.7656 5.0284 11.2192 11.2192 d 9.5455 0.2139 6.6552 0.1457 0.4330 0.4330 16 activation energy value of 92.91±2.51 kj mol-1, expressed the effect of temperature on the diffusion coefficient. the effect of forced convection drying at 40, 50 and 60 °c on the main quality criteria of punica granatum legrelliae’ s flowers was studied. accordingly, the solar drying at 40 °c is of great importance because it relatively preserved the bright color of fresh samples, as well as active compounds because this temperature kept the polyphenol content high (154.69±3.01 mg equivalent gallic acid/g dm). moreover, dried samples at this temperature were endowed with the highest antioxidant activity with an ic 50 of 1190.54±11.13 μg/g dm. this drying temperature was also optimal for the enzyme since polyphenoloxidase and peroxidase activities recorded the highest values (850.49±17.11 and 184.01±13.02 ue/mg protein/min, respectively). 5. tables and figures gab modified henderson peleg modified halsey modified oswin enderby des. r 0.9819 0.9661 0.9847 0.9745 0.9761 0.9629 sem 1.7945 0.1026 1.9771 0.0787 2.3731 2.8614 mre (%) 12.0542 22.2729 10.5050 13.2845 16.3544 22.0315 ads. r 0.9958 0.9729 0.9916 0.9766 0.9859 0.9960 sem 1.0429 0.0873 1.1692 0.0803 1.4105 1.0122 mre (%) 17.8972 29.1219 12.1983 13.1607 12.9449 18.0610 tab. 1. statistical parameters: r, sem, and mre of the six models fitted to the sorption isotherms. model’s expression temperatures 30 °c 40 °c 50 °c des. ads. des. ads. des. ads. a 15.6804 25.3289 16.9795 39.2313 41.5141 41.5141 b 45.8227 9.6476 24.8467 8.0212 10.9518 10.9518 c 0.6162 2.4406 0.7656 5.0284 11.2192 11.2192 d 9.5455 0.2139 6.6552 0.1457 0.4330 0.4330 tab. 2. peleg’ s coefficients for sorption isotherms of punica granatum legrelliae’s flowers. experiment dv ± 0.001 θ ± 0.5 rh x0± 0,001 xeq± 0,001 xf ± 0,001 time fig. 4: determination of the optimal water activity for storage of punica granatum legrelliae’s flowers, sdmax=1.08. 9 mre of 10.5050% and 12.1983% respectively for desorption and adsorption isotherms. the peleg coefficients (a, b, c, d) are dependent on temperature and they are presented in tab. 2. 3.3. determination of the optimum conditions for the storage all experimental data of desorption and adsorption at 30, 40 and 50 °c were gathered on the same graph (fig. 4). the sorption isotherm may particularly be described by a 3rd degree polynomial. the polynomial fitting is presented in the same figure and its relative equation is expressed by eq. (5). (5) this curve allowed to determine the value at which the second derivative of xeq equals to zero (inflection point) and consequently the optimum value of water activity for storage. the found value for punica granatum legrelliae’ s flowers (awop=0.35±0.005) is in agreement with the general stability domain of biological products that is between 0.2 and 0.4 (le meste et al., 2001). this result reveals that relative humidity for storage and conservation should be less than 35±0.5% in order to ensure better stability of the product. 3.4. net isosteric heat the net isosteric heat of sorption (∆hd) or differential enthalpy stands for the binding energy of the water to the substrate. this energy must be added to the heat vaporization energy (∆hvap) for dehydratation (eq. (6)). the net isosteric heat of sorption was calculated from the experimental data using the clausius clapeyron equation given by eq. (7) (beristain et al., 1996): (6) (7) this equation involves determining the isotherms at different temperatures in order to calculate the logarithmic variation of the water activity as a function of inverse temperature for fixed equilibrium moisture content. it is assumed that isosteric heat does not vary with the temperature. hence, ∆hd is determined from the slope -∆hd/r as it is given in eq. (8). (8) 9 mre of 10.5050% and 12.1983% respectively for desorption and adsorption isotherms. the peleg coefficients (a, b, c, d) are dependent on temperature and they are presented in tab. 2. 3.3. determination of the optimum conditions for the storage all experimental data of desorption and adsorption at 30, 40 and 50 °c were gathered on the same graph (fig. 4). the sorption isotherm may particularly be described by a 3rd degree polynomial. the polynomial fitting is presented in the same figure and its relative equation is expressed by eq. (5). (5) this curve allowed to determine the value at which the second derivative of xeq equals to zero (inflection point) and consequently the optimum value of water activity for storage. the found value for punica granatum legrelliae’ s flowers (awop=0.35±0.005) is in agreement with the general stability domain of biological products that is between 0.2 and 0.4 (le meste et al., 2001). this result reveals that relative humidity for storage and conservation should be less than 35±0.5% in order to ensure better stability of the product. 3.4. net isosteric heat the net isosteric heat of sorption (∆hd) or differential enthalpy stands for the binding energy of the water to the substrate. this energy must be added to the heat vaporization energy (∆hvap) for dehydratation (eq. (6)). the net isosteric heat of sorption was calculated from the experimental data using the clausius clapeyron equation given by eq. (7) (beristain et al., 1996): (6) (7) this equation involves determining the isotherms at different temperatures in order to calculate the logarithmic variation of the water activity as a function of inverse temperature for fixed equilibrium moisture content. it is assumed that isosteric heat does not vary with the temperature. hence, ∆hd is determined from the slope -∆hd/r as it is given in eq. (8). (8) 9 mre of 10.5050% and 12.1983% respectively for desorption and adsorption isotherms. the peleg coefficients (a, b, c, d) are dependent on temperature and they are presented in tab. 2. 3.3. determination of the optimum conditions for the storage all experimental data of desorption and adsorption at 30, 40 and 50 °c were gathered on the same graph (fig. 4). the sorption isotherm may particularly be described by a 3rd degree polynomial. the polynomial fitting is presented in the same figure and its relative equation is expressed by eq. (5). (5) this curve allowed to determine the value at which the second derivative of xeq equals to zero (inflection point) and consequently the optimum value of water activity for storage. the found value for punica granatum legrelliae’ s flowers (awop=0.35±0.005) is in agreement with the general stability domain of biological products that is between 0.2 and 0.4 (le meste et al., 2001). this result reveals that relative humidity for storage and conservation should be less than 35±0.5% in order to ensure better stability of the product. 3.4. net isosteric heat the net isosteric heat of sorption (∆hd) or differential enthalpy stands for the binding energy of the water to the substrate. this energy must be added to the heat vaporization energy (∆hvap) for dehydratation (eq. (6)). the net isosteric heat of sorption was calculated from the experimental data using the clausius clapeyron equation given by eq. (7) (beristain et al., 1996): (6) (7) this equation involves determining the isotherms at different temperatures in order to calculate the logarithmic variation of the water activity as a function of inverse temperature for fixed equilibrium moisture content. it is assumed that isosteric heat does not vary with the temperature. hence, ∆hd is determined from the slope -∆hd/r as it is given in eq. (8). (8) 18 h. el ferouali, a. zoukit, n. zehhar, f. benkhalti, h. bouamama, s. doubabi, n. abdenouri (8) fig. 5 presents the isosteric heat of sorption of punica granatum legrelliae’s flowers at temperatures ranging between 30 °c and 50 °c. this curves show that the isosteric heat is higher for small values of the moisture content indicating the highest binding energy for water removal, and it decreased along with the increase of the xeq. meel’s method (van meel, 1958). a polynomial model was found to be the best fitting of the dimensionless experimental data (eq. (10)). the used criteria to evaluate goodness of fit are the correlation coefficient r= 0.96191 and the reduced chisquare χ²= 0.0105. (10) modeling of the drying curves several mathematical models are used to describe the macroscopic behavior of the products. in this work, the most models describing drying kinetics were used: newton, page, henderson and pabis, 9 mre of 10.5050% and 12.1983% respectively for desorption and adsorption isotherms. the peleg coefficients (a, b, c, d) are dependent on temperature and they are presented in tab. 2. 3.3. determination of the optimum conditions for the storage all experimental data of desorption and adsorption at 30, 40 and 50 °c were gathered on the same graph (fig. 4). the sorption isotherm may particularly be described by a 3rd degree polynomial. the polynomial fitting is presented in the same figure and its relative equation is expressed by eq. (5). (5) this curve allowed to determine the value at which the second derivative of xeq equals to zero (inflection point) and consequently the optimum value of water activity for storage. the found value for punica granatum legrelliae’ s flowers (awop=0.35±0.005) is in agreement with the general stability domain of biological products that is between 0.2 and 0.4 (le meste et al., 2001). this result reveals that relative humidity for storage and conservation should be less than 35±0.5% in order to ensure better stability of the product. 3.4. net isosteric heat the net isosteric heat of sorption (∆hd) or differential enthalpy stands for the binding energy of the water to the substrate. this energy must be added to the heat vaporization energy (∆hvap) for dehydratation (eq. (6)). the net isosteric heat of sorption was calculated from the experimental data using the clausius clapeyron equation given by eq. (7) (beristain et al., 1996): (6) (7) this equation involves determining the isotherms at different temperatures in order to calculate the logarithmic variation of the water activity as a function of inverse temperature for fixed equilibrium moisture content. it is assumed that isosteric heat does not vary with the temperature. hence, ∆hd is determined from the slope -∆hd/r as it is given in eq. (8). (8) drying kinetics of punica granatum legrelliae’s flowers the experiments were performed at three air temperatures (40, 50 and 60 °c), under a drying air flow of 0.083 m3· s-1 and at an ambient relative humidity varying from 41 to 46%. the moisture content of the product at each drying time is calculated by eq. (9). (9) the initial moisture content of the product ranged from 304.8 to 325.0 (% d.b), and it was reduced to the final moisture content which varies from 4.8 to 28.6 (% d.b) (tab. 3). xeq were determined from the desorption isotherms. an increase in the drying air temperature had led to a significant reduction in the drying time; from 310 min for a temperature of 40 °c to 70 min for a temperature of 60 °c (fig. 6). it can be noticed the absence of phase 0, or the increasing drying rate period, and also the absence of phase 1, the constant drying rate period. there is only the presence of the falling drying rate period (phase 2) which is governed by the water diffusion in the material (bimbenet et al., 1985). fig. 7 represents the characteristic drying curve; the variation of dimensionless drying rate f versus moisture ratio as given by van fig. 5: net isosteric heat of desorption and adsorption of punica granatum legrelliae’s flowers versus equilibrium moisture content, sdmax=1.37. 10 fig. 5 presents the isosteric heat of sorption of punica granatum legrelliae’s flowers at temperatures ranging between 30 °c and 50 °c. this curves show that the isosteric heat is higher for small values of the moisture content indicating the highest binding energy for water removal, and it decreased along with the increase of the xeq. 3.5. drying kinetics of punica granatum legrelliae’s flowers the experiments were performed at three air temperatures (40, 50 and 60 °c), under a drying air flow of 0.083 m3.s-1 and at an ambient relative humidity varying from 41 to 46 %. the moisture content of the product at each drying time is calculated by eq. (9). (9) the initial moisture content of the product ranged from 304.8 to 325.0 (% d.b), and it was reduced to the final moisture content which varies from 4.8 to 28.6 (% d.b) (tab. 3). xeq were determined from the desorption isotherms. an increase in the drying air temperature had led to a significant reduction in the drying time; from 310 min for a temperature of 40 °c to 70 min for a temperature of 60 °c (fig. 6). it can be noticed the absence of phase 0, or the increasing drying rate period, and also the absence of phase 1, the constant drying rate period. there is only the presence of the falling drying rate period (phase 2) which is governed by the water diffusion in the material (bimbenet et al., 1985). fig. 7 represents the characteristic drying curve; the variation of dimensionless drying rate f versus moisture ratio as given by van meel’ s method (van meel, 1958). a polynomial model was found to be the best fitting of the dimensionless experimental data (eq. (10)). the used criteria to evaluate goodness of fit are the correlation coefficient r= 0.96191 and the reduced chisquare χ²= 0.0105. (10) 3.6. modeling of the drying curves several mathematical models are used to describe the macroscopic behavior of the products. in this work, the most models describing drying kinetics were used: newton, page, henderson and pabis, logarithmic, two term, two term exponential, wang and singh, tab. 3: drying conditions during the experiments in the solar dryer. experiment dv ± 0.001 θ ± 0.5 rh x0 ± 0,001 xeq ± 0,001 xf ± 0,001 time (m3· s-1) (°c) (%) (%d.b) (%d.b) (%d.b) (min) 1 0.083 40 42 304.8 11.2 28.6 310 2 0.083 50 46 325.0 11.9 25.0 120 3 0.083 60 41 304.8 7.4 4.8 70 10 fig. 5 presents the isosteric heat of sorption of punica granatum legrelliae’s flowers at temperatures ranging between 30 °c and 50 °c. this curves show that the isosteric heat is higher for small values of the moisture content indicating the highest binding energy for water removal, and it decreased along with the increase of the xeq. 3.5. drying kinetics of punica granatum legrelliae’s flowers the experiments were performed at three air temperatures (40, 50 and 60 °c), under a drying air flow of 0.083 m3.s-1 and at an ambient relative humidity varying from 41 to 46 %. the moisture content of the product at each drying time is calculated by eq. (9). (9) the initial moisture content of the product ranged from 304.8 to 325.0 (% d.b), and it was reduced to the final moisture content which varies from 4.8 to 28.6 (% d.b) (tab. 3). xeq were determined from the desorption isotherms. an increase in the drying air temperature had led to a significant reduction in the drying time; from 310 min for a temperature of 40 °c to 70 min for a temperature of 60 °c (fig. 6). it can be noticed the absence of phase 0, or the increasing drying rate period, and also the absence of phase 1, the constant drying rate period. there is only the presence of the falling drying rate period (phase 2) which is governed by the water diffusion in the material (bimbenet et al., 1985). fig. 7 represents the characteristic drying curve; the variation of dimensionless drying rate f versus moisture ratio as given by van meel’ s method (van meel, 1958). a polynomial model was found to be the best fitting of the dimensionless experimental data (eq. (10)). the used criteria to evaluate goodness of fit are the correlation coefficient r= 0.96191 and the reduced chisquare χ²= 0.0105. (10) 3.6. modeling of the drying curves several mathematical models are used to describe the macroscopic behavior of the products. in this work, the most models describing drying kinetics were used: newton, page, henderson and pabis, logarithmic, two term, two term exponential, wang and singh, fig. 7: characteristic drying curve of punica granatum legrelliae’s flowers, for x*(-): sdmax=0.02, for f(-): sdmax=0.06. fig. 6: variation of moisture content as a function of time, sdmax=7.73. hygroscopic and biochemical characterization of dried punica granatum legrelliae 19 logarithmic, two term, two term exponential, wang and singh, diffusion approach, verma et al. and midilli-kucuk. the models’ expressions are given in literature (menges and ertekin, 2006; yaldiz et al., 2001). the moisture ratio and the dimensionless drying rate of punica granatum legrelliae’s flowers during the thin layer drying experiments were calculated respectively by using eqs. (11) and (12): (11) (12) the appropriate model was selected according to the highest correlation coefficient (r), the lowest mean bias error (mbe) and the lowest reduced chisquare (χ²). these parameters are given as following: (13) (14) where x*exp,i stands for the experimental moisture ratio found in the measurements; x*pre,i is the predicted moisture ratio for this measurement; nob is the number of observations; and nc is the number of a model’s constants. the statistical parameters of the ten studied models are summarized in tab. 4. midilli-kucuk model was selected as the most convenient model to represent the drying behavior of punica granatum legrelliae’s flowers. the coefficients (a, k, n and b) of the chosen model at different temperatures are given in tab. 5. fig. 8 compares the experimental data with those predicted by midilli-kucuk model. the predicted data generally banded around the straight line. these observations highlighted the ability of this model to better simulate the change in water content in the solar drying punica granatum legrelliae’s flowers. hence, this product presents a weak external resistance to heat and mass transfer (midilli et al., 2002). tab. 4: statistical parameters r, χ², and mbe of the ten models applied to the drying curves. model r χ² mbe newton 0.9910 0.00187 0.00042 page 0.9983 0.00042 0.00343 henderson and pabis 0.9931 0.00158 0.00676 logarithmic 0.9984 0.00048 4.3122.10-8 two term 0.9979 0.00083 0.00357 two term exponentiel 0.9981 0.00046 0.0036 wang and singh 0.9974 0.00077 0.00207 approximation of diffusion 0.9978 0.00060 0.00186 verma et al. 0.9979 0.00056 0.00019 midilli-kucuk 0.9990 0.00038 0,00018 tab. 5: midilli-kucuk coefficients for punica granatum legrelliae’s flowers. model’s expression coefficients temperatures 40°c 50°c 60°c a 0.98732 0.99509 0.99692 k 0.00161 0.00777 0.03864 n 1.208 1.25159 1.11238 b -4.73821.10-4 -4.1336.10-5 -2.86821.10-4 11 diffusion approach, verma et al. and midilli-kucuk. the models’ expressions are given in literature (menges and ertekin, 2006; yaldiz et al., 2001). the moisture ratio and the dimensionless drying rate of punica granatum legrelliae’ s flowers during the thin layer drying experiments were calculated respectively by using eqs. (11) and (12): (11) (12) the appropriate model was selected according to the highest correlation coefficient (r), the lowest mean bias error (mbe) and the lowest reduced chisquare (χ²). these parameters are given as following: (13) (14) where x*exp,i stands for the experimental moisture ratio found in the measurements; x*pre,i is the predicted moisture ratio for this measurement; nob is the number of observations; and nc is the number of a model’ s constants. the statistical parameters of the ten studied models are summarized in tab. 4. midillikucuk model was selected as the most convenient model to represent the drying behavior of punica granatum legrelliae’ s flowers. the coefficients (a, k, n and b) of the chosen model at different temperatures are given in tab. 5. fig. 8 compares the experimental data with those predicted by midilli-kucuk model. the predicted data generally banded around the straight line. these observations highlighted the ability of this model to better simulate the change in water content in the solar drying punica granatum legrelliae’ s flowers. hence, this product presents a weak external resistance to heat and mass transfer (midilli et al., 2002). 3.7. effective moisture diffusivity and activation energy 11 diffusion approach, verma et al. and midilli-kucuk. the models’ expressions are given in literature (menges and ertekin, 2006; yaldiz et al., 2001). the moisture ratio and the dimensionless drying rate of punica granatum legrelliae’ s flowers during the thin layer drying experiments were calculated respectively by using eqs. (11) and (12): (11) (12) the appropriate model was selected according to the highest correlation coefficient (r), the lowest mean bias error (mbe) and the lowest reduced chisquare (χ²). these parameters are given as following: (13) (14) where x*exp,i stands for the experimental moisture ratio found in the measurements; x*pre,i is the predicted moisture ratio for this measurement; nob is the number of observations; and nc is the number of a model’ s constants. the statistical parameters of the ten studied models are summarized in tab. 4. midillikucuk model was selected as the most convenient model to represent the drying behavior of punica granatum legrelliae’ s flowers. the coefficients (a, k, n and b) of the chosen model at different temperatures are given in tab. 5. fig. 8 compares the experimental data with those predicted by midilli-kucuk model. the predicted data generally banded around the straight line. these observations highlighted the ability of this model to better simulate the change in water content in the solar drying punica granatum legrelliae’ s flowers. hence, this product presents a weak external resistance to heat and mass transfer (midilli et al., 2002). 3.7. effective moisture diffusivity and activation energy 11 diffusion approach, verma et al. and midilli-kucuk. the models’ expressions are given in literature (menges and ertekin, 2006; yaldiz et al., 2001). the moisture ratio and the dimensionless drying rate of punica granatum legrelliae’ s flowers during the thin layer drying experiments were calculated respectively by using eqs. (11) and (12): (11) (12) the appropriate model was selected according to the highest correlation coefficient (r), the lowest mean bias error (mbe) and the lowest reduced chisquare (χ²). these parameters are given as following: (13) (14) where x*exp,i stands for the experimental moisture ratio found in the measurements; x*pre,i is the predicted moisture ratio for this measurement; nob is the number of observations; and nc is the number of a model’ s constants. the statistical parameters of the ten studied models are summarized in tab. 4. midillikucuk model was selected as the most convenient model to represent the drying behavior of punica granatum legrelliae’ s flowers. the coefficients (a, k, n and b) of the chosen model at different temperatures are given in tab. 5. fig. 8 compares the experimental data with those predicted by midilli-kucuk model. the predicted data generally banded around the straight line. these observations highlighted the ability of this model to better simulate the change in water content in the solar drying punica granatum legrelliae’ s flowers. hence, this product presents a weak external resistance to heat and mass transfer (midilli et al., 2002). 3.7. effective moisture diffusivity and activation energy 11 diffusion approach, verma et al. and midilli-kucuk. the models’ expressions are given in literature (menges and ertekin, 2006; yaldiz et al., 2001). the moisture ratio and the dimensionless drying rate of punica granatum legrelliae’ s flowers during the thin layer drying experiments were calculated respectively by using eqs. (11) and (12): (11) (12) the appropriate model was selected according to the highest correlation coefficient (r), the lowest mean bias error (mbe) and the lowest reduced chisquare (χ²). these parameters are given as following: (13) (14) where x*exp,i stands for the experimental moisture ratio found in the measurements; x*pre,i is the predicted moisture ratio for this measurement; nob is the number of observations; and nc is the number of a model’ s constants. the statistical parameters of the ten studied models are summarized in tab. 4. midillikucuk model was selected as the most convenient model to represent the drying behavior of punica granatum legrelliae’ s flowers. the coefficients (a, k, n and b) of the chosen model at different temperatures are given in tab. 5. fig. 8 compares the experimental data with those predicted by midilli-kucuk model. the predicted data generally banded around the straight line. these observations highlighted the ability of this model to better simulate the change in water content in the solar drying punica granatum legrelliae’ s flowers. hence, this product presents a weak external resistance to heat and mass transfer (midilli et al., 2002). 3.7. effective moisture diffusivity and activation energy fig. 8: comparison of experimental moisture ratio with predicted moisture ratio from the midilli-kucuk model, for x*exp(-): sdmax=0.02, for x*pre(-): sdmax=0.01. 17 (m3.s-1) (°c) (%) (%d.b) (%d.b) (%d.b) (min) 1 0.083 40 42 304.8 11.2 28.6 310 2 0.083 50 46 325.0 11.9 25.0 120 3 0.083 60 41 304.8 7.4 4.8 70 tab. 3. drying conditions during the experiments in the solar dryer. model r χ² mbe newton 0.9910 0.00187 0.00042 page 0.9983 0.00042 0.00343 henderson and pabis 0.9931 0.00158 0.00676 logarithmic 0.9984 0.00048 4.3122.10-8 two term 0.9979 0.00083 0.00357 two term exponentiel 0.9981 0.00046 0.0036 wang and singh 0.9974 0.00077 0.00207 approximation of diffusion 0.9978 0.00060 0.00186 verma et al. 0.9979 0.00056 0.00019 midilli-kucuk 0.9990 0.00038 0,00018 tab. 4. statistical parameters r, χ², and mbe of the ten models applied to the drying curves. model’s expression coefficients temperatures 40°c 50°c 60°c a 0.98732 0.99509 0.99692 k 0.00161 0.00777 0.03864 n 1.208 1.25159 1.11238 b -4.73821.10-4 -4.1336.10-5 -2.86821.10-4 tab. 5. midilli-kucuk coefficients for punica granatum legrelliae’s flowers. effective moisture diffusivity and activation energy fick’s second law can be used to describe the drying process on which diffusion is dominant. the solution of fick’s second law in slab geometry is given by eq. (15) (crank, 1979), with the assumptions of moisture migration being by diffusion, shrinkage is negligible, and the temperature and the diffusion coefficient maintain almost the same value: (15) for a sufficiently long drying time, all terms of the above series are negligible compared with the first term. thus, eq. (15) becomes: (16) the effective moisture diffusivity deff was calculated using the slope of eq. (16) (eq. 17)), with h (the half thickness of the dried samples). it is found that 2h=0.017±0.0019 cm: 12 fick’ s second law can be used to describe the drying process on which diffusion is dominant. the solution of fick’ s second law in slab geometry is given by eq. (15) (crank, 1979), with the assumptions of moisture migration being by diffusion, shrinkage is negligible, and the temperature and the diffusion coefficient maintain almost the same value: (15) for a sufficiently long drying time, all terms of the above series are negligible compared with the first term. thus, eq. (15) becomes: (16) the effective moisture diffusivity deff was calculated using the slope of eq. (16) (eq. 17)), with h (the half thickness of the dried samples). it is found that 2h=0.017±0.0019 cm: (17) the activation energy was calculated using arrhenius type equations (akpinar et al., 2003) (eq. (18)): (18) drying of punica granatum legrelliae’ s flowers occurs only in the falling rate period, so liquid diffusion controls the process. fig. 9 illustrated the effects of temperature on the effective diffusion coefficient of the product. graphs were drawn between ln(x*) against the drying time according to fick’ s diffusion model. the effective moisture diffusivities (deff) values are (2.480±0.06) x 10-11, (7.794±0.2) x 10-11 and (2.111±0.19) x 10-10 m2 s-1 respectively for 40, 50 and 60 °c. hence, the increase in the drying air temperature increased the value of deff. in fact, when samples were dried at higher temperature, the activity of water molecules increased leading to higher moisture diffusivity (xiao et al., 2010). the values of the obtained deff lie within general range 10-8 to 10-12 m2.s-1 for drying of aromatic and medicinal plants (zogzas et al., 1996). the activation energy was calculated from the slop of ln(deff) as function of 1/t (fig. 10); its value is 92.91±2.51 kj mol-1, this 12 fick’ s second law can be used to describe the drying process on which diffusion is dominant. the solution of fick’ s second law in slab geometry is given by eq. (15) (crank, 1979), with the assumptions of moisture migration being by diffusion, shrinkage is negligible, and the temperature and the diffusion coefficient maintain almost the same value: (15) for a sufficiently long drying time, all terms of the above series are negligible compared with the first term. thus, eq. (15) becomes: (16) the effective moisture diffusivity deff was calculated using the slope of eq. (16) (eq. 17)), with h (the half thickness of the dried samples). it is found that 2h=0.017±0.0019 cm: (17) the activation energy was calculated using arrhenius type equations (akpinar et al., 2003) (eq. (18)): (18) drying of punica granatum legrelliae’ s flowers occurs only in the falling rate period, so liquid diffusion controls the process. fig. 9 illustrated the effects of temperature on the effective diffusion coefficient of the product. graphs were drawn between ln(x*) against the drying time according to fick’ s diffusion model. the effective moisture diffusivities (deff) values are (2.480±0.06) x 10-11, (7.794±0.2) x 10-11 and (2.111±0.19) x 10-10 m2 s-1 respectively for 40, 50 and 60 °c. hence, the increase in the drying air temperature increased the value of deff. in fact, when samples were dried at higher temperature, the activity of water molecules increased leading to higher moisture diffusivity (xiao et al., 2010). the values of the obtained deff lie within general range 10-8 to 10-12 m2.s-1 for drying of aromatic and medicinal plants (zogzas et al., 1996). the activation energy was calculated from the slop of ln(deff) as function of 1/t (fig. 10); its value is 92.91±2.51 kj mol-1, this 20 h. el ferouali, a. zoukit, n. zehhar, f. benkhalti, h. bouamama, s. doubabi, n. abdenouri (17) the activation energy was calculated using arrhenius type equations (akpinar et al., 2003) (eq. (18)): (18) drying of punica granatum legrelliae’s flowers occurs only in the falling rate period, so liquid diffusion controls the process. fig. 9 illustrated the effects of temperature on the effective diffusion coefficient of the product. graphs were drawn between ln(x*) against the drying time according to fick’s diffusion model. the effective moisture diffusivities (deff) values are (2.480±0.06) × 10-11, (7.794±0.2) × 10-11 and (2.111±0.19) × 10-10 m2 s-1 respectively for 40, 50 and 60 °c. hence, the increase in the drying air temperature increased the value of deff. in fact, when samples were dried at higher temperature, the activity of water molecules increased leading to higher moisture diffusivity (xiao et al., 2010). the values of the obtained deff lie within general range 10-8 to 10-12 m2· s-1 for drying of aromatic and medicinal plants (zogzas et al., 1996). the activation energy was calculated from the slop of ln(deff) as function of 1/t (fig. 10); its value is 92.91±2.51 kj mol-1, this range corresponds to the activation energy of other food products in a general range reported by several researchers (xiao et al., 2010; zogzas et al., 1996). effect of solar convective drying at different temperatures on the flowers’ color the cie lab color space was used for color analyses based on imaging process of samples. the l*, a* and b* color parameters have been widely used to describe color changes during thermal processing of agricultural products. l* represents lightness, ranging from 0 (black) to 100 (white), it indicates how dark/light are the samples. the parameter a* ranges between -128 (green) and 127 (red), and b* ranges between -128 (blue) and 127 (yellow). fig. 11 shows the effect of convective solar drying at 40, 50 and 60 °c on l*, a* and b* color parameters of punica granatum legrelliae’s flowers. the color of the fresh and dried flowers was determined. the fresh samples’ color is red-orange, its (l*, a*, b*) had an average value of almost (47.4±0.8944, 55±1.0801, 45.8±0.4472). the effect of temperature on the samples’ color is that the (l*, a*, b*) de creased by increasing temperature. in fact they decreased from an average of (41.8±1.3874, 54±1.2360, 34.8±1.7013) at 40 °c to an average of (27.6±0.8944, 40±1.5495, 25.2±1.9235) at 60 °c. hence, by drying at higher temperature, the color became darker, and the red and yellow colors decreased. 12 fick’ s second law can be used to describe the drying process on which diffusion is dominant. the solution of fick’ s second law in slab geometry is given by eq. (15) (crank, 1979), with the assumptions of moisture migration being by diffusion, shrinkage is negligible, and the temperature and the diffusion coefficient maintain almost the same value: (15) for a sufficiently long drying time, all terms of the above series are negligible compared with the first term. thus, eq. (15) becomes: (16) the effective moisture diffusivity deff was calculated using the slope of eq. (16) (eq. 17)), with h (the half thickness of the dried samples). it is found that 2h=0.017±0.0019 cm: (17) the activation energy was calculated using arrhenius type equations (akpinar et al., 2003) (eq. (18)): (18) drying of punica granatum legrelliae’ s flowers occurs only in the falling rate period, so liquid diffusion controls the process. fig. 9 illustrated the effects of temperature on the effective diffusion coefficient of the product. graphs were drawn between ln(x*) against the drying time according to fick’ s diffusion model. the effective moisture diffusivities (deff) values are (2.480±0.06) x 10-11, (7.794±0.2) x 10-11 and (2.111±0.19) x 10-10 m2 s-1 respectively for 40, 50 and 60 °c. hence, the increase in the drying air temperature increased the value of deff. in fact, when samples were dried at higher temperature, the activity of water molecules increased leading to higher moisture diffusivity (xiao et al., 2010). the values of the obtained deff lie within general range 10-8 to 10-12 m2.s-1 for drying of aromatic and medicinal plants (zogzas et al., 1996). the activation energy was calculated from the slop of ln(deff) as function of 1/t (fig. 10); its value is 92.91±2.51 kj mol-1, this 12 fick’ s second law can be used to describe the drying process on which diffusion is dominant. the solution of fick’ s second law in slab geometry is given by eq. (15) (crank, 1979), with the assumptions of moisture migration being by diffusion, shrinkage is negligible, and the temperature and the diffusion coefficient maintain almost the same value: (15) for a sufficiently long drying time, all terms of the above series are negligible compared with the first term. thus, eq. (15) becomes: (16) the effective moisture diffusivity deff was calculated using the slope of eq. (16) (eq. 17)), with h (the half thickness of the dried samples). it is found that 2h=0.017±0.0019 cm: (17) the activation energy was calculated using arrhenius type equations (akpinar et al., 2003) (eq. (18)): (18) drying of punica granatum legrelliae’ s flowers occurs only in the falling rate period, so liquid diffusion controls the process. fig. 9 illustrated the effects of temperature on the effective diffusion coefficient of the product. graphs were drawn between ln(x*) against the drying time according to fick’ s diffusion model. the effective moisture diffusivities (deff) values are (2.480±0.06) x 10-11, (7.794±0.2) x 10-11 and (2.111±0.19) x 10-10 m2 s-1 respectively for 40, 50 and 60 °c. hence, the increase in the drying air temperature increased the value of deff. in fact, when samples were dried at higher temperature, the activity of water molecules increased leading to higher moisture diffusivity (xiao et al., 2010). the values of the obtained deff lie within general range 10-8 to 10-12 m2.s-1 for drying of aromatic and medicinal plants (zogzas et al., 1996). the activation energy was calculated from the slop of ln(deff) as function of 1/t (fig. 10); its value is 92.91±2.51 kj mol-1, this fig. 10: ln(deff) versus 1/t, sdmax=0.08. fig. 9: effects of temperature on the effective diffusion coefficient of punica granatum legrelliae’s flowers, sdmax=0.32. effect of drying on biochemical parameters effect on total polyphenol content the highest total polyphenol content was assigned to the fresh sample (233.30±13.94 mg equivalent gallic acid/g dm) (fig. 12). a very small decrease of total polyphenol content was noticed by increasing the drying temperature from 40 °c to 60 °c. in fact, their values at 40 °c, 50 °c and 60 °c are respectively 154.69±3.01, 150.40±1.95 and 146.98±3.08 mg equivalent gallic acid/g dm. larrauri et al. (1997) also observed a decrease in the total polyphenol content in dried red grape pomace peels at high temperatures (100 °c, 140 °c) compared to dried samples at a lower temperature (60 °c). in fact, high temperatures affect the quality and quantity of these phenolic compounds due to their thermal decomposition. other studies have suggested that the reduction in the polyphenol content is due to polymerizations of the antioxidant molecules caused by the high temperatures (calín-sánchez et al., 2013; henríquez et al., 2014). fig. 11: effect of convective solar drying at 40, 50 and 60 °c on l*, a* and b* color parameters of punica granatum legrelliae’s flowers, sdmax=1.92. hygroscopic and biochemical characterization of dried punica granatum legrelliae 21 effect on polyphenoloxidase and peroxidase activities enzymatic browning is the result of the oxidation of o-diphenol to quinone by ppo in the presence of oxygen. according to fig. 13, polyphenoloxidases activity is very high in fresh flowers (1550.14±39.14 ue/mg protein/min) compared to dried ones. the highest enzymatic activity (ppo) among the dried samples was recorded for dried samples at 40 °c and, it decreased by increasing temperature from 850.49±17.11 ue/mg protein/min (at 40 °c) to 656.12±19.87 ue/mg protein/min (at 60 °c). the highest po activity was recorded by dried samples at 40 °c (184.01±13.02 ue/mg protein/min) (fig. 14). the obtained values of po activity at 50 °c and 60 °c were lower; they are respectively 127.55±10.12 and 115.38±12.08 ue/mg protein/min. this reduction of polyphenoloxidase and peroxidase activities was also observed by tan et al. (2015) after convective drying of mulberry leaves at 50 °c and 100 °c. according to baltacioğlu et al. (2015); ppo is very active at 40 °c, but this activity decreases after heat treatment between 50 °c and 70 °c. in general, inactivation or reduction of ppo and po during drying is due to the denaturation of enzymes by heat, as well as to the reduction of water activity of the plant material during drying. the inactivation of these enzymes thus leads to the inhibition and the degradation of the phenolic compounds (tan et al., 2015). finally, the high ppo and po activities at 40 °c indicate that this temperature is the optimum one for the enzyme. effect on antioxidant activity according to dpph radical scavenging ability, fresh samples had a strong antioxidant activity compared to the dried samples with an ic50 of 869.86±10.14 μg/g dm (fig. 15). convective dried samples at 40 °c recorded the highest antioxidant activity, with an ic 50 of 1190.54±11.13 μg/g dm. dried samples at 50 °c and 60 °c had lower activity, with respectively ic 50 of 1638.84±17.98 and 1696.63±56.11 μg/g dm. calín-sánchez et al. (2013) reported that convective drying of pomegranate arils and rind resulted in a reduction in antioxidant activity from 45.1 mg eq trolox/g dm for fresh samples to 24.5 mg eq trolox/g dm for dried ones at 60 °c. otherwise, according to wojdyło et al. (2016), jujube fruit showed better antioxidant activity when they are dried at low temperature (ranging from 23.9 mmol trolox/100 g dm at 50 °c up to 9.6 mmol trolox/100 g dm at 70 °c). similarly, rodríguez et al. (2013) observed a significant decrease in antioxidant capacity of thyme by the increase of drying temperature from 40 °c to 60 °c. hence, drying at high temperatures destroys some phenolic compounds and leads to the loss of the antioxidant activity. this could be explained by the degradation of phenolic monoterpenes and volatile aromatic compounds that significantly contribute to enhance the antioxidant capacity (rodriguez et al., 2013). it thus appears that drying at 40 °c is more preservative of the antioxidant activity for the studied samples. fig. 15: antioxidant activity ic50 (μg/g dm) of punica granatum legrelliae’s flowers, sdmax=56.11. fig. 12: total polyphenol content (mg equivalent gallic acid / g dm) of punica granatum legrelliae’s flowers, sdmax=13.94. fig. 13: polyphenoloxydase activity (ue/mg protein/min) of punica granatum legrelliae’s flowers, sdmax=39.14. fig. 14: peroxidase activity (ue/mg protein/min) of punica granatum legrelliae’s flowers, sdmax=32.71. 22 h. el ferouali, a. zoukit, n. zehhar, f. benkhalti, h. bouamama, s. doubabi, n. abdenouri conclusions the present paper highlights links between the hygroscopic behavior, the solar drying characterization and the dried punica granatum legrelliae’s flowers’ quality. the quality of the dried product was spotted in terms of its biochemical composition of polyphenols, antioxidant activity, polyphenoloxydase and peroxydase activities, and also regarding the color change. the modeling and the hygroscopic characteristics were determined for desorption and adsorp tion of punica granatum legrelliae’s flowers. evaluation of sorption isotherm provided valuable information for drying and storing this seasonal plant. these isotherms presented a sigmoidal shape of type ii in the iupac classification and among the six models chosen to fit the sorption curves, the peleg model provided the best description of the sorption isotherms. a polynomial fitting of the experimental sorption isotherms data led to disclose the optimal relative humidity for the storage and conservation, it should be less than 35±0.5% for better stability of the dried product. the net isosteric heat of sorption was evaluated, it is in the range of 70 kj mol-1 for small values of the moisture content (xeq=8 % d.b.), and it decreased along with the increase of the xeq; this thermodynamic quantity estimates the required energy for dehydration processes. according to the drying kinetics study, among the three drying periods, only the falling rate period exists. furthermore, the results of regression analysis showed that midilli-kucuk drying model predicts the drying kinetics of punica granatum legrelliae’s flowers, which means that heat and mass transfer are more limited in intern than in extern of the product. the effective moisture diffusivity values were obtained from the fick’s diffusion model, and the increase in air temperature raised the value of deff from (2.480±0.06) × 10-11 m2 s-1 at 40 °c to (2.111±0.19) × 10-10 m2 s-1 at 60 °c. the arrhenius relation, with an activation energy value of 92.91±2.51 kj mol-1, expressed the effect of temperature on the diffusion coefficient. the effect of forced convection drying at 40, 50 and 60 °c on the main quality criteria of punica granatum legrelliae’s flowers was studied. accordingly, the solar drying at 40 °c is of great importance because it relatively preserved the bright color of fresh samples, as well as active compounds because this temperature kept the polyphenol content high (154.69±3.01 mg equivalent gallic acid/g dm). moreover, dried samples at this temperature were endowed with the highest antioxidant activity with an ic 50 of 1190.54± 11.13 μg/g dm. this drying temperature was also optimal for the enzyme since polyphenoloxidase and peroxidase activities recorded the highest values (850.49±17.11 and 184.01±13.02 ue/mg protein/ min, respectively). acknowlegement this work was supported by the research institute for solar energy and new 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233-240. doi: 10.1016/j.biosystemseng.2009.11.001 yaldiz, o., ertekin, c., uzun, h.i., 2001: mathematical modeling of thin layer solar drying of sultana grapes. energy 26, 457-465. doi: 10.1016/s0360-5442(01)00018-4 zhang, l., fu, q., zhang, y., 2011: composition of anthocyanins in pomegranate flowers and their antioxidant activity. food chem. 127, 1444-1449. doi: 10.1016/j.foodchem.2011.01.077 zogzas, n.p., maroulis, z.b., marinos-kouris, d., 1996: moisture diffusivity data compilation in foodstuffs. drying technol. 14, 22252253. doi: 10.1080/07373939608917205 address of the author: h. el ferouali, a. zoukit, s. doubabi, n. abdenouri*: lset, cadi ayyad university, marrakech 4000 morocco *e-mail: n.abdenouri@uca.ma n. zehhar, f. benkhalti, h. bouamama: biotechnologie de la valorisation et la protection des agro-ressources chimie bio-organique et macromoléculaire, cadi ayyad university, marrakech 4000 morocco © the author(s) 2018. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 89, 82 88 (2016), doi:10.5073/jabfq.2016.089.010 1erciyes university department of horticulture, melikgazi, kayseri, turkey 2 fruit research institute, egirdir, isparta, turkey 3plant protection central research institute, ankara, turkey 4ataturk university department of horticulture, erzurum, turkey determination of genetic relatedness among turkish apple germplasm based on issr markers aydın uzun1, serif ozongun2, osman gulsen1, kadir ugurtan yılmaz1, suat kaymak3, sezai ercisli4* (received october 4, 2015) * corresponding author summary apple (malus domestica borkh.) is one of the most economically important pome fruits worldwide and turkey is within origin center of apple. in this research, inter-simple sequence repeat (issr) markers were used to determine relationships among the turkish apple accessions and some selected foreign cultivars and species. fourteen issr primers produced a total of 111 fragments and 76 of them were polymorphic. the number of average polymorphic fragments per primer was 5.4. the mean polymorphism information content (pic) was 0.37. the unweighted pair group method arithmetic average (upgma) analysis demonstrated that the accessions had a similarity range from 0.79 to 0.98. all accessions studied were discriminated and many subgroups were determined in the dendrogram based on the upgma analysis. high level of variation among the turkish apples existed. foreign cultivars, m.baccata, m. prunifolia and m. sylvestris accessions studied mix-clustered among the turkish accessions. for sub-structuring bayesian analysis, 71 loosely or uncorrelated markers with less than 10 % missing data were used. this indicated absence of subpopulations, meaning well and equal introgression of genetic backgrounds or species available among the accessions. it can be concluded that turkey was rich in apple genetic diversity, which may provide opportunities for apple breedind programs. introduction fruit, which contains phytochemicals that are being studied for added health benefits, has been recognized as a good source of vitamins and minerals, and for their role in preventing vitamin c and vitamin a deficiencies (bacvonkralj et al., 2014; rop et al., 2014). among fruits, apple has special importance because it is one of the most produced fruit crops among the temperate fruits with over 75 million tons of production per year (fao, 2012). domesticated apples (malus domestica borkh.) have been cultivated since ancient times and are now produced in a range of area from siberia with freezing temperatures during winter as low as -40 oc to some equatorial locations with high temperatures (janick et al., 1996). four origin centers were reported for apples including east asia, middle asia, east asia-europe and north america. turkey belongs to east asia-europe origin center and has considerable diversity (janick et al., 1996). in earlier time, breeding programs were based only on selections from naturally growing apple trees. then, hybridization became a more preferable tool for obtaining economically important new cultivars. origin of domesticated apples was probably based on m. sieversii known as wild apple in central asia (harris et al., 2002; coart et al., 2006 ). it was argued that m. sylvestris, the wild apple of western europe, might have contributed little or even nothing to the domesticated apple gene pool at least as maternal ancestor (robinson et al., 2001). similarly, m. sieversii was reported as the main contributor to the genome of the cultivated apple and m. sylvestris, in particular, determined as the secondary contributor. evolution of domesticated apples occurred over a long time period and involved more than one wild species (cornille et al., 2012). but some findings regarding to this issue that three most frequent chloroplast haplotypes of m. domestica and m. sylvestris were nearly absent in the analysed m. sieversii accessions concluded complex origin of domesticated apples. also high level of cpdna diversity was detected among the genus malus cultivars, which was explained by the hypothesis of the complex hybrid origin of m. domestica (coart et al., 2006). genetic diversity and phylogenetic studies on apples have been carried out using various marker systems. simple sequence repeats (ssrs) markers were the most reliable system for this kind of studies (patzak et al., 2012; garkava-gustavsson et al., 2013). in addition, amplified fragment length polymorphism (aflp) (kenis and keulemans, 2005; ning et al., 2007); chloroplast dna (cpdna) (coart et al., 2006) markers were used for estimating apple genetic diversity, relationships and constructing genetic maps of apples. inter-simple sequence repeat (issr) markers were reported to have higher reproducible rates due to the use of longer primers (16-25mers) than the rapd primers (10-mers). this marker system is cost effective because multiple loci are amplified during pcr amplification (zietkiewicz et al., 1994). it was previously used for apple genetic studies (goulao and oliveira, 2001; he et al., 2011). turkey is both within origin center of apples and a major apple producer with 2.88 million tons of production (fao, 2012). hence there is considerable genetic diversity of apples in turkey. in addition, introductions from foreign countries are available in apple genetic resources of turkey. conservation and characterization of this genetic pool are required for breeding programs and future utilization. in present study, genetic variation and relationships among the apple accessions collected from different parts of turkey and some selected foreign cultivars were investigated. materials and methods plant material and dna isolation one hundred and fifty-eight apple accessions were used for this study including 152 m. domestica cultivars and local genotypes, three m. sylvestris accessions, two m. baccata and one m. prunifola accession (tab. 1). the rest included common apple cultivars such as golden delicious, granny smith and red chief. for dna extractions, leaf tissues of all accessions were obtained from the collection located fruit research station in egirdir of isparta, turkey. total genomic dna was extracted from young leaves by the ctab method as described by doyle and doyle (1990). dna concentration was measured with a spectrophotometer (biotek instruments, inc. vinooski, united states) and 10 ng/ml dna templates were made using te (10 mm tris-hcl,1 mm edta, ph 8.0). tab. 1: name of apple accessions utilized in this study 1 lodiearlygolden 41 180887(5.2) 81 e73 121 42.bs.5(almila) 2 yazelmasi(2484) 42 190887(1.4) 82 e2411 122 42.e.2(ankaraguz.) 3 yazelmasi(2482) 43 180887(4.4) 83 e65 123 42.c.5(yazamasya) 4 beyazelma(2575) 44 200887(1.2) 84 e5 124 42.e.6(kabaelma) 5 ferik 45 blackjon 85 e392e 125 42.ko.(yaylapinari) 6 karanfil(2570) 46 daldabir 86 e24 126 42.e.4(mayhosy.k.) 7 beyelmasi(2477) 47 220887(3.2) 87 batum 127 42.e.3(hanimteni) 8 kiselmasi(2590) 48 230887(1.2) 88 e14 128 42.e.7(yildizkiran) 9 sahelmasi(2600) 49 250887(1.10) 89 e13 129 42.kp.3(karapinar) 10 gelinelmasi(2475) 50 yaztavsanbasi 90 sarigobek 130 42.c.3(tatlitavsanb.) 11 sekerelması(2551) 51 210887(1.1) 91 candir 131 32e1 12 tatlielma(2511) 52 e220887 92 e25 132 yenice 13 sarielma 53 e180887(2.1) 93 e11 133 orak 14 gobek(2455) 54 e210887(1.4) 94 uzunyumra 134 pancarlik 15 sogutelma(2480) 55 e170887(2.5) 95 e32 135 samsun 16 susuzelma(2500) 56 e210887(2.1) 96 cigit 136 harim 17 mektepelmasi(2565) 57 e130887(2.3) 97 e383e 137 gelendost 18 altinokelmasi(2490) 58 reinettetardiva 98 karpuz 138 inebolu 19 elma(2590) 59 e70 99 portakal 139 golden delicious 20 demir(2486) 60 e42 100 e33 140 jonagold 21 rizedemir 61 e71 101 gurcu 141 ozark gold 22 sandik 62 e40 102 e2 142 royal gala 23 petek(2577) 63 e55 103 542e 143 elstar 24 oltuelmasi(2594) 64 e52 104 e1 144 melrose 25 cincik(2471) 65 e51 105 cidagut 145 idared 26 mahsusaelmasi 66 e50 106 e6 146 fuji 27 elma(2523) 67 e49 107 sinap 147 red chief 28 petevrekelmasi 68 e82 108 384e 148 gloster 29 tavsanbasi(2531) 69 e57 109 e4 149 granny smith 30 tatlielma(2492) 70 e78 110 seker 150 e72 31 pasaelmasi 71 e66 111 e35 151 cooper43 32 lazelmasi(2507) 72 e47 112 e10 152 e33 33 hüryemez 73 e56 113 piraziz 153 m. prunifolia 34 kadirhatice 74 e37 114 gümüshane 154 m. baccata 1 35 güztavsanbasi 75 e63 115 gemlik.3 155 m. baccata 2 36 180887(5.1) 76 e81 116 tokat.1 156 m. sylvestris 1 37 sivanorelmasi 77 e9 117 tokat.3 157 m. sylvestris 2 38 kalkandelen 78 e76 118 tokat.4 158 m. sylvestris 3 39 karasaki 79 e48 119 42.kp.1 40 gurcu 80 e67 120 42.a.1 issr analysis fourteen issr primers previously evaluated by fang and roose (1997) and gulsen et al. (2010) were used for all apple accessions (tab. 2). pcr reaction components and pcr cycling parameters were performed as described by uzun et al. (2009). pcr products were separated on 2 % agarose gel in 1 x tbe buffer (89 mm tris, 89 mm boric acid, 2 mm edta) at 115 v for 2.5-3 h. the fragment patterns were photographed under uv light for further analysis. a 100 bp standard dna ladder (generuler, fermentas) was used for estimating size of issr fragments. data analysis each band was scored as present (1) or absent (0) and data were analyzed with the numerical taxonomy multivariate analysis system (ntsys-pc version 2.1) software package (rohlf, 2000). a similarity matrix was constructed based on dice’s coefficient (dice, 1945), which considers only one to one matches between two taxa for similarity. the similarity matrix was used to construct a dendrogram using the unweighted pair group method arithmetic average (upgma) to determine genetic relationships in the germplasm studied. goodness of fit test called mantel test was performed by using 84 a. uzun, s. ozongun, o. gulsen, k.u. yılmaz, s. kaymak, s. ercisli ultrametric distance matrix obtained from the dendrogram and similarity matrix (mantel, 1967). the result of this test is a cophenetic correlation coefficient, r, indicating how well the dendrogram represents similarity data. polymorphic information content (pic) for dominant markers was calculated as: pic = 1− [f 2 + (1− f)2], where ‘f’ is the frequency of the marker in the data set. pic for dominant markers is a maximum of 0.5 for ‘f’ = 0.5 (de riek et al., 2001). pic provides an estimate of the discriminatory power of a locus by taking into account not only the number of alleles that are expressed but also the relative frequencies of those alleles (smith et al., 1997). a principal coordinate analysis (pcoa) was performed based on the variance covariance matrix calculated from issr data. pcoa is a computational alternative to principle coordinate analysis (pca). pca is used for similarities and pcoa for dissimilarities. the data matrix was used to calculate distance matrix, then the distance matrix was double-centered, the double-centered matrix was then factored and a plot was made (rohlf, 2000). results and discussion issr analysis yielded 111 fragments and 76 of them (68.5 %) were polymorphic. number of bands scored per primer varied between 4 (hvh(ca)7t) and 14 (gaca4), with a mean of 7.9. the genalex ver. 6.5 program was employed, to determine allele frequency (p and q), no of effective alleles (ne), shannon’s information index (i), expected (he) and unbiased expected heterozygosity (uhe) (peakall and smouse, 2012). the pic values for the 21 primer combinations ranged from 0.02 (hvh(ca)7t and dbda(ca7) to 0.27 (gaca4) with a mean of 0.15 (tab. 2). cophenetic correlation between ultrametric similarities of tree and similarity matrix was found to be relatively high (r = 0.76, p < 0.01). values for effective alleles (ne) ranged from 1.02 ((tcc)5ry) to 1.48 ((ag)7yc) (average 1.28), for shannon’s information index from 0.04 ((tcc)5ry) to 0.43 ((gt)8ya) (average 0.28), for expected heterozygosity (he) and unbiased expected heterozygosity (uhe) from 0.02 ((tcc)5ry) to 0.28 ((gt)8ya) (average 0.18) (tab. 3). a dendrogram was constructed by using the upgma analysis based on 111 issr markers. the apple accessions studied had similarity values ranging from 0.79 to 0.98 indicating a high level of variation (fig. 1). similarly, goulao and oliveira (2001) found similarity levels of ~ 0.75-1.00 among 41 apples according to issr data. on the other hand, gulsen et al. (2010) determined higher variation among 192 apple accessions using pogp (peroxidase genebased polymorphism) markers. in the present study, all accessions were distinguished. the dendrogram consisted of many subgroups. foreign cultivars and three apple species studied (m. baccata, m. prunifolia and m. sylvestris) nested mixed with turkish accessions. pca was performed based on the genetic distance matrix to better tab. 2: results on issr primers used for apple accessions primers total fragments polymorphic fragments polymorphism (%) pic resolving power (ag)7yc 8 6 75 0.18 12.7 (agc)6g 12 8 67 0.14 16.5 (ca)8r 6 3 50 0.06 10.1 (caa)6 10 10 100 0.26 8.5 (cac)3gc 6 4 67 0.26 8.4 (ct)8tg 7 6 86 0.24 5.7 (ga)8yg 5 3 60 0.04 9.8 (gaca)4 14 12 86 0.27 11.1 (gt)6gg 10 8 80 0.22 10.1 (gt)8ya 9 8 89 0.26 9.9 hvh(tcc)7 6 3 50 0.04 10.1 (tcc)5ry 8 2 25 0.03 12.3 dbda(ca)7 6 2 33 0.02 11.9 hvh(ca)7t 4 1 25 0.02 7.9 mean 7,9 5,4 68,5 0.15 10.3 total 111 76 tab. 3: issr primers studied, their estimated allele frequency (p & q), no of effective alleles (ne), shannon’s information index (i), expected (he) and unbiased expected heterozygosity (uhe). primers p q ne i he uhe (ag)7yc 0.66 0.34 1.48 0.39 0.27 0.27 (agc)6g 0.60 0.40 1.26 0.27 0.17 0.17 (ca)8r 0.80 0.20 1.13 0.14 0.09 0.09 (caa)6 0.30 0.70 1.40 0.41 0.26 0.26 (cac)3gc 0.57 0.43 1.42 0.37 0.25 0.25 (ct)8tg 0.39 0.61 1.37 0.37 0.23 0.23 (ga)8yg 0.90 0.10 1.24 0.26 0.16 0.16 (gaca)4 0.29 0.71 1.34 0.33 0.21 0.21 (gt)6gg 0.38 0.62 1.38 0.38 0.24 0.24 (gt)8ya 0.41 0.59 1.47 0.43 0.28 0.28 hvh(tcc)7 0.79 0.21 1.13 0.17 0.10 0.10 (tcc)5ry 0.76 0.24 1.02 0.04 0.02 0.02 dbda(ca)7 0.94 0.06 1.14 0.15 0.10 0.10 hvh(ca)7t 0.93 0.07 1.15 0.19 0.11 0.11 mean 0.62 0.38 1.28 0.28 0.18 0.18 diversity analysis of turkish apple germplasm 85 fig 1: dendrogram of 158 apple accessions based on the issr markers and the upgma method. understand genetic relationships. fig. 2 presents the distribution of different genotypes according to two principal axes of variation using pcoa, which revealed the variation among the accessions similar to the upgma analysis. among the apples studied ‘sandik’ and ‘demir (2486)’ were the most distinct accessions with similarity value of 0.79. these two apples were collected from northeast anatolia and east black sea region of turkey (cetiner, 1981). in another study, ‘demir (2486)’ was clearly separated from the other apples (gulsen et al., 2010). ‘reinette tardiva’ was also apart from other apples with similarity 86 a. uzun, s. ozongun, o. gulsen, k.u. yılmaz, s. kaymak, s. ercisli of 0.84. ‘cigit’, ‘daldabir’, ‘tatlielma (2492)’, ‘beyazelma (2575)’ were nested in the same subgroup while ‘mektep’ apple, ‘rizedemir’ and ‘e55’ apples were in the same cluster. ‘m. baccata 1’ was clustered with ‘altinok’ and ‘samsun’ accessions. two m. baccata accessions studied in present research were distinguished from each other and clustered with the turkish local accessions. ‘portakal’ and ‘542e’, two little acidulated accessions were in the same subgroup. ‘mahsusa elmasi’, ‘e47’, ‘130887’, ‘180887’, ‘e66’, ‘e52’ and ‘e71’ were clustered closely. another subgroup consisted of ‘42e6 kabaelma’, ‘42a1 yazelması’ sampled from konya province, ‘32e1’, ‘gelendost’ sampled from isparta province and ‘tokat1’, ‘tokat3’. the upgma clustering grouped the geno types into meaningful clusters. three apples ‘kalkandelen’, ‘sogutelma (2486)’ and ‘beyelmasi (2477)’ having similar fruit characteristics with sourish flavour were clustered together. in the dendrogram describing the accessions above generated many different groups (a). in the dendrogram, the rest of 127 apple accessions were grouped into two main groups, b and c. group b had more accessions and were separated into two subgroups. two foreign red skin cultivars (‘gloster’ and ‘red chief’) and five local turkish accessions were clustered in the smaller group of b. foreign cultivars were not grouped apart from the turkish accessions. they were mixed clustered with local apples, indicating similar genetic backgrounds. accordingly, sonmezoglu and kutuk (2014) found that 23 local apple genotypes collected from karaman, turkey and three foreign cultivars were divided into two major groups and numerous sub groups, revealing a rich variation among the apple genotypes. on the other hand, pereira-lorenzo et al. (2008) found genetic diffe rence between spanish apple accessions and non-native cultivars. similarly, gasi et al. (2010) found that traditional bosnia and herzegovina cultivars were differentiated quite clearly from foreign cultivars, except for few genotypes. differences between the present study and others may be because of genetic backround of local apple accessions studied. large subgroup of group b divided into eight subclusters. one of them consisted of sourish flavour accessions including ‘sarielma’, ‘sarigobek’, ‘karpuz’, ‘hanimteni (42e3)’, ‘karapinar (42kp3)’. last two apples also found in the same cluster in previous study based on pogp markers (gulsen et al., 2010). these two accessions were collected from the same region of turkey. one of the foreign cultivars, ‘golden delicious’ was apart from the other common cultivars and clustered with three local apples. in group b, the largest subcluster consisted of 30 apple accessions. similarity of these accessions was between 0.89 and 0.98. in this subcluster, three foreign cultivars, ‘granny smith’, ‘cooper’ and ‘fuji’, three m. sylvestris accessions and many local accessions were nested. some of the local apples, ‘gelin’, ‘42ko1 yaylapinari’ and ‘42kp1 mayhostavsanbasi’ shared the same fruit characteristics such as soury flavor. ‘granny smith’ and ‘fuji’ were closely related based on issr data. similarly, these two cultivars were grouped closely according to ssr (gasi et al., 2010) and pogp markers (gulsen et al., 2010). on the other hand, goulao and oliveira (2001) found that ‘granny smith’ and ‘fuji’ were distinct based on issr data. three m. sylvestris accessions studied nested in this subcluster and were nearly identical. they were closely related to the turkish local apple accessions belong to m. domestica. coart at al. (2006) found high levels of haplotype sharing between m. sylvestris and m. domestica and assumed an interspecific gene flow, which is probably bidirectional and brought about by the use of (local) wild malus genotypes for the (local) cultivation process of apple. four local apples, ‘candir’, ‘güztavsanbasi’, ‘tavsanbasi’, ‘seker’ and ‘cincik’ were grouped closely in the dendrogram. two of them ‘güztavsanbasi’ and ‘tavsanbasi’ were collected from the same province of turkey (cetiner, 1981). most of foreign cultivars studied in the present study (‘idared’, ‘elstar’, ‘royal gala’, ‘melrose’, ‘ozark gold’ and ‘jonagold’) were grouped together. five local turkish accessions were also nested in this group. ‘elstar’, ‘jonagold’ and ‘gala (galaxy)’ were clustered based on ssr data (gasi et al., 2010). in another study, ‘royal gala’, ‘jonagold’ and ‘ozark gold’ were clustered closely but ‘idared’ and ‘melrose’ nested in the same group and slightly distinct from these three cultivars (goulao and oliveira, 2001). two apple species (m. baccata (no:1) and m. prunifolia) were closely rela ted. they were clustered with several turkish accessions such as ‘huryemez’, ‘karasaki’, ‘gurcu’, ‘cidagut’, ‘harim’, ‘pancarlik’ and ‘ferik’. two m. baccata accessions studied were apart from each other. the similar results reported by hokanson et al. (2001) and yao et al. (2010) indicated variation among the m. baccata samples. only one m. prunifolia accession was used in this study and it was highly similar to m. baccata (no:1) but not identical to fig. 2: principal coordinate analysis diagram showing the relationships among 158 apple accessions (numbers of accessions were identifed in tab. 1). diversity analysis of turkish apple germplasm 87 m. baccata (no:2). malus prunifolia and m. baccata were found in diverse clusters in a previous study (forte et al. 2002). similarly, harris et al. (2002) concluded that these two species were apart from each other according to nuclear ribosomal internal transcribed spacer gene. in addition, zhou and li (2000) assumed that m. prunifolia originated from hybridization between m. sieversii and m. baccata. the last subcluster of large subgroup of group b consis ted of 14 apple accessions including ‘petevrekelmasi’, ‘sivanora’, ‘lazelmasi’, ‘pasaelmasi’, ‘sekerelmasi’, ‘oltuelmasi’, ‘susuzelma’, ‘petek’, ‘karanfil’, ‘yaztavsanbasi’, ‘yazelmasi’, ‘e82’, ‘e220887’ and ‘210887 (1.1)’. in the dendrogram, 16 turkish accessions and two foreign cultivars (‘blackjon’ and ‘lodi early golden’) were clustered in group c. ‘lodi early golden’ was very similar to ‘yazelmasi (2484)’ and both were early maturing cultivars. conclusions several significant results were obtained from this study. issr markers confirmed efficiency for characterization of apple germplasm and cultivar identification. all of 158 apple accessions were distinguished from each other. the turkish apple accessions were closely related to the other known species, m. sylvestris, m. prunifolia and m. baccata. these three species were clearly separated from each other and they were mixed grouped with the turkish accessions. this verified gene flow among apple species and local apple genotypes. turkey has considerable morphological and molecular diversity in its apple genetic resources. turkey is located in east asia-europe origin center for apple and the middle of three important continents (asia, africa and europe). this region including turkey and iran was important in apple domestication and their transfer from central asia to the western countries (ghargani et al., 2009). the accessions studied in present study are maintained in the germplasm plots and are being investigated for important agronomic characters to exploit potential interest. acknowledgements the authors thank scientific research projects unit of erciyes university for funding and supporting the project with the project number of fba-10-3299. references bacvonkralj, m., jug, t., komel, e., fajt, n., jarni, k., zivkovic, j., mujic, i., trutic, n., 2014: effects of ripening degree and sample preparation on peach aroma profile characterization by headspace solidphase microextraction. turk. j. agric. for. 38, 676-687. cetiner, e., 1981: turkish plant genetic resources fruit and vineyard inventory. ege bölge zirai ars. enst. yay. no: 19. 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and molecular diversification of lemon selections. sci. hortic. 120, 473-478. yao, l., zheng, x., cai, d., gao, y., wang, k., cao, y., teng, y., 2010: exploitation of malus est-ssrs and the utility in evaluation of genetic © the author(s) 2016. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). diversity in malus and pyrus. genet. res. crop evol. 57, 841-851. zietkiewicz, e., rafalski, a., labuda, d., 1994: genome fingerprinting by simple sequence repeat (ssr) anchored polymerase chain reaction amplification. genomics 20, 176-183. zhou, z.q., li, y.n., 2000: the rapd evidence for the phylogenetic relationship of the closely related species of cultivated apple. genet. res. crop evol. 47, 353-357. address of the corresponding author: e-mail: sercisli@gmail.com journal of applied botany and food quality 92, 7 14 (2019), doi:10.5073/jabfq.2019.092.002 1department of botany, bharathiar university, coimbatore, tamil nadu, india 2department of biotechnology, k.s. rangasamy college of technology, tiruchengode, namakkal district, tamil nadu, india 3department of biotechnology, vit university, vellore, tamil nadu, india architectural effect of different tea clones on the development of blister blight disease ponnusamy ponmurugan1, balasubramanian mythili gnanamangai2*, kolandaisamy manjukarunambika3 (submitted: april 8, 2017; accepted: february 20, 2018) * corresponding author summary an attempt has been made to analyze the architectural traits of six elite tea (camellia sinensis) clones representing the three principal taxa assam, china and cambod with respect to the correlation of blister blight disease (exobasidium vexans) development. in order to analyze the architecture, branching habit and flushing behavior were observed and subsequently compared with disease incidence. all the clones followed similar architectural pattern irrespective of the cultivar but varied with levels of disease severity. the number of branches was higher in china when compared to assam and cambod, branch length was bigger in assam followed by cambod and china. branch angle of all the clones lay well within the described range of theoretical value of 45 to 90°. in general, internodal length was bigger in assam followed by cambod and china. these architectural characteristics determined the number of harvestable tea shoots in the bush canopy. china cultivars exhibited an erectophile type of leaf angle, which influenced effective net photosynthesis, transpiration rates and light penetration in leaves. these factors are playing important roles in a disease development strategy. this study should be useful for clonal selection for new clearings and re-planting areas. moreover, plants breeding programmes for studying the yield and tea quality losses due to blister blight disease benefit from the findings herein. keywords: blister blight, exobasidium vexans incidence, plant architecture, tea bush pattern. introduction tea plant architecture is the spatial distribution of different leaf composition covering scale leaf, scar of scale leaf, fish leaf, main tenance foliage, mother leaf, 1st, 2nd and 3rd leaves and a bud, stems and flowers on that plant at a given time (fig. 1). tea (camellia sinensis) is an evergreen plant that undergoes many cultural ope rations like plucking, pruning, tipping and centering. plucking of young harvestable shoots containing 2-3 leaves and a bud is being done and subjected to manufacture for tea powder. young shoots are continuously harvested at 10-14 day intervals which affects the bush health significantly. tea bushes are pruned to attack many pests and diseases (barthakur, 2011). after 4-5 years, depending upon the nature of tea cultivars and climate, tea bushes undergo pruning which gives rise to new shoots. those shoots are physiologically effective. some other factors determining the bush health are altitude of the tea garden, climate, pruning and tipping heights, length of the pruning cycle and the system of plucking. tea plant architecture depends on geometric, environmental factors and tea estate elevations influencing growth, including plucking patterns like manual plucking and shear harvesting and monsoon seasons. it is determined by an interaction between the intrinsic architecture defined by its genome and the characteristics of the population in which it grows (plant density). tea plants are planted by single, double and triple hedge methods to get the maximum crop yield per hectare. these planting styles regulate the architecture of tea plant. tea plant canopies are complex depending upon the style of planting (planting layout), tipping height and pruning types like light (80-90 cm from the ground level), medium (60-70 cm) and heavy pruning (15-25 cm). several parameters can be used to characterize the tea canopy architecture which includes leaf area index, leaf distribution and number of harvestable shoots on plucking surface (girardin et al., 1999). tea being a monoculture crop is susceptible to a number of de structive diseases and provides a stable microclimate for various diverse phytopathogens (premkumar et al., 2008). among the leaf diseases, blister blight disease caused by a fungus exobasidium vexans massee is one of the most damaging diseases of tea plants worldwide (sinniah et al., 2016). it is an important foliar disease incapable of causing enormous crop loss throughout the tea growing regions of asia, especially in india, sri lanka, indonesia and japan. e. vexans completes its life cycle in a short span of 11-28 days (premkumar and baby, 2005). thus, many generations of the fungus are completed during the favorable condition of the monsoon. the pathogen infects young succulent harvestable shoots leading to severe crop loss. the crop loss varies with the geographical locations and nature of tea clones and seedlings. it was estimated to be 33% in sri lanka (de silva et al., 1992), 20-25% in indonesia and as high as 35% in india (radhakrishnan and baby, 2004). in addition to crop loss the disease adversely affects the quality of made tea (ponmurugan et al., 2016). variation in several architectural traits combines to give species a distinctive visual appearance. but these different architectures were well correlated with different lightinterception properties (falster and westoby, 2003) rather its other metabolism and conduciveness to disease. commercial tea population lies under three principal taxa, ‘china’ (camellia sinensis (l.) o. kuntze), ‘assam’ (c. assamica ssp. assa mica (masters) wight) and ‘cambod’ (c. assamica ssp. lasiocalyx (planch ex watt) wight) (barua, 1965). plant architecture is the visible, morphological expression of the genetic blue print of a tree (halle, 1978). architectural analysis of tea clones will facilitate selection of the better traits against high yield, pests and disease resistance. the objective of the present study is to analyze the architectural features of different tea clones with respect to the development of blister blight disease over the course of the growing seasons. materials and methods six elite tea clones viz., upasi-1 and upasi-3 (assam), upasi-9 and upasi-15 (china) and upasi-17 and tri-2025 (cambod) maintained in upasi tea research institute experimental garden under similar cultural conditions were examined for various architectural parameters. the choice of the six tea cultivars was 8 p. ponmurugan, b.m. gnanamangai, k. manjukarunambika selected on the basis of differences in architectural features, green leaf yield potential and tea quality parameters. moreover, studies were undertaken to assess the specific effect of the architectural traits on epidemic blister blight development, tea cultivars with similar levels of susceptibility and resistance to blister blight were chosen. all the clones were planted in 1969 at 120 × 120cm spacing, in contour single hedge planting system. observations on canopy architecture, floral characteristics and leaf morphology were made with unplucked tea plants maintained in the breeding plots and plants under regular cultural operations. data on position of trunk, branch angle, branch length and number of branches were recorded. branches were designated as the first order (n+1), the second order (n+2) and so on till seventh order (n+7). internodal length was measured in the well-developed shoots which are about to be plucked. shoot components were designated as a bud, first (7th order), second (6th order), third (5th order), mother (4th order), fish (3rd order), and scale (2nd order) leaves and maintenance foliage (1st order). branch and internodal lengths were measured with a metric scale. branch and leaf angles were measured with a circular ocular meter (honda and fisher, 1978). leaf area was also calculated conventionally. all the observations were replicated 10 times in separate bushes and the data were subjected to statistical analysis. blister blight disease incidence was assessed every plucking round in all the three experimental blocks covering assam, china and cambod tea cultivars. young tender shoots containing three leaves and a bud were collected randomly from the harvest baskets and each shoot was examined individually for the incidence of blister blight. disease incidence was quantified on a percentage basis. the sequence of lesion development was divided into five stages such as 1) occurrence of translucent lesions, 2) well-defined lesions, 3) incipient stage of spore forming lesions, 4) vigorously sporulating lesions and 5) necrotized lesions and were carefully documented while examining the incidence of blister blight disease. healthy and infected harvestable shoots were collected from all the bushes of six tea cultivars falling under assam, china and cambod traits periodically. the freshly harvested leaves were used for chlorophyll estimations. a 100 mg of harvested tea plant material was taken in preparing the leaf disc using the conventional paper puncher. the prepared leaf disc was submerged in 5 ml of dmso (dimethyl sulfoxide) and the chlorophyll was estimated by following wellburn (1994). for all other biochemical parameters the 1 gm of harvested shoots was extracted and concentrated with 80% ethanol, and the final extract was filtered and used for estimation of total sugars (aoac, 1990), nitrogen (dubois, 1956), amino acids (moore and stein, 1948), polyphenols (dev choudhury and goswami, 1983) and catechins (swain and hillis, 1959). the data obtained were subjected to analysis of variance (anova) and the significant means were segregated by critical difference (cd) at various levels of significance (gomez and gomez, 1984). results wide variations were observed in the canopy architecture among tea cultivars (tab. 1). assam tea cultivars were orthotrophic with ‘de current’ or ‘deliquescent’ branches i.e., terminal leader eventually disappears and lateral branches tend to grow upwards. china tea cultivars were plagiotrophic with excurrent branches i.e., a prominent terminal leader with horizontal drooping lateral branches while cambod tea cultivars were neither orthotrophic nor plagiotropic. all the clones bear identical branching dynamics with non-rhythmic or continuous growth without endogenous periodicity of extension. in china cultivars, the number of branches (second to fourth order) was significantly higher than in assam and cambod cultivars (tab. 2). significant variation in the branch length was observed between assam and china tea cultivars while it was not significant either between cambod and assam or cambod and china cultivars (tab. 2). branch angle lies well within the range of 45 to 90° (tab. 2). tea leaves follow a special pattern of arrangement, ‘anisophylly’. each shoot comprises one or two scale leaves, one fish leaf, mother leaf and three to four normal leaves with a terminal bud (fig. 1). no significant variation was observed between the shape and size of maintenance foliage, mother leaf and third leaf. internodal length increased gradually from maintenance foliage to third leaf and de clined in second and first leaves. among the clones, no significant variation was observed in the intermodal lengths of maintenance tab. 1: comparison of architectural parameters of different tea cultivars parameters tea cultivars assam china cambod habit tree 10-15m tall big shrub 1-3m tall erect shrub 5-6m tall growth habit under trees with virgate branches vigorous shrub with erect, openly branched shrubs compound branches branching pattern semi-orthotrophic arise above the semi-plagiotropic compact, semi-orthotropic and semi ground, excellent spread arise from the ground plagiotropic with profuse branching, arise from the base. no. of branches in each order 2.8 to 6.0 3.5 to 8.7 2.8 to 6.6 branch length (cm) 11 to 36 7 to 25 8 to 30 branch angle(°) 50 to 70 40 to 47 50 to 70 internode length (cm) 2 to 6 1 to 4 1 to 5 stem circumference (cm) 37.5 30.5 33.3 length of main stem (cm) 37.5 28.8 32.5 leaves large, horizontal, broad, light green small, narrow, erect, dark green medium size, broad, light green, semi-erect leaf angle type planophile (>70°) erectophile (<50°) oligophile (50-70°) flowering sparse profuse medium dense plucking surface (cm2) 9788 9044 9358 plucking points/sq.ft 155.5 125.5 141.0 yield potential high low moderate tea quality medium superior superior blister blight due to leaf architechture 9 bud mother leaf fish leaf scale leaf maintenance leaf third leaf second leaf first leaf assam (9788 cm2, 155.5/sq.ft; respectively) followed by cambod (9358 cm2 and 141/sq.ft) and china (9044 cm2 and 125.5/sq.ft). they may be attributed to leaf size and leaf distribution in the canopy. assam cultivars had a higher leaf angle followed by cambod and china (tab. 3). flowers are solitary, pleonanthic type where they are produced on lateral branches and thus the shoot are not limited by flowering. flowering is always on old branches (rhamiflory) and so does not affect the shoot construction during its life cycle as observed in many species. sympodial branching, sylleptic growth habit, semiorthotropic and semi-plagiotropic branching, anisophylly, peonanthic flowers and rhamiflory are characteristic to camellia spp. (fig. 3b). blister blight infection was more in assam cultivar tea leaves than in cambod and china, which is coinciding with the result of leaf angle and leaf area. leaf angle of assam cultivars was planophile (> 70°) type which were horizontal, whereas, the same in china and cambod cultivars was erectophile (< 50°) and oligophile (50-70°) types; respectively. an average of leaf angle in china cultivars recorded was 28.1° in upasi-9 and 27.7° in upasi-15. whereas, it was 54.2° and 56°; respectively, with assam upasi-1 and upasi-3. the observations made in different tea cultivars with respect to blister blight disease incidence were presented in the tab. 3. dif ferent stages of blister blight lesions were recorded in tea shoots. the lesions were fully matured and ready for spore dispersal in the third leaf (fig. 3c) subsequently the lesion got necrotized (fig. 3d). on the second leaf, however, the lesions were immature and slightly lesser in number. the first leaf showed fewer lesions, which were in their early stages of development. the pattern remained the same in all the three tea cultivars. blister blight infection was registered the maximum with assam upasi-3 followed by cambod tri-2025 tab. 2: branching pattern parameters of various tea clones branch parameters assam china cambod c.d. at p = 0.05 upasi-1 upasi-3 upasi-9 upasi-15 upasi-17 tri-2025 no. of branches first order 5.7 5.5 6.7 6.5 6.6 4.5 1.3 second order 6.3 6.7 8.7 8.3 6.4 5.7 2.1 third order 4.1 3.7 6.8 6.5 4.6 3.5 1.3 fourth order 4.2 4.6 7.2 5.4 4.6 3.4 1.5 fifth order 2.7 3.0 3.9 3.4 3.5 2.8 1.1 average 4.6 4.7 6.7 6.0 5.1 4.0 1.5 branch length (cm) first order 23.8 22.7 19.9 24.0 28.4 20.2 6.3 second order 20.8 23.7 29.4 32.5 34.4 24.5 7.5 third order 31.0 28.7 22.4 28.5 25.2 24.2 6.5 fourth order 10.7 12.5 19.3 20.5 16.4 14.6 6.2 fifth order 08.5 07.5 10.5 15.3 08.4 08.8 3.3 average 19.0 19.0 20.3 24.2 22.6 18.5 6.0 branch angle(°) first order 67.3 53.5 44.5 43.5 52.3 65.7 18.3 second order 70.5 57.8 44.3 43.5 62.7 70.7 25.2 third order 72.8 63.6 43.6 45.0 53.0 55.5 17.2 fourth order 51.5 53.5 42.5 40.0 52.5 60.7 13.5 fifth order 63.5 58.5 42.5 45.6 50.7 55.5 16.5 average 65.1 57.4 43.5 43.5 54.2 61.6 18.1 fig. 1: shoot structure of a periodic tea shoot containing different types of leaves leaf to scale leaf, scale leaf to fish leaf and fish leaf to mother leaf (tab. 3). tea canopies in terms of number of plucking points and plucking surface among tea cultivars vary at different levels. both numbers of plucking points and plucking surface were found to be higher in average 34.4 72.9 15.1 51.2 24.8 61.1 16.1 figure 1. shoot structure of a periodic tea shoot containing different types of leaves first order lateral figure 2. branching pattern of a tea bush bud mother leaf fish leaf scale leaf maintenance leaf third leaf second leaf first leaf third order lateral second order lateral internode banji new growth 10 p. ponmurugan, b.m. gnanamangai, k. manjukarunambika and minimum with china upasi-15. these tea clones were highly susceptible to blister blight infection. in contrast, tea clones such as assam upasi-1, cambod upasi-17 and china upasi-9 were tolerant to blister blight. observations on the various stages of blister blight infections in tea leaves such as translucent, welldefined, spore forming, sporulating and necrotized lesions, there is no significant difference recorded (tab. 3). various degrees of biochemical constituents were observed in all the six tea cultivars (fig. 4). healthy and infected clone of the same cultivar showed significant reduction in all biochemical constituents, especially the chlorophyll, polyphenols and catechins. the difference was high between the healthy and infected clones of upasi-15 followed by tri-2025 and upasi-3. in contrast the difference was comparatively low in all the biochemical parameters including sugar and nitrogen in upasi-1, upasi-9 and upasi-17 clones. all the biochemical constituents in healthy conditions registered no significant difference among the six tea cultivars except for polyphenols which is high in upasi -15. discussion china cultivars having more branches indicate the vulnerability of the disease incidence. pruning induced more number of proleptic and sylleptic branches with high shrubby appearance (zhou and hara, 1990) which lead to die back of weaker branches (ponmurugan et al., 2000). branch length was more in the case of second and third tab. 3: leaf architectural parameters of various tea clones and blister blight disease incidence leaf parameters assam china cambod c.d. at p = 0.05 upasi-1 upasi-3 upasi-9 upasi-15 upasi-17 tri-2025 leaf angle(0) first order 55.3 b 52.3 b 27.8 a 27.5 a 45.7 b 43.3 b 10.7 second order 58.5 bc 60.5 bc 30.5 a 30.5 a 50.6 bc 43.5 b 11.6 third order 55.7 bd 63.2 bd 31.5 a 28.8 a 42.5 bc 45.6 bc 10.6 fourth order 72.5 bd 62.2 bd 27.0 a 25.7 a 45.2 bc 43.8 bc 13.3 fifth order 56.5 bcd 65.5 bd 40.5 a 42.7 abc 55.6 bcd 60.5 bd 15.3 sixth order 50.7 bc 52.6 bc 20.5 a 22.5 a 48.5 bc 47.3 bc 14.7 seventh order 30.5 bcd 35.7 bd 18.7 a 16.0 a 23.5 abc 25.6 abcd 11.3 average 54.2 56.0 28.1 27.7 44.5 44.2 12.5 leaf area (cm2) maintenance foliage (mf) 23.5ab 30.5c 22.2a 20.9a 20.5a 27.5bc 3.0 scale leaf (sl) 00.8a 01.3a 00.7a 00.7a 01.3a 01.3a 1.1 fish leaf (fl) 00.7a 02.2b 01.3ab 01.1ab 01.5ab 01.5ab 1.1 mother leaf (ml) 19.6a 29.9d 19.9a 19.0a 19.5ac 23.0bc 2.5 iiird leaf (3l) 20.0b 29.0d 18.0a 19.0ab 18.0a 23.0c 1.6 iind leaf (2l) 15.0bc 19.0d 13.5ab 11.1a 16.0b 17.5cd 2.5 ist leaf (1l) 07.3bc 11.5be 06.8bc 04.9a 07.5bcd 08.3bd 1.3 average 12.4 17.6 11.8 11.0 12.0 14.6 1.9 internodal length (cm) mf><sl 2.7bc 3.0bc 2.0ac 1.5a 2.7bc 2.5ac 1.0 sl><fl 3.0bcd 3.5bde 2.5ac 2.0a 3.0bcd 4.0be 0.5 fl><ml 4.5b 4.0b 3.3a 3.0a 4.0b 4.0b 0.3 ml><3l 4.9be 4.3bcde 3.7ac 3.0a 4.7bde 4.0bcd 0.7 3l><2l 7.5b 6.0bc 4.7ac 3.7a 6.0bc 5.5ac 2.2 2l><1l 4.5cd 4.7d 2.3ab 2.0a 3.3abc 3.5bcd 1.3 1l><bud 3.3c 2.5bc 1.3a 1.0a 1.2a 1.5ab 1.0 average 4.3 4.0 2.8 2.3 3.6 3.6 1.0 blister blight infection(%) translucent lesion 37.7bc 75.5e 17.7a 52.5cd 27.0ab 65.5de 18.8 well-defined lesion 36.5b 78.5d 15.5a 55.5c 28.5ab 64.5cd 17.5 spore-forming lesion 38.0bc 75.5be 16.6a 57.7bd 27.5ac 66.0bde 15.5 speculating lesion 34.8b 77.5d 15.0a 57.0c 25.0ab 64.5cd 16.5 necrotized lesion 25.0bc 57.5f 10.5a 33.5cd 15.9ab 45.0de 12.0 average 34.4 72.9 15.1 51.2 24.8 61.1 16.1 blister blight due to leaf architechture 11 order branches of all the clones. this may be due to the lack of air space and less competition between the branches for absorption of light, photosynthesis and exchange of gases (sarlikioti et al., 2011). branch angle determined the spread of the bush and it was most often influenced by planting style. high density planting leads to formation of weak branches and less supporting wood (balasubramanian, 2010). in the case of assam type, overall branch angle was com paratively higher which might have enabled the better spread of the bush and increased plucking points (barthelemy and caraglio, 2007). first order lateral lateral internode second order lateral third order banji newgrowth fig. 2: branching pattern of a tea bush fig. 3: architectural characters of tea plants and blister blight infection. a, different tea cultivar harvestable shoots; b, a typical tea bush; c, sporulating lesions of blister blight infected tea shoots and; d, necrotized lesions of blister blight infected tea shoot). 12 p. ponmurugan, b.m. gnanamangai, k. manjukarunambika with no significant variation among the tea clones between the shape and size of maintenance foliage, mother leaf and third leaf, smaller leaf area of china cultivars may be attributed to branch angles and its length and number of branches besides its genetic potential (owuor et al., 2008). in general, variation in the proportion of leaf and internode in tea shoot will influence the recovery percentage and quality of made tea. plant canopies are complex with dynamic structures which depend on growth stage, plant density, planting layout, growth pattern and branching habit (girardin et al., 1999). according to tivoli et al. (1996) the density of stems per unit area affected disease development and severity by changing the movement of the pathogen within the canopy and/or the microclimate. as per banerjee (1992), assam, china and cambod cultivars of the present investigation can be grouped into planophile, erectophile and oligophile, respectively (fig. 3a). the angle of inclination facilitates trapping of light energy, thereby promoting photosynthesis and productivity (ando et al., 2007). electrophiles are more efficient in photosynthesis as the lamina is fully exposed to the sun light. leaves act as natural spore traps where the planophiles enhance the deposition of the inoculum and thereby promoting disease incidence (le may et al., 2009). higher susceptibility of assam clones to foliar diseases and pests can be attributed to its leaf angle. based on the result of flowering patterns, cultivated camellia fall under attim’s model (fig. 3b) irrespective of the cultivar (halle, 1978). recently paraheliotropism of leaves were reported to have photoinhibition resulting in deleterious effects on plant (puglielli et al., 2017). leaf angle of china cultivars were erect, which determined effective physiological parameters, especially in net photosynthesis, trans piration rates and light penetration in leaf tissues (balasubramanian, 2010). erect position of leaves played a key role in determining disease development (onfroy et al., 2007). due to these characteristic features, the incidence of blister blight is very less in china cultivars which registered at 15.1% in resistant upasi-9 clone and 44.4% in susceptible upasi-15 clone (tab. 3). the results clearly showed that there was a direct correlation between leaf angle and blister blight infection in tea plants. the results on the blister blight incidence coincide with a similar observation made by nakamura (1991) and opined that tea cultivars have been found to react differently to blister blight and they sig nificantly vary in their degree of resistance and susceptibility to the disease severity. moreover, the nature resistance in tea cultivars to blister blight is due to defense response mechanism. in accordance with the report of le may et al. (2009), the behavior of various pea cultivars with respect to ascochyta blight disease has been studied and disease dynamics exhibited to depend on plant morphology. ideal plant architecture can optimize canopy architecture, improve photosynthetic efficiency, and prevent lodging, thus resulting in overall high yield (pan et al., 2017). similar results for biochemical constituents were reported earlier in grafted and ungrafted tea clones by balasubramanian et al., 2010. the results suggest that there is no influence of architecture for biochemical constituents among the tea cultivars except for polyphenols which is mainly attributed to the quality of brewed tea. and it also infers that since the bush canopy of some tea cultivars fig. 4: biochemical parameters of the harvestable tea shoot with respect to clones and blister blight infection. blister blight due to leaf architechture 13 favors disease development, the infected clones showed significant difference in all biochemical constituents as compared to healthy bush. these results were in agreement with works of ponmurugan et al., 2016, where all the biochemical constituents were significantly reduced in foliar diseased shoots of tea plants. conclusion to conclude, this study will be of more useful in clonal selection in new clearings and re-planting programmes in the tea estates. plant architecture study is a method in which cultivar characteristics and disease development with respect to architectural traits are correlated. canopy structure is thus an important factor as it is directly involved in controlling disease development during the crop season consequently on yield attributes. moreover, this investigation will also be useful for tea plant breeders to manipulate plant architecture such as a way to facilitate disease avoidance, which may be a valuable alternate approach to mitigate blister blight disease severity. acknowledgements the authors are thankful to the director, upasi tea research institute, valparai, national tea research foundation (ntrf), kolkata, and dst-fist fund for improvement of s&t infrastructure (sr/ fst/college-235/2014), and dbt-star college scheme (bt/hrd/ 11/09/2018), new delhi india for supporting financially to conduct field experiments. references ando, k., grumet, r., terpstra, k., kelly, j.d., 2007: manipulation of plant architecture to enhance crop disease control. perspectives agric. veter. sci. nutr. nat. res. 2, 1-8. doi: 10.1079/pavsnnr20072026 aoac, 1990: estimation of various biochemical parameters. in: helrich, k. 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accepted: may 22, 2018) * corresponding author summary light can affect the yields of alkaloid and essential oil in the synthesis of secondary metabolites directly or indirectly through plant growth. despite mahonia breviracema being an endemic medicinal species in china, research on the influence of light on production of alkaloid and essential oil is scarce. thus, this research evaluated the influence of various lighting conditions on alkaloid yields and the composition and yields of the essential oils of m. breviracema. the results revealed significant differences in alkaloid yields, oil yields and chemical characteristics of m. breviracema grown in four dif ferent light intensities from 10 to 100% full sun shine. the total amount of alkaloids in plants under i30 and i50 was higher than that under i10 and i100 due to the higher biomass of plants. oil yield of m. breviracema leaf increased linearly with the increase of light incidence. plants grown under i10 had less plastoglobuli, which coincided with the lowest oil yield (1.91 g kg-1). the plastoglobuli in chloroplasts increased when the irradiance levels increased, resul ting in the highest oil yields under i100 (4.53 g kg-1). the principal components in the leaves of m. breviracema were hexadecanoic acid (10.54-72.19%) and α-ionone (1.25-42.39%). the highest hexadecanoic acid content was obtained under i50, followed by i30, and the highest α-ionone content was obtained under i100. therefore, it is ne cessary to control the light environment to obtain raw materials with high quality. keywords: mahonia breviracema; irradiance; chloroplast; alkaloids; essential oil introduction mahonia is a genus of about 60 species which are distributed in southeast and east asia. a total of 35 species of mahonia have been found in china, 20 of which are regarded as medicinal plants (he and mu, 2015). the roots and stems are used as raw materials “gonglaomu” recorded in the chinese pharmacopoeia (china pharmacopeia commission, 2010). it is a medicinal plant traditio nally used to treat aridity, clear heat, and relieve cough and phlegm (china pharmacopeia commission, 2010). the leaves of mahonia plants are the raw material “gonglaoye” (ye, 2009), which has curative effects of clearing heat, relieving cough, and reducing sputum antioxidant and anti-malignant tumor. the major bioactive components of “gonglaomu” are alkaloids, including jatrorrhizine, palmatine and berberine. “gonglaoye” contain abundant essential oil (liu et al., 2010a, 2010b; zeng et al., 2006). medicinal plants have been increasingly used, which promotes the loss and exploitation of species. about 40% flora faces the danger of extinction because of human activities, such as excessive collection and extraction of certain species (oliveira, 2010). mahonia breviracema y.s. wang & p.g. xiao. is genus mahonia (berberidaceae.), and it is an endemic genus to southwest china. m. breviracema is a short shrub mainly distributed in guangxi, and grows in, coniferous forests, deciduous and evergreen forests. it has been reported that m. breviracema contains high amounts of alkaloids (kong et al., 2011). in recent years, the number of m. breviracema in wild distribution areas has sharply decreased because m. breviracema is not only sold as “gonglaomu” and m. breviracema but also used as ornamental plants. plants in the environment different from their natural habitat may show quantitative and/or qualitative changes in their composition in terms of special metabolites. therefore, explo ring the influence of irradiation intensity on growth and development of plants, particularly on the accumulation of secondary metabolites, is important to cultivate medicinal plants. irradiance is an important environmental factor influencing normal physiological functions, plant growth and secondary metabolic pro duct accumulation (ma et al., 2010). high light intensity enhances the accumulation and synthesis of total flavonoids and phenolics in young ginger (ghasemzadeh et al., 2010). however, excessively high light intensity leads to low flavonoid contents and weak photosynthetic capability in anoectochilus (ma et al., 2010), whereas a decrease in the content of essential oil at low light intensity has been reported in ocimum basilicum l. (chang et al., 2008). the content of sabinene in origanum vulgare l. ssp. vulgare essential oil is decreased by lower light intensity (de falco et al., 2013). kong et al. (2016) reported that there were obvious differences in the plastoglobules of mahonia bodinieri (gagnep.) laferr. leaves at different light intensity conditions, and m. bodinieri leaf oils are complex systems with varying compositions (dong et al., 2008). the influence of light intensity on the composition of essential oils and the content of alkaloid in m. breviracema has not been reported. therefore, m. breviracema is used as the material to analyze the content changes of volatile oil and alkaloids under different light conditions through botanical microtechnique, hplc and gc-ms. moreover, the optimal cultivating condition of m. breviracema is discussed in the pre sent study. the study result provides technical support and theoretical basis for the breeding and cultivation of m. breviracema. materials and methods plant material and growth conditions the experiment was carried out at guangxi institute of botany in yanshan, guilin, china. on marth 15th, 2011, the seeds of m. breviracema were sown. experimental design was performed according to the methods of kong et al. (2016). on may 15th, 2014, seedlings of m. breviracema with uniform size (40-50 cm in height) were transferred to pots containing peat soil 172 y. li, d. kong, h.-l. liang, h. wu and limestone mountain soil (1:1, v/v; ph at 5.8). after one month, the plants were subjected to four treatments of light irradiance (10% (i10), 30% (i30), 50% (i50) and 100% (i100) light-full sun) for 6 months. the full sun treatment in an experiment conducted on the farm was about 2000 ± 20 μmol photons m-2 s-1 at noon, which is determined by a li-6400 portable photosynthesis system (li-6400, licor, lincoln, ne, usa). light intensity was controlled through the modified method proposed by liao et al. (2005). a total of 20 pots included in each treatment were arranged from the north-west to the south-east every day. biomass five samples of plants were weighted after being separated into leaves, stems and roots to calculate their total dry biomass. the dry biomass was obtained by using an oven with air ventilation at 60 °c, until constant weight. hplc analysis five plants were collected randomly in each treatment to determine the content of alkaloid. the leaves were dried in oven for 20 min at 105 °c to deactivate enzymes and then cooled to 55 °c quickly. afterwards, stems, roots and leaves were dried for at least 48 h in a oven at 55 °c. the samples were further ground (60 mesh) and preserved in a drying oven. each sample was accurately weighed as 0.50 g and placed into a 250 ml conical flask; 100 ml of a hydrochloric acid-methanol solution (1:100, v/v) was added and the mixture was heated by reflux for 15 min at 55 °c. after reflux, the mixture was extracted with ultrasonication for 45 min; the mixtures were then shaken and cooled to room temperature and filtered. the residue was adjusted to 100 ml of hydrochloric acid-methanol (1:100, v/v), and the above procedure was repeated. finally, the two filtrates were combined and the mixture was filtered. the filtered fluid was then dried, re-dissolved, and filtered through a 0.22 μm syringe filter prior to hplc-dad analysis. an agilent 1100 hplc system (agilent technologies, palo alto, usa) consisting of a vacuum degasser, a g1311a quaternary pump, a g1315a diode array detector (dad) and a g1329a autosampler was utilized to obtain hplc chromatograms. the system was controlled by agilent chemstation software (agilent technologies, palo alto, usa). chromatography was operated on a gemini c18 reversed-phase column (4.6 mm × 250 mm, 5 μm) maintained at 25 ℃. the mobile phase was performed by using solvent a (0.05 mol l-1 kh2po4 buffer solution, ph 3.0 adjusted with h3po4) and b (acetonitrile) (72:28, v/v) at a flow rate of 1.0 ml min-1. the pump of the hplc equipment was carefully washed by 10 % isopropyl alcohol for 10 min before determining the samples, and 10 min was required to equilibrate the column by mobile phase after each sample. the uv spectra were recorded at 265 nm. identification of jatrorrhizine, palmatine and berberine was performed by comparing the retention times of chromatographic signals between detected samples and those of the three alkaloids standards under the same experimental conditions. twenty μl of each sample was injected and hplc analyses were performed in triplicate for statistical analysis. preparation of samples and extraction of essential oil fresh leaves were randomly collected from five plants of m. breviracema in each sample. leaves were dried in shade for 15 days at room temperature. the dried samples were also ground (60 meshes) and preserved in a drying oven. the essential oil was extracted through hydrodistillation according to the methods recorded in the pharmacopoeia of the people’s republic of china (china pharmacopeia commission, 2010). the leaves (15 g each) were conducted with hydrodistillation in a sealed vessel for 5 hours (li et al., 2013). we determined the yield of essential oils in triplicate, and the mean values were expressed as the results. the volatile oils were kept at 4 °c for further analysis. the average concentration of essential oil was calculated as the weight of oil (g) per 1 kg of dry leaf biomass and essential oil yield per plant (mg plant-1). the plots were analyzed through origin software. gc–ms analysis the essential oil was analyzed by gas chromatograph (finnigan trace gc-2000)-mass spectrometer (thermo finnigan, american). a 30 m × 0.25 mm idhp-1 bonded-phase fused-silica capillary column with a film thickness of 1 μm was used. the temperature of the injector was 150 °c. the initial temperature was maintained at 100 °c for 4 min and then increased to 130 °c at the rate of 5 °c/ min; afterwards, the temperature was maintained at 130 °c for 20 min. linear velocity of the helium carrier gas was 1.2 ml.min-1 at the split ratio of 30:1; ei was used as the ion source at 230 °c. sector mass analyzer scanned from 30 amu to 550 amu. diluted samples (25 μg/ml) were prepared through methylene dichloride, and 0.4 μl samples were injected. the chemical constituents were identified by comparing with the national institute of standards and technology (nist05.lib) library spectra and the literature (adams, 2001). the retention indices were calculated by injecting a series of n-alkanes in the same conditions. light microscopy and transmission electron microscopy the tissue of leaves was cut into pieces with the size of 1 mm × 1 mm × 0.5 mm. the specimens were processed through the method proposed by liu et al. (2012). sections (1 μm thick) were stained with 0.5% toluidine blue o and photographed through a leica em uc6 ultramicrotome (leica, germany). the chloroplasts number per mm2 was counted and averaged within 15 1000 × 1000 mm2 squares randomly selected from the cross sections. for the ultrastructural observations, ultrathin sections (70-90 nm in thickness) were stained with lead citrate and uranyl acetate. a philips fei-technai 12 microscope was used to observe the sections. statistical analysis data were reported as mean ± standard deviation of at least three experiments. one-way analysis of variance (anova) was subjected to using spss version 18.0 (spss inc., chicago, usa). duncan’s multiple range test was performed to detect differences between treatments on each variable (p < 0.05). results biomass the results show that light conditions can significantly influence the biomass of root, stem and leaf and total biomass (fig. 1), which were obviously higher under i30, followed by i50. there was no significant difference between i30 and i50. however, the total biomass under both i30 and i50 was statistically higher than that under i10 and i100. alkaloids analyses in roots and stems, higher contents of jatrorrhizine and palmatine in m. breviracema were observed under moderate light intensity. plants grown under i30 and i50 had higher concentrations of jatrorrhizine and palmatine in roots, i.e., 10.83 and 10.84 mg·g-1, and 2.04 and 1.86 mg·g-1, respectively. higher concentrations of jatrorrhizine and palmatine in the stems were observed under i50, (15.71 and 10.58 mg·g-1, respectively), followed by i30 (13.49 and 10.40 mg·g-1) (fig. 2, influence of light on alkaloid content and essential oil composition in mahonia breviracema 173 fig. 1: dry biomass of m. breviracema plant grown under different light intensities. the values are the average ± se, and different letters indicate that there are obvious differences in the shade treatments (p<0.05). a and c). the highest concentration of berberine were obtained under i10 (11.80 mg·g-1) followed by i50 (11.21 mg·g-1) in the roots (fig. 2a). while the concentration of berberine in the stems decreased with the increase of light irradiance, the highest values of berberine were up to 10.83 mg·g-1 under i10 (fig. 2c). we estimated the changes in total amount of alkaloid in root, stem, leaf and each plant further study the data. the variation of total amount of alkaloids with higher production in the roots and stems was obtained under the conditions of intermediate irradiance (i30 and i50), which showed low values under i10 and i100 (fig. 2, b and d), although it was not statistically significant among the various light intensities for roots. moreover, the concentrations of jatrorrhizine and berberine were obviously higher than palmatine in the roots, while palmatine content in the stems was significantly higher than that in the roots. in particular, the concentrations of palmatine under i30 and i50 were higher than berberine (fig. 2, a and c). the concentrations of jatrorrhizine (0.65-0.35 mg·g-1), palmatine (0.66-0.36 mg·g-1), and berberine in leaves were significantly lower compared to those in the roots and stems (fig. 2e). jatrorrhizine was only detected under i50 and i100 (0.65 and 0.35 mg·g-1, respectively), and berberine was not detected under all light treatments (fig. 2e). leaf structure there were notable changes taking place in leaf anatomical characteristics induced by light intensity. the results showed that the thickness of the entire lamina, palisade parenchyma and spongy parenchyma increased with the increase of light incidence (tab. 1; fig. 3). at the same time, chloroplast sizes and numbers in the palisade parenchyma decreased with increasing light intensity (tab. 2). to study the distribution and accumulation of oil in m. breviracema leaves, we investigated the microstructure of the leaves under different light intensities. chloroplast structure of m. breviracema were obviously influenced by light intensity (fig. 4). most chloroplasts in leaves grown under i10 and i30 had large size and showed normal ultrastructural organization, with a typical arrangement of stroma and grana thylakoids (fig. 4, c and f). few plastoglobules were observed under i10 (fig. 4, a and b). there were more electron-transparent plastoglobules in the chloroplasts under i30 (fig. 4, d and e). abnormal chloroplast structure with irregular and blurring grana lamellae arrangement was found under i50 (fig. 4i). electron-dense plastoglobules was significantly increased for i50 (fig. 4, g and h). under i100, the grana were totally ruptured (fig. 4, k and l), but the chloroplasts were filled with abundant plastoglobules (fig. 4, j and k). yield of essential oil the content of essential oil in leaves of m. breviracema grown under different light conditions changed significantly. the contents and yields of essential oil in m. breviracema leaves were sensitive to irradiance with a rising linear behavior, with the highest values found in plants grown under i100 (4.53 g kg-1), followed by i50 (3.12 g kg-1). plants grown under i10 had the lowest essential oil yield (1.91 g kg-1) (fig. 5a). the total amount of essential oil also followed a similar trend (fig. 5b). composition of essential oils the irradiance affected the essential oil composition of m. breviracema eliciting a variation of 29, 31, 28 and 28 identified components at i10, i30, i50 and i100, respectively, representing 91.97-97.34% of total essential oils and 26 common peaks (tab. 3). the study showed that hexadecanoic acid (72.19-10.54%) and α-ionone (1.25-42.39%) were found in greater quantities in m. breviracema leaf oils. the content of hexadecanoic acid was the largest under i50, followed by i30, and the lowest content was found under i100, i.e., only 10.54% of hexadecanoic acid. interestingly, the α-ionone constituents were significantly increased under i100, leading to a significant increase in the α-ionone content (42.39%); the other treatments only showed 1.25-2.32%. the contents of hexadecanoic acid, methyl ester and hexadecanoic acid, ethyl ester under i10 and i100 (1.48 and 1.83%, 1.20 and 1.77%, respectively) were higher than that of i30 and i50 (0.46 and 0.60%, 0.72 and 0.79%, respectively). moreover, the changes in the contents of octadecanoic acid, ethyl ester and methyl linolenate with a higher production under intermediate irradiance (i30 and i50) demonstrated that this component was also influenced by irradiance. nerolidol increased in the treatments with 50% and 100% light. these results showed that the 4 samples mainly contained large le vels of monoterpenes (2.69-42.95%) and oxygenated sesquiterpenes (13.27-73.33%). interestingly, the result showed that the variation of monoterpenes and oxygenated sesquiterpenes were inversely related, i.e. a rise of the monoterpenes content accompanied the decrease in the content of oxygenated sesquiterpenes. the leaf oils had the highest content of monoterpenes (42.95 %) under i100, while the highest oxygenated sesquiterpenes content (73.33 %) was observed under i50. discussion irradiance is essential for plant growth, since it affects the primary metabolism providing energy for photosynthesis and generating signals that regulate their development and even interfere with the translocation of assimilates among different plant organs (lima et al., 2010). plants grown in full light conditions absorb much more light energy in the leaves than the plants require and utilize for photosynthetic co2 fixation (wilhelm and selmar, 2011). high irradiation often is co-occurring with water deficiencies (kleinwächter and selmar, 2015). therefore, high light levels may reduce plant growth (tang et al., 2015). the foliar thickness, the ratio of palisade and spongy tissue thickness, as well as palisade and spongy tissue thickness increased in m. breviracema with increasing irradiance to cope with these light stresses (aleric and kirkman, 2005). the adaptation of plant morphology, anatomy to the changes in light intensity may influence the accumulation of secondary metabolites (ma et al., 2010). similarly, irradiance can influence the production of alkaloids 174 y. li, d. kong, h.-l. liang, h. wu fig. 2: levels of three alkaloids in m. breviracema roots (a), stems (c) and leaves (e) at various light levels. estimated total amounts of alkaloid in the roots (b), stems (d) and leaves (f) of a plant under different light intensities (g per plant). the values are the average ± se. different letters demonstrate that there are obvious differences in the shade treatments (p<0.05) tab. 1: the changes of anatomical characteristics of m. breviracema leaves under different light intensity. light intensity lamina thickness (μm) palisade tissue (μm) spongy parenchyma (μm) palisade/spongy i10 525.39±16.05d 65.09±2.05d 413.50±2.11d 0.157±0.02c i30 576.78±9.61c 76.09±2.13c 459.05±10.45c 0.166±0.01bc i50 682.56±4.53b 91.50±0.91b 503.09±4.49b 0.182±0.01b i100 742.90±2.14a 131.02±2.57a 571.50±9.87a 0.229±0.01a the values represent the mean ± se, and the same letters indicated no significant differences in four shade treatment (p<0.05) influence of light on alkaloid content and essential oil composition in mahonia breviracema 175 both directly and indirectly by increasing plant biomass (kong et al., 2016). li et al. (2009) reported that the contents of berberine, jatrorrhizine and palmatine increased with the light incidence for amur corktree, while the production of biomass was the highest at 75% full-sunlight treatment. in this study, we reveled that the irradiance levels significantly affected the plant biomass and alkaloid content in different plant organs of m. breviracema. notably, roots and stems showed higher contents of jatrorrhizine, palmatine and berberine, which were very low or even undetected in the leaves. jatrorrhizine, palmatine and berberine showed similar trends in roots and stems. however, the content of palmatine in stems was much higher than that in roots. in addition, jatrorrhizine was not detected under i10 and i30 in leaves, which indicated that higher light intensity promotes the synthesis of alkaloids (wilhelm and selmar, 2011). the highest contents of jatrorrhizine and palmatine in roots and stems were all observed under i30 and i50, and the content of berberine in roots and stems under i10 was the highest. however, the plant biomass was significantly higher under i30 and i50 than that under i10 and i100. thus, the total amount of jatrorrhizine, palmatine and berberine, and the total alkaloid yields in stems, roots and each plant under i30 and i50 were much higher compared with other treatments. in many cases excessive light under nutrient or water limitation stimulates the synthesis of secondary metabolites (wilhelm and selmar, 2011). research on the yield of essential oil under different shade conditions indicates that species have different responses to light intensity, like lippia sidoides cham. (souza et al., 2007) and ocimum basilicum (figueiredo et al., 2008) showed the increase of essential oil yields when grown at high light intensities. kong et al. (2016) revealed that plastoglobules in the chloroplasts were significantly increased at higher light intensity for m. bodinieri. this study showed that not only the numbers of grana lamellae, grana and chloroplasts decreased with the increase of light irradiance intensity, but also the plastoglobules were obviously affected by the light intensity for m. breviracema. notably, we revealed a positive correlation between the number of plastoglobules with the yield of essential oil at different growth stages in m. breviracema. the smaller amounts of plastoglobules were accumulated under i10, which was accordance with a lower yield of essential oil. our research showed significant chloroplast structural damage and increased plastoglobules with increasing light intensity. in particular, the grana totally ruptured and disappeared, but the chloroplasts were filled with abundant plastoglobules. the yield of essential oil rose sharply to 4.53 g kg-1 under i100. these results are in accordance with that of souza et al. (2007) and figueiredo et al. (2008) in that a higher light intensity enhan ced the accumulation and synthesis of essential oils. accumulation and synthesis essential oils in m. breviracem mainly occur in chloroplasts. furthermore, these results further confirmed that the increase of plastoglobules were closely related to the reduced chloroplast function and senescence under high light intensity (biswal, 1995). in general, the composition of essential oils is very sensitive and can suffer numerous reactions under stress factors (selmar and kleinwächter, 2012; kleinwächter and selmar, 2015). in the current study, the major and most representative component in all analyzed samples is the saturated fatty acid hexadecanoic acid which is exhibited in high percentage in the four samples (10.54-72.19%). this result is similar to that obtained in m. bodinieri (liu et al., 2010a). thereinto, the intermediate irradiance (i50) was beneficial for increasing hexadecanoic acid. moreover, in all these samples hexade canoic acids were found in various forms such as ethyl ester, methyl ester. hexadecanoic acid isolated from a marine red alga may be a lead compound of anticancer drugs (harada et al., 2002) and pre sents important anti-inflammatory and analgesic activities properties (aparna et al., 2012; hamdi et al., 2018). indeed, hexadecanoic acid derivatives showed and anti-nociceptive anti-inflammatory activities (zitterl-eglseer et al., 1997; deciga-campos et al., 2007). such as, hexadecanoic acid, methyl ester is responsible for various pharmacological actions like antimicrobial and antioxidants activities (tapiero et al., 2002). the presence of phytochemicals could be considered as sources of quality raw materials for food and pharmaceutical industries. interesting, we found that α-ionone, another important composition in the oil of m. breviracema, showed highest content in full light conditions. ionone has been reported as a product of oxidative rupture of β-carotene (sánchez-contreras et al., 2000). it is possible to increase the leaf temperature in the high light conditions, which is beneficial to the accumulation of carotenoids, while the high temperature stimulates the degradation of carotenoids and the accumulation of aromatic substances (ionone) (kawakami and kobayashi, 2002; zepka and mercadante, 2009; zhan et al., 2012; ramel et al., 2012). carotenoids are sources of essential precursors for the biosynthesis of bioactive compounds in plants when oxidative cleavages occur to form carotenoids derivatives. these compounds serve as signal molecules (ramel et al., 2012) and they have been implicated in the interactions of plants with their environment (light and temperature) (walter and strack, 2011; nisar tab. 2: changes in number of mesophyll chloroplast (n mm-2) and structural characteristics of chloroplast in leaves of m. breviracema in various light conditions. light chloroplast chloroplast chloroplast intensity length (μm) width (μm) number (n/mm2) i10 7.14±0.23a 4.27±0.26b 5293±109.41a i30 7.96±0.14a 3.68±0.29b 4403±232.38b i50 7.80±0.47a 3.44±0.68b 3227±186.19c i100 8.18±0.30a 2.62±0.46b 2796±172.05c the values indicate the average ± se. the same letters show there is no ob vious difference in the four shade treatments (p<0.05) fig. 3: light micrographs of characteristic semi-thin cross-sections in the leaves of m. breviracema at different light intensities. i10 (a), i30 (b), i50 (c), i100 (d). 176 y. li, d. kong, h.-l. liang, h. wu fig. 4: ultrastructure of chloroplast in the leaves of m. breviracema at different light intensities. i10 (a–c), i30 (d–f), i50 (g–i), i100 (j–l). dp: electron-dense plastoglobules, g: granum, tp: electron-transparent plastoglobules. et al., 2015; briardo et al., 2016). thereby, carotenoids play an important role on sensing and signalling oxidative stress, as chemical oxidation of b-carotene by 1o2, forming a wide variety of products, such as b-cyc b-ionone and a-ionone (ramel et al., 2012). hexadecanoic acid and α-ionone are the major components of m. breviracema leaf oils, suggesting a chemotype different from that described by liu et al. (2010b), where the main component is 4-terpineol (43.73%) for mahonia duclouxiana gagnep. hexadecanoic acid is used to treat blood lipids and cardiovascular diseases (consultation, 2003). α-ionone is an important material in the flavors and fragrances industry (sell, 2006) therefore, m. breviracema has important development potential and high medicinal value. moreover, m. breviracema is a good ornamental plant. furthermore, the leaf oils also contained high levels of nerolidol and octadecanoic acid. it was reported that sesquiterpene indole (the main volatile of nerolidol) provided indirect plant defense against various herbivores (pacheco et al., 2016). octadecanoic acid is an inducible plant defense molecule against insects (marko-varga et al., 2008). thereby, the increase of these compounds in leaves under high light intensity can indicate that there is an effective defense system for the adverse environmental conditions. conclusion this research explores the significant differences in the content of alkaloid and chemical characteristics of leaf oil of m. breviracema grown under various light intensities. the contents of jatrorrhizine, palmatine and berberine were notably higher in root and stem samples compared to the leaf samples. by analyzing the variations of alkaloid contents and biomass, i30 and i50 are determined as the best influence of light on alkaloid content and essential oil composition in mahonia breviracema 177 tab. 3: chemical constituents (%) of essential oil in leaves of m. breviracema under different light intensities. no compound ria relative content (%)b identification i10 i30 i50 i100 1 2,2,4,6,6-pentamthyl heptanes 1030 0.23±0.05b 0.43±0.05b 0.17±0.02b 10.37±1.17a gc–ms, ri 2 2,6,8-trimethyldecane 1121 0.39±0.05b 0.06±0.02c 0.06±0.06c 1.52±1.52a gc–ms, ri 3 α-ionone 1366 1.92±0.18b 2.32±0.21b 1.25±0.16b 42.39±1.65a gc–ms, ri, co 4 2,6,10-trimethyl dodecane 1429 0.18±0.03b 0.26±0.01a 0.05±0.01c gc–ms, ri 5 geranyl acetone 1434 0.22±0.02a 0.11±0.01b 0.04±0.01c tr gc–ms, ri 6 2,6,10-trimethyltetradecane 1555 0.90±0.37a 0.1±0.04c 0.16±0.03bc 0.51±0.02ab gc–ms, ri 7 nerolidol 1567 0.75±0.06c 0.54±0.03c 1.14±0.09b 2.75±0.20a gc–ms, ri 8 hexadecane 1600 2.71±0.29a 0.52±0.11d 0.91±0.11c 1.47±0.12b gc–ms, ri 9 1,2,3-popanetricarboxylic acid, 2-hydroxy-, 1655 1.19±0.05a 0.26±0.04d 0.42±0.10c 0.63±0.07b gc–ms, ri triethyl ester 10 phytane 1809 0.91±0.02a 0.53±0.11b 0.66±0.08b 0.93±0.10a gc–ms, ri 11 6,10,14-trimethyl-2-pentadecanone 1843 1.43±0.11a tr 0.07±0.03b gc–ms, ri 12 hexadecanoic acid, methyl ester 1908 1.48±0.10a 0.46±0.08d 0.72±0.08c 1.20±0.20b gc–ms, ri 13 α-farnesylacetone 1914 2.46±0.08a 0.56±0.08d 0.96±0.10c 1.60±0.30b gc–ms, ri 14 methyl hexadecanoate 1924 2.50±0.04a 0.38±0.05d 0.64±0.04c 0.79±0.11b gc–ms, ri 15 hexadecanoic acid, ethyl ester 1968 1.83±0.16a 0.60±0.30b 0.79±0.09b 1.77±0.21a gc–ms, ri 16 hexadecanoic acid 1978 52.23±2.27b 60.05±7.02b 72.19±5.89a 10.54±2.74c gc–ms, ri, co 17 octadecanoic acid 2002 6.54±0.88a 1.07±0.09c 1.44±0.21c 3.73±0.65b gc–ms, ri 18 isochiapin b 2005 1.01±0.03a 0.45±0.12b 0.50±0.10b 0.54±0.10b gc–ms, ri 19 eicosane 2008 0.41±0.09a 0.12±0.06c 0.12±0.08c 0.27±0.05b gc–ms, ri 20 phytol 2042 0.28±0.07a 0.27±0.05a 0.11±0.04b gc–ms, ri 21 9,12,15-octadecatrienoic acid, methyl ester 2051 1.35±0.19a 1.01±0.19b 0.96±0.12b gc–ms, ri 22 methyl linoleate 2062 0.21±0.05b 0.44±0.56a 0.11±0.03b 0.10±0.02b gc–ms, ri 23 9-octadecenoic acid (z)-, methyl ester 2078 0.81±0.17a 0.18±0.06b tr 0.56±0.18a gc–ms, ri 24 octadecanoic acid, ethyl ester 2083 1.05±0.07b 12.94±3.01a 1.39±0.19b 0.76±0.22b gc–ms, ri 25 methyl linolenate 2102 0.33±0.11a 0.12±0.07b tr gc–ms, ri 26 ethyl linoleate 2165 1.81±0.38a 1.24±0.16b 0.71±0.29c 1.15±0.15bc gc–ms, ri 27 linolenic acid ethyl ester 2173 0.32±0.06a tr tr 0.22±0.01b gc–ms, ri 28 3,7,11,15-tetramethyl-, 2201 6.74±1.14b 9.01±0.63a 6.46±0.14b 3.07±0.18c gc–ms, ri [r-(r*,r*-(e))]-2-hexadedcen-1-ol 29 docosane 2208 0.55±0.10b 0.42±0.14b 0.36±0.04b 0.88±0.10a gc–ms, ri 30 carvacrol 2214 0.55±0.13a 0.25±0.10b 0.33±0.03b 0.56±0.02a gc–ms, ri 31 tricosane 2300 2.04±0.24a 0.66±0.23b 2.43±1.01a 1.93±0.17a gc–ms, ri monoterpenes 2.69 2.68 1.62 42.95 sesquiterpenoids 6.72 1.33 1.49 3.37 oxygenated sesquiterpenes 52.98 60.6 73.33 13.29 hydrocarbons 6.62 2.25 4.48 5.48 others 22.98 30.48 14.22 26.88 total 91.99 97.34 95.14 91.97 a ri=retention indices on the basis of a homologous series of normal alkanes. b data are represented as the average ± sd. values sharing the same small letter within a line are not obviously different at p<0.05; 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10.1007/s00468-012-0752-1 liu, s.x., liu, b.m., dong, x.m., zhang, k.y., lin, x., chen, l., he, k.j., 2010a: analysis of essential oil form mahonia bodinieri gagnep, j. guangxi acad. sci. 26, 216-217. liu, s.x., liu, b.m., he, k.j., lin, x., lu, w.j., 2010b: analysis of essential oil from mahonia duclouxiana. j. chin. med. mater. 33, 1099-1102. ma, z.q., li, s.s., zhang, m.j., 2010: light intensity affects growth, photosynthetic capability, and total flavonoid accumulation of anoectochilus plants. hortscience 45, 863-867. marko-varga, g., nilsson, j., laurell, t., 2008: octadecanoic acid (stearic acid). encyclopedia of genetics genomics proteomics & informatics, 1387-1387. nisar, n., li, l., lu, s., khin, n.c., pogson, b.j., 2015: carotenoid megrowth environment to get the highest total alkaloids yields and enhance the value of medicinal plants. this study initially provides some information about the composition of m. breviracema leaf essential oils. higher irradiance is conductive to increasing the yield of essential oil. the essential oil extracted from leaves was rich in hexadecanoic acid and α-ionone. moderate light conditions (i30 and i50) were suitable for accumulation and synthesis of hexadecanoic acid, and high light intensity (i100) was beneficial for the accumulation and synthesis of α-ionone. by analyzing the changes in oil yields and compositions, i50 and i100 are determined the optimal growth environment to get the highest of leaf oils yield or high quality hexadecanoic acid and α-ionone. these results can be used as a reference for industrial exploiters of m. breviracema essential oils. acknowledgements this study was supported by the nature science foundation of china (31500261), the science and technology innovation fund project on forestry of guangdong province (2017kjcx006), and the youth foundation of college of forestry and landscape architecture of south china agricultural university (201603). references adams, rp., 2001: identification of 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quality 90, 246 258 (2017), doi:10.5073/jabfq.2017.090.031 civil and environmental engineering department, environment laboratory university of perugia an ethnobotanical investigation of traditional knowledge and uses of edible wild plants in the umbria region, central italy aldo ranfa, mara bodesmo* (received january 26, 2017; accepted april 15, 2017) * corresponding author summary these days edible wild plants (ewps) play a fundamental role in the mediterranean diet, thanks to their content of mineral elements and bioactive compounds with proven benefits for human health. the present study aims to document ethnobotanical knowledge and uses of ewps in central italy so that this knowledge will not be lost. during various nature fairs and exhibitions in umbria three hundred subjects were interviewed face-to-face between march and may 2013-2015. the participants provided information on local plant names, where and when the plants were collected, part(s) used, categories used, folk medicinal uses, taste perception and other uses. the results were analyzed using two ethnobotanical indices: the relative frequency of citation (rfc) and relative importance index (rii). the 100 ewps mentioned by the respondents belonged to 23 families, asteraceae (33%), brassicaceae (17%) and lamiaceae (11%) being the most dominant. the part(s) used were leaves (49%), shoots (17%), flowers and inflorescences (10%). fourteen food use categories were cited, of which boiled 31%, 28% raw, 12% in vegetable soups, 11% fried in fat, without or with beaten eggs. twenty-seven plant species were also mentioned as having folk medicinal uses. keywords: central italy, umbria region, edible wild plants, ethnobotanical knowledge, folk medicinal uses, taste perception introduction plants are an essential part of food intake; these days the bulk of our food requirements is satisfied by a few plant species of cultivated crops. in particular only seven species supply 90% of calories pro-capita worldwide, among them wheat, maize, rice and potatoes. however, it should not be forgotten that thousands of plant species are eaten locally, of which very few have been partially domesticated; indeed most are collected in the wild and belong to the immense patrimony of edible wild plants (ewps) present on our planet (heywood, 2011). over the last two decades there has been a growing interest in ewps which has prompted researchers to record local knowledge of various uses to preserve traditions and disseminate information. numerous research projects have shown how the european continent, for example countries such as poland (łuczaj and szymański, 2007), slovakia (łuczaj, 2012), the iberian peninsula (tardío et al., 2006; pardo de santayana et al., 2007), italy (ranfa, 2005; signorini et al., 2009; ranfa et al., 2011; guarrera and savo, 2013; ranfa, 2014; sansanelli and tassoni, 2014; ranfa et al., 2015) and greece (della et al., 2006), possess a rich and varied culture with respect to ewp uses (pardo de santayana et al., 2010; łuczaj et al., 2012). these species are in the centre of a new approach concerning food which focuses on health and uncontaminated food sources (łuczaj et al., 2012). especially in the mediterranean area, numerous studies have explored the extraordinary richness of wild species and the traditional uses associated with them (della et al., 2006; dogan, 2012), as well as looking into their importance as a source of bioactive compounds (sanchez mata et al., 2012). indeed in the mediterranean area there is still a strong tradition in the use of ewps. currently, around 2 300 wild plant and mushroom species are used directly in human food consumption or to prepare condiments and drinks (caneva et al., 2013). in italy there are at present 828 edible species belonging to 98 botanical families whose ethnobotanical uses have been documented (romojaro et al., 2013). furthermore these species are arousing much interest thanks to their nutraceutical value and the benefits that derive from frequent, habitual use (nebel et al., 2006). some studies carried out in italy have shown that these species have significant nutraceutical values, as they are rich in mineral elements and bio active compounds, with proven benefits for human health (ranfa et al., 2011; guarrera and savo, 2013; ranfa et al., 2014; maurizi et al., 2015; ranfa et al., 2015) thanks to their high polyphenol and unsaturated fatty acid contents (de lorgeril and salen, 2007). in the past, most studies in umbria concentrated on recovering local knowledge regarding medicinal (leporatti et al., 1985; nardelli, 1987) and food uses (dalla ragione i, dalla ragione l., 2003), while more recent research has shown that, with respect to many cultivated species, ewps possess higher fibre content, are anti oxidant and flavonoid-rich, and have beneficial effects in preventing chronic modern-day diseases (ranfa et al., 2011; maurizi et al., 2015; ranfa et al., 2015). the aim of this study is to collect and document local knowledge of traditional ewp uses together with information on collecting, processing, cooking and folk medicinal uses. until now, in umbria little research, fragmented to say the least, has been carried out in this field. therefore, this study represents one of the first attempts to document and preserve an important part of our ethnobotanical heritage which risks being lost. field work the study was conducted in umbria, central italy, in an area of 8 456 km² ca. with 895 259 inhabitants (fig. 1). the river tiber flows through the region, which is typified by a wide range of physical and climatic features, thus determining a great floristic diversity with over 2 000 different plant species (conti et al., 2007). the landscape features extensive plains where anthropogenic activity is concentrated and where over the years a progressive reduction of floristic diversity has been observed. on the other hand, the hilly and mountainous areas maintain high naturalistic diversity with a high percentage of forest cover and a low percentage of urbanization (aa.vv., 2004). umbria is a region of rich biodiversity, having 95 sites of community importance (scis) and 5 special protection areas (spas) that cover 15% of the regional territory (ministero dell’ambiente e della tutela del territorio e del mare, 2015). there are still extensive rural areas dotted with small villages where the countryside has altered very little, and where the knowledge and use of ewps is still very much alive (fig. 1) (ranfa et al., 2011, 2014, 2015). ethnobotanical knowledge in central italy 247 material and methods ethnobotanical data were collected during some events centred on ewps which took place in umbria, in the perugia and spello municipalities, aiming to disseminate and promote knowledge of wild plant species. of particular importance are the 2013, 2014, and 2015 editions of the mostra delle erbe spontanee, organized by the perugia mycological and field naturalists’ association (circolo micologico natualistico perugino), where potted wild plants bearing labels that described botanical characteristics, food use and medicinal properties were on display, and also the 2014 and 2015 editions of the wild plants show (‘subasio con gusto rassegna delle erbe campagnole) held in the spello municipality where various events promoted information on and uses of ewps. the data collected during these themed events were representative of the entire umbria region as participation was high and the informants came from various localities throughout umbria. in both municipalities various field trips encouraged the general public to participate in the collection and identification of wild species. therefore, over the past few years these species, for example ‘raponzoli’ (campanula rapunculus l.), ‘caccialepre’ [reichardia picroides (l.) roth], ‘papavero’ (papaver rhoeas l.) and ‘borragine’ (borago officinalis l.) are beginning to appear more frequently in local street markets (fig. 2 a, b, c, d, e). moreover, local restaurants are including dishes based on wild plants in their menus, one of the most recent being a ‘pesto’ made with wild greens (fig. 2-f). research on the uses of ewps began in 2012 (ranfa et al., 2013) and continued in 2013, 2014 and 2015. with respect to data collected in 2012, information relative to the aforementioned species has been integrated to include food use categories, taste perception and other uses. furthermore 55 new species have been indicated. ethnobotanical information was collected by means of standard ethnobotanical tools (alexiades, 1996), through 881 open anonymous and face-to-face interviews. the 300 informants were represented by 180 females and 120 males, aged between 45 and 80 years, of which only 10 informants were under 55 years of age. each informant, chosen at random, filled in more than one form and supplied information on 2-3, and in some cases 3-4, wild plant species, inclu ding local name, places of collection, period of collection, part(s) used, categories used, folk medicinal uses, taste perception and knowledge of particular anecdotes and recipes. for the data in the manuscript, the local and national guidelines have been used and appropriate permission for the study were requested. regarding local names, the informants indicated the names commonly used in their own areas. however, only the informants over 65 years of age supplied typically local names because mainly this group still conserves this know fig. 1: field work. perugia and spello, the municipalities where the themed events were organized. (source: umbria geo database, umbria region) fig. 2: exhibition of wild plants: field work to collect and identify wild plants, spello, during the ‘subasio con gusto’ event (a); wild edible plants on sale in a local market: ‘raponzoli’ (campanula rapunculus l.) (b); common edible wild plants in the umbria region, for example ‘caccialepre’ [reichardia picroides (l.) roth] (c), ‘papavero’ (papaver rhoeas l.) (d) and ‘borragine’ (borago officinalis l.) (e); pesto made with wild edible greens (f). 248 a. ranfa, m. bodesmo ledge, while the others gave more widely-known names which were similar, if not identical, to the italian scientific names, probably due to the large amount of information available through various channels (i.e. the internet, many thematic books, etc). the voucher specimens collected have been deposited in the plant bioresources for the environment laboratory of civil and environmental engineering department, university of perugia, italy. these samples constitute the voucher specimens used in this research, and the herbarium will be enriched and extended over time with the addition of further species identified by future informants. successively, the international plant names index (http://www.ipni. org/) was consulted to verify the accepted nomenclature for each species. two indices were calculated for data analysis: the relative frequency of citation (rfc) (tardío and pardo-de-santayana, 2008) and the relative importance index (rii) (pardo-de-santayana, 2003; tardío and pardo-de-santayana, 2008). the relative frequency of citation (rfc) is obtained by dividing the number of informants who mention the use of the species which correspond to the frequency of citation (fc) by the total number of informants participating in the investigation (n) (eq. 1). rfcs= fcs\n (1) where fc is the frequency of citation of the species, that is, the number of informants who cited that species, and n is the total number of informants who participated in the study. this index varies from 0 if the species was not cited to 1 if the species was cited by all informants. the relative importance index (rii) regards both the number of informants and the number of uses. in this case only food use was taken into consideration because the informants cited it most frequently (eq. 2): ris= rfcs(max) +rnfus(max)\2 (2) where rfcs(max) is the relationship between the frequency of citation of a given species (fcs) divided by the maximum number of informants citing any species and rnfus(max) is the relationship between number of food uses (nfu) given for that species by the total number of food uses given for all species. a correlation analysis of the two indices was then carried out using pearson’s correlation index (r) which represents a measure of the strength of the linear relationship between two variables. results a total of 100 uses for ewps (tab. 1) was documented. all species belong to 23 botanical families, most frequently represented by asteraceae (33%), followed by brassicaceae (17%) and lamiaceae (11%). part(s) used tab. 2 shows the percentages of part(s) used. leaves are mainly eaten when young and tender as a raw vegetable or cooked in traditional dishes, for example as a filling for ravioli or in savoury pies. lepidium latifolium leaves are used as mustard or pepper. mentha aquatica, mentha pulegium, mentha suaveolens, origanum vulgare and origanum majorana leaves are used for infusions or dried and used to season meat and fish. bellis perennis, borago officinalis, calendula arvensis, campanuula rapucunculus, viola odorata flowers or inflorescences are eaten raw in salads, used to make jam or else candied. ranunculus ficaria, urospermum. dalechampii and taraxacum officinale flower buds are used in place of capers. smilax aspera flowers are eaten in salads. young shoots are eaten raw or boiled; melissa officinalis shoots are used in alcoholic beverages, smilax aspera shoots are preserved in oil or pickled, while rubus ulmifolius shoots are used like asparagus in omelettes. chenopodium album, alliaria petiolata and diplotaxis tenuifolia are all eaten; foeniculum vulgare seeds are used in alcoholic beverages, papaver rhoeas seeds are added to bread and cookies, and sinapis alba seeds are used to make mustard. in 5% of the plants the stems are eaten, especially in brassica nigra and in foeniculum vulgare in which stems are eaten for their refreshing properties. in some species the roots are roasted as a coffee substitute (i.e.: chondrilla juncea, cichorium inthybus, reichardia picroides, sonchus spp., taraxacum officinale). muscari comosum bulbs are boiled, preserved in oil or pickled. ragadiolus stellatus rhizomes are also eaten. food use categories the most frequent method is boiling (b) to produce ‘cooked greens’ (tab. 2) which are then seasoned with oil, salt and lemon juice, the most commonly used species being sonchus spp., cichorium intybus, crepis spp., rumex spp. and urospermum dalechampii. about 28% of ewps are eaten raw (r) in mixed-leaf salads where the sweeter herbs such as plantago lanceolata and borago officinalis mitigate the bitterness of cichorium intybus, taraxacum officinale and helminthotheca echioides. in raw salads sanguisorba minor, campanula rapunculus, tordylium apulum, reichardia picroides, daucus carota, and capsella bursa-pastoris are indicated as being very tasty. about 12% of the species are used in vegetable soups (vs), such as achillea millefolium, arctium lappa, cardamine hirsuta and foeniculum vulgare. 11% are fried in fat, with or without beaten eggs (“frittata”) (f), for example asparagus acutifolius, clematis vitalba, calamintha ne peta, humulus lupulus and sonchus aspera. about 6% are used as a filling in ravioli or savoury pies (fil) especially borago officinalis and urtica dioica, and 2% in risotto such us silene vulgaris, allium spp. and diplotaxis muralis. some species are used in alcoholic beverages (ab). for example, achillea millefolium seeds are placed in wine barrels to improve wine preservation. other uses included, for example, allium neapolitanum used to make repellents for moths, beetles and other insects, dried calendula arvensis petals are used to aromatize wine, which after being left in the sun for ten days becomes excellent vinegar. clematis vitalba roots are smoked, parietaria officinalis leaves are used to clean wine flasks, while taraxacum officinale leaves are used in infusions and the flowers used to make jam. details are shown in tab. 2. taste perception the ewps with a bitter taste were urospermum dalechampii, clematis vitalba, cichorium intybus, crepis spp. and taraxacum officinale. many of the informants stated that flavor depends greatly on when the plants are collected, and that they are less bitter before flowering. ewps with a spicy taste are sanguisorba minor that tastes like fresh walnuts, tordylium apulum that tastes a little like cucumber, and then there are the aromatic plants such as origanum spp. and thymus spp. camapanula rapunculus roots are sweet while the leaves are rather bitter, tragopogon pratensis leaves are bitter while the root tastes like walnuts. the ewps which are reputed to have a sweet taste are lactuca spp., malva sylvestris, papaver rhoeas, ragadiolus stellatus, silene vulgaris and sonchus spp. local dishes the informants provided various traditional recipes. in particular, a popular springtime salad consists of ‘caccialepre’ (reichardia picroides) leaves which are dressed with heated olive oil, salted anhttp://www.ipni.org/ http://www.ipni.org/ ethnobotanical knowledge in central italy 249 ta b. 1 : l is t o f t he e di bl e w ild p la nt s ci te d in th e st ud y ar ea . sc ie nt ifi c na m es f am ily l oc al n am es p ar t( s) u se d f oo d us e ta st e f ol k m ed ic in al u se s o th er u se s n um be r of ca te go ri es * pe rc ep ti on ci ta ti on s 1 a ch ill ea m ill ef ol iu m l . a st er ac ea e ac hi lle a, m ill ef og lie le av es a b , r , v s bi tte r bo ile d le av es a pp lie d se ed s tie d in a c ot to n ba g 15 to c ut s an d w ou nd s pl ac ed in w in e ba rr el s to fo r h ea lin g im pr ov e w in e pr es er va tio n 2 a ju ga r ep ta ns l . l am ia ce ae bu gu la , c on so lid a, e rb a sh oo ts , y ou ng le av es b , r bi tte r 5 di s . l or en zo , e rb a m or a 3 a lli ar ia p et io la ta c av ar a b ra ss ic ac ea e al lia ri a co m un e, e rb a ag lin a se ed s, y ou ng le av es b , r , v s, ta st y le av es a pp lie d to g um s se ed s us ed in th e sa m e 3 & g ra nd e o th . to tr ea t g en gi vi tis , s ee ds w ay a s m us ta rd s ee ds st im ul at e ap pe tit e 4 a lli um n ea po lit an um a lli ac ea e ag lio b ia nc o, le av es , y ou ng b ul bs b , r , f , v s sp ic y us ed a s an in se ct a nd 13 c ir ill o ag lio n ap ol et an o m ot h re pe lle nt 5 a lli um tr iq ue tr um l . a lli ac ea e ag lio a ng ol ar e, a gl io le av es , y ou ng b ul bs b , r , f , v s sp ic y re du ce s hy pe rt en si on , 5 tr ig on o, a gl io s el va tic o an tib io tic , d is in fe ct an t, re du ce s gl yc em ia , ca rd io st im ul an t 6 a lli um u rs in um l . a lli ac ea e ag lio o rs in o le av es , y ou ng b ul bs b , r , f , v s sp ic y fr es h le av es a pp lie d to th e 6 sk in a ct a s a ru bi fa ci en t, po un de d an d us ed a s a po ul tic e to s oo th e ab sc es se s an d bo ils 7 a rc tiu m la pp a l . a st er ac ea e ba rd an a m ag gi or e, la pp a le av es , r hi zo m es , s te m s, b , r , v s bi tte r in fu si on s tim ul at es 10 ba rd an a, la pp ol a yo un g sh oo ts ha ir g ro w th 8 a sp ar ag us a cu tif ol iu s l . l ili ac ea e as pa ra go , a sp ar ag o de i tu ri on es b , f , r bi tte r 25 bo sc hi 9 b ar ba re a vu lg ar is r . b r. b ra ss ic ac ea e er ba d i s . b ar ba ra c om un e, flo w er s, le av es , s ho ot s b , v s sp ic y 3 ru co la p al us tr e 1 0 b el lis p er en ni s l . a st er ac ea e m ar gh er iti na , p ra to lin a flo w er s, le av es b , r , o th . bi tte r le av es a pp lie d to th e gu m s flo w er s us ed fo r l ud ic 25 to s oo th e sm al l c ut s, ac tiv iti es ‘l ov es m e, lo ve s ch ew ed to re fr es h th e m e no t’ ; l ea ve s us ed m ou th as a te a su rr og at e 1 1 b or ag o of fic in al is l . b or ag in a bo rr ag in e, b or ag in e flo w er s, le av es b , f , f il ., r sw ee t flo w er s m ac er at ed in w in e 40 ce ae fo r a w ee k m ak e an e xc el le nt b od ycl ea ns in g dr in k; flo w er s m ac er at ed in w hi te vi ne ga r t ur n bl ue ; fl ow er s fr oz en in ic e cu be s 1 2 b ra ss ic a ni gr a (l .) b ra ss ic ac ea e ca vo lo s en ap ene ra , flo w er s, le av es , s ee ds , b , f , r , o th . ta st y se ed s in th e w at er fo r 5 w .d .j . k oc h ra pa s el va tic a st em s fo ot b at hs 1 3 b un ia s er uc ag o l . b ra ss ic ac ea e ca sc el lo ra , c as ce llo re le av es , y ou ng s ho ot s b , r sp ic y le av es a re d iu re tic 8 co m un e, c as se lla , co te ca cc hi e, n av on e se lv at ic o 250 a. ranfa, m. bodesmo 1 4 c al am in th a ne pe ta (l .) l am ia ce ae ca la m en to , m en tu cc ia flo w er s, y ou ng le av es b , f ta st y st im ul at es b ile p ro du ct io n, 20 sa vi co m un e, n ep et el la s el va tic a, st im ul at es a pp et ite , l ea ve s po le gg io s el va tic o ru bb ed o n in se ct b ite s 1 5 c al en du la a rv en si s l . a st er ac ea e ca le nd ul a de i c am pi , flo w er s, le av es b , r sw ee t le av es h ea l w ou nd s dr ie d pe ta ls g iv e ar om a to 10 fio rr an ci o se lv at ic o w in e w hi ch , a ft er te n da ys in th e su n, b ec om es a n ex ce lle nt v in eg ar 1 6 c am pa nu la r ap un cu lu s l . c am pa nu la ra po nz ol o, ra m po nz ol o, flo w er s, le av es , r oo ts o r b , r ro ot s w ee t, us ed to tr ea t i nfl am m at io n 25 ce ae ra pe ro nz ol o, ra pu nz ol i ro ot st oc ks , y ou ng s ho ot s le av es b itt er of th e or al c av ity , l ea ve s us ed to tr ea t w ar ts , i nf us io n of fl ow er s us ed a s a ga rg le 1 7 c ap se lla b ur sa -p as to ri s b ra ss ic ac ea e bo rs a di p as to re , le av es , y ou ng s ho ot s b , f , r , v s ta st y 20 (l .) m ed ik . s ub sp . bo rs ac ch in a, e rb a bo rs a, bu rs apa st or is er ba c io cc a, e rb a ra pe ri na 1 8 c ar da m in e hi rs ut a l . b ra ss ic ac ea e bi lle ri p ri m at ic ci o, flo w er s, le av es b , r , v s ta st y 2 ca rd am in e 1 9 c ar lin a ac au lis l . s .l. a st er ac ea e ca rc io fo d i m on ta gn a, c ar do fl ow er h ea ds b , r bi tte r 3 di s an p el le gr in o 2 0 c en tr an th us r ub er (l .) d c . v al er ia na ba rb a di g io ve , c am ar ez za yo un g le av es b , r , v s bi tte r 5 su bs p. r ub er ce ae co m un e, s ao ni na , v al er ia na ro ss a 2 1 c he no po di um a lb um l . c he no po di a ab iti llo , f ar in ac ci o, le av es , y ou ng s ho ot s b , r , f il. , v s bi tte r 5 ce ae fa ri ne llo c om un e 2 2 c he no po di um c he no po di a fa ri ne llo b uo nen ri co , le av es , y ou ng s ho ot s b , r bi tte r 5 bo nu she nr ic us l . ce ae fa ri ne llo tu tta b uo na 2 3 c ho nd ri lla ju nc ea l . a st er ac ea e er ba p iz zu ta , g in es tr el la , le av es , y ou ng s ho ot s b , f , r , o th . ta st y fr es h le av es a pp lie d di re ct ly 15 la tta jo la , m as tr ic i, pi ol e, he al w ou nd s, c ut s an d ul ce rs , pi ol et ta ac ne a nd e cz em a 2 4 c ic ho ri um in ty bu s l . a st er ac ea e ci co ri a, c ic or ie tta , r ad ic ch io l ea ve s, le av es s ta lk s, ro ot , b , f il ., r , bi tte r to as te d ro ot s ub st itu te s 30 se lv at ic o, ra di ci a m ar e yo un g sh oo ts o th . co ff ee 2 5 c ir si um a rv en se (l .) sc op . a st er ac ea e ca rd o ca m pe st re , le av es , y ou ng s ho ot s b , f , v s bi tte r 5 sc ar da cc io ne , s to pp io ne , st op po lo ne 2 6 c le m at is v ita lb a l . r an un cu la cl em at id e, v ita bb ia yo un g sh oo ts f, r is ., r , bi tte r ro ot s sm ok ed in th e sa m e 38 ce ae v s w ay a s to ba cc o 2 7 c re pi s sa nc ta (l .) a st er ac ea e cr ep id e, d ol ce tta , yo un g le av es b , r bi tte r 10 b ab c. s ub sp . s an ct a ra di cc hi el la d i t er ra sa nt a 2 8 c re pi s ve si ca ri a l . a st er ac ea e co ta , c re pi de v es ci co sa , yo un g le av es b , r bi tte r 3 ra di cc hi el la v es ci co sa , ra di cc hi o sc ol te lla to 2 9 d au cu s ca ro ta l . a pi ac ea e ca po b ia nc o, c ar ot a ro ot s, y ou ng le av es b , r sw ee t tr ea tm en t f or b ro nc hi tis in 10 se lv at ic a, g al lin ac ci , ho rs es , t he in fu si on is a pa st ic ci on a st ro ng d iu re tic ethnobotanical knowledge in central italy 251 3 0 d ip lo ta xi s er uc oi de s (l .) b ra ss ic ac ea e m ar ai uo le , r uc he tta yo un g le av es b , r ta st y 10 d c . s ub sp . e ru co id es vi ol ac ea , r uc he tto ne 3 1 d ip lo ta xi s m ur al is (l .) d c . b ra ss ic ac ea e ru ch et ta d ei m ur i yo un g le av es r ta st y 5 3 2 d ip lo ta xi s te nu ifo lia (l .) b ra ss ic ac ea e er ba d ia vo la , r uc ol a, le av es , s ee ds r , v s, o th . ta st y se ed s us ed in th e sa m e 10 d c . ru co le tta , r uc ol et ta d i w ay a s m us ta rd ca m po , r uc he tta s el va tic a 3 3 e ch iu m v ul ga re l . b or ag in ac ea e er ba v ip er in a, v ip er in a le av es b sw ee t in fu si on o f s ee ds 3 az zu rr a st im ul at es la ct at io n 3 4 e ru ca s at iv a m ill er b ra ss ic ac ea e ru co la c om un e le av es b , r ta st y 5 3 5 f oe ni cu lu m v ul ga re m ill . a pi ac ea e fin oc ch io c om un e le av es , s ee ds , s te m s, b , f , v s, ta st y in fu si on c al m s hi cc ou gh s 18 yo un g sh oo ts o th . 3 6 g eu m u rb an um l . r os ac ea e ca ri ofi lla ta c om un e, yo un g le av es r , f il. , v s bi tte r 3 ga ro fa ni no 3 7 h el m in th ot he ca e ch io id es a st er ac ea e as pr ag gi ne v ol ga re , e rb a le av es b bi tte r 3 (l .) h ol ub br us ca , e rb a br us ci a, sp ra gg in e 3 8 h um ul us lu pu lu s l . c an na ba ce ae br us ca nd ol i, lu pp ol o yo un g sh oo ts b , f , r is . bi tte r 10 co m un e 3 9 h yo se ri s ra di at a l . s ub sp . a st er ac ea e ra di cc hi o se lv at ic o, yo un g le av es b , r bi tte r 3 ra di at a tr in et te , t ri nc ia te lla 4 0 h yp oc ha er is a ch yr op ho ru s a st er ac ea e co st ol in a an nu al e yo un g le av es b , r bi tte r 4 l . 4 1 h yp oc ha er is r ad ic at a l . a st er ac ea e co st ol e d’ as in o, c os to lin a yo un g le av es b , f il ., r ta st y ro ot s fe d to p ig s 5 gi un co lin a, in gr as sa po rc i, pi at te llo 4 2 k na ut ia a rv en si s (l .) c ou lt. d ip sa ca ce ae am br et ta c om un e, le av es r , v s bi tte r 7 ve do ve lla , v ed ov in a 4 3 la ct uc a m ur al is (l .) a st er ac ea e la ttu ga d ei b os ch i yo un g le av es r , v s sw ee t 3 g ae rt n. 4 4 la ct uc a pe re nn is l . s ub sp . a st er ac ea e la ttu ga p er en ne , yo un g le av es b , r bi tte r 3 pe re nn is la ttu ga ru pe st re 4 5 la ct uc a se rr io la l . a st er ac ea e er ba b us so la , l at to na , le av es b , r sw ee t 5 la ttu ga s el va tic a, la ttu gh el la 4 6 la m iu m p ur pu re um l . l am ia ce ae fa ls a or tic a pu rp ur ea , yo un g sh oo ts f, r , v s sw ee t 6 or tic a ch e no n pu ng e 4 7 la ps an a co m m un is l . a st er ac ea e ca vo lo s el va tic o, e rb a de lle yo un g le av es b , f il . bi tte r 2 su bs p. c om m un is m am m el le , g re sp ig no lo am ar o 4 8 le on to do n au tu m na lis l . a st er ac ea e de nt e di le on e au tu nn al e, yo un g le av es b , r , v s bi tte r 2 de nt e di le on e ra m os o 252 a. ranfa, m. bodesmo 4 9 le on to do n cr is pu s v ill . a st er ac ea e de nt e di le on e cr es po , yo un g le av es b , r bi tte r 3 su bs p. c ri sp us sp iz zi ca po lli 50 le on to do n hi sp id us l . a st er ac ea e de nt e di le on e co m un e yo un g le av es b , r bi tte r 3 51 le pi di um d ra ba l . b ra ss ic ac ea e co co la , l at to na se ed s, y ou ng le av es b , f , v s, sp ic y sp ic y se ed s as a s ub st itu te 3 su bs p. d ra ba o th . fo r p ep pe r 52 le pi di um la tif ol iu m l . b ra ss ic ac ea e le pi di o la tif og lio , le av es , y ou ng s ho ot s r , o th . sp ic y sl ic es o f t he b ul b ap pl ie d to 3 m os ta rd in a, p ep er el la th e te m pl es s oo th e he ad ac he 53 m al va s yl ve st ri s l . m al va ce ae m al va s el av tic a flo w er s, le av es , f, r , r is ., sw ee t in fu si on s fo r t he re lie f o f 15 su bs p. s yl ve st ri s yo un g sh oo ts v s he ar tb ur n an d in di ge st io n 54 m el is sa o ffi ci na lis l . l am ia ce ae ce dr on el la , c itr on el la , e rb a le av es , s ho ot s a b , f , r , ta st y 10 lim on a, m el is sa v er a r is . 55 m en th a aq ua tic a l . l am ia ce ae m en ta d ’a cq ua , flo w er s, le av es , s te m s a b , f , o th . ta st y 5 su bs p. a qu at ic a m en ta st ro d ’a cq ua 56 m en th a pu le gi um l . l am ia ce ae m en ta p ol eg gi o le av es , s te m s r , o th . ta st y 3 su bs p. p ul eg iu m 57 m en th a su av eo le ns e hr h. l am ia ce ae m en ta a fo gl ie ro to nd e, le av es r , o th . ta st y 3 m en ta s el va tic a 5 8 m us ca ri c om os um (l .) l ili ac ea e ci po lla cc io , l am pa gi on e, bu lb s b , o th . bi tte r 5 m ill . la m pa sc io ne , m us ca ri se lv at ic o 5 9 m ya gr um p er fo lia tu m l . b ra ss ic ac ea e m ia gr o lis ci o st em s, y ou ng le av es b , f il ., r ta st y 5 6 0 n as tu rt iu m o ffi ci na le b ra ss ic ac ea e cr es ci on e d’ ac qu a, flo w er s, le av es , r ta st y 10 r . b r. su bs p. o ffi ci na le cr es ci on e de lle fo nt an e, yo un g st em s cr es ci on e di s or ge nt e 6 1 o ri ga nu m m aj or an a l . l am ia ce ae am ar ic o, e rb a pe rs a, flo w er s, le av es r , v s, f , ta st y so ot he s ea ra ch e 6 m ag gi or an a, o ri ga no o th . m ag gi or an a, p er si a 6 2 o ri ga nu m v ul ga re l . l am ia ce ae ac ci ug he ro , e rb a ac ci ug a, flo w er s, le av es r , v s, f , ta st y or ig an ofla vo ur ed w in e 12 su bs p. v ul ga re er ba ro ss a, m ag gi or an a o th . pe lo sa , o ri ga no c om un e, re ga m o 6 3 p ap av er r ho ea s l . pa pa ve ra ce ae p ap av er o co m un e, p at at in a, se ed s, y ou ng le av es , b , f il ., sw ee t po ds u se d by c hi ld re n 15 su bs p. r ho ea s ro so la cc io yo un g sh oo ts r , v s, o th . as ‘s ta m ps ’ 6 4 p ar ie ta ri a of fic in al is l . u rt ic ac ea e ve tr io la c om un e yo un g le av es , y ou ng s ho ot s b , v s sw ee t le av es u se d to w as h 10 gl as s bo ttl es 6 5 p as tin ac a sa tiv a l . a pi ac ea e pa st in ac a co m un e ro ot s, y ou ng le av es b , r bi tte r 10 6 6 p et as ite s hy br id us (l .) a st er ac ea e fa rf ar ac ci o m ag gi or e le av es s ta lk s b , f il ., r bi tte r in fu si on o f l ea ve s 5 p. g ae rt n. , b . m ey er e t ca lm s co ug hs sc he rb . s ub sp . h yb ri du s 6 7 p ic ri s hi er ac io id es l . a st er ac ea e as pr ag gi ne c om un e, yo un g le av es b , r bi tte r 10 er ba b ru sc a ethnobotanical knowledge in central italy 253 6 8 p la nt ag o co ro no pu s l . pl an ta gi na , ba rb a de l c ap pu cc in o, yo un g le av es b , f bi tte r de co ct io n of ro ot s as a n 8 su bs p. c or on op us ce ae ba rb a de l f ra te , c or on op o, an tid ot e to v ip er b ite er ba s ae tta , e rb a st el la 6 9 p la nt ag o la nc eo la ta l . pl an ta gi na la nc iu ol a, li ng ua d i c an e, yo un g le av es b , f il ., r bi tte r 9 ce ae pi an ta gg in e m in or e, pl an ta go 7 0 p or tu la ca o le ra ce a l . po rt ul ac ea er ba g ra ss a, p or ca cc hi a, yo un g le av es b , r bi tte r 5 ce ae po rc el la na c om un e 7 1 r an un cu lu s fic ar ia l . r an un cu la ce lid on ia m in or e, e rb a fa va , bu lb s, fl ow er s, b , r , o th . bi tte r le av es c he w ed to c le an 8 ce ae fic ar ia , r an un co lo fl av ag el lo y ou ng le av es te et h an d re fr es h th e m ou th 7 2 r ap ha nu s ra ph an is tr um b ra ss ic ac ea e ra fa no , r am ol ac ci o le av es , r oo ts , s te m s b , f il ., r ta st y in cr ea se s bi le s ec re tio n 6 l . s .l. se lv at ic o, ra pa st el lo , ra va ne llo s el va tic o 7 3 r ei ch ar di a pi cr oi de s (l .) a st er ac ea e ca cc ia le pr e, c re pa te rr a, flo w er s, le av es , r oo ts b , r , o th . bi tte r 40 r ot h sc ac ci al ep re , g ra tta lin gu a 7 4 r ha ga di ol us s te lla tu s (l .) a st er ac ea e er ba c or ne tta , r ad ic ch io yo un g le av es b , r sw ee t 5 g ae rt n. st el la to , r ag ag gi ol o, ra gg io lo 7 5 r ub us u lm ifo liu s sc ho tt. r os ac ea e ro go , r ov o co m un e yo un g sh oo ts b , f bi tte r 5 7 6 r um ex a ce to sa l . po ly go na ce ae a ce to sa , e rb a br us ca , yo un g le av es f, f il ., v s bi tte r 5 su bs p. a ce to sa ro m ic e ac et os a 7 7 r um ex a ce to se lla l . po ly go na ce ae a ce to se lla , yo un g le av es b , f il ., r , bi tte r 2 ro m ic e ac et os el la v s 7 8 r um ex c ri sp us l . po ly go na ce ae r om ic e cr es po le av es b , f il ., v s bi tte r 6 7 9 r us cu s ac ul ea tu s l . l ili ac ea e pi cc as or ci , p un gi to po , tu ri on es b , f , r bi tte r 1 ru sc ol o 8 0 sa lv ia p ra te ns is l . s ub sp . l am ia ce ae ch ia re lla , s al vi a de i p ra ti, le av es b , f , v s bi tte r 3 pr at en si s sa lv ia p ra te ns e 8 1 sa ng ui so rb a m in or s co p. r os ac ea e bi bi ne lla , b ip in el la , le av es , y ou ng s ho ot s r , v s ta st y 20 m el on ce llo , p im pi ne lla , sa lv as tr el la m in or e, ve llu tin o ro ss o 8 2 sc an di x pe ct en -v en er is l . a pi ac ea e ac ic ul a m in or e, e rb a le av es , y ou ng s te m s r bi tte r 5 sp ill et ta 8 3 si le ne v ul ga ri s (m oe nc h) c ar yo ph yl la bu bb ol in i, co nc ig li, e rb a le av es , y ou ng s ho ot s f, r is ., r , sw ee t, ta st y flo w er s pr es se d on th e sk in 6 g ar ck e ce ae de l c uc co , s tr ig ol i, st ri co li v s to m ak e sm al l ‘ ex pl os io ns ’ 8 4 si na pi s al ba l . b ra ss ic ac ea e se na pe b ia nc a se ed s, y ou ng le av es b , o th ., v s ta st y 4 8 5 si na pi s ar ve ns is l . s ub sp . b ra ss ic ac ea e se na pe s el va tic a st em s, y ou ng le av es b , v s, f il . ta st y oi l e xt ra ct ed fr om th e 3 ar ve ns is se ed s us ed in la m ps 254 a. ranfa, m. bodesmo 8 6 sm ila x as pe ra l . l ili ac ea e ed er a sp in os a, s al sa le av es , y ou ng s ho ot s b , f , r , o th . bi tte r 3 pa es an a, s al as ap ar ig lia , st ra cc ia br ag he 8 7 so nc hu s as pe r (l .) h ill a st er ac ea e cr es pi gn o sp in os o, le av es , r oo ts , y ou ng s ho ot s b , f il ., r , sw ee t in se ct b ite s or a s a re m ed y 15 cr es pi gn ol a o th ., v s fo r m ou th u lc er s 8 8 so nc hu s ol er ac eu s l . a st er ac ea e gr us pi gn o, c ru sp in o, le av es , r oo ts , y ou ng s ho ot s b , f il ., r , sw ee t ro ot ro as te d as a c of fe e 25 cr is pi gn o, c ru sp ig no , o th ., v s su rr og at e gr es pi gn o, c re sp ig no 8 9 st el la ri a m ed ia (l .) v ill . c ar yo ph yl la ce nt oc ch io c om un e, le av es , y ou ng s ho ot s b , f , r sw ee t 5 ce ae ce nt oc ch io , e rb a ga lli ne lla 9 0 su lla c or on ar ia (l .) m ed ik . fa ba ce ae gu ar da ru bi o, s ul la c om un e flo w er s, s te m s, b , f , r sw ee t as tr in ge nt , r ed uc es b lo od 3 yo un g le av es ch ol es te ro l 9 1 ta ra xa cu m o ffi ci na le a st er ac ea e de nt e di le on e, p is ci ac an e, flo w er s, le av es , r oo ts b , r , f il ., bi tte r ro ot ro as te d as a s ur ro ga te 20 (g ro up ) pi sc ia lle tto , r ad ic ch io d ei v s, o th . fo r c of fe e pr at i, so ffi on e, v ol ar in a 9 2 th ym us s er py llu m s .l. l am ia ce ae pe po lin o, s er pi llo , le av es r , v s, o th . ta st y 6 se rp ol lin o, ti m o se rp ill o 9 3 to rd yl iu m a pu lu m l . a pi ac ea e om br el lin i d i p ra to , le av es b , r sw ee t, ta st y in fu si on re du ce s ha ir lo ss 12 om br el lin i p ug lie si , pi m pi ne llo ne , s ap or ite lla 9 4 tr ag op og on p ra te ns is l . a st er ac ea e ba ci ap re ti, b ar ba d i b ec co ro ot s, s te m s, y ou ng le av es b , f , r , o th . bi tte r cl ea ns es th e bo dy , c al m s w at er d is til le d fr om th e 4 co m un e, b ar ba d i p re te , co ug hs , r oo t c on ta in s pl an t u se d to ‘d ry c le an ’ sa ls efi ca in su lin , i s su ita bl e fo r le at he r di ab et ic s 9 5 tr ag op og on p or ri fo liu s l . a st er ac ea e ba rb a di b ec co v io le tta , ro ot s, y ou ng le av es b , r bi tte r 2 ra pe ro nz ol o se lv at ic o, sa ls efi ca 9 6 tu ss ila go fa rf ar a l . a st er ac ea e fa rf ar ac ci o, fa rf ug io , p iè le av es b , r bi tte r us ed to c al m c ou gh s 2 d’ as in o, u gn a d’ as in o 9 7 u ro sp er m um d al ec ha m pi i a st er ac ea e am ar ag o, c ic or ia m at ta , flo w er s, le av es , r oo ts b , f il ., r bi tte r bu ds u se d in th e sa m e w ay 15 (l .) f .w . s ch m id t co te ca cc hi a, g ru gn o, as c ap er s gr ug no le , l at tu ga cc io 9 8 u rt ic a di oi ca l . s ub sp . u rt ic ac ea e or tic a, u rt ic a le av es , y ou ng s ho ot s b , f , f il ., sw ee t ru n ha nd s th ro ug h ha ir 6 di oi ca r is . be fo re to uc hi ng th e pl an t to a vo id p ri ck s an d st in gs 9 9 ve ro ni ca b ec ca bu ng a l . sc ro ph ul ar ia er ba g ra ss a, yo un g le av es , y ou ng s ho ot s r bi tte r 6 ce ae ve ro ni ca b ec ca bu ng a 1 00 vi ol a od or at a l . v io la ce ae vi ol a m am m ol a flo w er s r , o th . sw ee t 4 to ta l 88 1 *a bb re vi at io n fo od u se s: a b : a lc oh ol ic b ev er ag es ; b : b oi le d ; f : f ri ed in fa t, w ith ou t o r w ith b ea te n eg gs (“ f ri tta ta ”) ; f il . : r av io li fil lin g or s av ou ry p ie fi lli ng ; o th : o th er (u se d as m us ta rd , p ep pe r, se ed in br ea d an d co ok ie s, p ic kl ed o r in o il, r oo t r oa st ed a s a co ffe e su bs tit ut e, d ri ed , i nf us io n) ; r is : r is ot to ; r : r aw ; v s: v eg et ab le s ou ps . ethnobotanical knowledge in central italy 255 chovies and vinegar and then tossed by hand to mix the condiments well. risotto with ‘strigoli’ (silene vulgaris) is also popular, while ‘vitabbia’ (clematis vitalba), ‘asparago’ (asparagus acutifolius) and ‘pungitopo’ or ‘piccasorci’ (ruscus aculeatus) are used in omelettes. another local traditional dish is a flatbread cooked on a hot stone slab (torta al testo), then split open and filled with cooked greens (called ‘erba cotta’) made up of a mixture of several species, including ‘crespigno’ (sonchus spp.), ‘cicoria’ (cichorium inthybus) and ‘grugno’ (urospermum dalechampii). folk medicinal uses twenty-seven plant species were also mentioned as having therapeutic effects (see tab. 1). achillea millefolium, bellis perennis and calendula arvensis leaves were cited as being used to heal cuts; calendula arvensis leaves are particularly effective in the treatment of bedsores, while chondrilla juncea is used to treat ulcers, acne and eczema. alliaria petiolata leaves are used to soothe inflammation of the gums and mouth, bellis perennis leaves are chewed to refresh the mouth and an infusion of cichorium intybus acts as a powerful laxative and diuretic. fresh allium ursinum leaves are applied to the skin as a rubefacient or pounded and used as a cataplasm to soothe abscesses and boils. an infusion of arctium lappa was reported to favor hair regrowth. ballota nigra leaves are used for footbaths, campanula rapunculus leaves soothe inflammation of the oral cavity and reduce warts, while an infusion of the flowers is used as a gargle. infusions of malva sylvestris were indicated for the relief of heartburn and indigestion. daucus carota is used to treat bronchitis in horses, while the infusion acts as a strong diuretic. an infusion of echium vulgare seeds stimulate milk production in lactating women, while an infusion of foeniculum vulgare seeds calm hiccups and aid digestion. lactuca spp. were indicated as having sedative properties, indeed in the past the latex was extracted (in particular from lactuca virosa) and made into small balls to make ‘lattucario,’ similar to chewing gum, which was then given to hyperactive children. sliced lepidium latifolium bulbs are applied to the temples to relieve headache. infusions of nasturtium officinale, mentha pulegium, petasites hybridus and tussilago farfara are used to calm coughs and origanum majorana is used to alleviate earache. a decoction of plantago coronopus roots is used as an antidote for viper bites. ranunculus ficaria leaves are chewed to clean teeth and refresh the mouth, and sonchus asper is employed against insect bites or as a remedy for mouth ulcers. tordylium apulum and urtica dioica infusions reduce hair loss and brighten natural hair color. calamintha nepeta leaves are rubbed on insect bites. borago officinalis flowers macerated in wine for a week produce an excellent purifying and diuretic beverage. other uses various other uses were also reported. small cotton bags containing achillea millefolium seeds are put into wine barrels to improve wine preservation. alliaria petiolata seeds are used to make mustard, while lepidium draba seeds are used like pepper. an infusion of bellis perennis leaves produces a drink similar to tea, and the leaves left to macerate in vinegar give it a bluish tinge. the flowers are also added to ice cubes. dried calendula arvensis petals are used to aromatize wine, which, after having been left in the sun for ten days, becomes excellent vinegar. in the past large pieces of fresh clematis vitalba vine were dried and then smoked like cigarettes. parietaria officinalis leaves were used to wash glass bottles. some ludic uses were cited: bellis perennis flowers were used to play ‘he loves me, he loves me not’, and children used papaver rhoeas seed pods as stamps. silene vulgaris flowers were squeezed to ‘explode’ on the skin like tiny bombs. tab. 2: ewps studied: percentages of parts used, food use categories and taste perception. part(s) used leaves (49%), shoots (17%), flowers and inflorescences (10%), stems (7%), roots (6%), seeds (4%) and bulbs (3%) food use boiled (31%), raw (28%), vegetable soups (12%), fried categories without or with eggs (11%), filling (ravioli or savoury pie) (6%), risotto (2%), alcoholic beverage (1%), other (9%) taste bitter (48%), tasty (27%), sweet (25%) perception discussion the comparison of the present study with studies other researches carried out in the mediterranean area shows that the majority of the ewps used for human consumption belong to the asteraceae family as they are considered to be particularly appetizing and above all widely-known (forbes, 1976; della et al., 2006; dogan, 2012). as other studies (ghirardini et al., 2007) have also shown, the data demonstrate that collecting and consuming ewps is still an important local activity. moreover, many species are also known and collected for their medicinal properties (e.g. guarrera et al., 2005; passalacqua et al., 2007; guarrera and savo, 2013). in central italy in particular ethnobotanical knowledge is very much alive (guarrera and leporatti, 2007), while nutraceutical properties have been studied extensively (ranfa et al., 2014, 2015; maurizi et al., 2015). with reference to the informants, there was a greater number of women, who provided more details than the male informants, probably because collecting and cooking wild plants is almost exclusively a female occupation, and it is them who possess the greatest knowledge of ewps, in agreement with other studies (forbes, 1976; ranfa et al., 2014; sansanelli and tassoni, 2014). some studies have shown a greater male presence, but this can be explained by the fact that the questionnaires were distributed in places such as cafes and social clubs where mainly men gather. access to private homes, where women tend to spend the whole day, is usually more difficult, as the women themselves are diffident and shy of strangers (forbes, 1976). the data collected confirm that species such as cichorium intybus, sonchus asper and borago officinalis are among the most widely known, as has also been reported in other studies (sansanelli and tassoni, 2014). the parts most frequently used are the leaves (49%), see for example sansanelli and tassoni (2014). in reference to food uses, the species are most commonly consumed boiled or eaten raw (ranfa et al., 2014; bodesmo et al., 2015). the raw consumption of these species is predominant in some parts of the mediterranean area such as in the turkish aegean region and in greece, where they are seasoned with olive oil or yoghurt (della et al., 2006; dogan, 2012). regarding taste perception, 48% of the ewps were indicated as having a bitter taste (see tab. 2), the most bitter ones were urospermum dalechampii, clematis vitalba, cichorium intybus, crepis spp. and taraxacum officinale, as also indicated by sansanelli and tassoni (2014). ghirardini et al. (2014) state that taraxacum officinale (dandelion) also known as ‘pisciacane’ or ‘piscialetto’, is one of the most bitter species, and its flavor is particularly appreciated in central and southern italy. the data obtained in this research show that not only food, but also 256 a. ranfa, m. bodesmo folk medicinal uses, are widespread, already shown by other studies carried out in central italy, in latium and the abruzzo region in particular (guarrera et al., 2005). some species listed in tab. 1 have also been recorded in other studies, for example bellis perennis and calendula arvensis leaves used to heal cuts, confirmed by other researchers (passalacqua et al., 2007), cichorium intybus used as a powerful laxative and diuretic, as reported in other studies (lentini and raimondo, 1990; passalacqua et al., 2007). infusions of malva sylvestris were indicated for the relief of heartburn and indigestion. use in gastro intestinal disorders has been reported by other authors (dogan and ugulu, 2013; leto et al., 2013). daucus carota is used to treat bronchitis in horses, while the infusion acts as a strong diuretic (leto et al., 2013). other studies have mentioned the hypnotic properties of lactuca spp. (yakoot et al., 2011; guarrera and savo, 2013). urtica dioica was often indicated as one of the most widely-known species in central italy for the treatment of various ailments, with herpes zoster in first position (uncini manganelli et al., 2005). calamintha nepeta leaves are rubbed on insect bites (passalacqua et al., 2007). borago officinalis flowers macerated in wine for a week produced an excellent purifying and diuretic beverage (leto et al., 2013). with reference to the rfc, the most frequently cited and most frequently used species are borago officinalis, reichardia picroides (rfc=0.05), clematis vitalba (rfc=0.04), asparagus acutifolius, bellis perennis, campanula rapunculus, cichorium inthybus and sonchus oleraceus (rfc=0.03). it has also been demonstrated that borago officinalis is one of the most frequently cited and used species in both the southern and the northern italian sites (ghirardini et al., 2007). on the basis of the rii calculation, borago officinalis and scandix pecten-veneris (rii=0.32), cichorium inthybus (rii=0.30), and sonchus oleraceus (rii=0.29) are the species with the greatest number of food uses, and therefore the most versatile. the two indices calculated by the pearson coefficient correlation (0.51) showed a positive correlation, thus confirming that the most frequently cited species corresponded to those having the greatest number of food uses. details are shown in tab. 3. conclusion the study shows that in central italy, and in the umbria region in particular, knowledge and use of ewps is still very much alive, not only in food use, but also for medicinal and ludic purposes. however this knowledge is principally in the hands of the elderly who rarely, and with great difficulty, manage to transmit it to the younger generations due to lack of interest on their part, as was pointed out by more than one informant. as the fao recognizes, nutrition and biodiversity converge towards a common objective of making uncontaminated food available within a policy of sustainable development, and in this context wild species play a key role in safe global nutrition (fao, 2009). the economic aspect must not be underestimated, as ewps, which are sold mainly in local markets, are included in the diet of a billion people worldwide. regulated markets do not exist, so despite their nutrition value, ewps are excluded from official statistics on economic values of natural resources although in many countries they represent an important supplement to income. furthermore a renewed interest in these species would stimulate the study of local flora and also contribute to disseminating knowledge, thus encouraging the conservation of local customs and traditions as well as deepening the understanding of local communities’ attitudes towards, and management of, their own resources, particularly the use of plants as food or medicine. this would permit these same communities to continue to draw sustainable benefit from their local ecosystems. moreover the importance of biodiversity conservation is also a central issue in pope francis’ encyclical letter ‘laudato si’ on care for our common home, which says: “…but a sober look at our world shows that the degree of human intervention, often in the service of business interests and consumerism, is actually making our earth less rich and beautiful, ever more limited and grey, even as technological advances and consumer goods continue to abound limitlessly. we seem to think that we can substitute an irreplaceable and irretrievable beauty with something which we have created ourselves…. ” (pope francis, 2015). tab. 3: synthesis of the most relevant data most bitter species cichorium intybus, clematis vitalba, crepis spp., taraxacum officinale, urospermum dalechampii most frequently cited and asparagus acutifolius, bellis perennis, borago officinalis, frequently used species campanula rapunculus, cichorium inthybus, clematis vitalba, reichardia picroides, sonchus oleraceus species with the greatest borago officinalis, cichorium inthybus, number of food uses scandix pecten-veneris, sonchus oleraceus bellis perennis, calendula arvensis used to heal cuts borago officinalis flowers macerated in wine for a week produced an excellent purifying and diuretic beverage calamintha nepeta leaves rubbed on insect bites cichorium intybus a powerful laxative and diuretic daucus carota used to treat bronchitis in horses, while the infusion acts as a strong diuretic malva sylvestris indicated for the relief of heartburn and indigestion urtica dioica often indicated as one of the most widely-known species in central italy for the treatment of various ailments, with herpes zoster in first position species with most folk medicinal uses http://www.sciencedirect.com/science/article/pii/s0378874104004453 ethnobotanical knowledge in central italy 257 authors’ contributions mb wrote the manuscript and processed statistical data and analysis. mb and ar collected ethnobotanical data, conducted interviews and wrote up the results. both authors read and approved the final manuscript. acknowledgements the authors would like to thank the fondazione cassa di risparmio of perugia for financial support for the study. we wish to thank the perugia mycological and field naturalists association (circolo micologico naturalistico perugino) and the spello municipality for having collaborated in collecting ethnobotanical data and for organizing themed ewp events. references alexiades, m.n., 1996: collecting ethnobotanical data: an introduction to basic concepts and techniques. in: alexiades, m.n. 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[internet]. may 24, 2015. available from: http://www.papalencyclicals.net/franc/index.htm. address of author: dr. aldo ranfa, dr. mara bodesmo, department of civil and envirnomental engineering, environment laboratory university of perugia, borgo xx giugno, 06100 perugia e-mail: bodesmo@gmail.com e-mail: aldo.ranfa@unipg.it © the author(s) 2017. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). http://dx.doi.org/10.5073/jabfq.2015.088.036 http://www.umbriageo.regione.umbria.it/ http://www.umbriageo.regione.umbria.it/ mailto:bodesmo@gmail.com mailto:aldo.ranfa@unipg.it journal of applied botany and food quality 91, 271 280 (2018), doi:10.5073/jabfq.2018.091.035 1department of plant physiology and biochemistry, west pomeranian university of technology in szczecin, szczecin, poland 2department of horticulture, west pomeranian university of technology in szczecin, szczecin, poland 3department of fruit and vegetable processing, wroclaw university of environmental and life science, wrocław, poland the influence of effective microorganisms and number of buds per cane in viticulture on chemical composition in fruits alicja auriga1*, ireneusz ochmian2, jacek wróbel1, jan oszmiański3 (submitted: april 6, 2018; accepted: september 13, 2018) * corresponding author summary as a result of climate warming, wine-growing zones have moved to the north, where conditions exist may result in poor fruit quality. fruits may develop significant amounts of tannin compounds, which are not acceptable to all consumers. the aim of this study was to demonstrate the influence of selected factors on the quality and content of polyphenols in grapevine fruits. the differentiating factors were as follows: two grapevine cultivars, varied number of buds per cane, and treatment with effective microorganisms (em). to determine the total content of polyphenols and individual polyphenolic compounds in the tested fruits, the uplc-pda-ms method was used. the results indicated that the studied factors had no effect on total soluble solids and titratable acidity in grapes. the experiment revealed that polyphenol content was most dependent on the cultivar, followed by the number of buds per cane; em treatment had the least effect. the fruit of the ‘regent’ cultivar was characterised by higher polyphenol content. ‘cabernet cortis’ berries had higher levels of phenolic acids and flavan-3-ols, while ‘regent’ berries were higher in anthocyanins and flavonols. em treatment had a large impact on the reduction of tannic acid compounds. fruits from untreated plants with four buds per cane had a significantly increased content of polyphenols, including flavan-3-ols. key words: polyphenol content, grapevine, effective microorga nisms, buds per cane, tannin introduction extensive chemisation of agricultural fields has resulted in progressive environmental pollution, and the agents applied have exerted negative effects on the human body. therefore, natural cultivation methods and preparations are being sought to ensure both bountiful and high-quality harvests (javaid and bajwa, 2011). effective microorganisms (em) are biological prepara tions that contain selected, naturally occurring microbes, such as lactobacillus casei, rhodopseudomonas palustris, saccharomyces albus, streptomyces albus, and aspergillus oryzae (higa, 1989; hu and qi, 2013). the microorganisms in the preparations are capable of high levels of antioxidant production, and as such, they naturally aid the plant protection system. the em technology was developed by teruo higa in the 1980s. the concept of using microbiological preparations in agriculture and environmental protection is thought to be an environmentally friendly alternative to commonly applied chemical agents (higa, 1989). grapevine pruning and shrub training with a specific number of buds is the basic ampelotechnical treatment aimed at yielding highquality fruits in vineyards (brighenti et al., 2017). pruning creates favourable conditions for the setting of properly sized fruits that are the right colour and have appropriate solid content. in addition, the procedure prevents damage that can be caused by shrubs becoming overloaded with too many fruits (senthilkumar and vijayakumar, 2015). polyphenols are one of the most frequently studied groups of biologically active compounds due to their health-promoting pro perties. at a time when cancer and cardiovascular disease are in creasingly on the rise, polyphenols are a natural adjuvant in the treatment, as well as prevention, of such diseases. the biological activity and medicinal properties of polyphenols are associated with the diversity of their structures. the compounds classified as polyphenols demonstrate high antioxidant, anti-inflammatory, antibiotic, and anticarcinogenic activity (mijowska et al., 2016). the most frequently mentioned sources of this valuable group of compounds include red grapes and their processed products, primarily wines (mijowska et al., 2017a). according to the inter national organisation of vine and wine’s 2017 statistical report on world vitiviniculture, the harvested output is approximately 77 million tons a year worldwide, of which 36% is consumed as fresh fruit and 47% is dedicated to wine production. the polyphenol content in individual parts of the grapevine fruit is variable; the highest value is in the seeds at 60%-70%, with 28%35% in the skin and approximately 10% in the fruit pulp (godjevac et al., 2010). the structures of phenolic compounds in grapes have a significant effect on the formation of flavour and the aromatic properties of wines, as well as their colour, whereas anthocyanins play the most important role in the colour of grapes and wines (he and giusti, 2010). the composition of the polyphenolic profile in grapes depends on many factors, such as the vine cultivar, degree of fruit ripeness, climatic and soil conditions, and physiological state of the plant. interactions among the aforementioned agents make it difficult to isolate a specific effect of one agent on polyphenol content. therefore, scientists and winemakers are unceasingly conducting research to investigate these factors (pantelić et al., 2016). from the consumer’s point of view, a higher content of polyphenols is beneficial for health reasons. however, due to the wines taste properties, it would be advisable to obtain fruit with a reduced flavan-3-ol content. the aim of this experiment was to examine the effect of em and the number of buds per cane on the quality of fruit grown in north-western poland, including the content and profile of polyphenols. the study also investigated the relationships between the studied factors, which of the cultivation methods most effectively reduced the content of tannic compounds. material and methods location the analysed material came from a non-irrigated five-year-old vineyard at the research station of west pomeranian university of technology in szczecin. the site is in the north-western part of poland called the szczecin lowland, approximately 90 km from the baltic sea. in this area, there are numerous hills the remnants of 272 a. auriga, i. ochmian, j. wróbel, j. oszmiański the frontal moraine that are 20-60 m above sea level. these hills affect the distribution and intensity of rainfall, number of sunlight hours, temperature, and wind speed. the climate is also significantly affected by the presence of large water basins (szczecin lagoon, dąbie lake, and the odra river), which provide additional moisture during the vegetation period. the majority of the west pomeranian province belongs to zone 7a on heinz and schreiber’s ‘map of zones of plant resistance to frost’. however, in szczecin and in the nearby northern region, minimum temperatures range from -12 °c to -15 °c, which correspond to values typical of zone 7b (mijowska et al., 2016). tab. 1 indicates changes in the weather for the years 2014 and 2015, as well as significant deviations from the average growing season during the multi-year period from 1951 to 2012. during the 2014 and 2015 growing seasons, average temperatures and sun hours were similar. unusually low precipitation was observed during the 2015 growing season (242 mm), compared to the years 1951-2012 and 2014 (391 and 448 mm, respectively). the period from april to july 2015 was characterised by lower average temperatures and less sun hours relative to the same period in 2014. the largest weather deviation in 2015 took place in august, when rainfall was extremely low (14.7 mm) compared to the average rainfall for the growing season during the multi-year period and in 2014 (74.2 and 104.6 mm, res pectively) (mijowska et al., 2016). the orchard contained an agricultural soil with a natural profile, developed from silt loam with an unusually low density of 1.12 mg m-3, a ph of 6.7, and a high water capacity of 31.3% (w w-1). it also contained a high level of organic matter (29.2 g kg-1 soil). regardless of the site, the soils were characterised by similar low salinity (ec 0.37 ms cm-1). in comparison to optimal soil mineral content as determined by sadowski et al. (1990), the soil in which the plants were grown, regardless of the stand, was characterised by high levels of phosphorus at 84 mg kg-1 (optimum 20-40 mg kg-1), potassium at 93 mg kg-1 (optimum 50-80 mg kg-1), and magnesium at 44 mg kg-1 (optimum 25-40 mg kg-1). every spring, nitrogen fertilisation was applied at a dose of 60 kg n per hectare, and a calcium fertiliser was applied at a dose of 80 kg ca per hectare. grape samples two red grapevine cultivars, ‘regent’ and ‘cabernet cortis’, were included in this research. the vines were planted with a north-south row orientation at 1 × 2.6 m and pruned with a guyot (one-cane) training system. during cultivation, plants were treated with em1, a beneficial microorganisms and molasses solution prepared according to the manufacturer’s instructions. em1 stock solution was diluted 1:1,000 (em:water) and applied in spray form every second week, starting at the appearance of leaf buds and continuing until harvest. additionally, the cultivars were grown with either four or eight buds per cane. the berry samples were collected in 2014 and 2015 at technological maturity. sampling was performed by picking grape berries randomly distributed throughout both cultivars. each sample consisted of three replications each of 100 randomly selected berries on both sides of the canopy and from different parts of the clusters. the collected samples were immediately frozen at -25 °c until analysis. physicochemical parameters total soluble solid (tss) content of grapes was determined as degrees brix (°bx) using a pal-1 (atago, tokyo, japan) refractometer. titratable acidity (ta) was determined by titration of a water extract of juice with 0.1 n naoh to an end point of ph 8.1. extraction procedure grape berries were prepared according to the method described in oszmiański et al. (2013). the thawed berries were separated with methanol acidified with 2.0% formic acid. the separation was conducted two times by incubating the samples for 20 min under sonication (sonic-6d, polsonic, warsaw, poland), followed by shaking every 4 min. subsequently, the suspension was centrifuged at 19,000 g for 10 min. prior to the analysis, the supernatant was additionally purified with a 0.20 μm hydrophilic ptfe membrane (millex samplicity filter, merck). the polyphenol content in each extract was determined by means of the ultraperformance liquid chromatography-photodiode array detector-mass spectrometry (uplc-pda-ms) method. all separa tions were conducted three times. identification of phenolic compounds by the uplc-pda-ms method analyses were performed according to the method described by mijowska et al. (2016). in fruit extracts for both cultivars, polyphenols were identified using an acquity uplc system appointed with a binary solvent manager, a pda detector (waters corporation, milford, ma, usa) and a g2 q-t of micro mass spectrometer (waters corporation, manchester, uk) equipped with an electrospray ionisation source operating in the negative and positive modes. individual polyphenols were separated using a uplc beh c18 tab. 1: weather conditions during the vegetative season (april-october) in the years 2014-2015 with reference to the average growing season during the multiyear period 1951-2012. month iv v vi vii viii ix x year average temperature (°c) mean 2014 10.8 13.4 16.3 21.3 17.5 15.4 11.8 15.2 2015 8.7 12.5 15.6 18.6 21.1 14.1 13.7 14.9 1951-2012 8.0 13.0 16.4 18.2 17.6 13.8 9.2 13.7 rainfall (mm) total 2014 47.5 85.3 26.5 70.8 104.6 80.9 32.8 448 2015 29.0 48.0 32.8 62.0 14.7 34.4 22.1 242 1951-2012 39.7 62.9 48.2 69.6 74.2 58.7 37.3 391 sun hours total 2014 210 213 189 224 123 133 99 1191 2015 136 161 159 197 278 126 131 1188 effect of em and pruning on grape composition 273 column (1.7 μm, 2.1 mm × 100 mm, waters corporation, milford, ma, usa). based on the data obtained, software was developed to scan multiple samples for defined substances. the various data analyses were monitored at the following wavelengths: flavan-3-ols at 280 nm, phenolic acids at 320 nm, flavonol glycosides at 360 nm, and anthocyanins at 520 nm. the pda spectra were measured over a wavelength range of 200 600 nm in 2 nm increments. finally, the retention times and spectra were compared with authenticated standards. statistical analysis analysis of variance (anova) was carried out in order to estimate the influence of three different factors (cultivar, number of buds, and em) on the physicochemical attributes of grapes. this method also allowed the statistical significance of the physical effect of particular factors, measured as contribution percentage, to be calculated (davim and reis, 2003). mean comparisons were performed using tukey’s least significant difference (lsd) test with significance set at p < 0.05. the statistical analyses were performed using the statistica 12.5 software. results and discussion total soluble solids (tss) and titratable acidity (ta) statistical analysis demonstrated that the performed treatments (i.e., the number of buds per cane and em treatment) had no signi ficant effect on tss (tab. 2). the average tss content in the fruits of both cultivars ranged from 16.85°bx to 17.95°bx, with the ta ranging from 0.525 to 0.640 mg 100 g-1 (tab. 3). tss and ta are very important parameters in evaluating the usefulness of the fruit in processing. in poland’s climatic conditions, fruits of the cultivated vine cultivars contain 18% sugar on average, representing approximately 22% of tss (angelov et al., 2015). leão et al. (2016) concluded that the cultivar selected determines the method of shrub training (i.e., the number of buds per cane). gladstones (1992) demonstrated that fruits that develop on vines with loose crowns feature a higher concentration of sugar in the juice and a better acid balance, unlike fruits that develop in shaded crown conditions. the results obtained in this experiment indicated that increased crown density had no significant effect on tss and ta. polyphenols the identification of 36 compounds categorised as anthocyanins, phenolic acids, flavanols, and flavan-3-ols was based on a com parison of their retention times, ms data, and ms/ms data with available standards and published data (tab. 5-6). the research indicated that the studied factors had a significant effect on the total content of polyphenols in fruits (tab. 4). the most signifi cant factor that differentiated polyphenol content was the cultivar (p = 69.8%), followed by the number of buds (p = 10.8%) and the use of em (p = 7.9%). significant interaction was demonstrated between the ampelotechnical treatments applied for both vine cul tivars. a significantly lower polyphenol content was found in fruits from plants treated with em and trained with eight buds per cane 382.4 mg 100 g−1 of fresh weight (fw) for ‘regent’ and 317.29 mg 100 g−1 fw for ‘cabernet cortis’, as compared to those from plants in the control conditions and rooted with four buds per cane (528.73 mg 100 g−1 fw for ‘regent’ and 382.42 mg 100 g−1 fw for ‘cabernet cortis’) (tab. 5). for vines rooted with four buds, as compared to vines with eight buds, the average total polyphenol content was higher by approximately 10.4% for ‘regent’ and 11.4% for ‘cabernet cortis’. the error value (p = 10.2%) indicates the magnitude of the effect exerted by other untested external factors on the measured parameters (tab. 4). the ‘regent’ cultivar features a significantly higher total content of polyphenols in fruits (472.01 mg 100 g−1 fw), as compared to the ‘cabernet cortis’ cultivar (349.83 mg 100 g−1 fw), regardless of the treatments performed (fig. 1). these values were more than twice as high as those of the fruits of ‘cabernet sauvignon’ (2356 mg kg-1) and ‘tempranillo’ (1489 mg kg-1) cultivated in southern europe (guerrero et al., 2009). the quantity of polyphenols and tab. 2: anova table for total soluble solids and titratable acidity. total soluble solids titratable acidity source of variance a 0.943 0.06 0.581 3.86 b 0.966 0.02 0.937 0.08 c 0.720 1.56 0.937 0.08 a × b 0.579 3.79 0.987 0.00 a × c 0.943 0.06 0.861 0.38 b × c 0.874 0.30 0.715 1.67 a × b × c 0.579 3.79 0.836 0.53 error 90.42 93.40 total 100.00 100.00 a – cultivar; b – number of buds per cane; c – em treatment; p – probability of error, p (%) – percentage of contribution p p (%) p p (%) tab. 3: effect of the performed treatments on the total soluble solids (in °bx) and titratable acidity (in g l-1) in the fruit of the tested vine cultivars. means with same letter were not significantly different by tukey’s comparison at p < 0.05 level. lowercase letters (a) indicate group means; capital letters (a) indicate group averages. ‘regent’ ‘cabernet cortis’ average average c em c em total soluble solids (°bx) 4 16.45 a 17.65 a 17.05 a 18.15 a 17.65 a 17.90 a 8 18.05 a 17.85 a 17.95 a 15.85 a 17.85 a 16.85 a average 17.25 a 17.75 a 17.00 a 17.75 a titratable acidity (g l-1) 4 0.595 a 0.660 a 0.627 a 0.580 a 0.585 a 0.525 a 8 0.645 a 0.620 a 0.632 a 0.600 a 0.580 a 0.590 a average 0.620 a 0.640 a 0.590 a 0.583 a c – control treatment; em – treatment with em; 4,8 – number of buds per cane 274 a. auriga, i. ochmian, j. wróbel, j. oszmiański tab. 4: anova table for polyphenol content. polyphenols anthocyanins phenolic acids flavonols flavan-3-ols source of variance a 0.000 69.82 0.000 87.87 0.000 28.46 0.000 52.79 0.000 28.56 b 0.000 10.79 0.000 3.09 0.394 0.55 0.036 1.89 0.000 41.42 c 0.000 7.85 0.001 2.65 0.002 8.79 0.000 26.22 0.001 8.56 a × b 0.474 0.22 0.116 0.48 0.000 43.20 0.636 0.09 0.110 1.55 a × c 0.122 1.09 0.009 1.47 0.739 0.08 0.000 9.57 0.034 2.85 b × c 0.875 0.01 0.555 0.06 0.295 0.83 0.410 0.27 0.023 3.32 a × b × c 0.898 0.01 0.579 0.06 0.337 0.69 0.849 0.01 0.544 0.21 error 10.20 4.31 17.39 9.16 13.53 total 100.00 100.00 100.00 100.00 100.00 a – cultivar; b – number of buds per cane; c – em treatment; p – probability of error; p (%) – percentage of contribution their composition in fruits depends on the cumulative parts (i.e., peel, flesh, and seeds). this is characteristic for a given cultivar (pantelić et al., 2016). as indicated in this experiment, the better lighting conditions of vines rooted with a smaller number of buds resulted in a higher polyphenol content in the fruits. this corresponds to the findings of other authors (degu et al., 2016; haselgrove et al., 2000). due to the process-related usefulness of the yield, the content of polyphenol compounds is one of the most important parameters of quality in grapes and wines. polyphenols have a direct impact on the organoleptic characteristics of wines, such as taste, sourness, bitterness, and colour (garrido and borges, 2013). they are also strong antioxidants, which are valuable to human health. therefore, the results obtained after treatment with em contradict the expectations of both producers and consumers. the use of em in cultivation caused a significant decrease in total polyphenol content in grapes, regardless of the selected cultivar and type of pruning. from a physiological point of view, this reduction creates favourable conditions for plants. em is a natural protective preparation designed to relieve physiological plant stress caused by various external factors (higa, 1989; talaat, 2014). however, increased levels of antioxidants in plants often indicate initiation of defence processes as a response to the intensified action of free radicals (talaat et al., 2015). anthocyanins anthocyanins were the largest group of polyphenolic compounds analysed in the fruits of both cultivars (tab. 6-7). anthocyanins, classified as a flavonoid subgroup, are natural plant pigments that may appear mainly as violet, dark blue, and red. anthocyanin accumulation in grape skins was significantly higher at 20 °c than p p (%) p p (%) p p(%) p p(%) p p(%) tab. 5: effect of the performed treatments on polyphenol content (in mg 100 g−1 fw) in the fruits of the tested vine cultivars. means with same letter were not significantly different by tukey’s comparison at p < 0.05 level. ‘regent’ ‘cabernet cortis’ average average c em c em polyphenols (mg 100 g−1 fw) 4 528.73 a 469.98 ab 499.35 a 382.42 cd 357.01 cde 369.71 c 8 471.28 ab 418.03 bc 444.66 b 342.57 de 317.29 e 329.93 d average 500.01 a 444.00 b 362.50 c 337.15 c anthocyanins (mg 100 g−1 fw) 4 374.28 a 333.06 bc 353.67 a 206.38 d 207.50 d 206.94 c 8 338.81 ab 297.14 c 317.98 b 198.01 d 184.77 d 191.39 c average 356.55 a 315.10 b 202.20 c 196.14 c phenolic acids (mg 100 g−1 fw) 4 23.13 a 22.18 a 22.66 a 36.37 c 33.14 cd 34.76 c 8 30.83 bcd 26.34 ab 28.59 b 29.02 bc 25.64 ab 27.33 b average 26.98 ab 24.26 a 32.69 c 29.39 bc flavonols (mg 100 g−1 fw) 4 24.33 a 15.62 b 19.98 a 13.73 bc 11.34 c 12.54 b 8 22.06 a 14.60 bc 18.33 a 12.28 bc 10.67 c 11.47 b average 23.20 a 15.11 b 13.00 bc 11.01 c flavan-3-ole (mg 100 g−1 fw) 4 106.99 a 99.11 a 103.05 a 125.94 c 105.02 a 115.48 c 8 79.58 b 79.94 b 79.76 b 103.27 a 96.21 a 99.74 a average 93.28 ab 89.53 a 114.60 c 100.62 b effect of em and pruning on grape composition 275 at 30 °c, and the most temperature-sensitive stage was from one to three weeks after colouring began (yamane et al., 2006). the analysis of variance showed that the total content of anthocyanins was significantly affected by the cultivar, the number of buds per cane, and em treatment, as well as the interaction between the number of buds and em treatment (tab. 4). the cultivar was found to have the highest percentage share (87.9%) of influence. the shares for the other factors (i.e., em and the number of buds) were 2.65% and 3.1%, respectively. as in the case of total polyphenols, the error value (p = 4.3%) was higher than the p values for em treatment and the number of buds; this indicates that untested factors had a greater influence on anthocyanin content. apart from its bioactive properties, anthocyanins are particularly important in the technology of red wine production (mijowska et al., 2017a). for the tested grapes, 19 groups of these compounds were identified. in fruits of the ‘regent’ cultivar, the compounds amounted to more than 70% and, for ‘cabernet cortis’, 53.8% to 58.2% of all polyphenolic compounds (tab. 6-7). the most abundant anthocyanins in the ‘regent’ cultivar fruits were the 3-o-glucoside forms of petunidin, peonidin, delphinidin, malvidin, and cyanidin. these compounds are among the most important anthocyanins in grapes (ivanova et al., 2010). according to other authors, malvidin derivatives are the largest group of anthocyanin compounds in red grape cultivars (figueiredo-gonzález et al., 2012). the total anthocyanin content in the ‘regent’ cultivar fruits was 335.83 mg 100 g−1 fw and, for ‘cabernet cortis’, 199.17 mg 100 g−1 fw (fig. 2). they represented 71.2% and 56.9%, respectively, of the polyphenol content in fruits of the tested vine cultivars. for the ‘regent’ cultivar, the highest quantity of anthocyanins (374.28 mg 100 g−1 fw) was found in the fruits of vines rooted with four buds per cane and in controlled conditions. a significant decrease was observed after em treatment: from 338.81 to 297.14 mg 100 g−1 fw with eight buds and from 374.27 to 333.06 mg 100 g−1 fw with four buds (tab. 6). for ‘cabernet cortis’, the performed treatments produced no significant changes in anthocyanin content (tab. 6). environmental factors have a greater influence on the levels of anthocyanins than on their composition, which is more closely related to the vine cultivar. anthocyanins are the main cause of the red colour of fruits, and they must be extracted from such fruits. they are accumulated primarily in the fruit skin and, in some cultivars, also in the flesh (flamini et al., 2013). this study confirms that anthocyanin composition is characteristic of the cultivar, as determined by the research conducted by other authors. shrubs with different numbers of buds feature different crown microclimates. the type of pruning applied results in changes in the levels of solar radiation, temperature, humidity, and wind (smart, 1987), affecting the composition and quality of grapes. in the completed experiment, thinning out the vine crown had a positive effect on the content of anthocyanins in fruits. similar results were obtained by aselgrove et al. (2000) and mijowska et al. (2016). according to talaat (2014), em preparations support the detoxi fication mechanism in plants exposed to stress. in plants subjected to em, an increased ability to sweep h2o2 in the glutathione-ascorbate cycle was observed. this can effectively attenuate the plants’ defensive reaction to stress. in this research, the cabernet cortis cultivar exhibited similar behaviour after em treatment. however, from the producer’s and consumer’s viewpoints, a lower anthocyanin content in fruits is undesirable. phenolic acids the profile of phenolic acids in the fruits of the studied cultivars was diversified and depended on the agrotechnical treatments performed. phenolic acids represented 5.4% of the total polyphenol content for the ‘regent’ cultivar and 8.9% for the ‘cabernet cortis’ cultivar. the cultivar, em treatment, and the interaction between the cultivar and the number of buds per cane had a significant effect on the content of phenolic acids (tab. 4). the greatest influence was exerted by the interaction between the cultivar and the number of buds (p = 43.2%), followed by the cultivar (p = 28.5%) and treatment with em (p = 8.8%). pruning the ‘regent’ cultivar with eight buds in controlled conditions increased the phenolic acid content in the fruits. in turn, pruning the ‘cabernet cortis’ cultivar with a higher number of buds per cane and treating with em resulted in a significant decrease in the content of phenolic acids (tab. 5-6). the average concentration of phenolic acids in both vine cultivars was determined by the number of buds per cane, not by treatment with em. for the ‘regent’ cultivar with eight buds, significantly higher levels of phenolic acids were found, while a comparable result was obtained for ‘cabernet cortis’ vines with four buds per cane (fig. 3). similar relationships were observed by mijowska et al. (2016), who demonstrated that less shading of the shrub crown significantly decreased the phenolic acid content in the ‘regent’ cultivar. ehrhardt et al. (2014) found that the level of trans-caftaric acid depended on the cultivation site, with 12.70 mg kg-1 fw in ‘regent’ grapes harvested in italy and a much higher value (35.98 mg kg-1 fw) in the colder climate of germany. in the authors’ own research, the results for the ‘regent’ fruits ranged from 18.84 mg 100 g−1 fw (four buds plus em treatment) to 26.32 mg 100 g−1 fw (eight buds). flavonols flavonols are yellow pigments masked by anthocyanins in red wines; however, the pigments affect their colour by co-pigmentation (castillo-muñoz et al., 2010). as a result, the extraction of anthocyanins during wine production is enhanced (castillomuñoz et al., 2007). the greatest biosynthesis of flavonols is observed during the intensive ripening of fruits that occurs postveraison (mattivi et al., 2006). in the fruits of the ‘regent’ and ‘cabernet cortis’ cultivars, flavonols represented 4.1% and 3.4%, respectively, of the total polyphenols detected. the analysis of variance revealed that the performed treatments had a significant impact on the flavonol content in fruits. an interaction was observed between the cultivar and em treatment (tab. 4-5). once again, the most crucial factor in determining the content of flavonols was the vine cultivar (p = 52.8%), followed by treatment with em (p = 26.2%) and the interaction (p = 9.6%). the number of buds per cane (p = 1.9%) had a small but significant fig. 1: effect of the performed treatments on total polyphenol content (in mg 100 g-1 fw) in the fruits of the tested vine cultivars. 276 a. auriga, i. ochmian, j. wróbel, j. oszmiański influence (tab. 4). after em treatment, a significant decrease in flavonol content was found in the ‘regent’ cultivar (tab. 5-7, fig. 4). as in the research by mijowska et al. (2016), this study found that the crown structure (pruning type) did not affect the content of flavonols in that cultivar. in turn, neither treatment procedure had a significant influence on flavonol content for the ‘cabernet cortis’ cultivar (tab. 5). the flavonol group includes, in particular, quercetin and its derivatives, myricetin, kaempferol, and isorhamnetin. the quantity of flavonols largely depends on the vine cultivar (liang et al., 2011). the ‘regent’ fruits were more abundant in quercetin derivatives, including rutin, as compared to the fruits of the ‘cabernet cortis’ cultivar. taking into account the significant decrease in flavonol content in the ‘regent’ fruits after em treatment and the previously mentioned reports by talaat (2014) relating to the detoxifying properties of the preparation, a hypothesis can be constructed that quercetin derivatives have a significant effect on the glutathioneascorbate cycle in the plant. moreover, this may indicate a significant tab. 6: polyphenol content (in mg 100 g−1 fw) of grape cultivar ‘regent’. means with same letter were not significantly different by tukey’s comparison at p < 0.05 level. r8 r4 r8 em r4 em compounds (mg 100 g−1) delphinidin 3.5-diglc 5.39 ab 6.33 b 4.79 a 5.67 ab delphinidin 3-o-glc;petunidin 3.5-diglc 147.08 ab 161.01 b 127.64 a 142.45 ab peonidin 3.5-diglc;malvidin 3.5-diglc;cyanidin 3-o-glc 112.78 ab 136.19 c 97.93 a 117.23 bc petunidin 3-o-glc 21.27 b 20.78 b 13.68 a 11.09 a peonidin 3-o-glc 16.22 b 13.58 a 12.88 a 13.75 a malvidin 3-o-glc 22.25 a 20.53 a 27.94 b 28.74 b delphinidin 3-o-acetyl-glc 0.90 a 1.39 b 0.79 a 1.23 b delphinidin 3-o-caffeoyl-glc 0.71 ab 0.95 c 0.62 a 0.84 bc petunidin 3-o-acetyl-glc 0.51 ab 0.64 c 0.45 a 0.56 bc cyanidin 3-o-caffeoyl-glc 0.11 ab 0.13 b 0.10 a 0.11 ab petunidin 3-o-caffeoyl-glc 0.44 ab 0.57 c 0.39 a 0.51 bc malvidin 3-o-acetyl-glc 0.24 ab 0.30 c 0.21 a 0.27 bc delphinidin 3-o-coumaroyl-glc 3.28 b 3.68 c 2.89 a 3.25 b peonidin 3-o-coumaroyl-glc 0.82 c 0.70 b 0.72 b 0.62 a peonidin 3-o-caffeoyl-glc 0.11 a 0.34 b 0.10 a 0.30 b malvidin 3-o-caffeoyl-glc 1.53 ab 1.82 c 1.35 a 1.61 bc cyanidin 3-o-coumaroyl-glc 1.64 bc 1.72 c 1.44 a 1.52 ab petunidin 3-o-coumaroyl-glc 0.07 ab 0.08 b 0.06 a 0.07 ab malvidin 3-o-coumaroyl-glc 3.47 a 3.52 a 3.19 a 3.23 a anthocyanins sum 338.81 b 374.27 c 297.14 a 333.06 b grp (cisand trans-isomers 3.32 c 2.77 ab 2.92 bc 2.45 a caftaric acid (cisand trans-isomers) 26.32 c 19.35 a 22.37 b 18.84 a coutaric acid (cisand trans-isomers 0.30 b 0.22 a 0.27 b 0.20 a fertaric acid 0.07 a 0.11 b 0.06 a 0.10 b galic acid 0.82 c 0.68 ab 0.72 b 0.60 a phenolic acids sum 30.83 c 23.13 ab 26.34 bc 22.18 a myricetin glucoside 4.50 b 5.18 c 3.95 a 4.58 b myricetin glucuronidec 0.82 b 0.75 b 0.39 a 0.45 a rutin 1.66 a 1.63 a 1.46 a 1.44 a quercetin-3-oglucoside 1.10 ab 1.29 b 0.97 a 1.14 ab quercetin glucuronide 13.90 b 15.38 b 7.76 a 7.92 a quercetin 0.09 ab 0.11 b 0.08 a 0.09 ab flavonols sum 22.06 b 24.33 b 14.60 a 15.62 a dimer b1 1.10 b 0.66 a 2.08 c 0.58 a catechin 3.56 c 2.74 ab 3.13 bc 2.42 a dimer b2 11.26 ab 12.43 b 9.90 a 11.48 ab epicatechin 51.06 a 78.58 b 53.51 a 73.23 b galloylated dimer 4.51 ab 5.12 b 3.97 a 4.52 ab dimer b4 8.08 b 7.46 ab 7.35 ab 6.88 a flavan-3-ols sum 79.58 a 106.99 c 79.94 a 99.11 b total 471.29 b 528.73 c 418.03 a 469.98 b c – control treatment; em – treatment with em; 4,8 – number of buds per cane effect of em and pruning on grape composition 277 tab. 7: polyphenol content (in mg 100 g−1 fw) of grape cultivar ‘cabernet cortis’. means with same letter were not significantly different by tukey’s comparison at p < 0.05 level. cc8 cc4 cc8 em cc4 em compounds (mg 100 g−1) delphinidin 3.5-diglc 4.78 c 3.41 ab 3.65 b 2.97 a delphinidin 3-o-glc;petunidin 3.5-diglc 81.43 ab 94.52 c 75.29 a 89.21 bc peonidin 3.5-diglc;malvidin 3.5-diglc;cyanidin 3-o-glc 49.78 b 50.89 b 46.66 a 56.07 c petunidin 3-o-glc 14.21 c 10.21 a 11.87 ab 12.73 ab peonidin 3-o-glc 5.16 a 7.37 b 5.05 a 9.45 c malvidin 3-o-glc 22.30 b 18.22 a 23.97 b 18.68 a delphinidin 3-o-acetyl-glc 3.90 b 4.61 c 3.21 a 3.82 b delphinidin 3-o-caffeoyl-glc 1.24 a 3.38 b 1.11 a 1.32 a petunidin 3-o-acetyl-glc 1.29 a 1.03 a 1.26 a 1.00 a cyanidin 3-o-caffeoyl-glc 2.27 b 1.96 a 2.02 a 1.96 a petunidin 3-o-caffeoyl-glc 0.14 a 0.19 b 0.14 a 0.15 a malvidin 3-o-acetyl-glc 0.26 ab 0.29 b 0.22 a 0.25 ab delphinidin 3-o-coumaroyl-glc 5.62 c 4.91 ab 5.18 b 4.88 a peonidin 3-o-coumaroyl-glc 0.56 a 0.70 b 0.55 a 0.56 a peonidin 3-o-caffeoyl-glc 0.19 b 0.15 a 0.19 b 0.16 a malvidin 3-o-caffeoyl-glc 0.68 b 0.64 b 0.51 a 0.61 b cyanidin 3-o-coumaroyl-glc 1.15 a 1.07 a 1.12 a 1.02 a petunidin 3-o-coumaroyl-glc 0.11 a 0.18 b 0.11 a 0.10 a malvidin 3-o-coumaroyl-glc 2.92 b 2.67 a 2.66 a 2.58 a anthocyanins sum 198.01 ab 206.38 b 184.77 a 207.50 b grp (cisand trans-isomers 1.95 b 1.74 a 1.92 b 1.71 a caftaric acid (cisand trans-isomers) 26.19 ab 33.82 c 22.85 a 30.67 bc coutaric acid (cisand trans-isomers 0.41 a 0.42 a 0.39 a 0.37 a fertaric acid 0.05 a 0.05 a 0.08 b 0.04 a galic acid 0.42 b 0.34 a 0.41 b 0.35 a phenolic acids sum 29.02 b 36.37 c 25.64 a 33.14 bc myricetin glucoside 4.35 a 5.15 b 5.29 b 3.89 a myricetin glucuronidec 0.91 b 0.82 ab 0.48 a 0.80 a rutin 0.36 a 0.51 c 0.45 bc 0.38 ab quercetin-3-oglucoside 0.52 a 0.55 a 0.50 a 0.48 a quercetin glucuronide 6.04 cd 6.65 d 3.87 a 5.74 bc quercetin 0.10 b 0.05 a 0.09 b 0.05 a flavonols sum 12.28 ab 13.73 b 10.67 a 11.34 ab dimer b1 1.42 b 1.44 b 1.28 a 1.30 a catechin 2.05 b 1.99 ab 1.59 a 1.85 ab dimer b2 11.56 a 13.11 b 10.30 a 11.12 a epicatechin 76.70 b 98.59 c 67.60 a 76.28 b galloylated dimer 7.37 a 9.19 b 9.28 b 8.61 b dimer b4 4.18 b 1.61 a 6.16 c 5.86 c flavan-3-ols sum 103.27 ab 125.94 c 96.21 a 105.02 b total 342.57 b 383.76 c 317.29 a 357.92 b cc – ‘cabernet cortis’; 4,8 – numbers of buds per cane; em – treatment with em. effect of em on quercetin derivatives. this hypothesis could also justify the strong correlation between the cultivar and em treatment. varying the number of buds in the experiment did not have a sig nificant influence on the content of flavonols. different results were obtained by spayd et al. (2002), who showed that well insolated fruits of the ‘merlot’ cultivar contained almost 10 times the total concentration of flavonols as those harvested from shaded clusters. in addition, baiano et al. (2015), feng et al. (2015), and mijowska et al. (2016) presented the beneficial effect of thinning out the vine crown on flavonol content. considering the error value of p = 6.4%, the lack of influence of the number of buds on flavonol content during the experiment may have been caused by other external factors excluded from the research. flavan-3-ols flavan-3-ols are a group of tannin compounds that plentifully occur mainly in red grapes. they are the cause of the ‘structure’ of wine, 278 a. auriga, i. ochmian, j. wróbel, j. oszmiański its astringency, and its bitterness. they also play a very important role in stabilising the red colour of wine (guerrero et al., 2009). during the maceration process, the compounds undergo extraction and pass on to the must. in wine prepared from the ‘regent’ cultivar, flavan-3-ol content was approximately 70.4 mg 100 g−1 (mijowska et al., 2017b). this research found that the total content of flavan-3ols in the ‘regent’ fruits was 91.4 mg 100 g−1 fw, and 107.61 mg 100 g−1 fw for ‘cabernet cortis’. therefore, the compounds from this group represented 19.4% and 30.8%, respectively, of the total polyphenol content detected in the fruits of these vine cultivars. the analysis of variance demonstrated that total flavan-3-ol content was significantly influenced by the cultivar (p = 28.6%), the number of buds (p = 41.4%), em treatment (p = 8.6%), and the interaction between pruning and em treatment (p = 3.3%) as well as between the cultivar and em treatment (p = 2.9%) (tab. 4). in the fruits of both vine cultivars trained with a larger number of buds, the average flavan-3-ol content was significantly lower than the fruits of shrubs trained with fewer buds per cane (fig. 5). em treatment resulted in a significant decrease in flavan-3-ol content in the ‘cabernet cortis’ cultivar. the content of flavan-3-ols in grapes depends on the cultivar; in the individual parts of the fruit, the content is highest in the seeds, followed in descending order by the skin, stick, and flesh (pantelić et al., 2016). reduced shrub crown density contributed to an increase in flavan-3-ol content in fruits, as reported by mijowska et al. (2016) and reynolds and vanden heuvel (2009). furthermore, degu et al. (2016) concluded that light had no effect on the content of flavan-3-ols. as reported by li et al. (2008), flavan-3-ols are recog nised as the most sensitive of all flavonoids to non-enzymatic degradation processes. during fermentation, such compounds (e.g., catechins) may undergo partial splitting into phenolic units with a lower molecular mass. this process is mainly due to a temperature rise (garrido and borges, 2013). a change in the quantity of these compounds was also observed during exposure to uvc radiation (mijowska et al., 2017a). the compounds of this group most fre quently found in grapes are epicatechins, with levels ranging from 51.06 to 78.58 mg 100 g−1 fw for the ‘regent’ cultivar, depending on the agrotechnical treatments applied. for ‘cabernet cortis’ fruits, the content was higher (67.60 to 98.59 mg 100 g−1 fw) (tab. 6-7). the high content of the compound in fruits was confirmed by the research completed by ehrhardt et al. (2014) and mijowska et al. (2017a). daly and stewart (1999) concluded that em treatment can improve fig. 2: effect of the performed treatments on anthocyanin content (in mg 100 g-1 fw) in the fruits of the tested vine cultivars. fig. 3: effect of the performed treatments on phenolic acid content (in mg 100 g-1 fw) in the fruits of the tested vine cultivars. -1 fig. 5: effect of the performed treatments on flavan-3-ol content (in mg 100 g-1 fw) in the fruits of the tested vine cultivars. fig. 4: effect of the performed treatments on flavonol content (in mg 100 g-1 fw) in the fruits of the tested vine cultivars. effect of em and pruning on grape composition 279 growth and yield by increasing photosynthesis and producing bioactive substances, such as hormones and enzymes. therefore, the reduction in flavan-3-ol content in fruits by means of em may be justified, once again, by the antioxidant properties of the preparation. conclusion the experiment demonstrated that the number of buds per cane and the use of em had a significant effect on polyphenol content in the fruits of the wine grape cultivars studied. however, these factors had no significant effect on ta and tss. the polyphenol content in the fruits was determined mainly by the cultivar and, to a lesser extent, the number of buds per cane and the smallest use of em. the fruits of the ‘regent’ cultivar were characterised by a higher polyphenol content compared to those of the ‘cabernet cortis’ cultivar. ‘cabernet cortis’ berries had higher levels of phenolic acids and flavan-3-ols, while those of ‘regent’ had higher levels of anthocyanins and flavonols. pruning plants with four buds per cane increased the content of all polyphenol groups studied, with the exception of phenolic acids in the ‘regent’ cultivar. the use of em reduced polyphenols, especially tannin compounds, in the fruits of both grape cultivars. from the point of view of wine production in a cold climate, this phenomenon is desirable and beneficial. keeping the vines at four buds per cane without using em mostly increased the levels of polyphenols, including the flavan-3-ols. however, leaving eight buds per cane and using em contributed to a significant reduction of these compounds, as well as to a lower concentration of flavan-3-ols. selecting the proper cultivar and pruning the vine shrubs with a smaller number of buds per cane is conducive to obtaining fruits of higher quality and polyphenol content. author’s contribution: aa experimental design, field work and data collection, laboratory work, data analysis, redaction of manuscript; io supervision, experimental design, data analysis; jw supervision, data analysis; jo laboratory work, data collection. conflict of interest disclosure: the authors declare no conflict of interest related to this work. references angelov, l., stalev, b., ochmian, i., mijowska, k., chelpinski, p., 2015: comparison of processing fruit quality of several grape varieties cultivated in climatic conditions of poland and bulgaria. 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(ed.), winetitles: adelaide, 205-211. spayd, s.e., tarara, j.m., mee, d.l., ferguson, j.c., 2002: separation of sunlight and temperature effects on the composition of vitis vinifera cv. merlot berries. am. j. enol. vitic. vol. 53. american society of enologists. retrieved from http://www.ajevonline.org/content/53/3/171 talaat, n.b., 2014: effective microorganisms enhance the scavenging capacity of the ascorbate-glutathione cycle in common bean (phaseolus vulgaris l.) plants grown in salty soils. plant physiol. biochem. 80, 136143. doi: 10.1016/j.plaphy.2014.03.035 talaat, n.b., ghoniem, a.e., abdelhamid, m.t., shawky, b.t., 2015: effective microorganisms improve growth performance. alter nutrients acquisition and induce compatible solutes accumulation in common bean (phaseolus vulgaris l.) plants subjected to salinity stress. plant growth regu. 75, 281-295. doi: 10.1007/s10725-014-9952-6 yamane, t., jeong, s.t., goto-yamamoto, n., koshita, y., kobayashi, s., 2006: effects of temperature on anthocyanin biosynthesis in grape berry skins. am. j. enol. vitic. 57, 54-59. address of the corresponding author: e-mail: alicja.auriga@zut.edu.pl © the author(s) 2018. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 90, 159 164 (2017), doi:10.5073/jabfq.2017.090.020 1 department of horticulture, west pomeranian university of technology szczecin, poland 2 department of fruit, vegetable and plant nutraceutical technology, wrocław university of environmental and life sciences, poland rootstock effects on polyphenol content in grapes of ‘regent’ cultivated under cool climate condition kamila mijowska1*, ireneusz ochmian1, jan oszmiański2 (received december 21, 2016) * corresponding author summary rootstocks are well known as the most efficient way to limit phyl loxera. however, they can be useful in order to improve grape quality. this study aimed to compare the content of polyphenol compounds in vine fruits of the cultivar ‘regent’ grafted on ‘couderc 161-49’, ‘sori’, ‘kober 125aa’, ‘börner’ or ‘kober 5bb’ rootstocks, or planted on own-roots. grape samples were collected in three consecutive seasons (2013-2015) at a research station of the west pomeranian university of technology szczecin in poland. thirty-three phenolic compounds were determined in the juice of examined samples using ultra-pressure liquid chromatography with photodiode array and mass spectrometry (uplc-pda/ms) method. a significant influence of rootstock on the content of polyphenols in grapes has been proven. the highest content of polyphenols was shown in fruits from a scion grafted on ‘sori’ and ‘kober 125aa’ rootstocks (675 and 643 mg · 100 g-1 fw, respectively). ‘börner’ and ‘kober 5bb’ rootstocks did not have a significant influence on the creation of polyphenol compounds in comparison to own-root plants. in addition, the use of the ‘börner’ rootstock resulted in fruits with an especially low content of phenolic acids. introduction it has been reported that there is an inverse association between the consumption of some fruits and vegetables and the mortality from age-related diseases. this can be partially attributed to a diet rich in antioxidants, especially phenolic compounds (dudonne et al., 2009). polyphenols are compounds that occur naturally in large amounts in some food products, including fruits. until now, 8,000 structures of those compounds have been discovered. natural polyphenols are safer and more acceptable by the consumers than synthetic antioxidants like butylated hydroxyanisole (bha) or bu tylated hydroxytoluene (bht), which is why they constitute an important ingredient of our everyday diet (liu et al., 2015). grapes belong to the most frequently eaten fruits in the world, both in their fresh and processed forms. furthermore, they have one of the highest content of phenolic compounds (manach et al., 2005) and a high bioactive potential due to their antioxidant, anti-inflammatory, anticancer and antimicrobial properties (gris et al., 2011). these health benefits have been associated with some groups of polyphenol compounds such as flavonoids, phenolic acids or stilbenes. among flavonoids, the most important ones are: anthocyanins, flavonols, flavones, flavanones (espín et al., 2007; stintzing and carle, 2004), catechins epicatechins, and procyanidins (wang et al., 2002; fuleki and ricardo-da-silva, 2003). the health benefits of polyphenols depend on the amounts consumed and their bioavailability (manach et al., 2004). the grapevine, as a plant, has been cultivated since ancient times; it was used for consumption and religious purposes (censi et al., 2014). in the 19th century, the plantation in europe was almost completely destroyed due to phylloxera (garnett et al., 2001). a solution to the problem was found in using rootstocks of the vitis sp. kind. a rootstock is a root system of a phylloxera resistant grapevine onto which a scion of a chosen cultivar is grafted (ozden et al., 2010). until now, it is still the most efficient way to limit phylloxera (vršič et al., 2015). on the areas “free from” phylloxera, rootstocks are used in order to improve the quality of the harvest and to limit the influence of adverse soil and climate conditions (reynolds and wardle, 2001). however, due to the effects of global warming, the area of pest occurrence has been shifted more to the north. this means that the use of rootstocks may also protect northern plantations against phylloxera in the future. nowadays, the interest in grapevine cultivation in poland is increasing, and new vineyards have been established where new varieties with resistances to major pests, like downy and powdery mildews dominate. grapevine cultivar ‘regent’, which is the subject of this study, was developed in 1967, and it is characterised by the dark red colour of its fruit skin (ehrhardt et al., 2014). the studies focused on the evaluation of the influence of rootstock upon the content of polyphenols in grapes of ‘regent’. materials and methods characteristics of the area of research and plant material the experiment was conducted in three consecutive years (20132015) at a research station of west pomeranian university of technology in szczecin. the research station is located in subzone 7a (heinze and schreiber, 1984) in the north-western part of poland in the szczecin lowland at a distance of approx. 65 km from the baltic sea (53°40’ n, 14°46’ e). the soil in the orchard was an agricultural soil with a natural profile, developed from silt loam (sand 42.7 %, silt 52.9 %, clay 4.4 %) with considerably lower density of 1.25 mg · m-3, ph 6.8-6.9 and higher water capacity of 46.2 [% ww-1]. it also contained much more organic matter – 34.3 g in kg of soil. regardless of the site, the soil was characterised by similarly low salinity – ec 0.33-0.42 ms cm-1. at depth of 20-40 cm it was characterised by a high content of p (72 mg kg-1), k (157 mg kg-1) and mg (47 mg kg-1). in turn, ca content was 455 mg kg-1 for 20-40 cm of depth, 1336 mg kg-1 for 60-80 cm, and 1577 mg kg-1 for 120-140 cm. ground water level was 140-160 cm. the study involved the dark-skinned grapevine cultivar ‘regent’, which is a german cultivar with interspecific hybrids in ancestry. the vines were planted in 2010 with a north-south row orientation at 2.3 × 1 m. ‘regent’ vines grafted onto five rootstocks, viz. ‘couderc 161-49’, ‘sori’, ‘kober 125aa’, ‘börner’ and ‘kober 5bb’ were grown, while own-root ‘regent’ vines served as a control. the vines were pruned with a guyot (one arm) training system and vertically positioned with eight shoots and two clusters each. other standard vineyard management practices, including pest treatment, were performed during all growing seasons. the experimental treatments were arranged in a randomised complete block design. each experimental unit was comprised of 6 vines. fruits were collected at physiological maturity on the first decade of october in successive seasons. 160 k. mijowska, i. ochmian, j. oszmiański reagents and standards formic acid and methanol were purchased from sigma-aldrich (steinheim, germany). acetonitrile was purchased from merck (darmstadt, germany). quercetin-3-o-glucoside, quercetin-3-orutinoside, kaempferol-3-o-glucoside, myricetin-3-o-glucoside, iso rhamnetin-3-o-glucoside, cyanidin-3-o-glucoside, peonidin-3-oglucoside, (-)-epicatechin, (+)-catechin, procyanidins, and gallic acid were purchased from extrasynthese (lyon, france). extraction procedure three replicates of 25 randomly chosen berries were kept frozen at -27 °c until analysis, and then prepared according to the methodo logy of oszmiański et al. (2013). the fruits were extracted with methanol acidified with 2.0 % formic acid. the separation was conducted twice by incubation for 20 min under ultrasonic treatment (sonic 6d, polsonic, warsaw, poland) followed by shaking from time to time (a few times or rarely). subsequently, the suspension was centrifuged mpw-251 (mpw med. instruments, warsaw, poland) at 19,000 × g for 10 min. prior to analysis, the supernatant was additionally purified with a hydrophilic ptfe 0.20 μm mem brane (millex samplicity filter, merck). the polyphenol content in each extract was specified by means of the ultra-performance liquid chromatographyphoto-diode array detector-mass spectrometry (lcpda-ms, waters corporation, milford, ma usa) method. all extractions were carried out in triplicate. identification of phenolic compounds by the uplc-pda/ms method analyses were performed by the methodology oszmiański et al. (2013). in ‘regent’ grapes extracts’ polyphenols identification was executed by using an acquity ultra performance lc system appointed with a binary solvent manager’s, a photodiode array detector (waters corporation, milford, ma) and a g2 q-tof micro mass spectrometer (waters, manchester, uk) equipped with an electrospray ionisation (esi) source operating in following modes: negative and positive. individual polyphenols separations were executed by using a uplc beh c18 column (1.7 μm, 2.1 mm × 100 mm, waters corporation, milford, ma) at 30 °c. the elution of injected samples (10 μl) was fulfilled in 15 min followed by a sequence of linear gradients and isocratic flow rates of 0.45 ml min-1. the mobile phase consisted of solvent a (0.1 % formic acid, v/v) and solvent b (100 % acetonitrile). the examination commenced with and initial isocratic elution of 99 % solvent a (0-1 min), while applying a linear gradient for 12 min uncovered lowering solvent a to 0 %. next, at 12.5 to 13.5 min, the gradient was returned back to the initial composition (99 % a), with the tore-equilibrate column being kept constant. the overall analysis was based on full data-dependant ms scanning with a m/z range from 100 to 2500. the reference compound used for the examination was leucine encephalin, at a concentration of 500 pg/μl and a flow rate of 2 μl/ min. the [m − h]− ion at 554.2615 da and [m + h]+ ion at 556.2771 were detected. the [m − h]−/[m + h]+ ions were recognised during a 15 min analysis performed within esi-ms accurate mass experiments, which were constantly introduced via the lockspray channel using a hamilton pump. the mass spectrometer operated in both negative-and positive-ion modes, adjusted to base peak intensity (bpi) chromatograms, scaled to 12,400 counts per second (cps) (100 %) within a locked mass correction of ± 1.000 for the mass window. the ms conditions were optimised according to the following parameters: a capillary voltage of 2500 v, a cone voltage of 30 v, a source temperature of 100 °c, a desolvation temperature of 300 °c and a desolvation gas (nitrogen) flow rate of 300 l/h. collision-induced fragmentation experiments were performed using argon as the collision gas, with voltage ramping cycles from 0.3 to 2 v. characterisation of each and every single component was conducted via retention time and accurate molecular masses while individual compounds were optimised to their estimated molecular mass in both negative and positive modes, prior to and as a result of fragmentation. afterwards, the data collected from uplc-ms were uploaded to the masslynx 4.1 chromalynx application manager software (masslynx 4.1 scn802, waters corporation, milford, ma usa). quantification was achieved by injection of solutions of known concentrations ranging from 0.05 to 5 mg/ml (r2 ≤ 0.9998) of phenolic compounds as standards. the results were expressed as mg per 100 ml for must. basing on the delivered data the software itself is developed to scan multiple samples for defined substances. the various data analysis runs were monitored at the following wavelengths: flavan-3-ols at 280 nm, phenolic acids at 320 nm, flavonol glycosides at 360 nm and anthocyanins at 520 nm. the pda spectra were measured over the wavelength range of 200600 nm in steps of 2 nm. finally the retention times and spectra were compared with authentic standards. statistical analysis all statistical analyses were performed with statistica 12.5 (statsoft polska, cracow, poland). the data were subjected to one and two factor variance analysis (anova). mean comparisons were performed using tukey’s least significant difference (lsd) test; significance was set at p < 0.05. to determine the relation between the rootstock and phenolic content the results obtained were subjected to an agglomerative cluster analysis and classified into groups in a hierarchical order by means of the ward’s method. results and discussion general identification of polyphenol compounds belonging to anthocyanins, phenolic acids, flavonols and flavan-3-ols was based on a comparison of their retention times, ms and ms/ms data with available standards and published data. the identification results are presented in our previous paper (mijowska et al., 2016). the results obtained in our study, as well as by other authors (jogaiah et al., 2015; koundouras et al., 2009; suriano et al., 2016), showed an important influence of the rootstock on the content of polyphenols in grapes. however, in the study of koundouras et al. (2009), rootstocks affected only seed phenolic concentrations. basic seasonal weather characteristics are shown in tab. 1. in 2015, the temperatures were significantly different from what they typically are in szczecin and its surrounding areas. in may and june of 2015, the temperatures recorded were 0.5 °c and 0.8 °c lower than averages of years 1951-2012, respectively. then in august of 2015, the air temperature was 3.5 °c higher than the mean value of the multi-year period. during the 2015 growing period, the rainfall was 38 % lower than normal, which, together with the high temperature, created highly atypical weather conditions. regardless of the rootstock used, the content of polyphenols was significantly higher in grapes harvested in 2015 (fig. 1). the highest content of polyphenols was shown in grapes with the ‘sori’ and ‘kober 125aa’ rootstocks in all studied years. a cluster analysis conducted using ward’s method (fig. 2) permitted the isolation of a separate group of these two rootstocks with similar influence on polyphenols in the fruits. another group was formed out of the single ‘börner’ rootstock, whose fruits had the lowest content of polyphenols in three years average. additionally, ‘börner’ and ‘kober 5bb’ rootstocks resulted in statistically lower level of polyphenols in grapes or without rootstock effects on grape polyphenols 161 significant differentiation compared to own-rooted plants in all studied years (fig. 1). the average polyphenol compound concentrations of years 20132015 are shown in tab. 2. the sum of polyphenols in grapes of ‘regent’ ranged from 554 to 675 mg · 100 g-1 fw, for ‘börner’ and ‘sori’ respectively. statistically similar content of polyphenols as in the case of ‘sori’ was shown in the fruits of ‘kober 125aa’ rootstock (643 mg · 100 g-1 fw). on the other hand, grapes from ‘kober 5bb’ rootstock and own-root plants showed no significant diversity compared with fruits of ‘börner’ rootstock. according to our results published previously (mijowska and ochmian, 2015), grapes from ‘sori’ and ‘kober 125aa’ rootstocks were also characterised by higher total soluble solids, while fruits from ‘börner’ and ‘kober 5bb’ rootstocks were classified as two with lower concentrations. these parameters could depend on the differences of vigour. refer to other authors findings, ‘sori’ is suggested as the low vigour rootstock (schmidt et al., 2005), while ‘kober 5bb’ and ‘börner’ are served as a medium/high vigour (cousins, 2005; schreiner, 2003). grapevine fruits are a rich source of polyphenolic compounds; however, they contain half of the total amount of compounds as compared to the blue-berried honeysuckle (oszmiański et al., 2016). anthocyanins in the grapes studied, fifteen anthocyanin compounds were identi fied. depending on the type of the rootstock, they constituted 60 64 % of all polyphenols identified. the highest contents of anthocyanins were found in grapes from plants with the ‘sori’ and ‘kober 125aa’ rootstocks (respectively: 423, 400 mg · 100 g-1 fw), and the lowest, in the fruit from own-root plants, as well as with the ‘börner’ and ‘kober 5bb’ rootstocks (respectively: 350, 355, 360 mg · 100 g-1 fw). in turn, suriano et al., 2016, observed the highest levels of anthocyanins in berries of vines ‘greco nero’ grafted onto ‘775 paulsen’ and ‘kober 5bb’. in the study of ehrhardt et al. (2014), the ‘regent’ cultivar fruits cultivated in germany and italy had 120130 mg · 100 g-1 fw level of anthocyanins, respectively. the most frequently found anthocyanin compounds in the fruits of ‘regent’ studied were the 3-o-glucoside forms of petunidin, peonidin, delphinidin, malvidin and cyanidin, in order. those compounds tab. 1: weather conditions during the vegetative season (april-october) in the years 2013-2015 with reference to the average growing season during the multiyear period (1951-2012). month iv v vi vii viii ix x year average temperature (°c) mean 2013 8.4 14.4 16.9 19.3 18.7 13.0 10.9 14.5 2014 10.8 13.4 16.3 21.3 17.5 15.4 11.8 15.2 2015 8.7 12.5 15.6 18.6 21.1 14.1 13.7 14.9 1951-2012 8.0 13.0 16.4 18.2 17.6 13.8 9.2 13.7 rainfall (mm) total 2013 20.8 88.1 112.5 50.4 35.9 43.9 45.8 397 2014 47.5 85.3 26.5 70.8 104.6 80.9 32.8 448 2015 29.0 48.0 32.8 62.0 14.7 34.4 22.1 242 1951-2012 39.7 62.9 48.2 69.6 74.2 58.7 37.3 391 fig. 1: total polyphenol content in mg · 100 g-1 fw of grapes of ‘regent’ in three years studied (2013-2015), as related to rootstocks and ownroot vines. means having same letter were not significantly different by tukey’s comparison at p < 0.05 level. lowercase letters (a) indicate the means of 2013, italic letters (a) of 2014, and underlined letters (a) of 2015. fig. 2: dendrogram of cluster analysis for rootstocks based on average for phenolic compositions. the vertical line (linkage distance 37) indicate the cut-off used to form the groups. 162 k. mijowska, i. ochmian, j. oszmiański tab. 2: polyphenol compound concentrations in grapes of ‘regent’ depending on rootstock [mg · 100 g-1 fw] – as the average of years 2013-2015. compounds rootstock couderc 161-49 sori kober 125aa börner kober 5bb own-root mean cyanidin-3-o-glucoside 56.1±4.9ab* 68.8±3.0c 66.9±4.7c 50.0±4.2a 67.5±3.5c 64.2±5.3bc 62.3 delphinidin-3-o-glucoside 71.1±4.3b 82.9±5.4c 64.8±6.1a 67.1±5.7ab 69.2±6.3ab 65.0±7.2a 70.0 malvidin-3-o-glucoside 59.7±6.0ab 69.1±4.4c 70.7±7.8d 65.4±5.8bc 51.7±5.2a 58.6±5.7ab 62.5 peonidin-3-o-glucoside 71.4±7.7b 79.2±6.8c 80.9±7.2c 56.7±5.4a 70.9±6.5b 64.7±7.5a 70.6 petunidin-3-o-glucoside 77.8±6.8b 83.8±7.2c 80.7±6.5bc 77.1±7.0b 71.8±5.5a 67.5±8.3a 76.5 cyanidin-3-o-acethyl-glucoside 1.07±0.09ab 1.32±0.12c 0.99±0.08ab 1.16±0.08bc 0.91±0.10a 1.67±0.13d 1.19 delphinidin-3-o-acethyl-glucoside 1.36±0.11ab 1.35±0.12ab 1.20±0.08a 1.58±0.09bc 1.08±0.07a 1.84±0.12c 1.40 malvidin-3-o-acethyl-glucoside 6.30±0.44b 6.37±0.48b 5.23±0.31a 6.18±0.25b 4.76±0.23a 5.26±0.36a 5.68 peonidin-3-o-acethyl-glucoside 0.99±0.06b 1.15±0.08c 1.13±0.08c 0.90±0.07b 0.55±0.04a 0.41±0.03a 0.86 petunidin-3-o-acethyl-glucoside 1.27±0.08c 0.87±0.07a 1.10±0.08b 1.37±0.06c 0.80±0.05a 1.04±0.07b 1.08 cyanidin-3-o-(6-p-coumaroyl)-glucoside 5.79±0.24bc 6.17±0.32c 5.91±0.22bc 5.44±0.21b 4.20±0.19a 3.60±0.21a 5.19 delphinidin-3-o-(6-p-coumaroyl)-glucoside 1.99±0.09ab 2.17±0.11b 2.35±0.10b 2.92±0.13c 1.59±0.09a 2.98±0.15c 2.33 malvidin-3-o-(6-p-coumaroyl)-glucoside 7.52±0.23c 7.20±0.27bc 6.99±0.28b 7.53±0.30c 5.68±0.22a 6.16±0.25a 6.85 peonidin-3-o-(6-p-coumaroyl)-glucoside 5.51±0.27c 5.29±0.19c 5.14±0.21c 4.64±0.18b 4.48±0.17b 3.20±0.15a 4.71 petunidin-3-o-(6-p-coumaroyl)-glucoside 6.88±0.38de 7.14±0.41e 5.79±0.30c 6.49±0.32cd 4.90±0.25b 3.84±0.27a 5.84 anthocyanins 375ab 423c 400bc 355a 360a 350a cis-caftaric acid 1.73±0.13d 1.33±0.09b 1.68±0.11cd 1.08±0.08a 1.53±0.11c 1.80±0.12d 1.53 trans-caftaric acid 2.55±0.20d 1.40±0.11b 1.72±0.15b 1.02±0.12a 2.18±0.20c 2.70±0.22d 1.93 cis-coutaric acid 2.73±0.16cd 2.29±0.15b 2.52±0.15bc 1.94±0.14a 2.93±0.17d 1.82±0.15a 2.37 trans-coutaric acid 0.58±0.03a 0.72±0.05c 0.64±0.04b 0.58±0.02a 0.66±0.04b 0.53±0.04a 0.62 cis-fertaric acid 0.79±0.05c 0.40±0.03a 0.56±0.04b 0.45±0.03a 0.76±0.05c 0.91±0.07d 0.65 trans-fertaric acid 0.64±0.05b 0.72±0.05bc 0.83±0.04d 0.53±0.03a 0.94±0.05e 0.76±0.05c 0.74 gallic acid 1.36±0.08b 1.04±0.05a 1.52±0.08c 1.03±0.06a 1.31±0.06b 1.72±0.08d 1.33 phenolic acid 10.38c 7.90b 9.47c 6.63a 10.31c 10.24c myricetin-3-o-glucoside 4.32±0.27b 6.21±0.33d 5.05±0.28c 3.93±0.19ab 4.16±0.22b 3.24±0.15a 4.49 quercetin-3-o-rutinoside 2.79±0.11a 4.07±0.15c 3.59±0.15bc 3.04±0.14ab 3.03±0.12ab 3.63±0.16bc 3.36 quercetin-3-o-glucoside 9.06±0.38b 12.15±0.53d 10.54±0.48c 7.20±0.37a 9.13±0.33b 7.91±0.46a 9.33 quercetin-3-o-glucuronide 7.75±0.28a 11.84±0.35d 11.10±0.31cd 8.76±0.30ab 7.52±0.22a 9.79±0.36bc 9.46 kaempferol-3-o-glucoside 0.53±0.03a 0.73±0.05c 0.74±0.04c 0.63±0.05b 0.45±0.03a 0.68±0.04bc 0.63 isorhamnetin-3-o-glucoside 2.09±0.11bc 3.04±0.13d 3.09±0.11d 2.37±0.09c 1.55±0.04a 1.76±0.05ab 2.32 flavonols 26.54a 38.04b 34.11b 25.93a 25.84a 27.01a procyanidin b1 17.61±0.55c 16.94±0.51c 13.78±0.58b 8.88±0.36a 12.35±0.43b 14.86±0.49b 14.07 procyanidin b2 27.54±0.94a 34.09±1.98b 28.37±1.07a 23.67±1.23a 25.45±1.45a 37.97±3.11b 29.52 procyanidin b3 10.98±0.34c 9.31±0.22bc 8.22±0.30b 4.45±0.17a 5.85±0.19a 7.82±0.24b 7.77 (+)-catechin 81.90±5.43a 99.54±4.89bc 105.63±5.11c 94.43±3.94b 81.22±3.62a 77.17±4.40a 89.98 (-)-epicatechin 46.41±2.92c 45.89±2.30c 42.84±2.45bc 34.65±2.03a 40.22±1.88b 62.88±3.19d 45.48 flavan-3-ols 184b 206c 199bc 166a 165a 201c total polyphenols 596b 675c 643c 554a 561ab 588ab * means in the same row followed by the same letter are not significantly different at p < 0.05 according to tukey test; ±sd: standard deviation belong to the most important anthocyanins in grapes (ivanova et al., 2010). their average content ranged from 62.3 to 76.5 mg · 100 g-1 fw, and their highest level was found in the fruits from plants with the ‘sori’ and ‘kober 125aa’ rootstocks. delphinidin-3-o-glucoside was an exception as its content was the lowest in the case of the ‘kober 125aa’ rootstock. own-root plants also had a similarly low content of this compound. the content of other anthocyanin compounds was significantly lower than the 3-o-glucoside forms and, on average, it varied from 0.86 (peonidin-3-o-acethyl-glucoside) to 6.85 mg · 100 g-1 fw (malvidin-3-o-(6-p-coumaroyl)-glucoside). according to other authors (río segade et al., 2009; figueiredogonzález et al., 2012), the largest group of anthocyanin compounds rootstock effects on grape polyphenols 163 of red varieties of grapes were derivatives of malvidin – 40.6-84.9 %. peonidins also constituted a large group of 22-44 % anthocyanins (figueiredo-gonzález et al., 2012). in the case of the grapes of ‘regent’, the distribution of anthocyanins was different. derivatives of petunidin constituted the largest group (83.31 mg · 100 g-1 fw), which was equal to 22 %. the remaining groups of anthocyanins occurred in similar numbers. the content of cyanidinswas the lowest (68.57 mg · 100 g-1 fw); however, it constituted as much as 18 % of all anthocyanins identified. phenolic acids the profile of phenolic acids in the fruit from the cultivar ‘regent’ was diversified and depended on the rootstock used. the lowest amounts of phenolic acids were found in grapes growing with the ‘börner’ rootstock (6.63 mg · 100 g-1 fw). the remaining plants had a significantly higher content of those compounds in the fruit (7.910.38 mg · 100 g-1 fw). in the case of grapes of the cultivar ‘regent’, seven different compounds belonging to the group of phenolic acids were identified. the ones occurring in the largest amounts were ciscoutaric acid (2.37 mg · 100 g-1 fw) and trans-caftaric acid (1.93 mg · 100 g-1 fw). according to ehrhardt et al. (2014), the level of trans-caftaric acid depends on the location of cultivation. authors noted that in the fruit of the grapevine ‘regent’ cultivated in italy, it was 1.27 mg · 100 g-1 fw, while in the cooler climate of germany it was significantly higher: 3.60 mg · 100 g-1 fw. additionally, our previous study on the evaluation of cluster zone leaf removal on grapes cultivar ‘regent’ polyphenol content showed that berries under shaded condition achieved higher level of phenolic acids. the amount of trans-caftaric acid was even five times higher compared to grapes from defoliated vines (mijowska et al., 2016). flavonols six compounds among the flavonols were identified in the fruits. similarly, as for the case of anthocyanins, the highest level of fla vonols was found in fruits harvested from plants with the ‘sori’ (38.04 mg · 100 g-1 fw) and ‘kober 125aa’ rootstocks (34.11 mg · 100 g-1 fw). grapes growing on own-roots or with other type of rootstock were characterised by a low content of flavonols (25.8427.01 mg · 100 g-1 fw). in a study by satisha et al. (2008), the flavonol content of ‘thompson seedless’ grapes was higher in the case of vines grafted on rootstocks as compared with ungrafted ones. derivatives of myricetin, quercetin, kaempferol and isorhamnetin were found in the flavonol group of grapes, and the most frequently found ones were quercetin-3-o-glucuronide (9.46 mg · 100 g-1 fw) and quercetin-3-o-glucoside (9.33 mg · 100 g-1 fw). flavan-3-ols the highest content of flavan-3-ols was found in grapes with the ‘sori’ rootstock (206 mg · 100 g-1 fw) and own-root plants (201 mg · 100 g-1 fw), while the lowest was found in plants with the ‘kober 5bb’ (165 mg · 100 g-1 fw) and ‘börner’ (166 mg · 100 g-1 fw) rootstocks. as reported by suriano et al. (2016), ‘kober 5bb’ seemed the rootstock that let the genotype ‘greco nero’ to better express flavans both in grapes and in derived wines. among flavan3-ols, b type procyanidins as well as (+)-catechin and (-)-epicatechin were identified. the most frequent compounds occurring in grapes from that group were (+)-catechin (89.98 mg · 100 g-1 fw) and (-)-epicatechin (45.48 mg · 100 g-1 fw), which is consistent with reports of other authors (ehrhardt et al., 2014; antoniolli et al., 2015). the highest content of (+)-catechin was recorded in fruits of plants with the ‘kober 125aa’ and ‘sori’ rootstocks (105.63 and 99.54 mg · 100 g-1 fw respectively). grapes harvested from ownroot plants had the lowest content of (+)-catechin and the highest content of (-)-epicatechin (62.88 mg · 100 g-1 fw). conclusions the study showed an important influence of rootstock on the content of polyphenols in grapes of ‘regent’. the content of polyphenols was significantly higher in grapes harvested in the year 2015, which was characterised by a low amount of rainfall and a high temperature during the growing season. the use of ‘sori’ and ‘kober 125aa’ rootstocks in cultivation of ‘regent’ on silt loam soil was beneficial for obtaining fruits with the highest content of polyphenol compounds. such a correlation has been shown in studies from all years. furthermore, ‘sori’ and ‘kober 125aa’ rootstocks had the most significant influence on the increase in the anthocyanin and flavonol content in grapes. additionally, ‘sori’ rootstock, next to own-rooted plants, enabled to achieve the highest concentration of flavan-3-ols in berries. ‘börner’ and ‘kober 5bb’ rootstocks did not have a significant influence on the level of polyphenol in fruits when compared to own-root plants. in addition, it has been shown that using the ‘börner’ rootstock reduces the content of phenolic acids in fruits. among all polyphenols determined, the majority of them were anthocyanin compounds, which occured most frequently in a form of 3-o-glucoside. the contents of derivatives of petunidin, peonidin, delphinidin, malvidin and cyanidin were at a similar level (18-22 %). acknowledgments research work and funds for covering the costs to publish in open access were supported by grants of west pomeranian university of technology szczecin: 517-07-014-5362/17 and 518-07-014-317603/18. conflict of interest: the authors declare that they have no competing interests. references antoniolli, a., fontana, a.r., piccoli, p., bottini, r., 2015: characterization of polyphenols and evaluation of antioxidant capacity in grape pomace of the cv. malbec. food chem. 178, 172-178. doi: 10.1016/j.foodchem.2015.01.082 censi, p., saiano, f., pisciotta, a., tuzzolino, n., 2014: geochemical behavior of rare earths in vitis vinifera grafted onto different rootstock and growing on several soils. sci. total environ. 473-474, 597-608. doi: 10.1016/j.scitotenv.2013.12.073 cousins, p., 2005: evolution, genetics, and breeding: viticultural applications of the origins of our rootstocks. in: cousins, p., striegler, r.k. 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(vitis vinifera l.). sci. hortic., 209, 309-315. doi: 10.1016/j.scienta.2016.07.004 vršič, s., pulko, b., kocsis, l., 2015: factors influencing grafting success and compatibility of grape rootstocks. sci hortic-amsterdam, 181, 168-173. doi: 10.1016/j.scienta.2014.10.058 wang, y., catana, f., yang, y., roderick, r., van breemen, r.b., 2002: an lc-ms method for analyzing total resveratrol in grape juice, cranberry juice and in wine. j. agric. food chem. 50, 431-435. doi: 10.1021/jf010812u address of the author: kamila mijowska, ireneusz ochmian: department of horticulture, west pomeranian university of technology szczecin, słowackiego 17, 71-434 szczecin, poland e-mail: kamila.mijowska@zut.edu.pl jan oszmiański: department of fruit, vegetable and plant nutraceutical technology, wrocław university of environmental and life sciences, chełmońskiego 37, 51-630 wrocław, poland © the author(s) 2017. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 91, 56 60 (2018), doi:10.5073/jabfq.2018.091.008 1agroscope, competence division for plants and plant products, ecotoxicology, wädenswil zh, switzerland 2 weingut kastanienberg, hainfeld, germany diversity of arbuscular mycorrhizal fungi in no-till and conventionally tilled vineyards fritz oehl1, bruno koch2 (submitted: january 23, 2018; accepted: february 16, 2018) * corresponding author summary the use of a permanent vegetation cover or frequent tillage in vineyards may affect soil water budget, nutrient availability, soil compaction, soil erosion and soil microbe biodiversity, and through all these and other factors also yield and wine quality parameters. the abundance and diversity of arbuscular mycorrhizal fungi (amf) might also be influenced, but so far effects on amf by permanent vege tation cover (= no-tillage systems) or repeated chiseling and rotarytillaging have rarely been compared in vineyards. the objective of this on-farm study was to determine amf species richness and diversity in two adjacent vineyards in palatinate (sw germany). in both vineyards, grown on fertile luvisols, the var. “pinot gris” was grown for 39 years, but with different soil cultivation and different fertilization strategies. in one vineyard, soil was maintained periodically without vegetation by passing rotatory cultivator and chiseling between the grapevine rows (‘inter-rows’) several times per year, preferably during spring and summer and in dependency of rainfall and ‘weed’ growth, and fertilization was mainly by organic fertilizers in the last ten years before soil sampling. in the other vineyard, a permanent vegetation has been established since planting, dominated by lolium perenne, and mineral fertilizers were exclusively applied. despite of similar high nutrient availability in both soils, in particular of phosphorus, astonishing high amf species richness and diversity were found in both vineyards. in the no-tillage inter-rows, 34 amf species were found, with a species composition typically for central european permanent grasslands (shannon diversity index 2.45). in the tillage system 24 amf species were found with a composition as known for extensively used, cultivated central european croplands (diversity index 2.26). we conclude that above all soil cultivation has affected amf diversity in these central european vineyards, while the level and type of fertilization affected the amf communities only on a minor level. key words: grapes; green cover strategies; glomeromycotina; mycorrhiza; soil management introduction permanent vegetation cover or no-tillage between rows in vineyards has always been practiced in different grapevine regions. permanent cover is common above all in sloped areas or in regions with relatively high annual precipitation, in order to prevent soil erosion and soil loss. in drier, semi-arid to arid, often mediterranean areas, however, soils are periodically tilled or left completely bare during the vege tation period of the grapevine, similar to those in olive plantations (turrini et al., 2017). in such climates, natural grasses and herbs, as well as green cover plants have often been considered as grapevine ‘weeds’ competing for water and soil nutrients, and thus possibly affecting negatively grapevine yields and wine quality (rodriguezlovelle et al., 2000; guerra and steenwerth, 2011). the management of such natural to intentionally seeded permanent vegetations has become major importance, especially in respect to on-going or expected climatic changes (rupp, 1996; koch and oehl, 2018). arbuscular mycorrhizal fungi (amf) of the fungal subphylum glomeromycotina represent an important group of beneficial soil microorganisms that have positive effects on grapevine growth and health (petgen et al., 1997; schreiner, 2007; scandellari, 2017) as well as on soil aggregate formation and prevention of soil erosion (rillig and mumey, 2006). it can be assumed that they can also affect grapevine quality positively, especially in drier years or in more arid regions, where water periodically is scarce and might restrict optimum grapevine development. however, these fungi are obligatory biotroph, which means that they need living plants for their maintenance at their habitats (smith and read, 2008). keeping the vineyard soil free of weeds and grassland plant species, as it is sometimes practiced not only in the grapevine rows, but also in the whole inter-spaces (‘inter-rows’), must thus affect development and reproduction of this fungal group, although in such situations they may remain important symbiotic partners of the deep-rooting grapevine plants. the objective of the present study was to determine the species richness and diversity of am fungi in two adjacent vineyards, but applying different soil cultivation and fertilization strategies. the vineyards, both grown for 39 years with the white wine variety “pinot gris”, were located in palatinate (southwestern germany), which represents one of the most traditional, largest wine growing areas in the north of the alps, where grapevine has been produced since the roman times (schultz and jones, 2010). it represents also one of the driest and warmest regions in germany (koch and oehl, 2018), climatically belonging to the european temperate zone, but both with an atlantic, and also a continental influence, well documented by the presence of castanea sativa l. grown on cambisols and of tschernosem/phaeosem soils in close vicinity to each other. our results may help to better understand and improve viticultural systems, increase natural soil fertility and biologically mediated nutrient availability, and to increase soil microbes biodiversity in vineyards. material and methods study site and soil sampling the two vineyards are located at about 700 m southeast of the village hainfeld (palatinate, germany; 49°c15’16’’n; 8°06’24’’e) established on haplic luvisol soils according fao/iuss soil classification. mean annual temperature is approximately 11.5°c, and mean annual rainfall is ca. 670 mm in a bi-modal distribution, with gene rally less rainfall in spring and early autumn than during summer and winter (koch and oehl, 2018). the rootstocks with grafted “pinot gris” were planted in rows in june 1975, choosing 2.0 m distances between the rows and 1.20 m within the rows. differences between both vineyards were in soil cultivation strategy of the inter-rows of the grapevine plants, which was no-tillage in one vineyard since the first year of planting, while in the other vineyard, the soil was periodically cultivated by rotary cultivator or chisel several times per year, depending on rainfall and ‘vineyard weed’ growth. the use of synthetic pesticides for plant protection against pathogens and pests diversity of arbuscular mycorrhizal fungi in vineyards 57 was similar in both vineyards during the 39 years and was carried out according to good agricultural practice. no herbicides were used beneath the rootstocks in the last 10 years before soil sampling, while before herbicides were mainly based on glyphosate or glufosinate, and before 1985 also on paraquat and diquat. both vineyards received about the same quantity of fertilizers according to the regional recommendations for grapevine production (since the year 2000 approx. 50-60 kg n, 10 kg p, 60-80 kg k, 15 kg mg ha-1 a-1). however, the tilled vineyard received composted horse manure, derived from an agricultural farm situated about 5 km west to the vineyards, instead of mineral fertilizers, which were applied in the no-tillage vineyard. soil sampling (0-10 cm depth) was performed in early springtime 2014 in five selected plots of 5 × 20 m2 per vineyard as described in oehl et al. (2005b) in the middle of the inter-rows at about 1.0 m distance to the pinot gris rootstocks; this represented five replicates per site. in each replicate plot, six subsamples were taken and pooled, totaling in about 1.2 kg soil per plot. soil samples were carefully airdried, sieved at 5 mm and stored as described in oehl et al. (2005b), before they were analyzed for selected chemical soil parameters and amf spore density and diversity. amf spore isolation and identification amf spores were extracted from the soil samples by wet sieving and sucrose density gradient centrifugation (sieverding, 1991). spores, spore clusters and sporocarps were picked from the extractions in petri dishes without pre-selection using a leica s8apo and mounted together on microscope slides using polyvinyl-lactic acidglycerol (plvg) or plvg mixed 1:1 (v/v) with melzer’s reagent. the slides were systematically examined under a leica dm750 compound microscope at up to 400-fold magnification to identify and count all morphologically distinct amf spore types present. morphological spore identification was based on two identification manuals (schenck and pérez, 1990; błaszkowski, 2012) and all newer existing amf species descriptions. taxonomic classification was based on the glomeromycota system of oehl et al. (2011) published by the international mycological association (ima), with a few updates (e.g. sieverding et al., 2014; błaszkowski et al., 2015; 2017; oehl et al., 2016). statistical analyses student t-test was applied to test for significant differences between the amf populations in the two vineyards in amf spore density, species richness and diversity, and for spore abundance for each amf species found in both vineyards. results and discussion chemical soil parameters both vineyards had quite similar chemical soil parameters (tab. 1) with an adequate organic carbon content (24.6-26.1 g kg-1) and slightly acidic to neutral ph values (6.6-6.8, measured in 1n kcl, 7.0-7.4 in water), which corresponds well with the similar level of nutrient application, despite of using quite different types of fertilizers. available nutrient values were all classified as high to very high and reflect the excessive fertilization recommendations of the past, especially before 1985. in particular, phosphorus levels were extremely high so that low amf spore density, species richness and diversity could be expected, since it is generally assumed that at higher plant available p levels plants depend less on mycorrhiza (smith and read, 2008). the values also suggest that p fertilization would not be needed, or should be further reduced in the next years, and the same might apply also for k and mg fertilization. the relatively high copper contents in both soils may reflect the intensive use of copper-based fungicides during the last hundred years of grapevine production in order to suppress downy mildew, which − together with powdery mildew − has been the major pathogen in the grapevine growing areas of the region during the last century. amf spore density in the no-tillage inter-rows, amf spore density was approximately 60% higher (22 g-1 soil) than in the periodically cultivated inter-rows (14 g-1 soil, fig. 1a). these values represent similar numbers for the no-till inter-rows, when compared to values from permanent grasslands studied in the upper rhine region between basel and mainz, where 15-40 spores g-1 soil where found in de-carbonated soils in early spring (e.g. oehl et al., 2003, 2005b, 2010, 2017). the spore densities found in the cultivated inter-rows were slightly above the densities reported for conventionally or organically managed croplands of similar soil ph in central europe (oehl et al., 2003; maurer et al., 2014). one reason for this finding might be that soil treatment by vineyard chisels and rotary cultivators is generally shallower (515 cm) than by ploughing in arable lands (generally 15-25 cm in low input croplands, in the past sometimes up to 25-30 cm in croplands; oehl et al., 2005b). thus, the sub-soil amf hyphal network and amf communities in vineyards might have been less disturbed than in croplands (oehl et al., 2005b). the high fertilization levels did not lead to low spore densities in the two vineyards. soil cultivation had a stronger effect than nutrient availability on amf spore density, which is in accordance with avio et al. (2013) and säle et al. (2015), and in general vineyards might have higher spore densities than croplands, which is in accordance with oehl et al. (2005b). amf species richness and diversity total amf species richness was about 40% higher in the no-tillage (34 species) than in the cultivated inter-rows (24 species, tab. 2), and mean species richness per plot was approximately 50% higher in the no-tillage vineyard (27 versus 18 species; fig. 1b). moreover, amf tab. 1: chemical soil parameters in no-tillage and periodically tilled interrows of two 39-years old “pinot gris” vineyards soil parameter no-till tillage unit nutrient availability level according to current recommendations in germany organic carbon 26.1 24.6 g kg-1 ph (h2o) 7.0 7.4 ph (1n kcl) 6.6 6.8 humus (%) 45 42 g kg-1 ca 4.6 3.5 g kg-1 medium p (na-acetat)* 69 104 mg kg-1 very high p (dl)* 203 257 mg kg-1 very high p (citrat)* 428 467 mg kg-1 very high k (na-acetat) 415 479 mg kg-1 very high k (dl) 515 562 mg kg-1 very high mg (dl) 315 246 mg kg-1 very high cu 43 43 mg kg-1 very high zn 112 35 mg kg-1 high *based on different methods to determine p-availability in european soils (neyroud et al., 2003). 58 f. oehl, b. koch diversity expressed as the shannon index (h-value) was also higher in the no-tillage (2.45) than in the cultivated inter-rows (2.26; fig. 1 c). this is in accordance with lumini et al. (2010), who found higher amf diversity in pastures and covered vineyards than in tilled vineyards in a mediterranean environment of italy. our species numbers and diversity indices are remarkable, as they are, despite of the high nutrient availability values, in the same range of numbers found in extensively managed grasslands (26-39 species and 2.29-2.54 h-value) and extensively to intensively managed, cultivated croplands (21-28 and 1.72-2.45) in central europe (oehl et al., 2003, 2010; maurer et al., 2014; wetzel et al., 2014; säle et al., 2015). those sites had much lower nutrient availability values. so far, the opinion was that high nutrient availability would counteract the development and diversity of amf. our new data suggest that many, if not all amf species, naturally occurring in extensively managed or natural permanent grasslands can somehow litigate with high nutrient (and even copper) values also in permanent covered inter-rows of vineyards. they will not disappear under grapevine production, especially not in inter-rows with permanent green cover. this is encouraging, as there is no measurable loss of amf biodiversity in vineyards, as a clear difference to intensively used croplands (e.g. oehl et al., 2003, 2010). we found, however, low spore densities and species richness of acaulosporaceae, ambisporaceae and gigasporales species (in to 1 2 3 fig. 1: amf spore density, species richness and shannon-weaver diversity in no-tillage and 4 conventional tillage inter-rows of two 39-years old “ pinot gris” vineyards. 5 6 fig. 1: amf spore density, species richness and shannon-weaver diversity in no-tillage and conventional tillage inter-rows of two 39-years old “pinot gris” vineyards. tab. 2: spore density for amf species in no-till and periodically tilled interrows of two “pinot gris” vineyards amf species spore density per 100 g p-value (standard deviation) no tilled tilled equal abundant in both vineyards claroideoglomus luteum 18 (1.4) 18 (3.0) 0.500 rhizoglomus fasciculatum 10 (0.7) 10 (1.6) 0.500 glomus spinosum 2 (0.2) 2 (0.3) 0.500 rhizoglomus invermaium 30 (2.2) 22 (3.8) 0.333 funneliformis mosseae 46 (3.5) 35 (6.0) 0.263 claroideoglomus claroideum 15 (1.2) 10 (1.8) 0.118 more abundant in the periodically tilled vineyard glomus diaphanum 21 (1.6) 112 (19.2) 0.005 archaeospora trappei 38 (2.9) 58 (10.0) 0.092 more abundant in the no-till vineyard septoglomus constrictum 82 (6.3) 12 (2.1) 0.002 diversispora epigaea 10 (0.8) 3 (1.5) 0.004 paraglomus occultum 14 (1.1) 2 (0.4) 0.005 palaeospora spaineae 6 (0.4) 2 (0.4) 0.008 rhizoglomus irregulare 30 (2.3) 14 (2.3) 0.009 dominikia compressa 39 (3.0) 8 (1.4) 0.011 glomus badium 13 (1.0) 1 (0.1) 0.017 funneliformis fragilistratus 13 (1.0) 5 (0.8) 0.017 sclerocystis sinuosa 399 (31.3) 140 (23.9) 0.021 diversispora celata 10 (0.8) 1 (0.1) 0.021 dominikia aurea 91 (7.1) 3 (0.5) 0.026 funneliformis geosporus 131 (10.2) 73 (10.4) 0.029 dominikia bernensis 139 (10.8) 36 (6.2) 0.045 sclerocystis sp. 33 (2.6) 9 (1.5) 0.054 paraglomus turpe 7 (0.6) 3 (0.5) 0.071 rhizoglomus intraradices 10 (0.8) 6 (1.0) 0.089 exclusively found in the no-till vineyard glomus macrocarpum 45 (3.5) rhizoglomus aggregatum 15 (1.2) glomus microcarpum 6 (0.5) acaulospora longula 3 (0.2) claroideoglomus etunicatum 3 (0.2) ambispora gerdemannii 2 (0.2) glomus heterosporum 2 (0.2) cetraspora armeniaca 1 (0.1) acaulospora laevis 1 (0.1) dominikia sp. br11 1 (0.1) total number of spores identified 100g-1 soil total amf species richness at site 34 24 average and standard deviation of five plots per treatment; p-values <0.05 show significant differences between the two vineyards according to student t-test. no-till inter-rows tillage inter-rows 1286 585 diversity of arbuscular mycorrhizal fungi in vineyards 59 tal only seven spores of acaulospora laevis, a. longula, ambispora gerdemannii and cetraspora armeniaca were detected), which is in accordance with findings of schreiner and mihara (2009). all these fungal taxa are known to react negatively not only on soil disturbance, but also to high fertilization levels (oehl et al., 2010, 2011, 2017; maurer et al., 2014). the high nutrient levels in the vineyards thus can explain, why no high spore abundances and species richness of these specific amf genera and families were found, although these amf taxa are frequent members of central european soils with ph 5.0-7.0 (błaszkowski, 1993; oehl et al., 2004, 2010; maurer et al., 2014). extensive future studies in central european vineyards have to prove if our assumptions are valid. the situation might be quite different even within growing areas, such as in palatinate, as grapevine areas are often geologically quite diverse and have a very wide range of soil types respective different ‘terroir’ (van leeuven and seguin, 2006; koch and oehl, 2018), and thus, may naturally have quite diverse amf communities (oehl et al., 2010, 2017). of the 34 amf species detected in the present study, six species were found in similar spore abundancies in both vineyards, among them claroideoglomus claroideum and cl. luteum, rhizoglomus fasciculatum and rh. invermaium, and funneliformis mosseae. two amf species were more frequent in the cultivated inter-rows than in the notillage inter-rows (glomus diaphanum and archaeospora trappei), while 16 species were, more or less clearly, more abundant in the notillage inter-rows than in the cultivated inter-rows (e.g. septoglomus constrictum, glomus badium, sclerocystis sinuosa, diversispora celata and dominikia compressa). the lasting ten species were exclusively detected in the no-tillage inter-rows (e.g. glomus macrocarpum), but some of them with a few spores only (e.g. cetraspora armeniaca and acaulospora laevis). these results again fit well with the former results from tilled and of non-tilled croplands, or vineyard in the areas of baden and rheinhessen in germany (e.g. oehl et al., 2005a, 2005b, 2010; maurer et al., 2014; wetzel et al., 2014). however, we can only assume, according to smith and read (2008) and all other literature about am fungi and their symbiosis, that all the amf species detected in our inter-rows are also symbionts of “pinot gris” plants themselves. only detail study in the greenhouse or molecular root analyses can elucidate the amf compositions within the grape roots (e.g. schreiner and mihala, 2009; lumini et al., 2010; likar et al., 2013), when compared to those in the roots of the different green cover plants. conclusions arbuscular mycorrhizal research has got a major importance during the last decades, but the majority of the am fungal species worldwide have not yet been discovered. moreover, we are still far from understanding and using the potential of the beneficial effects of amf for agronomical important crops. we do not know what even the most intensively investigated amf species can provide for the plants, soils, entire habitats and complex ecosystems. it has become a challenging task to figure out, how we can use these fungi for agricultural purposes or environmental benefits. we assume that amf and their diversity might be important for high quality grapevine and wine production, especially in regions of scarce rainfall, due to their capacity to deliver nutrients and water to the plants, and to protect soils against erosion and compaction during and after heavy rains, respectively. for their potential future use in vineyards, it is a precondition to know the effects of specific management practices and cultivation techniques on the development and biodiversity of these am fungi, and to know how we can maximize the ecosystem services, which they provide in our soil-plant systems, such as for grapevine and wine production. in vineyards under semi-arid conditions, no-till temporary and permanent plant cover in the grapevine inter-rows is a viable alternative to traditional soil tillage systems, but one of the challenges will be to establish and sustain beneficial plant and microbial communities that deal with the periodical water scarcity and do not compete but promote water and nutrient uptake, and thus grapevine growth and quality. acknowledgements we are thankful to the continued financial support of snsf (bern, switzerland) to our research studies (here iz73z0_152740). references avio, l., castaldini, m., fabiani, a., bedini, s., sbrana, c., turrini, a., giovannetti, m., 2013: impact of nitrogen fertilization and soil tillage on arbuscular mycorrhizal fungal communities in a mediterranean agroecosystem. soil biol. biochem. 67, 285-294. doi: 10.1016/j.soilbio.2013.09.005 błaszkowski, j., 1993: comparative studies on the occurrence of arbuscular fungi and mycorrhizae (glomales) in cultivated and uncultivated soils of poland. acta mycol. 28, 93-140. doi: 10.5586/am.1993.013 błaszkowski, j., 2012: glomeromycota. w. szafer institute of botany, polish academy of sciences. błaszkowski, j., chwat, g., góralska, a., ryska, p., kovács, g.m., 2015: two new genera, dominikia and kamienska, and d. disticha sp. nov. in glomeromycota. nova hedwigia 100, 225-238. doi: 10.1127/nova_hedwigia/2014/0216 błaszkowski, j., kozłowska, a., crossay, t., symanczik, s., alyahya’ei, m.n., 2017: a new family, pervetustaceae with a new genus, pervetustus, and p. simplex sp. nov. 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respiration and native mycorrhizal potential compared with soil tillage in a high-density olive orchard in a long term study. appl. soil ecol. 116, 70-78. doi: 10.1016/j.apsoil.2017.04.001 van leeuwen, c., seguin, g., 2006: the concept of terroir in viticulture. j.wine res. 17, 1-10. doi: 10.1080/09571260600633135 wetzel, k., silva, g., matczinski, u., oehl, f., fester, t., 2014: superior differentiation of arbuscular mycorrhizal fungal communities from till and no-till plots by morphological spore identification when compared to t-rflp. soil biol. biochem. 72, 88-96. doi: 10.1016/j.soilbio.2014.01.033 address of the authors: fritz oehl, agroscope, competence division for plants and plant products, ecotoxicology, schloss 1, 8820 wädenswil zh, switzerland e-mail: fritz.oehl@agroscope.admin.ch bruno koch, weingut kastanienberg, weinstrasse, 76835 hainfeld, germany © the author(s) 2018. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 90, 25 30 (2017), doi:10.5073/jabfq.2017.090.005 department of medicinal and aromatic plants, szent istván university, budapest, hungary effect of genotype and age on essential oil and total phenolics in hyssop (hyssopus officinalis l.) éva németh-zámbori*, péter rajhárt, katalin inotai (received october 22, 2016) * corresponding author summary five hyssopus officinalis l. accessions (german, hungarian and polish ones) were compared over three years with regard to their development and secondary metabolite production, in an open field experiment. the hungarian variety ‘sophie’ produced the highest essential oil (eo) yield (up to 2.037 ml/100 g). in general, one-year old individuals accumulated the most volatile organic compounds (vocs) and the accumulation was influenced significantly by genotype and year. a total of 47 components were identified in all of the oils. in all accessions cisand trans-pinocamphones were most frequently the major compounds, but there were quantitative differences among genotypes. highest proportions of these two components together appeared in ‘erfurter ysop’ (70.7 %). the third main compound was β-pinene that accumulated in the hungarian accessions in the highest proportions (11-19 %). the cultivation year did not have a considerable influence on the eo composition. significant difference in the total phenolic content was evident among genotypes, and ranged from 443.64 mg/g dw (‘erfurter ysop’) and 329.32 mg/g dw (‘hyzop lekarsky’) calculated as gallic acid. the effect of the year was not significant, although we detected a significant variety × year interaction. in general, the selected hyssop cultivars showed an advance to commercial batches. keywords: intraspecific variability, effect of year, flowering time, essential oil, pinocamphone, total phenolics, plant age introduction hyssop (hyssopus officinalis l.) herb has been traditionally used as a spice, a preservative, antiseptic and flavoring agent in the food industry, in cosmetics and in pharmacognosy in preparations against cough, asthma and spasms of the digestive system. although the shrub is native to the mediterranean sea and the middle east, it is cultivated in several temperate regions of the world. according to the official list of the community plant variety office (cpvo), 28 hyssop varieties have been announced since 1972, among which only five are currently officially registered. the huge majority of the selected genotypes are accessions of different petal colour, selected supposedly for ornamental purposes, while cultivars for production of high quality drug are hardly available. the plant is aromatic and an essential oil (eo), characterised mainly by monoterpenes, is obtained from the aerial parts. the majority of references describe pinocamphone (cis and trans) as main compounds while β-pinene and pinocarvone occur frequently in high concentrations (chialva et al., 1983; galambosi et al., 1993; koller and range, 1997). sesquiterpenes are less abundant, among which germacrene-d seems to be the most prevalent constituent (némethzámbori, 2015). the eo yield of the herb varies from 0.17 % (hodzsimatov and ramazanova, 1974) to 1.77 % (zawislak, 2011). there are considerable differences in the eo yields of different genotypes, and the accumulation seems to be influenced by the climate. a strong negative correlation between the amount of precipitation and the accumulation level of volatile organic compounds (vocs) in the flowering shoots has been demonstrated: rain during the development of flowers seems to have negative effect on the yield (németh et al., 2001). the peak accumulation of vocs could be measured at the time of full flowering, while it was significantly lower before and after flowering (németh et al., 2001; zawislak, 2011). although hyssop is known as a species rich in polyphenols (veres et al., 1995; chrpová et al., 2010), the number of adequate references on their levels and composition in this plant is limited. ferulic acid, isoquercitrin and rutin have been reported as main components of the phenolic fraction (vlase et al., 2014). in addition to flavonoids and tannins (zawislak, 2011), the herb contains rosmarinic acid at concentrations up to 0,24 % (veres et al., 1995). however, variability in the concentrations of phenolic compounds in hyssop has hardly been studied until now and the influencing factors are practically unknown. the goal of this study was to widen the knowledge on factors influencing the variability of active ingredients in hyssop and thus, influencing the quality of the drug. for this reason we followed the growth, eo and phenolics of five hyssop accessions of known origin over three consecutive years. materials and methods plant material: five accessions of hyssopus officinalis l. of different origin were investigated throughout 2012-2014 (tab. 1). seeds were sown in propagation trays on 22 march, 2012 and grown in a greenhouse. the seedlings of appr. 15 cm height were planted into open field plots on 15 may of the same year. each plot consisted of 25 plants and the layout of the plots followed was a random block design with two replications. during the experiment the same plots were investigated over three years with first, secondand three-year old plant individuals, respectively. growing conditions and open field measurements: the soil is slightly calcareous (ph 7.78), loamy-sand (clay content below <15 %) with low humus content (1.14 %). content of the main nutrients in the 0-30 cm soil layer are as follows, no3-n: 10.02 mg/kg, p2o5: 322 mg/ kg, k2o: 180 mg/kg, mg: 82.5 mg/kg. no additional fertilization was applied except for top-dressing with nitrogen (nh4no3, 40 kg/ha) after the harvest each year. weather conditions of the experimental periods are presented in tab. 2. before sampling the experimental site received only moderate amounts of precipitation and the weather was relatively warm. in 2014, the temperatures rose somewhat higher during this period than in former years. the stands received natural precipitation and irrigation was applied only in the very dry periods, however, this additional watering occurred only after sampling had taken place each year (tab. 1). rainfall in the period before harvest may significantly influence the accumulation of volatile compounds 26 é. németh-zámbori, p. rajhárt, k. inotai (németh et al., 2001), however, the conditions of the experimental years were practically similar to each other. weed control was assured mechanically and no other plant protection measures were needed. in the experimental years, the time of flowering for each accession was determined by counting the number of individuals with opened flowers on each plot, at three different times, and expressing the counts as a percentage of the total number of individuals for each period. in the first year before harvesting time, we observed also the colour of the petals for each individual, to evaluate the homogeneity of the populations. the number of plants with flower colours other than blue, was noted for each plot. sampling: the harvests (samples) were adjusted to the phenological phase of the plants: each sample was taken at full flowering stage, which occurred in slightly different dates (tab. 1). the flowering shoots above the woodened part of the stem were cut from randomly selected individuals and bulk samples to form four replications (two replication/plot) were prepared to assure representativeness. the samples were dried at room temperature and processed for determination of eo yield and composition, and total phenolic content. isolation of the essential oil: each sample (50 g) was hydrodistilled for three hours in a clevenger-type apparatus as recommended by the vii. hungarian pharmacopoeia [pharmacopoeia hungarica, 1986]. the eo content was calculated as volume (ml) of essential oil per 100 g of dried weight (three hours, at 105 °c). the collected oil was dried over anhydrous sodium sulfate and stored in glass vials at +4 °c in the dark prior to gas chromatography (gc) analysis. analysis of the essential oil: gc-flame ionisation detection (gcfid) analysis was carried out using an agilent technologies 6890n gc system instrument, equipped with a hp-5 (5 % phenyl methyl siloxane) capillary column (length: 30 m, d = 350 μm, film thickness: 0.25 μm), programmed as follows: initial temperature 50 °c (10 min), from 50 to 150 °c at a rate of 4 °c min-¹; from 150 to 220 °c at a rate of 12 °c min-¹ and 220 °c (10 min). carrier gas: helium (constant flow rate 0.5 ml min-¹), injector and detector temperatures: 250 °c, split ratio: 22.6:1, injected quantity: 0.2 μl. the percentage composition of the essential oil was computed from the gc peak areas. gas chromatography-mass spectrometry (gc-ms) analysis was carried out from the eos using the instrument mentioned above, equipped with an agilent technologies ms 5975 inert mass selective detector. the temperature programme was as follows: initial tem perature 60 °c (10 min), from 60 to 240 °c at a rate of 3 °c min-¹, held for 5 min. carrier gas: helium (constant flow rate: 1 ml min-¹); injector: 230 °c, split ratio: 30:1, transfer line: 240 °c. ionization energy was 70 ev. injected quantity: 0.2 μl. eo compounds were identified by matching their recorded mass spectra with those in mass spectral library references (nist and wiley) and by comparing their linear retention indices (lri) relative to the elution ranking of n-alkanes (c9-c20) with those of authentic compounds under the same conditions. in case of eo composition the average values of the two replicates are presented and discussed. total phenolic content: for the determination of the total phenolic (tpc) content, 1 g dried and powdered plant material was extracted with 100 ml boiling distilled water, which was allowed to stand for 24 h. then the extracts were filtered and stored in a freezer until the assay was conducted. tab. 1: the examined accessions of hyssop and their harvest times nr. denomination origin date of harvest 2012 2013 2014 1. ’sophie’ registered variety, breeder: dept. of medicinal and aromatic plants, 9august 21 june 22 june corvinus university 2. ’erfurter ysop’ commercial item from n. l. chrestensen gmbh (erfurt, germany) 9august 21 june 5 june 3. ’blankyt’ commercial item from pharmasaat gmbh (artern, germany) 9august 1 july 5 june 4. ’hyzop lekarsky’ commercial item from pnos ożarów mazowiecki (poland) 30 july 21 june 5 june 5. ’cyrano’ selected strain from genebank stock material, breeder as for 1. 9august 1 july 22 june tab. 2: average temperatures and sum of precipitation during the experimental years presented by decades (data from sprouting till the last harvest) month 2012 2013 2014 decades mean decades mean decades mean i. ii. iii.* i. ii. iii.* i. ii. iii.* april 8.0 9.0 13.8 14 3.9 10.9 16.1 10.3 11.8 10.7 14.6 12.4 may 16.1 12.7 16.0 19 16.7 14.5 12.1 14.4 13.3 13.1 18.2 14.9 june 17.2 19.3 20.2 22 14.7 21.0 17.1 17.6 19.2 20.0 18.4 19.2 july 25.3 18.7 20.1 23 20.1 18.9 21.8 20.3 20.6 22.1 21.9 21.5 aug 21.9 sum sum sum april 12.6 2.6 6.2 20 24.8 5.4 0.6 30.8 9.2 4.4 25.4 39.0 may 14.8 13.8 30.0 45 34.6 15.6 28.0 78.2 40.0 74.8 10.0 124.8 june 22.8 9.0 27.6 60 29.2 0.0 15.8 45.0 12.2 0.0 15.2 27.4 july 6.0 29.2 29.6 75 0.0 0.0 1.4 1.4 24.0 54.6 82.0 160.6 aug 0.0 *in months with 31 days it indicates 11 days. te m pe ra tu re pr ec ip ita tio n essential oil and phenolics in hyssop 27 the total phenolic content was determined by using the method of singleton and rossi (1965) with slight modifications. sample solution (0.5 ml) was transferred to a test tube and then 2.5 ml folin-ciocalteau’s reagent (sigma aldrich kft., budapest) in 10 % v/v was added. after 1 min of incubation, 2 ml of sodium carbonate (0.7 m) was added. the absorbance was measured at 760 nm in a thermo evolution 201 spectrophotometer after a 5 min incubation period in hot water (50 °c). gallic acid (0.3 m) was used as chemical standard for calibration. the total phenolic content of the sample was expressed as mg of gallic acid equivalents per g of dry weight of extract (gae mg/g dw). a blank, containing distilled water instead of extract, was prepared. triplicate measurements were made for each sample. statistical analysis: the results were analysed with the ibm spss statistics 22 software using the manova method to evaluate statistical differences among treatments. wilk’s lambda as unexplained variance rate was tested. homogeneity of variances was tested by levene’s method and genotypes/years were separated by tukey’s post hoc test. results during the experiment, each accession developed flowers already in the year of propagation. concerning the colour of the petals, blue flowers appeared on the majority of the individuals in each accession. in ‘erfurter ysop’ 4 % of individuals displayed a pink petal colour and in ‘cyrano’ we found a single plant with a white flower. the hungarian ‘sophie’, the german ‘blankyt’, and the polish material consisted of homogenous blue flowering individuals. the examined accessions displayed considerable differences in flowering time (fig. 1). the earliest one was the polish commercial material (accession 4), which reached full flowering 10-15 days before the others. the latest one each year was ‘cyrano’, which never flowered before mid-june. the other three accessions flowered intermediate to the two mentioned. it was, however, characteristic for each of the genotypes, that flowering happened earlier as the plants became older (tab. 1). for eo yield, the multivariate test was significant (p=0.000), both for variety and year, as well as for their interaction (tab. 3). evaluating the effect of the variety (genotype), the yield of eo was outstanding in the case of the variety ‘sophie’ (up to 2.037 ml/100 g). this indicates that this variety has been selected specifically for its high eo yield. comparably high values (up to 2.250 %) were reported formerly only as a result of treatment with gamma-rays (gille and floria, 2000). the lowest eo yield (0.565 %-0.987 ml/100 g) was determined in the german varieties (tab. 4). the statistical analysis showed significant differences between ‘sophie’ and each ‘erfurter ysop’, ‘blankyt’ and ‘hyzop lekarsky’. similarly, ‘cyrano’ proved to be significantly different from these three varieties, but not from ‘sophie’. during the three year experimental period, the vegetation year had a significant effect on the eo yield (tab. 3). interaction between genotype and year was also found to be significant. in general, the accumulation levels of the vocs showed a decreasing tendency throughout 2012-14 (tab. 4). the mean value of the five accessions in the third year of their cultivation represented only 62 % of the value measured in the first year. this tendency was obvious for three of the studied genotypes. ‘hyzop lekarsky’ exhibited a sudden drop of eo content in the second year but no further decrease in the third one. a totally opposite tendency was observable only in case of ‘blankyt’: the samples of the third year showed the highest values. nevertheless, this increase (0.2 ml/100 g dw) did not reach the size of the decrease observed in the other genotypes. altogether 47 components were identified in all the eo samples, and among them, 23 were present in all of the oils. among these compounds, only 13 compounds were present at concentrations above 1 % of the total and the variation of these compounds was further investigated (tab. 5). when evaluating these compounds, no characteristic qualitative differences could be established between the accessions. highest proportions of cis-pinocamphone, trans-pinocamphone and β-pinene were present in all the accessions. the characteristic differences among the accessions are quantitative ones. in ‘erfurter ysop’ and ‘hyzop lekarsky’, cis-pinocamphone was pre sent in high concentrations (up to 73.6 and 64.4 %, respectively), therefore these varieties can be described as cis-pinocamphone chemotypes. a similar profile was reported by other authors (koller and range, 1997; bernotienė and butkienė, 2010). cis-pinocamphone is also the major compound of ‘sophie’ (51.8-55.9 %) an ‘blankyt’ (52.2-54.8 %), and in ‘sophie’, relatively high levels (6.98.9 %) of pinocarvone are also present. in ‘cyrano’, almost equal amounts of cisand trans-pinocamphones were present, which is different from all of the other materials (tab. 5) and similar to the majority of accessions studied by galambosi et al. (1993) and chalchat et al. (2001). the highest proportions of the pinocamphone isomers together were found in the genotype ‘erfurter ysop’ (70.7 %), while the lowest percentages were measured in ’blankyt’ (59.3 %). these two terpenic ketons especially cis-pinocamphone were reported to have strong antifungal activity (fraternale et al., 2004). the lower limit for the two ketons (33 %) fixed in the inter national standard on hyssop oil (iso 9841:2013) was reached by each accession while the upper limit (70 %) was slightly exceeded in some cases by the samples of ‘erfurter ysop’. tab. 3: results of the multivariate test for eo yield and total phenolics content data wilks’ lambda essential oil total phenolics f df sign. level f df sign. variety 0.039 87.725 4 0.000 25.082 4 0.000 year 0.169 98.853 2 0.000 1.472 2 0.240 variety × year 0.147 16.737 8 0.000 4.202 8 0.001 fig. 1: proportion of flowering individuals of one-year old hyssop accessions at different observation times (2012) 28 é. németh-zámbori, p. rajhárt, k. inotai another characteristic difference among our populations was the concentration of β-pinene which ranged from 11-19 % in the hungarian accessions, while the others contained 8-9 % of the compound. according to most references, all the other monoterpenes present in the investigated oils are common components of hyssop (chialva et al., 1983; lawrence, 1992; piccaglia et al., 1999; chalcat et al., 2001; zawislak, 2013b). the concentrations of some of them might vary on a large scale as reported e.g. for 1.8-cineole up to 53 % (vallejo et al., 1995). camphor, which was present at a concentration of more than 20 % in the samples of schulz and stahl-biskup (1991) and limonene and methyl eugenol reported previously at 16 % and 44 %, respectively, by piccaglia et al., (1999), were not detected in the investigated oils. sesquiterpenes were in the minority in the eo from each genotype; their levels do not exceed 10 % in any of them. according to available data, primarily camphor and limonene might contribute significantly to the antifungal activities of the eo (fraternale et al., 2004). the vegetation year did not have a significant effect on the composition as reflected by the marginal values in tab. 5. for the tpc, the multivariate test was significant (p=0.000) for variety and variety × year interaction, but not for year (tab. 3). in contrast to the eo yield, the content of polyphenols reached higher values in the german genotypes, than in the hungarian and po lish ones. the highest value was measured in ‘erfurter ysop’ (443.64 gae mg/g dw) and it differed significantly from each of the other accessions, with the exception of ‘blankyt’ (tab. 4). in contrast, the lowest accumulation level (329.32 gae mg/g dw) was established in samples of ‘hyzop lekarsky’, which differed significantly from the other four accessions. the vegetation year had no significant influence on the accumulation of phenolics in hyssop. the mean values of the five accessions indicate, that in the second year of their cultivation (2013), the plants produced slightly (by 6.4 % and 5.2 %) higher contents compared to 2012 and 2014, respectively. however, not each of the studied genotypes followed this tendency, what is reflected also in the significant variety × year interaction (tab. 3). discussion differences in petal colour of hyssop are well known (galambosi et al., 1993), heterogeneity in this respect may be considered as low in our accessions. the difference in flowering time has never been demonstrated in a comparison trial until now and reveals an additional feature of intraspecific variability in hyssop. the measured yield values of the eos in the experimental populations can be evaluated as advantageous compared to the majority of references (hodzsimatov and ramazanova, 1974; koller and range, 1997; piccaglia et al., 1999; bernotienė and butkienė, 2010). the statistical similarity of ‘cyrano’ and ‘sophie’ and their differences from the other three varieties may be a reflection of the fact that the hungarian variety ‘sophie’ is the result of long year selection from the stock material of ‘cyrano’, thus, these two ones are related taxa. no such relationship is known for the other three accessions, although it cannot be excluded. the eo yield of ‘blankyt’ according to the homepage of the firm pharmasaat, is between 0.4 tab. 4: yield of essential oil and total phenolic content for the examined accessions during three vegetation years (mean values) accession essential oil (ml/100g dw) total phenolics (gae mg/g dw) 2012 2013 2014 mean 2012 2013 2014 mean sophie 2.037a 1.457a 1.020a 1.507a 309.33c 406.63b 410.77a 375.58b erfurter ysop 0.933c 0.893 b 0.565c 0.797b 440.31a 445.10a 431.66a 443.64a blankyt 0.781c 0.862b 0.987a 0.847b 436.02a 406.61b 385.53b 409.39ab hyzop lekarsky 1.317b 0.674c 0.759b 0.916b 342.63b 337.11c 308.23c 329.32c cyrano 1.940a 1.224a 1.066a 1.411a 358.23b 353.27c 372.69b 361.74b mean 1.401 1.022 0.880 377.30 401.82 381.77 different letters indicate significant differences in the coloumns tab. 5: proportions (as gc peak area %) of the main components (present in >1%) of the studied hyssop essential oils during the three years compound lri a ki b ‘sophie’ ‘erfurter ysop’ ‘blankyt’ ‘hyzop lekarsky’ ‘cyrano’ sabinene 976 975 1.5-2.0 tr-1.0 1.1-1.2 tr-0.1 1.4-1.5 β-pinene 981 979 16.0-19.1 8.2-8.5 8.0-8.2 8.6-8.9 11.7-14.2 β-myrcene 995 990 0.9-1.5 tr-1.2 1.1-1.3 1.0-1.2 1.4-1.6 β-phellandrene 1029 1029 1.2-2.3 3.0-6.6 2.6-3.0 4.2-4.9 3.3-5.0 unidentified 3.0-3.5 tr-3.5 3.8-5.0 1.6-2.0 tr-0.8 trans-pinocamphone 1163 1162 1.3-5.0 5.2-11.6 4.4-8.3 0.3-2.5 27.1-35.0 pinocarvone 1166 1164 6.9-8.9 tr-0.8 2.9-5.7 2.9-4.5 3.3-4.8 cis-pinocamphone 1170 1175 51.8-55.9 65.5-73.6 52.2-54.8 58.1-64.4 27.7-38.6 myrtenol 1194 1195 1.0-1.7 tr-1.1 0.6-0.8 1.2-2.2 2.1-2.7 germacrene-d 1482 1481 0.6-1.0 0.5-1.2 1.3-1.8 0.3-0.9 0.3-1.1 bicyclogermacrene 1497 1500 0.5-0.9 0.9-2.2 2.3-2.6 0.9-1.8 0.3-1.5 elemol 1553 1549 0.2-0.8 0.5-1.8 1.0-1.8 0.4-1.2 0.2-1.2 β-bisabolol 1671 1675 0.3-0.5 0.2-1.4 1.8-2.4 1.8-2.9 0.5-1.0 a linear retention indices (lri) calculated relative to the elution ranking of n-alkanes on hp-5 column, b kovats retention index according to adams (2007) essential oil and phenolics in hyssop 29 and 0.7 ml/100 g, thus the data measured at our experimental station represents better values than this. the polish material has previously been investigated by zawislak (2011) who found similar high values (up to 1.7 %) in one-year old populations. the age of the plants might have an important effect on voc content of the flowers, with decreasing tendency in the older plantations. there are very few former reports concerning this aspect. our data seem to be in accordance with the findings of hodzsimatov and ramazanova (1974). on the other hand, gille and floria (2000) demonstrated a significant elevation in eo yield in a local pink flowering population called ‘de ciorani’ from the second to the third year. the authors concluded on the most advantageous meteorological conditions as a reason for this finding. galambosi et al. (1993) reported interaction between the effects of age and genotype, however, not each accession has been investigated in each year in that study. we could systematically ascertain this statement calculating the significant interaction between genotype and year. it shows, that the degree of influence of the year on the eo yield is dependent on the variety. obviously, vegetation year and weather conditions are associated closely with each other. previously we demonstrated that in years of higher precipitation during the development of the flowers, the eo yield tends to decrease (németh et al., 2001). in the recent experiment, there was only moderate rainfall in the flowering period, and in none of the years was more than 30 mm precipitation registered in the decades before harvests (tab. 2). thus, a large influence of the weather may not be concluded. it was established, that each of the experimental accessions produced cisand trans-pinocamphones as main constituents of their vocs (lawrence, 1992; galambosi et al., 1993; koller and range, 1997; fraternale et al., 2004; zawislak, 2013a). the composition of the eo proved to be qualitatively similar for each accessions. the main quantitative differences were found in the proportion of the pinocamphone isomers. zawislak (2013a) reported a significant change in the proportions cisand trans-pinocamphones from vegetative to full flowering phase. nevertheless, all of our samples were harvested when in full flower, therefore the detected chemical variability is probably a genetic characteristic. the very low proportions of sesquiterpenes described in our experimental samples seems to be a common feature of hyssopus officinalis (chialva et al., 1983; vallejo et al., 1995; piccaglia et al., 1999; zawislak, 2013b). in contrast to our findings regarding the eo yield, the data demonstrated that the year did not have a considerable influence on the eo composition. this coincides with several former findings for other species (németh-zámbori, 2015). there are only few reports on the polyphenolic content of hyssop and no comparisons of different accessions of the species. zawislak (2011) reported 0.32-0.55 % flavonoids and 0.34-0.75 % tannins in the herb. although the genotype was identical to the one examined by us (‘hyzop lekarsky’ from firm pnos), the analytical methods based on polish pharmacopoeia vii are different, thus, data can not be directly compared. similarly to the eo composition, the vegetation year seems to have less influence on this phytochemical characteristics than does the genotype. the three year-long study on five hyssop accessions demonstrated that selected materials exhibit a better quality and morphological homogeneity compared to trade items of genetically uncertain origin, thus, a more intensive breeding of hyssop would be necessary to support cultivation. however, to optimize voc and phenolic production in parallel, a well oriented selection is needed for both traits. conflict of interest: the authors declare that they have no conflict of interest. references adams, r.p., 2007: identification of essential oil components by gas chromatography/mass spectrometry. 4th edn. allured publ. corp, carol stream, il usa. bernotienė, g., butkienė, r., 2010: essential oils of hyssopus officinalis l. cultivated in east lithuania. chemija, 21, 135-138. chalchat, j., adamovic, d., gorunovic, m.s., 2001: composition of oils of three cultivated forms of hyssopus officinalis endemic in yugoslavia: f. albus, f. cyaneus and f. ruber. j. essent. oil res. 13, 419-421. doi:10.1080/10412905.2001.9699712. chialva, f., liddle, p.a.p., doglia, g., 1983: chemotaxonomy of wormwood (artemisia absinthium l.). z. lebensmittel-untersuchung und -forschung, 176, 363-366. doi:10.1007/bf01057728. chrpová, d., kouřimská, l., gordon, m.h., heřmanová, v., roubíčková, i., pánek, j., 2010: antioxidant activity of selected phenols and herbs used in diets for medical conditions. czech j. food sci. 28, 317-325. fraternale, d., ricci, d., epifano, f., curini, m., 2004: composition and antifungal activity of two essential oils of hyssop. j. essent. oil res. 16, 617-622. doi: 10.1080/10412905.2004.9698810. galambosi, b., svoboda, k,p., deans, a.g., héthelyi, é., 1993: agronomical and phytochemical investigation of hyssopus officinalis. agric. sci. finl. 2, 293-302. gille, e., floria, e., 2000: a comparative morphological study of some varieties of hyssopus officinalis l. treated with gamma irradiation. proc. of the first conference on medicinal and aromatic plants of southeast european countries, may 29 june 3, arandelovac, jugoslavia, 387-393. hodzsimatov, k.h., ramazanova, n., 1974: some biological characteristics of essential oil accumulation and composition in hyssop cultivated in taskent region. (in russian). rastitelnije resursi, 11, 238-242. koller, w.d., range, p., 1997: geruchsprägende inhaltsstoffe von fenchel und ysop, z. arzn. gew. pfl. 2, 73-80. lawrence, b.m., 1992: progress in essential oils. perfum. flav. 17, 54-55. németh, é., varga, e., franke, r., 2001: variabilität des ätherischen ölgehaltes und deren ursachen in hyssopus officinalis l. z. arzn. gew. pfl. 6, 29-34. németh-zámbori, é., 2015: natural variability of essential oil components. in: baser, k.h.c, buchbauer, g. (eds.), handbook of essential oils, 87126. crc press taylor and francis group llc, boca-raton u.s. piccaglia, r., pace, l., tammaro, e., 1999: characterisation of essential oils from three italian ecotypes of hyssop (hyssopus officinalis ssp. artistatus briq.). j. essent. oil res. 11, 693-699. doi: 10.1080/10412905.1999.9711998. schulz, g., stahl-biskup, e., 1991: essential oils and glycosidic bound volatile from leaves, stems, flowers and roots of hyssopus officinalis. flavour frag. j. 6, 69-73. doi: 10.1002/ffj.2730060109. vallejo, m.c.g., herraiz, j.g., pérez-alonso, m.j., velasconegueruela, a., 1995: volatile oil of hyssopus officinalis l. from spain. j. essent. oil res. 7, 567-568. doi: 10.1080/10412905.1995.9698590. veres, k., varga, e., hajdú, zs., máthé, i., janicsák, g., dobos, á., tarján, g., 1995: changes of chemical composition of hyssopus offi-cinalis l. during vegetation period. summaries of the 9th hungarian medicinal plant conference and 4th conference on phytotherapy, (october, 18-20. szeged, hungary), 51. vlase, l., benedec, d., hanganu, d., damian, g., csillag, i., sevastre, b., mot, a.c., silaghi-dumitrescu, r., tilea, i., 2014: evaluation of antioxidant and antimicrobial activities and phenolic profile for hyssopus officinalis, ocimum basilicum and teucrium chamaedrys. molecules, 19, 5490-5507. doi: 10.3390/molecules19055490. zawislak, g., 2011: hyssop herb yield and quality depending on harvest term and plant spacing. acta sci. pol. hortorum cultus, 10, 331-342. zawislak, g., 2013a: the chemical composition of essential hyssop oil depending on plant growth stage. acta sci. pol. hortorum cultus, 12, 161170. 30 é. németh-zámbori, p. rajhárt, k. inotai zawislak, g., 2013b: morphological characters of hyssopus officinalis l. and chemical composition of its essential oil. modern phytomorhology, 4, 93-95. address of the corresponding author: dr. éva németh-zámbori, department of medicinal and aromatic plants, szent istván university, 1518 budapest, p.o. box 53, hungary e-mail: zamborine.nemeth.eva@kertk.szie.hu © the author(s) 2017. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 92, 15 23 (2019), doi:10.5073/jabfq.2019.092.003 1 recursos genéticos y productividad-genética, colegio de postgraduados, mexico 2 departamento de fitotecnia-universidad autónoma chapingo, edo. méxico, mexico 3 laboratorio de análisis bioquímico e instrumental, cinvestav, guanajuato, mexico metabolomic study of volatile compounds in the pigmented fruit from mexico crataegus genotypes maría dolores pérez-lainez1, tarsicio corona-torres1, maría del rosario garcía-mateos2*, robert winkler3, alejandro f. barrientos-priego2, raúl nieto-ángel2, víctor hebert aguilar-rincón1, josé armando garcía-velázquez1 (submitted: july 12, 2018; accepted: december 30, 2018) * corresponding author summary crataegus is distributed worldwide and presents a phenotypic diversity in size, shape, color and aroma of the fruit. the objective of this study was to identify genotypes of crataegus with a similar profile of volatile compounds by means of a metabolomic study. in addition, the content of pigment was evaluated to contribute to the agronomic, medicinal and chemotaxonomic value. color determination, total carotenoids (tc) and total anthocyanins (ta) were determined in the exocarp and mesocarp of fresh fruits by means of spectrophotometry. the volatile compounds were determined by low temperature plasma coupled to mass spectrometry (ltpms). a total of 75 volatile compounds were detected, according to abundance and mass-to-charge ratio, which by means of principal component analysis (pca) and selection of variables; genotypes were grouped according to size and origin. the pigment content was related to the physical color of the fruit. the highest concentration of carotenoids was 42.35 μg·g-1 fw. in the genotype po5, and 992.34 μg·g-1 fw for anthocyanins in the genotype ch18, concen trations of both compounds found in the exocarp of the fruit. keywords: anthocyanins, carotenoids, crataegus sp., volatile com pounds. introduction the genus crataegus (family rosaceae) displays wide phenotypic and genetic diversity grouping approximately 150 species, of which 55 are located in the euro-asian continent (europe, the middle east, east asia), and 95 in the american continent (phipps, 1997; phipps et al., 2003). in mexico there are 15 species reported (eggleston, 1909; phipps, 1997; núñez-colín et al., 2011), which are called “tejocote” (mexican hawthorn), a word that derives from nahuatl “te-xocotl” in reference to the hardness and acidity of the fruit (cabrera, 1992). the phenotypic diversity of this genus is appreciated in the different organs of the tree, among them the fruit, which vary in shape, size and color of the exocarp (skin) (yellow, orange, red and black), with different tonality and intensity. the mesocarp (pulp) goes from yellow, orange, greenish white, reddish white to non-homogeneous diffuse red (nieto-angel and borys, 1992; phipps et al., 2003). the fruit is also characterized by its flavor (flavor and taste) particular property of this species; in china, it has an intense and unique aroma that influences the acceptability and taste of food products, such as juices and jams due to the presence of some volatile compounds (zhao et al., 2015). attributes such as color, aroma, taste, texture, appearance, food safety and the nutritional value of fresh fruit are factors critical to consumer (barret et al., 2010). in tejocote, specifically the color and aroma influence the quality of the fruit and importance of its agro-industrial use (preparation of beverages with and without liquor, fruit paste and syrups); as well as, its consumption as fresh fruit. in mexico, since prehispanic times, different parts of the mexican hawthorn tree have been used in traditional medicine (edwards et al., 2012), current ly, this fruit is used preferably in traditional offerings, religious ceremonies and christmas (borys and leszczyñska-borys, 1994). morphological and molecular studies (nieto, 2007; betancourtolvera et al., 2017) of some genotypes and species of the genus crataegus have contributed to the taxonomic characterization and conservation of this under-utilized resource. many studies of some species located in china, turkey, europe and the united states report the presence of bioactive compounds (simple phenols, polyphenols, carotenoids, anthocyanins and flavonoids, among others) (caliskan et al., 2012; kostić et al., 2012; veberic et al., 2015; liu et al., 2016); which justify its medicinal properties (hypertension, angina pectoris, atherosclerosis, indigestion and ab dominal distension) (chang et al., 2002) and pharmacological properties of the cardiovascular system, as well as its antioxidant activity (cui et al.,2006). despite the great genetic and phenotypic diversity, few metabolites are identified in mexican species (banderas-tarabay et al., 2015; garcía et al., 2012). the variation of volatile compounds and pig ment content (anthocyanins and carotenoids) among these species is hitherto unknown; the profile of these elements could be considered the chemical fingerprint of each species, keys for the understanding of their genetic variation. on the other hand, metabolomic studies of bioactive compounds have been used to describe the phenotypic variation of a wide range of phytochemicals or changes in plant chemical composition. therefore, the objective of this study was to identify genotypes of crataegus that presented a similar profile of volatile compounds by means of a metabolomic study, and to evaluate the pigment content to contribute to the agronomic, medicinal and mainly chemotaxonomic value. materials and methods plant material hawthorn fruit was randomly collected at a commercial maturity stage in september-october 2016 and 2017 from the ex situ germ plasm bank at the university of chapingo (uach), estado de mexico, mexico; located at 19° 29’n and 98° 53’ w, at 2249 m height. the climate is classified as c (wo) (w)b (i’) g, corresponds to temperate sub-humid, with a mean annual precipitation of 645 mm and an average annual temperature of 15 ± 2 °c (garcía, 1988). the material was placed in paper bags previously labeled, in a coo ler at 4 °c. the samples harvested in 2016 were stored at -4 °c until analysis. the material harvested in 2017 was stored at -20 ± 2 °c until study. tab. 1 shows the site of origin of the genotypes studied, identified and grouped according to their morphological characte ristics (nieto, 2007; betancourt-olvera et al., 2017). before the analysis of metabolites, the equatorial diameter of 10 fruits was 16 m.d. pérez-lainez, t. corona-torres, m. del rosario garcía-mateos, r. winkler, a.f. barrientos-priego, raúl nieto-ángel, v.h. aguilar-rincón, j.a. garcía-velázquez measured using a vernier, an interval was obtained from the measurements; based on that fruits were grouped by size as follows: small (12.60 18.40 mm), medium (18.50 28.00 mm) and large (29.00 34.00 mm). fig. 1 shows the size and color of each of the genotypes studied. preparation of the sample three fruits fruit of each genotype (eu) were analyzed separately, without pre-treatment, each fruit was cut longitudinally, immediately the surface of the cut was exposed directly to the mass spectrometry team for analysis. for this study we used the methodology pro posed by martínez-jarquín and winkler (2013), with a lowtemperature plasma ionization source coupled to mass spectrometer (ltp-ms) lcq fleet (thermo scientific, waltham, massachusetts, usa). the advantage of this technique allows to analyze the volatile compounds of a sample without the previous extraction, avoiding low yields and losses of some compounds during the extraction, as well as the detection of these metabolites present in low concentrations (martínez-jarquín and winkler, 2017). recently, the method has been applied for the analysis of volatile compounds in coffee, tequila and mezcal (gamboa-becerra et al., 2017; martínez-jarquín et al., 2017). detection of volatile compounds the fresh fruit cut was exposed directly to the plasma beam coupled to the mass spectrometer until obtaining 20 mass spectra in a range of 50 to 500 m/z per cut in a fruit. three fruits were analyzed separately, finally 60 spectra were obtained by genotype. helium was used as discharge gas, the capillary temperature was 80 ± 2 °c, voltage of 10 kv and a frequency of 10 khz. the equipment’s previous calibra tion was previously performed as reported by martínez-jarquín and winkler (2013). measurement of color parameters for the color analysis (physical color measurement), 23 genotypes were used because the genotype ch69 was not fruitful in the year of collection (2017). the color of the exocarp and mesocarp of fresh fruit was determined by means of the evaluation of l* (luminosity), hue angle (hue) and color purity or chromaticity index (chroma) with a miniscan® ez 4500l spectrophotometer (hunterlab, vir ginia, usa). the readings of a* and b* were obtained to identify the color differences between tissue and genotypes in numerical form. variables were estimated with the following equations: hue = tan-1 (a/b); chroma = (a2+ b2) ½ (mc. guire, 1992). three fruits of each genotype with similar commercial maturity stage were selected; for each fruit three different points of the exocarp were studied. subsequently, for the measurement of pulp color, the fruit was cut into three portions longitudinally, each measurement was made in triplicate. measurement of total carotenoids the total quantification of carotenoids (tc) was carried out separately in the exocarp and mesocarp of the fresh fruit as indicated by méndez-iturbide et al. (2013) with minor modifications. to obtain the sample, between three and twenty fruits were used per genotype, depending on the size of the fruit. the tissue (2 g) tab. 1: geographical characteristics of 24 genotypes of the mexican hawthorn (crataegus sp.). genotype species* statea originb lat long h(m) mdc ch04 sulfurea chiapas s c de las casas 16.75 92.67 2300 oct-dec ch08 tracyi chiapas s c de las casas 16.75 92.67 2300 oct-nov ch10 tracyi chiapas s c de las casas 16.75 92.67 2300 sept-nov ch13 tracyi chiapas rancho nuevo 16.67 92.57 2400 dec-jan ch15 aurescens chiapas mitzitan 16.65 92.55 2380 jan-feb ch16 tracyi chiapas mitzitan 16.65 92.55 2380 nov-jan ch18 tracyi chiapas rancho robelo 16.67 92.45 2250 nov-feb ch19 baroussana chiapas mitzitan 16.65 92.55 2380 dec-jan ch22 rosei chiapas mitzitan 16.65 92.55 2380 oct-nov ch27 gracillior chiapas rancho robelo 16.67 92.45 2250 jan-feb ch43 greggiana chiapas rancho robelo 16.67 92.45 2250 jan-feb ch44 gracillior chiapas mitzitan 16.65 92.55 2380 oct-nov ch51 mexicana chiapas mitzitan 16.65 92.55 2380 nov-feb ch69 cuprina chiapas candelaria 16.70 92.53 2320 oct-nov ch72 baroussana chiapas s j yashitinin 16.65 92.45 2350 nov-dec ch83 tracyi chiapas s c de las casas 16.75 92.67 2300 sept-nov p02 tracyi puebla rancho nuevo 19.14 98.57 2620 sept-oct p05 aurescens puebla mitzitan 19.14 98.50 2628 sept-oct p06 tracyi puebla mitzitan 19.14 98.50 2625 sept-oct p25 cuprina puebla origen 19.10 98.47 2420 dec-feb p55 sulfurea puebla s c de las casas 19.17 98.40 2280 dec-feb p86 mexicana puebla huejotzingo 19.17 98.40 2280 nov-dec p100 mexicana puebla huejotzingo 19.17 98.40 2280 oct-dec em66 tracyi edo. mex. s c de las casas 19.48 98.77 2700 sept-oct a s c de las casas = san cristóbal de las casas; s j yashitinin = san josé yashitinin; s c del monte = santa catarina del monte. b edo. mex. = estado de méxico. c md = commercial maturity date (nieto, 2007). metabolomic study of volatile compounds in mexican crataegus genotypes 17 was ground in a mortar with 20 ml of hexane, acetone and ethanol (50:25:25 v/v), the solution was kept at 4 °c in the dark for 30 min, then it was filtered. each extraction was made in triplicate. the absorbance of the organic phase was measured at 450 nm in a multiscan® go spectrophotometer (thermo scientific, waltham, massachusetts, usa). hexane was used as a blank solution. to calculate the concentration (μg) of total carotenoids the following equation was used: μg = a × v (ml) × 106/ ε × 100; where ε = extinction coefficient of β-carotenoid (2505); v = volume of sample; a = absorbance obtained (méndez-iturbide et al., 2013). results are expressed in μg·g1 of fresh weight (fw): μg·g-1 = μg / weight of sample (g). measurement of total anthocyanins the concentration of total anthocyanins (ta) was determined by the differential ph method (giusti and wrolstad, 2001). the extrac tion of anthocyanins was carried out in the exocarp and mesocarp of the fresh fruit, in triplicate. for each replication, 2.0 g of fresh tissue was ground in a mortar with 20 ml of methanol acidified with hcl at 0.01 (v/v %) in a 10:1 ratio. then the extract was allowed to rest for 24 h in the dark at 4 °c. two test tubes were prepared with 0.5 ml of the extract, for each sample; then 25 ml of kcl and 25 ml of ch3coona·3h2o solution were added to one tube. the tubes were allowed to rest for 15 min in the dark to stabilize the reactions. the absorbance of each sample was measured at 520 and 700 nm in a multiscan® go spectrophotometer (thermo scientific, waltham, massachusetts, usa). the blank solution used was acidified methanol. the concentration of anthocyanins was expressed as cyanidin3-glucoside with the equation: ta (mg·l-1) = a × mw × df × 1000 ε-1; where ta (mg·l-1) is the concentration of total anthocyanins, mw = molecular weight of cyanidin-3-glucoside (445.2), df = dilution factor, ε = extinction coefficient (26 900) and a = absorbance obtained with the following equation: abs = (a520-a700)ph1(a520-a700)ph4.5 (wrolstad et al., 2005). statistic analysis the r program version 3.4.3 (http://www.rproject.org) with the rstudio interface (http://www.rstudio.com) was used for the data analysis (volatile compounds and physical-chemical color variables). the mass spectra obtained in the analysis of volatile compounds in a range of 50 to 500 m/z in format .raw were changed to the numerical format .mzml using the program msconver. the package maldiquant was used to process the mass data of the 60 spectra obtained by genotype, to obtain the final mass spectrum. subsequently, the package pheatmap generated the heat map with the abundance of the volatile compounds and the principal components analysis (pca) was obtained by means of the prcomp, to integrate the metabolomic analysis (grace and hudson, 2016). the color variables (l, hue, chroma, ta and tc) studied in the exocarp and mesocarp of the fruit were subjected to a one-way anova and to the analysis of the anova assumptions and a comparison of means (p < 0.05). due to the fact that not all the variables fulfilled the data normality test, for the variable hue in the exocarp, and ta and tc in the mesocarp, a data transformation was carried out using the logarithm method. the tc variables in the exocarp and hue in the mesocarp were analyzed by means of the kruskal-wallis test (p < 0.05). the packages used in this study were: agricolae, to obtain tukey’s mean comparison (p < 0.05); car, to perform the levene test in the analysis of assumptions and pgirmes to carry out the kruskalwallis test. results and discussion volatile compounds the analysis of volatile compounds by ionization with low-ttemperature plasma coupled to mass sspectrometry (ltp-ms) allowed to detect 75 compounds with increased relative intensity (%). their low molecular weight, 50 to 500 m/z, suggests that those volatile compounds are associated with the aroma of the fruit. fig. 2 shows only the mass spectra of two genotypes (p86, spectrum a and ch44, spectrum b) due to reasons of space. a principal components analysis (pca) was carried out with the 75 metabolites detected (compounds of the fruit aroma). the results were interpreted based on their eigenvalues and eigenvectors. the eigenvalues and the variance explained for each of the compounds are shown in tab. 2, where it is observed that five compounds justify 81.96% of the total variance. it was also found that the volatile compounds that provided 34.89% of the total variance in the first component (pc1) were those with m/z ratio: 144.98, 173.01, 270.8, 344.74, 117.03, 187.01, 200.99, 284.8, 159.0, 298.77, 256.79, 278.97, 358.74, 330.73, 235.1, 218.48, 103.06, 345.67, 204.12, 223.04 and 400.76. the volatile compounds of m/z: 288.74, 260.78, 98.99, 274.77, 117.03, 232,86, 302.68, 56.95, 81.01, 246.76, 153.12, 92.92, 127.06 and 250.91 provided 23.61% of the total variance of the se cond component (pc2). in the third component (pc3) the volatile compounds 228.87, 214.90, 219.0, 262.87, 258.86 and 230.79 pro vided 22.18% of the total variance. from the pca, the dispersion of the study genotypes in the first two principal components was obtained graphically (fig. 3), where a cluster was observed with the genotypes ch08, ch10 and ch51, from the state of chiapas. the second group corresponded to the genotypes pa06, pa05, pa02 and p100 from atexcac and huejotzfig. 1: fruit image of 24 hawthorn (crataegus sp.) genotypes. 18 m.d. pérez-lainez, t. corona-torres, m. del rosario garcía-mateos, r. winkler, a.f. barrientos-priego, raúl nieto-ángel, v.h. aguilar-rincón, j.a. garcía-velázquez exocarp of some genotypes. group i grouped the genotypes of small size (12.60 18.40 mm), more than 50% with red exocarp and 92% are from chiapas, with the exception of the genotype p25 (yellow exocarp from puebla). group ii has genotypes from three states (chiapas, puebla and estado de mexico) that were characterized by medium to large fruits (18.50 34.00 mm), with yellow, yelloworange and red exocarp. the variation in the size and in some cases the origin explain the differences between groups, the coloration of the exocarp of the fruit, as the species were not determining factors. the clusters showing high similarity (p100-p02, ch08-ch10, ch44-p86, ch13-ch72, ch15-ch27) in their volatile profile had in common the origin, species and/or color of the fruit, the exception was the cluster with the genotypes ch83 and p05. on the other hand, the volatile compounds of greater abundance are shown on the heat map (fig. 4), this is represented by the color scale where yellow corresponds to the lowest abundance up to red which corresponds to greater abundance. the five most abundant volatiles were only present in some genotypes (tab. 3) of the two groups of the dendrogram, e. g. the compound 260.78 m/z was found only in the genotypes of group ii, with different abundance. in contrast, the compound 144.98 m/z with different abundance was identified in all genotypes of groups i and ii. in this regard, two structural metabolites (hexyl acetate and butyl butanoate) with the same m/z (144,21) have been reported in different species (c. aestivalis, c. opaca and fig. 2: metabolomic profile of the volatile compounds obtained by ltp-ms. a) genotype p86 (c. mexicana); b) genotype ch44 (c. gracillior). tab. 2: eigenvalues and variance proportion explained by the principal component analysis of volatile compounds from the genotypes of crataegus. compound eigen values vpc (%) cv (%) 1 26.170 34.89 34.89 2 17.707 23.61 58.50 3 8.385 22.18 69.68 4 5.930 7.91 77.59 5 3.279 4.37 81.96 vpc: variance per compound. cv: cumulative variance. fig. 3: principal components of crataegus sp. genotypes by volatile compounds. ingo, puebla. in this same group, we found the genotypes ch83 and ch18 from two different regions of the state of chiapas (fig. 3). in the dendrogram located in the upper part of fig. 4 there are two groups (i and ii) with the 24 genotypes evaluated; in the lower part, the heat map is shown containing the abundance (relative intensity %) and the presence of the 37 volatile compounds (m/z ratio) selected by the principal component analysis. the color row of the dendrogram represents the color of each genotype, e.g. the red boxes are associated with the red coloration of the metabolomic study of volatile compounds in mexican crataegus genotypes 19 c. rufula) of crataegus from the united states by horvat and chapman (1991) and cha et al. (2011), however the last metabolite was found in the aroma of a different species (c. pinnatifida) cultivated in china (lingyun and bijun, 1997). both compounds have also been identified in the aroma of apple (espino-díaz et al., 2016). there are few studies that report the volatile profile in the crataegus fruit; of the 37 compounds reported in this study only seven coincide with those reported in the fruit (tab. 4), however volatile compounds have been reported in flowers (kovaleva et al., 2009) and leaves (lakache et al., 2014). in this study, even though the detection of volatiles with the ltpms technique did not allow the structural identification of the compounds identified by their m/z ratio, the heat map allowed to prove a different chemical profile for each genotype. color parameters the color parameters evaluated provide quality attributes that are important in consumer preferences. the lower values (< 52) of chroma (purity of color) obtained in the exocarp of the red fruits showed great variability in the intensity of color in comparison with yellow fruits which showed higher values (> 66) (tab. 4). the fruits with yellow exocarp (p55 and p05) and mesocarp (ch72 and ch83) had the highest values (> 61 and > 71, respectively) of luminosity (tissues with brighter colors) and hue (yellow exocarp and mesocarp), compared to red exocarp fruits with a luminosity of 32.46 to 47. 49 h 26 to 32 ° (tab. 5). it is important to mention that yellow and larger fruits (p55, p02 and p100), grown in puebla (first producer state at a national level with a production of 3500 t per year), are the most demanded in mexico due to their agroindustrial and cultural use (sagarpa, 2015). content of carotenoids carotenoids are natural pigments metabolized by plants, are responsible for the colors yellow, orange and red in some fruits and vegetables. the apocarotenoids (derivatives) are the result of the breaking of the carotenoids present in the aroma of flowers and fruits (namitha and negi, 2010). the exocarp of the genotypes p55 and p05 showed the highest concentrations of carotenoids (42.09 and 42.35 μg·g-1 fw, respectively), which coincides with the higher values found for hue in the present study. however, despite of having small fruits, the mesocarp of genotype p25 had the highest concentration of carotenoids (20.92 μg·g-1 fw), but also a considerable concentration in the exocarp (38.17 μg·g-1 de fw), this non-commercial genotype from puebla was the only one in group i, where most of the fig. 4: the heat map shows the abundance (color intensity) of each of the volatile compounds listed in the right column (m/z ratio). the dendrogram (upper part) shows the clusters of the genotypes by coloration of the hawthorn (crataegus sp.) fruit. 20 m.d. pérez-lainez, t. corona-torres, m. del rosario garcía-mateos, r. winkler, a.f. barrientos-priego, raúl nieto-ángel, v.h. aguilar-rincón, j.a. garcía-velázquez genotypes from chiapas were grouped. concentrations in the exocarp of the two genotypes (p100 and p02) from c. mexicana were higher (38.68 and 33.61 μg·g-1 fw) than that reported (26.4 μg·g-1 fw) by méndez-iturbide et al. (2013), but in lyophilized exocarp of the same species from the state of tlaxcala, mexico (tab. 5). the differences in the contents of both studies could be explained mainly by environmental factors, different stage of fruit ripening and postharvest management (reilly, 2013), as has been reported in other fruits (lópez-vidal et al., 2014; mesejo et al., 2011). the content of carotenes in hawthorn was lower than that reported for melon tab. 3: the five volatile compounds with increased relative intensity (%) by genotype. genotype m/z (% abundance) ch4 173.01 (4.31) 270.80 (4.11) 144.98 (4.01) 242.81 (3.99) 98.99 (3.94) ch8 270.80 (5.18) 173.01 (5.12) 344.74 (2.74) 98.99 (2.87) 144.98 (2.47) ch10 173.01 (5.02) 270.80 (4.39) 344.74 (2.87) 144.98 (2.57) 187.01 (2.51) ch13 173.01 (5.03) 144.98 (4.14) 270.80 (3.44) 200.99 (2.37) 288.74 (2.34) ch15 144.98 (4.95) 270.8 0 (4.22) 173.01 (3.70) 98.99 (3.34) 288.74 (3.21) ch16 173.01 (6.53) 144.98 (4.53) 270.80 (2.73) 344.74 (2.63) 288.74 (2.36) ch18 144.98 (4.11) 173.01 (3.79) 270.80 (2.16) 98.99 (2.11) 288.74 (2.02) ch19 98.99 (3.19) 173.01 (3.15) 144.98 (2.87) 270.80 (2.83) 200.99 ( 2.44) ch22 144.98 (6.91) 288.74 (6.82) 274.77 (2.68) 260.78 (2.54) 173.01 (2.13) p25 144.98 (5.45) 173.01 (4.62) 288.74 (3.32) 270.80 (2.99) 98.99 (2.44) ch27 144.98 (4.62) 173.01 (4.35) 270.80 (4.01) 288.74 (3.08) 98.99 (2.56) ch43 144.98 (4.28) 173.01 (3.87) 270.80 (3.75) 98.99 (3.14) 288.74 (2.11) ch44 288.74 (12.25) 260.78 (8.12) 144.98 (7.85) 274.77 (3.72) 117.03 (3.10) ch51 173.01 (4.46) 270.80 (4.42) 187.01 (2.97) 98.99 (2.81) 144.98 (2.75) p55 288.74 (6.45) 144.98 (6.07) 260.78 (4.11) 274.77 (3.82) 117.03 (2.46) em66 144.98 (5.62) 288.74 (4.70) 173.01 (2.99) 270.80 (2.52) 260.78 (2.46) ch69 173.01 (4.56) 144.98 (4.02) 270.80 (2.29) 200.99 (2.24) 117.03 (2.07) ch72 173.01 (5.95) 270.80 (3.98) 144.98 (3.72) 344.74 (3.11) 200.99 (2.30) ch83 144.98 (5.95) 98.99 (3.19) 28.84 (3.02) 173.01 (2.19) 270.80 (1.56) p86 288.74 (8.03) 144.98 (6.74) 260.78 (5.21) 117.03 (2.73) 274.77 (2.65) p100 144.98 (6.55) 288.74 (4.56) 98.99 (2.30) 260.78 (1.85) 117.03 (1.77) p02 144.98 (5.52) 288.74 (3.81) 98.99 (2.25) 173.01 (1.62) 117.03 (1.61) p05 144.98 (6.46) 288.74 (3.52) 98.99 (2.59) 173.01 (1.69) 117.03 (1.45) p06 144.98 (4.58) 98.99 (4.52) 173.01 (2.06) 117.03 (2.00) 270.8 (1.72) tab. 4: volatile compounds identified by gc-ms in the crataegus fruit name formula m/z m w species source 2-hexenal c6h10o 98.99 98.145 c. aestivalis, lingyun and bijun, 1997 c. opaca, horvat and chapman, 1991 c. rufula y c. piinnatifida benzaldehyde c7h6o 106.99 106.124 c. aestivalis, lingyun and bijun, 1997 c. opaca, robertson et al., 1993 c. rufula y c. piinnatifida hexyl acetate c8h16o2 144.98 144.214 c. aestivalis, lingyun and bijun, 1997 c. opaca, horvat and chapman, 1991 c. rufula y c. piinnatifida butyl butanoate c8h16o2 144.98 144.214 c. aestivalis y cha et al., 2011 c. opaca citral c10h16o 152.06 152.237 c. piinnatifida lingyun and bijun, 1997 hexyl hexanoate c12h24o2 200.99 200.322 c. aestivalis, horvat and chapman, 1991 c. opaca y c. rufula β-caryophyllene c15h24 204.12 204.357 c. monogyna robertson et al., 1993 m/z: mass-to-charge ratio; mw: molecular weight. metabolomic study of volatile compounds in mexican crataegus genotypes 21 (185.0 μg·g-1 fw) by martínez-valdivieso et al. (2014), but higher than that found in peach (12.0 μg·g-1fw) (cambell and padilla, 2013). therefore, the consumption of hawthorn fruits represent a source of carotenoids in the human diet, important in the prevention of vitamin a deficiencies, cancer, cardiovascular diseases, age-rela ted macular degeneration and cataract formation; beside this fruit has an important antioxidant activity (arathi et al., 2015). anthocyanin content the anthocyanins comprise another type of pigments responsible for the coloring of fruits from red to purple, are phenolic with antioxidant properties, which justify their nutraceutical properties (delgado-vargas et al., 2000). it was found that the genotypes with intense and dark red exocarp (ch18, ch51 and ch19) had the highest concentrations (992.34, 844.69 and 747.69 μg·g-1fw, respectively) (tab. 4) which corresponds to the smallest values of hue (< 28 °) and chroma (< 52), concentrations higher than those reported by froehlicher et al. (2009) (58.0 mg ·100 g-1 fw) in the exocarp of c. monogyna; however, lower than those reported for cranberries (206.0 mg·100 g fw) (ribera et al., 2010) and raspberry (28.7 55.6 mg·100 g-1 fw) (peña-varela et al., 2006). in contrast, yellow exocarp fruits showed very small concentrations (5.56 30.3 μg·g-1 fw). in the mesocarp of all genotypes a lower concentration of these pigments was observed (6.95 29.22 μg·g-1 fw), the exceptions were the genotypes ch10 and ch18, which had the highest anthocyanin values (29.22 and 28.38 μg·g-1) and higher hue values (> 51°) (tab. 5). it should be noted that in most of the genotypes the highest concentration of both pigments predominated in the exocarp, the exception was the genotypes p25 and ch18, where the concentrations in the fruit (exocarp + mesocarp) were important from the nutraceutical and medicinal point of view, because their consumption could provide high content of antioxidant pigments, for health benefits. conclusions the profiles of chemical volatile compounds were different among the 24 genotypes of the mexican crataegus. the metabolomic study allowed to cluster the genotypes according to their chemical profile. the differences in the volatile profile were related to the size of the fruit, and in some genotypes to their origin (state of chiapas). no relationship of volatile compounds by color or by species was found. the highest concentration of carotenoids was observed in the exocarp in comparison with the mesocarp of yellow fruits, in contrast the red fruits showed the highest concentrations in the exocarp. the ltp-ms technique allowed to detect volatile compounds of low abundance when analyzing the sample without pretreatment. the results of the present research could be used for the development of products derived from hawthorn with high levels of bioactive compounds that would justify their medicinal properties. acknowledgments we thank dr. maria teresa carrillo rayas (cinvestav unidad irapuato, cui), and maria isabel cristina elizarraraz anaya (cui), thermo and waters mexico for technical support. the project was funded by the conacyt fronteras project 2015-2/814 and the bi lateral grant conacyt-dfg 2016/277850. mdpl acknowledges her conacyt postgraduate scholarship. references arathi, b.p., sowmya, p.r., vijay, kbaskaran, v., lakshminarayana, r., 2015: metabolomics of carotenoids: the challenges and prospects – a review. trends food sci. tech. 45, 105-117. doi: 10.1016/j.tifs.2015.06.003 barrett, d.m., beaulieu, j.c., shewfelt, r., 2010: color, flavor, texture, and nutritional quality of fresh-cut fruits and vegetables: desirable le vels, instrumental and sensory measurement, and the effects of processing. crit. rev. food sci. nutr. 50, 369-389. doi: 10.1080/10408391003626322. banderas-tarabay, j.a., cervantes-rodríguez, m., grada-sánchez, m., espindola-lozano, m., cuevas-romero, e., navarro-ocaña, a., mendez-iturbide, d., 2015: antioxidant-mediated protective effect of hawthorn 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characteristics, biosynthesis, processing, and stability. crit. rev. food sci. nutr. 40, 173289. doi: 10.1080/10408690091189257 edwards, j.e., brown, p.n., talent, n., dickinson, t.a., shipley, p.r., 2012: a review of the chemistry of the genus crataegus. phytochemistry 79, 5-26. doi: 10.1016/j.phytochem.2012.04.006 echeverría, g., fuentes, m.t., graell, j., lópez, m.l., 2003: rela tionships between volatile production, fruit quality and sensory evaluation of fuji apples stored in different atmospheres by means of multi variate analysis. j. sci. food agric. 84, 5-20. doi: 10.1002/jsfa.1554 espino-díaz, m., roberto sepúlveda, d., gonzález-aguilar, g., olivas, g.i., 2016: biochemistry of apple aroma: a review. food technol. biotechnol. 54, 375-394. doi: 10.17113/ftb.54.04.16.4248 eggleston, w.w., 1909: the crataegi of mexico and central america. bulletin of the torrey botanical club 36, 501-514. froehlicher, t., hennebelle, t., martin-nizard, f., cleenewerck, p., hilbert, j.l., trotin, f., grec, s., 2009: phenolic profiles and anti oxidative effects of hawthorn cell suspensions, fresh fruits, and medicinal dried parts. food chem. 115, 897-903. doi: 10.1016/j.foodchem.2009.01.004 gamboa-becerra, r., montero-vargas, j.m., martínez-jarquín, s., gálvez-ponce, e., moreno-pedraza, a., winkler, r., 2017: rapid 22 m.d. pérez-lainez, t. corona-torres, m. del rosario garcía-mateos, r. winkler, a.f. barrientos-priego, raúl nieto-ángel, v.h. aguilar-rincón, j.a. garcía-velázquez ta b. 5 : c ol or p ar am et er s, to ta l c ar ot en oi d co nt en t ( t c ) a nd to ta l a nt ho cy an in s (t a ) i n th e ex oc ar p an d m es oc ar p of 2 3 ge no ty pe s of th e ge nu s c ra ta eg us . e xo ca rp m es oc ar p g en ot yp e l um in os it y h ue c hr om a t c * ta * l um in os it y h ue c hr om a t c * ta * (μ g· g1 ) (μ g· g1 ) (μ g· g1 ) (μ g· g1 ) c h 4 60 .7 3 a 64 .1 4 a b 66 .2 1 a bc 23 .8 0 ab 5. 56 e 65 .4 3 d ef g 65 .7 5 a b 59 .0 2 a 15 .6 1 a b 22 .2 6 a bc c h 8 35 .9 5 e f 29 .9 1 e fg 54 .8 8 g hj 16 .4 1 a b 56 3. 59 c 64 .0 7 e fg 62 .4 2 a b 49 .1 7 b cd 9. 85 b cd 16 .7 0 a bc c h 10 37 .1 0 d ef 28 .7 3 fg 54 .6 0 h i 11 .8 4 a b 36 8. 77 d 61 .1 5 fg 51 .0 8 b c 51 .8 3 a bc 4. 51 f g 29 .2 2 a c h 13 47 .4 9 b c 37 .3 1 d 62 .1 5 b cd ef 28 .7 0 a b 23 9. 35 d 68 .4 7 b cd ef 66 .8 8 a b 50 .1 4 b c 7. 39 c de f 16 .7 0 a bc c h 15 64 .3 5 a 67 .2 5 a 68 .2 7 a 13 .7 7 a b 6. 95 e 65 .8 7 c de fg 68 .5 4 a b 54 .3 0 a b 9. 78 b cd 10 .4 3 a bc c h 16 42 .8 0 c d 32 .4 1 e 57 .5 1 fg hi 11 .9 9 a b 28 5. 27 d 67 .9 1 b cd ef 67 .6 0 a b 46 .5 1 b cd 10 .5 7 b cd 14 .6 1 a bc c h 18 32 .4 6 f 26 .9 9 g 45 .0 5 j 19 .5 4 a b 99 2. 34 a 70 .0 8 a bc de 71 .9 3 a b 48 .1 5 b cd 5. 04 e fg 28 .3 8 a b c h 19 34 .6 8 e f 28 .0 6 fg 51 .9 4 i 5. 71 b 74 7. 69 b 67 .7 4 bc de f 70 .2 2 a b 47 .5 4 b cd 3. 95 g 18 .1 7 a bc c h 22 50 .7 3 b 55 .3 1 c 57 .7 8 e fg h 22 .2 6 a b 16 .1 9 e 61 .4 9 fg 69 .9 5 a b 45 .6 9 b cd 7. 28 d ef 12 .0 1 a bc p2 5 52 .4 8 b 59 .3 8 b c 60 .9 4 a bc 38 .1 7 a b 23 .6 6 e 52 .9 8 h 64 .6 9 ab 52 .0 2 a bc 20 .9 2 a 16 .7 0 a bc c h 27 52 .1 3 b 61 .4 7 a bc 60 .5 6 c de fg 14 .4 1 a b 15 .8 3 e 60 .0 0 g h 66 .9 7 a b 49 .2 0 b cd 7. 77 c de 14 .9 6 a bc c h 43 59 .9 7 a 63 .0 9 a b 63 .7 6 a bc d 10 .7 1 a b 23 .6 6 e 66 .2 1 c de fg 70 .9 1 a b 52 .8 6 a b 9. 55 b cd 9. 73 ab c c h 44 64 .4 2 a 63 .4 7 a b 66 .1 6 a bc 16 .9 2 a b 10 .4 3 e 66 .7 2 c de fg 70 .2 1 a b 45 .6 0 b cd 6. 67 d ef g 8. 35 ab c c h 51 34 .8 3 e f 28 .7 7 fg 52 .5 9 h i 15 .3 7 a b 84 4. 69 a b 60 .1 3 g h 57 .3 5 b 43 .9 3 c d 9. 6 b cd 26 .4 4 a bc p5 5 64 .9 3 a 63 .0 8 a b 67 .3 6 a b 42 .0 9 a 12 .5 1 e 71 .9 4 a bc d 69 .5 8 a b 47 .6 2 b cd 8. 98 b cd 6. 95 c e m 66 60 .6 4 a 62 .7 5 a b 65 .1 1 a bc 13 .3 8 a b 6. 96 e 68 .3 9 b cd ef 65 .8 1 a b 53 .6 0 a b 9. 11 b cd 16 .7 0 a bc c h 72 40 .9 3 d e 32 .6 2 e 58 .0 0 d ef gh 38 .1 0 a b 31 5. 89 d 71 .6 0 a bc d 72 .6 5 a b 52 .4 3 a bc 7. 37 d ef 15 .3 0 a bc c h 83 40 .3 1 d e 31 .3 8 e f 55 .7 6 g hi 22 .0 6 a b 52 8. 80 c 73 .2 2 a bc 73 .3 1 a b 46 .9 4 b cd 7. 87 c de 13 .9 2 a bc p8 6 62 .2 3 a 60 .7 5 a bc 65 .5 2 a bc 13 .5 3 a b 12 .5 2 e 69 .6 6 b cd ef 72 .4 8 a b 46 .1 2 b cd 4. 74 e fg 23 .6 6 a bc p1 00 61 .7 9 a 62 .2 3 a b 62 .8 6 a bc de f 33 .6 1 a b 25 .0 4 e 71 .7 1 a bc d 69 .3 4 a b 53 .8 5 a b 12 .8 4 a bc 8. 35 bc p0 2 62 .4 4 a 61 .9 4 a bc 67 .2 8 a b 38 .6 8 ab 18 .0 9 e 70 .1 8 a bc de 69 .3 2 a b 53 .9 3 a b 5. 22 e fg 20 .8 7 a bc p0 5 61 .2 6 a 59 .2 6 b c 67 .4 4 a b 42 .3 5 a 20 .8 7 e 75 .9 9 a 79 .8 0 a 41 .2 3 d 10 .3 4 b cd 11 .1 3 a bc p0 6 60 .8 9 a 62 .0 5 a bc 63 .4 2 a bc de 22 .0 6 a b 30 .3 0 e 74 .7 4 a b 79 .7 1 a 40 .4 1 d 9. 83 b cd 9. 67 ab c h sd 6. 26 5. 81 5. 79 8. 09 15 8. 92 7. 42 8. 89 8. 89 5. 57 24 .0 4 * μg ·g -1 o f f re sh ti ss ue . m ea ns w ith th e sa m e le tte rs a re n ot s ta tis tic al ly d iff er en t ( p < 0. 05 ). h sd : h on es tly s ig ni fic an t d iff er en ce . 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(eds.), advances in applied biotechnology. © springer-verlag berlin heidelberg. new york. doi: 10.1007/978-3-662-46318-5_55 addresses of authors: maría dolores pérez-lainez, tarsicio corona-torres, victor hebert aguilarrincón, josé armando garcía-velázquez. colegio de postgraduados. texcoco, estado de mexico. postal code 56230. mexico. maría del rosario garcía-mateos*, alejandro f. barrientos-priego, raúl nieto ángel. universidad autónoma chapingo. texcoco, estado de méxico. postal code 56230. mexico * rosgar08@hotmail.com (corresponding author) robert winkler. laboratorio de análisis bioquímico e instrumental. cinvestav. irapuato, guanajuato. postal code 36824. mexico © the author(s) 2019. this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/deed.en). journal of applied botany and food quality 91, 211 218 (2018), doi:10.5073/jabfq.2018.091.028 1*instituto de horticultura, universidad autónoma chapingo, carretera méxico-texcoco, chapingo, estado de méxico, méxico 2instituto de alimentos, universidad autónoma chapingo, carretera méxico-texcoco, chapingo, estado de méxico, méxico 3tecnológico nacional de méxico, instituto tecnológico de conkal, avenida tecnológico s/n conkal, yucatán, méxico nutraceutical components, antioxidant activity, and color of 11 varieties of prickly pear (opuntia sp.) m. ramírez-ramos1, k. medina-dzul3, r. garcía-mateos1*, j. corrales-garcía2, c. ybarra-moncada2, a.m. castillo-gonzález1 (submitted: october 13, 2017; accepted: june 26, 2018) * corresponding author summary in mexico, there are 50 recorded varieties of the prickly pear fruit. national production covers only the white-pulp fruit, but other red varieties have export potential; however, their nutraceutical properties are unknown. the pulp and peel (underutilized tissue) of the pigmented fruits of the genus opuntia sp. are marketed on a limited basis. they represent an alternative source of stable pigments (betalains), which are associated with antioxidant properties, for the agroindustry. the objective was to assess the content of nutra ceutical components, antioxidant activity, and peel and pulp color of 11 varieties of the prickly pear fruit that are marketed on a small scale. statistical analysis revealed that roja villanueva peel had the highest betalain content (39.97 mg 100 g-1 fw). alteña blanca peel demonstrated the highest concentration of phenolic compounds (618.39 mg gae 100 g-1 fw), whereas alteña roja had the highest ascorbic acid content (37.14 mg aae 100 g-1 fw). the greatest nutraceutical potential was observed in the pulp of the non-marketed tzaponopal rojo variety of the species o. robusta var. larreyi, due to the high antioxidant activity (0.0183 mg ml-1), as well as the darkest color (‹ hue value, 12.31) and the lowest lightness (‹ luminosity, 19.31), which coincides with the highest betalain concentration. keywords: antioxidant components, betalains, pulp, peel. introduction mexico is considered the center of origin of the family cactaceae, which includes approximately 1500 species of the genus opuntia (anoop et al., 2012), both wild and cultivated (sáenz, 2006). the subgenera opuntia and nopalea are the main producers of the edible fruit known as prickly pear fruit, grouped by its pigmentation into purple, red, orange, yellow, and white fruit (segura et al., 2007; caruso et al., 2010). prickly pear grows in arid and desert regions. it has a photosynthetic metabolism of crassulacean type called crassulacean acid metabolism (cam), which allows the production of biomass in the arid and drought conditions characteristic of its habitat (andreu et al., 2017). during the photosynthetic process, cam plants open their stomata during the desert nights and close them during the hot, dry days, thus efficiently using night moisture (taiz and zeiger, 2002). in mexico, white-pulp and green-peel prickly pears are highly appreciated for their market value. in 2014, prickly pear produc tion in the country reached approximately 588 000 t (siap, 2014); however, despite the great production and variability of this fruit in the country, the levels of antioxidant components in most prickly pear species are unknown. nonetheless, these components in vari ous other fruits are known to provide important nutraceutical benefits to consumers (tsao and akhtar, 2005; anoop et al., 2012). consumption of these other fruits prevents some chronic-degenera tive diseases, due to the presence of certain metabolites (phenolic compounds, flavonoids, ascorbic acid, and betalains) (sumaya martínez et al., 2011; abou-elella and ali, 2014). biosynthetically, betalamic acid-derived betalains, which are pigments used in the agri-food industry as natural colorants (strack et al., 2003), also have antioxidant properties (livrea and tesoriere, 2006; albano et al., 2015). moreover, color is part of fruit quality and may therefore be a factor in its preference, acceptance, or rejection, and play a decisive role in the failure or success of prickly pear marketing. the wide genetic diversity of the mexican prickly pear justifies the study of nutraceutical content, particularly in pigmented prickly pear varieties. the objective of the present study was to assess the content of nutraceutical components, antioxidant activity, and pulp and peel color of 11 varieties of prickly pear marketed on a small scale, and to identify those with the greatest nutraceutical attributes. such nutraceutical information will be of benefit to consumers. material and methods plant material collection healthy and pest-free fruits were harvested in the dr. facundo barrientos pérez experimental station at the universidad autó noma chapingo in chapingo, state of mexico (tab. 1), located at 19º 29́ n and 98º 53́ w, at an altitude of 2240 m. the climate is c (wo) (w) b (i’) g, considered the driest of the subhumid climates, with summer rains and mild temperatures. the area has a mean annual temperature of 17.8 °c and rainfall of 644.8 mm per year. the harvest criteria were the visual parameters used commercially in the producing region for each variety (completely characteristically developed fruit, with fruit weight ranging from 125 g to 230 g, depending on the variety) (corrales et al., 2006). after cutting, fruits were stored for 15 days. the pulp was then separated from the seed and peel, and the tissue (pulp and peel) was fractionated into small portions and frozen by direct immersion in liquid nitrogen. the samples were then stored at -20 ± 2 °c for later analysis. quantification of total betalains (betacyanins and betaxanthins) pigment content was determined according to the methodology described by castellanos-santiago and yahia (2008). the frozen sample (1 g) was mixed with 10 ml distilled water, sub jected to sonication for 20 min, and centrifuged at 2200 g for 20 min. absorbance of the extracts was measured in a genesys 10s spectrophotometer (thermo scientific, florida, usa) at 483 nm (betaxanthins) and 538 nm (betacyanins). in order to estimate the pigment concentration, the following equation was used: betacyanins or betaxanthins (mg g-1 fw) = (a × df × mw × v) (ε × l × fw)-1, where a = absorbance; df = dilution factor; mw = molecular weight (betacyanin: 550 g mol-1, betaxanthin: 308 g mol-1); v = extract 212 m. ramírez-ramos, k. medina-dzul, r. garcía-mateos, j. corrales-garcía, c. ybarra-moncada, a.m. castillo-gonzález volume (ml); ε = molar extinction coefficient (betacyanin: 60 000 l mol cm-1; betaxanthin: 48 000 l mol cm-1); fw = fresh sample weight (g); and l = cell length (1 cm). total betalain content was expressed as mg per 100 g fresh weight (fw). extraction of total phenolic compounds and flavonoids the sample (1 g) was dissolved in 25 ml ethanol (95% v/v) in an ultrasonic bath (cole-parmer 8892, illinois, usa) for 15 min. after 24 h, the volume was brought to 25 ml with ethanol (80% v/v) and centrifuged at 1409 g. this extract was subsequently used to determine the content of total phenolic compounds and flavonoids. quantification of total phenolic compounds the method proposed by waterman and mole (1994) was fol lowed. first, 10 ml na2co3 (10% w/v) were added to 0.5 ml ethanolic extract, and the mixture was then incubated at 38 °c for 15 min. subsequently, 3 ml water and 1 ml folin-ciocalteu reagent:water (1:1 v/v) were added to 1 ml of this mixture, which was left to stand for 15 min in the dark, and absorbance was then measured at 760 nm in a genesys 10s spectrophotometer. total phenolic compound content in the extract was expressed as mg gallic acid equivalents per 100 g fresh weight (mg gae 100 g-1 fw). quantification of flavonoids flavonoid content was quantified according to the method proposed by chang et al. (2002). first, 1.5 ml ethanol (95% v/v), 0.1 ml alcl3 (10% w/v), 0.1 ml 1 m potassium acetate, and 2.8 ml distilled water were added to 0.5 ml ethanolic extract. the mixture remained at room temperature for 30 min, and absorbance was measured in a genesys 10s spectrophotometer at 415 nm. results were expressed as mg quercetin equivalents per 100 g fresh weight (mg qe 100 g-1 fw). quantification of ascorbic acid the sample (1 g) was homogenized in 3 ml metaphosphoric acid (3% v/v) for 3 min. the mixture was filtered, and 1 ml supernatant was brought to 10 ml with metaphosphoric acid (3% v/v). then 2 ml ph 4 buffer solution (glacial acetic acid:sodium acetate [5% w/v, 1:1]), 3 ml dichloroindophenol, and 15 ml xylene were added to 2 ml of the mixture, which was shaken vigorously in a vortex (thermolyne type 6700, usa). absorbance was measured in a spectrophotometer at 520 nm. ascorbic acid was calculated with the following equation: mg total ascorbic acid = (c × v × 100) / (a × w), where c = ascorbic acid in the sample; v = capacity volume; a = aliquot of the solution taken (ml); and w = sample weight or volume. results were expressed as mg ascorbic acid equivalents per 100 g fresh weight (mg aae 100 g-1 fw). antioxidant activity assessment the analysis was performed using the dpph (2, 2-diphenyl-1picrylhydrazyl, sigma-aldrich) free radical method described by amico et al. (2008). for analysis, 10 g of sample were macerated in methanol and subjected to sonication for 20 min. the mixture was filtered, and the supernatant was concentrated in a büchi r-210 rotary evaporator (flawil, switzerland). from the methanol extract, the following solutions were prepared in methanol by serial dilution to obtain 0.2, 0.15, 0.1, and 0.05 mg ml-1 concentrations. as references, 0.1, 0.001, and 0.0001 mg ml-1 concentrations of quercetin and ascorbic acid were individually prepared. next, 3 ml dpph solution (0.1 mm) were added to 1 ml of each concentration of extracts and the individual references. mixtures were left at room temperature for 30 min, and then absorbance readings were taken at 516 nm. dpph percentage was determined by the following formula: % dpph = (ablank − asample) × 100 / ablank, where ablank is the control absorbance (dpph 0.1 mm) and asample is the absorbance obtained for each sample after 30 min with dpph 0.1 mm. the antioxidant activity of the samples was determined by calculating the mean inhibitory concentration (ic50), which is the concentration required by the sample to decrease dpph absorbance to 50%. low absorbance of the reaction mixture indicated high antioxidant activity. the standard curve was built with 3.93 mg dpph dissolved in 100 ml methanol to obtain a 0.1 mm concentration (stock solution). the following concentrations were prepared from this solution: 0.01, 0.02, 0.04, 0.06, 0.08, and 0.1 mm dpph. absorbance was measured at 516 nm in a genesys 10s spectrophotometer, and readings were taken in triplicate; methanol was used as a blank. tab. 1: descriptions of 11 varieties and species of prickly pear (opuntia sp.) grown at the universidad autónoma chapingo experimental station, chapingo, mexico. species variety origin color market value* opuntia sp. alteña blanca celaya, gto. yellow-greenish lmp opuntia sp. alteña roja celaya, gto. orange amp o. ficus-indica (l.). mill huatusco huatusco, ver. yellow lmp o. ficus-indica (l.). mill solferino chapingo, méx. orange amp o. ficus-indica (l.). mill roja villanueva villanueva, pue. orange amp o. ficus-indica (l.). mill jade desconocido pink lmp o. ficus-indica (l.). mill copena ceii zacatecas, zac. pink amp o. ficus-indica (l.). mill copena vi chapingo, méx. red purple lmp o. megacantha salm morada san martín de las pirámides, méx. red ampp o. megacantha salm plátano ojuelos, jal. yellow lmp o. robusta var. larreyi tzaponopal rojo san martín de las pirámides, méx. red amp *lmp: low market potential; amp: average market potential; ampp: average market potential as a source of pigments (corrales-garcía and hernándezsilva, 2005). nutraceutical components of prickly pear (opuntia sp.) 213 measurement of color parameters pulp and peel color were determined by assessing lightness (l), tone angle (hue) and purity of color or chromaticity index (chroma) with a hunterlab colorimeter (miniscan xe plus 45/ 0-l, reston, virginia, usa). readings from a* and b* were obtained to clearly quantify color differences among the tissues and varieties. variables were calculated with the following equations: hue = tan-1 (a/b) and chroma = (a2 + b2) ½ (mc guire, 1992). statistical analysis an asymmetric 11 × 2 factorial design, in which the pulp or peel of each variety was considered as a treatment, was used, with four replications. results were analyzed using an analysis of variance and tukey’s range test (p ≤ 0.05). the least significant difference (lsd) (α = 0.05) and principal component analysis were obtained with the sas (statistical analysis system 9.0). results and discussion total betalain content (betacyanins and betaxanthins) total betalain concentration was higher in red prickly pear fruits than in white and orange ones, which showed lower concentrations in pulp and peel (tab. 2). the total betalain levels (92.08 mg 100 g-1 fw) in the pulp of the tzaponopal rojo variety surpassed those of the other varieties. kuti (2005) reported a betalain concentration in opuntia spp. fruits similar to that found in the present research (81.5 mg 100 g-1 fw). betalains are responsible for the array of fruit colors in the many species and varieties of the genus opuntia (stintzing and carle, 2007). these metabolites derive biosynthetically from betalamic acid and are categorized as either betacyanins or betaxanthins. betacyanins are red-purple, and beta xanthins are responsible for the orange-yellow color of the pulp and rind of these fruits (zrÿd and christinet, 2004; stintzing and carle, 2007). the alteña blanca variety (yellow-green color) had the lowest content of betacyanins (responsible for red-purple color) and betaxanthins (responsible for yellow-orange color) in both pulp and peel; by contrast, the highest betacyanin content was found in the pulp of the tzaponopal rojo (red), copena vi (red-purple), and jade (pink) varieties. the highest betaxanthin content was found in the peel of roja villanueva and the pulp of tzaponopal rojo. these results support the view that the high content of pigments in the pulp and peel of these varieties might make them good sources of natural pigments for use in the food industry. values for both pigments found in the present study (0.46 mg 49 mg 100 g-1 fw for betacyanins and 0.5 mg -19.91 mg 100 g-1 fw for betaxanthins) were superior to those found by lópez et al. (2015) for the cambray genotype “xoconostle” (sour prickly pear fruit), which had values of 26.05 mg 100 g-1 fw tab. 2: content of total betalains, betacyanins, and betaxanthins in the fruit of 11 varieties and species of prickly pear (opuntia sp.) harvested at the universidad autónoma chapingo experimental station, chapingo, mexico. species variety tissue* total betalains betacyanins betaxanthins (mg 100 g-1 fw) (mg 100 g-1 fw) (mg 100 g-1 fw) opuntia sp. alteña blanca peel 0.99k 0.50g 0.50h pulp 1.03k 0.46g 0.57h opuntia sp. alteña roja peel 19.20hj 12.46ed 6.74dg pulp 23.08gi 14.36cd 8.73cf o. ficus indica huatusco peel 4.25jk 2.24eg 2.01gh pulp 4.34jk 1.81fg 2.53gh o. ficus indica solferino peel 22.98gi 15.24cd 7.74dg pulp 19.08hj 11.35df 7.74dg o. ficus indica roja villanueva peel 39.97cg 23.97bc 16.01ab pulp 35.30be 20.62bd 14.67ac o. ficus indica jade peel 21.21ih 14.89cd 6.42eh pulp 41.91bc 30.13b 11.79be o. ficus indica copena ceii peel 31.57eh 21.78bc 9.79cf pulp 40.36bd 29.04b 11.32be o. ficus indica copena vi peel 38.85dg 27.14b 11.71be pulp 43.73b 30.91b 12.82bd o. megacantha morada peel 17.03hk 12.67ed 4.36fh pulp 27.86fi 20.37bd 7.49dg o. megacantha plátano peel 14.02ik 2.19efg 11.83be pulp 11.48ik 1.54fg 9.95bf o.robusta/larreyi tzaponopal rojo peel 33.41eh 23.50bc 9.90bf pulp 69.06a 49.15a 19.91a cv 21.62 23.89 26.27 *fresh weight; cv = coefficient of variation. means with the same letters in a column are not significantly different (tukey, p ≤ 0.05). 214 m. ramírez-ramos, k. medina-dzul, r. garcía-mateos, j. corrales-garcía, c. ybarra-moncada, a.m. castillo-gonzález for betacyanins and 9.01 mg 100 g-1 fw for betaxanthins. khatabi et al. (2016) demonstrated that differences in betalain content in the juice and pulp of prickly pear fruits could be attributed to variability in prickly pear ecotypes, physiologies, and growth conditions. pigments identified in prickly pear (opuntia) fruits are stable, while the betacyanins in pitaya (stenocereus pruinosus) tend to decompose rapidly when they are isolated from the fruit (stintzing and carle, 2007). nevertheless, the rate of degradation of the pigments in opuntia and stenocereus fruits is unknown. these pigments exhibit important antioxidant activity and are non-toxic to humans (sumaya-martínez et al., 2011). moreover, high levels of betalains help prevent cancer and lipid oxidation of membranes (livrea and tesoriere, 2006). additionally, fruit quality is based partly on color, which may be a factor in its preference, acceptance, or rejection, thus playing a decisive role in the failure or success of prickly pear marketing. total phenolic compound content phenolic compounds are another group of secondary metabolites, identified in some fruits of the opuntia genus (pimienta-barrios et al., 2008; osorio-esquivel et al., 2011; andreu et al., 2017; mena et al., 2018), that protects plants from oxidative stress, and their consumption contributes to preventing disease in humans. it is important to point out that in every variety, phenolic compound levels were much higher in the peel than in the pulp; this is consistent with the work of moussa-ayoub et al. (2011), who observed similar behavior in o. macrorhiza fruits. alteña blanca peel had the highest concentration of these metabolites (618.39 mg gae 100 g-1 fw); on the other hand, copena ceii pulp had the lowest concentration (106.60 mg gae 100 g-1 fw) (tab. 3). results were superior to those obtained by lópez et al. (2015) for 15 xoconostle cultivars whose phenolic compound contents ranged between 132.83 mg and 231.37 mg gae 100 g-1 fw. yahia and mondragón-jacobo (2011) also reported total phenol values in different varieties of prickly pear (106.6 mg -130.0 mg gae 100 g-1 fw), and concentrations of phenolic compounds measured by garcía-cruz et al. (2013) in red pitaya (106.0 mg gae 100 g-1 fw) were lower than those found in prickly pear fruits. the differences observed among species and varieties of different genera may be due to (a) genetic factors (moussa-ayoub et al., 2014; osorioesquivel et al., 2011), and (b) stress caused by harvest and handling of fresh fruits, which alters physiology and stimulates responses that cause phenolic compounds to accumulate (pirovani et al., 2009). the effect of genotypic differences on the phenolic profiles of prickly pear fruits has also been investigated (moussa-ayoub et al., 2014; stintzing et al., 2005). however, several studies point out that the concentration of phenolic compounds in prickly pear depends on the environmental conditions, as well as the part of the cactus plant under consideration (khatabi et al., 2016; moussa-ayoub et al., 2014; stintzing et al., 2005). there was no relationship between the content of these phytotab. 3: content of total phenolic compounds, flavonoids, and ascorbic acid in the fruit of 11 varieties and species of prickly pear (opuntia sp.) harvested at the universidad autónoma chapingo experimental station, chapingo, mexico. species variety tissue* phenolic compounds flavonoids ascorbic acid (mg gae 100 g-1 fw) (mg qe 100 g-1 fw) (mg aae 100-1 g fw) opuntia sp. alteña blanca peel 618.39ª 27.73a 2.26de pulp 165.56ef 1.34f 3.74ce opuntia sp. alteña roja peel 486.79b 23.88ab 2.26de pulp 120.75f 4.37df 37.14a o. ficus indica huatusco peel 425.58bc 20.13ab 0.56e pulp 131.96ef 2.60ef 0e o. ficus indica solferino peel 404.59bc 22.41ab 6.80cd pulp 148.6ef 3.23df 4.62ce o. ficus indica roja villanueva peel 242.98ed 20.05ab 0e pulp 118.39f 9.60ce 0e o. ficus indica jade peel 374.88bc 20.05ab 14.85b pulp 122.52f 6.69df 2.80ce o. ficus indica copena ceii peel 322.99cd 20.74ab 0e pulp 106.60f 8.34df 0e o. ficus indica copena vi peel 425.59bc 23.96ab 16.59b pulp 143.74ef 8.23df 8.04c o. megacantha morada peel 389.03bc 17.76bc 0e pulp 107.19f 6.00df 0e o. megacantha plátano peel 400.82bc 21.43ab 15.83b pulp 111.90f 6.19df 15.49b o.robusta/larrey tzaponopal rojo peel 219.22ef 21.79ab 0e pulp 161.43ef 11.21cd 0e cv 16.59 22.1 34.88 *fresh weight; cv = coefficient of variation. means with the same letters in a column are not significantly different (tukey, p ≤ 0.05). nutraceutical components of prickly pear (opuntia sp.) 215 chemicals and the pulp and peel colors of the different varieties studied; this behavior was also observed by sumaya-martínez et al. (2011), who point out that when investigating the fruit colors of three groups of prickly pear cultivars (purple, yellow, and white), no relationship with total phenol content was found. however, khatabi et al. (2016) reported that the red prickly pear contains higher amounts of polyphenols than those of the yellow variety. assessment of the phenolic profile of prickly pear has been limited. phenolic composition of different parts of o. ficus-indica had been previously addressed (mena et al., 2018; moussa-ayoub et al., 2014). mena et al. (2018) identified the phytochemical profiles (41 compounds, mainly phenolics) of four botanical parts from six different prickly pear cultivars of o. ficus-indica. results obtained in the present study suggest that the prickly pear is an important source of phenolic compounds and can be used as a functional and nutraceutical food. its consumption could help in the prevention of some chronic or degenerative diseases (sotohernández et al., 2017). flavonoid content the flavonoid concentrations in all varieties were lower than the phenolic compound content, probably due to the presence of pro cyanidins (condensed tannins) and phenolic acids (chlorogenic acid and ferulic acid), which also occurs in other fruits (mena et al., 2018). it is important to highlight that procyanidin content has not been studied in prickly pear. another explanation for the loss of flavonoids may be that during the ripening of fruits these metabolites are transformed into phenolic compounds (barz and hoesel, 1977). no significant differences in flavonoid content were found among varieties (tab. 3). in all varieties, higher concentrations of these metabolites were observed in the peel than in the pulp. according to brielman et al. (2006), some types of flavonoids are responsible for the white or yellow pigmentation of some plant tissues, which agrees with the results obtained in the present study. few studies describe the presence of flavonoids in prickly pear fruits (moussa-ayoub et al., 2011; mena et al., 2018). these metabolites are also important as they have antioxidant, anti-inflammatory, and anticancer pro perties (crozier et al., 2009). furthermore, the results of this study can contribute knowledge of a food resource used ancestrally and still a part of the cultural identity of some states in the mexican republic, but with little recognized potential today. ascorbic acid content significant differences (p ≤ 0.05) were found in ascorbic acid content in the peel of the plátano, jade, and copena vi varieties as compared to the remaining varieties (tab. 3). in the peel of the roja villanueva, morada, copena ceii, tzaponopal rojo, and huatusco varieties, no ascorbic acid was detected. despite to the unstable structure of this metabolite in the presence of oxygen (de ancos et al., 2009), its possible degradation can be ruled out because all the samples were processed simultaneously; therefore, the difference may be due to genetic factors. latocha et al. (2011) point out that genotype is one of the most important factors in the identification of fruits with high ascorbic acid content. alteña roja pulp presented the highest ascorbic acid content (37.14 mg aae 100 g-1 fw), which is similar to that reported by corral-aguayo et al. (2008) (40 mg aae 100 g-1 fw) but inferior to that reported by kuti (2004) in o. ficusindica fruits (45.8 mg aae 100 g-1 fw). figueroa et al. (2010) reported lower levels for the mango (o. albicarpa) and cacalote (o. cochinera) cultivars (5.31 mg aae 100 g-1 fw and 25 aae mg 100 g-1 fw, respectively). values found in the present study support the idea that the content of this metabolite in some prickly pear varieties could contribute to antioxidant activity in consumers’ diets (albano et al., 2015). differences in ascorbic acid content reported by other authors could be attributable to crop conditions, taking into account the tolerance of prickly pear species to extreme climatic conditions, because when opuntia species (cam plants) are grown under limiting soil nutri ent and water conditions, their chemical composition may change (sumaya-martínez et al., 2011). antioxidant activity alteña blanca peel presented the highest antioxidant activity (lowest concentration of sample required to inhibit 50% of the dpph radical concentration) (fig. 1). roja villanueva peel had the lowest antioxidant activity. in addition, some significant differences (p ≤ 0.05) were found between the ic50 of the pulp and peel of this variety compared to that of the same tissues of the other varieties (fig. 1). however, other methods that more completely reflect antioxidant capacity were not evaluated. among the advantages of cultivating opuntia fruits are the presence of antioxidant compounds and the high content of stable pigments (betalains) in the pulp and peel of some varieties (tzaponopal rojo, copena ceii, and copena vi), which are important to the food industry, and their low water requirements. these advantages make them an option for agriculture in arid and semiarid regions of the country. brat et al. (2007) mention that the antioxidant activity of fruits and vegetables is not only associated with phenolic compounds but is also attributed to the content of other metabolites such as vitamin c, carotenoids, and sulfur compounds; therefore, the antioxidant activity found in some varieties in the present study may be due to the possible synergistic effect of a different set of phytochemicals. ávila-nava et al. (2014) used the dpph method to evaluate the antioxidant activity of o. ficus-indica fruits. healthy individuals first consumed an antioxidant-poor diet and then consumed 300 g of o. ficus-indica. at the end of the study, a significant increase in antioxidant activity in plasma and blood (20% and 5%, respectively) resulted. fig. 1: antioxidant activity in the fruit of 11 varieties and species of prickly pear (opuntia sp.) harvested at the universidad autónoma chapingo experimental station, chapingo, mexico. different letters in the bars are significantly different (tukey, p ≤ 0.05). color parameters in most varieties, significant differences between pulp and peel lightness were observed (tab. 4). alteña blanca pulp (lightest color) presented the greatest l, followed in descending order by the 216 m. ramírez-ramos, k. medina-dzul, r. garcía-mateos, j. corrales-garcía, c. ybarra-moncada, a.m. castillo-gonzález huatusco and plátano varieties. in the exact same order, the above varieties recorded the lowest total betalain content and are thus considered to be low-pigmented varieties. the tzaponopal rojo variety had the darkest color pulp and also recorded the lowest l. the pulp of this variety recorded the highest total betalain content, whereas its ic50 was the lowest. alteña blanca had the clearest peel color since it presented the greatest l; however, the peel of this variety also had the lowest ic50, (i.e., the highest antioxidant activity per unit weight). this could indicate that phytochemicals other than pigments were involved and contributed to the antioxidant activity. copena ceii, copena vi, and tzaponopal rojo presented the lowest peel l; these varieties consistently have dark colors (purple-red) and in this analysis showed low hue values (tone angle). most varieties exhibited lower hue values in the pulp than in the peel (i.e., the pulp was redder than the peel). the peel and pulp showed similar hue values only in some varieties (huatusco, morada, and tzaponopal rojo). pulp hue values followed a trend very similar to that described for peel; the lowest hue values (less than 9°) were observed in the pulp of the copena ceii and copena vi varieties, which consistently had the highest total betalain content. peel hue values could be divided into four distinct groups: (a) the alteña blanca variety, which had a high value (greater than 100°) and consistently presented the lowest total betalain content in the peel; (b) the plátano and huatusco varieties, with medium values (52°55°) and relatively low total betalain content; (c) most other varieties, with low values (18°20°) and relatively high total betalain content; and (d) the roja villanueva, which had a very low value (less than 5°) and consistently presented the highest total betalain content. except for the alteña blanca variety, chroma values (color purity) were higher in the pulp than in the peel. the chroma in descending order was as follows: alteña roja > solferino > plátano > tzaponopal rojo > roja villanueva > morada > copena vi > jade > copena ceii > huatusco > alteña blanca. the greater the chroma, the more vibrant and attractive the colors. results indicate that the pulp in varieties with higher chroma values has more attractive color than that in varieties with lower chroma values. the highest chroma values in peel were exhibited by the yellow varieties (alteña blanca and plátano), meaning that these had more defined and brighter colors; however, they showed the lowest total betalain content, which indicates that different pigments are possibly responsible for their colors. the roja villanueva variety also ex hibited a relatively high chroma value, which, in this case, may be associated with high total betalain content. neither in the peel nor in the pulp was there any clear association between color purity (chroma) and pigment content or antioxidant activity. in the international trade of prickly pear, major markets demand colorful red and yellow fruits, the brighter the better. the typical colors of the fruits are due to the presence of certain pigments (betalains, anthocyanins, and carotenes), which have antioxidant capacity. therefore, it is important to analyze color and antioxidant content to identify phytogenetic materials with high nutraceutical tab. 4: color parameters of the fruit of 11 varieties and species of prickly pear (opuntia sp.) harvested at the universidad autónoma chapingo experimental station, chapingo, mexico. species variety tissue* (l) (hue) (chroma) opuntia sp. alteña blanca peel 38.23b 100.96a 17.90eg pulp 45.83a 88.87b 12.03hj opuntia sp. alteña roja peel 23.51e 9.91hj 10.39jl pulp 18.73gh 21.25e 27.43a o. ficus indica huatusco peel 25.82ed 52.57c 10.54il pulp 36.11b 54.14c 14.74fh o. ficus indica solferino peel 22.83ef 18.52ef 11.49hk pulp 18.62gi 13.12fi 27.39a o. ficus indica roja villanueva peel 19.81fg 4.73 j 14.21gi pulp 19.67fh 15.75eg 23.35bc o. ficus indica jade peel 19.71fh 13.52 fi 7.89 km pulp 15.29ik 9.01hj 20.38ce o. ficus indica copena ceii peel 19.31gh 14.02fh 6.32m pulp 14.68jk 8.54 hj 18.47df o. ficus indica copena vi peel 19.32gh 12.01gi 6.97 lm pulp 16.31hj 7.98ij 21.70cd o. megacantha morada peel 19.39fh 12.21gi 8.24km pulp 15.52ik 13.27fi 23.12bc o. megacantha plátano peel 29.25cd 54.91c 14.52gh pulp 31.08c 43.08d 25.56ab o. robusta/larreyi tzaponopal rojo peel 19.31gh 12.31gi 6.95 lm pulp 12.60k 11.75gi 25.56ab cv 5.79 8.09 9.02 *fresh weight; lightness (l); tone (hue); purity (chroma); cv = coefficient of variation. means with the same letters in a column are not significantly different (tukey, p ≤ 0.05). nutraceutical components of prickly pear (opuntia sp.) 217 potential. according to the results of the present study, peels of the roja villanueva, copena ceii, copena vi, and tzaponopal rojo varieties could be outstanding raw material for the food industry because of their high betacyanin content. principal component analysis was used to group the varieties by type of antioxidant components (jolliffe, 2005). the principal com ponents (pc 1 and pc 2) explained 69.6% of data variability (40.9% and 28.8%, respectively) (fig. 2). fig. 2 depicts the makeup of the pulp of four groups of prickly pear fruit varieties: group i (copena vi and tzaponopal rojo varieties) had a high betalain content and the highest ic50; group ii (jade, morada, copena ceii, plátano, and huatusco varieties) presented an intermediate total phenol content and lc50; group iii (roja villanueva variety) had the lowest ic50; and group iv (alteña blanca, alteña roja, and solferino varieties) had the highest phenol and vitamin c concentrations, respectively. finally, it is recommended that research on the nutraceutical quality of other genotypes be intensified, aiming to increase their demand, generate new spaces for commercialization, and contribute to conservation of the country’s cultural identity. and flavonoid content, as well as the highest antioxidant activity (ic50); however, no ascorbic acid was detected in it. in conclusion, the extraction of pigments from the pulp and peel of the tzaponopal rojo variety could be useful mainly in the food industry. references abou-elella, f.m., mohammed-ali, r.f., 2014: antioxidant and anticancer activities of different constituents extracted from egyptian prickly pear cactus (opuntia ficus-indica) peel. biochem. anal. biochem. 3, 1-9. doi: 10.4172/2161-1009.1000158 albano, c., negro, c., tommasi, n., gerardi, c., mita, g., miceli, a., de bellis, l., blando, f., 2015: betalains, phenols and antioxidant capacity in cactus pear [opuntia ficus-indica (l.) mill.] fruits from apulia (south italy) genotypes. antioxidants. 4, 269-280. doi: 10.3390/antiox4020269 amico, v., chillemi, r., mangiafico, s., spatafora, c., tringali, c., 2008: polyphenol-enriched fractions from sicilian grape pomace: hplc-dad analysis and antioxidant activity. bioresource technol. 99, 5960-5966. doi: org/10.1016/j.biortech.2007.10.037 andreu, l., nuncio-jáuregui, n., carbonell-barrachina, a.a. legua, p., hernández, f., 2017: antioxidant properties and chemical characterization of spanish opuntia ficus-indica mill. cladodes and fruits. j. sci. food agric. 98, 1566-1573. doi: 10.1002/jsfa.8892 anoop, a.s., rana, m.k., preetham, s.p., 2012: cactus: a medicinal food. j. food sci. technol. 49, 530-536. doi: 10.1007/s13197-011-0462-5 ávila-nava, a., calderón-oliver, m., medina-campos, o.m., zou, t., gu, l., torres, n., tovar, a.r., pedraza-chaverri, j., 2014: extract of cactus (opuntia ficus-indica) cladodes scavenges reactive oxygen species in vitro and enhances plasma antioxidant capacity in humans. j. func. foods. 10, 13-24. doi: 10.1016/j.jff.2014.05.009 barz, w., hoesel, w., 1977: metabolism and degradation of phenolic compounds in plants. phytochemistry 12, 339-369. brat, p., georgé, s., bellany, a., chaufaut, d.u., mennen, l., scalbert, a., amiot-carlin, m.j., 2007: determination of the polyphenol content of fruit and vegetables. establishment of a database and estimation of the polyphenol intake. in: desjardins, y. 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(eds.) natural products from plants. boca raton, fl. usa. crc-taylor and francis. caruso, m., curro, s., las casas, g., la malfa, s., gentile, a., 2010: microsatellite markers help to assess genetic diversity among opuntia ficus-indica cultivated genotypes and their relation with related species. plant sys. evol. 290, 85-97. doi: 10.1007/s00606-010-0351-9 castellanos-santiago, e., yahia, e.m., 2008: identification and quantification of betalains from the fruits of 10 mexican prickly pears cultivar by high performance liquid chromatography and electrospray ionization mass spectrometry. j agr. food chem. 56, 5758-5764. doi: 10.1021/jf800362t chang, c., yang, m., wen, h., chern, j., 2002: estimation of total flavonoids content in propolis by two complementary colorimetric methods. j. food drug anal. 10, 176-182. corral-aguayo, r., yahia, e., carrillo-lópez, a., gonzálezaguilar, g., 2008: correlation between some nutritional components and the total antioxidant capacity measured with six different assays in eight horticultural crops. j. agric. food chem. 56, 10498-10504. doi: 10.1021/jf801983r crozier, a., jaganath, i.b., clifford, m.n., 2009: dietary phenolics: chemistry, bioavailability and effects on health. nat. prod. rep. 26, 1001-1043. doi: 10.1039/b802662a fig. 2: scatterplot of the principal component analysis of the pulp of 11 varieties of prickly pear by antioxidant metabolite type. alteña blanca (alb), alteña roja (alr), copena ceii (ceii), copena vi (cvi), huatusco (hua), jade (jad), tzaponopal rojo (tzr), morada (mor), plátano (plt), roja villanueva (rov), and solferino (sol). conclusions total betalain concentration was the highest in the pulp and peel of red-purple fruits, and l and hue values were the lowest in darker prickly pears; by contrast, betacyanin content was lowest in the pulp and peel of white and orange prickly pears and highest in redpurple fruits, while betaxanthin content was the greatest in white prickly pears. the peel of white prickly pears presented the greatest amount of phenolic compounds and flavonoids, as well as the highest antioxidant activity. the peel of alteña blanca, considered to have low marketing potential, had the highest antioxidant activity, an attribute that could help increase its marketing strength. currently, the tzaponopal rojo variety is not considered an important source of pigments; however, its pulp was found to have the highest betalain content. the alteña roja variety had the highest ascorbic acid content in its pulp. of all the varieties and species studied, the pulp of the tzaponopal rojo variety of the o. robusta var. larreyi species had the best nutraceutical attributes, as it exhibited the highest betalain 218 m. ramírez-ramos, k. medina-dzul, r. garcía-mateos, j. corrales-garcía, c. ybarra-moncada, a.m. castillo-gonzález corrales, g.j., franco, m., rodríguez, c.j., 2006: fruit characterization of twenty cactus pear (opuntia spp.) accessions i, physical and chemical characteristics 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(ed.), plant pigment. address of the corresponding author: e-mail: rosgar08@hotmail.com © the author(s) 2018. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 90, 274 279 (2017), doi:10.5073/jabfq.2017.090.034 1university of hamburg, department of biology, biocenter klein flottbek and botanical garden, hamburg, germany 2university of yaounde i, higher teachers training college department of biological sciecne, yaounde, cameroon 3university of hamburg, department of chemistry, institute of food chemistry, division of food microbiology and biotechnology, biocenter klein flottbek and botanical garden, hamburg, germany german cacao of cameroon − new facts on a traditional variety fallen into oblivion lina stoll1, nicolas niemenak2*, bernward bisping3, reinhard lieberei1 (received january 2, 2017; accepted july 27, 2017) * corresponding author summary “german” cacao cultivated in cameroon has emerged from a mixture of different gene pools with a large proportion of trinitario and with a pronounced content of polyphenols. in order to characterize this old genotype, polyphenols and polyphenol oxidase were compared with hybrid selected genotypes. epicatechin (25 mg/g 52 mg/g fat free dm) and catechin (0.5 1.9 mg/g fat free dm) content of german cacao seeds were of similar range with hybrid investigated samples. german cacao is characterized by its high content of anthocyanins especially cyanidine-3-arabinoside which ranges from 8.84 mg/g to 17.51 mg/g fat free dm. hybrid genotypes displayed 1 mg/g to 6.4 mg/g fat free dm of cyanidine-3-arabinoside. ppo activity was 10 to 20-fold higher in german cacao seeds compared to hybrid. anthocyanin and ppo through the oxidation of phenols to quinone are involved in colour development and pests and diseases resistance. pigment is one of the most important factors for the colour of cocoa powder. we discuss the high content of anthocyanin and ppo activity in german cacao in relation with the reddish colour of cocoa powder derived from cameroonian cacao. key words: anthocyanin, polyphenol oxidase, polyphenols, cacao, cameroon introduction cacao was first introduced to africa in the beginning of the 18th century by the portuguese. they brought cacao of the amelonado type (lower amazon forastero) from bahia (brazil) to the island of principe around 1822 (aikpokpodion, 2012). it arrived in sao tomé in 1850 (bartley, 2005). in 1854 the spanish brought cacao probably from sao tomé to the island of fernando po (now bioko, equatorial guinea) to the coast of cameroon. cacao from fernando po became the major source of planting material into the mainland of west africa (aikpokpodion, 2012). the first species of recorded cacao reached cameroon in 1876. it was probably introduced from trinidad (aikpokpodion, 2012; efombagn et al., 2008). thirteen plants imported by british missionaries from the royal botanic gardens of england were planted on cameroon mountains in the south western region. towards 1892, under the german colonial administration, lower amazon forastero (amelonado) from sao tomé and fernando po was introduced by preuss, the curator of the victoria (now limbe) botanical garden (efombagn et al., 2008; aikpokpodion, 2012). around 1895, forastero plants (332 plants) from trinidad were introduced (aikpokpodion, 2012; preuss, 1901). in the year 1900 several varieties of the forastero plants originating from south and central america were introduced by preuss (e.g. ‘puerto-cabello’, ‘venezuela’, ‘maracaibo’ and ‘la guira’ from venezuela, ‘guayaquil’ from ecuador, ‘soconusco’ from mexico, ‘nueva grenada’ from colombia, ‘suriname’, ‘criollo’ and ‘fo rastero’ (aikpokpodion, 2012)). at the beginning of the twentieth century the cacao diversity in cameroon had enlarged to one of the most diverse collections (bartley, 2005). the first cacao breeding process in cameroon took place in the 1950s by selection of the dominating local trinitario population. these were identified as selections from the nkoemvone (snk) accessions (research station, southern cameroon, efombagn et al., 2008). in the 1950s and 1960s the local cacao germplasm was broadened by introducing the upper amazon forastero types (efombagn et al., 2008). in the following years, the local trinitario species was crossed with this upper amazon forastero. in the 1970s and 1980s hybrids with a high yield potential and precocity were selected and established in cameroon. in addition, some cacao genotypes were selected and cultivated by smallholders because of their low susceptibility to diseases (efombagn et al., 2008). therefore these cacao trees were only slowly replaced by new breeding materials. because cacao was first introduced in cameroon under the german colonial administration, this traditional cacao is known as “german” cacao. the german cacao presents a high level of admixture of diverse genes. genes of lower amazon cacao (54%) were found to be predominant followed by upper amazon cacao (33%) and criollo (7%) (efombagn et al., 2008). only a low content of trinitario was found in the examined samples (efombagn et al., 2008). studies using 12 microsatellite loci suggest that 25.5% of cacao from 400 farm accessions is still closely related to the traditional amelonado variety (efombagn et al., 2009; parentage analysis). this is probably due to a relatively small number of hybrids (efombagn et al., 2006). another 46.3% were found to be direct descendants from 24 parental clones used in biclonal seed gardens, and 28.3% appeared to have come from uncontrolled pollination events in cacao farms. this could be the effect of a common practice of cacao growers, who use seeds collected in their own farm for new planting (efombagn et al., 2009). preuss probably got the trinitario varieties from venezuela but also from west indies. they had distinctive red podded fruits and an unusually red coloured powder (preuss, 1901; wood, 1991; laird et al., 2007). these trinitario varieties are still cultivated in the region around mount cameroon (laird et al., 2007), whereas in south and east cameroon these varieties were natural hybridized with amelonado genotypes from fernando po (laird et al., 2007). genetic diversity studies found a higher consistency with local trinitario cacao in the east region of cameroon and upper amazon cacao and its hybrids in the centre-south regions (efombagn et al., 2006). the genetic diversity of the farmers’ planting material is not very high, and it is genetically close to parental genotypes available in genebanks (efombagn et al., 2006). the “german” cacao has small seeds and takes a long time to grow, but it is described as durable and more resistant to pests and diseases (laird et al., 2007). many farmers prefer the “german” cacao for new planting because of its constantly running harvest, its longevity and its low susceptibility to pod rot (phytophthora megakarya, paulin cameroon: german cacao 275 et al., 2003). new bred varieties of cacao do indeed have a much higher yield than the local “german” cacao, but farmers criticize their higher susceptibility to black pod disease and their shorter life span. for this reason, the replacement of traditional varieties by new varieties is slow. in cameroon, cacao has been increasingly grown since the 1960s. today, about 400 000 hectares of cacao are cultivated in more than 200 000 farms (efombagn et al., 2006). mostly, these are very small family farms. on average, cacao plantations are about 6.5 hectares in size, and averagely 1200 trees are planted per hectare (paulin et al., 2003). the annual production is about 200 000 tonnes (icco, 2012). the majority of plantations are more than 30 years old (about 50%, paulin et al., 2003). according to jagoret et al. (2011), nearly 70% of cacao plantations in cameroon are more than 40 years old, and 40% of these plantations in southern cameroon were planted before 1960 (paulin et al., 2003). they have very low yields (e.g. 150 to 300 kg dry cacao per hectare) (efombagn et al., 2006). in well managed plantations with high performance clones, significant ly higher yields of up to 2500 kg per ha can be achieved. the objective of the current paper is to describe the characteristics of the traditional “german” cacao of cameroon. as already described, some traditional cacao varieties of cameroon have distinctive red coloured seeds. for a more detailed analysis of these characteristics, the content of epicatechin and anthocyanins were analyzed and the activity of the polyphenoloxidase was determined. phenolic compounds as well as oxidative enzymes contribute to the quality of raw cacao. material and methods material traditional “german” cacao varieties were collected from different cacao plantations from the region of yaounde: · mbangasina about 100 km north of yaounde, a dry region. pods with a yellowish, light brown colour. · awae about 50 km east of yaounde. pods with a yellow colour. · ngat about 40 km south east of yaounde. according to the owners description, the trees of this plantation were at least 50 years old. the fruits were much less affected by pests than the fruits of the other plantations. the fruits were classified in red and yellow according to their colour. hybrid varieties were collected in seed garden from sodecao in meigang. fruits were transported to hamburg and freeze dried (tab. 1). methods before further analyses, freeze-dried seeds were defatted. first, the testa and the radicals were removed and 10 ml of n-hexane was added to approximately 2 g seed material and it was crushed in a ball mill by shaking for 10 minutes at a frequency of 25/s. the homogenate was rinsed out of the ball mill with about 75 ml of petroleum ether (bp 4060 °c), then filtered and dried in a vacuum drying oven. polyphenols analysis 50 mg of defatted cocoa powder was stirred with 3 ml methanol for 30 sec with an ultraturrax (t25, janke & kunkel). after 15 min on ice with constant homogenisation, the samples were centrifuged for 10 min at 5000 rpm. the supernatant was decanted into 50 ml conical flasks. this extraction was repeated twice for only two minutes on ice and 20 seconds by an ultraturrax (t25, janke & kunkel). the samples were concentrated by drying on a rotary evaporator at 40 °c and 100 mbar, and then resuspended in 1.5 ml methanol (gradient grade). after subsequent filtration through a 0.45 μm filter (multoclear 25 mm ptfe, cschromatographie service), 20 μl of each sample were injected to the hplc. the polyphenols were measured by a pda-detector (photo-diodearray-detector, waters 996) within a wavelength range from 225 nm to 540 nm. the polyphenols were quantified at a wave length of 280 nm and the anthocyanins at 540 nm. a lichrocart 100 rp-18 end-capped column (particle size 5 μm, length 250 mm, inner dia meter 4 mm) with a lichrospher 100 rp-18 end-capped pre-column was used for separation. the column temperature was 26 °c and the eluents: a: 2% acetic acid; b: acetonitrile / deionized water / acetic acid (v / v / v 400: 90: 10) (tab. 2). catechin, epicatechin, cyanidin3-galactoside and cyanidin-3-arabinoside were used as standard for quantification. tab. 1: different clones used for analysis comparison with the german cacao. waa snk 13 ics 40 t 79/501 snk 64 ics 84 german cacao catongo n = 11 3 3 3 3 3 10 3 tab. 2: rp-hplc gradient used for the separation of polyphenols time flow rate a b (min) (ml min-1) (%) (%) 0 1.2 90 10 8 1.2 90 10 38 1.1 77 23 50 1 60 40 70 1 10 90 73 1 10 90 78 1.2 90 10 93 1.2 90 10 polyphenoloxidase (ppo) for each determination of the ppo activity, the cotyledons of one seed were used. the weights were determined and after adding 0.1 g polyvinylpolypyrrolidone and 10 ml iced cyanide sörensen buffer ph 6.4 (sörensen buffer: 738 ml kh2po4-solution 67 mmol/l, 262 ml na2hpo4-solution 67 mmol/l, ph 6.4. for cyanide sörensen buffer, the buffer contained 5 mmol/l kcn and 1.7 mmol/l h3po4), samples were homogenized by an ultraturrax (t25, janke & kunkel). subsequently, the samples were centrifuged for 30 min at 14000 g (rc5c, sorvall instruments). the ppo activity was measured in the pellet and the supernatant. 50-100 mg of the pellet were suspended in 1 ml sörensen buffer and further diluted, depending on the level of ppo activity. to measure the ppo activity in the supernatant, the cyanide-containing supernatant was purified from the cyanide with a pd10 column (sephadex™g-25m, amersham biosciences). for this purpose, the pd-10 column was first equilibrated with 25 ml sörensen buffer. subsequently, 2.5 ml of the sample solution were added to the column, which were eluted with 3.5 ml of sörensen buffer. the cyanide was added in order to protect the enzymes to tanning reactions by quinones produced during extraction (stoll, 2010). 276 l. stoll, n. niemenak, b. bisping, r. lieberei the measurements were performed at 25 °c with air-saturated sörensen-buffer (6.4) and 4-methylcatechol (56 mg 4-methylcatechol in 2 ml deionized water) as standard substrate. autoxidation of methylcatechol was measured in a solution containing 2.9 ml sörensen-buffer and 100 μl 4-methylcatechol solution. the ppo activity was evaluated within a solution containing 2.7 ml sörensen buffer, 200 μl enzyme suspension, 100 μl 4-methylcatechol solution. the enzyme reaction was started after two minutes of stirring the enzyme suspension in the buffer by adding the substrate solution. the oxygen consumption was recorded over a period of 6 min with a flat bed recorder. the ppo activity was determined from the increase of the curve 45 seconds after addition of substrate, by applying a tangent to the curve. the slope was calculated as a percentage of oxygen consumption per unit of time. of these, the rate of the autoxidation was subtracted. according to meyer (1975), the oxygen consumption of 1% per hour correlates with 6.7 nmol per hour. results and discussion according to the barry callebaut’s communication officer at the ism (international supplier trade fair for the sweets and snacks industry) launch in cologne (germany) 2012, the milk chocolates created with the red cacao from cameroon are of outstanding quality. the reddish colour of the cameron cacao is world-wide known and the derived cacao powders are perfectly suited for use in bakery application. however, the scientific and biological backgrounds underlying this specific property are not well uncovered. in incubation experiments (kadow et al., 2013), german cacao showed distinctly stronger red coloured medias by a leakage of anthocyanins from the cotyledons than other examined genotypes (data not shown). based on this observation, further analyses with traditional cacao seeds were carried out. fresh seeds of german cacao contain a very high content of polyphenols, especially anthocyanins. fig. 2a shows the epicatechin content of different cacao genotypes. the epicatechin content varies between 25 mg/g ffdm in seeds of t 79/501 and 52 mg/g ffdm in seeds of snk 64. the epicatechin content of german cacao lies in a similar range. there is no significant difference to the epicatechin content of the other analyzed geno types. the catechin content of the german cacao seeds was also found to be in a similar range as the content of the other investigated samples. the samples examined contained between 0.5 mg/g ffdm and 1.9 mg/g ffdm catechin (fig. 2b). the polyphenol content of german cacao seeds of different regions in cameroon was compared fig. 1: root aspect of traditional german cacao, gc (a) and hybrid ics40 clone (b) 6 days after germination in distilled water. the high accumulation of anthocyanin in the root is observed in gc. 3       fig. 2: epicatechin (a) and catechin (b) content from traditional german cacao (gc) and hybrid varieties. milled defatted cacao seeds were extracted in methanol and polyphenol analysis was done by hplc. mean values (n = 6) with different letters differed significantly at p < 0.05, post hoc test: fisher lsd, p < 0.05. mean is the result of two analyses from three independents cacao pods. e pi ca te ch in [m g g1 ff d m ] 3       c at ec hi n [m g g1 ff d m ] a b cameroon: german cacao 277 (data not shown). the highest content of all polyphenols were found in german cacao of awae. the lowest contents were found in german cacao of the dry area of mbangassina. the polyphenols quality of raw cacao beans is rarely diverse. however, their quantity rely on many factors such as the area of trees cultivation, bean maturity, macroand micro-environment, the harvest period and store time after harvest (niemenak et al., 2006; oracz et al., 2015). the content of cyanidin-3-galactoside and cyanidin-3-arabinoside in german cacao seeds were significantly higher than in the seeds of the other samples examined (fig. 3). this high anthocyanin content in cotyledon tissue corresponds to a high coloration of the root during germination (fig. 1). furthermore, it is a typical trait in german cacao and represents a potential phenotypic descriptor to morphologically identify the german cacao group. different developmental processes are regulated by flavonoids among which lateral root initiation well highlighted in arabidopsis and tomato using transgenes and mutants. in tomato mutants with reduced anthocyanin synthesis displayed reduction in lateral roots at the onsets of root initiation (negi et al., 2008; 2010). in addition to the high polyphenol content, fresh german cacao seeds contain an approximately 12-fold higher ppo activity (fig. 4) than seeds of other varieties examined. fresh german cacao seeds contain in the supernatant an activity between 0.01 and 0.14 μkat/g fresh weight (fw) and in the pellet an activity between 5.2 and 6.7 μkat/g fw. whereas in controlled samples only an activity between 0.003 and 0.026 μkat/g fw in the supernatant and between 0.1 and 0.7 μkat/g fw in the pellet were found. in both cases, most of the ppos were membrane bound. ppo catalyses the oxidation of ortho (o)-phenols to o-quinone at the expense of molecular o2. the o-quinone is a highly reactive electrophilic molecule and links with functional groups of proteins such as sulfhydryl, amine, amide, indole and amidazole substituents, forming protein-bound phenol (bittner, 2006). this catalytic action of ppo has an enormous impact on food quality and results in alteration of colour (browning), flavour, texture and nutritional value (vamos-vigyazo, 1981). however, in cacao, this browning has beneficial effects in improving chocolate flavour precursor’s development in fermented and dried beans. indeed, during the cacao cut-test chart the browning of the beans is one of the criteria to recognize good fermented cacao. further, the german cacao is well known by farmers to be blackpod resistant. the contribution of ppo and polyphenols to this feature may be significant. in fact, paulin et al. (2003) and omokolo et al. (1996) showed that cacao genotypes resistant against pod rot are rich in polyphenols. the most frequently suggested role for ppo and polyphenols in plants has been in defence against herbivores and pathogens. the o-quinone-protein complex is known to reduce the nutritional value of the tissue and thereby reduce predation and can also participate in the formation of structural barriers against inva ding pathogens. recently, araji et al. (2014) showed that silencing ppo in walnut (juglans regia) caused major alterations in the meta bolism of phenolic compounds and in the expression of phenylpropanoid pathway genes, suggesting a ppo role in secondary meta bolism. the main advantage of german cacao in comparison to other cacao varieties is its very high resistance against pathogens, which is 4       a b c y an id in -3 -g al ac to si d e [m g g -1 f fd m ] c y an id in -3 -a ra b in o si d e [ m g g -1 f fd m ] fig. 3: cyanidin-3-galactoside (a) and cyanidin-3-arabinoside (b) content from traditional german cacao (gc) and hybrid varieties. milled defatted cacao seeds were extracted in methanol and polyphenol analysis was done by hplc. mean values (n = 6) with different letters differed significantly at p < 0.05, post hoc test: fisher lsd, p < 0.05. mean is the result of two analyses from three independents cacao pods. c ya ni di n3ar ab in os id e [m g g1 ff d m ] c ya ni di n3ga la ct os id e [m g g1 ff d m ] 278 l. stoll, n. niemenak, b. bisping, r. lieberei mainly related to high ppo activity. this study highlights the positive impact of high anthocyanin content on reddish colour development necessary for producing premium cacao powder for chocolate related food. acknowledgment the study was partly supported by the alexander-von-humboldt stiftung (www.humboldt-stiftung.de) via grant to nicolas niemenak (3.4-cmr/1115305 stp). the authors express gratitude to thomas tumforde and detlef böhm for their assistance during experiments. references aikpokpodion, p.o., 2012: defining genetic diversity in the chocolate tree, theobroma cacao l. grown in west and central africa. edited by mahmut çalişkan, in tech, rijeka, croatia, 185-212. araji, s., grammer, t.a., gertzen, r., anderson, s.d., mikulicpetkovsek, m., veberic, r., phu, m.l., solar, a., leslie, c.a., dandekar, a.m., escobar, m.a., 2014: novel roles for the polyphenol oxidase enzyme in secondary metabolism and the regulation of cell death in 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[μ ka t g -1 fr es h w ei gh t] pp o a ct iv ity in p el le t [ μk at g -1 fr es h w ei gh t] fig. 4: polyphenol oxidase activity in pellet (a, bound ppo) and supernatant (b, solubilized ppo) from extracted fresh seed of traditional german cacao (gc) and hybrid varieties. fresh seeds were extracted in sörensen buffer and ppo estimated by using a polarographic method based on the consumption of oxygen (meyer et al., 1975). mean values (n = 6) with different letters differed significantly at p < 0.05, post hoc test: fisher lsd, p < 0.05. mean is the result of two analyses from three independents cacao pods. cameroon: german cacao 279 and outcrossing patterns in cacao (theobroma cacao l.) farms in cameroon. heredity 103, 46-53. efombagn, m.i.b., sounigo, o., nyassé, s., manzanares-dauleux, m., cilas, c., eskes, m.a.b., kolesnikova-allen, m., 2006: genetic diversity in cocoa germplasm of southern cameroon revealed by simple sequences repeat (ssrs) markers. afr. j. biotechnol. 5, 1441-1449. 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m.g., muday, g.k., 2008: ethylene regulates lateral root formation and auxin transport in arabidopsis thaliana. plant j. 55, 175-187. negi, s., sukumar, p., liu, x., cohen, j.d., muday, g.k., 2010: genetic dissection of the role of ethylene in regulating auxin-dependent lateral and adventitious root formation in tomato. plant j. 61, 3-15. niemenak, n., rohsius, c., elwers, s., ndoumou, d., lieberei, l., 2006: comparative study of different cocoa (theobroma cacao l.) clones in terms of their phenolic and anthocyanins contents. j. food compos. anal. 19, 612-619. omokolo, n.d., tsala, n.g., djocgoue, p.f., 1996: changes in carbohydrate, amino acid and phenol contents in cocoa pods from three clones after infection with phytophthora megakarya bra. and grif. ann. botany 77, 153-158. oracz, j., zyzelewicz, d., nebesny, e., 2015: the content of polyphenolic compounds in cocoa beans (theobroma cacao l.), depending on variety, growing region, and processing operations: a review. crit. rev. food sci. nutr. 55, 1176-1192. paulin, d., snoeck, l., nyasse, s., 2003: survey on the growing practices and planting material used for cacao growing in the central region of cameroon. ingenic newsletter 8, 5-8. preuss, p., 1901: expedition nach centralund südamerika 1899/1900. kolonial-wirtschaftliches komitee, berlin. stoll, l., 2010: biochemische indikatoren für keimung und fermentation in samen von kakao (theobroma cacao l.). url:http//ediss.sub.unihamburg.de, 559-560. vamos-vigyazo, l., haard, n.f., 1981: polyphenol oxidase and peroxidase in fruits and vegetables. crit. rev. food sci. nutr. 15, 49-127. wood, g.a.r., 1991: a history of early cocoa introductions. cocoa growers’ bulletin 44. address of the authors: lina stoll, reinhard lieberei, university of hamburg, department of biology, biocenter klein flottbek and botanical garden, ohnhorststraße 18, 22609 hamburg, germany nicolas niemenak, university of yaounde i, higher teachers training college department of biological sciecne, po box 47, yaounde, cameroon e-mail: niemenak@yahoo.com bernward bisping, university of hamburg, department of chemistry, insti tute of food chemistry, division of food microbiology and biotechnology, biocenter klein flottbek and botanical garden, ohnhorststraße 18, 22609 hamburg, germany © the author(s) 2017. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 91, 126 134 (2018), doi:10.5073/jabfq.2018.091.018 1university of belgrade, faculty of biology, institute of botany and botanical garden “jevremovac”, belgrade, serbia 2institute for medicinal plant research “dr. josif pančić”, belgrade, serbia antineurodegenerative, antioxidant and antibacterial activities and phenolic components of origanum majorana l. (lamiaceae) extracts sonja duletić-laušević1, ana alimpić aradski1*, stoimir kolarević1, branka vuković-gačić1, mariana oalđe1, jelena živković2, katarina šavikin2, petar d. marin1 (submitted: july 4, 2017; accepted: march 7, 2018) * corresponding author summary the aim of this study was to examine chemical composition, as well as antineurodegenerative, antioxidant and antibacterial activity of aqueous and ethanolic extracts of origanum majorana l. (lamia ceae) originating from serbia, greece, egypt and libya. total phenolics and flavonoids, antioxidant activities, and acetylcholinesterase and tyrosinase inhibitory activities were measured spectrophotome trically. determination of phenolic compounds in extracts was done using hplc-dad technique. antibacterial activity included determination of minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc) using the microdilution method. the highest phenolic and flavonoid contents were recorded in the ethanolic extract of the egyptian sample and in aqueous extract of serbian sample. the hplc analysis showed high content of rosmarinic acid, with the highest amount found in the ethanolic extract of the plants from egypt. water extracts showed prevalently better antioxidant and antineurodegenerative activity in applied tests than the ethanolic extracts. gram-positive bacterial strains showed higher sensitivity to tested extracts. according to the obtained results, sweet marjoram samples from serbia and egypt can be marked as more promising, due to the highest content of total phenolics and flavonoids and the best antioxidant, antibacterial and tyrosinase inhibitory activity. keywords: origanum majorana, extracts, chemical composition, antioxidant activity, antibacterial activity, antineurodegenerative activity list of abbreviations aae ascorbic acid equivalent; abts 2,2-azino-bis(3-ethyl benzothiazoline-6-sulfonic acid) diammonium salt; ache acetylcholinesterase; ad alzheimer disease; bha 2(3)-t-butyl-4-hydroxyanisole; bhi brain-heart infusion; bht 3,5-di-tert-butyl4-hydroxytoluene; dmso dimethylsulfoxide; dpph 2,2-dyphenyl-1-pikrylhydrazyl (dpph); dtnb 5,5’-dithiobis(2-nitrobenzoic acid); frap ferric reducing ability of plasma; gae gallic acid equivalent; l-dopa 3,4-dihydroxy-l-phenylalanine; mbc minimal bactericidal concentration; mhb mueller-hinton broth; mic minimal inhibitory concentration; qe quercetin equivalent; tfc total flavonoid content; tpc total phenolic content; tptz 2,4,6-tripyridil-s-triazin; tyr tyrosinase; β-cb assay β-carotene bleaching assay introduction the mint family (lamiaceae) includes aromatic plants widely used for culinary, medicinal, cosmetic and ornamental purposes, such as basil, rosemary, sage, oregano, lavender, thyme and mint (raja, 2012). the genus origanum is composed of 42 species and 18 hybrids widely distributed in eurasia and north africa (ietswaart, 1980), being native to the mountainous areas of mediterranean and asia (chishti et al., 2013). species belonging to the genus origanum are used since the ancient times as spices, medicinal, aromatic and ornamental plants (meyers, 2005). in vitro pharmacological investigations showed their antibacterial, antifungal, antioxidant, antispasmodic, antimutagenic, antitumoral, analgesic, antithrombin and antihyperglycaemic activities (chishti et al., 2013). origanum majorana l. (sweet marjoram) is herbaceous perennial shrub, inhabiting dry slopes and rocky places, native to cyprus and eastern mediterranean area (ietswaart, 1980). it is used as a medicinal plant, against cold, as a spasmolytic, antirheumatic, diuretic, and antiasthmatic drug, and as a culinary herb, for flavouring sausages, sauces, soups and condiments (chishti et al., 2013). the essential oil of marjoram of different origin was previously analyzed for the composition and biological activities (teixeira et al., 2013; hajlaoui et al., 2016). various extracts were studied for antioxidant (jun et al., 2001; vági et al., 2005a; el-maati et al., 2012; roby et al., 2013; ayari et al., 2013; benchikha et al., 2013; vasudeva et al., 2014; afifi et al., 2014; erenler et al., 2015) and antimicrobial activities (vági et al., 2005b; leeja and thoppil, 2007; abdel-massih et al., 2010). in the presented manuscript for the first time the marjoram extracts were tested for the antineurodegenerative effects, which is of growing scientific and public interest. the marjoram spices used in the analyses were produced by respectable companies for culinary herbs and spices, while libyan marjoram was purchased at the local market. in several papers the commercially purchased marjoram material was subjected to various analyses (mossa et al., 2015; wahby et al., 2015; fernandes et al., 2016), because of importance of testing the commercial, widely used spices in daily diet for possible benefits for consumers health. considering the insufficiency of data on medicinal properties of o. majorana extracts, the goals of this study were chemical analysis as well as investigation of antibacterial, antioxidant and antineurodegenerative activity of marjoram aqueous and ethanolic extracts obtained from plants cultivated in serbia and three mediterranean countries (greece, egypt, libya). material and methods chemicals and reagents methanol, ethanol, distilled water, glacial acetic acid, hydrochloric acid and chloroform were purchased from zorka pharma, šabac (serbia). gallic acid, quercetin, ascorbic acid, 2(3)-t-butyl-4-hydroxyanisole (bha), 3,5-di-tert-butyl-4-hydroxytoluene (bht), 2,2-dyphenyl-1-pikrylhydrazyl (dpph), 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (abts), 2,4,6-tripyridil-striazin (tptz), potassium acetate (c2h3ko2), potassium-persulfate (k2s2o8), dimethylsulfoxide (dmso), sodium carbonate anhydrous (na2co3), aluminium nitrate nonahydrate (al(no3)39h2o), sodium acetate (c2h3nao2), iron(iii) chloride (fecl3), iron(ii)-sulfate heptahydrate (feso4∙7h2o), β-carotene, folin-ciocalteu phenol reagent, sodium phosphate monobasic (nah2po4), sodium phosphate bioactivities of origanum majorana extracts 127 dibasic (na2hpo4), 5,5’-dithiobis(2-nitrobenzoic acid) (dtnb), acetylcholinesterase from electrophorus electricus (electric eel) (ache), acetylcholine iodide, galanthamine hydrobromide from lycoris sp., kojic acid, tyrosinase from mushroom and 3,4-dihydroxyl-phenylalanine (l-dopa), streptomycin, rifampicin and resazurin sodium salt (>90% (lc) were purchased from sigma chemicals co., st. louis, mo (usa), while tween 40 and linoleic acid were purchased from acros organics (belgium). brain-heart infusion (bhi) and mueller-hinton broth (mhb) were purchased from himedia (india). plant material dried and crushed plant material of o. majorana, which was used in the experimental procedure, originating from serbia, greece, egypt and libya, was commercially purchased. marjoram is cultivated at appropriate field and collected, stored and controlled by the respectable companies. the plant material from serbia is a product of institute for medicinal plant research “dr josif pančić“, the sample from greece is produced by “kagia“ company, from egypt is a product of “kotanyi“ company, while the plant material from libya was purchased from the local market. preparation of extracts grinded plant material (50 g) was extracted during 24 h at room temperature (5% w/v) using two solvents, hot water and 96% etha nol. the mixture was exposed to ultrasound 1 h before and after 24 h to improve extraction process. subsequently, extracts were filtered through filter paper (whatman no.1) and evaporated under reduced pressure (buchi rotavapor r-114). during the process of concentration of ethanolic extracts high temperatures were not used in order to prevent the loss of analytes and artifact formation. the obtained crude extracts were stored in a refrigerator at +4 °c for further experiments. determination of total phenolic and flavonoid contents the total phenolic (tpc) and flavonoid contents (tfc) were measured using jenway 6305uv/vis spectrophotometer as described before (alimpić et al., 2015, 2017). phenolic content of extracts was calculated from gallic acid curve equation and expressed as gallic acid equivalents (mg gae/g dry extract). flavonoid content of extracts was calculated from quercetin curve equation and expressed as quercetin equivalents (mg qe/g dry extract). values were presented as mean ± standard deviation averaged from three measurements. hplc analysis phenolic compounds in the tested extracts were determined by comparing the retention times and absorption spectra (200-400 nm) of unknown peaks with the reference standards. hplc-dad analysis was performed on an agilent 1200 series hplc (agilent technologies, palo alto, ca, usa) equipped with lichrospher® 100 rp 18e column (5 μm, 250 × 4 mm). mobile phase a was formic acid in water (0.17%), while mobile phase b was acetonitrile. the injection volume was 10 μl, and flow rate 0.8 ml/min with gradient program (0-53 min 0-100% b). stop time of the analysis was 55 min. the investigated samples were analyzed in triplicate. evaluation of antioxidant activity dpph free radical scavenging assay was performed according to previously described experimental protocol (alimpić et al., 2015, 2017). bha, bht and ascorbic acid were used as antioxidant standards. results were expressed as ic50 values (μg/ml) averaged from three measurements. abts free radical scavenging assay was performed according to alimpić et al. (2015, 2017). the extracts and antioxidant standards bha and bht were tested in concentration of 1 mg/ml and 0.1 mg/ml, respectively. abts activity was calculated from ascorbic acid calibration curve and expressed as ascorbic acid equivalents per gram of dry extract (mg aae/g), averaged from three measure ments. frap assay (ferric reducing ability of plasma), evaluating total antioxidant power of the sample, was performed according to alimpić et al. (2015, 2017). the extracts were tested in concentration of 0.5 mg/ml. bha, bht and ascorbic acid (0.1 mg/ml) were used as reference antioxidants. frap values of samples were calcula ted from standard curve equation and expressed as μmol feso4 × 7 h2o/g dry extract and presented as mean ± standard deviation averaged from three measurements. β-carotene bleaching (β-cb) assay was performed according to alimpić et al. (2017). crude extracts and standards bha, bht and ascorbic acid were tested in concentration of 0.5 mg/ml. the absorbances were measured using tecan sunrise sn microplate reader. the antioxidant activity of the extracts was evaluated in term of β-carotene bleaching using the following formula: [(a120-c120)/(c0c120)] × 100%, where a120 and c120 were the absorbance values measured at 120 minutes for sample and control, respectively, while c0 is absorbance of control at 0 minutes. antibacterial assay the antibacterial activity of extracts and standard antibiotics streptomycin and rifampicin was tested against four gram-negative (escherichia coli atcc 25922, pseudomonas aeruginosa atcc 15442, salmonella enteritidis atcc 12076, shigella flexneri atcc 9199) and four gram-positive bacterial strains (enterococcus fecalis atcc 29212, staphylococcus aureus atcc 25923, bacillus subtilis atcc 6633 and listeria innocua atcc 33090). the lowest concentrations without visible growth, i.e. minimal inhibitory concentrations (mics) were determined according to kolarević et al. (2016). antineurodegenerative activities acethylcholinesterase (ache) and tyrosinase (tyr) inhibitory activity assays were performed according to spectrophotometric method using 96-well plates as described before (alimpić et al., 2017). the applied concentrations of extracts and standards (galanthamine and kojic acid) were 25, 50 and 100 μg/ml. the absorbances were measured using tecan sunrise sn microplate reader equipped by xfluor4 software. the results are expressed as percents of inhibition of samples comparing to controls. statistical analysis differences between the group means and their significance were verified by one-way anova using the software package statistica v.7.0. the significance of differences was evaluated using bonferroni’s test and statistical significance was set at p<0.05. pearson’s correlation coefficients were calculated between content of phenolic components and antioxidant assays and interpreted according to taylor (1990). calculations and constructing of the charts were performed using ms office excel (2013). results and discussion yield of extracts, total phenolic and flavonoid contents the yields of extracts varied depending on the plant origin and applied solvent (tab. 1), with higher yields in aqueous extracts. among aqueous extracts, the extract of the egyptian o. majorana had the highest yield (29.51%), while the lowest yield was obtained for the 128 s. duletić-laušević, a. alimpić aradski, s. kolarević, b. vuković-gačić, m. oalđe, j. živković, k. šavikin, p.d. marin greek sample (16.47%). the highest yield among ethanolic extracts was recorded for the libyan sample (13.49%), and the lowest for the serbian plants (8.51%). in the study of vági et al. (2005a) the yield of ethanolic extract of plants from egypt was higher compared to our results (28.99% and 11.91%, respectively). triantaphyllou et al. (2001), applied different extraction methods and obtained a yield as low as 0.88%, in the water extract of greek o. majorana. in the study of benchikha et al. (2013) ethanolic extract of algerian o. majorana yielded 8.16%, which is close to the serbian sample in this study. total phenolic and flavonoid contents of extracts were measured spectrophotometrically and presented as gallic acid (mg gae/g of dry extract) and quercetin (mg qe/g of dry extract) equivalents, respectively (tab.1). all tested aqueous extracts of o. majorana, except for the one originating from egypt, showed higher phenolic content comparing to ethanolic extracts (tab. 1). the highest content of phenolics was found in aqueous extracts of the serbian o. majorana (122.71 mg gae/g dry extract), and in ethanolic extracts of the egyptian plant (139.98 mg gae/g dry extract). vági et al. (2005a) obtained higher percentage of phenolics in ethanolic extracts of o. majorana from egypt compared to the plants from hungary. they explained the results by influence of different climate conditions, while many more sunny days in egypt could cause higher production of self-defense compounds against radiation and microbes. ethanolic extract of egyptian marjoram showed higher amount of total pheno lic and flavonoid compounds, similar to the results of el-maati et al. (2012), who analyzed extracts of five egyptian medicinal plants and obtained higher amounts of phenolics and flavonoids in ethanolic extracts of o. majorana compared to aqueous extracts. benchikha et al. (2013) studied ethanolic extracts of algerian oregano species and obtained higher content of phenolics in o. majorana than in o. vulgare extract. flavonoids were detected in higher amounts in all tested ethanolic extracts compared to the aqueous extracts (tab. 1). ethanolic extract of the plant originating from egypt was the most abundant in flavonoids (20.05 mg qe/g dry extract) compared to other samples. elmaati et al. (2012) also obtained significantly higher amounts of flavonoids in ethanolic extracts of the egyptian o. majorana than in the aqueous extract. when comparing the percentage of flavonoids in ethanolic extracts of hungarian and egyptian o. majorana vági et al. (2005a) obtained lower values in ethanolic extracts of o. majorana from egypt. benchikha et al. (2013) found a high phenolic content in the ethanolic extract of o. majorana from algeria (266.86 mg gae /g), but the extract was poor in flavonoids. hplc analysis of the extracts chemical composition of o. majorana extracts was determined using hplc-dad (tab. 2) in order to determine components responsible for certain types of bioactivities. among phenolic acids rosmarinic acid was present in a significant amount, especially in ethanolic extracts of all tested samples. the ethanolic extract of the egyptian plant contained the highest amount of rosmarinic acid (41.54 mg/g). rosmarinic acid is restricted to the subfamily nepetoideae of the lamiaceae family (petersen and simmonds, 2003). this acid was previously reported as a dominant phenolic acid of the lamiaceae (wang et al., 2004; lee, 2010; embuscado, 2015), that has a range of biological activities (al-dhabi et al., 2014). in the comparative study of rosmarinic and caffeic acids contents of 76 lamiaceae species using tlc-densitometric method, janicsak et al. (1999) have found low content of rosmarinic acid (0.87-2.40 mg/g) and caffeic acid (0.09-0.27 mg/g) in tested origanum species. erenler et al. (2015) studied the phytochemical content of the turkish o. majorana water-soluble ethyl acetate extract and also detected rosmarinic acid and arbutin. in our samples, arbutin was, generally, more abundant in ethanolic extracts with the higest contab. 1: yields, total phenolic and flavonoid content of o. majorana extracts sample origin yield (%) tpc (mg gae/g) tfc (mg qe/g) aqueous extracts serbia 22.54 122.71±2.68a 15.64±0.52ad greece 16.47 111.10±1.32b 15.13±0.55abd egypt 29.51 95.33±4.61c 13.51±0.42b libya 24.81 84.76±1.84d 11.03±0.46c ethanol extracts serbia 8.51 84.38±2.23d 16.33±0.44d greece 10.36 83.25±0.85d 18.85±0.74e egypt 11.91 139.98±1.14e 20.05±1.11e libya 13.49 58.62±0.51f 14.87±0.29abd means followed with different letters in the same column are significantly different at p<0.05. tab. 2: phenolic components identified in o. majorana extracts sample origin content of phenolic components (mg/g dry extract) rosmarinic acid caffeic acid chlorogenic acid arbutin luteolin-7-o-glucoside aqueous extracts serbia 20.09±0.81a 1.02±0.03a 4.52±0.15a 14.73±0.52a 0.99±0.02a greece 9.37±0.19b 0.54±0.02e 3.91±0.10c 2.22±0.06e 3.82±0.13d egypt 23.98±0.79e 0.90±0.02f 4.42±0.17a 7.47±0.21f 5.57±0.19e libya 8.06±0.30b 1.88±0.07c 3.82±0.14c 3.33±0.09c 1.66±0.07b ethanol extracts serbia 22.47±0.75ae 0.13±0.00b 0.04±0.00bd 10.02±0.38b 1.10±0.03a greece 27.71±1.02d 0.49±0.01e 0.34±0.01be 15.46±0.49a 0.65±0.02c egypt 41.54±1.42f 0.26±0.01d 0.62±0.02e 25.67±1.03g 1.90±0.06b libya 13.11±0.26c 0.32±0.01d 0.03±0.00d 13.73±0.42d 0.40±0.01c means followed with different letters in the same column are significantly different at p<0.05. bioactivities of origanum majorana extracts 129 tent in egyptian sample. on the contrary, serbian sample contained greater amount of arbutin in water extract. arbutin has been shown to have antityrosinase (ye et al., 2010), antimicrobial (kundaković et al., 2014), antioxidant, antihyperglycaemic and antihyperlipidemic activities (shahaboddin et al., 2011). water extracts of analyzed samples contained much more chlorogenic acid compared to ethanolic extracts. chlorogenic acid showed several beneficial health effects, such as antioxidant, neuroprotective, antiinflamatory, hypoglycemic, antifungal, etc. (upadhyay and rao, 2013). this acid is an ester of caffeic and quinic acids, but caffeic acid showed stronger antioxidant activity when compared to chlorogenic acid in in vitro and in vivo experiments (sato et al., 2011). flavone luteolin and its glycosides have been reported to possess a wide range of bioactivities involved in prevention and treatment of various diseases (lopez-lazaro, 2009). among flavonoids, luteolin-7-o-glucoside was detected in all extracts with the highest content in aqueous extract of egyptian sample. after comparison of hplc-dad chromatograms of water and ethanolic extracts at 280 and 330 nm we have not detected any unusual peak that might suggest formation of artifacts as a result of using ethanol as extraction solvent. evaluation of antioxidant activity of extracts considering the facts that oxidative stress has been involved in the development of different human diseases and occurrence of various side effects of synthetic antioxidants, researchers have focused on the beneficial health effects of phytochemicals (embuscado, 2015), including those isolated from origanum species. antioxidant activity of o. majorana extracts was measured using four assays (tab. 3). tested aqueous extracts showed better activi ty against dpph radicals compared to ethanolic extracts. among aqueous extracts, efficiency varied from the serbian sample which showed the best activity (28.25 μg/ml) to the libyan sample with low activity (51.84 μg/ml). among ethanolic extracts the best activity was obtained for extract of the egyptian plants (54.15 μg/ml) which was also the most abundant in phenolics content, while the libyan extract showed the lowest efficiency (114.47 μg/ml). in the study of vági et al. (2005a) the ethanolic extract of the egyptian herb showed stronger antioxidant properties when compared to the hungarian one, exhibiting antioxidant power comparable to that of bht, probably caused by differences in the amounts of phenolic compounds. ramadan et al. (2014) obtained a higher percen tage of inhibition of dpph radicals for ethanol than methanol extract of egyptian o. majorana at analyzed concentrations. our results showed the weakest activity of libyan extracts, while benchikha et al. (2013) obtained significant results for the geographically close algerian o. majorana and o. vulgare ethanolic extracts, where all samples had better antioxidant activity than used controls (bha and α-tocopherol), with better results for the extract of o. majorana. antioxidant activity of tested o. majorana extracts was also determined using abts radical decolorization assay and expressed as ascorbic acid equivalents (tab. 3). the tested aqueous extracts in abts assay, with exception of the egyptian sample, demonstrated better activity than the ethanolic ones (tab. 3). the aqueous extract of the serbian sample showed the best activity (2.06 mg aae/g), and the ethanolic extract of the egyptian plant was the most powerful against abts radicals (2.25 mg aae/g), while the lowest results for both extracts were obtained for the libyan sample. el-maati et al. (2012) obtained better abts activity using ethanolic extracts compared to aqueous extracts of o. majorana from egypt (81.1 and 33.3%, respectively), which is in accordance with our results for the egyptian plant. ramadan et al. (2014) also studied egyptian marjoram and obtained high abts scavenging capacity of ethanolic extract (854 μm trolox/g). palaniswamy and padma (2011) obtained high percentage of inhibition of abts radicals for aqueous leaf extract, but lower than the methanolic extract (78.31% and 88.96%, respectively). in the frap assay tested aqueous extracts showed much better activity than the ethanolic ones, except for the egyptian plant extract (tab. 3). among aqueous extracts, the serbian sample showed the strongest activity (826.45 μmol fe (ii)/g), while the egyptian plants showed the lowest activity (554.66 μmol fe (ii)/g). however, similarly to the results recorded in dpph and abts assays, among ethano lic extracts, the strongest activity was obtained for the egyptian plants (796.25 μmol fe (ii)/g), while extract of the libyan plants showed the lowest activity (260.32 μmol fe (ii)/g). this assay was previously applied to o. vulgare, which revealed various results, better activity of the aqueous extracts of o. vulgare compared to the ethanolic ones in portuguese samples (teixeira et al., 2013), or tab. 3: antioxidant activities of o. majorana extracts sample origin dpph (ic50, μg/ml) abts (mg aae/g) frap (μmol fe(ii)/g) β-cb assay (% inhibition) aqueous extracts serbia 28.25±0.56a 2.06±0.03a 826.45±8.14a 83.24±2.40a greece 35.27±0.81a 1.88±0.09b 754.59±6.32b 46.54±0.81b egypt 50.90±1.54b 1.52±0.06c 554.66±3.50c 83.24±2.48a libya 51.84±1.33bc 1.31±0.04d 602.06±9.55d 71.28±2.97c ethanol extracts serbia 59.07±0.60c 1.52±0.07c 362.00±7.64e 61.70±2.98df greece 74.27±0.77d 1.52±0.06c 288.23±6.39f 63.30±1.60df egypt 54.15±3.60cb 2.25±0.02e 796.25±7.52g 60.90±1.99def libya 114.47±6.15e 1.30±0.04d 260.32±7.64h 66.49±2.79dc standards bht 17.94±0.17f 2.78±0.02f 445.34±5.77i 53.72±2.26ebf bha 13.37±0.43f 2.82±0.01f 583.72±5.26d 57.71±3.39f ascorbic acid 5.11±0.14g 180.81±8.61j 17.82±1.13g means followed with different letters in the same column are significantly different at p<0.05. 130 s. duletić-laušević, a. alimpić aradski, s. kolarević, b. vuković-gačić, m. oalđe, j. živković, k. šavikin, p.d. marin on the contrary, lower activity of the aqueous extracts of o. vulgare from tunisia than the ethanolic ones (béjaoui et al., 2013). similar to the results obtained in the other applied tests, in the β carotene bleaching assay most of the aqueous extracts showed a higher activity than ethanolic ones, except for the extracts of the plants originating from greece (tab. 3). among aqueous extracts, the strongest activity was shown by the egyptian and serbian samples (83.24%), while the lowest activity was recorded for the greek plants (46.54%). the strongest activity among ethanolic extracts showed the libyan plants (66.49%), while the etanolic extract of marjoram originating from egypt showed the lowest activity (60.90%). du ring the analysis of egyptian o. majorana extracts, el-maati et al. (2012) found a relatively low percentage of β-carotene inhibition, obtaining a better result for the ethanolic extract. the ethanolic extract of the egyptian plants showed considerable antioxidant activity in applied tests, probably due to the high content of rosmarinic acid, which is considered as a component with significant antioxidant activity (tepe, 2008; erenler et al., 2015). correlation between antioxidant activity and phenolic components content correlation between results obtained for antioxidant activity and phenolics content is presented as correlation coefficient (tab. 4). antioxidant activity was more strongly correlated to total phenolic than total flavonoid content which is in accordance to wojdyło et al. (2007). chlorogenic acid content was strongly correlated to dpph and frap values, rosmarinic acid and arbutin to abts values, while caffeic and chlorogenic acids were correlated to β-carroten bleaching assay. chlorogenic, caffeic and rosmarinic acids could be responsible for the antioxidant activity of tested extracts. antioxidant properties of these phenolic acids were also proved by raudonis et al. (2008), sato et al. (2011) and upadhyay and rao (2013). strong correlation is established between dpph and frap as well as abts and frap assays, while correlations between other assays are assessed as moderate to weak. results obtained in dpph and abts assays showed moderate negative correlation between each other as it was previously found for brasilian spices by barros mariutti et al. (2008). in this study, abts and frap assays showed strong correlation which is consistent with hossain et al. (2008). antibacterial activity of extracts search for natural antimicrobials is growing in current studies because of the undesirable health impact of synthetic antimicrobial food preservatives and the occurrence of pathogenic microorganisms resistant to pharmaceuticals (tajkarimi et al., 2010). in addition to the flavoring effect, origanum species are proved to have antimicrobial activity on human and plant pathogens (vági et al., 2005b; leeja and thoppil, 2007; ashraf et al., 2011; jaber et al., 2012; chishti et al., 2013; teixeira et al., 2013). the results of antibacterial activity of extracts against gram-positive bacteria are presented in tab. 5. the most sensitive strains among gram-positive bacteria were b. subtilis and l. innocua, both for aqueous and ethanolic extracts, especially for o. majorana originating from serbia and egypt (tab. 5). marjoram extracts showed almost no activity on tested concentrations against gram-negative bacterial strains, except aqueous extract of libyan sample which produced bacteriostatic and bactericidal effect on s. flexneri. gram-positive bacterial strains showed higher sensitivity against tested extracts compared to the gram-negative ones, which has also been proved by ayari et al. (2013). they found that some of the tested bacterial strains showed higher sensitivity towards methanolic extracts at lower values than antibiotics used as positive controls, as in the case of b. subtilis, l. innocua and s. aureus. antibacterial effect of extracts of other origanum species was analyzed by seve ral authors. ashraf et al. (2011) conducted the comparative analysis of antibacterial activity of o. vulgare chloroform, methanol and aqueous extracts, using agar well diffusion method and obtained promising results for chloroform and methanol extracts, but aqueous extract was not active against most of the studied strains. jaber et al. (2012) compared the antibacterial activity of aqueous, ethanolic and methanolic extracts of o. vulgare against three gram-positive and three gram-negative bacterial strains, using the well diffusion method. the ethanolic extract had higher activity than the aqueous one. in determination of the minimum inhibitory concentration the best activity against b. subtilis was obtained using the ethanolic extract, followed by the methanolic and aqueous extracts. teixeira et al. (2013) also reported on higher ability of the o. vulgare ethanolic extract for the inhibition of growth of seven bacterial strains compared to the hot and cold water extracts, which showed almost no inhibition. antineurodegenerative activities of extracts cholinesterase inhibition is a commonly used approach for treating the symptoms of alzheimer disease (ad), which is a widely distri buted neurological disorder. various plants could be employed as a new strategy in the treatment of neurodegenerative diseases, inclu tab. 4: correlation between antioxidant assays and content of phenolic components in o. majorana extracts dpph assay abts assay frap assay β-cb assay rosmarinic acid -0.03a 0.56b 0.12a 0.07a caffeic acid -0.36b -0.26a 0.31a 0.48b chlorogenic acid -0.72c 0.16a 0.65b 0.41b arbutin 0.23a 0.53b 0.05a 0.07a luteolin-7-o-glucoside -0.44b 0.10a 0.37b 0.09a total phenolic content -0,74c 0.95c 0.89c -0.03a flavonoid content 0,08a 0.61b 0.03a -0.35a dpph assay 1.00 -0.59b -0.82c -0.10a abts assay 1.00 0.79c -0.13a frap assay 100 0.06a β-cb assay 1.00 according to taylor (1990): ar≤0.35 weak correlation; b0.36<r<0,67 moderate correlation; c0.68<r<1 strong correlation. bioactivities of origanum majorana extracts 131 ding several lamiaceae species (perry et al., 2000; orhan et al., 2010, 2012). o. majorana extracts were tested for the inhibition of acetylcholin esterase and tyrosinase (tab. 6 and 7). analyzed aqueous extracts showed higher percentage of acethylcholinesterase inhibition than the ethanolic ones, with the exception of aqueous and ethanolic extracts of serbian marjoram (tab. 6). aqueous and ethanolic extracts of the greek plant showed the highest percentage of ache inhibition (22.80-37.29%), but lower than galanthamine (42.38-57.11%). the obtained results were not concentration dependent, contrary to used standard. the egyptian (mossa and nawwar, 2010) and tunisian (hajlaoui et al., 2016) marjoram essential oils showed significant antiacethylcholinesterase activity, but extracts were rarely exa mined. chung et al. (2001) in screening of 139 spices have obtained the highest inhibitory effect of marjoram ethanol extract, finding the ursolic acid as an active component and potent ache inhibitor. ali-shtayeh et al. (2014) detected much higher inhibition of ache for ethanolic extract of palestinian marjoram, while methanolic extract of iranian o. majorana showed significantly lower inhibition of ache (gholamhoseinian and razmi, 2012), when compared to our results. several studies presented results for antiacethylcholinesterase acti vity of o. vulgare extracts. vladimir-knežević et al. (2014) tested ethanolic extracts of 26 croatian medicinal plants for their inhibitory activity against ache, including o. vulgare which showed around 30% inhibition of enzyme activity at the concentration of 1 mg/ml. orhan et al. (2010) found 35.84% inhibition at 100 μg/ml for the acetone extract of algerian o. vulgare var. glandulosum, while at 25 and 50 μg/ml no activity was recorded. tyrosinase inhibitors are of great concern due to the role of ty rosinase in mammalian melanogenesis, fruit browning, and also in production of dopamine quinone derivatives involved in the degene ration of nigrostriatal dopaminergic neurons in parkinson’s disease (hasegawa, 2010). the ethanolic extracts of marjoram had higher activity than the aqueous ones in testing tyrosinase inhibitory activity (tab. 7). among aqueous extracts, sample from serbia showed the highest percen tage of tyrosinase inhibition (7.77-16.38%), while among ethanolic extracts the highest activity was exibited by the egyptian herb (15.1123.09%). mentioned extracts with the highest activities contained the highest level of arbutin, which is used as strong tyrosinase inhibitor and whitening agent in cosmetic products (lim et al., 2009; sarikurkcu et al., 2015). the obtained results showed dependence on the concentration of extracts. the used standard, kojic acid, showed stronger activity than the examined extracts (33.93-51.81%). there are very few literature data on the tyrosinase inhibitory activi ty of origanum extracts and essential oils. lin et al. (2011) found that antityrosinase activities were 21.8% for o. majorana and 4.7% for o. vulgare from china. significant tyrosinase inhibition activity of the methanolic extract and origanol a of the indian o. vulgare was recorded by rao et al. (2011). sarikurkcu et al. (2015) examined essential oils of two o. vulgare subspecies and obtained significantly better results for o. vulgare subsp. hirtum when compared to o. vulgare subsp. vulgare, probably due to high linalool content. essential oil of italian o. vulgare signi ficantly inhibited mushroom tyrosinase in a dose-depended manner (fiocco et al., 2016). conclusion literature data related to o. majorana bioactivities are mostly limi ted to essential oils, but this study showed that water and ethanolic extracts could also be promising natural sources of bioactive compounds. the aqueous extracts generally showed better antioxidant, antimicrobial and antineurodegenerative activity than the ethanota b. 5 : a nt ib ac te ri al a ct iv ity o f o . m aj or an a ex tr ac ts p re se nt ed a s m ic (m g/ m l ) a nd m b c (m g/ m l ) g ra m -p os iti ve b ac te ri a g ra m -n eg at iv e ba ct er ia sa m pl e e xt ra ct s b . s ub til is e . f ae ca lis l. in no cu a s. a ur eu s e . c ol i p. a er ug in os a s. fl ex ne ri s. e nt er iti di s m ic m b c m ic m b c m ic m b c m ic m b c m ic m b c m ic m b c m ic m b c m ic m b c se rb ia a qu eo us ≤ 0. 62 5 0. 62 5 ≤ 5 5 ≤ 2. 5 2. 5 > 5 > 5 > 5 > 5 ≤ 2. 5 2. 5 ≤ 5 5 e th an ol ic ≤ 0. 62 5 0. 62 5 ≤ 5 5 0. 62 5 1. 25 0. 62 5 1. 25 > 5 > 5 > 5 > 5 > 5 > 5 > 5 > 5 g re ec e a qu eo us ≤ 1. 25 1. 25 5 ≥ 5 > 5 > 5 ≤ 5 5 > 5 > 5 > 5 > 5 ≤ 2. 5 2. 5 ≤ 5 5 e th an ol ic ≤ 2. 5 2. 5 > 5 > 5 1. 25 2. 5 1. 25 2. 5 > 5 > 5 > 5 > 5 5 ≥ 5 > 5 > 5 e gy pt a qu eo us ≤ 1. 25 1. 25 0. 62 5 1. 25 ≤ 5 5 > 5 > 5 > 5 > 5 2. 5 ≥ 2. 5 ≤ 5 5 e th an ol ic ≤ 2. 5 2. 5 > 5 > 5 1. 25 2. 5 0. 62 5 1. 25 > 5 > 5 > 5 > 5 5 ≥ 5 > 5 > 5 l ib ya a qu eo us ≤ 1. 25 1. 25 ≤ 2. 5 2. 5 0. 62 5 1. 25 1. 25 2. 5 > 5 > 5 > 5 > 5 1. 25 2. 5 ≤ 5 5 e th an ol ic ≤ 2. 5 2. 5 > 5 > 5 1. 25 2. 5 1. 25 2. 5 > 5 > 5 > 5 > 5 5 ≥ 5 > 5 > 5 st re pt om yc in * ≤ 3. 12 5 3. 12 5 ≤ 20 0 20 0 ≤ 25 25 ≤ 1. 53 2 3. 12 5 ≤ 12 .5 12 .5 25 50 ≤ 1. 56 2 1. 56 2 ≤ 3. 12 5 3. 12 5 r if am pi ci n* ≤ 3. 12 5 50 ≤ 3. 90 6 7. 81 2 * m ic a nd m b c o f a nt ib io tic s w er e pr es en te d as μ g/ m l . or ig in 132 s. duletić-laušević, a. alimpić aradski, s. kolarević, b. vuković-gačić, m. oalđe, j. živković, k. šavikin, p.d. marin lic ones, with the exception of the egyptian sample in some tests. among tested marjoram samples originated from serbia, greece, egypt and libya, the most promising were plants from serbia and egypt, which contained high level of rosmarinic acid. this acid was found to be the predominant constituent in investigated extracts and presumably had a substantial influence on their antioxidant and antineurodegenerative activities. acknowledgements the authors are gratefull to the ministry of education, science and technological development of serbia [grant number 173029], [grant number 172058], [grant number 46013]. references abdel-massih, r.m., fares, r., bazzi, s., el-chami, n., baydoun, e., 2010: the apoptotic and anti-proliferative activity of origanum 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access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 90, 205 213 (2017), doi:10.5073/jabfq.2017.090.026 1dipartimento di scienze agrarie, forestali e alimentari, università degli studi di torino, torino, italy 2mention agriculture tropicale et développement durable ecole supérieure des sciences agronomiques, université d’antananarivo, antananarivo, madagascar 3dipartimento di scienze della vita e biologia dei sistemi, università degli studi di torino, torino, italy biodiversity and traditional medicinal plants from madagascar: phytochemical evaluation of brachylaena ramiflora (dc.) humbert decoctions and infusions dario donno*1, denis randriamampionona2, harilala andriamaniraka2, valeria torti3, maria gabriella mellano1, cristina giacoma3, gabriele loris beccaro1 (received december 23, 2016; accepted february 28, 2017) * corresponding author summary madagascar is characterized by one of the highest rates of endemism and biodiversity in the world. brachylaena preparations are extensively used in malagasy folk medicine for treating gastrointestinal diseases and blenorrhagia. the aim of this study was a preliminary phytochemical fingerprint of brachylaena ramiflora leaves infusions and bark decoctions, in order to characterize this species as source of biologically active compounds and their relative antioxidant activity by high-performance liquid chromatography-diode array detector. sixteen and twenty-three biomarkers (molecules with health properties selected for their demonstrated positive role on human organism) were identified in b. ramiflora leaf infusions and bark decoctions, respectively: the main compounds identified in the infusions were quinic acid (334.55±0.99 mg/100 gfw), chlorogenic acid (208.27± 7.74 mg/100 gfw), and g-terpinene (144.19±1.00 mg/100 gfw), while the major components in the decoctions were castalagin (2002.64± 13.96 mg/100 gfw), citric acid (1171.81±1.05 mg/100 gfw), and chlorogenic acid (646.44±2.31 mg/100 gfw). b. ramiflora could be considered as a promising source of natural antioxidants that may provide health-benefits. the development of pharmaceuticals based on a sustainable exploitation of wild medicinal plants or their cultivation by local villagers could offer a number of benefits to a wide range of people as an alternative source of income and a natural and accessible health remedy. keywords: medicinal tree-species, antioxidant activity, phytochemical fingerprint, ethnobotany, endemism. list of abbreviations: 1,2-phenylenediamine dihydrochloride: opda; ascorbic acid: aa; dehydroascorbic acid: dhaa; botanical and zoological park of tsimbazaza: bzpt; normal atmosphere: n.a.; relative humidity: r.h.; high-performance liquid chromatography: hplc; diode array detector: dad; total polyphenolic content: tpc; gallic acid equivalents: gae; fresh weight: fw; total anthocyanin content: tac; cyanidin-3-o-glucoside: c3g; ferric reducing antioxidant power: frap; polytetrafluoroethylene: ptfe; fluorophore 3-(1,2-dihydroxyethyl)furo(3,4-b)quinoxalina-1-one: dfq; total bioactive compound content: tbcc; pearson’s correlation co efficients: r introduction the world health organization estimates that nearly 80% of the population in developing countries depends mainly on traditional medicine for the treatment of ailments (randriamiharisoa et al., 2015; razafindraibe et al., 2013). the dependence on remedies derived from medicinal plants is particularly important in these countries, as madagascar, where modern medicine is often absent or simply too expensive (novy, 1997): economic devaluation of the developing countries leads to higher prices of pharmaceuticals and makes medicinal plants and traditional medicine more attractive (randriamiharisoa et al., 2015). additionally, some prefer traditional medicine for several reasons including familiarity, tradition and perceived safety (van andel and carvalheiro, 2013). traditional malagasy medicine makes use of a wide variety of plants to treat gastrointestinal disorders as diarrhea and intestinal parasites, which are particularly prevalent in rural areas of the country (leutscher and bagley, 2003). these diseases are rarely associated with mortality (gastrointestinal bleeding), but they cause significant morbidity as impaired physical and mental development (areeshi et al., 2013). madagascar, located approximately 400 km off of the coast of mozambique in southeastern africa, is the fourth largest island in the world (rasoanaivo, 1990). due to such long isolation and to its tropical location, madagascar is characterized by one of the highest rates of endemism and biodiversity in the world (norscia and borgognini-tarli, 2006): indeed, current floristic calculations indicate that madagascar hosts many endemic plant species (90% of vascular plant species and 96% of tree species) (rasoanaivo, 1990; razafindraibe et al., 2013). for this reason, it is not surprising that malagasy flora can provide a wide variety of medicinal plants as an affordable alternative to expensive western medicine. in any case, only an estimated 10% of the malagasy plant species has been screened for any biological activity until now (hudson et al., 2000). the literature provides evidences on antiplasmodial and antimicrobial properties of different ethnomedicinal plant species from madagascar (norscia and borgognini-tarli, 2006; rasoanaivo et al., 2004; rakotoniriana et al., 2010), but the available survey on malagasy medicinal plants is far from being exhaustive (novy, 1997; rasoanaivo, 1990). rural communities of madagascar still practice and often prefer traditional medicine, especially for treating common and infectious diseases (razafindraibe et al., 2013): in particular, about one third of the plants is used for the treatment of gastrointestinal disorders, one third in case of malaria/fever, and the remaining third in order to treat rheumatisms, cold, skin illnesses and inflammations (norscia and borgognini-tarli, 2006). moreover, reproductive, prenatal and postpartum health are the most frequently cited uses for medicinal plants in women’s health. brachylaena, a member of the subfamily inulae, is a genus of 20 species that occurs in tropical africa and is questionably native of the mascarenes. five species occur in madagascar, and b. ramiflora is widespread in highly disturbed areas, while in rainforest environments it is common from 500 to 2000 m of elevation, and it may 206 d. donno, d. randriamampionona, h. andriamaniraka, v. torti, m.g. mellano, c. giacoma, g.l. beccaro persist in secondary vegetation, due to the corky bark and stool shoots. b. ramiflora (dc.) humbert (family asteraceae) is also one of the most diffused medicinal plant of madagascar, mainly used by local population as purgative and against stomach-ache; barks, however, are also used against blenorrhagia. in some biological tests the extracts were not distinctly toxic (rasoanaivo et al., 1999). depending on the ethnic group and language, several names for b. ramiflora occur: hazotokana (“isolated tree”) in merina and bet sileo: 19 specimens; mananotra/mananitra in betsileo, 7 specimens; merana in betsileo, 3 specimens; kanda (near ifanadiana), 2 specimens. as all of the malagasy species of brachylaena, the species ramiflora may reach 20 m in height and yields are very durable timber known to be termite resistant (trunks for construction) (chaturvedula et al., 2002). leaves drop when new leaves develop, either at or just after anthesis. the stalks to the capitula range considerably in length. the variant species with subglabrous leaves occurs scattered over the whole distribution area, with no obvious association with any particular habitat. the biological value of the genus brachylaena has been documented (chaturvedula et al., 2002): it has been described as a rich source of a high number of polyphenolic, organic and terpenic compounds with antioxidant, anti-inflammatory and antibacterial activity (vieira et al., 1991). leaves are often the most used items for medicinal treatment, followed by bark and the entire plant, while decoctions and infusions are the most used methods of preparation (to be taken 3 times/day). infusion is the extraction process of phytochemical compounds from plant material in a solvent as water, oil or alcohol, by allowing the material to remain suspended in the solvent over time; the process of infusion is different from decoction, which involves boiling the plant material (capasso et al., 2006). as the research on malagasy medicinal plants resulted in the discovery of valuable drugs, the aim of this study was a preliminary phytochemical investigation on b. ramiflora leaves’ and barks’ infusions and decoctions, in order to characterize this species as source of biologically active compounds and adding new information on malagasy ethnobotanical species, indentifying and quantifying the main biologically active compounds and their relative antioxidant activity. a better understanding of ethnopharmacological knowledge is crucial to madagascar progress towards an improved self-sufficiency in health care and to the discovery of new natural treatments for glo bally significant diseases (randriamiharisoa et al., 2015). materials and methods study area and plant material the maromizaha forest (18°56’49” s; 48°27’55” e) is located in eastern madagascar, in the alaotra-mangoro region (moramanga district), within both the rural municipalities of andasibe and ambatovola. maromizaha is a 1,880 ha new protected area of largely contiguous forest located 140 km east of antananarivo and 225 km from toamasina: it is bordered to the north by the route nationale 2, to the east by the befody hills, to the west by the madiorano river and to the south by the ankazomirahavavy river (fig. 1). the forest ranges between elevations of 794 m and 1,224 m. the maromizaha forest is surrounded by three forest blocks, including the special reserve analamazaotra to the northwest, vohimana forest to the northeast and vohidrazana forest to the east. this region of madagascar is characterized by a tropical/sub-tropical climate tempered by altitude with high rainfall and a specific rainy season. the analyzed samples of b. ramiflora (leaves and bark) are shown in fig. 2. leaves and bark of b. ramiflora were identified and authenticated by botanists of the botanical and zoological park of tsimbazaza (bzpt, flora department) in madagascar; a voucher specimen (reference: j.s. miller, number 3760) was prepared and deposited in maromizaha forest fig. 1: geographical location of brachylaena ramiflora. hplc fingerprint of brachylaena ramiflora extracts 207 the herbarium section of the bzpt. samples were collected in november 2015 in the region of maromizaha forest where the plant is usually harvested for medicinal use by local population. sampling was made in triplicate. according to local malagasy traditions, infusions from leaves and decoctions from bark were prepared. in the infusion process, 200 ml of water was brought to an appropriate temperature (80 °c) and then poured over 5 g of leaves which were then allowed to steep in the liquid for 10 min. each herbal infusion was filtered (whatman filter paper, hardened ashless circles, 185 mm ø) and then stored at n.a., 4 °c and 95% r.h until analysis. in the decoction process, 20 g of bark were put in 200 ml of boiling water for 20 min; each sample was filtered (whatman filter paper, hardened ashless circles, 185 mm ø) and then stored at n.a., 4 °c and 95% r.h until analysis. materials, solvents and chemicals sodium carbonate, folin–ciocalteu phenol reagent, sodium acetate, citric acid, potassium chloride, hydrochloric acid, iron(iii) chloride hexahydrate, 2,4,6-tripyridyl-s-triazine, 1,2-phenylenediamine dihydrochloride (opda), all polyphenolic and terpenic standards, potassium dihydrogen phosphate, phosphoric acid and hplc-grade methanol and acetonitrile were purchased from sigma-aldrich (st. louis, mo, usa). acetic acid, ethanol, standards of organic acids and hplc-grade formic acid were purchased from fluka biochemika, buchs, switzerland. ethylenediaminetetraacetic acid disodium salt was purchased from amresco (solon, oh, usa). sodium fluoride was purchased from riedel-de haen (seelze, germany). cetyltrimethylammonium bromide (cetrimide), ascorbic acid (aa) and dehydroascorbic acid (dhaa) were purchased from extrasynthése (genay, france). milliq ultrapure water was produced by sartorius stedim biotech mod. arium (sartorius, göttingen, germany). spectrophotometric analysis the amount of total polyphenolic content (tpc) was determined following the folin-ciocalteu colorimetric method (slinkard and singleton, 1977) and results were expressed as mg of gallic acid equivalents (gae) per 100 g of fresh weight (fw). the total anthocyanin content (tac) in the extracts was determined using the ph-differential method (lee et al., 2005). tac was expressed as milligrams of cyanidin-3-o-glucoside (c3g) per 100 grams of fresh weight (mgc3g/100 gfw). antioxidant activity was evaluated by ferric reducing antioxidant power (frap) assay (benzie and strain, 1999) and results were expressed as millimoles of ferrous iron (fe2+) equivalents per kilogram (solid food) of fw. chromatographic analysis sample preparation protocols for hplc analysis small portions (2 ml) of obtained infusions and decoctions were filtered with circular pre-injection filters (0.45 μm, polytetrafluoroethylene membrane, ptfe) before hplc-dad analysis. in the case of vitamin c analysis, a c18 cartridge for solid phase extraction (sep-pak® c-18, waters, milford, ma, usa) was used to absorb the polyphenolic fraction. then, 250 μl of opda solution (18.8 mmol·l-1) were added to 750 μl of samples for dhaa derivatization into the fluorophore 3-(1,2-dihydroxyethyl)furo(3,4-b) quinoxalina-1-one (dfq) (gonzalez-molina et al., 2008). standard calibration the external standard method was used for quantitative determinations. twenty ml aliquot manual injections were performed in triplicate for each concentration level. the calibration curves were obtained by plotting the peak area (y) of the compound at each level versus the sample concentration (x). apparatus and chromatographic conditions an agilent 1200 high performance liquid chromatograph coupled to an agilent uv-vis diode array detector (agilent technologies, santa clara, ca, usa), was used for the chromatographic analysis. five chromatographic methods were used: a kinetex – c18 column (4.6 × 150 mm, 5 μm, phenomenex, torrance, ca, usa) was used to achieve the bioactive compound separation. several mobile phases were used for the biomarker identification and uv spectra were recorded at different wavelengths. the chromatographic conditions of each method were reported in tab. 1, while the main analytical method validation data are summarized in tab. 2. identification and quantification of bioactive compounds in the extracts all the samples were analyzed in triplicate, and standard deviations are given in order to assess the repeatability of the used methods. all single compounds were identified in samples by comparison and combination of their retention times and uv spectra with those of authentic standards in the same chromatographic conditions. total bioactive compound content (tbcc) was determined as the sum of the most important classes of selected biomarkers with an important role in the positive effects on human organism (“multimarker approach”) (mok and chau, 2006). by single bioactive compound profile, phytochemicals were grouped into different bioactive classes to evaluate the contribution of each class to total phytocomplex composition. biomarkers were selected for their demonstrated positive healthy properties and antioxidant activity by literature in relation to the use of this medicinal plant by local population. five polyphenolic classes were considered: benzoic acids, catechins, cinnamic acids, flavonols, and tannins. monoterpenes, organic acids, and vitamin c (as sum of ascorbic and dehydroascorbic acids) were also considered to obtain a complete analytical fingerprint. mass spectrometry data of some selected phytochemicals in b. ramiflora fig. 2: morphological traits of brachylaena ramiflora leaves (a) and bark (b). 208 d. donno, d. randriamampionona, h. andriamaniraka, v. torti, m.g. mellano, c. giacoma, g.l. beccaro decoctions and infusions were reported in supplementary materials (fig. s1). all the results were expressed as mg per 100 g of fresh weight (fw). statistical analysis all samples were prepared and analyzed in triplicate. results were subjected to t-student test and anova test for mean comparison (spss 22.0 software) and hsd tukey multiple range test (p < 0.05). the relationships between the tpc and antioxidant activity were investigated using pearson’s correlation coefficient (r). results and discussion the beneficial effects of bark and leaves of malagasy medicinal plants on human health have already been established in several stu dies (randriamiharisoa et al., 2015): in particular, the amount of total phenolics could be responsible for a great number of these effects. nevertheless, studies on tree-species plant bioactive compounds (botanicals) are still very scarce: in this study, the phytochemical value of infusions and decoctions of different parts of brachylaena ramiflora was investigated by chromatographic and spectrophotometric analyses (by determination of vitamins and organic acids, simple phenolics, flavonoids, anthocyanins and tannins, and of the antioxidant activity). this is one of the first reports which evaluates the b. ramiflora leaf infusion and bark decoction for their chemical parameters, phytochemical profile and antioxidant activity. total phenolics and anthocyanins phenols and related compounds, as anthocyanins, have antioxidant potential owing to hydroxyl groups, which allow free ion pairs to be donated easily. the tpc determination in plant infusions and decoctions was carried out by the folin-ciocalteu method, which depends on electron transfer from phenolic compounds to the folinciocalteu reagent in alkaline medium (erenler et al., 2016; donno et al., 2015b), while the tac was directly determined using the phdifferential method: the colored oxonium form of anthocyanin predominates at ph 1.0, and the colorless hemiketal form at ph4.5; the ph-differential method is based on the reaction producing oxonium forms (donno et al., 2015d). tpc and tac, spectrophotometrically estimated in the decoctions and infusions, are shown in tab. 3. the results of the tpc revealed that infusions and decoctions have different phenolic content which ranged significantly from 11.15±3.17 to 96.10±1.77 mggae/100 gfw, respectively. with regard to the tac, the infusion has shown a significantly higher content (27.86±13.99 mgc3g/100 gfw) compared to decoction (3.03±0.64 mgc3g/100 gfw). the results demonstrated that the highest polyphenol contents were found in the decoctions as reported in other studies (razakarivelo et al., 2015; karimi et al., 2010), even if these polyphenolic compounds were not anthocyanins: the preparation method could have influence on polyphenolic components, which leads to more efficient extraction for decoction method, but could also affect the phenolic molecules during the continuous heat to which decoctions were subjected as reported by ammar et al. (2015). phytochemical profile and phytocomplex synergistic or additive therapeutic effects of several phytochemicals, rather than a single compound, could contribute to disease prevention and reduce the risk of addiction and toxicity, producing a more complete and less drastic pharmacological effect than that of one or a few of its components taken separately: the so-called phytocomplex consists of a combination of different substances, both active principles and other plant components, which contribute to the overall therapeutic effect (donno et al., 2015a). since the biological activity of b. ramiflora infusions and decoctions is due to the sum of their bioactive components (phytocomplex), chemical composition of these preparations was analyzed by hplc fingerprint: the main constituents identified in the present study (polyphenolic and terpenic compounds, organic acids, and vitamins) are known bioactive compounds. sixteen and twenty-three biomarkers were identified in the b. ramiflora leaf infusions and bark decoctions, respectively (tab. 4): the main compounds identified by hplc-dad in the infusions were quinic acid (334.55±0.99 mg/100 gfw), chlorogenic acid (208.27± 7.74 mg/100 gfw), and γ-terpinene (144.19±1.00 mg/100 gfw), while the major components in the decoctions were castalagin (2002.64± 13.96 mg/100 gfw), citric acid (1171.81±1.05 mg/100 gfw), and chlorogenic acid (646.44±2.31 mg/100 gfw). overall, infusions and decoctions have shown different phenolic profile. seven (leaf infusions) and fourteen (bark decoctions) phetab. 1: chromatographic conditions of each used method (donno et al., 2015c). method compounds of interest stationary phase mobile phase flow wavelenght (ml min-1) (nm) a cinnamic acids, flavonols kinetex – c18 column a: 10 mm kh2po4/h3po4, ph=2.8 1.5 330 (4.6 × 150 mm, 5 μm) b: ch3cn b benzoic acids, catechins, kinetex – c18 column a: h2o/ch3oh/hcooh (5:95:0.1 v/v/v), ph=2.5 0.6 280 tannins (4.6 × 150 mm, 5 μm) b: ch3oh/hcooh (100:0.1 v/v) c monoterpenes kinetex – c18 column a: h2o 1.0 210, 220, (4.6 × 150 mm, 5 μm) b: ch3cn 235, 250 d organic acids kinetex – c18 column a: 10 mm kh2po4/h3po4, ph=2.8 0.6 214 (4.6 × 150 mm, 5 μm) b: ch3cn e vitamins kinetex – c18 column a: 5 mm c16h33n(ch3)3br/50 mm kh2po4, ph=2.5 0.9 261, 348 (4.6 × 150 mm, 5 μm) b: ch3oh elution conditions method a / gradient analysis: 5% b to 21% b in 17 min + 21% b in 3 min (2 min conditioning time) method b / gradient analysis: 3% b to 85% b in 22 min + 85% b in 1 min (2 min conditioning time) method c / gradient analysis: 30% b to 56% b in 15 min + 56% b in 2 min (3 min conditioning) method d / isocratic analysis ratio of phase a and b: 95:5 in 13 min (2 min conditioning time) method e / isocratic analysis: ratio of phase a and b: 95:5 in 10 min (5 min conditioning time) hplc fingerprint of brachylaena ramiflora extracts 209 t ab . 2 : m ai n va lid at io n pa ra m et er s of th e us ed m et ho ds fo r e ac h ca lib ra tio n st an da rd ( d o n n o e t a l., 2 01 5c ). m et ho d c la ss st an da rd id c od ea r et en ti on w av el en gh t c al ib ra ti on c ur ve e qu at io n r 2 c al ib ra ti on c ur ve r an ge l o d b l o q c ti m e (t r ) (m in ) (n m ) (m g l -1 ) (m g l -1 ) (m g l -1 ) a c in na m ic a ci ds ca ff ei c ac id 1 4. 54 33 0 y = 59 .0 46 x + 20 0. 6 0. 99 6 11 1 5 00 0. 30 5 1. 01 6 ch lo ro ge ni c ac id 2 3. 89 33 0 y = 13 .5 83 x + 76 0. 05 0. 98 4 11 1 5 00 0. 94 0 3. 13 4 co um ar ic a ci d 3 6. 74 33 0 y = 8. 93 42 x + 21 7. 4 0. 99 7 11 1 5 00 2. 90 7 9. 69 0 fe ru lic a ci d 4 7. 99 33 0 y = 3. 39 63 x 4 .9 52 4 1. 00 0 11 1 5 00 1. 24 5 4. 15 0 fl av on ol s hy pe ro si de 5 10 .8 9 33 0 y = 7. 13 22 x 4 .5 83 0. 99 9 11 1 5 00 3. 37 2 11 .2 41 is oq ue rc itr in 6 11 .2 4 33 0 y = 8. 30 78 x + 26 .6 21 0. 99 9 11 1 5 00 0. 25 2 0. 84 0 qu er ce tin 7 17 .6 7 33 0 y = 3. 40 95 x 9 8. 30 7 0. 99 8 11 1 5 00 4. 05 5 13 .5 18 qu er ci tr in 8 13 .2 8 33 0 y = 2. 74 13 x + 5. 63 67 0. 99 8 11 1 5 00 5. 45 6 18 .1 87 ru tin 9 12 .9 5 33 0 y = 6. 58 08 x + 30 .8 31 0. 99 9 11 1 5 00 2. 93 7 9. 79 0 b b en zo ic a ci ds el la gi c ac id 10 18 .6 5 28 0 y = 29 .9 54 x + 18 4. 52 0. 99 8 62 .5 25 0 0. 61 1 2. 03 5 ga lli c ac id 11 4. 26 28 0 y = 44 .9 96 x + 26 1. 86 0. 99 9 62 .5 25 0 0. 43 5 1. 45 1 c at ec hi ns ca te ch in 12 10 .3 1 28 0 y = 8. 91 97 x + 66 .9 52 1. 00 0 62 .5 25 0 2. 34 3 7. 80 9 ep ic at ec hi n 13 14 .3 0 28 0 y = 12 .8 8x 43 .8 16 0. 99 9 62 .5 25 0 0. 76 3 2. 54 3 ta nn in s ca st al ag in 14 16 .3 5 28 0 y = 4. 23 6x 8. 53 5 1. 00 0 62 .5 25 0 1. 00 9 3. 36 3 ve sc al ag in 15 17 .2 5 28 0 y = 4. 93 9x 1. 23 2 1. 00 0 62 .5 25 0 0. 60 3 2. 01 0 c m on ot er pe ne s lim on en e 16 3. 35 25 0 y = 0. 18 94 x 5 .4 20 0. 99 9 12 5 1 00 0 8. 65 4 28 .8 47 ph el la nd re ne 17 3. 57 21 0 y = 8. 78 3x 14 5. 3 0. 99 8 12 5 1 00 0 0. 56 2 1. 87 4 sa bi ne ne 18 3. 45 22 0 y = 18 .1 4x 10 04 0. 99 8 12 5 1 00 0 0. 09 4 0. 31 4 γte rp in en e 19 3. 28 23 5 y = 0. 48 86 x 2 3. 02 0. 99 9 12 5 1 00 0 17 .5 77 58 .5 90 te rp in ol en e 20 4. 83 22 0 y = 26 .5 2x + 8 76 .8 0. 99 9 12 5 1 00 0 0. 24 1 0. 80 4 d o rg an ic a ci ds ci tr ic a ci d 21 5. 30 21 4 y = 1. 06 03 x 2 2. 09 2 1. 00 0 16 7 1 00 0 18 .8 05 62 .6 82 m al ic a ci d 22 4. 03 21 4 y = 1. 41 5x 80 .2 54 0. 99 6 16 7 1 00 0 15 .7 21 52 .4 04 ox al ic a ci d 23 7. 85 21 4 y = 6. 45 02 x + 6. 15 03 0. 99 8 16 7 1 00 0 0. 55 0 1. 83 5 qu in ic a ci d 24 3. 21 21 4 y = 0. 80 87 x 3 8. 02 1 0. 99 8 16 7 1 00 0 26 .1 06 87 .0 21 su cc in ic a ci d 25 3. 46 21 4 y = 0. 92 36 x 8 .0 82 3 0. 99 5 16 7 1 00 0 7. 13 5 23 .7 83 ta rt ar ic a ci d 26 5. 69 21 4 y = 1. 84 27 x + 15 .7 96 1. 00 0 16 7 1 00 0 8. 52 0 28 .4 01 e v ita m in s as co rb ic a ci d 27 4. 14 26 1 y = 42 .7 1x + 2 7. 96 9 0. 99 9 10 0 1 00 0 0. 83 6 2. 78 6 de hy dr oa sc or bi c ac id 28 3. 41 34 8 y = 4. 16 28 x + 14 0. 01 0. 99 9 30 30 0 1. 09 5 3. 64 9 a i d c od e = id en tifi ca tio n co de ; b l o d = li m it of d et ec tio n; c l o q = li m it of q ua nt ifi ca tio n 210 d. donno, d. randriamampionona, h. andriamaniraka, v. torti, m.g. mellano, c. giacoma, g.l. beccaro nolic compounds were detected, identified, and quantified by their retention times and uv spectra compared with those of analytical standards analyzed in the same chromatographic conditions using a hplc-dad: polyphenols represented 46.43% of the infusion phytocomplex and 66.08% of the decoction phytocomplex (fig. 3), accor-ding to other researches (rasoanaivo et al., 2004; razakarivelo et al., 2015). phenolic compounds may be found in plant infusions and decoctions by boiling water particularly due to solution of hydrolysable tannins and flavonoids and water-soluble lignin fragments solved under acidic conditions (erenler et al., 2016). the main polyphenolic compounds found in infusions and decoctions of b. ramiflora leaves and bark were phenolic acids (31.88%) and tannins (28.95%), respectively (tab. 5). concerning the phenolic acids, they were identified as cinnamic and benzoic acids and tentatively identified as caffeic, chlorogenic, coumaric, and ferulic acids and ellagic and gallic acids, respectively. the presence of phenolic acids, as caffeic, ferulic and coumaric acids, has been already reported in plant material of different species with the same therapeutic effects (ammar et al., 2015). the present study did not report the presence of phenolic acid derivatives even if these compounds are present mainly as water soluble glycosides. moreover, the leaf infusion has revealed a considerably lower tannin content than the bark decoction: indeed, bark was expected to contain higher mass fraction of tannins since these compounds have a defensive role (majic et al., 2015). the presence of tannins in adequate amounts in bark extracts could be advantageous as they are able to very effectively quench free radicals (ammar et al., 2015). the identified biomarkers have a pharmaceutical and medicinal importance: in general, the antioxidant effects of phenolic compounds have been studied in relation to the prevention of coronary diseases and cancer, as well as age-related degenerative brain disorders (canterino et al., 2012). in addition, phenolic compounds, associated with antioxidant activity, play an important role in stabilizing lipid peroxidation (beyhan et al., 2010). terpenic compounds were dominant in b. ramiflora infusions in contrast to the decoctions: as shown in tab. 5, monoterpenes represented 17.33% of the infusion phytocomplex and 1.63% of the decoction phytocomplex. the main terpenic compounds of infusions were γ terpinene (144.19±1.00 mg/100 gfw) and sabinene (20.37±0.59 mg/ 100 gfw) followed by phellandrene (13.83±0.40 mg/100 gfw), while the only identified monoterpene of decoctions was phellandrene (116.67±17.17 mg/100 gfw) as reported in tab. 4. this difference in terpenic compound composition is probably due to the different extraction methods (piry et al., 1995), in particular to the temperature: these molecules are volatile compounds with anti-inflammatory activities (de cassia da silveir et al., 2013) and the obtained results could explain the differences in the antioxidant activity between infusions and decoctions. the monoterpene separation was very diffig. 3: phytocomplex representation of the brachylaena ramiflora extracts. mean value of each analyzed sample is given (n = 3). tab. 3: total polyphenolic content (tpc), antioxidant activity, and total anthocyanin content (tac) data in the analyzed plant extracts. tpc antioxidant activity tac mean value±sd mean value±sd mean value±sd (mggae/100 gfw) (mmol fe2+/kg) (mgc3g/100gfw) infusion 11.15±3.17a 1.07±0.03a 27.86±13.99b decoction 96.10±1.77b 6.42±0.08b 3.03±0.64a mean value and standard deviation of each sample is given (n = 3). different letters in superscript for each sample indicate the significant differences at p < 0.05. agae = gallic acid equivalents; bc3g = cyanidin-3-o-glucoside; cfw = fresh weight tab. 4: phytochemical fingerprint of analyzed samples. class bioactive marker infusion decoction mean value±sd mean value±sd (mg/100gfw) (mg/100gfw) cinnamic acids caffeic acid 13.10±0.63 5.44±0.34 chlorogenic acid 208.27±7.74 646.44±2.31 coumaric acid 90.20±8.30 426.86±2.52 ferulic acid n.d. 58.30±3.13 flavonols hyperoside n.d. 12.62±2.39 isoquercitrin 9.67±0.54 418.39±17.95 quercetin 120.44±0.86 293.47±7.47 quercitrin n.d. 54.44±0.93 rutin n.d. 85.98±7.17 benzoic acids ellagic acid 13.13±0.41 447.95±4.82 gallic acid 21.45±1.36 58.32±8.32 catechins catechin n.d. 149.24±6.43 epicatechin n.d. n.d. tannins castalagin n.d. 2002.64±13.96 vescalagin n.d. 71.77±1.21 monoterpenes limonene n.d. n.d. phellandrene 13.83±0.40 116.67±1.17 sabinene 20.37±0.59 n.d. γ-terpinene 144.19±1.00 n.d. terpinolene 9.79±0.94 n.d. organic acids citric acid n.d. 1171.81±1.05 malic acid n.d. 476.41±1.57 oxalic acid 4.46±0.30 85.18±0.65 quinic acid 334.55±0.99 16.00±0.18 succinic acid 34.83±0.15 14.15±0.10 tartaric acid n.d. 536.80±1.35 vitamins ascorbic acid 12.38±0.09 12.51±0.06 dehydroascorbic acid 7.31±0.14 0.71±0.04 mean value and standard deviation of each sample is given (n = 3). an.d. = not detected; bfw = fresh weight hplc fingerprint of brachylaena ramiflora extracts 211 tab. 5: phytocomplex of brachylaena ramiflora infusions and decoctions. infusions decoctions mean value±sd phytocomplex percentage mean value±sd phytocomplex percentage bioactive class (mg/100gfw) (%) (mg/100gfw) (%) cinnamic acids 311.56±8.53e 28.69 1137.04±6.34f 15.87 flavonols 130.11±0.58c 11.98 864.90±14.86e 12.07 benzoic acids 34.57±1.62b 3.18 506.28±3.81d 7.07 catechins n.d. / 149.24±6.43c 2.08 tannins n.d. / 2074.41±13.17g 28.95 anthocyanins 27.86±5.17ab 2.57 3.03±0.64a 0.04 monoterpenes 188.17±0.33d 17.33 116.67±1.17b 1.63 organic acids 373.85±0.90f 34.43 2300.35±1.80h 32.10 vitamins 19.69±0.16a 1.81 13.21±0.02a 0.18 tbcc 1085.81±12.21 7165.13±11.64 mean value and standard deviation of each sample is given (n = 3). different letters in superscript for each sample indicate the significant differences at p < 0.05. an.d. = not detected; bfw = fresh weight ficult (not so much high resolution), and for this reason the separation was also achieved by four different wavelengths. organic acids and vitamin c (as sum of ascorbic acid and dehydroascorbic acid) are another important antioxidant components with multi-purpose uses in pharmacology (eyduran et al., 2015). in b. ramiflora infusions they represented 34.43% and 1.81% of the total phytocomplex, respectively; in the decoctions, instead, organic acids represented 32.10% of the phytocomplex, while vitamin c participated in the phytocomplex with only 0.18% (fig. 3) because of the high extraction temperature that degrades the most heat-sensitive molecules. antioxidant activity antioxidant capacity is widely used as a parameter for medicinally bioactive and functional components in plant material and derivedproducts: the reducing capacity of specific compounds may serve as a significant indicator of total potential antioxidant activity (donno et al., 2013). the mechanisms of antioxidant activity of polyphenolic compounds and vitamins, as flavonoids and vitamin c, are well discussed but the mechanisms and structural requirements have not been fully understood (amić et al., 2003). the antioxidant properties of monoterpenes and organic acids have been also referred to by several authors (eyduran et al., 2015). in this study, b. ramiflora leaf infusions and bark decoctions have been evaluated for their activity as free radical scavengers by frap assay. the presence of antioxidant molecules in plant samples brings about the reduction of the fe3+ complex to the ferrous form: in this assay, the yellow color of the test solution changes to different shades of green and blue depending on the reducing power of antioxidant samples. the antioxidant compounds, present in analyzed extracts, reduced the fe3+ complex to the fe2+ form in different way depending on extraction method: the antioxidant capacity ranged from 1.07±0.03 mmol fe2+/kg (infusions) to 6.42±0.08 mmol fe2+/kg (decoctions) (tab. 3), according to similar studies (karimi et al., 2010). most of the identified compounds possess hydroxyl groups which have the ability of formation complexes with iron metal: the hydroxyl groups of the molecules donate electron pairs to the iron metal with a coordinate covalent bond to form metal complexes. the antioxidant activity of b. ramiflora leaves and bark could be attributed to the hydroxyl groups of the molecules that donate electron pairs to the metal, as also reported by erenler et al. (2016). the high pearson’s correlation coefficients between the polyphenolic content and antioxidant activity (rinfusions = 0.99 and rdecoctions = 0.62) confirmed a strong positive linear relationship between these two variables according to similar researches (majic et al., 2015). conclusions folk medicine represents an important tool to spot plants of pharmacological interest, since it can indicate potential sources of bioactive compounds. it can be inferred that the forest of maromizaha is a source of important raw materials for plant-derived pharmaceuticals. medicinal plant exploitation have a link with biodiversity conservation. the valorization of medicinal plants may increase local incentives to preserve and manage the habitat. it is hoped that further studies will generate an interest in the proper sustainable production, processing, and commercialization of b. ramiflora for medicinal purposes. indeed, according to the results of this study, it may be concluded that b. ramiflora could be considered as a promising source of natural antioxidants that may contribute to health benefits: the analyzed preparations revealed an important polyphenol content, in particular flavonoids and tannins, and the decoctions presented the highest levels. the phytochemical profile was also characterized by the presence of monoterpenes, organic acids and vitamin c. furthermore, agreements between local institutions and pharmaceutical companies may encourage a further development of prospective medicines and natural remedies based on b. ramiflora and other local medicinal plants. as the quantity of wild growing plant species is continuously declining, a major effort should be done to strengthen conservation policies and strategies. in this perspective, this preliminary survey would give a contribute to the knowledge of malagasy flora: the outcomes of this preliminary phytochemical investigation may provide a contribution to the identification and quantification of lead compounds responsible for traditional therapeutic claims, but a further quantitative evaluation on the basis of their chemical structures with hplc coupled to mass spectrometry is necessary. finally, further studies providing more ecophysiological and pharmacological information are necessary to have a more complete picture on b. ramiflora, highlighting the importance of biodiversity on the health and wellbeing of local communities. 212 d. donno, d. randriamampionona, h. andriamaniraka, v. torti, m.g. mellano, c. giacoma, g.l. beccaro declaration of interest the authors declare that they have no conflict of interest. references amić, d., davidović-amić, d., bešlo, d., trinajstić, n., 2003: structureradical scavenging activity relationships of flavonoids. croat. chem. acta 76, 55-61. ammar, i., ennouri, m., bouaziz, m., ben amira, a., attia, h., 2015: phenolic profiles, phytchemicals and mineral content of decoction and infusion of opuntia ficus-indica flowers. plant foods hum. nutr. 70, 388-394. doi: 10.1007/s11130-015-0505-6 areeshi, m., dove, w., papaventsis, d., gatei, w., combe, p., gros jean, p., leatherbarrow, h., hart, c., 2013: cryptosporidium species causing acute diarrhoea in children in antananarivo, madagascar. ann. trop. med. parasitol. 102, 309-315. doi: 10.1179/136485908x278793 benzie, i.f., strain, j.j., 1999: ferric reducing/antioxidant power assay: direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration. methods enzymol. 299, 15-27. doi: 10.1016/s0076-6879(99)99005-5 beyhan, ö., elmastas, m., gedikli, f., 2010: total phenolic compounds and antioxidant capacity of leaf, dry fruit and fresh fruit of feijoa (acca sellowiana, myrtaceae). j. med. plants res. 4, 1065-1072. doi: 10.5897/jmpr10.008 canterino, s., donno, d., mellano, m.g., beccaro, g.l., bounous, g., 2012: nutritional and sensory survey of citrus sinensis (l.) cultivars grown at the most northern limit of the mediterranean latitude. j. food qual. 35, 108-118. doi: 10.1111/j.1745-4557.2012.00435.x capasso, f., grandolini, g., izzo, a.a., 2006: fitoterapia – impiego razionale delle droghe vegetali. springer, milano. chaturvedula, v.p., schilling, j.k., miller, j.s., andriantsiferana, r., rasamison, v.e., kingston, d.g., 2002: two new triterpene esters from the twigs of brachylaena ramiflora from the madagascar rainforest. j. nat. prod. 65, 1222-1224. doi: 10.1021/np0201220 de cassia da silveira e sa, r., andrade, l.n., de sousa, d.p., 2013: a review on anti-inflammatory activity of monoterpenes. molecules (basel, switzerland) 18, 1227-1254. doi: 10.3390/molecules18011227 donno, d., beccaro, g.l., cerutti, a.k., mellano, m.g., bounous, g., 2015a: bud extracts as new phytochemical source for herbal preparations: quality control and standardization by analytical fingerprint. in: venket rao, a., rao, l.g. 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phenol analysis: automation and comparison with manual methods. am. j. enol. vitic. 28, 49-55. van andel, t., carvalheiro, l.g., 2013: why urban citizens in developing countries use traditional medicines: the case of suriname. evid. based complement. alternat. med. 2013, 1-13. doi: 10.1155/2013/687197 vieira, p.c., himejima, m., kubo, i., 1991: sesquiterpenoids from brachylaena hutchinsii. j. nat. prod. 54, 416-420. doi: 10.1021/np50074a011 address of the corresponding author: e-mail: dario.donno@unito.it © the author(s) 2017. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). i supplementary material     gallic acid chemical formula: c7h6o5 molecular weight: 170,12 m/z: 170.02 (100.0%), 171.02 (7.6%), 172.03 (1.3%)       γ-terpinene chemical formula: c10h16 molecular weight: 136,23 m/z: 136.13 (100.0%), 137.13 (11.0%)         fig. s1: mass spectrometry data of some selected phytochemicals in b. ramiflora decoctions and infusions supplementary material ii     terpinolene chemical formula: c10h16 molecular weight: 136,23 m/z: 136.13 (100.0%), 137.13 (11.0%)       citric acid chemical formula: c6h8o7 molecular weight: 192,12 m/z: 192.03 (100.0%), 193.03 (6.8%), 194.03 (1.6%)           oxalic acid chemical formula: c2h2o4 molecular weight: 90,03 m/z: 90.00 (100.0%), 91.00 (2.3%)       tartaric acid chemical formula: c4h6o6 molecular weight: 150,09 m/z: 150.02 (100.0%), 151.02 (4.6%), 152.02 (1.3%)       fig.  s1:  mass  spectrometry  data  of  some  selected  phytochemicals  in  b.  ramiflora  decoctions  and  infusions iii supplementary material journal of applied botany and food quality 91, 202 210 (2018), doi:10.5073/jabfq.2018.091.027 1 instituto de ciencias básicas, universidad veracruzana, xalapa ver., méxico 2 red de estudios moleculares avanzados, clúster biomimic®, instituto de ecología, a.c., xalapa, ver., méxico 3 facultad de instrumentación electrónica, universidad veracruzana, xalapa, ver., méxico phytochemical characterization of izote (yucca elephantipes) flowers naida juárez-trujillo1#, juan luis monribot-villanueva2#, víctor manuel jiménez-fernández3, rosaelena suárez-montaño1, ángel sahid aguilar-colorado2, josé antonio guerrero-analco2*, maribel jiménez1* (submitted: april 24, 2018; accepted: june18, 2018) * corresponding authors # authors contributed equally to this work summary flowers of the izote (yucca elephantipes) are traditionally consumed in different dishes in the mexican cuisine. although the use of the flowers in salvador, guatemala and méxico is quite popular, there are no scientific reports of their physicochemical properties and phytochemical composition of petals, carpels and stamens. as part of our research program on characterization of edible wild plants, we have analysed the composition and content of phenolic compounds in methanol crude extracts of petals, carpels and stamens from y. elephantipes. the petals exhibited eighteen phenolic compounds, including 4-coumaric acid, rutin, ferulic acid, 4-hydroxybenzoic acid, caffeic acid, quercetin 3-glucoside, trans-cinnamic acid, among others. the principal phenolic compound found in petals, carpels and stamens was 4-coumaric acid, with 1154.20, 526.19 and 484.50 μg/g, respectively. in addition, carpel and petals were found to be rich in fatty acids, including linoleic, oleic, and palmitic acid. the petals also contained the highest amount of total dietary fiber. based on these results, the flowers of y. elephantipes appear to be a good source of phenolic compounds. this information may be useful in identifying these types of flowers and contribute in future research related to their use in the food area. keywords: izote; phenolic compounds; wild flower; food composition; functional food; total dietary fiber. introduction edible flowers are becoming more popular, as evidenced by an increased number of edible flower cookbooks, culinary magazine articles and television shows. consumers can purchase packaged flowers for use in meals, as a garnish, or as an ingredient in salads, soups, entrees, desserts and drinks. the combination of flowers with common vegetables or other foods could generate new and interesting flavours (chambers and koppel, 2013; benvenuti et al., 2016). they improve the appearance, taste and aesthetic value of food, aspects that consumers appreciate, justifying the increasing trend of fresh top quality flowers’ sales worldwide (kelley et al., 2001). however, consumers also demand foods with beneficial health properties, in addition to the nutrients they contain, looking for functional qualities such as phenolic compounds and unsaturated fatty acids (fernandes et al., 2017). however, some edible flowers may contain toxic and hazardous chemical compounds. so that, more investigations are needed in order to know either beneficial or adverse properties of these products. in the case of describing nutraceutical properties, these can offer additional values of edible plants bio diversity for feeding requirements. the biological activity of some edible flowers has been attributed to their antioxidant content, including polyphenols, vitamin c, vitamin e, beta-carotene and other important phytochemicals (barros et al., 2010). in addition, unsaturated fatty acids such as linoleic and linolenic, present in some edible flowers, which cannot be synthesized by humans and must be acquired through dietary intake. unsaturated fatty acids synthesized by these types of flowers are now important dietary sources of these essential compounds. yucca elephantipes belongs to the agavaceae family, and is native to central america and méxico, where it grows at an altitude of 20003500 m a. s. l. it is also known as giant yucca, common yucca or y. guatemalensis. y. elephantipes is often used as an ornamental plant and tree fence, and generally, petals and carpels are separated from the flower to prepare specific dishes. petals and carpels (stigma, style, ovules and ovaries) of the flowers are consumed in different dishes in méxico, including “chileatole” with egg, with meat and some other regional dishes. its traditional use in ethnomedicine has been documented for many years, although little clinical evidence is available. in méxico and central america the flowers are used as diuretics and for the treatment of kidney disease. however, it is important to highlight that no systematic studies have been conducted to determine the bioactive ingredients of y. elephantipes flowers, which may play an important role in developing and making this edible flower popular on a global scale. even though the flowers can only be consumed within a short annual blooming period during the summertime, they still play an important role in the diet of low income populations (sotelo et al., 2007). all parts of the flower have a characteristic bitter taste, mainly due to their steroidal saponins content, particularly tigogenin and sarsasapogenin (galvez, 1996). unlike other flowers, the carpel and pistil carpel of y. elephantipes can be consumed separately. as part of our research program of characterization of edible wild plants and with the main goal to contribute to the knowledge on nutritional properties and phytochemical composition of y. elephantipes flowers, the aim of this study was to evaluate the content of phenolic compounds, unsaturated fatty acids as well as other para meters in the different parts of the flower (petal, carpel and stamen). materials and methods standards and reagents the solvents used for the extraction and liquid chromatography-mass spectrometry procedures were hplc and ms grade, respectively and they were purchased from sigma-aldrich (st louis, mo, usa). all the stock solutions, samples, solvents and reagents were filtered through 0.20 μm ptfe membrane filters (phenomenex, usa) before separation or injection in the instrument. samples whole flowers of y. elephantipes were collected during summer (july-august) in 2014 and 2015 around the city of xalapa in the state of veracruz, méxico (18°n, 96°o, 1200 m a.s.l.), according to phytochemicals of yucca flowers 203 recommendations by local consumers. the harvest indicator consi dered was that the panicle had more than 90% of flowers completely open. immediately after harvest, the petals, carpels and stamens were manually separated, washed and dried in air oven at 60 °c until approximately 90% moisture was removed. the dried samples were then ground and stored in air-free bags at -40 °c until use. samples from two years (2014 and 2015) were analysed in triplicate and separately, but no significant differences were found in the physicochemical and antioxidant properties of the two years evaluated (data not shown), so in this work the data were analysed as a single sample with six replicates. physicochemical properties one hundred fresh flowers were randomly selected. the petals, carpels and stamens of the flowers were weighed with an accuracy of 0.001 g. the proportion mass of each flower part was calculated as a percentage. titratable acidity and ph, as well as the moisture, ash, protein, fat, reducing sugars and soluble solid content was determined in the different parts of the flower (petals, carpels and stamens) following the methodology described by the association of official analytical chemists (aoac, 2000). the vitamin c content was determined by using a second-order derivative spectrophotometric method. a calibration curve was generated from different standard ascorbic acid solutions, which were measured using a spectrophotometer (8453; agilent technologies, santa clara, ca, usa) at 250-350 nm (pfendt et al., 2003). the colour measurement was made with a colorflex v1-72 colorimeter (hunter lab, reston, va, usa), using 0°/45° geometer specular excluded, spectral with a range of 400-700 nm, an spectral resolution < 3 nm, effective bandwidth of 10 nm equivalent triangular with a photometric range from 10 to 150%. the light source was a pulsed xenon lamp. the external colour of each part of the flower was analysed by measuring the l*, a* and b* parameters, and subsequently calculating the secondary hue angle (h°) and chroma. phenolic compounds extractions and phytochemical analysis petals (0.5 kg) were extracted three times, each with 3 l of 85% methanol for 48 hours at room temperature, 150 rpm. the extracts were filtered through whatman no. 1 filter paper under vacuum, and then they were concentrated in a rotary evaporator at 35 °c (r-210; buchi, flawil, switzerland). these extracts were used to determine the phenolic content of y. elephantipes. for the phytochemical ana lysis, extracts were prepared using an accelerated solvent extraction system (ase 350, thermo scientific, usa). briefly, 3.0 g of dry material was dispersed in 1.0 g of diatomaceous earth and placed in a 34 ml cell. the cell was filled up with meoh up to a pressure of 1,500 psi and heated at 60 °c during 5 min. then, the cell was washed off with 30% of cell volume. the extract was concentrated by rotary evaporation (büchi rii, switzerland). 10 mg of the crude extract was re-dissolved in 1.0 ml of meoh with 0.1% of formic acid (both ms grade, sigma-aldrich), filtered and placed in a 1.5 ml uplc vial. samples were analysed by triplicate. the identification and quantitation of individual phenolic compounds was performed with an ultra high performance liquid chromatography (uplc, agilent 1290 series) coupled to a triple quadrupole mass spectrometer (ms-ms, agilent 6460). the chromatographic analysis was carried out on a zorbax sb-c18 co lumn (1.8 μm, 2.1 × 50 mm) (agilent technologies) with the column oven set at 40 °c. the mobile phase consisted of (a) water containing 0.1% formic acid and (b) acetonitrile (90%) containing 0.1% formic acid. the gradient conditions of the mobile phase were: 0 min 1% b, 0.1-40 min linear gradient 1-40% b, 40.1-42 min linear gradient 40-90% b, 42.1-44 min isocratic 90% b isocratic, 44.1-46 min linear gradient 90-1% b, 46.1-47 min 1% b isocratic (total run time 47 min). the flow rate was 0.1 ml/min, and 1 μl of sample injection volume. a dynamic multiple reactions monitoring (dmrm) acquisition method was established. the mrm transitions for each compound were searched in public databases as metlin and were corroborated experimentally in our laboratory (tab. 1). the precursor and product ions were considered as qualifier ions and the product ion was considered as the quantifier ion. the esi source was operated in positive and negative ionization modes with the desolvation temperature of 300 °c, the cone gas (n2) flow of 5 l/min, the nebulizer pressure of 45 psi, the sheath gas temperature of 250 °c, the sheath gas flow of 11 l/min, the capillary voltage (positive and negative) was 3,500 v and the nozzle voltage (positive and negative) of 500 v. the retention time of each compound was determined experimentally and the search window was set in maximum 1 min. the cell accelerator voltage was 7 v for each compound. for quantitation, a calibration curve for each phenolic compound with 10 concentration points (0.5, 1, 3, 5, 7, 9, 11, 13, 15 and 17 μm) was prepared. each concentration point was injected twice and the respective areas considered for the quadratic regressions (tab. 1). the coefficient of determination (r2) values were higher than 0.99 for each compound (tab. 1). the data was obtained with the masshunter workstation software version b.06.00 (agilent technologies) and the results were expressed as the average ± standard deviation of μg/g of sample (dry weight). fatty acid profile the fatty acid profile was determined in the oil extracted from the petals, carpel and stamens with hexane hplc grade using a soxhlet system and converting the oil into methyl esters by the addition of bf3, in accordance with the method described by lopez et al. (2001). the hexane extract obtained from the esterification process was analysed in a gcd plus gas chromatograph coupled to a mass spectro meter, hewlett-packard model 1800 b, with the following para meters: the initial temperature of both the injector and the detector was 250 °c; the temperature was adjusted as follows: initial temperature of 80 °c for 5 min, then later elevated by 30 °c/min reaching to 250 °c. a carbowax column with a length of 30 m, a diameter of 0.25 mm and a film thickness of 0.25 um was used, with helium as the carrier gas at a flow of 1 ml/min. the mass spectra were obtained by means electron ionization at 70 ev. the identification was performed by comparison of the mass spectra obtained for each compound with a database (hp chemstation-nist 05 mass spectral search program, version 2.0d). in addition to the comparison with authentic commercial standards (fame mix, c8:c22, catalogue no. 18920-1amp, sigma-aldrich) analysed under the same conditions. statistical analysis the analysis of the sample (2014 and 2015) was performed in triplicate. variables responses were transformed to square root and ana lysed with a unifactorial multivariate design (manova, glm) testing by each part of the flower and verified that the assumptions of normality and homogeneity of variances were met. within each test, comparisons among the each part of the flower were performed by a post-hoc tukey analysis with α 0.05 and average and standard error was back transformed from square root. all statistical analyses were performed using statistica 7.0 © statsoft, inc. 1984-2004. results and discussion physicochemical properties the average weight of one flower of y. elephantipes was 0.39-0.45 g. the part that contributed most to the mass of the flowers was the car204 n. juárez-trujillo, j.l. monribot-villanueva, v.m. jiménez-fernández, r. suárez-montaño, á.s. aguilar-colorado, j.a. guerrero-analco, m. jiménez ta b. 1 : a na ly tic al c on di tio ns b y u pl c -m sm s fo r i de nt ifi ca tio n an d qu an tifi ca tio n of p he no lic c om po un ds . m r m tr an si ti on s c hr om at og ra ph ic d at a m as s sp ec tr om et er c on di ti on s q ua nt ifi ca ti on # c om po un d na m e p re cu rs or p ro du ct r et en ti on r et en ti on f ra gm en to r c ol lis io n p ol ar it y r an ge r eg re ss io n r2 io n io n ti m e w in do w vo lt aj e en er gy (μ m ) ty pe (m in ) (± m in ) (v ) (v ) 1 sh ik im ic a ci d1 17 3. 1 11 1. 1 1. 7 0. 5 10 0 10 n eg at iv e 0. 5 1 7 q ua dr at ic 0. 99 2 g al lic a ci d2 16 9 12 5. 2 3. 8 0. 5 10 0 10 n eg at iv e 0. 5 1 7 q ua dr at ic 0. 99 3 pr ot oc at ec hu ic a ci d1 15 3 10 9. 1 6. 4 0. 25 10 0 10 n eg at iv e 0. 5 1 7 q ua dr at ic 0. 99 4 4h yd ro xy be nz oi c ac id 1 13 7. 1 92 .8 9. 11 0. 5 10 0 10 n eg at iv e 0. 5 1 7 q ua dr at ic 0. 99 5 g en tis ic a ci d1 15 3 10 9 9. 2 0. 25 10 0 10 n eg at iv e 0. 5 1 7 q ua dr at ic 0. 99 6 ()e pi ga llo ca te ch in 3 30 5. 1 12 5 10 .4 4 0. 5 14 0 20 n eg at iv e 0. 5 1 7 q ua dr at ic 0. 99 7 4h yd ro xy ph en yl ac et ic a ci d4 10 7. 1 77 10 .5 2 0. 5 14 0 20 po si tiv e 0. 5 1 7 q ua dr at ic 0. 99 8 (+ )c at ec hi n2 29 1 13 8. 9 11 .2 0. 5 10 0 10 po si tiv e 0. 5 1 7 q ua dr at ic 0. 99 9 v an ill ic a ci d1 16 9 93 .0 3 11 .8 1 0. 5 10 0 10 po si tiv e 0. 5 1 7 q ua dr at ic 0. 99 10 sc op ol in 5 35 5. 1 19 3 11 .8 7 0. 5 10 0 20 po si tiv e 1 1 7 q ua dr at ic 0. 99 11 c hl or og en ic a ci d1 35 5. 1 16 3. 03 12 .1 2 0. 3 10 0 10 po si tiv e 0. 5 1 7 q ua dr at ic 0. 99 12 c af fe ic a ci d1 18 1. 04 16 3. 03 12 .2 4 0. 5 10 0 10 po si tiv e 0. 5 1 7 q ua dr at ic 0. 99 13 pr oc ya ni di n b 21 57 7. 1 42 5. 1 13 .6 9 0. 5 10 0 10 n eg at iv e 0. 5 1 7 q ua dr at ic 0. 99 14 k ur om an in 1 44 9 28 6. 9 14 .2 4 0. 5 14 0 30 po si tiv e 1 1 7 q ua dr at ic 0. 99 15 ()e pi ca te ch in 2 29 1 13 8. 8 14 .4 8 0. 5 10 0 10 po si tiv e 0. 5 1 7 q ua dr at ic 0. 99 16 v an ill in 1 15 3 12 4. 9 14 .9 9 0. 5 10 0 10 po si tiv e 0. 5 1 7 q ua dr at ic 0. 99 17 m an gi fe ri n2 42 3 30 2. 8 15 .1 8 0. 5 10 0 10 po si tiv e 1 1 7 q ua dr at ic 0. 99 18 k er ac ya ni n2 59 5. 2 28 7. 1 15 .2 0. 5 10 0 20 po si tiv e 1 1 7 q ua dr at ic 0. 99 19 4c ou m ar ic a ci d1 16 5. 05 14 7. 04 16 .2 2 0. 25 10 0 10 po si tiv e 0. 5 1 7 q ua dr at ic 0. 99 20 ()g al lo ca te ch in g al la te 2 45 8. 9 13 9 16 .5 7 0. 5 80 20 po si tiv e 0. 5 1 7 q ua dr at ic 0. 99 21 u m be lli fe ro ne 1 16 3 10 7 17 .1 0. 5 10 0 30 po si tiv e 1 1 7 q ua dr at ic 0. 99 22 q ue rc et in -3 ,4 -d io -g lu co si de 1 62 7 30 2. 9 17 .8 7 0. 5 10 0 10 po si tiv e 0. 5 1 7 q ua dr at ic 0. 99 23 sc op ol et in 1 19 3 13 3 18 .4 4 0. 5 10 0 10 po si tiv e 1 1 7 q ua dr at ic 0. 99 24 3c ou m ar ic a ci d1 16 5. 05 14 7. 04 18 .7 0. 25 10 0 10 po si tiv e 0. 5 1 7 q ua dr at ic 0. 99 25 fe ru lic a ci d1 19 5. 1 14 5. 02 19 .1 8 0. 5 10 0 20 po si tiv e 0. 5 1 7 q ua dr at ic 0. 99 26 si na pi c ac id 1 22 5. 1 20 7. 1 19 .5 7 0. 5 10 0 10 po si tiv e 0. 5 1 7 q ua dr at ic 0. 99 27 e pi ca te ch in g al la te 3 44 3. 1 12 3 19 .9 1 0. 5 10 0 10 po si tiv e 0. 5 1 7 q ua dr at ic 0. 99 28 m yr ic itr in 1 46 5 31 8. 9 20 .2 5 0. 5 10 0 10 po si tiv e 0. 5 1 7 q ua dr at ic 0. 99 29 e lla gi c ac id 1 30 1 14 5 20 .3 6 0. 5 10 0 40 n eg at iv e 0. 5 1 7 q ua dr at ic 0. 99 30 q ue rc et in -3 -d -g al ac to si de 2 46 5 30 2. 9 20 .6 0. 5 10 0 10 po si tiv e 0. 5 1 7 q ua dr at ic 0. 99 t he c el l a cc el er at or v ol ta ge w as 7 v fo r e ac h co m po un d. c om po un ds p ur ch as ed fr om 1 e xt ra sy nt he se (l yo n, f ra nc e) , 2 si gm a a ld ri ch (s t. l ou is , u sa ) 3 c ay m an c he m ic al c om pa ny (m ic hi ga n, u sa ), ki nd ly d on at ed by 4 d r. t ho r a rn as on (u ni ve rs ity o f o tta w a) , i so la te d 5 i n ho us e an d ki nd ly d on at ed b y 6 d r. se rg io p er az a sá nc he z (s ci en tifi c r es ea rc h c en te r o f y uc at an ). phytochemicals of yucca flowers 205 31 r ut in 2 61 1 30 2. 9 20 .6 5 0. 5 10 0 10 po si tiv e 0. 5 1 7 q ua dr at ic 0. 99 32 q ue rc et in -3 -g lu co si de 2 46 5 30 3 21 .1 9 0. 5 10 0 10 po si tiv e 0. 5 1 7 q ua dr at ic 0. 99 33 l ut eo lin -7 -o -g lu co si de 1 44 9 28 7 21 .4 0. 6 10 0 10 po si tiv e 0. 5 1 7 q ua dr at ic 0. 99 34 pa ni si c ac id 4 15 3. 1 10 9 22 .2 1 0. 5 12 0 5 po si tiv e 0. 5 1 7 q ua dr at ic 0. 99 35 pe nt ao -g al lo yl -β -d -g lu co se 2 77 1. 1 15 3 22 .2 7 0. 5 10 0 20 po si tiv e 0. 5 1 7 q ua dr at ic 0. 99 36 2, 4di m et ho xy -6 -m et hy lb en zo ic a ci d2 19 7 17 9 23 .4 3 0. 5 80 5 po si tiv e 1 1 7 q ua dr at ic 0. 99 37 k ae m pf er ol -3 -o -g lu co si de 1 44 9 28 6. 9 23 .6 4 0. 5 10 0 10 po si tiv e 0. 5 1 7 q ua dr at ic 0. 99 38 n ar in gi n1 27 3 15 3 23 .6 4 0. 5 12 0 10 po si tiv e 0. 5 1 7 q ua dr at ic 0. 99 39 q ue rc itr in 1 44 9. 1 30 3. 1 23 .7 1 0. 5 10 0 10 po si tiv e 0. 5 1 7 q ua dr at ic 0. 99 40 h es pe ri di n1 60 9. 1 30 1. 1 24 .5 5 0. 5 10 0 20 n eg at iv e 0. 5 1 7 q ua dr at ic 0. 99 41 m yr ic et in 1 31 7 17 9 24 .6 4 0. 5 10 0 10 n eg at iv e 0. 5 1 7 q ua dr at ic 0. 99 42 r os m ar in ic a ci d1 36 1. 1 16 3 24 .8 5 0. 5 10 0 10 po si tiv e 0. 5 1 7 q ua dr at ic 0. 99 43 ph lo ri dz in 1 43 5 27 2. 9 25 .2 1 0. 5 10 0 10 n eg at iv e 0. 5 1 7 q ua dr at ic 0. 99 44 tr an sre sv er at ro l2 22 9. 1 13 5 25 .8 6 0. 5 10 0 10 po si tiv e 0. 5 1 7 q ua dr at ic 0. 99 45 tr an sci nn am ic a ci d1 14 9. 1 13 1 28 .5 0. 5 10 0 10 po si tiv e 0. 5 1 7 q ua dr at ic 0. 99 46 c ir si m ar in 5 47 7 31 4. 9 29 .4 0. 5 10 0 10 po si tiv e 0. 5 1 7 q ua dr at ic 0. 99 47 q ue rc et in 2 30 2. 9 15 3. 1 29 .7 0. 5 10 0 35 po si tiv e 1 1 7 q ua dr at ic 0. 99 48 l ut eo lin 1 28 7. 1 15 3 30 .0 9 0. 3 10 0 30 po si tiv e 1 1 7 q ua dr at ic 0. 99 49 ps or al en 1 18 7 13 1. 1 31 .4 0. 5 10 0 20 po si tiv e 0. 5 1 7 q ua dr at ic 0. 99 50 a ng el ic in 2 18 7 13 1. 1 32 .7 4 0. 5 10 0 20 po si tiv e 0. 5 1 7 q ua dr at ic 0. 99 51 n ar in ge ni n1 27 1 15 1 32 .8 3 0. 5 10 0 10 n eg at iv e 0. 5 1 7 q ua dr at ic 0. 99 52 a pi ge ni n1 27 1 15 3 34 .1 0. 5 10 0 30 po si tiv e 1 1 7 q ua dr at ic 0. 99 53 k ae m pf er ol 1 28 7. 1 15 3 34 .7 9 0. 3 10 0 30 po si tiv e 0. 5 1 7 q ua dr at ic 0. 99 54 h es pe re tin 1 30 3. 1 17 7. 1 34 .8 0. 5 10 0 20 po si tiv e 0. 5 1 7 q ua dr at ic 0. 99 55 c itr op te n4 20 7. 06 19 2. 04 36 .1 1 0. 5 10 0 20 po si tiv e 0. 5 1 7 q ua dr at ic 0. 99 56 n or di hy dr og ua ia re tic a ci d1 30 3 19 3. 1 42 .3 6 0. 5 10 0 10 po si tiv e 0. 5 1 7 q ua dr at ic 0. 99 57 c hr ys in 1 25 5. 1 15 3 43 .1 3 0. 5 10 0 40 po si tiv e 1 1 7 q ua dr at ic 0. 99 58 k ae m pf er id e1 30 1 25 8. 2 43 .5 0. 5 10 0 20 po si tiv e 0. 5 1 7 q ua dr at ic 0. 99 59 e m od in 1 26 9 22 5 44 .2 0. 5 15 0 20 n eg at iv e 0. 5 1 5 q ua dr at ic 0. 99 60 c hr ys op ha no l1 25 5. 1 15 3 45 .3 0. 2 10 0 40 po si tiv e 0. 5 1 7 q ua dr at ic 0. 99 t he c el l a cc el er at or v ol ta ge w as 7 v fo r e ac h co m po un d. c om po un ds p ur ch as ed fr om 1 e xt ra sy nt he se (l yo n, f ra nc e) , 2 si gm a a ld ri ch (s t. l ou is , u sa ) 3 c ay m an c he m ic al c om pa ny (m ic hi ga n, u sa ), ki nd ly d on at ed by 4 d r. t ho r a rn as on (u ni ve rs ity o f o tta w a) , i so la te d 5 i n ho us e an d ki nd ly d on at ed b y 6 d r. se rg io p er az a sá nc he z (s ci en tifi c r es ea rc h c en te r o f y uc at an ). ta b. 1 c on ti nu ed : a na ly tic al c on di tio ns b y u pl c -m sm s fo r i de nt ifi ca tio n an d qu an tifi ca tio n of p he no lic c om po un ds . m r m tr an si ti on s c hr om at og ra ph ic d at a m as s sp ec tr om et er c on di ti on s q ua nt ifi ca ti on # c om po un d na m e p re cu rs or p ro du ct r et en ti on r et en ti on f ra gm en to r c ol lis io n p ol ar it y r an ge r eg re ss io n r2 io n io n ti m e w in do w vo lt aj e en er gy (μ m ) ty pe (m in ) (± m in ) (v ) (v ) 206 n. juárez-trujillo, j.l. monribot-villanueva, v.m. jiménez-fernández, r. suárez-montaño, á.s. aguilar-colorado, j.a. guerrero-analco, m. jiménez pels (78.4 g/100 g), followed by the petals (13.4 g/100 g) and stamens (7.4 g/100 g). tab. 2 shows the results of the proximate analysis and the physicochemical properties of the different parts of the flower. water was the main constituent of the flowers. the initial moisture content of the whole flower, expressed as a percentage of the fresh weight, was about 70 g/100 g. the moisture content decreased after drying, being for petals, carpels and dry stamens of 5.36 g/100 g, 8.36 g/100 g and 7.67 g/100 g, respectively. these moisture values prevent the deterioration and spoilage of the samples by microorganisms and chemical reactions (bell, 2008). total dietary fiber content in the petals, carpel and stamens was 19.23, 15.28 and 14.34 g/100 g, respectively. these values were similar to observed by other edible flowers (navarro-gonzalez et al., 2015) and that previously reported for some species of algae which have been used to supplement the diet (rupérez and saura-calixto, 2001), indicating that this flower may be considered as a good source of fiber. in addition, it has been reported that these flowers contain saponins (galvez, 1996) which may contribute to the reduction of cholesterol (san jose et al., 2016) and explain its traditional use for hypercholesterolemia. however, these should be used with caution since it has been reported that they are not absorbed in the intestine and therefore decrease the absorption of iron and zinc (hon et al., 1988). the fat content varied from 12.40 to 19.88 g/100 g, being carpels the ones with the highest concentration. these values are higher than those reported for other edible flowers (barros et al., 2010). stamens showed a higher content of reducing sugars of around 12.20 g/100 g, possibly because they contain pollen grains with a higher concentration of simple su gars and pollen to favor pollination. the edible petals had a bright white colour (l* = 57.56, a* = 6.07, b* = 26.98), while the carpels (l* = 54.64, a* = 2.62, b* = 26.08) and stamens (l* = 39.77, a* = 8.42, b* = 22.60) presented a typical light green colour. all samples exhibited a lower protein concentration (0.37 g/100 g) than that previously reported for the flowers of y. filifera (1.62 g/ 100 g) (sotelo et al., 2007). unlike other edible flowers such as rosa micrantha, protein was the least abundant nutritional component in all of the examined parts of the flower, whereas the total dietary fiber was the most abundant component. the results revealed that petals (151.05 mg/100 g) and stamens (126.00 mg/100 g) contain about twice the vitamin c concentration than carpels (65 mg/100 g), but all three samples contain more than the recommended minimum daily value of the food and drug administration of 45-90 mg/100g (fao, 2018). these values are consistent with data reported for other edible flowers (lara-cortés et al., 2014). according to these data, petals and stamens can be considered excellent vitamin c resources. vitamin c is an antioxidant and anticarcinogenic compound and their lack causes scurvy (grosso et al., 2013). our research represents an initial contribution to the study of this edible flower necessary to consider its use in traditional mexican and central american food and to encourage its use in international cuisine. phenolic compounds analysis phenolic compounds are the main active ingredients in edible flo wers (he et al., 2015; li et al., 2014). the interest in edible flowers is probably continuously increasing due to their potential health effects that are related with their chemical composition (fernandes et al., 2017). in order to determine the compounds that may be responsible for the traditional therapeutic effects, we investigated the chemical constituents of the petals, carpel and stamens (tab. 3). for this, we used an analytical method by uplc-ms-ms that includes a large number of well-known phenolic compounds which have been widely described in edible and medicinal plants (tab. 1) and that we have partially reported previously (jimenez et al., 2018; lascurain et al., 2018; reyes-ramírez et al., 2018). twenty compounds were identified and quantified based on their retention time and mass spectrometry data compared with authen tic standards in the different parts of the flower (fig. 1). from the meoh extract, the most abundant compound identified in petals, carpel and stamens was 4-coumaric acid with 1154.21, 526.19 and 484.50 μg/g, respectively. along with 4-coumaric acid, in petals it was identified other seventeen compounds including rutin (348.78 μg/g), ferulic acid (72.83 μg/g), 4-hydroxybenzoic acid (60.66 μg/g), caffeic acid (67.71 μg/g), quercetin 3-glucoside (37.40 μg/g), kaempferol (25.81 μg/g), trans-cinnamic acid (23.40 μg/g), among others. the carpels exhibited ten compounds besides 4-coumaric acid (526.19 μg/g), among them vanillic acid (75.34 μg/g), 4-hydroxybenzoic acid (45.81 μg/g) and trans-cinnamic acid (28.00 μg/g). stamens presen ted fifteen compounds more besides 4-coumaric acid being vanillic acid (110.21 μg/g) the second most abundant. 4-coumaric acid is a member of the phenolic compounds and it has been well reported its antioxidant effect, it has been used as natural antioxidant because it inhibits lipid peroxidation, decreases low density lipoprotein peroxidation and plays an important role in immune regulation in human (kilic and yesiloglu, 2013). in addition, phenolic compounds are known to be capable of electron delocalization, which is the cause of their free radical scavenging activity (rice-evans et al., 1996). therefore, these compounds may contribute to the antioxidant acti vity and therapeutic effects of this flower in the mexican traditional medicine. it has been reported that the bioactivity of some edible flowers is strongly correlated with the phenolic compounds composition (koike et al., 2015). the radical scavenging activity of the phenolic compounds mainly depends on the number and position of hydroxyl groups in the molecules. the phenolic compounds identified in y. elephatipes petals were similar to that reported in other edible flowers (he et al., 2015), but the concentration was specific for tab. 2: proximal analysis and physicochemical properties of the different parts of the izote (yucca elephantipes) flowers. property petal carpel stamens moisture (g/100 g) 5.36 ± 0.38a 8.36 ± 0.25b 7.67 ± 0.39b aw (t=25 °c) 0.449 ± 0.02a 0.610 ± 0.01b 0.660 ± 0.01c total ash (g/100 g ) 1.60 ± 0.04a 3.19 ± 0.99c 2.69 ± 0.01b total dietary fiber (g/100 g) 19.23 ± 1.15b 15.28 ± 1.25b 14.34 ± 2.50a proteins (g/100 g) 0.31 ± 0.05a 0.37 ± 0.04a 0.26 ± 0.09a ph 6.44 ± 0.34b 4.60 ± 0.20a 4.73 ± 0.08a fat (g/100 g) 17.55 ± 0.36c 19.88 ± 0.29b 12.40 ± 0.80a reducing sugar (g/100 g) 9.77 ± 1.20a 9.27 ± 1.50a 12.20 ± 2.30b colour l* 57.56 ± 1.61b 54.64 ± 1.12b 39.77 ± 0.70a a* 6.07 ± 0.23b 2.62 ± 0.20a 8.42 ± 0.26c b* 26.98 ± 0.34b 26.05 ± 0.23b 22.60 ± 0.65a hue 77.28 ± 0.60b 84.25 ± 0.47c 69.56 ± 0.17a chroma 27.59 ± 1.29a 26.18 ± 0.90a 24.12 ± 0.70a vitamin c (mg/100 g) 151.05 ± 3.20c 65.60 ± 1.90a 126.00 ± 2.05b results are expressed as the mean (n=6) ± sd. values with different letters within the same row indicate significant differences by tukey’s test (p < 0.05). titratable acidity (meq citric acid/100 g) 0.31 ± 0.05a 0.40 ± 0.07a 0.52 ± 0.07b phytochemicals of yucca flowers 207 this flower, so these results suggest that the flower can be considered a good source of 4-coumaric acid. fatty acid profile fifteen, thirteen and ten fatty acids were identified in the oil extracted from petals, carpel and stamens, respectively. the fatty acids varied from c12: 0 to c23: 0 and their individual relative area (%) is shown in tab. 4. the three oil extracts presented high concentration of unsaturated fatty acids corresponding to 9,12-octadecadienoic (lino leic) and 9-octadecenoic (oleic) acids, representing more than 70% in petals and carpel and about 50% in stamens. according with the recommendations of european food safety authority (efsa), the daily intake of saturated fatty acids should be as lowest as possible (efsa, 2010). all samples also contained hexadecanoic (palmitic) acid, but in lower proportions. petals presented the higher number of the fatty acids, corresponding to linoleic acid with a value of 36.07%, oleic acid with a value of 35.17% and palmitic acid with a value of 18.78%, while carpel exhibited a higher ratio of linoleic (38.40%) and linolenic (5.20%) acids, both of them considered essential fatty acids, suggesting that these samples could be used to supplement foods with low levels of these compounds. the results obtained were similar to those reported in malva silvestris flowers (barros et al., 2009). the saturated/unsaturated fatty ratio was 0.318, 0.276 and 0.630 for petal, carpel and stamens, respectively and they are comparable to those found in the oil of some varieties of pomegranate as well (amri et al., 2017). all samples showed a similar fatty acid profile considering the presence of linoleic (omega-6) and oleic (omega-9) acids in a higher proportion and linolenic acid (omega-3) in a lower proportion. these results suggest that y. elephantipes flowers are good options for consume of unsaturated fatty acids according to the recommendations of world health organization (who, 2008). conclusions we found that petals, carpels and stamens of the y. elephantipes flowers have different chemical and nutritional composition which provides information to consumers to make decisions about their consumption. our study revealed that petals contain a greater number of phenolic compounds, but in lower concentration compared to carpels and stamens. 4-coumaric acid was the compound found in greater concentration in carpels followed by stamens and finally in petals. carpels also had the highest concentration of free fatty acids, specifically linoleic and linolenic acid. in this regard, y. elephantipes flowers could potentially be a valuable natural source of phenolic compounds, with possible applications in functional foods. their physicochemical properties, combined with their chemical composition, suggest their suitability as a good source of fiber for human consumption, which may be used in many kinds of foods, beverage and supplements. acknowledgments the authors acknowledge the support to the l-idea, (conacyt) and financial support from sagarpa to the department of ad vanced molecular studies (remav) from institute of ecology (inecol). tab. 3: phenolic compounds (μg/g of dry sample) present in methanol extract from the dried petals, carpels and stamens of izote (yucca elephantipes) flower. # compound petals carpels stamens 1 gallic acid 2.91 ± 0.02 --2.37 ± 0.02* 2 protocatechuic acid 13.37 ± 0.15 2.94 ± 0.03 4.83 ± 0.12 3 gentisic acid 1.92 ± 0.1* 5.53 ± 0.05 4.86 ± 0.12 4 4-hydroxybenzoic acid 60.66 ± 0.85b 45.81 ± 0.79a 62.74 ± 1.94 5 epigallocatechin ----8.16 ± 0.15 6 (+)-catechin ----57.94 ± 0.57 7 vanillic acid 19.73 ± 0.24a 75.34 ± 0.95b 110.21 ± 1.64c 8 chlorogenic acid 8.18 ± 0.15a 3.58 ± 0.1* 35.76 ± 0.71 9 caffeic acid 67.71 ± 0.70b 2.44 ± 0.09* 3.95 ± 0.44a 10 4-coumaric acid 1154.21 ± 25.00 526.19 ± 15.97c 484.50 ± 6.10 11 quercetin 3,4-di-o-glucoside 9.55 ± 0.29a --19.69 ±0.39b 12 ferulic acid 72.83 ± 0.59c 26.14 ± 0.31a 41.62 ± 0.34b 13 3-coumaric acid 5.73 ± 0.40a ---- 14 rutin 348.78 ± 6.87c 12.20 ± 0.16a 32.22 ± 0.49b 15 quercetin 3-d-galactoside 19.71 ± 0.59a ---- 16 quercetin 3-glucoside 37.40 ± 0.57a ---- 17 kaempferol 3-o-glucoside 9.41 ± 0.13a ---- 18 trans-cinnamic acid 23.40 ± 0.33a 28.00 ± 0.07b 9.55 ± 0.04c 19 quercetin 9.54 ± 0.15a ---- 20 kaempferol 25.81 ± 0.36b --11.35 ± 0.15a *identification and quantitation was confirmed by ultra high performance liquid and dynamic multiple reaction monitoring (dmrm), using authentic standard for retention time mapping and calibration curve for 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authors: naida juárez-trujillo, rosaelena suárez-montaño, maribel jiménez: instituto de ciencias básicas, universidad veracruzana, dr. luis castelazo s/n, col. industrial-animas, xalapa ver., méxico. postal code 91190. e-mail: maribjimenez@uv.mx juan luis monribot-villanueva, ángel sahid aguilar-colorado, josé antonio guerrero-analco: red de estudios moleculares avanzados, clúster biomimic®, instituto de ecología, a.c., carretera antigua a coatepec núm. 351. el haya, xalapa, ver., méxico, postal code 91070. e-mail: joseantonio.guerrero@inecol.mx © the author(s) 2018. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). víctor manuel jiménez-fernández: facultad de instrumentación electrónica, universidad veracruzana, col. zona universitaria, xalapa, ver., méxico. postal code 91000. journal of applied botany and food quality 91, 287 295 (2018), doi:10.5073/jabfq.2018.091.037 1technology transfer office, research administration and development department, university of limpopo, sovenga, south africa 2medicinal plants and economic development (mped) research centre, department of botany, university of fort hare, alice, south africa ethnobotanical study of plants used medicinally by bapedi traditional healers to treat sinusitis and related symptoms in the limpopo province, south africa sebua silas semenya1, 2*, alfred maroyi2 (submitted: march 17, 2018; accepted: april 27, 2018) * corresponding author summary traditional uses of plants treating sinusitis are under-studied in africa and elsewhere. the present study therefore explored ethnobotanical practices of 132 bapedi traditional healers (ths) pertinent to these diseases in the three districts of limpopo province, south africa. information was accrued using a semi-structured questionnaire in personal interviews, complemented by field observations. a total of 44 plant species belonging to 43 genera and 29 botanical families, mostly the fabaceae (n=5 spp.) and asteraceae (n=4 spp.) were documented as being used by ths for sinusitis and related symptoms. trees (40.9%), followed by herbs (36.3%) and shrubs (22.7%) occupied the highest floristic composition. roots (43.1%) and leaves (22.7%) were the most preferred parts from these habits for herbal preparation. the most popular species across the surveyed districts for treating sinusitis and related symptoms were adansonia digitata, clerodendrum ternatum, cryptocarya transvaalensis, enicostema axillare, kalanchoe brachyloba, lasiosiphon caffer, moringa oleifera, sclerocarya birrea, siphonochilus aethiopicus and stylochaeton natalensis, each scoring the highest fidelity level and use value indexes. most species used by interviewed ths are recorded in this study for the first time as medicines for these ailments. key words: bapedi traditional healers, respiratory tract infections, sinusitis, south africa, traditional medicine introduction according to rightdiagnosis (2016), sinusitis is a long-term ongoing or recurring inflammation of the air-filled spaces that are located within the bones in and around the nose. this inflammation leads to blockade of the normal sinus drainage pathways, which in turn results in mucus retention, hypoxia, decreased mucociliary clearance and predisposition to bacterial growth (radojicic, 2012). approximately 0.5% of all upper respiratory tract infections worldwide are complicated by this affliction (worrall, 2011). therefore, sinusitis is an important global public health problem. in 2001, this condition represented 13.6 million outpatient visits in the united states of america (spiegel, 2004). it is estimated that 134 million indian populations are diagnosed with sinusitis annually (sunali and gharpure, 2012). its prevalence in seven cities in mainland china ranged from 4.8% to 9.7% in 2015 (shi et al., 2015). nazri et al. (2013) reported higher incidence of between 14.8% and 37% in australia. in pakistan over 25% of children suffer from sinusitis (sami et al., 2013), and from july 2000 to june 2001, 6.3 million new diagnoses of this ailment were identified in germany (bachert et al., 2003). little evidence exists suggesting that sinusitis is also one of the most common and significant health care problems in some african countries. for instance, its episode of 11.7% in nigeria was noted by iseh and makusidi (2010). mavale-manuel (2007) who conducted an epidemiological study incorporating 27 schools in suburban and semi-rural areas around maputo, found that the incidence of sinusitis was 23% and 8.8%, respectively. in south africa, an earlier study by nriagu (1999) reported the prevalence of 16–27% of this ailment amongst the children residing in kwazulu-natal province. risk factors of sinusitis such as hiv/aids, exposure to pollution, poverty and poor health infrastructures (olusesi and tshipukane, 2011) are prevalent in south africa, thus suggesting that this disorder might be common and also on the rise across the various geographical areas of the country. typical symptoms of sinusitis include cough, facial pain, fever, fatigue, halitosis, headache, irritability, nausea and rhinorrhoea (slavin et al., 2005), and its complications encompasses blindness, brain abscess, meningitis, orbital abscess and osteomyelitis, amongst the others (reuler et al., 1995). to avoid these complications it is therefore obligatory that sinusitis be treated or managed as earliest as possible. treatment of sinusitis and related symptoms by traditional doctors of various cultures is mainly based on the prescriptions of herbal medicine. this fact is highlighted by general ethnobotanical stu dies conducted amongst diverse ethnic groups residing in countries such as india (rameshkumar and ramakritinan, 2013), brazil (agra et al., 2008), pakistan (kayani et al., 2015), vietnam (minh et al., 2014), yemen (bhuwan et al., 2015). similar surveys carried amongst african traditional healers (ths) practicing in various countries including ethiopia (abera, 2014), cameroon (agbor and naidoo, 2014), nigeria (awoyemi et al., 2012) and lesotho (kose et al., 2015) emphasised this. in south africa same studies was executed amongst cultures such as zulu (coopoosamy and naidoo, 2012) and xhosa (mbeng, 2013), tswati (manzini, 2005) and rastafarian (nzue, 2009), and found that ths/people do treat sinusitis with medicinal plant remedies. however, nothing is known about the medicinal plant remedies used by bapedi ths residing in the limpopo province (south africa). overall, despite the glimpse of evidence suggesting that ths do treat sinusitis, there are still no ethnobotanical studies focusing on the documentation of indigenous knowledge pertinent to this condition in south africa, africa as a condition and elsewhere. this study was therefore initiated to bridge this gap in knowledge. materials and methods study area and population the present study was conducted in the municipalities of capricorn, sekhukhune and waterberg districts of the limpopo, south africa (fig. 1). five rural settlements from each of these municipalities were selected as a study site. 288 s.s. semenya, a. maroyi the local inhabitants of the studied areas, the bapedi, speak a dialect of sepedi in the sotho language group. this culture occupies a large part of limpopo province, with an estimated 52.9% of the population (southafrica.info, 2016). overall, the studied areas are poorly developed by government and very few services are available to the people. the vast majority of the houses are made of locally sourced natural materials (i.e., timber, grass and mud). most of the villages lack health centres, and as such, people have to pay for expensive and scarce transportation to access the adjacent centre. for most ailments, th’s services are the only accessible and affordable healing treatments. ethnobotanical survey and data collection before the survey commenced, permission to conduct the study was granted from the community leader of each sampled village, and ths who agreed to participate in our survey were requested to sign a consent form. the objective of the study was explained to both local tribal leaders and ths in their native language of sepedi. ethnobotanical enquiry and data gathering was carried out from may 2017 to october 2017 through face-to-face interviews guided by a semi-structured questionnaire and direct observation, with 132 ths who were conveniently sampled (i.e., with the help of local community leaders and ths). this questionnaire was designed to provide information on the pedi vernacular names of medicinal plants used for sinusitis and related symptoms, used plant part/s, methods of herbal preparation and administration, amongst other data. generally, interviews were conducted individually with each healer. field trips for plant collection and observation were conducted in the company of each interviewed ths. plant species were identified by healers via vernacular name/s, and subsequently researchers collected specimen for scientific identification. the plant specimens were collected using standard taxonomical procedures (e.g. taking specimens with flowers and fruits whenever possible). each plant sample was numbered and deposited at the larry leach herbarium (university of limpopo) for identification of botanical name by taxonomist. voucher specimens of each species were stored in this herbarium for future references. data analysis micro soft excel and statistical package for the social sciences (spss) ethnobotanical data gathered from the interview session and field tours with ths were entered in to microsoft excel 2000 spreadsheet, and spss version 14.0, afterwards analysed using descriptive statistics such as frequencies and percentages. fidelity level (fl) the uniformity and degree of therapeutic exploitation amongst ths with respect to an individual species against sinusitis and/or related symptoms was determined using fl as described by al-quran (2009), following the formula: np fl (%) = × 100 n where np was the number of ths who claim a use of a plant species to treat sinusitis and/or related symptom, and n was the total number of ths who mentioned the use of species as a medicine to treat any given condition (i.e, sinusitis and/related symptom/s). according to friedman et al. (1986), fl index reflects the extent of preference an individual species is given by users in the management of a particular ailment. fidelity level values show the percentage of ths claiming the use of particular plant species as herbal medicine for sinusitis. fig. 1: map of limpopo province indicating the studied areas (districts and municipalities). plants used for sinusitis and related symptoms 289 therefore, medicinal plants widely used by ths against sinusitis have higher fl values than those that are not widely used. use value (uv) use values (uv) are calculated for individual plant to give a quantitative measure of its relative importance to the informants’ objectively. the extent of utilisation of each species used therapeutically by bapedi ths for sinusitis and related symptoms were determined via uv, following phillips and gentry (1993) index: u uv = � n from the above formulation, u was the number of citations per species, where n represented the total number of ths. generally, plant with broad therapeutic uses or those that are highly accepted as cure of a particular ailment will score a high uv. results and discussion diversity of used plant species forty-four plant species (38 indigenous and 6 exotics) belonging to 43 genera and 29 botanical families were pointed out by 132 interviewed bapedi ths as treatment of sinusitis and related symptoms (tab. 1). amongst these families were the fabaceae, asteraceae, euphorbiaceae and lamiaceae with at least three species used against sinusitis. there is presently no ethnobotanical study focusing on traditional treatment of sinusitis which we can compare our finding with. however, members of the asteraceae, euphorbiaceae and lamiaceae were previously reported as conspicuous therapies for respiratory infections in northern maputaland, kwazulu-natal province, south africa (york, 2012), thus suggesting that their pharmacological potential and safety against these infections have been ethnically tested in different areas by different culture and perchance found to be effective, hence they are highly preferred by bapedi ths. in addition to this conjecture, the supremacy of the aforesaid families in our study might be attributed to ths’ knowledge regarding uses of their members as sinusitis and related conditions medications compared to other families. the remaining botanical families encountered in this survey had between two and one species, thus were least presented. poorly represented as these families were, the high degree and frequency of uses of most of their members by bapedi ths appeared to be worthy of further in-depth investigations with respect to pharmacological activities (tab. 1). plant habit trees (40.9%, n=18), followed by herbaceous species (36.3%, n=16) and shrubs (22.7%, n=10), respectively formed the highest floristic composition of 44 medicinal plants inventoried in the present study. distinct partialities of the first habit by interviewed ths might be attributed to chronic nature of sinusitis which requires the incessant availability of medications. this speculation is based on the fact that trees are available year round as they are not affected by seasonal changes. distribution of used plants with in the municipalities and districts of all (n=44) the species documented in this study, only 22.7% (n=10), namely adansonia digitata, clerodendrum ternatum, cryptocarya transvaalensis, enicostema axillare, kalanchoe brachyloba, lasiosiphon caffer, moringa oleifera, sclerocarya birrea, siphonochilus aethiopicus and stylochaeton natalensis were widely used by all ths across the three studied sites, thus suggesting that their knowledge as sinusitis and its ailed symptoms cures is common in bapedi traditional sector. medical information pertinent to 6.8% (n=3) of species (nicotiana tabacum, encephalartos transvenosus and lippia javanica) were sparsely distributed amongst ths in certain municipalities within the three surveyed districts (supplementary file: tab. 2). variability in use of these taxa across the studied sites is possibly due to the uneven distribution of knowledge among ths regarding their application as sinusitis cure. this supposition might be true especially since the acquisition and development of knowledge about medicinal plants is a lifelong process and more difficult to learn than other ethnobotanical uses (phillips and gentry, 1993). similar sentiment can be said for taxa which were used by ths practicing in certain municipalities located in two of the three studied districts (acacia senegal, adenia spinosa, aloe spp., artemisia afra, eucalyptus camaldulensis, peltophorum africanum, protea caffra, schkuhria pinnata, senna italica, sorghum bicolor, vernonia natalensis and warburgia salutaris), and those used by ths in some municipalities under one of these districts (acacia tortilis, citrus limon, clematis brachiata, euphorbia schinzii, lantana rugosa, mentha longifolia, plumbago zeylanica, psiadia punctulata and schinus molle). ethnomedical knowledge of the remaining species encompassing acokanthera rotundata, cannabis sativa, carissa bispinosa, croton gratissimus, cyperus sexangularis, erythrina lysistemon, eucomis pallidiflora, ptaeroxylon obliquum, solanum panduriforme and spirostachys africana were restricted to ths operating in a single municipality within one of the three surveyed districts, a finding which indicate that these species are less popular amongst the interviewed ths. however, it is also probable that the limited uses of such taxa might be ascribed to their effectiveness against sinusitis, presently known to few ths who retaliate to share it with colleagues in trepidation of competition. species utilisation and comparison with ethnobotanical literature amongst the 44 plant species recorded in this study, vast majority (59%, n=26) were used by ths to exclusively treat sinusitis, 11.3% (n=4) cured this ailment and the following two conditions; fatigue and fever. the remaining plants (31.8%, n=14) was implicated in the treatment of both these disorders. generally, the above-mentioned findings demonstrate the depth of bapedi ths’ indigenous knowledge on local plants and their application as cure for sinusitis and allied symptoms. plants used exclusively by ths in this study to treat sinusitis were aloe spp., a. rotundata, a. senegal, a. tortilis, c. bispinosa, c. brachiata, c. gratissimus, c. transvaalensis, c. ternatum, e. axillare, e. lysistemon, e. pallidiflora, e. schinzii, k. brachyloba, l. caffer, s. aethiopicus, s. birrea, l. javanica, n. tabacum, p. africanum, p. caffra, s. africana, s. italica, s. natalensis, s. panduriforme and w. salutaris. not surprisingly, amongst these species, only c. brachiata (robert, 1990) and w. salutaris (coopoosamy and naidoo, 2012), as well as s. aethiopicus (manzini, 2005) were previously reported as being used by other south african cultures, specifically the zulu and swazi ths, respectively as treatment of this disease. this came as no surprise due to the fact that currently there is no ethnobotanical study that focused on the medicinal plants used by ths or lay people for sinusitis. however, the fact that there is literature based proof highlighting on the use of above three stated species for this ri by ths of afore-mentioned cultures, suggests that other south african healers do treat the condition, and thus a through exploration and documentation of their related ethnobotanical knowledge should be prioritised before it is irretrievably lost together with the holders. clerodendrum ternatum, c. transvaalensis, e. axillare, k. brachyloba, l. caffer, s. birrea, s. aethiopicus and s. natalensis were used by all interviewed bapedi ths (n=132) who treated sinusitis in this study, thus indicating that they are pharmacologically active and principal in the treatment of this condition. 290 s.s. semenya, a. maroyi 2 t ab . 1 : m ed ic in al p la n ts u se d b y b ap ed i tr ad it io n al h ea le rs to s in u si ti s an d r el at ed s y m p to m s in th e l im p o p o p ro v in ce , s o u th a fr ic a f re qu en cy o f u se ; n =t h s (1 32 ) b o ta n ic al f am il y sp ec ie s na m e v er na cu la r na m e h ab it u se d pl an t pa rt s st at e of u se m et ho ds o f h er ba l p re pa ra ti on a nd ad m in is tr at io n a lim en t/ s tr ea te d u m % f l u v a n ac ar d ia ce ae *s ch in us m ol le l . t h o b a/ m o k w ep er e t re e l ea f f re sh b o il ed f o r 5 – 7 m in u te s. s te am i s in h al ed ( n as al ly ) u n d er b la n k et . t h ri ce a d ay f ev er 10 7. 5 10 0 0. 07 a n ac ar d ia ce ae sc le ro ca ry a bi rr ea ( a .r ic h .) h o ch st . su b sp . ca ff ra ( s o n d .) m o ru la /m o k an o t re e f ru it f re sh ju ic e is sq u ee ze d (r aw ), d ri ed an d p o u n d ed . p o w d er is p o u re d in th e h o t w at er . s te am is in h al ed ( n as al ly ) u n d er b la n k et . t h ri ce a d ay s in u si ti s 13 2 10 0 10 0 1 a p o cy n ac ea e a co ka nt he ra r ot un da ta ( c o d d ) k u p ic h a m o et h i s h ru b r o o t d ry b o il ed f o r 3 – 6 m in u te s. s te am i s in h al ed ( n as al ly ) u n d er b la n k et . t h ri ce a d ay s in u si ti s 4 3 10 0 0. 03 a p o cy n ac ea e c ar is sa b is pi no sa ( l .) d es f. e x b re n an l ep u tl o /m o th o k o lo /m o ⌃h u k u d u /m o th o lo t re e r o o t d ry p o u n d ed a n d m ix ed w it h d ri ed p o w d er ed r o o t o f e . ly si st em on . t ak en o ra ll y w it h w ar m w at er . t h ri ce a d ay s in u si ti s 1 0. 7 10 0 0. 00 a ra ce ae st yl oc ha et on n at al en si s s ch o tt m o k u n y a/ m o k u ⌃h et e h er b r o o t d ry b o il ed f o r 5 m in u te s. s te am i n h al ed ( n as al ly ) u n d er b la n k et . t h ri ce a d ay s in u si ti s 13 2 10 0 10 0 1 a sp h o d el ac ea e a lo e sp p . t h o g o /m ar o b ad ib o g al e s h ru b l ea f f re sh m ac er at ed i n w ar m w at er f o r 2 4 h rs . d ec o ct io n i s ta k en o ra ll y . t h ri ce a d ay s in u si ti s 5 3. 7 10 0 0. 03 m ix ed w it h d ri ed l ea f o f l. j av an ic a. b o il ed f o r 5 m in u te s. e x tr ac t is t ak en o ra ll y . t h ri ce a d ay s in u si ti s 1 0. 7 50 0. 00 a st er ac ea e a rt em is ia a fr a ja cq . ex w il ld . v ar . af ra l eg an a/ m o il an ⌃i h er b l ea f f re sh b o il ed f o r 4 – 5 m in u te s. s te am i s in h al ed ( n as al ly ) u n d er b la n k et . t h ri ce a d ay f ev er 1 0. 7 50 0. 00 a st er ac ea e p si ad ia p un ct ul at a (d c .) v at k e l es o tl an e/ m o n o tl et ⌃a n e/ le so d i s h ru b r o o t d ry p o u n d ed a n d t ak en o ra ll y w it h w ar m w at er . t h ri ce a d ay f ev er 10 7. 5 10 0 0. 07 a st er ac ea e *s ch ku hr ia p in na ta ( l am .) k u n tz e ex t h el l. ⇧ at h u m e/ m o ⌃a ⌃a n e/ s er al an e h er b w h o le p la n t f re sh b o il ed f o r 5 m in u te s. s te am i s in h al ed ( n as al ly ) u n d er b la n k et . t h ri ce a d ay f at ig u e 5 3. 7 10 0 0. 03 s in u si ti s 6 4. 5 37 .5 a st er ac ea e v er no ni a na ta le ns is s ch .b ip . e x w al p . m o ⌃u h la h er b l ea f d ry p o u n d ed a n d t ak en o ra ll y w it h w ar m w at er . t h ri ce a d ay f ev er 10 7. 5 62 .5 0. 12 c an el la ce ae w ar bu rg ia s al ut ar is ( g .b er to l. ) c h io v . m o la k a t re e b ar k d ry b o il ed f o r 4 – 1 1 m in u te s. e x tr ac t is t ak en o ra ll y . t h ri ce a d ay s in u si ti s 4 3 10 0 0. 03 3 c an n ab ac ea e *c an na bi s sa tiv a l . v ar . in d ic a (l am .) w eh m er l eb ak e/ p at ⌃e h er b l ea f d ry p o u n d ed a n d t ak en o ra ll y w it h w ar m w at er . t h ri ce a d ay f ev er 1 0. 7 10 0 0. 00 c ra ss u la ce ae k al an ch oe b ra ch yl ob a w el w . ex b ri tt en m o et h i/ m o ⌃i m an ew an ag a/ m o ri t⌃ ik an a s h ru b l ea f f re sh r u b b ed ( ra w ) b et w ee n h an d s an d v ap o u r is i n h al ed (n as al ly ). t h ri ce a d ay s in u si ti s 13 2 10 0 10 0 1 c y p er ac ea e c yp er us s ex an gu la ri s n ee s m o h la h la h er b r o o t d ry p o u n d ed a n d t ak en o ra ll y w it h w ar m w at er . t h ri ce a d ay f ev er 1 0. 7 10 0 0. 00 e u p h o rb ia ce ae c ro to n gr at is si m us b u rc h . v ar . gr at is si m us m o o lo lo g a/ s el o g an e t re e r o o t d ry b o il ed f o r 6 – 1 0 m in u te s. e x tr ac t is t ak en o ra ll y . t h ri ce a d ay o r p o u n d ed a n d t ak en o ra ll y w it h w ar m w at er . t h ri ce a d ay s in u si ti s 5 3. 7 10 0 0. 03 e u p h o rb ia ce ae e up ho rb ia s ch in zi i p ax n g ak ad ia n y a h er b r o o t d ry b o il ed f o r 5 – 8 m in u te s. s te am i s in h al ed ( n as al ly ) u n d er b la n k et . t h ri ce a d ay s in u si ti s 7 5. 3 10 0 0. 05 e u p h o rb ia ce ae sp ir os ta ch ys a fr ic an a s o n d . m o re k u ri /m o ta m p o ta t re e r o o t d ry b u rn ed f o r ab o u t 7 – 8 m in u te . s m o k e in h al ed (n as al ly ). t h ri ce a d ay s in u si ti s 2 1. 5 10 0 0. 01 f ab ac ea e a ca ci a se ne ga l ( l .) w il ld . v ar . ro st ra ta b re n an m o k g ar ip e t re e r o o t d ry b o il ed f o r 6 – 1 1 m in u te s. e x tr ac t is t ak en o ra ll y . t h ri ce a d ay s in u si ti s 4 3 10 0 0. 03 f ab ac ea e a ca ci a to rt ili s (f o rs sk .) h ay n e su b sp . h et er ac an th a (b u rc h .) b re n an m o ⌃h w an a t re e r o o t d ry b o il ed f o r 5 m in u te s. s te am i s in h al ed ( n as al ly ) u n d er b la n k et . t h ri ce a d ay s in u si ti s 11 8. 3 10 0 0. 08 f ab ac ea e e ry th ri na ly si st em on h u tc h . s eb al o /m m al e t re e b ar k d ry p o u n d ed a n d m ix ed w it h d ri ed p o w d er ed r o o t o f c . bi sp in os a. t ak en o ra ll y w it h w ar m w at er . t h ri ce a d ay s in u si ti s 1 0. 7 10 0 0. 00 f ab ac ea e p el to ph or um a fr ic an um s o n d . m o se h la t re e b ar k d ry b o il ed fo r 5 – 1 1 m in u te s. e x tr ac ts ta k en o ra ll y . t h ri ce a d ay s in u si ti s 7 5. 3 10 0 0. 05 f ab ac ea e se nn a ita lic a m il l. s u b sp . ar ac h o id es ( b u rc h .) l o ck m o ro te la d it ⌃h o ⌃i h er b r o o t d ry b o il ed fo r 4 – 8 m in u te s. e x tr ac t is ta k en o ra ll y . t h ri ce a d ay s in u si ti s 5 3. 7 10 0 0. 03 g en ti an ac ea e e ni co st em a ax ill ar e (l am .) a .r ay n al s u b sp . a x il la re m ak g o n o t⌃ o h le /m p h ed u -y ath ab a h er b w h o le p la n t d ry b o il ed f o r 5 – 1 4 m in u te s. e x tr ac t is t ak en o ra ll y . t h ri ce a d ay o r p o u n d ed a n d t ak en o ra ll y w it h w ar m w at er . t h ri ce a d ay s in u si ti s 13 2 10 0 10 0 1 ta b. 1 : m ed ic in al p la nt s us ed b y b ap ed i t ra di tio na l h ea le rs t o si nu si tis a nd re la te d sy m pt om s in t he l im po po p ro vi nc e, s ou th a fr ic a plants used for sinusitis and related symptoms 291 4 h y ac in th ac ea e e uc om is p al lid ifl or a b ak er s u b sp . p o le -e v an si i (n .e .b r. ) r ey n ek e ex j. c .m an n in g m at h u b ad if al a h er b b u lb f re sh b o il ed f o r 5 – 1 0 m in u te s. e x tr ac t is t ak en o ra ll y . t h ri ce a d ay s in u si ti s 1 0. 7 10 0 0. 00 l am ia ce ae c le ro de nd ru m te rn at um s ch in z s eb o k an e h er b w h o le p la n t d ry b o il ed f o r 5 m in u te s. s te am i n h al ed ( n as al ly ) u n d er b la n k et . t h ri ce a d ay s in u si ti s 13 2 10 0 10 0 1 l am ia ce ae m en th a lo ng ifo lia l . m o m in ti h er b w h o le p la n t d ry b o il ed a n d e x tr ac t is t ak en o ra ll y . t h ri ce a d ay f ev er 10 7. 5 10 0 0. 07 b o il ed f o r 5 – 9 m in u te s. e x tr ac t is t ak en o ra ll y . t h ri ce a d ay s in u si ti s 41 l au ra ce ae c ry pt oc ar ya tr an sv aa le ns is b u rt t d av y k g o su p sa t re e b ar k d ry p o u n d ed an d p o u re d in b o il ed w at er . s te am is in h al ed ( n as al ly ) u n d er b la n k et . t h ri ce a d ay s in u si ti s 91 10 0 10 0 1 m al v ac ea e a da ns on ia d ig ita ta m o g o o t re e r o o t d ry b o il ed f o r 6 – 1 0 m in u te s. e x tr ac t is t ak en o ra ll y . t h ri ce a d ay f at ig u e 13 2 10 0 10 0 1 m o ri n g ac ea e *m or in ga o le ife ra s en su e x el l & m en d o n m o ri n g k a t re e l ea f d ry p o u n d ed a n d t ak en o ra ll y w it h w ar m w at er . t h ri ce a d ay f at ig u e 13 2 10 0 10 0 1 s in u si ti s 1 0. 7 9 m y rt ac ea e e uc al yp tu s ca m al du le ns is d eh nh m o p il ik o m o t re e b ar k d ry b o il ed f o r 5 m in u te s. s te am i s in h al ed ( n as al ly ) u n d er b la n k et . t h ri ce a d ay f ev er 10 7. 5 90 .9 0. 08 p o u n d ed a n d t ak en o ra ll y w it h w ar m w at er . t h ri ce a d ay f at ig u e 6 4. 5 10 0 p as si fl o ra ce ae a de ni a sp in os a b u rt t d av y m o n n aap ar e/ p is ay ab at ⌃u m i/ m o th em a s h ru b s te m d ry b o il ed fo r 5 m in u te s. s te am is in h al ed (n as al ly ) u n d er b la n k et . t h ri ce a d ay s in u si ti s 10 7. 5 10 0 0. 12 p lu m b ag in ac ea e p lu m ba go z ey la ni ca l . m a⌃ im ab e/ m a⌃ eg o m ab e s h ru b r o o t d ry b o il ed f o r 6 – 1 3 m in u te s. e x tr ac t is t ak en o ra ll y . t h ri ce a d ay f ev er 10 7. 5 10 0 0. 07 p o ac ea e so rg hu m b ic ol or ( l .) m o en ch su b sp . ar u n d in ac eu m ( d es v .) d e w et & h ar la n m ab el eth o ro h er b s ee d d ry p o u n d ed a n d t ak en o ra ll y w it h m ag eu ® d ri n k o r so ft p o rr id g e. t h ri ce a d ay f at ig u e 18 13 .6 10 0 0. 13 p ro te ac ea e p ro te a ca ffr a m ei sn . su b sp . ca ff ra m o d u m el a t re e r o o t d ry p o u n d ed a n d t ak en o ra ll y w it h w ar m w at er . t h ri ce a d ay s in u si ti s 2 1. 5 10 0 0. 01 p ta er o x y la ce ae p ta er ox yl on o bl iq uu m ( t h u n b .) r ad lk . m o g ab al et sw an a t re e r o o t d ry p o u n d ed a n d t ak en o ra ll y w it h w ar m w at er . t h ri ce a d ay f ev er 4 3 10 0 0. 03 r an u n cu la ce ae c le m at is b ra ch ia ta t h u n b . m o tl em ap o o /m ak g w at ia n e h er b r o o t d ry b u rn ed f o r ab o u t 5 – 6 m in u te s. s m o k e is i n h al ed s in u si ti s 10 7. 5 10 0 0. 07 3 c an n ab ac ea e *c an na bi s sa tiv a l . v ar . in d ic a (l am .) w eh m er l eb ak e/ p at ⌃e h er b l ea f d ry p o u n d ed a n d t ak en o ra ll y w it h w ar m w at er . t h ri ce a d ay f ev er 1 0. 7 10 0 0. 00 c ra ss u la ce ae k al an ch oe b ra ch yl ob a w el w . ex b ri tt en m o et h i/ m o ⌃i m an ew an ag a/ m o ri t⌃ ik an a s h ru b l ea f f re sh r u b b ed ( ra w ) b et w ee n h an d s an d v ap o u r is i n h al ed (n as al ly ). t h ri ce a d ay s in u si ti s 13 2 10 0 10 0 1 c y p er ac ea e c yp er us s ex an gu la ri s n ee s m o h la h la h er b r o o t d ry p o u n d ed a n d t ak en o ra ll y w it h w ar m w at er . t h ri ce a d ay f ev er 1 0. 7 10 0 0. 00 e u p h o rb ia ce ae c ro to n gr at is si m us b u rc h . v ar . gr at is si m us m o o lo lo g a/ s el o g an e t re e r o o t d ry b o il ed f o r 6 – 1 0 m in u te s. e x tr ac t is t ak en o ra ll y . t h ri ce a d ay o r p o u n d ed a n d t ak en o ra ll y w it h w ar m w at er . t h ri ce a d ay s in u si ti s 5 3. 7 10 0 0. 03 e u p h o rb ia ce ae e up ho rb ia s ch in zi i p ax n g ak ad ia n y a h er b r o o t d ry b o il ed f o r 5 – 8 m in u te s. s te am i s in h al ed ( n as al ly ) u n d er b la n k et . t h ri ce a d ay s in u si ti s 7 5. 3 10 0 0. 05 e u p h o rb ia ce ae sp ir os ta ch ys a fr ic an a s o n d . m o re k u ri /m o ta m p o ta t re e r o o t d ry b u rn ed f o r ab o u t 7 – 8 m in u te . s m o k e in h al ed (n as al ly ). t h ri ce a d ay s in u si ti s 2 1. 5 10 0 0. 01 f ab ac ea e a ca ci a se ne ga l ( l .) w il ld . v ar . ro st ra ta b re n an m o k g ar ip e t re e r o o t d ry b o il ed f o r 6 – 1 1 m in u te s. e x tr ac t is t ak en o ra ll y . t h ri ce a d ay s in u si ti s 4 3 10 0 0. 03 f ab ac ea e a ca ci a to rt ili s (f o rs sk .) h ay n e su b sp . h et er ac an th a (b u rc h .) b re n an m o ⌃h w an a t re e r o o t d ry b o il ed f o r 5 m in u te s. s te am i s in h al ed ( n as al ly ) u n d er b la n k et . t h ri ce a d ay s in u si ti s 11 8. 3 10 0 0. 08 f ab ac ea e e ry th ri na ly si st em on h u tc h . s eb al o /m m al e t re e b ar k d ry p o u n d ed a n d m ix ed w it h d ri ed p o w d er ed r o o t o f c . bi sp in os a. t ak en o ra ll y w it h w ar m w at er . t h ri ce a d ay s in u si ti s 1 0. 7 10 0 0. 00 f ab ac ea e p el to ph or um a fr ic an um s o n d . m o se h la t re e b ar k d ry b o il ed fo r 5 – 1 1 m in u te s. e x tr ac ts ta k en o ra ll y . t h ri ce a d ay s in u si ti s 7 5. 3 10 0 0. 05 f ab ac ea e se nn a ita lic a m il l. s u b sp . ar ac h o id es ( b u rc h .) l o ck m o ro te la d it ⌃h o ⌃i h er b r o o t d ry b o il ed fo r 4 – 8 m in u te s. e x tr ac t is ta k en o ra ll y . t h ri ce a d ay s in u si ti s 5 3. 7 10 0 0. 03 g en ti an ac ea e e ni co st em a ax ill ar e (l am .) a .r ay n al s u b sp . a x il la re m ak g o n o t⌃ o h le /m p h ed u -y ath ab a h er b w h o le p la n t d ry b o il ed f o r 5 – 1 4 m in u te s. e x tr ac t is t ak en o ra ll y . t h ri ce a d ay o r p o u n d ed a n d t ak en o ra ll y w it h w ar m w at er . t h ri ce a d ay s in u si ti s 13 2 10 0 10 0 1 292 s.s. semenya, a. maroyi 4 h y ac in th ac ea e e uc om is p al lid ifl or a b ak er s u b sp . p o le -e v an si i (n .e .b r. ) r ey n ek e ex j. c .m an n in g m at h u b ad if al a h er b b u lb f re sh b o il ed f o r 5 – 1 0 m in u te s. e x tr ac t is t ak en o ra ll y . t h ri ce a d ay s in u si ti s 1 0. 7 10 0 0. 00 l am ia ce ae c le ro de nd ru m te rn at um s ch in z s eb o k an e h er b w h o le p la n t d ry b o il ed f o r 5 m in u te s. s te am i n h al ed ( n as al ly ) u n d er b la n k et . t h ri ce a d ay s in u si ti s 13 2 10 0 10 0 1 l am ia ce ae m en th a lo ng ifo lia l . m o m in ti h er b w h o le p la n t d ry b o il ed a n d e x tr ac t is t ak en o ra ll y . t h ri ce a d ay f ev er 10 7. 5 10 0 0. 07 b o il ed f o r 5 – 9 m in u te s. e x tr ac t is t ak en o ra ll y . t h ri ce a d ay s in u si ti s 41 l au ra ce ae c ry pt oc ar ya tr an sv aa le ns is b u rt t d av y k g o su p sa t re e b ar k d ry p o u n d ed an d p o u re d in b o il ed w at er . s te am is in h al ed ( n as al ly ) u n d er b la n k et . t h ri ce a d ay s in u si ti s 91 10 0 10 0 1 m al v ac ea e a da ns on ia d ig ita ta m o g o o t re e r o o t d ry b o il ed f o r 6 – 1 0 m in u te s. e x tr ac t is t ak en o ra ll y . t h ri ce a d ay f at ig u e 13 2 10 0 10 0 1 m o ri n g ac ea e *m or in ga o le ife ra s en su e x el l & m en d o n m o ri n g k a t re e l ea f d ry p o u n d ed a n d t ak en o ra ll y w it h w ar m w at er . t h ri ce a d ay f at ig u e 13 2 10 0 10 0 1 s in u si ti s 1 0. 7 9 m y rt ac ea e e uc al yp tu s ca m al du le ns is d eh nh m o p il ik o m o t re e b ar k d ry b o il ed f o r 5 m in u te s. s te am i s in h al ed ( n as al ly ) u n d er b la n k et . t h ri ce a d ay f ev er 10 7. 5 90 .9 0. 08 p o u n d ed a n d t ak en o ra ll y w it h w ar m w at er . t h ri ce a d ay f at ig u e 6 4. 5 10 0 p as si fl o ra ce ae a de ni a sp in os a b u rt t d av y m o n n aap ar e/ p is ay ab at ⌃u m i/ m o th em a s h ru b s te m d ry b o il ed fo r 5 m in u te s. s te am is in h al ed (n as al ly ) u n d er b la n k et . t h ri ce a d ay s in u si ti s 10 7. 5 10 0 0. 12 p lu m b ag in ac ea e p lu m ba go z ey la ni ca l . m a⌃ im ab e/ m a⌃ eg o m ab e s h ru b r o o t d ry b o il ed f o r 6 – 1 3 m in u te s. e x tr ac t is t ak en o ra ll y . t h ri ce a d ay f ev er 10 7. 5 10 0 0. 07 p o ac ea e so rg hu m b ic ol or ( l .) m o en ch su b sp . ar u n d in ac eu m ( d es v .) d e w et & h ar la n m ab el eth o ro h er b s ee d d ry p o u n d ed a n d t ak en o ra ll y w it h m ag eu ® d ri n k o r so ft p o rr id g e. t h ri ce a d ay f at ig u e 18 13 .6 10 0 0. 13 p ro te ac ea e p ro te a ca ffr a m ei sn . su b sp . ca ff ra m o d u m el a t re e r o o t d ry p o u n d ed a n d t ak en o ra ll y w it h w ar m w at er . t h ri ce a d ay s in u si ti s 2 1. 5 10 0 0. 01 p ta er o x y la ce ae p ta er ox yl on o bl iq uu m ( t h u n b .) r ad lk . m o g ab al et sw an a t re e r o o t d ry p o u n d ed a n d t ak en o ra ll y w it h w ar m w at er . t h ri ce a d ay f ev er 4 3 10 0 0. 03 r an u n cu la ce ae c le m at is b ra ch ia ta t h u n b . m o tl em ap o o /m ak g w at ia n e h er b r o o t d ry b u rn ed f o r ab o u t 5 – 6 m in u te s. s m o k e is i n h al ed s in u si ti s 10 7. 5 10 0 0. 07 5 r an u n cu la ce ae c le m at is b ra ch ia ta t h u n b . m o tl em ap o o /m ak g w at ia n e h er b r o o t d ry b u rn ed f o r ab o u t 5 – 6 m in u te s. s m o k e is i n h al ed (n as al ly ). t h ri ce a d ay s in u si ti s 10 7. 5 10 0 0. 07 r u ta ce ae *c itr us li m on ( l .) b u rm .f . m o sw ir i t re e b ar k d ry p o u n d ed a n d t ak en o ra ll y w it h w ar m w at er . t h ri ce a d ay f ev er 10 7. 5 10 0 0. 07 s o la n ac ea e *n ic ot ia na ta ba cu m l . m o fo la s h ru b l ea f d ry p o u n d ed a n d a p in ch o f a fi n g er i s sn u ff ( n as al ly ). t h ri ce a d ay s in u si ti s 3 2. 2 10 0 0. 02 s o la n ac ea e so la nu m p an du ri fo rm e e .m ey . m o th o la -o -m o se ro lw an e h er b r o o t d ry p o u n d ed a n d t ak en o ra ll y w it h w ar m w at er . t h ri ce a d ay s in u si ti s 4 3 10 0 0. 03 t h y m el ae ac ea la si os ip ho n ca ffe r m ei sn . n k ek o lo g e s h ru b r o o t d ry p o u n d ed an d p o u re d in b o il ed w at er . s te am is in h al ed ( n as al ly ) u n d er b la n k et . t h ri ce a d ay s in u si ti s 13 2 10 0 10 0 1 v er b en ac ea e la nt an a ru go sa t h u n b . b o k o k o ta n e/ m o k o k o ta n e s h ru b l ea f f re sh b o il ed f o r 5 – 1 0 m in u te s. s te am i s in h al ed ( n as al ly ) u n d er b la n k et . t h ri ce a d ay f ev er 10 7. 5 10 0 0. 07 m ix ed w it h f re sh l ea f o f a . a fr a. b o il ed f o r 5 m in u te s. e x tr ac t is t ak en o ra ll y . t h ri ce a d ay s in u si ti s 1 v er b en ac ea e li pp ia ja va ni ca ( b u rm .f .) s p re n g . m o ⌃u n k w an e/ m o tl ab ad ip o o s h ru b l ea f f re sh b o il ed fo r 5 – 9 m in u te s. e x tr ac t is ta k en o ra ll y . t h ri ce a d ay s in u si ti s 10 8. 3 10 0 0. 08 z am ia ce ae e nc ep ha la rt os tr an sv en os us s ta p f & b u rt t d av y m o fa k a t re e r o o t d ry b o il ed f o r 5 m in u te s. e x tr ac t is t ak en o ra ll y . t h ri ce a d ay f at ig u e 81 61 .3 10 0 0. 61 z in g ib er ac ea e si ph on oc hi lu s ae th io pi cu s (s ch w ei n f. ) b .l .b u rt t s er o k o lo h er b b u lb f re sh b o il ed f o r 5 – 6 m in u te s. s te am i s in h al ed ( n as al ly ) u n d er b la n k et . t h ri ce a d ay s in u si ti s 13 2 10 0 10 0 1 k ey : e x o ti c p la n t sp ec ie s: a st er is k ( *) , fi d el it y l ev el ; f l , u se m en ti o n ; u m a n d u se v al u e; u v plants used for sinusitis and related symptoms 293 therapies for sinusitis and accompanying symptoms as noted earlier species multi-used by bapedi for sinusitis and perceived symptoms (fatigue and fever) made-up 11.3%. fatigue was cured with a single species (a. spinosa), and fever with three species namely a. afra, e. camaldulensis and v. natalensis. traditional uses of a. spinosa as fatigue and sinusitis therapies are recorded in our study for the first time. this might be due to the fact that this species is only localised in selected areas of the limpopo province (foden and potter, 2005). amongst the species used by bapedi for fever and sinusitis, only their use for the first condition was previously reported in african literature. our finding regarding use of a. afra as fever medicine concur with that of thring and weitz (2005), who worked with the coloureds (mixed-race) people residing in the bredasdorp/elim region of the southern overberg, western cape province of south africa. similar to bapedi ths, the shona people of zimbabwe use e. camaldulensis to treat this condition (maroyi, 2011). medicinal application of v. natalensis for fever was previously highlighted by githens (1948) in south africa. thus therapeutic claims made on the aforesaid three plant species by bapedi ths can be taken to be credible, given that they have identical uses elsewhere. overall uses of these species in the treatment of fever and sinusitis in our study might be attributed to their strong aromatic flavour. this speculation is ascribed to the fact that most of the interviewed ths disclosed that all plant species with aromatic smell has the potential in the treatment of respiratory infections, particularly fever and sinusitis. the remaining species recorded in this study were used exclusively for fever (l. rugosa, m. longifolia, p. punctulata, p. zeylanica and s. molle) and fatigue (a. digitata, e. transvenosus, m. oleifera, s. bicolor and s. pinnata) in patients diagnosed with sinusitis. most of the aforesaid plants used by bapedi ths to relieve fever are in line with findings of previous studies conducted in south africa. for instance, vhavenda ths practicing in lwamondo area, limpopo province also use l. rugosa as fever medicine (mahwasane et al., 2013). utilisation of m. longifolia by bapedi ths coincides with findings of van wyk et al. (1997) who worked with the diverse south african cultures. comparably to our results, dold and cocks (2000), found that xhosa ths operating in the grahamstown and peddie districts of the eastern cape province, south africa also prescribe s. molle as medicine to relieve fever. the use this species by bapedi might be attributed to its evergreen nature which supplies them with fresh leaves through the year for herbal preparation. no ethnobotanical record of the remaining two fever therapies; p. punctulata and p. zeylanica was found in south africa. however, both p. punctulata (kokwaro, 1976) and p. zeylanica (tyagi and menghani, 2014) are also valued as fever medicines in east africa, thus emphasising their importance as traditional medicine to heal this condition by interviewed bapedi ths. in addition, it shows that the species are widely distributed in various geographical areas across africa. amongst the medicinal plants documented as being used by bapedi ths to heal fatigue in patients diagnosed with sinusitis, only a. digitata (wickens, 1979) and m. oleifera (metha et al., 2011) are well supported by literature. the remaining species namely e. transvenosus, s. bicolor and s. pinnata are recorded for the first time in the present survey as fatigue medicine. overall, new records of commonly known medicinal plants for sinusitis and perceived related symptoms in our study are an indication that the indigenous medical knowledge amongst the diverse south african cultures is poorly documented. fidelity level (fl) and use value (uv) analysis of all the 44 documented species in this study according to fl revealed c. ternatum, c. transvaalensis, e. axillare, k. brachyloba, l. caffer, s. birrea, s. aethiopicus and s. natalensis (um=132 and fl=100; sinusitis for each), a. digitata and m. olei fera, (um=132 and fl=100; fatigue, for each), e. transvenosus (um=81 and fl=100; fatigue) and s. bicolor (um=18 and fl=100; fatigue) as the most preferred. with exclusion of e. transvenosus and s. bicolor all these taxa scored the highest uvs of 1 against the mentioned ailments. plant parts used, mode of preparations, administrations and dosages anatomical part/s of the plant predominantly used in the present study to prepare herbal remedies included roots (43.1%, n=19), leaves (22.7%, n=10), bark (13.6%, n=6), whole plant (9%, n=4), bulb (4.5%, n=2), fruit, seed and stem (2.2%, n=1, for each), respectively. this finding reflects the extent of utilisation of these plant parts by bapedi ths, a practice which could partly be attributed to either the effectiveness or local availability of such parts throughout the year. these plant parts were processed mainly in their dry (77.2%, n=34) state than when still drench (22.7%, n=10). a total of 50 recipes were prepared by bapedi ths in this study. amongst these formulas, mono therapies (92%, n=46) based on pre parations made from a single plant part were distinctively favoured. herbal remedies which contained a mixture of plant parts (8%, n=4) harvested from various species was rarely used. the prevalence of mono therapies in this study might be attributed to its ease of preparation and safety, since there are no adverse drug interactions. in addition it might be attributed to its convenience for the patients, thus ensuring adherence in drug preparation. the plant parts combination during herbal preparation was reported by some ths in this study as augmenting the efficacy of medicine, a claim which is well supported by scientific studies (metha et al., 2011; york et al., 2012). the present study revealed that boiling (54%, n=27) and pounding (38%, n=19) was the most frequently employed method preparation for sinusitis and related symptoms treatments. other techniques such as burning (4%, n=2), maceration, rubbing, pounding and squeezing (2%, n=1, for each) were less frequently utilised in this study. in general, boiling and burning durations of plant parts for medical production in this study varied amongst ths (tab. 1). however, pre paration of remedies via the remaining techniques was comparable amongst ths. harvested plant parts were pounded by all ths with grinding stones. maceration involved freshly harvested plant parts and the process was done by all ths overnight (24 hrs). similarly, prescription which was made through rubbing and squeezing only involved fresh plant materials (tab. 1). oral (62%, n=31) and nasal (38%, n=19), respectively were the only used means of administering prepared herbal remedies/recipes in the present study. however, nasal was the most flexible route of taking these recipes compared to oral. for instance, ths used it to administer medicines made via all the earlier noted techniques of herbal preparations in this study, while oral was only employed for recipes made via pounding and boiling (tab. 1). there was no standardized measure on the dose of most herbal remedies documented in this study. uniformity in dosage strength amongst ths was only observed for oral drugs prepared via boiling; all bapedi ths prescribed this medicine with a metal (500 ml) thrice a day. the recommended dosages of the remaining medications va ried amongst ths. for instance, remedies prepared using part/s obtained from similar species was prescribed in different doses to treat similar complaints. this might be due to either ths preferences or experience. conclusions the present study is the first to provide a comprehensive baseline data on the traditional practices associated with therapeutic plants for treatment of sinusitis and related symptoms in south africa, 294 s.s. semenya, a. maroyi thus suggesting that there is still considerable scope for field work to explore and document these practices. species documented in this study were described for the first time in ethnobotanical literature as treatment of sinusitis and related conditions. therapeutic claims made by bapedi ths on some plants are well supported by ethnobotanical literature and therefore, to some extent, corroborate the reliability of ethnobotanical information documented in this study. acknowledgements special thanks to the traditional healers in the capricorn, sekhukhune and waterberg districts of the limpopo province, south africa, for sharing their knowledge on plant they use to treat sinusitis. the financial support from the south african national research foundation (nrf) is gratefully acknowledged. references abera, b., 2014: medicinal plants used in traditional medicine by oromo people, ghimbi district, southwest ethiopia. j. ethnobiol. ethnomed. 10, 40. doi: 10.1186/1746-4269-10-40 agbor, m.a., naidoo, s., 2015: ethnomedicinal plants used by traditional healers to treat oral health problems in cameroon. evid. based complement alternat. med. 2015. doi: 10.1155/2015/649832 agra, m.d.f., silva, n.k., basilio, i.j.l.d., freitas, p.f.d.f., barbosafilho, j.m., 2008: survey of medicinal plants used in the region northeast of brazil. braz. j. pharmacog. 18, 472-508. doi: 10.1590/s0102-695x2008000300023 al-quran, s., 2009: ethnopharmacological survey of wild medicinal plants in showbak, jordan. j. ethnopharmacol. 123, 45-50. awoyemi, o.k., ewa, e.e., abdulkarim, i.a., aduloju, a.r., 2012: ethnobotanical assessment of herbal plants in south western nigeria. acad. res. int. 2, 50-57. bachert, c., hormann, k., mosges, r., rasp, g., riechelmann, h., muller, r., 2003: an update on the diagnosis and treatment of sinusitis and nasal polyposis. allergy. 58, 176-91. bhuwan, k.c., ali, n.a.a., setzer, w.n., 2015: a survey of chemical compositions and biological activities of yemeni aromatic medicinal plants. med. 2, 67-92. coopoosamy, r.m., naidoo, k.k., 2012: an ethnobotanical study of medicinal plants used by traditional healers in durban, south africa. afr. j. pharm. pharmacol. 6, 818-823. dold, a.p., cocks, m.l., 2000: the medicinal use of some weeds, problem and alien plants in the grahamstown and peddie districts of the eastern cape, south africa. s. afr. j. sci. 96, 467-473. foden, w., potter, l., 2005: adenia spinosa burtt davy. national assess ment: red list of south african plants version 2015. retrieved feb. 7 2018, from http://redlist.sanbi.org/species.php?species=2712-12. friedman, j., yaniva, z., dafnib, a., palewitch, d., 1986: a preliminary classification of the healing potential of medicinal plants, based on a rational analysis of an ethnopharmacological field survey among bedouins in the negev desert, israel. j. ethnopharmacol. 16, 275-287. githens, t.s., 1948: drug plants of africa; 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(ed.), taxonomic aspects of african economic botany. a.e.t.f.a.t. las palmas de gran canaria. worrall, g., 2011: acute sinusitis. can. fam. physician. 57, 565-567. york t., 2012: an ethnopharmacological study of plants used for treating respiratory infections in rural maputaland. podium presentation, faculty of science and agriculture annual post-graduate symposium. university of zululand, oct. 27 2011 york, t., van vuuren, s.f., de wet, h., 2012: an antimicrobial evaluation of plants used for the treatment of respiratory infections in rural maputaland, kwazulu-natal, south africa. j. ethnopharmacol. 31, 118-127. doi: 10.1016/j.jep.2012.08.038 address of the corresponding author: e-mail: sebuasemenya@gmail.com/sebua.semenya@ul.ac.za © the author(s) 2018. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). i supplementary material su pp le m en ta ry fi le : t ab . 2: u se o f s pe ci es to tr ea t s in us iti s (s ) an d re la te d sy m pt om s w ith in th e di st ric ts a nd m un ic ip al iti es d is tr ic ts a nd m un ic ip al iti es c ap ri co rn s ek hu kh un e w at er be rg s pe ci es n am e aganang blouberg lepelle-nkumpi molemole polokwane sum of ailment (fc) elias motsoaledi ephrime mogale fetakgomo makhudumathamaga tubatse sum of ailment (fc) bela-bela lephalale modimolle mogalakwena mookgophong thabazimbi sum of ailment (fc) sum of overall ailment treated per species a ca ci a se ne ga l s :1 1 0 s :3 3 4 a ca ci a to rt ili s s :1 s : 1 0 11 0 0 11 a co ka nt he ra r ot un da ta 0 s :4 4 0 4 a da ns on ia d ig ita ta f a : 1 4 f a : 1 5 f a :1 1 f a : 1 3 f a : 1 5 68 f a :1 3 f a :8 f a :6 f a :8 f a :6 41 f a :5 f a :1 f a :4 f a :8 f a :5 23 13 2 0 f a :6 6 0 6 a de ni a sp in os a s :7 s :1 s :2 10 0 0 10 a lo e sp p. s :1 s :1 s :2 4 s :1 1 0 5 0 0 f e :1 1 1 a rt em is ia a fr a 0 s :1 ” 1 0 1 c an na bi s sa tiv a 0 0 f e :1 1 1 c ar is 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7 0 0 7 k al an ch oe b ra ch yl ob a s :1 4 s :1 5 s :1 1 s :1 3 s : 1 5 68 s :1 3 s :8 s :6 s :8 s :6 41 s : 5 s : 1 s :5 s :8 s :5 23 13 2 la nt an a ru go sa 0 0 f e :1 f e :1 f e :4 f e :4 10 10 li pp ia ja va ni ca s :2 2 s :1 ” s :1 s :3 4 s :5 5 11 la si os ip ho n ca ffe r s :1 4 s :1 5 s :1 1 s :1 3 s :1 5 68 s :1 3 s :8 s :6 s :8 s :6 41 s :5 s :1 s :4 s :8 s :5 23 13 2 m en th a lo ng ifo lia 0 0 f e :1 f e :1 f e :4 f e :4 10 10 m or in ga o le ife ra f a :1 4 f a :1 5 f a :1 1 f a :1 3 f a :1 5 68 f a :1 3 f a :8 f a :6 f a :8 f a :6 41 f a :5 f a :1 f a :4 f a :8 f a :5 23 13 2 n ic ot ia na ta ba cu m s :1 1 s :1 1 s :1 1 3 p el to ph or um a fr ic an um s :2 2 s :5 5 0 7 p lu m ba go z ey la ni ca 0 0 f e :1 f e :1 f e :4 f e :4 10 10 p ro te a ca ffr a 0 s :1 1 s :1 1 2 p si ad ia p un ct ul at a 0 0 f e :1 f e :1 f e :4 f e :4 10 10 p ta er ox yl on o bl iq uu m 0 0 f e :4 4 4 s ch in us m ol le 0 0 0 f e :1 f e :1 f e :4 f e :4 10 10 s ch ku hr ia p in na ta f a :1 1 0 f a :4 4 5 s cl er oc ar ya b irr ea s :1 4 s :1 5 s :1 1 s :1 3 s :1 5 68 s :1 3 s :8 s :6 s :8 s :6 41 s :5 s :1 s :4 s :8 s :5 23 13 2 s en na it al ic a 0 s :2 s :1 3 s :2 2 5 s ip ho no ch ilu s ae th io pi cu s s :1 4 s :1 5 s :1 1 s :1 3 s :1 5 68 s :1 3 s :8 s :6 s :8 s :6 41 s :5 s :1 s :4 s :8 s :5 23 13 2 s ol an um p an du rif or m e 0 s :4 4 0 4 s or gh um b ic ol or f a :2 f a :8 f a :2 12 f a :6 6 0 18 s pi ro st ac hy s af ric an a 0 s : 2 2 0 2 s ty lo ch ae to n na ta le ns is s :1 4 s :1 5 s :1 1 s :1 3 s :1 5 68 s :1 3 s :8 s :6 s :8 s :6 41 s :5 s :1 s :4 s :8 s :5 23 13 2 0 0 f e :1 f e :1 f e :4 f e :4 10 10 v er no ni a na ta le ns is 0 s :6 6 0 6 w ar bu rg ia s al ut ar is s :1 1 s :3 3 0 4 k ey : f at ig ue : f a , fe ve r: f e , l ac k of a pp et ite : l a , s in us iti s: s , s or e th ro at : s t. p la in n um er ic in di ca te n um be r of h ea le r/ s w ho u se a s pe ci es to tr ea t a n ai lm en ts w hi ls t n um er ic w ith a q uo ta tio n m ar k in di ca te n um be r of h ea le r/ s w ho u se a s pe ci es in co m bi na tio n to tr ea t a n ai lm en t/s journal of applied botany and food quality 90, 224 231 (2017), doi:10.5073/jabfq.2017.090.028 1research institute for biotechnology and bioengineering, isfahan university of technology, isfahan, iran 2 department of agronomy and plant breeding, college of agriculture, isfahan university of technology, isfahan, iran the effects of salt stress on physio-biochemical traits, total phenolic and mucilage content of plantago ovata forsk under in vitro conditions pooran golkar1, farzad amooshahi2, ahmad arzani2* (received january 13, 2017; accepted april 19, 2017) * corresponding author summary plantago ovata forsk (psyllium) is an important source of mucilage which is an ingredient in certain drugs and foodstuffs. the calli of 14 genotypes of psyllium were cultured on ms (murashige and skoog, 1962) medium containing 0, 100, and 200 mm nacl, for four weeks, and the effects of salt stress on the following callus traits were evaluated: growth rate (cgr), relative growth rate (rgr), relative water content (rwc), na+ concentration, k+ concentration, k+/na+ ratio, proline content, total phenolic compounds (tpc), and mucilage content. a reducing trend was observed in gr, rgr, rwc and k+/ na+ of the callus cultured in the medium with 100 mm nacl, comparing to nacl-free medium, while an increasing trend was observed in na+ content, proline content, and tpc under the same conditions. mucilage content of callus was found to increase in the medium containing 100 mm nacl (0.13 g g-1 dw) but decreased afterwards at 200 mm nacl (0.117 g g-1 dw), albeit with significant variations among genotypes. the results showed that among evaluated genotypes, isfahan-1 was the most salt tolerant genotype at cellular level. in addition, the highest mucilage content was obtained in khor-biabanak genotype when the calli grown at 100 mm nacl. it was postulated that mucilage content likely to be associated with salt tolerance and could be exploited to counteract the negative osmotic potential in callus affected by salt stress in p. ovata. keywords: callus induction, medicinal plant, mucilage, psyllium, tissue culture introduction global agricultural and food production are seriously threatened by high soil salt levels (parvaiz and satyawati, 2008; arzani and ashraf 2016), which affects many of the plant biochemical and physiological processes at the cellular or whole plant level (ashraf and harris, 2004; gupta and huang, 2014; arzani and ashraf, 2016). plants’ response to salt stress comprises numerous processes in cells that coordinately operate to alleviate hyperosmolarity and reestablish ionic homeostatic conditions (arzani and ashraf, 2016). development of salt tolerance in plants is, hence, a complex pheno menon (rai et al., 2011). saline-induced osmotic stress triggers a wide range of perturbations ranging from growth and development disruption to modification of the ion transport and uptake systems (basu et al., 2002; parihar et al., 2015). moreover, intracellular solutes and certain inorganic ions like k+ and na+ play intrinsic roles in plant response to salt stress (basu et al., 2002; arzani and ashraf, 2016). the synthesis and accumulation of compatible osmolyte such as proline is one of the effective mechanisms of adaptation to salt stress by stabilizing membranes and macromolecular structures (ashraf and foolad, 2007), detoxifying reactive oxygen species, and adjusting cellular osmosis (zhu, 2002).to this may be added the fact that actively growing cells, like those experiencing salt stress, may be characterized by a high phenolic content (lim et al., 2012). moreover, many studies have shown that changes in the levels of secondary metabolites, including phenolic compounds, enhance plant defense mechanisms against stress, particularly against oxidative stress induced by high salt concentrations (matkowski, 2008; lim et al., 2012). the study of salt tolerance mechanism is preferably conducted at the cellular level because it provides the means to study plant physiological and genetic processes independently of the regulatory mechanisms occurring at the whole plant level (arzani, 2008; lokhande et al., 2010; shibli et al., 2011). the in vitro salt stress has been studied in such different species as rice (basu et al., 2002), medicago sativa (ehsanpour and fatahian, 2003), wheat (arzani, 2008), cucurbitacea (cano et al., 1998; shibli et al., 2011), date palm (alkhairy, 2002), oil seeds (alvarez et al., 2003; soheilikhah et al., 2013), sugar cane (gandonou et al., 2006), and medicinal plants such as foeniculum vulgare (khorami and safarnejad, 2011) and thymus vulgaris (zia et al., 2010). plantago ovata forsk, commonly known as psyllium, is an annual medicinal herb plant with beneficial health properties, whose seeds have found important medicinal applications in treating high blood pressure, high cholesterol, diabetes, and hemorroid (sharma, 2004). the leaves, seeds, and seed husks of psyllium are rich sources of phytochemical substances such as mucilage (talukder et al., 2015). the plant is native to the mediterranean region and is found in india, pakistan, and iran (sharma, 2004). plantago genus is generally considered to be moderately tolerant to salt, though depending on the genotype and salt stress level (karimi and haghighi-pak, 2012). to the best of the authors’ knowledge, no published work has been reported on the biochemical and physiological characterization and mucilage production of psyllium, especially with respect to its mechanisms for combating salt stress at the cellular level. moreover, the efficient and economical production of mucilage from the psyllium calli through in vitro salt stress culture optimization was one of our desired goals. we tested this hypothesis in a series of experiments, in which the types of growth regulators combinations and concentrations have already been tested. thus, the experiment described here is the final experiment of the series to investigate the physiological and biochemical adaptive mechanisms of different genotypes of psyllium involved in responding to in vitro salt stress. in addition, the effects of nacl treatments on the phenolic and mucilage contents produced as secondary metabolites were assessed at the callus level in response to exogenous elicitor “nacl”. materials and methods plant material and growth conditions fourteen plantago ovata genotypes comprising 12 iranian (origi nated from different geographical regions of iran) and two exotic (from pakistan and india) genotypes were used in this study. the seeds were obtained from seed and plant improvement institute, karaj, iran. the seeds of 14 genotypes were surface sterilized in 20% (v/v) sodium hypochlorite solution containing 0.1 ml of tween-20 per 100 ml for 15 min, followed by treatment in 95% ethanol for effects of in vitro salt stress on plantago ovata 225 1-2 min. the murashige and skoog (1962) basal salt medium (sigma-aldrich) adjusted to ph 5.8, supplemented with 3% sucrose and 0.8% agar (sigma -aldrich), and then autoclaved for 20 min at 121 °c and 1.1 kg cm-2. the sterilized seeds were place on the medium and incubated in a growth chamber in the 16/8 day/night photoperiod at a temperature of 25±2 °c for up to two weeks until full germination. callus induction and in vitro salt stress treatment the calli were induced from hypocotyl explants cultured in ms medium (sigmaaldrich) that supplemented with 3% sucrose, 0.8% agar (w/v), 2,4d (0.5) mgl-1 and kin (1) mgl-1. the hypocotyl derived calli were incubated at 24±2 °c for 4 weeks under dark conditions. then, the calli were transferred onto solid ms medium containing the same concentrations of growth regulators as above and different concentrations of nacl: 0 (control), 100 and 200 (mm) [0, 0.6 and 1.2% nacl (w/v)] for 4 weeks (rai et al., 2011). the cultures were transferred to fresh medium every 2 weeks. after 4 weeks of salt treatment, different physiological and biochemical traits were measured. physiological traits relative growth rate (rgr) of callus was calculated as (w1-w0)/w0 × 100, where w0 is the initial callus fresh weight and w1 was con sidered as the final fresh weight of callus after 4 weeks of salt treatment (errabii et al., 2007). relative water content (rwc) was calculated as [(callus fresh weight – callus dry weight)/callus dry weight × 100] (lutts et al., 2004). callus samples of known fresh weight were dried in an oven set at 65 ºc for 48 h, after which they were reweighted and the difference in the initial and final mass determined. the callus growth rate (cgr) was calculated by measuring callus growth (mm/day) at 0, 15 and 30 days after callus transferring to salt containing media induction according to compton (1994). the callus diameter (di) was calculated by root square of (callus length × callus width) (compton, 1994). biochemical traits ion assay (na+ and k+) flame photometry method was used for measuring sodium and potassium concentration of the treated calli based on the method described by skoog et al. (2007). first, calli were collected and dried at 60 °c for at least 5 days, then 10 mg of each powder of dried callus was digested with 10 ml 3% (w/v) aqueous sulfosalicylic acid for 24 h at 4 °c, sample extract purified with whatman no. 1 filter paper and na+ and k+ concentrations were measured. the standard solutions of nacl and kcl prepared from reagent grade salts were used to estimate the ions concentrations by flame photometer (jenway model pep7, uk). proline content the modified bates et al. (1973) method was used for proline determination. in this method, 40 mg of callus tissue was homogenized in 1.7 ml of 3% (w/v) aqueous sulfosalicylic acid. the extractions were transferred to centrifuge tubes and centrifuge at 14000 g for 20 min. then 1 ml of each supernatant transferred into a 10 ml test tube, 1 ml of glacial acetic acid and 1 ml of the ninhydrin reagent added to each tube and heated for 1 h at 100 °c. tubes were then cooled using cold water and 2 ml of toluene added to each tube, sealed with aluminum foil and vortexed at 30 rpm for 2 min. the phase separation was completed after 30 min and the upper phase was used for measuring the absorbance at 520 and 490 nm by spectrophotometer (shimadzu uv-120-02, japan). total phenolic compound the extracts was prepared from 25 g 4 weeks old friable calli powdered in liquid nitrogen and immediately suspended in 50 ml of methanol and kept at room temperature for 24 hrs with periodic shaking. the solution was then filtered through whatman filter paper no. 41, the methanolic extract was concentrated until dry. the dried methanolic extract was dissolved in least amount of methanol and kept at refrigerator temperature 2-4 °c till further use (umesh, 2014). total soluble phenolic compounds (tpc) were determined by using folin-ciocalteu reagent according to the method of makkar et al. (1997). aliquots of extract (200 μl) was taken from each sample, and the volume was made up to 1 ml with methanol. then, 0.5 ml of folin-ciocalteu reagent (1:1 with water) and 2.5 ml of sodium carbonate solution (20%) were added sequentially in each tube. after vortexing, all the tubes were incubated at room temperature in the dark for 40 min. absorbance was measured at 725 nm against the reagent blank. the results are means of three repetitions expressed in the form of gallic acid equivalents (gae) per gram of dry mass. mucilage assay mucilage was extracted from callus according to gupta et al. (2015) with minor modification in psyllium. statistical analysis the experiments were conducted as either completely randomized design (crd) or factorial experiment based on crd with five replications for each treatment. five calli per replication were randomly selected and analyzed for free proline, total phenolic compounds, mucilage content, ion concentrations (na+ and k+) and physiological traits. the data was analyzed using analysis of variance (anova) by proc glm of sas (sas institute, 2011). mean comparisons were conducted according to fisher’s least significant difference (lsd) test at the 0.05 level of probability. principal component analysis was performed using proc factor (method = prin) with, that is, eigen value ≥ 1.0 in sas 9.3 (sas institute, 2011). then, a genotypeby-trait biplot was constructed by using the first two trait-focused scaling principal components (pc1 and pc2) derived from principal component analysis (pca) of a genotype-by-trait matrix containing standardized trait data. a simple regression and a multivariate backward stepwise regression analyses were also performed using proc reg of sas version 9.3 (sas institute, 2011). results and discussion physiological traits the results of analysis of variance showed significant differences among the genotypes investigated and the significant effects of salt treatments on the physiological traits (tab. 1). the genotype × salinity interaction was not significant for rwc, cgr and rgr (tab. 1). addition of nacl to psyllium callus cultures led to a significant decline in the callus physiological traits rwc, rgr and cgr under in vitro salt stress (fig. 1a, b) in p. ovata calli. karimi and haghighatpak (2012) reported the non-significant reduction of seedling-related traits of psyllium genotypes at 200 mm nacl under nacl stress. the inconsistency between the in vitro and in vivo responses may be explained by the multiplicity of the mechanisms involved in whole plant salt tolerance (arzani, 2008; rai et al., 2011). it has been established that the water potential gradient between the cell and the nutrient medium caused by nacl results in dehydrated cells and reduced callus fresh weight (al-khayri, 2002; rafiq et al., 2008; lokhande et al., 2010; shibli et al., 2011) as well as declining callus growth rate (cano et al., 1998; ashraf and ahmad, 2000; ehsanpour and fatahian, 2003; lutts et al., 2004). 226 p. golkar, f. amooshahi, a. arzani rwc decreased significantly with increasing salt stress, but no significant differences were observed in this parameter between the two 100 and 200 mm nacl levels (fig. 1a). the changes observed as a result of the reducing relative water content at 100 mm nacl might have been due to the fast reaction of callus cells to the de clining water availability in cells and the resulting loss of turgor pressure (al-khayri, 2002). reduced rwc values due to in vitro salt stress have been previously reported for different glycophytes (gandonou et al., 2006) and halophytes such as sesuvium portulacastrum (lokhande et al., 2010) and suaeda nudiflora (cherian and reddy, 2003). callus growth started to be appreciably inhibited at 100 mm nacl compared to that in the absence of nacl. the reduced callus growth could be compromised from a reduction in the osmotic potential of the medium culture that enhances the requirement for maintaining the turgor of the growing callus cells, a process that consumes energy and, eventually, leads to the declining callus growth (shibli et al., 2011). to gain a better understanding of salt-tolerance and salt-sensitivity, some researchers have used the ionic selectivity and osmoprotectant accumulation as traits to classify genotypes regarding their response to salt and also to select tolerant cell lines. the genotypes used in this study showed different physio-biochemical responses to in vitro salt stress (tab. 2). the mean comparisons of the genotypes evaluated for their callus growth and water related traits showed that salt stress had its greatest inhibitory effect on reducing callus growth in all the genotypes investigated at 200 mm concentration of nacl (fig. 2 and tab. 2). the highest (0.61 mm day-1) and the lowest (-0.19 mm day-1) values of cgr averaged over all the salt levels tested were obtained with behbahan and ghazvin genotypes, respectively (tab. 2). the highest values of rwc (35.55%) and rgr (82%) were obtained with isfahan-1, whereas the least rwc (11%) and rgr (47%) values belonged to ghazvin and isfahan-3, respectively (tab. 2). on the other hand, the genotypes with sensitive callus had severely reduced callus rwc and rgr under salt treatments relative to the control. similar results have been reported for other species (basu et al., 2002; ehsanpour and fatahian, 2003; gandonou et al., 2006). rwc was significantly correlated with cgr, rgr and mucilage content under 200 mm nacl conditions as evident by their vector angles in the biplot presented in fig. 3. a similar relationship was previously observed between rwc and cgr (basu et al., 2002; ahmad et al., 2009). this relationship could be explained by the osmotic effect of nacl which results in reduction of water content in cytosol causing in turn decline in cgr (arzani and ashraf, 2016). in addition, the increase in salt concentration may lead to the reduction of osmotic potential of the medium, results in reduced turgor of the growing cells, and eventually leads to a decrease in callus growth (cgr) (hamedi et al., 2016). biochemical traits ion content (k+ and na+) k+ ion reportedly plays an important role in enzyme activation (tester and davenport, 2003) although the relationship between k+ content and salt stress may vary from one species to another (alkhayri, 2002). the results of analysis of variance showed significant differences among the genotypes and the significant effects of salt treatments for ion content (k+, na+ and k+/na+) (tab. 1). the genotype × salt interaction was only significant for k+ concentration (tab. 1). in the present study, increased salt at 100 and 200 mm (nacl) led to a significant decrease in k+ concentration (fig. 2a). similar to these findings, k+ reportedly declined steadily in response to increasing salt concentration of callus cultures (chauhan and prathapasenan, 2000; basu et al., 2002; gandonou et al., 2006; lokhande et al., 2010; soheilikhah et al., 2013). other studies, however, have reported callus cultures to exhibit an initial increase in their k+ levels in response to low nacl levels (such as 25 mm) which later declined steadily at higher nacl levels (al-khayri, 2002). the reduction in k+ concentration in callus cells under salt stress could be explained by the alterations in expression and/or function of transporters as well as the ion channels especially those related to k+ tab. 1: the results of analysis of variance for callus related traits of psyllium genotypes subjected to salinity stress. trait¥ genotype salinity g × s residual (g) (s) df: 13 2 26 168 rwc 778.99** 5117.9** 160.03 96.21 cgr 0.93** 14.77** 0.142 0.10 rgr 0.23** 2.39** 0.031 0.021 k+ 7222.9** 2492.1** 2182.4** 288.75 na+ 2194.7** 701025 ** 1459.8 960.6 k+/na+ 1.65** 76.23** 1.12 0.86 proline 0.047** 18.07** 0.033** 0.005 tpc 6.2** 32.7** 1.31** 0.009 mucilage 0.003** 0.005** 0.0017* 0.0009 ** and * significant at p < 1% and 5%, respectively. ¥ df: degree of freedom; rwc: relative water content; cgr: callus growth rate; rgr: relative growth rate; tpc: total phenolic compound. fig. 1: effect of in vitro nacl stress on the callus rwc and rgr (a) callus growth rate (b) of plantago ovata. means by common letter do not significantly differ at the lsd5%. effects of in vitro salt stress on plantago ovata 227 ta b. 2 : t he m ea n co m pa ri so ns o f d iff er en t g en ot yp es o f p . o va ta fo r d iff er en t p hy si obi oc he m ic al tr ai ts a nd m uc ila ge c on te nt u nd er in v itr o sa lt st re ss . r w c c g r r g r n a+ k + k + / n a+ pr ol in e t pc m uc ila ge (% )* (m m (% ) (m m ol (m m ol g -1 d w ) (m g g1 fw ) (m g g a e g1 d w ) (g g -1 d w ) da y1 ) g1 d w ) n ac l ( m m ) a vg . a vg . a vg . a vg . 0 10 0 20 0 a vg . 0 10 0 20 0 0 10 0 20 0 0 10 0 20 0 g en ot yp e is fa ha n1 35 .5 5a ** 0. 29 be 82 a 13 5. 5d 24 2a 10 4b 78 a 1. 84 ab 0. 05 cd e 0. 59 a 1. 4a 1. 90 e 2. l0 f 2. 32 g 0. 11 ad 0. 11 bc 0. 11 ab c m as hh ad 22 .2 0d 0. 21 dg 75 ab 17 0. 6 a 20 8b c 11 0a 68 ab 1. 41 ab c 0. 08 ae 0. 40 c 1. 06 c 2. 00 de 2. 10 f 3. 30 d 0. 14 a 0. 14 b 0. 14 a sh ir az 12 .3 4e f 0. 05 fg h 48 c 14 0. 6c d 19 2c de 80 cd e 50 bc d 1. 66 ab c 0. 10 ae 0. 50 b 1. 08 c 2. 80 b 3. 8a 4. 04 b 0. 12 ab c 0. 13 bc d 0. 13 ab g ha zv in 11 .0 f -0 .1 9i 53 c 14 2. 6b cd 21 8b 90 bc 52 bc d 2. 47 a 0. 10 ae 0. 50 b 0. 90 d 1. 90 e 2. 50 e 3. 60 c 0. 09 cd 0. 10 c 0. 10 bc a ra n b id go l 26 .0 1b c 0. 47 ab c 69 b 15 2. 8 ad 15 2h 80 cd e 52 bc d 1. 09 bc 0. 10 ae 0. 50 b 1. 20 b 2. 10 cd 2. 8d 3. 04 e 0. 13 ab 0. 14 b 0. 14 a k ho r b ia ba na k 26 .1 4b c 0. 42 ad 50 c 15 8. 8 ab c 10 4i 74 cf 54 bc d 0. 97 bc 0. 13 ab c 0. 40 c 1. 02 c 2. 0d e 1. 9g 2. 80 f 0. 08 d 0. 20 a 0. 09 cd pa ki st an 18 .4 4d e 0. 25 cf 50 c 16 5. 4 a 20 0b cd 72 cf 56 bc d 1. 53 ab c 0. 03 e 0. 40 c 1. 00 c 1. 01 g 1. 08 i 4. 80 a 0. 10 bc d 0. 12 bc 0. 11 ab c a hv az 22 .6 3b cd 0. 50 ab 51 c 15 1. 5a d 16 0g h 70 de f 38 de 1. 15 bc 0. 14 ab 0. 50 b 1. 20 b 1. 40 f 1. 7h 2. 40 g 0. 10 bc d 0. 11 bc 0. 11 ab c in di a 27 .4 0b 0. 01 ef g 51 c 14 1. 7 cd 12 4i 64 ef 48 cd 0. 79 bc 0. 04 de 0. 40 c 1. 20 b 2. 20 c 3. 08 c 4. 00 b 0. 12 ab c 0. 14 b 0. 13 ab b eh ba ha n 19 .1 4c de 0. 61 a 76 ab 17 3. 2a 10 8i 56 f 28 e 0. 74 c 0. 16 a 0. 40 c 1. 07 c 2. 20 c 3. 81 a 4. 30 b 0. 10 bc d 0. 12 bc 0. 12 ab c c ha ha rm al b ak ht ia ri 13 .6 6e f 0. 02 gh i 72 ab 16 5. 93 a 11 2i 70 de f 38 de 1. 01 bc 0. 07 be 0. 55 ab 1. 20 b 3. 00 a 3. 5b 3. 90 b 0. 11 ad 0. 11 bc 0. 11 ab c is fa ha n2 24 .4 5b cd -0 .0 01 gh i 52 c 16 4. 8 ab 16 4f gh 88 bc d 64 ab c 0. 98 bc 0. 10 ae 0. 50 b 0. 90 d 2. 00 de 2. 9d 3. 00 e 0. 12 ab c 0. 13 bc d 0. 13 ab is fa ha n3 12 .7 1e f -0 .1 6i 47 c 15 7. 8a bc 18 5d ef 10 4b 64 ab c 1. 65 ab c 0. 07 be 0. 40 c 0. 80 d 1. 50 f 2. 4e 2. 80 f 0. 12 ab c 0. 14 b 0. 13 ab is fa ha n4 13 .8 2e f 0. 29 be 71 ab 16 2. 3a bc 17 4e fg 84 cd 54 bc d 1. 51 ab c 0. 12 ad 0. 40 c 1. 01 c 1. 0g 1. 1i 2. 00 h 0. 10 bc d 0. 11 cb 0. 06 d g ra nd m ea n 16 7. 3 81 .6 9 53 .1 4 0. 10 0. 46 1. 07 1. 93 2. 47 3. 30 0. 11 0. 13 0. 12 *m ea ns in e ac h co lu m n fo llo w ed b y th e sa m e le tte r a re n ot s ig ni fic an tly d iff er en t a t t he 5 % p ro ba bi lit y le ve l. ** r w c : r el at iv e w at er c on te nt ; c g r : c al lu s g ro w th r at e; r g r : r el at iv e gr ow th ra te ; t pc : t ot al p he no lic c om po un ds . 228 p. golkar, f. amooshahi, a. arzani (arzani and ashraf, 2016). the patterns of k+ content in response to increasing nacl levels (fig. 2a) were parallel to the trends of callus growth and callus water related traits. for instance, the highest callus growth was achieved with a culture medium containing no nacl, that is, the same concentration at which the highest k+ uptake was observed. furthermore, the inhibitory concentration of psyllium callus growth was identified to be 100 mm nacl (fig. 1b), which is the same concentration at which potassium concentration significantly reduced relative to the control (fig. 2a). na+ concentration in the callus was observed to increase significantly with increasing salt concentration (fig. 2a). the sharpest increase in na+ concentration was observed when the callus was cultured on a medium containing 100 mm nacl (fig. 2a). the rising trend of na+ concentration observed in this study was similar to those reported for such other plants as safflower (soheilikhah et al., 2013), foeniculum vulgare (khorami and safarnejad, 2011), and date palm (al-khayri, 2002). experimental nacl concentrations (i.e., 100 and 200 mm) concurrently led to an increase in plant na+ content (fig. 2a) although no significant changes were observed in the values of callus rgr (fig. 1a). this suggests that the elevated nutritional uptake of both na+ and k+ might have led to the retention of water in the callus (chaudhary et al., 1997). maintaining the cellular k+/na+ homeostasis is pivotal for plant survival in saline environments (arzani and ashraf, 2016). as a result of increasing na+ concentration in the medium, na+ ions compete with k+ ones during salt stress for the transporter as they both share the same transport mechanisms, thereby decreasing the uptake of k+. accordingly, the k+/na+ ratio was observed in this study to decrease significantly from 3.25 (the control) to 0.24 (at 200 mm nacl) (fig. 2b). this result is in agreement with those reported elsewhere (chaudhary et al., 1997; al-khayri et al., 2002). since the k+/ na+ ratio is critical for salt tolerance, increasing this ratio at 100 mm nacl in plantago ovata will be a promising area of future research. the genotypes examined were found to differ with respect to their intracellular ions under control conditions (tab. 1); all the genotypes, however, accumulated more na+ ions than did the control. the na+ content averaged over the salt level tested varied from 173.1 (mmol g-1 dw) in the behbahan genotype to 135.5 (mmol g-1 dw) in isfahan-1 (tab. 2). however, compared to those with salt-sensitive callus fig. 3: biplot drawn based on the first (pc1) and second (pc2) components obtained from principal component analysis using cgr, rwc, rgr, tpc, mucilage na+, k+, and k+/na+ ratio of psyllium genotypes under 200 mm (nacl) under in vitro conditions. fig. 2: effect of in vitro nacl stress on the k+ and na+ content (a) and k+/na+ ratio of plantago ovata callus. mean with one or more letter in common are not significantly different at the 5% probability level as tested by lsd5% test. effects of in vitro salt stress on plantago ovata 229 (i.e., indian and pakistani), the genotypes with salt-tolerant calli (i.e., isfahan-1) accumulated less na+ but more k+ ions. the imposition of nacl-shock caused an injury to the tissue leading to excessive leaching and poor retention of k+ in the callus in genotypes with lower salt stress tolerance, demonstrating a very sharp reduction in k+ content with increasing salt from control to higher levels. this confirms that the k+ inclusion mechanism, which is an indicator of salt tolerance at the whole plant level (karimi and haghighat-pak, 2012), was also expressed at the cellular level in psyllium. furthermore, it is seen that, compared to those with susceptible calli, the genotypes with tolerant calli (such as isfahan-1) accumulated less na+ and maintained higher levels of k+, rather than the genotypes with sensitive calli (such as ahvaz). this may be one reason under lying the better growth of the isfahan-1 calli under nacl. the different genotypes and their calli exhibited significant differences in their intracellular ion accumulation (tab. 2). the evaluated genotypes showed variations in their k+/na+ ratios from 0.74 (in behbahan) to 2.47 (in ghazvin) (tab. 2). the highest k+/na+ ratio recorded for the ghazvin genotype demonstrated its high salt tolerance and capacity for maintaining an ionic equilibrium between na+ and k+ ions. the harmful effect of na+ depends on its accumulation site. indeed, its accumulation is toxic to salt-sensitive callus in which it is carried into the cytoplasm; it is, however, nontoxic to salt-tolerant callus since it accumulates in their cell vacuoles (volkov, 2015). in this case, the vacuolar sequestration in different genotypes would play an important role in maintaining the water balance in the callus by increasing the osmotic pressure in the cells and keeping a high k+/na+ ratio. the k+ content and k+/na+ ratio was found to be correlated with proline content in callus affected by salt stress (fig. 3). these results were in agreement with those of other researchers who reported a positive and significant correlation between k+ content with k+/na+ ratio (basu et al., 2002; ehsanpour and fatahian, 2003). according to cherian and reddy (2003), the reduction in k+ concentration is capable of inhibiting growth as a result of reducing plant capacity for osmotic adjustment and turgor maintenance, or alternatively, by adversely affecting metabolic functions. in the current study, no significant relationship was found between k+ and other physiological traits (cgr, rwc and rgr) (fig. 2). on the other hand, positive relationship was observed between na+ with some of these traits. it is expectable that the enhanced na+ content would not only disturb plant nutrient balance and osmotic regulation but also reduce growth (gupta and huang, 2014). proline content proline acts as osmotica that protects the cytosol from dehydration as symptomatic salt stress damage (ashraf and foolad, 2007). the analysis of variance showed significant differences between evaluated genotypes, salt treatments and genotype × salt interaction for proline content (tab. 1). in psyllium callus, the proline content averaged over all the genotypes was found to increase dramatically with increasing salt from 0.1 mg g-1 fw in control to 1.1 mg g-1 fw in 200 mm (nacl) (tab. 2). the increase in proline content practically occurred at 100 mm nacl, which indicates that salt stress was problematic to cellular functions at this point. endogenous, free proline accumulation with increasing medium salt concentration has been corroborated in various in vitro culture systems subjected to salt stress (al-khayri, 2002; ehsanpour and fatahian, 2003; alvarez et al., 2003; lokhande et al., 2010). there was a significant difference among the genotypes for proline accumulation, as reported in tab. 2. clearly, proline content increased sharply in all the genotypes as the elevated concentration of nacl. genotypic differences in proline accumulation under salt stress have also been reported in medicago sativa (ehsanpour and fatahian, 2003), safflower (soheilikhah et al., 2013). proline content did not correlate with cgr, rwc and rgr under 200 mm nacl conditions (fig. 2). although the role of proline accumulation in tolerance to salt stress remains controversial, it is thought play role in osmotic phase of salt stress at either early stages of salt treatment or at mild salt stress conditions (arzani, 2008). nonetheless, some authors reported a negative correlation between proline accumulation and callus growth traits in tomato (cano et al., 1998) and date palm (al-khayri, 2002). there was a negative and significant correlation between k+ and proline content (fig. 2). it could be explained that proline and k+ involve in different pathways influencing salt tolerance in plant cells as stated in part in the preceding sentence. total phenolic compound the synthesis and accumulation of polyphenols in in vitro cultures are generally stimulated in response to salt stress (ksouri et al., 2007). the reduction in growth induced by salt stress might have resulted in a new pattern of resource providing additional carbon skeletons for phenolic biosynthesis (ksouri et al., 2007). the analysis of variance showed significant differences between evaluated genotypes, salt levels and genotype × salt interaction for tpc content (tab. 1). total phenolic content increased in term of general mean of the genotypes with increasing salt from control 1.93 (mg gaeg-1 dw) to higher levels including 100 mm (2.47 mg gaeg-1 dw) and 200 mm (3.3 mg gaeg-1 dw) (tab. 2). these results could be explained by the hypothesis that salt stress might be associated with increasing total phenolic compounds due to antioxidant activities (giri et al., 2012; lim et al., 2012). elevated phenol contents under salt stress have also been reportedly observed in the callus cultures of other species such as salvadora persica (sharma and ramawat, 2014). however, accumulation of phenolic compounds in plants due to salt stress may be species depend (lim et al., 2012). in addition, it depends on the salt concentrations, i.e. while phenolic compounds has not affected by moderate salt concentration; high salt stress caused a remarkable reduction in tpc in the seedlings of rice (chunthaburee et al., 2014). it was found a co-ordinate change in proline content and tpc of callus in this study, while this relationship was not significant (fig. 3). it has been demonstrated that accumulation of high amounts of proline in the cell increases tpc through the biosynthetic pathway of proline-linked pentose phosphate, which, in turn, enhances the phenyl propanoid synthesis (giri et al., 2012). mucilage content the variation in mucilage content was evaluated under in vitro salt stress. the analysis of variance showed significant differences between evaluated genotypes, salt levels and genotype × salt interaction for mucilage content (tab. 1). grand mean comparison of mucilage content showed slight increase from control (0.11 g g-1 dw) to 0.13 g g-1 dw at 100 mm nacl (tab. 2). the increase in mucilage (as a secondary metabolite) content under salt stress has been reported in such few species as salicornia brachiate (jaiswar and kazi, 2016) under in vivo conditions. a decrease in mucilage content at 200 mm nacl (0.115 g g-1 dw), rather than 100 mm nacl (0.13 g g-1 dw), may suggest the inhibitory effect of nacl at 200 mm on in vitro mucilage synthesis in psyllium. no steady trend was observed in the variations of mucilage content in the callus subjected to different salt treatments (tab. 2). mucilage content increased in all the genotypes as salt increased from control (0 mm) to 100 mm nacl and decreased afterward at 200 mm nacl (tab. 2). there were significant variations among genotypes for in vitro mucilage content of callus grown at control, 100 mm and 230 p. golkar, f. amooshahi, a. arzani 200 mm nacl treatments. the highest mucilage content at 100 mm (nacl) (0.20 g g-1 dw) was produced by khor-biabanak genotype. mucilage is a polysaccharide mixture with highly variable chemical constituents, which may have played an important role in the salt tolerance through modulating water retention and ion homeostasis in plants (ghanem et al., 2010). a strong and positive relationship was found between mucilage content and na+ content affected by 200 mm nacl at cellular level (fig. 3). this finding may be a clue to the physiological role of mucilage in salt tolerance of mucilage-containing plants and deserve further investigation. furthermore, it is in keeping with our goal of the eliciting effects of salt stress on the mucilage content at the cellular level. in vitro mucilage extraction and preparation could be an alternative and possible way to produce this medicinal substance. conclusions substantial variations were detected in the cellular responses of the different genotypes of psyllium to in vitro nacl stress. although, our data provides evidence of increasing mucilage content under in vitro mild salt stress in a mucilage-containing plant, dissecting the phy siological and molecular role of mucilage in regulating ion and water status deserves further attentions. acknowledgments the financial support of this research was provided by research institute for biotechnology and bioengineering at isfahan university of technology, iran under grant #93-02-20. references ahmad, m.s.a., javed, f., javed, s., alvi, a.k., 2009: relationship between callus growth and mineral nutrients uptake in salt-stressed indica rice callus. j. plant nutr. 32, 382-394. doi: 10.1080/01904160802660719 al-khayri, j.m., 2002: growth, proline accumulation, and ion content in sodium chloride-stressed callus of date palm. in vitro cell. dev. biol. plant 38, 79-82. doi: 10.1079/ivp2001258 alvarez, i., tomaro, l.m., bernavides, p.m., 2003: changes in polyamines, proline and ethylene in sunflower calluses treated with nacl. plant cell 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ion transport starting from halophytes and stepping to genetic and protein engineering for manipulating ion fluxes. front plant sci. 6, 873. doi: 10.3389/fpls.2015.00873 zhu, j.k., 2002: salt and drought stress signal transduction in plants. ann. rev. plant biol. 53, 247-273. doi: 10.1146/annurev.arplant.53.091401.143329 zia, a., rezanejad, f., safarnejad, a., 2010: in vitro selection for nacl tolerance in thymus vulgaris l. j. cell mol. res. 2, 86-92. address of the corresponding author: ahmad arzani, department of agronomy and plant breeding, college of agriculture, isfahan university of technology, isfahan 84156-83111, iran e-mail: a_arzani@cc.iut.ac.ir © the author(s) 2017. this is an open access article distributed under the terms of the creative commons attribution share-alike license (http://creativecommons.org/licenses/by-sa/4.0/). journal of applied botany and food quality 91, 281 286 (2018), doi:10.5073/jabfq.2018.091.036 1federal university of são francisco valley, center of agrarian sciences, agronomic engineering course, petrolina, pernambuco, brazil 2produtiva consulting, juazeiro, bahia, brazil a new approach to induce mango shoot maturation in brazilian semi-arid environment ítalo herbert lucena cavalcante1*, guilherme neves ferreira dos santos1, marcelle almeida da silva1, rogério dos santos martins2, augusto miguel nascimento lima1, pedro igor rodrigues modesto1, adilson monteiro alcobia1, tullyus rubens de souza silva1, renata araújo e amariz1, márkilla zunete beckmann-cavalcante1 (submitted: may 15, 2018; accepted: july 26, 2018) * corresponding author summary the shoot maturation phase is important for growing mangoes because it precedes the floral induction, when plants are under stress caused by high temperatures and low water availability. abiotic stress could be alleviated by using plant biostimulant which alters the phytohormone and carbohydrates biosynthesis. thus, an experiment was conducted to evaluate the use a plant biostimulant con taining ascophyllum nodosum to induce shoot maturation of mango cv. palmer grown in semi-arid environment. the experimental design consisted of randomized blocks with four treatments, ten replications and five plants per parcel. treatments consisted of: t1) biostimulant foliar spray + k fertilizer; t2) foliar spray with bio stimulant alternating with k fertilizer; t3) individual foliar sprays with magnesium sulfate, potassium sulfate, sulfur and calcium ferti lizers, potassium fertilizer and ethrel®; and t4) control treatment. the total soluble carbohydrates in leaves, buds and shoots, n, k and s leaf concentrations and fruit production were recorded. the carbohydrate concentrations, nitrogen, sulphur and potassium leaf concentrations and fruit production of mango depend on shoot maturation strategy. shoot maturation strategy using biostimulant containing ascophyllum nodosum alternating with k fertilizer from 30 days after pbz could be recommended for the production of mango cv. palmer. key words: mangifera indica l., biostimulant, fruit production, carbohydrate, algae extract. introduction mango (mangifera indica) is a very important fruit crop for brazil, which is the seventh most producing country and one of the largest mango exporter countries (fao, 2017). in brazil, mango is especially grown in são francisco valley, where more than 90% of brazilian exported mango is produced (alicew eb, 2017). mango flowering has been demonstrated in field and in the scientific literature to be a complex event dependent of many factors concerning to climate conditions detaching that temperature is the most influential climatic factor (la x m a n et al., 2016), nutritional status (cava lca n t e et al., 2016; ca r n ei ro et al., 2017), pruning (asr ey et al., 2013), hormone balance (ra m í r e z et al., 2014), carbohydrate concentration (pr asa d et al., 2014; ku m a r et al., 2014) and shoot maturation of the last vegetative flush, which has been measured as age of the last flush of vegetative stems, and it appears to be the primary factor regulating floral induction in warm climates (ra m í r e z et al., 2010). mango shoot suitable for flowering induction was studied by dav en p ort et al. (2006) who concluded that terminal stems (terminal intercalary units) must have attained a dark green color and achieved a minimum age of 4 months since the previous limp, redleaf stage in easily induced cultivars and 5 months for the more re calcitrant cultivars to obtain a reproductive shoot response in the low-latitude tropics; and ra m í r e z et al. (2010) that stem age was the key factor correlated with flowering in the tropical conditions. in addition, it is necessary to stablish the physiological characteristics of a mango shoot suitable for flowering, which is defined as a ‘mature shoot’. shoot maturation does not occur uniformly throughout the tree ś canopy and it is affected by shoot age, climate conditions and orchard management (non-published data), that could present crucial importance, especially for mango trees grown under adverse climate as occurs in são francisco valley. in this region mango has been grown under very high temperature and low air humidity averages, reaching 36.8 °c and 25.6% especially from september to december months (la bm et, 2016). this is a critical phase because it coincides with the pre-flowering-induction stage of those mango trees planned to produce fruits in april-may of the next year. in addition, during pre-flowering-induction phase mango is irrigated with lower water amounts, which is necessary to stimulate ethylene production and improve mango flowering (ra m í r e z and dav en p ort, 2016). under adverse climate conditions, the use of biostimulants could be an option to improve shoot maturation, especially those containing algae extracts such as ascophyllum nodosum, that promotes bene fic effects on plant physiology but the more substantial improve ments are associated with improving tolerance toward abiotic stresses, including drought stress (spann and little, 2011), ion toxicity (mancuso et al., 2006) and high temperature (zhang and ervin, 2008). indeed, according to wally et al. (2013) a. nodosum leaf sprays enhanced total concentration of cytokinins, abscisic acid (aba) and aba catabolite levels, whereas auxin levels reduction. the plant nutritional status also affects mango flowering (gen ú and pi n to, 2002), then the fertilizing management, especially for potassium, sulphur and nitrogen is crucial during the phase that precedes flowering induction, i.e., the shoot maturation. the key effect of potassium in plants is related to photosynthesis, plant respiration and solute translocations (ma rsch n er, 2012); and sulphur is used on yang cycle for ethylene biosynthesis (pe ssa r a k li, 2014). on the other hand, at the time of flowering induction nitrogen concentrations should be in the lower average limit of adequate supply in order to avoid new vegetative flushes instead of reproductive phase (dav enp ort et al., 2003). hence, the present study aimed to use a plant biostimulant containing ascophyllum nodosum extract to induce shoot maturation of mango cv. palmer grown in brazilian semi-arid environment. material and methods plant material and growth conditions ‘palmer’ mango (mangifera indica l.) plants with uniform size and vigor, that were four years old thus in the first production cycle, were used in this study. the study was conducted from 2016 to 2017 in two experimental orchards located in sebastião da manga farm in casa nova county 282 í.h.l. cavalcante, g.n.f. dos santos, m.a. da silva, r.s. martins, a.m.n. lima, p.i.r. modesto, a.m. alcobia, t.r.s. silva, r. araújo e amariz, m.z. beckmann-cavalcante (09°11’ s and 41º59’ w; at an altitude of 400.3 m above sea level), bahia state, brazil. the climate of this region is classified as bswh (köeppen), which corresponds to a semi-arid region. the average air temperature and air humidity ranged from 20.7 ºc to 35.4 ºc and from 61.4% and 88.7%, respectively, with accumulated precipitation of 194.8 mm. the plants, spaced with 5.0 m between the rows and 2.3 m between the plants, were daily drip-irrigated with one emitter at each 0.5 m, for a flow of 2.5 l h-1 each. all management practices such as pruning, control of weeds, pests and diseases, plant growth regulators for gibberellin inhibition (cultar®) and break dormancy (calcium nitrate) were performed following the instructions of gen ú and pi n to (2002). the nutrient management was performed through a fertirrigation system, according to plant demand (gen ú and pi n to, 2002). treatments and experimental design the experimental design consisted of randomized blocks with four treatments, ten replications and five plants in each parcel, reaching 50 plants per treatment. treatments consisted of four strategies for shoot maturation of mango, as follows: t1) biostimulant foliar spray (2.5 ml l) + k fertilizer (2.5 ml l, 30% k2o); t2) foliar spray with biostimulant (2.5 ml l) alternating with k fertilizer (2.5 ml l, 30% k2o); t3) individual foliar sprays [magnesium sulfate (20 g l, 14% s and 10% mg), potassium sulfate (25 g l, 17% s, 48% of k2o and 1.2% mg), sulfur and calcium fertilizers (8 g l, 50% s and 5% ca), potassium fertilizer (25 ml l, 55% k2o), and ethrel® (3ml l)]; and t4) control treatment. the biostimulant used contains n (1.3%), k2o (1.0%), ca (3.0%), mg (3.0%), s (3.56%), mo (0.5%), organic carbon (2.69%), ascophyllum nodosum extract (1.5%), amino acids (5.0%) and fulvic acids (2.0%). the treatments were applied according to the number of the days after the gibberellin inhibitor was applied, paclobutrazol (pbz) ([(2rs, 3rs)-1-(4-clorofenil)-4,4-dimetil-2-(1h-1,2,4-triazol-1-il) pentan-3-ol]), in a ten days interval, beginning at 30 days after pbz (t2) and 60 days after pbz (t1 and t3), based on the traditional shoot maturation sprays (t3) used commercially. data gathered and statistical analysis according to recommendations of si lva (2009) leaf samples were collected in shoots from the middle part of the canopy to perform the potassium, sulphur [k and s at the beginning, middle and end of shoot maturation (flower induction)] and nitrogen (at the end of shoot maturation, which coincides with flowering induction) leaf concentrations. leaves were chemically analyzed after they were washed and rinsed with distilled water and dried at 65 oc until reach constant weight following methodology described by si lva (2009). the k concentrations were determined by flame photometry, the n concentrations by kjeldahl method and the s contents were measured by turbidometry. the total soluble carbohydrates (tsc) concentrations were quantified in leaves, buds and shoots from the beginning of shoot maturation until flowering induction, reaching five evaluation dates, following the methodology described by du bois et al. (1956). at harvest the number of fruits per plant, fruit production and fruit yield (t ha-1) was calculated. only commercial fruits were manually harvested in a single day when they reached the physiological maturity which was characterized through pulp color (yellow cream), following the fruit selection parameter recommended by the br a z i li a n p rogr a m for hort icu lt u r e moder n i z at ion (2004) for commercial farms. statistical analyses included analysis of variance (anova) using combined data from the two experimental areas. all calculations were performed using the assistat 7.7 software, and terms were considered significant at p < 0.01. results and discussion all variables studied were affected by shoot maturation strategies, including those variables evaluated with time elapsing. total soluble carbohydrates depended on shoot maturation strategy independently of the plant part (shoot, leaf or bud) (fig. 2). as can be seen in fig. 2a, leaf carbohydrate concentrations of all treatments increased from the first to the second evaluation date but only t4 presented a peak at the third date. a similar data distribution presented in fig. 2 was also recorded by ur ba n et al. (2006) in study about season effects on leaf nitrogen partitioning and photosynthetic water use efficiency in mango. indeed, from the third to the forth date it was registered a huge decrease in this variable especially for t4 (346%), t1 (250%) and t3 (154%), followed by stabilization at the end of shoot maturation (flower induction), when t2 was statistically higher than other treatments. the higher leaf carbohydrate concentration of t2 at the end of shoot maturation stage (flower induction) could be explained by the positive effects of ascophyllum nodosum extract applied. according to wa lly et al. (2013) such products contain growth regulators (auxins, cytokinins and gibberellins), betain, amino acids and low concentrations of inorganic elements that influence cell growth and division cycle, expansion, nutrition and maturity. on the other hand, kh a n et al. (2009) explained that the mechanism(s) of actions of seaweed extract-elicited physiological responses are largely unknown and the algae extract effect on plants depends on plant species, soil, climate and crop management at all. the leaf carbohydrate concentration of fig. 2a are close to that recorded by ur ba n et al. (2006) and higher than average value quoted by pr asa d et al. (2014) for the same phenological phase of the pre sent study. in shoots (stem of the last vegetative flush) total soluble carbohydrates concentration also presented the same tendency for t2 and t4, i.e., average enhancement until the third evaluation and stabilization for the last evaluation date (fig. 2b). carbohydrate concentrations in shoots were lower than those registered in leaves, independently of the evaluation date, but at the end of shoot maturation phase t2 also presented significantly higher carbohydrate concentration that fig. 1: time scale of the main events during the experiments. flowering induction t1 spray t1 spray t1 spray t1 spray wbr begins t3 spray t3 spray t3 spray t3 spray t2 spray t2 spray t2 spray t2 spray t2 spray t2 spray wbr ends fruit harvest 30 40 50 60 70 80 90 240 days after pbz application wbr begins = water blade reduction begins; wbr ends = water blade reduction ends a new approach to induce mango shoot maturation 283 is a positive scenario since source-to-sink transport of sugar is one of the major determinants of plant growth and relies on the efficient and controlled distribution of sucrose (and some other sugars such as raffinose and polyols) across plant organs through the phloem. however, sugar transport through the phloem can be affected by many environmental factors that alter source sink relationships (lemoi n e et al., 2013). it is important to note the lower carbohydrate concentrations decrease recorded for t2 in both leaves and shoots from the middle to the end of shoot maturation phase, showing that plants of this treatment at the flowering induction (end of the shoot maturation) presented higher soluble carbohydrate concentrations. the other hand, soluble carbohydrates in buds (fig. 2c) presented a different data distribution as a function of the dates during shoot maturation, except for t2. it is relevant to point the extremely higher average recorded to the non-treated plants at the end of shoot maturation, because these plants presented very poor or no flowers, showing that for mango high carbohydrate concentration in buds is not a key for good flowering and fruit production. whether compared to pr asa d et al. (2014) data it is verified that average value recoded for t4 (control treatment) is extremely higher. leaf concentrations for potassium, sulphur and nitrogen depended on shoot maturation strategy (fig. 3). independently of the treatment, k leaf concentrations enhanced with time elapsing until reach a peak at the end of shoot maturation when t2 and t3 were above the other treatments. during shoot maturation phase the key effect of potassium in plants is related to photosynthesis, plant respiration and solute translocations (ma rsch n er, 2012). whether compared with ma lavolta et al. (1997) adequate range of supply (4.0-6.0 g kg-1), it verifies that at the end of shoot matu ration (flower induction) plants of all treatments presented higher averages. the other way, for the adequate range of supply reported by pi m plask er and bh a rgava (2003), i.e. 1.02-2.01% (or 10.220.1 g kg-1), plants of all treatments had adequate k leaf concentrations. comparatively, the average foliar potassium values presented in fig. 3a were higher than those recorded by cava lca n t e et al. (2016) in study about k fertilizing of ‘palmer’ mango under semiarid environment. sulphur leaf concentrations of t1, t2 and t4 were similar among them and presented a soft variation during shoot maturation phase (fig. 3b), but, t3 promoted a s leaf concentration peak at the middle of shoot maturation, followed by a huge decay of 300% until the end of this phase. this data distribution could be caused by the foliar spray with sulphates performed in t3 followed by ethrel® leaf fig. 2: total soluble carbohydrates concentrations in leaves (a), shoots (b) and buds (c) of mango cv. palmer as a function of branch maturation strategy in five evaluation dates. points with the same letters do not differ in among themselves by tukey’s test at 1% probability in each evaluation date. t1) biostimulant foliar spray + k fertilizer; t2) foliar spray with biostimulant alternating with k fertilizer; t3) individual foliar sprays (magnesium sulfate, potassium sulfate, sulfur and calcium fertilizers, potassium fertilizer, and ethrel®; and t4) control treatment. fm = fresh mass. 30 50 70 90 110 t1 t2 t3 t4 01 11 18 11 28 11 07 12 16 12 a b a a a b b b a a b c ar bo hy dr at es in s ho ot s (m g g1 o f f m ) evaluation dates ! evaluation dates ! evaluation dates 50 100 150 200 250 300 t1 t2 t3 t4 01 11 18 11 28 11 07 12 16 12 a a a a b b a a a c ar bo hy dr at es in le av es (m g g1 o f f m ) b 0 50 100 150 200 250 t1 t2 t3 t4 c ar bo hy dr at es in b ud s (m g g -1 of f m ) a a a a a b a b b a 01 11 18 11 28 11 07 12 16 12 a b c 30 50 70 90 110 t1 t2 t3 t4 01 11 18 11 28 11 07 12 16 12 a b a a a b b b a a b c ar bo hy dr at es in s ho ot s (m g g1 o f f m ) evaluation dates ! evaluation dates ! evaluation dates 50 100 150 200 250 300 t1 t2 t3 t4 01 11 18 11 28 11 07 12 16 12 a a a a b b a a a c ar bo hy dr at es in le av es (m g g1 o f f m ) b 0 50 100 150 200 250 t1 t2 t3 t4 c ar bo hy dr at es in b ud s (m g g -1 of f m ) a a a a a b a b b a 01 11 18 11 28 11 07 12 16 12 a b c 284 í.h.l. cavalcante, g.n.f. dos santos, m.a. da silva, r.s. martins, a.m.n. lima, p.i.r. modesto, a.m. alcobia, t.r.s. silva, r. araújo e amariz, m.z. beckmann-cavalcante spray, an ethylene precursor, in which production cycle uses s for yang cycle (pe ssa r a k li, 2014). independently of the shoot maturation strategy studied, all treatments were within the stated adequate range of supply 1.1-1.7 g kg-1 properly described by pi m plask er and bh a rgava (2003). as can be seen in fig. 4c, n leaf concentrations at the end of shoot maturation (flower induction) also depended on shoot maturation strategies, with statistically high average recorded for t4 (control treatment). according to pi m plask er and bh a rgava (2003) the n leaf concentration considered as adequate for mangoes range from 8.9 to 19.3 g kg-1, but there is controversy because according to dav en p ort et al. (2003) the leaf n levels for mango should be at 11 to 14 g kg-1 at the time of flowering induction in order to avoid new vegetative flushes instead of reproductive phase, i.e., n leaf concentration should be in maximum 14 g kg-1. on the other hand, in the present study t1, t2 and t3 presented commercial fruit yields with leaf n concentrations of 14.4, 14.7 and 15.1 g kg-1 respectively, while t4 presented no fruit production with n leaf concentration of 18.1 g kg-1 showing that adequate maximum n leaf concentration at flowering induction depends on mango cultivar and the production system used. fruit production variables depended on treatments studied (fig. 4), and that plants of control treatment presented a very poor fruit production, including lots of parcels presented no fruit production making not possible to perform statistical analysis for this treatment. fruit production variables presented the same data tendency, i.e., t2 promoted the higher number of fruits per plant, fruit production and fruit yield (fig. 4). for number of fruits per plant t2 presented an average almost 46% higher than t3, the second higher average, and it reached 94.8 commercial fruits per plants. cava lca n t e et al. (2016) recorded a maximum number of commercial fruits per plant of 294, but in eleven years old plants, that’s different from the current study which used four years old plants, in the first production cycle. fruit production (kg per plant) ranged from 28.14 kg plant (t1) to 38.62 kg plant, which is lower than other scientific results in the literature such as cava lca n t e et al. (2016), wa h da n et al. (2011) in egypt, 82.71-88.86 kg plant reported by ye sh i t ela et al. (2005) in south africa, qu i ja da et al. (2009) in venezuela and sa r a n and ku m a r (2011) in india. on the other hand, except for cava lca n t e et al. (2016), all authors quote the total fruit production per plant and not a commercial fruit production, in orchards with wide spaces between plants were, naturally, the plants are higher and produce more fruits, so the crucial comparison must refer to fruit yield. following the same tendency of the number of fruits per plant and fruit production (kg plant), the commercial fruit yield (t ha) was significantly higher to t2 which promoted an average of 33.6 t ha (fig. 4c), thus 20% and 27% higher than those quoted to t3 (26.89 t ha) and t1 (24.48 t ha) respectively. the fruit yield promoted by t2 is compatible to average value recorded by ba r bosa et al. (2016) (36.6 t ha) for ‘palmer’ and higher than 27 t ha of cava lca n t e et al. (2016) for ‘palmer’, 10.5 ton ha-1 of plegu e z u elo et al. (2012) in spain as well as higher than in the main worldwide producer countries such as brazil (16.1 t ha), china (8.2 t ha), india (7.3 t ha) and mexico (8.9 t ha) (fao, 2017). 0 1 2 3 4 5 6 7 t1 t2 t3 t4 01 11 28 11 19 12 s le af c on ce nt ra tio n (g k g1 ) 14.14 b 14.7 b 15.07 b 18.11 a 0 2 4 6 8 10 12 14 16 18 20 t1 t2 t3 t4 n le af c on ce nt ra tio n (g k g1 ) b branchmaturation strategy c 4 6 8 10 12 14 16 t1 t2 t3 t4 01 11 28 11 19 12 k le af c on ce nt ra tio n (g k g1 ) a a a a a a b b evaluation dates ba evaluation dates fig. 3: leaf concentrations of potassium (a), sulphur (b) at the beginning, middle and end of shoot maturation; and nitrogen (c) at the end of shoot maturation of mango cv. palmer as a function of shoot maturation strategy. points or bars with the same letters do not differ in among themselves by tukey’s test at 1% probability in each evaluation date. t1) biostimulant foliar spray + k fertilizer; t2) foliar spray with biostimulant alternating with k fertilizer; t3) individual foliar sprays (magnesium sulfate, potassium sulfate, sulfur and calcium fertilizers, potassium fertilizer, and ethrel®; and t4) control treatment. a new approach to induce mango shoot maturation 285 the superiority recorded for the treatment with algae extract leaf spray applied individually could also be explained by the anti-stress characteristics of this product since during maturation phase the temperatures were high (36.8 ºc) the air humidity was low (25.6%) and mango plants were exposed to water blade reduction. this way, la n e et al. (2006) argue that ascophyllum nodosum contain the polysaccharides laminaran, fucoidan, and alginate and laminarin has been shown to stimulate natural defense responses in plants and is involved in the induction of genes encoding various pathogenesisrelated proteins with antimicrobial properties. in a general form, positive effects of ascophyllum nodosum extracts were previously reported by mor a le s-paya n (2013) during seedling formation phase and moh a m ed and el-seh r aw y (2013), who recorded better plant nutrition and fruit retention of mango. conclusion thus, the results of this study indicate that: i) carbohydrate concentrations, nitrogen, sulphur and potassium leaf concentrations and fruit production of mango ‘palmer’ depend on shoot maturation strategy; ii) under the soil, climate and plant conditions of this study, shoot maturation strategy using foliar spray with biostimulant containing ascophyllum nodosum alternating with k fertilizer from 30 days after pbz use could be recommended for the production of mango ‘palmer’ in the semi-arid environment; 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